Novel DNA groove binding alkylators: design, synthesis, and biological evaluation

A group of oligopeptides has been synthesized that are structurally related to the natural antiviral antitumor agents netropsin and distamycin but which bear alkylating functions. Cytostatic activity against both human and murine tumor cell lines as well as their in vitro activity against a range of viruses is reported. The biological activity of these agents is discussed both in terms of their alkylating reactivity and of their structural differences. The incorporation of alkylating moieties into these minor groove DNA binders results in up to 45-fold increase in cytostatic activity compared with netropsin and up to 18 times the activity of distamycin.     

Structure-activity relationship of novel oligopeptide antiviral and antitumor agents related to netropsin and distamycin

A group of oligopeptides have been synthesized that are structurally related to the natural antiviral antitumor agents netropsin and distamycin. Cytostatic activity against both human and murine tumor cell lines as well as their in vitro activity against a range of viruses is reported. The biological activity of these agents is discussed both in terms of their structural differences and, in particular, in relation to their observed base- and sequence-dependent minor-groove binding to duplex oligonucleotides.     

Anti-HIV-I activity of linked lexitropsins.

Five groups of lexitropsin oligopeptides have been synthesized that are structurally related to the natural antiviral agents netropsin and distamycin and bearing two such moieties joined by flexible or rigid linkers. Inhibitory activity of these types of agents against murine leukemia retrovirus (MuLV) led to an evaluation of their inhibition of HIV-I in cell culture. The antiretroviral activity of the five different classes of lexitropsins is discussed in terms of their structural differences.     

Design, synthesis, DNA binding, and biological activity of a series of DNA minor-groove-binding intercalating drugs

A group of pseudopeptides, molecular combination of the natural antitumor agents distamycin or netropsin and the anilinoacridine chromophore (which is related to the synthetic antileukemic drug amsacrine) has been synthesized. Their DNA binding properties were determined and discussed in terms of their structural differences and in relation to their observed base-dependent binding. Binding data are consistent with a model in which the acridine nucleus occupies an intercalation site and the netropsin or distamycin residue resides in the DNA minor groove. Cytostatic and cytotoxic activities against a murine cell line are reported, as well as significant differences in the inhibition of DNA synthesis.     

Synthesis, DNA binding and antiviral activity of distamycin analogues containing different heterocyclic moieties

A series of novel distamycin analogues possessing an additional benzene or heterocyclic ring have been synthesised and tested for selective DNA binding properties and antiviral activity. Inhibition of herpes virus in cell culture appears to be related to AT base pair specificity. Some of the new compounds are also more potent than the parent distamycin against Moloney sarcoma virus.     

Synthesis, DNA binding, and biological evaluation of synthetic precursors and novel analogues of netropsin

A series of oligopeptides have been synthesized that are structurally related to the natural agent netropsin. The binding constants to double-stranded polynucleotides as well as the cytostatic activity against both murine human tumor cell lines and the in vitro activity against a range of DNA and RNA viruses have been determined for these novel compounds and some of their synthetic precursors. 1-Methyl-5-nitropyrrole-2-carboxylic acid methyl ester (4), N-[[1-methyl-4-(1-methyl-4-nitropyrrole-2-carboxamido)pyrrol-2- yl]carbonyl]-L-alanine tert-butyl ester (28), and N-[[1-methyl-4-(1-methyl-4-nitropyrrole-2-carboxamido)pyrrol-2- yl]carbonyl]-L-alanyl-L-alanine tert-butyl ester (29) showed modest inhibitory effect on tumor cell proliferation (CD50 = 26-85 micrograms/mL). Of all the compounds that were evaluated, 28 proved the most potent antiviral agent. It was inhibitory to parainfluenza-3 virus and Coxsackie virus B4 in Vero cells at a concentration of 20 micrograms/mL.     

Cytotoxics derived from distamycin A and congeners

Distamycin A is an antibiotic, characterised by an oligopeptidic pyrrolocarbamoyl frame ending with an amidino moiety, which binds reversibly to DNA minor groove with high selectivity for TA-rich sequences and shows antiviral and antiprotozoal activity. Distamycin was used as DNA sequence selective vector of alkylating functions, leading to a substantial increase of cytotoxicity in comparison to that, very weak, of distamycin itself. The benzoyl nitrogen mustard derivative of desformyldistamycin, tallimustine, was selected as a candidate antineoplastic drug in view of its strong activity against a series of experimental tumors. Tallimustine, like distamycin, shows DNA selective binding to TA-rich sequences but its cytotoxicity is not associated with DNA strand breaks and interstrand crosslinking, at variance with classical phenyl nitrogen mustards. Tallimustine represents an important model for the design of new minor groove alkylating agents derived from distamycin and analogues, including the so-called lexitropsins, sequence-reading oligopeptides in which one or more N-methyl-pyrrole units of distamycin are replaced by imidazole or other rings. The structural features of the alkylating moieties and binding frames, the antitumor activities and the mechanism of action of most representative cytotoxics derived from distamycin and congeners are discussed. Some of these, recently reported, show an activity profile apparently superior to tallimustine. Finally, a concise survey of representative hybrid compounds in which known non-alkylating cytotoxic agents or their active moieties have been tethered to distamycin and congeners is presented and briefly discussed. These compounds witness the attention paid to this approach on the basis of the interest for the DNA binding features of distamycin A.     

Design,synthesis, and biological activity of hybrid compounds between uramustine and DNA minor groove binder distamycin A

The design, synthesis, characterization, DNA binding properties, and cytotoxic activity of a novel series of hybrids, namely, a molecular combination of the natural antibiotic distamycin A and the antineoplastic agent uramustine, are reported, and the structure-activity relationships are discussed. This homologous series 29-34 consisted of the minor groove binder distamycin A joined to uramustine (uracil mustard) by suitable aliphatic carboxylic acid moieties containing a flexible polymethylene chain that is variable in length [(CH(2))(n)(), where n = 1-6). All the hybrid compounds in this series exhibit enhanced activity compared to both distamycin A and uramustine derivatives 22-27 used for conjugation, giving IC(50) values in the range 7.26-0.07 microM following a 1 h exposure of human leukemic K562 cells, with maximal activity shown when n = 6. The distance between the uramustine and distamycin frame is crucial for the cytotoxicity, with compounds having linker lengths of four to six being at least 20-fold more cytotoxic than linker lengths one to three. Taq polymerase stop experiments demonstrated selective covalent binding of uramustine-distamycin hybrids to A/T rich DNA sequences, which was again more efficient with compounds 32-34 with a longer linker length. Two consequences can be derived from our study: (a) the distamycin moiety directs binding to the minor groove of A/T rich DNA sequences and, consequently, is responsible for the alkylation regioselectivity found in footprinting studies; (b) the higher flexibility due to a longer linker between the distamycin and uracil moieties allows the formation of complexes with the mustard moiety situated more deeply in the minor groove and, hence, with better alkylating properties.     

Carbocyclic analogues of netropsin and distamycin: DNA-binding properties and inhibition of DNA topoisomerases

Inhibition of DNA topoisomerase and DNA-binding properties of a series of benzene-containing and C-terminus-modified analogues of distamycin and netropsin are described. These analogues contain two or three benzene units, respectively. Dibenzene analogues did not inhibit the topoisomerases, I and II. In this case, relaxation of DNA was inhibited with tribenzene analogues. Data from the ethidium displacement assay showed that these compounds were able to bind in the minorgroove binding mode in AT sequences of DNA. Molecular modelling experiments were performed to rationalize the lower binding affinity of tribenzene analogues of distamycin and netropsin, 3 and 4, compared to dibenzene analogues, 1 and 2. The superior DNA-binding afforded by 1 and 2 in comparison to 3 and 4 results from their more effective penetration into the minor groove of DNA and smaller perturbation of molecular structure upon complex formation.     

Minor groove binders 1998 – 2004

Several classes of compounds, including heterocyclic polyamides, aryl benzimidazoles, pyrrolobenzodiazepines, amidino arylfurans and benzofurans, have been found to bind to the minor groove of DNA with sequence selectivity and potential therapeutic consequences. Many such compounds are synthetic analogues of natural products such as distamycin, netropsin, CC-1065, duocarmycin and anthramycin. Biological activity has been demonstrated in antibacterial, antifungal, antiviral and anticancer applications, and several compounds are in clinical trials. This review covers patents since 1998 and reviews the structures, mechanisms and biological activity of minor groove binders in the context of the current scientific understanding described in the journal literature.     

Pyrazole analogues of the bispyrrolecarboxamide anti-tumour antibiotics: synthesis, DNA binding and anti-tumour properties.

Four bispyrazole compounds have been prepared as potentially more stable analogues of the DNA minor groove binding polypyrrole compounds netropsin and distamycin, which are susceptible to oxidative breakdown. These compounds bind less strongly to DNA, and show much lower specificity for binding to AT-rich DNA sequences in comparison with distamycin. N.m.r. studies show that two of these compounds cause a downfield shift of the DNA imino proton resonances on interaction with the oligonucleotide d(ATATATATAT)2, suggesting that these isomers can adopt low-energy conformations similar to that shown by distamycin in its DNA minor groove binding site. The benzoic acid mustard analogue of one of the minor groove binding bispyrazoles was prepared, and showed in vitro cytotoxicity comparable with that of the previously-reported distamycin mustard, but only a low level of activity in vivo.     

Antiherpes activity of the immunomodulator OK-432, a streptococcal preparation, in immunosuppressed mice

The antiviral activity of OK-432, an antitumor agent originating from Streptococcal preparations, against herpes simplex virus type 2 (HSV-2) was investigated in mice immunosuppressed by cyclophosphamide (CY). Intraperitoneal administration of OK-432 to mice 1 day after treatment with 200 mg CY/kg prevented death due to HSV-2 encephalitis in a dose-dependent manner. When the immunosuppressed mice were given OK-432 prior to HSV-2 infection, both by the intraperitoneal route, virus growth in the peritoneal cavity was significantly suppressed. Following with OK-432, the number of macrophages in immunosuppressed mice was increased to a significantly greater extent than the numbers of lymphocytes and polymorphonuclear leukocytes. The intrinsic antiviral activity of macrophages against HSV-2 as well as the natural killer (NK) activity against YAC-1 target cells was significantly enhanced by OK-432 in immunosuppressed mice.     

Structure-activity relationships of pyrrole amidine antiviral antibiotics III: preparation of distamycin and congocidine derivatives based on 2,5-disubstituted pyrroles

Isomers of distamycin A and tripyrrole congocidine containing 2,5-disubstituted pyrroles were synthesized along with distamycin and congocidine homologs containing a single pyrrole ring. Selected compounds were evaluated for their cytotoxicity and antiviral activity. All of the tripyrrole derivatives tested in this series were nontoxic but were less active than distamycin A. The monopyrrole derivative, N-methyl-5-nitropyrrole-2-carboxamido-beta-propionamidine hydrochloride, was nontoxic and was almost as active antivirally as distamycin A.     

Synthesis, DNA-binding properties, and antitumor activity of novel distamycin derivatives

A group of potential alkylating agents have been synthesized that are structurally related to the oligopeptide antiviral antibiotic distamycin. All derivatives form complexes with native calf-thymus DNA but compounds 2, 3, and 6 give rise to covalent adducts. Cytostatic activity against both human and murine tumor cell lines in vitro is displayed by the new compounds. Compounds 3 and 4 are active on melphalan-resistant L1210 leukemia in mice.     

Distamycin derivatives as potential anticancer agents

Distamycin A was used as DNA minor groove sequence-selective vector of alkylating functions and led to the synthesis of compounds endowed with relevant cytotoxic and antitumor activity. In particular, tallimustine (Pharmacia), a benzoic acid nitrogen mustard derivative of distamycin, showed excellent antitumor activity against murine transplanted solid tumors and human xenografts, and, in the early 1990s, prompted several groups to search for new cytotoxic agents derived from distamycin. Unfortunately, tallimustine showed severe myelotoxicity and its clinical development was discontinued. Nevertheless this compound has represented an important model for the design of new cytotoxic minor groove binders derived from distamycin and distamycin-like frames. Some recently reported derivatives of distamycin-like frames with high cytotoxicity and antitumor activity also show significantly improved cytotoxicity/myelotoxicity ratio, which promises drugs with clinical efficacy. Distamycin-derived cytotoxics have been extensively reviewed in the recent past. This review will focus on cytotoxics derived structurally from distamycin or distamycin-like frames disclosed between 1997 and the third quarter of 2000.     

Oligopyrrole carboxamides linked with a nucleobase as potential DNA minor groove binding ligands: synthesis, DNA binding and biological evaluation

The synthesis of a series of new oligopyrrole carboxamides closely related to netropsin and distamycin A, linked with a nucleobase is reported. The new compounds possess similar structure elements as the known peptide nucleic acids which are interesting sequence reading DNA ligands. Cytotoxicity in vitro, the DNA binding characteristics and the inhibition of topoisomerase I were studied. Four of the compounds, 27, 31, 33 and 37 bind to DNA probably at AT sequences like netropsin or distamycin A in the minor groove. Surprisingly, no cytotoxicity and no inhibition of topoisomerase I was found.     

Design, synthesis, DNA binding, and biological evaluation of water-soluble hybrid molecules containing two pyrazole analogues of the alkylating cyclopropylpyrroloindole (CPI) subunit of the antitumor agent CC-1065 and polypyrrole minor groove binders

We have synthesized and evaluated a series of hybrids, denoted 22--27, for in vitro cytotoxic activity against a variety of cancer cell lines. These hybrids represent a molecular combination of polypyrrole minor groove binders structurally related to the natural antitumor agent distamycin A and two pyrazole analogues of the left-hand segment called cyclopropylpyrroloindole (CPI) of the potent antitumor antibiotic (+)-CC-1065. These novel water-soluble hybrids have been designed to enhance the minor groove binding ability of alkylating units 20 and 21, which should increase their clinical appeal by overcoming the administration problems of (+)-CC-1065 derivatives. The DNA alkylating and cytotoxic activities against several tumor cell lines are reported and discussed in terms of their structural differences in relation to both the number of N-methyl pyrrole rings and the type of the alkylating unit tethered to the oligopeptidic frame. It may be noted that, in general, and especially for 22--24, the cytotoxicity of the hybrids was much greater than that of the alkylating units alone. In only one case, compound 27, did the hybrid have cytotoxic activity comparable to that of the alkylating unit alone against FM3A/0 cells. The broadest spectrum of activity and greatest potency was shown by the hybrid 24, in which the alkylating unit 20 and the deformyl distamycin A are tethered by 1-methyl 2,5-dicarbonyl pyrazole, with IC(50) values for the different tumor cell lines ranging from 7 to 71 nM. For compounds 22--24, the increase of the length of the pseudopeptidic moiety from one to three N-methylpyrrole residues led to an increased cytotoxicity. Among the hybrids tested for their inhibitory effects on the proliferation of murine L1210 leukemia cell line, compound 24 proved to be the most active (IC(50) = 7.4 nM), and in the sequencing gel experiments, it showed the strongest and most highly sequence-specific DNA alkylation activity. For compounds 22-24, the sequence specificity of DNA alkylation appears to be affected by the modification of the number of pyrrole rings, and the correlation between cytotoxicity and alkylation pattern suggests that 24 exerts its cytotoxicity through DNA sequence-specific alkylation of the third adenine located in the sequence 5'-ACAAAAATCG-3'. The two other hybrids 22 and 23 were slightly less active for tumor cell proliferation, with IC(50) values of 58 and 19 nM, respectively. With only one exception, none of the compounds was endowed with antiviral activity at subtoxic concentrations. Compound 24 inhibited the effect of vaccinia virus at a concentration that was significantly lower than its minimum cytotoxic concentration for the E(6)SM host cells. These compounds gave distinct patterns of alkylation in AT-rich sequences, indicating that minor structural changes produced marked alterations in sequence selectivity.     

Synthesis of newer N-furylidene and N-acetophenonylidene-5-chlorodiphenylamine-2-carboxylic acid hydrazides as antiviral and antibacterial agents

Five N-furylidene and fifteen N-acetophenonylidene-5-chlorodiphenylamine-2-carboxylic acid hydrazides were synthesized by the condensation of 5-chlorodiphenylamine-2-carboxylic acid hydrazides with furfuraldehyde and appropriate acetophenones. All the N-acetophenonylidenes thus synthesized were screened for their antiviral activity against animal viruses (Ranikhet disease virus and vaccinia virus in a stationary culture of chorioallantoic membrane of chick embryo) and against plant virus, namely gomphrena mosaic virus (GMV) in vitro as well as in vivo. Whereas five N-furylidenes were tested against gomphrena mosaic virus only in vitro and in vivo for their antiviral activity. Maximum significant protection was observed to the extent of 20% against Ranikhet disease virus. The majority of these compounds showed highly significant antiviral activity up to the extent of 96% in vitro and 89% in vivo against gomphrena mosaic virus. These compounds were also screened for their antibacterial activity against two micro-organisms; Bacillus subtilis and Staphylococcus aureus. Almost all of them were found to exhibit significant inhibition against B. subtilis while only a fewer showed significant inhibition against S. aureus.     

Antiviral activity of hop constituents against a series of DNA and RNA viruses

We investigated whether crude hop extracts and purified hop components representing every major chemical class of hop compound have antiviral activity. These hop constituents were tested for antiviral activity against bovine viral diarrhea virus (BVDV) as a surrogate model of hepatitis C virus (HCV), human immunodeficiency virus (HIV), influenza A virus (FLU-A), influenza B virus (FLU-B), rhinovirus (Rhino), respiratory syncytial virus (RSV), yellow fever virus (YFV), cytomegalovirus (CMV), hepatitis B virus (HBV), and herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2). The extracts all failed to prevent the replication of HIV, FLU-A, FLU-B, RSV and YFV. A xanthohumol-enriched hop extract displayed a weak to moderate antiviral activity against BVDV (therapeutic index (TI)=6.0), HSV-2 (TI=>5.3), Rhino (TI=4.0) and HSV-1 (TI=>1.9) with IC(50) values in the low microg/ml range. Pure iso-alpha-acids demonstrated low to moderate antiviral activity against both BVDV (TI=9.1) and CMV (TI=4.2) with IC(50) values in the low microg/ml range. No antiviral activity was detected using beta-acids or a hop oil extract. Ultra-pure preparations (>99% pure) were used to show that xanthohumol accounted for the antiviral activity observed in the xanthohumol-enriched hop extract against BVDV, HSV-1 and HSV-2. Xanthohumol was found to be a more potent antiviral agent against these viruses than the isomer iso-xanthohumol. With Rhino, the opposite trend was observed with iso-xanthohumol showing superior antiviral activity to that observed with xanthohumol. Xanthohumol also showed antiviral activity against CMV, suggesting that it might have a generalized anti-herpesvirus antiviral activity. Again, superior antiviral activity was observed with the xanthohumol isomer against CMV. In summary, iso-alpha-acids and xanthohumol were shown to have a low-to-moderate antiviral activity against several viruses. These hop constituents might serve as interesting lead compounds from which more active anti-HCV, anti-Rhino and anti-herpesvirus antiviral agents could be synthesized.