1. Field of the Invention

The present invention relates to 2-aryl— 4- quinolone compounds, and to use of the compounds as anti-tumor agents.

2. References

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3. Background of the Invention

Quinolones have been widely studied as anti¬ bacterial agents. The antibacterial activity of quinolones appears to be related, at least in part, to the ability of the compounds to bind to and inhibit DNA gyrase. The inhibitory activity of the compound can be altered by the R-group substituents at ring positions 6, 7, and 8 (seen in Figure 2A) . Fluorine in position 6 is particularly effective in enhancing antibacterial ability (Neu) .

One quinolone which has been studied as an anti¬ bacterial agent is 2-phenyl-4-quinolone, which can tautomerize to 2-phenyl-4-hydroxyquinoline. Several methods of synthesis of 2-phenyl-hydroxyquinoline have been proposed. For example condensation of an arylamine with ethyl benzoylacetate in the presence of polyphosphoric acid yields small amounts of the corresponding beta-arylaminocinna ates, which can then be cyclized to 2-phenylhydroxyquinoline in the presence of polyphosphoric acid (Staskun, et al. ; see also Venturella, et al. , 1975). 4-Methoxy-2- quinoline, in particular, can be thermally demethylated to yield 4-quinolone.

Alternate methods proposed by Fuson, et al . involve heating ethyl anthranilate with phenyl ether,

or anthranilic acid with acetophenone diethyl acetal, or ethyl anthranilate and acetophenone. Cyclization to yield the quinoline is accomplished by Claisen condensation (Fuson, et al . ) . In particular, the preparation of 2-phenyl-7-methoxy-4-quinolone from ethyl benzoyl acetate is discussed in the literature (Venturella, et al. , 1970).

A method for synthesizing 4-quinolones directly involves a variation of the von Niementowski synthesis and has been proposed by Chong, et al. An anthranilamide is converted to an imine which is then treated with LDA to yield various substituted 4- quinolones (Chong, et al.).

4. Summary of the Invention

In accordance with one aspect of the invention, it has been discovered that 2-ary1-4-quinolone compounds are effective in treating tumor cells, by inhibiting tumor cell growth. In this aspect, the invention includes a method of treating a tumor in a mammalian subject, by administering to the subject, a 2-ary1-4-quinolone compound, in an amount effective to reduce tumor growth in the subject.

In one general embodiment, the 2-ary1-4- quinolone compound has one of the forms:

wherein o, R R ₂ , and R ₃ are each selected from the group consisting of H, F, Cl, Br, I, OH, OCH ₃ , OCF ₃ , OCH ₂ CH ₃ , CH ₃ , CF ₃ , CH ₂ CH ₃ , SCH ₃ , SCH ₂ CH ₃ , NH ₂ , NHCH ₃ , N(CH ₃ ) ₂ , NHCH ₂ CH ₂ N(CH ₃ ) ₂ , NHC(0)CH ₃ ,

O. N Λ H HH and H NCH ,

R,, R ₅ , _$ , R ₇ , and R ₈ are each selected from the group consisting of H, F, Cl, Br, I, OH, OCH ₃ , OCF ₃ , OCH ₂ CH ₃ , CH ₃ , CF ₃ , CH ₂ CH ₃ , SCH ₃ , SCH ₂ CH ₃ , NH ₂ , NHCH ₃ , N(CH ₃ ) ₂ , OCH ₂ Ph; or two of said R ₄ , R ₅ , ^ R _7/ and R ₈ together are OCH ₂ CH ₂ 0, OCH ₂ CHPhO, or NCH ₂ CH ₂ 0; and X is 0, S, or NH.

In another general embodiment, the 2-aryl-4- quinolone is a dioxalane 2-aryl-4-quinolone having one of the forms:

wherein R ₄ , R ₅ , Rg, R ₇ , and R _g are each selected from the group consisting of H, F, Cl, Br, I, OH, 0CH ₃ , OCF ₃ , OCH ₂ CH ₃ , CH ₃ , CF ₃ , CH ₂ CH ₃ , SCH ₃ , SCH ₂ CH ₃ , NH ₂ , NHCH ₃ , N(CH ₃ ) ₂ , OCH ₂ Ph; or two of said R ₄ , R ₅ , R^ R ₇ , and R ₈ together are OCH ₂ CH ₂ 0, OCH ₂ CHPhO, or NCH ₂ CH ₂ 0; and X is 0, S, or NH.

In another aspect of the invention, it has been discovered that 2-ary1-4-quinolone compounds are effective to overcome multiple drug resistance in tumor cells, effectively inhibiting tumor cells which are drug-resistant to doxorubicin, vincristine, and/or VP-16 (an etoposide anti-tumor agent) . In this aspect, the invention includes, in a treatment regimen for inhibiting growth of a tumor in a subject, in which the tumor has become progressively more refractory to inhibition of growth by doxorub¬ icin, vincristin, or VP-16, a method for inhibiting the growth of the tumor, by administering to the subject, a 2-aryl-4-quinolone compound, in an amount effective to inhibit tumor growth in the treated subject.

The invention also includes a pharmaceutical composition which contains a 2-aryl-4-quinolone compound of the invention, for use in inhibiting the growth of tumor cells.

In another aspect, the invention includes novel anti-tumor compounds having structures I-VIII shown above. The hovel compounds include structures I-IV having alkylated amino groups at the 5, 6, 7, or 8 quinolone ring position, and the dioxalane quinolone compounds having structures V-VIII.

These and other objects and features of the invention will become more fully apparent when the

following detailed description of the invention is read in conjunction with the accompanying drawings.

Brief Description of the Drawings Figure 1 shows a general structure for the compounds useful in the method of the invention;

Figures 2A-2D illustrate four embodiments of the Fig. 1 structure which are useful for treatment of tumor cells (structures I-IV) ; Figures 3A-3D illustrate four additional embodiments which are useful for treatment of tumor cells (structures V-VIII) ;

Figure 4 illustrates one preferred method of synthesis of compound I shown in Figure 1A; Figure 5 shows the synthetic method for preparing 2-phenol-4-quinolone having a hydroxyl group at the 6, 7, or 8 ring position;

Figure 6 illustrates a method of synthesis of a pyridine analog of benzoyl acetate ethyl ester for use in the reaction scheme of Figure 4, for synthesis of a 2-pyridino-4-quinolone compound;

Figure 7 shows an alternate synthesis of a 2- pheny1-4-quinolone compound;

Figure 8 shows another synthesis scheme which is useful for preparing 2-aryl-4-quinolone compounds of the invention;

Figure 9 illustrates a method of synthesis of a compound having structure V shown in Figure 3A;

Figures 10A and 10B illustrate two exemplary dioxalane 2-aryl-4-quinolone compounds of the invention; and

Figure 11 illustrates methods for alkylating 2- phenol-4-quinolone at the ring nitrogen and 4- position oxygen.

Detailed Description of the Invention I. 2-Aryl-4-0uinolone Compounds

This section describes synthetic methods for preparing 2-aryl-4-quinolone compounds useful for treatment of tumors.

The 2-ary1-4-quinolone compounds of the invent¬ ion have the general structure shown in Figure 1. With reference to Figure l, the compounds includes a quinolone fused-ring structure, substituent groups attached to ring atoms 5, 6, 7, and 8, and a selected aryl group (Ar) attached to ring atom 2.

Figures 2A-2D show four general embodiments of the structure shown in Figure 1. In each embodiment, substituents Ro through R ₃ on ring atoms 5, 6, 7, and 8 are each selected from the group consisting of H, F, Cl, Br, I, OH, OCH ₃ , OCF ₃ , OCH ₂ CH ₃ , CH ₃ , CF ₃ , CH ₂ CH ₃ , SCH ₃ , SCH ₂ CH ₃ , NH ₂ , NHCH ₃ , N(CH ₃ ) ₂ , NHCH ₂ CH ₂ N(CH ₃ ) ₂ ,

In structure (I) shown in Figure 2A, the 2-aryl group is a phenyl ring in which substituents R, through R ₈ are selected from the group consisting of H, F, Cl, Br, I, OH, OCH ₃ , OCF ₃ , OCH ₂ CH ₃ , CH ₃ , CF ₃ , CH ₂ CH ₃ , SCH ₃ , SCH ₂ CH ₃ , NH ₂ , NHCH ₃ , N(CH ₃ ) ₂ , 0CH ₂ Ph. Figures 2B through 2D show additional structures, wherein the aryl group is a pyridine ring (Fig. 2B) , an indole ring (Fig. 2C) , and a furan (X = O) , thiophene (X = S) , or pyrrole (X = N) ring (Fig. 2D) . Figures 3A-3D show four additional embodiments of the Figure 1 structure, wherein a methylenedioxy moiety (-OCH ₂ 0-) forms a five-me bered ring with the 6- and 7-ring atoms of the quinolone backbone (structures V-VIII) . These compounds are referred to as "dioxalane, 2-aryl-4-quinolones". The 2-aryl

groups in these compounds are the same as those in compounds I-IV.

Also as discussed below, substitution at either the ring nitrogen (1 position) or the C-4 oxygen reduces or largely inactivates the tumor-cell cytotoxicity of the compounds. This indicates that the 1-position secondary amine and the 4-position keto group are important for anti-tumor activity.

Studies conducted in support of the invention, and reported in Section II below, indicate that anti- tumor activity, as evidenced by cytotoxicity and inhibition of cell proliferation against a variety of tumor cell lines, is retained by a number of exemplary 2-aryl-4-quinolone compounds. Considering general methods for synthesizing the compounds of the invention. Figure 4 shows one general synthetic scheme for producing compounds of the type illustrated in Figure 2A. This approach is detailed in Example 1. Briefly, aniline (IX) is condensed with benzoyl acetate ethyl ester (X) to produce the intermediate product XI, followed by a thermal cyclization in diphenyl ether at 240-250°C, to produce the desired 2-aryl-4-quinolone (I _H ) .

Using the method shown in Figure 4, 6-subst- ituted 2-aryl-4-quinolones and 8-substituted 2-aryl- 4-quinolones can be prepared from p-substituted anilines and o-substituted anilines, respectively. 5- and 7-Substituted 2-phenyl-4-quinolones can be prepared from m-substituted aniline starting material. Details of these syntheses are given in Example 1.

Figure 5 illustrates synthesis of 2-phenyl-4- quinolone compounds having an OH substitution at the 5, 6, 7, or ,8 position (XII). As seen, these

compounds can be prepared directly by demethylation of the corresponding methoxy precursors (XIII) with KOH in ethylene glycol (Venturella) . Details are given in Example 2. The methoxy precursors can be prepared as described in Example 1.

Table 1 identifies several exemplary 2-phenyl-4- quinolones (structure I in Figure 2A) formed by the above methods. In this table, R _N indicates substit¬ utions at the ring nitrogen, and RQ, substitutions at the 4'-position of the pendant ring. R,, R ₂ , and R ₃ indicate substituents at the 6-8 ring positions, respectively (Figure 1) , and Re indicates substitu¬ tions on the 2-phenyl ring.

Table 1

2-aryl-4-quinolone compounds in which the pendant aryl group is a pyridine ring can be prepared by similar methods. With reference to Figure 6, the pyridine analog (XV) of benzoyl acetate ethyl ester can be formed by reaction of nicotinic acid ethyl ester (XIV) with ethyl acetate in ethanol, in the presence of sodium ethoxide, according to published methods (Hurd) . Compound XV is then reacted with aniline or a substituted aniline, as illustrated in Figure 4, to yield the desired 2-pyridy1-4-quinolone compound (III) . The position of the ring nitrogen in the compound is determined by the ring position of

the ethyl ester group on the pyridine ring, as can be appreciated.

Figure 7 illustrates an alternative method for producing 2-aryl-4-quinolone compounds, and in part- icular, compounds substituted in the pendant phenyl ring. In this method, the hydroxyl group of p- hydroxyacetophenone (XVII) is protected by treatment with methoxymethyl chloride in the presence of NaH to give p-methoxymethoxy acetophenone (XVIII) . Treatment of XVIII with lithium diisopropylamine

(LDA) at -30°C generates the enolate form of XVIII, which is then reacted with N-methylisatoic anhydride (XIX) at -65°C to yield 2-(4'-methoxymethoxyphenyl)- l-methyl-4-quinolone (XXII) . The structure of XX was established based on its ^-NMR, IR, and MS spectral data. Further treatment of compound XX with concentrated HC1 at room temperature, followed by neutralization with NaHC0 ₃ gave the target compound 2-(4'-hydroxyphenyl)-l-methyl-4-quinolone (XXI). The identity of the compound (compound 50 in Table I) with an authentic sample of reevesianine (Wu) was established by a direct spectral comparison. Details of the method are given in Example 4 for the synthesis of compounds 49 and 50 in Table 1. Figure 8 illustrates a synthesis scheme for preparing 2-aryl-4-quinolone compounds which contain alkylated amine substituents (e.g., dimethylamine, morpholine, piperidine, and the like) attached at ring positions 5, 6, 7, and 8. With reference to Figure 8, for synthesizing a quinolone compound having a dimethylamino group at ring position 6 (R _j = N(CH ₃ ) ₂ ) , 5-chloro-2-nitroacetophenone (XXXI) is reacted with dimethylamine to form 5-dimethylamino acetophenone derivative XXXII. Following reduction

of the nitro group in XXXII by hydrogenation over palladium/carbon catalyst, the 2-amino-5-dimethyl- a inoacetophenone product (XXXIII) is reacted with 3- methylbenzoyl chloride to form the condensation product shown at structure XXXIV. Treatment of XXXIV with base (e.g., basic tert-butyl alcohol) leads to cyclization of XXXIV to form the 2-aryl-4-quinolone product XXXV.

It can be appreciated that a variety of other quinolone compounds can be synthesized using the synthesis method shown in Figure 8. Use of 3-chloro- 2-nitroacetophenone in place of compound XXXI leads to formation of an 8-dimethylamino quinolone compound. Use of 4-chloro-2-nitroacetophenone or 6- chloro-2-nitroacetophenone leads to formation of the corresponding 7- or 5-dimethylamino quinolone compound. It can be appreciated that compounds having other alkylated amine substituents at ring positions 5, 6, 7, and 8 can be prepared by using the desired alkylated amine in place of dimethylamine in the first reaction shown in Figure 8. For example, use of morpholine in place of dimethylamine in Figure 8 leads to formation of a 6-morpholino-2-aryl-4- quinolone. Finally, the synthesis scheme in Figure 8 is useful for preparing quinolone compounds having a variety of 2-aryl groups. For preparing 2-phenyl compounds having a non-hydrogen substituent at the 2 ' - or 6'-ring position of the 2-phenyl group, an ortho-substituted benzoyl chloride compound is used in place of the 3-methyl benzoyl chloride compound shown in Figure 8. Likewise,- for preparing a quinolone compound having a p-substituted 2-phenyl group, the appropriate p-substituted benzoyl chloride compound can be used. For preparing compounds having

structure III (Figure 2C) , indole-3-carboxylic acid chloride can be used instead of a benzoyl chloride compound. Likewise, for preparing a 2-furan, 2- thiophene, or 2-pyrrole quinolone compound, the appropriate acid chloride derivative is used.

Synthesis of the dioxalane 2-aryl-4-quinolone compound V shown in Figure 3A was carried out according to published procedures (Chong) , as illustrated in Figure 9. Typically, the dioxalane anthranilamide (XX) is converted into an intermediate imine (XXIV) by reaction with arylmethylketone (XXIII) . The imine is then converted to the desired dioxalane 2-ary1-4-quinolone compound (V) by reaction with lithium diisopropylamine (LDA) . Figures 10A and 10B show two exemplary dioxalane

2-aryl-4-quinolone compounds of the invention. Compound XXV, having a methoxy group at the 3' position of the 2-phenyl group was prepared, as described above, by reaction of dioxalane anthranilamide (XXII) with 3'-methoxy acetophenone, followed by treatment with LDA. Compound XXVI, having a dimethylamine group at the 3' position of the pendant phenyl group was similarly prepared, using 3'-(N,N-dimethylamino) acetophenone starting material.

Figure 11 illustrates modifications of 2-phenyl- 4-quinolone (I) to produce alkylation with various R groups (see Table 2) at the ring nitrogen or the 4- position oxygen. Treatment of I with NaH in DMF followed by methylation with Mel led to the formation of XXVIII (nvp 145-147°C) and XXIX (R = Me, p 42- 44°C) in a ratio of 2:3. Elemental and mass spectral (m/z 235, M+) data established the molecular formula of C ₁₆ H ₁₃ NO for both compounds, suggesting that they

are possibly the N- and O-methyl derivatives. The presence of a W-methyl group at 53.35, a proton at S6.23 (H-3) , a one-proton doublet at 58.46 (J = 8.0 Hz) (H-5) , and eight aromatic protons at 57.24-7.81 (m) led to the assignment of XXVIII (compound 35 in Table 1) as N-methyl-2-phenyl-4-quinolone (Venturella, et al. , 1975; and Venturella, et al . , 1970) . The assignment of XXIX (compound 36 in Table 2) as 4-methoxy-2-phenylquinolone (Goodwin, et al . ; and Staskun et al . ) was based on NMR spectral evidence, which includes the appearance of an O- methyl signal at 54.03, an H-3 proton at 57.11 instead of at 56.10-6.60 (which is usually seen for 2-pheny1-4-quinolones) . However, when I _H was treated with ethylhalide or a higher alkylhalide in the same way, only one product, which was confirmed to be the -alkylated compound (Table 2) , was obtained.

When compound I was treated with methanesulfonyl chloride or p-toluenesulfonyl chloride in pyridine, the mesylate (compound 44 in Table 2) or the tosylate (compound 45 in Table 2) was produced, indicated generally at XXX in Figure 11.

Table 2 identifies several exemplary O- substituted 2-pheny1-4-quinolones formed by the above methods, which are detailed for compounds 44, 45 and 46 in Example 4. Here, R identifies the alkyl moiety of a 4-position -OR group.

Table 2

II. Treatment Method

The invention includes a method of treating a tumor in a mammalian subject, by administering to the subject, a 2-ary1-4-quinolone compound, in an amount effective to inhibit tumor cells in the subject.

In one embodiment, the 2-ary1-4-quinolone compound has the structure given in Figures 2A-2D, including substitutions at ring positions 5-8, and on the pendant (2-position) phenyl group. In another general embodiment, the compounds have the general structure given in Figure 3A-3D, including substituents on the pendant phenyl group.

A. Tumor-Cell Cytotoxicity

Selected 2-phenyl-4-quinolones and related compounds (22-50 in Tables 1 and 2) were assayed for cytotoxicity in vitro against six tumor cell lines, including human lung carcinoma (A-549) , ileocecal carcinoma (HCT-8) , melanoma (-RPMI-7951) , and epidermoid carcinoma of the nasopharynx (KB) , and two murine leukemia lines (P-388 and L1210) . Details of

the cytotoxicity results are shown in Table 3, and provided in Example 6A.

Table 3

As shown in Table 3, compounds 22-30 and compound 33. demonstrated potent cytotoxicity, with EC ₅₀ values <1.0 μg/ml in virtually all cases. This indicates that several substituents at position 6 (Ri) (F, Cl, OH, CH ₃ , and OCH ₃ ) , position 7 (R ₂ ) (F, Cl, 0CH ₃ , and OH) , and position 8 (R ₃ ) (F) of the basic 2-phenyl-4-quinolone structure (e.g. 22) are possible with minimal effects in general cytotoxicity obtained with this class of compound. More

generally, a preferred embodiment of the invention contemplates 2-ary1-4-quinolone compounds in which R _j and R ₂ are each H, F, Cl, OH, CH ₃ , or OCH ₃ , and R ₃ is H, F, or Cl. With respect to position 8, introduction of an

OCH ₃ group at position 8 (R ₃ ) led to a compound (31) with selective cytotoxicity (only cytotoxic to HCT-8 with EC _JO = -0.55 μg/ml) . In contrast, the 6-hydroxy compound (32) showed selectivity towards the KB and L1210 lines. Introduction of an OH group at C-8 (34) or the introduction of alkyl groups at N-l (35 and 50) and at the C-4 oxygen (36-43) all led to poorly active compounds. The C-4 O-mesylated compound (44) was also minimally cytotoxic. Following initial identification of the cytotoxic properties of the 2-phenyl-4-quinolones, several of these compounds were submitted to the National Cancer Institute (NCI) for testing in its in vitro disease oriented antitumor screen (Boyd; Monks, et al . ) . This assay involves determination of a test agent's effect of growth parameters against a panel of approximately 60 cell lines derived from human cancers, which consist largely of solid tumors and a few leukemia lines. Assay results are reported as both dose response curves and pictographically as "mean graphs".

Compounds 24, 26, and 27 have their most notable effects in the screen on lines from the colon panel and on one line from the CNS panel, as seen in Table 4. In the table, "TGI" is the log concentration of molarity of compound which reduces viable cell number to the level at the start of the experiment; "LC ₅₀ " is the log compound molarity which reduces the viable

cell number to 50% of the level at the start of the experiment.

Table 4

All three compounds exhibit a growth-inhibition effect at concentrations between about 1-10 μM (-5 and -6) for the cell lines indicated in the table. Compound 26 appears most potent of the three compounds based on the LC ₅₀ values obtained with the three cell lines.

The three compounds were further tested against the cell lines MRC-3 (normal human lung fibroblast) , HTBD _U -145 (human prostate carcinoma) , HE _p -2 (human lung carcinoma) , BT549 (human breast carcinoma) , HeLaS ₂ (human cervical carcinoma) , A549 (human lung carcinoma) , SW480 (human colon carcinoma) , HepG ₂ (human liver carcinoma) , and HepG ₂ -T ₁₄ (Hep-G ₂ transfected with HBV) . Details of the compound test method are given in Example 6B. The results are seen in Table 5, which shows that all three compounds show about the same cytotoxicity potency to the several cell lines.

Table 5

The anti-tumor activity of dioxalane compounds

XXV and XXVI (Figures 7A and 7B, respectively) was compared with that of compound 27 against the tumor cell lines shown at the left-hand column in Table 6 below. Various concentrations of compounds XXV, XXVI, and 27 were added into 96 well multi-dish in triplicate. 1 x 10 ⁴ cells were added to each well. After three days of continuous exposure of the cells to the drugs, a final concentration of 0.5 mg/ml MTT in PBS was added and then these were incubated at 37°C Four hours later, the cells were lysed with isopropanol containing 0.06 N HC1 and read by an ELISA reader with wavelength set at 570 nm for attached cells and 570 nm-630 nm for suspension cells. Inhibition of cell proliferation was expressed as the O.D. of drug treated mean/O.D. of the control mean. IC ₅₀ was estimated by the curve plotted by concentration of the drug versus percentage of control.

Table 6

The two dioxalane compounds show high activity against the cell lines tested. Compound XXV showed highest activity in general, with compounds XXVI being substantially more active than compound 27 in the first two cell lines.

B. Activity Against Drug-Resistant Cells One major obstacle to effective chemotherapy, in many types of cancers, is the development of drug resistance to one or more anti-tumor compounds. Many types of tumors develop specific or broad spectrum resistance to otherwise effective anti-tumor agents such as doxorubicin (Tsuruo, et al . ) , vinblastine

(Barnett, et al . ) , and etoposides (Issell, et al . ) . It was therefore of interest to test the ability of the 2-pheny1-4-quinolone compounds to inhibit cell growth on a variety of drug-resistant tumor cell lines.

In a first series of tests, compounds 24, 26, and 27, and compounds XXV and XXVI were tested for cytotoxicity against non-resistant BK cells

(epidermal carcinoma of the nasopharynx) KB cells resistant to VP-16 and vincristine (KB _VP _ι _6R and KP _VCR ) , murine leukemia cells (P _38g ) , and murine leukemia cells resistant to doxorubicin (P _adr and P _SPR ) or resistant to VP-16 (P ₂₅ and P ₂ . ₅ ) . Cytotoxicity was measured after exposure to the test compound as described in Example 6B, with the results shown in Table 7 below. As seen, comparable levels of cytotoxicity activity were observed for both resistant and non-resistant cell lines, indicating that the compounds are not cross-resistant with doxorubicin, VP-16 or vincristine.

Table 7

In a secon series o es s, compoun s 24, 26, and 27 were tested for cytotoxicity against non- resistant human nasopharyngeal carcinoma (KB) cells and KB cells resistant to VP-16 (KB _REV and KB ₇ ) and KB cells resistant to vincristine (KB _VCR ) . Cytotoxicity was measured after exposure to the test compound as described in Example 6B, with the results shown in Table 8 below. As seen, comparable levels of cytotoxicity activity were observed for both resistant and non-resistant cell lines, indicating

that the compounds are not cross-resistant with vincristine or VP-16.

Table 8

C Interaction of 2-phenyl-4-guinolones with Tubulin

Studies conducted in support of the present invention indicate that one mode of action of the 2- phenyl-4-quinolone compounds, as anti-tumor agents, is an ability to bind to tubulin and inhibit cell mitosis. Compounds 24, 26, and 27 were all found to inhibit in vitro tubulin polymerization, and compound 26 was demonstrated to inhibit mitosis in HL-60 human leukemia cells at cytotoxic concentrations. In addition, compound 26 inhibited the binding of radiolabeled colchicine to tubulin, and, as occurs with most colchicine site drugs (Hamel, et al . , 1984) , stimulated tubulin-dependent GTP hydrolysis at concentrations that inhibit the polymerization reaction.

In order to examine structure-function relationships between the 2-pheny1-4-quinolone compounds and inhibition of tubulin polymerization, the compounds identified in Table 9 were tested for inhibition of tubulin polymerization and for inhibition of colchicine binding to tubulin, according to methods detailed in Examples 7A and 7B,

respectively. Table 9 presents the results, with a direct comparison performed at the same time with the potent antimiotic compounds colchicine, podophyllotoxin, and combretastatin A-4. These three natural products all bind at the colchicine site of tubulin (Hamel, 1990; Lin, et al . , 1990).

Table 9

The unsubstituted compound 22 had significant inhibitory activity in the tubulin polymerization assay, with an IC ₅₀ value of 7.3 μK, measured for the extent of polymerization after 20 minutes. Among the analogs tested, maximum enhancement of activity occurred with a substitution at position 6. Compared

with the unsubstituted 22 there was a 2-3 fold increase in inhibition, in terms of IC ₅₀ values, whether the substituent group was methoxy (26) , chloride (24) , or fluoride (23) . While the 6-methoxy substituent appeared to be slightly more inhibitory than the compounds with halides at position 6, the differences between the compounds were within the experimental error of the assay. A loss of activity, relative to 22, occurred with a hydroxyl substituent at position 6 (32, IC ₅₀ value, 16 μM) .

A substituent at position 7 also enhanced activity about 2-fold relative to the unsubstituted 22. Compounds 27, 29, and 33, with chloride, methoxy, and hydroxyl groups, respectively, at position 7 had IC ₅₀ values of 3.7, 4.8, and 3.4 μM. With chloride and especially methoxy substituents, somewhat lower IC ₅₀ values were obtained when the substituent was at position 6 as compared with position 7. With the hydroxyl substituent, in contrast, position 7 appeared to be more favorable (IC ₅₀ value of 3.4 μM for 33 as compared with 16 μM for 32) .

The OCH ₃ substitution at position 8 was available (31) , and it had little activity as an inhibitor of tubulin polymerization. Similarly, a single agent (35) with a substituent at position 1 was evaluated, and it, too, was noninhibitory. Further modification of 35 by addition of a hydroxyl group at position 4' (50) did not restore activity. Compounds 24, 26, and 27, three of the more potent inhibitors of polymerization, were also examined for effects on the binding of radiolabeled colchicine to tubulin (Table 9) . In this assay, combretastatin A-4 nearly completely inhibits binding

of colchicine to tubulin and podophyllotoxin inhibits the reaction 85-90% when present in equimolar concentration with the radiolabeled colchicine (Lin, et al . , 1988). In contrast, an equimolar concentration of the three 2-phenyl-4-quinolones had little effect. Only by increasing the concentration of the latter three compounds 10-fold was a significant effect on colchicine binding observed. Of the three compounds evaluated in this assay, the greatest inhibitory effect was observed with 26.

D. Treatment Method

In the treatment method of the invention, a 2- aryl-4-quinolone compound of the type described above is administered in a pharmaceutically effective amount, to reduce tumor growth in a mammalian subject. By pharmaceutically effective amount is meant a concentration at the tumor site or in the bloodstream which is effective to inhibit growth of tumor cells. This concentration can be determined from EC ₅₀ values from in vitro growth inhibition studies, such as those described, using either known tumor cell lines, and related to the patient's tumor type. The studies reported herein show that a compound concentration of between about 0.05 to 0.5 μg/ml is effective to reduce tumor growth for a large number of tumor-cell types. Doses effective to achieve these concentrations in the blood in human patients, either by parenteral, e.g., intravenous, or oral administration can be readily determined from animal model studies, using known dose relationships between dose and pharmacokinetics between animal models and humans.

During tumor treatment, the patient will typically receive periodic doses, e.g., biweekly doses of the drug, with the effectiveness of treatment being monitored by tumor biopsy, radiological methods, or blood enzyme levels, according to standard methods.

In a treatment regiment in which doxorubicin, a related anthracyline anti-tumor compound, VP-16 or a related etoposide compound, or vincristine or a related vinca alkaloid drug is used initially, and resistance to the anti-tumor compound is observed, the present invention provides a method of inhibiting tumor cell growth in the subject. In this method, a 2-aryl-4-quinolone compound is administered in an amount effective to inhibit tumor growth.

Drug resistance in the initial phase of the regimen is evidenced by an increasingly larger drug dose needed to inhibit tumor growth. That is, the tumor becomes increasingly more refractory to growth inhibition by the drug. In the second phase of the regimen, in which a 2-ary1-4-quinolone compound is administered, either alone or in combination with the initial-phase drug, the effective drug dose of aryl quinolone is generally no higher than that required before drug resistance develops.

From the foregoing, it can be appreciated how the treatment method of the invention offers advantages in tumor treatment. The compound itself is readily synthesized, and can be administered either orally or parenterally, e.g., by intravenous administration. The mode of action of the compound, inhibiting tubulin formation necessary for cell mitosis, would tend to confine the effects of the compound to actively dividing cell. In this regard, studies carried out in support of the invention show

that exemplary compounds 24, 26, and 27 produced no detectable breaks in protein-linked DNA, unlike VP- 16.

Further, the compounds do not appear to share cross-resistance with a number of common anti-tumor agents, including doxorubicin, vincristine, and VP- 16, and thus are useful particularly in a treatment regimen where anti-tumor resistance has developed, or in combination with other anti-tumor compounds, such as doxorubicin, cis-platin, vincristin, or VP-16.

The following examples illustrate methods of making the compounds of their invention, and biological assays of the compounds. The examples are in no way intended to limit the scope of the invention.

Methods Melting points were determined in open-ended capillary tubes on a Thomas-Hoover apparatus and are uncorrected. Infrared (IR) spectra were recorded on a Shimadzu IR 440 spectrometer in KBr. Nuclear magnetic resonance (NMR) spectra were taken at 90 MHz on a JEOL FZ 90Q and Varian VXR-300 with tetramethyl- silane (TMS) as an internal standard. Splitting patterns are designated as follows: s, singlet; br, broad; d, doublet; t, triplet; q, quartet; m, multiplet. Mass spectra (MS) were measured with an Hp 5995 GC-MS instrument and a JEOL JMS-D-30 mass spectrometer. Elemental analyses were performed by the Chung San Institute of Science Technology and National Cheng-Kang University, Taiwan, Republic of China.

Example 1

Synthesis of Compounds 22-31 A. 2-Phenyl-4-quinolone (22) 2-Pheny1-4-quinolone (22) was synthesized according to published methods (Goodwin, et al . ; Kasahara, et al . ; Cheng, R.T., et al . , 1986; Chen, et al . ) .

Aniline (1.86 g, 0.02 mole) and ethyl benzoylacetate (3.84 g, 0.02 mole) were dissolved in EtOH (150 ml) . Acetic acid (5 drops) was added at 50°C. After 24 hours the mixture was evaporated to dryness. THe residue was added with stirring in one portion to diphenyl ether (50 ml) at 240°C The temperature was raised to 250°C After 10 minutes, the mixture was cooled to room temperature and diluted with n-hexane (80 ml) . The precipitate was collected, washed with CHC1 ₃ , and purified by chromatography on a silica gel column. Elution with CHCl ₃ -EtOH and recrystallization from CHCl ₃ -EtOH afforded compound 22 (Table 1) .

B. 6-Substituted-2-phenyl-4-guinolones (Compounds 23-26) p-Substituted anilines (0.02 mole) and ethyl benzoylacetate (3.84 g, 0.02 mole) were allowed to react as in the preparation of 22, to afford 23-26 (Table I) .

C 7-Fluoro-2-phenyl-4-guinolone (Compound 27) m-Fluoroaniline (2.22 g, 0.02 mole) and ethyl benzoylacetate (3.84 g, 0.02 mole) were dissolved in EtOH (150 ml) . Acetic acid (5 drops) was added at 50°C The reaction mixture was stirred at 50°C for 24 hours and then evaporated to dryness. The residue was washed with H ₂ 0 and purified by chromatography on a silica gel column. Elution with CHC1 ₃ yielded

ethyl β-(m-fluorophenylamino) cinnamate (17) (3.6 g, 63%), mp 54-56: IR (KBr) cm ^"1 1700 (C ^* =0) , 1600 (C=C) . 'H-NMR (CDC1 ₃ ) 51.36 (3H, t, J=7.0 Hz, CH ₃ ) , 4.20 (2H, q, J=7.0 Hz, -OCH ₂ -) , 5.01 (1H, s, >C=CH-COO-) , 6.20- 7.40 (9H, m, aromatic protons), 10.30 (1H, br, NH) . High resolution MS m/z. M+ calculated for C _π H ₁₆ FN0 ₂ : 285.1165. Found: 285.1163.

The condensation intermediate (3.0 g, 0.01 mole) as a fine powder was added with stirring in one portion to diphenyl ether (50 ml) maintained at

240°C. The temperature was raised to 250°C After 10 minutes the mixture was cooled to room temperature and diluted with n-hexane (80 ml) . The precipitate was collected and washed with CHCl ₃ -EtOH and recrystallization from CHCl ₃ -EtOH afforded 27 (Table I).

D. 7-Chloro-2-phenyl-4-guinolone (Compound 28) (Fuson, et al . ) . m-Chloroanaline (2.55 g, 0.02 mole) and ethyl benzoylacetate (3.84 g, 0.02 mole) were allowed to react as in the preparation of 22 to afford 28 (Table I).

E. 7-Methoxy-2-phenyl-4-quinolone (Compound

29) (Venturella. et al . . 1975). m-Methoxyaniline (2.46 g, 0.02 mole) and ethyl benzoylacetate (2.84 g, 0.02 mole) were allowed to react as in the preparation of 22 to afford 29 (Table I) .

F. 8-Substituted-2-phenyl-4-guinolones (Compounds 30 and 31)

0-substituted anilines (9, (0.02 mole) and ethyl benzoylacetate (3.84 g, 0.02 mole) were allowed to

react as in the preparation of 22 to afford 30 and 31 (Table I) .

Example 2 Synthesis of Compounds 32 and 33

A. 6-Hvdroχy-2-pheny1-4-guinolone (Compound 32) . A mixture of Compound 26 (100 mg, 0.004 mole),

KOH (1 g, 0.018 mole) and ethylene glycol (10 ml) was boiled under reflux for 24 hours. The reaction mixture was poured into ice-water and filtered. The filtrate was neutralized with dilute HC1, and the precipitate was collected. The crude product was purified by chromatography on silica gel. Elution with CHCl ₃ -EtOH yielded Compound 32 (Table 1) .

B. 7-Hyroxγ-2-phenyl-quinolσne (Compound 33) (Venturella, et al . . 1970).

Compound 29 (100 mg, 0.0004 mole) was reacted with KOH (1 g, 0.018 mole) as described for the preparation of 32 to afford Compound 33 (Table I) .

Example 3

Synthesis of Compounds 34-38 Compound 22 (2.21 g, 0.01 mole) was dissolved in dry DMF (50 _. ml) and NaH (80% in oil, 0.3 g, 0.01 mole) was added portion-wise with stirring for 30 minutes at room temperature. Alkyl halides (Mel,

EtI, or PrI) (0.01 mole) were then added dropwise at 30-40°C Stirring was continued for an additional 30 minutes, and the reaction mixture was poured into ice water and extracted with CHC1 ₃ . The organic layer was washed with water, dried over MgS0 ₄ , and

evaporated. The residue was purified by chromatography on silica gel. Elution with benzene yielded Compound 35 (Coppola; Singh, et al . ) (Table I) and 36-43 (Table II) .

Example 4 Synthesis of Compounds 44, 45; and 50

A. 2-Phenylquinolin-4-yl mesylated (Compound

44) . Compound 22 (1.10 g, 0.005 mole) was dissolved in dry pyridine (10 ml) . Methanesulfonyl chloride (1.15 g, 0.01 mole) was added dropwise at 0°C The reaction mixture was stirred for 24 hours, and poured into ice-water (40 ml) . The precipitated solid was extracted with ether, and the extract washed with H ₂ 0 and dried over MgS0 ₄ . The solvent was evaporated, and the residue was purified by chromatography on silica gel. Elution with benzene yielded 44 (Table II).

B. 2-Phenylquinoline-4-yl tosylate (Compound

45) .

Compound 22 (1.10 g, 0.005 mole) was reacted with p-toluenesulfonyl chloride (1.90 g, 0.01 mole) as described for the preparation of 44 to afford 45 (Table II) .

C. N-Methyl-2-(4 -hvdroxyphenyl)-4-quinolone (Compound 50) (Wu) . p-Hydroxyacetophenone (46) (13.6 g, 0.1 mole) was dissolved in dry tetrahydrofuran (THF) (200 ml) , and NaH (80% in oil, 3.0 g, 0.1 mole) was added. Chloromethyl-methylether (δ.r g, 0.1 mole) in dry THF (20 ml) was added dropwise at 50°C. The reaction mixture was stirred at 50°C for an additional 1 hour

and then evaporated to dryness. The residue was purified by chromatography on a silica gel column. Elution with benzene yielded p- methoxymethoxyacetophenone (47) (12 g, 67%) as a viscous liquid, IR (Nujol) , cm" ¹ 1685 (C=0) . *H-NMR (CDC1 ₃ ) 5 - 2.57 (3H, S, -COCH ₃ ) , 3.48 (3H, s. OCH ₃ ) , 5.22 (2H, S. -0-CH2-0) , 7.03 (2H, d. J=9.0 Hz, H-3 and H-5) , 7.88 (2 H, d. J=9.0 Hz, H-2 and H-6) , high resolution, MS m/z : M+ calculated for 180.0786. Found: 180.0789. Diisopropylamine (2.0 g, 0.02 mole) was dissolved in dry THF (75 ml) and n- butyllithium (1.6 M in hexane, 1.28 g, 0.02 mole) was added at -30°c After cooling to -65°C, a solution of 47 (3.60 g, 0.02 mol) in THF (10 ml) was added dropwise, and the mixture was kept at this temperature for 1 hour. A solution of N- methylisatoic anhydride (48) (1.8 g, 0.01 mole) in THF (40 ml) was added dropwise at -65°C and the resulting suspension was stirred for 5 hours. The reaction mixture was quenched with saturated ammonium chloride solution and extracted with methylene chloride. The organic phase was combined and dried over MgS0 ₄ . The solvent was removed under reduced pressure, and the residue was purified by column chromatography on silica gel. Elution with CHC1 ₃ -

EtOH yielded 2-(4'-methoxymethoxyphenyl)-l-methyl-4- quinolin (49) (Table I). Compound 49 (1.48 g, 0.05 mole) was suspended in concentrated HC1 (10 ml) and stirred at 30°C for 30 minutes. The reaction mixture was neutralized with saturated NaHC0 ₃ and the precipitate was collected. The crude product was washed with CHC1 ₃ and recrystallized from EtOAc to afford Compound 50 (Table I) .

Example 5 Synthesis of dioxalane 2-aryl-4-quinolone compounds 4,5-methylenedioxy-anthranilamide (370 mg, 1.57 mmol) and 3-(N,N'-dimethyl)amino-acetophenone (256 mg, 157 mmol) , and a catalytic amount of p- toluenesulfonic acid in benzene were heated under reflux for 24 h with azeotropic removal of water. The mixture was then concentrated, and the oily residue was directly used for the next step. The corresponding imine in 30 ml of THF was treated with 2.0 equivalent of LDA (2.0 M solution in C ₆ Hι ₂ ) at 0°C under Ar, then at room temperature for 12 h to yield the 3'-(N,N ^/ -dimethyl) amino-6,7-methylene-dioxy-2- phenyl-4-quinolone.

Example 6 Tumor-Cell Cytotoxicity Activity A. Cytotoxicity Assay #1

Candidate compounds (22-50) were assayed for in vitro cytotoxicity with a panel of human and murine tumor cell lines at the School of Medicine, UNC-CH. These cell lines include lung carcinoma (A-549) , ileocecal carcinoma (HCT-8) , epidermoid carcinoma of the nasopharynx (KB) , melanoma (RPMI-7951) , and murine leukemia (P-388 and L-1210) . All cell lines were obtained from the American Type Culture Collection, Rockeville, MD, and were adapted to grow in antibiotic-free RPMI-1640 medium supplemented with 10 fetal calf serum. The assay principles followed those described by

Monks et al . except that tetrazolium was used to estimate live cell number in each well of tissue culture plate before and after addition of chemicals (Mosmann) . The EC ₅₀ value for each compound was

calculated mathematically from 2 data points across 50% inhibition in cell growth. Compounds with high efficacy to these human and murine tumor cell lines were further examined and new derivatives synthesized. Compounds with promising potential against solid tumors were then submitted to NCI for further in vitro tumor cell line assay and for further studies of molecular mechanism of action.

Cytotoxicity studies in human and murine tumor cell lines were performed in a 96 well format. Each well was deposited with approximately 1 x 10 ⁵ cells and overnight stabilization, testing chemicals, in appropriate dilution, were added to each of the wells (each concentration performed in quadruplicate) . Control wells received medium alone. After 3-4 days cultivation, the remaining cell number in each well was estimated by the formazan pigment formation assay. MTT was added to each well, and the plates were incubated overnight. The pigment formed was dissolved in DMSO, and the plates were analyzed on a Dynatech 360 ELISA reader at 570 nm. Actinomycin (Sigma Chemical Co., St. Louis, MO) was used as a positive control. The ED _S0 of actinomycin for these cell lines is usually less than 0.01 μg/ml.

B. Cytotoxicity Assay #2

Various concentrations of the test compound were added together with 10 ⁴ cells into 96 well multi dishes. After three days of continuous exposure of the cells to the compound, a final concentration of 0.5 mg/ml MTT (Mosmann) was added, with further incubation for 4 hours. At the end of this incubation, the monolayer cells were lysed with

isopropanol containing 0.01N HCl, after aspiration of the medium, and read at 570 nm by an ELISA reader. For cell suspensions, isopropanol containing 0.01N HCl was added directly to the cells, and the cells were read at 570-630 nm by an ELISA reader after storage in the dark overnight at room temperature. Inhibition of cell proliferation is expressed as IC ₅₀ by comparisons of the compound treated optical density with control (untreated cells) optical density.

Example 7 Tubulin Polymerization Activity Tubulin was purified from bovine brain as described previously (Hamel, et al . , 1984).

Nonradiolabeled colchicine was obtained from Sigma, podophyllotoxin from Aldrich, and [ ³ H]colchicine from DuPont. Combretastatin A-4 was a generous gift of Dr. G.R. Pettit of Arizona State University. Commercial monosodium glutamate, from Sigma, was repurified by acid precipitation and reneutralized (to pH 6.6, in a 2 M stock solution) with ultrapure NaOH obtained from Alfa (Huang, A.B., et al . , 1985). Minimal residual contamination by Mg ²⁺ and several other cations was confirmed by atomic absorption spectroscopy.

A. Tubulin Polymerization Assay Each reaction mixture contained in a 0.24 mL volume 1.0 mg/mL (10 μM) tubulin, 1.0 M monosodium glutamate, 1.0 mM MgCl ₂ , 4% (v/v) dimethyl sulfoxide, and varying concentrations of drugs (all concentrations, however, refer to final reaction volume of0.25 mL) . Reaction mixtures were

preincubated at 37°C for 15 minutes and chilled on ice, and 10 μL of 10 mM GTP (required for polymerization) was added to each mixture. Reaction mixtures were transferred to cuvettes in Gilford spectrophotometers held at 0°C by electronic temperature controllers. Baseline absorbances at 350 nm were established, and the reaction was initiated by a temperature jump to 37°C (the temperature rose at a rate of about 0.5°C/s). The reactions were followed for 20 minutes, and IC ₅₀ values, defined as the drug concentration required to inhibit the extent of polymerization by 50% after a 20 minute incubation, were determined graphically.

At least three independent experiments were performed with each drug, except that inactive compounds (defined as IC ₅₀ value greater than 40 μM) were generally evaluated only twice. Four spectrophotometers (sixteen samples) were used in each experiment. Each experiment had two control reaction mixtures, with the turbidity readings generally within 5% of each other.

Repurified glutamate was used in the studies with the 2-pheny1-4-quinolones because several of these agents caused aberrant turbidity readings when commercial glutamate was used, as described previously (Paull, et al . ; Getahun, et al . ) with other colchicine site drugs. Although we initially believed (Getahun, et al . ) this phenomenon was caused by Mg ⁺ contamination in the commercial glutamate, we were unable to reproduce the phenomenon by adding MgCl ₂ to the repurified glutamate. Besides eliminating spurious high turbidity readings with several of the phenylquinolone derivatives, use of the repurified glutamate resulted in our obtaining

significantly lower IC ₅₀ values with colchicine, podophyllotoxin, and combretastatin A-4 than we previously obtained with commercial glutamate (Lin, et al . , 1988; Muzaffar, et al . ) . The presumptive contaminant(s) in commercial glutamate is (are) unknown.

B. Inhibition of the Binding of Radiolabeled Colchicine to Tubulin. Each 0.1 L reaction mixture contained 0.1 mg

(1.0 μM) tubulin, 1.0 M commercial monosodium glutamate (pH 6.6 with HCl), 1 mM MgCl ₂ , 0.1 mM GTP, 5.0 μM [ ³ H]colchicine, 5% (v/v) dimethyl sulfoxide, and inhibitor as indicated. Incubation was for 20 minutes at 37°C Each reaction mixture was filtered under reduced vacuum through a stack of two DEAE- cellulose paper filters, washed with water, and radioactivity quantitated in a liquid scintillation counter.

Although the invention has been described with respect to particular embodiments, it will be appreciated that various changes and modifications can be made without departing from the invention.