HK1129208A

HK1129208A - Preparation of pegylated conjugates of vla-4 antagonists via a mitsunobu's reaction

Info

Publication number: HK1129208A
Application number: HK09108998.6A
Authority: HK
Inventors: Andrei W. Konradi; Jenifer L. Smith; Michael S. Dappen; Christopher M. Semko
Original assignee: 艾伦药物公司
Priority date: 2005-07-08
Filing date: 2006-07-07
Publication date: 2009-11-20

Description

Preparation of pegylated conjugates of VLA-4 antagonists via Mitsunobu reaction

Technical Field

The present invention relates to a process for preparing polymeric conjugates of therapeutic, agricultural and food additive compounds. More particularly, the present invention relates to a method of using Mitsunobu reaction conditions to prepare conjugates useful for the treatment of various diseases and disorders in mammals, particularly humans, as well as for agriculture and as food additives. In certain aspects, the invention relates to the formation of desired conjugates using Mitsunobu conditions with alcohols (particularly alcohol-containing polymers) and amines.

Background

The binding of biologically active compounds to polymers has received great attention and has become a versatile method of controlling a variety of properties, such as the biodistribution, pharmacokinetics, and toxicity of these compounds. A polymer often selected for preparing polymeric conjugates of biologically active compounds is polyethylene glycol (PEG). Both small and large biologically active molecules are widely used as covalent modifiers. See eur.polym.j for a discussion of these conjugates.19No.12, page 1177-1183 (Zaplnsky et al, 1983), et al, Journal of Controlled Release10(1989)145-154(Veronese et al, 1989), and Advanced Drug delivery reviews,16，157-182(Zaplinsky，1995)。

recently, it has been found that₄β₁Polymeric conjugates of (VLA-4) antagonists have greatly improved serum half-life. These polymeric compounds can be prepared using a variety of synthetic methods, including by reacting an ester of the reactive molecule with a polymeric amine to form a carboxamide, a carbamate between an amine of the reactive molecule and a polymeric chloroformate, or a carbamate between an isocyanate of the reactive molecule and a polymeric alcohol. The overall yield of these processes is generally unsatisfactory and often involves multiple steps and purification means. It would therefore be desirable to devise a method for isolating conjugates of VLA-4 inhibitors in quantitative or near quantitative yields.

The importance of these polymeric conjugates suggests the need for convenient and efficient synthesis of these materials.

Disclosure of Invention

The present invention provides improved synthesis of polymeric conjugates of agricultural, therapeutic and food additive compounds. The process of the present invention produces the final conjugated product in excellent yields. In a preferred aspect, the present invention provides a method of preparing a conjugate by condensation polymerizing an alcohol with an amine using Mitsunobu reaction conditions. In another preferred aspect, the present invention provides a method of preparing a conjugate by condensation polymerizing an amine with an alcohol using Mitsunobu reaction conditions.

In one aspect, the present invention provides a process for preparing a conjugate of an active compound, the process comprising: a) reacting a polymeric alcohol with a nucleophile to form a conjugate in the presence of a trivalent phosphine and a suitable azodicarbonyl compound, such as diethyl azodicarboxylate or azodicarbonyl dipiperidine; and b) isolating the conjugate.

In a particular aspect, the active compounds and resulting conjugates exhibit VLA-4 antagonist properties.

In another aspect, the invention provides a method for preparing a conjugate of formula I:

b is a biocompatible polymer moiety;

q is from about 1to about 100;

in each case A is independently a compound having biological or agricultural activity or A is a food additive compound.

The method comprises

a) Reacting a polymeric alcohol of formula Ia

Reacting a nucleophile of formula H-Nu in the presence of a trivalent phosphine and an appropriately substituted azodicarbonyl compound to form a compound of formula I, wherein Nu is a group corresponding to formula a above and H is an acidic hydrogen on Nu; and

b) isolating the compound of formula I.

Detailed Description

As noted above, the present invention provides a process for preparing conjugates of agricultural, therapeutic and food additive compounds (hereinafter "active compounds"). The conjugates include one or more polymer moieties covalently linked to one or more active compounds, wherein the resulting conjugate has the same type of activity as the active compound. In one aspect, the active compounds and resulting conjugates are capable of inhibiting leukocyte adhesion, particularly by at least partially binding to alpha₄A compound for integrin-mediated leukocyte adhesion.

In a preferred aspect, the A group in the conjugate of formula I may be represented by formula II

Wherein

J is selected from:

a) a group of formula (a):

wherein R is³¹Is a covalent bond with a polymer moiety optionally comprising a linking group, or R³¹is-H, R^31′、-NH₂、-NHR^31′or-N (R)^31′)₂、-NC₃-C₆Cyclyl, -OR^31′、-SR^31′Wherein each R is^31′Independently is optionally substituted straight or branched chain C₁-C₆Alkyl, optionally substituted C₃-C₆Cycloalkyl, optionally substituted aryl, optionally substituted heteroaryl,

and R is³²Is a covalent bond with a polymer moiety optionally comprising a linking group, or R³²is-H, -NO₂Haloalkyl or group-N (MR)⁴¹)R⁴²Wherein M is a covalent bond, -C (O) -, or-SO₂-，R⁴¹Is R^41′、N(R^41′)₂OR-OR^41′Wherein each R is^41′Independently hydrogen, optionally substituted straight or branched C₁-C₆Alkyl, optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted heterocycle or optionally substituted heteroaryl, wherein the optional substituents are halide, C₁-C₆Alkyl, or-OC₁-C₆An alkyl group, a carboxyl group,

and R is⁴²Is hydrogen or R^41′(ii) a And

b) a group of formula (b):

wherein R is selected from the group consisting of a covalent bond, an amino group, a substituted amino group, an alkyl group, and a substituted alkyl group attached to the polymer moiety, wherein each amino group, substituted amino group, alkyl group, and substituted alkyl group is optionally covalently attached to the polymer moiety, wherein, in each case, the polymer moiety optionally includes a linking group that is covalently attached to the polymer moiety;

Ar¹selected from the group consisting of aryl, substituted aryl, heteroaryl, and substituted heteroaryl, wherein each aryl, substituted aryl, heteroaryl, and substituted heteroaryl is optionally covalently attached to a polymer moiety, wherein the polymer moiety optionally includes a linking group that links the polymer moiety to Ar¹Covalent attachment;

Ar²selected from the group consisting of aryl, substituted aryl, heteroaryl and substituted heteroaryl, wherein each of aryl, substituted aryl, heteroaryl and substituted heteroaryl is optionally co-polymerized with a polymer moietyA valency linkage, wherein the polymer moiety optionally includes a linking group that links the polymer moiety to Ar²Covalent attachment;

x is selected from-NR¹-、-O-、-S-、-SO-、-SO₂And optionally substituted-CH₂-, wherein R¹Selected from hydrogen and alkyl;

t is selected from;

a) a radical of the formula (c)

Wherein Y is selected from the group consisting of-O-and-NR¹-, wherein R¹Selected from hydrogen and alkyl;

w is selected from the group consisting of a covalent bond with a polymer moiety optionally including a linking group, and-NR²R³Wherein R is²And R³Independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, and wherein R²And R³Together with the nitrogen atom to which they are bound, form a heterocyclic ring or substituted heterocyclic ring, wherein each alkyl, substituted alkyl, heterocyclic ring and substituted heterocyclic ring is optionally covalently bound to a polymer moiety, which further optionally comprises a linking group;

m is an integer equal to 0,1 or 2;

n is an integer equal to 0,1 or 2; and

b) a radical of the formula (d)

Wherein G is an optionally substituted aryl or an optionally substituted heteroaryl 5 or 6 membered ring containing 0 to 3 nitrogens, wherein said aryl or heteroaryl optionally further comprises a covalent bond to a polymer moiety optionally comprising a linking group;

R⁶is a covalent bond with a polymer moiety optionally comprising a linking group, or R⁶is-H;

R⁵⁵selected from the group consisting of alkoxy, substituted alkoxy, cycloalkoxy, substituted cycloalkoxy, aryloxy and substituted aryloxy, and-OH;

with the following conditions:

A.J、Ar¹、Ar²and at least one of T comprises a covalent bond with a polymer moiety;

B. when J is covalently linked to the polymer moiety, n is 1 and X is not-O-, -S-, -SO-or-SO₂-; and

C. when X is-O, then m is 2.

In a preferred embodiment, only J, Ar¹、Ar²And one of T contains a covalent bond with a polymer moiety.

The compound H-Nu used in the conjugation reaction is a compound containing an acidic hydrogen covalently linked to Nu. Preferred compounds of formula H-Nu may be represented by formula II.1

Wherein

J and Ar²Is as defined for formula II;

t is a group bearing an acidic hydrogen; and is

R⁵⁵Is an acidic protecting group.

Suitable T groups include heterocyclic groups, imides, phenols, phosphate mono-and diesters, carboxylic acids, hydroxamates, thiols, thioamides, β -ketoesters, and 1, 3-diketones.

Preferred T groups are groups of the formula (c)

w is an acidic hydrogen containing group, hydrogen preferably on the nitrogen atom adjacent to the carbonyl group, wherein the W group is optionally linked to y (co) via a linking group.

Another preferred T group is a group of the formula (d)

Wherein G is an optionally substituted aryl or an optionally substituted heteroaryl 5 or 6 membered ring containing 0 to 3 nitrogens; and is

R⁶Is hydrogen.

It will be appreciated that the value of q is calculated from the ratio of the number of polymer moieties to the number of A-moieties. In other words, when q is 1.5, then it is expected to be, for example, the following formula I':

preferred conjugates of formula I prepared by the present invention include those compounds of formula Ia:

and pharmaceutically acceptable salts thereof, wherein

B is a polymer moiety optionally covalently bound to a carrier;

q is from about 1to about 100;

a is independently at each occurrence a compound of formula IIa, and pharmaceutically acceptable salts thereof

Wherein

R is selected from the group consisting of a covalent bond, an amino group, a substituted amino group, an alkyl group, and a substituted alkyl group bound to the polymer moiety, wherein each amino group, substituted amino group, alkyl group, and substituted alkyl group is optionally covalently attached to the polymer moiety, wherein, in each case, the polymer moiety optionally includes a linking group covalently attached to the polymer moiety;

Ar²selected from the group consisting of aryl, substituted aryl, heteroaryl, and substituted heteroaryl, wherein each aryl, substituted aryl, heteroaryl, and substituted heteroaryl is optionally covalently attached to a polymer moiety, wherein the polymer moiety optionally includes a linking group that links the polymer moiety to Ar²Covalent attachment;

y is selected from the group consisting of-O-and-NR¹-, wherein R¹Selected from hydrogen and alkyl;

w is selected from the group consisting of a polymer moiety optionally including a linking groupA linked covalent bond, and-NR²R³Wherein R is²And R³Independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, and wherein R²And R³Together with the nitrogen atom to which they are bound, form a heterocyclic ring or substituted heterocyclic ring, wherein each alkyl, substituted alkyl, heterocyclic ring and substituted heterocyclic ring is optionally covalently bound to a polymer moiety, which further optionally comprises a linking group;

m is an integer equal to 0,1 or 2;

n is an integer equal to 0,1 or 2;

with the following conditions:

A.R、Ar¹、Ar²w and-NR²R³At least one of (a) contains a covalent bond with a polymer moiety;

B. when R is covalently linked to the polymer moiety, n is 1 and X is not-O-, -S-, -SO-or-SO₂-；

C. When X is-O-or-NR¹-when then m is 2; and

D. the molecular weight of the conjugate of formula Ia does not exceed 100,000.

Preferred conjugates of formula I prepared by the present invention include those of formula Ib:

wherein each A is independently a compound of formula IIb:

and wherein q is from about 1to about 100;

b is a polymer moiety optionally covalently bound to a carrier;

Ar¹selected from the group consisting of aryl, substituted aryl, heteroaryl, and substituted heteroaryl, wherein each aryl, substituted aryl, heteroaryl, and substituted heteroaryl is optionally covalently attached to a polymer moiety, wherein the PEG moiety optionally comprises a linking group that links the PEG moiety to Ar¹Covalent attachment;

with the proviso that Ar¹、Ar²W and-NR²R³Is covalently bound to a polymer moiety optionally comprising a linking group;

and further with the proviso that the molecular weight of the conjugate of formula Ib does not exceed 100,000.

Preferred conjugates of formula I prepared by the process of the invention include those of formula Ic below:

wherein each A is independently a compound of formula IIc, below, and pharmaceutically acceptable salts thereof:

and wherein q is from about 1to about 100;

b is a polymer moiety optionally covalently bound to a carrier;

w is selected from the group consisting of a covalent bond with a polymer moiety optionally including a linking group, and-NR²R³Wherein R is²And R³To which nitrogen atom one is boundTo form a heterocycle or substituted heterocycle, wherein each alkyl, substituted alkyl, heterocycle and substituted heterocycle is optionally covalently bound to a polymer moiety, said polymer moiety further optionally comprising a linking group;

n is an integer equal to 0,1 or 2;

provided that R, Ar¹、Ar²W and-NR²R³At least one of (a) is covalently bound to a polymer moiety, which optionally comprises a linking group;

and with the further proviso that the molecular weight of the conjugate of formula Ic does not exceed 100,000.

Preferred conjugates of formula I include those of formula Id below.

Wherein each A is independently a compound of formula IId below, and pharmaceutically acceptable salts thereof:

and wherein q is from about 1to about 100;

b is a polymer moiety optionally covalently bound to a carrier;

Ar¹selected from the group consisting of aryl, substituted aryl, heteroaryl and substituted heteroaryl, wherein each aryl, substitutedOptionally covalently linked to a polymer moiety, wherein the polymer moiety optionally comprises a linking group that links the polymer moiety to Ar¹Covalent attachment;

R²and R³Independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, and wherein R²And R³Together with the nitrogen atom to which they are bound, form a heterocyclic ring or substituted heterocyclic ring, wherein each alkyl, substituted alkyl, heterocyclic ring and substituted heterocyclic ring is optionally covalently bound to a polymer moiety, which further optionally comprises a linking group;

n is an integer equal to 0,1 or 2;

provided that R, Ar¹、Ar²and-NR²R³Is covalently bound to a polymer optionally comprising a linking group;

and further provided that the molecular weight of the conjugate of formula Id does not exceed 100,000.

Preferred conjugates of formula I include those of formula Ie below:

wherein each A is independently a compound of formula IIe below, and pharmaceutically acceptable salts thereof:

and wherein q is from about 1to about 100;

b is a polymer moiety optionally covalently bound to a carrier;

with the proviso that Ar¹、Ar²and-NR²R³At least one of (a) is covalently bound to a polymer moiety, which optionally comprises a linking group;

and further provided that the molecular weight of the conjugate of formula Ie does not exceed 100,000.

Preferred conjugates of formula I include those of formula If:

wherein each A is independently a compound of formula IIf:

and wherein q is from about 1to about 100;

b is a polymer moiety optionally covalently bound to a carrier;

R⁴covalently attached to a polymer moiety that optionally includes a linking group;

R⁵selected from alkyl and substituted alkyl;

Ar³selected from the group consisting of aryl, substituted aryl, heteroaryl, and substituted heteroaryl;

m is an integer equal to 0,1 or 2;

n is an integer equal to 0,1 or 2;

with the following conditions:

A. when R is covalently linked to the polymer moiety, n is 1 and X is not-O-, -S-, -SO-or-SO₂-；

B. When X is-O-or-NR¹-when then m is 2; and

C. the molecular weight of the conjugate of formula If does not exceed 100,000.

Preferred conjugates of formula I include those of formula Ig:

wherein each A is independently a compound of formula IIg, below, and pharmaceutically acceptable salts thereof:

and wherein q is from about 1to about 100;

b is a polymer moiety optionally covalently bound to a carrier;

R⁵selected from alkyl and substituted alkyl;

n is an integer equal to 0,1 or 2;

provided that the molecular weight of the conjugate of formula Ig does not exceed 100,000.

Preferred conjugates of formula I include those of formula Ih below:

wherein each A is independently a compound of formula IIh:

and wherein q is from about 1to about 100;

R⁴covalently linked to a polymer moiety, the polymer moiety optionally comprising a linking group;

provided that the molecular weight of the conjugate of formula Ih does not exceed 100,000.

Preferred conjugates of formula I include those of formula Ii below:

wherein each A is independently a compound of formula IIi:

or a pharmaceutically acceptable salt thereof,

and provided that the molecular weight of the conjugate of formula Ii does not exceed 100,000.

Preferably, when Ar is¹Ar in formulae IIa to IIe when not bound to a polymer moiety¹And Ar in the formulae IIf to IIh³Selected from:

a phenyl group,

4-methyl-phenyl group, and the compound is shown in the specification,

4-tert-butylphenyl group (I) having a general structure,

2, 4, 6-trimethylphenyl group,

2-fluorophenyl group is used as a catalyst,

3-fluorophenyl group is used as a catalyst,

the reaction product of 4-fluorophenyl and the fluorine-containing compound,

2, 4-difluorophenyl group, and a salt thereof,

the reaction product of 3, 4-difluorophenyl,

a3, 5-difluorophenyl group is added,

2-chlorphenyl, a phenyl group,

3-chlorphenyl, a phenyl group,

4-chlorphenyl, a phenyl group,

3, 4-dichlorophenyl group, a salt of the compound,

3, 5-dichlorophenyl group, a salt of the compound,

3-chloro-4-fluorophenyl, and the like,

4-bromophenyl group, and a pharmaceutically acceptable salt thereof,

2-methoxy phenyl group, and the compound is shown in the specification,

3-methoxy phenyl group, and the compound is shown in the specification,

4-methoxy phenyl group, and the compound is shown in the specification,

a3, 4-dimethoxyphenyl group,

4-tert-butoxyphenyl group,

4- (3' -dimethylamino-n-propoxy) -phenyl,

2-a group of a 2-carboxyphenyl group,

2- (methoxycarbonyl) phenyl and (2-methoxy-carbonyl) phenyl,

4-(H₂NC (O) phenyl group(s),

4-(H₂NC (S)) -) phenyl group,

a 4-cyanophenyl group, a phenyl group,

4-trifluoromethyl-phenyl group, and the salt thereof,

4-trifluoro-methoxy-phenyl, and the compound is shown in the specification,

3, 5-bis- (trifluoromethyl) phenyl,

4-nitro-phenyl, and the amino acid sequence of the amino acid sequence is shown in the specification,

4-amino-phenyl group, and the amino group,

4-(CH₃c (O) NH-) phenyl,

4- (phenylNHC (O) NH-) phenyl,

a 4-amidinophenyl group, which is a phenyl group,

a 4-methylaminophenylgroup, which is a phenyl group,

4-[CH₃SC(＝NH)-]a phenyl group,

4-chloro-3- [ H₂NS(O)₂-]A phenyl group,

a 1-naphthyl group, which is a group,

a 2-naphthyl group, which is a group,

(ii) a pyridin-2-yl group,

(ii) a pyridin-3-yl group,

(ii) a pyridin-4-yl group,

a pyrimidin-2-yl group, a pyrimidine-2-yl group,

a quinoline-8-yl group, which is a cyclic quinoline,

2- (trifluoroacetyl) -1, 2,3, 4-tetrahydroisoquinolin-7-yl,

a 2-thienyl group,

5-chloro-2-thienyl, or a pharmaceutically acceptable salt thereof,

2, 5-dichloro-4-thienyl,

1-N-methylimidazol-4-yl,

1-N-methylpyrazol-3-yl,

1-N-methylpyrazol-4-yl,

1-N-butylpyrazol-4-yl,

1-N-methyl-3-methyl-5-chloropyrazol-4-yl,

1-N-methyl-5-methyl-3-chloropyrazol-4-yl,

2-thiazolyl and

5-methyl-1, 3, 4-thiadiazol-2-yl.

Preferably, when A is of formulae IIa, IIb, IIc, IId and IIe, and Ar¹When combined with a polymer moiety, then Ar¹Having the formula:

-Ar¹-Z-(CH₂CHR⁷O)_PR⁸

wherein

Ar¹Selected from the group consisting of aryl, substituted aryl, heteroaryl and substituted heteroaryl,

z is selected from the group consisting of a covalent bond, a linker of from 1to 40 atoms, -O-and-NR⁹-, wherein R⁹Selected from the group consisting of hydrogen and alkyl,

R⁷selected from hydrogen and methyl;

R⁸selected from A, - (L)_w-polymer carrier, hydrogen, alkyl, substituted alkyl, aryl, substituted aryl and-CH₂CHR⁷NR¹⁰R¹¹Wherein R is⁷As defined above, R¹⁰And R¹¹Independently selected from hydrogen and alkyl, a is represented by any one of formulas IIa to IIh above, L is a linking group of 1to 40 atoms, and w is 0 or 1; and is

p is an integer from about 100 to 2200, preferably about 200 and 1360.

When A is of formula IIa or IIf and R is not bound to a polymer moiety, a substituent having the formula:

wherein R is⁵X, m and n are as defined above, preferably selected from azetidinyl, thiazolidinyl, piperidinyl, piperazinyl, morpholino, thiomorpholinyl, pyrrolidinyl, 4-hydroxypyrrolidinyl, 4-oxopyrrolidinyl, 4-fluoropyrrolidinyl, 4-difluoropyrrolidinyl, 4- (thiomorpholin-4-yl C (O) O-) pyrrolidinyl, 4- [ CH₃S(O)₂O-]Pyrrolidinyl, 3-phenylpyrrolidinyl, 3-thiophenylpyrrolidinyl, 4-amino-pyrrolidinyl, 3-methoxypyrrolidinyl, 4-dimethylpyrrolidinyl, 4-N-Cbz-piperazinyl, 4- [ CH₃S(O)₂-]Piperazine derivativesA group, 5-dimethylthiazolidin-4-yl, 1-dioxo-thiazolidinyl, 1-dioxo-5, 5-dimethylthiazolidin-2-yl and 1, 1-dioxothiomorpholinyl.

Preferably, when A is of formula IIa, and a substituent of formula:

when combined with a PEG moiety, preferred substituents are of the formula:

wherein

m is an integer equal to 0,1 or 2;

z is selected from the group consisting of a covalent bond, a 1to 40 atom linker, -O-and-NR⁹-, wherein R⁹Selected from the group consisting of hydrogen and alkyl,

R⁷selected from hydrogen and methyl;

p is an integer from about 100 to 2200, preferably about 200 and 1360.

When A is of the formulae IIa, IIb, IIc, IId, IIe, and Ar²When not bound to a polymer moiety, Ar is preferred²Selected from phenyl, substituted phenyl, 2-pyridyl, 3-pyridyl, 4-pyridyl and 4-pyridin-2-onyl.

When A is of the formulae IIa, IIb, IIc, IId, IIe, and when Ar²When combined with a polymer moiety, then Ar²Preferably represented by the formula:

wherein Ar is²Selected from the group consisting of aryl, substituted aryl, heteroaryl, and substituted heteroaryl;

R⁷selected from hydrogen and methyl;

p is an integer from about 100 to 2200, preferably about 200 and 1360.

In a preferred embodiment of the conjugate prepared by the method of the present invention, -YC (O) W is-OC (O) NR²R³。

When A is of the formula IIa, IIb or IIc, -YC (O) W is-OC (O) NR²R³And R is²And R³None of which is bound to a polymer moiety, then-OC (O) NR is preferred²R³Selected from:

(CH₃)₂NC(O)O-，

(piperidin-1-yl) -C (O) O-,

(piperidin-4-yl) -C (O) O-,

(1-methylpiperidin-4-yl) -C (O) O-,

(4-hydroxypiperidin-1-yl) -C (O) O-,

(4-formyloxypiperidin-1-yl) -C (O) O-,

(4-ethoxycarbonylpiperidin-1-yl) -C (O) O-,

(4-carboxypiperidin-1-yl) -C (O) O-,

(3-hydroxymethylpiperidin-1-yl) -C (O) O-,

(4-hydroxymethylpiperidin-1-yl) -C (O) O-,

(4-phenyl-1-Boc-piperidin-4-yl) -C (O) O-,

(4-piperidone-1-ylethyleneketal) -C (O) O-,

(piperazin-4-yl) -C (O) O-,

(1-Boc-piperazin-4-yl) -C (O) O-,

(4-methylpiperazin-1-yl) -C (O) O-,

(4- (2-hydroxyethyl) piperazin-1-yl) -C (O) O-,

(4-phenylpiperazin-1-yl) -C (O) O,

(4- (pyridin-2-yl) piperazin-1 ] -yl) -C (O) O-,

(4- (4-trifluoromethylpyridin-2-yl) piperazin-1-yl) -C (O) O-,

(4- (pyrimidin-2-yl) piperazin-1-yl) -C (O) O-,

(4-acetylpiperazin-1-yl) -C (O) O-,

(4- (phenyl-C (O) -) piperazin-1-yl) -C (O) O-,

(4- (pyridin-4' -yl-C (O) -) piperazin-1-yl) -C (O) O-,

(4- (phenyl-NHC (O) -) piperazin-1-yl) -C (O) -O-,

(4- (phenyl-NHC (S) -) piperazin-1-yl) -C (O) -,

(4-methanesulfonylpiperazin-1-yl) -C (O) O-,

(4-trifluoromethanesulfonylpiperazin-1-yl) -C (O) O-,

(morpholin-4-yl) -C (O) O-,

(thiomorpholin-4-yl) -C (O) O-,

(thiomorpholin-4' -ylsulfone) -C (O) O-,

(pyrrolidin-1-yl) -C (O) O-,

(2-methylpyrrolidin-1-yl) -C (O) O-,

(2- (methoxycarbonyl) pyrrolidin-1-yl) -C (O) O-,

(2- (hydroxymethyl) pyrrolidin-1-yl) -C (O) O-,

(2- (N, N-dimethylamino) ethyl) (CH₃)NC(O)O-，

(2- (N-methyl-N-toluene-4-sulfonylamino) ethyl) (CH₃)N-C(O)O-，

(2- (Morpholin-4-yl) ethyl) (CH₃)NC(O)O-，

(2- (hydroxy) ethyl) (CH)₃)NC(O)O-，

Bis (2- (hydroxy) ethyl) NC (O) O-,

(2- (formyloxy) ethyl) (CH₃)NC(O)O-，

(CH₃OC(O)CH₂) HNC (O) O-, and

2- (phenylNHC (O) O-) ethyl- ] HNC (O) O-.

When A is of the formula IIa, IIb or IIc, -YC (O) W is-OC (O) NR²R³And R is²And/or R³When combined with a PEG moiety, the PEG moiety is preferably represented by the formula:

-Z′-(CH₂CHR⁷O)_pR⁸

z' is selected from a covalent bond and a linking group of 1to 40 atoms;

R⁷selected from hydrogen and methyl;

R⁸selected from A, - (L)_w-polymer carrier, hydrogen, alkyl, substituted alkyl, aryl, substituted aryl and-CH₂CHR⁷NR¹⁰R¹¹Wherein R is⁷As defined above, R¹⁰And R¹¹Independently selected from hydrogen and alkyl, a is represented by formula II above, L is a linking group of 1to 40 atoms, and w is 0 or 1; and is

p is an integer from about 100 to 2200.

Preferably, the T group in formula II.1 has the following formula (d).

Wherein R is⁶Is hydrogen.

Preferred (D) groups include those shown in table D below.

Table D

Wherein

R⁶⁶Is a covalent bond with a polymer moiety optionally comprising a linking group, or R⁶⁶Is hydrogen or straight or branched C₁-C₆An alkyl group;

R⁷⁷is a covalent bond with a polymer moiety optionally comprising a linking group, or

R⁷⁷Is hydrogen, halogen or straight or branched C₁-C₆An alkoxy group; and is

R⁸⁸Is hydrogen.

Other (D) groups useful in the present invention include those shown in table D1.

TABLE D1

Other preferred compounds of formula II.1 for use in the method of the present invention are of formula II.1-a:

and pharmaceutically acceptable salts thereof, wherein

R⁵⁵Is an acid protecting group, preferably C₁-C₆Alkoxy, more preferably C₂-C₄An alkoxy group;

Ar¹selected from the group consisting of alkyl, substituted alkyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, heterocycle, substituted heterocycle, heteroaryl, and substituted heteroaryl; and is

R⁶Is hydrogen.

Preferred compounds of formula 11.1-a include those compounds of the following: wherein Ar is¹Is phenyl or a 5-or 6-membered heteroaryl group having at least one nitrogen atom, each optionally substituted with halogen, hydroxy, C₁-C₆Alkoxy radical, C₁-C₆Alkyl, nitro, trifluoromethyl, amino, mono-or di (C)₁-C₆) Alkylamino radical, amino radical (C)₁-C₆) Alkyl radical, C₂-C₆Acyl radical, C₂-C₆Acylamino, or amino (C)₁-C₆) Acyl substitution。Ar¹Is optionally substituted by halogen, hydroxy, C₁-C₆Alkyl radical, C₁-C₆Alkoxy, nitro, trifluoromethyl, amino, mono-or di (C)₁-C₆) Alkylamino radical, amino radical (C)₁-C₆) Alkyl radical, C₂-C₆Acyl radical, C₂-C₆Acylamino, or amino (C)₁-C₆) An acyl substituted pyridyl group. Particularly preferred compounds of formula 11.1-a include those compounds of the following: wherein Ar is¹Is optionally substituted with C₁-C₆Alkyl, halogen, hydroxy, C₁-C₆Alkoxy, nitro, trifluoromethyl, amino, or mono-or di (C)₁-C₆) An alkylamino substituted pyridyl group.

Other preferred compounds of formula 11.1 are also those of formula 11.2-a:

and pharmaceutically acceptable salts thereof, wherein

R⁶is hydrogen.

Preferred compounds of formula 11.2-a include those of the following: wherein R is³¹Is amino or mono-or di (C)₁-C₆) An alkylamino group; and R is³²is-H, -NO₂Or haloalkyl, more preferably trifluoromethyl.

Other preferred compounds of the formula 11.2-a are those in which

R³¹Is amino or mono-or di (C)₁-C₆) An alkylamino group; and is

R³²is-N (MR)⁴¹)R⁴²(ii) a Wherein M is-SO₂-or-CO-;

R⁴¹is C optionally substituted with₁-C₆Alkyl groups: halogen, hydroxy, C₁-C₆Alkoxy, amino, or mono-or di (C)₁-C₆) An alkylamino group; or phenyl or 5-or 6-membered heteroaryl containing at least one nitrogen, each optionally substituted with halogen, hydroxy, C₁-C₆Alkyl radical, C₁-C₆Alkoxy radical, C₃-C₇Cycloalkyl, amino, nitro, trifluoromethyl, or mono-or di (C)₁-C₆) Alkyl amino substitution; and is

R⁴²Is hydrogen, C₁-C₆Alkyl or C₃-C₇A cycloalkyl group.

Further preferred compounds of formula 11.2-a include those wherein

R in formula 11.2-a⁴¹The radicals being C optionally substituted by₁-C₄Alkyl groups: halogen, hydroxy, C₁-C₆Alkoxy, amino, or mono-or di (C)₁-C₆) An alkylamino group; or pyridyl or pyrimidyl, each optionally substituted with halogen, hydroxy, C₁-C₃Alkyl radical, C₁-C₃Alkoxy, amino, or mono-or di (C)₁-C₄) Alkyl amino substitution; and is

R⁴²Is hydrogen, C₁-C₄Alkyl or C₃-C₇A cycloalkyl group.

In one embodiment, the conjugate of the invention is divalent and is represented by formula III:

wherein each A is independently as defined above, and B 'is-Z' - (CH)₂CHR⁷O)_p-Z '-, wherein each Z' is independently a covalent bond or a linking group, R⁷Is hydrogen or methyl, and p is an integer from about 100 to 1360.

In another embodiment, the conjugates of the invention are trivalent to decavalent, and are preferably represented by formula IV:

wherein each a is independently as defined above and t is an integer from 3 to 10.

The process of the present invention employs a Mitsunobu reaction, i.e. an alcohol condensation reaction in the presence of a triarylphosphine or trialkylphosphine and a suitable azodicarboxylate. In a preferred reaction, a polymeric alcohol, such as polyethylene glycol, is reacted with a nucleophile in the presence of a triaryl-or trialkylphosphine and a diazotizing agent to form a conjugate. Nucleophiles are compounds having an acidic hydrogen (i.e., a moiety suitable for donating an electron). The bond formed by the reaction of the polymeric alcohol with the nucleophile can be varied, for example, carbon-oxygen bond formation, carbon-nitrogen bond formation, carbon-sulfur bond formation, carbon-halogen bond formation, and carbon-carbon bond formation.

Thus, the non-derivatized reactive compound may have a variety of functional groups that are reactive with the polymeric alcohol. Examples include carboxylic acids, alcohols, beta-ketoesters, amines, thiols, alkyl halides, acid halides, beta-diketones, and the like. Other examples of nucleophiles that can be used in the methods of the present invention can be found in organic reactions, 1992, 42, 335-.

Examples of triaryl-or trialkylphosphines are described in Synthesis, 2003, 3, 317-; tetrahedron lett, 1998, 39, 7787; chem.commun.1997, 759; and nucleic acids Nucleotides, 1999, 18, 727, all of which are incorporated herein by reference, and include triphenylphosphine, trimethylphosphine, triethylphosphine, tributylphosphine, and 1, 2-bis- (diphenylphosphino) ethane. The phosphines may also be polymer supported or water soluble. The preferred triarylphosphine is triphenylphosphine.

Diazotizing agents are typically esters or amides of azodicarboxylic acids and include Synthesis, 2003, 3, 317-; tetrahedron lett, 1999, 40, 7359; and those mentioned in bull. chem.soc.jpn., 1984, 57, 2675. Specific examples of these diazo compounds are diethyl azodicarboxylate, diisopropyl azodicarboxylate, 4-methyl-1, 2, 4-triazolidine-3, 5-dione, N, N, N ', N' -tetramethylazodicarboxamide, dipiperidine azodicarboxylate, bis (N-4-methylpiperazin-1-yl) azodicarboxamide, dimorpholinoazo dicarboxamide and di-tert-butyl azodicarboxylate.

Representative conjugates made by the process of the present invention, including pharmaceutically acceptable salts thereof, are shown in the following table:

TABLE I

Wherein, in each structure, the sum of all p' is 100-.

Definition of

As used herein, "alkyl" refers to a monovalent saturated aliphatic hydrocarbon group having 1to 5 carbon atoms, more preferably 1to 3 carbon atoms. Exemplary groups of this term are methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, n-pentyl and the like.

"substituted alkyl" refers to an alkyl group having 1to 3, preferably 1to 2, substituents selected from the group consisting of alkoxy, substituted alkoxy, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aryl, substituted aryl, aryloxy, substituted aryloxy, cyano, halogen, hydroxy, nitro, carboxy ester, cycloalkyl, substituted cycloalkyl, spirocycloalkyl, heteroaryl, substituted heteroaryl, heterocyclic ring, and substituted heterocyclic ring.

"alkylene" means a divalent saturated aliphatic hydrocarbon group, preferably having 1to 5, more preferably 1to 3 carbon atoms, which is a straight or branched chain group. Exemplary groups of this term are methylene (-CH)₂-) ethylene (-CH₂CH₂-) and n-propylidene (-CH)₂CH₂CH₂-) isopropylidene (-CH₂CH(CH₃) -) and the like.

"alkoxy" means an "alkyl-O-" group, examples of which include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentoxy, and the like.

"substituted alkoxy" refers to a "substituted alkyl-O-" group.

"acyl" refers to the groups H-C (O) -, alkyl-C (O) -, substituted alkyl-C (O) -, alkenyl-C (O) -, substituted alkenyl-C (O) -, alkynyl-C (O) -, substituted alkynyl-C (O) -, cycloalkyl-C (O) -, substituted cycloalkyl-C (O) -, aryl-C (O) -, substituted aryl-C (O) -, heteroaryl-C (O) -, substituted heteroaryl-C (O) -, heterocycle-C (O) -, and substituted heterocycle-C (O) -, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl-C (O) -, substituted cycloalkyl-C (O) -, and substituted heterocycle-C (O) -, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, and heteroaryl are each independently substituted, Substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycle, and substituted heterocycle are as defined herein.

"aminoacyl" refers to the group-C (O) NR¹⁰R¹⁰Wherein each R is¹⁰Are each independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, heteroaryl, substituted heteroaryl, heterocycle, substituted heterocycle, and combinations thereofEach R in¹⁰And the nitrogen atom, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein.

"acyloxy" refers to the group alkyl-C (O) O-, substituted alkyl-C (O), alkenyl-C (O), O-, substituted alkenyl-C (O), O-, alkynyl-C (O), O-, substituted alkynyl-C (O), O-, aryl-C (O), substituted aryl-C (O), O-, cycloalkyl-C (O), O-, substituted cycloalkyl-C (O), O-, heteroaryl-C (O), O-, substituted heteroaryl-C (O), O-, heterocycle-C (O) O-, and substituted heterocycle-C (O) O-, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, Cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycle, and substituted heterocycle are as defined herein.

"alkenyl" means an alkenyl group having 2 to 6 carbon atoms, preferably 2 to 4 carbon atoms, having at least 1, preferably 1to 2 sites of alkenyl unsaturation. Examples of such groups are vinyl, allyl, but-3-en-1-yl and the like.

"substituted alkenyl" refers to alkenyl groups having 1to 3 substituents, preferably 1to 2 substituents, selected from the group consisting of alkoxy, substituted alkoxy, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aryl, substituted aryl, aryloxy, substituted aryloxy, cyano, halogen, hydroxy, nitro, carboxy ester, cycloalkyl, substituted cycloalkyl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic, provided that any hydroxy substitution is not attached to the vinyl (unsaturated) carbon atom.

"alkynyl" means an alkynyl group having 2 to 6 carbon atoms, preferably 2 to 3 carbon atoms, and having at least 1, preferably 1to 2 sites of alkynyl unsaturation.

"substituted alkynyl" refers to alkynyl groups having 1to 3 substituents, preferably 1to 2 substituents, selected from the group consisting of alkoxy, substituted alkoxy, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aryl, substituted aryl, aryloxy, substituted aryloxy, cyano, halogen, hydroxy, nitro, carboxy ester, cycloalkyl, substituted cycloalkyl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic.

"amino" means-NH₂A group.

"cyano" refers to the group-CN.

"substituted amino" refers to the group-NR 'R', wherein R 'and R' are both independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, heteroaryl, substituted heteroaryl, heterocycle, substituted heterocycle, and wherein R 'and R' are joined together with the nitrogen to which they are attached to form a heterocycle or substituted heterocycle, with the proviso that neither R 'nor R' is hydrogen. When R' is hydrogen and R "is alkyl, substituted amino is sometimes referred to herein as alkylamino. When R' and R "are alkyl, substituted amino is sometimes referred to herein as alkylamino. When referring to a monosubstituted amino, it means that either R' or R "is hydrogen but not both. When referring to disubstituted amino, it means that neither R' or R "is hydrogen.

"aminoacyl" refers to the group-NR¹¹C (O) alkyl, -NR¹¹C (O) substituted alkyl, -NR¹¹C (O) cycloalkyl, -NR¹¹C (O) substituted cycloalkyl, -NR¹¹C (O) alkenyl, -NR¹¹C (O) substituted alkenyl, -NR¹¹C (O) alkynyl, -NR¹¹C (O) substituted alkynyl, -NR¹¹C (O) aryl, -NR¹¹C (O) substituted aryl, -NR¹¹C (O) heteroaryl, -NR¹¹C (O) substituted heteroaryl, -NR¹¹C (O) heterocyclic ring and-NR¹¹C (O) substituted heterocycle, wherein R¹¹Is hydrogen or alkyl, and wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedAlkynyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycle, and substituted heterocycle are as defined herein.

"nitro" means-NO₂A group.

"aryl" or "Ar" refers to a monovalent aromatic carbocyclic group having 6 to 14 carbon atoms, having a single ring (e.g., phenyl) or multiple condensed rings (e.g., naphthyl or anthracenyl) which may or may not be aromatic (e.g., 2-benzoxazolinone, 2H-1, 4-benzoxazin-3 (4H) -one-7-yl, and the like), provided that the connection is at one aromatic carbon atom. Preferred aryl groups include phenyl and naphthyl.

"substituted aryl" refers to aryl having 1to 3 substituents, preferably 1to 2 substituents, selected from the group consisting of hydroxy, acyl, acylamino, acyloxy, alkyl, substituted alkyl, alkoxy, substituted alkoxy, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, amino, substituted amino, aminoacyl, aryl, substituted aryl, aryloxy, substituted aryloxy, carboxy, carboxyester, cyano, thiol, thioalkyl, substituted thioalkyl, thioaryl, substituted thioaryl, thioheteroaryl, substituted thioheteroaryl, thiocycloalkyl, substituted thiocycloalkyl, thioheterocyclic, substituted thioheterocyclic, cycloalkyl, substituted cycloalkyl, halogen, nitro, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic, heteroaryloxy, substituted heteroaryloxy, Heterocyclyloxy, substituted heterocyclyloxy, aminosulfonyl (NH)₂-SO₂-) and substituted aminosulfonyl groups.

"aryloxy" refers to an aryl-O-group, examples of which include phenoxy, naphthoxy, and the like.

"substituted aryloxy" refers to a substituted aryl-O-group.

"carboxy" means-COOH or a salt thereof.

"carboxy ester" refers to the groups-C (O) O-alkyl, -C (O) O-substituted alkyl, -C (O) -aryl, and-C (O) O-substituted aryl, wherein alkyl, substituted alkyl, aryl, and substituted aryl are as defined herein.

"cycloalkyl" means a cyclic alkyl group having 3 to 10 carbon atoms and having a single ring or multiple rings, and examples thereof include adamantyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, and the like.

"cycloalkenyl" refers to a cyclic alkenyl group of 4 to 10 carbon atoms having a single ring or multiple rings, and further having at least 1, preferably 1to 2, ethylenic or vinyl (> C ═ C <) unsaturated internal sites.

"substituted cycloalkyl" and "substituted cycloalkenyl" refer to cycloalkyl or cycloalkenyl groups having 1to 5 substituents selected from the group consisting of oxy (═ O), thio (═ S), alkoxy, substituted alkoxy, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aryl, substituted aryl, aryloxy, substituted aryloxy, cyano, halogen, hydroxy, nitro, carboxyl ester, cycloalkyl, substituted cycloalkyl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic.

"Cycloalkoxy" refers to an-O-cycloalkyl group.

"substituted cycloalkoxy" refers to an-O-substituted cycloalkyl group.

"halo" or "halogen" refers to fluoro, chloro, bromo and iodo, preferably fluoro or chloro.

"hydroxy" means an-OH group.

"heteroaryl" refers to an aromatic group consisting of 1to 10 carbon atoms and 1to 4 heteroatoms selected from oxygen, nitrogen and sulfur in the ring. Such heteroaryl groups can have a single ring (e.g., pyridyl or furyl) or multiple condensed rings (e.g., indolizinyl or benzothienyl) wherein the condensed rings may or may not be aromatic and/or contain heteroatoms, provided that the point of attachment is at an atom of the aromatic heteroaryl group. Preferred heteroaryl groups include pyridyl, pyrrolyl, indolyl, thiophenyl and furanyl.

"substituted heteroaryl" refers to heteroaryl substituted with 1to 3 substituents selected from the same group as defined for substituted aryl.

"heteroaryloxy" refers to an-O-heteroaryl group, and "substituted heteroaryloxy" refers to an-O-substituted heteroaryl group.

"heterocycle" or "heterocyclic" or "heterocycloalkyl" or "heterocyclyl" refers to a saturated or unsaturated group having a single or multiple fused rings, having from 1to 10 carbon atoms in the ring and from 1to 4 heteroatoms selected from nitrogen, sulfur or oxygen, wherein in the fused ring system, one or more rings can be cycloalkyl, aryl or heteroaryl, provided that the point of attachment is on the heterocycle.

"substituted heterocycle" or "substituted heterocycloalkyl" or "substituted heterocyclyl" refers to a heterocyclyl group that is substituted with 1to 3 of the same substituents as defined for substituted cycloalkyl.

Examples of heterocyclyl and heteroaryl groups include, but are not limited to, azetidine, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, indoline, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphtylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, benzanthracene, phenanthroline, isothiazole, phenazine, isoxazole, phenoxazine, phenothiazine, imidazolidine, imidazoline, piperidine, piperazine, indoline, phthalimide, 1, 2,3, 4-tetrahydro-isoquinoline, 4,5, 6, 7-tetrahydrobenzo [ b ] thiophene, thiazole, thiazolidine, thiophene, benzo [ b ] thiophene, morpholinyl, thiomorpholinyl (also known as thiomorpholinyl), piperidinyl, pyrrolidine, tetrahydrofuranyl, and the like.

"thiol" refers to the-SH group.

"thioalkyl" or "alkylthioether" or "thioalkoxy" refers to an-S-alkyl group.

"substituted thioalkyl" or "substituted alkylthioether" or "substituted thioalkoxy" refers to an-S-substituted alkyl group.

"Thioaryl" refers to an-S-aryl group, wherein aryl is as defined above.

"substituted thioaryl" refers to an-S-substituted aryl group, wherein the substituted aryl group is as defined above.

"Thioaryl" refers to an-S-heteroaryl group, wherein heteroaryl is as defined above.

"substituted thiaheteroaryl" refers to an-S-substituted heteroaryl group, wherein substituted thiaheteroaryl is as defined above.

"Thioaterocycle" refers to an-S-heterocyclic group and "substituted Thioaterocycle" refers to an-S-substituted heterocyclic group in which heterocycle and substituted heterocycle are as defined above.

"heterocyclyloxy" refers to a heterocyclyl-O-group, and "substituted heterocyclyl-O-" refers to a substituted heterocyclyl-O-group, where heterocyclyl and substituted heterocyclyl are as defined above.

"Thiocycloalkyl" refers to an-S-cycloalkyl group, and "substituted Thiocycloalkyl" refers to an-S-substituted cycloalkyl group, wherein cycloalkyl and substituted cycloalkyl are as defined above.

The terms "compound" and "active compound" are used to refer to the VLA-4 antagonist portion of the conjugate of the invention or the VLA-4 antagonist present prior to conjugation to the polymer.

The term "linker", "linker" or "1 to 40 atom linker" refers to the following groups: (1) covalently linking the polymer to the active compound and/or (2) covalently linking the polyalkylene oxide moieties of the polymer to each other. A linking group linking the polyalkylene oxide moieties of the polymer together within any particular conjugate, and a reactive compound linking the polymer to the reactive compoundThe linking groups to which the conjugates are bonded may be the same or different (i.e., may have the same or different chemical structures). Representative functional group linkages (which may have one or more linking groups therein) are amide, ether, carbamate, thiocarbamate, urea, thiourea, amino groups, carbonyl groups, alkoxy groups, and the like. The linking group can be homologous or heterologous in its atomic content (e.g., a linking group containing only carbon atoms or a linking group containing carbon atoms and one or more heteroatoms present on the linking group). Preferably, the linking group contains 1to 25 carbon atoms and 0 to 15 substituents selected from oxygen, NR²²Sulfur, -S (O) -and-S (O)₂A heteroatom of (A), wherein R²²As defined above. The linking group may also be chiral or achiral, linear, branched or cyclic.

For insertion between functional group linkages or bonds within the linking group, the linking group can further comprise a spacer group including, but not limited to, a spacer selected from the group consisting of alkyl, substituted alkyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, heteroaryl, substituted heteroaryl, heterocycle, substituted heterocycle, and combinations thereof. The spacer can be homologous or heterologous in its atomic content (e.g., the spacer contains only carbon atoms or the spacer contains carbon atoms and one or more heteroatoms present on the spacer). Preferably, the spacer contains 1to 25 carbon atoms and 0 to 15 substituents selected from oxygen, NR²²Sulfur, -S (O) -and-S (O)₂A heteroatom of (A), wherein R²²As defined above. The spacer may also be chiral or achiral, linear, branched or cyclic.

Non-limiting examples of spacers are straight or branched alkylene chains, phenylene, biphenylene, and the like, rings, all of which can carry one or more functional groups capable of forming a linkage with the reactive compound and one or more polyalkylene oxide moieties. A specific example of a polyfunctional linker-spacer is lysine, which may be substituted with C₄The two amino groups substituted on the alkylene chain link any active compound to the two polymer moieties. It is composed ofNon-limiting examples of which include p-aminobenzoic acid and 3, 5-diaminobenzoic acid, which have 2 and 3 functional groups for forming linkages, respectively. Other such multifunctional linkages plus spacers will be readily apparent to those skilled in the art.

The term "polymer" refers to a biocompatible, water-soluble, substantially non-immunogenic polymer that is capable of coupling to more than one VLA-4 antagonist of formula II. Preferably the polymer is non-ionic and biocompatible and is non-toxic as measured at the dosages used. These polymers also include multiple copies of the polymer attached to the carrier.

Examples of suitable polymers include, but are not limited to: polyoxyalkylene polymers such as polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), polyacrylamide (PAAm), polydimethylacrylamide (PDAAm), polyvinyl alcohol (PVA), dextrose, poly (L-glutamic acid) (PGA), Styrene Maleic Anhydride (SMA), poly-N- (2-hydroxypropyl) methacrylamide (HPMA), polydivinyl ether maleic anhydride (DIVEMA) (Kameda, Y. et al, Biomaterials 25: 3259. Invitro. 3266, 2004; Thanou, M. et al, Current Opinion in investment drugs4 (6): 701. 709, 2003; Veronese, F.M. et al, II Farmaco 54: 497. 516, 1999).

The preferred polymer is a polyoxyalkylene. "polyoxyalkylene" means a macromolecule comprising at least one polyalkylene oxide moiety which is optionally covalently linked to one or more additional polyalkylene oxides, wherein the polyalkylene oxides are the same or different. Non-limiting examples include polyethylene glycol (PEG), polypropylene glycol (PPG), polyisopropylene glycol (PIPG), PEG-PEG, PEG-PPG, PPG-PIPG, and the like. Polymers also included in the definition of polyoxyalkylene are macromolecules in which polyalkylene oxide moieties are linked to one another optionally via linking groups. Illustrative examples are PEG-linker-PEG, PEG-linker-PIPG and the like. More specific examples include commercially available poly [ di (ethylene glycol) adipate, poly [ di (ethylene glycol) phthalate glycol, and the like. Other examples are block copolymers of alkylene oxide, polyethylene glycol, polypropylene glycol and polyoxyethylenated polyol units.

Covalent attachment of the polymer to the non-polymer substituted compound of formula II, via at least one of its ends, optionally via a linking group, using the methods of the invention, provides covalent attachment of the polymer to the non-polymer substituted compound of formula II.

When a linking group is employed, the linking group is covalently attached to at least one polymer terminus that is otherwise covalently attached to the non-polymer substituted compound of formula II. It will of course be appreciated that if suitable substituents are found on the non-polymer substituted compound of formula II, then the optional linking group may not be required as the polymer may be directly linked to the non-polymer substituted compound of formula II.

Preferred examples of the linking group include-O-, -NR-, and the like²²-、-NR²²C(O)O-、-OC(O)NR²²-、-NR²²C(O)-、-C(O)NR²²-、-NR²²C(O)NR²²-, - (alkylene) -NR²²C (O) O-, -alkylene-NR²²C(O)NR²²-, - (alkylene) -OC (O) NR²²-, - (alkylene) -NR²²-, - (alkylene) -O-, -alkylene-NR²²C (O) -, -alkylene-C (O) NR²²-、-NR³C (O) O-alkylene-, -NR²²C(O)NR²²Alkylene-, -OC (O) NR²²Alkylene, -NR²²-alkylene-, -O-alkylene-, -NR²²C (O) -alkylene-, -C (O) NR²²Alkylene-, -alkylene-NR²²C (O) O-alkylene-, -alkylene-NR³C(O)NR²²Alkylene-, -alkylene-OC (O) NR²²Alkylene-, -alkylene-NR²²Alkylene-, alkylene-O-alkylene-, -alkylene-NR²²C (O) -alkylene-, -C (O) NR²²-alkylene-, and

wherein

Selected from aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, heteroaryl, substituted heteroaryl, heterocycle and substituted heterocycle, and D and E are independently selected from the group consisting of a bond, -O-, CO, -NR²²-、-NR²²C(O)O-、-OC(O)NR²²-、-NR²²C(O)-、-C(O)NR²²-、-NR²²C(O)NR²²-, - (alkylene) -NR²²C (O) O-, -alkylene-NR²²C(O)NR²²-, - (alkylene) -OC (O) NR²²-, - (alkylene) -NR²²-, - (alkylene) -O-, -alkylene-NR²²C (O) -, alkylene-C (O) NR²²-、-NR²²C (O) O-alkylene-, -NR²²C(O)NR²²Alkylene-, -OC (O) NR²²-alkylene-, -NR²²-alkylene-, -O-alkylene-, -NR²²C (O) -alkylene-, -C (O) NR²²Alkylene-, -alkylene-NR²²C (O) O-alkylene-, -alkylene-NR²²C(O)NR²²Alkylene-, -alkylene-OC (O) NR²²Alkylene-, -alkylene-NR²²Alkylene-, alkylene-O-alkylene-, -alkylene-NR²²C (O) -alkylene-, and-C (O) NR²²-alkylene-, wherein R²²As defined above.

Preferred alkylene groups in the above-mentioned linking groups include C₁-C₁₅Alkylene, more preferably C₁-C₆Alkylene, and most preferably C₁-C₃An alkylene group. Preferred heterocyclic groups include piperazinyl, piperidinyl, homopiperazinyl, homopiperidinyl, pyrrolidinyl, and imidazolidinyl.

The conjugates of the invention may be incorporated into a carrier to which 1to 19 additional conjugates may be attached. The term "carrier" refers to an optional component or scaffold to which multiple conjugates can be bound without affecting the basic immunogenicity or toxicity when added to the conjugates of the invention. Such carriers are preferably mono-to decavalent materials containing multiple functional groups for attaching the polymer. These functional groups may be homogeneous or heterogeneous; but preferably the same kind of functional group.

Examples of commercially available carriers comprising the same type of functional group include, by way of example only, catechol, resorcinol, 1, 2-phenylenediamine, 1, 3-phenylenediamine, 1, 4-phenylenediamine; phthalic acid, isophthalic acid, 1, 3-propanediol, glycerol, 1, 2, 4-benzenetriol, pentaerythritol, glucose (in its pyranose form), 1, 3-phenyl diisocyanate, 1, 4-phenyl diisocyanate, isophthalaldehyde, phthalaldehyde, 1, 3-cyclopentanediol, ethylenediamine tetraacetic acid, and the like.

Representative structures for use as carriers or scaffolds include the following (where PEG is used for exemplary purposes only):

Tri-PEG-Glycerol

tetra-PEG-pentaerythritol

bis-PEG resorcinol

Examples of commercially available carriers comprising heterologous functional groups include, by way of example only, 6-hydroxyhexanoic acid, amino acids, salicylic acid, 3-or 4-aminosalicylic acid, 1, 3-diamino-2-hydroxypropane, 2-aminoethanol, 3-aminopropanol, glucosamine, sialic acid, amino acids, and the like.

Representative structures produced by conjugation of polymers using these carriers or scaffolds include the following (where PEG is used for exemplary purposes only):

Tri-PEG-1, 3-diamino-2-hydroxypropane

bis-PEG 3-aminophenol

The carrier may optionally contain one or more copies of a optionally linked to the carrier by a linking group, provided that at least one further functional group may be bound to another copy of a. For example, each of the following structures is considered a support or scaffold because there is also at least one additional functional group to bind an additional A substituent:

wherein L, w and A are as defined above.

The term "oxyalkylene" means-OCH₂CHR^d-, wherein R^dIs an alkyl group. By polymeric alkylene oxide is meant a polyoxyalkylene, polyalkylene oxide or polyalkylene glycol, non-limiting examples of which include PEG, polypropylene glycol, polybutylene glycol, polyisopropylene glycol and the like.

These polymers are optionally mono-terminated with a substituent preferably selected from the group consisting of alkyl, aryl, substituted alkyl, substituted aryl and the supports described above. Also included in these polymers are diamino terminated polyoxyalkylene polymers known in the art as. These polymers may still further beOptionally containing one or more non-oxyalkylene units such as commercially available poly [ di (ethylene glycol) adipate, poly [ di (ethylene glycol) phthalate glycol and the like. Also included are block copolymers of alkylene oxide, polyethylene glycol, polypropylene glycol, and polyoxyethylenated polyol units.

Polyoxyalkylenes, such as PEG, are typically provided as water-soluble waxy solids. Generally, as the molecular weight of a polymer increases, its viscosity and freezing point also increase. Commercial formulations are generally characterized by the "average molecular weight" of the polymer component.

The average molecular weight of the total amount of polymer from the single or multiple polymer moieties in the conjugates of the invention is generally between about 100 and 100,000; preferably about 10,000 to 80,000; more preferably from about 20,000 to about 70,000.

Also, other suitable polymers, such as polyvinylpyrrolidone (PVP), polyacrylamide (PAAm), polydimethylacrylamide (PDAAm), polyvinyl alcohol (PVA), dextrose, poly (L-glutamic acid) (PGA), Styrene Maleic Anhydride (SMA), poly-N- (2-hydroxypropyl) methacrylamide (HPMA), polydivinyl ether maleic anhydride (divma), are well known in the art, having a molecular weight of about 100 to 100,000; preferably from about 10,000 to 80,000; more preferably from about 20,000 to about 70,000.

"pharmaceutically acceptable salt" refers to salts that retain the biological effects and properties of the compounds of the present invention and which do not have biological or other undesirable properties. In many cases, the compounds of the invention are capable of forming acidic and/or basic salts due to the presence of amino and/or carboxyl groups or groups analogous thereto.

Pharmaceutically acceptable basic addition salts can be prepared from inorganic and organic bases. Salts from inorganic bases include, by way of example only: sodium, potassium, lithium, ammonium, calcium, and magnesium salts. Salts from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, such as alkylamines, dialkylamines, trialkylamines, substituted alkylamines, di (substituted alkyl) amines, tri (substituted alkyl) amines, alkenylamines, dienylamines, trienylamines, substituted alkenylamines, di (substituted alkenyl) amines, tri (substituted alkenyl) amines, cycloalkylamines, di (cycloalkyl) amines, tri (cycloalkyl) amines, substituted cycloalkylamines, di-substituted cycloalkylamines, tri-substituted cycloalkylamines, cycloalkenylamines, di (cycloalkenyl) amines, tri (cycloalkenyl) amines, substituted cycloalkenylamines, di-substituted cycloalkenylamines, tri-substituted cycloalkenylamines, arylamines, diarylamines, triarylamines, heteroarylamines, diheteroarylamines, heterocyclylamines, diheterocyclic amines, tri-heterocyclic amines, mixed di-and tri-amines in which at least two of the substituents on the amines are different, the substituents are selected from the group consisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, heteroaryl, heterocycle, and the like. Also included are amines in which two or three substituents together with the amino nitrogen form a heterocycle or heteroaryl.

Examples of suitable amines include, by way of example only, isopropylamine, trimethylamine, diethylamine, tri (isopropyl) amine, tri (N-propyl) amine, ethanolamine, 2-dimethylaminoethanol, tromethamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, N-alkylglucosamine, theobromine, purine, piperazine, piperidine, morpholine, N-ethylpiperidine, and the like. It is also understood that other carboxylic acid derivatives, such as carboxylic acid amides, including carboxamides, lower alkyl carboxamides, dialkyl carboxamides, and the like, may also be used in the practice of the present invention.

Pharmaceutically acceptable acid addition salts may be prepared from inorganic and organic acids. Salts from inorganic acids include hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, and the like. Salts from organic acids include acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like.

The term "pharmaceutically acceptable cation" refers to a cation of a pharmaceutically acceptable salt.

It is to be understood that, among all substituents defined herein, a polymer obtained with a substituent defined with a substituent further substituted as such (e.g., with a substituted aryl group having a substituted aryl group as a substituent, i.e., itself substituted with a substituted aryl group, etc.) is not intended herein to be included. In this case, the maximum number of such substituents is 3. That is, each of the above definitions is subject to certain limitations, e.g., substituted aryl is defined as-substituted aryl- (substituted aryl).

It is also to be understood that the above definitions are not intended to include impermissible substitution patterns (e.g., methyl substituted with 5 fluoro groups or hydroxyl alpha to an ethylenically or acetylenically unsaturated hydrocarbon). Such impermissible substitution is well known to those skilled in the art.

Preparation of compounds

The starting active compounds used in the process of the present invention can be prepared from readily available starting materials using well known procedures and readily available starting materials, or when starting materials are not known or are not commercially available, such materials can be readily prepared using procedures described in the literature. It is to be understood that where typical or preferred process conditions (i.e., reaction temperatures, times, molar proportions of reactants, solvents, pressures, etc.) are specified, other process conditions may also be used unless otherwise specified. The optimal reaction conditions may vary depending on the particular reactants or solvents used, but these conditions can be determined by one skilled in the art by routine optimization procedures.

In addition, it will be apparent to those skilled in the art that some conventional protecting groups may be required to protect certain functional groups from undesirable reactions. Suitable protecting groups for a wide variety of functional groups and suitable conditions for protecting and deprotecting particular functional groups are well known in the art. For example, a number of Protecting Groups are described in t.w.greene and g.m.wuts, Protecting Groups in Organic Synthesis (Protecting Groups in Organic Synthesis), second edition, Wiley, New York, 1991 and references cited herein.

Furthermore, the compounds of the present invention typically contain one or more chiral centers. Thus, these compounds may be prepared or isolated as pure stereoisomers, i.e., individual enantiomers or diastereomers, or as mixtures enriched in stereoisomers, if desired. Unless otherwise indicated, all such stereoisomers (and enriched mixtures) are included within the scope of the present invention. Pure stereoisomers (or enriched mixtures) may be prepared using, for example, optically active starting materials or stereoselective reagents well known in the art. Alternatively, racemic mixtures of these compounds can be separated using, for example, chiral column chromatography, chiral solvents, and the like.

Conjugates prepared according to the present invention preferably comprise a polymer moiety containing from about 1to about 100 substituents of formula II:

in particular, the polymer moiety may be covalently bonded to Ar¹Substituent, R substituent, Ar²Substituents are attached and/or linked within the T substituents, wherein the polymer moiety may be directly linked or linked via a linking group. In turn, the polymer moiety can optionally be bound to a carrier to which multiple copies of the polymer are attached.

The compounds of formula II can be prepared by first linking the heterocyclic amino acid 1 with the appropriate arylsulfonyl chloride as shown in scheme I below:

FIG. 1 schematically shows

R, Ar therein¹X, m and n are as defined above.

Specifically, in scheme 1 above, heterocyclic amino acids1(ii) an equivalent or excess (preferably about 1.1 to about 2 equivalents) of a stoichiometric amount of an arylsulfonyl halide2Mixing is carried out in a suitable inert diluent such as methylene chloride and the like. Typically the reaction is carried out at a temperature in the range of from about-70 ℃ to about 40 ℃ until the reaction is substantially complete, typically within 1to 24 hours. Preferably, the reaction is carried out in the presence of a suitable base to scavenge the acid produced in the reaction. Suitable bases include, by way of example, tertiary amines such as triethylamine, diisopropylethylamine, N-methyl-morpholine, and the like. Alternatively, the reaction is carried out under Schotten-Baumann-type conditions using an aqueous alkaline solution (e.g., aqueous sodium hydroxide solution, aqueous phosphate buffered to pH7.4, and the like). The product obtained3It can be recovered by conventional methods such as chromatography, filtration, evaporation, crystallization and the like or, alternatively, used in the next step without purification and/or separation.

The heterocyclic amino acid 1 used in the above reaction is a known compound or a compound which can be prepared from a known compound by a conventional synthetic procedure. Examples of suitable amino acids for use in this reaction include, but are not limited to, L-proline, trans-4-hydroxy-L-proline, cis-4-hydroxy-L-proline, trans-3-phenyl-L-proline, cis-3-phenyl-L-proline, L- (2-methyl) proline, L-pipecolic acid, L-azetidine-2-carboxylic acid, L-thiazolidine-4-carboxylic acid, L- (5, 5-dimethyl) thiazolidine-4-carboxylic acid, L-thiomorpholine-3-carboxylic acid. If desired, amino acids1The corresponding carboxylic acid esters of (a), such as methyl, ethyl, t-butyl and the like, may be used with arylsulfonyl chlorides in the above-described reactions. The ester group is then hydrolyzed to the carboxylic acid using conventional reagents and conditions, i.e., treatment with an alkali metal hydroxide in an inert diluent (e.g., methanol/water) then affords the N-sulfonylamino acid3。

Also, in the above-mentioned opposite directionArylsulfonyl chloride to be used therein2Are known compounds or may be prepared from known compounds by conventional synthetic procedures. Such compounds are generally derived from the corresponding sulfonic acids, i.e. from compounds of the formula Ar¹SO₃H compound (wherein Ar is¹As defined above) was prepared using phosphorus trichloride and phosphorus pentachloride. The reaction is generally carried out by contacting the sulfonic acid with about 2 to 5 molar equivalents of phosphorus trichloride and phosphorus pentachloride in a solvent-free or inert solvent (e.g., methylene chloride) at a temperature in the range of about 0 ℃ to about 80 ℃ for about 1to about 48 hours to provide the sulfonyl chloride. Alternatively, arylsulfonyl chlorides2From the corresponding thiol compounds, i.e. Ar¹-SH compound (wherein Ar¹As defined herein) by the use of chlorine (Cl)₂) And water under conventional reaction conditions.

Alternatively, arylsulfonyl chlorides used in the above-mentioned reactions2May be prepared by using Cl-SO₃H is prepared by acylating substituted benzene or heterocyclic alkyl chlorosulfonyl.

Examples of arylsulfonyl chlorides suitable for use in the present invention include, but are not limited to, benzenesulfonyl chloride, 1-naphthalenesulfonyl chloride, 2-naphthalenesulfonyl chloride, p-toluenesulfonyl chloride, o-toluenesulfonyl chloride, 4-acetamidobenzenesulfonyl chloride, 4-tert-butylbenzenesulfonyl chloride, 4-bromobenzenesulfonyl chloride, 2-carboxybenzenesulfonyl chloride, 4-cyanobenzenesulfonyl chloride, 3, 4-dichlorobenzenesulfonyl chloride, 3, 5-dichlorobenzenesulfonyl chloride, 3, 4-dimethoxybenzenesulfonyl chloride, 3, 5-bistrifluoromethylbenzenesulfonyl chloride, 4-fluorobenzenesulfonyl chloride, 4-methoxybenzenesulfonyl chloride, 2-methoxycarbonylbenzenesulfonyl chloride, 4-methylamino-benzenesulfonyl chloride, 4-nitrobenzenesulfonyl chloride, 4-trifluoromethyl-benzenesulfonyl chloride, 4-trifluoromethoxy-benzenesulfonyl chloride, 4-trifluoromethyl, 2, 4, 6-trimethylbenzenesulfonyl chloride, 2-thiophenesulfonyl chloride, 5-chloro-2-thiophenesulfonyl chloride, 2, 5-dichloro-4-thiophenesulfonyl chloride, 2-thiazolesulfonyl chloride, 2-methyl-4-thiazolesulfonyl chloride, 1-methyl-4-imidazolesulfonyl chloride, 1-methyl-4-pyrazolesulfonyl chloride, 5-chloro-1, 3-dimethyl-4-pyrazolesulfonyl chloride, 3-pyridinesulfonyl chloride, 2-pyrimidinesulfonyl chloride and the like. If desired, sulfonyl fluorides, bromides or anhydrides may be used in place of sulfonyl chlorides in the above reactionsTo form N-sulphonylamino acids3。

Then, as shown in scheme 2 below, N-arylsulfonyl amino acids3Coupling with commercially available tyrosinates:

FIG. 2 is a schematic view of

R, Ar therein¹X, m and n are as defined above, R^aIs hydrogen or alkyl, but preferably alkyl such as tert-butyl, Z represents an optional substituent on the aromatic ring, and o is 0,1 or 2.

This coupling reaction is generally carried out using well-known coupling agents such as carbodiimides, BOP reagent (benzotriazol-1-yloxy-tris (dimethylamino) -phosphino hexafluorophosphonate) and the like. Examples of suitable carbodiimides include Dicyclohexylcarbodiimide (DCC), 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC), and the like. Polymer-supported forms of carbodiimide coupling agents may also be used if desired, including, for example, those described in tetrahedron letters, 34(48), 7685 (1993). In addition, known coupling cocatalysts, such as N-hydroxysuccinimide, 1-hydroxybenzotriazole and the like, may be used to accelerate the coupling reaction.

Such coupling reactions are typically carried out by reacting N-sulfonyl amino acids3With about 1to about 2 equivalents of coupling agent and at least 1 equivalent, preferably about 1to about 1.2 equivalents of tyrosine derivative4In an inert diluent such as methylene chloride, chloroform, acetonitrile, tetrahydrofuran, N-dimethylformamide and the like. Typically, this reaction is carried out at a temperature in the range of about 0 ℃ to about 37 ℃ for about 12 to about 24 hours. After completion of the reaction, the compound is recovered by conventional methods including neutralization, evaporation, extraction, precipitation, chromatography, filtration and the like5。

Or, N-sulfonylaminoAcid(s)3Can be converted into an acidic halide which is then reacted with a compound4Coupling to give the compound5. The acid halide can be prepared by reacting a compound3With an inorganic acid halide such as thionyl chloride, phosphorus trichloride, phosphorus tribromide or phosphorus pentachloride, or preferably oxalyl chloride, under conventional conditions. This reaction is typically carried out using about 1to 5 molar equivalents of an inorganic acid halide or oxalyl chloride in the absence of a solvent or in an inert solvent (such as methylene chloride or carbon tetrachloride) at a temperature in the range of about 0 ℃ to about 80 ℃ for about 1to about 48 hours. A catalyst such as DMF may also be used in the reaction.

Then N-sulfonylamino acids3With at least 1 equivalent, preferably from about 1.1 to about 1.5 equivalents, of the tyrosine derivative4In an inert diluent such as methylene chloride, at a temperature in the range of about-70 ℃ to about 40 ℃ for about 1to about 24 hours. Preferably, the reaction is carried out in the presence of a suitable base to scavenge the acid produced during the reaction. Examples of suitable bases include tertiary amines such as triethylamine, diisopropylethylamine, N-methylmorpholine, and the like. Alternatively, the reaction may be carried out under Schotten-Baumann type conditions using an aqueous alkaline solution (e.g., sodium hydroxide and the like). After completion of the reaction, the compound is recovered by conventional methods including neutralization, evaporation, extraction, precipitation, chromatography, filtration and the like5。

Alternatively, the derivatives can be prepared by first forming the diamino acid derivative and then reacting the diamino acid with an aryl sulfonyl halide2Coupling preparation of compounds5As shown in schematic 3 below;

schematic diagram 3

R, R therein^a、Ar¹X, Z, m, n and o are as defined above.

Diamino acids6Is easy to openReducing amino acid1With amino acids4Prepared using conventional amino acid coupling techniques and reagents (e.g., carbodiimide, BOP reagents, and the like) coupling as described above. Sulfonyl chloride may then be used2Sulfonated diamino acids6And adopting the above-mentioned synthesis steps to obtain the compound7。

Tyrosine derivatives for use in the above reactions4Are known compounds or may be prepared from known compounds by conventional synthetic procedures. For example, tyrosine derivatives suitable for use in the above reactions4Including, but not limited to, L-tyrosine methyl ester, L-tyrosine tert-butyl ester, L-3, 5-diiodotyrosine methyl ester, L-3-iodotyrosine methyl ester, beta- (4-hydroxy-naphthalen-1-yl) -L-alanine methyl ester, beta- (6-hydroxy-naphthalen-2-yl) -L-alanine methyl ester, and the like. Other esters or amides of the above compounds may of course be used if desired.

N-arylsulfonyl-heterocyclic amino acid-tyrosine derivatives7Can be used as a starting point for attaching a polymer moiety to Ar by using the method of the present invention²On the radical.

Amine moieties located on other parts of the molecule can be used in the manner described above to covalently attach polymer groups to the molecule. For example, at Ar¹On a heterocyclic amino acid or Ar²The above amines, may likewise be derivatized for PEG substitution using the methods of the invention. Amine moieties may be included in these substituents during synthesis, if appropriate protected. Alternatively, amine precursors may be used.

In addition, amino substituents can be added to the heterocyclic amino acid functionality and then derivatized to include a polymer moiety. For example, it is described in U.S. Pat. No. 6,489,300 that the heterocyclic amino acid functional group can be 2-carboxypiperazine. Alternatively, commercially available 3-or 4-hydroxyproline may be oxidized to the corresponding ketone and then reductively aminated with aqueous ammonia in the presence of sodium cyanoborohydride to form the corresponding amine moiety. Still further, 4-cyanoproline can be reduced to give the formula-CH₂NH₂By a substituted alkyl group ofThe amine is derivatized.

Still further, an amine moiety may be added to Ar²Among the functional groups. Preferably, the amine moiety is present as an amine precursor, e.g. with Ar²Attached nitro or cyano groups.

The non-derivatised compounds of formulae IIa to IIh are then coupled to the polymer using the method of the present invention. Schematic figure 4 depicts an embodiment of the invention:

FIG. 4 is a schematic view of

In scheme 9, in phosphine (here PPh)₃) And azodicarboxylic acid esters (e.g. diisopropyl azodicarboxylate), PEG alcohols100With nucleophilic reagents105Treatment to form protected esters110. The ester is then hydrolyzed to form the desired conjugate115。

The reaction takes place under mild and substantially neutral conditions. The reaction is preferably carried out in at least one suitable solvent. Examples include halogenated solvents such as dichloromethane, aromatic hydrocarbon solvents such as benzene or toluene, or ether solvents such as tetrahydrofuran and diethyl ether. Other suitable solvents include ethyl acetate, acetonitrile and DMF. Most preferably, chlorinated solvents or ether solvents are used. In a most preferred embodiment, the solvent is dichloromethane or tetrahydrofuran.

The reaction temperature is generally in the range of about-100 to 100 deg.C, preferably in the range of about-20 to 50 deg.C, and even more preferably in the range of from about 0 deg.C to about room temperature. In a particularly preferred embodiment, the reaction temperature is between about-10 and 10 ℃.

The reaction time ranges from about 5 minutes to about 100 hours, preferably from about 30 minutes to about 50 hours. More preferably, the reaction is carried out between about 45 minutes and about 10 hours to completion.

As mentioned above, examples of triaryl-or trialkylphosphines include triphenylphosphine, trimethylphosphine, triethylphosphine, tributylphosphine and 1, 2-bis- (diphenylphosphino) ethane. The phosphine may also be polymer-supported or water-soluble. The preferred triarylphosphine is triphenylphosphine.

Examples of azo compounds are diethyl azodicarboxylate, diisopropyl azodicarboxylate, 4-methyl-1, 2, 4-triazolidine-3, 5-dione, N, N, N ', N' -tetramethylazodicarboxamide, dipiperidine azodicarboxylate, bis (N-4-methylpiperazin-1-yl) azodicarboxamide, dimorpholinoazodicarboxylamide and di-tert-butyl azodicarboxylate.

It is understood that other suitable polymeric alcohols may be used in place of PEG, and one of ordinary skill in the art will be readily able to modify the reaction scheme below to add these other polymers. In some cases, the PEG moiety may be introduced directly to Ar²On a group, and in other cases, a PEG moiety can be introduced through attachment of a linking group moiety.

Other polymers suitable for conjugation with the compound of formula II include, but are not limited to, polyvinylpyrrolidone (PVP), polyacrylamide (PAAm), polydimethylacrylamide (PDAAm), polyvinyl alcohol (PVA), dextrose, poly (L-glutamic acid) (PGA), Styrene Maleic Anhydride (SMA), poly-N- (2-hydroxypropyl) methacrylamide (HPMA), polydivinyl ether maleic anhydride (divma). By way of example, PVP, PAAm and PDAAm may be functionalized by the introduction of comonomers during free radical polymerization. Both PVA and dextrose contain primary hydroxyl groups (OH) suitable for conjugation. Methods for the synthesis of these biopolymers and methods for conjugating them to biological substances are well known in the art (see, e.g., published U.S. patent application No. 20040043030; U.S. patent No. 5,177,059; U.S. patent No. 6,716,821; U.S. patent No. 5,824,701; U.S. patent No. 6,664,331; U.S. patent No. 5,880,131; Kameda, Y. et al, Biomaterials 25: 3259. 3266, 2004; Thanou, M. et al, Current Opinion in Investigational Drugs4 (6): 701-.

Representative polymers suitable for use in the present invention include:

HO (alkylene-O)_ppR^bb	Mono-capped monohydroxy PEG (mPEG)
HO (alkylene-O)_ppR^bb	Mono-capped monohydroxy PEG (mPEG)	H₂N (alkylene-O)_ppR^bb	Mono-capped monoamino PEG

HO (alkylene-O)_ppR-OH	Uncapped dihydroxy PEG
HO (alkylene-O)_ppR-OH	Uncapped dihydroxy PEG	H₂N (alkylene-O)_ppR-OH	Uncapped monoamino PEG
HO (alkylene-O)_ppR^bb	Branched monohydroxy PEG	H₂N (alkylene-O)_ppR-OH	Uncapped monoamino PEG
HO (alkylene-O)_ppR^bb	Branched monohydroxy PEG	HO (alkylene-O)_ppR^bb	Dendritic monohydroxy PEG

Wherein pp and alkylene are as defined herein, R^bbPreferably selected from alkyl, substituted alkyl, aryl and substituted aryl.

Other suitable polymers are shown below:

mono-capped monohydroxy PEG (mPEG)

Mono-capped monoamino PEG

Uncapped dihydroxy PEG

Branched PEG:

PEG reagent (20kDa 4-arm) from NOF

20kDa 4-arm PEG tetramine

Diglycerol core

Cat#Sunbright DG-200PA

PEG reagent (40kDa 8-arm) from Nektar

40kDa 8-arm PEG

Hexaglycerol core

Cat#0 J000T08

Dendritic PEG:

PEG reagent (40kDa 4-arm) from NOF

40kDa 4-arm PEG alcohol 40kDa 4-arm PEG tetramine

Pentaerythritol core

cat.#Sunbright PTE-40000 cat.#Sunbright PTE-400PA

PEG reagent (40kDa 3-arm) from NOF

40kDa 3-arm PEG 40kDa 3-arm PEG triamine

Glycerol core

cat#Sunbright GL-40000 cat#Sunbright GL-400PA

PEG reagents (40 and 20kDa) from SunBio

Y-PEG series (aspartic acid core)

Y-PEG amine (40kDa) Y-PEG nitrophenyl alcohol carbamate (40kDa)

Cat# PYAM-40 Cat# PYNPC-40

6-arm series (sorbitol core)

Lower molecules available in the sorbitol 6-arm series

Amounts include 10, 15 and 20 kDa. Derivatisation other than alcohols

The molecule is conjugated.

40kDa 6-arm PEG

Customized product

These PEG polymers may be further modified by chain extension with PEG diamines via appropriate linking groups, such as urethane or urea.

PEG diamine

A variety of nucleophilic compounds having an acidic hydrogen can be used in the process of the present invention. These compounds may be biologically active compounds, i.e. therapeutic compounds (drugs) and agricultural chemicals (pesticides, herbicides, and plant growth stimulants such as fertilizers) or food additive compounds. Examples of the group having an acidic nitrogen which can be added to these compounds are shown above; see the structures listed in tables D and D1.

Pharmaceutical formulations

When used as a medicament, the conjugates of the invention are typically administered in the form of a pharmaceutical composition. These conjugates can be administered by a variety of routes including oral, rectal, transdermal, subcutaneous, intravenous, intramuscular, sublingual, ophthalmic, or inhalation (including administration by nasal or oral inhalation). Preferred routes of administration include subcutaneous, intravenous and inhalation. These compositions may be prepared in a manner well known in the pharmaceutical art and include at least one conjugate.

The invention also provides conjugates comprising a conjugate according to the invention, e.g. of formula I, and₄β₇pharmaceutical compositions of the inhibitor alone in combination. These compositions also include a pharmaceutically acceptable carrier or excipient and can be administered as described elsewhere herein.

The invention also includes pharmaceutical compositions comprising one or more conjugates of formula I as active ingredients and a pharmaceutically acceptable carrier. In preparing the compositions of the present invention, the active ingredient is generally mixed with an excipient, diluted with an excipient or enclosed within a carrier, which may be in the form of sterile injectable solutions or sterile packaged powders. For subcutaneous administration, simple carriers may include water, Na₂HPO₄、NaH₂PO₄And NaCl in a sterile solution in a ratio to provide an isotonic and physiologically acceptable pH, known as PBS or phosphate buffered saline. Other options are known to those skilled in the art, including mixed solvent systems, which can affect the rate of absorption and the total amount of contact. These options include a mixed solvent system containing glycerol, polyethylene glycol 400 and cottonseed oil. Ethanol, N' -dimethylacetamide, propylene glycol and benzyl alcohol, which can be used to manipulate permeability increase and hypertonicity, are also possible.

During the preparation of the formulation, it may be necessary to grind the active compound to obtain a suitable particle size before mixing with the other ingredients. If the active compound is substantially insoluble, it is usually ground to a particle size of less than 200 mesh. If the active compound is substantially water soluble, the particle size is generally adjusted by milling, for example to about 40 mesh, so that it is substantially uniformly distributed in the formulation.

Some examples of suitable excipients include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, and methyl cellulose. The formulation may additionally comprise: lubricants, such as talc, magnesium stearate and mineral oil; a humectant; emulsifying agents and suspending agents; preservatives, such as methyl-and propylhydroxy-benzoate; a sweetener; and a flavoring agent. The compositions of the present invention may be formulated so that the active ingredient is released rapidly, permanently or with a delay after administration to the patient by methods known in the art.

The administration of therapeutic agents by subcutaneous or intravenous formulation is well known in the pharmaceutical industry. Subcutaneous or intravenous formulations should have certain properties in addition to being soluble in the therapeutic drug in the composition. For example, the dosage form should promote overall stability of the active ingredient, and the formulation should also be cost-effective to manufacture. All of these factors ultimately determine the overall success and utility of the intravenous formulation.

Other auxiliary additives which may be included in the pharmaceutical formulations of the compounds of the present invention are the following: solvent: ethanol, glycerol, propylene glycol; a stabilizer: EDTA (ethylenediaminetetraacetic acid), citric acid; antibacterial preservative: benzyl alcohol, methyl paraben, propyl paraben; buffering agent: citric acid/sodium citrate, potassium hydrogen tartrate, sodium hydrogen tartrate, acetic acid/sodium acetate, maleic acid/sodium maleate, sodium hydrogen phthalate, phosphoric acid/potassium dihydrogen phosphate, phosphoric acid/disodium hydrogen phosphate; and a tonicity modifier: sodium chloride, mannitol, dextrose.

The presence of a buffer is required to maintain the aqueous solution pH in the range of about 4 to about 8, more preferably in the range of about 4 to about 6. The buffer system is typically a mixture of weak acids and soluble salts thereof, such as sodium citrate/citric acid; or mono-or dicationic salts of dibasic acids, for example, potassium hydrogen tartrate; sodium hydrogen tartrate, phosphoric acid/potassium dihydrogen phosphate, and phosphoric acid/disodium hydrogen phosphate.

The amount of buffer system used depends on (1) the desired pH; and (2) the amount of drug. The amount of buffer generally used is the buffer in the formulation: alendronate (where the moles of buffer are taken from the combined moles of buffer components, e.g. sodium citrate and citric acid) is used in a molar ratio of 0.5: 1to 50: 1to maintain the pH in the range of 4 to 8, typically in a molar ratio of buffer (combined) to drug present of 1: 1to 10: 1.

One buffer useful in the present invention is sodium citrate/citric acid in the range of 5 to 50mg citric acid per ml to 1to 15mg citric acid per ml sufficient to maintain the pH of the aqueous composition at 4-6.

Buffers may also be present to prevent precipitation of the drug by forming soluble metal complexes with dissolved metal ions (e.g., Ca, Mg, Fe, Al, Ba), which may have evolved from glass containers or rubber stoppers or be present in ordinary tap water. Such agents may act as competitive complexing agents for the drug and produce soluble metal complexes that produce unwanted particles.

In addition, the presence of an agent (e.g., sodium chloride at about 1-8 mg/ml) may be required to adjust the tonicity to the same value as human blood to avoid adverse side effects of red blood cell swelling or contraction, such as nausea or diarrhea and possibly related blood disorders, that occur during administration of the intravenous formulation. Typically, the tonicity of the formulation is matched to human blood and is in the range 282 to 288mOsm/kg, typically 285mOsm/kg, which corresponds to the osmotic pressure of a 0.9% sodium chloride solution.

The intravenous formulation may be administered by direct intravenous injection, intravenous bolus drip, or may be administered by infusion into a suitable infusion solution, such as 0.9% sodium chloride injection or other compatible infusion solution.

The compositions are preferably formulated in unit dosage forms containing from about 5 to about 100mg, more commonly from about 10 to about 30mg, of the active ingredient per dose. The term "unit dosage form" refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient.

The conjugate is effective over a wide dosage range and is usually administered in a pharmaceutically effective amount. It is to be understood, however, that the amount of conjugate actually administered will be determined by a physician, in the light of the relevant circumstances, including the condition to be treated, the chosen route of administration, the actual compound administered, the age, weight, and response of each patient, the severity of the patient's symptoms, and the like.

To prepare solid compositions, such as tablets, the principal active ingredient is mixed with a pharmaceutical excipient to form a solid preformulation composition containing a homogeneous mixture of a compound of the present invention. When referring to these preformulation compositions as homogeneous, it is meant that the active ingredient is dispersed evenly throughout the composition so that the composition can be readily subdivided into equally effective unit dosage forms such as tablets, pills and capsules. The solid pre-formulation is then subdivided into unit dosage forms of the type described above containing, for example, from 0.1 to about 500mg of the active ingredient of the invention.

The tablets or pills of the invention may be coated or otherwise formulated into a compound dosage form to provide long-lasting benefits. For example, a tablet or pill may include both an internal dosage form and an external dosage form component, the latter being in the form of a coating on the former. The two components may be separated by an enteric layer (enteric layer) which serves to resist disintegration in the stomach, leaving the inner component intact in the duodenum or delayed in release. A variety of materials may be used for such enteric layers or coatings, including a wide variety of polymeric acids, and mixtures of polymeric acids with materials such as shellac, cetyl alcohol and cellulose acetate.

Liquid dosage forms which may be incorporated into the novel compositions of the present invention for oral or injectable administration include aqueous syrups, aqueous or oily suspensions of suitable taste, and flavored emulsions with edible oils such as cottonseed oil, sesame oil, coconut oil or peanut oil, as well as elixirs and similar pharmaceutical vehicles.

Compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable aqueous or organic solvents, or mixtures thereof, and powders. The liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as previously described. Preferably, the composition is administered by the oral or nasal inhalation route to exert local or systemic effects. Compositions in preferred pharmaceutically acceptable solvents may be administered by spraying using an inert gas. The nebulized solution may be inhaled directly from the nebulizing device, or the nebulizing device may be attached to a mask vent or intermittent positive pressure ventilator. The solution, suspension or powder composition may be administered, preferably orally or nasally, from a device that delivers the formulation in a suitable manner. For administration by inhalation or insufflation, the total molecular weight of the conjugate is preferably between about 10,000 daltons and 70,000 daltons, more preferably between about 20,000 daltons and 45,000 daltons.

Polymer conjugates

The compounds of the invention formulated and administered as above are polymer conjugates. Polymer conjugates are expected to provide many benefits over non-conjugated polymers, such as increased solubility and in vivo stability.

As such, a single polymer molecule may be used to conjugate the compounds of the invention, although it is also contemplated that more than one polymer molecule may be attached, typically via a carrier. The conjugated compounds of the present invention find utility in both in vivo and non-in vivo applications. In addition, it is to be appreciated that other groups, moieties, or other conjugated species may be used in the conjugated polymer depending on the end application. As an example, it may be beneficial in some applications to functionalize the polymer to render it reactive and to enable it to be conjugated with compounds of formula II to enhance various properties or characteristics of the total conjugated material. Thus, the polymer may contain any functional group, repeating group, linkage, or other constitutive structure that does not interfere with the intended use of the conjugated compounds of the present invention.

Illustrative polymers for achieving these desired characteristics are described above, and in PCT WO01/54690(Zheng et al), which is incorporated herein by reference in its entirety. The polymer may be linked (preferably via a linker moiety) to a compound of the invention to form a stable bond that is not significantly cleaved by human enzymes. The proportion of bonds connecting the polymer and the compound of the invention attached to the polymer that need to be cleaved within 24 hours, as determined by techniques standard in the art, including but not limited to High Pressure Liquid Chromatography (HPLC), will generally not exceed 20% for bonds that are not "significantly" cleaved.

Typically, the compounds of the invention contain at least about two compounds of formula II attached to a polymer. The final amount is balanced between maximizing the reaction and minimizing non-specific modification of the product, while specifying that the chemical entity maintains optimal activity while optimizing the half-life of the compounds of the invention. Preferably, at least about 50% of the biological activity of the compounds of the invention is retained, more preferably 100%.

As noted above in the preferred practice of the invention, C is advantageously added to the target polymer system₂-C₄The polyalkylene glycol residues of alkylpolyalkylene glycols, preferably polyethylene glycol (PEG) or poly (oxy) alkylene glycol residues of these ethylene glycols. Thus, the polymer to which the compounds of the present invention are attached can be a homopolymer of polyethylene glycol (PEG) or a polyoxyethylenated polyol, provided that the polymer is soluble in water at room temperature in all cases. Non-limiting examples of such polymers include polyalkylene oxide homopolymers such as PEG or polypropylene glycols, polyoxyethylenated glycols, copolymers thereof, and block copolymers thereof, provided that the water solubility of the block copolymer is retained.

Examples of polyoxyethylated polyols include, but are not limited to, polyoxyethylated glycerol, polyoxyethylated sorbitol, polyoxyethylated glucose, or the like. The glycerol backbone of polyoxyethylated glycerol is the same as the natural backbone, for example in animal and human mono-, di-and triglycerides. Thus, such branches may not necessarily be considered foreign in vivo.

It will be understood by those of ordinary skill in the art that the foregoing list is illustrative only, and that all polymeric materials having the properties described herein are contemplated. The polymer need not have a particular molecular weight, but preferably has a molecular weight of between about 100 and 100,000, preferably about 10,000 to 80,000; more preferably from about 20,000 to about 70,000. Especially polymers of size 20,000 or more are most effective in preventing product loss due to renal filtration.

PEG derivatives refer to polyethylene glycol polymers in which one or both terminal hydroxyl groups on the polyethylene glycol itself have been modified. Examples of suitable modifications include replacing one or both of the hydroxyl groups with additional functional groups, which may or may not be protected with low molecular weight ligands or other macromolecules or polymers. Modification of the terminal hydroxyl groups of polyethylene glycol can be accomplished by reacting the polyethylene glycol with a compound that includes complementary reactive functional groups, including functional groups that react with the hydroxyl groups in the polyethylene glycol. PEG derivatives of the compounds of the invention may contain one or more polyethylene glycol (PEG) substituents covalently linked thereto via a linking group.

The following formulation examples illustrate the pharmaceutical compositions of the present invention.

Formulation example 1

Hard gelatin capsules were prepared containing the following ingredients:

quality of

Ingredients (mg/capsule)

Active ingredient 30.0

Starch 305.0

Magnesium stearate 5.0

The above ingredients were mixed together and filled into hard gelatin capsules in a mass of 340 mg.

Formulation example 2

Tablets were prepared using the following ingredients:

quality of

Ingredients (mg/tablet)

Active ingredient 25.0

Cellulose, microcrystalline 200.0

Colloidal silica 10.0

Stearic acid 5.0

These ingredients were mixed and tableted to give tablets, each weighing 240 mg.

Formulation example 3

Preparing a dry powder inhalation formulation comprising:

the weight of the ingredients

Active ingredient 5

Lactose 95

The active ingredient is mixed with lactose and the mixture is added to a dry powder inhalation device.

Formulation example 4

Tablets each containing 30mg of active ingredient were prepared as follows:

quality of

Ingredients (mg/tablet)

Active ingredient 30.0mg

Starch 45.0mg

Microcrystalline cellulose 35.0mg

Polyvinylpyrrolidone 4.0mg

(10% solution in sterile water)

Sodium carboxymethyl starch 4.5mg

Magnesium stearate 0.5mg

Talc 1.0mg

A total of 120mg

The active ingredient, starch and cellulose were passed through a 20 mesh u.s. sieve and mixed thoroughly. A solution of polyvinylpyrrolidone was mixed with the resulting powder and then passed through a 16 mesh u.s. sieve. The resulting granules were dried at 50 to 60 ℃ and passed through a 16 mesh u.s. Sodium carboxymethyl starch, magnesium stearate and talc, which had previously passed through a 30 mesh u.s. sieve, were then added to the granules, mixed and then compressed on a tablet press to give tablets weighing 120mg each.

Formulation example 5

Capsules each containing 40mg of drug were prepared as follows:

quality of

Ingredients (mg/capsule)

Active ingredient 40.0mg

Starch 109.0mg

Magnesium stearate 1.0mg

A total of 150.0mg

The active ingredient, starch and magnesium stearate were mixed, passed through a 20 mesh u.s. sieve and filled into hard gelatin capsules in a mass of 150 mg.

Formulation example 6

Each suppository containing 25mg of active ingredient was prepared as follows:

the amount of the components

Active ingredient 25mg

Saturated fatty acid glycerides amounted to 2,000mg

The active ingredient was passed through a 60 mesh u.s. sieve and suspended in saturated fatty acid glycerides (which previously required a small amount of heat to melt them). The mixture was then poured into suppository molds of nominal 2.0g capacity and allowed to cool.

Formulation example 7

Suspensions containing 50mg of drug per 5.0ml dose were prepared as follows:

the amount of the components

Active ingredient 50.0mg

Xanthan gum 4.0mg

Sodium carboxymethylcellulose (11%)

Microcrystalline cellulose (89%) 50.0mg

Sucrose 1.75g

Sodium benzoate 10.0mg

Appropriate amount of flavoring and coloring (q.v.)

Pure water up to 5.0ml

The active ingredient, sucrose and xanthan gum were mixed, passed through a 10 mesh u.s. sieve and then mixed with a solution of microcrystalline cellulose and sodium carboxymethylcellulose previously prepared in water. Sodium benzoate, flavors and colors were diluted with some water, added and stirred. Sufficient water was then added to obtain the desired capacity.

Formulation example 8

Quality of

Ingredients (mg/capsule)

Active ingredient 15.0mg

Starch 407.0mg

Magnesium stearate 3.0mg

A total of 425.0mg

The active ingredient, starch and magnesium stearate were mixed, passed through a 20 mesh u.s. sieve and filled into hard gelatin capsules in a mass of 425.0 mg.

Formulation example 9

Subcutaneous formulations were prepared as follows:

mass of the ingredients

Active ingredient 50mg.mL

Phosphate buffered saline 1.0ml

Formulation example 10

Topical formulations were prepared as follows:

mass of the ingredients

1-10g of active ingredient

Emulsifying wax 30g

20g of liquid paraffin

White vaseline up to 100g

White petrolatum is heated to melt. Adding liquid paraffin and emulsifying wax, and stirring to dissolve. The active ingredient is added and stirring is continued until dispersed. The mixture was then cooled to a solid.

Formulation example 11

Intravenous formulations were prepared as follows:

mass of the ingredients

250mg of active ingredient

Isotonic saline 100ml

Another preferred formulation for use in the method of the present invention employs a transdermal delivery device ("patch"). Such transdermal patches may be used for continuous or discontinuous infusion of the compounds of the present invention in controlled amounts. The construction and use of transdermal patches for delivering agents is well known in the art. See, for example, U.S. Pat. No. 5,023,252 issued to 1991, 6, 11, which is incorporated herein by reference. Such patches may be made to deliver the agent continuously, in pulses, or as needed.

It is often necessary or desirable to introduce pharmaceutical compositions directly or indirectly into the brain. Direct techniques typically involve placing a drug delivery catheter into the ventricular system of the host to bypass the blood brain barrier. One such implantable delivery system for the transport of biological factors to specific anatomical sites in the body is described in U.S. patent No. 5,011,472, which is incorporated herein by reference.

Indirect techniques, which often involve formulating the composition to provide drug latentiation by converting the hydrophilic drug to a lipid soluble drug, are generally preferred. Latency is generally achieved by the following method: the hydroxyl, carbonyl, sulfate and primary amine groups existing on the medicine are sealed, so that the medicine has higher lipid solubility and can be transported through a blood brain barrier. Alternatively, delivery of hydrophilic drugs can be enhanced by intra-arterial infusion of hypertonic solutions, which temporarily opens the blood-brain barrier.

Other dosage forms suitable for use in the present invention may be found in Remington's pharmaceutical Sciences, machine Publishing Company, Philadelphia, Pa., 17 th edition (1985).

As noted above, the compounds described herein are suitable for use in the various drug delivery systems described above. Alternatively, to enhance the serum half-life of the administered compound in vivo, the compound may be encapsulated, introduced into a liposome cavity, formulated as a colloid, or other conventional techniques may be used to extend the serum half-life of the compound. Liposomes can be prepared by a variety of methods, such as those described in U.S. Pat. Nos. 4,235,871, 4,501,728, and 4,837,028 to Szoka et al, each of which is incorporated herein by reference.

Applications of

The conjugates of the invention are VLA-4 antagonists. Some conjugates are also at least on alpha₄β₇Integrins have partial affinity. The conjugates have an extended residence time in vivo compared to non-conjugated compounds. The extended residence time of the conjugate in the body reduces the amount of drug required, thereby resulting in fewer adverse effects and less potential for toxicity. In addition, the pharmaceutical formulation can be administered to a patient fewer times to achieve a similar or greater therapeutic effect.

By suppressing alpha₄β₁Or alpha₄β₇Mediated by binding to cellular receptors (e.g., VCAM-1, fibronectin, and MadCAM), the conjugates of the invention can enhance inhibition of leukocyte adhesion to endothelial cells in vivo. Preferably, the conjugates of the invention may be administered for treatment by alpha-therapy, for example by infusion or by subcutaneous injection or orally₄β₁And/or alpha₄β₇(commonly referred to as leukocyte adhesion) mediated diseases. The conjugates of the invention are useful in the treatment of a variety of inflammatory brain diseases, particularly central nervous system diseases in which endothelial cell/leukocyte adhesion mechanisms cause destruction of healthy brain tissue. Thus, the conjugates of the invention are useful, for example, in the treatment of Experimental Autoimmune Encephalomyelitis (EAE), Multiple Sclerosis (MS), meningitis and encephalitis.

The conjugates of the invention are also useful for treating disorders and diseases caused by tissue damage in other organ systems, i.e., where tissue damage also occurs via adhesion mechanisms leading to leukocyte migration or activation. Examples of such diseases in mammalian patients are inflammatory diseases such as asthma, alzheimer's disease, atherosclerosis, AIDS dementia, diabetes (including acute juvenile onset diabetes), inflammatory bowel disease (including ulcerative colitis and crohn's disease), rheumatoid arthritis, tissue transplant rejection, tumor metastasis, stroke, and other brain trauma, nephritis, retinitis, atopic dermatitis, psoriasis, myocardial ischemia, and leukocyte-mediated acute lung injury such as that which occurs in adult respiratory distress syndrome.

Other diseases treatable using the conjugates of the invention also include erythema nodosum, allergic conjunctivitis, optic neuritis, uveitis, allergic rhinitis, ankylosing spondylitis, psoriatic arthritis, vasculitis, reiter's syndrome, systemic lupus erythematosus, progressive systemic sclerosis, polymyositis, dermatomyositis, wegener's granulomatosis, aortic inflammation, sarcoidosis, lymphopenia, temporal arteritis, pericarditis, myocarditis, congestive heart failure, polyarteritis nodosa, hyperresponsive syndrome, allergy, hypereosinophilic syndrome, allergic granulomatous vasculitis (Churg-Strauss syndrome), chronic obstructive pulmonary disease, allergic pneumonia, chronic active hepatitis, interstitial cystitis, autoimmune endocrine dysfunction, primary biliary cirrhosis, autoimmune aplastic anemia, chronic inflammatory bowel disease, chronic, Chronic persistent hepatitis and thyroiditis.

The invention also provides methods of treating a patient at least partially with alpha₄A method of treating a disease caused or exacerbated by integrin-mediated leukocyte binding, which method comprises co-administering an effective amount of a conjugate of the invention, e.g., a conjugate of formula I, and an effective amount of a conjugate that is α₄β₇Individual compounds of the inhibitor. The co-administration may be simultaneous or sequential. For example, administration of the conjugates of the invention may be preceded by an alpha₄β₇The inhibitor is administered for a few minutes or hours. Or, a₄β₇The inhibitor may be administered prior to the conjugate of the invention.

Suitable in vivo model demonstrating efficacy of treatment of inflammatory responseTypes include EAE (Experimental autoimmune encephalomyelitis) mice, rats, guinea pigs or primates, and others that are alpha dependent₄Inflammatory models of integrins.

Inflammatory bowel disease is a general term for two similar diseases known as crohn's disease and ulcerative colitis. Crohn's disease is a primary, chronic, ulcerative inflammatory disease characterized by well-defined boundaries that generally involve transmural granulomatous inflammatory reactions in all levels of the intestinal wall. Any segment of the gastrointestinal tract from the mouth to the anus can be affected, although the disease most commonly affects the terminal ileum and/or colon. Ulcerative colitis is an inflammatory response primarily localized to the colonic mucosa and submucosa. Within the inflammatory bowel disease pathology are large numbers of lymphocytes and macrophages, which can cause inflammatory damage.

Asthma is characterized by an increased responsiveness of the tracheobronchial tree to various stimuli, causing paroxysmal contractions of the bronchial airways. The stimuli cause the release of a variety of inflammatory mediators from IgE-coated mast cells, including histamine, eosinophils and neutrophiles, leukotrienes, prostaglandins, and platelet activating factor. The release of these factors recruits basophils, eosinophils and neutrophils, causing inflammatory damage.

Atherosclerosis is a disease of the arteries (e.g., coronary, carotid, aorta and iliac). The basic lesion is an atheroma, consisting of a raised focal plaque (focalpareque) within the intima, which has a lipid core and an outer fibrous cap. Atheroma compensates for arterial blood flow, weakening the affected artery. Myocardial and cerebral infarctions are the major consequences of this disease. Macrophages and leukocytes are recruited to the plaque and cause inflammatory damage.

Rheumatoid arthritis is a chronic, recurrent inflammatory disease that causes mainly joint damage and joint destruction. Rheumatoid arthritis usually affects the small joints of the extremities first, but then can involve the wrists, elbows, ankles and knees. Arthritis is caused by the interaction of synovial cells with leukocytes which infiltrate from the circulation into the synovial lining of the joint (synovitis lining). See, e.g., Paul, "Immunology" (3d ed., Raven Press, 1993).

Another indication for the conjugates of the invention is the treatment of organ or transplant rejection mediated by VLA-4. In recent years, the efficiency of surgical techniques for tissue and organ (e.g., skin, kidney, liver, heart, lung, pancreas, and bone marrow) transplantation has increased considerably. Perhaps the main prominent problem is the lack of satisfactory drugs to induce immune tolerance in the recipient to the transplanted allograft or organ. When allogeneic cells or organs are transplanted into a host (i.e., the donor and recipient are different individuals of the same species), the host immune system may mount an immune response to foreign antigens in the transplant (host versus graft disease), causing destruction of the transplanted tissue. CD8⁺Cells, CD4 cells and monocytes are all involved in rejection of transplanted tissue. Conjugates of the invention can be conjugated to alpha₄Integrin binding is particularly useful for blocking alloantigen-induced immune responses in the recipient, thereby preventing these cells from participating in the destruction of the transplanted tissue or organ. See, e.g., Paul et al, Transplant International9, 420-425 (1996); georczynski et al Immunology 87, 573-580 (1996); georcyznski et al, transplant. Immunol.3, 55-61 (1995); yang et al, Transplantation 60, 71-76 (1995); anderson et al, APMIS 102, 23-27 (1994).

A related use of the VLA-4 binding conjugates of the invention is in modulating the immune response involved in "graft versus host" disease (GVHD). See, e.g., Schlegel et al, J.Immunol.155, 3856-. GVHD is a potentially lethal disease that occurs when immunocompetent cells are transferred to allogeneic recipients. In this case, the immunocompetent cells of the donor may attack the tissue in the recipient. Tissues of the skin, intestinal epithelium and liver are frequent targets and can be destroyed during GVHD. This disease presents a particularly serious problem when immune tissue is transplanted, as in bone marrow transplantation; however, it has also been reported that GVHD is less severe in other cases, including heart and liver transplants. The therapeutic agents of the invention are particularly useful in blocking the activation of donor T cells, thereby interfering with their ability to lyse target cells in the host.

The formulations of the invention are particularly useful in the treatment of multiple sclerosis, rheumatoid arthritis and asthma.

A further use of the conjugates of the invention is in the inhibition of tumor metastasis. Several tumor cells have been reported to express VLA-4, and compounds that bind to VLA-4 block adhesion of these cells to endothelial cells. Steinback et al, Urol. Res.23, 175-83 (1995); orosz et al, int.j. cancer 60, 867-71 (1995); freedman et al, Leuk. Lymphoma 13, 47-52 (1994); okahara et al, Cancer Res.54, 3233-6 (1994).

A compound having a desired biological activity can be modified as desired to provide a desired property, such as an increase in a pharmacological property (e.g., in vivo stability, bioavailability), or the ability to be detected in a diagnostic application. Stability can be determined in a variety of ways, such as by determining the half-life of the protein during incubation with peptidases or human plasma or serum. Many such protein stability assays have been described (see, e.g., Verhoef et al, Eur.J.DrugMetab.Pharmacokinet, 1990, 15 (2): 83-93).

A further use of the conjugates of the invention is in the treatment of multiple sclerosis. Multiple sclerosis is a progressive neuroautoimmune disease estimated to be 250,000 to 350,000 patients in the united states. Multiple sclerosis is believed to be the result of a specific autoimmune response in which certain leukocytes attack and cause destruction of myelin, the insulating sheath that covers nerve fibers. In animal models of multiple sclerosis, murine monoclonal antibodies directed against VLA-4 have been shown to block the adhesion of leukocytes to endothelial cells and thus prevent inflammation of the central nervous system and subsequent paralysis of the animal¹⁶。

The pharmaceutical compositions of the present invention are suitable for use in a variety of drug delivery systems. Suitable dosage forms for use in the present invention may be found in Remington's pharmaceutical sciences, machine Publishing Company, Philadelphia, Pa., 17 th edition (1985).

The amount administered to a patient will depend on the substance being administered, the purpose of the administration (e.g., prophylactic or therapeutic), the condition of the patient, the mode of administration, and the like. In therapeutic applications, the compositions may be administered to a patient already suffering from a disease in an amount sufficient to cure or at least partially relieve the symptoms of the disease and its complications. An amount sufficient to achieve this goal is referred to as a "therapeutically effective dose". The amount effective for this purpose will depend on the condition being treated and the judgment of the attending physician in view of factors such as the severity of the inflammation, the age, weight and general condition of the patient, and the like.

The composition administered to the patient may be in the form of a pharmaceutical composition as described above. These compositions may be sterilized by conventional sterilization techniques, or may be sterile filtered. The resulting aqueous solution may be packaged for use, or lyophilized, the lyophilized formulation being combined with a sterile aqueous carrier prior to administration.

The therapeutic dosage of the conjugates of the invention will vary depending, for example, on the particular use being treated, the mode of administration of the conjugate, the health and condition of the patient, and the judgment of the prescribing physician. For example, for intravenous administration, the dosage will generally range from about 20 μ g to about 2000 μ g/kg body weight, preferably from about 20 μ g to about 500 μ g, more preferably from about 100 μ g to about 300 μ g/kg body weight. Suitable dosage ranges for intranasal administration are generally from about 0.1pg to 1mg/kg body weight. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems.

The conjugates of the invention also bind to or antagonize alpha₆β₁、α₉β₁、α₄β₇、α_dβ₂、α_eβ₇Effect of integrin (although alpha)₄β₁And alpha₉β₁Preferred in the present invention). Thus, the conjugates of the invention may also be used to prevent or reverse symptoms, disorders or diseases induced by the binding of these integrins to their respective ligands.

For example, International application WO 98/53817 published at 3.12.1998 (the disclosure of which is incorporated herein by reference in its entirety) and references cited therein describe a₄β₇A mediated disease. This reference also describes the determination of antagonistic alpha₄β₇Assay for dependent binding of VCAM-Ig fusion protein.

In addition, with α_dβ₂And alpha_eβ₇Integrin-bound compounds are particularly useful in the treatment of asthma and related pulmonary diseases. See, e.g., M.H.Grayson et al, J.exp.Med.1998, 188(11) 2187-2191. And alpha_eβ₇Integrin-bound compounds are also useful for treating systemic lupus erythematosus (see, e.g., M.Pang., et al, Arthritis Rheum.1998, 41(8), 1456-; crohn's disease, ulcerative colitis, and Inflammatory Bowel Disease (IBD) (see, e.g., d.elewaut et al, Scand j.gastroentenol 1998, 33(7) 743-; sjogren's syndrome (see, e.g., U.S. kroneld et al, scanand j.gastroenterol 1998, 27(3), 215-; and rheumatoid arthritis (see, e.g., Scand J. gastroenterol 1996, 44(3), 293-. And with alpha₆β₁The bound compounds can be used to prevent fertilization (see, e.g., H.Chen et al, chem.biol.1999, 6, 1-10).

In another aspect of the invention, the conjugates and compositions described herein are useful for inhibiting migration of immune cells from the bloodstream to the central nervous system, or to areas where inflammation-induced myelin destruction is caused, for example, in the case of multiple sclerosis. Preferably, these agents inhibit immune cell migration by inhibiting demyelination and may further promote remyelination. These agents may also prevent demyelination and promote remyelination in the central nervous system for congenital metabolic diseases in which infiltrating immune cells primarily affect myelin development in the CNS. It is also preferred to reduce paralysis when these agents are administered to a subject who is suffering from a demyelinating disease or disorder to induce paralysis.

Included among the inflammatory diseases to be treated by the compositions, conjugates, and methods disclosed herein are diseases associated with demyelination in general. Histologically, myelin abnormalities are demyelination or dysmyelination. Demyelination indicates destruction of myelin. Myelination disorders refer to defects in the formation or maintenance of myelin due to oligodendrocyte dysfunction. Preferably, the compositions and methods disclosed herein are contemplated to treat diseases and conditions associated with demyelination and to aid remyelination. Other diseases or conditions contemplated for treatment include meningitis, encephalitis and spinal cord injury, as well as conditions that often induce demyelination due to an inflammatory response. The conjugates, compositions, and methods disclosed herein are not directed to diseases and disorders in which, for example, abnormal myelin formation, such as dysmyelination, is caused by a genetic defect.

The compositions, conjugates, and mixtures (cocktails) disclosed herein are contemplated for use in the treatment of conditions and diseases associated with demyelination. Diseases and conditions involving demyelination include, but are not limited to, multiple sclerosis, congenital metabolic diseases (e.g., phenylketonuria, Tay-Sachs disease, Niemann-Pick disease, Gaucher's disease, Hurler's syndrome, Krabbe's disease, and other leukodystrophies), neuropathy with abnormal myelination (e.g., Guillain-barre syndrome, Chronic Immune Demyelinating Polyneuropathy (CIDP), multifocal CIDP, anti-MAG syndrome, op syndrome, anti-sulfatide antibody syndrome, anti-GM 2 antibody syndrome, POEMS syndrome, perineuritis, IgM anti-GD 1b antibody syndrome), drug-related demyelination (e.g., caused by administration of chloroquine, FK506, perhexiline, procainamide and zimelidine (zimeldine)), other genetic demyelinating diseases (e.g. carbohydrate-deficient glycoproteins, cokaench heddleSympathy (Cockayne's syndrome), congenital dysmyelination, congenital muscular dystrophy, Fabry's disease, Marinesco-Sjgren syndrome, metachromatic leukodystrophy, Pelizaeus-merzbacher disease, Refsum disease, prion-related diseases, and sala disease, and other demyelinating disorders (e.g., meningitis, encephalitis, or spinal cord injury) or diseases.

There are many disease models available to study these diseases in vivo. For example, animal models include, but are not limited to:

TABLE III

Disease model	Species of species
Disease model	Species of species	EAE	Mouse, rat, guinea pig
Myelin Oligodendrocyte Glycoprotein (MOG) -induced EAE	Rat	EAE	Mouse, rat, guinea pig
Myelin Oligodendrocyte Glycoprotein (MOG) -induced EAE	Rat	Transgenic model of demyelination TNF-alpha	Mouse

Multiple sclerosis

The most common demyelinating disease is multiple sclerosis, but many other metabolic and inflammatory diseases can cause defective or abnormal myelination. MS is a chronic neurological disease that occurs in early adulthood, with most cases progressing to overt disability. There are approximately 350,000 MS cases in the united states alone. In addition to trauma, MS is the most common cause of early to mid-term neurological disability in adults.

The reason for MS still needs to be further determined. MS is characterized by chronic inflammation, demyelination and gliosis (scarring). Demyelination can cause negative or positive effects on axonal conduction. Positive conduction abnormalities include axonal conduction slowing, variable conduction blocks (which occur in the presence of high frequency but not low frequency impulse sequences) or complete conduction blocks. Positive conduction abnormalities include spontaneous or ectopic impulses generated after mechanical stress and abnormal "cross-talk" between demyelinated nodules (exon).

It has been observed that the reactivity of T cells to myelin proteins (myelin basic protein (MBP) or myelin Protein Lipoprotein (PLP)) mediates CNS inflammation in experimental allergic encephalomyelitis. Elevated levels of CNS immunoglobulins (Ig) have also been observed in patients. Some of the tissue damage observed in MS may also be mediated by cytokine products that activate T cells, macrophages or astrocytes.

Currently, 80% of patients diagnosed with MS survive 20 years after the onset of disease. Treatment of MS includes: (1) treatment for changes in the course of disease, including treatment for acute exacerbations and long-term suppression for disease; (2) treating symptoms of MS; (3) prevention and treatment of medical complications; and (4) to address secondary interpersonal and social problems,

the onset of MS may be so severe or moderate that the patient is not attentive to the physician. The most common symptoms include weakness of one or more limbs, blurred vision due to optic neuritis, sensory disturbances, double vision and ataxia. The course of the disease can be divided into three major groups: (1) relapsing MS, (2) chronic progressive MS, and (3) inactive MS. Recurrent MS is characterized by recurrent episodes of neurological dysfunction. Episodes of MS typically occur over several days to several weeks, followed by possible complete, partial or no recovery. Recovery from peak periods of symptoms after onset usually occurs in weeks to months, although recovery lasting 2 or more years is rare.

Chronic progressive MS can cause progressive deterioration gradually, with no stationary or remission periods. This form occurs in patients with a prior history of relapsing MS, although 20% recall that there was no relapse. Acute relapse can also occur during the course of the disease.

The third form is an inactive MS. Inactive MS is characterized by varying degrees of fixed neurological deficits. Most patients with inactive MS have an earlier past history of relapsing MS.

The course of the disease also depends on the age of the patient. For example, preferred prognostic factors include early onset (except childhood), relapse, and little disability 5 years after onset. In contrast, poor prognosis is associated with a later age of onset (i.e., 40 years of age or older) and progressive course of disease. These variables are interdependent in that the age at which chronic progressive MS begins has a tendency to be later than relapsing MS. Disability in patients with chronic progressive MS is usually caused by progressive paraplegia or quadriplegia (paralysis). In one aspect of the invention, it is preferred that the patient is treated when in remission, rather than during the relapsing phase of the disease.

Short term use of adrenocorticotropic hormone or oral corticosteroids (e.g., oral prednisone or intravenously administered methylprednisolone) is the only specific therapeutic measure for treating patients with acute exacerbations of MS.

Newer treatments for MS include interferon beta-1 b, interferon beta-1 a and(formerly copolymer 1) the patient is treated. These three drugs have been shown to significantly reduce the recurrence rate of the disease. These drugs may be self-administered intramuscularly or subcutaneously.

However, there is currently no treatment that inhibits demyelination, either alone to promote or induce spontaneous remyelination or to reduce paralysis. One aspect of the invention contemplates the treatment of MS with the agents disclosed herein alone or in combination with other standard treatment modalities.

Congenital metabolic disease

Congenital metabolic diseases include phenylketonuria and other amino acid uropathies, tay-sachs disease, niemann-pick disease, gaucher disease, heller's syndrome, krabbe disease, and other leukodystrophies that may affect myelin development, as described in more detail below.

PKU is a genetic metabolic error caused by a deficiency in phenylalanine hydroxylase. The lack of this enzyme causes mental retardation, organ damage, abnormal posture, and serious pregnancy can be endangered when the pregnant woman develops PKU. A mouse model for studying PKU has been discovered. Infants identified with PKU are preferably kept on a no or low phenylalanine diet. One aspect of the present invention uses this diet in conjunction with the conjugates and compositions disclosed herein to prevent demyelination and damage to remyelinated cells due to PKU.

Typical tay-sachs disease occurs in subjects at about 6 months of age and will eventually cause death of the subject by the age of 5 years. The disease is due to the lack of hexosaminidase a (hex a), an enzyme that is required for the degradation of certain fatty substances in brain and nerve cells. In the absence of this enzyme, these substances accumulate, causing nerve cell destruction. Another form of hex a enzyme deficiency occurs later in life and is referred to as juvenile, chronic and adult onset hex a deficiency. Symptoms resemble the features of classical tay-sachs disease. There are also adult onset forms of enzyme deficiency. Currently there are no cure cases or treatments for this disease/defect, only intrauterine preventive tests that detect fetal disease. Thus, the conjugates and compositions disclosed herein may be used to ameliorate or prevent the destruction of nerve cells in such patients.

Niemann-pick disease is divided into three types: acute infantile, type B, is a less common chronic, non-neurological type, and type C is a biochemical and genetic idiotype of the disease. In normal individuals, cellular cholesterol is transported to lysosomes for processing and then released. Cells collected from subjects with niemann-pick disease show a defect in the process of releasing cholesterol from lysosomes. This causes an excessive accumulation of cholesterol inside lysosomes, causing processing disorders. NPC1 was found to have a known sterol-sensitive region similar to that in other proteins, which is thought to play an important role in regulating cholesterol transport. No successful treatment has been established for type A and C of Nippon-Pico. For type C, patients are recommended to enter a low cholesterol diet. Thus, the conjugates and compositions disclosed herein can be used to ameliorate or prevent the destruction of cells.

Gaucher disease is a genetic disease caused by genetic mutations. Generally, this gene is responsible for an enzyme called glucocerebrosidase, which is required by the body to break down fat, glucocerebrosidase. In patients with gaucher's disease, the body is unable to produce the enzyme normally and the fat is unable to be broken down. Like tay-sachs disease, gaucher's disease is very common in offspring from eastern ohseh (deutsche), although individuals of any species may be involved. In the german jewish population, gaucher's disease is the most common genetic disease, with an incidence of about 1/450 people. Among the public, gaucher's disease affects approximately 1/100,000 people.

In 1991, enzyme replacement therapy was the first effective treatment for gaucher's disease. The method of treatment comprises administering intravenously a modified form of glucocerebrosidase. It is to be understood that the compositions and conjugates disclosed herein may be used alone or, more preferably, in combination with glucocerebrosidase to treat diseased subjects.

Herrller's syndrome, also known as type I mucopolysaccharidosis, is an overlapping disease. Common to these genetic diseases is the accumulation of glycosaminoglycan cells in fibroblasts. These diseases are genetically distinguishable. Fibroblast and bone marrow transplantation seem to be of little help, and therefore conjugates and compositions are needed that can be used to improve disease severity and progression. The conjugates and compositions disclosed herein can be administered to a subject to improve the progression and/or severity of the disease.

Krabbe's disease (also known as globulocytic leukodystrophy) is an autosomal recessive disease caused by a deficiency in galactosylceramidase (or galactocerebrosidase), which catabolizes the major lipid component of myelin. The incidence in france is estimated to be 1:150,000 births. The disease causes demyelination in the central and peripheral nervous systems. Usually the disease occurs within the first year of birth and progresses rapidly, but juvenile, adolescent or adult onset types have also been reported with more variable rates of progression. The diagnosis was established from an enzyme assay (galactosylceramidase deficiency). There are several natural animal models (mouse, dog, monkey). Krabbe's disease, like all leukodystrophy, has no cured case or effective treatment. One embodiment of the present invention is the use of the compositions and conjugates disclosed herein to treat or ameliorate krabbe's disease and other leukodystrophies.

Leukodystrophy is a group of genetically defined progressive diseases that involve the brain, spinal cord and peripheral nerves. These include Adrenoleukodystrophy (ALD), Adrenomyeloneuropathy (AMN), Aicardi-Goutiers syndrome, Alexander's disease, CACH (i.e., infantile ataxia with reduced CNS myelin formation or leukopenia), CADASIL (i.e., autosomal dominant cerebral arterial disease with subcortical infarction and leukoencephalopathy), Canavan disease (spongiform degeneration), tendinopathy (CTX), krabbe's disease (as described above), Metachromatic Leukodystrophy (MLD), neonatal adrenoleukodystrophy, ovarian leukodystrophy syndrome (ovarioleukysysythesis), Pelizaeus-merzbacheresis (X-linked spastic paraplegia), Refsum disease (Refsum disease), van derKnaap syndrome (vacuolar leukodystrophy with subcortical cysts), and Zellweger syndrome (Zellweger syndrome). There is no effective treatment for these diseases, let alone cured cases. Accordingly, there is a need for methods of treating or ameliorating the symptoms of these diseases, such as by using the compositions and conjugates disclosed herein.

Neuropathy with abnormal myelination

There are a number of chronic immune polyneuropathies that can cause demyelination in patients. The age of onset of these diseases varies depending on the condition. There are standard methods of treatment for these diseases and may be used in combination with the compositions and conjugates disclosed herein. Alternatively, the compositions and conjugates disclosed herein may be used alone. The standard treatment methods available include the following:

TABLE IV

Neuropathy	Clinical characteristics	Method of treatment
Neuropathy	Clinical characteristics	Method of treatment	Chronic Immune Demyelinating Polyneuropathy (CIDP)	Onset between 1-80 years of age, characterized by weakness, sensory deficits, and hyperproliferation.	T-cell immunosuppression using prednisone, cyclosporin A or methotrexate, HIG, and plasmapheresis
Multi-focal CIDP	Onset between 28 and 58 years of age, characterized by asymmetric weakness, loss of sensation, and a slowly progressive or relapsing-remitting course.	T cell immunosuppression with prednisone, Human Immunoglobulin (HIG)	Chronic Immune Demyelinating Polyneuropathy (CIDP)

Multifocal Motor Neuropathy (MMN)	The age of onset ranges from 25 to 70 years, with males being twice as old as females. Features include weakness, muscle atrophy, fasciculation and cramping, which are progressive over a period of 1-30 years.	HIG B-cell immunosuppression with plasmapheresis, cyclophosphamide, Rituxan
Multifocal Motor Neuropathy (MMN)			Neuropathy with IgM binding to myelin-associated glycoprotein (MAG)	Onset is usually above 50 years of age and is characterized by sensory deficits (100%), weakness, disturbance of weight gain, tremor, all of which are slowly progressive.	B cell immunosuppressive plasma replacement of cyclophosphamide Rituxan alpha-interferon cladribine (cladribine) or fludarabine (fludarabine) prednisone
GALIP syndrome (abnormal gait, autoantibodies, older age, onset, polyneuropathy)	Gait abnormalities with polyneuropathy	HIG plasmapheresis cyclophosphamide
	Gait abnormalities with polyneuropathy	HIG plasmapheresis cyclophosphamide	POEMS syndrome (polyneuropathy, organ enlargement, endocrinopathy, M-protein and skin changes) is also known as Crow-Fukase syndrome and Takatsuki disease	Onset between 27 and 80 years of age, with weakness and weakness, loss of sensation, weakened or missing tendon reflexes, skin disorders and other manifestations.	Radiation therapy is used for osteopetrosis. Chemotherapy (melphalan and prednisone) is used for widespread (widespread) lesions.

Drug and radiation induced demyelination

Certain drugs and radiation can induce demyelination in a subject. Drugs that cause demyelination include, but are not limited to, chloroquine, FK506, piperacillin, procainamide, and zimepiride.

Radiation can also induce demyelination. Central Nervous System (CNS) toxicity caused by radiation is thought to be caused by the following causes: (1) damage to vascular structures, (2) removal of oligodendrocyte-2 astrocyte progenitors and mature oligodendrocytes, and (3) removal of neural stem cell populations in the hippocampus, cerebellum, and cortex, broadly altering cytokine expression. Most radiation damage is caused by radiation therapy in the treatment of certain cancers. See review by Belka et al, 2001 br.j. cancer 85: 1233-9. However, exposure to radiation may also be one of the causes for astronauts (Hopewell, 1994 adv. space Res.14: 433-42), also in the case of exposure to radioactive substances.

For patients who have received a drug or who have been exposed to occasional or intentional exposure to radiation, it may be beneficial to prevent demyelination or to promote remyelination by administration of one of the conjugates or compositions disclosed herein.

Diseases involving demyelination

Other genetic syndromes/diseases that cause demyelination include Cockayne syndrome (Cockayne's syndrome), congenital dysmyelination, fabry's disease, metachromatic leukodystrophy, pelizaeus-Merzbacher disease, Refsum disease, prion-related diseases, and Salla disease.

Cokahn's Syndrome (CS) is a rare genetic disorder that is sensitive to sunlight, short in stature, and premature in appearance. In the typical form of Cokahn's syndrome (type I), symptoms are progressive, with symptoms usually evident after 1 year of age. Early onset or congenital (type II) form of cockayne syndrome is evident at birth. Interestingly, unlike other DNA repair diseases, cockayne syndrome is not associated with cancer. CS is a multisystemic disease that causes profound growth disorders in the body and brain, as well as progressive cachexia, retinal, cochlear and neurodegenerative diseases, with leukodystrophy and demyelinating neuropathy, but without increasing the incidence of cancer. After exposure to UV (e.g., sunlight), patients with cockayne syndrome no longer develop transcriptionally coupled repair. Two gene defects have now been identified in Cokahn's syndrome, CSA and CSB. The CSA gene is found on chromosome 5. Both genes encode proteins that interact with transcriptional structural components and DNA repair proteins.

Currently, no cure or effective treatment for the patient is found. Accordingly, one aspect of the present invention is the treatment of this disease with the conjugates and compositions disclosed herein.

Congenital dysmyelination has several names, including congenital dysmyelination neuropathy, congenital dysmyelination polyneuropathy, congenital dysmyelination (onion bulb-like) polyneuropathy, congenital dysmyelination neuropathy, congenital neuropathy caused by dysmyelination, dysmyelination neuropathy, and CHN. Among the most common genetic diseases in humans, hereditary peripheral neuropathy is a complex, clinically and genetically heterogeneous group of diseases that can produce progressive deterioration of peripheral nerves. Congenital dysmyelination is one of this group of diseases. This group of diseases includes hereditary neuropathy prone to compression paralysis, Charcot-Marie-Tooth disease, demo-Sottas syndrome, and congenital dysmyelination neuropathy. There are no known cure cases or effective treatments for these diseases.

Fabry disease has several names, including: phaeosphaceloma, ceramidase deficiency, acid ceramidase deficiency, AC deficiency, N-lauryl sphingosine deacylase deficiency, and N-acylsphingosine aminohydrolase. As some names suggest, the disease is caused by a deficiency in acid ceramidase (also known as ceramide amino hydrolase, ASAH). Enzyme deficiency causes accumulation of unsulfonated acidic mucopolysaccharides in neurons and glial cells. Patients with this disease usually die before the age of 2 years.

Metachromatic Leukodystrophy (MLD) is a hereditary disease caused by a deficiency in arylsulfatase a. It is one of a group of genetic diseases known as leukodystrophy that affects myelin growth. MLD has three forms: late infant, juvenile, and adult types. In the most common advanced infant forms, onset of symptoms begins between 6 months and 2 years. Infants are usually normal at birth, but eventually lose the ability to acquire before. Symptoms include hypotonia (low muscle tone), abnormal speech, loss of mental capacity, blindness, rigidity (i.e., uncontrolled muscle tone), spasticity, swallowing impairment, paralysis, and dementia. Symptoms of juvenile onset begin to appear between the ages of 4 and 14 and include poor school performance, mental deficits, ataxia, seizures (seizure) and dementia. In adult forms, symptoms begin to occur after age 16 and may include attention deficit, depression, psychiatric disorders, ataxia, tremor, and dementia. Seizures can occur in adult forms, but are less common than others. In all three forms, mental abolishness is usually the first sign to appear.

Pelizaeus-merzbacher disease (also known as peripartum sudang leukodystrophy) is an X-linked genetic disease that causes proteolipid protein abnormalities. This abnormality usually causes death of the infant before the age of 1 year. There is no known treatment or cure for the disease.

Refsum disease (also known as phytane oxidase deficiency, hereditary motor disorders of polyneuritis type or hereditary motor and sensory neuropathy type IV, HMSN IV) is caused by a mutation in a gene encoding phytanoyl-coa hydroxylase (PAHX or PHYH). The main clinical manifestations are retinitis pigmentosa, chronic polyneuropathy and cerebellar signs. Phytanic acid, a rare branched-chain fatty acid (3, 7, 11, 15-tetramethyl-hexadecanoic acid), accumulates in the interstitial and body fluids of patients with this disease and cannot be metabolized due to a PAHX deficiency. The once or twice monthly plasmapheresis method effectively removes the acid from the body and allows relaxation of dietary restrictions on limiting phytanic acid intake.

Prion-related diseases include Gerstmann-Straussler disease (GSD), Creutzfeldt-Jakob disease (CJD), familial fatal insomnia, and abnormal subtypes of prion proteins as infectious agents in these diseases as well as kuru and scrapie (the diseases found in sheep). The term prion is from "protein infectives" (Prusiner, Science 216: 136-44, 1982). Proteolytic cleavage of prion-associated protein (PRP) produces amyloid peptide (amyloidogenic peptide) which polymerizes into insoluble fibers.

Salla disease and other types of sialuria (sialuria) are diseases that involve sialic acid storage problems. They are autosomal recessive neurodegenerative diseases that can exist in either severe infantile (i.e., ISSD) or slowly progressive adult (more common in finland) forms (i.e., Salla disease). The main symptoms are hypotonia, cerebellar ataxia and mental retardation. Palliative or improved treatments for these conditions and diseases are also contemplated.

Other conditions that cause demyelination include post-infectious encephalitis (also known as acute disseminated encephalomyelitis, ADEM), meningitis, and damage to the spinal cord. The compositions and conjugates disclosed herein are also contemplated for use in treating these other demyelinating disorders.

The following synthetic and biological examples are provided to illustrate the invention, but are not to be construed as limiting the scope of the invention in any way. All temperatures are degrees celsius unless otherwise noted.

Examples

In the following examples, the following abbreviations have the following meanings. If an abbreviation is not defined, it has a generally accepted meaning.

ACN ═ acetonitrile

bs as wide singlet

d is doublet peak

double doublet of dd

Et 3N-triethylamine

g is g ═ g

h and hr are hours

HPLC ═ high performance (or pressure) liquid chromatography

kg is kg

kDa ═ kilodaltons

L is liter

m is multiplet

M is equal to mole

mg ═ mg

min is minutes

mL to mL

mm-mm

mM ═ millimole

mmol ═ mmol

s ═ singlet

sat. (saturated)

t is triplet

TFA ═ trifluoroacetic acid

TLC or tic ═ thin layer chromatography

Ts ═ tosyl group

μ L ═ microliter

Microgram of mug

Mum ═ micron

The general method comprises the following steps: protons were obtained using a Gemini 2000 or Bruker Avance 300 spectrometer (¹H) And carbon (C)¹³C) Nuclear magnetic resonance spectroscopy (NMR). The presence of polyethylene glycol (PEG) was detectable by a large, broad single peak at 3.6 ppm. The integral of the signal may vary depending on the size of the PEG moiety. The presence of the conjugated VLA-4 antagonist may also be in the conjugate¹Detected on the H NMR spectrum. In silica 60F₂₅₄(EMD15341-1) Pre-coated plate or Pre-coated MKC18F silica 60(Whatman 4803-110) thin layer chromatography was performed. Mass spectrometry was performed in cationic single quadrupole mode (single quad mode) on an Agilent mass spectrometer (LC/MSD VL).

HPLC method of PEG product and PEG conjugate:

preparative reverse phase HPLC was performed at 210nm using a Varian Prep Star (model SD-1) module with a Varian UV detector. The method A comprises the following steps: at Vydac C18, 300Samples of PEG product and PEG conjugate were purified on a pore size column (250mm X21.2 mm) using reverse phase HPLC, typically using a gradient of 35-50% ACN + 0.1% TFA over 100min at a rate of 20 mL/min. The method B comprises the following steps: at Vydac C18, 300Samples of PEG product and PEG conjugate were purified on a pore size column (250mm x 50mm) using reverse phase HPLC, typically using a gradient of 35-50% ACN + 0.1% TFA, at a rate of 60mL/min over 100 min. The method C comprises the following steps: purity of the PEG product and conjugate was confirmed by reverse phase analytical HPLC using a device equipped with a Waters Symmetry 300Aperture, 3.5 μ C18 column (150 mm. times.4.6 mm) Agilent Series 1100 quaternary system using a gradient of 40-50% ACN w/0.1% TFA at a flow rate of 1.5mL/min was connected to an Agilent 1100 variable wavelength detector set at 210nm and a Sedex 75 evaporative light scattering detector (40 ℃, gain 5).

PEG reagent: the following PEG starting materials were obtained by NOF Corporation (Yebsiu Garden plant Tower, 20-3Ebisu 4-chome, Shibuya-ku, Tokyo 150-: 40kDa 4-arm PEG alcohol (NOF Cat. Sunbright PTE-40000); 40kDa 3-arm PEG alcohol (NOF Cat. Sunbright GL-400).

Example 1

Sodium hydroxide (10g, 0.25m) was dissolved in water (300 ml). To this solution was added 4-nitrophenylalanine (50.3g, 0.22m) and stirred until complete dissolution. To the resulting solution was added sodium carbonate (28.8g, 0.26m) and the stirred suspension was cooled to +8 ℃ in an ice bath. Benzyl chloroformate (44.7g, 0.26m) was added dropwise, stirred vigorously, maintaining the internal temperature in the range of +6 ℃ to +9 ℃. The mixture was stirred at +6 ℃ for an additional 1hr and transferred to a separatory funnel, rinsed with ether (2 × 150 ml). The aqueous phase was placed in a large erlenmeyer flask (2L), acidified carefully with dilute aqueous HCl to pH 2 and extracted with ethyl acetate (4 × 500 ml). The combined extracts were washed with water and MgSO₄And (5) drying. The solution was filtered, the filtrate was evaporated, and the residue was dissolved in ethyl acetate (150ml) and diluted with hexane (500 ml). The crystalline material was filtered off, rinsed with cold solvent and dried in air to give Cbz-4-nitrophenylalanine, 75g (99.5% yield).¹H-NMR，DMSO-d6，(δ)：12.85(bs，1H)，8.12(d，2H，J＝9Hz)，7.52(d，2H，J＝9Hz)，7.30(m，5H)，4.95(s，2H)，4.28(m，1H)，3.32(bs，1H)，3.10(m，2H).¹³C-NMR(δ)：173.1，156.3，146.6，137.3，130.8，128.5，128.0，127.8，123.5，65.6，55.1，36.6.MS(m/z)：367.1[M+23].

Cbz-4-nitrophenylalanine (75g, 0.22m) was dissolved in dioxane (300 ml). The resulting stirred solution was cooled to-20 ℃ (internal) in a dry ice bath. Liquefied isobutene (approximately 290ml) was added, followed by three aliquots of concentrated sulfuric acid (35ml) each at 30min intervals. The addition of acid is a very strongly exothermic process, accompanied by a major polymerization. Efficient mechanical agitation is important at this stage. The resulting mixture was stirred for 20hr, then warmed to room temperature, carefully poured into saturated aqueous sodium carbonate (2L) and diluted with ethyl acetate (600 ml). The organic layer was separated and the aqueous layer was extracted with ethyl acetate (2 × 200 ml). The combined extracts were washed with water and dried over sodium sulfate. The solution was filtered and evaporated to dryness. The residue was poured into an ethyl acetate/hexane mixture (500 ml; 1:1) and filtered through a plug of silica gel (ca. 2X 2 in). Additionally using a certain amount ofThe same solvent (2L total) rinses the silica and the filtrate is evaporated to give the fully protected 4-nitrophenylalanine as a viscous oil, 73g (83% after two steps).¹H-NMR，CDCl₃，(δ)：8.12(d，2H，J＝8.4Hz)，7.36(m，7H)，5.35(m，1H)，5.10(m，2H)，4.57(m，1H)，3.31(m，2H)，1.43(s，9H).¹³C-NMR，CDCl₃，(δ)：169.7，155.3，146.9，143.9，136.0，130.2，128.4，128.2，128.0，123.3，82.9，66.9，54.7，38.2，31.4，27.8，13.9.MS(m/z)：423.1[M+23].

Protected 4-Nitrophenylalanine (73g, 0.18m) was dissolved in ethanol (500ml), and a platinum oxide catalyst (1.5g) was added. The resulting solution was stirred vigorously under hydrogen (50-60psi) at room temperature until hydrogen adsorption ceased (3 hr). The catalyst was filtered off, the filtrate evaporated to dryness, the residue taken up in ethyl acetate (200ml), filtered through a plug of silica gel (2 × 2in) and the silica rinsed with an ethyl acetate-hexane mixture (3:2, 2L). The filtrate was concentrated to about 200ml and hexane (500ml) was added. The crystalline product was filtered off, rinsed with cold solvent and air dried. Yield-56 g, 84%.¹H-NMR，CDCl₃，(δ)：7.30(bs，5H)，6.92(d，2H，J＝8.1Hz)，6.58(d，2H，J＝8.1Hz)，5.21(m，1H)，5.10(d，2H，J＝2.1Hz)，4.46(m，1H)，3.59(bs，2H)，2.97(s，2H，J＝5.4Hz)，1.42(s，9H).¹³C-NMR，CDCl₃，(δ)：170.6，145.1，136.3，130.2，128.3，127.9，125.6，115.0，81.9，66.6，55.2，37.4，27.8MS(m/z)：393.1[M+23].

Example 2

The product of example 1, 4-aminophenylalanine (20g, 0.054m) was dissolved in ethanol (200ml) and treated with Hunig's base (21g, 0.162m, 3 equivalents) and 2-chloro-3-nitropyridine (10.3g, 0.65m, 1.2 equivalents). The resulting solution was stirred under nitrogen and heated to reflux for 24 hr. LC analysis showed that there was a small amount ofThe amine of the reaction is present. A small amount of chloronitropyridine (1.1g, 0.13 eq.) was added and reflux continued for 24 hr. The reaction mixture was cooled and evaporated to dryness. The residue was dissolved in ethyl acetate (600ml), and the resulting solution was washed with water (1 × 200ml), diluted aqueous citric acid (0.2N, 2 × 200ml), brine (1 × 200ml), and dried over sodium sulfate. The solid was filtered off and the filtrate was evaporated to give 37g of a dark red oil containing the expected product with an excess of chloronitropyridine. The impure product was purified by flash chromatography (Biotage 75L system) eluting with a mixture of ethyl acetate: hexane (3: 17). Fractions containing pure product were combined and evaporated to give the product as a dark red, viscous oil, 26g (99%).¹H-NMR，CDCl₃，(δ)：10.10(s，1H)，8.49(m，2H)，7.57(d，2H，J＝9Hz)，7.35(bs，5H)，7.19(d，2H，J＝9Hz)，6.84(m，1H)，5.30(m，1H)，5.13(d，2H，J＝3Hz)，4.57(m，1H)，3.11(m，2H)，1.45(s，9H).¹³C-NMR，CDCl₃，(δ)：170.4，155.5，155.1，150.0，136.7，136.3，135.4，132.4，129.9，128.5，128.3，128.0，127.9，122.2，113.7，82.2，66.7，55.1，37.7，27.8，20.9.MS(m/z)：493.1[M+1]，515.1[M+23].

The red nitro compound (26g, 0.054m) was dissolved in THF (350ml) and platinum oxide catalyst (1.35g) was added. The resulting mixture was stirred vigorously under an atmosphere of hydrogen (50-60psi) until hydrogen adsorption ceased (2 hr). The catalyst was filtered off and the filtrate was evaporated to dryness. The residue was dissolved in ethyl acetate (100ml), which was diluted with hexane (50ml) until crystallization started. The mixture was further diluted with an ethyl acetate/hexane (1:1) mixture (300ml) and allowed to stand in a refrigerator for 3 hr. The crystalline solid was filtered off, rinsed with cold solvent and air dried to give the product, 23g, 94%.¹H-NMR，CDCl₃，(δ)：7.81(dd，1H，J1＝1.5Hz，J2＝4.8Hz)，7.33(bs，5H)，7.17(d，2H，J＝8.4Hz)，7.03(d，2H，J＝8.4Hz)，6.96(dd，1H，J1＝1.5Hz，J2＝7.5Hz)，6.75(dd，1H，J1＝5.0Hz，J2＝7.7Hz)，6.22(s，1H)，5.31(m，1H)，5.09(bs，2H)，4.50(m，1H)，3.41(bs，2H)，3.02(m，2H)，1.43(s，9H).¹³C-NMR，CDCl₃，(δ)：170.6，155.6，145.5，140.21，138.8，136.3，130.8，129.9，128.5，128.3，127.9，123.4，118.2，117.0，82.0，66.6，55.2，37.4，27.9.MS(m/z)：407.1[M-56]，463.1[M+1]，485.1[M+23].

Aminopyridine (19g, 0.041m) was suspended in dichloromethane (200ml) and CDI (12g, 0.074m, 1.8 eq) was added. The resulting mixture was stirred at room temperature for 20 hr. The reaction mixture was washed with saturated aqueous bicarbonate (2 × 100ml), brine (1 × 100ml) and dried over sodium sulfate. The solid was filtered off and the filtrate evaporated to dryness. The residue was dissolved in ethyl acetate (hot, 300ml) and crystallized. The crystalline product was filtered off, rinsed with cold ethyl acetate and air dried to give 19.9g, 81% of imidazolone.¹H-NMR，CDCl₃，(δ)：10.63(s，1H)，8.06(d，1H，J＝3Hz)，7.66(d，2H，J＝9Hz)，7.32(m，8H)，7.05(m，1H)，5.36(m，1H)，5.13(s，2H)，4.59(m，1H)，3.17(m，2H)，1.45(s，9H).¹³C-NMR，CDCl₃，(δ)：170.4，155.6，154.3，143.8，141.0，136.2，135.8，131.8，130.2，128.3，128.0，125.9，122.2，118.3，116.0，82.4，66.8，55.0，37.7，27.8.MS(m/z)：433.1[M-56]，489.2[M+1]，511.2[M+23].

Example 3

Pyridine-3-sulfonic acid (125g, 0.78m) was placed in a 1L 3-necked flask equipped with a mechanical stirrer, reflux condenser, thermometer and nitrogen inlet. Next, phosphorus pentachloride (250g, 1.19m, 1.5 eq) was added, followed immediately by phosphorus oxychloride (330ml, 3.8m, 4.5 eq). The contents of the starting flask were stirred at room temperature for 30min, then slowly subjected to mild reflux (internal temperature about 110 ℃) for 1 hour, held at this temperature for about 3.5hr, and then cooled to room temperature over the following 12 hr. During this time gas evolution was observed. Removing volatile, yellow semisolid residue under reduced pressure (12mmHg/40 deg.C)The residue was diluted with DCM (1L). The slurry was slowly poured into a stirred ice-cooled saturated aqueous bicarbonate solution, maintaining the pH at 7. Gas evolution was observed. The organic layer was separated and the aqueous layer was back-extracted with DCM. The combined extracts were washed with cold saturated aqueous bicarbonate, brine and dried over magnesium sulfate. The solid was filtered off and the filtrate was evaporated to give pyridine-3-sulfonyl chloride as a pale yellow oily liquid, 123g (93% purity; theoretical 88%).¹H-NMR，CDCl₃，(δ)：9.26(d，1H)，8.98(dd，1H)，8.34(m，1H)，7.62(m，1H).¹³C-NMR，CDCl₃，(δ)：155.3，147.4，140.9，134.6，124.2.

MS(m/z)：178.0[M+1].

L-penicillamine (150g, 1.0m) was dissolved in DI water (1500ml) and stirred, cooled to +8 ℃ in an ice bath, and treated with formalin (150ml, 37% aqueous solution). The reaction mixture was stirred at +8 ℃ for 2hr, then the cooling bath was removed and stirring continued for 12 hr. The clear solution was concentrated under reduced pressure (14mmHg/50 ℃) to give a white residue. The solid was resuspended, then dissolved in hot MeOH (2500ml) and allowed to stand at room temperature for 12 hr. The white fluffy precipitate was filtered off and rinsed with cold methanol. The filtrate was concentrated and crystallized again. The collected precipitate was combined with the first harvested product and dried in a vacuum oven at 55 deg.C under 45mmHg for 24 hr. The yield of (R) -5, 5-dimethylthiazolidine-4-carboxylic acid was 138g of (C:)>99% purity; theoretical value 86%).¹H-NMR，DMSO-d6，(δ)：4.25(d，1H)，4.05(d，1H)，3.33(s，1H)，1.57(s，3H)，1.19(s，3H).¹³C-NMR，DMSO-d6，(δ)：170.8，74.4，57.6，51.8，28.9，27.9.MS(m/z)：162.3[M+1].

In a 4L reactor equipped with a mechanical stirrer and thermometer, a buffer solution was prepared with potassium dihydrogen phosphate (43g, 0.31m) and dipotassium hydrogen phosphate (188.7g, 1.08m) in DI water (2L). (R) -5, 5-dimethylthiazolidine-4-carboxylic acid (107g, 0.675m) was added and stirred until completely dissolved. The solution was cooled to +8 ℃ in an ice bath. A separately prepared solution of pyridine-3-sulfonyl chloride (124g, 0.695m)/DCM (125ml) was added dropwise to the reactor and stirred vigorously for 1 hr. Monitoring systemAfter 4hr, the pH of the mixture was found to be 5, and solid bicarbonate was added to adjust the pH to 6. The mixture was warmed to room temperature over 18 hr. The pH was adjusted to 2 with dilute aqueous sulfuric acid, stirred for 1hr, and the precipitated yellow solid was filtered off and rinsed to neutrality with water. The solid cake was transferred to a 2L Erlenmeyer flask, suspended in DCM (500ml), shaken back occasionally for 5min, and filtered again. The filter cake was washed with DCM and air dried. The yield of the title compound (R) -5, 5-dimethyl-3- (pyridin-3-ylsulfonyl) thiazolidine-4-carboxylic acid was 148.9g (98% purity; theoretical 73%).¹H-NMR，DMSO-d6，(δ)：9.05(d，1H)，8.89(m，1H)，8.32(m，1H)，7.69(m，1H)，4.68(q，2H)，4.14(s，1H)，1.35(s，3H)，1.29(s，3H).¹³C-NMR，DMSO-d6，(δ)：170.0，154.3，147.9，135.8，134.1，124.8，72.6，54.3，50.2，29.4，25.0.MS(m/z)：303.2[M+1].

Example 4

Example 2 the product (52g, 0.106m) was slurried in MeOH (450ml) and hydrogenation catalyst (8.7g, 5% Pd/C, Degussa) was added and the mixture stirred under hydrogen (60psi) until no further adsorption (ca.2 hr). THF (150ml) was added to dissolve the precipitated solid, the solution was filtered through a plug of Celite, and the filter was washed with DCM. The filtrate was evaporated to dryness, redissolved in DCM (300ml) and stripped again (stripped). This operation was repeated 2 times. The foamy solid was maintained under high vacuum for 3 hr. The yield of the title compound is 38.3g (101% of theory).¹H-NMR，CDCl₃，(δ)：8.08(m，1H)，7.56(AB q，4H)，7.37(m，1H)，7.06(m，1H)，3.68(m，1H)，2.03(m，2H)，1.49(s，9H).¹³C-NMR，CDCl₃，(δ)：173.8，154.6，143.9，141.0，137.4，131.5，130.2，126.1，122.3，118.0，116.1，81.4，56.0，40.6，27.9.MS(m/z)：299.3[M-56]，355.4[M+1]，377.4[M+23].

Example 5

The product of example 4 (38.3g, say 0.106m) was dissolved in DCM (500ml) and treated with the following compounds in sequence: n-methylmorpholine (27g, 30ml, 0.266 m; 2.5 equiv.), HOBt (17.3g, 0.128 m; 1.2 equiv.), and the product of example 3 (33.8g, 0.112 m; 1.06 equiv.). The heterogeneous solution was cooled to +4 ℃ in an ice bath and treated with the addition of EDC (22.5g, 0.117 m; 1.1 eq.) in one portion. The reaction mixture was stirred, allowed to warm to room temperature over the next 4hr, and then held for an additional 18 hr. The solvent was stripped and the residue dissolved in ethyl acetate (1.2L), washed with saturated aqueous bicarbonate (2X 250ml), water (250ml), brine (300ml) and dried over magnesium sulfate. The solution was filtered and evaporated to dryness to give a pale orange yellow, viscous oily product, 76g of (A)>>100%). The crude product was purified by flash chromatography on silica gel (Biotage 75L in ethyl acetate/methanol (3%) mixture). The fractions containing the pure product were combined and evaporated to give 54g of the title compound (83% yield).¹H-NMR，CDCl₃，(δ)：10.37(s，1H)，9.11(s，1H)，8.87(m，1H)，8.19(m，1H)，8.05(m，1H)，7.56(AB q，4H)，7.52(m，1H)，7.36(m，1H)，7.06(m，2H)，4.83(m，1H)，4.58(AB a，2H)，3.96(s，1H)，3.19(m，2H)，1.49(s，9H)，1.22(s，3H)，1.18(s，3H).¹³C-NMR，CDCl₃，(δ)：169.7，167.6，153.9，148.4，143.8，140.9，135.8，135.6，132.9，131.9，130.2，125.9，123.8，122.1，118.0，115.9，82.8，73.6，60.3，54.8，53.7，50.6，37.8，29.1，27.8，23.9，14.1.MS(m/z)：583.3[M-56]，639.4[M+1]，661.3[M+23].

Example 6

To an ice-cooled solution of ethyl trifluorobutyrate (15g, 89mmol) and ethyl formate (36mL, 444mmol) in THF (200mL) was added N₂1MKOtBu/THF (107mmol, 107mL) was added over 25 minutes. After 15 minutes, the ice bath was removed and the reaction mixture was stirred at room temperature for 1 hour. Additional ethyl formate (18mL, 222mmol) was then added and the reaction mixture was stirred overnight. The reaction mixture was concentrated and the residue was extracted between cold ether (100mL) and cold water (300 mL). The pH of the aqueous phase was adjusted to 2 with concentrated HCl. The product was extracted with dichloromethane (1X 100mL, 45X 75mL), the combined organic extracts washed with brine (1X 100mL), dried (MgSO)₄) Filtration and concentration gave the title compound as a thick oil which solidified on standing at 10.2g (58.5%). MS (M/z) ═ 198(M + H)⁺。

Example 7

To a solution of the product of example 6 (10g, 51mmol) and diethylguanidine sulfate (8.3g, 25.2mmol) in EtOH (60mL) in N₂A solution of NaOEt in 21% EtOH (20.7mL, 55.5mmol) was added over 10 minutes. The reaction mixture was then heated to reflux for 5 hours. The heterogeneous solution was cooled and poured into cold water (100mL) to give a homogeneous solution. The pH of the solution was adjusted to about 3.5 with concentrated HCl and 1N HCl. The solid precipitated from the solution was collected by filtration. The pale brown solid was washed with water and air dried to give 2.9g, (23%) of the title compound. MS (M/z) ═ 250(M + H)⁺.¹H NMR(300MHz，CD₃OD)δ 7.65(br s，1H)，3.55(q，4H)，3.30(q，2H)，1.25(t，6H).

Example 8

The product of example 7 (2.0g, 8.02mmol), DIEA (1.5mL, 8.83mmol), DMAP (.98g, 0.8mmol) anddichloromethane (30mL) was charged to the flask. The mixture was cooled to 0 deg.C and trifluoroacetic anhydride (1.5mL, 8.83mmol) was added. The reaction became homogeneous and was stirred at 0 ℃ for 3 hours. The mixture was washed with saturated NaHCO₃Quenched and extracted with dichloromethane. The organic phase was washed with 0.2N citric acid and Na₂SO₄Drying, filtration and concentration in vacuo afforded 2.87g (94%) of the title compound as a brown solid.¹H NMR(300MHz，CDCl₃)δ 8.28(s，1H)，3.65-3.52(m，4H)，3.29-3.19(q，2H)，1.22-1.17(t，6H).

Example 9

Example 8 product (1.3g, 3.5mmol), H-Phe (p-NO)₂) OtBu (1.1g, 4.2mmol) and DIEA (0.9mL, 5.3mmol) in CH₃CN (14mL) solution in N₂The mixture was heated to reflux overnight. The next day, additional H-Phe (p-NO) was added₂) OtBu (0.8g, 3mmol), and refluxing was continued for 3 days. The reaction mixture was then cooled and concentrated. The residue was poured into EtOAc (50mL) and the organic portion was washed with 0.5N KHSO₄Washed (3X 50mL), water (1X 50mL), brine (1X 10mL), dried (MgSO 4)₄) Filtered and concentrated to a brown gum. The crude material was purified by flash chromatography (5:1 hexanes/EtOAc) to give 640mg (38%) of the title compound as a gold gum. TLC 3:1 Hexane/EtOAc, R_f＝0.30，MS(m/z)＝498(M+H)⁺，¹H NMR，(300MHz，CDCl₃)δ 8.19(d，2H)，7.80(s，1H)，7.25(d，2H)，5.19(br d，1H)，4.95(q，1H)，3.70-3.50(m，4H)，3.45-3.25(m，2H)，3.10(q，2H)，1.40(s，9H)，1.05(t，6H).

Example 10

The product of example 9 (635mg, 1.27 mm)ol) was dissolved in pure EtOH (5mL) to which was added 35mg Pd/C, 10 wt%. Reaction hydrogenation (45psi H₂) 2.5H, during which 50mg Pd/C, 10 wt.% were added and the reaction mixture was again hydrogenated (45psi H)₂) Overnight. The reaction mixture was filtered through a pad of celite and the filtrate was concentrated to yield 452mg (76%) of the title compound. MS (M/z) ═ 468(M + H)⁺，¹H NMR(300MHz，CDCl₃)δ 7.75(s，1H)，6.90(d，2H)，6.60(d，2H)，5.05(br d，1H)，4.80(q，1H)，3.70-3.45(m，6H)，3.10-2.90(m，4H)，1.40(s，9H)，1.15(t，6H).

Example 11

EXAMPLE 10A solution of the product (598mg, 1.28mmol), 2-chloro-3-nitropyridine (243mg, 1.53mmol) and DIEA (0.67mL, 3.83mmol) in EtOH (5mL) in N₂Heating and refluxing. The next day, the reaction was cooled and 2-chloro-3-nitropyridine (40mg, 0.25mmol) and DIEA (0.11mL, 0.60mmol) were added and the reaction heated to reflux for 1 day. The reaction mixture was then concentrated and the residue was taken up in EtOAc (20 mL). The organic phase was washed with water (2 × 20 mL). The combined aqueous washes were back-extracted with EtOAc (2 × 10 mL). The combined organic extracts were extracted with 0.2N citric acid (3X 20mL), water (1X 10mL), saturated NaHCO₃Washed (3X 20mL), brine (1X 10mL), dried (MgSO₄) Filtered and stripped to an orange-yellow gum. The crude product was purified by flash chromatography using 4:1 hexane/EtOAc (R)_f0.14) to yield 610mg (81%) of the title compound as a red oil. MS (M/z) ═ 590(M + H)⁺，¹H NMR(300MHz，CDCl₃)δ 10.10(s，1H)，8.55(d，1H)，8.50(m，1H)，7.79(s，1H)，7.75(d，2H)，7.15(d，2H)，6.80(q，1H)，5.10(br d，1H)，4.90(m，1H)，3.70-3.45(m，4H)，3.25(m，2H)，3.10(q，2H)，1.40(s，9H)，1.10(t，6H).

Example 12

To the product of example 11 (610mg, 1.03mmol) in pure EtOH (5mL) was added 60mg Pd/C, 10 wt%. Hydrogenation of the mixture (45psi H₂) Overnight. The next day, the reaction mixture was filtered through celite, and the filtrate was concentrated to yield 500mg (87%) of the title compound. MS (M/z) ═ 560(M + H)⁺，¹H NMR(300MHz，CDCl₃)δ 7.85(d，2H)，7.80(s，1H)，7.20(d，2H)，7.05(d，2H)，7.00(d，1H)，7.75(m，1H)，6.20(br s 1 H)，5.15(br s，1H)，4.85(m，1H)，3.75-3.45(m，4H)，3.40(br s，2H)，3.15(m，2H)，3.05(q，2H)，1.40(s，9H)，1.15(t，6H).

Example 13

EXAMPLE 12 product (141mg, 0.250mmol) and CDI (62mg, 0.378mmol) in CH₂Cl₂The solution was stirred overnight (3 mL). The next day, additional CDI (30mg, 0.185mmol) was added and the reaction stirred for an additional 1 day. The reaction mixture was then concentrated, poured into EtOAc (10mL), and the organic portion was washed with 0.2N citric acid (3X 5mL), water (1X 5mL), saturated NaHCO₃Washed (3X 5mL), brine (1X 5mL), dried (MgSO 5₄) Filtered and concentrated to give 69mg (47%) of the title compound as a foam, which was used without further purification. MS (M/z) ═ 586(M + H)⁺，¹H NMR(300MHz，CDCl₃)δ 8.20(br s，1H)，8.05(d，1H)，7.80(s，1H)，7.65(d，2H)，7.90(m，3H)，7.05(m，1H)，5.15(br d，1H)，4.95(m，1H)，3.70-3.45(m，4H)，3.25(app d，2H)，3.10(q，2H)，1.40(s，9H)，1.15(t，6H).

Example 14

To a solution of 4, 6-dichloro-5-aminopyrimidine (5.0g, 30.7mmol)/DMSO (30mL) was added Na₂S·9H₂O (7.4g, 30.8 mmol). The mixture was stirred at room temperature overnight. Water (40mL) was then added to the mixture and the solution was evaporated under reduced pressure to approximately 6 mL. To this solution was added concentrated HCl (0.5mL) and water to precipitate the product. The solution was filtered and the orange-yellow solid washed with water and dried to give 4.3g (86%) of the title compound.¹H NMR(300MHz，DMSO-d₆)δ 5.84(2H，s)，7.79(1H，s)，14.37(1H，br s)；MS(m/z)：MH⁺＝162.

Example 15

To dissolve in concentrated NH₄To the product of example 14 (4.3g, 26mmol) in OH (4mL) was added EtOH (40 mL). To this solution Raney nickel (in excess) was added in portions. The reaction was stirred at room temperature overnight and then heated at 80 ℃ for 2 hr. The mixture was filtered through celite and the filtrate was concentrated. The crude product was purified by flash chromatography on silica using EtOAc/hexanes to give 1.6g (47%) of the title compound as a yellow solid.¹H NMR(300MHz，DMSO-d₆)δ 5.90(2H，s)，8.20(2H，s)；MS(m/z)MH⁺＝130.

Example 16

To the product of example 15 (0.51g, 3.9mmol) in MeOH (20mL) and HOAc (0.5mL) was added CH₃CHO (0.52mL, 9.2 mmol). Then adding NaBH in one time₃CN (590mg, 9.2 mmol). The reaction was stirred at room temperature overnight and HOAc, CH were added₃CHO andNaBH₃and (C) CN. The reaction was stirred overnight, concentrated and the residue was taken up in EtOAc and saturated NaHCO₃In (1). The separated aqueous layer was back extracted with EtOAc. The combined organic layers were dried and concentrated to a residue. The residue was dissolved in MeOH and HOAc, CH were used as described above₃CHO and NaBH₃And (5) CN treatment. After the above work-up procedure, the crude product was purified by flash chromatography on silica using EtOAc/hexanes to give 0.35g (57%) of the title compound as a yellow oil.¹HNMR(300MHz，CDCl₃)δ 1.35(3H，q，J＝12Hz)，3.29(2H，m)，4.21(1H，bs)，8.04(1H，s)，8.36(1H，s)；MS(m/z)：MH⁺＝158.

Example 17

To the product of example 16 (70mg, 0.45mmol) dissolved in DMF (1mL) was added TEA (93. mu.L) and isonicotinoyl chloride (0.12g, 0.67 mmol). The reaction mixture was stirred at room temperature for 2 days, then in EtOAc and saturated NaHCO₃And (4) extracting. The separated aqueous layer was back extracted with EtOAc. The combined organic layers were dried and concentrated to give 67mg (57%) of the title compound, which was used without further purification.¹H NMR(300MHz，CDCl₃) δ 1.26(3H), 3.65-3.69(1H), 4.21(1H), 7.17(2H), 8.43(1H), 8.54(2H), 8.86(1H) note:¹h NMR shows that all peaks are very broad and indicate that rotamers exist; MS (m/z): MH⁺＝263。

Example 18

To a solution of the product from example 17 (0.11g, 0.42mmol) and the product from example 8 (0.135g, 0.38mmol) in IPA (2.5ml) was added DIEA (0.35ml, 1.9 mmol). The reaction mixture was stirred in a sealed tube at 130 deg.CAnd 2 days. The crude mixture was concentrated and the oil was purified by flash column chromatography using 0-10% MeOH/CH₂Cl₂Purification by solvent gradient gave the title compound as an oil.¹H NMR(300MHz，CDCl₃) δ 1.16(1.2H, m), 1.26-1.31(1.8H, m), 1.50-1.53(9H, d, J ═ 9Hz), 3.0(1H, m), 3.2(0.8H, m), 3.36(1.2H, m), 4.12-4.18(1.2H, m), 4.96-5.10(.8H, m), 5.80-5.95(1H, m), 6.93-6.96(1H, m), 7.07(1H, m), 7.31-7.45(5H, m), 7.66-7.75(3H, m), 8.06(1H, m), 8.44-8.51(2H, m); HPLC/MS: 1.29min Single Peak, MH⁺＝581.

Example 19

At 0 ℃ under N₂To 2, 4-dichloro-5-nitropyrimidine (2.0g, 10.3mmol) in MeOH (7mL) was added NaOMe (0.5M in MeOH, 25mL) dropwise. After the addition was complete, the reaction mixture was stirred at 0 ℃ for 15 min. Diethylamine (5mL) was then added and the mixture was stirred at room temperature overnight. The reaction mixture was concentrated and the residue was taken up in EtOAc and H₂And (4) extracting between O. The organic layer was dried and concentrated to a residue, which was purified by flash chromatography on silica using EtOAc/hexanes to give the title compound as an off-white solid (1.1g, 4.9mmol, 47% yield).¹HNMR(300MHz，CDCl3)δ 1.26(6H，t，J＝6.6Hz)，3.70(4H，m)，4.08(3H，s)，9.01(1H，s)；HPLC/MS：MH⁺＝227.

Example 20

EXAMPLE 19 product (1.1g, 4.9mmol) in MeOH/EtOAc (1:1, 20mL) with Pd/C (5% degussa, 0.5g) and H₂(50psi) was reduced overnight on a Parr shaker. The reaction mixture was filtered and the filtrate was concentrated under reduced pressure to give the title compound as a solidMaterial (0.85g, 4.3mmol, 88.5% yield).¹H NMR(300MHz，CDCl₃)δ 1.18(6H，t，J＝6.9Hz)，3.03(2H，br)，3.57(6H，t，J＝6.9Hz)，3.96(3H，s)，7.71(1H，s)；HPLC/MS：MH⁺＝197.

Example 21

To CH containing the product of example 20 (0.85g, 4.3mmol)₂Cl₂Isonicotinoyl chloride hydrochloride (1.13g, 6.3mmol) was added (15mL) and TEA (1.4mL, 10 mmol). After 15min, TLC showed no starting material. The mixture was washed with EtOAc and saturated NaHCO₃Extraction is carried out in between. The aqueous layer was washed twice with EtOAc. The combined organic layers were washed with saturated NaHCO₃And a saline flush. Over MgSO₄Dried and filtered. The filtrate was concentrated to give the title compound as a brown solid (1.3g, 4.3mmol, 100% yield).¹H NMR(300MHz，CDCl₃)δ 1.20(6H，t，J＝6.9Hz)，3.60(4H，q，J＝6.9Hz)，3.96(3H，s)，7.72(2H，d，J＝6.0Hz)，7.75(1H，bs)，8.80(2H，d，J＝6.0Hz)，8.89(1H，s)；HPLC/MS：MH⁺＝302.

Example 22

To the product of example 21 (100mg, 0.33mmol) in THF (1mL) was added KOtBu (1M in THF, 0.5mL) followed by slow addition of EtI (40. mu.L, 0.5 mmol). The reaction mixture was stirred at room temperature overnight. TLC showed the disappearance of starting material. The mixture was in EtOAc and H₂And extracting between O. The aqueous layer was washed with EtOAc. The combined organic layers were washed with saturated NaHCO₃And a saline flush. Concentration by drying afforded the title compound (90mg, 0.27mmol, 83%) which was used without further purification.¹H NMR(300MHz，CDCl₃)δ 1.10(9H，m)，3.47(5H，m)，3.92(1H，m)，7.14(2H，d，J＝6.0Hz)，7.78(1H，bs)，8.44(2H，d，J＝6.0Hz)；HPLC/MS：MH⁺＝330.

Example 23

To the product of example 22 (200mg, 0.61mmol) in DMF (4mL) was added EtSNa (66mg, 0.79mmol) and the reaction mixture was heated at 100 ℃ for 1 hr. LC/MS showed that the starting material was still present. Another portion of NaSEt (66mg, 0.79mmol) was added and the reaction heated for an additional 2 hr. LC/MS showed only product. DMF was removed under reduced pressure, water (10mL) was added, followed by concentrated HCl (0.132 mL). The solvent was evaporated to give a residue. The residue was dissolved in EtOH and filtered. The filtrate was concentrated to give the title compound (190mg, 100%) which was used without further purification.¹H NMR(300MHz，CD₃OD)δ 1.24(9H，m)，3.60(4H，m)，3.60-4.00(2H，br)，8.12(3H，d，J＝5.7Hz)，8.92(2H，d，J＝5.7Hz)；HPLC/MS：MH⁺＝316.

Example 24

To the product of example 23 (70mg, 0.22mmol)/POCl₃To (3mL) was added diethylaniline (30. mu.L) at room temperature. The reaction mixture was heated to 100 ℃ for 30 min. And then concentrated. The residue was in EtOAc and H₂And extracting between O. H for organic layer₂And washing twice with O. Then dried and concentrated to give the title compound (50mg, 0.15mmol, 68%) which was used in the next reaction without further purification. HPLC/MS: MH⁺＝334。

Example 25

To a solution of the product from example 24 (50mg, 0.15mmol) and the product from example 8 (60mg, 0.17mmol) in IPA (0.75mL) was added DIEA (0.15mL, 0.8 mmol). The reaction mixture was stirred in a sealed tube at 130 ℃ for 7 days. The crude mixture was concentrated and the residue was purified by preparative HPLC and flash chromatography on silica gel to give an off-white solid (10 mg). 1HNMR (300MHz, CDCl3) δ 1.10-1.30(9H, m), 1.48(4.5H, s), 1.51(4.5H, s), 2.80-3.38(3H, m), 3.53(4H, m), 4.05-4.30(1H, m), 4.83(0.5H, m), 4.96(0.5H, m), 5.15-5.50(1H, m), 6.95-7.10(2H, m), 7.25-7.50(5H, m), 7.69(0.5H, d, J ═ 8.4Hz), 7.76(0.5H, d, J ═ 8.4Hz), 8.08(1H, d, J ═ 5.1Hz), 8.51(2H, m), 8.83(0.5H, 8.95, br H, 0.5H, br);

HPLC/MS：MH+＝652.

example 26

Step A

Example 26A

40kDa 3-arm PEG alcohol (0.25g, 0.00625mmol), the product of example 5 (0.04g, 0.056mmol) and triphenylphosphine (0.025g, 0.094mmol) were dried by azeotropic distillation from toluene (5 mL). Half of the volume was distilled off (2.5mL) and the mixture was cooled to room temperature. Adding CH₂Cl₂The reaction was homogeneous (0.5 mL). Diethyl azodicarboxylate (0.015mL, 0.094mmol) was added dropwise and the reaction stirred for 48 hours. HPLC method C showed complete disappearance of the original PEG alcohol. The reaction was concentrated in vacuo to give the tert-butyl ester of example 26A as a white solid.

Example 26B

Example 26A (0.2g, 0.005mmol) was dissolved in formic acid (3 m)L), the mixture was heated at 40 ℃ for 24 hours. The reaction was concentrated in vacuo and purified according to HPLC method A to afford example 26B as a white solid, 0.1g (48%). Determination of conjugates by HPLC method C>99% purity (retention time ═ 8.1 minutes).¹H NMR(CDCl₃)δ 9.08(bs，3H)，8.84(bs，3H)，8.18-8.16(d，3H)，8.02-8.00(d，3H)，7.67-7.61(m，6H)，7.47-7.38(m，9H)，7.08-7.04(m，3H)，6.91(m，3H)，4.88(m，3H)，4.62-4.49(dd，6H)，4.13(m，6H)，3.64(bs，5919H PEG)，3.23(m，6H)，1.25-1.24(d，18H).

Step B

A mixture of GL400 Sunbright PEG (50.0g, NOF lot # M4N594), the product of example 5 (7.19g, 9 equivalents compared to GL400, 3 equivalents/hydroxy) and triphenylphosphine (2.95g, 9 equivalents) was charged to toluene (300mL) and water was removed by azeotropic distillation. The mixture was cooled to room temperature and a further amount of remaining toluene was removed by rotary evaporation. The mixture was again dissolved in anhydrous dichloromethane (180mL) and cooled in an ice bath. Diisopropyl azodicarboxylate (DIAD, 2.27g, 2.17mL, 9 equivalents) was added over 1 hour via an injection drive. The mixture was stirred in the ice bath for 1.5h, during which HPLC analysis indicated a complete conversion to example 26A.

The viscous reaction mixture was added slowly and with stirring to a 75:25MTBE/IPA (4.0L) mixture via a fine-necked funnel and stirred for 1 hour. The precipitate was collected by vacuum filtration, washed with MTBE (200mL), and dried in vacuo to afford purified example 26A (51.5 g).

Example 26A (51.4g) was charged to formic acid (250mL) and heated to near reflux for 1.0h, during which HPLC analysis indicated complete deprotection. The mixture was cooled to room temperature and a portion of the formic acid (-100 mL) was removed by rotary evaporation. The mixture was diluted with dichloromethane (50 mL). The viscous reaction mixture was then added to a 75:25MTBE/IPA (4.0L) mixture with stirring and stirred for 45min (Note 4). The precipitate was collected by vacuum filtration, washed with MTBE (300ml) and dried in vacuo (<1Torr, 24h) to give example 26B (50.0 g).

The material was taken up in dichloromethane (400mL) and filtered through a sintered glass buchner funnel ("polish filtration"). The filtrate was concentrated by rotary evaporation to a volume of-200 mL and added slowly and with stirring to a 75:25 mixture of MTBE/IPA (4.0L). The precipitate was collected by vacuum filtration, washed with MTBE (300ml), and dried in vacuo (<1Torr, 3 days) to afford example 26B (48.8g,. about.94%).

The following conjugates were synthesized using a similar method:

example 27

Example 27

A40 kDa 4-arm PEG alcohol was coupled to the product of example 5 and deprotected to the final product using a method analogous to example 26. The product was purified according to HPLC method a. Determination of conjugates by HPLC method C>95% purity (retention time 7.5-8.1 min).¹H NMR(CDCl₃)δ 9.08(bs，4H)，8.84(bs，4H)，8.18-8.16(d，4H)，8.02-8.00(d，4H)，7.67-7.61(m，8H)，7.47-7.38(m，12H)，7.08-7.04(m，4H)，6.91(m，4H)，4.88(m，4H)，4.62-4.49(dd，8H)，4.13(m，8H)，3.64(bs，10101H PEG)，3.23(m，8H)，1.25-1.24(d，24H).

Example 28

The 40kDa 3-arm PEG alcohol was coupled with the tert-butyl ester product from example 18 (shown below) and deprotected to the final product using a method analogous to that of example 26. The product was purified according to HPLC method a. Determination of conjugates by HPLC method C>95% purity (retention time 7.3 min).¹H NMR(CDCl₃)δ 8.66(bs，3H)，8.44(bs，3H)，8.04-8.02(d，3H)，7.75-7.30(m，24H)，7.10-7.06(m，3H)，6.93(s，3H)，5.60-5.50(m，3H)，4.15(m，6H)，3.66(bs，4270H PEG)，3.00(m，3H)，3.40-3.20(m，6H)，1.27(d，9H).

Example 29

Example 29A

The 40kDa 3-arm PEG alcohol (0.00625mmol), the product from example 13 (0.056mmol) and triphenylphosphine (0.094mmol) were dried from toluene (5mL) by azeotropic distillation. Half of the volume was distilled off (2.5mL) and the mixture was cooled to room temperature. Adding CH₂Cl₂The reaction was homogeneous (0.5 mL). Diethyl azodicarboxylate (0.094mmol) was added dropwise and the reaction stirred for 48 hours. The reaction was concentrated in vacuo to give the tert-butyl ester of example 29A.

Example 29B

Example 29A (0.005mmol) was dissolved in formic acid (3mL) and heated at 40 ℃ for 24 h. The reaction was concentrated in vacuo and purified according to HPLC method a to afford example 29B.

Example 30

A40 kDa 3-arm PEG alcohol was coupled with the tert-butyl ester product from example 25 and deprotected to form the final product using a method analogous to example 26. The product was purified according to HPLC method a.

Reference to the literature

The following publications, patents and patent applications are incorporated herein by reference in their entirety:

1 Hemler and Takada, European patent application publication No. 330,506, published on 30/8 in 1989

2 Elices et al, Cell, 60: 577-584(1990)

3 Springer，Nature，346：425-434(1990)

4 Osborn，Cell，62：3-6(1990)

5 Vedder et al, Surgery, 106: 509(1989)

6 Pretolani et al, j.exp.med., 180: 795(1994)

7 Abraham et al, j.clin.invest, 93: 776(1994)

8 Mulligan et al, j. immunology, 150: 2407(1993)

9 Cybutsky et al, Science, 251: 788(1991)

10 Li et al, ariterioscler, thromb, 13: 197(1993)

11 Sasseville et al, am.j.path, 144: 27(1994)

12 Yang et al, proc.nat.acad.science (USA), 90: 10494(1993)

13 Burkly et al, Diabetes, 43: 529(1994)

14 Baron et al, j.clin.invest, 93: 1700(1994)

15 Hamann et al, j.immunology, 152: 3238(1994)

16 Yednock et al, Nature, 356: 63(1992)

17 Baron et al, j.exp.med., 177: 57(1993)

18 van Dinther-Janssen et al, J.immunology, 147: 4207(1991)

19 van Dinther-Janssen et al, Annals. 672(1993)

20 Elces et al, J.Clin.Invest., 93: 405(1994)

21 Postigo et al, J.Clin.Invest., 89: 1445(1991)

22 Paul et al, transfer. 813(1993)

23 okarrara et al, can.res., 54: 3233(1994)

24 Paavonen et al, int.j.can., 58: 298(1994)

25 Schadendorf et al, j.path, 170: 429(1993)

26 Bao et al, diff., 52: 239(1993)

27 Lauri et al, British j. cancer, 68: 862(1993)

28 Kawaguchi et al, japan j. cancer res, 83: 1304(1992)

29 Kogan et al, U.S. Pat. No. 5,510,332, granted on 23/4/1996

30 International patent application publication WO 96/01644

31 Thorsett et al, U.S. patent No. 6,489,300, granted at 3.12.2002 and Konradi et al, U.S. patent No. 66,492,372, granted at 10.12.2002.

All publications, patents and patent applications above are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

Claims

1. A method of preparing a conjugate of an active compound comprising:

a) reacting a polymeric alcohol with a nucleophilic active compound having an acidic hydrogen under Mitsunobu conditions; and

b) the conjugate is isolated.

2. The process of claim 1 wherein said conditions comprise a trivalent phosphine and an azodicarbonyl compound.

3. A method of preparing a conjugate of formula I:

b is a biocompatible polymer moiety optionally covalently bound to a stem molecule having branched arms;

q is from about 1to about 100;

in each case A is independently a compound of the formula II

Or a pharmaceutically acceptable salt thereof, wherein

J is selected from:

a) a group of formula (a):

and R is³²Is a covalent bond with a polymer moiety optionally comprising a linking group, or R³²is-H, -NO₂Haloalkyl or group-N (MR)⁴¹)R⁴²Wherein M is a covalent bond, -C (O) -, or-SO₂-，R⁴¹Is R^41′、N(R^41′)₂OR-OR^41′，

Wherein each R^41′Independently hydrogen, optionally substituted straight or branched C₁-C₆Alkyl, optionally substitutedSubstituted cycloalkyl, optionally substituted aryl, optionally substituted heterocycle or optionally substituted heteroaryl, wherein the optional substituents are halogen, C₁-C₆Alkyl, or-OC₁-C₆An alkyl group, a carboxyl group,

and R is⁴²Is hydrogen or R^41′(ii) a And

b) a group of formula (b):

wherein R is selected from the group consisting of a covalent bond to a polymer moiety, an amino group, a hydroxyl group, a substituted amino group, an alkyl group, an alkoxy group, an aryloxy group, a heteroaryloxy group, a heterocyclyloxy group, a thiol group, an arylthio group, a heteroarylthio group, a heterocyclylthio group, and a substituted alkyl group, wherein each amino group, substituted amino group, alkyl group, and substituted alkyl group is optionally covalently attached to the polymer moiety, wherein, in each case, the polymer moiety optionally includes a linking group covalently attached to the polymer moiety;

t is selected from;

a) a radical of the formula (c)

w is selected from the group consisting of a covalent bond with a polymer moiety optionally including a linking group, and-NR²R³Wherein R is²And R³Independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, and wherein R²And R³Together with the nitrogen atom to which they are bound, form a heterocyclic ring or substituted heterocyclic ring, wherein each of the alkyl, substituted alkyl, heterocyclic ring and substituted heterocyclic ring is optionally covalently bound to a polymer moiety, said polymer moiety optionally further comprising a linking group;

m is an integer equal to 0,1 or 2;

n is an integer equal to 0,1 or 2; and

b) a radical of the formula (d)

R⁶is a covalent bond with a polymer moiety optionally comprising a linking group, or R⁶is-H, alkyl, substituted alkyl or-CH₂C(O)R¹⁷Wherein R is¹⁷is-OH, -OR¹⁸or-NHR¹⁸Wherein R is¹⁸Is alkyl, substituted alkyl, aryl or substituted aryl;

R⁵⁵selected from the group consisting of amino, substituted amino, alkoxy, substituted alkoxy, cycloalkoxy, substituted cycloalkoxy, aryloxy, and substituted aryloxy, and-OH;

with the following conditions:

A.R、Ar¹、Ar²and at least one of T comprises a covalent bond with a polymer moiety;

B. when R is covalently bound to a polymer moietyWhen linked, n is 1 and X is not-O-, -S-, -SO-or-SO₂-；

C. When X is-O-or-NR¹-when then m is 2; and is

D. The molecular weight of the conjugate of formula I is no more than 100,000;

the method comprises the following steps:

c) reacting a polymeric alcohol of formula Ia

Wherein B is as described above in formula PR₃To a nucleophile of formula H-Nu in the presence of a trivalent phosphine of (a) and a dialkyl azodicarboxylate to form a compound of formula I, wherein Nu is a group of formula a above; and isolating the compound of formula I.

4. The method of claim 1, wherein J, Ar²And only one of T contains a covalent bond with the polymer moiety.

5. The method of claim 1, wherein q is an integer from 1to about 20.

6. The method of claim 1, wherein q is an integer from 1to about 8.

7. The process according to claim 1, wherein in each case A is independently a compound of the formula IIa

8. The method of claim 1, wherein a is independently at each occurrence a compound of formula lib:

9. the method of claim 1, wherein a is independently at each occurrence a compound of formula IIc:

10. the method of claim 1 wherein said nucleophilically active compound is a compound of the formula

Wherein

J is selected from:

a) a group of formula (a):

Wherein each R^41′Independently hydrogen, optionally substituted straight or branched C₁-C₆Alkyl, optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted heterocycle or optionally substituted heteroaryl, wherein the optional substituents are halogen, C₁-C₆Alkyl, or-OC₁-C₆An alkyl group, a carboxyl group,

and R is⁴²Is hydrogen or R^41′(ii) a And

b) a group of formula (b):

t is a group bearing an acidic hydrogen; and is

R⁵⁵Is an acid protecting group.

11. The method of claim 10 wherein said nucleophilically active compound has the formula:

wherein R is⁵⁵Is an acid protecting group.

12. The method of claim 11, wherein R⁵⁵Is C₁-C₆An alkoxy group.

13. The method of claim 11, wherein m is 1, X is S, and R is independently selected at each occurrence from hydroxyl, alkoxy, alkyl, or a covalent bond with a polymer moiety.

14. The method of claim 12, wherein n is 2 and R is methyl in both instances.

15. The method of claim 1 wherein said nucleophilically active compound has the formula:

wherein R is⁵⁵Is an acid protecting group.

16. The method of claim 15, wherein R⁵⁵Is C₁-C₆An alkoxy group.

17. The method of claim 15, wherein G is pyridyl and R³¹Is hydrogen or dialkylamino, and R³²Is a sulfonamide, amide or urea.

18. The method of claim 1, wherein the nucleophilic active compound is:

or

19. The method of claim 1 wherein said polymeric alcohol is

(total Mw of conjugate is about 41,500), (total Mw of conjugate is about 42,000), or

(the total Mw of the conjugate was about 41,500).

20. The method of claim 1, wherein the polymeric alcohol is selected from the group in column a and the nucleophilic active compound is selected from the group in column B:

(Total Mw of the conjugate is about 41,500)

(Total Mw of conjugate is about 42,000)

(Total Mw of the conjugate is about 41,500)

21. The process according to claim 1, wherein the polymeric alcohol is added to the nucleophile of formula H-Nu in the presence of a trivalent phosphine and a dialkyl azodicarboxylate in at least one solvent.

22. The process of claim 21 wherein said dialkyl azodicarboxylate is selected from the group consisting of diethyl azodicarboxylate, diisopropyl azodicarboxylate, 4-methyl-1, 2, 4-triazolidine-3, 5-dione, N' -tetramethylazodicarboxamide, dipiperidine azodicarboxylate, bis (N-4-methylpiperazin-1-yl) azodicarboxamide, dimorpholinoazodicarboxamide, and di-tert-butyl azodicarboxylate.

23. The method of claim 22 wherein the dialkyl azodicarboxylate is selected from the group consisting of diethyl azodicarboxylate, diisopropyl azodicarboxylate, 4-methyl-1, 2, 4-triazolidine-3, 5-dione, N' -tetramethyl azodicarboxamide, dipiperidine azodicarboxylate, and di-tert-butyl azodicarboxylate.

24. The method of claim 21, wherein the solvent is a chlorinated solvent or an ether solvent.

25. The process of claim 24 wherein the solvent is dichloromethane or tetrahydrofuran.

26. The method of claim 21, wherein the trivalent phosphine is selected from the group consisting of triphenylphosphine, trimethylphosphine, triethylphosphine, tributylphosphine, 1, 2-bis- (diphenylphosphino) ethane, polymer-bound phosphine, and water-soluble phosphine.

27. The method of claim 26 wherein the trivalent phosphine is triphenylphosphine.

28. The method of claim 1, wherein the conjugate is prepared using a polymeric alcohol/nucleophilic active compound combination 1, 2, or 3:

combinatorial polymeric alcohol nucleophilic active compounds

(Total Mw of the conjugate is about 41,500)

(Total Mw of conjugate is about 42,000)