BRPI0716812A2 - PRO POLYMERIC DRUGS DIRECTED CONTAINING MULTIFUNCTIONAL LEADERS - Google Patents

Description

PRO POLYMERIC DRUGS DIRECTED CONTAINING LEADERS

MULTIFUNCTIONAL

CROSS REFERENCE WITH RELATED ORDERS

This patent application claims priority of US Patent Application No. 60 / 844,943 filed September 15, 2006, the teachings of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

Many of the low molecular weight anti-cancer agents are known to have significant toxicity. Over the years, several attempts to reduce toxicity and improve the effectiveness of these low molecular weight anti-cancer agents have been presented.

An attempt to reduce toxicity and improve the effectiveness of these low molecular weight anti-cancer agents has been directed to drug delivery systems focused on targeting agents. For example, the targeting agent, such as single chain antibody, RGD peptides or folic acid, may direct the transport of the therapeutic agent and enhance its therapeutic effect. At the same time, targeted transport of very potent chemotherapy reduces its toxicity. The strategy has been validated, for example, by Mylotarg® approval of Wyeth's gentuzumab zogamycin, a humanized anti-CD33 antibody linked to calicheamicin cytotoxin, for FDA acute myeloid leukemia (AML).

Traditionally, immunoconjugates include three components: a targeting agent such as a monoclonal antibody, a cytotoxin and a ligand.

However, this strategy requires the antibody to maintain a high degree of its antigen binding affinity after drug conjugation. Moreover, antibody-drug conjugates need to be stable in buffers or plasma and should not release the toxin prematurely during the journey. They also need to be internalized considering that the antibody binds to its antigen on the cell surface. Even so, the drug molecule needs to be released intact into the desired tumor cell at a desired rate as well.

Single chain antibodies (SCAs) or single chain antigen-binding antibodies include the binding domain of a complete antibody only one quarter of its size. However, ACS can bind to the antigen specifically with high affinity. A description of the theory and production of single chain antigen-binding proteins can be found, for example, in US Nos. 4,946,778, 5,260,203, 5,455,030 and 5,518,889. The single chain antigen-binding proteins produced by the processes cited in the above-cited US patent documents have binding specificity and affinity substantially similar to the corresponding Fab fragment, their teachings being incorporated herein by reference. More recently, US Patent No. 6,872,393 has disclosed polyalkylene oxide single chain polypeptides. The teachings of each of the following patents are incorporated herein by reference.

Despite attempts and advances, there is still a need to provide polymeric drug delivery systems focused on targeting agents having desired therapeutic activity and less toxicity. The present invention focuses on this need and others.

SUMMARY OF THE INVENTION

In order to overcome the aforementioned problems and improve the technology for polymer drug delivery, novel and advantageous compounds which employ the use of both targeting agent and pegylation technologies as well as improved therapeutic techniques are provided. Accordingly, according to one aspect of the present invention there are provided compounds of Formula (I):

D- (Li) a-L2- (L3) b-D2

THE

on what:

R1 is a substantially non-antigen water soluble polymer;

A is a protection group or

D1- (L1) a-L2- (L3) b-D2.

5th

each Dι is independently selected from steering moieties, functional groups, and leaving groups, preferably steering moieties; each D2 is independently selected from biologically active moieties, functional groups and leaving groups, preferably biologically active moieties;

each Li is independently a permanent binder or a labile binder, preferably a permanent binder; L2 is a multifunctional binder;

each L3 is independently a permanent binder or a labile binder, preferably a labile binder; and

(a) and (b) are independently zero or a positive integer, preferably zero or 1.

In one aspect of the invention, the polymeric residue is a polyethylene oxide that can be conjugated to both small drug molecules and single chain antibodies (SCA) via labile ligands and permanent ligands via multifunctional ligands. In this manner, compounds are provided that provide an SCA-PEG-Ligand-Drug transport platform for targeted delivery of cytotoxic compounds that are small molecules. In addition, it is advantageous that the charge of both small molecule and SCA per binding site is increased compared to traditional SCA-drug conjugates. Thus, they are presented for targeted SCA drugs that are cost-effective and low cost. Still further, the prodrugs of the present invention provide methods for regulating the circulation half-life of drugs, for example by increasing the circulation half-life, when desired, through the use of appropriate binders.

In a general aspect of the present invention, without limitation, it is described in the structure

below, down, beneath, underneath, downwards, downhill:

Targeting— (permanent linker) - (multifunctional Iinker) - (releasable linker) - Drug Moiety -

Cêeg)

where: "targeting moiety" = targeting moiety, "multifunctional linker" = multifunctional linker, "releasable linker" = labile linker and "drug" = drug,

wherein PEG is bound to both SCA and drug, such as a cytotoxic agent, by means of a centrally positioned multifunctional binder; the binder with the SCA is permanent whereas the binder with the cytotoxic agent is labile and can be configured to be stable in the blood but easily degraded in the presence of a site-specific enzyme. In a preferred aspect, the SCA is bound via a permanent binder such as one containing a maleiimide group and the cytotoxic agent via a labile binder such as a peptidyl binder (VaI-Cit) which may be specifically degraded by captesin B. In a preferred embodiment, the cytotoxic agent is SN38.

Additional aspects of the present invention include methods for making polymers

activators containing multifunctional linkers, methods for preparing conjugates containing them as well as treatment methods based on administering effective amounts of the conjugates containing a biologically active portion to the patient (mammal) in need thereof. One of the advantages of the present invention is that the transport systems

The polymeric compounds described herein are stable and thus increase the bioavailability of drugs.

Another advantage is that polymeric transport systems can release intact drugs into target tumor cells.

Yet another advantage is that the drug release ratio can be modified to achieve a desired speed.

A further advantage of the polymeric systems corresponding to the present invention is that they are suitable for low molecular weight drugs and oligonuneotides such as low antisense interference RNAs (siRNA) or LNA compounds.

Other advantages and additional advantages will become apparent from the description below.

follow.

For purposes of the present invention, the term "residue" shall be understood to mean a part of the compound to which it refers, i.e. cytotoxin, SN38, permanent binder, multifunctional binder, labile binder, etc. which remains after the compound has undergone substitution reaction with another compound.

For purposes of the present invention, the term "polymeric residue" or "PEG residue" shall be understood to mean the portion of the polymer or PEG remaining after it has been reacted with other compounds, portions, etc.

For purposes of the present invention substituted alkyls include carboxyalkyls, aminoalkyls, dialkylaminos, hydroxyalkyls and mercaptoalkyl; substituted alkenyls include carboxyalkenyls, aminoalkenyls, dialkenylamines, hydroxyalkenyls and mercaptoalkenyls; substituted alkynyls include carboxyalkynyls, aminoalkynyls, dialkylamines, hydroxyalkylyls and mercaptoalquinyls; Substituted cycloalkyls include moieties such as 4-chlorocyclohexyl; aryls include moieties such as naphthyl; substituted aryls include moieties such as 3-bromo phenyl; aralkyls include moieties such as tolyl; heteroalkyls include moieties such as ethylthiophene; substituted heteroalkyls include moieties such as 3-methoxythiophene; alkoxy includes portions such as methoxy; and phenoxy includes moieties such as 3-nitrophenoxy. Halo should be understood to include fluorine, chlorine, iodine and bromine.

For purposes of the present invention "nucleic acid" or "nucleotide" shall be understood to include deoxyribonucleic acid (DNA), both single stranded and double stranded ribonucleic acid (RNA), unless otherwise specified, and chemical modifications. of the same.

For the purposes of the present invention, the terms "a biologically active moiety" and "a biologically active moiety residue" shall be understood to mean the part of the biologically active compound remaining after the compound has undergone a substitution reaction in which the carrier portion was attached.

For purposes of ease of description, and not limitation, it should be understood that the term "biologically active portions" is interchangeable with "drugs", "cytotoxic agents", "cytotoxins".

For purposes of ease of description, and not limitation, it is to be understood that the term "small molecules" is interchangeable with "pharmaceutically active compounds".

Unless otherwise defined for the purposes of the present invention:

The term "alkyl" is to be understood to include, for example, halo, alkoxy, and nitro-C1-12 straight, branched or substituted alkyls, substituted C3.8 cycloalkyls or substituted cycloalkyls, etc .;

the term "substituted" shall be understood to include the addition or exchange of one or more atoms contained within a functional group or composed of one or more different atoms;

the term "substituted alkyls" includes carboxyalkyls, aminoalkyls, dialkylamines, hydroxyalkyls and mercaptoalkyls; the term "substituted cycloalkyl" includes moieties such as 4-chlorocyclohexyl; aryls include moieties such as naphthyl; substituted aryls include moieties such as 3-bromophenyl; aralkyls include moieties such as toluyl; heteroalkyls include moieties such as ethylthiophene;

the term "substituted heteroalkyl" includes moieties such as 3-methoxythiophene; alkoxy includes portions such as methoxy; and phenoxy includes moieties such as 3-nitrophenoxy;

the term "halo" shall be understood to include fluorine, chlorine, iodine and bromine; and

The terms "sufficient amounts" and "effective amounts" for the purposes of the present invention are to be understood to mean an amount that will achieve a therapeutic effect and such effect should be understood by those skilled in the art.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 schematically illustrates synthetic methods described in Examples 1-5.

FIG. 2 schematically illustrates synthetic methods described in Examples 6-7.

FIG. 3 schematically illustrates synthetic methods described in Examples 8-9.

FIG. 4 schematically illustrates synthetic methods described in Examples 10-14.

FIG. 5 schematically illustrates synthetic methods described in Examples 15-17.

FIG. 6 schematically illustrates synthetic methods described in Examples 18-19.

FIG. 7 schematically illustrates synthetic methods described in Examples 20-23.

FIG. 8 schematically illustrates synthetic methods described in Examples 24-25.

FIG. 9 schematically illustrates synthetic methods described in Examples 26-28.

FIG. 10 schematically illustrates synthetic methods described in Examples 29-32.

FIG. 11 schematically illustrates synthetic methods described in Examples 33-35.

FIG. 12 schematically illustrates synthetic methods described in Examples 36-38.

FIG. 13 schematically illustrates synthetic methods described in Examples 39-40.

DETAILED DESCRIPTION OF THE INVENTION A. OVERVIEW

In one aspect of the present invention there are provided compounds of (I): D1- (L1) a-L2- (L3) b-D2

R

ι

THE

on what:

R1 is a substantially non-antigen water soluble polymer; A is a protection group or

D1- (L1) a-L2- (L3) b-D2.

>

each D is independently selected from steering moieties, functional groups and leaving groups, preferably steering moieties;

each D2 is independently selected from biologically active moieties, functional groups and leaving groups, preferably biologically active moieties;

each Li is independently a permanent binder or a labile ligand, preferably a permanent binder; L2 is a multifunctional binder;

each L 3 is independently a permanent binder or a labile binder, preferably labile binder; and

(a) and (b) are independently zero or a positive integer, preferably zero or 1.

In a preferred aspect of the invention, targeted polymeric carrier systems include compounds of Formula:

R

A-R1-N

Ll (lia),

A-R1-N

R

ι ■

L3-D2

d2 L1-D1 (Ilb),

L3-D2

A-R1-N

-IN-L-I-U1

d3 R12 (Hc) 5

A-R1-N

LrD1 (Hd)

LrD1 (He), (Hg)

A-R1-N

AIR

A-R1-N

(IIh),

U-Do

-L1-D1

(IIi) and

L1-D1

-L3-D3

(Hj)

on what

R 2 and R '2 are independently selected from hydrogen, C 1-6 alkyl, C 2-6.

alkenyl, C2.6 alkynyl, C3.19 branched alkyl, C3.8 cycloalkyl, C1-6 substituted alkyl, C2-6 substituted alkenyl, C2.6 substituted alkynyl, C.v8 substituted cycloalkyl, aryl, substituted aryl, heteroaryl, heteroraila substituted C6-6 heteroalkyl, substituted C6-6 heteroalkyl, C6-6 alkoxy, aryloxy, C6-6 heteroalkoxy, heteroaryloxy, C2-6 alkanoyl, arylcarbonyl, C2-6-alkoxycarbonyl, aryloxycarbonyl, C2-6 alkanoyloxy, substituted C2-6 arylcarbonyl, .6 substituted alkanoyloxy, substituted aryloxycarbonyl, substituted C2.6 alkanoyloxy and substituted arylcarbonyloxy;

(cl), (c2), (c3), (c4), (c5), (c6), (c'6), (c "6), (c7) and (c8) are independently zero or an integer positive, preferably zero or an integer from about 1 to about 10, and more preferably zero, 1 or 2;

(dl), (d2), (d3), (d4), (d5) and (d7) are independently zero or a positive integer, preferably zero or an integer from about 1 to about 10, and more preferably. zero or an integer from about 1 to about 4; and all other variables are previously defined. In another aspect, the polymeric systems described herein are both protected in a

their terminals with a CH3 group, i.e. mPEG as well as, in other embodiments, bis-activated PEGs are provided, such as those of Formula: H

A-R1-N

-L3-D2 -L1-D1

THE

ο O

D2-L3 ^ η H / -L3-D2

) —N — R1 — N— (D1-L1- ^ \ L1-Di

O O

ο

V-L3-D2 A-R1-NHV O

A-R1-NH

A-R1-NH

L1-D1

HN-L1-D1,

.0 0 D2-L3 ^ VL3-D2

OVNH-R1-NH4 O

D1-L <

.0

D9-L

D1-L1

NH-R1-NH

LrD1,

3-υ2

L1-D1

7th

D2-L3- ^ i / L3-D2

-N-R1-NH-

D1-L1-NH

HN-L

A-R1-NH

LrD1,

Λ

L3-D2

A-R1 — N

0 P

L1-D1 .0

ρΛ

L3-D2

A — R1-N

M

THE

LrD1,

P

D2-L3- f VlS-D2

-NH-R1-NH-

D1-L1

L1-D1

\

L3-D2

Ο

K

Dr li

N — R1 — N

Li-D1, Ο.

D2-L3

.0

THE

M

You/

N — R1-N

D1-L1

M

L3-D2

.0

LrD1,

As-d2

A-R1-NH

D9-L

2 - "- 3

L3-D2 D2-L3.

, L3-D2

-THE

NH-R1-NH

"THE'

L3-C 10

A-R1-NH

L3-D9

Do-l

2 "L3

Li-D1

D1-L1

NH-R1-NH O.

AIR

R2 N-

L1-D1

-O-L3-D2

-S-L1-D

1,

D1-L1

L3-D5

R2

-N

L1-D1

LrD1,

O — L3-D2 —S-L1-D1

R2

-N

THE

, 0

O-L1-D1

—S-L3-D

D1-L1-O-

3, and D3-L3 S

R2

-N-

-R1 — N-

> -0 — L1-D1 ^ -S-L3-D3.

Other optional protecting groups include H, NH 2, OH, CO 2 H, C 1-6 alkoxy and C 1-6 alkyl. Preferred protecting groups include methoxy and methyl.

In most aspects of the present invention, the polymers included herein are generally described as substantially non-antigenic polymers. Within the polymer genre, polyalkylene oxides are preferred and polyethylene glycols (PEGs) are most preferred. For purposes of ease of description rather than, the invention is sometimes described using PEG as the prototype polymer. It should be understood, however, that the scope of the invention applies to a wide variety of polymers which may be linear, substantially linear, branched, etc.

In another aspect of the invention, the biological portions include -NH 2, -OH and SH-containing moieties.

20

B. MULTIFUNCTIONAL LINKERS (L2)

In view of the structures of the present invention, the multifunctional binder allows bonds (labile or permanent) of 3 or more components, i.e. a targeting agent, a polymer and a biologically active cytotoxic compound such as SN38. One skilled in the art will appreciate that other molecules including at least three independent functional groups may also be used. A preferred multifunctional binder may be an aspartic acid residue or a lysine.

L2 having at least three functional sites can be selected from: <Μ

I-N

d4

4

(Id),

(Ig), and

on what

R2 and R2 are independently selected from hydrogen, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3.19 branched alkyl, C3.8 cycloalkyl, substituted Cn6 alkyl, C2-6 substituted alkenyl, C2-6 substituted alkynyl, C3 .8 substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, Cn6 heteroalkyl, Cu6 substituted heteroalkyl, Cu6 alkoxy, aryloxy, Cu6 heteroalkoxy, heteroaryloxy, C2.6 alkanoyl, arylcarbonyl, C2.6 alkoxycarbonyl, aryloxycarbonyl, C2.6 alkoxycarbonyl, arylcarbonyloxy, substituted C 2-6 alkanoyl, substituted arylcarbonyl, substituted C 2-6 alkanoyloxy, substituted aryloxycarbonyl, substituted C 2-6 alkanoyloxy and substituted arylcarbonyloxy;

(cl), (c2), (c3), (c4), (c5), (c6), (c'6), (c "6), (c7) and (c8) are previously defined, and (dl ), (d2), (d3), (d4), (d5) and (d7) are also previously defined Preferred embodiments include: O.

Qs s

The. THE

H /*"

1 ' \ ,

HNl

-N-

C. Substantially non-antigenic water soluble polymers (R1)

The prodrugs of the present invention include a polymeric residue preferably preferably water soluble and substantially non-antigenic polymers. Suitable examples of such polymers include polyalkylene oxides (PAO) such as polyethylene glycols. Some preferred polymers are polyethylene glycols such as mPEG. A non-limiting list of such polymers thus includes polyalkylene oxide homopolymers such as polyethylene glycol (PEG) or polypropylene glycols, polyoxyethylenated polyols, copolymers thereof and block copolymers thereof.

The polymeric portion of the compounds described herein has an average molecular weight of from about 2,000 to about 100,000 daltons, preferably from about 5,000 to about 60,000 daltons. In some aspects, the polyalkylene oxide may be from about 5,000 to about 25,000, and preferably from about 12,000 to about 20,000 daltons when proteins or oligonucleotides are attached or alternatively from about 20,000 to about 45,000 daltons, and preferably from about 30,000 to about 40,000 daltons when pharmaceutically active compounds (small molecules such as those having average molecular weight less than about 1,500 daltons) are employed in the compounds described herein.

Polyethylene glycol (PEG) is generally represented by the structure: -O- (CH2CH2O) n-

where (n) is an integer from about 10 to about 2,300, and is dependent on the number of polymeric arms when multi-arm polymers are used. It will be understood that the water soluble polymer will be functionalized for binding with binders. Alternatively, the portion of the polyethylene glycol (PEG) residue of the invention may be represented by the structure:

-Y7I- (CH2CH2O) n-CH2CH2Y7I-. -Y71- (CH2CH2O) n-CH2C (= Y72) -Y71-,

-Y7i-C (= Y72) - (CH2) a71 -Y73- (CH2CH2O) n-CH2CH2-Y73- (CH2) a7i-C (= Y72) -Y7-1, -Y71- (CR7IR72) a72-Y73- ( CH2) b71-O- (CH2CH2O) n- (CH2) b71-Y73- (CR71R72) a72-Y7-, -Y71- (CH2CH2O) n-CH2CH2-, -Y71- (CH2CH2O) n-CH2Q = Y72) -,

-C (= Y72) - (CH 2) a 71-Y73- (CH 2 CH 20) n-CH 2 CH 2 -Y73 - (CH 2) a 71 -C (= Y 72) -, and - (CR 7IR 72) a 72 Y Y- (CH 2) b 71 -O - (CH2CH2O) n- (CH2) b7rY73- (CR71R72) a72-, wherein:

Y7I and Y73 are independently O, S, SO, SO2, NR73 or a bond; Y72 is O, S, or NR74;

R71-74 are independently the same portions that may be used for R2; (a71), (a72), and (b71) are independently zero or a positive integer, preferably 0-6, and more preferably 1; and

(n) is an integer from about 10 to about 2300.

As an example, PEG can be functionalized as follows, non-limiting:

-C (= Y74) - (CH 2) m - (CH 2 CH 20) n -, -C (= Y74) - Y - (CH 2) m - (CH 2 CH 20) n -, -C (= Y74) -NR, - ( CH2) m- (CH2CH2O) n-, -CR75R76- (CH2) m- (CH2CH2O) n- wherein

R75 and R76 are independently selected from H, Cu6 alkyl, aryl, substituted aryl, aralkyl, heteroalkyl, substituted heteroalkyl and C1-6 substituted alkyl; m is zero or is a positive integer, preferably 1; Y74 is O or S; and

η represents the degree of polymerization. Although the prodrugs of the present invention may be formed using any of the substantially non-antigenic polymers described herein, some preferred polyalkylene oxides include:

CH 3 O-PEG-O- (CH 2) m -CO 2 H; PEG-NHS;

CH 3 O-PEG-O- (CH 2) m -NR 11 R 12; and CH 3 O-PEG-O- (CH 2) 2 -S- (CH 2) m -CO 2 H; SC-PEG; or

Any of the activated PEGs known in the art. In particular, mono-activated polyethylene glycols (PEGs), C 1-4 alkyl terminated PAOs such mono-methyl polyethylene glycols (mPEGs) are preferred when monosubstituted polymers are desired; Bis-activated polyethylene oxides are preferred when disubstituted prodrugs are desired.

Mono- or di-acid activated polymers such as PEG acids or PEG diacids are used to provide the desired bond. Suitable PAO acids can be synthesized by converting mPEG-OH to an ethyl ester. See also Gehrhardt, H., et al., Polymer Bulletin 18: 487 (1987) and Veronese, F.M. et al., J. Controlled Release 10; 145 (1989). Alternatively, PAO acid may be synthesized by converting an mPEG-OH to a t-butyl ester. See, for example, U.S. Pat. 5,605,976 and US No. 5,965,566. The teachings of these documents are incorporated herein by reference.

Branched derivatives or U-PEG are described in US Pat. Nos. 5,643,575, 5,919,455, 6,113,906 and 6,566,506, the teachings of which are incorporated herein by reference. A non-limiting list of these polymers corresponds to polymeric systems (i) - (vii) having the following structures:

Il

mPEG — O — C ^ C | _ | 2

H2 i61

mPEG — O — C. I

N

H (i), O

H Il

m-PEG-N-C2

CH- (Υ63ΟΗ2) „610 (= 0) - H / m-PEG — Ν — C

0 (ϋ),

THE

Il H

m-PEG-O — C — Ν,

m-PEG-0 — C — N 'Il H

(CH2) 4

, CH- (Y63CH2) 6iC (= 0) -

O (iü),

THE

m-PEG-0 — C — NH \

THE

(CH2) w62 N-

Ç

w61

- (CH2) W64C (= 0) -

, (CH2) w63

m-PEG-0 — C-N

Il H

O O

Il H

m-PEG-O — C — N

\

(CH2) w62

m-PEG-O — C-N

Il H

HÇ- (Y63CH2) w61C (O) -

, (CH2) w63

o (v), and

THE

m-PEG — C-NH

\

(CH2) W62

m-PEG — C-N '

Il H

HÇ- (V63CH2) w61 C (= 0) -

(CH2) w63

I saw him)

wherein: Yo-62 are independently O, S or NR6I;

Y63 is O, NR62, S, SO or SO2

(w62), (w63) and (w64) are independently O or a positive integer, preferably from about 0 to about 10, more preferably from about 1 to about 6;

(w61) is O or 1;

mPEG is methoxy PEG

wherein PEG is previously defined and the total molecular weight of the polymer moiety is from about 2,000 to about 100,000 daltons; and Rói and R62 are independently selected from hydrogen, C1-6 alkyl, C2.6

alkenyl, C 2-6 alkynyl, C 3-9 branched alkyl, C 3,8 cycloalkyl, substituted C 1-6 alkyl, C 2-9. 6 substituted alkenyl, C2.6 substituted alkynyl, C3. substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, C1-6 heteroalkyl, substituted C6-6 heteroalkyl, C1-6 alkoxy, aryloxy, C1-6 heteroalkoxy, heteroaryloxy, C2-6 alkanoyl, arylcarbonyl, C2-6 alkoxycarbonyl. 6 alkanoyloxy, arylcarbonyloxy, substituted C2.6 alkanoyl, substituted arylcarbonyl, substituted C2.6 alkanoyloxy, substituted aryloxycarbonyl, substituted C2.6 alkanoyloxy, and substituted arylcarbonyloxy.

In yet another aspect, the polymers include multi-arm PEG-OH or "PEG-star" products such as those described in the uNOF Corp. catalog. Drug Delivery System catalog, Ver. 8, April 2006 ", the contents of which are incorporated herein by reference. The multi-arm polymer conjugates have four or more polymer arms and preferably four or eight polymer arms.

For purposes of illustration and not limitation, the multi-arm polyethylene glycol (PEG) residue may be H2C-O- (CH2CH2O) nH

I

HC-0 - (CH 2 CH 2 O) nH

CH2

I * I

CH2

HC-O - (CH 2 CH 2 O) nH

CH2

r *

ο

CH2

HC-O- (CH 2 CH 2 O) nH

H2C-O- (CH2CH2O) nH

on what:

(χ) is 0 and a positive integer, i.e. from about 0 to about 28; and (n) is the degree of polymerization. In a particular embodiment of the present invention, the multi-arm PEG has the

structure:

H2C-O- (CH2CH2O) nH

HC-O- (CH 2 CH 2 O) nH

CH2

ο- ι

CH2

HC-O- (CH 2 CH 2 O) nH

CH2

r <

THE

CH2

HC-O- (CH 2 CH 2 O) nH

H2C-O- (CH2CH2O) nH

where η is a positive integer. In a preferred embodiment of the invention, the polymers have a total molecular weight of from about 5,000 Da to about 60,000 Da, and preferably from 12,000 Da to 40,000 Da.

In yet another particular embodiment, the multi-arm PEG has the structure: 10

15

or

(OCH2CH2) nOH (OCH2CH2) 'nOH

H0- (CH2CH2O) n- HO- (CH2CH2O) r, ''

where η is a positive integer. In a preferred embodiment of the invention, the polymerization degree for multi-arm polymer (n) is from about 28 to about 350 to provide polymers having a total molecular weight of about 5,000 Da to about 60,000 Da, and preferably from about 65 to about 270 (12,000-45,000 daltons) to provide polymers having a total molecular weight of about 12,000 Da to 45,000 Da. This represents the number of repeater units in the polymer chain and is dependent on the molecular weight thereof.

The polymers may be converted to appropriately activated polymers using activation techniques described in US Nos. 5,122,614 or 5,808,096. Specifically, such PEGs may be of Formula:

(CHaCH2O) ti,

Hoch ^^ och ^ /;

uVTVu--

^ H2CH2

THE

j ^ CH2CH2OJ- pjjp „

Q - PI-LnH O "2O.

-u O2LJi ^ jOCH2 CH2Ju ■

Star

or

O -CH 2 CH 2 - (OCH 2 CH 2 X 10 -0 ° -CH 3 ZHz ^ OCH 2 CHZ 1 1.0

Multi-arms

O - (CH ^ CH ^ -CH ^ H ^

ICHiCHjPJlJ-CH2CH2-O-

on what:

(u ') is an integer from about 4 to about 455; and up to 3 terminal portions of the residue is / are protected with methyl or other lower alkyl. In some preferred embodiments, all four arms of the PEG may be converted to appropriate activating groups to facilitate the attachment of aromatic groups. Such compounds prior to conversion include:

H3C- (OCH2CH2) u.

THE

H3C3 (OCH2CH2)

, (CH2CH2OW CT CH2CH2 ^ qj_ |

CX

(CH 2 CH 20) u.

-oh

, (CH2CH2O) u CH2CH2-

HaC- (OCH2CH2) o

^ (CH 2 CH 2 O) 1,

H3C- (OCH2CH2) / ° 0H

HnC-

- (OCH2CH2) u, \

HO-

-CH2CH2 - (OCH2CH2) u

(CH 2 CH 2 O) u, - ^ O '2 2 CH 2 CH 2 -

(CH 2 CH 2 O) -CH 2 CH 2.

OH

-OH

^ (CH2CH2O) u- ^

H 0 ^ CH 2 CH 2 ^ (OCH 2 CH 2) u ch ^ -OH

(CH2CH20) -ch2CH2 ^ CH2CH2 - (OCH2CH2) U ^ 0H

H3C- (OCH2CH2) u-O

H3C- (OCH2CH2) L

0— (CH2CH2O) U-CH2CH2 — OH

- (CH2CH2O) u-CH3

H3C- (OCH2CH2) U-O

H3C- (OCH2CH2) U.-

THE

O- (CH2CH2O) u-CH3

THE-

- (CH2CH2O) u-CH2CH2-OH H3C- (OCH2CH2) u-O

H3C- (OCH2CH2) u.

-THE

THE

O- (CH2CH2O) u-CH2CH2-OH

"(CH2CH2O) u'-CH2CH2-OH

HO-CH2CH2- (OCH2CH2) U-O

H3C- (OCH2CH2) u

-THE

THE

0— (CH2CH2O) u-CH2CH2 — OH

THE-

"(CH 2 CH 2 O) u CH 3

H3C- (OCH2CH2) u.-0

HO-CH2CH2- (OCH2CH2) U-

-0

O- (CH2CH2O) u-CH2CH2-OH

"(CH2CH2O) u-CH3

H3C- (OCH2CH2) u-O

HO-CH 2 CH 2 - (OCH 2 CH 2) U.

-0

0

0— (CH2CH2O) u-CH2CH2 -— OH

0-

"(CH2CH2O) u. — CH2CH2 — OH

HO-CH2CH2- (OCH2CH2) U-O H3C- (OCH2CH2) u-0

0

O- (CH2CH2O) u-CH2CH2-OH

0-

- (CH2CH2O) u-CH2CH2-OH

HO-CH2CH2- (OCH2CH2) U.-0

HO-CH 2 CH 2 - (OCH 2 CH 2) U.

-THE

THE

O- (CH2CH2O) U-CH2CH2-OH

THE-

"(CH2CH2O) u-CH2CH2-OH

The polymeric substances included herein are preferably water soluble at room temperature. A non-limiting list of such polymers includes polyalkylene oxide homopolymers, such as pilethylene glycol (PEG) or polypropylene glycols, polyoxyethylenated polyols, copolymers thereof and block copolymers thereof, provided that the water solubility of the block copolymers is maintained. In still another embodiment, and as an alternative to PAO base polymers, one or more effective non-antigenic materials such as dextran, polyvinyl alcohols, carbohydrate-based polymers, hydroxypropyl methacrylamide (HPMA), polyalkylene oxides, and / or copolymers thereof can be used. See also US Patent No. 6,153,655, the teachings of which are incorporated herein by reference. It will be understood by one of ordinary skill in the art that the same type of activation may be employed, as described herein for PAOs such as PEG. Those skilled in the art will further notice that the following list is illustrative only and that any polymeric materials having the qualities described herein are contemplated. For purposes of the present invention "substantially or effectively non-antigenic" means any material known in the art to be non-toxic and not to trigger an appreciable immunogenic response in mammals.

In some aspects, polymers having terminal amino groups may be employed to make the compounds described herein. Methods for preparing polymers containing high purity amino termini are described in US Patent Nos. 11,508,507 and 11,537,172, the teachings of which are incorporated herein by reference. For example, azide-containing polymers react with phosphine-based reducing agents such as triphenylphosphine or an alkali metal boroid reducing agent such as NaBH4. Alternatively, polymers including leaving groups react with amine salts such as methyl tert-butyl imidodicarbonate potassium salt (KNMeBoc) or di-tert-butyl imidodicarbonate potassium salt (KNBoc2) followed by deprotection of the protected amino group . The purity of the terminal amine-containing polymers formed by these processes is greater than about 95% and preferably greater than 99%.

In alternative aspects, polymers containing terminal carboxylic acid groups may be employed in the polymeric transport systems described herein. Methods for preparing polymers having US No. 11 / 328,662, the teachings of which are incorporated herein by reference. Methods include first preparing a tertiary alkyl ester of a polyalkylene oxide followed by conversion to the carboxylic acid derivative thereof. The first step of preparing the PAO carboxylic acids from the process includes forming an intermediate such as the polyalkylene oxide carboxylic acid t-butyl ester. This intermediate is formed by reacting PAO with a t-butyl haloacetate in the presence of a base such as potassium t-butoxide. Once the t-butyl ester intermediate is formed, the polyalkylene oxide carboxylic acid derivative can readily be obtained in purity exceeding 92%, preferably exceeding 97%, more preferably exceeding 99% and even more preferably exceeding 99.5%. of purity.

5th

D. DIRECTING AGENTS (D1)

Targeting agents may be attached to the polymeric compounds described herein to guide the conjugates to the target area in vivo. Targeting agents allow biologically active moieties such as pharmaceutically active compounds and oligonucleotides to have therapeutic efficacy in the target area, i.e. the tumor site. Targeted transport in vivo increases the cellular uptake of these molecules for improved therapeutic efficacy. In certain aspects, some cell penetrating peptides may be exchanged for a variety of targeting peptides for targeted transport to the tumor site.

In one aspect of the invention, the targeting moiety, such as a chain antibody

(SCA) or single-chain antigen-binding antibody, monoclonal antibody, cell adhesion peptides such as RGD and Selectin peptides, cell penetration peptides (CPPs) such as TAT, Penetratin and (Arg) 9, receptor alloys, Targeting carbohydrates or lectins, oligonucleotides, oligonucleotide derivatives such as blocked nucleic acids (LNA) and aptamers, or the like, allow cytotoxic drugs to be specifically targeted to target regions. See Curr Opin Pharmacol. 2006 0ct; 6 (5): 509-14, "Cell-penetrating peptides as vectors for peptide, protein and oligonucleotide delivery"; see also J Pharm Sci. 2006 Sep; 95 (9): 1856-72 "Cell adhesion molecules for targeted drug delivery", the teachings of which are incorporated herein by reference.

The targeting moieties may be labeled with biotinylated compounds, fluorescent compounds, radiolabeled compounds. An appropriate label is prepared by binding any appropriate moiety, eg, an amino acid residue, to any prior art isotope emission, radio-opaque marker, magnetic resonance marker, or other suitable non-radioactive isotopic imaging markers. by magnetic resonance imaging, fluorescent-type markers, markers displaying visible colors and / or capable of fluorescing under ultraviolet, infrared or electrochemical stimulation, to allow imaging of tumor tissue during surgical procedure and so on. Optionally, the diagnostic marker is incorporated into and / or bound to a conjugated therapeutic moiety, allowing monitoring of the therapeutic delivery of a biologically material within an animal or human patient.

In still a further embodiment of the present invention, labeled conjugates are readily prepared by methods known in the art with any appropriate marker, including, e.g., radioisotopic markers. Simply by way of example, these include 1331 Iodine, 123Iodine, 99mTechnium and / or 111Indium to produce radioimmunointigraphic agents for selective uptake in tumor cells in vivo. For example, there are a number of methods known in the art for binding peptides to Tc-99m, including, by way of example only, those shown in US Pat. 5,328,679; 5,888,474; 5,997,844 and 5,997,845, incorporated herein by reference.

Preferred targeting moieties are single chain antibodies (SCAs) or variable fragments of single chain antibodies (sFv). An SCA has antibody domains that can bind to or recognize specific target tumor cell molecules. In addition to maintaining an antigen binding site, a PEGylated ACS can, by ligands, reduce antigenicity and increase the half-life of ACS in the bloodstream.

The terms "single chain antibody" (SCA), "molecule or antibody that binds

SimpleS chain antigen "or" Single chain Fv "(sFv) are used interchangeably. Single chain antibody has antigen binding affinity. Single chain antibody (SCA) or single chain sFv were constructed in several A description of the theory and production of single chain antigen binding proteins can be found in US Patent Applications No. 10 / 915,069 and US No. 6,824,782, the teachings of which are incorporated herein by reference.

Typically, SCA or Fv domains may be selected from monoclonal antibodies known in the literature by their abbreviations 26-10, MOPC 315, 741F8, 520C9, McPC 603, Dl.3, murine phOx, human phOx, RFL3.8 sTCR, 1A6, Sel55 -4.18-2- 3,4-4-20,7Α4-1, B6.2, CC49.3C2.2c, MA-15C5 / K12G0, Ox, etc. (see, Huston, JS, et al., Proc. Natl. Acad. Sci. USA 85: 5879-5883 (1988); Huston, JS, et al., SIM News 38 (4) (Supp): 11 (1988); McCartney, J. et al., ICSU Short Reports 10: 114 (1990); McCartney, JE et al., Unpublished results (1990); Nedelman, MA et al., J. Nuclear Med. 32 (Supp.): 1005 (1991); Huston, JS and collaborators, In: Molecular Design and Modeling: Concepts and Applications, Part B, edited by JJ Langone, Methods in Enzymology 203: 46-88 (1991); Huston, JS and collaborators, In: Advances in the Applications of Monoclonal Antibodies in Clinical Oncology, Epenetos, AA (Ed.), London, Chapman & Hall (1993); Bird, RE et al., Science 242: 423-426 (1988); Bedzyk, WD et al., J. Biol. Chem 265: 18615-18620 (1990); Colcher, D. et al., J. Nat. Cancer Inst. 82: 1191-1197 (1990); Gibbs, RA et al., Proc. Natl. Acad. Sci. USA 88: 4001-4004 (1991); Milenic, DE and collaborators s, Cancer Research 51: 6363-6371 (1991); Pantoliano, M. W. et al., Biochemistry 30: 10117-10125 (1991); Chaudhary, V. K. et al., Nature 339: 394-397 (1989); Chaudhary, V. K. et al., Proc. Natl. Acad. Know. USA 87: 1066-1070 (1990); Batra, J. K. et al., Biochem. Biophys. Res. Comm. 171: 1-6 (1990); Batra, J. K. and colleagues. J. Biol. Chem. 265: 15198-15202 (1990); Chaudhary, V. K. et al., Proc. Natl. Acad I know. USA 87: 9491-9494 (1990); Batra, J. K. et al., Mol. Cell. Biol. 11: 2200-2205 (1991); Brinkmann, U. et al., Proc. Natl. Acad. Know. USA 88: 8616-8620 (1991); Seetharam, S. et al., J. Biol. Chem. 266: 17376-17381 (1991); Brinkmann, U. et al., Proc. Natl. Acad. Know. USA 89: 3075-3079 (1992); Glockshuber, R. et al., Biochemistry 29: 1362-1367 (1990); Skerra, A. et al., Bio / Technol. 9: 273-278 (1991); Pack, P. et al., Biochemistry 31: 1579-1534 (1992); Clackson, T. et al., Nature 352: 624-628 (1991); Marks, J. D. and colleagues, J. Mol. Biol. 222: 581-597 (1991); Iverson, B. L. et al., Science 249: 659-662 (1990); Roberts, V. A. et al., Proc. Natl. Acad. Know. USA 87: 6654-6658 (1990); Condra, J. H. et al., J. Biol. Chem. 265: 2292-2295 (1990); Laroche, Y. et al., J. Biol. Chem. 266: 16343-16349 (1991); Holvoet, P. et al., J. Biol. Chem. 266: 19717-19724 (1991); Anand, Ν. N. et al., J. Biol. Chem. 266: 21874-21879 (1991); Fuchs, P. et al., Biol Technol. 9: 1369-1372 (1991); Breitling, F. et al., Gene 104: 104-153 (1991); Seehaus, T. et al., Gene 114: 235-237 (1992); Takkinen, K. et al., Protein Engng. 4: 837-841 (1991); Dreher, M. L. and co-workers, J. Immunol. Methods 139: 197-205 (1991); Mottez, E. et al., Eur. J. Immunol. 21: 467-471 (1991); Traunecker, A. et al., Proc. Natl. Acad. Know. USA 88: 8646-8650 (1991); Traunecker, A. et al., EMBO J. 10: 3655-3659 (1991); Hoo, W.F.S. et al., Proc. Nati. Acad. Know. USA 89: 4759-4763 (1993)). Each of these publications has been incorporated by reference.

Alternatively, the targeting moieties may be oligonucleotides or

derivatives thereof. Oligonucleotides (analogs) are not limited to a single species but, on the contrary, are designed to function with a variety of such portions, it being understood that ligands may bind to one or more 3'- or 5'- groups, usually groups. PO4 or SO4 of a nucleotide. Oligonucleotides include antisense or complementary oligonucleotides, low interference RNA (siRNA), blocked nucleic acid (LNA), micro RNA (miRNA), peptide nucleic acid (PNA), olligo morpholine phosphorodiamidate (PMO), and aptamer, etc.

For example, a non-limiting list of targeting groups include vascular endothelial cell growth factor, FGF2, somatostatin and somatostatin analogs, transferrin, melanotropin, ApoE and ApoE peptides, von Willebrand factor and Willebrand factor peptides, fiber protein. adeniviral and adenoviral fiber protein peptides, PD1 and PD1 peptides, EGF and EGF peptides, RGD peptides, folate, etc. Other optional targeting agents noted by the skilled artisan may also be employed in the compounds described herein. Preferably, the targeting agents include single chain antibody.

(SCA), RGD peptides, selectin, TAT, penetratin, Oligo (Arg), preferably (Arg) 9, folic acid, etc. For purposes of the present invention, the term "TAT" may be understood to mean a transactivator moiety of the transcription activation protein including the peptide sequence YGRKKRRQRRR, for example. The list of peptide sequences and structures used in the descriptive report and examples includes: C-TAT: (SEQ ID NO: 1) CYGRKKRRQRRR; C- (Arg) 9: (SEQ ID NO: 2) CRRRRRRRRR; RGD can be linear or cyclic: folic acid

(Arg) 9 may include a cysteine for conjugation such as CRRRRRRRRR and TAT

You can add an extra cysteine to the end of the peptide such as CYGRKKRRQRRRC.

E. BIOLOGICALLY ACTIVE PORTIONS (D2)

A wide variety of biologically active moieties may be attached to the activated polymers described herein. Biologically active moieties include pharmaceutically active compounds, enzymes, proteins, oligonucleotides, antibodies, monoclonal antibodies, single chain antibodies and peptides.

Any cytotoxic or chemotherapeutic agent, i.e. folic acid, etc. Capable of binding to a polymer, including but not limited to those of biologically active moieties, are described in US Patent No. 5,965,566, the teachings of which are incorporated herein by reference.

In one aspect of the invention, biologically active compounds are suitable for medical or diagnostic use in the treatment of animals, e.g., mammals including humans, for conditions in which such treatment is desired. In another aspect, biologically active moieties containing amino-, hydroxyl- or thiol-

are within the scope of the present invention. The only limitation on the types of biologically active moieties suitable for inclusion is that at least one amino-, hydroxyl- or thiol- group is available to react and bind with a carrier moiety and that there is no substantial loss in bioactivity in the form of the moiety. in conjunction with the polymeric transport systems described herein.

Alternatively, suitable precursor compounds for incorporation into the carrier polymer conjugate compounds of the present invention may be active after hydrolytic release of the bound compound, or inactive after hydrolytic release of the bound compound but active after further processing / reaction. For example, an anticancer drug that is carried into the bloodstream by the polymeric transport system will remain inactive until it enters the cancer or tumor cell, where it will be activated by cell chemistry, e.g., by an enzymatic reaction that is unique to that cell.

In a preferred embodiment the polymeric carrier systems described herein include pharmaceutically active compounds.

For purposes of the present invention it should be understood that the pharmaceutically compounds include low molecular weight molecules. Typically, pharmaceutically acceptable compounds have a molecular weight of less than about 1,500 daltons. A non-limiting list of these compounds includes topoisomerase I DNA inhibitors such as camptothecin and paclitaxel analogs, taxanes and derivatives, nucleosides including AZT and acyclovir, anthracycline compounds including daunorubicin and doxorubicin, anti-metabolite related compounds including Ara-C ( cytosine arabinoside) and gemcitabine, etc.

1. Taxanes and Taxane Derivatives

One class of the compounds included in the prodrug compositions of the present invention is taxanes. For purposes of the present invention, the term "taxane" includes all compounds within the terpene family of taxanes. Thus, taxol® (paclitaxel), 3'-substituted te / Y-butoxycarbonyl amine derivatives (taxotere®) and the like as well as other available analogs of, for example Sigma Chemical from St. Louis, Missouri, are within the scope of the present invention.

These compounds have proven to be effective anti-cancer agents. Several studies indicate that the agents have activity against several neoplasms. Until now, their use was severely due, among other reasons, to their poor availability, poor water solubility and hypersensitivity. It is to be understood that other taxanes, including 7-aryl carbamates and 7-carbazates disclosed in US Pat. 5,622,986 and 5,547,981 may also be included among the prodrugs of the present invention. The teachings of these documents are incorporated herein by reference.

While the examples describe inter alia paclitaxel for illustrative purposes, it should be understood that the methods described herein are suitable for all taxanes and related molecules.

2. Camptothecin and Related Topoisomerase I Inhibitors

Camptothecin is a water-insoluble cytotoxic alkaloid produced by Chinese natural camptotheca accuminata trees and nothapodytes foetida from India. Camptothecin, related compounds and the like are also known to be potential anticancer or antitumor agents and have been shown to be active in vitro and in vivo. Camptothecin and related compounds are also candidates for conversion to prodrugs of the present invention. See, for example, US Patent No. 5,004,758 and the Hawkins Publication, iiOncology, December 1992, pp. 17-23. Camptothecin and related analogs are, for example, Topotecan, Irinotecan (CPT-11) or SN38.

Additional camptothecin analogs include those reported in the literature including 10-hydroxycamptothecins, 11-hydroxycamptothecins and / or 10,11-dihydroxycamptothecins, 7- and / or 9-alkyl, substituted alkyl, cycloalkyl, alkoxy, alkenyl, aminoalkyl, etc. camptothecins, A-ring substituted camptothecins such as 10,11-alkylenedioxycamptothecins, such as those described in US Patent No. 5,646,159, the teachings of which are incorporated herein by reference, etc.

In a preferred embodiment of the present invention the cytotoxic agent is SN38.

3. Oligonucleotides

In order to fully understand the scope of the present invention, the following terms are defined. The person skilled in the art will understand that the terms "nucleic acid" or "nucleotide" apply to deoxyribonucleic acid ("DNA"), both single stranded and double stranded ribonucleic acid ("RNA"), unless otherwise specified, and any chemical modifications thereof. An "oligonucleotide" is generally a small polynucleotide, e.g., ranging in size from about 2 to about 200 nucleotides, or more preferably from about 10 to about 30 nucleotides in length. Oligonucleotides according to the present invention are generally synthetic nucleic acids and are single stranded unless otherwise specified. The terms "polynucleotide" and "polynucleic acid" may be used herein synonymously.

Modifications to the oligonucleotides contemplated by the invention include, for example, the addition or substitution of selected nucleotides with functional groups or moieties that allow covalent attachment of an oligonucleotide to a desired polymer, and / or the addition or substitution of charge-incorporating functional moieties. polarizability, hydrogen bonding, electrostatic interaction and oligonucleotide functionality. Such modifications include, but are not limited to, sugar 2'-position modifications, pyrimidine 5-position modifications, purine 8-position modifications, exocyclic amine modifications, 4-thiouridine substitution, 5- bromine or 5-iodouracil, backbone modifications, methylations, pair and base combinations such as isocytidine and isoguanidine isobases, and analogous combinations. Oligonucleotide modifications may also include 3 'and 5' modifications such as protection. A non-limiting list of nucleoside analogs includes: t

THE-

-THE-

B

THE

O = P-S "

Phosphotioate

THE-

THE

THE

O = P-O "/

2-0-Methyl

B

ο-ι _ B

ο 0

0 = Y-0 "

ζ

Ί-MOE

THE-

B

0 xF

O = P-O "

/

2'-Fluoro

2'-AP

NH2

HNA

Scene

B

N'H

-f O

NAP

y,

O = P-N

\

Morpholine

F B

ο

O = II-O "2'-F-ANA

0

0-

B

ο

O-P-O 2'-β-hydroxy) propyl

λ

OH

0-

B

N

O = P-O "

s

3'-Phosphoramidate

0-

B

ο

O = P-BH3 "Boranophosphates

10

See more examples of nucleoside analogs described in Freier &Altmann; Nucl. Acid Res., 1997, 25, 4429-4443 and Uhlmann; Curr. Opinion in Drug Development 2000, 3 (2), 293-213, the teachings of which are incorporated herein by reference.

The term "antisense" as used herein refers to sequence and nucleotides that are complementary to specific DNA or RNA sequences that encode a gene product or that encode a control sequence. The term "antisense tape" is used to refer to a nucleic acid tape that is complementary to the "sense" tape. In normal operation of cellular metabolism, the sense strand of the DNA molecule is the strand that encodes polypeptides and / or other gene products. The sense strand serves as a template for the synthesis of transcribed messenger RNA ("mRNA") (an antisense strand) which, in turn, directs the synthesis of any encoded gene product. Antisense nucleic acid molecules may be produced by methods known in the art, including synthesis by ligating the gene (s) of reverse orientation into a viral promoter that allows the synthesis of a complementary strand. Once introduced into the cell, this transcribed tape combines with natural sequences produced by the cell to form duplexes. These duplexes then block both later transcription and translation. In this way mutant phenotypes can be generated. The designations "negative" or (-) are also known in the art to refer to antisense tape and "positive" or (+) to refer to sense tapes.

In a preferred embodiment, the choice of conjugation is an oligonucleotide (or "polynucleotide") and, after conjugation, the target is referred to as an oligonucleotide residue. Oligonucleotides may be selected from any phosphodiester or phosphorothioate backbone oligonucleotides and oligodeoxynucleotides known in the art.

Oligonucleotide or oligonucleotide derivatives may include from about 10 to about 1000 nucleic acids and preferably relatively small polynucleotides, eg, ranging in size from about 2 to about 200 nucleotides, or more preferably from about 10 to about 100 nucleotides. 30 nucleotides in length.

Additionally, oligonucleotides and oligodeoxynucleotides useful in accordance with the present invention include, but are not limited to, the following:

Oligonucleotides and oligodeoxynucleotides with natural phosphodiester or phosphotioate backbones or any modified backbone analogs;

LNA (blocked nucleic acid);

PNA (nucleic acid with peptide backbone);

Low interference RNA (siRNA);

microRNA (miRNA);

Nucleic acid with peptide backbone (PNA);

Morphino phosphodiamidate (PMO) oligonucleotides;

tricyclo-DNA;

ODN (double-stranded oligonucleotide); catalytic RNA sequence (RNAi);

ribozymes;

aptameros;

spiegelomers (L-conformation oligonucleotides); CpG oligomers, and the like, as described in:

Tides 2002, "Oligonucleotide and Peptide Technology Conferences", May 6-8, 2002, Las Vegas, NV and "Oligonucleotide & Peptide Technologies", 18th & 19th November 2003, Hamburg, Germany, the contents of which are incorporated herein by reference.

Oligonucleotides according to the present invention may optionally include any suitable analogs or derivatives known in the art, including those listed in Table 1 below:

TABLE 1 Representative Nucleotide Analogs and Derivatives 4-Acetylcytidine 5-methoxyminomethyl-2-thiouridine 5- (carboxyhydroxymethyl) uridine beta, D-mannosylqueuosin 2'-0-methylcytidine 5-methoxycarbonylmethyl-2-thiouridine-5-carboxymethyl-2-thiomethylamine -methoxycarboni Imethiuridine 5-carboxymethylaminomethyluridine 5-methoxyuridine Dihydrouridine 2-methylthio-N6-isopentenyladenosine 2'-0-methylpseudouridine N - ((9-beta-D-ribofuranosyl-2-methylthio) -thiazine-thiazine-2-yl) - ((9-beta-D-ribofuranosylpurin-6-yl) N-methylcarbamoyl) threonine 2'-0-methylguanosine Uridine-5-oxiacetic acid methylester Inosine Uridine-5-oxyacetic acid N6-isopentenyladenosine Wibutoxosin 1-methyladenosine Pseudine -methylpseudouridine Queuosine 1-methylguanosine 2-thiocytidine 1-methylinosine 5-methyl-2-thiouridine 2,2-dimethylguanosine 2-thiouridine 2-methyladenosine 4-thiouridine 5-methyluridine 3-methylcitidine N - ((9-beta- D-ribofuranosylpurine-6-yl) carbamoyl) threonine 5-methylcytidine 2'-0-methyl-5-methyluridine N6-methyladenosine 2'-0-methyluridine 7-methylguanosine wibutin 5-methylaminomethyluridine 3- (3-amino-3-carboxypropyl) uridine Blocked adenosine Blocked guidine Thymine blocked blocked uridine blocked methylcytidine

Preferably, the oligonucleotide surrounds the target tumor cells or regulates the protein involved in tumor cell resistance to the anti-cancer therapeutic compound. For example, any protein known in the art, such as BCL-2, for antisense oligonucleotide regulation for cancer therapy may be used for the present invention. See US Patent Application No. 10 / 822,205, filed April 9, 2004, the teachings of which are incorporated herein by reference. A non-limiting list of preferred therapeutic oligonucleotides includes HIF-1a antisense oligonucleotides and oligonucleotides. Survivine antisense. In a preferred embodiment, the oligonucleotide may be, for example, a

oligonucleotide having substantially a nucleotide sequence substantially similar to that of Genasense (w / w / a oblimersen sodium, produced by Genta Inc., Berkeley Heights, NJ). Genasense is an 18-unit phosphotioate antisense oligonucleotide, TCTCCCAGCGTGCGCCAT (SEQ ID NO: 6), complementary to the first six codons of the human bcl-2 mRNA primer (bcl-2 human mRNA is known in the art), and is described , eg, as SEQ ID NO: 19 in US Patent No. 6,414,134, incorporated herein by reference). The U.S. Food and Drug Administration (FDA) gave Genasense the Orphan Drug classification in August 2000. Preferred modalities include: (i) Survivin antisense LNA (SEQ ID NO: 3)

mCs-Ts-mCs-As.as-ts-cs-cs-as-ts-gs-gs-mCs-As-Gs-c;

where the capital letters represent LNA, the "s" represents a phosphotioate backbone;

(ii) Bcl2 antisense siRNA:

SENSE 5'- GCAUGCGGCCUCUGUUUGAdTdT-3 '(SEQ ID NO: 4)

ANTISENSE 3'-dTdTCGUACGCCGGAGACAAACU-5 '(SEQ ID NO: 5) where dT represents DNA;

(iii) Genasense (antisense phosphotioate oligonucleotide): (SEQ ID NO: 6) ts-cs-ts-cs-cs-as-gs-cs-gs-cs-gs-cs-cs-cs -as-t where the lower case letters represent DNA and "s" represent the phosphotioate backbone;

(iv) HIFla antisense LNA (SEQ ID: 7)

5'- sTsGsGscsasasgsgsastscscsTsGsTsa -3 '(SEQ ID NO: 7) where the capital letters represent LNA, the "s" represents a phosphotioate backbone;

LNA includes 2'-0.4'-C methylene bicyclonucleotide as shown below:

where "monomer" = monomer and "configuration" = configuration.

See detailed description of survivin LNA in US Patent Nos. 11 / 272,124, entitled "LNA Oligonucleotides and the Treatmemt of Cancer" and 10 / 776,934, entitled "Oligomeric Compounds for the Modulation Survivin Expression", the teachings of which are incorporated herein by reference. See also US patent applications Nos. 10 / 407,807 entitled "Oligomeric Compounds for the HIF-I Alpha Expression Modulation" and 11 / 271,686 entitled "Potent LNA Oligonucleotides for Inhibition of HIF-IA Expression", the contents of which are incorporated herein by reference.

The oligonucleotides employed in the compounds described herein can be modified with amino (CH2) w linkers at the 5 'or 3' termini of the oligonucleotides, where w, in that aspect, is a positive integer preferably from about 1 to about 10, preferably 6. Modified oligonucleotides contemplated in the compounds described herein may be NH- (CH2) w-Oligonucleotide.

In a preferred embodiment, the 5 'termination of the same siRNA tape is modified. For example, the siRNA employed in the polymer conjugates is modified with a 5'-C6-NH2. A particular embodiment of the present invention employs Bcl2-siRNA having the sequence of

1

8 LNA Monomer β-D configuration

SENSE 5 '- (NH2-C6) GCAUGCGGCCUCUGUUUGAdTdT-3' ANTISENSE 3'-dTdTCGUACGCCGGAGACAAACU-5 '. In another preferred embodiment, the compounds described herein may include hindered ester-containing oligonucleotides containing amino (CH2) linkers. See US Patent Application No. 60 / 844,942 entitled "Polyalkylene Oxide Having Hindered Ester-Based Biodegradable Linkers", the teachings of which are incorporated herein by reference. Polymeric compounds can release oligonucleotides without the amino tail. For example, oligonucleotides may have the structure:

preferably about 6.

In yet another preferred embodiment, the oligonucleotides may be modified with sulfhydryl (thio oligonucleotide) ligands (CH 2). Thio oligonucleotides may be used for direct conjugation to the cysteine residue of the positively charged peptide via the maleimidyl group. Thio oligonucleotides may have SH- (CH2) w-Oligonucleotide structure. Thio oligonucleotides may further include hindered esters having the structure:

10

(where "oligonucleotide" =

oligonucleotide).

20

Examples of modified oligonucleotides include:

(i) C6-NH2 modified tailed genasense:

5'- NH2- C6- stsCstscscscsasgscsgstsgscsgscscsast -3 '

s

Il

O-P-T-sC-sT-sC-sC-sC-sA-sG-sC-sG-sT-sG-sC-sG-sC-sC-sA-sT Cr

25

(ii) C6-NH2 tail modified antisense HIFla LNA: 5'-NH2-C6-sTsGsGscsasasgscsastscscsTsGsTsa -3 ';

(iii) Survivin C6-NH2 tail modified antisense LNA: 5'-NH2-C6-smCsTsmCsAsastscscsastsgsgsmCsAsGsc -3 ';

(iv) Survivin C6-SH tail modified antisense LNA:

5'-HS-C6-smCsTsmCsAsastscscsastsgsgsmCsAsGsc -3 ';

(v) Modified Genasense with hindered ester tail:

THE

O — T-C-T-C-C-C-G-C-G-T-G-C-G-C-C-A-T

4. Additional Biologically Active Portions

In addition, the compounds of the present invention may be prepared using many other compounds. For example, biologically active compounds such as cisplatin derivatives containing OH groups, floxuridine, podophyllotoxin and related compounds may be included.

Other useful precursor compounds include, for example, some biologically active low molecular weight proteins, enzymes and peptides, including glycan peptides, as well as other anti-tumor agents, cardiovascular agents such as forskolin, antineoplastics such as combretastatin, vinblastine, vincristine , doxorubicin, AraC, maytansine, etc. anti-infectives such as vancomycin, etc. antifungal agents such as nystatin or amphoteracin B, antiinflammatory agents, steroidal agents and the like.

The following are biologically active portions that are suitable for the prodrugs of the present invention.

F. PERMANENT AND LEVEL BONDS: (L1 and L3)

Ligands L1 and L3 include bifunctional ligands. Bifunctional may be permanent or labile binders. Bifunctional ligands include amino acids or amino acid derivatives. Amino acids can be either naturally occurring or non-naturally occurring amino acids. Naturally occurring amino acid derivatives and analogs, as well as various non-naturally occurring amino acids known in the art (D or L), hydrophobic or otherwise, are also contemplated within the scope of the invention. A suitable non-limiting list of non-naturally occurring amino acids includes 2-aminoadipic acid, 3-aminoadipic acid, beta-alanine, beta-aminopropionic acid, 2-aminobutyric acid, 4-aminobutyric acid, piperidinic acid, 6-aminocaproic acid, 2-aminoheptanoic acid, 2-aminoisobutyric acid, 3-aminoisobutyric acid, 2-aminopimelic acid, 2,4-aminobutyric acid, desmosine, 2,2-diaminopimelic acid, 2,3-diaminopropionic acid, N-ethylglycine, N-ethylilasparagine, 3-hydroxyproline, 4-hydroxyproline, isodesmosin, allo-isoleucine, N-methylglycine, sarcosine, N-methyl-isoleucine, 6-N-methyl-lysine, N-methylvaline, norvaline, norleucine, and ornithine. Some preferred amino acid residues are selected from glycine, alanine, methionine and sarcosine, and more preferably glycine. Alternatively, Li and L3 can be selected from: - [C (= 0)] v (CR22R23) t [C (= 0)] v-, - [C (= 0)] v (CR22R23) r0 [C (= 0)] v -, - [C (= 0)] v (CR22R23) t-NR26 [C (= 0)] v-, - [C (= 0)] v0 (CR22R23) t [C (= 0) ] v -, - [C (= 0)] v0 (CR22R23) t0 [C (= 0)] v-, - [C (= 0)] v0 (CR22R23) tNR26 [C (= 0)] v-, - [C (= 0)] vNR2, (CR22R23) t [C (= 0)] v-, - [C (= 0)] vNR21 (CR22R23) t0 [C (= 0)] v-, - [C (= 0)] vNR21 (CR22R23) tNR26 [C (= 0)] v-, - [C (= 0)] v (CR22R23) t0- (CR28R29) r [C (= 0)] v-, - [ C (= 0)] v (CR22R23) tNR26- (CR28R29) t. [C (= 0)] v-, - [C (= 0)] v (CR22R23) tS- (CR28R29), [C (= 0 )] v-, - [C (= 0)] v0 (CR22R23) t0- (CR28R29) t [C (= 0)] v-, - [C (= 0)] v0 (CR22R23) tNR26- (CR28R29) f [C (= 0)] v.-, - [C (= 0)] v0 (CR22R23) tS- (CR28R29) t [C (= 0)] v-, - [C (= 0)] vNR21 ( CR22R23) t0- (CR28R29) t [C (= 0)] v-, - [C (= 0)] vNR21 (t CR22R23) tNR26- (CR28R29) t [C (= 0)] v-, - [C (= 0)] vNR21 (CR22R23) tS- (CR28R29) t. [C (= 0)] v-, - [C (= 0)] v (CR22R23CR28R290) tNR26 [C (= 0)] v-, - [C (= 0)] v (CR22R23CR28R290) t [C (= 0)] v-, - [C (-0)] v0 (CR22R23CR28R290) tNR26 [C (= 0)] v-, - [C (= 0)] v0 (CR22R23CR28R290) t [C (= 0)] v -, - [C (= 0)] vNR21 (CR22R23CR28R290) tNR26 [C (= 0)] v-, - [C (= 0)] vNR2I (CR22R 23CR28R290) t [C (= 0)] v-, - [C (= 0)] v (CR22R23CR28R290) t (CR24R25) f [C (= 0)] v-, - [C (= 0)] v0 ( CR22R23CR28R290) t (CR24R25) t [C (= 0)] v-,

- [C (= 0)] vNR21 (CR22R23CR28R290) t (CR24R25) t [C (= 0)] v-, - [C (= 0)] v (CR22R23CR28R290) t (CR24R25) r0 [C (= 0) ] v-, - [C (= 0)] v (CR22R23) t (CR24R25CR28R290) t [C (= 0)] v-, - [C (= 0)] v (CR22R23) t (CR24R25CR28R290) t'NR26 [C (= 0)] V'-, - [C (= 0)] v0 (CR22R23CR28R290) t (CR24R25) tO [C (= 0)] v-,

- [C (= 0)] v 0 (CR22R23) t (CR24R25CR28R290) t [C (= 0)] v-, - [C (= 0)] v0 (CR22R23) t (CR24CR25CR28R290) t NR26 [C (= 0 )] - - [C (= 0)] VNR21 (CR22R23CR28R29O) t (CR24R25) 1 0 [C (= 0)] ν -, - [C (= 0)] vNR2i (CR22R23), (CR24R25CR28R29O) t - [C (= 0)] v -, - [C (= 0)] vNR21 (CR22R23) t (CR24R25CR28R290) tNR26 [C (= 0)] v-,

THE

- [C (= 0)] v0 (CR22R23) tx * - (CR24R25) t.NR26 [C (= 0)] v, -

-THE-

- [C (= 0)] vNR21 (CR22R23) hr (CR24R25) t.NR26 [C (= 0)] v '

-THE-

and

20

- [C (= 0)] v NR 2 i (CR 22 R 23) t \\ (CR 24 R 25) t, 0 [C (= 0)] V.

wherein: R-21-29 Are independently selected from hydrogen, Cu6 alkyls, C3-12 branched alkyls, C3.8 cycloalkyls, C1-6 substituted alkyls, C3.8 substituted alkyls, aryls, substituted aryls, aralkyls, C1-6 heteroalkyls Substituted C 1-6 heteroalkyl, C 1-6 alkoxy, phenoxy and C 1-6 heteroalkoxy;

(t) and (f) are independently zero or a positive integer, preferably zero or r an integer from about 1 to about 12, more preferably an integer from about 1 to about 8 and even more preferably 1. or 2; and

(v) and (v ') are independently zero or 1.

Preferably, bifunctional ligands may be selected from:

- [C (= 0)] rNH (CH 2) 2 CH = N-NHC (= 0) - (CH 2) 2-,

- [C (= 0)] rNH (CH 2) 2 (CH 2 CH 20) 2 (CH 2) 2 NH [C (= 0)] r -,

- [C (= 0)] rNH (CH 2 CH 2) (CH 2 CH 20) 2 NH [C (= 0)] r-,

- [C (= 0)] rNH (CH 2 CH 2) s NH (CH 2 CH 2) s [C (= 0)] r -,

- [C (= 0)] r NH (CH 2 CH 2) s S (CH 2 CH 2) s [C (= 0)] r-,

- [C (= 0)] rNH (CH 2 CH 2) (CH 2 CH 20) [C (= 0)] r -,

- [C (= 0)] rNH (CH 2 CH 2) s 0 (CH 2 CH 2) s [C (= 0)] r-,

- [C (= 0)] rNH (CH 2 CH 2 O) (CH 2) NH [C (= 0)] r-,

- [C (= 0)] rNH (CH 2 CH 20) 2 (CH 2) [C (= 0)] r-,

- [C (= 0)] rNH (CH 2 CH 20) s (CH 2) s [C (= 0)] r-,

- [C (= 0)] rNHCH2CH2NH [C (= 0)] r-,

- [C (= 0)] rNH (CH 2 CH 2) 20 [C (= 0)] r · -,

- [C (= 0)] rNH (CH 2 CH 2 O) [C (= 0)] r-,

- [C (= 0)] rNH (CH 2 CH 2 O) 2 [C (= 0)] r-,

- [C (= 0)] rNH (CH 2) 3 [C (= 0)] r-,

- [C (= 0)] r0 (CH2CH20) 2 (CH2) [C (= 0)] r-,

- [C (= 0)] r 0 (CH 2) 2 NH (CH 2) 2 [C (= 0)] r -,

- [C (= 0)] r0 (CH 2 CH 2 O) 2 NH [C (= 0)] r-,

- [C (= 0)] r0 (CH2) 20 (CH2) 2 [C (= 0)] r-,

- [C (= 0)] r0 (CH2) 2S (CH2) 2 [C (= 0)] r-,

- [C (= 0)] r0 (CH2CH2) NH [C (= 0)] r -, - [C (= 0)] r0 (CH2CH2) 0 [C (= 0)] r-, - [C ( = 0)] r0 (CH2) 3NH [C (= 0)] r -, - [C (= 0)] r0 (CH2) 30 [C (= 0)] r-, - [C (= 0)] r0 (CH2) 3 [C (= 0)] r-, - [C (= 0)] rCH2NHCH2 [C (= 0)] r-, - [C (= 0)] rCH20CH2 [C (= 0)] r -, - [C (= 0)] rCH2SCH2 [C (= 0)] r -, - [C (= 0)] rS (CH2) 3 [C (= 0)] r-, - [C (= 0)] r (CH2) 3 [C (= 0)] r-,

- [C (= 0)] r0CH2 ^ - ^^ CH2 NH [C (= 0)] r.-

9th

- [C (= 0)] r0CH2 ^^^ - CH20 [C (= 0)] r--

9th

- [C (= 0)] rNHCH2 - ^^ - CH2NH [C (= 0)] r - - [C (= 0)] rNHCH2 - ^^> - CH20 [C (= 0)] r.—

wherein (r) and (r ') are independently zero or 1, provided that both (r) and (r') are not simultaneously zero.

1. labile binders

In a preferred embodiment of the present invention, the compounds described herein have a biologically active moiety attached to a labile linker. An advantage of the invention is that the biologically active moiety can be released in a controlled manner.

Among the labile ligands may be benzyl elimination based ligands, blocked trialkyl based ligands (or trialkylated lactonized blocking ligands), bicin-based ligands, labile acid ligands, lysosomally cleavable peptides and captepsin B cleavable peptides. labile may be disulfide bonding, hydrazone containing binders and thiopropionate containing binders.

Alternatively, labile ligands are intracellular labile ligands, extracellular ligands and labile acidic ligands. Labile acid binders, such as hydrazone bonds, may be hydrolyzed in the acid environment of the lysosome. Some suitable labile ligands are oligopeptides including Val-Cit, Ala-Leu-Ala-Leu, Gly-Phe-Leu-Gly and Phe-Lys. A preferred labile binder is a peptidyl binder (Val-Cit) which may be specifically degraded by captesin B. Preferably, L3 includes labile binder. Preferred labile binders include:

L1

al

oh "

Ç-

R31

I

-Y12-Ar-I-C-

bl I

v32

V4

Il

-Y13-C-

γιτ | > —I-C

Liaj-O- Ϊ1Ϊ j11

R

38

C (CR44R45)

111 ^ 40

Y

A51 (J ') x'1 1- (L14) q1-

Y

1I

-C - (L1) -O--

L Jp1N 7 ° 11

R39 -R42

C (CR46R47) m11

19

R43

k11 N-C-C- (J) x11 ^

/ R41

n11

-> - s — s -> -

^ 50

I

Ν — N—

R

51 O

Val-Cit-,

-Gly-Phe-Leu-Gly-, -AIa-Leu-Wing-Leu-, -Phe-Lys-,

THE

H / == ■

-VaI — Cit — C-Ν ^ λ λ

Il H / = \ Phe-Lys-C-N - (^ J) —v

THE

Il H

-Val-Cit — λ w O- ^ T 0 O,

O O,

Val-Cit-C (= 0) -CH20CH2-C (= 0) -, Val-Cit-C (= 0) -CH2SCH2-C (= 0) -, and -NHCH (CH3) -C (= 0) -NH (CH 2) 6 -C (CH 3) 2 -C (= 0) - wherein,

Y1-19 are independently O, S or NR48;

R 31-48, R 50-51, and A 5I are independently selected from hydrogen, C 1-6 alkyls, C 3-12 branched alkyls, C 3-8 cycloalkyls, C 1-6 substituted alkyls, C 3-8 substituted alkyls, aryls, substituted aryls, aralkyls, C 1-6 heteroalkyl, substituted C 1-6 heteroalkyl, C 1-6 alkoxy, phenoxy and C 1-6 heteroalkoxy; Ar is an aryl or heteroaryl moiety;

Ln-I5 are independently selected bifunctional spacers; J and J '

are independently selected from actively transported target cell moieties, hydrophobic moieties, bifunctional ligand moieties and combinations thereof;

(cll), (hll), (kl 1), (111), (mil) and (η 11) are independently selected positive integers, preferably 1; (ali), (el 1), (g11), (J11), (o 11) and (q11) are independently either zero or a positive integer, preferably 1; and

(bll), (xll), (x'll), (fl 1), (il 1) and (pl 1) are independently zero or one.

Several labile leagues. benzyl elimination based or blocked trialkyl based are described, for example, in US Pat. 6,180,095, 6,720,306, 5,965,119, 6,624,142 and 6,303,569, the contents of which are incorporated herein by reference. Bicine-based binders are also described in US Pat. 7,122,189 and 7,087,229 and US Patent Nos. 10 / 557,522, 11 / 502,108 and 11 / 011,818, the contents of which are incorporated herein by reference.

Preferably, biologically active compounds including oligonucleotides are attached to polymeric moieties of the compounds described herein via labile acid linkers. Without being bound by any theory, labile acid ligands facilitate the release of oligonucleotides from polymeric compounds into cells and specifically into the lysosome, endosome or macropinosome.

2. Permanent Binders

In preferred aspects of the invention, the targeting moiety such as SCA is attached to the multifunctional binder by a permanent binder. The permanent ligands are capable of conjugating to steering portions and the multifunctional ligand. A preferred permanent binder may be a maleimidyl containing molecule which may provide a thioether bond.

Permanent ligands containing maleimidyl groups can be selected from:

OOO In an alternative embodiment, the Permanent Ligands include structures corresponding to those shown above, but instead of a maleimidyl group having groups such as vinyl, sulfone residues, amino, carboxy, mercapto, hydrazide, carbamate alike rather than maleimidyl.

5th

G. EXIT GROUPS AND FUNCTIONAL GROUPS

In some aspects, suitable leaving groups include, without limitation, halogens (Br, Cl), activated carbonate, carbonylimidazole, cyclic imidione, isocyanate, N-hydroxysuccinimidyl, para-nitrophenoxy, N-hydroxyphthalimide, N-hydroxybenzotriazolyl, imidazole, tosylate, mesylate, tresylate, nosylate, C1 -C6 alkyloxy, C1 -C6 alkanoyl, arylcarbonyloxy, ortho-nitrophenoxy, N-hydroxybenzotriazolyl, imidazole, pentafluorophenoxy, 1,3,5-trichlorophenoxy, and 1,3,5-trifluoromethyl groups which will be apparent to a person skilled in the art.

For purposes of the present invention, leaving groups should be understood as groups capable of reacting with a nucleophile found on the desired target, i.e. a biologically active moiety and a targeting moiety.

In some preferred embodiments, functional groups to be attached to the polymeric carrier systems include maleimidyl, vinyl, sulfone, amino, carboxy, mercapto, hydrazide, carbazate residues and the like, which may further be conjugated to an active group or steering group.

For purposes of the present invention, when D1 or D2 are functional groups, Li and L3 groups are not functional moieties.

In a preferred embodiment, the functional group may be maleimidyl and the leaving groups may be selected from OH, methoxy, tert-butoxy, para-nitrophenoxy and N-hydroxysuccinimidyl.

H. PREFERRED EMBODIMENTS CORRESPONDING TO FORMULA (I)

Some particular embodiments prepared by the methods described herein include:

30 O

-VaI — Cit — C-D2

O _ ο

D2 / - {/> - Ν — C-Cit-Val

^ —Ο ^ NH Η

HN '

-Go — Cit-C-

THE

ο = 'B' PEG f "/ ........ j O

THE

H O

0K

H 7

mPE G ^ 1-nYS

0

Il

-VaI — Cit — C-D2

0 Ο.

D2-C-Cit-Val

mPEG

D2

/ —— cit-va |.

THE

THE

HN ^ Val-Cit-C — ΝΗΛχ

Of

The Q

-X ^ -D1

THE

The Val — Cit-C — NH

^ t ~ \ D2

mPEG

HN / Val ~ -Cit-C ~ D2

THE

-N

O H

THE

THE

D2-C-Cit-Val

H

O o (

THE

w, li

-VaI — Cit — C —- D

-N 'Ò H

υ

THE

-VaI — Cit — C-D2

mPEG

D2 — C-Cit-Val

THE

-VaI — Cit — C-D2

8 M.

^ VaI — Cit — C-D2

mPEG

THE

THE

THE

Peg

THE

THE

mPEG

THE

• Vai — Cit — C-NH-

The f ^ N '* 52

A.S.

W //

D1

THE

D2 O

D2

mPEG

THE

^ /) - NH-C-Cit-Val

R52 ^ n'R51h o '

N

NH

Peg

THE

THE

Il

Phe-Lys-C-

The r ^ N '* 52

No. on what

mPEG has Formula: CH 3 -O (CH 2 CH 2 O) n-; PEG has Formula -O (CH 2 CH 2 O) n-;

(n) is a positive integer from about 10 to about 2,300; R51 and R52 are independently selected from hydrogen, C1-6 alkyls, C3-12 branched alkyls, C3.8 cycloalkyls, Cu6 substituted alkyls, C3.8 substituted alkyls, aryls, substituted aryls, aralkyls, CN6 heteroalkyls, Cu6 substituted heteroalkyls, C C,. 6 alkoxy, C 1-6 alkyloxycarbonyl, aryloxycarbonyl, phenoxy and C 1-6 heteroalkoxy; D1 is a directing moiety, a functional group or an leaving group; and D 2 is a biologically active moiety, a functional group or an leaving group. Preferred polymeric compounds according to the present invention include:

mPEG ^ N

SN38

nY ^ = - V

O O

SN38

C — SN38

mPEG ^ N

THE

SN38 — C-

-Cit — Val

"NH I H

H hV

THE

Il / = -VaI — Cit-C

^ //

SN38

THE

Il

\\ / -C-Cit-Val

SN38 NH

η Hr

THE

-VaI — Cit — C-

W //

0

SN38

mPEG

O H

THE

SN38 -k.

THE

THE'

0

Il

SN38 j— & /) - N — C-Cit-Val

H NH

^ VpEG-γΝ ^

U0

3-SN38

mPEG

SN38

The

Il

SN38C-Cit-Val

η 7

peg ^ nY% Q

THE

THE

Il

-VaI — Cit — C-SN38 H hV

mPEG ^ NY "S

THE

Il / = -VaI — Cit — C

^ //

"the O.

SN38

\ /> - C-Cit-Val

SN38 ^ NH

SN38

mPEG

THE

HN / Val-Cit-C — ΝΗΛχ ^

O o.

SN38

THE

THE

THE

THE

SN38 / - (\ /> - NH-C-Cit-Val

the cA ^ n

Γ

PEG 'O O

HN '

THE

Il

-VaI — Cit — C-NH-

THE

W

SN38

THE

THE

Il

-VaI — Cit — C-SN38

H HN mPEG ^ N O

THE

NO2,

THE

Il

SN38 C-Cit-Val

NH

NHBoch o ^ VN ^ PEG

• Vai — Cit — C-SN38

NHBoc N

NO2

NO2 O

H "SN ^ SN38

mPEG (YNVx) NHBoc N '

S JL λ .s. 1

THE

AT THE,

THE

Il

SN38

-Cit-Val

NHBoc ο

-S A, v

NO2

Peg

NO2

mPEG ^ Y

SN38

NO2

(i) —NH-C-Cit-Val

Peg

The Il

-VaI — Cit — C-NH

Ό NHBoc N

THE

SN38

THE

Il

, Phe-Lys-C-

-SN38

ι

NHBoc N '

NO2,

THE

Il

SN38 C-Lys-Phe

THE

Il

NH

Peg

HN '

, Phe — Lys-C-SN38

THE

NO2, O

H "ϊ" SN38

NHBoc N '

0

NO2

THE

Il

\ /) - C-Lys — Phe

SN38 ^ NH Η

NHBCCh 0 ^ YNY ^ peg

NO2

mPEG

THE

Il / - \

Phe-Lys-C-NH— (J> —χ SN38

H 0- ^

No

0

THE

AT THE,

NO2,

SN38 / - <! ) —NH-C-Lys-Phe

^ 0 i: τ η

υ "" "N NHBoc O ^ Vn Y ^ PEG-

O O

THE

THE

Hn

/ Phe-Lys-C-NH— (J

Ό NHBoc N

THE

M

SN38

THE

NO2

THE

Il

-VaI — Cit — C-SN38

mPEG ^ ff γ '^ O

The L ^^ ^ NH2

THE

Il

SN38C-Cit-Val

THE

Il

^ VaI — Cit — C-SN38

Y-PEG-V-A,

O-LNA LNA-O

Peg

0

Il

-VaI — Cit — C-SN38

mPEG ^ YN ^ 0

0

Il

SN38 C-Cit-Val

SCA

S-SCA

Peg

O-LNA

SN38

S-SCA

SN38

mPEG ^ N- 0

S-SCA

0

Il

SN38 — C-Cit-Val

SCA-S

nY-PEG

3 — SN38

SCA O

mPEG

/ Val-Cit-C- ^ H ι SN38

THE

SN38

SCA-S

SN38

S-SCA

mPEG ^ Y

THE

Λ /) - N-C-Cit-Val

SN38 - ^ H NH

° 0

H, Ν.

H

, Nv

^ Val-Cit-C-N- ^ „

SN38

THE

SCA-S

Jx

THE

0 "· Λ H 0

w ~ Ν O H .Ο.

S-SCA

H 7

THE

THE

Il

-VaI — Cit-C-SN38

Ο Ο F

THE

Il

SN38-C-Cit-Val

-VaI — Cit — C-SN38

-Nv ^ -S-SCA The mPEG

Hn

THE

Il / = ■ -VaI — Cit-C

O ο.

^ //

SN38

vV

^ Ny / S-SCA O

THE

<\ /> - C-Cit-Val

SN38 NH

SCA-S-V Μ. - J 0 O

HN / Val-Cit-C ^ x //

mPEG ^^ O

SN38

-NH — C-Cit — Val

SN38— ^ NH

Peg

3-SN38

mPEG

SN38-C-Cit-Val

Peg

• Vai — Cit - (: - SN38 O

Il

Λ / al — Cit — C-

-SN38

ι

mPEG ^^ Y ^ O Η NH2

S-SCA

THE

SN38-

SCA-S '

-Cit-Val

NH

2 η o ^ Vr ^ T

^ -N ^^ J O O

SN38

S-SCA

THE

H

mPEG ^ fl ^ y ^ O H NH 2

The L ^ N ^

THE

SN38

SCA-S

Peg

THE

THE

THE

mPEG

S-SCA

SN38

SN38

THE

THE

Á /) - NH-C-Cit-Val

NH2 H O 'N

NH

SCA-S '

PEG 'O O

HN '

THE

Val-Cit-C-NH-

0H NH2

THE

SN38

THE

^ S-SCA

O O

Il

, Phe-Lys-C-SN38

mPEG ^ YNY ^ 0 H

O ^ N ^^ - Folic acid

THE

SN38-C-Lys-Phe

Folie acid ^^ - NO

THE

Il

Phe-Lys-C-SN38

THE

Il

, Phe-Lys-C-SN38

mPEG ^ f ^ Y ^ O NH 2

The L ^ N. AT-

S-SCA

THE

THE

Il

THE

SN38-C-Lys-Phe

Phe-Lys-C-SN38

SCA-S

"S-SCA

THE

mPEG

THE

SN38

/ Phe-Lys-C - <(>

SN38

SCA-S

S-SCA O

, Phe — Lys-C — NH—)

π

H NH2

SN38

THE

"S-SCA

THE

\) —NH-C-Lys-Phe

SN38 — W NH

The KlLJ J ^, N

THE

SCA-S

NH2 H o

^ PEG O Ò

Hn

THE

, Phe — Lys-C — NH — i)

H NH 2 N-

SN38

THE

THE

"S-SCA

C-SN38

S-RGD

SN38-C-Cit-Val

RGD-S

Peg

C-SN38

S-RGD

THE

Il

-VaI — Cit — C — SN38

mPEG ^ YNy ^ O

THE

S-RGD

THE

Il

SN38 — C-Cit-Val

RGD

Hn

THE

Il

-Go — Cit — C — SN38

o o (

RGD mPEG

SN38

THE'

^ y-S-RGD

SN38

RGD

_ 0 λ V-C-Cit-Val

NH

0 ^ X ^ Y ^ "peg ^ T

H O

SN38

S-RGD

mPEG

HN '

THE

Il

-VaI — Cit — C-SN38

THE

O ο.

.N ^ V-S-RGD O

SN38

Peg

Val — Cit — C-SN38

S-RGD mPEG

SN38

\ / - - C-Cit-Val

SN38 "NH

H H

.0 O ^ r ^ Y ^ PEG ^ f ^ V ^ O Ο.

THE

rgd-s-λ ^ ν.

THE

SN38

THE

-N ^^ - S-RGD O

Peg

HN '

THE

Il

Λ / al — Cit — C-SN38

THE

THE

N -___, - NH-RGD

Ο ο

ο

SN38-C-Cit-Val .Val — Cit — C-SN38

NH Η Η HN

ι_ι ο \ νΥ ^ ρεο ^ υν> Λο η

NH-RGD

T T

ο ο O

Il

MaS — Cit-C-SN38

NH2

O, N ^ A. - S-

SN38

RGD-S '

S-RGD

C-SN38

nY-PEG-YV O

'S-RGD

THE

mPEG

THE

SN38

THE

Il

Λ /) - C-Cit-Val

RGD-S '

NH H | _ |

η OsS-nY ^ PEOTn

O O

THE

Il

-VaI — Cit-c — NH — 4}

mPEG

-SN38

THE

S-RGD O

Il

Phe-Lys-C-SN38

mPEG

Il °

SN38-C-Lys-Phe / Phe-Lys-C-SN38

NH η η HN

RGD-NI-K ___ ν

π

ο

O ^^^ O O

3-SN38

mPEG

S-RGD

SN38-C-Lys-Phe

RGD-S '

Peg

Phe-Lys-C-SN38

THE

mPEG

THE

THE

THE

Il

\ /> - C-Lys — Phe

SN38 W NH

S-RGD

THE

RGD-S '

2 H 0 ^ N ^ PEG

O O

Phe-Lys-C-NH- (J)

Hi

NH 2

SN38

"S-RGD

THE

SN38

mPEG

LNA-O

O-LNA

H H

H-YnY-PEG-Ynt-O Foic Acid. Acid

Π Π

O O

O-LNA

mPEG

LNA-O

H

-N ^ O

NH

H

Folie Acid ^^^ ν

¡

THE

on what

S-SCA is a single chain antibody;

"r ^ -V ^ A η

O O

O-LNA

N ^ Folie Acid

¡

The RGD is

HS

LNA is blocked nucleic acid; "Folie acid" is the residue of

OH

SN38 is 7-ethyl-10-hydroxycamptothecin;

mPEG has Formula: CH 3 -O (CH 2 CH 2 O) n-;

PEG has Formula -O (CH 2 CH 2 O) n-; and

(n) is a positive integer from about 10 to about 2,300.

I. METHODS FOR PREPARING THE CONJUGATE

Generally, conjugates may be made by sequential addition of polymer, cytotoxic agent and targeting moiety to the multifunctional binder. The exact order of addition is not limited to this order and, as will be apparent to one skilled in the art, there are aspects where PEG will be added first to the multifunctional ligand, followed by the addition of the targeting agent such as a monoclonal antibody. Details concerning some preferred aspects of this embodiment are set forth in the Examples below.

In one aspect of the invention, the polymeric compound having a multifunctional binder may be prepared by conjugating a polymeric compound having an OH group or terminal leaving group to a nucleophile containing a cytotoxic agent linked via an optional binder. The person skilled in the art may use less nucleophile as compared to the number of leaving groups in the polymer to form an intermediate containing both cytotoxic agent and leaving groups. This intermediate may further react with a targeting moiety to form the multi-substituted polymer conjugate with cytotoxic agent and targeting agent. Alternatively, the polymer may be activated with different groups to give different chemical reactivity to various nucleophilic moieties. For example, different protecting groups such as tert-Bu ester and carboxylic acid terminal methyl ester may be selectively deprotected and stepwise to generate varying degrees of active groups to be conjugated to biologically active agents such as cytotoxic agents and directing agents. As shown in FIG. 1, the maleimidyl group and succinimidyl ester may react selectively with SH or NH 2 containing moieties, respectively.

All chemical reactions described herein are reactions with necessary conditions and steps known to a person skilled in the art. The synthetic reactions described herein do not require unnecessary experimentation.

Methods for preparing polymeric conjugates containing multifunctional linkers include:

React a compound of Formula (IIIa):

M1- (L1) to-L2-M2

Laughs

A21 (IIIa)

with a compound containing a biologically active portion of Formula (IIIb): M3- (L3) b — D22

under conditions sufficient to form a compound of Formula (IIIc):

M1- (L1) a-L2- (L3) b-D22

I

í1

a22 (IIIC)

on what

R1 is a substantially non-antigen water soluble polymer;

A2I is a protection group or A22 is a protection group or

M1- (L1) a-L2- (L3) b-D22.

Mi is a functional group;

D22 is a biologically active portion;

M2 is OH or a leaving group;

M3 is OH, NH2, or SH;

Li is a permanent binder or a labile binder;

L2 is a multifunctional binder;

L3 is a permanent binder or a labile binder; and

(a) and (b) are independently zero or a positive integer.

The methods still include

React the compound of Formula (IIIc):

M1- (L1) a-L2- (L3) b-D22

f

A22 (IIIC)

with a nucleophile-containing moiety having Formula (IIId) D2-M4 (I] Id)

under conditions sufficient to form a compound of Formula (IIIe) D2- (L1) a-L2- (L3) b-D22

I

Laughs

A23 (IIIe)

on what:

A23 is a protection group or

D2- (L1) a-L2- (L3) b-D22.

Mi is a functional group; D2I is a directing moiety; and M4 is OH, NH2, or SH. Binding of a nucleophilic compound such as those of Formula (IIIb) to PEG or another polymer may be done using standard organic synthesis techniques well known to one skilled in the art. The activated polymeric moiety such as SC-PEG, PEG-amine, PEG acids, etc. It can be obtained from both commercial sources and synthesized by the person skilled in the art without unnecessary experimentation.

Binding of nucleophilic compound such as those of Formula (IIIb) to polymeric moieties is preferably conducted in the presence of a coupling agent. A non-limiting list of coupling agents includes 1,3-diisopropylcarbodiimide (DIPC), any dialkyl carbodiimide, 2-halo-1-pyridinium halides (Mukaiyama's rulers), 1- (3-dimethylaminopropyl) -3 -ethyl carbodiimide (EDC), propanophosphonic acid cyclic anhydride (PPACA) and phenyl dichlorophosphates, etc. which are available, for example, from commercial sources such as Sigma-Aldrich Chemical, or synthesized using known techniques.

Preferably, the reactions are conducted in an inert solvent such as dichloromethane, chloroform, DMF or mixtures thereof. The reactions may preferably be conducted in the presence of a base such as dimethylaminopyridine (DMAP), diisopropylethylamine, pyridine, triethylamine, etc. to neutralize any acids generated. Reactions may be conducted at temperatures from about 0 ° C to about 22 ° C (room temperature). Some particular embodiments prepared by the methods described herein include:

H. TREATMENT METHODS

In alternative aspects of the invention, methods are also disclosed for treating a mammal comprising administering an effective amount of a pharmaceutical composition containing a compound of the present invention of Formula (I) to a patient in need thereof.

In a preferred aspect of the invention, methods are also provided for treating a patient having a cancer or cancer comprising administering an effective amount of a pharmaceutical composition containing a compound of the present invention of Formula (I) to a patient in need thereof. In some aspects, the cancer being treated may be one or more of the following: solid tumors, lymphomas, small cell lung cancer, acute lymphocyte leukemia (ALL), pancreatic cancer, glioblastoma, ovarian cancer, gastric cancers, etc. The compositions are useful for treating neoplastic diseases, reducing tumor burden, preventing metastases or neoplasms, and preventing tumor recurrences / neoplastic growth.

Another aspect of the present invention provides methods of treating various medical conditions in mammals. Briefly, any biologically active moiety that can be attached to the PEG polymer can be administered to a mammal in need of such treatment. Any oligonucleotide, etc. which has therapeutic effect in its unconjugated state may be used in its conjugated form, as described herein.

The amount of composition to be administered will depend on the precursor molecule included therein. Generally, the amount of prodrug used in treatment methods is an amount that effectively achieves the therapeutic outcome in mammals. Of course, the doses of various drug compounds will vary depending on the precursor compound, in vivo ratio, polymer molecular weight, etc. One skilled in the art will determine the optimal dose of polymeric transport conjugates based on clinical experience and treatment indication. True dosages will be apparent to one of ordinary skill in the art without unnecessary experimentation.

The compounds of the present invention may be included in one or more pharmaceutical compositions for administration to mammals. The pharmaceutical compositions may be based on a solution, suspension, tablet, capsule or the like, prepared according to methods well known in the art. It is also contemplated that such compositions may be for oral and / or parenteral routes, depending on the needs of the person skilled in the art. A solution and / or suspension of the composition may be used, for example, as a vehicle for injection or infiltration of the composition by prior art techniques, e.g., intravenous, intramuscular, intraperitoneal, subcutaneous injection and the like. Such administration may also be by infusion into a hole or cavity, as well as by inhalation or intranasal routes. In preferred aspects of the invention, however, polymeric conjugates are administered parenterally to mammals in need thereof.

In yet another aspect of the present invention, methods are provided for administering polynucleotides (oligonucleotides), preferably antisense oligonucleotides to mammalian cells. The methods include transporting an effective amount of the conjugate prepared as described herein to the condition being treated and will depend on the effectiveness of polynucleotides for such conditions. For example, if the unconjugated oligonucleotide (e.g. BCL2 antisense oligonucleotides, Survivin antisense oligonucleotides) is effective against certain cancers or neoplastic cells, the method will include transporting a polymer conjugate containing the oligonucleotides to cells having susceptibility to native oligonucleotides. Transport can be done in vivo, as part of an appropriate composition, or directly to cells in an ex vivo environment. In a particular treatment, polymeric conjugates including oligonucleotides (SEQ ID NO. 1, SEQ ID NOs: 2 and 3, and SEQ ID NO: 4) may be used.

EXAMPLES

The following examples serve for a better understanding of the invention but are by no means restricting the scope of the invention. The bold numbers cited in the Examples correspond to those shown in Fig. 1. Abbreviations were used in all examples, such as DCC (dicyclohexylcarbodiimide), DCM (dichloromethane), DCU (dicyclohexylurea), DIEA (diisopropylethylamine), DIPC (diisopropylcarbodiimide), DMAP (4-dimethylaminopyridine), DMF (Δ, Ν'-dimethylformamide), DSC (disuccinimidyl carbonate), EDC (1- (3-dimethylaminopropyl) -3-ethyl carbodiimide), EEDQ (2-ethoxy-1-ethoxycarbonyl-1 , 2-dihydroquinoline), IPA (isopropanol), NHS (N-hydroxysuccinimide), NMM (N-methylmorpholine), PEG (polyethylene glycol), SCA-SH (single chain antibody), SN38 (7-ethyl-10-hydroxycamptothecin ), TBDPS (tert-butyl dipropylsilyl), and TEA (triethylamine).

General Procedures. All reactions were conducted in a dry nitrogen or argon atmosphere. Commercial reagents were used without further purification. All PEG compounds were dried in vacuo or by azeotropic distillation with toluene prior to use. 1 H NMR spectra were obtained at 300 MHz and 13 C NMR spectra at 75.46 MHz using a Varian Mercury 300 spectrometer and deuterated chloroform were used as a solvent unless otherwise specified. Chemical displacements (δ) were reported in parts per million (ppm) from the tetramethyl silane (TMS) displacement.

HPLC method. The reaction mixture and purity of intermediates and end products were monitored on a Beckman Coulter System Gold® HPLC instrument. It employs a ZORBAX® 300SB C8 (150 χ 4.6 mm) or Phenomenex Jupiter® 300A Cl8 (150 χ 4.6 mm) reverse phase column with UV 168 Diode Array detector using 10-90% acetonitrile gradient 0 0.05% trifluoroacetic acid (TFA) at a flow rate of 1 mL / min.

Example 1. N- (Methoxycarbonyl) maleimide (compound 1).

Methylchloroformate (4.4 mL, 56.7 mmol, 1 eq) was added to a solution of maleimide (5.5 g, 56.7 mmol, 1 eq) and NMM (6.2 mL, 56.7 mmol, 1 µM). eq) in 200 mL of EtOAc at 0 ° C. The suspension was stirred at 0 ° C for 30 minutes, filtered and washed with EtOAc. The filtrate and washings were combined and washed with cold water over anhydrous Na 2 SO 4. After filtration and evaporation under vacuum, a pink solid was obtained. Purification by silica gel column chromatography (Hexane-EtOAc, 1: 1, v / v) gave the product (4.8 g, 55%).

Example 2. NE-Maleoyl-α- (Boc) -L-lysine (compound 2).

N- (Methoxycarbonyl) maleimide (315 mg, 2.03 mmol, 1 eq) was added to a solution of Boc-L-Lysine (500 mg, 2.03 mmol, Ieq) in 10 mL of saturated aqueous NaHCO 3 at 0 ° C. ° C. The mixture was stirred vigorously at 0 ° C for 40 minutes at room temperature for a further hour. After cooling to 0 ° C, the solution was acidified to pH 3.0 with concentrated H2SO4 before extraction with ethyl acetate. The organic phases were combined and dried over anhydrous MgSO4. After removal of the solvent in vacuo, the residue was purified by silica gel column chromatography using a mixture of CHCl3-MeOH (5: 1, v / v) to give the product (458 mg, 69%): 1 H NMR δ 1 , 39-1.87 (6 δ, m, (CH 2) 3), 1.44 (9H, s, Boc), 3.52 (2H, t, J = 7.2 Hz, NCH 2), 4.25 -4.30 (1H, m, CH), 5.15 (1H, d, NH), 6.70 (2H, s, maleimide).

Example 3. NE-Maleoyl-α-L-lysine (compound 3). A solution of NE-maleoyl-α- (Boc) -L-lysine (172 mg, 0.53 mmol) in 5 mL of anhydrous DCM was treated with 10 mL of 2N HCl solution in ethyl ether for one hour at room temperature before adding 10 ml of anhydrous ethyl ether. The resulting solid was filtered to give 82 mg (59%) yield of N '-Maleoyl-aL-lysine HCl salt: 1 H NMR (CD 3 OD) δ 1.36-1.54 (2 Η, m, CH 2) 1.65 (2 Η, q, J = 7.2 Hz, CH 2), 1.83-2.02 (2 Η, m, CH 2), 1.44 (9H, s, Boc), 3.53 (2H, t, J = 6.9 Hz, NCH 2), 3.95 (1H, t, 6.2 Hz, CH), 6.82 (2H, s, maleimide); 13 C NMR (CD 3 OD) δ 23.17, 29.03, 31.01, 37.96, 135.26, 172.34, 175.20.

Example 4. 5KmPEG-amide-Lys (NE-amino) -COOH (compound 4).

To a solution of 51SnPEGCOONHS (805 mg, 0.16 mmol, 1 eq) in 8 mL of anhydrous DCM was added NE-maleoyl-α-L-lysine (3.82 mg, 0.31 mmol, 2 eq) in 2 mL of DMF followed by DIEA (109 µg, 0.62 mmol, 4 eq). The reaction mixture was stirred at room temperature overnight, filtered and evaporated under vacuum. The residue was first precipitated with DCM / ether and then recrystallized with CH 3 CN / IPA. 13 C NMR (CDCl 3): δ 22.53, 28.02, 31.69, 37.32, 51.18, 58.86, 70.11-71.73 (PEG), 133.84, 169.51, 170 , 38, 172.41.

Example 5. 5KmPEG-amide-Lys (NE-maleoyl) -NHS (compound 5).

5KmPEG-Lys (NE-maleoyl) -COOH (4) and NHS in DCM at 0 ° C were treated with DCC. The mixture was stirred at room temperature overnight. The solvent was evaporated under vacuum and the residue precipitated with DCM / ether and then recrystallized with CH 3 CN / IPA.

Example 6. SKmPEG-carbamate-Lys (NE-amino) -COOH (compound 6).

To a solution of 5KmPEGCOONHS (5 g, 0.98 mmol, 1 eq) in 45 mL of anhydrous DCM was added NE-maleoyl-α-L-lysine (3.514 mg, 1.95 mmol, 2 eq) in 5 mL. mL of DMF followed by DIEA (0.7 mL, 3.92 mmol, 4 eq). The reaction mixture was stirred at room temperature overnight, filtered and evaporated under vacuum. The residue was first precipitated with DCM / ethyl ether and then recrystallized with CH 3 CN / IPA to give compound 6: 13 C NMR δ 22.30, 28.01, 31.89, 37.29, 53.26, 58.89, 64 , 06, 69.31-71.77 (PEG), 133.87, 155.60, 170.42, 172.82. Example 7. 5KmPEG-carbamate-Lys (Ne-maleoyl) -NHS (compound 7).

To a solution of 5KmPEG-carbamate-Lys (Ne-maleoyl) -COOH (6.1 g, 0.19 mmol, 1 eq) and NHS (88 mg, 0.76 mmol, 4 eq) in 10 mL of DCM a At 0 ° C DIPC (118 µg, 0.76 mmol, 4 eq) was added. The mixture was stirred at room temperature overnight. The solvents were evaporated under vacuum and the residue was precipitated with DCM / ether and then recrystallized with CH 3 CN / IPA to give compound 7: 13 C NMR δ 22.31, 25.90, 28.25, 32.39, 37.44, 52.42, 59.30, 64.92, 69.64-72.18 (PEG), 134.25, 155.59, 167.75, 168.55, 170.91.

Example 8. Compound 8.

To a solution of 5'extGS (5 mg, 0.85 μιηοΐ) in PBS buffer (2.5 mL, pH 7.8) was added compound 7 (92 mg, 17 μιηοΐ, 20 eq) and the mixture was added. stirred at room temperature for 2 hours. The reaction mixture was diluted to 10 mL with water, filtered and poured into a strong ion exchange Poros HQ column (10 mm χ 1.5 mm, bed volume ~ 16 mL) which was pre-equilibrated with Tris-HCI 20 buffer. mM, pH 7.0 (buffer A). The column was washed with 3-4 column volumes of buffer A to remove excess PEG ligand. Then, the product was eluted with a 0 to 100% IM NaCl gradient in 20 mM Tris-HCl buffer, pH 7.0, buffer B within 10 minutes, followed by 100% buffer B for 10 minutes at a flow rate of 10%. mL / min. The eluted product was desalted using a HiPrep desalination column (50 mL) and lyophilized to give 8 (1 mg oligo equivalent, 40% yield).

Example 9. Compound 9.

To a solution of compound 8 (1 mg oligo equivalent) in PBS buffer (1 mL, pH 7.0) was added the cRGDfC peptide (1 mg, 1.7 μιηοΐ) and the mixture was stirred at room temperature for 2 hours. . The reaction mixture was diluted to 20 mL with water and poured into a strong ion exchange Poros HQ column (10 mm χ 1.5 mm, bed volume ~ 16 mL) which was pre-equilibrated with 20 mM Tris-HCl buffer. pH 7.0 (buffer A). The column was washed with 3-4 column volumes of buffer A to remove excess PEG ligand. Then, the product was eluted with a gradient from 0 to 100% IM NaCl in 20 mM in Tris-HCl buffer, pH 7.0, buffer B for 10 minutes, followed by 100% buffer B for 10 minutes with flow rate. 10 mL / min. The eluted product was desalted using the HiPrep desalination column (50 mL) and lyophilized to give compound 9 (0.78 mg oligo equivalent, 78% yield).

Example 10. Compound 10.

To a solution of p-amino benzyl alcohol (1 g, 8.12 mmol, 1 eq) in 80 mL of anhydrous THF was added DIEA (1.4 mL, 8.12 mmol, 1 eq) and Boc 2 O (1 9 mL, 8.12 mmol, 1 eq). The mixture was heated at reflux overnight and then cooled and evaporated under vacuum. The residue was dissolved in EtOAc. The organic phase was washed with 0.1N HCl solution, dried over MgSO4, filtered and evaporated under vacuum. The crude product was purified by silica gel column chromatography (Hex-EtOAc, 1: 1, v / v) to give 1.85 g of product (quantitative yield): 1 H NMR δ 1.49 (9H, s), 2 , 17 (1H, s), 4.53 (2H, s), 6.83 (1H, s), 7.19 (2H, d, J = 8.5 Hz), 7.28 (2H, d, J = 8.2 Hz); 13 C NMR δ 28.28, 64.54, 80.37, 118.49, 127.59, 135.31, 137.46, 152.72.

Example 11. Compound 11.

To a -20 ° C solution of compound 10 (220 mg, 0.98 mmol, 1 eq) and DIEA (188 μί, 1.08 mmol, 1.1 eq) in 8 mL of anhydrous DCM was added. solution of MsCl (84 μί, 1.08 mmol, 1.1 eq) in 2 mL of DCM andro dropwise. The mixture was allowed to reach room temperature and then stirred for 1 hour. The reaction mixture was washed with saturated NaHCO 3 solution, saturated NH 4 Cl solution and brine, dried over MgSO 4, filtered and evaporated under vacuum. The crude compound was used in the next step without further purification.

Example 12. Compound 12.

Compound 11 (0.98 mmol, 2 eq) was dissolved in 15 mL of anhydrous DMF and SN38 (192 mg, 0.49 mmol, 1 eq) and K 2 CO 3 (68 mg, 0.49 mmol, 1 eq) were added. ). The reaction mixture was heated to a temperature of 60 ° using a preheated oil bath. After 2 hours, HPLC showed total disappearance of SN38 and substantial product formation. The reaction mixture was evaporated under vacuum and the residue redissolved in EtOAc and washed with water, saturated NH 4 Cl and brine, dried over MgSO 4, filtered and evaporated under vacuum. The crude product was purified by silica gel column chromatography (CH2Cl2-EtOAc, 1: 1, v / v) to give 140 mg of product (48% yield): 1 H NMR (DMSO-Rf6) δ 0.88 (3H, t, J = 7.0 Hz), 1.27 (3H, t, J = 7.3 Hz), 1.47 (9H, s), 1.85-1.88 (2H, m), 3, 15-3.17 (2H, m), 5.25 (2H, s), 5.26 (2H, s), 5.42 (2H, s), 6.49 (1H, s), 7.26 (1H, s), 7.41-7.56 (6H, m), 8.05 (1H, d, J = 9.1 Hz), 9.39 (1H, s); 13 C-NMR (DMSO-ε) δ 7.65, 13.31, 22.09, 27.96, 30.11, 49.31, 65.05, 69.47, 72.17, 78.84, 95.74 , 103.39, 117.71, 117.92, 122.35, 127.44, 127.99, 128.43, 129.59, 131.07, 139.07, 143.46, 144.06, 145 , 92, 149.21, 149.69, 152.37, 156.46, 156.82, 172.11.

Example 13. Compound 13.

A 15% (v / v) TFA solution in DCM was added to compound 12 (535 mg) and the mixture was stirred at room temperature for 1h or until HPLC showed complete disappearance of the starting material. Ethyl ether (200 mL) was added and the resulting solid was filtered and washed with more ether (307 mg, 58% yield).

15

Example 14. Compound 14.

Compound 13 (48 mg, 0.1 mmol, 1 eq) and 15 (36 mg, 0.1 mmol, 1 eq) were treated with EEDQ (48 mg, 0.2 mmol, 2 eq). The mixture was stirred in the dark at room temperature overnight. The solvents were removed under vacuum and the solid residue was triturated with 10 mL of ethyl ether. The solid was filtered and purified by silica gel column chromatography (CHCl 3: MeOH, 15: 1 to 9: 1) to give the desired product (8 mg, 9% yield): 13 C NMR (DMSO-40 δ 7.8, 13.49, 18.11, 18.17, 18.58, 19.13, 19.25, 22.25, 26.51, 26.72, 28.19, 29.59, 30.26, 30, 41, 30.50, 49.49, 51.67, 51.80, 52.90, 55.98, 59.26, 59.67, 63.03, 65.21, 69.54, 72.33, 77.91, 78.01, 95.92, 103.63, 118.10, 118.95, 122.52, 127.63, 128.21, 128.58, 131.17, 131.27, 138, 53, 143.69, 144.25, 146.10, 149.43, 149.86, 155.15, 155.26, 156.63, 156.97, 158.48, 158.66, 170.39, 171.15, 171.33, 172.15, 172.29.

Example 15. Boc-VaI-Cit-OH (Compound 15).

Boc-Val-NHS (10 g, 31.81 mmol, 1 eq) in 80 mL DME was added to a solution of L-citrulline (5.85 g, 33.40 mmol, 1.05 eq) in 20 mL of THF and NaHCO 3 (2.8g, 33.40 mmol, 1.05 eq) in 80 mL of water. The mixture was stirred at room temperature overnight. Aqueous citric acid (200 mL of 15% solution in water) was added and the mixture was extracted with 10% IPA / EtOAc (2 x 200 mL). The suspension was washed with water (2 x 200 mL) and the solvent evaporated under vacuum. The resulting solid was triturated with 200 mL of ethyl ether and filtered to give the product (6.08 g, 51% yield): 1 H NMR (CD 3 OD) δ 0.92 (3H, d, J = 6.7 Hz), 0 , 97 (3H, d, J = 6.7 Hz), 1.44 (9H, s), 1.51-2.06 (5H, m), 3.12 (2H, t, J = 6.7 Hz), 3.90 (1H, d, J = 7.0 Hz), 4.37-4.41 (1H, m). 13 C NMR (CD 3 OD): δ 18.58, 19.83, 27.58, 28.75, 30.03, 32.09, 40.48, 53.33, 61.38, 80.49, 157.75, 162.05, 174.30, 174.69.

Example 16. Compound 16 (Boc-Val-Cit-PAB).

Boc-Val-Cit (1 g, 2.67 mmol, leq) and p-aminobenzyl alcohol (362 mg, 2.94 mmol, 1.1 eq) in 2: 1 DCM / MeOH (26 mL / 13 mL) were treated with EEDQ (1.32 g, 5.34 mmol, 2 eq). The mixture was stirred in the dark at room temperature overnight. The solvents were removed under vacuum and the resulting solid triturated with 10 mL of ethyl ether. The solid was collected by filtration and washed with ethyl ether to give the product (900 mg, 70%): 1 H NMR (CD 3 OD) δ 0.93 (3H, d, J = 7.0 Hz), 0.97 (3H, d, J = 7.0 Hz), 1.44 (9H, s), 1.49-2.06 (5H, m), 3.07-3.29 (2H, m), 3.91 (1H , d, J = 6.7 Hz), 4.50-4.55 (3H, m), 7.29 (2H, d, J = 8.5 Hz), 7.54 (2H, d, J = 8.5 Hz), 8.22 (1H, d, J = 7.3 Hz); 13 C NMR (CD 3 OD) δ 18.64, 19.85, 27.84, 28.75, 30.61, 31.92, 40.28, 54.90, 54.99, 61.73, 64.78, 80 , 62, 121.09, 128.44, 138.47, 138.55, 158.01, 162.11, 172.00, 174.42, 174.52.

20

Example 17. Compound 14 (Boc-Val-Cit-PABE-SN38).

To a suspension of Boc-Val-Cit-PAB (214 mg, 0.45 mmol, 1 eq) in 3 mL of CH 3 CN was added DIEA (0.23 mL, 1.35 mmol, 3 eq) followed by addition. dropwise from a solution of MsCl (0.1 mL, 1.35 mmol, 3 eq) in 1 mL CH 3 CN. After 1 hour, TLC showed no starting material left (CHCl 3 -MeOH, 5: 1, v / v). The reaction mixture was evaporated under vacuum and the resulting residue was dissolved in EtOAc. The organic phase was washed with saturated NH 4 Cl, saturated NaHCO 3 and brine; dried over MgSO4, filtered and evaporated under vacuum. The crude residue was dissolved in 5 mL of DMF and SN38 (88 mg, 0.22 mmol, 0.5 eq) and K 2 CO 3 (31 mg, 0.22 eq, 0.5 eq) were added and the mixture was heated at 60 ° C for 3 hours. Product formation was monitored and conformed to the product prepared in Example 14 by HPLC. Example 18. Compound 17.

Compound 14 in DCM was treated with 2N HCl solution in ether. After completion of the reaction, additional ether was added and the resulting solid was filtered off and washed with ether to give Val-Cit-PABE-SN38. To a solution of compound 5 in anhydrous DCM was added HCl-Val-Cit-PABE-SN38 in anhydrous DMF followed by D1EA. The mixture was stirred at room temperature overnight. The solvent was removed under vacuum and the resulting solid was precipitated with DCM / ether and recrystallized with CH 3 CN / IPA to give the product.

Example 19. Compound 18.

To a solution of compound 17 (Ieq) in a DCM / DMF mixture was added RGDC (2 eq). The reaction mixture was stirred at room temperature overnight and then the solvent was evaporated under vacuum. The residue was precipitated with DCM / ethyl ether and recrystallized from DMF / IPA to give the product.

Example 20. Compound 19.

To a solution of 4-arm 20kPEGSC (7 g, 0.35 mmol, 1 eq) in 60 mL of anhydrous DCM was added Lys (Boc) -OH (690 mg, 2.8 mmol, 2 eq) in 15 mL. mL of DMF followed by DIEA (1 mL, 5.6 mmol, 4 eq). The reaction mixture was stirred at room temperature overnight, filtered and evaporated under vacuum. The residue was first filtered off with DCM / ethyl ether and recrystallized from CH 3 CN / IPA to give compound 19: 13 C NMR δ 21.88, 27.97, 29.04, 31.57, 39.60, 44.98, 53, 00, 63.44, 68.86-70.37 (PEG), 78.04, 155.26, 172.89.

Example 21. Compound 20.

To a solution of 4-arm PEG-Lys (Boc) -OH in 25 mL of anhydrous DCM was added a 4N HCl solution in dioxane (25 mL). The reaction was stirred at room temperature for 4 hours and then evaporated under vacuum. The residue was precipitated by the addition of ethyl ether. The solid was filtered and washed with ether to give 4-arm PEG-Lys-OH (1.77 g): 13 C NMR δ 21.97, 26.54, 31.22, 39.63, 45.11, 53, 16, 63.66, 69.88-70.51 (PEG), 78.04, 155.59, 172.78. Example 22. Compound 21.

A solution of BocCys (NPys) -OH (300 mg, 0.80 mmol, 1 eq) and NHS (97 mg, 0.84 mmol, 1.05 eq) in 10 mL of anhydrous DCM at 0 ° C was treated with DCC (173 mg, 0.84 mmol, 1.05 eq). The mixture was allowed to warm to room temperature and stirred overnight. Solid DCU side product was filtered. To the filtrate, compound 20 (1.7 g, 0.082 mmol, 1 eq) was added followed by DIEA (114 µg, 0.66 mmol, 8 eq). The reaction mixture was stirred at room temperature overnight and evaporated under vacuum. The residue was first precipitated with DCM / ether and then recrystallized from CH 3 CN / IPA to give compound 21 (1.5 g): 13 C NMR δ 22.32, 25.52, 28.27, 28.83, 31.90 , 39.03, 42.45, 45.38, 53.41, 53.58, 63.91, 64.37, 69.30-72.38 (PEG), 80.11, 120.98, 133, 94, 142.54, 153.34, 155.51, 155.71, 157.28, 169.98, 173.32.

Example 23. Compound 22.

To a solution of compound 21 (1.5 g, 0.07 mmol, 1 eq) and NHS (125 mg, 1.09 mmol, 16 eq) in 15 mL of DCM at 0 ° C was added DIPC (168 μΐ, 1.09 mmol, 16 eq). The mixture was stirred at room temperature overnight. The solvents were evaporated under vacuum and the residue precipitated with DCM / ether and then recrystallized with CH 3 CN / IPA to give the product (1.3 g): 13 C NMR δ 22.05, 25.76, 25.93, 28, 68, 28.92, 32.01, 38.89, 42.49, 45.79, 52.40, 53.97, 64.78, 69.57-71.77 (PEG), 80.47, 121 , 32, 134.29, 142.93, 153.75, 155.71, 157.60, 168.18, 169.14, 169.71, 170.56.

Example 24. Compound 23.

(BocPheLys (Fmoc) -OH). To a solution of BocPheOSu (1.5g, 4.14mmol, 1eq) in DCM (10.3 mL) and DMF (10.3 mL) was added H-Lys (Fmoc) -OH (1.84g, 4.55 mmol, 1.1 eq) followed by DIEA (2.9 mL, 16.56 mmol, 4 eq). The resulting reaction mixture was stirred overnight and diluted with ethyl acetate. The solution was washed with water (30 mL χ 2) and brine (30 mL χ 2). The organic phases were combined, dried over MgSO 4 and concentrated in vacuo to give a yellow residue. The crude residue was then chromatographed on silica gel using CHCl 3 -MeOH (3: 1, v / v) to give the product. MS [M + 1] + 616.

Example 25. Compound 24. Compound 13 (111 mg, 0.22 mmol, Ieq) and 16 (208 mg, 0.34 mmol, 1.5 eq) were treated with EEDQ (167 mg, 0.67 mmol, 3 eq). The mixture was stirred in the dark at room temperature overnight. The solvents were removed under vacuum and the resulting residue triturated with 10 mL of ethyl ether. The solid was filtered and purified by silica gel column chromatography (CH 2 Cl 2 -EtOAc, 1: 1 to 1: 2, v / v) to give the desired product (20 mg, 8% yield). MS [M + 1] + 1095.

Example 26. Compound 25.

Compound 24 (8 eq) was treated with 4N HCl solution in dioxane. The reaction was stirred at room temperature for 4 hours. Ethyl ether was added and the solid was filtered. This solid was dissolved in DMF and added to a solution of compound 22 (Ieq) in DCM. DIEA (16 eq) was added to this mixture and the reaction was stirred at room temperature overnight. The solvent was removed under vacuum and the resulting solid precipitated with DCM / ethyl ether and recrystallized from DMF / IPA to give the product.

Example 27. Compound 26.

To a solution of compound 25 in DCM, piperidine was added. The reaction mixture was stirred for 4 hours. The solvent was removed under vacuum and the resulting solid precipitated with DCM / ethyl ether and recrystallized from DMF / IPA to give the product.

Example 28. Compound 27.

To a solution of compound 26 (Ieq) in a DCM / DMF mixture was added RGDC (2 eq). The reaction mixture was stirred at room temperature overnight and then the solvent was evaporated under vacuum. The residue was precipitated with DCM / ether and recrystallized from DMF / IPA to give the product.

Example 29. FmocVaI-Cit-OH (Compound 28).

Fmoc-Val-NHS (2.5 g, 5.73 mmol, 1 eq) in 15 mL DME was added to a solution of L-citrulline (1.05 g, 6.01 mmol, 1.05 eq) in 8 mL THF and NaHCO 3 (505 mg, 6.01 mmol, 1.05 eq) in 15 mL water. The reaction mixture was stirred at room temperature overnight. Aqueous citric acid (75 mL of a 15% solution in water) was added and the mixture was extracted with 10% IPA / EtOAc (2 x 100 mL). The organic phases were washed with water (3 x 100 mL) and the solvent was evaporated under vacuum. The resulting solid was triturated with 100 mL of ethyl ether and filtered to give the product (1.98 g, 70% yield): 1 H NMR (DMSO-J 6) δ 0.86 (3H, d, J = 6.7 Hz) 0.90 (3H, d, J = 7.0 Hz), 1.40-1.48 (2H, m), 1.51-1.75 (2H, m), 1.98 (1H, sext) , J = 6.8 Hz), 2.95 (2H, q, J = 6.2 Hz), 3.93 (1H, dd, J = 7.3, 8.8 Hz), 4.14-4 , 29 (4H, m), 5.40 (2H, broad signal), 5.96 (1H, t, J = 5.6Hz), 7.32 (2H, t, J = 7.5Hz) 7.39-7.44 (3H, m), 7.75 (2H, t, J = 6.3 Hz), 7.89 (2H, d, J = 7.3 Hz), 8.19 ( 1H, d, J = 7.3 Hz); 13 C NMR (DMSO-J 6) δ 18.31, 19.25, 26.75, 28.40, 30.59, 46.68, 51.91, 59.81, 64.92, 65.65, 1119, 98, 125.30, 126.94, 127.52, 140.55, 143.61, 143.76, 155.88, 158.58, 171.12,173,25.

Example 30. FmocVal-Cit-PAB (Compound 29).

A solution of compound 28 (1 g, 2.01 mmol, leq) and p-aminobenzyl alcohol (273 mg, 2.21 mmol, 1.1 eq) in a DCM / MeOH mixture (20 mL / 10 mL) was treated. with EEDQ (996 mg, 4.03 mmol, 2 eq). The mixture was stirred in the dark at room temperature overnight. The solvents were removed under vacuum and the resulting solid was triturated with 10 mL of ethyl ether. The solid was collected by filtration and washed with ethyl ether to give the product (925 mg, 76% yield): 1 H NMR (DMSO-J 6) δ 0.87 (6H, d, t = 7.5 Hz), 1.36 -1.47 (2H, m), 1.51-1.75 (2H, m), 1.99 (1H, sext, J = 6.7 Hz), 2.90-3.04 (3H, m ), 3.93 (1H, dd, J = 7.3, 8.8 Hz), 4.23-4.38 (4 H, m), 4.43 (2H, d, J = 5.3 Hz ), 5.13 (1H, t, J = 5.6 Hz), 5.43 (2H, broad signal), 5.99 (1H, t, J = 5.6 Hz), 7.23 (2H, d, J = 8.5 Hz), 7.32 (2H, t, J = 7.3 Hz), 7.39-7.48 (3H, m), 7.54 (2H, d, J = 8 0.5 Hz), 7.74 (2H, t, J = 6.7 Hz), 7.89 (2H, d, J = 7.6 Hz), 8.12 (1H, d, J = 7.3 Hz); 13 C NMR (DMSO-J 6) δ 18.34, 19.28, 26.84, 29.56, 30.47, 46.67, 53.05, 60.05, 62.55, 65.65, 118.71 , 119.98, 125.23, 126.79, 126.94, 127.51, 137.27, 137.36, 140.55, 143.60, 143.73, 155.93, 158.69, 170 , 17, 171.06.

Example 31. Val-Cit-PAB (Compound 30).

To a solution of compound 29 (622 mg, 1.03 mmol) in 10 mL of anhydrous DMF was added piperidine (2 mL). The mixture was stirred at room temperature overnight and then the solvents evaporated under vacuum. The residue was triturated with DCM and the resulting solid was filtered and washed with DCM to give product (283 mg, 72% yield): 1 H NMR (DMSO-J6) δ 0.79 (3Η, d, J = 6.7 Hz) 0.89 (3Η, d, J = 6.7 Hz), 1.38-1.42 (2H, m), 1.52-1.68 (2H, m), 1.94 (1H, sext) , J = 6.3 Hz), 2.89-3.01 (2H, m), 3.05 (1H, d, J = 4.7 Hz), 4.46 (2H, s), 5.09 (1H, broad signal), 5.40 (2H, s), 5.98 (1H, t, J = 5.3 Hz), 7.23 (2H, d, J = 8.2 Hz), 7, 54 (2H, d, J = 8.5 Hz), 8.14 (1H, broad signal), 10.03 (1H, s); 13 C-NMR (DMSO-J6) δ

16.80, 19.33, 26.53, 29.95, 31.10, 52.31, 59.33, 62.35, 118.61, 126.59, 137.06, 137.14,

158.44, 170.06, 173.69.

Example 32. Compound 31.

To a solution of compound 22 (1.3 g, 0.06 mmol, 1 eq) in 15 mL anhydrous DCM and 3 mL anhydrous DMF was added compound 30 (175 mg, 0.46 mmol, 8 eq). ). The reaction mixture was stirred overnight at room temperature and the solvent was evaporated under vacuum. The residue was precipitated with DCM / ether and recrystallized with CH 3 CN / IPA to give the product (1.1 g): 13 C NMR (CDCl 3 / CD 3 OD) δ 18.48, 19.46, 22.85, 26.74, 28.54, 28.94, 29.50, 30.47,

31.81, 39.21, 42.04, 45.79, 53.51, 54.06, 55.32, 59.67, 64.32, 65.04, 69.54-70.99 (PEG), 80 , 70, 120.23, 121.44, 127.64, 134.31, 137.16, 142.94, 153.86, 155.86, 156.85, 160.44,

170.45, 171.02, 171.87, 173.19.

Example 33. Compound 32.

To a solution of compound 31 (1 eq) in a DCM / DMF mixture (4/1, v / v), DSC (16 eq) was added and the mixture was cooled to 0 ° C. Then pyridine (16 eq) was added and the mixture gradually warmed to room temperature and stirred overnight. The solvent was evaporated under vacuum and the residue was precipitated with DCM / ethyl ether and recrystallized with CH 3 CN / IPA to give the product.

Example 34. Compound 33.

To a solution of compound 32 was added doxorubicin. The reaction mixture was stirred at room temperature overnight. The solvent was evaporated under vacuum and the residue was precipitated with DCM / ethyl ether and recrystallized from DMF / IPA to give the product.

Example 35. Compound 34. To a solution of compound 33 (Ieq) in a DCM / DMF mixture was added RGDC (2 eq). The reaction mixture was stirred at room temperature overnight. The solvent was evaporated under vacuum and the residue was precipitated with DCM / ethyl ether and recrystallized from DMF / IPA to give the product.

Example 36. Compound 35.

To a solution of compound 19 (6.14 g, 0.307 mmol, 1 eq) and NHS (283 mg, 2.456 mmol, 8 eq) in 31 mL of anhydrous 0 ° C DCM was added DCC (507 mg, 2.456 mmol, 2 eq). The reaction mixture was stirred at room temperature overnight, filtered under a Celite® plate and evaporated under vacuum. The residue was precipitated with DCM / ethyl ether and recrystallized with CH 3 CN / IPA to give the product (5.69 g, 86%): 13 C NMR δ 21.59, 25.21, 28,11,29,03,31.42 , 39.36, 45.13,51.80, 64.07, 68.90-70.53 (PEG), 155.15, 155.58, 168.17, 168.54.

Example 37. Compound 36

To a solution of compound 35 (5.g, 0.232 mmol, 1 eq) in 23 mL of anhydrous DCM and anhydrous DMF was added HCl-NH 2 Cys (NPys) -OH (0.77 g, 2.78 mmol, 12 eq). ) followed by DIEA (0.65 mL, 3.71 mmol, 16 eq). The reaction mixture was stirred at room temperature overnight and evaporated under vacuum after filtration under a Celite® plate. The residue was precipitated with DCM / ethyl ether and recrystallized with CH 3 CN / IPA to give the product (4.88 g, 95% yield): 13 C NMR δ 22.17, 28.22, 29.27, 31.85, 39, 92, 45.22, 51.85, 54.49, 63.98, 69.06-70.62 (PEG), 78.56, 120.87, 132.88, 142.27, 153.44, 155 , 67, 156.47, 162.14, 170.74, 171.20.

Example 38. Compound 37.

To a solution of compound 36 (4.38 g, 0.197 mmol, 1 eq) in DCM (44 mL) was added 8.8 mL of TFA and the reaction mixture was allowed to stir overnight at room temperature. After 20 hours the reaction mixture was evaporated under vacuum and the residue was precipitated with DCM / ethyl ether and recrystallized with CH 3 CN / IPA to give the product (2.1 g, 49%): 13 C NMR δ 21.47, 26.41 , 31.64, 39.47, 45.25, 52.32, 54.11, 63.75, 69.12-70.65 (PEG), 120.85, 133.46, 142.25, 153, 67, 155.64, 156.39, 171.53, 171.79. Example 39. Compound 38.

To a solution of folic acid (124mg, 0.28mmol, leq) in 3.1 mL DMSO at room temperature was added NHS (71 mg, 0.616 mmol, 2.2 eq) followed by addition of DCC (64 mg 0.308 mmol, 1.1 eq) and 63 µl triethylamine. The reaction mixture was stirred overnight in the dark and filtered under a Celite® plate to give a yellow FA-NHS solution which was used for the next binder coupling step. To a solution of H2N-Lys (4-arm PEG) CysNPys (37.381 mg, 0.0175mmol, leq) in 4 mL of anhydrous DCM and anhydrous DMF was added a solution of FA-NHS DMSO (prepared as shown above) followed by DIEA (49 μι, 0.28 mmol, 16 eq). The reaction mixture was allowed to stir overnight in the dark at room temperature. She was then dialyzed for 4 days with distilled water. The yellow solution was then lyophilized to give 197 mg of the final product.

Example 40. Compound 39.

To a solution of C6-thio-LNA-survivin (OligoSH) in phosphate buffer pH 6.5 was added 38 and the solution was stirred for 1 hour at room temperature. The progress of the reaction was verified by anion exchange HPLC. The reaction mixture was filtered with 0.2 micron filter and poured into a Poros anion exchange column. The product was eluted with a gradient using 20 mM Tris buffer system. 2M HCl NaCl pH 7.0. The product was desalted and lyophilized. LIST OF SEQUENCES <110> ENZON PHARMACEUTICALS, INC

5th

<120> PRO DIRECT POLYMERIC DRUGS CONTAINING MULTIFUNCTIONAL LEADERS

<130> 213.12 63-PCT

10

<140> <141>

<150> 60 / 884,943 <151> 15-09-2006

<160> 7

<170> PatentIn Ver. 3.3

<210> 1 <211> 12 <212> PRT <213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Synthetic peptide <4 00> 1

Cys Tyr Gly Arg Lys Arg Lys Arg Arg Gln Arg Arg Arg 1 5 10

<210> 2 <211> 10 <212> PRT

<213> Artificial sequence 40 <220>

<223> Description of Artificial Sequence: Synthetic peptide <400> 2

Cys Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg 10

45

<210> 3 <211> 16 <212> DNA 50 <213> Artificial Sequence <220> <223> Sequence Description <220>

<221> miscellanous aspect <222> (1) (16) <223> Thioskeleton

<220>

<221> modified base <222> (1)

<223> Methylated cytosine <22 0>

<221> miscellaneous aspect <222> (1) <223> LNA

<2 2 0>

<221> miscellaneous aspect

<222> (2)

<223> LNA

<220>

<221> modified base

<222> (3)

<223> Methylated cytosine <220>

<221> miscellaneous aspect

<222> (3)

<223> LNA

<220>

<221> miscellaneous aspect <222> (4) <223> LNA

<220>

40 <221> modified base <222> (13)

<223> Methylated cytosine <220>

45 <221> miscellaneous aspect <222> (13) <223> LNA

<220>

50 <221> miscellaneous aspect <222> (14) <223> LNA

2/4

artificial: Synthetic oligonucleotide 15

<220>

<221> miscellaneous aspect

<222> (15)

<223> LNA

<400> 3

ctcaatccat ggcagc ΐ6

10

<210> 4 <211> 21 <212> DNA

<213> Artificial sequence <220>

<223> Description of Combined DNA / RNA Molecule: Synthetic Oligonucleotide

<220>

<223> Description of Artificial Sequence: Synthetic oligonucleotide <220>

<221> miscellaneous aspect <222> (1) (19) <223> RNA

<400> 4

gcaugcggcc ucuguuugat t ??? 21

<210> 5 <211> 21 <212> DNA

<213> Artificial sequence

<220>

<223> Description of combined DNA / RNA molecule: 40 Synthetic oligonucleotide

<220>

<223> Description of Artificial Sequence: Synthetic oligonucleotide

45 <220>

<221> miscellaneous aspect <222> (1) (19) <223> RNA

50 <400> 5

ucaaacagag gccgcaugct t 21 <210> 6 <211> 18 <212> DNA <213> Artificial Sequence

<22 0>

<223> Description of Artificial Sequence: Synthetic oligonucleotide

<220>

<221> miscellaneous aspect

<222> (1) .. (18)

<223> Thioskeleton

<400> 6

tctcccagcg tgcgccat 18

<210> 7

<211> 16

<212> DNA

<213> Artificial sequence <220>

<223> Description of Artificial Sequence: Synthetic oligonucleotide <220>

<221> miscellaneous aspect

<222> (1) (16)

<223> Thioskeleton

<220>

<221> miscellaneous aspect <222> (1) (3) <223> LNA

<220>

<221> miscellaneous aspect <222> (13) (15) 40 <223> LNA

<400> 7

tggcaagcat cctgta

16

Claims (29)

1.Composite, characterized in that it is of formula (I) wherein: Ri is a substantially non-antigen water soluble polymer; A is a protecting group or <formula> formula see original document page 90 </formula> each D1 is independently selected from the group consisting of steering moieties, functional groups, and leaving groups; each D2 is independently selected from the group consisting of biologically active moieties, functional groups and leaving groups; each L | is independently a permanent binder or a selected labile binder; L2 is a multifunctional binder; each L3 is independently a permanent ligand or a selected labile ligand, (a) and (b) are independently zero or a positive integer. A compound according to claim 1, characterized in that L2 is selected from the group consisting of: <formula> formula see original document page 90 </formula> <formula> formula see original document page 91 </formula> in wherein R2 and R2 are independently selected from the group consisting of hydrogen, C1-6 alkyl, C2.6 alkenyl, C2.6 alkynyl, C3-19 branched alkyl, C3.8 cycloalkyl, substituted Cn6 alkyl, C2.6 substituted alkenyl, C2-6 substituted alkynyl, C3.8 substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, C6-6 heteroalkyl, substituted C6-6 heteroalkyl, aryloxy, C1-6 heteroalkoxy, heteroaryloxy, C2-6 alkanoyl, arylcarbonyl, alkoxycarbonyl, aryloxycarbonyl, C2.6 alkanoyloxy, arylcarbonyloxy, C2.6 substituted alkanoyl, substituted arylcarbonyl, C2.6 substituted alkanoyloxy, substituted aryloxycarbonyl, C2.6 substituted alkanoyloxy and substituted arylcarbonyloxy; (cl), (c2), (c3), (c4), (c5), (c6), (c'6), (c "6), (c7) and (c8) are independently zero or an integer positive, and (dl), (d2), (d3), (d4), (d5) and (d7) are independently zero or a positive integer. A compound according to claim 2, characterized in that L2 is selected from the group consisting of: <formula> formula see original document page 91 </formula> A compound according to claim 2, characterized in that it is selected from the group consisting of: <formula> formula see original document page 92 </formula> A compound according to claim 4, characterized in that it is selected from the group consisting of: <formula> formula see original document page 92 </formula> <formula> formula see original document page 93 </formula> <formula> formula see original document page 94 </formula> A compound according to claim 1, characterized in that the biologically active portion is selected from the group consisting of portions containing -NH 2, -OH and -SH. A compound according to claim 1, characterized in that the biologically active portion is selected from the group consisting of pharmaceutically active compounds, enzymes, proteins, oligonucleotides, antibodies, monoclonal antibodies, single chain antibodies and peptides. A compound according to claim 7, characterized in that the pharmaceutically active compounds are selected from the group consisting of topoisomerase DNA inhibitors, microtubule inhibiting drugs, DNA damaging agents, antimetabolites, nucleoside analogues and anti-cancer drugs. . A compound according to claim 1, characterized in that the biologically active moiety is an oligonucleotide. Compound according to claim 9, characterized in that the oligonucleotide is selected from the group consisting of antisense oligonucleotides, blocked nucleic acids (LNA), low interference RNA (siRNA), microRNA (miRNA), aptameros, nucleic acids peptides (PNA), and morpholino phosphodiamidate (PMO) oligonucleotides. A compound according to claim 9, characterized in that the oligonucleotide is selected from the group consisting of antisense bcl-2 oligonucleotides, antisense HIF-1a oligonucleotides and antisense Survivin oligonucleotides. A compound according to claim 1, wherein the targeting agent is selected from the group consisting of monoclonal antibodies, single chain antibodies, cell adhesion peptides, cell penetration peptides, receptor ligands, carbohydrate targeting molecules. or targeting lectins and oligonucleotides. A compound according to claim 1, characterized in that the targeting agent is selected from the group consisting of RGD peptide, selectin, TAT, penetratin, (Arg) 9 and folic acid. Compound according to claim 1, characterized in that the labile ligand is selected from the group consisting of benzyl elimination based ligands, trialkyl blocking based ligands, bicine based ligands, acid labile ligands, lysosomally cleavable peptides. and captepsin B cleavable peptides. A compound according to claim 1, characterized in that the labile binders are independently selected from the group consisting of: <formula> formula see original document page 95 </formula> <formula> formula see original document page 96 </ Val-Cit-, -Gly-Phe-Leu-Gly-, -Ala-Leu-Ala-Leu- -Phe-Lys-, <formula> formula see original document page 96 </formula> Val-Cit- C (= 0) -CH20CH2-C (= 0) -, Val-Cit-C (= 0) -CH2SCH2-C (= 0) -, and -NHCH (CH3) -C (= 0) -NH (CH2 ) 6-C (CH 3) 2-C (= 0) -, wherein Y11-19 are independently O, S or NR48; R31-48, R-50, R110 and A5I are independently selected from the group consisting of hydrogen, C1-6 alkyls, C3.12 branched alkyls, C3.8 cycloalkyls, C1-6 substituted alkyls, C3-8 alkyls substituted aryls, substituted aryls, aralkyl, C 1-6 heteroalkyls, substituted C 1-6 heteroalkyls, C 1-6 alkoxy, C 1-6 alkyloxycarbonyl, phenoxy and C 1-6. heteroalkoxy; Ar is an aryl or heteroaryl moiety; LiM5 are independently selected bifunctional spacers; ZeZ 'are independently selected from the group consisting of moieties actively transported to a target cell, hydrophobic moieties, bifunctional ligand moieties and combinations thereof; (cll), (hll), (kl 1), (111), (mil) and (nll) are independently selected positive integers; (ali), (ell), (gll), (jH), (oll) and (qll) are independently either zero or a positive integer; and C b1), (x11), (x12), (f11), (ill) and (p1) are independently zero or one. A compound according to claim 1, characterized in that the permanent ligands are independently selected from the group consisting of: <formula> formula see original document page 97 </formula> A compound according to claim 1, characterized in that L1 and L3 are independently selected from the group consisting of an amino acid, an amino acid derivative and a peptide. A compound according to claim 1, characterized in that R 1 comprises a terminally branched or multi-arm linear polyalkylene oxide. A compound according to claim 18, characterized in that the polyalkylene oxide is selected from the group consisting of polyethylene glycol and polypropylene glycol. A compound according to claim 18, characterized in that the polyalkylene oxide is selected from the group consisting of -Y2I- (CH2CH2O) n-CH2CH2Y2I-, -Y2, - (CH2CH20) n-CH2C (= Y22) -Y2, -, -Y2i-C (= Y22) - (CH2) a2-Y23- (CH2CH20) n-CH2CH2-Y23- (CH2) a2-C (= Y22) Y23- (CH2) b2-O- (CH2CH2O) n- (CH2) b2-Y23- (CR51R52) a2-Y2I-, wherein: Y21 and Y23 are independently O, S, SO, SO2, NR53 or a bond; Y22 is O, S, or NR53; R51.53 are independently selected from the group for R2; (a2) and (b2) are independently zero or a positive integer; and (n) is an integer from about 10 to about 2300. A compound according to claim 18 wherein the polyalkylene oxide is a polyethylene glycol Formula wherein -n- (CH 2 CH 2 O) n- is an integer from about 10 to about 2,300. A compound according to claim 1, characterized in that R 1 has an average molecular weight of from about 2,000 to about 100,000 daltons. Compound according to claim 1, characterized in that R 1 has an average molecular weight of from about 5,000 to about 60,000 daltons. Compound according to claim 1, characterized in that R 1 has an average molecular weight of from about 5,000 to about 25,000 daltons or from about 20,000 to about 45,000 daltons. A compound according to claim 1, characterized in that it is selected from the group consisting of: <formula> formula see original document page 99 </formula> <formula> formula see original document page 100 </formula> <formula> formula see original document page 101 </formula> <formula> formula see original document page 102 </formula> <formula> formula see original document page 103 </formula> where mPEG has Formula: CH3-O (CH2CH2O) n- ; PEG has Formula -O (CH 2 CH 2 O) n-; (n) is a positive integer from about 10 to about 2,300; R5I and R52 are independently selected from the group consisting of hydrogen, C1-6 alkyls, C3-12 branched alkyls, C3.8 cycloalkyls, substituted C6-6 alkyls, substituted C3-6 alkyls, aryls, substituted aryls, aralkyl, C1-6 heteroalkyls, Substituted C1-6 heteroalkyl, C1-6 alkoxy, C1-6 alkyloxycarbonyl, aryloxycarbonyl, phenoxy and C1 heteroalkoxy; D1 is a directing moiety, a functional group or an leaving group; and D 2 is a biologically active moiety, a functional group or an leaving group. A compound according to claim 1, characterized in that it is selected from the group consisting of: <formula> formula see original document page 104 </formula> <formula> formula see original document page 105 </formula> <formula> formula see original document page 106 </formula> <formula> formula see original document page 107 </formula> <formula> formula see original document page 108 </formula> <formula> formula see original document page 109 </formula> < formula> formula see original document page 110 </formula> <formula> formula see original document page 111 </formula> <formula> formula see original document page 112 </formula> <formula> formula see original document page 113 </ formula > <formula> formula see original document page 114 </formula> <formula> formula see original document page 115 </formula> <formula> formula see original document page 116 </formula> <formula> formula see original document page 117 < / formula> <formula> formula see original document page 118 </formula> <formula> formula see original document page 119 </formula> S-SCA is a single chain antibody; RGD is <formula> formula see original document page 120 </formula> mPEG has Formula: CHrO (CH2CH2O) n-; PEG has Formula -O (CH 2 CH 2 O) n-; and (n) is a positive integer from about 10 to about 2,300. A method for preparing a polymeric conjugate containing a multifunctional binder comprising: reacting a compound of Formula (IIIa): M1- (L1) a-L2-M2 <formula> formula see original document page 120 </ formula > with a biologically active moiety containing a compound having Formula (IIIb): M3— (L3) b-D22 under conditions sufficient to form the compound of Formula (IIIc): M1- (L1) a-L2- (L3) b Wherein R1 is a substantially non-antigen water soluble polymer; A2I is a protecting group or <formula> formula see original document page 121 </formula> A22 is a protecting group or <formula> formula see original document page 121 </formula> Mi is a functional group; D22 is a biologically active portion; M2 is OH or a leaving group; M3 is OH, NH2, or SH; L is a permanent binder or a labile binder; L2 is a multifunctional binder; L3 is a permanent binder or a labile binder; and (a) and (b) are independently zero or a positive integer. A method according to claim 26 further comprising: reacting the compound of Formula (IIIc): (IIIC) with a nucleophilic moiety having Formula (IIId) 20 D21-M4 (H] d) under conditions sufficient to form a compound of Formula (IIIe): wherein: A23 is a protecting group or <formula> formula see original document page 122 </formula> Μ1 is a functional group; D21 is a directing moiety; M4 is OH, NH2, or SH; and all other variables are as described in claim 27. A method of treating a mammal comprising administering an effective amount of a compound of Formula (I) to a patient in need thereof. A method for administering polynucleotides to mammalian cells comprising carrying an effective amount of a compound of Formula (I) to a cell requiring such treatment.