Synthesis and biological evaluation of potent benzoselenophene and heteroaromatic analogues of (S)-1-(chloromethyl)-8-methoxy-2,3-dihydro-1H-benzo[e]indol-5-ol (seco-MCBI)

A diverse series of compounds (18a–x) were synthesized from (S)-1-(chloromethyl)-8-methoxy-2,3-dihydro-1H-benzo[e]indol-5-ol (seco-MCBI) and benzoselenophene or heteroaromatic acids. These new compounds were evaluated for their cytotoxicity against the human gastric NCI-N87 and human ovarian SK-OV3 cancer cell lines. The incorporation of a methoxy substituent at the C-7 position of the seco-CBI unit enhances the cytotoxicity through its additional van der Waals interaction and gave a much higher potency than the corresponding seco-CBI-based analogues. Similarly, the seco-MCBI-benzoselenophene conjugates (18h–x) exhibited substitution effects on biological activity, and the N-butyramido and N-methylthiopropanamido analogues are highly potent, possessing >77- and >24-fold better activity than seco-MCBI-TMI for the SK-OV3 and NCI-N87 cell lines, respectively.


Introduction
Cancer is considered one of the major causes of death worldwide. 1 Tremendous resources are being invested worldwide to develop preventive, diagnostic, and therapeutic strategies for cancer. 2 In recent years, antibody-drug conjugates (ADCs) have become an effective class of therapeutic agents for cancer therapy. 3 The concept was introduced over 30 years ago to improve the therapeutic index of anticancer drugs. 4 ADCs have potential target selectivity towards tumour cells compared to conventional chemotherapy; 5 however, their use is complicated in practice because cell-surface antigens are oen limited and the process of internalization is inefficient. Assuming all steps involved in the mechanism of ADC have an efficiency of 50%, only 1-2% of the administered drug will reach tumour cells. This makes the choice of a cytotoxin particularly important because it is required to be highly efficacious at very low concentrations. 6 The cyclopropylpyrrolo [e]indolone (CPI)-containing alkaloids, i.e., CC-1065, and the duocarmycin class of compounds [7][8][9][10][11] (Fig. 1) attracted our interest due to their higher antitumour activity. They are effective at picomolar concentrations against L1210 cell assay. These natural products have biological properties and therapeutic efficacy that are determined by their capacity for characteristic duplex DNA alkylation and DNA binding affinity. [12][13][14][15][16] The study of natural products and their synthetic derivatives has dened the fundamental features that control the selectivity and efficiency of DNA alkylation. [17][18][19][20] In our earlier study, we synthesized and evaluated the in vitro cytotoxicity of benzoselenophene analogues of seco-CBI. 21 It has been demonstrated that the benzoselenophene was a good substitute as a DNA binding unit for the indole moiety of duocarmycin analogues, which helps to improve the biological activity through increased curvature and hydrophobicity. To further enhance the biological activity, we used (S)-1-(chloromethyl)-8-methoxy-2,3-dihydro-1H-benzo [e] indol-5-ol (i.e., seco-MCBI) as a DNA alkylating agent in this study. The magnitude of the electronic effect of C-7 methoxy substituent of seco-CBI affects the reactivity of DNA alkylation and the solvolysis rate, providing additional noncovalent interactions. 22 These features encourage us to prepare seco-MCBI-benzoselenophene conjugates (Fig. 2). Similarly, the substituents attached to the DNA binding unit not only provide DNA binding affinity and selectivity, but they also affect the rate and efficiency of DNA alkylation and biological activity. 23 To evaluate the substituent effect on activity, we designed and synthesized differently substituted benzoselenophene analogues. We also aimed to develop a hydrophilic drug that does not compromise the cytotoxicity but that will instead help to improve the aqueous solubility for preparing non-aggregated ADCs. It can be achieved by attaching a hydrophilic substituent at the benzoselenophene unit or by introducing a hetero atom at a selected position in the side chain, which maintains both binding affinity and aqueous solubility. In view of the abovementioned observation and our goal of preparing highly potent candidates, we describe the syntheses and anticancer activities of benzoselenophene and heteroaromatic conjugates of seco-MCBI.

Chemistry
In our previous work, we synthesized various selenophenefused aromatic compounds with the aim of using them as DNA binding units. 21,24 Because the substitution at the C-5 position of the DNA binding unit in duocarmycin analogues is more effective for enhancing the cytotoxicity against cancer cell lines, we mostly focused on the synthesis of C-5-substituted benzoselenophene analogues. 25,26 The nitro analogue 1 is the key substrate to prepare a diverse series of N-substituted amido analogues. First, palladium-catalysed reduction of nitro group was performed to acquire amine 2. Then, the resultant amine 2 was converted into N-substituted amino and various amido analogues, which hydrolysed to provide their corresponding acids (3-7) with good overall yields. Other required benzoselenophene carboxylic acids were prepared by our previously reported method (Scheme 1). 21,24 To prepare hydrophilic benzoselenophene analogues, the 6methoxy-substituted nitro compound 8 was used as a starting material. It was converted to the corresponding amine 9 in a palladium-catalysed nitro reduction. Then, the 6-methoxy substituted acetamido analogue 10 was prepared by treating 9 with acetic anhydride in pyridine. The resultant intermediate 10 was hydrolysed to acquire acid 11. To prepare other 6-alkoxysubstituted acetamido analogues, demethylation of 10 was accomplished by BCl 3 and tetra(n-butyl)ammonium iodide, yielding the free phenol 12. The resultant analogue 12 was treated with 2-chloro-N,N-dimethylethanamine under basic conditions to obtain an N,N-dimethylethoxy derivative of benzoselenophene ester, which was hydrolysed to generate carboxylic acid 13. Similarly, pegylation of analogue 12 was performed by treatment with the tosyl protected Peg 5 -OH under basic conditions, and then the resultant ester intermediate was hydrolysed to give 14 (Scheme 2).
To prepare seco-MCBI, we rst synthesized intermediate 15 from 3-methoxy benzaldehyde according to a previously reported procedure. 22 Initially, we used a known method to make intermediate 16 from 15 by following three different previously described reactions. 27 The overall yield was low because of rst step, in which the iodo intermediate was found to be unstable and degraded during purication. To overcome this problem, we performed one-pot synthesis of 16 from 15 through a sequence of iodination, alkylation and cyclization, which provided the desired product with an excellent yield (82%). Aer obtaining intermediate 16, we followed the reported procedure to prepare N-Boc-MCBI 17 by mesylation, debenzylation and cyclization reactions. [28][29][30] Next, the heteroaryl-seco-MCBI conjugate, 18a-w, were synthesized by N-Boc deprotection of 17 in 4 N HCl in ethyl acetate and by coupling with different carboxylic acids using EDCI as a coupling reagent (Scheme 3).

Biological activity
Different derivatives of seco-MCBI-benzoselenophene, seco-MCBI-heteroaromatic analogues (18a-x) along with seco-MCBI-TMI, 22 seco-CBI-TMI 31 and various seco-CBI-heteroaromatic analogues (19a-d) were examined for their in vitro activity against the human gastric NCI-N87 and human ovarian SK-OV3 cancer cell lines. The cells were seeded in 384-well plates at 500 cells per well and were then treated with compounds in ve-fold serial dilution. Aer 3 days of incubation at 37 C, the cell viability was checked using an adenosine triphosphate monitoring system based on rey luciferase. The 5,6,7-trimethoxyindole (TMI) derivatives of seco-CBI and seco-MCBI were considered highly potent candidates of the duocarmycin class of compounds and used for activity comparison. 13 Although a previous study explained the electronic effect of the C-7 methoxy group of seco-CBI (i.e., seco-MCBI) on the functional reactivity of the agents, it had little or no perceptible effect on the biological activities against L1210 cell lines compared to the corresponding seco-CBI-based agent. 22 In our study, the seco-MCBI-TMI was 6 and 12 times more potent than seco-CBI-TMI in the SK-OV3 (IC 50 ¼ 5.4 versus 30 pM) and NCI-N87 (IC 50 ¼ 11 versus 130 pM) cell assays, respectively (Table 1). However, a signicant activity difference was observed between heteroaromatic analogues (selenophene-fused pyridine, furan and thiophene) of seco-CBI and seco-MCBI. All heteroaromatic conjugates of seco-MCBI 18a-c have been found to be much more effective against both cell lines than the corresponding seco-CBI analogues 19a-c (Table 1). It was also observed that the selenophene-fused analogues of seco-MCBI and seco-CBI are more active against the SK-OV3 cell line than against the NCI-N87 cell line.
The methoxy substitution effect on the biological activity of seco-MCBI-benzoselenophene analogues occurred in the order C-5 > C-6 > C-7 for the SK-OV3 cell line and C-7 z C-5 > C-6 for the NCI-N87 cell line ( Table 2). The activity for SK-OV3 was similar to that of the corresponding moieties of seco-CBI-indole analogues against L1210 leukaemia cell lines. 26 Compound 18h with C-5 OMe was only 1.4-and 2.3-fold less potent than the seco-MCBI-TMI analogue against SK-OV3 (IC 50 ¼ 7.7 versus 5.4 pM) and NCI-N87 (IC 50 ¼ 26 versus 11 pM) cell lines, respectively ( Table 2). These results indicate that the C-5-substituted benzoselenophene analogues are more effective at enhancing their cytotoxicity than the C-6 and C-7 substituted benzoselenophene analogues are.
In our preliminary study, we found that the N-substituted benzoselenophene analogues at the C-5 position are more cytotoxic. 21 Therefore, we prepared different N-substitute seco-MCBI-benzoselenophene analogues and examined their cytotoxicity (Table 3). In a previously reported study, 26 the seco-CBIindole analogues with NO 2 , NMe 2 , NHCOMe and NHCOPr substituents at the C-5 position have similar activities (IC 50 ¼ 20-40 pM against L1210) in our study, we observed a signicant difference in the biological activity for the corresponding benzoselenophene analogues. For example, 18l, a nitro-substituted analogue, was less cytotoxic (IC 50 ¼ 22 700 and 5000 pM against NCI-N87 and SK-OV3, respectively) than 18m with an NMe 2 substituent (IC 50 ¼ 490 and 65 pM against NCI-N87 and SK-OV3, respectively). 18n, with an N-acetamido moiety (IC 50 ¼ 1.7 and 0.2 pM against NCI-N87 and SK-OV3, respectively), was highly potent, surpassing the cytotoxicity of seco-MCBI-TMI. To increase the hydrophilic properties of the cytotoxic agent, we prepared analogues 18p-q with N,N-dimethylethoxy and Peg 5 substituents, respectively, at the C-6 position of the acetamido analogue 18n, but diminished activities were observed compared to that of analogue 18n. Interestingly, for the compound 18r with a N-butyramido substituent, 5-and 3-fold enhancements in activities were observed against NCI-N87 and SK-OV3 cells, respectively, compared to 18n, but no improvements were observed for 18s, with an N-hexanamide substituent. To achieve further enhancement in the activity, we replaced the g-carbon of the N-butyramide substituent of 18r with an N,N-dimethyl amine and S-Me group, resulting in analogues 18t and 18u, respectively. The 18t has signicantly reduced potency against both cell lines, while 3.5-fold improvement was observed for 18u against the SK-OV3 cell line. The substituted pyrrole 18v was found to be 2 times less potent than the 18n against the SK-OV3 cell line, but it was >10 times more potent than the seco-MCB-TMI analogue against both cell lines. The activity was dramatically reduced in sulfonamide derivatives 18w-x, which were >2200 times less potent than seco-MCBI-TMI. This may due to the poor interaction of the sp 3 hybridized sulfonyl group in the minor groove. Overall, in this series of N-substituted benzoselenophene analogues, the Nbutyramido (18r) and methylthiopropanamido (18u) analogues were found to be the most potent, exhibiting IC 50 values < 1 pM against both cell lines in the current assay.

General materials and methods
All reagents were obtained from commercial suppliers and used without further purication, unless specied. The starting carboxylic acids of the analogues 18c-d and 18f-g i.e. thieno [3,2- 3 CN gradient over 20 min). For preparative HPLC, a C18 column (5 mm, 10 Â 250 mm) was employed at a ow rate of 4 mL min À1 using the gradient condition B. High resolution mass spectra (HRMS) were recorded using two different instruments: (i) fast atom bombardment ionization using a double-focusing magnetic sector mass analyzer (ii) electrospray ionization using an ion trap analyzer. All reactions were monitored by thinlayer chromatography (TLC) performed on glass packed silica gel plates (60F-254) with UV light and visualized with ninhydrin, p-anisaldehyde, phosphomolybdic acid or KMnO 4 solution stains. Column chromatography was performed with silica gel (100-200 mesh) with the indicated solvent system.
Synthetic procedure for amide and urea derivatives of benzoselenophene (2). The synthetic procedure for compound 2 is described in our previous work. 21 5 . To a solution of ethyl 5-aminobenzo[b]selenophene-2-carboxylate (2) (200 mg, 0.75 mmol) in 5 mL CH 3 CN, 36.5% formaldehyde solution in H 2 O (1 mL) was slowly added at 0 C. Aer 5 min stirring, NaBH 3 CN (9 mg, 0.15 mmol) and acetic acid (0.1 mL) were added and the reaction mixture was stirred continuously at room temperature for 24 h. Aer complete conversion of starting compound, the reaction mixture was diluted with CH 2 Cl 2 -H 2 O (10 mL, 1 : 1) mixture. The organic layer was separated and dried over the MgSO 4 and ltered. The ltrate was concentrated under reduced pressure to provide the crude product, which was puried by silica column chromatography using 30% ethyl acetate in hexane as an eluent to afford the desired ethyl 5-(dimethylamino) benzo[b]selenophene-2-carboxylate as a brown solid (213 mg, 97%). 1 , 0.68 mmol) was dissolved in 5 mL MeOH and then 5 mL 3 N NaOH solution was added. The mixture was stirred at room temperature for 24 h. Aer complete hydrolysis, the reaction mixture was concentrated under reduced pressure to obtain a crude residue which was acidied with 2 N HCl solution. The crude product was extracted with CH 2 Cl 2 (3 Â 10 mL), concentrated under reduced pressure and then puried by silica column chromatography using 5% MeOH in CH 2 Cl 2 as an eluent to afford the desired product 3 as a brown solid (150 mg, 83%). 1 (5). The mixture of 3-(dimethylamino)propanoic acid (103 mg, 0.67 mmol), N-(3-dimethylaminopropyl)-N 0 -ethylcarbodiimide hydrochloride EDCI (257 mg, 1.34 mmol) and DMAP (109 mg, 0.89 mmol) were dissolved in 5 mL dry DMF. The reaction mixture was stirred at 0 C for 15 min and then the solution of compound 2 (120 mg, 0.45 mmol) in 0.5 mL DMF was slowly added under a N 2 atmosphere. The reaction mixture was stirred continuously at room temperature for 17 h until complete conversion of starting material was conrmed by TLC. The reaction mixture was diluted with 10 mL water and extracted with CH 2 Cl 2 (3 Â 10 mL). The combined organic layers was washed with brine, dried over the MgSO 4 , ltered and concentrated under reduced pressure. The crude product was puried by silica column chromatography using 10% MeOH in CH 2 Cl 2 as an eluent to provide the desired ethyl 5-(3-(dimethylamino) propanamido)benzo[b]selenophene-2-carboxylate as a brown solid (116 mg, 71%). 1
The crude product was puried by silica column chromatography using 10% MeOH in CH 2 Cl 2 as an eluent. The desired product was obtained as a brown solid (157 mg, 76%). 1  , and then 20% TFA in CH 2 Cl 2 was added slowly at 0 C. The reaction mixture was stirred continuously at room temperature for 3 h. Aer completion the reaction, the solvent was removed under reduced pressure. The residue was dissolved in 5 mL dry CH 2 Cl 2, and then acetyl chloride (110 mg, 1.41 mmol) and TEA (237 mg, 2.34 mmol) were added at 0 C. The reaction mixture was stirred at room temperature for 5 h until complete conversion of starting material was conrmed by TLC. The reaction mixture was diluted with 10 mL water and extracted with CH 2 Cl 2 (3 Â 10 mL). The combined organic layers was washed with brine, dried over the MgSO 4 , ltered and concentrated under reduced pressure. The crude product was puried by silica column chromatography using 5% MeOH in CH 2 Cl 2 as an eluent. The desired ethyl 5-(4-acetamido-1-methyl-1H-pyrrole-2-carboxamido)benzo[b]selenophene-2-carboxylate was obtained as a yellow solid (90 mg, 45%). 1 166.7, 164.6, 159.9, 141.5, 138.5, 137.4,  137.1, 133.9, 126.1, 122.5, 122.3, 120.9, 119.1, 118.2, 105.3, 36.3 Ethyl 5-amino-6-methoxybenzo[b]selenophene-2-carboxylate (9). To a solution of ethyl 6-methoxy-5-nitrobenzo[b]selenophene-2-carboxylate 24 (1.8 g, 4.36 mmol) in 25 mL dry ethyl acetate, 10% Pd/C was added under a N 2 atmosphere. The reaction mixture was stirred under a H 2 atmosphere for 6 h. On complete conversion, the mixture was ltered through a Celite pad followed by washing with ethyl acetate (3 Â 25 mL). The ltrate was concentrated under reduced pressure to obtain the crude product, which was puried by silica column chromatography using 30% ethyl acetate in hexane as an eluent to provide the desired product 9 as an oily liquid (1.54 g, 94%) 1  Ethyl 5-acetamido-6-methoxybenzo[b]selenophene-2-carboxylate (10). Pyridine (1.49 mL, 18.5 mmol) was added to the solution of 9 (1.1 g, 3.69 mmol) in 10 mL dry CH 2 Cl 2 . The reaction mixture was stirred for 15 min at room temperature, aer that acetic anhydride (0.5 mL, 3.87 mmol) was added slowly under a N 2 atmosphere. The reaction mixture was stirred continuously at room temperature for 8 h. The reaction mixture was quenched with 10 mL water and desired product was extracted with CH 2 Cl 2 (3 Â 10 mL). The combined organic layer was washed with brine and dried over MgSO 4 , ltered and concentrated under reduced pressure. The crude product was puried by silica column chromatography using 50% ethyl acetate in hexane as an eluent to afford the desired 10 as a brown solid (1.13 g, 90%). 1  5-Acetamido-6-methoxybenzo[b]selenophene-2-carboxylic acid (11). Compound 10 (200 mg, 0.59 mmol) was hydrolysed by similar method used for the synthesis of 3. The desired acid 11 was obtained as a yellow solid aer purication by silica column chromatography using 5% MeOH in CH 2 Cl 2 as an eluent (170 mg, 93%). 1 (12). Compound 10 (0.8 g, 2.35 mmol) was dissolved in 10 mL anhydrous CH 2 Cl 2 , and then tetra n-butylammonium iodide (2.17 g, 5.87 mmol) was added under a N 2 atmosphere at À78 C. Aer 10 min stirring, 5.9 mL of BCl 3 (1 M CH 2 Cl 2 solution, 5.87 mmol) was slowly added, and then the reaction mixture was stirred for 2 h at 0 C. The reaction mixture was quenched with 25 mL ice water and desired product was extracted with CH 2 Cl 2 (3 Â 50 mL). The combined organic layer was washed with brine, dried over MgSO 4 , ltered and concentrated under reduced pressure to obtain a crude product 0.620 g, which was used without purication for next step reaction. HRMS (ESI): m/z calcd for (C 13 H 13 NNaO 4 Se) [M + Na] + 349.9907, found 349.9902.

Synthesis of the intermediate (16)
A solution of 9 (5.0 g, 13.17 mmol) in anhydrous THF (350 mL) was cooled to À78 C, then treated with catalytic amount H 2 SO 4 (85 mL) in THF (10 mL). Aer 15 min stirring, a solution of NIS (3.55 g, 15.82 mmol) in THF (20 mL) was added and the reaction mixture was stirred at À78 C for 2 h, and then at room temperature for 30 min. The progress of the reaction was monitored by TLC. Aer complete conversion of starting material, NaH (60% dispersion in mineral oil, 4.3 g, 105.42 mmol) was added in portion under N 2 atmosphere at 0 C and then stirred the reaction mixture at room temperature for 30 min. (S)-Glycidyl nosylate (4.10 g, 15.82 mmol) was added under N 2 atmosphere and the mixture was stirred for 3 to 5 h at room temperature. On complete conversion of intermediate, 3 M solution of EtMgBr in diethyl ether (13.15 mL, 39.53 mmol) was added slowly and stirred continuously for 2 h. The reaction mixture was quenched with saturated NH 4 Cl at 0 C, and then extracted with ethyl acetate (3 Â 200 mL). The combine organic layer was washed with aqueous NaCl and dried over Na 2 SO 4 , ltered and concentrated under reduced pressure to get crude residue, which was puried by ash column chromatography on silica gel using 40% ethyl acetate in hexane as an eluent to provide the desired product 16 (4.74 g, 82%).

General procedures for the synthesis of seco-MCBI derivatives (18a-x)
To 17 (30 mg, 0.09 mmol) in a round bottom ask, 4 mL saturated solution of HCl in ethyl acetate was added at À78 C. The reaction mixture was stirred at the À78 C for 30 min and then room temperature for 1 h. Aer salt formation was observed on TLC, ethyl acetate was evaporated under nitrogen ow and then completely dried under high vacuum for 1 h. The resulting residue was dissolved in anhydrous DMF (0.2 mL) and added to the mixture of acid (1.1 eq.) and EDCI (52 mg, 0.27 mmol) in anhydrous DMF (0.5 mL) at 0 C. The reaction mixture was stirred at 0 C for 3 h and then at room temperature for 5 h. Aer completion the reaction, the reaction mixture was diluted with water and extracted with ethyl acetate (3 Â 15 mL). The combined organic layer was washed with brine, dried over MgSO 4 , ltered and concentrated. The crude product was puried by column chromatography to afford the desired product. USA)) were seeded 384-well plates at 500 cells per well. Aer 2 h plating, cells were treated with toxins in 5-fold and 14-point serial dilution series in quadruplicate. Aer 3 days of incubation at 37 C in a 5% CO 2 humidied incubator, cell viability was checked using an adenosine triphosphate monitoring system based on rey luciferase (ATPliteTM 1step, Perki-nElmer, MA, USA). IC 50 values were calculated as an average of quadruplicated experiments (GraphPad Prism 5.0, CA, and USA).

Conclusions
In summary, a series of benzoselenophene and heteroaromatic analogues of seco-MCBI (18a-w) were synthesized, and their cytotoxicities against the human gastric NCI-N87 and human ovarian SK-OV3 cancer cell lines were evaluated. The incorporation of a methoxy group at the C-7 position in seco-CBI enhances the cytotoxicity through additional van der Waals interactions, and it was found to be much more potent than a seco-CBI-based analogue. The seco-MCBI-benzoselenophene analogues (18h-x) exhibited substitution effects on the biological activity and allowed for detailed study of the structureactivity relationship (SAR). Among the series of N-substituted analogues (18h-x), the 18n, 18r and 18u-v analogues were more potent than seco-MCBI-TMI and other compounds. The higher potency of 18r than 18n results from the extended length of the C-5 substituents of the benzoselenophene unit, which has greater hydrophobicity and van der Waals interactions in the DNA minor groove. The potency of N-butyramido analogue 18r was diminished aer substitution with the hydrophilic N,Ndimethyl amino group, but it maintained or slightly increased the activity aer substitution with S-methyl group. The activity was reduced in hydrophilic analogues 18p (IC 50 ¼ 190, 37 pM against NCI-N87 and SK-OV3, respectively) and 18q (IC 50 ¼ 1000, 260 pM against NCI-N87 and SK-OV3, respectively). However, the activities of hydrophilic analogues 18p and 18t are sufficiently high for use as a cytotoxic agent in ADCs. Overall, we successfully synthesized and screened potent candidates of benzoselenophene analogues of duocarmycin that can be used to develop effective therapeutics for advanced chemotherapy.

Conflicts of interest
There are no conicts to declare.