Molecular properties prediction, synthesis and antimicrobial activity of some newer oxadiazole derivatives

European Journal of Medicinal Chemistry 45 (2010) 5862e5869

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Original article

Molecular properties prediction, synthesis and antimicrobial activity of some newer oxadiazole derivatives Mohammed Afroz Bakht a, *, M. Shahar Yar b, Sami Gaber Abdel-Hamid a, Saleh I. Al Qasoumi a, Abdul Samad a a b

Department of Pharmaceutical Chemistry, College of Pharmacy in Al-Kharj, King Saud University, P.O. Box 2457, Riyadh, Saudi Arabia Departments of Pharmaceutical Chemistry, Faculty of Pharmacy, Hamdard University, New Delhi 110062, India

a r t i c l e i n f o

a b s t r a c t

Article history: Received 8 April 2010 Received in revised form 1 July 2010 Accepted 2 July 2010 Available online 1 October 2010

In present investigation a series of 28 oxadiazole analogues (AB1eAB28) were subjected to molecular properties prediction, drug-likeness by Molinspiration (Molinspiration, 2008) & MolSoft (MolSoft, 2007) softwares, lipophilicity and solubility parameters using ALOGPS 2.1 program. Out of 28 analogues only 16 were chosen on the basis of Lipinski “Rule of Five” (Ro5) for the synthesis and antimicrobial screening as oral bioavailable drugs/leads. Maximum drug-likeness model score (1.22) was found to be of compound AB13. Selected compounds (AB1eAB2), (AB5eAB9), (AB12eAB16), (AB18eAB21) were synthesized and characterized by IR, NMR and mass spectral analysis followed by antibacterial and antifungal screening. It was observed that compounds showed moderate to good antibacterial activity, but their antifungal activity was somewhat moderate. Compounds AB13 and AB20 showed pronounced activity against all bacterial and fungal strains. We had noticed that compounds (AB13, AB20) bearing OH group at one of the phenyl ring of oxadiazole exhibited good antimicrobial properties and their drug-likeness model score were also predicted maximum among the series. Ó 2010 Elsevier Masson SAS. All rights reserved.

Keywords: 1,3,4-Oxadiazole Molecular properties prediction Lipinski-Rule of 5 Drug-likeness Antimicrobial screening

1. Introduction Antimicrobial agents are considered “miracle drugs” that are our leading weapons in the treatment of infectious diseases. Antimicrobial resistance is the ability of certain microorganisms to withstand attack by antimicrobials, and the uncontrolled rise in resistant pathogens threatens lives and wastes limited healthcare resources. Life-treating infectious diseases caused by multidrug-resistant Gram-positive and Gram-negative pathogen bacteria increased an alarming level around the world. Owing to this increased microbial resistance, new classes of antibacterial agents with novel mechanisms are crucial need to combat with the multidrug-resistant infections. The widespread use of 1,3,4-oxadiazoles as a scaffold in medicinal chemistry establishes this moiety as a member of the privileged structures class [1]. 1,3,4-Oxadiazole ring is associated with many types of biological properties such as anti-inflammatory [2e4], hypoglycemic [5], antifungal, antibacterial [6e8] and insecticidal [9,10] activities. The major side effects in the use of aryl alkanoic acids is their gastric irritation, which is partly due to the corrosive nature of carboxylic acid group present in them. In order to reduce or * Corresponding author. . Tel.: þ966 553753763; fax: þ966 14670560. E-mail address: [email protected] (M.A. Bakht). 0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.ejmech.2010.07.069

mask the side effects of carboxylic moiety we planned to synthesize different 2,5-disubstituted-1,3,4-oxadiazoles via the condensation of appropriate acid hydrazides with various chalcones containing free carboxylic acid group in presence of phosphorus oxychloride in the hope of getting potent antimicrobial agents. In the development of drugs intended for oral use, good drug absorption and appropriate drug delivery are very important [11]. About 30% of oral drugs fail in development due to poor pharmacokinetics [12]. Among the pharmacokinetic properties, a low and highly variable bioavailability is indeed the main reason for stopping further development of the drug [13]. Thus, predictions of bioavailability and bioavailability-related properties, such as solubility, lipophilicity are important before actual synthesis, in order to reduce enormous wastage of expensive chemicals and precious time. An in silico model for predicting oral bioavailability is very important, both in the early stage of drug discovery to select the most promising compounds for further optimization and in the later stage to identify candidates for further clinical development [13]. In present investigation a series of 28 oxadiazole analogues (AB1eAB28) were subjected to molecular properties prediction, drug-likeness by Molinspiration [32] & MolSoft (MolSoft, 2007) softwares, lipophilicity and solubility parameter by using ALOGPS 2.1 program to filter the compounds for further synthesis and antimicrobial screening.

M.A. Bakht et al. / European Journal of Medicinal Chemistry 45 (2010) 5862e5869

2. Molecular properties and drug-likeness A molecular property is a complex balance of various structural features which determine whether a particular molecule is similar to the known drugs. It generally means “molecules which contain functional groups and/or have physical properties consistent with most of the known drugs”. These properties, mainly hydrophobicity, molecular size, flexibility and presence of various pharmacophoric features influence the behavior of molecules in a living organism, including bioavailability. Computational chemists have a wide array of tools and approaches available for the assessment of molecular diversity. Diversity analysis has been shown to be an important ingredient in designing drugs. So, computational sensitivity analysis and structural analysis have been used to study the drug-likeness of the candidate drug. As good bioavailability can be achieved with an appropriate balance between solubility and partitioning properties. Thus in order to achieve good oral drugswe have subjected a series of 1,3,4-oxadiazole derivatives (AB1eAB28) for the prediction of lipophilicity, solubility and Lipinski “Rule of Five” [14] and other properties for filtering compounds for subsequent synthesis and antimicrobial screening.

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at Syracuse Research Corporation [24]. The XLOGP2 is an atomadditive method applying corrections [25,26]. Computed partition coefficients (XLOGP2 method) for drugs studied varied between 3.71e6.80 and by KoW-WIN method varied between 2.75 and 6.00 (Table 1). The LogKow (Kow-WIN) method is best supported for most of the compounds on the basis of lipophilicity (5) to consider an oral drug/lead. 2.2. Solubility An insufficient aqueous solubility is likely to impede bioavailability of the drugs. Drug solubility is one of the important factors, which affect the movement of a drug from the site of administration into the blood. It is well known that insufficient solubility of drugs can lead to poor absorption [27]. Investigation of the ratelimited steps of human oral absorption of 238 drugs [27] showed that the absorption of a drug is usually very low if the calculated solubility is <0.0001 mg/l. As per this criterion compounds of series (AB1eAB28) almost fulfill the requirements of solubility (ALOGPS) and could be considered as the candidate drugs (Table 1). 2.3. Absorption, polar surface area, and “Rule of five” properties

2.1. Lipophilicity Poor solubility and poor permeability are amongst the main causes for failure during drug development [15e17]. It is therefore, important to determine these physico-chemical properties associated with a drug, before synthetic work is undertaken. The computed logP values (P is the partition coefficient of the molecule in the watereoctanol system) are shown in Table 1. The ALOGPS method is part of the ALOGPS 2.1 program [18] used to predict lipophilicity [19] and aqueous solubility [20,21] of compounds. The lipophilicity calculations within this program are based on the associative neural network approach and the efficient partition algorithm. The LogKow (Kow-WIN) program [22,23] estimates the log octanol/water partition coefficient (logP) of organic chemicals and drugs using an atom/fragment contribution method developed Table 1 Calculated partition coefficients and solubilities of the oxadiazoles investigated. Compounds

ALOGPS

KoW-WIN

XLOGP2

AB1 AB2 AB3 AB4 AB5 AB6 AB7 AB8 AB9 AB10 AB11 AB12 AB13 AB14 AB15 AB16 AB17 AB18 AB19 AB20 AB21 AB22 AB23 AB24 AB25 AB26 AB27 AB28

4.54 (11.87 mg/l) 4.70 (8.83 mg/l) 4.47 (16.74 mg/l) 4.59 (11.89 mg/l) 4.60 (10.61 mg/l) 4.27 (22.82 mg/l) 4.53 (12.99 mg/l) 4.52 (13.49 mg/l) 4.67 (10.15 mg/l) 4.40 (20.99 mg/l) 4.54 (14.14 mg/l) 4.56 (12.61 mg/l) 4.22 (27.65 mg/l) 4.47 (16.03 mg/l) 4.28 (22.45 mg/l) 4.46 (15.97 mg/l) 4.23 (30.12 mg/l) 4.39 (19.44 mg/l) 4.35 (19.63 mg/l) 4.02 (42.58 mg/l) 4.23 (27.11 mg/l) 4.61 (11.16 mg/l) 4.74 (8.95 mg/l) 4.51 (16.61 mg/l) 4.61 (12.44 mg/l) 4.66 (10.26 mg/l) 4.42 (18.12 mg/l) 4.56 (13.43 mg/l)

6.00 4.46 4.60 3.53 4.26 3.23 3.74 3.80 4.44 4.69 3.61 4.34 3.32 3.82 3.23 3.88 4.12 3.05 3.78 2.75 3.26 3.53 4.18 4.42 3.35 4.08 3.05 3.56

3.71 6.62 6.80 5.89 6.44 5.59 5.68 5.91 6.54 6.71 5.81 6.35 5.51 5.60 5.59 6.22 6.39 5.49 6.03 5.19 5.28 5.89 6.51 6.69 5.78 6.33 5.49 5.57

High oral bioavailability is an important factor for the development of bioactive molecules as therapeutic agents. Good intestinal absorption, reduced molecular flexibility (measured by the number of rotatable bonds), low polar surface area or total hydrogen bond count (sum of donors and acceptors), are important predictors of good oral bioavailability [28,29]. Molecular properties such as membrane permeability and bioavailability is always associated with some basic molecular descriptors such as logP (partition coefficient), molecular weight (MW), or hydrogen bond acceptors and donors counts in a molecule [30]. Lipinski [14] used these molecular properties in formulating his “Rule of Five”. The rule states that most molecules with good membrane permeability have logP 5, molecular weight 500, number of hydrogen bond acceptors 10, and number of hydrogen bond donors 5. This rule is widely used as a filter for drug-like properties. Table 2 contains calculated percentage of absorption (%ABS), molecular polar surface area (PSA) and Lipinski parameters of the investigated compounds of the series (AB1eAB28). Magnitude of absorption is expressed by the percentage of absorption. Absorption percent was calculated [25] using the expression: %ABS ¼ 109  0.345 PSA. Polar surface area (PSA) was determined by the fragment-based method of Ertl and coworkers [31,32]. A poor permeation or absorption is more likely when there are more than 5H bond donors, 10 H-bond acceptors. Hydrogen-bonding capacity has been also identified as an important parameter for describing drug permeability [29]. The series (AB1eAB28) under investigation has most of the compounds possessing less number of hydrogen bond donors (5) but do possess considerable number of acceptors (10) as shown in Table 2. Number of rotatable bonds is important for conformational changes of molecules under study and ultimately for the binding of receptors or channels. It is revealed that for passing oral bioavailability criteria, number of rotatable bond should be 10 [28]. The compounds in this series (AB1eAB28) in general possess high number of rotatable bonds (7e10) and therefore, exhibit large conformational flexibility. Molecular polar surface area (PSA) is a very useful parameter for the prediction of drug transport properties. PSA is a sum of surfaces of polar atoms (usually oxygen, nitrogen and attached hydrogen) in a molecule. PSA and volume is inversely proportional to %ABS. Compound AB1, AB2, AB3 and AB5 have maximum absorption (88.28%) as their corresponding polar surface area and volume are least among the series (Table 2). Drug-likeness model score

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Table 2 Calculated absorption (%ABS), polar surface area (PSA), Lipinski parameters and drug-likeness model score of the oxadiazoles investigated. Compounds

%ABS

Volume (A3)

PSA (A2)

NROTB

HBA

HBD

log P, Calcd.

Formula weight

Drug-likeness model score

AB1 AB2 AB3 AB4 AB5 AB6 AB7 AB8 AB9 AB10 AB11 AB12 AB13 AB14 AB15 AB16 AB17 AB18 AB19 AB20 AB21 AB22 AB23 AB24 AB25 AB26 AB27 AB28

88.28 88.28 88.28 76.76 88.28 82.33 85.61 85.49 85.39 85.39 73.87 85.39 79.44 82.77 82.33 82.33 82.33 69.59 82.33 76.38 79.66 76.76 76.76 76.35 65.23 76.76 70.81 74.09

410.19 427.38 432.04 435.18 431.13 421.34 440.44 442.03 459.23 463.89 467.02 462.97 453.18 472.29 420.74 437.93 442.59 450.93 441.68 431.89 450.99 435.18 452.37 459.41 460.16 456.12 446.33 465.43

60.07 60.07 60.07 93.47 60.07 77.31 67.80 68.15 68.45 68.45 101.85 68.45 85.69 76.19 77.33 77.33 77.33 114.25 77.33 94.57 85.07 93.47 93.47 94.66 126.87 93.47 110.71 101.21

8 8 9 8 8 10 9 9 9 10 9 10 11 8 8 8 9 8 8 11 9 9 9 10 8 9 12 8

6 6 6 8 6 7 7 7 7 7 9 7 8 8 7 7 7 9 7 8 8 8 8 7 10 8 9 9

0 0 0 0 0 1 0 0 0 0 0 0 1 0 1 1 1 2 1 2 1 0 0 1 0 0 1 0

3.99 4.68 4.88 3.89 4.45 3.55 2.96 4.05 4.73 4.94 3.95 4.51 3.60 3.01 3.53 4.22 4.42 3.38 3.99 3.08 2.50 3.89 4.58 4.97 3.79 4.35 3.45 2.86

412 446 490 457 426 428 442 442 476 522 487 456 458 472 428 464 507 473 442 444 458 457 492 537 502 471 473 487

0.55 0.39 0.02 0.68 0.44 0.92 0.82 0.70 0.42 0.09 0.60 0.45 1.22 0.90 0.63 0.40 0.09 0.20 0.44 1.06 0.83 0.68 0.71 1.77 0.87 8.15 0.30 0.48

(a combined effect of physico-chemical properties, pharmacokinetics and pharmacodynamics of a compound and is represented by a numerical value) was computed by MolSoft (MolSoft, 2007) software for the 28 molecules under study. As shown in Fig. 1 the green color indicates non drug-like behavior and those fall under blue color are considered as drug-like. Computed drug-likeness scores are presented in Table 2. Compounds having zero or negative value should not be considered as drug-like. Maximum drug-likeness score was found out to be 1.22 for compound AB13. After successful prediction of molecular properties of the series (AB1eAB28) under investigation, 16 compounds were selected out of 28 for the next steps of synthesis and antimicrobial screening. On the basis of drug-likeness model score compounds (AB3, AB4, AB10, AB11, AB17 and AB22eAB28) failed to be treated like candidate drugs. So it was observed that those compounds (AB3, AB4, AB10, AB11, AB17) having nitro/bromo substituted phenyl group at 5th position of 1,3,4-oxadiazole moiety and compounds (AB22eAB28) having nitro substituted phenyl group at 2nd position of oxadiazole moiety failed to comply with the parameters of Lipinski-Rule of five (Ro5) and their drug-likeness model score was also not adequate to be treated like candidate drugs.

values 8.3e15.7 Hz. Similarly a singlet appeared at d 3.7e3.6 owing to the protons of the methoxy group and the OCH2 protons resonated as a singlet between d 4.8e4.2. The mass spectrum of compounds are characterized by molecular ion peak at m/z (Mþ1or M1), which is in agreement with their respective molecular

3. Results and discussion 3.1. Chemistry The compounds (IIIeV) in 1H NMR spectrum exhibited two doublets with J values between 13.0 and 12.6 Hz conforming the trans coupling. Formation of 1,3,4-oxadiazoles {(AB1eAB2), (AB5eAB9), (AB12eAB16), (AB18eAB21)} was confirmed by recording their IR, 1 H NMR and mass spectra. In general IR spectrum of compounds showed absorption at 1561e1549 cm1 due to the C]N group, band at 1171e1155 cm1 due to stretching of oxadiazole ring. The 1H NMR spectrum of compounds in general showed multiplet in the region of d 8.7e6.8 due to aromatic proton. At d 7.2e6.1 appeared as two doublets due to alkenyl proton with J

Fig. 1. Graph showing compound (AB13) of maximum drug-likeness model score using MolSoft 2007.

M.A. Bakht et al. / European Journal of Medicinal Chemistry 45 (2010) 5862e5869

formulas. The spectral values for all the compounds and C, H, N analysis are given in the Experimental part. 3.2. Pharmacology 3.2.1. Antibacterial activity The investigation of antibacterial screening data revealed that all the tested compounds showed moderate to good bacterial inhibition. Compounds AB6, AB13, AB14, AB15 and AB20 showed good bacterial inhibition. Most of them exhibited good antibacterial activity largely against Escherichia coli and Pseudomonas aeruginosa. Compound AB13 showed almost equivalent antibacterial activity to standard Ciprofloxacin. MBC of compound AB13 was found to be same as MIC, against Staphylococcus aureus, but in most of the compounds MBC was two, three or four folds higher than their corresponding MIC values. Compound AB13 showed good antibacterial activity against almost all bacterial strains. 3.2.2. Antifungal activity Antifungal screening data indicates that most of the compounds showed moderate activity. Among the screened compounds, AB13 and AB20 showed good inhibition against both of selected fungal strains i.e. Aspergillus niger and Candida albicans. MFC of most of the compounds was found to be two to four folds higher than their corresponding MIC results. 4. Conclusion A series (AB1eAB28) of 1,3,4-oxadiazoles were subjected for the prediction of molecular properties and drug-likeness by different softwares in order to find suitable molecules for the synthesis and antimicrobial screening. Among the series only 16 compounds {(AB1eAB2), (AB5eAB9), (AB12eAB16), (AB18eAB21)} were chosen on the basis of molecular properties and drug-likeness score for oral bioavailability. Selected compounds {(AB1eAB2), (AB5eAB9), (AB12eAB16), (AB18eAB21)} were subjected for the synthesis and antimicrobial screening. The synthesis of 1,3,4-oxadiazole derivatives {(AB1eAB2), (AB5eAB9), (AB12eAB16), (AB18eAB21)} was governed by reacting newly formed chalcones (IIIeV) which were previously prepared by reacting 2-(4-formyl-2-methoxyphenoxy) acetic acid (I) and acetophenones (II). Condensation of newly formed chalcones (IIIeV) with different acid hydrazides yielded respective oxadiazole derivatives {(AB1eAB2), (AB5eAB9), (AB12eAB16), (AB18eAB21)}. All the synthesized compounds were screened for antibacterial and antifungal activity by adopting standard protocol. On the basis of

results obtained from antimicrobial screening it was found that compound AB13, (E)-3-(4-((5-(2-hydroxyphenyl)-1,3,4-oxadiazol-2-yl) methoxy)-3-methoxyphenyl)-1-(4-methoxy phenyl)prop-2-en-1one and AB20, (E)-3-(4-((5-(2-hydroxyphenyl)-1,3,4-oxadiazol-2-yl) methoxy)-3-methoxyphenyl)-1-(4-hydroxyphenyl)prop-2-en-1-one were most active of the series. Examining closely on substitutions, it may be concluded that role of electron donating groups (eOH) on the phenyl ring of 1,3,4-oxadiazole has great influence on antimicrobial activity. This result is further supported by the fact that their molecular property and drug-likeness model score were maximum among the series. Finally it is conceivable that further derivatization of such compounds will be of great interest with the hope to get more selective antimicrobial agents. 5. Chemistry The synthesis of 1,3,4-oxadiazole derivatives {(AB1eAB2), (AB5eAB9), (AB12eAB16), (AB18eAB21)}, described in this study are outlined in Scheme 1 and physical data is presented in Table 3. The chalcones (IIIeV) were prepared by reacting 2-(4-formyl-2methoxyphenoxy) acetic acid (I) and appropriate acetophenone (II) in the presence of a base by conventional ClaiseneSchmidt condensation. Reaction between newly synthesized chalcones with appropriate acid hydrazide in 5 ml of phosphorus oxychloride (POCl3) gave 1,3,4-oxadiazoles in good yield. The compounds were recrystallized from ethanol. The purity of the compounds was checked by TLC. Spectral data 1H NMR, IR of all the synthesized compounds and Mass spectra of selected compounds were recorded and found in full agreement with the proposed structures. The elemental analysis results were within 0.4% of the theoretical values. 6. Pharmacology 6.1. Antibacterial studies The newly prepared compounds were screened for their antibacterial activity against E. coli (ATCC-25922), S. aureus (ATCC25923), P. aeruginosa (ATCC-27853) and Bacillus subtilis (ATCC6633) (recultured) bacterial strains by disc-diffusion method [33,34]. A standard inoculum (1e2  107 c.f.u./ml 0.5 McFarland standards) was introduced on to the surface of sterile agar plates, and a sterile glass spreader was used for even distribution of the inoculum. The discs measuring 6.25 mm in diameter were prepared from Whatman no. 1 filter paper and sterilized by dry heat at 140  C HX

HO

O

+ O

O

I

CH3

O

KOH / CH3OH

H3C

CHO

5865

R

HB

O

O

II

HO

O O

R

(III-V)

R = H, OCH 3, OH

CH3

R'CONHNH2 POCl3 O

HX

N

HB

O

N O

O

CH3

R'

(AB1-AB2), (AB5-AB9), (AB12-AB16), (AB18-AB21) Scheme 1. Schematic representation for synthesis of 1,3,4-oxadiazoles.

R

5866

M.A. Bakht et al. / European Journal of Medicinal Chemistry 45 (2010) 5862e5869

Table 3 Physical constants of the synthesized compounds. Compound No.

R

R0

Yield (%)

Molecular Weight

Melting point ( C)

AB1 AB2 AB5 AB6 AB7 AB8 AB9 AB12 AB13 AB14 AB15 AB16 AB18 AB19 AB20 AB21

H H H H H OCH3 OCH3 OCH3 OCH3 OCH3 OH OH OH OH OH OH

C6H5 p-CleC6H4 p-CH3eC6H4 o-OHeC6H4 C6H5eOCH2 C6H5 p-CleC6H4 p-CH3eC6H4 o-OHeC6H4 C6H5eOCH2 C6H5 p-CleC6H4 p-NO2eC6H4 p-CH3eC6H4 o-OHeC6H4 C6H5eOCH2

91 84 79 73 76 68 72 78 83 77 82 71 89 69 71 78

412 446 426 428 442 442 476 456 458 472 428 463 473 442 444 458

175e177 168e170 170e172 175e177 176e178 164e166 146e148 161e163 129e131 103e105 162e164 157e159 160e162 167e169 120e122 130e132

for 1 h. The sterile discs previously soaked with the test compound solution in DMSO of specific concentration 100 mg and 200 mg/disc were carefully placed on the agar culture plates. The plates were inverted and incubated for 24 h at 37  C. Ciprofloxacin was used as a standard drug. Inhibition zones were measured and compared with the controls. The bacterial zones of inhibition values are given in Table 4. Minimum inhibitory concentrations (MICs) were determined by broth dilution technique. The nutrient broth, which contained logarithmic serially two fold diluted amount of test compound and controls were inoculated with approximately 5  105 c.f.u. of actively dividing bacteria cells. The cultures were incubated for 24 h at 37  C and the growth was monitored visually Table 4 Antibacterial zone of inhibition (mm) of oxadiazoles. Compounds

Conc. (mg/ml) Zone of inhibition (mm) S. aureus B. subtilis E. coli P. aeruginosa

AB1

100 200 AB2 100 200 AB5 100 200 AB6 100 200 AB7 100 200 AB8 100 200 AB9 100 200 AB12 100 200 AB13 100 200 AB14 100 200 AB15 100 200 AB16 100 200 AB18 100 200 AB19 100 200 AB20 100 200 AB21 100 200 Std. (Ciprofloxacin) 100 200

16 18 12 14 15 16 17 19 15 17 16 18 14 15 15 17 21 23 17 19 17 18 13 15 12 13 15 17 19 20 17 18 23 24

13 14 9 11 12 14 15 16 12 14 12 14 12 13 12 14 18 20 13 16 13 15 11 13 10 11 12 14 16 18 14 16 21 22

19 20 16 18 19 21 20 22 18 20 19 21 17 19 19 21 25 26 21 22 21 23 16 18 15 17 19 20 23 24 20 22 26 27

23 25 19 22 23 25 25 27 23 24 25 26 22 24 23 24 30 32 26 28 25 27 22 23 21 23 23 25 27 28 25 27 32 33

and spectrophotometrically. The lowest concentration (highest dilution) required to arrest the growth of bacteria was regarded as minimum inhibitory concentration (MIC). To obtain the minimum bacterial concentration (MBC), 0.1 ml volume was taken from each tube and spread on agar plates. The number of c.f.u. was counted after 18e24 h of incubation at 35  C. MBC was defined as the lowest drug concentration at which 99.9% of the inoculum was killed. The minimum inhibitory concentration and minimum bactericidal concentration are given in Table 5. 6.2. Antifungal studies The newly prepared compounds were screened for their antifungal activity against C. albicans and A. niger in DMSO by agar diffusion method [35,36]. Sabourauds agar media was prepared by dissolving peptone (1 g), D-glucose (4 g) and agar (2 g) in distilled water (100 ml) and adjusting pH to 5.7. Normal saline was used to make a suspension of spore of fungal strain for lawning. A loopful of particular fungal strain was transferred to 3 ml saline to get a suspension of corresponding species. Twenty milliliters of agar media was poured into each Petri dish. Excess of suspension was decanted and the plates were dried by placing in an incubator at 37  C for 1 h. Using an agar punch, wells were made and each well was labeled. A control was also prepared in triplicate and maintained at 37  C for 3e4 days. The fungal activity of each compound was compared with voriconazole as a standard drug. Inhibition zones were measured and compared with the controls. The fungal zones of inhibition values are given in Table 6. The nutrient broth, which contained logarithmic serially two fold diluted amount of test compound and controls was inoculated with approximately 1.6  104e6  104 c.f.u./ml. The cultures were incubated for 48 h at 35  C and the growth was monitored. The lowest concentration (highest dilution) required to arrest the growth of fungus was regarded as minimum inhibitory concentration (MIC). To obtain the minimum fungicidal concentration (MFC), 0.1 ml volume was taken from each tube and spread on agar plates. The number of c.f.u. was counted after 48 h of incubation at 35  C. MFC was defined as the lowest drug concentration at which 99.9% of the inoculums were killed. The minimum inhibitory concentration and minimum fungicidal concentration are given in Table 7.

Table 5 MIC and MBC results of oxadiazoles. Comp.

AB1 AB2 AB5 AB6 AB7 AB8 AB9 AB12 AB13 AB14 AB15 AB16 AB18 AB19 AB20 AB21 Std

S. aureus

B. subtilis

E. coli

MIC

MBC

MIC

MBC

MIC

MBC

MIC

P. aeruginosa MBC

12.5 12.5 6 6 6 12.5 12.5 12.5 6 12.5 25 25 25 25 6 6 6

50 25 25 12.5 12.5 25 50 25 6 25 50 100 50 50 12.5 12.5 12.5

25 6 6 6 12.5 6 12.5 12.5 6 12.5 12.5 12.5 12.5 12.5 6 12.5 6

50 12.5 12.5 12.5 25 12.5 50 25 12.5 12.5 25 25 50 25 12.5 25 12.5

12.5 12.5 6 6 12.5 12.5 12.5 6 6 25 12.5 25 50 12.5 12.5 6 6

25 25 25 12.5 12.5 25 100 25 12.5 12.5 50 50 100 25 25 12.5 12.5

25 25 12.5 6 12.5 12.5 12.5 12.5 12.5 25 25 25 12.5 25 6 12.5 6

50 50 50 12.5 25 50 100 25 25 12.5 50 100 100 100 12.5 25 12.5

MIC (mg/ml) ¼ minimum inhibitory concentration, i.e., the lowest concentration to completely inhibit bacterial growth; MBC (mg/ml) ¼ minimum bactericidal concentration, i.e., the lowest concentration to completely kill bacteria. Ciprofloxacin is used as standard drug.

M.A. Bakht et al. / European Journal of Medicinal Chemistry 45 (2010) 5862e5869 Table 6 Antifungal zone of inhibition (mm) of oxadiazoles. Compounds

Conc. (mg/ml)

AB1

100 200 100 200 100 200 100 200 100 200 100 200 100 200 100 200 100 200 100 200 100 200 100 200 100 200 100 200 100 200 100 200 100 200

AB2 AB5 AB6 AB7 AB8 AB9 AB12 AB13 AB14 AB15 AB16 AB18 AB19 AB20 AB21 Std. (Voriconazole)

Zone of inhibition (mm) Aspergillus niger

Candida albicans

21 25 18 20 20 22 22 25 22 25 23 24 19 21 20 23 25 26 16 19 22 24 18 21 17 19 20 23 27 29 22 24 28 30

16 18 12 15 15 16 18 20 16 18 15 17 16 17 16 18 19 21 11 14 16 18 14 17 13 15 15 17 21 24 17 19 24 26

7.1.1.1. 2-(2-Methoxy-4-((E)-3-oxo-3-phenylprop-1-enyl)phenoxy) acetic acid (III). IR: (KBr) cm1: 3174 (COOH), 1706e1692 (2C]O), 1560 (C]C). 1H NMR (DMSO-d6) ppm: 10.1 (1H, s, COOH); 8.3e7.2 (8H, m, Ar-H); 6.8 (1H, d, HB, J ¼ 12.6 Hz); 6.6 (1H, d, Hx, J ¼ 12.9 Hz); 4.5 (2H, s, OCH2); 3.7(3H, s, OCH3); m/z: 313 (Mþ1). 7.1.1.2. 2-(2-Methoxy-4-((E)-3-(4-methoxyphenyl)-3-oxoprop-1-enyl) phenoxy)acetic acid (IV). IR: (KBr) cm1: 3188 (COOH), 1706e1695 (2C]O), 1540 (C]C). 1H NMR (DMSO-d6) ppm: 10.5 (1H, s, COOH); 8.3e7.2 (7H, m, Ar-H); 7.1 (1H, d, HB, J ¼ 12.7 Hz); 7.0 (1H, d, Hx, J ¼ 12.7 Hz); 4.5 (2H, s, OCH2); 3.8 (6H, s, 2  OCH3); m/z: 341(M1).

7.1. Chemistry All the chemicals used were laboratory grade and procured from E. Merck (Germany) and S.D. Fine Chemicals (India). Melting points were determined by open tube capillary method and are

Table 7 MIC and MFC Results of oxadiazoles.

AB1 AB2 AB5 AB6 AB7 AB8 AB9 AB12 AB13 AB14 AB15 AB16 AB18 AB19 AB 20 AB21 Std (Voriconazole)

A. niger

uncorrected. Thin layer chromatography (TLC) plates (silica gel G), were used to confirm the purity of the commercial reagents used, compounds synthesized and to monitor the reactions as well. Two different solvent systems, toluene: ethyl acetate: formic acid (5:4:1) and petroleum ether: toluene: acetic acid (5:4:1), were used to run the TLC. The spots were located under iodine vapors/UV light. IR spectra were obtained on a PerkineElmer 1720 FT-IR spectrometer (KBr Pellets). 1H NMR spectra were recorded on a Bruker AC 400 MHz, spectrometer-using TMS as internal standard in DMSO-d6. The FAB mass spectra were recorded on a JEOLSX 102/ DA-6000 Mass Spectrometer. 7.1.1. General procedure for the synthesis of chalcones (IIIeV) A mixture of 2-(4-formyl-2-methoxyphenoxy) acetic acid (I) (0.01 mol) and appropriate acetophenone (II) (0.01 mol) is reacted as per ClaiseneSchmidt condensation, in absolute methanol were stirred at room temperature in the presence of base (alcoholic solution of potassium hydroxide 30%; 5 mL) till completion of the reaction. The reaction mixture was allowed to stand overnight and then poured into ice-cold water followed by neutralization with HCl. The solid separated was filtered, dried and crystallized from ethanol. The purity of the chalcone was checked by TLC.

7. Experimental

Comp.

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C.albicans

MIC

MFC

MIC

MFC

12.5 12.5 e 6 6 12.5 12.5 12.5 6 6 25 e 25 25 6 6 6

25 25 e 12.5 12.5 25 50 25 12.5 12.5 50 e 100 50 12.5 12.5 12.5

12.5 25 12.5 6 12.5 6 e 12.5 6 12.5 12.5 12.5 e 25 6 12.5 6

25 50 25 12.5 25 25 e 25 12.5 25 25 25 e 100 12.5 25 12.5

MIC (mg/ml) ¼ minimum inhibitory concentration, i.e., the lowest concentration to completely inhibit fungal growth; MFC (mg/ml) ¼ minimum fungicidal concentration, i.e., the lowest concentration to completely kill the fungi.

7.1.1.3. 2-(4-((E)-3-(4-hydroxyphenyl)-3-oxoprop-1-enyl)-2-methoxyphenoxy) acetic acid (V). IR: (KBr) cm1: 3550 (OH), 3092 (COOH), 1706e1692 (2C]O) 1539 (C]C). 1H NMR (DMSO-d6) ppm: 9.8 (1H, s, COOH); 9.1(1H, s, OH); 7.8e7.2 (7H, m, Ar-H); 7.0 (1H, d, HB, J ¼ 13 Hz); 6.9 (1H, d, Hx, J ¼ 13 Hz); 4.5 (2H, s, OCH2); 3.8 (3H, s, OCH3); m/z: 329 (Mþ1). 7.1.2. General procedure for synthesis of 1,3,4-oxadiazole derivatives (AB1eAB2), (AB5eAB9), (AB12eAB16), (AB18eAB21) To an equimolar mixture (0.001 mol) of chalcone derivative (III, IV, V) and appropriate acid hydrazide in 5 ml of phosphorus oxychloride (POCl3) was added and the reaction mixture was refluxed for 18e20 h. After completion of reaction the mixture was poured onto crushed ice (20 g) and neutralized with aqueous sodium hydroxide solution. The product so obtained was filtered, washed with water and recrystallized from ethanol. 7.1.2.1. (E)-3-(4-((5-phenyl-1,3,4-oxadiazol-2-yl)methoxy)-3-methoxyphenyl)-1-phenylprop-2-en-1-one (AB1). IR: (KBr) cm1: 1682 (C] O), 1654 (C]C), 1560 (C]N), 1167 (CeOeC). 1H NMR (DMSO-d6) ppm: 7.8e7.2 (13H, m, Ar-H); 6.8e6.6 (Alkenyl proton, 2H, 2d, J ¼ 12.6, 12.9 Hz); 4.5 (2H, s, OCH2); 3.7 (3H, s, OCH3); m/z: 413 (Mþ1); Anal. Calcd. for C25H20N2O4: C, 72.80; H, 4.89; N, 6.79%. Found: C, 72.82; H, 4.90; N, 6.80%. 7.1.2.2. (E)-3-(4-((5-(4-chlorophenyl)-1,3,4-oxadiazol-2-yl)methoxy)3-methoxyphenyl)-1-phenylprop-2-en-1-one (AB2). IR: (KBr) cm1: 1682 (C]O),1654 (C]C), 1560 (C]N), 1166 (CeOeC). 1H NMR (DMSO-d6) ppm: 8.3e7.0 (12H, m, Ar-H); 6.8e6.5 (Alkenyl proton, 2H, 2d, J ¼ 8.8, 10.9 Hz); 4.5 (2H, s, CH2); 3.8 (3H, s, OCH3); m/z: 448

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(Mþ1); Anal. Calcd. for C25H19ClN2O4: C, 67.19; H, 4.29; N, 6.27%. Found: C, 67.18; H, 4.30; N, 6.29%. 7.1.2.3. (E)-3-(4-((5-p-tolyl-1,3,4-oxadiazol-2-yl)methoxy)-3-methoxyphenyl)-1-phenylprop-2-en-1-one (AB5). IR: (KBr) cm1: 1680 (C] O), 1654 (C]C), 1560 (C]N), 1167 (CeOeC). 1H NMR (DMSO-d6) ppm: 8.2e7.1 (12H, m, Ar-H); 6.8e6.6 (Alkenyl proton, 2H, 2d, J ¼ 12.2, 15.7 Hz); 4.5 (2H, s, OCH2); 3.8 (3H, s, OCH3); 2.2 (3H, s, CH3); m/z: 427 (Mþ1); Anal. Calcd. for C26H22N2O4: C, 73.22; H, 5.20; N, 6.57%. Found: C, 73.25; H, 5.21; N, 6.59%. 7.1.2.4. (E)-3-(4-((5-(2-hydroxyphenyl)-1,3,4-oxadiazol-2-yl)methoxy)3-methoxyphenyl)-1-phenylprop-2-en-1-one (AB6). IR: (KBr) cm1: 3536 (OH), 1682 (C]O), 1654 (C]C), 1556 (C]N), 1166 (CeOeC). 1H NMR (DMSO-d6) ppm: 9.1 (1H, s, OH); 8.3e7.1 (12H, m, Ar-H); 6.7e6.5 (Alkenyl proton, 2H, 2d, J ¼ 12.4, 12.6 Hz); 4.6 (2H, s, OCH2); 3.7 (3H, s, OCH3); m/z: 429 (Mþ1); Anal. Calcd. for C25H20N2O5: C, 70.08; H, 4.71; N, 6.54%. Found: C, 70.09; H, 4.71; N, 6.56%. 7.1.2.5. (E)-3-(4-((5-(phenoxymethyl)-1,3,4-oxadiazol-2-yl)methoxy) -3-methoxyphenyl)-1-phenylprop-2-en-1-one (AB7). IR: (KBr) cm1: 1686 (C]O), 1654 (C]C), 1560 (C]N), 1171 (CeOeC). 1H NMR (DMSO-d6) ppm: 8.2e6.8 (13H, m, Ar-H); 6.6e6.2 (Alkenyl proton, 2H, 2d, J ¼ 10.6, 10.7 Hz); 4.7e4.5(4H, s, 2  OCH2); 3.8 (3H, s, OCH3); m/z: 443(Mþ1); Anal. Calcd. for C26H22N2O5: C, 70.58; H, 5.01; N, 6.33%. Found: C, 70.59; H, 5.03; N, 6.36%. 7.1.2.6. (E)-3-(4-((5-phenyl-1,3,4-oxadiazol-2-yl)methoxy)-3-methoxyphenyl)-1-(4-methoxyphenyl)prop-2-en-1-one (AB8). IR: (KBr) cm1: 1682 (C]O), 1654 (C]C), 1561 (C]N), 1157 (CeOeC). 1H NMR (DMSO-d6) ppm: 8.0e7.2 (12H, m, Ar-H); 7.1e6.9 (Alkenyl proton, 2H, 2d, J ¼ 10.1, 10.8 Hz); 4.5 (2H, s, OCH2); 3.9e3.8 (6H, s, 2OCH3); m/z: 443(Mþ1); Anal. Calcd. for C26H22N2O5: C, 70.58; H, 5.01; N, 6.33%. Found: C, 70.59; H, 5.03; N, 6.36%. 7.1.2.7. (E)-3-(4-((5-(4-chlorophenyl)-1,3,4-oxadiazol-2-yl)methoxy)3-methoxyphenyl)-1-(4-methoxyphenyl)prop-2-en-1-one (AB9). IR: (KBr) cm1: 1682 (C]O), 1654 (C]C), 1559 (C]N), 1158 (CeOeC). 1 H NMR (DMSO-d6) ppm: 8.2e7.1 (11H, m, Ar-H); 6.7e6.6 (Alkenyl proton, 2H, 2d, J ¼ 10.1, 10.8 Hz); 4.5 (2H, s, OCH2); 3.8e3.7 (6H, s, 2  OCH3); m/z: 478 (Mþ1); Anal. Calcd. for C26H21ClN2O5: C, 65.48; H, 4.44; N, 5.87%. Found: C, 65.49; H, 4.48; N, 5.86%. 7.1.2.8. (E)-3-(4-((5-p-tolyl-1,3,4-oxadiazol-2-yl)methoxy)-3-methoxyphenyl)-1-(4-methoxyphenyl)prop-2-en-1-one (AB12). IR: (KBr) cm1: 1682 (C]O), 1654 (C]C), 1560 (C]N), 1158 (CeOeC). 1H NMR (DMSO-d6) ppm: 8.0e7.2 (11H, m, Ar-H); 6.7e6.3 (Alkenyl proton, 2H, 2d, J ¼ 12.1, 12.2 Hz); 4.4 (2H, s, OCH2); 3.7e3.6 (6H, s, 2  OCH3); 2.3 (3H, s, CH3); m/z: 457(Mþ1); Anal. Calcd. for C27H24N2O5: C, 71.04; H, 5.30; N, 6.14%. Found: C, 71.03; H, 5.33; N, 6.16%. 7.1.2.9. (E)-3-(4-((5-(2-hydroxyphenyl)-1,3,4-oxadiazol-2-yl)methoxy)3-methoxyphenyl)-1-(4-methoxyphenyl)prop-2-en-1-one (AB13). IR: (KBr) cm1: 3535 (OH), 1682 (C]O), 1654 (C]C), 1561 (C]N), 1158 (CeOeC). 1H NMR (DMSO-d6) ppm: 9.2 (1H, s, OH); 8.1e7.2 (11H, m, Ar-H); 6.8e6.5 (Alkenyl proton, 2H, 2d, J ¼ 10.3, 10.7 Hz); 4.7 (2H, s, OCH2); 3.7e3.6 (6H, s, 2  OCH3); m/z: 459(Mþ1); Anal. Calcd. for C26H22N2O6: C, 68.11; H, 4.84; N, 6.11%. Found: C, 68.13; H, 4.83; N, 6.14%. 7.1.2.10. (E)-3-(4-((5-(phenoxymethyl)-1,3,4-oxadiazol-2-yl)methoxy)3-methoxyphenyl)-1-(4-methoxyphenyl)prop-2-en-1-one (AB14). IR: (KBr) cm1: 1682 (C]O), 1654 (C]C), 1560 (C]N), 1157 (CeOeC). 1 H NMR (DMSO-d6) ppm: 7.9e6.9 (12H, m, Ar-H); 6.9e6.8 (Alkenyl

proton, 2H, 2d, J ¼ 8.3, 8.4 Hz); 4.8e4.6 (4H, s, 2  OCH2); 3.7e3.6 (6H, s, 2  OCH3); m/z: 473 (Mþ1); Anal. Calcd. for C27H24N2O6: C, 68.63; H, 5.12; N, 5.93%. Found: C, 68.6; H, 5.13; N, 5.94%. 7.1.2.11. (E)-3-(4-((5-phenyl-1,3,4-oxadiazol-2-yl)methoxy)-3-methoxyphenyl)-1-(4-hydroxyphenyl)prop-2-en-1-one (AB15). IR: (KBr) cm1: 3550 (OH), 1680 (C]O), 1654 (C]C), 1559 (C]N), 1156 (CeOeC). 1 H NMR (DMSO-d6) ppm: 9.2 (1H, s, OH); 8.5e7.3 (12H, m, Ar-H); 7.2e6.7 (Alkenyl proton, 2H, 2d, J ¼ 12.3, 15.0 Hz); 4.4 (2H, s, OCH2); 3.8 (3H, s, OCH3); m/z: 429 (Mþ1); Anal. Calcd. for C25H20N2O5: C, 70.08; H, 4.71; N, 6.54%. Found: C, 70.05; H, 4.73; N, 6.54%. 7.1.2.12. (E)-3-(4-((5-(4-chlorophenyl)-1,3,4-oxadiazol-2-yl)methoxy)3-methoxy phenyl)-1-(4-hydroxyphenyl)prop-2-en-1-one (AB16). IR: (KBr) cm1: 3550 (OH), 1682 (C]O), 1654 (C]C), 1560 (C]N), 1155 (CeOeC). 1H NMR (DMSO-d6) ppm: 9.2 (1H, s, OH); 8.2e7.7 (11H, m, Ar-H); 6.8e6.7 (Alkenyl proton, 2H, 2d, J ¼ 14.7, 15.6 Hz); 4.5 (2H, s, OCH2); 3.8 (3H, s, OCH3); m/z: 464 (Mþ1); Anal. Calcd. for C25H19ClN2O5: C, 64.87; H, 4.14; N, 6.05%. Found: C, 64.85; H, 4.13; N, 6.07%. 7.1.2.13. (E)-3-(4-((5-(4-nitrophenyl)-1,3,4-oxadiazol-2-yl)methoxy)3-methoxy phenyl)-1-(4-hydroxyphenyl)prop-2-en-1-one (AB18). IR: (KBr) cm1: 3550 (OH), 1686 (C]O), 1654 (C]C), 1560 (C]N), 1158 (CeOeC). 1H NMR (DMSO-d6) ppm: 9.2 (1H, s, OH); 8.3e7.0 (11H, m, Ar-H); 6.7e6.5 (Alkenyl proton, 2H, 2d, J ¼ 12.8, 13.2 Hz); 4.4 (2H, s, OCH2); 3.6 (3H, s, OCH3); m/z: 474 (Mþ1); Anal. Calcd. for C25H19N3O7: C, 63.42; H, 4.05; N, 8.88%. Found: C, 63.43; H, 4.07; N, 8.91%. 7.1.2.14. (E)-3-(4-((5-p-tolyl-1,3,4-oxadiazol-2-yl)methoxy)-3-methoxyphenyl)-1-(4-hydroxyphenyl)prop-2-en-1-one (AB19). IR: (KBr) cm1: 3550 (OH), 1682 (C]O), 1654 (C]C), 1559 (C]N), 1156 (CeOeC). 1H NMR (DMSO-d6) ppm: 8.5 (1H, s, OH); 8.3e7.1 (11H, m, Ar-H); 7.0e6.8 (Alkenyl proton, 2H, 2d, J ¼ 12.4, 12.3 Hz); 4.4 (2H, s, OCH2); 3.8 (3H, s, OCH3); 2.3 (3H, s, CH3); m/z: 443 (Mþ1); Anal. Calcd. for C26H22N2O5: C, 70.58; H, 5.01; N, 6.33%. Found: C, 70.57; H, 5.03; N, 6.36%. 7.1.2.15. (E)-3-(4-((5-(2-hydroxyphenyl)-1,3,4-oxadiazol-2-yl)methoxy)3-methoxy phenyl)-1-(4-hydroxyphenyl)prop-2-en-1-one (AB20). IR: (KBr) cm1: 3550e3536 (2OH), 1680 (C]O), 1654 (C]C), 1549 (C] N), 1155 (CeOeC). 1H NMR (DMSO-d6) ppm: 10.8 (2H, s, 2  OH); 8.0e6.9 (11H, m, Ar-H); 6.7e6.1 (Alkenyl proton, 2H, 2d, J ¼ 10.4, 9.6 Hz); 4.4 (2H, s, OCH2); 3.7 (3H, s, OCH3); m/z: 445 (Mþ1); Anal. Calcd. for C25H20N2O6: C, 67.56; H, 4.54; N, 6.30%. Found: C, 67.57; H, 4.57; N, 6.33%. 7.1.2.16. (E)-3-(4-((5-(phenoxymethyl)-1,3,4-oxadiazol-2-yl)methoxy)3-methoxy phenyl)-1-(4-hydroxyphenyl)prop-2-en-1-one (AB21). IR: (KBr) cm1: 3550 (OH), 1682 (C]O), 1654 (C]C), 1559 (C]N), 1156 (CeOeC). 1H NMR (DMSO-d6) ppm: 9.4 (1H, s, OH); 8.7e7.0 (12H, m, Ar-H); 6.9e6.5 (Alkenyl proton, 2H, 2d, J ¼ 12.5, 13.4 Hz); 4.4e4.2 (4H, s, 2  OCH2); 3.8 (3H, s, OCH3); m/z: 459 (Mþ1); Anal. Calcd. for C26H22N2O6: C, 68.11; H, 4.84; N, 6.11%. Found: C, 68.13; H, 4.87; N, 6.13%. Acknowledgments The authors wish to express the gratitude to the staff of Jamia Hamdard for providing necessary facility to carry out the research work. References [1] S.J. Dolman, F. Gosselin, P.D. O’Shea, I.W. Davies, J. Org. Chem. 71 (2006) 9548e9551.

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