Synthesis and antimicrobial activity of azepine and thiepine derivatives J. Serb. Chem. Soc. 80 (7) 839–852 (2015) UDC 547.71.8+547.639.5+546.98+ JSCS–4763 542.913:615.281/.282 Original scientific paper 839 Synthesis and antimicrobial activity of azepine and thiepine derivatives NINA BOŽINOVIĆ1#, IRENA NOVAKOVIĆ2#, SLAĐANA KOSTIĆ RAJAČIĆ2#, IGOR M. OPSENICA1*# and BOGDAN A. ŠOLAJA1**# 1Faculty of Chemistry, University of Belgrade, Studentski trg 16, P. O. Box 51, 11158, Belgrade, Serbia and 2Institute of Chemistry, Technology, and Metallurgy, University of Belgrade, Njegoševa 12, 11000 Belgrade, Serbia (Received 16 January, revised 2 February, accepted 6 February 2015) Abstract: A series of new pyridobenzazepine and pyridobenzothiepine deri- vatives was synthesized by Pd-catalyzed formation of C–N and C–S bonds. All synthesized compounds were tested for their in vitro antimicrobial activity. The pyridobenzazepine derivatives showed better antibacterial and antifungal activity than the corresponding dipyridoazepine analogue. Among the synthe- sized azepines, derivative 8 displayed potent activity against the tested bacteria (MIC ranged 39–78 µg mL-1), while azepine 12 showed promising antifungal activity (MIC ranged 156–313 µg mL-1). The synthesized thiepine derivatives exhibited weak antibacterial activity, but showed pronounced antifungal activity. Keywords: azepines; thiepines; heterocycles; palladium; antibacterials; anti- fungal. INTRODUCTION The tricyclic moieties of 5H-dibenz[b,f]azepine (1)1 and dibenzo[b,f]thiepine (2)2 are important heterocyclic pharmacophores in a number of drugs. Carbam- azepine (3) and opipramol (4) belong to the dibenzazepine group of heterocyclic compounds. Carbamazepine (3) is an anticonvulsant used to treat seizures, nerve pain and bipolar disorder,1a while opipramol (4) is a tricyclic antidepressant (TCA) and is used to treat generalized anxiety disorders.1b The dibenzothiepine zotepine (5) is an atypical antipsychotic, and it is used to treat schizophrenia (Fig. 1).3 Over the past few decades, several different strategies were developed for the synthesis of 5H-dibenz[b,f]azepines4 and dibenzo[b,f]thiepines.2,4e,5 The use of palladium-catalyzed reactions is an efficient procedure for the synthesis of 5H- *,** Corresponding authors. E-mails: (*)igorop@chem.bg.ac.rs; (**)bsolaja@chem.bg.ac.rs # Serbian Chemical Society member. doi: 10.2298/JSC150116013B _________________________________________________________________________________________________________________________ (CC) 2015 SCS. All rights reserved. Available on line at www.shd.org.rs/JSCS/ 840 BOŽINOVIĆ et al. -dibenz[b,f]azepines,6 and methods based on the double N-arylation reaction are of particular relevance.7 Hitherto, only one method employing a Pd-catalyzed reaction for the construction of the dibenzothiepine core has been reported.8 The Mizoroki–Heck cyclisation of the corresponding diaryl thioether was used for the synthesis of dibenzothiepine 2. Fig. 1. Tricyclic 5H-dibenz[b,f]azepines and diben- zo[b,f]thiepines. Recently, a simple and efficient Pd-catalyzed method was developed for the synthesis of 5H-pyrido[4,3-b:3',4'-f]benzazepine and 5H-dipyrido[4,3-b][1]aze- pine compounds (Scheme 1).9 Scheme 1. Pd-catalyzed synthesis of 5H-pyrido[4,3-b][1]benzazepine and 5H-dipyrido[4,3-b:3',4'-f]azepine compounds. The protocol is based on a Pd-catalyzed double amination reaction of the corresponding stilbenes. Additionally, as an expansion of the methodology, for the first time Pd-catalyzed formation of C–S bonds was applied to the ring closure of a thiepine derivatives from the corresponding stilbene precursors and an S-nucleophile (Scheme 1). Formerly, the synthesized azepines and thiepines are shown in Fig. 2. _________________________________________________________________________________________________________________________ (CC) 2015 SCS. All rights reserved. Available on line at www.shd.org.rs/JSCS/ SYNTHESIS AND ANTIMICROBIAL ACTIVITY OF AZEPINE AND THIEPINE DERIVATIVES 841 Herein, the synthesis of some new pyridobenzazepine and pyridobenzo- thiepine derivatives using the previously described methodology is presented. All synthesized compounds were evaluated for their in vitro antimicrobial activity against eight bacterial and three fungal pathogenic strains. Fig. 2. Structures of the synthesized azepine and thiepine derivatives. RESULTS AND DISCUSSION Chemistry The Wittig reaction between phosphonium salt 229 and aldehydes 1910 and 2111 provided the corresponding Z-stilbenes 24 and 25, respectively, in high yield. For the preparation of ethylene derivative 26, commercially available 2-bromo-5-fluorobenzaldehyde 23 was used (Scheme 2). Scheme 2. The synthesis of Z-stilbenes 24–26. _________________________________________________________________________________________________________________________ (CC) 2015 SCS. All rights reserved. Available on line at www.shd.org.rs/JSCS/ 842 BOŽINOVIĆ et al. The syntheses of new iminostilbene compounds 27–30 were achieved using the previously described reaction conditions for Pd-catalyzed double amination reactions (Scheme 3).9 Scheme 3. The synthesis of new pyridobenzazepine and dipyridoazepine compounds. The reactions of Z-stilbenes 6, 7 and 24–26 with potassium thioacetate (1.2 equiv.) in the presence of a catalyst composed from Pd2(dba)3 (5 mol %) and dppf (10 mol %) under microwave-mediated heating afforded the thiepine derivatives 16, 17 and 31–33 in moderate yields (Scheme 4). It should be noted that higher proportions of KSAc (2.4 eqiuv.) resulted in significantly better yields of 31 and 32, whereas the yield of thiepines 16 and 33 did not improve. In the case of stilbene 7, the higher load of KSAc resulted in a complex reaction mixture. Scheme 4. The synthesis of thiepine derivatives. _________________________________________________________________________________________________________________________ (CC) 2015 SCS. All rights reserved. Available on line at www.shd.org.rs/JSCS/ SYNTHESIS AND ANTIMICROBIAL ACTIVITY OF AZEPINE AND THIEPINE DERIVATIVES 843 In the next synthetic step, the thiepine compound 31 was chemically trans- formed into its tetrahydro and biphenyl derivatives 34 and 35, respectively. After N-methylation, and NaBH4 reduction, the tetrahydro derivative 34 was obtained in 66% yield. The Suzuki–Miyaura reaction on thiepine 31 with phenylboronic acid gave derivative 35 in moderate yield (Scheme 5). The coupling reaction was performed with the catalytic system Pd(OAc)2/SPhos–K3PO4 in toluene. These transformations of thiepine 31 opened up new possibilities for the preparation of structurally diverse substituted thiepines. Scheme 5. The transformations of thiepine compound 31. Antimicrobial activity The synthesized azepine derivatives were screened for their antibacterial and antifungal activities against five Gram-negative bacteria (Escherichia coli, Pro- teus hauseri, Pseudomonas aeruginosa, Salmonella enterica subsp. enterica serovar Enteritidis and Klebsiella pneumoniae), three Gram-positive bacteria (Staphylococcus aureus, Micrococcus luteus ATCC 10240 and M. luteus ATCC 4698) and three fungal strains (Candida albicans, Saccharomyces cerevisiae and Aspergillus brasiliensis). Amikacin (AMK) and chloramphenicol (CHL) were used as standard antibacterials, and nystatin (NYT) and fluconazole (FLC) were used as antifungal reference compounds. The minimum inhibitory concentration (MIC) was determined as the lowest concentration of the compound that resulted in inhibition of bacterial, respectively fungal growth, using a broth microdilution method. The results of antibacterial activities of azepine derivatives are given in Table I. The azepines 8–15 and 27–30 exhibited lower antibacterial activity with respect to amikacin (Table I). Compound 8 was more potent than chloram- phenicol against three Gram-negative bacteria (E. coli, P. hauseri and P. aeru- ginosa) and one Gram–positive bacteria (M. luteus ATCC 4698). All pyridobenzazepine derivatives (8, 10, 12, 14 and 27) showed higher inhibitory activity than the corresponding dipyridoazepine analogues (9, 11, 13, 15 and 28) against all bacteria. Azepine 8 with an N,N-dimethyl substituent was 4 times more potent than the corresponding N,N-diethyl substituted analogue 10. Additionally, 27, which incorporates the side chain nitrogen in pyrrolidine ring was more potent than 10, but less active than 8. The results of the antibacterial screening for compounds 8 and 14 revealed that the introduction of an oxygen _________________________________________________________________________________________________________________________ (CC) 2015 SCS. All rights reserved. Available on line at www.shd.org.rs/JSCS/ 844 BOŽINOVIĆ et al. atom in the side chain significantly decreased the antibacterial activity. The substituted azepines 29 and 30 showed lower antibacterial potency than 8 against all the screened bacteria. TABLE I. Antibacterial minimal inhibitory concentrations (MIC / µg mL-1) of the azepine derivatives Cmpd. Gram-negative bacteria Gram-positive bacteria E. coli P. hauseri P. aerugi- nosa S. enterica K. pneu- moniae S. aureus M. luteus ATCC 10240 M. luteus ATCC 4698 8 39 78 78 78 78 39 78 39 9 1250 1250 1250 1250 625 1250 1250 2500 10 156 313 313 625 625 156 313 313 11 1250 1250 1250 1250 1250 1250 1250 1250 12 39 313 313 313 313 39 156 78 13 1250 2500 1250 1250 1250 2500 1250 2500 14 313 625 625 625 625 313 625 313 15 1250 1250 1250 1250 1250 1250 1250 1250 27 78 156 313 313 313 156 156 156 28 625 625 625 625 625 625 625 625 29 78 156 313 313 313 156 313 156 30 156 313 313 313 625 313 313 313 AMK 5 7 50 8 8 11 2 2 CHL 62 125 250 43 62 15 31 125 Finally, among the synthesized azepines, derivative 8 was the most active one and showed a broad spectrum of antibacterial activity (MIC ranged 39–78 µg mL–1). The minimum inhibitory concentrations (MIC) of the synthesized azepines against three fungal strains are presented in Table II. Compounds 12 and 27 showed excellent activity (MIC = 156 µg mL–1) against C. albicans and S. cere- visiae; they were more potent than nystatin and fluconazole. Compounds 12 and 27 are 16 times more active than nystatin against C. albicans, and 8 times more potent against S. cerevisiae than nystatin. In addition, compound 8 was more potent than nystatin and fluconazole against S. cerevisiae, while derivative 29 was more active than the reference compounds against the C. albicans strain. Compounds 12 and 30 showed a four-fold greater potency (MIC = 313 µg mL–1) than nystatin in inhibiting the growth of the A. brasiliensis strain, but were less active when compared to fluconazole. Again, as with the antibacterial activity, it was observed that the pyridobenzazepine derivatives (8, 10, 12, 14 and 27) showed better antifungal activity than the corresponding dipyridoazepine analogues (9, 11, 13, 15 and 28). The synthesized thiepines were screened for their antibacterial and anti- fungal activities against four Gram-negative bacteria (E. coli, P. hauseri, P. aer- uginosa and Salmonella enterica subsp. enterica serovar Enteritidis), four Gram- _________________________________________________________________________________________________________________________ (CC) 2015 SCS. All rights reserved. Available on line at www.shd.org.rs/JSCS/ SYNTHESIS AND ANTIMICROBIAL ACTIVITY OF AZEPINE AND THIEPINE DERIVATIVES 845 -positive bacteria (Clostridium sporogenes, S. aureus, M. luteus ATCC 10240 and Kocuria rhizophila) and three fungal strains (C. albicans, S. cerevisiae and A. brasiliensis), using a disk diffusion method. Amikacin (AMK) was used as the standard antibacterial drug, and nystatin (NYT) was used as the antifungal reference compound. The antimicrobial activity was evaluated based on the diameter of the zone of inhibition. TABLE II. Antifungal minimal inhibitory concentrations (MIC / µg mL-1) of the azepine derivatives Cmpd. C. albicans S. cerevisiae A. brasiliensis 8 2500 156 1250 9 2500 1250 1250 10 625 625 625 11 1250 1250 1250 12 156 156 313 13 2500 1250 1250 14 625 313 625 15 1250 1250 1250 27 156 156 625 28 625 625 1250 29 156 313 625 30 313 313 313 NYT 2500 1250 1250 FLC 313 313 156 The results of antimicrobial activities of thiepine derivatives (Table III) revealed that all the tested thiepines displayed weak antibacterial activity with inhibition zones of 10–20 mm. TABLE III. Antibacterial activity expressed as inhibition diameter zones in millimetres (mm) of thiepine derivatives Cmpd.a Gram–negative bacteria Gram–positive bacteria E. coli P. hauseri P. aeru- ginosa S. enterica C. sporo- genes S. aureus M. luteus ATCC 10240 K. rhizophila 16 N.A.b N.A. N.A. N.A. N.A. N.A. N.A. N.A. 17 14 10 N.A. 10 16 N.A. N.A. N.A. 31 14 N.A. N.A. N.A. 20 N.A. N.A. 10 32 14 10 N.A. 10 14 N.A. N.A. N.A. 33 N.A. N.A. N.A. N.A. N.A. N.A. N.A. N.A. 34 10 14 N.A. N.A. 10 N.A. N.A. N.A. 35 N.A. N.A. N.A. N.A. N.A. N.A. N.A. N.A. AMKc 26 26 25 25 24 24 27 23 a Compounds concentration 1 mg disk-1; b N.A.: no activity (inhibition zone <10 mm); c AMK concentration 30 μg disk-1 _________________________________________________________________________________________________________________________ (CC) 2015 SCS. All rights reserved. Available on line at www.shd.org.rs/JSCS/ 846 BOŽINOVIĆ et al. On the other hand, with the exception of 31, all compounds exhibited pronounced antifungal activity against the three fungal strains (Table IV). The investigation of antifungal screening revealed that at a concentration of 1000 µg disk–1, compounds 16, 17, 32, 33, 34 and 35 were very potent, and showed complete growth inhibition against the C. albicans and S. cerevisiae strains. Among synthesized thiepines, compound 32 showed excellent antifungal activity particularly on the C. albicans strain with an inhibition zone of 50 mm at 250 µg disk–1, 28 mm at 125 µg disk–1 and 14 mm at 62.5 µg disk–1 concentrations. In addition, compound 32 at a concentration 125 µg disk–1 displayed moderate activity against the A. brasiliensis and S. cerevisiae strains (growth inhibition zones 12–18 mm). All the tested thiepine derivatives were completely inactive against the fungal strains at a concentration 31.3 µg disk–1. TABLE IV. Antifungal activity of the thiepine derivatives expressed as diameter of the inhibition zones in millimetres (mm) Cmpd. C. albicansa S. cerevisiaeb A. brasiliensisa Concentration, µg disk-1 1000 500 250 125 1000 500 250 125 1000 500 250 125 16 C.I.c 12 10 N.A.d C.I. 18 15 11 18 14 12 10 17 C.I. C.I. 30 N.A. C.I. 20 16 12 C.I. 16 12 10 31 N.A. N.A. N.A. N.A. N.A. N.A. N.A. N.A. N.A. N.A. N.A. N.A. 32 C.I. C.I. 50 28 C.I. 30 22 18 C.I. 30 20 12 33 C.I. 14 N.A. N.A. C.I. 22 16 12 12 10 N.A. N.A. 34 C.I. C.I. 10 N.A. C.I. 26 18 N.A. C.I. 10 N.A. N.A. 35 C.I. 14 N.A. N.A. C.I. 16 10 N.A. 12 N.A. N.A. N.A. aNystatin concentration 30 μg disk-1, 30 mm including disk; bnystatin concentration 30 μg disk-1, 54 mm including disk; ccomplete inhibition; dno activity (inhibition zone <10 mm) EXPERIMENTAL Instrumentation Microwave reactions were performed in a Biotage Initiator 2.5 microwave reactor. Melting points were determined using a Boetius PMHK apparatus (Carl Zeiss, Germany) and are not corrected. The IR spectra were recorded on a Perkin-Elmer spectrophotometer FTIR 1725X. The NMR spectra were recorded on a Bruker Ultrashield Advance III spectrometer (500 MHz) using TMS as the internal standard. The chemical shifts are expressed in ppm (δ) values and coupling constants (J) in Hz. The ESI–MS (HRMS) spectra were acquired on an Agilent Technologies 1200 Series instrument equipped with a Zorbax Eclipse Plus C18 column and a DAD detector in combination with a 6210 Time-of-Flight LC/MS instrument in the positive ion mode. The samples were dissolved in MeOH. GC/MS spectra were acquired on an Agilent Technologies 7890A instrument equipped with a DB-5 MS column and 5975C MSD and FID detector. Lobar LichroPrep Si 60 or LichroPrep RP-18 columns (Merck, Ger- many), coupled to a Waters RI 401 detector, were used for preparative column chromato- graphy. Thin-layer chromatography was performed on pre-coated Merck silica gel 60 F254 and Merck RP-18 F254 plates. The solution MeOH (NH3) stands for a combination MeOH/ /NH3 aq. = 9:1. The compounds were analyzed for purity using an Agilent 1200 HPLC system _________________________________________________________________________________________________________________________ (CC) 2015 SCS. All rights reserved. Available on line at www.shd.org.rs/JSCS/ SYNTHESIS AND ANTIMICROBIAL ACTIVITY OF AZEPINE AND THIEPINE DERIVATIVES 847 equipped with Quat pump (G1311B) and DAD detector 1260 VL (other details are presented in the Supplementary material to this paper). All compounds were >95 % pure. Chemistry (2-Bromo-5-chlorophenyl)methanediyl diacetate. A mixture of 1-bromo-4-chloro-2- -methylbenzene (1.0 g, 4.9 mmol), acetic anhydride (6.4 mL), acetic acid (5.0 mL) and concentrated sulphuric acid (1.5 mL) was cooled to 0 °C in an ice bath. Then an acetic acid (5.0 mL) solution of CrO3 (1.8 g, 18.0 mmol) was dropwisely added into the stirred mixture over 1.5 h. The mixture was stirred for the next 2 h at 0 °C. The product was filtered, washed with water (50 mL) and dried under reduced pressure. 2-Bromo-5-chlorobenzaldehyde (19). (2-Bromo-5-chlorophenyl)methanediyl diacetate (1.5 g, 4.7 mmol) was refluxed in MeOH–H2O (15 mL, 1/1 V/V) containing H2SO4 (1.6 mL) for 30 min. The reaction mixture was then diluted with H2O (15 mL) and extracted with EtOAc (3×20 mL). The combined organic layers were washed with H2O (20 mL) and brine (20 mL), dried over Na2SO4 and concentrated under reduced pressure. The residue and 1 M hydrochloric acid (4.0 mL) were heated for 3 h in THF (15 mL) under reflux. The solvent was removed under reduced pressure. The residue was purified by column chromatography (SiO2, hexane/EtOAc = 95/5). Yield: 0.84 g, 83 %. 2-Bromo-5-methoxybenzaldehyde (21). To the solution of meta-anisaldehyde (3.03 g, 22.3 mmol) in AcOH (5.0 mL), Br2 (1.4 mL, 26.8 mmol, 1.2 eq.) was drop wisely added, and the reaction mixture was stirred for 36 h at room temperature. Upon completion, the reaction was quenched with a saturated solution of Na2SO3 (25 mL), then poured into water (10 mL), and extracted with EtOAc (3×25 mL). The combined organic layers were washed with water (3×20 mL) and brine (15 mL), dried (Na2SO4), and concentrated to give the desired 2-bromo- -5-methoxybenzaldehyde (21, 3.99 g, 83 %). 3-[(Z)-2-(2-Bromo-5-chlorophenyl)ethenyl]-4-chloropyridine (24). To a suspension of phosphonium salt 22 (0.55 g, 1.2 mmol) in THF (12 mL) KOt-Bu (0.16 g, 1.4 mmol) was added. After 30 min, a solution of 2-bromo-5-chlorobenzaldehyde (0.26 g, 1.2 mmol) in THF (3 mL) was added over 5 min. The reaction mixture was stirred at room temperature for 16 h, when it was quenched with sat. aqueous soln. of NaHCO3. The aqueous phase was separated and extracted with EtOAc (3×25 mL). The organic extracts were combined, dried over Na2SO4, concentrated under vacuum and purified by column chromatography (SiO2, hex- ane/EtOAc = 95/5) to yield the Z-isomer (270 mg, 69 %). 3-[(Z)-2-(2-Bromo-5-methoxyphenyl)ethenyl]-4-chloropyridine (25). To a suspension of phosphonium salt 22 (0.14 g, 0.30 mmol) in THF (1.6 mL) was added KOt-Bu (40 mg, 0.36 mmol). After 30 min, a solution of 2-bromo-5-methoxybenzaldehyde (65 mg, 0.30 mmol) in THF (2 mL) was added over 5 min. The reaction mixture was stirred at room temperature and after 18 h, it was quenched with NaHCO3. The aqueous phase was separated and extracted with EtOAc (3×10 mL). The organic extracts were combined, dried over Na2SO4, concen- trated under vacuum and purified by preparative column chromatography (RP, MeOH/H2O = = 8:2) to yield the Z-isomer (60 mg, 82 %). 3-[(Z)-2-(2-Bromo-5-fluorophenyl)ethenyl]-4-chloropyridine (26). To a suspension of phosphonium salt 22 (0.45 g, 0.96 mmol) in THF (12 mL) was added KOt-Bu (0.13 g, 1.2 mmol). After 30 min, a solution of 2-bromo-5-fluorobenzaldehyde (0.19 g, 0.96 mmol) in THF (2 mL) was added over 5 min. The reaction mixture was stirred at room temperature and after 18 h, it was quenched with saturated aqueous solution of NaHCO3 (15 mL). The aqueous phase was separated and extracted with EtOAc (3×20 mL). The organic extracts were com- _________________________________________________________________________________________________________________________ (CC) 2015 SCS. All rights reserved. Available on line at www.shd.org.rs/JSCS/ 848 BOŽINOVIĆ et al. bined, dried over Na2SO4, concentrated under vacuum and purified by preparative column chromatography (RP, MeOH/H2O = 8:2) to yield compound 26 (196 mg, 65 %). General procedure for Pd-catalyzed amination A reaction tube containing a stirring bar was evacuated and backfilled with argon. The tube was then charged with Pd(OAc)2 (5 mol %), JohnPhos (10 mol %) and NaOt-Bu (2.8 eq.) and filled with argon. Toluene was added. After stirring at room temperature for 5 min, an aryl halide (1 eq.) and amine (3 eq.) were added, the tube was filled with argon and capped. Reaction mixture was heated to 100 °C and stirred at the same temperature. Products were purified by preparative column chromatography: SiO2, CH2Cl2/MeOH(NH3) = 9/1. 5-[3-(Pyrrolidin-1-yl)propyl]-5H-pyrido[4,3-b][1]benzazepine (27). Following the gen- eral procedure, a mixture of 3-[(Z)-2-(2-bromophenyl)ethenyl]-4-chloropyridine (24 mg, 0.080 mmol), 3-(pyrrolidin-1-yl)propan-1-amine (31 µL, 0.24 mmol), sodium tert-butoxide (22 mg, 0.23 mmol), Pd(OAc)2 (0.9 mg, 5 mol %), JohnPhos (2.4 mg, 10 mol %) and toluene (1.5 mL) was stirred at 100 °C for 48 h. Yield: 17 mg, 68 %. 5-[3-(Pyrrolidin-1-yl)propyl]-5H-dipyrido[4,3-b:3′,4′-f]azepine (28). Following the general procedure, a mixture of 3,3′-(Z)-ethene-1,2-diylbis(4-chloropyridine) (20 mg, 0.080 mmol), 3-(pyrrolidin-1-yl)propan-1-amine (31 µL, 0.24 mmol), sodium tert-butoxide (22 mg, 0.23 mmol), Pd(OAc)2 (0.9 mg, 5 mol %), JohnPhos (2.4 mg, 10 mol %) and toluene (1.5 mL) was stirred at 100 °C for 24 h. Yield: 17 mg, 69 %. 3-(8-Chloro-5H-pyrido[4,3-b][1]benzazepin-5-yl)-N,N-dimethylpropan-1-amine (29). Following the general procedure, a mixture of 3-[(Z)-2-(2-bromo-5-chlorophenyl)ethenyl]-4- -chloropyridine (26 mg, 0.080 mmol), 3-(dimethylamino)-1-propylamine (30 µL, 0.24 mmol), sodium tert-butoxide (22 mg, 0.23 mmol), Pd(OAc)2 (0.9 mg, 5 mol %), JohnPhos (2.4 mg, 10 mol %) and toluene (1.5 mL) was stirred at 100 °C for 48 h. Yield: 18 mg, 70 %. 3-(8-Methoxy-5H-pyrido[4,3-b][1]benzazepin-5-yl)-N,N-dimethylpropan-1-amine (30). Following the general procedure, a mixture of 3-[(Z)-2-(2-bromo-5-methoxyphenyl)ethenyl]- -4-chloropyridine (26 mg, 0.080 mmol), 3-(dimethylamino)-1-propylamine (30 µL, 0.24 mmol), sodium tert-butoxide (22 mg, 0.23 mmol), Pd(OAc)2 (0.9 mg, 5 mol %), JohnPhos (2.4 mg, 10 mol %) and toluene (1.5 mL) was stirred at 100 °C for 48 h. Yield: 10 mg, 40 %. General procedure for the synthesis of the thiepine derivatives A reaction tube containing a stirring bar was evacuated and backfilled with argon. The tube was charged with tris(dibenzylideneacetone)dipalladium (Pd2dba3, 5 mol %), dppf (10 mol %), NaOt-Bu (1.2 eq.), aryl halide (1 eq.) and KSCOCH3 (1.2 eq.) and evacuated and backfilled with argon. The flask was capped with a rubber septum, and toluene was added. The reaction mixture was heated in a Biotage initiator 2.5 microwave at 170 °C for 60 min. After completion of the reaction, the mixture was cooled to room temperature. The products were purified by column chromatography: SiO2, hexane/EtOAc = 8/2. [1]Benzothiepino[3,2-c]pyridine (16). Following the general procedure, a mixture of 3-[(Z)-2-(2-bromophenyl)ethenyl]-4-chloropyridine (35 mg, 0.12 mmol), KSCOCH3 (16 mg, 0.14 mmol), sodium tert-butoxide (14 mg, 0.14 mmol), Pd2dba3 (5.4 mg, 5 mol %), dppf (6.6 mg, 10 mol %) and toluene (1.5 mL) was heated in a Biotage Initiator 2.5 microwave at 170 °C for 60 min. Yield: 13 mg, 51 %. Pyrido[3′,4′:6,7]thiepino[3,2-c]pyridine (17). Following the general procedure, a mix- ture of 3,3′-(Z)-ethene-1,2-diylbis(4-chloropyridine) (30 mg, 0.12 mmol), KSCOCH3 (16 mg, 0.14 mmol), sodium tert-butoxide (14 mg, 0.14 mmol), Pd2dba3 (5.4 mg, 5 mol %), dppf (6.6 _________________________________________________________________________________________________________________________ (CC) 2015 SCS. All rights reserved. Available on line at www.shd.org.rs/JSCS/ SYNTHESIS AND ANTIMICROBIAL ACTIVITY OF AZEPINE AND THIEPINE DERIVATIVES 849 mg, 10 mol %) and toluene (1.5 mL) was heated in a Biotage initiator 2.5 microwave at 170 °C for 60 min. Yield: 15 mg, 60 %. 8-Chloro[1]benzothiepino[3,2-c]pyridine (31). Following the general procedure, a mix- ture of 3-[(Z)-2-(2-bromo-5-chlorophenyl)ethenyl]-4-chloropyridine (29 mg, 0.088 mmol), KSCOCH3 (12 mg, 0.11 mmol), sodium tert-butoxide (10 mg, 0.11 mmol), Pd2dba3 (4.0 mg, 5 mol %), dppf (4.9 mg, 10 mol %) and toluene (1.1 mL) was heated in a Biotage initiator 2.5 microwave at 170 °C for 60 min. Yield: 6.8 mg, 32 %. 8-Methoxy[1]benzothiepino[3,2-c]pyridine (32). Following the general procedure, a mixture of 3-[(Z)-2-(2-bromo-5-methoxyphenyl)ethenyl]-4-chloropyridine (60 mg, 0.18 mmol), KSCOCH3 (25 mg, 0.22 mmol), sodium tert-butoxide (21 mg, 0.22 mmol), Pd2dba3 (8.5 mg, 5 mol %), dppf (10 mg, 10 mol %) and toluene (2.3 mL) was heated in a Biotage initiator 2.5 microwave at 170 °C for 60 min. Yield: 8.4 mg, 19 %. 8-Fluoro[1]benzothiepino[3,2-c]pyridine (33). Following the general procedure, a mix- ture of 3-[(Z)-2-(2-bromo-5-fluorophenyl)ethenyl]-4-chloropyridine (30 mg, 0.096 mmol), KSCOCH3 (13 mg, 0.12 mmol), sodium tert-butoxide (11 mg, 0.12 mmol), Pd2dba3 (4.4 mg, 5 mol %), dppf (5.3 mg, 10 mol %) and toluene (1.2 mL) was heated in a Biotage Initiator 2.5 microwave at 170 °C for 60 min. Yield: 7.6 mg, 35 %. 8-Chloro-2-methyl-1,2,3,4-tetrahydro[1]benzothiepino[3,2-c]pyridine (34). A solution of 8-chloro[1]benzothiepino[3,2-c]pyridine (17 mg, 0.068 mmol) in MeCN (3 mL) was refluxed with an excess of methyl iodide (25 µL, 0.27 mmol, 4 eq.). After 2 h, the solvent was removed under reduced pressure. The resulting yellow solid was dissolved in dry methanol (3 mL) and NaBH4 (6.0 mg, 0.13 mmol) was added under an inert atmosphere at room tem- perature. After 15 min, the MeOH was removed under reduced pressure. The crude residue was dissolved in EtOAc and washed with H2O. The organic layer was dried over anhydrous Na2SO4, concentrated under vacuum and purified by column chromatography (SiO2, EtOAc/ /MeOH = 1:1) to yield the product (11.8 mg, 66 %). 8-Phenyl[1]benzothiepino[3,2-c]pyridine (35). A reaction tube containing a stirring bar was evacuated and backfilled with Ar. The tube was then charged with Pd(OAc)2 (0.9 mg, 5 mol %), SPhos (3.2 mg, 10 mol %), phenylboronic acid (12 mg, 0.095 mmol, 1.2 eq.) and anhydrous K3PO4 (34 mg, 0.16 mmol, 2.0 eq.). The tube was capped with a rubber septum and filled with argon. Dry toluene (1.0 mL) was added through the septum and the resulting mixture was stirred at room temperature for 2 min. 8-Chloro[1]benzothiepino[3,2-c]pyridine (20 mg, 0.079 mmol) was added and the tube was sealed. The reaction mixture was heated at 100 °C for 18 h. The reaction mixture was allowed to cool to room temperature. The product was purified by column chromatography (SiO2, hexane/EtOAc = 8/2). Yield: 11.5 mg, 50 %. Antimicrobial evaluation Microbroth dilution method. The antimicrobial activity was evaluated using a broth microdilution method according to NCCLS (National Committee for Clinical Laboratory Standards (2000) Approval standard document M7-A5, Villanova, PA, USA). The following Gram-negative bacterial strains used were: Escherichia coli (ATCC 25922), Proteus hauseri (ATCC 13315), Pseudomonas aeruginosa (ATCC 9027), Salmonella enterica subsp. enterica serovar Enteritidis (ATCC 13076) and Klebsiella pneumoniae (ATCC 10031). The Gram- -positive bacterial strains used were: Staphylococcus aureus (ATCC 6538), Micrococcus lut- eus (ATCC 10240) and M. luteus (ATCC 4698). The employed fungal species were: Candida albicans (ATCC 10231), Saccharomyces cerevisiae (ATCC 9763) and Aspergillus brasilien- sis (ATCC 16404). MIC determination was performed by a serial dilution method in sterile 96-well microtitre plates. Fresh Mueller–Hinton broth (for bacteria) and Sabouraud dextrose _________________________________________________________________________________________________________________________ (CC) 2015 SCS. All rights reserved. Available on line at www.shd.org.rs/JSCS/ 850 BOŽINOVIĆ et al. broth (for fungi) were used. Stock solutions of the compounds were prepared in dimethyl sulphoxide (DMSO), and then serial dilutions of the compounds were made in the concen- tration range from 10,000 to 4.9 µg mL-1. Amikacin (AMK) and chloramphenicol (CHL) were used as positive controls for the bacteria, while nystatin (NYT) and fluconazole (FLC) were used as positive controls for the fungi. The solvent (DMSO) served as negative control. In each well of the plate, ten microlitres of bacterial cultures (106 cells mL-1) for antibacterial activity and 10 mL of fungal cultures (105 spores mL-1) were inoculated. The microtiter plates were incubated at 37 °C for 24 h for the bacteria or at 28 °C for 48 h for the fungi. The MIC was determined as the lowest concentration that resulted in inhibition of bacterial or fungal growth. Disk diffusion method. Antimicrobial activity was evaluated using a disk diffusion method according to NCCLS (National Committee for Clinical Laboratory Standards (1997) Approval standard document M2-A6 Performance standards for antibacterial disk suscept- ibility test, Wayne, PA, USA). Antibacterial activity. The antibacterial activity was evaluated using four different strains of Gram-negative bacteria: E. coli (ATCC 25922), P. hauseri (ATCC 13315), P. aeruginosa (ATCC 9027) and S. enterica subsp. enterica serovar Enteritidis (ATCC 13076), and four different strains of the Gram-positive bacteria: Clostridium sporogenes (ATCC 19404), S. aureus (ATCC 6538), M. luteus (ATCC 10240) and Kocuria rhizophila (ATCC 9341). The determination of antibacterial activity was performed using the disk diffusion method. In each Petri dish (90 mm diameter), 22 mL of nutrient agar and 100 µL of bacterial suspension were added. The test substances were dissolved in CH2Cl2 (1 mg 100 µL -1) and then 100 µL of solution was applied to a filter paper disk (8 mm in diameter) and the solvent was evaporated. The loaded disks were placed on the surface of the medium and left for 30 min at room temperature for compound diffusion. Amikacin 30 µg per filter paper disk (8 mm in diameter) was used as the positive control, while the disks of the same diameter impregnated with 100 µL of CH2Cl2 were used as the negative control. The plates were incubated for 24 h at 37 °C. The zones of inhibition were recorded in millimetres. Antifungal activity. The antifungal activity was tested against three different strains: C. albicans (ATCC 10231), S. cerevisiae (ATCC 9763) and A. brasiliensis (ATCC 16404). Sab- ouraud dextrose agar was prepared according to the manufacture’s instruction. Into each sterile Petri dish (90 mm diameter), 22 mL of previously prepared agar suspension was poured and 100 µL of fungi was added. The test compounds were dissolved in CH2Cl2 and applied on filter paper disk (8 mm in diameter) at final concentrations 1000, 500, 250, 125, 62.5 and 31.3 µg/disk. Nystatin (30 µg disk-1) was used as a positive control while a disk impregnated with CH2Cl2 was used as the negative control. Petri dishes were incubated for 48 h at 28 °C. The zone of inhibition was measured in millimetres, including the disk. CONCLUSIONS New pyridobenzazepine and pyridobenzothiepine derivatives were syn- thesized using a methodology for Pd-catalyzed formation of C–N and C–S bonds.9 Additionally, the successful transformations of thiepine 31 to tetrahydro and biphenyl derivatives opened up new possibilities for the preparation of structurally diverse substituted derivatives. All newly and previously synthesized compounds were evaluated for their in vitro antimicrobial activity against eight bacterial and three fungal pathogenic strains. All pyridobenzazepine derivatives _________________________________________________________________________________________________________________________ (CC) 2015 SCS. All rights reserved. Available on line at www.shd.org.rs/JSCS/ SYNTHESIS AND ANTIMICROBIAL ACTIVITY OF AZEPINE AND THIEPINE DERIVATIVES 851 showed better antibacterial and antifungal activity than the corresponding dipyridoazepine analogues. Among the synthesized azepines, derivative 8 was the most active and showed a broad spectrum of antibacterial activity (MIC ranged 39–78 µg mL–1). The synthesized thiepine derivatives exhibited weak antibacterial activity but, on the other hand, with the exception of 31, all thie- pines showed pronounced antifungal activity. SUPPLEMENTARY MATERIAL Analytical and spectral data of the compounds, as well as copies of the corresponding 1H-NMR and 13C-NMR spectra of the products and HPLC purity chromatograms are available electronically from http://www.shd.org.rs/JSCS/, or from the corresponding author on request. Acknowledgments. This research was financially supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia (Grant No. 172008) and the Serbian Academy of Sciences and Arts. И З В О Д СИНТЕЗА И АНТИМИКРОБНА АКТИВНОСТ АЗЕПИНСКИХ И ТИЕПИНСКИХ ДЕРИВАТА НИНА БОЖИНОВИЋ1, ИРЕНА НОВАКОВИЋ2, СЛАЂАНА КОСТИЋ РАЈАЧИЋ2, ИГОР М. ОПСЕНИЦА1 и БОГДАН А. ШОЛАЈА1 1Хемијски факултет, Универзитет у Београду, Студентски трг 16, п. пр 51, 11158, Београд и 2Институт за хемију, технологију и металургију, Универзитет у Београду, Његошева 12, 11000 Београд Синтетисана је серија пиридобензазепинских и пиридобензотиепинских деривата формирањем C–N и C–S веза помоћу катализатора на бази Pd и испитана је њихова in vitro антимикробна активност. Синтетисани пиридобензазепински деривати показују већу антибактеријску и антифунгалну активност у поређењу са одговарајућим дипири- доазепинским дериватима. Азепин 8 показао је највећу антибактеријску активност (MIC у опсегу 39–78 μg mL-1), а азепин 12 показао се као најактивнији дериват према испи- таним сојевима гљива (MIC у опсегу 156–313 μg mL-1). Синтетисани тиепински деривати имају слабу антибактеријску активност, али са друге стране имају добру антифунгалну активност. (Примљено 16. јануара, ревидирано 2. фебруара, прихваћено 6. фебруара 2015) REFERENCES 1. a) B. LeDuc, in Foye's Principles of Medicinal Chemistry, 6th ed.; T. L. Lemke, D. A. Williams, Eds., Lippincott Williams & Wilkins, Philadelphia, PA, 2007, p. 521; b) K. C. Miles, Emergency medicine: a comprehensive study guide, 6th ed., J. E. Tintinalli, G. D. Kelen, J. S. Stapczynski, Eds., McGraw–Hill, New York, NY, 2004, p. 1025 2. M. Protiva, J. Heterocycl. Chem. 33 (1996) 497, and references cited therein 3. I. Ueda, Y. Sato, S. Maeno, S. Umio, Chem. Pharm. Bull. 26 (1978) 3058 4. a) L. J. Kricka, A. Ledwith, Chem. Rev. 74 (1974) 101, and references cited therein; b) A. 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