WO1988004658A1

WO1988004658A1 - 10-(substituted phenyl)-3-alkylflavin derivatives having antiprotozoal, antiproliferative and antiinflammatory activity

Info

Publication number: WO1988004658A1
Application number: PCT/AU1987/000428
Authority: WO
Inventors: William Butler Cowden; Ian Albert Clark
Original assignee: The Australian National University
Priority date: 1986-12-17
Filing date: 1987-12-17
Publication date: 1988-06-30

Abstract

Compounds of general Formula (I), wherein Y is selected from alkyl, alkenyl, alkynyl, phenylalkyl or substituted or unsubstituted phenyl; n is an integer from 1 to 5; R1, R2, R3, R4 and X, which may be the same or different, are selected from -Y, -O-Y, -S-Y (wherein Y is as defined above or is unsubstituted or alkyl substituted cycloalkyl and cycloalkenyl), cyclic or non-cyclic amino moieties, halogeno, hydroxyl, thiol or cyano. The 5 N-oxide, dihydro and tetrahydro derivatives are disclosed. Preparation of the above compounds, compositions including the above compounds and methods of using such compositions for antiprotozoal, antiproliferative and/or antiinflammatory treatment of a human or animal are disclosed.

Description

10-(SUBSTITUTED PHENYL)-3-ALKYLFLAVIN DERIVATIVES HAVING ANTIPROTO- ZOAL, ANTIPROLIFERATIVE AND ANTIINFLAMMATORY ACTIVITY

This invention relates to 10-(substituted phenyl)-3-alkylflavin compounds. In particular, the present invention relates to the use of these compounds as selective antiproliferative and antiinflammatory compounds, and as antiprotozoal, more particularly antimalarial, compounds.

The spread of chloroquine and multidrug- resistant strains of Plasmodium falciparum (the primary causative agent of lethal malaria infections in man) has prompted a search for new approaches to antimalarial therapy, and in one approach the antimalarial effects of chronic riboflavin deficiency in animals (1) support an observation made in man(2). Recently, a number of riboflavin antagonists have been shown to have potent antimalarial properties in vitro (3), although depletion of riboflavin in 48 and 96 hour cultures is apparently without effect (4).

These same riboflavin antagonists were also shown to have considerable anticoccidial activity in vivo

(5). Following this approach, it has recently been suggested (6) that inhibitors of riboflavin metabolism are worth investigating as potential antimalarial drugs.

It has now been found that certain

10-(substituted phenyl)-3-alkylflavin compounds, including some compounds of this group which are novel per se, have significant antimalarial activity in vivo.

According to a first aspect of the present invention, there is provided a group of compounds having antiprotozoal, antiproliferative and/or antiinflammatory activity, said compounds comprising compounds of the general formula I:

wherein Y is selected from the group consisting of alkyl of from 1 to 8 carbon atoms, alkenyl of from 2 to 8 carbon atoms, alkynyl of from 2 to 8 carbon atoms, phenylalkyl of from 7 to 12 carbon atoms, phenyl, alkylphenyl of from 7 to 14 carbon atoms, mono-, di- and trihalogenophenyl, and mono-, di- and tri(trihalogenomethyl)phenyl; n is an integer of from 1 to 5; and R ¹, R², R³, R⁴ and X, which may be the same or different, are each selected from the group consisting of:

(a) H, alkyl of from 1 to 8 carbon atoms, alkenyl of from 2 to 8 carbon atoms, alkynyl of from 2 to 8 carbon atoms, unsubstituted and alkyl-substituted cycloalkyl and cycloalkenyl of from 3 to 10 carbon atoms, phenylalkyl of from 7 to 12 carbon atoms, mono-, di- and trihalogenoalkyl of from 1 to 3 carbon atoms, phenyl, alkylphenyl of from

7 to 14 carbon atoms, mono- di- and tri(trihalogenomethyl)phenyl and mono-, di- and trihalogenophenyl;

(b) amino moieties of the formula

where (i) A and B are each selected from the group consisting of hydrogen, alkyl of from 1 to 8 carbon atoms, alkenyl of from 2 to 8 carbon atoms, alkynyl of from 2 to 8 carbon atoms, unsubstituted and alkylsubstituted cycloalkyl and cycloalkenyl of from 3 to 10 carbon atoms, or

(ii) A and B together with the nitrogen atom to which they are attached represent a heterocyclic moiety selected from the group consisting of aziridinyl, azetidinyl, pyrrolidinyl, piperidinyl, hexahydroazepinyl, heptamethyleneiminyl, morpholinyl, thiomorpholinyl, piperazinyl,

4-alkyl- and 4-arylpiperazinyl, octamethyleneiminyl, each of the said heterocyclic moieties optionally having attached as substituents on carbon atoms thereof from 1 to 3 alkyl groups of from 1 to 8 carbon atoms;

(c) halogeno, including F, Cl, Br, and I; (d) alkoxy, aryloxy, alkylthio or arylthio moieties of the formulae -O-Z or -S-Z, wherein Z is alkyl of from 1 to 8 carbon atoms, alkenyl of from 2 to 8 carbon atoms, alkynyl of from 2 to 8 carbon atoms unsubstituted and alkylsubstituted cycloalkyl and cycloalkenyl of from 3 to 10 carbon atoms, phenylalkyl of from 7 to 12 carbon atoms, phenyl, alkylphenyl of from 7 to 14 carbons atoms, mono-, di-and tri(trihalogenomethyl)phenyl and mono-, di- and trihalogenophenyl;

(e) hydroxy or thiol; and (f) cyano; or the 5 N-oxide derivatives thereof of the formula la

the dihydro derivatives thereof of the formula lb

or the tetrahydro derivatives thereof of the formula Ic

wherein in the formulae la , lb, and Ic , Y, n, R¹ , R² , R ³ , R ⁴ and X are as defined above.

In this aspect, this invention provides a method for the antiprotozoal, antiproliferative and/or antiinflammatory treatment of a human or animal, which comprises the administration to the human or animal of an effective amount of a compound of the general formulae I, la, lb or Ic.

This invention also provides a pharmaceutical or veterinary composition for antiprotozoal, antiproliferative and/or antiinflammatory treatment of a human or animal which comprises a compound of the general formulae I, la, lb or Ic, together with a pharmaceutical or veterinary carrier therefor.

In yet another aspect of the invention there is provided a class of compounds which are novel per se. These compounds are compounds of the general formula II:

wherein Y, n, R¹, R², R³, R⁴ and X are as defined above, or the 5N-oxide, dihydro or tetrahydro derivatives thereof, with the proviso that (X)_n is not 4'-chloro, 3'-methyl, 4'-methyl, 2'-ethyl or 2',6'-dimethyl, when Y is methyl and R ¹, R², R³ and R⁴ are hydrogen.

Particularly preferred compounds of the general formulae I and II are compounds in which Y represents methyl or ethyl. Also preferred are compounds in which the substituent in the 10 position in general formulae I and II may, for example, be selected from:

2'-chlorophenyl

3'-chlorophenyl 3'-bromophenyl

4'-bromophenyl

3'-fluorophenyl

4'-fluorophenyl

4'-chloro-2'-methylphenyl 2',4'-dichlorophenyl

2',5'-dichlorophenyl

3',5'-dichlorophenyl 4'-chloro-3'-trifluoromethylphenyl 3'-trifluoromethylphenyl 4'-butylphenyl 3'-methoxyphenyl 4'-methoxyphenyl

3'-ethylphenyl 3',5'-dimethylphenyl 4'-diethylaminophenyl 4'-hydroxyphenyl 3'-methylthiophenyl

4'-methylthiophenyl Preferred compounds are compounds wherein the phenyl group is di-substituted. Particularly preferred compounds are 10-(3',5'-dichlorophenyl) -3-methylflavin and 10-(3',5'-dimethylphenyl) -3-methylflavin.

All of the above compounds are novel and hence fall within both general formula I and general formula II. Other compounds which fall within general formula I include the compounds:

10-(4'-chlorophenyl)-3-methylflavin 10-(3'-methylphenyl)-3-methylflavin 10-(4'-methylphenyl)-3-methylflavin 10-(phenyl)-3-methylflavin

10-(2'-ethylphenyl)-3-methylflavin 10-(2',6'-dimethylphenyl)-3-methylflavin. The antiprotozoal, antiproliferative and/or antiinflammatory activity of these compounds, was not known prior to the present invention.

Compounds of the general formulae I and II may be made by either of two general methods known in the art for the preparation of 10-phenylflavins. The first of these known methods (7-10) involves the condensation of an alloxan of the general formula III, with a 2-aminodiphenyl amine of the general formula IV:

The second method (11) involves the condensation of a 6-anilinouracil of the general formula V, with a nitrosobenzene of the general formula VI:

The 5 N-oxide derivatives (la) can be prepared by direct oxidation of the parent compounds (I) with peroxycarboxylic acid reagents (such as m-chloroperoxybenzoic, peroxymaleic or trifluoroperoxyacetic acids) by methods found in the literature (e.g. 12). The di- and tetrahydro derivatives (lb and Ic) can be prepared by reductions of the parent compounds (I) with catalyst in the presence of hydrogen or by chemical or electrochemical means by methods found in the literature (e.g. 13-17).

Further features of the present invention will be apparent from the following Examples which illustrate, by way of example only, the preparation and biological activity of compounds of this invention.

EXAMPLE 1

Preparation of 10-(substituted ρhenyl)-3- methyl or -ethylflavins

A number of 10-(halogenophenyl)-3-methyl and

-ethylflavins were prepared by the action of nitrosobenzene on appropriate 3-methyl or 3-ethyl-6- anilinouracils in the presence of acetic anhydride. 10-(4'-chlorophenyl)-3-methylflavin was prepared according to the method of Yoneda et al

(11). Melting points are uncorrected. Analyses were performed and C,H,N values were within +/- 0.4% of the theoretical. Nitrosobenzene, 6-aminouracil and the anilines used herein were obtained from Aldrich Chemical Co., U.S.A. or EGA-Chemie, F.R.G. (a) 6-Arylamino-3-methyluracils An intimate mixture of

6-chloro-3-methyluracil (1.6g., 10mmol) and the appropriate aniline (30mmol) was heated in an oil bath at 170-175° for 15min. (155-160° for 10min. for compound le), cooled briefly and poured into ethanol (35ml) and stirred for 15min. The solid was filtered off, washed with ether (2 x 30ml), recrystallized from acetic acid and dried (Table 1). Analytical samples were prepared from MeOH. 6-Arylamino-4-ethyluracils are prepared by analogous methods. (b) 10-HalogenophenyIflavins The appropriate 6-anilino-3-methyl or -ethyluracil (10mmol) and nitrosobenzene (3.21g, 30mmol) were refluxed in a mixture of acetic anhydride (16ml) and acetic acid (6ml) for 35min. The volume of the reaction mixture was then reduced by ca 50% under reduced pressure and ethanol (15ml) added. After crystallization was complete the solid was filtered off, washed with ethanol and ether and recrystallized from acetic acid to give the compounds of Table 2. Analytical samples were prepared from MeOH.

(c) 10-(2',4'-Dichlorophenyl)-3-methylflavin 6-(2',4'-Dichloroanilino)-3-methyluracil (1.42g, 5mmol) and nitrosobenzene (1.6g, 15mmol) were refluxed in a mixture of acetic anhydride (10ml) and acetic acid (25ml) for 1.5h. The volume was reduced to ca 10ml under reduced pressure and the mixture treated as above to give 10-(2'-,4'- dichlorophenyl)-3-methylflavin. The analytical sample was prepared from acetic acid. (d) Other 10-(substituted phenyl)-3-methyl and -ethylflavins listed in Table 2 have been prepared by analogous methods.

EXAMPLE 2

Biological Activity

Female CBA/CaH mice, 6-8 weeks old, were used in all in vivo experiments. Stock cultures of P.vinckei vinckei (strain V52, from F.E.G. Cox) were stored in liquid nitrogen and had been passaged several times in CBA mice before use. All infections were initiated by intraperitoneal injection with 5 x 10⁵ parasitised red blood cells and monitored by examiing stained thin smears (Diff-Quik stain set, Australian Hospital Supply, Sydney). Infections became patent on the fourth day and rose exponentially to reach 40-50% parasitemia by day 7, death occurring within a further 2 days. In vitro studies were performed with parasites of a Papua New Guinea (PNG) strain of Plasmodium falciparum (FC27). These were maintained in routine culture using group O erythrocytes cultured in 10% (v/v) O serum in RPMI 1640 (Gibco Ltd.) supplemented with 25mM HEPES, 26mM sodium bicarbonate and 100μg/ml gentamicin (Garamycin, Flow Ltd.). Flasks were gassed with 5% CO₂, 5% O₂, 90% N₂ and the medium renewed daily. Fresh erythrocytes were added every two to three days. (a) In vivo drug trials In order to screen for activity, the drugs were finely powdered, then suspended in propylene glycol and administered to groups of 5, 10 or 20 mice in doses of 25, 50, 75, 100 or 150mg/kg by gavage (50μl). Control animals received propylene glycol alone. A single dose was given when parasitemias reached 30-40% and the efficacy determined by examining stained thin smears at 24 and 48 hours post treatment. Animals were considered to be cured when no parasites were evident 60 days later. Further experiments were conducted including treatment orally with multiple doses. The drugs were also administered by intraperitoneal and subcutaneous injection as a suspension in olive oil (50μl), control animals receiving olive oil alone. Using these methods the following tabularized results were obtained and are presented as examples of the efficacy of 10-(substituted phenyl)-3-alkylflavins against plasmodia.

TABLE 3A Percent of cured mice out of 10 treated with a single intraperitoneal dose of 10-(substituted phenyl)-3- methylflavin.

TABLE 3B Percent of cured mice out of 10 treated with a single oral dose of 10-(substituted phenyl)-3-methylflavin.

*out of 20 treated mice

Drugs: 3a: 10-(4'-chlorophenyl)-3-methylflavin

3b: 10-(4'-bromophenyl)-3-methylflavin

3c: 10-(3',4'-dichlorophenyl)-3-methylflavin

3d: 10-(3',5'-dichlorophenyl)-3-methylflavin 3e: 10-(3',5'-dimethylphenyl)-3-methylflavin

3f: 10-(3'-trifluoromethylphenyl)-3- methylflavin

(b) In vitro drug trials The drugs were dissolved in dimethyl sulfoxide and incubated overnight at 37°C with group O human serum. Small volumes of this drug-treated serum (1-10μl, containing 3.5 to 35μg/ml) were added to triplicate flat bottom wells of micro-culture trays (Linbro) containing 100μl of a 1% suspension of parasitised erythrocytes (0.5-5% parasitemia, asynchronous parasites) in culture medium without other serum. Normal untreated serum was added to wells containing less than 10% (v/v) serum to bring the final concentration to this amount. Serum amount with the equivalent volume of dimethyl sulfoxide (maximum of 40μl/ml) was added to control wells.

In experiments involving radioactive uptake of a metabolic label, 20μl of [³H]-hypoxanthine

(1/100 dilution of 1 mCi/ml, 2.8 Ci/mmol, Amersham

Australia) in RPMI was added to the wells. At the end of culture, the cells were harvested onto filters with a "skatron" cell harvester using water as the washing fluid. The filters were then counted by liquid scintillation. Inhibition of parasite growth was calculated from the level of incorporation of [³H]hypoxanthine in control wells. In some experiments parasite growth was assessed in

Giemsa-stained smears made from the contents of culture wells.

The micro-culture trays were incubated for 24 or 48 hours in the usual gas mixture or in one containing a higher oxygen level, namely 5% CO₂ in air (19% O₂).

Using these methods it was demonstrated by way of example that the in vitro growth of P.falciparum was inhibited by 10-(4'-chlorophenyl) -3-methylflavin at a concentration of 4-8μM.

Inhibition increased from 53 to 83% at 8μM when the incubation was increased from 24 to 48 hours. The results are shown in Figure 1 which plots antimalarial activity of 10-(4'-chlorophenyl)-3- methylflavin against P.falciparum in 24 hour (◊) and

48 hour cultures as assessed by incorporation of

[³H]-hypoxanthine. Points are means of triplicate wells in four separate experiments. At higher oxygen levels (19%) the inhibition was not significantly increased ( 10%) at 4μM and 0.8μM in 48 hour cultures (3 experiments, data now shown). There was no obvious hemolysis of parasitised cells in drug treated cultures but parasite development was retarded as assessed on Giemsa-stained smears, and a high percentage of abnormal forms, mostly undeveloped rings or trophozoites, were observed. (c) Other biological activity (i) The compounds of this invention have cytostatic and cytoxic activity against a rat mammary adenocarcinoma cell line in culture. The rat mammary adenocarcinoma cells, MAT 13762, were originally from the Mason Research Laboratories, Worcester, MA. and were routinely cultured in RPMI 1640 medium supplemented with 10% heat-inactivated foetal calf serum. These cells were poorly adherent and grew mainly in suspension. By way of example, when cultured in the presence of 10-(4'-chlorophenyl)-3-methylflavin at 4μM concentration, the tumour cells ceased growing after 24 hours as assessed by cytometry. The same compound at 10μM and higher concentrations was cytostatic in 24 hour cultures and obviously cytotoxic in 48 hour and longer cultures as assessed cytometrically. (ii) The compounds of this invention also inhibit phorbol myristate acetate induced chemiluminescence in both rat and human polymorphonuclear leucocytes in culture and thus have potential antiinflammatory activity.

Rat peritoneal exudate cells were elicited with 12% sodium caseinate and after 16 hours sacrificed and the cells removed. The cells were layered onto percoll gradient d 1.070/1.079/1.080 and spun at 600 x g for 20 minutes. A 99.8% pure polymorphonuclear (PMN) population was isolated from the bottom of the tube. The cells were washed twice and resuspended at 1 x 10⁶ cells per ml in phosphate buffered saline/glucose (5μM). The cell suspension was treated with luminol (113μM) and incubated for 5 minutes.

At this point, the test compound in 2μl of

DMSO (test) or 2μl of DMSO alone (control) was added to the cell suspension. After 1 minute of incubation phorbol myristate acetate (10^-7M) was added and the resulting chemiluminescence measured.

By way of example, 10-(4'-chlorophenyl)-3- methylflavin produced 50% inhibition at 2μM and 74% inhibition at 8μM, 10-(3',5'-dichlorophenyl)

-3-methylflavin produced 52% inhibition at 10μM,

10-(3',5'-dimethylphenyl)-3-methylflavin produced 37% inhibition of 10μM and 10-(3'-trifluoromethyl- phenyl)-3-methylflavin produced 40% inhibition at 10μM.

REFERENCES

1. Kakai, P. and Thurnham, D.I., (1983). Trans. R. Soc.Med.Hvg.. 77. 680-686.

2. Thurnham, D.I., Oppenheimer, S.J. and Bull, R., (1983). Trans. R.Soc.Trop.Med.Hyg., 77. 423-424.

3. Geary, T.G., Divo, A.A. and Jensen, J.B., (1985). J.Protozool., 32, 65-69.

4. Divo, A.A. Geary, T.G., Davis, N.L. and Jensen, J.B., (1985). J.Protozool., 32, 59-64.

5. Graham, D.W., Brown, J.E., Ashton, W.T., Brown, R.D. and Rogers, E.F., (1977). Experientia, 33, 1274-1276.

6. Dutta, P., Pinto, J. and Rivlin, R., (1985). Lancet, ii, 1040-1043.

7. Kuhn, R. and Weygard, R., (1935). Chem.Ber., 68, 1282.

8. Mair, L., Kasperek, G.J. and Bruice, T.C, (1972), Biochemistry. 11, 3991.

9. Knappe, W.R., (1974). Chem.Ber., 107. 1614.

10. Smith, S.B., and Bruice, T.C, (1975), J.Amer .Chem.Soc., 97, 2875.

11. Yoneda, F., Shinozuka, K., Tsukuda, K. and Koshiro, A., (1979). J. Heterocyclic Chem., 16, 1365.

12. Yoneda, Sakuma, Ichiba and Shinomura, (1976) J.Amer.Chem.Soc., 98, 830.

13. Manousek and Jiricka, (1961), Chem. Tech. (Berlin), 13, 169.

14. Kuhn and Strobele, (1937), Chem.Ber. 70, 753

15. Michaelis, Schubert and Smythe, (1937) J.Biol.Chem., 116, 587.

16. Michaelis and Schwarzenbach, (1938) J.Biol. Chem. 123, 527.

17. Hemmerich and Erlenmeyer (1957), Helv.Chim. Acta. 40, 180.

Claims

CLAIMS :

1. A method for the antiprotozoal, antiproliferative and/or antiinflammatory treatment of a human or animal, which comprises the administration of an effective amount of a compound of the general formula I:

(a) H, alkyl of from 1 to 8 carbon atoms, alkenyl of from 2 to 8 carbon atoms, alkynyl of from 2 to 8 carbon atoms, unsubstituted and alkyl-substituted cycloalkyl and cycloalkenyl of from 3 to 10 carbon atoms, phenylalkyl of from 7 to 12 carbon atoms, mono-, di- and trihalogenoalkyl of from 1 to 3 carbon atoms, phenyl, alkylphenyl of from 7 to 14 carbon atoms, mono- di- and tri(trihalogenomethyl)phenyl and mono-, di- and trihalogenophenyl;

(b) amino moieties of the formula

(ii) A and B together with the nitrogen atom to which they are attached represent a heterocyclic moiety selected from the group consisting of aziridinyl, azetidinyl, pyrrolidinyl, piperidinyl, hexahydroazepinyl, heptamethyleneiminyl, morpholinyl, thiomorpholinyl, piperazinyl, 4-alkyl- and 4-arylpiperazinyl, octamethyleneiminyl, each of the said heterocyclic moieties optionally having attached as substituents on carbon atoms thereof from 1 to 3 alkyl groups of from 1 to 8 carbon atoms;

(c) halogeno, including F, Cl, Br, and I;

(d) alkoxy, aryloxy, alkylthio or arylthio moieties of the formulae -O-Z or -S-Z, wherein Z is alkyl of from 1 to 8 carbon atoms, alkenyl of from 2 to 8 carbon atoms, alkynyl of from 2 to 8 carbon atoms unsubstituted and alkylsubstituted cycloalkyl and cycloalkenyl of from 3 to 10 carbon atoms, phenylalkyl of from 7 to 12 carbon atoms, phenyl, alkylphenyl of from 7 to 14 carbons atoms, mono-, di-and tri(trihalogenomethyl) phenyl and mono-, di- and trihalogenophenyl;

the dihydro derivatives thereof of the formula lb

or the tetrahydro derivatives thereof of the formula Ic

wherein in the formulae la, lb, and Ic, Y, n, R ¹, R² , R³ , R⁴ and X are as defined above,

2. A method according to claim 1, wherein in the compound of the general formulae I, la, lb or Ic, n is 2.

3. A method according to claim 1, wherein in the compound of the general formulae I, la, lb or Ic, y represents methyl or ethyl.

4. A method according to claim 1, which comprises the administration of an effective amount of a compound selected from 10-(3',5'-dichlorophenyl) -3-methylflavin and 10-(3',5'-dimethylphenyl)-3- methylflavin.

5 . Use of a compound of the general formulae I, la, lb or Ic as defined in claim 1, for the manufacture of a medicament for the antiprotozoal, antiproliferative and/or antiinflammatory treatment of a human or animal.

6. A pharmaceutical or veterinary composition for antiprotozoal, antiproliferative and/or antiinflammatory treatment of a human or animal which comprises a compound of the general formulae I, la, lb or Ic, as defined in claim 1, together with a pharmaceutical or veterinary carrier therefor.

7. A compound of the general formula II:

wherein Y, n, R ¹, R² , R³, R⁴ defined in claim 1 or a 5N-oxide, dihydro or tetrahydro derivative thereof, with the proviso that

(X)_n is not H, 4'-chloro, 3'-methyl, 4'-methyl,

2'-ethyl or 2',6'-dimethyl, when Y is methyl and R ¹, R², R³ and R⁴ are hydrogen.

8. A compound according to claim 7 wherein n is 2.

9. A compound according to claim 7, wherein Y represents methyl or ethyl.

10. A compound according to claim 7, selected from 10-(3',5'-dichlorophenyl)-3-methylflavin and 10-93',5'-dimethylphenyl)-3-methylflavin.