Maria V. Papadopoulou

Novel 3-Nitro-1H-1,2,4-triazole-Based Amides and Sulfonamides as Potential Antitrypanosomal Agents

A series of novel 3-nitro-1H-1,2,4-triazole-based (and in some cases 2-nitro-1H-imidazole-based) amides and sulfonamides were characterized for their in vitro antitrypanosomal and antileishmanial activities as well as mammalian toxicity. Out of 36 compounds tested, 29 (mostly 3-nitro-1H-1,2,4-triazoles) displayed significant activity against Trypanosoma cruzi intracellular amastigotes (IC(50) ranging from 28 nM to 3.72 μM) without concomitant toxicity to L6 host cells (selectivity 66-2782). Twenty-three of these active compounds were more potent (up to 58-fold) than the reference drug benznidazole, tested in parallel. In addition, nine nitrotriazoles which were moderately active (0.5 μM ≤ IC(50) < 6.0 μM) against Trypanosoma brucei rhodesiense trypomastigotes were 5-31-fold more active against bloodstream-form Trypanosoma brucei brucei trypomastigotes engineered to overexpress reduced nicotinamide adenine dinucleotide dependent nitroreductase. Finally, three nitrotriazoles displayed a moderate activity against the axenic form of Leishmania donovani . Therefore, 3-nitro-1H-1,2,4-triazole-based amides and sulfonamides are potent antitrypanosomal agents.

Novel 3-Nitro-1H-1,2,4-triazole-based Aliphatic and Aromatic Amines as anti-Chagasic Agents

A series of novel 2-nitro-1H-imidazole- and 3-nitro-1H-1,2,4-triazole-based aromatic and aliphatic amines were screened for antitrypanosomal activity and mammalian cytotoxicity by the Drugs for Neglected Diseases initiative (DNDi). Out of 42 compounds tested, 18 3-nitro-1,2,4-triazoles and one 2-nitroimidazole displayed significant growth inhibitory properties against T. cruzi amastigotes (IC(50) ranging from 40 nM to 1.97 μM), without concomitant toxicity toward the host cells (L6 cells), having selectivity indices (SI) 44-1320. Most (16) of these active compounds were up to 33.8-fold more potent than the reference drug benznidazole, tested in parallel. Five novel 3-nitro-1,2,4-triazoles were active against bloodstream-form (BSF) T. b. rhodesiense trypomastigotes (IC(50) at nM levels and SI 220-993). An NADH-dependent nitroreductase (TbNTR) plays a role in the antiparasitic activity because BSF T. b. brucei trypomastigotes with elevated TbNTR levels were hypersensitive to tested compounds. Therefore, a novel class of affordable 3-nitro-1,2,4-triazole-based compounds with antitrypanosomal activity has been identified.

4-[3-(2-Nitro-1-imidazolyl)propylamino]-7-chloroquinoline hydrochloride (NLCQ-1), a novel bioreductive compound as a hypoxia-selective cytotoxin.

A novel weakly DNA-intercalative bioreductive compound. 4-[3-(2-nitro-1-imidazolyl)-propylamino]-7-chloroquinoline hydrochloride (NLCQ-1). has been synthesized and studied as a hypoxia-selective cytotoxin in vitro. NLCQ-1, which shares a similar structure with the DNA-intercalative antimalarial drug chloroquine, bound more strongly to DNA than the nonchlorinated analog NLQ-1 (4-[3-(2-nitro-1-imidazolyl)propylamino]-quinaldine hydrochloride). Thus, NLCQ-1 exhibited a C50 [concentration for 50% displacement of the ethidium bromide (EB) from a DNA-EB complex] of 44 microM, whereas a C50 value could not be reached for NLQ-1 up to 225 microM. NLCQ-1 demonstrated significant hypoxic selectivity in several rodent (V79, EMT6, SCCVII) or human (A549, OVCAR-3) tumor cell lines. Its potency as a hypoxic cytotoxin (expressed as the product of exposure time and concentration for 50% survival) ranged between 10 and 136 microM x h, for the cell lines tested, at 30 microM input concentration. Because uptake in all cell lines was similar, the differences in potency may reflect differences in the enzymatic profile or damage repair processes among the cell lines. In addition, however, the most striking feature of NLCQ-1 was that hypoxic selectivity increased with exposure time, a common feature normally found in only bis-bioreductive agents carrying two moieties with different redox potentials. Thus, hypoxic selectivity of NLCQ-1 in V79 cells at 50% survival was increased from fivefold up to 388-fold by increasing exposure time from 1 to 4.5 h, as the result of a concomitant increase and decrease in its hypoxic and aerobic potency, respectively, over time. Because the nonchlorinated analog NLQ-1 did not demonstrate similar behavior, we hypothesized that the C-7 chlorine of NLCQ-1 might play a significant role in this phenomenon.

Novel 3-nitro-1H-1,2,4-triazole-based compounds as potential anti-Chagasic drugs : in vivo studies

BACKGROUND Chagas disease is caused by the parasite Trypanosoma cruzi, is endemic in Latin America and leads to an estimated 14,000 deaths per year and around 100 million people at risk of infection. Drugs currently used in the treatment of Chagas are old, partially effective and have numerous side effects. METHODOLOGY We have previously reported that 3-nitro-1H-1,2,4-triazole-based compounds demonstrate significant and selective activity against T. cruzi amastigotes in infected L6 cells via activation of a type I nitroreductase, specific to trypanosomatids. In the present work we evaluated in vivo 13 of these compounds based on their high in vitro potency against T. cruzi (IC50 < 1 µM) and selectivity (SI: toxicity to L6 cells/toxicity against T. cruzi amastigotes > 200). Representative compounds of different chemical classes were included. A fast luminescence assay with transgenic parasites that express luciferase, and live imaging techniques were used. A total of 11 out of 13 compounds demonstrated significant antichagasic activity when administered intraperitoneally for 5-10 days at relatively small doses. The best in vivo activity was demonstrated by amides and sulfonamide derivatives. ADMET studies were performed for specific compounds. CONCLUSION At least three compounds were identified as effective, non-toxic antichagasic agents suitable for further development.

4-[3-(2-Nitro-1-imidazolyl)propylamino]-7-chloroquinoline hydrochloride (NLCQ-1), a novel bioreductive agent as radiosensitizer in vitro and in vivo: comparison with tirapazamine.

The novel hypoxia-selective cytotoxin NLCQ-1, which is a weak DNA intercalator, was studied in conjunction with radiation against V79 cultured cells and EMT6 or SCCVII tumors in their syngeneic mice and compared with tirapazamine (TPZ). NLCQ-1 was a very potent and efficient radiosensitizer of hypoxic V79 cells, providing SER values of 2.27-2.56 at 20-80 microM concentration (measured at 10% survival level). Its C1.6 (concentration for an SER of 1.6 to be obtained) was 7.2+/-0.2 microM. Its in vitro therapeutic index (ThI, defined as CT50(Air),/C1.6) varied by the exposure time from 57 (1-h exposure) to 145 (4.5-h exposure). The corresponding C1.6 value for TPZ was 16.9 microM whereas its in vitro therapeutic index was 49 (3-h exposure). A schedule-dependent synergistic interaction was observed between NLCQ-1 or TPZ and 20 Gy of radiation in both tumor models examined, by using the in vivo-in vitro assay as endpoint. Optimal synergism (> 1 log) was observed in EMT6 tumors when each bioreductive drug was given between 45 and 60 min before irradiation. NLCQ-1 alone had no significant antitumor activity at 10 mg/kg (28% of its single LD50), whereas a 0.4 surviving fraction was obtained by TPZ at 30 mg/kg (38% of its single LD50). SER values of 1.52 and 1.25 were obtained with 10 mg/kg NLCQ-1 and 30 mg/kg TPZ, respectively, in EMT6 tumors. An SER value of 1.58 was obtained for both hypoxia-selective cytotoxins, at equitoxic doses, in SCCVII tumors, by using a fractionated regimen. These results suggest a possible use of NLCQ-1 or TPZ as adjuvants to radiotherapy.

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.

Optimizing the use of combined radioimmunotherapy and hypoxic cytotoxin therapy as a function of tumor hypoxia

Combined radioimmunotherapy (RAIT) and hypoxic cytotoxin therapy (SR4233 or NLCQ‐1) have been evaluated with both modalities administered on the same day with only moderate improvement compared with the effects of RAIT alone. In a series of studies using oxygen electrodes, immunohistochemistry and radiotracers, we have demonstrated that RAIT induces a prolonged state of hypoxia in most tumors, without affecting the pO2 levels in normal tissues. Using serial microelectrode measurements through subcutaneous (s.c.) GW‐39 human colonic xenografts, we established that the median pO2 was unrelated to the initial size of the tumor, over a range of sizes from 1.0 to 4.0 cm. Fourteen days after mice were given a 240‐μCi dose of 131I‐MN‐14 anti‐carcinoembryonic antigen immunoglobulin G, their median pO2 declined from 26.1 ± 9.6 mmHg to 9.8 ± 3.9 mmHg (p < 0.001). Using the radiotracer 3H‐MISO that accumulates in hypoxic regions, uptake in GW‐39, LoVo and LS174T s.c. human colonic tumors increased 3.0‐ to 4.2‐fold from day 14 through day 28 post‐RAIT, but uptake of 3H‐MISO in CALU‐3 tumors remained unchanged after RAIT. Normal tissue (liver, kidney, lung) uptake of 3H‐MISO did not exhibit significant changes. The increase in tumor hypoxia was also demonstrated visually using anti‐PIMO staining of tumor sections. We postulated that sequential delivery of the 2 therapeutic agents, with the hypoxic cytotoxin given 2 weeks after RAIT when tumor pO2 levels were at their nadir, would improve the therapeutic response above either modality alone or above the 2 agents delivered on the same day. Tumor growth was compared in mice given either RAIT or cytotoxin alone, the combined treatment on the same day or with the cytotoxin delivered 14 days after RAIT. Tumor size on day 35 for RAIT‐treated and SR4233‐treated GW‐39 were 3.56 ± 0.40 and 7.98 ± 2.50 cm3. When RAIT + SR4233 were delivered on the same day, tumor size dropped to 2.78 ± 0.80 cm3. If RAIT was given on day 0 and SR4233 on day 14, size further declined further to 1.74 ± 0.32 cm3 (p < 0.05 compared with same day delivery). For LS174T, tumor size on day 28 for RAIT‐treated and SR4233‐treated tumors were 1.14 ± 0.36 cm3 and 3.65 ± 0.78 cm3, respectively. When RAIT + SR4233 were delivered on the same day, size was 0.51 ± 0.174 cm3. If RAIT was dosed on day 0 and SR4233 was given on day 14, tumor size was 0.13 ± 0.07 cm3 (p < 0.05). Similar results were obtained for LoVo, but not for CALU‐3 tumors. Another hypoxic cytotoxin, NLCQ‐1, was also more efficacious 2 weeks after RAIT, compared with same‐day dosing. Thus, information on tumor hypoxia after radioantibody therapy could be important for ascertaining a window of opportunity when the surviving tumor regions are most responsive to hypoxic cytotoxins.

Novel 3-Nitrotriazole-Based Amides and Carbinols as Bifunctional Antichagasic Agents

3-Nitro-1H-1,2,4-triazole-based amides with a linear, rigid core and 3-nitrotriazole-based fluconazole analogues were synthesized as dual functioning antitrypanosomal agents. Such compounds are excellent substrates for type I nitroreductase (NTR) located in the mitochondrion of trypanosomatids and, at the same time, act as inhibitors of the sterol 14α-demethylase (T. cruzi CYP51) enzyme. Because combination treatments against parasites are often superior to monotherapy, we believe that this emerging class of bifunctional compounds may introduce a new generation of antitrypanosomal drugs. In the present work, the synthesis and in vitro and in vivo evaluation of such compounds is discussed.

Novel nitro(triazole/imidazole)-based heteroarylamides/sulfonamides as potential antitrypanosomal agents.

We have previously shown that 3-nitro-1H-1,2,4-triazole-based arylamides and arylsulfonamides demonstrate significant activity in vitro against Trypanosoma cruzi, the causative parasite of Chagas disease. More importantly, several such analogs displayed significant antichagasic activity in vivo, superior to that of benznidazole, the current clinical standard. We now report the synthesis and in vitro evaluation of a small series of novel nitro(triazole/imidazole)-based heteroarylamides/sulfonamides (including 3-nitrotriazole-, 2- and 4-nitroimidazole-based compounds) as potential antitrypanosomal agents. All nitrotriazoles displayed significant growth inhibitory properties against T. cruzi with the most potent generating IC50 values of <1 μM and up to >1400-fold selectivity toward the parasite. The 2-nitroimidazole-based derivatives were moderately active against T. cruzi and displayed selectivity <50, while the 4-nitroimidazoles were mostly inactive. Several 3-nitrotriazole-based analogs showed activity against Trypanosoma brucei rhodesiense but none of the tested compounds displayed activity toward Leishmania donovani. From the detailed SARs presented here, we identified the 3-nitrotriazole-based chlorinated thiophene/benzothiophene sulfonamides/amides as being the most active antichagasic compounds, displaying up to 14-fold higher potency against T. cruzi than the reference compound benznidazole.

NLCQ-1, a novel hypoxic cytotoxin: potentiation of melphalan, cisDDP and cyclophosphamide in vivo

PURPOSE To investigate in vivo interactions between the recently developed bioreductive agent 4-[3-(2-nitroimidazolyl)-propylamino]-7-chloroquinoline hydrochloride (NLCQ-1) and the chemotherapeutic agents melphalan (L-PAM), cis-platin (cisDDP) and cyclophosphamide (CPM). METHODS AND MATERIALS EMT6 and FSaIIC tumor cells were inoculated (subcutaneously) into the leg(s) of female Balb/c and male C3H mice, respectively. Treatment was initiated at 10 mm (EMT6) and 5 mm (FSaIIC) mean tumor diameter. The in vivo-in vitro and tumor regrowth assays were used, respectively, as endpoints. Bone marrow toxicity studies were also performed when the in vivo-in vitro assay was used. Drugs were given by i.p. injection. Tumors were excised 18-h after chemotherapeutic drug administration (Balb/c mice) or measured daily until three times their original size (C3H mice). The optimum administration schedule for potentiation between NLCQ-1 and each chemotherapeutic drug, as well as dose modification factors (DMF) at the optimum time, were determined with the in vivo-in vitro assay. When the tumor regrowth assay was used, each chemotherapeutic agent was given either as a single dose or as a split dose over two consecutive days at the optimum administration time after a 10 mg/kg NLCQ-1 i.p. injection. RESULTS NLCQ-1 (at 0.33 times MTD), strongly potentiated the antitumor effect of L-PAM, cisDDP and CPM without concurrent enhancement in bone marrow toxicity. Potentiation was strictly schedule dependent and the optimum effect (1.5 to 2 logs killing beyond additivity) was observed when NLCQ-1 was given 60-, 45-, and 110-min before L-PAM, cisDDP, and CPM, respectively. The DMF values at 30% survival were 2.5, 1.9, and 3.8 for L-PAM, cisDDP, and CPM, respectively. DMF values for bone marrow toxicity at 50% survival were ca. 1 for all chemotherapeutic drugs. Pretreatment with NLCQ-1 resulted in 4-12 days extra delay in the regrowth of FSaIIC tumors. CONCLUSIONS These results support the clinical investigation of NLCQ-1 as a chemosensitizer.