RU2790175C2 - Cyclic di-nucleotide compounds for treatment of cancer - Google Patents

Abstract

FIELD: organic chemistry; pharmaceutics.

SUBSTANCE: group of inventions relates to the field of organic chemistry and pharmaceutics; it is aimed at production of new compounds, which are useful in the treatment of cancer. Compounds of the formula (III) or their pharmaceutically acceptable salts are disclosed, where R_1a is selected from a group consisting of H, -OH, and -F; R_1b is selected from a group consisting of H, -OH, and F, where at least one of R_1a and R_1b is -H; R_4a is selected from a group consisting of -H, -OH, and -F; R_4b is selected from a group consisting of -H, -OH, and -F, where at least one of R_4a and R_4b is -H; P₁ and P₂, each independently, has S or R stereochemical configuration; Z is -O- or -NH-; X_1a and X_2a are the same or different and are independently selected from =O or =S; X_1b and X_2b are the same or different and are independently selected from -OR₅ and -SR₅; where R₅ is selected from H and -CH₂OC(O)OC_1-6alkyl; L₁ in the formula (III) is four, five, or six carbon atoms long; where X₁₀, X₁₁, X₁₂, X₁₃, X₁₄, and X₁₅ are independently selected from a bond, -CH₂-, or -CH-, where -CH₂- or -CH- is unsubstituted or substituted with (i) -OH, (iv) -NH₂, or (v) -D, and, if X₁₀ or X₁₅ is a bond, such a bond is not a double bond or a triple bond; and where any two adjacent members of the group, including X₁₀, X₁₁, X₁₂, X₁₃, X₁₄, and X₁₅, may optionally form, with additional atoms, C₃ cycloalkyl or C₃ heterocycloalkyl, the specified C₃ heterocycloalkyl includes N atom; where B₁ and B₂ are independently selected from structures represented in cl.1. In addition, a pharmaceutical composition for the treatment of cancer, containing pharmaceutically effective amount of the specified compounds, the use of compounds for the production of a pharmaceutical composition, and methods for the treatment of cancer, which provide administration of presented compounds to a patient, are disclosed.

EFFECT: group of inventions provides efficient treatment of cancer.

37 cl, 13 dwg, 14 tbl, 114 ex

Description

Link to related applications

The present application claims priority under Provisional Application US No. 62/460562, filed Feb. 17, 2017; U.S. Provisional Application No. 62/479169, filed March 30, 2017; U.S. Provisional Application No. 62/551645, filed August 29, 2017; U.S. Provisional Application No. 62/551647, filed August 29, 2017; and U.S. Provisional Application No. 62/551668, filed Aug. 29, 2017. All of these applications are incorporated by reference as if they were fully transcribed herein.

Background of the Invention

STING (interferon gene stimulator) is a signaling molecule in the innate response to dsDNA in the cytosol. STING deletion has been described in numerous human cancers. In addition, deregulation of STING signaling in human cancers has also been described in melanoma (Xia T, et al., "Recurrent Loss of STING Signaling in Melanoma Correlates with Susceptibility to Viral Oncolysis" Cancer Res. 2016) and colon cancer. . (Xia T, et al., "Deregulation of STING Signaling in Colorectal Carcinoma Constrains DNA Damage Responses and Correlates With Tumorigenesis" Cell Rep. 2016; 14:282-97). Interestingly, in these studies, the results of genomic analysis showed that the lower expression of STING is not due to a gene deletion or mutation, but to epigenetic changes. (Xia, Cancer Res. 2016; Xia, Cell Rep. 2016). The protective activity of STING against cancer is also confirmed by evidence obtained from studies in mouse models. STING knockout mice have been shown to be characterized by impaired tumor control. (Woo SR, et al. "STING-dependent cytosolic DNA sensing mediates innate immune recognition of immunogenic tumors" Immunity 2014; 41:830-42).

In addition, the role of STING in protecting ontogeny has been demonstrated in several spontaneous mouse models, including glioma (Ohkuri T, et al., "Protective role of STING against gliomagenesis: Rational use of STING agonist in anti-glioma immunotherapy" Oncoimmunology. 2015;4 :e999523) and colon cancer (Zhu Q, et al., "Cutting edge: STING mediates protection against colorectal oncogenesis by regulating the amount of intestinal inflammation" J. Immunol. 2014; 193:4779-82). This antitumor effect may be related to its ability to overcome NF-kB and STAT3 overactivation. (Okihuri 2015). Pathway activation involving STING is also characterized by high activity in preclinical mouse tumor models. (Woo 2014; Chandra D, et al. "STING ligand c-di-GMP improves cancer vaccination against metastatic breast cancer" Cancer Immunol Res. 2014;2:901-10; Corrales L, et al., "Direct Activation of STING in the Tumor Microenvironment Leads to Potent and Systemic Tumor Regression and Immunity" Cell Rep. 2015; 11:1018-30; Curran E, et al. "STING Pathway Activation Stimulates Potent Immunity against Acute Myeloid Leukemia" Cell Rep. 2016; 15: 23 57-66; Tang CH, et al. "Agonist-Mediated Activation of STING Induces Apoptosis in Malignant B Cells" Cancer Res. 2016; 76:2137-52). Said antitumor activity is likely associated with a disruption of the tumor vasculature and occurs after the induction of an adaptive immune response. (Corrales L, et al., "The host STING pathway at the interface of cancer and immunity" J. Clin. Invest. 2016; 126:2404-11). Accordingly, direct stimulation of STING in the tumor microenvironment by an agonist may represent a novel approach in the treatment of numerous types of cancer.

Brief description of the present invention

Embodiments may provide a compound of formula (I):

означает простую связь или двойную связь.(where P ₁ is lower left phosphorus and P ₂ is upper right phosphorus as shown above) containing substituents and stereochemistry as indicated in Table 1 below, or a pharmaceutically acceptable salt thereof.

means a single bond or a double bond.

If a phosphorus atom contains four substituents that are different, that phosphorus atom will be a stereocenter. SpSp/RpRp/SpRp/RpSp refers to phosphorus stereochemistry as noted.

Embodiments may further provide a compound of formula (II):

containing substituents and stereochemistry as noted in Table 2 below, or a pharmaceutically acceptable salt thereof. If a phosphorus atom contains four substituents that are different, that phosphorus atom will be a stereocenter.

Embodiments may provide a compound of formula (III):

or a pharmaceutically acceptable salt thereof, wherein

R _1a is selected from the group consisting of -H, -OH and -F;

R _1b is selected from the group consisting of -H, -OH and -F, where at least one of R _1a and R _1b is -H;

R _4a is selected from the group consisting of -H, -OH and -F;

R _4b is selected from the group consisting of -H, -OH and -F, where at least one of R _4a and R _4b is -H;

P ₁ and P ₂ each independently has the S or R stereochemical configuration;

Z is -O- or -NH-;

X _1a and X _2a are the same or different and independently selected from ═O or ═S;

X _1b and X _2b are the same or different and independently selected from -OR ₅ and -SR ₅ ;

where R ₅ is selected from the group consisting of -H, C _1-6 alkyl, -C(O)C _1-6 alkyl and -CH ₂ OC(O)OC _1-6 alkyl;

L ₁ in formula (III) is four, five or six carbon atoms long and is

означает простую связь, двойную связь или тройную связь и где (i) или 0, или 1 встречаемость

в L₁ означает тройную связь; или (ii) 0, 1 или 2 встречаемости

в L₁ означает двойную связь, где геометрия вокруг каждой двойной связи является цис или транс; и (iii) где, если 1 встречаемость

в L₁ означает тройную связь, 0 встречаемостей

в L₁ означает двойную связь; и (iv) где, если 2 встречаемости

в L₁ означает двойную связь, такие двойные связи являются или смежными связями, или чередующимися связями;Where

means single bond, double bond or triple bond and where (i) is either 0 or 1 occurrence

in L ₁ means a triple bond; or (ii) 0, 1 or 2 occurrences

in L ₁ means a double bond, where the geometry around each double bond is cis or trans; and (iii) where if 1 occurrence

in L ₁ means triple bond, 0 occurrences

in L ₁ means a double bond; and (iv) where if 2 occurrences

in L ₁ means a double bond, such double bonds are either adjacent bonds or alternating bonds;

where X ₁₀ , X ₁₁ , X ₁₂ , X ₁₃ , X ₁₄ and X ₁₅ are independently selected from the bond, -CH ₂ - or -CH-, where -CH ₂ - or -CH- is unsubstituted or substituted by (i) -OH , (ii) -F, (iii) -Cl, (iv) -NH ₂ or (v) -D and if X ₁₀ or X ₁₅ is a bond, such a bond is not a double bond or a triple bond;

and where any two adjacent group members including X ₁₀ , X ₁₁ , X ₁₂ , X ₁₃ , X ₁₄ and X ₁₅ may optionally form with additional C ₃ atoms cycloalkyl or C ₃ heterocycloalkyl, said C ₃ heterocycloalkyl includes N or O atom;

where B ₁ and B ₂ are independently selected from:

, где связи в точках q и r на В₁ и В₂ присоединены в точках q и r на формуле (III).

, where bonds at points q and r on B ₁ and B ₂ are attached at points q and r on formula (III).

In some embodiments, where L ₁ includes a triple bond or more than one double bond, L ₁ may be, for example,

Embodiments may also provide a compound of formula (III):

or a pharmaceutically acceptable salt thereof, wherein

R _1a is selected from the group consisting of -H and -F;

R _1b is selected from the group consisting of -H and -F, where R _1a and R _1b cannot both be -F;

R _4a is selected from the group consisting of -H and -F;

R _4b is selected from the group consisting of -H and -F, where R _4a and R _4b cannot both be -F;

P ₁ and P ₂ each independently has the S or R stereochemical configuration;

X _1a and X _2a are the same or different and independently selected from ═O or ═S;

X _1b and X _2b are the same or different and independently selected from -OR ₅ and -SR ₅ ;

where R ₅ is selected from the group consisting of -H, C _1-6 alkyl and -C(O)C _1-6 alkyl;

L ₁ in formula (III) is four or five carbon atoms long and is

означает простую связь или двойную связь и где или 0, или 1 встречаемость

в L₁ означает двойную связь, где геометрия вокруг двойной связи является цис или транс;Where

means single bond or double bond and where either 0 or 1 occurrence

in L ₁ means a double bond, where the geometry around the double bond is cis or trans;

where X ₁₀ and X ₁₄ are independently selected from a bond, -CH- or -CH ₂ - and where, if X ₁₀ or X ₁₄ is a bond, such a bond is not a double bond;

where B ₁ and B ₂ are independently selected from:

где связи в точках q и r на B₁ и В₂ присоединены в точках q и r на формуле (III).

where bonds at points q and r on B ₁ and B ₂ are attached at points q and r on formula (III).

Embodiments may provide a compound of formula (IV):

or a pharmaceutically acceptable salt thereof, wherein

R _1a is selected from the group consisting of -H, -OH and -F;

R _1b is selected from the group consisting of -H, -OH and -F, where at least one of R _1a and R _1b is -H;

R _4a is selected from the group consisting of -H, -OH and -F;

R _4b is selected from the group consisting of -H, -OH and -F, where at least one of R _4a and R _4b is -H;

P ₁ and P ₂ each independently has the S or R stereochemical configuration;

X _1a and X _2a are the same or different and independently selected from ═O or ═S;

X _1b and X _2b are the same or different and independently selected from -OR ₅ and -SR ₅ ;

where R ₅ is selected from the group consisting of -H, C _1-6 alkyl, -C(O)C _1-6 alkyl and -CH ₂ OC(O)OC _1-6 alkyl;

L ₁ in formula (IV) is four, five or six carbon atoms long and is

означает простую связь, двойную связь или тройную связь и где (i) или 0, или 1 встречаемость

в L₁ означает тройную связь; или (ii) 0, 1 или 2 встречаемости

в L₁ означает двойную связь, где геометрия вокруг каждой двойной связи является цис или транс; и (iii) где, если 1 встречаемость

в L₁ означает тройную связь, 0 встречаемостей

в L₁ означает двойную связь; и (iv) где, если 2 встречаемости

в L₁ означает двойную связь, такие двойные связи являются или смежными связями, или чередующимися связями;Where

means single bond, double bond or triple bond and where (i) is either 0 or 1 occurrence

in L ₁ means a triple bond; or (ii) 0, 1 or 2 occurrences

in L ₁ means a double bond, where the geometry around each double bond is cis or trans; and (iii) where if 1 occurrence

in L ₁ means triple bond, 0 occurrences

in L ₁ means a double bond; and (iv) where if 2 occurrences

in L ₁ means a double bond, such double bonds are either adjacent bonds or alternating bonds;

where X ₁₀ , X ₁₁ , X ₁₂ , X ₁₃ , X ₁₄ and X ₁₅ are independently selected from the bond, -CH ₂ - or -CH-, where -CH ₂ - or -CH- is unsubstituted or substituted by (i) -OH , (ii) -F, (iii) -Cl, (iv) -NH ₂ or (v) -D and if X ₁₀ or X ₁₅ is a bond, such a bond is not a double bond or a triple bond;

and where any two adjacent group members including X ₁₀ , X ₁₁ , X ₁₂ , X ₁₃ , X ₁₄ and X ₁₅ may optionally form with additional C ₃ atoms cycloalkyl or C ₃ heterocycloalkyl, said C ₃ heterocycloalkyl includes N or O atom;

where B ₁ and B ₂ are independently selected from:

, где связи в точках q и r на В₁ и В₂ присоединены в точках q и r на формуле (IV).

, where bonds at points q and r on B ₁ and B ₂ are attached at points q and r on formula (IV).

Embodiments may also provide a compound of formula (IV):

or a pharmaceutically acceptable salt thereof, wherein

R _1a is selected from the group consisting of -H and -F;

R _1b is selected from the group consisting of -H and -F, where R _1a and R _1b cannot both be -F;

R _4a is selected from the group consisting of -H and -F;

R _4b is selected from the group consisting of -H and -F, where R _4a and R _4b cannot both be -F;

P ₁ and P ₂ each independently has the S or R stereochemical configuration;

X _1a and X _2a are the same or different and independently selected from ═O or ═S;

X _1b and X _2b are the same or different and independently selected from -OR ₅ and -SR ₅ ;

where R ₅ is selected from the group consisting of -H, C _1-6 alkyl and -C(O)C _1-6 alkyl;

L ₁ in formula (IV) is four or five carbon atoms long and is

означает простую связь или двойную связь и где или 0, или 1 встречаемость

в L₁ означает двойную связь, где геометрия вокруг двойной связи является цис или транс;Where

means single bond or double bond and where either 0 or 1 occurrence

in L ₁ means a double bond, where the geometry around the double bond is cis or trans;

where X ₁₀ and X ₁₄ are independently selected from a bond, -CH- or -CH ₂ - and where, if X ₁₀ or X ₁₁ is a bond, such bond is not a double bond;

where B ₁ and B ₂ are independently selected from:

где связи в точках q и r на B₁ и В₂ присоединены в точках q и r на формуле (IV).

where bonds at points q and r on B ₁ and B ₂ are attached at points q and r on formula (IV).

Embodiments may provide a compound of formula (V):

or a pharmaceutically acceptable salt thereof, wherein

R _1a is selected from the group consisting of -H, -OH and -F;

R _1b is selected from the group consisting of -H, -OH and -F, where at least one of R _1a and R _1b is -H;

R _4a is selected from the group consisting of -H, -OH and -F;

R _4b is selected from the group consisting of -H, -OH and -F and where at least one of R _4a and R _4b is -H;

P ₁ and P ₂ each independently has the S or R stereochemical configuration;

X _1a and X _2a are the same or different and independently selected from ═O or ═S;

X _1b and X _2b are the same or different and independently selected from -OR ₅ and -SR ₅ ;

where R ₅ is selected from the group consisting of -H, C _1-6 alkyl, -C(O)C _1-6 alkyl and -CH ₂ OC(O)OC _1-6 alkyl;

L ₁ in formula (V) is four, five or six carbon atoms long and is

означает простую связь, двойную связь или тройную связь и где (i) или 0, или 1 встречаемость

в L₁ означает тройную связь; или (ii) 0, 1 или 2 встречаемости

в L₁ означает двойную связь, где геометрия вокруг каждой двойной связи является цис или транс; и (iii) где, если 1 встречаемость

в L₁ означает тройную связь, 0 встречаемостей

в L₁ означает двойную связь; и (iv) где, если 2 встречаемости

в L₁ означает двойную связь, такие двойные связи являются или смежными связями, или чередующимися связями;Where

means single bond, double bond or triple bond and where (i) is either 0 or 1 occurrence

in L ₁ means a triple bond; or (ii) 0, 1 or 2 occurrences

in L ₁ means a double bond, where the geometry around each double bond is cis or trans; and (iii) where if 1 occurrence

in L ₁ means triple bond, 0 occurrences

in L ₁ means a double bond; and (iv) where if 2 occurrences

in L ₁ means a double bond, such double bonds are either adjacent bonds or alternating bonds;

where X ₁₀ , X ₁₁ , X ₁₂ , X ₁₃ , X ₁₄ and X ₁₅ are independently selected from the bond, -CH2- or -CH-, where -CH ₂ - or -CH- is unsubstituted or substituted by (i) -OH, (ii) -F, (iii) -Cl, (iv) -NH ₂ or (v) -D and if X ₁₀ or X ₁₅ is a bond, such bond is not a double bond or a triple bond;

and where any two adjacent group members including X ₁₀ , X ₁₁ , X ₁₂ , X ₁₃ , X ₁₄ and X ₁₅ may optionally form with additional C ₃ atoms cycloalkyl or C ₃ heterocycloalkyl, said C ₃ heterocycloalkyl includes N or O atom;

where B ₁ and B ₂ are independently selected from:

, где связи в точках q и r на В₁ и В₂ присоединены в точках q и r на формуле (V).

, where bonds at points q and r on B ₁ and B ₂ are attached at points q and r on formula (V).

Embodiments may also provide a compound of formula (V):

or a pharmaceutically acceptable salt thereof, wherein

R _1a is selected from the group consisting of -H and -F;

R _1b is selected from the group consisting of -H and -F, where R _1a and R _1b cannot both be -F;

R _4a is selected from the group consisting of -H and -F;

R _4b is selected from the group consisting of -H and -F, where R _4a and R _4b cannot both be -F;

P ₁ and P ₂ each independently has the S or R stereochemical configuration;

X _1a and X _2a are the same or different and independently selected from ═O or ═S;

X _1b and X _2b are the same or different and independently selected from -OR ₅ and -SR ₅ ;

where R ₅ is selected from the group consisting of -H, C _1-6 alkyl and -C(O)C _1-6 alkyl;

L ₁ in formula (V) is four or five carbon atoms long and is

означает простую связь или двойную связь и где или 0, или 1 встречаемость

в L₁ означает двойную связь, где геометрия вокруг двойной связи является цис или транс;Where

means single bond or double bond and where either 0 or 1 occurrence

in L ₁ means a double bond, where the geometry around the double bond is cis or trans;

where X ₁₀ and X ₁₄ are independently selected from a bond, -CH- or -CH ₂ - and where, if X ₁₀ or X ₁₄ is a bond, such a bond is not a double bond;

where B ₁ and B ₂ are independently selected from:

где связи в точках q и r на B₁ и В₂ присоединены в точках q и r на формуле (V).

where bonds at points q and r on B ₁ and B ₂ are attached at points q and r on formula (V).

в L₁ означает двойную связь, где геометрия вокруг двойной связи является транс; и (iii) Z представляет собой -O-.According to some embodiments of the compounds and/or salts as reported in the formulas above, (i) the stereochemical configuration of P ₁ and P ₂ is both R, and the stereochemical configuration of P ₁ is R and P ₂ is S, or the stereochemical configuration P ₁ is S and P ₂ is R; (ii) one occurrence

in L ₁ means a double bond, where the geometry around the double bond is trans; and (iii) Z is -O-.

. Согласно некоторым вариантам осуществления X_1a и Х_2а оба представляют собой =O и X_1b и X_2b оба представляют собой -SH. Согласно некоторым вариантам осуществления L₁ представляет собой

Additional embodiments of compounds and/or salts, as reported in the formulas above, can be found in other aspects of the present invention. For example, some embodiments provide a compound or a pharmaceutically acceptable salt, wherein R _1a and R _4a are each -F. In some embodiments, R _1b and R _4b are each -F. In some embodiments, B ₁ and B ₂ are each

. In some embodiments, X _1a and X _2a are both =O and X _1b and X _2b are both -SH. In some embodiments, L ₁ is

In some embodiments, the linker is four carbons long. In some embodiments, the linker is five carbons long.

Some embodiments provide a compound selected from the group consisting of:

or a pharmaceutically acceptable salt thereof.

According to embodiments, a compound or pharmaceutically acceptable salt with one or more of (i) an EC ₅₀ value below 100 micromoles in a cell reporter assay that expresses a STING HAQ genetic variant can be provided; (ii) EC ₅₀ values below 100 micromoles in reporter cells expressing the human STING AQ variant; (iii) EC ₅₀ values below 100 micromoles in reporter cells expressing the human STING WT variant; and (iv) EC ₅₀ values below 100 micromoles in reporter cells expressing the STING REF variant. According to one embodiment, a connection is provided having the following structure:

or a pharmaceutically acceptable salt thereof.

According to additional embodiments, a pharmaceutically acceptable salt of a compound described herein may be provided, wherein the salt is a diammonium salt. According to additional embodiments, the compounds described herein may be provided as a triethylamine (TEA) salt. In accordance with additional embodiments, a pharmaceutical composition may be provided containing a compound or salt described herein and a pharmaceutically acceptable excipient.

According to embodiments, a method of treating cancer may be provided comprising administering to a patient a compound, pharmaceutically acceptable salt, or pharmaceutical composition described herein.

The use of a compound or a pharmaceutically acceptable salt thereof described herein is contemplated for the preparation of a pharmaceutical composition for the treatment of cancer.

According to embodiments, a compound, pharmaceutically acceptable salt, or pharmaceutical composition described herein for the treatment of cancer may be provided.

According to embodiments, a method for treating cancer may be provided, comprising: identifying an individual having a cancer to be treated with a compound, pharmaceutically acceptable salt, or pharmaceutical composition described herein; and administering to said individual a pharmaceutically effective amount of the compound, pharmaceutically acceptable salt, or pharmaceutical composition by which the cancer has been identified as being treatable.

In some embodiments, an individual is identified as having a cancer to be treated with a compound, pharmaceutically acceptable salt, or pharmaceutical composition described herein as a result of the presence of the STING REF variant allele in the patient.

In accordance with some embodiments, a method of treating cancer in a patient having the STING REF allele is provided, comprising administering to said patient a compound, pharmaceutically acceptable salt, or pharmaceutical composition described herein.

In accordance with some embodiments, a method of treating cancer in a patient having the STING WT allele is provided, comprising administering to said patient a compound, pharmaceutically acceptable salt, or pharmaceutical composition described herein.

In accordance with some embodiments, a method of treating cancer in a patient having the STING AQ allele is provided, comprising administering to said patient a compound, pharmaceutically acceptable salt, or pharmaceutical composition described herein.

In accordance with some embodiments, a method of treating cancer in a patient having the STING HAQ allele is provided, comprising administering to said patient a compound, pharmaceutically acceptable salt, or pharmaceutical composition described herein.

In some embodiments, the cancer is selected from the group consisting of lymphoma, melanoma, colorectal cancer, breast cancer, acute myeloid leukemia, colon cancer, liver cancer, prostate cancer, pancreatic cancer, kidney cancer, and glioma. In accordance with some embodiments, the cancer is metastatic.

In some embodiments of the compounds of the formulas provided herein, one of the bonds in L ₁ is a double bond. In additional embodiments, such a double bond has a trans geometry. In additional embodiments, L ₁ is saturated. In certain embodiments, L ₁ includes five carbon atoms. In other embodiments, L ₁ includes 4 carbon atoms.

Embodiments may further provide mixtures of compounds as described herein, including mixtures of stereoisomers of such compounds. For example, a mixture of Compound 11 and Compound 12 may be provided, or a mixture of Compound 2 and Compound 4 may be provided. Of course, these are non-limiting examples and other mixtures are possible.

Specific embodiments are set forth in Table 3 below.

In the table above, compounds 8, 11 and 12 are depicted using the same structural formula and refer to three separate stereoisomers. However, the Applicant will note that the phosphorus chirality of Compound 8 is not necessarily the same as the phosphoric chirality of other compounds labeled "Stereoisomer I", such as Compound 21. The same is true of other stereoisomers.

Embodiments may refer to C ₄ -C ₆ linkers, which may be covalently linked at either end to purine or pyrimidine bases that form part of a cyclic dinucleotide. In an embodiment, the linkers are butene, pentene, or hexene linkers linked at either end to purine bases. In another embodiment, the linkers are butene linkers linked at either end to purine bases; in another embodiment, the linkers are transbutene linkers with a double bond located between the central two carbon atoms.

The following numbered embodiments are illustrative of the use of such C ₄ -C ₆ linkers:

1. Compound of formula (X):

or a pharmaceutically acceptable salt thereof, wherein:

A ₁ and A ₂ are sugar moieties and may be the same or different;

B ₃ and B ₄ are purine or pyrimidine bases, which may be the same or different, and which form nucleotides with, respectively, A ₁ and A ₂ ;

L is an alkyl linker;

X ₁₉ and X ₂₀ are the same or different and are selected from the group consisting of -O-, -CH-, -NH- and -S-, where -CH- and -NH- may be substituted or unsubstituted.

2. A compound or pharmaceutically acceptable salt of numbered embodiment 1 wherein -CH- and -NH- X ₁₉ and X ₂₀ may be substituted with C _1-6 alkyl.

3. The compound or pharmaceutically acceptable salt of numbered embodiment 1, wherein L is butene, pentene, or hexane.

4. The compound or pharmaceutically acceptable salt of numbered embodiment 3, wherein L is a transbutene linker with a double bond at its center.

The compounds or pharmaceutically acceptable salts of the numbered embodiments may be useful, for example, in the treatment of cancer. A ₁ , A ₂ , B ₃ and B ₄ may be further substituted, for example, with hydroxyl, halogen or methoxy. Each phosphorus in formula (X) may be substituted with, for example, -SH, -OH, ═O, or ═S, until its valency is saturated.

Examples of cyclic dinucleotide analogs that may benefit from the linkers of the present disclosure include, without limitation, the analogs defined in US Patent Application No. 2014/0205653 A1; US Patent Application No. 2014/0329889 A1; US Patent Application No. 2014/0341976 A1; US Patent Application No. 2015/0056224 A1; US Patent Application No. 2016/0362441 A1; US Patent Application No. 2017/0158724 A1; US Patent Application No. 2017/044206 A1; US patent No. 5547941; US patent No. 7569555 B2; US patent No. 7592326 B2; US patent No. 7709458 B2; US patent No. 9549944 B2; WO 2009/133560 A1; WO 2015/074145 A1; WO 2015/077354 A1; WO 2015/185565 A1; WO 2016/100261 A1; WO 2016/120305 A1; WO 2016/145102 A1; WO 2017/027645 A1; WO 2017/027646 A1; WO 2017/075477 A1; WO 2017/093933A1; WO 2017/123657 A1; WO 2017/175156 A1; EP 1740.192 B1; CN 102199183A; Corrales, L. et al., "Direct Activation of STING in the Tumor Microenvironment Leads to Potent and Systemic Tumor Regression and Immunity", Cell Reports, 11: 1018-1030 (2015); and Lioux, T. et al., "Design, Synthesis and Biological Evaluation of Novel Cyclic Adenosine-Inosine Monophosphate (cAIMP) Analogs That Activate Stimulator of Interferon Genes (STING)", J. Med. Chem., 59: 10253-10267 (2016). All such documents, including the compounds of the present invention, are incorporated by reference into the present description; if any component of any of such documents conflicts with or is otherwise inconsistent with any of the present disclosure, then the present disclosure is the control.

In some embodiments, a compound described herein is provided as the free acid. In some embodiments, the compound is present as the NH ₄ salt or as the triethylamine (TEA) salt.

Embodiments may provide a method of treating cancer in a patient in need of such treatment, which comprises administering to the patient a therapeutically effective amount of a compound described herein, or a pharmaceutically acceptable salt thereof, as described above.

In some embodiments, the compound is administered as the free acid. In some embodiments, the compound is administered as the diammonium salt (NH ₄ ). Pharmaceutical compositions are provided containing a compound of formula I, formula II, formula III, formula IV, formula V, Table 3, or a pharmaceutically acceptable salt thereof, as well as a pharmaceutically acceptable excipient. Embodiments may provide a compound of Formula I wherein n is 1; the geometry around the double bond is trans; X ₁ and X ₂ are each SH; stereochemistry at P ₁ is S; and the stereochemistry at P ₂ is R; or a pharmaceutically acceptable salt thereof.

According to embodiments, a method of treating cancer in a patient may be provided, comprising administering to said patient a compound or a pharmaceutically acceptable salt or pharmaceutical composition thereof, as described herein. The cancers to be treated described herein may be metastatic cancers. They may be selected from, for example, lymphoma, melanoma, colorectal cancer, breast cancer, acute myeloid leukemia, colon cancer, liver cancer, prostate cancer, pancreatic cancer, kidney cancer and glioma. Also contemplated are the uses of the compounds, salts and pharmaceutical compositions for the treatment of cancer and/or the preparation of a medicament for the treatment of cancer.

In accordance with embodiments, a method of treating cancer may be provided, comprising identifying an individual having a cancer to be treated with a compound, pharmaceutically acceptable salt, or pharmaceutical composition described herein, and administering to the individual a compound, pharmaceutically acceptable salt, or pharmaceutical composition, with by which the patient was identified as eligible for treatment. In some embodiments, an individual is identified as having a cancer to be treated with a compound, pharmaceutically acceptable salt, or pharmaceutical composition described herein as a result of the presence of a human STING REF variant allele in a patient.

According to embodiments, a method of treating cancer in a patient having the STING REF allele, comprising administering to said patient a compound or a pharmaceutically acceptable salt or pharmaceutical composition thereof, described herein, may be provided.

According to embodiments, a method of treating cancer in a patient having the STING WT allele, comprising administering to said patient a compound or a pharmaceutically acceptable salt or pharmaceutical composition thereof, described herein, may be provided.

According to embodiments, a method of treating cancer in a patient having the STING AQ allele, comprising administering to said patient a compound or a pharmaceutically acceptable salt or pharmaceutical composition thereof, described herein, may be provided.

According to embodiments, a method of treating cancer in a patient having the STING HAQ allele can be provided, comprising administering to said patient a compound, or a pharmaceutically acceptable salt or pharmaceutical composition thereof, described herein.

Brief description of the drawings

On FIG. 1 shows the synthesis of Compound 1a and Compound 2a.

On FIG. 2A and FIG. 2B depicts an alternative synthesis of Compound 1 and Compound 1a. This alternative synthesis is also depicted in FIG. 2C - Fig. 2E.

On FIG. 3 shows the ¹ H NMR spectrograph for Compound 1.

On FIG. 4A, Fig. 4B and FIG. 4C shows the results of X-ray crystallography (ORTEP drawings) for an asymmetric crystal of Compound 1, a first molecule from an asymmetric crystal, and a second molecule from an asymmetric crystal, respectively.

On FIG. 4D shows the results of X-ray crystallography (drawing ORTEP) for a crystal of Compound 2.

On FIG. 5A and FIG. 5B depicts the synthesis route for Compounds 18, 19 and 20.

On FIG. 6 shows an expression vector map for STING WT (pLenti-STING WT human-Puro).

Fig. 7 and FIG. 8 accompany Example 108 and depict the therapeutic activity of Compound 1a in the dual CT26 tumor model.

Fig. 9 accompanies Example 109 and shows a plot of tumor volume for treated tumors as well as a survival curve.

Fig. 10 accompanies Example 110 and shows a plot of tumor volume for treated tumors as well as a survival curve.

On FIG. 11 is an image of the X-ray crystallographic structure of human STING WT complexed with Compound 1.

On FIG. 12 shows the C-terminal domain of STING REF in complex with Compound 1.

On FIG. 13 shows an example of the synthesis of Compound 38 and Compound 39.

Detailed description of embodiments

Provided herein are compounds that may be useful in the treatment of cancer. These compounds can activate the interferon gene stimulator (STING).

According to some embodiments, said compound is provided as the free acid. According to some embodiments, said compound is provided, for example, as an NH4 salt or a TEA salt. Referencing the compound number that follows the "a" will refer to the diammonium salt of the particular compound. For example, "Compound 1a" means the diammonium salt of Compound 1.

In accordance with embodiments, a method of treating cancer in a patient in need thereof may be provided, comprising administering to the patient a therapeutically effective amount of a compound of Formula I, Formula II, Formula III, Formula IV, or Formula V, or a pharmaceutically acceptable salt thereof.

According to some embodiments, said compound is administered as the free acid. In some embodiments, the compound is administered, for example, as a diammonium salt (NH ₄ ). Can also be provided pharmaceutical compositions for the treatment of cancer containing the compound described herein or its pharmaceutically acceptable salt, as well as a pharmaceutically acceptable excipient.

The embodiments described herein may be used in the treatment of cancer or in the preparation of drugs used in the treatment of cancer. The term "cancer" may include, without limitation, colon cancer, liver cancer, melanoma, colorectal cancer, breast cancer, acute myeloid leukemia, and glioma.

Those skilled in the art will recognize that if the substituents associated with phosphorus atoms (P ₁ , P ₂ ) contain both single and double bonds, they may be subject to tautomerization. For example, compounds can tautomerize at equilibrium. One example is shown below:

Such tautomers are to be considered as being within the scope of the claims. A structural representation of any tautomer for a given compound will represent the same compound.

In some embodiments, a compound selected from the group consisting of compounds described herein is provided as the free acid or a pharmaceutically acceptable salt thereof. In some embodiments, a compound selected from the group consisting of compounds described in the present invention is provided as an NH ₄ salt, which may be a diammonium salt.

As used herein, “C _1-6 alkyl” or “C ₁ -C ₆ ” alkyl is intended to include C ₁ , C ₂ , C ₃ , C ₄ , C ₅ or C ₆ straight chain (straight) saturated aliphatic hydrocarbon groups; and C ₃ , C ₄ , C ₅ or C ₆ branched chain saturated aliphatic hydrocarbon groups. For example, C _1-6 alkyl is contemplated to include C ₁ , C ₂ , C ₃ , C ₄ , C ₅ or C ₆ alkyl groups. Examples of alkyl include fragments containing from one to six carbon atoms, such as, without limitation, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, n-pentyl, sec-pentyl or n -hexyl. Similarly, "C1-3 alkyl" or "C1-C3 alkyl" is contemplated to include C ₁ , C ₂ or C ₃ straight chain (linear) saturated aliphatic hydrocarbon groups and C ₃ branched chain saturated aliphatic hydrocarbon groups.

Used in the present description, the term "C _3-6 cycloalkyl" or "C ₃ -C ₆ cycloalkyl" refers to a saturated or unsaturated non-aromatic hydrocarbon ring containing from 3 to 6 carbon atoms (for example, C ₃ -C ₆ ). Examples of cycloalkyl include, without limitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclopentenyl, and cyclohexenyl. The term "C _5-6 cycloalkyl" or "C ₅ -C ₆ cycloalkyl" refers to a saturated or unsaturated non-aromatic hydrocarbon ring containing 5 or 6 carbon atoms (eg C ₅ -C ₆ ).

The term "C _5-6 heterocycloalkyl" or "C ₅ -C ₆ heterocycloalkyl" refers to a saturated or unsaturated non-aromatic 5-6 membered monocyclic containing one or more heteroatoms (such as O, N or S), unless specifically noted otherwise. . The term "C _4-6 heterocycloalkyl" or "C ₄ -C ₆ heterocycloalkyl" refers to a saturated or unsaturated non-aromatic 4-6 membered monocyclic containing one or more heteroatoms (such as O, N or S) unless specifically noted otherwise. . Examples of heterocycloalkyl groups include, without limitation, piperidinyl, piperazinyl, pyrrolidinyl, dioxanyl, tetrahydrofuranyl, isoindolinyl, indolinyl, imidazolidinyl, pyrazolidinyl, oxazolidinyl, isoxazolidinyl, triazolidinyl, oxiranyl, azetidinyl, oxetanyl, thietanyl, 1,2,3,6-tetrahydropyridinyl, tetrahydropyranyl, tetrahydrothiophene, dihydropyranyl, pyranyl, morpholinyl, 1,4-diazepanyl, 1,4-oxazepanyl, and the like.

Additional examples of heterocycloalkyl groups include, without limitation, acridinyl, azocinyl, benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzoxazolinyl, benzthiazolyl, benztriazolyl, benztetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl, chromium , decahydroquinolinyl, 2H,6H-1,5,2-dithiazinyl, dihydrofuro[2,3-b]tetrahydrofuran, furanyl, furazanil, imidazolidinyl, imidazolinyl, imidazolyl, 1H-indazolyl, indolenyl, indolinyl, indolizinyl, indolyl, 3H-indolyl , isathionyl, isobenzofuranyl, isochromanyl, isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl, isothiazolyl, isoxazolyl, methylenedioxyphenyl, morpholinyl, naphthyridinyl, octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl; 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, 1,2,4-oxazol5(4H)-one, oxazolidinyl, oxazolyl, oxindolyl, pyrimidinyl, phenanthridinyl, phenanthrolinyl, phenazinyl, phenothiazinyl, phenoxatinil, phenoxazinyl, phthalazinyl , piperazinyl, piperidinyl, piperidonyl, 4-piperidonyl, piperonyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridooxazole, pyridoimidazole, pyridothiazole, pyridinyl, pyridyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, , quinazolinyl, quinolinyl, 4H-quinolizinyl, quinoxalinyl, quinuclidinyl, tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl, tetrazolyl, 6H-1,2,5-thiadiazinyl, 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2 ,5-thiadiazolyl, 1,3,4-thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thienothiazolyl, thienooxazolyl, thienoimidazolyl, thiophenyl, triazinyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,2,5 -triazolyl, 1,3,4-triazolyl and xanthenyl.

As used herein, the term "C _5-6 aryl" or "C ₅ -C ₆ aryl" refers to an aromatic hydrocarbon ring containing from 5 to 6 carbon atoms (for example, C ₅ -C ₆ ) that does not contain any heteroatom in ring structure.

The term "C _3-6 heteroaryl" or "C ₃ -C ₆ heteroaryl" refers to an aromatic 3-6 membered monocyclic containing one or more heteroatoms (such as O, N or S), unless specifically noted otherwise, except that the heteroaryl ring will include no more than one oxygen atom or one sulfur atom.

The term "C _2-6 alkenyl" includes unsaturated aliphatic groups containing 2, 3, 4, 5, or 6 carbon atoms and which contain at least one double bond. For example, the term "C _2-6 alkenyl" includes straight chain alkenyl groups (eg, ethenyl, propenyl, butenyl, pentenyl, hexenyl) and straight chain alkenyl groups. In certain embodiments, a straight chain or branched alkenyl group has six or fewer carbon atoms in its backbone (eg, C ₂ -C ₆ for straight chain, C ₃ -C ₆ for branched chain). The term "C _2-6 alkenyl" includes alkenyl groups containing two to six carbon atoms.

The term "C _2-6 alkynyl" includes unsaturated aliphatic groups containing 2, 3, 4, 5 or 6 carbon atoms, but which contain at least one triple bond. For example, "alkynyl" includes straight chain alkynyl groups (eg, ethynyl, propynyl, butynyl, pentynyl, hexynyl) and branched alkynyl groups. In certain embodiments, a straight chain or branched alkynyl group has six or fewer carbon atoms in its backbone (eg, C ₂ -C ₆ for straight chain, C ₃ -C ₆ for branched chain). The term "C _2-6 alkynyl" includes alkynyl groups containing two to six carbon atoms.

Methods of treatment

According to embodiments, a method of treating cancer in a patient in need thereof may be provided, comprising administering to the patient a therapeutically effective amount of a compound described herein, or a pharmaceutically acceptable salt thereof.

In some embodiments, the administered compound is provided as the free acid or a pharmaceutically acceptable salt thereof. In some embodiments, the administered compound is provided as an NH ₄ salt, the free acid, or a pharmaceutically acceptable salt thereof. According to some embodiments, said compound is provided as an NH ₄ salt.

The term "optionally substituted" refers to a moiety having designated substituents replacing one or more hydrogen atoms on one or more hydrogen-bearing atoms in the moiety.

Chemical compounds designated or described include all naturally occurring isotopes of atoms occurring in the compounds of the present invention. Isotopes include atoms having the same atomic number but different mass numbers. By way of general example and without limitation, ¹ H isotopes include tritium and deuterium, and ¹² C carbon isotopes include ¹³ C and ¹⁴ C.

Doses

The optimal dose for the treatment of cancer can be determined empirically for each individual using known methods and will depend on a number of factors, including the activity of the funds; age, body weight, general health, sex and nutrition of the individual; time and method of administration; and other drugs that the individual is taking. Optimal doses can be established using standard testing and procedures that are well known in the art. The introduction of the above compounds can occur using any suitable method.

The term "pharmaceutically acceptable salt" as used herein refers to acid addition salts or base addition salts of the compounds in this disclosure. A pharmaceutically acceptable salt is any salt that retains the activity of the parent compound and does not have any unduly deleterious or undesirable effect on the subject to whom it is administered and in the context in which it is administered. Pharmaceutically acceptable salts include, without limitation, metal complexes and salts of both inorganic and carboxylic acids. Pharmaceutically acceptable salts also include metal salts such as aluminum, calcium, iron, magnesium and complex salts. In addition, pharmaceutically acceptable salts include, without limitation, acid salts such as acetic, aspartic, alkylsulfonic, arylsulfonic, axetyl, benzenesulfonic, benzoic, bicarboxylic, bubbly, bivinic, butyric, calcium edetate, camsyl, carboxylic, chlorobenzoic, citric , ethylenediaminetetraacetic, edisyl, estol, ezyl, ezilin, formic, fumaric, gluceptive, gluconic, glutamine, glycolic, glycolilarsanil, hexamine, hexylresorcinol, hydrabamic, hydrobromic, hydrochloric, hydroiodic, hydroxynaphthoic, isethic, lactic, lactobionic, malic, malic , almond, methanesulfonic, methylnitrogen, methylsulfur, mucus, muconic, napsilic, nitrogenous, oxalic, p-nitromethanesulfonic, pamoic, pantothenic, phosphoric, monohydrophosphoric, dihydrophosphoric, phthalic, polygalacturonic, propionic, salicylic, stearic, succinic, sulfamic, sulfanilic, sul phonic, sulfuric, tannic, tartaric, theocline, toluenesulfonic, etc. Sodium salts and potassium salts can also be prepared.

In embodiments, diammonium salts may be provided. Pharmaceutically acceptable salts can be derived from amino acids, including but not limited to cysteine. Methods for preparing compounds in the form of salts are known to those skilled in the art (see, for example, Stahl et al., Handbook of Pharmaceutical Salts: Properties, Selection, and Use, Wiley-VCH; Verlag Helvetica Chimica Acta, Zurich, 2002; Berge et al., J. Pharm. Sci. 66:1, 1977).

The term "effective amount" of a therapeutic agent is an amount sufficient to produce an observable therapeutic benefit compared to cancer that has not been treated in a subject or patient.

The active agents described herein can be combined with a pharmaceutically acceptable carrier to obtain their pharmaceutical compositions. The specific choice of carrier and composition will depend on the particular route of administration for which the composition is intended.

The term "pharmaceutically acceptable carrier" as used herein refers to a non-toxic carrier, adjuvant, or vehicle that does not interfere with the pharmacological activity of the compound with which it is formulated. Pharmaceutically acceptable carriers, adjuvants, or bases that may be used in the compositions of the present invention include, but are not limited to, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, sodium hydrogen phosphate, potassium hydrogen phosphate, chloride sodium, zinc salts, colloidal silicon dioxide, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene glycol and wool grease.

The compositions of the present invention may be suitable for parenteral, oral, inhalation spray, topical, rectal, nasal, buccal, vaginal, or implantable reservoir administration. In accordance with some embodiments, the composition contains ingredients that come from natural or non-natural sources. In accordance with some embodiments, the formulation or carrier may be provided in a sterile form. Non-limiting examples of a sterile carrier include endotoxin-free water or pyrogen-free water.

The term "parenteral" as used herein includes subcutaneous, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intrahepatic, intralesional, and intracranial injection or infusion routes. In accordance with a certain embodiment, the compounds are administered intravenously, orally, subcutaneously, or via intramuscular injection. Sterile injectable forms of the compositions of the present invention may be in the form of an aqueous or oily suspension. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent. Among the acceptable vehicles and solvents that may be used are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile fixed oils are traditionally used as a solvent or suspending medium.

For this purpose, any bland fixed oil can be used, including synthetic mono- or diglycerides. Fatty acids and their glyceride derivatives are suitable for injectables because they are natural pharmaceutically acceptable oils such as olive or castor oil, especially in their polyoxyethylated versions. These oily solutions or suspensions may also contain a long chain alcoholic diluent or dispersant such as carboxymethyl cellulose or similar dispersants which are widely used in the formulation of pharmaceutically acceptable dosage forms, including emulsions and suspensions. Other widely used surfactants such as Tween, Span and other emulsifying agents which are widely used in the preparation of pharmaceutically acceptable solid, liquid or other dosage forms can also be used for formulation purposes.

For oral administration, the compound or salt may be provided in an acceptable oral dosage form, including, without limitation, capsules, tablets, aqueous suspensions, or solutions. In the case of tablets for oral administration, commonly used carriers include lactose and cornstarch. Lubricants such as magnesium stearate may also be added. For oral administration in capsule form, suitable diluents include lactose and dried cornstarch. If aqueous suspensions are required for oral administration, the active ingredient may be combined with emulsifying and suspending agents. Certain sweetening, flavoring or coloring agents may also be added, if desired. In addition, preservatives may be added. Suitable examples of pharmaceutically acceptable preservatives include, without limitation, various antibacterial and antifungal agents such as diluents such as ethanol, propylene glycol, benzyl alcohol, chlorobutanol, quaternary ammonium salts, and parabens (such as methylparaben, ethylparaben, propylparaben, etc.) .

"Immediate release" means a standard release, in which the release of the drug begins immediately after administration. As used herein, the term "immediate release" includes dosage forms that promote dissolution of the drug in the contents of the gastrointestinal tract, while not intended to delay or prolong the dissolution or absorption of the drug. The aim is to release the drug quickly after administration, for example, to be able to release at least 80% of the drug within about 30 minutes after the start of dissolution in the dissolution test.

“Sustained release” or “extended release” refers to dosage forms whose temporal and/or spatial release characteristics of the drug are chosen for therapeutic purposes or for convenience, not provided by standard dosage forms, such as a solution or immediate release dosage form.

The term "steady state" means that the plasma level of a specific active agent has been reached and maintained with subsequent doses of the active agent at a level that is at or above the minimum effective therapeutic level and below the minimum toxic plasma level for a particular active agent. .

The term "unit formulation" as used herein refers to a single carrier or vehicle formulated to deliver effective amounts of both therapeutic agents to a patient. A single carrier is designed to deliver an effective amount of each of the agents along with any pharmaceutically acceptable carriers or excipients. According to some embodiments, the base is a tablet, capsule, pill, or patch.

The term "unit dose" is used herein to refer to the simultaneous administration of both agents together, in one dosage form, to the patient to be treated. In accordance with some embodiments, the unit dose is a unit formulation. In certain embodiments, the unit dose contains one or more bases, whereby each base contains an effective amount of the agent along with pharmaceutically acceptable carriers and excipients. In some embodiments, a unit dose is one or more tablets, capsules, pills, or patches administered to a patient at the same time.

The term "dose range" as used herein refers to the upper and lower limits of the allowable variation in the amount of analyte. In a typical case, the dose of funds in any amount within the defined range can be administered to patients undergoing treatment.

The term "treat" is used herein to alleviate, alleviate or normalize at least one symptom of a disease in a subject. For example, in relation to cancer, the term "treat" can mean stopping, delaying the onset (ie, the period before clinical manifestation of a disease or symptom of a disease), and/or reducing the risk of developing or exacerbating a symptom of a disease. The term "provide a protective effect" as used herein means preventing, delaying or treating or all, if necessary, the development or continuation or exacerbation of symptoms of a disease in a subject.

The term "subject" or "patient" includes animals that are capable of suffering from or being affected by cancer. Examples of subjects or patients include mammals, such as humans, dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits, rats, and transgenic non-human animals. In certain embodiments, the subject is a human, such as a human suffering from, at risk of suffering from, or potentially capable of suffering from cancer.

The term "about or about" generally means within 20%, more preferably within 10%, and most preferably even within 5% of a specified value or range. Alternatively, especially in biological systems, the term "about" means about within a logarithm (ie, an order of magnitude), preferably within a factor of two from a certain value.

The use of terms in the singular in the context of the description of the present invention (especially in the context of the following claims) is intended to include the singular and plural, unless otherwise indicated herein or clearly contrary to context. The terms "comprising", "having", "including" and "including" are considered as open terms (ie, meaning "including without limitation"), unless otherwise indicated. The enumeration of ranges of values in this document is intended to be used only as a shorthand way of separately referring to each individual value within the range, unless otherwise noted in this document, and each individual value is included in the description as if it were separately mentioned. in this document.

Illustrative disorders of cell proliferation that can be treated with one or more of the compounds disclosed herein include, without limitation, cancer, precancerous or precancerous conditions, and metastatic lesions in tissues and organs in the body. Cell proliferation disorders may include hyperplasia, metaplasia, and dysplasia.

The compound disclosed herein, or a pharmaceutically acceptable salt thereof, may be used to treat or prevent a cell proliferation disorder, or to treat or prevent cancer in a subject having an increased risk of developing cancer relative to the population, or used to identify suitable candidates for such purposes. .

Pharmaceutical formulations and routes of administration

Provided herein are pharmaceutical compositions containing a compound or a pharmaceutically acceptable salt thereof for the treatment of cancer. Pharmaceutical compositions may additionally contain a carrier or excipient, a stabilizer, a flavoring agent and/or a coloring agent.

The compound, or a pharmaceutically acceptable salt thereof, may be administered via a number of routes of administration known to those skilled in the art. Routes of administration include oral administration. According to certain embodiments, the pharmaceutical formulation containing the compound or a pharmaceutically acceptable salt thereof may be taken orally in the form of a liquid, syrup, tablet, capsule, powder, aerosol, chewable tablet, or dissolvable disc. Alternatively, the pharmaceutical compositions of the present invention may be administered intravenously or transdermally. Additional routes of administration are known to those skilled in the art (see, for example, Remington's Pharmaceutical Sciences, Gennaro A. R., Ed., 20th Edition, Mack Publishing Co., Easton, Pa.).

In some embodiments, the compound or pharmaceutically acceptable salt is formulated as a paste, gel, or suspension. For example, the drug is dissolved, trapped, or suspended in the form of drug particles, microencapsulated particles, or drug-polymer particles in a gel solution or soft form. An oral gel formulation has the advantage of being easier to administer to patients who have difficulty swallowing tablets, capsules or pills. In certain embodiments, the compound is thoroughly mixed and suspended in an appropriate medium to form a paste or gel. Additional tools may optionally be mixed to obtain a flavor during oral administration. Raspberry or sweetener flavored peanut butter or alginate are examples of a variety of suitable drug taste masking agents. According to various embodiments, the paste or gel may also be formulated with suitable binders or excipients known in the art for topical application.

Methods for preparing sustained release formulations in the form of tablets, capsules or pills are known in the art. In some embodiments, a sustained release formulation is obtained by coating an active drug ingredient with a polymer, preferably a water-insoluble polymer. For example, the water-insoluble polymer is used in the pharmaceutical field as a sustained release coating agent, an enteric coating agent, or an enteric coating agent. The water-insoluble polymer may include, for example, ethyl cellulose, purified shellac, white shellac, aminoalkyl methacrylate copolymer RS, hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose acetate succinate, methacrylic acid copolymer L, methacrylic acid copolymer LD, methacrylic acid copolymer S, aminoalkyl methacrylate copolymer E, or polyvinyl acetal diethylaminoacetate. .

The type, degree of substitution, and molecular weight of the water-insoluble polymers may depend on the solubility of the active ingredient in water or alcohol, the level of sustained release desired, and the like. The water-insoluble polymers may be used either singly or in combination. They can be further included in hydrogenated oil, stearic acid or cetanol as a coating aid and medium chain triglyceride, triacetin, triethyl citrate or cetanol as a plasticizer.

According to some embodiments, the sustained release formulation is a matrix type tablet or granule. The active ingredient can be coated with up to 3 different types of polymers. These three different types of polymers may include: 1) a water-insoluble polymer such as ethyl cellulose; 2) a pH-independent gelling polymer such as hydroxypropyl methylcellulose; and 3) a pH dependent gelling polymer such as sodium alginate. These three different types of polymers can be used together to slow the release rate of drugs.

Dosage Forms: Release Properties

Sustained release formulations can achieve a degree of delayed effect. At the same time, the content and/or bioavailability of the active ingredient may vary based on a number of factors such as, for example, the absorption window, the carriers or excipients used in the formulation, the mode of delivery of the formulation, and/or the transit time of the active ingredient through the patient's gastrointestinal tract. .

The medicament may contain at least one sustained release portion to provide a sustained release function and one immediate release portion to provide an immediate release function. In accordance with certain embodiments, if the drug is in unit dosage form, it may be in the form of tablets formed from a mixture of sustained release granules containing a sustained release portion and immediate release granules containing an immediate release portion. , a capsule preparation obtained by filling a capsule with sustained-release granules and immediate-release granules, or compressed-coated tablets in which an outer layer consisting of an immediate-release portion is formed on an inner layer containing a sustained-release portion. At the same time, there is no limitation with respect to the above embodiments.

Moreover, there are no specific restrictions on the retention state of the drug in the composition or immediate release portion or sustained release portion; the compound may be dispersed uniformly in the composition, the immediate release portion or the sustained release portion, or may be contained in only one composition portion, the immediate release portions or the sustained release portions, or may be contained in such a manner that a concentration gradient occurs.

The sustained release portion of the composition of the present invention contains at least one pH-independent polymeric substance or one pH-dependent polymeric substance to control drug release.

The pH-independent polymeric substance used herein may be a polymeric substance whose state of charge changes very little under the pH conditions typically encountered in the gastrointestinal tract, in particular from pH 1 to pH 8. This means , for example, a polymeric substance that does not have functional groups whose charge state changes depend on pH, for example, basic functional groups such as amino groups or acidic functional groups such as carboxylic acid groups. It should be noted that a pH-independent polymeric substance may be included for the purpose of imparting a sustained release function to the composition of the present invention, but may also be included for another purpose. Moreover, the pH-independent polymeric substance used in the present invention may be water-soluble, or may swell in water or dissolve in water to form a gel.

Examples of water-insoluble, pH-independent polymeric materials include, without limitation, cellulose ethers, cellulose esters, and copolymers of methacrylic acid and acrylic acid (trade name Eudragit, manufactured by Rohm GmbH & Co. KG, Darmstadt, Germany). Examples include, without limitation, cellulose alkyl ethers such as ethyl cellulose (trade name Ethocel, manufactured by Dow Chemical Company, USA), ethyl methyl cellulose, ethyl propyl cellulose and isopropyl cellulose and butyl cellulose, aralkyl cellulose ethers such as benzyl cellulose, cyanoalkyl cellulose ethers such as cyanoethyl cellulose, esters cellulose acetate and organic acids such as cellulose acetate butyrate, cellulose acetate, cellulose propionate or cellulose butyrate and cellulose acetopropionate, copolymers of ethyl acrylate and methyl methacrylate (trade name Eudragit NE, manufactured by Rohm GmbH & Co. KG, Darmstadt, Germany) and aminoalkyl methacrylate copolymer RS (trade names Eudragit RL, Eudragit RS). There are no particular restrictions on the average particle diameter of the water-insoluble polymer used in the present invention, however, in general, the lower the average particle diameter, the more effective the performance, and the average particle diameter is preferably from 0.1 to 100 μm, more preferably from 1 to 50 µm, particularly preferably 3 to 15 µm, most preferably 5 to 15 µm. Moreover, examples of water-soluble or water-swellable pH-independent polymer materials include, without limitation, polyethylene oxide (trade name Polyox, manufactured by Dow Chemical Company, molecular weight 100,000 to 7,000,000), low substituted hydroxypropyl cellulose (trade name L-HPC , manufactured by Shin-Etsu Chemical, Japan), hydroxypropyl cellulose (trade name HPC, manufactured by Nippon Soda, Co., Ltd, Japan), hydroxypropyl methylcellulose (trade names Metolose 60SH, 65SH, 90SH, manufactured by Shin-Etsu Chemical, Japan) and methyl cellulose ( trade name Metolose SM, manufactured by Shin-Etsu Chemical, Japan).

In some embodiments, the composition may contain a single pH-independent polymeric material or may contain multiple pH-independent polymeric materials. The pH-independent polymeric material, when used in accordance with the embodiments described herein, may be a water-insoluble polymeric material, most preferably ethyl cellulose, an ethyl acrylate-methyl methacrylate copolymer (trade name Eudragit NE), or an aminoalkyl methacrylate RS copolymer (trade name Eudragit RL, Eudragit RS). Particularly preferred is one of ethyl cellulose and an aminoalkyl methacrylate copolymer RS. Most preferred is ethyl cellulose. There are no specific restrictions on the amount of pH-independent polymeric substance contained in the composition; this amount can be adjusted appropriately according to the purpose, such as controlled sustained release of the drug.

The pH-dependent polymeric substance that can be used in the embodiments described herein can be a polymeric substance whose state of charge changes under pH conditions typically encountered in the gastrointestinal tract, in particular from pH 1 to pH 8. This means, for example, a polymeric substance having functional groups whose charge state changes depend on pH, for example basic functional groups such as amino groups or acidic functional groups such as carboxylic acid groups. The pH-dependent functional groups of the pH-dependent polymeric substance are preferably acidic functional groups, with the pH-dependent polymeric substance most preferably having carboxylic acid groups.

The pH-dependent polymer material used in the present invention may be water soluble or may swell in water or dissolve in water to form a gel. Examples of pH-dependent polymeric substances used in the present invention include, without limitation, enteric polymeric substances. Examples of enteric polymeric materials include, without limitation, methacrylic acid-methyl methacrylate copolymers (Eudragit L100, Eudragit S100, manufactured by Rohm GmbH & Co. KG, Darmstandt, Germany), methacrylic acid-ethyl acrylate copolymers (Eudragit L100-55, Eudragit L30D-55, manufactured by Rohm GmbH & Co. KG, Darmstadt, Germany), hydroxypropyl methylcellulose phthalate (HP-55, HP-50, manufactured by Shin-Etsu Chemical, Japan), hydroxypropyl methylcellulose acetate succinate (AQOAT, manufactured by Shin-Etsu Chemical, Japan), carboxymethyl cellulose ( CMEC, manufactured by Freund Corporation, Japan) and cellulose acetate phthalate.

Examples of pH-dependent polymeric substances that swell in water or dissolve in water to form a gel include, but are not limited to, alginic acid, pectin, carboxyvinyl polymer, and carboxymethylcellulose. In accordance with the present invention, the composition may contain one pH-dependent polymeric substance or may contain several pH-dependent polymeric substances. The pH dependent polymeric substance used in the present invention is preferably an enteric polymeric substance, more preferably a copolymer of methacrylic acid and ethyl acrylate, a copolymer of methacrylic acid and methyl methacrylate, hydroxypropyl methylcellulose phthalate or hydroxypropyl methylcellulose acetate succinate, particularly preferably a copolymer of methacrylic acid and ethyl acrylate.

When using pH dependent polymeric substances in the method for producing a composition according to the present invention, a commercially available powder type or granular type or suspension type product in which the pH dependent polymeric substance has been dispersed in a solvent in advance can be used as it is, or such a commercially available product can be used dispersed in water or an organic solvent. The lower the particle diameter of the pH-dependent polymeric substance, the more effective the performance, and the pH-dependent polymeric substance is preferably of the powder type. In the case of a copolymer of methacrylic acid and ethyl acrylate, an example is Eudragit L100-55. There are no particular restrictions on the average particle diameter of the pH-dependent polymeric substance used in the present invention, however, the average particle diameter is preferably 0.05 to 100 µm, more preferably 0.05 to 70 µm, most preferably 0.05 to 50 µm. Moreover, there are no particular restrictions on the amount of pH-dependent polymeric substance, for example, in the case of an enteric polymeric substance, the amount is generally from 1 to 90 mass parts, preferably from 1 to 70 mass parts, more preferably from 5 to 60 mass parts, particularly preferably from 10 to 50 mass parts, based on 100 mass parts of the composition.

The medicament according to the embodiments described herein may further contain any of various adjuvants such as any of pharmacologically acceptable diluents, lubricants, binders, and disintegrants, as well as preservatives, colors, sweeteners, plasticizers, film-forming agents. etc., if necessary. Examples of diluents include, without limitation, lactose, mannitol, dicalcium phosphate, starch, pregelatinized starch, crystalline cellulose, non-compacted colloidal silica, synthetic aluminum silicate, magnesium aluminate metasilicate, and the like. Examples of lubricants include, without limitation, magnesium stearate, calcium stearate, talc, sodium stearyl fumarate, and the like. Examples of binders include, but are not limited to, hydroxypropylcellulose, methylcellulose, sodium carboxymethylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, and the like. Examples of disintegrants include, but are not limited to, carboxymethyl cellulose, calcium carboxymethyl cellulose, sodium croscarmellose, sodium carboxymethyl starch, low substituted hydroxypropyl cellulose, and the like. Examples of preservatives include, without limitation, paraoxybenzoic acid esters, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, sorbic acid, and the like. Preferred examples of colorants include, without limitation, water-insoluble color pigments, natural pigments (e.g., beta-carotene, chlorophyll, iron oxide red), iron oxide yellow, iron oxide red, iron oxide yellow, etc. Preferred examples of sweeteners include, without limitation, sodium saccharin, dipotassium glycyrrhizate, aspartame, stevia, or the like. Examples of plasticizers include, without limitation, glycerol fatty acid esters, triethyl citrate, propylene glycol, polyethylene glycol, or the like. Examples of film-forming agents include, but are not limited to, hydroxypropyl methylcellulose, hydroxypropyl cellulose, and the like.

How to get

In order to obtain the embodiments described herein, one standard method or a combination of standard methods can be used. For example, when preparing drug-containing granules as a sustained release portion or an immediate release portion, granulation is the main operation, but it can be combined with other operations such as mixing, drying, screening and classification. As the granulation method, for example, a wet granulation method in which a binder and a solvent are added to the powder and granulation is performed, a dry granulation method in which the powder is compressed and granulation is performed, a melt granulation method in which a binder that melts when heated , added and heated and granulated, or similar methods can be used.

In addition, according to the granulation method, an operating method such as a mixing granulation method using a planetary mixer, a screw mixer or the like, a high-speed mixing granulation method using a Henschel mixer, a Super mixer or the like can be used. , an extrusion granulation method using a cylindrical granulator, a rotary granulator, a screw extrusion granulator, a briquette granulator or the like, a high shear wet granulation method, a fluid bed granulation method, a post-compression granulation method, a crush granulation method, or a granulation method spraying. After granulation, drying with a dryer, fluidized bed and the like, crushing and screening can be carried out to obtain granules or fine granules for use. Moreover, a granulation solvent can be used in the preparation of a composition according to the present invention. There are no particular restrictions on such a granulation solvent, which may be water or any of the organic solvents, for example, water, a lower alcohol such as methanol or ethanol, a ketone such as acetone or methyl ethyl ketone, methylene chloride, or mixtures thereof.

In the case of the sustained release granules contained in the embodiments, at least one drug and at least one selected from pH-independent polymeric substances and pH-dependent polymeric substances are mixed together, diluent and binder are added if necessary, and performing granulation to obtain a granular substance. The obtained granular substance is dried with a tray dryer, a fluidized bed dryer, and the like, and screened with a mill or an oscillator, whereby sustained release granules can be obtained. Alternatively, at least one drug, at least one selected from pH-independent polymeric substances and pH-dependent polymeric substances, and, if necessary, a diluent and a binder, can be added as a method for producing sustained-release granules in the present invention. substance by a dry press or a roller press or a tablet filling machine, and compress into a mold while mixing, and then granulate by crushing to a suitable size. The granular substance produced by such a granulator can be used as it is in the form of granules or fine granules according to the present invention, or can be further ground with an electric mill, a roller granulator, a speed mill and sieved to obtain sustained release granules. . It should be noted that sustained release granules can also be prepared as sustained release granules.

The compressible preparation can be prepared as a sustained release drug portion or an immediate release portion, or as a composition as described herein, using one standard method or a combination of standard methods. For example, at least one drug is used, at least one selected from pH-independent polymeric substances and pH-dependent polymeric substances, a diluent such as mannitol or lactose, a binder such as polyvinylpyrrolidone or crystalline cellulose, a disintegrant such as such as carmellose sodium or crospovidone, and a lubricant such as magnesium stearate or talc, and tabletting is carried out by a standard method, whereby a compressible preparation can be obtained. In this case, tabletting is the main operation in the process of obtaining compressed into the form of a preparation, however, it can be combined with other operations, such as mixing, drying, sugar coating and coating shell.

Examples of the tabletting method include, without limitation, direct mold compression in which at least one drug and pharmacologically acceptable adjuvants are mixed together and then the mixture is directly mold-compressed into tablets in a tablet machine, as well as dry granule compression or wet granule compression. in which the sustained release granules or immediate release granules according to the present invention are compressed into a mold after the addition of a lubricant or a disintegrant, if necessary. There are no specific restrictions on the tablet machine used in mold compression; for example, a single-die tablet machine, a rotary tablet machine, or a coated tablet machine can be used.

The sustained-release or immediate-release drug-containing granules or compressible preparation according to the embodiments herein may be used as is, in the form of granules or tablets in the form of a composition, but may also be further processed with obtaining a composition. For example, the compressible preparation or granules can be film-coated with a film-based substance such as ethyl cellulose, casein, methyl cellulose, hydroxypropyl methylcellulose, methacrylic acid copolymer L, cellulose acetate phthalate, shellac or the like, or sugar-coated with using a sugar coating liquid containing sucrose, sugar alcohol, gum arabic powder, talc or the like, to form film-coated tablets or sugar-coated tablets. One solvent in this coating technique may be purified water, but an organic solvent such as an alcohol, ketone, ether, or chlorinated hydrocarbon, or a mixture thereof, may also be used. For example, ethanol, acetone, methylene chloride, or the like. can be used as an organic solvent. In addition, as the coating machine, a machine commonly used in drug coating techniques can be used, examples include a spray coating machine in which coating can be performed by spraying a coating liquid or the like, and a fluid bed rotary granulator for layering.

In the case of capsule preparations, capsule preparations can be prepared by filling the sustained release granules or immediate release granules described above or minitablets into hard gelatin capsules or HPMC capsules using an automatic capsule filling machine. Alternatively, in the case of gavage or dry syrup preparations to be used mixed with water or the like when taken, the sustained release granules or immediate release granules described above may be mixed with a thickening or dispersing agent to disperse these granules, then the mixture is converted into granules or tablets. In addition, the liquid or gel may be formed with water and substances selected from dispersants, emulsifiers, thickeners, preservatives, pH adjusters, sweeteners, flavors, flavors, and the like. At the same time, with respect to other production methods, there are no restrictions on the above.

In order for the embodiments described herein to be more fully understood, examples are set forth below. It should be understood that these examples are provided for illustrative purposes only and are not intended to be limiting.

Examples

The following abbreviations may be used in any of the examples.

All: allyl

BOP: (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate

DMT: 4,4'-Dimethoxytrityl

(DMTO-:

Bz: benzoyl

ib: isobutyryl

Hunig's base: i-Pr ₂ NEt (diisopropylethylamine)

AllylOH: allyl alcohol

OAll: -OCH ₂ CHCH ₂

ACN: acetonitrile

All: -CH2CHCH2

2-NitroBnBr: 2-nitrobenzyl bromide

Bz: benzoyl

ib: isobutyryl

i-Pr: isopropyl

CE: cyanoethyl

DEAD: diethyl azodicarboxylate

DIAD: diisopropyl azodicarboxylate

DCM: dichloromethane

DDTT: N,N-dimethyl-N'-(3-thioxo-3H-1,2,4-dithiazol-5-yl)formimidamide

DMOCP: 2-chloro-5,5-dimethyl-1,3,2-dioxaphosphinane 2-oxide

TBS: t-butyldimethylsilyl

3H-benzo[c][1,2]dithiol-3-one:

Example 1 Synthesis of Compound 1a

A complete scheme of such synthesis is available in Fig. 1.

Stage A

To a mixture of (2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-fluoro- 3-yl(2-cyanoethyl)diisopropylphosphoramidite (compound 100) (mixture of phosphorus diastereomers; 80.0 g, 91.332 mmol, 1 eq., ChemGenes Corporation Cat. No. ANP-9151), allyl alcohol (9.63 ml, 142 mmol , 1.55 eq.) and triphenylphosphine (38.3 g, 146 mmol, 1.60 eq.) in THF (1.1 L) was added DEAD (40 wt.% solution in toluene; 54.2 ml, 137 mmol , 1.5 eq.) at ambient temperature. Stirring was continued at ambient temperature and the reaction was monitored by LC/MS. After completion (19 h) the mixture was concentrated in vacuo (35° C.) and the resulting mixture was purified by silica gel column chromatography (800 g x 2 columns, 40-60% EtOAc in n-heptane buffered with 0.5% triethylamine) to give compound 101 as a white foam (84.2 g, quantitative yield, mixture of phosphorus diastereomers).

¹ H NMR (3:2 mixture of phosphorus diastereomers, 400 MHz, CDCl ₃ ) δ 1.14-1.21 (m, 12 H) 2.40 (t, J=6.2 Hz, 1.2H) 2.59 (t, J=6.2 Hz, 0.8H ) 3.27 (d, J=8.6 Hz, 1H) 3.52-3.66 (m, 5H) 3.78 (s 2.4H) 3.79 (s 3.6H) 4.28-4.34 (m, 1H) 4.84-4.96 (m, 0.4H) 4.99 (d, J=5.5 Hz, 2H) 4.95-5.10 (m, 0.6H) 5.05 (d, J=10.9 Hz, 1H) 5.22 (br d, J=17.6 Hz, 1H) 5.64 (br d, J=53.2 Hz, 0.6H) 5.70 (br d, J=51.6Hz, 0.4H) 5.96-6.75 (m, 1H) 6.20 (d, J=16.0Hz, 0.6H) 6.24 (d, 1=17.2Hz, 0.4H) 6.74-6.79 (m, 4H) 7.02-7.06 (m, 2H) 7.17-7.24 (m, 8H) 7.32-7.34 (m, 2H) 7.41-7.44 (m, 2H) 8.11 (s, 1H) 8.52 (s, 0.4H) 8.54(s, 0.6H).

Stage B

To a solution of compound 101 (3.00 g, 3.28 mmol, 1 eq.) in acetonitrile (30 ml) was added water (0.118 ml, 6.55 mmol, 2.0 eq.) and pyridine trifluoroacetate salt (0.759 g, 3.93 mmol, 1.2 eq.). After stirring at ambient temperature for 1 minute, tert-butylamine (14.5 g, 21.0 ml, 0.20 mol, 60 eq.) was added. After cleavage of the cyanoethyl group was complete (monitored by LC/MS), the reaction mixture was concentrated in vacuo and azeotroped twice with acetonitrile. The crude mixture was dissolved in DCM (45.0 ml) and treated with water (0.118 ml, 6.55 mmol, 2.0 eq.) and NaHSO ₄ -SiO ₂ (1.18 g, 6.55 mmol, 2 eq.) at ambient temperature. After cleavage of the DMT group was complete (observed by LC/MS, approx. 1 hour), the reaction mixture was filtered and washed twice with DCM/MeOH (9/1, 20 ml). The combined filtrates were concentrated in vacuo and treated with 1:1 n-heptane/toluene (~30 ml). The top layer was removed by decantation. The same operation was repeated once more with n-heptane/toluene (1/1, 30 ml) and the bottom layer was azeotroped twice with acetonitrile to give compound 102 (100% expected theoretical yield). The product was used in the next step without further purification.

Stage C

To a mixture of compound 102 (1.56 g, 3.27 mmol, 1 eq.; and compound 101 (3.00 g, 3.28 mmol, 1 eq.) in acetonitrile (30 ml) was added pyridine trifluoroacetate salt (azeotropically dried with pyridine, 0.760 g, 3.94 mmol, 1.25 eq. DDTT (0.840 g, 4.09 mmol, 1.30 eq., ChemGenes Corporation cat. no. RN-1588) was added after 5 minutes and after completion sulfurization (observed by LC/MS) the reaction mixture was concentrated in vacuo The residue was dissolved in DCM (30 ml) and treated with water (0.57 ml, 32 mmol, 10 eq.) and 6% dichloroacetic acid (1.56 ml, 18.9 mmol, 6.0 eq.) in DCM (30 ml) After 20 minutes the reaction was quenched with pyridine (20 ml) and concentrated in vacuo The residue was azeotroped with pyridine to give compound 103 (3.22 g, 100% predicted theoretical yield) The product was used in the next step without further purification.

Stage D

DMOCP (1.45 g, 7.88 mmol, 2.50 eq.) at ambient temperature. After completion of the macrocyclization (monitored by LC/MS), water (1.7 ml, 94.5 mmol, x10 times DMOCP) was added followed by 3H-benzo[c][1,2]dithiol-3- he (0.795 g, 4.73 mmol, 1.5 eq.). After completion of sulfurization (approximately 40 minutes) the reaction mixture was partially concentrated in vacuo to approximately 15 ml and poured into a mixture of saturated aqueous NaHCO ₃ (50 ml) and water (30 ml). After 10 minutes of stirring at ambient temperature, the mixture was extracted with 1:1 EtOAc/MTBE (60 ml x 3 times). The organic layers were combined, washed with brine (25 ml), dried over Mg ₂ SO ₄ and concentrated in vacuo. The residue was purified by silica gel column chromatography (0-20% MeOH in DCM) to give compound 104 (3.31 g, 3.20 mmol, 100% theoretical yield) as a brown oil. The product was used in the next step without further purification.

Stage E

To a solution of compound 104 (3.31 g, 3.20 mmol, 1 eq.) in acetonitrile (66.2 ml) was added 2-nitrobenzyl bromide (2.42 g, 11.2 mmol, 3.50 eq.) and triethylamine (1.78 ml, 12.8 mmol, 4.00 eq.). After completion of the reaction (observed by LC/MS, approximately 20 hours at ambient temperature), the reaction mixture was concentrated in vacuo and purified by silica gel column chromatography (60% ethyl acetate/n-heptane to 100% ethyl acetate) to give 0.568 g of product in as a mixture of phosphorus diastereomers. Separation of the diastereomers by preparative HPLC gave compound 105 (SR isomer; 0.225 g, 0.180 mmol, 5.6% overall yield from compound 101) and compound 106 (RR isomer; 0.187 g, 0.149 mmol, 4.7% overall yield from compound 1 ).

Compound 105 (SpRp) ¹ H NMR (400 MHz, CDCl ₃ ) δ=8.63 (s, 1H), δ=8.61 (s, 1H), 8.04-8.00 (m, 2H), 7.99 (s, 1H), 7.90 (s, 1H), 7.65-7.44 (m, 8H), 7.40-7.31 (m, 4H), 7.25-7.21 (m, 4H), 6.15-5.89 (m, 5H), 5.61 (dd, J=52.0, 5.1 Hz, 1H), 5.55 (ddd, J=51.2, 4.7, 2.7 Hz, 1H) 5.51-5.42 (m, 1H), 5.31-5.22 (m, 2H), 5.11 (dd, J=3.9, 9.8 Hz, 2H), 5.04-4.95 (m, 4H), 4.55-4.37 (m, 7H), 4.29-4.12 (m, 3H)

Compound 106 (RpRp) ¹ H NMR (400 MHz, CDCl ₃ ) δ=8.65 (s, 2H), 8.06 (dd, J=1.4, 8.0 Hz, 2H), 7.98 (s, 2H), 7.57-7.52 (m , 6Н), 7.47-7.32 (m, 6Н), 7.25-7.21 (m, 4Н), 6.15 (d, J=18.7 Hz, 2Н), 6.09-5.99 (m, 2Н), 5.82-5.76 (m, 2Н ), 5.60 (dd, J=51.8, 4.9 Hz, 2Н), 5.27 (dd, J=1.2, 17.2 Hz, 2Н), 5.12 (dd, J=1.0, 10.4 Hz, 2Н), 5.06-4.96 (m, 4H), 4.55-4.40 (m, 4H), 4.36-4.24 (m, 4H), 4.21-4.02 (m, 2H)

Preparative HPLC conditions:

Stage F

(519 mL) added 2nd generation Hoveid-Grubbs™ catalyst ((1,3-bis-(2,4,6-trimethylphenyl)-2-imidazolidinylidene)dichloro(ortho-isopropoxyphenylmethylene)ruthenium; available from SIGMA-ALDRITCH® catalog no. 569755; CAS 301224-40-8; 91 mg, 0.15 mmol, 0.35 eq) and quinone (0.102 ml, 1.243 mmol, 3.0 eq). The mixture was heated to reflux temperature and the progress of the reaction was monitored by LC/MS. After 3 hours additional catalyst (91 mg, 0.15 mmol, 0.35 eq.) was added and the reaction continued for another 3 hours. After cooling, the mixture was treated with DMSO (0.59 ml, 8.3 mmol, 20 eq.) at ambient temperature for 15 hours, concentrated in vacuo and purified by silica gel column chromatography (SiO ₂ 25 g, 66% ethyl acetate in n -heptane to 100% ethyl acetate) to give compound 107 (200 mg, 0.163 mmol, 39% yield) as a brown dry foam.

¹ H NMR (400 MHz, CDCl ₃ ) δ=8.19 (s, 1H), 8.12 (dd, J=7.8 Hz, 1.9 Hz, 1H), 8.10 (s, 1H), 8.02 (d, J=8.2 Hz, 1H), 7.89 (s, 1H), 7.63 (br d, J=7.0 Hz, 1H), 7.53-7.41 (m, 10H), 7.35-7.30 (m, 2H), 7.25-7.20 (m, 4H), 6.23 (d, J=17.6 Hz, 1H), 6.14 (d, J=18.8 Hz, 1H), 5.86-5.75 (m, 1H), 5.75 (dt, J=15.3, 5.0 Hz, 1H), 5.67 (dt , J=15.3, 4.7 Hz, 1H), 5.60 (dd, J=52.0, 3.9 Hz. 1H), 5.48 (dd, J=50.4, 3.9 Hz. 1H) 5.50-5.39 (m, 1H), 4.91-4.64 (m, 4H), 4.57-4.25 (m, 9H), 4.15 (d, J=7.03 Hz, 1H), 4.11 (d, J=7.03 Hz, 1H).

Stage G

thiophenol (0.88 ml, 8.55 mmol, 119 eq.) and triethylamine (0.88 ml, 6.31 mmol, 88 eq.) were added. The resulting mixture was stirred at ambient temperature. After completion of the reaction (observed by LC/MS, 13 hours), methanol (5.28 ml) and 28% ammonium hydroxide (3.52 ml) were added and the resulting mixture was heated to 50°C. After completion of the reaction (observed by LC/MS, 5 hours), the mixture was cooled to ambient temperature and the resulting brownish slurry was filtered and washed with water (15 ml). The filtrate was again filtered to remove additional solids. The final filtrate was extracted twice with a 1:1 mixture of toluene and heptane (30 ml). The aqueous layer was concentrated in vacuo and then resuspended in water (6 ml). The resulting solid was filtered off and the filtrate was subjected to preparative HPLC to give compound 1 diammonium salt (also referred to as compound 1a) (39 mg, 0.050 mmol, 70% yield) as a white solid.

Compound 1a (SpRp, trans) ¹ H NMR (400 MHz, CD ₃ OD) δ=9.05 (s, 1H), 8.33 (s, 1H), 8.25 (s, 1H), 8.12 (s, 1H), 6.34 ( br s, 2Н), 5.88 (br s, 2H), 5.66 (br d, J=51.6 Hz, 1H), 5.59 (br d, J=52.2 Hz, 1H) 5.01 (br s, 2H), 4.68-4.34 (m, 6H), 4.07-3.82 (m, 2H), 3.79-3.55 (m, 2H); ³¹ P NMR (162 MHz, CD ₃ OD) δ=55.48 (s, 1P), 55.16 (s, 1P).

Compound 1a. Preparative HPLC conditions:

Example 1.1 - Alternative synthesis for compound 1a

An alternative synthesis route for compound 1a is shown in FIG. 2A and FIG. 2B, as well as in FIG. 2C and presented below.

Stage 1

Compound 129 (570 g, 1.53 mol, 1 wt, 1 vol, 1 eq) was dissolved in pyridine (2.85 L, 35.2 mol, 4.89 wt, 5.0 vol). , 23 eq.). The mixture was cooled to 2.6°C and treated with 4,4'-dimethoxytrityl chloride (DMTCl; 543 g, 1.60 mol., 0.953 wt., 1.05 eq.). The mixture was stirred at 0-5° C. for 2 hours and then allowed to warm to ambient temperature. The reaction was monitored by LC/MS and complete conversion was confirmed after stirring overnight. The reaction mixture was cooled below 5°C and quenched by treatment with MeOH (124 ml, 3.05 mol., 0.172 wt., 0.217 vol., 2.0 eq.) for 15 minutes. The mixture was co-evaporated with touol (2.00 L, 3.04 wt, 3.51 vol) in vacuo and then diluted with a mixture of EtOAc (2.850 L, 4.5 wt, 5.0 vol) and n- heptane (2.85 L, 3.42 wt., 5.0 vol.). The organic layer was washed with saturated NaHCO ₃ (9 wt.% solution in water; 2.0 l, 3.5 vol.). Additional EtOAc (2.85 L, 4.5 wt., 5.0 vol.) was added to completely dissolve the crude product. After stirring for 5 minutes, the two layers were separated. The organic layer was washed with water (2.0 L, 3.5 wt., 3.5 vol.). The solid began to slowly precipitate from the organic layer. The aqueous layer was separated. The organic layer was then concentrated to approximately 1 vol. The crude product was slurried with a mixture of n-heptane (2.00 L, 2.40 wt., 3.51 vol.) and toluene (0.50 L, 0.76 wt., 0.88 vol.). After stirring for 15 minutes, a pale yellow solid was collected by vacuum filtration. The filter cake was washed successively with: (1) a mixture of n-heptane (0.60 L, 0.72 wt., 1.05 vol.) and toluene (0.30 L, 0.46 wt., 0.53 vol.) and then (2) n-heptane (3.00 L, 3.6 wt., 5.26 vol.). The solid was dried without heat for 30 minutes and then transferred to drying trays at 50° C. in a vacuum oven overnight to give compound 130 as a pale yellow solid (996.7 g, 1.47 mol, 1 .75 wt., 97% yield).

¹ H NMR (400 MHz, CHLOROFORM-d) δ=8.99 (s, 1H), 8.76 (s, 1H), 8.21 (s, 1H), 8.04-8.00 (m, 2H), 7.64-7.59 (m, 1H ), 7.57-7.50 (m, 2Н), 7.41-7.36 (m, 2Н), 7.32-7.15 (m, 7Н), 6.83-6.76 (m, 4Н), 6.31 (dd, J=2.5, 17.0 Hz, 1H ), 5.68 (ddd, J=2.3, 4.7, 52.7 Hz, 1H), 4.88-4.77 (m, 1H), 4.26-4.21 (m, 1H), 3.77 (s, 6H), 3.57 (dd, J=3.1 , 10.9 Hz, 1H), 3.43 (dd, J=4.1, 10.7 Hz, 1H), 2.60 (br s, 1H)

Stage 1'

Compound 129 (430 g, 1.15 mol, 1 wt, 1 vol, 1 eq) and imidazole (118 g, 1.73 mol, 0.274 wt, 1.50 eq) were dissolved in DMF ( 1.72 l, 3.78 wt., 4.0 vol.) and the resulting mixture was cooled to 5°C. TBS-Cl (191 g, 1.27 mol, 0.444 wt, 1.10 eq) was added. The mixture was stirred at 0 to 11° C. for 2 h, allowed to warm slowly to ambient temperature (progress was monitored by LCMS). The reaction was completed 6 h after the addition of TBS-Cl, stirred at ambient temperature for another 20 h. The mixture was cooled to 2°C and treated with methanol (93 ml, 74 g, 2.3 mol., 0.17 wt. ,22 wt., 2.0 eq.) for 10 minutes. The reaction mixture was diluted with a mixture of MTBE (1.72 L, 1.23 kg, 2.96 wt., 4.0 vol.) and EtOAc (1.72 L, 1.55 kg, 3.60 wt., 4.0 vol.), and then saturated NH ₄ Cl (28 wt.% solution in water; 2.15 l, 5.0 vol.). Solids slowly began to fall out of solution. The mixture was allowed to warm to 24° C. and water (1.08 L, 1.08 kg, 2.5 wt., 2.5 vol.) was added thereto (T-internal = 22° C.). More solids began to precipitate out of the mixture. Additional water (1.08 L, 1.08 kg, 2.5 wt., 2.5 vol.) and MTBE (1.40 L, 1.04 kg, 2.4 wt., 3.0 wt.) were added to the mixture. 3 rev.). An off-white solid was collected by vacuum filtration. The reactor was washed with water (320 mL, 0.74 vol) followed by MTBE (1.80 L, 1.33 kg, 3.10 wt, 4.19 vol) to transfer any remaining solid to the filter. The filter cake was washed successively with: (1) water (1.80 L, 1.80 kg, 4.2 wt., 4.2 vol.), (2) water (1.80 L, 1.80 kg, 4. 2 wt., 4.2 vol.), (3) a mixture of MTBE (0.90 l, 0.67 kg, 1.5 wt., 2.1 vol.) and n-heptane (0.90 l, 0 .62 kg, 1.4 wt., 2.1 vol.), (4) a mixture of MTBE (0.90 l, 0.67 kg, 1.5 wt., 2.1 vol.) and n-heptane ( 0.90 l, 0.62 kg, 1.4 wt., 2.1 vol.). The recovered solid was dried in vacuo at 40° C. for 2 days to give compound 133 as a white solid (483 g, 0.991 mol, 1.12 wt, 86% yield).

¹ H NMR (400 MHz, CHLOROFORM-d) δ=8.97 (s, 1H), 8.82 (s, 1H), 8.36 (s, 1H), 8.04-8.00 (m, 2H), 7.64-7.58 (m, 1H ), 7.56-7.51 (m, 2Н), 6.40 (dd, J=2.3, 16.0 Hz, 1H), 5.45 (ddd, J=2.7, 4.3, 53.1 Hz, 1H), 4.75-4.66 (m, 1H), 4.22-4.17 (m, 1H), 4.07 (dd, J=2.3, 11.7 Hz, 1H), 3.91 (dd, J=2.7, 11.7 Hz, 1H), 2.38 (dd, J=2.7, 7.0 Hz, 1H) , 0.92 (s, 9Н), 0.11 (s, 3Н), 0.11 (s, 3Н).

Stage 2

Compound 130 (993 g, 1.47 mol, 1 wt, 1 vol, 1 eq) and imidazole (150 g, 2.20 mol, 0.151 wt, 1.5 eq) were dissolved in DMF ( 3.48 l, 3.28 kg, 3.3 wt., 3.5 vol.) and the mixture was cooled to 5°C. TBS-Cl (244 g, 1.62 mol, 0.245 wt, 1.10 eq) was added. The reaction mixture was stirred at 0 to 5° C. for 2 h, allowed to warm slowly to ambient temperature and monitored by LCMS. After 17 hours, additional imidazole (100 g, 1.47 mol, 0.10 wt, 1.0 eq) and TBS-Cl (111 g, 735 mmol, 0.112 wt, 0.50 eq) were added. and stirring was continued at ambient temperature for 2 hours and at 35°C for 2 hours. The resulting mixture was cooled to 13.6°C and treated with MeOH (119 ml, 2.94 mol., 2 eq.) for 10 minutes. Ice (5 kg, 5 wt.) and saturated NH ₄ Cl (28 wt.% solution in water; 5.0 L, 5 vol.) were added to a separate reactor. The reaction mixture was added to ice/NH ₄ Cl. An off-white solid immediately began to precipitate out of solution. An additional 2 kg of ice (2 kg, 2 wt.) and water (3.0 L, 3 vol.) were added to the mixture. The reaction vessel was washed with water (0.50 L, 0.5 vol) and a wash sample was added to the mixture. N-heptane (2.00 L, 2 vol) was added to the mixture and stirring was continued for 10 minutes. An off-white solid was collected by vacuum filtration. The filter cake was washed with: (1) water (4.0 L, 4.0 vol.), (2) water (4.0 L, 4.0 vol.), (3) n-heptane (4.0 L, 4.0 vol.), (4) n-heptane (4.0 L, 4.0 vol.). The recovered solid was dried in vacuo at 45° C. for 4 days to give compound 131 as an off-white solid (1.095 kg, 1.39 mol, 1.10 wt, 94% yield).

¹ H NMR (400 MHz, CHLOROFORM-d) δ=9.09 (s, 1H), 8.78 (s, 1H), 8.28 (s, 1H), 8.02 (d, J=7.4 Hz, 2H), 7.63-7.59 ( m, 1H), 7.55-7.50 (m, 2H), 7.37 (d, J=7.1 Hz, 2H), 7.29-7.17 (m, 7H), 6.79 (d, J=7.9 Hz, 4H), 6.29 (dd , J=2.9, 16.2 Hz, 1H), 5.60 (ddd, J=2.7, 3.9, 53.1 Hz, 1H), 4.78 (ddd, J=4.7, 6.4, 15.8 Hz, 1H), 4.26-4.22 (m, 1H ), 3.77 (s, 6Н), 3.58 (dd, J=3.1, 10.9 Hz, 1H), 3.26 (dd, J=3.7, 10.7 Hz, 1H), 0.85 (s, 9Н), 0.10 (s, 3Н) , 0.02 (s, 3H)

Stage 3

Compound 131 (1000 g, 1.27 mol, 1 wt, 1 vol, 1 eq) and trans-2-butene-1,4-diol (olefin geometry confirmed by ¹ H-NMR; 335 g, 3 ,80 mol 0.335 wt, 3.0 eq) was azeotroped twice with THF (3.0 L, 3.0 vol). The residue was dissolved in a mixture of THF (10 L, 10 vol.) and toluene (15 L, 15 vol.). Triphenylphosphine (432 g, 1.65 mol., 0.432 wt., 1.3 eq.) was added, and then the reaction mixture was cooled to -5°C. DIAD (0.320 L, 1.65 mol, 333 g, 0.333 wt, 0.320 vol, 1.3 eq) was added slowly over 20 minutes while maintaining T-internal below 5°C. The reaction mixture was stirred at 0-5°C for 1 h and observed using LCMS. The ice bath was removed and the mixture was allowed to warm to rt. After stirring overnight (17 h), triphenylphosphine (83 g, 0.32 mol, 0.083 wt, 0.25 eq) and DIAD (62 ml, 0.32 mol, 64 g, 0.064 wt, 0.062 vol., 0.25 eq.). After an additional 1 hour at room temperature the reaction mixture was diluted with MTBE (10 L, 10 vol.), washed twice with half-saturated NaCl (18 wt.% solution in water; 2×4 L) and concentrated in vacuo to a thick oil. The mixture was redissolved in a mixture of MTBE (4.00 L, 4 vol) and n-heptane (0.50 L, 0.5 vol) and then cooled to 0°C. A seed crystal of triphenylphosphine oxide was added to the solution. Solids slowly began to precipitate out of solution and stirred overnight. The white solid was collected by vacuum filtration and washed with MTBE (2 L, 2 vol) to isolate 540 g of triphenylphosphine oxide. The filtrate was concentrated and purified on Biotage 150L KP-Sil (SiO ₂ 5 kg; pre-treated with 1% TEA in Hep/etOAc; eluents: heptane/EtOAc (48 L 33% EtOAc with 1% TEA, 24 L 50% EtOAc with 1% TEA, 24 L 66% EtOAc with 1% TEA) → 100% EtOAc with 1% TEA). The column was monitored by TLC (2:1 EtOAc/n-heptane). The pure product fractions were combined and concentrated in vacuo to give compound 132 as a pale white foamy solid (634 g containing 14 wt% DIAD of the resulting by-product, pure 545 g, 0.63 mol, 50% calculated yield). The mixture fractions were combined and concentrated in vacuo to give a pale yellow foamy solid (750 g) which was repurified with Biotage 150M HP-Sphere (2.5 kg SiO ₂ ; pre-treated with 1% TEA in Hep/EtOAc; loaded toluene sample, eluents: Hep/EtOAc/1% TEA (12 L 50% EtOAc with 1% TEA, 16 L 66% EtOAc with 1% TEA) → EtOAc with 1% TEA). The column was monitored by TLC (2/1/0.03 EtOAc/n-hep/TEA). Pure product fractions were combined and concentrated in vacuo to give additional compound 132 as a pale white foamy solid (206 g, 0.24 mol, 18% yield).

¹ H NMR (400 MHz, CHLOROFORM-d) δ=8.58 (s, 1H), 8.10 (s, 1H), 7.43-7.37 (m, 2H), 7.32-7.28 (m, 2H), 7.24-7.15 (m , 8Н), 7.03-6.98 (m, 2Н), 6.78-6.73 (m, 4Н), 6.18 (dd, J=2.7, 17.2 Hz, 1H), 5.88 (td, J=5.5, 15.6 Hz, 1H), 5.77 (td, J=5.1, 15.6 Hz, 1H), 5.60 (ddd, J=2.7, 4.3, 53.1 Hz, 1H), 5.03-4.96 (m, 2H), 4.91 (ddd, J=4.5, 6.6, 16.6 Hz, 1H), 4.18-4.14 (m, 1H), 3.88-3.82 (m, 2H), 3.78 (s, 6H), 3.52 (dd, J=2.7, 10.9 Hz, 1H), 3.14 (dd, J= 3.5, 10.9 Hz, 1H), 0.85 (s, 9H), 0.10 (s, 3H), 0.01 (s, 3H).

Stage 4

1,07 мол., 0,652 масс., 1,15 экв.) азеотропически сушили при помощи THF (2×3 л, 2×3,8 об.) и повторно растворяли в THF (9,60 л, 8,45 кг, 12,0 об.) при к.т. Трифенилфосфин (317 г, 1,21 мол., 0,396 масс., 1,30 экв.) добавляли и смесь охлаждали ниже -5°С. Добавляли DIAD (226 мл, 1,16 мол., 235 г, 0,294 масс., 0,283 об., 1,25 экв.), Т-внутренняя ниже 7°С. Реакционную смесь оставляли нагреваться до к.т. медленно. За реакцией наблюдали при помощи LCMS. Через 21 ч реакционную смесь концентрировали в вакууме до густого масла, азеотропировали н-гептаном (2,00, 1,37 кг, 1,71 масс., 2,50 об.), а затем повторно растворяли в смеси МТВЕ (2,40 л, 1,78 кг, 2,2 масс., 3,0 об.) и н-гептана (800 мл, 547 г, 0,68 масс., 1,0 об.). Раствор затравляли трифенилфосфиноксидом и охлаждали до 5°С, разбавляли н-гептаном (400 мл, 274 г, 0,34 масс., 0,50 об.) и перемешивали при 5°С в течение 30 минут. Осадок белого твердого вещества собирали вакуумной фильтрацией и промывали 2:1 (объем/объем) смесью МТВЕ и н-гептана (1,8 л) с получением трифенилфосфиноксида (455 г). Фильтрат концентрировали в вакууме и очищали при помощи Biotage 150 L KP-Sil (SiO2 5 кг; предварительно обрабатывали 1% TEA; загружали пробы растворением в толуоле, элюенты: 9:1 гептан/EtOAc (16 л) и 15 TEA, 3.6:1 (46 л), 2:1 (20 л) и 1% TEA, 1:1 (30 л) и 1% TEA и 100% EtOAc (16 л) и 1% TEA). Объединенные чистые фракции продукта концентрировали в вакууме с получением соединения 134 в виде грязно-белой твердой пены (662,2 г). Фракции смеси объединяли и концентрировали в вакууме (480 г). Белое нерастворимое твердое вещество, образованное разбавлением толуолом (300 мл) перед загрузкой в Biotage 150L, удаляли вакуумной фильтрацией. Вещество, растворимое в толуоле, очищали при помощи Biotage 150М HP-Sphere (SiO₂ 2,5 кг (предварительно обработанное 1% TEA); загрузка пробы при помощи толуола; элюенты: 2:1 гептан/EtOAc (26 л) масс/ 1% TEA, 1:1 (25 л) масс/ 1% TEA, 1:4 (34 л) масс/ 1% TEA). За колонкой наблюдали при помощи TLC (1:1 гептан/EtOAc). Объединенные чистые фракции продукта концентрировали в вакууме с получением дополнительного соединения 134 в виде грязно-белой твердой пены (165,5 г. Всего 662,2+165,5 г=827,7 г, 930 ммоль, 1,03 масс., 67% выход).

1.07 mol, 0.652 wt, 1.15 eq) was azeotropically dried with THF (2 x 3 L, 2 x 3.8 vol) and redissolved in THF (9.60 L, 8.45 kg , 12.0 vol.) at r.t. Triphenylphosphine (317 g, 1.21 mol., 0.396 wt., 1.30 eq.) was added and the mixture was cooled below -5°C. DIAD (226 ml, 1.16 mol, 235 g, 0.294 wt, 0.283 vol, 1.25 eq) was added, t-int. below 7°C. The reaction mixture was allowed to warm to rt. slowly. The reaction was monitored by LCMS. After 21 hours, the reaction mixture was concentrated in vacuo to a thick oil, azeotroped with n-heptane (2.00, 1.37 kg, 1.71 wt., 2.50 vol.), and then redissolved in a mixture of MTBE (2.40 l, 1.78 kg, 2.2 wt., 3.0 vol.) and n-heptane (800 ml, 547 g, 0.68 wt., 1.0 vol.). The solution was seeded with triphenylphosphine oxide and cooled to 5°C, diluted with n-heptane (400 ml, 274 g, 0.34 wt., 0.50 vol.) and stirred at 5°C for 30 minutes. The white solid precipitated was collected by vacuum filtration and washed with a 2:1 (v/v) mixture of MTBE and n-heptane (1.8 L) to give triphenylphosphine oxide (455 g). The filtrate was concentrated in vacuo and purified with Biotage 150 L KP-Sil (SiO2 5 kg; pre-treated with 1% TEA; samples were loaded by dilution in toluene, eluents: 9:1 heptane/EtOAc (16 L) and 15 TEA, 3.6:1 (46 L), 2:1 (20 L) and 1% TEA, 1:1 (30 L) and 1% TEA and 100% EtOAc (16 L) and 1% TEA). The combined pure product fractions were concentrated in vacuo to give compound 134 as an off-white solid foam (662.2 g). Fractions of the mixture were combined and concentrated in vacuo (480 g). A white insoluble solid formed by dilution with toluene (300 ml) before loading into Biotage 150L was removed by vacuum filtration. Toluene soluble material was purified with Biotage 150M HP-Sphere (SiO ₂ 2.5 kg (pre-treated with 1% TEA); sample loading with toluene; eluents: 2:1 heptane/EtOAc (26 L) w/1 % TEA, 1:1 (25 L) w/ 1% TEA, 1:4 (34 L) w/ 1% TEA). The column was monitored by TLC (1:1 heptane/EtOAc). The combined pure product fractions were concentrated in vacuo to give additional compound 134 as an off-white solid foam (165.5 g. Total 662.2 + 165.5 g = 827.7 g, 930 mmol, 1.03 wt., 67 % exit).

¹ H NMR (400 MHz, CHLOROFORM-d) δ=8.47 (s, 1H), 8.39 (s, 1H), 8.20 (s, 1H), 8.01 (s, 1H), 7.38-7.31 (m, 5H), 7.27-7.19 (m, 6H), 7.14-7.06 (m, 3H), 6.93-6.87 (m, 2H), 6.76 (d, J=8.6 Hz, 4H), 6.26 (dd, J=2.0, 16.0 Hz, 1H), 6.15 (dd, J=2.7, 17.2 Hz, 1H), 5.86 (dd, J=4.7, 15.2 Hz, 1H), 5.80 (dd, J=4.7, 15.2 Hz, 1H), 5.51 (ddd, J =2.7, 4.3, 52.8 Hz, 1H), 5.31 (ddd, J=2.0, 4.3, 52.8 Hz, 1H), 4.87 (d, J=4.7 Hz, 2H), 4.85-4.81 (m, 1H), 4.79 ( d, J=4.3 Hz, 2H), 4.71-4.59 (m, 1H), 4.20-4.13 (m, 2H), 4.06 (dd, J=2.7, 11.3 Hz, 1H), 3.90 (dd, J=2.7, 11.7 Hz, 1H), 3.77 (s, 6H), 3.52 (dd, J=3.1, 10.9 Hz, 1H), 3.18 (dd, J=3.9, 10.9 Hz, 1H), 0.92 (s, 9H), 0.84 ( s, 9H), 0.10 (s, 3H), 0.09 (s, 6H), 0.07 (s, 3H)

Stage 5-6

To a solution of compound 134 (410.7 g, 309 mmol, 1 wt., 1 vol., 1 eq.) in pyridine (1.23 L, 1.21 kg, 15.2 mol., 2.9 wt., 3, 0 vol., 49 eq.) diphenyl phosphite (90 ml, 109 g, 0.46 mol., 0.26 wt., 0.22 vol., 1.5 eq.) was added. The reaction mixture was stirred at rt. and observed with LCMS. After 2 hours (80% conversion), additional diphenyl phosphite (29.9 ml, 36.2 g, 155 mmol, 0.088 wt., 0.073 vol., 0.50 eq.) was added. After an additional 1 hour, additional diphenyl phosphite (6.0 ml, 7.2 g, 31 mmol, 0.018 wt., 0.015 vol., 0.10 eq.) was added and the reaction was continued for another 0.5 h (98% conversion). The reaction mixture was added to a mixture of saturated NaHCO ₃ (9 wt.% solution in water; 2.1 l, 5 vol.) and water (1.0 l ml, 2.5 vol.) while maintaining T-internal 4.7- 12°C. The reactor was washed with a small volume of EtOAc. Stirring was continued at rt. for 30 minutes and the reaction was monitored by LCMS (100% conversion). The reaction mixture was extracted twice with a 1:1 mixture of EtOAc and MTBE (2×8.2 L, 2×20 vol.). The combined organic layers were washed with water (4.1 L, 10 vol), concentrated in vacuo and azeotroped with toluene (3×4.1 L, 3×10 vol; continuous feed) to remove pyridine to give compound 135 (0.55 equivalent of the remaining pyridine).

Step 6 - Crude compound 135 was dissolved in dichloromethane (3.08 L, 4.07 kg, 9.9 wt., 7.5 vol.) at ambient temperature. Water (55.7 ml, 0.136 vol., 10 eq.) was added followed by dichloroacetic acid solution (77 ml, 120 g, 0.93 mol., 0.29 wt., 0.19 vol., 3.0 equiv.) in DCM (3.08 L, 7.5 vol.) while maintaining internal T below 25°C. (turned into an orange solution). Triethylsilane (Et ₃ SiH; 494 mL, 359 g, 3.09 mol, 0.875 wt, 1.20 vol, 10.0 eq) was added after 30 min (T-int. changed from 18.2°C to 17°C) and stirring was continued for 20 minutes. Triethylamine (431 ml, 313 g, 3.09 mol., 0.762 wt., 1.05 vol., 10.0 eq.) was added (T-internal changed from 17.8°C to 22°C). The mixture was concentrated to 1.55 kg (3.8 wt), redissolved in EtOAc (6.2 L, 5.5 kg, 14 wt, 15 vol), washed successively with: (1) water (1.0 L , 2.5 vol.) and saturated NaHCO ₃ (9 wt.% solution in water, 0.82 l, 2.0 vol.). The crude EtOAc product solution was stored at -20°C overnight (0.82 L, 2.0 vol.), and the next day the solution was concentrated in vacuo at 25°C. The crude mixture thus obtained (654 g) was triturated with: (1) n-heptane (3.01 L, 7.5 vol.), (2) n-heptane mixture (2.46 L, 6.0 vol. .) and toluene (0.82 l, 2.0 vol.). Part of the solution (supernatant) was decanted and the remaining solid at the bottom was dissolved in acetonitrile (4.1 L, 10 vol.). The mixture was concentrated in vacuo at 25°C and azeotroped with acetonitrile twice to give compound 136. The product was used in the next step without purification (allowable theoretical 100% yield).

Stage 7

Step 7a Compound 136 (337 g, 309 mmol, 1 wt, 1 vol, 1 eq) was dissolved in anhydrous pyridine (13.5 L, 13.2 kg, 39 wt, 40 vol) at RT . Triethylamine (129 ml, 927 mmol, 94 g, 0.28 wt., 0.38 vol., 3.0 eq.) was added, followed by 2-chloro-5,5-dimethyl-1,3,2-dioxophosphinane 2-oxide (DMOCP; 103 g, 556 mmol, 0.31 wt., 1.80 eq.). The resulting mixture was stirred at ambient temperature for 30 minutes and observed by LCMS (100% conversion) to form compound 137.

Step 7b TEA (129 ml, 927 mmol, 94 g, 0.28 wt., 0.38 vol., 3.0 eq.), water (100 ml, 5.56 mol., 0.30 wt., 0 30 wt., 18 eq.) and sulfur (34.7 g, 1.08 mol., 0.10 wt., 3.5 eq.) were added to the above mixture of compound 137. After 90 minutes (100% conversion) added NaHCO ₃ (9 wt.% solution in water; 3.37 l, 10 vol.) while maintaining the T-internal below 30°C (16.6°C-27°C). The resulting mixture was filtered to remove salts. The filtrate was concentrated in the mixture in vacuo, diluted with MTBE (5.1 L, 15 vol.) and washed twice with NaCl (30 wt.% solution in water; 2×1.35 L, 2×4 vol.). Insoluble solids were filtered off and the filtrate was concentrated in vacuo and azeotroped with toluene (4.0 L, 12 vol). The resulting solid was removed by filtration and the crude mixture was dissolved in toluene and purified with Biotage 150L KP-Sil (SiCh 5 kg; pre-treated with Hep/EtOAc/TEA (1.5/1.5/0.03 CV); eluted: EtOAc/TEA (3/0 .03 CV), EtOAc/MeOH/TEA (4/0.2/0.04 CV), EtOAC/MeOH/TEA (2/0.2/0.02CV) 3a column was monitored by TLC (EtOAC/MeOH/ TEA = 9/1/0.1) Fractions containing the Sp isomer were combined and concentrated in vacuo to give compound 138 as a light pink foamy solid (Sp isomer; 154 g, 128 mmol, 0.46 wt., 41.3% yield = 9/1/0.1). Fractions containing the Rp isomer were combined and concentrated in vacuo to give compound 240 as a light pink foamy solid (Rp isomer; 64 g, 53 mmol, 0.19 wt., 17% yield).

Compound 138 (Sp isomer):

¹ H NMR (400 MHz, CHLOROFORM-d) δ=8.51 (s, 1H), 8.50 (s, 1H), 8.22 (s, 1H), 8.14 (s, 1H), 7.49-7.44 (m, 2H), 7.38-7.27 (m, 4H), 7.25-7.21 (m, 2H), 7.14 (t, J=7.1 Hz, 2H), 6.44 (dd, J=2.5, 13.9 Hz, 1H), 6.18 (d, J= 15.2Hz, 1H), 5.78 (td, J=6.3, 15.6Hz, 1H), 5.69 (td, J=4.7, 15.6Hz, 1H), 5.56 (dd, J=3.9, 50.8Hz, 1H), 5.20- 5.06 (m, 1H), 4.95-4.79 (m, 4H), 4.69 (dd, J=4.3, 16.0 Hz, 1H), 4.54-4.38 (m, 3H), 4.35 (d, J=5.5 Hz, 1H) , 4.32-4.29 (m, 1H), 4.05 (dd, J=1.6, 11.7 Hz, 1H), 3.91 (dd, J=3.1, 11.7 Hz, 1H), 3.14-3.06 (m, 6H), 1.30 (t , J=7.4 Hz, 9Н), 0.91 (s, 9Н), 0.90 (s, 9Н), 0.12 (s, 3Н), 0.08 (s, 3Н), 0.06 (s, 3Н), 0.05 (s, 3Н)

Compound 240 (Rp isomer):

¹ H NMR (400 MHz, CHLOROFORM-d) δ=8.54 (s, 1H), 8.38 (s, 1H), 8.33 (s, 1H), 8.01 (s, 1H), 7.39-7.09 (m, 10H), 6.39 (dd, J=2.3, 14.1Hz, 1H), 6.13 (d, J=17.2Hz, 1H), 5.72 (d, J=3.1Hz, 2H), 5.68 (dd, J=4.3, 51.2Hz, 1H ), 5.43-5.29 (m, 1H), 5.10-4.96 (m, 3H), 4.90-4.83 (m, 2H), 4.78-4.72 (m, 1H), 4.52 (ddd, J=3.9, 6.6, 17.2 Hz , 1H), 4.44-4.35 (m, 2H), 4.31-4.26 (m, 1H), 4.20-4.12 (m, 2H), 3.87 (dd, J=3.5, 11.7 Hz, 1H), 3.79-3.77 (m , 1H), 3.15-3.09 (m, 6H), 1.33 (t, J=7.4 Hz, 9H), 0.94 (s, 9H), 0.89 (s, 9H), 0.13 (s, 3H), 0.12 (s, 3H), 0.10 (s, 3H), 0.09 (s, 3H)

Stage 8

Compound 138 (221 g, 183 mmol, 1 wt., 1 vol., 1 eq.) was dissolved in a mixture of pyridine (530 ml, 6.56 mol., 519 g, 2.3 wt., 2.4 vol.) and TEA (2.65 L, 19.0 mole, 1.93 kg, 8.7 wt., 12 vol., 104 eq.). Triethylamine trihydrofluoride (264 mL, 1.62 mol, 262 g, 1.2 wt, 1.2 vol, 8.9 eq as complex, 27 eq HF) was added and the mixture was stirred at rt. , while the transformation was monitored by LCMS. After 3 hours (97% conversion), methoxytrimethylsilane (TMSOMe; 1.40 L, 10.2 mol, 1.06 kg, 4.8 wt, 6.3 vol, 55 eq) was added and stirring was continued for 30 minutes. Reactor with sticky hard coating. Part of the solution (supernatant) was decanted. The solid was triturated twice with toluene (2 x 2.2 L, 2 x 10 vol; supernatant decanted). The crude solid remaining in the reactor was dissolved in dichloromethane (2.2 L, 10 vol.) and washed with NH ₄ Cl (28 wt.% solution in water; 2.2 L, 10 vol.). The aqueous layer was back extracted with dichloromethane (2.2 L, 10 vol). The combined organic layers were washed with a mixture of NaCl (36 wt.% solution in water; 1.1 L, 5 vol.) and water (1.1 L, 5 vol.), and then concentrated in vacuo to obtain compound 139 as a brown dry foam (152 g, 155 mmol, 0.70 wt., 85% yield). The crude product was carried to the next step without purification.

Stage 9

Compound 139 (150 g, 153 mmol, 1 wt, 1 vol, 1 eq) was azeotroped with acetonitrile (4 L, 27 vol) and then redissolved in acetonitrile (1.05 L, 0.83 kg, 5.5 wt., 7.0 vol.) at r.t. 2-Nitrobenzyl bromide (44.4 g, 205 mmol, 0.30 wt., 1.34 eq.) was added at rt. and the reaction was monitored by LCMS. After 23 h (100% conversion) EtOAc (1.50 L, 10 vol.), NH ₄ Cl (28 wt.% solution in water; 300 ml, 2 vol.) and water (300 ml, 2 vol.) were added (pH=6) and the resulting mixture was partially concentrated in vacuo at 25°C to a weight of 1.11 kg. EtOAc (2.25 L, 15 vol) was added and the mixture was stirred for 5 minutes. The two layers were separated. The aqueous layer was extracted with ethyl acetate (750 ml, 5 vol). The combined organic layers were washed successively with: (1) a mixture of NaCl (36 wt.% solution in water; 300 ml, 2 vol.) and water (300 ml, 2 vol.) and (2) water (600 ml, 4 vol.) . The organic layer was then concentrated in vacuo and azeotroped with n-heptane (1.50 L, 10 vol). MTBE (0.95 L, 6.3 vol.) was added to the crude solid and the mixture was heated at 40°C. The mixture was diluted with EtOAc (300 ml, 2 vol) and slowly cooled to 0°C. The dense solid was allowed to settle and the supernatant was siphoned off through a fritted filter tube. The solid was washed twice with MTBE (2 x 300 mL, 2 x 2 vol; supernatant was siphoned off through a fritted filter tube each time) and dried in vacuo at 40° C. overnight to give compound 140 as a pale yellow solid (156 g ). The filtrate was concentrated in vacuo to give a brown oil (17.8 g) which was purified with Biotage Snap-Ultra 340 g (eluents: 0-5% MeOH in EtOAc) to give additional compound 140 as a pale yellow solid ( 5.8 g). Total 156 g + 5.8 g = 161.8 g (pure 152 mmol, 95% pure, 99% yield)

¹ H NMR (400 MHz, CHLOROFORM-d) δ=8.46 (s, 1H), 8.15 (s, 1H), 8.10 (s, 1H), 8.09-8.06 (m, 1H), 7.89 (s, 1H), 7.54-7.51 (m, 1H), 7.49-7.45 (m, 4H), 7.37-7.28 (m, 3H), 7.24-7.19 (m, 3H), 7.16-7.11 (m, 2H), 6.22 (d, J =16.8 Hz, 1H), 6.14 (dd, J=2.7, 17.2 Hz, 1H), 5.83-5.61 (m, 3H), 5.60-5.48 (m, 1H), 5.07 (dd, J=3.5, 51.6 Hz, 1H), 5.06-4.96 (m, 1H), 4.79 (dd, J=4.9, 15.8 Hz, 1H), 4.69 (d, J=5.9 Hz, 2H), 4.67-4.56 (m, 1H), 4.48-4.40 (m, 3H), 4.37-4.30 (m, 1H), 4.27 (d, J=5.9 Hz, 2H), 4.19-4.13 (m, 1H), 3.93-3.85 (m, 1H), 3.85-3.78 (m , 1Н)

Stage 10-11

об., 1 экв.) и 2-цианоэтил-N,N,N',N'-тетраизопропилфосфородиамидит (25,3 мл, 79,5 ммоль, 0,33 масс., 0,35 об., 1,10 экв.) азеотропировали с безводным ацетонитрилом трижды (3×2 л), повторно растворяли в дихлорметане (0,73 л, 10 об.) и охлаждали до 0-5°С. Добавляли диизопропиламмония тетразолид (6,19 г, 36,1 ммоль, 0,085 масс., 0,50 экв.). Полученную реакционную смесь перемешивали при 0°С в течение 10 ч, нагревали до 10°С в течение 2 ч, сохраняли при 10°С в течение 10 ч и нагревали до к.т. в течение 2 ч. За реакцией наблюдали при помощи LCMS и TLC (EtOAc с 0,5% TEA). Через 18 ч добавляли безводный ацетонитрил (0,73 л, 10 об.) и смесь хранили при -20°С в течение 3 суток.

vol., 1 eq.) and 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite (25.3 ml, 79.5 mmol, 0.33 wt., 0.35 vol., 1.10 eq. .) were azeotroped with anhydrous acetonitrile three times (3×2 L), redissolved in dichloromethane (0.73 L, 10 vol.) and cooled to 0-5°C. Diisopropylammonium tetrazolide (6.19 g, 36.1 mmol, 0.085 wt, 0.50 eq) was added. The resulting reaction mixture was stirred at 0°C for 10 h, heated to 10°C for 2 h, kept at 10°C for 10 h and heated to rt. for 2 hours. The reaction was monitored by LCMS and TLC (EtOAc with 0.5% TEA). After 18 h, anhydrous acetonitrile (0.73 L, 10 vol) was added and the mixture was stored at -20° C. for 3 days.

Step 11a The mixture from Step 10 was warmed to ambient temperature and added via addition funnel in portions (100 mL every 30 minutes for 9 h) to the pyridine trifluoroacetate salt mixture (azeotroped twice beforehand with pyridine; 41.9 g, 217 mmol, 0 .57 wt., 3.0 eq.) and acetonitrile (5.85 L, 80 vol.). The reaction was monitored by LCMS. After 13 h, a solution of 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite (5.8 ml, 18 mmol, 0.25 eq.) in acetonitrile (24 ml) was added over 4 h. The amount of additional reagent was determined based on remaining compound 140 (~30% based on LCMS). More conversion of the diol was observed after 6 hours.

Step 11b ((Dimethylaminomethylidene)amino)-3H-1,2,4-dithiazolin-3-thione (DDTT; 20.8 g, 101 mmol, 0.28 wt., 1.4 eq) was added and stirring was continued for over 1 h. The reaction mixture was partially concentrated to ~800 ml and diluted with MTBE (1.46 L, 20 vol.), NaHCO ₃ (9 wt.% solution in water; 1.1 L, 15 vol.) and water (0 .37 l, 5 vol.). pH=8. The layers were separated and the aqueous layer was extracted with a mixture of MTBE (1.46 L, 20 vol.) and EtOAc (1.10 L, 15 vol.). The combined organic layers were washed twice with 30% aq. NaCl (2×0.73 L, 2×10 vol.), concentrated in vacuo at 35°C and azeotroped with toluene (1.46 L, 20 vol.). LCMS and TLC (EtOAc) showed the ratio of compound 143 (SpRp, required): compound 241 (SpSp) = 5:1.

The crude product was purified on Biotage 150M KP-Sil, (SiO ₂ 2.5 kg; eluents: EtOAc/Hep: 2:1 (4 CV), 3:1 (2.5 CV), 4:1 (2.5 CV), 100% EA (3 CV), 5-10% MeOH in EA 4 CV) to give compound 143 (36 g, 31.5 mmol, 44% yield).

Compound 143 (SpRp): ¹ H NMR (400 MHz, CHLOROFORM-d) δ=8.59 (s, 1H), 8.10 (s, 1H), 8.03-7.99 (m, 1H), 7.91 (s, 1H), 7.56 -7.53 (m, 2H), 7.49-7.40 (m, 5H), 7.35-7.28 (m, 2H), 7.24-7.16 (m, 4H), 6.92 (s, 1H), 6.29 (d, J=14.9 Hz , 1H), 6.08 (d, J=20.7 Hz, 1H), 5.97-5.83 (m, 1H), 5.76 (td, J=4.7, 15.6 Hz, 1H), 5.61-5.51 (m, 2Н), 5.40 ( d, J=4.3 Hz, 1H), 5.29-5.17 (m, 1H), 4.91 (dd, J=7.4, 14.9 Hz, 1H), 4.86-4.75 (m, 3H), 4.63 (dd, J=3.7, 9.2 Hz, 1H), 4.58-4.43 (m, 5H), 4.34-4.19 (m, 4H), 2.79 (td, J=5.9, 16.8 Hz, 1H), 2.66 (td, J=6.3, 16.8 Hz, 1H ).

Compound 241 (SpSp) ¹ H NMR (400 MHz, CHLOROFORM-d) δ=8.11 (s, 1H), 8.03 (d, J=8.2 Hz, 1H), 7.94 (s, 1H), 7.90 (s, 1H) , 7.61 (s, 1H), 7.56-7.40 (m, 7H), 7.33-7.28 (m, 2H), 7.23-7.17 (m, 4H), 6.22 (d, J=17.6 Hz, 1H), 6.15 (d , J=18.8 Hz, 1H), 5.85 (dd, J=3.5, 51.2 Hz, 1H), 5.75-5.45 (m, 5H), 4.95-4.23 (m, 14H), 2.82 (t, J=6.1 Hz, 2H).

Stage 12

Compound 143 (71.6 g, 62.6 mmol, 1 wt., 1 vol., 1 eq.) was dissolved in 1,4-dioxane (0.43 L, 6 vol.). Thiophenol (215 mL, 2.09 mol, 230 g, 3.2 wt, 3 vol, >30 eq) was added followed by triethylamine (215 mL, 1.54 mol, 156 g, 2.2 wt., 3 vol.). Some exotherm was observed (T-internal increased by ~7°C), so a water/ice bath was used to cool and control T-internal below 27°C. The reaction was monitored by LCMS. After 2 h, MeOH (0.57 L, 8 vol.) and NH ₄ OH (28 wt.%; 15 mol., 0.57 L, 8 vol., >200 eq.) were added. The resulting mixture was heated at 50°C for 5 h, cooled to rt. and stirred overnight. After 14 hours, water (0.72 L, 10 vol) was added (no solid observed) and the mixture was extracted three times 1:1 (v/v) with a mixture of n-heptane and toluene (3 x 0.86 L, 3 x 12 vol.), and then toluene (0.57 l, 8 vol.). The aqueous layer was concentrated in vacuo at 40-50° C. and diluted with water (1.07 L, 15 vol). The resulting suspension was kept overnight at room temperature. The resulting solid was filtered off, washed with water (0.36 L, 5 vol). The filtrate was still cloudy and filtered through Celite and a Kuno filter. The turbidity was still present. HCl (1.0 M solution in water; 132 ml, 132 mmol, 2.1 eq.) was added over 1 h and the pH value was checked (pH<2). Stirring was continued at rt. for 1 h and the mixture was filtered. The filter cake was washed with water (8×0.20 L), dried in a vacuum oven at 35°C for 2 days and without heat for 1 day to give Compound 1 as a pale orange solid (44.88 g, 60 .1 mmol, 0.63 wt., 96% yield).

Stage 13

To the free acid compound 1 (22.42 g, 1 eq.) was added ammonia (2.0 M solution in MeOH; 220 ml, 440 mmol, 10 vol., 15 eq.). EtOH (55 ml, 2.5 vol.) was added and the resulting solution was filtered through a Kuno filter (0.45 micron; PTFE), washed 1:1 (v/v) with a mixture of MeOH and EtOH (90 ml, 4 vol.). The filtrate was concentrated in vacuo at 30° C. to give an off-white solid which was dried at RT. all night, crushed with a spatula (easy to break) and dried additionally in a vacuum at rt. The isolated solid was then suspended in toluene (250 ml) and stirred at rt. within 30 minutes. The solid was then collected by vacuum filtration and washed twice with toluene (2×50 ml). The solid was then dried under vacuum in a vacuum oven to give 22.4 g of compound 1a (diammonium salt of compound 1).

Recrystallization: Compound 1a (22.14 g, 28.36 mmol, 1 wt, 1 vol., 1 eq.) was dissolved in a mixture of water (664 ml, 30 vol.) and ammonium hydroxide (28 wt.%; 2.5 ml, 18 mmol, 0.63 eq.) (pH=9-10) and extracted thrice with toluene (3×300 ml, 3×14 vol.), thrice with EtOAc (3×200 ml, 3×9 vol.) and three times with toluene (3×300 ml, 3×14 vol.). The resulting aqueous layer was treated with HCl (1.0 M solution in water; 90 ml, 90 mmol, 3.2 eq.) for 3.5 hours (pH≤2). The mixture was stirred for 30 minutes and then the solid precipitated was collected by vacuum filtration. The filter cake was washed three times with water (3×200 ml, 3×9 vol.) and dried in vacuum overnight. Ammonia (2.0 M solution in MeOH; 250 ml, 500 mmol, 17.6 eq.) and ethanol (100 ml) were added to the solid and the resulting mixture was concentrated in vacuo until crystals appeared (~100 ml), at which time the concentration was stopped and the mixture was stirred for 20 minutes. Ethanol (45 ml) was added and the mixture was partially concentrated (45 ml was removed). The same operation was repeated two more times, and then the mixture was cooled to 0°C and stirred for 3.5 hours. The white solid was collected by vacuum filtration and washed with cold ethanol (20 ml) and then with ethyl acetate (2×50 ml). The white solid was dried in vacuo at rt for 3 days to give compound 1a as a white solid (16.6 g, 21.3 mmol, 0.75 wt., 75% yield). The filtrate was concentrated in vacuo and dried in vacuo at rt for 3 days to give compound 1a as an off-white solid (4.16 g, 5.3 mmol, 18% yield).

Example 1.2 - ¹ H NMR analysis of compound 1

¹ H NMR spectrogram of compound 1a is shown in Fig.3. Received spectrum:

¹ H-NMR spectrum (400 MHz, DMSO-d ₆ , δ _H 2.49 ppm, 80°C)

δ (ppm): 3.05-3.13 (4H, m), 3.70 (1H, dd, J=13.5 Hz), 3.78 (1H, dd, J=12.4 Hz), 4.21-4.24 (2H, m) , 4.28 (1H, m), 4.38 (1H, m), 4.53-4.68 (2H, m), 5.22 (1H, m), 5.76 (2H, s), 5.78 (1H, m), 6.26 (1H, m ), 6.29 (1Н, m), 8.13 (1Н, s), 8.14 (1Н, s), 8.36 (1Н, brs), 8.59 (1Н, brs).

Example 1.3 - X-Ray Analysis of Compound 1

Approximately 2 mg of compound 1 was dissolved in 600 μl of water. 120 μl of this solution was placed in another glass vessel, and then this vessel was stored in a fixed container with 3 ml of MeCN at room temperature for 1 week. This is an H ₂ O/MeCN vapor diffusion method to obtain a sample.

A colorless block single crystal (0.1 x 0.1 x 0.1 mm) obtained in crystallization solution was dispersed in liquid Parabar 10312 and mounted on Dual-Thickness MicroMounts™ (MiTeGen). Diffraction data were collected at -160° C. on an XtaLAB PRO P200 MM007HF (Rigaku) using the ω-axis oscillation method using Cu-Kα multilayer specular monochromatic radiation.

In FIG. 4A shows the ORTEP figure of compound 1 molecules in an asymmetric unit along with the number of random water molecules. In FIG. 4B shows the crystal structure of one of the molecules of compound 1 of FIG. 4A. In FIG. 4C shows the crystal structure of another molecule of compound 1 shown in FIG. 4A.

The crystal structure of compound 1 was explained using a final R-factor of 0.1354. The Flack parameter was approximately zero (0.083(17)), denoting that the absolute configuration of Compound 1 is (R, S). Crystal structure analysis also shows that many water molecules were present in the large channel of compound 1, which means that water molecules are able to easily escape from the channel. The analysis also confirms that the conformations of both crystallographically independent molecules of the asymmetric unit were almost identical.

Additional X-ray diffraction parameters are shown below:

Example 2 Synthesis of Compound 2

Compound 106 (RpRp isomer of compound 105), obtained from example 1, step E, was treated separately as in example 1, step F and example 1, step G, to obtain compound 2a (RR isomer of compound 1a):

7.99 (s, 2H), 7.96 (s, 2H), 7.65-7.48 (m, 10Н), 7.38-7.33 (m, 2H), 7.26-7.24 (m, 4H), 6.22 (d, J=17.6 Гц, 2H), 5.95-5.84 (m, 2H), 5.71 (dd, J=50.8, 3.9 Гц, 2H) 5.73-5.71 (m, 2H), 4.88-4.77 (m, 4H), 4.59-4.38 (m, 8H), 4.19 (m, 2H).

7.99 (s, 2H), 7.96 (s, 2H), 7.65-7.48 (m, 10H), 7.38-7.33 (m, 2H), 7.26-7.24 (m, 4H), 6.22 (d, J=17.6 Hz, 2H), 5.95-5.84 (m, 2H), 5.71 (dd, J=50.8, 3.9 Hz, 2H) 5.73-5.71 (m, 2H), 4.88-4.77 (m, 4H), 4.59-4.38 (m, 8H ), 4.19 (m, 2H).

Compound 2a (RpRp, trans) ¹ H NMR (400 MHz, CD ₃ OD) δ=8.70 (s, 1H), 8.49 (s, 1H), 8.23 (s, 1H), 8.09 (s, 1H), 6.34 ( br s, 2Н), 5.83 (br s, 2H), 5.73-5.53 (m, 2H), 5.38-5.01 (m, 2H), 4.76-4.32 (m, 6H), 3.95 (br s, 2H), 3.69 -3.64 (m, 2H); ³¹ P NMR (162 MHz, CD ₃ OD) δ=55.57 (s, 1P), 55.32 (s, 1P).

Example 2.1 - X-Ray Analysis of Compound 2

Compound 2 (0.5 mg) was weighed and dissolved in acetonitrile/28% ammonia solution. This solution was then stored at room temperature with the cap loosely attached. After 2 weeks, a columnar crystal appears.

A colorless block single crystal (0.1×0.1×0.5 mm) obtained in crystallization solution was dispersed in liquid Parabar 10312 and mounted on Dual-Thickness MicroMounts™ (MiTeGen). Diffraction data were collected at -160° C. on an XtaLAB PRO P200 MM007HF (Rigaku) using the off-axis oscillation method using Cu-Kα multilayer specular monochromatic radiation.

In FIG. 4D shows the ORTEP molecular figure of compound 2.

Additional parameters for this X-ray diffraction analysis are shown below:

Example 3 Synthesis of Compound 3

Compound 109 (SpSp isomer of compound 105), obtained from example 1, step E, was treated separately as in example 1, step F and example 1, step G, to obtain compound 3a (SS isomer of compound 1a):

Compound 109 (SpSp isomer of compound 105): ¹ HNMR (400 MHz, CHLOROFORM-d) δ=8.52 (s, 2H), 7.99 (d, J=8.2 Hz, 2H), 7.91 (s, 2H), 7.63-7.40 (m, 10H), 7.35-7.28 (m, 2H), 7.23-7.16 (m, 4H), 6.10-5.93 (m, 4H), 5.92-5.75 (m, 2H), 5.62-5.50 (m, 2H) , 5.26-5.16 (m, 2H), 5.09-5.03 (m, 2H), 4.98-4.91 (m, 4H), 4.61-4.25 (m, 10H)

Compound 3а (SpSp, trans): ¹ H NMR (400 MHz, CD ₃ OD) δ=8.97 (br s, 1H), 8.84 (br s, 1H), 8.21 (br s, 2H), 6.31 (br s, 2H), 6.08 (br d, J=53.5 Hz, 1H), 5.89 (br s, 2H), 5.63 (br d, J=52.4 Hz, 1H), 5.13-4.96 (m, 2H), 4.72-4.32 ( m, 6H), 4.01 (br d,J=9.8 Hz, 2H), 3.67 (br s, 2H).

Example 4 Synthesis of Compound 4a

Compound 107 obtained from example 2, step F, was isolated as a single cis-isomer using silica gel chromatography, treated separately as in example 1, step G, to obtain compound 4a.

Compound 107: ¹ H NMR (400 MHz, CHLOROFORM-d) δ=8.13 (s, 2H), 8.12-8.08 (m, 2H), 7.91 (s, 2H), 7.65-7.55 (m, 4H), 7.54- 7.45 (m, 6H), 7.33-7.27 (m, 2H), 7.23-7.16 (m, 4H), 6.14 (d, J=17.6 Hz, 2H), 5.88 (dd, J=3.9, 50.8 Hz, 2H) , 5.76-5.61 (m, 4H), 5.21-4.99 (m, 4H), 4.60-4.46 (m, 4H), 4.45-4.37 (m, 2H), 4.30-4.13 (m, 4H), 3.49 (d, J=5.1Hz, 2H)

Compound 4a (RpRp, cis 1a, Compound 4a): ¹ H NMR (400 MHz, METHANOL-d ₄ ) δ=8.48 (br s, 2H), 8.02 (br s, 2H), 6.29 (d, J=14.8 Hz , 2H), 5.99 (br s, 2H), 5.43 (d, J=51.2 Hz, 2H), 5.03-4.88 (m, 2H), 4.43 (br d, J=11.7 Hz, 2H), 4.32 (br d , J=9.4 Hz, 2H), 4.27-4.17 (m, 2H), 4.21-4.02 (m, 2H), 3.97 (br dd, J=6.1, 12.3 Hz, 2H).

Example 5 Synthesis of Compound 5

The cis isomer obtained from example 1, step F was treated separately as in example 1, step G, to give compound 5a.

Compound 111 (cis isomer of compound 107): ¹ H NMR (400 MHz, CHLOROFORM-d) δ=8.48 (s, 1H), 8.15-8.09 (m, 1H), 8.03 (d, J=8.2 Hz, 1H) , 7.94 (s, 1H), 7.75 (s, 1H), 7.65-7.06 (m, 11H), 6.07 (d, J=17.2 Hz, 1H), 5.98 (d, J=20.3 Hz, 1H), 5.97- 5.79 (m, 1H), 5.84 (dd, J=3.5, 51.2 Hz, 1H), 5.54-5.47 (m, 1H), 5.50 (dd, J=3.9, 52.0 Hz, 1H), 5.38-5.21 (m, 2H), 5.18-5.02 (m, 2H), 5.02-4.95 (m, 1H), 4.78-4.69 (m, 1H), 4.60-4.16 (m, 10H)

Compound 5a (SpRp, cis 1a): ¹ H NMR (400 MHz, METHANOL-d ₄ ) δ=8.88 (br s, 1H), 8.51 (br s, 1H), 8.16 (s, 1H), 8.05 (s, 1H), 6.38 (d, J=15.6 Hz, 1H), 6.33 (d, J=14.1 Hz, 1H), 6.14-6.09 (m, 2H), 6.01 (d, J=49.2 Hz, 1H), 5.42 ( d, J=49.6 Hz, 1H), 5.02-4.87 (m, 2H), 4.76-3.92 (m, 8H), 3.75-3.56 (m, 2H)

Example 6 Synthesis of Compound 6a (SpRp)

To a solution of compound 1a (1.5 mg, 2.0 µmol) in MeOH/H ₂ O (0.6/0.5 ml) at ambient temperature was added palladium hydroxide on a carbon carrier (20 wt.% dry weight, 2 mg). The resulting mixture was treated with hydrogen (balloon) while the reaction was monitored by LCMS. After consumption of the starting material was complete, the mixture was filtered and washed with methanol until all product was purified. The filtrate was concentrated in vacuo and the residue was dissolved in water (1 ml). Compound 6a (0.9 mg) was obtained by RHPLC purification.

LCMS (MS m/z 749.2 [M+H] ⁺ )

Example 7 Synthesis of Compound 9

Ammonium hydroxide (1 ml) was added to a solution of compound 107 (3.7 mg, 3.02 μmol) in EtOH (1.5 ml). The resulting mixture was heated at 50°C for 8 hours and cooled to ambient temperature. The solvent was removed under reduced pressure. The residue was treated with 2 ml of water and the resulting solid was filtered off. The filtrate was subjected to preparative HPLC to give compound 9a (2.5 mg).

Compound 9a: ¹ H NMR (400 MHz, METHANOL-d ₄ ) δ=8.67 (br s, 1H), 8.50 (br s, 1H), 8.25 (br s, 1H), 8.12 (br s, 1H), 6.47 -6.28 (m, 2H), 5.92-5.79 (m, 2H), 5.74 (d, J=50.8 Hz, 1H), 5.32 (d, J=52.4 Hz, 1H), 5.16-4.95 (m, 2H), 4.71-4.25 (m, 6H), 4.17-3.97 (m, 2H), 3.83-3.61 (m, 2H).

LCMS: MS m/z 715.2 [M+H] ⁺ .

Example 8 Synthetic routes for compounds 8a, 11a and 12a

With compound 112 and compound 102 as starting materials, compounds 8, 11 and 12 were prepared with the same reaction sequences as described in example 1.

Preparative HPLC Conditions for Fractions A and B

Compound 8a: Fraction A was treated as in step g, Example 1 to give two isomers which were separated by HPLC. Compound 8a was the fastest rotation isomer (retention time: 4.1 min) and compound 37a (retention time: 4.5 min) was the slower rotation isomer.

¹ H NMR (400 MHz, METHANOL-d ₄ ) δ=9.01 (s, 1H), 8.61 (s, 1H), 8.22 (s, 1H), 8.20 (s, 1H), 6.34 (d, J=10.2 Hz , 1H), 6.31 (d, J=10.9 Hz, 1H), 5.92 (dd, J=2.7, 51.2 Hz, 1H), 5.85-5.71 (m, 2Н), 5.37 (d, J=51.6 Hz, 1H) , 4.81-4.56 (m, 6H), 4.52 (br d, J=12.1 Hz, 1H), 4.42 (br d, J=9.4 Hz, 3H), 4.05 (dd, J=4.1, 11.9 Hz, 1H), 3.95 (br dd, J=4.3, 12.1 Hz, 1H), 3.94-3.84 (m, 1H), 3.62 (br dd, J=4.9, 15.4 Hz, 1H), 2.71-2.58 (m, 1H), 2.46- 2.33(m, 1H)

³¹ P NMR (162 MHz, METHANOL-d ₄ ) δ=55.36 (s, 1P), 55.18 (s, 1P)

LCMS: MS m/z 761.2 [M+H] ⁺

Compound 37a: ¹ H NMR (400 MHz, METHANOL-d ₄ ) δ=8.95 (s, 1H), 8.40 (s, 1H), 8.18 (s, 1H), 8.15(brs, 1H), 6.34 (d, J =5.5 Hz, 1H), 6.31 (d, J=6.6 Hz, 1H), 5.96-5.68 (m, 3H), 5.40 (dd, J=2.7, 51.6 Hz, 1H), 4.85 (s, 3H), 4.58 (br d, J=12.1 Hz, 1H), 4.49 (br d, J=12.1 Hz, 1H), 4.43-4.33 (m, 3H), 4.29 (dd, J=8.6, 14.1 Hz, 1H), 4.05 ( dd, J=4.5, 11.9 Hz, 1H), 3.95 (dd, J=4.9, 12.3 Hz, 1H), 3.94-3.86 (m, 1H), 2.67-2.55 (m, 1H), 2.52-2.40 (m, 1H).

Compound 8a/37a Preparative HPLC conditions:

Fraction B was treated as in step g, Example 1 to give two isomers which were separated by prep. -HPLC described below. Compound 11a was the fastest rotation isomer (retention time: 11.2 minutes) and compound 12a was the slowest rotation isomer (retention time: 12.1 minutes)

Compound 11a: ¹ H NMR (400 MHz, METHANOL-d ₄ ) δ=8.63 (br s, 2H), 8.18 (br s, 1H), 8.17 (br s, 1H), 6.34 (d, J=13.3 Hz, 1H), 6.32 (d, J=12.9 Hz, 1H), 5.86-5.65 (m, 2H), 5.48 (d, J=48.5 Hz, 1H), 5.35 (d, J=43.8 Hz, 1H), 4.84- 4.53 (m, 6H), 4.47-4.36 (m, 2H), 4.05-3.91 (m, 2H), 3.96-3.85 (m, 1H), 3.70-3.54 (m, 1H), 2.66-2.54 (m, 1H ), 2.43-2.30 (m, 1H)

Compound 12a: ¹ H NMR (400 MHz, METHANOL-d ₄ ) δ=8.56 (br s, 1H), 8.41 (s, 1H), 8.17 (s, 1H), 7.95 (br s, 1H), 6.35 (d , J=14.8 Hz, 1H), 6.31 (d, J=15.2 Hz, 1H), 5.82-5.65 (m, 2H), 5.50 (d, J=51.2 Hz, 1H), 5.36 (d, J=53.9 Hz , 1H), 4.74-4.33 (m, 8H), 4.26-4.16 (m, 1H), 4.07-3.93 (m, 3H), 2.64-2.44 (m, 2H).

Compound 11a/12a Preparative HPLC conditions:

Example 9 Synthetic Routes for Compounds 13 and 14

With Compound 114 and Compound 115 as starting materials, compounds 13 and 14 were prepared with the same reaction sequences as described in Example 1 plus the TBS deprotection step.

Compound 16: ¹ H NMR (400 MHz, CHLOROFORM-d) δ=9.36 (s, 1H), 9.18 (s, 1H), 8.77 (s, 1H), 8.76 (s, 2H), 8.07 (br dd, J =1.8, 7.6 Hz, 3H), 8.05-8.00 (m, 2H), 7.99 (s, 1H), 7.87-7.82 (m, 1H), 7.70-7.65 (m, 1H), 7.63-7.55 (m, 3H ), 7.55-7.41 (m, 6H), 7.39-7.35 (m, 1H), 7.30-7.26 (m, 1H), 6.37 (d, J=8.6 Hz, 1H), 5.89 (d, J=4.7 Hz, 1H), 5.36-5.27 (m, 2H), 5.15 (ddd, J=3.7, 8.4, 11.7 Hz, 1H), 4.64-4.43 (m, 4H), 4.43-4.38 (m, 1H), 4.37-4.30 ( m, 1H), 4.23-4.13 (m, 2H), 4.11-3.98 (m, 2H), 3.97-3.86 (m, 1H), 0.97 (s, 9H), 0.83 (s, 9H), 0.25 (s, 3Н), 0.18 (s, 3Н), 0.16 (s, 3Н), -0.13 (s, 3Н).

Compound 13a: ¹ H NMR (400 MHz, METHANOL-d ₄ ) δ=8.78 (br s, 1H), 8.47 (br s, 1H), 8.21 (br s, 1H), 8.08 (br s, 1H), 6.44 -6.21 (m, 1H), 6.19-6.03 (m, 1H), 6.01-5.78 (m, 2H), 5.38-4.95 (m, 2H), 4.67-4.45 (m, 5H), 4.45-4.35 (m, 1H), 4.34-4.29 (m, 1H), 4.25 (br d, J=11.3 Hz, 1H), 4.18-3.89 (m, 2H), 3.88-3.54 (m, 2H).

³¹ P NMR (162 MHz, METHANOL-d ₄ ) δ=56.89 (br s, 1P), 56.37 (br s, 1P)

Compound 117 (Rp ₁ Rp ₂ isomer of compound 116): ¹ H NMR (400 MHz, CHLOROFORM-d) δ=9.36 (br s, 1H), 9.11 (br s, 1H), 8.98 (s, 1H), 8.87 ( s, 1H), 8.78 (s, 1H), 8.14-7.96 (m, 6H), 7.72-7.27 (m, 13H), 6.44 (d, J=8.6 Hz, 1H), 5.84 (d, J=6.6 Hz , 1H), 5.59-5.50 (m, 1H), 5.48-5.44 (m, 1H), 5.33-5.24 (m, 1H), 4.75-3.85 (m, 11H), 0.90 (s, 9H), 0.79 (s , 9H), 0.20 (s, 3H), 0.11 (s, 3H), 0.07 (s, 3H), -0.21 (s, 3H)

Compound 14а (Rp ₁ Rp ₂ isomer of compound 13а)

LCMS: MS m/z 743.17 [M+H] ⁺

Example 10 Synthetic Routes for Compounds 15 and 16

To a solution of compound A (2.05 g, 2.40 mmol) in MeCN (26.3 ml) was added tert-butylamine (13.15 ml, 124.1 mmol). After stirring for 1 hour at ambient temperature, the reaction mixture was concentrated in vacuo and azeotroped with MeCN. The residue was dissolved in MeCN (52.6 ml) and treated with 1-(bromomethyl)-2-nitrobenzene (1.064 g, 4.925 mmol) and triethylamine (0.755 ml, 5.42 mmol). After completion of the reaction (observed by LC/MS), the reaction mixture was concentrated in vacuo and purified by silica gel column chromatography (66% ethyl acetate/n-heptane to 100% ethyl acetate) to give 0.226 g of compound 118. The isolated product was dissolved in DMF (5 ml) and allyl bromide (0.046 ml, 0.528 mmol) and potassium carbonate (0.073 g, 0.528 mmol) were added. The resulting mixture was stirred at ambient temperature while progress was monitored by LCMS. After 19 hours 1/1 MTBE/EtOAc (12/12 ml), saturated aqueous NH ₄ Cl solution (15 ml) and water (10 ml) were added. The organic layer was separated, washed with brine (5 ml) twice, dried over MgSO ₄ and concentrated in vacuo. The residue was purified by silica gel column chromatography (50% ethyl acetate/n-heptane to 100% ethyl acetate) to give 37 mg of the bis-allylated product. The isolated product (37 mg, 0.027 mmol) was dissolved in toluene (55 ml) and heated to moderate reflux temperature (oil bath 120-125°C). A solution of 2nd generation Hoveid-Grubbs catalyst ((1,3-bis-(2,4,6-trimethylphenyl)-2-imidazolidinylidene)dichloro(ortho-isopropoxyphenylmethylene)ruthenium; available from SIGMA-ALDRITCH® cat. no. 569755; CAS 301224-40-8; 8.5 mg, 0.014 mmol) and quinone (5.9 mg, 0.054 mmol) in toluene (10 ml). The mixture was heated to reflux temperature and the progress of the reaction was monitored by LC/MS. After consumption of starting material was complete, the mixture was cooled to ambient temperature and treated with DMSO (0.059 mL, 0.81 mmol) for 15 hours. The resulting mixture was concentrated in vacuo and purified by silica gel column chromatography (SiO ₂ 10 g, 66% ethyl acetate in n-heptane to 100% ethyl acetate) to give compound 119 (2.2 mg).

Compound 119: ¹ H NMR (400 MHz, CHLOROFORM-d) δ=8.49 (s, 1H), 8.12 (d, J=7.8 Hz, 1H), 8.05-8.02 (m, 1H), 8.02 (s, 1H) , 7.82 (s, 1H), 7.70-7.10 (m, 17H), 6.27 (d, J=16.8 Hz, 1H), 6.15-6.05 (m, 1H), 5.97 (d, J=8.2 Hz, 1H), 5.94-5.72 (m, 3H), 4.94 (br s, 2H), 4.86-4.69 (m, 2H), 4.67-4.60 (m, 1H), 4.60-4.44 (m, 4H), 4.39-4.33 (m, 1H), 4.31-4.10 (m, 6H), 0.96 (s, 9H), 0.25 (s, 3H), 0.19 (s, 3H)

Compound 119 was treated as in step G in the example followed by deprotection of TBS with TEA 3HF to give compound 15a.

Compound 15a: ¹ H NMR (400 MHz, METHANOL-d ₄ ) δ=8.76 (br s, 1H), 8.44 (br s, 1H), 8.24 (br s, 1H), 8.07 (br s, 1H), 6.45 -6.19 (m, 2H), 6.12-5.75 (m, 2H), 5.54 (br d, J=51.6 Hz, 1H), 5.67-5.06 (m, 2H), 4.66-4.37 (m, 3H), 4.47- 4.37 (m, 1H), 4.36-4.22 (m, 2H), 4.17-3.94 (m, 2H), 3.94-3.80 (m, 1H) 3.80-3.59 (m, 2H)

Compound 16 was obtained from compound B (RR isomer of compound A) with the same sequences as described for compound 15.

Compound 120 (Rp ₁ Rp ₂ isomer of compound 119): ¹ H NMR (400 MHz, CHLOROFORM-d) δ=8.89 (s, 1H), 8.10 (d, J=8.2 Hz, 1H), 8.01 (d, J= 8.2 Hz, 1H), 7.85-7.74 (m, 4H), 7.69-7.61 (m, 2H), 7.58 (s, 1H), 7.55-7.48 (m, 1H), 7.45-7.30 (m, 6H), 7.22 -7.15 (m, 1H), 7.13-7.04 (m, 3H), 6.76-6.68 (m, 1H), 6.31-6.22 (m, 1H), 6.22 (d, J=16.0 Hz, 1H), 6.12-5.99 (m, 2Н), 5.94 (d, J=8.2 Hz, 1H), 5.92-5.81 (m, 1H), 5.67 (dd, J=3.5, 51.2 Hz, 1H), 5.08-4.93 (m, 2Н), 4.87-4.77 (m, 1H), 4.60-4.22 (m, 8H), 4.21-4.16 (m, 1H), 4.11-4.02 (m, 1H), 3.94 (br d, J=11.7 Hz, 1H), 3.66 (dd, J=4.7, 11.3 Hz, 1H), 0.90 (s, 9H), 0.09 (s, 3H), 0.08 (s, 3H)

Compound 16a: ¹ H NMR (400 MHz, METHANOL-d ₄ ) δ=8.54 (br s, 1H), 8.24 (br s, 1H), 8.17 (br s, 1H), 8.08 (br s, 1H), 6.44 -6.23 (m, 2H), 6.07-5.68 (m, 2H), 5.43 (d, J=50.4 Hz, 1H), 5.31-5.05 (m, 2H), 4.69-4.26 (m, 6H), 4.19-3.90 (m, 2H), 3.90-3.57 (m, 3H)

Example 11 Synthesis Routes for Adenine/Guanine (A/G) Analog Compounds Compound 21, Compound 22 and Compound 23

With Compound 121 and Compound 101 as starting materials, adenine/guanine analogs were prepared with the same reaction sequences as described in Example 1.

Preparative HPLC Conditions for the Separation of Compounds C, D, and E

Compound D: ¹ H NMR (400 MHz, CHLOROFORM-d) δ=8.60 (s, 1H), 8.27 (s, 1H), 8.04-8.01 (m, 1H), 8.01 (s, 1H), 7.99-7.95 ( m, 1H), 7.80 (s, 1H), 7.63-7.51 (m, 5H), 7.46-7.41 (m, 1H), 7.39-7.30 (m, 3H), 7.25-7.20 (m, 2H), 6.96- 6.84 (m, 1H), 6.19-5.99 (m, 4H), 5.67 (dd, J=4.3, 52.3 Hz, 1H), 5.47 (dd, J=1.4, 17.4 Hz, 1H), 5.36-5.24 (m, 4Н), 5.14-5.09 (m, 1H), 5.08 (d, J=5.5 Hz, 2Н), 5.04-4.98 (m, 2Н), 4.51-4.30 (m, 10Н), 4.21-4.13 (m, 1H) , 2.83-2.67(m, 1H), 1.16 (d, J=7.0 Hz, 3H), 1.14 (d, J=6.6 Hz, 3H)

Compound D was treated as in steps F and G of example 1 to give compound 22.

Compound 22: LCMS: MS m/z 763.07 [M+H] ⁺

Compound C: ¹ H NMR (400 MHz, CHLOROFORM-d) δ=9.08 (s, 1H), 8.60 (s, 1H), 8.07-8.02 (m, 2H), 8.02 (s, 1H), 7.80 (s, 1H), 7.55-7.30 (m, 9H), 7.24-7.19 (m, 2H), 6.58-6.45 (m, 1H), 6.23-5.97 (m, 4H), 5.97-5.86 (m, 1H), 5.63- 5.57 (m, 1H), 5.54-5.47 (m, 1H), 5.48-5.44 (m, 1H), 5.35-5.30 (m, 1H), 5.29-5.22 (m, 1H), 5.18-5.13 (m, 2H ), 5.11-5.07 (m, 1H), 5.02-4.97 (m, 2Н), 4.51-4.22 (m, 9Н), 4.14-4.07 (m, 1H), 2.99-2.85 (m, 1H), 1.20 (d , J=6.6 Hz, 3H), 1.17 (d, J=7.0 Hz, 3H)

Compound C was treated as in steps F and G in Example 1 to give Compound 21

Compound 21: LCMS: MS m/z 763.13 [M+H] ⁺

Compound E: ¹ H NMR (400 MHz, CHLOROFORM-d) δ=8.99 (s, 1H), 8.57 (s, 1H), 8.02-7.94 (m, 2H), 7.93 (s, 1H), 7.82 (s, 1H), 7.68-7.63 (m, 1H), 7.60-7.55 (m, 1H), 7.54-7.49 (m, 2H), 7.45-7.31 (m, 5H), 7.25-7.21 (m, 2H), 6.22- 5.94 (m, 6H), 5.56 (br dd, J=5.1, 51.5 Hz, 1H), 5.54-5.46 (m, 1H), 5.34-5.29 (m, 1H), 5.29-5.22 (m, 2H), 5.16 -5.08 (m, 3H), 5.02-4.96 (m, 2H), 4.62-4.54 (m, 1H), 4.52-4.26 (m, 9H), 2.93-2.81 (m, 1H), 1.21 (d, J= 2.3Hz, 3H), 1.20 (d, J=2.0Hz, 3H)

Compound E was treated as in steps F and G in Example 1 to give Compound 23

Compound 23: LCMS: MS m/z 763.18 [M+H] ⁺

Example 12 Synthesis of Compound 24

With compound F, compound 24a was prepared with the same reaction sequences as described in example 1.

Compound 24: ¹ H NMR (400 MHz, METHANOL-d ₄ ) δ=8.95 (br s, 1H), 8.29 (br s, 1H), 8.23 (br s, 1H), 8.08 (br s, 1H), 6.31 (br d, J=12.9 Hz, 2H), 6.23-6.07 (m, 1H), 6.04-5.84 (m, 2H), 5.71 (br d, J=50.8 Hz, 1H), 5.27-4.32 (m, 7H ), 4.27-4.16 (m, 1H), 4.12 (dd, J=5.7, 11.9 Hz, 1H), 4.07-3.98 (m, 1H), 3.78-3.62 (m, 2H), 1.40 (d, J=7.0 Hz, 3H)

Example 13 - Synthesis of Compound 18, Compound 19 and Compound 20

The way of such synthesis is shown in Fig. 3.

To a solution of 9-((2R,3R,4R,5R)-3-fluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-9H-purin-6-ol (100 mg, 37 mmol) and Hunig's base (0.129 ml, 0.74 mmol) in DMF (1 ml) was added BOP (327 mg, 74 mmol) at 20°C. The mixture was stirred at 20° C. overnight. The completion of the reaction was observed by UPLC-MS. The solvent was removed on a highvac rotary evaporator. The residue was dissolved in DCM and purified using Biotage (24 g Si-gel column, EtOAc in heptane = 0-100%, 10 vol, 100%, 10 vol) to give 105 mg BOP adduct in 73% yield.

¹ H NMR (400 MHz, METHANOL-d ₄ ) δ ppm 3.74-3.84 (m, 1H) 3.92-4.00 (m, 1H) 4.12-4.19 (m, 1H) 4.62-4.74 (m, 1H) 5.37-5.57 ( m, 1H) 6.41-6.52 (m, 1H) 7.47-7.55 (m, 1H) 7.57-7.64 (m, 2H) 8.03-8.14 (m, 1H) 8.36-8.46 (m, 1H) 8.86 (s, 1H)

To a solution of (2R,3R,4R,5R)-5-(6-((1H-benzo[d][1,2,3]triazol-1-yl)oxy)-9H-purin-9-yl)- 4-fluoro-2-(hydroxymethyl)tetrahydrofuran-3-ol (135 mg, 0.349 mmol) in allyl alcohol (3 ml) was added cesium carbonate (500 mg, 1.535 mmol) at 20°C. The mixture was stirred at 20° C. for 1 hour. Completion of the reaction was observed by UPLC-MS. The reaction mixture was treated with us. NHCO ₃ /brine and extracted with EtOAc/Hept. The organic layer was dried over Na ₂ SO ₄ and filtered. The solvent and volatile organic substances in the filtrate were removed on a rotary evaporator. The residue was purified using Biotage (12 g Si-gel column, EtOAc in heptane = 0-100%, 10 vol, 100%, 10 vol) to give 100 mg of the desired allyl adduct in 92% yield.

¹ H NMR (400 MHz, METHANOL-d ₄ ) δ ppm 8.56 (s, 1H) 8.47 (s, 1H) 6.29-6.41 (m, 1H) 6.06-6.21 (m, 1H) 5.33-5.53 (m, 2H) 5.22-5.31 (m, 1H) 5.02-5.14 (m, 2H) 4.56-4.71 (m, 1H) 4.10-4.18 (m, 1H) 3.88-3.96 (m, 1H) 3.70-3.79 (m, 1H)

To a solution of diisopropylphosphoramidite (150 mg, 0.164 mmol) and (2R,3R,4R,5R)-5-(6-(allyloxy)-9H-purin-9-yl)-4-fluoro-2-(hydroxymethyl)tetrahydrofuran- 3-ol (76 mg, 0.246 mmol) in acetonitrile (1069 μl, 20.47 mmol) molecular sieves (3 A, 150 mg) were added at 20°C. The mixture was stirred at 20°C for 1 hour before adding 1H-imidazole-4,5-dicarbonitrile (38.7 mg, 0.328 mmol) at rt. The mixture was stirred at rt. within 1 h. Using UPLC-MS observed the completion of the reaction. (E)-N,N-dimethyl-N'-(3-thioxo-3H-1,2,4-dithiazol-5-yl)formimidamide (37.0 mg, 0.18 mmol) was added. Sulfurization was observed to be complete in 30 minutes using UPLC-MS. The reaction mixture was treated with us. NHCO ₃ /brine and extracted with EtOAc/Hept. The organic layer was dried over Na ₂ SO ₄ and filtered. The solvent and volatile organic substances in the filtrate were removed on a rotary evaporator. The residue was purified using Biotage (25 g Si-gel column, EtOAc in heptane = 0-100%, 15 vol., 100%, 10 vol.).

¹ H NMR (500 MHz, METHANOL-d ₄ , two ~1:1 diastereomers) δ ppm 8.62-8.63 (m, 1H) 8.57-8.58 (m, 1H) 8.48-8.50 (m, 1H) 8.44-8.46 (m , 1H) 8.40-8.41 (m, 1H) 8.38-8.39 (m, 1H) 8.33-8.35 (m, 1H) 8.25-8.27 (m, 1H) 7.22-7.33 (m, 11H) 7.11-7.20 (m, 17H) ) 7.05-7.11 (m, 4H) 6.83-6.92 (m, 6H) 6.65-6.77 (m, 11H) 6.28-6.41 (m, 5H) 6.23-6.28 (m, 2H) 6.10-6.22 (m, 5H) 6.00 -6.10 (m, 4H) 5.89-6.00 (m, 5H) 5.52-5.57 (m, 2H) 5.45-5.52 (m, 3H) 5.41-5.45 (m, 1H) 5.24-5.34 (m, 4H) 5.13-5.17 (m, 3H) 5.07-5.12 (m, 5H) 4.93-4.98 (m, 4H) 4.88-4.93 (m, 5H) 4.73-4.82 (m, 3H) 4.50-4.59 (m, 2H) 4.36-4.48 (m , 4H) 4.27-4.36 (m, 5H) 4.16-4.27 (m, 5H) 3.83-4.04 (m, 5H) 3.73-3.78 (m, 13H) 3.64-3.73 (m, 4H) 3.52-3.58 (m, 1Н) 3.45-3.51 (m, 2Н) 3.35-3.41 (m, 2Н) 3.16-3.23 (m, 2Н) 3.08-3.15 (m, 2Н) 2.84-2.93 (m, 2Н) 2.79-2.83 (m, 2Н) 2.70-2.77 (m, 2H) 2.61-2.69 (m, 3H)

метоксифенил)(фенил)метокси)метил)-4-фтортетрагидрофуран-3-ил) O-(((2R,3R,4R,5R)-5-(6-(аллилокси)-9Н-пурин-9-ил)-4-фтор-3-гидрокситетрагидрофуран-2-ил)метил)-O-(2-цианоэтил)фосфоротиоат (120 мг, 0,104 ммоль) в ацетонитриле (4171 мкл, 79,852 ммоль) добавляли молекулярные сита (3А, 1 масс.) при 20°С. Смесь перемешивали при 20°С в течение 1 ч перед добавлением трипирролидинофосфина (71,6 мкл, 0,311 ммоль). 0,45 М тетразол в ACN (253 мкл, 0,114 ммоль) затем добавляли в 7 частях с 2 мин интервалом. TLC (EtOAc/Hept=2:1, RfSM=0,7, RfProd=0,0-0,6) было неполным. Добавляли 2× фосфорилирующего реагента и тетразола. Реакционную смесь перемешивали при к.т. В течение 10 мин. Ни при помощи UPLC-MS, ни TLC не наблюдали остатка SM. Реакционную смесь переносили в колбу, содержащую ацетонитрил (20,8 мл), воду (104 мкл, 5,776 ммоль) и пиридиновую трифторацетатную соль (421 мг, 2,178 ммоль). Смесь перемешивали в течение 10 мин. При помощи UPLC-MS наблюдали образование требуемого продукта. Реакционную смесь смешивали с EtOAc и промывали HCl [0,1 н]/солевым раствором, а затем нас. NHCO₃/солевым раствором для предотвращения снятия защитных групп с DMT. Водный слой снова экстрагировали EtOAc (1×). Объединенный органический слой сушили над Na₂SO₄, а затем фильтровали. Растворители и летучие вещества в фильтрате удаляли на ротационном испарителе с получением неочищенного продукта, который использовали на следующей стадии без дополнительной очистки.

methoxyphenyl)(phenyl)methoxy)methyl)-4-fluorotetrahydrofuran-3-yl) O-(((2R,3R,4R,5R)-5-(6-(allyloxy)-9H-purin-9-yl)- 4-fluoro-3-hydroxytetrahydrofuran-2-yl)methyl)-O-(2-cyanoethyl)phosphorothioate (120mg, 0.104mmol) in acetonitrile (4171µl, 79.852mmol) molecular sieves (3A, 1wt) were added at 20°C. The mixture was stirred at 20°C for 1 hour before the addition of tripyrrolidinophosphine (71.6 μl, 0.311 mmol). 0.45 M tetrazole in ACN (253 μl, 0.114 mmol) was then added in 7 parts at 2 min intervals. TLC (EtOAc/Hept=2:1, RfSM=0.7, RfProd=0.0-0.6) was incomplete. 2x phosphorylating reagent and tetrazole were added. The reaction mixture was stirred at rt. Within 10 min. Neither UPLC-MS nor TLC observed an SM residue. The reaction mixture was transferred to a flask containing acetonitrile (20.8 ml), water (104 μl, 5.776 mmol) and pyridine trifluoroacetate salt (421 mg, 2.178 mmol). The mixture was stirred for 10 minutes. UPLC-MS was used to observe the formation of the desired product. The reaction mixture was mixed with EtOAc and washed with HCl [0.1 N]/brine followed by sat. NHCO ₃ /brine to prevent deprotection of DMT. The aqueous layer was back extracted with EtOAc (1x). The combined organic layer was dried over Na ₂ SO ₄ and then filtered. Solvents and volatiles in the filtrate were removed on a rotary evaporator to give a crude product which was used in the next step without further purification.

The crude hydrophosphonate intermediate was dissolved in DCM (2635 µl, 40.946 mmol) and water (29.5 µl, 1.638 mmol) before adding dichloroacetic acid (135 µl, 1.638 mmol) in DCM (2635 µl, 40.946 mmol) at 20°C. The mixture was stirred at 20° C. for 5 minutes. The completion of the reaction was observed by UPLC-MS. The reaction was neutralized with pyridine (510 μl, 6.308 mmol). The volatiles were removed on a rotary evaporator and then on a highvac rotary evaporator. The residue was azeotroped with pyridine once more before being used for cyclization.

Crude unprotected DMT hydrogen phosphonate (80 mg, 0.087 mmol) was dissolved in pyridine (1831 µl, 22.639 mmol) before addition of 2-chloro-5,5-dimethyl-1,3,2-dioxaphosphinan 2-oxide (48.2 mg , 0.261 mmol) at r.t. The mixture was stirred at rt. within 10 min. No SM peak was observed by UPLC at 2.1 min. The reaction was quenched by the addition of water (47.1 μl, 2.612 mmol) and then 3H-benzo[c][1,2]dithiol-3-one (21.97 mg, 0.131 mmol) was added immediately. After 10 minutes, it was observed by UPLC that no starting material remained. The reaction mixture was stirred at rt. within 1 hour. No changes were observed by UPLC-MS. The reaction mixture was diluted with EtOAc and washed with sat. NaHCO ₃ and saline. The organic layer was dried over Na ₂ SO before filtration. The solvent evaporation residue was dissolved in MeOH before NH ₃ -H ₂ O was added. The mixture was stirred for 5 minutes before all solvent and volatiles were removed on a rotary evaporator and a highvac rotary evaporator.

ммоль) и 1,4-диоксане (496 мкл, 5,797 ммоль) перед добавлением TEA (39,6 мкл, 0,284 ммоль) и 1-(бромметил)-2-нитробензола (49,1 мг, 0,227 ммоль) при 20°С. Смесь перемешивали при 20°С в течение 16 ч. При помощи UPLC-MS наблюдали, что реакция не завершилась. Ни при помощи TLC (EtOAc), ни HPLC не наблюдали, что остался орто-нитробензилбромид. Добавляли 1-(бромметил)-2-нитробензол (49,1 мг, 0,227 ммоль) и TEA (39,6 мкл, 0,284 ммоль). Реакционную смесь перемешивали в течение 5 ч. Реакционную смесь обрабатывали нас. NHCO₃/солевым раствором и экстрагировали EtOAc/гепт. Органический слой сушили над Na₂SO₄ и фильтровали. Растворитель и летучие органические вещества в фильтрате удаляли на ротационном испарителе. Остаток очищали с применением Biotage (12 г Si-gel колонка, EtOAc в гептане = 0-100%, 15 об., 100%, 10 об., 10% МеОН в EtOAc, 6 об.). Фракции, содержащие требуемый продукт, объединяли. Выпариванием растворителя получали требуемый продукт. Преп.-TLC (EtOAc) использовали для выделения наиболее полярного изомера, что приводило к конечному продукту соединения 18. Другие два изомера разделяли с применением преп.-TLC (EtOAc/DCM=1:1, проводили 3 раза). Более полярный изомер превращали в соединение 19, а менее полярный изомер превращали в соединение 20.

mmol) and 1,4-dioxane (496 µl, 5.797 mmol) before adding TEA (39.6 µl, 0.284 mmol) and 1-(bromomethyl)-2-nitrobenzene (49.1 mg, 0.227 mmol) at 20°C . The mixture was stirred at 20° C. for 16 hours. It was observed by UPLC-MS that the reaction was not complete. Neither TLC (EtOAc) nor HPLC observed that ortho-nitrobenzyl bromide remained. 1-(bromomethyl)-2-nitrobenzene (49.1 mg, 0.227 mmol) and TEA (39.6 μl, 0.284 mmol) were added. The reaction mixture was stirred for 5 hours. The reaction mixture was treated with us. NHCO ₃ /brine and extracted with EtOAc/hept. The organic layer was dried over Na ₂ SO ₄ and filtered. The solvent and volatile organic substances in the filtrate were removed on a rotary evaporator. The residue was purified using Biotage (12 g Si-gel column, EtOAc in heptane = 0-100%, 15 vol., 100%, 10 vol., 10% MeOH in EtOAc, 6 vol.). Fractions containing the desired product were pooled. Evaporation of the solvent gave the desired product. Prep-TLC (EtOAc) was used to isolate the most polar isomer, resulting in the final product compound 18. The other two isomers were separated using prep-TLC (EtOAc/DCM=1:1, run 3 times). The more polar isomer was converted to compound 19 and the less polar isomer was converted to compound 20.

The most polar product from the first p-TLC (EtOAc), designated as compound 128-1: ¹ H NMR (500 MHz, chloroform-d) δ ppm 8.62 (s, 1H) 8.55 (s, 1H) 8.00-8.07 (m, 2Н) 7.99 (s, 1Н) 7.96 (s, 1Н) 7.56-7.64 (m, 2Н) 7.48-7.56 (m, 3Н) 7.42-7.48 (m, 1Н) 7.34-7.42 (m, 2Н) 7.29-7.34 ( m, 1H) 7.18-7.25 (m, 2H) 6.09-6.21 (m, 3H) 5.98-6.09 (m, 2H) 5.63-5.75 (m, 1H) 5.52-5.63 (m, 1H) 5.42-5.51 (m, 2H) 5.32 (dd, J=10.27, 0.98 Hz, 1H) 5.27 (dd, J=17.12, 1.47 Hz, 1H) 5.13 (d, J=5.87 Hz, 2 H) 5.09-5.12 (m, 1H) 4.95- 5.05 (m, 2H) 4.36-4.54 (m, 7H) 4.23-4.31 (m, 2H) 4.15-4.21 (m, 1H)

The more polar product from the second p-TLC, designated compound 128-2 (EtOAc/DCM=1:1, 3X) ¹ H NMR (400 MHz, methanol-d ₄ ) δ ppm 8.75 (s, 1H) 8.59 (s, 1Н) 8.43 (s, 1Н) 8.39 (s, 1Н) 8.01-8.07 (m, 2Н) 7.38-7.56 (m, 7Н) 7.27-7.35 (m, 1Н) 7.15-7.26 (m, 3Н) 6.45 (d, J=19.91 Hz, 1H) 6.38 (d, J=19.52 Hz, 1H) 5.95-6.26 (m, 4H) 5.83 (dd, J=51.54, 4.69 Hz, 1H) 5.73 (dd, J=51.54, 4.69 Hz, 1Н) 5.46-5.57 (m, 1Н) 5.28-5.36 (m, 1Н) 5.19-5.28 (m, 1Н) 5.16 (dt, J=5.56, 1.51 Hz, 2Н) 5.03-5.10(m, 1Н) 4.95 (br d, J=5.50 Hz, 2H) 4.41-4.62 (m, 4H) 4.28-4.40 (m, 2H) 4.12-4.27 (m, 4H)

Less polar product from the second p-TLC, designated compound 128-3 (EtOAc/DCM=1:1, 3X) ¹ HNMR (400 MHz, chloroform-d) δ ppm 8.53 (s, 1H) 8.36 (s, 1H) 7.95-8.02 (m, 2H) 7.95 (s, 1H) 7.89 (s, 1H) 7.53-7.61 (m, 2H) 7.44-7.53 (m, 4H) 7.36-7.44 (m, 2H) 7.25-7.31 (m, 1H) 7.12-7.20 (m, 2H) 5.91-6.18 (m, 4H) 5.85-5.91 (m, 1H) 5.72-5.79 (m, 1H) 5.61-5.72 (m, 1H) 5.47-5.57 (m, 1H) 5.40-5.47 (m, 1H) 5.25-5.32 (m, 1H) 5.15-5.23 (m, 1H) 4.99-5.14 (m, 3H) 4.92 (d, J=5.47 Hz, 2H) 4.52-4.60 (m, 2H) ) 4.37-4.52 (m, 4H) 4.35 (d, J=3.13 Hz, 1H) 4.30 (d, J=5.47 Hz, 1H) 4.25 (d, J=2.74 Hz, 1H) 4.20 (d, J=4.69 Hz , 1Н)

To a refluxed solution of 2-nitrobenzyl-protected CDN (10 mg, 8.696 µmol) in toluene (20 ml, 187.761 mmol) was added a solution of 2nd generation Hoveid-Grubbs catalyst ((1,3-bis-(2,4 ,6-trimethylphenyl)-2-imidazolidinylidene)dichloro(ortho-isopropoxyphenylmethylene)ruthenium available from SIGMA-ALDRITCH® cat. no. 569755; CAS 301224-40-8; mmol) in toluene (2 ml). The resulting solution was heated under reflux for 4 h (temp, oil bath 120-125°C). It was observed by TLC (EtOAc, Rf _sm =0.8, Rf _Prod =0.3) and UPLC-MS that the reaction was not complete. 1 ml Ru catalyst solution was added. The reaction mixture was heated at reflux for another 2 hours. There was still a lot of unreacted SM. Another 1 ml Ru catalyst solution was added. The reaction mixture was heated at reflux for another 2 hours. No SM was observed on reverse phase HPLC. The reaction mixture was cooled to rt. and quenched with DMSO (0.019 ml, 0.261 mmol). Evaporation of the solvent gave the crude product, which was purified on a silica gel column (10 g Si-gel column, eluent 50 to 100%, 15 vol, 100%, 10 vol). Fractions containing products with required. MS, were combined and evaporated on a rotary evaporator. The residue was purified again by prep-TLC (EtOAc).

Thiophenol (0.25 ml, 2.428 mmol) and TEA (0.25 ml, 1.794 mmol) at 20°C. The mixture was stirred at rt. within 3 h. Using UPLC, the completion of the transformation was observed.

Methanol (1.5 ml) was added followed by the addition of 29% NH ₃ in H ₂ O (1.0 ml). The resulting mixture was heated at 50°C for 6 h and cooled to rt.

The suspension of the reaction mixture was filtered and washed with water (25 ml). Precipitates formed in the filtrate after washing with water. The filtrate was again filtered to remove some of the solid.

The resulting filtrate was extracted with a mixture of tol./hep. (3×, 1/1, 25 ml each). The aqueous layer was concentrated in vacuo and then dissolved in water (6 ml). The precipitates were filtered again. UPLC was used to observe the formation of the desired product. The product was purified by reverse phase HPLC using the same method to purify other major closed CDN analogs.

For Compound 18, LCMS: MS m/z 748.11 [M+H] ⁺ 746.15 [M-H] ^-

For Compound 19, LCMS: MS m/z 748.06 [M+H] ⁺ 746.17 [M-H] ^-

For Compound 20, LCMS: MS m/z 748.09 [M+H] ⁺ 746.21 [M-H] ^-

Example 13 Synthesis of Compound 26

Compound 151

To a solution of compound 150 (0.70 g, 2.256 mmol) in pyridine (10.5 ml) at 0°C was added 4,4'-dimethoxytrityl chloride (0.803 g, 2.369 mmol). The resulting solution was stirred overnight while warming to ambient temperature. Upon completion (monitored by LCMS), sat. NH ₄ Cl solution (10 ml) and MTBE (20 ml) were added. The layers were separated and the aqueous layer was extracted with MTBE/EtOAc (3/1, 12 ml). The combined organic layers were washed with 30% aqueous NaCl (5 ml), dried over MgSO ₄ , filtered and concentrated in vacuo. Purification of the crude product by silica gel column chromatography (SiO ₂ 25 g, 50%-70% EtOAc in n-heptane) gave 0.942 g of compound 151.

¹ H NMR (400 MHz, CHLOROFORM-d) δ=8.47 (s, 1H), 8.11 (s, 1H), 7.43-7.35 (m, 2H), 7.30-7.26 (m, 4H), 7.25-7.17 (m , 3Н), 6.82-6.75 (m, 4Н), 6.28 (dd, J=2.3, 17.6 Hz, 1H), 6.22-6.10 (m, 1H), 5.63 (ddd, J=2.7, 4.7, 53.1 Hz, 1H ), 5.47 (qd, J=1.4, 17.3 Hz, 1H), 5.31 (dd, J=1.2, 10.6 Hz, 1H), 5.13 (t, J=1.4 Hz, 1H), 5.12 (t, J=1.2 Hz , 1H), 4.89-4.76 (m, 1H), 4.21 (td, J=3.2, 6.8 Hz, 1H), 3.78 (s, 6Н), 3.55 (dd, J=2.7, 10.9 Hz, 1H), 3.44 ( dd, J=4.3, 10.9 Hz, 1H), 2.29-2.21 (m, 1H).

Compound 152

To a solution of compound 151 (0.942 g, 1.538 mmol) in DMF (7.5 ml) at ambient temperature was added 3-((bis(diisopropylamino)phosphino)oxy)propanenitrile (0.927 g, 3.075 mmol), 0.45 M 1 ,2,3,4-tetrazole (2.8 ml, 1.2 mmol) and 1-methylimidazole (30 μl, 0.38 mmol). The resulting solution was stirred at ambient temperature for 4 hours. After completion (observed by LCMS), TEA (0.50 ml, 3.6 mmol), DMF (11.3 ml) and water (1.9 ml) were added. The resulting mixture was extracted three times with n-heptane (3 ml each time). The DMF layer was diluted with water (4 ml) and extracted with a mixture of toluene/n-heptane (1/1, 10 ml). The combined organic layers were washed twice with 30% aqueous NaCl solution (3 ml each), dried over MgSO ₄ , filtered and concentrated in vacuo. Purification of the residue by column chromatography on silica gel (SiO ₂ 50 g, 33%-40% EtOAc in n-heptane with 1% TEA) gave 1.115 g of compound 152 (3:2 diastereomeric mixture) as a white foamy solid.

Compound 153

дважды азеотропировали MeCN (20 мл каждый раз). К полученному остатку добавляли MeCN (20,0 мл) и 1Н-имидазол-4,5-дикарбонитрил (0,243 г, 2,058 ммоль). После завершения реакции (наблюдали при помощи LCMS) реакционную смесь обрабатывали (Е)-N,N-диметил-N'-(3-тиоксо-3Н-1,2,4-дитиазол-5-ил)формимидамидом (0,422 г, 2,058 ммоль). После завершения сульфурирования добавляли насыщенный раствор NaHCO₃ (20 мл) и МТВЕ (30 мл). Слои разделяли и водный слой экстрагировали смесью МТВЕ/EtOAc (15/15 мл). Объединенные органические слои промывали 30% водным раствором NaCl (10 мл), сушили над MgSO₄, фильтровали и концентрировали в вакууме. Очисткой остатка методом колоночной хроматографии на силикагеле (SiO₂ 50 г, 33% - 100% EtOAc в н-гептане с 1% TEA) получали 0,88 г соединения 153.

azeotroped twice with MeCN (20 ml each time). MeCN (20.0 ml) and 1H-imidazole-4,5-dicarbonitrile (0.243 g, 2.058 mmol) were added to the resulting residue. After completion of the reaction (observed by LCMS), the reaction mixture was treated with (E)-N,N-dimethyl-N'-(3-thioxo-3H-1,2,4-dithiazol-5-yl)formimidamide (0.422 g, 2.058 mmol). After completion of the sulfurization was added a saturated solution of NaHCO ₃ (20 ml) and MTBE (30 ml). The layers were separated and the aqueous layer was extracted with a mixture of MTBE/EtOAc (15/15 ml). The combined organic layers were washed with 30% aqueous NaCl (10 ml), dried over MgSO ₄ , filtered and concentrated in vacuo. Purification of the residue by column chromatography on silica gel (SiO ₂ 50 g, 33% - 100% EtOAc in n-heptane with 1% TEA) gave 0.88 g of compound 153.

LC/MS (ESI) m/z 1076.48 [M+Na] ⁺ .

Compound 154

температуре окружающей среды добавляли дифенилфосфит (0,323 мл, 1,67 ммоль). Реакционную смесь перемешивали в течение 1 ч и наблюдали при помощи LCMS. После завершения смесь добавляли в смесь насыщенного водного раствора NaHCO₃ (13,2 мл) и воды (4,4 мл), при этом поддерживали внутреннюю Т ниже 30°С, промывали EtOAc (8,8 мл). Полученную смесь перемешивали при температуре окружающей среды и гидролиз наблюдали при помощи LCMS. После завершения смесь дважды экстрагировали смесью EtOAc/МТВЕ (1/1, 26 мл). Объединенные органические слои промывали 30% водным раствором NaCl (3 мл), сушили над MgSO₄, фильтровали и концентрировали в вакууме. Остаток дважды азеотропировали толуолом (7 мл каждый раз). Неочищенный продукт растворяли в дихлорметане (6,6 мл) и обрабатывали водой (0,15 мл, 8,35 ммоль) и 6% дихлоруксусной кислотой (0,41 мл, 5,0 ммоль) в дихлорметане (6,6 мл) при температуре окружающей среды. После завершения снятия защитных групп с DMT (наблюдали при помощи LCMS) добавляли триэтилсилан (2,7 мл, 17 ммоль). Через 10 мин перемешивания полученную смесь обрабатывали пиридином (4,4 мл) и TEA (1 мл) и концентрировали в вакууме. Остаток трижды растирали в порошок с н-гептаном (8,8 мл каждый раз) и дважды азеотропировали MeCN. Неочищенный продукт (соединение 154) использовали на следующей стадии без дополнительной очистки.

diphenyl phosphite (0.323 mL, 1.67 mmol) was added at ambient temperature. The reaction mixture was stirred for 1 h and observed using LCMS. After completion, the mixture was added to a mixture of saturated aqueous NaHCO ₃ (13.2 ml) and water (4.4 ml), while maintaining the internal T below 30°C, washed with EtOAc (8.8 ml). The resulting mixture was stirred at ambient temperature and hydrolysis was monitored by LCMS. Upon completion, the mixture was extracted twice with EtOAc/MTBE (1/1, 26 ml). The combined organic layers were washed with 30% aqueous NaCl (3 ml), dried over MgSO ₄ , filtered and concentrated in vacuo. The residue was azeotroped twice with toluene (7 ml each time). The crude product was dissolved in dichloromethane (6.6 ml) and treated with water (0.15 ml, 8.35 mmol) and 6% dichloroacetic acid (0.41 ml, 5.0 mmol) in dichloromethane (6.6 ml) at ambient temperature. After completion of the deprotection of DMT (monitored by LCMS), triethylsilane (2.7 ml, 17 mmol) was added. After 10 min stirring, the resulting mixture was treated with pyridine (4.4 ml) and TEA (1 ml) and concentrated in vacuo. The residue was triturated with n-heptane three times (8.8 ml each) and azeotroped twice with MeCN. The crude product (compound 154) was used in the next step without further purification.

LC/MS (ESI) m/z 814.29 [M-H] ^- .

Compound 155

To a solution of compound 154 (0.681 g, 0.835 mmol) in pyridine (68 ml) at ambient temperature was added 2-chloro-5,5-dimethyl-1,3,2-dioxaphosphorinan-2-oxide (0.462 g, 2.505 mmol) . The resulting solution was stirred for 1 hour at ambient temperature and monitored by LCMS. Upon completion, water (0.45 ml, 25 mmol) (10 eq. DMOCP) and sulfur (0.134 g, 4.175 mmol) were added. After completion of sulfurization (monitored by LCMS), the reaction mixture was treated with saturated aqueous NaHCO ₃ (13.6 ml) and concentrated in vacuo. The residue was treated with EtOAc (27 ml) and water (13.6 ml). The layers were separated and the aqueous layer was extracted with a mixture of EtOAc/MTBE (1/1, 27 ml). The combined organic layers were washed with 30% aqueous NaCl (13.6 ml), dried over MgSO ₄ , filtered and concentrated in vacuo. Purification of the residue by column chromatography on silica gel (SiO ₂ 25 g, 0% - 10% MeOH in EtOAc) gave 0.693 g (theoretical yield) of compound 155.

LC/MS (ESI) m/z 830.23 [M+H] ⁺ .

Compound 156

To a solution of compound 155 (0.693 g, 0.835 mmol) in acetonitrile (14 ml) at ambient temperature was added tert-butylamine (14 ml, 131 mmol). The resulting solution was stirred for 10 min and observed by LCMS. Upon completion, the reaction mixture was concentrated in vacuo and azeotroped twice with MeCN. Acetonitrile (14 ml) and 2-nitrobenzyl bromide (0.541 g, 2.51 mmol) were added to the residue. After stirring overnight, the reaction mixture was concentrated in vacuo and purified by silica gel column chromatography (SiO ₂ 100 g, 75% - 100% EtOAc in n-heptane and 0% - 10% MeOH in EtOAc) to give 0.103 g of compound 156.

¹ H NMR (400 MHz, CHLOROFORM-d) δ=8.59 (s, 2H), 8.05 (br d, J=8.2 Hz, 2H), 8.03 (s, 2H), 7.58 (d, J=7.4 Hz, 2H ), 7.48 (t, J=7.4 Hz, 2H), 7.45-7.37 (m, 2H), 6.20 (br d, J=18.8 Hz, 2H), 6.21-6.08 (m, 2H), 5.98-5.83 (m , 2H), 5.69 (dd, J=4.7, 52.0 Hz, 1H), 5.47 (br d, J=17.2 Hz, 2H), 5.31 (br d, J=10.6 Hz, 2H), 5.14 (br d, J =5.5 Hz, 4H), 4.54 - 4.46 (m, 2H), 4.45-4.40 (m, 2H), 4.37-4.26 (m, 4H), 4.21-4.11 (m, 2H).

Based on the synthesis procedure, NMR data (symmetrical) and HPLC retention time (slowest eluting isomer), Applicants believed that compound 156 has RR phosphorus stereochemistry. Such a stereochemical determination will be subject to confirmation by X-ray crystallography.

Compound 157

To a solution of compound 156 (0.103 g, 0.098 mmol) in toluene (103 ml) under reflux was added a solution of the 2nd generation Hoveyd-Grubbs catalyst ((1,3-bis-(2,4,6-trimethylphenyl)-2- imidazolidinylidene)dichloro(ortho-isopropoxyphenylmethylene)ruthenium available from SIGMA-ALDRITCH® cat. no. 569755; CAS 301224-40-8; 31 mg, 0.049 mmol) and quinone (32 mg, 0.295 mmol) in toluene (10 mL). The mixture was stirred at reflux for 2 h and additional catalyst (16 mg, 0.025 mmol) was added. Upon completion, the reaction mixture was cooled to ambient temperature and treated with DMSO (0.14 ml, 2.0 mmol) overnight. The resulting mixture was concentrated in vacuo and purified by silica gel column chromatography (SiO ₂ 10 g, 0%-10% methanol in EtOAc) to give the desired product, which was further purified by prep. TLC (EtOAc) to give 3.6 mg of compound 157.

¹ H NMR (400 MHz, CHLOROFORM-d) δ=8.11 (dd, J=1.2, 8.2 Hz, 2H), 8.09 (s, 2H), 7.68 (s, 2H), 7.67-7.58 (m, 4H), 7.52-7.48 (m, 2H), 6.26 (d, J=17.6 Hz, 2H), 6.02-5.97 (m, 2H), 5.97-5.88 (m, 2H), 5.68 (dd, J=3.9, 50.8 Hz, 2Н), 5.15 (br d, J=10.9 Hz, 2Н), 4.99 (br d, J=10.9 Hz, 2H), 4.56-4.44 (m, 8Н), 4.25 (br t, J=6.1 Hz, 2H) .

Compound 26

To compound 157 (3.6 mg, 3.5 µmol) were added 1,4-dioxane (0.11 ml), thiophenol (0.045 ml, 0.44 mmol) and TEA (0.054 ml, 0.39 mmol). The resulting mixture was stirred at ambient temperature while the reaction was monitored by LCMS. After completion of the transformation, water (0.5 ml) was added. The resulting mixture was extracted three times with n-heptane/toluene (1/1, 0.4 ml each) and then with toluene (0.3 ml). The aqueous layer was concentrated in vacuo and treated with water (0.5 ml). The resulting solid was filtered off, washed with water (0.5 ml). Freeze drying of the combined filtrates gave 2.0 mg of the bis-TEA salt of Compound 26 as a white foamy solid.

¹ H NMR (400 MHz, METHANOL-d ₄ ) δ=8.67 (s, 2H), 8.18 (s, 2H), 6.41 (d, J=14.8 Hz, 2H), 6.07-6.01 (m, 2H), 5.56 (dd, J=3.1, 51.2 Hz, 2H), 5.48-5.41 (m, 2H), 5.08 (br d, J=12.1 Hz, 2H), 4.99-4.88 (m, 2H), 4.52 (br d, J =12.5 Hz, 2H), 4.40 (br d, J=9.8 Hz, 2H), 3.98 (dd, J=5.7, 12.3 Hz, 2H), 3.14 (q, J=7.4 Hz, 12H), 1.27 (t, J=7.4Hz, 18H).

Example 15 Synthesis of Compound 27

Compound 27 was obtained from compound 159, a by-product of step 11b of the pathway for compound 1 shown in FIG. 2B.

To a solution of compound 159 (2.0 g, 1.75 mmol) in methanol (24 ml) was added 28% ammonium hydroxide (12 ml). The resulting mixture was stirred at 50° C. for 5 hours and cooled to ambient temperature. Upon completion (observed by LCMS), the reaction mixture was cooled to ambient temperature, treated with water (20 ml) and extracted three times with a mixture of toluene/EtOAc (1/1, 24 ml each time). The aqueous layer was acidified with 1.0 M hydrochloric acid (3.5 ml, 3.5 mmol), stirred at ambient temperature for 30 minutes and 0° C. for 30 minutes. The resulting suspension was filtered, washed with water (20 ml). The cake was dried in a vacuum oven at 35° C. overnight and dissolved in a mixture of 28% NH ₄ OH (2 ml) and MeOH (20 ml). The resulting solution was concentrated in vacuo and treated with EtOH (4 ml) to obtain a suspension. The resulting solid was collected by filtration and dried in vacuo. Received 70 mg of compound 27 as a white solid.

¹ H NMR (400 MHz, METHANOL-d ₄ ) δ=9.03 (br s, 1H), 8.31 (br s, 1H), 8.26 (br s, 1H), 8.13 (br s, 1H), 6.43-6.24 ( m, 2H), 5.97-5.83 (m, 2H), 5.71 (br d, J=51.6 Hz, 1H), 5.64 (br d, J=50.0 Hz, 1H), 5.12-4.99 (m, 1H), 4.96 -4.90 (m, 1H), 4.68-4.32 (m, 6H), 4.14-3.97 (m, 2H), 3.77-3.62 (m, 2H).

Example 16 Synthesis of Compound 28

Compound 28 was prepared from compound 160, the product of step 11 of the pathway for compound 1 shown in Figure 2B, using the same method for compound 27.

¹ H NMR (400 MHz, METHANOL-d ₄ ) δ=8.70 (br s, 1H), 8.49 (br s, 1H), 8.24 (br s, 1H), 8.10 (br s, 1H), 6.52-6.26 ( m, 2H), 5.94-5.78 (m, 2H), 5.69 (br d, J=54.7 Hz, 1H), 5.48-5.26 (m, 1H), 5.14 (br d, J=53.1 Hz, 2H), 5.06 -4.94 (m, 1H), 4.74-4.19 (m, 4H), 4.10-3.93 (m, 2H), 3.76-3.58 (m, 2H)

Example 17 Synthesis of Compound 29

Compound 162

Compound 161 (5.0 g, 6.329 mmol) and but-2-yn-1,4-diol (1.907 g, 22.153 mmol) azeotroped twice with THF. The residue was dissolved in THF (50.0 ml) and toluene (75 ml). Triphenylphosphine (2.158 g, 8.228 mmol) was added and the resulting solution was cooled below 5°C. DIAD (1.60 ml, 8.23 mmol) was added dropwise while keeping the internal T below 10°C. The resulting solution was stirred at 0-5° C. while progress was monitored by LCMS. Upon completion, the reaction mixture was concentrated in vacuo to remove most of the THF. The residue was diluted with MTBE (50.0 ml) and washed twice with 30% aqueous NaCl solution (40.0 ml each) and twice with water (25 ml each). The organic solution was concentrated in vacuo and purified by silica gel column chromatography (SiO ₂ 100 g, 50%-70% EtOAc in n-heptane buffered with 1% TEA) to give 4.671 g of compound 162.

¹ H NMR (400 MHz, CHLOROFORM-d) δ=8.61 (s, 1H), 8.14 (s, 1H), 7.43 (d, J=7.2 Hz, 2H), 7.33-7.27 (m, 3H), 7.24- 7.18 (m, 8H), 7.05-7.01 (m, 2H), 6.76 (d, J=8.6 Hz, 4H), 6.20 (dd, J=2.7, 17.2 Hz, 1H), 5.59 (td, J=3.1, 53.1 Hz, 1H), 5.19 (br d, J=2.3 Hz, 2H), 4.93-4.83 (m, 1H), 4.18-4.15 (m, 1H), 4.01 (br d, J=6.3 Hz, 2H), 3.78 (s, 6H), 3.53 (dd, J=2.7, 10.9 Hz, 1H), 3.17 (dd, J=3.7, 11.1 Hz, 1H), 0.85 (s, 9H), 0.10 (s, 3H), 0.02 (s, 3H).

Compound 164

Compound 162 (4.671 g, 5.444 mmol), compound 163 (3.32 g, 6.805 mmol) and triphenylphosphine (1.856 g, 7.077 mmol) were dissolved in THF (56.1 ml) and cooled below 5°C. To the resulting solution was added DIAD (1.3 ml, 6.8 mmol), while maintaining the internal T below 10°C. After consumption of compound 162 was complete (monitored by LCMS), the reaction mixture was concentrated in vacuo and purified by silica gel column chromatography (SiO ₂ 340 g, 55%-70% EtOAc in n-heptane buffered with 1% TEA) to give 3.879 g of compound 164 as a white foamy solid.

¹ H NMR (400 MHz, CHLOROFORM-d) δ=8.51 (s, 1H), 8.45 (s, 1H), 8.20 (s, 1H), 8.05 (s, 1H), 7.41 (d, J=7.1 Hz, 2Н), 7.36-7.32 (m, 4Н), 7.28-7.13 (m, 8Н), 6.98 (t, J=7.8 Hz, 2Н), 6.79-6.74 (m, 4Н), 6.29 (dd, J=2.0, 16.4Hz, 1H), 6.19 (dd, J=2.7, 17.2Hz, 1H), 5.53 (ddd, J=2.7,4.7, 53.1Hz, 1H), 5.33 (ddd, J=2.0, 3.9, 53.1Hz, 1H ), 5.23-5.21 (m, 1H), 5.05-5.01 (m, 2Н), 4.99-4.94 (m, 2Н), 4.81 (ddd, J=4.3, 6.7, 16.7 Hz, 1H), 4.71-4.60 (m , 1H), 4.21-4.14 (m, 2H), 4.08-4.03 (m, 1H), 3.89 (dd, J=3.1, 11.7 Hz, 1H), 3.77 (s, 6H), 0.91 (s, 9H), 0.84 (s, 9Н), 0.09 (s, 3Н), 0.08 (s, 6Н), 0.00 (s, 3Н).

Compound 165

To a solution of compound 164 (1.5 g, 1.13 mmol) in pyridine (12.0 ml) was added diphenyl phosphite (0.35 ml, 1.8 mmol) at ambient temperature. The resulting solution was stirred at ambient temperature, this was observed using LCMS. After completion, the reaction mixture was added to a mixture of saturated aqueous NaHCO ₃ (22.5 ml) and water (7.5 ml), while maintaining the internal T below 30°C. After hydrolysis was complete (monitored by LCMS), the resulting mixture was extracted twice with EtOAc/MTBE (30.0/30.0 ml). The combined organic layers were washed with 30% aq. NaCl (15.0 ml), dried over MgSO4, filtered and concentrated in vacuo. The residue was azeotroped three times with toluene and dissolved in dichloromethane (11 ml), water (0.20 ml, 11 mmol) and 6% dichloroacetic acid (0.57 ml, 6.9 mmol) in dichloromethane (11 ml) at ambient temperature. After removal of the DMT group was complete (monitored by LCMS), triethylsilane (1.8 mL, 11 mmol) was added. After 10 min stirring, TEA (1.6 ml, 11 mmol) was added and the resulting mixture was concentrated in vacuo. The residue was dissolved in EtOAc (22.5 ml), washed with water (3.8 ml) and saturated aqueous NaHCO ₃ (8%) (3.0 ml), dried over MgSO ₄ , filtered and concentrated in vacuo. The crude product was triturated twice with n-heptane (33.8 ml) and a mixture of n-heptane (9.0 ml) and toluene (3.0 ml). The supernatant was decanted and the solid remaining at the bottom was dissolved in acetonitrile. The resulting solution was concentrated in vacuo and azeotroped twice in acetonitrile. The crude product, compound 165, was used in the next step without purification (allowable theoretical yield 100%).

LC/MS (ESI) m/z 1089.74 [M+H] ⁺ .

Compound 166

To a solution of compound 165 (1.231 g, 1.13 mmol) in pyridine (100 ml) at ambient temperature was added TEA (0.47 ml, 3.4 mmol) and DMOCP (0.375 mg, 2.03 ml). The resulting solution was stirred at ambient temperature, this was observed using LCMS. Upon completion, water (0.41 ml, 22.6 mmol) was added followed by sulfur (0.181 g, 5.651 mmol). After completion of the sulfurization (monitored by LCMS), saturated aqueous NaHCO ₃ (8%) (24.6 ml) and water (10 ml) were added. The resulting mixture was concentrated in vacuo to ~50 ml and extracted twice with MTBE/EtOAc (31/31 ml) each time. The combined organic layers were washed with 30% aq. NaCl (25 ml), dried over MgSO ₄ , filtered, concentrated in vacuo and purified by silica gel column chromatography (SiO ₂ 100 g, 0-10% methanol in ethyl acetate buffered with 1% TEA) to give compound 166 (0.484 g) .

LC/MS (EST) m/z 1103.65 [M+H] ⁺ .

Compound 167

Et ₃ N 3HF (0.581 ml, 3.563 mmol) was added to a solution of compound 166 (0.484 g, 402 mmol) in pyridine (1.2 ml) at ambient temperature. The resulting mixture was stirred at ambient temperature, this was observed using LCMS. After completion of the conversion, methoxytrimethylsilane (3.9 ml, 28 mmol) was added. After 20 minutes of stirring at ambient temperature, the supernatant was decanted. Toluene (5 ml) was added to the residue, and after 10 minutes of stirring, the toluene layer was decanted. The same operation was repeated once more. The obtained crude product was dissolved in dichloromethane (10 ml), washed with saturated aqueous NH ₄ Cl (27 wt.%) (5 ml) and 30% aq. NaCl (2.4 ml) and dried over MgSO ₄ . Filtration after concentration of the resulting organic layer gave 0.386 g of a pale brown solid which was azeotroped twice with MeCN. The resulting crude product was dissolved in MeCN (4.6 mL) and treated with 2-nitrobenzyl bromide (0.103 g, 0.475 mmol) at ambient temperature. After alkylation was complete (monitored by LCMS), the reaction mixture was concentrated in vacuo and purified by silica gel column chromatography (SiO ₂ 25 g, 0-5% MeOH in EtOAc) to give 0.251 g of compound 167 as a white solid.

¹ H NMR (400 MHz, CHLOROFORM-d) δ=8.42 (br s, 1H), 8.31 (br s, 1H), 8.22 (br s, 1H), 8.10-8.04 (m, 1H), 7.93 (s, 1H), 7.53-7.37 (m, 8H), 7.32-7.27 (m, 3H), 7.24-7.16 (m, 2H), 6.18 (br d, J=16.8 Hz, 1H), 6.26-6.14 (m, 1H ), 5.56 (d, J=53.1 Hz, 1H), 5.48-5.37 (m, 1H), 5.31 (brs, 1H), 5.15 (brd, J=17.6 Hz, 1H), 4.92 (d, J=17.6 Hz , 1H), 4.82 (d, J=17.2 Hz, 1H), 4.88-4.77 (m, 1H), 4.76-4.66 (m, 1H), 4.58-4.33 (m, 3H), 4.29 (br d, J= 5.9 Hz, 1H), 4.21 (br s, 1H), 4.13–4.06 (m, 1H), 3.93–3.79 (m, 2H).

Compound 168

Compound 167 (0.224 g, 0.222 mmol) and 3-

(bis(diisopropylamino)phosphinooxy)propanenitrile (0.081 ml, 0.255 mmol) was dissolved in MeCN (5 ml) and concentrated. The same operation was repeated two more times. The resulting mixture was dissolved in dichloromethane (2.2 ml), cooled to 0° C. and treated with diisopropylammonium tetrazolide (0.019 g, 0.111 mmol). The reaction mixture was brought to ambient temperature overnight and diluted with MeCN (2.2 ml). The resulting solution was added over 10 hours via a syringe pump to a solution of the pyridine trifluoroacetate salt (0.129 g, 0.665 mmol) in MeCN (18 ml). After additional stirring for 1 h, 3-(bis(diisopropylamino)phosphinooxy)propanenitrile (0.04 ml, 0.12 mmol) in MeCN (1 ml) was added over 4 h. (E)-N,N-dimethyl -N'-(3-thioxo-3H-1,2,4-dithiazol-5-yl)formimidamide (0.064 g, 0.311 mmol). After completion of the sulfurization (monitored by LCMS), the reaction mixture was concentrated in vacuo and the resulting residue was taken up in MTBE (4.5 ml). The resulting organic solution was washed with saturated aqueous NaHCO ₃ (8%) (4 ml) and water (2 ml). The combined aqueous layers were back extracted with MTBE/EtOAc (4/4 mL). The combined organic layers were washed twice with 30% aqueous NaCl (2 ml each), dried over MgSO ₄ , filtered, concentrated in vacuo. The residue was purified by silica gel column chromatography (SiO ₂ 25 g, 50%-100% EtOAc in n-heptane) to give 64 mg of compound 168.

¹ H NMR (400 MHz, CHLOROFORM-d) δ=8.57 (s, 1H), 8.18 (s, 1H), 8.06-8.01 (m, 1H), 7.91 (s, 1H), 7.62-7.56 (m, 2H ), 7.55-7.50 (m, 2Н), 7.49-7.43 (m, 3Н), 7.42-7.34 (m, 2Н), 7.30-7.18 (m, 5Н), 6.33 (d, J=15.2 Hz, 1H), 6.10 (d, J=20.7 Hz, 1H), 5.95-5.77 (m, 1H), 5.54 (dd, J=4.3, 52.8 Hz, 1H), 5.46 (dd, J=3.9, 50.8 Hz, 1H), 5.32 -5.18 (m, 1H), 5.10 (d, J=17.6 Hz, 1H), 4.89 (d, J=17.6 Hz, 1H), 4.85-4.81 (m, 2H), 4.78 (br d, J=12.1 Hz , 1H), 4.64 (br dd, J=4.1, 9.2 Hz, 1H), 4.55 (dd, J=2.7, 12.1 Hz, 1H), 4.52-4.44 (m, 3Н), 4.38-4.20 (m, 4Н) , 2.75 (td, J=6.3, 17.6 Hz, 1H), 2.58 (td, J=5.9, 17.2 Hz, 1H).

Compound 29

To a solution of compound 168 (40 mg, 0.035 mmol) in 1,4-dioxane (0.5 ml) was added thiophenol (0.24 ml, 2.3 mmol) and TEA (0.24 ml, 1.7 mmol). The resulting mixture was stirred at ambient temperature while the reaction was monitored by LCMS. Upon completion, methanol (0.64 ml) and 28% ammonium hydroxide (0.64 ml) were added. The resulting mixture was heated to 50°C, stirred for 5 h and cooled to ambient temperature. After deprotection was completed (monitored by LCMS), the resulting mixture was treated with water (0.80 ml) and extracted three times with n-heptane/toluene (1/1, 0.5 ml each time) followed by toluene (0.3 ml). The aqueous layer was concentrated in vacuo at 40-50° C. and treated with water (1 ml). The resulting solid was filtered off, washed with water (0.3 ml). The combined filtrates were treated with 1.0 M HCl (0.07 ml, 0.07 mmol). The resulting slurry was filtered and washed with water (1 ml). The filter cake was dissolved in 2.0 M ammonia (1.0 ml, 2.0 mmol) in MeOH. The resulting solution was concentrated in vacuo to 0.5 ml and treated with EtOH (0.5 ml). The same operation was repeated two more times. EtOAc (0.9 ml) was added dropwise to the resulting solution. There was precipitation. The resulting solid was collected by filtration, washed with 4/1 EtOAc/EtOH (0.4 ml) and dried under vacuum overnight at ambient temperature. 12 mg of compound 29 were obtained.

¹ H NMR (400 MHz, METHANOL-d ₄ ) δ=8.88 (br s, 1H), 8.50 (br s, 1H), 8.20 (br s, 1H), 8.12 (br s, 1H), 6.42-6.17 ( m, 2H), 5.84-5.54 (m, 1H), 5.52 (d, J=51.6 Hz, 1H), 5.06-4.78 (m, 2H), 4.77-4.55 (m, 3H), 4.49 (br d, J =10.2 Hz, 1H), 4.42 (br s, 2H), 4.09-3.77 (m, 4H).

Same reaction sequence

Example 18 Synthesis of Compound 25

Compound 170

To a solution of the 2nd generation Hoveid-Grubbs catalyst (0.996 g, 1.585 mmol) in dichloromethane (40.0 mL) was added prop-2-en-d5-1-ol (10.0 g, 158 mmol) at reflux. After 1 h, an additional 1 mol. % Hoveid-Grubbs 2nd generation catalyst (0.996 g, 1.585 mmol). The resulting solution was stirred at reflux overnight, cooled to ambient temperature and concentrated in vacuo. The residue was purified by silica gel column chromatography (Si02 340 g 0%-10% MeOH in EtOAc) to give 4.2 g of compound 170 as a brown oil.

¹³ C NMR (101 MHz, METHANOL-d ₄ ) δ=130.87 (t, J=23.8 Hz, 2C), 62.22 (quintal, J=21.9 Hz, 2C).

Compound 171

With compound 170 as starting material, compound 171 was prepared by the same reaction sequence (steps 1-11) as described in the synthetic route for compound 1 in FIG. 2A and FIG. 2B.

¹ H NMR (400 MHz, CHLOROFORM-d) δ=8.59 (s, 1H), 8.10 (s, 1H), 8.02-7.96 (m, 1H), 7.93 (s, 1H), 7.58-7.50 (m, 2H ), 7.48-7.37 (m, 5H), 7.34-7.27 (m, 2H), 7.24-7.14 (m, 4H), 6.90 (s, 1H), 6.28 (d, J=14.8 Hz, 1H), 6.14- 6.03 (m, 1H), 5.97-5.81 (m, 1H), 5.49 (dd, J=4.7, 52.3 Hz, 2Н), 5.43 (dd, J=3.9, 50.8 Hz, 1H), 5.30-5.15 (m, 1H), 4.75 (br d, J=12.1 Hz, 1H), 4.61 (brdd, J=4.3, 9.4 Hz, 1H), 4.53 (dd, J=2.9, 11.9 Hz, 1H), 4.47-4.41 (m, 3Н), 4.32-4.18 (m, 4Н), 2.79 (td, J=7.0, 17.2 Hz, 1H), 2.66 (td, J=6.3, 16.8 Hz, 1H)

Compound 25

With compound 171 as starting material, compound 25 was prepared by the same reaction sequence (steps 12-13) as described in FIG. 2A and FIG. 2B.

¹ H NMR (400 MHz, CD ₃ OD) δ=9.03 (br s, 1H), 8.30 (br s, 1H), 8.24 (br s, 1H), 8.10 (br s, 1H), 6.44-6.13 (m , 2H), 0.00 (d, J=52.4 Hz, 1H), 0.00 (d, J=51.2 Hz, 1H), 4.71-4.32 (m, 6H), 4.09-3.98 (m, 1H), 3.97-3.87 ( m, 1H).

Example 19 - Synthesis of compound 31 (either RpRp or SpSp)

Compound 17

A mixture of (E)-but-2-ene-1,4-diol (0.10 g, 1.135 mmol) and compound 172 (2.52 g, 2.55 mmol) was azeotroped twice with THF and dissolved in THF (5.0 ml) and toluene (7.5 ml). Triphenylphosphine (0.714 g, 2.724 mmol) was added to the resulting solution. The resulting solution was cooled below 5°C and treated with DIAD (0.53 ml, 2.72 mmol) while maintaining the internal T below 10°C. The resulting reaction mixture was stirred overnight while warming to ambient temperature. After completion of the reaction (monitored by LCMS), the reaction mixture was concentrated in vacuo and purified by silica gel column chromatography (SiO ₂ 50 g, pre-treated with 1% TEA in n-heptane/EtOAc (1/1), 50% - 66% EtOAc in n-heptane) to give 2.46 g of compound 173 as a white foamy solid. This material was used in the next step without further purification.

Compound 174

To a solution of compound 173 (2.387 g, 1.177 mmol) in acetonitrile (28.6 ml) was added water (0.085 ml, 4.7 mmol), pyridine trifluoroacetate salt (0.500 g, 2.589 mmol) at ambient temperature. After 1 min, 2-amino-2-methylpropane (19.1 ml, 182 mmol) was added. The resulting mixture was stirred at ambient temperature while the reaction was monitored by LCMS. Upon completion, the reaction mixture was concentrated in vacuo and azeotroped with MeCN. The residue was dissolved in dichloromethane (24 ml) and treated with water (0.42 ml) and 6% dichloroacetic acid (1.2 ml, 14 mmol) in dichloromethane (24 ml). After completion of the deprotection of DMT (monitored by LCMS), the reaction was quenched with pyridine (12 ml) and the reaction mixture was concentrated in vacuo. The resulting residue was treated with a mixture of n-heptane/toluene (15/15 ml) and the upper layer was decanted. The same operation was repeated once more. Drying the remaining residue in vacuo gave compound 174, which was used in the next step without further purification.

LC/MS (ESI) m/z 1151.64 [M+H] ⁺ .

Compound 175

To a solution of compound 174 (1.355 g, 1.177 mmol) in pyridine (271 ml) at ambient temperature was added TEA (0.98 ml, 7.1 mmol) and 2-chloro-5,5-dimethyl-1,3,2 -dioxaphosphinan 2-oxide (0.869 g, 4.708 mmol). The resulting solution was stirred at ambient temperature until all starting material was consumed (monitored by LCMS). After completion of the reaction, water (0.85 ml, 47 mmol) (10 eq. DMOCP) was added followed by sulfur (0.377 g, 11.77 mmol). The resulting mixture was stirred at ambient temperature while the reaction was monitored by LCMS. After completion of sulfurization, the reaction mixture was treated with saturated aqueous NaHCO ₃ (27 ml) and water (10 ml) and concentrated in vacuo. To the resulting residue were added MTBE/EtOAc (34/34 ml), saturated aqueous NaHCO ₃ (27 ml) and water (20 ml). The layers were separated and the aqueous layer was extracted with a mixture of EtOAc/MTBE (34/34 ml). The combined organic layers were washed with 30% aqueous NaCl (7 ml), dried over MgSO ₄ , filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (SiO ₂ 50 g, 0%-20% MeOH in EtOAc with 1% TEA) to give 56 mg of compound 175 (symmetrical based on NMR; either RpRp or SpSp isomer).

¹ H NMR (400 MHz, CHLOROFORM-d) δ=9.09 (s, 2H), 8.46 (s, 2H), 7.56-7.05 (m, 10H), 6.37 (d, J=8.2 Hz, 2H), 6.28- 6.22 (m, 2H), 5.29 (ddd, J=4.7, 7.8, 11.7 Hz, 2H), 5.00 (br d, J=13.7 Hz, 2H), 4.84 (d, J=4.3 Hz, 2H), 4.76 ( br d, J=13.3 Hz, 2H), 4.31 (br s, 2H), 4.28-4.20 (m, 2H), 4.16-4.08 (m, 2H), 3.00-2.77 (m, 12H), 1.12-0.99 ( m, 18H), 0.96 (s, 18H), 0.34 (s, 6H), 0.25 (s, 6H).

Compound 31

To a solution of compound 175 (56 mg, 0.0405 mmol) in methanol (1.1 ml) was added 28% ammonium hydroxide (0.22 ml). The resulting mixture was stirred at 50° C. for 5 hours, cooled to ambient temperature, concentrated in vacuo and azeotroped twice with MeCN. Pyridine (0.22 ml), TEA (0.11 ml) and TEA 3HF (0.11 ml, 0.69 mmol) were added to the resulting residue. The resulting mixture was stirred at 50° for 5 h, cooled to ambient temperature, treated with methoxytrimethylsilane (0.90 ml, 6.50 mmol) for 30 min. The resulting mixture was concentrated in vacuo and dissolved in water (5 ml). The resulting aqueous solution was extracted three times with toluene (5 ml each) and concentrated in vacuo. The crude product was dissolved in 1 ml of water, filtered through a syringe filter and acidified with 1 N HCl until pH<3. The resulting mixture was stored in a freezer (5°C) for 2 days and the resulting solid was collected by filtration. The solid was dissolved in 2 M NH ₃ in MeOH (1.5 ml) and concentrated in vacuo to give 2.8 mg of compound 31 as a mixture of NH ₃ and TEA salt. The product was dissolved in EtOH (1 ml), treated with 0.1 ml TEA, concentrated to 0.5 ml and stored in a freezer (-20°C) overnight. The supernatant was removed and the remaining solid was further dried in vacuo to give 0.8 mg of the bis TEA salt of Compound 31 (symmetrical based on NMR; either RpRp or SpSp isomer).

¹ H NMR (400 MHz, METHANOL-d ₄ ) δ=8.26-7.83 (m, 4H), 6.37 (br s, 2H), 5.89 (br s, 2H), 5.36-5.07 (m, 2H), 4.95 ( br d, J=3.5 Hz, 2H), 4.58 (br dd, J=6.3, 10.6 Hz, 2H), 4.43 (br s, 2H), 4.12-4.00 (m, 4H), 3.89-3.70 (m, 2H ), 3.19 (q, J=7.2Hz, 12H), 1.30 (t, J=7.4Hz, 18H)

Example 20 Synthesis of Compound 32

Compound 178

To a solution of compound 177 (2.0 g, 2.532 mmol) and (+)-2,3-ortho-isopropylidene-L-threitol (compound 176) (1.232 g, 7.595 mmol) in THF (20.0 ml) and toluene (30.0 ml) triphenylphosphine (0.930 g, 3.544 mmol) was added. The resulting solution was cooled below 5°C and treated with DIAD (0.64 ml, 3.3 mmol). The reaction mixture was heated to ambient temperature. After 8 hours stirring at ambient temperature, additional DIAD (0.64 ml) and PPh ₃ (0.93 g) were added. After stirring overnight at ambient temperature, the reaction mixture was stirred at 40° C. for 2 days and concentrated in vacuo. Purification of the residue by column chromatography on silica gel (SiO ₂ 100 g, pretreated with 1% TEA in n-heptane/EtOAc (1/1), 50%-70% EtOAc in n-heptane) gave 0.524 g of compound 178 as a white foamy solid.

¹ H NMR (400 MHz, CHLOROFORM-d) δ=8.62 (s, 1H), 8.09 (s, 1H), 7.40-7.09 (m, 12H), 7.01-6.92 (m, 2H), 6.80-6.73 (m , 4Н), 6.16 (dd, J=2.7, 16.8 Hz, 1H), 5.49 (ddd, J=3.1, 4.3, 53.1 Hz, 1H), 4.85-4.75 (m, 2Н), 4.63 (dd, J=3.9 , 14.8 Hz, 1H), 4.30-4.19 (m, 2Н), 4.18-4.12 (m, 1H), 3.94 (tt, J=3.5, 8.2 Hz, 1H), 3.84 (td, J=5.5, 12.1 Hz, 1H), 3.78 (s, 6Н), 3.53 (dd, J=2.7, 10.9 Hz, 1H), 3.17 (dd, J=3.5, 10.9 Hz, 1H), 2.94 (br t, J=5.5 Hz, 1H) , 0.89 (s, 6Н), 0.83 (s, 9Н), 0.08 (s, 3Н), -0.01 (s, 3Н)

Compound 180

To a solution of compound 178 (0.409 g, 0.84 mmol) in THF (6 ml) was added triphenylphosphine (0.191 g, 0.728 mmol) and DIAD (0.142 ml, 0.728 mmol). After 40 h stirring at ambient temperature, the reaction mixture was concentrated in vacuo and purified by column chromatography (SiO ₂ 50 g, pretreated with 1% TEA in n-heptane/EtOAc (1/1), 50%-66% EtOAc in n- heptane) to give 0.533 g of compound 180.

LC/MS: LRMS (ESI) m/z 1403.86 [M+H] ⁺ .

With compound 180 as starting material, compound 181 was prepared by the same reaction sequence (step 5-11) as described in FIG. 2A and FIG. 2B.

¹ H NMR (400 MHz, CHLOROFORM-d) δ=8.72 (s, 1H), 8.14-8.06 (m, 1H), 7.93 (s, 1H), 7.69 (s, 1H), 7.62-7.53 (m, 6H ), 7.53-7.45 (m, 1H), 7.43-7.33 (m, 2H), 7.30-7.24 (m, 3H), 7.19-7.12 (m, 2H), 6.11 (d, J=16.8 Hz, 1H), 6.05 (d, J=19.5 Hz, 1H), 5.89 (dd, J=3.5, 52.3 Hz, 1H), 5.57-5.46 (m, 1H), 5.41 (dd, J=3.5, 53.1 Hz, 1H), 5.18 -5.04 (m, 1H), 4.83 (dd, J=4.7, 14.4 Hz, 1H), 4.78-4.73 (m, 1H), 4.70 (br d, J=12.1 Hz, 1H), 4.64-4.48 (m, 4Н), 4.46-4.35 (m, 4Н), 4.33-4.27 (m, 2Н), 4.16-4.06 (m, 2Н), 4.05-3.96 (m, 1H), 2.55 (t, J=6.1 Hz, 2H) , 1.26 (s, 6H).

Compound 182

To a solution of compound 181 (3.2 mg, 2.629 µmol) in dichloromethane (0.5 ml) was added tert-butylamine (0.5 ml, 4.7 mmol). After 30 minutes of stirring, the reaction solution was concentrated in vacuo and azeotroped twice with MeCN. The residue was dissolved in acetonitrile (1 ml) and treated with 1-(bromomethyl)-2-nitrobenzene (1.7 mg, 7.9 μmol). After completion of the alkylation (observed by LCMS), the reaction mixture was concentrated by purging with nitrogen. Purification of the residue by column chromatography on silica gel (SiO ₂ 4 g, 80%-100% EtOAc in n-heptane) gave 2 mg of compound 182.

¹ H NMR (400 MHz, CHLOROFORM-d) δ=8.12 (dd, J=1.4, 8.0 Hz, 1H), 8.03-8.00 (m, 1H), 8.00 (s, 1H), 7.93 (s, 1H), 7.70 (s, 1H), 7.70-7.29 (m, 13H), 7.21 (dt, J=2.7, 7.6 Hz, 4H), 6.07 (d, J=19.9 Hz, 1H), 5.94 (d, J=21.1 Hz , 1H), 5.91-5.77 (m, 1H), 5.81 (dd, J=4.3, 51.5 Hz, 1H), 5.62-5.49 (m, 1H), 5.54 (dd, J=3.1, 52.7 Hz, 2H), 4.83 (q, J=5.9 Hz, 1H), 4.72-4.63 (m, 2H), 4.61-4.28 (m, 13H), 1.42 (s, 3H), 1.28 (s, 3H).

Compound 32

Acetic acid (0.8 ml) and water (0.2 ml) were added to compound 182 (2.0 mg, 1.5 μmol). The resulting mixture was stirred at ambient temperature for 14 hours and at 45-50° C. for 24 hours, concentrated in vacuo and azeotroped twice with toluene. To the residue was added 1,4-dioxane (0.12 ml), thiophenol (60 μl) followed by TEA (60 μl). The resulting mixture was stirred at ambient temperature while the reaction was monitored by LCMS. Upon completion, methanol (160 μl) and 28% ammonium hydroxide (160 μl) were added. The resulting mixture was stirred at 50° C. until debenzoylation was complete (monitored by LCMS). Upon completion, water (0.3 ml) was added. The resulting solid was filtered off, washed with water (0.2 ml). The filtrate was extracted twice with toluene (0.5 ml each time) and concentrated in vacuo at 40-50°C. The residue was treated with water (0.5 ml) and the resulting solid was filtered off, washed with water (0.1 ml), HPLC purification of the combined aqueous layers under the conditions described below gave 1.5 mg of compound 32.

LC/MS: LRMS (ESI) m/z 781.23 [M+H] ⁺ .

Preparative HPLC conditions for compound 32:

Example 21 Synthesis of Compound 33 and Compound 34

To a solution of 9-((2R,3R,4R,5R)-3-fluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-1,9-dihydro-6H-purin-6-one, compound 183 (5.00 g, 18.5 mmol) in pyridine (75 ml) Ac ₂ O (7.0 ml, 74 mmol) and DMAP (0.565 g, 4.626 mmol) were added at 0°C. The resulting mixture was warmed to ambient temperature and stirred while the reaction was monitored by LCMS. Upon completion, the reaction mixture was concentrated in vacuo and treated with EtOAc (200 ml) and water (50 ml). Sedimentation took place. The resulting solid was collected by filtration and washed with MTBE. Drying in vacuo at 40° C. overnight gave 5.81 g of compound 193 as a white solid.

¹ H NMR (400 MHz, DMSO-d ₆ ) δ=12.53 (br s, 1H), 8.33 (s, 1H), 8.15 (d, J=4.3 Hz, 1H), 6.37 (dd, J=2.7, 19.1 Hz, 1H), 5.86 (ddd, J=2.7, 5.1, 51.6 Hz, 1H), 5.62 (ddd, J=5.5, 7.0, 16.0 Hz, 1H), 4.49-4.38 (m, 2H), 4.26 (dd, J=4.7, 12.1 Hz, 1H), 2.19 (s, 3H), 2.04 (s, 3H).

Compound 184

To a solution of compound 193 in dichloromethane (40.0 ml) at 0° C. were slowly added DMF (1.3 ml, 16.9 mmol) and thionyl chloride (1.28 ml, 17.5 mmol). The resulting mixture was heated to reflux and stirred until all starting material was consumed (monitored by LCMS). Upon completion, the reaction mixture was cooled to ambient temperature and treated with saturated aqueous NaHCO ₃ (40 ml). The layers were separated and the aqueous layer was extracted twice with dichloromethane (30 ml each). The combined organic layers were dried over MgSO ₄ , filtered and concentrated in vacuo to give 2.81 g of compound 184 as a pale brown oil.

¹ H NMR (400 MHz, CHLOROFORM-d) δ=8.79 (s, 1H), 8.30 (s, 1H), 6.29 (dd, J=2.0, 18.0 Hz, 1H), 5.79 (ddd, J=1.6, 4.7 , 51.6 Hz, 1H), 5.53 (ddd, J=5.1, 7.6, 17.8 Hz, 1H), 4.58-4.48 (m, 2Н), 4.33 (dd, J=3.9, 12.5 Hz, 1H), 2.20 (s, 3Н), 2.07 (s, 3Н).

Compound 194

To a solution of compound 184 (0.22 g, 0.59 mmol) in a mixture of THF (7.7 ml) and NMP (0.77 ml) at ambient temperature was added iron (III) acetylacetonate (0.021 g, 0.059 mmol) and 0.5 M 3-butenylmagnesium bromide (1.77 mL, 0.885 mmol) in THF. After completion of the reaction (monitored by LCMS), MTBE (10 ml) and 0.1 N HCl (10 ml) were added. The resulting mixture was stirred at ambient temperature for 10 minutes. The layers were separated and the aqueous layer was extracted with a mixture of EtOAc/MTBE (1/1, 10 ml). The combined organic layers were washed with 30% aqueous NaCl (5 ml), dried over MgSO ₄ and concentrated in vacuo. The crude product was used in the next step without further purification.

¹ H NMR (400 MHz, CHLOROFORM-d) δ=8.90 (s, 1H), 8.16 (s, 1H), 6.26 (dd, J=2.0, 18.4 Hz, 1H), 5.98-5.85 (m, 1H), 5.90-5.73 (m, 1H), 5.63 (ddd, J=4.9, 7.5, 17.7 Hz, 1H), 5.12-5.05 (m, 1H), 4.98 (dd, J=1.6, 10.2 Hz, 1H), 4.54- 4.50 (m, 2H), 4.31 (dd, J=5.1, 12.9 Hz, 1H), 3.30 (t, J=7.4 Hz, 2H), 2.70-2.64 (m, 2H), 2.19 (s, 3H), 2.05 (s, 3H).

Compound 185

The crude product of compound 194 (0.232 g, 0.590 mmol in theory) was dissolved in methanol (1.5 ml) and treated with 2.0 M ammonia (1.5 ml, 3.0 mmol) in MeOH at ambient temperature. After completion of the diacetylation, the reaction mixture was concentrated in vacuo and purified by silica gel column chromatography (SiO ₂ 10 g, 0%-5% MeOH in EtOAc) to give 0.17 g of compound 185.

LCMS: MS (ESI) m/z 309.20 [M+H] ⁺

Compound 195

To a solution of compound 185 (1.22 g, 2.928 mmol) in pyridine (9.0 ml) was added 4,4'-(chloro(phenyl)methylene)bis(methoxybenzene) (1.042 g, 3.075 mmol) at 0°. The reaction mixture was allowed to warm to ambient temperature while the reaction was monitored by LCMS. Upon completion, MTBE (18 ml) and saturated aqueous NaHCO ₃ (9.0 ml) were added. After 10 minutes of stirring, the layers were separated and the aqueous layer was extracted with MTBE (18.0 ml). The combined organic layers were washed with 30% aqueous NaCl (10 ml), filtered and concentrated in vacuo. Purification by silica gel column chromatography (SiO ₂ 50 g, 50%-100% ethyl acetate in heptane with 1% TEA) gave 1.53 g of compound 195 as an orange solid.

¹ H NMR (400 MHz, CHLOROFORM-d) δ=8.86-8.78 (m, 1H), 8.21 (s, 1H), 7.40-7.36 (m, 2H), 7.31-7.18 (m, 7H), 6.79 (d , J=8.6 Hz, 4H), 6.29 (dd, J=2.3, 17.2 Hz, 1H), 5.92 (tdd, J=6.4, 10.4, 17.0 Hz, 1H), 5.70 (ddd, J=2.7, 4.7, 52.8 Hz, 1H), 5.09 (dd, J=1.6, 17.2 Hz, 1H), 4.97 (dd, J=1.6, 10.2 Hz, 1H), 4.90-4.77 (m, 1H), 4.26-4.19 (m, 1H) , 3.78 (s, 6H), 3.56 (dd, J=3.1, 10.9 Hz, 1H), 3.43 (dd, J=4.3, 10.9 Hz, 1H), 3.33-3.28 (m, 2H), 2.70-2.64 (m , 2H), 2.20 (dd, J=2.5, 7.2 Hz, 1H).

Compound 186

To a solution of compound 195 (1.530 g, 2.505 mmol) in dichloromethane (23 mL) at ambient temperature were added 3-(bis(diisopropylamino)phosphinooxy)propanenitrile (1.20 mL, 3.76 mmol) and diisopropylammonium tetrazolide (0.493 g, 2.881 mmol). The reaction mixture was stirred at ambient temperature while progress was monitored by LCMS. Upon completion, the reaction mixture was concentrated in vacuo and purified by silica gel column chromatography (SiO ₂ 50 g, 40%-66% EtOAc in n-heptane with 1% TEA) to give 1.80 g of compound 186 as a pale orange foamy solid substances.

Compounds 196 and 197

Compound 186 (1.8 g, 2.22 mmol) and compound 185 (1.110 g, 2.664 mmol) were dissolved in acetonitrile (21.6 ml) and concentrated in vacuo. The same operation was repeated once more. The resulting mixture was dissolved in acetonitrile (21.6 mL) and treated with pyridinium trifluoroacetate (0.514 g, 2.664 mmol) at ambient temperature. After completion of the reaction (observed by LCMS), ((dimethylaminomethylidene)amino)-3H-1,2,4-dithiazolin-3-thione (0.911 g, 4.439 mmol) was added and the resulting mixture was stirred at ambient temperature. After completion of the sulfurization (monitored by LCMS), saturated aqueous NaHCO ₃ solution (30 ml) was added and the resulting mixture was extracted three times with MTBE (30 ml each time). The combined organic layers were washed with 30% aqueous NaCl (20 ml), dried over MgSO ₄ , filtered and concentrated in vacuo. Purification by column chromatography on silica gel (SiO ₂ 100 g, 0%-10% MeOH in EtOAc with 1% TEA) gave 0.481 g of compound 196 and 1.315 g of compound 197.

Compound 196: LCMS: MS (ESI) m/z 1073.25 [M+Na] ⁺

Compound 197: LCMS: MS (ESI) m/z 995.23 [MH] ^-

Connection188

To a TEA solution of compound 197 salt (1.315 g, 1.197 mmol) in MeCN (20 ml) at ambient temperature was added 2-nitrobenzyl bromide (0.388 g, 1.796 mmol). The resulting solution was stirred at ambient temperature while the reaction was monitored by LCMS. Upon completion, the reaction mixture was concentrated in vacuo and purified by silica gel column chromatography (SiO ₂ 100 g, 0%-5% MeOH in EtOAC) to give 0.90 g of compound 188.

LCMS: MS (ESI) m/z 1154.29 [M+Na] ⁺

Compound 198

To a solution of compound 188 (1.35 g, 1.192 mmol) in toluene (540 ml) was added a solution of 2nd generation Hoveyd-Grubbs catalyst (0.187 g, 0.298 mmol) at gentle reflux (110-112°C internal T). ) and quinone (0.322 g, 2.981 mmol)) in toluene (20 mL). The resulting solution was stirred at 110-112° C. while the reaction was monitored by LCMS. After 4 hours additional catalyst (0.10 g, 0.16 mmol) was added and stirring was continued at reflux. Upon completion, the reaction mixture was cooled to ambient temperature and treated with DMSO (2.54 ml, 35.8 mmol). After 3 h stirring, the reaction mixture was concentrated in vacuo and purified by silica gel column chromatography (SiO ₂ 50 g, 0%-15% MeOH in EtOAC) to give a mixture of compound 198 and compound 199. The mixture was dissolved in dichloromethane (2 ml) and worked up water (0.021 ml, 1.2 mmol) and 6% dichloroacetic acid (0.098 ml, 1.2 mmol) in dichloromethane (2 ml). After 10 min the reaction was quenched with pyridine (1 ml) and concentrated in vacuo. The residue was dissolved in dichloromethane (50 ml) and washed twice with 30% aqueous NaCl solution (15 ml each) and dried over MgSO ₄ . Purification of the crude product by silica gel column chromatography (SiO ₂ 50 g, 5%-10% MeOH in DCM) gave 0.25 g of compound 198.

Compound 199: LCMS: MS (ESI) m/z 1104.31 [M+H] ⁺

Compound 198: LCMS: MS (ESI) m/z 802.16 [M+H] ⁺

Compound 190

ммоль) и 3-(бис(диизопропиламино)фосфиноокси)пропаннитрил (0,111 мл, 0,349 ммоль) растворяли в MeCN (10 мл) и концентрировали в вакууме. Ту же операцию повторяли еще два раза. Полученную смесь растворяли в дихлорметане (2,3 мл), охлаждали до 0-5°С и обрабатывали диизопропиламмонием тетразолидом (0,025 г, 0,145 ммоль). Реакционную смесь нагревали до температуры окружающей среды всю ночь, а затем разбавляли ацетонитрилом (2,3 мл). Полученный раствор добавляли в течение 7 ч при помощи шприцевого насоса к раствору пиридиновой трифторацетатной соли (0,168 г, 0,872 ммоль) в MeCN (18,6 мл). Затем добавляли 3-(бис(диизопропиламино)фосфиноокси)пропаннитрил (40 мг, 0,133 ммоль) в MeCN (2 мл) в течение 2 часов. После 1 ч перемешивания добавляли (E)-N,N-диметил-N'-(3-тиоксо-3Н-1,2,4-дитиазол-5-ил)формимидамид (0,119 г, 0,581 ммоль) и реакционный раствор перемешивали до завершения сульфурирования (наблюдали при помощи LCMS). После завершения смесь концентрировали в вакууме, растворяли в МТВЕ (5 мл) и обрабатывали насыщенным водным NaHCO₃ (3,50 мл) и водой (1,2 мл). Слои разделяли и водный слой экстрагировали смесью МТВЕ/EtOAc (5/3 мл). Объединенные органические слои промывали дважды 30% водным раствором NaCl (2,3 мл), сушили над MgSO₄, фильтровали и концентрировали в вакууме. Очисткой неочищенного продукта методом колоночной хроматографии на силикагеле (SiO₂ 25 г, 0%-20% МеОН в EtOAc) получали 119 мг соединения 190 (58 мг более быстро элюирующих изомеров и 61 мг более медленно элюирующих изомеров).Compound 198 (0.233 g,

mmol) and 3-(bis(diisopropylamino)phosphinooxy)propanenitrile (0.111 ml, 0.349 mmol) were dissolved in MeCN (10 ml) and concentrated in vacuo. The same operation was repeated two more times. The resulting mixture was dissolved in dichloromethane (2.3 ml), cooled to 0-5° C. and treated with diisopropylammonium tetrazolide (0.025 g, 0.145 mmol). The reaction mixture was warmed to ambient temperature overnight and then diluted with acetonitrile (2.3 ml). The resulting solution was added over 7 hours using a syringe pump to a solution of the pyridine trifluoroacetate salt (0.168 g, 0.872 mmol) in MeCN (18.6 ml). Then 3-(bis(diisopropylamino)phosphinooxy)propanenitrile (40 mg, 0.133 mmol) in MeCN (2 ml) was added over 2 hours. After 1 hour stirring, (E)-N,N-dimethyl-N'-(3-thioxo-3H-1,2,4-dithiazol-5-yl)formimidamide (0.119 g, 0.581 mmol) was added and the reaction solution was stirred until completion of sulfurization (monitored by LCMS). Upon completion, the mixture was concentrated in vacuo, dissolved in MTBE (5 ml) and treated with saturated aqueous NaHCO ₃ (3.50 ml) and water (1.2 ml). The layers were separated and the aqueous layer was extracted with a mixture of MTBE/EtOAc (5/3 ml). The combined organic layers were washed twice with 30% aqueous NaCl (2.3 ml), dried over MgSO ₄ , filtered and concentrated in vacuo. Purification of the crude product by column chromatography on silica gel (SiO ₂ 25 g, 0%-20% MeOH in EtOAc) gave 119 mg of compound 190 (58 mg of faster eluting isomers and 61 mg of slower eluting isomers).

Compound 191 (SpRp isomer)

To a solution of the more rapidly eluting isomers of compound 190 (58 mg, 0.062 mmol) in dichloromethane (1.8 ml) was added tert-butylamine (1.2 ml, 11 mmol). The resulting solution was stirred at ambient temperature until all starting material was consumed (monitored by LCMS). Upon completion, the reaction mixture was concentrated in vacuo, azeotroped twice with acetonitrile and dissolved in acetonitrile (1.7 ml). To the resulting solution was added 1-(bromomethyl)-2-nitrobenzene (40.3 mg, 0.187 mmol). The reaction mixture was stirred at ambient temperature while the reaction was monitored by LCMS. After alkylation was completed, the reaction mixture was concentrated in vacuo and purified by silica gel column chromatography (SiO ₂ 10 g, 0%-5% MeOH in EtOAC) to give 19 mg of compound 191 (SpRp isomer).

¹ H NMR (400 MHz, CHLOROFORM-d) δ=8.42 (s, 1H), 8.27 (s, 1H), 8.13 (dd, J=1.2, 8.2 Hz, 1H), 8.05 (s, 1H), 7.98 ( d, J=7.8 Hz, 1H), 7.69-7.67 (m, 1H), 7.61-7.34 (m, 5H), 6.33 (d, J=17.2 Hz, 1H), 6.19 (d, J=19.9 Hz, 1H ), 6.09-5.93 (m, 1H), 5.82-5.66 (m, 2H), 5.67 (dd, J=3.5, 50.8 Hz, 1H), 5.35-5.32 (m, 2H), 4.62-4.29 (m, 11H ), 3.35-3.28 (m, 1H), 3.18-3.14 (m, 2H), 3.01-2.91 (m, 1H), 2.84-2.78 (m, 1H), 2.60-2.49 (m, 3H).

Compound 192 (RpRp isomer)

обрабатывали такой же реакционной последовательностью, как описано для соединения 191, с получением 14 мг соединения 192 и 7 мг соединения 191.The slower eluting isomer of compound 190

treated with the same reaction sequence as described for compound 191 to give 14 mg of compound 192 and 7 mg of compound 191.

¹ H NMR (400 MHz, CHLOROFORM-d) δ=8.18 (s, 2H), 8.10 (s, 2H), 8.10 (dd, J=1.0, 8.0 Hz, 2H), 7.66-7.57 (m, 4H), 7.53-7.46 (m, 2H), 6.31 (d, J=17.6 Hz, 2H), 6.19-6.05 (m, 2H), 5.84 (dd, J=4.3, 50.8 Hz, 2H), 5.39-5.30 (m, 2Н), 4.58-4.46 (m, 6Н), 4.45-4.39 (m, 2Н), 4.19 (ddd, J=3.1, 6.3, 10.2 Hz, 2Н), 3.32 (td, J=3.5, 15.6 Hz, 2Н) , 3.06 (td, J=6.3, 15.2 Hz, 2H), 2.67-2.57 (m, 4H).

Based on the synthetic procedure, NMR data (symmetrical), HPLC retention time (slowest eluting isomer), and biological activities of compound 34, which was derived from compound 192, we believed that compound 192 has RR phosphorus stereochemistry. Such a stereochemical determination will be subject to confirmation by X-ray crystallography.

Compound 33

добавляли тиофенол (0,26 мл, 2,5 ммоль), а затем TEA (0,26 мл, 1,9 ммоль). Полученную смесь перемешивали при температуре окружающей среды для завершения снятия защитных групп (наблюдали при помощи LCMS). После завершения добавляли воду (2 мл). Полученную смесь трижды экстрагировали толуолом (2 мл каждый раз). Водный слой концентрировали в вакууме при 40-50°С и растворяли в воде (2 мл). Полученную смесь экстрагировали четыре раза смесью EtOAc/МТВЕ (1/1 мл каждый раз). Водный слой концентрировали в вакууме с получением бис-ТЕА соли соединения 33.To a solution of compound 192 (26 mg, 0.026 mmol) in 1,4-dioxane

thiophenol (0.26 ml, 2.5 mmol) was added followed by TEA (0.26 ml, 1.9 mmol). The resulting mixture was stirred at ambient temperature to complete deprotection (monitored by LCMS). Upon completion, water (2 ml) was added. The resulting mixture was extracted three times with toluene (2 ml each time). The aqueous layer was concentrated in vacuo at 40-50°C and dissolved in water (2 ml). The resulting mixture was extracted four times with a mixture of EtOAc/MTBE (1/1 ml each time). The aqueous layer was concentrated in vacuo to give the bis-TEA salt of compound 33.

¹ H NMR (400 MHz, METHANOLS) δ=9.06 (s, 1H), 8.73 (s, 1H), 8.56 (s, 1H), 8.47 (s, 1H), 6.43 (d, J=14.4 Hz, 1H) , 6.38 (d, J=15.2 Hz, 1H), 6.27 (dd, J=3.5, 51.1 Hz, 1H), 5.63 (dd, J=2.7, 51.5 Hz, 1H), 5.30-5.14 (m, 2H), 5.06-4.91 (m, 2H), 4.58 (br d, J=12.5 Hz, 1H), 4.52-4.39 (m, 3H), 4.07 (dd, J=4.7, 11.7 Hz, 1H), 3.98 (dd, J =5.1, 12.5 Hz, 1H), 3.43-3.34 (m, 2H), 3.22 (q, J=7.2 Hz, 12H), 2.98-2.86 (m, 2H), 2.79-2.45 (m, 4H), 1.32 ( t, J=7.4Hz, 18H)

Compound 34

With compound 192 as starting material, compound 34 was prepared with the same reaction sequence as described for compound 33.

¹ H NMR (400 MHz, METHANOL-d ₄ ) δ=8.77 (s, 2H), 8.49 (s, 2H), 6.39 (d, J=15.2 Hz, 2H), 5.71 (dd, J=3.1, 51.2 Hz , 2Н), 5.24-5.13 (m, 2Н), 5.02 (dtd, J=3.1, 9.0, 25.4 Hz, 2Н), 4.54 (br d, J=12.1 Hz, 2Н), 4.42 (br d, J=9.8 Hz, 2H), 3.99 (dd, J=5.9, 12.1 Hz, 2H), 3.20 (q, J=7.3 Hz, 12H), 2.90 (ddd, J=3.5, 10.2, 14.1 Hz, 2H), 2.81-2.70 (m, 2H), 2.55-2.43 (m, 2H), 1.30 (t, J=7.4 Hz, 18H).

Example 22 Synthesis of Compound 35 and Compound 36

Compound 201

To the disodium salt of compound 200 (0.10 g, 0.126 mmol) was added 2-nitrobenzyl bromide (0.068 g, 0.316 mmol) and MeCN (2.0 ml). The resulting mixture was stirred at ambient temperature while the reaction was monitored by LCMS. Upon completion, the reaction mixture was concentrated in vacuo and purified by silica gel column chromatography (SiO ₂ 10 g, 0%-5% MeOH in DCM) to give 115 mg of compound 201.

¹ H NMR (400 MHz, CHLOROFORM-d) δ=8.13 (dd, J=0.8, 8.2 Hz, 1H), 8.06 (s, 1H), 8.03 (dd, J=1.0, 8.0 Hz, 1H), 7.80 ( br s, 1H), 7.75 (s, 1H), 7.70-7.65 (m, 1H), 7.62-7.54 (m, 2H), 7.53-7.47 (m, 2H), 7.45-7.39 (m, 1H), 7.34 -7.27 (m, 1H), 6.23 (br d, J=16.8 Hz, 1H), 6.14 (brd, J=18.8 Hz, 1H), 5.95 (br s, 2H), 5.77 (td, J=5.1, 15.2 Hz, 2H), 5.71 (td, J=5.1, 15.6 Hz, 1H), 5.63-5.55 (m, 1H), 5.47 (br d, J=11.7 Hz, 1H), 4.58-4.27 (m, 11H), 4.18-4.10 (m, 1H), 4.05-3.71 (m, 2H)

Compound 202

To a solution of compound 201 (77 mg, 0.076 mmol) in 2,2,2-trifluoroethanol (2 ml) was added O-(2,4-dinitrophenyl)hydroxylamine (36.5 mg, 0.183 mmol) and bis[rhodium(α, α,α',α'-tetramethyl-1,3-benzenedipropionic acid)] (5.74 mg, 0.015 mmol) one part. The mixture was stirred at ambient temperature overnight, diluted with DCM (10 ml) and treated with saturated aqueous NaHCO ₃ (10 ml). The layers were separated and the aqueous layer was extracted twice with DCM (10 ml each). The combined organic layers were dried over MgSO ₄ and purified by silica gel column chromatography (SiO ₂ 10 g, 0%-15% MeOH in EtOAc) to give 3.3 mg of compound 202.

LCMS: MS m/z 1032.07 [M+H] ⁺

Compound 35 and Compound 36

To a solution of compound 202 (3.3 mg, 3.2 μmol) in 1,4-dioxane (0.40 ml) was added thiophenol (0.2 ml, 1.9 mmol) and TEA (0.2 ml, 1. 4 mmol). The resulting mixture was stirred at ambient temperature while the reaction was monitored by LCMS. Upon completion, the reaction mixture was treated with water (2 ml) and extracted three times with toluene (2 ml each time). The aqueous layer was concentrated in vacuo at 40-50°C and dissolved in water (1.5 ml). The resulting solid was filtered off, washed with water (0.5 ml). HPLC separation of the combined filtrates under the conditions described below gave compound 35 (retention time: 8.4 min) and compound 36 (retention time: 8.9 min).

Compound 35

LCMS: MS m/z 762.08 [M+H] ⁺

Compound 36

LCMS: MS m/z 762.22 [M+H] ⁺

Compound 35/Compound 36. Preparative HPLC conditions:

Example - 23 - Synthesis of compound 38 and compound 39

In FIG. 13 shows an example of the synthesis of compound 38 and compound 39.

Stage 1

To a solution of compound 203 (5.82 g, 9.314 mmol) and allyl alcohol (0.95 ml, 14 mmol) in THF (50 ml) at 0°C was added a solution of DIAD (2.4 ml, 11.6 mmol) and triphenylphosphine (2.93 g, 11.2 mmol) in toluene (25 ml), while maintaining the internal T below 10°C. The resulting solution was warmed to ambient temperature and stirred while the reaction was monitored by LCMS. After completion (5 h), the mixture was concentrated in vacuo and purified by silica gel column chromatography (100 g, 20%-60% EtOAc in n-heptane) to give 4.56 g of compound 204 as a pinkish oil.

Compound 204: ¹ H NMR (400 MHz, CHLOROFORM-d) δ=8.55 (s, 1H), 8.24 (s, 1H), 7.46 (d, J=8.2 Hz, 2H), 7.31-7.23 (m, 1H) , 7.20-7.10 (m, 2Н), 6.02 (d, J=5.1 Hz, 1H), 6.07-5.95 (m, 1H), 5.21 (dd, J=1.6, 17.2 Hz, 1H), 5.05 (dd, J =1.4, 10.4 Hz, 1H), 5.00 (d, J=5.5 Hz, 2H), 4.82 (t, J=4.9 Hz, 1H), 4.22-4.16 (m, 1H), 4.05-4.00 (m, 2H) , 3.82 (dd, J=2.5, 11.5 Hz, 1H), 0.95 (s, 9Н), 0.81 (s, 9Н), 0.14 (s, 3Н), 0.13 (s, 3Н), 0.00 (s, 3Н), -0.23 (s, 3H).

Stage 2

To compound 204 (4.56 g, 6.858 mmol) was added acetic acid (80 ml) and water (20 ml). The resulting mixture was stirred at ambient temperature overnight and at 35° C. for 1 day. The resulting mixture was concentrated in vacuo and purified by silica gel column chromatography (SiO ₂ , 50 g, 33%-60% EtOAc in n-heptane) to give 3.22 g of compound 205 as a white foamy solid.

Compound 205: ¹ H NMR (400 MHz, CHLOROFORM-d) δ=8.56 (s, 1H), 7.88 (s, 1H), 7.51-7.47 (m, 2H), 7.32-7.27 (m, 1H), 7.20- 7.16 (m, 2H), 6.06-5.95 (m, 1H), 5.72 (d, J=7.8 Hz, 1H), 5.26 (dd, J=5.5, 7.8 Hz, 1H), 5.20 (dd, J=1.6, 17.2 Hz, 1H), 5.06 (dd, J=1.6, 10.2 Hz, 1H), 5.03-4.98 (m, 2H), 4.23 (d, J=5.5 Hz, 1H), 4.14 (s, 1H), 3.92 ( dd, J=1.6, 12.9 Hz, 1H), 3.68 (br d, J=12.9 Hz, 1H), 0.75 (s, 9H), -0.14 (s, 3H), -0.62 (s, 3H).

Stage 3

To a solution of compound 205 (3.22 g, 5.847 mmol) in THF (120 ml) at ambient temperature was added water (30 ml) and triphenylphosphine (2.454 g, 9.355 mmol). The resulting mixture was stirred at ambient temperature while the reaction was monitored by LCMS. After completion (18 h) the reaction mixture was concentrated in vacuo and azeotroped with MeCN three times. The residue was purified by silica gel column chromatography (SiO ₂ (NH) 55 g, 20%-100% EtOAc in n-heptane) to give 4.82 g of compound 206 (63% acceptable purity). The product was used in the next step without further purification.

Compound 206: ¹ H NMR (400 MHz, CHLOROFORM-d) δ=8.56 (s, 1H), 7.92 (s, 1H), 7.54 (qd, J=1.6, 7.6 Hz, 2H), 7.31-7.27 (m, 1H), 7.19-7.15 (m, 2H), 6.07-5.94 (m, 1H), 5.84 (d, J=6.6 Hz, 1H), 5.50 (brd, J=10.6 Hz, 1H), 5.20 (dd, J =1.6, 17.2 Hz, 1H), 5.05 (dd, J=1.4, 10.4 Hz, 1H), 5.02-4.98 (m, 2H), 4.95-4.91 (m, 1H), 4.16-4.08 (m, 2H), 3.96 (dd, J=1.6, 12.9 Hz, 1H), 3.73 (dd, J=2.0, 5.5 Hz, 1H), 3.73-3.67 (m, 1H), 0.78 (s, 9H), -0.20 (s, 3H ), -0.46 (s, 3Н).

Stage 4

To a solution of compound 206 (4.82 g, 63% pure, 5.72 mmol) in pyridine (39.0 ml) at ambient temperature were added TEA (1.3 ml, 8.6 mmol) and trityl-Cl (1.753 g, 6.289 mmol). After completion of the reaction (observed by LC/MS) was added a saturated solution of NaHCO ₃ (60 ml). The resulting mixture was extracted three times with a mixture of MTBE/EtOAc (1/1, 70 ml each time). The combined organic layers were washed with 30% aqueous NaCl (30 ml) and dried over MgSO ₄ , filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (SiO ₂ 50 g, pre-treated with 1% TEA in EtOAc/n-heptane, 20%-100% EtOAc in n-heptane with 1% TEA) to give 1.484 g of compound 207 as a white solid substances.

Compound 207: ¹ H NMR (400 MHz, CHLOROFORM-d) δ=8.49 (s, 1H), 8.10 (s, 1H), 7.50-7.46 (m, 8H), 7.31-7.22 (m, 7H), 7.20- 7.14 (m, 5H), 6.11 (d, J=6.3 Hz, 1H), 6.07-5.94 (m, 1H), 5.20 (dd, J=1.2, 17.2 Hz, 1H), 5.06 (dd, J=1.4, 10.4 Hz, 1H), 5.03-4.98 (m, 2H), 4.84 (dd, J=3.1, 10.6 Hz, 1H), 4.52 (t, J=5.7 Hz, 1H), 3.78 (d, J=1.6 Hz, 1H), 3.64-3.57 (m, 1H), 3.26-3.16 (m, 3H), 0.77 (s, 9H), -0.18 (s, 3H), -0.63 (s, 3H).

Stage 5

To a solution of compound 207 (0.50 g, 0.652 mmol) in MeCN (6 mL) at ambient temperature was added 3-((bis(diisopropylamino)phosphaneyl)oxy)propanenitrile (0.393 g, 1.304 mmol) and pyridinium trifluoroacetate (0.113 g , 0.587 mmol). The resulting solution was stirred at ambient temperature and monitored by LCMS. After completion (1 h), the resulting solution was treated with gaseous hydrogen sulfide (sparging) for 1 min, followed by pyridinium trifluoroacetate (0.252 g, 1.304 mmol). The resulting solution was stirred at ambient temperature and monitored by LCMS. After completion (1 h) the resulting mixture was diluted with MTBE (60 ml). The resulting solution was washed with water (15 ml each time) twice and with 30% NaCl aqueous solution (15 ml) and dried over Na ₂ SO ₄ , filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (SiO ₂ 25 g, 25%-40% EtOAc in n-heptane) to give 0.411 g of compound 208 (1:1 P diastereomeric mixture) as a white foamy solid.

Compound 208 (1:1 P diastereomeric mixture) ¹ H NMR (400 MHz, CHLOROFORM-d) δ=8.49 (s, 1H), 8.49 (s, 1H), 8.11 (s, 1H), 8.06 (s, 1H) , 7.54-7.47 (m, 4H), 7.46-7.38 (m, 14H), 7.32-7.27 (m, 2H), 7.23-7.04 (m, 20H), 6.11-5.99 (m, 2H), 5.98 (d, J=2.0 Hz, 1H), 5.91 (d, J=1.6 Hz, 1H), 5.27 (dd, J=1.6, 6.3 Hz, 1H), 5.23 (dd, J=1.6, 6.3 Hz, 1H), 5.12 ( dd, J=1.6, 6.3 Hz, 1H), 5.09 (dd, J=1.2, 6.3 Hz, 1H), 5.02 (br d, J=5.5 Hz, 4H), 4.57-4.45 (m, 2H), 4.43- 4.34 (m, 1H), 4.32-4.04 (m, 8H), 3.23-3.19 (m, 1H), 3.10 (br s, 2H), 3.06-2.99 (m, 1H), 2.96-2.89 (m, 2H) , 2.73 (dt, J=1.4, 6.4 Hz, 2H), 2.65 (t, J=6.3 Hz, 2H), 0.85 (s, 9H), 0.83 (s, 9H), 0.01 (s, 3H), -0.09 (s, 6H), -0.11 (s, 3H).

Stage 6

To a solution of compound 208 (0.411 g, .457 mmol) and compound 206 (0.293 g, 0.502 mmol) in acetonitrile (5 ml) at ambient temperature were added DIEA (0.16 ml, 0.91 mmol) and CCl ₄ (0 .18 ml, 1.8 mmol). The reaction mixture was stirred at ambient temperature and monitored by LCMS. After completion (1 h), the resulting mixture was concentrated in vacuo and purified by silica gel column chromatography (SiO ₂ 25 g, 33%-100% EtOAc in n-heptane) to give 0.461 g of compound 210 (1:1 P diastereomeric mixture) in form of a white foamy solid.

Compound 210: ¹ H NMR (400 MHz, CHLOROFORM-d) δ=8.52 (s, 1H), 8.51 (s, 1H), 8.49 (s, 1H), 8.49 (s, 1H), 8.24 (s, 1H) , 8.19 (s, 1H), 8.13 (s, 1H), 8.01 (s, 1H), 7.55-7.37 (m, 20H), 7.33-6.99 (m, 30H), 6.09-5.97 (m, 4H), 5.96 (d, J=2.7Hz, 1H), 5.87 (d, J=1.2Hz, 1H), 5.82 (d, J=4.7Hz, 1H), 5.80 (d, J=3.9Hz, 1H), 5.28-5.17 (m, 4H), 5.11-5.04 (m, 4H), 5.03-4.96 (m, 8H), 4.75 (t, J=5.1 Hz, 1H), 4.70 (t, J=4.5 Hz, 1H), 4.46- 4.33 (m, 3H), 4.31-4.18 (m, 5H), 4.12-3.96 (m, 8H), 3.89 (br d, J=11.7 Hz, 1H), 3.78-3.67 (m, 3H), 3.57 (dd , J=2.9, 4.1 Hz, 1H), 3.56-3.44 (m, 2H), 3.08-3.01 (m, 2H), 2.99-2.93 (m, 1H), 2.91 (d, J=8.6 Hz, 1H), 2.77-2.73 (m, 1H), 2.72-2.53 (m, 4H), 0.84 (s, 9H), 0.84 (s, 9H), 0.84 (s, 9H), 0.82 (s, 9H), 0.00 (s, 3Н), -0.05 (s, 6Н), -0.08 (s, 3Н), -0.12 (s, 3Н), -0.15 (s, 3Н), -0.18 (s, 3Н), -0.18 (s, 3Н) .

Stage 7

To a solution of compound 210 (0.461 g, 0.324 mmol) in MeCN (5.5 ml) at ambient temperature was added 3-((bis(diisopropylamino)phosphaneyl)oxy)propanenitrile (0.195 g, 0.648 mmol) and pyridinium trifluoroacetate (0.050 g , 0.259 mmol). The resulting solution was stirred at ambient temperature and monitored by LCMS. After completion (40 min), the resulting solution was sparged with gaseous hydrogen sulfide for 1 min and then treated with pyridinium trifluoroacetate (0.138 g, 0.713 mmol). Upon completion (observed by LCMS), the resulting solution was diluted with MTBE (30 ml), washed with water (10 ml each) twice and with 30% NaCl aqueous solution (10 ml), dried over Na ₂ SO ₄ , filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (SiO ₂ 25 g, 33%-60% EtOAc in n-heptane) to give 0.329 g of compound 211 as a white foamy solid.

Compound 211: LC/MS (ESI) m/z 1555.48 [M+H] ⁺ .

Stage 8

To a solution of compound 211 (0.329 g, 0.211 mmol) in dichloromethane (7.2 ml) at ambient temperature were added water (0.032 ml, 1.8 mmol) and dichloroacetic acid (0.29 ml, 3.6 mmol). The resulting mixture was stirred at ambient temperature and monitored by LCMS. After completion (30 min) the reaction mixture sat. NaHCO ₃ solution (25 ml). The resulting mixture was extracted twice with DCM (30 ml each time). The combined organic layers were washed with 30% aqueous NaCl (20 ml), dried over MgSO ₄ , filtered and concentrated in vacuo. The crude product of compound 212 (0.278 g theoretical yield) was used in the next step without further purification.

Compound 212: LC/MS (ESI) m/z 1313.40 [M+H] ⁺ .

Stage 9

Triethylamine (1.0 ml, 7.2 mmol) and CCl ₄ (1.0 ml, 10 mmol) were added to a solution of compound 212 (0.278 g, 0.212 mmol) in MeCN (55.6 ml) at ambient temperature. The resulting solution was stirred at ambient temperature, this was observed using LCMS. Upon completion (30 min), the reaction mixture was concentrated to ~20 ml in vacuo and diluted with MTBE (30 ml). The resulting mixture was washed with water (10 ml) and 30% aqueous NaCl solution (10 ml each) twice, dried over MgSO ₄ , filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (SiO ₂ 25 g, 20%-75% EtOAc in n-heptane) to give 71 mg of compound 213 (SpRp isomer), 43 mg of compound 214 (either RpRp or SpSp isomer, higher rf on TLC (SiO ₂ )) and 24 mg of compound 215 (either RpRp or SpSp isomer, lower rf on TLC (SiO ₂ )).

Compound 213 (SpRp isomer) ¹ H NMR (400 MHz, CHLOROFORM-d) δ=8.58 (s, 1H), 8.44 (s, 1H), 8.42 (s, 1H), 8.12 (s, 1H), 7.46 (dd , J=7.2, 15.4 Hz, 4H), 7.36-7.27 (m, 2H), 7.18 (q, J=7.6 Hz, 4H), 6.09 (s, 1H), 5.98 (s, 1H), 6.06-5.95 ( m, 2Н), 5.25 (d, J=11.3 Hz, 1H), 5.21 (dd, J=1.4, 10.4 Hz, 1H), 5.12-5.03 (m, 3Н), 5.00-4.92 (m, 3Н), 4.87 (d, J=4.7 Hz, 1H), 4.60 (br d, J=11.7 Hz, 1H), 4.55 (d, J=3.9 Hz, 1H), 4.44 (br d, J=11.3 Hz, 1H), 4.39 -4.32 (m, 2H), 4.26-4.08 (m, 8H), 3.75 (dd, J=11.3, 16.0 Hz, 1H), 3.49 (dd, J=8.2, 10.6 Hz, 1H), 2.82-2.72 (m , 3Н), 2.61 (td, J=5.9, 17.2 Hz, 1H), 0.99 (s, 9Н), 0.96 (s, 9Н), 0.32 (s, 3Н), 0.23 (s, 6Н), 0.22 (s, 3H).

Compound 214 (either RpRp or SpSp isomer, higher rf on TLC (SiO ₂ ) isomer): ¹ H NMR (400 MHz, CHLOROFORM-d) δ=8.60 (s, 2H), 8.18 (s, 2H), 7.50 (d, J=7.0 Hz, 4H), 7.37-7.31 (m, 2H), 7.25-7.21 (m, 4H), 6.05-5.93 (m, 4H), 5.22 (dd, J=1.6, 17.2 Hz, 2H ), 5.09 (dd, J=1.0, 10.4 Hz, 2Н), 5.00-4.83 (m, 6Н), 4.61 (dd, J=1.2, 11.7 Hz, 2Н), 4.25 (dq, J=4.3, 10.8 Hz, 2Н), 4.09 (br dd, J=3.7, 10.4 Hz, 2H), 4.04 (td, J=5.5, 10.9 Hz, 2Н), 3.99-3.88 (m, 4Н), 3.70 (dd, J=11.9, 15.0 Hz, 2H), 2.71 (td, J=5.5, 17.1 Hz, 2H), 2.49–2.37 (m, 2H), 0.98 (s, 18H), 0.25 (s, 6H), 0.24 (s, 6H).

Compound 215 (either RpRp or SpSp isomer, lower rf on TLC (SiO ₂ ) isomer): ¹ H NMR (400 MHz, CHLOROFORM-d) δ=8.54 (s, 2H), 8.06 (s, 2H), 7.47 -7.44 (m, 4H), 7.32-7.27 (m, 2H), 7.20-7.15 (m, 4H), 6.06-5.95 (m, 2H), 5.92 (d, J=3.5 Hz, 2H), 5.22 (dd , J=1.2, 17.2 Hz, 2Н), 5.07 (dd, J=1.4, 10.4 Hz, 2Н), 5.04-4.94 (m, 6Н), 4.62-4.52 (m, 2Н), 4.46-4.40 (m, 2Н ), 4.40-4.22 (m, 8Н), 3.81 (dd, J=6.6, 12.1 Hz, 2Н), 2.89-2.71 (m, 4Н), 0.88 (s, 18Н), 0.08 (s, 6Н), -0.07 (s, 6H).

Stage 10

To a solution of compound 213 (71 mg, 0.054 mmol) in toluene (28.4 ml) under reflux was added a solution of the 2nd generation Hoveid-Grubbs catalyst (17.0 mg, 0.027 mmol) and P-benzoquinone (11.70 mg , 0.108 mmol) in toluene (8 ml). The mixture was heated to reflux temperature and the progress of the reaction was monitored by LC/MS. After 3 hours additional catalyst (8.5 mg, 0.0135 mmol) in toluene (2.5 ml) was added and the reaction was continued for another 2.5 hours. After cooling, the mixture was concentrated in vacuo and purified by silica gel column chromatography (SiO ₂ 10 g, 33%-66% ethyl acetate in n-heptane) to give 17 mg of compound 216 (trans/cis=5/1) as a brown dry foam .

Compound 216: ¹ H NMR (trans-isomer only, 400 MHz, CHLOROFORM-d) δ=8.37 (s, 1H), 8.34 (s, 1H), 8.24 (s, 1H), 8.20 (s, 1H), 7.41 (d, J=7.1 Hz, 2H), 7.36-7.27 (m, 3H), 7.24-7.13 (m, 5H), 5.97 (d, J=5.9 Hz, 2H), 5.75 (td, J=5.5, 15.2 Hz, 1H), 5.69 (td, J=5.5, 15.6 Hz, 1H), 5.07 (d, J=3.9 Hz, 1H), 5.03 (dd, J=5.1, 15.2 Hz, 1H), 4.94 (t, J =5.5 Hz, 2H), 4.85 (dd, J=5.1, 15.2 Hz, 1H), 4.74 (d, J=3.9 Hz, 1H), 4.67 (br d, J=12.1 Hz, 1H), 4.45 (br d , J=10.2 Hz, 1H), 4.42-4.34 (m, 2H), 4.28-4.19 (m, 2H), 4.19-4.06 (m, 4H), 3.96-3.84 (m, 1H), 3.82-3.68 (m , 1H), 3.56 (br dd, J=12.1, 14.5 Hz, 1H), 3.33 (dd, J=9.0, 10.9 Hz, 1H), 2.83-2.78 (m, 2H), 2.72 (td, J=5.6, 16.6 Hz, 1H), 2.39 (td, J=6.3, 17.2 Hz, 1H), 1.00 (s, 18H), 0.42 (s, 3H), 0.40 (s, 3H), 0.34 (s, 3H), 0.30 ( s, 3H).

Compound 217

Compound 214 obtained in step 9 was treated separately based on step 10 to give compound 217 (5/1 mixture of trans/cis isomers).

¹ H NMR (trans-isomer only, 400 MHz, CHLOROFORM-d) δ=8.47 (s, 2H), 8.14 (s, 2H), 7.43-7.38 (m, 4H), 7.36-7.29 (m, 2H), 7.22 (t, J=7.0 Hz, 4H), 5.83 (s, 2H), 5.77 (t, J=3.3 Hz, 2H), 5.17 (d, J=3.5 Hz, 2H), 5.11-5.04 (m, 2H ), 4.70 (br dd, J=3.5, 16.4 Hz, 2Н), 4.62 (br d, J=12.1 Hz, 2Н), 4.16-4.08 (m, 6Н), 3.93 (br dd, J=3.7, 11.9 Hz , 2Н), 3.74-3.64 (m, 2Н), 3.49 (t, J=12.9 Hz, 2H), 2.42 (td, J=5.9, 18.4 Hz, 2Н), 2.13 (ddd, J=5.5, 7.8, 16.8 Hz, 2H), 1.00 (s, 18H), 0.40 (s, 6H), 0.34 (s, 6H).

Compound 218

Compound 215 obtained in step 9 was treated separately based on step 10 to give compound 218.

¹ H NMR (400 MHz, CHLOROFORM-d) δ=8.14 (s, 2H), 8.12 (s, 2H), 7.49-7.43 (m, 2H), 7.36-7.27 (m, 4H), 7.19-7.12 (m , 4Н), 5.96 (s, 2Н), 5.67-5.54 (m, 2Н), 4.95-4.82 (m, 4Н), 4.69 (d, J=4.3 Hz, 2Н), 4.60-4.09 (m, 12Н), 3.63 (dd, J=8.8, 16.2 Hz, 2H), 2.93 (td, J=5.9, 17.2 Hz, 2H), 2.82-2.72 (m, 2H), 0.99 (s, 18H), 0.30 (s, 6H) , 0.27 (s, 6H).

Stage 11

To compound 216 (trans/cis=5/1, 17 mg, 0.013 mmol) was added a solution of methylamine solution (2 ml) (33% in EtOH) at ambient temperature. The resulting solution was stirred at ambient temperature as observed by LCMS. After completion (1 h) the reaction mixture was concentrated in vacuo. Pyridine (0.9 ml), TEA (0.45 ml) and triethylamine trihydrofluoride (0.36 ml, 2.2 mmol) were added to the residue. The resulting mixture was stirred at 50-60° C. for 4 hours and cooled to ambient temperature. After deprotection of TBS was completed (monitored by LCMS), the reaction mixture was treated with methoxytrimethylsilane (1.5 ml, 12 mmol) and stirred for 1 h. Water (3 ml) was added and the resulting mixture was extracted twice with toluene (3 ml each time ) and twice with EtOAc (2 ml each time). The aqueous layer was filtered with a syringe filter and the filtrate was subjected to preparative HPLC to obtain 5.3 mg of compound 38 and 1.1 mg of compound 220.

Compound 38 (SpRp, trans): ¹ H NMR (400 MHz, METHANOL-d ₄ ) δ=9.27 (br s, 1H), 8.47 (br s, 1H), 8.21 (br s, 1H), 8.09 (br s , 1H), 6.19-5.96 (m, 2H), 5.94-5.71 (m, 2H), 5.13-4.68 (m, 2H), 4.55-4.39 (m, 2H), 4.38-4.23 (m, 1H), 4.20 -3.91 (m, 5H), 3.75-3.50 (m, 4H).

Compound 220 (SpRp, cis): ¹ H NMR (400 MHz, METHANOL-d ₄ ) δ=9.08 (s, 1H), 8.72 (br s, 1H), 8.55 (s, 1H), 8.24 (s, 1H) , 8.18 (s, 1H), 6.22-6.14 (m, 2H), 6.10 (d, J=1.6 Hz, 2H), 5.01 (d, J=3.9 Hz, 1H), 4.41 (br d, J=9.8 Hz , 1H), 4.34-4.25 (m, 3H), 4.24-4.18 (m, 1H), 4.15 (dd, J=6.1, 11.5 Hz, 1H), 4.08-3.99 (m, 4H), 3.71-3.57(m , 3H).

Compound 39 and Compound 222

Compound 217 was treated separately based on step 11 to give compound 39 and compound 222.

Compound 39 (trans-isomer): ¹ H NMR (400 MHz, METHANOL-d ₄ ) δ=8.83 (br s, 1H), 8.43 (br s, 1H), 8.17 (br s, 1H), 8.11 (br s , 1H), 6.17-5.95 (m, 2H), 5.93-5.63 (m, 2H), 5.10-4.78 (m, 2H), 4.69-4.53 (m, 1H), 4.52-4.35 (m, 2H), 4.12 -3.92 (m, 5H), 3.76-3.44 (m, 4H).

Compound 222 (cis-isomer): ¹ H NMR (400 MHz, METHANOL-d ₄ ) δ=8.71 (br s, 2H), 8.18 (s, 2H), 6.17-6.06 (m, 4H), 4.37 (br d , J=9.8 Hz, 2H), 4.33 (d, J=3.9 Hz, 2H), 4.27 (br dd, J=7.0, 13.7 Hz, 2H), 4.06 (dd, J=7.0, 11.3 Hz, 2H), 4.19-4.03 (m, 2H), 4.01 (br d, J=9.8 Hz, 2H), 3.74 (ddd, J=3.9, 6.4, 10.5 Hz, 2H).

Compound 40

Compound 218 was treated separately based on step 11 to give compound 40: ¹ H NMR (400 MHz, METHANOL-d ₄ ) δ=9.34-8.88 (m, 2H), 8.73 (br s, 1H), 8.34-8.02 (m, 2H), 6.20-5.96 (m, 2H), 5.89 (br s, 2H), 5.25-4.95 (m, 2H), 4.76-4.64 (m, 1H), 4.47-4.22 (m, 2H), 4.20-4.07 (m, 2H), 4.07-3.93 (m, 2H), 3.82-3.55 (m, 3H), 3.54-3.38 (m, 2H)

Example 24 Synthesis of Compound 30

Compound 2 (1.1 mg, 1.4 μmol) was added to a solution of iodomethylisopropyl carbonate (13.75 mg, 0.056 mmol) in acetone/water (0.4/0.10 ml) at ambient temperature. The resulting mixture was stirred at ambient temperature in the dark while the reaction was monitored by LCMS. After completion (2 h) the reaction mixture was diluted with water (0.4 ml) and extracted with n-heptane three times (0.5 ml each). Purification of the crude product in the aqueous layer gave 0.5 mg of compound 30.

¹ H NMR (400 MHz, CHLOROFORM-d) δ=7.92 (s, 2H), 7.72 (br s, 2H), 6.22 (d, J=17.2 Hz, 2H), 6.19-6.03 (m, 2H), 5.98 (br t, J=6.3 Hz, 2H), 5.82-5.77 (m, 2H), 5.68 (dd, J=2.3, 51.6 Hz, 1H), 5.54 (dd, J=10.9, 13.7 Hz, 2H), 5.48 (dd, J=10.9, 12.9 Hz, 2H), 4.94 (quin., J=6.2 Hz, 2H), 4.71-4.63 (m, 2H), 4.63-4.56 (m, 2H), 4.50-4.43 (m, 2Н), 4.22-4.12 (m, 2Н), 4.05-3.90 (m, 2Н), 1.33 (d, J=4.3 Hz, 6Н), 1.32 (d, J=4.3 Hz, 6Н).

Example 103 Reporter Assay of STING HAQ Agonist Activity

THP1-Dual™ cells (InvivoGen, Cat # thpd-nfis) were used to determine EC ₅₀ . THP1 Dual™ cells were characterized by Invivogen (Insight 201402-1) as carrying the STING HAQ genotype. Cells were grown and maintained under conditions recommended by the manufacturer. Interferon regulatory factor (IRF) pathway induction, as described in the manufacturer's manual, was monitored to determine EC ₅₀ . Briefly, cells were seeded and treated with various concentrations of the compound for 20 hours while incubated at 37° C., 5% CO ₂ . Cells were resuspended and QUANTI-Luc™ solution (Cat. No.: rep-qlc1) was added. The resulting light emission was measured with a luminometer (Envision, Perkin Elmer). The received signals were plotted and the EC ₅₀ was calculated using the GraphPad Prism7 computer program.

EC ₅₀ values are described in Table. 4-8 below. EC ₅₀ values may be presented based on a single assay or as an average of many assays. Each table is preceded by a structure used to describe that table.

Human STING EC50 (μM) was measured using the ammonium salt form of each compound in Table. 4.

Human STING EC50 (μM) was measured using the ammonium salt form of each compound shown in Table. 5.

Human STING EC50 (μM) was measured using the ammonium salt form of each compound shown in Table. 6, except for Compound 26, Compound 31, Compound 33 and Compound 34, all of which were tested as bis-TEA salts.

Human STING EC50 (μM) was measured using the ammonium salt form of each compound in Table. 7.

Human STING EC50 (μM) was measured using the ammonium salt form of each compound in Table. 8.

Example 104 STING Variant-Specific Reporter Analysis

Human STING has 4 main variants, including WT, HAQ, REF and AQ variants. REF-STING, also referred to as R232H, for example, occurs in approximately 14% of the human population. Compared to the wild-type allele, R232H was characterized by a reduced response to cyclic dinucleotides of bacteria and metazoans. Details regarding these 4 major variants, as well as other rare variants, are described by Yi G, et al., "Single nucleotide polymorphisms of human STING can affect innate immune response to cyclic dinucleotides" PLoS One 2013; 8:e77846. STING variant-specific reporter cell lines were determined using THP1-Dual™ KO-STING cells (InvivoGen, Cat # thpd-kostg) and expression vectors for the three STING protein variants. An expression vector map for STING WT is shown in FIG. 6. For the other two expression vectors, sequences from different STING variants were used in this vector, with STING WT replaced with the appropriate nucleotide sequence.

STING variant-expressing vectors for WT-STING, REF-STING, and AQ-STING were prepared and stably transformed into THP1-Dual™ KO-STING cells to prepare STING variant-specific reporter assays for WT-STING, REF-STING, and AQ -STING respectively. EC ₅₀ values were determined as described above in Example 103 for a reporter analysis of STING HAQ agonist activity. The results are shown below in Table. 9. The DNA sequences used for these STING variants are shown in SEQ ID NO: 1 (nucleotide sequence of human STING WT), SEQ ID NO: 2 (nucleotide sequence of human STING REF), and SEQ ID NO: 3 (nucleotide sequence of human STING AQ ).

STING WT human:

STING REF of a person:

STING AQ human:

Example 105 Mouse STING Agonist Activity Reporter Assay

RAW-Lucia™ ISG cells (InvivoGen, Cat # rawl-isg) were used for reporter analysis of mouse STING agonists. EC ₅₀ values were determined as described above in Example 103 in the STING HAQ agonist activity reporter assay. The results are shown below in Table. 9.

Example 106 Differential Scanning Fluorimetry (DSF) Analysis

The DSF-based assay was used to measure the physical interaction between the compound and the recombinant STING protein. The truncated recombinant STING protein (a.k. 155-341) (SEQ ID NO: 4) was expressed in E. coli and isolated for analysis as described below. The assay matrix was prepared in 384-well plates to a final volume of 10 μl per well, consisting of 1 μM recombinant STING protein (a.a. 155-341) (SEQ ID NO: 4), 100 mM PBS, pH 7.4, with addition of 100 mM KCl, 5X SYPRO orange dye, and 50 μM compound (final conc. DMSO 0-1%). Analyzes were performed on a QuantStudio 12K Flex real-time PCR system using a temperature gradient from 25°C to 95°C at a rate of 0.05°C/min, and excitation and emission filters at 470 and 586 nm, respectively. According to the fluorescence derivative curves determined using the Applied Biosystems® Protein Thermal Shift computer program (algorithm version 1.3.), the melting point (Tm) of the unbound and ligand bound recombinant STING protein was calculated and the melting point difference (dTm D) was calculated.

As a rule, compounds with ΔTm values greater than 0 were considered as those in which there was a physical interaction with the protein under study, and the ΔTm value is positively associated with the binding affinity of the compounds. Here, Compound 1a had a ΔTm of 17.6 (Table 9), indicating a physical interaction with the STING protein.

Example 107 Ex Vivo PBMC Stimulation Assay

Human blood from 5 healthy donors was collected using 10.0 ml BD Vacutainer Sodium heparin tubes (Cat. No. 367874). Isolation of peripheral blood mononuclear cells (PBMCs) was performed using 50 ml SIGMA ACCUSPIN (Cat. No. A2055) and Sigma ACCUSPIN System-HISTOPAQUE-1077 (Cat. No. A7054) tubes using the manufacturer's protocol. The PBMC layer was removed and washed with 1x phosphate buffered saline (PBS) as suggested by Sigma. PBMCs were counted and finally suspended at 1 x 10e6/ml in RPMI (Corning cat. no. 10-041-CV) supplemented with 10% fetal bovine serum (FBS) (Gibco cat. no. 20140.79). 1 ml of cells (1×10e6) were transferred into a 5 ml Falcon round bottom polypropylene laboratory tube (Cat. No. 352063) and stimulated with various concentrations (0, 0.1, 1, 10 μM) for 24 hours in 5% CO2 incubator at 37°C.

After 24 hours of incubation, the tubes were centrifuged at 1400 rpm. within 5 minutes and the supernatants were removed. Supernatants were stored at -80°C for subsequent measurement of IFNβ. IFNβ measurement was performed using the Human IFN-β Core Kit (Cat. No. Meso Scale Diagnostics K151ADA) using the manufacturer's protocol. IFN-beta evaluation was performed by reading the assay plate into a MESO SECTOR Imager 2400 and using the MSD Discovery Workbench 4.0 software. After 24 hours, the IFNβ protein was analyzed. The results showed that Compound 1a can induce IFNβ production in primary human PBMCs in a dose dependent manner.

The results shown in Table. 10 represent the average of measurements taken with five different donors.

To quantify IFNβ mRNA, total RNA was isolated using the RNeasy Mini kit (Qiagen, Germany) according to the manufacturer's protocol. IFNβ mRNA was quantified by qPCR analysis. Briefly, total RNA (400 ng to 1000 ng) was converted to cDNA in a 60 μl reaction volume using the SuperScript VILO MasterMix (Life Technologies, USA). The resulting cDNA (10 ng) was then amplified using Applied Biosystems TaqMan expression assays using RNA specific primers for IFNB1 (Hs01077958_s1) and GAPDH (Hs99999905_m1). qPCR analysis was performed using TaqMan Fast Advanced Master Mix (Life Technologies, USA) on an Applied Biosystems Quantstudio 12K Flex real-time PCR system, with an initial 2-minute step at 50°C followed by the following conditions: 95°C for 2 sec. and 40 cycles at 95°C for 1 sec. and 60°C for 20 sec. Relative gene expression was calculated after normalization to the reference GAPDH gene using the 2-ΔΔCT method. Calculations were performed using the Applied Biosystems Quantstudio 12K Flex v1.2.2 software. The fold change of IFNβ mRNA relative to vehicle-treated samples is shown in Table. 11. The results showed that Compound 1a can induce IFNβ mRNA in primary PBMCs in a dose- and time-dependent manner. In Table. 11 shows the average calculated from five different donors.

Example 108 Antitumor Effect of Compound 1a in Double Tumor

models CT26

Compound 1a was tested for antitumor activity in the CT26 dual tumor model, which is a mouse colon cancer model. Balb/cJ female mice aged 5-6 weeks (Jackson Labs, Bar Harbor, Maine) were implanted subcutaneously with CT26 tumor cells on both sides of each animal, 10 ⁵ cells per side. For study A, treatment was started 5 days (1.25 mg/kg, 2.5 mg/kg and 5 mg/kg) after tumor implantation, when mean tumor volumes reached about 100 mm ³ . For study B, treatment was started 8 days (0.6 mg/kg and 10 mg/kg) after tumor implantation, when mean tumor volumes reached about 120 mm ³ . The processing scheme is described in Table. 12 and Tab. 13.

All mice in the study had two subcutaneous CT26 tumors. "Treated tumor" means a tumor that has been directly administered a compound, while "untreated tumor" means a tumor that has not been directly administered a compound. Tumor volume was monitored throughout the experiment. Tumor volume was measured daily after the start of the experiment. Tumor burden was calculated from caliper measurements using the formula for the volume of an elongated ellipsoid (L×W ² )/2, where L and W are the respective orthogonal length and width (mm).

Compound 1a was characterized by high and therapeutic activity in the dual tumor model CT26 (Fig. 7 and Fig. 8). For treated tumors, a cure rate of 20% was found even at the lowest dose tested in the study (FIG. 8, dose 0.6 mg/kg). At the same time, the highest dose (10 mg/kg) resulted in 100% cure of the indicated tumor in the animals at the end of the study. In the case of untreated tumors, a dose-dependent antitumor effect was also evident. The highest dose group (10 mg/kg) had 80% therapeutic effects; ever lower doses also exhibited tumor growth inhibitory activity. Thus, a therapeutic window of 0.6 mg/kg to 10 mg/kg for Compound 1a was observed, with antitumor activity being observed not only locally but also systemically based on effects in the uninjected distal tumor lesion. In conclusion, these results indicate that local administration of Compound 1a can induce both local and systemic (inhibitory) antitumor activity.

Example 109 Antitumor Effect of Compound 1a in the CT26 Liver Metastasis Model

Compound 1a was investigated for its antitumor activity in the CT26 liver metastasis model. Anesthetized 5-6 week old female BALB/cJ mice (Jackson Labs, Bar Harbor, Maine) were intrasplenicly implanted with luciferase-expressing CT26 tumor cells (5 x 10 ⁵ cells per mouse). During a subsequent ten minute period, the tumor cells were allowed to enter the bloodstream into the livers of the animals. The spleens were then removed and the animals were sutured and allowed to recover. Three days later, CT26 tumor cells (10 ⁵ cells per mouse) were re-implanted, this time subcutaneously (sc) in the area under the right forelimb, promoting the development of a tumor mass for the administration of the compounds. Nine days after the intrasplenic injection, the compound (10 mg/kg) was injected intratumorally, once, into the sc tumor.

The local antitumor effect of a compound was measured by its effect on tumor sc, with the inhibitory effect of the compound assessed by overall survival of treated mice compared to base treated controls, based on the adverse effect of growing tumor mass in the liver of each mouse. Compound 1a was characterized as high activity against local sc tumors, as well as therapeutic systemic activity in 9 out of 10 treated animals (Fig. 9). These results indicate that local administration of Compound 1a can induce both local and systemic (inhibitory) antitumor activity, including against distant lesions, for example, in the liver.

Example 110 Antitumor Effect of Compound 1a in GL261 Orthotopic Brain Model

Compound 1a was investigated for its antitumor activity in the GL261 orthotopic brain model. GL261 is a mouse glioma cell line. Luciferase-expressing GL261 mouse glioma cells (2×10 ⁴ cells/mouse) were intracranially implanted in 5-6 week old female B6 albino mice (Jackson Labs, Bar Harbor, Maine). After 3-4 days, GL261 cells were implanted subcutaneously (106 cells/mouse) in the area under the right forelimb in order to promote the development of a tumor mass for the introduction of compounds. Ten days after intracranial implantation, the compound (10 mg/kg) was injected intratumorally, once, into the sc tumor. The local antitumor effect of a compound was measured by its effect on tumor sc, with the inhibitory effect of the compound assessed by overall survival of treated mice compared to base treated controls, based on the adverse effect of the growing tumor mass in the brain of each mouse. Compound 1a was characterized by both high activity against local sc tumors, as well as therapeutic systemic activity in 5 of 8 treated animals (Fig. 10). These results indicate that local administration of Compound 1a can induce both local and systemic (inhibitory) antitumor activity, including against distant lesions, for example, in the brain.

Example 111 X-ray crystallographic structure confirming the complex with STING WT

In order to further understand the mechanism of binding to the target of new compounds under study, the X-ray crystallographic structure of STING WT in complex with the compounds was determined.

A. Expression and purification of the STING WT C-terminal domain (residue 155-341)

The DNA sequence encoding the human STING WT protein from amino acids 155 to 341 (SEQ ID NO: 4) was cloned into the pET21b vector, after a His-TEV-Sumo tag at its N-terminus (SEQ ID NO: 5). The pET21b sequence has been deposited with Addgene and is available at addgene.org/vector-database/2550/; said sequence is incorporated herein by reference.

BL21 (DE3) Codon Plus E. coli cells were transformed with the indicated plasmid and expression of the recombinant protein was induced with 0.1 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG). The protein was purified from the soluble fraction of the cell lysate using Ni-NTA affinity chromatography. The His-TEV-Sumo tag was removed with Sumo protease and separated from the untagged 155-341 STING WT sequence using a second Ni-NTA affinity chromatography column. The protein was further purified by anion exchange chromatography and stored in a buffer containing 20 mM Tris⋅HCl, pH 7.5, and 150 mM NaCl at a concentration of 35 mg/ml.

B. Crystallization and structural determination of the STING WT C-terminal domain complexed with Compound 1

In order to co-crystallize the 155-341 STING WT sequence with Compound 1, the STING WT protein was diluted to 10 mg/ml with storage buffer (20 mM Tris⋅HCl, pH 7.5, and 150 mM NaCl) and mixed with Compound 1 (100 mM stock solution in DMSO) in a molar ratio of 1:5. The mixture was incubated for 4 hours at 4° C. and centrifuged at 13,000 rpm. for 20 minutes before crystallization. Mesh pallets for crystallization were installed using the hanging drop method by diffusion in vapor at 18°C. Crystals were grown by mixing 1 μl of STING WT/Compound 1 solution with an equal volume of well solution containing 100 mM HEPES, pH 7.5, 200 mM CaCl2 and 15% (w/v) PEG 8000. 20% (w/w) ./vol.) PEG 400 was used as a cryoprotective reagent for flash freezing in liquid nitrogen. Diffraction datasets were collected using a Pilatus detector with an SSRF BL19U1 beam and processed using the HKL3000 and the SCALEPACK2MTZ program in the CCP4 software package.

в пространственной группе Р212121 с использованием элементарной ячейки, измеренной при а=33,820, b=78,110, с=132,212, α=90,00, β=90,00, γ=90,00. Две копии последовательности 155-341 STING WT идентифицировали в каждой асимметричной ячейке, связанной с одной молекулой Соединения 1 на поверхности раздела димера.The structure of the 155-341 STING WT sequence associated with Compound 1 was determined using the PHASER molecular replacement program (maximum likelihood molecular replacement) using PDB ID 4F9E as the initial search model. The presence of Compound 1 between the STING WT dimer interface was confirmed in the Fo-Fc difference map calculated using model phases. The model was built and completed manually with the Coot program and refined with the Refmac5 program in the CCP4 software package. The final improved structure was described at a resolution of 2.38

in space group P212121 using unit cell measured at a=33.820, b=78.110, c=132.212, α=90.00, β=90.00, γ=90.00. Two copies of the 155-341 STING WT sequence were identified in each asymmetric cell associated with one Compound 1 molecule at the dimer interface.

C. Interaction of Compound 1 with STING WT observed in X-ray crystal structure

On FIG. 11 shows the X-ray crystallographic structure of human STING WT complexed with Compound 1. The X-ray crystallographic structure of human STING WT complexed with Compound 1, which was co-crystallized from a sample of Compound 1a, was examined. The compound bound in an interface pocket formed by the STING WT protein dimer. Two faces of the adenine base of the compound formed a π-π stacking interaction with Tyr240 and the guanine group of Arg238, respectively. The trans-olefin linker formed a van der Waals interaction with the aliphatic portion of the Arg238 side chain. The fluorine substituent at the C2' position of the ribose group of the compound was inserted into the hydrophobic hole identified by Thr263, Pro264 and Tyr163. The negatively charged triphosphate group of the compound formed a salt bridge using Arg238 and hydrogen bonds to Ser162 and Thr267, respectively. In addition, the triophosphate group also formed an electrostatic interaction with the guanidine group of Arg232. The LID STING WT loop region, consisting of residues 226-243, spanned two base groups and a trans-oleic linker.

Example 112 Determination of the X-ray crystallographic structure of STING REF complexed with Compound 1.

A. Expression and purification of the C-terminal domain of STING REF (residue 155-341, SEQ ID NO: 6)

The DNA sequence encoding the human STING REF protein from amino acids 155 to 341 (SEQ ID NO: 6) was cloned into the pET21b vector, after a His-TEV-Sumo tag at its N-terminus (SEQ ID NO: 7). The pET21b sequence has been deposited with Addgene and is available at addgene.org/vector-database/2550/; said sequence is incorporated herein by reference.

BL21 (DE3) Codon Plus E. coli cells were transformed with the indicated plasmid and expression of the recombinant protein was induced with 0.1 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG). The protein was purified from the soluble fraction of the cell lysate using Ni-NTA affinity chromatography. The His-TEV-Sumo tag was removed with Sumo protease and separated from the untagged 155-341 STING REF sequence using a second Ni-NTA affinity chromatography column. The protein was further purified by anion exchange chromatography and stored in a buffer containing 20 mM Tris⋅HCl, pH 7.5, and 150 mM NaCl at a concentration of 24 mg/ml.

B. Crystallization and structural determination of the C-terminal domain of STING REF complexed with Compound 1

In order to co-crystallize the 155-341 STING REF sequence with Compound 1, the STING REF protein was diluted to 10 mg/ml with storage buffer (20 mM Tris⋅HCl, pH 7.5, and 150 mM NaCl) and mixed with Compound 1 (100 mM stock solution in DMSO) in a molar ratio of 1:5. The mixture was incubated for 4 hours at 4° C. and centrifuged at 13,000 rpm. for 20 minutes before crystallization. Mesh pallets for crystallization were installed using the hanging drop method by diffusion in vapor at 18°C. Crystals were grown by mixing 1 µl of STING REF/Compound 1 solution with an equal volume of well solution containing 100 mM HEPES, pH 7.5, 200 mM CaCl2 and 15% (w/v) PEG 8000. 20% (w/w) ./vol.) PEG 400 was used as a cryoprotective reagent for flash freezing in liquid nitrogen. Diffraction datasets were collected using a Pilatus detector with an SSRF BL18U1 beam and processed using the HKL3000 and the SCALEPACK2MTZ program in the CCP4 software package. This structure is shown in Fig. 12.

в пространственной группе Р212121 с использованием элементарной ячейки, измеренной при а=33,733, b=77,831, с=131,689, α=90,00, β=90,00, γ=90,00. Две копии последовательности 155-341 STING REF идентифицировали в каждой асимметричной ячейке, связанной с одной молекулой Соединения 1 на поверхности раздела димера.The sequence structure of 155-341 STING REF associated with Compound 1 was determined using the PHASER molecular replacement program (maximum likelihood molecular replacement) using the previously determined sequence structure of 155-341 STING WT (as described above) as the initial search model. The presence of Compound 1 between the STING REF dimer interface was confirmed in the Fo-Fc difference map calculated using model phases. The model was built and completed manually with the Coot program and refined with the Refmac5 program in the CCP4 software package. The final improved structure was described at a resolution of 2.76

in space group P212121 using unit cell measured at a=33.733, b=77.831, c=131.689, α=90.00, β=90.00, γ=90.00. Two copies of the 155-341 STING REF sequence were identified in each asymmetric cell associated with one Compound 1 molecule at the dimer interface.

C. Interaction of Compound 1 with STING REF observed in X-ray crystal structure

On FIG. 12 shows the X-ray crystallographic structure of human STING REF complexed with Compound 1, which was co-crystallized from a sample of Compound 1a. The compound bound in an interface pocket formed by the STING protein dimer. Two faces of the adenine base of the compound formed a π-π stacking interaction with Tyr240 and the guanine group of Arg238, respectively. The trans-olefin linker formed a van der Waals interaction with the aliphatic part of the Arg238 side chain, while the guanidine part of the Arg238 side chain formed a π-π stacking interaction with the imidazole group of the His232 side chain on the outside. The olefin linker was in contact with an interacting pair of Arg238 and His232 side chains. The fluorine substituent at the C2' position of the ribose group of the compound was inserted into the hydrophobic hole identified by Thr263, Pro264 and Tyr163. The negatively charged triphosphate group of the compound formed a salt bridge using Arg238 and hydrogen bonds to Ser162 and Thr267, respectively. The LID STING REF loop region, consisting of residues 226-243, spanned two base groups and a transoleic linker.

Example 113 Comparisons

EC50 values were calculated for human STING assays for STING WT, STING HAQ, STING AQ and STING REF in direct comparisons using Compound 1a of the present study, natural STING ligand (2'3' cGAMP) and putative STING agonist ML RR-S2 CDA described in Corrales, et al., "Direct Activation of STING in the Tumor Microenvironment Leads to Potent and Systemic Tumor Regression and Immunity," Cell Reports (2015) 11:1018-1030, which is incorporated herein by reference. Analyzes were performed as described in the examples above. It should be noted that the values in Table. 14 were limited to analyzes performed in head-to-head comparisons and may not reflect the mean values found in more of the studies described in Table. 5 or elsewhere. Additionally, note that "2'3' cGAMP" means the same as "ML cGAMP", as described in the Cell Reports publication.

In Table. 14 also describes the dissociation constants (Kd) for the binding of human STING WT to each of the three test compounds, as measured by isothermal titration calorimetry (ITC). ITC is a microcalorimetric titration technique that measures thermodynamic properties associated with intermolecular interactions. Based on these studies, Compound 1a appears to have formed the strongest association with STING WT of the compounds tested.

materials

Recombinant human wild-type protein STING (a.k. 139-379, H232R) was generated by expression of a construct in E. coli encoding the cytosolic domain of human WT STING containing amino acids 139-379.

Reagents

The sources of reagents used in this study are shown below.

Protein buffer preparation

The STING protein was stored at -60° C. in 90 μl and 100 μl aliquots, each at 3.0 mg/ml and 20 mg/ml, respectively, in PBS, pH 7.5, containing 5% glycerol. On the day of analysis, protein aliquots were thawed, diluted into 400 µl, and buffer exchanged in PBS using an Amicon Ultra centrifuge filter block (MW cutoff 10k, 0.5 ml) using at least four 10 minute 14,000 xg centrifugations using Eppendorf microcentrifuge and then finally diluted to 20-30 μM (depending on the experiment) with 1X PBS. The protein concentration was determined using a Nanodrop 2000 spectrophotometer and the protein extinction coefficient was 22140 (M ^-1 cm ^-1 ).

Sample preparation

Two hundred ml of 1 mM stock solutions of Compound 1a, 2'3' cGAMP and ML RR-S2CDA were placed in 1.5 ml microcentrifuge tubes. Before each experiment, the samples were diluted to concentrations of 200-500 μM (depending on the experiment).

Ways

The assays were performed on an Affinity ITC unit (TA Instruments, no. 609003.901) equipped with an ITC cleaning tool (TA Instruments, no. 601800.901). An approximately 400 μl STING protein solution containing 20-30 μM STING protein was pipetted into a 185 μl calorimeter well, providing some allowable excess. The reference cell contained an equivalent amount of Milli-Q water. Incubation was performed at 25° C. with 20 x 2.5 μl injections of 100-300 μM compounds. ITC Run Ver. 3.3.0.0 (TA Instruments) was used to obtain thermograms consisting of multiple initial heat peaks (µcal/sec) representing the heat flux at each injection. Analytical software Nano Analyze Ver. 3.70 (TA Instruments) was used to correct for baseline, correct for blank heat of dilution, or heat of dilution of the sample (at saturation), and to integrate heat flux peaks resulting in "Q" values on the graphs. The resulting isotherms were fitted with an independent model to obtain thermodynamic parameters.

Received and described the values of Kd and n (molar ratios at the points of inflection of the curve). Optimal conditions for protein and ligand concentrations were obtained from preliminary experiments.

results

The heat flux thermograms and the binding isotherms of the studied compounds with the recombinant human wild-type protein STING (a.k. 139-379, H232R) were determined. The binding of each compound to STING was endothermic, as indicated by negative heat fluxes with an exothermic (positive direction) heat of dilution (observed after the compound had reached protein saturation). It was shown that 2', 3' cGAMP resulted in a similar endothermic response against different STING variants. Compound 1a had the lowest Kd of 0.04 μM followed by 2'3' cGAMP with a Kd of 0.07 μM and then ML RR-S2 CDA with a Kd of 0.40 μM. All compounds had n values close to 0.5, indicating that the STING protein was present as a dimer and was associated with 1 mole of compound per 2 moles of STING.

Example 114 Identification of Potential Metabolites

Compound 1a was incubated in hepatocytes of CD-1 mice, Sprague Dawley rats, Beagle dogs, cynomolgus monkeys, and humans to assess the formation of major metabolites.

materials

Cryopreserved pooled hepatocytes were purchased from ThermoFisher Scientific (Waltham, MA), Xenotech, LLC (Kansas City, KS), and In Vitro ADMET Laboratories (Columbia, MD) and appropriate media were purchased from In Vitro ADMET Laboratories (Columbia, MD) and Life Technologies (Carlsbad, California). AOPI staining solution and phosphate buffer were purchased from Corning Life Sciences (Tewkesbury, MA) and Nexcelom Bioscience (Lawrence, MA), respectively. All chemicals, reagents, and solvents used in the assay were analytical grade or HPLC grade.

Schemes and procedures of experiments

Incubation of hepatocytes

Compound 1a was weighed and dissolved in HPLC water containing 0.12% formic acid in PBS to give 1020 mmol/l. The solution was then diluted 2.5 times separately to 4 mmol/l and then further diluted 21,000 times with Williams E medium containing 0.1% human serum albumin and 2 mmol/l L-glutamine to form a working stock solution with a concentration of 20 µmol/l.

Before incubation, cryopreserved hepatocytes were thawed in water at 37°C. One tube of cryopreserved hepatocytes was added to each 50 ml conical tube of cryopreserved hepatocyte recovery medium (UCRM) obtained from In Vitro ADMET Laboratories (Columbia, Maryland). Cells were centrifuged in a Beckman centrifuge (Brea, CA) with a GH 3.8 rotor at 740 rpm. for 10 minutes at room temperature 4°C. The supernatant was removed and the cells were resuspended in culture medium for counting. After the cells were resuspended in the culture medium, 20 µl of the resuspension was transferred and mixed with 20 µl of the AOPI staining solution. The solution was gently mixed and cells were counted using a Cellometer (Nexcelom, Lawrence, MA). After counting, cells were resuspended at 1 or 2 million viable cells/ml in Williams E medium containing 2 mmol/l L-glutamione (pH 7.4).

A suspension of hepatocytes (50 μl/well) was added to a 48-well plate. Fifty microliters of working stock solution containing Compound 1a (20 μmol/l) was added to the beginning of the reaction. The plate was placed in a tissue culture incubator (humidified air atmosphere 5% CO2/95% and 37°C) and reactions were terminated by adding 200 µl of a stop solution of 100% methanol/acetonitrile (1/1, v/v) since 2010 ng/ml furosemide and 0.2 µmol/l (R)-propranolol at 5, 30, 60, 120, 180 and 240 minutes. The mixture was centrifuged and filtered and the supernatant was collected for analysis. Final concentrations of cryopreserved hepatocytes were 1×10 ⁶ cells/ml. The final incubation concentration of Compound 1a was 10 µmol/l.

LC-MS/MS Conditions for Metabolite Identification

The LC-MS/MS system consisted of a Shimadzu and AB-SCIEX TripleTOF 5600 Hybrid Quadrupole HPLC system and a TOF mass spectrometer (Framingham, MA). The Shimadzu HPLC system (Kyoto, Japan) consisted of a communication bus module (CBM-20A), an autosampler (SIL-30AC) with an attached rack changer (Rack Changer II), two pumps (LC-30AD) and a column thermostat (CTO -30A). The mass spectrometer was calibrated with both negative and positive AB-SCIEX APCI calibration solutions (Framingham, MA). Samples resulting from incubation with hepatocytes were analyzed in both negative and positive scan modes. The main analytical method and instrumental conditions are presented below. The modification of the spectrometer settings depended on the needs of the analyte.

LC-MS/MS Conditions:

Activity data analysis

Mass spectrometry data were obtained using AB-Sciex Analyst TF (version 1.5.1; Framingham, MA). Chromatograms and spectra were obtained using AB-Sciex PeakView (version 2.2.0.1; Framingham, MA). Comparison of relative peak areas for extraction ion chromatograms was based on ±0.0002 Da expected exact mass-to-charge ratio (m/z) for each analyte of interest.

results

As a result of incubation with hepatocytes, no metabolite was detected. Under the assay conditions shown, Compound 1a had a retention time of about 7.8 minutes. In the negative scan mode, Compound 1a was characterized by m/z of the deprotonated molecular ion 745 (C ₂₄ H ₂₅ F ₂ N ₁₀ O ₈ P ₂ S ₂ ^- ) and m/z of the doubly deprotonated molecular ion 372 (C ₂₄ H ₂₄ F ₂ N ₁₀ O ₈ P ₂ S ₂ ^2- ). The main MS/MS product ions were observed with m/z 533 (C ₁₉ H ₁₉ FN ₁₀ O ₄ PS ^- ) and m/z 186 (C ₉ H ₈ N ₅ ^- ). Under positive scanning conditions, Compound 1а was characterized by m/z of the deprotonated molecular ion 747 (C ₂₄ H ₂₇ F ₂ N ₁₀ O ₈ P ₂ S ₂ ⁺ ) and the main MS/MS product ions with m/z 651 (C ₂₄ H ₂₆ F ₂ N ₁₀ O ₆ PS ⁺ ), m/z 252 (C ₁₀ H ₁₁ FN ₅ O ₂ ⁺ ) and m/z 188 (C ₉ H ₁₀ N ₅ ⁺ ). MS and MS/MS data confirmed the structure of Compound 1a.

Compound 1a was stable when incubated with mouse, rat, dog, monkey and human hepatocytes. No visible metabolite of Compound 1a was identified in this study. In samples resulting from incubation with hepatocytes, only Compound 1a itself could be detected and confirmed using tandem mass spectrometry (MS/MS) fragments.

All documents referred to in this disclosure are incorporated herein by reference, although in the event that any incorporated document conflicts with a textual description, then said textual description must be verified. Those skilled in the art will appreciate that various changes and modifications may be made to the material provided herein and that the material is within the scope and spirit of the present disclosure.

Claims (166)

1. Compound of formula (III)

or a pharmaceutically acceptable salt thereof, wherein R _1a is selected from the group consisting of H, -OH and -F; R _1b is selected from the group consisting of H, -OH and F, where at least one of R _1a and R _1b represents -H; R _4a is selected from the group consisting of -H, -OH and -F; R _4b is selected from the group consisting of -H, -OH and -F, where at least one of R _4a and R _4b is -H; P ₁ and P ₂ each independently has the S or R stereochemical configuration; Z is -O- or -NH-; X _1a and X _2a are the same or different and independently selected from ═O or ═S; X _1b and X _2b are the same or different and independently selected from -OR ₅ and -SR ₅ ; where R ₅ is selected from H and -CH ₂ OC(O)OC _1-6 alkyl; L ₁ in formula (III) is four, five or six carbon atoms long and is

, Where

means single bond, double bond or triple bond and where (i) is either 0 or 1 occurrence

in L ₁ means a triple bond; or (ii) 0, 1 or 2 occurrences

in L ₁ mean a double bond, where the geometry around each double bond is cis or trans; and (iii) where if 1 occurrence

in L ₁ means triple bond, 0 occurrences

in L ₁ means a double bond; and (iv) where if 2 occurrences

in L ₁ means a double bond, such double bonds are either adjacent bonds or alternating bonds; where X ₁₀ , X ₁₁ , X ₁₂ , X ₁₃ , X ₁₄ and X ₁₅ are independently selected from the bond, -CH ₂ - or -CH-, where -CH ₂ - or -CH- is unsubstituted or substituted by (i) -OH , (iv) -NH ₂ or (v) -D and if X ₁₀ or X ₁₅ is a bond, such bond is not a double bond or a triple bond; and where any two adjacent group members, including X ₁₀ , X ₁₁ , X ₁₂ , X ₁₃ , X ₁₄ and X ₁₅ , may optionally form with additional C ₃ atoms cycloalkyl or C ₃ heterocycloalkyl, said C ₃ heterocycloalkyl includes N atom ; where B ₁ and B ₂ are independently selected from:

where bonds at points q and r on B ₁ and B ₂ are attached at points q and r on formula (III). 2. A compound or pharmaceutically acceptable salt according to claim 1, where R _1a is selected from the group consisting of H and -F; R _1b is selected from the group consisting of H and F, where R _1a and R _1b cannot both be -F; R _4a is selected from the group consisting of H and -F; R _4b is selected from the group consisting of -H and -F, where R _4a and R _4b cannot both be -F; P ₁ and P ₂ each independently has the S or R stereochemical configuration; X _1a and X _2a are the same or different and independently selected from ═O or ═S; X _1b and X _2b are the same or different and independently selected from -OR ₅ and -SR ₅ ; where R ₅ is selected from the group consisting of -H, C _1-6 alkyl and -C(O)C _1-6 alkyl; L ₁ in formula (III) is four or five carbon atoms long and is

Where

means single bond or double bond and where either 0 or 1 occurrence

in L ₁ means a double bond, where the geometry around the double bond is cis or trans; where X ₁₀ and X ₁₁ are independently selected from a bond, -CH-, or -CH ₂ - and where, if X ₁₀ or X ₁₄ is a bond, such a bond is not a double bond; where B ₁ and B ₂ are independently selected from:

where bonds at points q and r on B ₁ and B ₂ are attached at points q and r on formula (III). 3. Compound of formula (IV)

or a pharmaceutically acceptable salt thereof, wherein R _1a is selected from the group consisting of -H, -OH and -F; R _1b is selected from the group consisting of H, -OH and -F, where at least one of R _1a and R _1b is -H; R _4a is selected from the group consisting of -H, -OH and -F; R _4b is selected from the group consisting of -H, -OH and -F, where at least one of R _4a and R _4b is -H; P ₁ and P ₂ each independently has the S or R stereochemical configuration; X _1a and X _2a are the same or different and independently selected from ═O or ═S; X _1b and X _2b are the same or different and independently selected from -OR ₅ and -SR ₅ ; where R ₅ is selected from H and -CH ₂ OC(O)OC _1-6 alkyl; L ₁ in formula (IV) is four, five or six carbon atoms long and is

Where

means single bond, double bond or triple bond and where (i) is either 0 or 1 occurrence

in L ₁ means a triple bond; or (ii) 0, 1 or 2 occurrences

in L ₁ mean a double bond, where the geometry around each double bond is cis or trans; and (iii) where if 1 occurrence

in L ₁ means triple bond, 0 occurrences

in L ₁ means a double bond; and (iv) where if 2 occurrences

in L ₁ means a double bond, such double bonds are either adjacent bonds or alternating bonds; where X ₁₀ , X ₁₁ , X ₁₂ , X ₁₃ , X ₁₄ and X ₁₅ are independently selected from the bond, -CH ₂ - or -CH-, where -CH ₂ - or -CH- is unsubstituted or substituted by (i) -OH or (v) -D and if X ₁₀ or X ₁₅ is a bond, such bond is not a double bond or a triple bond; where B ₁ and B ₂ are independently selected from:

where bonds at points q and r on B ₁ and B ₂ are attached at points q and r on formula (IV). 4. Compound of formula (IV)

or a pharmaceutically acceptable salt thereof, wherein R _1a is selected from the group consisting of H and -F; R _1b is selected from the group consisting of -H and -F, where R _1a and R _1b cannot both be -F; R _4a is selected from the group consisting of -H and -F; R _4b is selected from the group consisting of H and F, where R _4a and R _4b cannot both be F; P ₁ and P ₂ each independently has the S or R stereochemical configuration; X _1a and X _2a are the same or different and independently selected from ═O or ═S; X _1b and X _2b are the same or different and independently selected from -OR ₅ and -SR ₅ ; where R ₅ represents -H; L ₁ in formula (IV) is four or five carbon atoms long and is

Where

means single bond or double bond and where either 0 or 1 occurrence

in L ₁ means a double bond, where the geometry around the double bond is cis or trans; where X ₁₀ and X ₁₁ are independently selected from a bond, -CH- or -CH ₂ - and where, if X ₁₀ or X ₁₄ is a bond, such a bond is not a double bond; where B ₁ and B ₂ are independently selected from:

where bonds at points q and r on B ₁ and B ₂ are attached at points q and r on formula (IV). 5. Compound of formula (V)

or a pharmaceutically acceptable salt thereof, wherein R _1a is selected from the group consisting of -H, -OH and -F; R _1b is selected from the group consisting of -H, -OH and -F, where at least one of R _1a and R _1b is -H; R _4a is selected from the group consisting of H, -OH; R _4b is selected from the group consisting of -H, -OH and where at least one of R _4a and R _4b is -H; P ₁ and P ₂ each independently has the S or R stereochemical configuration; X _1a and X _2a are ═O; X _1b and X _2b are -SR ₅ ; where R ₅ represents -H; L ₁ in formula (V) is four, five or six carbon atoms long and is

Where

means a single bond or a double bond and where (i) or 0 or 1 or (ii) 0, 1 or 2 occurrences

in L ₁ mean a double bond, where the geometry around each double bond is cis or trans; and (iii) where if 1 occurrence

in L ₁ means triple bond, 0 occurrences

in L ₁ means a double bond; and (iv) where if 2 occurrences

in L ₁ means a double bond, such double bonds are either adjacent bonds or alternating bonds; where X ₁₀ , X ₁₁ , X ₁₂ , X ₁₃ , X ₁₄ and X ₁₅ are independently selected from the bond, -CH ₂ - or -CH-, where -CH ₂ - or CH is unsubstituted or substituted by (i) OH and, if X ₁₀ or X ₁₅ is a bond, such a bond is not a double bond or a triple bond; and where any two adjacent group members including X ₁₀ , X ₁₁ , X ₁₂ , X ₁₃ , X ₁₄ and X ₁₅ may optionally form with additional C ₃ atoms cycloalkyl or C ₃ heterocycloalkyl, said C ₃ heterocycloalkyl includes N or O atom; where B ₁ and B ₂ are independently selected from:

where bonds at points q and r on B ₁ and B ₂ are attached at points q and r on formula (V). 6. Compound of formula (V)

or a pharmaceutically acceptable salt thereof, wherein R _1a is selected from the group consisting of -H and -F; R _1b is selected from the group consisting of -H and -F, where R _1a and R _1b cannot both be -F; R _4a is -H; R _4b is -H; P ₁ and P ₂ each independently has the S or R stereochemical configuration; X _1a and X _2a are =O X _1b and X _2b are -SR ₅ ; where R ₅ represents H; L ₁ in formula (V) is four or five carbon atoms long and is

Where

means single bond or double bond and where either 0 or 1 occurrence

in L ₁ means a double bond, where the geometry around the double bond is cis or trans; where X ₁₀ and X ₁₄ are independently selected from a bond, -CH- or -CH ₂ - and where, if X ₁₀ or X ₁₄ is a bond, such a bond is not a double bond; where B ₁ and B ₂ are independently selected from:

where bonds at points q and r on B ₁ and B ₂ are attached at points q and r on formula (V). 7. Connection or pharmaceutically acceptable salt according to any one of paragraphs. 1-6, where (i) the stereochemistry of P ₁ and P ₂ is both R, the stereochemistry of P ₁ is R and P ₂ is S, or the stereochemistry of P ₁ is S and P ₂ is R; (ii) one occurrence

in L ₁ means a double bond, where the geometry around the double bond is trans; And (iii) Z is -O-. 8. Connection or pharmaceutically acceptable salt according to any one of paragraphs. 1-7, where R _1a and R _4a are each -F. 9. Connection or pharmaceutically acceptable salt according to any one of paragraphs. 1-7, where R _1b and R _4b are each -F. 10. Connection or pharmaceutically acceptable salt according to any one of paragraphs. 1-9, where B ₁ and B ₂ each represents

11. Connection or pharmaceutically acceptable salt according to any one of paragraphs. 1-10, where X _1a and X _2a are both =O and where X _1b and X _2b are both -SH. 12. Connection or pharmaceutically acceptable salt according to any one of paragraphs. 1-11, where L ₁ represents

. 13. A compound or pharmaceutically acceptable salt according to claim 1 or 2, where L ₁ is five carbon atoms in length. 14. Connection or pharmaceutically acceptable salt according to any one of paragraphs. 1-11, where L ₁ is four carbon atoms long. 15. A compound or a pharmaceutically acceptable salt thereof, selected from the group consisting of:

or their pharmaceutically acceptable salts. 16. A compound or a pharmaceutically acceptable salt thereof according to claim 15, selected from the group consisting of:

or their pharmaceutically acceptable salts. 17. Connection or pharmaceutically acceptable salt according to any one of paragraphs. 1-16, wherein the compound or pharmaceutically acceptable salt is characterized by (i) an EC ₅₀ value below 100 micromoles in reporter cells expressing a human STING HAQ variant; (ii) an EC ₅₀ value below 100 micromoles in reporter cells expressing the human STING AQ variant; (iii) an EC ₅₀ value below 100 µM in reporter cells expressing the human STING WT variant; or (iv) an EC ₅₀ value below 100 micromoles in reporter cells expressing the human STING REF variant. 18. A compound or pharmaceutically acceptable salt, wherein the compound is

or a pharmaceutically acceptable salt thereof. 19. A compound or pharmaceutically acceptable salt, wherein the compound is

or a pharmaceutically acceptable salt thereof. 20. A compound or pharmaceutically acceptable salt, wherein the compound is

or a pharmaceutically acceptable salt thereof. 21. A compound or pharmaceutically acceptable salt, wherein the compound is

or a pharmaceutically acceptable salt thereof. 22. A compound or pharmaceutically acceptable salt, wherein the compound is

or a pharmaceutically acceptable salt thereof. 23. Pharmaceutically acceptable salt according to any one of paragraphs. 1-22, where the salt is a diammonium salt. 24. Pharmaceutical composition for the treatment of cancer, containing a pharmaceutically effective amount of a compound according to any one of paragraphs. 1-22 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient. 25. A method of treating cancer, which involves administering to the patient a compound according to any one of paragraphs. 1-22 or a pharmaceutically acceptable salt according to any one of paragraphs. 1-23 or a pharmaceutical composition according to claim 24. 26. The use of a compound according to any one of paragraphs. 1-22 or a pharmaceutically acceptable salt according to any one of paragraphs. 1-23 to obtain a pharmaceutical composition for the treatment of cancer. 27. The use of a compound according to any one of paragraphs. 1-22 or a pharmaceutically acceptable salt according to any one of paragraphs. 1-23. 28. The use of a pharmaceutical composition according to claim 24 for the treatment of cancer. 29. A method for treating cancer, which includes: identifying an individual having a cancer to be treated with a compound according to any one of paragraphs. 1-22, a pharmaceutically acceptable salt according to any one of paragraphs. 1-23 or the pharmaceutical composition according to claim 24; And administering to said individual a pharmaceutically effective amount of a compound, pharmaceutically acceptable salt, or pharmaceutical composition by which the cancer has been identified as being treatable. 30. The method according to p. 29, in which the specified individual is identified as having a cancer to be treated with a compound according to any one of paragraphs. 1-22, a pharmaceutically acceptable salt according to any one of paragraphs. 1-23 or the pharmaceutical composition of claim 24 as a result of the presence of the STING REF variant allele in the patient. 31. The method according to claim 25 or 29, wherein the cancer is selected from the group consisting of lymphoma, melanoma, colorectal cancer, breast cancer, acute myeloid leukemia, colon cancer, liver cancer, prostate cancer, pancreatic cancer, cancer kidneys and gliomas. 32. A method of treating cancer in a patient having a REF STING allele, which involves administering to said patient a compound according to any one of paragraphs. 1-22, a pharmaceutically acceptable salt according to any one of paragraphs. 1-23 or a pharmaceutical composition according to claim 24. 33. A method of treating cancer in a patient having the WT STING allele, which involves administering to said patient a compound according to any one of paragraphs. 1-22, a pharmaceutically acceptable salt according to any one of paragraphs. 1-23 or a pharmaceutical composition according to claim 24. 34. A method of treating cancer in a patient having an AQ STING allele, which involves administering to said patient a compound according to any one of paragraphs. 1-22, a pharmaceutically acceptable salt according to any one of paragraphs. 1-23 or a pharmaceutical composition according to claim 24. 35. A method of treating cancer in a patient having the HAQ STING allele, which involves administering to said patient a compound according to any one of paragraphs. 1-22, a pharmaceutically acceptable salt according to any one of paragraphs. 1-23 or a pharmaceutical composition according to claim 24. 36. The method according to any one of paragraphs. 32-35, wherein the cancer is selected from the group consisting of melanoma, colorectal cancer, breast cancer, acute myeloid leukemia, colon cancer, liver cancer, and glioma. 37. The method according to any one of paragraphs. 25 or 32-35, wherein said cancer is metastatic.

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