Chapter I Section A

Synthesis and Screening of Some Bioactive Heterocycles

Chapter I Section A

Novel fusion of 1,2,3 triazole, piperidine and quinoxaline rings and evaluation of their antifungal activity INTRODUCTION The emergence of highly resistant eukaryotic microorganisms Fungi to drugs in the past decades has challenged the research group’s world over to design, synthesize and evaluate the new molecules.1 Fungi are vital opportunistic pathogens of humans, and are becoming drug-resistant to the approved compounds most prominent of them include Cryptococcus neoformans and species of Candida and Aspergillus. The Candida albicans, could cause superficial or invasive infections or both in immune compromised individuals.2 Therefore, it is necessary to find new and efficacious drugs to solve these problems. Quinoxaline derivatives have gained considerable interest from pharmaceutical perspectives as

they are significant

3

intermediates for the synthesis of API’s. They have been shown to possess wide range of pharmacological activities like antiviral, antibacterial, anti-inflammatory, antitumor, antidepressant and Pim kinases inhibitory activities.4,5 The 1,2,3-triazole and its derivatives forms an important class of heterocycles that has pivotal position especially in medicinal chemistry. The 1,2,3triazoles analogues have chemotherapeutic value, 6 which are used as potent antineoplastic,7 antimicrobial, anticonvulsant,

12

antimalarial,

8

analgesic, 13

9

antiinflammatory,10 local anesthetic,

11

and antiHIV agents.14 Some of the 1,2,3-triazole

derivatives are used as DNA cleaving agents15 and as potential antitubercular agents.16 Piperidine nucleus is another important heterocycle which forms an important core of many drug molecules. Piperidine and its analogues are reported in literature for varied pharmacological activities like antibacterial, agents.

17

antihistaminics

18

and antitubercular

19

After extensive literature search, it was observed that, till date enough efforts have not been made to combine these three moieties as a single molecular scaffold and to study their biological activity. Recently, our investigation has been directed towards the synthesis of heterocyclic molecules and studying their biological, especially antifungal activity. In continuation with our earlier work,

20

to identify and

design new antifungal molecules which are potent, selective and less toxic, we report herein the synthesis and antimicrobial evaluation of some novel structural scaffolds incorporating both the quinoxaline moiety with 1,2,3-triazoles, and piperidine. This University Department of Chemical Technology, Dr. Babasaheb Ambedkar Marathwada University

23

Chapter I Section A

Synthesis and Screening of Some Bioactive Heterocycles

combination was selected in an attempt to investigate the influence of such fusion and structure variation on the probable biological activities, hoping to add some biological significance to the antifungal activity. RESULTS AND DISCUSSION The reaction sequence for different title compounds is as outlined in (Scheme 1 and Scheme 2). The starting material tert-butyl 4-azidopiperidine-1carboxylate (1), was prepared according to our reported procedure.20 Scheme 1. Synthesis of tert-butyl 4-(4-(((5-bromoquinoxalin-6-yl)amino) methyl) -1H-1,2,3-triazol-1-yl)piperidine-1-carboxylate (5).

The compound (1) was cyclized using propargyl alcohol in dimethyl formamide to give tert-butyl 4-(4-(hydroxymethyl)-1H-1,2,3-triazol-1-yl)piperidine1-carboxylate (2) with 92 % yield. The compound (2) was subjected to O-mesylation using methane sulphonyl chloride to give the corresponding O-mesyl derivative (3) with 95% yield. The tert-butyl 4-(4-(((5-bromoquinoxalin-6-yl)amino)methyl)-1H1,2,3-triazol-1-yl)piperidine-1-carboxylate (5), was prepared by the nucleophilic substitution reaction using 5-bromoquinoxalin-6-amine in acetonitrile at 60oC with 84 % yield (Scheme 1). Compound (5) was then subjected to deprotection to give 5bromo-N-((1-(piperidin-4-yl)-1H-1,2,3-triazol-4-yl)methyl) quinoxalin-6- amine (6a) by using trifluoro acetic acid (TFA) in dichloromethane with 90 % yield. The 5bromo-N-((1-(piperidin-4-yl)-1H-1,2,3-triazol-4-yl)methyl) quinoxalin-6-amine (6a) University Department of Chemical Technology, Dr. Babasaheb Ambedkar Marathwada University

24

Synthesis and Screening of Some Bioactive Heterocycles

Chapter I Section A

was subjected to substitution reaction by using acyl chlorides/ alkyl chlorides to get the target derivatives (6b-l) in dichloromethane with 80-90 % yield (Table 1, Scheme 2). Herein the N-substitution occurred, on the nitrogen of piperazine ring which was sterically less hindered compared to nitrogen attached to the aromatic ring. The nitrogen attached to the aromatic ring is in sterically crowded environment with aromatic ring on one side and the triazole ring blocking the other side for substitution. Scheme 2. Synthesis of 5-bromo-N-((1-(piperidin-4-yl)-1H-1,2,3-triazol-5yl)methyl) quinoxalin-6-amine derivatives (6a-l).

Table 1. Synthesis of 5-bromo-N-((1-(piperidin-4-yl)-1H-1,2,3-triazol-5-yl) methyl)quinoxalin-6-amine derivatives (6a-l).a,c Sr. no.

Substituent (-R)

Time ( hr.)

Yieldc (%)

Melting point (°C)

6a

Hydrogen

2.0

90

162-164

6b

Methyl

2.5

89

178-180

6c

Ethyl

2.5

87

170-172

6d

Isopropyl

2.5

90

184-186

6e

Butyl

3.5

82

156-158

6f

Acetyl

2.0

89

164-166

6g

Methane sulphonyl

2.0

90

179-181

6h

Benzoyl

3.0

90

191-193

6i

Benzyl

3.5

80

185-187

6j

p-Pyridine

5.0

81

174-176

6k

p-Phenol

4.5

82

192-194

6l

p-Tolyl

5.0

81

189-191

a. Reaction condition (6a): Compound (5) (10 mmol), TFA (2 mmol) in (10 mL) of DCM at room temperature. b. Reaction condition (6b-l): Compound (6a) (10 mmol), alkyl or acyl halides (10 mmol) in 10 mL of DCM, triethyl amine (20 mmol) and CuI (2 mmol) at 0-5°C. c. Isolated yields.

ANTIFUNGAL ACTIVITY All the synthesized compounds were screened for in-vitro antifungal activity. The antifungal activity was evaluated against different fungal strains such as University Department of Chemical Technology, Dr. Babasaheb Ambedkar Marathwada University

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Synthesis and Screening of Some Bioactive Heterocycles

Chapter I Section A

Candida albicans (NCIM3471), Fusarium oxysporum (NCIM1332), Aspergillus flavus (NCIM539), Aspergillus niger (NCIM1196) and Cryptococcus neoformans (NCIM576). Minimum inhibitory concentration (MIC) values were determined using standard agar dilution method as per CLSI guidelines.21-24 Miconazole was used as a standard for the comparison of antifungal activity. Dimethyl sulfoxide was used as solvent control. MIC values of the tested compounds are presented in (Table 2). STRUCTURE ACTIVITY RELATIONSHIP From the antifungal activity data (Table 2), it was observed that compound (6a, 6f and 6g) are the most active among all tested compounds. Compounds (2, 3 and 5) are the important intermediates in the synthesis of target compounds and were also tested for antifungal activity. Cyclic alcohol compound (2) shows moderate activities. Corresponding O- mesylated compound (3) shows slight increments in activity. Introduction of substituted quinoxaline ring by replacing -OMs leads to the formation of amine compound (5). These variations shows increased antifungal activity. Deprotection of the -boc of nitrogen in piperidine (6a) enhances the antifungal activity against all the tested organisms except C. neoformans. Compound (6a) is equipotent with miconazole against C. albicans and F.oxysporum. Table 2. Antifungal activity of the synthesized compounds (2, 3, 5 and 6a-l) MIC Values (μg/mL)a

Compound C. Albicans

F. Oxysporum

A. Flavus

A. Niger

C. Neoformans

2

50

50

30

40

*

3

45

40

25

30

150

5

30

30

20

25

125

6a

25

25

15

15

100

6b

40

40

25

25

150

6c

50

80

50

50

*

6d

70

100

70

80

*

6e

90

*

100

150

*

6f

20

20

15

15

80

6g

15

20

10

10

30

6h

50

40

60

80

100

6i

60

50

70

100

*

6j

35

30

40

60

80

6k

100

100

150

100

150

6l

150

150

*

150

*

Miconazole

25

25

12.5

12.5

25

*- Activity not reported up to 200 μg/mL

a - Values are average of three readings.

University Department of Chemical Technology, Dr. Babasaheb Ambedkar Marathwada University

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Chapter I Section A

Synthesis and Screening of Some Bioactive Heterocycles

Substitution of methyl group (6b) on nitrogen decreases the antifungal activity compared with unsubstituted nitrogen (6a). The further increase in the alkyl chain decreases antifungal activity resulting in enhanced MIC (6c, 6d, 6e). Introduction of acetyl group on nitrogen (6f) increases the antifungal activity compared with unsubstituted analogue against all tested organisms except against C. neoformans. This modification gives rise to one of the active compound in the series. Compound (6f) is more potent than miconazole against C. albicans and F. oxysporum whereas it is almost equipotent with miconazole against A. flavus and A. niger. Introduction of methyl sulfone on piperidine nitrogen gave most active compound of the series i.e. (6g). Compound (6g) was more potent than miconazole against all tested organisms except against C. neoformans. Introduction of –COPh (benzoyl) and – CH2Ph (benzyl) on piperidine nitrogen (6h, 6i) leads to decrease in activity compared to its methyl substituted analogue. Introduction of pyridine nucleus on nitrogen (6j) was more beneficial than its corresponding phenyl substituted analogues. Introduction of phenol and toluene on the piperidine nitrogen led to decrease in antifungal activity. EXPERIMENTAL The propargyl alcohol, copper iodide, triethyl amine, trifluoro acetic acid, substituted alkyl, acyl halides and 5-bromoquinoxalin-6-amine used were commercially available. Melting points were recorded on SRS Optimelt, melting point apparatus and are uncorrected. 1H NMR spectra were recorded on a 400 MHz Bruker spectrometer and 13C NMR spectra were recorded on a 100 MHz Bruker spectrometer are reported as parts per million (ppm) downfield from a tetramethylsilane internal standard. The following abbreviations are used; singlet (s), doublet (d), triplet (t), quartet (q), multiplate (m) and broad (br). Mass spectra were taken with MicromassQUATTRO-II of WATER mass spectrometer. Synthesis of tert-butyl 4-(4-(hydroxymethyl)-1H-1,2,3-triazol-1-yl)piperidine-1carboxylate (2) In a 50 mL RBF, propargyl alcohol (10 mmol), triethyl amine (20 mmol) and compound (1) (10 mmol) was added in dimethyl formamide (5 ml), followed by CuI (2 mmol) and the resulting solution was stirred at room temperature for 10 hr. After completion (monitored by TLC, 20 % Ethyl acetate: n-hexane), solvent was removed in-vacuo and ethyl acetate (10 mL) was added to the residue and then extracted with 3 portions (3 x 10 mL) of distilled water. The combined organic layers were dried over University Department of Chemical Technology, Dr. Babasaheb Ambedkar Marathwada University

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Chapter I Section A

Synthesis and Screening of Some Bioactive Heterocycles

anhydrous Na2SO4 and concentrated in-vacuo to get the desired compound (2), which was recrystallized using ethanol as solvent. Pale Yellow Solid, Yield: 92 %, Melting Point: 173-175oC ES-MS m/z (%): 283 (M+H) 1 H NMR (400 MHz, CDCl3):  ppm 1.51 (s, 9H), 2.10-2.20 (m, 4H), 2.45-2.55 (m, 4H), 3.72 (m, 1H), 3.95(s, 1H), 4.72 (d, 2H), 7.82 (s, 1H). 13 C NMR (100 MHz, CDCl3):  ppm 27.1, 29.4, 45.7, 55.7, 61.2, 82.3, 124.7, 144.1, 160.8. Synthesis of tert-butyl 4-(4-(((methylsulfonyl)oxy)methyl)-1H-1,2,3-triazol-1-yl) piperidine-1-carboxylate (3) In a 50 mL RBF, compound (2) (10 mmol) and triethyl amine (15 mmol) was added in 10 mL dichloromethane, stirred at room temperature for 5 minutes and then cooled to 0-5°C. Methane sulphonyl chloride (12 mmol) was added drop wise at 0-5°C, after addition the reaction mixture was allowed to attain room temperature and then stirred at room temperature for 2 hr. After completion (monitored by TLC, 30 % Ethyl acetate: n-hexane), the reaction was quenched by addition of distill water (10 mL).The organic layer was extracted with 3 portions (3 x 10 mL) of water and was washed with saturated NaHCO3 (3 x 10 mL), the dichloromethane layer after extraction was separated, dried over anhydrous Na2SO4 and concentrated in-vacuo to afford compound (3), which was recrystallized using ethanol as solvent. Off White Solid, Yield: 95 %, Melting Point: 210-212oC ES-MS m/z (%): 361 (M+H) 1 H NMR (400 MHz, CDCl3):  ppm 1.54 (s, 9H), 2.14-2.27 (m, 4H), 2.38-2.51 (m, 4H), 3.25 (s, 3H), 3.78 (m, 1H), 4.65 (d, 2H), 7.80 (s, 1H). 13 C NMR (100 MHz, CDCl3):  ppm 27.8, 29.7, 40.1, 45.3, 61.8, 65.1, 81.6, 123.9, 143.7, 160.1. Synthesis of tert-butyl 4-(4-(((5-bromoquinoxalin-6-yl)amino)methyl)-1H-1,2,3triazol-1-yl)piperidine-1-carboxylate (5) In a 50 mL RBF, compound (3) (10 mmol), triethyl amine (20 mmol) and 5bromoquinoxalin-6-amine (4) (10 mmol) in 10 mL of acetonitrile was added at room temperature. The resulting solution was then heated to 60oC for 8 hr. After completion (monitored by TLC, 20 % Ethyl acetate: n-hexane), solvent was removed in-vacuo and ethyl acetate (10 mL) was added to the residue and then extracted with 3 portions (3 x 10 mL) of distilled water. The organic layer was dried over anhydrous Na2SO4 and concentrated in-vacuo to get the desired compound (5), which was recrystallized using ethanol as solvent. Light Brown Solid, Yield: 84 %, Melting Point: 188-190 oC University Department of Chemical Technology, Dr. Babasaheb Ambedkar Marathwada University

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Chapter I Section A

Synthesis and Screening of Some Bioactive Heterocycles

ES-MS m/z (%): 489 (M+H) 1 H NMR (400 MHz, CDCl3):  ppm 1.52 (s, 9H), 2.20-2.45 (m, 4H), 2.75-2.95 (m, 4H), 3.91 (m, 1H), 7.51 (s, 1H), 7.95 (d, 1H), 9.41 (s, 1H), 9.70 (s, 1H). 13 C NMR (100 MHz, CDCl3):  ppm 27.1, 29.7, 42.4, 46.8, 61.3, 81.2, 98.5, 120.9, 124.3, 126.4, 128.0, 131.2, 143.6, 152.1, 153.9, 157.4, 160.8. Synthesis of 5-bromo-N-((1-(piperidin-4-yl)-1H-1,2,3-triazol-4-yl)methyl)quinoxalin -6-amine (6a) In a 50 mL RBF, compound (5) (10 mmol) and trifluoro acetic acid (2 mmol) was added in 10 mL of dichloromethane. The solution was stirred for 2 hr. at room temperature. After completion (monitored by TLC, 40 % Ethyl acetate: n-hexane), the mixture was extracted with saturated NaHCO3 (3 x 10 mL). The organic layers was, dried over anhydrous Na2SO4 and evaporated to get the desired product (6a) (Table 1, entry 1) in 90 % yield, which was recrystallized using ethanol as solvent. Synthesis of 5-bromo-N-((1-(1-methylpiperidin-4-yl)-1H-1,2,3-triazol-4-yl)methyl) quinazolin-6-amine (6b-l) In a 50 mL RBF, compound (6a) (10 mmol) in 10 mL of dichloromethane, triethyl amine (20 mmol) and CuI (2 mmol) was added and stirred at room temperature for 5 minutes and then cooled to 0-5°C. To this cooled solution, different substituted alkyl or acyl halides (10 mmol) were added. The resulting solution was stirred for 2-5 hr. After completion (monitored by TLC, 40 % Ethyl acetate: n-hexane), extracted with distilled water (3x10mL). The organic layer was dried over anhydrous Na2SO4 and on concentration in-vacuo gave the desired compounds (6b-l) in 80-90 % yield (Table 1, entry 2-12), which were recrystallized using ethanol as solvent. Spectral characterization 5-bromo-N-((1-(piperidin-4-yl)-1H-1,2,3-triazol-4-yl)methyl)quinoxalin-6-amine (6a): Pale Yellow Solid, ES-MS m/z (%): 389 (M+H). 1

H NMR (400 MHz, CDCl3):  ppm 2.04 (s, 1H), 2.10-2.25 (m, 4H), 2.58-2.72 (m, 4H), 3.72 (m, 1H), 4.04 (d, 1H), 4.60 (d, 2H), 6.75 (d, 1H), 7.44 (s, 1H), 7.82 (d, 1H), 8.56 (d, 2H). 13 C NMR (100 MHz, CDCl3):  ppm 27.1, 42.4, 46.8, 59.6, 97.8, 121.3, 124.7, 126.8, 128.3, 132.1, 151.3, 144.8, 154.5, 158.8. 5-bromo-N-((1-(1-methylpiperidin-4-yl)-1H-1,2,3-triazol-4-yl)methyl)quinoxalin-6amine (6b): Pale Yellow Solid, ES-MS m/z (%): 403 (M+H). 1 H NMR (400 MHz, CDCl3):  ppm 2.15-2.31 (m, 4H), 2.40 (s, 3H), 2.60-2.76 (m, 4H), 3.68 (m, 1H), 4.14 (d, 1H), 4.42 (d, 2H), 6.69 (d, 1H), 7.32 (s, 1H), 7.71 (d, 1H), 8.62 (d, 2H). University Department of Chemical Technology, Dr. Babasaheb Ambedkar Marathwada University

29

Chapter I Section A

Synthesis and Screening of Some Bioactive Heterocycles

13

C NMR (100 MHz, CDCl3):  ppm 25.3, 42.9, 58.1, 60.2, 98.4, 121.7, 125.2, 127.4, 128.9, 132.9, 146.2, 152.4, 156.1, 159.4. 5-bromo-N-((1-(1-ethylpiperidin-4-yl)-1H-1,2,3-triazol-4-yl)methyl)quinoxalin-6amine (6c): Pale Yellow Solid, ES-MS m/z (%): 417 (M+H). 1

H NMR (400 MHz, CDCl3):  ppm 1.31 (t, 3H), 2.30-2.40 (m, 4H), 2.56-2.70 (m, 4H), 3.20 (q, 2H), 3.55 (m, 1H), 4.02 (d, 1H), 4.34 (d, 2H), 6.75 (d, 1H), 7.25 (s, 1H), 7.67 (d, 1H), 8.67 (d, 2H). 13 C NMR (100 MHz, CDCl3):  ppm 15.1, 28.1, 43.7, 51.2, 56.8, 60.9, 97.9, 122.2, 124.7, 127.8, 128.5, 131.5, 145.6, 149.6, 155.1, 158.3. 5-bromo-N-((1-(1-isopropylpiperidin-4-yl)-1H-1,2,3-triazol-4-yl)methyl)quinoxalin6-amine (6d): Pale Yellow Solid, ES-MS m/z (%): 431 (M+H). 1

H NMR (400 MHz, CDCl3):  ppm 1.18 (d, 6H), 2.32-2.45 (m, 4H), 2.50-2.65 (m, 4H), 2.85 (m, 1H), 3.69 (m, 1H), 4.07 (d, 1H), 4.24 (d, 2H), 6.81 (d, 1H), 7.18 (s, 1H), 7.52(d, 1H), 8.51(d, 2H). 13 C NMR (100 MHz, CDCl3):  ppm 20.3, 28.7, 42.9, 52.6, 57.1, 59.7, 61.7, 98.4, 121.8, 122.9, 126.1, 129.3, 133.7, 146.9, 151.3, 154.8, 157.1. 5-bromo-N-((1-(1-butylpiperidin-4-yl)-1H-1,2,3-triazol-4-yl)methyl)quinoxalin-6amine (6e): Pale Yellow Solid, ES-MS m/z (%): 445 (M+H). 1

H NMR (400 MHz, CDCl3):  ppm 0.99 (q, 3H), 1.45 (m, 4H), 2.18-2.32 (m, 4H), 2.62-2.75 (m, 4H), 3.12 (m, 2H), 3.82 (m, 1H), 4.08 (d, 1H), 4.68 (d, 2H), 7.18 (d, 1H), 7.72 (s, 1H), 7.85 (d, 1H), 8.44 (d, 2H). 13

C NMR (100 MHz, CDCl3):  ppm 15.4, 19.2, 28.1, 31.1, 42.8, 53.7, 58.3, 62.4, 98.1, 122.2, 125.4, 127.6, 128.7, 132.7, 142.8, 148.4, 152.4, 157.3. 1-(4-(4-(((5-bromoquinoxalin-6-yl)amino)methyl)-1H-1,2,3-triazol-1-yl)piperidin-1yl)ethanone (6f): Pale Yellow Solid, ES-MS m/z (%): 431 (M+H). 1 H NMR (400 MHz, CDCl3):  ppm 2.26 (s, 3H), 2.40-2.55 (m, 4H), 3.48-3.60 (m, 4H), 3.80 (m, 1H), 4.05 (d, 1H), 4.31 (d, 2H), 6.77 (d, 1H), 7.12 (s, 1H), 7.43 (d, 1H), 8.47 (d, 2H). 13

C NMR (100 MHz, CDCl3):  ppm 23.2, 28.5, 43.5, 45.2, 61.2, 98.8, 120.3, 121.6, 126.7, 129.5, 132.8, 145.2, 152.4, 156.1, 157.1, 175.3. 5-bromo-N-((1-(1-(methylsulfonyl)piperidin-4-yl)-1H-1,2,3-triazol-4-yl)methyl) quinoxalin-6-amine (6g): Pale Yellow Solid, ES-MS m/z (%): 467 (M+H). 1 H NMR (400 MHz, CDCl3):  ppm 2.31-2.40 (m, 4H), 2.62-2.75 (m, 4H), 2.90 (s, 3H), 3.44 (m, 1H), 4.10 (d, 1H), 4.48 (d, 2H), 6.95 (d, 1H), 7.41 (s, 1H), 7.73 (d, 1H), 8.42 (d, 2H). 13 C NMR (100 MHz, CDCl3):  ppm 26.4, 40.2, 42.8, 52.8, 62.1, 99.5, 121.4, 122.5, 127.3, 129.5, 131.4, 144.1, 149.9, 152.5, 156.9. University Department of Chemical Technology, Dr. Babasaheb Ambedkar Marathwada University

30

Chapter I Section A

Synthesis and Screening of Some Bioactive Heterocycles

(4-(4-(((5-bromoquinoxalin-6-yl)amino)methyl)-1H-1,2,3-triazol-1-yl)piperidin-1yl)(phenyl)methanone (6h): Pale Yellow Solid, ES-MS m/z (%): 493 (M+H). 1 H NMR (400 MHz, CDCl3):  ppm 2.40-2.50 (m, 4H), 2.65–2.75 (m, 4H), 3.54 (m, 1H), 4.05 (d, 1H), 4.28 (d, 2H), 6.72 (d, 1H), 7.25 (s, 1H), 7.56 (d, 1H), 7.70-7.85 (m, 5H), 8.59 (d, 2H). 13

C NMR (100 MHz, CDCl3):  ppm 27.5, 42.7, 45.1, 62.6, 99.5, 120.3, 122.1, 125.4, 126.5, 128.1, 129.4, 131.1, 136.7, 144.2, 149.3, 153.9, 157.5, 170.2. 5-bromo-N-((1-(1-phenylpiperidin-4-yl)-1H-1,2,3-triazol-4-yl)methyl)quinoxalin-6amine (6i): Pale Yellow Solid, ES-MS m/z (%): 465 (M+H). 1 H NMR (400 MHz, CDCl3):  ppm 2.20-2.35 (m, 4H), 2.42-2.60 (m, 4H), 3.80 (s, 3H), 3.18 (m, 1H), 4.05 (d, 1H), 4.52 (d, 2H), 6.85 (d, 1H), 7.20-7.35 (m, 5H), 7.50 (s, 1H), 7.61 (d, 1H), 8.51 (d, 2H). 13 C NMR (100 MHz, CDCl3):  ppm 27.8, 43.1, 51.3, 61.7, 67.7, 98.7, 117.1, 120.1, 122.3, 122.9, 125.2, 126.1, 127.4, 128.5, 129.8, 132.5, 134.7, 139.6, 143.8, 151.0, 153.9, 157.5. 5-bromo-N-((1-(1-(pyridin-4-yl)piperidin-4-yl)-1H-1,2,3-triazol-4-yl)methyl) quinoxalin-6-amine (6j): Pale Yellow Solid, ES-MS m/z (%): 466 (M+H). 1 H NMR (400 MHz, CDCl3):  ppm 2.35-2.50 (m, 4H), 2.65-2.80 (m, 4H), 3.65 (m, 1H), 4.12 (d, 1H), 4.62 (d, 2H), 6.90 (d, 1H), 7.15 (d, 2H), 7.51 (s, 1H), 7.95 (d, 1H), 8.33 (d, 2H), 8.48 (d, 2H). 13

C NMR (100 MHz, CDCl3):  ppm 28.2, 43.7, 52.7, 61.5, 99.5, 107.5, 120.7, 122.5, 126.2, 127.9, 131.7, 143.1, 145.2, 148.3, 152.1, 153.4, 156.3. 4-(4-(4-(((5-bromoquinoxalin-6-yl)amino)methyl)-1H-1,2,3-triazol-1-yl)piperidin-1yl)phenol (6k): Pale Yellow Solid, ES-MS m/z (%): 481 (M+H). 1 H NMR (400 MHz, CDCl3):  ppm 2.40-2.55 (m, 4H), 2.62-2.75 (m, 4H), 3.75 (m, 1H), 4.02 (d, 1H), 4.54 (d, 2H), 5.72 (s, 1H), 6.62 (d, 2H), 6.85(d, 2H), 6.90 (d, 1H), 7.65 (s, 1H), 7.90 (d, 1H), 8.41 (d, 2H). 13

C NMR (100 MHz, CDCl3):  ppm 27.9, 43.3, 51.2, 61.8, 99.8, 115.5, 118.2, 121.3, 122.7, 125.6, 127.6, 130.9, 141.8, 143.1, 148.8, 149.4, 153.1, 158.4. 5-bromo-N-((1-(1-(p-tolyl)piperidin-4-yl)-1H-1,2,3-triazol-4-yl)methyl)quinoxalin6-amine (6l): Pale Yellow Solid, ES-MS m/z (%): 479 (M+H). 1 H NMR (400 MHz, CDCl3):  ppm 2.12 (s, 3H), 2.52-2.65 (m, 4H), 2.70-2.83 (m, 4H), 3.82 (m, 1H), 4.06 (d, 1H), 4.59 (d, 2H), 6.58 (d, 2H), 6.85 (d, 1H), 7.12 (d, 2H), 7.68 (s, 1H), 7.92 (d, 1H), 8.52(d, 2H). 13 C NMR (100 MHz, CDCl3):  ppm 22.0, 27.5, 42.8, 51.0, 61.6, 99.7, 114.2, 120.4, 121.5, 122.3, 125.4, 127.1, 128.4, 130.4, 131.1, 132.7, 143.4, 149.8, 152.4, 153.4, 158.9.

University Department of Chemical Technology, Dr. Babasaheb Ambedkar Marathwada University

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Synthesis and Screening of Some Bioactive Heterocycles

Chapter I Section A 1

H NMR spectrum of compound (6b):

N N

N

N H

N N

N Br

N N

13

N

N N

N H

N Br

C NMR spectrum of compound (6b):

N N

N

N N

N H

N Br

University Department of Chemical Technology, Dr. Babasaheb Ambedkar Marathwada University

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Synthesis and Screening of Some Bioactive Heterocycles

Chapter I Section A

Mass spectrum of compound (6b):

N N

N

N N

N H

N Br

University Department of Chemical Technology, Dr. Babasaheb Ambedkar Marathwada University

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Chapter I Section A

Synthesis and Screening of Some Bioactive Heterocycles

REFRENCES 1. Hawksworth, D. L. Mycol. Res. 2001, 105, 1422-1432. 2. Odds, F. C.; Gow, N. Trends in Microbiology 2003, 11, 272-279. 3. Sato, N. Comprehensive Heterocyclic Chemistry II, Oxford, 1996, 6, 233. 4. Sakata, G.; Makino, K.; Kuraswa, Y. Heterocycles 1988, 27, 2481-2515. 5. Gavara, L.; Moreau P. Eur. J. Med. Chem. 2010, 45, 5520-5526. 6. Passananti, A.; Lauria, A.; Cirrincione, G. Heterocycles 1998, 48, 1229-1235. 7. Chen, M. D.; Du, X. L. Heterocyclic Comm. 2000, 6, 421-426. 8. Holla, B. S.; Kumari, N. S. Eur. J. Med. Chem. 2005, 40, 1173-1178. 9. Banu, K. M.; Ananthanarayanan, C. Ind. J. Pharm. Sci., 1999, 4, 202-205. 10. Cunha, A. C.; Barreiroa, E. J. Bioorg. Med. Chem. Lett., 2003, 11, 2051-2059. 11. Saemian, N.; Matloubi, H. J. Radioanal. Nucl. Chem., 2006, 268, 545-548. 12. Jilino, M.; Stevens, F. G. J. Chem. Soc, Perkin Trans., 1998, 1, 1677-1680. 13. Velarquez, S.; Camarasa, M. J. Antivir. Chem. Chemother. 1998, 9, 481-489. 14. Danoun, S.; Tomas, V. A. Heterocyclic Comm. 1998, 4, 45-51. 15. Biagi, G.; Martelli, A.; Nardi, A. Il Farmaco. 2004, 59, 397-404. 16. Weis, R.; Seebacher, W. Bioorg. Med. Chem. Lett. 2008, 16, 10326-10331. 17. Sheng, R.; Yang, B.; He, Q.; Hu, Y. Eur. J. Med. Chem., 2009, 44 , 7-17. 18. Aridoss, G.; Jeong, Y. T.; Bioorg. Med. Chem. Lett. 2008, 18, 6542-6548. 19. Bogatcheva, E.; Propova, M.; Bioorg. Med. Chem. Lett. 2011, 21, 5353-5357. 20. a) Sangshetti, J. N.; Chabukswar, A. R.; Shinde, D. B. Bioorg. Med. Chem. Lett. 2011, 21, 444-448; b) Sangshetti, J. N.; Shinde, D. B. Eur. J. Med. Chem. 2011, 46, 1040-1045. 21. Cruickshank, R.; Duguid, J. P.; Marmion, B. P.; Swain, R. H. Eds., 2 th Ed. Medicinal Microbiology; Churchill Livingstone: London, 1975, 2. 22. Collins, A. H. Microbiological Methods, 2 nd Ed. Butterworth: London, 1976. 23. Khan, Z. K. In vitro and vivo screening techniques for bioactivity screening and evaluation, Proc. Int. Workshop UNIDO-CDRI, 1997, 210-211. 24. a) Duraiswamy, B.; Mishra, S. K.; Subhashini, V.; Dhanraj, S. A.; Suresh, B. Ind. J. Pharm. Sci., 2006, 68, 389-391; b) Saundane, A. R.; Rudresh, K.; Satynarayan, N. D.; Hiremath, S. P. Ind. J. Pharm. Sci. 1998, 60, 379-383; c) Therese, K. L.; Bhagylaxmi, R.; Madhavan, H. N.; Deepa, P. Ind. J. Med. Microbiol. 2006, 24, 273-279. University Department of Chemical Technology, Dr. Babasaheb Ambedkar Marathwada University

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Chapter I Section B

Synthesis and Screening of Some Bioactive Heterocycles

Chapter I Section B

A novel amalgamation of 1,2,3 triazoles, piperidines and thieno pyridine rings and their antifungal screening INTRODUCTION The eukaryotic microorganisms Fungi, are vital opportunistic pathogens of humans, and are becoming drug-resistant to the approved compounds most prominent of them include Cryptococcus neoformans and species of Candida and Aspergillus, with Candida albicans, which could cause superficial or invasive infections or both in immune compromised individuals.1,2 In other words, they are becoming drug-resistant to the approved compounds and some therapies later in clinical development. The emergence of such drug resistant Fungi in the past decades has propelled the researchers around the globe to find new and efficacious drugs to solve these problems by designing, synthesizing and biological evaluation of the developed molecules.

3,4

Therefore, it is necessary to design new molecules which

are not yet developed and to determine their biological activity. The literature survey revealed, that till date the novel fusion of terahydrothieno pyridine, 1,2,3-triazole and piperidine has skipped through the vigilant eyes of the research groups around the globe. The lack of efforts of such fusion of the three moieties into a single molecule, containing all of these three important cores and to study its biological activity propelled us to carry out this work. The past decade has seen rise in development of sulfur containing heterocycles. The sulfur containing heterocycles have emerged as an important class of molecule from the industrial perspective due to their biodiversities. Tetrahydro thienopyridine is one of such sulfur containing heterocycles having varied biological activities like antiinflammatory, vasodilators and as a blood platelet aggregation inhibitory action.5 The other nucleus which is under study is piperidine and its analogues which are reported in literature for varied pharmacological activities like antibacterial,

6

AChE inhibitors, 7 antihistaminics

8

and antitubercular agents. 9 The

piperidine nucleus is also an important core of many drug molecules. The antifungal property possed by the triazole analogues makes it a hot topic of research, especially in the industries. The 1,2,3-triazole and its derivatives enjoy a central position especially in medicinal chemistry attracting an large number of researchers. The 1,2,3potent antineoplastic,10 antimicrobial,11

triazoles analogues are established as

University Department of Chemical Technology, Dr. Babasaheb Ambedkar Marathwada University

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Synthesis and Screening of Some Bioactive Heterocycles

Chapter I Section B

analgesic, 12 antiinflammatory,13 local anesthetic, 14 anticonvulsant, 15 antimalarial, 16 and antiHIVagents.17 More recently there are reports of 1,2,3-triazole derivatives being used as DNA cleaving agents18 and antitubercular agents. 19 In continuation of our work, 20 on the synthesis and biological evaluation of new heterocyclic molecules, we herein report the synthesis and antifungal

evaluation of some novel structural scaffolds having 1,2,3-triazoles,

piperidine and tetrahydrothieno pyridine. RESULT AND DISCUSSION The reaction sequence for different title compounds is as outlined in (Scheme 1 and Scheme 2). The starting material tert-butyl 4-azidopiperidine-1carboxylate (1), was prepared according to our reported procedure.20 Scheme 1. Synthesis of tert-butyl 4-(4-(((methylsulfonyl)oxy)methyl)-1H-1,2,3triazol-1-yl)piperidine-1-carboxylate.

N3

N boc

N N N Propargyl alcohol TEA CuI, DMF

N N

N boc

N

N boc

TEA,DCM

3 N N

OSO2CH3 N

N N

S

TEA

+ HN N boc 3 Mesyl compound

OSO2CH3

Methanesulphonyl chloride

2

1 N N

OH

S

Acetonitrile

4 4,5,6,7-tetrahydrothieno [3,2-c]pyridine

N boc 5

The compounds (2, 3) were prepared as per Chapter I Section A (Scheme 1). The tertbutyl 4-(4-((4,5-dihydrothieno[2,3-c]pyridin-6(7H)-yl)methyl)-1H-1,2,3-triazol-1-yl) piperidine-1-carboxylate (5) was prepared by nucleophilic substitution reaction using O-mesyl derivative (3) and 4,5,6,7-tetrahydrothieno[2,3-c]pyridine (THTP) in acetonitrile at 60 oC for 6 hr. with 85 % yield (Scheme 1). The compound (5) was then subjected to deprotection to give 5-((1-(piperidin-4-yl)-1H-1,2,3-triazol-4-yl)methyl)University Department of Chemical Technology, Dr. Babasaheb Ambedkar Marathwada University

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Synthesis and Screening of Some Bioactive Heterocycles

Chapter I Section B

4,5,6,7-tetrahydrothieno[3,2-c]pyridine (6a) by using trifluoro acetic acid (TFA) in dichloromethane with 87 % yield. Scheme 2. Synthesis of 5-bromo-N-((1-(piperidin-4-yl)-1H-1,2,3-triazol-5-yl) methyl) quinoxalin-6-amine derivatives.

The 5-bromo-N-((1-(piperidin-4-yl)-1H-1,2,3-triazol-4-yl)methyl) quinoxalin-6-amine quinoxalin-6-amine (6a) was subjected to substitution reaction in dichloromethane by using acyl chlorides/ alkyl chlorides to get the target derivatives (6b-k) in 82-91 % yield (Table 1, Scheme 2). Table 1. Synthesis of 5-bromo-N-((1-(piperidin-4-yl)-1H-1,2,3-triazol-5-yl) methyl)quinoxalin-6-amine derivatives (6a-k).a,b Sr. no.

Substituent (-R)

Time (min)

Yieldc ( %)

Melting point (°C)

6a

Hydrogen

60

87

185-187

6b

Methyl

120

89

178-180

6c

Ethyl

100

85

184-186

6d

Acetyl

80

87

156-158

6e

Propionyl

150

86

191-193

6f

Isobutyryl

100

89

179-181

6g

Benzoyl

100

90

164-166

6h

p- Chlorobenzoyl

150

84

170-172

6i

Methane sulphonyl

60

91

189-191

6j

p-methylbenzene sulphonyl

60

85

192-194

6k

Benzyl

180

82

174-176

a. Reaction condition (6a): Compound (5) (10 mmol), TFA (2 mmol) in (10 mL) of DCM at room temperature b. Reaction condition (6b-k): Compound (6a) (10 mmol), alkyl or acyl halides (10 mmol) in 10 mL of DCM, triethyl amine (20 mmol) and CuI (2 mmol) at 0-5°C. c. Isolated yields.

ANTIFUNGAL ACTIVITY All the synthesized compounds were screened for in vitro antifungal activity. The antifungal activity was evaluated against different fungal strains such as University Department of Chemical Technology, Dr. Babasaheb Ambedkar Marathwada University

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Synthesis and Screening of Some Bioactive Heterocycles

Chapter I Section B

Candida albicans (NCIM3471), Fusarium oxysporum (NCIM1332), Aspergillus flavus (NCIM539), Aspergillus niger (NCIM1196) and Cryptococcus neoformans (NCIM576). Minimum inhibitory concentration (MIC) values were determined using standard agar dilution method as per CLSI guidelines.21-24 Miconazole was used as a standard for the comparison of antifungal activity. Dimethyl sulfoxide was used as solvent control. MIC values of the tested compounds are presented in (Table 2) Table 2. Antifungal activity of the synthesized compounds (6a-k). MIC Values (μg/mL)a

Compound C. Albicans

F. Oxysporum

A. Flavus

A. Niger

C. Neoformans

6a

50

90

60

100

100

6b

40

70

40

*

*

6c

90

100

35

47.5

150

6d

40

45

*

100

*

6e

50

60

30

*

*

6f

100

100

35

47.5

150

6g

90

*

65

*

100

6h

45

40

40

42.5

45

6i

50

30

40

40

25

6j

60

70

45

37.5

40

6k

50

75

60

100

90

Miconazole

25

25

12.5

12.5

25

*- Activity not reported up to 200 μg/mL

a - Values are average of three readings.

EXPERIMENTAL The propargyl alcohol, copper iodide, triethyl amine, trifluoro acetic acid, substituted alkyl and acyl halides, tetrahydrothieno pyridine used were commercially available. Melting points were recorded on SRS Optimelt, melting point apparatus and are uncorrected. 1H NMR spectra were recorded on a 400 MHz Bruker spectrometer and 13C NMR spectra were recorded on a 100 MHz Bruker spectrometer are reported as parts per million (ppm) downfield from a tetramethylsilane internal standard. The following abbreviations are used; singlet (s), doublet (d), triplet (t), quartet (q), multiplate (m) and broad (br). Mass spectra were taken with MicromassQUATTRO-II of WATER mass spectrometer. Note: The compounds (2 and 3) were prepared as per Chapter I Section A. Synthesis of tert-butyl 4-(4-((4,5-dihydrothieno[2,3-c]pyridin-6(7H)-yl)methyl)-1H1,2,3-triazol-1-yl)piperidine-1-carboxylate (5) In a 50 mL RBF, compound (3) (10 mmol), triethyl amine (20 mmol) and THTP (4) University Department of Chemical Technology, Dr. Babasaheb Ambedkar Marathwada University

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Chapter I Section B

Synthesis and Screening of Some Bioactive Heterocycles

(10 mmol) was added in acetonitrile (10 mL) at room temperature. The resulting solution was then heated to 60°C for 6 hr. After completion (monitored by TLC, 30 % Ethyl acetate: n-hexane), solvent was removed in-vacuo and ethyl acetate (10 mL) was added to the residue and then extracted with 3 portions (3 x 10 mL) of distilled water. The combined organic layers were dried over anhydrous Na2SO4 and concentrated in-vacuo to get the desired compound (5), which was recrystallized using ethanol as solvent. Pale Yellow Solid, Yield: 85 %, Melting Point: 214-216°C ES-MS m/z (%): 404 (M+H) 1 H NMR (400 MHz, CDCl3):  ppm 1.67 (s, 9H), 2.22-2.35 (m, 4H), 2.68-2.80 (m, 4H), 3.05-3.20 (m, 4H), 3.54 (d, 2H), 3.84 (m, 1H), 4.14 (s, 2H), 6.82 (d, 1H), 7.42 (s, 1H), 7.65 (d, 1H). 13 C NMR (100 MHz, CDCl3):  ppm 22.5, 25.8, 27.1, 28.3, 29.0, 29.7, 42.3, 46.2, 57.6, 59.2, 60.4, 61.3, 81.5, 114.5, 120.9, 124.1, 131.2, 133.6, 134.1, 161.3. General procedure for synthesis of 5-((1-(piperidin-4-yl)-1H-1,2,3-triazol-4yl)methyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridine (6a) In a 50 mL RBF, compound (5) (10 mmol) and trifluoro acetic acid (2 mmol) was added in dichloromethane (10 mL). The solution was stirred for 2 hr. at room temperature. After completion (monitored by TLC, 40 % Ethyl acetate: n-hexane), the mixture was extracted with saturated NaHCO3 (3 x 10 mL). The organic layers was, dried over anhydrous Na2SO4 and evaporated to get the desired product (6a) (Table 1, entry 1), which was recrystallized using ethanol as solvent in 87 % yield. General procedure for synthesis of 5-((1-(piperidin-4-yl)-1H-1,2,3-triazol-4yl)methyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridine derivatives (6b-k) In a 50 mL RBF, compound (6) (10 mmol) and triethyl amine (20 mmol) was added in dichloromethane (10 mL) and CuI (2 mmol) was added and stirred at room temperature for 5 minutes and then cooled to 0-5°C. To this cooled solution, different substituted alkyl or acyl halides (10 mmol) were added. The resulting solution was stirred for 2-3 hr. After completion (monitored by TLC, 40 % Ethyl acetate: nhexane), was extracted with distilled water (3 x 10mL). The organic layer was dried over anhydrous Na2SO4 and on concentration in-vacuo gave the desired compounds (6b-6k) (Table 1, entry 2-11), which were recrystallized using ethanol as solvent in 82-91 % yield. Spectral characterization 5-((1-(piperidin-4-yl)-1H-1,2,3-triazol-4-yl)methyl)-4,5,6,7-tetrahydrothieno[3,2c]pyridine (6a): Pale Yellow Solid, ES-MS m/z (%): 304 (M+H). University Department of Chemical Technology, Dr. Babasaheb Ambedkar Marathwada University

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Chapter I Section B

Synthesis and Screening of Some Bioactive Heterocycles

1

H NMR (400 MHz, CDCl3):  ppm 1.97 (s, 1H), 2.28-2.42 (m, 4H), 2.72-2.85(m, 4H), 3.10-3.25 (m, 4H), 3.65 (s, 2H), 3.74 (m, 1H), 3.91 (s, 2H), 6.85 (d, 1H), 7.30 (d, 1H), 7.82 (s, 1H). 13 C NMR (100 MHz, CDCl3):  ppm 23.7, 27.1, 28.6, 42.4, 46.3, 54.7, 56.2, 60.1, 61.5, 122.9, 124.2, 125.5, 131.0, 133.9, 135.6. 5-((1-(1-methylpiperidin-4-yl)-1H-1,2,3-triazol-4-yl)methyl)-4,5,6,7-tetrahydro thieno[3,2-c]pyridine (6b): Pale Yellow Solid, ES-MS m/z (%): 318 (M+H). 1 H NMR (400 MHz, CDCl3):  ppm 2.15 (s, 3H), 2.25-2.40 (m, 4H), 2.64-2.78 (m, 4H), 3.10-3.24 (m, 4H), 3.58 (s, 2H), 3.71 (m, 1H), 3.86 (s, 2H), 6.83 (d, 1H), 7.24 (d, 1H), 7.76 (s, 1H). 13 C NMR (100 MHz, CDCl3):  ppm 24.1, 27.4, 28.3, 47.1, 54.2, 55.8, 57.5, 58.6, 59.8, 61.7, 122.5, 124.5, 125.7, 131.4, 134.3, 135.8. 5-((1-(1-ethylpiperidin-4-yl)-1H-1,2,3-triazol-4-yl)methyl)-4,5,6,7-tetrahydro thieno[3,2-c]pyridine (6c): Pale Yellow Solid, ES-MS m/z (%): 332 (M+H). 1 H NMR (400 MHz, CDCl3):  ppm 1.30 (s, 3H), 2.08-2.31 (m, 4H), 2.60-2.75 (m, 4H), 2.95 (q, 2H), 3.17-3.40 (m, 4H), 3.56 (s, 2H), 3.72 (m, 1H), 3.81 (s, 2H), 6.87 (d, 1H), 7.27 (d, 1H), 7.82 (s, 1H). 13 C NMR (100 MHz, CDCl3):  ppm 14.1, 23.7, 27.2, 28.0, 49.5, 53.7, 55.1, 56.2, 57.4, 58.6, 61.4, 122.7, 124.8, 126.2, 131.8, 134.5, 135.6. 1-(4-(4-((6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl)methyl)-1H-1,2,3-triazol-1yl)piperidin-1-yl)ethanone (6d): Pale Yellow Solid, ES-MS m/z (%): 346 (M+H). 1 H NMR (400 MHz, CDCl3):  ppm 2.05-2.26 (m, 4H), 2.40 (s, 3H), 3.25-3.40 (m, 4H), 3.45-3.60 (m, 4H), 3.65 (s, 2H), 3.75 (m, 1H), 3.86 (s, 2H), 6.80 (d, 1H), 7.32 (d, 1H), 7.74 (s, 1H). 13 C NMR (100 MHz, CDCl3):  ppm 22.8, 24.3, 27.5, 28.3, 42.1, 43.2, 55.4, 56.5, 57.4, 61.6, 122.9, 125.0, 126.5, 132.6, 134.1, 135.3, 175.4. 1-(4-(4-((6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl)methyl)-1H-1,2,3-triazol-1yl)piperidin-1-yl)propan-1-one (6e): Pale Yellow Solid, ES-MS m/z (%): 360 (M+H). 1 H NMR (400 MHz, CDCl3):  ppm 1.30 (s, 3H), 2.15-2.29 (m, 4H), 2.46 (q, 2H), 3.05-3.20 (m, 4H), 3.35-3.54 (m, 4H), 3.62 (s, 2H), 3.78 (m, 1H), 3.91 (s, 2H), 6.83 (d, 1H), 7.42 (d, 1H), 7.68 (s, 1H). 13 C NMR (100 MHz, CDCl3):  ppm 12.1, 24.0, 26.7, 27.6, 28.5, 42.6, 43.7, 55.1, 57.1, 58.2, 62.1, 123.1, 124.6, 125.7, 132.0, 133.5, 134.7, 175.9. 1-(4-(4-((6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl)methyl)-1H-1,2,3-triazol-1yl)piperidin-1-yl)-2-methylpropan-1-one (6f): Pale Yellow Solid, ES-MS m/z (%): 374 (M+H). 1 H NMR (400 MHz, CDCl3):  ppm 1.27 (s, 6H), 2.04-2.21 (m, 4H), 2.54 (m, 1H), 2.68-2.80 (m, 4H), 3.20-3.35 (m, 4H), 3.67 (s, 2H), 3.74 (s, 2H), 3.84 (m, 1H), 6.87 (d, 1H), 7.50 (s, 1H), 7.71 (d, 1H). 13 C NMR (100 MHz, CDCl3):  ppm 22.4, 24.3, 27.2, 27.9, 36.5, 43.1, 44.3, 55.2, 57.3, 58.5, 61.3, 123.3, 124.5, 125.8, 131.8, 133.7, 135.0, 172.5. University Department of Chemical Technology, Dr. Babasaheb Ambedkar Marathwada University

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Chapter I Section B

Synthesis and Screening of Some Bioactive Heterocycles

(4-(4-((6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl)methyl)-1H-1,2,3-triazol-1-yl) piperidin-1-yl)(phenyl)methanone (6g): Pale Yellow Solid, ES-MS m/z (%): 408 (M+H). 1 H NMR (400 MHz, CDCl3):  ppm 2.12 -2.28 (m, 4H), 2.72-2.85 (m, 4H), 3.24-3.40 (m, 4H), 3.60 (s, 2H), 3.72 (s, 2H), 3.87 (m, 1H), 6.81 (d, 1H), 7.42-7.58 (m, 5H), 7.68 (s, 1H), 7.78 (d, 1H). 13 C NMR (100 MHz, CDCl3):  ppm 24.3, 27.0, 28.1, 43.2, 44.0, 55.5, 57.3, 58.4, 61.0, 123.1, 124.2, 125.4, 126.2, 127.0, 128.4, 130.4, 132.5, 134.2, 135.6, 174.7. (4-chlorophenyl)(4-(4-((6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl)methyl)-1H-1,2,3triazol-1-yl)piperidin-1-yl)methanone (6h): Pale Yellow Solid, ES-MS m/z (%): 443 (M+H). 1 H NMR (400 MHz, CDCl3):  ppm 2.15-2.30 (m, 4H), 2.65-2.80 (m, 4H), 3.34-3.48 (m, 4H), 3.63 (s, 2H), 3.77 (s, 2H), 3.87 (m, 1H), 6.78 (d, 1H), 7.22-7.40 (m, 4H), 7.65 (s, 1H), 7.80 (d, 1H). 13 C NMR (100 MHz, CDCl3):  ppm 24.5, 27.4, 28.1, 43.0, 43.8, 55.2, 56.3, 57.1, 61.0, 123.2, 124.0, 125.1, 128.1, 129.4, 130.4, 131.2, 132.6, 134.3, 135.4, 171.2. 5-((1-(1-(methylsulfonyl)piperidin-4-yl)-1H-1,2,3-triazol-4-yl)methyl)-4,5,6,7tetrahydrothieno[3,2-c]pyridine (6i): Pale Yellow Solid, ES-MS m/z (%): 382 (M+H). 1 H NMR (400 MHz, CDCl3):  ppm 1.98 (s, 3H), 2.14-2.32 (m, 4H), 2.65-2.77 (m, 4H), 3.05-3.20 (m, 4H), 3.50 (s, 2H), 3.75 (m, 1H), 4.05 (s, 2H), 6.90 (d, 1H), 7.46 (s, 1H), 7.72 (d, 1H). 13 C NMR (100 MHz, CDCl3):  ppm 23.1, 26.0, 27.3, 40.3, 45.7, 46.2, 56.4, 57.2, 58.0, 61.5, 121.7, 123.6, 126.4, 131.2, 133.6, 134.1. 5-((1-(1-tosylpiperidin-4-yl)-1H-1,2,3-triazol-4-yl)methyl)-4,5,6,7-tetrahydrothieno [3,2-c]pyridine (6j): Pale Yellow Solid, ES-MS m/z (%): 458 (M+H). 1 H NMR (400 MHz, CDCl3):  ppm 1.97 (s, 3H), 2.12 -2.30 (m, 4H), 2.55-2.70 (m, 4H), 3.05-3.20 (m, 4H), 3.47 (s, 2H), 3.71 (m, 1H), 4.12 (s, 2H), 6.87 (d, 1H), 7.34 (s, 1H), 7.49-7.60 (m, 4H), 7.65 (d, 1H). 13 C NMR (100 MHz, CDCl3):  ppm 21.7, 23.4, 25.6, 26.3, 43.9, 44.7, 56.1, 57.4, 58.3, 60.7, 122.9, 123.8, 126.1, 127.0, 128.6, 129.8, 131.2, 133.6, 134.5, 138.2, 145.2. 5-((1-(1-benzylpiperidin-4-yl)-1H-1,2,3-triazol-4-yl)methyl)-4,5,6,7-tetrahydro thieno[3,2-c]pyridine (6k): Pale Yellow Solid, ES-MS m/z (%): 394 (M+H). 1 H NMR (400 MHz, CDCl3):  ppm 2.08-2.21 (m, 4H), 2.51-2.65 (m, 4H), 3.05-3.22 (m, 4H), 3.44 (s, 2H), 3.61 (s, 2H), 3.75 (m, 1H), 4.12 (s, 2H), 6.81 (d, 1H), 7.37 (s, 1H), 7.50-7.65 (m, 5H), 7.71 (d, 1H). 13 C NMR (100 MHz, CDCl3):  ppm 22.7, 27.3, 28.5, 51.2, 52.1, 54.3, 57.6, 58.7, 60.3, 64.8, 122.7, 124.1, 125.0, 126.7, 127.6, 128.5, 129.4, 130.5, 131.2, 133.4, 135.1, 138.2.

University Department of Chemical Technology, Dr. Babasaheb Ambedkar Marathwada University

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Synthesis and Screening of Some Bioactive Heterocycles

Chapter I Section B 1

H NMR spectrum of compound (6c):

N

13

N N N

S N

C NMR spectrum of compound (6c):

N

N N N

S N

University Department of Chemical Technology, Dr. Babasaheb Ambedkar Marathwada University

42

Synthesis and Screening of Some Bioactive Heterocycles

Chapter I Section B

Mass spectrum of compound (6c):

N

N N N

S N

University Department of Chemical Technology, Dr. Babasaheb Ambedkar Marathwada University

43

Chapter I Section B

Synthesis and Screening of Some Bioactive Heterocycles

REFERENCES 1. Kontoyiannis, D. P. Lancet. 2002, 359, 1135-1144. 2. Odds, F. C.; Brown, A.; Gow, N. Trends in Microbiology 2003, 11, 272-279. 3. Hawksworth, D. L. Mycol. Res. 2001, 105, 1422-1432. 4. Morschhauser, J. Fungal Genet. Biol. 2010, 47, 94-106. 5. Rene, A.; Castaigne, R. 1977, U S Patent no. 4,051,141. 6. Sheng, R.; Xu; Y.; He, Q.; Hu, Y. Eur. J. Med. Chem. 2009, 44 , 7-17. 7. Ravula, S. B.; Yu, J.; Beaton, G. Bioorg. Med. Chem. Lett. 2012, 22, 421-426. 8. Aridoss, G.; Jeong, Y. T. Bioorg. Med. Chem. Lett. 2008, 18, 6542-6548. 9. Bogatcheva, E.; Propova, M.; Bioorg. Med. Chem. Lett. 2011, 21, 5353-5357. 10. Matsuoka, M.; Kitao, T. J. Heterocyclic Chem. 1992, 29, 439-443. 11. Kim, Y. B.; Kim, S. K. Bioorg. Med. Chem. Lett. 2004, 14, 541-544. 12. Gavara, L.; Moreau P. Eur. J. Med. Chem. 2010, 45, 5520-5526. 13. Passannanti, A.; Cirrincione, G. Heterocycles 1998, 48, 1229-1235. 14. Chen, M. D.; Yang, S. Y.; Du, X. L. Heterocyclic Comm. 2000, 6, 421-426. 15. Sherement, E. A.; Berestovitsk, V. Russ. J. Org. Chem. 2004, 40, 594-595. 16. Holla, B. S.; Kumari, N. S. Eur. J. Med. Chem. 2005, 40, 1173-1178. 17. Cunha, A. C.; Barreiroa , E. J. Bioorg. Med. Chem. Lett. 2003, 11, 2051-2059. 18. Banu, K. M.; Ananthnarayanan, C. Ind. J. Pharm. Sci. 1999, 4, 202-205. 19. Velarquez, S.; Camarasa, M. J. Antivir. Chem. Chemother. 1998, 9, 481-489. 20. a) Sangsheti, J. N.; Shinde, D. B. Bioorg. Med. Chem. Lett. 2011, 21, 444-448; b) Sangsheti, J. N.; Shinde, D. B. Eur. J. Med. Chem. 2011, 46, 1040-1044. 21. Cruickshank, R. ; Duguid, J. P.; Marmion, B. P. ; Swain, R. H. Eds., 2th Ed. Medicinal Microbiology; Churchill Livingstone: London, 1975, 2. 22. Collins, A. H. Microbiological Methods, 2 nd Ed.; Butterworth: London, 1976. 23. Khan, Z. K. In vitro and vivo screening techniques for bioactivity screening and evaluation, Proc. Int. Workshop UNIDO-CDRI, 1997, 210-211. 24. a) Duraiswamy, B.; Mishra, S. K.; Subhashini, V.; Dhanraj, S. A.; Suresh, B. Ind. J. Pharm. Sci. 2006, 68, 389-391; b) Saundane, A. R.; Rudresh, K.; Satynarayan, N. D.; Hiremath, S. P. Ind. J. Pharm. Sci. 1998, 60, 379-383; c) Therese, K. L.; Madhavan, H. N.; Deepa, P. Ind. J. Med. Microbiol. 2006, 24, 273-279. University Department of Chemical Technology, Dr. Babasaheb Ambedkar Marathwada University

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Chapter I Section C

Synthesis and Screening of Some Bioactive Heterocycles

Chapter I Section C

Ultrasound mediated, sodium bisulfite catalyzed, solvent free synthesis of 6-amino-3-methyl-4-substitued-2,4-dihydropyrano[2,3-c] pyrazole-5-carbonitrile INTRODUCTION The multicomponent reactions is an emerging trend in the synthetic transformations due to its operational simplicity, occurring with less or minimum side products giving higher yields of the desired products. The advantage of the multi component reactions over the multi step reaction is simple experimental procedure, occurring in a single step giving higher yields from easily available starting materials without isolation of any intermediate, thus in turn, saving time, energy and raw materials required for the reaction making the protocol economically attractive and environment friendly.1 In past decade, several methods have been developed using ultrasound irradiation as the unconventional source of energy, accelerating the synthetic reactions. Ultrasound irradiation being advantageous over the conventional thermal reactions, reducing the time and increases the yield of the product generally by minimizing the side products associated with the prolonged heating and provide an convenient practical ecofriendly protocol for the reaction.1 In recent years, pyrano[2,3-c]pyrazole molecule is an emerging class of heterocycles and is widely explored as it is an important core of the emerging drugs along with wide medicinal applications as, potential inhibitors of human CHK1 kinase,2 antiinflammatory,3 anticancer,3 analgesic,3 molluscicidal activity4 and as antimicrobial.5 The synthesis of pyrano[2,3-c]pyrazoles can be accomplished in several

ways,

like

by the

use

of

3-methyl-1-phenylpyrazolin-5-one

and

tetracyanoethylene in the presence of triethylamine.6 Literature reveals methods employing, piperazine,7 piperidine,7 β- cyclodextrin,8 γ-alumina8 and L-proline8 as catalysts for the synthesis of 6-amino-4-alkyl/aryl-3-methyl-2,4-dihydropyrano[2,3c]pyrazole-5-carbonitriles. Even though, the reported methods are effective, they have their own limitations in terms of toxicity arising by the use of piperazine and piperidine.6 Secondly, these methods employ expensive catalyst for the reaction8 requiring solvent for the reaction,7,8 making the work up procedure tedious. The appealing biological application of the substituted pyrano[2,3c]pyrazole molecule drives us to develop new methodology by minimizing or University Department of Chemical Technology, Dr. Babasaheb Ambedkar Marathwada University

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Chapter I Section C

Synthesis and Screening of Some Bioactive Heterocycles

overcoming the drawbacks of the literature methods. In continuation of our research on development of new methodologies for the synthesis of biologically important heterocycles keeping the eco- friendly approach in mind,9 herein, we wish to report the ultrasound assisted synthesis of substituted pyrano[2,3-c]pyrazole, by simple, convenient and practical green synthesis protocol using sodium bisulfite as a catalyst. The key features of this methodology are operational simplicity, shorter time and excellent yields making the protocol environment friendly and economically lucrative. RESULTS AND DISCUSSION Our continued interests for the development of efficient and environmentally friendly procedures for the synthesis of heterocyclic compounds,

9

led us to explore the synthesis of substituted pyrano[2,3-c]pyrazole, in a one-pot reaction in the presence of sodium bisulfite, which is easily available at low cost. To screen the catalyst and to explore the effect of catalyst on reaction conditions, initially we examined catalyst free reaction condition. The product was formed but it took longer reaction time and the yield was also not significant (Table 1, entry 1) when compared to the literature method (83 % yield in 10 min).7 Encouraged by our initial observation, we explored the catalytic activity of various catalysts by using ultrasound as mentioned in (Table 1, entry 2-14). Scheme 1. Synthesis of substituted pyrano [2,3-c]pyrazole catalyzed by sodium bisulfite under ultrasonic irradiation

By evaluating the results obtained in (Table 1) piperidine, DMAP, pyrrolidine, L-proline and sodium bisulfite showed promising results in terms of yields. The testing of the catalyst on this parameter of time revealed that DMAP, Lproline and sodium bisulfite were the best in presence and absence of solvent (Table 1, entry 7, 8, 11, 12, 13, 14). In comparison with DMAP and L- proline the sodium bisulfite was found to be far superior for this reaction both in terms of the time and yield. To study the optimum catalytic loading for the reaction, sodium bisulfite was used in combination of various solvents like water, acetonitrile, ethanol-water (50 %), University Department of Chemical Technology, Dr. Babasaheb Ambedkar Marathwada University

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Synthesis and Screening of Some Bioactive Heterocycles

Chapter I Section C

ethanol and in solvent free conditions. Table 1. Optimization of catalyst for the synthesis of 6-amino-2,4-dihydro-3methyl-4-phenylpyrano[2,3-c]pyrazole-5-carbonitrile (5a).a Catalyst (Mol %)

Solvent

Time (min)

Yieldb (%)

No Catalyst

0

Ethanol

60

75

Et3N

50

Ethanol

5

80

3

Et2NH

50

Ethanol

5

82

4

K2CO3

50

Ethanol

1

80

5

Pyridine

50

Ethanol

3

78

6

Piperidine

50

Ethanol

2

88

7

DMAP

50

Ethanol

1

90

8

DMAP

50

Solvent Free

45 sec

92

9

Pyrrolidine

50

Ethanol

2

82

10

Morpholine

50

Ethanol

5

75

11

L-Proline

50

Ethanol

1

92

12

L-Proline

50

Solvent Free

45 sec

94

13

Sodium bisulfite

50

Ethanol

1

97

Sr. No.

Catalyst

1 2

14 a.

b.

Sodium bisulfite 50 Solvent Free 30 sec 99 Reaction condition: Ethyl acetoacetate (10 mmol), hydrazine hydrate (10 mmol), malononitrile (10 mmol), benzaldehyde (10 mmol), and various catalysts under ultrasonic waves in solvent-free condition at 30°C. Isolated yields.

Surprisingly, it was observed that reaction proceeded smoothly in absence of solvent, which might be due to the ultrasonic cavitations creating microscopic internal high pressure and high temperature.10 Table 2. Optimization of catalyst loading for the synthesis of 6-amino-2,4dihydro-3-methyl-4-phenylpyrano[2,3-c]pyrazole-5-carbonitrile (5a).a Sr. no.

Catalyst (Mol %)

Solvent

Time (min)

Yieldb (%)

1

50

Water

3

88

2

50

Acetonitrile

5

75

3

50

Ethanol-Water (50 %)

2

90

4

50

Ethanol

1

92

5

50

Solvent Free

30 sec

99

6

40

Solvent Free

30 sec

99

7

30

Solvent Free

30 sec

99

8

20

Solvent Free

30 sec

99

9

10

Solvent Free

30 sec

99

10 5 Solvent Free 45 sec 84 a. Reaction condition: Ethyl acetoacetate (10 mmol), hydrazine hydrate (10 mmol), malononitrile (10 mmol), benzaldehyde (10 mmol), and various concentrations of sodium bisulfite as catalyst under ultrasonic waves in solvent-free condition at 30°C. b. Isolated yields. University Department of Chemical Technology, Dr. Babasaheb Ambedkar Marathwada University

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Synthesis and Screening of Some Bioactive Heterocycles

The results from (Table 2) indicate that the reaction can proceed to completion without the use of solvent in shorter reaction time and better yield compared to the solvents enlisted above. The 10 mol% of sodium bisulfite was found to be suitable for the completion of the reaction in shorter reaction time and better yields, lower concentration of sodium bisulfite results in increase in reaction time and decrease in yield. Also, higher concentration of sodium bisulfite does not appreciably improve the yield and time of reaction. After optimizing the reaction conditions, the generality of this method was evaluated by using substituted aromatic aldehydes containing electron withdrawing and electron donating group and heterocyclic aldehydes to give the corresponding products as shown in (Table 3). Table 3. Synthesis of substituted pyrano[2,3-c]pyrazole (5a-l) catalyzed by sodium bisulfite under ultrasonic irradiation.a Sr. no.

Aldehyde

5a

Benzaldehyde

Time (sec) 30

Yieldb (%)

Melting point (°C) Observed

Reference

244-246

244-2468

99

8

99

5b

4-metylbenzaldehyde

30

205-207

206-208

5c

4-methoxybenzaldehyde

30

210-212

210-2128

99

222-224

224-226

8

99

8

97

5d

4-hydroxybenzaldehyde

30

5e

4-nitrobenzaldehyde

40

250-252

251-253

5f

3-nitrobenzaldehyde

30

193-195

193-1958

98

232-234

234-236

8

98

8

98

5g

4-chlorobenzaldehyde

30

5h

2-chlorobenzaldehyde

30

144-146

145-147

5i

4-bromobenzaldehyde

30

178-180

178-1808

99

172-174

175-177

8

98

190-191

8

98

5j

Furan-2-carbaldehyde

5k 5l a.

b.

Thiophene-2-carbaldehyde

35 40

187-189

Isonicotinaldehyde 40 215-217 218-2198 97 Reaction condition: Ethyl acetoacetate (10 mmol), hydrazine hydrate (10 mmol), malononitrile (10 mmol), substituted aldehydes (10 mmol), and sodium bisulfite (10 mol%) under ultrasonic waves in solvent-free condition at 30°C. Isolated yields.

The advantage of this method over the reported methods,7,8 is easily available and inexpensive catalyst and operational simplicity. Also, the time required for the completion of reaction is less (30 sec) as compared to that of the literature method8 which requires more than 50-70 min for its completion and the yields ranging from 80-90%. Herein, we propose a possible mechanism for the synthesis of pyrano[2,3-c]pyrazole (Scheme 2), in a one-pot reaction in the presence of sodium bisulfite. University Department of Chemical Technology, Dr. Babasaheb Ambedkar Marathwada University

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Synthesis and Screening of Some Bioactive Heterocycles

Chapter I Section C

Scheme 2. Plausible reaction mechanism for the synthesis of substituted pyrano[2,3-c]pyrazole catalyzed by sodium bisulfite under ultrasonic irradiation. O

O O

NaHSO3 )))) -C2H5OH

O NH2NH H Na HSO 3

O

NC

O H

R

-H2O NH NH2 Cyclization

N H Na HSO

CN H OH

+ NaHSO3 R )))) N

O N N H Pyrazolone NC

-H2O

Knoevenagel Condensation

CN H

R

Ylidenemalononitrile

3

H N N H

Keto-Enol

O

Tautomerization

N N H

R

HH O + NC

CN

Michael Addition Reaction

N

H

Tautomerization

CN H

N

HN N

O

R

R

R

NH2

N

O

NH

Cyclization

CN N

N H

OC N

Firstly, there is knoevenagel condensation between the aldehyde and the malononitrile leading to the formation of the ylidenemalononitrile by loss of a water molecule. Simultaneously the reaction occurring between the hydrazine hydrate and ethyl acetoacetate yields pyrazolone with the elimination of ethanol and water molecule. This ylidenemalononitrile and pyrazolone undergoes the michael addition reaction followed by the ring closure and consequent tautomerization to give the pyrano [2,3-c]pyrazole as the product. In conclusion, we have developed a highly efficient environment friendly, economically lucrative, convenient and practical method for ultrasound assisted one-pot synthesis of substituted pyrano[2,3-c]pyrazole catalyzed by sodium bisulfite. The developed method is simple, robust and practical, which proceeds without any special handling technique and requiring routine reagents. Thus, this one-pot green chemistry protocol is advantageous requiring lesser University Department of Chemical Technology, Dr. Babasaheb Ambedkar Marathwada University

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Chapter I Section C

Synthesis and Screening of Some Bioactive Heterocycles

reaction time, operational simplicity with ease of execution to give high yields. EXPERIMENTAL The ethyl acetoacetate, hydrazine hydrate, malononitrile, substituted aldehydes and sodium bisulfite used were commercially available. Melting points were recorded on SRS Optimelt melting point apparatus and are uncorrected. Ultrasonication was performed in an Ultrasonic Bath Sonicator of PCI Analytics,® having ultrasound cleaner with a frequency of 35 kHz and a nominal power of 200 W. The reaction flask was located in close proximity of the maximum energy area in the cleaner such that the reaction vessel was slightly lower than the water level and the temperature of the water bath was controlled at 30 °C. 1H NMR spectra were recorded on a 400 MHz Varian-Gemini spectrometer and 13C NMR spectra were recorded on a 100 MHz Bruker spectrometer are reported as parts per million (ppm) downfield from a tetramethylsilane internal standard. The following abbreviations are used, singlet (s), doublet (d), triplet (t), quartet (q), multiplate (m) and broad (br). Mass spectra were taken with Micromass-QUATTRO-II of WATER mass spectrometer. General Procedure for the synthesis of substituted pyrano[2,3-c]pyrazole (5a-l): In a 50 mL RBF, ethyl acetoacetate (10 mmol), hydrazine hydrate (10 mmol), malononitrile (10 mmol), substituted aldehydes (10 mmol), and sodium bisulfite (10 mol %) were added. The mixture was irradiated using ultrasound radiation for 30-40 sec at 30°C. After completion (monitored by TLC, 20 % Ethyl acetate: n-hexane), the solid obtained was filtered and washed with water (2 x 5 ml). The crude product was recrystallized from ethanol. The pure products were collected in 97–99 % yields (Table 3). Spectral characterization 6-amino-2,4-dihydro-3-methyl-4-phenylpyrano[2,3-c]pyrazole-5-carbonitrile (5a): Yellow Solid, ES-MS m/z (%): 253 (M+H). 1 H NMR (400 MHz, CDCl3): δ ppm 1.87 (s, 3H), 4.90 (s, 1H), 6.75 (s br, 2H), 7.107.65 (m, 5H), 12.35(s, 1H). 13 C NMR (100 MHz, CDCl3): δ ppm 15.2, 26.4, 60.4, 115.9, 119.4, 128.8, 130.1, 131.1, 132.4, 133.0, 137.8, 141.5, 166.2, 179.3. 6-amino-3-methyl-4-(p-tolyl)-2,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile (5b): Yellow Solid, ES-MS m/z (%): 267 (M+H). 1 H NMR (400 MHz, CDCl3): δ ppm 1.97 (s, 3H), 2.54 (s, 3H), 4.95 (s, 1H), 6.75 (s br, 2H), 7.32-7.55 (m, 4H), 12.23(s, 1H). University Department of Chemical Technology, Dr. Babasaheb Ambedkar Marathwada University

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Chapter I Section C

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13

C NMR (100 MHz, CDCl3): δ ppm 15.7, 21.7, 26.1, 60.7, 114.3, 119.9, 127.1, 128.2, 132.7, 133.9, 134.6, 139.8, 143.0, 167.5, 178.1. 6-amino-4-(4-methoxyphenyl)-3-methyl-2,4-dihydropyrano[2,3-c]pyrazole-5carbonitrile (5c): Yellow Solid, ES-MS m/z (%): 283 (M+H). 1 H NMR (400 MHz, CDCl3): δ ppm 1.92 (s, 3H), 3.90 (s, 3H), 4.87 (s, 1H), 6.90 (s br, 2H), 7.12-7.35 (m, 4H), 12.20(s, 1H). 13 C NMR (100 MHz, CDCl3): δ ppm 15.7, 26.2, 58.7, 61.2, 112.3, 114.1, 117.3, 121.7, 128.1, 131.7, 132.1, 143.0, 161.5, 168.7, 180.8. 6-amino-4-(4-hydroxyphenyl)-3-methyl-2,4-dihydropyrano[2,3-c]pyrazole-5carbonitrile (5d): Yellow Solid, ES-MS m/z (%): 269 (M+H). 1

H NMR (400 MHz, CDCl3): δ ppm 1.94 (s, 3H), 4.89 (s, 1H), 5.72 (s, 1H), 6.95 (s br, 2H), 7.22-7.48 (m, 4H), 12.45(s, 1H). 13 C NMR (100 MHz, CDCl3): δ ppm 15.9, 27.1, 61.3, 113.6, 117.0, 118.7, 121.4, 130.1, 133.4, 136.8, 144.5, 156.1, 168.5, 179.8. 6-amino-3-methyl-4-(4-nitrophenyl)-2,4-dihydropyrano[2,3-c]pyrazole-5carbonitrile (5e): Yellow Solid, ES-MS m/z (%): 298 (M+H). 1 H NMR (400 MHz, CDCl3): δ ppm 1.90 (s, 3H), 4.94 (s, 1H), 6.91 (s br, 2H), 7.527.74 (d, 2H), 8.05-8.14 (d, 2H), 12.31(s, 1H). 13 C NMR (100 MHz, CDCl3): δ ppm 15.7, 27.5, 61.7, 117.0, 123.1, 126.7, 127.9, 133.4, 136.1, 143.0, 146.1, 149.7, 169.0, 180.1 6-amino-3-methyl-4-(3-nitrophenyl)-2,4-dihydropyrano[2,3-c]pyrazole-5carbonitrile (5f): Yellow Solid, ES-MS m/z (%): 298 (M+H). 1 H NMR (400 MHz, CDCl3): δ ppm 1.94 (s, 3H), 4.91 (s, 1H), 6.85 (s br, 2H), 7.487.60 (d, 2H), 8.09-8.21 (d, 2H), 12.38(s, 1H). 13

C NMR (100 MHz, CDCl3): δ ppm 15.6, 25.2, 61.7, 117.1, 121.1, 123.5, 124.1, 135.7, 137.8, 139.1, 143.2, 150.1, 168.4, 180.4. 6-amino-4-(4-chlorophenyl)-3-methyl-2,4-dihydropyrano[2,3-c]pyrazole-5carbonitrile (5g): Yellow Solid, ES-MS m/z (%): 288 (M+H). 1 H NMR (400 MHz, CDCl3): δ ppm 1.90 (s, 3H), 4.96 (s, 1H), 6.91 (s br, 2H), 7.187.30 (m, 4H), 12.41 (s, 1H). 13 C NMR (100 MHz, CDCl3): δ ppm 15.5, 25.9, 59.1, 117.5, 121.0, 128.4, 129.7, 131.2, 133.0, 134.2, 135.7, 144.6, 168.7, 179.0. 6-amino-4-(3-chlorophenyl)-3-methyl-2,4-dihydropyrano[2,3-c]pyrazole-5carbonitrile (5h): Yellow Solid, ES-MS m/z (%): 288 (M+H). 1 H NMR (400 MHz, CDCl3): δ ppm 1.94 (s, 3H), 4.91 (s, 1H), 6.98 (s br, 2H), 7.327.55 (m, 4H), 12.46 (s, 1H). University Department of Chemical Technology, Dr. Babasaheb Ambedkar Marathwada University

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Chapter I Section C

Synthesis and Screening of Some Bioactive Heterocycles

13

C NMR (100 MHz, CDCl3): δ ppm 15.4, 26.8, 62.1, 117.4, 121.1, 128.4, 129.5, 130.9, 132.7, 136.4, 138.1, 144.9, 167.7, 181.4. 6-amino-4-(4-bromophenyl)-3-methyl-2,4-dihydropyrano[2,3-c]pyrazole-5carbonitrile (5i): Yellow Solid, ES-MS m/z (%): 332 (M+H). 1 H NMR (400 MHz, CDCl3): δ ppm 1.91 (s, 3H), 4.94 (s, 1H), 6.92 (s br, 2H), 7.327.55 (d, 2H), 7.70-7.85 (d, 2H), 12.32 (s, 1H). 13 C NMR (100 MHz, CDCl3): δ ppm 15.4, 24.8, 59.8, 113.4, 119.7, 122.5, 130.4, 131.9, 133.7, 135.9, 137.5, 144.1, 168.2, 180.3. 6-amino-4-(furan-2-yl)-3-methyl-2,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile (5j): Yellow Solid, ES-MS m/z (%): 243 (M+H). 1

H NMR (400 MHz, CDCl3): δ ppm 1.90 (s, 3H), 4.97 (s, 1H), 6.27 (d, 1H), 6.67 (t, 1H), 6.86 (s br, 2H), 7.30 (d, 1H), 12.30 (s, 1H). 13 C NMR (100 MHz, CDCl3): δ ppm 15.7, 29.4, 63.1, 109.8, 113.5, 117.2, 121.2, 143.9, 147.6, 155.6, 168.0, 178.9. 6-amino-3-methyl-4-(thiophen-2-yl)-2,4-dihydropyrano[2,3-c]pyrazole-5carbonitrile(5k): Yellow Solid, ES-MS m/z (%): 259 (M+H). 1 H NMR (400 MHz, CDCl3): δ ppm 1.95 (s, 3H), 4.88 (s, 1H), 6.70 (d, 1H), 6.82 (t, 1H), 6.97 (s br, 2H), 7.55 (d, 1H), 12.24 (s, 1H). 13 C NMR (100 MHz, CDCl3): δ ppm 15.5, 29.1, 64.4, 117.0, 122.4, 124.2, 127.4, 130.7, 143.7, 145.1, 168.7, 178.1. 6-amino-3-methyl-4-(pyridin-4-yl)-2,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile (5l): Yellow Solid, ES-MS m/z (%): 254 (M+H). 1 H NMR (400 MHz, CDCl3): δ ppm 1.93 (s, 3H), 4.87 (s, 1H), 6.92 (s br, 2H), 7.287.40 (d, 2H), 8.15-8.32 (d, 2H), 12.42 (s, 1H). 13

C NMR (100 MHz, CDCl3): δ ppm 15.7, 28.9, 63.1, 117.5, 121.8, 124.0, 127.4, 142.9, 148.2, 152.1, 154.1, 167.8, 178.5.

University Department of Chemical Technology, Dr. Babasaheb Ambedkar Marathwada University

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Synthesis and Screening of Some Bioactive Heterocycles

Chapter I Section C 1

H NMR spectrum of compound (5a):

N HN N

13

O

NH2

C NMR spectrum of compound (5a):

N HN N

O

NH2

University Department of Chemical Technology, Dr. Babasaheb Ambedkar Marathwada University

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Synthesis and Screening of Some Bioactive Heterocycles

Chapter I Section C

Mass spectrum of compound (5a):

N HN N

O

NH2

University Department of Chemical Technology, Dr. Babasaheb Ambedkar Marathwada University

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Chapter I Section C

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REFERENCES 1. a) Ganem, B. Acc. Chem. Res. 2009, 42, 463-472; b) Sunderhaus, J. D., Martin, S. F. Chem. Eur. J. 2009, 15, 1300-1308; c) Satyanarayana, V. S., Sivakumar A. 2011, 18, 917-922; d) Mason, T. J. Chem. Soc. Rev. 1997, 26, 443-451. 2. Foloppe, N., Fisher, L. M., Howes, R., Potter, A., Robertson, A. G., Surgenor, A. E. Bioorg. Med. Chem. 2006, 14, 4792-4802. 3. a) Kuo, S. C., Huang, L. J., Nakamura, H. J. Med. Chem. 1984, 27, 539-544; b) Wang, J. L., Liu, D., Zheng, Z. J., Shan, S., Han, X., Srinivasula, S. M., Croce, C. M, Alnemri, E. S., Huang, Z., Proc. Natl. Acad. Sci. 2000, 97, 71247129. 4. Abdelrazek, F. M., Metz, P., Kataeva, O., Jager, A., Elmahrouky, S. F. Arch. Pharm. 2007, 340, 543-548. 5. Eltamany, E. S., Elshahed, F. A., Mohamed, B. H. J. Serb. Chem. Soc. 1999, 64, 9-18. 6. Almatar, H. M., Khalil, K. D., Adam, A. Y., Elnagdi, M. H. Molecules 2010, 15, 6619-6629. 7. a) Peng, Y., Song, G., Dou R. Green Chem. 2006, 8, 573-575; b) Vasuki, G., Kumaravel, K. Tet. Lett. 2008, 49, 5636-5638. 8. a) Kanagaraj, K., Pitchumani, K. Tet. Lett. 2010, 51, 3312-3316; b) Mecadon, H., Rohman, M. R., Kharbangar, I., Laloo, B. M., Kharkongor, I., Rajbangshi, M., Myrboh, B. Tet. Lett. 2011, 52, 3228-3231; c) Mecadon, H., Rohman, M. R., Rajbangshi, M., Myrboh, B. Tet. Lett. 2011, 52, 2523-2525. 9. a) Sangshetti, J. N., Kokare, N. D., Kotharkar, S. A., Shinde, D. B. Monatsh. Chem. 2008, 139, 125-127; b) Sangshetti, J. N., Chabukswar, A. R., Shinde, D. B. Bioorg. Med. Chem. Lett. 2011, 21, 444-448. 10. Wang, S. X., Li, Z. Y., Zhang, J. C., Li, J. T. Ultra. Sonochem. 2008, 15, 677680.

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