Synthesis, Clastogenic and Cytotoxic Potential, and In Vivo Antitumor Activity of a Novel N-Mustard Based on Indole-3-carboxylic Acid Derivative

Compound T1089-a novel nitrogen mustard based on an indole-3-carboxylic acid derivative (ICAD)-has been synthesized. The ICAD used as the basis for T1089 is a TLR agonist capable of activating an antitumor immune response. This study describes the synthesis method and presents the results of preliminary investigations of this compound. This research included an assessment of acute toxicity in mice, in vivo clastogenic activity evaluated via the bone marrow chromosome aberration (BMCA) test in mice, in vitro cytotoxicity determined by the MTT assay against human lung carcinoma A549 cells, and in vivo antitumor effects (ATEs) in models of conventional chemotherapy (CCT) of solid tumors in mice. The bifunctional alkylating agent cyclophosphamide (CPA) was used as a reference drug. <b>Toxicological</b> studies revealed that T1089 belongs to toxicity class III (moderately toxic), with acute toxicity values (LD₁₆ and LD₅₀) in mice following intraperitoneal (i.p.) administration being 191 and 202 mg/kg, respectively. The alkylating activity and clastogenic potential of T1089 were demonstrated by its effects in the BMCA test, which were comparable to those of CPA. A single i.p. administration of CPA and T1089 at a dose of 0.064 mmol/kg induced similar stimulation of structural mutagenesis associated with DNA strand breaks. The frequency of karyocytes with aberrations increased 20-fold compared to the control, primarily due to a rise in chromatid breaks and fragments, and to a lesser extent, due to an increase in exchange-type aberrations. <b>In vitro</b> cytotoxicity studies indicated differences in the mechanisms of alkylating activity between CPA and T1089. According to the MTT assay, the cytotoxic effects of CPA were observed only at concentrations exceeding 2 mM (IC₅₀ = 4.2 ± 0.3 mM), corresponding to lethal in vivo doses, which is expected since the formation of CPA's alkylating metabolite requires hepatic microsomal enzymes. In contrast, significant cytotoxic effects of T1089 were observed at much lower concentrations (15-50 μM, IC₅₀ = 33.4 ± 1.3 μM), corresponding to safe in vivo doses. <b>Differences</b> were also observed in the in vivo ATEs of CPA and T1089 in the Ehrlich solid carcinoma (ESC) CCT model. Following seven i.p. administrations at 48 h intervals (33 mg/kg), both compounds exhibited increasing toxicity, manifested as cumulative body weight loss in treated mice. However, despite the aggressive CCT regimen, ESC showed low sensitivity to CPA. The ATE of CPA developed slowly, reaching a significant level only after four injections, and even after seven administrations, tumor inhibition (TI) did not exceed 30%. In contrast, ESC was significantly more sensitive to T1089 under the same CCT conditions. The ATE of T1089 exhibited a cumulative pattern but developed more rapidly and to a greater extent. A significant antitumor effect was observed after just two injections, with maximal efficacy (TI = 53%) achieved after four injections and sustained until the end of the observation period. <b>A high</b> ATE of T1089 was also observed in the B-16 melanoma CCT model. Following six i.p. administrations at 48 h intervals (28 mg/kg), T1089 treatment was associated with minimal toxicity. Despite this mild CCT regimen, melanoma exhibited high sensitivity to T1089. Maximal ATE (TI = 56%) was achieved after two injections, and subsequent administrations maintained a consistently high efficacy (TI = 52-55%) until the end of the study. <b>In summary</b>, preliminary findings demonstrate that T1089 possesses alkylating activity characteristic of bifunctional agents, accompanied by high in vitro cytotoxicity and in vivo ATEs in CCT models (at high doses). Given that the ICAD used as the basis for T1089 is a TLR agonist capable of stimulating antitumor immunity, T1089 can be considered a dual-action alkylating agent with combined antitumor effects. These results justify further investigation of T1089 in conventional and metronomic chemotherapy regimens, particularly in combination with immune checkpoint inhibitors and antitumor vaccines.