Synthesis and cytotoxic activity of 3-[2-(1 H -Indol-3-yl)-1,3-thiazol-4-yl]-1 H - pyrrolo[3,2-c ]pyridine hydrobromides, analogues of the marine alkaloid nortopsentin

A new series of thiazole nortopsentin analogues with a 5-azaindole moiety was conveniently synthesized in good to excellent yields by an Hantzsch reaction between thioamides and α -bromoacetyl compounds. The cytotoxic activity of the new derivatives was tested against different human tumor cell lines of the NCI full panel. All tested compounds were active against all of the investigated cell lines showing GI 50 values from micro to submicromolar levels. Some of the new analogues exhibited good selectivities against different NCI sub-panels


Introduction
The marine environment covers approximately 70% of the earth's surface and represents a rich source of compounds with a wide range of biological activities. 1 For this reason several efforts have been made aiming to exploit the enormous potential of marine natural products, developing their total synthesis in laboratory or synthesizing derived molecules using their scaffolds as leads.Up to 2019, the clinical marine pharmaceutical pipeline consisted of 31 marine-derived compounds in active clinical trials and 9 approved marine-derived compounds. 2Among the approved drugs, many compounds found application as anticancer drugs such as cytarabine used for the therapy of malignant acute myeloid, lymphocytic and myelogenous leukemia, trabectedin for tissue sarcoma, midostaurine for the acute myeloid leukemia, eribulin mesylate for breast cancer and liposarcoma. 3Marine alkaloids constitute one of the most attractive class of natural products 4 and in particular bis-indolyl alkaloids, characterized by two indole units connected to a spacer through their 3 position, constitute a group of deep-sea sponge metabolites with very interesting pharmacological activities such as antiproliferative, 5 antiinflammatory, 6 antimicrobial, 7 and antiviral. 8Nortopsentins A-C (Chart 1), isolated from the Halichondride sponge Spongosorites ruetzleri from deep water in the Bahamas, are the only family of bis-indolyl alkaloids bearing an imidazolediylbis[indole] skeleton.They exhibited in vitro cytotoxicity against P388 leukemia cells (IC50, 4.5− 20.7 μM) and inhibited the growth of Bacillus subtilis and Candida albicans.Their methylated derivatives (Figure 1) showed a significant improvement in cytotoxicity against P388 cells compared to that of the parent compounds (IC50, 0.8−2.1 μM). 9 Furthermore, nortopsentin C inhibited neural nitric oxide synthase (bNOS) and calcineurin activities, suggesting its probable action against calmodulin, a common co-factor of these two enzymes. 6More recently, the antiviral activity against tobacco mosaic virus (TMV) and anti-phytopathogenic-fungus property of nortopsentins A-C and their analogues were also reported. 10][13][14][15][16][17] In this effort, our research group synthesized a large library of analogues in which the imidazole moiety of nortopsentins A-C was replaced by a thiazole core and one indole portion by an azaindole ring, leading to compounds that showed antiproliferative activity against a wide range of human tumor cell lines with GI50 values in the micro-submicromolar range.Among of them, the indolyl-7-azaindolyl thiazoles (Figure 2) resulted the most active derivatives, exhibiting antiproliferative activity in the micro-submicromolar range, CDK1 inhibition (IC50 0.64-0.89µM) and significant tumor volume inhibition in mouse xenograft models. 18 recent study demonstrated that the treatment of colorectal cancer stem cells (CR-CSCs) with indolyl-7-azaindolyl thiazoles induces reduction of CSCs viability, making them sensitive to conventional chemotherapy drugs, such as oxaliplatin and 5FU.Moreover, the combination therapy of these derivatives with CHK1 inhibitor Rabusertinib showed a synergistic effect, abrogating CR-CSCs proliferative and clonogenic potential. 19n addition to the frequently used 7-azaindoles, the 5-azaindole ring is a promising pharmacophore moiety found in different antitumor molecules, despite its uncommon presence in marine natural products. 20,21Thus, continuing our studies on bioactive nitrogen heterocyclic systems 22,23 and to complete the structure-activity relationship (SAR) analysis of the nortopsentin azaindole analogues, herein we report a new series of indolyl-5-azaindolyl thiazoles of type 1 (Figure 2).We also describe the NCI's in vitro disease-oriented antitumor screen of the new synthesized analogues.
The isolated thiazoles 1c,d,g,h,k-v were submitted to the National Cancer Institute (NCI; Bethesda, MD), and were prescreened according to the NCI protocol at a 10 −5 M dose (data not shown) on the full panel of approximately 55 human cancer cell lines derived from 9 human cancer cell types that have been grouped into disease subpanels including leukemia, non-small cell lung, colon, central nervous system, melanoma, ovarian, renal, prostate, and breast tumor cell lines.All tested thiazoles satisfied the criteria set by the NCI for activity in this assay and were selected for further screenings at five concentrations at 10-fold dilution (10 −4 −10 −8 M) on the full panel.The growth inhibition activity of compounds was defined in terms of the GI50 value (which represents the molar concentration of the compound that inhibits 50% net cell growth).
The thiazoles 1c,d,g,h,k-v were active against the total number of cell lines investigated, showing antitumor activity in the micromolar -submicromolar range (GI50 0.18-26.3µM) (Table2).
All derivatives were efficacious against the leukemia sub-panel (Table 2), with particularly selectivity towards K-562 cell line, eliciting GI50s in the range 0.24-2.09µM.In addition, compounds 1l and 1v also exhibited good selectivity against CCRF-CEM cells of the same sub-panel, with GI50 values of 0.36 µM and 0.42 µM, respectively (Table 2).
Moreover, all compounds also proved to be active towards MDA-MB-468 and MCF7 cell lines of breast cancer sub-panel, for which thiazoles 1c and 1d were the most potent compounds, with GI50 values lower than or equal to 0.4 µM (Table 2).Regarding MDA-MB-468 cell line, also thiazoles 1g and 1q exhibited good GI50 values (0.44 µM and 0.23 µM, respectively).
Likewise, towards HCT-116 cell line of the colon cancer sub-panel, the GI50s were registered in the low micromolar range, with values of 0.93 µM and 0.18 µM for the most active compounds 1d and 1l, respectively.[a] The molar concentration that inhibits 50% net cell growth.nd : not determined

Conclusions
A new series of thiazole nortopsentin analogues of type 1, in which the imidazole moiety of nortopsentins A-C was replaced by a thiazole core and one indole unit by a 5-azaindole ring, was efficiently synthesized in good to excellent yields.The new nortopsentin derivatives 1c,d,g,h,k-v were active against the totality of the about 55 human tumor cell lines of NCI full panel, showing good antiproliferative activity in the microsubmicromolar range (GI50 0.18-26.3µM).Thiazoles 1k, 1l and 1v were particularly efficacious against leukemia sub-panel (GI50 in the range 0.24-1.71µM, 0.24-1.57µM and 0.35-2.13µM, respectively).Compound 1d proved to be the most active against breast cancer sub-panel (GI50 in the range 0.27-2.16µM).Furthermore, analogues 1d and 1l showed a good selectivity against HCT-116 cell line of the colon cancer subpanel (GI50 of 0.93 µM and 0.18 µM, respectively).The encouraging biological results found for this new series confirmed the advantageous influence of the thiazole central core, in comparison with the other fivemembered heterocycles, on the antiproliferative activity of this class of compounds.The reason of this improved activity could be attributed to low lying C−S σ* orbitals that, conferring small regions of low electron density on sulfur (σ-holes), may play an important role in the interaction with the biological target. 24

Experimental Section
General.All melting point were taken on a Büchi-Tottoly capillary apparatus and are uncorrected.IR spectra were determined in bromoform with a Shimadzu FT/IR 8400S spectrophotometer. 1 H and 13 C NMR spectra were measured at 200 and 50.0 MHz, respectively, in DMSO-d6 solution, using a Bruker Avance II series 200 MHz spectrometer.Thiazoles 1c,d,g,h,k-v were characterized only by 1 H NMR spectra, as due to their poor solubility t 13 C NMR spectroscopy was not perfomed.Column chromatography was performed with Merk silica gel 230-400 mesh ASTM or with Büchi Sepacor chromatography module (prepacked cartridge system).Elemental analyses (C, H, N) were within ± 0.4% of theoretical values and were performed with a VARIO EL III elemental analyzer.General procedures, analytical and spectroscopic data for intermediates 2d,3a-d,4a-c,5d,6a-d and 7a-c were previously reported. 18

General synthesis of 2-bromo-1-(1H-pyrrolo[3,2-c]pyridin-3-yl)-ethanones (9a,b).
To a solution of the appropriate 5-azaindoles 8a,b (4.2 mmol) in dry DCM (20 mL), anhydrous aluminum chloride (2.0 g, 14.8 mmol) was slowly added.The reaction mixture was heated under reflux and a solution of bromoacetyl bromide (0.37 mL, 4.2 mmol) in anhydrous DCM (2 mL) was added dropwise.The resulting solution was allowed to stir under reflux for 40 min (for derivative 9a) or 15 min (for derivative 9b).After cooling, water/ice were slowly added and the obtained precipitate was filtered off to give the pure desired compounds (9a,b).General procedure for the synthesis of thiazoles (1a-p).A suspension of the proper thioamides 5d,6a-d,7a-c (2.5 mmol) and α-bromoacetyl derivatives 9a,b (2.5 mmol) in ethanol (10 mL) was heated under reflux for 1 h.After cooling, the precipitate obtained, was filtered off and dried.Thiazoles 1c,d,g,h,k-p were recrystallized from ethanol to give the pure compounds as hydrobromide salts.Thiazoles 1a,b,e,f,i,j were very unstable and were immediately used for the next step without purification and characterization.

General procedure for the synthesis of thiazoles (1q-v).
To a suspension of appropriate thiazole 1a,b,e,f,i,j (0.38 mmol) in dichloromethane (10 mL), trifluoacetic acid (0.5 mL) was added.The reaction was heated at reflux for 24 h.The solvent was dried (Na2SO4), evaporated under reduced pressure and the residue recrystallized with ethanol to afford the desired thiazoles 1q-v.

Figure 1 .
Figure 1.Structures of nortopsentins A-C and their methylated derivatives.

Table 1 .
Substituted a ND: not determined.The crude was used in the step v without further purification.b

Table 2 .
In vitro inhibition of cancer cell lines growth by thiazoles 1c