1252
J.Med. Chem. 1990,33,1252-1257
6.24(dd, 1 H, J = 4.5 Hz, J = 1.5 Hz), 7.08 (dd, 1 H, J = 4.5 Hz, J = 4.5Hz), 7.95 (d, 1 H, J = 6.0Hz),8.19 (d, 1 H, J = 12.0 Hz), 8.77 (s, 1 H); mass spectrum, m/z 316 (M + HI+; IR (KBr) 3420 (OH), 1720 (C=O) cm-'. In Vitro Antibacterial Activity. The in vitro antibacterial activity of the new compounds was tested in a side-by-side comparison with ciprofloxacin (le)and determined by conventional agar dilution procedures. The organisms were grown overnight in brain-heart infusion (BHI) broth (Difco 0037-01-6)at 36 OC. Twofold dilutions of the stock solution (2000rg/mL) of the test
compound were made in BHI agar to obtain the teat concentration ranging from 200 to 0.005 pg/mL. The plate was inoculated with approximately lo'organisms. It was then incubated at 36 OC for 18 h. The minimum inhibitory concentrations (MIC, pg/mL) were the lowest concentrations of the test compounds that yielded no visible growth on the plate.
Acknowledgment. We thank Dwight Hardy and the staff of the MicrobiologicalTeam for their in vitro testing and Cyndy Davis for typing the manuscript.
Analogues of 1,5-Bis(4-amidinophenoxy)pentane(Pentamidine)in the Treatment of Experimental Pneumocystis carinii Pneumonia Richard R. Tidwell,*it Susan Kilgore Jones,+J. Dieter Geratz,+Kwasi A. Ohemeng,t Michael Cory,$ and James Edwin Hall8 Division of Medicinal and Biological Chemistry, Department of Pathology, School of Medicine and Department of Parasitology, School of Public Health, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, and Burroughs- Wellcome Company, Cornwallis Road, Research Triangle Park, North Carolina 27709. Received July 24, 1989
A series of 33 analogues of the anti-Pneumocystis carinii drug 1,5-bis(4-amidinophenoxy)pentane (pentamidine) was synthesized for screening against a rat model of P . carinii pneumonia (PCP). Twenty-five of the compounds showed efficacy against PCP when compared to a saline-treated control group. Two compounds, 1,4-bis(4amidinophen0xy)butane (butamidine, 6) and 1,3-bis(4-amidino-2-methoxyphenoxy)propane (DAMP, 16), were statistically more effective than the parent drug in treating PCP in the rat model of infection. In addition to their activity against PCP, the compounds were also evaluated for antitrypsin activity, ability to inhibit thymidylate synthetase, affinity for DNA, and toxicity. No correlation was observed between the tested molecular interactions of the diamidines and their effectiveness against PCP.
An aromatic diamidino compound, pentamidine, was discovered as early as 1957 to be an effective agent for the treatment of P. carinii pneumonia (PCP).' Since then the drug has seen continued use for the treatment of PCP despite an extensive list of adverse reactions that include nephrotoxicity, hepatotoxicity, hypotension, and sterile abscesses at the injection ~ i t e However, . ~ ~ ~ pentamidine was a distant second to the relatively nontoxic diaminopyrimidinesulfonamide combinations for the treatment of PCP.4 This preference changed drastically with the clinical upsurge of cases of PCP caused by the acquired immune deficiency syndrome (AIDS) and the observation that trimethoprim-sulfamethoxazole caused a high frequency of adverse reactions in patients with AIDS-related PCP.5p6 This unfortunate circumstance, combined with the finding that PCP is the leading cause of morbidity and mortality in AIDS patients,7i8 has caused an increased dependency on the use of pentamidine in treatment of AIDS-related PCP. Recent studies have shown that the toxicity of pentamidine can be greatly reduced and drug efficacy increased by aerosol administration?J" Despite these findings there is still an urgent need for a safe and effective drug that can be given either by oral or by parenteral administration for treatment of PCP associated with AIDS. There is published record of only a handful of pentamidine-related compounds as having been tested against PCP. The screening of large numbers of drugs against PCP has been limited due to the lack of a dependable in vitro assay system. Therefore, the evaluation of anti-P. carinii drugs has depended on a somewhat cumbersome Department of Pathology, University of North Carolina at Chapel Hill. Burroughs-Wellcome. *Department of Parasitology, University of North Carolina at Chapel Hill.
*
and expensive model utilizing immunosuppressed animals. The model involves the administration of corticosteroids to rats for a period of 6-8 weeks, resulting in the spontaneous induction of PCP.11-14 A number of drug studies have shown that the rat model of PCP is an effective predictor of drug efficacy in humans."J2 An early report demonstrated that a diamidine derivative, hydroxystilbamidine, showed some activity against P. carinii in the rat model of the disease." Two recent studies demonstrated that several dicationic molecules with structures related (1) Ivady, G.; Paldy, L. Monutsschr. Kinderheilkd. 1957,106,lO. ( 2 ) Pearson, R. D.; Hewlett, E. L. Ann. Intern. Med. 1985,103, 782. ( 3 ) MacFarlane, J. T.; Finch, R. G. Thorax 1985,40,561. (4) Walzer, P. D.; Kim, C. K.; Cushion, M. T. In Parasitic Znfections in the Compromised Host; Walzer, P. D., Genta, R. M.,
Eds.; Marcel Dekker, Inc.: New York, 1989 pp 83-178. (5) Jaffe, H. S.; Abrams, D. I.; Ammann, A. J.; Lewis, B. J.; Golden, J. A. Lancet 1983,2,1109. (6) Gordin, F. M.; Simon, G. L.; Wofsy, C. B.; Mills, J. Ann. Zntern. Med. 1984,100,495. (7) Niedt, G. W.; Schinella, R. A. Arch. Pathol. Lab. Med. 1985, 109,727. (8) Moskowitz, L.; Hensley, G. T.; Chan, J. C.; Adams, K. Arch. Pathol. Lab. Med. 1985,109,735. (9) Montgomery, A. B.; Luce, J. M.; Turner, J.; Lin, E. T.; Debs, R. J.; Corkery, K. J.; Brunette, E. N.; Hopewell, P. C. Lancet 1987,ii, 480. (10) Debs, R. J.; Blumenfeld, W.; Brunette, E. N.; Straubinger, R. M.; Montgomery,A. B.; Lm,E.; Agabian, N.; Papahadjopoulca, D. Antimicrob. Agents Chemother. 1987,31,37. (11) Frenkel, J. K.; Good, J. T.; Shultz, J. R. Lab. Invest. 1966,15, 1559. (12) Walzer, P. D.; Kim, C. K.; Foy, J.; Linke, M. J.; Cushion, M. T. Antimicrob. Agents Chemother. 1988,32,896. (13) Hughes, W. T.; McNabb, P. C.; Makers, T. D.; Feldman, S. Antimicrob. Agents Chemother. 1974,5,289. (14) Tidwell, R. R.; Kilgore, S. G.; Ohemeng, K. A.; Geratz, J. D.; Hall, J. E. J. Protozool. 1989,36,74s.
0022-2623/90/1833-1252$02.50/00 1990 American Chemical Society
Journal of Medicinal Chemistry, 1990, Vol. 33, No. 4
Analogues of 1,5-Bis(4-amidinophenoxy)pentane
1253
Scheme I1
Scheme I
gOH " +
n = 2-6
2
3n
a) R=H, X=F
n=3-6
b) R=N02, X=CI
Na, EtOH
i
i A
1. HCI, EtOH/
2. EtOH, NH,
H,N-(CH2),-NH2
A
2
1 -
R = H,CI, Br, or -OCH,
+
f f i e x
Br-(CHd,-Br
R
I \
I
Et,N,DMSO
Ac,O, TFA, HNO,
(where R=H, p-CN)
n=2-6 R=H. NO?
I. HCI, EtOH 2. NH,, EtOH O - ( C IH ~ ) . _ O - @ - C ~ 1. HC1, EtOH 2. NHz-(CH2),-NH, where n=S, R=H, p-CN
I
W
NO2
"2
2Qa v
II
I
H,. Pd/C
iv
n=4,6 Hz, PdC (where n=3,5, R=N02)
"
1. HCI, EtOH 2. NH,, EtOH
2Ul
to pentamidine also possessed activity against rat PCP.I2J3 These studies, while showing promise for pentamidine type compounds against PCP, added few data to determine the structure-activity correlations or the mechanism of activity of this class of compounds against P. carinii. This paper describes the synthesis and the anti-P. c a d i activity of 33 analogues of pentamidine against the rat model of the disease. Also, we have determined the anti-protease activity, thymidylate synthetase inhibitory activity, and the DNA-binding ability of selected analogues to look for correlations between these activities and anti-P. carinii potency. Chemistry. For the present study, all of the compounds were synthesized in our laboratory. The syntheses of compounds 5-12,14,and 15 have been previously reported, and they were prepared according to the established route as outlined in Scheme I.l6JS While the activity of compound 28 against Trypanosoma rhodesiense and Plasmodium berghei has been reported," the synthesis has been detailed only in a final report in an Army contract.'* The compound was synthesized by replacing ammonia with diaminoethane according to Scheme I, route ii. The dinitro analogues of pentamidine (20-22)were prepared by dinitration (Scheme I, route iii) of the corresponding dinitriles (3)followed by conversion of nitrile derivatives 19 to the respective amidines (Scheme I, route iv). DiGeratz, J. D.; Whitmore, A. C.; Cheng, M. C. F.; Piantadosi, C. J. Med. Chem. 1973,16,970. Geratz, J. D.; Cheng, M. C.; Tidwell, R. R. J. Med. Chem. 1975, 18, 477. Steck, E. A.; Kinnamon, K. E.; Rane, D. S.; Hanson, W. L. Exp. Parasitol. 1981,52, 404. Elsanger, E. F.; Final report, Department of the Army contract no. DADA 49-193-MD-2754, 1970. Berger, B. J.; Hall, J. E.; Tidwell, R. R. J . Chromatogr. 1989, 494,191. Cory, M.; McKee, D. D.; Kagan, J.; Henry, D. W.; Miller, J. A. J. Am. Chem. SOC.1985,107,2528. Dolnick, B. J., Cheng, Y. C. J. Biol. Chem. 1978, 253, 3563.
amino derivatives 23-27 were prepared from the corresponding dinitro compounds 20-22 by hydrogenation with Pd/C (Scheme I, route v). The synthesis of cu,w-bis(4amidinoani1ino)alkanes 32-34 was accomplished by nucleophilic displacement reaction (Scheme 11, route i) of 4-fluorobenzonitrile (29a)with the appropriate diaminoalkane (30)to give the corresponding dibenzonitrile derivative 31. The nitriles were converted to amidines in the usual manner (Scheme 11, route ii) to give the desired products 32-34. Similarly, 4-chloro-3-nitrobenzonitrile (29b)was used as the starting reagent to give the desired cu,(~~bis(4-amidino-2-nitroanilino)alkanes 35 and 36. 1,3Bis(4-amidino-2-aminoanilino)propane(38)and 1,bbis(4-amidino-2-aminoanilino)pentane (40) were prepared by reduction (Scheme 11, route iv) of the corresponding nitro derivatives 35 and 36 using Pd/C, while the four- and six-carbon analogues 39 and 41 were synthesized by first reducing (Scheme 11, route iii) the desired a,w-bis(4cyano-2-nitroanilino)alkane31 to the corresponding amine 37. The nitriles were then converted to the amidine products (Scheme 11, route v). Since the intermediate 1,2-bis(4-cyanoanilino)ethane was insoluble in any appropriate solvent, the synthesis of 1,2-bis(4-amidino-2aminoani1ino)ethane (45)was prepared according to the steps outlined in Scheme III. 4-Chloro-3-nitrobenzonitrile (29)was reacted with a 10-fold excess of ethylenediamine (30)at 25 "C (Scheme 111, route i) to give monosubstituted product 42. The nitro group was reduced, and the product was reacted with a second mole of 29 (Scheme 111, route ii) to give 1-(2-amino-4-cyanoanilino)-2-(4-cyano-2-nitroanilino)ethane. The diamidine was prepared, followed by reduction of the second nitro group (Scheme 111, route iii) to give 44. Results and Discussion Thirty-one analogues of pentamidine (Table I) were synthesized and screened for their ability to reduce the
1254 Journal of Medicinal Chemistry, 1990, Vol. 33, No. 4
Tidwell et al.
Table I. Structure and Chemical Data of Pentamidine Analogues HN
n-xa&c~NH
@X--(CHd
)'c H2N no. 4 5 (propamidine) 6 (butamidine) 7 (pentamidine) 8 9 10 11 12 13 14 15 16 (DAMP) 17 18 20 21 22 23 24 25 26 27 32 33 34 35 36 38 39 40 41 44 28
X
n
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 N N N N N N
2 3 4 5
N N N N
I
ti
R H H
H H
6
H
3 4 5
H H H H
6 4
5 5 3 4 5 4 5 6
2 3 4
c1 c1
Br OCH3 OCHB OCH3
NO2 NO2 NO2 "2
5
"2 "2 "2
6
"2
3 4 5 3 5 3 4 5 6 2
H H H NO2 NO2 "2 "2
"2 "2 "2
\ "2
am para para para Para para meta meta meta meta para para Para Para para para para para para para para para para para para para para para para para Para Para para para
m n "C >300 144-145 dec 267-270 247 252 300 257 133-134 268 >300 247-248 254-255 293 297-299 258-259 298 255 287-288 dec >300 273 dec 285 270 290 dec 298-300 >300 295 295 dec 293-294 229 274-275 dec 300-303 285 dec >300 147
formulao
I
ti
Elemental analysis (C,H, N) within &0.4% of the theoretical value.
severity of PCP in immunosuppressed rats as measured by histologic scoring of Grocott's methenamine silver (GMS) stained lung sections. Animals treated with the test compounds were compared to those treated with the parent drug (pentamidine) and a saline-treated control group. A typical experiment included six groups of animals (eight animals/group) consisting of four test groups, a pentamidine control group, and a saline control group. Since no statistical variation was observed in the histological lung scores of either control group from experiment to experiment, the test results were combined, and the results are reported in Table 11. Each compound was tested at 10 mg/kg per day by iv injection unless low solubility or high toxicity necessitated a reduced test dose. Twenty-five of the compounds tested (compounds 4 and 15 could not be tested due to insolubilities) produced a statistically significant reduction in lung PCP (regardless of the test dose). Only one compound (44) was inactive when tested at the optimal dose level of 10 mg/kg. The other compounds (12,13,20, and 35) not exhibiting activity against PCP were tested at lower doses due to the lack of solubility. While 15 compounds proved statistically equal to pentamidine in reducing the extent of PCP, only two analogues (6 and 16) produced a mean histologic score that was significantly lower than that of the parent drug. The activity observed for 1,3-bis(4-amidino-2-methoxyphenoxylpropane (16) was especially noteworthy because this
analogue produced a mean histologic score that was significantly lower than pentamidine at one-half the dosage level of the parent drug. Two other methoxy-substituted analogues (17 and 18) also showed good activity against PCP; however, compound 18 proved to be highly toxic at the standard dose of 10 mg/ kg. Substitution of the amidino groups of pentamidine by imidazoline moieties gave a compound (28) that was equally as potent as pentamidine and caused no toxic side effects. The effectiveness of the imidazoline moiety in experimental PCP has been previously reported for imidocarb.12 Dianilino analogues 32-36, 38-41, and 44 were not appreciably more effective than the respective diphenoxy derivatives. However, several compounds in this series proved to be highly toxic. Likewise, substitution of the aromatic rings with nitro groups (compounds 20-22, 35, and 36) produced compounds that were highly toxic relative to the unsubstituted analogues. Amino substitution of the aromatic rings (compounds 23-27) caused no significant change in activity or toxicity. Halogen substitution (compounds 13-15) produced molecules of very low solubility that exhibited no activity against PCP at the highest soluble concentration. Since little is known about the mechanism of action of pentamidine against PCP, we investigated the possibility that a correlation existed between one of the reported molecular interactions of amidine type molecules and their
Analogues of 1,5-Bis(4-amidinophenoxy)pentane
Table 11. Extent of Disease by Histologic Score' no. of animals per scoring group mean no.b n score 0.5 1 2 3 4 toxicity saline 72 3.2 1 2 10 25 34 0 4 not tested in animals-insoluble 1 0 0 + 5 80.9' 3 4 6 8 0.5'~~ 8 0 0 0 0 0 7 63 1.1' 21 28 12 2 0 ++ 8 7 0.9' 3 3 1 0 0 +++ 9 8 1.6c*e 2 1 4 1 0 0 10 7 1.9'9d 2 1 1 3 0 0 11 16 1 . 7 ' ~ ~ 4 3 6 2 1 + 12 81.6' 1 4 1 2 0 + l$ 8 2.78 0 1 2 3 2 0 14' 83.58 0 0 0 4 4 0 15 not tested in animals-insoluble 16h 8 0.6'~~ 7 1 0 0 0 0 17h 80.9' 3 4 1 0 0 0 18 14 1.6'~~ 2 4 7 1 0 ++++ 20' 8 3.18 0 0 3 1 4 ++++ 21' 8 2.0'8 0 2 4 2 0 +++ 22j 6 0.8' 4 1 1 0 0 ++++ 23 80.7' 4 4 0 0 0 0 24 71.0' 2 4 1 0 0 0 25 81.0' 2 5 1 0 0 0 26 15 0.9' 8 5 1 1 0 + 27h 8 2.2f9 0 1 4 3 0 +++ 28 80.7c 5 3 0 0 0 0 32 8O.gc 5 1 2 0 0 + 33 4 0.6' 3 1 0 0 0 ++++ 34 7 1.6'~~ 1 2 3 1 0 +++ 35h 8 3.78 0 0 0 2 6 ++ 36h 7 1.9c.d 1 3 0 2 1 +++ 38 71.2' 1 4 2 0 0 + 39 8 1.6'~~ 0 4 3 1 0 + 40 81.2' 3 2 3 0 0 + 41 7 1.5'~~ 3 1 2 0 1 + 44 82.68 0 0 4 3 1 +
" Histologic scoring: 0.5 =
