Synthesis of Potential Antimicrobial/Antitubercular s ... · Rahul V. Patel et al : Synthesis of Potential Antimicrobial/Antitubercular s-triazine Scaffolds Endowed with Quinoline

Rahul V. Patel et al : Synthesis of Potential Antimicrobial/Antitubercular s-triazine Scaffolds Endowed with Quinoline and… 733

Synthesis of Potential Antimicrobial/Antitubercular s-Triazine Scaffolds Endowed with Quinoline and Quinazoline Heterocycles

Rahul V. Patela, Premlata kumari1, Dhanji P. Rajani2, Kishor H. Chikhalia3*

1Applied Chemistry Department, S.V. National Institute of Technology, Surat-395007, India. 2Microcare Laboratory, Surat-395007, India. 3School of Science, Department of Chemistry, Gujarat University, Ahmedabad-380025, India.

ABSTRACT: Two series of 2–(4–cyanophenyl amino)–4–quinoline (quinazoline)–4–yloxy–6–piperazinyl (piperidinyl)–1,3,5–triazines were

synthesized so as to investigate their antimicrobial and antitubercular action. Newer analogues were characterized by IR, 1H NMR spectroscopy and elemental analyses. Pharmacological screening against eight bacteria (S. aureus, B. cereus, E. coli, P. aeruginosa, K. pneumoniae, S. typhi, P. vulgaris, S. flexneria), four fungi (A. niger, A. fumigatus, A. clavatus, C. albicans) and Mycobacterium tuberculosis H37Rv was examined and the effects of various substituents on biological profiles (MIC, 6.25-50 µg/mL) of final analogues were investigated. Some of the final analogues displayed good antimycobacterial activity (MIC, 12.5 µg/mL).

KEYWORDS: s-Triazine; quinoline, quinazoline; antimicrobial activity; antituberculosis activity.

Introduction Since last 10 years it has been observed that the evolution and spread of multidrug resistant microorganisms is of grave concern to global health care. These organisms possessed the ability to withstand attack by antimicrobial drugs currently available, and the uncontrolled rise in resistant pathogens threatens lives. Such infections most commonly affect immunocompromised individuals, patients with malignancies and transplant recipients1. A potential approach to overcome the resistance problem is to design innovative agents with a different mode of action so that no cross-resistance with the present therapeuticals can occur. According to the World Health Organization (WHO), there were 9.4 million new TB cases (including 3.3 million women) in 20092. Moreover, the development of drug-resistant strains of mycobacterium species, has contributed to the inefficiency of the conventional antituberculosis therapy, thus, it is still necessary to search for new antimycobacterial agents. When a patient develops bacterial resistant to the first-line drugs: isoniazid, rifampicin, ethambutol and pyrazinamide3, problems in the chemotherapy of tuberculosis arise, which seriously threatens the progress in antituberculosis medical care.

Consequently, identifying and developing novel drug entities is apparent in light of the significant problems associated with current drugs.

The design of inhibitors that can accommodate potency to multiple biological targets remains an intriguing scientific endeavour. 1,3,5-Triazine nucleus have attracted a great deal of attention among chemists due to its diverse biological activities such as antimicrobial4,5, antiprotozoal6, anticancer7, antimalarial8 and antiviral9 activity. Profound medicinal applications associated with piperazine heterocycle render them as useful structural units in drug research10-14 while the piperazine-quinoline combination is found in the structure of many well known antimicrobial drugs like ciprofloxacin, norfloxacin, ofloxacin, pefloxacin, rufloxacin, enrofloxacin etc. In our previous study, we have introduced various piperazine and piperidine derivatives to the s-triazine core and indentified the compounds with good antibacterial activity, in an order to expand the structure activity relationship we carried out this research work aiming to the discovery of the similar type of scaffolds with selective piperazine bases, that were activity in the previous research work15,16. Prompted by these observations it was contemplated to envisage the combination of above mentioned biolabile components of significant activities in a compact system to identify new candidates that may be value in designing new biologically active agents.

* For correspondence: Kishor H. Chikhalia,

Tel: +919427155529

Email: [email protected]

739

International Journal of Drug Design and Discovery

Volume 3 • Issue 1 • January – March 2012. 739-730

740 International Journal of Drug Design and Discovery Volume 3 • Issue 1 • January – March 2012

Results and Discussion

Chemistry

The triazines described were synthesized starting from cyanuric chloride (2,4,6-trichloro-1,3,5-triazine) and different nucleophiles (Scheme 1). The nucleophiles can selectively displace the different chlorines by control of the reaction temperature17. In general, the first chlorine can be displaced when the temperature is maintained at 0°C, the second between 25oC-50°C, and the third substitution at above 60°C and due to reactivity the temperature can

exceed 80°C. Another important factor that has to be considered for the preparation of the different derivatives is the nature of the reactive group and the order of entry of the group. Next, different amino/phenoxy groups were introduced, a compound with strong nucleophile (OH) was introduced after the substitution of compound with weak nucleophile (-NH2) at the first chlorine atom. In addition, a wide range of cyclic amines were introduced to the third reactive chlorine atom because of the ease of reaction condition carried at reflux temperature.

N

N

N

Cl Cl

Cl C

N

NH2

THF, Et3N

N N

N

Cl

Cl

CN

HN

2,4,6-Trichloro-1,3,5-triazine 4-Amino-benzonitrile

1

1

N

X

OH

THF, NaH N

N

N

Cl

CN

NH

N

X O

X=H: 4-Hydroxyquinoline (2a)

X=N: 4-Hydroxyquinazoline (2b)

X=H: 4-Hydroxyquinoline (3a)

X=N: 4-Hydroxyquinazoline (3b)

3a, 3b 4a-j1,4-Dioxane

K2CO3

N

N

N

R

CN

NH

N

X O

X=H: 4-Hydroxyquinoline (5a-j)

X=N: 4-Hydroxyquinazoline (6a-j)

O-5 °C

R.T - 50 °C

Reflux

4-(4,6-dichloro-1,3,5-triazin-2-ylamino)benzonitrile

1

23

4

5

67

8

R=


HN N

HN N

HN N C

HN

HN N

HN N

HN N

Cl

Cl Cl

O

CH3

CH3

CH3

HN N CH

ClF

F

CF3

HN N OCH3

4a

4b

4c

4d

4e

4f

4g

4h

4j

HN N CH2

H3CO OCH3

OCH34i

Scheme 1 Synthesis of target compounds.

Hence, in the present study the first step comprises formation of intermediate 1 in very good yield by the nucleophilic displacement of one chlorine atom of s-triazine ring by 4-amino-benzonitrile. Compound 1 displayed an absorption band at 2223 cm-1 confirming the presence of a C≡N group, and a strong band near 3288 cm-1 further confirmed the presence of an -NH group. The synthesis of disubstituted s-triazine intermediates (3a, 3b) was achieved in 80-85% of yield by the reaction between (1) and 4-hydroxyquinoline (2a) or 4-Hydroxy quinazoline (2b) in the presence of 60% NaH at 45-50°C. A characteristic band appeared at 1251 cm-1 and 1256 cm-1

corresponded to the C-O-C linkage in the FT-IR spectra of compound 3. Subsequent coupling of the so formed compound 3 with the desired cyclic amines under basic conditions in 1,4-dioxane solvent at 70-80°C formed the corresponding 5a-j and 6a-j18 and this reaction proceeded in good yields. The correct synthesis of 5a-j and 6a-j was confirmed on the basis of IR and 1H NMR spectral analysis, and the purity was ascertained by elemental analysis.

Biological activity

Investigation on antimicrobial screening data (Table 1 and 2) showed some of the compounds showed excellent activity against all the mentioned microorganisms. From the bioassay it can be stated that the final analogues with the substitutions of 4-hydroxyquinazoline demonstrated improved efficacy as compared to analogues bearing 4-hydroxyquinoline. Both the type of scaffolds displayed

inhibitory efficacy in a good range of MIC as well as inhibition zones. All the newer analogues showed 6.25-25 µg/ml of MIC against bacteria, 25-50 µg/ml against fungi 12.5 µg/ml of lowest MIC against mycobcatria. From these results, it was also possible to make a number of correlations regarding the relationship between the structure of the newer scaffolds and their antimicrobial activities as:

(i) Analogues with 4-hydroxyquinoline substituent and cyclic amines bearing electron withdrawing halogen atom(s) were effective against K. pneuminiae and S. flexneria, with electron donating alkoxy functionality showed inhibition towards A. niger, where as analogues with methyl substituent displayed activity against E. coli.

(ii) Analogues with 4-hydroxyquinazoline substituent and cyclic amines bearing electron withdrawing halogen atom(s) demonstrated activity against S. typhi bacteria and A. fumigatus fungi, with electron donating alkoxy functionality displayed inhibitory action against S. aureus, P. vulgaris bacteria and C. albicans fungi, while analogues with methyl substituent were effective against P. aeruginosa.

(iii) Newer analogues with both the type of, 4-hydroxyquinoline and 4-hydroxyquinazoline moieties were mixdly active against B. cereus bacteria, A. clavatus fungi as well as against mytcobacteria H37Rv.


Table 1 In vitro antibacterial activity of compounds 5a-6j.

Compound (100 µg/disc)

Zone of inhibition [mm (MIC in µg/ml)] Gram (+) Gram (-)

S.a B.c E.c P.a K.p S.t P.v S.f 5a 16 (100) 17 (100) <10 (100) 13 (100) 23 (25) 15 (100) 13 (100) 14 (100) 5b 18 (100) 18 (100) 16 (100) 14 (100) 19 (100) 18 (100) 14 (100) 15 (100) 5c 17 (100) 19 (50) 24 (25) 20 (50) 15 (100) 13 (100) 19 (100) 14 (100) 5d 21 (25) 22 (12.5) 20 (100) 22 (25) 15 (100) 17 (100) 16 (100) 13 (100) 5e 17 (100) 17 (100) 17 (100) 15 (100) 22 (50) 22 (50) 15 (100) 11 (100) 5f 19 (50) 19 (100) 16 (100) 13 (100) 23 (25) 20 (100) 15 (100) 18 (100) 5g 15 (100) 22 (12.5) 17 (100) 16 (100) 21 (50) 23 (50) 15 (100) 17 (100) 5h 22 (25) 21 (25) 20 (50) 17 (100) 19 (100) 20 (100) 16 (100) 24 (25) 5i 25 (12.5) 20 (50) 21 (50) 20 (50) 15 (100) 14 (100) 22 (50) 14 (100) 5j 20 (50) 18 (100) 21 (50) 17 (100) 13 (100) 15 (100) 20 (100) 17 (100) 6a 14 (100) 16 (100) 12 (100) 14 (100) 22 (50) 14 (100) 11 (100) 13 (100) 6b 20 (50) 19 (50) 18 (100) 13 (100) 17 (100) 21 (50) 13 (100) 12 (100) 6c 16 (100) 20 (25) 23 (50) 21 (25) 17 (100) 17 (100) 20 (100) 16 (100) 6d 22 (25) 22 (12.5) 22 (50) 23 (12.5) 14 (100) 15 (100) 19 (100) 14 (100) 6e 19 (50) 18 (100) 19 (100) 14 (100) 21 (50) 24 (25) 17 (100) 14 (100) 6f 20 (50) 20 (50) 15 (100) 17 (100) 22 (50) 19 (100) 17 (100) 19 (100) 6g 17 (100) 21 (25) 18 (100) 15 (100) 20 (50) 24 (25) 15 (100) 19 (100) 6h 24 (12.5) 22 (12.5) 21 (50) 18 (100) 19 (100) 22 (50) 18 (100) 23 (50) 6i 25 (6.25) 20 (50) 22 (50) 21 (50) 16 (100) 16 (100) 23 (25) 16 (100) 6j 21 (25) 20 (50) 20 (100) 19 (100) 15 (100) 15 (100) 21 (50) 17 (100)

Ciprofloxacin (100 µg/disc)

30 (1.56) 31 (0.39) 32 (1.56) 33 (0.78) 33 (0.78) 30 (0.39) 31 (0.39) 32 (1.56)

DMSO – – – – – – – –

Table 2 In vitro antifungal and antitubercular activity of compounds 5a-6j.


Antifungal activity Antituberculosis activity Zone of inhibition [mm (MIC in µg/ml)] MIC

(µg/ml) % Inhibition

A.n A.f A.c C.a H37Rv 5a 13 (100) 17 (100) 16 (100) 12 (100) 100 96

5b 15 (100) 19 (100) 18 (100) 11 (100) 62.5 97

5c 18 (100) 13 (100) 12 (100) 18 (100) 25 98

5d 17 (100) 12 (100) 15 (100) 19 (50) 12.5 99

5e 16 (100) 16 (100) 22 (50) 15 (100) 62.5 97

5f 17 (100) 17 (100) 19 (100) 12 (100) 50 98

5g 17 (100) 18 (100) 20 (100) 14 (100) 100 95

5h 19 (100) 19 (100) 22 (50) 15 (100) 12.5 99

5i 22 (25) 13 (100) 20 (100) 20 (50) 12.5 99

5j 20 (100) 11 (100) 19 (100) 18 (50) 50 97

6a 15 (100) 17 (100) 16 (100) 14 (100) 50 97

Table 2 Contd…



Antifungal activity Antituberculosis activity Zone of inhibition [mm (MIC in µg/ml)] MIC

(µg/ml) % Inhibition

6b 15 (100) 21 (50) 17 (100) 15 (100) 12.5 99 6c 19 (100) 15 (100) 11 (100) 18 (100) 12.5 99 6d 18 (100) 14 (100) 22 (50) 20 (50) 12.5 99 6e 13 (100) 16 (100) 20 (100) 16 (100) 100 95 6f 16 (100) 18 (100) 18 (100) 14 (100) 50 97 6g 17 (100) 18 (100) 19 (100) 17 (100) 50 96 6h 19 (100) 21 (50) 22 (50) 18 (100) 12.5 99 6i 21 (50) 15 (100) 19 (100) 21 (25) 25 97 6j 19 (100) 16 (100) 22 (50) 20 (50) 50 96

Kitaconazole (100 µg/disc)

30 (1.56) 29 (0.78) 31 (0.78) 33 (1.56) - -

Pyrazinamide – – – – 6.25 99 DMSO – – – – – –

Conclusions Two series of 4-hydroxyquinoline and 4-hydroxyquinazoline based s-triazinyl piperazines were synthesized and screened against wide range of pathogenic bacteria, fungi and mycobacteria. The bioassay results demonstrated that some analogues were potential active against all the microorganisms at MICs 6.25-50 µg/ml. Analogues with 4-hydroxyquinazoline constituent displayed improved activity and can be highlighted as new active leads that provide a powerful incentive for further research in this area. Overall, The MIC values of these novel compounds evidenced that the presence of halogen atom(s) and alkyl or alkoxy substituent gave rise to a better pharmacological potency, whereas from the present bioassay the activity of the final analogues lies in the order antitubercular>antibacterial>antifungal. Moreover, we believe that findings of the present study will have a good impact on medicinal chemists to synthesize similar compounds which will probably indicate greater biological potency.

Experimental Section Melting points were determined in open capillaries on a Veego electronic apparatus VMP–D and are uncorrected. IR spectra of synthesized compounds were recorded on a Shimadzu 8400–S FT–IR spectrophotometer using KBr pellets. Thin layer chromatography was performed on object glass slides (2 x 7.5 cm) coated with silica gel–G and spots were visualized under UV irradiation. 1H NMR spectra were recorded on a Varian 400 MHz model spectrometer. Elemental analyses (C, H, N) were performed using a Heraeus Carlo Erba 1180 CHN analyzer (Hanau, Germany).

4–[4,6–Dichloro–1,3,5–triazin–2–ylamino]– benzonitrile (1) To a stirred solution of 2,4,6–trichloro–1,3,5–triazine (10 g, 0.054 mole) in anhydrous THF (150 ml) 4–amino–benzonitrile (6.41 g, 0.054 mole) was drop wise added at 0oC-5°C. The resulting reaction mixture was stirred at this temperature for 2 hours. Then triethyl amine (5.48 g, 0.054 mole) was added and stirring was continued for another 2 hours. The reaction mixture was then treated with crushed ice, followed by neutralization with dilute HCl, and filtered, dried, and recrystallized from acetone to afford 1. Light Yellow solid, yield: 88%, melting point 248oC-250°C (dec.); IR: 2223 (C≡N), 3288 (–NH).

4–[4–Chloro–6–(quinoline–4–yloxy)–1,3,5–triazine–2–ylamino]–benzonitrile (3a) and 4–[4–Chloro–6–(quinazolin–4–yloxy)–1,3,5–triazine–2–ylamino]–benzonitrile (3b)

To a stirred solution of 4–hydroxyquinoline or 4–hydroxyquinazoline (8 g, 0.055 mole) in anhydrous THF (150 ml) 60% NaH (1.32 g, 0.055 mole) was added at room temperature during 1 h and 1 (14.63 g for 4-hydroxyquinoline and 14.66 g for 4-hydroxyquinazoline, 0.055 mole) was then added to the mixture. Stirring was continued for another 20 hours at 45°C. Progress of the reaction was monitored by TLC using toluene: acetone (95:5 v/v) as eluent. The mixture was treated with crushed ice, filtered and dried to afford 3a or 3b.

(3a): Yellowish brown solids, yield: 84%, melting point 275oC-277°C; IR: 2222 (C≡N), 1251 (C–O–C).

(3b) Yellow Solid, Yield: 81%, melting point 249oC-251°C; IR: 2224 (C≡N), 1256 (C–O–C).


General procedure for preparation of compounds 5a-j and 6a-j To a solution of 3a or 3b (0.01 mole) in 1,4–dioxane (30 ml), the respective substituted piperazine or piperidine derivative was added and the reaction mixture was refluxed for 11-19 hours. Potassium carbonate (0.01 mole) was used for neutralization of the reaction mixture. Progress of the reaction was monitored by TLC using toluene: acetone (97:3 v/v) as eluent. The mixture was then treated with crushed ice and neutralized by dilute HCl. The precipitate thus obtained was filtered off, dried and recrystallized from THF to afford the desired compounds 5a-j and 6a-j.

Characterization of compounds 5a-j and 6a-j 4–[4–(3–Chloro–phenyl)–piperazin–1–yl]–6–(quinoline–4–yloxy)–1,3,5–triazin–2–ylamino]–benzonitrile (5a). Yield 80%, melting point 212oC-214°C; IR: 3286 (–NH), 2220 (C≡N), 1255 (C–O–C), 829 cm–1 (s–triazine), 759 (C–Cl); 1H NMR: δ 9.11 (s, 1H,–NH, Ctri–NH–), 8.79 (d, J = 8.4 Hz, 1H), 8.67 (s, 1H), 8.43 (dd, J = 7.2, 1.2 Hz, 1H), 8.15 (dd, J = 8.1, 1.6 Hz, 1H), 7.70–7.64 (m, 2H), 7.40–7.32 (m, 7H, Ar–H), 6.59 (dd, J = 7.4, 1.4 Hz, 1H, –CH), 3.86 (br s, 4Hpip), 3.51 (br s, 4Hpip). Analysis: Calculated for C29H23ClN8O: C, 65.10; H, 4.33; N, 20.94. Found: C, 65.31; H, 4.22; N, 20.82.

4–[4–[4–(2,3–Dichloro–phenyl)–piperazin–1–yl]–6–(quinoline–4–yloxy)–1,3,5–triazin–2–ylamino]–benzonitrile (5b): Yield 79%, melting point 251oC-254°C; IR: 3282 (–NH), 2222 (C≡N), 1250 (C–O–C), 834 cm–1 (s–triazine), 754 (C–Cl); 1H NMR: δ 8.99 (1H, s,–NH, Ctri–NH–), 8.70 (d, J = 8.0 Hz, 1H), 8.54 (s, 1H), 8.50 (dd, J = 7.4, 1.1 Hz, 1H), 8.07 (dd, J = 7.9, 1.3 Hz, 1H), 7.63–7.59 (m, 2H), 7.46–7.37 (m, 4H, Ar-H), 7.08 (dd, J = 7.7, 1.5 Hz, 1H, -CH), 6.91 (t, J = 7.2 Hz, 1H), 6.50 (dd, J = 7.0, 1.0 Hz, 1H, –CH), 3.72 (br s, 4Hpip), 3.39 (br s, 4Hpip). Analysis: Calculated for C29H22Cl2N8O: C, 61.17; H, 3.89; N, 19.68. Found: C, 61.24; H, 3.71; N, 19.50.

4–[4–(4–Acetyl–piperazin–1–yl)–6–(quinoline–4–yloxy)–1,3,5–triazin–2–ylamino]– benzonitrile (5c): yield 75%, melting point 238oC-239°C; IR: 3284 (–NH), 2223 (C≡N), 1702 (–C=O), 1482 (–CH3), 1254 (C–O–C), 837 cm–1 (s–triazine); 1H NMR: δ 9.25 (1H, s,–NH, Ctri–NH–), 8.82 (d, 1H, J = 8.1 Hz), 8.60 (s, 1H), 8.33 (dd, J = 7.1, 1.0 Hz, 1H), 8.16 (dd, J = 7.9, 1.6 Hz, 1H), 7.62–7.57 (m, 2H), 7.41–7.33 (m, 4H, Ar-H), 3.77 (br s, 4Hpip), 3.50 (br s, 4Hpip), 2.19 (s, 3H, COCH3). Analysis: Calculated for C25H22N8O2: C, 64.37; H, 4.75; N, 24.02. Found: C, 64.15; H, 4.59; N, 23.92.

4–[4–(3,5–Dimethyl–piperidin–1–yl)–6–(quinoline–4–yloxy)–1,3,5–triazine–2–ylamino]–benzonitrile (5d): Yield 81%, melting point 279oC-281°C; IR: 3280 (–NH), 2222

(C≡N), 1251 (C–O–C), 839 (s–triazine); 1H NMR: δ 8.99 (1H, s,–NH, Ctri–NH–), 8.65 (d, J = 7.1 Hz, 1H), 8.59 (s, 1H), 8.29 (dd, J = 7.3, 1.4 Hz, 1H), 7.93 (dd, J = 7.0, 1.2 Hz, 1H), 7.59–7.55 (m, 2H), 7.45–7.32 (m, 5H, Ar-H), 3.71 (dd, J = 12.1, 7.3 Hz, 2Hpip), 2.98 (dd, J = 12.4, 7.5 Hz, 2Hpip), 1.80–1.73 (m, 3Hpip), 1.49 (d, J = 6.2 Hz, 6H, 2CH3). Analysis: Calculated for C26H25N7O: C, 69.16; H, 5.58; N, 21.71. Found: C, 69.00; H, 5.77; N, 21.96.

4–[4–{4–[(4–Chloro–phenyl)–phenyl–methyl]–piperazin–1–yl}–6–(quinoline–4–yloxy)–1,3,5–triazine–2–ylamino]–benzonitrile (5e): Yield 78%, melting point 243oC-245°C; IR: 3286 (–NH), 2220 (C≡N), 1247 (C–O–C), 830 (s–triazine); 1H NMR: δ 9.14 (s, 1H,–NH, Ctri–NH–), 8.73 (d, J = 8.2 Hz, H2, 1H), 8.64 (s, 1H), 8.39 (dd, J = 7.6, 1.6 Hz, 1H), 8.15 (dd, J = 7.9, 1.3 Hz, 1H), 7.65–7.60 (m, 2H), 7.49–7.25 (m, 13H, Ar-H), 3.90 (s, 1H, N–CH), 3.86 (br s, 4Hpip), 3.49 (br s, 4Hpip). Analysis: Calculated for C36H29ClN8O: C, 69.17; H, 4.68; N, 17.93. Found: C, 69.02; H, 4.79; N, 17.71.

4–[4–[4–(2–Fluoro–phenyl)–piperazin–1–yl]–6–(quinoline–4–yloxy)–1,3,5–triazine–2–ylamino]–benzonitrile (5f): Yield 86%, melting point 251oC-253°C; IR: 3279 (–NH), 2223 (C≡N), 1258 (C–O–C), 839 (s–triazine); 1H NMR: δ 8.99 (s, 1H,–NH, Ctri–NH–), 8.70 (d, J = 7.3 Hz, 1H), 8.57 (s, 1H), 8.30 (dd, J = 7.4, 1.5 Hz, 1H), 8.11 (dd, J = 7.4, 1.2 Hz, 1H), 7.62–7.58 (m, 2H), 7.53–7.40 (m, 10H, 10CH), 6.91 (dd, J = 12.5, 6.8 Hz, 2H), 6.70–6.61 (1H, m), 6.49 (dd, J = 12.7, 6.5 Hz, 1H), 3.84 (br s, 4Hpip), 3.45 (br s, 4Hpip). Analysis: Calculated for C29H23FN8O: C, 67.17; H, 4.47; N, 21.61. Found: C, 66.98; H, 4.31; N, 21.42.

4–[4–[4–(4–Fluoro–phenyl)–piperazin–1–yl]–6–(quinoline–4–yloxy)–1,3,5–triazine–2–ylamino]–benzonitrile (5g): Yield 82%, melting point 266oC-267°C; IR: 3289 (–NH), 2219 (C≡N), 1254 (C–O–C), 829 (s–triazine); 1H NMR: δ 8.98 (s, 1H,–NH, Ctri–NH–), 8.77 (d, J = 7.5 Hz, 1H), 8.64 (s, 1H), 8.43 (dd, J = 8.2, 1.9 Hz, 1H), 8.15 (dd, J = 7.4, 1.3 Hz, 1H), 7.59–7.56 (m, 2H), 7.50–7.39 (m, 10H, Ar-H), 7.12 (dd, J = 12.9, 7.3 Hz, 2H), 6.60–6.54 (m, 1H), 6.50 (dd, J = 12.3, 6.1 Hz, 1H), 3.88 (br s, 4Hpip), 3.54 (br s, 4Hpip). Analysis: Calculated for C29H23FN8O: C, 67.17; H, 4.47; N, 21.61. Found: C, 67.36; H, 4.22; N, 21.79.

4–{4–(Quinoline–4–yloxy)–6–[4–(3–trifluoromethyl–phenyl)–piperazin–1–yl]–1,3,5–triazine–2–ylamino}–benzonitrile (5h): Yield 72%, melting point 285oC-286°C; IR: 3291 (–NH), 2218 (C≡N), 1259 (C–O–C), 829 (s–triazine); 1H NMR: δ 8.94 (s, 1H,–NH, Ctri–NH–), 8.61 (d, J = 6.9 Hz, 1H), 8.50 (s, 1H), 8.41 (dd, J = 7.7, 1.4 Hz, 1H), 8.01 (dd, J = 6.8, 1.0 Hz, 1H), 7.64–7.60 (m, 2H), 7.51–7.33 (m, 8H, Ar-H), 3.82 (br s, 4Hpip), 3.50 (br s,


4Hpip). Analysis: Calculated for C30H23F3N8O: C, 63.38; H, 4.08; N, 19.71. Found: C, 63.54; H, 3.91; N, 19.93.

4–{4–(Quinoline–4–yloxy)–6–[4–(2,3,4–trimethoxy–benzyl)–piperazin–1–yl]–1,3,5–triazine–2–ylamino}–benzonitrile (5i): Yield 75%, melting point 289oC-291°C; IR: 3277 (–NH), 2224 (C≡N), 1486 (–CH2), 1255 (C–O–C), 834 (s–triazine); 1H NMR: δ 9.17 (s, 1H,–NH, Ctri–NH–), 8.78 (d, J = 8.8 Hz, 1H), 8.71 (s, 1H), 8.39 (dd, J = 7.8, 1.9 Hz, 1H), 8.01 (dd, J = 7.1, 1.0 Hz, 1H), 7.69–7.67 (m, 2H), 745–7.26 (m, 6H, Ar-H), 6.82 (d, J = 7.1 Hz, 1H), 6.61 (d, J = 7.5 Hz, 1H), 3.87 (br s, 4Hpip), 3.64 (s, 9H, 3OCH3), 3.51 (br s, 4Hpip). Analysis: Calculated for C33H32N8O4: C, 65.55; H, 5.33; N, 18.53. Found: C, 65.31; H, 5.45; N, 18.69.

4–[4–[4–(4–Methoxy–phenyl)–piperazin–1–yl]–6–(quinoline–4–yloxy)–1,3,5–triazine–2–ylamino]–benzonitrile (5j): Yield 80%, melting point 242oC-243°C; IR: 3285 (–NH), 2222 (C≡N), 1249 (C–O–C), 835 (s–triazine); 1H NMR: δ 8.97 (s, 1H,–NH, Ctri–NH–), 8.77 (d, J = 7.5 Hz, 1H), 8.57 (s, 1H), 8.35 (dd, J = 8.0, 1.8 Hz, 1H), 8.11 (dd, J = 7.6, 1.3 Hz, 1H), 7.75–7.72 (m, 2H), 7.45–7.31 (m, 5H, Ar-H), 7.10 (d, J = 7.9 Hz, 1H), 6.69 (d, J = 7.1 Hz, 1H), 4.12 (s, 3H, OCH3), 3.85 (br s, 4Hpip), 3.47 (br s, 4Hpip). Analysis: Calculated for C30H26N8O2: C, 67.91; H, 4.94; N, 21.12. Found: C, 67.70; H, 4.77; N, 21.29.

4–[4–(3–Chloro–phenyl)–piperazin–1–yl]–6–(quinazolin–4–yloxy)–1,3,5–triazin–2–ylamino]–benzonitrile (6a): Yield: 85%; melting point 252oC-254°C; IR: 3281 (NH), 2222 (C≡N), 1252 (C–O–C), 833 cm–1 (s–triazine), 754 (C–Cl); 1H NMR: δ 8.78 (s, 1H, Ctri–NH–), 8.40 (s, 1H, N–CHqui–N), 8.01 (dd, J = 11.1, 7.4 Hz, 2H), 7.75 (t, J = 7.4 Hz, 1H), 7.50–7.29 (m, 6H, Ar–H), 7.03 (dd, J = 7.5, 6.8 Hz, 1H), 6.77 (dd, J = 10.8, 4.1 Hz, 1H), 6.67 (d, J = 7.4 Hz, 1H), 3.86 (br s, 4Hpip), 3.47 (br s, 4Hpip). Analysis: Calculated for C28H22ClN9O: C, 62.74; H, 4.14; N, 23.52. Found: C, 62.92; H, 3.92; N, 23.39.

4–[4–[4–(2,3–Dichloro–phenyl)–piperazin–1–yl]–6–(quinazolin–4–yloxy)–1,3,5–triazin–2–ylamino]–benzonitrile (6b): Yield: 78%; melting point 261oC-264°C; IR: 3277 (NH), 2224 (C≡N), 1255 (C–O–C), 830 cm–1 (s–triazine), 754 (C–Cl); 1H NMR: δ 8.97 (s, 1H, –NH, Ctri–NH–), 8.41 (s, 1H, N–CHqui–N), 8.11 (dd, J = 10.8, 7.9 Hz, 2H), 7.70 (t, J = 6.9 Hz, 1H), 7.47–7.39 (m, 3H, Ar–H), 7.16 (d, J = 7.4 Hz, 2H), 7.07–6.99 (m, 1H), 6.90 (t, J = 7.4 Hz, 1H), 6.51 (dd, J = 7.4, 0.8 Hz, 1H), 3.80 (br s, 4Hpip), 3.45 (br s, 4Hpip). Analysis: Calculated for C28H21Cl2N9O: C, 58.96; H, 3.71; N, 22.10. Found: C, 58.83; H, 3.59; N, 21.93.

4–[4–(4–Acetyl–piperazin–1–yl)–6–(quinazolin–4–yloxy)–1,3,5–triazin–2–ylamino]– benzonitrile (6c): Yield: 86%; melting point 228oC-230°C; IR: 3280 (NH), 2220 (C≡N), 1700 (–C=O), 1475 (–CH3), 1257 (C–O–C), 839 cm–1 (s–triazine). 1H NMR: δ 8.99 (s, 1H, Ctri–NH–), 8.30 (s, 1H, N–CHqui–N), 8.10 (dd, J = 10.3, 7.8 Hz, 2H), 7.81 (t, J = 7.2 Hz, 1H), 7.47–7.34 (m, 5H, Ar–H), 3.89 (br s, 4Hpip), 3.50 (br s, 4Hpip), 2.09 (s, 3H, COCH3). Analysis: Calculated for C24H21N9O2: C, 61.66; H, 4.53; N, 26.97. Found: C, 61.82; H, 4.26; N, 26.84.

4–[4–(3,5–Dimethyl–piperidin–1–yl)–6–(quinazolin–4–yloxy)–1,3,5–triazine–2–ylamino]–benzonitrile (6d): Yield: 83%; melting point 269oC-271°C; IR: 3280 (NH), 2221 (C≡N), 1256 (C–O–C), 838 (s–triazine); 1H NMR: δ 8.90 (s, 1H, Ctri–NH–), 8.42 (s, 1H, N–CHqui–N), 8.13 (dd, J = 11.6, 5.5 Hz, 2H), 7.67 (t, J = 6.6 Hz, 1H), 7.49–7.37 (m, 6H, Ar–H), 3.66 (dd, J = 12.4, 7.6 Hz, 2Hpip), 3.08 (dd, J = 12.4, 7.6 Hz, 2Hpip), 1.83–1.76 (m, 3Hpip), 1.45 (d, J = 6.4 Hz, 6H, 2CH3). Analysis: Calculated for C25H24N8O: C, 66.36; H, 5.35; N, 24.76. Found: C, 66.21; H, 5.20; N, 24.82.

4–[4–{4–[(4–Chloro–phenyl)–phenyl–methyl]–piperazin–1–yl}–6–(quinazolin–4–yloxy)–1,3,5–triazine–2–ylamino]–benzonitrile (6e): Yield: 78%; melting point 258oC-260°C; IR: 3284 (NH), 2222 (C≡N), 1255 (C–O–C), 837 (s–triazine); 1H NMR: δ 8.82 (s, 1H, Ctri–NH–), 8.41 (s, 1H, N–CHqui–N), 8.20 (dd, J = 11.3, 5.6 Hz, 2H), 7.70 (t, J = 7.1 Hz, 1H), 7.55–7.27 (m, 14H, Ar–H), 3.90 (s, 1H, N–CH), 3.77 (br s, 4Hpip), 3.43 (br s, 4Hpip). Analysis: Calculated for C35H28ClN9O: C, 67.14; H, 4.51; N, 20.13. Found: C, 66.97; H, 4.40; N, 20.29.

4–[4–[4–(2–Fluoro–phenyl)–piperazin–1–yl]–6–(quinazolin–4–yloxy)–1,3,5–triazine–2–ylamino]–benzonitrile (6f): Yield: 81%; melting point 265oC-266°C; IR: 3272 (NH), 2220 (C≡N), 1260 (C–O–C), 834 (s–triazine); 1H NMR: δ 8.95 (s, 1H, Ctri–NH–), 8.32 (s, 1H, N–CHqui–N), 7.98 (dd, J = 10.9, 7.0 Hz, 2H), 7.76 (t, J = 7.2 Hz, 1H), 7.51–7.29 (m, 5H, Ar–H), 6.92 (dd, J = 12.6, 6.7 Hz, 2H), 6.76–6.65 (m, 1H), 6.51 (dd, J = 12.6, 6.3 Hz, 1H), 3.82 (br s, 4Hpip), 3.55 (br s, 4Hpip). Analysis: Calculated for C28H22FN9O: C, 64.73; H, 4.27; N, 24.26. Found: C, 64.88; H, 4.16; N, 24.40.

4–[4–[4–(4–Fluoro–phenyl)–piperazin–1–yl]–6–(quinazolin–4–yloxy)–1,3,5–triazine–2–ylamino]–benzonitrile (6g): Yield: 70%; melting point 282oC-284°C; IR: 3284 (NH), 2218 (C≡N), 1258 (C–O–C), 829 (s–triazine); 1H NMR: δ 8.96 (s, 1H, Ctri–NH–), 8.44 (s, 1H, N–CHqui–N), 8.18 (dd, J = 11.1, 7.5 Hz, 2H), 7.83 (t, J = 7.9 Hz, 1H), 7.59–7.32 (m, 5H, Ar–H), 7.02 (dd, J = 12.8, 6.9 Hz, 2H), 6.67–6.64 (m, 1H), 6.49 (dd, J = 12.1, 6.0 Hz, 1H), 3.44 (br s, 4Hpip), 3.85 (br s, 4Hpip). Analysis:


Calculated for C28H22FN9O: C, 64.73; H, 4.27; N, 24.26. Found: C, 64.66; H, 4.42; N, 24.17.

4–{4–(Quinazolin–4–yloxy)–6–[4–(3–trifluoromethyl–phenyl)–piperazin–1–yl]–1,3,5–triazine–2–ylamino}–benzonitrile (6h): Yield: 77%; melting point >300°C; IR: 3294 (NH), 2223 (C≡N), 1187, 1134, 1109 (C–F), 1251 (C–O–C), 839 (s–triazine); 1H NMR: δ 8.91 (s, 1H, Ctri–NH–), 8.51 (s, 1H, N–CHqui–N), 8.24 (dd, J = 10.3, 7.2 Hz, 2H), 7.71 (t, J = 7.1 Hz, 1H), 7.55–7.20 (m, 9H, Ar–H), 3.48 (br s, 4Hpip), 3.83 (br s, 4Hpip). Analysis: Calculated for C29H22F3N9O: C, 61.16; H, 3.89; N, 22.13. Found: C, 60.98; H, 3.79; N, 22.01.

4–{4–(Quinazolin–4–yloxy)–6–[4–(2,3,4–trimethoxy–benzyl)–piperazin–1–yl]–1,3,5–triazine–2–ylamino}–benzonitrile (6i): Yield: 81%; melting point 265oC-267°C; IR: 3289 (NH), 2219 (C≡N), 1482 (–CH2), 1258 (C–O–C), 830 (s–triazine); 1H NMR: δ 8.82 (s, 1H, Ctri–NH–), 8.49 (s, 1H, N–CHqui–N), 8.17 (dd, J = 10.1, 6.9 Hz, 2H), 8.07 (t, J = 7.6 Hz, 1H), 7.63–7.25 (m, 27H, Ar–H), 6.89 (d, J = 7.4 Hz, 1H), 6.71 (d, J = 7.6 Hz, 1H), 3.71 (s, 9H, 3OCH3), 3.78 (br s, 4Hpip), 3.41 (br s, 4Hpip). Analysis: Calculated for C32H31N9O4: C, 63.46; H, 5.16; N, 20.81. Found: C, 63.60; H, 5.00; N, 20.94.

4–[4–[4–(4–Methoxy–phenyl)–piperazin–1–yl]–6–(quinazolin–4–yloxy)–1,3,5–triazine–2–ylamino]–benzonitrile (6j): Yield: 84%; melting point 260oC-261°C; IR: 3281 (NH), 2222 (C≡N), 1249 (C–O–C), 835 (s–triazine); 1H NMR: δ 8.88 (s, 1H, Ctri–NH–), 8.41 (s, 1H, N–CHqui–N), 8.09 (dd, J = 9.9, 6.1 Hz, 2H), 8.11 (t, J = 7.8 Hz, 1H), 7.56–7.29 (m, 7H, Ar–H), 7.03 (d, J = 7.7 Hz, 1H), 6.66 (d, J = 7.1 Hz, 1H), 4.02 (s, 3H, OCH3), 3.85 (br s, 4Hpip), 3.47 (br s, 4Hpip). Analysis: Calculated for C29H25N9O2: C, 65.53; H, 4.74; N, 23.71. Found: C, 65.37; H, 4.87; N, 23.62.

Antimicrobial activity The synthesized derivatives (6a–k, 7a–k) were examined for antimicrobial activity against several bacteria (Staphylococcus aureus MTCC 96, Bacillus cereus MTCC 619, Escherichia coli MTCC 739, Pseudomonas aeruginosa MTCC 741, Klebsiella pneumoniae MTCC 109, Salmonella typhi MTCC 733, Proteus vulgaris MTCC 1771, Shigella Flexneria MTCC 1457) and fungi (Aspergillus niger MTCC 282, Aspergillus fumigatus MTCC 343, Aspergillus clavatus MTCC 1323, Candida albicans MTCC 183) species using paper disc diffusion technique19 and MIC of the compound was determined by agar streak dilution method20 as described earlier21. Antituberculosis screening for test compounds was performed against M. tuberculosis H37Rv using L. J. (Lowenstein and Jensen) MIC method22 for the

measurement of MIC. The MIC values were evaluated at concentration range of 0.39–100 µg/ml and each value presented in Table 1 and 2 is the mean of three independent experiments.

Acknowledgements The authors are thankful to Applied Chemistry Department of S. V. National Institute of Technology, Surat for the scholarship, encouragement and facilities. The authors wish to offer their deep gratitude to Microcare Laboratory, Surat, India for carrying out the biological screenings. We are also thankful to Centre of Excellence, Vapi, India for carrying out FT–IR and 1H NMR analysis.

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[18] Patel, R.V.; Kumari, P.; Rajani, D.P.; Chikhalia, K.H. J. Fluorine Chem. 2011, 132, 617.

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[22] Isenberg, H.D. Clinical microbiology procedures handbook, vol. 1, American Society for Microbiology, Washington, D.C. 1992.

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Synthesis of Potential Antimicrobial/Antitubercular s ... · Rahul V. Patel et al : Synthesis of Potential Antimicrobial/Antitubercular s-triazine Scaffolds Endowed with Quinoline