Synthesis and bioactivity of novel bis(heteroaryl)piperazine (BHAP) reverse transcriptase inhibitors: structure-activity relationships and increased metabolic stability of novel substituted pyridine analogs.

The major route of metabolism of the bis(heteroaryl)piperazine (BHAP) class of reverse transcriptase inhibitors (RTIs), atevirdine and delavirdine, is via oxidative N-dealkylation of the 3-ethyl- or 3-isopropylamino substituent on the pyridine ring. This metabolic pathway is also the predominant mode of metabolism of (alkylamino)piperidine BHAP analogs (AAP-BHAPs), compounds wherein a 4-(alkylamino)piperidine replaces the piperazine ring of the BHAPs. The novel AAP-BHAPs possess the ability to inhibit non-nucleoside reverse transcriptase inhibitor (NNRTI) resistant recombinant HIV-1 RT and NNRTI resistant variants of HIV-1. This report describes an approach to preventing this degradation which involves the replacement of the 3-ethyl- or 3-isopropylamino substituent with either a 3-tert-butylamino substituent or a 3-alkoxy substituent. The synthesis, bioactivity and metabolic stability of these analogs is described. The majority of analogs retain inhibitory activities in enzyme and cell culture assays. In general, a 3-ethoxy or 3-isopropoxy substituent on the pyridine ring, as in compounds 10, 20, or 21, resulted in enhanced stabilities. The 3-tert-butylamino substituent was somewhat beneficial in the AAP-BHAP series of analogs, but did not exert a significant effect in the BHAP series. Lastly, the nature of the indole substitution sometimes plays a significant role in metabolic stability, particularly in the BHAP series of analogs.

Novenamines as inhibitors of two independent enzymes during DNA replication in a toluenized Escherichia coli cell system.

The amphiphilic novenamines described in this report have been shown previously to be specific inhibitors of human immunodeficiency virus type 1 reverse transcriptase-associated ribonuclease, which they inhibit when they are in the micellar state but not when they are monomeric. These compounds also inhibit the bacterial enzyme DNA gyrase, which is essential for DNA replication. Hence, the present studies were initiated to determine whether the molecular species inhibiting the gyrase reaction was the monomeric or the micellar form. For this purpose, the rate of DNA replication was measured in a toluenized Escherichia coli cell system in the presence of increasing concentrations of novenamines. The resulting concentration-response curves proved anomalous, suggesting the involvement of micelles or some other, noncovalently aggregated forms of the inhibitors. The results were analyzed in terms of a variety of kinetic schemes and were found to be most consistent with the model where novenamines inhibit replicative DNA synthesis predominantly as cooperative dimers and, to a lesser extent, as monomers, but not as highly aggregated micelles. Based on this analysis and the knowledge that novobiocin and all novenamine-containing analogs are powerful gyrase inhibitors, we conclude that the target of the cooperative, dimeric inhibition is the gyrase, whereas the monomers of the novenamines inhibit another enzyme species involved in the bacterial DNA replication process.

The amphiphilic properties of novenamines determine their activity as inhibitors of HIV-1 RNase H.

Few inhibitors of the RNase H function associated with the HIV-1 reverse transcriptase have been discovered to date. We observed that three novenamines, U-34445, U-35122, and U-35401, are specific inhibitors of the HIV-1 RT RNase H function. All three compounds are strong amphiphiles and contain one ionizable group. Hence, a priori, in aqueous solutions the inhibitors might exist in at least four different physical states, namely protonated monomers, ionized monomers, protonated micelles, and ionized micelles. The three inhibitors all yielded anomalous dose-response curves, indicating that the four molecular species have different inhibitory potentials. In order to identify the inhibitory species, the amphiphilic properties of these compounds were studied. It was established that in alkaline solutions, around pH 8, all compounds are ionized and form micelles at concentrations above their CMC. Both the protonated and the ionized forms of these molecules form stable insoluble monomolecular layers at the air/water interface. The anomalies of the dose-response curves can be resolved by taking into account the fact that, in solution, the relative proportion of these molecules in each physical state depends on the pH and on their analytical concentration. Thus interpreted, the results indicate that RNase H is inhibited only by the ionized micellar form of these compounds and not by their monomeric form. Around their pKa (approximately pH 5), the three compounds reproducibly form uniformly sized, self-emulsified colloidal particles that may be used as an efficient drug delivery system.

The benzylthio-pyrimidine U-31,355, a potent inhibitor of HIV-1 reverse transcriptase.

U-31,355, or 4-amino-2-(benzylthio)-6-chloropyrimidine is an inhibitor of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) and possesses anti-HIV activity in HIV-1-infected lymphocytes grown in tissue culture. The compound acts as a specific inhibitor of the RNA-directed DNA polymerase function of HIV-1RT and does not impair the functions of the DNA-catalyzed DNA polymerase or the Rnase H of the enzyme. Kinetic studies were carried out to elucidate the mechanism of RT inhibition by U-31,355. The data were analyzed using Briggs-Haldane kinetics, assuming that the reaction is ordered in that the template:primer binds to the enzyme first, followed by the addition of dNTP, and that the polymerase is a processive enzyme. Based on these assumptions, a velocity equation was derived that allows the calculation of all the essential forward and backward rate constants for the reactions occurring between the enzyme, its substrates, and the inhibitor. The results obtained indicate that U-31,355 acts as a mixed inhibitor with respect to the template:primer and dNTP binding sites associated with the RNA-directed DNA polymerase domain of the enzyme. The inhibitor possessed a significantly higher binding affinity for the enzyme-substrate complexes, than for the free enzyme and consequently did not directly affect the functions of the substrate binding sites. Therefore, U-31,355 appears to impair an event occurring after the formation of the enzyme-substrate complexes, which involves either inhibition of the phosphoester bond formation or translocation of the enzyme relative to its template:primer following the formation of the ester bond. Moreover, the potency of U-31,355 depends on the base composition of the template:primer in that the inhibitor showed a much higher binding affinity for the enzyme-poly (rC):(dG)10 complexes than for the poly (rA):(dT)10 complexes.

U-90152, a potent inhibitor of human immunodeficiency virus type 1 replication.

Bisheteroarylpiperazines are potent inhibitors of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT). We describe a novel bisheteroarylpiperazine, U-90152 [1-(5-methanesulfonamido-1H-indol-2-yl-carbonyl)-4-[3-(1-methyl eth yl-amino)pyridinyl]piperazine], which inhibited recombinant HIV-1 RT at a 50% inhibitory concentration (IC50) of 0.26 microM (compared with IC50s of > 440 microM for DNA polymerases alpha and delta). U-90152 blocked the replication in peripheral blood lymphocytes of 25 primary HIV-1 isolates, including variants that were highly resistant to 3'-azido-2',3'-dideoxythymidine (AZT) or 2',3'-dideoxyinosine, with a mean 50% effective dose of 0.066 +/- 0.137 microM. U-90152 had low cellular cytotoxicity, causing less than 8% reduction in peripheral blood lymphocyte viability at 100 microM. In experiments assessing inhibition of the spread of HIV-1IIIB in cell cultures, U-90152 was much more effective than AZT. When approximately 500 HIV-1IIIB-infected MT-4 cells were mixed 1:1,000 with uninfected cells, 3 microM AZT delayed the evidence of rapid viral growth for 7 days. In contrast, 3 microM U-90152 totally prevented the spread of HIV-1, and death and/or dilution of the original inoculum of infected cells prevented renewed viral growth after U-90152 was removed at day 24. The combination of U-90152 and AZT, each at 0.5 microM, also totally prevented viral spread. Finally, although the RT amino acid substitutions K103N (lysine 103 to asparagine) and Y181C (tyrosine 181 to cysteine), which confer cross-resistance to several nonnucleoside inhibitors, also decrease the potency of U-90152, this drug retains significant activity against these mutant RTs in vitro (IC50s, approximately 8 microgramM).

Steady-state kinetic studies with the non-nucleoside HIV-1 reverse transcriptase inhibitor U-87201E.

The multifunctional HIV-1 RT (human immunodeficiency virus type 1-reverse transcriptase) enzyme possesses three main functions including the RNA- and DNA-directed DNA polymerases and the RNase H. The bisheteroarylpiperazine U-87201E inhibits the two polymerase functions but not the RNase H. Enzymatic kinetic studies of the HIV-1 RT-catalyzed RNA- and DNA-directed DNA polymerase activities were carried out in order to determine if the inhibitor interferes with either the template:primer or the deoxyribonucleotide triphosphate (dNTP)-binding sites of the enzyme. The data were analyzed using steady-state kinetics, considering that the polymerase reaction is ordered in that the template:primer is added first, followed by the dNTP and that the enzyme functions processively. The data were consistent with the model. The steady-state rate constants for the forward and backward reactions were of similar magnitude for both the RNA- and DNA-catalyzed DNA polymerases and suggest that both functions share the same substrate-binding sites. The dissociation constants for the enzyme-inhibitor and enzyme-substrate-inhibitor complexes were somewhat higher for the DNA-directed DNA polymerase function as compared to the RNA directed one. This indicates that U-87201E is a more potent inhibitor for the RNA-directed DNA polymerase than the DNA-directed DNA polymerase. The pattern of inhibition exerted by U-87201E was noncompetitive with respect to both the nucleic acid and nucleotide-binding sites of the RT enzyme for both the RNA- and DNA-directed DNA polymerases. Hence, U-87201E inhibits these functions by interacting with a site distinct from the template:primer and dNTP-binding sites. HIV-2 RT was insensitive to U-87201E, demonstrating the unique sensitivity of HIV-1 RT to this inhibitor.

Enzymatic kinetic studies with the non-nucleoside HIV reverse transcriptase inhibitor U-9843.

The polymer of ethylenesulfonic acid (U-9843) is a potent inhibitor of HIV-1 RT (reverse transcriptase) and the drug possesses excellent antiviral activity at nontoxic doses in HIV-infected lymphocytes grown in tissue culture. The drug also inhibits RTs isolated from other species such as AMV and MLV retroviruses. Enzymatic kinetic studies of the HIV-1 RT catalyzed RNA-directed DNA polymerase function, using synthetic template:primers, indicate that the drug acts generally noncompetitively with respect to the template:primer binding site but the specific inhibition patterns change somewhat depending on the drug concentration. The inhibitor acts noncompetitively with respect to the dNTP binding sites. Hence, the drug inhibits this RT polymerase function by interacting with a site distinct from the template:primer and dNTP binding sites. In addition, the inhibitor also impairs the DNA-dependent DNA polymerase activity of HIV-1 RT and the RNase H function. This indicates that the drug interacts with a target site essential for all three HIV RT functions addressed (RNA- and DNA-directed DNA polymerases, RNase H).

Nonnucleoside reverse transcriptase inhibitors that potently and specifically block human immunodeficiency virus type 1 replication.

Certain bis(heteroaryl)piperazines (BHAPs) are potent inhibitors of the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) at concentrations lower by 2-4 orders of magnitude than that which inhibits normal cellular DNA polymerase activity. Combination of a BHAP with nucleoside analog HIV-1 RT inhibitors suggested that together these compounds inhibited RT synergistically. In three human lymphocytic cell systems using several laboratory and clinical HIV-1 isolates, the BHAPs blocked HIV-1 replication with potencies nearly identical to those of 3'-azido-2',3'-dideoxythymidine or 2',3'-dideoxyadenosine; in primary cultures of human peripheral blood mononuclear cells, concentrations of these antiviral agents were lower by at least 3-4 orders of magnitude than cytotoxic levels. The BHAPs do not inhibit replication of HIV-2, the simian or feline immunodeficiency virus, or Rauscher murine leukemia virus in culture. Evaluation of a BHAP in HIV-1-infected SCID-hu mice (severe combined immunodeficient mice implanted with human fetal lymph node) showed that the compound could block HIV-1 replication in vivo. The BHAPs are readily obtained synthetically and have been extensively characterized in preclinical evaluations. These compounds hold promise for the treatment of HIV-1 infection.

Coumarins as inhibitors of bacterial DNA gyrase.

Chlorobiocic acid and 3-(carbobenzoxyamino)-4,7-dihydroxy-8-methylcoumarin were identified as two new inhibitors of Micrococcus luteus DNA gyrase. Both compounds possess weak antibacterial activity against whole M. luteus cells which indicates that they probably lack efficient transport functions to penetrate the cell envelope.

Novenamine is the active moiety in novobiocin.

Novobiocin inhibits semiconservative DNA replication in procaryotes and more specifically DNA gyrase, an enzyme essential for DNA replication. Chemically, novobiocin consists of three distinct entities: the sugar noviose, a coumarin residue and a benzoic acid derivative. The subentity consisting of noviose plus the coumarin residue is referred to as novenamine; the subentity consisting of the coumarin plus the benzoic acid derivative is referred to as novobiocic acid. These subentities as well as noviose and the benzoic acid residue are essentially devoid of inhibitory activity against whole bacterial cells. These fragments were tested for their ability to inhibit DNA replication in a permeabilized Escherichia coli cell system and as potential inhibitors of DNA gyrase. Of all the fragments tested, only novenamine was found to inhibit DNA replication and DNA gyrase. The potency of novenamine essentially equaled that of novobiocin. This subunit thus represents the minimal structural entity necessary to interact with DNA gyrase.

New antibiotic U-64846: fermentation, isolation and characterization.

Antibiotic U-64846 is a new entity with the molecular formula C18H35C1N4O9 (MW 486). It is a very water soluble, reddish solid which decomposes above 300 degrees C and which is air-sensitive. The antibiotic is produced by Streptomyces braegensis and it inhibits a variety of Gram-positive bacteria. Acidic hydrolysis gave 3,7-diaminoheptanoic acid. The antibiotic gives 1H NMR, 13C NMR, IR and UV spectra which indicate it is not closely related to known antibiotic families.

Fermentation, isolation, characterization and structure of nitrosofungin.

The new antifungal agent nitrosofungin was isolated in high yields from a mixed culture of two organisms consisting of a bacterium of the genus Alcaligenes (UC 9152) and Streptomyces plicatus UC 8272. The bacterium produces the agent, the streptomycete enhances the production by providing a precursor or an inducer. Nitrosofungin in high concentrations inhibits a broad variety of pathogenic fungi in vitro. The agent is relatively non-toxic in small laboratory animals and high blood levels are obtained after either oral or systemic administration. Nitrosofungin is only the second N-nitrosohydroxylamine isolated from microbial sources to date. It has been identified as 2-N-nitrosohydroxylamino-1-propanol, an acidic and highly water-soluble compound.

Desertomycin: purification and physical-chemical properties.

Desertomycin was isolated from Streptomyces macronensis Dietz sp. nov. UC 8271. Extensive spectroscopic work led us to place desertomycin in the macrocyclic lactone family which contains monazomycin, scopafungin, primycin, azalomycin F4a and niphithricins A and B. The apparent molecular formula was determined by fast atom bombardment mass spectroscopy to be C57H109NO24 (MW = 1,191). Mild acid hydrolysis yielded mannose but contrary to published reports, glutamic acid is not a constituent of desertomycin.

Fermentation, isolation, characterization and structure of antibiotic U-58,431.

A new antibiotic U-58,431 has been isolated from the fermentation broth of Streptomyces helicus DIETZ and LI, sp. n. (UC-5837) and the structure of this antibiotic, namely, 6-amino-3,4,5,8-tetrahydro-4,9-dihydroxy-3-methyl-5,8-dioxo-1,4-ethano-1H-2-benzopyron-7-carboxamide has been determined by X-ray crystallography. The antibiotic inhibits a variety of Gram-positive and Gram-negative bacteria in vitro. However, it is toxic to mice and does not protect experimentally infected animals when administered at the maximum tolerated dose.

Inhibition of ribosomal and RNA polymerase functions by rubradirin and its aglycone.

The antibiotic rubradirin is structurally related to the ansamycin family of antibiotics. Most members of this group act as specific inhibitors of bacterial RNAP. Rubradirin and its aglycone possess diverging modes of action. Rubradirin inhibits ribosomal functions related to the peptide chain initiation process. It does not inhibit RNAP. By contrast the aglycone of rubradirin retains moderate inhibitory activity towards ribosomal functions but acts essentially as an extremely potent inhibitor of RNAP.