Design, Synthesis, and Evaluation of the Anticancer

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IC50, half maximal inhibitory concentration. Month 2018. Anticancer Agents of β-Lactam Quinone Hybrids. Journal of Heterocyclic Chemistry. DOI 10.1002/jhet ...
Design, Synthesis, and Evaluation of the Anticancer Properties of Novel Quinone Bearing Carbamyl β-Lactam Hybrids

Month 2018

Nagaraju Payili,a,b Satyanarayana Yennam,a*

Santhosh Reddy Rekula,a Challa Gangu Naidu,b Yamini Bobde,c and Balaram Ghoshc

a

Chemistry Services, GVK Biosciences Pvt. Ltd., Survey Nos: 125 (part) and 126, IDA Mallapur, Hyderabad 500076, Telangana, India b Vignan’s Foundation for Science, Technology and Research University (VFSTRU), Vadlamudi, Guntur 522213, Andhra Pradesh, India c Department of Pharmacy, Birla Institute of Technology and Science, Pilani, Hyderabad Campus, Shameerpet, Hyderabad 500078, Telangana, India *E-mail: [email protected] Received November 10, 2017 DOI 10.1002/jhet.3169 Published online 00 Month 2018 in Wiley Online Library (wileyonlinelibrary.com).

This paper describes a simple and practical protocol for the direct synthesis of a series of new hybrid molecules of carbamyl β-lactam derivatives bearing quinone moiety via [2 + 2] cycloaddition of Staudinger reaction. The β-lactam ring is tolerating carbamylation and further leads to a variety of quinone hybrid derivatives. The structures of the compounds were characterized by IR, MS, nuclear magnetic resonance, and high-resolution mass spectra analysis. All the new synthesized compounds were screened for their in vitro antiproliferative activity using an MTT assay analysis. Out of 14 derivatives synthesized in the current study, compounds 9h, 9k, 9i, and 9b exhibited the very good anticancer activities in B16F10 cell line. J. Heterocyclic Chem., 00, 00 (2018).

INTRODUCTION The available anticancer drugs have distinct mechanisms of action that may vary in their effects on different types of normal and cancer cells. A single “cure” for cancer has proved elusive because there is not a single type of cancer but as many as different types of cancer. In addition, there are very few demonstrable biochemical differences between cancerous cells and normal cells. For this reason, the effectiveness of many anticancer drugs is limited by their toxicity to normal rapidly growing cells in the intestinal and bone marrow areas. A final problem is that cancerous cells that are initially suppressed by a specific drug may develop a resistance to that drug. For this reason, cancer chemotherapy may consist of using several drugs in combination for varying lengths of time. In cancer chemotherapy, there is currently much interest in the design of small molecules that bind to DNA with sequence selectivity and noncovalent interactions [1]. Quinone drugs attract the attention of chemists and biologists because of their unique structure and biological properties [2,3]. Several antitumor agents, frequently employed in the treatment of different forms of cancer, quinone containing motifs also discovered to have marked cytotoxic effects. These quinone derivatives are

important and common building blocks, and convenient precursors for many biological compounds [4,5]. The concept of synthesizing natural product hybrids and analogues, containing two different pharmacophoric subunits, has been recently devised [6]. Currently, novel strategies are the optimization of therapies with available drugs, repurposing of medicines, developing analogs of existing drugs and the evaluation and use of chemosensitizers [7]. There is, however, another strategy that has gained much attention in the field of contemporary medicinal chemistry. It involves the combination of two biologically active molecules (pharmacophores) into one single hybrid entity with a dual mode of action. The concept of “hybrid drugs” has been gaining popularity in medicine. Because a single drug is not always able to adequately control the illness, the combination of drugs with different pharmacotherapeutic profile may be needed [8]. Drugs involving the incorporation of two drug pharmacophores in a single molecule with the intention of exerting dual drug action have been described [9]. These novel hybrid molecules have the potential to enhance efficacy, improve safety, be cost-effective, and reduce the propensity to elicit resistance relative to the parent drugs (Fig. 1) [10]. There are many investigations

© 2018 Wiley Periodicals, Inc.

N. Payili, S. Yennam, S. R. Rekula, C. G. Naidu, Y. Bobde, and B. Ghosh

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Figure 1. Examples of previously published quinone and diaziridinyl quinone bearing five-membered heterocyclic hybrids.

available independently into various types of β-lactams and quinones [11], but no information is available regarding the synthesis of compounds involving the combination of carbamyl β-lactams and quinone ring system. As part of our ongoing research in the synthesis of quinone hybrid molecules [12], we wish to report a general and efficient synthesis of quinone bearing carbamyl β-lactams by [2 + 2] cycloaddition of Staudinger reaction as key step and their characterization, which can be regarded as hybrids of the pharmacologically relevant subunits of carbamyl β-lactam and quinone ring system. There are many synthetic methods known for the construction of the β-lactam rings. In general, the βlactam ring is formed through either ketene-imine cyclizations [13] (the Staudinger reaction) or ester enolateimine condensations [14] (the Gilman–Speeter reaction). Other notable methods are sometimes employed, including photoinduced rearrangements [15] and radical cyclizations [16]. However, the Staudinger reaction is still the most frequently used and is considered to be the most effective. The Staudinger reaction involves the cycloaddition between an acid chloride (or equivalent) and an imine, in the presence of a tertiary base, and the stereochemistry of the resulting βlactam can be cis, trans, or a mixture of cis and trans.

RESULTS AND DISCUSSION The target compounds 9a–k were synthesized as outlined in Scheme 1. The compound 3 was prepared by treating 2,5-dimethoxybenzaldehyde (1) with aniline (2) in the presence of catalytic amount of acetic acid in methanol at reflux temperature for 2 h in very good yield (87%). The 1H nuclear magnetic resonance (NMR) spectra of crude compound (3) showed the presence of imine C─H proton at 8.89 ppm as singlet with 92% GC– MS purity. Our study was initiated with the optimization of Staudinger reaction based on reported literature [17]. In an initial experiment, treatment of imine (3) with acetoxyacetyl chloride (4) under general condition using Et3N and CH2Cl2 as solvent at room temperature provided complex mixture. Further, the systematic study of this reaction carried out by variation of temperature, solvents, and bases. The Staudinger reaction between imine (3) and acetoxyacetyl chloride (4) using Et3N as base at 78°C to room temperature for 16 h provided βlactam derivatives in good yield. The synthesized β-lactam compound (5) showed mixture of cis/trans isomers (75.4:22.9) by HPLC analysis at 5.1 and 5.5 retention time, respectively. The separation of cis/trans isomers by flash column

Scheme 1. Synthesis of quinone bearing carbamyl β-lactam hybrids.

Journal of Heterocyclic Chemistry

DOI 10.1002/jhet

Month 2018

Anticancer Agents of β-Lactam Quinone Hybrids

chromatography was unsuccessful due to very close retention time. However, by using preparative HPLC in normal phase, both isomers (5a and 5b; Fig. 3) were separated and confirmed by NOE studies (Supporting Information). In 1H NMR, the H-3 and H-4 protons of cis β-lactams appear at 6.10 and 5.74 ppm as two doublet signals because of the adjacent C─H coupling, whereas trans β-lactams do not have adjacent C─H coupling and appear as two singlet signals at 5.68 and 5.23 ppm. The β-lactam compound (5) that was further confirmed by 13C NMR of cis/trans isomeric mixture showed signal at 169.26 and 168.74 ppm for carbonyl peak of β-lactams ring and 56.1 and 75.4 ppm for C-3 and C-4 carbon, respectively. The mass spectrum shows a molecular ion peak at 342, which confirms the assigned molecular mass for the β-lactam compound 5. The mechanism of the β-lactam formation is complicated, and two paths have been proposed to explain the nature of the products. The first path involves the formation of the ketene by the reaction of acid chloride or equivalent in the presence of a base, attack of the ketene (A) by the imine (3) and subsequent cyclization of the intermediate to the β-lactam (Fig. 2). Support for the ketene mechanism has been derived from spectroscopic study [18] and by trapping experiments [19]. The second mechanism proposes an acylation of the imine by the acid chloride to form N-acyliminium chloride (C) that produces the zwitterionic intermediate (B), and then, it forms the β-lactam through a ring closure reaction. Like the ketene mechanism, the acylation of imine route has also been proposed by a number of investigators [8]. Deacetylation of compound (5) upon treatment with 1.0 eq of sodium hydroxide in THF/water (9:1) at room temperature resulted in hydroxy β-lactam derivative (6) in 75% yield. In 1H NMR, ─OH proton of β-lactam showed broad singlet at 2.69 ppm, and the H-3 and H-4 protons of β-lactams appear at ~5.43–5.42 ppm and ~5.22– 5.21 ppm for cis isomer and ~4.79 and ~4.27 ppm for

trans isomer, respectively, observed as two doublet signals for cis isomer and singlet signals for trans isomer because of the two adjacent ─CH─ occurs coupling forming an AB system. After obtaining the key intermediate 6, we explored the displacement reaction with various nitrogen nucleophiles. Thus, treatment of compound 6 with amines 7a–k using triphosgene in the presence of trimethylamine in dichloromethane at 10°C to room temperature for 2 h gave the carbamyl β-lactam derivatives (8a–k) in a very good yield (65%). This mild carbamylation procedure tolerates the β-lactam ring opening. The compounds (8a–k) were confirmed by IR, 1 H NMR, and 13C NMR. The cis/trans isomers ratio was identified by UPLC reverse phase column [ACQUITY BEH 18 (50 × 2.1 mm, 1.7 μm)] using formic acid/water (Supporting Information). The carbamyl β-lactam derivative (8a–k) was treated with cerium ammonium nitrate in acetonitrile/water (8:2) at 10°C for 2 h and produced targeted molecules of quinone carbamyl β-lactam hybrids (9a–k) in very good yield (55–70%) (Scheme 2). Importantly, the βlactam ring stereochemistry was unaffected. The cis/trans stereochemistry of the four-membered ring is set during the cyclization step to form the 2-azetidinone ring, and it is transferred unaltered during the further synthetic steps. This methodology offers the possibility of achieving 2zetidinone quinones in racemic forms. All the compounds (9a–k) were well characterized by 1H NMR, IR, MS, 13C NMR, and high-resolution mass spectra (HRMS) data. The 1 H NMR data of title compounds (9a–k) showed the presence of mixture of cis/trans isomer of β-lactams. In 1H NMR, the H-3 and H-4 protons of β-lactams appear at ~6.04–6.03 ppm and ~5.58–5.57 ppm for cis isomer and ~5.43–5.43 ppm and ~5.10–5.09 ppm for trans isomer, respectively. These are observed as two doublet signals for cis isomer and singlet signal for trans isomer due to the two adjacent ─CH─ occurs coupling forming an AB system. The title compounds (9a–k) were further confirmed by 13 C NMR, and the mixture of cis/trans isomers showed

Figure 2. Proposed mechanism for the formation of β-lactam ring via ketene formation by Staudinger reaction protocol.

Journal of Heterocyclic Chemistry

DOI 10.1002/jhet

N. Payili, S. Yennam, S. R. Rekula, C. G. Naidu, Y. Bobde, and B. Ghosh

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Scheme 2. Synthesis of carbamyl β-lactam quinone hybrids.

signal at 186.39 and 185.55 ppm for two carbonyl peaks that validate the benzoquinone moiety. In IR spectra of the final compounds (9a–k), there is a strong absorption at 1780 cm1 due to the presence of C═O of β-lactams ring, 1709 cm1 of carbamyl C═O, and 1654 cm1of quinone C═O and a weak absorption at 2932 cm1 for CH─N stretching vibration. The cis/trans isomers ratio of compounds 9a–k was identified by normal phase HPLC [column: ethylpyridine (250 × 4.6 mm)] using n-Hexane/ IPA. The ratio of compounds (9a–k) was furnished in Scheme 2. The details of analytical reports were given in the Supporting Information. All the benzoquinones derivatives are characteristic yellowish solid. Further, to confirm the cis/trans isomers, one of the final compound 9b-1/9b-2 was successfully separated by

preparative TLC and obtained 97.98% purity of cis isomer and 99.58% purity of trans isomer (Fig. 3). The NOE spectroscopic data were used to confirm these cis/ trans isomers. In cis isomer (9b-1), when we irradiate C3-attached hydrogen at δ 6.07 ppm, the C4-attached hydrogen was enhanced. Whereas in trans isomer (9b-2), when we irradiate C3-attached hydrogen at δ 5.42 ppm, both the C4-attached hydrogen and quinone proton at δ 6.63 were enhanced. The cis/trans isomers were further supported by HSQC experiments. Anticancer evaluation. Cell culture and MTT assay procedure. Anticancer activity of the new compounds

was screened using murine melanoma cell line (B16F10). The B16F10 cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with fetal bovine serum

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Figure 3. Cis and trans isomers of 5a and 5b and 9b-1 and 9b-2.

Figure 4. Anticancer activity of new compounds by MTT assay. B16F10 cells were treated with all compounds at two doses; 100 and 10 μM in triplicate. Data represent mean ± SD.

and antibiotic. All reagents were purchased from Himedia Laboratories Pvt. Ltd., Mumbai, India. The cells were incubated at 37°C in humidified atmosphere and 5% CO2; 1 × 104 cells were seeded in 96-well plate and incubated overnight. The cells were treated with synthesized compounds at two different doses (100 and 10 μM) for 24 h. After that, cells were treated with 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT) for 4 h, and then, DMSO was added into the wells, and absorbance was measured by SpectraMax (Molecular Devices, USA). Half maximal inhibitory concentration values were determined for some best active compounds where cell culture, compound treatment with other assay reagents had been performed following the same procedure as described earlier. The selected four compounds were tested at 10 different concentrations such as 400, 200, 100, 50, 25, 12.5, 6.25, 3.125, 1.562, and 0.781 μM. The experiment was repeated with two different batches of cells. With the synthesized compounds (9a–k, 5, and 9b0 and 9b″) in hand, we evaluated their anticancer activity by using MTT assay, which is an enzyme-based colorimetric assay method (Fig. 4). From the results, it was found that all carbamyl compounds possess good anticancer activity at 100 μM concentration, whereas at 10 μM concentration, some of them showed moderate to less activity. From the experimental results, it was found that the carbamyl derivative (9b) with methyl group at 4-position of piperidine ring showed better activity. However, in the

absence of methyl group at 4-position of piperidine ring, 9i showed moderate activity. The carbamyl azetidine derivative (9k) showed less activity than 4-methyl piperidine derivative (9b) but more activity than without methyl group at 4-position of piperidine (9i). The carbamyl derivative of N-methoxymethanamine (9h) also showed good activity (Fig. 5; Table 1). It has been observed that five-membered pyrrolidine derivatives showed less activity than six-membered piperidine ring derivatives, and this may be due to more planar and stability of six-membered ring system. Introduction of methyl group at para position of piperidine results in IC50 value 24.40 μM and found to be the most active compound in the series.

Figure 5. Anticancer activity of compounds (9h, 9k, 9i, and 9b) by MTT assay. B16F10 cells were treated with compounds at concentration range of 0.781–400 μM (n = 2). Data represent mean ± SD. IC50, half maximal inhibitory concentration.

Journal of Heterocyclic Chemistry

DOI 10.1002/jhet

N. Payili, S. Yennam, S. R. Rekula, C. G. Naidu, Y. Bobde, and B. Ghosh Table 1 IC50 values for carbamyl β-lactam quinone hybrids (9a–k, 5, and 9b0 and 9b″) on B16F10 cells. Nos

Compound

IC50 (μM)

1 2 3 4 5 6 7 8 9 10 11 12a 13b 14

9a 9b 9c 9d 9e 9f 9g 9h 9i 9j 9k 9b0 9b″ 5

>60 24.40 >60 >60 >60 >60 >60 36.94 54.05 >60 57.82 >60 >60 >60

IC50, half maximal inhibitory concentration. Cis isomer of 9b. b Trans isomer of 9b. a

CONCLUSION To the best of our knowledge, this is the first example of the preparation of hybrid products containing the pharmacologically relevant subunits of quinone bearing carbamyl β-lactam ring system. These compounds might have useful biological and therapeutic activities. The structures of the title compounds were confirmed by IR, 1 H NMR, MS, and elemental analysis. These new synthesized compounds were tested for their in vitro antiproliferative activity using an MTT assay. Out of the 14 derivatives prepared in the current study, compounds 9h, 9k, 9i, and 9b exhibited good anticancer properties tested against B16F10 cells.

EXPERIMENTAL General information. Melting points were determined in open capillary tubes on Cintex melting point apparatus and are uncorrected. IR (KBr) spectra were recorded on Perkin-Elmer FT/IR-4000 using ATR (υmax in cm1) in the frequency range of 600–4000 cm1. 1H NMR and 13 C NMR spectra were recorded in CDCl3 on a Bruker DRX-400 (400 MHz FT NMR). Chemical shifts are presented in δ ppm employing TMS as internal reference. Splitting patterns were reported as s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; and br, broad. HRMS were recorded with an Agilent Technologies 6510 Q-TOF spectrometer. General procedure for the synthesis of quinone compounds To a stirred solution of compound 8a–8k 9a–k.

(1.0 mmol) in acetonitrile (6 Vol), water (4 Vol), CAN (2.0 mmol) was added at 10°C and stirred at RT for

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2 h. TLC analysis showed completion of the reaction. The progress of the reaction was monitored by TLC (40% EA/pet ether). The reaction mixture was quenched with water (25 mL) and extracted with EtOAc (2 × 20 mL). The combined organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to afford crude compound. The crude was purified by column chromatography or prep TLC to afford corresponding quinone derivatives 9a–k. 2-(3,6-Dioxocyclohexa-1,4-dien-1-yl)-4-oxo-1-phenylazetidin3-yl 2-methyl pyrrolidine-1-carboxylate (mixture of cis/trans isomers) (9a). Yield = 68%; brown solid, MP 91–95°C;

IR (KBr): 3420, 2970, 1772, 1714, 1658, 1499, 1376, 1121, 968, 838, 757, 686, 650, and 538 cm1; 1H NMR (500 MHz, CDCl3): δ = 7.35–7.25 (m, 7H), 7.16–7.12 (m, 2H), 6.86 (d, 2H, J = 10.0 Hz), 6.78 (d, 2H, J = 10.0 Hz), 6.65–6.61 (m, 2H), 6.19–6.0 (m, 1H), 5.59–5.46 (m, 2H), 5.11–5.09 (m, 1H), 4.11–3.61 (m, 3H), 3.48–3.34 (m, 4H), 2.12–1.79 (m, 6H), 1.25–1.15 (m, 6H); 13C NMR (126 MHz, CDCl3): δ = 186.34, 136.71, 136.65, 136.62., 136.58, 136.54, 136.47, 136.44, 136.01, 135.96, 133.38, 132.28, 129.42, 125.15, 117.28, 116.98, 77.25, 56.87, 54.14, 53.86, 53.83, 53.70, 46.92, 46.79, 46.45, 46.15, 46.12, 32.94, 32.34, 32.20, 23.53, 23.46, 23.41, 22.72, 22.61, 20.61, 20.55, 19.51, 19.42; HRMS calcd C21H21N2O5 381.1450; found: 381.1454 (M + H)+; purity: 53% + 42%. 2-(3,6-Dioxocyclohexa-1,4-dien-1-yl)-4-oxo-1-phenylazetidin3-yl 4-methyl piperidine-1-carboxylate (mixture of cis/trans isomers) (9b). Yield = 65%; brown solid, MP 95–99°C;

IR (KBr): 3418, 2924, 1773, 1712, 1657, 1501, 1376, 1231, 1111, 754, 687, 646, 562, and 529 cm1; 1H NMR (500 MHz, CDCl3): δ = 7.36–7.25 (m, 5H), 7.17–7.12 (m, 1H), 6.88 (d, 1H, J = 11.0 Hz), 6.78 (d, 1H, J = 10.0 Hz), 6.64–6.62 (m, 1H), 6.09–5.98 (m, 1H), 5.60–5.56 (m, 1H), 4.10–3.99 (m, 2H), 3.88–3.81 (m, 1H), 2.77–2.60 (m, 3H), 1.67–1.47 (m, 2H), 1.25–0.98 (m, 1H), 097–0.87(m, 5H); 13C NMR (126 MHz, CDCl3): δ = 186.39, 186.23, 185.68, 161.85, 140.68, 136.78, 136.68, 136.47, 136.07, 135.98, 133.40, 132.55, 129.51, 129.13, 125.28, 125.16, 117.36, 117.07, 57.08, 54.45, 54.21, 44.61, 44.51, 44.10, 33.83, 33.67, 33.41, 30.60, 21.69, 21.54; HRMS calcd C22H23N2O5 395.1607; found: 395.1620 (M + H)+; purity: 68% + 21%. 2-(3,6-Dioxocyclohexa-1,4-dien-1-yl)-4-oxo-1-phenylazetidin3-yl diethylcarbamate (mixture of cis/trans isomers) (9c).

Yield = 62%; brown solid, MP 114–118°C; IR (KBr): 3578, 3051, 2977, 2930, 1776, 1707, 1654, 1499, 1378, 1270, 1169, 1077, 932, 757, 686, 648, and 533 cm1; 1H NMR (400 MHz, CDCl3): δ = 7.36–7.29 (m, 4H), 7.18– 7.14 (m, 1H), 6.87 (d, 1H, J = 10.0 Hz), 6.77 (dd, 1H, J = 2.4 Hz), 6.65–6.64 (m, 1H), 6.04 (d, 1H, J = 5.2 Hz), 5.59 (d, 1H, J = 5.2 Hz), 3.24–3.16 (m, 3H), 3.07–3.04 (m, 1H), 1.05–0.96 (m, 7H); 13C NMR (126 MHz, CDCl3): δ = 186.19, 185.56, 161.80, 153.01, 140.71, 136.69,

Journal of Heterocyclic Chemistry

DOI 10.1002/jhet

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Anticancer Agents of β-Lactam Quinone Hybrids

136.51, 135.96, 133.49, 129.51, 125.27, 117.08, 54.33, 42.28, 41.46, 13.97, 13.15; HRMS calcd C20H21N2O5 369.1448; found: 369.1450 (M + H)+; purity: 99%. 1-(2-(3,6-Dioxocyclohexa-1,4-dien-1-yl)-4-oxo-1-phenylazetidin3-yl) 2-methyl pyrrolidine-1,2-dicarboxylate (mixture of cis/trans isomers) (9d). Yield = 71%; brown solid, MP 92–96°C;

IR (KBr): 3420, 3069, 2924, 2380, 1643, 1629, 1588, 1369, 1277, 1193, 1091, 1011, 949, 868, 773, 702, 636, 578, and 527 cm1; 1H NMR (400 MHz, CDCl3): δ = 7.35–7.27 (m, 5H), 7.21–7.13 (m, 1H), 6.90–6.78 (m, 2H), 6.68–6.58 (m, 1H), 6.49–6.48 (m, 1H), 6.11–5.97 (m, 1H), 5.60–5.49 (m, 1H), 4.34–4.11 (m, 1H), 3.67 (s, 3H), 3.57–3.43 (m, 3H), 2.18–1.80 (m, 5H); 13C NMR (126 MHz, CDCl3): δ = 186.57, 185.74, 171.95, 171.89, 161.39, 139.51, 136.73, 136.67, 136.56, 136.44, 136.0, 133.99, 129.48, 125.27, 125.19, 117.35, 117.11, 117.10, 117.02, 116.99, 59.23, 59.07, 58.46, 54.98, 54.15, 52.37, 52.33, 52.18, 47.20, 46.43, 30.75, 29.69, 29.57, 24.10, 24.01, 23.40, 23.01; HRMS calcd C22H21N2O7 425.1349; found: 425.1353 (M + H)+; purity: 78% + 20%. 2-(3,6-Dioxocyclohexa-1,4-dien-1-yl)-4-oxo-1-phenylazetidin3-yl morpholine-4-carboxylate (mixture of cis/trans isomers) Yield = 60%; brown solid, MP 146–150°C; IR (9e).

(KBr): 3418, 3086, 3053, 2962, 2855, 1780, 1711, 1656, 1598, 1151, 1498, 1377, 1240, 1110, 856, 759, 684, 637, 569, and 532 cm1; 1H NMR (400 MHz, CDCl3): δ = 7.37–7.29 (m, 5H), 7.18–7.13 (m, 1H), 6.90–6.86 (m, 2H), 6.82–6.79 (m, 1H), 6.78–6.62 (m, 1H), 6.10 (d, 1H, J = 5.2 Hz), 5.57–5.45 (m, 1H), 3.69–3.24 (m, 11H); 13C NMR (126 MHz, CDCl3): δ = 186.29, 186.11, 185.70, 185.60, 161.40, 153.08, 152.37, 141.97, 140.57, 136.77, 136.72, 136.63, 136.59, 135.94, 135.88, 133.55, 132.58, 129.55, 125.39, 125.28, 117.35, 117.05, 81.52, 66.44, 66.10, 56.96, 54.47, 44.27, 44.13; HRMS calcd C20H19N2O6 383.1243; found: 383.1253 (M + H)+; purity: 85% + 12%. 2-(3,6-Dioxocyclohexa-1,4-dien-1-yl)-4-oxo-1-phenylazetidin3-yl dipropylcarbamate (mixture of cis/trans isomers) (9f).

Yield = 62%; brown solid, MP 114–118°C; IR (KBr): 3423, 3060, 2963, 2926, 1768, 1718, 1657, 1501, 1376 1239, 1160, 1111, 1032, 854, 758, 667, 589, and 544 cm1; 1H NMR (400 MHz, CDCl3): δ = 7.36–7.28 (m, 7H), 7.16–7.13 (m, 1H), 6.78 (d, 1H, J = 10.4 Hz), 6.77 (d, 1H, J = 10.4 Hz), 6.64 (s, 1H), 6.05 (d, 1H, J = 5.6 Hz), 5.57 (d, 1H, J = 5.2 Hz), 3.19–2.92 (m, 5H), 1.48–1.32 (m, 7H), 0.86–0.72 (m, 8H); 13C NMR (126 MHz, CDCl3): δ = 186.18, 185.58, 161.85, 153.49, 140.71, 136.67, 136.50, 135.96, 133.49, 129.51, 125.25, 117.08, 117.05, 54.42, 49.61, 48.83, 21.78, 20.94, 11.17, 11.07; HRMS calcd C22H25N2O5 397.1763; found: 397.1769 (M + H)+; purity: 92% + 4%. 2-(3,6-Dioxocyclohexa-1,4-dien-1-yl)-4-oxo-1-phenylazetidin3-yl dimethyl carbamate (mixture of cis/trans isomers) (9g).

Yield = 64%; brown solid, MP 116–120°C; IR (KBr): 3420, 3404, 3058, 2927, 1776, 1714, 1657, 1498, 1378,

1174, 1105, 908, 855, 757, 689, 613, and 533 cm1; 1H NMR (400 MHz, CDCl3): δ = 7.36–7.29 (m, 5H), 7.17–7.14 (m, 1H), 6.87 (d, 1H, J = 10.0 Hz), 6.79 (d, 1H, J = 10.4 Hz), 6.65 (s, 1H), 6.09 (d, 1H, J = 5.6 Hz), 5.56 (d, 1H, J = 5.2 Hz), 2.84 (s, 3H), 2.76 (s, 3H); 13C NMR (126 MHz, CDCl3): δ = 186.39, 186.25, 185.69, 161.94, 152.55, 151.87, 142.23, 140.82, 136.76, 136.68, 136.58, 136.06, 136.01, 133.47, 132.42, 129.51, 129.21, 125.24, 125.14, 117.35, 117.04, 81.35, 57.03, 54.45, 46.55, 46.51, 46.11, 45.80, 25.65, 25.54, 24.83, 24.72; HRMS calcd C18H17N2O5 341.1137; found: 341.1133 (M + H)+; purity: 82% + 12%. 2-(3,6-Dioxocyclohexa-1,4-dien-1-yl)-4-oxo-1-phenylazetidin3-yl methoxy(methyl) carbamate (mixture of cis/trans isomers) Yield = 68%; brown solid, MP 108–112°C; IR (9h).

(KBr): 3423, 3058, 2930, 1777, 1717, 1656, 1598, 1501, 1375, 1310 1165, 1035, 960, 907, 836, 756, 686, and 612 cm1; 1H NMR (400 MHz, CDCl3): δ = 7.37–7.29 (m, 6H), 7.19–7.15 (m, 1H), 6.88 (d, 1H, J = 9.6 Hz), 6.81–6.77 (m, 1H), 6.66–6.65 (m, 1H), 6.15 (d, 1H, J = 5.6 Hz), 5.55 (d, 1H, J = 5.6 Hz), 3.54 (s, 3H), 3.06 (s, 3H); 13C NMR (126 MHz, CDCl3): δ = 186.15, 185.85, 161.07, 154.24, 140.49, 136.76, 136.73, 136.61, 136.6, 136.50, 135.94, 133.68, 133.48, 132.60, 129.56, 129.5, 125.38, 125.31, 125.25, 117.37, 117.05, 81.44, 61.63, 56.94, 54.60, 54.33, 42.27, 41.54, 35.38; HRMS calcd C18H17N2O6 357.1087; found: 357.1101 (M + H)+; purity: 89% + 9%. 2-(3,6-Dioxocyclohexa-1,4-dien-1-yl)-4-oxo-1-phenylazetidin3-yl piperidine-1-carboxylate (mixture of cis/trans isomers) Yield = 72%; brown solid, MP 136–140°C; IR (9i).

(KBr): 3411, 3053, 2932, 2856, 1780, 1709, 1595, 1500, 1379, 1230, 1143, 1027, 850, 753, 686, 628, 569, and 531 cm1; 1H NMR (400 MHz, CDCl3): δ = 7.37–7.29 (m, 4H), 7.17–7.12 (m, 1H), 6.89–6.85 (m, 1H), 6.80– 6.76 (m, 1H), 6.65–6.63 (m 1H), 6.03 (d, 1H, J = 5.2 Hz), 5.57 (d, 1H, J = 5.2 Hz), 3.48–3.22 (m, 5H), 1.41–1.25 (m, 5H); 13C NMR (126 MHz, CDCl3): δ = 186.39, 186.24, 185.68, 185.54, 161.84, 161.77, 153.11, 152.33, 142.23, 140.69, 136.77, 136.69, 136.46, 136.07, 135.97, 133.41, 132.53, 129.50, 129.23, 125.27, 125.14, 117.35, 117.06, 81.56, 57.07, 54.33, 45.26, 45.14, 44.95, 25.61, 25.43, 24.10, 23.97; HRMS calcd C21H21N2O5 381.1450; found: 381.1463 (M + H)+; purity: 71% + 25%. 2-(3,6-Dioxocyclohexa-1,4-dien-1-yl)-4-oxo-1-phenylazetidin3-yl pyrrolidine-1-carboxylate (mixture of cis/trans isomers) Yield = 74%; brown solid, MP 118–122°C; IR (9j).

(KBr): 3417, 3055, 2967, 2878, 1777, 1712, 1655, 1594, 1498, 1372, 1175, 1116, 906, 860, 754, 688, and 583 cm1; 1H NMR (400 MHz, CDCl3): δ = 7.37–7.29 (m, 5H), 7.17–7.14 (m, 1H), 6.87 (d, 1H, J = 10.4 Hz), 6.78 (d, 1H, J = 10.0 Hz), 6.65 (s, 1H), 6.12 (d, 1H, J = 5.6 Hz), 5.56 (d, 1H, J = 5.2 Hz), 3.45–3.25 (m, 4H), 3.02–2.96 (m, 1H), 1.91–1.76 (m, 5H); 13C NMR (126 MHz, CDCl3): δ = 186.20, 185.69, 161.78, 153.61, 140.75, 136.78, 136.72, 136.62, 135.99, 133.48, 132.51,

Journal of Heterocyclic Chemistry

DOI 10.1002/jhet

N. Payili, S. Yennam, S. R. Rekula, C. G. Naidu, Y. Bobde, and B. Ghosh 129.53, 125.29, 125.19, 117.38, 117.07, 81.67, 57.03, 54.48, 36.76, 35.81, 29.68; HRMS calcd C20H19N2O5 367.1294; found: 367.1291 (M + H)+; purity: 85% + 14%. 2-(3,6-Dioxocyclohexa-1,4-dien-1-yl)-4-oxo-1-phenylazetidin3-yl pyrrolidine-1-carboxylate (mixture of cis/trans isomers) Yield = 69%; brown solid, MP 90–94°C; IR (9k).

(KBr): 3427, 2962, 2891, 1772, 1723, 1595, 1498, 1373, 1120, 1038, 908, 827, 754, 687, 587, and 845 cm1; 1H NMR (400 MHz, CDCl3): δ = 7.36–7.28 (m, 6H), 7.17– 7.14 (m, 3H), 6.91–6.80 (m, 3H), 6.63–6.60 (m, 1H), 6.07 (d, 1H, J = 5.2 Hz), 5.53 (d, 1H, J = 5.2 Hz), 4.18– 3.72 (m, 8H), 2.29–2.17 (m, 3H); 13C NMR (126 MHz, CDCl3): δ = 186.34, 186.24, 185.72, 161.75, 161.66, 152.95, 142.09, 140.59, 136.71, 136.65, 136.58, 135.99, 133.61, 132.37, 129.52, 129.23, 125.27, 125.19, 117.36, 117.04, 80.98, 57.01, 54.50, 49.86, 49.22, 29.65, 15.83, 15.77; HRMS calcd C19H17N2O5 353.1137; found: 353.1138 (M + H)+; purity: 83% + 16%.

Acknowledgments. Authors are grateful to GVK Biosciences Pvt. Ltd. for the financial support and encouragement. Help from the analytical department for the analytical data is appreciated. We thank Dr. Sudhir Kumar Singh for his invaluable support and motivation.

REFERENCES AND NOTES [1] Baraldi, P. G.; Bovero, A.; Fruttarolo, F.; Preti, D.; Tabrizi, M. A.; Pavani, M. G.; Romagnoli, R. Med Res Rev 2004, 24, 475. [2] (a) Butler, J.; Hoey, B. M. Redox cycling drugs and DNA damage; In DNA and Free RadicalsHalliwell, B.; Aruoma, O. I. Eds.; Ellis Horwood: New York, 1993, pp. 243–273; (b) Holzer, H.; Glogner, P.; Sedlmayr, G. Biochem Z 1958, 330, 59; (c) Pratt, W. B.; Ruddon, R. W.; Ensminger, W. D.; Maybaum, J. The Anticancer Drugs; Oxford University Press: New York, 1994, pp. 17–18; (d) Smith, M. T. J Toxicol Environ Health, Part A 1985, 16, 665. [3] (a) Junior, E. N. S.; Moura, M. A. B. F.; Pinto, A. V.; Carmo, F. R.; Pinto, M.; Souza, M. C. B. V.; Araujo, A. J.; Pessoa, C.; Costa-Lotufo, L. V.; Montenegro, R. C.; de Moraes, M. O.; Ferreira, V. F.; Goulart, M. O. F. J Braz Chem Soc 2009, 20, 635; (b) Benchekroun, M. N.; Myers, C. E.; Sinha, B. K. Free Radic Biol Med 1994, 17, 191; (c) Huang, P.; Feng, L.; Oldham, E. A.; Keating, M. J.; Plunkett, W. Nature 2000, 407, 390; (d) Powis, G. Free Radic Biol Med 1989, 6, 63; (e) Moore, H. W.; Czerniak, R.; Hamdam, A. Drugs Exp Clin Res 1986, 12, 475; (f) Nohl, H.; Jordan, W.; Youngman, R. J. Adv Free Rad Biol Med 1986, 2, 211; (g) Schmitz, F. J.; Bloor, S. J. J Org Chem 1988, 53, 3922; (h) Alvi, K. A.; Rodriguez, J.; Diaz, M. C.; Moretti, R.; Wilhelm, R. S.; Lee, R. H.; Slate, D. L.; Crews, P. J Org Chem 1993, 58, 471. [4] (a) Gould, S. Chem Rev 1997, 97, 2499; (b) Liu, J. -K. Chem Rev 2006, 106, 2209; (c) Babula, P.; Mikelova, R.; Kizek, R.; Havel, L.; Sladky, Z. Ceska Slov Farm 2006, 55, 151; (d) Koyama, J. Recent Pat Antiinfect Drug Discov 2006, 1, 113; (e) Babula, P.; Adam, V.; Havel,

Vol 000

L.; Kizek, R. Ceska Slov Farm 2007, 56, 114; (f) Verma, R. P. Anti Cancer Agents Med Chem 2006, 6, 489; (g) Bishop, K. J. M.; Klajn, R.; Grzybowski, B. A. Angew Chem Int Ed 2006, 45, 5348. [5] (a) Miller, R. F.; Huang, S. J Antibiot 1995, 48, 520; (b) Zhang, B.; Salituro, G.; Szalkowski, D.; Li, Z.; Zhang, Y.; Royo, I.; Viella, D.; Diez, M. T.; Pelaez, F.; Ruby, C.; Kendall, R. L.; Mao, X.; Griffin, P.; Calaycay, J.; Zierath, J. R.; Heck, J. V.; Smith, R. G.; Möller, D. E. Science 1999, 284, 974; (c) Fotso, S.; Maskey, R. P.; Grün-Wollny, I.; Schulz, K.-P.; Munk, M.; Laatsch, H. J Antibiot 2003, 56, 931; (d) Coleman, R. S.; Felpin, F.-X.; Chen, W. J Org Chem 2004, 69, 7309; (e) Nikolovska-Coleska, Z.; Xu, L.; Hu, Z.; Tomita, Y.; Li, P.; Roller, P. P.; Wang, R.; Fang, X.; Guo, R.; Zhang, M.; Lippman, M. E.; Yang, D.; Wang, S. J Med Chem 2004, 47, 2430; (f) Viault, G.; Grée, D.; Das, S.; Yadav, J. S.; Grée, R. Eur J Org Chem 2011, 7, 1233. [6] Hoppen, S.; Emde, U.; Friedrich, T.; Grubert, L.; Koert, U. Angew Chem Int Ed 2000, 39, 2099. [7] (a) Schlitzer, M. Arch Pharm 2008, 341, 149; (b) Biamonte, M. A.; Wanner, J.; LeRoch, K. G. Bioorg Med Chem Lett 2013, 23, 2829; (c) Klein, E. Y. Int J Antimicrob Agents 2013, 41, 311; (d) Muregi, F. W.; Kirira, P. G.; Ishih, A. Curr Med Chem 2011, 18, 113; (e) Builders, M. I. Int J Pharm 2008, 70, 777. [8] Bisi, A.; Rampa, A.; Budriesi, R.; Gobbi, S.; Belluti, F.; Ioan, P.; Valoti, E.; Chiarini, A.; Valenti, P. Bioorg Med Chem 2003, 11, 1353. [9] Schellenberg, D.; Abdulla, S.; Roper, C. Curr Mol Med 2006, 6, 253. [10] (a) Walsh, J. J.; Bell, A. Curr Pharm Des 2009, 15, 2970; (b) Muregi, F. W.; Ishih, A. Drug Dev Res 2010, 71, 20; (c) Meunier, B. In Polypharmacology in Drug DiscoveryPeters, J.-U. Ed.; John Wiley & Sons, 2012, p. 423. [11] Alcaide, B.; Almendros, P.; Salgado, N. R. Tetrahedron Lett 2001, 42, 1503. [12] (a) Aitha, A.; Yennam, S.; Behera, M.; Jaya Shree, A. Tetrahedron Lett 2016, 57, 1507; (b) Jones, K.; Swapnaja, M.; Yennam, S.; Chavali, M.; Poornachandra, Y.; Kumar, C. G.; Muthusamy, K.; Jayaraman, V. B.; Arumugam, P.; Balasubramanian, S.; Sriram, K. K. D. Eur J Med Chem 2016, 117, 85; (c) Kumar, P. R.; Behera, M.; Raghavulu, K.; Jaya Shree, A.; Yennam, S. Tetrahedron Lett 2012, 53, 4108. [13] Palomo, C.; Aizpurua, J. M.; Ganboa, I.; Oiarbide, M. Eur J Org Chem 1999, 1999, 3223. [14] (a) Gilman, H.; Speeter, M. J Am Chem Soc 1943, 65, 2255; (b) Hart, D. J.; Ha, D. -C. Chem Rev 1989, 89, 1447; (c) Benaglia, M.; Cinquini, M.; Cozzi, F. Eur J Org Chem 2000, 2000, 563. [15] Toda, F.; Miyamoto, H.; Inoue, M.; Yasaka, S.; Matijasic, I. J Org Chem 2000, 65, 2728. [16] Ishibashi, H.; Kameoka, C.; Kodama, K.; Ikeda, M. Tetrahedron 1996, 52, 489. [17] Shi, J.; Linden, A.; Heimgartner, H. Helv Chim Acta 2013, 96, 1462. [18] Lynch, J. E.; Riseman, S. M.; Laswell, W. L.; Tschaen, D. M.; Volante, R. P.; Smith, G.; Shinkai, I. J Org Chem 1989, 54, 3792. [19] Georg, G. I.; Ravikumar, V. T. In The Organic Chemistry of βLactamsGeorg, G. I. Ed.; VCH publishers: New York, 1992, p. 295.

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Journal of Heterocyclic Chemistry

DOI 10.1002/jhet