HK40087274B - Kras g12c inhibitors and methods of using the same - Google Patents

Description

KRAS G12C Inhibitors and Their Usage

This application is a divisional application. The original application was filed on May 21, 2018, with application number 201880047910.1 (PCT/US2018/033714) and the invention title was "KRAS G12C Inhibitor and Method of Using Thereof".

Technical Field

This article provides information on KRAS G12C inhibitors, their compositions, and methods of use. These inhibitors can be used to treat a variety of conditions, including pancreatic cancer, colorectal cancer, and lung cancer.

Background Technology

KRAS gene mutations are common in pancreatic cancer, lung adenocarcinoma, colorectal cancer, gallbladder cancer, thyroid cancer, and bile duct cancer. KRAS mutations are also observed in approximately 25% of NSCLC patients, and some studies suggest that KRAS mutations are a poor prognostic factor for NSCLC patients. Recently, mutations in the V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) have been found to confer resistance to epidermal growth factor receptor (EGFR) targeted therapy in colorectal cancer; therefore, KRAS mutation status can provide important information before prescribing TKI therapy. In conclusion, there is a need for novel medical treatments for patients with pancreatic cancer, lung adenocarcinoma, or colorectal cancer, especially those diagnosed with these cancers characterized by KRAS mutations, and including those whose disease has progressed after chemotherapy.

Summary of the Invention

This article provides compounds having the structure of formula (I).

in

^E1 and ^E2 are each independently N or ^CR1 ;

^R1 is independently H, hydroxyl, _C1-6 alkyl, _C1-6 haloalkyl, _C1-6 alkoxy, NH- _C1-6 alkyl, N( _C1-6 alkyl) ₂ , cyano, or halo.

^R2 is a halogroup, _C1-6 alkyl, _C1-6 haloalkyl, OR', N(R') ₂ , _C2-3 alkenyl, C2-3 ynyl, _C0-3 alkylene- _C3-14 cycloalkyl, _C0-3 alkylene-C2-14 heterocycloalkyl, aryl, heteroaryl, _C0-3 alkylene aryl, or _C0-3 alkylene heteroaryl, and each R' is independently H, _{C1-6 alkyl} , _{C1-6 haloalkyl} , _C3-14 cycloalkyl, _C2-14 heterocycloalkyl, _C2-3 alkenyl, _C2-3 ynyl, aryl, or heteroaryl, or two R' substituents together with the nitrogen atom to which they are attached form a 3-7 membered ring;

^R3 is a halogroup, _C1-3 alkyl, _C1-2 haloalkyl, _C1-3 alkoxy, _C3-4 cycloalkyl, _C2-3 alkenyl, _C2-3 alkynyl, aryl, or heteroaryl.

^R4 is

Ring A is a single ring (4-7 members), a double ring, a bridged ring, a fused ring, or a spiral ring (6-11 members).

L is a bond, a _C1-6 alkylene group, a -OC _0-5 alkylene group, a -SC _0-5 alkylene group, or a -NH-C _0-5 alkylene group, and for _C2-6 alkylene groups, a -OC _2-5 alkylene group, a -SC _2-5 alkylene group, and an NH-C _2-5 alkylene group, one carbon atom of the alkylene group may optionally be replaced by O, S, or NH;

R ^4' is H, _C1-6 alkyl, C2-6 ynyl, _C1-6 alkylene- _OC1-4 alkyl, _C1-6 alkylene-OH, _C1-6 haloalkyl, cycloalkyl, heterocycloalkyl, _C0-3 alkylene- _C3-4 cycloalkyl, _C0-3 alkylene- _C2-14 heterocycloalkyl, aryl, heteroaryl, _C0-3 alkylene- _C6-14 aryl, or selected from H, _C1-6 alkyl, C2 ...

^R5 and ^R6 are each independently H, a halogroup, _C1-6 alkyl, _C2-6 ynyl, _C1-6 alkylene- _OC1-4 alkyl, C1-6 alkylene-OH, _C1-6 haloalkyl, _C1-6 alkyleneamine, _C0-6 alkylene-amide, _C0-3 alkylene-C(O)OH, _C0-3 alkylene-C(O) _OC1-4 alkyl, _C1-6 alkylene-O-aryl, _C0-3 alkylene-C(O) _C1-4 alkylene-OH, cycloalkyl, heterocycloalkyl, _aryl , heteroaryl, C0-3 alkylene- _C3-14 cycloalkyl, _C0-3 alkylene- _C2-14 heterocycloalkyl, _C0-3 alkylene- _{C6-14 aryl} , _C0-3 alkylene- _C2-14 heteroaryl or cyano, or ^R5 and R ^{The 6-} membered ring, together with the atoms attached to it, forms a 4-6 membered ring; and

^R7 is an H or _C1-8 alkyl group, or ^R7 and ^R5 together with the atoms to which they are attached form a 4-6 membered ring, or a pharmaceutically acceptable salt thereof.

In another embodiment, compounds having the structure of formula (I) are provided herein.

in

^E1 and ^E2 are each independently N or ^CR1 ;

^R1 is independently H, hydroxyl, _C1-6 alkyl, _C1-6 haloalkyl, _C1-6 alkoxy, NH- _C1-6 alkyl, N( _C1-4 alkyl) ₂ , cyano, or halo.

^R2 is a halogroup, _C1-6 alkyl, _C1-6 haloalkyl, OR', N(R') ₂ , _C2-3 alkenyl, _{C2-3 ynyl, C0-3 alkylene} - _C3-14 cycloalkyl, _C0-3 alkylene- _C2-14 heterocycloalkyl, aryl, heteroaryl, _C0-3 alkylene- _C6-14 aryl or C0-3 alkylene- _C2-14 heteroaryl, and each R' is independently H, _{C1-6 alkyl} , _C1-6 haloalkyl, _C3-14 cycloalkyl, _C2-3 alkenyl, _C2-3 ynyl, aryl or heteroaryl, or two R' substituents together with the nitrogen atom to which they are attached form a 3-7 membered ring;

^R3 is a halogroup, _C1-3 alkyl, _C1-2 haloalkyl, _C1-3 alkoxy, _C3-14 cycloalkyl, _C2-3 alkenyl, _C2-3 alkynyl, aryl, or heteroaryl.

^R4 is

Ring A is a single ring (4-7 members), a double ring, a bridged ring, a fused ring, or a spiral ring (6-11 members).

L is a bond, a _C1-6 alkylene group, a -OC _0-5 alkylene group, a -SC _0-5 alkylene group, or a -NH-C _0-5 alkylene group, and for _C2-6 alkylene groups, a -OC _2-5 alkylene group, a -SC _2-5 alkylene group, and an NH-C _2-5 alkylene group, one carbon atom of the alkylene group may optionally be replaced by O, S, or NH;

^R5 and ^R6 are each independently H, a halogroup, _C1-8 alkyl, _C2-8 ynyl, _C1-6 alkylene- _OC1-4 alkyl, _C1-6 alkylene-OH, C1-6 haloalkyl, _C1-6 alkyleneamine, _C0-6 alkylene-amide, _C0-3 alkylene-C(O)OH, _C0-3 alkylene-C(O) _OC1-4 alkyl, _C1-6 alkylene-O-aryl, _C0-3 alkylene-C(O) _C1-4 alkylene-OH, cycloalkyl, heterocycloalkyl, _aryl , heteroaryl, C0-3 alkylene- _C3-14 cycloalkyl, _C0-3 alkylene- _C2-14 heterocycloalkyl, _C0-3 alkylene- _{C6-14 aryl} , _C0-3 alkylene- _C2-14 heteroaryl or cyano, or ^R5 and R ^{The 6-} membered ring, together with the atoms attached to it, forms a 4-6 membered ring; and

^R7 is an H or _C1-6 alkyl group, or ^R7 and ^R5 together with the atoms to which they are attached form a 4-6 membered ring, or a pharmaceutically acceptable salt thereof.

Additionally, compounds having formula (II) or pharmaceutically acceptable salts thereof are provided:

^E1 and ^E2 are each independently N or ^CR1 ; J is N, ^NR10 , or ^CR10 ; M is N, ^NR13 , or ^CR13 ; and can be single or double bonds as needed to ensure each atom exhibits its normal valence state; ^R1 is independently H, hydroxyl, _C1-6 alkyl, _C1-4 haloalkyl, _{C1-4 alkoxy, NH-C1-4 alkyl} , N( _C1-4 alkyl) ₂ , cyano, or halo; ^R2 is halo, _C1-6 alkyl, _C1-6 haloalkyl, OR', N(R') ₂ , _C2-3 alkenyl, _C2-3 alkynyl, _C0-3 alkylene- _C3-14 cycloalkyl, _C0-3 alkylene- _C2-14 heterocycloalkyl, aryl, heteroaryl, _C0-3 alkylene- _C6-14 aryl, or C _0-3 alkylene- _C2-14 heteroaryl, wherein each R' is independently H, _C1-6 alkyl, _C1-6 haloalkyl, _C3-14 cycloalkyl, _C2-14 heterocycloalkyl, _C2-3 alkenyl, _C2-3 ynyl, aryl or heteroaryl, or two R' substituents together with the nitrogen atom to which they are attached form a 3-7 membered ring; ^R3 is haloyl, _C1-3 alkyl, _C1-2 haloalkyl, _C1-3 alkoxy, _C3-4 cycloalkyl, _C2-14 heterocycloalkyl, _C2-3 alkenyl, _C2-3 ynyl, aryl or heteroaryl;

^R4 is a ring A, which is a monocyclic 4-7 membered ring or bicyclic, bridged, fused, or spirocyclic 6-11 membered ring; L is a bond, _C1-6 alkylene, -OC _0-5 alkylene, -SC _0-5 alkylene, or -NH-C _0-5 alkylene, and for _C2-6 alkylene, -OC _2-5 alkylene, -SC _2-5 alkylene, and NH-C _2-5 alkylene, one carbon atom of the alkylene group may optionally be replaced by O, S, or NH; R4 ^' is H, _C1-8 alkyl, _C2-8 alkynyl, _C1-6 alkylene-OC _1-4 alkyl, _C1-6 alkylene-OH, _C1-6 haloalkyl, cycloalkyl, heterocycloalkyl, C _0-3 alkylene-C _3-14 cycloalkyl, C _0-3 alkylene-C _2-14 heterocycloalkyl, aryl, heteroaryl, C _0-3 alkylene-C _6-14 aryl, or selected from

_R5 and ^R6 are each independently H, a halogroup, _C1-6 alkyl, _C2-6 ynyl, _C1-6 alkylene- _OC1-4 alkyl, C1-6 alkylene-OH, _C1-6 haloalkyl, _C1-6 alkyleneamine, _C0-6 alkylene-amide, _C0-3 alkylene-C(O)OH, _C0-3 alkylene-C(O) _OC1-4 alkyl, _C1-6 alkylene-O-aryl, _C0-3 alkylene-C(O) _C1-4 alkylene-OH, cycloalkyl, heterocycloalkyl, _aryl , heteroaryl, C0-3 alkylene- _C3-14 cycloalkyl, _C0-3 alkylene- _C2-14 heterocycloalkyl, _C0-3 alkylene- _{C6-14 aryl} , _C0-3 alkylene- _C2-14 heteroaryl or cyano, or ^R5 and R ^R6 , together with its attached atoms, forms a 4-6 membered ring; ^R7 is H or a _C1-8 alkyl, or ^R7 and ^R5, together with their attached atoms ^{, form a 4-6 membered ring; Q is CR8R9, C=CR8R9 , C =} O, C=S, or C= ^NR8 ; ^R8 and ^R9 are each independently H, _C1-3 alkyl, hydroxyl, _C1-3 alkoxy, cyano, nitro, or _C3-6 cycloalkyl, or ^R8 and ^R9 , together with their attached carbon atoms, can form a 3-6 membered ring; ^R10 is a _C1-8 alkyl, _C0-3 alkylene- _C6-14 aryl, _C0-3 alkylene- _C3-14 heteroaryl, _C0-3 alkylene- _C3-14 cycloalkyl, _C0-3 alkylene- _C2-14 heterocycloalkyl, C _1-6 alkoxy, OC _0-3 alkylene-C _6-14 aryl, OC _0-3 alkylene-C _3-14 heteroaryl, OC _0-3 alkylene-C _3-14 cycloalkyl, OC _0-3 alkylene-C _2-14 heterocycloalkyl, NH-C _1-8 alkyl, N(C _1-8 alkyl) ₂ , NH-C _0-3 alkylene-C _6-14 aryl, NH-C _0-3 alkylene-C _2-14 heteroaryl, NH-C _0-3 alkylene-C _3-14 cycloalkyl, NH-C _0-3 alkylene-C _2-14 heterocycloalkyl, halogen, cyano, or C _1-6 alkylene-amine; and

R ¹³ is a _C1-6 alkyl, _C1-6 haloalkyl, _C1-6 alkyleneamine, or _C3-14 cycloalkyl.

Or its pharmaceutically acceptable salt, subject to the following limitations:

(1) When J is NR ¹⁰ , M is N or CR ¹³ ;

(2) When M is NR ¹³ , J is N or CR ¹⁰ ;

(3) When J is CR ¹⁰ , M is N or NR ¹³ ; and

(4) When M is CR ¹³ , J is N or NR ¹⁰ .

In some embodiments, when Q is C=O and ^E1 and ^E2 are each ^CR1 , then (1) ^R10 is a _C1–3 alkylene aryl, _C1–3 alkylene heteroaryl, _C0–3 alkylene- _C3–8 cycloalkyl, _C1–3 alkylene- _C2–7 heterocycloalkyl, or haloyl; or (2) ^R13 is a _C1–3 haloalkyl or _C3–5 cycloalkyl. In various embodiments, J is ^NR10 and M is ^CR13 . In some embodiments, J is ^CR10 and M is ^NR13 . In some embodiments, J is N and M is ^NR13 . In various embodiments, J is ^NR10 and M is N.

Additionally, compounds having the structure of formula (II) are provided.

in

^E1 and ^E2 are each independently N or ^CR1 ;

J is N, NR ¹⁰ , or CR ¹⁰ ;

M is N, NR ¹³ , or CR ¹³ ;

Single or double bonds can be formed as needed to ensure that each atom exhibits its normal valence state;

^R1 is independently H, hydroxyl, _C1-4 alkyl, _C1-4 haloalkyl, _C1-4 alkoxy, NH- _C1-4 alkyl, N( _C1-4 alkyl) ₂ , cyano, or halo.

^R2 is a halogroup, _C1-6 alkyl, _C1-6 haloalkyl, OR', N(R') ₂ , _C2-3 alkenyl, _{C2-3 ynyl, C0-3 alkylene} - _C3-14 cycloalkyl, _C0-3 alkylene- _C2-14 heterocycloalkyl, aryl, heteroaryl, _C0-3 alkylene- _C6-14 aryl or C0-3 alkylene- _C2-14 heteroaryl, and each R' is independently H, _{C1-6 alkyl} , _C1-6 haloalkyl, _C3-14 cycloalkyl, _C2-14 heterocycloalkyl, _C2-3 alkenyl, _C2-3 ynyl, aryl or heteroaryl, or two R' substituents together with the nitrogen atom to which they are attached form a 3-7 membered ring;

^R3 is a halogroup, _C1-3 alkyl, _C1-2 haloalkyl, _C1-3 alkoxy, _C3-4 cycloalkyl, _C2-3 alkenyl, _C2-3 alkynyl, aryl, or heteroaryl.

^R4 is

Ring A is a single ring (4-7 members), a double ring, a bridged ring, a fused ring, or a spiral ring (6-11 members).

L is a bond, a _C1-6 alkylene group, a -OC _0-5 alkylene group, a -SC _0-5 alkylene group, or a -NH-C _0-5 alkylene group, and for _C2-6 alkylene groups, a -OC _2-5 alkylene group, a -SC _2-5 alkylene group, and an NH-C _2-5 alkylene group, one carbon atom of the alkylene group may optionally be replaced by O, S, or NH;

^R5 and ^R6 are each independently H, a halogroup, _C1-6 alkyl, _C2-6 ynyl, _C1-6 alkylene- _OC1-4 alkyl, C1-6 alkylene-OH, _C1-6 haloalkyl, _C1-6 alkyleneamine, _C0-6 alkylene-amide, _C0-3 alkylene-C(O)OH, _C0-3 alkylene-C(O) _OC1-4 alkyl, _C1-6 alkylene-O-aryl, _C0-3 alkylene-C(O) _C1-4 alkylene-OH, cycloalkyl, heterocycloalkyl, _aryl , heteroaryl, C0-3 alkylene- _C3-14 cycloalkyl, _C0-3 alkylene- _C2-14 heterocycloalkyl, _C0-3 alkylene- _{C6-14 aryl} , _C0-3 alkylene- _C2-14 heteroaryl or cyano, or ^R5 and R ^{The 6-} membered ring, together with the atoms attached to it, forms a 4-6 membered ring;

^R7 is H or _C1-8 alkyl, or ^R7 and ^R5 together with the atoms attached to them form a 4-6 membered ring;

Q is CR ⁸ R ⁹ , C=CR ⁸ R ⁹ , C=O, C=S or C=NR ⁸ ;

^R8 and ^R9 are each independently H, _C1-3 alkyl, hydroxyl, _C1-3 alkoxy, cyano, nitro or _C3-6 cycloalkyl, or ^R8 and ^R9 together with the carbon atom to which they are attached can form a 3-6 membered ring;

R ¹⁰ is _C1-8 alkyl, _C0-3 alkylene- _C6-14 aryl, C0-3 alkylene-C3-14 heteroaryl, _C0-3 alkylene- _C3-14 cycloalkyl, _C0-3 alkylene- _C2-14 heterocycloalkyl, _C1-6 alkoxy, _OC0-3 alkylene- _C6-14 aryl, OC0-3 alkylene- _C3-14 heteroaryl, _OC0-3 alkylene- _C3-14 cycloalkyl, _OC0-3 alkylene- _C2-14 heterocycloalkyl, NH- _C1-8 alkyl, N _{( C1-8} alkyl) ₂ , NH- _C0-3 alkylene- _{C6-14 aryl} , NH- _C0-3 alkylene- _C2-14 heteroaryl, NH _{-C0-3 alkylene} - _C3-14 cycloalkyl, NH-C _0-3 alkylene-C _2-14 heterocyclic alkyl, halogen, cyano or C _1-6 alkylene-amine;

Its restrictions are:

(1) When J is NR ¹⁰ , M is N or CR ¹³ ;

(2) When M is NR ¹³ , J is N or CR ¹⁰ ;

(3) When J is CR ¹⁰ , M is N or NR ¹³ ; and

(4) When M is CR ¹³ , J is N or NR ¹⁰ .

In some embodiments, when Q is C=O and ^E1 and ^E2 are each ^CR1 , then (1) ^R10 is a _C1–3 alkylene aryl, _C1–3 alkylene heteroaryl, _C0–3 alkylene- _C3–8 cycloalkyl, _C1–3 alkylene- _C2–7 heterocycloalkyl, or haloyl; or (2) ^R13 is a _C1–3 haloalkyl or _C3–5 cycloalkyl. In various embodiments, J is ^NR10 and M is ^CR13 . In some embodiments, J is ^CR10 and M is ^NR13 . In some embodiments, J is N and M is ^NR13 . In various embodiments, J is ^NR10 and M is N.

Additionally, compounds having formula (III) or (III’) or pharmaceutically acceptable salts thereof are provided:

^E1 and ^E2 are each independently N or ^CR1 ;

^R1 is independently H, hydroxyl, _C1-6 alkyl, _C1-4 haloalkyl, _C1-4 alkoxy, NH- _C1-4 alkyl, N( _C1-4 alkyl) ₂ , cyano, or halo.

^R2 is a halogroup, _C1-6 alkyl, _C1-6 haloalkyl, OR', N(R') ₂ , _C2-3 alkenyl, _{C2-3 ynyl, C0-3 alkylene} - _C3-14 cycloalkyl, _C0-3 alkylene- _C2-14 heterocycloalkyl, aryl, heteroaryl, _C0-3 alkylene- _C6-14 aryl or C0-3 alkylene- _C2-14 heteroaryl, and each R' is independently H, _{C1-6 alkyl} , _C1-6 haloalkyl, _C3-14 cycloalkyl, _C2-14 heterocycloalkyl, _C2-3 alkenyl, _C2-3 ynyl, aryl or heteroaryl, or two R' substituents together with the nitrogen atom to which they are attached form a 3-7 membered ring;

^R3 is a halogroup, _C1-3 alkyl, _C1-2 haloalkyl, _C1-3 alkoxy, _C3-4 cycloalkyl, _C2-14 heterocycloalkyl, _C2-3 alkenyl, _C2-3 alkynyl, aryl, or heteroaryl.

^R4 is

Ring A is a single ring (4-7 members), a double ring, a bridged ring, a fused ring, or a spiral ring (6-11 members).

L is a bond, a _C1-6 alkylene group, a -OC _0-5 alkylene group, a -SC _0-5 alkylene group, or a -NH-C _0-5 alkylene group, and for _C2-6 alkylene groups, a -OC _2-5 alkylene group, a -SC _2-5 alkylene group, and an NH-C _2-5 alkylene group, one carbon atom of the alkylene group may optionally be replaced by O, S, or NH;

R ^4' is H, _C1-8 alkyl, _C2-8 ynyl, C1-6 alkylene- _OC1-4 alkyl, _C1-6 alkylene-OH, _C1-6 haloalkyl, cycloalkyl, heterocycloalkyl, _C0-3 alkylene- _C3-14 cycloalkyl, _C0-3 alkylene- _C2-14 heterocycloalkyl, aryl, heteroaryl, _C0-3 alkylene-C6-14 aryl, or selected from H, _C1-8 alkyl, _C2-8 ynyl ...

^R5 and ^R6 are each independently H, a halogroup, _C1-6 alkyl, _C2-6 ynyl, _C1-6 alkylene- _OC1-4 alkyl, C1-6 alkylene-OH, _C1-6 haloalkyl, _C1-6 alkyleneamine, _C0-6 alkylene-amide, _C0-3 alkylene-C(O)OH, _C0-3 alkylene-C(O) _OC1-4 alkyl, _C1-6 alkylene-O-aryl, _C0-3 alkylene-C(O) _C1-4 alkylene-OH, cycloalkyl, heterocycloalkyl, _aryl , heteroaryl, C0-3 alkylene- _C3-14 cycloalkyl, _C0-3 alkylene- _C2-14 heterocycloalkyl, _C0-3 alkylene- _{C6-14 aryl} , _C0-3 alkylene- _C2-14 heteroaryl or cyano, or ^R5 and R ^{The 6-} membered ring, together with the atoms attached to it, forms a 4-6 membered ring;

^R7 is H or _C1-8 alkyl, or ^R7 and ^R5 together with the atoms attached to them form a 4-6 membered ring;

Q is CR ⁸ R ⁹ , C=CR ⁸ R ⁹ , C=O, C=S or C=NR ⁸ ;

^R8 and ^R9 are each independently H, _C1–6 alkyl, hydroxyl, _C1–6 alkoxy, cyano, nitro or _C3–14 cycloalkyl, or ^R8 and ^R9 together with the carbon atom to which they are attached can form a 3-6 membered ring;

R ¹⁰ is _C1-8 alkyl, _C0-3 alkylene- _C6-14 aryl, C0-3 alkylene-C3-14 heteroaryl, _C0-3 alkylene- _C3-14 cycloalkyl, _C0-3 alkylene- _C2-14 heterocycloalkyl, _C1-6 alkoxy, _OC0-3 alkylene- _C6-14 aryl, OC0-3 alkylene- _C3-14 heteroaryl, _OC0-3 alkylene- _C3-14 cycloalkyl, _OC0-3 alkylene- _C2-14 heterocycloalkyl, NH- _C1-8 alkyl, N _{( C1-8} alkyl) ₂ , NH- _C0-3 alkylene- _{C6-14 aryl} , NH- _C0-3 alkylene- _C2-14 heteroaryl, NH _{-C0-3 alkylene} - _C3-14 cycloalkyl, NH-C _0-3 alkylene-C _2-14 heterocyclic alkyl, halogen, cyano or C _1-6 alkylene-amine.

Additionally, compounds having formula (III) or (III’) or pharmaceutically acceptable salts thereof are provided:

in

^E1 and ^E2 are each independently N or ^CR1 ;

^R1 is independently H, hydroxyl, _C1-6 alkyl, _C1-4 haloalkyl, _C1-4 alkoxy, NH- _C1-4 alkyl, N( _C1-4 alkyl) ₂ , cyano, or halo.

^R2 is a halogroup, _C1-6 alkyl, _C1-6 haloalkyl, OR', N(R') ₂ , _C2-3 alkenyl, _{C2-3 ynyl, C0-3 alkylene} - _C3-14 cycloalkyl, _C0-3 alkylene- _C2-14 heterocycloalkyl, aryl, heteroaryl, _C0-3 alkylene- _C6-14 aryl or C0-3 alkylene- _C2-14 heteroaryl, and each R' is independently H, _{C1-6 alkyl} , _C1-6 haloalkyl, _C3-14 cycloalkyl, _C2-14 heterocycloalkyl, _C2-3 alkenyl, _C2-3 ynyl, aryl or heteroaryl, or two R' substituents together with the nitrogen atom to which they are attached form a 3-7 membered ring;

^R3 is a halogroup, _C1-3 alkyl, _C1-2 haloalkyl, _C1-3 alkoxy, _C3-4 cycloalkyl, _C2-14 heterocycloalkyl, _C2-3 alkenyl, _C2-3 alkynyl, aryl, or heteroaryl.

R ⁴ is a ring A is a single ring 4-7 elemental ring or double ring, bridged, fused or spiral ring 6-11 elemental ring;

L is a bond, a _C1-6 alkylene group, a -OC _0-5 alkylene group, a -SC _0-5 alkylene group, or a -NH-C _0-5 alkylene group, and for _C2-6 alkylene groups, a -OC _2-5 alkylene group, a -SC _2-5 alkylene group, and an NH-C _2-5 alkylene group, one carbon atom of the alkylene group may optionally be replaced by O, S, or NH;

^R5 and ^R6 are each independently H, a halogroup, _C1-6 alkyl, _C2-6 ynyl, _C1-6 alkylene- _OC1-4 alkyl, C1-6 alkylene-OH, _C1-6 haloalkyl, _C1-6 alkyleneamine, _C0-6 alkylene-amide, _C0-3 alkylene-C(O)OH, _C0-3 alkylene-C(O) _OC1-4 alkyl, _C1-6 alkylene-O-aryl, _C0-3 alkylene-C(O) _C1-4 alkylene-OH, cycloalkyl, heterocycloalkyl, _aryl , heteroaryl, C0-3 alkylene- _C3-14 cycloalkyl, _C0-3 alkylene- _C2-14 heterocycloalkyl, _C0-3 alkylene- _{C6-14 aryl} , _C0-3 alkylene- _C2-14 heteroaryl or cyano, or ^R5 and R ^{The 6-} membered ring, together with the atoms attached to it, forms a 4-6 membered ring;

^R7 is H or _C1-8 alkyl, or ^R7 and ^R5 together with the atoms attached to them form a 4-6 membered ring;

Q is CR ⁸ R ⁹ , C=CR ⁸ R ⁹ , C=O, C=S or C=NR ⁸ ;

^R8 and ^R9 are each independently H, _C1–6 alkyl, hydroxyl, _C1–6 alkoxy, cyano, nitro or _C3–14 cycloalkyl, or ^R8 and ^R9 together with the carbon atom to which they are attached can form a 3-6 membered ring;

R ¹⁰ is _C1-8 alkyl, _C0-3 alkylene- _C6-14 aryl, C0-3 alkylene-C3-14 heteroaryl, _C0-3 alkylene- _C3-14 cycloalkyl, _C0-3 alkylene- _C2-14 heterocycloalkyl, _C1-6 alkoxy, _OC0-3 alkylene- _C6-14 aryl, OC0-3 alkylene- _C3-14 heteroaryl, _OC0-3 alkylene- _C3-14 cycloalkyl, _OC0-3 alkylene- _C2-14 heterocycloalkyl, NH- _C1-8 alkyl, N _{( C1-8} alkyl) ₂ , NH- _C0-3 alkylene- _{C6-14 aryl} , NH- _C0-3 alkylene- _C2-14 heteroaryl, NH _{-C0-3 alkylene} - _C3-14 cycloalkyl, NH-C _0-3 alkylene-C _2-14 heterocyclic alkyl, halogen, cyano or C _1-6 alkylene-amine.

In some embodiments, these compounds have the structure of formula (III). In other embodiments, these compounds have the structure of formula (III').

The compounds having formula (II) or (III) disclosed herein may have one or more of the following characteristics. In some embodiments, Q is C=O. In some embodiments, Q is C=S. In some embodiments, Q is C= ^NR8 . In various embodiments, ^R8 is a _C1-2 alkyl group. In some embodiments, Q is ^CR8R9 . In various embodiments, Q is C= ^CR8R9 . In some embodiments, ^R8 and ^R9 together with ^the carbon atoms to which they are attached form ^a 3–4 membered ring. In some embodiments, ^R8 is a _C1-2 alkyl group, and ^R9 is H.

Compounds having formula (IV) or (IV’) or their pharmaceutically acceptable salts are also provided:

^E1 and ^E2 are each independently ^CR1 or N;

^R1 is independently H, hydroxyl, _C1-6 alkyl, _C1-6 haloalkyl, _C1-6 alkoxy, NH- _C1-6 alkyl, N( _C1-6 alkyl) ₂ , cyano, or halo.

^R2 is a halogroup, _C1-6 alkyl, _C1-6 haloalkyl, OR', N(R') ₂ , _C2-3 alkenyl, _{C2-3 ynyl, C0-3 alkylene} - _C3-14 cycloalkyl, _C0-3 alkylene- _C2-14 heterocycloalkyl, aryl, heteroaryl, _C0-3 alkylene- _C6-14 aryl or C0-3 alkylene- _C2-14 heteroaryl, and each R' is independently H, _{C1-6 alkyl} , _C1-6 haloalkyl, _C3-14 cycloalkyl, _C2-14 heterocycloalkyl, _C2-3 alkenyl, _C2-3 ynyl, aryl or heteroaryl, or two R' substituents together with the nitrogen atom to which they are attached form a 3-7 membered ring;

^R3 is a halogroup, a _C1-2 haloalkyl group, a _C1-3 alkoxy group, a _C3-4 cycloalkyl group, a _C2-3 alkenyl group, a _C2-3 alkynyl group, an aryl group, or a heteroaryl group;

^R4 is

Ring A is a single ring (4-7 members), a double ring, a bridged ring, a fused ring, or a spiral ring (6-11 members).

L is a bond, a _C1-6 alkylene group, a -OC _0-5 alkylene group, a -SC _0-5 alkylene group, or a -NH-C _0-5 alkylene group, and for _C2-6 alkylene groups, a -OC _2-5 alkylene group, a -SC _2-5 alkylene group, and an NH-C _2-5 alkylene group, one carbon atom of the alkylene group may optionally be replaced by O, S, or NH;

R ^4' is H, _C1-8 alkyl, _C2-8 ynyl, C1-6 alkylene- _OC1-4 alkyl, _C1-6 alkylene-OH, _C1-6 haloalkyl, cycloalkyl, heterocycloalkyl, _C0-3 alkylene- _C3-14 cycloalkyl, _C0-3 alkylene- _C2-14 heterocycloalkyl, aryl, heteroaryl, _C0-3 alkylene-C6-14 aryl, or selected from H, _C1-8 alkyl, _C2-8 ynyl ...

^R5 and ^R6 are each independently H, a halogroup, _C1-6 alkyl, _C2-6 ynyl, _C1-6 alkylene- _OC1-4 alkyl, C1-6 alkylene-OH, _C1-6 haloalkyl, _C1-6 alkyleneamine, _C0-6 alkylene-amide, _C0-3 alkylene-C(O)OH, _C0-3 alkylene-C(O) _OC1-4 alkyl, _C1-6 alkylene-O-aryl, _C0-3 alkylene-C(O) _C1-4 alkylene-OH, cycloalkyl, heterocycloalkyl, _aryl , heteroaryl, C0-3 alkylene- _C3-14 cycloalkyl, _C0-3 alkylene- _C2-14 heterocycloalkyl, _C0-3 alkylene- _{C6-14 aryl} , _C0-3 alkylene- _C2-14 heteroaryl or cyano, or ^R5 and R ^{The 6-} membered ring, together with the atoms attached to it, forms a 4-6 membered ring;

^R7 is H or _C1-8 alkyl, or ^R7 and ^R5 together with the atoms attached to them form a 4-6 membered ring;

^R8 is H, _C1-3 alkyl, hydroxyl, _C1-3 alkoxy, halogen, cyano, nitro, _C3–14 cycloalkyl, or ^{NR11R12 ;}

^R11 and ^R12 are each independently H, _C1–8 alkyl, or _C3–14 cycloalkyl; and

R ¹⁰ is _C1-8 alkyl, _C0-3 alkylene- _C6-14 aryl, C0-3 alkylene-C2-14 heteroaryl, _C0-3 alkylene- _C3-14 cycloalkyl, _C0-3 alkylene- _C2-14 heterocycloalkyl, _C1-6 alkoxy, _OC0-3 alkylene- _C6-14 aryl, OC0-3 alkylene- _C2-14 heteroaryl, _OC0-3 alkylene- _C3-14 cycloalkyl, _OC0-3 alkylene- _C2-14 heterocycloalkyl, NH- _C1-8 alkyl, N _{( C1-8} alkyl) ₂ , NH- _C0-3 alkylene- _{C6-14 aryl} , NH- _C0-3 alkylene- _C2-14 heteroaryl _{, NC0-3} alkylene- _C3-14 cycloalkyl, NC _0-3 alkylene-C _2-14 heterocyclic alkyl, halogen, cyano or C _1-6 alkylene-amine;

In some embodiments, the compounds disclosed herein have the structure of formula (IV). In various embodiments, the compounds disclosed herein have the structure of formula (IV'). In some embodiments, ^E1 and ^E2 are each ^CR1 , and ^R8 is a hydroxyl, halogen, nitro, or _C3-6 cycloalkyl group.

In some embodiments, ^R8 is a methyl group.

Additionally, compounds having the structure of formula (IV) or (IV’) are provided:

in

^E1 and ^E2 are each independently ^CR1 or N;

^R1 is independently H, hydroxyl, _C1-6 alkyl, _C1-6 haloalkyl, _C1-6 alkoxy, NH- _C1-6 alkyl, N( _C1-6 alkyl) ₂ , cyano, or halo.

^R2 is a halogroup, _C1-6 alkyl, _C1-6 haloalkyl, OR', N(R') ₂ , _C2-3 alkenyl, _{C2-3 ynyl, C0-3 alkylene} - _C3-14 cycloalkyl, _C0-3 alkylene- _C2-14 heterocycloalkyl, aryl, heteroaryl, _C0-3 alkylene- _C6-14 aryl or C0-3 alkylene- _C2-14 heteroaryl, and each R' is independently H, _{C1-6 alkyl} , _C1-6 haloalkyl, _C3-14 cycloalkyl, _C2-14 heterocycloalkyl, _C2-3 alkenyl, _C2-3 ynyl, aryl or heteroaryl, or two R' substituents together with the nitrogen atom to which they are attached form a 3-7 membered ring;

^R3 is a halogroup, a _C1-2 haloalkyl group, a _C1-3 alkoxy group, a _C3-14 cycloalkyl group, a _C2-3 alkenyl group, a _C2-3 alkynyl group, an aryl group, or a heteroaryl group;

^R4 is

Ring A is a single ring (4-7 members), a double ring, a bridged ring, a fused ring, or a spiral ring (6-11 members).

L is a bond, a _C1-6 alkylene group, a -OC _0-5 alkylene group, a -SC _0-5 alkylene group, or a -NH-C _0-5 alkylene group, and for _C2-6 alkylene groups, a -OC _2-5 alkylene group, a -SC _2-5 alkylene group, and an NH-C _2-5 alkylene group, one carbon atom of the alkylene group may optionally be replaced by O, S, or NH;

^R5 and ^R6 are each independently H, a halogroup, _C1-6 alkyl, _C2-6 ynyl, _C1-6 alkylene- _OC1-4 alkyl, C1-6 alkylene-OH, _C1-6 haloalkyl, _C1-6 alkyleneamine, _C0-6 alkylene-amide, _C0-3 alkylene-C(O)OH, _C0-3 alkylene-C(O) _OC1-4 alkyl, _C1-6 alkylene-O-aryl, _C0-3 alkylene-C(O) _C1-4 alkylene-OH, cycloalkyl, heterocycloalkyl, _aryl , heteroaryl, C0-3 alkylene- _C3-14 cycloalkyl, _C0-3 alkylene- _C2-14 heterocycloalkyl, _C0-3 alkylene- _{C6-14 aryl} , _C0-3 alkylene- _C2-14 heteroaryl or cyano, or ^R5 and R ^{The 6-} membered ring, together with the atoms attached to it, forms a 4-6 membered ring;

^R7 is H or _C1-8 alkyl, or ^R7 and ^R5 together with the atoms attached to them form a 4-6 membered ring;

^R8 is H, _C1-3 alkyl, hydroxyl, _C1-3 alkoxy, halogen, cyano, nitro, _C3-14 cycloalkyl, or ^{NR11R12 ;}

^R11 and ^R12 are each independently H, _C1–8 alkyl, or _C3–15 cycloalkyl; and

R ¹⁰ is _C1-8 alkyl, _C0-3 alkylene- _C6-14 aryl, C0-3 alkylene-C3-14 heteroaryl, _C0-3 alkylene- _C3-14 cycloalkyl, _C0-3 alkylene- _C2-14 heterocycloalkyl, _C1-6 alkoxy, _OC0-3 alkylene- _C6-14 aryl, OC0-3 alkylene- _C3-14 heteroaryl, _OC0-3 alkylene- _C3-14 cycloalkyl, _OC0-3 alkylene- _C2-14 heterocycloalkyl, NH- _C1-8 alkyl, N _{( C1-8} alkyl) ₂ , NH- _C0-3 alkylene- _{C6-14 aryl} , NH- _C0-3 alkylene- _C2-14 heteroaryl, NH _{-C0-3 alkylene} - _C3-14 cycloalkyl, NH-C _0-3 alkylene-C _2-14 heterocyclic alkyl, halogen, cyano or C _1-6 alkylene-amine;

Or its pharmaceutically acceptable salt.

In some embodiments, the compounds disclosed herein have the structure of formula (IV). In various embodiments, the compounds disclosed herein have the structure of formula (IV'). In some embodiments, ^E1 and ^E2 are each ^CR1 , and ^R8 is a hydroxyl, halogen, nitro, or _C3-6 cycloalkyl group.

In some embodiments, ^R8 is a methyl group.

Additionally, compounds having the structure of formula (V) or pharmaceutically acceptable salts thereof are provided:

in

^E1 and ^E2 are each independently ^CR1 or N;

^R1 is independently H, hydroxyl, _C1-6 alkyl, _C1-6 haloalkyl, _C1-6 alkoxy, NH- _C1-6 alkyl, N( _C1-6 alkyl) ₂ , cyano, or halo.

^R2 is a halogroup, _C1-6 alkyl, _C1-6 haloalkyl, OR', N(R') ₂ , _C2-3 alkenyl, _{C2-3 ynyl, C0-3 alkylene} - _C3-14 cycloalkyl, _C0-3 alkylene- _C2-14 heterocycloalkyl, aryl, heteroaryl, _C0-3 alkylene- _C6-14 aryl or C0-3 alkylene- _C2-14 heteroaryl, and each R' is independently H, _{C1-6 alkyl} , _C1-6 haloalkyl, _C3-14 cycloalkyl, _C2-14 heterocycloalkyl, _C2-3 alkenyl, _C2-3 ynyl, aryl or heteroaryl, or two R' substituents together with the nitrogen atom to which they are attached form a 3-7 membered ring;

^R3 is a halogroup, _C1-6 alkyl, _C1-6 haloalkyl, _C1-6 alkoxy, _C3-14 cycloalkyl, _C2-6 alkenyl, _C2-6 alkynyl, aryl, or heteroaryl.

^R4 is

Ring A is a single ring (4-7 members), a double ring, a bridged ring, a fused ring, or a spiral ring (6-11 members).

L is a bond, a _C1-6 alkylene group, a -OC _0-5 alkylene group, a -SC _0-5 alkylene group, or a -NH-C _0-5 alkylene group, and for _C2-6 alkylene groups, a -OC _2-5 alkylene group, a -SC _2-5 alkylene group, and an NH-C _2-5 alkylene group, one carbon atom of the alkylene group may optionally be replaced by O, S, or NH;

R ^4' is H, _C1-8 alkyl, _C2-8 ynyl, C1-6 alkylene- _OC1-4 alkyl, _C1-6 alkylene-OH, _C1-6 haloalkyl, cycloalkyl, heterocycloalkyl, _C0-3 alkylene- _C3-14 cycloalkyl, _C0-3 alkylene- _C2-14 heterocycloalkyl, aryl, heteroaryl, _C0-3 alkylene-C6-14 aryl, or selected from H, _C1-8 alkyl, _C2-8 ynyl ...

^R5 and ^R6 are each independently H, a halogroup, _C1-6 alkyl, _C2-6 ynyl, _C1-6 alkylene- _OC1-4 alkyl, C1-6 alkylene-OH, _C1-6 haloalkyl, _C1-6 alkyleneamine, _C0-6 alkylene-amide, _C0-3 alkylene-C(O)OH, _C0-3 alkylene-C(O) _OC1-4 alkyl, _C1-6 alkylene-O-aryl, _C0-3 alkylene-C(O) _C1-4 alkylene-OH, cycloalkyl, heterocycloalkyl, _aryl , heteroaryl, C0-3 alkylene- _C3-14 cycloalkyl, _C0-3 alkylene- _C2-14 heterocycloalkyl, _C0-3 alkylene- _{C6-14 aryl} , _C0-3 alkylene- _C2-14 heteroaryl or cyano, or ^R5 and R ^{The 6-} membered ring, together with the atoms attached to it, forms a 4-6 membered ring;

^R7 is an H or _C1-8 alkyl group, or ^R7 and ^R5 together with their attached atoms form a 4-6 membered ring; and

R ¹⁰ is _C1-8 alkyl, _C0-3 alkylene- _C6-14 aryl, C0-3 alkylene-C3-14 heteroaryl, _C0-3 alkylene- _C3-14 cycloalkyl, _C0-3 alkylene- _C2-14 heterocycloalkyl, _C1-6 alkoxy, _OC0-3 alkylene- _C6-14 aryl, OC0-3 alkylene- _C3-14 heteroaryl, _OC0-3 alkylene- _C3-14 cycloalkyl, _OC0-3 alkylene- _C2-14 heterocycloalkyl, NH- _C1-8 alkyl, N _{( C1-8} alkyl) ₂ , NH- _C0-3 alkylene- _{C6-14 aryl} , NH- _C0-3 alkylene- _C2-14 heteroaryl, NH _{-C0-3 alkylene} - _C3-14 cycloalkyl, NH-C _0-3 alkylene-C _2-14 heterocyclic alkyl, halogen, cyano or C _1-6 alkylene-amine; or pharmaceutically acceptable salts thereof.

Additionally, compounds having the structure of formula (V) are provided:

in

^E1 and ^E2 are each independently ^CR1 or N;

^R1 is independently H, hydroxyl, _C1-6 alkyl, _C1-6 haloalkyl, _C1-6 alkoxy, NH- _C1-6 alkyl, N( _C1-6 alkyl) ₂ , cyano, or halo.

^R2 is a halogroup, _C1-6 alkyl, _C1-6 haloalkyl, OR', N(R') ₂ , _C2-3 alkenyl, _{C2-3 ynyl, C0-3 alkylene} - _C3-14 cycloalkyl, _C0-3 alkylene- _C2-14 heterocycloalkyl, aryl, heteroaryl, _C0-3 alkylene- _C6-14 aryl or C0-3 alkylene- _C2-14 heteroaryl, and each R' is independently H, _{C1-6 alkyl} , _C1-6 haloalkyl, _C3-14 cycloalkyl, _C2-14 heterocycloalkyl, _C2-3 alkenyl, _C2-3 ynyl, aryl or heteroaryl, or two R' substituents together with the nitrogen atom to which they are attached form a 3-7 membered ring;

^R3 is a halogroup, _C1-6 alkyl, _C1-6 haloalkyl, _C1-6 alkoxy, _C3-14 cycloalkyl, _C2-8 alkenyl, _C2-8 alkynyl, aryl, or heteroaryl.

^R4 is

Ring A is a single ring (4-7 members), a double ring, a bridged ring, a fused ring, or a spiral ring (6-11 members).

L is a bond, a _C1-6 alkylene group, a -OC _0-5 alkylene group, a -SC _0-5 alkylene group, or a -NH-C _0-5 alkylene group, and for _C2-6 alkylene groups, a -OC _2-5 alkylene group, a -SC _2-5 alkylene group, and an NH-C _2-5 alkylene group, one carbon atom of the alkylene group may optionally be replaced by O, S, or NH;

^R5 and ^R6 are each independently H, a halogroup, _C1-6 alkyl, _C2-6 ynyl, _C1-6 alkylene- _OC1-4 alkyl, C1-6 alkylene-OH, _C1-6 haloalkyl, _C1-6 alkyleneamine, _C0-6 alkylene-amide, _C0-3 alkylene-C(O)OH, _C0-3 alkylene-C(O) _OC1-4 alkyl, _C1-6 alkylene-O-aryl, _C0-3 alkylene-C(O) _C1-4 alkylene-OH, cycloalkyl, heterocycloalkyl, _aryl , heteroaryl, C0-3 alkylene- _C3-14 cycloalkyl, _C0-3 alkylene- _C2-14 heterocycloalkyl, _C0-3 alkylene- _{C6-14 aryl} , _C0-3 alkylene- _C2-14 heteroaryl or cyano, or ^R5 and R ^{The 6-} membered ring, together with the atoms attached to it, forms a 4-6 membered ring;

^R7 is an H or _C1-8 alkyl group, or ^R7 and ^R5 together with their attached atoms form a 4-6 membered ring; and

R ¹⁰ is _C1-8 alkyl, _C0-3 alkylene- _C6-14 aryl, C0-3 alkylene-C3-14 heteroaryl, _C0-3 alkylene- _C3-14 cycloalkyl, _C0-3 alkylene- _C2-14 heterocycloalkyl, _C1-6 alkoxy, _OC0-3 alkylene- _C6-14 aryl, OC0-3 alkylene- _C3-14 heteroaryl, _OC0-3 alkylene- _C3-14 cycloalkyl, _OC0-3 alkylene- _C2-14 heterocycloalkyl, NH- _C1-8 alkyl, N _{( C1-8} alkyl) ₂ , NH- _C0-3 alkylene- _{C6-14 aryl} , NH- _C0-3 alkylene- _C2-14 heteroaryl, NH _{-C0-3 alkylene} - _C3-14 cycloalkyl, NH-C _0-3 alkylene-C _2-14 heterocyclic alkyl, halogen, cyano or C _1-6 alkylene-amine; or pharmaceutically acceptable salts thereof.

The compounds having formulas (I), (II), (III), (III'), (IV), (IV'), or (V) disclosed herein may have one or more of the following characteristics. In some embodiments, each of ^E1 and ^E2 is ^CR1 . In other embodiments, ^E1 is ^CR1 and ^E2 is N. In some embodiments, ^E1 is N and ^E2 is ^CR1 . In various embodiments, each of ^E1 and ^E2 is N.

The compounds having formulas (II), (III), (III'), (IV), (IV'), or (V) disclosed herein may have one or more of the following characteristics. In various embodiments, R ¹⁰ is a _C1-6 alkyl, aryl, heteroaryl, _C3-14 cycloalkyl, _C2-14 heterocycloalkyl, _C1-6 alkoxy, OC _0-6 alkylene- _{C6-14 aryl, OC 0-6 alkylene-C2-14 heteroaryl} , OC _0-6 alkylene- _C3-14 cycloalkyl, OC _0-6 alkylene- _C2-14 heterocycloalkyl, NC _1-8 alkyl, N(C _1-8 alkyl) _{2 ,} NH-C _0-6 alkylene- _C6-14 aryl, NH-C _0-6 alkylene- _C2-14 heteroaryl, NH-C _0-6 alkylene- _C3-14 cycloalkyl, or NH-C _0-6 alkylene- _C2-14 heterocycloalkyl. In various embodiments, R ¹⁰ is a _C1-8 alkyl. In some embodiments, ^R10 is a _C0-3 alkylene- _C6-14 heteroaryl. In some embodiments, ^R10 is a _C0-3 alkylene- _C2-14 heteroaryl. In some embodiments, R10 is a _C0-3 alkylene- _C3-14 cycloalkyl. In some embodiments, ^R10 is a _C0-3 alkylene- _C2-14 heterocycloalkyl. In other embodiments, ^R10 is a _C0-6 alkyleneamine. For example, ^R10 can be i-Pr, t-Bu, phenyl, benzyl, _OCH3 , Cl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, ^etc.

In some embodiments, R ¹⁰ comprises an ortho-substituted aryl group, an ortho-substituted heteroaryl group, or a 2-substituted cyclohexyl group. For example, R ¹⁰ may be...

The compounds having formulas (I), (II), (III), (III'), (IV), (IV'), or (V) disclosed herein may have one or more of the following characteristics. In some embodiments, ^R1 is H. In some embodiments, ^R1 is F. In some embodiments, ^R1 is methyl.

Compounds having formulas (I), (II), (III), (III'), (IV), (IV'), or (V) disclosed herein may have one or more of the following characteristics. In various embodiments, ^R2 is an aryl group. In some embodiments, ^R2 is a heteroaryl group. In various embodiments, ^R2 is phenyl, naphthyl, pyridinyl, indazole, indolyl, azaindolyl, dihydroindolyl, benzotriazolyl, benzoxadiazolyl, imidazolyl, cenolinyl, imidazopyridyl, pyrazolopyridyl, quinolinyl, isoquinolinyl, quinazolinyl, quinazolinone, indolone, isoindolone, tetrahydronaphthyl, tetrahydroquinolinyl, or tetrahydroisoquinolinyl. For example, ^R2 may be Cl, Br, _CF3 , cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, piperidine, pyrrolidine, azacyclobutane, _OCH3 , _OCH2CH3 , _phenyl ,

In various embodiments, ^R2 can be bromine, and

The compounds having formulas (I), (II), (III), (III'), (IV), (IV'), or (V) disclosed herein may have one or more of the following characteristics. In various embodiments, ^R3 is a halogroup. In various embodiments, ^R3 is Cl. In various embodiments, ^R3 is F. In some embodiments, ^R3 is a _C1-2 alkyl group. In some embodiments, ^R3 is methyl. In some embodiments, ^R3 is a _C1-2 haloalkyl group. In various embodiments, ^R3 is _CF3 .

The compounds having formulas (I), (II), (III), (III'), (IV), (IV'), or (V) disclosed herein may have one or more of the following characteristics. In some embodiments, ^R4 is in various embodiments, ^R4 is in some embodiments, ^R4 is in some embodiments, ^R4 is in some embodiments, ^R4 is in some embodiments ^.

In some embodiments, ^R4 can be

In various embodiments, R ^4' is H, _C1-8 alkyl, _C2-8 ynyl, _C1-6 alkylene- _OC1-4 alkyl, _C1-6 alkylene-OH, _C1-6 haloalkyl, _C0-3 alkylene- _C3-8 cycloalkyl, _C0-3 alkylene- _C2-7 heterocycloalkyl, _C0-3 alkylene- _C6-14 aryl, or selected from H.

In various embodiments, it can be...

In some embodiments, ring A is

In some embodiments, ring A comprises piperidinyl, piperazine, pyrrolidinyl, or aziridine. In some embodiments, ring A comprises piperidinyl.

In various embodiments, ring A can be

In various embodiments, ring A can be

The compounds having formulas (I), (II), (III), (III’), (IV), (IV’) or (V) disclosed herein may have one or more of the following characteristics.

In some embodiments, L is a key.

In some embodiments, L is a _C1-2 alkylene group.

In various embodiments, L is O. In some embodiments, L is S.

In each embodiment, L is NH.

In some embodiments, ^R5 is H or a halogen.

In some embodiments, ^R5 is H, Br, Cl, F, CN, CH3, _CF3 , _CH2Br , _CH2OH , _CH2CH2OH , _{CH2OCH2phenyl} , _cyclopropyl , phenyl, _CH2phenyl , _CH2OCH3 , _CH2N ( _CH3 ) ₂ , _{CH2N (CH2CH3)2 , CH2CO2H , CH2CO2CH3} , _{CH2NHC ( O ) CH3 , CH2C} (O) _NHCH3 , _CH2OC (O) _{CH3 , or}

In some embodiments, ^R6 is a _C1-6 alkyl, _C1-6 alkylene- _OC1-6 alkyl, _C1-6 alkylene-OH, _C1-3 haloalkyl, _C1-6 alkylene-amine, _C0-6 alkylene-amide, _C0-1 alkylene-C(O) _OC1-3 alkyl, _C0-1 alkylene- _C2-14 heterocyclic alkyl, _C0-1 alkylene- _C3-14 cycloalkyl, or _C0-3 alkylene- _C6-14 aryl.

In various embodiments, ^R6 is a _C0-6 alkyleneamine or a _C0-3 alkyleneamide and is _CH2NH2 , CH( _CH3 ) _NH2 , CH(CH3 _{)2NH2 , CH2CH2NH2} , CH2CH2N(CH3) ₂ , _CH2NHCH3 , C( _O ) _NHCH3 , _{C (} O) _N ( _{CH3 ) 2} , _CH2C (O _{) NHphenyl} , _{CH2NHC ( O ) CH3 , CH2NHCH2CH2OH , CH2NHCH2CO2H , CH2NH ( CH3 ) CH2CO2CH3} , _{CH2NHCH2CH2OCH3 , CH2NH ( CH3 ) CH2CH2OCH3 , CH2} NH(CH ₃ )CH ₂ C(O)N(CH ₃ ) ₂ , CH ₂ NH(CH ₃ )CH ₂ C(O)NHCH ₃ , CH ₂ NMe ₂ , CH ₂ NH(CH ₃ )CH ₂ CH ₂ OH, CH ₂ NH(CH ₃ )CH ₂ CH ₂ F, CH ₂ N ⁺ (CH ₃ ) ₃ , CH ₂ NHCH ₂ CHF _2. CH ₂ NHCH ₂ CH ₃ .

In various embodiments, ^R6 is _phenyl , cyclopropyl, CH3, _CF3 , _CH2CH3 , _CH2NH2 , CH( _CH3 ) _NH2 , CH( _CH3 ) _2NH2 , _CH2Cl , _CH2Br , _CH2OCH3 , _CH2Ophenyl , _CH2OH , CO2H, _CO2CH2CH3 , _CH2CO2H , _CH2CH2NH2 , _CH2CH2OH , CH2CH2N _{(CH3 )2 , CH2NHCH3} , _{C (} O) _{NHCH3 ,} C _{( O ) N} ( _CH3 ) ₂ , _CH2C ( _{O )} NHphenyl, _{CH2CHF2 , CH2F} , _{CHF2 , CH2NHC ( O} ) _{CH3 , CH2NHCH 2} CH ₂ OH, CH ₂ NHCH ₂ CO ₂ H, CH ₂ NH(CH ₃ )CH ₂ CO ₂ CH ₃ , CH ₂ NHCH ₂ CH ₂ OCH ₃ , CH ₂ NH(CH ₃ )CH ₂ CH ₂ OCH ₃ , CH ₂ NH(CH ₃ )CH ₂ C(O)N(CH ₃ ) ₂ , CH ₂ NH(CH ₃ )CH ₂ C(O)NHCH ₃ , CH ₂ CH ₂ CCH, CH ₂ NMe ₂ , CH ₂ NH(CH ₃ )CH ₂ CH ₂ OH, CH ₂ NH(CH ₃ )CH ₂ CH ₂ F, CH ₂ N ⁺ (CH ₃ ) ₃ , CH ₂ NHCH ₂ CHF ₂ , CH ₂ NHCH ₂ CH ₃ ,

In various embodiments, ^R5 and ^R6 together are

In some embodiments, each of ^R5 and ^R6 is H.

In some embodiments, R ⁷ is H.

In some embodiments, ^R7 is a methyl group.

In various embodiments, ^R7 and ^R5 together are _-CH2- or -C(O) _CH2- .

The compounds disclosed herein may be in pharmaceutically acceptable salt form. The provided compounds may be formulated into pharmaceutical preparations comprising the compounds disclosed herein and pharmaceutically acceptable excipients.

A method for inhibiting KRAS G12C in cells is also provided, the method comprising contacting the cells with a compound or composition disclosed herein. Additionally, a method for treating a subject with cancer is provided, the method comprising administering to the subject a therapeutically effective amount of a compound or composition disclosed herein. In some embodiments, the cancer is lung cancer, pancreatic cancer, or colorectal cancer.

Detailed Implementation

definition

Abbreviations: The following abbreviations may be used in this article:

Unless otherwise indicated, the use of the terms "a" and "the" and similar references in the context of describing the invention (especially in the context of the claims) should be interpreted to cover both the singular and the plural. Unless otherwise indicated herein, statements herein relating to ranges of values are intended only as a way of individually referring to each independent value within that range, and each independent value is incorporated into the specification as if it were individually stated herein. Unless otherwise required, the use of any and all instances or exemplary language (e.g., "such as") provided herein is intended to better illustrate the invention and is not intended to limit the scope of the invention. No language in this specification should be construed as indicating any non-claimed element essential to the practice of the invention.

As used herein, the term "alkyl" refers to a straight-chain and branched _C1 - _C8 hydrocarbon group, including but not limited to methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, n-pentyl, 2-methylbutyl, 3-methylbutyl, 2,2-dimethylpropyl, n-hexyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, 3,3-dimethylbutyl, and 2-ethylbutyl. The term C _{_mn} indicates that the alkyl group has "m" to "n" carbon atoms. The term "alkylene" refers to an alkyl group with substituents. Alkyl (e.g., methyl) or alkylene (e.g., _-CH₂- ) groups may be substituted by one or more, and typically one to three, independently selected from, for example, the following groups: halogen, trifluoromethyl, trifluoromethoxy, hydroxyl, alkoxy, nitro, cyano, alkylamino, C₁ _-8 alkyl, C₂ _-8 alkenyl, C₂ _-8 ynyl, -NC, amino, _-CO₂H , _-CO₂C₁ - _C₈ alkyl, -OCOC₁- _C₈ alkyl, _{C₃ -C₁₀ cycloalkyl, C₃-C₁₀ heterocycloalkyl , C₅ - C₁₀ aryl} , and _C₅ - _C₁₀ heteroaryl. The term "halogenated alkyl" specifically refers to an alkyl group in which at least one (e.g., 1 to 6) or all of the hydrogen atoms of the alkyl group are substituted by a halogen atom.

The terms "alkenyl" and "alkynyl" refer to alkyl groups that also include double and triple bonds, respectively.

As used herein, the term "halogen" refers to fluorine, chlorine, bromine, and iodine. The term "alkoxy" is defined as -OR, where R is an alkyl group.

As used herein, the terms “amino” or “amine” can be used interchangeably to refer to the -NR ₂ group, wherein each R is ^, for example, H or a substituent. In some embodiments, the amino group is further substituted to form an ammonium ion, such as NR ₃₊ . The ammonium portion is specifically included in the definition of “amino” or “amine”. Substituents can be, for example, alkyl, alkoxy, cycloalkyl, heterocycloalkyl, amide, or carboxylic acid ester. The R group can be further substituted, for example, by one or more (e.g., one to four) groups selected from: halogen, cyano, alkenyl, alkynyl, alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, urea, carbonyl, carboxylic acid ester, amine, and amide. The term “amide” or “acylamino” can be used interchangeably to refer to a group similar to an amine or amino group but additionally including a C(O), such as -C(O)NR ₂ . Some anticipated amino or acylamino groups (some with optional alkylene groups, such as alkylene-amino or alkylene-acylamino) include: _CH₂NH₂ , CH _{( CH₃ ) NH₂} , CH _{( CH₃} ) _₂NH₂ , CH₂CH₂NH₂, CH₂CH₂N(CH₃ _{) ₂} , _CH₂NHCH₃ , C( _O ) _NHCH₃ , _{C(O)N(CH₃)₂ , CH₂C ( O ) NHphenyl , CH₂NHC ( O ) CH₃ , CH₂NHCH₂CH₂OH} , _{CH₂NHCH₂CO₂H} , _{CH₂NH ( CH₃ ) CH₂CO₂CH₃ , CH₂NHCH₂CH₂OCH₃} , _{CH₂NH ( CH₃ ) CH₂CH₂OCH₃ , CH₂} NH(CH ₃ )CH ₂ C(O)N(CH ₃ ) ₂ , CH ₂ NH(CH ₃ )CH ₂ C(O)NHCH ₃ , CH ₂ CH ₂ CCH, CH ₂ NMe ₂ , CH ₂ NH(CH ₃ )CH ₂ CH ₂ OH, CH ₂ NH(CH ₃ )CH ₂ CH ₂ F, CH ₂ N ⁺ (CH ₃ ) ₃ , CH ₂ NHCH ₂ CHF ₂ , CH ₂ NHCH ₂ CH ₃ ,

As used herein, the term "aryl" refers to a _C6-14 monocyclic or polycyclic aromatic group, preferably a _C6-10 monocyclic or bicyclic aromatic group, or a _C10-14 polycyclic aromatic group. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, fluorenyl, azulel, anthraceneyl, phenanthryl, pyrene, biphenyl, and triphenyl. Aryl also refers to a _C10-14 bicyclic or tricyclic carbocyclic ring, where one ring is aromatic and the other rings are saturated, partially unsaturated, or aromatic, such as dihydronaphthyl, indene, dihydroindene, or tetrahydronaphthyl (tetrahydronaphthyl). Unless otherwise indicated, the aryl group may be unsubstituted or substituted by one or more, and particularly one to four, independently selected from, for example, the following groups: halogen, _C1-8 alkyl, _C2-8 alkenyl, _C2-8 alkynyl, _-CF3 , _-OCF3 , _-NO2 , -CN, -NC, -OH, alkoxy, amino, _-CO2H , -CO2C1 _{-C8 alkyl} , -OCOC1 _{- C8} alkyl, _C3 - _C10 cycloalkyl, _C3 - _C10 heterocycloalkyl, _{C5 - C10} aryl, and _C5 - _C10 heteroaryl.

As used herein, the term "cycloalkyl" refers to a monocyclic or polycyclic nonaromatic carbon ring, wherein the polycyclic ring may be fused, bridged, or spirocyclic. The carbon ring may have 3 to 10 carbon ring atoms. Carbon rings considered include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, and cyclononyl.

As used herein, the term "heterocyclic alkyl" means a monocyclic or polycyclic (e.g., bicyclic), saturated or partially unsaturated ring system containing three or more (e.g., 3 to 12, 4 to 10, 4 to 8, or 5 to 7) total atoms, wherein 1 to 5 (e.g., 1, 2, 3, 4, or 5) atoms are independently selected from nitrogen, oxygen, and sulfur. Non-limiting examples of heterocyclic alkyls include azirrocyclobutane, pyrrolyl, piperidinyl, piperazine, dihydropyrrolyl, morpholinyl, thiomorpholinyl, dihydropyridinyl, oxoheptanyl, dioxoheptanyl, thioheptanyl, and diazaheptanyl.

Unless otherwise indicated, cycloalkyl or heterocycloalkyl groups may be unsubstituted or substituted with one or more, and particularly one to four, groups. Some anticipated substituents include halogens, _C1-8 alkyl, _C2-8 alkenyl, _C2-8 alkynyl, -OCF3, _-NO2 , _-CN , -NC, -OH, alkoxy, amino _{, -CO2H} , -CO2C1 _-C8 alkyl, -OCOC1- _C8 alkyl, _C3 - _C10 cycloalkyl, _C3 - _C10 heterocycloalkyl, _C5 - _C10 aryl, and _{C5 - C10 heteroaryl} .

As used herein, the term "heteroaryl" refers to a monocyclic or polycyclic system (e.g., bicyclic) containing one to three aromatic rings and one to four (e.g., 1, 2, 3, or 4) heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the heteroaryl group has 5 to 20, 5 to 15, 5 to 10, or 5 to 7 ring atoms. Heteroaryl also refers to _C10-14 bicyclic and tricyclic rings, wherein one ring is aromatic and the other rings are saturated, partially unsaturated, or aromatic. Examples of heteroaryl groups include, but are not limited to, furanyl, imidazolyl, isothiazolyl, isoxazolyl, oxadiazolyl, oxazolyl, pyrazinyl, pyrazolyl, pyridinyl, pyrimidinyl, pyrroloyl, thiadiazolyl, thiazolyl, thiophene, tetrazolyl, triazinyl, triazolyl, benzofuranyl, benzimidazolyl, benzisoxazolyl, benzopyranyl, benzothiadiazolyl, benzothiazolyl, benzothiophene, benzobenzenethio, and benzene. Triazolyl, benzoxazolyl, furanopyridyl, imidazopyridyl, imidazothiazolyl, indoleazinyl, indole, indazole, isobenzofuranyl, isobenzothiophenyl, isoindole, isoquinolinyl, isothiazolyl, naphridyl, oxazolylpyridyl, phthalazinyl, pteridinyl, purine, pyridinylpyridyl, pyrrolopyridyl, quinolinyl, quinoxalinyl, quinazolinyl, thiadiazopyrimidinyl, and thienenopyridyl. Unless otherwise indicated, heteroaryl groups may be unsubstituted or substituted with one or more, and particularly one to four, or one or two substituents. Substituents considered include halogens, _C1-8 alkyl, _C2-8 alkenyl, _C2-8 alkynyl, -OCF3, _-NO2 , _-CN , -NC, -OH, alkoxy, amino, -CO2H, -CO2C1 _{- C8} alkyl, -OCOC1 _{- C8} alkyl, _C3 - _C10 cycloalkyl, _C3 - _C10 heterocycloalkyl, _{C5 -C10 aryl} , and _{C5 - C10} heteroaryl.

As used in this article, the term Boc refers to structure

As used in this article, the term Cbz refers to the structure

As used in this article, the term Bn refers to the structure

As used herein, the term trifluoroacetamide refers to a structure...

As used herein, the term triphenylmethyl refers to the structure

As used herein, the term toluenesulfonyl refers to the structure

As used in this article, the term Troc refers to structure.

As used in this article, the term Teoc refers to structure

As used in this article, the term Alloc refers to the structure

As used herein, the term Fmoc refers to the compound disclosed herein.

This article presents KRAS inhibitors having one of the structures of formulas I-V, which will be discussed in more detail below.

The compounds disclosed herein include all pharmaceutically acceptable isotopically labeled compounds, wherein one or more atoms of the disclosed compounds are replaced with atoms having the same number of atoms but with atomic masses or mass numbers different from those normally found in nature. Examples of isotopes that may be incorporated into the disclosed compounds include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, chlorine, and iodine, such as ^2H , ^3H , ^11C , ^13C , 14C, ^13N , ^15N , ^15O , ^17O , ^18O , ^31P , ^32P , ^35S , ^18F , ^36Cl , ^123I , and ^125I , respectively. These radiolabeled compounds can be used to help determine or measure the efficacy of compounds by characterizing, for example, the site or mode of action, or the binding affinity to pharmacologically important sites of action. Certain ^isotopically labeled compounds disclosed herein, such as those incorporating radioisotopes, can be used in drug and/or substrate tissue distribution studies. Radioactive isotopes tritium ( ^3H ) and carbon-14 ( ^14C ) are particularly suitable for this purpose due to their ease of incorporation and existing detection methods.

Substitution with heavier isotopes, such as deuterium (i.e., ^2H ), can provide certain therapeutic benefits due to higher metabolic stability, such as increased in vivo half-life or reduced dose requirements, and is therefore preferred in some cases.

Substitution with positron-emitting isotopes, such as ^11C , ^18F , ^15O , and ^13N , can be used in positron emission tomography (PET) studies to examine substrate acceptor occupancy. Isotopically labeled structural (I) compounds can generally be prepared using conventional techniques known to those skilled in the art, or by methods similar to those described in the preparations and examples below, using a suitable isotopically labeled reagent instead of the previously used unlabeled reagent.

The isotopically labeled compounds disclosed herein can generally be prepared using conventional techniques known to those skilled in the art, or methods similar to those described in the appended examples and schemes, by using an appropriate isotopically labeled reagent instead of the previously used unlabeled reagent.

Some of the compounds disclosed herein can exist as stereoisomers (i.e., isomers that differ only in the spatial arrangement of atoms), including optical isomers and conformational isomers (or conformations). The compounds disclosed herein include all stereoisomers as pure individual stereoisomer formulations and enriched formulations of each, as well as racemic mixtures of such stereoisomers and individual diastereomers and enantiomers that can be separated according to methods known to those skilled in the art. Furthermore, the compounds disclosed herein include all tautomeric forms of these compounds.

Some of the compounds disclosed herein can exist as transisomers, which are conformational stereoisomers that occur when rotation around the single bonds in the molecule is blocked or significantly slowed down due to steric interactions with other parts of the molecule. The compounds disclosed herein include all transisomers as pure individual transisomer formulations, enriched formulations of each, or nonspecific mixtures of each. If the rotational barrier around the single bond is sufficiently high and the interconversion between conformations is sufficiently slow, then the separation and differentiation of isomer species can be permitted. For example, groups such as, but not limited to, the following R ¹⁰ groups may exhibit restricted rotation.

This disclosure provides a compound having the structure of formula (I).

Where ^E1 and ^E2 are each independently N or ^CR1 ; ^R1 is independently H, hydroxyl, _C1-4 alkyl, _C1-4 haloalkyl, _C1-4 alkoxy, NH- _C1-4 alkyl, N( _C1-4 alkyl) ₂ , cyano, or halo; ^R2 is halo, _C1-6 alkyl, _C1-6 haloalkyl, OR', N(R') ₂ , _C2-3 alkenyl, _C2-3 alkynyl, _C0-3 alkylene- _C3-8 cycloalkyl, _C0-3 alkylene- _C2-7 heterocycloalkyl, _C0-3 alkylene aryl, or _C0-3 alkylene heteroaryl, and each R' is independently H, _C1-6 alkyl, _C1-6 haloalkyl, _C3-4 cycloalkyl, _C2-3 alkenyl, C _2-3- alkynyl, aryl, or heteroaryl, or two R' substituents together with their attached nitrogen atom to form a 3-7 membered ring; ^R3 is a halogroup, _C1-3 alkyl, _C1-2 haloalkyl, _C1-3 alkoxy, _C3-4 cycloalkyl, _C2-3 alkenyl, _C2-3 alkynyl, aryl, or heteroaryl; ^R4 is...

Ring A is a monocyclic 4-7 membered ring or a bicyclic, bridged, fused, or spirocyclic 6-11 membered ring; L is a bond, _C1-6 alkylene, -OC _0-5 alkylene, -SC _0-5 alkylene, or -NH-C _0-5 alkylene, and for _C2-6 alkylene, -OC _2-5 alkylene, -SC _2-5 alkylene, and NH-C _2-5 alkylene, one carbon atom of the alkylene group may optionally be replaced by O, S, or NH; R ^4' is H, _C1-8 alkyl, _C2-8 alkynyl, _C1-6 alkylene-OC _1-4 alkyl, _C1-6 alkylene-OH, _C1-6 haloalkyl, _C0-3 alkylene- _C3-8 cycloalkyl, _C0-3 alkylene- _C2-7 heterocyclic alkyl, _C0-3 alkylaryl, or selected from ^R5 and ^R6 , each independently being H, a halogen, C _1-8 alkyl, _C2-8 ynyl, _{C1-6 alkylene-OC1-4} alkyl, _C1-6 alkylene-OH, _C1-6 haloalkyl, _C1-6 alkyleneamine, _C0-6 alkyleneamide, _C0-3 alkylene-C(O)OH, _C0-3 alkylene-C(O) _OC1-4 alkyl, _C1-6 alkylene-O-aryl, _C0-3 alkylene-C(O) _C1-4 alkylene-OH, _C0-3 alkylene- _C3-8 cycloalkyl, _C0-3 alkylene _{- C2-7} heterocycloalkyl, _C0-3 alkylene aryl or cyano, or ^R5 and ^R6 together with the atoms to which they are attached to form a 4-6 membered ring; and ^R7 is H or _C1-3 alkyl, or ^R7 and R ^{The 5-} membered ring, together with the atoms attached to it, forms a 4-6 membered ring, or a pharmaceutically acceptable salt thereof.

Compounds of formula I may be in the form of formula (I-A), (I-B), (I-C), or (I-D):

This disclosure also provides a compound having the structure of formula (II).

Where ^E1 and ^E2 are each independently N or ^CR1 ; J is N, ^NR10 , or ^CR10 ; M is N, ^NR13 , or ^CR13 ; single or double bonds are used as needed to apply the normal valence of each atom; ^R1 is independently H, hydroxyl, _C1-4 alkyl, _C1-4 haloalkyl, _C1-4 alkoxy, NH- _C1-4 alkyl, N( _C1-4 alkyl) ₂ , cyano, or halo; ^R2 is halo, _C1-6 alkyl, _C1-6 haloalkyl, OR', N(R') ₂ , _C2-3 alkenyl, _C2-3 alkynyl, _C0-3 alkylene- _C3-8 cycloalkyl, _C0-3 alkylene- _C2-7 heterocycloalkyl, _C0-3 alkylene aryl, or _C0-3 alkylene heteroaryl, and each R' is independently H, C _1-6 alkyl, _C1-6 haloalkyl, _C3-4 cycloalkyl, _C2-3 alkenyl, _C2-3 ynyl, aryl or heteroaryl, or two R' substituents together with the nitrogen atom attached thereto form a 3-7 membered ring; ^R3 is a halogroup, _C1-3 alkyl, _C1-2 haloalkyl, _C1-3 alkoxy, _C3-4 cycloalkyl, _C2-3 alkenyl, _C2-3 ynyl, aryl or heteroaryl; ^R4 is...

Ring A is a monocyclic 4-7 membered ring or a bicyclic, bridged, fused, or spirocyclic 6-11 membered ring; L is a bond, _C1-6 alkylene, -OC _0-5 alkylene, -SC _0-5 alkylene, or -NH-C _0-5 alkylene, and for _C2-6 alkylene, -OC _2-5 alkylene, -SC _2-5 alkylene, and NH-C _2-5 alkylene, one carbon atom of the alkylene group may optionally be replaced by O, S, or NH; R ^4' is H, _C1-8 alkyl, _C2-8 alkynyl, _C1-6 alkylene-OC _1-4 alkyl, _C1-6 alkylene-OH, _C1-6 haloalkyl, _C0-3 alkylene- _{C3-8 cycloalkyl} , _C0-3 alkylene- _C2-7 heterocyclic alkyl, _C0-3 alkylaryl, or selected from ^R5 and ^R6 , each independently being H, a halogen, C _1-8 alkyl, _C2-8 ynyl, _{C1-6 alkylene-OC1-4} alkyl, _C1-6 alkylene-OH, _C1-6 haloalkyl, _C1-6 alkyleneamine, _C0-6 alkyleneamide, _C0-3 alkylene-C(O)OH, _C0-3 alkylene-C(O) _OC1-4 alkyl, _C1-6 alkylene-O-aryl, _C0-3 alkylene-C(O) _C1-4 alkylene-OH, _C0-3 alkylene- _C3-8 cycloalkyl, _C0-3 alkylene _{- C2-7} heterocycloalkyl, _C0-3 alkylene aryl or cyano, or ^R5 and ^R6 together with their attached atoms forming a 4-6 membered ring; ^R7 is H or _C1-3 alkyl, or ^R7 and ^R5 together with their attached atoms forming a 4-6 membered ring; Q is ^{CR8 R9} , C= ^CR8 , C=O, C=S, or C= ^NR8 ; ^{R8 and R9} are each independently H, _C1-3 alkyl, hydroxyl, _C1-3 alkoxy, cyano, nitro, or _C3-6 cycloalkyl, or ^R8 and ^R9 together with their attached carbon atoms can form a 3-6 membered ring; ^R10 is _C1-8 alkyl, _C0-3 alkylene aryl, _C0-3 alkylene heteroaryl, _{C0-3 alkylene-C3-8 cycloalkyl, C0-3 alkylene - C2-7} heterocycloalkyl, C1-6 alkoxy, _OC0-3 alkylene aryl, _OC0-3 alkylene heteroaryl, _OC0-3 alkylene _{-C3-8 cycloalkyl, OC0-3 alkylene aryl, OC0-3 alkylene - C2-7 heterocycloalkyl} , NH-C _1-8 alkyl, N(C _1-8 alkyl) ₂ , NH-C _0-3 alkylene aryl, NH-C _0-3 alkylene heteroaryl, NH-C _0-3 alkylene-C _3-8 cycloalkyl, NH-C _0-3 alkylene-C _2-7 heterocycloalkyl, halogen, cyano or C _1-6 alkyleneamine; and R ¹³ is C _1-4 alkyl, C _1-3 haloalkyl, C _1-3 alkyleneamine and C _3-5 cycloalkyl, or a pharmaceutically acceptable salt thereof, subject to the following limitations: (1) when J is NR ¹⁰ , M is N or CR ¹³ ; (2) when M is NR ¹³ , J is N or CR ¹⁰ ; (3) when J is CR ¹⁰ , M is N or NR ¹³ ; and (4) when M is CR ¹³ , J is N or NR ¹⁰ .

In various embodiments, J is NR ¹⁰ and M is CR ^13. In some embodiments, J is CR ¹⁰ and M is NR ^13. In some embodiments, J is CR ¹⁰ and M is N. In various embodiments, J is N and M is NR ^13. In some embodiments, J is N and M is CR ^13. Some particularly desirable R ^13s include methyl, ethyl, propyl, isopropyl, butyl, sec- _butyl , trifluoromethyl, _CH₂NH₂ , and cyclopropyl. In some embodiments, J is NR ¹⁰ and M is N. In some embodiments, when Q is C=O and each of ^E1 and ^E2 is ^CR1 , then (1) ^R10 is a _C1-3 alkylene aryl, _C1-3 alkylene heteroaryl, _C0-3 alkylene- _C3-8 cycloalkyl, _C1-3 alkylene- _C2-7 heterocycloalkyl or haloyl; or (2) ^R13 is a _C1-3 haloalkyl or _C3-5 cycloalkyl.

Compounds of formula II may be in the form of formula (II-A), (II-B), (II-C), (II-D), (II-E), (II-F), (II-G), (II-H), (II-J), (II-K), (II-L), (II-M), (II-N), (II-O), (II-P), or (II-Q):

This disclosure also provides a compound having a structure of formula (III) or formula (III’):

Each ^R1 is independently H, hydroxyl, _C1-4 alkyl, _C1-4 haloalkyl, _C1-4 alkoxy, NH- _C1-4 alkyl, N( _C1-4 alkyl) ₂ , cyano, or halo; ^R2 is halo, C1-6 alkyl, C1-6 haloalkyl, OR', N(R') ₂ , _C2-3 alkenyl, C2-3 alkynyl, _C0-3 alkylene- _C3-8 cycloalkyl, C0-3 alkylene- _C2-7 heterocycloalkyl, _C0-3 alkylene aryl, or C0-3 alkylene heteroaryl, and each R' is independently H, C1-6 alkyl, C1-6 haloalkyl, _C3-4 cycloalkyl, _C2-3 alkenyl, _C1-6 alkyl, _C1-6 alkyl, C1-7 cycloalkyl, _C2-3 alkenyl, C1-6 alkyl, _C1-6 alkyl, C1-6 alkyl, _C1-7 cycloalkyl, C1-8 alkyl, C1-6 alkyl, C1-6 alkyl, _C1-7 alkyl, C1-8 alkyl, _C1-9 alkyl, C1-1 ... _2-3- alkynyl, aryl, or heteroaryl, or two R' substituents together with their attached nitrogen atom to form a 3-7 membered ring; ^R3 is a halogroup, _C1-3 alkyl, _C1-2 haloalkyl, _C1-3 alkoxy, _C3-4 cycloalkyl, _C2-3 alkenyl, _C2-3 alkynyl, aryl, or heteroaryl; ^R4 is...

Ring A is a monocyclic 4-7 membered ring or a bicyclic, bridged, fused, or spirocyclic 6-11 membered ring; L is a bond, _C1-6 alkylene, -OC _0-5 alkylene, -SC _0-5 alkylene, or -NH-C _0-5 alkylene, and for _C2-6 alkylene, -OC _2-5 alkylene, -SC _2-5 alkylene, and NH-C _2-5 alkylene, one carbon atom of the alkylene group may optionally be replaced by O, S, or NH; R ^4' is H, _C1-8 alkyl, _C2-8 alkynyl, _C1-6 alkylene-OC _1-4 alkyl, _C1-6 alkylene-OH, _C1-6 haloalkyl, _C0-3 alkylene- _{C3-8 cycloalkyl} , _C0-3 alkylene- _C2-7 heterocyclic alkyl, _C0-3 alkylaryl, or selected from ^R5 and ^R6 , each independently being H, a halogen, C _1-8 alkyl, _C2-8 ynyl, _{C1-6 alkylene-OC1-4} alkyl, _C1-6 alkylene-OH, _C1-6 haloalkyl, _C1-6 alkyleneamine, _C0-6 alkyleneamide, _C0-3 alkylene-C(O)OH, _C0-3 alkylene-C(O) _OC1-4 alkyl, _C1-6 alkylene-O-aryl, _C0-3 alkylene-C(O) _C1-4 alkylene-OH, _C0-3 alkylene- _C3-8 cycloalkyl, _C0-3 alkylene _{- C2-7} heterocycloalkyl, _C0-3 alkylene aryl or cyano, or ^R5 and ^R6 together with their attached atoms forming a 4-6 membered ring; ^R7 is H or _C1-3 alkyl, or ^R7 and ^R5 together with their attached atoms forming a 4-6 membered ring; Q is ^{CR8 R9} , C= ^CR8 , C=O, C=S, or C= ^NR8 ; ^{R8 and R9} are each independently H, _C1-3 alkyl, hydroxyl, _C1-3 alkoxy, cyano, nitro, or _C3-6 cycloalkyl, or ^R8 and ^R9 together with their attached carbon atoms can form a 3-6 membered ring; and ^R10 is _C1-8 alkyl, _C0-3 alkylene aryl, _C0-3 alkylene heteroaryl, _{C0-3 alkylene-C3-8 cycloalkyl, C0-3 alkylene - C2-7} heterocycloalkyl, _C1-6 alkoxy, _C1-6 alkoxy, _OC0-3 alkylene aryl, _OC0-3 alkylene heteroaryl, _OC0-3 alkylene- _C3-8 cycloalkyl, _OC0-3 alkylene- _C2-7 heterocycloalkyl, NH-C _1-8 alkyl, N(C _1-8 alkyl) ₂ , NH-C _0-3 alkylene aryl, NH-C _0-3 alkylene heteroaryl, NH-C _0-3 alkylene-C _3-8 cycloalkyl, NH-C _0-3 alkylene-C _2-7 heterocycloalkyl, halogen, cyano or C _1-6 alkyleneamine, or a pharmaceutically acceptable salt thereof.

Compounds of formula III may be in the form of formula (III-A), (III-B), (III-C), or (III-D):

Compounds having formula III’ can be in the form of formula (III-A’), (III-B’), (III-C’), or (III-D’):

This disclosure also provides a compound having a structure of formula (IV) or formula (IV’):

^E1 and ^E2 are each independently ^CR1 or N; ^R1 is independently H, hydroxyl, _C1-4 alkyl, _C1-4 haloalkyl, _C1-4 alkoxy, NH- _C1-4 alkyl, N( _C1-4 alkyl) ₂ , cyano, or halo; ^R2 is halo, _C1-6 alkyl, _C1-6 haloalkyl, OR', N(R') ₂ , _C2-3 alkenyl, _C2-3 alkynyl, _C0-3 alkylene- _C3-8 cycloalkyl, _C0-3 alkylene- _C2-7 heterocycloalkyl, _C0-3 alkylene aryl, or _C0-3 alkylene heteroaryl, and each R' is independently H, _C1-6 alkyl, _C1-6 haloalkyl, _C3-4 cycloalkyl, _C2-3 alkenyl, C _2-3 alkynyl, aryl, or heteroaryl, or two R' substituents together with their attached nitrogen atom to form a 3-7 membered ring; ^R3 is a halogroup, _C1-2 haloalkyl, _C1-3 alkoxy, _C3-4 cycloalkyl, _C2-3 alkenyl, _C2-3 alkynyl, aryl, or heteroaryl; ^R4 is a ring A is a monocyclic 4-7 membered ring or bicyclic, bridged, fused, or spirocyclic 6-11 membered ring; L is a bond, _C1-6 alkylene, -OC _0-5 alkylene, -SC _0-5 alkylene, or -NH-C _0-5 alkylene, and for _C2-6 alkylene, -OC _2-5 alkylene, -SC _2-5 alkylene, and NH-C _2-5 alkylene, one carbon atom of the alkylene group may optionally be replaced by O, S, or NH; ^R4' is H, _C1-8 alkyl, _C2-8 alkynyl, C _1-6 alkylene-OC _1-4 alkyl, C _1-6 alkylene-OH, C _1-6 haloalkyl, C _0-3 alkylene-C _{3-8 cycloalkyl} , C _0-3 alkylene-C _2-7 heterocycloalkyl, C _0-3 alkylene aryl, or selected from R ⁵ and R ⁶ , each independently being H, halogen, C _1-8 alkyl, C _2-8 ynyl, C _1-6 alkylene-OC _1-4 alkyl, C _1-6 alkylene-OH, C _1-6 haloalkyl, C _1-6 alkyleneamine, C _0-6 alkyleneamide, C _0-3 alkylene-C(O)OH, C _0-3 alkylene-C(O)OC _1-4 alkyl, C _1-6 alkylene-O-aryl, C _0-3 alkylene-C(O)C _1-4 alkylene-OH, C _0-3 alkylene-C _3-8 cycloalkyl, C R10 is a _C1-3 alkylene- _C2-7 heterocyclic alkyl, _C0-3 alkylene aryl or cyano, or ^R5 and ^R6 together with their attached atoms form a 4-6 membered ring; ^R7 is H or _C1-3 alkyl, or ^R7 and ^R5 together with their attached atoms form a 4-6 membered ring; ^R8 is _C1-3 alkyl, hydroxyl, _C1-3 alkoxy, halogroup, cyano, nitro, _C3-6 cycloalkyl or ^NR11R12 ; ^R11 and ^R12 are each independently H, _C1-4 alkyl or ^C3-5 _cycloalkyl ; and ^R10 is _C1-8 alkyl, _C0-3 alkylene aryl, _C0-3 alkylene heteroaryl, _C0-3 alkylene _{-C3-8 cycloalkyl, C0-3 alkylene-C2-7 heterocyclic alkyl, C1-6 alkoxy , OC 0-3} alkylene aryl, OC _0-3 alkylene heteroaryl, OC _0-3 alkylene- _C3-8 cycloalkyl, OC _0-3 alkylene- _C2-7 heterocycloalkyl, NH- _C1-8 alkyl, N( _C1-8 alkyl) ₂ , NH- _C0-3 alkylene aryl, NH- _C0-3 alkylene heteroaryl, NH- _C0-3 alkylene- _C3-8 cycloalkyl, NH- _C0-3 alkylene- _C2-7 heterocycloalkyl, halogen, cyano, or _C1-6 alkyleneamine, or a pharmaceutically acceptable salt thereof. In some embodiments, ^E1 and ^E2 are each ^CR1 , and ^R8 is a hydroxyl, halogen, nitro, or _C3-6 cycloalkyl. In some embodiments, ^R8 is methyl. The compound may have the structure of formula (IV-A), (IV'-A), (IV-B), (IV'-B), (IV-C), (IV'-C), (IV-D), or (IV'-D):

This article also provides compounds having the structure of formula (V):

^E1 and ^E2 are each independently ^CR1 or N; ^R1 is independently H, hydroxyl, _C1-4 alkyl, _C1-4 haloalkyl, _C1-4 alkoxy, NH- _C1-4 alkyl, N( _C1-4 alkyl) ₂ , cyano, or halo; ^R2 is halo, _C1-6 alkyl, _C1-6 haloalkyl, OR', N(R') ₂ , _C2-3 alkenyl, _C2-3 alkynyl, _C0-3 alkylene- _C3-8 cycloalkyl, _C0-3 alkylene- _C2-7 heterocycloalkyl, _C0-3 alkylene aryl, or _C0-3 alkylene heteroaryl, and each R' is independently H, _C1-6 alkyl, _C1-6 haloalkyl, _C3-4 cycloalkyl, _C2-3 alkenyl, C _2-3 alkynyl, aryl, or heteroaryl, or two R' substituents together with their attached nitrogen atom to form a 3-7 membered ring; ^R3 is a halogroup, _C1-3 alkyl, _C1-2 haloalkyl, _C1-3 alkoxy, _C3-4 cycloalkyl, _C2-3 alkenyl, _C2-3 alkynyl, aryl, or heteroaryl; ^R4 is a ring A is a monocyclic 4-7 membered ring or bicyclic, bridged, fused, or spirocyclic 6-11 membered ring; L is a bond, _C1-6 alkylene, -OC _0-5 alkylene, -SC _0-5 alkylene, or -NH-C _0-5 alkylene, and for _C2-6 alkylene, -OC _2-5 alkylene, -SC _2-5 alkylene, and NH-C _2-5 alkylene, one carbon atom of the alkylene group may optionally be replaced by O, S, or NH; R4 ^' is H, _C1-8 alkyl, C _2-8 ynyl, _C1-6 alkylene- _OC1-4 alkyl, _C1-6 alkylene-OH, _C1-6 haloalkyl, _C0-3 alkylene- _{C3-8 cycloalkyl} , _C0-3 alkylene- _C2-7 heterocycloalkyl, _C0-3 alkylene aryl, or selected from...

^R5 and ^R6 are each independently H, a halogroup, _C1-8 alkyl, _C2-8 alkynyl, _C1-6 alkylene- _OC1-4 alkyl, C1-6 alkylene _{-OH, C1-6 haloalkyl, C1-6 alkyleneamine, C0-6 alkyleneamide, C0-3 alkylene-C(O)OH, C0-3 alkylene - C (} O) _OC1-4 alkyl, _C1-6 alkylene-O-aryl, _C0-3 alkylene-C(O) _C1-4 alkylene-OH, _C0-3 alkylene- _C3-8 cycloalkyl, _C0-3 alkylene- _C2-7 heterocycloalkyl, _C0-3 alkylene aryl or cyano, or ^R5 and ^R6 together with the atoms to which they are attached form _a 4-6 membered ring; ^R7 is H or _C1-3 alkyl, or ^R7 and R ⁵ , together with the atoms to which it is attached, forms a 4-6 membered ring; and R ¹⁰ is a _C1-8 alkyl, _C0-3 alkylene aryl, _C0-3 alkylene heteroaryl, _C0-3 alkylene- _C3-8 cycloalkyl, _C0-3 alkylene- _C2-7 heterocycloalkyl, _C1-6 alkoxy, OC0-3 alkylene aryl, _OC0-3 alkylene heteroaryl, _OC0-3 alkylene- _C3-8 cycloalkyl, _OC0-3 alkylene- _C2-7 heterocycloalkyl, NH- _C1-8 alkyl, _NC1-8 alkyl, NH- _C0-3 alkylene aryl, NH- _C0-3 alkylene heteroaryl, NH- _{C0-3 alkylene-C3-8 cycloalkyl} , NH- _C0-3 alkylene _{-C2-7 heterocycloalkyl} , halogen, cyano, or C _1-6 alkyleneamines; or pharmaceutically acceptable salts thereof.

For compounds having formulas (II), (III), and (III'): In some embodiments, Q is C=O. In some embodiments, Q is C=S. In some embodiments, Q is C= ^NR8 . ^R8 may be a _C1-2 alkyl group, such as methyl.

Q can be ^CR8R9 or C= ^CR8R9 . ^R8 and ^{R9 ,} together with the carbon atoms attached to them ^, can form a 3-4 membered ring, such as a cyclopropyl ring. In some embodiments, ^R8 is a _C1-2 alkyl group (e.g., methyl) and ^R9 is H.

For compounds having formulas (II), (III), (III'), (IV), (IV'), and (V): In various embodiments, ^R10 is a _C1-4 alkyl, aryl, heteroaryl, _C3-6 cycloalkyl, _C3-6 heterocycloalkyl, _C1-4 alkoxy, or aryloxy. In various embodiments, ^R10 is a _C1-8 alkyl, _C1-5 alkyl, or _C1-3 alkyl. In various embodiments, ^R10 is a _C0-3 alkylene aryl, _C0-1 alkylene aryl, or phenyl. In various embodiments, ^R10 is a _C0-3 alkylene heteroaryl or _C0-1 alkylene heteroaryl, and the heteroaryl group may be, for example, pyridyl. In various embodiments, ^R10 is a _C0-3 alkylene- _C3-8 cycloalkyl, _C0-1 alkylene- _C3-8 cycloalkyl, or _C3-8 cycloalkyl, and the cycloalkyl group may be, for example, cyclohexyl. In various embodiments, ^R10 is a _C0-3 alkylene- _C3-8 heterocycloalkyl or a _C0-1 alkylene- _C3-8 heterocycloalkyl. In various embodiments, ^R10 is a _C0-6 alkyleneamine or a _C0-3 alkyleneamine or amine. Some particularly anticipated ^R10s include i-Pr, t-Bu, phenyl, benzyl, _OCH3 , Cl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc.

^R10 may comprise an ortho-substituted aryl group, an ortho-substituted heteroaryl group, or a 2-substituted cyclohexyl group. For example, in another embodiment, ^R10 may comprise...

For all compounds:

^R1 can be a smaller fraction. For example, ^R1 can be H, _C1–2 alkyl (e.g., methyl), _C1–2 haloalkyl (e.g., _CF3 ), or a halogroup (e.g., F). Some particularly anticipated ^R1s include H, F, Me, Cl, and _CF3 .

^R2 can be _C1-3 alkyl, _C1-3 haloalkyl, _C1-3 alkoxy, _C0-1 alkylene- _C3-8 cycloalkyl, _C3-6 cycloalkyl, _C0-1 alkylene aryl (e.g., aryl), or _C0-1 alkylene heteroaryl (e.g., heteroaryl). Some particularly desirable ^R2 groups include phenyl, naphthyl, pyridinyl, indazole, indolyl, azaindolyl, dihydroindolyl, benzotriazolyl, benzoxadiazolyl, imidazolyl, cenolinyl, imidazopyridyl, pyrazolopyridyl, quinolinyl, isoquinolinyl, quinazolinyl, quinazolinone, indolone, isoindolone, tetrahydronaphthyl, tetrahydroquinolinyl, or tetrahydroisoquinolinyl. Some other specific ^R2s include Cl, Br, _CF3 , cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, piperidine, pyrrolidine, azacyclobutane, _OCH3 , _OCH2CH3 , _phenyl ,

And in some embodiments, ^R2 is

^R3 can be a halogroup (e.g., Cl, F), a _C1-2 alkyl group (e.g., methyl), or a _C1-2 haloalkyl group (e.g., _CF3 ). Some particularly anticipated R3 ^groups include Cl, F, Me, _CF3 , OMe, Et, C= _CH2 , and cyclopropyl.

L is a bond, a _C1-6 alkylene group, a -OC _0-5 alkylene group, _{a -SC 0-5 alkylene group, or a -NH-C 0-5 alkylene group} , and for _C2-6 alkylene groups, a carbon atom _of the alkylene group may optionally be substituted with O, S, or NH. For example, when the carbon atom on the _C2 alkylene group is substituted with NH, L may be _-CH2 -NH-, or when the carbon atom on the _OC3 alkylene group is substituted with O, it may be –O- _CH2CH2 - _O- . Other options for substitution of _C3 , _C4 , _C5 , or _C6 alkylene groups with O, S, or NH are also considered. In some embodiments, L is a _C1-2 alkylene group, O, S, or NH. In some embodiments, L is a bond.

Ring A is a monocyclic 4-7 membered ring or a bicyclic, bridged, fused, or spirocyclic 6-11 membered ring. Some particularly anticipated rings include cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, pyrrolyl, piperidinyl, azacycloheptyl, imidazoalkyl, hexahydropyrimidinyl, hexahydropyridazinyl, tetrahydrofuranyl, tetrahydrothiofuranyl, azacyclobutyl, spiroheptyl, spiroctyl, spirononyl, spirodecyl, diazabicyclodecyl, diazabicyclononyl, diazabicyclooctyl, diazabicycloheptyl, hexahydropyrrolopyridinyl, octahydropyrrolopyridinyl, and octahydropyrrolopyrimidinyl. In various embodiments, ring A may comprise piperidinyl, piperazinyl, pyrrolyl, or azacyclobutyl. In some embodiments, ring A comprises piperidinyl. Ring A may be further substituted with one to three substituents. Some non-limiting examples of substitution on ring A include one to three substituents selected from the following: alkyl, alkenyl, alkynyl, hydroxyalkyl, carboxylic acid or ester, haloalkyl, alkylamine, C(O) _NH2 , oxo, halogen, cyano, and isocyano.

When R ⁴ is , ring A can be, for example, more specifically, when R ⁴ is , ring A can be, for example,

When R ⁴ is true, it can be more specifically defined in such embodiments as ring A being, for example,

^R5 and ^R6 are substituents on the acrylamide moiety of the KRAS inhibitors disclosed herein. In some embodiments, each of ^R5 and ^R6 is H. Some particularly anticipated ^R5 substituents include H, Br, Cl, F, CN, _CH3 , _CF3 , _CH2Br , _CH2OH , _CH2CH2OH , _{CH2OCH2phenyl} , cyclopropyl, phenyl, _CH2phenyl , _CH2OCH3 , _CH2N ( _CH3 ) _{2 , CH2N ( CH2CH3} ) ₂ , _CH2CO2H , _CH2CO2CH3 , _CH2NHC ( _O ) _CH3 , _CH2C ( _O ) _NHCH3 , _CH2OC (O _{) CH3 , or}

Some specifically anticipated R- ⁶ substituents include phenyl, cyclopropyl, CH3, _CF3 , _CH2CH3 , _CH2NH2 , CH( _CH3 )NH2, CH( _{CH3 ) 2NH2, CH2Cl , CH2Br , CH2OCH3} , _CH2Ophenyl , CH2OH, _CO2H , _CO2CH2CH3 , _CH2CO2H , _CH2CH2NH2 , CH2CH2OH, CH2CH2N _{( CH3 )2 , CH2NHCH3} , _{C ( O ) NHCH3} , C _{( O ) N} ( _CH3 ) ₂ , _CH2C ( _{O ) NHphenyl} , _CH2CHF2 , _CH2F , _{CHF2 , CH2NHC (} O) _{CH3 , CH2} NHCH ₂ CH ₂ OH, CH ₂ NHCH ₂ CO ₂ H, CH ₂ NH(CH ₃ )CH ₂ CO ₂ CH ₃ , CH ₂ NHCH ₂ CH ₂ OCH ₃ , CH ₂ NH(CH ₃ )CH ₂ CH ₂ OCH ₃ , CH ₂ NH(CH ₃ )CH ₂ C(O)N(CH ₃ ) ₂ , CH ₂ NH(CH ₃ )CH ₂ C(O)NHCH ₃ , CH ₂ CH ₂ CCH, CH ₂ NMe ₂ , CH ₂ NH(CH ₃ )CH ₂ CH ₂ OH, CH ₂ NH(CH ₃ )CH ₂ CH ₂ F, CH ₂ N ⁺ (CH ₃ ) ₃ , CH ₂ NHCH ₂ CHF ₂ , CH ₂ NHCH ₂ CH ₃ ,

^R5 and ^R6, together with the atoms they are attached to, can form 4-6 membered rings, such as 5- or 6-membered rings. These rings include ^R5 and ^R6 together...

In most embodiments, ^R7 is H. However, in some embodiments, ^R7 is methyl. In other embodiments, ^R7 and ^R5 together are _-CH2- or -C(O) _CH2- .

Some of the specially anticipated options include

and

Some particularly anticipated ^R4 substituents include

and

Some specifically anticipated R ^4' substituents may include

In another embodiment, the present invention discloses compounds having structures selected from the following:

In another embodiment, the present invention discloses compounds having structures selected from the following:

And its stereoisomers, its transisomers, its pharmaceutically acceptable salts, pharmaceutically acceptable salts of its stereoisomers or its transisomers, or pharmaceutically acceptable salts of their transisomers.

In another embodiment, the present invention discloses compounds having structures selected from the following:

Or its stereoisomer, its transisomer, its pharmaceutically acceptable salt, its pharmaceutically acceptable salt, or its transisomer's pharmaceutically acceptable salt.

In another embodiment, the present invention discloses compounds having structures selected from the following:

Or its stereoisomer, its transisomer, its pharmaceutically acceptable salt, its pharmaceutically acceptable salt, or its transisomer's pharmaceutically acceptable salt.

In another embodiment, the present invention discloses compounds having structures selected from the following:

Or its stereoisomer, its transisomer, its pharmaceutically acceptable salt, its pharmaceutically acceptable salt, or its transisomer's pharmaceutically acceptable salt.

In another embodiment, the present invention discloses compounds having structures selected from the following:

Or its stereoisomer, its transisomer, its pharmaceutically acceptable salt, its pharmaceutically acceptable salt, or its transisomer's pharmaceutically acceptable salt.

In another embodiment, the present invention discloses compounds having structures selected from the following:

Or its stereoisomer, its transisomer, its pharmaceutically acceptable salt, its pharmaceutically acceptable salt, or its transisomer's pharmaceutically acceptable salt.

In another embodiment, the present invention discloses compounds having structures selected from the following:

Or its stereoisomer, its transisomer, its pharmaceutically acceptable salt, its pharmaceutically acceptable salt, or its transisomer's pharmaceutically acceptable salt.

In another embodiment, the present invention discloses compounds having structures selected from the following:

Or its stereoisomer, its transisomer, its pharmaceutically acceptable salt, its pharmaceutically acceptable salt, or its transisomer's pharmaceutically acceptable salt.

In another embodiment, the present invention discloses compounds having structures selected from the following:

Or its stereoisomer, its transisomer, its pharmaceutically acceptable salt, its pharmaceutically acceptable salt, or its transisomer's pharmaceutically acceptable salt.

In another embodiment, the present invention discloses compounds having structures selected from the following:

Or its stereoisomer, its transisomer, its pharmaceutically acceptable salt, its pharmaceutically acceptable salt, or its transisomer's pharmaceutically acceptable salt.

In another embodiment, the present invention discloses compounds having structures selected from the following:

Or its stereoisomer, its transisomer, its pharmaceutically acceptable salt, its pharmaceutically acceptable salt, or its transisomer's pharmaceutically acceptable salt.

In another embodiment, the present invention discloses compounds having structures selected from the following:

Or its stereoisomer, its transisomer, its pharmaceutically acceptable salt, its pharmaceutically acceptable salt, or its transisomer's pharmaceutically acceptable salt.

In another embodiment, the present invention discloses compounds having structures selected from the following:

Or its stereoisomer, its transisomer, its pharmaceutically acceptable salt, its pharmaceutically acceptable salt, or its transisomer's pharmaceutically acceptable salt.

In another embodiment, the present invention discloses compounds having structures selected from the following:

Or its stereoisomer, its transisomer, its pharmaceutically acceptable salt, its pharmaceutically acceptable salt, or its transisomer's pharmaceutically acceptable salt.

In another embodiment, the present invention discloses compounds having structures selected from the following:

Or its stereoisomer, its transisomer, its pharmaceutically acceptable salt, its pharmaceutically acceptable salt, or its transisomer's pharmaceutically acceptable salt.

In another embodiment, the present invention discloses compounds having structures selected from the following:

Or its stereoisomer, its transisomer, its pharmaceutically acceptable salt, its pharmaceutically acceptable salt, or its transisomer's pharmaceutically acceptable salt.

In another embodiment, these compounds may be used as intermediates in a method for preparing the compounds of this application.

In another embodiment, these compounds may be in the form of pharmaceutically acceptable salts.

In another embodiment, these compounds may be in the form of a pharmaceutical formulation comprising any one or more compounds and pharmaceutically acceptable excipients.

In another embodiment, these compounds can be used in a method for inhibiting KRAS G12C in cells, the method comprising contacting the cells with any of these compounds or with the pharmaceutical formulation.

In another embodiment, these compounds can be used in a method of treating a subject's cancer, the method comprising administering to the subject a therapeutically effective amount of any one or a combination of these compounds.

In another embodiment, the cancer is lung cancer, pancreatic cancer, or colorectal cancer.

In another embodiment, the cancer is lung cancer.

In another embodiment, the cancer is pancreatic cancer.

In another embodiment, the cancer is colorectal cancer.

In another embodiment, the method further includes administering a therapeutically effective amount of another pharmaceutically active compound to a patient in need.

In another embodiment, the other pharmaceutically active compound is carfilzomib.

In another embodiment, the other pharmaceutically active compound is cytarabine.

In another embodiment, the invention includes the use of one or more compounds for treating cancer in a subject.

In another embodiment, the present invention includes the use of one or more compounds for the preparation of a medicament for treating cancer.

In another embodiment, the cancer is a blood malignancy.

In another embodiment, the invention includes the use of one or more compounds for treating cancer, wherein the cancer is a blood malignancy.

Examples 1-11 below use the classical system notation, where the first number indicates the method used to synthesize the compound, the second number is an identification number, and, if present, the third number indicates the elution order of the compound in the chromatographic separation procedure. If the third number is absent, the compound is a single compound or a mixture of isomers. Examples 12-53 use the classical system, where the first number is an identification number, and, if present, the second number indicates the elution order of the compound in the chromatographic separation procedure. If the second number is absent, the compound is a single compound or a mixture of isomers. The sequential numbering of the examples is discontinuous, and some example numbers have been intentionally omitted for formatting purposes. “-” indicates no change, or no entry in the relevant box. Compounds specifically anticipated include those listed in Table 1 and Table 1(a):

Table 1

Table 1(a)

The compounds disclosed in the synthesis

The compounds disclosed in this article can be synthesized via a variety of specific methods. The following outlines examples of specific synthetic routes and general schemes intended to provide guidance to ordinary synthetic chemists, who will find them easy to understand. Modifications to solvents, concentrations, reagents, protecting groups, the order of synthetic steps, time, temperature, etc., can be made as needed, entirely within the skill and judgment of a person of ordinary technical ability.

Method 1

Method 1 Synthesis: The compound having formula (I) disclosed herein can be synthesized as briefly described in Method 1. In step 1, a suitable aromatic or heteroaromatic acid is reacted with a halogenating agent to form a halogenated aromatic or heteroaromatic acid. Then, in step 2, the acid is reacted with an amidating agent to form an amide intermediate. Then, in step 3, the amide intermediate is reacted with a sulfurizing agent to form a thioamide intermediate. Next, in step 4, the thioamide intermediate is reacted with an oxidizing agent to form the thiazole ring shown. Then, in step 5, the amine of the thiazole is converted into a leaving group using an activating agent. Then, as shown in step 6, the leaving group is replaced with a protected ^R4 group. Then, in step 7, the ^R2 moiety is introduced by cross-coupling a suitable ^R2 (protected) reagent with the X halide on the thiazole intermediate. Then, in step 8, depending on the protecting group used, the ^R4 group is deprotected under appropriate conditions, then the ^R4 group is acylated to introduce the acrylamide moiety as shown, and finally, the ^R2 group is deprotected. Suitable protecting groups and deprotecting agents are known to those skilled in the art, for example, as discussed in Greene's Protective Groups in Organic Synthesis.

The halogenating agents considered include, but are not limited to, chlorine, bromine, N-chlorosuccinimide, and N-bromosuccinimide, optionally in the presence of a catalyst, such as iron or aluminum. It will be readily understood by ordinary synthetic chemists that other halogenating agents and catalysts can be used.

Considered amidating agents include, but are not limited to, N,N'-diisopropylcarbodiimide, N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide, benzotriazol-1-yl-oxytripyridylphosphonium hexafluorophosphate, O-(benzotriazol-1-yl)-N,N,N',N'-tetramethylureaium hexafluorophosphate, thionyl chloride, isobutyl chloroformate, diethyl cyanophosphonate, carbonyl diimidazole, and polyphosphonic anhydride. It will be readily understood by ordinary synthetic chemists that other amidating agents can be used.

The sulfiding agents considered include, but are not limited to, sulfur, phosphorus pentasulfide, and Lawson's reagent. It will be readily understood by ordinary synthetic chemists that other sulfiding agents can be used.

Oxidizing agents considered include, but are not limited to, hydrogen peroxide, diacetate iodobenzene, tert-butyl hydrogen peroxide, N-bromosuccinimide, and ammonium peroxydisulfate. Ordinary synthetic chemists will readily understand that other oxidizing agents can be used.

The activators considered include, but are not limited to, sodium nitrite and tert-butyl nitrite. It will be readily understood by ordinary synthetic chemists that other activators can be used.

The cross-coupling reactions considered include, but are not limited to, Suzuki coupling, Negishi coupling, Hiyama coupling, Kumada coupling, and Stille coupling. It will be readily understood by the average chemist that couplings as shown in Method 1 can be carried out under a variety of conditions.

Method 2

Method 2 Synthesis: Method 2 provides an alternative method for forming the compound having formula (I) disclosed herein. Following halogenation in step 1, a protected ^R4 group is introduced in step 2 by reacting with an acid in a coupling reaction. In step 3, the oxo group is converted to sulfur using a sulfiding agent. Then, in step 4, a thiazole ring is formed in the presence of an oxidizing agent. The remaining steps 5–8 are similar to steps 7 and 8 of Method 1 described above.

Method 3

Method 3 Synthesis: Method 3 provides an alternative method for forming the compound having formula (I) disclosed herein. The ^R4 group of the isothiazole intermediate is deprotected and acylated in step 1 to introduce the acrylamide moiety. Then, in step 2, the ^R2 moiety is introduced by cross-coupling a suitable ^R2 (protected) reagent with the X halide on the isothiazole intermediate. Finally, the ^R2 group is deprotected in step 3.

Method 4

Method 4 Synthesis: Method 4 provides an alternative method for forming the compound having formula (I) disclosed herein. Following substitution of the leaving group on the isothiazole intermediate with a protected ^R4 group as described in step 1, the ^R4 group intermediate is deprotected and acylated in step 2 to introduce the acrylamide moiety. Similar to Method 1, the ^R2 moiety is introduced in step 3 via a cross-coupling reaction, and the ^R2 group is deprotected in step 4.

Method 5

Method 5 Synthesis: Method 5 provides an alternative method for forming compounds having formula (I) disclosed herein. In this alternative method, the ^R2 moiety is first introduced by cross-coupling with the X halide on the aromatic or heteroaromatic amide intermediate shown in step 1. Then, in step 2, the amide intermediate is reacted with a sulfurizing agent to form a thioamide intermediate. In step 3, this intermediate is oxidized to give an isothiazole ring. Then, in step 4, the amino group is converted into a leaving group and subsequently, in step 5, substituted with a protected ^R4 group. Finally, in step 6, the ^R4 group is deprotected and reacted with an acylation agent, and then the ^R2 group is deprotected.

Method 6

Method 6 Synthesis: Method 6 provides an alternative method for forming the compound having formula (I) disclosed herein. In this alternative method, an isothiazole intermediate is reacted with a metallizing agent to activate the X halide. Then, an ^R2 group is introduced by reacting the activated intermediate with a suitable ^R2 (protected) reagent. In the final step, the ^R4 group is deprotected and acylated to introduce the acrylamide moiety.

The anticipated metallizing agents include, but are not limited to, bis(pinacolyl)diboron, magnesium, zinc, hexamethyldistinane, and n-butyllithium. Other metallizing agents and catalysts will be readily understood by synthetic chemists with general skills.

Method 7

Method 7 Synthesis: Method 7 provides an alternative method for forming compounds of formula (I) disclosed herein. The ^R2 moiety is first introduced by cross-coupling with the X halide on the aromatic or heteroaromatic acid intermediate shown in step 1. Then, in step 2, the acid moiety is reacted with a suitable ^R4 (protected) reagent in the presence of an amidating agent. Then, in step 3, the carbonyl group of the acid derivative is converted to a thiocarbonyl group using a sulfiding agent. Then, in step 4, the thioacid derivative is reacted with an oxidizing agent to form an isothiazole intermediate. Finally, the ^R4 group is deprotected and acylated to introduce the acrylamide moiety, and the ^R2 group is deprotected.

Method 8

Method 8 Synthesis: The compounds having formula (II) disclosed herein can be synthesized as briefly described in Method 8. In step 1, a suitable aromatic or heteroaromatic acid is reacted with an amidating agent to form a major amide intermediate. The amide is then reacted with an isocyanate forming agent and ^R10 -substituted amine to form a urea intermediate. Considered isocyanate forming agents include oxalyl chloride, thionyl chloride, and phosphorus oxychloride. In step 3, the urea intermediate is then reacted with a cyclizing agent to form the quinazolinidone ring shown. Considered cyclizing agents include, but are not limited to, bases such as potassium hexamethyldisilamide, potassium tert-butoxide, sodium hydride, and phosphazene bases. In step 4, the ^R2 moiety is introduced by cross-coupling a suitable ^R2 (protected) agent with the X halide on the quinazolinidone intermediate. In step 5, an activator is used to convert the oxo group of the quinazolinidone into a leaving group. The activators considered include, but are not limited to, thionyl chloride, trifluoromethanesulfonic anhydride, phosphorus oxychloride, and phosphorus pentachloride. Then, as shown in step 6, the leaving group is replaced with a protected ^R4 group to form a substituted quinazolinone. The remaining deprotection-acylation-deprotection steps shown in steps 7-9 are similar to step 8 in method 1.

Method 9

Method 9 Synthesis: Method 9 provides an alternative method for forming compounds of formula (II) disclosed herein. In step 1, the oxo group of the quinazolinidone is converted into a leaving group. Step 2 involves the introduction of an ^R4 (protected) group, deprotection of the ^R4 group, and acylation of the free ^R4 group. In step 3, the ^R2 group is introduced by cross-coupling a suitable ^R2 (protected) reagent with the X halide on the quinazolinidone intermediate. Finally, the ^R2 group is deprotected.

Method 10

Method 10 Synthesis: The compound having formula (V) disclosed herein can be synthesized as briefly described in Method 10. As shown in step 1, a suitable acid anhydride is reacted with hydrazine to form an phthalazine dione ring. In step 2, the ^R2 moiety is introduced by cross-coupling a suitable ^R2 reagent with the X halide on a quinazolinone intermediate. Then, in step 3, the ^R2 group is protected. The phthalazine dione ring is halogenated twice. Halogenating agents considered include thionyl chloride, phosphorus oxychloride, and oxalyl chloride. Then, as shown in step 5, a halogen group is substituted with the protected ^R4 group to form a substituted phthalazine ring. Then, in steps 6 and 7, depending on the protecting group used, the ^R4 group is deprotected under suitable conditions, and then the free ^R4 group is acylated to introduce an acrylamide moiety. In step 8, the ^R2 group is deprotected. Finally, in step 9, the ^R10 moiety is introduced by cross-coupling an appropriate ^R10 reagent with the X halide on the phthalazine intermediate.

Method 11

Method 11 Synthesis: Method 11 provides an alternative method for forming compounds having formula (II) disclosed herein. In step 1, the oxo group of the quinazolinidone is converted into a leaving group. In step 2, an ^R4 (protected) group is introduced. In step 3, an R2 group is introduced by cross-coupling a suitable ^R2 (protected) reagent with the X halide on the quinazolinidone intermediate. Finally, in steps 4 and 5, ^{the R4} group is deprotected and subsequently acylated.

Drug composition, administration and route of administration

This document also provides pharmaceutical compositions comprising compounds as disclosed herein and pharmaceutically acceptable excipients, such as diluents or carriers. Compounds and pharmaceutical compositions suitable for use in this invention include those that can be administered in an effective amount to achieve their intended purpose. The administration of the compound will be described in more detail below.

The appropriate pharmaceutical formulation can be determined by a technician based on the route of administration and the required dosage. See, for example, Remington’s Pharmaceutical Sciences, 1435-712 (18th edition, Mack Publishing Co., Easton, PA, 1990). The formulation can affect the physical state, stability, or rate of release and clearance in vivo of the administered drug. Depending on the route of administration, the appropriate dosage can be calculated based on body weight, body surface area, or organ size. Those skilled in the art can refine the calculations required to determine the appropriate therapeutic dosage in a conventional manner, without conducting excessive experiments, particularly based on the dosage information and measurements disclosed herein and pharmacokinetic data available from animal or human clinical trials.

The phrase "pharmaceutically acceptable" or "pharmacologically acceptable" refers to molecular entities and compositions that do not produce adverse reactions, allergic reactions, or other adverse effects when administered to animals or humans. As used herein, "pharmaceutically acceptable" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption-delaying agents. The use of such excipients in pharmaceutically active substances is well known in the art. Their use in therapeutic compositions is considered except in cases where any conventional media or agents are incompatible with the therapeutic composition. Complementary active ingredients may also be incorporated into the composition. In an exemplary embodiment, the formulation may comprise corn syrup solids, high-oleic safflower oil, coconut oil, soybean oil, L-leucine, tricalcium phosphate, L-tyrosine, L-proline, L-lysine acetate, DATEM (emulsifier), L-glutamine, L-valine, dipotassium hydrogen phosphate, L-isoleucine, L-arginine, L-alanine, glycine, L-asparagine monohydrate, L-serine, potassium citrate, L-threonine, sodium citrate, magnesium chloride, L-histidine, L-methionine, ascorbic acid, Calcium carbonate, L-glutamic acid, L-cysteine dihydrochloride, L-tryptophan, L-aspartic acid, choline chloride, taurine, m-inositol, ferrous sulfate, ascorbyl palmitate, zinc sulfate, L-carnitine, α-tocopherol acetate, sodium chloride, nicotinamide, mixed tocopherols, calcium pantothenate, ketone sulfate, thiamine chloride hydrochloride, vitamin A palmitate, manganese sulfate, riboflavin, pyridoxine hydrochloride, folic acid, β-carotene, potassium iodide, phylloquinone, biotin, sodium selenate, chromium chloride, sodium molybdate, vitamin D3, and cyanocobalamin.

Compounds may be present in pharmaceutical compositions as pharmaceutically acceptable salts. As used herein, "pharmaceutically acceptable salts" include, for example, base addition salts and acid addition salts.

Pharmaceutically acceptable base addition salts can be formed using metals or amines (e.g., alkali metals and alkaline earth metals or organic amines). Pharmaceutically acceptable salts of compounds can also be prepared using pharmaceutically acceptable cations. Suitable pharmaceutically acceptable cations are well known to those skilled in the art and include alkali metals, alkaline earth metals, ammonium, and quaternary ammonium cations. Carbonates or bicarbonates are also possible. Examples of metals used as cations are sodium, potassium, magnesium, ammonium, calcium, or ferric iron. Examples of suitable amines include isopropylamine, trimethylamine, histidine, N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, dicyclohexylamine, ethylenediamine, N-methylglucosamine, and procaine.

Pharmaceutically acceptable acid addition salts include inorganic or organic acid salts. Examples of suitable acid salts include hydrochlorides, formates, acetates, citrates, salicylates, nitrates, and phosphates. Other suitable pharmaceutically acceptable salts are well known to those skilled in the art and include, for example, formic acid, acetic acid, citric acid, oxalic acid, tartaric acid or mandelic acid, hydrochloric acid, hydrobromic acid, sulfuric acid or phosphoric acid; and those with organic carboxylic acids, sulfonic acids, sulfonic acids or phosphates or N-substituted aminosulfonic acids, such as acetic acid, trifluoroacetic acid (TFA), propionic acid, glycolic acid, succinic acid, maleic acid, hydroxymaleic acid, methylmaleic acid, fumaric acid, malic acid, tartaric acid, lactic acid, oxalic acid, gluconic acid, glucuronic acid, glucuronic acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, salicylic acid, 4-aminosalicylic acid, 2-benzene Salts of oxybenzoic acid, 2-acetoxybenzoic acid, pyric acid, nicotinic acid, or isonicotinic acid; and salts of amino acids, such as the 20 α-amino acids involved in protein synthesis in nature, such as glutamic acid or aspartic acid, and salts of phenylacetic acid, methanesulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, ethane-1,2-disulfonic acid, benzenesulfonic acid, 4-methylbenzenesulfonic acid, naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 2-phosphoglyceric acid or 3-phosphoglyceric acid, glucose-6-phosphate, N-cyclohexylaminosulfonic acid (for the formation of cyclohexylaminosulfonate), or other acidic organic compounds, such as ascorbic acid.

Pharmaceutical compositions containing the compounds disclosed herein can be manufactured in a conventional manner, such as by conventional mixing, dissolving, granulation, sugar-coated pelleting, grinding, emulsification, encapsulation, trapping, or lyophilization processes. Appropriate formulations depend on the chosen route of administration.

For oral administration, suitable compositions can be readily formulated by combining the compounds disclosed herein with pharmaceutically acceptable excipients (e.g., carriers) well known in the art. Such excipients and carriers enable the formulation of the compounds of the present invention into tablets, pills, sugar-coated pills, capsules, liquids, gels, syrups, slurries, suspensions, etc., for oral ingestion by a patient to be treated. Pharmaceutical formulations for oral use can be obtained by adding a solid excipient as disclosed herein to a compound, optionally grinding the resulting mixture, and, if desired, processing the granular mixture with the addition of a suitable excipient to obtain a tablet or sugar-coated pill core. Suitable excipients include, for example, fillers and cellulose preparations. Disintegrants may be added if necessary. Pharmaceutically acceptable ingredients for various types of formulations are well known and can be, for example, binders (e.g., natural or synthetic polymers), lubricants, surfactants, sweeteners and flavoring agents, coating materials, preservatives, dyes, thickeners, adjuvants, antimicrobial agents, antioxidants, and carriers for various types of formulations.

When administered orally in a therapeutically effective amount of the compounds disclosed herein, the compositions are typically in the form of solids (e.g., tablets, capsules, pills, powders, or lozenges) or liquid formulations (e.g., aqueous suspensions, solutions, elixirs, or syrups).

When administered in tablet form, the composition may additionally contain functional solids and/or solid carriers, such as gelatin or adjuvants. Tablets, capsules, and powders may contain about 1% to about 95% of the compound, and preferably about 15% to about 90% of the compound.

When applied in liquid or suspension form, a functional liquid and/or liquid carrier, such as water, petroleum, or oil of animal or plant origin, may be added. The liquid form of the composition may further contain aqueous saline solution, sugar alcohol solution, dextrose or other sugar solution, or glycol. When applied in liquid or suspension form, the composition may contain about 0.5% to about 90% by weight of the compounds disclosed herein, and preferably about 1% to about 50% by weight of the compounds disclosed herein. In one embodiment considered, the liquid carrier is non-aqueous or substantially non-aqueous. For application in liquid form, the composition may be supplied as a rapidly dissolving solid formulation for dissolution or suspension immediately prior to application.

When a therapeutically effective amount of the compounds disclosed herein is administered via intravenous, transdermal, or subcutaneous injection, the composition is in the form of a pyrogen-free, parenterally acceptable aqueous solution. The preparation of such parenterally acceptable solutions should take into account pH, isotonicity, stability, etc., and is within the scope of the art. In addition to the compounds disclosed herein, preferred compositions for intravenous, transdermal, or subcutaneous injection typically contain an isotonic agent. Such compositions can be prepared for administration as a solution in water of a free base or a pharmacologically acceptable salt appropriately mixed with a surfactant (e.g., hydroxypropyl cellulose). Dispersions can also be prepared in glycerol, liquid polyethylene glycol, mixtures thereof, and in oils. Under normal storage and use conditions, these formulations may optionally contain preservatives to prevent microbial growth.

Injectable compositions may include sterile aqueous solutions, suspensions, or dispersions, as well as sterile powders for the ad hoc preparation of sterile injectable solutions, suspensions, or dispersions. In all embodiments, the form must be sterile and the flowability must be sufficient to allow for easy injection. It must be stable under manufacturing and storage conditions and must resist microbial (e.g., bacterial and fungal) contamination by optionally including preservatives. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. In one considered embodiment, the carrier is non-aqueous or substantially non-aqueous. Suitable flowability can be maintained, for example, by using coatings, such as lecithin; in dispersion embodiments, by maintaining the desired particle size of the compound; and by using surfactants. Prevention of microbial action can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenols, sorbic acid, thimerosal, etc. In many embodiments, including isotonic agents (e.g., sugars or sodium chloride) is preferred. Extended absorption of injectable compositions can be achieved by using absorption delay agents (e.g., aluminum monostearate and gelatin) in the composition.

Sterile injectable solutions are prepared by incorporating the active compound in the desired amount into a suitable solvent containing, as needed, various other components listed above, followed by filtration and sterilization. Dispersions are typically prepared by incorporating various sterilized active ingredients into a sterile medium containing a base dispersion medium and other desired components from those listed above. In embodiments for preparing sterile powders for sterile injectable solutions, preferred preparation methods include vacuum drying and freeze-drying techniques, which produce powders of the active ingredient plus any other desired components from its pre-sterilely filtered solution.

Slow-release or sustained-release formulations can also be prepared to achieve controlled release of the active compound upon contact with bodily fluids in the gastrointestinal tract and to provide substantially constant and effective levels of the active compound in plasma. For example, release can be controlled by one or more of dissolution, diffusion, and ion exchange. Additionally, slow-release methods can promote absorption through saturable or restricted pathways within the gastrointestinal tract. For this purpose, for example, the compound can be encapsulated in a polymer matrix of a biodegradable polymer, a water-soluble polymer, or a mixture of both, and optionally a suitable surfactant. In this case, encapsulation can mean incorporating microparticles into the polymer matrix. Controlled-release formulations can also be obtained by encapsulating dispersed microparticles or emulsion-coated droplets via known dispersion or emulsion coating techniques.

For inhalation administration, the compounds of the present invention are conveniently delivered from pressurized packaging or nebulizers using a suitable propellant in the form of an aerosol spray. In pressurized aerosol embodiments, a valve can be provided to determine the dosage unit for delivery of a metered amount. Capsules and cartridges, such as gelatin, for use in inhalers or blowpipes can be formulated as powder mixtures containing the compound with a suitable powder matrix (e.g., lactose or starch).

The compounds disclosed herein can be formulated for parenteral administration via injection (e.g., by bolus or continuous infusion). Injectable formulations can be presented in unit dosage forms (e.g., in ampoules or in multi-dose containers) and may contain preservatives. Compositions can take the form of suspensions, solutions, or emulsions in oily or aqueous media and may contain formulations such as suspending agents, stabilizers, and/or dispersants.

Pharmaceutical formulations for parenteral administration include aqueous solutions of compounds in water-soluble form. Alternatively, suspensions of the compounds can be prepared as suitable oily injectable suspensions. Suitable lipophilic solvents or mediators include fatty oils or synthetic fatty acid esters. Aqueous injectable suspensions may contain substances that increase the viscosity of the suspension. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compound and allow for the preparation of highly concentrated solutions. Alternatively, the compositions of the present invention may be in powder form for preparation with a suitable mediator (e.g., sterile, pyrogen-free water) prior to use.

The compounds disclosed herein can also be formulated into rectal compositions, such as suppositories or retention enemas (e.g., containing a conventional suppository matrix). In addition to the formulations previously described, the compounds can also be formulated as long-acting preparations. Such long-acting formulations can be administered by implantation (e.g., subcutaneous or intramuscular) or by intramuscular injection. Thus, for example, the compounds can be formulated with suitable polymeric or hydrophobic materials (e.g., as emulsions in acceptable oils) or ion exchange resins, or as slightly soluble derivatives (e.g., as slightly soluble salts).

Specifically, the compounds disclosed herein can be administered orally, buccally, or sublingually in the form of tablets containing excipients (e.g., starch or lactose), or in capsules or ovules alone or in combination with excipients, or in elixirs or suspensions containing flavoring agents or coloring agents. Such liquid formulations can be prepared with pharmaceutically acceptable additives (e.g., suspensions). The compounds can also be administered parenterally, such as intravenously, intramuscularly, subcutaneously, or intracoronaryly. For parenteral administration, the compounds are preferably used in the form of sterile aqueous solutions, which may contain other substances, such as salts or sugar alcohols (e.g., mannitol) or glucose, to make the solution isotonic with blood.

For veterinary use, the compounds disclosed herein are administered as suitably acceptable formulations according to standard veterinary practice. Veterinarians can readily determine the most appropriate dosing regimen and route of administration for a particular animal.

In some embodiments, all necessary components for treating KRAS-related disorders using compounds as disclosed herein (alone or in combination with another agent or intervention conventionally used to treat this disease) may be packaged into a kit. Specifically, the present invention provides a kit for a therapeutic intervention for a disease comprising a packaged kit of pharmaceuticals, including the compounds disclosed herein and buffer solutions and other components for preparing said pharmaceuticals in a deliverable form; and/or a means for delivering such pharmaceuticals; and/or any agent for combination therapy with the compounds disclosed herein; and/or instructions for the treatment of the disease packaged with the pharmaceuticals. The instructions may be affixed to any tactile medium, such as printed paper, or computer-readable magnetic or optical media, or instructions referencing a remote computer data source, such as a World Wide Web webpage accessible via the Internet.

"Therapeutic effective dose" means the amount that effectively treats or prevents the development of existing symptoms in a treated subject or alleviates existing symptoms. In particular, the determination of an effective dose is entirely within the capabilities of those skilled in the art, based on the detailed disclosure provided herein. Generally, a "therapeutic effective dose" refers to the amount of compound that results in the desired effect. For example, in a preferred embodiment, a therapeutically effective dose of the compound disclosed herein reduces KRAS activity by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% compared to a control group.

The amount of compound administered can depend on the subject being treated, the subject's age, health status, sex, and weight, the type of concurrent treatment (if any), the severity of the condition, the nature of the desired effect, the manner and frequency of treatment, and the prescribing physician's judgment. The frequency of administration can also depend on the pharmacodynamic effect against arterial oxygen pressure. However, the optimal dose can be adjusted for individual subjects, as understood by those skilled in the art and determined without tolerance experiments. This typically involves adjusting the standard dose (e.g., reducing the dose if the patient is underweight).

While individual needs may vary, the determination of the optimal range of effective amounts of the compounds is within the scope of the art. For human administration in the curative or preventative treatment of the conditions and disorders identified herein, for example, typical doses of the compounds of the present invention may be from about 0.05 mg/kg/day to about 50 mg/kg/day, such as at least 0.05 mg/kg, at least 0.08 mg/kg, at least 0.1 mg/kg, at least 0.2 mg/kg, at least 0.3 mg/kg, at least 0.4 mg/kg, or at least 0.5 mg/kg, and preferably 50 mg/kg or less, 40 mg/kg or less, 30 mg/kg or less, 20 mg/kg or less, or 10 mg/kg or less, for example, it may be from about 2.5 mg/day (0.5 mg/kg x 5 kg) to about 5000 mg/day (50 mg/kg x 100 kg). For example, the dosage of the compound can be from about 0.1 mg/kg/day to about 50 mg/kg/day, from about 0.05 mg/kg/day to about 10 mg/kg/day, from about 0.05 mg/kg/day to about 5 mg/kg/day, from about 0.05 mg/kg/day to about 3 mg/kg/day, from about 0.07 mg/kg/day to about 3 mg/kg/day, from about 0.09 mg/kg/day to about 3 mg/kg/day, or from about 0.05 mg/kg/day to about 0.1 mg/day. /kg/day, about 0.1mg/kg/day to about 1mg/kg/day, about 1mg/kg/day to about 10mg/kg/day, about 1mg/kg/day to about 5mg/kg/day, about 1mg/kg/day to about 3mg/kg/day, about 3mg/day to about 500mg/day, about 5mg/day to about 250mg/day, about 10mg/day to about 100mg/day, about 3mg/day to about 10mg/day, or about 100mg/day to about 250mg/day. These dosages can be administered as a single dose or divided into multiple doses.

Methods using KRAS G12C inhibitors

This disclosure provides a method for inhibiting RAS-mediated cell signaling, the method comprising contacting cells with an effective amount of one or more compounds disclosed herein. Inhibition of RAS-mediated signal transduction can be assessed and confirmed by a variety of methods known in the art. Non-limiting examples include exhibiting: (a) decreased GTPase activity of RAS; (b) decreased GTP-binding affinity or increased GDP-binding affinity; (c) increased K-dissociation of GTP or decreased K-dissociation of GDP; (d) decreased levels of downstream signal transduction molecules in the RAS pathway, such as pMEK, pERK, or pAKT; and/or (e) decreased binding of the RAS complex to downstream signal transduction molecules (including, but not limited to, Raf). One or more of the above can be determined using kits and commercially available assays.

This disclosure also provides methods for treating diseases using the compounds or pharmaceutical compositions disclosed herein, including but not limited to diseases involving G12C KRAS, HRAS, or NRAS mutations (e.g., cancer).

In some embodiments, a method of treating cancer is provided, the method comprising administering to a subject in need an effective amount of any of the aforementioned pharmaceutical compositions comprising compounds as disclosed herein. In some embodiments, the cancer is mediated by a KRAS, HRAS, or NRAS G12C mutation. In various embodiments, the cancer is pancreatic cancer, colorectal cancer, or lung cancer. In some embodiments, the cancer is gallbladder cancer, thyroid cancer, and bile duct cancer.

In some embodiments, this disclosure provides a method for treating a barrier in a subject in need, wherein the method includes determining whether the subject has a KRAS, HRAS, or NRAS G12C mutation, and if the subject is determined to have a KRAS, HRAS, or NRAS G12C mutation, administering to the subject a therapeutically effective dose of at least one compound as disclosed herein or a pharmaceutically acceptable salt thereof.

The disclosed compounds inhibit anchorage-independent cell growth and therefore have the potential to inhibit tumor metastasis. Therefore, another embodiment of this disclosure provides a method for inhibiting tumor metastasis, the method comprising administering an effective amount of the compounds disclosed herein.

KRAS, HRAS, or NRAS G12C mutations have also been identified in hematologic malignancies (e.g., cancers affecting the blood, bone marrow, and/or lymph nodes). Therefore, some embodiments involve administering the disclosed compounds (e.g., in the form of a pharmaceutical composition) to a patient requiring treatment for a hematologic malignancy. Such malignancies include, but are not limited to, leukemia and lymphoma. For example, the currently disclosed compounds can be used to treat diseases such as acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), chronic myeloid leukemia (CML), acute monocytic leukemia (AMoL), and/or other leukemias. In other embodiments, these compounds can be used to treat lymphomas, such as all subtypes of Hodgkin lymphoma or non-Hodgkin lymphoma. In various embodiments, these compounds can be used to treat plasma cell malignancies, such as multiple myeloma, mantle cell lymphoma, and Waldenström macroglobulinemia.

Determining whether a tumor or cancer contains a G12C KRAS, HRAS, or NRAS mutation can be done by assessing the nucleotide sequence encoding the KRAS, HRAS, or NRAS protein, by assessing the amino acid sequence of the KRAS, HRAS, or NRAS protein, or by assessing the characteristics of a putative KRAS, HRAS, or NRAS mutant protein. Sequences of wild-type human KRAS, HRAS, or NRAS are known in the art (e.g., accession number NP203524).

Methods for detecting mutations in the nucleotide sequences of KRAS, HRAS, or NRAS are known to those skilled in the art. These methods include, but are not limited to, polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assays, polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) assays, real-time PCR assays, PCR sequencing, mutant allele-specific PCR amplification (MASA) assays, direct sequencing, primer extension reactions, electrophoresis, oligonucleotide ligation assays, hybridization assays, TaqMan assays, SNP genotyping assays, high-resolution melting assays, and microarray analysis. In some embodiments, samples are evaluated for G12C KRAS, HRAS, or NRAS mutations using real-time PCR. In real-time PCR, fluorescent probes specific to KRAS, HRAS, or NRAS G12C mutations are used. When a mutation is present, the probe binds and fluorescence is detected. In some embodiments, direct sequencing of specific regions (e.g., exon 2 and/or exon 3) in the KRAS, HRAS, or NRAS genes is used to identify KRAS, HRAS, or NRAS G12C mutations. This technology will identify all possible mutations in the sequenced region.

Methods for detecting mutations in KRAS, HRAS, or NRAS proteins are known to those skilled in the art. These methods include, but are not limited to, the use of binding agents (e.g., antibodies) specific to mutant proteins to detect KRAS, HRAS, or NRAS mutants, protein electrophoresis and Western blotting, and direct peptide sequencing.

Methods for determining whether a tumor or cancer contains G12C KRAS, HRAS, or NRAS mutations can use a variety of samples. In some embodiments, the sample is taken from a subject with a tumor or cancer. In some embodiments, the sample is a fresh tumor/cancer sample. In some embodiments, the sample is a frozen tumor/cancer sample. In some embodiments, the sample is a formalin-fixed and paraffin-embedded sample. In some embodiments, the sample is a circulating tumor cell (CTC) sample. In some embodiments, the sample is processed into a cell lysis product. In some embodiments, the sample is processed into DNA or RNA.

This disclosure also relates to methods for treating hyperplasia disorders in mammals, the method comprising administering to said mammal a therapeutically effective amount of a compound as disclosed herein or a pharmaceutically acceptable salt thereof. In some embodiments, the method relates to treating a subject with cancer, such as acute myeloid leukemia, juvenile cancer, childhood adrenocortical carcinoma, AIDS-related cancers (e.g., lymphoma and Kaposi's sarcoma), anal cancer, appendiceal cancer, astrocytoma, atypical teratoma, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain stem cell glioma, brain tumor, breast cancer, bronchial tumor, Burkitt lymphoma, carcinoid tumor, atypical teratoma, embryonal tumor, germ cell tumor, primary lymphoma, cervical cancer, childhood cancer, chordoma, cardiac tumor, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia. Leukemia (CML), Chronic Myelodysplastic Disorder, Colon Cancer, Colorectal Cancer, Craniopharyngioma, Cutaneous T-cell Lymphoma, Extrahepatic Ductal Carcinoma in Situ (DCIS), Embryoma, CNS Cancer, Endometrial Cancer, Ependymoma, Esophageal Cancer, Nasal Glioma, Ewing Sarcoma, Extracranial Ectodermal Tumor, Gonadal Ectodermal Tumor, Ocular Cancer, Osteofibrous Histiocytoma, Gallbladder Cancer, Gastric Cancer, Gastrointestinal Carcinoid Tumor, Gastrointestinal Stromal Tumor (GIST), Germ Cell Tumor, Gestational Trophoblastoma, Pilocytic Leukemia, Head and Neck Cancer, Heart Cancer, Liver Cancer, Hodgkin's Lymphoma, Hypopharyngeal Cancer, Intraocular Melanoma, Islet Cell Tumor, Pancreatic Neuroendocrine Tumor Tumors, kidney cancer, laryngeal cancer, lip and oral cavity cancer, liver cancer, lobular carcinoma in situ (LCIS), lung cancer, lymphoma, metastatic and occult primary squamous neck cancer, midline carcinoma, oral cancer, multiple endocrine tumor syndrome, multiple myeloma/plasma cell tumor, mycosis fungoides, myelodysplastic syndrome, spinal dysplasia/myeloproliferative neoplasm, multiple myeloma, Merkel cell carcinoma, malignant mesothelioma, malignant fibrous histiocytoma and osteosarcoma of bone, nasal cavity and paranasal sinus cancer, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer (NSCLC), oral cancer, lip and oral cavity cancer, oropharyngeal cancer, ovarian cancer, pancreas Cancer, papilloma, paraganglioma, paranasal sinus and nasal cavity cancer, parathyroid carcinoma, penile cancer, pharyngeal cancer, pleural pulmonary blastoma, primary central nervous system (CNS) lymphoma, prostate cancer, rectal cancer, transitional cell carcinoma, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, skin cancer, stomach/gastric cancer, small cell lung cancer, small bowel cancer, soft tissue sarcoma, T-cell lymphoma, testicular cancer, laryngeal cancer, thymoma and thymic carcinoma, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, trophoblastoma, rare childhood cancers, urethral cancer, uterine sarcoma, vaginal cancer, vulvar cancer, or virus-induced cancer. In some embodiments, the method relates to treating non-cancerous hyperplasia disorders, such as benign skin hyperplasia (e.g., psoriasis), restenosis, or prostate cancer (e.g., benign prostatic hyperplasia (BPH)).

In some embodiments, treatment methods relate to treating lung cancer, and these methods include administering an effective amount of any of the above-described compounds (or pharmaceutical compositions comprising the compound) to a subject in need. In some embodiments, the lung cancer is non-small cell lung cancer (NSCLC), such as adenocarcinoma, squamous cell lung cancer, or large cell lung cancer. In some embodiments, the lung cancer is small cell lung cancer. Other lung cancers that can be treated with the disclosed compounds include, but are not limited to, adenomas, carcinoid tumors, and undifferentiated carcinomas.

This disclosure further provides a method for modulating the activity of G12C mutant KRAS, HRAS, or NRAS proteins by contacting the protein with an effective amount of the compound disclosed herein. Modulation may be to inhibit or activate protein activity. In some embodiments, this disclosure provides a method for inhibiting protein activity by contacting a G12C mutant KRAS, HRAS, or NRAS protein with an effective amount of the compound disclosed herein in solution. In some embodiments, this disclosure provides a method for inhibiting the activity of G12C mutant KRAS, HRAS, or NRAS proteins by contacting cells, tissues, or organs expressing the protein of interest. In some embodiments, this disclosure provides a method for inhibiting protein activity in subjects, including but not limited to rodents and mammals (e.g., humans), by administering an effective amount of the compound disclosed herein to the subject. In some embodiments, the percentage of modulation exceeds 25%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%. In some embodiments, the percentage of inhibition exceeds 25%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%.

In some embodiments, this disclosure provides a method for inhibiting the activity of KRAS, HRAS, or NRAS G12C in cells by contacting the cells with an amount of the disclosed compound sufficient to inhibit the activity of KRAS, HRAS, or NRAS G12C in the cells. In some embodiments, this disclosure provides a method for inhibiting the activity of KRAS, HRAS, or NRAS G12C in tissues by contacting the tissue with an amount of the disclosed compound sufficient to inhibit the activity of KRAS, HRAS, or NRAS G12C in the tissue. In some embodiments, this disclosure provides a method for inhibiting the activity of KRAS, HRAS, or NRAS G12C in an organism by contacting the organism with an amount of the disclosed compound sufficient to inhibit the activity of KRAS, HRAS, or NRAS G12C in the organism. In some embodiments, this disclosure provides a method for inhibiting the activity of KRAS, HRAS, or NRAS G12C in animals by contacting the animals with an amount of the disclosed compound sufficient to inhibit the activity of KRAS, HRAS, or NRAS G12C in the animals. In some embodiments, this disclosure provides a method for inhibiting the activity of KRAS, HRAS, or NRAS G12C in mammals by contacting the mammals with an amount of the disclosed compound sufficient to inhibit the activity of KRAS, HRAS, or NRAS G12C in the mammals. In some embodiments, this disclosure provides a method for inhibiting the activity of KRAS, HRAS, or NRAS G12C in humans by contacting the humans with an amount of the disclosed compound sufficient to inhibit the activity of KRAS, HRAS, or NRAS G12C in the humans. This disclosure provides a method for treating a disease mediated by KRAS, HRAS, or NRAS G12C activity in a subject requiring such treatment.

Combination therapy:

This disclosure also provides methods for combination therapy, wherein agents known to modulate other pathways or other components of the same pathway, or even overlapping sets of target enzymes, are used in combination with compounds of this disclosure or pharmaceutically acceptable salts thereof. In one aspect, such therapy includes, but is not limited to, combinations of one or more compounds of this disclosure with chemotherapeutic agents, therapeutic antibodies, and radiation therapy to provide synergistic or additive therapeutic effects.

Many chemotherapeutic agents are currently known in the art and can be used in combination with the compounds disclosed herein. In some embodiments, the chemotherapeutic agents are selected from the group consisting of: mitosis inhibitors, alkylating agents, antimetabolites, intercalating antibiotics, growth factor inhibitors, cell cycle inhibitors, enzymes, topoisomerase inhibitors, bioreaction modifiers, antihormonal agents, angiogenesis inhibitors, and antiandrogens. Non-limiting examples include chemotherapeutic agents, cytotoxic agents, and non-peptide small molecules such as imatinib mesylate, carfilzomib, bortezomib, Casodex (bicalutamide), gefitinib, and adriamycin, as well as a variety of other chemotherapeutic agents. Non-limiting examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquinone, meturedopa, and uredopa; ethyleneimine and methylamelamine, including hexamethylmelamine, triethylenethiophospharamide, triethylenethiophospharamide, and trimethylolomelamine; nitrogen mustards such as chlorambucil, naphthiamethoxam, cholophosphamide, estradiol, ifosfamide, dichloromethyldiethylamine, mechlorethamine oxide hydrochloride, melphalan, and neo-embezzin. Mbichin, phenesterine, prednimustine, trafosamide, uracil mustard; nitrosoureas, such as carmustine, chlorhexidine, formustine, lomustine, nimustine, ramustine; antibiotics, such as aclacinomycin, actinomycin, autramycin, diazoserine, bleomycin, cactinomycin, calichemycin micin), carabicin, erythromycin, carzinophilin, Casodex™, chromomycin, cypromycin, daunomycin, detoxin, 6-diazo-5-oxo-L-leucine, doxorubicin, epirubicin, isorubicin, idarubicin, marcellomycin, mitomycin, mycophenolic acid, nogamycin, oligomycin, pepromycin, potfiromycin, puromycin, triamcinolone acetonide Quelamycin, Rodobicin, Streptomycin, Streptozotocin, Tuberculin, Ubenimex, Azotocin, Zolarubicin; antimetabolites, such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs, such as denopterin, methotrexate, pteroxate, trimethoprim; purine analogs, such as fludarabine, 6-mercaptopurine, thiopurine, thioguanine; pyrimidine analogs, such as azacitidine, azacitidine, 6-azauridine, carmoflurane. fluorouridine, cytarabine, dideoxyuridine, deoxyfluorouridine, enoxabin, fluorouridine; androgens such as capprotestone, dromostanolone propionate, cyclothrostanol, meandrosten, testosterone; antiadrenergic drugs such as aminoglutethimide, mitotane, trelostan; folic acid supplements such as frolinic acid; aceglucuronolactone; aldehyde phosphoramide glycoside; aminolevulinic acid; acridine; betrabucil; bisperidone; idatraxa (edatraxate); defofamine; colchicine; diazinon; elfomithine; elifonitrile; etogluconate; gallium nitrate; hydroxyurea; lentinan; chlordamine; mitoxantrone; mitoxantrone; mopiperol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazine; procarbazine; PSK; propylimine; [unclear text - possibly a typo, should be removed] Zoran; germanospiramine; Alternaria alternifolia ketoacid; triaminoquinone; 2,2',2"-trichlorotriethylamine; urethan; vindesine; dacarbazine; mannomustine; dibromomannitol; dibromoeusine; piperbbromide; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxanes, such as taxol and docetaxel; retinoids; esperamicins; capecitabine; and pharmaceutically acceptable salts, acids, or derivatives of any of the above.

Suitable chemotherapeutic cell opsonizers also include antihormonal agents used to modulate or inhibit the effects of hormones on tumors, such as antiestrogens, including, for example, tamoxifen (Nolvadex™), raloxifene, aromatase inhibitor 4(5)-imidazole, 4-hydroxytamoxifen, trivoxifen, keoxifene, LY 117018, onanasone, and toremifene (Faldon); and antiandrogens, such as flutamide, nilumid, bicalutamide, leuprorelin, and goserelin; phenylbutyrate nitrogen Mustard; Gemcitabine; 6-thioguanine; Mercaptopurine; Metapterin; Platinum analogs, such as cisplatin and carboplatin; Vincristine; Platinum; Etoposide (VP-16); Ifosfamide; Mitomycin C; Mitoxantrone; Vincristine; Vinorelbine; Novantrone; Teniposide; Donomycin; Aminopterin; Xeloda; Ibandronate; Camptothecin-11 (CPT-11); Topoisomerase inhibitor RFS 2000; Difluoromethylornithine (DMFO).

If desired, the compounds or pharmaceutical compositions disclosed herein may be used in combination with commonly prescribed anticancer drugs, such as ABVD, AVICINE, Abagovomab, Acridine carboxamide, Adecatumumab, 17-N-allylamino-17-demethoxygerdomyl, Alpharadin, Alvocidib, 3-aminopyridine-2-carboxaldehyde thiourea, Amonafide, Anthracenedione, antiCD22 immunotoxins, antitumor drugs, antitumorigenic herbs, Apaziquone, Atiprmod, Azathioprine, and Belotecan. Can), Bendamustine, BIBW 2992, Biricodar, Brostallicin, Bryostatin, Buthionine sulfoximine, CBV (chemotherapy), Calyculin, cell cycle nonspecific antitumor agents, dichloroacetic acid, Discodermolide, Elsamitrucin, Enocitabine, Epothilone, Eribulin, Everolimus, Exatecan, Exisulind, Ferruginol, Forodesine, Phosphorus Fosfestrol, ICE chemotherapy regimen, IT-101, Imexon, Imiquimod, Indolocarbazole, Irofulven, Laniquidar, Larotaxel, Lenalidomide, Lucanthone, Lurtotecan, Mafosfamide, Mitozolomide, Nafoxidine, Nedaplatin, Olaparib, Ortataxel, PAC-1, Pawpaw, Pixantrone, proteasome inhibitors, Rebeccamyc Cin), Resiquimod, Rubitecan, SN-38, Salinosporamide A, Sapacitabine, Stanford V, Swainsonine, Talaporfin, Tariquidar, Tegafur-uracil, Temodar, Tesetaxel, Triplatin tetranitrate, Tri(2-chloroethyl)amine, Troxacitabine, Uramustine, Vadimozan, Vinflunine, ZD6126, or Zosuquidar.

This disclosure further relates to methods for inhibiting abnormal cell growth or treating hyperplasia disorders in mammals using a combination of the compounds or pharmaceutical compositions provided herein with radiotherapy. Techniques for administering radiotherapy are known in the art, and these techniques can be used in the combination therapies described herein. The administration of the compounds disclosed herein in such combination therapies can be determined as described herein.

Radiation therapy can be administered through one or a combination of several methods, including but not limited to external beam therapy, internal radiation therapy, implantation radiation, stereotactic radiosurgery, whole-body radiation therapy, radiotherapy, and permanent or temporary interstitial brachytherapy. As used herein, the term “brachytherapy” refers to radiation therapy delivered by a spatially confined radioactive material inserted into or near a lesion site in the body, such as a tumor or other proliferative tissue. The term is intended to include, without limitation, exposure to radioactive isotopes (e.g., At-211, I-131, I-125, Y-90, Re-186, Re-188, Sm-153, Bi-212, P-32, and Lu). Suitable radiation sources used as cytomodulation agents in this disclosure include both solid and liquid forms. By way of non-limiting examples, the radioactive source can be a radionuclide, such as I-125, I-131, Yb-169, Ir-192 as solid sources, I-125 as a solid source, or other radionuclides that emit photons, beta particles, gamma radiation, or other therapeutic rays. The radioactive material can also be a fluid prepared from any solution of one or more radionuclides (e.g., a solution of I-125 or I-131), or the radioactive fluid can be generated using a suitable fluid slurry containing small particles of a solid radionuclide (e.g., Au-198, Y-90). Furthermore, one or more radionuclides can be embedded in gels or radioactive microspheres.

The compounds or pharmaceutical compositions disclosed herein may be used in combination with a certain amount of one or more substances selected from the following: anti-angiogenic agents, signal transduction inhibitors, anti-proliferative agents, glycolysis inhibitors, or autophagy inhibitors.

Antiangiogenic agents can be used in combination with the compounds disclosed herein and the pharmaceutical compositions described herein. These antiangiogenic agents are, for example, MMP-2 (matrix metalloproteinase 2) inhibitors, MMP-9 (matrix metalloproteinase 9) inhibitors, and COX-11 (cyclooxygenase 11) inhibitors. Antiangiogenic agents include, for example, rapamycin, temsirolimus (CCI-779), everolimus (RAD001), sorafenib, sunitinib, and bevacizumab. Examples of useful COX-II inhibitors include alecoxib, vardicoxib, and rofecoxib. Examples of useful matrix metalloproteinase inhibitors are described in the following patents: WO 96/33172, WO 96/27583, European Patent Publication EP 0818442, European Patent Publication EP1004578, WO 98/07697, WO 98/03516, WO 98/34918, WO 98/34915, WO 98/33768, WO 98/30566, European Patent Publication 606046, European Patent Publication 931788, WO WO 90/05719, WO 99/52910, WO 99/52889, WO 99/29667, WO 1999007675, European Patent Publication EP 1786785, European Patent Publication EP1181017, US Patent Publication US 20090012085, US Patent Publication US 5863949, US Patent Publication US5861510 and European Patent Publication EP 0780386, all of which are incorporated herein by reference in their entirety. Preferred MMP-2 and MMP-9 inhibitors are those with minimal or no activity against MMP-1. More preferably, those that selectively inhibit MMP-2 and/or AMP-9 relative to other matrix metalloproteinases (i.e., MAP-1, MMP-3, MMP-4, MMP-5, MMP-6, MMP-7, MMP-8, MMP-10, MMP-11, MMP-12, and MMP-13). Some specific examples of MMP inhibitors that can be used in this disclosure are AG-3340, RO 32-3555, and RS 13-0830.

The compounds of this invention can also be used in co-treatment with other antitumor agents, such as acemannan, arubicin, interleukin, alemtuzumab, alitretinoin, hexamethylmelamine, amifostine, aminolevulinic acid, arubicin, acridine, anagrelide, anastrozole, ANCER, ancestim, argalabin, arsenic trioxide, and BAM 002 (Nov). Elos), bexarotene, bicalutamide, bromouridine, capecitabine, simmointerleukin, cetrorex, cladribine, clotrimazole, cytarabine octodecyl phosphate, DA 3030 (Dong-A), daclizumab, denileukin diftitox, deslorelin, dexrazoxen, dilazep, docetaxel, Doxorubicin, doxorcalciferol, deoxyfluorouridine, doxorubicin, bromocriptine, carmustine, cytarabine, fluorouracil, HIT (diclofenac), interferon-alpha, doxorubicin, retinoic acid, edifoxetine, eczolosin, eflornithine, ethirimol, epirubicin, erythropoietin beta, etoposide phosphate, exemestane, exisulind, fazodazole, filgrastim, finasteride, fludarabine phosphate Formestan, formestane, gallium nitrate, gemcitabine, gemtuzumab/oczogamicin, gimeracil/oteracil/tegafur combination, glycopine, goserelin, heptaplatin, human chorionic gonadotropin, human fetal alpha-fetoprotein, ibandronic acid, idarubicin, imiquimod, interferon-alpha, interferon-alpha, natural interferon-alpha-2, stem cells Interferon-α-2a, Interferon-α-2b, Interferon-α-N1, Interferon-α-n3, Interferon alfacon-1, Interferon α, Natural Interferon β, Interferon β-1a, Interferon β-1b, Interferon γ, Natural Interferon γ-1a, Interferon γ-1b, Interleukin-1β, Iodobenzylguanidine, Irinotecan, Isogratin, Lanreotide, LC 9018 (Yakult), Leflunomide, Lenograstim ), lentinan sulfate, letrozole, leukocyte alpha interferon, leuprorelin, levamisole + fluorouracil, liarozole, lobaplatin, chlordamine, lovastatin, masorofol, melarisol, metoclopramide, mifepristone, mitefocin, milistatin, mismatched double-stranded RNA, mitoxantrone, dibromoceroxyl, mitoxantrone, molgramostim, nafarelin, naloxone + pentazocine, nartograstim, nedaplatin, nilutamide, nacoside Ding, novel erythropoiesis-stimulating protein, NSC 631570 octreotide, oprelvekin, oxatatropine, oxaliplatin, paclitaxel, pamidronic acid, pegaspargase, pegylated interferon-α-2b, pentosan polysulfate sodium, pentostatin, picibanil, pirarubicin, rabbit anti-thymocyte polyclonal antibody, pegylated interferon -α-2a, porphyrin sodium, raloxifene, raltitrexed, rasburiembodiment, rhenium ethodole (Re 186), RII retinamide, rituximab, romotide, samarium (153Sm), sargramostim, cizonan, sobuzosen, sonermin, strontium chloride-89, suramin, tasonemin Tasonermin, Tazarotene, Tegafur, Temoporfin, Temozolomide, Teniposide, Tetrachlorodecoxide, Thalidomide, Thymofasin, Thyroid-stimulating hormone alpha, Topotecan, Toremifene, Tositumomab-Iodine-131, Trastuzumab, Triosmectin, Retinoic acid, Tralostan, Trimethoprim, Triptorelin, Tumor necrosis factor alpha, Natural ubenimex, Bladder cancer vaccine, Maruyama vaccine, Melanoma lysate vaccine, Valrubicin, Vertepofen, Vinorelbine, VIRULIZIN, Zinostatin stimalamer, or zoledronic acid; Abaric; AE 941 (Aeterna), Ambamustine, antisense oligonucleotides, Bcl-2 (Genta), APC 8015 (Dendreon), Cetuximab, Decitabine abine, dexaminoglutethimide, diazinon, EL 532 (Elan), EM 800 (Endorecherche), enuracil, estanidazole, fenretinide, filgrastim SD01 (Amgen), fulvestrant, gallotabine, gastrin-17 immunogen, HLA-B7 gene therapy (Vical), granulocyte-macrophage colony stimulation Factor, histamine dihydrochloride, teimomab, ilomasta, IM 862 (Cytran), interleukin-2, iproxifene, LDI 200 (Milkhaus), leridistim, lintuzumab, CA 125 MAb (Biomira), cancer MAb (Japan Pharmaceutical Development Co., Ltd.) HER-2 and Fc MAb (Medarex), idiotype 105AD7 MAb (CRC Technology), idiotype CEA MAb (Trilex), LYM-1-iodine-131 MAb (Techniclone), polymorphic epithelial mucin-yttrium-90 MAb (Antisoma), marimastat, and other related drugs. Noriol, Mitumomab, Motexafin Gadolinium, MX 6 (Galderma), Nelarabine, Nolatrexed, P30 Protein, Pegvisomant, Pemetrexed, Porfiromycin, Prinomastat, RL 0903 (Shire), Lupi Tetraplatin, Satraplatin, Sodium Phenylacetate, Sparfosic Acid, SRL 172 (SR Pharma), SU 5416 (SUGEN), TA 077 (Tanabe), Tetrathiomolybdate, Thaliblastine, Thrombopoietin, Tin Ethiopurpurin, Tirazamine, Cancer Vaccine (Barmira), Melanoma Vaccine (New York University), Melanoma Vaccine (Sloan Kettering Institute), Melanoma Tumor Lysis Vaccine (New York Medical College), Viral Melanoma Cell Lysis Vaccine (Royal Newcastle Hospital), or Valspodar.

The compounds of this invention can be further used in combination with VEGFR inhibitors. Other compounds described in the following patents and patent applications can be used in combination therapies: US 6,258,812, US 2003/0105091, WO 01/37820, US 6,235,764, WO 01/32651, US 6,630,500, US 6,515,004, US 6,713,485, US 5,521,184, US 5,770,599, US 5,747,4 98. WO 02/68406, WO 02/66470, WO 02/55501, WO 04/05279, WO04/07481, WO 04/07458, WO 04/09784 , WO 02/59110, WO 99/45009, WO 00/59509, WO 99/61422, US 5,990,141, WO 00/12089 and WO 00/02871.

In some embodiments, the combination comprises a combination of the composition of the present invention with at least one anti-angiogenic agent. Agents include, but are not limited to, chemical compositions synthesized in vitro, antibodies, antigen-binding regions, radionuclides, and combinations thereof, and conjugates. Agents may be agonists, antagonists, allosteric modulators, toxins, or more generally, may be used to inhibit or stimulate their targets (e.g., receptor or enzyme activation or inhibition), thereby promoting cell death or inhibiting cell growth.

Exemplary anti-angiogenic agents include ERBITUX ^™ (IMC-C225), KDR (kinase domain receptor) inhibitors (e.g., antibodies and antigen-binding regions that specifically bind to kinase domain receptors), anti-VEGF agents (e.g., antibodies or antigen-binding regions that specifically bind to VEGF, or soluble VEGF receptors or their ligand-binding regions) (e.g., AVASTIN ^™ or VEGF-TRAP ^™ ), and anti-VEGF receptor agents (e.g., antibodies or antigen-binding regions that specifically bind to them), EGFR inhibitors (e.g., antibodies or antigen-binding regions that specifically bind to them) (e.g., Vectibix (panitumumab), IRESSA ^™ (gefitinib), TARCEVA ^™) . (Erlotinib), anti-Ang1 agents and anti-Ang2 agents (e.g., antibodies or antigen-binding regions that specifically bind to or to their receptors (e.g., Tie2/Tek), and anti-Tie2 kinase inhibitors (e.g., antibodies or antigen-binding regions that specifically bind to them). The pharmaceutical compositions of the present invention may also comprise one or more agents that specifically bind to and inhibit the activity of growth factors (e.g., antibodies, antigen-binding regions, or soluble receptors), such as antagonists of hepatocyte growth factor (HGF, also known as scattering factor), and antibodies or antigen-binding regions that specifically bind to their receptor “c-met.”

Other anti-angiogenic agents include Camppath, IL-8, β-FGF, Tek antagonists (Ceretti et al., U.S. Publication No. 2003/0162712; U.S. Patent No. 6,413,932), anti-TWEAK agents (e.g., those that specifically bind to antibody or antigen-binding regions, or soluble TWEAK receptor antagonists; see Wiley, U.S. Patent No. 6,727,225), and ADAM de-integrin domains for antagonizing the binding of integrin to its ligands (Fanslow et al., U.S. Publication No. 2002/00). 42368), antibodies or antigen-binding regions that specifically bind to anti-eph receptors and/or anti-ephrin (US Patent Nos. 5,981,245; 5,728,813; 5,969,110; 6,596,852; 6,232,447; 6,057,124 and members of their respective patent families), and anti-PDGF-BB antagonists (e.g., antibodies or antigen-binding regions that specifically bind to PDGF-BB ligands), as well as antibodies or antigen-binding regions that specifically bind to PDGF-BB ligands and PDGFR kinase inhibitors (e.g., antibodies or antigen-binding regions that specifically bind to them).

Other anti-angiogenic/anti-tumor agents include: SD-7784 (Pfizer, USA); cilengitide (Merck KGaA, EPO 770622); pegaptaniboctasodium (Gilead Sciences, USA); alphastatin (BioActa, UK); M-PGA (Celgene, US 5712291); ilomastastatin (Arriva, US 5892112); emaxanib (Pfizer, US 5792783); vatalanib. lanib (Novartis, Switzerland); 2-methoxyestradiol (EntreMed, USA); TLC ELL-12 (Elon, Ireland); anecrolaveacetate (Alcon, USA); α-D148 Mab (Amgen, USA); CEP-7055 (Cephalon, USA); anti-Vn Mab (Crucell, Netherlands); DAC: anti-angiogenic agent (ConjuChem, Canada); angiocidin (InKine Pharmaceuticals, USA); KM-2550 (Kyowa Hakko Kogyo Co., Ltd., Japan). Yowa Hakko); SU-0879 (Pfizer, USA); CGP-79787 (Novartis, Switzerland, EP 970070); ARGENT technology (Ariad, USA); YIGSR-Stealth (Johnson & Johnson, USA); Fibrinogen-E fragment (Baxter, UK); Angiogenesis inhibitor (Trigen, UK); TBC-1635 (Encysive Pharmaceuticals, USA); SC-236 (Pfizer, USA); ABT-567 (Abbott, USA); Metastatin (Antrimester, USA); Angiogenesis inhibitor (T... ripep); mammary filament inhibitor (maspin) (Sosei, Japan); 2-methoxyestradiol (Oncology Sciences Corporation, USA); ER-68203-00 (IVAX, USA); flufenoxuron (Lane Labs, USA); Tz-93 (Tsumura, Japan); TAN-1120 (Takeda, Japan); FR-111142 (Fujisawa, Japan, JP 02233610); platelet factor 4 (RepliGen, USA, EP 407122); vascular endothelial growth factor antagonist (Bore, Denmark). bevacizumab (pINN) (Genentech, USA); angiogenesis inhibitors (Sugum, USA); XL 784 (Exelixis, USA); XL 647 (Exelixis, USA); second-generation MAbα5β3 integrin (Applied Molecular Evolution Foundation and MedImmune, USA); gene therapy for retinopathy (Oxford BioMedica, UK); enzastaurin hydrochloride (USAN) (Lilly, USA); CEP 7055 (Servallon, USA and Sanofi, France) Sanofi-Synthelabo; BC 1 (Genoa Institute of Cancer Research, Italy); angiogenesis inhibitors (Alchemia, Australia); VEGF antagonists (Regeneron, USA); rBPI 21 and BPI-derived anti-angiogenic agents (XOMA, USA); PI 88 (Progen, Australia); pINN (Merck, Germany; Munich Technical University, Germany; Scripps Clinic and Research Foundation, USA). ch Foundation); Cetuximab (INN) (Aventis, France); AVE 8062 (Ajinomoto, Japan); AS 1404 (Cancer Research Laboratory, New Zealand); SG 292 (Telios, USA); Endostatin (Boston Children's Hospital, USA); ATN 161 (Attenuon, USA); Angiostatin (Boston Children's Hospital, USA); 2-Methoxyestradiol (Boston Children's Hospital, USA); ZD 6474 (AstraZeneca, UK); ZD 6126 (Angel, UK) Angiogene Pharmaceuticals; PPI2458 (Praecis, USA); AZD 9935 (AstraZeneca, UK); AZD 2171 (AstraZeneca, UK); Vatalani (pINN) (Novartis, Switzerland and Schering AG, Germany); Tissue factor pathway inhibitor (Antrimed, USA); Pigantanib (Pinn) (Gilead Sciences, USA); Xanthorrhizol (Yonsei University, South Korea); Gene-based VEGF-2 vaccine (Scripps Clinic and Research Foundation, USA); SPV5.2 (Supratek, Canada); SDX 10 3 (University of California, San Diego); PX 478 (ProlX, Inc., USA); Transfer inhibitor (Antrimed, Inc., USA); Troponin I (Harvard University, USA); SU 6668 (Sugen, Inc., USA); OXI 4503 (OXiGENE, Inc., USA); o-guanidine (Dimensional Pharmaceuticals, Inc., USA); motuporamine C (University of British Columbia, Canada); CDP 791 (Celltech, UK) Group); Atemod (pINN) (GlaxoSmithKline, UK); E 7820 (Eisai, Japan); CYC 381 (Harvard University, USA); AE 941 (Etna, Canada); Angiogenesis Vaccine (Antrimed, USA); Urokinase Plasminogen Activator Inhibitor (Dandelion, USA); Oglufanide (pINN) (Melmotte, USA); HIF-1α Inhibitor (Xenova, UK); CEP 5214 (Selfallon, USA); BAY RES 2622 (Bayer, Germany); Angixidin (Incoin, USA); A6 (Angstrom, USA) KR 31372 (Korea Research Institute of Chemical Technology, South Korea); GW 2286 (GlaxoSmithKline, UK); EHT 0101 (ExonHit, France); CP 868596 (Pfizer, USA); CP 564959 (OSI, USA); CP 547632 (Pfizer, USA); 786034 (GlaxoSmithKline, UK); KRN 633 (Kirin Brewery, Japan); Intraocular 2-methoxyestradiol drug delivery system (Antrimed, USA); Anginex (Maastricht University, Netherlands) University of Minnesota, USA; ABT 510 (Abbott Laboratories, USA); AAL 993 (Novartis, Switzerland); VEGI (ProteomTech, USA); Tumor necrosis factor-α inhibitor (National Institute on Aging, USA); SU 11248 (Pfizer and Sugen, USA); ABT 518 (Abbott Laboratories, USA); YH16 (Yantai Rongchang, China); S-3APG (Boston Children's Hospital and Antrimed, USA); MAb,KDR (ImClone Systems, USA); MAbα5β 1 (Protein Design, Inc., USA); KDR kinase inhibitors (Cell Technologies Group, UK and Johnson & Johnson, USA); GFB 116 (South Florida University and Yale University, USA); CS 706 (Sankyo, Japan); comprehendingin A4 prodrug (Arizona State University, USA); chondroitinase AC (IBEX, Canada); BAY RES 2690 (Bayer, Germany); AGM 1470 (Harvard University, USA and Takeda Corporation, Japan) The following are listed: AG 13925 (Agouron, USA); Tetrathiomolybdate (University of Michigan, USA); GCS 100 (Wayne State University, USA); CV 247 (Ivy Medical, UK); CKD 732 (Chong Kun Dang, South Korea); Vascular endothelial growth factor MAb (Nova, UK); Isopridine (INN) (Nippon Shinyaku, Japan); RG 13577 (Aventis, France); WX 360 (Wilex, Germany). ); Squalamine (pINN) (Genaera, USA); RPI 4610 (Sirna, USA); Cancer Therapy (Marinova, Australia); Heparinase Inhibitor (InSight, Israel); KL 3106 (Kolon, South Korea); Honokiol (Emory University, USA); ZK CDK (Schriner, Germany); ZK Angio (Schriner, Germany); ZK 229561 (Novartis, Switzerland and Schriner, Germany); XMP 300 (Drama, USA); VGA 1102 (Taisho, Japan); VEGF Receptor Modulator (Pharmacopeia, United States Pharmacopeia); VE - cadherin-2 antagonists (Inclone Systems, Inc., USA); Vasostatin (National Institutes of Health, USA); Flk-1 vaccine (Inclone Systems, Inc., USA); TZ 93 (Tsumura Industries, Inc., Japan); Tumor stomatine (Beth Israel Hospital, USA); Truncated soluble FLT1 (vascular endothelial growth factor receptor 1) (Merck & Co., Ltd., USA); Tie-2 ligand (Regeneron Corporation, USA); and thromboretin 1 inhibitors (Allegheny Health, Education and Research Foundation, USA).

Autophagy inhibitors include, but are not limited to, chloroquine, 3-methyladenine, hydroxychloroquine (Plaquenil ^™ ), bafilomycin A1, 5-amino-4-imidazolamide ribonucleoside (AICAR), okadaic acid, autophagy-inhibiting alginate toxins that inhibit type 2A or type 1 protein phosphatases, cAMP analogs, and drugs that increase cAMP levels such as adenosine, LY204002, N6-mercaptopurine ribonucleoside, and vincristine. Additionally, antisense or siRNAs that inhibit protein expression, including but not limited to ATG5 (which is involved in autophagy), can be used.

Other pharmaceutically active compounds/pharmaceuticals that can be used to treat cancer and can be used in combination with one or more compounds of the present invention include: epoetin alfa; darbepoetin alfa; panitumumab; pegfilgrastim; palifermin; filgrastim; denosumab; anxistatin; AMG 102; AMG 386; AMG 479; AMG 655; AMG 745; AMG 951; and AMG 706 or pharmaceutically acceptable salts thereof.

In some embodiments, the compositions provided herein are administered in combination with chemotherapeutic agents. Suitable chemotherapeutic agents may include natural products such as vinca alkaloids (e.g., vincristine, vinorelbine, and vinorelbine), paclitaxel, epidipodophyllotoxin (e.g., etoposide and teniposide), antibiotics (e.g., dextrin (actinomycin D), doxorubicin, doxorubicin, and idarubicin), anthracycline antibiotics, mitoxantrone, bleomycin, procainoxantrone (scintillan), mitomycin, and enzymes (e.g., L-asparaginase, which is systemically metabolized). L-Asparagine and deprive cells that do not have the ability to synthesize their own asparagine, antiplatelet agents, antiproliferative/antimitotic alkylating agents (e.g., nitrogen mustard, such as dichloromethyldiethylamine, cyclophosphamide and analogues, melphalan and chlorambucil), ethyleneimine and methylmelamine (e.g., hexaamethylmelaamine and thiotepa), CDK inhibitors (e.g., seliciclib, UCN-01, P1446A-05, PD-03). 32991, dinaciclib, P27-00, AT-7519, RGB286638 and SCH727965), alkyl sulfonates (e.g., busulfan), nitrosoureas (e.g., carmustine (BCNU) and analogues and streptozotocin), triazine-dacarbazine (DTIC), antiproliferative/antimitotic antimetabolites such as folic acid analogues (e.g., methotrexate), pyrimidine analogues (e.g.) Examples of inhibitors include fluorouracil, fluorouracil, and cytarabine; purine analogs and related inhibitors (e.g., mercaptopurine, thioguanine, pentostatin, and 2-chlorodeoxyadenosine); aromatase inhibitors (e.g., anastrozole, exemestane, and letrozole); platinum coordination complexes (e.g., cisplatin and carboplatin); procarbazine; hydroxyurea; mitotane; aminoglutethimide; histone deacetylase (HDAC) inhibitors (e.g., trachomatis, sodium butyrate, aspirin, hydroamic acid, vorinostat, LBH 589, romidepsin, ACY-1215, and panobinostat); mTor inhibitors (e.g., tesimolimus, everolimus, ridaforolimus, and sirolimus); and KSP (Eg5) inhibitors (e.g., Array). 520), DNA binding agents (e.g., Zalypsis), PI3Kδ inhibitors (e.g., GS-1101 and TGR-1202), PI3Kδ and γ inhibitors (e.g., CAL-130), multi-kinase inhibitors (e.g., TG02 and sorafenib), hormones (e.g., estrogens) and hormone agonists such as luteinizing hormone-releasing hormone (LHRH) agonists (e.g., goserelin, leuprorelin, and triptorelin), BAFF neutralizing antibodies (e.g., LY2127399), IKK inhibitors, p38MAPK inhibitors, anti-IL-6 (e.g., CNTO328), telomerase inhibitors (e.g., GRN 163L), aurora kinase inhibitors (e.g., MLN8237), cell surface monoclonal antibodies (e.g., anti-CD38 (HUMAX-CD38), anti-CS1 (e.g., elotuzumab), HSP90 inhibitors (e.g., 17 AAG and KOS 953), P13K/Akt inhibitors (e.g., perifosine), Akt inhibitors (e.g., GSK-2141795), PKC inhibitors (e.g., enzatolin), FTIs (e.g., Zarnestra ^™) ), anti-CD138 (e.g., BT062), Torc1/2 specific kinase inhibitors (e.g., INK128), kinase inhibitors (e.g., GS-1101), ER/UPR targets (e.g., MKC-3946), cFMS inhibitors (e.g., ARRY-382), JAK1/2 inhibitors (e.g., CYT387), PARP inhibitors (e.g., olaparib and veliparib (ABT-888)), BCL-2 antagonists. Other chemotherapeutic agents may include dichloromethyldiethylamine, camptothecin, ifosfamide, tamoxifen, raloxifene, gemcitabine, noviben, sorafenib, or any analogues or derivative variants of the foregoing.

The compounds of this invention can also be used in combination with radiotherapy, hormone therapy, surgery and immunotherapy, which are well known to those skilled in the art.

In some embodiments, the pharmaceutical compositions provided herein are administered in combination with steroids. Suitable steroids may include, but are not limited to, 21-acetoxypregnenolone, aclomethasone, alpha-pregnane, anxine, beclomethasone, betamethasone, budesonide, chloroprednisone, clobetasol, clocotropin, chlorprednisolone, corticosteroids, cortisone, cortisol, divazoline, dextromethorphan, desonide, hydroxymethasone, dexamethasone, diflorasone, diflucortolone, difuprednate, glycyrrhetinic acid, fluzacrolimus, and fluclochlor. Flucloronide, flumethasone, fluocinolone acetonide, fluocinolone acetate, fluocortinbutyl ester, fluclocolone, fluorometholone, fluperolone acetate, fluprednidene acetate, fluprednisolone, flurandrenolide, fluticasone propionate, Formocortal, halcinonide, halobetasol propionate, halometasone, hydrocortisone, loteprednol etabonate, horseprednisolone, methylprednisolone, methylprednisolone, mometasone furoate, peramisone, prednicarbamate, prednisolone The compounds include prednisolone 2,5-diethylaminoacetate, prednisolone sodium phosphate, prednisone, prednival, prednylidene, limexolo, tixocortol, triamcinolone, triamcinolone acetonide, triamcinolone benetonide, triamcinolone hexacetonide, and their salts and/or derivatives. In one particular embodiment, the compounds of the present invention may also be used in combination with other pharmaceutically active agents for treating nausea. Examples of agents that can be used to treat nausea include: dronabinol, granisetron, metoclopramide, ondansetron, and prochlorperazine, or pharmaceutically acceptable salts thereof.

The compounds or pharmaceutical compositions disclosed herein may also be used in combination with one or more substances selected from the following: EGFR inhibitors, MEK inhibitors, PI3K inhibitors, AKT inhibitors, TOR inhibitors, and immunotherapies, including anti-PD-1, anti-PDL-1, anti-CTLA4, anti-LAG1 and anti-OX40 agents, GITR agonists, CAR-T cells, and BiTE.

EGFR inhibitors include, but are not limited to, small molecule antagonists, antibody inhibitors, or specific antisense nucleotides or siRNAs. Useful EGFR antibody inhibitors include cetuximab (Erbitux), panitumumab (Vectib), zalutumumab, nimotuzumab, and matuzumab. Small molecule antagonists of EGFR include gefitinib, erlotinib (Tarceva), and more recently, lapatinib (TykerB). See, for example, Yan L et al., Pharmacogenetics and Pharmacogenomics In Oncology Therapeutic Antibody Development, BioTechniques 2005; 39(4):565-8, and Paez J G et al., EGFR mutations in Lung Cancer Correlation With Clinical Response To Gefitinib Therapy, Science 2004; 304(5676):1497-500.

Non-limiting examples of small molecule EGFR inhibitors include any EGFR inhibitor described in the following patent publications, and all pharmaceutically acceptable salts and solvates of said EGFR inhibitors: European Patent Application EP 520722, published December 30, 1992; European Patent Application EP 566226, published October 20, 1993; PCT International Publication WO 96/33980, published October 31, 1996; and US Patent No. 5,747,498. Granted on May 5, 1998; PCT International Publication WO 96/30347, published on October 3, 1996; European Patent Application EP 787772, published on August 6, 1997; PCT International Publication WO 97/30034, published on August 21, 1997; PCT International Publication WO 97/30044, published on August 21, 1997; PCT International Publication WO 97/38994, published on October 23, 1997; PC International Patent Application Publication No. WO 97/49688, published December 31, 1997; European Patent Application No. EP 837063, published April 22, 1998; International Patent Application Publication No. WO 98/02434, published January 22, 1998; International Patent Application Publication No. WO 97/38983, published October 23, 1997; International Patent Application Publication No. WO 95/19774, published July 27, 1995; International Patent Application Publication No. WO 95/1 Application No. 9970 was published on July 27, 1995; PCT International Publication WO 97/13771 was published on April 17, 1997; PCT International Publication WO 98/02437 was published on January 22, 1998; PCT International Publication WO 98/02438 was published on January 22, 1998; PCT International Publication WO 97/32881 was published on September 12, 1997; German Application DE 19629652 was published on January 29, 1998. Publication; PCT International Publication WO 98/33798, published August 6, 1998; PCT International Publication WO 97/32880, published September 12, 1997; PCT International Publication WO 97/32880, published September 12, 1997; European Patent Application EP 682027, published November 15, 1995; PCT International Publication WO 97/02266, published January 23, 1997; PCT International Publication WO 97 /27199, published on July 31, 1997; PCT International Publication WO 98/07726, published on February 26, 1998; PCT International Publication WO 97/34895, published on September 25, 1997; PCT International Publication WO 96/31510’, published on October 10, 1996; PCT International Publication WO 98/14449, published on April 9, 1998; PCT International Publication WO 98/14450, published on 1998. Publication dated April 9, 1998; PCT International Publication WO 98/14451, published April 9, 1998; PCT International Publication WO 95/09847, published April 13, 1995; PCT International Publication WO 97/19065, published May 29, 1997; PCT International Publication WO98/17662, published April 30, 1998; US Patent No. 5,789,427, granted August 4, 1998; US Patent No. 5,650,4 15, granted July 22, 1997; U.S. Patent No. 5,656,643, granted August 12, 1997; PCT International Publication WO 99/35146, published July 15, 1999; PCT International Publication WO 99/35132, published July 15, 1999; PCT International Publication WO 99/07701, published February 18, 1999; and PCT International Publication WO 92/20642, published November 26, 1992. Other non-limiting examples of small molecule EGFR inhibitors include any EGFR inhibitor described in Traxler, P., 1998, Exp. Opin. Ther. Patents [Therapeutic Patent Expert Commentary] 8(12):1599-1625.

Antibody-based EGFR inhibitors include any anti-EGFR antibody or antibody fragment that can partially or completely block the activation of EGFR by its natural ligands. Non-limiting examples of antibody-based EGFR inhibitors include those described in the following literature: Modjtahedi, H. et al., 1993, Br.J. Cancer 67:247-253; Teramoto, T. et al., 1996, Cancer 77:639-645; Goldstein et al., 1995, Clin. Cancer Res. 1:1311-1318; Huang, S.M. et al., 1999, Cancer Res. 15:59(8):1935-40; and Yang, X. et al., 1999, Cancer Res. 59:1236-1243. Therefore, EGFR inhibitors can be monoclonal antibodies such as Mab E7.6.3 (Yang, 1999, ibid.), or Mab C225 (ATCC accession number HB-8508), or antibodies or antibody fragments with their binding specificity.

MEK inhibitors include, but are not limited to, CI-1040, AZD6244, PD318088, PD98059, PD334581, RDEA119, ARRY-142886, ARRY-438162, and PD-325901.

PI3K inhibitors include, but are not limited to, womanpem, 17-hydroxywomanpem analogs described in WO 06/044453, 4-[2-(1H-indazol-4-yl)-6-[[4-(methanesulfonyl)piperazin-1-yl]methyl]thieno[3,2-d]pyrimidin-4-yl]morpholine (also known as GDC 0941 and described in PCT publications WO 09/036,082 and WO 09/055,730), 2-methyl-2-[4-[3-methyl-2-oxo-8-(quinolin-3-yl)-2,3-dihydroimidazo[4,5-c]quinolin-1-yl]phenyl]propionitrile (also known as BEZ 235 or NVP-BEZ 235 and described in PCT publication WO 06/122806), (S)-1-(4- ((2-(2-aminopyrimidin-5-yl)-7-methyl-4-morpholinothieno[3,2-d]pyrimidin-6-yl)methyl)piperazin-1-yl)-2-hydroxyprop-1-one (described in PCT Publication No. WO 2008/070740), LY294002 (2-(4-morpholino)-8-phenyl-4H-1-benzopyran-4-one, available from Exxon Medical Chemicals Co., Ltd.) (Axon Medchem) PI103 hydrochloride (3-[4-(4-morpholinylpyridino-[3’,2’:4,5]furano[3,2-d]pyrimidin-2-yl]phenol hydrochloride, available from Axon Medical Chemicals) PIK 75 (N’-[(1E)-(6-bromoimidazo[1,2-a]pyridin-3-yl)methylene]-N,2-dimethyl -5-nitrobenzenesulfonyl-hydrazide hydrochloride, available from Exxon Medical Chemicals, Inc.), PIK 90 (N-(7,8-dimethoxy-2,3-dihydro-imidazo[1,2-c]quinazolin-5-yl)-nicotinamide, available from Exxon Medical Chemicals, Inc.), GDC-0941 dimethylsulfonate (2-(1H-indazole-4-yl)-6-(4-methanesulfonyl-piperazin-1-ylmethyl) (1-[1-[5-(4-fluoro-2-hydroxy-phenyl)-furan-2-yl]-methyl-(Z)-ylidene]-thiazolidin-2,4-dione, available from Exxon Medical Chemicals), AS-252424 (5-[1-[5-(4-fluoro-2-hydroxy-phenyl)-furan-2-yl]-methyl-(Z)-ylidene]-thiazolidin-2,4-dione, available from Exxon Medical Chemicals), and TGX-221 (7-methyl-2-(4-morpholino)-4-yl-thieno[3,2-d]pyrimidine dimethylsulfonate, available from Exxon Medical Chemicals) and TGX-221 (7-methyl-2-(4-morpholino)-4-yl-thieno[3,2-d]pyrimidine dimethylsulfonate, available from Exxon Medical Chemicals). (Linyl)-9-[1-(phenylamino)ethyl]-4H-pyrido-[1,2-a]pyrimidin-4-one, available from Exon Medical Chemicals, Inc.), XL-765, and XL-147. Other PI3K inhibitors include demethoxyviridin, perifoxine, CAL101, PX-866, BEZ235, SF1126, INK1117, IPI-145, BKM120, XL147, XL765, Palomid 529, GSK1059615, ZSTK474, PWT33597, IC87114, TG100-115, CAL263, PI-103, GNE-477, CUDC-907, and AEZS-136.

AKT inhibitors include, but are not limited to, Akt-1-1 (inhibiting Akt1) (Barnett et al. (2005) Biochem.J., 385(Pt.2), 399-408); Akt-1-1,2 (inhibiting Ak1 and 2) (Barnett et al. (2005) Biochem.J., 385(Pt.2), 399-408); API-59CJ-Ome (e.g., Jin et al. (2004) Br.J. Cancer, 91, 1808-12); 1-H-imidazo[4,5-c]pyridyl compounds (e.g., WO05011700); indole-3-carbinol and its derivatives (e.g., US Patent No. 6,656,963); Sarkar and Li (2004) J Nutr. [Journal of Nutrition] 134 (12 Supplement), 3493S-3498S); perifoxine (e.g., interfering with Akt membrane localization; Dasmahapatra et al. (2004) Clin. Cancer Res. [Clinical Cancer Research] 10 (15), 5242-52, 2004); phosphatidylinositol ether lipid analogs (e.g., Gills and Dennis (2004) Expert. Opin. Investig. Drugs [Research Drug Expert Review] 13, 787-97); and tricitabine (TCN or API-2 or NCI identifier: NSC 154020; Yang et al. (2004) Cancer Res. [Cancer Research] 64, 4394-9).

TOR inhibitors include, but are not limited to, AP-23573, CCI-779, everolimus, RAD-001, rapamycin, tesiromolimus, ATP-competitive TORC1/TORC2 inhibitors, including PI-103, PP242, PP30, and Torin 1. Other TOR inhibitors include FKBP12 enhancers; rapamycin and its derivatives, including: CCI-779 (tesirobolimus), RAD001 (everolimus; WO 9409010) and AP23573; rapamycin analogues, such as those disclosed in WO98/02441 and WO 01/14387, such as AP23573, AP23464 or AP23841; 40-(2-hydroxyethyl)rapamycin, 40-[3-hydroxy(hydroxymethyl)methylpropionate]-rapamycin (also known as CC1779), 40-epi-(tetrazole)-rapamycin (also known as ABT578), 32-deoxyrapamycin, 16-pentyneoxy-32(S)-dihydrorapamycin and other derivatives disclosed in WO05005434; and derivatives disclosed in the following patents. U.S. Patent Nos. 5,258,389, WO 94/090101, WO 92/05179, 5,118,677, 5,118,678, 5,100,883, 5,151,413, 5,120,842, WO 93/111130, WO 94/02136, WO 94/ 02485, WO 95/14023, WO 94/02136, WO 95/16691, WO 96/41807, WO 96/41807 and U.S. Patent No. 5,256,790; phosphorus-containing rapamycin derivatives (e.g., WO 05016252); 4H-1-benzopyran-4-one derivatives (e.g., U.S. Provisional Application No. 60/528,340).

Immunotherapy includes, but is not limited to, anti-PD-1 agents, anti-PDL-1 agents, anti-CTLA-4 agents, anti-LAG1 agents, and anti-OX40 agents. Exemplary anti-PD-1 antibodies and their methods of use are described in the following literature: Goldberg et al., Blood 110(1):186-192 (2007); Thompson et al., Clin. Cancer Res. 13(6):1757-1761 (2007); and Korman et al., International Application No. PCT/JP 2006/309606 (Publication No. WO 2006/121168A1), each of which is expressly incorporated herein by reference. Including: Yervoy ^™ (ipilimumab) or tremelimumab (for CTLA-4), galiximab (for B7.1), BMS-936558 (for PD-1), MK-3475 (for PD-1), AMP224 (for B7DC), BMS-936559 (for B7-H1), MPDL3280A (for B7-H1), MEDI-570 (for ICOS), AMG557 (for B7H2), MGA271 (for B7H3), IMP321 (for LAG-3), BMS-663513 (for CD137), PF-05082566 (for CD137), CDX-1127 (for CD27), anti-OX40 (Providence Health) Services), huMAbOX40L (for OX40L), Atacicept (for TACI), CP-870893 (for CD40), Lucarumumab (for CD40), Dacetuzumab (for CD40), Muromonab-CD3 (for CD3), Ipilimumab (for CTLA-4). Immunotherapy also includes genetically engineered T cells (e.g., CAR-T cells) and bispecific antibodies (e.g., BiTE).

GITR agonists include, but are not limited to, GITR fusion proteins and anti-GITR antibodies (e.g., bivalent anti-GITR antibodies), such as GITR fusion proteins described in the following patents: U.S. Patent No. 6,111,090box.c, European Patent No. 090505B1, U.S. Patent No. 8,586,023, PCT Publications Nos. WO 2010/003118 and 2011/090754; or anti-GITR antibodies described in, for example, the following patents: U.S. Patent No. 7,025,962, European Patent No. 1947183B1, U.S. Patent No. 7,812,135, U.S. Patent No. 8,388,967, U.S. Patent No. 8,591,886, European Patent No. EP 186633. 9. PCT Publication Nos.: WO 2011/028683, WO 2013/039954, WO 2005/007190, WO 2007/133822, WO 2005/055808, WO99/40196, WO 2001/03720, WO 99/20758, WO2006/083289, WO 2005/115451, US Patent No. 7,618,632, and PCT Publication No.: WO2011/051726.

In some embodiments, a compound having a structure or its stereoisomer, its transisomer, its pharmaceutically acceptable salt, a pharmaceutically acceptable salt of its stereoisomer or its transisomer, and a therapeutically effective amount of other pharmaceutically active compounds are administered to a patient in need, such as cytarabine, proteasome inhibitors (e.g., carfilzomib or opzomib), BC1-2 inhibitors (e.g., venetumola), MCl-1 inhibitors (e.g., AMG-176), monoclonal antibodies (e.g., raltolumab), and immunomodulatory imide drugs (IMiDs) (e.g., thalidomide, lenalidomide, pomalidomide, and apremilast).

In some embodiments, a compound having a structure or its stereoisomer, its transisomer, its pharmaceutically acceptable salt, a pharmaceutically acceptable salt of its stereoisomer or its transisomer, and a therapeutically effective amount of other pharmaceutically active compounds are administered to a patient in need, such as cytarabine, proteasome inhibitors (e.g., carfilzomib or opzomib), BC1-2 inhibitors (e.g., venetumola), MCl-1 inhibitors (e.g., AMG-176), monoclonal antibodies (e.g., raltolumab), and immunomodulatory imide drugs (IMiDs) (e.g., thalidomide, lenalidomide, pomalidomide, and apremilast).

In some embodiments, a compound having a structure or its stereoisomer, its transisomer, its pharmaceutically acceptable salt, a pharmaceutically acceptable salt of its stereoisomer or its transisomer, and a therapeutically effective amount of other pharmaceutically active compounds are administered to a patient in need, such as cytarabine, proteasome inhibitors (e.g., carfilzomib or opzomib), BC1-2 inhibitors (e.g., venetumola), MCl-1 inhibitors (e.g., AMG-176), monoclonal antibodies (e.g., raltolumab), and immunomodulatory imide drugs (IMiDs) (e.g., thalidomide, lenalidomide, pomalidomide, and apremilast).

In some embodiments, a compound having a structure or its stereoisomer, its transisomer, its pharmaceutically acceptable salt, a pharmaceutically acceptable salt of its stereoisomer or its transisomer, and a therapeutically effective amount of other pharmaceutically active compounds are administered to a patient in need, such as cytarabine, proteasome inhibitors (e.g., carfilzomib or opzomib), BC1-2 inhibitors (e.g., venetumola), MCl-1 inhibitors (e.g., AMG-176), monoclonal antibodies (e.g., raltolumab), and immunomodulatory imide drugs (IMiDs) (e.g., thalidomide, lenalidomide, pomalidomide, and apremilast).

In some embodiments, a compound having a structure or its stereoisomer, its transisomer, its pharmaceutically acceptable salt, a pharmaceutically acceptable salt of its stereoisomer or its transisomer, and a therapeutically effective amount of other pharmaceutically active compounds are administered to a patient in need, such as cytarabine, proteasome inhibitors (e.g., carfilzomib or opzomib), BC1-2 inhibitors (e.g., venetumola), MCl-1 inhibitors (e.g., AMG-176), monoclonal antibodies (e.g., raltolumab), and immunomodulatory imide drugs (IMiDs) (e.g., thalidomide, lenalidomide, pomalidomide, and apremilast).

In some embodiments, a compound having a structure or its stereoisomer, its transisomer, its pharmaceutically acceptable salt, a pharmaceutically acceptable salt of its stereoisomer or its transisomer, and a therapeutically effective amount of other pharmaceutically active compounds are administered to a patient in need, such as cytarabine, proteasome inhibitors (e.g., carfilzomib or opzomib), BC1-2 inhibitors (e.g., venetumola), MCl-1 inhibitors (e.g., AMG-176), monoclonal antibodies (e.g., raltolumab), and immunomodulatory imide drugs (IMiDs) (e.g., thalidomide, lenalidomide, pomalidomide, and apremilast).

In some embodiments, a compound having a structure or its stereoisomer, its transisomer, its pharmaceutically acceptable salt, a pharmaceutically acceptable salt of its stereoisomer or its transisomer, and a therapeutically effective amount of other pharmaceutically active compounds are administered to a patient in need, such as cytarabine, proteasome inhibitors (e.g., carfilzomib or opzomib), BC1-2 inhibitors (e.g., venetumola), MCl-1 inhibitors (e.g., AMG-176), monoclonal antibodies (e.g., raltolumab), and immunomodulatory imide drugs (IMiDs) (e.g., thalidomide, lenalidomide, pomalidomide, and apremilast).

In some embodiments, a compound having a structure or its stereoisomer, its transisomer, its pharmaceutically acceptable salt, a pharmaceutically acceptable salt of its stereoisomer or its transisomer, and a therapeutically effective amount of other pharmaceutically active compounds are administered to a patient in need, such as cytarabine, proteasome inhibitors (e.g., carfilzomib or opzomib), BC1-2 inhibitors (e.g., venetumola), MCl-1 inhibitors (e.g., AMG-176), monoclonal antibodies (e.g., raltolumab), and immunomodulatory imide drugs (IMiDs) (e.g., thalidomide, lenalidomide, pomalidomide, and apremilast).

In some embodiments, a compound having a structure or its stereoisomer, its transisomer, its pharmaceutically acceptable salt, a pharmaceutically acceptable salt of its stereoisomer or its transisomer, and a therapeutically effective amount of other pharmaceutically active compounds are administered to a patient in need, such as cytarabine, proteasome inhibitors (e.g., carfilzomib or opzomib), BC1-2 inhibitors (e.g., venetumola), MCl-1 inhibitors (e.g., AMG-176), monoclonal antibodies (e.g., raltolumab), and immunomodulatory imide drugs (IMiDs) (e.g., thalidomide, lenalidomide, pomalidomide, and apremilast).

In some embodiments, a compound having a structure or its stereoisomer, its transisomer, its pharmaceutically acceptable salt, a pharmaceutically acceptable salt of its stereoisomer or its transisomer, and a therapeutically effective amount of other pharmaceutically active compounds are administered to a patient in need, such as cytarabine, proteasome inhibitors (e.g., carfilzomib or opzomib), BC1-2 inhibitors (e.g., venetumola), MCl-1 inhibitors (e.g., AMG-176), monoclonal antibodies (e.g., raltolumab), and immunomodulatory imide drugs (IMiDs) (e.g., thalidomide, lenalidomide, pomalidomide, and apremilast).

In some embodiments, a compound having a structure or its stereoisomer, its transisomer, its pharmaceutically acceptable salt, a pharmaceutically acceptable salt of its stereoisomer or its transisomer, and a therapeutically effective amount of other pharmaceutically active compounds are administered to a patient in need, such as cytarabine, proteasome inhibitors (e.g., carfilzomib or opzomib), BC1-2 inhibitors (e.g., venetumola), MCl-1 inhibitors (e.g., AMG-176), monoclonal antibodies (e.g., raltolumab), and immunomodulatory imide drugs (IMiDs) (e.g., thalidomide, lenalidomide, pomalidomide, and apremilast).

In some embodiments, a compound having a structure or its stereoisomer, its transisomer, its pharmaceutically acceptable salt, a pharmaceutically acceptable salt of its stereoisomer or its transisomer, and a therapeutically effective amount of other pharmaceutically active compounds are administered to a patient in need, such as cytarabine, proteasome inhibitors (e.g., carfilzomib or opzomib), BC1-2 inhibitors (e.g., venetumola), MCl-1 inhibitors (e.g., AMG-176), monoclonal antibodies (e.g., raltolumab), and immunomodulatory imide drugs (IMiDs) (e.g., thalidomide, lenalidomide, pomalidomide, and apremilast).

In some embodiments, a compound having a structure or its stereoisomer, its transisomer, its pharmaceutically acceptable salt, a pharmaceutically acceptable salt of its stereoisomer or its transisomer, and a therapeutically effective amount of other pharmaceutically active compounds are administered to a patient in need, such as cytarabine, proteasome inhibitors (e.g., carfilzomib or opzomib), BC1-2 inhibitors (e.g., venetumola), MCl-1 inhibitors (e.g., AMG-176), monoclonal antibodies (e.g., raltolumab), and immunomodulatory imide drugs (IMiDs) (e.g., thalidomide, lenalidomide, pomalidomide, and apremilast).

In some embodiments, a compound having a structure or its stereoisomer, its transisomer, its pharmaceutically acceptable salt, a pharmaceutically acceptable salt of its stereoisomer or its transisomer, and a therapeutically effective amount of other pharmaceutically active compounds are administered to a patient in need, such as cytarabine, proteasome inhibitors (e.g., carfilzomib or opzomib), BC1-2 inhibitors (e.g., venetumola), MCl-1 inhibitors (e.g., AMG-176), monoclonal antibodies (e.g., raltolumab), and immunomodulatory imide drugs (IMiDs) (e.g., thalidomide, lenalidomide, pomalidomide, and apremilast).

In some embodiments, a compound having a structure or its stereoisomer, its transisomer, its pharmaceutically acceptable salt, a pharmaceutically acceptable salt of its stereoisomer or its transisomer, and a therapeutically effective amount of other pharmaceutically active compounds are administered to a patient in need, such as cytarabine, proteasome inhibitors (e.g., carfilzomib or opzomib), BC1-2 inhibitors (e.g., venetumola), MCl-1 inhibitors (e.g., AMG-176), monoclonal antibodies (e.g., raltolumab), and immunomodulatory imide drugs (IMiDs) (e.g., thalidomide, lenalidomide, pomalidomide, and apremilast).

The compounds described herein may be used in combination with the agents disclosed herein or other suitable agents, depending on the condition being treated. Therefore, in some embodiments, one or more of the compounds disclosed herein will be co-administered with other agents as described above. When used in combination therapy, the compounds described herein may be administered simultaneously or separately from a second agent. Such combination administration may include simultaneous administration of two agents in the same dosage form, simultaneous administration in separate dosage forms, and separate administration. That is, the compounds described herein and any of the agents described above may be formulated together in the same dosage form and administered simultaneously. Alternatively, the compounds disclosed herein and any of the agents described above may be administered simultaneously, wherein the two agents are present in separate formulations. In another alternative, any of the agents described above may be administered immediately after administration of the compounds disclosed herein, or vice versa. In some embodiments of the separate administration regimen, the administration of the compounds disclosed herein and any of the agents described above may be spaced minutes, hours, or days apart.

Since one aspect of the invention contemplates the treatment of diseases/conditions with combinations of pharmaceutically active compounds that can be administered separately, the invention further relates to the combination of individual pharmaceutical compositions in the form of a kit. The kit contains two separate pharmaceutical compositions: the compound of the invention and a second pharmaceutical compound. The kit includes containers for containing the individual compositions, such as separate bottles or separate foil pouches. Other examples of containers include syringes, boxes, and bags. In some embodiments, the kit includes instructions for use of the individual components. The kit format is particularly advantageous when the individual components are preferably administered in different dosage forms (e.g., oral and parenteral), at different dose intervals, or when the prescribing healthcare professional requires titration of individual components in the combination.

Example

Method 1

Example 1-1: 1-(4-(6-(2-bromo-5-hydroxyphenyl)-5-chloro-7-fluorobenzo[c]isothiazo-3-yl)piperazin-1-yl)prop-2-en-1-one

Step 1: 2-Amino-4-bromo-5-chloro-3-fluorobenzoic acid (Intermediate A). A mixture of 2-amino-4-bromo-3-fluorobenzoic acid (3.91 g, 16.71 mmol, Apollo Scientific Ltd., Stockport, UK) and N-chlorosuccinimide (1.36 mL, 16.7 mmol) in N,N-dimethylformamide (33 mL) was stirred at 70 °C for 20 h. The reaction mixture was then allowed to cool to room temperature, ice water (40 mL) was added, and the resulting mixture was stirred for 1 h. The resulting precipitate was collected by filtration, washed with water, and dried under vacuum to give 2-amino-4-bromo-5-chloro-3-fluorobenzoic acid. ^¹H NMR (400 MHz, DMSO- _d₆ ) δ 7.69 (¹H, d, J = 2.0 Hz), 6.48–7.23 (²H, brs). ¹⁹ F NMR (376MHz, DMSO-d ₆ ) δ–119.70 (1F, s). m/z(ESI,+ve)270.0(M+H) ⁺ .

Step 2: 2-Amino-4-bromo-5-chloro-3-fluorobenzamide (Intermediate B). Ammonium chloride (1.10 g, 20.6 mmol) and diisopropylethylamine (5.13 mL, 29.5 mmol) were sequentially added to a mixture of 2-amino-4-bromo-5-chloro-3-fluorobenzoic acid (Intermediate A, 3.96 g, 14.7 mmol) and TBTU (4.97 g, 15.5 mmol, Advanced ChemTech, Louisville, Kentucky, USA) in N,N-dimethylformamide (30 mL), and the mixture was stirred at room temperature for 30 min. The reaction mixture was then added to a saturated aqueous solution of sodium bicarbonate and stirred for 15 min. The resulting precipitate was collected by filtration, washed with water, and dried under vacuum to give 2-amino-4-bromo-5-chloro-3-fluorobenzamide. ¹ H NMR (400MHz, DMSO-d ₆ ) δ 8.03 (1H, br s), 7.72 (1H, d, J = 2.0 Hz), 7.47 (1H, br s), 6.86 (2H, s). ¹⁹ F NMR (376MHz, DMSO-d ₆ ) δ–120.79 (1F, s). m/z(ESI,+ve)268.9(M+H) ⁺ .

Step 3: 2-Amino-4-bromo-5-chloro-3-fluorobenzothioamide. Lawson's reagent (2.81 g, 6.95 mmol) was added to 2-amino-4-bromo-5-chloro-3-fluorobenzamide (intermediate B, 3.10 g, 11.59 mmol) in THF (77 mL), and the resulting mixture was stirred at room temperature for 1 h. The reaction mixture was then diluted with EtOAc (75 mL) and washed sequentially with 2 M HCl aqueous solution (50 mL), saturated sodium bicarbonate aqueous solution (50 mL), and brine ( ₅₀ mL). The organic extract was then dried over _Na₂SO₄ , collected by filtration, and concentrated under vacuum. Chromatographic purification of the residue (silica gel, 0–3% MeOH in DCM) yielded 2-amino-4-bromo-5-chloro-3-fluorobenzothiamide: ^¹H NMR (400 MHz, DMSO- _d⁶ ) δ 9.93–10.15 (¹H, m), 9.63 (¹H, brs), 7.28 (¹H, d, J = 1.96 Hz), 6.34 (²H, s). ^¹⁹ F NMR (376 MHz, DMSO- _d⁶ ) δ -119.52 (¹F, s). m/z (ESI, +ve) 284.8 (M+H) ^⁺ .

Step 4: 6-Bromo-5-chloro-7-fluorobenzo[c]isothiazol-3-amine Hydrogen peroxide (30 wt% in water, 2.93 mL, 28.7 mmol) was added dropwise to an ice-cooled solution of 2-amino-4-bromo-5-chloro-3-fluorobenzothiamide (2.71 g, 9.55 mmol) in pyridine (32 mL), and the resulting mixture was then allowed to warm to room temperature and stirred for 24 h. Water (50 mL) was added, and the precipitated solid was collected by filtration, washed with water, and dried under vacuum to give 6-bromo-5-chloro-7-fluorobenzo[c]isothiazol-3-amine: ^¹H NMR (400 MHz, DMSO- _d₆ ) δ 8.12–8.26 (2H, m), 7.95–8.06 (1H, m). ¹⁹ FNMR (376MHz, DMSO-d ₆ ) δ–114.32 (1F, s). m/z(ESI,+ve)283.0(M+H) ⁺ .

Step 5: 6-Bromo-3,5-dichloro-7-fluorobenzo[c]isothiazolium (intermediate C) was slowly added to an ice-cooled mixture of 6-bromo-5-chloro-7-fluorobenzo[c]isothiazolium-3-amine (2.47 g, 8.78 mmol), water (12 mL), and concentrated hydrochloric acid (37 wt%, 12 mL, 395 mmol) in a solution of sodium nitrite (0.788 g, 11.4 mmol) in water (2.0 mL). The resulting mixture was stirred at 0 °C for 2.5 h, and then copper chloride (I) (1.39 g, 14.1 mmol) in a mixture of concentrated hydrochloric acid (37 wt%, 12 mL, 395 mmol) was added at 0 °C. The reaction mixture was then allowed to warm to room temperature and stirred for 20 h. The reaction mixture was diluted with water (50 mL), and the precipitated solid was collected by filtration and dried under vacuum. The collected material was absorbed into (3:1) DCM:MeOH (200 mL) and washed sequentially with water (200 mL) and brine (100 mL). The organic layer was then dried over _Na₂SO₄ , filtered, and concentrated under vacuum. Chromatographic purification of the residue (0–20% EtOAc in silica gel and heptane) yielded 6-bromo-3,5-dichloro- ₇ -fluorobenzo[c]isothiazol: ^¹H NMR (400 MHz, DMSO- _d₆ ) δ 7.99 (¹H, d, J = 1.57 Hz). ^¹⁹ F NMR (376 MHz, DMSO- _d₆ ) δ – 111.48 (¹F, s). m/z (ESI, +ve) 425.0 (M + H) ⁺ .

Step 6: 4-(6-bromo-5-chloro-7-fluorobenzo[c]isothiazol-3-yl)piperazine-1-carboxylic acid tert-butyl ester (Intermediate D) A mixture of 6-bromo-3,5-dichloro-7-fluorobenzo[c]isothiazol (Intermediate C, 150 mg, 0.497 mmol) and 1-Boc-piperazine (204 mg, 1.09 mmol) in N,N-dimethylformamide (2.0 mL) was stirred at room temperature for 20 h. The reaction mixture was then adsorbed onto silica gel and purified chromatographically (silica gel, 0-20% EtOAc in heptane) to provide 4-(6-bromo-5-chloro-7-fluorobenzo[c]isothiazol-3-yl)piperazine-1-carboxylic acid tert-butyl ester. ^¹H NMR (400MHz, chloroform-d) δ 7.60 (¹H, d, J = 1.56Hz), 3.68–3.79 (⁴H, m), 3.40–3.51 (⁴H, m), 1.26 (⁹H, s). m/z (ESI, +ve) 451.8 (M + H) ⁺ .

Step 7: 4-(6-(2-bromo-5-methoxyphenyl)-5-chloro-7-fluorobenzo[c]isothiazol-3-yl)piperazine-1-carboxylic acid tert-butyl ester: A mixture of 4-(6-bromo-5-chloro-7-fluorobenzo[c]isothiazol-3-yl)piperazine-1-carboxylic acid tert-butyl ester (intermediate D, 111 mg, 0.247 mmol), 2-bromo-5-methoxyphenylboronic acid (0.114 mL, 0.494 mmol), sodium carbonate (0.041 mL, 0.988 mmol), and tetra(triphenylphosphine)palladium (14.3 mg, 0.012 mmol) in 1,4-dioxane (1.6 mL) and water (0.4 mL) was heated at 90 °C for 21 h. The reaction mixture was then concentrated under vacuum, adsorbed onto silica gel, and purified by column chromatography (0-20% (3:1) EtOAc/EtOH in silica gel, heptane) to supply tert-butyl 4-(6-(2-bromo-5-methoxyphenyl)-5-chloro-7-fluorobenzo[c]isothiazo-3-yl)piperazine-1-carboxylate: m/z(ESI,+ve)558.1(M+H) ⁺ .

Step 8: 1-(4-(6-(2-bromo-5-hydroxyphenyl)-5-chloro-7-fluorobenzo[c]isothiazol-3-yl)piperazin-1-yl)prop-2-en-1-one Hydrogen chloride (4M in 1,4-dioxane, 2.0 mL, 8.0 mmol) was added to a mixture of tert-butyl 4-(6-(2-bromo-5-methoxyphenyl)-5-chloro-7-fluorobenzo[c]isothiazol-3-yl)piperazin-1-carboxylate (107 mg, 0.192 mmol) and methanol (2.0 mL), and the resulting mixture was stirred at room temperature for 1 h. The reaction mixture was then concentrated under vacuum to give 6-(2-bromo-5-methoxyphenyl)-5-chloro-7-fluoro-3-(piperazin-1-yl)benzo[c]isothiazol: m/z(ESI,+ve) 458.0(M+1) ⁺ .

Add N,N-diisopropylethylamine (0.101 mL, 0.578 mmol) to this material (88 mg) in dichloromethane (2 mL), and cool the resulting mixture to 0 °C. Add acryloyl chloride (0.26 M in DCM, 0.75 mL, 0.19 mmol), and stir the resulting mixture at 0 °C for 10 min. Concentrate the reaction mixture under vacuum to provide 1-(4-(6-(2-bromo-5-methoxyphenyl)-5-chloro-7-fluorobenzo[c]isothiazolyl-3-yl)piperazin-1-yl)prop-2-en-1-one: m/z(ESI,+ve)512.0(M+H) ⁺ .

For compounds without a methyl ether protecting group, the crude material is purified at this stage. For compounds with a methyl ether protecting group, the crude material is used directly in subsequent transformations without purification.

The obtained 1-(4-(6-(2-bromo-5-methoxyphenyl)-5-chloro-7-fluorobenzo[c]isothiazolyl-3-yl)piperazin-1-yl)prop-2-en-1-one was absorbed in 1,2-dichloroethane (2.0 mL) and cooled to 0 °C. Boron tribromide solution (1.0 M in hexane, 0.97 mL, 0.97 mmol) was added, and the resulting mixture was stirred at 0 °C for 1 h. The reaction mixture was then added to a saturated sodium bicarbonate aqueous solution (2.0 mL) and extracted with (2:1) DCM/MeOH ( ₁₀ mL). The organic extract was dried over _Na₂SO₄ , filtered, and concentrated under vacuum. Chromatographic purification of the residue (silica gel, 0–3% MeOH in DCM) yielded 1-(4-(6-(2-bromo-5-hydroxyphenyl)-5-chloro-7-fluorobenzo[c]isothiazo-3-yl)piperazin-1-yl)prop-2-en-1-one: ^¹H NMR (400 MHz, DMSO- _d6 ) δ 9.99 (br s, 1H), 8.04 (s, 1H), 7.55 (d, J = 8.7 Hz, 1H), 6.81–6.94 (m, 2H), 6.79 (d, J = 2.9 Hz, 1H), 6.19 (dd, J = 16.7, 2.2 Hz, 1H), 5.77 (dd, J = 10.5, 2.2 Hz, 1H), 3.87 (br d, J = 19.5 Hz, 4H), 3.63 (br d, J = 19.5 Hz, 4H). t,J=5.1Hz,4H). ¹⁹ F NMR (376MHz, DMSO-d ₆ ) δ–124.16 (1F, s). m/z(ESI,+ve)498.0(M+H) ⁺

Table 1(b): Compounds 1-2 to 1-28 were prepared following the procedure described in steps 1-8 of Method 1 above:

Method 2

Example 2-1: 1-(4-(5-chloro-6-(3-hydroxy-1-naphthyl)[1,2]thiazo[3,4-b]pyridin-3-yl)-1-piperazinyl)-2-propen-1-one

Step 1: 2-Amino-6-bromo-5-chloronicotinic acid. N-chlorosuccinimide (2.78 g, 20.8 mmol) was added to a solution of 2-amino-6-bromonicotinic acid (4.51 g, 20.8 mmol, Ark Pharmaceuticals, Arlington Heights, ILLU, USA) in DMF (75 mL), and the resulting mixture was heated at 70 °C for 2.5 h. Heating was then stopped, and stirring was continued for 16 h. The reaction mixture was then poured into ice water. After the ice had melted, the resulting slurry was filtered through a sintered glass funnel. The collected solids were air-dried to provide 2-amino-6-bromo-5-chloronicotinic acid: ^¹H NMR (400 MHz, DMSO- _d⁶ ) δ 8.05 (s, 1H), 7.64 (br.s, 2H). m/z (ESI, +ve) 250.9 (M+H) ^⁺ .

Step 2: 4-(2-amino-6-bromo-5-chloronicotinyl)piperazine-1-carboxylic acid tert-butyl ester. TBTU (1.93 g, 6.0 mmol) was added to a solution of 2-amino-6-bromo-5-chloronicotinic acid (1.12 g, 4.5 mmol) in DMF (14 mL). After 5 min, the reaction was treated sequentially with 1-Boc-piperazine (912 mg, 4.9 mmol) and DIPEA (2.33 mL, 13.4 mmol). The resulting solution was stirred at room temperature for 25 h, saturated _NaHCO3 aqueous solution (75 mL) was added, and the resulting mixture was extracted with DCM. The organic layer was separated and washed sequentially with water (2×), dried over anhydrous sodium sulfate, and concentrated under vacuum. Chromatographic purification of the residue (silica gel, 0 to 7% MeOH in DCM) 4-(2-amino-6-bromo-5-chloronicotinyl)piperazine-1-carboxylic acid tert-butyl ester: ^¹H NMR (400 MHz, DMSO- _d⁶ ) δ 7.58 (s, 1H), 6.66 (s, 2H), 3.33 (s, 8H), 1.40 (s, 9H). m/z (ESI, +ve) 419.0 (M+H) ⁺ .

Step 3: 4-(2-amino-6-bromo-5-chloropyridine-3-thiocarbonyl)piperazine-1-carboxylate tert-butyl ester. Lawson's reagent (353 mg, 0.87 mmol) was added to a solution of 4-(2-amino-6-bromo-5-chloronicotinyl)piperazine-1-carboxylate (610 mg, 1.45 mmol) in THF (7.5 mL), and the resulting solution was stirred at 50 °C for 2.5 h. The reaction mixture was then allowed to cool to room temperature and treated sequentially with water (10 mL) and 1N HCl aqueous solution (4 mL). The resulting mixture was extracted with EtOAc (2×), and the combined extracts were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. Chromatographic purification of the residue (silica gel, 0–6% MeOH in DCM) yielded tert-butyl 4-(2-amino-6-bromo-5-chloropyridin-3-thiocarbonyl)piperazine-1-carboxylate: ^¹H NMR (400 MHz, DMSO- _d⁶ ) δ 7.47 (s, 1H), 6.58 (br. s, 2H), 4.30 (ddd, J = 13.3, 6.3, 3.3 Hz, 1H), 4.01–4.13 (m, 2H), 3.68–3.77 (m, 1H), 3.51–3.59 (m, 1H), 3.40–3.50 (m, 3H), 1.41 (s, 9H). m/z (ESI, +ve) 434.9 (M+H ⁾

Step 4: 4-(5,6-dichloroisothiazo[3,4-b]pyridin-3-yl)piperazine-1-carboxylate tert-butyl ester. NCS (116 mg, 0.87 mmol) was added to a solution of 4-(2-amino-6-bromo-5-chloropyridin-3-thiocarbonyl)piperazine-1-carboxylate (343 mg, 0.79 mmol) in THF (8 mL), and the resulting solution was stirred at room temperature for 20 min. Then, a mixture of water (10 mL) and 1 M sodium sulfite aqueous solution (5 mL) was added, and the resulting mixture was extracted with EtOAc (2×). The combined extracts were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. Chromatographic purification of the residue (silica gel, 0–4% MeOH in DCM) yielded 4-(5,6-dichloroisothiazo[3,4-b]pyridin-3-yl)piperazine-1-carboxylic acid tert-butyl ester: ^¹H NMR (400 MHz, chloroform-d) δ 8.10 (s, 1H), 3.69–3.80 (m, 4H), 3.50–3.57 (m, 4H), 1.51 (s, 9H). m/z (ESI, +ve) 389.0 (M+H) ⁺ .

Step 5: 4-(5-chloro-6-(3-methoxynaphthyl-1-yl)isothiazo[3,4-b]pyridin-3-yl)piperazine-1-carboxylic acid tert-butyl ester. A mixture of 4-(5,6-dichloroisothiazo[3,4-b]pyridin-3-yl)piperazine-1-carboxylic acid tert-butyl ester (154 mg, 0.36 mmol), (3-methoxynaphthyl-1-yl)boronic acid (287 mg, 1.42 mmol), and cesium carbonate (463 mg, 1.42 mmol) in 1,4-dioxane (8 mL) and water (2 mL) was bubbled with argon, followed by the addition of tetra(triphenylphosphine)palladium (41 mg, 0.04 mmol). The reaction mixture was bubbled with argon again and then heated in a sealed tube at 100 °C for 25 h. After cooling to room temperature, the reaction mixture was diluted with brine (40 mL) and extracted with EtOAc (2×). The combined extracts were dried over sodium sulfate, filtered, and concentrated under vacuum. Chromatographic purification of the residue (silica gel, 0–3.5% MeOH in DCM) yielded tert-butyl 4-(5-chloro-6-(3-methoxynaphthyl-1-yl)isothiazo[3,4-b]pyridin-3-yl)piperazine-1-carboxylate: m/z(ESI,+ve)511.1(M+H) ⁺ .

Step 6: 5-Chloro-6-(3-methoxynaphthyl-1-yl)-3-(piperazin-1-yl)isothiazo[3,4-b]pyridine. Trifluoroacetic acid (560 μL, 7.6 mmol) was added to a solution of tert-butyl piperazine-1-carboxylate (155 mg, 0.30 mmol) in DCM (6 mL), and the resulting solution was stirred at room temperature for 2.3 h, and then concentrated under vacuum. Chromatographic purification of the residue (silica gel, 0-25% MeOH in DCM) was performed using 5-chloro-6-(3-methoxynaphthyl-1-yl)-3-(piperazin-1-yl)isothiazo[3,4-b]pyridine as a TFA salt: ^¹H NMR (400 MHz, DMSO- _d⁶ ) δ 8.78 (s, 1H), 7.94 (d, J = 8.2 Hz, 1H), 7.46–7.53 (m, 2H), 7.31 (d, J = 3.7 Hz, 2H), 7.19 (d, J = 2.4 Hz, 1H), 3.95 (s, 3H), 3.76–3.83 (m, 4H), 3.35–3.43 (m, 4H). m/z (ESI, +ve) 411.0 (M+H) ⁺ .

Step 7: 1-(4-(5-chloro-6-(3-methoxy-1-naphthyl)[1,2]thiazo[3,4-b]pyridin-3-yl)-1-piperazinyl)-2-propen-1-one. DIPEA (100 μL, 0.57 mmol) and acryloyl chloride (23 μL, 0.29 mmol) were sequentially added to an ice-cooled slurry of 5-chloro-6-(3-methoxynaphthyl)-3-(piperazin-1-yl)isothiazo[3,4-b]pyridine (TFA salt; 100 mg, 0.19 mmol) in DCM (5 mL). The resulting solution was stirred at 0 °C for 70 min, and saturated _NaHCO3 aqueous solution (15 mL) was added. The resulting mixture was extracted with DCM (3×), and the combined extracts were dried over sodium sulfate, filtered, and concentrated under vacuum. Chromatographic purification of the residue (silica gel, 0 to 7% MeOH in DCM) yielded 1-(4-(5-chloro-6-(3-methoxy-1-naphthyl)[1,2]thiazo[3,4-b]pyridin-3-yl)-1-piperazinyl)-2-propen-1-one: ^1H NMR (400MHz, DMSO- _d6) )δ8.73(s,1H),7.93(d,J＝8.2Hz,1H),7.45-7.54(m,2H),7.25-7.39(m,2H),7.19(d,J＝2.5Hz,1H),6.86(dd,J＝16.7,10. 3Hz, 1H), 6.19 (dd, J＝16.7, 2.3Hz, 1H), 5.77 (dd, J＝10.5, 2.3Hz, 1H), 3.94 (s, 3H), 3.81-3.94 (m, 4H), 3.69-3.76 (m, 4H). m/z(ESI,+ve)465.0(M+H) ⁺ .

Step 8: 1-(4-(5-chloro-6-(3-hydroxy-1-naphthyl)[1,2]thiazo[3,4-b]pyridin-3-yl)-1-piperazinyl)-2-propen-1-one. Boron tribromide (1.0 M in hexane, 400 μL, 0.40 mmol) was added dropwise to an ice-cooled solution of 1-(4-(5-chloro-6-(3-methoxynaphthyl)isothiazo[3,4-b]pyridin-3-yl)piperazin-1-yl)propen-2-one (37.3 mg, 0.08 mmol) in 1,2-dichloroethane (4 mL), and the resulting mixture was stirred at 0 °C for 2.3 h. Then, saturated _NaHCO3 aqueous solution (5 mL) was added, and the resulting mixture was extracted with (4:1) DCM:MeOH (2×). The combined extracts were dried over sodium sulfate, filtered, and concentrated under vacuum. Chromatographic purification of the residue (silica gel, 0–6% MeOH in DCM) yielded 1-(4-(5-chloro-6-(3-hydroxy-1-naphthyl)[1,2]thiazo[3,4-b]pyridin-3-yl)-1-piperazinyl)-2-propen-1-one: ^1H NMR (400MHz, DMSO-d6 ₎ )δ9.97(br.s,1H),8.72(s,1H),7.79(d,J＝8.6Hz,1H),7.42(t,J＝7.1Hz,1H),7.17-7.28(m,3H),7.09(d,J＝2.1Hz,1H) ,6.86(dd,J＝16.7,10.5Hz,1H),6.19(dd,J＝16.7,2.3Hz,1H),5.74-5.79(m,1H),3.81-3.95(m,4H),3.68-3.76(m,4H). m/z(ESI,+ve)451.0(M+H) ⁺ .

Table 2: Compounds 2-2 to 2-6 were prepared following the procedures described in steps 1-8 of Method 2 above:

Method 3

Example 3-1: 1-(4-(5-chloro-7-fluoro-6-(3-hydroxynaphthyl-1-yl)benzo[c]isothiazo-3-yl)piperazin-1-yl)prop-2-en-1-one

Step 1: 1-(4-(6-bromo-5-chloro-7-fluorobenzo[c]isothiazol-3-yl)piperazin-1-yl)prop-2-en-1-one. 0.2 M acryloyl chloride (1.240 mL, 0.248 mmol) from DCM was added to an ice-cooled solution of 6-bromo-5-chloro-7-fluoro-3-(piperazin-1-yl)benzo[c]isothiazolium (intermediate D, 87 mg, 0.248 mmol) and N,N-diisopropylethylamine (0.129 mL, 0.744 mmol) in dichloromethane (2.3 mL), and the resulting mixture was stirred at 0 °C for 10 min. The mixture was then concentrated under vacuum, and the residue was sonicated in MeOH (2 mL). The suspended solids were collected by filtration, washed with MeOH, and dried under vacuum to provide 1-(4-(6-bromo-5-chloro-7-fluorobenzo[c]isothiazo-3-yl)piperazin-1-yl)prop-2-en-1-one: ^¹H NMR (400 MHz, DMSO- _d⁶ ) δ 8.13 (¹H, d, J = 1.56 Hz), 6.84 (¹H, dd, J = 10.47, 16.73 Hz), 6.17 (¹H, dd, J = 2.35, 16.63 Hz), 5.66–5.82 (¹H, m), 3.73–3.93 (4H, m), 3.55–3.67 (4H, m). ^¹⁹F NMR (376 MHz, DMSO- _d⁶ ) δ–113.39 (s, ¹F). m/z(ESI,+ve)405.8(M+H) ⁺ .

Step 2: 1-(4-(5-chloro-7-fluoro-6-(3-methoxynaphthyl-1-yl)benzo[c]isothiazol-3-yl)piperazin-1-yl)prop-2-en-1-one (intermediate E). A mixture of 1-(4-(6-bromo-5-chloro-7-fluorobenzo[c]isothiazol-3-yl)piperazin-1-yl)prop-2-en-1-one (intermediate D, 79 mg, 0.20 mmol), (3-methoxynaphthyl-1-yl)boronic acid (47.3 mg, 0.234 mmol), tetrakis(triphenylphosphine)palladium (22.5 mg, 0.020 mmol) and sodium carbonate (83 mg, 0.78 mmol) in water (0.500 mL) and 1,4-dioxane (2.0 mL) was heated at 100 °C for 16 h. The reaction mixture was then adsorbed onto silica gel and purified by chromatography (silica gel, 0-3% MeOH in DCM) to give 1-(4-(5-chloro-7-fluoro-6-(3-methoxynaphthyl-1-yl)benzo[c]isothiazo-3-yl)piperazin-1-yl)prop-2-en-1-one: m/z(ESI,+ve)482.0(M+H) ⁺ .

Step 3: 1-(4-(5-chloro-7-fluoro-6-(3-hydroxynaphthyl-1-yl)benzo[c]isothiazo-3-yl)piperazin-1-yl)prop-2-en-1-one. Boron tribromide (1.0 M in hexane, 0.664 mL, 0.664 mmol) was added to an ice-cooled solution of 1-(4-(5-chloro-7-fluoro-6-(3-methoxynaphthyl-1-yl)benzo[c]isothiazo-3-yl)piperazin-1-yl)prop-2-en-1-one (64 mg, 0.13 mmol) in 1,2-dichloroethane (2.0 mL), and the resulting mixture was stirred at 0 °C for 1 h. The reaction mixture was then added to a saturated aqueous solution of sodium bicarbonate (2.0 mL), and the resulting mixture was extracted with (2:1) DCM:MeOH (10 mL). The organic extract was dried _{over Na₂SO₄} , filtered, and concentrated under vacuum. Chromatographic purification of the residue (silica gel, 0–3% MeOH in DCM) yielded 1-(4-(5-chloro-7-fluoro-6-(3-hydroxynaphthyl-1-yl)benzo[c]isothiazo-3-yl)piperazin-1-yl)prop-2-en-1-one: ^¹H NMR (400 MHz, DMSO- _d₆). )δ9.90-10.04(1H,m),8.10(1H,s),7.80(1H,d,J＝8.41Hz),7.43(1H,ddd,J＝1.96,6.11,8.17Hz),7.16-7.31(3H,m),7.07(1H ,d,J＝2.35Hz),6.87(1H,dd,J＝10.47,16.73Hz),6.19(1H,dd,J＝2.25,16.73Hz),5.77(1H,dd,J＝2.25,10.47Hz),3.88(4H,br d,J＝19.56Hz),3.61-3.72(4H,m). ¹⁹ F NMR (376MHz, DMSO-d ₆ ) δ–123.78 (s, 1F). m/z(ESI,+ve)468.0(M+H) ⁺ .

Substitutional synthesis of intermediate E

1-(4-(5-chloro-7-fluoro-6-(3-methoxynaphthyl-1-yl)benzo[c]isothiazol-3-yl)piperazin-1-yl)prop-2-en-1-one (intermediate E, substitution synthesis): 1-(piperazin-1-yl)prop-2-en-1-one bis(2,2,2-trifluoroacetate) (961 mg, 2.61 mmol, eNovation Chemicals LLC, Bridgewater, NJ, USA) in N,N-dimethylformamide (5.6 mL) and N,N-diisopropylethylamine (1.243 mL, 7.12 mmol) were added sequentially to a solution of 6-bromo-3,5-dichloro-7-fluorobenzo[c]isothiazol (intermediate C, 715 mg, 2.37 mmol) in N,N-dimethylformamide (5.6 mL). The resulting mixture was stirred at room temperature for 1 h, and then heated at 50 °C for 22 h. After cooling to room temperature, the reaction mixture was added to ice water (10 mL), and the resulting precipitate was collected by filtration and washed with water. The collected solid was adsorbed onto silica gel and purified by chromatography (silica gel, 0-3% MeOH in DCM) to supply 1-(4-(6-bromo-5-chloro-7-fluorobenzo[c]isothiazo-3-yl)piperazin-1-yl)prop-2-en-1-one.

Table 3: Compounds 3-2 to 3-24 were prepared following the procedures described in steps 1-3 of Method 3 above:

Method 4

Example 4-1: 1-(6-(5-chloro-7-fluoro-6-(3-hydroxynaphthyl-1-yl)benzo[c]isothiazo-3-yl)-2,6-diazaspiro[3.3]hept-2-yl)prop-2-en-1-one.

Step 1: 6-(6-bromo-5-chloro-7-fluorobenzo[c]isothiazol-3-yl)-2,6-diazaspiro[3.3]heptane-2-carboxylic acid tert-butyl ester. A mixture of 6-bromo-3,5-dichloro-7-fluorobenzo[c]isothiazol (intermediate C, 169 mg, 0.562 mmol) and 2-Boc-2,6-diazaspiro[3.3]heptane (212 mg, 1.07 mmol, Esther Company, Bristol, PAN, USA) in DMF (3.5 mL) was stirred at room temperature for 5 h. Ice water (5 mL) was added, and the resulting mixture was stirred for 15 min. The resulting precipitate was collected by filtration, washed with water, and dried under vacuum to provide tert-butyl 6-(6-bromo-5-chloro-7-fluorobenzo[c]isothiazolyl-3-yl)-2,6-diazaspiro[3.3]heptane-2-carboxylate: ^¹H NMR (400MHz, DMSO- _d⁶ ) δ 7.52–7.74 (¹H, m), 4.55 (4H, s), 4.09 (4H, s), 1.38 (9H, s). ^¹⁹F NMR (376MHz, DMSO- _d⁶ ) δ–113.55 (¹F, s). m/z (ESI, +ve) 464.0 (M+1).

Step 2: 1-(6-(6-bromo-5-chloro-7-fluorobenzo[c]isothiazol-3-yl)-2,6-diazaspiro[3.3]heptane-2-yl)prop-2-en-1-one. Hydrogen chloride solution (4 M in 1,4-dioxane, 5.0 mL, 20 mmol) was added to tert-butyl 6-(6-bromo-5-chloro-7-fluorobenzo[c]isothiazol-3-yl)-2,6-diazaspiro[3.3]heptane-2-carboxylate (249 mg, 0.538 mmol) in methanol (10 mL), and the resulting mixture was stirred at room temperature for 2 h. The reaction mixture was then concentrated under vacuum to provide 6-bromo-5-chloro-7-fluoro-3-(2,6-diazaspiro[3.3]heptane-2-yl)benzo[c]isothiazolium: m/z(ESI,+ve)363.8(M+1) ⁺ .

Add N,N-diisopropylethylamine (0.281 mL, 1.61 mmol) to dichloromethane (3.0 mL) and cool the resulting mixture to 0 °C. Then add acryloyl chloride (0.2 M in DCM, 2.69 mL, 0.538 mmol) and stir the resulting mixture at 0 °C for 10 min. The reaction mixture was then concentrated under vacuum, and the residue was purified by chromatography (0-10% (3:1) EtOAc/EtOH in silica gel, DCM) to provide 1-(6-(6-bromo-5-chloro-7-fluorobenzo[c]isothiazolyl-3-yl)-2,6-diazaspiro[3.3]heptane-2-yl)prop-2-en-1-one: ^¹H NMR (400MHz, DMSO- _d6 ) δ 7.65 (¹H,d, J = 1.4Hz), 6.25-6.36 (¹H,m), 6.10 (¹H,dd, J = 17.0, 2.3Hz), 5.64-5.72 (¹H,m), 4.58 (4H,s), 4.47 (2H,s), 4.18 (2H,s). ¹⁹ F NMR (376MHz, DMSO-d ₆ ) δ–113.54 (1F, s). m/z(ESI,+ve)418.0(M+H) ⁺ .

Step 3: 1-(6-(5-chloro-7-fluoro-6-(3-methoxynaphthyl-1-yl)benzo[c]isothiazo-3-yl)-2,6-diazaspiro[3.3]heptane-2-yl)prop-2-en-1-one. A mixture of 1-(6-(6-bromo-5-chloro-7-fluorobenzo[c]isothiazolyl-3-yl)-2,6-diazaspiro[3.3]heptan-2-yl)prop-2-en-1-one (102 mg, 0.245 mmol), (3-methoxynaphthyl-1-yl)boronic acid (59.3 mg, 0.294 mmol), tetra(triphenylphosphine)palladium (28.3 mg, 0.024 mmol), and sodium carbonate (104 mg, 0.979 mmol) in water (0.5 mL) and 1,4-dioxane (2.0 mL) was heated at 100 °C for 1 h. The reaction mixture was then adsorbed onto silica gel and purified by chromatography (silica gel, 0-5% MeOH in DCM). The purified material was sonicated in MeOH, and the suspended solids were collected by filtration, washed with MeOH, and dried under vacuum to provide 1-(6-(5-chloro-7-fluoro-6-(3-methoxynaphthyl-1-yl)benzo[c]isothiazolyl)-2,6-diazaspiro[3.3]heptane-2-yl)prop-2-en-1-one: ^¹H NMR (400MHz, DMSO- _d6) )δ7.93(1H,d,J＝8.4Hz),7.67(1H,s),7.45-7.57(2H,m),7.23-7.36(2H,m),7.16(1H,d,J＝2.5Hz),6.27-6.39(1 H,m),6.11(1H,dd,J＝17.0,2.2Hz),5.65-5.76(1H,m),4.58-4.67(4H,m),4.50(2H,s),4.22(2H,s),3.93(3H,s). ¹⁹ FNMR (376MHz, DMSO-d ₆ ) δ–123.88 (1F, s). m/z(ESI,+ve)494.0(M+H) ⁺ .

Step 4: 1-(6-(5-chloro-7-fluoro-6-(3-hydroxynaphthyl-1-yl)benzo[c]isothiazolyl)-2,6-diazaspiro[3.3]heptan-2-yl)prop-2-en-1-one. Boron tribromide (1.0 M in hexane, 0.638 mL, 0.638 mmol) was added to ice-cooled 1-(6-(5-chloro-7-fluoro-6-(3-methoxynaphthyl-1-yl)benzo[c]isothiazolyl)-2,6-diazaspiro[3.3]heptan-2-yl)prop-2-en-1-one (63 mg, 0.128 mmol) in 1,2-dichloroethane (2.0 mL), and the resulting mixture was stirred at 0 °C for 2 h. The reaction mixture was then added to a saturated aqueous sodium bicarbonate solution (2.0 mL), and the resulting mixture was extracted with (2:1) DCM:MeOH (10 mL). The organic extract was _dried over _Na₂SO₄ , filtered, and concentrated under vacuum. Chromatographic purification of the residue (silica gel, 0–2% MeOH in DCM (containing 2 M ammonia)) gave 1-(6-(5-chloro-7-fluoro-6-(3-hydroxynaphthyl-1-yl)benzo[c]isothiazolyl)-2,6-diazaspiro[3.3]heptane-2-yl)prop-2-en-1-one: ^1H NMR (400 MHz, DMSO- _d6) )δ9.82-10.04(1H,m),7.79(1H,d,J＝8.2Hz),7.66(1H,s),7.43(1H,dt,J＝8.3,4.0Hz),7.26(1H,d,J＝2.3Hz),7.22(2H,d,J＝3.7Hz),7 .05(1H,d,J＝2.3Hz),6.26–6.38(1H,m),6.12(1H,dd,J＝16.8,2.2Hz),5.66–5.72(1H,m),4.58–4.67(4H,m),4.50(2H,s),4.22(2H,s). ¹⁹ F NMR (376MHz, DMSO-d ₆ ) δ–123.98 (1F, s). m/z(ESI,+ve)480.0(M+H) ⁺ .

Table 4: Compounds 4-2 to 4-9 were prepared following the procedure described in steps 1-4 of Method 4 above:

Method 5

Example 5-1: N-(1-(5-chloro-7-fluoro-6-(3-hydroxy-1-naphthyl)-2,1-benzothiazo-3-yl)-3-azacyclobutyl)-N-methyl-2-acrylamide

Step 1: 2-Amino-5-chloro-3-fluoro-4-(3-methoxynaphthyl-1-yl)benzamide. A mixture of (3-methoxynaphthyl-1-yl)boronic acid (2.04 g, 10.1 mmol), 2-amino-4-bromo-5-chloro-3-fluorobenzamide (intermediate B (1.93 g, 7.20 mmol)), tetrakis(triphenylphosphine)palladium (0.832 g, 0.720 mmol), sodium carbonate (1.2 mL, 28.8 mmol) in water (9.6 mL) and 1,4-dioxane (38.4 mL) was heated at 90 °C for 2 days. The reaction mixture was then filtered through a diatomaceous earth mat and washed with EtOAc. The filtrate was diluted with a saturated aqueous solution of _NaHCO3 (50 mL) and extracted with EtOAc (3 × 50 mL). The organic extract was washed with brine (30 mL) and _dried over _Na2SO4 . The solution was then filtered and the filtrate concentrated under vacuum. The residue was suspended in MeOH (5 mL), and the suspended solids were collected by filtration, washed with MeOH, and dried to give 2-amino-5-chloro-3-fluoro-4-(3-methoxynaphth-1-yl)benzamide. Chromatographic purification of the concentrated filtrate (0% to 100% (3:1) EtOAc-EtOH in silica gel, heptane) yielded additional 2-amino-5-chloro-3-fluoro-4-(3-methoxynaphth-1-yl)benzamide. ¹ H NMR (400MHz, DMSO-d ₆ )δ8.01-8.17(m,1H),7.92(d,J＝8.2Hz,1H),7.75(s,1H),7.43-7.55(m, 3H),7.23-7.34(m,2H),7.10(d,J＝2.5Hz,1H),6.73(s,2H),3.93(s,3H). m/z(ESI,+ve)345.0(M+H) ⁺ .

Step 2: 2-Amino-5-chloro-3-fluoro-4-(3-methoxynaphthyl-1-yl)thiobenzamide. Lawson's reagent (1.49 mL, 3.67 mmol) was added to a solution of 2-amino-5-chloro-3-fluoro-4-(3-methoxynaphthyl-1-yl)benzamide (2.11 g, 6.12 mmol) in tetrahydrofuran (41 mL), and the resulting mixture was stirred at room temperature for 1 h. The reaction mixture was then diluted with EtOAc (60 mL) and washed sequentially with 2 M HCl (60 mL), a saturated aqueous solution of _NaHCO3 (60 mL) _, and brine (60 mL). The organic extract was dried over _Na2SO4 , filtered, and concentrated under vacuum. The residue was sonicated in DCM (5 mL), and the resulting precipitate was collected by filtration, washed with DCM, and dried under vacuum to provide 2-amino-5-chloro-3-fluoro-4-(3-methoxynaphthyl-1-yl)thiobenzamide. Chromatographic purification of the filtrate (0% to 100% (3:1) EtOAc-EtOH in silica gel, heptane) gave additional 2-amino-5-chloro-3-fluoro-4-(3-methoxynaphthyl-1-yl)thiobenzamide: m/z (ESI, +ve) 361.0 (M + H) ⁺ .

Step 3: 5-Chloro-7-fluoro-6-(3-methoxynaphthyl-1-yl)benzo[c]isothiazolyl-3-amine. Hydrogen peroxide solution (30% in water, 2.2 mL, 21.3 mmol) was slowly added to an ice-cooled solution of 2-amino-5-chloro-3-fluoro-4-(3-methoxynaphthyl-1-yl)thiobenzamide (1.92 g, 5.33 mmol) in pyridine (18 mL). The resulting mixture was allowed to warm to room temperature and stirred at room temperature for 18 h. The reaction mixture was then diluted with water (60 mL), and the resulting precipitate was collected by filtration, washed sequentially with water and MeOH, and dried under vacuum to give 5-chloro-7-fluoro-6-(3-methoxynaphthyl-1-yl)benzo[c]isothiazolyl-3-amine: ^¹H NMR (400 MHz, DMSO- _d⁶ ) δ 8.14 (s, 2H), 7.99–8.03 (m, 1H), 7.93 (d, J = 8.3 Hz, 1H), 7.48–7.55 (m, 1H), 7.47 (d, J = 2.3 Hz, 1H), 7.31 (d, J = 3.9 Hz, 2H), 7.16 (d, J = 2.5 Hz, 1H), 3.94 (s, 3H). ^¹⁹F NMR (376 MHz, DMSO- _d⁶ ) δ -124.71 (s, 1F). m/z(ESI,+ve)359.0(M+H) ⁺ .

Step 4: 3,5-Dichloro-7-fluoro-6-(3-methoxynaphthyl-1-yl)benzo[c]isothiazolium. 5-chloro-7-fluoro-6-(3-methoxynaphthyl-1-yl)benzo[c]isothiazol-3-amine (1.55 g, 4.31 mmol) was added fractionally over 15 min at 65 °C to a suspension of copper(II) chloride (0.870 g, 6.47 mmol) and tert-butyl nitrite (0.77 mL, 6.47 mmol) in acetonitrile (43 mL). The resulting mixture was stirred at 65 °C for 30 min, then cooled to ambient temperature and diluted with ice water (50 mL). The precipitated solid was collected by filtration, washed with water, and dried under vacuum. The residue was sonicated in DCM (10 mL), and the suspended solids were collected by filtration, washed with DCM, and dried under vacuum to recover unreacted 5-chloro-7-fluoro-6-(3-methoxynaphthyl-1-yl)benzo[c]isothiazol-3-amine. The filtrate was concentrated under vacuum to give 3,5-dichloro-7-fluoro-6-(3-methoxynaphthyl-1-yl)benzo[c]isothiazol. ^¹H NMR (400 MHz, DMSO- _d⁶ ) δ 7.98 (s, 1H), 7.96 (d, J = 8.2 Hz, 1H), 7.49–7.56 (m, 2H), 7.28–7.36 (m, 2H), 7.24–7.28 (m, 1H), 3.95 (s, 3H). ¹⁹ FNMR (376MHz, DMSO-d ₆ )δ–122.17 (s, 1F). m/z(ESI,+ve)378.0(M+H) ⁺ .

Step 5: (1-(5-chloro-7-fluoro-6-(3-methoxynaphthyl-1-yl)benzo[c]isothiazol-3-yl)azacyclobutane-3-yl)(methyl)carbamate tert-butyl. A mixture of 3,5-dichloro-7-fluoro-6-(3-methoxynaphthyl-1-yl)benzo[c]isothiazol (100 mg, 0.264 mmol), DIPEA (0.14 mL, 0.793 mmol), and 3-Boc-3-methylaminoazacyclobutane (0.098 mL, 0.529 mmol, Beta Pharma Scientific, Inc.) in DMF (1.3 mL) was stirred at room temperature for 18 h. Then ice water (3 mL) was added, and the resulting mixture was stirred for 15 min. The precipitated solid was then collected by filtration, washed with water, and dried under vacuum to supply (1-(5-chloro-7-fluoro-6-(3-methoxynaphthyl-1-yl)benzo[c]isothiazolyl)azacyclobutane-3-yl)(methyl)carbamate tert-butyl ester: m/z(ESI,+ve)528.0(M+H) ⁺ .

Step 6: N-(1-(5-chloro-7-fluoro-6-(3-hydroxy-1-naphthyl)-2,1-benzothiazol-3-yl)-3-azacyclobutane-3-yl)-N-methyl-2-acrylamide. The title compound was prepared in three steps from (1-(5-chloro-7-fluoro-6-(3-methoxynaphthyl-1-yl)benzo[c]isothiazol-3-yl)azacyclobutane-3-yl)(methyl)carbamate (131.1 mg, 0.248 mmol): ^¹H NMR (400 MHz, DMSO- _d6) )δ9.89-10.10(m,1H),7.79(d,J＝8.4Hz,1H),7.73(s,1H),7.43(ddd,J＝8.2,5.1,2.9Hz,1H),7.20-7.30(m,3H),7.05(d,J＝2.2Hz,1H),6.81 (dd,J＝16.7,10.5Hz,1H),6.10-6.23(m,1H),5.69-5.81(m,1H),5.37-5.59(m,1H),4.63-4.74(m,3H),4.53-4.61(m,1H),3.14-3.23(m,3H). ¹⁹ F NMR (376MHz, DMSO-d ₆ ) δ-124.10 (s, 1F). m/z(ESI,+ve)468.0(M+H) ⁺ .

Table 5: Compounds 5-2 to 5-9 were prepared following the procedures described in steps 1-6 of Method 5 above:

Method 6

Example 6-1: 1-(4-(6-(6-amino-3-chloro-2-pyridyl)-5-chloro-7-fluoro-2,1-benzothiazo-3-yl)-1-piperazinyl)-2-propen-1-one

Step 1: 4-(5-chloro-7-fluoro-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)benzo[c]isothiazo-3-yl)piperazine-1-carboxylic acid tert-butyl ester. A mixture of 4-(6-bromo-5-chloro-7-fluorobenzo[c]isothiazo-3-yl)piperazine-1-carboxylic acid tert-butyl ester (intermediate D, 1.10 g, 2.45 mmol), bis(pinacolyl)diboron (1.86 g, 7.34 mmol), potassium acetate (0.61 mL, 9.8 mmol), and Pd(dppf) _Cl₂ ·DCM (0.537 g, 0.734 mmol) in 1,4-dioxane (12 mL) was heated at 100 °C for 40 h. The reaction mixture was then concentrated under vacuum and purified chromatographically (0% to 100% (3:1) EtOAc-EtOH in silica gel, heptane) to provide tert-butyl 4-(5-chloro-7-fluoro-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)benzo[c]isothiazolyl)piperazine-1-carboxylate: ^¹H NMR (400 MHz, DMSO- _d⁶ ) δ 7.85 (s, 1H), 3.59 (br d, J = 4.7 Hz, 4H), 3.44–3.54 (m, 4H), 1.43 (s, 9H), 1.35 (s, 5H), 1.15 (s, 7H). ^¹⁹F NMR (376 MHz, DMSO- _d⁶ ) δ -125.11 (s, 1F). m/z(ESI,+ve)498.0(M+H) ⁺ .

Step 2: 4-(6-(6-amino-3-chloropyridin-2-yl)-5-chloro-7-fluorobenzo[c]isothiazo-3-yl)piperazine-1-carboxylic acid tert-butyl ester. A mixture of 4-(5-chloro-7-fluoro-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)benzo[c]isothiazo-3-yl)piperazine-1-carboxylic acid tert-butyl ester (99.5 mg, 0.200 mmol), SPhos Pd G3 (17.3 mg, 0.020 mmol), 6-bromo-5-chloropyridin-2-amine (Combilux, San Diego, USA, 124 mg, 0.6 mmol), sodium carbonate (85 mg, 0.80 mmol) in water (0.25 mL) and 1,2-DCE (0.75 mL) was heated at 50 °C for 2 h. The reaction mixture was concentrated under vacuum and purified by chromatography (0% to 100% (3:1) EtOAc-EtOH in silica gel, heptane) to give tert-butyl 4-(6-(6-amino-3-chloropyridin-2-yl)-5-chloro-7-fluorobenzo[c]isothiazolyl)piperazine-1-carboxylate: m/z(ESI,+ve)498.0(M+H) ⁺ .

Step 3: 1-(4-(6-(6-amino-3-chloro-2-pyridyl)-5-chloro-7-fluoro-2,1-benzothiazo-3-yl)-1-piperazinyl)-2-propen-1-one. The title compound was prepared in two steps from tert-butyl 4-(6-(6-amino-3-chloropyridin-2-yl)-5-chloro-7-fluorobenzo[c]isothiazo-3-yl)piperazine-1-carboxylate (31.6 mg, 0.063 mmol) following the procedure reported in step 8 of Method 1: ^¹H NMR (400 MHz, DMSO- _d⁶ ) δ 7.97–8.10 (m, 1H), 7.60 (d, J = 8.9 Hz, 1H), 6.86 (dd, J = 16.6, 10.6 Hz, 1H), 6.57 (d, J = 8.9 Hz, 1H), 6.38 (s, 2H), 6.19 (dd, J = 16.8, 2.3 Hz, 1H), 5.71–5.84 (m, 1H), 3.86 (br⁶) δ ... d, J＝19.9Hz, 4H), 3.63 (br d,J＝1.0Hz, 4H). ¹⁹ F NMR (376MHz, DMSO-d ₆ ) δ-126.04 (s, 1F). m/z(ESI,+ve)452.0(M+H) ⁺ .

Table 6: Compound 6-2 was prepared following the procedure described in steps 1-3 of Method 6 above:

Method 7

Example 7-1: 1-((3R)-4-(5-chloro-7-fluoro-6-(3-hydroxy-1-naphthyl)-2,1-benzothiazol-3-yl)-3-(difluoromethyl)-1-piperazinyl)-2-propen-1-one|1-((3S)-4-(5-chloro-7-fluoro-6-(3-hydroxy-1-naphthyl)-2,1-benzothiazol-3-yl)-3-(difluoromethyl)-1-piperazinyl)-2-propen-1-one

Step 1: 2-Amino-5-chloro-3-fluoro-4-(3-methoxynaphth-1-yl)benzoic acid. Prepared from intermediate A using a procedure similar to that described in Step 7 of Method 1: m/z(ESI,+ve)346.0(M+H) ⁺ .

Step 2: 4-(2-amino-5-chloro-3-fluoro-4-(3-methoxynaphthyl-1-yl)benzoyl)-3-(difluoromethyl)piperazine-1-carboxylic acid tert-butyl ester. A mixture of 2-amino-5-chloro-3-fluoro-4-(3-methoxynaphthyl-1-yl)benzoic acid (0.150 g, 0.434 mmol), TBTU (0.188 g, 0.586 mmol), 3-(difluoromethyl)piperazine-1-carboxylic acid tert-butyl ester (0.123 g, 0.521 mmol), and DIPEA (0.23 mL, 1.302 mmol) in DMF (4 mL) was stirred at ambient temperature for 3 h. The reaction mixture was then washed with a saturated aqueous solution _{of NaHCO3} , and the aqueous washings were extracted with EtOAc. _The combined organic layers were dried over _Na2SO4 , filtered, and concentrated under vacuum. Chromatographic purification of the residue (silica gel, 0-40% EtOAc/heptane) yielded tert-butyl 4-(2-amino-5-chloro-3-fluoro-4-(3-methoxynaphth-1-yl)benzoyl)-3-(difluoromethyl)piperazine-1-carboxylate: m/z(ESI,+ve)586(M+Na) ⁺ .

Step 3: 4-(2-amino-5-chloro-3-fluoro-4-(3-methoxynaphthyl-1-yl)phenylthiocarbonyl)-3-(difluoromethyl)piperazine-1-carboxylate tert-butyl ester. Lawson's reagent (0.041 mL, 0.10 mmol) was added to a solution of 4-(2-amino-5-chloro-3-fluoro-4-(3-methoxynaphthyl-1-yl)benzoyl)-3-(difluoromethyl)piperazine-1-carboxylate tert-butyl ester (0.095 g, 0.168 mmol) in THF (4 mL), and the resulting mixture was stirred at 50 °C for 18 h. The reaction mixture was then concentrated under vacuum and purified by column chromatography (silica gel, 0-30% EtOAc/heptane) to give tert-butyl 4-(2-amino-5-chloro-3-fluoro-4-(3-methoxynaphthyl-1-yl)phenylthiocarbonyl)-3-(difluoromethyl)piperazine-1-carboxylate: m/z(ESI,+ve)602.2(M+Na) ⁺ .

Step 4: 4-(5-chloro-7-fluoro-6-(3-methoxynaphthyl-1-yl)benzo[c]isothiazolyl-3-yl)-3-(difluoromethyl)piperazine-1-carboxylate tert-butyl ester. NBS (0.022 g, 0.17 mmol) was added to a solution of 4-(2-amino-5-chloro-3-fluoro-4-(3-methoxynaphthyl-1-yl)phenylthiocarbonyl)-3-(difluoromethyl)piperazine-1-carboxylate tert-butyl ester in THF (7 mL), and the resulting mixture was stirred at ambient temperature for 15 min. The reaction mixture was diluted with water and washed with 10% sodium thiosulfate. The aqueous washing solution was extracted with EtOAc, and the combined organic layers were then concentrated under vacuum to obtain tert-butyl 4-(5-chloro-7-fluoro-6-(3-methoxynaphthyl-1-yl)benzo[c]isothiazo-3-yl)-3-(difluoromethyl)piperazine-1-carboxylate: m/z(ESI,+ve)578.2(M+H) ⁺ .

Step 5: 1-((3R)-4-(5-chloro-7-fluoro-6-(3-hydroxy-1-naphthyl)-2,1-benzothiazol-3-yl)-3-(difluoromethyl)-1-piperazinyl)-2-propen-1-one|1-((3S)-4-(5-chloro-7-fluoro-6-(3-hydroxy-1-naphthyl)-2,1-benzothiazol-3-yl)-3-(difluoromethyl)-1-piperazinyl)-2-propen-1-one. Prepared using a procedure similar to that described in Step 8 of Method 1: ^¹H NMR (400 MHz, DMSO- _d⁶ ) δ 10.13 (br.s., ¹H) 8.12 (d, J = 2.2 Hz, ¹H) 7.80 (d, J = 8.2 Hz, ¹H) 7.43 (br.s., ¹H) t,J＝7.0Hz,1H)7.20-7.30(m,3H)7.08(dd,J＝5.8,2.2Hz,1H)6.78-6.91(m,1H)6.27-6.70(m,1H)6.20(dd,J ＝16.6,2.0Hz,1H)5.76-5.84(m,1H)4.73-4.87(m,1H)4.19-4.72(m,2H)3.55-3.90(m,3H)3.36-3.47(m,1H). m/z(ESI,+ve)518.0(M+H) ⁺ .

Table 7: Compounds 7-2 and 7-3 were prepared following the procedure described in steps 1-5 of Method 7 above:

Method 8

Example 8-1: 6-Chloro-7-(2-fluoro-6-hydroxyphenyl)-1-(2-(2-propyl)phenyl)-4-(4-(2-acryloyl)-1-piperazinyl)-2(1H)-quinazolinone

Step 1: 4-Bromo-5-chloro-2-fluorobenzamide. A mixture of 4-bromo-5-chloro-2-fluorobenzoic acid (23.3 g, 92 mmol) and thionyl chloride (67 mL, 0.92 mol) was stirred at 70 °C under a reflux condenser for 1 h. The reaction mixture was then concentrated under vacuum, and the residue was absorbed in 1,4-dioxane (200 mL), treated with ammonium hydroxide (30% aqueous solution, 82 mL, 0.64 mol), and stirred at room temperature for 15 min. The reaction mixture was concentrated under vacuum to give 4-bromo-5-chloro-2-fluorobenzamide: m/z(ESI,+ve)251.8(M+H) ⁺ .

Step 2: 4-Bromo-5-chloro-2-fluoro-N-((2-isopropylphenyl)carbamoyl)benzamide. A mixture of 4-bromo-5-chloro-2-fluorobenzamide (5.90 g, 23.4 mmol) and oxalyl chloride (1 M in DCM; 12.9 mL, 25.7 mmol) in DCE (100 mL) was stirred at 80 °C under a reflux condenser for 1 h. The reaction mixture was then cooled to room temperature and 2-isopropylaniline (6.62 mL, 46.7 mmol) was added. The resulting mixture was stirred at room temperature for 15 min and then cooled to 0 °C. The precipitated solid was removed by filtration, and the collected filtrate was concentrated under vacuum to obtain 4-bromo-5-chloro-2-fluoro-N-((2-isopropylphenyl)carbamoyl)benzamide: ^¹H NMR (400 MHz, DMSO- _d⁶ ) δ 11.06 (br.s., 1H) 10.31 (s, 1H) 7.97–8.05 (m, 2H) 7.82 (d, J = 7.2 Hz, 1H) 7.32–7.38 (m, 1H) 7.14–7.25 (m, 2H) 3.11 (spt, J = 6.8 Hz, 1H) 1.24 (d, J = 6.8 Hz, 6H). ^¹⁹ F NMR (376 MHz, DMSO- _d⁶ ) δ–113.6 (s, 1F). m/z(ESI,+ve)412.7 and 414.6(M+H) ⁺ .

Step 3: 7-Bromo-6-chloro-1-(2-isopropylphenyl)quinazolin-2,4(1H,3H)-dione (intermediate F). KHMDS (1M in THF, 8.30 mL, 8.30 mmol) was added to a mixture of 4-bromo-5-chloro-2-fluoro-N-((2-isopropylphenyl)carbamoyl)benzamide (1.56 g, 3.77 mmol) in THF (19 mL) at -20 °C, and the resulting mixture was allowed to warm to room temperature for 1 _h . The reaction mixture was then diluted with EtOAc (150 mL) and washed with saturated ammonium chloride aqueous solution (2 × 100 mL). The organic layer was dried over _Na₂SO₄ , filtered, and concentrated under vacuum. The residue was suspended in DCM (5 mL), sonicated, collected by filtration, and dried under vacuum to give 7-bromo-6-chloro-1-(2-isopropylphenyl)quinazolin-2,4(1H,3H)-dione: ^¹H NMR (400 MHz, _CDCl₃ ) δ 9.43 (br.s., 1H) 8.29 (s, 1H) 7.55–7.59 (m, 2H) 7.39–7.44 (m, 1H) 7.16 (d, J = 7.8 Hz, 1H) 6.75 (s, 1H) 2.59–2.77 (m, 1H) 1.17–1.24 (m, 3H) 1.11 (d, J = 6.8 Hz, 3H). m/z (ESI, +ve) 392.9 and 395.0 (M+H) ^⁺ .

Step 4: 6-Chloro-7-(2-fluoro-6-methoxyphenyl)-1-(2-isopropylphenyl)quinazoline-2,4(1H,3H)-dione. A mixture of 7-bromo-6-chloro-1-(2-isopropylphenyl)quinazoline-2,4(1H,3H)-dione (intermediate F, 1.17 g, 2.96 mmol), (2-fluoro-6-methoxyphenyl)boric acid (2.02 g, 11.9 mmol), SPhos Pd G3 (0.128 g, 0.148 mmol), and potassium carbonate (2 M in water, 4.45 mL, 8.90 mmol) in DME (30 mL) was stirred at 85 °C for 16 _h . The reaction mixture was then diluted with EtOAc (150 mL) and washed with a saturated aqueous solution of _NaHCO3 (3 × 100 mL). The organic layer was dried over _Na2SO4 , filtered, and concentrated under vacuum. Chromatographic purification of the residue (silica gel, 0-50% EtOAc in heptane) yielded 6-chloro-7-(2-fluoro-6-methoxyphenyl)-1-(2-isopropylphenyl)quinazolin-2,4(1H,3H)-dione: ^1H NMR (400MHz, DMSO-d6 ₎ )δ11.90(d,J＝1.2Hz,1H)8.11(d,J＝3.3Hz,1H)7.53-7.59(m,1H)7.48(t t,J＝7.0,2.2Hz,1H)7.38-7.44(m,1H)7.32-7.37(m,2H)6.93(dd,J＝8.4, 4.3Hz, 1H) 6.86 (t, J = 8.7Hz, 1H) 6.15 (s, 1H) 3.66 (d, J = 30Hz, 3H) 2.73 (dq, J = 14.2, 7.0Hz, 1H) 1.11 (t, J = 7.1Hz, 3H) 1.03 (dd, J = 12.7, 6.8Hz, 3H). ¹⁹ F NMR (376MHz, DMSO-d ₆ ) δ – 113.8 (s, 1F) – 115.2 (s, 1F). m/z(ESI,+ve)439.1(M+H) ⁺ .

Step 5: 4,6-Dichloro-7-(2-fluoro-6-methoxyphenyl)-1-(2-isopropylphenyl)quinazoline-2(1H)-one. Add phosphorus oxychloride (0.503 mL, 5.40 mmol) to a solution of 6-chloro-7-(2-fluoro-6-methoxyphenyl)-1-(2-isopropylphenyl)quinazoline-2,4(1H,3H)-dione (0.395 g, 0.900 mmol) and Et ₃ N (0.753 mL, 5.40 mmol) in acetonitrile (9 mL), and stir the resulting solution at 80 °C for 1.5 h. The reaction mixture was concentrated under vacuum to give 4,6-dichloro-7-(2-fluoro-6-methoxyphenyl)-1-(2-isopropylphenyl)quinazolin-2(1H)-one: m/z(ESI,+ve)457.1(M+H) ⁺ .

An alternative procedure for step 5 (used as described in the table below): Phosphorus oxychloride (6.0 equivalents) was added to a stirred mixture of the product of step 4 (1.0 equivalents), triethylamine (18.0 equivalents), and 1H-benzo[d][1,2,3]triazole (12 equivalents) in acetonitrile (0.07 M), and the resulting reaction mixture was stirred at 80 °C for 3.5 h. The reaction mixture was then slowly poured into rapidly stirred water (100 mL) at 10 °C. The aqueous suspension was stirred for 15 min and then extracted with EtOAc (100 mL). The organic layer was washed with brine (100 mL), dried over _MgSO4 , filtered, and concentrated under vacuum to give the benzotriazole adduct intermediate, which was used directly in step 6.

Step 6: 4-(6-chloro-7-(2-fluoro-6-methoxyphenyl)-1-(2-isopropylphenyl)-2-oxo-1,2-dihydroquinazoline-4-yl)piperazine-1-carboxylate tert-butyl ester. A solution of 4,6-dichloro-7-(2-fluoro-6-methoxyphenyl)-1-(2-isopropylphenyl)quinazoline-2(1H)-one (obtained from Step 5 of Method 8), tert-butyl piperazine-1-carboxylate (0.335 g, 1.80 mmol), and _Et3N (0.753 mL, 5.40 mmol) in DCE (9 mL) was stirred at 60 °C for 20 _min . The reaction mixture was diluted with EtOAc (100 mL) and washed with saturated _NaHCO3 aqueous solution (3 × 75 mL). The organic layer was dried over _Na2SO4 and concentrated under vacuum. Chromatographic purification of the residue (silica gel, 0-60% (3:1) EtOAc-EtOH in heptane) yielded 4-(6-chloro-7-(2-fluoro-6-methoxyphenyl)-1-(2-isopropylphenyl)-2-oxo-1,2-dihydroquinazolin-4-yl)piperazine-1-carboxylic acid tert-butyl ester: m/z (ESI,+ve) 607.3 (M+H) ⁺ .

Note: When using (S)-1-(3-methylpiperazin-1-yl)prop-2-en-1-one 2,2,2-trifluoroacetate, it is synthesized as follows:

(S)-1-(3-methylpiperazin-1-yl)prop-2-en-1-one 2,2,2-trifluoroacetate

Step 6-a: (S)-tert-butyl-4-acryloyl-2-methylpiperazine-1-carboxylate. Acryloyl chloride (1.34 mL, 16.5 mmol) was added to a solution of (S)-1-boc-2-methylpiperazine (3.00 g, 15.0 mmol, Boc Sciences, Shirley, NY) in THF (30.0 mL) at -10 °C, and the resulting mixture was stirred at -10 °C for 5 min. Triethylamine (6.26 mL, 44.9 mmol) was then slowly added, and the resulting mixture was stirred at -10 °C for 15 min, and then allowed to warm to room temperature. The reaction mixture was partitioned between EtOAc and a saturated aqueous solution of _NaHCO3 . The aqueous layer was extracted with EtOAc (3×), and the organic layers were combined, dried over _MgSO4 , filtered, and concentrated under vacuum. Chromatographic purification of the residue (silica gel, 0-100% EtOAc in heptane) Supply (S)-tert-butyl-4-acryloyl-2-methylpiperazine-1-carboxylate: ^¹H NMR (400MHz, DMSO- _d⁶ ) δ 6.72–6.85 (m, 1H) 6.10–6.18 (m, 1H) 5.68–5.76 (m, 1H) 4.08–4.32 (m, 2H) 3.68–4.03 (m, 2H) 2.86–3.14 (m, 2H) 2.66–2.80 (m, 1H) 1.38–1.43 (s, 9H) 0.96–1.04 (m, 3H). m/z (ESI, +ve) 277.3 (M+Na) ⁺ .

Step 6-b: (S)-1-(3-methylpiperazin-1-yl)prop-2-en-1-one 2,2,2-trifluoroacetate. A mixture of (S)-tert-butyl-4-acryloyl-2-methylpiperazin-1-carboxylate (3.21 g, 12.62 mmol) and TFA (4.7 mL, 63.1 mmol) in DCM (16 mL) was stirred at room temperature for 24 h. The reaction mixture was then concentrated under vacuum to give (S)-1-(3-methylpiperazin-1-yl)prop-2-en-1-one 2,2,2-trifluoroacetate: ^¹H NMR (400 MHz, DMSO- _d⁶ ) δ 8.70–8.99 (m, 1H) 6.74–6.91 (m, 1H) 6.12–6.26 (m, 1H) 5.70–5.84 (m, 1H) 4.25–4.44 (m, 1H) 4.07–4.25 (m, 1H) 3.49–3.53 (m, 1H) 3.22–3.32 (m, 2H) 2.92–3.08 (m, 2H) 1.14–1.29 (m, 3H). m/z (ESI, +ve) 155.1 (M+H) ⁺ .

Step 7: 6-Chloro-7-(2-fluoro-6-methoxyphenyl)-1-(2-isopropylphenyl)-4-(piperazin-1-yl)quinazolin-2(1H)-one. A solution of 0.594 g (0.978 mmol) of tert-butyl piperazine-1-carboxylate in 4 mL of TFA was stirred at ambient temperature for 30 min. The reaction mixture was concentrated under vacuum to give 6-chloro-7-(2-fluoro-6-methoxyphenyl)-1-(2-isopropylphenyl)-4-(piperazin-1-yl)quinazolin-2(1H)-one: m/z(ESI,+ve)507.2(M+H) ⁺ .

Step 8: 4-(4-Acryloylpiperazin-1-yl)-6-chloro-7-(2-fluoro-6-methoxyphenyl)-1-(2-isopropylphenyl)quinazoline-2(1H)-one. Acryloyl chloride (0.079 mL, 0.98 mmol) was added to an ice-cooled solution of 6-chloro-7-(2-fluoro-6-methoxyphenyl)-1-(2-isopropylphenyl)-4-(piperazin-1-yl)quinazoline-2(1H)-one and DIPEA (0.85 mL, 4.9 mmol) in DCM (10 mL), and the resulting mixture was stirred at 0 °C for 30 min. The reaction mixture was then diluted with EtOAc (100 mL) and washed with saturated _NaHCO3 aqueous solution (3 × 75 mL). The organic layer was dried over _{Na2SO4 ,} decanted, and concentrated under vacuum. Chromatographic purification of the residue (silica gel, 0-100% (3:1) EtOAc-EtOH in heptane) yielded 4-(4-acryloylpiperazin-1-yl)-6-chloro-7-(2-fluoro-6-methoxyphenyl)-1-(2-isopropylphenyl)quinazolin-2(1H)-one: ^¹H NMR (400 MHz, _CDCl₃ ) δ 7.86 (d, J = 1.2 Hz, 1H) 7.41-7.54 (m, 2H) 7.29-7.37 (m, 2H) 7.14 (dt, J = 7.8, 1.7 Hz, 1H) 6.70-6.79 (m, 2H) 6.58-6.68 (m, 1H) 6.50 (d, J = 7.4 Hz, 1H) 6.39 (dd, J＝16.8,1.8Hz,1H)5.75-5.84(m,1H)3.79-4.06(m,8H)3.75(s,2H)3.66(s,1 H) 2.69 (tt, J = 13.4, 6.8 Hz, 1H) 1.20-1.24 (m, 3H) 1.07 (dd, J = 6.8, 3.9 Hz, 3H). ¹⁹ F NMR (377MHz, CDCl ₃ ) δ – 113.05 (s, 1F) – 113.55 (s, 1F). m/z(ESI,+ve)561.2(M+H) ⁺ .

Step 9: 6-Chloro-7-(2-fluoro-6-hydroxyphenyl)-1-(2-(2-propyl)phenyl)-4-(4-(2-acryloyl)-1-piperazinyl)-2(1H)-quinazolinone. BBr ₃ (1M in DCE, 3.3 mL, 3.3 mmol) was added to an ice-cooled solution of 4-(4-acryloylpiperazin-1-yl)-6-chloro-7-(2-fluoro-6-methoxyphenyl)-1-(2-isopropylphenyl)quinazolin-2(1H)-one (0.372 g, 0.663 mmol) in DCE (1.7 mL), and the resulting mixture was stirred at 0 °C for 20 min, then allowed to warm to room temperature and stirred at room temperature for 2 h. A saturated aqueous solution of NaHCO ₃ was added to the reaction mixture, followed by EtOAc (150 mL). The organic layer was separated and washed with a saturated aqueous solution of _NaHCO3 (3 × 100 mL). The organic layer was then dried over _Na2SO4 , filtered, and concentrated under vacuum. Chromatographic purification of the residue (silica gel, 0-100% (3:1) EtOAc-EtOH in heptane) yielded 6-chloro-7-(2-fluoro- ₆ -hydroxyphenyl)-1-(2-(2-propyl)phenyl)-4-(4-(2-acryloyl)-1-piperazinyl)-2(1H)-quinazolinone: ^1H NMR (400 MHz, DMSO-d6 ₎ )δ10.06(br.d.,J＝15.1Hz,1H)8.03(d,J＝1.2Hz,1H)7.51–7.56(m,1H)7.45(t,J＝7.6Hz,1H)7.33(tdd,J＝7.5,7.5,3.8,1.4Hz,1H)7.14–7.25(m ,2H)6.84(dd,J＝16.8,10.4Hz,1H)6.62–6.74(m,2H)6.14–6.26(m,2H)5 .71–5.78(m,1H)3.71–3.99(m,8H)2.52–2.59(m,1H)1.02–1.12(m,6H). ¹⁹ F NMR (377MHz, DMSO-d ₆ ) δ – 113.6 (s, 1F) – 114.8 (s, 1F). m/z(ESI,+ve)547.1(M+H) ⁺ .

Table 8: Compounds 8-2 to 8-6 were prepared following the procedures described in steps 1-9 of Method 8 above:

Method 9

Example 9-1: 6-Chloro-7-(2,3-dichloro-5-hydroxyphenyl)-4-((2S)-2-methyl-4-(2-acryloyl)-1-piperazinyl)-1-(2-(2-propane)phenyl)-2(1H)-quinazolinone

Step 1: 7-Bromo-4,6-dichloro-1-(2-isopropylphenyl)quinazoline-2(1H)-one. Phosphorus oxychloride (0.915 mL, 5.97 mmol) was added to a mixture of 7-bromo-6-chloro-1-(2-isopropylphenyl)quinazoline-2,4(1H,3H)-dione (intermediate F, 470 mg, 1.194 mmol) and DIPEA (0.623 mL, 3.58 mmol) in acetonitrile (11.4 mL). The resulting mixture was heated at 80 °C for 2 h, then cooled to ambient temperature and concentrated under vacuum to give 7-bromo-4,6-dichloro-1-(2-isopropylphenyl)quinazoline-2(1H)-one: m/z(ESI,+ve) 413.0(M+H) ⁺ .

Step 2: (S)-4-(4-Acryloyl-2-methylpiperazin-1-yl)-7-bromo-6-chloro-1-(2-isopropylphenyl)quinazoline-2(1H)-one. A mixture of 7-bromo-4,6-dichloro-1-(2-isopropylphenyl)quinazoline-2(1H)-one (492 mg, 1.19 mmol), (S)-4-N-boc-2-methylpiperazine (478 mg, 2.39 mmol), and DIPEA (0.623 mL, 3.58 mmol) in DMF (2.3 mL) was stirred at room temperature for 10 min. Then, ice water (10 mL) was added, and the resulting mixture was stirred for 15 min. The precipitated solid was collected by filtration, washed with water, and dried under vacuum to give (S)-tert-butyl 4-(7-bromo-6-chloro-1-(2-isopropylphenyl)-2-oxo-1,2-dihydroquinazolin-4-yl)-3-methylpiperazine-1-carboxylate: m/z(ESI,+ve)577.1(M+H) ⁺ .

TFA (2.0 mL, 26.8 mmol) was added to a solution of (S)-tert-butyl-4-(7-bromo-6-chloro-1-(2-isopropylphenyl)-2-oxo-1,2-dihydroquinazoline-4-yl)-3-methylpiperazin-1-carboxylate (297 mg, 0.516 mmol) in DCM (2.0 mL), and the resulting mixture was stirred at room temperature for 15 min. The resulting mixture was concentrated under vacuum to provide (S)-7-bromo-6-chloro-1-(2-isopropylphenyl)-4-(2-methylpiperazin-1-yl)quinazoline-2(1H)-one: m/z(ESI,+ve) 477.0(M+H) ⁺ .

Acryloyl chloride (0.258 M in DCM, 4.0 mL, 1.031 mmol) was added to an ice-cooled mixture of (S)-7-bromo-6-chloro-1-(2-isopropylphenyl)-4-(2-methylpiperazin-1-yl)quinazolin-2(1H)-one and DIPEA (0.269 mL, 1.547 mmol) in DCM (2.0 mL), and the resulting mixture was stirred at 0 °C for 20 min. The residue was concentrated under vacuum and then purified by chromatography (silica gel, 0-100% (3:1) EtOAc-EtOH in heptane) to give (S)-4-(4-acryloyl-2-methylpiperazin-1-yl)-7-bromo-6-chloro-1-(2-isopropylphenyl)quinazolin-2(1H)-one: ^1H NMR (400MHz, DMSO-d6) δ 7.91-8.08 (m, 1H), 7.49-7.67 (m, 2H), 7.41 (br d, J = 5.8Hz, 1H), 7.21 (br s, 1H), 6.76-6.98 (m, 1H), 6.52-6.67 (m, 1H), 6.09-6.29 (m, 1H), 5.75 (br d, J = 5.8Hz ... s,1H),4.61-4.96(m,1H),4.23-4.48(m,1H),3.93-4.21(m,2H),3.50-3.7 7(m,1H),3.33-3.49(m,1H),3.23-3.28(m,1H),2.94-3.24(m,1H),1.27(br d,J＝9.3Hz,6H),1.09(br s,3H). m/z(ESI,+ve)531.1(M+H) ⁺ .

Step 3: (S)-4-(4-acryloyl-2-methylpiperazin-1-yl)-6-chloro-7-(2,3-dichloro-5-methoxyphenyl)-1-(2-isopropylphenyl)quinazolin-2(1H)-one. A mixture of (S)-4-(4-acryloyl-2-methylpiperazin-1-yl)-7-bromo-6-chloro-1-(2-isopropylphenyl)quinazolin-2(1H)-one (120 mg, 0.226 mmol), 2-(2,3-dichloro-5-methoxyphenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborane (82 mg, 0.272 mmol), _Na₂CO₃ (96 _mg , 0.906 mmol), and Pd( _PPh₃ ) _₄ (26.2 mg, 0.023 mmol) in 1,4-dioxane (1.6 mL) and water (0.4 mL) was heated at 90 °C for 17 h. The reaction mixture was then concentrated under vacuum and purified by chromatography (0-100% (3:1) EtOAc-EtOH in silica gel, heptane) to provide (S)-4-(4-acryloyl-2-methylpiperazin-1-yl)-6-chloro-7-(2,3-dichloro-5-methoxyphenyl)-1-(2-isopropylphenyl)quinazolin-2(1H)-one: m/z(ESI,+ve)627.0(M+H) ⁺ .

Step 4: 6-Chloro-7-(2,3-dichloro-5-hydroxyphenyl)-4-((2S)-2-methyl-4-(2-acryloyl)-1-piperazinyl)-1-(2-(2-propane)phenyl)-2(1H)-quinazolinone. BBr ₃ (1M in hexane, 0.32 mL, 0.320 mmol) was added to an ice-cooled mixture of (S)-4-(4-acryloyl-2-methylpiperazin-1-yl)-6-chloro-7-(2,3-dichloro-5-methoxyphenyl)-1-(2-isopropylphenyl)quinazolin-2(1H)-one (40 mg, 0.064 mmol) and DCE (1.0 mL), and the resulting mixture was stirred at 0 °C for 30 min. Add 2.0 mL of saturated _NaHCO3 aqueous solution and extract the resulting mixture with (2:1) DCM/MeOH (5 mL). Dry the organic extract _{in Na2SO4} , filter, and concentrate under vacuum. Chromatographic purification of the residue (silica gel, 0–10% MeOH in DCM) yielded 6-chloro-7-(2,3-dichloro-5-hydroxyphenyl)-4-((2S)-2-methyl-4-(2-acryloyl)-1-piperazinyl)-1-(2-(2-propane)phenyl)-2(1H)-quinazolinone: ^¹H NMR (400 MHz, DMSO-d6) δ 10.42 (br d, J = 17.0 Hz, 1H), 7.86–8.11 (m, 1H), 7.50–7.63 (m, 1H), 7.47 ...47 (br d, J = 17.0 Hz, 1H), 7.86–8.11 (m, 1H), 7.50–7.63 (m, 1H), 7.47 (br d, J = 17 t,J＝6.0Hz,1H),7.36(t,J＝7.5Hz,1H),7.15-7.26(m,1H),7.05(d,J＝2.3 Hz,1H),6.78-6.96(m,1H),6.44-6.58(m,1H),6.11-6.29(m,2H),5.71-5 .82(m,1H),4.68-4.98(m,1H),3.96-4.52(m,3H),3.52-3.85(m,2H),3.3 4-3.51(m,1H),2.95-3.26(m,1H),1.27-1.41(m,3H),0.95-1.13(m,6H). m/z(ESI,+ve)611.0(M+H) ⁺ .

Table 9: Compounds 9-2 to 9-14 were prepared following the procedures described in steps 1-4 of Method 9 above:

Method 10

Example 10-1: 1-(4-(7-chloro-6-(2-fluoro-6-hydroxyphenyl)-4-(2-methylphenyl)-1-phthalazinyl)-1-piperazinyl)-2-propen-1-one.

Step 1: 6,7-Dichloro-2,3-dihydrophthalazine-1,4-dione (Intermediate G). Hydrazine (0.232 mL, 10.1 mmol) was added to a mixture of 5,6-dichloroisobenzofuran-1,3-dione (2.00 g, 9.22 mmol, TCI America, Portland, Oregon, USA) and ethanol (30 mL), and the resulting mixture was heated under reflux for 2 h, followed by cooling to room temperature. The resulting precipitate was collected by filtration and washed with water to give 6,7-dichloro-2,3-dihydrophthalazine-1,4-dione: m/z(ESI,+ve)231.1(M+H) ⁺ .

Step 2: 6-Chloro-7-(2-fluoro-6-hydroxyphenyl)-2,3-dihydrophthalazine-1,4-dione. A mixture of 6,7-dichloro-2,3-dihydrophthalazine-1,4-dione (intermediate G, 3.80 g, 16.45 mmol), 2-fluoro-6-hydroxyphenylboronic acid (10.26 g, 65.8 mmol, Combibloc, San Diego, California, USA), SPhos Pd G3 (1.423 g, 1.645 mmol), and _{2M Na₂CO₃} aqueous solution (32.9 mL, 65.8 mmol) in DME (60 mL) was stirred at 80 °C for 16 h. The reaction mixture was cooled to room temperature and diluted with water (200 mL) and EtOAc (300 mL). The aqueous layer was separated, acidified with 5N HCl, and extracted with EtOAc (300 mL). The combined organic layers were washed with brine (200 mL), dried over _MgSO₄ , filtered, and concentrated under vacuum. The residue was suspended in DCM (50 mL) and collected by filtration to give 6-chloro-7-(2-fluoro-6-hydroxyphenyl)-2,3-dihydrophthalazine-1,4-dione: m/z(ESI,+ve)307.0(M+H) ⁺ .

Step 3: 6-(2-((tert-butyldiphenylsilyl)oxy)-6-fluorophenyl)-7-chloro-2,3-dihydrophthalazine-1,4-dione. Tert-butyl(chloro)diphenylsilane (2.67 mL, 10.25 mmol) was added to an ice-cooled mixture of 6-chloro-7-(2-fluoro-6-hydroxyphenyl)-2,3-dihydrophthalazine-1,4-dione (2.62 g, 8.54 mmol) and TEA (4.75 mL, 34.2 mmol) in acetonitrile (40 mL). The resulting mixture was stirred at 0 °C for 15 min, then heated to room temperature and stirred for 1.5 h. Additional tert-butyl(chloro)diphenylsilane (2.67 mL, 10.25 mmol) was added, and the resulting mixture was stirred at room temperature for 16 h. The reaction mixture was then diluted with water (300 mL), acidified with 5 N HCl, and extracted with EtOAc (300 mL). The organic layers were separated and washed sequentially with brine (250 mL), dried over _MgSO4 , filtered, and concentrated under vacuum. The residue was absorbed into DCM (200 mL), TFA (20 mL) was added, and the resulting mixture was stirred at room temperature for 45 min. The reaction mixture was then diluted with a saturated aqueous solution of _NaHCO3 (200 mL) and extracted with DCM (2 × 250 mL). The combined organic extracts were dried over _MgSO4 , filtered, and concentrated under vacuum to give 6-(2-((tert-butyldiphenylsilyl)oxy)-6-fluorophenyl)-7-chloro-2,3-dihydrophthalazine-1,4-dione: m/z(ESI,+ve)545.2(M+H) ⁺ .

Step 4: 6-(2-((tert-butyldiphenylsilyl)oxy)-6-fluorophenyl)-1,4,7-trichlorophthalazine. Pyridine (1.45 mL, 17.1 mmol) was added to a mixture of 6-(2-((tert-butyldiphenylsilyl)oxy)-6-fluorophenyl)-7-chloro-2,3-dihydrophthalazine-1,4-dione (4.66 g, 8.55 mmol) and phosphorus oxychloride (6.39 mL, 68.4 mmol), and the resulting mixture was heated at 100 °C for 1.5 h. The reaction mixture was then cooled to room temperature and slowly poured into stirred water (300 mL) while maintaining an internal temperature <10 °C. After stirring for 15 min, the resulting mixture was extracted with EtOAc (400 mL), and the organic extract was washed sequentially with brine (250 mL), dried over _MgSO4 , filtered, and concentrated under vacuum. Chromatographic purification of the residue (silica gel, 0-25% EtOAc in heptane) yielded 6-(2-((tert-butyldiphenylsilyl)oxy)-6-fluorophenyl)-1,4,7-trichlorophthalazine: m/z(ESI,+ve)581.1(M+H) ⁺ .

Step 5: 4-(6-(2-((tert-butyldiphenylsilyl)oxy)-6-fluorophenyl)-4,7-dichlorophthalazine-1-yl)piperazine-1-carboxylic acid tert-butyl ester (intermediate H). 1-Boc-piperazine (5.00 g, 26.9 mmol) was added to a mixture of 6-(2-((tert-butyldiphenylsilyl)oxy)-6-fluorophenyl)-1,4,7-trichlorophthalazine (5.21 g, 8.95 mmol) and triethylamine (3.77 mL, 26.9 mmol) in DCM (35 mL), and the resulting mixture was stirred at room temperature for 19 h. The reaction mixture was then partitioned between DCM (300 mL) and a saturated aqueous solution of _NaHCO3 (200 mL). The organic layer was separated, dried over _MgSO4 , filtered, and concentrated under vacuum. Chromatographic purification of the residue (silica gel, 0-50% EtOAc in heptane) yielded a mixture of tert-butyl 4-(6-(2-((tert-butyldiphenylsilyl)oxy)-6-fluorophenyl)-4,7-dichlorophthalazin-1-yl)piperazin-1-carboxylate and tert-butyl 4-(7-(2-((tert-butyldiphenylsilyl)oxy)-6-fluorophenyl)-4,6-dichlorophthalazin-1-yl)piperazin-1-carboxylate. Individual positional isomers were purified by chiral SFC (OJ-H column (30×250 mm, 5 μm), 15% (20 mM _NH3 in MeOH) in supercritical _CO2 ), providing 4-(6-(2-((tert-butyldiphenylsilyl)oxy)-6-fluorophenyl)-4,7-dichlorophthalazine-1-yl)piperazine-1-carboxylic acid tert-butyl ester as the second eluting isomer: ^¹H NMR (400 MHz, CDCl3 _). )δ8.27(s,1H)8.17(s,1H)7.56-7.61(m,4H)7.40-7.46(m,2H)7.31-7.37(m,4H)6.99-7.07(m,1H)6.7 7(t,J=8.61Hz,1H)6.42(d,J=8.22Hz,1H)3.72-3.77(m,4H)3.53-3.59(m,4H)1.51(s,9H)0.66(s,9H). m/z(ESI,+ve)731.2(M+H) ⁺ .

Step 6: 6-(2-((tert-butyldiphenylsilyl)oxy)-6-fluorophenyl)-4,7-dichloro-1-(piperazin-1-yl)phthalazine. Trifluoroacetic acid (2 mL, 26.8 mmol) was added to a stirred solution of 4-(6-(2-((tert-butyldiphenylsilyl)oxy)-6-fluorophenyl)-4,7-dichlorophthalazin-1-yl)piperazine-1-carboxylic acid tert-butyl ester (intermediate H, 1.21 g, 1.654 mmol) in DCM (10 mL), and the resulting mixture was stirred at room temperature for 1.5 h. The reaction mixture was then diluted with saturated _NaHCO3 aqueous solution (75 mL) and extracted with DCM (2 × 100 mL). The combined organic extracts were dried over _MgSO4 , filtered, and concentrated under vacuum to give 6-(2-((tert-butyldiphenylsilyl)oxy)-6-fluorophenyl)-4,7-dichloro-1-(piperazin-1-yl)phthalazine: m/z(ESI,+ve)631.3(M+H) ⁺ .

Step 7: 1-(4-(6-(2-((tert-butyldiphenylsilyl)oxy)-6-fluorophenyl)-4,7-dichlorophthalazin-1-yl)piperazin-1-yl)prop-2-en-1-one. Acryloyl chloride (0.148 mL, 1.81 mmol) was added to a mixture of 6-(2-((tert-butyldiphenylsilyl)oxy)-6-fluorophenyl)-4,7-dichloro-1-(piperazin-1-yl)phthalazine (1.04 g, 1.647 mmol) and triethylamine (0.694 mL, 4.94 mmol) in DCM (10 mL), and the resulting mixture was stirred at room temperature for 45 min. Saturated _NaHCO3 aqueous solution (75 mL) was added, and the resulting mixture was extracted with DCM (3 × 100 mL). The combined organic extracts were dried over _MgSO4 , filtered, and concentrated under vacuum to give 1-(4-(6-(2-((tert-butyldiphenylsilyl)oxy)-6-fluorophenyl)-4,7-dichlorophthalazin-1-yl)piperazin-1-yl)prop-2-en-1-one: m/z(ESI,+ve)685.1(M+H) ⁺ .

Step 8: 1-(4-(4,7-dichloro-6-(2-fluoro-6-hydroxyphenyl)phthalazin-1-yl)piperazin-1-yl)prop-2-en-1-one (intermediate I). TBAF (1M in THF, 3.3 mL, 3.30 mmol) was added to a solution of 1-(4-(6-(2-((tert-butyldiphenylsilyl)oxy)-6-fluorophenyl)-4,7-dichlorophthalazin-1-yl)piperazin-1-yl)prop-2-en-1-one (1.13 g, 1.648 mmol) in THF (10 mL), and the resulting mixture was stirred at room temperature for 15 min. The reaction mixture was concentrated under vacuum, and the residue was purified by column chromatography (silica gel, 0-100% EtOAc in heptane) to give 1-(4-(4,7-dichloro-6-(2-fluoro-6-hydroxyphenyl)phthalazin-1-yl)piperazin-1-yl)prop-2-en-1-one: ^¹H NMR (400MHz, DMSO-d6) δ 10.26 (br s, 1H) 8.31 (s, 1H) 8.14 (s, 1H) 7.31-7.40 (m, 1H) 6.78-6.92 (m, 3H) 6.17 (dd, J = 16.63, 2.35Hz, 1H) 5.74 (dd, J = 10.37, 2.35Hz, 1H) 3.79-3.92 (m, 4H) 3.46-3.55 (m, 4H). m/z(ESI,+ve)447.0(M+H) ⁺ .

Step 9: 1-(4-(7-chloro-6-(2-fluoro-6-hydroxyphenyl)-4-(o-tolyl)phthalazin-1-yl)piperazin-1-yl)prop-2-en-1-one. A mixture of 1-(4-(4,7-dichloro-6-(2-fluoro-6-hydroxyphenyl)phthalazin-1-yl)piperazin-1-yl)prop-2-en-1-one (intermediate I, 25 mg, 0.056 mmol), 2-tolylboronic acid (30.4 mg, 0.224 mmol, Frontier Sciences, Logan, Utah, USA), Pd( _PPh3 ) ₄ (6.46 mg, 5.59 μmol, Strem Chemicals Inc., NewburyPort, Massachusetts, USA) and 2M _Na2CO3 aqueous solution (0.084 _mL , 0.168 mmol) in 1,4-dioxane (0.3 mL) was stirred at 40 °C for 18 h. The reaction mixture was then diluted with EtOAc (20 mL) and washed with water (15 mL). The organic layer was separated and washed sequentially with brine (15 mL), dried over _MgSO₄ , filtered, and concentrated under vacuum. Chromatographic purification of the residue (silica gel, 0-100% EtOAc in heptane) was performed with 1-(4-(7-chloro-6-(2-fluoro-6-hydroxyphenyl)-4-(o-tolyl)phthalazin-1-yl)piperazin-1-yl)prop-2-en-1-one: ^¹H NMR (400 MHz, DMSO- _d₆ ) δ 10.15 (br) s,1H)8.33(s,1H)7.36-7.45(m,2H)7.24-7.36(m,4H)6.90(dd,J＝16.63,10.37Hz,1H)6.70-6.80(m,2H)6.18( dd,J＝16.73,2.25Hz,1H)5.75(dd,J＝10.56,2.15Hz,1H)3.83-3.97(m,4H)3.47-3.62(m,4H)1.98-2.06(m,3H). m/z(ESI,+ve)503.1(M+H) ⁺ .

Table 10: Compounds 10⁻² to 10⁻¹³ were prepared following the procedures described in steps 1-9 of Method 10 above:

Method 11

Example 11-1: 6-Chloro-7-(5-methyl-1H-indazol-4-yl)-1-(2-(2-propyl)phenyl)-4-(4-(2-acryloyl)-1-piperazinyl)-2(1H)-quinazolinone

Step 1: 4-(1H-benzo[d][1,2,3]triazol-1-yl)-7-bromo-6-chloro-1-(2-isopropylphenyl)quinazoline-2(1H)-one. Phosphorus oxychloride (1.204 mL, 7.85 mmol) was added to a stirred mixture of 7-bromo-6-chloro-1-(2-isopropylphenyl)quinazoline-2,4(1H,3H)-dione (intermediate F, 515 mg, 1.308 mmol), triethylamine (3.31 mL, 23.55 mmol), and 1H-benzo[d][1,2,3]triazole (2.01 g, 16.87 mmol) in acetonitrile (15 mL). The reaction mixture was heated to 80 °C and stirred for 1 h. The reaction mixture was cooled to room temperature and filtered. The filtrate was then slowly poured into rapidly stirred water (150 mL) at approximately 10 °C. The aqueous suspension was stirred for 15 min, then extracted twice with EtOAc (150 mL). The organic layers were combined, washed with brine (150 mL), dried over _MgSO4 , filtered, and concentrated under vacuum to give crude 4-(1H-benzo[d][1,2,3]triazol-1-yl)-7-bromo-6-chloro-1-(2-isopropylphenyl)quinazolin-2(1H)-one. m/z (ESI) M+H: 494.0.

Step 2: 4-(7-bromo-6-chloro-1-(2-isopropylphenyl)-2-oxo-1,2-dihydroquinazolin-4-yl)piperazine-1-carboxylate tert-butyl ester. Piperazine-1-carboxylate tert-butyl ester (268 mg, 1.438 mmol) was added to a stirred mixture of crude 4-(1H-benzo[d][1,2,3]triazol-1-yl)-7-bromo-6-chloro-1-(2-isopropylphenyl)quinazolin-2(1H)-one (647 mg, 1.308 mmol) and triethylamine (3.68 mL, 26.2 mmol) in dimethyl sulfoxide (6 mL). The reaction mixture was stirred at 80 °C for 30 min. The reaction mixture was diluted with EtOAc (100 mL) and washed with water (75 mL). The organic layer was separated, washed with brine (75 mL), dried over _MgSO4 , filtered, and concentrated under vacuum. Chromatographic purification of the residue (silica gel, 0 to 100% EtOAc in heptane) yielded tert-butyl 4-(7-bromo-6-chloro-1-(2-isopropylphenyl)-2-oxo-1,2-dihydroquinazolin-4-yl)piperazine-1-carboxylate. ¹ H NMR(400MHz, chloroform-d)δ7.79(1H,s)7.49-7.59(2H,m)7.36-7.42(1H,m)7.11(1H,d,J＝7.63Hz)6.80(1H,s)3.79-3.92 (4H,m)3.62-3.73(4H,m)2.60(1H,spt,J＝6.80Hz)1.49-1.54(9H,m)1.22(3H,d,J＝6.85Hz)1.08(3H,d,J＝6.85Hz). m/z(ESI)M+H:561.0.

Step 3: 4-(6-chloro-1-(2-isopropylphenyl)-7-(5-methyl-1H-indazol-4-yl)-2-oxo-1,2-dihydroquinazolin-4-yl)piperazine-1-carboxylic acid tert-butyl ester. 4-(7-bromo-6-chloro-1-(2-isopropylphenyl)-2-oxo-1,2-dihydroquinazolin-4-yl)piperazine-1-carboxylic acid tert-butyl ester (115 mg, 0.205 mmol), 4-boronyl-5-methyl-1h-indazole (0.144 mL, 0.819 mmol, Ark Pharmaceuticals, Arlington Heights, .I., USA), Sphos Pd G3 (0.016 mL, 0.020 mmol), and sodium carbonate (2 M aqueous solution, 0.409 mL, 0.819 mmol) were mixed in a sealed vial under argon atmosphere in 1,2-dimethoxyethane (1 mL). The reaction mixture was stirred at 100 °C for 24 h. The reaction mixture was cooled to room temperature and diluted with EtOAc (50 mL) and water (40 mL). The organic layer was separated, washed with brine (40 mL), dried over _MgSO4 , filtered, and concentrated under vacuum. Chromatographic purification of the residue (silica gel, 0 to 50% (3:1 EtOAc/EtOH) in heptane) yielded tert-butyl piperazine-1-carboxylate. m/z (ESI) M+H: 613.2.

Step 4: 6-Chloro-1-(2-isopropylphenyl)-7-(5-methyl-1H-indazol-4-yl)-4-(piperazin-1-yl)quinazoline-2(1H)-one. Trifluoroacetic acid (0.5 mL, 6.71 mmol) was added to a stirred mixture of 4-(6-chloro-1-(2-isopropylphenyl)-7-(5-methyl-1H-indazol-4-yl)-2-oxo-1,2-dihydroquinazoline-4-yl)piperazin-1-carboxylic acid tert-butyl ester (78 mg, 0.127 mmol) in dichloromethane (1 mL). The reaction mixture was stirred at room temperature for 1 h. The reaction mixture was concentrated under vacuum to give crude 6-chloro-1-(2-isopropylphenyl)-7-(5-methyl-1H-indazol-4-yl)-4-(piperazin-1-yl)quinazoline-2(1H)-one. m/z(ESI)M+H:513.2.

Step 5: 6-Chloro-7-(5-methyl-1H-indazol-4-yl)-1-(2-(2-propyl)phenyl)-4-(4-(2-acryloyl)-1-piperazinyl)-2(1H)-quinazolinone. Acryloyl chloride (10.33 μl, 0.127 mmol) was added to a stirred mixture of 6-chloro-1-(2-isopropylphenyl)-7-(5-methyl-1H-indazol-4-yl)-4-(piperazin-1-yl)quinazolin-2(1H)-one (65 mg, 0.127 mmol) and triethylamine (0.178 mL, 1.267 mmol) in dichloromethane (2 mL). The reaction mixture was stirred at 0 °C for 20 min. Additional acryloyl chloride (5.17 μl, 0.064 mmol) was added, and the reaction mixture was stirred again at 0 °C for 20 min. The reaction mixture was diluted with DCM (25 mL) and quenched with a saturated sodium bicarbonate aqueous solution (20 mL). The organic layer was separated, dried over _MgSO4 , filtered, and concentrated under vacuum. Chromatographic purification of the residue (silica gel, 0 to 80% (3:1 EtOAc/EtOH) in heptane) yielded an impure product. Further chromatographic purification of the impure product (silica gel, 0 to 100% acetone in heptane) yielded the separated diastereomers. 6-Chloro-7-(5-methyl-1H-indazol-4-yl)-1-(2-(2-propyl)phenyl)-4-(4-(2-acryloyl)-1-piperazinyl)-2(1H)-quinazolinone (Example 11-1-1) was the first diastereomer to be eluted. ^¹H NMR (400 MHz, chloroform-d) δ 10.28 (¹H, br s)7.94(1H,s)7.35-7.49(4H,m)7.25-7.31(2H,m)7.11(1H,d,J＝7.67Hz)6.64(1H,dd,J＝16.79,10.57Hz)6.54(1H,s)6.41(1H,dd,J＝16.79, 1.87Hz) 5.81 (1H, dd, J = 10.57, 1.66Hz) 3.83-4.07 (8H, m) 2.74 (1H, spt, J = 6.84Hz) 2.13 (3H, s) 1.23 (3H, d, J = 6.84Hz) 1.04 (3H, d, J = 6.84Hz). m/z(ESI)M+H:567.2. The second diastereomer to be eluted was further purified by column chromatography (silica gel, 0 to 80% (3:1 EtOAc/EtOH) in heptane) to give 6-chloro-7-(5-methyl-1H-indazol-4-yl)-1-(2-(2-propyl)phenyl)-4-(4-(2-acryloyl)-1-piperazinyl)-2(1H)-quinazolinone (Example 11-1-2). ^¹H NMR (400 MHz, chloroform-d) δ 10.37 (¹H, br s)7.94(1H,s)7.34-7.50(4H,m)7.21-7.31(2H,m)7.13(1H,d,J＝7.67Hz)6.64(1H,dd,J＝16.90,10.68Hz)6.55(1H,s)6.41(1H,dd,J＝16.79, 1.66Hz)5.81(1H,dd,J＝10.47,1.55Hz)3.83-4.08(8H,m)2.70(1H,spt,J＝6.84Hz)2.13(3H,s)1.22(3H,d,J＝6.84Hz)1.03(3H,d,J＝6.84Hz). m/z(ESI)M+H:567.2.

Follow steps 1-5 of method 11 above:

Part 2 - Individual Cases

Example 121-(4-(7-chloro-4-cyclopropyl-6-(2-fluoro-6-hydroxyphenyl)-1-phthalazinyl)-1-piperazinyl)-2-propen-1-one

Step 1: 4-(6-(2-((tert-butyldiphenylsilyl)oxy)-6-fluorophenyl)-7-chloro-4-cyclopropylphthalazin-1-yl)piperazin-1-carboxylate tert-butyl ester. Add the complex of [1,1'-bis(diphenylphosphino)ferrocene]palladium(II) dichloride with dichloromethane (0.033 g, 0.041 mmol) and 2-methyltetrahydrofuran (2.0 mL) to a 20 mL vial containing 4-(6-(2-((tert-butyldiphenylsilyl)oxy)-6-fluorophenyl)-4,7-dichlorophthalazin-1-yl)piperazin-1-carboxylate (intermediate H, 0.060 g, 0.082 mmol). The resulting mixture was capped and stirred at room temperature for 10 min, then cyclopropyl zinc bromide (0.5 M in THF, 0.820 mL, 0.410 mmol; Rieke Metals, Lincoln, Nebraska, USA) was added via syringe. The reaction mixture was heated at 80 °C for 3 h, then cooled to room temperature and partitioned between EtOAc (30 mL) and water (10 mL). The aqueous layer was extracted once more with EtOAc (20 mL). The combined organic layers were dried over _MgSO4 , filtered, and concentrated under vacuum. The crude product was purified by column chromatography (24 g silica gel, 0 to 30% acetone in heptane) to obtain tert-butyl 4-(6-(2-((tert-butyldiphenylsilyl)oxy)-6-fluorophenyl)-7-chloro-4-cyclopropylphthalazin-1-yl)piperazin-1-carboxylate. ¹ H NMR(chloroform-d)δ:8.31-8.38(m,1H),8.15-8.23(m,1H),7.55-7.64(m,4H),7.39-7. 47(m,2H),7.29-7.38(m,4H),6.99-7.09(m,1H),6.74-6.85(m,1H),6.36-6.47( m,1H),3.68-3.79(m,4H),3.37-3.51(m,4H),2.37-2.48(m,1H),1.48-1.54(m,9 H),1.37-1.45(m,1H),1.30-1.33(m,1H),1.00-1.15(m,2H),0.61-0.71(m,9H). m/z(ESI)M+H:737.4.

Step 2: 6-(2-((tert-butyldiphenylsilyl)oxy)-6-fluorophenyl)-7-chloro-4-cyclopropyl-1-(piperazin-1-yl)phthalazine. Trifluoroacetic acid (0.316 mL, 4.10 mmol) was added to a solution of tert-butyl piperazine-1-carboxylate in DCM (0.7 mL). The resulting mixture was capped and stirred at room temperature for 30 min. The reaction mixture was diluted with DCM (10 mL) and alkalized with a saturated aqueous solution of _NaHCO3 (5 mL). The aqueous layer was extracted once more with DCM (10 mL). The combined organic layers were dried with _MgSO4 , filtered, and concentrated under vacuum to obtain 6-(2-((tert-butyldiphenylsilyl)oxy)-6-fluorophenyl)-7-chloro-4-cyclopropyl-1-(piperazin-1-yl)phthalazine. ¹ H NMR(chloroform-d)δ:8.30-8.36(m,1H),8.18-8.24(m,1H),7.55-7.64(m,4H),7.40 -7.46(m,2H),7.33(q,J＝7.1Hz,4H),6.97-7.09(m,1H),6.74-6.83(m,1H),6 .36-6.46(m,1H),3.45-3.55(m,4H),3.16-3.26(m,4H),2.35-2.49(m,1H), 1.37-1.46(m,1H),1.30-1.33(m,1H),1.06-1.12(m,2H),0.61-0.70(m,9H). m/z(ESI)M+H:637.2.

Step 3: 1-(4-(6-(2-((tert-butyldiphenylsilyl)oxy)-6-fluorophenyl)-7-chloro-4-cyclopropylphthalazin-1-yl)piperazin-1-yl)prop-2-en-1-one. Triethylamine (16 μl, 0.114 mmol) and dichloromethane (1.0 mL) were added to a 20 mL vial containing 6-(2-((tert-butyldiphenylsilyl)oxy)-6-fluorophenyl)-7-chloro-4-cyclopropyl-1-(piperazin-1-yl)phthalazine (0.023 g, 0.036 mmol). The resulting mixture was capped and stirred at room temperature for 10 min, after which acryloyl chloride (4.0 μl, 0.049 mmol) was added via syringe. The reaction mixture was capped and stirred at room temperature for another 20 min. The reaction was quenched with saturated _NaHCO3 aqueous solution (3 mL) and diluted with DCM (10 mL). The aqueous layer was extracted once more with DCM (5 mL). The combined organic layers were dried over _MgSO₄ , filtered, and concentrated under vacuum to obtain 1-(4-(6-(2-((tert-butyldiphenylsilyl)oxy)-6-fluorophenyl)-7-chloro-4-cyclopropylphthalazin-1-yl)piperazin-1-yl)propyl-2-en-1-one. ^¹H NMR (chloroform-d) δ: 8.32–8.38 (m, 1H), 8.16–8.24 (m, 1H), 7.55–7.65 (m, 4H), 7.40–7.48 (m, 2H), 7.31–7.38 (m, 4H), 6.98–7.10 (m, 1H), 6.75–6.84 (m, 1H), 6.60–6.72 (m, 1H), 6.41–6.47 (m, 1H). m,1H),6.31-6.40(m,1H),5.72-5.82(m,1H),3.79-4.08(m,4H),3.44-3.62(m,4H),2.38- 2.49(m,1H),1.40-1.45(m,1H),1.33-1.37(m,1H),1.04-1.13(m,2H),0.62-0.68(m,9H). m/z(ESI)M+H:691.2.

Step 4: 1-(4-(7-chloro-4-cyclopropyl-6-(2-fluoro-6-hydroxyphenyl)-1-phthalazinyl)-1-piperazinyl)-2-propen-1-one. Add 2.0 mL of tetrahydrofuran to a 20 mL vial containing 0.022 g (0.032 mmol) of 1-(4-(6-(2-((tert-butyldiphenylsilyl)oxy)-6-fluorophenyl)-7-chloro-4-cyclopropylphthalazin-1-yl)piperazin-1-yl)propen-2-en-1-one (2.0 mL), followed by tetrabutylammonium fluoride (1.0 M THF solution, 0.070 mL, 0.070 mmol). Cap the vial and stir at room temperature for 30 min. Concentrate the reaction mixture under vacuum. The crude product was purified by column chromatography (24 g silica, 0 to 5% MeOH in DCM) to obtain 1-(4-(7-chloro-4-cyclopropyl-6-(2-fluoro-6-hydroxyphenyl)-1-phthalazinyl)-1-piperazinyl)-2-propen-1-one. ¹ H NMR(chloroform-d)δ:8.30-8.37(m,1H),8.11-8.18(m,1H),7.29-7.38(m,1H),6.9 6-7.18(m,1H),6.88-6.94(m,1H),6.76-6.85(m,1H),6.59-6.72(m,1H),6. 31-6.42(m,1H),5.73-5.84(m,1H),3.73-4.05(m,4H),3.35-3.62(m,4H),2 .40-2.52(m,1H),1.35-1.42(m,1H),1.29-1.34(m,1H),1.03-1.14(m,2H). m/z(ESI)M+H:453.2.

Example 13

1-(4-(4-anilino-7-chloro-6-(2-fluoro-6-hydroxyphenyl)-1-phthalazinyl)-1-piperazinyl)-2-propen-1-one

Step 1: 4-(6-(2-((tert-butyldiphenylsilyl)oxy)-6-fluorophenyl)-7-chloro-4-(phenylamino)phthalazin-1-yl)piperazin-1-carboxylate tert-butyl ester. Dimethyl sulfoxide (2.0 mL) was added to a 20 mL vial containing 4-(6-(2-((tert-butyldiphenylsilyl)oxy)-6-fluorophenyl)-4,7-dichlorophthalazin-1-yl)piperazin-1-carboxylate (0.060 g, 0.082 mmol), followed by aniline (0.075 mL, 0.820 mmol). The vial was capped and refluxed at 80 °C for 3 h. The reaction was cooled to room temperature and partitioned between EtOAc (30 mL) and water (10 mL). The organic layer was separated and washed with water (2 × 10 mL). The organic layer was dried over _MgSO₄ , filtered, and concentrated under vacuum. The crude product was purified by column chromatography (40 g silica, 0 to 30% EtOAc in heptane) to obtain tert-butyl 4-(6-(2-((tert-butyldiphenylsilyl)oxy)-6-fluorophenyl)-7-chloro-4-(phenylamino)phthalazin-1-yl)piperazine-1-carboxylate. ¹ H NMR(chloroform-d)δ:8.17-8.25(m,1H),7.76-7.81(m,1H),7.60-7.69(m,5H),7.50-7.55(m,2H),7.40-7.46(m,2H),7.31-7.37(m,5H),7.06-7.11( m,2H),6.76-6.83(m,1H),6.57-6.66(m,1H),6.39-6.50(m,1H),3.66- 3.81(m,4H),3.32-3.43(m,4H),1.51-1.53(m,9H),0.69-0.75(m,9H). m/z(ESI)M+H:788.2.

Step 2: 7-(2-((tert-butyldiphenylsilyl)oxy)-6-fluorophenyl)-6-chloro-N-phenyl-4-(piperazin-1-yl)phthalazin-1-amine. Similar to Step 2 in Example 12, the reaction of tert-butyl 4-(6-(2-((tert-butyldiphenylsilyl)oxy)-6-fluorophenyl)-7-chloro-4-(phenylamino)phthalazin-1-yl)piperazin-1-carboxylate produces 7-(2-((tert-butyldiphenylsilyl)oxy)-6-fluorophenyl)-6-chloro-N-phenyl-4-(piperazin-1-yl)phthalazin-1-amine. ¹ H NMR(chloroform-d)δ:8.19-8.26(m,1H),7.75-7.80(m,1H),7.60-7.68(m,5H),7.49-7.55(m,2H),7.39-7.46(m,3H),7.32-7.37(m,5H),7. 02-7.11(m,2H),6.75-6.84(m,1H),6.59-6.67(m,1H),6.43-6.53(m,1H),3.35-3.47(m,4H),3.16-3.27(m,4H),0.70-0.76(m,9H). m/z(ESI)M+H:688.2.

Step 3: 1-(4-(6-(2-((tert-butyldiphenylsilyl)oxy)-6-fluorophenyl)-7-chloro-4-(phenylamino)phthalazin-1-yl)piperazin-1-yl)prop-2-en-1-one. Similar to Step 3 in Example 12, the reaction of 7-(2-((tert-butyldiphenylsilyl)oxy)-6-fluorophenyl)-6-chloro-N-phenyl-4-(piperazin-1-yl)phthalazin-1-amine produces 1-(4-(6-(2-((tert-butyldiphenylsilyl)oxy)-6-fluorophenyl)-7-chloro-4-(phenylamino)phthalazin-1-yl)piperazin-1-yl)prop-2-en-1-one. ¹ H NMR(chloroform-d)δ:8.16-8.24(m,1H),7.77-7.84(m,1H),7.62-7.67(m,4H),7.5 2-7.55(m,1H),7.41-7.46(m,3H),7.32-7.38(m,6H),7.02-7.11(m,2H),6. 77-6.84(m,1H),6.65-6.71(m,1H),6.46-6.51(m,1H),6.30-6.39(m,2H),5 .73-5.81(m,1H),3.86-4.05(m,4H),3.37-3.53(m,4H),0.69-0.75(m,9H). m/z(ESI)M+H:742.3.

Step 4: 1-(4-(4-anilino-7-chloro-6-(2-fluoro-6-hydroxyphenyl)-1-phthalazinyl)-1-piperazinyl)-2-propen-1-one. Similar to Step 4 in Example 12, the reaction of 1-(4-(6-(2-((tert-butyldiphenylsilyl)oxy)-6-fluorophenyl)-7-chloro-4-(phenylamino)phthalazin-1-yl)piperazin-1-yl)propen-2-one yields 1-(4-(4-anilino-7-chloro-6-(2-fluoro-6-hydroxyphenyl)-1-phthalazinyl)-1-piperazinyl)-2-propen-1-one. ¹ H NMR(chloroform-d)δ:7.96-8.09(m,2H),7.46-7.57(m,2H),7.37-7.44(m,1H),7.29-7.33(m,1H),7.20-7.26(m,1H),6.96-7.07(m,1H),6. 81-6.87(m,1H),6.70-6.77(m,1H),6.54-6.67(m,1H),6.29-6.41(m,1H),5.68-5.82(m,1H),3.74-3.96(m,4H),3.12-3.43(m,4H). m/z(ESI)M+H:504.2.

Example 14

1-(4-(7-chloro-4-cyclopentyl-6-(2-fluoro-6-hydroxyphenyl)-1-phthalazinyl)-1-piperazinyl)-2-propen-1-one

Step 1: 4-(6-(2-((tert-butyldiphenylsilyl)oxy)-6-fluorophenyl)-7-chloro-4-cyclopentylphthalazin-1-yl)piperazin-1-carboxylate tert-butyl ester. Similar to Step 1 in Example 12, the reaction of 4-(6-(2-((tert-butyldiphenylsilyl)oxy)-6-fluorophenyl)-4,7-dichlorophthalazin-1-yl)piperazin-1-carboxylate tert-butyl ester (intermediate H) with cyclopentyl zinc bromide (0.5 M in THF, Regimetal, Lincoln, Nebraska) produces 4-(6-(2-((tert-butyldiphenylsilyl)oxy)-6-fluorophenyl)-7-chloro-4-cyclopentylphthalazin-1-yl)piperazin-1-carboxylate tert-butyl ester. ^¹H NMR (chloroform-d) δ: 8.18–8.22 (m, ¹H), 8.12–8.16 (m, ¹H), 7.60–7.66 (m, ²H), 7.50–7.56 (m, ²H), 7.39–7.47 (m, ²H), 7.34–7.38 (m, ²H), 7.28–7.33 (m, ²H), 7.09 (br) d,J＝1.2Hz,1H),6.75-6.82(m,1H),6.37-6.44(m,1H),3.72-3.78(m,4H),3.44-3.51(m,4H),2 .03-2.23(m,4H),1.87-1.96(m,2H),1.67-1.79(m,3H),1.51-1.54(m,9H),0.62-0.67(m,9H). m/z(ESI)M+H:765.2.

Step 2: The reaction of 6-(2-((tert-butyldiphenylsilyl)oxy)-6-fluorophenyl)-7-chloro-4-cyclopentyl-1-(piperazin-1-yl)phthalazine. Similar to Step 2 in Example 12, the reaction of 4-(6-(2-((tert-butyldiphenylsilyl)oxy)-6-fluorophenyl)-7-chloro-4-cyclopentylphthalazin-1-yl)piperazin-1-carboxylic acid tert-butyl ester produces 6-(2-((tert-butyldiphenylsilyl)oxy)-6-fluorophenyl)-7-chloro-4-cyclopentyl-1-(piperazin-1-yl)phthalazine. ¹ H NMR (chloroform-d) δ: 8.17-8.21 (m, 1H), 8.12-8.16 (m, 1H), 7.61-7.66 (m, 2H), 7.51-7.56 (m, 2H), 7.40-7.46 (m, 2H), 7.34-7.38 (m, 2H), 7.29-7.33 (m, 2H), 6.99-7.08 (m, 1H), 6. 74-6.82(m,1H),6.37-6.45(m,1H),3.58-3.67(m,4H),3.27-3.36(m,4H),2.18-2.22 (m,1H),2.08-2.12(m,2H),1.86-1.91(m,3H),1.69-1.77(m,3H),0.59-0.67(m,9H). m/z(ESI)M+H:665.2.

Step 3: 1-(4-(6-(2-((tert-butyldiphenylsilyl)oxy)-6-fluorophenyl)-7-chloro-4-cyclopentylphthalazin-1-yl)piperazin-1-yl)prop-2-en-1-one. Similar to Step 3 in Example 12, the reaction of 6-(2-((tert-butyldiphenylsilyl)oxy)-6-fluorophenyl)-7-chloro-4-cyclopentyl-1-(piperazin-1-yl)phthalazine yields 1-(4-(6-(2-((tert-butyldiphenylsilyl)oxy)-6-fluorophenyl)-7-chloro-4-cyclopentylphthalazin-1-yl)piperazin-1-yl)prop-2-en-1-one. ¹ H NMR (chloroform-d) δ: 8.18-8.25 (m, 1H), 8.13-8.17 (m, 1H), 7.61-7.67 (m, 2H), 7.50-7.57 (m, 2H), 7.39-7.48 (m, 2H), 7.28-7.37 (m, 4H), 6.99-7.10 (m, 1H), 6.75-6.83 (m, 1H), 6. 62-6.71(m,1H),6.33-6.43(m,2H),5.73-5.81(m,1H),3.84-4.07(m,4H),3.71-3.82 (m,1H),3.49-3.65(m,4H),1.80-1.96(m,4H),1.67-1.77(m,4H),0.62-0.67(m,9H). m/z(ESI)M+H:719.2.

Step 4: 1-(4-(7-chloro-4-cyclopentyl-6-(2-fluoro-6-hydroxyphenyl)-1-phthalazinyl)-1-piperazinyl)-2-propen-1-one. Similar to Step 4 in Example 12, the reaction of 1-(4-(6-(2-((tert-butyldiphenylsilyl)oxy)-6-fluorophenyl)-7-chloro-4-cyclopentylphthalazin-1-yl)piperazin-1-yl)propen-2-one yields 1-(4-(7-chloro-4-cyclopentyl-6-(2-fluoro-6-hydroxyphenyl)-1-phthalazinyl)-1-piperazinyl)-2-propen-1-one. ¹ H NMR(chloroform-d)δ:8.10-8.22(m,2H),7.29-7.38(m,1H),6.86-6.93(m,1H),6.77-6.85(m,1H),6.61-6.72(m,1H),6.33-6.44(m,1H),5. 74-5.85(m,1H),3.82-4.05(m,4H),3.75-3.82(m,1H),3.40-3.63(m,4H),2.06-2.24(m,4H),1.81-1.96(m,2H),1.67-1.79(m,2H). m/z(ESI)M+H:481.2.

Example 15

1-(4-(7-chloro-6-(2-fluoro-6-hydroxyphenyl)-4-(1-piperidinyl)-1-phthalazinyl)-1-piperazinyl)-2-propen-1-one

Step 1: 4-(7-chloro-6-(2-fluoro-6-hydroxyphenyl)-4-(piperidin-1-yl)phthalazin-1-yl)piperazin-1-carboxylic acid tert-butyl ester. Piperidine (1.0 mL, 10.10 mmol) was added to a 20 mL vial containing 4-(6-(2-((tert-butyldiphenylsilyl)oxy)-6-fluorophenyl)-4,7-dichlorophthalazin-1-yl)piperazin-1-carboxylic acid tert-butyl ester (intermediate H, 0.060 g, 0.082 mmol). The vial was capped and heated at 80 °C for 2 h. The reaction was cooled to room temperature and partitioned between EtOAc (30 mL) and water (10 mL). The organic layer was separated and washed with water (2 x 10 mL). The combined organic layers were dried over _MgSO₄ , filtered, and concentrated under vacuum to obtain tert-butyl 4-(7-chloro-6-(2-fluoro-6-hydroxyphenyl)-4-(piperidin-1-yl)phthalazin-1-yl)piperazin-1-carboxylate. ^¹H NMR (chloroform-d) δ: 8.09–8.14 (m, 1H), 7.97–8.03 (m, 1H), 7.28–7.35 (m, 1H), 6.75–6.88 (m, 2H), 3.65–3.76 (m, 4H), 3.30–3.44 (m, 8H), 1.72–1.81 (m, 4H), 1.61–1.71 (m, 3H), 1.48–1.53 (m, 9H). m/z (ESI) M+H: 542.2.

Step 2: 2-(7-chloro-1-(piperazin-1-yl)-4-(piperidin-1-yl)phthalazin-6-yl)-3-fluorophenol. Similar to Step 2 in Example 12, the reaction of tert-butyl 4-(7-chloro-6-(2-fluoro-6-hydroxyphenyl)-4-(piperidin-1-yl)phthalazin-1-yl)piperazin-1-carboxylate produces 2-(7-chloro-1-(piperazin-1-yl)-4-(piperidin-1-yl)phthalazin-6-yl)-3-fluorophenol. ¹ H NMR(chloroform-d)δ:8.09-8.13(m,1H),7.95-8.03(m,1H),7.28-7.38(m,1H),6.83-6.89(m,1H),6.75-6.82( m,1H),3.39-3.48(m,4H),3.31-3.38(m,4H),3.12-3.21(m,4H),1.75-1.80(m,4H),1.64-1.69(m,2H). m/z(ESI)M+H:442.2.

Step 3: 1-(4-(7-chloro-6-(2-fluoro-6-hydroxyphenyl)-4-(1-piperidinyl)-1-phthalazinyl)-1-piperidinyl)-2-propen-1-one. Similar to Step 3 in Example 12, the reaction of 2-(7-chloro-1-(piperidin-1-yl)-4-(piperidin-1-yl)phthalazin-6-yl)-3-fluorophenol produces 1-(4-(7-chloro-6-(2-fluoro-6-hydroxyphenyl)-4-(1-piperidinyl)-1-phthalazinyl)-1-piperidinyl)-2-propen-1-one. ¹ H NMR(chloroform-d)δ:8.08-8.15(m,1H),7.98-8.05(m,1H),7.29-7.39(m,1H),6.86-6.94(m,1H),6.76-6.85(m,1H),6.59-6.70(m,1H),6. 30-6.43(m,1H),5.72-5.84(m,1H),3.77-4.05(m,4H),3.40-3.56(m,4H),3.32-3.38(m,4H),1.73-1.85(m,4H),1.64-1.70(m,2H). m/z(ESI)M+H:496.2.

Example 16

1-(4-(7-chloro-6-(2-fluoro-6-hydroxyphenyl)-4-phenoxy-1-phthalazinyl)-1-piperazinyl)-2-propen-1-one

Step 1: 4-(7-chloro-6-(2-fluoro-6-hydroxyphenyl)-4-phenoxyphthalazin-1-yl)piperazin-1-carboxylate tert-butyl ester. Phenol (0.130 g, 1.381 mmol) and tetrahydrofuran (3.0 mL) were packed into a dry 50 mL round-bottom flask. The mixture was cooled to 0 °C, and then potassium tert-butoxide (0.153 g, 1.367 mmol) was added. The mixture was stirred at 0 °C for 10 min, then heated to room temperature and stirred for 30 min. 4-(6-(2-((tert-butyldiphenylsilyl)oxy)-6-fluorophenyl)-4,7-dichlorophthalazin-1-yl)piperazin-1-carboxylate tert-butyl ester (intermediate H, 0.100 g, 0.137 mmol) was added, and the resulting mixture was heated at 60 °C for 2 h. The reaction was cooled to room temperature and quenched with water. The resulting mixture was partitioned between EtOAc (30 mL) and water (15 mL). The aqueous layer was extracted once more with EtOAc (20 mL). The combined organic layers were dried over _MgSO4 , filtered, and concentrated under vacuum. The crude product was purified by column chromatography (40 g silica, 10 to 50% acetone) to obtain tert-butyl 4-(7-chloro-6-(2-fluoro-6-hydroxyphenyl)-4-phenoxyphthalazin-1-yl)piperazine-1-carboxylate: m/z(ESI)M+H: 551.2.

Step 2: 2-(7-chloro-4-phenoxy-1-(piperazin-1-yl)phthalazin-6-yl)-3-fluorophenol. Similar to Step 2 in Example 12, the reaction of 4-(7-chloro-6-(2-fluoro-6-hydroxyphenyl)-4-phenoxyphthalazin-1-yl)piperazin-1-carboxylate produces 2-(7-chloro-4-phenoxy-1-(piperazin-1-yl)phthalazin-6-yl)-3-fluorophenol. ¹ H NMR(chloroform-d)δ:8.37-8.42(m,1H),8.14-8.19(m,1H),7.37-7.45(m,2H),7.29-7.34(m,1H),7. 19-7.25(m,2H),6.89-6.98(m,1H),6.76-6.87(m,4H),3.36-3.45(m,4H),3.13-3.22(m,4H). m/z(ESI)M+H:451.2.

Step 3: 1-(4-(7-chloro-6-(2-fluoro-6-hydroxyphenyl)-4-phenoxy-1-phthalazinyl)-1-piperazinyl)-2-propen-1-one. Similar to Step 3 in Example 12, the reaction of 2-(7-chloro-4-phenoxy-1-(piperazin-1-yl)phthalazin-6-yl)-3-fluorophenol produces 1-(4-(7-chloro-6-(2-fluoro-6-hydroxyphenyl)-4-phenoxy-1-phthalazinyl)-1-piperazinyl)-2-propen-1-one. ¹ H NMR(chloroform-d)δ:8.41-8.45(m,1H),8.17-8.20(m,1H),7.40-7.45(m,2H),7.28-7.37(m,2H),7.20-7.26(m,1H),6.78-6.87( m,2H),6.59-6.70(m,1H),6.31-6.41(m,1H),5.97-6.06(m,1H),5.74-5.81(m,1H),3.76-4.03(m,4H),3.38-3.53(m,4H). m/z(ESI)M+H:505.2.

Examples 17-1 and 17-2

(2E)-1-(4-(5-chloro-7-fluoro-6-(3-methoxy-1-naphthyl)-2,1-benzothiazol-3-yl)-1-piperazinyl)-4-(dimethylamino)-2-buten-1-one (Example 17-1) and (2E)-1-(4-(5-chloro-7-fluoro-6-(3-hydroxy-1-naphthyl)-2,1-benzothiazol-3-yl)-1-piperazinyl)-4-(dimethylamino)-2-buten-1-one (Example 17-2)

Step 1: 4-(5-chloro-6-(3-methoxynaphthyl-1-yl)benzo[c]isothiazolyl)piperazine-1-carboxylate tert-butyl ester. A slurry of 4-(6-bromo-5-chloro-7-fluorobenzo[c]isothiazolyl)piperazine-1-carboxylate (intermediate D, 459 mg, 1.02 mmol), (3-methoxynaphthyl-1-yl)boronic acid (823 mg, 4.07 mmol), and cesium carbonate (1.33 g, 4.07 mmol) in a mixture of 1,4-dioxane (8 mL) and water (2 mL) was degassed with argon. Tetra(triphenylphosphine)palladium (118 mg, 0.10 mmol) was added, and the mixture was degassed again with argon. The reaction mixture was sealed and heated at 100 °C for 23 h. The reaction was allowed to cool to room temperature, diluted with brine (60 mL), and extracted twice with EtOAc. The combined organic layers were dried over anhydrous sodium sulfate and concentrated. The residue was purified by silica gel chromatography (elution: 0–2% MeOH in DCM) to provide tert-butyl 4-(5-chloro-6-(3-methoxynaphthyl-1-yl)benzo[c]isothiazo-3-yl)piperazine-1-carboxylate. m/z (ESI) M+H: 528.0.

Step 2: 5-Chloro-6-(3-methoxynaphthyl-1-yl)-3-(piperazin-1-yl)benzo[c]isothiazolium. Trifluoroacetic acid (1.04 mL, 13.9 mmol) was added to a solution of tert-butyl piperazine-1-carboxylate (327 mg, 0.56 mmol) in DCM (6 mL) via syringe. The resulting yellow solution was stirred at room temperature for 4 h and then concentrated. The residue was purified by silica gel chromatography (elution: 0-25% MeOH in DCM) to provide a single TFA salt of 5-chloro-6-(3-methoxynaphthyl-1-yl)-3-(piperazin-1-yl)benzo[c]isothiazolium. m/z (ESI) M+H: 428.0.

Step 3: (2E)-1-(4-(5-chloro-7-fluoro-6-(3-methoxy-1-naphthyl)-2,1-benzothiazol-3-yl)-1-piperazinyl)-4-(dimethylamino)-2-buten-1-one. Thionyl chloride (41 μL, 0.69 mmol) was added to a solution of 5-chloro-6-(3-methoxynaphthyl)-3-(piperazin-1-yl)benzo[c]isothiazolium (74 mg mono-TFA salt, 0.14 mmol) and trans-4-dimethylaminocrotonate (38 mg, 0.23 mmol) in DMA (2 mL) using a syringe. The resulting brown solution was stirred at room temperature for 2.5 h. The reaction mixture was quenched with water (50 mL) and extracted with 8:1 DCM/MeOH. The organic layer was dried over anhydrous sodium sulfate and concentrated. The residue was purified by silica gel chromatography (elution: 0-15% MeOH in DCM) to provide (2E)-1-(4-(5-chloro-7-fluoro-6-(3-methoxy-1-naphthyl)-2,1-benzothiazol-3-yl)-1-piperazinyl)-4-(dimethylamino)-2-buten-1-one. ¹ H NMR (400MHz, DMSO-d ₆ )δ8.10(s,1H),7.94(d,J＝8.4Hz,1H),7.46-7.55(m,2H),7.27-7.35(m,2H),7.19(d,J＝2.5Hz,1H),6.6 1-6.72(m,2H),3.94(s,3H),3.80-3.93(m,4H),3.62-3.68(m,4H),3.07(d,J＝4.3Hz,2H),2.18(s,6H). m/z(ESI)M+H:539.2.

Step 4: (2E)-1-(4-(5-chloro-7-fluoro-6-(3-hydroxy-1-naphthyl)-2,1-benzothiazol-3-yl)-1-piperazinyl)-4-(dimethylamino)-2-buten-1-one. Boron tribromide (1.0 M in hexane, 218 μL, 0.22 mmol) was added dropwise to a solution of (2E)-1-(4-(5-chloro-7-fluoro-6-(3-methoxy-1-naphthyl)-2,1-benzothiazol-3-yl)-1-piperazinyl)-4-(dimethylamino)-2-buten-1-one (23.5 mg, 0.044 mmol) in 1,2-dichloroethane (4 mL) using a syringe at 0 °C. The resulting yellow slurry was stirred at 0 °C for 2.75 h and then quenched with a saturated aqueous solution of _NaHCO3 (4 mL). The mixture was extracted twice with a 4:1 mixture of DCM and MeOH. The combined organic layers were dried over anhydrous sodium sulfate and concentrated. The residue was purified by silica gel chromatography (elution: 0-18% MeOH in DCM) to provide (2E)-1-(4-(5-chloro-7-fluoro-6-(3-hydroxy-1-naphthyl)-2,1-benzothiazol-3-yl)-1-piperazinyl)-4-(dimethylamino)-2-buten-1-one. ¹ H NMR (400MHz, DMSO-d ₆ )δ9.97(s,1H),8.10(s,1H),7.80(d,J＝8.4Hz,1H),7.40-7.46(m,1H),7.19-7.30(m,3H),7.07(d,J＝2. 4Hz, 1H), 6.62-6.71 (m, 2H), 3.80-3.93 (m, 4H), 3.62-3.69 (m, 4H), 3.07 (d, J = 4.1Hz, 2H), 2.17 (s, 6H). m/z(ESI)M+H:525.0.

Examples 18-1 to 18-3

1-(4-(5-chloro-7-fluoro-6-(3-methoxy-1-naphthyl)-2,1-benzothiazol-3-yl)-1-piperazinyl)-2-(hydroxymethyl)-2-propen-1-one (Example 18-1) and 2-(bromomethyl)-1-(4-(5-chloro-7-fluoro-6-(3-hydroxy-1-naphthyl)-2,1-benzothiazol-3-yl)-1-piperazinyl)-2-propen-1-one (Example 18-2) and 1-(4-(5-chloro-7-fluoro-6-(3-hydroxy-1-naphthyl)-2,1-benzothiazol-3-yl)-1-piperazinyl)-2-(hydroxymethyl)-2-propen-1-one (Example 18-3)

Step 1: 1-(4-(5-chloro-7-fluoro-6-(3-methoxy-1-naphthyl)-2,1-benzothiazol-3-yl)-1-piperazinyl)-2-(hydroxymethyl)-2-propen-1-one. A solution of 1-(4-(5-chloro-7-fluoro-6-(3-methoxynaphthyl)benzo[c]isothiazol-3-yl)piperazin-1-yl)prop-2-en-1-one (intermediate O, 29 mg, 0.06 mmol) in tert-butanol (0.4 mL) and water (0.4 mL) was placed in a vial. Phenol (5.7 mg, 0.06 mmol), DABCO (20.3 mg, 0.18 mmol), and formaldehyde (37% aqueous solution, 24 μL, 0.24 mmol) were added sequentially. The resulting solution was sealed and heated at 55 °C for 29 h. The reaction was cooled to room temperature and partitioned between water (6 mL) and 10:1 DCM/MeOH. The organic layer was separated, and the aqueous layer was extracted twice more with 10:1 DCM/MeOH. The combined organic layers were dried over anhydrous sodium sulfate and concentrated. The residue was purified by silica gel chromatography (elution: 0–3.5% MeOH in DCM) to provide 1-(4-(5-chloro-7-fluoro-6-(3-methoxy-1-naphthyl)-2,1-benzothiazol-3-yl)-1-piperazinyl)-2-(hydroxymethyl)-2-propen-1-one. ¹ H NMR (400MHz, DMSO-d ₆ )δ8.12(s,1H),7.94(d,J＝8.2Hz,1H),7.47-7.55(m,2H),7.25-7.34(m,2H),7.19(d,J＝2.5Hz,1H),5.43(br.s,1H) ,5.20(br.s,1H),5.14(t,J＝5.8Hz,1H),4.12(d,J＝5.7Hz,2H),3.94(s,3H),3.78-3.85(m,4H),3.54-3.66(m,4H). m/z(ESI)M+H:512.0.

Step 2: 2-(bromomethyl)-1-(4-(5-chloro-7-fluoro-6-(3-hydroxy-1-naphthyl)-2,1-benzothiazol-3-yl)-1-piperazinyl)-2-propen-1-one and 1-(4-(5-chloro-7-fluoro-6-(3-hydroxy-1-naphthyl)-2,1-benzothiazol-3-yl)-1-piperazinyl)-2-(hydroxymethyl)-2-propen-1-one. A boron tribromide solution (1.0 M in hexane, 167 μL, 0.17 mmol) was added dropwise to a solution of 1-(4-(5-chloro-7-fluoro-6-(3-methoxy-1-naphthyl)-2,1-benzothiazol-3-yl)-1-piperazinyl)-2-(hydroxymethyl)-2-propen-1-one (17.1 mg, 0.033 mmol) in 1,2-dichloroethane (4 mL) using a syringe at 0 °C. The resulting slurry was stirred at 0 °C for 40 min, followed by quenching with a saturated aqueous solution of _NaHCO3 (5 mL). The mixture was extracted twice with a 4:1 mixture of DCM and MeOH. The combined organic layers were dried over anhydrous sodium sulfate and concentrated. The residue was purified by silica gel chromatography (elution: 0-7% MeOH in DCM) to give two products.

First elution peak: 2-(bromomethyl)-1-(4-(5-chloro-7-fluoro-6-(3-hydroxynaphthyl-1-yl)benzo[c]isothiazo-3-yl)piperazin-1-yl)prop-2-en-1-one. ^¹H NMR (400MHz, DMSO- _d⁶ ) δ 9.96 (br.s, 1H), 8.13 (s, 1H), 7.80 (d, J = 8.2Hz, 1H), 7.40–7.47 (m, 1H), 7.19–7.29 (m, 3H), 7.07 (d, J = 2.4Hz, 1H), 5.78 (s, 1H), 5.41 (s, 1H), 4.38 (s, 2H), 3.84–3.93 (m, 4H), 3.62–3.72 (m, 4H). m/z(ESI)M+H:560.0

Second elution peak: 1-(4-(5-chloro-7-fluoro-6-(3-hydroxynaphthyl-1-yl)benzo[c]isothiazo-3-yl)piperazin-1-yl)-2-(hydroxymethyl)prop-2-en-1-one. ¹ H NMR (400MHz, DMSO-d ₆ )δ9.98(br.s,1H),8.11(s,1H),7.79(d,J＝8.2Hz,1H),7.37-7.48(m,1H),7.17-7.28(m,3H),7.07(d,J＝2.4Hz, 1H),5.43(br.s,1H),5.20(br.s,1H),5.07-5.14(m,1H),4.12(br.s,2H),3.78-3.86(m,4H),3.57-3.66(m,4H). m/z(ESI)M+H:498.0

Examples 19-1 to 19-3

1-(4-(5-chloro-7-fluoro-6-(5-methoxy-1-methyl-1H-indazol-7-yl)-2,1-benzothiazol-3-yl)-1-piperazinyl)-2-propen-1-one (Example 19-1) and 1-(4-(5-chloro-7-fluoro-6-(5-hydroxy-1-methyl-1H-indazol-7-yl)-2,1-benzothiazol-3-yl)-1-piperazinyl)-2-propen-1-one (Example 19-2) and 1-(4-(5-chloro-7-fluoro-6-(5-hydroxy-2-methyl-2H-indazol-7-yl)-2,1-benzothiazol-3-yl)-1-piperazinyl)-2-propen-1-one (Example 19-3)

Step 1: 4-(5-chloro-7-fluoro-6-(5-methoxy-1H-indazole-7-yl)benzo[c]isothiazolyl)piperazine-1-carboxylic acid tert-butyl ester. A slurry of intermediate D (232 mg, 0.51 mmol), 5-methoxy-7-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)-1H-indazole (535 mg, 1.95 mmol, see synthesis below), and cesium carbonate (636 mg, 1.95 mmol) in a mixture of 1,4-dioxane (8 mL) and water (2 mL) was degassed with argon. Tetra(triphenylphosphine)palladium (59 mg, 0.05 mmol) was added, and the mixture was degassed again with argon. The reaction mixture was sealed and heated at 100 °C for 18 h. The reaction was allowed to cool to room temperature and partitioned between brine (40 mL) and EtOAc. The aqueous layer was extracted twice with EtOAc, and the combined organic layers were dried over anhydrous sodium sulfate and concentrated. The residue was purified by silica gel chromatography (elution: 0–4.5% DCM/MeOH) to provide tert-butyl 4-(5-chloro-7-fluoro-6-(5-methoxy-1H-indazol-7-yl)benzo[c]isothiazolyl)piperazine-1-carboxylate. LCMS-ESI (POS.) m/z: 518.2 (M+H) ⁺ . ¹ H NMR (400MHz, DMSO-d ₆ )δ12.90(br.s,1H),8.06(s,1H),8.03(s,1H),7.31(d,J＝1.4Hz,1H),6.99(d, J＝2.2Hz,1H),3.83(s,3H),3.61-3.69(m,4H),3.54-3.60(m,4H),1.45(s,9H).

5-Methoxy-7-(4,4,5,5-Tetramethyl-1,3,2-dioxaborane-2-yl)-1H-indazole. A suspension of 7-bromo-5-methoxy-1H-indazole (1.00 g, 4.40 mmol, Ark Pharmaceuticals, Arlington Heights, .I., USA), potassium acetate (1.30 g, 13.2 mmol), and bis(pinacolyl)diboron (1.23 g, 4.84 mmol) in 1,4-dioxane (18 mL) was degassed with argon. A complex of [1,1'-bis(diphenylphosphine)ferrocene]palladium(ii) dichloride with dichloromethane (108 mg, 0.13 mmol) was added, and degassed again with argon. The reaction mixture was sealed and heated at 80 °C for 2 days. The reaction was allowed to cool to room temperature and partitioned between water (50 mL) and EtOAc. The aqueous layer was extracted twice with EtOAc, and the combined organic layers were dried over anhydrous sodium sulfate and concentrated. The residue was purified by silica gel chromatography (elution: 2%–65% EtOAc/heptane) to provide 5-methoxy-7-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)-1H-indazole. LCMS-ESI (POS.) m/z: 275.1 (M+H) ⁺ .

Step 2: 4-(5-chloro-7-fluoro-6-(5-methoxy-1-methyl-1H-indazole-7-yl)benzo[c]isothiazol-3-yl)piperazine-1-carboxylate tert-butyl ester and 4-(5-chloro-7-fluoro-6-(5-methoxy-2-methyl-2H-indazole-7-yl)benzo[c]isothiazol-3-yl)piperazine-1-carboxylate tert-butyl ester. Sodium hydride (60% dispersion in mineral oil, 44.5 mg, 1.1 mmol) was added to a solution of 4-(5-chloro-7-fluoro-6-(5-methoxy-1H-indazole-7-yl)benzo[c]isothiazol-3-yl)piperazine-1-carboxylate (115 mg, 0.22 mmol) in THF (5 mL). After 10 min, iodomethane (69 μL, 1.1 mmol) was added and the reaction was stirred for another 15 min at room temperature. The mixture was then partitioned between a saturated ammonium chloride aqueous solution (10 mL) and DCM. The aqueous layer was extracted twice with DCM, and the combined organic layers were dried over anhydrous sodium sulfate and concentrated to give a mixture of tert-butyl 4-(5-chloro-7-fluoro-6-(5-methoxy-1-methyl-1H-indazole-7-yl)benzo[c]isothiazolyl)piperazine-1-carboxylate and tert-butyl 4-(5-chloro-7-fluoro-6-(5-methoxy-2-methyl-2H-indazole-7-yl)benzo[c]isothiazolyl)piperazine-1-carboxylate. The crude mixture was used directly in the next step without purification. LCMS-ESI (POS.) m/z: 532.0 (M+H) ⁺ .

Step 3: 5-chloro-7-fluoro-6-(5-methoxy-1-methyl-1H-indazol-7-yl)-3-(piperazin-1-yl)benzo[c]isothiazole (intermediate J) and 5-chloro-7-fluoro-6-(5-methoxy-2-methyl-2H-indazol-7-yl)-3-(piperazin-1-yl)benzo[c]isothiazole (intermediate K). Trifluoroacetic acid (484 μL, 6.5 mmol) was added to a crude mixture of 143 mg of 4-(5-chloro-7-fluoro-6-(5-methoxy-1-methyl-1H-indazole-7-yl)benzo[c]isothiazolyl-3-yl)piperazine-1-carboxylate and 4-(5-chloro-7-fluoro-6-(5-methoxy-2-methyl-2H-indazole-7-yl)benzo[c]isothiazolyl-3-yl)piperazine-1-carboxylate in DCM (6 mL) via syringe. The resulting solution was stirred at room temperature for 25 min and then concentrated. The residue was purified by silica gel chromatography (elution: 0-25% DCM/MeOH).

First elution peak: a single TFA salt (intermediate J) of 5-chloro-7-fluoro-6-(5-methoxy-1-methyl-1H-indazol-7-yl)-3-(piperazin-1-yl)benzo[c]isothiazolium. LCMS-ESI (POS.) m/z: 432.0 (M+H) ⁺ . ^1H NMR (400MHz, DMSO-d ₆ ) δ 8.16 (s, 1H), 8.03 (s, 1H), 7.35 (d, J＝2.4Hz, 1H), 6.99 (d, J＝2.4Hz, 1H), 3.84 (s, 3H), 3.67–3.76 (m, 4H), 3.56 (s, 3H), 3.36–3.42 (m, 4H).

Second elution peak: Single TFA salt of 5-chloro-7-fluoro-6-(5-methoxy-2-methyl-2H-indazol-7-yl)-3-(piperazin-1-yl)benzo[c]isothiazolium (intermediate K). LCMS-ESI (POS.) m/z: 432.0 (M+H) ⁺ .

Step 4: 1-(4-(5-chloro-7-fluoro-6-(5-methoxy-1-methyl-1H-indazol-7-yl)-2,1-benzothiazol-3-yl)-1-piperazinyl)-2-propen-1-one. DIPEA (104 μL, 0.60 mmol) was added dropwise to an ice-cold slurry of intermediate J containing a single TFA salt (108 mg, 0.20 mmol) in DCM (5 mL) using a syringe, followed by the addition of acryloyl chloride (24 μL, 0.30 mmol). The resulting solution was stirred at 0 °C for 3 h, then quenched with a saturated _NaHCO3 aqueous solution (15 mL) and extracted twice with DCM. The combined organic layers were dried over anhydrous sodium sulfate and concentrated. The residue was purified by silica gel chromatography (elution buffer: 0-7% DCM/MeOH) to provide 1-(4-(5-chloro-7-fluoro-6-(5-methoxy-1-methyl-1H-indazol-7-yl)-2,1-benzothiazol-3-yl)-1-piperazinyl)-2-propen-1-one. LCMS-ESI (POS.) m/z: 486.0 (M+H) ⁺ . ¹ H NMR (400MHz, DMSO-d ₆ )δ8.13(s,1H),8.02(s,1H),7.33(d,J＝2.2Hz,1H),6.99(d,J＝2.4Hz,1H),6.85(dd,J＝16.6,10.6Hz,1H),6.18(dd, J＝16.7,2.3Hz,1H),5.76(dd,J＝10.5,2.3Hz,1H),3.85-3.95(m,4H),3.84(s,3H),3.62-3.72(m,4H),3.56(s,3H).

Step 5: 1-(4-(5-chloro-7-fluoro-6-(5-hydroxy-1-methyl-1H-indazol-7-yl)-2,1-benzothiazol-3-yl)-1-piperazinyl)-2-propen-1-one. Boron tribromide solution (1.0 M in hexane, 746 μL, 0.75 mmol) was added dropwise to an ice-cooled solution of 1-(4-(5-chloro-7-fluoro-6-(5-methoxy-1-methyl-1H-indazol-7-yl)benzo[c]isothiazol-3-yl)piperazin-1-yl)propen-2-en-1-one (72.5 mg, 0.15 mmol) in 1,2-dichloroethane (5 mL) using a syringe. The resulting slurry was stirred at 0 °C for 3.75 h, then quenched with a saturated aqueous solution of _NaHCO3 (5 mL) and extracted twice with a 4:1 mixture of DCM/MeOH. The combined organic layers were dried over anhydrous sodium sulfate and concentrated. The residue was purified by silica gel chromatography (elution: 0–6% DCM/MeOH) to provide 1-(4-(5-chloro-7-fluoro-6-(5-hydroxy-1-methyl-1H-indazol-7-yl)benzo[c]isothiazolyl-3-yl)piperazin-1-yl)prop-2-en-1-one. LCMS-ESI (POS.) m/z: 472.0 (M+H) ⁺ . ¹ H NMR (400MHz, DMSO-d ₆ )δ9.40(s,1H),8.12(s,1H),7.92(s,1H),7.12(d,J＝2.2Hz,1H),6.81-6.91(m,2H),6.18(dd,J＝1 6.7, 2.5Hz, 1H), 5.76 (dd, J = 10.4, 2.4Hz, 1H), 3.81-3.94 (m, 4H), 3.62-3.70 (m, 4H), 3.52 (s, 3H).

Synthesis of 1-(4-(5-chloro-7-fluoro-6-(5-hydroxy-2-methyl-2H-indazol-7-yl)-2,1-benzothiazolyl-3-yl)-1-piperazinyl)-2-propen-1-one.

Using intermediate K from step 3, steps 4 and 5 are performed as described above to produce 1-(4-(5-chloro-7-fluoro-6-(5-hydroxy-2-methyl-2H-indazol-7-yl)-2,1-benzothiazolyl-3-yl)-1-piperazinyl)-2-propen-1-one. LCMS-ESI(POS.) m/z: 472.0(M+H) ⁺ . ¹ H NMR (400MHz, DMSO-d ₆ )δ9.28(s,1H),8.11(s,1H),8.01(s,1H),6.95(d,J＝2.0Hz,1H),6.77-6.90(m,2H),6.18(dd,J＝1 6.7, 2.5Hz, 1H), 5.76 (dd, J = 10.4, 2.2Hz, 1H), 4.03 (s, 3H), 3.80-3.94 (m, 4H), 3.58-3.66 (m, 4H).

Example 20

1-(4-(5-chloro-7-fluoro-6-(3-hydroxy-1-naphthyl)-2,1-benzothiazo-3-yl)-1-piperazinyl)-4-hydroxy-2-methylene-1-butanone

Step 1: 4-((tert-butyldiphenylsilyl)oxy)-1-(4-(5-chloro-7-fluoro-6-(3-methoxynaphthyl-1-yl)benzo[c]isothiazo-3-yl)piperazin-1-yl)-2-methylenebut-1-one. At 0 °C, 2 M oxaloyl chloride solution (0.21 mL, 0.43 mmol) was added to a solution of 4-((tert-butyldiphenylsilyl)oxy)-2-methylenebutyric acid (101 mg, 0.29 mmol, prepared according to the following references: Pihko, PM, J. Org. Chem., 2006, 71, 2538-2541 and Greaney, MF, Org. Lett., 2007, 9, 1931-1934) in DCM (2 mL), followed by the addition of a catalytic amount of DMF (5 μL). The reaction mixture was allowed to warm to room temperature and stirred for 2 h. The reaction mixture was concentrated under vacuum, then diluted with DCM (1 mL) and added to a solution of 5-chloro-7-fluoro-6-(3-methoxynaphthyl-1-yl)-3-(piperazin-1-yl)benzo[c]isothiazolium (intermediate N, 122 mg, 0.29 mmol), triethylamine (0.20 mL, 1.43 mmol), and DCM (2 mL). The reaction mixture was allowed to warm to room temperature and DMAP (2 mg, 0.016 mmol) was added. The reaction mixture was stirred at room temperature for 15 h, then concentrated under vacuum and purified by silica gel column chromatography (elution: 0-50% EtOAc:heptane) to give 4-((tert-butyldiphenylsilyl)oxy)-1-(4-(5-chloro-7-fluoro-6-(3-methoxynaphthyl-1-yl)benzo[c]isothiazolyl-3-yl)piperazin-1-yl)-2-methylenebut-1-one. ¹ H NMR (400MHz, DMSO-d ₆ )δ8.06(s,1H),7.94(d,J=8.0Hz,1H),7.63-7.61(m,4H),7.52-7.49(m,2H),7.47-7.40(m,6H),7.33-7.28(m,2H),7. 20-7.19(m,1H),5.37(s,1H),5.24(s,1H),3.94(s,3H),3.83-3.76(m,6H),3.53(brs,2H),3.31(s,4H),1.01(s,9H). m/z(ESI)M+H:764.

Step 2: 1-(4-(5-chloro-7-fluoro-6-(3-hydroxy-1-naphthyl)-2,1-benzothiazol-3-yl)-1-piperazinyl)-4-hydroxy-2-methylene-1-butanone. At 0°C, add BBr 3 (0.28 mL, 0.56 mmol) to a solution of 4-((tert-butyldiphenylsilyl)oxy)-1-(4-(5-chloro-7-fluoro-6-(3-methoxynaphthyl)benzo[c]isothiazol-3-yl)piperazin-1-yl)-2-methylenebutanone (85 mg, 0.11 mmol) and DCM ( ₂ mL) in a 2 M solution of DCM. The reaction mixture was quenched with water, concentrated under vacuum, and subjected to silica gel column chromatography (eluting with 0-50% heptane/3:1 EtOAc:EtOH) to give 1-(4-(5-chloro-7-fluoro-6-(3-hydroxy-1-naphthyl)-2,1-benzothiazol-3-yl)-1-piperazinyl)-4-hydroxy-2-methylene-1-butanone. ^1H NMR(400MHz,DMSO-d6)δ9.93(br s,1H),8.11(s,1H),7.80(d,J＝12Hz,1H),7.43(m,1H),7.26-7.20(m,3H),7.07(s,1H),5.32(s,1H),5.16(s,1H),3.83(br s,4H),3.63(br s, 4H), 3.53 (t, J = 8.0Hz, 2H), 2.42 (t, J = 8.0Hz, 2H). ¹⁹ FNMR (377MHz, DMSO-d ₆ ) δ-123.8 (s, 1F). m/z(ESI)M+H:512.

Example 21

1-(4-(5-chloro-7-fluoro-6-(7-hydroxy-5-quinolinyl)-2,1-benzothiazo-3-yl)-1-piperazinyl)-2-propen-1-one

1-(4-(5-chloro-7-fluoro-6-(7-hydroxy-5-quinolinyl)-2,1-benzothiazol-3-yl)-1-piperazinyl)-2-propen-1-one was prepared from intermediate D by method 1 using 7-methoxy-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)quinoline (synthesized below), wherein the following changes were made: in step 7, S-Phos Pd G3, an aqueous solution of potassium carbonate, and DME were used; in step 8-1, TFA/DCM was used; in step 8-2, DCE was used as a solvent; and in step 8-3, a boron tribromide solution (1.0 M in DCE) was used to obtain 1-(4-(5-chloro-7-fluoro-6-(7-hydroxy-5-quinolinyl)-2,1-benzothiazol-3-yl)-1-piperazinyl)-2-propen-1-one. ¹ H NMR (400MHz, CDCl ₃ )δ8.81(dd,J＝4.2,1.3Hz,1H)7.72-7.78(m,2H)7.64(s,1H)7.28(d,J＝2.2Hz,1H)7.16(dd,J＝8.4,4.3Hz,1H)6.5 6-6.66(m,1H)6.40(dd,J＝16.8,1.6Hz,1H)5.78-5.87(m,1H)4.01(br.s.,2H)3.89(br.s.,2H)3.50-3.60(m,4H). ¹⁹ F NMR (376MHz, CDCl ₃ ) δ–121.33 (s, 1F). MS(ESI,+ve)m/z:469.1(M+1) ⁺ .

7-Methoxy-5-(4,4,5,5-Tetramethyl-1,3,2-dioxaborane-2-yl)quinoline. A solution of 5-bromo-7-methoxyquinoline (0.407 g, 1.71 mmol, OxChem, Wooddale, Illinois, USA), 4,4,4',4',5,5,5',5'-octamethyl-2,2'-bis(1,3,2-dioxaborane) (0.912 g, 3.59 mmol), _PdCl₂ (dppf) (0.051 g, 0.070 mmol), and potassium acetate (0.503 g, 5.13 mmol) in DMF (9 mL) was stirred at 90 °C for 1 h, followed by stirring at 100 °C for 45 min. The reaction mixture was diluted with EtOAc (100 mL) and washed with saturated sodium bicarbonate aqueous solution (2 × 75 mL). The organic layer was separated, dried over _{anhydrous Na₂SO₄} , and concentrated under vacuum. The crude product was adsorbed onto silica and purified by column chromatography (silica gel, 0-80% heptane/EtOAc) to give 7-methoxy-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)quinoline. MS (ESI,+ve) m/z: 286.1 (M+1) ⁺ .

Example 22

1-(5-chloro-7-fluoro-6-(3-hydroxy-1-naphthyl)-2,1-benzothiazo-3-yl)-4-(2-acryloyl)-2-piperazinic acid

Methyl 4-acryloyl-1-(5-chloro-7-fluoro-6-(3-hydroxy-1-naphthyl)benzo[c]isothiazo-3-yl)piperazin-2-carboxylic acid (Example 7-3, 0.022 g, 0.042 mmol) in THF/EtOH (1:1; 6 mL) was added to NaOH (5N aqueous solution; 1.0 mL, 5.0 mmol), and the resulting mixture was stirred at 0 °C for 5 min. The reaction was acidified with 5N HCl at 0 °C, extracted with EtOAc, and purified by HPLC to give 1-(5-chloro-7-fluoro-6-(3-hydroxy-1-naphthyl)-2,1-benzothiazo-3-yl)-4-(2-acryloyl)-2-piperazincarboxylic acid. m/z(ESI,+ve)512.0(M+H) ⁺ . ¹ H NMR (400MHz, DMSO-d ₆ )δ3.14-3.28(m,1H)3.52-3.87(m,3H)4.15-5.03(m,2H)5.15-5.23(m,1H)5.77-5.83(m,1H)6.13-6.24(m,1H)6.86(br.s.,1H )7.06-7.12(m,1H)7.20-7.30(m,3H)7.38-7.49(m,1H)7.76-7.84(m,1H)8.07-8.13(m,1H)9.98(br.s.,1H)13.42(br.s.,1H).

Example 23

1-(4-(5-chloro-6-(5-cyclopropyl-1H-indazol-4-yl)-7-fluoro-2,1-benzothiazo-3-yl)-1-piperazinyl)-2-propen-1-one

Example 23 is prepared as described in Method 1, using (5-cyclopropyl-1H-indazole-4-yl)boronic acid in step 7 (see Synthesis below), omitting steps 8-3. m/z(ESI,+ve)482.0(M+H) ⁺ . ¹ H NMR (400MHz, DMSO-d ₆ )δ12.92-13.19(1H,m),8.02-8.21(1H,m),7.47-7.60(2H,m),7.02-7.09(1H,m),6.80-6.93(1H,m),6.15-6.25(1H,m ),5.71-5.82(1H,m),3.80-3.96(4H,m),3.60-3.72(4H,m),1.55-1.74(1H,m),0.72-0.79(2H,m),0.58-0.71(2H,m).

(5-Cyclopropyl-1H-indazol-4-yl)boronic acid

Step 1: 2-Bromo-1-cyclopropyl-4-fluorobenzene. 2-Bromo-4-fluoro-1-iodobenzene (22 g, 73.1 mmol) and cyclopropylboronic acid (12.6 g, 146 mmol) in 1.1 L of cyclopentyl methyl ether were added to a 2- _L round-bottom flask at ambient temperature. _Na₂CO₃ (2 M aqueous solution; 183 mL) was added, and the reaction mixture was degassed with _N₂ gas for 20 min. Tetrakis (8.45 g, 7.31 mmol) was added, and the reaction mixture was degassed again with _N₂ gas for 20 min. The reaction mixture was then transferred to a 5-L autoclave under _N₂ atmosphere and heated to 130 °C for 40 h. The reaction mixture was cooled to ambient temperature, filtered through a diatomaceous earth mat, and washed with ether (200 mL). Water (500 mL) was added to the filtrate, and the organic layer was separated. The aqueous layer was extracted with diethyl ether (2 × 300 mL), and the combined organic layers were dried over anhydrous sodium sulfate and evaporated under reduced pressure. The crude material was adsorbed onto a silica gel stopper and purified chromatographically (silica gel, 100% petroleum ether) to provide 2-bromo-1-cyclopropyl-4-fluorobenzene. GC-MS m/z: 214/216 ^¹H NMR (400 MHz, _CDCl₃ ) δ 7.36–7.23 (m, 1H), 6.95 (dt, J = 7.0, 1.5 Hz, 2H), 2.09 (ddd, J = 13.8, 8.5, 5.4 Hz, 1H), 1.12–0.88 (m, 2H), 0.76–0.50 (m, 2H).

Step ₂ : 2-Bromo-3-cyclopropyl-6-fluorobenzaldehyde. 2-Bromo-1-cyclopropyl-4-fluorobenzene (6.5 g, 30.2 mmol) in tetrahydrofuran (130 mL) was added dropwise to a 500 mL round-bottom flask under a nitrogen atmosphere. LDA (18.1 mL, 36.3 mmol, 2 M in THF, 1.2 equivalents) was added dropwise at -78 °C (internal temperature maintained between -65 °C and -70 °C), and the reaction mixture was stirred for 1 h. DMF (6 mL) was then added dropwise to the reaction mixture (internal temperature maintained between -65 °C and -70 °C), and the reaction was stirred again at -78 °C for 3 h. The reaction was quenched with saturated ammonium chloride aqueous solution (100 mL) and the temperature was slowly increased to ambient temperature. The mixture was diluted with diethyl ether (200 mL), and the organic layer was separated and washed with brine solution (2 × 50 mL). The combined organic layers were dried over _{anhydrous Na₂SO₄} and evaporated under reduced pressure. The crude material was adsorbed onto a silica gel stopper and purified chromatographically (silica gel, 0–2% EtOAc/hexane) to provide 2-bromo-3-cyclopropyl-6-fluorobenzaldehyde. GC-MS m/z: 242 ^1H NMR (400MHz, _CDCl₃ ) δ 10.43 (d, J = 1.5 Hz, 1H), 7.26–7.12 (m, 1H), 7.06 (t, J = 9.3 Hz, 1H), 2.15 (td, J = 8.4, 4.3 Hz, 1H), 1.17–0.94 (m, 2H), 0.78–0.52 (m, 2H).

Step 3: 4-Bromo-5-cyclopropyl-1H-indazole. Add 2-bromo-3-cyclopropyl-6-fluorobenzaldehyde (4 g, 16.5 mmol) and hydrazine hydrate (4.0 mL, 82 mmol) to a 100 mL sealed tube in ethylene glycol (40 mL). Stir the reaction mixture at 90 °C for 2 h, then heat to 150 °C for 16 h. Cool the reaction mixture to ambient temperature and add ice-cold water (40 mL) and EtOAc (50 mL). Separate the organic layers and extract the aqueous layer with EtOAc (2 × 40 mL). Wash the combined organic layers with water (2 × 40 mL) and a brine solution (40 mL), dry over anhydrous sodium sulfate, and concentrate under vacuum. Adsorb the crude material onto a silica gel stopper and chromatographically purify it (silica gel, 0–20% EtOAc/hexane) to provide 4-bromo-5-cyclopropyl-1H-indazole. The compound was purified by reversed-phase preparative liquid chromatography (YMC: _C18 , 150 × 20 mm, 5 μm; mobile phase: water and 0.1% TFA in acetonitrile; flow rate: 15 mL/min) to obtain pure compound. MS (ESI cation) m/z: 237/239.0 (M+1). ^1H NMR (400 MHz, DMSO- _d6 ) δ 13.31 (s, 1H), 7.97 (s, 1H), 7.46 (d, J = 8.6 Hz, 1H), 6.97 (d, J = 8.6 Hz, 1H), 2.21 (tt, J = 8.5, 5.3 Hz, 1H), 1.24–0.87 (m, 2H), 0.93–0.33 (m, 2H).

Step 4: 5-Cyclopropyl-1H-indazole-4-yl)boronic acid. Add 0.62 g (2.6 mmol) of 4-bromo-5-cyclopropyl-1H-indazole and 0.996 g (3.92 mmol) of bis(pinacol)diboron from 25 mL of 1,4-dioxane to a 100 mL round-bottom flask; add potassium acetate (0.77 g, 7.84 mmol), and degas the reaction mixture with _N2 gas for 10 min. Add _PdCl2 (dppf)·DCM adduct (0.213 g, 0.261 mmol) to the reaction mixture; degas the reaction mixture again with _N2 gas for 10 min, and then heat to 100 °C for 16 h. Cool the reaction mixture to ambient temperature, filter through a diatomaceous earth mat, and wash with EtOAc (50 mL). The filtrate was concentrated under vacuum, and the crude material was adsorbed onto a silica gel stopper and purified chromatographically (silica gel, 0-50% EtOAc/hexane). The compound was further purified by reversed-phase preparative liquid chromatography (Grace column; 0-70% MeCN/water) to provide 5-cyclopropyl-1H-indazole-4-yl)boronic acid. MS (ESI cation) m/z: 285.2 (M+1). ¹ HNMR (400MHz, DMSO-d ₆ )δ12.88(s,1H),8.13(q,J＝1.3Hz,1H),7.50(d,J＝8.7Hz,1H),6.83(dd,J＝8.8,1.4Hz, 1H), 2.78–2.60 (m, 1H), 1.38 (d, J = 1.4Hz, 12H), 1.07–0.85 (m, 2H), 0.75–0.48 (m, 2H).

Example 24

1-(4-(5-chloro-7-fluoro-6-(3-(methylamino)-1-isoquinolinyl)-2,1-benzothiazo-3-yl)-1-piperazinyl)-2-propen-1-one

Step 1: 1-(5-chloro-7-fluoro-3-(piperazin-1-yl)benzo[c]isothiazo-6-yl)-N-methylisoquinoline-3-amine. At 0 °C, a solution of isopropyl magnesium chloride (2.0 M solution in tetrahydrofuran, 0.050 mL, 0.100 mmol) was added to a solution of 4-(6-bromo-5-chloro-7-fluorobenzo[c]isothiazo-3-yl)piperazin-1-carboxylic acid tert-butyl ester (intermediate D, 30 mg, 0.067 mmol) in tetrahydrofuran (0.6 mL). The mixture was stirred for 5 min, then zinc chloride (1.9 M solution in 2-methyltetrahydrofuran, 0.053 mL, 0.100 mmol) was added, and the reaction mixture was heated to room temperature and stirred for 40 min. The reaction mixture was then transferred to a vial containing Sphos Pd G3 (5.76 mg, 6.66 μmol) and (1-bromoisoquinoline-3-yl)(methyl)carbamate tert-butyl ester (24.7 mg, 0.073 mmol, see Synthesis below) and heated to 70 °C overnight. The crude reaction mixture was diluted with saturated _NH4Cl aqueous solution (50 mL) and EtOAc (50 mL). The organic layer was separated, _dried over _Na2SO4 , filtered, and concentrated. Purification by silica gel column chromatography (eluting with 6%–20% MeOH in DCM) yielded 1-(5-chloro-7-fluoro-3-(piperazin-1-yl)benzo[c]isothiazolyl-6-yl)-N-methylisoquinoline-3-amine. m/z (ESI, +ve) 428.1 (M + H) ⁺ .

Synthesis of (1-bromoisoquinoline-3-yl)(methyl)carbamate tert-butyl ester: At room temperature, sodium bis(trimethylsilyl)amide (1M solution in tetrahydrofuran, 1.79 mL, 1.79 mmol) was added to a solution of 1-bromoisoquinoline-3-amine (200 mg, 0.897 mmol, Maybridge Chemical Co., Altrincham, UK) in 5 mL of tetrahydrofuran. The mixture was stirred for 10 min, followed by the addition of Boc-anhydride (0.208 mL, 0.897 mmol) in 1 mL of THF. The reaction mixture was stirred for 5 min, followed by dilution with saturated aqueous _NH₄Cl solution (50 mL) and EtOAc (50 mL). The organic layer was separated, _dried over _Na₂SO₄ , filtered, and concentrated. (1-Bromoisoquinoline-3-yl)carbamate tert-butyl ester was provided by silica gel column chromatography (eluting with 0-20% EtOAc in heptane). m/z (ESI,+ve) 345.0 (M+Na) ⁺ .

Sodium hydride (60% dispersion in mineral oil, 22.52 mg, 0.563 mmol) was added to a solution of (1-bromoisoquinoline-3-yl)carbamate tert-butyl ester (140 mg, 0.433 mmol) in tetrahydrofuran (3 mL) at room temperature. The mixture was stirred for 15 min, followed by the addition of iodomethane (0.033 mL, 0.520 mmol). After stirring overnight, the reaction mixture was diluted with saturated aqueous _NH₄Cl solution (50 mL) and EtOAc (50 mL). The organic layer was separated, _dried over _Na₂SO₄ , filtered, and concentrated. (1-bromoisoquinoline-3-yl)(methyl)carbamate tert-butyl ester was obtained by silica gel column chromatography (eluting with 0–10% EtOAc in heptane). ^¹H NMR (400MHz, methanol- _d⁴ ) δ 8.25 (d, J = 8.61Hz, 1H), 7.88–7.95 (m, 2H), 7.79 (t, J = 7.5Hz, 1H), 7.70 (t, J = 7.6Hz, 1H), 3.42 (s, 3H), 1.54 (s, 9H). m/z (ESI, +ve) 359.1 (M+H) ⁺ .

Step 2: 1-(4-(5-chloro-7-fluoro-6-(3-(methylamino)-1-isoquinolinyl)-2,1-benzothiazo-3-yl)-1-piperazinyl)-2-propen-1-one. Similar procedure to Step 8-2 of Method 1. Purified by silica gel column chromatography (eluting with 0-14% MeOH in DCM) for 15 min. ¹ H NMR (400MHz, methanol-d ₄ )δ7.90(s,1H),7.63(d,J＝8.5Hz,1H),7.43(t,J＝7.4Hz,1H),7.24(d,J＝8.5Hz,1H),7.05(t,J＝7.4Hz,1H),6.72 -6.84(m,1H),6.67(s,1H),6.15-6.28(m,1H),5.68-5.81(m,1H),3.87-3.97(m,4H),3.63(m,4H),2.90(s,3H). m/z(ESI,+ve)482.0(M+H) ⁺ .

Example 25

1-(4-(6-(3-amino-1-isoquinolinyl)-5-chloro-7-fluoro-2,1-benzothiazo-3-yl)-1-piperazinyl)-2-propen-1-one

Step 1: 4-(6-(3-((tert-butoxycarbonyl)amino)isoquinolin-1-yl)-5-chloro-7-fluorobenzo[c]isothiazolyl-3-yl)piperazine-1-carboxylate tert-butyl ester. A procedure similar to Step 1 in Example 25, except that a solution of 1.3 M lithium isopropyl chloride in THF is used instead of the magnesium isopropyl chloride solution, and bis(2-methyl-2-propane)(1-bromo-3-isoquinolinyl)-2-iminodicarbonate (synthesized below) is used instead of (1-bromoisoquinolin-3-yl)(methyl)carboxylate tert-butyl ester. m/z(ESI,+ve) 614.2(M+H) ⁺ .

Synthesis of bis(2-methyl-2-propane)(1-bromo-3-isoquinolinyl)-2-iminodicarbonate: A solution of 1-bromoisoquinolinyl-3-amine (1.0 g, 4.48 mmol, Mabray Chemicals, Altlingham, UK) in DCM (50 mL) was added to Boc-anhydride (3.12 mL, 13.45 mmol) and DMAP (0.055 g, 0.448 mmol). The reaction mixture was heated to room temperature and stirred overnight. The reaction mixture was then diluted with saturated _NH4Cl aqueous solution (100 mL) and DCM (50 mL). The organic layer was separated, _dried over _Na2SO4 , filtered, and concentrated. Purification by silica gel column chromatography (eluting with 0–10% EtOAc in heptane) for 15 min yielded bis(2-methyl-2-propane)(1-bromo-3-isoquinolinyl)-2-iminodicarbonate. ^¹H NMR (400MHz, methanol- _d⁴ ) δ 8.36 (d, J = 8.5Hz, 1H), 8.03 (d, J = 8.1Hz, 1H), 7.77–7.92 (m, 3H), 1.44 (s, 18H). m/z (ESI, +ve) 267.0 (M+H) ⁺ .

Step 2: 1-(4-(6-(3-amino-1-isoquinolinyl)-5-chloro-7-fluoro-2,1-benzothiazol-3-yl)-1-piperazinyl)-2-propen-1-one. The procedure is similar to steps 8-1 and 8-2 of Method 1, except that in step 8-1, TFA in DCM is used instead of 4M HCl in dioxane/MeOH. Purification is performed by silica gel column chromatography (eluting with 0-12% MeOH in DCM). This material is then subjected to SFC purification: a diol column (21.2 x 250 mm, 5 μm) using 17% (20 mM NH3 in MeOH) in _{supercritical CO2} (total flow rate 7 g/min) to give 1-(4-(6-(3-amino-1-isoquinolinyl)-5-chloro-7-fluoro-2,1-benzothiazol-3-yl)-1-piperazinyl)-2-propen-1-one. ¹ H NMR (400MHz, methanol-d ₄ )δ7.85(s,1H),7.53(d,J＝8.5Hz,1H),7.39(t,J＝7.6Hz,1H),7.23(d,J＝8.5Hz,1H),7.03(t,J＝7.8Hz,1H),6.82(s,1H),6.7 1(dd,J＝10.8,16.8Hz,1H),6.2(dd,J＝1.5,16.8Hz,1H),5.70(dd,J＝1.5,10.8Hz,1H),3.82-3.93(m,4H),3.50-3.66(m,4H). m/z(ESI,+ve)468.0(M+H) ⁺ .

Example 26

1-(4-(6-(2-amino-4-quinolinyl)-5-chloro-7-fluoro-2,1-benzothiazo-3-yl)-1-piperazinyl)-2-propen-1-one

Step 1: 4-(5-chloro-7-fluoro-6-(tributyltinyl)benzo[c]isothiazolyl)piperazine-1-carboxylic acid tert-butyl ester. A solution of 4-(6-bromo-5-chloro-7-fluorobenzo[c]isothiazolyl)piperazine-1-carboxylic acid tert-butyl ester (intermediate D, 320 mg, 0.710 mmol), 1,1,1,2,2,2-hexabutyldistannane (824 mg, 1.420 mmol), and tetra(triphenylphosphine)palladium (0) (82 mg, 0.071 mmol, Strehm Chemical Company, Newburyport, Massachusetts, USA) in N,N-dimethylacetamide (5 mL) was heated in a sealed vial at 160 °C for 40 min in a microwave oven. The reaction mixture was diluted with saturated _NaHCO3 aqueous solution (50 mL), brine (50 mL), and EtOAc (100 mL). The organic layer was separated, _dried over _Na₂SO₄ , filtered, and concentrated. Purification by silica gel column chromatography (eluting with 0-30% EtOAc in heptane) yielded tert-butyl 4-(5-chloro-7-fluoro-6-(tributyltinyl)benzo[c]isothiazo-3-yl)piperazine-1-carboxylate. m/z (ESI,+ve) 662.2 (M+H) ⁺ .

Step 2: 2-Methyl-2-propane 4-(6-(2-(bis((((2-methyl-2-propane)oxy)carbonyl)amino)-4-quinolinyl)-5-chloro-7-fluoro-2,1-benzothiazolyl-3-yl)-1-piperazine carboxylate. (4-bromoquinolin-2-yl)-2-iminodicarbonate di-tert-butyl ester (19.2 mg, 0.045 mmol, prepared in a manner similar to bis(2-methyl-2-propane)(1-bromo-3-isoquinolinyl)-2-iminodicarbonate in Example 26, using 4-bromoquinolin-2-amine (Ark Pharmaceuticals, Arlington Heights, .I., USA) as the starting material), 4-(5-chloro-7-fluoro-6-(tributyltinyl)benzo[c]isothiazolyl) A solution of tert-butyl 3-(triphenylphosphine)piperazine-1-carboxylate (20 mg, 0.030 mmol), tetra(triphenylphosphine)palladium (0) (6.99 mg, 6.05 μmol, Strehm Chemical Company, Newburyport, Massachusetts, USA), copper iodide (I) (1.153 mg, 6.05 μmol), and cesium fluoride (13.79 mg, 0.091 mmol) in DMF (0.5 mL) was heated at 60 °C for 30 min in a sealed vial. The crude reaction mixture was diluted with saturated _NaHCO3 aqueous solution (50 mL) and EtOAc (100 mL). The organic layer was separated, _dried over _Na2SO4 , filtered, and concentrated. 2-Methyl-2-propane 4-(6-(2-(bis((((2-methyl-2-propane)oxy)carbonyl)amino)-4-quinolinyl)-5-chloro-7-fluoro-2,1-benzothiazo-3-yl)-1-piperazincarbamate was provided by silica gel column chromatography (eluting with 0-50% EtOAc in heptane). m/z (ESI,+ve) 714.2 (M+H) ⁺ .

Step 3: 1-(4-(6-(2-amino-4-quinolinyl)-5-chloro-7-fluoro-2,1-benzothiazol-3-yl)-1-piperazinyl)-2-propen-1-one. The procedure is similar to steps 8-1 and 8-2 of Method 1, except that in step 8-1, TFA in DCM is used instead of 4M HCl in dioxane/MeOH. ¹ H NMR (400MHz, methanol-d ₄ )δ7.90(s,1H),7.53(d,J＝8.2Hz,1H),7.42-7.49(m,1H),7.10(d,J＝8.0Hz,1H),7.03-7.08(m,J＝7.6Hz,1H),6.6 7-6.81(m,2H),6.19(dd,J＝1.8,16.6Hz,1H),5.72(dd,J＝1.8,10.6Hz,1H),3.87-3.93(m,4H),3.56-3.66(m,4H). m/z(ESI,+ve)468.0(M+H) ⁺ .

Example 27

1-(4-(3-(2-fluoro-6-hydroxyphenyl)-2-methyl-5-(2-(2-propyl)phenyl)pyrido[2,3-d]pyridazin-8-yl)-1-piperazinyl)-2-propen-1-one

Step 1: 6,7-Dihydropyrido[2,3-d]pyridazine-5,8-dione. Hydrazine (1.26 mL, 40.2 mmol) was added to a stirred solution of 2,3-pyridinedicarboxylic anhydride (4.00 g, 26.8 mmol) in ethanol (100 mL). The reaction mixture was refluxed for 16 h, then cooled to room temperature and concentrated under vacuum to give crude 6,7-dihydropyrido[2,3-d]pyridazine-5,8-dione, which was used directly in the next step. m/z (ESI) M+H: 164.1.

Step 2: 5,8-Dichloropyrido[2,3-d]pyridazine. Pyridine (4.57 mL, 53.7 mmol) was added to a mixture of crude 6,7-dihydropyrido[2,3-d]pyridazine-5,8-dione (4.38 g, 26.8 mmol) and phosphorus oxychloride (v) (20.1 mL, 215 mmol). The reaction mixture was stirred at 100 °C for 2 h. The reaction mixture was cooled and slowly poured into rapidly stirred water (250 mL) at about 10 °C. The aqueous suspension was stirred for 15 min and then extracted with EtOAc (250 mL). The organic layer was separated, washed with brine (200 mL), dried over _MgSO4 , filtered, and concentrated under vacuum. Chromatographic purification of the residue (0 to 100% EtOAc in silica gel and heptane) gave 5,8-dichloropyrido[2,3-d]pyridazine. ^¹H NMR (400MHz, chloroform-d) δ 9.41 (¹H,dd,J＝4.30, 1.56Hz) 8.65 (¹H,dd,J＝8.41, 1.56Hz) 8.02 (¹H,dd,J＝8.41, 4.30Hz). m/z (ESI) M+H: 200.0.

Step 3: 3,5-Dichloropyrido[2,3-d]pyridazine-8(7H)-one and 3,8-dichloropyrido[2,3-d]pyridazine-5(6H)-one. N-chlorosuccinimide (1268 mg, 9.50 mmol, TCI USA, Portland, Oregon, USA) was added to a stirred solution of 5,8-dichloropyrido[2,3-d]pyridazine (950 mg, 4.75 mmol) in acetic acid (20 mL), and the reaction mixture was heated to 100 °C for 16 h. Additional N-chlorosuccinimide (1268 mg, 9.50 mmol, TCI USA, Portland, Oregon, USA) was added, and the reaction mixture was stirred at 100 °C for another 4 h. Additional N-chlorosuccinimide (634 mg, 4.75 mmol, TCI USA, Portland, Oregon, USA) was added, and the reaction mixture was stirred for another 4 h. The reaction mixture was then diluted with water (75 mL) and extracted three times with EtOAc (100 mL). The combined organic layers were washed with brine (150 mL), dried over _MgSO4 , filtered, and concentrated under vacuum. Chromatographic purification of the residue (0 to 75% EtOAc in silica gel and heptane) yielded a mixture of 3,5-dichloropyrido[2,3-d]pyridazine-8(7H)-one compounds and positional isomers of 3,8-dichloropyrido[2,3-d]pyridazine-5(6H)-one. m/z (ESI) M+H: 215.9.

Step 4: 3,5,8-Trichloropyrido[2,3-d]pyridazine. Pyridine (2.024 mL, 23.79 mmol) was added to a mixture of isomers of 3,5-dichloropyrido[2,3-d]pyridazine-8(7H)-one and 3,8-dichloropyrido[2,3-d]pyridazine-5(6H)-one (2.57 g, 11.90 mmol) in phosphorus oxychloride (8.90 mL, 95 mmol). The reaction mixture was stirred at 100 °C for 1.5 h. The reaction mixture was cooled and slowly poured into rapidly stirred water (150 mL) at about 10 °C. The aqueous suspension was stirred for 15 min and then extracted with EtOAc (200 mL). The organic layer was separated, washed with brine (150 mL), dried over _MgSO4 , filtered, and concentrated under vacuum. Chromatographic purification of the residue (silica gel, 0 to 50% EtOAc in heptane) yielded 3,5,8-trichloropyridino[2,3-d]pyridazine. ^¹H NMR (400 MHz, chloroform-d): δ 9.27 (¹H,d, J = 2.35 Hz), 8.58 (¹H,d, J = 2.35 Hz). m/z (ESI): M+H: 233.9.

Step 5: 4-(3,5-Dichloropyrido[2,3-d]pyridazin-8-yl)piperazine-1-carboxylic acid tert-butyl ester. 1-Boc-piperazine (278 mg, 1.494 mmol) was added to a stirred mixture of 3,5,8-trichloropyrido[2,3-d]pyridazine (292 mg, 1.245 mmol) and triethylamine (0.350 mL, 2.491 mmol) in dimethyl sulfoxide (5 mL). The reaction mixture was stirred at room temperature for 3 h, then diluted with EtOAc (75 mL) and washed with saturated aqueous sodium bicarbonate solution (75 mL). The organic layer was separated, washed with brine (50 mL), dried over _MgSO₄ , filtered, and concentrated under vacuum. Chromatographic purification of the residue (silica gel, 0 to 25% acetone in heptane) yielded tert-butyl 4-(3,5-dichloropyridano[2,3-d]pyridazin-8-yl)piperazine-1-carboxylate, which was eluted first by the two positional isomers. ^¹H NMR (400 MHz, chloroform-d): δ 9.01 (¹H,d, J = 2.54 Hz), 8.43 (¹H,d, J = 2.54 Hz), 4.04–4.15 (4H,m), 3.64–3.70 (4H,m), 1.50 (9H,s). m/z (ESI): M+H: 384.0.

Step 6: 4-(3-chloro-5-(2-isopropylphenyl)pyrido[2,3-d]pyridazin-8-yl)piperazin-1-carboxylic acid tert-butyl ester. 4-(3,5-dichloropyrido[2,3-d]pyridazin-8-yl)piperazin-1-carboxylic acid tert-butyl ester (199 mg, 0.518 mmol), 2-isopropylphenylboronic acid (93 mg, 0.570 mmol, Alfaesa, Haverhill, Massachusetts, USA), tetra(triphenylphosphine)palladium (59.8 mg, 0.052 mmol, Strehm Chemical Company, Newburyport, Massachusetts, USA), and sodium carbonate (2 M aqueous solution, 1.036 mL, 2.072 mmol) were mixed in 1,4-dioxane (4 mL) under an argon atmosphere. The reaction mixture was stirred at 40 °C for 16 h. The reaction mixture was cooled to room temperature, diluted with EtOAc (50 mL), and washed with water (40 mL). The organic layer was separated, washed with brine (50 mL), dried over _MgSO₄ , filtered, and concentrated under vacuum. Chromatographic purification of the residue (0 to 50% EtOAc in silica gel and heptane) yielded a mixture of the starting material and the desired product. The mixture was subjected to the initial reaction conditions again, using a small amount of 2-isopropylphenylboronic acid (56 mg, 0.342 mmol, Alfaisa Company, Haverhill, Massachusetts, USA). The mixture was stirred at 40 °C for 16 h. Additional 2-isopropylphenylboronic acid (28 mg, 0.171 mmol, Alfaisa Company, Haverhill, Massachusetts, USA) was added, and the reaction mixture was stirred again for 6 h. The reaction mixture was cooled to room temperature, diluted with EtOAc (50 mL), and washed with water (40 mL). The organic layer was separated, washed with brine (50 mL), dried over _MgSO₄ , filtered, and concentrated under vacuum. Chromatographic purification of the residue (silica gel, 0 to 50% EtOAc in heptane) yielded tert-butyl 4-(3-chloro-5-(2-isopropylphenyl)pyrido[2,3-d]pyridazin-8-yl)piperazine-1-carboxylate. ¹ H NMR(400MHz, chloroform-d)δ8.95(1H,d,J＝2.35Hz)7.72(1H,d,J＝2.54Hz)7.45-7.53(2H,m)7.26-7.33(1H,m)7.16-7.21(1H,m) 4.04-4.23(4H,m)3.66-3.73(4H,m)2.67(1H,spt,J＝6.75Hz)1.48(9H,s)1.16(3H,d,J＝6.85Hz)1.03(3H,d,J＝6.85Hz). m/z(ESI)M+H:468.2.

Step 7: 4-(3-chloro-5-(2-isopropylphenyl)-2-methylpyrido[2,3-d]pyridazin-8-yl)piperazin-1-carboxylate tert-butyl ester. Methyllithium (1.6 M solution in diethyl ether, 0.137 mL, 0.219 mmol) was added to a stirred solution of 4-(3-chloro-5-(2-isopropylphenyl)pyrido[2,3-d]pyridazin-8-yl)piperazin-1-carboxylate (93 mg, 0.199 mmol) in tetrahydrofuran (1 mL). The reaction mixture was stirred at -78 °C for 5 min, then allowed to rise to 0 °C and stirred for 30 min. The reaction mixture was cooled back to -78 °C, and additional methyllithium (1.6 M solution in diethyl ether, 0.068 mL, 0.109 mmol) was added. The reaction mixture was stirred at -78°C for 5 min, then allowed to rise to 0°C and stirred for another 15 min. The reaction mixture was quenched with water (20 mL) and extracted with EtOAc (30 mL). The organic layer was separated, washed with brine (20 mL), dried over _MgSO4 , filtered, and concentrated under vacuum to give crude tert-butyl 4-(3-chloro-5-(2-isopropylphenyl)-2-methyl-1,2-dihydropyrido[2,3-d]pyridazin-8-yl)piperazine-1-carboxylate. m/z (ESI) M+H: 484.3.

4,5-Dichloro-3,6-dioxo-1,4-cyclohexadiene-1,2-dionitrile (45.0 mg, 0.198 mmol) was added to a stirred mixture of crude 4-(3-chloro-5-(2-isopropylphenyl)-2-methyl-1,2-dihydropyrido[2,3-d]pyridazin-8-yl)piperazine-1-carboxylic acid tert-butyl ester (96 mg, 0.198 mmol) in dichloromethane (2 mL). The reaction mixture was stirred at room temperature for 10 min. The reaction mixture was diluted with DCM (30 mL) and washed with water (20 mL). The organic layer was separated, dried over _MgSO4 , filtered, and concentrated under vacuum. Chromatographic purification of the residue (silica gel, 0 to 50% EtOAc in heptane) yielded tert-butyl 4-(3-chloro-5-(2-isopropylphenyl)-2-methylpyridano[2,3-d]pyridazin-8-yl)piperazine-1-carboxylate. ¹ H NMR(400MHz, chloroform-d)δ7.72(1H,s)7.51-7.55(2H,m)7.32-7.37(1H,m)7.22-7.27(1H,m)4.08-4.25(4H,m)3.71 -3.79(4H,m)2.87(3H,s)2.73(1H,spt,J＝6.68Hz)1.54(9H,s)1.21(3H,d,J＝6.85Hz)1.07(3H,d,J＝6.85Hz). m/z(ESI)M+H:482.1.

Step 8: 4-(3-(2-fluoro-6-hydroxyphenyl)-5-(2-isopropylphenyl)-2-methylpyridano[2,3-d]pyridazin-8-yl)piperazin-1-carboxylic acid tert-butyl ester. 4-(3-chloro-5-(2-isopropylphenyl)-2-methylpyridano[2,3-d]pyridazin-8-yl)piperazin-1-carboxylic acid tert-butyl ester (78 mg, 0.162 mmol), (2-fluoro-6-hydroxyphenyl)boronic acid (101 mg, 0.647 mmol, Combilot), Sphos Pd G3 (14.00 mg, 0.016 mmol), and sodium carbonate (2 M aqueous solution, 0.324 mL, 0.647 mmol) were mixed in 1,2-dimethoxyethane (1 mL) under an argon atmosphere and then heated at 80 °C for 2.5 h. The reaction mixture was cooled, diluted with EtOAc (30 mL), and washed with water (25 mL). The organic layer was separated, washed with brine (25 mL), dried over _MgSO4 , filtered, and concentrated under vacuum. Chromatographic purification of the residue (0 to 50% EtOAc in silica gel and heptane) gave tert-butyl 4-(3-(2-fluoro-6-hydroxyphenyl)-5-(2-isopropylphenyl)-2-methylpyridano[2,3-d]pyridazin-8-yl)piperazine-1-carboxylate (66 mg, 0.118 mmol, 73.1% yield). m/z (ESI) M+H: 558.2.

Step 9: 3-Fluoro-2-(5-(2-isopropylphenyl)-2-methyl-8-(piperazin-1-yl)pyrido[2,3-d]pyridazin-3-yl)phenol. Trifluoroacetic acid (0.2 mL, 2.68 mmol) was added to a stirred solution of 4-(3-(2-fluoro-6-hydroxyphenyl)-5-(2-isopropylphenyl)-2-methylpyrido[2,3-d]pyridazin-8-yl)piperazin-1-carboxylic acid tert-butyl ester (64 mg, 0.115 mmol) in dichloromethane (0.5 mL). The reaction mixture was stirred at room temperature for 30 min. The reaction mixture was diluted with DCM (30 mL) and quenched with saturated sodium bicarbonate aqueous solution (20 mL). The organic layer was separated, dried over _MgSO4 , filtered, and concentrated under vacuum to obtain crude 3-fluoro-2-(5-(2-isopropylphenyl)-2-methyl-8-(piperazin-1-yl)pyrido[2,3-d]pyridazin-3-yl)phenol. m/z(ESI)M+H: 458.1.

Step 10: 1-(4-(3-(2-fluoro-6-hydroxyphenyl)-5-(2-isopropylphenyl)-2-methylpyridano[2,3-d]pyridazin-8-yl)piperazin-1-yl)prop-2-en-1-one. Acryloyl chloride (9.45 μl, 0.116 mmol) was added to a stirred mixture of 3-fluoro-2-(5-(2-isopropylphenyl)-2-methyl-8-(piperazin-1-yl)pyridano[2,3-d]pyridazin-3-yl)phenol (53 mg, 0.116 mmol) and triethylamine (0.049 mL, 0.348 mmol) in dichloromethane (1 mL) at 0 °C. The reaction mixture was stirred at 0 °C for 10 min. The reaction mixture was diluted with DCM (25 mL) and quenched with a saturated aqueous sodium bicarbonate solution (20 mL). The organic layer was separated, dried over _MgSO₄ , filtered, and concentrated under vacuum. Chromatographic purification of the residue (silica gel, 0 to 100% EtOAc in heptane) gave 1-(4-(3-(2-fluoro-6-hydroxyphenyl)-2-methyl-5-(2-(2-propyl)phenyl)pyrido[2,3-d]pyridazin-8-yl)-1-piperazinyl)-2-propen-1-one. ^¹H NMR (400 MHz, chloroform-d) δ 9.51 (0.6 H, br s) 8.98 (0.4 H, br s) s)7.63(0.4H,s)7.58(0.6H,s)7.35-7.43(2H,m)7.10-7.26(3H,m)6.78(1H,dd,J＝16.63,8.22Hz)6.59-6.71(2H,m)6.36(1H,dd,J＝16.82,1 .57Hz)5.78(1H,dd,J＝10.56,1.37Hz)4.10-4.38(4H,m)3.80-4.03(4H,m)2.60-2.72(1H,m)2.61(1.2H,s)2.59(1.8H,s)0.91-1.08(6H,m). m/z(ESI)M+H:512.3.

Example 28

1-(4-(7-chloro-6-(2-fluoro-6-hydroxyphenyl)-4-((1R)-1-phenylethyl)-1-phthalazinyl)-1-piperazinyl)-2-propen-1-one and 1-(4-(7-chloro-6-(2-fluoro-6-hydroxyphenyl)-4-((1S)-1-phenylethyl)-1-phthalazinyl)-1-piperazinyl)-2-propen-1-one

A mixture of α-methylbenzyl zinc bromide (0.5 M in THF, 492 μl, 0.246 mmol), tetrakis(triphenylphosphine)palladium (5.68 mg, 4.92 μmol, Strehm Chemical Company, Newburyport, Massachusetts, USA) and 1-(4-(4,7-dichloro-6-(2-fluoro-6-hydroxyphenyl)phthalazin-1-yl)piperazin-1-yl)prop-2-en-1-one (intermediate I, 22 mg, 0.049 mmol) was stirred in a sealed vial at 60 °C for 16 h. The reaction mixture was concentrated, and the residue was purified by chromatography (0 to 100% EtOAc in silica gel and heptane) to give a mixture of 1-(4-(7-chloro-6-(2-fluoro-6-hydroxyphenyl)-4-((1R)-1-phenylethyl)-1-phthalazinyl)-1-piperazinyl)-2-propen-1-one and 1-(4-(7-chloro-6-(2-fluoro-6-hydroxyphenyl)-4-((1S)-1-phenylethyl)-1-phthalazinyl)-1-piperazinyl)-2-propen-1-one). ¹ H NMR (400Mhz, methanol-d ₄ )δ8.27(1H,s)8.15(0.33H,s)8.10(0.67H,s)7.19-7.31(5H,m)7.10-7.16(1H,m)6.86(1H,dd,J＝16.73,10.66Hz)6.62-6.78(2H,m)6.2 7(1H,dd,J＝16.82,1.96Hz)5.80(1H,dd,J＝10.66,1.86Hz)4.94-5.01(1H,m)3.93-4.03(4H,m)3.49-3.60(4H,m)1.81(3H,d,J＝7.04Hz). m/z(ESI)M+H:517.1.

Example 29

1-(4-(7-chloro-4-(4-fluorobenzyl)-6-(2-fluoro-6-hydroxyphenyl)-1-phthalazinyl)-1-piperazinyl)-2-propen-1-one

In a sealed vial under an argon atmosphere, 0.089 mL (0.044 mmol) of 0.5 M zinc chloride in THF was added to a stirred mixture of 1-(4-(4,7-dichloro-6-(2-fluoro-6-hydroxyphenyl)phthalazin-1-yl)piperazin-1-yl)prop-2-en-1-one (intermediate I, 18 mg, 0.040 mmol) and tetra(triphenylphosphine)palladium (4.65 mg, 4.02 μmol, Strehm Chemical Company, Newburyport, Massachusetts, USA) in tetrahydrofuran (0.1 mL). The reaction mixture was stirred at room temperature for 2 h, then heated to 40 °C for 3 h. Additional 0.089 mL (0.044 mmol) of 4-fluorobenzyl zinc chloride was added, and the reaction mixture was stirred again at 40 °C for 16 h. Additional 4-fluorobenzyl zinc chloride (0.089 mL, 0.044 mmol) was added, and the reaction mixture was heated to 60 °C and stirred for 6 h. The reaction mixture was concentrated under vacuum. Chromatographic purification of the residue (0 to 100% EtOAc in silica gel and heptane) gave 1-(4-(7-chloro-4-(4-fluorobenzyl)-6-(2-fluoro-6-hydroxyphenyl)-1-phthalazinyl)-1-piperazinyl)-2-propen-1-one. ^¹H NMR (400MHz, methanol- _d⁴ ) δ 8.32 (¹H, s) 8.19 (¹H, s) 7.26-7.34 (³H, m) 6.98 (²H, t, J = 8.71Hz) 6.69-6.91 (³H, m) 6.28 (¹H, dd, J = 16.92, 1.86Hz) 5.82 (¹H, dd, J = 10.56, 1.76Hz) 4.54-4.65 (²H, m) 3.99 (⁴H, m) 3.58 (⁴H, m). m/z (ESI) M+H: 521.2.

Examples 30 and 31

2-(1-(4-acryloyl-1-piperazinyl)-7-chloro-4-phenyl-6-phthalazinyl)-3-fluorophenol (Example 30) and 2-(4-(4-acryloyl-1-piperazinyl)-7-chloro-1-phenyl-6-phthalazinyl)-3-fluorophenol (Example 31)

Step 1: 1,4,6,7-Tetrachlorophthalazine (Intermediate L). Pyridine (431 μl, 5.28 mmol) was added to a stirred mixture of 6,7-dichloro-2,3-dihydrophthalazine-1,4-dione (Intermediate G, 610 mg, 2.64 mmol) in phosphorus oxychloride (2.4 mL, 26.4 mmol). The reaction mixture was heated to 100 °C for 2 h, then cooled and slowly poured into rapidly stirred water (75 mL) at about 10 °C. The resulting suspension was filtered, and the solid was washed with water to give 1,4,6,7-tetrachlorophthalazine. ^¹H NMR (400 MHz, chloroform-d) δ 8.43 (2H, s). m/z (ESI) M+H: 266.9.

Step 2: 4-(4,6,7-trichlorophthalazine-1-yl)piperazine-1-carboxylic acid tert-butyl ester (intermediate M). 1-Boc-piperazine (340 mg, 1.824 mmol) was added to a stirred mixture of 1,4,6,7-tetrachlorophthalazine (intermediate L, 543 mg, 2.027 mmol) and triethylamine (0.846 mL, 6.08 mmol) in dichloromethane (8 mL). The reaction mixture was stirred at room temperature for 2 days. An additional 1-boc-piperazine (340 mg, 1.824 mmol) was added, and the reaction mixture was stirred again at room temperature for 23 h. The reaction mixture was quenched with saturated sodium bicarbonate aqueous solution (20 mL) and extracted with DCM (30 mL). The organic layer was separated, washed with brine (20 mL), dried over _MgSO₄ , filtered, and concentrated under vacuum. Chromatographic purification of the residue (silica gel, 0–50% EtOAc in heptane) yielded tert-butyl 4-(4,6,7-trichlorophthalazine-1-yl)piperazine-1-carboxylate. ^¹H NMR (400 MHz, chloroform-d): δ 8.35 (¹H, s) 8.12 (¹H, s) 3.68–3.75 (4H, m) 3.45–3.52 (4H, m) 1.51 (9H, s). m/z (ESI): M+H: 417.0.

Step 3: 4-(6,7-Dichloro-4-phenylphthalazin-1-yl)piperazin-1-carboxylate tert-butyl ester. 4-(4,6,7-trichlorophthalazin-1-yl)piperazin-1-carboxylate tert-butyl ester (intermediate M, 95 mg, 0.227 mmol), tetra(triphenylphosphine)palladium (26.3 mg, 0.023 mmol, Strehm Chemical Company, Newburyport, Massachusetts, USA), phenylboronic acid (27.7 mg, 0.227 mmol), and sodium carbonate (2M aqueous solution, 0.341 mL, 0.682 mmol) were mixed in a sealed vial in 1,4-dioxane (1 mL) under an argon atmosphere. The reaction mixture was stirred at 40 °C for 24 h. Additional tetra(triphenylphosphine)palladium (26.3 mg, 0.023 mmol) and phenylboronic acid (13.5 mg, 0.113 mmol) were added, and the reaction mixture was stirred again at 40 °C for 24 h. The reaction mixture was quenched with saturated sodium bicarbonate aqueous solution (20 mL) and extracted with EtOAc (25 mL). The organic layer was separated, washed with brine (20 mL), dried over _MgSO4 , filtered, and concentrated under vacuum. Chromatographic purification of the residue (0 to 100% EtOAc in silica gel and heptane) gave tert-butyl 4-(6,7-dichloro-4-phenylphthalazin-1-yl)piperazine-1-carboxylate. ^¹H NMR (400 MHz, chloroform-d) δ 8.13 (1H, s) 8.07 (1H, s) 7.62–7.67 (2H, m) 7.50–7.55 (3H, m) 3.65–3.74 (4H, m) 3.44–3.53 (4H, m) 1.47 (9H, s). m/z (ESI) M+H: 459.1.

Step 4: 6,7-Dichloro-1-phenyl-4-(piperazin-1-yl)phthalazine. Tert-butyl 4-(6,7-dichloro-4-phenylphthalazin-1-yl)piperazin-1-carboxylate (68 mg, 0.148 mmol) was stirred in trifluoroacetic acid (1 mL, 13.46 mmol) at room temperature for 20 min. The reaction mixture was quenched with saturated sodium bicarbonate aqueous solution (20 mL) and extracted twice with DCM (25 mL). The organic layer was separated, dried over _MgSO₄ , filtered, and concentrated under vacuum to give crude 6,7-dichloro-1-phenyl-4-(piperazin-1-yl)phthalazine, which was used directly in the next step. m/z (ESI) M+H: 359.0.

Step 5: 1-(4-(6,7-dichloro-4-phenylphthalazin-1-yl)piperazin-1-yl)prop-2-en-1-one. Acryloyl chloride (0.013 mL, 0.162 mmol) was added to a stirred mixture of 6,7-dichloro-1-phenyl-4-(piperazin-1-yl)phthalazine (53 mg, 0.148 mmol) and triethylamine (0.062 mL, 0.443 mmol) in dichloromethane (1 mL). The reaction mixture was stirred at room temperature for 30 min. The reaction mixture was quenched with saturated aqueous sodium bicarbonate solution (15 mL) and extracted with DCM (20 mL). The organic layer was separated, dried over _MgSO4 , filtered, and concentrated under vacuum. Chromatographic purification of the residue (silica gel, 0 to 100% EtOAc in heptane) gave 1-(4-(6,7-dichloro-4-phenylphthalazin-1-yl)piperazin-1-yl)prop-2-en-1-one. ^¹H NMR (400 MHz, chloroform-d) δ 8.20 (¹H, s) 8.14 (¹H, s) 7.66–7.75 (2H, m) 7.54–7.62 (3H, m) 6.66 (¹H, dd, J = 16.63, 10.37 Hz) 6.37 (¹H, dd, J = 16.82, 1.96 Hz) 5.78 (¹H, dd, J = 10.56, 1.96 Hz) 3.85–4.04 (¹H, m) 3.53–3.72 (¹H, m). m/z(ESI)M+H:431.2.

Step 6: 2-(1-(4-acryloyl-1-piperazinyl)-7-chloro-4-phenyl-6-phthalazinyl)-3-fluorophenol and 2-(4-(4-acryloyl-1-piperazinyl)-7-chloro-1-phenyl-6-phthalazinyl)-3-fluorophenol. 1-(4-(6,7-dichloro-4-phenylphthalazin-1-yl)piperazin-1-yl)prop-2-en-1-one (43 mg, 0.104 mmol), 2-fluoro-6-hydroxyphenylboronic acid (17.84 mg, 0.114 mmol, Combilux, San Diego, California, USA), Sphos Pd G3 (9.00 mg, 10.40 μmol), and sodium carbonate (2 M aqueous solution, 0.156 mL, 0.312 mmol) were mixed in a sealed vial in 1,2-dimethoxyethane (0.5 mL) under an argon atmosphere. The reaction mixture was stirred at 60 °C for 3 h. Additional 2-fluoro-6-hydroxyphenylboronic acid (8.92 mg, 0.057 mmol, Combilux, San Diego, California, USA) and SPhos Pd G3 (9.00 mg, 10.40 μmol) were added, and the reaction mixture was stirred at 60 °C for another 2 h. The reaction mixture was quenched with saturated sodium bicarbonate aqueous solution (15 mL) and extracted with EtOAc (20 mL). The organic layer was separated, washed with brine (10 mL), dried over _MgSO4 , filtered, and concentrated under vacuum. Chromatographic purification of the residue (0 to 100% EtOAc in silica gel, heptane) gave a mixture of two positional isomers. Reversed-phase preparative chromatography (XBridge Prep C185 μm OBD, 150 × 30 mm; 35 to 55% (0.1% TFA in water) in (0.1% TFA in acetonitrile); flow rate = 30 mL/min) gave the separated positional isomers. Fractions containing individual positional isomers were neutralized with saturated aqueous sodium bicarbonate solution and extracted with DCM, and the organic extract was concentrated under vacuum. The separated positional isomers were further purified individually by column chromatography (silica gel, 0 to 100% EtOAc in heptane). 2-(1-(4-acryloyl-1-piperazinyl)-7-chloro-4-phenyl-6-phthalazinyl)-3-fluorophenol (Example 30) is the first positional isomer eluted from reversed-phase preparative chromatography. ¹ H NMR(400MHz, chloroform-d)δ8.21(1H,s)8.06(1H,s)7.62-7.69(2H,m)7.45-7.51(3H,m)7.24-7.32(1H,m)6.81-6.90(1H,m)6.75(1H,t,J＝8.41 Hz)6.65(1H,dd,J＝16.82,10.56Hz)6.38(1H,dd,J＝16.82,1.76Hz)5.79(1H,dd,J＝10.56,1.76Hz)3.86-4.02(4H,m)3.57-3.76(4H,m). m/z(ESI)M+H:489.0. 2-(4-(4-acryloyl-1-piperazinyl)-7-chloro-1-phenyl-6-phthalazinyl)-3-fluorophenol (Example 31) is the second-position isomer eluted from the reverse-phase column. ¹ H NMR(400MHz, chloroform-d)δ8.15(1H,s)8.12(1H,s)7.68-7.73(2H,m)7.53-7.58(3H,m)7.30(1H,brtd,J＝8.22,6.65Hz)6.88(1H,d,J＝8.22Hz)6.78(1H, t,J＝8.61Hz)6.57(1H,dd,J＝16.82,10.56Hz)6.28(1H,dd,J＝16.73,1.66Hz)5.71(1H,dd,J＝10.56,1.56Hz)3.78-3.89(4H,m)3.51-3.73(4H,m). m/z(ESI)M+H:489.1.

Examples 32 and 33

2-(1-(4-acryloyl-1-piperazinyl)-7-chloro-4-methoxy-6-phthalazinyl)-3-fluorophenol (Example 32) and 2-(4-(4-acryloyl-1-piperazinyl)-7-chloro-1-methoxy-6-phthalazinyl)-3-fluorophenol (Example 33)

Examples 32 and 33 were prepared using a method similar to that of Examples 30 and 31, but step 3 was changed as follows:

Step 3: 4-(6,7-dichloro-4-methoxyphthalazin-1-yl)piperazin-1-carboxylate tert-butyl ester. 4-(4,6,7-trichlorophthalazin-1-yl)piperazin-1-carboxylate tert-butyl ester (intermediate M, 198 mg, 0.474 mmol) and sodium methoxide (25% solution in methanol, 2 mL, 8.75 mmol) were mixed in a sealed vial. The reaction mixture was stirred at 60 °C for 2 h. The reaction mixture was quenched with saturated sodium bicarbonate aqueous solution (25 mL) and extracted with EtOAc (25 mL). The organic layer was separated, washed with brine (20 mL), dried over _MgSO4 , filtered, and concentrated under vacuum. Chromatographic purification of the residue (silica gel, 0 to 50% EtOAc in heptane) gave 4-(6,7-dichloro-4-methoxyphthalazin-1-yl)piperazin-1-carboxylate tert-butyl ester. ^¹H NMR (400MHz, chloroform-d) δ 8.29 (¹H, s) 8.08 (¹H, s) 4.22 (³H, s) 3.68–3.73 (⁴H, m) 3.33–3.38 (⁴H, m) 1.51 (⁹H, s). m/z (ESI) M+H: 413.1.

From step 6: First elution position isomer: 2-(1-(4-acryloyl-1-piperazinyl)-7-chloro-4-methoxy-6-phthalazinyl)-3-fluorophenol (Example 32) ¹ H NMR(400MHz, chloroform-d)δ8.23(1H,s)8.11(1H,s)7.32(1H,td,J＝8.31,6.46Hz)6.88(1H,d,J＝8.22Hz)6.77-6.83(1H,m)6.65(1H,dd,J ＝16.82,10.56Hz)6.37(1H,dd,J＝16.82,1.76Hz)5.79(1H,dd,J＝10.47,1.86Hz)4.18(3H,s)3.79-4.05(4H,m)3.34-3.54(4H,m). m/z(ESI)M+H:443.1.

Second elution position isomer: 2-(4-(4-acryloyl-1-piperazinyl)-7-chloro-1-methoxy-6-phthalazinyl)-3-fluorophenol (Example 33). ¹ H NMR(400MHz, chloroform-d)δ8.32(1H,s)8.01(1H,s)7.32(1H,td,J＝8.27,6.55Hz)6.89(1H,d,J＝8.22Hz)6.77-6.83(1H,m)6.60(1H,dd,J ＝17.02,10.56Hz)6.30(1H,dd,J＝16.82,1.76Hz)5.75(1H,dd,J＝10.56,1.76Hz)4.22(3H,s)3.67-3.98(4H,m)3.25-3.55(4H,m). m/z(ESI)M+H:443.1.

Example 34:

1-(4-Acryloyl-1-piperazinyl)-4-benzyl-6,7-dichlorophthalazine

Example 34 was prepared using a method similar to that of Examples 30 and 31, except that step 6 was omitted, and steps 2 and 3 were modified as follows:

Steps 2 and 3: 4-(4-benzyl-6,7-dichlorophthalazine-1-yl)piperazine-1-carboxylic acid tert-butyl ester. Benzyl zinc bromide (0.5 M in THF, 1.926 mL, 0.963 mmol) was added under an argon atmosphere to a vial containing 1,4,6,7-tetrachlorophthalazine (intermediate L, 258 mg, 0.963 mmol) and tetra(triphenylphosphine)palladium (111 mg, 0.096 mmol, Strehm Chemical Company, Newburyport, Massachusetts, USA). The reaction mixture was stirred at room temperature for 16 h. 1-Boc-piperazine (1.79 g, 9.63 mmol) was added, and the reaction mixture was stirred at 60 °C for 5 h. The reaction mixture was quenched with a saturated aqueous sodium bicarbonate solution (40 mL) and extracted with EtOAc (50 mL). The organic layer was separated, washed with brine (40 mL), dried over _MgSO₄ , filtered, and concentrated under vacuum. Chromatographic purification of the residue (silica gel, 0 to 50% EtOAc in heptane) gave tert-butyl 4-(4-benzyl-6,7-dichlorophthalazine-1-yl)piperazine-1-carboxylate. ^¹H NMR (400 MHz, chloroform-d) δ 8.11 (¹H, s) 8.10 (¹H, s) 7.27–7.35 (4H, m) 7.20–7.25 (¹H, m) 4.59 (2H, s) 3.69–3.74 (4H, m) 3.44–3.49 (4H, m) 1.52 (9H, s). m/z (ESI) M+H: 473.1.

From step 5: 1-(4-Acryloyl-1-piperazinyl)-4-benzyl-6,7-dichlorophthalazine. ^¹H NMR (400MHz, chloroform-d) δppm 8.13 (¹H,s) 8.12 (¹H,s) 7.28-7.36 (4H,m) 7.20-7.26 (¹H,m) 6.65 (¹H,dd,J＝16.82,10.56Hz) 6.37 (¹H,dd,J＝16.82,1.57Hz) 5.78 (¹H,dd,J＝10.56,1.56Hz) 4.61 (2H,s) 3.83-4.01 (4H,m) 3.48-3.62 (4H,m). m/z (ESI) M+H: 427.1.

Examples 35 and 36:

2-(1-(4-acryloyl-1-piperazinyl)-4-benzyl-7-chloro-6-phthalazinyl)-3-fluorophenol (Example 35) and 2-(4-(4-acryloyl-1-piperazinyl)-1-benzyl-7-chloro-6-phthalazinyl)-3-fluorophenol (Example 36)

1-(4-(4-benzyl-6,7-dichlorophthalazin-1-yl)piperazin-1-yl)prop-2-en-1-one. 2-Fluoro-6-hydroxyphenylboronic acid (12.77 mg, 0.082 mmol, Combibloc, San Diego, California, USA), SPhos Pd G3 (7.09 mg, 8.19 μmol), and sodium carbonate (2 M aqueous solution, 0.123 mL, 0.246 mmol) were mixed in a sealed vial under argon atmosphere in 1,2-dimethoxyethane (0.3 mL). The reaction mixture was stirred at 60 °C for 1 h. The reaction mixture was quenched with saturated sodium bicarbonate aqueous solution (15 mL) and extracted with EtOAc (20 mL). The organic layer was separated, washed with brine (10 mL), dried over _MgSO4 , filtered, and concentrated under vacuum. Chromatographic purification of the residue (silica gel, 0 to 100% EtOAc in heptane) yielded a mixture of two positional isomers. Reversed-phase preparative chromatography (XBridge Prep C18 5 μm OBD, 150 × 30 mm; 20 to 90% (0.1% TFA in water) in (0.1% TFA in acetonitrile); flow rate = 30 mL/min) yielded partially separated positional isomers. The fraction containing the positional isomers was neutralized with saturated aqueous sodium bicarbonate solution and extracted with DCM, and the organic extract was concentrated under vacuum. 2-(1-(4-acryloyl-1-piperazinyl)-4-benzyl-7-chloro-6-phthalazinyl)-3-fluorophenol (Example 35) was the first positional isomer eluted during reversed-phase preparative chromatography and contained approximately 36% of the second positional isomer to be eluted. ^1H NMR (400MHz, chloroform-d) δ 8.13 (1H,s) 8.11 (1H,s) 7.12-7.37 (6H,m) 6.91 (1H,d,J＝8.22Hz) 6.77 (1H,t,J＝8.61Hz) 6.64 (1H,dd,J＝16.82,10.56Hz) 6.37 (1H,dd,J＝16.82,1.76Hz) 5.79 (1H,dd,J＝10.56,1.96Hz) 4.55 (2H,s) 3.34-4.01 (8H,m). m/z(ESI)M+H:503.1. 2-(4-(4-acryloyl-1-piperazinyl)-1-benzyl-7-chloro-6-phthalazinyl)-3-fluorophenol (Example 36) is the second-position isomer to be eluted. ^¹H NMR (400MHz, chloroform-d) δ 8.12 (¹H, s) 8.05 (¹H, s) 7.26-7.36 (⁵H, m) 7.19-7.24 (¹H, m) 6.93 (¹H, d, J = 8.41Hz) 6.76 (¹H, t, J = 8.31Hz) 6.58 (¹H, dd, J = 16.82, 10.76Hz) 6.28 (¹H, dd, J = 16.82, 1.76Hz) 5.75 (¹H, dd, J = 10.56, 1.76Hz) 4.54 (²H, s) 3.32-3.93 (⁸H, m). m/z (ESI) M+H: 50 3.1.

Example 37

1-(4-Acryloyl-1-piperazinyl)-4-benzyl-6-chloro-7-(5-methyl-1H-indazol-4-yl)phthalazine and 1-(4-Acryloyl-1-piperazinyl)-4-benzyl-7-chloro-6-(5-methyl-1H-indazol-4-yl)phthalazine

Example 37 was prepared using a method similar to Examples 35 and 36, but with 5-methyl-1h-indazole-4-ylboronic acid (Combilux Corporation, San Diego, California, USA) instead of 2-fluoro-6-hydroxyphenylboronic acid. In this example, the two positional isomers were not separated. ^¹H NMR (400 MHz, chloroform-d) δ 8.23 (0.6H, s) 8.22 (0.4H, s) 8.02 (0.4H, s) 8.00 (0.6H, s) 7.19-7.57 (8H, m) 6.68 (0.4H, dd, J = 16.82, 10.56 Hz) 6.60 (0.6H, dd, J = 16.82, 10.56 Hz) 6.38 (0.4H, dd, J = 16.63, 1.76 H) z)6.32(0.6H,dd,J＝16.82,1.76Hz)5.79(0.4H,dd,J＝10.56,1.76Hz)5.73(0.6H,dd,J＝10.56,1.7 6Hz)4.67(1.2H,s)4.60(0.8H,s)3.74-4.06(4H,m)3.46-3.70(4H,m)2.21(1.8H,s)2.06(1.2H,s). m/z(ESI)M+H:523.

Example 38

6-Chloro-1-(cyclopropylmethyl)-7-(2-fluoro-6-hydroxyphenyl)-4-(4-(2-acryloyl)-1-piperazinyl)pyrido[2,3-d]pyrimidin-2(1H)-one

The starting materials used in Example 38 were prepared using steps 1-4 of Method 8 with reagents 2,5,6-trichloronicotinic acid (step 1), aminomethylcyclopropane (step 2), 2-fluoro-6-hydroxyphenylboronic acid (step 4, Combibloc, San Diego, California, USA) and sodium carbonate (step 4).

Step 1: 7-(2-((tert-butyldiphenylsilyl)oxy)-6-fluorophenyl)-6-chloro-1-(cyclopropylmethyl)pyrido[2,3-d]pyrimidine-2,4(1H,3H)-dione. Tert-butylchlorodiphenylsilane (0.036 mL, 0.139 mmol) was added to a stirred mixture of 6-chloro-1-(cyclopropylmethyl)-7-(2-fluoro-6-hydroxyphenyl)pyrido[2,3-d]pyrimidine-2,4(1H,3H)-dione (42 mg, 0.116 mmol) and triethylamine (0.065 mL, 0.464 mmol) in acetonitrile (0.5 mL). The reaction mixture was stirred at room temperature for 1 h. The reaction mixture was quenched with saturated NH4Cl aqueous solution (25 mL) and extracted with EtOAc (30 mL). The organic layer was separated, washed with brine (25 mL), dried over _MgSO₄ , filtered, and concentrated under vacuum to obtain crude 7-(2-((tert-butyldiphenylsilyl)oxy)-6-fluorophenyl)-6-chloro-1-(cyclopropylmethyl)pyrido[2,3-d]pyrimidine-2,4(1H,3H)-dione, which was used directly in the next step. m/z (ESI) M+H: 599.8.

Step 2: 4-(4-Acryloylpiperazin-1-yl)-7-(2-((tert-butyldiphenylsilyl)oxy)-6-fluorophenyl)-6-chloro-1-(cyclopropylmethyl)pyrido[2,3-d]pyrimidin-2(1H)-one. Phosphorus oxychloride (0.087 mL, 0.933 mmol) was added to a stirred mixture of crude 7-(2-((tert-butyldiphenylsilyl)oxy)-6-fluorophenyl)-6-chloro-1-(cyclopropylmethyl)pyrido[2,3-d]pyrimidin-2,4(1H,3H)-dione (70 mg, 0.117 mmol), triethylamine (0.295 mL, 2.099 mmol), and 1H-benzo[d][1,2,3]triazole (167 mg, 1.400 mmol) in acetonitrile (2 mL). The reaction mixture was stirred at 80 °C for 4 h. The reaction mixture was concentrated under vacuum. The resulting residue was absorbed into 1,2-dichloroethane (2 mL), and triethylamine (0.295 mL, 2.099 mmol) and 1-(piperazin-1-yl)prop-2-en-1-one (32.7 mg, 0.233 mmol, Ennover Chemicals, Bridgewater, NJ, USA) were added. The reaction mixture was stirred at room temperature for 16 h. Additional triethylamine (0.148 mL, 1.050 mmol) and 1-(piperazin-1-yl)prop-2-en-1-one (32.7 mg, 0.233 mmol, Ennover Chemicals, Bridgewater, NJ, USA) were added, and the reaction mixture was stirred at room temperature for 1 h, then heated to 60 °C and stirred for 4 h. The reaction mixture was diluted with saturated _NaHCO3 aqueous solution (40 mL) and extracted with DCM (50 mL). The organic layer was separated, dried over _MgSO4 , filtered, and concentrated under vacuum. The resulting residue was reabsorbed into 1,2-dichloroethane (2 mL), and triethylamine (0.295 mL, 2.099 mmol) and 1-(piperazin-1-yl)prop-2-en-1-one (32.7 mg, 0.233 mmol, Ennover Chemicals, Bridgewater, NJ, USA) were added. The reaction mixture was stirred at 60 °C for 6 h. The reaction mixture was diluted with saturated _NaHCO3 aqueous solution (40 mL) and extracted with DCM (50 mL). The organic layer was separated, dried over _MgSO4 , filtered, and concentrated under vacuum. Chromatographic purification of the residue (silica gel, 0 to 100% (3:1 EtOAc/EtOH) in heptane) yielded 4-(4-acryloylpiperazin-1-yl)-7-(2-((tert-butyldiphenylsilyl)oxy)-6-fluorophenyl)-6-chloro-1-(cyclopropylmethyl)pyrido[2,3-d]pyrimidin-2(1H)-one, which was used in the next step without further purification. m/z (ESI) M+H: 721.8.

Step 3: 4-(4-Acryloylpiperazin-1-yl)-6-chloro-1-(cyclopropylmethyl)-7-(2-fluoro-6-hydroxyphenyl)pyrido[2,3-d]pyrimidin-2(1H)-one. Tetrabutylammonium fluoride (1.0 M solution in tetrahydrofuran, 0.025 mL, 0.025 mmol) was added to a stirred mixture of 4-(4-acryloylpiperazin-1-yl)-7-(2-((tert-butyldiphenylsilyl)oxy)-6-fluorophenyl)-6-chloro-1-(cyclopropylmethyl)pyrido[2,3-d]pyrimidin-2(1H)-one (6 mg, 8.31 μmol) in tetrahydrofuran (0.2 mL). The reaction mixture was stirred at room temperature for 20 min, and then concentrated under vacuum. Chromatographic purification of the residue (silica gel, 0 to 100% (3:1 EtOAc/EtOH) in heptane) yielded 6-chloro-1-(cyclopropylmethyl)-7-(2-fluoro-6-hydroxyphenyl)-4-(4-(2-acryloyl)-1-piperazinyl)pyrido[2,3-d]pyrimidin-2(1H)-one. ¹ H NMR (400MHz, chloroform-d) δ8.04 (1H, s) 7.26-7.33 (1H, m) 6.82 (1H, d, J = 8.29Hz) 6.71 (1H, t, J = 8.91Hz) 6.51 (1H,dd,J＝16.79,10.57Hz)6.30(1H,dd,J＝16.79,1.45Hz)5.72(1H,dd,J＝10.47,1.55Hz)4.15(2H,br d,J=6.43Hz)3.69-3.90(8H,m)1.14-1.27(4H,m)0.73-0.88(1H,m). m/z(ESI)M+H:483.8.

Example 39

6-Chloro-7-(2-fluoro-6-hydroxyphenyl)-1-(2-methyl-6-(2-propyl)phenyl)-4-((2S)-2-methyl-4-(2-acryloyl)-1-piperazinyl)pyrido[2,3-d]pyrimidin-2(1H)-one

Step 1: 2,5,6-Trichloronicotinamide (Intermediate P). 1,1’-carbonyldiimidazole (40 g, 247 mmol) was added in portions to 2,5,6-trichloronicotinic acid (50.7 g, 224 mmol, Combi-blocks, San Diego, California, USA) in THF (400 mL), with gas release stopped between each addition. The resulting mixture was stirred for 5 min and then degassed under vacuum and purged with nitrogen (×2). The resulting mixture was heated to 50 °C for 60 min, then diluted with toluene (100 mL) and concentrated to half volume. The resulting mixture was cooled to 0 °C and ammonium hydroxide (60 mL, 437 mmol) was slowly added via syringe. The reaction was stirred at room temperature for 10 min, diluted with EtOAc (200 mL), and washed with water (3 × 100 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated. The residue was suspended in 9:1 heptane/EtOAc (300 mL) and filtered. Collect the filtered solids and partially evaporate the remaining mother liquor to half its volume, cool to 0°C, and filter. Combine the two batches of filtered solids to provide 2,5,6-trichloronicotinamide.

Step 2: 2,5,6-Trichloro-N-((2-isopropyl-6-methylphenyl)aminoformyl)nicotinamide. Oxaloyl chloride (2.7 mL, 5.4 mmol in 2M solution of DCM) was added to a mixture of 2,5,6-trichloronicotinamide (intermediate P, 1.13 g, 5.0 mmol) in THF (30 mL). The resulting slurry was heated at 65 °C for 40 min, then heating was stopped and the reaction was allowed to cool to room temperature. 2-Isopropyl-6-methylaniline (0.80 mL, 5.36 mmol, Enamine, Monmouth Junction, NJ, USA) was added and the reaction was stirred at room temperature for 14 h. The reaction was concentrated and the residue was partitioned between EtOAc (50 mL) and a saturated aqueous solution of sodium bicarbonate (10 mL). The organic layer was washed with brine (10 mL), dried over anhydrous sodium sulfate, and concentrated. The residue was suspended in 5:1 heptane/EtOAc (10 mL) and filtered. The filtered solids were collected to provide 2,5,6-trichloro-N-((2-isopropyl-6-methylphenyl)carbamoyl)nicotinamide. ^¹H NMR (400 MHz, chloroform-d) δppm 9.63 (s, 1H), 9.35 (br s, 1H), 8.25 (s, 1H), 7.19–7.26 (m, 2H), 7.13 (d, J = 7.3 Hz, 1H), 3.14 (quin, J = 6.9 Hz, 1H), 2.29 (s, 3H), 1.23 (d, J = 6.8 Hz, 6H). m/z (ESI, +Ve ions): 400.0 (M + H) ⁺ .

Step 3: 6,7-Dichloro-1-(2-isopropyl-6-methylphenyl)pyrido[2,3-d]pyrimidine-2,4(1H,3H)-dione. KHMDS (1M solution in THF, 7.5 mL, 7.5 mmol) was added to a mixture of 2,5,6-trichloro-N-((2-isopropyl-6-methylphenyl)carbamoyl)nicotinamide (1.45 g, 3.6 mmol) in THF (20 mL). After stirring at room temperature for 30 min, the reaction was concentrated to 1/3 volume and quenched with saturated ammonium chloride aqueous solution (10 mL). The mixture was extracted with EtOAc (40 mL). The organic layer was washed with brine (10 mL), dried over anhydrous sodium sulfate, and concentrated to provide 6,7-dichloro-1-(2-isopropyl-6-methylphenyl)pyrido[2,3-d]pyrimidine-2,4(1H,3H)-dione. This material was used in the next step without further purification. m/z (ESI, +Ve ions): 364.0 (M+H) ⁺ .

Step 4: (S)-tert-butyl-4-(6,7-dichloro-1-(2-isopropyl-6-methylphenyl)-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-4-yl)-3-methylpiperazine-1-carboxylate. DIPEA (1.50 mL, 8.6 mmol) was added to a mixture of crude 6,7-dichloro-1-(2-isopropyl-6-methylphenyl)pyrido[2,3-d]pyrimidin-2,4(1H,3H)-dione (3.6 mmol) and acetonitrile (10 mL), followed by the addition of phosphorus oxychloride (0.50 mL, 5.3 mmol). The resulting mixture was heated at 80 °C for 1 h, then cooled to room temperature and concentrated. The residue was dissolved in DMF (15 mL) and treated with DIPEA (1.50 mL, 8.6 mmol) followed by (S)-4-N-Boc-2-methylpiperazine (900 mg, 4.5 mmol, ArkPharm Inc., Arlington Heights, Illinois, USA). The resulting solution was stirred at room temperature for 14 h and then diluted with EtOAc (30 mL). The mixture was washed with water (10 mL) and brine (10 mL), and the organic layer was dried over anhydrous magnesium sulfate and concentrated. The residue was purified by silica gel chromatography (elution: 10%–50% 3:1 EtOAc-EtOH/heptane) to provide (S)-tert-butyl-4-(6,7-dichloro-1-(2-isopropyl-6-methylphenyl)-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-4-yl)-3-methylpiperazine-1-carboxylate. ¹ H NMR (400MHz, MeOH-d ₄ ) δppm 8.45 (s, 1H), 7.34-7.43 (m, 2H), 7.23 (d, J = 7.3Hz, 1H), 4.97 (br s, 1H), 4.34 (br d, J = 13.3Hz, 1H), 4.15 (br d,J＝12.0Hz,1H),4.01(br d,J＝13.7Hz,1H),3.80(br s, 1H), 3.09-3.32 (m, 2H), 2.49-2.59 (m, 1H), 1.99 (d, J = 3.7 Hz, 3H), 1.55 (s, 9H), 1.50 (dd, J = 1.7, 6.6 Hz, 3H), 1.18 (dd, J = 6.7, 1.8 Hz, 3H), 1.09 (dd, J = 6.8, 2.3 Hz, 3H). m/z (ESI, +Ve ions): 546.1 (M+H) ⁺ .

Step 5: (3S)-tert-butyl4-(6-chloro-7-(2-fluoro-6-hydroxyphenyl)-1-(2-isopropyl-6-methylphenyl)-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-4-yl)-3-methylpiperazine-1-carboxylate. Add (S)-tert-butyl4-(6,7-dichloro-1-(2-isopropyl-6-methylphenyl)-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-4-yl)-3-methylpiperazine-1-carboxylate (8.3 g, 15.19 mmol), potassium acetate (7.50 g, 76 mmol), and the complex of [1,1'-bis(diphenylphosphino)ferrocene]-dichloropalladium(II) with dichloromethane (0.495 g, 0.606 mmol) to a round-bottom flask. Add 1,4-dioxane (40 mL) and water (8 mL) and heat the mixture to 90 °C. Add potassium trifluoroborate (2-fluoro-6-hydroxyphenyl) (intermediate Q, 7.45 g, 34.2 mmol) and then add the complex of [1,1'-bis(diphenylphosphino)ferrocene]-dichloropalladium(II) with dichloromethane (0.176 g). Stir the resulting mixture at room temperature for 2.5 h, then cool to room temperature, dilute with EtOAc (200 mL), and wash with water (1×) and brine (1×). Dry the organic layer with anhydrous sodium sulfate and concentrate. The residue was purified by silica gel chromatography (elution buffer: 5%-40% 3:1 EtOAc-EtOH/heptane) to provide (3S)-tert-butyl 4-(6-chloro-7-(2-fluoro-6-hydroxyphenyl)-1-(2-isopropyl-6-methylphenyl)-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-4-yl)-3-methylpiperazine-1-carboxylate. ¹ H NMR (400MHz, chloroform-d) δppm 8.68 (br s, 1H), 8.14 (br s,1H),7.33-7.45(m,2H),7.25(d,J＝6.3Hz,2H),6.64-6.73(m,2H),3.91-5.15(m,4H),3.67(br s,1H),3.32(br s,2H),2.49-2.76(m,1H),1.95-2.08(m,3H),1.53(s,12H),1.14-1.29(m,3H),0.95-1.07(m,3H). ¹⁹ F NMR (377MHz, chloroform-d) δ ppm - 104.56 (br s, 1F). m/z (ESI, +ve ions): 622.1 (M+H) ⁺ .

Step 6: 6-Chloro-7-(2-fluoro-6-hydroxyphenyl)-1-(2-methyl-6-(2-propane)phenyl)-4-((2S)-2-methyl-4-(2-acryloyl)-1-piperazinyl)pyrido[2,3-d]pyrimidin-2(1H)-one. Trifluoroacetic acid (1.5 mL, 19.6 mmol) was added to a solution of (3S)-tert-butyl-4-(6-chloro-7-(2-fluoro-6-hydroxyphenyl)-1-(2-isopropyl-6-methylphenyl)-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-4-yl)-3-methylpiperazin-1-carboxylate (680 mg, 1.1 mmol) in DCM (10 mL). The reaction was stirred at room temperature for 1.5 h and then concentrated. The residue was partitioned between EtOAc (40 mL) and saturated sodium bicarbonate aqueous solution (2 × 15 mL). The organic layer was washed with brine (5 mL), dried over anhydrous sodium sulfate, and concentrated. The residue was dissolved in DCM (10 mL) and treated with DIPEA (0.5 mL, 2.9 mmol), followed by acryloyl chloride (0.09 mL, 1.1 mmol). The reaction was stirred at room temperature for 10 min, then diluted with EtOAc (30 mL) and washed with saturated sodium bicarbonate aqueous solution (10 mL) and brine (5 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated. The residue was purified by silica gel chromatography (elution buffer: 10%-60% 3:1 EtOAc-EtOH/heptane) to provide 6-chloro-7-(2-fluoro-6-hydroxyphenyl)-1-(2-methyl-6-(2-propane)phenyl)-4-((2S)-2-methyl-4-(2-acryloyl)-1-piperazinyl)pyrido[2,3-d]pyrimidin-2(1H)-one. ¹ H NMR (400MHz, DMSO-d ₆ ) δppm 10.07 (s, 1H), 8.40 (br s,1H),7.17-7.25(m,3H),7.06-7.13(m,1H),6.79-6.91(m,1H),6.69(d,J＝8.0Hz,1H),6.65(t,J＝8.8Hz,1H),6.20(br d,J＝16.8Hz,1H),5.73-5.78(m,1H),4.91(br s,1H),4.21-4.46(m,2H),3.95-4.20(m,1H),3.42-3.80(m,2H),3.03-3.27(m,1H),2.53-2.63(m,1H),1.85(br s,3H),1.32(br t,J＝5.9Hz,3H), 1.05(d,J＝6.8Hz,3H), 0.91(br d,J＝6.6Hz,3H). ¹⁹ F NMR (377MHz, DMSO-d ₆ ) δ ppm -115.71--115.50 (m, 1F), -116.16 (br s, 1F). m/z(ESI,+ve ion):576.0(M+H) ⁺ .

Potassium trifluoroborate (2-fluoro-6-hydroxyphenyl) (intermediate Q) was added to a suspension of (2-fluoro-6-hydroxyphenyl)boric acid (30 g, 192 mmol, Combi-blocks, San Diego, California, USA) in acetonitrile (750 mL). The mixture was stirred for 2 min, and then L-(+)-tartaric acid (72.2 g, 481 mmol) in THF (375 mL) was added via a feeding funnel over a period of 10 min. The mixture was vigorously stirred with a mechanical stirrer for 1 h, and the resulting suspension was filtered, and the filtered solid was washed with a small amount of THF. The solid was discarded and the filtrate was partially concentrated until the solid began to precipitate from the solution. The mixture was then cooled to -20 °C and stirred for 16 h. The reaction was slowly heated and 2-propanol (20 mL) was added. The resulting suspension was filtered, and the filtered solid was washed with 2-propanol. The filtrate was further concentrated until a suspension was formed, and then cooled to -20°C and stirred for 20 min. The resulting suspension was diluted with 2-propanol and filtered, and the filtered solids were washed with 2-propanol. The two batches of solids were combined to provide potassium 2-fluoro-6-hydroxyphenyl trifluoroborate (intermediate Q). ^¹H NMR (400 MHz, DMSO- _d⁶ ) δppm 8.07 (q, J = 14.7 Hz, 1H) 6.93 (q, J = 7.5 Hz, 1H) 6.30–6.38 (m, 2H).

Example 40

6-Chloro-7-(2-fluorophenyl)-1-(4-methyl-2-(2-propyl)-3-pyridyl)-4-((2S)-2-methyl-4-(2-acryloyl)-1-piperazinyl)pyrido[2,3-d]pyrimidin-2(1H)-one

Step 1: 2-Isopropyl-4-methylpyridin-3-amine (Intermediate R). A complex of [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) and dichloromethane (79 mg, 0.10 mmol) was added to a slurry of 3-amino-2-bromo-4-methylpyridine (360 mg, 1.9 mmol, Combi-blocks, San Diego, California, USA) in THF (4 mL). The resulting slurry was deoxygenated with argon for 2 min and then 2-propylzinc bromide (0.5 M solution in THF, 5.40 mL, 2.7 mmol, Sigma-Aldrich, St. Louis, Missouri) was added. The resulting solution was heated at 60 °C for 17 h, then heating was stopped and the reaction was allowed to cool to room temperature. The reaction mixture was quenched with water (10 mL) and 1N NaOH solution (20 mL) and then extracted with EtOAc (2x). The combined organic layers were dried over anhydrous sodium sulfate and concentrated. The residue was purified by silica gel chromatography (elution: 0–15% MeOH/DCM) to provide 2-isopropyl-4-methylpyridin-3-amine. ^¹H NMR (400 MHz, DMSO- _d⁶ ) δppm 7.66 (d, J = 4.6 Hz, 1H), 6.78 (d, J = 4.8 Hz, 1H), 4.72 (br s, 2H), 3.14–3.25 (m, 1H), 2.08 (s, 3H), 1.14 (d, J = 6.8 Hz, 6H). m/z (ESI, +Ve ion): 151.1 (M+H) ^⁺ .

Step 2: 2,5,6-Trichloro-N-((2-isopropyl-4-methylpyridin-3-yl)carbamoyl)nicotinamide. Oxaloyl chloride (2M solution in DCM, 7.4 mL, 14.7 mmol) was slowly added via syringe to a slurry of 2,5,6-trichloronicotinamide (intermediate P, 3.10 g, 13.8 mmol) in THF (46 mL) at -78 °C. The resulting slurry was heated at 60 °C for 3.5 h, then heating was stopped and the reaction was cooled to -78 °C. Triethylamine (6.0 mL, 42.6 mmol) was added, followed by a solution of 2-isopropyl-4-methylpyridin-3-amine (intermediate R, 2.12 g, 14.1 mmol) via a catheter. The resulting slurry was heated to room temperature and stirred for 1 h, then partitioned between water (120 mL) and EtOAc (175 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated. The residue was suspended in 9:1 heptane/EtOAc and filtered. The filtered solids were collected to provide 2,5,6-trichloro-N-((2-isopropyl-4-methylpyridin-3-yl)carbamoyl)nicotinamide. ^¹H NMR (400MHz, DMSO- _d⁶ ) δppm 11.31 (s, 1H), 9.54 (s, 1H), 8.66 (s, 1H), 8.34 (d, J = 4.8Hz, 1H), 7.16 (d, J = 5.0Hz, 1H), 3.24–3.33 (m, 1H), 2.22 (s, 3H), 1.17 (d, J = 6.6Hz, 6H). m/z (ESI, +Ve ions): 400.9 (M+H) ^⁺ .

Step 3: 6,7-Dichloro-1-(2-isopropyl-4-methylpyridin-3-yl)pyrido[2,3-d]pyrimidin-2,4(1H,3H)-dione. KHMDS (1M solution in THF, 23.5 mL, 23.5 mmol) was slowly added via syringe to an ice-cold solution of 2,5,6-trichloro-N-((2-isopropyl-4-methylpyridin-3-yl)carbamoyl)nicotinamide (4.71 g, 11.7 mmol) in 55 mL of THF. After 10 min, the ice bath was removed, and the resulting solution was stirred at room temperature for another 30 min. The reaction was quenched with saturated ammonium chloride aqueous solution (125 mL) and extracted with EtOAc (250 mL). The organic layer was washed with brine (1x), dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica gel chromatography (elution buffer: 0–11% MeOH/DCM) to provide 6,7-dichloro-1-(2-isopropyl-4-methylpyridin-3-yl)pyrido[2,3-d]pyrimidine-2,4(1H,3H)-dione. ^¹H NMR (400 MHz, DMSO- _d⁶ ) δppm 12.27 (br s, 1H), 8.59 (s, 1H), 8.52 (d, J = 5.0 Hz, 1H), 7.28 (d, J = 5.0 Hz, 1H), 2.82–2.92 (m, 1H), 2.04 (s, 3H), 1.08 (d, J = 6.6 Hz, 3H), 1.01 (d, J = 6.8 Hz, 3H). m/z (ESI, +Ve ion): 365.0 (M+H) ^⁺ .

Step 4: 4,6,7-Trichloro-1-(2-isopropyl-4-methylpyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one. DIPEA (1.80 mL, 10.3 mmol) was added to a slurry of 6,7-dichloro-1-(2-isopropyl-4-methylpyridin-3-yl)pyrido[2,3-d]pyrimidin-2,4(1H,3H)-dione (2.52 g, 6.9 mmol) in acetonitrile (45 mL), followed by slow addition of phosphorus oxychloride (1.58 mL, 10.3 mmol) via syringe. The resulting mixture was heated at 80 °C for 1.75 h, then cooled to room temperature and concentrated to provide 4,6,7-trichloro-1-(2-isopropyl-4-methylpyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one. The material is used in the next step without further purification.

Step 5: (S)-tert-butyl-4-(6,7-dichloro-1-(2-isopropyl-4-methylpyridin-3-yl)-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-4-yl)-3-methylpiperazine-1-carboxylate. DIPEA (3.61 mL, 20.7 mmol) was added to a solution of 4,6,7-trichloro-1-(2-isopropyl-4-methylpyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one (2.64 g, 6.9 mmol) in THF (40 mL), followed by the addition of (S)-4-N-Boc-2-methylpiperazine (2.07 g, 10.3 mmol, Combi-Blocks, Inc., San Diego, California, USA). The resulting solution was stirred at room temperature for 1.5 h, and then ice water (60 mL) was added. The mixture was stirred for another 5 min and then extracted with EtOAc (3x). The combined organic layers were dried over anhydrous sodium sulfate and concentrated. The residue was purified by silica gel chromatography (elution: 0-11% MeOH/DCM) to provide (S)-tert-butyl-4-(6,7-dichloro-1-(2-isopropyl-4-methylpyridin-3-yl)-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-4-yl)-3-methylpiperazine-1-carboxylate. ¹ H NMR (400MHz, DMSO-d ₆ )δppm8.46(dd,J＝11.6,5.2Hz,2H),7.25(d,J＝4.8Hz,1H),4.79-4.93(m,1 H),4.10-4.24(m,1H),3.87-4.05(m,1H),3.77-3.87(m,1H),3.62-3.76(m, 1H),2.99-3.25(m,2H),2.55-2.69(m,1H),1.94(d,J＝2.5Hz,3H),1.45(s, 9H), 1.32 (brt, J = 5.7Hz, 3H), 1.06 (d, J = 6.8Hz, 3H), 1.00 (d, J = 6.6Hz, 3H). m/z (ESI, +ve ions): 547.2 (M+H) ⁺ .

Step 6: (S)-tert-butyl-4-(6-chloro-7-(2-fluorophenyl)-1-(2-isopropyl-4-methylpyridin-3-yl)-2-oxo-1,2-dihydropyridino[2,3-d]pyrimidin-4-yl)-3-methylpiperazine-1-carboxylate. Potassium acetate (914 mg, 9.3 mmol) and (2-fluorophenyl)boronic acid (313 mg, 2.2 mmol, Sigma-Aldrich, St. Louis, Missouri, USA) were added to a solution of (S)-tert-butyl-4-(6,7-dichloro-1-(2-isopropyl-4-methylpyridin-3-yl)-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-4-yl)-3-methylpiperazine-1-carboxylate (1.02 g, 1.8 mmol) in 1,4-dioxane (17 mL). The mixture was purged with argon, and then the complex of [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) with dichloromethane (76 mg, 0.093 mmol) was added. The mixture was purged with argon again and heated at 90 °C. After 30 seconds, three drops of water were added to the reaction mixture. The mixture was heated at 90 °C for 40 min, and then allowed to cool to room temperature. Water (50 mL) and brine (4 mL) were added, and the resulting mixture was extracted with EtOAc (2x). The combined organic layers were washed with brine (1x), dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica gel chromatography (elution: 0–9% MeOH/DCM) to provide (S)-tert-butyl 4-(6-chloro-7-(2-fluorophenyl)-1-(2-isopropyl-4-methylpyridin-3-yl)-2-oxo-1,2-dihydropyridino[2,3-d]pyrimidin-4-yl)-3-methylpiperazine-1-carboxylate. ¹ H NMR (400MHz, DMSO-d ₆ )δppm8.43(d,J＝2.5Hz,1H),8.39(d,J＝4.8Hz,1H),7.47-7.55(m,1H),7.16-7.33(m,4H),4.86-4.97(m,1H),4.21-4.30(m,1H),3 .90-4.06(m,2H),3.80-3.89(m,1H),3.67-3.78(m,1H),3.04-3.16(m,1H),2.65-2.75(m,1H),1.93(s,3H),1.48(s,9H),1.36(br d,J＝6.6Hz,3H), 1.06(d,J＝6.6Hz,3H), 0.94(dd,J＝6.6,2.1Hz,3H). m/z (ESI,+ve ions): 607.0(M+H) ⁺ .

Step 7: 6-Chloro-7-(2-fluorophenyl)-1-(4-methyl-2-(2-propyl)-3-pyridinyl)-4-((2S)-2-methyl-4-(2-acryloyl)-1-piperazinyl)pyrido[2,3-d]pyrimidin-2(1H)-one. Trifluoroacetic acid (4.0 mL, 53.9 mmol) was added to a solution of (S)-tert-butyl4-(6-chloro-7-(2-fluorophenyl)-1-(2-isopropyl-4-methylpyridin-3-yl)-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-4-yl)-3-methylpiperazin-1-carboxylate (935 mg, 1.54 mmol) in DCM (20 mL). The resulting solution was stirred at room temperature for 1.5 h and then concentrated. The residue was dissolved in DCM (12 mL), cooled to 0 °C, and treated with DIPEA (0.807 mL, 4.62 mmol), followed by acryloyl chloride (0.131 mL, 1.62 mmol; added dropwise via syringe). The resulting solution was stirred at 0 °C for 35 min, then quenched with saturated sodium bicarbonate aqueous solution (35 mL) and extracted with DCM (2x). The combined organic layers were dried over anhydrous sodium sulfate and concentrated. The residue was purified by silica gel chromatography (elution: 0–11% MeOH/DCM) to provide 6-chloro-7-(2-fluorophenyl)-1-(4-methyl-2-(2-propyl)-3-pyridyl)-4-((2S)-2-methyl-4-(2-acryloyl)-1-piperazinyl)pyrido[2,3-d]pyrimidin-2(1H)-one. ¹ HNMR (400MHz, DMSO-d ₆ ) δppm 8.46 (br d,J＝4.6Hz,1H),8.39(d,J＝4.8Hz,1H),7.47-7.55(m,1H),7.16-7.34(m,4H),6.78-6.94(m,1H),6.15-6.26(m,1H),5.73-5.80(m,1H),4.95(br s,1H),4.36-4.45(m,0.5H),4.24-4.36(m,1.5H),4.11-4.21(m,0.5H), 3.98-4.08(m,0.5H),3.71-3.85(m,1H),3.60-3.69(m,0.5H),3.41-3.53 (m,0.5H),3.06-3.27(m,1H),2.65-2.75(m,1H),1.94(d,J＝1.7Hz,3H),1 .34(d,J＝6.2Hz,3H), 1.07(d,J＝6.6Hz,3H), 0.94(dd,J＝6.5,0.9Hz,3H). m/z (ESI, +Ve ions): 560.9 (M+H) ⁺ .

Example 41

6-Fluoro-7-(2-Fluoro-6-hydroxyphenyl)-1-(4-methyl-2-(2-propyl)-3-pyridyl)-4-((2S)-2-methyl-4-(2-acryloyl)-1-piperazinyl)pyrido[2,3-d]pyrimidin-2(1H)-one

Step 1: 2,6-Dichloro-5-fluoronicotinamide (Intermediate S). Oxaloyl chloride (2M solution in DCM, 11.9 mL, 23.8 mmol) was added to a mixture of 2,6-dichloro-5-fluoronicotinic acid (4.0 g, 19.1 mmol, AstaTech Inc., Bristol, PA) in dichloromethane (48 mL), followed by a catalytic amount of DMF (0.05 mL). The reaction was stirred overnight at room temperature and then concentrated. The residue was dissolved in 1,4-dioxane (48 mL) and cooled to 0 °C. Ammonium hydroxide solution (28.0%–30% NH3, 3.6 mL, 28.6 mmol) was slowly added via syringe. The resulting mixture was stirred at 0 °C for 30 min and then concentrated. The residue was diluted with a 1:1 EtOAc/heptane mixture and stirred for 5 min, then filtered. The filtered solids were discarded, and the remaining mother liquor was partially concentrated to half its volume and filtered. The filtered solid was washed with heptane and dried overnight in a vacuum oven (45 °C) to provide 2,6-dichloro-5-fluoronicotinamide. ^¹H NMR (400 MHz, DMSO- _d⁶ ) δppm 8.23 (d, J = 7.9 Hz, ¹H) 8.09 (br s, ¹H) 7.93 (br s, ¹H). m/z (ESI, +Ve ions): 210.9 (M+H) ^⁺ .

Step 2: 2,6-Dichloro-5-fluoro-N-((2-isopropyl-4-methylpyridin-3-yl)aminocarbamoyl)nicotinamide. Oxaloyl chloride (2M solution in DCM, 14.4 mL, 28.8 mmol) was slowly added via syringe to an ice-cold slurry of 2,6-dichloro-5-fluoronicotinamide (intermediate S, 5.0 g, 23.9 mmol) in 20 mL of THF. The resulting mixture was heated at 75 °C for 1 h, then heating was stopped, and the reaction was concentrated to half its volume. After cooling to 0 °C, 20 mL of THF was added dropwise via a tube, followed by a solution of 2-isopropyl-4-methylpyridin-3-amine (intermediate R, 3.59 g, 23.92 mmol) in 10 mL of THF. The resulting mixture was stirred at 0 °C for 1 h and then quenched with a 1:1 mixture of brine and saturated ammonium chloride aqueous solution. The mixture was extracted with EtOAc(3x), and the combined organic layers were dried over anhydrous sodium sulfate and concentrated to provide 2,6-dichloro-5-fluoro-N-((2-isopropyl-4-methylpyridin-3-yl)carbamoyl)nicotinamide. This material was used in the next step without further purification. m/z (ESI, +Ve ions): 385.1 (M+H) ⁺ .

Step 3: 7-Chloro-6-fluoro-1-(2-isopropyl-4-methylpyridin-3-yl)pyrido[2,3-d]pyrimidin-2,4(1H,3H)-dione. KHMDS (1M solution in THF, 50.2 mL, 50.2 mmol) was slowly added via syringe to an ice-cold solution of 2,6-dichloro-5-fluoro-N-((2-isopropyl-4-methylpyridin-3-yl)carbamoyl)nicotinamide (9.2 g, 24.0 mmol) in 40 mL of THF. The ice bath was removed and the resulting mixture was stirred at room temperature for 40 min. The reaction was quenched with saturated ammonium chloride solution and extracted with EtOAc (3x). The combined organic layers were dried over anhydrous sodium sulfate and concentrated. The residue was purified by silica gel chromatography (elution buffer: 0-50% 3:1 EtOAc-EtOH/heptane) to provide 7-chloro-6-fluoro-1-(2-isopropyl-4-methylpyridin-3-yl)pyrido[2,3-d]pyrimidine-2,4(1H,3H)-dione. ^¹H NMR (400MHz, DMSO- _d⁶ ) δppm 12.27 (brs, 1H), 8.48-8.55 (m, 2H), 7.29 (d, J = 4.8 Hz, 1H), 2.87 (quin, J = 6.6 Hz, 1H), 1.99-2.06 (m, 3H), 1.09 (d, J = 6.6 Hz, 3H), 1.01 (d, J = 6.6 Hz, 3H). ^19F NMR (376MHz, DMSO- _d⁶ ) δ: -126.90 (s, 1F). m/z (ESI, +Ve ions): 349.1 (M+H) ^⁺ .

Step 4: 4,7-Dichloro-6-fluoro-1-(2-isopropyl-4-methylpyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one. Phosphorus oxychloride (1.63 mL, 17.5 mmol) was added dropwise to a solution of 7-chloro-6-fluoro-1-(2-isopropyl-4-methylpyridin-3-yl)pyrido[2,3-d]pyrimidin-2,4(1H,3H)-dione (4.7 g, 13.5 mmol) and DIPEA (3.5 mL, 20.2 mmol) in acetonitrile (20 mL). The resulting mixture was heated at 80 °C for 1 h, then cooled to room temperature and concentrated to provide 4,7-dichloro-6-fluoro-1-(2-isopropyl-4-methylpyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one. This material was used in the next step without further purification. m/z (ESI, +Ve ions): 367.1 (M+H) ⁺ .

Step 5: (S)-tert-butyl 4-(7-chloro-6-fluoro-1-(2-isopropyl-4-methylpyridin-3-yl)-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-4-yl)-3-methylpiperazine-1-carboxylate. DIPEA (7.1 mL, 40.3 mmol) was added to an ice-cold solution of 4,7-dichloro-6-fluoro-1-(2-isopropyl-4-methylpyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one (13.5 mmol) in acetonitrile (20 mL), followed by the addition of (S)-4-N-Boc-2-methylpiperazine (3.23 g, 16.1 mmol, Combi-Blocks, Inc., San Diego, California, USA). The resulting mixture was heated to room temperature and stirred for 1 h, then diluted with cold saturated sodium bicarbonate aqueous solution (200 mL) and EtOAc (300 mL). The mixture was stirred again for 5 min, the layers were separated, and the aqueous layer was extracted again with EtOAc (1x). The combined organic layers were dried over anhydrous sodium sulfate and concentrated. The residue was purified by silica gel chromatography (elution: 0-50% EtOAc/heptane) to provide (S)-tert-butyl-4-(7-chloro-6-fluoro-1-(2-isopropyl-4-methylpyridin-3-yl)-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-4-yl)-3-methylpiperazine-1-carboxylate. m/z (ESI, +ve ion): 531.2 (M+H) ⁺ .

Step 6: (3S)-tert-butyl 4-(6-fluoro-7-(2-fluoro-6-hydroxyphenyl)-1-(2-isopropyl-4-methylpyridin-3-yl)-2-oxo-1,2-dihydropyridino[2,3-d]pyrimidin-4-yl)-3-methylpiperazine-1-carboxylate. The mixture of (S)-tert-butyl-4-(7-chloro-6-fluoro-1-(2-isopropyl-4-methylpyridin-3-yl)-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-4-yl)-3-methylpiperazine-1-carboxylate (4.3 g, 8.1 mmol), potassium trifluoro(2-fluoro-6-hydroxyphenyl)borate (intermediate Q, 2.9 g, 10.5 mmol), potassium acetate (3.2 g, 32.4 mmol), and the complex of [1,1'-bis(diphenylphosphine)ferrocene]dichloropalladium(II) with dichloromethane (661 mg, 0.81 mmol) in 1,4-dioxane (80 mL) was degassed for 1 min under nitrogen. Deoxygenated water (14 mL) was added, and the resulting mixture was heated at 90 °C for 1 h. The reaction was allowed to cool to room temperature, quenched with a semi-saturated aqueous sodium bicarbonate solution, and extracted with EtOAc (2x) and DCM (1x). The combined organic layers were dried over anhydrous sodium sulfate and concentrated. The residue was purified by silica gel chromatography (elution: 0-60% 3:1 EtOAc-EtOH/heptane) to provide (3S)-tert-butyl-4-(6-fluoro-7-(2-fluoro-6-hydroxyphenyl)-1-(2-isopropyl-4-methylpyridin-3-yl)-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-4-yl)-3-methylpiperazine-1-carboxylate. ¹ H NMR (400MHz, DMSO-d ₆ ) δppm 10.19 (br s,1H),8.38(d,J＝5.0Hz,1H),8.26(dd,J＝12.5,9.2Hz,1H),7.23-7.28(m,1H),7.18(d, J＝5.0Hz,1H),6.72(d,J＝8.0Hz,1H),6.68(t,J＝8.9Hz,1H),4.77-4.98(m,1H),4.24(br t,J＝14.2Hz,1H),3.93-4.08(m,1H),3.84(br d,J＝12.9Hz,1H),3.52-3.75(m,1H),3.07-3.28(m,1H),2.62-2.74(m,1H),1.86-1.93(m,3H),1.43-1.48(m,9 H), 1.35 (dd, J＝10.8, 6.8Hz, 3H), 1.26-1.32 (m, 1H), 1.07 (dd, J＝6.6, 1.7Hz, 3H), 0.93 (dd, J＝6.6, 2.1Hz, 3H). ¹⁹ F NMR (376MHz, DMSO-d ₆ ) δ: -115.65 (s, 1F), -128.62 (s, 1F). m/z(ESI,+ve ion):607.3(M+H) ⁺ .

Step 7: 6-Fluoro-7-(2-fluoro-6-hydroxyphenyl)-1-(4-methyl-2-(2-propyl)-3-pyridinyl)-4-((2S)-2-methyl-4-(2-acryloyl)-1-piperazinyl)pyrido[2,3-d]pyrimidin-2(1H)-one. Trifluoroacetic acid (25 mL, 324 mmol) was added to a solution of (3S)-tert-butyl4-(6-fluoro-7-(2-fluoro-6-hydroxyphenyl)-1-(2-isopropyl-4-methylpyridin-3-yl)-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-4-yl)-3-methylpiperazin-1-carboxylate (6.3 g, 10.4 mmol) in DCM (30 mL). The resulting mixture was stirred at room temperature for 1 h and then concentrated. The residue was dissolved in DCM (30 mL), cooled to 0 °C, and treated sequentially with solutions of DIPEA (7.3 mL, 41.7 mmol) and acryloyl chloride (0.849 mL, 10.4 mmol) in DCM (3 mL; added dropwise via syringe). The reaction was stirred at 0 °C for 10 min, then quenched with a semi-saturated aqueous sodium bicarbonate solution and extracted with DCM (2x). The combined organic layers were dried over anhydrous sodium sulfate and concentrated. The residue was purified by silica gel chromatography (elution: 0-100% 3:1 EtOAc-EtOH/heptane) to provide 6-fluoro-7-(2-fluoro-6-hydroxyphenyl)-1-(4-methyl-2-(2-propyl)-3-pyridyl)-4-((2S)-2-methyl-4-(2-acryloyl)-1-piperazinyl)pyrido[2,3-d]pyrimidin-2(1H)-one. ¹ H NMR (400MHz, DMSO-d ₆ )δppm10.20(s,1H),8.39(d,J＝4.8Hz,1H),8.24-8.34(m,1H),7.23-7.32(m,1H),7.19(d,J＝5. 0Hz,1H),6.87(td,J＝16.3,11.0Hz,1H),6.74(d,J＝8.6Hz,1H),6.69(t,J＝8.6Hz,1H),6.21(br d,J＝16.2Hz,1H),5.74-5.80(m,1H),4.91(br s, 1H), 4.23-4.45 (m, 2H), 3.97-4.21 (m, 1H), 3.44-3.79 (m, 2H), 3.11-3.31 (m, 1H), 2.67-2.77 (m, 1H), 1.91 (s, 3H), 1.35 (d, J = 6.8 Hz, 3H), 1.08 (d, J = 6.6 Hz, 3H), 0.94 (d, J = 6.8 Hz, 3H). ¹⁹ F NMR (376 MHz, DMSO- _d⁶ ) δppm -115.64 (s, 1F), -128.63 (s, 1F). m/z (ESI, +Ve ions): 561.2 (M+H) ^⁺ .

Example 42

1-(2-Cyclopropyl-4-methyl-3-pyridyl)-6-fluoro-7-(2-fluoro-6-hydroxyphenyl)-4-((2S)-2-methyl-4-(2-acryloyl)-1-piperazinyl)pyrido[2,3-d]pyrimidin-2(1H)-one

Step 1: 2-Cyclopropyl-4-methylpyridin-3-amine (Intermediate T). Cyclopropyl zinc bromide (0.5 M solution in THF, 68.4 mL, 34.2 mmol, Sigma-Aldrich, St. Louis, Missouri) was slowly added via a feeding funnel to a slurry of 3-amino-2-bromo-4-methylpyridine (4.0 g, 21.4 mmol, Combi-blocks, San Diego, California, USA) and dichloro[1,1'-bis(diphenylphosphino)ferrocene]palladium(II) dichloromethane adduct (1.78 g, 2.1 mmol) in 100 mL of THF. The resulting mixture was allowed to heat at 70 °C for 6 h, then heating was stopped and the reaction was allowed to cool to room temperature. The reaction mixture was quenched with 5 N NaOH solution and extracted with EtOAc (1x). The organic layer was dried over anhydrous magnesium sulfate and concentrated. The residue was purified by silica gel chromatography (elution buffer: 0-50% 3:1 EtOAc-EtOH/heptane) to provide 2-cyclopropyl-4-methylpyridin-3-amine. ^¹H NMR (400 MHz, chloroform-d) δppm 7.84 (d, J = 4.8 Hz, 1H), 6.82 (d, J = 4.8 Hz, 1H), 3.82 (br s, 2H), 2.17 (s, 3H), 1.85 (quin, J = 6.7 Hz, 1H), 0.92-0.99 (m, 4H). m/z (ESI, +Ve ion): 149.1 (M+H) ⁺ .

Step 2: 2,6-Dichloro-N-((2-cyclopropyl-4-methylpyridin-3-yl)carbamoyl)-5-fluoronicotinamide. Oxaloyl chloride (2M solution in DCM, 10.7 mL, 21.5 mmol) was slowly added via syringe to a solution of 2,6-dichloro-5-fluoronicotinamide (intermediate S, 3.0 g, 14.4 mmol) in THF (30 mL). The resulting mixture was heated at 65 °C for 2 h, then heating was stopped and the reaction was concentrated. The residue was dissolved in THF (30 mL) and added via tube to a solution of 2-cyclopropyl-4-methylpyridin-3-amine (intermediate T, 2.1 g, 14.4 mmol) in THF (105 mL). The resulting mixture was stirred at room temperature for 3 h, then quenched with water and extracted with EtOAc (3x). The combined organic layers were dried over anhydrous sodium sulfate and concentrated. The residue was purified by silica gel chromatography (elution buffer: 0-50% EtOAc/heptane) to provide 2,6-dichloro-N-((2-cyclopropyl-4-methylpyridin-3-yl)carbamoyl)-5-fluoronicotinamide. ^¹H NMR (400 MHz, chloroform-d) δppm 9.85 (s, 1H), 9.68 (s, 1H), 8.29 (d, J = 4.8 Hz, 1H), 7.97 (d, J = 7.0 Hz, 1H), 6.99 (d, J = 4.8 Hz, 1H), 2.29 (s, 3H), 2.08–2.16 (m, 1H), 1.08–1.13 (m, 2H), 0.95–1.02 (m, 2H). m/z (ESI, +Ve ion): 383.0 (M+H) ⁺ .

Step 3: 7-Chloro-1-(2-cyclopropyl-4-methylpyridin-3-yl)-6-fluoropyrido[2,3-d]pyrimidin-2,4(1H,3H)-dione. KHMDS (1M solution in THF, 17.5 mL, 17.5 mmol) was slowly added via syringe to an ice-cold solution of 2,6-dichloro-N-((2-cyclopropyl-4-methylpyridin-3-yl)carbamoyl)-5-fluoronicotinamide (3.35 g, 8.7 mmol) in 30 mL of THF. The ice bath was removed and the resulting mixture was stirred at room temperature for 14 h. The reaction was quenched with a saturated aqueous ammonium chloride solution and extracted with EtOAc (3x). The combined organic layers were dried over anhydrous sodium sulfate and concentrated to provide 7-chloro-1-(2-cyclopropyl-4-methylpyridin-3-yl)-6-fluoropyrido[2,3-d]pyrimidine-2,4(1H,3H)-dione. This material was used in the next step without further purification. m/z (ESI, +ve ions): 347.0 (M+H) ⁺ .

Step 4: 4,7-Dichloro-1-(2-cyclopropyl-4-methylpyridin-3-yl)-6-fluoropyrido[2,3-d]pyrimidin-2(1H)-one. Phosphorus oxychloride (1.22 mL, 13.1 mmol) was added dropwise to a solution of 7-chloro-1-(2-cyclopropyl-4-methylpyridin-3-yl)-6-fluoropyrido[2,3-d]pyrimidin-2,4(1H,3H)-dione (8.7 mmol) and DIPEA (2.32 mL, 13.1 mmol) in acetonitrile (25 mL). The resulting mixture was heated at 80 °C for 3 h, then cooled to room temperature and concentrated to provide 4,7-dichloro-1-(2-cyclopropyl-4-methylpyridin-3-yl)-6-fluoropyrido[2,3-d]pyrimidin-2(1H)-one. This material was used in the next step without further purification. m/z (ESI, +Ve ions): 365.0 (M+H) ⁺ .

Step 5: (S)-tert-butyl 4-(7-chloro-1-(2-cyclopropyl-4-methylpyridin-3-yl)-6-fluoro-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-4-yl)-3-methylpiperazine-1-carboxylate. DIPEA (7.74 mL, 43.7 mmol) was added to an ice-cold solution of 4,7-dichloro-1-(2-cyclopropyl-4-methylpyridin-3-yl)-6-fluoropyrido[2,3-d]pyrimidin-2(1H)-one (8.7 mmol) in DCM (25 mL), followed by (S)-4-N-Boc-2-methylpiperazine (1.75 g, 8.7 mmol, Combi-Blocks, Inc., San Diego, California, USA). The mixture was stirred at 0 °C for 2 h, then quenched with water and extracted with EtOAc (3x). The combined organic layers were dried over anhydrous sodium sulfate and concentrated. The residue was purified by silica gel chromatography (elution: 0-60% EtOAc/heptane) to provide (S)-tert-butyl4-(7-chloro-1-(2-cyclopropyl-4-methylpyridin-3-yl)-6-fluoro-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-4-yl)-3-methylpiperazine-1-carboxylate. ^¹H NMR (400 MHz, DMSO- _d⁶ ) δppm 8.31-8.39 (m, 2H), 7.18 (d, J = 4.8 Hz, 1H), 4.82 (br s, 1H), 4.10-4.21 (m, 1H), 3.96 (br s, 1H), 3.82 (br s, 1H). d, J = 13.3 Hz, 1H), 3.62-3.72 (m, 1H), 3.19-3.52 (m, 2H), 1.94 (s, 3H), 1.62 (dq, J = 8.2, 4.0 Hz, 1H), 1.45 (s, 9H), 1.32 (dd, J = 6.5, 3.6 Hz, 3H), 0.87-0.98 (m, 1H), 0.69-0.84 (m, 2H), 0.57-0.68 (m, 1H). m/z (ESI, +Ve ions): 529.0 (M+H) ⁺ .

Step 6: (3S)-tert-butyl 4-(1-(2-cyclopropyl-4-methylpyridin-3-yl)-6-fluoro-7-(2-fluoro-6-hydroxyphenyl)-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-4-yl)-3-methylpiperazine-1-carboxylate. Argon gas was introduced into a mixture of (S)-tert-butyl-4-(7-chloro-1-(2-cyclopropyl-4-methylpyridin-3-yl)-6-fluoro-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-4-yl)-3-methylpiperazine-1-carboxylate (987 mg, 1.87 mmol), (2-fluoro-6-hydroxyphenyl)boronic acid (524 mg, 3.36 mmol, Combi-blocks, San Diego, California, USA), potassium acetate (916 mg, 9.33 mmol), and a complex of [1,1'-bis(diphenylphosphine)ferrocene]dichloropalladium(II) with dichloromethane (152 mg, 0.19 mmol) in 1,4-dioxane (10 mL), and the mixture was heated at 80 °C. After 2 min, three drops of water were added to the reaction mixture, and the temperature was raised to 90 °C. The mixture was heated at 90°C for 1 hour, and then allowed to cool to room temperature. Water was added and the resulting mixture was extracted with EtOAc (3x). The combined organic layers were washed with brine (1x), dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica gel chromatography (elution: 0-65% EtOAc/heptane) to provide (3S)-tert-butyl-4-(1-(2-cyclopropyl-4-methylpyridin-3-yl)-6-fluoro-7-(2-fluoro-6-hydroxyphenyl)-2-oxo-1,2-dihydropyridino[2,3-d]pyrimidin-4-yl)-3-methylpiperazine-1-carboxylate. ¹ H NMR (400MHz, DMSO-d ₆ ) δppm 10.22(s,1H),8.19-8.31(m,2H),7.23-7.32(m,1H),7.10(d,J＝5.0Hz,1H),6.74(d, J＝8.3Hz,1H),6.69(t,J＝8.8Hz,1H),4.76-4.98(m,1H),4.15-4.31(m,1H),3.99(br s, 1H), 3.78-3.89 (m, 1H), 3.55-3.77 (m, 1H), 2.99-3.29 (m, 2H), 1.91 (d, J = 2.7 Hz, 3H), 1.68 (td, J = 8.0, 4.5 Hz, 1H), 1.45 (s, 9H), 1.35 (dd, J = 18.7, 6.6 Hz, 3H), 0.82-0.89 (m, 1H), 0.71-0.82 (m, 2H), 0.57-0.66 (m, 1H). m/z (ESI, +Ve ions): 605.0 (M+H) ⁺ .

Step 7: 1-(2-Cyclopropyl-4-methyl-3-pyridyl)-6-fluoro-7-(2-fluoro-6-hydroxyphenyl)-4-((2S)-2-methyl-4-(2-acryloyl)-1-piperazinyl)pyrido[2,3-d]pyrimidin-2(1H)-one. Trifluoroacetic acid (2.17 mL, 28.1 mmol) was added to a solution of (3S)-tert-butyl-4-(1-(2-cyclopropyl-4-methylpyridin-3-yl)-6-fluoro-7-(2-fluoro-6-hydroxyphenyl)-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-4-yl)-3-methylpiperazine-1-carboxylate (850 mg, 1.41 mmol) in DCM (10 mL). The resulting mixture was stirred at room temperature for 3 h and then concentrated. The residue was dissolved in DCM (10 mL), cooled to 0 °C, and treated dropwise with DIPEA (1.23 mL, 7.03 mmol) followed by acryloyl chloride (0.103 mL, 1.27 mmol) via syringe. The reaction was stirred at 0 °C for 2 h, then quenched with water and extracted with DCM (3x). The combined organic layers were dried over anhydrous sodium sulfate and concentrated. The residue was purified by silica gel chromatography (elution: 0-80% 3:1 EtOAc-EtOH/heptane) to provide 1-(2-cyclopropyl-4-methyl-3-pyridyl)-6-fluoro-7-(2-fluoro-6-hydroxyphenyl)-4-((2S)-2-methyl-4-(2-acryloyl)-1-piperazinyl)pyrido[2,3-d]pyrimidin-2(1H)-one. ¹ H NMR (400MHz, DMSO-d ₆ ) δppm 8.32 (br t,J＝10.1Hz,1H),8.28(d,J＝5.0Hz,1H),7.51-7.60(m,1H),7.27-7.38(m,3H),7.14(d,J＝4.8Hz,1H),6.80-6.92(m,1H),6.20(br d,J＝16.6Hz,1H),5.69-5.80(m,1H),4.92(br d,J＝1.5Hz,1H),4.24-4.46(m,2H),3.97-4.19(m,1H),3.71(br s,1H),3.42-3.66(m,1H),3.05-3.30(m,1H),1.97(s,3H),1.65(br s,1H),1.33(d,J＝6.6Hz,3H),0.90(td,J＝5.4,2.6Hz,1H),0.80-0.87(m,1H),0.70-0.79(m,1H),0.60-0.70(m,1H). m/z(ESI,+ve ion):559.0(M+H) ⁺ .

Example 43

6-Chloro-1-(4,6-di(2-propyl)-5-pyrimidinyl)-7-(2-fluorophenyl)-4-((2S)-2-methyl-4-(2-acryloyl)-1-piperazinyl)pyrido[2,3-d]pyrimidin-2(1H)-one

Step 1: 4,6-Diisopropylpyrimidine-5-amine (Intermediate U). A solution of 4,6-dichloro-5-aminopyrimidine (3.00 g, 18.29 mmol, Combilux, San Diego, California, USA) in THF (18 mL) was deoxygenated for 5 min by bubbling argon into the mixture. Zinc 2-propyl bromide (0.5 M solution in THF, 91.0 mL, 45.5 mmol, Sigma-Aldrich, St. Louis, Missouri, USA) was added via syringe, followed by XantPhos PdG3 (434 mg, 0.46 mmol, Sigma-Aldrich, St. Louis, Missouri, USA). The resulting mixture was stirred at room temperature for 16 h and then filtered through a diatomaceous earth mat. The filter cake was washed with EtOAc, and the filtrate was collected and concentrated to give 4,6-diisopropylpyrimidine-5-amine (3.45 g). This material was used in the next step without further purification. m/z (ESI, +Ve ions): 180.2 (M+H) ⁺ .

Step 2: 2,5,6-Trichloro-N-((4,6-diisopropylpyrimidin-5-yl)aminoformyl)nicotinamide. A solution of 2,5,6-trichloronicotinamide (intermediate P, 3.30 g, 14.6 mmol) in 1,2-dichloroethane (49 mL) was treated with oxalyl chloride (2 M solution in DCM, 11.0 mL, 22.0 mmol). The mixture was heated at 80 °C for 45 min, then heating was stopped and the reaction was concentrated. The residue was dissolved in acetonitrile (49 mL), cooled to -10 °C, and a solution of 4,6-diisopropylpyrimidin-5-amine (intermediate U, 3.15 g, 17.6 mmol) in acetonitrile (5 mL) was added via a tube. The resulting mixture was stirred overnight at room temperature and then concentrated. The residue was suspended in a 10:1 heptane/EtOAc solution (110 mL) and filtered. The filtrate was concentrated and the residue purified by silica gel chromatography (elution: 0-40% EtOAc/heptane) to provide 2,5,6-trichloro-N-((4,6-diisopropylpyrimidin-5-yl)carbamoyl)nicotinamide. ^¹H NMR (400 MHz, DMSO- _d⁶ ) δppm 11.30-11.46 (m, 1H), 9.66 (br s, 1H), 8.95-9.01 (m, 1H), 8.65-8.72 (m, 1H), 3.26 (s, 2H), 1.17 (d, J = 6.6 Hz, 12H). m/z (ESI, +Ve ions): 432.0 (M+H) ^⁺ .

Step 3: 6,7-Dichloro-1-(4,6-diisopropylpyrimidin-5-yl)pyrido[2,3-d]pyrimidin-2,4(1H,3H)-dione. KHMDS (1M solution in THF, 12.2 mL, 12.2 mmol) was added to a solution of 2,5,6-trichloro-N-((4,6-diisopropylpyrimidin-5-yl)carbamoyl)nicotinamide (2.10 g, 4.9 mmol) in THF (49 mL) at -20 °C. The cooling bath was removed and the resulting mixture was stirred at room temperature for 2 h. The reaction mixture was quenched with a saturated aqueous ammonium chloride solution (50 mL), diluted with brine, and extracted with 3:1 EtOAc/MeOH (1x). The layers were separated, and the aqueous layer was extracted again with EtOAc (1x). The combined organic layers were dried over anhydrous magnesium sulfate and concentrated. The residue was suspended in heptane/EtOAc and filtered. The filtrate was concentrated to provide 6,7-dichloro-1-(4,6-diisopropylpyrimidin-5-yl)pyrido[2,3-d]pyrimidin-2,4(1H,3H)-dione. ^¹H NMR (400MHz, DMSO- _d⁶ ) δppm 12.33(s,1H), 9.18(s,1H), 8.61(s,1H), 2.90–3.02(m,2H), 1.10(d,J＝6.6Hz,6H), 0.99(d,J＝6.6Hz,6H). m/z (ESI, +VE ions): 394.0(M+H) ^⁺ .

Step 4: (S)-tert-butyl 4-(6,7-dichloro-1-(4,6-diisopropylpyrimidin-5-yl)-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-4-yl)-3-methylpiperazine-1-carboxylate. Phosphorus oxychloride (0.255 mL, 2.74 mmol) was slowly added via syringe to a solution of 6,7-dichloro-1-(4,6-diisopropylpyrimidin-5-yl)pyrido[2,3-d]pyrimidin-2,4(1H,3H)-dione (900 mg, 2.28 mmol) and DIPEA (0.518 mL, 2.97 mmol) in acetonitrile (15 mL). The resulting mixture was heated at 80 °C for 45 min and then cooled to -10 °C. DIPEA (1.2 mL, 6.88 mmol) was added, followed by the addition of a solution of (S)-4-N-Boc-2-methylpiperazine (1.37 g, 6.85 mmol, Combi-Blocks, Inc., San Diego, California, USA) in acetonitrile (5 mL) via a catheter. The resulting mixture was heated to room temperature and stirred for 10 min, and then DIPEA (1.2 mL, 6.88 mmol) was added again. The reaction mixture was poured into ice water and extracted with EtOAc (2x). The combined organic layers were washed with brine (1x), dried over anhydrous magnesium sulfate, and concentrated. The residue was purified by silica gel chromatography (elution: 0-80% EtOAc/heptane) to provide (S)-tert-butyl 4-(6,7-dichloro-1-(4,6-diisopropylpyrimidin-5-yl)-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-4-yl)-3-methylpiperazine-1-carboxylate. ¹ H NMR (400MHz, DMSO-d ₆ ) δppm 9.15 (s, 1H), 8.48 (s, 1H), 5.75 (s, 1H), 4.90 (br s, 1H), 4.21 (brd, J = 14.1Hz, 1H), 3.91-4.06 (m, 1H), 3.83 (br d,J＝13.3Hz,1H),3.73(br t,J＝10.6Hz,1H),3.03-3.19(m,1H),2.69(dq,J＝13.4,6.7Hz,2H),1.45(s,9H),1.31-1.36(m,3H),1.09(d,J＝6.6Hz,6H),1.00(d,J＝6.6Hz,6H). m/z (ESI, +Ve ions): 576.2 (M+H) ⁺ .

Step 5: (S)-tert-butyl 4-(6-chloro-1-(4,6-diisopropylpyrimidin-5-yl)-7-(2-fluorophenyl)-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-4-yl)-3-methylpiperazine-1-carboxylate. The mixture of (S)-tert-butyl 4-(6,7-dichloro-1-(4,6-diisopropylpyrimidin-5-yl)-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-4-yl)-3-methylpiperazine-1-carboxylate (500 mg, 0.87 mmol) and potassium acetate (426 mg, 4.34 mmol) in 1,4-dioxane (4.3 mL) was deoxygenated for 5 min by bubbling argon gas into the mixture. A complex of [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) with dichloromethane (63 mg, 0.087 mmol) was added and the mixture was heated at 90 °C for 10 min. A solution of 2-fluorophenylboronic acid (243 mg, 1.735 mmol, Combi-Blocks, Inc., San Diego, California, USA) in 1,4-dioxane (2 mL) was slowly added, followed by 6 drops of water. The resulting mixture was heated at 90 °C for 1 h. The reaction mixture was adsorbed onto a silica gel stopper and purified by silica gel chromatography (elution: 0–8% MeOH/DCM) to provide (S)-tert-butyl 4-(6-chloro-1-(4,6-diisopropylpyrimidin-5-yl)-7-(2-fluorophenyl)-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-4-yl)-3-methylpiperazine-1-carboxylate. ¹ H NMR (400MHz, DMSO-d ₆ ) δppm 9.05 (s, 1H), 8.46 (s, 1H), 8.15 (s, 1H), 7.49-7.54 (m, 1H), 7.27-7.32 (m, 1H), 7.12-7.16 (m, 1H), 4.93 (br s,1H),4.29(br d,J＝13.9Hz,1H),4.07(br d,J＝4.6Hz,1H),3.85(br d,J＝13.7Hz,1H),3.75(br t, J = 11.0 Hz, 1H), 3.56 (s, 2H), 3.08–3.22 (m, 3H), 2.67–2.78 (m, 2H), 1.45 (s, 9H), 1.08 (d, J = 6.6 Hz, 6H), 0.92 (d, J = 6.6 Hz, 6H). m/z (ESI, +Ve ions): 636.2 (M+H) ⁺ .

Step 6. 6-Chloro-1-(4,6-di(2-propyl)-5-pyrimidinyl)-7-(2-fluorophenyl)-4-((2S)-2-methyl-4-(2-acryloyl)-1-piperazinyl)pyrido[2,3-d]pyrimidin-2(1H)-one. Trifluoroacetic acid (0.176 mL, 2.36 mmol) was added to a solution of (S)-tert-butyl4-(6-chloro-1-(4,6-diisopropylpyrimidin-5-yl)-7-(2-fluorophenyl)-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-4-yl)-3-methylpiperazin-1-carboxylate (150 mg, 0.24 mmol) in DCM (2.4 mL). The resulting mixture was heated at 38 °C for 2 h and then concentrated. The residue was dissolved in DCM (2.4 mL), cooled to 0 °C, and treated with DIPEA (0.494 mL, 2.83 mmol). After 2 min, acryloyl chloride (0.019 mL, 0.24 mmol) was added dropwise via syringe, and the reaction was stirred at 0 °C for another 10 min. The reaction mixture was concentrated and the residue was partitioned between a saturated aqueous sodium bicarbonate solution and EtOAc (2x). The combined organic layers were dried over anhydrous magnesium sulfate and concentrated. The residue was purified by silica gel chromatography (elution: 0–100% EtOAc/heptane, followed by 0–8% MeOH/DCM) to provide 6-chloro-1-(4,6-di(2-propyl)-5-pyrimidinyl)-7-(2-fluorophenyl)-4-((2S)-2-methyl-4-(2-acryloyl)-1-piperazinyl)pyrido[2,3-d]pyrimidin-2(1H)-one. ¹ H NMR (400MHz, DMSO-d ₆ ) δppm 9.06(s,1H),8.46-8.52(m,1H),7.48-7.55(m,1H),7.26-7.34(m,2H),7.17(td,J＝7.4,1.6Hz,1H),6.82-6.93(m,1H),6.22(br d,,J＝16.6Hz,1H),5.75-5.80(m,1H),5.00(br s,1H),4.31-4.43(m,2H),4.02-4.21(m,1H),3.81(br d,J＝8.9Hz,1H),3.45-3.70(m,1H),3.10-3.30(m,1H),2.73(br d, J＝6.4Hz, 2H), 1.36 (d, J＝6.6Hz, 3H), 1.09 (d, J＝6.6Hz, 6H), 0.93 (d, J＝6.4Hz, 6H). m/z(ESI,+ve ion):590.2(M+H) ⁺ .

Example 44

6-Chloro-1-(2-cyclopropyl-4-methyl-3-pyridyl)-7-(2-fluoro-6-hydroxyphenyl)-4-((2S)-2-methyl-4-(2-acryloyl)-1-piperazinyl)pyrido[2,3-d]pyrimidin-2(1H)-one

Step 1: 2,5,6-Trichloro-N-((2-cyclopropyl-4-methylpyridin-3-yl)carbamoyl)nicotinamide. Oxaloyl chloride (2M solution in DCM, 11.6 mL, 23.3 mmol) was slowly added via syringe to a solution of 2,5,6-trichloronicotinamide (intermediate P, 3.5 g, 15.5 mmol) in THF (30 mL). The resulting mixture was heated at 65 °C for 2 h, then heating was stopped and the reaction was concentrated. The residue was dissolved in THF (30 mL) and treated via catheter with a solution of 2-cyclopropyl-4-methylpyridin-3-amine (intermediate T, 2.5 g, 17.1 mmol) in THF (15 mL). The resulting mixture was stirred at room temperature for 2 h, then quenched with water and extracted with EtOAc (3x). The combined organic layers were dried over anhydrous sodium sulfate and concentrated to provide 2,5,6-trichloro-N-((2-cyclopropyl-4-methylpyridin-3-yl)carbamoyl)nicotinamide. This material was used in the next step without further purification. ^¹H NMR (400 MHz, DMSO- _d⁶ ) δppm 11.30 (br s, 1H), 9.44–9.73 (m, 1H), 8.64 (s, 1H), 8.21 (d, J = 5.0 Hz, 1H), 7.07 (d, J = 5.0 Hz, 1H), 2.22 (s, 3H), 0.92 (s, 2H), 0.91 (d, J = 3.5 Hz, 2H). m/z (ESI, +Ve ions): 400.9 (M+H) ^⁺ .

Step 2: 6,7-Dichloro-1-(2-cyclopropyl-4-methylpyridin-3-yl)pyrido[2,3-d]pyrimidin-2,4(1H,3H)-dione. KHMDS (1M solution in THF, 2.30 mL, 2.30 mmol) was slowly added via syringe to an ice-cold solution of 2,5,6-trichloro-N-((2-cyclopropyl-4-methylpyridin-3-yl)carbamoyl)nicotinamide (460 mg, 1.15 mmol) in 5 mL of THF. The resulting mixture was stirred at 0 °C for 2 h, then quenched with a saturated aqueous ammonium chloride solution and extracted with EtOAc (3x). The combined organic layers were dried over anhydrous sodium sulfate and concentrated. The residue was purified by silica gel chromatography (elution buffer: 0-40% 3:1 EtOAc-EtOH/heptane) to provide 6,7-dichloro-1-(2-cyclopropyl-4-methylpyridin-3-yl)pyrido[2,3-d]pyrimidine-2,4(1H,3H)-dione. ¹ H NMR (400MHz, DMSO-d ₆ ) δppm 12.26 (br s,1H),8.59(s,1H),8.36(d,J＝5.0Hz,1H),7.19(d,J＝5.0Hz,1H),2.05(s,3H),1.86-1. 96(m,1H),0.88-0.95(m,1H),0.79-0.87(m,1H),0.73-0.79(m,1H),0.62-0.70(m,1H). m/z(ESI,+ve ion):362.9(M+H) ⁺ .

Step 3: 4,6,7-Trichloro-1-(2-cyclopropyl-4-methylpyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one. Phosphorus oxychloride (1.77 mL, 19.0 mmol) was added to a solution of 6,7-dichloro-1-(2-cyclopropyl-4-methylpyridin-3-yl)pyrido[2,3-d]pyrimidin-2,4(1H,3H)-dione (4.60 g, 12.7 mmol) and DIPEA (3.32 mL, 19.0 mmol) in acetonitrile (25 mL). The resulting mixture was heated at 80 °C for 3 h, then cooled to room temperature and concentrated to provide 4,6,7-trichloro-1-(2-cyclopropyl-4-methylpyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one. This material was used in the next step without further purification. m/z (ESI, +Ve ions): 380.9 (M+H) ⁺ .

Step 4: (S)-tert-butyl-4-(6,7-dichloro-1-(2-cyclopropyl-4-methylpyridin-3-yl)-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-4-yl)-3-methylpiperazine-1-carboxylate (intermediate V). DIPEA (11.1 mL, 63.3 mmol) was added to an ice-cold solution of 4,6,7-trichloro-1-(2-cyclopropyl-4-methylpyridin-3-yl)pyrido[2,3-d]pyrimidin-2(1H)-one (12.7 mmol) in DCM (40 mL), followed by the addition of (S)-4-N-Boc-2-methylpiperazine (2.54 g, 12.7 mmol, Combi-Blocks, Inc., San Diego, California, USA). The resulting mixture was stirred at 0 °C for 1 h, then quenched with water and extracted with DCM (3x). The combined organic layers were dried over anhydrous sodium sulfate and concentrated. The residue was purified by silica gel chromatography (elution: 0-50% 3:1 EtOAc-EtOH/heptane) to provide (S)-tert-butyl-4-(6,7-dichloro-1-(2-cyclopropyl-4-methylpyridin-3-yl)-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-4-yl)-3-methylpiperazine-1-carboxylate. ¹ H NMR (400MHz, DMSO-d ₆ ) δppm 8.44 (d, J = 7.0Hz, 1H), 8.33 (d, J = 5.0Hz, 1H), 7.17 (d, J = 5.2Hz, 1H), 4.85 (br s, 1H), 4.16 (br d, J = 11.0Hz, 1H), 3.96 (br dd,J＝3.4,2.2Hz,1H),3.82(br d,J＝13.3Hz,1H),3.69(q,J＝12.0Hz,1H),3.19-3.29(m,1H),3.03-3.19(m,1H),1.95(d,J＝3.9Hz,3H),1.58-1.68(m,1H) ), 1.45 (s, 9H), 1.32 (dd, J = 6.6, 2.5Hz, 3H), 0.88-0.97 (m, 1H), 0.77-0.86 (m, 1H), 0.70-0.77 (m, 1H), 0.59-0.69 (m, 1H). m/z(ESI,+ve ion):544.9(M+H) ⁺ .

Step 5: (3S)-tert-butyl 4-(6-chloro-1-(2-cyclopropyl-4-methylpyridin-3-yl)-7-(2-fluoro-6-hydroxyphenyl)-2-oxo-1,2-dihydropyridino[2,3-d]pyrimidin-4-yl)-3-methylpiperazine-1-carboxylate. The mixture of (S)-tert-butyl-4-(6,7-dichloro-1-(2-cyclopropyl-4-methylpyridin-3-yl)-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-4-yl)-3-methylpiperazin-1-carboxylic acid tert-butyl ester (intermediate V, 992 mg, 1.82 mmol), (2-fluoro-6-hydroxyphenyl)boronic acid (510 mg, 3.27 mmol, Combi-blocks, San Diego, California, USA), potassium acetate (892 mg, 9.09 mmol), and the complex of [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) with dichloromethane (119 mg, 0.15 mmol) in 1,4-dioxane (16 mL) was degassed under argon and heated to 80 °C. After 2 min, two drops of water were added to the reaction mixture and the temperature was raised to 90 °C. The mixture was heated at 90°C for 1 h and then allowed to cool to room temperature. Water was added and the resulting mixture was extracted with EtOAc (3x). The combined organic layers were dried over anhydrous magnesium sulfate and concentrated. The residue was purified by silica gel chromatography (elution: 0-50% 3:1 EtOAc-EtOH/heptane) to provide (3S)-tert-butyl-4-(6-chloro-1-(2-cyclopropyl-4-methylpyridin-3-yl)-7-(2-fluoro-6-hydroxyphenyl)-2-oxo-1,2-dihydropyridino[2,3-d]pyrimidin-4-yl)-3-methylpiperazine-1-carboxylate. ¹ H NMR (400MHz, DMSO-d ₆ ) δppm 10.04-10.22 (m, 1H), 8.31-8.45 (m, 1H), 8.23 (d, J = 5.0Hz, 1H), 7.19-7.31 (m, 1H), 7.09 (br s,1H),6.60-6.76(m,2H),4.75-5.02(m,1H),4.10-4.36(m,1H),3.92-4.06(m,1H),3.56-3.89(m,2H),2.91-3.30(m,2H),1.90(br d, J = 19.3 Hz (3H), 1.61-1.77 (m, 1H), 1.45 (s, 9H), 1.30-1.40 (m, 3H), 0.59-0.90 (m, 4H). m/z (ESI, +ve ions): 620.9 (M+H) ⁺ .

Step 6: 1-(2-Cyclopropyl-4-methyl-3-pyridyl)-6-chloro-7-(2-fluoro-6-hydroxyphenyl)-4-((2S)-2-methyl-4-(2-acryloyl)-1-piperazinyl)pyrido[2,3-d]pyrimidin-2(1H)-one. Trifluoroacetic acid (2.47 mL, 33.2 mmol) was added to a solution of (3S)-tert-butyl4-(6-chloro-1-(2-cyclopropyl-4-methylpyridin-3-yl)-7-(2-fluoro-6-hydroxyphenyl)-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-4-yl)-3-methylpiperazin-1-carboxylate (1.03 g, 1.66 mmol) in DCM (10 mL). The resulting mixture was stirred at room temperature for 3 h and then concentrated. The residue was dissolved in DCM (10 mL), cooled to 0 °C, and treated dropwise with DIPEA (1.45 mL, 8.29 mmol) followed by acryloyl chloride (0.120 mL, 1.49 mmol) via syringe. The reaction was stirred at 0 °C for 2 h, then quenched with water and extracted with DCM (3x). The combined organic layers were dried over anhydrous sodium sulfate and concentrated. The residue was purified by silica gel chromatography (elution: 0-50% 3:1 EtOAc-EtOH/heptane) to provide 6-chloro-1-(2-cyclopropyl-4-methyl-3-pyridyl)-7-(2-fluoro-6-hydroxyphenyl)-4-((2S)-2-methyl-4-(2-acryloyl)-1-piperazinyl)pyrido[2,3-d]pyrimidin-2(1H)-one. ¹ H NMR (400MHz, DMSO-d ₆ ) δppm 10.06-10.26 (m, 1H), 8.33-8.49 (m, 1H), 8.23 (d, J = 4.8Hz, 1H), 7.20-7.34 (m, 1H), 7.09 (br d,J＝2.1Hz,1H),6.79-6.92(m,1H),6.63-6.78(m,2H),6.16-6.29(m,1H),5.76(dd,J＝10.4,2.3Hz,1H),4.77-5.08(m,1H), 4.21-4.47(m,2H),3.98-4.20(m,1H),3.39-3.92(m,2H),2.92-3.28(m,1H),1.85-1.99(m,3H),1.62-1.79(m,1H),1.34(br d,J＝19.5Hz,3H),0.74-0.88(m,3H),0.56-0.68(m,1H). m/z (ESI, +Ve ions): 574.9 (M+H) ⁺ .

Example 45

6-Chloro-1-(2-cyclopropyl-4-methyl-3-pyridyl)-7-(2-fluorophenyl)-4-((2S)-2-methyl-4-(2-acryloyl)-1-piperazinyl)pyrido[2,3-d]pyrimidin-2(1H)-one

Step 1: (S)-tert-butyl 4-(6-chloro-1-(2-cyclopropyl-4-methylpyridin-3-yl)-7-(2-fluorophenyl)-2-oxo-1,2-dihydropyridino[2,3-d]pyrimidin-4-yl)-3-methylpiperazine-1-carboxylate. The mixture of (S)-tert-butyl-4-(6,7-dichloro-1-(2-cyclopropyl-4-methylpyridin-3-yl)-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-4-yl)-3-methylpiperazine-1-carboxylate (intermediate V, 500 mg, 0.92 mmol), (2-fluorophenyl)boronic acid (269 mg, 1.92 mmol, Combi-blocks, San Diego, California, USA), potassium acetate (450 mg, 4.58 mmol), and the complex of [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) with dichloromethane (75 mg, 0.09 mmol) in 1,4-dioxane (7 mL) was degassed with argon and heated to 80 °C. After 2 min, three drops of water were added to the reaction mixture and the temperature was raised to 90 °C. The mixture was heated at 90°C for 1 hour and then allowed to cool to room temperature. Water was added and the resulting mixture was extracted with EtOAc (3x). The combined organic layers were dried over anhydrous magnesium sulfate and concentrated. The residue was purified by silica gel chromatography (elution: 0-40% 3:1 EtOAc-EtOH/heptane) to provide (S)-tert-butyl-4-(6-chloro-1-(2-cyclopropyl-4-methylpyridin-3-yl)-7-(2-fluorophenyl)-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-4-yl)-3-methylpiperazine-1-carboxylate. ¹ H NMR (400MHz, DMSO-d ₆ ) δppm 8.41 (d, J = 2.5 Hz, 1H), 8.25 (d, J = 5.0 Hz, 1H), 7.49-7.56 (m, 1H), 7.25-7.35 (m, 3H), 7.11 (d, J = 4.8 Hz, 1H), 4.90 (br d,J＝1.5Hz,1H),4.24(br d,J＝13.7Hz,1H),3.93-4.07(m,1H),3.85(br d, J = 13.9 Hz, 1H), 3.65-3.79 (m, 1H), 3.21-3.30 (m, 1H), 3.09-3.20 (m, 1H), 1.96 (s, 3H), 1.60-1.70 (m, 1H), 1.45 (s, 9H), 1.36 (d, J = 6.6 Hz, 3H), 0.86-0.93 (m, 1H), 0.72-0.83 (m, 2H), 0.61-0.71 (m, 1H). m/z (ESI, +Ve ions): 605.0 (M+H) ⁺ .

Step 2: 6-Chloro-1-(2-cyclopropyl-4-methyl-3-pyridinyl)-7-(2-fluorophenyl)-4-((2S)-2-methyl-4-(2-acryloyl)-1-piperazinyl)pyrido[2,3-d]pyrimidin-2(1H)-one. Trifluoroacetic acid (1.28 mL, 17.2 mmol) was added to a solution of (S)-tert-butyl4-(6-chloro-1-(2-cyclopropyl-4-methylpyridin-3-yl)-7-(2-fluorophenyl)-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-4-yl)-3-methylpiperazin-1-carboxylate (521 mg, 0.86 mmol) in DCM (10 mL). The resulting mixture was stirred at room temperature for 3 h and then concentrated. The residue was dissolved in DCM (10 mL), cooled to 0 °C, and treated dropwise with DIPEA (0.762 mL, 4.31 mmol) followed by acryloyl chloride (0.440 mL, 0.86 mmol) via syringe. The reaction was stirred at 0 °C for 2 h, then quenched with water and extracted with DCM (1x). The organic layer was dried over anhydrous sodium sulfate and concentrated. The residue was purified by silica gel chromatography (elution: 0-50% 3:1 EtOAc-EtOH/heptane) to provide 6-chloro-1-(2-cyclopropyl-4-methyl-3-pyridinyl)-7-(2-fluorophenyl)-4-((2S)-2-methyl-4-(2-acryloyl)-1-piperazinyl)pyrido[2,3-d]pyrimidin-2(1H)-one. ¹ H NMR (400MHz, DMSO-d ₆ ) δppm 8.44 (br s,1H),8.25(d,J＝5.0Hz,1H),7.48-7.56(m,1H),7.24-7.35(m,3H),7.11(d,J＝5.0Hz,1H),6.78-6.92(m,1H),6.20(br d,J＝16.2Hz,1H),5.72-5.79(m,1H),4.94(br s,1H),4.25-4.44(m,2H),3.99-4.20(m,1H),3.43-3.69(m,2H),3.02-3.15(m ,1H),1.96(d,J＝3.3Hz,3H),1.60-1.71(m,1H),1.34(d,J＝6.8Hz,3H),0.89(br dd,J=8.3,4.6Hz,1H),0.72-0.84(m,2H),0.61-0.71(m,1H). m/z(ESI,+ve ion):559.0(M+H) ⁺ .

Example 46

6-Chloro-1-(2-ethyl-4-methyl-3-pyridyl)-7-(2-fluorophenyl)-4-((2S)-2-methyl-4-(2-acryloyl)-1-piperazinyl)pyrido[2,3-d]pyrimidin-2(1H)-one.

Step 1: 2-Ethyl-4-methylpyridin-3-amine (intermediate W). Ethyl magnesium bromide (3.5 mL, 10.5 mmol, in 3 M solution in diethyl ether) was slowly added via syringe to a solution of zinc chloride (0.5 M in THF, 18 mL, 9.0 mmol). This addition was exothermic. The solution was stirred at room temperature for 10 min, and then 3-amino-2-bromo-4-methylpyridinium (1.5 g, 8.0 mmol, Combi-blocks, San Diego, California, USA) and dichloro[1,1'-bis(diphenylphosphino)ferrocene]palladium(ii)dichloromethane adduct (120 mg, 0.16 mmol) were added. The resulting mixture was heated at 50 °C for 20 min, then heating was stopped and the reaction was allowed to cool to room temperature. The reaction mixture was quenched with 10% ammonium hydroxide solution (30 mL) and extracted with EtOAc (80 mL, 2 × 40 mL). The combined organic layers were dried over anhydrous sodium sulfate and concentrated. The residue was purified by silica gel chromatography (elution: 1%–5% MeOH/DCM) to provide 2-ethyl-4-methylpyridin-3-amine. ^¹H NMR (400 MHz, chloroform-d) δppm 7.92 (d, J = 5.0 Hz, 1H), 6.87 (d, J = 4.8 Hz, 1H), 3.59 (br s, 2H), 2.74 (q, J = 7.6 Hz, 2H), 2.19 (s, 3H), 1.33 (t, J = 7.6 Hz, 3H). m/z (ESI, +Ve ions): 137.1 (M+H) ⁺ .

Step 2: 2,5,6-Trichloro-N-((2-ethyl-4-methylpyridin-3-yl)aminocarbamoyl)nicotinamide. Oxaloyl chloride (2M solution in DCM, 5.4 mL, 10.8 mmol) was slowly added via syringe to a solution of 2,5,6-trichloronicotinamide (intermediate P, 2.5 g, 11.1 mmol) in THF (20 mL). The resulting mixture was heated at 65 °C for 1.5 h, then heating was stopped and the reaction was allowed to cool to room temperature. 2-Ethyl-4-methylpyridin-3-amine (intermediate W, 1.5 g, 10.7 mmol) was added via catheter to a solution of THF (15 mL). The resulting mixture was stirred at room temperature for 1 h, and then partially concentrated to remove most of the THF. The residue was partitioned between a saturated aqueous sodium bicarbonate solution (30 mL) and EtOAc (50 mL). The organic layer was washed with brine (1 x), dried over anhydrous sodium sulfate, and concentrated. The residue was suspended in 10:1 heptane/EtOAc (60 mL) and filtered to provide 2,5,6-trichloro-N-((2-ethyl-4-methylpyridin-3-yl)carbamoyl)nicotinamide. The product was used directly in the next step.

Step 3: 6,7-Dichloro-1-(2-ethyl-4-methylpyridin-3-yl)pyrido[2,3-d]pyrimidin-2,4(1H,3H)-dione. KHMDS (1M solution in THF, 16.0 mL, 16.0 mmol) was slowly added via syringe to an ice-cold solution of 2,5,6-trichloro-N-((2-ethyl-4-methylpyridin-3-yl)carbamoyl)nicotinamide (10.7 mmol) in THF (60 mL). The resulting solution was stirred at 0 °C for 10 min, then quenched with saturated ammonium chloride aqueous solution (20 mL) and extracted with EtOAc (60 mL). The organic layer was washed with brine (1x), dried over anhydrous sodium sulfate, and concentrated. The residue was suspended in 9:1 heptane/EtOAc (60 mL) and filtered to provide 6,7-dichloro-1-(2-ethyl-4-methylpyridin-3-yl)pyrido[2,3-d]pyrimidine-2,4(1H,3H)-dione. ^¹H NMR (400 MHz, DMSO- _d⁶ ) δppm 12.14–12.39 (m, 1H), 8.54 (s, 1H), 8.47 (d, J = 5.0 Hz, 1H), 7.28 (d, J = 5.0 Hz, 1H), 2.41–2.49 (m, 2H), 2.03 (s, 3H), 1.07 (t, J = 7.5 Hz, 3H). m/z (ESI, +Ve ions): 350.9 (M+H) ^⁺ .

Step 4: (S)-tert-butyl-4-(6,7-dichloro-1-(2-ethyl-4-methylpyridin-3-yl)-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-4-yl)-3-methylpiperazine-1-carboxylate. Phosphorus oxychloride (0.40 mL, 4.3 mmol) was added to a solution of 6,7-dichloro-1-(2-ethyl-4-methylpyridin-3-yl)pyrido[2,3-d]pyrimidin-2,4(1H,3H)-dione (1.0 g, 2.9 mmol) and DIPEA (1.5 mL, 8.6 mmol) in acetonitrile (8 mL). The resulting solution was heated at 80 °C for 30 min and then cooled to 0 °C. DIPEA (1.5 mL, 8.6 mmol) was added, followed by (S)-4-N-Boc-2-methylpiperazine (800 mg, 4.0 mmol, Combi-Blocks, Inc., San Diego, California, USA). The resulting mixture was heated to room temperature and stirred for 30 min, then diluted with EtOAc (30 mL) and washed with saturated sodium bicarbonate aqueous solution (10 mL) and brine (10 mL). The organic layer was dried over anhydrous magnesium sulfate and concentrated. The residue was purified by silica gel chromatography (elution: 10%-50% 3:1 EtOAc-EtOH/heptane) to provide (S)-tert-butyl-4-(6,7-dichloro-1-(2-ethyl-4-methylpyridin-3-yl)-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-4-yl)-3-methylpiperazine-1-carboxylate. ¹ H NMR (400MHz, DMSO-d ₆ ) δppm 8.46 (d, J = 5.0 Hz, 1H), 8.44 (s, 1H), 7.27 (d, J = 5.0 Hz, 1H), 4.85 (br s, 1H), 4.12-4.21 (m, 1H), 3.95 (br s, 1H), 3.82 (br d,J＝12.9Hz,1H),3.69(br d,J＝11.4Hz,1H),2.94-3.27(m,2H),2.25-2.43(m,2H),1.94(d,J＝1.7Hz,3H),1.45(s,9H),1.32(t,J＝6.2Hz,3H),1.05(t,J＝7.6Hz,3H). m/z (ESI, +Ve ions): 533.0 (M+H) ⁺ .

Step 5: (S)-tert-butyl 4-(6-chloro-1-(2-ethyl-4-methylpyridin-3-yl)-7-(2-fluorophenyl)-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-4-yl)-3-methylpiperazine-1-carboxylate. The mixture of (S)-tert-butyl-4-(6,7-dichloro-1-(2-ethyl-4-methylpyridin-3-yl)-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-4-yl)-3-methylpiperazine-1-carboxylate (450 mg, 0.84 mmol), (2-fluorophenyl)boronic acid (199 mg, 1.43 mmol, Combi-blocks, San Diego, California, USA), potassium acetate (250 mg, 2.55 mmol), and the complex of [1,1'-bis(diphenylphosphine)ferrocene]dichloropalladium(II) with dichloromethane (33 mg, 0.04 mmol) in 1,4-dioxane (5 mL) and water (1 mL) was degassed with nitrogen and heated at 65 °C for 1.5 h. The reaction was allowed to cool to room temperature, then diluted with EtOAc (40 mL) and washed with saturated sodium bicarbonate aqueous solution (10 mL) and brine (10 mL). The organic layer was dried over anhydrous magnesium sulfate and concentrated. The residue was purified by silica gel chromatography (elution: 10%-50% 3:1 EtOAc-EtOH/heptane) to provide (S)-tert-butyl-4-(6-chloro-1-(2-ethyl-4-methylpyridin-3-yl)-7-(2-fluorophenyl)-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-4-yl)-3-methylpiperazine-1-carboxylate. ¹ H NMR (400MHz, DMSO-d ₆ ) δppm 8.43 (s, 1H), 8.36 (d, J = 5.0Hz, 1H), 7.47-7.55 (m, 1H), 7.20-7.33 (m, 4H), 4.90 (br s, 1H), 4.24 (br s, 1H), 3.99 (br s,1H),3.85(br d,J＝13.3Hz,1H),3.72(br d, J = 11.6 Hz, 1H), 3.33–3.47 (m, 1H), 3.03–3.21 (m, 1H), 2.30–2.43 (m, 2H), 1.95 (s, 3H), 1.46 (s, 9H), 1.36 (t, J = 6.1 Hz, 3H), 1.04 (dt, J = 7.5, 2.3 Hz, 3H). ^19F NMR (377 MHz, chloroform-d) δppm -113.84–113.85 (2s, 1F). m/z (ESI, +Ve ions): 592.9 (M+H) ⁺ .

Step 6: 6-Chloro-1-(2-ethyl-4-methyl-3-pyridinyl)-7-(2-fluorophenyl)-4-((2S)-2-methyl-4-(2-acryloyl)-1-piperazinyl)pyrido[2,3-d]pyrimidin-2(1H)-one. Trifluoroacetic acid (3.0 mL, 40.3 mmol) was added to a solution of (S)-tert-butyl4-(6-chloro-1-(2-ethyl-4-methylpyridin-3-yl)-7-(2-fluorophenyl)-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-4-yl)-3-methylpiperazin-1-carboxylate (500 mg, 0.84 mmol) in DCM (10 mL). The resulting mixture was stirred at room temperature for 30 min and then concentrated. The residue was dissolved in DCM (10 mL) and treated with a solution of DIPEA (0.500 mL, 2.86 mmol) followed by acryloyl chloride (0.100 mL, 1.23 mmol) in DCM (2 mL). The reaction was stirred at room temperature for 30 min and then quenched with saturated sodium bicarbonate aqueous solution (10 mL) and brine (5 mL). The mixture was extracted with EtOAc (2 x 15 mL) and the combined organic layers were dried over anhydrous sodium sulfate and concentrated. The residue was purified by silica gel chromatography (elution: 10%-60% 3:1 EtOAc-EtOH/heptane) to provide 6-chloro-1-(2-ethyl-4-methyl-3-pyridyl)-7-(2-fluorophenyl)-4-((2S)-2-methyl-4-(2-acryloyl)-1-piperazinyl)pyrido[2,3-d]pyrimidin-2(1H)-one. ¹ H NMR (400MHz, chloroform-d) δppm 8.45 (d, J = 5.0Hz, 1H), 8.10 (s, 1H), 7.38-7.46 (m, 1H), 7.08-7.20 (m, 4H), 6.62 (br s,1H),6.42(dd,J＝16.7,1.6Hz,1H),5.82(dd,J＝10.5,1.8Hz,1H),4.24-5.21(m,3H),3.60-4.15(m, 3H),3.00-3.36(m,1H),2.41-2.60(m,2H),2.00-2.12(m,3H),1.45-1.61(m,3H),1.15-1.22(m,3H). ^19F NMR (377MHz, chloroform-d) δppm -111.94--113.08 (m, 1F). m/z (ESI, +Ve ions): 547.2 (M+H) ⁺ .

Example 47

6-Chloro-1-(4,6-diethyl-5-pyrimidinyl)-7-(2-fluorophenyl)-4-((2S)-2-methyl-4-(2-acryloyl)-1-piperazinyl)pyrido[2,3-d]pyrimidin-2(1H)-one

Step 1: 4,6-Divinylpyrimidine-5-amine. A mixture of 4,6-dichloro-5-aminopyrimidine (5.0 g, 30.5 mmol, Sigma Aldrich, St. Louis, Missouri), potassium vinyltrifluoroborate (16.3 g, 122 mmol, Sigma Aldrich, St. Louis, Missouri), Pd(dppf) _Cl₂ (1.12 g, 1.52 mmol), and cesium carbonate (49.7 g, 152 mmol) in 1,4-dioxane (130 mL) and water (13 mL) was degassed under nitrogen and heated at 100 °C for 4 h. The reaction was allowed to cool to room temperature and then diluted with water and extracted sequentially with EtOAc (3 × 100 mL) and DCM (5 × 100 mL). The combined organic layers were dried over anhydrous magnesium sulfate and concentrated. The residue was purified by silica gel chromatography (elution buffer: 0-20% 4:1 DCM-MeOH/DCM) to provide 4,6-divinylpyrimidine-5-amine. ^¹H NMR (400 MHz, chloroform-d) δppm 8.62 (s, 1H), 6.80-6.89 (m, 2H), 6.46 (dd, J = 17.0, 1.7 Hz, 2H), 5.71 (dd, J = 10.9, 1.8 Hz, 2H), 3.86 (br s, 2H). m/z (ESI, +VE ions): 148.1 (M+H) ⁺ .

Step 2: 4,6-Diethylpyrimidine-5-amine (Intermediate X). A solution of 4,6-divinylpyrimidine-5-amine (3.1 g, 21.1 mmol) in ethanol (50 mL) was treated with palladium (10 wt.% on activated carbon, 1.12 g, 1.05 mmol). The mixture (4x) was purified with 20 psi hydrogen and stirred at room temperature for 4 h under 20 psi hydrogen. The reaction mixture was filtered through a diatomaceous earth mat and concentrated to give 4,6-diethylpyrimidine-5-amine. ^¹H NMR (400 MHz, chloroform-d) δppm 8.59 (s, 1H), 3.62 (br s, 2H), 2.70 (q, J = 7.6 Hz, 4H), 1.34 (t, J = 7.6 Hz, 6H). m/z (ESI, +Ve ion): 152.2 (M+H) ⁺ .

Step 3: 2,5,6-Trichloro-N-((4,6-diethylpyrimidin-5-yl)aminoformyl)nicotinamide. Oxaloyl chloride (2M solution in DCM, 15.5 mL, 31.0 mmol) was added to a suspension of 2,5,6-trichloronicotinamide (intermediate P, 4.67 g, 20.7 mmol) in THF (100 mL). The resulting mixture was heated at 65 °C for 1 h, then heating was stopped, and the reaction was concentrated to one-third of its original volume. Toluene (100 mL) was added, and the mixture was cooled to 0 °C. A solution of 4,6-diethylpyrimidin-5-amine (intermediate X, 3.13 g, 20.7 mmol) in 1,2-dichloroethane (20 mL) was added dropwise via a tube. The resulting mixture was heated to room temperature and stirred for 15 min, and then concentrated. The residue was suspended in MTBE (50 mL) and filtered. The filtered solid was briefly dried in a vacuum oven at 50°C to provide 2,5,6-trichloro-N-((4,6-diethylpyrimidin-5-yl)carbamoyl)nicotinamide. m/z (ESI, +Ve ions): 402.1 (M+H) ⁺ .

Step 4: 6,7-Dichloro-1-(4,6-diethylpyrimidin-5-yl)pyrido[2,3-d]pyrimidin-2,4(1H,3H)-dione. KHMDS (1M solution in THF, 36.2 mL, 36.2 mmol) was slowly added via syringe to an ice-cold solution of 2,5,6-trichloro-N-((4,6-diethylpyrimidin-5-yl)carbamoyl)nicotinamide (7.28 g, 18.1 mmol) in 100 mL of THF. The resulting mixture was heated to room temperature and stirred for 5 min, then quenched with a saturated aqueous ammonium chloride solution and extracted with EtOAc (5 × 100 mL). The combined organic layers were dried over anhydrous magnesium sulfate and concentrated. The residue was dried overnight in a vacuum oven at 45°C to provide 6,7-dichloro-1-(4,6-diethylpyrimidin-5-yl)pyrido[2,3-d]pyrimidin-2,4(1H,3H)-dione. This material was used in the next step without further purification. m/z (ESI, +ve ions): 366.0 (M+H) ⁺ .

Step 5: (S)-tert-butyl 4-(6,7-dichloro-1-(4,6-diethylpyrimidin-5-yl)-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-4-yl)-3-methylpiperazine-1-carboxylate.

A solution of 6,7-dichloro-1-(4,6-diethylpyrimidin-5-yl)pyrido[2,3-d]pyrimidin-2,4(1H,3H)-dione (3.87 g, 10.6 mmol) in acetonitrile (30 mL) was treated with triethylamine (3.71 mL, 26.4 mmol) followed by phosphorus oxychloride (1.26 mL, 12.7 mmol). The resulting mixture was heated at 60 °C for 30 min and then cooled to 0 °C. Triethylamine (1.50 mL, 10.7 mmol) was added, followed by (S)-4-N-Boc-2-methylpiperazine (2.22 g, 11.1 mmol, Combi-Blocks, Inc., San Diego, California, USA). The reaction mixture was heated to room temperature and stirred for 30 min, and then partitioned between EtOAc (100 mL) and brine (40 mL). The organic layer was washed with water (30 mL) and brine (50 mL), dried over anhydrous magnesium sulfate, and concentrated. The residue was purified by silica gel chromatography (elution: 0-100% EtOAc/heptane) to provide (S)-tert-butyl-4-(6,7-dichloro-1-(4,6-diethylpyrimidin-5-yl)-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-4-yl)-3-methylpiperazine-1-carboxylate. ¹ H NMR (400MHz, DMSO-d ₆ ) δppm 9.09 (s, 1H), 8.48 (s, 1H), 4.88 (br s, 1H), 4.19 (br d, J = 13.7Hz, 1H), 3.90-4.00 (m, 1H), 3.83 (br d,J＝13.7Hz,1H),3.66-3.78(m,1H),3.05-3.35(m,2H),2.35-2.46(m,4H),1.45(s,9H),1.33(d,J＝6.6Hz,3H),1.08(t,J＝7.5Hz,6H). m/z(ESI,+ve ion):548.2(M+H) ⁺ .

Step 6: (S)-tert-butyl 4-(6-chloro-1-(4,6-diethylpyrimidin-5-yl)-7-(2-fluorophenyl)-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-4-yl)-3-methylpiperazine-1-carboxylate. The mixture of (S)-tert-butyl-4-(6,7-dichloro-1-(4,6-diethylpyrimidin-5-yl)-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-4-yl)-3-methylpiperazine-1-carboxylate (354 mg, 0.65 mmol), (2-fluorophenyl)boronic acid (108 mg, 0.78 mmol, Combi-blocks, San Diego, California, USA), potassium acetate (317 mg, 3.23 mmol), and the complex of [1,1'-bis(diphenylphosphine)ferrocene]dichloropalladium(II) with dichloromethane (23 mg, 0.03 mmol) in 1,4-dioxane (6.5 mL) was degassed with argon for 5 min. Four drops of water were added and the mixture was heated at 90 °C for 30 min. The reaction was allowed to cool to room temperature and then partitioned between water (10 mL) and EtOAc (20 mL). The aqueous layer was extracted with DCM (20 mL). The combined organic layers were washed with brine (20 mL), dried over anhydrous magnesium sulfate, and concentrated. The residue was purified by silica gel chromatography (elution: 0-30% 4:1 DCM-MeOH/DCM) to provide (S)-tert-butyl 4-(6-chloro-1-(4,6-diethylpyrimidin-5-yl)-7-(2-fluorophenyl)-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-4-yl)-3-methylpiperazine-1-carboxylate. ¹ H NMR (400MHz, DMSO-d ₆ ) δppm 9.01(s,1H),8.45(s,1H),7.49-7.57(m,1H),7.26-7.34(m,2H),7.20-7.26(m,1H),4.88-4.98(m,1H),4.27(br d,J＝13.7Hz,1H),3.93-4.03(m,1H),3.85(br d,J＝13.5Hz,1H),3.70-3.81(m,1H),3.05-3.35(m,2H),2.35-2.46(m,4H),1.46(s,9H),1.37(d,J＝6.6Hz,3H),1.06(td,J＝7.5,1.6Hz,6H). ¹⁹ F NMR (377MHz, DMSO-d ₆ ) δ ppm - 114.01 (s, 1F). m/z(ESI,+ve ion):608.2(M+H) ⁺ .

Step 7: 6-Chloro-1-(4,6-diethyl-5-pyrimidinyl)-7-(2-fluorophenyl)-4-((2S)-2-methyl-4-(2-acryloyl)-1-piperazinyl)pyrido[2,3-d]pyrimidin-2(1H)-one. Trifluoroacetic acid (3.0 mL, 38.9 mmol) was added to a solution of (S)-tert-butyl4-(6-chloro-1-(4,6-diethylpyrimidin-5-yl)-7-(2-fluorophenyl)-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-4-yl)-3-methylpiperazin-1-carboxylic acid (ester (327 mg, 0.54 mmol) in 5 mL DCM). The resulting mixture was stirred at room temperature for 15 min and then concentrated. The residue was dissolved in DCM (10 mL), cooled to 0 °C, and treated with DIPEA (0.470 mL, 2.69 mmol), followed by acryloyl chloride (0.048 mL, 0.59 mmol). The reaction was stirred at 0 °C for 10 min, and then DIPEA (0.300 mL, 1.71 mmol) was added, followed by acryloyl chloride (0.020 mL, 0.25 mmol). After maintaining the mixture at 0 °C for another 20 min, the reaction was quenched with saturated sodium bicarbonate aqueous solution (10 mL) and extracted with DCM (2 × 30 mL). The combined organic layers were dried over anhydrous magnesium sulfate and concentrated. The residue was purified by silica gel chromatography (elution buffer: 0-60% 4:1 DCM-MeOH/DCM) to provide 6-chloro-1-(4,6-diethyl-5-pyrimidinyl)-7-(2-fluorophenyl)-4-((2S)-2-methyl-4-(2-acryloyl)-1-piperazinyl)pyrido[2,3-d]pyrimidin-2(1H)-one. ^¹H NMR (400MHz, DMSO- _d⁶ ) δppm 9.01 (s, 1H), 8.48 (br⁶) d,J＝5.0Hz,1H),7.50-7.57(m,1H),7.27-7.35(m,3H),7.20-7.27(m,1H) ,6.79-6.93(m,1H),6.17-6.26(m,1H),5.65-5.80(m,1H),4.92-5.02(m, 1H),4.23-4.44(m,2H),3.99-4.20(m,1H),3.72-3.88(m,1H),3.40-3.71 (m,1H),2.35-2.48(m,4H),1.36(d,J＝6.6Hz,3H),1.06(t,J＝7.5Hz,6H). ^19F NMR (377MHz, DMSO- _d⁶ ) δppm -114.01 (s, 1F). m/z (ESI, +Ve ions): 562.1 (M+H) ^⁺ .

Example 48

6-Chloro-7-(2-fluoro-6-hydroxyphenyl)-4-((2S)-2-methyl-4-(2-acryloyl)-1-piperazinyl)-1-(4-(2-propane)-1,3-thiazolyl-5-yl)pyrido[2,3-d]pyrimidin-2(1H)-one

Step 1: 2,5,6-Trichloro-N-((4-isopropylthiazolyl-5-yl)carbamoyl)nicotinamide. Oxaloyl chloride (2M solution in DCM, 2.4 mL, 4.9 mmol) was added to a mixture of 2,5,6-trichloronicotinamide (intermediate P, 1.00 g, 4.5 mmol) in THF (20 mL). The resulting mixture was heated at 70 °C for 30 min, then heating was stopped and the reaction was allowed to cool to room temperature. 4-(propane-2-yl)-1,3-thiazolyl-5-amine (715 mg, 5.0 mmol, Enamine, Monmouth Junction, NJ, USA) was added. The reaction mixture was stirred at room temperature for 10 min and then concentrated. The residue was suspended in MeOH and filtered. The filtered solids were discarded and the filtrate was concentrated to provide 2,5,6-trichloro-N-((4-isopropylthiazolyl-5-yl)carbamoyl)nicotinamide. ^¹H NMR (400MHz, DMSO- _d⁶ ) δppm 11.69 (br s, 1H), 10.64 (br s, 1H), 8.71 (s, 1H), 8.58 (s, 1H), 3.13 (br d, J = 6.4Hz, 1H), 1.25 (d, J = 6.8Hz, 6H). m/z (ESI, +Ve ions): 393.0 (M+H) ^⁺ .

Step 2: 6,7-Dichloro-1-(4-isopropylthiazolyl-5-yl)pyrido[2,3-d]pyrimidin-2,4(1H,3H)-dione. KHMDS (1M solution in THF, 9.3 mL, 9.3 mmol) was added to an ice-cold solution of 2,5,6-trichloro-N-((4-isopropylthiazolyl-5-yl)carbamoyl)nicotinamide (1.75 g, 4.5 mmol) in 20 mL of THF. The resulting mixture was heated to room temperature and stirred for 30 min, then cooled to 0 °C and treated with KHMDS (1M solution in THF, 1.0 mL, 1.0 mmol). The reaction was heated to room temperature and stirred for 15 min, then quenched with a semi-saturated aqueous ammonium chloride solution (60 mL) and extracted with EtOAc (2 × 50 mL). The combined organic layers were washed with water (60 mL), eluted, dried, and concentrated using a Chem Elut extraction cartridge (Agilent Technologies, Santa Clara, California). The residue was purified by silica gel chromatography (elution: 5%–70% EtOAc/heptane) to provide 6,7-dichloro-1-(4-isopropylthiazolyl-5-yl)pyrido[2,3-d]pyrimidine-2,4(1H,3H)-dione. ^¹H NMR (400 MHz, DMSO- _d⁶ ) δppm 12.20 (s, 1H), 9.14 (s, 1H), 8.54 (s, 1H), 2.86 (quin, J = 6.8 Hz, 1H), 1.11 (t, J = 7.4 Hz, 6H). m/z (ESI, +Ve ion): 357.0 (M+H) ^⁺ .

Step 3: (S)-tert-butyl-4-(6,7-dichloro-1-(4-isopropylthiazo-5-yl)-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-4-yl)-3-methylpiperazine-1-carboxylate. DIPEA (4.40 mL, 25.3 mmol) was added to a mixture of 6,7-dichloro-1-(4-isopropylthiazo-5-yl)pyrido[2,3-d]pyrimidin-2,4(1H,3H)-dione (3.01 g, 8.4 mmol) and acetonitrile (40 mL), followed by the addition of phosphorus oxychloride (1.57 mL, 16.8 mmol). The resulting mixture was heated at 80 °C for 15 min, then cooled to room temperature and concentrated. The residue was dissolved in DMF (30 mL) and treated with DIPEA (7.34 mL, 42.2 mmol), followed by (S)-4-N-Boc-2-methylpiperazine (1.89 g, 9.5 mmol, Sigma-Aldrich, St. Louis, Missouri, USA). The resulting solution was stirred at room temperature for 5 min, then ice water (100 mL) was added and the mixture was stirred for another 15 min. The mixture was filtered and the filtered solids were dissolved in DCM, dried and concentrated by passing them through a Chem Elut extraction tube (Agilent Technologies, Santa Clara, California). The residue was purified by silica gel chromatography (elution buffer: 40%-100% EtOAc/heptane) to provide (S)-tert-butyl 4-(6,7-dichloro-1-(4-isopropylthiazo-5-yl)-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-4-yl)-3-methylpiperazine-1-carboxylate. ¹ H NMR (400MHz, DMSO-d ₆ ) δppm 9.10 (s, 1H), 8.40 (d, J = 1.7Hz, 1H), 4.85 (br s, 1H), 4.07-4.18 (m, 1H), 3.88-4.00 (m, 1H), 3.81 (br d,J＝13.9Hz,1H),3.64-3.77(m,1H),3.28(s,1H),3.01-3.19(m,1H,),2.65(t,J =13.3, 6.6Hz, 1H), 1.44 (s, 9H), 1.31 (dd, J = 6.4, 3.3Hz, 3H), 1.09-1.13 (m, 6H). m/z(ESI,+ve ion):539.2(M+H) ⁺ .

Step 4: (3S)-tert-butyl 4-(6-chloro-7-(2-fluoro-6-hydroxyphenyl)-1-(4-isopropylthiazolyl-5-yl)-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-4-yl)-3-methylpiperazine-1-carboxylate. The mixture of (S)-tert-butyl-4-(6,7-dichloro-1-(4-isopropylthiazo-5-yl)-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-4-yl)-3-methylpiperazine-1-carboxylate (2.03 g, 3.76 mmol), potassium acetate (1.86 g, 18.9 mmol), (2-fluoro-6-hydroxyphenyl)boronic acid (911 mg, 5.84 mmol, Combi-blocks, San Diego, California, USA), and the complex of [1,1'-bis(diphenylphosphine)ferrocene]-dichloropalladium(II) with dichloromethane (282 mg, 0.39 mmol) in 1,4-dioxane (36 mL) was degassed with argon for 5 min. A drop of water was added and the resulting mixture was heated to 90 °C for 1.5 h. The reaction was allowed to cool to room temperature and then partitioned between water (50 mL) and EtOAc (2 x 50 mL). The combined organic extracts were washed with water (60 mL), eluted, dried, and concentrated using a Chem Elut extraction tube (Agilent Technologies, Santa Clara, California). The residue was purified by silica gel chromatography (elution: 50%–100% EtOAc/heptane) to provide (3S)-tert-butyl 4-(6-chloro-7-(2-fluoro-6-hydroxyphenyl)-1-(4-isopropylthiazo-5-yl)-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-4-yl)-3-methylpiperazine-1-carboxylate. ¹ H NMR (400MHz, DMSO-d ₆ ) δppm 10.10 (s, 1H), 9.00 (s, 1H), 8.31-8.39 (m, 1H), 7.21-7.32 (m, 1H), 6.65-6.77 (m, 2H), 4.88 (br s,1H),4.11-4.33(m,1H),3.91-3.98(m,1H),3.83(br d,J＝13.9Hz,1H),3.64-3.78(m,1H),3.28(br s,1H),3.03-3.21(m,1H),2.63(br s,1H),1.45(s,9H),1.36(br s,3H),1.09(br d,J＝6.8Hz,3H), 1.01(d,J＝6.8Hz,3H). ¹⁹ F NMR (376MHz,DMSO-d ₆ )δppm-115.41(br s,1F). m/z (ESI,+ve ions):615.2(M+H) ⁺ .

Step 5: 6-Chloro-7-(2-fluoro-6-hydroxyphenyl)-4-((2S)-2-methyl-4-(2-acryloyl)-1-piperazinyl)-1-(4-(2-propane)-1,3-thiazolyl-5-yl)pyrido[2,3-d]pyrimidin-2(1H)-one. Trifluoroacetic acid (10 mL, 130 mmol) was added to a solution of (3S)-tert-butyl4-(6-chloro-7-(2-fluoro-6-hydroxyphenyl)-1-(4-isopropylthiazolyl)-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-4-yl)-3-methylpiperazin-1-carboxylate (2.15 g, 3.50 mmol) in DCM (20 mL). The reaction was stirred at room temperature for 30 min and then concentrated. The residue was dissolved in DCM (20 mL), cooled to 0 °C, and treated with DIPEA (3.05 mL, 17.5 mmol), followed by acryloyl chloride solution (0.258 M in DCM, 10.84 mL, 2.8 mmol). The reaction mixture was heated to room temperature and stirred for 15 min, then cooled to 0 °C and treated with acryloyl chloride solution (0.258 M in DCM, 2.0 mL, 0.52 mmol). After heating to room temperature and stirring for another 10 min, the reaction was concentrated. The residue was purified by silica gel chromatography (elution buffer: 40%-100% 3:1 EtOAc-EtOH/heptane) to provide 6-chloro-7-(2-fluoro-6-hydroxyphenyl)-4-((2S)-2-methyl-4-(2-acryloyl)-1-piperazinyl)-1-(4-(2-propane)-1,3-thiazolyl-5-yl)pyrido[2,3-d]pyrimidin-2(1H)-one. ¹ H NMR(400MHz, DMSO-d ₆ )δppm 10.10(s,1H),9.01(s,1H),8.37(br s,1H),7.22-7.32(m,1H),6.80-6.93(m,1H),6.66-6.77(m,2H),6.20(br d,J＝16.6Hz,1H),5.71-5.81(m,1H),4.83-5.04(m,1H),3.98-4.45(m,3H),3.56-3.87(m,2H),3.07(br dd,J＝4.1,2.9Hz,1H),2.59-2.70(m,1H),1.33(br d,J＝5.8Hz,3H), 1.07-1.14(m,3H), 1.01(brs,1F). ^19F NMR (376MHz,DMSO- _d6 )δppm-115.41(brs,1F). m/z (ESI,+ve ions): 569.2(M+H) ⁺ .

Example 49

6-Chloro-7-(2-fluoro-6-hydroxyphenyl)-1-(2-(2-methyl-2-propane)phenyl)-4-((2S)-2-methyl-4-(2-acryloyl)-1-piperazinyl)pyrido[2,3-d]pyrimidin-2(1H)-one

Step 1: N-((2-(tert-butyl)phenyl)carbamoyl)-2,5,6-trichloronicotinamide. Oxaloyl chloride (2.5 mL, 5.0 mmol, 2 M solution in DCM) was added to a mixture of 2,5,6-trichloronicotinamide (intermediate P, 1.02 g, 4.5 mmol) in THF (20 mL). The resulting mixture was heated at 70 °C for 40 min, then heating was stopped and the reaction was allowed to cool to room temperature. 2-tert-butylaniline (0.708 mL, 4.5 mmol, Ark Pharmaceuticals, Arlington Heights, .I., USA) was added. The reaction mixture was stirred at room temperature for 10 min and then concentrated. The residue was suspended in MeOH and filtered. The filtered solid was collected to provide N-((2-(tert-butyl)phenyl)carbamoyl)-2,5,6-trichloronicotinamide. ^¹H NMR (400MHz, DMSO- _d⁶ ) δppm: 11.43 (br s, 1H), 9.79–10.16 (m, 1H), 8.65 (s, 1H), 7.49 (br dd, J = 7.5, 1.7Hz, 1H), 7.43 (br d, J = 7.7Hz, 1H), 7.18–7.28 (m, 2H), 1.40 (s, 9H). m/z (ESI, +Ve ions): 422.0 (M + Na) ^⁺ .

Step 2: 1-(2-(tert-butyl)phenyl)-6,7-dichloropyrido[2,3-d]pyrimidine-2,4(1H,3H)-dione. KHMDS (1M solution in THF, 6.6 mL, 6.6 mmol) was added to an ice-cold mixture of N-((2-(tert-butyl)phenyl)carbamoyl)-2,5,6-trichloronicotinamide (1.26 g, 3.1 mmol) in THF (20 mL). The resulting mixture was heated to room temperature and stirred for 10 min, then quenched by adding a semi-saturated aqueous ammonium chloride solution (60 mL) and extracted with EtOAc (2 × 50 mL). The combined organic layers were washed with water (60 mL), eluted, dried, and concentrated using a Chem Elut extraction tube (Agilent Technologies, Santa Clara, California). The residue was suspended in MeOH and filtered. The filtered solids were collected to provide 1-(2-(tert-butyl)phenyl)-6,7-dichloropyrido[2,3-d]pyrimidine-2,4(1H,3H)-dione. ^¹H NMR (400MHz, DMSO- _d⁶ ) δppm 12.19(s,1H), 8.56(s,1H), 7.65(dd,J＝8.2,1.3Hz,1H), 7.41–7.46(m,1H), 7.33(td,J＝7.5,1.2Hz,1H), 7.21(dd,J＝7.8,1.3Hz,1H), 1.17(s,9H). m/z (ESI, +Ve ion): 364.0(M+H) ^⁺ .

Step 3: (S)-tert-butyl-4-(1-(2-(tert-butyl)phenyl)-6,7-dichloro-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-4-yl)-3-methylpiperazine-1-carboxylate. DIPEA (0.574 mL, 3.30 mmol) was added to a mixture of 1-(2-(tert-butyl)phenyl)-6,7-dichloropyrido[2,3-d]pyrimidin-2,4(1H,3H)-dione (925 mg, 2.54 mmol) and acetonitrile (20 mL), followed by the addition of phosphorus oxychloride (0.284 mL, 3.05 mmol). The resulting mixture was heated at 80 °C for 45 min and then cooled to 0 °C. DIPEA (1.33 mL, 7.62 mmol) was added, followed by (S)-4-N-Boc-2-methylpiperazine (0.522 g, 2.61 mmol, Sigma-Aldrich, St. Louis, Missouri, USA). The resulting solution was allowed to gradually warm to room temperature over 40 min, then poured into a cold saturated sodium bicarbonate solution (50 mL) and stirred for another 10 min. The mixture was extracted with EtOAc (2 × 30 mL), and the combined organic extracts were eluted, dried, and concentrated using Chem Elut extraction tubes (Agilent Technologies, Santa Clara, California). The residue was purified by silica gel chromatography (elution: 30%-80% EtOAc/heptane) to provide (S)-tert-butyl4-(1-(2-(tert-butyl)phenyl)-6,7-dichloro-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-4-yl)-3-methylpiperazine-1-carboxylate. ¹ H NMR (400MHz, DMSO-d ₆ ) δppm 8.33-8.48(m,1H),7.61(d,J＝7.9Hz,1H),7.36-7.42(m,1H),7.29(td,J＝7.5,1.2Hz,1H),7.00(dd,J＝7.7,1.0Hz,1H),4.68-4.89(m,1H),3.8 8-4.25(m,2H),3.77-3.86(m,1H),3.52-3.71(m,1H),3.09-3.26(m,1H ), 2.84-3.07 (m, 1H), 1.44 (s, 9H), 1.24 (s, 3H), 1.13 (d, J = 2.3Hz, 9H). m/z (ESI, +Ve ions): 546.2 (M+H) ⁺ .

Step 4: (3S)-tert-butyl 4-(1-(2-(tert-butyl)phenyl)-6-chloro-7-(2-fluoro-6-hydroxyphenyl)-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-4-yl)-3-methylpiperazine-1-carboxylate. The mixture of (S)-tert-butyl-4-(1-(2-(tert-butyl)phenyl)-6,7-dichloro-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-4-yl)-3-methylpiperazine-1-carboxylate (1.00 g, 1.83 mmol), potassium acetate (905 mg, 9.22 mmol), (2-fluoro-6-hydroxyphenyl)boronic acid (347 mg, 2.22 mmol, Combi-blocks, San Diego, California, USA), and the complex of [1,1'-bis(diphenylphosphine)ferrocene]-dichloropalladium(II) with dichloromethane (137 mg, 0.19 mmol) in 1,4-dioxane (20 mL) was degassed with argon for 5 min. A drop of water was added and the resulting mixture was heated to 90 °C for 1.5 h. The reaction was allowed to cool to room temperature and then partitioned between water (40 mL) and EtOAc (2 x 40 mL). The combined organic extracts were washed with water (40 mL), eluted, dried, and concentrated using a Chem Elut extraction tube (Agilent Technologies, Santa Clara, California). The residue was purified by silica gel chromatography (elution: 40%–100% EtOAc/heptane) to provide (3S)-tert-butyl-4-(1-(2-(tert-butyl)phenyl)-6-chloro-7-(2-fluoro-6-hydroxyphenyl)-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-4-yl)-3-methylpiperazine-1-carboxylate. ¹ H NMR (400MHz, DMSO-d ₆ ) δppm10.03-10.09(m,1H),8.31-8.39(m,1H),7.53(d,J＝8.1Hz,1H),7.25-7.32(m,1H),7.18-7.24(m,2H),6.94(br d,J＝6.6Hz,1H),6.61-6.73(m,2H),4.80(br d,J＝1.2Hz,1H),4.09-4.25(m,1H),3.92-4.01(m,1H),3.84(br d,J＝12.9Hz,1H),3.63(br d, J = 9.1 Hz, 1H), 2.99–3.26 m, 2H), 1.45 s, 9H), 1.29–1.37 m, 3H), 1.11 s, 9H. ^¹⁹ F NMR (376 MHz, DMSO- _d⁶ ) δppm -115.35–115.44 m, 1F. m/z (ESI, +Ve ions): 622.2 M + ^H⁺ .

Step 5: 6-Chloro-7-(2-fluoro-6-hydroxyphenyl)-1-(2-(2-methyl-2-propane)phenyl)-4-((2S)-2-methyl-4-(2-acryloyl)-1-piperazinyl)pyrido[2,3-d]pyrimidin-2(1H)-one. Trifluoroacetic acid (5.0 mL, 64.9 mmol) was added to a solution of (3S)-tert-butyl4-(1-(2-(tert-butyl)phenyl)-6-chloro-7-(2-fluoro-6-hydroxyphenyl)-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-4-yl)-3-methylpiperazin-1-carboxylate (984 mg, 1.58 mmol) in DCM (10 mL). The resulting mixture was stirred at room temperature for 30 min and then concentrated. The residue was dissolved in DCM (10 mL), cooled to 0 °C, and treated with DIPEA (1.38 mL, 7.91 mmol), followed by acryloyl chloride solution (0.258 M in DCM, 4.90 mL, 1.27 mmol). The reaction mixture was heated to room temperature and stirred for 30 min, then cooled to 0 °C and treated with acryloyl chloride solution (0.258 M in DCM, 1.0 mL, 0.26 mmol). After heating to room temperature and stirring for another 5 min, the reaction was concentrated. The residue was purified by silica gel chromatography (elution buffer: 40%-100% 3:1 EtOAc-EtOH/heptane) to provide 6-chloro-7-(2-fluoro-6-hydroxyphenyl)-1-(2-(2-methyl-2-propane)phenyl)-4-((2S)-2-methyl-4-(2-acryloyl)-1-piperazinyl)pyrido[2,3-d]pyrimidin-2(1H)-one. ¹ H NMR (400MHz, DMSO-d ₆ ) δppm 10.06 (br d, J＝5.4Hz, 1H), 8.38 (br d,J＝12.2Hz,1H),7.54(d,J＝7.7Hz,1H),7.17-7.32(m,3H),6.91-7.02(m,1H),6.83(br dd,J＝10.2,16.4Hz,1H),6.61-6.74(m,2H),6.20(br d,J＝16.6Hz,1H),5.72-5.80(m,1H),4.85(br d, J = 2.1 Hz, 1H), 4.07–4.51 (m, 3H), 3.59–3.81 (m, 3H), 1.24–1.37 (m, 3H), 1.06–1.13 (m, 9H). ^19F NMR (376 MHz, DMSO- _d⁶ ) δppm -115.42 (m, 1F). m/z (ESI, +Ve ions): 576.2 (M+H) ^⁺ .

Example 50

6-Chloro-7-(2-fluorophenyl)-4-((2S)-2-methyl-4-(2-acryloyl)-1-piperazinyl)-1-(2-(2-propyl)phenyl)-2(1H)-pterinone

Step 1: Methyl 3-amino-6-chloro-5-(2-fluorophenyl)pyrazine-2-carboxylate. A mixture of methyl 3-amino-5,6-dichloropyrazine-2-carboxylate (10.0 g, 45 mmol, Ark Pharmaceuticals, Arlington Heights, .I.), (2-fluorophenyl)boronic acid (6.93 g, 49.5 mmol, Combi-blocks, San Diego, .C.), and potassium carbonate (13.1 g, 95 mmol) in a 10:1 DME/water mixture (220 mL) was degassed for 5 min under nitrogen and then tetrakis(triphenylphosphine)palladium(0) (1.04 g, 0.90 mmol) was added. The reaction mixture was stirred at 90 °C for 16 h, then allowed to cool to room temperature and partitioned between EtOAc (200 mL) and 1N HCl (200 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated to provide methyl 3-amino-6-chloro-5-(2-fluorophenyl)pyrazine-2-carboxylate. m/z(ESI,+ve): 282.1(M+H) ⁺ .

Step 2: Methyl 3,6-dichloro-5-(2-fluorophenyl)pyrazine-2-carboxylate. Amyl nitrite (15.8 mL, 117 mmol) and copper chloride (12.6 g, 94 mmol) were added to a solution of methyl 3-amino-6-chloro-5-(2-fluorophenyl)pyrazine-2-carboxylate (22.0 g, 78 mmol) in t-BuOH (220 mL). The resulting mixture was stirred at 60 °C for 16 h and then partitioned between water (1 L) and EtOAc (2 L). The organic layer was dried over anhydrous sodium sulfate and concentrated. The residue was purified by silica gel chromatography (elution: 0-5% EtOAc/hexane) to provide methyl 3,6-dichloro-5-(2-fluorophenyl)pyrazine-2-carboxylate. m/z (ESI, +Ve ions): 300.9 (M+H) ⁺ .

Step 3: 3,6-Dichloro-5-(2-fluorophenyl)pyrazine-2-carboxamide. Add ammonium hydroxide solution (30%, 150 mL, 44.8 mmol) to a solution of methyl 3,6-dichloro-5-(2-fluorophenyl)pyrazine-2-carboxylate (13.5 g, 44.8 mmol) in THF (150 mL). Heat the resulting mixture at 50 °C for 4 h, then allow it to cool to room temperature and partition between water and EtOAc (500 mL). Dry the organic layer with anhydrous sodium sulfate and concentrate. Purify the residue by silica gel chromatography (elution: 0-30% EtOAc/hexane) to provide 3,6-dichloro-5-(2-fluorophenyl)pyrazine-2-carboxamide. ¹ H NMR (400MHz, DMSO-d ₆ ) δppm 8.32 (br s, 1H), 8.16 (br s, 1H), 7.64-7.77 (m, 2H), 7.41-7.47 (m, 2H). m/z(ESI,+ve):286.0(M+H) ⁺ .

Step 4: 3,6-Dichloro-5-(2-fluorophenyl)-N-((2-isopropylphenyl)aminoformyl)pyrazin-2-carboxamide. A mixture of 3,6-dichloro-5-(2-fluorophenyl)pyrazin-2-carboxamide (704 mg, 2.46 mmol) and oxalyl chloride solution (2 M in DCM, 1.35 mL, 2.71 mmol) in DCE (15 mL) was heated for 1 h at 80 °C. The reaction was cooled to room temperature and 2-isopropylaniline (0.35 mL, 2.46 mmol) was added. The resulting mixture was stirred at room temperature for 3 h and then concentrated. The residue was treated with EtOAc, sonicated, and filtered. The filtered solid was collected to provide 3,6-dichloro-5-(2-fluorophenyl)-N-((2-isopropylphenyl)aminoformyl)pyrazin-2-carboxamide. m/z(ESI,+ve):446.8(M+H) ⁺ .

Step 5: 6-Chloro-7-(2-fluorophenyl)-1-(2-isopropylphenyl)pteridine-2,4(1H,3H)-dione (intermediate Y). KHMDS (1M solution in THF, 0.93 mL, 0.93 mmol) was added to an ice-cold mixture of 3,6-dichloro-5-(2-fluorophenyl)-N-((2-isopropylphenyl)carbamoyl)pyrazine-2-carboxamide (208 mg, 0.46 mmol) in THF (4 mL). The resulting mixture was allowed to warm to room temperature and stirred for 2 h. The reaction was partitioned between EtOAc (10 mL) and saturated ammonium chloride aqueous solution (2 × 5 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated. The residue was purified by silica gel chromatography (elution buffer: 0–50% EtOAc/heptane) to provide 6-chloro-7-(2-fluorophenyl)-1-(2-isopropylphenyl)pteridine-2,4(1H,3H)-dione. ^¹H NMR (400 MHz, chloroform-d) δppm 8.50 (br s, 1H), 7.40–7.47 (m, 3H), 7.31 (dt, J = 8.3, 4.2 Hz, 1H), 7.17–7.24 (m, 2H), 7.09–7.16 (m, 2H), 2.69 (quin, J = 6.8 Hz, 1H), 1.22 (d, J = 6.8 Hz, 3H), 1.06 (d, J = 6.8 Hz, 3H). m/z (ESI, +ve): 410.9 (M+H) ⁺ .

Step 6: 6-Chloro-7-(2-fluorophenyl)-4-((2S)-2-methyl-4-(2-acryloyl)-1-piperazinyl)-1-(2-(2-propane)phenyl)-2(1H)-pteridone. DIPEA (0.269 mL, 1.54 mmol) was added to a solution of 6-chloro-7-(2-fluorophenyl)-1-(2-isopropylphenyl)pteridin-2,4(1H,3H)-dione (373 mg, 0.91 mmol) in acetonitrile (5 mL), followed by the addition of phosphorus oxychloride (0.127 mL, 1.36 mmol). The resulting solution was stirred at 80 °C for 30 min and then concentrated. The residue was dissolved in DMF (5 mL), cooled to 0 °C, and treated with a solution of DIPEA (0.54 mL, 3.08 mmol), followed by (S)-1-(3-methylpiperazin-1-yl)prop-2-en-1-one 2,2,2-trifluoroacetate (582 mg, 1.00 mmol) in DMF (0.5 mL). The resulting solution was stirred at 0 °C for 30 min and then allowed to gradually warm to room temperature. The reaction was partitioned between EtOAc (10 mL) and water (2 × 10 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated. The residue was purified by silica gel chromatography (elution: 0-3% MeOH/DCM) to provide 6-chloro-7-(2-fluorophenyl)-4-((2S)-2-methyl-4-(2-acryloyl)-1-piperazinyl)-1-(2-(2-propane)phenyl)-2(1H)-pterinone. ^¹H NMR (400MHz, DMSO- _d⁶ ) δppm 7.51-7.61 (m, 1H), 7.44 (dt, J = 7.9, 1.9Hz, 1H), 7.33-7.38 (m, 2H), 7.27-7.32 (m, 2H), 7.24 (brt, J = 7.7Hz, 1H), 7.13 (br dt, J = 7.9, 1.7Hz, 1H), 6.89 (br dt, J = 7.9, 1.7Hz, 1H), 6.89 (br dt, J = 7.9, 1.7Hz, 1H). dd,J＝16.4,10.4Hz,1H),6.22(brd,J＝16.4Hz,1H),5.76(dd,J＝10.2,1.2Hz,1H),4.68-5.54(m,1H),4.2 1-4.44(m,6H),2.69-2.83(m,1H),1.21-1.31(m,3H),1.10(t,J＝6.3Hz,3H),0.99(dd,J＝6.7,3.4Hz,3H). m/z(ESI,+ve):546.8(M+H) ⁺ .

Example 51

7-(2-Fluorophenyl)-6-methyl-4-((2S)-2-methyl-4-(2-acryloyl)-1-piperazinyl)-1-(2-(2-propane)phenyl)-2(1H)-pterinone

Step 1: 7-(2-fluorophenyl)-1-(2-isopropylphenyl)-6-methylpteridine-2,4(1H,3H)-dione. A mixture of 6-chloro-7-(2-fluorophenyl)-1-(2-isopropylphenyl)pteridine-2,4(1H,3H)-dione (intermediate Y, 930 mg, 2.27 mmol), a complex of [1,1'-bis(diphenylphosphino)ferrocene]-dichloropalladium(II) with dichloromethane (166 mg, 0.28 mmol), trimethylboroxane (0.64 mL, 4.54 mmol, Sigma-Aldrich, St. Louis, Missouri, USA), and potassium carbonate (0.63 g, 4.54 mmol) in 10:1 dioxane/water (11 mL) was added to a glass microwave reaction vessel. The reaction was degassed with nitrogen for 5 min and heated at 100 °C for 1.5 h in an Emrys Optimizer microwave reactor (Biotage, Uppsala, Sweden). The reaction was allowed to cool to room temperature and then partitioned between water (10 mL) and EtOAc (10 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated. The residue was purified by silica gel chromatography (elution: 0-40% EtOAc/heptane) to provide 7-(2-fluorophenyl)-1-(2-isopropylphenyl)-6-methylpteridine-2,4(1H,3H)-dione. ¹ H NMR (400MHz, chloroform-d) δppm 8.53 (br s,1H),7.38-7.48(m,3H),7.30(dt,J＝8.3,4.2Hz,1H),7.08-7.20(m,4H),2.71(qui n, J＝6.8Hz, 1H), 2.61 (d, J＝2.3Hz, 3H), 1.21 (d, J＝6.8Hz, 3H), 1.05 (d, J＝6.8Hz, 3H). m/z(ESI,+ve):391.0(M+H) ⁺ .

Step 2: 7-(2-fluorophenyl)-6-methyl-4-((2S)-2-methyl-4-(2-acryloyl)-1-piperazinyl)-1-(2-(2-propane)phenyl)-2(1H)-pteridone. DIPEA (0.252 mL, 1.44 mmol) was added to a solution of 7-(2-fluorophenyl)-1-(2-isopropylphenyl)-6-methylpteridin-2,4(1H,3H)-dione (330 mg, 0.85 mmol) in acetonitrile (5 mL), followed by the addition of phosphorus oxychloride (0.119 mL, 1.27 mmol). The resulting solution was stirred at 60 °C for 1 h and then concentrated. The residue was dissolved in DMF (5 mL), cooled to 0 °C, and treated via a catheter with a solution of DIPEA (0.51 mL, 2.88 mmol), followed by (S)-1-(3-methylpiperazin-1-yl)prop-2-en-1-one 2,2,2-trifluoroacetate (544 mg, 0.93 mmol) in DMF (0.5 mL). The resulting mixture was stirred at 0 °C for 30 min and then allowed to gradually warm to room temperature. The reaction was partitioned between EtOAc (10 mL) and water (2 × 10 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated. The residue was purified by silica gel chromatography (elution: 0-3% MeOH/DCM) to provide 7-(2-fluorophenyl)-6-methyl-4-((2S)-2-methyl-4-(2-acryloyl)-1-piperazinyl)-1-(2-(2-propane)phenyl)-2(1H)-pterinone. ¹ H NMR (400MHz, DMSO-d ₆ ) δppm 7.48-7.57(m,1H),7.42(dt,J＝7.7,1.9Hz,1H),7.32-7.37(m,2H),7.20-7.31(m,3H),7.10(br dt,J＝7.1,1.0Hz,1H),6.80-6.95(m,1H),6.20(br d,J＝16.6Hz,1H),5.75(dd,J＝10.3,2.0Hz,1H),4.66-5.57(m,1H),4.13- 4.46(m,3H),3.44-3.73(m,3H),2.56-2.70(m,1H),2.42(s,3H),1.25(br s, 3H), 1.09 (t, J = 6.1 Hz, 3H), 0.97 (dd, J = 6.6, 1.7 Hz, 3H). m/z(ESI,+ve):527.0(M+H) ⁺ .

Example 52

7-(2-fluoro-6-hydroxyphenyl)-6-methyl-4-((2S)-2-methyl-4-(2-acryloyl)-1-piperazinyl)-1-(2-(2-propane)phenyl)pyrido[2,3-d]pyrimidin-2(1H)-one

Step 1: 2,6-Dichloro-5-methylnicotinic acid. Ethyl 2,6-dichloro-5-methylnicotinic acid (6.49 g, 27.7 mmol, Pharmablock Inc., Sunnyvale, California, USA) was heated for 16 h in a mixture of TFA (30 mL) and 5N HCl (24 mL) at 90 °C. The reaction was allowed to cool to room temperature and then partially concentrated. Water was added and the mixture was filtered. The filtered solid was collected and dried under vacuum to provide 2,6-dichloro-5-methylnicotinic acid. m/z (ESI,+ve): 205.9 (M+H) ⁺ .

Step 2: 2,6-Dichloro-5-methylnicotinamide. Oxaloyl chloride (2M solution in DCM, 16.6 mL, 33.1 mmol) was added to an ice-cold mixture of 2,6-dichloro-5-methylnicotinic acid (4.55 g, 22.1 mmol) in DCM (30 mL), followed by a few drops of DMF. The reaction mixture was allowed to gradually heat to room temperature and stirred for 1 h, then concentrated. The residue was suspended in toluene (15 mL), cooled to 0 °C, and treated with ammonium hydroxide (30% , 9.1 mL, 62 mmol). The reaction was stirred at room temperature for 30 min and then filtered. The filtered solid was washed with water and dried under vacuum to provide 2,6-dichloro-5-methylnicotinamide. m/z (ESI, +ve): 204.9 (M + H) ⁺ .

Step 3: 2,6-Dichloro-N-((2-isopropylphenyl)carbamoyl)-5-methylnicotinamide. A mixture of 2,6-dichloro-5-methylnicotinamide (513 mg, 2.50 mmol) and oxaloyl chloride (2 M solution in DCM, 1.38 mL, 2.63 mmol) in THF (10 mL) was heated for 1 h at 65 °C. The reaction was cooled to room temperature and 2-isopropylaniline (0.36 mL, 2.63 mmol) was added. The resulting mixture was stirred at room temperature for 1 h and then concentrated. The residue was partitioned between EtOAc (50 mL) and a saturated aqueous sodium bicarbonate solution (15 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated. The residue was suspended in a 5:1 heptane/EtOAc mixture and filtered. The filtered solid was collected to provide 2,6-dichloro-N-((2-isopropylphenyl)carbamoyl)-5-methylnicotinamide. m/z(ESI,+ve):365.8(M+H) ⁺ .

Step 4: 7-Chloro-1-(2-isopropylphenyl)-6-methylpyrido[2,3-d]pyrimidin-2,4(1H,3H)-dione. KHMDS (1M solution in THF, 4.54 mL, 4.54 mmol) was added via syringe to an ice-cold mixture of 2,6-dichloro-N-((2-isopropylphenyl)carbamoyl)-5-methylnicotinamide (831 mg, 2.27 mmol) in THF (10 mL). The resulting mixture was allowed to warm to room temperature and stirred for 2 h. The reaction was partitioned between EtOAc (20 mL) and a saturated aqueous solution of ammonium chloride (2 × 10 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated. The residue was suspended in a 5:1 heptane/EtOAc mixture and filtered. The filtered solids were collected to provide 7-chloro-1-(2-isopropylphenyl)-6-methylpyridano[2,3-d]pyrimidine-2,4(1H,3H)-dione. ^¹H NMR (400 MHz, DMSO- _d⁶ ) δppm 8.36 (s, 1H), 7.49 (dd, J = 8.5, 1.4 Hz, 1H), 7.44 (td, J = 6.8, 1.2 Hz, 1H), 7.26–7.33 (m, 1H), 7.23 (dd, J = 7.9, 1.7 Hz, 1H), 2.68 (quin, J = 6.8 Hz, 1H), 2.34 (s, 3H), 1.08 (d, J = 6.8 Hz, 3H), 1.02 (d, J = 6.6 Hz, 3H). m/z(ESI,+ve):329.9(M+H) ⁺ .

Step 5: (S)-4-(7-chloro-1-(2-isopropylphenyl)-6-methyl-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-4-yl)-3-methylpiperazine-1-carboxylic acid tert-butyl ester. DIPEA (0.70 mL, 4.04 mmol) was added to a solution of 7-chloro-1-(2-isopropylphenyl)-6-methylpyrido[2,3-d]pyrimidin-2,4(1H,3H)-dione (444 mg, 1.35 mmol) in acetonitrile (5 mL), followed by the addition of phosphorus oxychloride (0.125 mL, 1.35 mmol). The resulting solution was stirred at 80 °C for 2 h and then concentrated. The residue was dissolved in acetonitrile (5 mL), cooled to 0 °C, and treated with DIPEA (0.70 mL, 4.04 mmol), followed by (S)-4-N-Boc-2-methylpiperazine (297 mg, 1.48 mmol, Combi-Blocks, Inc., San Diego, California, USA). The resulting mixture was stirred at 0 °C for 30 min and then allowed to gradually warm to room temperature. The reaction was partitioned between EtOAc (10 mL) and water (2 × 10 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated. The residue was purified by silica gel chromatography (elution buffer: 0-30% 3:1 EtOAc-EtOH/heptane) to provide (S)-4-(7-chloro-1-(2-isopropylphenyl)-6-methyl-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-4-yl)-3-methylpiperazine-1-carboxylic acid tert-butyl ester. ^¹H NMR (400MHz, DMSO- _d⁶ ) δppm 8.16 (d, J = 16.2Hz, ¹H), 7.47 (dd, J = 6.8, 1.2Hz, ¹H), 7.41 (br⁶). t,J＝7.3Hz,1H),7.28(dt,J＝7.7,1.2Hz,1H),7.09(dt,J＝7.8,1.6Hz,1H),4.70-4.90(m,1H),3.89-4.20(m,2H),3.97-4. 09(m,2H),3.76-3.88(m,1H),3.53-3.72(m,1H),2.44(td,J＝6.8,4.9Hz,1H),2.35(d,J＝0.8Hz,3H),1.45(s,9H),1.25(br s, 3H), 1.06 (d, J = 6.8, 3H), 1.02 (dd, J = 6.8, 1.7Hz, 3H). m/z(ESI,+ve):511.9(M+H) ⁺ .

Step 6: (3S)-4-(7-(2-fluoro-6-hydroxyphenyl)-1-(2-isopropylphenyl)-6-methyl-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-4-yl)-3-methylpiperazine-1-carboxylic acid tert-butyl ester. At 90 °C, a mixture of (S)-4-(7-chloro-1-(2-isopropylphenyl)-6-methyl-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-4-yl)-3-methylpiperazin-1-carboxylic acid tert-butyl ester (458 mg, 0.89 mmol), potassium acetate (439 mg, 4.47 mmol), (2-fluoro-6-hydroxyphenyl)boronic acid (418 mg, 2.68 mmol, Combi-blocks, San Diego, California, USA) and the complex of [1,1'-bis(diphenylphosphine)ferrocene]-dichloropalladium(II) with dichloromethane (65 mg, 0.09 mmol) in 1,4-dioxane (7 mL) and water (0.05 mL) was heated for 2 h. The reaction was allowed to cool to room temperature and then partitioned between saturated aqueous sodium bicarbonate solution (15 mL) and EtOAc (20 mL). The organic layer was washed with brine (10 mL), dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica gel chromatography (elution buffer: 0-30% 3:1 EtOAc-EtOH/heptane) to provide (3S)-4-(7-(2-fluoro-6-hydroxyphenyl)-1-(2-isopropylphenyl)-6-methyl-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-4-yl)-3-methylpiperazine-1-carboxylic acid tert-butyl ester. m/z (ESI,+ve): 587.9 (M+H) ⁺ .

Step 7: 7-(2-fluoro-6-hydroxyphenyl)-6-methyl-4-((2S)-2-methyl-4-(2-acryloyl)-1-piperazinyl)-1-(2-(2-propyl)phenyl)pyrido[2,3-d]pyrimidin-2(1H)-one. Trifluoroacetic acid (1.7 mL, 14.7 mmol) was added to a solution of (3S)-4-(7-(2-fluoro-6-hydroxyphenyl)-1-(2-isopropylphenyl)-6-methyl-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-4-yl)-3-methylpiperazine-1-carboxylic acid tert-butyl ester (288 mg, 0.49 mmol) in DCM (5 mL). The resulting mixture was stirred at room temperature for 30 min and then concentrated. The residue was dissolved in DCM (3 mL), cooled to 0 °C, and treated with a solution of DIPEA (0.34 mL, 1.96 mmol), followed by acryloyl chloride (0.042 mL, 0.51 mmol) in DCM (0.5 mL). The reaction mixture was heated to room temperature and stirred for 30 min, then quenched with a saturated aqueous sodium bicarbonate solution (5 mL) and extracted with EtOAc (10 mL). The organic layer was washed with brine (5 mL), dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica gel chromatography (elution: 0-50% 3:1 EtOAc-EtOH/heptane) to provide 7-(2-fluoro-6-hydroxyphenyl)-6-methyl-1-(2-methyl-6-(2-propyl)phenyl)-4-((2S)-2-methyl-4-(2-acryloyl)-1-piperazinyl)pyrido[2,3-d]pyrimidin-2(1H)-one. ¹ H NMR (400MHz, DMSO-d ₆ ) δppm 9.94 (s, 1H), 8.10-8.19 (m, 1H), 7.38 (dd, J＝7.5, 1.0Hz, 1H), 7.31 (td, J＝7.1, 0.8Hz, 1H), 7.16-7.24 (m, 2H), 7.06 (br dt,J＝7.9,1.9Hz,1H),6.75-6.97(m,1H),6.71(d,J＝8.1Hz,1H),6.64(t,J＝8.8Hz,1H),6.21(br dd,J＝16.9,5.7Hz,1H),5.76(dd,J＝10.2,2.5Hz,1H),4.76-4.98(m,1H),4.10-4.51(m,2H),3.24-3.81(m,4H ), 2.43-2.49(m,1H),2.12(d,J＝2.9Hz,3H),1.22-1.33(m,3H),1.05(d,J＝6.8Hz,3H),0.96(d,J＝6.8Hz,3H). m/z(ESI,+ve):541.8(M+H) ⁺ .

Example 53

6-Chloro-7-(2-fluorophenyl)-1-(4-methyl-1-(2-propane)-1H-pyrazol-5-yl)-4-((2S)-2-methyl-4-(2-acryloyl)-1-piperazinyl)pyrido[2,3-d]pyrimidin-2(1H)-one

Step 1: 2,5,6-Trichloro-N-((1-isopropyl-4-methyl-1H-pyrazol-5-yl)aminocarbamoyl)nicotinamide. At room temperature, a solution of oxalyl chloride (2M in DCM, 5.70 mL, 11.4 mmol) was added to a mixture of 2,5,6-trichloronicotinamide (intermediate P, 2.43 g, 10.8 mmol) in THF (21.5 mL). The resulting mixture was heated at 65 °C for 3 h, then heating was stopped and the reaction was allowed to cool to 0 °C. A solution of 1-isopropyl-4-methyl-1H-pyrazol-5-amine (1.50 g, 10.8 mmol, ChemBridge, San Diego, California, USA) in THF (15 mL) was added via a tube. The reaction mixture was stirred at 0 °C for 30 min, then heated to room temperature and stirred for another 16 h. The reaction mixture was filtered, and the filtered solid was collected and washed with acetonitrile (20 mL) to provide 2,5,6-trichloro-N-((1-isopropyl-4-methyl-1H-pyrazol-5-yl)carbamoyl)nicotinamide. ^¹H NMR (400 MHz, DMSO- _d⁶ ) δppm 11.39 (br s, 1H), 9.51 (br s, 1H), 8.62 (s, 1H), 7.29 (s, 1H), 4.33–4.44 (m, 1H), 1.87 (s, 3H), 1.33 (d, J = 6.6 Hz, 6H). m/z (ESI, +Ve ions): 389.9 (M+H) ^⁺ .

Step 2: 6,7-Dichloro-1-(1-isopropyl-4-methyl-1H-pyrazol-5-yl)pyrido[2,3-d]pyrimidin-2,4(1H,3H)-dione. KHMDS (1M solution in THF, 18.0 mL, 18.0 mmol) was slowly added via syringe to an ice-cold mixture of 2,5,6-trichloro-N-((1-isopropyl-4-methyl-1H-pyrazol-5-yl)carbamoyl)nicotinamide (3.52 g, 9.0 mmol) in THF (30 mL). After 10 min, the resulting reaction mixture was allowed to warm to room temperature and stirred for 18 h. The resulting slurry was quenched with saturated ammonium chloride aqueous solution (50 mL) and brine (50 mL), and then extracted with EtOAc (2 x 50 mL). The combined organic layers were washed with brine (50 mL), dried over sodium sulfate, and concentrated to provide 6,7-dichloro-1-(1-isopropyl-4-methyl-1H-pyrazol-5-yl)pyrido[2,3-d]pyrimidine-2,4(1H,3H)-dione. This material was used in the next step without further purification. ^¹H NMR (400 MHz, DMSO- _d⁶ ) δppm 12.22 (br s, 1H), 8.55 (s, 1H), 7.45 (s, 1H), 4.22–4.34 (m, 1H), 1.78 (s, 3H), 1.25 (t, J = 6.6 Hz, 6H). m/z (ESI, +Ve ions): 353.9 (M+H) ^⁺ .

Step 3: 4,6,7-Trichloro-1-(1-isopropyl-4-methyl-1H-pyrazol-5-yl)pyrido[2,3-d]pyrimidin-2(1H)-one. Phosphorus oxychloride (1.68 mL, 18.0 mmol) was added to a solution of 6,7-dichloro-1-(1-isopropyl-4-methyl-1H-pyrazol-5-yl)pyrido[2,3-d]pyrimidin-2,4(1H,3H)-dione (3.19 g, 9.0 mmol) and DIPEA (4.71 mL, 27.0 mmol) in acetonitrile (60 mL). The resulting mixture was heated at 80 °C for 30 min, then cooled to room temperature and concentrated to provide 4,6,7-trichloro-1-(1-isopropyl-4-methyl-1H-pyrazol-5-yl)pyrido[2,3-d]pyrimidin-2(1H)-one. This material was used in the next step without further purification.

Step 4: (S)-tert-butyl-4-(6,7-dichloro-1-(1-isopropyl-4-methyl-1H-pyrazol-5-yl)-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-4-yl)-3-methylpiperazine-1-carboxylate. DIPEA (4.71 mL, 27.1 mmol) was added to a solution of 4,6,7-trichloro-1-(1-isopropyl-4-methyl-1H-pyrazol-5-yl)pyrido[2,3-d]pyrimidin-2(1H)-one (3.36 g, 9.0 mmol) in THF (45 mL), followed by the addition of (S)-4-N-Boc-2-methylpiperazine (2.71 g, 13.6 mmol, Combi-Blocks, Inc., San Diego, California, USA). The resulting mixture was stirred at room temperature for 1 h, then quenched with ice-cold saturated sodium bicarbonate solution (100 mL) and extracted with EtOAc (100 mL, 2 × 50 mL). The combined organic layers were dried over anhydrous sodium sulfate and concentrated. The residue was purified by silica gel chromatography (elution: 0-50% 3:1 EtOAc-EtOH/heptane) to provide (S)-tert-butyl-4-(6,7-dichloro-1-(1-isopropyl-4-methyl-1H-pyrazol-5-yl)-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-4-yl)-3-methylpiperazine-1-carboxylate. ¹ HNMR (400MHz, DMSO-d ₆ ) δppm 8.43 (d, J＝19.1Hz, 1H), 7.42 (s, 1H), 4.77-4.98 (m, 1H), 4.15 (br dd,J＝26.7,13.5Hz,1H),3.61-4.07(m,4H),2.94-3.28(m,2H),1.72(d,J＝5.2Hz,3H),1.44(s,9H),1.32(br dd,J＝13.0,6.7Hz,3H),1.21-1.28(m,6H). m/z(ESI,+ve ion):536.0(M+H) ⁺ .

Step 5: (S)-4-(6-chloro-7-(2-fluorophenyl)-1-(1-isopropyl-4-methyl-1H-pyrazol-5-yl)-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-4-yl)-3-methylpiperazine-1-carboxylic acid tert-butyl ester. At 80 °C, a mixture of (S)-4-(6,7-dichloro-1-(1-isopropyl-4-methyl-1H-pyrazol-5-yl)-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-4-yl)-3-methylpiperazine-1-carboxylic acid tert-butyl ester (703 mg, 1.31 mmol), (2-fluorophenyl)boronic acid (330 mg, 2.36 mmol, Combi-blocks, San Diego, California, USA), anhydrous sodium carbonate (416 mg, 3.93 mmol), and tetrakis(triphenylphosphine)palladium (0) (151 mg, 0.13 mmol) in 1,4-dioxane (3.5 mL) and water (0.87 mL) was heated for 2 h. The reaction was concentrated and the residue was purified by silica gel chromatography (elution: 0-100% EtOAc/heptane) to provide (S)-4-(6-chloro-7-(2-fluorophenyl)-1-(1-isopropyl-4-methyl-1H-pyrazol-5-yl)-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-4-yl)-3-methylpiperazine-1-carboxylic acid tert-butyl ester. ¹ H NMR (400MHz, DMSO-d ₆ ) δppm 8.41 (d, J＝8.9Hz, 1H), 7.50-7.66 (m, 1H), 7.24-7.37 (m, 4H), 4.92 (br s, 1H), 4.24 (br t,J＝12.4Hz,1H),3.95-4.12(m,2H),3.67-3.89(m,2H),3.00-3.29(m,2 H), 1.70 (s, 3H), 1.45 (s, 9H), 1.36 (t, J = 6.6Hz, 3H), 1.14-1.28 (m, 6H). ¹⁹ F NMR (376MHz, DMSO-d ₆ ) δ ppm - 113.95 (d, J = 6.9 Hz, 1F). m/z (ESI, +ve ions): 596.0 (M+H) ⁺ .

Step 6: 6-Chloro-7-(2-fluorophenyl)-1-(4-methyl-1-(2-propyl)-1H-pyrazol-5-yl)-4-((2S)-2-methyl-4-(2-acryloyl)-1-piperazinyl)pyrido[2,3-d]pyrimidin-2(1H)-one. Trifluoroacetic acid (7.4 mL, 63.6 mmol) was added to a solution of (S)-4-(6-chloro-7-(2-fluorophenyl)-1-(1-isopropyl-4-methyl-1H-pyrazol-5-yl)-2-oxo-1,2-dihydropyrido[2,3-d]pyrimidin-4-yl)-3-methylpiperazin-1-carboxylic acid tert-butyl ester (532 mg, 0.74 mmol) in DCM (7.4 mL). The resulting mixture was stirred at room temperature for 1 h and then concentrated. The residue was dissolved in DCM (7.4 mL), cooled to 0 °C, and treated with DIPEA (1.28 mL, 7.35 mmol), followed by acryloyl chloride (0.2 M solution in DCM, 3.67 mL, 0.74 mmol). The resulting mixture was stirred at 0 °C for 20 min, then quenched with a saturated sodium bicarbonate aqueous solution (50 mL) and extracted with DCM (2 × 50 mL). The combined organic layers were dried over anhydrous sodium sulfate and concentrated. The residue was purified by silica gel chromatography (elution: 0-50% EtOAc/heptane, followed by 0-50% 4:1 DCM-MeOH/DCM) to provide 6-chloro-7-(2-fluorophenyl)-1-(4-methyl-1-(2-propane)-1H-pyrazol-5-yl)-4-((2S)-2-methyl-4-(2-acryloyl)-1-piperazinyl)pyrido[2,3-d]pyrimidin-2(1H)-one. ¹ H NMR (400MHz, DMSO-d ₆ ) δppm 8.44 (brd, J＝7.5Hz, 1H), 7.50-7.59 (m, 1H), 7.23-7.41 (m, 4H), 6.77-6.93 (m, 1H), 6.21 (br d,J＝16.4Hz,1H),5.71-5.81(m,1H),4.96(br s,1H),4.22-4.46(m,2H),3.96-4.21(m,2H),3.37-3.89(m,2H),3.00-3.2 8(m,1H),1.71(d,J＝2.5Hz,3H),1.34(t,J＝6.8Hz,3H),1.12-1.29(m,6H). ^19F NMR (376MHz, DMSO- _d⁶ ) δppm -113.96 (d, J = 9.5Hz, 1F). m/z (ESI, +Ve ions): 550.2 (M+H) ^⁺ .

Table 12: Examples of isolated compounds including stereoisomers, some of which are transisomers.

Table 12(b) lists examples of compounds including the isolation of the transisomer.

Table 13: Analysis Data of the General Program

Table 14: Analysis data for individual cases

Table 14(b): Analysis data for individual cases

Bioassay data

For the compounds in Table 15, the following determination conditions were used:

Coupled nucleotide exchange assay: Purified GDP-bound KRAS protein (aa 1-169) containing G12C and C118A amino acid substitutions and an N-terminal His tag was pre-incubated with the compound dose-response titration solution in assay buffer (25 mM HEPES pH 7.4, ₁₀ mM MgCl2, and 0.01% Triton X-100) for 2 hours. After compound pre-incubation, purified SOS protein (aa 564-1049) and GTP (Roche 10106399001) were added to the assay wells and incubated for another hour. To determine the degree of inhibition of SOS-mediated nucleotide exchange, purified GST-labeled cRAF (aa 1-149), nickel-chelating AlphaLISA receptor beads (PerkinElmer AL108R), and AlphaScreen glutathione donor beads (PerkinElmer 6765302) were added to the assay wells and incubated for 10 minutes. The assay discs were then read using a PerkinElmer EnVision multilabel reader, and the data were analyzed using a 4-parameter logic model to calculate the _IC50 values.

ERK1/2 phosphate MSD assay: MIA PaCa-2 (CRL-1420™) and A549 (CCL-185™) cells were cultured in RPMI 1640 medium (Thermo Fisher Scientific 11875093) containing 10% fetal bovine serum (Thermo Fisher Scientific 16000044) and 1x penicillin-streptomycin-glutamyl (Thermo Fisher ^{Scientific 10378016} ). Sixteen hours prior to compound treatment, MIA PaCa-2 or A549 cells were seeded at a density of 25,000 cells/well in 96-well cell culture dishes and incubated at 37°C and ₅ % CO2. The compound dose-response titration solution was diluted in growth medium and added to the appropriate wells of a cell culture dish, then incubated at 37°C and 5% _CO2 for 4 hours. After compound treatment, cells were stimulated for 10 min with 10 ng/mL EGF (Roche 11376454001), washed with ice-cold Dubekah phosphate-buffered saline (Thermo Fisher Scientific 14190144) free of Ca2 ⁺ or Mg2 ⁺ , and then dissolved in RIPA buffer (50 mM Tris-HCl pH 7.5, 1% Igepal, 0.5% sodium deoxycholate, 150 mM NaCl, and 0.5% sodium dodecyl sulfate) containing protease inhibitors (Roche 4693132001) and phosphatase inhibitors (Roche 4906837001). Cell lysates were cryopreserved overnight at -80°C. Following the manufacturer's protocol, ERK1/2 phosphorylation in the compound-treated lysates was determined using the ERK1/2 Phosphate Whole Cell Lysis Kit (Meso Scale Discovery K151DWD). The assay discs were read on a Meso Scale Discovery Sector Imager 6000, and the data were analyzed using a 4-parameter logic model to calculate the _IC50 values.

Table 15: Biochemistry and Cellular Activity of Compounds

For the compounds in Table 16, the following determination conditions were used:

Coupled nucleotide exchange assay: Purified GDP-bound KRAS protein (aa 1-169) containing G12C and C118A amino acid substitutions and an N-terminal His tag was pre-incubated for 5 min with the compound dose-response titration solution in assay buffer (25 mM HEPES pH 7.4, ₁₀ mM MgCl2, and 0.01% Triton X-100). After compound pre-incubation, purified SOS protein (aa 564-1049) and GTP (Roche 10106399001) were added to the assay wells and incubated for another 30 min. To determine the degree of inhibition of SOS-mediated nucleotide exchange, purified GST-labeled cRAF (aa 1-149), nickel-chelating AlphaLISA receptor beads (PerkinElmer AL108R), and AlphaScreen glutathione donor beads (PerkinElmer 6765302) were added to the assay wells and incubated for 5 minutes. The assay discs were then read using a PerkinElmer EnVision multilabel reader, and the data were analyzed using a 4-parameter logic model to calculate the _IC50 values.

ERK1/2 phosphate MSD assay: MIA PaCa-2 (CRL-1420™) and A549 (CCL-185™) cells were cultured in RPMI 1640 medium (ThermoFisher Scientific 11875093) containing 10% fetal bovine serum (ThermoFisher Scientific 16000044) and 1x penicillin-streptomycin-glutamyl (ThermoFisher ^Scientific 10378016). Sixteen hours before compound treatment, MIA PaCa-2 or A549 cells were seeded at a density of 25,000 cells/well in 96- ^well cell culture dishes and incubated at 37°C and ₅ % CO2. The compound dose-response titration solution was diluted in growth medium and added to the appropriate wells of a cell culture dish, then incubated at 37°C and 5% _CO2 for 2 hours. After compound treatment, cells were stimulated for 10 min with 10 ng/mL EGF (Roche 11376454001), washed with ice-cold Dubekah phosphate-buffered saline (Thermo Fisher Scientific 14190144) free of Ca2 ⁺ or Mg2 ⁺ , and then dissolved in RIPA buffer (50 mM Tris-HCl pH 7.5, 1% Igepal, 0.5% sodium deoxycholate, 150 mM NaCl, and 0.5% sodium dodecyl sulfate) containing protease inhibitors (Roche 4693132001) and phosphatase inhibitors (Roche 4906837001). According to the manufacturer's protocol, the phosphorylation of ERK1/2 in the compound-treated lysates was determined using the ERK1/2 Phosphate Whole Cell Lysis Kit (Meso ScaleDiscovery K151DWD). The assay disk was read on a Meso ScaleDiscovery Sector Imager 6000, and the data were analyzed using a 4-parameter logic model to calculate the _IC50 value.

Table 16: Biochemistry and Cellular Activity of Compounds

(-) = Untested

This invention has been described in conjunction with preferred embodiments. However, it should be understood that the invention is not limited to the disclosed embodiments. It should be understood that various modifications can be made by those skilled in the art based on the description of the embodiments of the invention herein. Such modifications are covered by the claims below.

Claims (15)

1. A compound or a pharmaceutically acceptable salt thereof, wherein the compound is selected from: 2. A pharmaceutical composition comprising the compound according to claim 1 and a pharmaceutically acceptable excipient. 3. Use of the compound according to claim 1 in the preparation of a medicament for treating cancers characterized by KRAS G12C mutations. 4. The use according to claim 3, wherein the cancer is non-small cell lung cancer, small intestine cancer, appendix cancer, colorectal cancer, endometrial cancer, pancreatic cancer, liver cancer, small cell lung cancer, cervical cancer, germ cell tumor, ovarian cancer, pancreatic neuroendocrine tumor, bladder cancer, spinal dysplasia or myeloproliferative tumor, head and neck cancer, esophageal cancer, soft tissue sarcoma, malignant mesothelioma, thyroid cancer, leukemia, skin cancer, gastric cancer, nasal cavity cancer or bile duct cancer. 5. The use according to claim 3, wherein the cancer is non-small cell lung cancer, small bowel cancer, appendix cancer, colorectal cancer, endometrial cancer, pancreatic cancer, skin cancer, gastric cancer, nasal cancer, or bile duct cancer. 6. The use according to claim 5, wherein the cancer is non-small cell lung cancer. 7. The use according to claim 5, wherein the cancer is colorectal cancer. 8. The use according to claim 5, wherein the cancer is pancreatic cancer. 9. A compound, wherein the compound is 10. A compound, wherein the compound is 11. A compound, wherein the compound is 12. A compound, wherein the compound is 13. A compound, wherein the compound is 14. A compound, wherein the compound is 15. A method for preparing 6-fluoro-7-(2-fluoro-6-hydroxyphenyl)-1-(4-methyl-2-(2-propyl)-3-pyridyl)-4-((2S)-2-methyl-4-(2-acryloyl)-1-piperazinyl)pyrido[2,3-d]pyrimidin-2(1H)-one or a pharmaceutically acceptable salt thereof, said method comprising providing a compound according to any one of claims 9-14, and converting the compound to 6-fluoro-7-(2-fluoro-6-hydroxyphenyl)-1-(4-methyl-2-(2-propyl)-3-pyridyl)-4-((2S)-2-methyl-4-(2-acryloyl)-1-piperazinyl)pyrido[2,3-d]pyrimidin-2(1H)-one or a pharmaceutically acceptable salt thereof: