BACKGROUND OF THE INVENTION Compounds that inhibit cell growth or cell proliferation are becoming increasingly important in the search for effective chemotherapeutic agents, particularly those indicated in the treatment of cancer.

Antiproliferative compounds also are potentially useful as immunosuppressive agents, which are indicated, for example, in the treatment of graft or tissue transplant rejection. It is known that certain vitamins K and analogs thereof possess the ability to inhibit all growth. Cell growth inhibition by vitamins K has been reported to involve a mechanism in which gamma glutamyl carboxylation is implicated. Wang, et al., Hepatology, Vol. 22, No. 3,876-882 (1995). Adducts of menadione have been reported to promote in vitro vitamin K- dependent gamma glutamyl carboxylation. Mack, et al. , J.

Biol. Chem. , 2656-2664 (1979). Synthetic vitamin K analogs having variations in the side chain at position 3 were studied, some of which demonstrated potent growth inhibitory activity against Hep3B human hepatoma cells.

Nishikawa, et al. , J. Biol. Chem. , Vol. 270, No. 47, 28304-28310 (1995). Thioalkyl derivatives of vitamin K3 and its epoxide-have been reported to inhibit Hep3B and Hep3G cells. Kerns, et al., Bioorg. Chem., Vol. 23,101-

108 (1995). It has also been reported that certain vitamins K inhibit the growth of Hep40 cells.

Recently, Ham, et al. , has investigated the inhibitory effect of menadione on protein tyrosine phosphatase, and has proposed that menadione inactivates cdc25 phosphatase irreversibly by forming a covalently bonded adduct with a thiol residue on the enzyme, which residue purportedly involves a cysteine molecule at the active site. Bioorgr. Chem. , Vol. 25,33-36 (1997). As protein tyrosine phosphatases have critical roles in many cellular functions, and cdc25 phosphatase is believed to exhibit oncogenic properties in mammalian cells, the inhibition of such phosphatases is potentially useful in the treatment of human cancer. Moreover, the identification of selective phosphatase inhibitors can produce new chemotherapeutic leads in cancer and immunosuppression applications which exhibit fewer side effects related to non-selective inhibition of cellular pathways. However, despite the ongoing research, it continues to be a serious challenge to identify new potent antiproliferative agents, particularly those which selectively inhibit cancer cell growth without disturbing normal cellular pathways.

Thus, there remains a need for new chemical compounds with potent inhibitory activity against cancer cell growth. Further, there exists a need for pharmaceutical compositions comprising such compounds, as well as a method of preparing such compounds. Moreover, there exists a need for methods of inhibiting cancer cell growth. The present invention provides such compounds, compositions, and associated methods. These and other

advantages of the present invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.

BRIEF SUMMARY OF THE INVENTION The present invention provides a compound selected from the group consisting of : wherein Rl and R4 are the same or different and are each H, Cl-C3 alkyl, phosphate, or Cl-C3 alkyl carboxylate; R2 and R3 are the same or different and are each H, a halogen, A-Z, S-Z, S-A-Z, N(B)-Z, N(B)-A-Z, O-Z, or O-A- Z, wherein : A is a Cl-C20 linear or branched saturated or unsaturated alkyl, haloalkyl, arylalkyl, cycloalkylalkyl, heterocycloalkyl, hydroxyalkyl, alkoxyalkyl, thioalkyl, carboxyalkyl, carboalkoxyalkyl, carboxyaminoalkyl, a 5- to 6-membered ring carbohydrate or the corresponding acyclic analog thereof, peptidyl, or aminoalkyl diradical ; B is H or Cl-C6 alkyl; Z is H, aryl, heterocyclic, or a heteroatom- containing functional group ; and Rs-R8 are the same or different and are each H, a halogen, heterocyclic, Z as defined above, A-Z as defined

above, or a heteroatom-containing functional group, provided that R-9-R"are not all H.

The present invention also provides a pharmaceutical composition, which composition includes a pharmaceutically acceptable carrier and a therapeutically effective amount of at least one compound of the present invention, as well as a method of treating or preventing cancer which method comprises administering an anticancer-effective amount of at least one compound of the present invention.

The present invention additionally provides a method of preventing or treating tissue transplant rejection in a mammal, which method involves administering an anti- rejection-effective amount of at least one compound of the present invention.

The present invention further provides a general method of inhibiting the growth of a cell which involves contacting the cell with a cell-growth-inhibitory- effective amount of at least one compound of the present invention.

The invention may best be understood with reference to the accompanying drawings and in the following detailed description of the preferred embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 illustrates general synthetic approaches to various heteroatom-containing benzene ring modifications starting from an aminonaphthoquinone of the present invention.

Figures 2A-2H illustrate examples of various benzene ring modifications to provide compounds of the present invention, derivatives, or precursors thereof.

Figures 3A-3D illustrate examples of various synthetic modifications of the quinone ring of the compounds of the present invention.

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention provides a compound selected from the group consisting of : wherein R1 and R4 are the same or different and are each H, Cl-C3 alkyl, phosphate, or Cl-C3 alkyl carboxylate ; R2 and R3 are the same or different and are each H, a halogen, A-Z, S-Z, S-A-Z, N(B)-Z, N(B)-A-Z, O-Z, or O-A- Z, wherein : A is a Cl-c2, linear or branched saturated or unsaturated alkyl, haloalkyl, arylalkyl, cycloalkylalkyl, heterocycloalkyl, hydroxyalkyl, alkoxyalkyl, thioalkyl, carboxyalkyl, carboalkoxyalkyl, carboxyaminoalkyl, a 5- to 6-membered ring carbohydrate or the corresponding acyclic analog thereof, peptidyl, or aminoalkyl diradical ; B is H or C1-C6 alkyl; Z is H, aryl, a heterocyclic substituent such as, for example, aziridine, epoxy, azetidine, oxetane, imidazole, imidazolidine, thiazole, thiazolidine, pyrazole, pyrrole, furane, dihydrofuran, tetrahydrofuran, pyrrolidine, pyrrolidone, pyrazoline, thiophene, oxazole,

oxazolidine, oxazolidone, isoxazol, isoxazolidine, pyridine, piperidine, pyridone, pyrimidine, pyrazine, piperazine, piperadone, triazine, or a heteroatom- containing functional group such as, for example, OR9, SR9, S(O)R9, SO2R9, SO2NR9R10, SO2N(OH)R9, NO2, NR9R10, N (OH) R9, NR9C (O) Rl°, NR9C (S) Rl°, N (OH) C (O) R9, N (OH) C (S) R9, NR9CO2R10, N(OH)CO2R9, NR9C(O)NHR10, NHC(O)NR9R10, <BR> <BR> <BR> NR9C (S) NHR"), NHC (S) NR9R'O, N (OH) C (O) NR9R'O, N (OH) C (S) NR9R'O, NR9C(O)N(OH)R10, NR9C(S)N(OH)R10, NR9SO2R10, NHSO2NR9R10, NR9SO2NHR'O, CR9=NR'O, CR9=N (OR'O), C02R9, C (0) SR9, C (0) R9, C(S)R9, C(O)NR9R10, C(S)NR9R10, C(O)N(OH)R9, C(S)N(OH)R9, or P (0) (OR9) (OR'O), wherein R9 and R are the same or different and are each H or Cl-c3 alkyl ; and R5-R'3 are the same or different and are each H, a halogen, a heterocycle such as, for example, aziridine, epoxy, azetidine, oxetane, imidazole, imidazolidine, thiazole, thiazolidine, pyrazole, pyrrole, furane, dihydrofuran, tetrahydrofuran, pyrrolidine, pyrrolidone, pyrazoline, thiophene, oxazole, oxazolidine, oxazolidone, isoxazol, isoxazolidine, aryl, pyridine, piperidine, pyridone, pyrimidine, pyrazine, piperazine, piperadone, triazine, Z as defined hereinabove, A-Z as defined hereinabove, or a heteroatom-containing functional group such as, for example, OR¹¹, SR¹¹, S(O)R¹¹, SO2R¹¹, SO2NR¹¹R¹², SO2N(OH)R¹¹, NO2, NR¹¹R¹² N(OH)R¹¹, NR¹¹C(O)R¹², NR¹¹C(S)R¹², N (OH) C (0) R", N (OH) C (S) R", NRllCo2R 12, N (OH) Co2R", NR¹¹C(O)NHR¹², NHC(O)NR¹¹R¹², NR¹¹C(S)NHR¹², NHC(S)NR¹¹R¹², <BR> <BR> <BR> N (OH) C (0) NR'-'R, N (OH) C (S) NR"R, NR"C (0) N (OH) R, <BR> <BR> <BR> <BR> <BR> NR C(S)N(OH)R , NR SO2R , NHSO2NR R , NR SO2NHR , <BR> <BR> <BR> <BR> <BR> CRll=NRl2, CRll=N(ORl2), CO2Rll, C(O)SRll, C(O)Rll, C(S)Rll, <BR> <BR> <BR> <BR> <BR> C(O)NR R , C(S)NRllRl2 C(O)N(OH)Rll C(S)N(OH)Rll

P (O) (ORll) (OR12), wherein Rll and R12 are the same or different and are each H or D-Z, wherein D is a Cl-Cl0 linear or branched saturated or unsaturated alkyl, haloalkyl, arylalkyl, heterocycloalkyl, hydroxyalkyl, alkoxyalkyl, thioalkyl, peptidyl, or aminoalkyl diradical ; and pharmaceutically acceptable salts thereof, provided that Rus-rare not all H.

All substituents defined herein can be used within the context of a substituent with one point of attachment (a single radical), or within the context of a linker with two points of attachment (a"diradical", as utilized herein). It will be appreciated that a substituent, as defined herein, with one point of attachment is the same as the corresponding diradical, except one of the points of attachment of the diradical is instead substituted with hydrogen.

The term"diradical"as utilized herein means any suitable linear or branched substituent, having two sites available for covalent bonding, with a primary chain defining the fundamental skeleton, not including substituents which do not constitute a link in the primary chain, ranging from 1 to about 20 carbon atoms.

Suitable diradicals include, for example, hydrocarbon substituents (saturated or unsaturated, substituted or unsubstituted), peptides, or cyclic or linear carbohydrate diradicals. Preferably, diradical A is a C1-C20 linear or branched saturated or unsaturated alkyl, haloalkyl, arylalkyl, heterocycloalkyl, hydroxyalkyl, alkoxyalkyl, thioalkyl, carboxyalkyl, carboalkoxyalkyl, carboxyaminoalkyl, a 5- to 6-membered ring carbohydrate or the corresponding acyclic analog thereof, peptidyl, or

aminoalkyl diradical. Substituent A can be covalently bonded to the compound of the present invention directly as represented by A-Z, wherein Z is defined below.

Substituent A also can be bonded to the compound of the present invention indirectly as represented by thiol S-Z, amine N (B) -A-Z, or ether O-A-Z.

The term"alkyl"as utilized herein means a straight-chain or branched-chain saturated or unsaturated alkyl substituent containing from 1 to about 20 carbon atoms in the primary chain, preferably from 1 to about 10 carbon atoms, more preferably from 1 to about 6 carbon atoms.

When A is alkyl, the primary chain is advantageously a Cl-c20 linear or branched saturated or unsaturated alkyl diradical including, for example, a linear alkyl diradical of the formula (CH2) n wherein n is an integer from 1 to 20, or a branched alkyl substituent of the formula : an unsaturated alkyl substituent of the formula : The term"haloalkyl"as used herein refers to an alkyl substituent as utilized herein, wherein at least one hydrogen atom is replaced by a halogen. Exemplary haloakyls include substituents of the formula :

The term"aryl"as utilized herein means an aromatic carbocyclic substituent, as is commonly understood in the art, and includes monocyclic and polycyclic aromatics such as, for example, phenyl and napththyl substituents, optionally substituted with one or more substituents selected from the group consisting of a halogen, an alkyl, an alkoxy, an amino, cyano, nitro.

The term"cycloalkyl"as utilized herein means a polycyclic alkyl substituent defined by 1 or more alkyl carbocyclic rings, which can be the same or different when the cycloalkyl is a polycyclic substituent, having 3 to about 10 carbon atoms in the carbocyclic skeleton of each ring, preferably about 4 to about 7 carbon atoms, more preferably 5 to 6 carbon atoms. Preferably, polycyclic cycloakyls have fewer than four rings, more preferably fewer than three rings, and are most preferably monocyclic or bicyclic. Examples of monocyclic cycloalkyl substituents include cyclopropyl, cyclobutal, cyclopentyl, cyclohexyl, cyclohexenyl, cyclododecil. Examples of polycyclic cycloalkyl radicals include decahydronaphthyl, bicyclo [5. 4. 0] undecyl, adamantyl.

The term"heterocycle"as utilized herein means a cycloakyl substituent as defined herein (including polycyclics), wherein at least one carbon which defines the carbocyclic skeleton is substituted with a heteroatom

such as, for example, 0, N, or S, optionally comprising one or more double bonds within the ring, including heteroaryl rings. The term heterocycle, as utilized herein is used synonymously herein with the term "heterocyclic substituent."Preferably, polycyclic heterocycles have fewer than four rings, more preferably fewer than three rings, and are most preferably monocyclic or bicyclic. The heterocycle preferably has 3 to about 10 atoms (members) in the cyclic skeleton of each ring, preferably about 4 to about 7 atoms, more preferably 5 to 6 atoms. Examples of heterocylic substituents include, for example, aziridine, epoxy, azetidine, oxetane, imidazole, imidazolidine, thiazole, thiazolidine, pyrazole, pyrrole, furan, dihydrofuran, tetrahydrofuran, pyrrolidine, pyrrolidone, pyrazoline, thiophene, oxazole, oxazolidine, oxazolidone, isoxazol, isoxazolidine, pyridine, piperidine, pyridone, pyrimidine, pyrazine, piperazine, piperadone, triazine, morpholine.

The term"arylalkyl"as utilized herein means alkyl as utilized herein, wherein at least one hydrogen atom is replaced with an aryl substituent as utilized herein.

Arylalkyls include, for example, substituents of the formula :

The term"cycloalkylalkyl"as used herein means any suitable linear or branched, saturated or unsaturated alkyl bearing a cycloalkyl ring, as utilized herein, having from 1 to about 20 carbons in the primary chain including, by way of example, substituents of the formula : The term"heterocycloalkyl"as used herein refers to any suitable linear or branched, saturated or unsaturated alkyl bearing a heterocyclic ring, as utilized herein, having from 1 to about 20 carbons in the primary chain including, by way of example, substituents of the formula : suitable derivatives thereof.

The term"hydroxyalkyl"as used herein refers to any suitable hydroxy-substituted linear or branched, saturated or unsaturated alkyl, as utilized herein, having from 1 to about 20 carbons in the primary chain including, by way of example, substituents of the formula :

The term"alkoxyalkyl"as used herein refers to any suitable alkoxy substituted linear or branched, saturated or unsaturated, alkyl, as utilized herein, having from 1 to about 20 carbons in the primary chain including, by way of example, substituents of the formula : The term"thioalkyl"as used herein refers to any suitable alkoxy-containing linear or branched, saturated or unsaturated alkyl, as utilized herein, having from 1 to about 20 carbons in the primary chain including, by way of example, substituents of the formula : The term "carboxyalkyl, as used herein, refers to any suitable linear or branched, saturated or unsaturated alkyl, as utilized herein, bearing a carboxy substituent, as is commonly understood in the art, having from 1 to about 20 carbons in the primary chain including, by way of example, substituents of the formula :

The term"carboalkoxyalkyl"as used herein refers to any suitable linear or branched, saturated or unsaturated alkyl, as utilized herein, having from 1 to about 20 carbons in the primary chain including, by way of example, substituents of the formula : Typically, the carboalkoxy portion of the carboalkoxyalkyl substituents define an ester linkage, which linkage can be included in the primary chain or can exist outside the primary chain of the substituent.

The term"carboxyaminoalkyl", as used herein, refers to any suitable linear or branched, saturated or unsaturated alkyl, as utilized herein, bearing a aminocarbonyl substituent, as is commonly understood in the art (e. g., amides and ureas), having from 1 to about 20 carbons in the primary chain including, by way of example, substituents of the formula :

The term"5- to 6-membered ring carbohydrate"as used herein refers to any suitable 5- to 6-membered ring carbohydrate, as is commonly understood in the art, including, by way of example, 5- to 6-membered ring carbohydrate substituents, or the corresponding acyclic analog thereof, having the formula : or the corresponding acyclic (ring-opened) analog thereof.

The term"peptidyl"as utilized herein means any suitable linear or branched peptide as commonly understood in the art, defined by at least two alpha- amino acids, natural or unnatural and, optionally, of D

or L configuration, or a D, L-mixture thereof, linked together by amide bonds, the primary chain having from 1 to about 20 atoms (carbon and nitrogen atoms combined) including, by way of example, peptide substituents of the formula -Gly-Gly-, -Gly-Ala-, -Gly-Phe-Ala-, -Phe-Pro- Arg-, -His-D- (thiazolyl) Ala-, -Leu-Val-Ile-, -Gly-L- (naphthyl) Ala-Gly-Gly-, and any other suitable combination of amino acids.

The term"aminoalkyl"as used herein refers to any suitable amino-containing linear or branched, saturated or unsaturated alkyl, as utilized herein, having from 1 to about 20 atoms (carbon and nitrogen atoms combined) in the primary chain including, by way of example, substituents of the formula : Substituent B refers to any suitable nitrogen-bonded substituent, and is preferably H or alkyl. More preferably, B is H or Cl-C6 alkyl.

Substituent Z includes any suitable organic or inorganic substituent occupying one of the bonding sites of diradical A including, for example, H, aryl, heterocyclic, and heteroatom-containing functional groups as defined herein. For example, when Z is H then AZ becomes AH.

Z also includes heterocyclic substituents, as utilized herein, optionally substituted with at least one substituent selected from fluorine, chlorine, bromine,

iodine, hydroxy, methoxy, C1-C6 alkyl groups. Preferred heterocyclic substituents include, for example, aziridine, epoxy, azetidine, oxetane, imidazole, imidazolidine, thiazole, thiazolidine, pyrazole, pyrrole, furane, dihydrofuran, tetrahydrofuran, pyrrolidine, pyrrolidone, pyrazoline, thiophene, oxazole, oxazolidine, oxazolidone, isoxazol, isoxazolidine, pyridine, piperidine, pyridone, pyrimidine, pyrazine, piperazine, piperadone, triazine, and suitable derivatives thereof.

Z additionally can be any suitable heteroatom- containing functional group such as, for example, hydroxy, ethers, thiols, thioethers, sulfoxides, sulfones, sulfamides, sulfonamides, N-hydroxysulfamides, N-hydroxysulfonamides, nitro, amines, hydroxylamines, amides, thioamides, N-hydroxyamides, N-hydroxythioamides, carbamates, N-hydroxycarbamates, ureas, thioureas, N- hydroxyureas, N-hydroxythioureas, imines, oximes, esters, thioesters, ketones, thioketones, phosphonates, and phosphonamides. Preferably, the heteroatom-containing functional group is OR9, SR9, S(O)R9, SO2R9, SO2NR9Rl°, <BR> <BR> <BR> <BR> S02N (OH) R9, NO., NR9R'O, N (OH) R9, NR9C (O) Rlo, NR9C (S) Rlo, <BR> <BR> <BR> <BR> <BR> <BR> N(OH)C(O)R9, N(OH)C(S)R9, NR9CO2R1°, N(OH)CO2R9, NR9C(o)NHRl0, NHC(O)NR9R10, NR9C(S)NHR10, NHC(S)NR9R10, N(OH)C(O)NR9R10, N(OH)C(S)NR9R10, NR9C(O)N(OH)R10, NR9C(S)N(OH)R10, NR9SO2R10, <BR> <BR> <BR> <BR> NHS02NR9R'O, NR9SO, NHR'-O, CR9=NR'O, CR9=N (OR'O), C02R9, C (0) SR9, <BR> <BR> <BR> <BR> <BR> <BR> C (0) R9, C (S) R9, C (0) NR9R'-O, C (S) NR9R'O, C (0) N (OH) R9, C(S)N(OH)R9, or P(O)(OR9)(ORl0), wherein R9 and Rl° are the same or different and are each H, alkyl, cycloalkyl, aryl, or heterocycloalkyl, as utilized herein.

Preferably, R9 and Rl° are the same or different and are each H or Cl-C3 alkyl.

Surprisingly, it has been discovered that modification of the benzene ring of naphthoquinones significantly enhances activity with respect to cell growth inhibition. Although not wishing to be bound by any one particular theory, it is believed that modification of the benzene ring of naphthoquinones improves activity with respect to phosphatase inhibition.

Thus provided are benzene ring-modified naphthoquinones, and derivatives thereof, that inhibit the cell growth of a broad range of cancer cell lines with unprecedented inhibitory potency.

The compounds of the present invention possess at least one benzene ring substituent, as represented by R5- R9 in structures I-III above. In the naphthoquinones of the present invention, R5-R8 can be the same or different and can each include any suitable substituent such as, for example, a halogen, an alkyl, cycloalkyl, an aryl, a heterocyclic substituent, Z, A-Z, or a heteroatom- containing functional group as defined herein.

Preferably R5-R8 are the same or different and are each H, F, C1, Br, I, a heterocycle which is aziridine, epoxy, azetidine, oxetane, imidazole, imidazolidine, thiazole, thiazolidine, pyrazole, pyrrole, furane, dihydrofuran, tetrahydrofuran, pyrrolidine, pyrrolidone, pyrazoline, thiophene, oxazole, oxazolidine, oxazolidone, isoxazol, isoxazolidine, aryl as defined herein, pyridine, piperidine, pyridone, pyrimidine, pyrazine, piperazine, piperadone, triazine, Z as defined herein, A-Z as defined herein, or a heteroatom-containing functional group which is OR¹¹, SR¹¹, S(O)R¹¹, SO2R¹¹, SO2NR¹¹R¹², SO2N(OH)R¹¹, NO2, NRR, N (OH) R, NRC (0) R, NRC (S) R, N (OH) C (0) R,

N(OH)C(S)R¹¹,NR¹¹CO2R¹², N(OH)CO2R¹¹, NR¹¹C(O)NHR¹², NHC(O)NR¹¹R¹², NR¹¹C(S)NHR¹², NHC(S)NR¹¹R¹² N(OH)C(O)¹¹R¹², N (OH) C (S) NR"R, NR"C (0) N (OH) R, NR"C (S) N (OH) R NR¹¹SO2R¹², NHSO2NR¹¹R¹², NR¹¹SO2NHR¹², CR¹¹=NR¹², CR¹23=N(OR12), CO2R¹¹, C(O)R¹¹, C(S)R¹¹, C(O)R¹¹, C(O)NR¹¹R¹², C(S)NR¹¹R¹², C (0) N (OH) R", C (S) N (OH) R", or P(O)(ORll)(ORl2) wh i R and R12 are the same or different and are each H or D-Z, wherein D is a Cl-Clo alkyl, haloalkyl, arylalkyl, heterocycloalkyl, hydroxyalkyl, alkoxyalkyl, thioalkyl, peptidyl, or aminoalkyl diradical as defined herein ; or a pharmaceutically acceptable salt, an ester, or a prodrug thereof, provided that R5-Rs are not all H.

Preferably, the antiproliferative naphthoquinone compound of the present invention is of the formula : wherein R2-R8 are the same or different and are each defined as hereinabove, or a pharmaceutically acceptable salt, an ester, or a prodrug thereof.

In one preferred embodiment, R'is H, C1-C3 alkyl, F, C1, or Br ; and R'is H, A-Z, S-Z, S-A-Z, N (B) -Z, N (B) -A- Z, O-Z, or O-A-Z, wherein A, B, and Z are defined herein, or a pharmaceutically acceptable salt, an ester, or a prodrug thereof. More preferably, R2 is H or Cl-C3 alkyl.

In another preferred embodiment, R² and R³ are the same or different and are each H, Cl-C3 alkyl, or S-A-Z, wherein A and Z are defined herein, or a pharmaceutically

acceptable salt, an ester, or a prodrug thereof. In this embodiment, it is particularly preferred that either of R2 or R3 is H or Cl-C3 alkyl, and the other is S-A-Z.

In yet another preferred embodiment, R2 and R3 are the same or different and are each H, A-Z, S-Z, S-A-Z, N(B)-Z, N(B)-A-Z, O-Z, or O-A-Z, as defined herein, wherein at least two of any of substituents R5-R8 are H.

More preferably, R2 is H, Cl-C3 alkyl, F, Cl, or Br ; and R3 is H, A-Z, S-Z, S-A-Z, N(B)-Z, N(B)-A-Z, O-Z, or O-A-Z, as defined herein, and at least two of any of substituents Rs-R8 are H. Still more preferably, R2 is H or C1-C3 alkyl.

In yet another preferred embodiment, R2 and R3 are the same or different and are each H, C1-C3 alkyl, or S-A- Z, as defined herein, and at least two of any of substituents Rs-R8 are H. More preferably, either of R2 or R3 is H or C1-C3 alkyl, and the other is S-A-Z, as defined herein.

The antiproliferative naphthoquinones and related compounds of the present invention have the unique structural feature of benzene ring substitution, which feature has not been previously disclosed in connection with improved cellular growth inhibition. The benzene ring substitution of the compounds of the present invention provides antiproliferative naphthoquinone compounds which are expected to have satisfactory, if not unprecedented, activity and/or selectivity in the inhibition of cancer cell growth. Detailed in vivo and in vitro structure activity studies will enable a person of ordinary skill in the art to modify the functional groups R1-R8 to design compounds with optimum potency and

selectivity against a broad range of different cancer cell lines. Since the compounds of the present invention are based on a vitamin K-type naphthoquinone carbocyclic skeleton, it is expected that the compounds will be safe and well-tolerated in vivo. Moreover, the compounds of the present invention can be designed in furtherance of other considerations such as, for example, cost, chemical and/or biological stability, bioavailability, and the ability of the compound to be formulated for a particular route of administration. Thus, the present invention provides compounds having great potential in the prophylactic and/or therapeutic management of mammalian cancers and of immunological diseases such as, for example, tissue transplant rejection and graft rejection.

A person of ordinary skill in the art will appreciate that numerous synthetic approaches are available to prepare the compounds of the present invention. One such approach involves direct substitution of the aromatic ring in an appropriately substituted, protected, or unsubstituted naphthoquinone. Aromatic substitution is well known in the chemical arts and can be accomplished, for example, by electrophilic or nucleophilic substitution, by free radical chemistry, or via a transition metal-mediated coupling reaction. A general description of electrophilic, nucleophilic, and free radical aromatic substitution reactions can be found, for example, in March, Advanced Organic Chemistry, John Wiley & Sons, New York (1985), chapters 11,13, and 14, respectively.

The following reaction schemes illustrate, by way of example, some of the numerous synthetic approaches

available in the art to introduce functional groups onto the benzene ring of naphthoquinones. One well-known example of electrophilic substitution is nitration, which provides access to four possible nitronaphthoquinone (nitro-NQ) analogs as exemplified in Scheme 1 below : Scheme 1 0 0 0 0 R3 Rs z R3 , 5 0 0 0 z 0 0 0l NAPHTHOQUINONE NITRO-NQ's The nitro group can be used to impart desirable biological and/or physical properties, or can be used as a versatile precursor for a variety of other compounds such as, for example, an aminonaphthoquinone (amino-NQ - structure 11 of Fig. 1) as indicated in Scheme 2 : Scheme 2 0 0 R3 R3 Reduction 0 o OZ Hz O O NITRO-NQ AMINO-NQ The aromatic amine, in turn, can be used to impart desirable biological properties and/or be used as a versatile precursor to a variety of other analogs such as, for example, iminonaphthoquinones (imino-NQ) and substituted aminonaphthoquinones (substituted amino-NQ) as indicated generally in Scheme 3 below : Scheme 3 0 0 0 3 3 R3 R ~ 2 2 RCH=N RCH2NH 0 0 0 AMINO-NQ IMINO-NQ SUBSTITUTED AMINO-NQ

Alternatively, aminonaphthoquinones can be used as a precursor for other compounds such as, for example, amides and sulfonamides, as illustrated in Scheme 4 below : Scheme 4 0 0 H2 X RCOC1 X 4 ° \ RSO2C1 H ° R (R 2 O , I* AMIDO-NQ R w 3 II 11 1 H O SULFONAMIDO-NQ Amino-NQ's also can be used as precursors for a variety of other functionally diverse compounds as illustrated in Figure 1, which is a general synthetic diagram representing some of the possible analogs which are synthetically accessible from an amino-NQ of the present invention. With reference to Figure 1, amino-NQ (11) can be treated with COX, (e. g., phosgene or carbonyldiimidazole) or CSX2 (e.g., thiophosgene or thiocarbonyldiimidazole) to provide isocyanate (12) or

thioisocyanate (14), respectively. Alternatively, amino- NQ (11) can be treated with SO2X2 (e.g., thionyl chloride) to provide sulfamoyl chloride 13. As illustrated in Figure 1, isocyanate 12 and thioisocyanate 14 are intermediates from which numerous diverse analogs can be obtained such as, for example, hydroxamates, ureas, and carbamates (from isocyanate (12) ), or thionocarbamates and thioureas (from thioisocyanate (14) ). Figure 1 also illustrates the conversion of intermediate sulfamoyl chloride 13 to provide other analogs such as, for example, sulfamates and sulfamides.

It will also be appreciated that numerous other electrophilic aromatic substitution reactions can be applied such as, for example, halogeneation, to provide halodyhidronaphthoquinones (halo-DHNQ), or halonaphthoquinones (halo-NQ) using additional transformations, as illustrated in Scheme 5, below : Scheme 5 OMe OMe0 Rs Ri R3 [ X FeCl z OMe OMe o HALO-DHNQ HALO-NQ (X = F, Cl, Br, I) Electrophilic alkylation and acylation also can be utilized, to provide alkyl naphthoquinones (alkyl-NQ) and acyl naphthoquinones (acyl-NQ), as illustrated in Scheme 6 below : Scheme 6 0 00 R3 R \ Ra \ Ra R-x. o ; I and/or or Lewis acid # R2 (R = alkyl or. acyl) 0 0 0 (alkyl- or acyl-NQ)

It will be appreciated that nucleophilic aromatic substitution reactions can be applied, to provide access to other diverse naphthoquinones of the present invention. For example, aminonapthoquinones can be converted to the corresponding diazonium salt (diazonium- NQ), which can be displaced by any suitable nucleophile (Nu :) to provide the corresponding nucleophilically- substituted naphthoquinone (Nu-NQ), as illustrated generally in Scheme 7 below : Scheme 7 0 0 0 3 3 3 Z t R R3 Rs ''N I \ z z N-N+ Nu 0 0 u DIAZONIUM-NQ Nu-NQ Any suitable nucleophile can be used in the nucleophilic substitution reaction (Scheme 7, above). Suitable nucleophiles include for example, water, thiols, alcohols, and halogens.

Any suitable carbon-carbon bond forming reaction can be used to introduce a substituent on the aromatic ring of the naphthoquinones of the present invention.

Suitable carbon-carbon bond forming reactions include olefination reactions such as, for example, the Stille

coupling, providing access to olefinated naphthoquinones (olefinated-NQ) from bromonaphthoquinone (Br-NQ) precursors. Optionally, the olefinated naphthoquinone can be functionalized by any suitable olefin reaction such as, for example, hydroboration and oxidation, to provide hydroxyalkyl naphthoquinones (hydroxyalkyl-NQ), or arylation such as, for example, the Heck reaction to provide aryl alkylnaphthoquinones (Ar-alkyl-NQ), as indicated in Scheme 8 below : Scheme 8 0 3 R HO 2 2 I O 1 ) R2BH O Br 3 HYDROXYALKYL-NQ Br (. ~ (PhP) 4Pd 2 (Stille) 0 BROMO-NQ OLEFINATED-NQ base A. r 2 0 x O AR-ALKYL-NQ Alternatively, direct arylation of the aromatic ring can be accomplished by any suitable arylation reaction such as, for example, the Suzuki coupling, to provide aryl naphthoquinones (Ar-NQ), as illustrated in Scheme 9 below : Scheme 9 0 0 R a Ar-B (OH) 2 Br (Ph3P) 4Pd Ar (Stille) o O 0 BROMO-NQ Ar-NQ

A person of ordinary skill in the art also will appreciate that commercially available compounds such as, for example, menadione (2-methyl-1,4-naphthoquinone) and 1, 4-naphthoquinone, available at Aldrich Chemical Co., can be used as precursors in the synthesis of compounds of the present invention. It will also be appreciated that the naphthoquinone ring can be optionally constructed from an appropriately substituted cyclic or acyclic precursor to provide compounds of the present invention. Moreover, a person of skill in the art will appreciate that the naphthoquinones of the present invention can exist in different oxidation states such as, for example, a dihydrohaphthoquinone (DH-NQ), a napthoquinone (NQ), or an epoxynaphthoquinone (NQ-oxide), as indicated generally in Scheme 10 below : Scheme 10 OH 00 oxidation oxidation () reduction (D reduction c> OH 00 DH-NQ NQ NQ-oxide Any suitable reaction can be employed to modify the quinone portion of the naphthoquinone ring. Suitable reactions include, for example, halogenation of an appropriately substituted dihydroquinone to provide a chlorodihydronaphthoquinone (Cl-DHNQ), which can be further converted to a chloronaphthoquinone (Cl-NQ), as illustrated in Scheme 11 below : Scheme 11

Other reactions for modifying the quinone ring include, for example, displacement of a leaving group at the alpha position with a suitably reactive nucleophile (Nu :), to provide the desired displacement product. Suitable leaving groups include, for example, halogens (e. g., chloride), as illustrated in Scheme 12 below : Scheme 12 Any suitable nucleophile can be used in the displacement reaction of the present invention, which nucleophiles include, for example, alcohols, amines, thiols, acetylides, carbanions (e. g., enolates, organometalic compounds), azide, and enolates.

A person of ordinary skill in the art will appreciate that the chemical reactions and synthetic transformations illustrated herein are not in any way exhaustive, but are merely illustrative of some of the possible structural variations encompassed within the scope of the present invention. Moreover, the compounds

of the present invention can be utilized as final compounds for a particular therapeutic application and/or for non-therapeutic applications (e. g., a bioassay standard). Moreover, the compounds of the present invention include pharmaceutically acceptable salts, esters, and prodrugs. The compounds of the present invention also can be used as synthetic precursors, intermediates, or derivatives of compounds to be used in a particular therapeutic application or for other applications (e. g., bioassay kits, medical research, and diagnostic devices). It will also be appreciated that substitution on more than one carbon of the benzene ring and/or the quinone ring (including the dihydroquinone and the epoxynaphthquinone) is encompassed within the scope of the present invention. Moreover, the synthetic reactions utilized in the present invention can be manipulated by varying the conditions (e. g., solvent, temperature, reagents, order of addition, neighboring group effect, and the like) to control aspects of product formation such as, for example, degree of substitution, rate of reaction, regioselectivity, stereoselectivity, and the like. Any suitable synthetic approach can be utilized in the context of the present invention including, for example, linear synthesis, convergent synthesis, biosynthesis, enzymatic reactions, and the like. It will also be appreciated that the chemistry encompassed within the scope of the present invention can be applied to any suitable substrate such as, for example, an appropriately substituted and/or unsubstituted naphthoquinone, derivative, and/or precursor thereof.

In accordance with the present invention there is provided a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective (which includes a prophylactic effective) amount of at least one compound of the present invention.

Preferably, the composition includes a therapeutically effective amount of a naphthoquinone of Formula I (above), wherein R-R"are the same or different and are each defined as hereinabove, or a pharmaceutically acceptable salt, ester, or prodrug thereof, provided that Rs-R8 are not all H.

In one preferred embodiment, the pharmaceutical composition of the present invention has, as the active component, a naphthoquinone of Formula I, wherein R2 is H, C1-C3 alkyl, F, C1, or Br ; and R3 is H, A-Z, S-Z, S-A- Z, N(B)-Z, N(B)-A-Z, O-Z, or O-A-Z, as defined hereinabove, and pharmaceutically acceptable salts thereof, provided that Rus-rare not all H. More preferably, R2 is H or Cl-C3 alkyl.

In another preferred embodiment, the pharmaceutical composition of the present invention has, as the active component, a naphthoquinone of Formula I, wherein R2 and R3 are the same or different and are each H, Cl-c3 alkyl, or S-A-Z, as hereinabove, and pharmaceutically acceptable salts thereof. More preferably, either of R2 or R3 is H or C1-C3 alkyl, and the other is S-A-Z.

In yet another preferred embodiment, the pharmaceutical composition of the present invention has, as the active component, a naphthoquinone of Formula I, wherein R2 and R3 are the same or different and are each H, A-Z, S-Z, S-A-Z, N(B)-Z, N(B)-A-Z, O-Z, or O-A-Z, as

defined hereinabove, and pharmaceutically acceptable salts thereof, wherein at least two of any of substituents Rs-R8 is H. More preferably, R2 is H, Cl-C3 alkyl, F, C1, or Br ; and R3 is H, A-Z, S-Z, S-A-Z, N (B) - Z, N(B)-A-Z, O-Z, or O-A-Z, as defined herein, and at least two of any of substituents Rs-R8 are H. Still more preferably, R2 is H or Cl-C3 alkyl.

In yet another preferred embodiment, the pharmaceutical composition of the present invention utilizes, as an active component, a naphthoquinone of Formula I, wherein R2 and R3 are the same or different and are each H, C1-C3 alkyl, or S-A-Z, as defined herein, provided that R5-R8 are not all H and at least two of any of substituents R5-R8 are H. More preferably, one of R2 or R3 is H or C1-C3 alkyl, and the other is S-A-Z.

The pharmaceutical compositions of the present invention may be in a form suitable for oral use such as, for example, tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, syrups or elixirs. Compositions intended for oral use may be prepared according to any method known in the art form the manufacture of pharmaceutical compositions, and such compositions can contain one or more agents such as, for example, sweetening agents, flavoring agents, coloring agents, and preserving agents in order to provide a pharmaceutically elegant and/or palatable preparation. Tablets can contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for manufacture of tablets. Such excipients can be, for example, inert diluents such as, for example, calcium

carbonate, lactose, calcium phosphate or sodium phosphate ; granulating and disintegrating agents such as, for example, maize starch or alginic acid ; binding agents such as, for example, starch, gelatine or acacia, and lubricating agents such as, for example, stearic acid or talc. The tablets may be uncoated, or they may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate alone or with a wax may be employed.

Formulations for oral use also can be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example arachis oil, peanut oil, liquid paraffin or olive oil.

Aqueous suspensions typically contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, for example, sodium carboxymethyl cellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gam acacia ; dispersing or wetting agents may be a natural- occurring phosphatide, for example, lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensation products of

ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyoxyethylene sorbitan mono- oleate. The aqueous suspensions also can contain one or more preservatives, for example, ethyl or n-propyl p- hydroxy benzoate, one or more coloring agents, one or more flavoring agents and one or more sweetening agents such as, for example, sucrose or saccharin.

Oily suspensions may be formulated by suspending the active ingredient in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oil suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents, such as those set forth above, and flavoring agents may be added to provide a palatable oral preparation. These compositions can be preserved by the addition of an antioxidant such as, for example, ascorbic acid.

Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients, for example sweetening, flavoring and coloring agents, also may be present.

The pharmaceutical compositions of the present invention also can be in the form of oil-in-water

emulsions. The oily phase can be a vegetable oil, for example, olive oil or arachis oils, or a mineral oil, for example liquid paraffin or mixtures of these. Suitable emulsifying agents may be naturally-occurring gums, for example gum acacia or gum tragacantn, naturally-occurring phosphatides, for example soya bean lecithin, and esters or partial esters derived from fatty acids and hexitol anhydrides, for example sorbitan mono-oleate, and condensation products of the said partial esters and ethylene oxide, for example polyoxyethylene sorbitan mono- oleate. The emulsions also can contain sweetening and flavoring agents.

The pharmaceutical compositions of the present invention can be in the form of syrups and elixirs, which are typically formulated with sweetening agents such as, for example, glycerol, sorbitol or sucrose. Such formulations also can contain a demulcent, a preservative and flavoring and coloring agents.

The pharmaceutical compositions can be in the form of a sterile injectable preparation, for example, as a sterile injectable aqueous or oleagenous suspension.

Suitable suspensions for parenteral administration can be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above. Formulations suitable for parenteral administration also can include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain anti-oxidants, buffers, bacteriostates, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non- aqueous sterile suspensions that can include suspending

agents, solubilizers, thickening agents, stabilizers, and preservatives. The sterile injectable preparation can be a solution or a suspension in a non-toxic parenterally- acceptable diluent or solvent, for example, as a solution in water or 1, 3-butanediol. Among the acceptable vehicles and solvents that can be employed, for example, are water, Ringer's solution and isotonic sodium chloride solution.

In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as, for example, oleic acid find use in the preparation of injectables.

The compounds of the present invention also can be administered in the form of suppositories for rectal administration of the drug. These compositions can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Such materials include, for example, cocoa butter and polyethylene glycols. Formulations suitable for vaginal administration can be presented as pessaries, tampons, creams, gels, pastes, and foams.

Formulations suitable for topical administration may be presented as creams, gels, pastes, or foams, containing, in addition to the active ingredient, such carriers as are known in the art to be appropriate.

The antiproliferative naphthoquinones of the present invention, alone or in combination with other suitable components, can be made into aerosol formulations to be

administered via inhalation. These aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like. They also can be formulated as pharmaceuticals for non-pressured preparations such as in a nebulizer or an atomizer.

The formulations can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid excipient, for example, water, for injections, immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.

Any suitable dosage level can be employed in the pharmaceutical compositions of the present invention. The dose administered to an animal, particularly a human, in the context of the present invention should be sufficient to effect a prophylactic or therapeutic response in the animal over a reasonable time frame. The amount of active ingredient that can be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. One skilled in the art will recognize that the specific dosage level for any particular patient will depend upon a variety of factors including, for example, the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination and the severity of the

particular disease undergoing therapy. The size of the dose will also be determined by the existence, nature, and extent of any adverse side-effects that might accompany the administration of a particular compound. Other factors which effect the specific dosage include, for example, bioavailability, metabolic profile, and the pharmacodynamics associated with the particular compound to be administered in a particular patient. Suitable doses and dosage regimens can be determined by comparisons to anticancer or immunosuppressive agents that are known to effect the desired growth inhibitory or immunosuppressive response. The preferred dosage is the amount which results in immunosuppression or inhibition of cancer cell proliferation, without significant side effects. In proper doses and with suitable administration of certain compounds, the present invention provides for a wide range of intracellular effects, e. g., from partial inhibition to essentially complete inhibition of cell proliferation. This is particularly important in the context of the present invention, as this differential inhibition can potentially be used to discriminate between cancer cells and highly proliferative non-malignant cells.

In the treatment of some individuals with the compounds of the present invention, it may be desirable to use a high dose regimen in conjunction with citrovorum factor rescue of non-malignant cells. In such treatment, any agent capable of rescue of non-malignant cells can be employed, such as citrovorum factor, folate derivatives, or leucovorin. Such rescue agents are well-known to those or ordinary skill in the art. A rescue agent is preferred

which does not interfere with the ability of the present inventive compounds to modulate cellular function.

The antiproliferative naphthoquinones of the present invention also can be administered in combination with other chemotherapeutic compounds such as, for example, doxifluridine, fluorouracil, methotrexate, hydroxyurea, cytarabine, cisplatin, carboplatin, mitimycins, cyclophosphamide, ifosphamide, chloroambucil, thiotepa, melphalan, doxorubicin, epirubicin, mitoxanthrone, bleomycin, daunorubicin, etoposide, vincristine, vindesine, tamoxifen, leuprolide, flutamide, goserelin, medroxyprogesterone, estramustine, megestrol acetate, and the like, as well as admixtures and combinations thereof.

The individual daily dosages for these combinations can range from about one-fifth of the minimally recommended clinical dosages to the maximum recommended levels for the entities when they are given singly.

The present invention provides a method of preventing or treating cancer in a mammal, which method involves administering a therapeutically effective amount of at least one compound of the present invention.

Preferably, the compound to be administered according to the present invention is a naphthoquinone of Formula I above, wherein Rl-Ra are the same or different and are each defined as hereinabove, and pharmaceutically acceptable salts thereof, provided that R5-R8 are not all H.

In one preferred embodiment the compound to be administered in the present method is a naphthoquinone of Formula I, wherein R2 is H, C1-C3 alkyl, F, C1, or Br ; and R3 is H, A-Z, S-Z, S-A-Z, N(B)-Z, N(B)-A-Z, O-Z, or O-A-

Z, as defined hereinabove, or a pharmaceutically acceptable salt, an ester, or a prodrug thereof, provided that R5-R8 are not all H. More preferably, R'is H or C1- C3 alkyl.

In another preferred embodiment, the compound to be administered in the present method is a naphthoquinone of Formula I, wherein R2 and R3 are the same or different and are each H, C1-C3 alkyl, or S-A-Z, as hereinabove, or a pharmaceutically acceptable salt, ester, or a prodrug thereof. More preferably, either of R2 or R3 is H or Cl-C3 alkyl, and the other is S-A-Z.

In yet another preferred embodiment, the compound to be administered in the present method is a naphthoquinone of Formula I, wherein R2 and R3 are the same or different and are each H, A-Z, S-Z, S-A-Z, N(B)-Z, N(B)-A-Z, O-Z, or O-A-Z, as defined hereinabove, and pharmaceutically acceptable salts thereof, wherein at least two of any of substituents R5-R8 is H. More preferably, R2 is H, Cl-C3 alkyl, F, Cl, or Br ; and R3 is H, A-Z, S-Z, S-A-Z, N (B) - Z, N (B) -A-Z, O-Z, or O-A-Z, as defined herein, and at least two of any of substituents R5-R8 are H. Still more preferably, R2 is H, Cl-C3 alkyl.

In yet another preferred embodiment, the compound to be administered in the present method is a naphthoquinone of Formula I, wherein R2 and R3 are the same or different and are each H, Cl-c3 alkyl, or S-A-Z, as defined herein, provided that R5-Ra are not all H and at least two of any of substituents R5-R8 are H. More preferably, either of R2 or R3 is H or Cl-C3 alkyl, and the other is S-A-Z.

The present method can be applied toward the treatment or prevention of any type of cancer, the growth

of which can be inhibited by the naphthoquinones of the present invention. Preferably, the present method is directed to the treatment or prevention of at least one cancer selected from the group consisting of hepatoma, breast cancer, lung cancer, ovarian cancer, and melanoma.

The present invention further provides a method of preventing or treating tissue transplant rejection in a mammal, which method involves administering an anti- rejection-effective amount of at least one compound of the present invention. The present method can be applied toward the treatment or prevention of any type of tissue transplant including, for example, skin grafts, arterial grafts, cardiac grafts, heart replacements, and the like.

Preferably, the present method of preventing or treating tissue transplant rejection is directed to cardiac tissue transplants.

The present invention further provides a general method of inhibiting the growth of a cell which comprises contacting the cell with a cellular growth inhibiting- effective amount of at least one compound of the present invention. The present method of inhibiting cell growth can be applied toward any suitable therapeutic and/or non-therapeutic application. Suitable non-therapeutic applications include, for example, an internal standard for a medical diagnostic kit or a medical device, an internal standard for a bioassay kit, or in research applications directed to cellular growth inhibition.

Among the preferred cell lines for which growth is inhibited according to the present method are hepatocyte cells and mammalian cancer cells, which are preferably selected from the group consisting of hepatoma cells,

breast cancer cells, lung cancer cells, ovarian cancer cells, and melanoma cells, in addition to immune cells such as, for example, lymphocytes.

Examples The following examples further illustrate the present invention but, of course, should not be construed as in any way limiting its scope.

Example 1 This example demonstrates nitration of the benzene ring of menadione to provide all four possible isomers of nitromenadione as illustrated in Fig. 2A. To a solution of menadione (201) (10. 58 g, 61. 5 mmol) in sulfuric acid (90 mL) at 0-5 °C was slowly added, with stirring, 45 mL of 90% nitric acid. After complete addition of nitric acid, the ice bath was removed and the mixture was stirred approximately 1. 5 hours at room temperature, after which the solution was poured over ice. The resulting precipitate was filtered, washed with water (0. 5-1 L), and dried under vacuum to give 13. 58 g of a yellow solid. The crude product was chromatographed on silica gel, eluting with hexame-25% ethyl acetate, and several fractions were collected. One fraction gave 0. 614 g of a bright yellow solid containing 6-nitromenadione and 7-nitromenadione, (203) and (204), respectively. A second fraction gave 5. 139 g of a yellow solid which was a mixture of isomers.

A third fraction gave 8. 832 g of a mixture of isomers containing a polar material which did not move from the origin by TLC (hexane-25% ethyl acetate). This third fraction was recrystallized from ethyl acetate/hexane to

give 2. 384 g of a yellow solid containing 5-nitromenadione and 8-nitromenadione, (202) and (205), respectively.

Example 2 This example demonstrates nitration of the benzene ring of 3-bromomenadione to provide all four possible isomers of nitromenadione as illustrated in Fig. 2A. One particular advantage to this approach is that the isolation of the pure 8-nitro species, namely 3-bromo-8- nitromenadione (210), can be accomplished without chromatography. To a solution of 3-bromomenadione (206) (10. 77 g, 43. 1 mmol) in sulfuric acid (60 mL) at 0 °C (bath temperature) was slowly added fuming nitric acid (20 mL). After complete addition, the color turned from red to orange, and a precipitate formed several minutes thereafter. After stirring 30-45 minutes at O °C, the resulting suspension was allowed to warm to room temperature and was poured over ice. The precipitate was then filtered and the filter cake washed with water (2L) and dried under vacuum to give 12. 95 g of a yellow solid containing predominantly the 5- and 8-nitro isomers, along with a smaller amount of the 6- and 7-nitro isomers. The crude solid was recrystallized from ethyl acetate/hexane to give 7. 16 g of a yellow solid containing mostly the 5- and 8-nitro isomers ( (207) and (210), respectively) along with a small amount of the 6- and 7-nitro isomers ( (208) and (209), respectively). A second recrystallization gave a mixture of, (207) and (210), the supernatant from which grew more crystals, consisting mainly of (210), which were subjected to a subsequent recrystallization from ethyl acetate/hexane to give 0. 225 g of pure (210) (m. p. 160-161

°C). Repeated recrystallizations of the crystals obtained from the second recrystallization afforded an additional 0. 365 g of pure (210). Fractions from repeated recrystallizations which were enriched in (210) were repeatedly recrystallized to afford an additional 0. 405 g of pure (210) (m.p. 160-161 °C, 92% pure by HPLC: 25h ethyl acetate/hexane, 1. 0 mL/min. , silica column : Microsorb MV-86-100-C5,5 mm, 100 A ; retention time (207) : approximately 5 min. ; retention time (210) : approximately 5. 9 min. The foregoing demonstrates that all four possible nitro isomers of 3-bromomenadione can be obtained via nitration and that the 8-nitro species can be isolated in pure form without using column chromatography.

Example 3 This example demonstrates the nitration of a mixture of 3-chloro-6-bromomenadione (211) and 3-chloro-7- bromomenadione (212) to give a mixture of 3-chloro-6- nitro-7-bromomenadione (213), 3-chloro-6-bromo-7- nitromenadione (214), 3-chloro-7-bromo-8-nitromenadione (215), and 3-chloro-5-nitro-6-bromomenadione (216), as illustrated in Fig. 2B. To a solution of (211) and (212) in conc. sulfuric acid (2 mL) at about 0 °C was added fuming nitric acid (0. 35 mL), after which the solution turned from dark red to orange and a precipitate formed after about 15 minutes of stirring. After stirring the mixture for a total of 1 hour at 0 °C, the mixture was poured over ice and the resulting solid was taken up in ethyl acetate to give a bright yellow organic layer, which was washed with water (3X), brine, dried over magnesium sulfate, and concentrated to give 0. 128 g of a bright

yellow solid. 1H NMR analysis of the crude product confirmed that the crude product was a mixture of four nitrated products, (213-216), in a ratio of 1 : 8. 5 (213) and (214) : (215) and (216).

Example 4 This example demonstrates the olefination of a mixture of compounds of the present invention using a Stille coupling, as illustrated in Fig. 2C. A dry toluene solution of 2-methyl-3-chloro-1,4-dimethoxy-6- bromonaphthalene (217), 2-methyl-3-chloro-1, 4-dimethoxy-7- bromonaphthalene (218), [CH3(CH2)2CH2]3SnCH=CH2, and (Ph3P), Pd (5. 1 mole'-.) was refluxed 20 hours, to give an olefinated product (73 yield) which was a mixture of 2- methyl-3-chloro-1, 4-dimethoxy-6-vinylnaphthalene (219) and 2-methyl-3-chloro-1,4-dimethoxy-7-vinylnaphthalene(220).

Example 5 This example demonstrates the olefination of a mixture of 3-(2-hydroxyethyl)thio-6-bromomenadione (221) and 3- (2-hydroxyethyl) thio-7-bromomenadione (222) via Stille coupling, as illustrated in Fig. 2D. A dry toluene solution of (221), (222), [CH3(CH2)2CH2]3SnCH=CH2, and (Ph3P)4Pd (7.2 mole %) was refluxed 20 hours, to give an olefinated product (13% yield) which was a mixture 3- (2- hydroxyethyl) thio-6-vinylmenadione (223) and 3- (2- hydroxyethyl)thio-7-vinylmenadione (224).

Example 6 This example demonstrates the olefination of a mixture of 3-n-butylthio-6-bromomenadione (225) and 3-n-

butylthio-7-bromomenadione (226) via Stille coupling, as illustrated in Fig. 2D. A dry toluene solution of (225), (226), [CH3(CH2)2CH2]3SnCH=CH2, and (Ph3P)4Pd (11 mole %) was heated at 90-100 °C for 20 hours, to give an olefinated product (50% yield) which was a mixture of 3-n-butylthio- 6-vinylmenadione (227) and 3-n-butylthio-7-vinylmenadione (228).

Example 7 This example demonstrates the arylation of the benzene ring of a mixture of compounds of the present invention via Suzuki coupling, as illustrated in Fig. 2E.

A dry toluene solution of 2-methyl-3-chloro-1,4-dimethoxy- 6-bromonaphthalene (217), 2-methyl-3-chloro-1,4-dimethoxy- 7-bromonaphthalene (218), C6HsB(OH) 21 and (Ph3P)4Pd (4.4 mole %) was heated at 85-90 °C for 17 hours in the presence of potassium carbonate to give an arylated product (83% yield) which was a mixture of 2-methyl-3- chloro-1,4-dimethoxy-6-phenylnaphthalene(229) and 2- methyl-3-chloro-1,4-dimethoxy-7-phenylnaphthalene(230).

Example 8 This example demonstrates arylation of the benzene ring in a mixture of compounds of the present invention via Suzuki coupling, as illustrated in Fig. 2F. A solution containing a mixture of 3-n-butylthio-6- bromomenadione (225) and 3-n-butylthio-7-bromomenadione (226) (0. 086 g, 0. 25 mmol) in dry toluene (5 mL) was added to a mixture of C6H, B (OH) 2 (0. 062 g, 0. 51 mmol), (Ph3P)4Pd (0. 015 g, 0. 013 mmol, 5. 2 mole %), and potassium carbonate (0. 052 g, 0. 38 mmol). The resulting mixture was heated at

85-90 °C for 18 hours, then for approximately 3. 5 hours at ambient temperature (22 °C) to provide a yellow-green suspension which was taken up in ethyl acetate and extracted with water (2X), dilute aqueous HC1, and brine.

The resulting organic layer was dried over sodium sulfate and concentrated to give a brown oil (0. 186 g), which was chromatographed on silica gel, eluting with hexane-5% ethyl acetate, to give a first set of impure fractions containing the desired arylated products with a small amount of impurity (0. 048 g, yellow oil) and a pure set of fractions (0. 036 g, orange oil). The impure fractions were purified on preparative TLC, eluting with hexane-5% ethyl acetate, to give the desired arylated products as a bright yellow oil (0. 038 g), affording the desired products in 87% overall yield as a mixture of 3-n- butylthio-6-phenylmenadione (231) and 3-n-butylthio-7- phenylmenadione (232).

Example 9 This example demonstrates halogenation of 2-methyl-3- chloro-1,4-dimethoxynaphthalene (233), as illustrated in Fig. 2G. To a suspension of Fe (0. 059 g, 1. 06 mmol) and (233) (0. 236 g, 1. 0 mmol) in CC14 (4 mL) at ambient temperature was added Br2 (0.078 mL, 1. 52 mmol). After stirring the mixture 16 hours at ambient temperature, the mixture was taken up in ether and the ogranic layer was washed with dilute aqueous Na2S203, water (2X), saturated aqueous NaHC03, brine, dried over magnesium sulfate, filtered, and evaporated to give an orange oil (0. 577 g), which was chromatograped on silica gel, eluting with hexane-5% ethyl acetate, to give 0. 264 g (83% yield) of a

white solid which was a mixture of 2-methyl-3-chloro-1, 4- dimethoxy-6-bromonaphthalene (217) and 2-methyl-3-chloro- 1,4-dimethoxy-7-bromonaphthalene (218), also containing a small amount of an uncharacterized impurity.

Example 10 This example demonstrates halogenation of 2-methyl-3- bromo-1,4-dimethoxynaphthalene (236), as illustrated in Fig. 2G. Applying the bromination procedure of Example 9, except replacing 2-methyl-3-chloro-1, 4- dimethoxynaphthalene with (236), a mixture of 2-methyl-3- bromo-1,4-dimethoxy-6-bromonaphthalene(237) and 2-methyl- 3-bromo-1,4-dimethoxy-7-bromonaphthalene(238) was obtained in 780 overall yield after chromatographic purification.

Example 11 This example demonstrates the preparation of amide- substituted naphthoquinones of the present invention in two steps via reduction of the nitronaphthoquinone to provide the corresponding aminonaphthoquinone, followed by acylation with an appropriate acylating agent to provide the corresponding amide, as illustrated in Fig. 2H. A suspension of 3-bromo-5-nitromenadione (207) (0. 226 g, 0. 76 mmol) and 5% Pd/C (0. 024 g) in methylene chloride (35 mL) was stirred over a hydrogen atmosphere (39 psi) for 9 hours, which resulted in a hydrogen pressure drop of about 2 psi. The resulting suspension was filtered through Celite, with ethyl acetate rinses, and concentrated to give 0. 226 g of a crude product (purple/black solid) containing of 3-bromo-5-aminomenadione (239). After

dissolving the crude product (containing 239) in methylene chloride (35 mL), acetic anydride was added (1. 2 mL, 12. 7 mmol) and the mixture was stirred 12 hours, after which additional acetic anhydride was added (0. 9 mL, 9. 5 mmol) and the mixture was stirred for an additional 7 hours, followed subsequently by two successive 1 mL additions of acetic anhydride (12. 2 mmol total) and continuation of stirring for an additional 12-36 hours following each successive addition. The resulting mixture was concentrated to give 1. 6 g of an orange solid suspended in liquid, and was chromatographed on silica, eluting with hexane-25% acetone, to give 0. 050 g of 3-bromo-5- acetamidomenadione (240) (21% yield, m. p. : 197-199 °C).

Example 12 This example demonstrates the preparation of an aminonaphthoquinone of the present invention via reduction of the corresponding nitronaphthoquinone. A suspension of 3-bromo-8-nitromenadione (210) (0. 302 g, 1. 01 mmol) and 10% Pd/C (0. 097 g) in methylene chloride (10 mL) was stirred over a hydrogen atmosphere (balloon, slightly above atmospheric pressure) for 5 hours, after which the mixture was filtered (Celite), with subsequent methylene chloride and acetone rinses of the Celite bed.

Concentration of the filtrate resulted in a crude product (0. 384 g, purple solid) which was a mixture of 3-bromo-8- aminomenadione (242) with residual starting material (210).

Example 13 This example demonstrates the acylation of 3-bromo-5- aminomenadione (239) with hexanoyl chloride as illustrated in Fig. 2H. To a solution of (239) in pyridine is added hexanoyl chloride and the mixture is stirred until the reaction is complete. The reacted mixture is taken up in ethyl acetate and the organic layer is washed with dilute aqueous acid to remove the pyridine, extracted with aqueous sodium bicarbonate, brine, dried over Na2SO4, filtered, and evaporated to give a crude product containing 3-bromo-5-n-hexanamidomenadione (241).

Example 14 This example demonstrates the acylation of 3-bromo-8- aminomenadione (242) with acetyl chloride as illustrated in Fig. 2H. To a solution of (242) in pyridine is added hexanoyl chloride and the mixture is stirred until the reaction is complete. The reacted mixture is taken up in ethyl acetate and the organic layer is washed with dilute aqueous acid to remove the pyridine, extracted with aqueous sodium bicarbonate, brine, dried over Na2SO4, filtered, and evaporated to give a crude product containing 3-bromo-8-acetamidomenadione (243).

Example 15 This example demonstrates the acylation of 3-bromo-8- aminomenadione (242) with hexanoyl chloride as illustrated in Fig. 2H. To a solution of 242 in pyridine is added hexanoyl chloride and the mixture is stirred until the reaction is complete. The reacted mixture is taken up in ethyl acetate and the organic layer is washed with dilute

aqueous acid to remove the pyridine, extracted with aqueous sodium bicarbonate, brine, dried over Na2SO4, filtered, and evaporated to give a crude product containing 3-bromo-8-n-hexanamidomenadione (244).

Example 16 This example demonstrates halogenation of the 3- position of 2-methyl-1,4-dimethoxymenadione (301), as illustrated in Fig. 3A. To a solution of (301) (0. 609 g, 3. 01 mmol) in acetic acid (5 mL) at ambient temperature was slowly added thionyl chloride (0. 3 mL, 3. 7 mmol) and the mixture was stirred 3 hours at ambient temperature.

The mixture was diluted with ether and the organic phase was extracted with water, saturated aqueous NaHC03, brine, dried over magnesium sulfate, filtered, and concentrated to give 0. 708 g of a crude product (yellow solid). The crude product was chromatographed on silica gel, eluting with hexane-5 ethyl acetate, followed by hexane-7% ethyl acetate, to give 0. 574 g of the desired product 2-methyl- 3-chloro-1,4-dimethoxynaphthalene (233) as a white solid in 81 yield.

Example 17 This example demonstrates halogenation of the 3- position of menadione (201), as illustrated in Fig. 3B.

To a solution of (201) (10. 32 g, 60 mmol) and sodium acetate (19. 7 g, 240 mmol) in acetic acid (200 mL) at ambient temperature, was slowly added Br2 (4 mL, 78 mmol) to give a red solution which was stoppered and stored in the dark for one week. The resulting mixture produced the desired product as bright yellow needles. The orange

supernatant was decanted and the yellow needles were dried under vacuum to give 3-bromomenadione (206) (10. 0 g, 66% yield, m.p.:151-152°C).

Example 18 This example demonstrates the oxidative conversion of a 1, 4-dimethoxynaphthalene of the present invention to a naphthoquinone of the present invention, as illustrated in Fig. 3C. To a solution containing a mixture of 2-methyl- 3-chloro-1,4-dimethoxy-6-bromonaphthalene(217) and 2- methyl-3-chloro-1, 4-dimethoxy-7-bromonaphthalene (218) (0. 105 g, 0. 33 mmol) in dry benzene (4 mL) at ambient temperature was added FeCl3 (0.215 g, 1. 33 mmol). After stirring the mixture 2 hours at ambient temperature, the mixture was diluted with ether and the organic phase was extracted with water (2X), saturated NaHC03, brine, dried over magnesium sulfate, and concentrated to give 0. 091 g of a mixture of 3-chloro-6-bromomenadione (211) and 3- chloro-7-bromomenadione (212) as a yellow solid in 96% yield.

Example 19 This example demonstrates synthetic modification of the 3-position of menadione derivatives via nucleophilic displacement of a halogen at the 3-position, as illustrated in Fig. 3D. To a solution of 3-bromo-5- nitromenadione (207) (0. 098 g, 0. 33 mmol) and imidazole (0. 028 g, 0. 41 mmol) in tetrahydrofuran (2 mL) at ambient temperature was added 2-mercaptoethanol (0. 028 mL, 0. 40 mmol) and the mixture was stirred for 1 hour. The mixture was diluted with ethyl acetate and the organic phase was

extracted with water (2X), dried over sodium sulfate, and concentrated to give a brown oil (0. 109 g).

Chromatography on silica gel, eluting with hexane-30% ethyl acetate, afforded 0. 072 g of the desired product 3- (2-hydroxyethyl) thio-5-nitromenadione (302) as a yellow solid (74k yield; m.p.: 117-118 °C).

Example 20 This example demonstrates synthetic modification of the 3-position of menadione derivatives via nucleophilic displacement of a halogen at the 3-position, as illustrated in Fig. 3D. To a solution of 3-bromo-8- nitromenadione (210) (0. 151 g, 0. 51 mmol) and imidazole (0. 034 g, 0. 49 mmol) in dry methylene chloride (1. 5 mL) at ambient temperature was added 2-mercaptoethanol (0. 035 mL, 0. 49 mmol) and the mixture was stirred for 0. 5 hours. The mixture was diluted with ethyl acetate and the organic phase was extracted with dilute aqueous acid, water (2X), brine, dried over sodium sulfate, and concentrated to give an orange/brown oil (0. 189 g). Chromatography on silica gel, eluting with hexane-35 ethyl acetate, afforded 0. 125 g of the desired product 3- (2-hydroxyethyl) thio-8- nitromenadione (303) as a yellow solid (83o yield ; m. p..

131-132 °C).

Example 21 This example demonstrates synthetic modification of the 3-position of menadione derivatives via nucleophilic displacement of a halogen at the 3-position, as illustrated in Fig. 3D. To a solution of 3-bromo-5- acetamidomenadione (240) (0. 050 g, 0. 16 mmol) and

imidazole (0. 017 g, 0. 25 mmol) in tetrahydrofuran (3 mL) at ambient temperature was added 2-mercaptoethanol (0. 023 mL, 0. 33 mmol) and the mixture was stirred for 2 hours.

Additional imidazole (0. 041 g, 0. 60 mmol) and 2- mercaptoethanol (0. 046 mL, 0. 66 mmol) was added and the mixture was stirred an additional 24 hours. The mixture was diluted with ethyl acetate and the organic phase was extracted with dilute aqueous HC1, water, brine, dried over sodium sulfate, and concentrated to give a brown oil (0. 058 g). Chromatography on silica gel, eluting with hexane-30% ethyl acetate, afforded 0. 014 g of the desired product 3- (2-hydroxyethyl) thio-5-acetamidomenadione (304) as an orange solid (29% yield; m.p.: 137-138 °C).

Example 22 This example demonstrates synthetic modification of the 3-position of menadione derivatives via nucleophilic displacement of a halogen at the 3-position, as illustrated in Fig. 3D. To a solution of 3-bromo-8- acetamidomenadione (243) (0. 062 g, 0. 20 mmol) and imidazole (0. 016 g, 0. 24 mmol) in tetrahydrofuran (2 mL) at ambient temperature was added 2-mercaptoethanol (0. 016 mL, 0. 23 mmol) and the mixture was stirred for 2-3 hours.

The mixture was diluted with ethyl acetate and the organic phase was extracted with water (2X), brine, dried over sodium sulfate, and concentrated to give a brown-yellow solid (0.068 g). Chromatography on silica gel, eluting with hexane-30% ethyl acetate, afforded 0. 024 g of the desired product 3- (2-hydroxyethyl) thio-8- acetamidomenadione (305) as an orange solid (40% yield ; m. p. : 130-136 °C).

Example 23 This example illustrates the antiproliferative activity of the compounds of the present invention.

Representative naphthoquinones of the present invention were tested for growth inhibition activity with respect to normal liver cells (Heps) and cancer cells (Hep 3B). The IDso's of the tested compounds with respect to the Heps cell line were determined by a standard DNA synthesis assay based on [3H] thymidine incorporation. The IDso's for the Hep 3B cancer cell line were determined by a standard cell growth inhibition assay, as disclosed in Nishikawa et al., J. Biol. Chem. , 270, No. 47,28304-29310 (1995). The inhibitory data is shown in below Table 1.

Table 1 Compound/ Entr Comments Structure Hep Heps y 3B 0 I SOH 23a Comp. 308 5. 6 16 (Fig. 3D) * 0 1 : 1 mixture of compounds 302 23b and 303 (Fig. 5 7 3D) o, N Y 0 1 : 1 mixture of compounds 306 O AS OH 23c and 307 4. 3 8 (Fig. 3D) H O 8 1 : 1 mixture of compounds 304 O S - OH 23d and 305 1. 5 0. 3 (Fig. 3D) I 1 : 1 mixture of isomers at 23e positions 6 and ar X ~ 7) õ 0 1 : 1 mixture of isomers at 23f positions 6 and o 1 : 1 mixture of isomers at FN AS - OH 23g positions 6 and 7) õ 0

The above data clearly demonstrate the potent antiproliferative activity of the naphthoquinones of the present invention. For example, entry 23d demonstrates that the mixture of compounds (304) and (305) (IDso = 1. 5 jj. M) of the present invention possess unprecedented antiproliferative activity against Hep 3B. Heretofore,

the most potent known napthtoquinone against the hepatoma cell line is (308) (IDso = 5.6 pM), in which the benzene ring is unsubstituted, and is nearly four fold less active than compounds (304) and (305). It is predicted that further modification of the benzene ring according to the present invention will further enhance antiproliferative activity, and that the compounds of the present invention will demonstrate in vivo efficacy in the prevention and treatment of numerous different types of cancers.

Example 24 This example demonstrates the activity of compound (308) (Fig. 3D), and two other related naphthoquinones, against a variety of different cancer cell lines. Using the cell growth inhibition assay described in Example 23, the compounds were tested against different cancer cell lines, including Huh 7, Mahlavu, Hep G2, PLC/PRF/5, H4E, HTC, and 7777. The IDso's are shown below in Table 2.

Table 2 IDso (tM) R3= R3= R3= Sw Sm S~ Line (comp. 308) Line Line Huh 7 10. 2 13. 5 6. 0 Mahlavu 5. 0 20. 2 5. 0 Hep G2 12. 2 8. 0 8. 1 PLC/PRF/24. 3 6. 7 12. 0 5 H4E 24. 4 20. 2 16. 0 HTC 16. 2 13. 5 24. 2 7777 22.3 18.8 15.0

The above data demonstrate that naphthoquinones without benzene ring substitution have the ability to inhibit the growth of a broad range of different types of cancer cells. It is predicted that the naphthoquinones of the present invention also will inhibit the growth of a broad range of cancer cells. As demonstrated in Example 23, compounds of the present invention will likely be more potent against such cell lines than the above-tested compounds lacking substitution on the benzene ring.

Example 25 This example demonstrates the effect of synthetic modifications at R3 on the antiproliferative activity of 2-methylnaphthoquinone. It is predicted that modifications of R3, in combination with synthetic modification of the benzene ring according to the present invention, will provide compounds with equipotent or

enhanced antiproliferative activity over similar compounds lacking modification of the benzene ring. The IDso's of the compounds tested in this example were determined for Hep 3B and Heps, according to Example 23. The results are shown below in Table 3.

Table 3 IDso ( pM ) Entry R3 Hep 3B Heps 25a'65 140 25b 16 34 H I 25c RN OH 35 87 ,/o / 25d'20 84 25e 0 60 50 25f ! s 14 37 s 25g 63 34 H 25h 6 18 25i SOMe '9 15 9 15 0 25j ;/So^ 13 17 o 25k ,/s > N, " 20 15 ! N H HN 251 10 22 o o

The above data demonstrate that diverse synthetic modification of R3 can be applied to naphthoquinones while maintaining micromolar antiproliferative activity. It is predicted that similar R3 modifications in combination with appropriate benzene ring modification will result in equipotent or enhanced antiproliferative activiy.

Synthetic modification of the compounds of the present invention also can be directed to refining the pharmacological properties of the molecule (e. g., bioavailability and enzymatic stability).

Example 26 This example demonstrates generally that naphthoquinones can retain activity as cell growth inhibitors when the naphthoquinone ring is in different oxidation states. It is predicted that the compounds of Formula (I), (II), and (III) also will exhibit potent antiproliferative activity irrespective of the oxidation state of the naphthoquinone ring. Using the assays described in Example 23, various compounds were tested against Hep 3B and Heps cell lines. The results are shown below in Table 4.

Table 4

Compound/ Entr Comments Structure Hep Heps y 3B 1, 4- 26a naphthoquino 15 28 ne o sw 26b naphthoquino O ° 29 - ne oxide 0 H dihydro- < so ~- 26c naphthoquino 35 ne OH OPOK dihydro- 26d naphthoquino < 11 - ne Opo3K2 diphosphate The above date clearly demonstrate that naphthoquinones can maintain micromolar cell growth inhibitory activity irrespective of the oxidation state of the naphthoquinone. The 1,4-napthoquinone (entry 26a), the corresponding oxide (entry 26b), and the corresponding dihydronaphthoquinone (entry 26c) exhibit micromolar antiproliferative activity against the Hep 3B cell line (15-35 tM). It is expected that the benzene ring- substituted compounds of the present invention will maintain superior antiproliferative activity irrespective of the oxidation state of the naphthoquinone ring.

Example 27 This example demonstrates that napthoquinones can maintain inhibitory activity against cell growth when R2

is modified. Using the assays described in Example 23, various compounds were tested against Hep 3B and Heps cell lines. The results are shown below in Table 5.

Table 5 Compound/ IDso (pM) Entr Comments Structure Hep Heps y 3B s 27a R2 = methyl < 15 28 o o 27b R2 = H 5 30 0 s o 27c Ruz - methyl OH S. 6 16 (comp. 308) o SOH 27d R2 = H X s OH 3 5 22 o 0 The above data clearly demonstrate that synthetic modification of R'can be applied to naphthoquinones generally without sacrificing antiproliferative activity.

For example, entries 27c and 27d above show that the 3- hydroxyethylthio naphthoquinones with and without a methyl group at Ruz exhibait potent antiproliferative activity against Hep 3B (5.6 µM and 3. 5 M, respectively). It is expected that R2 modification of the napthoquinones of the present invention can be applied to obtain optimum antiproliferative activity with respect to a particular cell line. R2 also can be modified to obtain other

desirable characteristics such as, for example, improved selectivity against cancer cells, improved solubility (for drug delivery), and/or improved bioavailability.

Example 28 This example demonstrates generally the inhibitory activity of naphthoquinones against lymphocyte proliferation, and further demonstrates the ability of naphthoquinones to prevent acute organ allograft rejection. Compound (308) (Fig. 3D) was tested for its cell growth inhibitory effect against lymphocytes in vitro and for its ability to prolong allograft survival (i. e., prevent allograft rejection). Variable concentrations of (308) were added to a mixed lymphocyte reaction and the cells were harvested at d5 post-culture for determination of [3H] thymidine incorporation. Complete (1000) inhibition of lymphocyte proliferation was discerned when (308) was used at a dose as low as 2ng/ml (8nM).

Heterotopic cardiac transplants were performed across C57BL/10 (H-2b)oC3H (H-2k) mice strain combinations : allograft function was monitored by periodic palpation and rejection was defined by cessation of cardiac impulses wheich was confirmed by laparotomy. The treatment and outcome in each group is depicted in Table 6 (below). The untreated control group (Group 28-I) was compared with the treated groups (Groups 28-11 and 28-III). The treated groups received 3 mg/kg of compound (308) intramuscularly at various time points after the transplant (Post Tx).

All the members of Group 28-11 were treated with compound (308) on the day of the transplant (dO-Post Tx). Each member of Group 28-III was treated with compound (308) at 1,0, 1, and 7 days, respectively, after the transplant.

The results of the allograft survival experiment are shown below in Table 6.

Table 6 n Treatment Graft Group (number of with Comp. Survival mice) 308 (days + SD) (3 mg/kg <BR> <BR> i. m.) <BR> <BR> <BR> <BR> 28-16control7.53.2 28-11 5 dO-Post Tx 23.2 + 12.5 28-III 4 d-1, 0, 1, and 21.0 + 3.6 7 post-Tx The above data clearly demonstrate that compound (308) exhibits profound antiproliferative activity in vitro (100o inhibition of lymphocyte proliferation at 2ng/mL). The in vitro activity translates to a significant in vivo prolongation of allograft survival (21-23 days vs. 7. 5 days without treatment). It is expected that the benzene ring-modified compounds of the present invention will exhibit potent activity against lymphocyte proliferation and will be potent inhibitors of acute transplant rejection.

Example 29 This example demonstrates the improved antiproliferative activity of the compounds of the compounds of the present invention, when compared with a naphthoquinone lacking modification of the benzene ring.

Applying the growth inhibition assay described in example 23, three different naphthoquinones where tested and compared for growth inhibition of hepatoma cells, the

results of which are graphically depicted in Figure 4.

Figure 4 is a graphical representation of the rate of hepatoma cell growth at different concentrations of each of the tested compounds. The symbol represents compound (308) (FIG. 3d), the symbol represents a 1 : 1 mixture of compounds (302) and (303), (Fig. 3D), the symbol ("") represents a 1 : 1 mixture of compounds (304) and (305). The data in figure 4 clearly demonstrate that modification of the benzene ring results in a significant improvement in anti-proliferative activity at low concentrations, for example, at less than 10 pM, the mixture of compounds (302) and (303), and the mixture of compounds (304) and (305), inhibited the growth of hepatoma cells down to 40%, whereas compound (308) (lacking substitution on the benzene ring) was virtually ineffective at inhibiting the growth of the hepatoma cells (95% of control).'It is predicted that further modification of the benzene ring will result in further enhancement of anti-proliferative activity against hepotoma and other cancer cell lines.

All of the references cited herein, including patents, patent applications, and publications, are hereby incorporated in their entireties by reference.

While this invention has been described with an emphasis upon preferred embodiments, it will be obvious to those of ordinary skill in the art that variations of the preferred embodiments may be used and that it is intended that the invention may be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications encompassed within the spirit and scope of the invention as defined by the following claims.