Nitrotriazole-Based Compounds as Antichagasic Agents in a Long-Treatment In Vivo Assay.

3-Nitrotriazole-based compounds belonging to various chemical subclasses were found to be very effective against Chagas disease both in vitro and in vivo after a short administration schedule. In this study, five compounds with specific characteristics were selected to be administered for longer periods of time to mice infected with the virulent Trypanosoma cruzi Y strain to further evaluate their effectiveness as antichagasic agents and whether or not potential adverse effects occur. Benznidazole was included for comparison purposes. Complete parasitemia depletion, weight gain, 100% survival, and a lack of myocardial inflammation were observed with four of the compounds and benznidazole administered intraperitoneally at 15 or 20 mg/kg of body weight/day for 40 days. There was a significant reduction in the number of treatment days (number of doses) necessary to induce parasitemia suppression with all four compounds compared to that required with benznidazole. Partial cures were obtained with only one compound tested at 15 mg/kg/day and on the schedule mentioned above but not with benznidazole. Taken together, our data suggest that these compounds demonstrate potent trypanocidal activity comparable to or better than that of the reference drug, benznidazole, when they are administered at the same dose and on the same schedule.

The antitubercular activity of various nitro(triazole/imidazole)-based compounds.

Twenty three 3-nitrotriazole- and five nitroimidazole-based compounds, mostly amides, were screened for antitubercular activity against Mycobacterium tuberculosis H37Rv (Mtb H37Rv) under aerobic or low oxygen conditions, intracellular activity in murine J774 macrophages or THP-1 cells, activity against resistant Mtb strains as well as cytotoxicity in normal cells. Compounds with a Minimum Inhibitory Concentration (MIC) less than 10µM and 10-50µM were characterized as active and moderately active, respectively, whereas compounds with a MIC >50µM were characterized inactive. Fifteen 3-nitrotriazole-based compounds were active or moderately active against aerobic Mtb and thirteen of them were bactericidal, however, only four 3-nitrotriazoles were moderately active against anaerobic Mtb. All examined 2-nitroimidazole-based compounds were inactive against aerobic Mtb, and from the ones examined against anaerobic Mtb, only one was found moderately active. All examined compounds demonstrated intracellular activity and lack of cross-resistance. The five active 3-nitrotriazoles demonstrated good selectivity for Mtb. In conclusion, these classes of 3-nitrotriazole-based compounds merit further investigation as potential antitubercular agents.

Discovery of potent nitrotriazole-based antitrypanosomal agents: In vitro and in vivo evaluation.

3-Nitro-1H-1,2,4-triazole- and 2-nitro-1H-imidazole-based amides with an aryloxy-phenyl core were synthesized and evaluated as antitrypanosomal agents. All 3-nitrotriazole-based derivatives were extremely potent anti-Trypanosoma cruzi agents at sub nM concentrations and exhibited a high degree of selectivity for the parasite. The 2-nitroimidazole analogs were only moderately active against T. cruzi amastigotes and exhibited low selectivity. Both types of compound were active against Leishmania donovani axenic amastigotes with excellent selectivity for the parasite, whereas three 2-nitroimidazole-based analogs were also moderately active against infected macrophages. However, no compound demonstrated selective activity against Trypanosoma brucei rhodesiense. The most potent in vitro anti-T. cruzi compounds were tested in an acute murine model and reduced the parasites to an undetectable level after five days of treatment at 13 mg/kg/day. Such compounds are potential inhibitors of T. cruzi CYP51 and, being excellent substrates for the type I nitroreductase (NTR) which is specific to trypanosomatids, work as prodrugs and constitute a new generation of effective and more affordable antitrypanosomal agents.

Nitrotriazole- and imidazole-based amides and sulfonamides as antitubercular agents.

Twenty-three 3-nitrotriazole-based and 2-nitroimidazole-based amides and sulfonamides were screened for antitubercular (anti-TB) activity in aerobic Mycobacterium tuberculosis H37Rv by using the BacTiter-Glo (BTG) microbial cell viability assay. In general, 3-nitrotriazole-based sulfonamides demonstrated anti-TB activity, whereas 3-nitrotriazole-based amides and 2-nitroimidazole-based amides and sulfonamides were inactive. Three 3-nitrotriazole-based sulfonamides (compounds 4, 2, and 7) demonstrated 50% inhibitory concentration (IC50), IC90, and MIC values of 0.38, 0.43, and 1.56 µM (compound 4), 0.57, 0.98, and 3.13 µM (compound 2), and 0.79, 0.87, and 3.13 µM (compound 7), respectively. For 3-nitrotriazole-based sulfonamides, anti-TB activity increased with lipophilicity, whereas the one-electron reduction potential (E1/2) did not play a role. 2-Nitroimidazole-based analogs, which were inactive in the BTG assay, were significantly more active in the low-oxygen assay and more active than the 3-nitrotriazoles. All active nitrotriazoles in the BTG assay were similarly active or more potent (lower MIC values) against resistant strains, with the exception of compounds 2, 3, 4, and 8, which demonstrated greater MIC values against isoniazid-resistant strains. Five 3-nitrotriazole-based sulfonamides demonstrated activity in infected murine J774 macrophages, causing log reductions similar to those seen with rifampin. However, some compounds caused toxicity in uninfected macrophages. In conclusion, the classes of 3-nitrotriazole-based amides and sulfonamides merit further investigation as potential antitubercular agents.

Novel 3-nitro-1H-1,2,4-triazole-based piperazines and 2-amino-1,3-benzothiazoles as antichagasic agents.

We have previously shown that 3-nitro-1H-1,2,4-triazole-based amines demonstrate significant trypanocidal activity, in particular against Trypanosoma cruzi, the causative parasite of Chagas disease. In the present work we further expanded our research by evaluating in vitro the trypanocidal activity of nitrotriazole-based piperazines and nitrotriazole-based 2-amino-1,3-benzothiazoles to establish additional SARs. All nitrotriazole-based derivatives were active or moderately active against T. cruzi; however two of them did not fulfill the selectivity criteria. Five derivatives were active or moderately active against Trypanosoma brucei rhodesiense while one derivative was moderately active against Leishmania donovani. Active compounds against T. cruzi demonstrated selectivity indexes (toxicity to host cells/toxicity to T. cruzi amastigotes) from 117 to 1725 and 12 of 13 compounds were up to 39-fold more potent than the reference compound benznidazole. Detailed SARs are discussed.

Nitroimidazole-based bioreductive compounds bearing a quinazoline or a naphthyridine chromophore.

In our search for novel bioreductive agents with weak DNA-binding characteristics, we have synthesized two new 2-nitroimidazolyl derivatives tethered to a fused aromatic-ring chromophore with two nitrogen atoms: 4-[3-(2-nitro-1-imidazolyl)-propylamino]-2-methyl-quinazoline hydrochloride (NLQZ-1) and 4-[3-(2-nitro-1-imidazolyl)-propylamino]-1,5-naphthyridine hydrochloride (NLPP-1). DNA binding was evaluated by using the ethidium bromide displacement assay. Cytotoxicity, radiosensitization, and interaction with chemotherapeutic agents were evaluated in V79 and A549 cells by using the clonogenic assay. Both compounds are not DNA intercalators and showed relatively low uptake characteristics in V79 cells. A slightly increasing hypoxic selectivity [HS = IC(50)(A)/IC(50)(H) (IC(50) is the product of a compound's concentration and the time necessary for 50% reduction in clonogenicity under aerobic (A) or hypoxic (H) conditions)] was observed with incubation time in the case of NLPP-1 (12-19 and 15-26 in V79 and A549 cells, respectively, with 1-3 h of incubation). The HS of NLQZ-1 was approximately 14, independently of incubation time. Good radiosensitization of hypoxic V79 cells was obtained with both compounds at nontoxic concentrations [the concentration for a sensitization enhancement ratio of 1.6 (C(1.6)) was 61.4 and 75.0 micromol/l for NLQZ-1 and NLPP-1, respectively]. For NLPP-1, a C(1.6) of 44.1 micromol/l was obtained in A549 cells. Both compounds interacted synergistically with cisplatin or melphalan in V79 cells, under hypoxic preexposure conditions and dose modification factors values of approximately 2.5 were obtained at 10% survival. It is concluded that although compounds that do not bind to DNA are in general less potent hypoxic cytotoxins, they can still show good HS values and interact synergistically with radiation/chemotherapeutic agents. Therefore, a further in-vivo evaluation of NLQZ-1 and NLPP-1 is worthwhile.

NLNQ-1, a 2-[3-(2-nitro-1-imidazolyl) propylamino]-3-chloro-1,4-naphthoquinone, as a hypoxia-selective cytotoxin and radiosensitizer.

BACKGROUND: Compounds bearing two independent redox centers are considered bis-bioreductive agents and usually demonstrate increased hypoxic selectivity with exposure time due to different requirements for reduction of each center. We have synthesized a novel 2-[3-(2-nitro-1-imidazolyl)propylamino]-3-chloro-1,4-naphthoquinone (NLNQ-1), through Michael addition. NLNQ-1, which combines a naphthoquinone (with a relatively high one electron reduction potential) with a 2-nitroimidazole (with a relatively low one electron reduction potential), could perform as a more potent hypoxia-selective cytotoxin and radiosensitizer. MATERIALS AND METHODS: NLNQ-1 was evaluated in V79 cells under hypoxic/normoxic conditions, alone or with radiation, by using the clonogenic assay. RESULTS: Clearly NLNQ-1 was a more potent cytotoxin than the 2-alkylsulfonyloxy-naphthoquinones (VH-compounds), developed previously in our lab, demonstrating hypoxic and aerobic IC50 values at microM rather than mM concentrations. As a radiosensitizer of hypoxic cells, NLNQ-1 was superior to the best bis-nitroimidazolic compound, NNB (which combines a 2-nitroimidazole with a 5-nitroimidazole), demonstrating a C1.6 value of 25.4 microM (ca. 25 fold lower than that of NNB), whereas its in vitro therapeutic index (IC50A/C1.6) ranged from 5.3-13.2. CONCLUSION: NLNQ-1 could be used as a novel scaffold for bis-bioreductive agents that can be properly modified for further optimization of their hypoxia-selective toxicity and radiosensitization properties.

Advantage of combining NLCQ-1 (NSC 709257) with radiation in treatment of human head and neck xenografts.

NLCQ-1 (NSC 709257), a hypoxia-selective cytotoxin that targets DNA through weak intercalation, was investigated for efficacy in combination with single or fractionated radiotherapy of human head and neck xenografts. A staged tumor experiment was performed in tumor-bearing female athymic nude mice that were locally irradiated with or without NLCQ-1. Tumor hypoxia was assessed by immunohistochemistry for pimonidazole adducts in tumors of varying weight. Fractionated radiation, depending on the dose, was administered either once daily for 4 days or once daily for 4 days followed by a 7-day rest and repeat. NLCQ-1 was administered i.p. at 15 mg/kg alone or 45 min before each radiation dose. Hypoxia (1-52%) was detected in all tumors and was positively correlated with tumor size. NLCQ-1 alone resulted in about 10 days of tumor growth delay, measured at sixfold the tumor's original size, without causing toxicity. All combination treatments with NLCQ-1 were more effective than treatments with radiation alone. Radiation at 1 Gy given once daily for 4 days on days 20 and 30 caused 3.5 days of tumor growth delay, whereas in combination with NLCQ-1 it caused 14.5 days of growth delay. Radiation at 5 Gy given in two doses 10 days apart resulted in 3.5 days of tumor growth delay, whereas more than 20 additional days of delay were observed in combination with NLCQ-1. Radiation given as a single dose of 10 Gy resulted in about 7 days of tumor growth delay, whereas in combination with NLCQ-1 about 30 additional days of delay were seen. These results suggest a significant advantage in combining radiation with NLCQ-1 in treatment of human head and neck tumors, which are known to have hypoxic areas.

NLCQ-1 and NLCQ-2, two new agents with activity against dormant Mycobacterium tuberculosis.

4-[3-(2-Nitro-1-imidazolyl)-propylamino]-7-chloroquinoline hydrochloride (NLCQ-1; NSC 709257) and 4-[4-(2-nitro-1-imidazolyl)-butylamino]-7-chloroquinoline hydrochloride (NLCQ-2), two weak DNA-intercalating nitroimidazole-based bioreductive prodrugs, have been tested against dormant Mycobacterium tuberculosis and demonstrated a significant activity comparable with that of the nitroimidazopyran PA-824. Minimum bactericidal concentrations (MBCs) of 3.1-18.4 and 4.9-9.8 microg/mL were obtained for NLCQ-1 and NLCQ-2, respectively. In the same test setting; the corresponding MBC range for PA-824 was 6.4-12.8 microg/mL. For rifampicin, isoniazid, minocycline, streptomycin, clarithromycin and capreomycin, the corresponding MBC values were 2.5, >100, >156.25, >12.5, >312.5 and 37.5 microg/mL, respectively. Toxicity against Vero cells provided 50% inhibitory concentrations (IC(50)) of 146.7, >640 and >640 microg/mL for NLCQ-1, NLCQ-2 and PA-824, respectively. Therefore, the selectivity index (SI) was 8-47.3, >65.3-130.6 and >50-100 for NLCQ-1, NLCQ-2 and PA-824, respectively. These results suggest the potential usefulness of these compounds in the therapy of the latent form of tuberculosis.

DNA repair mechanisms are involved in the hypoxia-dependent toxicity of NLCQ-1 (NSC 709257) and its synergistic interaction with alkylating agents.

BACKGROUND: The hypoxia selective cytotoxin NLCQ-1 significantly potentiates the antitumor effect of several alkylating agents in vitro and in vivo. Synergy in mice requires administration of NLCQ-1 ca. 1 h before the alkylating agent, a fact that may be related to an in vitro hypoxic pretreatment effect. Since NLCQ-1 targets DNA through weak intercalation, the induction of DNA lesions upon reductive metabolism may be a reasonable mechanism that predisposes cells to further damage by a subsequent alkylating agent. MATERIALS AND METHODS: To indirectly identify such lesions, cell lines defective in specific DNA repair genes (EM9 and UV41) and their repair-proficient parental AA8, were exposed to NLCQ-1 +/-L-PAM/cisDDP under hypoxic/aerobic conditions and appropriate administration routes, and assessed for clonogenicity. Selected comparisons with tirapazamine (TPZ) were also performed. RESULTS: EM9 cells, which lack the functional XRCC1 gene involved in base excision repair, and thus are unable to efficiently repair DNA single-strand breaks (ssbs), were 3.7-fold and 4.5-fold more sensitive to NLCQ-1 and TPZ, respectively, than the parental AA8 cells, under hypoxic conditions. UV41 cells, which are defective in repairing DNA interstrand cross-links (ERCC4/XPF), were 4.5-fold more sensitive than AA8 cells to NLCQ-1. In potentiation studies with melphalan or cisplatin, synergy was observed in AA8 cells but not in EM9 or UV41 cells, with either NLCQ-I or TPZ. CONCLUSION: These results suggest that NLCQ-1 is involved in the formation of DNA ssbs and interstrand crosslinks under hypoxic conditions. The synergistic interaction of NLCQ-1 with L-PAM or cisDDP is probably due to an enhancement in the L-PAM/cisDDP-induced DNA interstrand cross-links, possibly as a result of an inhibited repair mechanism of these lesions.

The antitrypanosomal and antitubercular activity of some nitro(triazole/imidazole)-based aromatic amines.

A limited number of novel 3-nitrotriazole- and 2-nitroimidazole-linked quinolines and quinazolines were synthesized and screened for in vitro antitrypanosomal and antitubercular activities as well as cytotoxicity in normal cells. All compounds were active against T. cruzi amastigotes, while all but one were active or moderately active against T. b. rhodesiense. However, only two chloroquinolines exhibited satisfactory selectivity indices (SI) against T. cruzi and only one of them demonstrated a satisfactory SI against T. b. rhodesiense. All tested compounds demonstrated a Minimum Inhibitory Concentration (MIC) ≥ 200 µM against aerobic Mtb. However, the 2-nitroimidazole-based analogs were active against hypoxic Mtb with MIC values 2.89-9.18 µM. The present data support our previous observations that 2-nitroimidazole-based aromatic amines are selectively active against nonreplicating Mtb, while 3-nitrotriazole-based aromatic amines are potent antichagasic agents.

Potentiation of alkylating agents by NLCQ-1 or TPZ in vitro and in vivo.

PURPOSE: To investigate potential synergistic interactions between bioreductive agents, either NLCQ-1 or tirapazamine (TPZ) and two alkylating chemotherapeutic drugs, and how such interactions compare in vitro and in vivo. MATERIALS AND METHODS: V79 cells (in vitro studies) and the SCCVII/C3H murine tumor model (in vivo studies) were used. The alkylating chemotherapeutic agents examined were cisplatin (cisDDP) and melphalan (L-PAM). In vivo, all agents were administered by i.p. injection wherein NLCQ-1 and TPZ were given at equitoxic doses of 10 and 23 mg/kg, respectively. Optimal administration schedules and dose modification factors (DMF) were determined in vivo for the antitumor effect or bone marrow toxicity by using the in vivo-in vitro clonogenic assay as the endpoint. RESULTS: A schedule-dependent synergistic interaction was observed between NLCQ-1/TPZ and each alkylating agent, both in vitro and in vivo, and an optimal potentiation was obtained when each bioreductive agent was administered prior to each chemotherapeutic drug. However, significant DMF values and an in vivo therapeutic index (TI) was obtained only with NLCQ-1. Limited mechanistic studies in V79 cells by using the alkaline comet assay demonstrated that hypoxic preincubation with NLCQ-1 increases the cross-links induced by subsequent aerobic exposure to cisDDP. CONCLUSIONS: These results verify our previous observations in EMT6 tumors and suggest a potential clinical use of NLCQ-1 as a synergistic adjuvant to chemotherapy with alkylating agents against solid tumors possessing hypoxic regions.

Novel fluorinated hypoxia-targeted compounds as Non-invasive probes for measuring tumor-hypoxia by 19F-magnetic resonance spectroscopy (19F-MRS).

BACKGROUND: 19F-labeled 2-nitroimidazoles bound to hypoxic cells in tumors are known to be useful probes for measuring hypoxia, since they can allow their non-invasive detection to be carried out by 19F nuclear magnetic resonance. MATERIALS AND METHODS: Seven different multifluorinated nitroimidazole-based compounds have been synthesized to furnish this aim: N,N-bis(m-trifluoromethylbenzyl)-3-(2-nitro-1-imidazolyl)propylamine hydrochloride (Bis-mTFN-1); N,N-bis(p-trifluoro-methylbenzyl)-3-(2-nitro-1-imidazolyl)propylamine hydrochloride (Bis-pTFN-1); N-3,5-di-trifluoro-methylbenzyl, 3-(2-nitro-1-imidazolyl)propylamine hydrochloride (DiCF3); N-(m-trifluoromethyl-benzyl)-3-(2-nitro-1-imidazolyl)-propylamine hydrochloride (mTFN-1); N-(p-trifluoromethylbenzyl)-3-(2-nitro-1-imidazolyl)propylamine hydrochloride (pTFN-1); N-(p-trifluoromethylbenzylcarbonyl)-3-(2-nitro-1-imidazolyl)propylamine (pTFA-1) and 5,6-dimethyl-4-[3-(2-nitro-1-imidazolyl)propylamino]-2-trifluoromethylpyrimidine hydrochloride (CF3PM). The compounds were studied in V79 cells in vitro, whereas selected compounds were tested for systemic toxicity in BALB/c mice. RESULTS: With the exception of pTFA-1, all compounds were soluble in water or saline. All compounds were stable at room temperature as solids, or at 0-5 degrees C as aqueous solutions. Very good uptake were obtained in aerobic V79 cells with selected compounds. Thus, intracellular vs. extracellular concentration ratios (Ci/Ce) were increasing with input concentration up to 200. All compounds behaved as hypoxia-selective cytotoxins in V79 cells, in vitro, with selectivity ranging between 2.0 (DiCF3) to 15.5 (CF3PM). No lethality or body weight loss was observed with all tested compounds. Some signs of neurotoxicity were seen with bis-pTFN-1, mTFN-1 and pTFN-1 at the higher tested i.p. doses. No adverse effects were observed with CF3PM at any tested dose. CONCLUSION: These results suggest that some of the above compounds could be utilized as 19F-MRS probes for measuring tumor-hypoxia, by accumulation and binding into the hypoxic regions of tumors.

Novel non-invasive probes for measuring tumor-hypoxia by 19F-magnetic resonance spectroscopy (19F-MRS). Studies in the SCCVII/C3H murine model.

BACKGROUND: 19F-labeled 2-nitroimidazoles bound to hypoxic cells in tumors are known to be useful probes for measuring hypoxia since they can allow for their non-invasive detection by 19F nuclear magnetic resonance, provided that they do not lose 19F during their hypoxia-mediated metabolism. Two such compounds, N-(m-trifluoromethylbenzyl)-3-(2-nitro-1-imidazolyl)-propylamine hydrochloride (mTFN-1) and 5,6-dimethyl-4-[3-(2-nitro-1-imidazolyl)-propylamino]-2-trifluoromethylpyrimidine hydrochloride (CF3PM) were selected from a series of analogs, for their in vivo evaluation, based on their high solubility in saline and low toxicity in mice. MATERIALS AND METHODS: MRS experiments were performed in anesthetized C3H mice bearing SCCVII tumors in their flanks. Fluorinated compounds, mTFN-1 or CF3PM, were injected intraperitoneally (i.p.) at a dose of 110 or 150 mg/kg, respectively, in 0.75 mL saline. A 0.9 cm surface coil tuned to fluorine frequency was positioned directly over the tumor, the head, or the liver and 1800 transients were collected over 20 min in a Bruker Omega 4.7 T instrument. Spectroscopic measurements were taken at 2, 7 and 19 h post injection of the fluorinated drug. RESULTS: CF3PM was detected in the plasma up to 2 h post injection with maximum concentration observed 30 min post administration. In the MRS studies, mTFN-1 signal in the tumor was 68.8, 86.8 and 27.2% of the reference at 1-2, 6-7 and 18-19 h post injection, respectively. The corresponding values in the brain were 0, 125.7 and 26.6%, respectively, whereas the corresponding values in the liver were 359.3, 307.7 and 0%, respectively. CF3PM signal in the tumor was 3.3, 57.7 and 7.1% of the reference at 1-2, 6-7 and 18-19 h post injection, respectively. The corresponding values in the liver were 267.6, 60.5 and 0%, respectively. No CF3PM signal was detected in the brain at any time interval. CONCLUSION: These results suggest that CF3PM could be used as a potential probe for measuring hypoxia in tumors by 19F-MRS.

Antitrypanosomal activity of 5-nitro-2-aminothiazole-based compounds.

A small series of 5-nitro-2-aminothiazole-based amides containing arylpiperazine-, biphenyl- or aryloxyphenyl groups in their core were synthesized and evaluated as antitrypanosomatid agents. All tested compounds were active or moderately active against Trypanosoma cruzi amastigotes in infected L6 cells and Trypanosoma brucei brucei, four of eleven compounds were moderately active against Leishmania donovani axenic parasites while none were deemed active against T. brucei rhodesiense. For the most active/moderately active compounds a moderate selectivity against each parasite was observed. There was good correlation between lipophilicity (clogP value) and antileishmanial activity or toxicity against L6 cells. Similarly, good correlation existed between clogP values and IC50 values against T. cruzi in structurally related subgroups of compounds. Three compounds were more potent as antichagasic agents than benznidazole but were not activated by the type I nitrorectusase (NTR).

Nitrotriazole-based acetamides and propanamides with broad spectrum antitrypanosomal activity.

3-Nitro-1H-1,2,4-triazole-based acetamides bearing a biphenyl- or a phenoxyphenyl moiety have shown remarkable antichagasic activity both in vitro and in an acute murine model, as well as substantial in vitro antileishmanial activity but lacked activity against human African trypanosomiasis. We have shown now that by inserting a methylene group in the linkage to obtain the corresponding propanamides, both antichagasic and in particular anti-human African trypanosomiasis potency was increased. Therefore, IC50 values at low nM concentrations against both T. cruzi and T. b. rhodesiense, along with huge selectivity indices were obtained. Although several propanamides were active against Leishmania donovani, they were slightly less potent than their corresponding acetamides. There was a good correlation between lipophilicity (clogP value) and trypanocidal activity, for all new compounds. Type I nitroreductase, an enzyme absent from the human host, played a role in the activation of the new compounds, which may function as prodrugs. Antichagasic activity in vivo was also demonstrated with representative propanamides.

Mechanisms involved in the potentiation of paclitaxel or 5-fU by the hypoxic cytotoxin NLCQ-1 (NSC 709257) in vitro.

BACKGROUND: 4-[3- (2-Nitro-1-imidazolyl)-propylamino]-7-chloroquinoline hydrochloride (NLCQ-1) is a novel, weakly DNA-intercalating hypoxia selective cytotoxin, which significantly potentiates the antitumor effect, but not the systemic toxicity, of commonly used chemotherapeutic agents. In the present study, we investigated if the same schedule-dependent synergism seen in vivo exists in vitro as well, between NLCQ-1 and paclitaxel or 5-fluorouracil (5FU), and some of the mechanisms involved in such interactions. MATERIALS AND METHODS: V79 cells, while in monolayers, were exposed to paclitaxel or 5FU under aerobic conditions at various time-intervals before or after their hypoxic exposure to NLCQ-1, while in suspension. Survival was assessed by the clonogenic assay and synergistic interactions were evaluated by using the fractional product analysis. Cells treated as above were examined for apoptosis, DNA damage and repair, as well as DNA, RNA and protein syntheses inhibition. RESULTS: A schedule-dependent synergistic interaction existed between NLCQ-1 and paclitaxel/5FU, similar to that seen in transplanted EMT6 murine tumors. Optimal potentiation was observed when NLCQ-1 was administered 2 or 4 h after the antimitotic paclitaxel or the thymidylate synthase inhibitor 5FU. Apoptosis induced by paclitaxel or 5FU alone was enhanced by NLCQ-1 up to 2.5- and 1.7-fold, respectively. DNA damage was detected as single-strand breaks (ssbs) in the combination treatments and was unrepairable at least up to 24 h post treatment. DNA, RNA and protein syntheses were inhibited by paclitaxel/5FU alone ca. 50% immediately post treatment and this inhibition was persistent up to 24 h post treatment. In combination with NLCQ-1, a slight synergistic and persistent inhibition was observed in all three syntheses. CONCLUSION: Enhancement in apoptosis, unrepairable DNA damage and inhibition of DNA, RNA and protein syntheses are some of the mechanisms involved in the potentiation of paclitaxel or 5FU by NLCQ-1.

NLCQ-1 (NSC 709257) in combination with radiation against human glioma U251 xenografts.

BACKGROUND: The efficacy of the weak DNA-intercalative hypoxia-selective cytotoxin NLCQ-1 (NSC 709257) was investigated in combination with single or fractionated doses of radiation against human glioma U251 xenografts. Two "advanced stage" experiments were performed in female athymic nude mice. MATERIALS AND METHODS: Tumor-bearing mice were allocated in groups of 8-10 (treated) or 10-20 (control) and irradiated in the presence or absence of NLCQ-1. Fractionated radiation was administered either qd x 4 or qd x 2, followed by a 9-day rest and repeated dosing. NLCQ-1 was administered i.p. 45 min before each radiation dose. RESULTS: NLCQ-1 alone did not show antitumor activity or toxicity. Radiation at the highest single dose used (5.0 Gy) showed antitumor activity without weight loss (optimal T/C = -45). Lower single radiation doses (2.0 or 3.0 Gy) were marginally effective (optimal T/C of 34 and 40, respectively). The addition of NLCQ-1 to the treatment with each single radiation dose provided better optimal T/C values (e.g., -64 with 5.0 Gy). Fractionated radiation at 1.0 Gy qd x 4 showed minimal effectiveness (T/C = 27) but, in combination with NLCQ-1, the T/C value was improved to 19. Radiation alone, given on a 3.0 Gy qd x 2, 9-day rest and repeat schedule was very effective (T/C = -57) without toxicity and resulted in 5 out of 10 complete regressions up to 42 days. When NLCQ-1 was added to the above protocol an optimal TIC value of -100 and 9 out of 10 complete regressions were obtained with a follow-up of 52 days. CONCLUSION: The above results suggest a significant advantage in combining radiation with NLCQ-1 against glioma tumors.

NLCQ-1 (NSC 709257): exploiting hypoxia with a weak DNA-intercalating bioreductive drug.

The development of weak DNA-intercalating bioreductive compounds is a new strategy to ensure DNA affinity high enough to produce toxicity yet low enough to permit efficient extravascular diffusion and penetration to hypoxic tumor tissue, as has been exemplified by the lead compound 4-[3-(2-nitro-1-imidazolyl)-propylamino]-7-chloroquinoline hydrochloride (NLCQ-1, NSC 709257). Indeed, because of its weak DNA-binding, NLCQ-1 demonstrates significant hypoxic selectivity in several rodent and human tumor cell lines that can be increased up to 388-fold with 4.5 h exposure. In vitro reduction studies suggest that cytochrome P450 and b(5) reductases play a significant role in NLCQ-1 bioreductive activation. NLCQ-1 synergistically enhances the effect of radiation against hypoxic cells in vitro and murine tumors in vivo and optimizes the effect of radioimmunotherapy in human xenografts. Importantly, NLCQ-1 substantially enhances, in a schedule-dependent manner, the antitumor effect of alkylating agents, as well as 5-fluorouracil and paclitaxel against murine tumors and human xenografts, without a concomitant enhancement in bone marrow or hypoxia-dependent retinal toxicity. In addition, NLCQ-1 exhibits good stability in human plasma and favorable pharmacokinetics in mice. The synthesis of NLCQ-1 has been successfully scaled-up and its excellent recovery from biological fluids has been established. Because of these results and the fact that NLCQ-1 compares favorably with the frontrunner, bioreductive compound tirapazamine, NLCQ-1 is about to enter a Phase I clinical trial.

3-Nitrotriazole-based piperazides as potent antitrypanosomal agents.

Novel linear 3-nitro-1H-1,2,4-triazole-based piperazides were synthesized and evaluated as antitrypanosomal agents. In addition, some bisarylpiperazine-ethanones which were formed as by-products were also screened for antiparasitic activity. Most 3-nitrotriazole-based derivatives were potent and selective against Trypanosoma cruzi parasites, but only one displayed these desired properties against Trypanosoma brucei rhodesiense. Moreover, two 3-nitrotriazole-based chlorophenylpiperazides were moderately and selectively active against Leishmania donovani. Although the bisarylpiperazine-ethanones were active or moderately active against T. cruzi, none of them demonstrated an acceptable selectivity. In general, 3-nitrotriazole-based piperazides were less toxic to host L6 cells than the previously evaluated 3-nitrotriazole-based piperazines and seven of 13 were 1.54- to 31.2-fold more potent antichagasic agents than the reference drug benznidazole. Selected compounds showed good ADMET characteristics. One potent in vitro antichagasic compound (3) was tested in an acute murine model and demonstrated antichagasic activity after a 10-day treatment of 15 mg/kg/day. However, neither compound 3 nor benznidazole showed a statistically significant P value compared to control due to high variability in parasite burden among the untreated animals. Working as prodrugs, 3-nitrotriazole-based piperazides were excellent substrates of trypanosomal type I nitroreductases and constitute a novel class of potentially effective and more affordable antitrypanosomal agents.