European Journal of Medicinal Chemistry 70 (2013) 419e426
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European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and antimicrobial activity of some novel hydrazide, benzochromenone, dihydropyridine, pyrrole, thiazole and thiophene derivatives Hala M. Refat a, *, A.A. Fadda b a b
Department of Chemistry, Faculty of Education, Suez Canal University, El-Gomhoria St., Al-Arish, Egypt Department of Chemistry, Faculty of Science, Mansoura University, ET-35516 Mansoura, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history: Received 18 May 2013 Received in revised form 29 August 2013 Accepted 1 September 2013 Available online 12 October 2013
As a part of ongoing studies in developing new potent antimicrobial agents, a novel synthesis of 2-cyanoN-(1-(3-oxo-3H-benzo[f]chromen-2-yl)ethylidene)acetohydrazide (3) has been reported. The latter compound was reacted with different reagents to give new heterocyclic compounds. The structures of the newly synthesized compounds were confirmed by elemental analysis, IR, 1H NMR, 13C NMR and mass spectral data. Representative compounds of the synthesized products were tested and evaluated as antimicrobial agents. Ó 2013 Elsevier Masson SAS. All rights reserved.
Keywords: Benzochromenone Acetohydrazide Hydrazideehydrazone Antimicrobial activities
1. Introduction Hydrazines and their derivatives constitute an important class of compounds that has found wide utility in organic synthesis [1,2]. Also, they constitute an important class of compounds for new drug development [3]. A number of hydrazideehydrazone derivatives have been claimed to possess interesting bioactivity such as antibacterial-antifungal [4e6], anticonvulsant [7,8], antiinflammatory [9,10], antimalarial [11] and antituberculosis activities [12,13]. Moreover, the coumarin nucleus is prevalent in numerous natural products and is extremely important in the chemistry of biological activities [14], which have found applications in treatment of antibacterial, antitumor, anti-inflammatory, antithrombotic, cardio protectors or enzymatic inhibitors antimicrobial and antifungal [15e19]. In view of the above-mentioned findings, and as a continuation of our effort to identify new candidates that may be valuable in designing new, potent, selective, and less toxic antimicrobial agents [20e25], we report herein the synthesis of a series of hydrazidee hydrazones together with their use in a series of heterocyclic
* Corresponding author. Tel: þ20 50 2343348, þ20 1061988989 (mobile). E-mail addresses: [email protected], [email protected] (H.M. Refat). 0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.ejmech.2013.09.003
transformations and their evaluation as antimicrobial agents. This combination was suggested to investigate the influence of such hybridization and structure variation on the anticipated biological activities, hoping to add some synergistic biological significance to the target molecules. 2. Results and discussion 2.1. Chemistry The synthetic strategies adopted for the synthesis of the intermediates and to target compounds are depicted in Schemes 1e3. In Scheme 1, synthesis of 2-cyano-N-(1-(3-oxo-3H-benzo[f]chromen-2-yl)ethylidene)acetohydrazide (3), in analogy with the reported literature [26] by the reaction of 2-acetyl-3H-benzo[f] chromen-3-one (1) [27] and 2-cyanoacetohydrazide (2). The assignment of structure 3 was supported by elemental analysis and spectral data. The IR spectrum displayed stretching vibration bands at 3193 and 2263 cm1 corresponding to NH and CN groups, in addition to the stretching vibration of two carbonyl groups at 1733 and 1679 cm1. Its 1H NMR spectrum (DMSO-d6) revealed the presence of singlet signals at d 2.28, 4.34 and 11.21 ppm assignable for CH3, CH2CN and NH protons. The mass spectrum showed the molecular ion peak at m/z ¼ 319 corresponding to the molecular formula (C18H13N3O3).
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O O
CH3 O
+
NC
NHNH 2
O
(1)
(2 )
EtOH
CH3
O CN
N N H A rC
O
HO
Ar
O (3)
Ar N 2 C =4 l -C H 3 -C 6H 4
CH3
O
O
O
CH3
DMFDMA
CN
N N H
O CN
N N H
Ar
O CH3
Ar
O CN
N N H
6, Ar= 4-Cl-C 6H4 7, Ar= 4-F-C 6H 4
O
O
8, Ar= 3-Pyridyl
H3C (5)
O
N
N N H
(4 )
CH3
Scheme 1. Synthetic route to hydrazideehydrazone.
Its 1H NMR spectrum (DMSO-d6) revealed the presence of signals at d 2.49 and 2.65 ppm assignable for two CH3 protons, also, two signals at d 10.25 and 11.68 ppm for two NH protons. The mass spectrum showed the molecular ion peak at m/z ¼ 437 corresponding to the molecular formula (C25H19N5O3). Also, treatment of compound 3 with dimethylformamide dimethylacetal (DMF-DMA) in dry xylene under reflux afforded 2cyano-3-(dimethylamino)-N-(1-(3-oxo-3H-benzo[f]chromen-2-yl)
Next, we studied the reactivity of the active methylene group present in compound 3 towards diazonium salt. Thus, the reaction of compound 3 with 4-methylbenzenediazonium chloride gave the hydrazone derivative 4. The assignment of structure 4 was supported by elemental analysis and spectral data. The IR spectrum displayed stretching vibration bands at 3388, 3193 and 2228 cm1 corresponding to two NH and CN groups, in addition to the stretching vibration of two carbonyl groups at 1734 and 1679 cm1.
CHO
CH3
OH
N N H
(3) O
CN Ar
O
O (9 )
X
X= COOEt
X= CN
CH3
O
CH3 CN
N N H O
O
O
Ar
O CN
N N H O
O N
EtOOC CN
C
Ar CN
- EtOH
CH3
O
O
O HO
CH3
CN
N N
Ar CN
12, Ar= 2,5-(OCH3)2-C 6H 3 13, Ar= 3-OH-C 6H 4
O CN
N N O
OH N 2
Ar CN
10, Ar= 2,5-(OCH3)2-C 6H 3 11, Ar= 4-F-C 6H 4
Scheme 2. Synthetic route to chromene and dihydropyridine.
O
H.M. Refat, A.A. Fadda / European Journal of Medicinal Chemistry 70 (2013) 419e426
421
CH3 TEA
CH3
O
N N H O
NH2 N Ph
O
OH N 2
NH2 (14)
PhNCS S
CH3
TEA, S
S
O
CN
N N
CN CN
O
S
O
N N H
(3)
O
(17)
O
S
NH2 CN
H 2N
(15) CH3 N N H O
O
NH2 S
O
CH3
PhCOCH2Br
S
N N
O O (18)
O CN
O Ph (16)
Scheme 3. Synthetic route to pyrrole, thiazole and thiophene.
ethylidene)acrylohydrazide (5). The assignment of structure 5 was supported by elemental analysis and spectral data. The IR spectrum displayed stretching vibration bands at 3194 and 2190 cm1 corresponding to NH and CN groups, in addition to the stretching vibration of two carbonyl groups at 1720 and 1679 cm1. Its 1H NMR spectrum (DMSO-d6) revealed the presence of signals at d 2.66, 3.19 and 3.23 ppm assignable for three CH3 protons, a singlet at d 8.23 ppm for vinylic proton, and a singlet at d 11.60 ppm for NH proton. The mass spectrum showed the molecular ion peak at m/ z ¼ 374 corresponding to the molecular formula (C21H18N4O3). Further elucidation for the structure of 3 was obtained through studying its chemical reactivity with some chemical reagents. Thus, the reaction of compound 3 with certain aromatic aldehydes gave the corresponding benzylidene derivatives 6e8. Analytical and spectral data for the later compounds were in agreement with the proposed structures. Generally, the IR spectra for these derivatives showed the presence of the NH group at around 3280 cm1, cyano group at around 2229 cm1 and two carbonyl groups at around 1735 and 1678 cm1. Whereas the 1H NMR spectra showed the absence of the active methylene protons and showed a singlet signals at d 2.66, 8.12 and 11.68 ppm assignable for CH3, vinylic and NH protons. In addition, the mass spectroscopic measurements of compounds 6, 7 and 8 showed the molecular ion peaks at m/z ¼ 441 (Mþ, 12.80%), 425 (Mþ, 22.2%) and 408 (Mþ, 17.9%), respectively, which are in agreement with their molecular formula. On the other hand, benzochromenone derivative 9 was obtained through reaction of 3 with 2-hydroxy-1-naphthaldehyde. The assignment of structure 9 was supported by elemental analysis and spectral data. Its IR spectrum displayed stretching vibration bands at 3245 cm1 corresponding to NH group, in addition to the stretching vibration of three carbonyl groups at 1734e1674 cm1. Its 1H NMR spectrum (DMSO-d6) revealed the presence of a singlet at d 2.66 ppm assignable for CH3 proton and a singlet at d 11.65 ppm for NH proton. The mass spectrum showed the molecular ion peak at m/z ¼ 474 corresponding to the molecular formula (C29H18N2O5). Next, we studied the reactivity of the hydrazideehydrazone derivative 3 towards cinnamonitrile derivatives. Thus, the reaction of compound 3 with either a-cyanocinnamonitrile or ethyl cinnamonitrile derivatives in DMF and catalytic amount of TEA afforded the corresponding pyridine derivatives 10e13, respectively (Scheme 2). The structures 10e13 were established by the correct elemental analysis and compatible spectroscopic data. In general,
the IR spectra of compounds 10 and 11 revealed absorption bands at 3440, 3352 and 2206 cm1 for NH2 and CN groups, in addition to the stretching vibration of two carbonyl groups at 1732 and 1626 cm1. Whereas the structures 12e13 revealed absorption bands at 3434 and 2218 cm1 for OH and CN groups, in addition to the stretching vibration of two carbonyl groups at 1735 and 1678 cm1. The 1H NMR spectra (DMSO-d6) of compound 10 displayed a singlet signals at d 2.66, 3.77 and 6.78 ppm assignable to CH3, two OCH3 and NH2 protons, respectively. Whereas the compound 12 displayed a singlet signals at d 2.66, 3.87 and 10.76 ppm assignable to CH3, two OCH3 and OH groups, respectively. In addition, the mass spectroscopic measurements of compounds 10e 13 showed the molecular ion peaks at m/z ¼ 531 (Mþ, 15.24%), 489 (Mþ, 15.5%), 532 (Mþ, 22.7%) and 488 (Mþ, 93.54%), respectively, which are in agreement with their molecular formulas. Also, condensation of 3 with malononitrile gave the corresponding 2-pyridinone derivative 14 (Scheme 3). The assignment of structure 14 was based on analytical and spectral data. Its IR spectrum displayed absorption bands at 3481, 3296, 2210, and 1724, 1643 cm1 corresponding to NH2, CN and two carbonyl groups, respectively. Its 1H NMR spectrum (DMSO-d6) revealed to signals at d 2.49, 4.81, 4.92 and 6.15 ppm assignable for the CH3, two NH2 and C5eH pyridinone protons, respectively. The mass spectrum showed the molecular ion peak at m/z ¼ 385 corresponding to the molecular formula (C21H15N5O3). The condensation of 3 with malononitrile and elemental sulfur in the presence of triethylamine gave the corresponding thiophene derivative 15. The assignment of structure 15 was supported by elemental analysis and spectral data. Its IR spectrum displayed absorption bands at 3418, 3327, 3212, 2197, 1713 and 1678 cm1 assignable to NH2, NH, CN and two carbonyl groups, respectively. Its 1 H NMR spectrum (DMSO-d6) revealed to signals at d 2.65, 6.98, 7.25 and 11.60 ppm assignable for the CH3, two NH2 and NH protons, respectively. The mass spectrum showed the molecular ion peak at m/z ¼ 417 corresponding to the molecular formula (C21H15N5O3S). Recently, we have reported the reaction of cyanoacetamide moiety with a-halocarbonyl compounds which represents a new, simple and efficient synthetic route for the synthesis pyrrole derivatives. Therefore, cyclocondensation of 3 with phenacyl bromide in DMF using triethylamine as a basic catalyst furnished the pyrrole derivative 16. The assignment of structure 16 was supported by
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elemental analysis and spectral data. Its IR spectrum displayed stretching vibration bands at 2227, 1734 and 1678 cm1 assignable to CN and two carbonyl groups. Its 1H NMR spectrum (DMSO-d6) revealed the presence of two doublet signals at d 4.54 and 5.65 ppm assignable for the two vicinal pyrroline protons H-3 and H-4, respectively, singlet at d 2.65 ppm for CH3 proton. The mass spectrum showed the molecular ion peak at m/z ¼ 419 corresponding to the molecular formula (C26H17N3O3). On the other hand, the Gewald reaction [28] of compound 3 with both elemental sulfur and phenyl isothiocyanate in warming DMF containing triethylamine as a basic catalyst led to functionalized thiazole derivative 17. The assignment of structure 17 was based on analytical and spectral data. Its IR spectrum displayed absorption bands at 3440, 3360 and 3220 cm1 assignable to NH2 and NH groups, in addition to the stretching vibration of two carbonyl groups at 1730 and 1658 cm1. Its 1H NMR spectrum (DMSO-d6) revealed the presence of signals at d 2.65, 6.82 and 11.65 ppm assignable for CH3, NH2 and NH protons. The mass spectrum showed the molecular ion peak at m/z ¼ 486 corresponding to the molecular formula (C25H18N4O3S2). Similarly, 2-amino-N-(1-(3-oxo-3H-benzo[f]chromen-2-yl)ethylidene)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carbohydrazide (18) could be achieved according to the method described by Gewald, by reaction of compound 3 with cyclohexanone and elemental sulfur. The assignment of structure 18 was based on analytical and spectral data. Thus, its IR spectrum displayed absorption bands at 3421, 3390 and 3264 cm1 assignable to NH2 and NH groups, in addition to the stretching vibration of two carbonyl groups at 1729 and 1655 cm1. Its 1H NMR spectrum (DMSO-d6) revealed the presence of signals at d 1.79e2.38 assignable for the benzothiophene, signals at d 2.65, 6.49 and 11.76 ppm assignable for CH3, NH2 and NH protons. The mass spectrum showed the molecular ion peak at m/z ¼ 431 corresponding to the molecular formula (C24H21N3O3S). 3. Pharmacology 3.1. Antimicrobial evaluation All the newly synthesized compounds 3e18 were initially evaluated for in vitro antibacterial activity against Gram-positive bacteria (Staphylococcus aureus) (MTCC-96) and Gram-negative bacteria (Escherichia coli) (MTCC-443) and fungal (Candida albicans) using conventional Broth dilution method [29]. Ampicillin and Clotrimazole were used as reference drugs. The results were recorded for each tested compound as the average diameter of inhibition zones (IZ) of bacterial or fungal growth around the discs in mm. The minimal inhibitory concentrations (MICs) for compounds that showed significant growth inhibition zones (>10 mm) were determined using two fold serial dilution method [30]. The inhibition zone diameters and MIC (mg/mL) values are recorded in Tables 1 and 2. It was observed that compounds 9 and 17 exhibited the highest activity against S. aureus and E. coli. When acetyl group in compound 1 was replaced by benzocoumarin ring (compound 9). The activity increased and displayed the highest activity at (MIC ¼ 62.5 mg/mL) against S. aureus and (MIC ¼ 93.7 mg/mL) against E. coli, while compounds 3, 4, 7, 8, 10, 11, 16 and 17 which contain electron withdrawing group or heterocyclic rings showed good activity (MIC ¼ 125 mg/mL) against S. aureus bacteria. On the other hand, compound 6 was equipotent to Ampicillin in inhibiting the growth of S. aureus (MIC ¼ 187.5 mg/mL). On the other hand, compounds 3, 4, 8, 10, 11, 13 and 14 exhibited equipotent to Ampicillin in inhibiting the growth of E. coli (MIC ¼ 125 mg/mL). Compounds 3e18 were also evaluated for their in vitro antifungal activity against C. albicans. All compounds exhibited a very weak
Table 1 Diameter of inhibition zone (mm) of the newly synthesized compounds. Compound no.
3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Ampicillin Clotrimazole
Diameter of inhibition zone (mm) bacteria Fungi Gram(þ) bacteria
Gram() bacteria
S. aureus
% Activity index
E. coli
% Activity index
C. albicans % Activity index
21 24 NA 23 22 22 27 23 21 13 12 8 NA 13 26 5 25 NA
84 96 NA 92 88 88 108 92 84 52 48 32 NA 52 104 20 100 NA
23 24 NA 17 20 23 28 23 21 20 24 23 NA 16 27 NA 27 NA
85.5 88.9 NA 63 74.1 85.2 104 85.2 77.8 74.1 88.9 85.2 NA 59.2 100 NA 100 NA
11 21 7 17 18 19 20 18 10 10 19 18 20 13 15 9 NA 30
36.7 70 23.3 56.7 60 63.3 66.7 60 33.3 33.3 63.3 60 66.7 43.3 50 30 NA 100
“NA”: no activity.
activity against C. albicans as compared to standard Clotrimazole (MIC ¼ 125e500 mg/mL). 3.2. Structure activity relationships (SAR’s) From the results of antimicrobial activity of the newly synthesized compounds 3e18, antimicrobial activity was considerably affected by electron withdrawing substituents and the incorporation of electron withdrawing groups is responsible for enhancing activity against the test microorganism. Compounds 9 and 17 which contain heterocyclic ring pyran and thiazole moieties with electron withdrawing properties were most potent against E. coli and S. aureus. Moreover, newly synthesized compounds have higher potency against Gram-negative bacteria than compared to Grampositive bacterial strains. In addition, the antibacterial activity showed that pyran and thiazole displayed higher activity than the other heterocyclic rings. On the other hand, antifungal activity Table 2 Minimal inhibitory concentration (MIC, mg/mL) of the newly synthesized compounds. Compound no.
3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Ampicillin Clotrimazole “NA”: no activity.
MIC, in mg/mL
Fungi
Gram(þ) bacteria
Gram() bacteria
S. aureus
E. coli
C. albicans
125 125 NA 187.5 125 125 62.5 125 125 250 250 375 NA 125 125 375 187.5 NA
125 125 NA 250 250 125 93.7 125 125 187.5 125 125 NA 250 93.7 NA 125 NA
250 125 750 125 125 125 187.5 125 375 250 187.5 125 125 250 125 500 NA 5.8
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displayed conflicting results where in compounds having electron withdrawing substituents revealed weak inhibition. 4. Conclusion Our attempts at exploring benzochromenone based pyran, pyridine, pyrrole, thiazole and thiophene derivatives have unexpectedly led to identification of a novel chemo type with substantial antimicrobial activity. Among the newly synthesized compounds 3e18, analogs 9 and 17 showed highest inhibition against nearly all of the tested bacteria, while all compounds were inactive against fungal strains. Results of antimicrobial activity clearly demonstrated that the presence of electron withdrawing groups/atoms attached to the benzochromenone ring is essential for enhancing antimicrobial activity. On the basis of structure activity relationship, electron withdrawing substituents (pyran and thiazole rings) are beneficial for antibacterial activity and inactive for antifungal activity. From the activity data, compound 9 and 17 showed highest antibacterial inhibition. Thus suggesting that the compounds from the present series with electron withdrawing groups can serve as important gateways for the design and development of new antimicrobial agents with potent activity and minimal toxicity. 5. Experimental Melting points were measured with a Gallenkamp apparatus are uncorrected. IR spectra were recorded KBr disc on a Mattson 5000 FTIR spectrophotometer at Microanalytical Unit, Faculty of Science, Mansoura University. The 1H NMR and 13C NMR spectra were measured on Bruker WP AC 300 (300 MHz) in DMSO-d6 as solvent, using tetramethylsilane (TMS) as an internal standard, and chemical shifts are expressed as dppm. Mass spectra were determined on Finnigan Incos 500 (70 eV). Elemental analyses were carried out at the Microanalytical Centre, Faculty of Science, Cairo University. 5.1. Synthesis of 2-cyano-N-(1-(3-oxo-3H-benzo[f]chromen-2-yl) ethylidene)acetohydrazide (3) To a solution of 2-cyanoacetohydrazide 2 (1.0 g, 0.01 mol) in ethanol (25 mL), 2-acetyl-3H-benzo[f]chromen-3-one 1 (2.38 g, 0.01 mol) was added. The reaction mixture was heated under reflux for 3 h, the obtained solid was filtered off while hot and recrystallized from a mixture of ethanol and DMF (2:1) to give compound 3. Pale yellow needle crystals; yield (94%); m.p. 276e278 C; IR (KBr): n/cm1 ¼ 3193 (NH), 2263 (CN), 1733, 1679 (2C]O); 1H NMR (300 MHz, DMSO-d6) d (ppm): 2.28 (s, 3H, CH3), 4.34 (s, 2H, CH2CN), 7.60e8.68 (m, 7H, AreH), 11.21 (s, 1H, NH, D2O exchangeable); 13C NMR (75 MHz, DMSO-d6) d (ppm): 23.3, 29.4, 112.3, 116.5, 117.2 (2C), 121.5, 123.4, 126.5, 129.4 (2C), 130.2 (2C), 136.1, 145.5, 155.8, 160.2, 172.3; MS (EI, 70 eV) m/z (%) ¼ 319 (Mþ, 54.4), 279 (88.9), 236 (58.9), 223 (73.3), 195 (36.7), 165 (100.0). Anal. Calcd. for C18H13N3O3 (319): C, 67.71; H, 4.10; N, 13.16%. Found: C, 67.66; H, 4.02; N, 13.11%. 5.2. Synthesis of 2-oxo-2-(2-(1-(3-oxo-3H-benzo[f]chromen-2-yl) ethylidene)hydrazinyl)-N-p-tolylacetohydrazonoyl cyanide (4) To a cold (0e5 C) solution of compound 3 (3.19 g, 0.01 mol) in pyridine (25 mL) was added the 4-methyl-benzenediazonium chloride [which was prepared by dissolving sodium nitrite (0.68 g, 0.01 mol) in water (2 mL) and adding to a cold solution of p-toluidine (1.07 g, 0.01 mol) containing the appropriate amount of hydrochloric acid with continuous stirring] portion wise over the
423
reaction mixture was kept in an ice box overnight and then diluted with water. The solid that precipitated was filtered off, washed with water and dried, and recrystallized from a mixture of ethanol and DMF (2:1) to give compound 4. Orange red crystals; yield (85%); m.p. 250e252 C; IR (KBr): n/ 1 cm ¼ 3388, 3193 (2NH), 2228 (CN), 1734, 1679 (2C]O); 1H NMR (300 MHz, DMSO-d6) d (ppm): 2.49, 2.65 (s, 6H, 2CH3), 7.54e8.62 (m, 11H, AreH), 10.25, 11.68 (s, 2H, 2NH, D2O exchangeable); 13C NMR (75 MHz, DMSO-d6) d (ppm): 21.3, 23.5, 106.8, 112.5, 115.5, 116.4 (4C), 121.5, 123.5, 126.6, 128.5 (2C), 129.2 (2C), 130.2 (3C), 136.2, 145 (2C), 155.6, 160.2, 172; MS (EI, 70 eV) m/z (%) ¼ 437 (Mþ, 16.0), 289 (12.8), 238 (6.4), 221 (10.6), 195 (9.6), 167 (8.5), 139 (14.9). Anal. Calcd. for C25H19N5O3 (437): C, 68.64; H, 4.38; N, 16.01%. Found: C, 68.61; H, 4.32; N, 15.98%. 5.3. Synthesis of 2-cyano-3-(dimethylamino)-N-(1-(3-oxo-3Hbenzo[f]chromen-2-yl)ethylidene)acrylohydrazide (5) To a solution of compound 3 (3.19 g, 0.01 mol) in dry xylene (25 mL), N,N-dimethylformamide-dimethylacetal (DMF-DMA) (1.32 mL, 0.01 mol) was added. The reaction mixture was heated under reflux for 3 h. After cooling, the precipitated product that formed was collected by filtration and recrystallized from a mixture of ethanol and DMF (2:1) to give compound 5. Orange crystals; yield (62%); m.p. 210e212 C; IR (KBr): n/ cm1 ¼ 3194 (NH), 2190 (CN), 1720, 1679 (2C]O); 1H NMR (300 MHz, DMSO-d6) d (ppm): 2.66 (s, 3H, CH3), 3.19 (s, 3H, NCH3), 3.23 (s, 3H, NCH3), 7.42e8.63 (m, 7H, AreH), 8.23 (s, 1H, CH]N), 11.60 (s, 1H, NH, D2O exchangeable); 13C NMR (75 MHz, DMSO-d6) d (ppm): 23.5, 42 (2C), 106.1, 112.3, 115.5, 116.6 (2C), 122.5, 123.5, 126.5, 129.1 (2C), 130 (2C), 136, 144.4, 156 (2C), 160.2, 172; MS (EI, 70 eV) m/z (%) ¼ 374 (Mþ, 23.2), 330 (23.2), 317 (42.7), 262 (65.9), 235 (100.0), 205 (30.5), 163 (28.0). Anal. Calcd. for C21H18N4O3 (374): C, 67.37; H, 4.85; N, 14.96%. Found: C, 67.30; H, 4.84; N, 14.89%. 5.4. General procedure for the synthesis of 3-(4-substituted)-2cyano-N-(1-(3-oxo-3H-benzo[f]chromen-2-yl)ethylidene) acrylohydrazide derivatives (6e8) and 3-oxo-N-(1-(3-oxo-3Hbenzo[f]chromen-2-yl)ethylidene)-3H-benzo[f]chromene-2carbohydrazide (9) Equimolecular mixture of 3 (3.19 g, 0.01 mol) and selected aldehyde (0.01 mol), in DMF (20 mL) containing piperidine (0.5 mL) was heated under reflux for 3 h. The reaction mixture was left to cool then poured onto ice/water containing few drops of hydrochloric acid and the formed solid product was collected by filtration and recrystallized from ethanol. 5.4.1. Synthesis of 3-(4-chlorophenyl)-2-cyano-N-(1-(3-oxo-3Hbenzo[f]chromen-2-yl)ethylidene)acrylohydrazide (6) Yellow powder; yield (83.7%); m.p. 184e185 C; IR (KBr): n/ cm1 ¼ 3280 (NH), 2229 (CN), 1735, 1678 (2C]O); 1H NMR (300 MHz, DMSO-d6) d (ppm): 2.66 (s, 3H, CH3), 7.41e8.68 (m, 11H, AreH), 8.12 (s, 1H, vinyliceH), 11.68 (s, 1H, NH, D2O exchangeable); 13 C NMR (75 MHz, DMSO-d6) d (ppm): 23.6, 107.1, 112.3, 115.5, 116.5, 117.6, 121.1, 123.4, 126.6, 128.4 (4C), 130.2 (3C), 132.2 (3C), 136.4, 150 (2C), 156.2, 160.2, 172.3; MS (EI, 70 eV) m/z (%) ¼ 441 (Mþ, 12.8), 312 (10.6), 275 (12.8), 242 (12.8), 221 (27.7), 193 (25.5), 168 (27.7), 139 (42.6). Anal. Calcd. for C25H16ClN3O3 (441): C, 67.95; H, 3.65; N, 9.51%. Found: C, 67.91; H 3.60; N 9.46%. 5.4.2. Synthesis of 2-cyano-3-(4-fluorophenyl)-N-(1-(3-oxo-3Hbenzo[f]chromen-2-yl)ethylidene)acrylohydrazide (7) Yellowish brown powder; yield (85.5%); m.p. 215e217 C; IR (KBr): n/cm1 ¼ 3280 (NH), 2229 (CN), 1735, 1678 (2C]O); 1H NMR
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(300 MHz, DMSO-d6) d (ppm): 2.66 (s, 3H, CH3), 7.45e8.66 (m, 11H, AreH), 8.14 (s, 1H, vinyliceH), 11.66 (s, 1H, NH, D2O exchangeable); 13 C NMR (75 MHz, DMSO-d6) d (ppm): 23.4, 107, 112, 115.5 (4C), 117.4, 121, 123, 126, 127.5, 128 (2C), 130 (4C), 136, 150 (2C), 155.2, 160, 162, 172; MS (EI, 70 eV) m/z (%) ¼ 425 (Mþ, 22.2), 383 (30.1), 365 (27.5), 340 (30.1), 266 (30.7), 241 (31.6), 184 (20.8). Anal. Calcd. for C25H16FN3O3 (425): C, 70.58; H, 3.79; N, 9.88%. Found: C, 70.51; H 3.74; N 9.85%. 5.4.3. Synthesis of 2-cyano-N-(1-(3-oxo-3H-benzo[f]chromen-2-yl) ethylidene)-3-(pyridin-3-yl)acrylohydrazide (8) Yellow powder; yield (78.6%); m.p. 198e200 C; IR (KBr): n/ cm1 ¼ 3280 (NH), 2229 (CN), 1735, 1678 (2C]O); 1H NMR (300 MHz, DMSO-d6) d (ppm): 2.66 (s, 3H, CH3), 7.44e8.66 (m, 11H, AreH), 8.15 (s, 1H, vinyliceH), 11.69 (s, 1H, NH, D2O exchangeable); 13 C NMR (75 MHz, DMSO-d6) d (ppm): 23, 107, 112.5, 115.8 (2C), 117.7, 121, 123 (2C), 126.6, 128.5 (2C), 130 (2C), 132.5 (2C), 136, 145, 148 (2C), 153, 155, 160.2, 172; MS (EI, 70 eV) m/z (%) ¼ 408 (Mþ, 17.9), 283 (15.4), 221 (100.0), 195 (51.3), 139 (87.2). Anal. Calcd. for C24H16N4O3 (408): C, 70.58; H, 3.95; N, 13.72%. Found: C, 70.50; H 3.98; N 13.67%. 5.4.4. Synthesis of 3-oxo-N-(1-(3-oxo-3H-benzo[f]chromen-2-yl) ethylidene)-3H-benzo[f]chromene-2-carbohydrazide (9) Yellowish brown powder; yield (71.5%); m.p. 194e196 C; IR (KBr): n/cm1 ¼ 3245 (NH), 1734, 1713, 1674 (3C]O); 1H NMR (300 MHz, DMSO-d6) d (ppm): 2.66 (s, 3H, CH3), 7.61e8.66 (m, 14H, AreH), 11.65 (s, 1H, NH, D2O exchangeable); 13C NMR (75 MHz, DMSO-d6) d (ppm): 23.6, 112.5 (2C), 115.5 (2C), 117.7 (2C), 121 (2C), 123.2 (2C), 126.8 (2C), 128 (4C), 130 (4C), 136, 143, 145 (2C), 155.3, 160 (2C), 172; MS (EI, 70 eV) m/z (%) ¼ 474 (Mþ, 66.39), 433 (42.62), 275 (47.54), 268 (54.0), 239 (77.87), 225 (45.08), 211 (64.75), 170 (46.72), 153 (60.66), 141 (77.05). Anal. Calcd. for C29H18N2O5 (474): C, 73.41; H, 3.82; N, 5.90%. Found: C, 73.31; H 3.74; N 5.85%. 5.5. General procedure for the synthesis of 6-amino-4-(substituted phenyl)-2-oxo-1-(1-(3-oxo-3H-benzo[f]chromen-2-yl) ethylideneamino)-1,2-dihydro-pyridine-3,5-dicarbonitrile derivatives (10e11) and 4-(substituted phenyl)-6-hydroxy-2-oxo-1(1-(3-oxo-3H-benzo[f]chromen-2-yl)ethylideneamino)-1,2dihydropyridine-3,5-dicarbonitrile derivatives (12e13) To a solution of compound 3 (3.19 g, 0.01 mol) in DMF (20 mL) containing triethylamine (0.5 mL), either a-cyanocinnamonitrile or ethyl cinnamonitrile (0.01 mol) was added. The reaction mixture, in each case was heated under reflux for 3 h. The reaction mixture was left to cool then poured onto ice/water containing few drops of hydrochloric acid and the formed solid product was collected by filtration and recrystallized from ethanol. 5.5.1. Synthesis of 6-amino-4-(2,5-dimethoxyphenyl)-2-oxo-1-(1(3-oxo-3H-benzo[f]chromen-2-yl)ethylideneamino)-1,2dihydropyridine-3,5-dicarbonitrile (10) Yellow powder; yield (86.7%); m.p. 130e132 C; IR (KBr): n/ cm1 ¼ 3440, 3352 (NH2), 2206 (CN), 1732, 1626 (2C]O); 1H NMR (300 MHz, DMSO-d6) d (ppm): 2.66 (s, 3H, CH3), 3.77 (s, 6H, 2OCH3), 6.78 (s, 2H, NH2, D2O exchangeable), 7.21e8.67 (m, 10H, AreH); 13C NMR (75 MHz, DMSO-d6) d (ppm): 23.4, 55.8, 56.3, 77, 112 (2C), 114.5, 115.3 (2C), 115.8 (2C), 116.3 (2C), 121 (2C), 123.4, 126.6, 128.5 (2C), 130.6 (2C), 136.2, 150 (2C), 153, 155.7 (2C), 160, 166, 172; MS (EI, 70 eV) m/z (%) ¼ 531 (Mþ, 15.2), 487 (14.1), 469 (23.2), 443 (37.2), 296 (100), 256 (46.9), 237 (17.7), 209 (34.2), 145 (65.8). Anal. Calcd. for C30H21N5O5 (531): C, 67.79; H, 3.98; N, 13.18%. Found: C, 67.70; H 3.94; N 13.15%.
5.5.2. Synthesis of 6-amino-4-(4-fluorophenyl)-2-oxo-1-(1-(3-oxo3H-benzo[f]chromen-2-yl)ethylideneamino)-1,2-dihydropyridine3,5-dicarbonitrile (11) Yellow powder; yield (82.7%); m.p. 205e206 C; IR (KBr): n/ cm1 ¼ 3440, 3357 (NH2), 2208 (CN), 1733, 1626 (2C]O); 1H NMR (300 MHz, DMSO-d6) d (ppm): 2.66 (s, 3H, CH3), 6.51 (s, 2H, NH2, D2O exchangeable), 7.15e8.65 (m, 11H, AreH); 13C NMR (75 MHz, DMSO-d6) d (ppm): 23.2, 77, 112.2, 115.3 (3C), 115.6 (2C), 116 (2C), 121.3, 123.2, 126.8, 128 (3C), 128.5 (2C), 130 (2C), 136.2, 150.3, 155.5 (2C), 160 (2C), 168, 172; MS (EI, 70 eV) m/z (%) ¼ 489 (Mþ, 15.5), 403 (19.3), 287 (25.2), 238 (16.8), 221 (100.0), 193 (73.1), 164 (46.2), 139 (38.7). Anal. Calcd. for C28H16FN5O3 (489): C, 68.71; H, 3.29; N, 14.31%. Found: C, 68.68; H 3.24; N 14.27%. 5.5.3. Synthesis of 4-(2,5-dimethoxyphenyl)-6-hydroxy-2-oxo-1(1-(3-oxo-3H-benzo[f]chromen-2-yl)ethylideneamino)-1,2dihydropyridine-3,5-dicarbonitrile (12) Yellow powder; yield (82%); m.p. 200e202 C; IR (KBr): n/ 1 cm ¼ 3436 (OH), 2227 (CN), 1735, 1678 (2C]O); 1H NMR (300 MHz, DMSO-d6) d (ppm): 2.66 (s, 3H, CH3), 3.87 (s, 6H, 2OCH3), 7.41e8.68 (m, 10H, AreH), 10.76 (s, 1H, OH, D2O exchangeable); 13C NMR (75 MHz, DMSO-d6) d (ppm): 23.4, 55.8, 56.8, 63, 112 (2C), 114.5, 115.3 (2C), 115.8 (2C), 116.5 (2C), 121 (2C), 123, 126, 128 (2C), 130 (2C), 136.5, 150 (2C), 153, 155, 160, 166, 172, 188; MS (EI, 70 eV) m/z (%) ¼ 532 (Mþ, 22.7), 302 (27.3), 215 (45.5), 182 (22.7), 175 (27.3), 139 (45.5). Anal. Calcd. for C30H20N4O6 (532): C, 67.67; H, 3.79; N, 10.52%. Found: C, 67.63; H 3.71; N 10.46%. 5.5.4. Synthesis of 6-hydroxy-4-(3-hydroxyphenyl)-2-oxo-1-(1-(3oxo-3H-benzo[f]chromen-2-yl)ethylideneamino)-1,2dihydropyridine-3,5-dicarbonitrile (13) Yellow powder; yield (86%); m.p. 216e218 C; IR (KBr): n/ cm1 ¼ 3500e3434 (2OH), 2218 (CN), 1723, 1676 (2C]O); 1H NMR (300 MHz, DMSO-d6) d (ppm): 2.66 (s, 3H, CH3), 7.64e8.66 (m, 11H, AreH), 9.80, 10,76 (2s, 2H, 2OH, D2O exchangeable); 13C NMR (75 MHz, DMSO-d6) d (ppm): 23.2, 58, 112 (2C), 115.3 (2C), 115.6 (2C), 116 (2C), 121 (2C), 123, 126.2, 128.3 (2C), 130.2 (3C), 134, 136, 150, 155, 160 (2C), 166, 172, 188; MS (EI, 70 eV) m/z (%) ¼ 488 (Mþ, 39.54), 443 (20.26), 395 (16.99), 326 (36.27), 278 (25.49), 221 (100), 167 (24.18). Anal. Calcd. for C28H16N4O5 (488): C, 68.85; H, 3.30; N, 11.47%. Found: C, 68.83; H 3.31; N 11.45%. 5.6. Synthesis of 4,6-diamino-2-oxo-1-(1-(3-oxo-3H-benzo[f] chromen-2-yl)ethylideneamino)-1,2-dihydropyridine-3-carbonitrile (14) A mixture of compound 3 (3.19 g, 0.01 mol) and malononitrile (0.66 g, 0.01 mol) in dimethylformamide (20 mL) containing triethylamine (0.5 mL) was refluxed for 3 h, and then allowed to cool, then poured onto ice/water containing few drops of hydrochloric acid and the formed solid product was collected by filtration and recrystallized from a mixture of ethanol and DMF (2:1) to give compound 14. Yellow powder; yield (82%); m.p. >300 C; IR (KBr): n/ cm1 ¼ 3481, 3296 (NH2), 2210 (CN), 1724, 1643 (2C]O); 1H NMR (300 MHz, DMSO-d6) d (ppm): 2.49 (s, 3H, CH3), 4.81, 4.92 (2s, 4H, 2NH2, D2O exchangeable), 6.15 (s, 1H, C5eH pyridinone ring) 7.52e 8.99 (m, 7H, AreH); 13C NMR (75 MHz, DMSO-d6) d (ppm): 23.1, 70, 84, 112, 115.5, 116.3 (2C), 121, 123, 126.2, 128.5 (2C), 130.4 (2C), 136.4, 145, 150, 155.3, 160, 172, 178; MS (EI, 70 eV) m/z (%) ¼ 385 (Mþ, 5.9), 239 (5.9), 195 (8.2), 180 (8.2), 170 (7.1), 157 (8.2). Anal. Calcd. for C21H15N5O3 (385): C, 65.45; H, 3.92; N, 18.17%. Found: C, 65.41; H 3.86; N 18.12%.
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5.7. Synthesis of 3,5-diamino-4-cyano-N-(1-(3-oxo-3H-benzo[f] chromen-2-yl)ethylidene)thiophene-2-carbohydrazide (15) To a solution of compound 3 (3.19 g, 0.01 mol) in absolute ethanol (25 mL) containing triethylamine (0.5 mL), malononitrile (0.66 g, 0.01 mol) and elemental sulfur (0.32 g, 0.01 mol) were added. The reaction mixture was heated under reflux for 2 h, and then allowed to cool, then poured onto ice/water and the formed solid product was collected by filtration and recrystallized from ethanol to give compound 15. Yellowish white powder; yield (68%); m.p. 230e232 C; IR (KBr): n/cm1 ¼ 3418, 3327 (NH2), 3212 (NH), 2197 (CN), 1713, 1678 (2C]O); 1H NMR (300 MHz, DMSO-d6) d (ppm): 2.65 (s, 3H, CH3), 6.98, 7.25 (2s, 4H, 2NH2, D2O exchangeable), 7.59e8.66 (m, 7H, Are H), 11.60 (s, 1H, NH, D2O exchangeable); 13C NMR (75 MHz, DMSOd6) d (ppm): 23, 81, 112, 115.6, 116, 117, 121, 123.4, 126.6, 128.5 (2C), 130.2 (2C), 136, 146, 150, 155.3, 158, 160 (2C), 174; MS (EI, 70 eV) m/z (%) ¼ 417 (Mþ, 14.78), 359 (63.98), 291 (81.74), 239 (52.17), 222 (73.04), 208 (46.96), 193 (48.70), 167 (56.52). Anal. Calcd. for C21H15N5O3S (417): C, 60.42; H, 3.62; N, 16.78%. Found: C, 60.40; H 3.58; N 16.74%. 5.8. Synthesis of 2-oxo-1-(1-(3-oxo-3H-benzo[f]chromen-2-yl) ethylideneamino)-5-phenyl-2,3-dihydro-1H-pyrrole-3-carbonitrile (16) A mixture of compound 3 (3.19 g, 0.01 mol) and phenacyl bromide (1.9 g, 0.01 mol) in dimethylformamide (20 mL) containing triethylamine (0.5 mL) was refluxed for 3 h, and then allowed to cool, then poured onto ice/water containing few drops of hydrochloric acid and the formed solid product was collected by filtration and recrystallized from ethanol to give compound 16. Yellow powder; yield (65%); m.p. 186e188 C; IR (KBr): n/ cm1 ¼ 2227 (CN), 1734, 1678 (2C]O); 1H NMR (300 MHz, DMSOd6) d (ppm): 2.65 (s, 3H, CH3), 4.54 (d, J ¼ 2.5 Hz, 1H, pyrrole H-3), 5.65 (d, J ¼ 2.5 Hz, 1H, pyrrole H-4), 7.61e8.65 (m, 12H, AreH); 13C NMR (75 MHz, DMSO-d6) d (ppm): 23, 38, 96, 112, 115.8, 116, 117, 121, 123.4, 126.5, 127.5, 128.4 (4C), 128.8 (2C), 130.2 (2C), 132, 136 (2C), 150, 155, 160, 172; MS (EI, 70 eV) m/z (%) ¼ 419 (Mþ, 15.80), 238 (30.20), 221 (59.60), 193 (32.80), 178 (19.40), 165 (31.60), 139 (38.00). Anal. Calcd. for C26H17N3O3 (419): C, 74.45; H, 4.09; N, 10.02%. Found: C, 74.44; H, 4.10; N 9.97%. 5.9. Synthesis of 4-amino-N-(1-(3-oxo-3H-benzo[f]chromen-2-yl) ethylidene)-3-phenyl-2-thioxo-2,3-dihydrothiazole-5carbohydrazide (17) To a solution of compound 3 (3.19 g, 0.01 mol) in absolute ethanol (25 mL) containing triethylamine (0.5 mL), elemental sulfur (0.32 g, 0.01 mol) and phenyl isothiocyanate (1.35 g, 0.01 mol) were added. The reaction mixture was heated under reflux for 3 h, and then allowed to cool, then poured onto ice/water and the formed solid product was collected by filtration and recrystallized from ethanol to give compound 17. Yellow powder; yield (68%); m.p. 180e182 C; IR (KBr): n/ cm1 ¼ 3440, 3360 (NH2), 3220 (NH), 1730, 1658 (2C]O); 1H NMR (300 MHz, DMSO-d6) d (ppm): 2.65 (s, 3H, CH3), 6.82 (s, 2H, NH2, D2O exchangeable), 7.59e8.66 (m, 12H, AreH), 11.65 (s, 1H, NH, D2O exchangeable); 13C NMR (75 MHz, DMSO-d6) d (ppm): 23, 73.8, 112, 115.6, 117.3, 121, 123, 126, 128 (3C), 129.4 (4C), 130 (2C), 134, 136.5, 150, 155, 160 (2C), 172, 186; MS (EI, 70 eV) m/z (%) ¼ 486 (Mþ 1, 90.82), 470 (58.16), 442 (58.16), 365 (87.76), 236 (71.43), 221 (74.90), 208 (77.55). Anal. Calcd. for C25H18N4O3S2 (486): C, 61.71; H, 3.73; N, 11.51%. Found: C, 61.63; H 3.76; N 11.49%.
425
5.10. Synthesis 2-amino-N-(1-(3-oxo-3H-benzo[f]chromen-2-yl) ethylidene)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carbohydrazide (18) To a solution of compound 3 (3.19 g, 0.01 mol) in absolute ethanol (25 mL) containing triethylamine (0.5 mL), elemental sulfur (0.32 g, 0.01 mol) and cyclohexanone (0.98 g, 0.01 mol) were added. The reaction mixture was heated under reflux for 3 h, and then allowed to cool, then poured onto ice/water and the formed solid product was collected by filtration and recrystallized from ethanol to give compound 18. Yellow powder; yield (58%); m.p. 220e222 C; IR (KBr): n/ cm1 ¼ 3421, 3390 (NH2), 3264 (NH), 1729, 1655 (2C]O); 1H NMR (300 MHz, DMSO-d6) d (ppm): 1.79e2.38 (m, 8H, 4CH2 benzothiophene), 2.65 (s, 3H, CH3), 6.49 (s, 2H, NH2, D2O exchangeable), 7.59e8.64 (m, 7H, AreH), 11.76 (s, 1H, NH, D2O exchangeable); 13C NMR (75 MHz, DMSO-d6) d (ppm): 17.3, 21, 23.2 (CH3), 23.5, 25.2 (4CH2), 112, 115.6 (2C), 117.4, 121, 123.2, 125.5, 126.4, 128.3 (2C), 130 (2C), 136.5, 139, 150, 155, 160, 162, 172; MS (EI, 70 eV) m/z (%) ¼ 431 (Mþ, 27.78), 277 (26.98), 263 (26.19), 252 (22.62), 219 (20.63), 204 (21.83), 193 (34.13), 165 (30.95). Anal. Calcd. for C24H21N3O3S (431): C, 66.80; H, 4.91; N, 9.74%. Found: C, 66.72; H 4.94; N 9.70%. 6. Antimicrobial evaluation The disks of Whatman filter paper were prepared with standard size (5.0 mm diameter) and kept into 1.0 Oz screw capped wide mouthed containers for sterilization. These bottles are kept into hot air oven at a temperature of 150 C. Then, the standard sterilized filter paper disks impregnated with a solution of the test compound in DMSO (1 mg/mL) were placed on nutrient agar plate seeded with appropriate test organism in triplicates. Standard conditions of 106 CFU/mL (Colony Forming U/mL) and 104 CFU/mL were used for antibacterial and antifungal assay, respectively. Pyrex glass petri dishes (9 cm in diameter) were used and two disks of filter paper were inoculated in each plate. The utilized test organisms were S. aureus as examples of Gram positive bacteria and E. coli as examples of Gram negative bacteria. They were also evaluated for their in vitro antifungal potential against C. albicans fungal strain. Ampicillin and Clotrimazole were used as standard antibacterial and antifungal agents, respectively. DMSO alone was used as control at the same above mentioned concentration and due this there was no visible change in bacterial growth. The plates were incubated at 36 C for 24 h for bacteria and for 48 h for fungi. Compounds that showed significant growth inhibition zones (>10 mm) using the two fold serial dilution technique, were further evaluated for their minimal inhibitory concentrations (MICs). 6.1. Minimal inhibitory concentrations (MIC) measurement The microdilution susceptibility test in Müller-Hinton Broth (Oxoid) and Sabouraud Liquid Medium (Oxoid) were used for the determination of antibacterial and antifungal activity, respectively. Stock solutions of the tested compounds, Ampicillin and Clotrimazole were prepared in DMSO at concentration of 1000 mg/mL. Each stock solution was diluted with standard method broth (Difco) to prepare serial two fold dilution at concentrations of (500, 250, ., 3.125 mg/mL), 10 mL of the broth containing about 106 CFU/mL of test bacteria was added to each well of 96-well microliter plate. The sealed microplates were incubated at 36 C for 24 h for antibacterial activity and at 36 C for 48 h for antifungal activity in a humid chamber. At the end of the incubation period, the minimal inhibitory concentrations (MICs) values were recorded as the lowest concentrations of the substance that had no visible turbidity.
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Control experiments with DMSO and uninoculated media were run parallel to the test compounds under the same conditions. Acknowledgment The authors are thankful to Department of Pharmacology, Faculty of Pharmacy, Mansoura University, Egypt for performing the antimicrobial evaluation. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.ejmech.2013.09.003. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10]
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European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and antimicrobial activity of some novel hydrazide, benzochromenone, dihydropyridine, pyrrole, thiazole and thiophene derivatives Hala M. Refat a, *, A.A. Fadda b a b
Department of Chemistry, Faculty of Education, Suez Canal University, El-Gomhoria St., Al-Arish, Egypt Department of Chemistry, Faculty of Science, Mansoura University, ET-35516 Mansoura, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history: Received 18 May 2013 Received in revised form 29 August 2013 Accepted 1 September 2013 Available online 12 October 2013
As a part of ongoing studies in developing new potent antimicrobial agents, a novel synthesis of 2-cyanoN-(1-(3-oxo-3H-benzo[f]chromen-2-yl)ethylidene)acetohydrazide (3) has been reported. The latter compound was reacted with different reagents to give new heterocyclic compounds. The structures of the newly synthesized compounds were confirmed by elemental analysis, IR, 1H NMR, 13C NMR and mass spectral data. Representative compounds of the synthesized products were tested and evaluated as antimicrobial agents. Ó 2013 Elsevier Masson SAS. All rights reserved.
Keywords: Benzochromenone Acetohydrazide Hydrazideehydrazone Antimicrobial activities
1. Introduction Hydrazines and their derivatives constitute an important class of compounds that has found wide utility in organic synthesis [1,2]. Also, they constitute an important class of compounds for new drug development [3]. A number of hydrazideehydrazone derivatives have been claimed to possess interesting bioactivity such as antibacterial-antifungal [4e6], anticonvulsant [7,8], antiinflammatory [9,10], antimalarial [11] and antituberculosis activities [12,13]. Moreover, the coumarin nucleus is prevalent in numerous natural products and is extremely important in the chemistry of biological activities [14], which have found applications in treatment of antibacterial, antitumor, anti-inflammatory, antithrombotic, cardio protectors or enzymatic inhibitors antimicrobial and antifungal [15e19]. In view of the above-mentioned findings, and as a continuation of our effort to identify new candidates that may be valuable in designing new, potent, selective, and less toxic antimicrobial agents [20e25], we report herein the synthesis of a series of hydrazidee hydrazones together with their use in a series of heterocyclic
* Corresponding author. Tel: þ20 50 2343348, þ20 1061988989 (mobile). E-mail addresses: [email protected], [email protected] (H.M. Refat). 0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.ejmech.2013.09.003
transformations and their evaluation as antimicrobial agents. This combination was suggested to investigate the influence of such hybridization and structure variation on the anticipated biological activities, hoping to add some synergistic biological significance to the target molecules. 2. Results and discussion 2.1. Chemistry The synthetic strategies adopted for the synthesis of the intermediates and to target compounds are depicted in Schemes 1e3. In Scheme 1, synthesis of 2-cyano-N-(1-(3-oxo-3H-benzo[f]chromen-2-yl)ethylidene)acetohydrazide (3), in analogy with the reported literature [26] by the reaction of 2-acetyl-3H-benzo[f] chromen-3-one (1) [27] and 2-cyanoacetohydrazide (2). The assignment of structure 3 was supported by elemental analysis and spectral data. The IR spectrum displayed stretching vibration bands at 3193 and 2263 cm1 corresponding to NH and CN groups, in addition to the stretching vibration of two carbonyl groups at 1733 and 1679 cm1. Its 1H NMR spectrum (DMSO-d6) revealed the presence of singlet signals at d 2.28, 4.34 and 11.21 ppm assignable for CH3, CH2CN and NH protons. The mass spectrum showed the molecular ion peak at m/z ¼ 319 corresponding to the molecular formula (C18H13N3O3).
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O O
CH3 O
+
NC
NHNH 2
O
(1)
(2 )
EtOH
CH3
O CN
N N H A rC
O
HO
Ar
O (3)
Ar N 2 C =4 l -C H 3 -C 6H 4
CH3
O
O
O
CH3
DMFDMA
CN
N N H
O CN
N N H
Ar
O CH3
Ar
O CN
N N H
6, Ar= 4-Cl-C 6H4 7, Ar= 4-F-C 6H 4
O
O
8, Ar= 3-Pyridyl
H3C (5)
O
N
N N H
(4 )
CH3
Scheme 1. Synthetic route to hydrazideehydrazone.
Its 1H NMR spectrum (DMSO-d6) revealed the presence of signals at d 2.49 and 2.65 ppm assignable for two CH3 protons, also, two signals at d 10.25 and 11.68 ppm for two NH protons. The mass spectrum showed the molecular ion peak at m/z ¼ 437 corresponding to the molecular formula (C25H19N5O3). Also, treatment of compound 3 with dimethylformamide dimethylacetal (DMF-DMA) in dry xylene under reflux afforded 2cyano-3-(dimethylamino)-N-(1-(3-oxo-3H-benzo[f]chromen-2-yl)
Next, we studied the reactivity of the active methylene group present in compound 3 towards diazonium salt. Thus, the reaction of compound 3 with 4-methylbenzenediazonium chloride gave the hydrazone derivative 4. The assignment of structure 4 was supported by elemental analysis and spectral data. The IR spectrum displayed stretching vibration bands at 3388, 3193 and 2228 cm1 corresponding to two NH and CN groups, in addition to the stretching vibration of two carbonyl groups at 1734 and 1679 cm1.
CHO
CH3
OH
N N H
(3) O
CN Ar
O
O (9 )
X
X= COOEt
X= CN
CH3
O
CH3 CN
N N H O
O
O
Ar
O CN
N N H O
O N
EtOOC CN
C
Ar CN
- EtOH
CH3
O
O
O HO
CH3
CN
N N
Ar CN
12, Ar= 2,5-(OCH3)2-C 6H 3 13, Ar= 3-OH-C 6H 4
O CN
N N O
OH N 2
Ar CN
10, Ar= 2,5-(OCH3)2-C 6H 3 11, Ar= 4-F-C 6H 4
Scheme 2. Synthetic route to chromene and dihydropyridine.
O
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421
CH3 TEA
CH3
O
N N H O
NH2 N Ph
O
OH N 2
NH2 (14)
PhNCS S
CH3
TEA, S
S
O
CN
N N
CN CN
O
S
O
N N H
(3)
O
(17)
O
S
NH2 CN
H 2N
(15) CH3 N N H O
O
NH2 S
O
CH3
PhCOCH2Br
S
N N
O O (18)
O CN
O Ph (16)
Scheme 3. Synthetic route to pyrrole, thiazole and thiophene.
ethylidene)acrylohydrazide (5). The assignment of structure 5 was supported by elemental analysis and spectral data. The IR spectrum displayed stretching vibration bands at 3194 and 2190 cm1 corresponding to NH and CN groups, in addition to the stretching vibration of two carbonyl groups at 1720 and 1679 cm1. Its 1H NMR spectrum (DMSO-d6) revealed the presence of signals at d 2.66, 3.19 and 3.23 ppm assignable for three CH3 protons, a singlet at d 8.23 ppm for vinylic proton, and a singlet at d 11.60 ppm for NH proton. The mass spectrum showed the molecular ion peak at m/ z ¼ 374 corresponding to the molecular formula (C21H18N4O3). Further elucidation for the structure of 3 was obtained through studying its chemical reactivity with some chemical reagents. Thus, the reaction of compound 3 with certain aromatic aldehydes gave the corresponding benzylidene derivatives 6e8. Analytical and spectral data for the later compounds were in agreement with the proposed structures. Generally, the IR spectra for these derivatives showed the presence of the NH group at around 3280 cm1, cyano group at around 2229 cm1 and two carbonyl groups at around 1735 and 1678 cm1. Whereas the 1H NMR spectra showed the absence of the active methylene protons and showed a singlet signals at d 2.66, 8.12 and 11.68 ppm assignable for CH3, vinylic and NH protons. In addition, the mass spectroscopic measurements of compounds 6, 7 and 8 showed the molecular ion peaks at m/z ¼ 441 (Mþ, 12.80%), 425 (Mþ, 22.2%) and 408 (Mþ, 17.9%), respectively, which are in agreement with their molecular formula. On the other hand, benzochromenone derivative 9 was obtained through reaction of 3 with 2-hydroxy-1-naphthaldehyde. The assignment of structure 9 was supported by elemental analysis and spectral data. Its IR spectrum displayed stretching vibration bands at 3245 cm1 corresponding to NH group, in addition to the stretching vibration of three carbonyl groups at 1734e1674 cm1. Its 1H NMR spectrum (DMSO-d6) revealed the presence of a singlet at d 2.66 ppm assignable for CH3 proton and a singlet at d 11.65 ppm for NH proton. The mass spectrum showed the molecular ion peak at m/z ¼ 474 corresponding to the molecular formula (C29H18N2O5). Next, we studied the reactivity of the hydrazideehydrazone derivative 3 towards cinnamonitrile derivatives. Thus, the reaction of compound 3 with either a-cyanocinnamonitrile or ethyl cinnamonitrile derivatives in DMF and catalytic amount of TEA afforded the corresponding pyridine derivatives 10e13, respectively (Scheme 2). The structures 10e13 were established by the correct elemental analysis and compatible spectroscopic data. In general,
the IR spectra of compounds 10 and 11 revealed absorption bands at 3440, 3352 and 2206 cm1 for NH2 and CN groups, in addition to the stretching vibration of two carbonyl groups at 1732 and 1626 cm1. Whereas the structures 12e13 revealed absorption bands at 3434 and 2218 cm1 for OH and CN groups, in addition to the stretching vibration of two carbonyl groups at 1735 and 1678 cm1. The 1H NMR spectra (DMSO-d6) of compound 10 displayed a singlet signals at d 2.66, 3.77 and 6.78 ppm assignable to CH3, two OCH3 and NH2 protons, respectively. Whereas the compound 12 displayed a singlet signals at d 2.66, 3.87 and 10.76 ppm assignable to CH3, two OCH3 and OH groups, respectively. In addition, the mass spectroscopic measurements of compounds 10e 13 showed the molecular ion peaks at m/z ¼ 531 (Mþ, 15.24%), 489 (Mþ, 15.5%), 532 (Mþ, 22.7%) and 488 (Mþ, 93.54%), respectively, which are in agreement with their molecular formulas. Also, condensation of 3 with malononitrile gave the corresponding 2-pyridinone derivative 14 (Scheme 3). The assignment of structure 14 was based on analytical and spectral data. Its IR spectrum displayed absorption bands at 3481, 3296, 2210, and 1724, 1643 cm1 corresponding to NH2, CN and two carbonyl groups, respectively. Its 1H NMR spectrum (DMSO-d6) revealed to signals at d 2.49, 4.81, 4.92 and 6.15 ppm assignable for the CH3, two NH2 and C5eH pyridinone protons, respectively. The mass spectrum showed the molecular ion peak at m/z ¼ 385 corresponding to the molecular formula (C21H15N5O3). The condensation of 3 with malononitrile and elemental sulfur in the presence of triethylamine gave the corresponding thiophene derivative 15. The assignment of structure 15 was supported by elemental analysis and spectral data. Its IR spectrum displayed absorption bands at 3418, 3327, 3212, 2197, 1713 and 1678 cm1 assignable to NH2, NH, CN and two carbonyl groups, respectively. Its 1 H NMR spectrum (DMSO-d6) revealed to signals at d 2.65, 6.98, 7.25 and 11.60 ppm assignable for the CH3, two NH2 and NH protons, respectively. The mass spectrum showed the molecular ion peak at m/z ¼ 417 corresponding to the molecular formula (C21H15N5O3S). Recently, we have reported the reaction of cyanoacetamide moiety with a-halocarbonyl compounds which represents a new, simple and efficient synthetic route for the synthesis pyrrole derivatives. Therefore, cyclocondensation of 3 with phenacyl bromide in DMF using triethylamine as a basic catalyst furnished the pyrrole derivative 16. The assignment of structure 16 was supported by
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elemental analysis and spectral data. Its IR spectrum displayed stretching vibration bands at 2227, 1734 and 1678 cm1 assignable to CN and two carbonyl groups. Its 1H NMR spectrum (DMSO-d6) revealed the presence of two doublet signals at d 4.54 and 5.65 ppm assignable for the two vicinal pyrroline protons H-3 and H-4, respectively, singlet at d 2.65 ppm for CH3 proton. The mass spectrum showed the molecular ion peak at m/z ¼ 419 corresponding to the molecular formula (C26H17N3O3). On the other hand, the Gewald reaction [28] of compound 3 with both elemental sulfur and phenyl isothiocyanate in warming DMF containing triethylamine as a basic catalyst led to functionalized thiazole derivative 17. The assignment of structure 17 was based on analytical and spectral data. Its IR spectrum displayed absorption bands at 3440, 3360 and 3220 cm1 assignable to NH2 and NH groups, in addition to the stretching vibration of two carbonyl groups at 1730 and 1658 cm1. Its 1H NMR spectrum (DMSO-d6) revealed the presence of signals at d 2.65, 6.82 and 11.65 ppm assignable for CH3, NH2 and NH protons. The mass spectrum showed the molecular ion peak at m/z ¼ 486 corresponding to the molecular formula (C25H18N4O3S2). Similarly, 2-amino-N-(1-(3-oxo-3H-benzo[f]chromen-2-yl)ethylidene)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carbohydrazide (18) could be achieved according to the method described by Gewald, by reaction of compound 3 with cyclohexanone and elemental sulfur. The assignment of structure 18 was based on analytical and spectral data. Thus, its IR spectrum displayed absorption bands at 3421, 3390 and 3264 cm1 assignable to NH2 and NH groups, in addition to the stretching vibration of two carbonyl groups at 1729 and 1655 cm1. Its 1H NMR spectrum (DMSO-d6) revealed the presence of signals at d 1.79e2.38 assignable for the benzothiophene, signals at d 2.65, 6.49 and 11.76 ppm assignable for CH3, NH2 and NH protons. The mass spectrum showed the molecular ion peak at m/z ¼ 431 corresponding to the molecular formula (C24H21N3O3S). 3. Pharmacology 3.1. Antimicrobial evaluation All the newly synthesized compounds 3e18 were initially evaluated for in vitro antibacterial activity against Gram-positive bacteria (Staphylococcus aureus) (MTCC-96) and Gram-negative bacteria (Escherichia coli) (MTCC-443) and fungal (Candida albicans) using conventional Broth dilution method [29]. Ampicillin and Clotrimazole were used as reference drugs. The results were recorded for each tested compound as the average diameter of inhibition zones (IZ) of bacterial or fungal growth around the discs in mm. The minimal inhibitory concentrations (MICs) for compounds that showed significant growth inhibition zones (>10 mm) were determined using two fold serial dilution method [30]. The inhibition zone diameters and MIC (mg/mL) values are recorded in Tables 1 and 2. It was observed that compounds 9 and 17 exhibited the highest activity against S. aureus and E. coli. When acetyl group in compound 1 was replaced by benzocoumarin ring (compound 9). The activity increased and displayed the highest activity at (MIC ¼ 62.5 mg/mL) against S. aureus and (MIC ¼ 93.7 mg/mL) against E. coli, while compounds 3, 4, 7, 8, 10, 11, 16 and 17 which contain electron withdrawing group or heterocyclic rings showed good activity (MIC ¼ 125 mg/mL) against S. aureus bacteria. On the other hand, compound 6 was equipotent to Ampicillin in inhibiting the growth of S. aureus (MIC ¼ 187.5 mg/mL). On the other hand, compounds 3, 4, 8, 10, 11, 13 and 14 exhibited equipotent to Ampicillin in inhibiting the growth of E. coli (MIC ¼ 125 mg/mL). Compounds 3e18 were also evaluated for their in vitro antifungal activity against C. albicans. All compounds exhibited a very weak
Table 1 Diameter of inhibition zone (mm) of the newly synthesized compounds. Compound no.
3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Ampicillin Clotrimazole
Diameter of inhibition zone (mm) bacteria Fungi Gram(þ) bacteria
Gram() bacteria
S. aureus
% Activity index
E. coli
% Activity index
C. albicans % Activity index
21 24 NA 23 22 22 27 23 21 13 12 8 NA 13 26 5 25 NA
84 96 NA 92 88 88 108 92 84 52 48 32 NA 52 104 20 100 NA
23 24 NA 17 20 23 28 23 21 20 24 23 NA 16 27 NA 27 NA
85.5 88.9 NA 63 74.1 85.2 104 85.2 77.8 74.1 88.9 85.2 NA 59.2 100 NA 100 NA
11 21 7 17 18 19 20 18 10 10 19 18 20 13 15 9 NA 30
36.7 70 23.3 56.7 60 63.3 66.7 60 33.3 33.3 63.3 60 66.7 43.3 50 30 NA 100
“NA”: no activity.
activity against C. albicans as compared to standard Clotrimazole (MIC ¼ 125e500 mg/mL). 3.2. Structure activity relationships (SAR’s) From the results of antimicrobial activity of the newly synthesized compounds 3e18, antimicrobial activity was considerably affected by electron withdrawing substituents and the incorporation of electron withdrawing groups is responsible for enhancing activity against the test microorganism. Compounds 9 and 17 which contain heterocyclic ring pyran and thiazole moieties with electron withdrawing properties were most potent against E. coli and S. aureus. Moreover, newly synthesized compounds have higher potency against Gram-negative bacteria than compared to Grampositive bacterial strains. In addition, the antibacterial activity showed that pyran and thiazole displayed higher activity than the other heterocyclic rings. On the other hand, antifungal activity Table 2 Minimal inhibitory concentration (MIC, mg/mL) of the newly synthesized compounds. Compound no.
3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Ampicillin Clotrimazole “NA”: no activity.
MIC, in mg/mL
Fungi
Gram(þ) bacteria
Gram() bacteria
S. aureus
E. coli
C. albicans
125 125 NA 187.5 125 125 62.5 125 125 250 250 375 NA 125 125 375 187.5 NA
125 125 NA 250 250 125 93.7 125 125 187.5 125 125 NA 250 93.7 NA 125 NA
250 125 750 125 125 125 187.5 125 375 250 187.5 125 125 250 125 500 NA 5.8
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displayed conflicting results where in compounds having electron withdrawing substituents revealed weak inhibition. 4. Conclusion Our attempts at exploring benzochromenone based pyran, pyridine, pyrrole, thiazole and thiophene derivatives have unexpectedly led to identification of a novel chemo type with substantial antimicrobial activity. Among the newly synthesized compounds 3e18, analogs 9 and 17 showed highest inhibition against nearly all of the tested bacteria, while all compounds were inactive against fungal strains. Results of antimicrobial activity clearly demonstrated that the presence of electron withdrawing groups/atoms attached to the benzochromenone ring is essential for enhancing antimicrobial activity. On the basis of structure activity relationship, electron withdrawing substituents (pyran and thiazole rings) are beneficial for antibacterial activity and inactive for antifungal activity. From the activity data, compound 9 and 17 showed highest antibacterial inhibition. Thus suggesting that the compounds from the present series with electron withdrawing groups can serve as important gateways for the design and development of new antimicrobial agents with potent activity and minimal toxicity. 5. Experimental Melting points were measured with a Gallenkamp apparatus are uncorrected. IR spectra were recorded KBr disc on a Mattson 5000 FTIR spectrophotometer at Microanalytical Unit, Faculty of Science, Mansoura University. The 1H NMR and 13C NMR spectra were measured on Bruker WP AC 300 (300 MHz) in DMSO-d6 as solvent, using tetramethylsilane (TMS) as an internal standard, and chemical shifts are expressed as dppm. Mass spectra were determined on Finnigan Incos 500 (70 eV). Elemental analyses were carried out at the Microanalytical Centre, Faculty of Science, Cairo University. 5.1. Synthesis of 2-cyano-N-(1-(3-oxo-3H-benzo[f]chromen-2-yl) ethylidene)acetohydrazide (3) To a solution of 2-cyanoacetohydrazide 2 (1.0 g, 0.01 mol) in ethanol (25 mL), 2-acetyl-3H-benzo[f]chromen-3-one 1 (2.38 g, 0.01 mol) was added. The reaction mixture was heated under reflux for 3 h, the obtained solid was filtered off while hot and recrystallized from a mixture of ethanol and DMF (2:1) to give compound 3. Pale yellow needle crystals; yield (94%); m.p. 276e278 C; IR (KBr): n/cm1 ¼ 3193 (NH), 2263 (CN), 1733, 1679 (2C]O); 1H NMR (300 MHz, DMSO-d6) d (ppm): 2.28 (s, 3H, CH3), 4.34 (s, 2H, CH2CN), 7.60e8.68 (m, 7H, AreH), 11.21 (s, 1H, NH, D2O exchangeable); 13C NMR (75 MHz, DMSO-d6) d (ppm): 23.3, 29.4, 112.3, 116.5, 117.2 (2C), 121.5, 123.4, 126.5, 129.4 (2C), 130.2 (2C), 136.1, 145.5, 155.8, 160.2, 172.3; MS (EI, 70 eV) m/z (%) ¼ 319 (Mþ, 54.4), 279 (88.9), 236 (58.9), 223 (73.3), 195 (36.7), 165 (100.0). Anal. Calcd. for C18H13N3O3 (319): C, 67.71; H, 4.10; N, 13.16%. Found: C, 67.66; H, 4.02; N, 13.11%. 5.2. Synthesis of 2-oxo-2-(2-(1-(3-oxo-3H-benzo[f]chromen-2-yl) ethylidene)hydrazinyl)-N-p-tolylacetohydrazonoyl cyanide (4) To a cold (0e5 C) solution of compound 3 (3.19 g, 0.01 mol) in pyridine (25 mL) was added the 4-methyl-benzenediazonium chloride [which was prepared by dissolving sodium nitrite (0.68 g, 0.01 mol) in water (2 mL) and adding to a cold solution of p-toluidine (1.07 g, 0.01 mol) containing the appropriate amount of hydrochloric acid with continuous stirring] portion wise over the
423
reaction mixture was kept in an ice box overnight and then diluted with water. The solid that precipitated was filtered off, washed with water and dried, and recrystallized from a mixture of ethanol and DMF (2:1) to give compound 4. Orange red crystals; yield (85%); m.p. 250e252 C; IR (KBr): n/ 1 cm ¼ 3388, 3193 (2NH), 2228 (CN), 1734, 1679 (2C]O); 1H NMR (300 MHz, DMSO-d6) d (ppm): 2.49, 2.65 (s, 6H, 2CH3), 7.54e8.62 (m, 11H, AreH), 10.25, 11.68 (s, 2H, 2NH, D2O exchangeable); 13C NMR (75 MHz, DMSO-d6) d (ppm): 21.3, 23.5, 106.8, 112.5, 115.5, 116.4 (4C), 121.5, 123.5, 126.6, 128.5 (2C), 129.2 (2C), 130.2 (3C), 136.2, 145 (2C), 155.6, 160.2, 172; MS (EI, 70 eV) m/z (%) ¼ 437 (Mþ, 16.0), 289 (12.8), 238 (6.4), 221 (10.6), 195 (9.6), 167 (8.5), 139 (14.9). Anal. Calcd. for C25H19N5O3 (437): C, 68.64; H, 4.38; N, 16.01%. Found: C, 68.61; H, 4.32; N, 15.98%. 5.3. Synthesis of 2-cyano-3-(dimethylamino)-N-(1-(3-oxo-3Hbenzo[f]chromen-2-yl)ethylidene)acrylohydrazide (5) To a solution of compound 3 (3.19 g, 0.01 mol) in dry xylene (25 mL), N,N-dimethylformamide-dimethylacetal (DMF-DMA) (1.32 mL, 0.01 mol) was added. The reaction mixture was heated under reflux for 3 h. After cooling, the precipitated product that formed was collected by filtration and recrystallized from a mixture of ethanol and DMF (2:1) to give compound 5. Orange crystals; yield (62%); m.p. 210e212 C; IR (KBr): n/ cm1 ¼ 3194 (NH), 2190 (CN), 1720, 1679 (2C]O); 1H NMR (300 MHz, DMSO-d6) d (ppm): 2.66 (s, 3H, CH3), 3.19 (s, 3H, NCH3), 3.23 (s, 3H, NCH3), 7.42e8.63 (m, 7H, AreH), 8.23 (s, 1H, CH]N), 11.60 (s, 1H, NH, D2O exchangeable); 13C NMR (75 MHz, DMSO-d6) d (ppm): 23.5, 42 (2C), 106.1, 112.3, 115.5, 116.6 (2C), 122.5, 123.5, 126.5, 129.1 (2C), 130 (2C), 136, 144.4, 156 (2C), 160.2, 172; MS (EI, 70 eV) m/z (%) ¼ 374 (Mþ, 23.2), 330 (23.2), 317 (42.7), 262 (65.9), 235 (100.0), 205 (30.5), 163 (28.0). Anal. Calcd. for C21H18N4O3 (374): C, 67.37; H, 4.85; N, 14.96%. Found: C, 67.30; H, 4.84; N, 14.89%. 5.4. General procedure for the synthesis of 3-(4-substituted)-2cyano-N-(1-(3-oxo-3H-benzo[f]chromen-2-yl)ethylidene) acrylohydrazide derivatives (6e8) and 3-oxo-N-(1-(3-oxo-3Hbenzo[f]chromen-2-yl)ethylidene)-3H-benzo[f]chromene-2carbohydrazide (9) Equimolecular mixture of 3 (3.19 g, 0.01 mol) and selected aldehyde (0.01 mol), in DMF (20 mL) containing piperidine (0.5 mL) was heated under reflux for 3 h. The reaction mixture was left to cool then poured onto ice/water containing few drops of hydrochloric acid and the formed solid product was collected by filtration and recrystallized from ethanol. 5.4.1. Synthesis of 3-(4-chlorophenyl)-2-cyano-N-(1-(3-oxo-3Hbenzo[f]chromen-2-yl)ethylidene)acrylohydrazide (6) Yellow powder; yield (83.7%); m.p. 184e185 C; IR (KBr): n/ cm1 ¼ 3280 (NH), 2229 (CN), 1735, 1678 (2C]O); 1H NMR (300 MHz, DMSO-d6) d (ppm): 2.66 (s, 3H, CH3), 7.41e8.68 (m, 11H, AreH), 8.12 (s, 1H, vinyliceH), 11.68 (s, 1H, NH, D2O exchangeable); 13 C NMR (75 MHz, DMSO-d6) d (ppm): 23.6, 107.1, 112.3, 115.5, 116.5, 117.6, 121.1, 123.4, 126.6, 128.4 (4C), 130.2 (3C), 132.2 (3C), 136.4, 150 (2C), 156.2, 160.2, 172.3; MS (EI, 70 eV) m/z (%) ¼ 441 (Mþ, 12.8), 312 (10.6), 275 (12.8), 242 (12.8), 221 (27.7), 193 (25.5), 168 (27.7), 139 (42.6). Anal. Calcd. for C25H16ClN3O3 (441): C, 67.95; H, 3.65; N, 9.51%. Found: C, 67.91; H 3.60; N 9.46%. 5.4.2. Synthesis of 2-cyano-3-(4-fluorophenyl)-N-(1-(3-oxo-3Hbenzo[f]chromen-2-yl)ethylidene)acrylohydrazide (7) Yellowish brown powder; yield (85.5%); m.p. 215e217 C; IR (KBr): n/cm1 ¼ 3280 (NH), 2229 (CN), 1735, 1678 (2C]O); 1H NMR
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(300 MHz, DMSO-d6) d (ppm): 2.66 (s, 3H, CH3), 7.45e8.66 (m, 11H, AreH), 8.14 (s, 1H, vinyliceH), 11.66 (s, 1H, NH, D2O exchangeable); 13 C NMR (75 MHz, DMSO-d6) d (ppm): 23.4, 107, 112, 115.5 (4C), 117.4, 121, 123, 126, 127.5, 128 (2C), 130 (4C), 136, 150 (2C), 155.2, 160, 162, 172; MS (EI, 70 eV) m/z (%) ¼ 425 (Mþ, 22.2), 383 (30.1), 365 (27.5), 340 (30.1), 266 (30.7), 241 (31.6), 184 (20.8). Anal. Calcd. for C25H16FN3O3 (425): C, 70.58; H, 3.79; N, 9.88%. Found: C, 70.51; H 3.74; N 9.85%. 5.4.3. Synthesis of 2-cyano-N-(1-(3-oxo-3H-benzo[f]chromen-2-yl) ethylidene)-3-(pyridin-3-yl)acrylohydrazide (8) Yellow powder; yield (78.6%); m.p. 198e200 C; IR (KBr): n/ cm1 ¼ 3280 (NH), 2229 (CN), 1735, 1678 (2C]O); 1H NMR (300 MHz, DMSO-d6) d (ppm): 2.66 (s, 3H, CH3), 7.44e8.66 (m, 11H, AreH), 8.15 (s, 1H, vinyliceH), 11.69 (s, 1H, NH, D2O exchangeable); 13 C NMR (75 MHz, DMSO-d6) d (ppm): 23, 107, 112.5, 115.8 (2C), 117.7, 121, 123 (2C), 126.6, 128.5 (2C), 130 (2C), 132.5 (2C), 136, 145, 148 (2C), 153, 155, 160.2, 172; MS (EI, 70 eV) m/z (%) ¼ 408 (Mþ, 17.9), 283 (15.4), 221 (100.0), 195 (51.3), 139 (87.2). Anal. Calcd. for C24H16N4O3 (408): C, 70.58; H, 3.95; N, 13.72%. Found: C, 70.50; H 3.98; N 13.67%. 5.4.4. Synthesis of 3-oxo-N-(1-(3-oxo-3H-benzo[f]chromen-2-yl) ethylidene)-3H-benzo[f]chromene-2-carbohydrazide (9) Yellowish brown powder; yield (71.5%); m.p. 194e196 C; IR (KBr): n/cm1 ¼ 3245 (NH), 1734, 1713, 1674 (3C]O); 1H NMR (300 MHz, DMSO-d6) d (ppm): 2.66 (s, 3H, CH3), 7.61e8.66 (m, 14H, AreH), 11.65 (s, 1H, NH, D2O exchangeable); 13C NMR (75 MHz, DMSO-d6) d (ppm): 23.6, 112.5 (2C), 115.5 (2C), 117.7 (2C), 121 (2C), 123.2 (2C), 126.8 (2C), 128 (4C), 130 (4C), 136, 143, 145 (2C), 155.3, 160 (2C), 172; MS (EI, 70 eV) m/z (%) ¼ 474 (Mþ, 66.39), 433 (42.62), 275 (47.54), 268 (54.0), 239 (77.87), 225 (45.08), 211 (64.75), 170 (46.72), 153 (60.66), 141 (77.05). Anal. Calcd. for C29H18N2O5 (474): C, 73.41; H, 3.82; N, 5.90%. Found: C, 73.31; H 3.74; N 5.85%. 5.5. General procedure for the synthesis of 6-amino-4-(substituted phenyl)-2-oxo-1-(1-(3-oxo-3H-benzo[f]chromen-2-yl) ethylideneamino)-1,2-dihydro-pyridine-3,5-dicarbonitrile derivatives (10e11) and 4-(substituted phenyl)-6-hydroxy-2-oxo-1(1-(3-oxo-3H-benzo[f]chromen-2-yl)ethylideneamino)-1,2dihydropyridine-3,5-dicarbonitrile derivatives (12e13) To a solution of compound 3 (3.19 g, 0.01 mol) in DMF (20 mL) containing triethylamine (0.5 mL), either a-cyanocinnamonitrile or ethyl cinnamonitrile (0.01 mol) was added. The reaction mixture, in each case was heated under reflux for 3 h. The reaction mixture was left to cool then poured onto ice/water containing few drops of hydrochloric acid and the formed solid product was collected by filtration and recrystallized from ethanol. 5.5.1. Synthesis of 6-amino-4-(2,5-dimethoxyphenyl)-2-oxo-1-(1(3-oxo-3H-benzo[f]chromen-2-yl)ethylideneamino)-1,2dihydropyridine-3,5-dicarbonitrile (10) Yellow powder; yield (86.7%); m.p. 130e132 C; IR (KBr): n/ cm1 ¼ 3440, 3352 (NH2), 2206 (CN), 1732, 1626 (2C]O); 1H NMR (300 MHz, DMSO-d6) d (ppm): 2.66 (s, 3H, CH3), 3.77 (s, 6H, 2OCH3), 6.78 (s, 2H, NH2, D2O exchangeable), 7.21e8.67 (m, 10H, AreH); 13C NMR (75 MHz, DMSO-d6) d (ppm): 23.4, 55.8, 56.3, 77, 112 (2C), 114.5, 115.3 (2C), 115.8 (2C), 116.3 (2C), 121 (2C), 123.4, 126.6, 128.5 (2C), 130.6 (2C), 136.2, 150 (2C), 153, 155.7 (2C), 160, 166, 172; MS (EI, 70 eV) m/z (%) ¼ 531 (Mþ, 15.2), 487 (14.1), 469 (23.2), 443 (37.2), 296 (100), 256 (46.9), 237 (17.7), 209 (34.2), 145 (65.8). Anal. Calcd. for C30H21N5O5 (531): C, 67.79; H, 3.98; N, 13.18%. Found: C, 67.70; H 3.94; N 13.15%.
5.5.2. Synthesis of 6-amino-4-(4-fluorophenyl)-2-oxo-1-(1-(3-oxo3H-benzo[f]chromen-2-yl)ethylideneamino)-1,2-dihydropyridine3,5-dicarbonitrile (11) Yellow powder; yield (82.7%); m.p. 205e206 C; IR (KBr): n/ cm1 ¼ 3440, 3357 (NH2), 2208 (CN), 1733, 1626 (2C]O); 1H NMR (300 MHz, DMSO-d6) d (ppm): 2.66 (s, 3H, CH3), 6.51 (s, 2H, NH2, D2O exchangeable), 7.15e8.65 (m, 11H, AreH); 13C NMR (75 MHz, DMSO-d6) d (ppm): 23.2, 77, 112.2, 115.3 (3C), 115.6 (2C), 116 (2C), 121.3, 123.2, 126.8, 128 (3C), 128.5 (2C), 130 (2C), 136.2, 150.3, 155.5 (2C), 160 (2C), 168, 172; MS (EI, 70 eV) m/z (%) ¼ 489 (Mþ, 15.5), 403 (19.3), 287 (25.2), 238 (16.8), 221 (100.0), 193 (73.1), 164 (46.2), 139 (38.7). Anal. Calcd. for C28H16FN5O3 (489): C, 68.71; H, 3.29; N, 14.31%. Found: C, 68.68; H 3.24; N 14.27%. 5.5.3. Synthesis of 4-(2,5-dimethoxyphenyl)-6-hydroxy-2-oxo-1(1-(3-oxo-3H-benzo[f]chromen-2-yl)ethylideneamino)-1,2dihydropyridine-3,5-dicarbonitrile (12) Yellow powder; yield (82%); m.p. 200e202 C; IR (KBr): n/ 1 cm ¼ 3436 (OH), 2227 (CN), 1735, 1678 (2C]O); 1H NMR (300 MHz, DMSO-d6) d (ppm): 2.66 (s, 3H, CH3), 3.87 (s, 6H, 2OCH3), 7.41e8.68 (m, 10H, AreH), 10.76 (s, 1H, OH, D2O exchangeable); 13C NMR (75 MHz, DMSO-d6) d (ppm): 23.4, 55.8, 56.8, 63, 112 (2C), 114.5, 115.3 (2C), 115.8 (2C), 116.5 (2C), 121 (2C), 123, 126, 128 (2C), 130 (2C), 136.5, 150 (2C), 153, 155, 160, 166, 172, 188; MS (EI, 70 eV) m/z (%) ¼ 532 (Mþ, 22.7), 302 (27.3), 215 (45.5), 182 (22.7), 175 (27.3), 139 (45.5). Anal. Calcd. for C30H20N4O6 (532): C, 67.67; H, 3.79; N, 10.52%. Found: C, 67.63; H 3.71; N 10.46%. 5.5.4. Synthesis of 6-hydroxy-4-(3-hydroxyphenyl)-2-oxo-1-(1-(3oxo-3H-benzo[f]chromen-2-yl)ethylideneamino)-1,2dihydropyridine-3,5-dicarbonitrile (13) Yellow powder; yield (86%); m.p. 216e218 C; IR (KBr): n/ cm1 ¼ 3500e3434 (2OH), 2218 (CN), 1723, 1676 (2C]O); 1H NMR (300 MHz, DMSO-d6) d (ppm): 2.66 (s, 3H, CH3), 7.64e8.66 (m, 11H, AreH), 9.80, 10,76 (2s, 2H, 2OH, D2O exchangeable); 13C NMR (75 MHz, DMSO-d6) d (ppm): 23.2, 58, 112 (2C), 115.3 (2C), 115.6 (2C), 116 (2C), 121 (2C), 123, 126.2, 128.3 (2C), 130.2 (3C), 134, 136, 150, 155, 160 (2C), 166, 172, 188; MS (EI, 70 eV) m/z (%) ¼ 488 (Mþ, 39.54), 443 (20.26), 395 (16.99), 326 (36.27), 278 (25.49), 221 (100), 167 (24.18). Anal. Calcd. for C28H16N4O5 (488): C, 68.85; H, 3.30; N, 11.47%. Found: C, 68.83; H 3.31; N 11.45%. 5.6. Synthesis of 4,6-diamino-2-oxo-1-(1-(3-oxo-3H-benzo[f] chromen-2-yl)ethylideneamino)-1,2-dihydropyridine-3-carbonitrile (14) A mixture of compound 3 (3.19 g, 0.01 mol) and malononitrile (0.66 g, 0.01 mol) in dimethylformamide (20 mL) containing triethylamine (0.5 mL) was refluxed for 3 h, and then allowed to cool, then poured onto ice/water containing few drops of hydrochloric acid and the formed solid product was collected by filtration and recrystallized from a mixture of ethanol and DMF (2:1) to give compound 14. Yellow powder; yield (82%); m.p. >300 C; IR (KBr): n/ cm1 ¼ 3481, 3296 (NH2), 2210 (CN), 1724, 1643 (2C]O); 1H NMR (300 MHz, DMSO-d6) d (ppm): 2.49 (s, 3H, CH3), 4.81, 4.92 (2s, 4H, 2NH2, D2O exchangeable), 6.15 (s, 1H, C5eH pyridinone ring) 7.52e 8.99 (m, 7H, AreH); 13C NMR (75 MHz, DMSO-d6) d (ppm): 23.1, 70, 84, 112, 115.5, 116.3 (2C), 121, 123, 126.2, 128.5 (2C), 130.4 (2C), 136.4, 145, 150, 155.3, 160, 172, 178; MS (EI, 70 eV) m/z (%) ¼ 385 (Mþ, 5.9), 239 (5.9), 195 (8.2), 180 (8.2), 170 (7.1), 157 (8.2). Anal. Calcd. for C21H15N5O3 (385): C, 65.45; H, 3.92; N, 18.17%. Found: C, 65.41; H 3.86; N 18.12%.
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5.7. Synthesis of 3,5-diamino-4-cyano-N-(1-(3-oxo-3H-benzo[f] chromen-2-yl)ethylidene)thiophene-2-carbohydrazide (15) To a solution of compound 3 (3.19 g, 0.01 mol) in absolute ethanol (25 mL) containing triethylamine (0.5 mL), malononitrile (0.66 g, 0.01 mol) and elemental sulfur (0.32 g, 0.01 mol) were added. The reaction mixture was heated under reflux for 2 h, and then allowed to cool, then poured onto ice/water and the formed solid product was collected by filtration and recrystallized from ethanol to give compound 15. Yellowish white powder; yield (68%); m.p. 230e232 C; IR (KBr): n/cm1 ¼ 3418, 3327 (NH2), 3212 (NH), 2197 (CN), 1713, 1678 (2C]O); 1H NMR (300 MHz, DMSO-d6) d (ppm): 2.65 (s, 3H, CH3), 6.98, 7.25 (2s, 4H, 2NH2, D2O exchangeable), 7.59e8.66 (m, 7H, Are H), 11.60 (s, 1H, NH, D2O exchangeable); 13C NMR (75 MHz, DMSOd6) d (ppm): 23, 81, 112, 115.6, 116, 117, 121, 123.4, 126.6, 128.5 (2C), 130.2 (2C), 136, 146, 150, 155.3, 158, 160 (2C), 174; MS (EI, 70 eV) m/z (%) ¼ 417 (Mþ, 14.78), 359 (63.98), 291 (81.74), 239 (52.17), 222 (73.04), 208 (46.96), 193 (48.70), 167 (56.52). Anal. Calcd. for C21H15N5O3S (417): C, 60.42; H, 3.62; N, 16.78%. Found: C, 60.40; H 3.58; N 16.74%. 5.8. Synthesis of 2-oxo-1-(1-(3-oxo-3H-benzo[f]chromen-2-yl) ethylideneamino)-5-phenyl-2,3-dihydro-1H-pyrrole-3-carbonitrile (16) A mixture of compound 3 (3.19 g, 0.01 mol) and phenacyl bromide (1.9 g, 0.01 mol) in dimethylformamide (20 mL) containing triethylamine (0.5 mL) was refluxed for 3 h, and then allowed to cool, then poured onto ice/water containing few drops of hydrochloric acid and the formed solid product was collected by filtration and recrystallized from ethanol to give compound 16. Yellow powder; yield (65%); m.p. 186e188 C; IR (KBr): n/ cm1 ¼ 2227 (CN), 1734, 1678 (2C]O); 1H NMR (300 MHz, DMSOd6) d (ppm): 2.65 (s, 3H, CH3), 4.54 (d, J ¼ 2.5 Hz, 1H, pyrrole H-3), 5.65 (d, J ¼ 2.5 Hz, 1H, pyrrole H-4), 7.61e8.65 (m, 12H, AreH); 13C NMR (75 MHz, DMSO-d6) d (ppm): 23, 38, 96, 112, 115.8, 116, 117, 121, 123.4, 126.5, 127.5, 128.4 (4C), 128.8 (2C), 130.2 (2C), 132, 136 (2C), 150, 155, 160, 172; MS (EI, 70 eV) m/z (%) ¼ 419 (Mþ, 15.80), 238 (30.20), 221 (59.60), 193 (32.80), 178 (19.40), 165 (31.60), 139 (38.00). Anal. Calcd. for C26H17N3O3 (419): C, 74.45; H, 4.09; N, 10.02%. Found: C, 74.44; H, 4.10; N 9.97%. 5.9. Synthesis of 4-amino-N-(1-(3-oxo-3H-benzo[f]chromen-2-yl) ethylidene)-3-phenyl-2-thioxo-2,3-dihydrothiazole-5carbohydrazide (17) To a solution of compound 3 (3.19 g, 0.01 mol) in absolute ethanol (25 mL) containing triethylamine (0.5 mL), elemental sulfur (0.32 g, 0.01 mol) and phenyl isothiocyanate (1.35 g, 0.01 mol) were added. The reaction mixture was heated under reflux for 3 h, and then allowed to cool, then poured onto ice/water and the formed solid product was collected by filtration and recrystallized from ethanol to give compound 17. Yellow powder; yield (68%); m.p. 180e182 C; IR (KBr): n/ cm1 ¼ 3440, 3360 (NH2), 3220 (NH), 1730, 1658 (2C]O); 1H NMR (300 MHz, DMSO-d6) d (ppm): 2.65 (s, 3H, CH3), 6.82 (s, 2H, NH2, D2O exchangeable), 7.59e8.66 (m, 12H, AreH), 11.65 (s, 1H, NH, D2O exchangeable); 13C NMR (75 MHz, DMSO-d6) d (ppm): 23, 73.8, 112, 115.6, 117.3, 121, 123, 126, 128 (3C), 129.4 (4C), 130 (2C), 134, 136.5, 150, 155, 160 (2C), 172, 186; MS (EI, 70 eV) m/z (%) ¼ 486 (Mþ 1, 90.82), 470 (58.16), 442 (58.16), 365 (87.76), 236 (71.43), 221 (74.90), 208 (77.55). Anal. Calcd. for C25H18N4O3S2 (486): C, 61.71; H, 3.73; N, 11.51%. Found: C, 61.63; H 3.76; N 11.49%.
425
5.10. Synthesis 2-amino-N-(1-(3-oxo-3H-benzo[f]chromen-2-yl) ethylidene)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carbohydrazide (18) To a solution of compound 3 (3.19 g, 0.01 mol) in absolute ethanol (25 mL) containing triethylamine (0.5 mL), elemental sulfur (0.32 g, 0.01 mol) and cyclohexanone (0.98 g, 0.01 mol) were added. The reaction mixture was heated under reflux for 3 h, and then allowed to cool, then poured onto ice/water and the formed solid product was collected by filtration and recrystallized from ethanol to give compound 18. Yellow powder; yield (58%); m.p. 220e222 C; IR (KBr): n/ cm1 ¼ 3421, 3390 (NH2), 3264 (NH), 1729, 1655 (2C]O); 1H NMR (300 MHz, DMSO-d6) d (ppm): 1.79e2.38 (m, 8H, 4CH2 benzothiophene), 2.65 (s, 3H, CH3), 6.49 (s, 2H, NH2, D2O exchangeable), 7.59e8.64 (m, 7H, AreH), 11.76 (s, 1H, NH, D2O exchangeable); 13C NMR (75 MHz, DMSO-d6) d (ppm): 17.3, 21, 23.2 (CH3), 23.5, 25.2 (4CH2), 112, 115.6 (2C), 117.4, 121, 123.2, 125.5, 126.4, 128.3 (2C), 130 (2C), 136.5, 139, 150, 155, 160, 162, 172; MS (EI, 70 eV) m/z (%) ¼ 431 (Mþ, 27.78), 277 (26.98), 263 (26.19), 252 (22.62), 219 (20.63), 204 (21.83), 193 (34.13), 165 (30.95). Anal. Calcd. for C24H21N3O3S (431): C, 66.80; H, 4.91; N, 9.74%. Found: C, 66.72; H 4.94; N 9.70%. 6. Antimicrobial evaluation The disks of Whatman filter paper were prepared with standard size (5.0 mm diameter) and kept into 1.0 Oz screw capped wide mouthed containers for sterilization. These bottles are kept into hot air oven at a temperature of 150 C. Then, the standard sterilized filter paper disks impregnated with a solution of the test compound in DMSO (1 mg/mL) were placed on nutrient agar plate seeded with appropriate test organism in triplicates. Standard conditions of 106 CFU/mL (Colony Forming U/mL) and 104 CFU/mL were used for antibacterial and antifungal assay, respectively. Pyrex glass petri dishes (9 cm in diameter) were used and two disks of filter paper were inoculated in each plate. The utilized test organisms were S. aureus as examples of Gram positive bacteria and E. coli as examples of Gram negative bacteria. They were also evaluated for their in vitro antifungal potential against C. albicans fungal strain. Ampicillin and Clotrimazole were used as standard antibacterial and antifungal agents, respectively. DMSO alone was used as control at the same above mentioned concentration and due this there was no visible change in bacterial growth. The plates were incubated at 36 C for 24 h for bacteria and for 48 h for fungi. Compounds that showed significant growth inhibition zones (>10 mm) using the two fold serial dilution technique, were further evaluated for their minimal inhibitory concentrations (MICs). 6.1. Minimal inhibitory concentrations (MIC) measurement The microdilution susceptibility test in Müller-Hinton Broth (Oxoid) and Sabouraud Liquid Medium (Oxoid) were used for the determination of antibacterial and antifungal activity, respectively. Stock solutions of the tested compounds, Ampicillin and Clotrimazole were prepared in DMSO at concentration of 1000 mg/mL. Each stock solution was diluted with standard method broth (Difco) to prepare serial two fold dilution at concentrations of (500, 250, ., 3.125 mg/mL), 10 mL of the broth containing about 106 CFU/mL of test bacteria was added to each well of 96-well microliter plate. The sealed microplates were incubated at 36 C for 24 h for antibacterial activity and at 36 C for 48 h for antifungal activity in a humid chamber. At the end of the incubation period, the minimal inhibitory concentrations (MICs) values were recorded as the lowest concentrations of the substance that had no visible turbidity.
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Control experiments with DMSO and uninoculated media were run parallel to the test compounds under the same conditions. Acknowledgment The authors are thankful to Department of Pharmacology, Faculty of Pharmacy, Mansoura University, Egypt for performing the antimicrobial evaluation. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.ejmech.2013.09.003. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10]
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