Some novel reserpine coupled 1,3,4-oxadiazole derivatives (RAH1-RAH12) were synthesized in good yields and evaluated for their possible antibacterial, anti-inflammatory activities. The structures of the synthesized compounds were confirmed on the basis of their spectral data and elemental analysis. Their antibacterial activities were evaluated by the disc diffusion method while their anti-inflammatory activities were evaluated by the carrageenan-induced rat paw edema test. All the tested compounds showed moderate to good antibacterial activity when compared with chloramphenicol and high to moderate anti-inflammatory activity when compared to indomethacin. The obtained results showed that the most active compounds could be useful as a template for future design, modification and investigation to produce more active analogs.
Keywords: Reserpine; 1,3,4-Oxadiazole; Anti-inflammatory activity; Antibacterial activity
The development of an effective therapeutic agent for the management of inflammation has undergone continual evolution leading to the emergence of more efficacious classes of drugs. Since the discovery of aspirin, much efforts have been devoted to the development of nonsteroidal anti-inflammatory drugs (NSAIDs), which are among the most widely prescribed medication in clinical practice despite their documented renal and gastrointestinal (GI) side effects. Conventional NSAIDs exert non-selective inhibition [1] of COX enzymes, the agents which catalyze the rate-limiting step in the formation of prostanoids from arachidonic acid. Such indiscriminate inhibition of COX-1 as well as COX-2 has been blamed for high incidence of GI irritation or, in the worst case, development of life threatening GI ulcers and bleeding in long term users of NSAIDs. Consequently, a second generation of NSAIDs has been developed which selectively inhibit COX-2. Being selective COX-2 inhibitors, these are expected to achieve the same antiinflammatory efficacy as traditional NSAIDs but minimize the risk of unwanted GI complications. Though the selective COX-2 inhibitors have minimal toxicity in the gastrointestinal tract, these agents can produce severe side effects in renal, hepatic, and cardiovascular systems.
The recent withdrawal of valdecoxib [2] and rofecoxib [3] has focused attention on the adverse cardiovascular effects of selective COX-2 inhibitors [4]. Thus, search for novel anti-inflammatory drugs with minimal GI side Discovery of potent and effective antibacterial agents represents one of the most important advances in therapeutics, not only in the control of serious infections, but also in the prevention and treatment of some infectious complications of other therapeutic modalities such as cancer chemotherapy and surgery. Over the past decade, microbial infection becomes an important complication and a major cause of morbidity and mortality in immuno-compromised individuals such as those suffering from tuberculosis, cancer and AIDS and in organ transplantation cases [5, 6]. Despite the availability of large number of antibacterial drugs, the increase in diseases attributed to drug-resistant and multidrug resistant (MDR) bacterial pathogens indicates a growing need for new antibacterial drugs. The most difficult types of infections to treat are those involving Gram-negative bacteria and mycobacteria, since these classes of bacteria often possess multiple mechanisms that make them resistant to entire classes of antibacterial drugs [7]. Consequently, the development of newer antimicrobial agents will remain an important challenging task for medicinal chemists.
It is reported that reserpine is having varied biological activity such as antihypertensive, sedative & hypnotics, tranquilizer, cure pain due to affection of bowels, in a labour to increase uterine contraction, asthma and dermatological disorders[8, 9]. Therefore, we have selected reserpine as a test molecule and prepared its semisynthetic analogues having hydrazide and 1,3,4- oxadiazole nucleus. 1,3,4- oxadiazole derivatives have like anti-tubercular, bacteriostatic, hypoglycemic, diuretic, anti-inflammatory, anti-viral, anti-fungal, sedative and hypnotic activity [10-13].
Till date the derivatives of reserpine having 1, 3, 4- oxadiazole nucleus has not been reported. Therefore it is thought worthwhile to prepare semisynthetic analogues of reserpine having 1, 3, 4-oxadiazole nucleus. The synthesized compounds have been characterized by various spectral analysis and these synthesized compounds were screened for anti-inflammatory and antibacterial activities.
All the starting materials and solvents used in the synthesis were obtained from Himedia Laboratories, Mumbai and S.D. Fine Chemicals and used without further purification. The homogeneity of the compounds was monitored by ascending thin layer chromatography (TLC) on silica gel-G (Merck) coated aluminum plates, visualized by iodine vapour/UV chamber. The melting points were determined in open capillary tubes. FT-IR 8400 Shimadzu Spectrometer (IR analysis), Waters Q-T of Micromass (ESI-MS) and Thermo Finnigan (elemental analysis) were used for analysis.
Solution of reserpic acid hydrazide (0.01 mole) in phosphorous oxychloride (3-5 mL) and several aromatic acid (0.01 mol) were added in the round bottom flask followed by refluxed for 5-8 hr. After completion of reaction (monitored by TLC), cooled to RT, mixture was neutralized with ice cold solution of 10% NaHCO , a solid 3 mass obtained was filtered and washed using ice cold water and crystallized from suitable solvents. The purity of synthesized compounds was checked by TLC.
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0 Nature, Brown solid; M.P. 316 C (decomposed); Yield: 71%; Mol. Formula: C29H32N4O4; Mol. Weight: 500.59; IR -1 (KBr, cm ): 3415 (OH), 3203 (NH str), 3068 (Ar-CH), 1556 + (C=N), 1118, 1067,989 (C-O-C) M/S 501.40 [M+1] ; % Anal. Cal. for C29H32N4O4; C, 69.58; N, 11.19; H, 6.44; Found: C, 60.445; N, 9.908; H, 5.332.
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(RAH2) 0 Nature, Brown solid; M.P.280 C (decomposed); Yield: 74 %; Mol. Formula: C29H31 BrN4O4; Mol. Weight: 578.15; IR -1 (KBr, cm ): 3303 (NH str), 3225(OH), 2938 (Ar-CH),1631 + (C=N), 1158, 1072, 1010 (C-O-C), M/S 579.58 [M+1] ; % Anal. Cal. for C H BrN O ; C, 60.11; N, 9.67; H, 5.39; Found: C, 60.445; N, 9.908; H, 5.332.
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(RAH ) 3 0 Nature, Blackish brown solid; M.P. 360 C (decomposed); Yield: 79%; Mol. Formula: C H N O ; Mol. Weight:
-1 545.23; IR (KBr, cm ): 3441 (NH str), 3374 (OH), 3126 (Ar-CH), 1709 (C=N), 1578 (NO ), 1136, 1120, 1067, 993 2 + (C-O-C); M/S5 46.58 [M+1] ; % Anal. Cal. for C H N O ; C, 63.84; N, 12.84; H, 5.73; Found: C, 62.565; N, 11.998; H, 6.5.117.
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(RAH ) 4 0 Nature: Dark brown powder; M.P. 296 C (decomposed); Yield: 86%; Mol. Formula: C H ClN O ; Mol. Weight:
-1 534.20; IR (KBr, cm ): 3219 (NH str), 2938 (OH), 2843 (Ar-CH), 1631 (C=N), 1157, 1092, 1013 (C-O-C), 739 (C- + Cl); M/S 535.43 [M+1] ; % Anal. Cal. for C H ClN O ; C, 65.10; N, 10.47; H, 5.84; Found: C, 65.790; N, 10.868; H, 5.587.
’ [2 ,3’:3,4]pyrido[1,2-b]isoquinolin-3-ol (RAH5) 0 Nature: Brown solid; M.P. 330 C (decomposed); Yield:56%; Mol. Formula: C30H34N4O4; Mol. Weight:
-1 514.26; IR (KBr, cm ): 3240 (NH str), 2940 (Ar-CH), 1630 (C=N),1467(C=C), 1160, 1088, 912 (C-O-C), 763 (C-H + bending);M/S 515.49 [M+1] ; % Anal. Cal. for C30H34N4O4; C, 70.02; N, 10.89; H, 6.66; Found: C, 70.340; N, 10.675; H, 6.989.
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(RAH6) 0 Nature: Dark brown solid; M.P. 346 C (decomposed); Yield: 72%; Mol. Formula: C29H32N4O5; Mol.
-1 Weight:516.24; IR (KBr, cm ): 3326(NH), 3240 (OH str), 2942 (Ar-CH), 1630 (C=N), 1477(C=C); 1344 (OH bending), 1104, 980 (C-O-C), 769 (C-H bending); M/S + 517.52 [M+1] ; % Anal. Cal. for C29H32N4O5; C, 67.43; N, 10.85; H, 6.24; Found: C,67.670; N, 11.108; H, 6.188.
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0 Nature: brown solid; M.P. 292 C (decomposed); Yield: 75%; Mol. Formula: C H N O ; Mol. Weight: 545.59; IR -1 (KBr, cm ): 3444 (OH str), 3369 (NH), 3076 (Ar-CH), 1658 (C=N), 1556 (NO ) ,1408 (C=C), 1346 (NO ), 1118, 989, 2 2 + 948 (C-O-C); M/S 546.54 [M+1] ; % Anal. Cal. for C H N O ; C, 63.84; N, 12.84; H, 5.73; Found; C, 62.433; N, 12.932; H, 5.142.
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0 Nature: Dark brown solid; M.P. 296 C (decomposed); Yield: 79%; Mol. Formula: C29H31BrN4O4; Mol. Weight:
-1 578.15; IR (KBr, cm ): 3284 (OH str), 3219 (NH), 2981 (Ar-CH), 1620 (C=N), 1423(C=C), 1199, 1134, 1072, 1034 -1 + (C-O-C), 865 (Ph), 651 (C-Br) Cm ; M/S 579.57 [M+1] ; % Anal. Cal. for C29H31BrN4O4; C, 60.11; N, 9.67; H, 5.39; Found: C, 60.580; N, 9.117; H, 5.098.
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(RAH9) 0 Nature: Yellowish brown solid; M.P. 297 C (decomposed); Yield: 71%; Mol. Formula: C29H31BrN4O4; Mol. Weight:
-1 578.15; IR (KBr, cm ): 3437 (OH str), 3143 (NH), 2981 (Ar-CH), 1664,1558 (C=N), 1415(C=C), 1120, 991 (C-O- + C); M/S 579.50 [M+1] ; % Anal. Cal. for C29H31BrN4O4; C, 60.11; N, 9.67; H, 5.39; Found: C, 60.250; N, 9.248; H, 5.757.
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10 0 Nature: Dark brown solid; M.P. 326 C (decomposed); Yield: 73%; Mol. Formula: C H N O ; Mol. Weight:
-1 512.24; IR (KBr, cm ): 3229 (NH), 2941 (Ar-CH), 1621, 1573 (C=N), 1460(C=C), 1163, 1082, 929 (C-O-C), 826 + (Ph); M/S 513.66 [M+1] ; % Anal. Cal. for C H N O ; C, 70.70; N, 10.64; H, 6.51; Found: C, 70.389; N, 10.255; H, 6.899.
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(RAH11) 0 Nature: Blackish brown solid; M.P.350 C (decomposed); Yield: 65%; Mol. Formula: C29H30N6O8; Mol. Weight:
-1 590.21; IR (KBr, cm ): 3379 (OH), 3298 (NH), 2981 (ArCH), 1600 (C=N), 1336 (NO2), 1168 (C-O-C str. oxadiazole), 1168 (C-O-C str. oxadiazole), 1066 (N-N str.
+ oxadiazole), 952 (C-O-C), 868 (Ph); M/S 591.57[M+1] ; % Anal. Cal. for C29H30N6O8; C, 58.98; N, 14.23; H, 5.12; Found: C, 57.230; N, 15.852; H, 5.128.
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(RAH12) 0 Nature: Yellowish brown solid; M.P. 310 C (decomposed); Yield: 77%; Mol. Formula: C27H30N4O5; Mol. Weight:
-1 490.55; IR (KBr, cm ): 3348 (OH), 3223 (NH), 2993 (ArCH), 1647 (C=N), 1568 (NO2), 1116, 1020, 1053, 987 (C- + O-C oxadiazole & furan), 864 (Ph); M/S 490.55 [M+1] ; % Anal. Cal. for C27H30N4O5; C, 66.11; N, 11.42; H, 6.16; Found: C, 65.175; N, 11.258; H, 6.257.
Wistar rats of one of either sex (180–200 g) were used excluding pregnant. In one group 6 animals are present. The animals were kept in RT and food and water were given to acclimatized for 2 days. This activity was done by the method described by winter et al [14] (paw oedema method). The tests were evaluated by studying inflammatory response in the rats by injecting Carrageenan, which produces edema. The compounds were evaluated by examining their ability to diminish or prevent the edema. Indomethacin was used as standard drug for this study.
Nutrient agar and Vogel Johnson agar plates were prepared by pouring 15-20 ml of the medium into each sterilized Petri 0 dishes (washed and dried in hot air oven at 160 C for 1 hour) and were allowed to solidify at room temp (RT). The medium was seeded with organism by spread plate method using sterile cotton swabs. Three cups were scooped in each plate using a sterile cork borer of 8 mm diameter. Then the solution of test compounds (0.06 ml, 0.08 ml and 0.10 ml) was added to the respective bores by using micropipettes. 0.06 ml, 0.08 ml and 0.10 ml of the chloramphenicol were taken as a standard reference. DMSO was used as control.
0 The Petri dishes were incubated at 37 C for 24 h and zoneinhibition was observed and measured using scale (in mm) for each organism [15, 16]. E. coli and S. aureus were used as microorganism for testing of the synthesized compounds. The media was used for both sub-culturing and also for estimating antibacterial activity. Chloramphenicol was used as standard for antibacterial activity.
Reserpic acid hydrazide (RAH) was synthesized by reacting reserpine with hydrazine hydrate as per reported method [17]. We expanded our series and synthesized twelve 1,3,4-oxadiazole derivatives of reserpic acid hydrazide (RAH1-RAH12). The compounds RAH1- RAH12 were obtained by reacting compound RAH with
various substituted aromatic acids (Scheme 1). The isolated yields of synthesized compounds were in the range of 71- 86 % and reactions were completed in about 5-8 hr (monitored by TLC). The compounds were purified by recrytallization and purity was checked by TLC method. Melting points E: ethyl acetate; H: n-hexane; C: chloroform; M: methanol.
were determined in open capillary tubes. The physical data for the compounds are presented in Table 1. The formation of synthesized compounds was confirmed by means of IR, Mass and elemental analysis spectroscopy and data suggested for proposed structures.
1 12 isoamyl alcohol, hydrazine hydrate, few drops of acetic acid Ar-COOH
N H CO 3 N H O Reserpine 36 hr reflux, N H CO 3 N H O C NHNH Reserpic acid hydrazide N H CO 3 N OH + H O C OCH 3 NHNH 2 POCl 3 Reflux N H3CO N H OH 3 H N OCH 3 N O Ar H CO OCH 3 3 H CO 3 C O O C OCH 3 OCH 3 H CO OCH 3 3 + OH H CO 3 OCH O 3 H NHN 2 2 3,4,5-tri-methoxy-benzoic acid hydrazide Where Ar 2) 1) 3) 4) Br O N 2 Cl OH 5) 6) 7) 8) C Br NO 2 9) 10) O N NO 2 2 O 11) 12) Br CH C H
1 12
Antiinflammatory activity was carried at time intervals 1hr (Table 2), 2hr (Table 3) and 3hr (Table 4). All compounds have shown anti-inflammatory activity.
activity when compared with standard drug (indomethacin) at the dose of 10 mg/kg after 1 hour. In the second hour of inhibition, compounds RAH4, RAH , RAH , RAH and RAH have shown increased inhibition as compared to the first hour. The remaining compounds have shown decrease in inhibition as compared to the first hour. In the third hour of inhibition, compounds RAH , RAH , RAH , RAH , 2 3 7 8 RAH9, RAH10 and RAH12 have shown the increased inhibition as compared to the second hour. The compounds RAH7 and RAH11 were found to show less activity among the derivatives. Overall result of antiinflammatory activity showed that the reserpine scaffold and 1,3,4-oxadiazole ring seem to be pharmacophore and other substituents may play a role in enhancing the activity. The % inhibition is plotted against the test compound to compare anti-inflammatory with standard indomethacin (Figure 1).
The synthesized compounds (RAH1- RAH12) have been screened for antibacterial against E. coli and S. aureus. The synthesized compounds have shown moderate to good antibacterial activity. The compounds RAH1, RAH4, RAH8 and RAH10 found to have a very good antibacterial activity for E. coli when compared with Chloramphenicol (standard drug). RAH2 has also shown equipotent activity against E. coli. The compound RAH3 has shown more potent activity than standard for E. coli. RAH8 has shown same activity at dose of 80 μg/ml against E. coli. The compounds RAH and RAH have shown comparable 2 8 activity for S. aureus when compared with Chloramphenicol (standard drug). Compound RAH has 3 shown same activity for S. aureus when compared with standard drug. Also compound RAH has shown broad 3 spectrum antibacterial activity when compared with standard Chloramphenicol.
1 12 1 12 RAH 3 5 6 OH RAH 3 5 7 NO 2 RAH8 6 9 Br RAH9 4 5 Br RAH 5 8 10 CH C H NO RAH11 2 1 3 NO 2 RAH12 O 2 5 Standard Chloramphenicol 7 9
A series of novel compounds titled RAH1-RAH12were prepared in good yield and purity was checked by TLC.
The synthesized compounds structures were established and characterized by spectral analysis like IR, Mass and elemental spectral analysis etc. The synthesized compounds were evaluated for anti-inflammatory activity and compared with standard Indomethacin. Most of the synthesized compounds were activity comparable to standard. The synthesized compounds were also tested for in vitro antibacterial activity by disc diffusion method against one Gram-negative microorganism viz., E. coli (NCTC 10418), and one Gram-positive microorganism viz., S. aureus (NCTC 6571). Chloramphenicol was used as standard drug for antibacterial activity. The synthesized compounds were shown broad spectrum of antibacterial activity. The compounds have shown moderate to good activity. Also compound RAH has 3 shown broad spectrum antibacterial activity when 7 2 4 6 6 5 7 8 7 4 7 10 5 1 3 6 8 1 2 5 research work can be further studied and optimized for development of new antibacterial, and anti-inflammatory agents.
The authors wish to express their gratitude to President, Prof. Javed Khan and Managing Director, Mr Waseem Khan, Oriental Education Society and Dr. (Mrs) Sudha Rathod, Principal, Oriental College of Pharmacy, Sanpada, Navi Mumbai for providing instrumental facility and other necessary support to carry out the experiment. Amjad Ali is also very much thankful to Principal, Dr. Ashish Kumar Sharma, Institute of Pharmacy, NIMS University, Jaipur, Rajasthan for providing all the facilities and support to carry out this research work. Our sincere acknowledgments to SAIF (Sophisticated Analytical Instrument Facility), IIT, Powai, Mumbai, and SAIF, Punjab University, Chandigarh for providing spectral analysis of my synthesized compounds.