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Syntheses of several carbamate analogues of 2,5-bis(4-amidinophenyl)furan (1) under mild conditions and their evaluation as prodrugs against Pneumocystis carinii pneumonia (PCP) in an immunosuppressed rat model are described. Thus, nine new bis-carbamates:  methoxycarbonyl (2), 2,2,2-trichloroethoxycarbonyl (3), ethylthiocarbonyl (4), benzyloxycarbonyl (5), (4-methyl-2-oxo-1,3-dioxol-4-en-5-yl)methoxycarbonyl (6), phenoxycarbonyl (7), 4-fluorophenoxycarbonyl (8), 4-methoxyphenoxycarbonyl (9), and (1-acetoxy)ethoxycarbonyl (10) and a bis-carbonate ethoxycarbonyloxy (11) of the bis-amidine 1 have been synthesized and evaluated. The in vivo results show that the 4-fluorophenyl carbamate 8 and the 4-methoxyphenyl carbamate 9 in this series had the best anti-PCP activity by both intravenous and oral administration at a dosage level of 22 mol and 33 μmol/kg/day, respectively. Compounds 3−7 were also more active than the parent drug (1) on oral administration. The acute toxicity usually exhibited by the parent amidine 1 at a dosage level of 22 μmol/kg/day on intravenous administration has been significantly reduced by the prodrug modifications, with the exception of compound 10 which exhibited some toxicity. This report also describes the synthesis of several aryl−alkyl and aryl−aryl carbonates (12−14, 16−23) as efficient reagents for the preparation of carbamate derivatives from bis-arylamidines.

Dicationic 2,4-bis(4-amidinophenyl)furans 5−10 and 2,4-bis(4-amidinophenyl)-3,5-dimethylfurans 14 and 15 have been synthesized. Thermal melting studies revealed high binding affinity of the compounds to poly(dA-dT) and to the duplex oligomer d(CGCGAATTCGCG)2. All of the new compounds were effective against Pneumocystis carinii pneumonia in the immunosuppressed rat model with up to 200-fold increase in activity compared to the control compound pentamidine. No toxicity was noted for 5, 7−10 at the dose of 10 μmol/kg/d; however, the isopropyl analogue 7 showed toxicity comparable to pentamidine at the dosage of 20 μmol/kg/d. Dimethylation of the parent compound on the furan ring resulted in reduced activity and increased toxicity.

Aromatic dicationic compounds, such as pentamidine, have potent antimicrobial activities. Clinical use of these compounds has been restricted, however, by their toxicity and limited oral activity. A novel approach, using amidoxime derivatives as prodrugs, has recently been proposed to overcome these limitations. Although results were presented for amidoxime derivatives of only one diamidine, pentamidine, the authors in the original proposal claimed that amidoxime derivatives would work as effective prodrugs for all pharmacologically active diamidines. Nine novel amidoxime derivatives were synthesized and tested in the present study for activity against Pneumocystis carinii in corticosteroid-suppressed rats. Only three of the nine compounds had significant oral anti-Pneumocystis activity. The bisbenzamidoxime derivatives of three direct pentamidine analogs had excellent oral and intravenous activities and reduced acute host toxicity. These compounds are not likely candidates for future drug development, however, because they have chronic toxic effects and the active amidine compounds have multiple sites susceptible to oxidative metabolism, which complicates their pharmacology and toxicology. Novel diamidoximes from three other structural classes, containing different groups linking the cationic moieties, lacked significant oral or intravenous anti-Pneumocystis activity, even though the corresponding diamidines were very active intravenously. Both active and inactive amidoximes were readily metabolized to the corresponding amidines by cell-free liver homogenates. Thus, the amidoxime prodrug approach may provide a strategy to exploit the potent antimicrobial and other pharmacological activities of selected, but certainly not all, aromatic diamidines.

The syntheses of 12 new 2,5-bis[4-(N-alkylamidino)phenyl]furans are reported. The interaction of these dicationic furans with poly(dA-dT) and with the duplex oligomer d(CGCGAATTCGCG)2 was determined by Tm measurements, and the effectiveness of these compounds against the immunosuppressed rat model of Pneumocystis carinii was evaluated. At the screening dose of 10 μmol/kg, 9 of the 14 N-alkylamidino furans described here are more active than the parent compound 1. Substitution of an alkyl group on the amidino nitrogen, except for in 9, 13, and 15, resulted in higher affinity for DNA than the parent compound as judged by the larger ΔTm values and suggests enhanced van der Waals interactions in the bis-amidine−DNA complex. Five of the compounds, 3, 5, 7, 10, and 12, yield cyst counts of less than 0.1% of control when administered at a dosage of 10 μmol/kg. Five compounds, 1, 6, 8, 10, and 12, show significant activity at a dosage of approximately 1 μmol/kg; 12 is the most active derivative, and it is approximately 100 times more effective than pentamidine in this animal model.

Crystal structures are reported of complexes of two novel furan derivatives of berenil with alkyl benzamidine groups bound to the DNA sequence d(CGCGAATTCGCG)2. They have both been determined to 2.2 Å resolution and refined to R factors of 16.9% and 18.6%. In both structures the alkyl substituents, cyclopropyl and isopropyl, are found to be orientated away from the floor of the minor groove. The drugs are located in the minor groove by two strong amidinium hydrogen bonds, to the O2 of the thymines situated at the 5‘ and 3‘ ends of the AT-rich region. The isopropyl-substituted derivative has a tight hydrogen-bonded water network in the minor groove at one amidine site, which alters the orientation of the isopropyl substituent. This compound has superior DNA-binding properties and activity against Pneumocystis carinii and Cryptosporidium parvum infections in vivo compared to the cyclopropyl derivative, which in turn is superior to the parent furan compound. We suggest that the nature and extent of the interactions of these compounds in the DNA minor groove play an important role in these activities, possibly in conjunction with a DNA-binding protein. The overall effect of these alkyl benzamidine substitutions is to increase the binding of the drugs to the minor groove.

Considerable evidence now indicates that DNA is the receptor site for dicationic benzimidazole anti-opportunistic infections agents (Bell, C. A.; Dykstra, C. C.; Naiman, N. A. I.; Cory, M.; Fairley, T. A.; Tidwell, R. R. Antimicrob. Agents Chemother. 1993, 37, 2668−2673. Tidwell, R. R.; Jones, S. K.; Naiman, N. A.; Berger, I. C.; Brake, W. R.; Dykstra, C. C.; Hall, J. E. Antimicrob. Agents Chemother. 1993, 37, 1713−1716). To obtain additional information on benzimidazole−receptor complexes, the syntheses and DNA interactions of series of symmetric benzimidazole cations, linked by alkyl or alkenyl groups, have been evaluated. Biophysical techniques, thermal denaturation measurement (ΔTm), kinetics, and circular dichroism (CD) have been used in conjunction with NMR and molecular modeling to evaluate the affinities, binding mode, and structure of complexes formed between these compounds and DNA. All of the compounds bind strongly to DNA samples with four or more consecutive AT base pairs, and they bind negligibly to GC rich DNA or to RNA. Spectral and kinetics characteristics of the benzimidazole complexes indicate that the compounds bind in the DNA minor groove at AT sequences. NMR and molecular modeling of the complex formed between an ethylene-linked benzimidazole derivative, 5, and the self-complementary oligomer d(GCGAATTCGC) have been used to establish structural details for the minor groove complex. These results have been used as a starting point for molecular mechanics calculations to refine the model of the minor groove−benzimidazole complex and to draw conclusions regarding the molecular basis for the effects of substituent changes on benzimidazole−DNA affinities.

The syntheses of nine new derivatives of 2,5-bis[4-(N-alkylamidino)phenyl]furans with extended aromatic systems are reported. The interaction of these dicationic furans with poly(dA)*poly(dT) and with the duplex oligomers d(CGCGAATTCGCG)2 and d(GCGAATTCGC)2 was determined by Tm measurement, and the effectiveness of these compounds against the immunosuppressed rat model of Pneumocystis carinii was evaluated. At a screening dose of 10 μmol/kg, 4 of the 12 amidino furans described here are more active than the parent compound 1. In general, extension of the aromatic system in the absence of a substitution of the amidino nitrogens resulted in higher affinity for DNA than the parent compound as judged by the larger ΔTm values and suggests enhanced van der Waals interactions in the amidino furan−DNA complex. Three of the compounds, 3, 5, and 11, yield cysts counts of less than 0.1% of control when administered at a dosage of 10 μmol/kg. Compound 3, which does not have an extended aromatic system, is the most active derivative. Although a direct correlation between anti-P. carinii activity and DNA binding affinity was not observed, all compounds which have significant activity have large ΔTm values.

The antiprotozoal/antifungal drug pentamidine [1,5-bis(4-amidinophenoxy)pentane] has been recently shown to be metabolized by rat liver fractions to at least six putative metabolites as detected by high-performance liquid chromatography. Two minor metabolites have been previously identified as N-hydroxypentamidine and N,N'-dihydroxypentamidine. In this study, the two major microsomal metabolites have been identified as the 2-pentanol and 3-pentanol analogs of pentamidine [1,5-di(4-amidinophenoxy)-2-pentanol; and 1,5-bis(4-amidinophenoxy)-3-pentanol]. As well, a seventh putative metabolite has been discovered and identified as para-hydroxybenzamidine, a fragment of the original drug. Whereas the cytochromes P-450 have been demonstrated as the enzyme system responsible for pentamidine metabolism, hydroxylation of the drug was not inducible by phenobarbital, beta-naphthoflavone, clofibrate, isosafrole, pregnenolone-16 alpha-carbonitrile, ethanol or pentamidine pretreatment of rats. The kinetics of the production of the two major microsomal metabolites has been determined as Km = 56 +/- 19 microM and Vmax = 126 +/- 21 pmol/min/mg microsomal protein for the 2-pentanol analog, and Km = 28 +/- 0.28 microM and Vmax = 195 +/- 2.4 pmol/min/mg microsomal protein for the 3-pentanol analog. Therefore, the mixed-function oxidases readily convert pentamidine to hydroxylated metabolites, but exactly which isozyme(s) of cytochrome P-450 is responsible is not clear.

A series of proteases of diverse substrate specificity were tested for their effect on respiratory syncytial virus-induced cytopathology. Three of the enzymes, thrombin, plasmin, and trypsin, were able to augment significantly the fusion of virus-infected A549 cells. On a concentration basis, thrombin was the most active promoter, followed by plasmin and then trypsin. Hirudin, a specific thrombin inhibitor, blocked the fusion-enhancing property of thrombin, yet had no influence on the basal rate of fusion in the absence of the enzyme. By contrast, the amidine-type inhibitors of trypsin-like proteases, bis(5-amidino-2-benzimidazolyl)-methane (BABIM), blocked not only the thrombin effect, but also the fusion in the thrombin-free controls. The suppressive activity of BABIM was observed at concentrations so low as to exclude any direct inhibitory effect on thrombin itself. These results make it seem very likely that thrombin advances cell fusion by activating a BABIM-sensitive protease. Plasmin and trypsin can be expected to act in a similar manner.

To evaluate the importance of proteolytic activity in the pathogenesis of cell injury by Rickettsia rickettsii, a series of four aromatic amidine inhibitors of trypsin-like proteases were introduced into the plaque model. The compounds were shown to be active toward plaque reduction with their order of effectiveness parallel to their antitrypsin activity. One of the compounds, bis(5-amidino-2-benzimidazolyl)-methane, at a concentration of 10(-5) M demonstrated complete inhibition of plaque formation on day 6. Bis(5-amidino-2-benzimidazolyl)methane at the same concentration reduced cell injury even when added to the system after 72 h of rickettsial infection. The reduction in morbidity in guinea pigs experimentally infected with R. rickettsii and treated with bis(5-amidino-2-benzimidazolyl)methane as compared with morbidity in infected, untreated animals, comprised delay in the onset of fever and slightly fewer febrile animals. Because bis(5-amidino-2-benzimidazolyl)methane had no effect on phospholipase A2, the enzyme activity associated with penetration-induced cell injury, it is likely that a trypsin-like protease also plays an essential role either in the physiology of R. rickettsii or as its pathogenic mechanism.

Tryptase is the major secretory protease of human mast cells and is proposed to be involved in neuropeptide processing and tissue inflammation. Exploration of the biology of tryptase has been hindered by the lack of potent, selective inhibitors. The current study explores the properties of aromatic diamidines as inhibitors of dog and human tryptase. The strongest inhibitors of tryptase in this series are bis(5-amidino-2-benzimidazolyl)methane (BABIM) and (5-amidino-2-benzimidazolyl)-(5-(N,N'-dimethylamidino)-2- benzimidazolyl)methane, which exhibit K(i) values of 1.8 and 1.4 nM, respectively, in blocking the hydrolysis of tosyl-L-Gly-Pro-Lys-4-nitroanilide by human tryptase. These compounds are approximately 10,000-fold more potent than benzamidine, and are the strongest reversible inhibitors of tryptase described to date. Other aromatic mono- and diamidines, including amiloride and pentamidine, are less potent. Nonetheless, they abolish tryptase activity at high inhibitor concentrations. The rank order of tryptase inhibitor potency parallels that of inhibitors tested against trypsin. BABIM, the only highly active member of this series whose potency against other targets has been examined previously, is a far stronger inhibitor of tryptase than of other trypsin-like serine proteases, including those involved with hemostasis, fibrinolysis and the complement system. Therefore, BABIM appears to have selective affinity for tryptase. In addition to inhibiting tryptase-induced hydrolysis of peptide-based chromogenic substrates, BABIM blocks completely the reversal of vasoactive intestinal peptide-induced relaxation of isolated trachea by dog tryptase. Thus, BABIM and related amidines are potent inhibitors of mast cell tryptases that may be useful in exploring mast cell protease biology.

Several aromatic mono- and diamidines were found to block cell fusion induced by respiratory syncytial virus. The best inhibitors were able to achieve complete suppression of syncytium formation at a concentration of 1.0 microM. Inhibition occurred in respiratory syncytial virus-infected HEp-2 and CV-1 cells, but the same inhibitors were ineffective in preventing fusion induced by parainfluenza virus type 3. The fusion inhibitors did not reduce single-cycle virus yields, but did reduce multiple-cycle yields. In addition, the active compounds caused a significant retardation of respiratory syncytial virus penetration. The mechanism by which amidines interfere with respiratory syncytial virus-host cell interactions is unknown, but parallels can be drawn between antiviral activity and the ability of the compounds to inhibit certain trypsin-like proteases.