Design, synthesis and in vitro cytotoxic studies of novel bis-pyrrolo[2,1][1,4] benzodiazepine-pyrrole and imidazole polyamide conjugates.

The design, synthesis and biological evaluation of novel pyrrolo [2,1][1,4] benzodiazepine (PBD) dimers 38-43 linked with pyrrole and imidazole polyamides from either side by a flexible methylene chain of variable length are described, which involved mercuric chloride mediated cyclization of the corresponding amino diethyl thioacetals. The compounds were prepared with varying numbers of pyrrole and imidazole containing polyamides to determine the structural requirements for optimal in vitro antitumor activity. These compounds were tested against a panel of 60 human cancer cells by the National Cancer Institute, and demonstrated that, of the compounds bis-PBD-pyrrole polyamides (38-40) and bis-PBD-imidazole polyamides (41-43) certain of the bis-PBD-pyrrole and imidazole polyamide conjugates are active for individual cancer cell lines (Table 1). However, this study found that bis-PBD-pyrrole and imidazole polyamide conjugates 38-43 in general are potent against many human cancer cell lines.

High-throughput genotyping of human platelet antigens using the 5'-nuclease assay and minor groove binder probe technology.

BACKGROUND AND OBJECTIVES: Human platelet antigen (HPA) genotyping is a valuable tool for typing platelets to assist in the management of alloimmunized thrombocytopenic patients. We describe, for the first time, 5' nuclease assays (NA) to genotype HPA-5 and -15, and improved 5'-NA to genotype HPA-1, -2 and -3, by utilizing minor groove binder (MGB) and non-fluorescent quencher (NFQ) technology. Superior probe specificity and fluorescent performance is attained through MGB-NFQ probe modifications compared with previous 5'-NA designs. MATERIALS AND METHODS: Primers and dye-labelled MGB-NFQ probes were designed and synthesized to detect the single nucleotide polymorphism responsible for each HPA-1, -2, -3, -5 and -15. One-hundred blood samples were tested for the combinations of HPA genotypes 1, 2, 3 and 5 by our traditional sequence-specific primer-polymerase chain reaction (SSP-PCR) method, and 41 blood samples were tested for HPA-15 by SSP-PCR at an external laboratory. These results were then compared with those obtained by using the new 5'-NA. RESULTS: There was complete concordance of results for all samples tested by SSP-PCR and 5'-NA. The 5'-NA offers distinct advantages over non-fluorescent genotyping methods. DNA amplification and allele discrimination occurs in a single closed tube for each antigen with no post-PCR manipulation required. This minimizes the risk of cross-contamination and mislabelling of samples, as well as making the assay less time-consuming to perform. In comparison with other fluorescent assays, the 5'-NA has the highest sample throughput, resulting from the use of a 96-well platform, identical cycling conditions for all assays and the potential for automation. CONCLUSIONS: Genotyping for HPA-1, -2, -3, -5, and -15 by the 5'-NA is suitable for routine analysis. The latest 5'-NA design, using MGB probe technology, ensures superior detection of all alleles and is the most versatile fluorescent assay, ideal for both urgent clinical samples and large-scale screening programs.

Prolonged profound abciximab associated immune thrombocytopenia complicated by transient multispecific platelet antibodies.

Patients receiving abciximab occasionally develop transient severe thrombocytopenia within a few hours of receiving the drug. Thrombocytopenia has been reported to resolve within 10 days of abciximab administration, but in this case profound thrombocytopenia lasted 21 days before a slow spontaneous recovery. Management was complicated by the presence of HLA antibodies and the transient production of antibodies directed at major platelet glycoproteins IIb/IIIa, Ib/IX, and Ia/IIa. The patient remained refractory to platelet transfusion and two courses of intravenous gammaglobulin for the duration of her admission.

Methionine sustituted polyamides are RNAse mimics that inhibit translation.

RNAse mimics are small molecules that can cleave RNA in a fashion similar to ribonucleases. These compounds would be very useful as gene specific reagents if their activities could be regulated and targeted. We demonstrate here that polyamides with methionine substituents show enhanced RNA cleavage activity relative to other polyamides. Conjugation of these compounds to aminoglycosides produced RNAse mimics that are capable of inhibiting eukaryotic protein synthesis. As a new class of compounds capable of interacting with nucleic acids, these novel aminoglycoside-polyamides constitute promising scaffolds for the construction of nuclease mimics with biological activity.

Synthesis and in vitro cytotoxicity studies of novel L-tryptophan-polyamide conjugates and L-tryptophan dimers linked with aliphatic chains and polyamides.

The synthesis and biological evaluation of novel L-tryptophan pyrrole, imidazole polyamide conjugates (16-21), L-tryptophan-glycosylated pyrrole polyamide conjugates (28-30), L-tryptophan dimers (37-42) with straight carbon links of varying length, and L-tryptophan dimers (68-73) linked with pyrrole and imidazole polyamide from both sides by a flexible methylene chain of variable length are described. The compounds were prepared with varying numbers of pyrrole- and/or imidazole-containing polyamides and glycosylated pyrrole polyamides to determine the structural requirements for optimal in vitro antitumor activity. The compounds listed in Table 1 have been evaluated in a three cell line, one dose primary anticancer assay. The compounds listed in Table 2 have been evaluated against nine panels of 60 human cancer cell lines including leukemia, non-small cell lung cancer, colon cancer, CNS cancer, melanoma, ovarian cancer, renal cancer, prostate cancer, and breast cancer. It is observed from the initial cytotoxic data (Table 1) that compounds 16-19, 28-30, 68-69, and 71-73 have varying cytotoxic potencies against the three cancer cell lines. It is also observed, from the biological data from Table 2 for compounds 20-21, 37-42, and 70 against the 60 different tumor cells, that the L-tryptophan dimers 37-42 linked by a different number of carbon chains are more active than the L-tryptophan dimers linked by pyrrole or imidazole polyamides. The cytotoxic potency in tryptophan dimers, linked by a different number of carbon atoms increased the number of carbons between the two L-tryptophan rings.

Design, synthesis and in vitro cytotoxicity studies of novel pyrrolo [2,1][1,4] benzodiazepine-glycosylated pyrrole and imidazole polyamide conjugates.

The design, synthesis and biological evaluation of novel pyrrolo [2,1][1,4] benzodiazepine-water insoluble 31-38 and water soluble 39-46 glycosylated pyrrole and imidazole polyamide conjugates are described that involved mercuric chloride mediated cyclization of the corresponding amino diethyl thioacetals. The compounds were prepared with varying numbers of pyrrole and imidazole containing polyamides and incorporating glucose moieties in order to improve the water solubility of PBD-polyamide conjugates and probe the structural requirements for optimal in vitro antitumor activity. These compounds were tested against a panel of 60 human cancer cells by the National Cancer Institute, and demonstrated that the water soluble PBD-polyamide compounds exhibited a higher level of cytotoxic activity than the existing natural and synthetic pyrrolo [2,1-c][1,4] benzodiazepines. The cytotoxic activities of these compounds dramatically increase after hydrolysis of their acetylated counterparts. The activity data summarized in Table 1 and Table 2 show that the solubility of the PBD-polyamides and also the type of heterocycle play important roles influencing the cytotoxic activity of the PBD-polyamide conjugates. The PBD-glycosylated polyamide (water soluble) conjugates 39-46 are highly cytotoxic against many human cancer cell lines in comparison with the PBD-polyamide (water insoluble version) conjugates.

Synthesis and cytotoxicity evaluation of novel C7-c7, C7-N3 and N3-N3 dimers of 1-chloromethyl-5-hydroxy-1,2-dihydro-3H-benzo[e]indole (seco-CBI) with pyrrole and imidazole polyamide conjugates.

The C7-C7, C7-N3 and N3-N3 dimers of 1-chloromethyl-5-hydroxy-1,2-dihydro-3H-benzo[e]indole (seco-CBI) with pyrrole and imidazole polyamides were synthesized and preliminary anti-cancer evaluation carried out by NCI against three types of cancer cells.

Recent developments in novel pyrrolo[2,1-c][1,4]benzodiazepine conjugates: synthesis and biological evaluation.

The biological activity of many low molecular weight antitumor compounds appear to be related to their mode and specificity of interaction with particular DNA sequences. Such small molecules are of considerable interest in chemistry, biology and medicine. Most of the anticancer drugs employed clinically exert their antitumor effect by inhibiting nucleic acid (DNA or RNA) or protein synthesis. Inhibition can occur for example through cross-linking of bases in DNA or binding to and inactivation of enzymes necessary for the synthetic processes. It is evident that DNA is an important cellular target for many anticancer agents. Much information has been obtained from molecular genetics, i.e. replication of DNA and its transcription to RNA, which provides the template for protein synthesis. DNA is a well-characterized intracellular target but its large size and sequential nature makes it an elusive target for selective drug action. Binding of low molecular weight ligands to DNA causes a wide variety of potential biological responses. In this context PBDs (pyrrolo[2,1-c][1,4]benzodiazepines), a group of potent naturally occurring antitumor antibiotics produced by various Streptomyces species, are one of the most promising types of lead compounds. They differ in the number, type and position of substituent in both their aromatic A-ring and Py C-rings, and in the degree of saturation of the C-rings which can be either fully saturated or unsaturated at either C2-C3 (endocyclic) or C2 (exocyclic). There is either an imine or carbinolamine methyl ether at the N10-C11 position. This latter is an electrophilic center responsible for alkylating DNA. In the search for compounds with better antitumor selectivity and DNA sequence specificity many PBD analogues have been synthesized in an attempt to increase their potency against tumor cells. We review here recent progress on pyrrolo[2,1-c][1,4]benzodiazepine (PBDs) analogues and their conjugates, also the progress and developments of PBD conjugates with polyamides (information reading molecules in the minor groove of DNA). For example, the cross-linking efficiency of PBD dimers is much greater than that of other cross linkers including cisplatin and melphalan. A large number of PBD dimers and polyamide conjugates with varying linker lengths and bearing different heterocycles at different positions in the PBD ring synthesized in our group and their pharmacological properties have been reviewed.

Synthesis and antitumor cytotoxicity evaluation of novel pyrrolo[2,1-c][1,4]benzodiazepine imidazole containing polyamide conjugates.

The design, synthesis, and biological evaluation of novel C-8 linked pyrrolo[2,1-c][1,4]benzodiazepine (PBD)-imidazole polyamide conjugates (1-5) are described that involve mercuric chloride-mediated cyclization of the corresponding amino diethyl thioacetals. The compounds were prepared with varying numbers of imidazole- and pyrrole-containing polyamides in order to probe the structural requirements for optimal in vitro antitumor activity. These compounds were tested against a panel of human cancer cells by the National Cancer Institute, demonstrating that the compounds exhibited a higher level of cytotoxic activity than the existing natural and synthetic pyrrolo[2,1-c][1,4]benzodiazepines. The data presented show that the length of the polyamides and also the type of heterocycle play important roles in this series of compounds for anticancer activity. Compounds 1, 2, 3, and 5 have significant cytotoxic activity against the various types of cancer cell lines. It appears that cytotoxic activity is related to both the length and the heterocycles of the polyamides. Compound 1 exhibited a wide spectrum of anticancer activities against all cell lines in nine cancer panels and was especially effective against colon cancer, melanoma, and renal cancer and breast cancer, however, compound 4 did not exhibit any significant anticancer activity. This study found that PBD-imidazole polyamide conjugates are highly cytotoxic against many human cancer cell lines in comparison with the PBD-pyrrole polyamide conjugates.

Inhibition of feline immunodeficiency virus (FIV) replication by DNA binding polyamides.

Two DNA minor-groove binding polyamides 1 and 2 were designed and synthesized and evaluated for inhibition of FIV-34TF10 replication. Both 1 and 2 decreased the replication of FIV-34TF10 by 75% by acting at the level of the virus but outside of the LTR or env region.

DNA sequence recognition of thiazole-containing cross-linked polyamides can be favored.

The binding ability of cross-linked thiazolated polyamides (containing the base sequence-reading elements thiazole(Th)-pyrrole(Py)-pyr-role(Py) and thiazole(Th)-imidazole(Im)-pyrrol(Py) to various DNA dodecamers has been investigated. CD titration experiments at high salt concentration demonstrate that the dimers with a heptanediyl linker (C7 dimer) show a significantly higher sequence specificity than their corresponding monomers. The dimer of Th-Py-Py primarily prefers binding to pure AT sequences and that of Th-Im-Py to the dodecamer sequences containing a GC pair within the central sequence (e.g. AACGTT). Surprisingly, the sequence binding ability is strongly influenced by the presence of a T-A step: e.g. Th-Py-Py has a similar affinity to the sequences TTTAAA and ATCGTA; likewise Th-Im-Py shows a preference for these sequences. The CD results correlate with footprinting data. Related biochemical studies on the effect of polyamides on DNA gyrase activity in vitro show that the C7 dimers most effectively inhibit the enzyme activity compared with the monomers and the natural reference minor groove binder distamycin. The highest inhibitory potency is observed for the Th-Py-Py-dimer. The role of the T-A step in binding of the cross-linked dimer to the minor groove is discussed in light of the sequence recognition of the TATA box binding protein.

Design and synthesis of bis 1-chloromethyl-5-hydroxy-1,2- dihydro-3H-benz[e]indole (seco-CBI)-pyrrole polyamide conjugates.

[carbohydrate structure: see text] The design and synthesis of bis 1-chloromethyl-5-hydroxy-1,2-dihydro-3H-benz[e]indole (seco-CBI)-pyrrole polyamide conjugates (13, 17) as DNA minor groove binding agents are described.

DNA sequence recognition in the minor groove by crosslinked polyamides: The effect of N-terminal head group and linker length on binding affinity and specificity.

Development of sequence-reading polyamides or "lexitropsins" with comparable DNA-binding affinities to cellular proteins raises the possibility of artificially regulated gene expression. Covalent linkage of polyamide ligands, with either a hairpin motif or crosslinking methylene bridge, has greatly improved binding affinity by ensuring their side-by-side register. Whereas hairpin polyamides have been investigated extensively, the optimized structure of crosslinked polyamides remains to be determined. This study examines a series of thiazole-imidazole-pyrrole (TIP) monomers and crosslinked dimers to evaluate the effects on selectivity and binding affinity of different N-terminal head groups attached to the leading thiazole ring and differing methylene linker lengths. Quantitative footprinting of a DNA sequence, containing potential match and mismatch sites for both maximum overlap and one-residue stagger binding modes, allowed measurement of binding constants at each putative site. Within an N-terminal amino TIP series, C7 and C8-linked compounds bound most strongly to these sites, whereas maximum binding affinity was observed for a C6 linker with a formyl head group. A C5 linker gave weak binding with either head group. A hydrogen or acetyl head group abrogated binding. Binding was confirmed by gel shift analyses. The highest specificity for the maximum overlap site over the one-residue stagger was observed with TIP-C7-amino. Selectivity of the leading thiazole was modulated by the head group, with N-terminal formyl TIP exhibiting up to 3-fold specificity for AGT over TGT, suggesting that N-formyl-thiazole may provide sequence discrimination of adenine over thymine. Moreover, the leading head group and methylene linker length significantly influences the binding characteristics of crosslinked polyamides.

Heterocyclic compounds as inflammation inhibitors.

Clinical use of non-steroidal anti-inflammatory drugs (NSAIDs) is associated with significant toxicity particularly in the gastrointestinal tract and kidney. Various approaches such as formulation co-administration (of agents to protect the stomach), chemical manipulation and synthesis of new safer anti-inflammatory drugs reported in the literature to overcome the toxicity of NSAIDs have been summarized. As far as synthesis of new more effective and safer anti-inflammatory drugs is concerned, we have reported recent findings in the area of synthesis of heterocyclic compounds such as pyrimidines, imidazole, benzimidazole, thiazole, thiazolidine, acridine, thiourea, alkanoic acid derivatives and other related heterocyclic compounds and their role as inflammation inhibitors.

A new all-season passive sampling system for monitoring H2S in air.

A new Maxxam All-Season Passive Sampling system for monitoring H2S in air has been developed. This passive sampling system employs the same approaches as the Maxxam All-Season Passive Sampling Systems for monitoring SO2, NO2, and O3 reported previously. This system has been extensively tested in the lab (temperature from -20 to 20 degrees C, relative humidity from 30 to 84%, and wind speed from 0.5 to 150 cm/s) and validated in field studies. Comparing measurements obtained with the use of the new passive sampling system with equivalent measurement with the use of an active filter pack H2S sampler yielded an accuracy of greater than 85%. The new H2S passive sampling system can be used to measure ambient H2S concentrations ranging from 0.02 to 7 ppb based on a 1-month exposure period. There is no significant interference found from other sulfur compounds in air. This system has been used in many air monitoring projects.

Recent developments in sequence selective minor groove DNA effectors.

DNA is a well characterized intracellular target but its large size and sequential nature make it an elusive target for selective drug action. Binding of low molecular weight ligands to DNA causes a wide variety of potential biological responses. In this respect the main consideration is given to recent developments in DNA sequence selective binding agents bearing conjugated effectors because of their potential application in diagnosis and treatment of cancers as well as in molecular biology. Recent progress in the development of cross linked lexitropsin oligopeptides and hairpins, which bind selectively to the minor groove of duplex DNA, is discussed. Bis-distamycins and related lexitropsins show inhibitory activity against HIV-1 and HIV-2 integrases at low nanomolar concentrations. Benzoyl nitrogen mustard analogs of lexitropsins are active against a variety of tumor models. Certain of the bis-benzimidazoles show altered DNA sequence preference and bind to DNA at 5'CG and TG sequences rather than at the preferred AT sites of the parent drug. A comparison of bifunctional bizelesin with monoalkylating adozelesin shows that it appears to have an increased sequence selectivity such that monoalkylating compounds react at more than one site but bizelesin reacts only at sites where there are two suitably positioned alkylation sites. Adozelesin, bizelesin and carzelesin are far more potent as cytotoxic agents than cisplatin or doxorubicin. A new class of 1,2,9,9a-tetrahydrocyclo-propa[c]benz[e]indole-4-one (CBI) analogs i.e., CBI-lexitropsin conjugates arising from the latter leads are also discussed.A number of cyclopropylpyrroloindole (CPI) and CBI-lexitropsin conjugates related to CC-1065 alkylate at the N3 position of adenine in the minor groove of DNA in a sequence specific manner, and also show cytotoxicities in the femtomolar range. The cross linking efficiency of PBD dimers is much greater than that of other cross linkers including cisplatin, and melphalan. A new class of PBD-lexitropsin conjugates is also discussed. Certain functional models of the bleomycins (BLMs) show outstanding DNA cleavage activity comparable with that of and positionally distinct from natural BLM.

Design, synthesis, and intracellular localization of a fluorescently labeled DNA binding polyamide related to the antibiotic distamycin.

The design and synthesis of the lipophilic (9) and fluorescent (10) conjugates of a structural analogue of distamycin and their in vitro cellular localization studies are reported. Confocal laser scanning microscopy (CLSM) indicates that 10 rapidly enters human ovarian adenocarcinoma (SKOV-3) cells with principal uptake in mitochondria and uniform cytoplasmic distribution.

RNase activity of a DNA minor groove binder with a minimalist catalytic motif from RNase A.

Imidazole and compounds containing imidazole residues have been shown to cleave RNA in an RNase A-mimicking manner. Di-imidazole lexitropsin is a compound which is derived from the polyamide drugs distamycin and netropsin essentially by the replacement of two pyrrole heterocycles with N-methyl-imidazole residues. This enables it to bind to the minor groove of B-DNA in a sequence-specific manner. We demonstrate here that this lexitropsin derivative has RNA cleavage activity, as tested on model RNAs. Optimal cleavage conditions and cleavage specificity resemble those known from other imidazole conjugates and are thus consistent with an RNase A type cleavage mechanism. The optimum concentration of the compound for cleavage is similar to previously investigated imidazole-based RNase mimics. As a whole new class of chemical compounds capable of interacting with nucleic acids through extensive hydrogen bonding, these imidazole containing compounds constitute promising scaffolds and ligands, for the construction of novel RNase mimics with high affinity.

Novel cyclopropylindole conjugates and dimers: synthesis and anti-cancer evaluation.

There is a considerable interest currently in the development of DNA sequence specific or selective agents for genetic targeting for the control of gene expression, for application in diagnosis or ultimately in therapy. In this context CC-1065 is one of the most impressive lead compounds isolated in trace quantities from the culture of Streptomyces zelensis at Upjohn in 1978. The unique structure was confirmed by single X-ray in 1981. However CC-1065 cannot be used in humans because it was found that it caused delayed deaths in experimental animals. In the search for compounds with better antitumor selectivity and DNA sequence specificity many CC-1065 analogs have been synthesized in an attempt to avoid the undesired side effects while retaining its potency against tumor cells. Two successful attempts in the modification in the active moiety of the parent natural product 1,2,8,8a-tetrahydro-7-methylcyclopropa[3,2-e]indole-4-one (CPI) and 1,2,9,9a-tetrahydrocyclopropa[c]benz[e]indole-4-one (CBI) have been made. We review here recent progress with the analogs of CPI and CBI and their conjugates both by solution and solid phase, also the progress and development of CPI and CBI conjugates with polyamides (information reading molecules in the minor groove of DNA). Since CPI-CPI dimers are significantly more potent than CC-1065 in vitro and in vivo, a large number of CBI-CBI dimers with varying linkers lengths and positions synthesized in our group and their pharmacological properties have been reviewed.