HK1250718B - Intermediates and methods for synthesizing calicheamicin derivatives - Google Patents

Description

Intermediates and methods for synthesizing calicheamicin derivatives

This application is a divisional application of the patent application with application number 201480060096.9, application date October 28, 2014, and invention name “Intermediates and methods for synthesizing calicheamicin derivatives”.

Technical Field

The present invention relates to the synthesis of calicheamicin derivatives. The present invention also relates to intermediates and linker molecules that can be used to prepare calicheamicin derivatives and to conjugate calicheamicin to biomacromolecules such as monoclonal antibodies.

Background Art

Gemtuzumab ozogamicin (gemtuzumab ozogamicin) consists of a monoclonal antibody against CD33 conjugated to calicheamicin via an acid-hydrolyzable linker. This product is marketed as the first antibody-targeted chemotherapy agent and is approved for the treatment of acute myeloid leukemia (AML) in elderly patients. Inotuzumab ozogamicin is a CD22 antibody linked to calicheamicin currently in clinical trials for the treatment of certain types of cancer.

An effective family of antibacterial and antitumor drugs, collectively known as calicheamicin or LL-E33288 complexes, is disclosed in U.S. Patent No. 4,970,198. U.S. Patent No. 5,053,394 also discloses methyl trisulfide antibacterial and antitumor drugs. These compounds in U.S. Patent No. 4,970,198 and U.S. Patent No. 5,053,394 contain a methyl trisulfide group that can react with suitable thiols to form disulfide bonds while introducing functional groups such as hydrazides or similar nucleophiles. An example of such a reaction with calicheamicin is given in U.S. Patent No. 5,053,394. U.S. Patent No. 5,770,701 relates to a method for preparing a targeted form of a disulfide compound of the LL-E33288 complex. The linker 4-(4-acetyl-phenoxy)butyric acid is condensed with N-acetyl gamma-calicheamicin dimethyl hydrazide to afford the carboxylic acid-hydrazone, which is further treated with N-hydroxysuccinimide to afford the OSu ester (N-succinimidyloxy), which is readily conjugated to selected biomacromolecules.

U.S. Patent No. 8,273,862 describes a synthetic method for constructing linker intermediate molecules (referred to herein as "trilinker-activated esters" or "trifunctional linker intermediates"). These linker intermediates can be conjugated to calicheamicin to produce calicheamicin derivatives, which can then be further conjugated to biomacromolecules, such as monoclonal antibodies. In the steps leading to the preparation of the linker intermediates, the synthetic method described in U.S. Patent No. 8,273,862 uses a thiol compound ("Compound 2 "), such as 3-methyl-3-mercaptobutyric acid hydrazide, as an intermediate.

WO 2008/147765 describes a method for synthesizing sulfhydryl-containing intermediates, such as 3-methyl-3-mercaptobutyric acid hydrazide, which can be used to prepare linker intermediate acids and calicheamicin derivatives as described in the above paragraphs. WO 2008/147765 also relates to 3-methyl-3-mercaptobutyric acid hydrazide as a "DMH linker." WO 2008/147765 notes that 3-methyl-3-mercaptobutyric acid hydrazide is the preferred sulfhydryl-containing N-acylhydrazine for the purpose of linking calicheamicin to monoclonal antibodies to prepare, for example, gemtuzumab ozogamicin or inotuzumab ozogamicin.

In WO 2008/147765, the compound 3-methyl-3-mercaptobutyric acid hydrazide is prepared by removing the benzyl protecting group from p-methoxybenzyl sulfide hydrazide under acidic conditions. To obtain p-methoxybenzyl sulfide hydrazide, WO 2008/147765 teaches that p-methoxybenzyl sulfide acid and oxalyl chloride are first reacted in dichloromethane to form p-methoxybenzyl sulfide chloride. The p-methoxybenzyl sulfide chloride is then added to a mixture of anhydrous hydrazine and dichloromethane to obtain p-methoxybenzyl sulfide hydrazide. However, as described in WO 2008/147765, the two reactants, p-methoxybenzyl sulfide acid and p-methoxybenzyl sulfide chloride, themselves together generate the undesirable byproduct bismethoxybenzyl sulfide hydrazide, resulting in lower yield and quality. Furthermore, as described in WO 2008/147765, due to the reactive and unstable nature of the acyl chloride molecule p-methoxybenzyl sulfide acyl chloride, anhydrous hydrazine and low temperatures, such as -70°C, must be used.

Another aspect of preparing calicheamicin derivatives involves chemically linking calicheamicin to a linker molecule. To conjugate calicheamicin to the linker intermediate of U.S. Patent No. 8,273,862, a final reaction is performed between calicheamicin and a "trilinker-activated ester" (or "trifunctional linker intermediate"). Structurally, as explained above, calicheamicin contains a trisulfide moiety used in its derivatized form, and the chemistry of this reaction between the trisulfide moiety of calicheamicin and the trilinker-activated ester is important in achieving good yields and purity. Previously used sulfur exchange reactions for calicheamicin derivatives resulted in complex reaction mixtures, multiple byproducts, and low yields.

Finally, the calicheamicin derivative is purified after its formation. The method of U.S. Patent No. 8,273,862 includes purifying a calicheamicin derivative, which method includes a normal phase chromatography step. The normal phase chromatography step in the purification method described in U.S. Patent No. 8,273,862 uses dichloromethane as a solvent, and as described above, exposure to dichloromethane above various minimum levels is considered to pose a potential health hazard. Therefore, when using a normal phase chromatography method that includes dichloromethane, care must be taken to limit exposure to dichloromethane.

Summary of the Invention

The present invention provides novel intermediates and methods for synthesizing and purifying linker intermediates that can be used to conjugate calicheamicin-class antitumor antibiotics to biomacromolecules, such as monoclonal antibodies. The present invention also provides novel methods for synthesizing calicheamicin derivatives comprising calicheamicin covalently bonded to a linker. The calicheamicin derivatives thus prepared can be conjugated to biomacromolecules, such as monoclonal antibodies, to prepare antibody-drug conjugates. For example, the intermediates and synthesis methods of the present invention can be used to prepare calicheamicin derivatives that are used to prepare gemtuzumab ozogamicin or inotuzumab ozogamicin.

The present invention provides improvements to prior art methods for synthesizing and purifying calicheamicin derivatives that overcome some of the problems associated with these prior art methods.

As explained above, WO 2008/147765 teaches that the intermediate p-methoxybenzyl sulfide acid is first reacted with oxalyl chloride in dichloromethane to form a p-methoxybenzyl sulfide chloride intermediate that can be used to synthesize calicheamicin derivatives. In WO 2008/147765, p-methoxybenzyl sulfide chloride is then added to a mixture of anhydrous hydrazine and dichloromethane to obtain a p-methoxybenzyl sulfide hydrazide intermediate. However, as described in WO 2008/147765, the two reactants p-methoxybenzyl sulfide acid and p-methoxybenzyl sulfide chloride themselves jointly generate the undesirable byproduct bis-methoxybenzyl sulfide hydrazide, resulting in lower yield and quality. The present invention solves this problem of bis-methoxybenzyl sulfide hydrazide byproduct by completely avoiding the use of p-methoxybenzyl sulfide chloride as an intermediate. By completely avoiding the acid chloride intermediate, p-methoxybenzyl sulfide chloride, the present invention now also allows the use of hydrated forms of hydrazine, which do not require the same specialized handling procedures as anhydrous hydrazine, and this new process also avoids the cumbersome requirement for low temperatures. Finally, because the use of p-methoxybenzyl sulfide chloride is avoided in the present invention, the use of dichloromethane and the safety precautions used with methoxybenzyl sulfide chloride are not required.

The present invention also improves the yield of the reaction between calicheamicin and a linker intermediate compared to existing methods, such as the method described in US Patent No. 8,273,862. The present invention improves the yield of the resulting calicheamicin derivative by including a carbodiimide in the reaction.

As described herein, it has also been discovered that a novel method for purifying calicheamicin derivatives can be obtained, comprising the use of reversed-phase high performance liquid chromatography (RP-HPLC), despite the presence of two water-labile groups (hydrazone and N-hydroxysuccinimide ester) on the calicheamicin derivatives. Thus, the present invention overcomes the aforementioned problems associated with the use of normal phase chromatography, e.g., using dichloromethane, as described in U.S. Patent No. 8,273,862.

The present invention provides a compound of formula I,

wherein R ¹² is selected from linear and branched C ₁ -C ₈ alkyl groups;

R ¹⁰ is each independently selected from hydrogen, R ¹² and -OR ¹² ;

R ⁸ and R ⁹ are each independently selected from hydrogen and linear and branched C ₁ -C ₈ alkyl, wherein said R ⁸ and R ⁹ alkyl are each independently optionally substituted with -NH ₂ , -NHR ¹¹ , -NR ¹¹ R ¹³ , -OR ¹¹ , -OH, or -SR ¹¹ , wherein each R ¹¹ and each R ¹³ are independently selected from linear and branched C ₁ -C ₅ alkyl;

r is an integer selected from 0 and 1;

G is oxygen or sulfur;

Z ¹ is H or a linear or branched C ₁ -C ₅ alkyl group;

Ar is 1,2-, 1,3- or 1,4-phenylene optionally substituted by 1, 2 or 3 groups independently selected from linear or branched C ₁ -C ₆ alkyl, -OR ¹⁴ , -SR ¹⁴ , halogen, nitro, -COOR ¹⁴ , -C(═O)NHR ¹⁴ , -O(CH ₂ ) _n COOR ¹⁴ , -S(CH ₂ ) _n COOR ¹⁴ , -O(CH ₂ ) _n C(═O)NHR ¹⁴ and -S(CH ₂ ) _n C(═O)NHR ¹⁴ , or Ar is 1,2-, 1,3-, 1,4-, 1,5-, 1,6-, 1,7-, 1,8-, 2,3-, 2,6- or 2,7-naphthylene optionally substituted by 1, 2, 3 or 4 groups independently selected from linear or branched C 1 -C 6 alkyl, ₁ -C ₆ alkyl, —OR ¹⁴ , —SR ¹⁴ , halogen, nitro, —COOR ¹⁴ , —C(═O)NHR ¹⁴ , —O(CH ₂ ) _n COOR ¹⁴ , —S(CH ₂ ) _n COOR ¹⁴ , —O(CH ₂ ) _n C(═O)NHR ¹⁴ , and —S(CH ₂ ) _n C(═O)NHR ¹⁴ ;

wherein each R ¹⁴ is independently selected from (C ₁ -C ₅ )alkyl, and each R ¹⁴ is independently optionally substituted with 1 or 2 groups selected from —OH, —(C ₁ -C ₄ )alkyl, and —S(C ₁ -C ₄ )alkyl;

Each n is an integer independently selected from 0, 1, 2, 3, 4 and 5;

W is selected from —O—, —S—, —C(═O)NH—, —NHC(═O)—, and —NR ¹⁵ —, wherein R ¹⁵ is (C ₁ -C ₅ )alkyl, and R ¹⁵ is optionally substituted with 1 or 2 groups selected from —OH, —(C ₁ -C ₄ )alkyl, and —S(C ₁ -C ₄ )alkyl; and

Y is a linear or branched (C ₁ -C ₆ )alkylene group or a linear or branched (C ₂ -C ₆ )alkenylene group.

Compounds of Formula 1 are useful as intermediates in the synthesis of linker intermediates and calicheamicin derivatives containing such linker intermediates, which in turn can be conjugated to biomacromolecules such as monoclonal antibodies.

In one embodiment of the invention, each R ¹⁰ on the compound of formula I is hydrogen. In another embodiment, each R ¹⁰ on the compound of formula I is hydrogen, and R ¹² is methyl.

In another embodiment of the present invention, the compound of formula I is a compound having the following structure:

In another embodiment of the invention, R ⁸ and R ⁹ on the compound of Formula I are both methyl, r is 0, G is oxygen, Z ¹ is methyl, Ar is 1,4-phenylene, W is —O—, and Y is —(CH ₂ ) ₃ —.

In another embodiment of the present invention, R ⁸ and R ⁹ on the compound of formula I are both methyl.

In another embodiment of the present invention, r in the compound of formula I is 0. In another embodiment of the present invention, r in the compound of formula I is 0, and G is oxygen. In another embodiment of the present invention, r in the compound of formula I is 0, and G is sulfur.

The present invention also provides a method for synthesizing the compound of formula I mentioned above, as described, the compound of formula I can be used as an intermediate for synthesizing a linker intermediate and a calicheamicin derivative containing the linker intermediate group. In one embodiment, the present invention provides a method for synthesizing a compound of formula I,

wherein R ¹² is selected from linear and branched C ₁ -C ₈ alkyl groups;

R ¹⁰ is each independently selected from hydrogen, R ¹² and -OR ¹² ;

R ⁸ and R ⁹ are each independently selected from hydrogen and linear and branched C ₁ -C ₈ alkyl, wherein said R ⁸ and R ⁹ alkyl are each independently optionally substituted with -NH ₂ , -NHR ¹¹ , -NR ¹¹ R ¹³ , -OR ¹¹ , -OH, or -SR ¹¹ , wherein each R ¹¹ and each R ¹³ are independently selected from linear and branched C ₁ -C ₅ alkyl;

r is an integer selected from 0 and 1;

G is oxygen or sulfur;

Z ¹ is H or a linear or branched C ₁ -C ₅ alkyl group;

Ar is 1,2-, 1,3- or 1,4-phenylene optionally substituted by 1, 2 or 3 groups independently selected from linear or branched C ₁ -C ₆ alkyl, -OR ¹⁴ , -SR ¹⁴ , halogen, nitro, -COOR ¹⁴ , -C(═O)NHR ¹⁴ , -O(CH ₂ ) _n COOR ¹⁴ , -S(CH ₂ ) _n COOR ¹⁴ , -O(CH ₂ ) _n C(═O)NHR ¹⁴ and -S(CH ₂ ) _n C(═O)NHR ¹⁴ , or Ar is 1,2-, 1,3-, 1,4-, 1,5-, 1,6-, 1,7-, 1,8-, 2,3-, 2,6- or 2,7-naphthylene optionally substituted by 1, 2, 3 or 4 groups independently selected from linear or branched C 1 -C 6 alkyl, ₁ -C ₆ alkyl, —OR ¹⁴ , —SR ¹⁴ , halogen, nitro, —COOR ¹⁴ , —C(═O)NHR ¹⁴ , —O(CH ₂ ) _n COOR ¹⁴ , —S(CH ₂ ) _n COOR ¹⁴ , —O(CH ₂ ) _n C(═O)NHR ¹⁴ , and —S(CH ₂ ) _n C(═O)NHR ¹⁴ ;

wherein each R ¹⁴ is independently selected from (C ₁ -C ₅ )alkyl, and each R ¹⁴ is independently optionally substituted with 1 or 2 groups selected from —OH, —(C ₁ -C ₄ )alkyl, and —S(C ₁ -C ₄ )alkyl;

Each n is an integer independently selected from 0, 1, 2, 3, 4 and 5;

W is selected from -O-, -S-, -C(=O)NH-, -NHC(=O)- and -NR ¹⁵ -, wherein R ¹⁵ is (C ₁ -C ₅ )alkyl, and R ¹⁵ is optionally substituted with 1 or 2 groups selected from -OH, -(C ₁ -C ₄ )alkyl and -S(C ₁ -C ₄ )alkyl; and Y is a linear or branched (C ₁ -C ₆ )alkylene group or a linear or branched (C ₂ -C ₆ )alkenylene group; the method comprises reacting a compound of formula II

wherein R ¹⁰ , R ¹² , R ⁸ , R ⁹ , r and G are as defined above, reacted with a compound of formula III,

wherein Z ¹ , Ar, W, and Y are as defined above. In one embodiment of the method of synthesizing the compound of Formula I, r is 0, G is oxygen, Z ¹ is methyl, Ar is 1,4-phenylene, W is -O-, and Y is -(CH ₂ ) ₃ -. In another embodiment of the method of synthesizing the compound of Formula I, R ⁸ and R ⁹ are methyl. In one embodiment of the method of synthesizing the compound of Formula I, each R ¹⁰ is hydrogen. In another embodiment of the method of synthesizing the compound of Formula I, each R ¹⁰ is hydrogen, and R ¹² is methyl.

In another embodiment of the process of the present invention for synthesizing the compound of formula I, R ⁸ and R ⁹ on the compound of formula II are both methyl.

In another embodiment of the method of the present invention for synthesizing the compound of formula I, r in the compound of formula II is 0. In another embodiment of the method of the present invention for synthesizing the compound of formula I, r in the compound of formula II is 0, and G is oxygen. In another embodiment of the method of the present invention for synthesizing the compound of formula I, r in the compound of formula II is 0, and G is sulfur.

The present invention also provides a method for synthesizing a compound of formula II,

wherein R ¹² is selected from linear and branched C ₁ -C ₈ alkyl groups;

R ¹⁰ is each independently selected from hydrogen, R ¹² and -OR ¹² ;

R ⁸ and R ⁹ are each independently selected from hydrogen and linear and branched C ₁ -C ₈ alkyl, wherein each of said R ⁸ and R ⁹ alkyl groups is independently optionally substituted with -NH ₂ , -NHR ¹¹ , -NR ¹¹ R ¹³ , -OR ¹¹ , -OH, or -SR ¹¹ , wherein each R ¹¹ and each R ¹³ are independently selected from linear and branched C ₁ -C ₅ alkyl;

r is an integer selected from 0 and 1; and

G is oxygen or sulfur;

The method comprises treating a compound of formula VII with an azole-based activator of formula IX in an organic solvent,

wherein R ¹⁶ is —C(═O)OH or —C(═V)SH, wherein V is oxygen or sulfur, or R ¹⁶ is —NH ₂ ;

wherein V' is oxygen or sulfur; and

Where E is

wherein m is an integer of 0, 1, 2 or 3; q is an integer of 0, 1 or 2; and p is an integer of 0, 1, 2, 3 or 4; and wherein each R ¹⁷ attached to E is independently selected from linear and branched (C ₁ -C ₆ )alkyl groups;

To form a compound of formula VIII,

wherein when R ¹⁶ is -C(=O)OH, r is 0 and G is oxygen; when R ¹⁶ is -C(=V)SH, r is 0 and G is V; and when R ¹⁶ is -NH ₂ , r is 1 and G is V';

The compound of formula VIII is then combined with hydrazine to form the compound of formula II. The compound of formula II is useful as an intermediate in the synthesis of linker intermediates and calicheamicin derivatives containing the linker intermediate group, which in turn can be conjugated to biomacromolecules such as monoclonal antibodies.

The azole activator is any compound containing an azole structure, designated as Formula IX, which, when reacted with a compound of Formula VII, yields a compound of Formula VIII, wherein E is as described above. Examples of azole activators useful in the present invention include carbonyldiimidazole; thiocarbonyldiimidazole; carbonylbis-pyrazole, wherein each pyrazole is optionally substituted with 1 to 3 (C ₁ -C ₆ )alkyl groups; carbonylbis-1,2,3-triazole; carbonylbis-benzotriazole; and carbonylbis-1,2,4-triazole. Preferably, the azole activator is carbonyldiimidazole.

The compound of formula VIII is optionally isolated and then combined with hydrazine. In one embodiment, the compound of formula VIII is not isolated prior to combining with hydrazine. In another embodiment, the compound of formula VIII is isolated and then combined with hydrazine.

Preferably, R ¹⁶ is —C(═O)OH, and the azole activator is carbonyldiimidazole.

In one embodiment of the method of the present invention for preparing a compound of Formula II, in the compound of Formula II, r is 0 and G is oxygen. In another embodiment of the method for preparing a compound of Formula II, in the compound of Formula II, r is 0 and G is oxygen, and R ¹⁶ on the compound of Formula VII is -C(=O)OH. In another embodiment of the method for preparing a compound of Formula II, in the compound of Formula II, r is 0 and G is oxygen, R ¹⁶ on Formula VII is -C(=O)OH, and the azole activator is carbonyldiimidazole.

In another embodiment of the process of the present invention for preparing the compound of formula II, the compound of formula VIII has the following structure:

In another embodiment of the method for preparing the compound of Formula II, the compound of Formula VIII has the structure:

The azole activator is carbonyldiimidazole.

In another embodiment of the process of the present invention for synthesizing the compound of formula II, R ⁸ and R ⁹ on the compound of formula VII are both methyl.

In another embodiment of the method of the present invention for synthesizing a compound of Formula II, the compound of Formula VII comprises R ¹⁶ which is -C(=V)SH, and V is oxygen or sulfur. It should be understood that when the compound of Formula VII comprises R ¹⁶ which is -C(=V)SH and V which is oxygen, such compounds of Formula VII may exist as tautomers which are the same compound of Formula VII but wherein R ¹⁶ is -(C=S)OH. When the compound of Formula VII comprises R ¹⁶ which is -C(=O)SH and tautomers thereof, wherein R ¹⁶ is -C(=S)OH, the resulting product compound of Formula II comprises G which is oxygen.

In another embodiment of the method of the present invention for synthesizing a compound of Formula II, the compound of Formula VII comprises R ¹⁶ which is -NH _2. When the compound of Formula VII in the method for synthesizing a compound of Formula II comprises R ¹⁶ which is -NH ₂ , r in the compound of Formula II obtained from the method is 1. In another embodiment of the method for synthesizing a compound of Formula II, the method comprises a compound of Formula VII wherein R ¹⁶ is -NH ₂ ; and a compound of Formula IX wherein V' is oxygen; the compound of Formula II obtained from the method comprises r which is 1 and G which is V' (i.e., oxygen). In another embodiment of the method for synthesizing a compound of Formula II, the method comprises a compound of Formula VII wherein R ¹⁶ is -NH ₂ ; and a compound of Formula IX wherein V' is sulfur; the compound of Formula II obtained from the method comprises r which is 1 and G which is V' (i.e., sulfur).

In another embodiment of the method for synthesizing a compound of formula II, the method includes an azole-based activator of formula IX, wherein V' is oxygen. In another embodiment of the method for synthesizing a compound of formula II, the method includes an azole-based activator of formula IX, wherein V' is sulfur.

In another embodiment of the method for synthesizing the compound of Formula II, the hydrazine is anhydrous hydrazine. In another embodiment of the method for synthesizing the compound of Formula II, the hydrazine is hydrazine monohydrate. In another embodiment, the hydrazine is an aqueous solution of hydrazine. In another embodiment, the hydrazine is a solution of hydrazine in tetrahydrofuran.

As explained above, compounds of formula I are useful as intermediates for the synthesis of linker intermediates and calicheamicin derivatives comprising said linker intermediate groups. Thus, the present invention provides methods for the synthesis of compounds of formula IV, which are also useful as intermediates for the synthesis of linker intermediates and calicheamicin derivatives comprising said linker intermediate groups. In one embodiment, the present invention provides methods for the synthesis of compounds of formula IV,

wherein R ⁸ and R ⁹ are each independently selected from hydrogen and linear and branched C ₁ -C ₈ alkyl, wherein said R ⁸ and R ⁹ alkyl are each independently optionally substituted with -NH ₂ , -NHR ¹¹ , -NR ¹¹ R ¹³ , -OR ¹¹ , -OH, or -SR ¹¹ , wherein each R ¹¹ and each R ¹³ are independently selected from linear and branched C ₁ -C ₅ alkyl;

r is an integer selected from 0 and 1;

G is oxygen or sulfur;

Z ¹ is H or a linear or branched C ₁ -C ₅ alkyl group;

Ar is 1,2-, 1,3- or 1,4-phenylene optionally substituted by 1, 2 or 3 groups independently selected from linear or branched C ₁ -C ₆ alkyl, -OR ¹⁴ , -SR ¹⁴ , halogen, nitro, -COOR ¹⁴ , -C(═O)NHR ¹⁴ , -O(CH ₂ ) _n COOR ¹⁴ , -S(CH ₂ ) _n COOR ¹⁴ , -O(CH ₂ ) _n C(═O)NHR ¹⁴ and -S(CH ₂ ) _n C(═O)NHR ¹⁴ , or Ar is 1,2-, 1,3-, 1,4-, 1,5-, 1,6-, 1,7-, 1,8-, 2,3-, 2,6- or 2,7-naphthylene optionally substituted by 1, 2, 3 or 4 groups independently selected from linear or branched C 1 -C 6 alkyl, ₁ -C ₆ alkyl, —OR ¹⁴ , —SR ¹⁴ , halogen, nitro, —COOR ¹⁴ , —C(═O)NHR ¹⁴ , —O(CH ₂ ) _n COOR ¹⁴ , —S(CH ₂ ) _n COOR ¹⁴ , —O(CH ₂ ) _n C(═O)NHR ¹⁴ , and —S(CH ₂ ) _n C(═O)NHR ¹⁴ ;

wherein each R ¹⁴ is independently selected from (C ₁ -C ₅ )alkyl, and each R ¹⁴ is independently optionally substituted with 1 or 2 groups selected from —OH, —(C ₁ -C ₄ )alkyl, and —S(C ₁ -C ₄ )alkyl;

Each n is an integer independently selected from 0, 1, 2, 3, 4 and 5;

W is selected from —O—, —S—, —C(═O)NH—, —NHC(═O)—, and —NR ¹⁵ —, wherein R ¹⁵ is (C ₁ -C ₅ )alkyl, and R ¹⁵ is optionally substituted with 1 or 2 groups selected from —OH, —(C ₁ -C ₄ )alkyl, and —S(C ₁ -C ₄ )alkyl; and Y is a linear or branched (C ₁ -C ₆ )alkylene group or a linear or branched (C ₂ -C ₆ )alkenylene group, the method comprising treating the compound of formula I with a strong acid,

wherein R ¹² is selected from linear and branched C ₁ -C ₈ alkyl groups;

R ¹⁰ is each independently selected from hydrogen, R ¹² and -OR ¹² ;

and R ⁸ , R ⁹ , r, G, Z ¹ , Ar, W and Y are as defined;

A mixture comprising a compound of formula IV is formed. The strong acid used in the method for synthesizing the compound of formula IV can be determined by one of ordinary skill in the art as it is any acid that removes the substituted phenyl-methylene group from the sulfur atom to obtain the compound of formula IV. In one embodiment, the strong acid used in the method for synthesizing the compound of formula IV of the present invention is selected from trifluoroacetic acid, sulfuric acid, trifluoromethanesulfonic acid, HCl, HBr, HI. In one embodiment of the method for synthesizing the compound of formula IV, R ⁸ and R ⁹ are methyl, r is 0, G is oxygen, Z ¹ is methyl, Ar is 1,4-phenylene, W is –O-, and Y is –(CH ₂ ) ₃ -. In another embodiment of the method for synthesizing the compound of formula IV, R ¹⁰ is each hydrogen. In another embodiment of the method for synthesizing the compound of formula IV, R ¹⁰ is each hydrogen, and R ¹² is methyl.

In another embodiment of the present invention for synthesizing the compound of formula IV, R ⁸ and R ⁹ on the compound of formula I are both methyl.

In another embodiment of the present invention for synthesizing compounds of formula IV, r in the compound of formula I is 0. In another embodiment of the present invention for synthesizing compounds of formula IV, r in the compound of formula I is 0, and G is oxygen. In another embodiment of the present invention for synthesizing compounds of formula IV, r in the compound of formula I is 0, and G is sulfur.

The present invention also provides a method for synthesizing a linker intermediate of formula V using an intermediate of formula I and an intermediate of formula IV. In one embodiment, the present invention provides a method for synthesizing a linker intermediate of formula V,

wherein R ⁸ and R ⁹ are each independently selected from hydrogen and linear and branched C ₁ -C ₈ alkyl, wherein said R ⁸ and R ⁹ alkyl are each independently optionally substituted with -NH ₂ , -NHR ¹¹ , -NR ¹¹ R ¹³ , -OR ¹¹ , -OH, or -SR ¹¹ , wherein each R ¹¹ and each R ¹³ are independently selected from linear and branched C ₁ -C ₅ alkyl;

r is an integer selected from 0 and 1;

G is oxygen or sulfur;

Z ¹ is H or a linear or branched C ₁ -C ₅ alkyl group;

Ar is 1,2-, 1,3- or 1,4-phenylene optionally substituted by 1, 2 or 3 groups independently selected from linear or branched C ₁ -C ₆ alkyl, -OR ¹⁴ , -SR ¹⁴ , halogen, nitro, -COOR ¹⁴ , -C(═O)NHR ¹⁴ , -O(CH ₂ ) _n COOR ¹⁴ , -S(CH ₂ ) _n COOR ¹⁴ , -O(CH ₂ ) _n C(═O)NHR ¹⁴ and -S(CH ₂ ) _n C(═O)NHR ¹⁴ , or Ar is 1,2-, 1,3-, 1,4-, 1,5-, 1,6-, 1,7-, 1,8-, 2,3-, 2,6- or 2,7-naphthylene optionally substituted by 1, 2, 3 or 4 groups independently selected from linear or branched C 1 -C 6 alkyl, ₁ -C ₆ alkyl, —OR ¹⁴ , —SR ¹⁴ , halogen, nitro, —COOR ¹⁴ , —C(═O)NHR ¹⁴ , —O(CH ₂ ) _n COOR ¹⁴ , —S(CH ₂ ) _n COOR ¹⁴ , —O(CH ₂ ) _n C(═O)NHR ¹⁴ , and —S(CH ₂ ) _n C(═O)NHR ¹⁴ ;

wherein each R ¹⁴ is independently selected from (C ₁ -C ₅ )alkyl, and each R ¹⁴ is independently optionally substituted with 1 or 2 groups selected from —OH, —(C ₁ -C ₄ )alkyl, and —S(C ₁ -C ₄ )alkyl;

Each n is an integer independently selected from 0, 1, 2, 3, 4 and 5;

W is selected from —O—, —S—, —C(═O)NH—, —NHC(═O)—, and —NR ¹⁵ —, wherein R ¹⁵ is (C ₁ -C ₅ )alkyl, and R ¹⁵ is optionally substituted with 1 or 2 groups selected from —OH, —(C ₁ -C ₄ )alkyl, and —S(C ₁ -C ₄ )alkyl;

Y is a linear or branched (C ₁ -C ₆ )alkylene group or a linear or branched (C ₂ -C ₆ )alkenylene group; and

Z is selected from the group consisting of

The method includes

a) treating a compound of formula I with a strong acid,

wherein R ¹² is selected from linear and branched C ₁ -C ₈ alkyl groups and each R ¹⁰ is independently selected from hydrogen, R ¹² and —OR ¹² ;

forming a mixture comprising a compound of Formula IV;

and

b) reacting a compound of formula IV with a compound ZH;

Thereby synthesizing the linker intermediate of formula V. In one embodiment of the method of synthesizing the linker intermediate of formula V, the strong acid is trifluoroacetic acid or sulfuric acid.

In another embodiment of the present invention for synthesizing the linker intermediate of formula V, R ⁸ and R ⁹ on the compound of formula I are both methyl.

In another embodiment of the present invention for the synthesis of the linker intermediate of formula V, r in the compound of formula I is 0. In another embodiment of the present invention for the synthesis of the linker intermediate of formula V, r in the compound of formula I is 0, and G is oxygen. In another embodiment of the present invention for the synthesis of the linker intermediate of formula V, r in the compound of formula I is 0, and G is sulfur.

In another embodiment of the method for synthesizing the linker intermediate of formula V, ZH is

In another embodiment of the method for synthesizing the linker intermediate of formula V, the linker intermediate is a compound having the structure:

In another embodiment of the method for synthesizing the linker intermediate of formula V, the compound of formula I used in the method is prepared by reacting a compound of formula II

With the compound of formula III obtained by reaction,

As explained, the linker intermediate can be used to prepare calicheamicin derivatives containing the linker intermediate group. The calicheamicin derivatives can, in turn, be conjugated to biomacromolecules such as monoclonal antibodies. Thus, the compounds of Formula I and the synthetic methods described in this application can be used to prepare such calicheamicin derivatives. Thus, the present invention provides a method for synthesizing the calicheamicin derivatives of Formula VI,

where J is

R ₁ is or CH ₃ ; R ₂ is or H;

R3 _is or H; _R4 is or H;

R ⁵ is -CH ₃ , -C ₂ H ₅ or -CH(CH ₃ ) ₂ ;

X is an iodine or bromine atom;

R ^5′ is hydrogen or a group RCO, wherein R is hydrogen, a branched or unbranched alkyl group of 1 to 10 carbon atoms, an alkylene group of 2 to 10 carbon atoms, an aryl group of 6 to 11 carbon atoms, a (C ₆ -C ₁₁ )aryl-alkyl (C ₁ -C ₅ ) group or a heteroaryl or heteroaryl-alkyl (C ₁ -C ₅ ) group, wherein heteroaryl is defined as 2- or 3-furyl, 2- or 3-thienyl, 2- or 3-(N-methylpyrrolyl), 2-, 3- or 4-pyridyl, 2-, 4- or 5-(N-methylimidazolyl), 2-, 4- or 5-oxazolyl, 2-, 3-, 5- or 6-pyrimidinyl, 2-, 3-, 4-, 5-, 6-, 7- or 8-quinolyl or 1-, 3-, 4-, 5-, 6-, 7- or 8-isoquinolyl, all of which are optionally substituted with one or more hydroxy, amino, carboxyl, halogen, nitro, (C ₁ -C ₃ )alkoxy or thioalkoxy of 1 to 5 carbon atoms;

_R6 and _R7 are each independently selected from H and

R ⁸ and R ⁹ are each independently selected from hydrogen and linear and branched C ₁ -C ₈ alkyl, wherein said R ⁸ and R ⁹ alkyl are each independently optionally substituted with -NH ₂ , -NHR ¹¹ , -NR ¹¹ R ¹³ , -OR ¹¹ , -OH, or -SR ¹¹ , wherein each R ¹¹ and each R ¹³ are each independently selected from linear and branched C ₁ -C ₅ alkyl;

r is an integer 0 or 1;

G is oxygen or sulfur;

Z ¹ is H or a linear or branched C ₁ -C ₅ alkyl group;

Ar is 1,2-, 1,3- or 1,4-phenylene optionally substituted by 1, 2 or 3 groups independently selected from linear or branched C ₁ -C ₆ alkyl, -OR ¹⁴ , -SR ¹⁴ , halogen, nitro, -COOR ¹⁴ , -C(═O)NHR ¹⁴ , -O(CH ₂ ) _n COOR ¹⁴ , -S(CH ₂ ) _n COOR ¹⁴ , -O(CH ₂ ) _n C(═O)NHR ¹⁴ and -S(CH ₂ ) _n C(═O)NHR ¹⁴ , or Ar is 1,2-, 1,3-, 1,4-, 1,5-, 1,6-, 1,7-, 1,8-, 2,3-, 2,6- or 2,7-naphthylene optionally substituted by 1, 2, 3 or 4 groups independently selected from linear or branched C 1 -C 6 alkyl, ₁ -C ₆ alkyl, —OR ¹⁴ , —SR ¹⁴ , halogen, nitro, —COOR ¹⁴ , —C(═O)NHR ¹⁴ , —O(CH ₂ ) _n COOR ¹⁴ , —S(CH ₂ ) _n COOR ¹⁴ , —O(CH ₂ ) _n C(═O)NHR ¹⁴ , and —S(CH ₂ ) _n C(═O)NHR ¹⁴ ;

wherein each R ¹⁴ is independently selected from (C ₁ -C ₅ )alkyl, and each R ¹⁴ is independently optionally substituted with 1 or 2 groups selected from —OH, —(C ₁ -C ₄ )alkyl, and —S(C ₁ -C ₄ )alkyl;

Each n is an integer independently selected from 0, 1, 2, 3, 4 and 5;

W is selected from —O—, —S—, —C(═O)NH—, —NHC(═O)—, and —NR ¹⁵ —, wherein R ¹⁵ is (C ₁ -C ₅ )alkyl, and R ¹⁵ is optionally substituted with 1 or 2 groups selected from —OH, —(C ₁ -C ₄ )alkyl, and —S(C ₁ -C ₄ )alkyl;

Y is a linear or branched (C ₁ -C ₆ )alkylene group or a linear or branched (C ₂ -C ₆ )alkenylene group; and

Z is selected from the group consisting of

The method includes

a) treating a compound of formula I with a strong acid,

wherein R ¹² is selected from linear and branched C ₁ -C ₈ alkyl groups,

and each R ¹⁰ is independently selected from hydrogen, R ¹² and -OR ¹² , forming a mixture comprising a compound of Formula IV;

b) reacting a compound of formula IV with a compound ZH;

forming a linker intermediate of formula V,

and

c) reacting the linker intermediate of formula V from step (b) with a methyl trisulfide compound CH ₃ -SSSJ;

Thus, the calicheamicin derivatives of Formula VI are synthesized. In one embodiment of the method of the present invention for synthesizing the calicheamicin derivatives of Formula VI, the strong acid is sulfuric acid or trifluoroacetic acid. In another embodiment of the method of the present invention for synthesizing the calicheamicin derivatives of Formula VI, R ⁸ and R ⁹ are methyl, r is 0, G is oxygen, Z ¹ is methyl, Ar is 1,4-phenylene, W is -O-, and Y is -(CH ₂ ) ₃ -. In another embodiment of the method of synthesizing the calicheamicin derivatives of Formula VI, each R ¹⁰ is hydrogen. In another embodiment of the method of synthesizing the calicheamicin derivatives of Formula VI, each R ¹⁰ is hydrogen, and R ¹² is methyl.

In another embodiment of the present invention for synthesizing the calicheamicin derivatives of Formula VI, R ⁸ and R ⁹ on the compound of Formula I are both methyl.

In another embodiment of the present invention for the synthesis of calicheamicin derivatives of Formula VI, r in the compound of Formula I is 0. In another embodiment of the present invention for the synthesis of calicheamicin derivatives of Formula VI, r in the compound of Formula I is 0, and G is oxygen. In another embodiment of the present invention for the synthesis of calicheamicin derivatives of Formula VI, r in the compound of Formula I is 0, and G is sulfur.

In another embodiment of the method for synthesizing the calicheamicin derivative of formula VI, the compound of formula I is prepared by reacting a compound of formula II

With the compound of formula III obtained by reaction,

In another embodiment of the method for synthesizing the calicheamicin derivative of formula VI, one of _R6 and _R7 is hydrogen and the other of _R6 and _R7 is

In one embodiment of the intermediates and synthetic methods of formula I of the present invention, Z ¹ is methyl. In another embodiment of the intermediates and synthetic methods of the present invention, r is 0, and G is oxygen. In another embodiment of the intermediates and synthetic methods of the present invention, Z ¹ is methyl, r is 0, and G is oxygen.

In another embodiment of the synthetic method of the present invention, J is an ozogamicin moiety.

In another embodiment of the synthetic method of the present invention, the calicheamicin derivative of formula VI has the following structure:

The calicheamicin derivatives of Formula VI synthesized by the methods of the present invention can be conjugated to biomacromolecules such as monoclonal antibodies. For example, the calicheamicin derivatives of Formula VI synthesized by the methods of the present invention can be conjugated to the monoclonal anti-CD22 antibody inotuzumab (an antibody that specifically binds to the CD22 antigen expressed on the surface of certain cancer cells) or the anti-CD33 antibody gemtuzumab (an antibody that specifically targets the anti-CD33 antigen expressed on the surface of certain cancer cells). In one embodiment, the calicheamicin derivatives of Formula VI synthesized by the methods of the present invention, when conjugated to the monoclonal antibody, have the following structure:

Wherein Ab is a monoclonal antibody. Examples of monoclonal antibodies Ab include, but are not limited to, gemtuzumab and inotuzumab.

The terms used in this specification generally have their ordinary meaning in the art, in the context of the invention, and in the specific context in which each term is used.

As used herein, unless otherwise indicated, the term "calicheamicin derivative" refers to a derivative of a compound of formula CH ₃ -SSSJ, wherein J is as defined herein, which derivative comprises the calicheamicin moiety -SSJ bonded to a linker intermediate group:

wherein R ⁸ , R ⁹ , r, G, Z ¹ , Ar, W, Y, and Z are as defined herein. The calicheamicin derivatives can also be conjugated (i.e., covalently bonded) to a biomacromolecule, such as a monoclonal antibody, at the terminus comprising a -C(=O)Z moiety. Examples of compounds of the formula CH 3 -SSSJ are described, for example, in U.S. Pat. No. 4,970,198 and U.S. Pat. No. 5,053,394, both of which are incorporated herein by reference in their entireties. An example of the compound CH ₃ -SSSJ is calicheamicin ozogamicin.

Unless otherwise indicated, the term "linker intermediate" as used herein refers to those isolated molecules of Formula V described herein that are capable of covalently bonding to a molecule of Formula CH ₃ -SSSJ at one end thereof and have a functional group at the other end (the -C(=O)Z end) that can be covalently bonded to a biomacromolecule, such as a monoclonal antibody. The isolated linker intermediate can be used as a component for preparing calicheamicin derivatives and calicheamicin linked to biomacromolecules, such as monoclonal antibodies.

Unless otherwise indicated, the term "hydrocarbyl," by itself or as part of another term, means a straight or branched, saturated or unsaturated hydrocarbon having the indicated number of carbon atoms (e.g., "C ₁ -C ₈ " hydrocarbyl refers to a hydrocarbyl group having 1 to 8 carbon atoms). When the number of carbon atoms is not indicated, the hydrocarbyl group has 1 to 8 carbon atoms (unless it is unsaturated, in which case the hydrocarbyl group has 2 to 8 carbon atoms). Representative straight-chain C ₁ -C ₈ hydrocarbon groups include, but are not limited to, methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, and n-octyl; while branched C ₁ -C ₈ hydrocarbon groups include, but are not limited to, -isopropyl, -sec-butyl, -isobutyl, -tert-butyl, -isopentyl, and -2-methylbutyl; and unsaturated C ₂ -C ₈ hydrocarbon groups include, but are not limited to, vinyl, allyl, 1-butenyl, 2-butenyl, isobutenyl, 1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl, 2,3-dimethyl-2-butenyl, 1-hexyl, 2-hexyl, 3-hexyl, ethynyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, and 3-methyl-1-butynyl.

Unless otherwise indicated, "alkylene" by itself or as part of another term refers to a saturated, branched, straight-chain, or cyclic hydrocarbon radical having the indicated number of carbon atoms, having two monovalent radical centers derived by removing two hydrogen atoms from the same or two different carbon atoms of a parent alkane. In one embodiment, "alkylene" refers to a saturated, branched, or straight-chain alkylene radical having the indicated number of carbon atoms, having two monovalent radical centers. Typical alkylene radicals include, but are not limited to, methylene ( _-CH2- ), _1,2 -ethylene _{( -CH2CH2-} ), 1,3-propylene ( _{-CH2CH2CH2- )} , 1,4-butylene ( _{-CH2CH2CH2CH2-} ), and the _like . _The term " _alkenylene " refers to a branched, straight-chain, or cyclic hydrocarbon radical having the indicated number of carbon atoms, but having at least two carbon atoms joined by a double bond, wherein the radical has two monovalent radical centers derived by removing two hydrogen atoms from two different carbon atoms of a parent alkane. In one embodiment, "alkylene" refers to a branched or straight chain group having the indicated number of carbon atoms, but having at least two carbon atoms joined by a double bond, wherein the group has two monovalent radical centers. Examples of alkenylene include, but are not limited to, -CH=CH-, _-CH2CH =CH-, and -CH( _CH3 )CH=CH.

Unless otherwise indicated, "aryl" by itself or as part of another term means a carbocyclic aromatic hydrocarbon radical of 6 to 20, preferably 6 to 14, carbon atoms derived by removing one hydrogen atom from each of one or more carbon atoms of a parent aromatic ring system. Typical aryl groups include, but are not limited to, those derived from benzene, naphthalene, anthracene, biphenyl, and the like. If indicated, aryl groups herein may be optionally substituted. The term "arylene" refers to a divalent radical derived from an aryl group as defined herein.

"Halogen" refers to a fluorine, chlorine, bromine or iodine atom.

The present invention also provides a method for synthesizing the calicheamicin derivative of formula VI,

where J is

R ₁ is or CH ₃ ; R ₂ is

or H;

R ₃ is or H; R ₄ is

or H;

R ⁵ is -CH ₃ , -C ₂ H ₅ or -CH(CH ₃ ) ₂ ;

X is an iodine or bromine atom;

R ^5′ is hydrogen or a group RCO, wherein R is hydrogen, a branched or unbranched alkyl group of 1 to 10 carbon atoms, an alkylene group of 2 to 10 carbon atoms, an aryl group of 6 to 11 carbon atoms, a (C ₆ -C ₁₁ )aryl-alkyl (C ₁ -C ₅ ) group or a heteroaryl or heteroaryl-alkyl (C ₁ -C ₅ ) group, wherein heteroaryl is defined as 2- or 3-furyl, 2- or 3-thienyl, 2- or 3-(N-methylpyrrolyl), 2-, 3- or 4-pyridyl, 2-, 4- or 5-(N-methylimidazolyl), 2-, 4- or 5-oxazolyl, 2-, 3-, 5- or 6-pyrimidinyl, 2-, 3-, 4-, 5-, 6-, 7- or 8-quinolyl or 1-, 3-, 4-, 5-, 6-, 7- or 8-isoquinolyl, all of which are optionally substituted with one or more hydroxy, amino, carboxyl, halogen, nitro, (C ₁ -C ₃ )alkoxy or thioalkoxy of 1 to 5 carbon atoms;

_R6 and _R7 are each independently selected from H and

R ⁸ and R ⁹ are each independently selected from hydrogen and linear and branched C ₁ -C ₈ alkyl, wherein each of said R ⁸ and R ⁹ alkyl groups is independently optionally substituted with -NH ₂ , -NHR ¹¹ , -NR ¹¹ R ¹³ , -OR ¹¹ , -OH, or -SR ¹¹ , wherein each R ¹¹ and each R ¹³ are independently selected from linear and branched C ₁ -C ₅ alkyl;

r is an integer 0 or 1;

G is oxygen or sulfur;

Z ¹ is H or a linear or branched C ₁ -C ₅ alkyl group;

Ar is 1,2-, 1,3- or 1,4-phenylene optionally substituted by 1, 2 or 3 groups independently selected from linear or branched C ₁ -C ₆ alkyl, -OR ¹⁴ , -SR ¹⁴ , halogen, nitro, -COOR ¹⁴ , -C(═O)NHR ¹⁴ , -O(CH ₂ ) _n COOR ¹⁴ , -S(CH ₂ ) _n COOR ¹⁴ , -O(CH ₂ ) _n C(═O)NHR ¹⁴ and -S(CH ₂ ) _n C(═O)NHR ¹⁴ , or Ar is 1,2-, 1,3-, 1,4-, 1,5-, 1,6-, 1,7-, 1,8-, 2,3-, 2,6- or 2,7-naphthylene optionally substituted by 1, 2, 3 or 4 groups independently selected from linear or branched C 1 -C 6 alkyl, ₁ -C ₆ alkyl, —OR ¹⁴ , —SR ¹⁴ , halogen, nitro, —COOR ¹⁴ , —C(═O)NHR ¹⁴ , —O(CH ₂ ) _n COOR ¹⁴ , —S(CH ₂ ) _n COOR ¹⁴ , —O(CH ₂ ) _n C(═O)NHR ¹⁴ , and —S(CH ₂ ) _n C(═O)NHR ¹⁴ ;

wherein each R ¹⁴ is independently selected from (C ₁ -C ₅ )alkyl, and each R ¹⁴ is independently optionally substituted with 1 or 2 groups selected from —OH, —(C ₁ -C ₄ )alkyl, and —S(C ₁ -C ₄ )alkyl;

Each n is an integer independently selected from 0, 1, 2, 3, 4 and 5;

W is selected from —O—, —S—, —C(═O)NH—, —NHC(═O)—, and —NR ¹⁵ —, wherein R ¹⁵ is (C ₁ -C ₅ )alkyl, and R ¹⁵ is optionally substituted with 1 or 2 groups selected from —OH, —(C ₁ -C ₄ )alkyl, and —S(C ₁ -C ₄ )alkyl;

Y is a linear or branched (C ₁ -C ₆ )alkylene group or a linear or branched (C ₂ -C ₆ )alkenylene group; and

Z is selected from the group consisting of

The method comprises making a linker intermediate of formula V

The calicheamicin derivative of formula VI is synthesized by reacting with a methyl trisulfide compound CH ₃ -SSSJ in the presence of a carbodiimide.

The present invention also provides a method for synthesizing a calicheamicin derivative of formula X,

where J is

R ₁ is or CH ₃ ; R ₂ is

or H;

R3 _is or H; _R4 is or H;

R ⁵ is -CH ₃ , -C ₂ H ₅ or -CH(CH ₃ ) ₂ ;

X is an iodine or bromine atom;

R ^5′ is hydrogen or a group RCO, wherein R is hydrogen, a branched or unbranched alkyl group of 1 to 10 carbon atoms, an alkylene group of 2 to 10 carbon atoms, an aryl group of 6 to 11 carbon atoms, a (C ₆ -C ₁₁ )aryl-alkyl (C ₁ -C ₅ ) group or a heteroaryl or heteroaryl-alkyl (C ₁ -C ₅ ) group, wherein heteroaryl is defined as 2- or 3-furyl, 2- or 3-thienyl, 2- or 3-(N-methylpyrrolyl), 2-, 3- or 4-pyridyl, 2-, 4- or 5-(N-methylimidazolyl), 2-, 4- or 5-oxazolyl, 2-, 3-, 5- or 6-pyrimidinyl, 2-, 3-, 4-, 5-, 6-, 7- or 8-quinolyl or 1-, 3-, 4-, 5-, 6-, 7- or 8-isoquinolyl, all of which are optionally substituted with one or more hydroxy, amino, carboxyl, halogen, nitro, (C ₁ -C ₃ )alkoxy or thioalkoxy of 1 to 5 carbon atoms;

_R6 and _R7 are each independently selected from H and

Y' is a linear and branched (C ₁ -C ₁₈ )alkylene group, a linear and branched (C ₂ -C ₁₈ )alkenylene group, an arylene group or a heteroarylene group, an arylene (C ₁ -C ₁₈ )alkylene group, an arylene (C ₂ -C ₁₈ )alkenylene group, a heteroarylene (C ₁ -C ₁₈ )alkylene group or a heteroarylene (C ₁ -C ₁₈ ) an alkenylene group, wherein the heteroarylene group is a divalent group derived from a furyl group, a thienyl group, an N-methylpyrrolyl group, a pyridyl group, an N-methylimidazolyl group, an oxazolyl group, a pyrimidinyl group, a quinolyl group, an isoquinolyl group, an N-methylcarbazolyl group, an aminocoumarinyl group, or a phenazinyl group, and wherein the Y′ may be optionally substituted with a dialkylamino group having 1 to 5 carbon atoms, an alkoxy group having 1 to 5 carbon atoms, a hydroxyl group, —SH, or an alkylthio group having 1 to 5 carbon atoms;

Q is selected from -C(=O)NHN=, -C(=S)NHN=, -NHC(=O)NHN=, -NHC(=S)NHN=, and -O-N=;

Z ¹ is H or a linear or branched C ₁ -C ₅ alkyl group;

Ar is 1,2-, 1,3- or 1,4-phenylene optionally substituted by 1, 2 or 3 groups independently selected from linear or branched C ₁ -C ₆ alkyl, -OR ¹⁴ , -SR ¹⁴ , halogen, nitro, -COOR ¹⁴ , -C(═O)NHR ¹⁴ , -O(CH ₂ ) _n COOR ¹⁴ , -S(CH ₂ ) _n COOR ¹⁴ , -O(CH ₂ ) _n C(═O)NHR ¹⁴ and -S(CH ₂ ) _n C(═O)NHR ¹⁴ , or Ar is 1,2-, 1,3-, 1,4-, 1,5-, 1,6-, 1,7-, 1,8-, 2,3-, 2,6- or 2,7-naphthylene optionally substituted by 1, 2, 3 or 4 groups independently selected from linear or branched C 1 -C 6 alkyl, ₁ -C ₆ alkyl, —OR ¹⁴ , —SR ¹⁴ , halogen, nitro, —COOR ¹⁴ , —C(═O)NHR ¹⁴ , —O(CH ₂ ) _n COOR ¹⁴ , —S(CH ₂ ) _n COOR ¹⁴ , —O(CH ₂ ) _n C(═O)NHR ¹⁴ , and —S(CH ₂ ) _n C(═O)NHR ¹⁴ ;

wherein each R ¹⁴ is independently selected from (C ₁ -C ₅ )alkyl, and each R ¹⁴ is independently optionally substituted with 1 or 2 groups selected from —OH, —(C ₁ -C ₄ )alkyl, and —S(C ₁ -C ₄ )alkyl;

Each n is an integer independently selected from 0, 1, 2, 3, 4 and 5;

W is selected from —O—, —S—, —C(═O)NH—, —NHC(═O)—, and —NR ¹⁵ —, wherein R ¹⁵ is (C ₁ -C ₅ )alkyl, and R ¹⁵ is optionally substituted with 1 or 2 groups selected from —OH, —(C ₁ -C ₄ )alkyl, and —S(C ₁ -C ₄ )alkyl;

Y is a linear or branched (C ₁ -C ₆ )alkylene group or a linear or branched (C ₂ -C ₆ )alkenylene group; and

Z is selected from the group consisting of

The method comprises making a linker intermediate of formula XI

The calicheamicin derivative of formula X is synthesized by reacting with a methyl trisulfide compound CH ₃ -SSSJ in the presence of carbodiimide.

In one embodiment of this method for synthesizing a calicheamicin derivative of Formula VI or Formula X in the presence of a carbodiimide, one of _R6 and _R7 is hydrogen, and the other of _R6 and _R7 is

In another embodiment of the method of the present invention for synthesizing the calicheamicin derivative of Formula VI or Formula X, the methyl trisulfide has an initial concentration of greater than about 3 g/L of the reaction mixture in the reaction with the linker intermediate of Formula V (or which may be a linker intermediate of Formula XI). In another embodiment, the methyl trisulfide has an initial concentration of between about 10 g/L and 110 g/L of the reaction mixture in the reaction with the linker intermediate of Formula V (or which may be a linker intermediate of Formula XI).

In another embodiment of this method for synthesizing the calicheamicin derivatives of Formula VI or Formula X, the carbodiimide is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. The carbodiimide in this method of synthesizing the calicheamicin derivatives of Formula VI or Formula X of the present invention can be any molecule containing a carbodiimide moiety, and such molecules are known in the art. Examples of carbodiimides that can be used in the present invention include, but are not limited to, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC); N,N'-dicyclohexylcarbodiimide (DCC); N,N'-diisopropylcarbodiimide (DIC); N-cyclohexyl-N'-(2-morpholinoethyl)carbodiimide; N-cyclohexyl-N'-[2-(4-methylmorpholin-4-yl)ethyl]carbodiimide tosylate; N-cyclohexyl-N'-[4-(diethylmethylammonio)cyclohexyl]carbodiimide tosylate; N,N'-bis(2,2-dimethyl-1,3-dioxolan-4-yl)methyl]carbodiimide; and N-benzyl-N'-isopropylcarbodiimide. Additional carbodiimides suitable for use in the methods of the present invention can be determined by one of ordinary skill in the art.

The present invention also provides a method for purifying a calicheamicin derivative of Formula VI or a calicheamicin derivative of Formula X, wherein Formula VI and Formula X are as defined above, the method comprising subjecting the calicheamicin derivative of Formula VI or the calicheamicin derivative of Formula X to a reversed-phase high performance liquid chromatography (RP-HPLC) purification protocol. It is surprising that a reversed-phase purification protocol can be used to purify calicheamicin derivative molecules of Formula VI or Formula X because these compounds have two water-labile groups, namely a hydrazone group and an N-hydroxysuccinimide (NHS) ester group, each of which has a different pH dependency for hydrolysis. In addition, the use of reversed-phase purification is advantageous over the normal-phase chromatography purification described in the prior art (see, U.S. Patent No. 8,273,862) because normal-phase chromatography uses toxic, environmentally unfriendly solvents such as dichloromethane and methanol. The reversed-phase high performance liquid chromatography (RP-HPLC) protocol ensures the use of a C-18 stationary phase that binds to the components of the reaction mixture. These components are then eluted and separated using a gradient consisting of an aqueous and organic mobile phase having a pH of about 4 to about 6 with optimal stability for the two hydrolyzable groups present. In one embodiment, the gradient comprises at least two phases. In another embodiment, the gradient comprises 1, 2, 3, 4 or 5 phases. In another embodiment, the gradient comprises 2 or 3 phases. In another embodiment, the gradient comprises 2 phases. Each phase can be organic, aqueous or a combination thereof. The gradient moves from aqueous properties to increased organic properties over a period of time. Aqueous phases as known in the art can be used in the present invention. Examples of aqueous phases that can be used in the present invention include, but are not limited to, sodium acetate (NaOAc), sodium succinate, N-methylmorpholine, sodium citrate and 2-(N-morpholino)ethanesulfonic acid. Organic phases as known in the art can be used in the present invention. Examples of organic phases that can be used in the present invention include, but are not limited to, acetonitrile, isopropyl alcohol, acetone, dimethoxyethane and N-methyl-2-pyrrolidone. As mentioned, any of the phases can comprise a mixture of aqueous and organic properties, such as a mixture of NaOAc and acetonitrile. For example, a mobile phase consisting of 55% 20 mM sodium acetate at pH 5 and 45% acetonitrile is an aqueous/organic mobile phase that can be used for RP-HPLC purification of the present invention. As another example, an example of a gradient that can be used in the present invention is a gradient comprising a first mobile phase consisting of 55% 20 mM sodium acetate at pH 5 and 45% acetonitrile, followed by a second mobile phase consisting of acetonitrile.

In one embodiment of the method of purifying a calicheamicin derivative of Formula VI or Formula X comprising an RP-HPLC protocol, the calicheamicin derivative of Formula VI or Formula X comprises Z selected from the group consisting of:

In another embodiment of the method of purifying a calicheamicin derivative of Formula VI or Formula X comprising an RP-HPLC protocol, the calicheamicin derivative of Formula VI or Formula X comprises Z selected from the group consisting of:

In another embodiment of the method of purifying a calicheamicin derivative of Formula VI or Formula X comprising an RP-HPLC protocol, the calicheamicin derivative of Formula VI or Formula X comprises Z, which is:

After reverse phase purification, the resulting purified calicheamicin derivative can be isolated, for example, by concentration and partitioning, as described in the art; or the purified calicheamicin derivative can be isolated by using a solid phase extraction (SPE) protocol. The solid phase extraction protocol has been identified as an effective alternative to dichloromethane partitioning for product isolation from RP-HPLC fractions. In solid phase extraction, the product is bound to a reverse phase resin by loading it with a weak solvent, washed to remove buffer salts (remaining from the RP-HPLC purification), and then eluted with an organic solvent, such as acetonitrile, to yield a concentrated product solution that is free or substantially free of salts.

Thus, additional embodiments of any of the methods of the invention described herein for synthesizing calicheamicin derivatives of Formula VI or Formula X further comprise purifying the synthesized calicheamicin derivative of Formula VI or Formula X by performing a reverse phase purification protocol. In additional embodiments of the invention, the calicheamicin derivative of Formula VI or Formula X from the reverse phase purification protocol is subsequently subjected to a solid phase extraction protocol.

DETAILED DESCRIPTION

The methods of the present invention for synthesizing the linker intermediates of Formula V are described in the following reaction schemes I-IV. In the chemical formulae in schemes I-IV, R ⁸ , R ⁹ , R ¹⁰ , R ¹² , Q, Ar, W, Y, Z ¹ and Z are as defined above.

Plan I

With respect to Scheme 1, (4-alkoxyphenyl)methanethiol 1 , wherein R ¹² is as described herein, is reacted with methyl senecioate 2 to give a carboxylic acid intermediate compound 3. A suitable organic solvent, such as tetrahydrofuran, and an azole-based activator of formula IX, such as carbonyldiimidazole (CDI), as described herein, are then added to intermediate 3 to give intermediate 3. This gives intermediate 3.5 . Following this reaction, 3.5 is combined with hydrazine. The hydrazine source may be anhydrous hydrazine as described in WO 2008/147765; however, preferably the hydrazine source is aqueous hydrazine, such as hydrazine monohydrate. This reaction gives intermediate compound 4. Compound p-methoxybenzyl sulfide hydrazide (which is an intermediate compound 4 wherein R ¹² is methyl and R ¹⁰ is each hydrogen) is described in WO 2008/147765, the entire contents of which are incorporated herein by reference.

Option II

In Scheme II, intermediate 4 is reacted with compound 5 (wherein ^Z is as described herein), such as 4-(4-acyl-phenoxy)butanoic acid, in an inert (in other words, non-reactive) solvent, optionally using an acidic catalyst, to provide intermediate 6. Examples of inert solvents that can be used in this reaction include, but are not limited to, alcohols (e.g., methanol), ethers, and esters, such as ethyl acetate. One of ordinary skill in the art can determine suitable inert solvents for this reaction. Acidic catalysts can also be determined by one of ordinary skill in the art; examples of acidic catalysts include, but are not limited to, acetic acid.

Option III

In Scheme III, intermediate compound 6 is deprotected to form compound 7. In this reaction, methoxybenzene and a strong acid, such as trifluoroacetic acid, optionally under heating, are added to compound 6 to provide intermediate compound 7. Other strong acids, such as sulfuric acid, may be used instead of trifluoroacetic acid.

Option IV

In Scheme IV, intermediate 7 is converted to compound 8 , which is an embodiment of the linker intermediate of Formula V described herein. Intermediate 7 can be converted to a linker intermediate, such as 8 , as described in the art, for example, as described in U.S. Patent No. 8,273,862 (the entire contents of which are incorporated herein by reference). However, preferably, as described in Scheme IV, intermediate 7 is reacted with a tertiary amine base, such as triethylamine (TEA) and trimethylacetyl chloride (PivCl), in the presence of an inert solvent, such as tetrahydrofuran. Subsequently, a compound of Formula ZH, such as N-hydroxysuccinimide, is introduced to provide linker intermediate 8 .

After synthesizing the linker intermediate of Formula V (e.g., compound 8 ), the calicheamicin derivative of Formula VI is then synthesized using the linker intermediate from Reaction Scheme IV and methods known in the art, such as those described in U.S. Patent No. 8,273,862. For example, the linker intermediate of Formula V can be first reacted with an alkali metal carbonate (including, but not limited to, sodium carbonate) and heated under mild reflux to form a sodium salt of the linker intermediate in acetonitrile. The sodium salt of the linker intermediate is then reacted with methyl trisulfide, CH ₃ -SSSJ, at about -15°C in an inert organic solvent (preferably acetonitrile) to provide the calicheamicin derivative of Formula VI. Preferably, the reaction is carried out in acetonitrile at about 0°C. Optionally, an organic base can replace the alkali metal carbonate, which preferably includes triethylamine in acetonitrile at about 0°C.

Alternatively, calicheamicin derivatives of Formula VI can be synthesized from linker intermediates of Formula V by reaction with a molecule of formula _CH3- SSSJ using carbodiimide-containing methods described herein.

The calicheamicin derivatives of Formula VI can also be conjugated to biomacromolecules such as monoclonal antibodies to form antibody-drug conjugates using techniques described in the art, such as the methods described in U.S. Pat. No. 5,053,394 and U.S. Pat. No. 5,770,701, the entire contents of which are incorporated herein by reference.

The following examples are provided to illustrate some embodiments of the present invention, but should not be construed as limiting the scope of the invention. Those skilled in the art will readily appreciate that known variations of the specific conditions of the following examples may be used.

Example 1

3-(4-Methoxybenzylthio)-3-methylbutanoic acid

85 g of 1 and 255 mL of 2-methyltetrahydrofuran were added to a reactor at 20-25° C. 125.05 g of 2 was added to the reactor, and the reactor was degassed by bubbling a stream of nitrogen through the stirred solution for 15-20 minutes. Tetrabutylammonium fluoride (1 M in THF, 0.05 eq, 36.1 mL) was added to the reactor, and the reaction was maintained at 20-30° C. for 2 hours.

A solution of calcium chloride dihydrate (0.35 equivalents, 28.060 g) in 255 mL of water was added. After stirring for 20 minutes, the lower aqueous phase was removed. To the upper organic phase was added a solution of 252 mL of methanol and 3 equivalents of NaOH in 252 mL of water. The reaction mixture was stirred until complete consumption of the intermediate ester (3) was observed.

The reaction was cooled to 15°C and 2-methyltetrahydrofuran (252 mL) was added, followed by 252 mL of water. Concentrated HCl (3.1 equivalents, 184 mL) was slowly added, maintaining the reaction within the range of 15-30°C. The lower aqueous phase was removed. The organic layer was washed with 1M HCl (252 mL) and heptane (1940 mL) was added. The solution was distilled to remove the 2-methyltetrahydrofuran. The resulting slurry was stirred for 1 hour, then filtered and dried. The mother liquor was concentrated to obtain a second batch of solids.

The total yield of the title compound acid 4 was 162.59 g (88.5%). ¹ H NMR (CDCl ₃ ): δ (ppm) 1.5 (s, 6H), 2.3 (s, 2H), 3.6 (m, 5H), 6.7 (d, 2H), 7.6 (d, 2H).

Example 2

3-(4-Methoxybenzylthio)-3-methylbutyric acid hydrazide

Acid 4 (81.67 g, 321 mmol) was added to 375 mL of THF (tetrahydrofuran). CDI (1.05 eq., 54.7 g) was added in three portions, and the reaction was stirred at 20°C for 2.5 hours. In a separate reactor, a solution of hydrazine monohydrate (2.5 eq., 40.19 g) in 200 mL of THF was prepared. The solution of intermediate 5 was added to the hydrazine hydrate solution, maintaining the internal temperature at 20°C. After the addition was complete, the reaction was stirred for 18 hours and then concentrated to 100 mL. 850 mL of EtOAc (ethyl acetate) was added, and the solution was washed three times with 500 mL of water and then with 200 mL _of brine. The organic layer was dried over _Na₂SO₄ , filtered through celite, and concentrated on a rotary evaporator to yield a white slurry. 300 mL of heptane was added, and 200 mL was removed on a rotary evaporator. An additional 200 mL of heptane was added and the mixture was stripped to yield a thick white slurry. The mixture was filtered, washed with heptane and dried. From 4, 83.15 g (96.5%) of the title compound 6 were obtained. MS 269 (M+1), 121, 120.

Example 3

4-(4-(1-(2-(3-(4-methoxybenzylthio)-3-methylbutanoyl)hydrazono)-ethyl)phenoxy)butanoic acid

68 g of 6 and 57.43 g of 7 were added to 680 mL of methanol (MeOH). 68 mL of acetic acid (HOAc) was added, and the mixture was heated at 45°C for 3 hours, cooled to 20°C, and held for 16 hours. The slurry was filtered, washed with methanol, and dried. 112.60 g of the title compound 8 was obtained as a mixture of E and Z isomers. ¹ H NMR (DMSO-d ₆ ): δ(ppm)1.5(m,6H),2.0(m,2H),2.2(m,3H),2.4(m,2H),2.7(s,1.1H),3.0(s,0.9H),3.7(m,3H),3.8(m,2H),4.0(m,2H),6.8(m 2H),6.9(m,2H),7.3(m,2H),7.7(m,2H),10.2&10.3(s,1H),12.1(s,1H). LC-MSm/z 473[M+H] ⁺ .

Example 4

4-(4-(1-(2-(3-mercapto-3-methylbutanoyl)hydrazono)ethyl)-phenoxy)butanoic acid

At 20-25°C, 290.5 g (614.7 mmol) of 8 and 1162 mL of anisole were charged to the reactor. Trifluoroacetic acid (1162 mL) was added and the reaction was heated to 65-70°C over 2 minutes for 2.5 hours. The reaction was cooled to 40°C. TFA (trifluoroacetic acid) was distilled under vacuum and replaced with 2-methyltetrahydrofuran (2905 mL). Distillation was continued until a thick slurry was observed (the final volume of the slurry was approximately 2 L). The slurry was cooled to 15°C and filtered. The crude product was reslurried in methanol (1836.6 mL), heated to 55°C, and then cooled to 20°C overnight. The slurry was filtered, washed with methanol, and dried. 171.80 g (93.54%) of the title compound 9 was obtained from 8 as a mixture of E and Z isomers. ¹ H NMR (DMSO-d ₆ ): δ (ppm) 1.5 (m, 6H), 2.0 (m, 2H), 2.2 (m, 3H), 2.4 (m, 2H), 2.7 (s, 1.1H), 3.0 (s, 0.9H), 3.3 (s 1H),4.0(m,2H),6.9(m,2H),7.7(m,2H),10.2&10.3(s,1H),12.1(s,1H).

Example 5

4-(4-(1-(2-(3-mercapto-3-methylbutanoyl)-hydrazono)ethyl)phenoxy)-butyric acid 2,5-dioxopyridine Pyrrolidin-1-yl ester

Reactor setup: 2-L jacketed reactor, Tr probe, nitrogen inlet.

60 g (170.3 mmol) was added to 2400 mL of THF and cooled to 10°C. Triethylamine (2 equivalents, 34.46 g) was added, followed by the slow addition of trimethylacetyl chloride (1.1 equivalents, 22.81 g) over 10 minutes, maintaining the temperature between 10-20°C. The mixture was stirred at 10-20°C for 30 minutes. N-hydroxysuccinimide (1.1 equivalents, 21.99 g) was added to the reactor and stirred at 20-25°C for 30 minutes.

The slurry was filtered to remove the TEA-HCl salt and concentrated in vacuo to a volume of approximately 800 mL. Hexane (780 mL) was slowly added to allow the product to crystallize. The slurry was stirred for 1.5 hours, then filtered, washed with heptane, and dried. 70.4 g (92% yield) of the title compound connector intermediate 10 was obtained from 9.

144 g of 10 was added to 2100 mL of THF, and the crude product was recrystallized and heated to 60°C. The mixture was filtered through Celite, and 2100 mL of hexane was slowly added. The mixture was cooled to 20°C over 1.5 hours. The slurry was filtered, washed with cold THF/hexane (1:1), then with hexane, and dried. 126 g (87.5% recovery) of the title compound, linker intermediate 10, was obtained from 9. ^1H NMR (DMSO- _d6 ): δ (ppm): 1.5 (m, 6H), 2.1 (m, 2H), 2.2 (m, 3H), 2.7 (s, 1.1H), 2.9 (M, 5H), 3.0 (s, 1.9H), 3.3 (s, 1H), 4.3 (m, 2H), 7.0 (m, 2H), 7.5 (m, 2H), 10.2 & 10.3 (s, 1H).

Example 6

Butyric acid, 3-[[(2E)-2-[(1R,4Z,8S)-8-[[2-O-[4-(acetylethylamino)-2,4-dideoxy-3- O-methyl-α-L-threo-pentopyranosyl]-4,6-dideoxy-4-[[[2,6-dideoxy-4-S-[4-[(6-deoxy-3-O-methyl [(1-(2-((4-thio)-β-D-pyranose)-1-methyl-3-((2-((4-((4-thio)-β-D-pyranose)-1-methyl-3-(((4-( ... [hexosyl]oxy]amino]-β-D-glucopyranosyl]oxy]-1-hydroxy-10-[(methoxycarbonyl)amino]-11-oxo Bicyclo[7.3.1]tridec-4,9-diene-2,6-diyn-13-ylidene]ethyl]dithio]-3-methyl-,2-[(1E)-1-[4- Preparation of [4-[(2,5-dioxo-1-pyrrolidinyl)oxy]-4-oxobutoxy]-phenyl]ethylidene]hydrazide

To a solution of N-acetyl-calicheamicin (50 mg, 0.035 mmol) in acetonitrile (1.0 mL) at room temperature was added the linker intermediate butyric acid, 3-mercapto-3-methyl-, 2-[(E)-1-[4-[4-[(2,5-dioxo-1-pyrrolidinyl)-oxy]-4-oxobutoxy]phenyl]ethylidene]hydrazide (31.9 mg, 0.07 mmol) in one portion, followed by 1,(3-dimethylaminopropyl)-3-ethylcarbodiimide (6.7 mg, 0.035 mmol) and then triethylamine (5.3 mg, 7.3 μL, 0.053 mmol). The reaction mixture (slurry) was stirred at room temperature for 1 hour, at which point it became a yellow solution. The yield was determined based on the area percentage relative to the derivative standard (60% yield).

Mass Spectrometry:

(M+Na)＝1801.4578

^1H NMR:

¹ H NMR (CDCl ₃ +5% CD ₃ OD, 400MHz): 8.69 (s, 1H, 18C-NH-N ₌ ), 7.75 (d, J = 8.7Hz, 2H, 22), 6.95 (d, J = 8.7Hz, 2H, 23), 6.38 (br t,J＝7Hz,1H,14),6.23(d,J＝1.5Hz,1H,8),5.90(d,J＝9.5Hz,1H,4),5.80(dd,J＝9.5,1.5Hz,1H, 5),5.72(d,J＝1.5Hz,1H,1D),5.63(brd,J＝2.2Hz,1H,1E),5.03(dd,J＝11.5,1.6Hz,1H,1B),4.7 0(m,1H,5E),4.61(d,J＝7.8Hz,1H,1A),4.6(m,1H,3E),4.49(m,1H,2D),4.31(m,1H,3B),4.20(m ,1H,5D),4.10(m,2H,25),4.07(m,1H,5B),4.03(m,1H,3A),3.91(m,1H,15),3.89(s,3H,2C-OCH ₃ ),3.84(s,3H,3C-OCH ₃ ),3.8(m,1H,3D),3.76(m,1H,15),3.75(m,1H,4B),3.65(m,1H,5A),3.63(bs,3H,10-NHCOOCH ₃ ),3.62(m,1H,2A),3.6(m,1H,4D),3.57(s,3H,3D-OCH ₃ ),3.4(m,1H,5E),3.37(s,3H,3E-OCH ₃ ),3.30(m,2H,4E-N-CH ₂ CH ₃ ),3.12(d,J＝17.6Hz,1H,12),3.0(m,1H,4E),2.85(m,2H,27),2.85(bs,4,30),2.72(d,J＝17.6Hz,1H,12), 2.5(m,2H,17),2.4(m,1H,2E flat),2.33(m,1H,4A),2.25(m,2H,26),2.18(s,3H,19),2.08(s,3H,4E-N-COCH ₃ ),2.0(m,1H,2B),1.8(m,1H,2B),1.50(s,3H,16a),1.44(s,3H,16b),1.42(d,J＝6.2Hz,3H,6B),1 .4(m,1H,2E axis),1.31(d,J＝6.1Hz,3H,6A),1.31(d,J＝6.1Hz,3H,6D),1.19(t,J＝7.2Hz,3H,4E-N-CH ₂ CH ₃ ).

Example 7

Butyric acid, 3-[[(2E)-2-[(1R,4Z,8S)-8-[[2-O-[4-(acetylethylamino)-2,4-dideoxy-3- O-methyl-α-L-threo-pentopyranosyl]-4,6-dideoxy-4-[[[2,6-dideoxy-4-S-[4-[(6-deoxy-3-O-methyl [(1-(2-((4-thio)-β-D-pyranose)-1-methyl-3-((2-((4-((4-thio)-β-D-pyranose)-1-methyl-3-(((4-( ... [hexosyl]oxy]amino]-β-D-glucopyranosyl]oxy]-1-hydroxy-10-[(methoxycarbonyl)amino]-11-oxo Bicyclo[7.3.1]tridec-4,9-diene-2,6-diyn-13-ylidene]ethyl]dithio]-3-methyl-,2-[(1E)-1-[4- Preparation of [4-[(2,5-dioxo-1-pyrrolidinyl)oxy]-4-oxobutoxy]-phenyl]ethylidene]hydrazide without using EDC

As a comparison to the process of the present invention illustrated in Example 6, the following reaction was performed: To a solution of N-acetyl-calicheamicin (50 mg, 0.035 mmol) in acetonitrile (1.0 mL) at room temperature was added the linker intermediate butyric acid, 3-mercapto-3-methyl-, 2-[(E)-1-[4-[4-[(2,5-dioxo-1-pyrrolidinyl)oxy]-4-oxobutoxy]phenyl]-ethylidene]-hydrazide (31.9 mg, 0.07 mmol) in one portion, followed by triethylamine (5.3 mg, 7.3 uL, 0.053 mmol). The reaction mixture (slurry) was stirred at room temperature for 1 hour, at which point it became a yellow solution. The yield was determined based on the area percentage relative to the derivative standard (35% yield).

Mass Spectrometry:

(M+Na)＝1801.4578

^1H NMR:

¹ H NMR (CDCl ₃ +5% CD ₃ OD, 400MHz): 8.69 (s, 1H, 18C-NH-N ₌ ), 7.75 (d, J = 8.7Hz, 2H, 22), 6.95 (d, J = 8.7Hz, 2H, 23), 6.38 (br t,J＝7Hz,1H,14),6.23(d,J＝1.5Hz,1H,8),5.90(d,J＝9.5Hz,1H,4),5.80(dd,J＝9.5,1.5Hz,1H, 5),5.72(d,J＝1.5Hz,1H,1D),5.63(brd,J＝2.2Hz,1H,1E),5.03(dd,J＝11.5,1.6Hz,1H,1B),4.7 0(m,1H,5E),4.61(d,J＝7.8Hz,1H,1A),4.6(m,1H,3E),4.49(m,1H,2D),4.31(m,1H,3B),4.20(m ,1H,5D),4.10(m,2H,25),4.07(m,1H,5B),4.03(m,1H,3A),3.91(m,1H,15),3.89(s,3H,2C-OCH ₃ ),3.84(s,3H,3C-OCH ₃ ),3.8(m,1H,3D),3.76(m,1H,15),3.75(m,1H,4B),3.65(m,1H,5A),3.63(bs,3H,10-NHCOOCH ₃ ),3.62(m,1H,2A),3.6(m,1H,4D),3.57(s,3H,3D-OCH ₃ ),3.4(m,1H,5E),3.37(s,3H,3E-OCH ₃ ),3.30(m,2H,4E-N-CH ₂ CH ₃ ),3.12(d,J＝17.6Hz,1H,12),3.0(m,1H,4E),2.85(m,2H,27),2.85(bs,4,30),2.72(d,J＝17.6Hz,1H,12), 2.5(m,2H,17),2.4(m,1H,2E flat),2.33(m,1H,4A),2.25(m,2H,26),2.18(s,3H,19),2.08(s,3H,4E-N-COCH ₃ ),2.0(m,1H,2B),1.8(m,1H,2B),1.50(s,3H,16a),1.44(s,3H,16b),1.42(d,J＝6.2Hz,3H,6B),1 .4(m,1H,2E axis),1.31(d,J＝6.1Hz,3H,6A),1.31(d,J＝6.1Hz,3H,6D),1.19(t,J＝7.2Hz,3H,4E-N-CH ₂ CH ₃ )

Example 8

Butyric acid, 3-[[(2E)-2-[(1R,4Z,8S)-8-[[2-O-[4-(acetylethylamino)-2,4-dideoxy-3- O-methyl-α-L-threo-pentopyranosyl]-4,6-dideoxy-4-[[[2,6-dideoxy-4-S-[4-[(6-deoxy-3-O-methyl [(1-(2-((4-thio)-β-D-pyranose)-1-methyl-3-((2-((4-((4-thio)-β-D-pyranose)-1-methyl-3-(((4-( ... [hexosyl]oxy]amino]-β-D-glucopyranosyl]oxy]-1-hydroxy-10-[(methoxycarbonyl)amino]-11-oxo Bicyclo[7.3.1]tridec-4,9-diene-2,6-diyn-13-ylidene]ethyl]dithio]-3-methyl-,2-[(1E)-1-[4- Large-scale preparation of [4-[(2,5-dioxo-1-pyrrolidinyl)oxy]-4-oxobutoxy]-phenyl]ethylidene]hydrazide, Then purify

To a solution of N-acetyl-calicheamicin (60.2 g, 42.7 mmol) in acetonitrile (900 mL) at 4°C was added the linker intermediate butyric acid, 3-mercapto-3-methyl-, 2-[(E)-1-[4-[4-[(2,5-dioxo-1-pyrrolidinyl)oxy]-4-oxobutoxy]-phenyl]ethylidene]hydrazide (38.4 g, 85.4 mmol) in one portion, followed by 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (8.2 g, 42.7 mmol). The bottle containing the linker and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide was rinsed with acetonitrile (300 mL) and added to the reaction mixture. Triethylamine (6.5 g, 8.9 mL, 64.1 mmol) was then added to the reaction mixture. The reaction mixture (slurry) was stirred at 4°C for 1 hour, at which point it became a yellow solution. The reaction mixture was diluted with acetonitrile (600 mL) and 20 mM sodium acetate buffer, pH 5.0 (1200 mL) and purified by reverse phase HPLC using a gradient of mobile phases A and B (mobile phase A: 55:45 (v/v) 20 mM NaOAc buffer, pH 5.0 (pH 4.5-5.5): acetonitrile; mobile phase B: acetonitrile). Fractions of the desired purity were collected and then subjected to solid phase extraction (SPE). In SPE, the purified fractions were loaded onto the column, washed with a water/acetonitrile mixture, and then eluted with acetonitrile to obtain concentrated fractions containing the product. The resulting fractions were concentrated in vacuo, dissolved in ethyl acetate, and precipitated by adding hexane. The solid was filtered and dried to obtain the title compound as a white solid with a purity of 96.9% (45.2 g, 60% yield) as determined by HPLC.

Mass Spectrometry:

(M+Na)＝1801.4578

^1H NMR:

¹ H NMR (CDCl ₃ +5% CD ₃ OD, 400MHz): 8.69 (s, 1H, 18C-NH-N ₌ ), 7.75 (d, J = 8.7Hz, 2H, 22), 6.95 (d, J = 8.7Hz, 2H, 23), 6.38 (br t,J＝7Hz,1H,14),6.23(d,J＝1.5Hz,1H,8),5.90(d,J＝9.5Hz,1H,4),5.80(dd,J＝9.5,1.5Hz,1H, 5),5.72(d,J＝1.5Hz,1H,1D),5.63(brd,J＝2.2Hz,1H,1E),5.03(dd,J＝11.5,1.6Hz,1H,1B),4.7 0(m,1H,5E),4.61(d,J＝7.8Hz,1H,1A),4.6(m,1H,3E),4.49(m,1H,2D),4.31(m,1H,3B),4.20(m ,1H,5D),4.10(m,2H,25),4.07(m,1H,5B),4.03(m,1H,3A),3.91(m,1H,15),3.89(s,3H,2C-OCH ₃ ),3.84(s,3H,3C-OCH ₃ ),3.8(m,1H,3D),3.76(m,1H,15),3.75(m,1H,4B),3.65(m,1H,5A),3.63(bs,3H,10-NHCOOCH ₃ ),3.62(m,1H,2A),3.6(m,1H,4D),3.57(s,3H,3D-OCH ₃ ),3.4(m,1H,5E),3.37(s,3H,3E-OCH ₃ ),3.30(m,2H,4E-N-CH ₂ CH ₃ ),3.12(d,J＝17.6Hz,1H,12),3.0(m,1H,4E),2.85(m,2H,27),2.85(bs,4,30),2.72(d,J＝17.6Hz,1H,12), 2.5(m,2H,17),2.4(m,1H,2E flat),2.33(m,1H,4A),2.25(m,2H,26),2.18(s,3H,19),2.08(s,3H,4E-N-COCH ₃ ),2.0(m,1H,2B),1.8(m,1H,2B),1.50(s,3H,16a),1.44(s,3H,16b),1.42(d,J＝6.2Hz,3H,6B),1 .4(m,1H,2E axis),1.31(d,J＝6.1Hz,3H,6A),1.31(d,J＝6.1Hz,3H,6D),1.19(t,J＝7.2Hz,3H,4E-N-CH ₂ CH ₃ )

Claims (4)

1. A method for preparing the compound izumab ozomicin, comprising the following steps: (a) Treatment of compounds of formula I with a strong acid: Compounds forming formula IV: (b) Reacting a compound of formula IV with 1-hydroxypyrrolidine-2,5-dione to form a compound of formula V: (c) Reaction of compound V with N-acetyl-galicarmycin in acetonitrile, followed by reaction with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide in the presence of triethylamine, forms the following linker-effective load: as well as (d) Conjugate the linker-load to izmuzumab. 2. A method for preparing the compound gemutuzumab ozomicin, comprising the following steps: (a) Treatment of compounds of formula I with a strong acid: Compounds forming formula IV: (b) Reacting a compound of formula IV with 1-hydroxypyrrolidine-2,5-dione to form a compound of formula V: (c) Reaction of compound V with N-acetyl-galicarmycin in acetonitrile, followed by reaction with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide in the presence of triethylamine, forms the following linker-effective load: as well as (d) Conjugate the linker-load to gemtuzumab. 3. A method for preparing the compound izumab ozomicin, comprising the following steps: (a) Compounds of formula V: The reaction with N-acetyl-galicarmycin in acetonitrile, followed by reaction with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide in the presence of triethylamine, forms the following linker-effective load: as well as (b) Conjugate the linker-load to izmuzumab. 4. A method for preparing the compound gemutuzumab ozomicin, comprising the following steps: (a) Make compound V: The reaction with N-acetyl-galicarmycin in acetonitrile, followed by reaction with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide in the presence of triethylamine, forms the following linker-effective load: as well as (b) Conjugate the linker-load to gemtuzumab.