JP2003508502A - Methods and compositions for the production of long-lasting antineoplastic agents - Google Patents

Abstract

  (57) [Summary] Derivatives of the antitumor agent are prepared. The derivative may form a covalent bond with one or more blood components, especially mobile blood components. Provided is the use of a composition for the manufacture of a medicament for use in the treatment of cancer, the composition comprising a derivative of taxol and its analogs, wherein the derivative comprises an amino group in a blood component, It includes a reactive group that reacts with a hydroxyl or thiol group to form a stable covalent bond, wherein the reactive group is selected from N-hydroxysuccinimide, N-hydroxy-sulfosuccinimide and maleimide groups.

Description

Detailed Description of the Invention

FIELD OF THE INVENTION The present invention relates to modified antitumor agents. In particular, the present invention relates to antitumor agents modified so that they can form covalent bonds with blood components in vivo.

BACKGROUND OF THE INVENTION Since more than 50 years ago the discovery of the antitumor properties of nitrogen mustard, cancer chemotherapy has
It has been an ever expanding area of scientific effort, and has been an important component of cancer treatment along with surgery and radiation therapy. Chemotherapy was once accepted as the only way to prolong survival for patients previously diagnosed as treatable by surgery and / or radiation therapy, but nowadays most of the more than 2,000 variants of cancer It is recognized as the mode of treatment in all.

Modern cancer chemotherapies typically involve the combination of two or three different anti-neoplastic agents, and advances in technology and medical knowledge have shown that patients with multiple forms of cancer have It has greatly improved the prospect of recovery. The role of anti-neoplastic agents in cancer treatment varies widely depending on the morphology of the cancer. For example, chemotherapy is often the major step of treatment in ovaries, testes, breast, bladder, and other cancers, leukemias and lymphomas, and is commonly used for sarcomas, melanomas, myelomas, and other Used in combination with radiation therapy in most procedures. In contrast, chemotherapy is often used as a last resort only or as a temporary treatment for many solid tumors (eg, pancreatic and lung cancers). Exceptions exist within each class of tumor or other neoplasm.

Natural antineoplastic agents suffer from poor bioavailability, widespread first metabolic transit, and short half-life, resulting in the need for these agents to be constantly infused into patients. . Moreover, although useful against cancer by killing cancer cells, anti-neoplastic agents are also often toxic to normal cells, which toxicity is associated with their uncontrolled biodistribution.

Accordingly, there is a need for methods to extend the half-life of antitumor agents. At least two publications convey this issue. King's European Patent Application No. 0554708A1 discloses a drug modified with a ligand attached to a drug using a hydrazone bond, and an agent attached for delivery via the hydrazone bond. Furthermore, Kratz's PCT application WO 98/10794 discloses at least one derived from a maleimide or N-hydroxysuccinimide ester compound.
Disclosed is a conjugate of transferrin, albumin and polyethylglycol with one HS group, HO group, H ₂ N group.

The conjugates disclosed in these publications are made using methods performed ex vivo. Such methods include obtaining human serum, purifying a desired carrier (eg, albumin or transferrin) from the serum, specific reactive groups (eg, “ligand” disclosed in King's application). And reacting the purified protein, and repurifying the conjugate to obtain a composition suitable for in vivo use. Such a method
Very invasive, lengthy, and expensive. Therefore, there is a need to develop anti-cancer drugs with better bioavailability that can be introduced in vivo without the need for a step of conjugation in vitro.

SUMMARY OF THE INVENTION The present invention relates to novel chemically reactive derivatives of antitumor agents, methods of synthesizing such derivatives, and methods of using such derivatives in vivo.
The anti-neoplastic drug derivatives of the present invention can react in vivo with available functional groups on mobile blood proteins to form covalent bonds, so that the resulting covalent conjugates are bound to these anti-cancer compounds. Substantially retain activity.

One aspect of the present invention relates to the use of the composition (the composition comprising a derivative of an antitumor drug and its analogs) in the manufacture of a medicament. This derivative contains reactive groups that react with amino, hydroxyl, or thiol groups on blood components to form stable covalent bonds. The reactive group is selected from N-hydroxyl succinimide, N-hydroxy-sulfosuccinimide and maleimide groups for use in treating cancer. Specific examples of anti-neoplastic agents used in the present invention include taxol, methotrexate, hydroxyurea, tamoxifen, mitomycin C, and analogs thereof. Particular examples of blood components that are reactive with the derivative include blood proteins, more specifically blood protein thiol groups, and even more particularly albumin.

Another aspect of the invention relates to a method for extending the half-life of an antitumor agent in vivo. These methods include the following steps: modifying the anti-neoplastic agent by attaching the reactive group to a site well away from the pharmacophore region, such that: An anti-tumor drug derivative substantially retains its therapeutic activity and is capable of forming a covalent bond with a functional group on the blood component; and between the reactive group and the functional group on the blood component. Forming a covalent bond with the anti-neoplastic agent derivative-blood component conjugate with the step of increasing the half-life of the anti-neoplastic agent. Specifically, the present invention is
The step of administration of the antineoplastic drug derivative occurs in vivo prior to formation of the covalent bond,
As a result, methods of forming anti-tumor drug derivative-blood component conjugates in vivo are included.

Yet another aspect of the invention includes antineoplastic drug derivatives and methods of making such derivatives. The anti-tumor drug derivative comprises a linking group with the anti-tumor drug and a reactive group capable of reacting with a functional group on the blood component (eg, maleimide or N-hydroxysuccinimide (NHS)). Specific antitumor drug derivatives include doxorubicin, taxol, methotrexate, hydroxyurea,
Included are derivatives of tamoxifen, mitomycin C, platinum complexes, vinca alkaloids, and their analogs.

By reacting with and binding to a blood component in vivo,
The anti-tumor drug derivative of the present invention can be delivered to an appropriate site or receptor via blood. The conjugated molecule extended the half-life in the bloodstream as compared to the unconjugated anti-tumor drug. Thus, the conjugated anti-neoplastic agent can maintain anti-cancer activity for an extended period of time compared to the unconjugated parent drug, and provide such activity with reduced side effects.

The present invention further comprises conjugates of antineoplastic drug derivatives and mobile blood components.
This is specific for mobile endogenous proteins such as serum albumin, IgG, proteins of stromal cells and blood bone cells using maleimide (maleimide-antitumor drug) -derived antitumor drug as a reactive group. Label, and NHS as reactive group (NHS antitumor drug) and sulfo-NHS as reactive group (sulfoN
HS-Anti-tumor drug) non-specific labeling of mobile blood proteins with an anti-tumor drug induced using. The invention further includes a method for providing in vivo anti-cancer activity comprising administering to a mammalian host a novel anti-tumor drug derivative or conjugate thereof.

The present invention also relates to the use of antibodies to localize and bind such conjugates, eg, to remove unwanted excess conjugates from the bloodstream of the host. The invention also relates to the use of the antibody to detect the level of anti-neoplastic drug conjugate in blood.

In general, an antitumor drug derivative according to the present invention is a compound of the formula: A-X-R-D wherein A is an anti-tumor drug and X is Selective functional groups, such as ethers, thioethers, secondary or tertiary amines, secondary or tertiary amides, esters, thioesters, imines, hydrazones, semicarbazones, acetals, hemiacetals, ketals, hemiketals,
Aminal, hemiaminal, sulfonic acid, sulfuric acid, sulfonamide, sulfonamidate, phosphoric acid, phosphoramide, phosphonate, or phosphoramidate, R is any linking group, eg, non- Presence (where X
Is directly bonded to D), any alkyl chain C _1-10 , any fluoroalkyl C _1-10 or any combination of fluoro-substituted alkyl chains C _1-10 ,-(Z-
_{CH 2 CH 2 -Z) n -} , - (Z-CF 2 CH 2 -Z) n -, - (Z-CH 2 CF 2 -Z
) _N - be any ether or thioether containing alkyl or fluoroalkyl chain, such as but not limited to, where a n = 1 to 4,
Then, Z is, O, or _{S, -Y-C 6 H 4} -, - Y-C 6 H 4 -Y- structure disubstituted ortho like, be either meta or para benzene, Here, Y is CH ₂ , O, S, NH, NR [R = H, alkyl, acyl], furan,
An ester or mixed anhydride or imidate, which is any combination of heterocycles disubstituted with thiophene, pyran, oxazole, or thiazole, and D includes phenolic ester, thiol ester, alkyl ester, phosphate ester, and the like. A reactive group such as a carboxyl group, a phosphoryl group, or an acyl group, and succimidyl such as N-hydroxysuccinimide (NHS) or N-hydroxy-sulfosuccinimide (sulfo-NHS) Groups and maleimide groups, maleimide groups such as, but not limited to, maleimidic acid (including but not limited to maleimidopropionic acid (MPA)) and maleinimide esters.

It has to be noted that the use of R as a linking group is optional. In some cases, it may be advantageous or even necessary to use a linking group to better retain the therapeutic activity of the anti-neoplastic agent. However, in other cases, the direct attachment of the reactive group (D) to the sensitive functional group (X) of the anti-neoplastic agent may well preserve the therapeutic activity of the anti-neoplastic agent, so the use of a linking group May not be necessary.

[0016]   Certain anti-tumor drug derivatives of the present invention have the formula:

[0017]

[Chemical 3] A doxorubicin derivative, such as a compound of the formula:

[0018]

[Chemical 4] A taxol derivative, such as a compound of the formula:

[0019]

[Chemical 5] A methotrexate derivative, such as a compound of the formula:

[0020]

[Chemical 6] A hydroxyurea derivative, such as a compound of the formula:

[0021]

[Chemical 7] A tamoxifen derivative, such as a compound of the formula:

[0022]

[Chemical 8] A mitomycin C derivative, such as a compound of the formula:

[0023]

[Chemical 9] Vincristine derivatives, such as the compounds of.

DETAILED DESCRIPTION OF THE INVENTION For the purposes of the present invention, and in order to ensure a full understanding of the invention, the following definitions are provided: Anti-tumor agent—Antineoplastic agent, eg , Fluoropyrimidines, pyrimidine nucleosides, purines, platinum analogs, anthracyclines / anthracenesions, podophyllotoxins, camptothecins, hormones and hormone analogs, enzymes, proteins and antibodies, vinca alkaloids, taxanes or colchicines,
Antihormonal agents, antifolates, antimicrotubule agents, alkylating agents (classical and nonclassical), antimetabolites, antibiotics, topoisomerase inhibitors, antiviral agents, and miscellaneous cytotoxic agents ( For example, anti-cancer agents such as hydroxyurea, mitotan, fusion toxins, PZA, bryostatin, retinoids, butyric acid and derivatives, pentosan, fumagillin and others). The purpose of all anti-neoplastic drugs is to eliminate (treat) or delay the growth and spread (remission) of cancer cells. Most of the anti-neoplastic agents listed above pursue this end by having predominantly cytotoxic activity, which affects and directly kills cancer cells. Other anti-neoplastic drugs stimulate the body's natural immunity, which affects cancer cell death.

The anti-tumor agents used in the present invention may have asymmetric carbon atoms or one or more asymmetric centers and thus give rise to optical isomers and diastereomers. When indicated without regard to stereochemistry, the anti-tumor agents used in the present invention include such optical isomers and diastereomers; and racemic and dissolved enantiomerically pure R and S stereoisomer; and other mixtures of R and S stereoisomers,
And the pharmaceutically acceptable salt is included. Each anti-neoplastic agent contains a pharmacophore region within the body that interacts with a target site (eg, a receptor).

Reactive group: A reactive group is a functional group capable of forming a covalent bond. Such reactive groups are linked to the antitumor agent of interest through a linking group (as defined below) and a hydrolytically stable bond, or optionally without a linking group. Or be combined. The reactive group is generally stable in an aqueous environment and is usually a carboxyl group, a phosphate group, or a convenient acyl group, either as an ester or as a mixed anhydride or imidate, Can form covalent bonds with functional groups such as amino, hydroxy or thiol groups at target sites on blood components. For the most part, the ester comprises a phenolic compound or may be a thiol ester, an alkyl ester, a phosphoric acid ester and the like. Examples of the reactive group include, but are not limited to, a succimidyl group such as N-hydroxysuccinimide (NHS) and N-hydroxy-sulfosuccinimide (sulfo-NHS), a maleimide group, and a maleimidic acid (maleimidopropionic acid (MPA)). ) And maleimide groups such as maleimide esters. In a further preferred embodiment of the invention, the functional group on the blood component is a thiol group and the reactive group is a maleimide group.

Sensitive Functional Group-A sensitive functional group is a group of atoms that represents a potential reactive site for a therapeutic agent. When present, sensitive functional groups can be selected as attachment points for linking group-reactive group modifications. Sensitive functional groups include carboxyl, amino, thiol, hydroxyl, ether, thioether, secondary or tertiary amine, secondary or tertiary amide, ester, thioester, imine, hydrazone, semicarbazone, acetal, hemiacetal, ketal, hemiketal. , Aminal, hemiaminal, sulfonic acid, sulfuric acid, sulfonamide, sulfonamidate, phosphoric acid, phosphoramide, phosphonate, or phosphonamidate,
It is not limited to these.

Functional group: A functional group is a group on blood components, including mobile and fixed proteins. Reactive groups on the anti-tumor drug react with it to form covalent bonds. The functional group is usually a hydroxyl group which is bound to an ester reactive group, a thiol group which is bound to a maleimide, an imidate and a thioester group, an activated carboxyl group, a phosphate group or any other acyl group. Including the amino group.

Blood component: The blood component can be either fixed or mobile. Immobilized blood components are immobile blood components, and are tissues, membrane receptors, interstitial proteins, fibrin proteins, collagen, platelets, endothelial cells, epithelial cells and associated membrane and membrane receptors, somatic cells, skeletal cells. And smooth muscle cells, neuronal components, osteocytes and osteoclasts, and all body tissues particularly associated with the circulatory and lymphatic systems. A mobile blood component is a blood component that does not have a fixed position for an extended period (generally no more than 5 minutes) (most often 1 minute). These blood components are not membrane associated and are present in blood for an extended period of time and are present at a minimum concentration of at least 0.1 μg / ml. Mobile blood components include serum albumin, transferrin, ferritin and immunoglobulins such as IgM and IgG. The mobile blood component has a half-life of at least about 12 hours.

Protecting group: A protecting group is a chemical moiety utilized to prevent an anti-tumor drug from reacting with itself. Specifically, the hydroxamine moieties of the anti-neoplastic agents used in the present invention include t-butyl, aryl, benzyl, tetrahydropyranyl, t
Protected using protecting groups including but not limited to -butyloxycarbonyl, t-butyldimethylsilyl and trimethylsilyl.

Linking group: A linking group is a chemical moiety that links or attaches a reactive group to an antitumor agent. The linking group is one or more alkyl groups, fluoroalkyl groups, alkoxy groups, alkenyl groups, alkynyl groups or amino groups substituted with alkyl groups, cycloalkyl groups, polycyclic groups, aryl groups, polyaryl groups, substituted aryl groups, It may include a heterocyclic group and a substituted heterocyclic group. The linking group can also be a polyethoxy amino acid (eg,
AEA ((2-amino) ethoxyacetic acid) or a preferred linking group AEEA ([2-
(2-amino) ethoxy]) ethoxyacetic acid).

Anti-tumor Drug Derivatives-Antineoplastic drug derivatives are anti-tumor drugs modified by attaching reactive groups. The reactive group can be attached to the anti-neoplastic agent via a linking group or optionally without a linking group. Generally, the anti-tumor drug derivative comprises an anti-tumor drug molecule and a linking group with a reactive group capable of reacting with a functional group on the blood component. The antineoplastic drug derivative may be administered in vivo, so that
Binding to blood components occurs in vivo, or they may be first bound to blood components in vitro, and the resulting conjugated antitumor agent (as defined below). It was administered in vivo.

Conjugated anti-tumor drug—The conjugated anti-tumor drug is linked to the anti-tumor drug either with or without the reactive group of the anti-tumor drug derivative (with or without a linking group). And a functional group of a blood component, the derivative of an antitumor drug conjugated to the blood component via a covalent bond formed. As used herein, the term "conjugated anti-tumor agent" is specifically created for a conjugated anti-tumor agent (eg, "conjugated doxorubicin" or "conjugated taxol"). It was By conjugation with blood components, the anti-neoplastic drug derivative can be delivered via the blood to the appropriate site or receptor in the patient.

In view of these definitions, the present invention relates to compositions that are derivatives of anti-neoplastic agents capable of reacting via covalent bonds with available reactive functional groups on blood components.
The invention also relates to such derivatives, combinations with such blood components, such methods of making derivatives, and methods for their use. These methods include the step of prolonging the effective therapeutic life of the anti-neoplastic agent in question compared to the administration of the anti-neoplastic agent itself to the patient.

To form a covalent bond with a functional group on a blood component, one of skill in the art will appreciate that a wide variety of active carboxyl groups may be used as reactive groups when the hydroxy moieties are physiologically acceptable at the required levels. In particular esters may be used. A number of different hydroxyl groups can be used, but the most convenient are N-hydroxysuccinimide (NHS), N-hydroxy-sulfosuccinimide (sulfo-NHS), maleimide, maleimidic acid (maleimidopropionic acid (MPA)). (Including but not limited to), as well as maleimide esters. In a preferred embodiment of the invention, the functional group on the blood component is a thiol group and the reactive group is maleimide.

The object of the present invention is to improve the bioavailability of these inhibitors on protein carriers via selective conjugation of the inhibitors, but without modifying their outstanding anti-cancer properties. The derivatization of anti-tumor agents to confer an extended half-life and better distribution. A preferred carrier for the present invention is, but not limited to, albumin, which is then conjugated via an anti-tumor agent derivatized with a maleimide moiety via its free thiol.

1. Antitumor Agents Used in the Present Invention Currently, there are about 20 recognized classes of approved antitumor drugs. This classification is generalized based on the common structure shared by a particular drug or the common mechanism of action by a drug. Some drugs fall into more than one class, but generally the accepted classifications are as follows (classifications are not listed in any particular order): Fluoropyrimidines , Pyrimidine nucleosides, purines, platinum analogs, anthracyclines / anthracenediones,
Podophyllotoxin, camptothecin, hormones and hormone analogs, enzymes, proteins and antibodies, vinca alkaloids, taxanes, antihormonal agents, antifolates, antimicrotubule agents, alkylating agents (classical and nonclassical) , Antimetabolites, antibiotics, topoisomerase inhibitors, antivirals, and miscellaneous cytotoxic agents as described in more detail below.

Some partial listings of commonly known commercially approved (or actively developing) anti-neoplastic agents organized by classification are as follows: Fluoro Pyrimidine analogs such as pyrimidines (eg, 5-FU, fluorodeoxyuridine, futrafur, 5′-deoxyfluorouridine, UFT, S-1 and capecitabine); deoxycytidine, cytosine, arabinoside, 5-azacytosine, 9-β-arabi. Arabinofuranosyl, gemcitabine (ge)
and pyrimidine nucleosides such as 5-azacytosine-arabinosine; 6-mercaptopurine, thioguanine, azathioprine, allopurinol, cladribine, fludarabine.
), Pentostatin, and purines and purine analogs such as 2-chloroadenosine; cisplatin, carboplatin, oxaliplatin, tetraplatin, platinum-
Platinum analogs and platinum coordination complexes such as DACH, olmaplatin, CI-973, and JM-216; anthracyclines or anthracenediones such as doxorubicin, daunorubicin, epirubicin, idaurubicin, and mitoxantrone; epidophyllo such as etoposide and teniposide. Tosicin; irinotecan, topotecan, 9-aminocamptothecin, 10,11-methylenedioxycamptothecin, 9-nitrocamptothecin, and TAS 103.
Camptothecins such as; diethylstilbestrol, tamoxifen, toremifene, tolmudex, thymitaq, flutamide, bicarutamide, finasteride, estradiol, trioxyphene, droloxifene.
xifene), medroxyprogestrone acetate, megestrol acetate, aminoglutethimide, test lactone and other hormones and hormone analogs; asparaginase, interleukins, interferons, leuprolide, pegaspargase and others Enzymes, proteins and antibodies of vincristine, vinblastine, vinorelbine
, Vinca alkaloids such as vindesine; taxanes such as paclitaxel and docetaxel and colchicine; antihormonal agents such as anastrozole; methotrexate (amethopterin), aminopterin, trimetrexate, trimethoprim, pyritrex (p), pyritrex (p), pyritrex (p). Pyrimethamine,
Antifolate drugs such as edatrexate and MDAM; anti-microtubule agents such as taxanes and vinca alkaloids; nitrogen mustard (mechlorethamine, chlorambucil, melphalan, uracil mustard), oxaazaphosphorine (ifosfamide, cyclophosphamide,
Alkylating agents (traditional and non-traditional) such as perphosphamide, trophosphamide), alkyl sulfonates (busulfan), nitrosoureas (carmustine, lomustine, streptozocin), thiotepa, dacarpazine and others; the purines listed above. Antibiotics such as, pyrimidine and nucleosides; antibiotics such as anthracycline / anthracenesion, bleomycin, dactinomycin, mitomycin, plicamycin, pentostatin, streptozocin, daunorubidine (rubimicin), and doxorubicin; camptothecin. (Topo I), epipodophyllotoxin (epipodo
phyllotoxin), m-AMSA, and ellipticine (Topo I)
I) such as topoisomerase inhibitors; AZT, zalcitabine, gemcitabine (gemc)
Itabine), didanosine, and other antiviral agents; as well as hydroxyurea, mitotanes, fused toxins, PZA, bryostatin (br
Miscellaneous cytotoxic agents such as yostatin), retinoids, butyric acid and derivatives, pentosan, fumagillin, and others.

A major difficulty and cause for any anti-tumor drug is its cytotoxicity on normal healthy cells. Drugs listed above (and these drugs currently in development)
Has the potential to mediate serious and often life-threatening toxic side effects even when given a therapeutically effective dose. Although extensive efforts have been made to develop antitumor agents that are safe to use in effective doses, the toxic side effects associated with such drugs are almost always present. The specific symptoms of toxic side effects are available in any of the numerous oncology textbooks, publications, patents, and other printed materials. The compounds of the invention have better bioavailability and therefore do not need to be administered in large doses, they have a better safety profile than underivatized antitumor agents.

2. Mechanism of Action Detailed mechanisms of action and toxicity (both proven and putative) are detailed throughout the prior art. An overview of these mechanisms is provided below to outline the metabolism of anti-neoplastic drugs, and their effects on malignant disease, as well as on healthy tissues.

A. Antihormones As the name suggests, antihormonal antitumor drugs exert cytotoxic activity by blocking hormonal action at terminal receptor organs. Several different types of neoplasms require hormonal stimulation in order to spread cell replication. By blocking hormonal action, antihormonal agents deprive neoplastic cells of the stimuli needed for replication. When cells reach the end of their life cycle, they usually die without dividing and producing additional malignant cells.

Antihormonal drugs have been used with varying degrees of success as treatments for malignancies of the breast, uterus, prostate, testis and other sex-specific organs. Antihormonal drugs generally target the androgen receptor in males and the estrogen or progesterone receptor in females. In recent years, much research has been directed to the development of aromatase inhibitors, which indirectly reduce blood levels of estrogen by blocking the conversion of androgens to estrogens. Antihormonal antitumor agents are essentially classified as steroids or non-steroids.

The toxicity of anti-hormonal anti-neoplastic agents is generally mild to moderate. Because they are usually close to analogs of natural physiological factors, they are relatively specific in their mechanism of action and do not inhibit DNA synthesis, which makes all anti-neoplastic agents Minimize the serious side effects found in most.

The manifestation of toxicity in antihormonal therapy includes its effects normally associated with hormone deficiency, such as hot flashes, weight gain (and weight loss), skin rashes, acne and the like. Although nausea and vomiting can occasionally occur, GI distress is usually mild to moderate and usually does not require dose reduction. Antihormonal therapy is usually
Limited to patients with a positive response to hormones.

(B. Folate Antagonist) A folate antagonist exerts a cytotoxic activity by inhibiting the action of dihydrofolate reductase (DHFR) (an enzyme important in cell replication). DHFR is well known to reduce dihydrofolate to its active, fully reduced tetrahydro form. Tetrahydrofolate acts as a coenzyme in the de novo synthesis of purines, which are important precursors to DNA. Depletion of the tetrahydrofolate pool also inhibits thymidylate biosynthesis, which blocks the conversion of deoxyuridine phosphate (dUMP) to deoxythymidine phosphate (dTMP) (DNA nucleotide precursor).

Although many antifolate drugs are licensed for use in the United States, only methotrexate is the only licensed antitumor drug on the market. Other licensed antifolates have bacterial DHs with far greater strength and frequency than human DHFR.
Due to its propensity to bind FR, it is mainly used as an anti-infective drug. Methotrexate itself is also used in the treatment of rheumatoid arthritis, psoriasis, and related inflammatory diseases, and for the treatment of cancers of the breast, uterus, testis, ovary and for various forms of leukemia (eg acute lymphocytic leukemia). Used in. One drug, methotrexate, is curative for choriocarcinoma, a rare type of cancer.

Antifolates produce a wide variety of toxic and other inadequate effects. Hematological toxicity is the major dose limiting toxicity associated with methotrexate and other antifolates. Other toxic and inadequate effects associated with antifolates include: neurotoxicity (which is sometimes severe and irreversible); gastrointestinal toxicity, mainly mucositis, Nausea, diarrhea, vomiting, loss of appetite, etc .;
Hepatotoxicity; Dermatological toxicity, often manifested by rash, hair loss, and tissue inflammation; Pulmonary toxicity; Nephrotoxicity; and other inappropriate toxic side effects. Antifolates are generally recognized as drugs with a low therapeutic index and a high incidence of toxic side effects.

C. Antimicrotubule Agents Microtubule agents interfere with cell division by disrupting the normal function of the microtubules of cells. Microtubules are an important element of cells, play an important role in segregating replicating chromosomes during cell division, and in many interphase cell functions such as maintaining cell shape and scaffolding, intracellular trafficking, Secretion, neurotransmission, etc.).

Two classes of anti-neoplastic agents that affect microtubules are taxanes (paclitaxel and docetaxel are representatives of this class) and vinca alkaloids (vincristine, vinblastine, and vinorelbine are members of this class). It is an authorized member).

Taxanes and vinca alkaloids are naturally occurring or semi-synthetically derivatized analogs of naturally occurring compounds of plant origin. In particular, the taxanes are needles and twigs of the European yew (Taxus baccata) or the Pacific yew (Taxus brevifoli) native to the West Coast of North America.
It is derived from the bark of a). The most widely known taxanes at this time are paclitaxel (Taxol®) and docetaxel (Taxotere®), which are widely known as antitumor agents.

Vinca alkaloids include vincristine (Oncovin®), vinblastine (Velban®), and vinorelbine (Na).
velbine®), and these are generally derived from the periwinkle plant (Cantharanthus roseus). These drugs are also widely known as antitumor agents.

Despite their different mechanisms of action, both taxanes and vinca alkaloids exert their biological effects on cellular microtubules. Taxanes act to promote the polymerization of tubulin, a protein subunit of spindle microtubules.
The end result of this is the inhibition of microtubule depolymerization, which leads to the formation of stable and non-functional microtubules. This disrupts the dynamic equilibrium in the microtubule system and arrests the cell cycle in late G ₂ and M phases, thereby inhibiting cell replication.

Like taxanes, vinca alkaloids also act to affect the intracellular microtubule system. In contrast to taxanes, vinca alkaloids bind to tubulin and inhibit or prevent polymerization of tubulin subunits into microtubules. Vinca alkaloids also induce microtubule depolymerization, thereby inhibiting microtubule assembly and mediating metaphase arrest of cells. Vinca alkaloids are also used in the synthesis of nucleic acids and proteins; amino acids, cyclic A
MP and glutathione synthesis; exerts an effect on cellular respiration; and
It exerts immunosuppressive activity at higher concentrations.

Both taxanes and vinca alkaloids are toxic compounds with a low therapeutic index. Neurotoxicity and myelosuppression are among the most commonly reported clinical toxicities of these drugs.

Taxanes have been shown to cause many different toxic and inadequate side effects in patients. The most well-known serious side effects of taxanes are neurotoxicity and hematological toxicity, especially severe neutropenia and thrombocytopenia. Taxanes also cause hypersensitivity reactions in a high percentage of patients at the recommended dosages; GI effects (nausea, diarrhea, and vomiting are common); hair loss;
And cause other inappropriate effects.

Vinca alkaloids are primarily associated with: neurotoxic effects (a major side effect in patients receiving vincristine); hematological toxicity (leukopenia
Often severe and dose limiting in the case of vinblastine); gastrointestinal effects (nausea, diarrhea, and vomiting); hair loss; tissue hypersensitivity and necrosis (especially at the injection site)
And other harmful and inappropriate effects.

D. Alkylating Agents As the name implies, alkylating agents generally exert cytotoxic activity by alkylating DNA, directly impeding the cell's replication cycle. Demonstrate. As described in the general listing above, antineoplastic alkylating agents fall into a number of subclasses based on their general structure and the mechanism by which they act to alkylate DNA.

Several subclasses of antitumor alkylating agents are commercially available as chemotherapeutic agents. The best known are the nitrogen mustards, which include chlorambucil, mechlorethamine, melphalan, uracil mustard, and the like.

Oxazaphosphorin is a subtype of nitrogen mustard and includes ifosfamide, cyclophosphamide, trophosphamide (tro
phosphamide) and the like are included. Other types of antitumor drugs that act as alkylating agents and are commercially available as of the filing date of this application include alkyl sulfonates (busulfan) and nitrosoureas (carmustine, lomustine, and to some extent streptozocin). To be Other anti-tumor drugs are also D
It has the ability to alkylate NA and / or RNA. Dacarbazine and procarbazine are currently approved drugs in the United States that act as DNA alkylators.

Antitumor alkylating agents produce a wide variety of toxicities and other inadequate side effects. Hematological toxicity is the major dose limiting toxicity associated with all alkylating agents and is often severe. Other toxic and inadequate effects associated with alkylating agents include: neurotoxicity; gastrointestinal toxicity, primarily nausea,
Diarrhea, vomiting, loss of appetite, etc .; hepatotoxicity; dermatological and topical sensitivity, often expressed by rash, hair loss, and tissue inflammation and necrosis; pulmonary effects;
Urogenital effects (hemorrhagic cystitis is often severe in patients receiving cyclophosphamide therapy); and other inadequate toxic side effects. Anti-tumor alkylating agents are widely recognized as drugs with a low therapeutic index and a high incidence of toxic side effects.

E. Antimetabolites Antimetabolites generally replace fraudulent nucleotides with cellular DNA, thereby disrupting cell division, or D
It exerts cytotoxic activity by inhibiting key cellular enzymes that interfere with NA replication. Antimetabolites are always composed of purine (guanine or adenosine) bases or pyrimidine (cytidine or thymidine) bases, with or without a sugar moiety attached.

Purine antimetabolites are widely used anti-leukemic drugs, especially in the treatment of various forms of childhood leukemia. 6-mercaptopurine (6-MP) has 19
It has been administered as a treatment for acute lymphoblastic leukemia (ALL) since the mid-40's. 6-Thioguanine (6-TG) is currently used as a remission inducer in acute myelogenous leukemia (AML). Azathioprine, which is a prodrug of 6-MP, is widely used as an immunosuppressant in clinical transplantation. In general, purine antimetabolites are not effective against solid tumors, probably due to increased lifespan of solid tumor cells when compared to leukemia.

Like other cytotoxic agents, purine antimetabolites also affect the function of normal healthy cells. The cells most affected by purine antimetabolites are:
Cells that grow rapidly, which have a relatively short lifespan, that is, cells found in the bone marrow and upper GI tract.

Purine antimetabolites generally produce a narrow spectrum of toxicity and other inadequate effects. Hematological toxicity is the major dose limiting toxicity associated with 6-mercaptopurine, azathioprine, 6-thioguanine, and other purine antimetabolites.
In particular, leukopenia, anemia, and thrombocytopenia are widely reported effects for these drugs. Other toxic and inadequate effects associated with purine antimetabolites include gastrointestinal toxicity, primarily nausea, diarrhea, and vomiting; liver toxicity; and other inadequate toxic side effects. Purine antimetabolites are generally recognized as drugs with a low therapeutic index and a high incidence of toxic side effects.

In general, all nucleoside analogs are phosphorylated in vivo, usually by deoxycytidine kinase, to their corresponding nucleotides. In this active form, these drugs are all potent antimetabolites, and DN
It is presumed to inhibit one or more enzymes required for A replication. Many anti-neoplastic nucleosides act in the S phase (and possibly at the G, S boundary) in a cell cycle specific manner. Some enzymes that antitumor antimetabolites may inhibit include ribonucleotide reductase, DNA polymerase, thymidylate synthetase,
Examples include uracil riboside phosphorylase and DNA primase. Many antitumor nucleosides also exhibit antiviral activity because of their inhibition of cell replication enzymes.

Nucleoside antimetabolites produce a wide variety of toxicity and other inadequate effects. Hematological toxicity is the major dose limiting toxicity associated with all anti-neoplastic nucleosides and is often severe. Other toxic and inappropriate effects associated with nucleosides include: neurotoxicity, which occasionally
Severe and irreversible; gastrointestinal toxicity, mainly nausea, diarrhea, vomiting, loss of appetite;
Hepatotoxicity; dermatological toxicity, often manifested by rash, hair loss, and tissue inflammation; pulmonary effects; and other inappropriate toxic side effects. Anti-tumor nucleosides are generally recognized as drugs with a low therapeutic index and a high incidence of toxic side effects.

F. Anthracyclines / Anthracenediones This class of antineoplastic drugs also possesses antibiotic activity and was originally developed for such purposes. Although the main mechanism of action of these compounds has not been determined, they do not form topoisomerase II (T) due to the formation of a cleavable complex.
It is presumed to act by inhibiting opo II). Other mechanisms have also been proposed to explain the broad spectrum of activity of anthracyclines.

Due to its broad spectrum of activity, anthracyclines and anthracenedionediones have been extensively studied and reported over the last 25 years. Currently, there are three of these agents licensed for use in the United States (doxorubicin, daunorubicin, and mitozantrone), and two more (epirubicin and idarubicin) are licensed for use in Europe.

Like other cytotoxic agents, anti-neoplastic anthracyclines and anthracenediones also affect normal healthy cell function. In particular, the use of these agents has been associated with a type of cardiotoxicity that has not been observed with other anti-neoplastic agents. Cardiotoxicity, although uncommon (about 20% suffers serious problems), can be chronic and life-threatening. Congestive heart failure and other equally severe heart diseases occasionally occur in patients receiving anthracycline chemotherapy.
In most cases, adverse cardiac effects are associated with cumulative dose, and in some cases,
This effect is irreversible.

Antitumor anthracyclines and anthracenediones can generally cause other toxic and inadequate effects. Hematological toxicity is the major dose limiting toxicity associated with all antineoplastic anthracyclines and anthracenediones.
In particular, leukopenia, anemia, and thrombocytopenia are widely reported effects for these drugs. Other toxic and inadequate effects associated with antineoplastic anthracyclines and anthracenediones include: gastrointestinal toxicity, mainly stomatitis (also frequently associated with nausea, diarrhea, and vomiting); hair loss ( This occurs in almost all patients); severe local tissue damage after extravasation; and other inadequate toxic side effects. Antitumor anthracyclines and anthracenediones have a very broad spectrum of activity, but are generally recognized as drugs with a low therapeutic index and a high incidence of toxic side effects.

No means existed before to combine the toxic effects of anthracyclines and anthracenediones. It was necessary to lower the dose of this drug or to discontinue it, which in turn affects the probability of remission or cure.
Recently, the experimental drug ICRF 187, which metabolizes to form the diamide analog of EDTA, has shown some efficacy in ameliorating the accumulated cardiotoxicity of anthracyclines and anthracenediones.

G. Other Antibiotics Antitumor antibiotics exert their cytotoxic activity through a variety of mechanisms. Pentostatin is a purine analog and acts as an antimetabolite of this class. Bleomycin is a glycoprotein with a molecular weight of approximately 1500 daltons and oxidatively cleaves DNA. Actinomycin (also known as dactinomycin) inhibits RNA and protein synthesis. Plicamycin (mitramycin) binds to DNA, and mitomycin and streptozocin are DNA alkylating agents.

The toxicity associated with antibiotics is similar to other drugs with similar mechanisms of action. Hematological toxicity is quite common with these drugs, and is sometimes severe and dose limiting. Most of these drugs are also highly irritating to tissues, thus local extravasation is a problem. Furthermore, mitramycin is associated with thrombocytopenia-induced hemorrhagic syndrome, especially with prolonged use or high doses of this drug.

Antitumor antibiotics are the oldest class of oncological drugs (actinomycin
It was first introduced clinically in 1954) and is still widely used in the treatment of pediatric neoplasms (actinomycin), testicular neoplasms (bleomycin), and many other solid tumors. Has been done.

H. Topoisomerase Inhibitors Topoisomerase inhibitors are relatively newly introduced antitumor agents, and more are currently in various stages of research and development. As more information about topoisomerases develops, more drugs that inhibit these enzymes will probably be studied.

Topoisomerase (hereinafter, Topo I and Topo II
Said to allow a transient rotation of the DNA strands so that a transient D
It is a ubiquitous cellular enzyme that initiates NA strand breaks. The function of these enzymes is D
Important for NA replication process. In the absence of these, the twisted strands in the DNA helix prevent free rotation, the DNA strands cannot properly separate, and the cell eventually dies without division. Topo I is DNA
Linked to the 3'end of the single-strand break, Topo II is
'Link to the end.

(Epipodophyllotoxin) Epipodophyllotoxin inhibits the action of Topo II. Natural podophyllotoxins are derived from the Podhirum and Mandrake plants, and extracts of these plants have been the source of folk remedies for centuries. The discovery of antitumor properties of both naturally occurring and semisynthetic epipodophyllotoxins goes back more than 30 years.

Naturally occurring podophyllotoxin has no glucoside ring and the C4 position in the E-ring is a methoxy moiety. The mechanism of action is not fully understood,
While the E-ring hydroxy is presumed to be responsible for inducing double-stranded DNA breaks by inhibiting Topo II, the glycoside moiety is believed to prevent the interference of synthetic drug derivatives with spindle microtubule assembly.

Etoposide (and etoposide phosphate, which is a water-soluble prodrug of etoposide), is currently used in a variety of cancers (testicular neoplasm, lung cancer, lymphoma, neuroblastoma, AIDS-related Kaposi's sarcoma, Wilms tumor, (Including various types of leukemia, etc.). Teniposide is a relatively new drug approved by the FDA and started to be marketed in the US in 1992 as a treatment for refractory childhood leukemia. Teniposide has also shown activity against bladder cancer, lymphoma, neuroblastoma, small cell lung cancer, and certain CNS tumors, but has not been approved for such use.

Both etoposide and teniposide are not sufficiently water soluble (10.mu.
g / mL), and must be formulated with an organic solvent for actual IV delivery. Etoposide is also available in oral dosage form as liquid filled capsules.

The major dose limiting toxicity of epipodophyllotoxin is neutropenia, which is often particularly severe among patients treated with other antineoplastic agents or radiation. . Other hematological toxicities have been commonly reported, including hair loss, GI distress, neurological toxicities, hypersensitivity reactions, and the like. In addition, some patients receiving etoposide suffer from congestive heart failure and / or myocardial infarction, which often result in large saline dilutions (and in some cases Then, the delivery rate).

Podophyllotoxin exhibits complex pharmacokinetic properties. Etoposide breaks down in vivo into at least six independent metabolites. At least two of these metabolites (glucuronide and transhydroxy acid metabolites) are known to have little cytotoxic activity and are presumed to be responsible for the hematological toxicity of the drug. Since etoposide phosphate is rapidly converted to etoposide, the same metabolite, toxicity, and pharmacokinetic properties also result from this prodrug. Teniposide has been studied as extensively as etoposide, but is presumed to have similar properties.

Camptothecin Camptothecin has been known for more than 30 years, but its usefulness as an antitumor agent has only recently been discovered. The gap between discovery and introduction is largely due to the very poor water solubility exhibited by these compounds. For nearly 20 years after the discovery of active compounds, research was directed to the discovery of the more water-soluble camptothecin to facilitate administration.

Researchers have formulated lipophilic drugs with sodium hydroxide in an attempt to increase the water solubility of the drug for administration. Unfortunately, these formulations were essentially inert and were also highly toxic in vivo. In the mid-1980s, camptothecin derivatives were hydrolyzed under basic pH conditions to give E-
It was discovered that the ring lactone opened to form a carboxylated anion. This form of the compound was found to have antitumor activity no more than 1/10 that of the lactone form and was also found to be highly toxic to healthy cells.

Much research has been devoted since this discovery to the development of water soluble camptothecin derivatives that retained their active lactone form. Along these lines, recently approved irinotecan (CPT-11) and topotecan were developed. Irinotecan is a CP known as SN38 (10-hydroxy-7-ethyl CPT).
It is a water-soluble prodrug of a highly active, highly lipophilic derivative of T. Topotecan incorporates the 9-dimethylaminoethyl moiety of the 10-hydroxy derivative of CPT.

As with many other antineoplastic agents, the major toxicities of CPT derivatives are hematological toxicity and GI distress. Irinotecan is associated with severe diarrhea, which is often dose limiting, as well as neutropenia, stomatitis, other GI distress, hair loss and elevated liver function. Topotecan has similar toxicity, but diarrhea is usually not severe.

Camptothecin is generally recognized as a drug with a low therapeutic index and a high incidence of toxic side effects. Co-administration of a protective agent that reduces the toxic side effects of camptothecin provides a safer and more effective chemotherapeutic regimen. This may further allow higher doses of anti-neoplastic drugs to be given, thereby increasing the probability of successful treatment.

(I. Platinum Complex) Since the discovery of these antineoplastic properties was more than 30 years ago, the platinum complex was
It has been developed as a therapeutic agent for many diverse types of solid tumors. Two such complexes (cisplatin and carboplatin) currently have widespread use (both as single agents and in combination therapy for tumors of the testes, ovaries, lungs, bladder, and other organs).

The mechanism of action of platinum complexes has been extensively studied. It has been discovered that platinum complexes covalently bind to DNA, thereby disrupting DNA function and leading directly to cell death. Platinum complexes also bind freely to proteins, and it is expected that protein-bound platinum may also affect DNA.

The onset of toxicity of cisplatin is quite different from that of carboplatin. Cumulatively, dose limiting nephrotoxicity is common with cisplatin treatment, while hematological toxicities similar to electrophilic alkylating agents are primarily carboplatin related. Both cisplatin and carboplatin are also associated with gastrointestinal disorders (mainly nausea and vomiting and neurotoxic effects).

Since platinum complexes are not extensively metabolized in vivo, the platinum species present in the body form hydroxylated and aquated complexes depending on the reactivity of the complex with water. Moreover, platinum complexes are largely excreted from the body via renal excretion, and the acidic conditions present in the kidneys lead to the formation of these generally (to neoplasia) and toxic species. Tends to support. In particular, the chlorine atom of cisplatin is easily replaced by the hydroxyl and aco moieties under acidic conditions, which often explains the severe nephrotoxicity associated with drugs.

Both cisplatin and carboplatin are highly lipophilic compounds,
This allows them to easily cross the cell membrane. The hydroxylated and aquated forms are forms of very low lipid solubility (in particular neutral or slightly alkaline pH). This explains these forms of the generally inactive drug. Moreover, the elimination of cisplatin occurs more rapidly than carboplatin. This explains the different signs of toxicity.

Other toxicities are associated with platinum complexes such as neurotoxicity, ototoxicity (especially in carboplatin), gastrointestinal disorders (mainly nausea and vomiting).

J. Other Drugs Similar toxic effects, namely myelosuppression, gastrointestinal disorders, hypertension, nephrotoxicity, hepatotoxicity,
Hematological toxicity, such as due to mucositis, is associated with most anti-neoplastic agents to some extent.
Because any cancer treatment regime of interest involves prolonging the patient's life and improving the quality of life, the signs of toxicity are carefully considered for alternatives.

(3. Anti-neoplastic Derivative) The anti-neoplastic agent derivative of the present invention comprises an anti-neoplastic agent modified by attaching a reactive group. The reactive group can be attached to the antineoplastic agent via a linking group, or optionally without a linking group. Generally, an antineoplastic derivative comprises a linking group having a reactive group capable of reacting with a functional group in the antineoplastic molecule and blood components. The anti-neoplastic agent derivative may be administered in vivo such that conjugates with blood components occur in vivo, or they may first be conjugated to blood components in vitro and administered in vivo. Results in a conjugated anti-neoplastic agent (as described below).

A wide variety of active carboxyl groups, especially esters, can be used as active groups to form covalent bonds with functional groups on proteins. Here, the hydroxyl moiety is pharmaceutically acceptable at the level required to derivatize the antineoplastic agent. A large number of diverse hydroxyl groups can be used for these linking agents, but the most convenient are N-hydroxysuccinimide (NHS), and N-hydroxy-sulfosuccinimide (sulfo-NHS).

Primary amines are the main targets for NHS esters, as illustrated in Scheme 1A below. An accessible α-amino group is present at the N-terminus of proteins that react with NHS esters. However, the α-amino group on the protein is
Not desirable for NHS coupling, or NHS coupling is not available. Five amino acids have nitrogens in their side chains, but only the ε-amine of lysine reacts significantly with NHS esters. As shown in Scheme 1A below, when the NHS ester conjugation reaction reacts with a primary amine, an amide bond is formed, releasing N-hydroxysuccinimide.

[0098]

[Chemical 10] In a preferred embodiment of the invention, the functional group on the protein is a thiol group and the reactive group is γ-maleimido-butyl aramid (GMBA) or M.
It is a maleimide containing a group such as PA. The maleimido group is most selective for sulfhydryl groups on the protein when the pH of the reaction mixture is maintained between 6.5 and 7.4, as shown in Figure 1B below. At pH 7.0, the rate of reaction of maleimide groups with sulfhydryls is 1000 times faster than reaction with amines. A stable thioester linkage is formed between the maleimide group and the sulfhydryl group, which cannot be cleaved under physiological conditions.

[0099]

[Chemical 11] The manner in which the antineoplastic derivative of the present invention is produced is very widespread, depending on the nature of the various elements, including the molecule. Synthetic procedures are selected to be simple, provide high yields, and allow highly purified products. Generally, the chemically reactive group is created as the last step (eg, esterification to form an active ester using a carboxyl group is the final step in the synthesis). Each anti-neoplastic agent is selected to undergo derivatization with a linker and reaction reagent and is modified according to the following criteria.

First, the location of the pharmacophore region was determined by those skilled in the art as previously published Structure-Activity-Relationships (Structure-Activity-Relation).
determined according to the ship (SAR) study. For example, the structure-activity relationships of taxol and docetaxel have been studied and well explored.
The bioconjugate is attached at the C2 'position in the form of an ester and remains active (F. Dosio et al., Journal of Controlled R).
Release, 1997, 47, 293-304; Shashoua et al., U.S. Patent No. 5,795,909, August 18, 1998). 3'of docetaxel
In position, the use of lipophilic amides maintains activity (F. Guerite-Voeg
olein et al. Med. Chem. , 1991, 34, 992-998; F
． Guerite-Voegolein et al., European Patent Application 253739, 19
July 16, 1987, and C.I. S. Swindell et al., Bioorg. Med
． Chem. Lett. , 1994, 4,1531-1536). In the case of doxorubicin, this modification is most removed from the pharmacophore region since all of the modifications are at the C14 position (B. Patelli et al., DE 2627146, 197).
December 30, 6; F. Arcamore et al., DE 2557537, 8 July 1976; H. King et al., European Patent Application 294294 A2, December 7, 1988). In the case of mitomycin C, the best position for modification is at position N-1a (N. Umemoto et al., J. Appl. Biochem., 1984, 6, 6.
297-307; Tanaka et al., Biol. Pharm. Bull. , 1
996, 19, 774-777 and R.M. L. Barton, European Patent Application 36
8668, November 10, 1988). In the case of vincristine, the best position for modification is at the C23 position (RL Barton, European Patent Application 368668, 1,
(November 10, 988).

The antineoplastic agent is then modified at a site well away from the pharmacophore region to prevent strong binding interference between the modified drug and the biological receptor, resulting in antineoplastic formation. The agent retains its therapeutic activity (ie, the therapeutic activity is reduced only 10-fold).

Finally, keeping the site of chemical modification constant, the antineoplastic biological activity is optimized by modifying the length and nature of the linker. Some examples of specific antineoplastic derivatives are given below.

[0103]   (A. Doxorubicin derivative)   One example of an antineoplastic derivative is the doxorubicin derivative of formula I:

[0104]

[Chemical 12] Where: X is an ether, thioether, secondary or tertiary amine, secondary amide or tertiary amide, ester, thioester, imine, hydrazone, semicarbazone, acetal, hemiacetal, ketal, hemiketal, aminal, hemiaminal, sulfonate. , Sulphates, sulphonamides, sulphonamidates, phosphates, phosphoramides, phosphonates, or phosphonamidates, preferably sensitive functional groups including but not limited to primary amides; R is a free radical as defined above. An optional linking group, eg, R, is absent (where X is directly attached to D), any alkyl chain C _1-10 , any fluoroalkyl C _1-10 or any combination of fluoro-substitutions. Alkyl chain C _1-10 ,-(Z
_{-CH 2 CH 2 -Z) n -} , - (Z-CF 2 CH 2 -Z) n -, - (Z-CH 2 CF 2 -Z
) _N - (where, n = 1-4 and Z is any ether or any thioether containing alkyl chain or fluoroalkyl chains, such as O or is either _{S), -Y-C 6 H} 4 _{-, - Y-C 6 H} 4 -Y- ( where, Y is, CH _2, O
, S, NH, NR [R = H, alkyl, acyl] in any combination) ortho, meta, or para disubstituted benzene, furan, thiophene, pyran, oxazole, or thiazole. _{May be,} but is not limited to, a substituted heterocycle, preferably an alkyl chain C _1-6 ; and D is a reactive group as defined above, preferably a maleimido group. The dashed C—X bond, as an alternative, indicates the presence of a single or double bond.

[0105]   Another example of an antineoplastic derivative is the doxorubicin derivative of formula II:

[0106]

[Chemical 13] Where: X is an ether, thioether, secondary or tertiary amine, secondary amide or tertiary amide, ester, thioester, imine, hydrazone, semicarbazone, acetal, hemiacetal, ketal, hemiketal, aminal, hemiaminal, sulfonate. , Sulphates, sulphonamides, sulphonamidates, phosphates, phosphoramides, phosphonates, or phosphonamidates, preferably sensitive functional groups including but not limited to primary amides; R is a free radical as defined above. An optional linking group, eg, R, is absent (where X is directly attached to D), any alkyl chain C _1-10 , any fluoroalkyl C _1-10 or any combination of fluoro-substitutions. Alkyl chain C _1-10 ,-(Z
_{-CH 2 CH 2 -Z) n -} , - (Z-CF 2 CH 2 -Z) n -, - (Z-CH 2 CF 2 -Z
) _N - (where, n = 1-4 and Z is any ether or any thioether containing alkyl chain or fluoroalkyl chains, such as O or is either _{S), -Y-C 6 H} 4 _{-, - Y-C 6 H} 4 -Y- ( where, Y is, CH _2, O
, S, NH, NR [R = H, alkyl, acyl] in any combination) ortho, meta, or para disubstituted benzene, furan, thiophene, pyran, oxazole, or thiazole. _{May be,} but is not limited to, a substituted heterocycle, preferably an alkyl chain C _1-6 ; and D is a reactive group as defined above, preferably a maleimido group. The dashed C—X bond, as an alternative, indicates the presence of a single or double bond.

[0107]   Another example of an antineoplastic derivative is the doxorubicin derivative of formula III:

[0108]

[Chemical 14] Where: V is O, N, S, or C; W is O, N, S, or C; n is 0 or 1; and Q is absent or oxygen, sulfur , Secondary or tertiary amines, secondary or tertiary amides, phosphorus oxides, or substituted or unsubstituted carbon atoms (preferably V is oxygen, Q is absent, and W is primary). R is an optional linking group as defined above, eg, R is absent (where X is directly attached to D), any alkyl chain C _1-10 , Any fluoroalkyl C _1-10 or any combination of fluoro-substituted alkyl chains C _1-10 ,-(Z
_{-CH 2 CH 2 -Z) n -} , - (Z-CF 2 CH 2 -Z) n -, - (Z-CH 2 CF 2 -Z
) _N - (where, n = 1-4 and Z is any ether or any thioether containing alkyl chain or fluoroalkyl chains, such as O or is either _{S), -Y-C 6 H} 4 _{-, - Y-C 6 H} 4 -Y- ( where, Y is, CH _2, O
, S, NH, NR [R = H, alkyl, acyl] in any combination), ortho, meta, or para disubstituted benzene, furan, thiophene, pyran, oxazole, or thiazole. substituted heterocycle, such as, preferably is -CH ₂ - obtained (but not limited to); and D is a reactive group, as defined above, preferably a maleimide group. The dashed C-X and C-V bonds, as an alternative, indicate the presence of a single or double bond.

B. Taxol Derivatives For the purposes of the present invention, derivatized taxol will attach a reactive group (ie, a free alcohol) at the ring opening position, even in the presence of evidence of reduced activity. Was synthesized by The bioconjugate is attached at the C2 'position in the form of an ester and activity is maintained (F. Dosio et al., Journal of Contr.
rolled Release, 1997, 47, 293-304). We believe that this bond is very weak and can be cleaved in vivo (Mag.
ri et al. Med. Chem. , 1989, 32, 788-792).

[0110]   One example of a taxol derivative according to the invention is a compound of formula IV:

[0111]

[Chemical 15] Where: X is an ether, thioether, secondary or tertiary amine, secondary amide or tertiary amide, ester, thioester, imine, hydrazone, semicarbazone, acetal, hemiacetal, ketal, hemiketal, aminal, hemiaminal, sulfonate. , Sulphate, sulphonamide, sulphonamidate, phosphate, phosphoramide, phosphonate or phosphonamidate, preferably a sensitive functional group including but not limited to -NH- or -CO-; R is defined above. An optional linking group such as R is absent (where X is directly bonded to D), any alkyl chain C _1-10 , any fluoroalkyl C _1-10 or any A fluoro-substituted alkyl chain C _1-10 ,-(Z
_{-CH 2 CH 2 -Z) n -} , - (Z-CF 2 CH 2 -Z) n -, - (Z-CH 2 CF 2 -Z
) _N - (where, n = 1-4 and Z is any ether or any thioether containing alkyl chain or fluoroalkyl chains, such as O or is either _{S), -Y-C 6 H} 4 _{-, - Y-C 6 H} 4 -Y- ( where, Y is, CH _2, O
, S, NH, NR [R = H, alkyl, acyl] in any combination) ortho, meta, or para disubstituted benzene, furan, thiophene, pyran, oxazole, or thiazole. A substituted heterocycle such as, but not limited to, an alkyl chain C _1-6 or — (O—CH ₂ —CH ₂ —O) _1-2 —CH ₂ —CH ₂ —; and D Is a reactive group as defined above, preferably a maleimide group.

[0112]   Another example of a taxol derivative according to the invention is a compound of formula V:

[0113]

[Chemical 16] Where: X is an ether, thioether, secondary or tertiary amine, secondary amide or tertiary amide, ester, thioester, imine, hydrazone, semicarbazone, acetal, hemiacetal, ketal, hemiketal, aminal, hemiaminal, sulfonate. , Sulfate, sulfonamide, sulfonamidate, phosphate, phosphoramide, phosphonate, or phosphonamidate, preferably a sensitive functional group including but not limited to -CO- (amide); R is defined above. An optional linking group such as R is absent (where X is directly attached to D), any alkyl chain C _1-10 , any fluoroalkyl C _1-10 or any Combined fluoro-substituted alkyl chains C _1-10 ,-(Z
_{-CH 2 CH 2 -Z) n -} , - (Z-CF 2 CH 2 -Z) n -, - (Z-CH 2 CF 2 -Z
) _N - (where, n = 1-4 and Z is any ether or any thioether containing alkyl chain or fluoroalkyl chains, such as O or is either _{S), -Y-C 6 H} 4 _{-, - Y-C 6 H} 4 -Y- ( where, Y is, CH _2, O
, S, NH, NR [R = H, alkyl, acyl] in any combination) ortho, meta, or para disubstituted benzene, furan, thiophene, pyran, oxazole, or thiazole. _{May be,} but is not limited to, a substituted heterocycle, preferably an alkyl chain C _1-6 ; and D is a reactive group as defined above, preferably a maleimido group.

[0114]   (C. Methotrexate derivative)   One example of a methotrexate derivative according to the invention is a compound of formula VI:

[0115]

[Chemical 17] Where: X is an ether, thioether, secondary or tertiary amine, secondary amide or tertiary amide, ester, thioester, imine, hydrazone, semicarbazone, acetal, hemiacetal, ketal, hemiketal, aminal, hemiaminal, sulfonate. , Sulfate, sulfonamide, sulfonamidate, phosphate, phosphoramide, phosphonate, or phosphonamidate, preferably a sensitive functional group including but not limited to -NH- (primary amide); R is defined above. An optional linking group such as R is absent (where X is directly bonded to D), any alkyl chain C _1-10 , any fluoroalkyl C _1-10 or any A fluoro-substituted alkyl chain C _1-10 ,-(Z
_{-CH 2 CH 2 -Z) n -} , - (Z-CF 2 CH 2 -Z) n -, - (Z-CH 2 CF 2 -Z
) _N - (where, n = 1-4 and Z is any ether or any thioether containing alkyl chain or fluoroalkyl chains, such as O or is either _{S), -Y-C 6 H} 4 _{-, - Y-C 6 H} 4 -Y- ( where, Y is, CH _2, O
, S, NH, NR [R = H, alkyl, acyl] in any combination) ortho, meta, or para disubstituted benzene, furan, thiophene, pyran, oxazole, or thiazole. _{May be,} but is not limited to, a substituted heterocycle, preferably an alkyl chain C _1-6 ; and D is a reactive group as defined above, preferably a maleimido group.

One example of a methotrexate derivative according to the present invention is a compound of formula VII:

[0117]

[Chemical 18] Where: X is a secondary or tertiary amine, secondary amide or tertiary amide, ester, thioester, imine, hydrazone, semicarbazone, acetal, hemiacetal, ketal, hemiketal, aminal, hemiaminal, sulfonate, sulfate, sulfone. Amide, sulfonamidate, phosphate, phosphoramide, phosphonate, or phosphonamidate, preferably a sensitive functional group including but not limited to -NH-; R is an optional linkage as defined above. A group, eg R, is absent (where X is directly attached to D), any alkyl chain C _1-10 , any fluoroalkyl C _1-10 or any combination of fluoro-substituted alkyl chains C _1-10 ,-(Z
_{-CH 2 CH 2 -Z) n -} , - (Z-CF 2 CH 2 -Z) n -, - (Z-CH 2 CF 2 -Z
) _N - (where, n = 1-4 and Z is any ether or any thioether containing alkyl chain or fluoroalkyl chains, such as O or is either _{S), -Y-C 6 H} 4 _{-, - Y-C 6 H} 4 -Y- ( where, Y is, CH _2, O
, S, NH, NR [R = H, alkyl, acyl] in any combination) ortho, meta, or para disubstituted benzene, furan, thiophene, pyran, oxazole, or thiazole. _{May be,} but is not limited to, a substituted heterocycle, preferably an alkyl chain C _1-6 ; and D is a reactive group as defined above, preferably a maleimido group.

[0118]   (D. Oxyurea derivative)   One example of an oxyurea according to the present invention is a compound of formula VIII:

[0119]

[Chemical 19] Where: X is an ether, thioether, secondary or tertiary amine, secondary amide or tertiary amide, ester, thioester, imine, hydrazone, semicarbazone, acetal, hemiacetal, ketal, hemiketal, aminal, hemiaminal, sulfonate. R'is an alkyl C; _1-6 , fluoroalkyl C _1-6 , substituted or unsubstituted aryl or heteroaryl; R is an optional linking group as defined above, eg, R is absent (wherein X is directly bonded to D), any Kill chain C _1-10, optionally fluoroalkyl C _1-10 or any combination fluoro-substituted alkyl chain C _1-10, - (Z
_{-CH 2 CH 2 -Z) n -} , - (Z-CF 2 CH 2 -Z) n -, - (Z-CH 2 CF 2 -Z
) _N - (where, n = 1-4 and Z is any ether or any thioether containing alkyl chain or fluoroalkyl chains, such as O or is either _{S), -Y-C 6 H} 4 _{-, - Y-C 6 H} 4 -Y- ( where, Y is, CH _2, O
, S, NH, NR [R = H, alkyl, acyl] in any combination) ortho, meta, or para disubstituted benzene, furan, thiophene, pyran, oxazole, or thiazole. _{May be,} but is not limited to, a substituted heterocycle, preferably an alkyl chain C _1-6 ; and D is a reactive group as defined above, preferably a maleimido group.

[0120]   (E. Tamoxifen derivative)   One example of tamoxifen according to the invention is a compound of formula IX:

[0121]

[Chemical 20] Where: X is an ether, thioether, secondary or tertiary amine, secondary amide or tertiary amide, ester, thioester, imine, hydrazone, semicarbazone, acetal, hemiacetal, ketal, hemiketal, aminal, hemiaminal, sulfonate. R'is a sensitive functional group including, but not limited to, Hydrogen, alkyl, aryl, or acyl, preferably methyl; R is an optional linking group as defined above, eg, R is absent (where X is directly bonded to D). ), Any alkyl chain C _1-10 , any full Oroalkyl C _1-10 or any combination of fluoro-substituted alkyl chains C _1-10 ,-(Z
_{-CH 2 CH 2 -Z) n -} , - (Z-CF 2 CH 2 -Z) n -, - (Z-CH 2 CF 2 -Z
) _N - (where, n = 1-4 and Z is any ether or any thioether containing alkyl chain or fluoroalkyl chains, such as O or is either _{S), -Y-C 6 H} 4 _{-, - Y-C 6 H} 4 -Y- ( where, Y is, CH _2, O
, S, NH, NR [R = H, alkyl, acyl] in any combination) ortho, meta, or para disubstituted benzene, furan, thiophene, pyran, oxazole, or thiazole. _{May be,} but is not limited to, a substituted heterocycle, preferably an alkyl chain C _1-6 ; and D is a reactive group as defined above, preferably a maleimido group.

[0122]   (F. Mitomycin C derivative)   One example of mitomycin C according to the present invention is a compound of formula X:

[0123]

[Chemical 21] Where: X is an ether, thioether, secondary or tertiary amine, secondary amide or tertiary amide, ester, thioester, imine, hydrazone, semicarbazone, acetal, hemiacetal, ketal, hemiketal, aminal, hemiaminal, sulfonate. , Sulfate, sulfonamide, sulfonamidate, phosphate, phosphoramide, phosphonate, or phosphonamidate, preferably a sensitive functional group including but not limited to -CO- (amide); R is defined above. An optional linking group such as R is absent (where X is directly attached to D), any alkyl chain C _1-10 , any fluoroalkyl C _1-10 or any Combined fluoro-substituted alkyl chains C _1-10 ,-(Z
_{-CH 2 CH 2 -Z) n -} , - (Z-CF 2 CH 2 -Z) n -, - (Z-CH 2 CF 2 -Z
) _N - (where, n = 1-4 and Z is any ether or any thioether containing alkyl chain or fluoroalkyl chains, such as O or is either _{S), -Y-C 6 H} 4 _{-, - Y-C 6 H} 4 -Y- ( where, Y is, CH _2, O
, S, NH, NR [R = H, alkyl, acyl] in any combination) ortho, meta, or para disubstituted benzene, furan, thiophene, pyran, oxazole, or thiazole. _{May be,} but is not limited to, a substituted heterocycle, preferably an alkyl chain C _1-6 ; and D is a reactive group as defined above, preferably a maleimido group.

(G.5-Fluorouracil) The compound of the present invention, which is a product of a derivative of 5-fluorouracil, is a mixture of four stereoisomers. These four isomers have the formula XI A, XI B, XI
C and XID compounds.

[0125]

[Chemical formula 22] Where: X is an ether, thioether, secondary or tertiary amine, secondary amide or tertiary amide, ester, thioester, imine, hydrazone, semicarbazone, acetal, hemiacetal, ketal, hemiketal, aminal, hemiaminal, sulfonate. R is a sensitive functional group including, but not limited to, An optional linking group as defined, eg, R, is absent (where X is directly attached to D), any alkyl chain C _1-10 , any fluoroalkyl C _1-10 or Any combination of fluoro-substituted alkyl chains C _1-10 ,-(Z
_{-CH 2 CH 2 -Z) n -} , - (Z-CF 2 CH 2 -Z) n -, - (Z-CH 2 CF 2 -Z
) _N - (where, n = 1-4 and Z is any ether or any thioether containing alkyl chain or fluoroalkyl chains, such as O or is either _{S), -Y-C 6 H} 4 _{-, - Y-C 6 H} 4 -Y- ( where, Y is, CH _2, O
, S, NH, NR [R = H, alkyl, acyl] in any combination) ortho, meta, or para disubstituted benzene, furan, thiophene, pyran, oxazole, or thiazole. _{May be,} but is not limited to, a substituted heterocycle, preferably an alkyl chain C _1-6 ; and D is a reactive group as defined above, preferably a maleimido group.

[0126]   (H. vincristine)   One example of a vincristine derivative according to the present invention is a compound of formula XII:

[0127]

[Chemical formula 23] Where: X is a secondary or tertiary amine, secondary amide or tertiary amide, ester, thioester, imine, hydrazone, semicarbazone, acetal, hemiacetal, ketal, hemiketal, aminal, hemiaminal, sulfonate, sulfate, sulfone R is a sensitive functional group including, but not limited to, amide, sulfonamidate, phosphate, phosphoramide, phosphonate, or phosphonamidate; R is an optional linking group as defined above, eg, R is , Absent (where X is directly bonded to D), any alkyl chain C _1-10 , any fluoroalkyl C _1-10 or any combination of fluoro-substituted alkyl chains C _1-10 ,-( Z
_{-CH 2 CH 2 -Z) n -} , - (Z-CF 2 CH 2 -Z) n -, - (Z-CH 2 CF 2 -Z
) _N - (where, n = 1-4 and Z is any ether or any thioether containing alkyl chain or fluoroalkyl chains, such as O or is either _{S), -Y-C 6 H} 4 _{-, - Y-C 6 H} 4 -Y- ( where, Y is, CH _2, O
, S, NH, NR [R = H, alkyl, acyl] in any combination) ortho, meta, or para disubstituted benzene, furan, thiophene, pyran, oxazole, or thiazole. (But not limited to) substituted heterocycles; and D is a reactive group as defined above, preferably a maleimide group.

4. Conjugated Anti-neoplastic Agent After the anti-neoplastic agent has been derivatized according to the present invention, it is used to form a conjugated anti-neoplastic agent either in vivo or in vitro. . Such a conjugate is
Via a covalent bond formed between the reactive group of the antineoplastic derivative (attached to the antineoplastic derivative with or without a linking group) and the functional group of the blood component Includes antineoplastic derivatives that are conjugated to blood components. The anti-neoplastic agent derivatives of the present invention can be used in conjugates with specific or non-specific labeling of blood components as described in more detail below.

A. Specific Labeling Preferably, the antitumor drug derivative of the present invention is designed to specifically react with a thiol group on a mobile blood protein. Such a reaction is preferably
Maleimide (eg, prepared from GMBS, MPA or other maleimide)
Is established by covalent attachment of a derivatized anti-tumor drug to thiol groups on mobile blood proteins (eg, serum albumin or IgG).

Under certain circumstances, specific labeling with maleimide is NHS or sulfo-NHS
Offers several advantages over non-specific labeling of mobile proteins with groups such as Thiol groups are less abundant in vivo than amino groups. Therefore, the maleimide derivative of the present invention covalently binds fewer proteins. For example, albumin, the most abundant blood protein, has only a single thiol group. Therefore, the anti-tumor drug-maleimide-albumin conjugate tends to contain a molar ratio of about 1: 1 anti-tumor drug: albumin. IgG in addition to albumin
Molecules (class II) also have free thiols. Since IgG molecules and serum albumin make up the majority of soluble proteins in blood, they also make up the majority of free thiol groups in blood available for covalent attachment to maleimide-antitumor drugs. .

Furthermore, even among blood proteins containing free thiol groups, specific labeling with maleimide leads to preferential formation of antitumor drug-maleimide-albumin conjugates due to the unique properties of albumin itself. . The single free thiol group of albumin is highly conserved among species and is located at amino acid residue 34 (Cys ³⁴ ).
It has recently been demonstrated that albumin Cys ³⁴ has increased reactivity compared to free thiols on other free thiol-containing proteins. This is partly due to the very low pK value of Cys ³⁴ for albumin (5.5). This is much lower than the typical pK value for common cysteine residues, which is typically around 8. Due to this low pK, under normal physiological conditions, Cys ³⁴ of albumin exists mainly in the ionized form, which dramatically increases its reactivity. In addition to the low pK value of Cys ^34, another factor which enhances the reactivity of Cys ³⁴ is,
Its position, which is close to the surface of one loop of region V of albumin (c
device). This position makes Cys ³⁴ highly available to ligands of all species, and is a key factor in Cys ³⁴ 's biological role as a trap for free radicals and a scavenger for free thiols. These properties make Cys ³⁴ highly reactive with the anti-tumor drug-maleimide, and the acceleration of this reaction rate is 1000 times that of the anti-tumor drug-maleimide with other free thiol-containing proteins. It can be about twice as large.

Another advantage of the antitumor drug-maleimide-albumin conjugate is its reproducibility, which is specific for Cys ³⁴ and is associated with a 1: 1 loading of the antitumor drug on albumin. Other techniques, such as the free amine glutaraldehyde, DCC, EDC and other chemical activations, lack this selectivity. For example, albumin is 52
Lysine residues, 25-30 of which are located on the surface of albumin,
And it is easy to use for joining. Activating these lysine residues, or modifying the antitumor drug to bind through these lysine residues, results in a heterogeneous population of conjugates. Even when a 1: 1 molar ratio of anti-tumor drug: albumin was used, the yield consisted of multiple conjugate products, some of which
There are 0, 1, 2 or more antitumor agents per albumin, each of which contains an antitumor agent randomly linked at any one of the 25-30 available lysine sites. Have. Given the many possible combinations, it is difficult to characterize the exact composition and properties of each batch, and batch-to-batch reproducibility is nearly impossible, making such conjugates desirable as therapeutic agents. I don't have it. Furthermore, conjugates via lysine residues of albumin appear to have the advantage of delivering many therapeutic agents per at least one albumin molecule, although studies have favored a 1: 1 ratio of therapeutic agent: albumin. Is shown. Steh
le et al., “The Loading Rate Determines Tum.
or Targeting Properties of Methotrex
ate-Albumin Conjugates in Rats ", Anti
In a paper by Cancer Drugs, Vol. 8, pp. 677-685 (1997), which is incorporated herein in its entirety, the authors describe a 1: 1 conjugation via glutaraldehyde. We report that a ratio of anti-cancer methotrexate: albumin produced the most desired results. These conjugates were taken up by tumor cells, whereas those with 5: 1-20: 1 methotrexate molecules had altered HPLC profiles and were rapidly taken up by the liver in vivo. It is speculated that at these high ratios, conformational changes to albumin diminish its effectiveness as a therapeutic agent carrier.

Controlled administration of maleimide-antitumor agents in vivo can control the specific labeling of albumin and IgG in vivo. In a typical administration,
80-90% of the administered maleimide-antitumor drug labeled albumin, 5
Less than% labels IgG. For example, trace labeling of free thiols such as glutathione also occurs. Such specific labels are preferred for in vivo applications. This is because it allows an accurate calculation of the estimated half-life of the administered drug.

In addition to providing controlled specific in vivo labeling, maleimide-antitumor agents can provide ex vivo specific labeling of serum albumin and IgG. Such ex vivo labeling involves adding a maleimide-antitumor drug to blood, serum or saline solution containing serum albumin and / or IgG. Once modified ex vivo with a maleimide-antitumor drug, these blood, serum or saline solutions can be readministered into the blood for in vivo treatment.

Compared to NHS-antitumor agents, maleimide-antitumor agents are generally stable in the presence of aqueous solutions and in the presence of free amines. Maleimide-antitumor agents are
Protecting groups to prevent the maleimide-antitumor drug from reacting with itself are generally not required as they only react with free thiols. In addition, its increased stability of anti-tumor agents allows the use of additional purification steps such as HPLC to prepare highly purified products suitable for in vivo use. Finally, this increased chemical stability provides the product with a longer shelf life.

B. Non-Specific Labeling The anti-tumor agents of the present invention can also be derivatized for non-specific labeling of blood components. Coupling to amino groups is commonly used for non-specific labeling, especially with the formation of amide bonds. To form such a bond, a wide variety of active carboxyl groups (particularly esters) can be used as chemically reactive groups attached to antitumor agents, where the hydroxyl moiety is Physiologically acceptable at the required level. Many different hydroxyl groups can be used in these linking agents, but the most convenient are N-hydroxysuccinimide (NHS) and N-hydroxysulfosuccinimide (sulfo-NHS).

Other linking agents that may be utilized are described in US Pat. No. 5,612,034, hereby incorporated by reference.

[0138] The various sites to which chemically reactive groups of non-specific anti-tumor agents may react in vivo include cells (especially erythrocytes and platelets) and proteins (eg immunoglobulins (IgG). And IgM), serum albumin, ferritin, steroid binding protein, transferrin, thyroxine binding protein, α-2-macroglobulin) and the like. Receptors to which these anti-tumor drug derivatives react and are not long-lived are generally cleared from the human host within about 3 days. The proteins shown above (including those of these cells) are especially relevant for blood concentration-based half-life,
It can persist in the bloodstream for at least 3 days and for 5 days or more (usually not more than 60 days, more usually not more than 30 days).

For the most part, the reaction is with mobile components in the blood, especially blood proteins and cells, more particularly blood proteins and red blood cells. By “mobility” is meant that the component does not have a constant position for any extended period of time (generally no more than 5 minutes, more usually no more than 1 minute). , Some of these blood components may be relatively stationary for long periods of time. First, there is a relatively heterogeneous population of labeled proteins and cells. However, for the most part, their population within days after administration varies substantially from their original population, depending on the half-life of the labeled protein in the bloodstream. Thus, typically within about 3 days or more, IgG becomes the predominant labeled protein in the bloodstream.

Typically, by day 5 post-administration, IgG, serum albumin, and red blood cells will be at least 60 mol%, and usually at least about 75 mol% of the conjugated components in blood. , IgG, IgM (to a substantially lesser extent) and serum albumin are at least about 50 mol%, usually at least about 75 mol%, more usually at least about 50% of the acellular conjugation component. It is 80 mol%.

These desired conjugates of non-specific anti-tumor agents to blood components can be prepared in vivo by direct administration of these anti-tumor agents to patients, where the patient is a human or other mammal. Can be. Administration can be in bolus form or can be slowly introduced over time by infusion, such as using metered flow.

If desired, the conjugate of interest may also combine blood with an anti-tumor drug derivative of the invention to allow covalent attachment of the anti-tumor drug derivative to a reactive functional group on a blood component,
They can then be prepared ex vivo by returning or administering these conjugated blood to the host. Furthermore, the above also purifies the individual blood components or a limited number of components (eg erythrocytes, immunoglobulins, serum albumin, etc.) first, and the component (s) is chemically reactive. It can be achieved by combining with these anti-tumor agents ex vivo. This labeled blood or blood component can then be returned to the host to provide the therapeutically effective conjugate of interest in vivo. Blood can also be treated to prevent aggregation during ex vivo handling.

5. Therapeutic Uses of Antitumor Drug Derivatives The compounds of the present invention, including but not limited to the compounds detailed in the Examples, have anticancer activity. As derivatives of anti-tumor agents with anti-cancer activity, the compounds of the invention are useful, for example, in the treatment of various diseases: breast, colon, rectum, lung,
Oropharynx, hypopharynx, esophagus, stomach, pancreas, liver, gallbladder, bile duct, small intestine, urinary tract (kidney,
Bladder and urothelium), female reproductive tract (including cervix, uterus, endometrium, ovary, choriocarcinoma and vestibular trophoblastic disease), male reproductive tract (prostate, sperm) Sac, testicular and germ cell tumors, endocrine glands (including thyroid gland, adrenal glands and pituitary gland), skin (hmangiomas), melanomas, sarcomas (derived from soft tissue bones) and Kaposi's sarcoma, Wilms tumor , Including rhabdomyosarcoma) solid tumors and carcinomas; head and neck;
Tumors of the brain, nerves, eyes and meninges (including astrocytomas, gliomas, glioblastomas, retinoblastomas, neuromas, neuroblastomas); marrow tumors and hematopoietic tumors, Solid tumors (including chloromas, plasmacytomas, mycosis fungoides plaques and tumors, and cutaneous T-cell lymphoma / leukemia) resulting from hematopoietic malignancies (eg, leukemia); acute lymphocytic leukemia, acute granulocytic leukemia And chronic granulocytic leukemia; lymphoma (including Hodgkin lymphoma and non-Hodgkin lymphoma); autoimmune disease (rheumatism,
Prevention of immune arthritis and osteoarthritis; ocular diseases (diabetic retinopathy,
Retinopathy of prematurity, corneal transplant rejection, post lens fibrosis, neovascular glaucoma, rubeosis, retinal neovascularization (due to macular degeneration and hypoxia); abnormal neovascularization of the eye; skin disease (Including psoriasis); vascular disease (including hemangiomas and capillary proliferation within atherosclerotic plaques); myocardial angiogenesis; plaque neovascularization; hemophilia arthritis; angiofibroma; wound granulation; Having angiogenesis as a pathological result including diseases characterized by excessive or abnormal stimulation (enteric adhesion, Crohn's disease, atherosclerosis, scleroderma and hyperplastic scars, and ulcers (Helicobacter pilori)) (Including diseases); rheumatoid arthritis,
Osteogenic sarcoma, osteoarthritis, osteopenia (eg, osteoporosis), periodontitis, gingivitis, corneal epidermal or gastric ulcer, tumor metastasis and invasion and proliferation, retinopathy, wound healing (eye inflammation, Diseases of soft and bone tissues, gingivitis / periodontal disease, vascular disease (restenosis) inflammation of the aneurysm, autoimmune diseases, and rare cancers (eg choriocarcinoma).

The compounds of the invention may also be used as described above, when used alone or in combination with radiotherapy and / or other chemotherapeutic treatments that are routinely administered to patients to treat angiogenic disorders. It is useful for preventing metastases from tumors of For example,
When used in the treatment of solid tumors, the compounds of the present invention may be used in combination with α-inferon, COMP (cyclophosphamide, vincristine, methotrexate and prednisone), etoposide, mBACOD (methotrexate, bleomycin, doxorubicin, cyclophosphamide). , Vincristine and dexamethasone), PROMACE / MOPP (prednisone, methotrexate, doxorubicin, cyclophosphamide, taxol, etoposide /
Mechloetamine, vincristine, prednisone and procarbazine), vincristine, vinblastine, angioinhibin, TNP-470, pentosan polysulfate, platelet factor-4, angiostatin, LM-609, SU-101, CM.
-101, techgalan, thalidomide, SP-PG and the like may be administered with chemotherapeutic agents. Other chemotherapeutic agents include alkylating agents such as nitrogen mustards (including mechlorethamine, melphan chlorambucil, cyclophosphamide and ifosfamide); nitrosoureas (including carmustine, lomustine, semustine and streptozocin). ); Alkyl sulfonic acid (including busulfan)); Triazine (including dacarbazine); Ethyleneimine (including thiotepa and hexamethylmelamine); Folic acid analog (including methotrexate); Pyrimidine analog (5-FU, cytosine) Arabinoside); purine analogues (including 6-mercaptopurine and 6-thioguanine); antitumor antibiotics (including actinomycin D); anthracyclines (doxorubicin, bleo) Isins, including mitomycin C and methramycin); hormones and hormone antagonists (including tamoxifen and corticosteroids) and bioscellaneous drugs (including cisplatin and brequinal); fragments of plasminogen (kringle-5) and other integrins. Examples include fragments derived from the binding substrate. For example, tumors are routinely treated by surgery, radiation therapy or chemotherapy with the administration of anti-tumor drug derivatives to spread the stasis of micrometastases and inhibit the growth of any remaining primary tumor. obtain.

6. Administration of Antitumor Drug Derivatives In accordance with the present invention, antitumor drug derivatives can be prepared by administration by any route or can be administered by any route. The anti-tumor drug derivative is a physiologically acceptable medium (eg, deionized water, phosphate buffered saline (PBS)
), Saline, aqueous ethanol or other alcohol, plasma, protein solution, mannitol, aqueous glucose, alcohol, vegetable oil, etc.). Other additives that may be included include buffers (vehicles generally buffered at a pH in the range of about 5-10, buffers generally in the concentration range of about 50-250 mM), salts (concentrations of the salts). Are generally in the range of about 5-500 mM), physiologically acceptable stabilizers and the like. These compositions can be lyophilized for convenient storage and shipping.

Anti-tumor drug derivatives of interest are for the most part parenteral (eg intravascular (I
V), intraarterial (IA), intramuscular (IM), subcutaneous (SC), etc.). Administration can be in the appropriate state by infusion. In some cases, where the reaction of the functional groups is relatively slow, the administration may be oral, nasal, vaginal, rectal, transdermal or aerosol, the nature of the conjugate of which is to the vasculature. Enable transportation. Usually a single injection is used, but more than one injection can be used if desired. The anti-tumor drug derivative can be administered by any convenient means, including syringes, trocars, catheters and the like. The particular form of administration will vary depending on the dose administered (whether a single bolus or continuous administration, etc.). Preferably the administration is intravascular and the site of introduction is not critical to the invention and is preferably the site where fast blood flow is present (eg intravenous, peripheral or central vein). Other routes may find use in which administration is combined with sustained release techniques or protective matrices. It is contemplated that the anti-tumor drug, analog or derivative is effectively dispersed in the blood so that it may react with blood components. Conjugate concentrations vary widely and generally range from about 1 pg / ml to 50 mg / ml. The total amount administered intravascularly will generally be in the range of about 0.1 mg / ml to about 10 mg / ml, more usually in the range of about 1 mg / ml to about 5 mg / ml.

Binding to long-lived components in the blood, such as immunoglobulins, serum albumin, red blood cells and platelets, results in many advantages. The activity of anti-tumor drug compounds ranges from days to weeks. Only one dose need be given during this period. Higher specificity can be achieved. This is because the active compound is predominantly bound to large molecules and is unlikely to be taken up intracellularly and interfere with other physiological processes.

The blood of a mammalian host can be monitored one or more times for the presence of anti-tumor drug compounds. A portion or sample of the host's blood may be removed to determine if the anti-tumor drug is bound to this long-lived blood component in an amount sufficient to be therapeutically active, and then the blood The level of the anti-tumor drug compound in can be determined. If desired, it can also be determined to which blood component the antitumor drug derivative molecule was bound. This is especially important when using non-specific anti-tumor drug derivatives. Calculating serum albumin and IgG half-lives for specific maleimide antitumor drug derivatives is much easier.

The invention therefore relates to conjugates of antitumor drug derivatives with such blood components, in particular blood proteins (eg albumin), as well as methods for their administration to human or other mammalian patients.

7. Concentration and Pharmacokinetic Determination of Antitumor Drug Derivatives Another aspect of the invention is the use of antibodies specific for antitumor drug derivatives and conjugates in biological samples (eg, Method for determining the concentration of derivatives and conjugates of antitumor agents in blood), and such antitumor agents, analogues and / or
Alternatively, it relates to the use of such antibodies as a treatment for the toxicity potentially associated with their derivatives or conjugates. This is advantageous because the increased stability and survival of an anti-tumor drug derivative in vivo in a patient creates new problems during treatment, including an increased likelihood of toxicity. . The use of anti-tumor drug antibodies (either monoclonal or polyclonal) with specificity for a particular anti-tumor drug derivative may help mediate any such problems. Antibodies may be generated or derived from a host immunized with a particular anti-tumor drug derivative or immunogenic fragment of this drug or a synthetic immunogen corresponding to an antigenic determinant of this drug. Preferred antibodies have high specificity and affinity for the native, derivative and conjugate forms of antitumor drug derivatives. Such antibodies may also be labeled with enzymes, fluorescent dyes or radioactive labels.

Antibodies specific for derivatized anti-tumor agents can be produced by using purified anti-tumor agents for the induction of derivatized anti-tumor agent-specific antibodies. The induction of antibodies contemplates not only stimulation of the immune response by injection into animals, but also similar steps in the production of synthetic antibodies or other specifically binding molecules (eg, screening of recombinant immunoglobulin libraries). It Both monoclonal and polyclonal antibodies can be produced by procedures well known in the art.

Anti-therapeutic agent antibodies may be used to treat toxicity induced by administration of anti-tumor agent derivatives and may be used ex vivo or in vivo. Ex vivo methods include immunodialysis treatment for toxicity using anti-therapeutic antibody immobilized on a solid support. In vivo methods include administration of the anti-therapeutic agent antibody in an amount effective to induce clearance of the antibody-drug complex.

The antibody can be used to remove antineoplastic drug derivatives ex vivo from the blood by contacting the antibody with the blood of a patient under sterile conditions. For example, the antibody can be immobilized or immobilized on a column matrix, and the patient's blood can be removed from the patient and passed over the matrix. The anti-tumor agent derivative or conjugate is conjugated to the antibody, and blood containing low concentrations of the anti-tumor agent derivative or conjugate can then be returned to the patient's circulatory system. The amount of anti-tumor drug compound removed can be controlled by adjusting the pressure and flow rate. Preferential removal of this antineoplastic derivative and conjugate from the plasma component of the patient's blood has been described by
It can be effective by first separating the plasma component from the cellular component by the use of a semi-permeable membrane or by methods known in the prior art to permeate the plasma component on a matrix containing anti-therapeutic antibodies. Alternatively, preferential removal of anti-tumor agent-conjugated blood cells (including red blood cells) collects and concentrates blood cells in the patient's blood and contacts these blood cells with immobilized anti-therapeutic antibody. And may be effective by eliminating serum components of the patient's blood.

The anti-therapeutic antibody may be administered to a patient parenterally in vivo. This patient has received a therapeutic anti-tumor agent derivative or conjugate. This antibody binds anti-tumor drug compounds and conjugates. Once bound, the antineoplastic derivative activity must be completely blocked, thereby reducing the biologically effective concentration of the antineoplastic derivative in the patient's bloodstream and minimizing adverse side effects. , Disturbed. In addition, the bound antibody-anti-tumor agent complex facilitates clearance of the anti-tumor agent compound and conjugate from the patient's bloodstream.

The ¹⁴ C radiolabel is the most effective method for determining the pharmacokinetic profile of antitumor drug derivatives of the present invention. Because such a small amount of drug
This is because it is necessary to show efficacy after administration. The pharmacokinetic profiles of the compounds described in this invention are described in terms of the area under the curve (AUC). The AUC is then measured using a commercially available ¹⁴ C-labeled drug when the reactive group is attached in one step or with a hydrolytically labile bond. When including a multi-step synthesis, it has a ¹⁴ C radiolabel on the reactive group and is attached to the antineoplastic agent either in the final step or at a very late step in the synthesis. Pharmacokinetic evaluation was performed by intraperitoneally intraperitoneally in the chest at a single bolus dose to rats with physiologically relevant solutions in radiolabeled antitumor drug derivatives at appropriate concentrations or, preferably, in the tail vein. It is done either intravenously or by first injecting the carotid artery. The measurements are taken at the following times: -2 hours, 0 hours, 0.25 hours, 0.5 hours, 1 hour, 2 hours, 4 hours, 2 hours.
4 hours, 48 hours. Here, the appropriate number of samples is used at each time point. This AUC is a count of radioactivity plotted against time for blood and various organs, which may or may not include liver, kidney, heart, lung, brain, fat, muscle and bone. Evaluated from. Even if the in vitro activity was reduced, the improved pharmacokinetic profile improved the pharmacodynamic effect and produced greater efficacy and reduced side effects.

The fully described invention is now illustrated by the following non-limiting examples. The invention will be more readily understood by reference to the following examples which illustrate the invention rather than limit the scope of the invention.

(Example 1) (Synthesis of NHS derivative from carboxylic acid having no other sensitive functional group in molecule) Compound drug to be modified containing carboxylic acid having no other sensitive functional group in the molecule (1 mmol ) and N- hydroxysuccinimide (1.1 mmol), in solution in anhydrous CH ₂ Cl ₂ (10mL), adding EDC (2.2 mmol).
The solution is stirred at room temperature for 20 hours or until complete. The reaction is then washed with water, saturated sodium chloride, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue is dissolved in a minimum amount of solvent and purified by chromatography or recrystallized from a suitable solvent system to give the NHS derivative.

Example 2 Synthesis of NHS Derivatives from Molecules Containing Amino and / or Thiol Functional Groups and Carboxylic Acids If free amino or thiol groups are present in the molecule, these It is preferred to protect the functional groups of (1) before the addition of the NHS derivative. For example, if the molecule contains a free amino group, it is necessary to convert this amine to an Fmoc or preferably tBoc protected amine before carrying out the chemistry described in Example 1. This amine functional group is not deprotected after preparation of the NHS derivative. Therefore, this method applies only to compounds that do not need to have an amine group free to induce the desired pharmacological effect. Example 3 where the amino group needs to be free to retain the original biological properties of the molecule.
Another type of chemistry, described in ~ 6, must be performed.

Example 3 Synthesis of NHS Derivatives from Molecules Containing Amino or Thiol Functional Groups and No Carboxylic Acids If the selected molecule is carboxylic acid free, then difunctional. An array of reactive linkers can be used to convert this molecule into a reactive NHS derivative. For example, ethylene glycol-bis (succinimidyl succinate) (EGS) and triethylamine are dissolved in DMF and free amino-containing molecules (EGF-rich 10: 1).
In the ratio of 1) to produce the mono-NHS derivative.

To produce NHS derivatives from thiol derivatized molecules, N- in DMF is used.
[4-Maleimidobutyloxy] succinimide ester (GMBS) and triethylamine may be used. The maleimide group reacts with the free thiol and the NHS derivative is purified from the reaction mixture by chromatography on silica gel or HPLC.

Example 4 Synthesis of NHS Derivatives from Molecules Containing Multiple Chemical Functional Groups Each case must be analyzed and solved in a different manner. However, because of the large array of commercially available protecting groups and bifunctional linkers, the present invention preferably uses only one step of chemistry to derivatize the molecule (as described in Example 1 or 3), Alternatively, two steps (as described in Example 2 and including prior protection of sensitive groups) or three steps (protection, activation, and deprotection) can be used to apply to any molecule. Only under exceptional circumstances it is necessary to use a multi-step (more than three steps) synthesis to convert the molecule into an active NHS or maleimide derivative.

Example 5 Synthesis of Maleimide Derivatives from Molecules Containing Free Amino Groups and Free Carboxylic Acids To generate maleimide derivatives from amino derivatized molecules, N- [4- in DMF was used. Maleimidobutyloxy] succinimide ester (GMBS) and triethylamine may be used. The succinimide ester group reacts with the free amino acid and the maleimide derivative is purified from the reaction mixture by crystallization or chromatography on silica or HPLC.

Example 6 Synthesis of Maleimide Derivatives from Molecules Containing Multiple Other Functional Groups and No Free Carboxylic Acids If the selected molecule is carboxylic acid free, then difunctional. An array of reactive crosslinkers can be used to convert the molecule into a reactive NHS derivative. For example, coupling maleimidopropionic acid (MPA) with a free amine to give HBT in DMF.
U / HOBt / DIEA activation can be used to generate maleimide derivatives by reaction of free amines with carboxylic acids of MPA.

[0164]   (Example 7)   (Preparation of doxorubicin derivative (MPA-AEEA-doxorubicin))

[0165]

[Chemical formula 24] In order to synthesize the doxorubicin derivative MPA-AEEA-doxorubicin,
Doxorubicin is first converted to 14-bromodoxorubicin by using the following procedure: Doxorubicin hydrochloride (226 mg) was dissolved in 4 mL anhydrous methanol,
Then mix with 0.2 mL methyl orthoformate and 8 mL dioxane. 0
． After addition of 83 mL bromine solution in methylene chloride (10% w / w), the reaction is carried out at room temperature for 40 minutes. The reaction mixture is poured into 50 mL dry diethyl ether and the precipitate that forms is collected by centrifugation. Discard this supernatant. After washing twice with 5 mL of dry ether, the precipitate was stirred 1
Treat with 2 mL of acetone at room temperature for 1 hour. The treated precipitate is collected by centrifugation, rinsed twice with ether and dried to give 226 mg of 14-bromodoxorubicin red powder (yield 87%).

The resulting 14-bromodoxorubicin is then treated with 14-bromodoxorubicin with amino-AEEA-MPA in N, N-dimethylformamide (DMF) in the presence of diisopropylethylamine (DIEA), and Conversion to MPA-AEEA-doxorubicin is carried out in a single step by heating at 60 ° C. for 1 hour. Specifically, DMC (2 mL) and diisopropylethylamine (
14-bromodoxorubicin (0.1 mmol) and ME in 0.2 mL)
EA-EDA. Stir HCl ([N- (2-amino) ethyl 2- (2-maleimido) ethoxyethoxyacetamide, 0.4 mmol] for 2 h or until complete (by TLC). The reaction mixture under reduced pressure. Concentrate and triturate the residue with ether.The product is purified by preparative HPLC using a gradient of 5-60% acetonitrile to give MPA-AEEA-doxorubicin. It is not limited to the use of a nitrogen atom to replace bromine.Practitioners in the art may also use alcoholates, secondary amines, primary or secondary amidates or primary or secondary sulfonamidates.

[0167]   (Example 8)   (Preparation of doxorubicin derivative (MPA-doxorubicin))

[0168]

[Chemical 25] To synthesize MPA-doxorubicin, 14-bromodoxorubicin (0.1 mmol) synthesized in Example 7 above is first converted to 14-azide by treatment with sodium azide in DMF. To synthesize the azide, 14-bromodoxorubicin is treated with Boc ₂ O (0.4 mmol) in MeOH for 60 minutes or until complete. The solvent was evaporated and the residue was
Used in the next step. In the next step, the crude product (0.1 mmol) and NaN ₃ (0.4 mmol) contained in this residue in DMF (0.1 mL) is stirred at room temperature for 16 hours or until complete (TLC). . The product is extracted with methylene chloride and the combined methylene chloride layers are washed with water and dried. The solvent is evaporated and the residue is purified by flash column chromatography on silica gel to give doxorubicin azide.

14-Azidoxorubicin is then converted to aminodoxorubicin using the following method: 1,3-propanedithiol (0.3 ml) is added to DMF (
To a solution of doxorubicin azide (0.1 mmol) in 1 ml) at room temperature. The reaction mixture is stirred for 16 hours or until complete. The solvent is evaporated under reduced pressure to give 14-aminodoxorubicin, which is used as crude product in the next step.

N-methylmorpholine (1 mmol) was added to aminodoxorubicin (0.1 mm
ol) and succinyl 3-maleimidopropanoate (3-MPA) in methylene chloride solution (10 mL). The reaction is stirred for 30 minutes or until complete. The methylene chloride is washed with water and dried (Na ₂ SO ₄ ). The solvent is evaporated and the product is purified by flash column chromatography. The product is then treated with TFA (0.5 mL) for 10 minutes. The TFA was evaporated and the residue was purified by preparative HPLC to give MPA
Obtain doxorubicin.

[0171]   (Example 9)   (Preparation of doxorubicin derivative (NHS-C6-Doxorubicin))

[0172]

[Chemical formula 26] To synthesize NHS-C6-Doxorubicin, 14-aminodoxorubicin prepared in Example 8 is reacted with di-N-hydroxysuccinyl adipate (0.14 mmol) in DMF (0.5 mL) for 1 hour at room temperature. To convert to a 6 carbon chain. The reaction mixture is then concentrated, the residue treated with TFA diluted with dichloromethane, concentrated again and the resulting NHS-C.
6-Doxorubicin is purified by HPLC.

Example 10 Preparation of Taxol Derivative (MPA-AEE-Taxol) The ether bond was chosen as the hydrolytically stable bond to prevent any cleavage. The synthesis of MPA-AEE-Taxol is described below from Taxol.

[0174]

[Chemical 27] Taxol is converted to the MPA-AEE-Taxol derivative by the following method: Taxol (0.02 mmol) and Ag ₂ CO ₃ (0.1 mmol) in acetonitrile (2 mL) are cooled to 0 ° C. To this mixture was added 2-maleimidoethyl-2'-iodoethyl ether (MPA-AEE-I) (0.02m).
mol) is added. The reaction is stirred for 2 hours or until complete. The precipitate is removed by filtration and washed with acetonitrile. The filtrate is evaporated to give a residue which is purified by preparative HPLC using a gradient of 5-60% acetonitrile to give the desired MPA-AEE-Taxol derivative and its regioisomer.

[0175]   (Example 11)   (Preparation of taxol derivative (MPA-taxol))

[0176]

[Chemical 28] To prepare MPA-Taxol, N, N-dimethylformamide (1 m
To a solution of taxol (0.02 mmol) in 1), N-methylmorpholine (0
． Succinyl 3-maleimidopropanoate (0.
A solution of 025 mmol) is added and the reaction mixture is stirred for 16 hours at room temperature. The solvent is slipped under reduced pressure and the residue is purified by HPLC using a gradient of 5-60% acetonitrile to give the desired MPA-taxol derivative and its regioisomer.

[0177]   (Example 12)   (Preparation of taxol derivative (MPA-EEA-taxol))

[0178]

[Chemical 29] To prepare MPA-EEA-docetaxel, by treating docetaxel with trifluoroacetic acid in dichloromethane,
First, it is converted to a free amino compound (docetaxel (0.1 mmol) is converted to TFA).
(Treat with 0.5 mL for 10 minutes). The TFA is evaporated and the residue containing aminodocetaxel is used in the next step.

N-Methylmorpholine is added to a THF solution (5 mL) containing aminodocetaxel (0.1 mmol) and succinyl 2- (2-maleimido) ethoxyethoxyacetate (MEEA succinate) (0.2 mmol). . The reaction is stirred for 30 minutes or until complete. Evaporate this solvent,
Then, the residue is purified by preparative HPLC to obtain MPA-EEA-docetaxel.

The process is not limited to this set of conditions and one skilled in the art will also know that the amino compound is dissolved in dichloromethane and treated with bis- (trimethylsilyl) -trifluoroacetamide to silylate the free alcohol. The conditions to be used can be used. Concentrated and redissolved in DMF, and MPA-EEA-CO ₂ H
And ethyl-dimethylaminopropylcarbodiimide (EDC) or dicyclohexylcarbodiimide (DCC) are added and allowed to react for 16 hours. The residue of this reaction is dissolved in methanol and a catalytic amount of TFA is added and the mixture is allowed to react for 2 hours. The reaction mixture is then concentrated and the residue is purified by HPLC to give MPA-EEA-docetaxel.

[0181]   (Example 13)   (Preparation of taxol derivative (C3'-NHS-glutamyl-docetaxel))

[0182]

[Chemical 30] Aminodocetaxel (0.1 mmol) synthesized in Example 12 above was added to
React with succinic anhydride (0.2 mmol) and N-methylmorpholine in THF (10 mL) and stir for 60 minutes or until complete. THF is evaporated and the residue is extracted with EtOAc. The combined EtOAc layers are washed with water and dried (Na ₂ SO ₄ ). The solvent is evaporated and the residue is purified by flash column chromatography to give the succinic acid derivative.

The residue (0.1 mmol) obtained above is treated with DCC (0.12 mmol) and N-hydroxysuccinimide (10 mmol) for 60 minutes or until complete. The product is extracted with EtOAc, and the extract is washed with water and dried (Na ₂ SO ₄ ). The solvent is evaporated and the residue is purified by preparative HPLC to give C3'-NHS-glutamyl-docetaxel.

[0184]   (Example 14)   (Preparation of taxol derivative (C3'-MPA-adipyl-docetaxel))

[0185]

[Chemical 31] Aminodocetaxel (0.1 mmol) synthesized in Example 12 was treated with THF.
Disuccinyl adipate (0.2 mmol) and N-methylmorpholine (
0.4 mmol) and stir at room temperature for 60 minutes or until complete. THF was evaporated and the residue was purified by preparative HPLC to give 3C.
'-MPA-adipyl-docetaxel is obtained.

[0186]   (Example 15)   (Preparation of methotrexate derivative (MPA-EEA-methotrexate))

[0187]

[Chemical 32]   In order to synthesize MPA-EEA-methotrexate, the methotrexate
The min group is treated with di-t-butyloxy in a suitable solvent (preferably methanol).
It is first protected as a Boc derivative by reaction with carbonyl anhydride. concrete
Was methotrexate (1 mmol) in methanol (10 mL) with Boc ₂ Treat with O (4 mmol) for 30 minutes. Evaporate the methanol, and
The crude product is purified by flash column chromatography on silica gel or preparative.
Purify by HPLC to give the protected methotrexate.

Next, the protected methotrexate (1 mmol) was added to Ac ₂ O (10 mL).
At 40 ° C. for 60 minutes. Excess Ac ₂ O is evaporated under reduced pressure. The ether is triturated to remove residual solvent, and the product, methotrexate anhydrate, is dried and purified. The crude product is used in the next step.

N-methylmorpholine (2 mmol) was added to crude methotrexate anhydride (1 m
mol) and MEEA-EDA. Add to methylene chloride solution (40 mL) containing HCl (1.2 mmol). The reaction is stirred at room temperature for 30 minutes. Methylene chloride is washed with HCl (1N) and water and dried (Na ₂ SO ₄ ).
This reaction produces a mixture of amide products. The first amide product is the result of the reaction at the distal carboxylate, and the second amide product is the result of the reaction at the proximal carboxylate. These compounds are separated by chromatography, then the individual compounds are deprotected with TFA diluted in dichloromethane, concentrated and the residue is purified by HPLC. Each of these compounds is treated with TFA and the product is purified by preparative HPLC to give the deprotected product as shown below.

[0190]

[Chemical 33] Example 16 (Preparation of Methotrexate Derivative (NHS-Methotrexate)) First, t-butyl methotrexate was added to Rosowsky et al. Med.
Chem. , 1991, 34, 574-579,
Prepared. The preferred site of attachment of the linker is the SAR-based position described in the above publications.

[0191]

[Chemical 34] The t-Bu-methotrexate is then converted to the di-Boc derivative using di-t-butoxycarbonyl anhydride as described in further detail below.
The methyl ester of caproic acid is then coupled to this diBoc derivative at room temperature in the presence of HOBT using DCC as the coupling agent to give compound C. The methyl ester of compound C was then hydrolyzed using dilute sodium hydroxide in methanol and water and acidified to pH 4, then extracted with ethyl acetate, dried (Na ₂ SO ₄ ). It is then concentrated to give a residue, which is converted to the succinate ester by first forming a mixed anhydride with isobutyl chloroformate in the presence of NMM in dichloromethane.
The residue is then deprotected using TFA diluted in dichloromethane and concentrated and the residue is purified by HPLC to give NHS-methotrexate.

[0192]

[Chemical 35] More specifically, t-Bu-methotrexate (1 mmol) and Boc ₂ O (24 mmol) in MeOH (10 mL) are stirred for 60 minutes or until complete. MeOH is evaporated and the residue is purified by flash column chromatography to give diBoc-methotrexate. The di-Boc-methotrexate (1 mmol), DCC (2 mmol) and HOBT (2 mmol) are stirred in dichloromethane (910 mL) at room temperature for 120 minutes. Then methyl 6-aminocaproate (2 mmol) is added. The reaction is stirred for 60 minutes or until complete. This reaction is
Quench with (1 mL). The precipitate is removed by filtration. The filtrate is washed with water and dried (Na ₂ SO ₄ ). The solvent is evaporated and the residue is purified by flash column chromatography to give compound C.

Compound C (1 mmol) is treated with NaOH (1N, 10 mL) for 30 minutes or until complete. The pH of this aqueous solution is adjusted to 4 with AcOH. The product is extracted with EtOAc. The combined extracts are washed with water and dried (Ns2S
O4). The solvent is evaporated and the residue is purified by flash column chromatography to give the carboxylic acid. This acid (1 mmol) is treated with isobutyl chloroformate (2 mmol) in the presence of N-methylmorpholine in dichloromethane (20 mL) at −20 ° C. for 30 minutes. N-hydroxysuccinimide (4 mmol) is added. The reaction is stirred at room temperature for 60 minutes. The methylene chloride solution is washed with HCl (1N) and dried. The solvent is evaporated and the residue is purified by flash column chromatography. The product is treated with TFA (5 mL) for 30 minutes. T
FA was evaporated and the residue was purified by preparative HPLC to give NHS-
Get Methotrexate.

[0194]   (Example 17)   (Preparation of hydroxyurea derivative (MPA-EEA-hydroxyurea))

[0195]

[Chemical 36] To prepare MPA-EEA-hydroxyurea, first, toluene (50
2- (2-maleimido) ethoxyethoxyacetamide (10 mmo in mL).
l) is treated with lead tetraacetate (20 mmol) for 2 hours or until complete. The reaction mixture is washed with water and dried (Na ₂ SO ₄ ). Toluene is evaporated and the residue is purified by flash column chromatography on silica gel to give the isocyanate derivative. The isocyanate derivative (5 mmol) is then cooled to -40 ° C in methylene chloride (30 mL) with hydroxyamine hydrochloride (5 mmol). To this reaction mixture is slowly added N-methylmorpholine (5 mmol). The reaction is warmed to room temperature.
The methylene chloride solution is washed with HCl (1N), water and dried (Na ₂ SO ₄ ). The product MPA-EEA-hydroxyurea is purified by flash column chromatography on silica gel.

[0196]   (Example 18)   (Preparation of tamoxifen derivative (MPA-tamoxifen))

[0197]

[Chemical 37] To prepare MPA-tamoxifen, mesyl chloride (12 mmol) was added to
2- [4- (1,2-diphenylbutenyl) phenoxy] ethanol (G.C.
Crawey EP 19377 B1, April 4, 1982) (10 mmol
) And triethylamine (12 mmol) in DMF (20 mL) solution,
Add at 0 ° C. The reaction is stirred for 60 minutes or until complete. NaN ₃ (30 mmol) is then added and the reaction mixture is stirred at 50 ° C. for 60 minutes. The solvent is evaporated under reduced pressure. The residue is purified by flash chromatography to give 2- [4- (1,2-diphenylbutenyl) phenoxy] ethyl azide.

Formic acid (95%, 50 mmol) was added to tamoxifen azide (10 m) at reflux.
mol) and Pd (OAc) ₂ (0.5 mmol) in a mixture of methanol. The reaction is stirred at reflux for 30 minutes. The crude tamoxifen amine product, which is the catalyst removed by filtration and the solvent evaporated to dryness, is used in the next step.

Diisopropylethylamine (12 mmol) was added to tamoxifen amine (1
0 mmol) and succinyl 3-maleimidopropanoate (12 mmol)
To a methylene chloride (30 mL) solution containing. The reaction is stirred for 60 minutes or until complete. The methylene chloride solution is washed with HCl, water and dried. The solvent is evaporated to give a residue which is purified by flash column chromatography to give MPA-tamoxifen.

[0200]   (Example 19)   (Preparation of tamoxifen derivative (NHS-adipyl-tamoxifen))

[0201]

[Chemical 38] Tamoxifen primary amine (1 mmol) prepared above, along with disuccinyl adipate (2 mmol) and N-methylmorpholine (2 mmol),
Stir in methylene chloride (10 mL) for 60 minutes or until the reaction is complete.
The methylene chloride is washed with water and dried. Evaporate this solvent,
The residue is then purified by flash column chromatography on silica gel to give N-hydroxysuccinate with 6 carbon chains.

[0202]   (Example 19)   (Preparation of mitomycin derivative (N-1a-MPA-mitomycin C))

[0203]

[Chemical Formula 39] To prepare N-1a-MPA-mitomycin C, mitomycin C (
1 mmol) is treated with succinyl 3-maleimidopropanoate (1.2 mmol) in methylene chloride (10 mL) for 16 hours or until complete. The methylene chloride is washed with water and dried (Na ₂ SO ₄ ). The solvent is evaporated and the residue is purified by preparative HPLC to give N-1a-MPA-mitomycin C.

Example 20 (Mitomycin derivative (N-1a-NHS-adipyl-mitomycin C)
Preparation)

[0205]

[Chemical 40] Mitomycin C is converted to a 6 carbon chain N-hydroxysuccinate by reacting with di-N-hydroxysuccinyl adipate in DMF using the following method.

Mitomycin C (1 mmol) is treated with disuccinyl adipate (1 mmol) in methylene chloride (10 mL) for 16 hours or until complete. The methylene chloride is washed with water and dried (Na ₂ SO ₄ ). The solvent is evaporated and the residue is purified by preparative HPLC to give N-1a-NHS-adipyl-mitomycin C.

Example 21 Preparation of Vincristine Derivative (MPA-Vincristine) Literature (BC Laguzza et al., US Pat. No. 4,801,688, 198).
31 January 1997, Culliman, U.S. Pat. No. 4,203,898, 198.
MPA-Vincristine is prepared by attaching a reactive group to the vincristine analog at the C23 carbonyl or C3 position on the ring using the hydrazide functional group described in (May 0).

[0208]

[Chemical 41] More specifically, vincristine (25 mg, 0.027 mmol) or, alternatively, vinblastine (having a methyl group at the N ₁ position instead of formyl) is dissolved in ethanol (0.5 mL), and anhydrous hydrazine is added. (0.2m
mol). The mixture is heated to 60 ° C. for 4 hours in a sealed test tube. The reaction mixture is concentrated under reduced pressure and under vacuum, and the residue is purified by flash column chromatography to give vincristine hydrazide. Vincristine hydrazide (0.01 mmol) was dissolved in DMF (0.5 ml) and a solution of succinyl 3-maleimidopropanoate (0.015 mmol) in DMF (0.1 ml) was added at room temperature and the reaction The mixture is stirred at room temperature for 16 hours. The reaction mixture is concentrated under reduced pressure and the residue is purified by HPLC using the conditions described above to give 4-desacetyl-MPA-
Get vincristine.

Next, 4-desacetyl-MPA-vincristine (0.005 mmol) in methylene chloride (30 ml) was added to acetic anhydride (0.5 M methylene chloride solution 20 μl).
) And triethylamine (20 μl of 0.5 M methylene chloride solution) at 0 ° C. The mixture is stirred at room temperature for 2 hours. The reaction mixture is diluted with methylene chloride and washed with saturated bicarbonate and brine, dried (Na ₂ SO ₄ ) and concentrated. The residue is purified by HPLC to give MPA-Vincristine and C3 Acetyl MPA-Vincristine. C3 acetyl MPA-Vincristine can be converted to MPA-Vincristine in the presence of wet silica (H
Argrove, U.S. Pat. No. 3,392,173, July 1968).

Example 22 In Vivo Half-Life The in vivo half-life of an anti-tumor agent derivative according to the invention is determined by the in vivo administration of the anti-tumor agent derivative and its binding to the endogenous protein in vivo. Can be determined by a survey of. In particular, rabbits are injected with a solution of the antitumor drug derivative according to the invention. Blood samples are taken and analyzed for the presence of reactive groups (eg maleimide) using gel electrophoresis. The same procedure is performed for the underivatized antitumor agent and serves as a control.

Alternatively, a radiolabeled anti-tumor agent derivative (eg, ¹²⁵ I-labeled anti-tumor agent derivative) can be injected into the test and control groups. Animals are given by slow injection (5 minutes) of 50 μg / Kg conjugate diluted in 5% glucose with 1% human serum albumin. One day after injection, giving animals 10μ Curie ^{12 5} I-labeled anti-tumor agent derivative (100 [mu] g per animal). One hour after injection of the antineoplastic derivative, the animals are sacrificed and various blood and tissue specimens are collected. Control animals ^{receive 125} I-labeled underivatized antitumor agent. Specific radioactivity was measured using a gamma counter and is expressed in cpm per ml blood or cpm per gram tissue.

Example 23 NHS-Antineoplastic Agent Binding to RBCs and Plasma Proteins To measure NHS-antineoplastic agent binding to RBCs and plasma proteins, 5 mg solubilized in DMSO (approximately 1 0.5 mg / kg) and 50 mg (about 15 mg / kg) of NHS-antitumor agents (eg NHS-C6-doxorubicin, C3′-NHS-glutamyl-docetaxel (C3′-NHS-glutam).
yl-docetaxel), NHS-methotrexate, N-1-a-NHS
-Adipyl-mitomycin C or NHS-adipyl-tamoxifen) may be injected into rabbits. Blood samples were then taken on the same day, 0.5 hours after injection, 1
Hours, 2 hours and 4 hours, then, for example, 1 day, 2 days after injection,
Samples will be collected after 3, 6, 9, 9, 13, 20, 27, 34, 41, 48 and 55 days. NHS-anti-tumor agent binding to RBCs is demonstrated by flow cytometry. NHS for plasma proteins
-The binding of antitumor agents is analyzed by immunoblotting. Plasma proteins are separated on a 10% polyacrylamide gel (Coomassie blue stain) under reducing conditions (10 mM DTT).

Example 24 MPA-Antineoplastic Agent Binding to RBCs and Plasma Proteins To measure MPA-antitumor agent binding to RBCs and plasma proteins, 5 mg solubilized in DMSO (approximately 1 0.5 mg / kg) and 50 mg (about 15 mg / kg) MPA-antitumor agent (eg MPA-doxorubicin, MP
A-AEEA-doxorubicin, MPA-AEE-taxol, MPA-taxol, MPA-EEA-docetaxel, MPA-EEA-methotrexate, M
PA-EEA-hydroxyurea, MPA-tamoxifen, N-1a-MPA
-Mitomycin C or MPA-Vincristine) may be injected into rabbits. The blood samples are then taken on the same day at 0.5, 1, 2 and 4 hours post injection, and then, for example, 1 day, 2 days, 3 days, 6 days, 9 days after injection. , 13 days, 20 days, 27 days, 34 days, 41 days, 48 days and 5 days
Collect on the 5th day. MPA-anti-tumor agent binding to RBCs is shown by flow cytometry. MPA-antitumor agent binding to plasma proteins is analyzed by immunoblotting. Plasma proteins are stored under reducing conditions (10m
MDTT) and separated on a 10% polyacrylamide gel (Coomassie blue stain).

Example 25 Antitumor Effect of Doxorubicin Derivatives The antitumor effect of doxorubicin derivatives according to the present invention can be determined by comparing the antitumor effect of doxorubicin in mice with the doxorubicin derivative according to the present invention. Primary kidney tumors are induced by subcapsular injection of kidney cancer cells in mouse kidney. Then doxorubicin and the doxorubicin derivatives according to the invention are administered and their antitumor effects are compared. Sample dosages that can be used include: 4 × 10 mmol for doxorubicin
/ Kg (4 × 6 mg / kg, equal to M _r = 580.0 g / mol) and 4 × 20 mmol / kg for doxorubicin derivative (4 × 16.7 mg / kg,
M _r = equal to 807.8 g / mol). After 24 hours, weight loss in both treatment groups is measured to determine the potency of doxorubicin derivatives compared to underivatized doxorubicin.

Although the present invention has been described in relation to particular embodiments thereof, it is generally in accordance with the principles of the present invention and as such may enter into known or customary practice in the art to which the invention pertains, and the present specification. Any variation, use or application, including such developments from the present disclosure, as may be applied to the essential features described above in the text, and in accordance with the scope of the appended claims It is understood that further modifications of the invention and their applications can be intended to be covered.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61P 35/00 A61P 35/00 // C07D 207/452 C07D 207/452 405/12 405/12 475/08 475/08 487/14 487/14 519/04 519/04 (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU , MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, K) E, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, A, BB, BG, BR, BY, BZ, CA, CH, CN, CR, CU, CZ, DE, DK, DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR , HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU , ZA, ZW (72) Inventor Rigger, Roger Canada J 4 W 2 2 M 9 Quebec, Brossard, Tissalon 6960 (72) Inventor Fan, Cetsey Canada H 9 J 3 X 2 Quebec, Kirkland, Donow 153 ( 72) Inventor Milner, Peter Gee. United States California 94022, Los Altos Hills, Manuela Road 14690 (72) Inventor Smith, Damon Canada H3W2Z4 Quebec, Montreal, Westmount, Holton Avenue 22 (72) Inventor Ezrin, Alan Em. United States California 94556, Moraga, Quintas Lane 110 F-term (reference) 4C050 AA01 BB01 CC04 DD01 EE02 FF02 GG03 HH04 4C063 AA01 BB08 CC72 DD04 EE01 4C069 AD08 CC04 4C072 QQ04 QQ07 UU01 4C086 CB07 MA14 NA14 BC14 NA14 NA02 BC01 NA14 BC02 MA14 NA02 BC02 MA14 NA02 ZB26

Claims (20)

[Claims] 1. Use of a composition for the manufacture of a medicament for use in the treatment of cancer, said composition comprising taxol and its analog derivatives.
Here, the derivative contains a reactive group which reacts with an amino group, a hydroxyl group or a thiol group in a blood component to form a stable covalent bond, and the reactive group contains N-hydroxysuccinimide or N-hydroxy group. -Use of a composition selected from sulfosuccinimide and maleimide groups. 2. Use of a composition according to claim 1, wherein the derivative reacts with blood proteins. 3. The derivative reacts with a thiol group on a blood protein,
Use of the composition according to claim 1. 4. The derivative reacts with albumin to form a covalent bond,
Use of the composition according to claim 1. 5. The reactive group is N-hydroxysuccinimide or N-
Use of the composition according to claim 1, which is hydroxysulfosuccinimide. 6. Use of the composition according to claim 1, wherein the reactive group is a maleimide group. 7. The derivative of taxol has the following formula: Use of the composition according to any one of claims 1 to 6, which is 8. A method for extending the longevity of taxol or an analogue thereof in vivo, comprising: (a) the taxol or analogue thereof being retained by a taxol derivative sufficiently to retain its therapeutic activity and a blood component. Modifying by reacting a reactive group at a site sufficiently distant from the pharmacophore region to form a taxol derivative so that a covalent bond can be formed with the above functional group; (b) the taxol derivative (C) forming a covalent bond in vivo between the reactive group and a functional group on a blood component to form a taxol derivative-blood component conjugate, thereby Prolonging the life of taxol or its analogs. 9. The method of claim 8, wherein the reactive group is a maleimide group. 10. The method of claim 9, wherein the reactive group is attached to the taxol via a linking group. 11. The method of claim 8, wherein the blood component is albumin. 12. A pharmaceutical composition for treating cancer, comprising an effective amount of taxol and its analog derivatives together with a pharmaceutically acceptable carrier.
Here, the derivative contains a reactive group which reacts with an amino group, a hydroxyl group or a thiol group in a blood component to form a stable covalent bond, and the reactive group contains N-hydroxysuccinimide or N-hydroxy group. A composition selected from the group consisting of sulfosuccinimide and maleimide groups. 13. The derivative of claim 1, wherein the derivative is covalently linked to a blood protein.
The composition according to 2. 14. The composition of claim 13, wherein the derivative reacts with thiol groups on blood proteins. 15. The composition of claim 14, wherein the blood protein comprises albumin. 16. The reactive group is N-hydroxysuccinimide or N.
-The composition of claim 12, which is -hydroxysulfosuccinimide. 17. The composition according to claim 12, wherein the reactive group is a maleimide group. 18. The derivative of taxol has the formula: 13. The composition of claim 12, which is 19. A conjugate comprising the taxol derivative according to claim 18 covalently bound to a blood protein. 20. The blood protein comprises serum albumin.
9. The conjugate according to item 9.