# Patent

## Title
Paclitaxel enhancer compounds

## Abstract

Disclosed is a compound represented by the Structural Formula (I):




    
    
        Y is a covalent bond, a phenylene group or a substituted or unsubstituted straight chained hydrocarbyl group. In addition, Y, taken together with both >C=Z groups to which it is bonded, is a substituted or unsubstituted aromatic group. Preferably, Y is a covalent bond or —C(R7R8)—.
        R1 and R2 are independently an aryl group or a substituted aryl group, R3 and R4 are independently —H, an aliphatic group, a substituted aliphatic group, an aryl group or a substituted aryl group.
        R5-R6 are independently —H, an aliphatic group, a substituted aliphatic group, an aryl group or a substituted aryl group.
        R7 and R8 are each independently —H, an aliphatic or substituted aliphatic group, or R7 is —H and R8 is a substituted or unsubstituted aryl group, or, R7 and R8, taken together, are a C2-C6 substituted or unsubstituted alkylene group.
        Z is ═O or ═S.
        Also disclosed are pharmaceutical compositions comprising the compound of the present invention and a pharmaceutically acceptable carrier or diluent.
        Also disclosed is a method of treating a subject with cancer by administering to the subject a compound of Structural Formula (I) in combination with Paclitaxel or an analog of Paclitaxel.

## Background
RELATED APPLICATIONS
This application is a continuation of U.S. application Ser. No. 10/846,152, filed May 14, 2004 now U.S. Pat. No. 7,037,940, which is a continuation of U.S. application Ser. No. 10/193,075, filed Jul. 10, 2002 now U.S. Pat. No. 6,800,660, which claims the benefit of U.S. Provisional Application No. 60/304,252, filed Jul. 10, 2001, and U.S. Provisional Application No. 60/361,946, filed Mar. 6, 2002. The entire teachings of these four applications are incorporated herein by reference.


BACKGROUND OF THE INVENTION
Many new drugs are now available to be used by oncologists in treating patients with cancer. Often, tumors are more responsive to treatment when anti-cancer drugs are administered in combination to the patient than when the same drugs are administered individually and sequentially. One advantage of this approach is that the anti-cancer agents often act synergistically because the tumors cells are attacked simultaneously with agents having multiple modes of action. Thus, it is often possible to achieve more rapid reductions in tumor size by administering these drugs in combination. Another advantage of combination chemotherapy is that tumors are more likely to be eradicated completely and are less likely to develop resistance to the anti-cancer drugs being used to treat the patient.
One serious limitation of combination chemotherapy is that anti-cancer agents generally have severe side effects, even when administered individually. For example, the well known anti-cancer agent Paclitaxel causes neutroperia, neuropathy, mucositis, anemia, thrombocytopenia, bradycardia, diarrhea and nausea. Unfortunately, the toxicity of anti-cancer agents is generally additive when the drugs are administered in combination. As result, certain types of anti-cancer drugs are generally not combined. The combined toxic side-effects of those anti-cancer drugs that are administered simultaneously can place severe limitations on the quantities that can be used in combination. Often, it is not possible to use enough of the combination therapy to achieve the desired synergistic effects. Therefore, there is an urgent need for agents which can enhance the desirable tumor attacking properties of anti-cancer agents without further increasing their undesirable side-effects.
It has now been found that certain bis[thio-hydrazide amide] compounds significantly enhance the anti-cancer activity of Paclitaxel. For example, Compound (1) was used in combination with Paclitaxel (TAXOL) to treat tumors induced in nude mice from the human breast tumor cell line MDA-435. The tumor volume was about five fold less after 24 days of treatment in mice which had been administered 5 mg/kg of Paclitaxel and 25 mg/kg of Compound (1) than in mice which had only been administered 5 mg/kg of Paclitaxel or in mice which had only been administered 50 mg/kg of Compound (1) (Example 13). These results are shown graphically in FIG. 1. The structure of Compound (1) is shown below:




It has also been found that these bis[thio-hydrazide amide] compounds have minimal toxic side effects. For example, the mice treated with Paclitaxel and Compound (1) showed little if any weight loss over the treatment period (see FIG. 2). Based on these results, novel compounds which enhance the anti-cancer activity of Paclitaxel, pharmaceutical compositions comprising these compounds and methods of treating a subject with cancer are disclosed herein,

One embodiment of the present invention is a compound represented by the Structural Formula (I):




Y is a covalent bond, a phenylene group or a substituted or unsubstituted straight chained hydrocarbyl group. In addition, Y, taken together with both >C=Z groups to which it is bonded, is a substituted or unsubstituted aromatic group. Preferably, Y is a covalent bond or —C(R7R8)—.
R1 and R2 are independently an aryl group or a substituted aryl group, R3 and R4 are independently —H, an aliphatic group, a substituted aliphatic group, an aryl group or a substituted aryl group.
R5-R6 are independently —H, an aliphatic group, a substituted aliphatic group, an aryl group or a substituted aryl group.
R7 and R8 are each independently —H, an aliphatic or substituted aliphatic group, or R7 is —H and R8 is a substituted or unsubstituted aryl group, or, R7 and R8, taken together, are a C2-C6 substituted or unsubstituted alkylene group.
Z is ═O or ═S.
In one aspect, R1 and R2 in the compound represented by Structural Formula (I) are not both phenyl when Y is —C(R7R8)—, R3 and R4 are both phenyl and R5—R8 are all —H.
Another embodiment of the present invention is a pharmaceutical composition comprising a pharmaceutically acceptable carrier or diluent and a compound represented by Structural Formula (I). Preferably, the pharmaceutical composition comprises an effective concentration of the compound.
Yet another embodiment of the present invention is a method of treating a subject with cancer. The method comprises administering to the subject an effective amount of Paclitaxel or a Paclitaxel analog and an effective amount of a compound represented by Structural Formula (I).
The disclosed compounds increase the anti-cancer activity of Paclitaxel and Paclitaxel analogs. In addition, these compounds have minimal toxic side-effects. Consequently, it is possible to increase the effectiveness of Paclitaxel and analogs thereof when used in combination with the disclosed compounds, even when approaching the highest tolerated doses of Paclitaxel. Thus, it is expected that combination therapy with the compounds of the present invention will provide improved clinical outcomes for patients with cancers that are being treated with Paclitaxel. By co-administering the disclosed compounds with Paclitaxel, it is also possible to achieve the same therapeutic effectiveness previously achieved with higher doses of Paclitaxel, thereby reducing the side-effects and improving the quality of life for the patient.



BRIEF DESCRIPTION OF THE DRAWINGS
The foregoing and other objects, features and advantages of the invention will be apparent from the following more particular description of preferred embodiments of the invention, as illustrated in the accompanying drawings in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating the principles of the invention.
FIG. 1 is a graph showing the average tumor volume in milliliters over time (in days) in nude mice treated with vehicle; Compound (1) (50 mg/kg); Paclitaxel (5 mg/kg); Compound (1) (25 mg/kg) and Paclitaxel (5 mg/kg); or Compound (1) (50 mg/kg) and Paclitaxel (5 mg/kg). The tumors were generated from the human breast tumor cell line MDA-435.
FIG. 2 is a graph showing the percent weight change over time in nude mice treated with vehicle; Compound (1) (50 mg/kg); Paclitaxel (5 mg/kg); Compound (1) (25 mg/kg) and Paclitaxel (5 mg/kg); or Compound (1) (50 mg/kg) and Paclitaxel (5 mg/kg). The mice were being treated for tumors generated from the human breast tumor cell line MDA-435.
FIG. 3 is the structure of Paclitaxel TAXOL
FIG. 4 is the structure of Docetaxel (TAXOTERE)
FIGS. 5-25 are each the structure of a Paclitaxel analog.
FIG. 26 is the structure of a polymer comprising a Paclitaxel analog group pendent from the polymer backbone. The polymer is a terpolymer of the three monomer units shown.



DETAILED DESCRIPTION OF THE INVENTION
In a first preferred embodiment, Y in Structural Formula (I), taken together with both >C=Z groups to which it is bonded, is a substituted or unsubstituted arylene group and the compound is represented by Structural Formula (II):




R1-R6 in Structural Formula (II) are as described in Structural Formula (I). Ar is a substituted or unsubstituted arylene group. Preferably, Ar is a nitrogen-containing heteroarylene group. Examples are shown below:




Ring A is substituted or unsubstituted.

In a second preferred embodiment, Y in Structural Formula (I) is a covalent bond or a substituted or unsubstituted straight chained hydrocarbyl group. R7 and R8 are as described for Structural Formula (I). Preferably, Y is a covalent bond, —C(R7R8)—, —(CH2CH2)—, trans-(CH═CH)—, cis-(CH═CH)—, —(CC)— or a 1,4-phenylene group. Even more preferably, Y is a covalent bond or —C(R7R8)—.
In a third preferred embodiment, Y in Structural Formula (I) is a covalent bond or —C(R7R8)— and the compound of the present invention is represented by Structural Formula (III):




R1-R8 are as described for Structural Formula (I). Y′ is a covalent bond or —C(R7R8)—. Preferably, R7 and R8 are both methyl; R7 and R8, taken together, are propylene or butylene; or R7 is —H and R8 is lower alkyl (preferably methyl), thienyl, phenyl, benzyl, or amino.

In a more preferred embodiment, R5—R8 in Structural Formula (III) are —H and the compound is represented by Structural Formula (IV):




R1-R4 in Structural Formula (IV) are as described in Structural Formula (I). Y′′ is a covalent bond or —CH2—.

In a first example of a compound represented by Structural Formula (IV), R3 and R4 are both a substituted or unsubstituted aliphatic group, preferably both a substituted or unsubstituted lower alkyl group and more preferably both a methyl group or ethyl. When R3 and R4 in Structural Formula (IV) are both a substituted or unsubstituted aliphatic group, then R1 and R2 are preferably both a substituted or unsubstituted aryl group (e.g., a substituted or unsubstituted heteroaryl group, a substituted or unsubstituted phenyl group, or a phenyl group with at least one substituent other than an aliphatic group).
In a second example of a compound represented by Structural Formula (IV), R3 and R4 are both a substituted or unsubstituted heteroaryl group. When R3 and R4 in Structural Formula (IV) are both a substituted or unsubstituted heteroaryl group, then R1 and R2 are preferably both: 1) a substituted or unsubstituted phenyl group; or 2) a substituted or unsubstituted heteroaryl group.
In a third example of a compound represented by Structural Formula (IV), R3 and R4 are both a substituted or unsubstituted phenyl group (e.g., a phenyl group substituted with at least one group other than an aliphatic group). When R3 and R4 in Structural Formula (IV) are both a substituted or unsubstituted phenyl group, then R1 and R2 are preferably both: 1) a substituted or unsubstituted phenyl group; or 2) a substituted or unsubstituted heteroaryl group.
In a fourth example of a compound represented by Structural Formula (IV), R1 and R2 are both a substituted or unsubstituted aryl group (e.g., a substituted or unsubstituted heteroaryl group, a substituted or unsubstituted phenyl group or a phenyl group substituted with at least one group other than an aliphatic group). More preferably, R3 and R4 are both methyl and the remainder of the variables are as described above.
In a fourth preferred embodiment, the compound of the present invention is represented by Structural Formula (III), wherein at least one of R1-R4 is a heteroaryl group, a substituted heteroaryl group, or a phenyl group substituted with at least one group other than an aliphatic group. Preferably, R5—R8 are all —H.
The following are specific examples of compounds represented by Structural Formula (IV): R1 and R2 are both phenyl, and R3 and R4 are both o-CH3-phenyl; R1 and R2 are both o-CH3C(O)O-phenyl, and R3 and R4 are phenyl; R1 and R2 are both phenyl, and R3 and R4 are both methyl; R1 and R2 are both phenyl, and R3 and R4 are both ethyl; R1 and R2 are both phenyl, and R3 and R4 are both n-propyl; R1 and R2 are both p-cyanophenyl, and R3 and R4 are both methyl; R1 and R2 are both p-nitro phenyl, and R3 and R4 are both methyl; R1 and R2 are both 2,5-dimethoxyphenyl, and R3 and R4 are both methyl; R1 and R2 are both phenyl, and R3 and R4 are both n-butyl; R1 and R2 are both p-chlorophenyl, and R3 and R4 are both methyl; R1 and R2 are both 3-nitrophenyl, and R3 and R4 are both methyl; R1 and R2 are both 3-cyanophenyl, and R3 and R4 are both methyl; R1 and R2 are both 3-fluorophenyl, and R3 and R4 are both methyl; R1 and R2 are both 2-furanyl, and R3 and R4 are both phenyl; R1 and R2 are both 2-methoxyphenyl, and R3 and R4 are both methyl; R1 and R2 are both 3-methoxyphenyl, and R3 and R4 are both methyl; R1 and R2 are both 2,3-dimethoxyphenyl, and R3 and R4 are both methyl; R1 and R2 are both 2-methoxy-5-chlorophenyl, and R3 and R4 are both ethyl; R1 and R2 are both 2,5-difluorophenyl, and R3 and R4 are both methyl; R1 and R2 are both 2,5-dichlorophenyl, and R3 and R4 are both methyl; R1 and R2 are both 2,5-dimethylphenyl, and R3 and R4 are both methyl; R1 and R2 are both 2-methoxy-5-chlorophenyl, and R3 and R4 are both methyl; R1 and R2 are both 3,6-dimethoxyphenyl, and R3 and R4 are both methyl; R1 and R2 are both phenyl, and R3 and R4 are both 2-ethylphenyl; R1 and R2 are both 2-methyl-5-pyridyl, and R3 and R4 are both methyl; or R1 is phenyl; R2 is 2,5-dimethoxyphenyl, and R3 and R4 are both methyl.
In a fifth preferred embodiment, Y in Structural Formula (I) is —C(R7R8)— and R5 and R6 are both —H. When Y is a covalent bond or —CR7R8— and R5 and R6 are both —H, the compound of the present invention is represented by Structural Formula (V):




R1-R4, R7 and R8 are as described for Structural Formula (I) and Y′ is a covalent bond or —CR7R8—. R7 and R8 are the same or different. Preferably, R7 and R8 are both methyl; R7 and R8, taken together, are propylene or butylene; or R7 is —H and R8 is lower alkyl (preferably methyl), thienyl, phenyl or benzyl.

In one example of a compound represented by Structural Formula (V), R1 and R2 are both aryl or substituted aryl groups and R3 and R4 are both a lower alkyl group or a substituted lower alkyl group; preferably, R1 and R2 are both aryl or substituted aryl groups, R3 and R4 are both methyl or ethyl, R7 is —H and R8 is —H or methyl. In another example of a compound represented by Structural Formula (V), R1 and R2 are both phenyl or substituted phenyl and R3 and R4 are both methyl, ethyl, phenyl, or thienyl. When R1 and R2 are both phenyl or substituted phenyl and R3 and R4 are both methyl, ethyl, phenyl, or thienyl, then preferably R7 and R8, taken together, are propylene or butylenes. In yet another example of a compound represented by Structural Formula (V), Y′ is a covalent bond or —CR7R8—; R1 and R2 are both a substituted or unsubstituted aryl group; R3 and R4 are both —H, methyl or ethyl; and R7 is —H and R8 is —H or methyl.
The following are specific examples of compounds represented by Structural Formula (V): R1 and R2 are both phenyl; R3 and R4 are both methyl; R7 is —H, and R8 is ethyl; R1 and R2 are both phenyl; R3 and R4 are both phenyl, and R7 and R8 are both methyl; R1 and R2 are both 2-thienyl; R3 and R4 are both phenyl, and R7 and R8 are both methyl; R1 and R2 are both 4-cyanophenyl; R3 and R4 are both methyl; R7 is —H, and R8 is methyl; R1 and R2 are both phenyl; R3 and R4 are both methyl; R7 is —H, and R8 is methyl; R1 and R2 are both phenyl; R3 and R4 are both methyl; R7 is —H, and R8 is benzyl; R1 and R2 are both phenyl; R3 and R4 are both methyl; R7 is —H, and R8 is ethyl; R1 and R2 are both phenyl; R3 and R4 are both ethyl; R7 is —H, and R8 is n-butyl; R1 and R2 are both 2,5-dimethoxyphenyl; R3 and R4 are both methyl; R7 is —H, and R8 is methyl; R1 and R2 are both phenyl; R3 and R4 are both methyl; R7 is —H, and R8 is iso-propyl; R1 and R2 are both 3-nitrophenyl; R3 and R4 are both methyl; R7 is —H, and R8 is methyl; R1 and R2 are both 4-chlorophenyl; R3 and R4 are both methyl; R7 is —H, and R8 is methyl; R1 and R2 are both phenyl; R3 and R4 are both methyl; R7 is —H, and R8 is 3-thienyl; R1 and R2 are both phenyl; R3 and R4 are both methyl, and R7 and R8, taken together, are propylene; R1 and R2 are both 2,3-dimethoxyphenyl; R3 and R4 are both methyl; R7 is —H, and R8 is methyl; R1 and R2 are both 2-chloro-5-methoxy phenyl; R3 and R4 are both methyl; R7 is —H, and R8 is methyl; R1 and R2 are both 2,5-difluorophenyl; R3 and R4 are both methyl; R7 is —H, and R8 is methyl; R1 and R2 are both 2,5-dichlorophenyl; R3 and R4 are both methyl; R7 is —H, and R8 is methyl; R1 and R2 are both 2,6-dimethoxyphenyl; R3 and R4 are both methyl; R7 is —H, and R8 is methyl; R1 and R2 are both 2,5-dimethylphenyl, R3 and R4 are both methyl; R7 is —H, and R8 is methyl; R1 and R2 are both 2,5-dimethoxyphenyl; R3 and R4 are both ethyl; R7 is —H, and R8 is methyl, and R1 and R2 are both 2,5-diethoxyphenyl; R3 and R4 are both methyl; R7 is —H, and R8 is methyl.
In a sixth preferred embodiment, Y in Structural Formula (I) is a covalent bond or —CH2—. When Y is a covalent bond or —CH2—, the compound of the present invention is represented by Structural Formula (VI):




R1-R6 in Structural Formula (VI) are as described for Structural Formula (I). R5 and R6 are the same or different. Y′′ is a covalent bond or —CH2—.

In one example of a compound represented by Structural Formula (VI), R5 and R6 are both a lower alkyl group (preferably methyl) or a phenyl group. When R5 and R6 are both a lower alkyl group or a phenyl group, then R1 and R2 are preferably both phenyl or substituted phenyl and R3 and R4 are preferably both a lower alkyl group.
The following are more specific examples of compounds of the present invention: R1 and R2 are both phenyl, R3 and R4 are both phenyl, R5 and R6 are both methyl, and R7 and R8 are both —H; R1 and R2 are both phenyl, R3 and R4 are both phenyl, R5 and R6 are both n-hexyl, and R7 and R8 are both —H; R1 and R2 are both phenyl, R3 and R4 are both methyl, R5 and R6 are both methyl, and R7 and R8 are both —H; R1 and R2 are both phenyl, R3 and R4 are both methyl, R5 and R6 are both methyl, and R7 is —H and R8 is methyl; R1 and R2 are both phenyl, R3 and R4 are both —H, R5 and R6 are both phenyl, R7 is —H, and R8 is methyl; R1 and R2 are both 4-chlorophenyl, R3 and R4 are both methyl, R5 and R6 are both methyl, and R7 and R8 are both —H.
In Structural Formulas (I)-(VI), R1 and R2 are the same or different; and/or R3 and R4 are the same or different. Preferably, R1 and R2 are the same, and R3 and R4 are the same.
A “straight chained hydrocarbyl group” is an alkylene group, i.e., —(CH2)x—, with one or more (preferably one) methylene groups optionally replaced with a linkage group. x is a positive integer (e.g., between 1 and about 10), preferably between 1 and about 6 and more preferably 1 or 2. A “linkage group” refers to a functional group which replaces a methylene in a straight chained hydrocarbyl. Examples of suitable linkage groups include a ketone (—C(O)—), alkene, alkyne, phenylene, ether (—O—), thioether (—S—), or amine [—N(Ra)]—, wherein Ra is defined below. A preferred linkage group is —C(R7R8)—, wherein R7 and R8 are defined above. Suitable substitutents for an alkylene group and a hydrocarbaryl group are those which do not substantially interfere with the reactions described herein. R7 and R8 are preferred substituents for an alkylene or hydrocarbyl group.
An aliphatic group is a straight chained, branched or cyclic non-aromatic hydrocarbon which is completely saturated or which contains one or more units of unsaturation. Typically, a straight chained or branched aliphatic group has from 1 to about 20 carbon atoms, preferably from 1 to about 10, and a cyclic aliphatic group has from 3 to about 10 carbon atoms, preferably from 3 to about 8. An aliphatic group is preferably a straight chained or branched alkyl group, e.g, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, tert-butyl, pentyl, hexyl, pentyl or octyl, or a cycloalkyl group with 3 to about 8 carbon atoms. A C1-C20 straight chained or branched alkyl group or a C3-C8 cyclic alkyl group is also referred to as a “lower alkyl” group.
Aromatic groups include carbocyclic aromatic groups such as phenyl, naphthyl, and anthracyl, and heteroaryl groups such as imidazolyl, thienyl, furanyl, pyridyl, pyrimidy, pyranyl, pyrazolyl, pyrroyl, pyrazinyl, thiazole, oxazolyl, and tetrazole.
Aromatic groups also include fused polycyclic aromatic ring systems in which a carbocyclic aromatic ring or heteroaryl ring is fused to one or more other heteroaryl rings. Examples include benzothienyl, benzofuranyl, indolyl, quinolinyl, benzothiazole, benzooxazole, benzimidazole, quinolinyl, isoquinolinyl and isoindolyl.
The term “arylene” refers to an aryl group which is connected to the remainder of the molecule by two other bonds. By way of example, the structure of a 1,4-phenylene group is shown below:




Substituents for an arylene group are as described below for an aryl group.

Non-aromatic heterocyclic rings are non-aromatic carbocyclic rings which include one or more heteroatoms such as nitrogen, oxygen or sulfur in the ring. The ring can be five, six, seven or eight-membered. Examples include tetrahydrofuranyl, tetrahyrothiophenyl, morpholino, thiomorpholino, pyrrolidinyl, piperazinyl, piperidinyl, and thiazolidinyl.
The terms “lower alkoxy”, “lower acyl”, “(lower alkoxy)methyl” and “(lower alkyl)thiomethyl” mean to —O-(lower alkyl), —C(O)-(lower alkyl), —CH2—O-(lower alkyl) and —CH2—S-(lower alkyl), respectively. The terms “substituted lower alkoxy” and “substituted lower acyl” mean —O-(substituted lower alkyl) and —C(O)-(substituted lower alkyl), respectively.
Suitable substituents on an aliphatic group, non-aromatic heterocyclic group, benzylic or aryl group (carbocyclic and heteroaryl) are those which do not substantially interfere with the ability of the disclosed compounds to enhance the anti-cancer activity of Paclitaxel and analogs thereof. A substituent substantially interferes with the ability of a disclosed compound to enhance anti-cancer activity when the enhancement is reduced by more than about 50% in a compound with the substituent compared with a compound without the substituent. Examples of suitable substituents include —OH, halogen (—Br, —Cl, —I and —F), —ORa, —O—CORa, —CORa, —CN, —NO2, —COOH, —SO3H, —NH2, —NHRa, —N(RaRb), —COORa, —CHO, —CONH2, —CONHRa, —CON(RaRb), —NHCORa, —NRCORa, —NHCONH2, —NHCONRaH, —NHCON(RaRb), —NRcCONH2, —NRcCONRaH, —NRcCON(RaRb), —C(═NH)—NH2, —C(═NH)—NHRa, —C(═NH)—N(RaRb), —C(═NRc)—NH2, —C(═NRc)—NHRa, —C(═NRc)—N(RaRb), —NH—C(═NH)—NH2, —NH—C(═NH)—NHRa, —NH—C(═NH)—N(RaRb), —NH—C(═NRc)—NH2, —NH—C(═NRc)—NHRa, —NH—C(═NRc)—N(RaRb), —NRdH—C(═NH)—NH2, —NRd—C(═NH)—NHRa, —NRd—C(═NH)—N(RaRb), —NRd—C(═NRc)—NH2, —NRd—C(═NRc)—NHRa, —NRd—C(═NRc)—N(RaRb), —NHNH2, —NHNHRa, —NHRaRb, —SO2NH2, —SO2NHRa, —SO2N(RaRb), —CH═CHRa, —CN═CRaRb, —CRc═CRaRb,—CRc═CHRa, —CRc═CRaRb, —CCRa, —SH, —SOkRa (k is 0, 1 or 2) and —NH—C(═NH)—NH2. Ra—Rb are each independently an aliphatic, substituted aliphatic, benzyl, substituted benzyl, aromatic or substituted aromatic group, preferably an alkyl, benzylic or aryl group. In addition, —N(RaRb), taken together, can also form a substituted or unsubstituted non-aromatic heterocyclic group. A non-aromatic heterocyclic group, benzylic group or aryl group can also have an aliphatic or substituted aliphatic group as a substituent. A substituted aliphatic group can also have a non-aromatic heterocyclic ring, a substituted a non-aromatic heterocyclic ring, benzyl, substituted benzyl, aryl or substituted aryl group as a substituent. A substituted aliphatic, non-aromatic heterocyclic group, substituted aryl, or substituted benzyl group can have more than one substituent.
Also included in the present invention are pharmaceutically acceptable salts of the compounds described herein. The compound of the present invention which possess a sufficiently acidic, a sufficiently basic, or both functional groups, and accordingly can react with any of a number of inorganic bases, and inorganic and organic acids, to form a salt. Acids commonly employed to form acid addition salts are inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, and the like, and organic acids such as p-toluenesulfonic acid, methanesulfonic acid, oxalic acid, p-bromophenyl-sulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, acetic acid, and the like. Examples of such salts include the sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caproate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-1,4-dioate, hexyne-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, sulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, gamma-hydroxybutyrate, glycolate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, mandelate, and the like.
Base addition salts include those derived from inorganic bases, such as ammonium or alkali or alkaline earth metal hydroxides, carbonates, bicarbonates, and the like. Such bases useful in preparing the salts of this invention thus include sodium hydroxide, potassium hydroxide, ammonium hydroxide, potassium carbonate, and the like.
Paclitaxel, also referred to as “TAXOL”, is a well-known anti-cancer drug which acts by inhibiting microtubule formation. Many analogs of Paclitaxel are known, including Docetaxel, the structure of which is shown in FIG. 4. Docetaxel is also referred to as “TAXOTERE”. The structure of other Paclitaxel analogs are shown in FIGS. 5-25. These compounds have the basic taxane skeleton as a common structure feature and have also been shown to have the ability to arrest cells in the G2-M phases due to stabilized microtubules. Thus, it is apparent from FIGS. 5-25 that a wide variety of substituents can decorate the taxane skeleton without adversely affecting biological activity. it is also apparent that zero, one or both of the cyclohexane rings of a Paclitaxel analog can have a double bond at the indicated positions. For clarity purposes, the basic taxane skelton is shown below in Structural Formula (VII):




Double bonds have been omitted from the cyclohexane rings in the taxane skeleton represented by Structural Formula (VII). It is to be understood that the basic taxane skeleton can include zero or one double bond in one or both cyclohexane rings, as indicated in FIGS. 5-25 and Structural Formulas (VIII) and (IX) below. A number of atoms have also omitted from Structural Formula (VII) to indicate sites in which structural variation commonly occurs among Paclitaxel analogs. For example, substitution on the taxane skeleton with simply an oxygen atom indicates that hydroxyl, acyl, alkoxy or other oxygen-bearing substituent is commonly found at the site. It is to be understood that these and other substitutions on the taxane skeleton can also be made without losing the ability to enhance and stabilize microtubule formation. Thus, the term “Paclitaxel analog” is defined herein to mean a compound which has the basic Paclitaxel skeleton and which promotes disassembly of microtubules.
Typically, the Paclitaxel analogs used herein are represented by Structural Formula (VIII) or (IX):




R10 is a lower alkyl group, a substituted lower alkyl group, a phenyl group, a substituted phenyl group, —SR19, —NHR19 or —OR19.
R11 is a lower alkyl group, a substituted lower alkyl group, an aryl group or a substituted aryl group.
R12 is —H, —OH, lower alkyl, substituted lower alkyl, lower alkoxy, substituted lower alkoxy, —O—C(O)-(lower alkyl), —O—C(O)-(substituted lower alkyl), —O—CH2—O-(lower alkyl) —S—CH2—O-(lower alkyl).
R13 is —H, —CH3, or, taken together with R14, —CH2—.
R14 is —H, —OH, lower alkoxy, —O—C(O)-(lower alkyl), substituted lower alkoxy, —O—C(O)-(substituted lower alkyl), —O—CH2—O—P(O)(OH)2, —O—CH2—O-(lower alkyl), —O—CH2—S-(lower alkyl) or, taken together with R20, a double bond.
R15 —H, lower acyl, lower alkyl, substituted lower alkyl, alkoxymethyl, alkthiomethyl, —OC(O)—O(lower alkyl), —OC(O)—O(substituted lower alkyl), —OC(O)—NH(lower alkyl) or —OC(O)—NH(substituted lower alkyl).
R16 is phenyl or substituted phenyl.
R17 is —H, lower acyl, substituted lower acyl, lower alkyl, substituted, lower alkyl, (lower alkoxy)methyl or (lower alkyl)thiomethyl.
R18 —H, —CH3 or, taken together with R17 and the carbon atoms to which R17 and R18 are bonded, a five or six membered a non-aromatic heterocyclic ring.
R19 is a lower alkyl group, a substituted lower alkyl group, a phenyl group, a substituted phenyl group.
R20 is —H or a halogen.
R21 is —H, lower alkyl, substituted lower alkyl, lower acyl or substituted lower acyl.
Preferably, the variables in Structural Formulas (VIII) and (IX) are defined as follows: R10 is phenyl, tert-butoxy, —S—CH2—CH—(CH3)2, —S—CH(CH3)3, —S—(CH2)3CH3, —O—CH(CH3)3, —NH—CH(CH3)3, —CH═C(CH3)2 or para-chlorophenyl; R11 is phenyl, (CH3)2CHCH2—, -2-furanyl, cyclopropyl or para-toluyl; R12 is —H, —OH, CH3CO— or —(CH2)2—N-morpholino; R13 is methyl, or, R13 and R14, taken together, are —CH2—;
R14 is —H, —CH2SCH3 or —CH2—O—P(O)(OH)2; R15 is CH3CO—;
R16 is phenyl; R17 —H, or, R17 and R18, taken together, are —O—CO—O—;
R18 is —H; R20 is —H or —F; and R21 is —H, —C(O)—CHBr—(CH2)13—CH3 or —C(O)—(CH2)14—CH3; —C(O)—CH2—CH(OH)—COOH, —C(O)—CH2—O—C(O)—CH2CH(NH2)—CONH2, —C(O)—CH2—O—CH2CH2OCH3 or —C(O)—O—C(O)—CH2CH3.
A Paclitaxel analog can also be bonded to or be pendent from a pharmaceutically acceptable polymer, such as a polyacrylamide. One example of a polymer of this type is shown in FIG. 26. The term “Paclitaxel analog”, as it is used herein, includes such polymers.
The disclosed compounds are enhancers of the anti-cancer activity of Paclitaxel and Paclitaxel analogs. A compound enhances the anti-cancer activity of Paclitaxel or a Paclitaxel analog when the activity of Paclitaxel or the Paclitaxel analog is greater when administered in combination with the compound than when administered alone. The degree of the increase in activity depends upon the amount of compound administered. The compounds of the present invention can therefore be used in combination with Paclitaxel or Paclitaxel analogs to treat subjects with cancers. Examples include colon cancer, pancreatic cancer, melanoma, renal cancer, sarcoma, breast cancer, ovarian cancer, lung cancer. stomach cancer, bladder cancer and cervical cancer.
A “subject” is a mammal, preferably a human, but can also be an animal in need of veterinary treatment, e.g., companion animals (e.g., dogs, cats, and the like), farm animals (e.g., cows, sheep, pigs, horses, and the like) and laboratory animals (e.g., rats, mice, guinea pigs, and the like).
In order to achieve an enhancement of the anti-cancer activity of Paclitaxel and Paclitaxel analogs, an effective amount of a compound of the present invention and an effective amount of Paclitaxel or analog of Paclitaxel are administered to the subject. With respect to Paclitaxel or an analog of Paclitaxel, an “effective amount” is a quantity in which anti-cancer effects are normally achieved. With respect to a compound of the present invention, an “effective amount” is the quantity in which a greater anti-cancer effect is achieved when the compound is co-administered with Paclitaxel or a Paclitaxel analog compared with when Paclitaxel or the Paclitaxel analog is administered alone. The compound and Paclitaxel (or Paclitaxel analog) can be co-administered to the subject as pan of the same pharmaceutical composition or, alternatively, as separate pharmaceutical compositions. When administered as separate pharmaceutical compositions, the compound or the present invention and Paclitaxel (or Paclitaxel analog) can be administered simultaneously or at different times, provided that the enhancing effect of the compound is retained.
The amount of compound and Paclitaxel (or Paclitaxel analog) administered to the subject will depend on the type and severity of the disease or condition and on the characteristics of the subject, such as general health, age, sex, body weight and tolerance to drugs. It will also depend on the degree, severity and type of cancer. The skilled artisan will be able to determine appropriate dosages depending on these and other factors. Effective dosages for Paclitaxel and Paclitaxel analog are well known and typically range from between about 1 mg/mm2 per day and about 1000 mg/mm2 per day, preferably between about 10 mg/mm2 per day and about 500 mg/mm2 per day. Effective amounts of a compound of the present invention typically range between about 1 mg/mm2 per day and about 10 grams/mm2 per day, and preferably between 10 mg/mm2 per day and about 5 grams/mm2.
The disclosed compounds are administered by any suitable route, including, for example, orally in capsules, suspensions or tablets or by parenteral administration, Parenteral administration can include, for example, systemic administration, such as by intramuscular, intravenous, subcutaneous, or intraperitoneal injection. The compounds can also be administered orally (e.g., dietary), topically, by inhalation (e.g., intrabronchial, intranasal, oral inhalation or intranasal drops), or rectally, depending on the type of cancer to be treated. Oral or parenteral administration are preferred modes of administration. Suitable routes of administration of Paclitaxel and Paclitaxel analogs are well known in the art and include by parenteral administration, as described above for the compounds of the present invention. Suitable routes of administration for Paclitaxel and analogs thereof are well known and include inter alia parenteral and oral administration.
The disclosed compounds can be administered to the subject in conjunction with an acceptable pharmaceutical carrier as part of a pharmaceutical composition for treatment of cancer. Formulation of the compound to be administered will vary according to the route of administration selected (e.g., solution, emulsion, capsule). Suitable pharmaceutical carriers may contain inert ingredients which do not interact with the compound. Standard pharmaceutical formulation techniques can be employed, such as those described in Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa. Suitable pharmaceutical carriers for parenteral administration include, for example, sterile water, physiological saline, bacteriostatic saline (saline containing about 0.9% mg/mi benzyl alcohol), phosphate-buffered saline, Hank's solution, Ringer's-lactate and the like. Methods for encapsulating compositions (such as in a coating of hard gelatin or cyclodextrasn) are known in the art (Baker, et al., “Controlled Release of Biological Active Agents”, John Wiley and Sons, 1986). Suitable formulations for Paclitaxel and Paclitaxel analogs are well known in the art.
The disclosed compounds can be prepared according to methods described in Examples 1-12 and also according to methods described in the co-pending U.S. Provisional Application entitled SYNTHESIS OF TAXOL ENHANCERS U.S. Provisional Application No. 60/304,318, filed Jul. 10, 2001. The entire teachings of this application are incorporated herein by reference.
The present invention is illustrated by the following examples, which are not intended to be limiting in any way.
EXEMPLIFICATION
Example 1




Preparation of Thiobenzoic acid N-methylhydrazino: Thiobenzoic acid N-methylhydrazide were prepared in 88% yield by slight modification of the prior art (Acta Chem. Scand. 1961, 1087-1096); 1H NMR (CDCl3) δ 3.3 (s, 3H), 6.0 (s, 2H), 7.3-7.4 (m, 5H); ESMS calcd (C8H10N2S): 166.1; found: 167.1 (M+H)+.
Example 2
Preparation of Thiobenzoic acid N-methylhydrazide: Bromobenzene (1.6 g, 10 mmol) was added into 25 ml anhydrous THF solution containing magnesium powder (0.3 g, 12.5 mmol), and refluxed for 2 hr. After it was cooled, the clear reaction solution was added into carbon disulfide (1 ml, 16.8 mmol) at 0° C., and stirred for 30 min at rt. The resulting mixture was then added into methylhydrazine (1.6 ml, 30 mmol) at 0° C., and stirred for another 2 hours. To this solution was added water (15 ml) and extracted with EtOAc (30 ml×3). The organic solution was concentrated to minimum volume, and subjected to silica gel column chromatography (eluant: 1:3-1:1 ethyl acetate: hexanes) to give thiobenzoic acid N1-methyl hydrazide (0.72 g, total yield: 48%). 1H NMR (CDCl3) δ 3.3 (s, 3H), 6.0 (s, 2H), 7.3-7.4 (m, 5H); ESMS calcd (C8H10N2S): 166.1; found: 167.1 (M+H)+.
Example 3




Preparation of 2,5-Dimethoxythiobenzoic acid N-methylhydrazine: DCC (4.5 g, 21.8 mmol) was added in one portion to a solution of 2,5-dimethoxybenzoic acid (3.6 g, 20 mol), methylhydrazine (1.2 ml, 23 mmol) and DMAP (30 mg, cat. ) in CH2Cl2 (60 ml) cooled in an ice bath. The reaction mixture was stirred overnight at room temperature. The slurry was cooled at −20° C. for 1 h and filtered. The CH2Cl2 solution was evaporated and the residue was dried in vacuum. The resulting crude product was dissolved in toluene (50 ml). To this solution was added Lawesson's reagent (5.8 g, 14 mmol). The mixture was refluxed for 40 min, cooled to room temperature, and directly subjected to silica gel column chromatography (eluent: 25% to 35% ethyl acetate in hexanes) to give the 2,5-dimethoxythiobenzoic acid N-methylhydrazide (3.7 g, yield: 82%) as off-white solid. 1H NMR (300 MHz, CDCl3): δ 6.88-6.80(m, 3H), 5.46 (s, 2H), 3.84(s, 3H), 3.82 (s, 3H), 3.28(s, 3H).
Example 4




Preparation of N-Malonyl-bis[N′-methyl-N′-(thiobenzoyl)hydrazide]: To a stirred solution of thiobenzoic acid N-methylhydrazide (0.166 g, 10 mmol), HOBt.H2O (0.15 g, 11 mmol) and malonic acid (0.052 g, 5 mmol) in DMF (2 mL) was added DCC (0.22 g, 10.7 mmol) at 0° C. The resultant suspension was stirred at 0° C. for 1 h and at room temperature for 3 h. Precipitated material was filtered off and washed with EtOAc (3×15 mL). Combined filtrate and washings was washed successively with H2O (2×20 mL), 5% citric acid (20 mL), H2O (20 mL), Saturated NaHCO3 (20 mL) and brine (20 mL). After being dried over Na2SO4, the solvent was removed under reduced pressure to afford the crude product as a yellow solid, which was washed with warm EtOAc. 0.16 g (yield 80%) of pure product was obtained as a yellow powder. Rf 0.3 (Hexane/EtOAc 1:1 v/v); 1H NMR (CDCl3) δ 3.1-3.8 (m, 6H), 3.4 (s, 2H), 7.1-7.45 (m, 10 H), 9.5-10.5 (m, 1H) ppm; ESMS calcd (C19H20N4O2S2): 400.1; found: 399.1 (M−H)+.
Preparation of N-(2-Methylmalonyl-bis{N′-methyl-N′-[(2,5-dimethoxy)thiobenzoyl]hydrazide]




DCC (4 g, 19 mmol) was added to a solution of 2,5-dimethoxythiobenzoic acid N-methylhydrazide (3.7 g, 16.4 mmol) and 2-methylmalonic acid (2 g, 17 mmol) in DMF (20 ml) with stirring at 0° C. The reaction mixture was stirred for 1 h at room temperature. The slurry was cooled at −20° C. for 1 h and filtered. The filtrate was diluted with EtOAc (300 ml), washed with water (50 ml×3), dried with Na2SO4. The EtOAc solution was concentrated to minimum volume, and subjected to silica gel colunm chromatography (eluent: 1:4 to 2:1, ethyl acetate: hexanes) to give the title compound (3.5 g, 80%) as yellow powder. 1H NMR (CDCl3) δ 10.12-9.14 (2H), 7.12-6.81 (m, 6H), 4.01-3.78(m, 6H), 3.75-3.22(m, 6H), 2.82-2.62(m, 1H), 1.12-0.11(m,3H); ESMS cacld (C24H30N4O6S2):534.16; found: 535.1 (M+H).
Example 5




Preparation of N-Malonyl-bis[N′-methyl-N′-(thiobenzoyl)hydrazide]: To a solution of thiobenzoic acid N-methylhydrazine (10 g) stirred at 0 C were added subsequently triethylamine (8.5 mL) and malonyl dichloride (3.05 mL). The reaction mixture was stirred for 10 min, washed with water (3×50 mL), dried over sodium sulfate and concentrated. Purification by recrystallization from methylene dichloride (35 mL) gave the product as light yellow crystals (9.0 g, 75%) which was identical to the product obtained in Example 6.
Example 6




Preparation of N-Malonyl-bis[N′-methyl-N′-(thiobenzoyl)hydrazide]: A stirred solution of thiobenzoic acid N-methylhydrazide (1.66 g, 10 mmol) and diphenyl malonate (1.30 g, 5.08 mmol) in dry THF (100 mL) was heated to reflux for 72 h. Volatile components were then removed under reduced pressure. The crude product was purified by column chromatography on silica gel using a mixture of hexane and EtOAc as eluant (gradient from 4:1 v/v to 1:1 v/v). 1.07 g (51% yield) of pure product N-malonyl-bis[N′-methyl-N′-(thiobenzoyl)hydrazide] was obtained as a yellow powder. Physical property was identical to that obtained in Example 5.
Example 7




A slurry of thiobenzoic acid N-methylhydrazide (1.0 g, 6 mmol), mono-tert-butyl malonate (1.0 mL, 6 mmol), HOBt.H2O (0.98 g, 7.2 mmol), and DCC (1.34 g, 6.5 mmol) in DMF (5 mL) was stirred at 0° C. for 3 h and then at room temperature for 3 h. Precipitated material was filtered off and washed with EtOAc (3×20 mL). Combined filtrate and washings was washed successively with H2O (2×20 mL), 5% citric acid (20 mL), H2O (20 mL), Saturated NaHCO3 (20 mL) and brine (20 mL). After being dried over Na2SO4, the solvent was removed under reduced pressure to afford the crude product as a solid, which was washed with Et2O. 0.94 g (yield 51%) of pure product N′-Methyl-N′-thiobenzoyl-hydrazinocarbonyl)-acetic acid tert-butyl ester was obtained as a yellow powder. 1H NMR (CDCl3) δ 1.6-1.7 (ds, 9H), 3.1-4.1 (m, 5 H), 7.3-7.7 (m, 5H), 9.7-10.3 (ds, 1H) ppm; ESMS calcd (C15H20N2O3S): 308; found: 307 (M−H)+.
A solution of N′-methyl-N′-thiobenzoyl-hydrazinocarbonyl)-acetic acid tert-butyl ester (0.19 g, 0.6 mmol) and TFA (0.12 mL, 1.6 mmol) in dry DCM (10 mL) was stirred at 10° C.-15° C. for 12 h (reaction was monitored by TLC). Volatile components were removed under reduced pressure (bath temperature below 5° C.). After being dried in vacuo, DMF (3 mL) was added followed by the addition of DCC (0.13 g, 0.6 mmol), HOBt.H2O (93 mg, 0.7 mmol) and thio-2,5-dimethoxybenzoic acid N-methylhydrazide (0.13 g, 0.57 mmol). The resultant solution was stirred at 0° C. for half an hour and then at room temperature for 3 h. Precipitated material was filtered off and washed with EtOAc (3×10 mL). Combined filtrate and washings was washed successively with H2O (2×10 mL), 5% citric acid (10 mL), H2O (10 mL), Saturated NaHCO3 (20 mL) and brine (20 mL). After being dried over Na2SO4, the solvent was removed under reduced pressure to afford the crude product as an oil, which was purified by SGC (4:1 hexane/EA to 2:1 EtOAc/Hexane). 0.14 g (yield 53%) of pure product was obtained as a yellow powder. 1H NMR (CDCl3) δ 3.1-3.9 (m, 18H), 6.7-7.4 (m, 9H) ppm; ESMS calcd (C21H24N4O4S2): 460.1; found: 461.1 (M+H)+.
Example 8




Preparation of N—N-malonyl-bis[N′-phenyl-N′-(thiobenzoyl)hydrazide]




A mixture of phenylhydrazine (30 mL) and ethyl malonate (in xylene (150 mL) was heated to reflux overnight. The reaction was cooled to room temperature. The precipitates were collected via filtration and washed with ethanol to give N-malonyl-bis(N′-phenylhydrazide) as a white solid (14 g). The hydrazide (3.4 g) was suspended in benzoic anhydride (50 g) with warming. To it was added dropwise perchloric acid (57% in water, 3 mL). The reaction mixture turned to clear solution initially and then quickly solidified. After standing at room temperature for 1 h, ether (50 mL) was added. The resulting slurry was filtered and washed with ether (2×00 mL) to give the perchlorate salts as a white solid (5.7 g). The salts were taken into acetone and added as a slurry over 5 min to Na2S (0.6 M in water, 90 mL) stirred at room temperature. After 30 min, the reaction was acidified with HCl(c) to afford a yellow slurry. The solid was collected via filtration and washed with water (20 mL) and ether (2×25 mL) to give N-malonyl-bis[N′-phenyl-N′-(thiobenzoyl)hydrazide] as an off-white solid (3.6 g). 1H NMR (CDCl3): δ 7.2 (m, 20H); 3.5 (br s, 2H). MS calcd for C29H24N4O2S2: 524.13: Found: 525.1 (M+H).
Example 9




Preparation of N-Malonyl-bis[N-methyl-N′-phenyl-N′-(thiobenzoyl)hydrazide]
To a stirred solution of N-malonyl-bis[N′-phenyl-N′-(thiobenzoyl)hydrazide] (180 mg, 0.34 mmol), MeOH (22 uL) and triphenylphosphine (200 mg, 0.64 mmol) in dry THF (10 mL) was added a solution of DEAD (0.12 mL) in THF (3 mL) dropwise. The resultant orange solution was stirred at room temperature for 12 h. After removal of the volatile components, the crude product was purified by SGC (3:1 Hexane/EtOAc) to afford 98 mg (52% yield) of the title compound as syrup. 1H NMR (CDCl3) δ 3.3-4.5 (m, 8H), 7.1-7.8 (m, 20 H) ppm; ESMS calcd (C31H28N4O2S2): 552; found: 551 (M−H)+.
Example 10




A stirred mixture of N-malonyl-bis[N′-phenyl-N′-(thioacetyl)hydrazide) starting material (0.1 g, 0.25 mmol) and Lawesson's reagent (0.15 g, 0.37 mmol) in dry benzene (20 mL) was heated to reflux for 1 h. After being cooled to room temperature, the mixture was filtered through a layer of silica gel, washed with THF (2×15 mL). The filtrate and washings were combined and concentrated under reduced pressure. Flush column chromatography on silica gel (hexane to 4:1 hexane/EtOAc to 2:1 hexane/EtOAc) afforded N-bisthiomalonyl-bis[N′-phenyl-N′-(thioacetyl)hydrazide as a clear syrup (16 mg, 15%).
1H NMR (CDCl3) δ 3.80-3.95 (m, 8H), 7.02-7.30 9m, 10 H). ESMS calcd (C19H20N4S4): 432.06; found: 433.0 (M+H)+.
Example 11
The compounds shown below were prepared by the procedures described above. Analytical data is provided for these compounds.




1H NMR (CDCl3) δ 3.1-3.8 (m, 6H), 3.4 (s, 2H), 7.1-7.45 (m, 10 H), 9.5-10.5 (m, 1H) ppm; ESMS calcd (C19H20N4O2S2): 400.1; found: 399.1 (M−H)+.




1H NMR (CDCl3) δ 1.0-1.35 (m, 6H), 3.0-4.3 (m, 6H), 7.05-7.40 (m, 10H), 9.1-10.1 (m, 2H); ESMS cacld (C21H24N4O2S2): 428.8; found: 427 (M−H)+. Anal Calc For C21H24N4O2S2 (428.13) C, 58.85; H, 5.64; N, 13.07; S, 14.96. Found: C, 58.73; H, 5.62; N, 12.97; S, 14.96.




1H NMR (CDCl3) δ 0.7-1.0 (m, 6H), 1.4-1.9 (m, 4H), 3.1-4.2 (m, 6H), 7.1-7.4 (m, 10H), 8.9-10.2 (m, 2H) ppm; ESMS (C23H28N4O2S2): 456.1; found: 455.1 (M−H)+.




mp 141-143° C.; 1H NMR (CDCl3) δ 0.6-1.05 (m, 6H), 1.1-1.9 (m, 8H), 3.0-4.2 (m, 6H), 7.0-7.35 (m, 10H), 8.9-11 (ms, 2H). ESMS (C25H32N4O2S2): 484.2; found: 483.1 (M−H)+. Anal Calc For C25H32N4O2S2 (484.2) C, 61.95; H, 6.65; N, 11.56; S, 13.23. Found: C, 61.98; H, 6.52; N, 11.26; S, 13.16.




1H NMR (DMSO-d6) δ 0.4-0.9 (dd, 3H, J=7), 2.7 (q, 1H), 3.1-3.6 (m, 6H), 7.1-7.5 (m, 10H), 10.9 (br, 2H) ppm; ESMS (C20H22N4O2S2): 414; found: 413 (M−H)+.




1H NMR (CDCl3) δ 0.5 (t, 3H, J=7), 1.1-1.6 (m, 2H), 2.7 (t, 1H, J=7), 3.1-3.3 (m, 6H), 7.0-7.3 (m, 10H), 10.25 (s, 2H) ppm; MS (C21H24N4O2S2): 428.1; found: 427.1 (M−H)+.




1H NMR (CDCl3) δ 0.5 (d, 6H, J=7), 0.9-1.2 (m, 1H), 3.0-41 (m, 7H), 7.1-7.4(M, 10H), 10.3 (s, 2H) ppm; ESMS (C22H26N4O2S2): 442.1; found: 441.1 (M−H)+.




1H NMR (CDCl3) δ 0.4-1.3 (m, 5H), 1.5-1.8 (m, 2H), 3.0-3.7 (m, 6H), 7.1-7.5 (m, 10H), 11 (s, 2H) ppm; ESMS (C23H28N4O2S2): 456.1; found: 455.1 (M−H)+.




1H NMR (CDCl3) δ 2.1 (d, 2H, J=7), 2.9 (t, 1H, J=7), 3.1-3.5 (m, 6H), 6.8-7.4 (m, 15 H), 11 (s, 2H) ppm; ESMS (C26H26N4O2S2): 490.1; found: 489.1 (M−H)+.




1H NMR (CDCl3) δ 0.4 (d, 3H, J=7), 1.0-1.4 (m, 6H), 2.75 (q, 1H), 3.0-4.3 (m, 4H), 7.1-7.4 (m, 10H), 10.6 (s, 2H); ESMS Calc For (C22H26N4O2S2): 442.1; found: 441.1 (M−H)+; Anal Calc For C22H26N4O2S2 (442.15) C, 59.70; H, 5.92; N, 12.66; S, 14.49. Found: C, 59.64; H, 5.92; N, 12.59; S, 14.47.




1H NMR (DMSO-d6) δ 3.20 (br, 2H), 7.1-7.6 (m, 20 H), 11.5 (s, 2H) ppm; ESMS calcd (C29H24N4O2S2): 524.1; found: 523.1 (M−H)+.




1H NMR (CDCl3) δ 3.0-4.3 (m, 14H), 6.6-7.5 (m, 8H), 10.4 (s, 2H) ppm; ESMS calcd (C21H24N4O2S2): 460.2; found: 461.2 (M+H)+.




1H NMR (CDCl3) δ 2.65-3.60 (m, 8H), 7.2-7.4 (m, 8H), 11.1 (br, 2H); ESMS calcd (C19H18C12N4O2S2): 468.0; found: 467.9 (M−H)+.




1H NMR (CDCl3) δ 0.4 (d, 3H, J=7), 2.7 (q, 1H, J=7), 3.0-3.8 (m, 6H), 7.2-8.2 (m, 8H), 10.5-10.7 (ms, 2H) ppm; ESMS calcd (C20H20Cl2N4O2S2): 482.0; found: 481.0 (M−H)+.




1H NMR (CDCl3) δ 2.9-3.8 (m, 6H), 7.3-7.7 (m, 4H), 8.0-8.3 (m, 4H), 10.9 (s, 2H); ESMS calcd (C10H18N6O6S2): 490.0; found: 489.0 (M−H)+.




1H NMR (CDCl3) δ 3.1-3.9 (m, 14H), 6.7-7.8 (m, 8H), 9.0-10 (m, 2H) ppm; ESMS calcd (C21H24N4O4S2): 460.1; found: 459.1 (M−H)+.




(SBR-11-5032): 1H NMR (CDCl3) δ 3.0-3.9 (m, 14H), 6.7-7.3 (m, 8H), 9.0-10 (m, 2H) ppm; ESMS calcd (C21H24N4O4S2): 460.1; found: 459.1 (M−H)+.




1H NMR (acetone-d6) δ 3.5 (s, 2H), 6.45 (d, 2H, J=5), 6.9 (d, 2H, J=5), 7.2-7.6 (m, 12H), 10.6 (s, 2H) ppm; ESMS calcd (C25H20N4O4S2): 504.1; found: 503.1 (M−H)+.




1H NMR (DMSO-d6) δ 2.60 (s, 6H), 3.05 (s, 6H), 3.40 (s, 2H), 7.15-7.50 (m, 8H) ppm; ESMS calcd (C27H24Cl2N6O4S2): 630.1; found: 629.1 (M−H)+.




1H NMR (CDCl3) δ 10.06-8.82 (2H), 7.16-6.81(m,6H), 4.01-3.81(m, 6H), 3.78-3.11(m,6H), 2.81-2.58(m,2H): ESMS cacld (C23H28N4O6S2): 520.15; found: 521 (M+H).




1H NMR (CDCl3) δ 10.38-9.01 (2H), 7.12-6.82 (m, 6H), 3.92-3.78(m, 12H), 3.75-3.06(m, 6H), 2.61-2.51 (m, 2H); ESMS cacld (C23H28N4O6S2): 520.15; found: 521 (M+H).




1H NMR (CDCl3) δ 9.45-8.63 (2H), 7.18-6.81 (m, 6H), 4.01-3.80(m, 6H), 3.78-3.24(m, 6H), 2.62-2.50(m, 1H), 1.74-0.11 (m, 3H); ESMS cacld (C24H30N4O6S2): 534.16; found: 535 (M+H).




1H NMR (CDCl3) δ 10.19-8.61 (2H), 7.26-6.52(m, 6H), 3.81-3.08(m, 8H), 3.01-2.88(m, 12H). ESMS cacld (C23H30N6O2S2): 486.19; found: 487 (M+H).




1H NMR (CDCl3) δ 9.92-8.80 (2H), 7.41-6.72 (m, 6H), 4.01-3.81(m,6H), 3.80-3.15 (m,6H), 2.76-2.42(m, 2H); ESMS cacld (C21H22Cl2N4O4S2): 528.05; found: 529(M+H).




1H NMR (CDCl3) δ 10.21-9.02(2H), 7.60-6.81 (m, 6H), 4.14-3.88(m, 6H), 3.87-3.18 (m,6H), 2.84-2.65(m, 1H),1.10-0.16 (m, 3H); ESMS cacld (C22H24Cl2N4O4S2): 542.06; found: 543(M+H).




1H NMR (CDCl3) δ 10.02-9.20 (2H), 7.63-7.01 (m, 6H), 4.21-3.22(m, 6H), 1.88-1.36 (m, 2H); ESMS cacld (C19H16F4N4O2S2): 472.07; found: 473 (M+H).




1H NMR (CDCl3) δ 7.93-7.61 (2H), 7.40-6.92 (m, 6H), 3.98-3.41 (m, 6H), 2.19-0.93 (m, 4H); ESMS cacld (C20H18F4N4O2S2): 486.08; found: 487 (M+H).




1H NMR (CDCl3) δ 10.12-9.21(2H), 7.67-7.23 (m, 6H), 3.94-3.22 (m, 6H), 2.01-1.21 (m, 2H); ESMS cacld (C19H16Cl4N4O2S2): 535.95; found: 537(M+H).




1H NMR (CDCl3) δ 7.78-7.23 (2H), 4.56-3.10 (m, 6H), 2.34-1.12 (m, 4H); ESMS cacld (C20H18Cl4N4O2S2): 549.96; found: 551 (M+H).




1H NMR (CDCl3) δ 9.92-9.01 (2H), 7.38-7.15 (m,3H), 6.66-6.51 (m,3H), 3.98-3.75 (m,12H), 3.72-3.21(m,6H), 2.01-0.42 (m, 4H); ESMS cacld (C24H30N4O6S2): 534.16; found: 535 (M+H).




1H NMR (CDCl3) δ 10.51-9.82 (2H), 7.42-6.80 (m, 6H), 3.92-3.04(m, 6H), 2.60-1.21 (m, 14H); ESMS cacld (C23H28N4O2S2): 456.17; found: 457(M+H).




1H NMR (CDCl3) δ 10.51-8.82 (2H), 7.11-6.89 (m, 6H), 3.81-3.02 (m, 6H), 2.40-1.02 (m, 16H); ESMS cacld (C24H30N4O2S2): 470.18; found: 471(M+H).




1H NMR (CDCl3) δ 9.86-8.42 (2H), 7.01-6.6 (m, 6H), 4.18-3.51 (m, 16H), 3.22-2.26 (2H), 1.40-1.04 (m, 6H); ESMS cacld (C25H32N4O6S2):548.18; found: 547 (M−H).




1H NMR (CDCl3) δ 9.99-8.41 (2H), 7.01-6.68 (m, 6H), 4.18-3.56 (m, 16H), 1.40-0.02 (m, 10H); ESMS cacld (C26H34N4O6S2): 562.19; found: 561(M−H).




1H NMR (CDCl3) δ 10.12-8.82 (2H), 7.03-6.62 (m, 6H), 4.21-3.87 (m, 8H), 3.84-3.01(m, 6H), 2.71-2.42 (m, 2H), 1.56-1.21 (m, 12H); ESMS cacld (C27H36N4O6S2): 576.21; found: 577(M+H).




1H NMR (CDCl3) δ 9.81-8.79 (2H), 7.01-6.64 (m, 6H), 4.21-3.81(m, 8H), 3.80-3.22 (m, 6H), 1.54-1.20 (m, 13H), 1.01-0.16 (m, 3H); ESMS cacld (C28H38N4O6S2): 590.22; found: 591(M+H).




1H NMR (DMSO-d6): δ 8.25 (d, J=8.1 Hz, 4H), 7.50 (d, J=8.1 Hz, 4H), 3.7-3.3 (m, 8H); ESMS cacld for C19H18N6O6S2: 490.1; Found: 489.0 (M−H).




1H NMR (CDCl3): δ 10.25 (m, 2H), 7.7-7.4 (m, 8H), 3.7 (m, 2H), 3.35 (m, 6H); ESMS cacld for C21H18N6O2S2: 450.1; Found: 449.0 (M−H).




1H NMR (CDCl3): δ 8.2 (s, 2H), 7.7-7.5 (m, 4H), 3.7-3.4 (m, 8H), 2.9-2.8 (m, 6H); ESMS cacld for C19H22N6O2S2: 430.1; Found: 431.1 (M+H).




1H NMR (CDCl3): δ 10.0-9.2 (m, 2H), 7.9-7.45 (m, 8H), 4.0-3.4 (m, 8H); ESMS cacld for C21H18N6O2S2: 450.1; Found: 451.0 (M+H).




1H NMR (CDCl3): δ 10.1-9.4 (2H), 7.5-7.2 (m, 8H), 3.9-3.3 (m, 8H); ESMS cacld for C19H18F2N4O2S2: 436.1; Found: 437.1 (M+H).




1H NMR (CDCl3): δ 3.3 (s, 2H), 3.6 (s, 6H), 5.25 (s, 4H), 7.05-7.3 (m, 16H), 7.6 (s, 2H), 7.9 (d, 2H, J=6), 10.56 (s, 2H)ppm; ESMS calcd (C37H34N6O2S2): 658.2; found: 659.2 (M+H)+.




1H NMR (DMSO) δ 11.98 (2H), 7.44-7.12 (m, 10H), 3.69-3.14(s, 6H). ESMS cacld (C18H18N4O2S2): 386.09: found: 387.1 (M+H).




1H NMR (CHCl3) δ 9.48-8.55 (2H), 7.56-7.20(m, 1OH), 3.80-3.31(m, 6H), 2.88-2.22(m, 4H). ESMS cacld (C20H22N4O2S2): 414.12; found: 415.1 (M+H).




1H NMR (300 MHz, CDCl3) δ 10.21-9.91 (m, 2H), 8.06-7.32 (m, 14H), 3.91-3.56 (m, 6H). ESMS cacld (C24H22N4O2S2): 462.12; found: 463 (M+H).




1H NMR (300 MHz, DMSO-d6) δ 11.60-11.40 (m, 2H), 7.48-6.46 (m, 12H), 3.64-3.3.30(m, 6H). ESMS cacld (C20H20N4O2S2): 412.10; found: 413 (M+H).




1H NMR (300 MHz, CDCl3) δ 7.58-7.20 (m, 12H), 3.68-3.20 (m, 6H). ESMS cacld (C20H20N4O2S2): 412.10; found: 413 (M+H).




1H NMR (300 MHz, CDCl3): δ 9.65-8.70 (2H), 8.01-7.21(m, 14H), 3.84-3.40(m, 6H). ESMS cacld (C24H22N4O2S2): 462.12: found: 463 (M+H).




1H NMR (CDCl3): δ 7.2 (m, 18H); 3.5 (br s, 2H); 2.4 (br s, 6H). MS calcd for C31H28N4O2S2: 552.2: Found: 553.2 (M+H).




1H NMR (CDCl3): δ 7.5 (br s, 18H), 3.4 (br s, 2H), 2.45 (s, 6H). ESMS cacld for C33H28N4O6S2: 640.1; Found 641.1 (M+H).




1H NMR (CDCl3-D2O): δ 7.45-7.15 (m, 20 H), 1.6 (br s, 6H). ESMS cacld for C31H28N4O2S2: 552.2; Found: 553.2 (M+H).




1H NMR (DMSO-d6): δ 11.3 (s, 2H), 7.75 (d, J=6.0 Hz, 2H), 7.5-7.4 (m, 12 H); 6.9 (m, 2H); ESMS cacld for C27H24N4O2S4: 564.1; Found: 565.2 (M+H).




1H NMR (300 MHz, CDCl3): δ 10.18-8.60 (m, 2H), 7.26-6.46 (m, 8H), 3.80-3.02(m, 6H), 3.00-2.80(m, 12H). 1.78-1.56(m, 2H). ESMS cacld (C23H30N4O2S2): 486.19; found: 487 (M+H).




1H NMR (300 MHz, DMSO): δ 10.90-10.81 (m, 2H), 7.50-7.21 (m, 10H), 3.78-3.36(m, 6H), 2.64-0.50(m, 10H). ESMS cacld (C20H28N4O2S2): 456.17; found: 457 (M+H).




1H NMR (300 MHz, CDCl3): δ 10.00-9.71 (m, 2H), 7.72-7.21(m, 8H), 3.80-3.26(m, 6H). ESMS cacld (C20H16N6O2S2): 436.08; found: 437 (M+H).




1H NMR (300 MHz, CDCl3): δ 10.60-9.41 (m, 2H), 7.15-6.23(m, 6H), 3.89-3.28(m, 6H), 3.76(S, 12H). ESMS cacld (C22H28N4O6S2): 506.13; found: 507 (M+H).




1H NMR (300 MHz, DMSO): δ 7.40-7.12 (m, 10H), 3.70-2.80(m, 6H), 1.84-0.72(m, 16H). ESMS cacld (C26H34N4O2S2): 498.21; found: 499 (M+H).




1H NMR (300 MHz, CDCl3): δ 10.42-9.53 (m, 2H), 7.55-6.87(m, 8H), 3.99-3.28(m, 6H), ESMS cacld (C18H10N4F2O2S2): 422.07; found: 423 (M+H).




1H NMR (300 MHz, DMSO): δ 12.08 (br. 2H), 8.27-7.24 (m, 8H), 3.70-3.15(m, 6H). ESMS cacld (C18H16N6O6S2): 476.06; found: 477 (M+H).




1H NMR (300 MHz, CDCl3): δ 10.12-9.83 (m, 2H), 7.15-6.63(m, 6H), 3.99-2.91(m, 6H), ESMS cacld (C22H26N4O6S2): 506.13; found: 507 (M+H).




1H NMR (300 MHz, DMSO): δ 11.12-10.54 (m, 2H), 8.27-7.18 (m, 10H), 4.26-3.72(m, 2H), 3.37-3.18(m, 2H). ESMS cacld (C17H16N4O2S2): 372.07; found: 371 (M−H).




1H NMR (300 MHz, DMSO): δ 11.52 (br, 2H), 7.95-7.33(m, 10H), 3.42-3.22(m, 6H), 2.48(m, 2H). ESMS cacld (C23H20N4O2S4): 512.05; found: 513 (M+H).




1H NMR (300 MHz, CDCl3): δ 7.81-7.28(m, 8H), 3.82(s, 6H). ESMS cacld (C22H18N4O2S4): 498.03; found: 499 (M+H).




1H NMR (300 MHz, CDCl3): δ 10.02-9.11 (m, 2H), 8.16-7.28(m, 8H), 3.99-3.08(m, 6H), 2.90-1.20(m, 2H). ESMS cacld (C23H24N4O6S2): 516.1 1; found: 517 (M+H).




1H NMR (300 MHz, DMSO): δ 7.99 (m, 8H), 8.16-7.28(m, 8H), 3.80-3.14(m, 6H), 1.80-1.21(m, 2H). ESMS cacld (C21H20N4O6S2): 488.08; found: 487 (M−H).




1H NMR (300 MHz, CDCl3): δ 10.82-10.55 (m, 2H), 7.91-7.29(m, 10H), 3.64-3.11(m, 6H), 1.90-1.40(m, 2H). ESMS cacld (C19H20N4O2S2): 400.19; found: 399 (M−H).
Example 12
Compound (1) Enhances the Anti-Cancer Activity of Paclitaxel In Vivo
General Procedure of In Vivo Anti-Tumor Study
The in vivo anti-cancer enhancing effect of novel compounds was assessed in tumor bearing mice using the tumor growth inhibition assay. Tumor cells were implanted by injection of a tumor cell suspension subcutaneously in the flank of a mouse. Treatment of the tumor with an experimental compound and Paclitaxel begun after the tumor had been established (volume was about 100 mm3). Animal then begun a multiple injection schedule where the compound and Paclitaxel were given by IV route of administration. Tumors were measured two times a week. During the course of this assay, animals were monitored daily for signs of toxicity including body weight loss.
Procedure
A supplemented media was prepared from 50% DMEM/Dulbecco Modified Eagle Medium (High Glucose), 50% RPMI 1640, 10% FBS/Fetal Bovine Serum (Hybridoma Tested; Sterile Filtered), 1% L-Glutamine, 1% Penicillin-Streptomycin, 1% MEM Sodium Pyruvate and 1% MEM Non-Essential Amino Acids. FBS was obtained from Sigma Chemical Co. and other ingredients were obtained from Invitrogen Life Technologies, USA). The supplemental media was warmed to 37° C. and 50 ml of media was added to a 175 cm2 tissue culture flask.
The cells used in the assay were MDA-435 Human Breast Carcinoma from the American Type Culture Collection. 1 vial of MDA-435 cells from the liquid nitrogen frozen cell stock was removed. The frozen vial of cells was immediately placed into a 37° C. water bath and gently swirled until thawed. The freeze-vial was wiped with 70% ethanol and cells were immediately pipetted into the 175 cm2 tissue culture flask containing supplemented media. The cells were incubated overnight and the media was removed and replaced with fresh supplemented media the next day. The flask was incubated until flask became about 90% confluent. This took anywhere from 5-7 days.
The flask was washed with 10 ml of sterile room temperature phosphate buffered saline (PBS). The cells were trypsinized by adding 5 ml of warmed Trypsin-EDTA (Invitrogen) to the flask of cells. The cells were then incubated for 2-3 minutes at 37° C. until cells begun to detach from the surface of the flask. An equal volume of supplemented media (5 ml) was added to the flask. All the cells were collected into 50 ml tube, and centrifuged at 1000 RPM for 5 minutes at 20° C. The supernatant was aspirated and the cell pellet was resuspended in 10 ml of supplemented media and the cells were counted. 1-3 million cells/flask were seeded into 5-7 tissue culture flasks (175 cm2). Each flask contained 50 ml of supplemented media. The flasks were incubated until about 90% confluent. The passaging of the cells was repeated until enough cells have been grown for tumor implantation.
The above procedure for trypsinizing and centrifuging the cells were followed. The supernatant was aspirated and the cell pellet was resuspended in 10 ml of sterile PBS and the cells were counted. The cells were centrifuged and then resuspended with appropriate volume of sterile PBS for injection of correct number of cells needed for tumor implantation. In the case of MDA-435, 100 million cells were suspended with 2.0 ml of sterile PBS to a final concentration of 50 million cells/ml in order to inject 5 million cells in 0.1 ml/mouse.
Mice (CD-1 nu/nu) were obtained from Charles River Laboratories: nomenclature: Crl:CD-1-nuBR, Age: 6-8 weeks. The mice were allowed to acclimate for 1 week prior to their being used in an experimental procedure.
Implantation of the MDA-435 tumor cell suspension took place into the corpus adiposum of the female CD-1 nu/nu mouse. This fat body is located in the ventral abdominal viscera of the mouse. Tumor cells were implanted subsutaneously into the fat body located in the right quadrant of the abdomen at the juncture of the os coxae (pelvic bone) and the os femoris (femur). 5 million MDA-435 cells in 0.1 ml of sterile PBS were injected using 27 G (1⁄2 inch) needle. MDA-435 tumors developed 2-3 weeks after implantation.
Compound stock solutions were prepared by dissolving the compound in cell-culture-grade DMSO (dimethyl sulfoxide) at the desired concentration. This stock solution in DMSO was sonicated in an ultrasonic water bath until all the powder dissolved.
The Formulation Solvent was prepared as follows: 20% of Cremophore RH40 (Polyoxyl 40 Hydrogenated Castor Oil obtained from BASF corp.) in water was prepared by first heating 100% Cremophore RH40 in a water bath at 50-60° C. until it liquefied and became clear. 10 ml of the 100% Cremophore RH40 aliquoted into a conical centrifuge tube containing 40 ml of sterile water (1:5 dilution of Cremophore RH40). The 20% Cremophore RH40 solution was reheated until it became clear again, and mixed by inverting the tube several times. This 20% Cremophore RH40 solution was stored at room temperature, and was kept for up to 3 months.
Preparation of Dosing Solution for Compound Administration: The compound stock solution was diluted 1:10 with 20% Cremophore RH40: 1) 2.0 ml of 10 mg/ml dosing solution of Compound (1) was prepared by diluting 100 mg/ml Compound Stock solution with 1.8 ml of 20% Cremophore RH40 water solution; and 2) a dosoing solution comprising 2.0 ml of 1 mg/ml of Paclitaxel (obtained from Sigma Chemical Co.) and 5 mg/ml of Compound (1) was obtained by mixing 0.1 ml of Compound 1 DMSO stock solution (50 mg/ml) and 0.1 ml of Paclitaxel DMSO stock solution (10 mg/ml) and diluting with 1.8 ml of 20% Cremophore RH40 water solution. The final formulation for the dosing solution was 10% DMSO, 18% Cremophore RH40 and 72% water.
The Dosing Solution (Dosing Volume: 0.01 ml/gram=10 ml/kg) was injected intravenously into the mice bearing MDA-435 human breast tumor.










PROTOCOL









Group
Compounds
(Dose)





1
Vehicle Only



2
Paclitaxel
(5 mg/kg)


3
Compound (1)
(50 mg/kg)


4
Paclitaxel
(5 mg/kg) + Compound (1) (25 mg/kg)


5
Paclitaxel
(5 mg/kg) + Compound (1) (50 mg/kg)









Dosing Schedule: 3 times a week (Monday, Wednesday, Friday) for 3 weeks 5 mice were used for each group
Results
FIG. 1 shows the effects of Compound (1) on enhancing anti-tumor activity of Paclitaxel (TAXOL). As can be seen from FIG. 1, Compound (1) significantly enhanced anti-tumor activity of Paclitaxel on human breast tumor MDA-435 in nude mice. FIG. 2 shows the effects of Compound (1) and Paclitaxel on the body weight of nude mice bearing MDA-435 human breast tumor. As can be seen from FIG. 2. Compound (1) significantly enhanced anti-tumor activity of Paclitaxel without increasing toxicity.
Example 13
Compounds (1) and (2) Enhance the Anticancer Activity of Paclitaxel In Vivo




The protocol described in Example 12 was used to test Compounds (1) and (2) for their ability to enhance the anti-cancer activity of paclitaxel in mice, except as modified as described below.










PROTOCOL









Group
Compounds
(Dose)





1
Vehicle Only



2
Paclitaxel
(2 mg/kg)


3
Paclitaxel
(5 mg/kg)


4
Compound (1)
(80 mg/kg)


5
Compound (2)
(80 mg/kg)


6
Paclitaxel
(2 mg/kg) + Compound (1) (80 mg/kg)


7
Paclitaxel
(5 mg/kg) + Compound (1) (80 mg/kg)


8
Paclitaxel
(2 mg/kg) + Compound (2) (80 mg/kg)


9
Paclitaxel
(5 mg/kg) + Compound (2) (80 mg/kg)









Dosing Schedule: 3 times a week (Monday, Wednesday, Friday) for 3 weeks 5 mice were used for each group.











RESULTS










Average Tumor




Volume (mm3)


Group
on Day 23
% Tumor Growth












1
301.3
100


2
259.8
86


3
164.8
55


4
270.0
90


5
305.8
101


6
193.3
64


7
106.2
35


8
148.4
49


9
60.6
20









Compounds (1) and (2) significantly enhanced anti-tumor activity of Paclitaxel at both of 2 mg/kg and 5 mg/kg without increasing toxicity.
Example 14
Compound (1) Enhances the Anticancer Activity of Paclitaxel In Vivo
The protocol described in Example 12 was used to test Compound (1) for its ability to enhance the anti-cancer activity of paclitaxel in mice, except modified as described below.










PROTOCOL









Group
Compounds
(Dose)





1
Vehicle Only



2
Paclitaxel
(10 mg/kg)


3
Compound (1)
(50 mg/kg)


4
Paclitaxel
(10 mg/kg) + Compound (1) (25 mg/kg)









Dosing Schedule:
3 times a week (Monday, Wednesday, Friday) for 3 weeks
5 mice were used for each group










RESULTS










Average Tumor
% Tumor Growth


Group
Volume [mm3]
Inhibition on Day 48












1
752.2
—


2
105.4
86%


3
754.9
 0%


4
0.59
>99.9%    









When 10 mg/kg of Paclitaxel was used, significant anti-tumor activity was observed. However, after drug treatment (Day 1 ̃20) terminated, the tumor started growing to become 105 mm3 volume on Day 43. On the other hand, average tumor volume after treatment of Paclitaxel (10 mg/kg) plus Compound (1) (25 mg/kg) was only 0.59 mm3 with more than 99.9% tumor growth inhibition.
Example 15
Compounds (3)-(5) Enhance the Anticancer Activity of Paclitaxel In Vivo




The protocol described in Example 12 was used to test Compounds (3)-(5) for their ability to enhance the anti-cancer activity of paclitaxel in mice, except modified as described below.










PROTOCOL









Group
Compounds
(Dose)





1
Vehicle Only



2
Paclitaxel
(5 mg/kg)


3
Paclitaxel
(5 mg/kg) + Compound (3) (50 mg/kg)


4
Paclitaxel
(5 mg/kg) + Compound (4) (100 mg/kg)


5
Paclitaxel
(5 mg/kg) + Compound (5) (100 mg/kg)









Dosing Schedule:
3 times a week (Monday, Wednesday, Friday) for 3 weeks
5 mice were used for each group










RESULTS











Average % Tumor



Group
Growth Inhibition on Day 27







2
19



3
76



4
66



5
79










Compounds (3)-(5) demonstrated significant enhancing effects of Paclitaxel anti-tumor activity.
While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

## Claims

1. A method of treating a subject with melanoma, said method comprising administering to the subject an effective amount of paclitaxel or a paclitaxel analog and an effective amount of a compound represented by the following structural formula:




or a pharmaceutically acceptable salt thereof, wherein:
Y is a covalent bond, phenylene group or a substituted or unsubstituted hydrocarbyl group, or, Y, taken together with both >C═Z groups to which it is bonded, is a substituted or unsubstituted aromatic group;
R1 and R2 are independently an aryl group or a substituted aryl group;
R3 and R4 are independently —H, an aliphatic group, a substituted aliphatic group, an aryl group or a substituted aryl group;
R5-R6 are independently —H, an aliphatic group, a substituted aliphatic group, an aryl group or a substituted aryl group;
and Z is ═O or ═S.

2. The method of claim 1 wherein Y is a covalent bond, phenylene group or a substituted or unsubstituted hydrocarbyl group, or, Y, taken together with both >C═Z groups to which it is bonded, is a substituted or unsubstituted aromatic group.

3. The method of claim 2 wherein Y, taken together with both >C═Z groups to which it is bonded, is a substituted or unsubstituted aromatic group.

4. The method of claim 3 wherein the compound is represented by the following structural formula:




or a pharmaceutically acceptable salt thereof wherein Ring A is substituted or unsubstituted and W is —CH— or —N—.

5. The method of claim 2 wherein Y is a covalent bond or a substituted or unsubstituted straight chained hydrocarbyl group.

6. The method of claim 2 wherein the compound is represented by the following structural formula:




or a pharmaceutically acceptable salt thereof, wherein Y′ is a covalent bond or —CR7R8— and R7 and R8 are each independently —H, an aliphatic or substituted aliphatic group, or R7 is —H and R8 is a substituted or unsubstituted aryl group, or, R7 and R8 taken together, are a C2—C6 substituted or unsubstituted alkylene group.

7. The method of claim 6 wherein the paclitaxel analog is represented by a structural formula selected from:




wherein:
R10 is a lower alkyl group, a substituted lower alkyl group, a phenyl group, a substituted phenyl group, —SR19, —NHR19 or —OR19;
R11 is a lower alkyl group, a substituted lower alkyl group, an aryl group or a substituted aryl group;
R12 is —H, —OH, lower alkyl, substituted lower alkyl, lower alkoxy, substituted lower alkoxy, —O—C (O)-(lower alkyl), —O—C(O)-(substituted lower alkyl), —O—CH2—O-(lower alkyl)—S—CH2—O-(lower alkyl);
R13 is —H, —CH3, or, taken together with R14, —CH2—;
R14 is —H, —OH, lower alkoxy, —O—C(O)-(lower alkyl), substituted lower alkoxy, —O—C(O)-(substituted lower alkyl), —O—CH2—O—P(O)(OH)2, —O—CH2—O-(lower alkyl), —O—CH2—S-(lower alkyl) or, taken together with R20, a double bond;
R15 —H, lower acyl, lower alkyl, substituted lower alkyl, alkoxymethyl, alkthiomethyl, —OC(O)—O(lower alkyl), —OC(O)—O(substituted lower alkyl), —OC(O)—NH(lower alkyl) or —OC(O)—NH(substituted lower alkyl);
R16 is phenyl or substituted phenyl;
R17 is —H, lower acyl, substituted lower acyl, lower alkyl, substituted, lower alkyl, (lower alkoxy)methyl or (lower alkyl)thiomethyl;
R18 —H, —CH3 or, taken together with R17 and the carbon atoms to which R17 and R18 are bonded, a five or six membered a non-aromatic heterocyclic ring;
R19 is a lower alkyl group, a substituted lower alkyl group, a phenyl group, a substituted phenyl group;
R20 is —H or a halogen; and
R21 is —H, lower alkyl, substituted lower alkyl, lower acyl or substituted lower acyl.

8. The method of claim 7 wherein:
R10 is phenyl, tert-butoxy, —S—CH2—CH—(CH3)2, —S—CH(CH3)3, —S—(CH2)3CH3, —O—CH(CH3)3, —NH—CH(CH3)3, —CH═C(CH3)2 or para-chlorophenyl;
R11 is phenyl, (CH3)2CHCH2—, -2-furanyl, cyclopropyl or para-toluyl;
R12 is —H, —OH, CH3CO— or —(CH2)2—N-morpholino;
R13 is methyl, or, R13 and R14, taken together, are—C —CH2—;
R14 is —H, —CH2SCH3 or —CH2—O—P(O)(OH)2;
R15 is CH3CO—;
R16 is phenyl;
R1 —H, or, R17 and R18, taken together, are —O—CO—O—;
R18 is —H;
R20 is H or —F; and
R21 is —H, —C(O)—CHBr—(CH2)13—CH3 or —C(O)—(CH2)14—CH3; —C(O)—CH2—CH(OH)—COOH, —C(O)—CH2—O—C(O)—CH2CH(NH2)—CONH2, —C(O)—CH2—O—CH2CH2OCH3 or —C(O)—O—C(O)—CH2CH3.

9. The method of claim 8 wherein the paclitaxel analog is selected from:

10. The method of claim 2 wherein the paclitaxel analog is the copolymer of N-(2-hydroxypropyl)methacrylamide,methacryloylglycine-2-hydroxypropylamide and
[2aR[2α,4β,4β,6β,9α(2R,3S),11β,12α,12α,12α]]-6,12b-diacetoxy-9-[3-benzamido-2-(methacryloyl-glycyl-L-phenylalanyl-L-leucyl.glycyloxy)-3-phenylpropionyloxy]-12-benzoyloxy-4,11-dihydroxy-4a,8,13,13-tetramethyl-2a,3,4,4a,5,6,9,10,11,12,12a, 12b-dodecahydro-1H-7,11-methanocyclodeca[3,4]benz[1,2-b]oxet-5-one.

11. The method of claim 2 wherein the subject is administered paclitaxel or docetaxel.

12. A method of treating a subject with melanoma, said method comprising administering to the subject an effective amount of paclitaxel or a paclitaxel analog and an effective amount of a compound represented by the following structural formula:




or a pharmaceutically acceptable salt thereof, wherein Y′ is a covalent bond or —CR7R8—;
R1 and R2 are independently an aryl group or a substituted aryl group;
R3 and R4 are independently —H, an aliphatic group, a substituted aliphatic group, an aryl group or a substituted aryl group; and
R7 and R8 are each independently —H, an aliphatic or substituted aliphatic group, or R7 is —H and R8 is a substituted or unsubstituted aryl group, or, R7 and R8 taken together, are a C2—C6 substituted or unsubstituted alkylene group.

13. The method of claim 12 wherein R7 and R8 are different.

14. The method of claim 12 where R1 and R2 are the same; R3 and R4are the same; and R7 and R8 are the same.

15. The method of claim 12 wherein R1 and R2 are both aryl or substituted aryl groups and R3 and R4 are both a lower alkyl group or a substituted lower alkyl group.

16. The method of claim 12 wherein R1 and R2 are both phenyl or substituted phenyl and R3 and R4 are methyl, ethyl, phenyl, or thienyl.

17. The method of claim 16 wherein R7 and R8 are both methyl or wherein R7 and R8, taken together, are propylene or butylene.

18. The method of claim 16 wherein R7 is —H and R8 is lower alkyl, thienyl, phenyl or benzyl.

19. The method of claim 16 wherein R1 and R2 are both phenyl substituted with one or more groups selected from —OH, —Br, —Cl, —I, —F, —ORa, —O—CORa, —CORa, —CN, —NO2, —COOH, —SO3H, —NH2, —NHRa, —N(RaRb), —COORa, —CHO, —CONH2, —CONHRa, —CON(RaRb), —NHCOR2, —NRCORa, —NHCONH2, —NHCONRaH, —NHCON(RaRb), —NRcCONH2, —NRcCONRaH, —NRcCON(RaRb), —C(═NH)—NH2, —C(═NH)—NHRa, —C(═NH)—N(RaRb), —C(═NRc)—NH2, —C(═NRc)—NHRa, —C(═NRc)—N(RaRb), —NH—C(═NH)—NH2, —NH—C(═NH)—NHRa, —NH—C(═NH)—N(RaRb), —NH—C(═NRc)—NH2, —NH—C(═NRc)—NHRa, —NH—C(═NRc)—N(RaRb), —NRd—C(═NH)—NH2, —NRd—C(═NH)—NHRa, —NRd—C(═NH)—N(RaR), —NRd—C(═NRc)—NH2, —NRd—C(═NRc)—NHRa, —NRd—C(═NRc)—N(RaRb), —NHNH2, —NHNHRa, —NHNRaRb, —SO2NH2, —SO2NHRa, —SO2NRaRb, —CH═CHRa, —CH═CRaRb, —CRc═CRaRb, —CRC═CHRa, —CRc═CRaRb, —CCRa, —SH, —SRa, —S(O)Ra, —S(O)2Ra, alkyl groups, non-aromatic heterocyclic group, benzyl group, aryl group wherein Ra—Rd are each independently an alkyl group, benzyl, aromatic group, or, —N(RaRb), taken together, can also form a non-aromatic heterocyclic group.

20. The method of claim 12 wherein the compound is represented by the following structural formula:



or a pharmaceutically acceptable salt thereof wherein:
a) R1 and R2 are both phenyl; R3 and R4 are both phenyl; R7 and R8 are both —H;
b) R1 and R2 are both phenyl; R3 and R4 are both methyl; R7 and R8 are both —H;
c) R1 and R2 are both phenyl; R3 and R4 are both ethyl; R7 and R8 are both —H;
d) R1 and R2 are both 4-cyanophenyl; R3 and R4 are both methyl; R7 is methyl; R8 is —H;
e) R1 and R2 are both 4-methoxyphenyl; R3 and R4 are both methyl; R7 and R8 are both —H;
f) R1 and R2 are both phenyl; R3 and R4 are both methyl; R7 is methyl;R8 is —H;
g) R1 and R2 are both phenyl; R3 and R4 are both ethyl; R7 is methyl; R8 is —H;
h) R1 and R2 are both 4-cyanophenyl; R3 and R4 are both methyl; R7 and R8 are both —H;
i) R1 and R2 arc both 2,5-dimethoxyphenyl; R3 and R4 are both methyl; R7 and R8 are both —H;
j) R1 and R2 are both 2,5-dimethoxyphenyl; R3 and R4 are both methyl; R7 is methyl; R8 is —H:
k) R1 and R2 are both 3-cyanophenyl; R3 and R4 are both methyl; R7 and R8 are both —H;
l) R1 and R2 are both 3-fluorophenyl; R3 and R4 are both methyl; R7 and R8 are both —H;
m) R1 and R2 are both 4-chlorophenyl; R3 and R4 are both methyl; R7 is methyl; R8 is —H;
n) R1 and R2 are both 2-dimethoxyphenyl; R3 and R4 are both methyl; R7 and R8 are both —H;
o) R1 and R2 are both 3-methoxyphenyl; R3 and R4 are both methyl; R7 and R8 are both —H;
p) R1 and R2 are both 2,3-dimethoxyphenyl; R3 and R4 are both methyl; R7 and R8 are both —H;
q) R1 and R2 are both 2,3-dimethoxyphenyl; R3 and R4 are both methyl; R7 is methyl; R8 is —H;
r) R1 and R2 are both 2,5-difluorophenyl; R3 and R4 are both methyl; R7 and R8 are both —H;
s) R1 and R2 are both 2,5-difluorophenyl; R3 and R4 are both methyl; R7 is methyl; R8 is —H;
t) R1 and R2 are both 2,5-dichlorophenyl; R3 and R4 are both methyl; R7 and R8 are both —H;
u) R1 and R2 are both 2,5-dimethylphenyl; R3 and R4 are both methyl; R7 and R8 are both —H;
v) R1 and R2 are both 2,5-dimethoxyphenyl; R3 and R4 are both methyl; R7 and R8 are both —H;
w) R1 and R2 are both phenyl; R3 and R4 are both methyl; R7 and R8 are both —H; or
x) R1 and R2 are both 2,5-dimethoxyphenyl; R3 and R4 are both methyl; R7 is methyl; R8 is —H.

21. The method of claim 12 wherein the compound is represented by the following structural formula:




or a pharmaceutically acceptable salt thereof, wherein:
a) R1 and R2 are both 4-cyanophenyl; R3 and R4 are both methyl; R7 is methyl; R8 is —H;
b) R1 and R2 are both 2,5-dimethoxyphenyl; R3 and R4 are both methyl; R7 and R8 are both —H;
c) R1 and R2 are both 2,5-dimethoxyphenyl; R3 and R4 are both methyl; R7 is methyl; R8 is —H;
d) R1 and R2 are both 3-fluorophenyl; R3 and R4 are both methyl; R7 and R8 are both —H;
e) R1 and R2 are both 2,3-dimethoxyphenyl; R3 and R4 are both methyl; R7 and R8 are both —H;
f) R1 and R2 are both phenyl; R3 and R4 are both methyl; R7 and R8 are both —H; and R1 and R2 are both phenyl; R3 and R4 are both phenyl; R7 and R8 are both —H.

22. A method of treating a subject with melanoma, said method comprising administering to the subject an effective amount of paclitaxel or a paclitaxel analog and an effective amount of a compound represented by the following structural formula:




or a pharmaceutically acceptable salt thereof.

23. The method of claim 22 wherein the paclitaxel analog is represented by a structural formula selected from:



R10 is a lower alkyl group, a substituted lower alkyl group, a phenyl group, a substituted phenyl group, —SR19, —NHR19 or —OR19;
R11 is a lower alkyl group, a substituted lower alkyl group, an aryl group or a substituted aryl group;
R12 is —H, —OH, lower alkyl, substituted lower alkyl, lower alkoxy, substituted lower alkoxy, —O—C(O)-(lower alkyl), —O—C(O)-(substituted lower alkyl), —O—CH2—O-(lower alkyl) or —S—CH2—O-(lower alkyl);
R13 is —H, —CH3, or, taken together with R14, is —CH2—;
R14 is —H, —OH, lower alkoxy, —O—C(O)-(lower alkyl), substituted lower alkoxy, —O—C(O)-(substituted lower alkyl), —O—CH2—O—P(O)(OH)2, —O—CH2—O-(lower alkyl), —O—CH2—S-(lower alkyl) or, taken together with R20, is a double bond;
R15 —H, lower acyl, lower alkyl, substituted lower alkyl, alkoxymethyl, alkthiomethyl, —OC(O)—O(lower alkyl), —OC(O)—O(substituted lower alkyl), —OC(O)—NH(lower alkyl) or —OC(O)—NH(substituted lower alkyl);
R16 is phenyl or substituted phenyl;
R17 is —H, lower acyl, substituted lower acyl, lower alkyl, substituted, lower alkyl, (lower alkoxy)methyl or (lower alkyl)thiomethyl;
R18 —H, —CH3 or, taken together with R17 and the carbon atoms to which R17 and R18 are bonded, is a five or six membered a non-aromatic heterocyclic ring;
R19 is a lower alkyl group, a substituted lower alkyl group, a phenyl group, or a substituted phenyl group;
R20 is —H or a halogen; and
R21 is —H, lower alkyl, substituted lower alkyl, lower acyl or substituted lower acyl.

24. The method of claim 23 wherein:
R10 is phenyl, tert-butoxy, —S—CH2—CH—(CH3)2, —S—CH(CH3)3, —S—(CH2)3CH3, —O—CH(CH3)3, —NH—CH(CH3)3, —CH═C(CH3)2 or para-chlorophenyl;
R11 is phenyl, (CH3)2CHCH2—, -2-furanyl, cyclopropyl or para-toluyl;
R12 is —H, —OH, CH3CO— or —(CH2)2—N-morpholino;
R13 is methyl, or, R13 and R14, taken together, are —CH2—;
R14 is —H, —CH2SCH3 or —CH2—O—P(O)(OH)2;
R15 is CH3CO—;
R16 is phenyl;
R17 —H, or, R17 and R18, taken together, are —O—CO—O—;
R18 is —H;
R20 is —H or —F; and
R21 is —H, —C(O)—CHBr—(CH2)13—CH3, —C(O)—(CH2)14—CH3, —C(O)—CH2—CH(OH)—COOH, —C(O)—CH2—O—C(O)—CH2CH(NH2)—CONH2, —C(O)—CH2—O—CH2CH2OCH3 or —C(O)—O—C(O)—CH2CH3.

25. The method of claim 24 wherein the paclitaxel analog is selected from:

26. The method of claim 25 wherein the subject is administered paclitaxel or docetaxel.

27. A method of treating a subject with melanoma comprising administering to the subject an effective amount of paclitaxel or docetaxel and an effective amount of a compound represented by the following structural formula:




or a pharmaceutically acceptable salt thereof.

28. A compound represented by the following structural formula:




or a pharmaceutically acceptable salt thereof, wherein;
Y is a covalent bond, phenylene group or a substituted or unsubstituted hydrocarbyl group;
R1 and R2 are independently an aryl group or a substituted aryl group;
R3 and R4 are independently —H, an aliphatic group, a substituted aliphatic group, an aryl group or a substituted aryl group;
R5-R6 are independently —H, an aliphatic group, a substituted aliphatic group, an aryl group or a substituted aryl group;
and Z is ═O or ═S; provided that when Y is —CH2—, R3 and R4 are both phenyl and R5 and R6 are both —H, then R1 and R2 are not both phenyl.

29. The compound of claim 28, wherein the compound is represented by the following structural formula:




or a pharmaceutically acceptable salt thereof, wherein Y′′ is —CH2—.

30. The compound of claim 29, wherein R3 and R4 are both a lower alkyl group or a substituted lower alkyl group.

31. The compound of claim 28, wherein the compound is represented by the following structural formula:




or a pharmaceutically acceptable salt thereof, wherein:
a) R1 and R2 are both 2,3-dimethoxyphenyl; R3 and R4 are both methyl; R7 and R8 are both —H; or
b) R1 and R2 are both phenyl; R3 and R4 are both methyl; R7 and R8 are both —H.