Chapter 2. Synthesis and Biological Evaluation of Some Pyrazole Derivatives

Synthesis and Biological Evaluation of Some Pyrazole Derivatives

CAPTER 2. Synthesis and Biological Evaluation of Some Pyrazole Derivatives 2.1. Synthesis of 4-functionalized pyrazoles 2.1.1. Motivation for the current work Incorporation of heterocyclic rings in general and pyrazoles, pyrazolines, thiazoles or coumarins in particular into prospective pharmaceutical candidates is an established strategy to improve activity and safety of active molecules. Amongst five membered nitrogen heterocycles, pyrazoles have attained a special status in the eyes of chemists and biologists owing to their easy methods of syntheses and enormous pharmacological applications. Although scarcely found in nature, this class represents a key motif in heterocyclic chemistry and enjoys a unique place in medicinal and pesticide chemistry due to its capability to exhibit a wide range of bioactivities such as antimicrobial, 1-5 anticancer, 6-8 anti-inflammatory, 9-12 13, 14 antidepressant, anticonvulsant, 14, 15 antihyperglycemic, 16 antipyretic, 17 antiviral, 18 selective enzyme inhibitory activities, 19 etc. Many of the pyrazole derivatives are potent broad spectrum 20-23 insecticides, fungicides and herbicides. In recent years, resistance developed by bacteria and microbial pathogens to currently available antimicrobial drugs has become a constant public health problem of potentially crisis proportions throughout the world. Uncontrolled widespread use of these drugs has made these pathogens increasingly resistant that has resulted in an increase in morbidity and mortality. Thus, the development of novel molecules with excellent broad spectrum antimicrobial profiles is still in demand. The discovery of the natural pyrazole C-glycoside pyrazofurin (1), 24 a broad spectrum antibiotic with pronounced antimicrobial and antiviral activities, led to the synthesis of several pyrazole derivatives with 25, 26 excellent antimicrobial profiles. Amongst a large array of medicinally important pyrazole derivatives, 4-functionalized pyrazoles occupy a unique position and their evaluation as antimicrobial agents has attracted much attention in the recent 27-30 past. 1 2 39

Pyrazole derivatives: Synthesis and biological evaluation Pyrazoles with various functional groups at position-4 such as cyano or oxime (2), 27 aldehyde or carboxylate (3), 28 etc., have been known to show good antimicrobial properties. It has been undoubtedly reported that association of benzenesulfonamide moiety with various heterocycles, has shown diverse range of bioactivities such as antimicrobial, 31,32 anti-inflammatory, 33 anticancer, 34,35 anti-iv, 36 etc. Motivated by these findings, we set out to synthesize some novel 1,3-diaryl-4- functionalizedpyrazoles bearing benzenesulfonamide moiety at position-1 and an aldehyde 4, carboxylic acid 5, cyano 6 or carbothioamide 7 moiety at position-4 for their evaluation as antibacterial and antifungal agents. 2 2 2 S 2 4 R = C aryl alkyl/carboxylate aryl 5 R = C 6 R = C 7 R = CS 2 C/C CC 2 5 /C R 2 3 Thus, the presence of an additional sulfonamide group at position-4 of the phenyl ring being the only difference between 2 (C) and 6, a direct comparison of the antimicrobial potential of 2 and 6 would be possible providing an opportunity to evaluate the potential of a sulfonamide group for antimicrobial activity. ere, we are reporting the synthesis and antimicrobial evaluation of carboxylic acids 5 and carbothioamides 7. 2.1.2. Synthetic discussion for 1,3-diaryl-4-functionalized pyrazoles (4-7) The synthetic route used to synthesize the 4-functionalized pyrazoles 4-7 is outlined in Scheme 2.1. First of all, 4-hydrazinobenzenesulfonamide hydrochloride (8) was prepared via diazotization of sulfanilamide followed by reduction of the corresponding diazonium salt with stannous chloride under strong acidic conditions. 37 The treatment of aqueous solution of 8 with sodium acetate generated the free hydrazine which on condensation with various substituted methyl ketones 9 in aqueous ethanol afforded corresponding hydrazones 10. Subsequent reactions of hydrazones 10 under Vilsmeier-aack conditions 38 afforded 4-formylpyrazoles 11 with protected sulfonamide group. While we independently synthesized and 40

characterized sulfonamide protected (11) and deprotected (4) 4-formylpyrazoles in early 2009, a report by Bekhit et al. 39 in 2009 revealed the parallel synthesis of 11 and 4 by their group albeit with notable differences as discussed in the relevant subsections. The deprotection of sulfonamide group to afford aldehyde 4 with free S 2 2 group was accomplished under basic conditions using methanolic solution of a with TF as co-solvent. xidation of aldehyde 4 underwent smoothly using KMn 4 in aqueous pyridine 40 to afford carboxylic acids 5. n the other hand, oxidation of 4 using ammonical solution of iodine 41 in TF afforded 4-2 2 S C 3. 2 Cl + C 3 C C 3 Ca C R R Et/ 2, reflux PCl 3 DMF, 55-60 C 8 9 10 C 2 S 2 2 S 2 2 S a/me/tf KMn 4 R rt R Pyridine/ 2, rt R C C C 11 4 5 I 2 / 3 /TF rt 2 2 S 2 2 S C R 2 S/Et 3 Pyridine R CS 2 6 7 R = C 3 C 3 F Cl Br 2 S a b c d e f g h Scheme 2.1. Synthesis of 1,3-diaryl-4-functionalizedpyrazoles 4-7 41

Pyrazole derivatives: Synthesis and biological evaluation cyanopyrazoles 6 which were further converted into the corresponding carbothioamides 7 by passing 2 S gas through the solution in pyridine in the presence of triethylamine. 42 The synthetic details for each step are given in the following text. 2.1.2.1. Synthesis of protected 4-formylpyrazoles (11) and deprotected 4-formylpyrazoles (4) Synthesis of 4-formylpyrazoles (11) from hydrazones (10) was performed following the well established Vilsmeier-aack reaction which initiates by the + PCl 3 Cl PCl 2 Cl PCl 2 12a Cl PCl 2 13a Cl PCl 2 12b Cl PCl 2 13b Scheme 2.2. Formation of Vilsmeier-aack reagent formation of an equilibrium mixture of iminium salts 12 and 13 by the reaction of dimethylformamide (DMF) with phosphoryl chloride (PCl 3 ) as shown in Scheme 2.2. 2 C Ar R R = C 6 4 S 2 2 10 R C 2 Cl Ar 12 R Cl Ar R 14 15 Ar R 16 Ar R 17 Ar R Cl 18 12 Ar R Ar Cl R Ar hydrolysis R Ar R = S 19 20 11 Scheme 2.3. Plausible mechanism for the synthesis of 4-formylpyrazoles Subsequently electrophilic Vilsmeier-aack reagent 12 or 13 reacts with the hydrazone 10 yielding the iminium intermediate 14 which loses a molecule of Cl to 42

provide intermediate 15 (Scheme 2.3). Intramolecular nucleophilic attack by group in intermediate 15 initiates the cyclization and the resulting pyrazoline 16 immediately loses Me 2 to give the more stable pyrazole derivative 18. The pyrazole 18 further reacts with another molecule of the electrophilic Vilsmeier-aack reagent 12 in an electrophilic substitution process giving an iminium salt 20, which subsequently hydrolyzes to the corresponding 4-formylpyrazoles 11 with protected sulfonamide group (Scheme 2.3). It is interesting, though not unexpected, to note that under the reaction conditions, the sulfonamide group of the benzenesulfonamide moiety also reacts with the electrophilic Vilsmeier-aack reagent 12 and gets converted into - [(dimethylamino)methylidene]sulfonamide group (23). The mechanism for this transformation is depicted in Scheme 2.4 taking a simple example of p- methylbenzenesulfonamide 21. Cl S 2 + Cl S 21 12 22 23 S Scheme 2.4. Protection of sulfonamide group under Vilsmeier-aack conditions Structure of novel sulfonamido protected formylpyrazoles 11 was assigned on the basis of their IR, 1 MR and 13 C MR spectra. It is pertinent to mention here that just after we finished the synthesis of eight novel 4-formylpyrazoles 11, and their subsequent deprotection to 4 in our lab in the beginning of 2009, an article by Bekhit et al. 39 also appeared in February 2009, revealing the synthesis and characterization of five of the eight compounds, namely 11a, 11b, 11e, 11f and 11g but with notable differences. While we neutralized the completed reaction mixture by saturated aqueous sodium bicarbonate solution after pouring into ice cold water for solid separation, their group boiled the aqueous mixture for obtaining the same. The IR spectra of -protected formylpyrazoles 11 showed a band at 2928-2960 cm -1 for C- stretchings of aldehydic proton, a band at 1682-1694 cm -1 for C= stretchings, a band at 1628-1636 cm -1 for C= stretchings, and two bands in the regions 1335-1343 cm -1 & 1142-1150 cm -1 for S 2 stretchings. The 1 MR spectra of 11 in general displayed a characteristic signal at δ 9.86-10.08 assigned for aldehydic proton and a 43

Pyrazole derivatives: Synthesis and biological evaluation singlet at δ 8.60-9.43 for C 5 - of pyrazole ring. The presence of C was further established by a signal at δ 184.5-184.9 in 13 C MR spectrum. The presence of - [(dimethylamino)methylidene]sulfonamide group in 1 MR was ascertained on the basis of a singlet at δ 8.10-8.24 for =C moiety and two singlets for three protons each at δ 3.10-3.18 and δ 2.86-3.07 corresponding to two methyl groups of dimethylaminomethylidene moiety whose presence was further confirmed by two characteristic signals in the aliphatic region δ 41.0-41.9 and δ 35.1-35.6 in 13 C MR. A singlet due to three protons at δ 2.45 and a signal at δ 20.9 in 1 MR and 13 C MR spectrum respectively showed the presence of methyl group in 11b while the presence of methoxy group in 1 MR spectrum of 11c was ascertained by a singlet at δ 3.80 due to three protons which was further confirmed by a signal at δ 55.2 in 13 C MR spectrum. The presence of fluorine at the para position of the aromatic ring in 11d was ascertained by the presence of three doublets at δ 162.8 (d, 1 J CF = 241.6 z), δ 130.9 (d, 3 J CF = 8.3 z) and δ 115.5 (d, 2 J CF = 21.8 z) in its 13 C MR spectrum. aving pyrazoles 11 in hand, the dimethylaminomethylidene group was removed under basic conditions to afford the corresponding 4-formylpyrazoles 4 with free sulfonamide group as outlined in Scheme 2.1. To achieve this, pyrazoles 11 were treated with methanolic solution of a using TF as co-solvent to make the reaction mixture homogeneous. The mechanism for this basic hydrolysis is depicted in Scheme 2.5. otably, Bekhit et al. 39 used acidic conditions for the deprotection of this dimethylaminomethylidene group. owever, the basic conditions adopted by us afforded higher yields (80-92%) as compared to the yields under acidic conditions (65-75%). S R S R 2 2 S R 11 24 4 R = C Scheme 2.5. Plausible mechanism for the deprotection of dimethylaminomethylidene group Ar 44

The appearance of a D 2 exchangeable singlet at around δ 7.2 integrating for two protons in the 1 MR spectra of 4-formylpyrazoles 4 ascertained the presence of a free S 2 2 group indicating that the dimethylaminomethylidene group has been cleaved off under basic conditions. The spectral data for 4-formylpyrazoles 4 was in good agreement with the proposed structure. In general, IR spectra of 4 displayed two absorption bands in the region 3100-3400 cm -1 characteristic of - stretchings indicating the availability of a free S 2 2 group. Besides this, IR spectra of 4 displayed a band at 2918-2939 cm -1 for C- stretchings of aldehydic group, a band at 1666-1686 cm -1 for C= stretchings, a band at 1595-1597 cm -1 for C= stretchings, and two bands in the regions 1327-1342 cm -1 & 1158-1165 cm -1 for S 2 stretchings. In 1 MR spectra of 4, a singlet at δ 10.02-10.06 was ascertained for aldehydic proton and another singlet at δ 8.99-9.50 for C 5 - of pyrazole ring. The presence of C was further confirmed by a signal at δ 184.6-185.1 in the 13 C MR spectra. A singlet due to three protons at δ 2.40 and a signal at δ 21.3 in 1 MR and 13 C MR spectrum respectively showed the presence of methyl group in 4b while a singlet due to three protons at δ 3.85 was ascertained for methoxy group in 1 MR spectrum of 4c which was further confirmed by a signal at δ 55.7 in 13 C MR. The presence of fluorine at the para position of the aromatic ring in 4d was ascertained by the presence of three doublets at δ 163.2 (d, 1 J CF = 249.1 z), δ 131.3 (d, 3 J CF = 8.3 z) and δ 115.9 (d, 2 J CF = 22.6 z) in its 13 C MR spectrum. A notable feature which we noticed here was that in general, C 5 - and aldehydic proton of 4 are more deshielded than those of 11 indicating that free sulfonamide group has more electron withdrawing influence than protected sulfonamide group towards pyrazole ring. 2.1.2.2. Synthesis of pyrazole-4-carboxylic acids (5), 4- cyanopyrazoles (6) and pyrazole-4-carbothioamides (7) aving 4-formylpyrazoles 4 in hand, these were converted into pyrazole-4- carboxylic acids (5). To achieve this, 4-formylpyrazoles 4 were oxidized using potassium permanganate (KMn 4 ) in aqueous pyridine 40 carboxylic acids (5) in moderate yields (Scheme 2.1). affording pyrazole-4- The structure of the carboxylic acids 5 was ascertained by analysis of their IR, 1 MR, 13 C MR and mass spectral data (DART-MS). The IR spectrum of 5a 45

Pyrazole derivatives: Synthesis and biological evaluation showed characteristic wide absorption band in the region 2550-3100 cm -1 corresponding to - group participating in hydrogen bonding, along with a sharp absorption band at 1674 cm -1 corresponding to C= stretching. The 1 MR spectrum of 5a displayed a characteristic broad exchangeable singlet at δ 12.68 due to C group along with a singlet at δ 9.20 due to C 5 - of pyrazole and another exchangeable singlet at δ 7.46 due to two protons of S 2 2 group, appearing in the aromatic region. 13 C MR spectrum of 5a displayed a characteristic signal at δ 163.5 due to C. The other pyrazole-4-carboxylic acids 5b-5h also showed spectral characteristics similar to that of 5a. The presence of a methyl group attached to aromatic ring in 5b was ascertained on the basis of its 1 MR and 13 C MR spectrum which displayed a singlet for three protons at δ 2.36 and a signal at δ 20.9 respectively. The presence of a singlet for three protons at δ 3.81 in the 1 MR spectrum and a signal at δ 55.1 in the 13 C MR spectrum of 5c confirmed the presence of C 3 group. The presence of fluorine at the para position of the aromatic ring in 5d was confirmed by the presence of three doublets at δ 162.4 (d, 1 J CF = 244.3 z), δ 131.3 (d, 3 J CF = 8.7 z) and δ 114.7 (d, 2 J CF = 21.1 z) in its 13 C MR spectrum. For synthesizing pyrazole-4-carbothioamides 7a-7h, 4-formylpyrazoles 4a-4h, were first converted into corresponding 4-cyanopyrazoles 6a-6h. To achieve this, first of all, 4-fomylpyrazoles 4 in TF were treated with aqueous ammonia followed by the addition of iodine (I 2 ) 41 during stirring. The dark brown solution became colorless R aq. 3, I 2, r.t. R C TF 4 6 R = 2 2 S Ar 3-2 -I R 25 I I -I R 26 I 3 Scheme 2.6. Plausible mechanism for formation of cyano functionality from an aldehyde functionality 46

as the reaction proceeded due to consumption of iodine and pure cyanopyrazoles 6 precipitate out after charging the colorless solution with hypo (aqueous a 2 S 2 3 ) to effectively neutralize excess I 2. This transformation utilizes iodine as an appropriate oxidant and presumably proceeds via an intermediate -iodoaldimine (26) which eliminates an I molecule in ammonia solution to afford the nitrile product 6 (Scheme 2.6). Structure of 4-cyanopyrazoles 6 was assigned on the basis of their IR, 1 MR and 13 C MR spectra. While we achieved the synthesis of eight 4- cyanopyrazoles 6a-6h from 4-formylpyrazoles 4 using a single step protocol in the beginning of 2009, a report in February 2009 described the synthesis of five of these 4-cyanopyrazoles (6a, 6b, 6e, 6f and 6g) by a two step protocol 39 involving the conversion of aldehyde to the oxime followed by dehydration of oxime using PCl 3. The single step protocol adopted by us seems to be advantageous over the reported two step protocol in terms of using milder and greener materials (I 2, 3 as compared to PCl 3 ) as well as in terms of yields which are generally higher. IR spectra of 6 in general displayed a characteristic absorption band at 2230-2236 cm -1 due to C stretchings along with other absorption bands in the region 3100-3400 cm -1 due to - stretchings, 1596-1605 cm -1 for C= stretchings, and 1335-1345 & 1160-1165 cm -1 for S 2 stretchings. Disappearance of a singlet due to C group was observed along with a singlet at δ 9.51-9.63 for C 5 - of pyrazole ring in 1 MR spectra of 6. Appearance of a characteristic signal at δ 91.1-92.7 in 13 C MR spectra of 6 was assigned to C which further confirmed the compound formation. Presence of a singlet due to three protons at δ 2.38 and a signal at δ 21.4 in 1 MR and 13 C MR spectrum respectively confirmed the methyl group in 6b. Similarly, methoxy group in 6c was assigned to the presence of a singlet due to three protons at δ 3.84 in 1 MR spectrum and a signal at δ 55.8 in 13 C MR spectrum. The presence of fluorine at the para position of the aromatic ring in 6d was ascertained by the presence of three doublets at δ 163.4 (d, 1 J CF = 248.3 z), δ 129.2 (d, 3 J CF = 9.0 z) and δ 116.6 (d, 2 J CF = 21.9 z) in its 13 C MR spectrum. After getting 4-cyanopyrazoles 6 in hand, these were converted into corresponding pyrazole-4-carbothioamides 7 following well established literature procedure. 42 4-Cyanopyrazoles 7 were dissolved in pyridine and a small amount of 47

Pyrazole derivatives: Synthesis and biological evaluation triethylamine was added whereupon 2 S gas was passed though the solution. Color of the solution appears to signify reaction progression as it changed first from yellow to green and finally to dark brown. Reaction mixture was kept as such for overnight whereupon it was poured into ice cold water and neutralized with cold dilute hydrochloric acid with vigorous stirring to obtain precipitates of pyrazole-4- carbothioamides 7 after crystallization. The mechanism of the reaction (Scheme 2.7) presumably involves an initial attack of 2 S on nitrile carbon of 6 in the presence of weak base (triethylamine) along with subsequent shifting of a proton from 2 S to nitrile nitrogen to form an intermediate tautomer 27 (S-C=). ne more shifting of proton from S to in 27 affords final carbothioamide 7 (S=C- 2 ). S R C - + - + + + R C + + R C 2 S S 6 27 7 R = 2 2 S Ar Scheme 2.7. Plausible mechanism for the synthesis of carbothioamides 7 The structure of the compound 7a was ascertained by analysis of its IR, 1 MR, 13 C MR and mass spectral data (DART-MS). The IR spectrum of 7a showed four absorption bands at 3441 cm -1, 3302 cm -1, 3225 cm -1 and 3101 cm -1 which were assigned to the - stretchings of S 2 2 group and CS 2 moiety. The functional group region of the spectrum also exhibited two characteristic absorption bands at 1651 cm -1 & 1597 cm -1 due to C= stretchings, one due to CS 2 moiety and another due to pyrazole ring along with absorption band at 1504 cm -1 due to - bending and at 1319 cm -1 & 1157 cm -1 due to S 2 stretchings. The 1 MR spectrum of 7a displayed two characteristic exchangeable singlets, at δ 9.85 and δ 9.29 corresponding to and S protons along with a singlet at δ 8.89 due to pyrazole C 5 - and another exchangeable singlet at δ 7.46 due to two protons of S 2 2 group, appearing in the aromatic region. 13 C MR spectrum of 7a displayed a characteristic signal at δ 194.2 due to CS 2. The other pyrazole-4-carbothioamides 7b-7f and 7h also showed spectral characteristics similar to that of 7a along with additional characteristic peaks for other groups like C 3, C 3 and F. 1-[4-48

(Aminosulfonyl)phenyl]-3-(4-nitrophenyl)-1-pyrazole-4-carbothioamide 7g could not be synthesized by the same method inspite of repeated attempts. We are at loss to explain this exceptional behavior in case of nitro substituent. 2.2. Synthesis of pyrazolylpyrazolines 2.2.1. Motivation for the current work Five membered heterocycles with a vicinal diaryl substitution pattern have occupied a central space in the rationale design for making diverse range of biologically active molecules including cyclooxygenase inhibitors, kinase inhibitors, GPCR antagonists and agonists, phosphatase inhibitors, and dopamine transporter inhibitors. 43 Since the spatial extent of the common underlying 1,2-diaryl substituted heterocycle by far exceeds that of a generic scaffold, and the nature of the underlying heterocycle is quite diverse, a versatile chemistry can be ruled out as the main reason why this structure type frequently occurs in biologically interesting low molecularweight compounds. 44 Drugs having a pyrazole ring bearing two adjacent aryl groups in a vicinal relation continue to occupy leading positions in the list of best selling pharmaceutical products. 45 Celecoxib (28) and tepoxalin (29) are two non-steroidal anti-inflammatory drugs bearing vicinal dirayls on pyrazole ring. 2 2 S Ar R CF 3 Cl 3 C S 2 2 28 29 30 Besides pyrazoles, pyrazoline derivatives are also found to possess a variety of medicinal applications such as anti-inflammatory, 46-48 antimicrobial, 49,50 antimycobacterial, 51 antiamoebic, 52 antidepressant, 53 anticancer, 54 anticonvulsant, 55 insecticidal, 56 etc. Recently, Rathish et al. 57 reported that 1,3,5-trisubstituted pyrazolines (30) bearing benzenesulfonamide group exhibit excellent anti-inflammatory activity. The prescription of co-administration of multiple drugs for treatment of 49

Pyrazole derivatives: Synthesis and biological evaluation inflammatory conditions associated with some microbial infections may inflict added health problems especially in patients with impaired liver or kidney functions. A monotherapy of an anti-inflammatory drug with antimicrobial properties will be better from the pharmaco-economic point of view. This will enhance patient compliance and in case that this anti-inflammatory antimicrobial agent shows minimum adverse effects and high safety margin, such a drug will be highly desirable. 58 Pyrazolylpyrazoline scaffold has only recently been investigated by Bekhit et al. 58 and us 30 as a novel template for dual antimicrobial anti-inflammatory agents. Appreciation of these findings and having 4-formylpyrazoles 4 in hand coupled with the realization that an aldehyde functionality is an important launching pad for building heterocyclic scaffolds, motivated us to further elaborate the aldehyde functionality into pyrazoline nucleus and synthesize two novel series of pyrazolylpyrazolines (31a-l and 32a-l) bearing benzenesulfonamide moiety at position-1 of pyrazole as a potential template for dual antimicrobial antiinflammatory agents. It must be noted that this scaffold potentially provides vicinal diaryl substitution pattern on both the pyrazole as well as pyrazoline nucleus (32). R 2 2 S R R 1 2 2 S 2 2 S 31 32 R 1 2.2.2. Synthetic discussion for pyrazolylpyrazolines (31 and 32) The synthesis of pyrazolylpyrazolines 31 and 32 was achieved in two steps from 4-formylpyrazoles 4 according to Scheme 2.8. The 4-formylpyrazoles 4 bearing benzenesulfonamide group at -1 of pyrazole, on base catalyzed Claisen-Schmidt condensation reaction 59 with appropriately substituted acetophenones 9 in methanol/tf smoothly afforded the corresponding chalcones 33. Finally, these chalcones were treated, in refluxing Et/TF containing catalytic amount of glacial acetic acid, with hydrazine hydrate to obtain corresponding unsubstituted 50

pyrazolylpyrazolines 31, and with 4-hydrazinobenzenesulfonamide hydrochloride (8) 37 to obtain -benzenesulfonamido-substituted pyrazolylpyrazolines 32. The structures of all the target pyrazolylpyrazolines 31 and 32 were assigned on the basis of their IR, 1 MR, 13 C MR and mass spectral data. The synthetic details for each step are described in the following text. 2 2 S R CC 3 C R + R' a/me TF, rt 2 2 S R' 4 R =, C 3, F 9 R' =, C 3, C 3, F 33 33 S 2 2 2 2. 2 Et/TF reflux, 6-7h 8. 2 Cl Et/TF reflux, 6-7h 2 2 S 2 2 S R 2 2 S R R' R' 31 32 31, 32 & 33 a b c d e f g h i j k l R C 3 C 3 C 3 C 3 F F F F R C 3 C 3 F C 3 C 3 F C 3 C 3 F Scheme 2.8. Synthesis of pyrazolylpyrazolines 31 & 32 51

Pyrazole derivatives: Synthesis and biological evaluation 2.2.2.1. Synthesis of chalcones (33) Chalcones 33 were readily prepared in moderate to good yields by base catalyzed Claisen-Schmidt condensation reaction 59 of 4-formylpyrazoles 4 with appropriately substituted acetophenones 9. The mechanism of base catalyzed Claisen- Schmidt condensation reaction (Scheme 2.9) of aromatic aldehydes 4 with aromatic methyl ketones 9 involves the base promoted enolate anion (34) formation which subsequently attacks the carbonyl carbon of the aldehyde 4 to afford β- hydroxycarbonyl compound 36. Subsequent dehydration of 36 under basic conditions affords the chalcone 33. Ar 1 2 C Ar 1 C 2 Ar 1 C 2 R 4 9 34 Ar 1 R Ar 1 R Ar 1 35 36 33 R R = 2 2 S Ar Scheme 2.9. Mechanism of the chalcone synthesis The structure of the chalcones 33 was assigned on the basis of their IR, 1 MR and 13 C MR spectra. The IR spectrum of 33a showed two absorption bands at 3351 cm -1 & 3245 cm -1 which were assigned to the - stretchings of S 2 2 group. The functional group region of the spectrum also exhibited absorption bands at 1651 cm -1 (C= stretching), 1597 cm -1 (C= stretching), 1531 cm -1 (C=C stretching), 1506 cm -1 (- bending), and 1327 cm -1 & 1151 cm -1 (S 2 stretchings). 1 MR spectrum of chalcone 33a displayed a doublet at δ 7.88 for one of the two vinyl protons (C=C) with coupling constant 15.6 z which confirmed the trans-stereochemistry. The signal for other vinyl proton was found to be merged with aromatic region of the spectrum. The pyrazole C 5 - appeared as a singlet at δ 9.53. Besides appropriate signals for the aromatic protons, another characteristic signal in the 1 MR spectrum 52

of 33a was an exchangeable singlet for two protons at δ 7.48 which can be ascribed to the protons of S 2 2 group. The other chalcones 33b-33l were also synthesized following the same procedure and displayed similar characteristics as that of 33a besides displaying additional peaks due to groups like C 3, C 3, F, etc. depending on the structure of the chalcones in the 1 MR & 13 C MR spectra. Complete spectral data assignments are listed in the experimental section. 2.2.2.2. Synthesis of pyrazolylpyrazolines (31 and 32) Condensation of chalcones with hydrazine and its derivatives is a well reported method in literature 48,60-62 for the synthesis of pyrazolines. In the present study, the chalcones 33 were reacted with hydrazine hydrate to obtain corresponding unsubstituted pyrazolylpyrazolines 31, and with 4-hydrazinobenzenesulfonamide hydrochloride 37 (8) to obtain -benzenesulfonamido-substituted pyrazolylpyrazolines 32 (Scheme 2.8). The general mechanism for the condensation of chalcones 33 with -substituted hydrazines is depicted in Scheme 2.10. The mechanism involves an initial attack of nuceolphilic nitrogen of hydrazine on the carbonyl carbon to form an intermediate 37, which on subsequent cyclization followed by rearrangement afforded pyrazoline ring 39. R = 2 2 S Ar Ar 1 R 33 2 R 1 Ar 1 R 1 R 1 R 1 - + R Ar 1 R + + Ar 1 R 37 38 39 Scheme 2.10. Mechanism for the synthesis of pyrazolines from chalcones and -substituted hydrazines The reaction of 4-{4-[(E)-3-oxo-3-phenyl-1-propenyl]-3-phenyl-1-pyrazol-1- yl}benzenesulfonamide (33a) with hydrazine hydrate was first to be investigated. Acidic solution of chalcone 33a and hydrazine hydrate in Et/TF (1 : 1) was refluxed which on reaction completion was concentrated and cooled to room temperature. Solid separated out was crystallized from ethanol to afford 4-[3-phenyl- 4-(3-phenyl-4,5-dihydro-1-pyrazol-5-yl)-1-pyrazol-1-yl]benzenesulfonamide (31a) in 80% yield. The structure of 31a was assigned on the basis of its IR, 1 MR, 13 C 53

Pyrazole derivatives: Synthesis and biological evaluation MR and mass spectral (ES-MS) data. The IR spectrum of 31a showed broad absorption band at 3271 cm -1 which was assigned to - stretchings of S 2 2 group and of pyrazoline, merging with each other. The functional group region of the spectrum also exhibited characteristic absorption bands for C= stretching, - bending and S 2 stretchings. 1 MR spectrum of pyrazolylpyrazoline 31a displayed an exchangeable singlet at δ 7.59 due to of pyrazoline along with a singlet at δ 8.72 due to characteristic C 5 - of the pyrazole ring and another exchangeable singlet in the aromatic region due to two amino protons corresponding to the S 2 2 group merging with aromatic protons. The three pyrazoline protons of 31a displayed a typical ABX type pattern of doublet of doublets. Methine proton (C 5 -) of pyrazoline resonates at δ 4.99 appearing as a triplet with coupling constant 10.5 z. Two methylene protons (C 4 -) of pyrazoline displayed two doublets of doublets at δ 3.52 and δ 2.99 with coupling constants 10.5 z and 16.5 z. The other protons appeared in the aromatic region as predicted. The structure of the compound 31a was further supported by its 13 C MR spectrum which displayed signals on expected lines 63 due to C 3 -pyrazoline at δ 151.7, C 4 -pyrazoline at δ 42.6 and C 5 -pyrazoline at δ 55.5. The other unsubstituted pyrazolylpyrazolines 31b-31l, were also synthesized following the same procedure as adopted for 31a and displayed spectral characteristics similar to that of 31a. The 1 MR spectrum of 31b displayed a singlet at δ 2.30 corresponding to the methyl group attached to the aromatic ring which was further confirmed by the presence of a signal in 13 C MR spectrum at δ 21.3. The 1 MR spectrum of 31c showed a singlet at δ 3.76 corresponding to the methoxy group attached to the aromatic ring which was further confirmed by the signal in its 13 C MR spectrum at δ 55.6. The presence of fluorine at the para position of the aromatic ring in 31d was confirmed by the presence of three doublets at δ 162.4 (d, 1 J CF = 246.4 z), 130.4 (d, 3 J CF = 8.6 z) and 115.8 (d, 2 J CF = 20.3 z) in its 13 C MR spectrum. The presence of a methoxy group in 31e, 31f and 31h was confirmed on the basis of a singlet for three protons in the narrow range δ 3.80-3.81 in their 1 MR spectra and a signal in the narrow range δ 55.5-55.6 in their 13 C MR spectra. Presence of a methyl group attached to aromatic ring in 31f was confirmed on the basis of a singlet at δ 2.30 in its 1 MR spectrum and a signal at δ 21.3 in its 13 C MR spectrum. The presence of two methoxy groups in 31g was confirmed on the 54

basis of two singlets δ 3.80 and 3.76 in its 1 MR spectrum and two signals at δ 55.6 and 55.5 in its 13 C MR spectrum. 13 C MR of 31h confirmed the presence of a fluorine atom in the molecule by displaying three doublets at δ 162.4 (d, 1 J CF = 245.3 z), 128.0 (d, 3 J CF = 8.3 z) and 115.8 (d, 2 J CF = 21.1 z) due to coupling between carbon and fluorine. 31i-31l also displayed similar group of signals due to the presence of fluorine atom(s). The presence of a methyl group attached to the aromatic ring in 31j was confirmed on the basis of a singlet at δ 2.31 in its 1 MR spectrum and a signal at δ 21.3 in its 13 C MR spectrum. The presence of a methoxy group in 31k was confirmed by a singlet at δ 3.76 in its 1 MR spectrum and a signal at δ 55.8 in its 13 C MR spectrum. To synthesize -benzenesulfonamido-substituted pyrazolylpyrazolines 32, reaction of 4-{4-[(E)-3-oxo-3-phenyl-1-propenyl]-3-phenyl-1-pyrazol-1- yl}benzenesulfonamide (33a) with 4-hydrazinobenzenesulfonamide hydrochloride (8) was performed under acidic conditions in ethanol/tf (1 : 1). n reaction completion, the reaction solution was concentrated and cooled to room temperature. The solid separated out was crystallized from ethanol/tf (1 : 1) to afford 4-{4-[1-[4- (aminosulfonyl)phenyl]-3-phenyl-4,5-dihydro-1-pyrazol-5-yl]-3-phenyl-1-pyrazol- 1-yl}benzenesulfonamide (32a) in 82% yield. The structure of 32a was assigned on the basis of its IR, 1 MR, 13 C MR and mass spectra (ES-MS). The IR spectrum of 32a showed absorption bands at 3340 cm -1 & 3263 cm -1 which were assigned to - stretchings of S 2 2. The functional group region of the spectrum also exhibited absorption bands at 1597 cm -1 (C= stretching), 1504 cm -1 (- bending), and 1327 cm -1 & 1157 cm -1 (S 2 stretchings). 1 MR spectrum of pyrazolylpyrazoline 32a displayed two characteristic exchangeable singlets appearing in the aromatic region, one near δ 7.42 and another near δ 7.04, both due to amino protons of two S 2 2 groups. C 5 - of the pyrazole ring appeared as a singlet at δ 8.49. Simliar to 31a, three pyrazoline protons of 32a displayed a typical ABX type pattern of doublet of doublets. Methine proton (C 5 -) of pyrazoline resonates at δ 5.69 appearing as a doublet of doublet with coupling constants 6.0 z and 12.0 z. Two methylene protons (C 4 -) of pyrazoline displayed two signals, a doublet of doublet at δ 4.11 with coupling constants 12.0 z and 17.7 z, and a doublet of doublet at δ 3.44 merging with water peak of the MR solvent, with coupling constants 6.0 z and 17.7 z. The 55

Pyrazole derivatives: Synthesis and biological evaluation other protons appeared in the aromatic region as expected. The structure of the compound 32a was further supported by its 13 C MR spectrum which displayed signals on expected lines 63 due to C 3 -pyrazoline at δ 151.5, C 4 -pyrazoline at δ 42.6 and C 5 -pyrazoline at δ 55.1. The other -benzenesulfonamido-substituted pyrazolylpyrazolines 32b-32l were also synthesized following the same procedure and displayed spectral characteristics similar to that of 32a including the ABX type pattern for three pyrazoline protons. Values indicate that benzenesulfonamide group at -1 of pyrazoline has a deshielding influence on the methine and methylene protons of pyrazoline when compared with unsubstituted pyrazolylpyrazolines 31. Besides signals characteristic of a basic pyrazolylpyrazolines skeleton, 32b-32l also displayed additional peaks on predicted lines ascribable to C 3, C 3 or F in 1 MR & 13 C MR spectra. 2.3. Synthesis of thiazolylhydrazinomethylidenepyrazoles 2.3.1. Motivation for the current work The occurrence of thiazole ring system in numerous biologically active molecules, natural as well as synthetic, fuelled the interest of chemists as well as biologists towards this moiety. Several thiazole containing synthetic compounds such as 40 64 and 41 65 have been reported in the recent past to possess antiinflammatory 64,66-68 or antimicrobial 65,69-71 activities. Meloxicam 72 (42), fanetizole 73 (43), sulfathiazole 74 (44) and abafungin (45) are some of the thiazole bearing marketed drugs. Ar R C 3 S R S 3 C S S 40 41 42 S 2 S S S 43 44 45 56

Coumarins as a class are also found abundantly in nature and nowadays have become indispensable units to the chemists and the biochemists due to their diverse biological profiles such as anti-inflammatory, 47,75,76 antiviral, 77 antimicrobial, 78 anticancer, 79,80 anti-iv, 81,82 etc. Many coumarin derivatives 75 have been found to be inhibitors of cyclooxygenase and lipooxygenase in the arachidonic acid pathway of inflammation. In the development of newer antimicrobials, coumarins 83 have been identified as target specific plant anti-bacterial agents with growth inhibitory potential particularly against Gram-positive species. Kalkhambkar et al. 84 synthesized a series of new fluorinated coumarins (46) and 1-azacoumarins (47) which displayed excellent anti-inflammatory and moderate analgesic activities. In addition, most of the compounds were found to be potent antibacterial as well as antifungal agents. F F R R 46 47 R = 6-Cl; 6-C 3 ; 7-C 3 ; 5,6-benzo; 7,8-benzo The incorporation of essential structural features of pyrazoles with thiazole and coumarin while retaining benzenesulfonamide moiety can potentially provide new derivatives with enhanced biological activities. Motivated by these findings, we envisioned the synthesis of a library of thiazolylhydrazinomethylidenepyrazoles (48) bearing coumarin moiety for their evaluation as dual antimicrobial antiinflammatory agents. 2 2 S S R R 1 48 57

Pyrazole derivatives: Synthesis and biological evaluation 2.3.2. Synthetic discussion for thiazolylhydrazinomethylidenepyrazoles (48) Target thiazolylhydrazinomethylidenepyrazoles 48 have been synthesized by the reaction of pyrazole-4-carbaldehyde thiosemicarbazones 50 with various 6- substituted-3-bromoacetylcoumarins 51 according to Scheme 2.11. Treatment of ethanolic solution of 4-formylpyrazoles 4 with thiosemicarbazide (49) in the presence of catalytic amount of acetic acid afforded the corresponding pyrazole-4-carbaldehyde thiosemicarbazones 85 50 which on subsequent reaction with various 6-substituted-3- bromoactylcoumarins 51 in Et : TF afforded the target thiazolylhydrazinomethylidenepyrazoles 48. The synthetic details for each step are given in the following text. R 2 2 S 2 2 S 4 + 2 2 49 S Et/ + reflux, 1h 2 S 50 a b c d e R C 3 C 3 F Cl R 2 2 S 50 + R 1 51 a b c R Cl Br Br C 3 Ca/Et:TF reflux, 2h R 1 S R 48 48 a b c d e f g h i j k l m n o R C 3 C 3 C 3 C 3 C 3 C 3 F F F Cl Cl Cl R 1 Cl Br Cl Br Cl Br Cl Br Cl Br Scheme 2.11. Synthesis of thiazolylhydrazinomethylidenepyrazoles 48 2.3.2.1. Synthesis of pyrazole-4-carbaldehyde thiosemicarbazones (50) Starting from 4-formylpyrazoles 4, pyrazole-4-carbaldehyde thiosemicarbazones 50 were obtained in excellent yields by refluxing an ethanolic 58

solution of 4-formylpyrazoles 4 and thiosemicarbazide (49) in presence of catalytic amount of acetic acid for 1 h followed by usual work-up. 85 The structure of the compound 50a was ascertained by analysis of its IR, 1 MR, 13 C MR and mass spectral data. The IR spectrum of 50a showed three absorption bands at 3464 cm -1, 3317 cm -1 and 3124 cm -1 which were assigned to the - stretchings of S 2 2 group and - moiety. The functional group region of the spectrum also exhibited absorption bands at 1597 cm -1 (C= stretching), 1543 cm - 1 (C= stretching), 1504 (- bending), and 1335 cm -1 & 1157 cm -1 (S 2 stretchings). The two absorption bands for C= stretching can be ascribed to C= bond of the pyrazole ring and C= bond of C=CS 2 moiety. The 1 MR spectrum of 50a displayed a singlet at δ 9.30 that was assigned to =C. The C 5 - of pyrazole moiety appeared as a singlet at δ 8.23. owever assignments of 1 MR signals of =C/C 5 - may be interchangeable. Two amino protons corresponding to the S 2 2 group were obtained as exchangeable singlet for two protons at δ 7.48. Besides the appropriate signals in the aromatic region, the other characteristic signals in the 1 MR spectrum of 50a are the three exchangeable singlets at δ 11.41, δ 8.33 and δ 7.81 which were assigned to the S,, and = protons indicating that in solution CS 2 moiety may exist in its tautomeric form (S-C=). The other thiosemicarbazones 50b-50e also showed spectral characteristics similar to that of 50a. The presence of a methyl group attached to aromatic ring in 50b was ascertained on the basis of its 1 MR and 13 C MR spectrum which displayed a singlet for three protons at δ 2.37 and a signal at δ 21.3 respectively. The presence of a singlet for three protons at δ 3.82 in the 1 MR spectrum and a signal at δ 55.7 in the 13 C MR spectrum of 50c confirmed the presence of C 3 group. The presence of fluorine at the para position of the aromatic ring in 50d was confirmed by the presence of three doublets at δ 162.9 (d, 1 J CF = 246.0 z), δ 130.7 (d, 3 J CF = 8.3 z) and δ 116.2 (d, 2 J CF = 21.1 z) in its 13 C MR spectrum. 2.3.2.2. Synthesis of thiazolylhydrazinomethylidenepyrazoles (48) After the successful synthesis of all five thiosemicarbazones 50, we turned our attention to their reaction with various 6-substituted-3-bromoacetylcoumarins 51 to afford corresponding thiazolylhydrazinomethylidenepyrazoles 48 bearing benzenesulfonamide and coumarin moieties following antzsch thiazole synthesis 59

Pyrazole derivatives: Synthesis and biological evaluation strategy. 86 The present synthesis involves the refluxing of 50 and 51 along with sodium acetate in Et : TF followed by crystallization of the separated solid from Et : DMF to afford target compounds 48. The general mechanism of antzsch thiazole synthesis is depicted in Scheme 2.12. The first step is the formation of an acyclic intermediate 54 by the nucleophilic attack of sulfur (53) on the halogencarrying carbon (52) resulting in the formation of the C-S-C bond. This attack occurs with the Walden inversion (S 2 -reaction). In the next step, an attack by the lone pair of nitrogen atom at the carbonyl carbon within 54 yields a cyclic intermediate 55 which on elimination of water molecule ultimately leads to the formation of the thiazole ring 56. R 2 X C + C C 2 S R 1 R 2 C S R 1 R 2 52 53 54 55 S R 1 R 2 S 56 R 1 Scheme 2.12. Mechanism for the antzsch thiazole synthesis The structure of the first synthesized target compound 48a was assigned on the basis of its IR, 1 MR and 13 C MR spectral data and was confirmed on the basis of its mass spectrum (ES-MS). The IR spectrum of 48a showed a broad absorption band at 3248 cm -1 arising due to the overlap of - stretchings of S 2 2 group and - moiety. The functional group region of the spectrum also exhibited a characteristic C= stretching band of lactone at 1705 cm -1 due to the presence of coumarin moiety along with other absorption bands at 1566 cm -1 (C= stretching), 1504 cm -1 (- bending), and 1335 cm -1 & 1157 cm -1 (S 2 stretchings). 1 MR spectrum of 48a displayed an exchangeable singlet for one proton at δ 12.04 which was assigned to. Another exchangeable singlet for two protons of S 2 2 was found to be merging with aromatic and coumarin protons. Two singlets for one proton each at δ 9.00 and δ 8.52 were assigned to the C= and coumarin C 4 - respectively. Pyrazole C 5 - appeared in the region δ 8.19-8.22 merging with other aromatic protons and thiazole C 5 - also merged with other aromatic protons in the region δ 7.76-7.85. The other thiazolylhydrazinomethylidenepyrazoles 48b-48o were also synthesized following the same procedure and showed spectral characteristics similar to 48a. All other target compounds (48b-48o) showed characteristic C= stretchings 60

of lactone nature at 1705-1713 cm -1 due to the presence of coumarin moiety. Besides displaying other appropriate signals in 1 MR spectra of 48b-48o, an exchangeable singlet displayed in the range δ 12.04-12.12 was attributed to, a singlet in the range δ 8.96-9.03 was assigned to C=, another singlet in the range δ 8.13-8.28, mostly merging with aromatic protons, was ascribed to pyrazole C 5 - and one more singlet in the range δ 7.73-7.85, sometimes merging with aromatic ones was assigned to thiazole C 5 -. An exchangeable singlet for two protons at about δ 7.47 in 1 MR was ascribed to S 2 2 group. Coumarin C 4 - in 48b-48o appeared as a singlet in the range δ 8.44-8.52. Coumarin C 5 - displayed as a singlet in the range δ 7.96-8.01 in 48b, 48e, 48h, 48k, 48n and δ 8.11-8.17 in 48c, 48f, 48i, 48l, 48o. A singlet due to three protons in the aliphatic region at δ 2.40 in 1 MR was assigned for C 3 group in 48d-48f which was further confirmed by a signal at δ 21.3 in 13 C MR. Similarly, a singlet due to three protons appeared at δ 3.84 in 1 MR was ascribed for C 3 group in 48g-48i which was further illustrated by a signal at δ 55.6-55.7 in 13 C MR. 13 C MR spectra of 48j-48l ascertained the presence of fluorine atom at the para position of the aromatic ring by displaying three doublets at δ 162.8 (d, 1 J CF = 246.1 z), 131.2 (d, 3 J CF = 8.3 z) and 115.9 (d, 2 J CF = 21.1-21.8 z). 2.4. Synthesis of pyrazolo[3,4-b]pyridines 2.4.1. Motivation for the current work Pyrazolo[3,4-b]pyridine ring system is an important member of the family of fused heterocycles which have shown promise in designing new pharmaceuticals with better pharmacological profiles. This fused system is associated with diverse biological activities such as antimicrobial, 87,88 anti-inflammatory, 89 analgesic, 90 antimalarial, 91 antiviral, 92 antitumour, 93 sedative/hypnotic, 90 etc. Cartazolate (57) and Etazolate (58) (anxiolytic), Riociguat (59) (antihypertensive), non-nucelotide reverse 2 F 2 Cl R 57 58 59 60 61 R = R = 2 61

Pyrazole derivatives: Synthesis and biological evaluation transcriptase inhibitors (RTIs) 94 60 and 61, are some of the drugs bearing pyrazolo[3,4-b]pyridine system. wing to its unique characteristics, a trifluoromethyl moiety can alter the activities of the parent compound in dramatic ways, 95,96 e.g. acidity, polarizability, lipophilicity, enhanced hydrophobic binding, stability against metabolic oxidation, etc. All these findings make CF 3 bearing aromatics and heteroaromatics as increasingly attractive targets in the search of novel pharmaceuticals. A perusal of literature also revealed that the synthesis of pyrazolo[3,4- b]pyridine ring system containing benzenesulfonamide and trifluoromethyl group for evaluation as antimicrobial agents is still in its infancy. 97 Expanding the earlier studies from our group on pyrazolopyridines 97 as potential antimicrobial agents, we synthesized a library of thirty differently substituted pyrazolo[3,4-b] pyridines bearing benzenesulfonamide and trifluoromethyl moieties (62, 63 & 64) and evaluated them for antibacterial and antifungal activities. 2 2 S R R 1 CF3 62 R = 63 R = C 3 64 R = Cl 2.4.2. Synthetic discussion for pyrazolo[3,4-b]pyridines (62, 63 and 64) Generally, two distinct routes are suggested for the synthesis of pyrazolo[3,4- b]pyridine systems in the literature. The first one (route A, Scheme 2.13) involves the formation of pyrazole ring on a 3-acetyl/cyanopyridine bearing a good leaving group at 2-position. owever, this method lacks versatility in terms of substitution as only pyridines containing methyl, aryl, hydroxyl and amino groups at position-3 can be attained. The second route (route B, Scheme 2.13) uses the condensation reaction of 5-amino-1-pyrazoles with bifunctional electrophiles to form the pyridine moiety, and offers a great diversity and flexibility in terms of substitution of pyrazole and pyridine rings, despite of multiple step synthesis, large reaction times and low/moderate yields. 62

R 3 R R 1 Route A R 2 R 2 X X = Cl, R 3 = CAr, CC 3, CC 3, C R 2 Route B R R, R 1, R 2 = alkyl, aryl,, 2 R 1 2 Scheme 2.13. Synthetic routes of pyrazolo[3,4-b]pyridines. We envisioned the synthesis of differently substituted pyrazolo[3,4- b]pyridines following route B. First of all, 5-aminopyrazoles 66 were synthesized by 2 2 S 2 2 S. 2 Cl + R C Et reflux, 6-7 h 2 R 8 65 a b c R C 3 Cl 66 a b c R C 3 Cl 2 2 S 2 2 S 66 + F 3 C R 1 67 gl. Ac reflux, 12 h 1 2 7a 7 3 3a R + 6 4 R 1 CF3 5 3 CC R Major Product (48-80%) Minor Product (6-25%) 62, 63, 64 68 a b c R C 3 Cl R 1 (67) C 3 CF 3 S R (66) C 3 C 3 F Cl Br 62a 62b 62c 62d 62e 62f 62g 62h 62i 62j C 3 63a 63b 63c 63d 63e 63f 63g 63h 63i 63j Cl 64a 64b 64c 64d 64e 64f 64g 64h 64i 64j Scheme 2.14. Synthesis of pyrazolo[3,4-b]pyridines 62, 63 and 64 63

Pyrazole derivatives: Synthesis and biological evaluation reacting 4-hydrazinobenzenesulfonamide hydrochloride 37 8 with various α- cyanoacetophenones 98 65 in refluxing ethanol which on subsequent reaction with differently substituted trifluoromethyl-β-diketones 99-101 67 in glacial acetic acid under reflux yielded the target pyrazolo[3,4-b]pyridines (62, 63 and 64) as major product along with acylated 5-aminopyrazoles 68 as minor byproduct which could be separated by fractional crystallization from ethanol (Scheme 2.14). Assignment of the position of CF 3 in pyrazolo[3,4-b]pyridines 62, 63 and 64 as well as synthetic details for each step have been discussed in the following text. 2.4.2.1. Synthesis of 5-aminopyrazoles (66) 5-Aminopyrazoles 66 were prepared by the condensation of 4- hydrazinobenzenesulfonamide hydrochloride 37 8 with α-cyanoacetophenones 98 65 in ethanol. To achieve this, ethanolic solution of appropriate α-cyanoacetophenone 65 and 4-hydrazinobenzenesulfonamide hydrochloride 8 along with catalytic amount of glacial acetic acid was refluxed for 6-7 h and subsequently concentrated. Crystalline solid that separated out was recrystallized from aqueous ethanol to afford 5-2 2 S 8 2 C 65 R 2 2 S C 69 R - 2 2 2 S C 70 R 2 2 S 71 R - + + 2 2 S 2 66 R Scheme 2.15. Plausible mechanism for 5-aminopyrazole synthesis 64

aminopyrazoles 66 in 65-70% yield. The plausible mechanism of the reaction (Scheme 2.15) involves an initial nucleophilic attack of 2 of 4- hydrazinobenzenesulfonamide (8) under mildly acidic conditions on carbonyl carbon of 65 to generate intermediate 69. Subsequent loss of 2 molecule from 69 generated a new intermediate 70. Second nucleophilic attack of on nitrile carbon within intermediate 70 takes place to generate intermediate 5-iminopyrazoline 71 which on rearrangement leads to the formation of required 5-aminopyrazoles 66. The structure of the compound 66a was confirmed by the comparison of its melting point and spectral data reported earlier in our lab. 97 Remaining two novel 5- aminopyrazoles 66b and 66c were identified by IR, 1 MR and 13 C MR spectra. The IR spectrum of 66b showed three absorption bands at 3356 cm -1, 3302 cm -1 and 3263 cm -1 which were assigned to the - stretchings of S 2 2 and 2 groups. The functional group region of the spectrum also exhibited absorption bands at 1628 cm -1 (C= stretching), 1597 cm -1 (C= stretching), 1512 (- bending), and 1327 cm -1 & 1165 cm -1 (S 2 stretchings). Presence of two bands for C= stretch, one ascribed to C= bond of the pyrazole ring and another to imino C=, shows the existence of the molecule in imino form as in 5-iminopyrazoline 71. The 1 MR spectrum of 66b displayed a characteristic upfield sharp singlet integrated for one proton at δ 5.93 that was assigned to pyrazole C 4 -. Two characteristic exchangeable singlets, both integrated for two protons appearing at δ 5.65 and δ 7.44, were assigned to the 2 group and S 2 2 group respectively. Besides the appropriate signals in the aromatic region, the presence of a methyl group attached to aromatic ring in 66b was ascertained on the basis of its 1 MR and 13 C MR spectrum which displayed a singlet for three protons at δ 2.32 and a signal at δ 21.3 respectively. The spectral characteristics of 66c were on similar lines as that of 66b. 2.4.2.2. Synthesis of pyrazolo[3,4-b]pyridines (62, 63 and 64) The reaction of 5-aminopyrazoles 66 with unsymmetrical 1,3-diketones especially trifluoromethyl-β-diketones 67 has been a subject matter of intense investigation in the recent past. Formation of two different regioisomers of pyrazolo[3,4-b]pyridine, with CF 3 either at 4-position 102 or at 6-position 97,103 6 R 1 2 7 5 4 3 o, p-cf 3 65

Pyrazole derivatives: Synthesis and biological evaluation of the pyrazolopyridine skeleton, has been reported in the literature under similar reaction conditions. 19 F MR spectroscopy has played a decisive role in establishing the structure of these isomeric compounds. It is reported in the literature 104 that the CF 3 group at o-position in pyridine displays higher negative values as compared to that of p-position in 19 F MR. The relative difference in values at o- and p-positions may be attributed to the presence of nitrogen at adjacent position attached via a double bond. A recent report of August 2012 105 has shown on the basis of 13 C, 19 F and MBC experiments that solvent mediated stepwise synthesis of pyrazolo[3,4- b]pyridine gave single regioisomer with CF 3 at 4-position. Earlier reports presumed that the reaction of 5-aminopyrazoles with trifluoromethyl-β-diketones is initiated exclusively by the attack of the amino group on the carbonyl carbon adjoining the CF 3 group. owever, it is now believed that carbonyl group adjacent to CF 3 remains in hydrated form in protic solvent and attack of amino group takes place on the other available carbonyl group, generating intermediate 73 which on cyclization followed dehydration yields a single cyclized isomer, pyrazolo[3,4-b]pyridine 75 bearing trifluoromethyl group at position-4 (Scheme 2.16). R R 2 72 R 1 R 2 CF 3 67 protic solvent R 2 73 R 1 CF 3 R R 1 R R 1 R 2 74 CF 3 CF 3 R 2 75 Scheme 2.16. Mechanism for pyrazolo[3,4-b]pyridine synthesis As far as the choice of solvent is concerned, the only solvent which worked 97,103, 106 fine in our hands for pyrazolopyridine formation is glacial acetic acid, an observation that is in consonance with another recent literature report. 107 Coupling of 66 with various trifluoromethyl-β-diketones 67 in refluxing glacial acetic acid provided two products, one desired regioisomer pyrazolo[3,4-b]pyridines with C 4 -CF 3 66

(62, 63 and 64) as major product along with acylated 5-aminopyrazoles (68a-68c) in minor amount. Proposed mechanism 108,109 for the synthesis of acylated aminopyrazoles (Scheme 2.17) involves the nucleophilic attack of amino group of 5-aminopyrazoles 66 on carboxyl carbon of acetic acid under reflux conditions leading to formation of ammonium salts 76 which ultimately loses a water molecule yielding acylated 5- aminopyrazoles 68. 2 2 S 2 2 S 2 66 R 3 C Solvent δ 2 3 C δ 76 R 2 2 S - 2 3 CC 68 R Scheme 2.17. Mechanism for acylation of 5-aminopyrazole While, to the best of our knowledge, we were the first to report the formation of acylated 5-aminopyrazoles (68) under the conditions of the reaction of 5- aminopyrazoles (66) and β-diketones to give fused pyrazolopyridines (62, 63, 64) and presented our results in the form of a research poster at Indian Science Congress 2012 dated January 3-7, 2012 under chemical sciences section, 110 a recent but later article also discloses similar findings. 105 We synthesized a library of thirty differently substituted pyrazolo[3,4-b]pyridines (62a-62j, 63a-63j and 64a-64j) following the procedure earlier adopted by us. 97 A solution of appropriate 5-aminopyrazole 66 and trifluoromethyl-β-diketone 67 in glacial acetic acid was refluxed. The crude solid material upon fractional crystallization from ethanol yielded two products: pyrazolo[3,4-b]pyridines (62, 63 or 64) as major product and 5-acetamidopyrazoles (68) in minor amount. The formation of side product like 68 in this reaction offers for the first time probable reason for a moderate to low yield reported in the 67

Pyrazole derivatives: Synthesis and biological evaluation literature 97,103,105,106 for the synthesis of fused pyrazolopyridines. The formation of 68 was conclusively proved by refluxing 5-aminopyrazoles (66) in glacial acetic acid in the absence of β-diketones where the sole product obtained was acylated 5- aminopyrazoles (68a-68c), identical with samples obtained from the synthesis of pyrazolopyridines. It reveals that both the reactions i.e. (i) nucleophilic attack of amino group on the carbonyl carbon, and (ii) amino acylation, go side by side and compete with each other. We observed that substitution of group R in 5- aminopyrazole plays an important role for controlling yield ratio of both the products. Amount of acylation follows the order -C 3 ( 10%) < - ( 15%) < -Cl ( 20%) revealing that acylation occurs to a lesser extent when R group is electron releasing (68b, C 3 ) in nature and more when R is electron withdrawing (68c, Cl). Acylated compounds (68a-68c) were easily separated from the desired pyrazolopyridines (62a- 62j, 63a-63j and 64a-64j) by fractional crystallization from ethanol without using column chromatography because of poor solubility of acylated product in ethanol. Spectral data ( 1 MR, 13 C MR, 19 F MR, IR and mass) of the newly synthesized compounds were in full agreement with the proposed structures. In general, IR spectra of pyrazolo[3,4-b]pyridines (62a-62j, 63a-63j and 64a-64j) exhibited two characteristic absorption bands in the region 3200-3400 cm -1 corresponding to - stretchings of S 2 2 group along with other absorption bands in the regions 1589-1597 cm -1 (C= stretchings), 1497-1512 cm -1 (- bendings), and two more bands, one near 1335 cm -1 and another near 1157 cm -1 corresponding to S 2 stretchings. Final structure of all fused pyrazolopyridines was supported by the presence of a singlet for pyridine C 5 - in 1 MR resonating in the range δ 7.75-7.79 for 62a, 63a and 64a; δ 8.99-9.00 for 62i, 63i and 64i; and δ 8.20-8.37 in others, sometimes merging with aromatic protons. Position of CF 3 (C 4 -CF 3 ) in pyrazolopyridines 62, 63 and 64 was identified with the help of 19 F MR which displayed a signal in the range δ 58.58 to 59.01 which was in agreement with those reported for similar structures in literature. 104,105 19 F MR signal of C 6 -CF 3 resonates in the narrow range δ 65.45 to 65.47 as in the case of 62b, 63b and 64b. Methyl group at C 6 -position in 62a, 63a and 64a was assigned by the presence of a singlet in the aliphatic region at δ 2.80-2.81 in 1 MR which was further confirmed by the presence of a signal at δ 25.1 in 13 C MR. p-tolyl group in 62d, 63d and 64d was assigned by the presence of a singlet in 68

the aliphatic region δ 2.42-2.50 which was further confirmed by the presence of a signal at δ 21.4 in 13 C MR whereas p-anisyl group in 62e, 63e and 64e was identified by the presence of a singlet at δ 3.86-3.87 and a signal at δ 55.2-55.8 in 1 MR and 13 C MR respectively. Acylated 5-aminopyrazoles, 68a-68c were identified by the presence a characteristic absorption band at 1666 cm -1 ascertained to C= stretching due to presence of acetamido group in IR which were finally 10.20 due to proton and another singlet at δ 2.03 due to three protons of C 3 group in 1 MR and two characteristic signals in 13 C MR, one at δ 169.5 corresponding to C and another at δ 23.3 corresponding to C 3. 2.5. Biological Testing Results confirmed by the presence of an exchangeable singlet in the narrow range δ 10.18-2.5.1. Anti-inflammatory activity Determination of the inhibition of swelling induced in rat paw edema is one of the most popular methods used for testing anti-inflammatory (AI) activity of a compound. In this method, a small amount of solution or suspension of a phlogistic agent (edemogen) is injectd into the planter tissues of the hind paw of a rat and the amount of swelling is measured by determining the paw volume using a plethysmometer (model 7140, Ugo Basile, Italy). The most widely used assay in this category is carrageenan induced paw edema in rats introduced by Winter et. al. 111 in 1962. Pyrazolylpyrazolines (31a-31l and 32a-32l) and thiazolylhydrazino- for their in vivo AI methylidenepyrazoles (48a-48o) were evaluated activity. 2.5.1.1. Pharmacological assay Male Wistar albino rats weighing 200-250 g were used throughout the study. They were kept in the animal house under standard conditions of light and temperature with free access to food and water. Food was withdrawn 12 h before and during experimental hours. The animals were randomly divided into groups each consisting of six rats. ne group of six rats was kept as control and received tween 80 (95 : 5). Another group received the standard drug indomethacin at a dose of 10 mg/kg body weight ip. ther groups of rats were administered the test compounds at a dose of 50 mg/kg body weight orally. A mark was made on the left hind paw just 69

Pyrazole derivatives: Synthesis and biological evaluation beyond the tibiotarsal articulation, so that every time the paw was dipped up to the fixed mark and constant paw volume was ensured. Paw volumes were measured using a plethysmometer (model 7140, Ugo Basile, Italy). Thirty minutes after administration of test compounds and standard drug, 0.1 ml of 1% w/v of carrageenan suspension in normal saline was injected into subplanter region of the left hind paw of all the animals. The initial paw volume was measured within 30 s of the injection and remeasured again 1, 2, 3, and 4 h after administration of carrageenan. The edema was expressed as an increase in the volume of paw and the percentage of edema inhibition. % 100 where V t = volume of edema at specific time interval and V = volume of edema at zero time interval. 0 2.5.2. Antimicrobial studies All the newly synthesized pyrazole derivatives, i.e. 4-functionalized-pyrazoles e.g. pyrazole-4-carboxylic acids (5a-5h) and pyrazole-4-carbothioamides (7a-7f, 7h); pyrazolylpyrazolines (31a-31l and 32a-32l); thiazolylhydrazinomethylidenepyrazoles (48a-48o); and pyrazolo[3,4-b]pyridines (62a-62j, 63a-63j and 64a-64j) were evaluated for their in vitro antibacterial activity against two Gram-positive and two Gram-negative bacteria. In addition to this, these compounds were also evaluated for their in vitro antifungal activity against two fungi. All the microbial cultures used in the present study were procured from the Microbial Type Culture Collection (MTCC), Institute of Microbial Technology (IMTEC), Chandigarh, IDIA. 2.5.2.1. Test microorganisms Four bacterial and two fungal strains for testing activity of each compound were selected on the basis of their clinical importance in causing diseases in humans. Staphylococcus aureus (S. aureus) (MTCC 96), Bacillus subtilis (B. subtilis) (MTCC 121) representing Gram-positive bacteria and Escherichia coli (E. coli) (MTCC 1652), Pseudomonas aeruginosa (P. aeruginosa) (MTCC 741) representing Gramnegative bacteria were used for evaluating antibacterial activity of the compounds. For evaluation of antifungal activity, either Aspergillus niger (A. niger) (MTCC 282) and 70

Aspergillus flavus (A. flavus) (MTCC 871) or Candida albicans (C. albicans) (MTCC 227) and Saccharomyces cerevisiae (S. cervisiae) (MTCC 170) representing pathogenic yeasts were used. 2.5.2.2. In vitro antibacterial assay The in vitro antibacterial activity of all the target compounds and antifungal activity (against yeasts, C. albicans and S. cerevisiae) of thiazolylhydrazinomethylidenepyrazoles 48, and pyrazolopyridines 62, 63 and 64 were evaluated by agar well diffusion method. 112,113 All the microbial cultures were adjusted to 0.5 McFarland standard, which is visually comparable to a microbial suspension of approximately 1.5 10 8 cfu/ml. 20 ml of Mueller inton agar media was poured into each petri plate and the agar plates were swabbed with 100 μl inocula of the test microorganisms and kept for 15 min for adsorption. Using sterile cork borer of 8 mm diameter, wells were bored into the seeded agar plates and these were loaded with a 100 μl volume with concentration of 4.0 mg/ml of each compound reconstituted in dimethylsulphoxide (DMS). All the plates were incubated at 37 C for 14 h. Antibacterial activity against bacteria, and antifungal activity against yeasts, indicated by an inhibition zone surrounding the well containing the compounds, was recorded if the zone of inhibition was greater than 8 mm by using zone reader (iantibiotic zone scale). The experiments were performed in triplicate. DMS was used as a negative control whereas ciprofloxacin was used as a positive control in case of bacteria, and amphotericin-b for fungal yeasts. 2.5.2.3. Determination of minimum inhibitory concentration (MIC) Minimum inhibitory concentration (MIC) is the lowest concentration of an antimicrobial compound that will inhibit the visible growth of a microorganism after overnight incubation. MIC of various compounds against bacterial strains was tested either through a macrodilution tube method (5, 7, 31, 32 and 48) as recommended by ational Committee for Clinical Laboratory Standards ( CCLS) 113,114 or through a modified agar well diffusion method 115 (62, 63 and 64). In macrodilution tube method, various test concentrations of compounds were prepared from 128 to 0.25 μg/ml in sterile tubes o. 1 to 10. 100 μl sterile Mueller inton Broth (MB) was poured in each sterile tube followed by addition of 200 μl 71

Pyrazole derivatives: Synthesis and biological evaluation solution of test compound in tube 1. Two fold serial dilutions were carried out from the tube 1 to the tube 10 and excess broth (100 μl) was discarded from the last tube o. 10. To each tube, 100 μl of standard inoculum (1.5 10 8 cfu/ml) was added. Turbidity was observed after incubating the inoculated tubes at 37 C for 24 h. aerobically at 37 o C for 24 h and observed for the inhibition zones. MIC, shown by a bacteria and amphotericin-b against fungi as well as yeasts while DMS as negative control. compounds. was used as inocula for evaluating antifungal activity of chemical measured and expressed as percentage mycelial inhibition determined by the formula given below: In modified agar well diffusion method, a two fold serial dilution of each tested compound was prepared by first reconstituting the compound in DMS followed by dilution in sterile distilled water to achieve a decreasing concentration range of 256 to 0.5µg/mL. A 100 µl volume of each dilution was introduced into wells (in triplicate) in the agar plates already seeded with 100 µl of standardized inoculum (10 6 cfu/ml) of the test microbial strain. All test plates were incubated clear zone of inhibition, was recorded for each test organism. In both the methods, ciprofloxacin was used as positive control against 2.5.2.4. In vitro antifungal assay The in vitro antifungal activity of compounds (5, 7, 31 and 32) against A. niger and A. flavus was evaluated by poisoned food method. 116 All the test molds were grown on Sabouraud Dextrose Agar (SDA) at 25 C for 7 days. ne week old culture of the mold 15 ml of the molten SDA (45 C) was poisoned by the addition of 100 μl volume having concentration of 4.0 mg/ml of each compound, reconstituted in the DMS, poured into the sterile petri plates and allowed to solidify at room temperature. The solidified poisoned SDA plates containing the test compounds were inoculated with fungal plugs (8 mm diameter) obtained from the actively growing margins of the fungal plates. Plates were incubated at 25 C for 7 days. DMS was used as a negative control whereas fluconazole was used as positive control. The experiments were performed in triplicates. Diameter of the fungal colonies was 72

% Where 100 dc = average diameter of fungal colony in negative control plates dt = average diameter of fungal colony in experimental plates 2.5.3. Results and discussion 2.5.3.1. 4-Functionalized-pyrazoles (carboxylic acids 5a-5h, and carbothioamides 7a-7f & 7h) 2.5.3.1.1. In vitro antibacterial activity Results revealed that pyrazole-4-carboxylic acids (5a-5h) and pyrazole-4- carbothioamides (7a-7f and 7h), in general, possessed moderate to good antibacterial activity against Gram-positive bacteria (S. aureus, B. subtilis). owever, none of them was found to be effective against any of the tested Gram-negative bacteria (E. coli, P. aeruginosa). n the basis of zone of inhibition against the test bacterium, compounds 7b and 7f were found to be the most effective against S. aureus showing the maximum zone of inhibition of 21.6 mm and compound 5e against B. subtilis producing 21.6 mm zone of inhibition (Table 2.1) as compared with the standard drug ciprofloxacin which showed the zone of inhibition 27.6 mm against S. aureus and 26.3 mm against B. subtilis. Table 2.1. I n vitro antibacterial activity and MIC of compounds 5 and 7 Compound a Diameter of growth of inhibition zone Minimum inhibitory b (mm) concentration (MIC) ( μg/ml) S. aureu s B. subtil is E. co li P. aeruginosa S. aureus B. subtilis 5a 15.3 16.6 128 128 5b 14.6 17.3 >128 64 5c 15.6 17.0 128 64 5d 15.0 18.6 128 64 5e 18.6 21.6 64 32 5f 15.6 14.3 128 >128 5g 14.6 15.0 >128 128 5h 18.6 15.6 64 128 7a 15.3 15.0 128 128 7b 21.6 16.3 32 128 7c 16.6 15.6 128 128 73

Pyrazole derivatives: Synthesis and biological evaluation 7d 17.6 15.3 64 128 7e 18.3 14.6 64 >128 7f 21.6 18.3 32 64 7h 20.3 17.6 64 64 Ciprofloxacin 27.6 26.3 25.3 25.0 5 5 a C oncentration 4.0 mg/ml. b Values, including diameter of the well (8 mm), are means of three replicates. o activity. Besides 7b and 7f, compounds 5e, 5h, 7e and 7h showed moderate antibacterial activity against S. aureus with zone of inhibition >18.0 mm while compounds 5d and 7f showed moderate antibacterial activity against B. subtilis with zone of inhibition >18.0 mm. owever, in terms of MIC, none of the compounds was found to possess appreciable antibacterial activity. Amongst all the compounds, the MIC ranged between 32 (7b, 7f, 5e) and 128 µg/ml against Gram-positive bacteria (Table 2.1). A comparison between the two series of compounds (5 and 7) indicates that in general, 4-carbothioamide derivatives (7) exhibit better activity against S. aureus while a reverse trend is observed against B. subtilis. Within the individual series, no correlation between the antibacterial activity with respect to the substituent on phenyl ring is observed. owever, in general, compounds containing a halogen substituent showed better antibacterial activity than the compounds with other substituents. A comparative study with corresponding 4-cyanoderivatives (6) 39 revealed that replacement of cyano group with carboxylic acid moiety (5) or carbothioamide moiety (7) resulted in complete loss of activity against Gram-negative bacteria and a decreased activity against Gram-positive bacteria. It is an established fact that Gram-negative bacteria are hard to target owing to their cell wall structure, however, it seems from the comparison that increased hydrophilicity due to the replacement of a cyano group with carboxylic acid moiety or carbothioamide moiety is detrimental to activity against Gram-negative bacteria. 2.5.3.1.2. In vitro antifungal activity When compared the antifungal activity of carboxylic acids 5 and carbothioamides 7 with the reference drug fluconazole which showed 75.3% & 74.6% inhibition against A. niger and A. flavus respectively, it was noted that compounds 5g, 5h, 7c and 7h showed moderate antifungal activity with 55% inhibition of mycelial 74

growth against A. niger and compounds 5a, 5g, 5h, 7c, 7d and 7h showed 55% inhibition of mycelial growth against A. flavus (Table 2.2). Table 2.2. In vitro antifungal activity of compounds 5 and 7 Compound a M ycelial growth inhibit ion (%) A. niger A. flavus 5a 50.0 55.5 5b 46.6 51.1 5c 44.4 44.4 5d 52.5 48.8 5e 45.5 44.4 5f 51.1 45.5 5g 61.1 57.7 5h 57.7 55.5 7a 50.0 53.3 7b 44.4 51.1 7c 55.5 57.7 7d 44.4 61.1 7e 50.0 52.5 7f 44.4 51.1 7h 61.1 55.5 Fluconazole 75.3 74.6 a Concentration 4.0 mg/ml. Although few compounds showed good antibacterial activity and few others showed moderate antifungal activity, none of the compounds from series 5 & 7 could show consistently good antibacterial as well as antifungal activity. 2.5.3.2. Unsubstituted pyrazolylpyrazolines (31a-31l) and - benzenesulfonamido-substituted pyrazolylpyrazolines (32a-32l) 2.5.3.2.1. In vivo anti-inflammatory activity ine of the newly synthesized unsubstituted pyrazolylpyrazolines (31a, 31c, 31d, 31e, 31g, 31h, 31i, 31k and 31l) and nine of the newly synthesized - benzenesulfonamido-substituted pyrazolylpyrazolines (32a, 32c, 32d, 32e, 32g, 32h, 32i, 32k and 32l) were selected for evaluation of their in vivo anti-inflammatory activity by carrageenan-induced paw edema method. 111 Increase in the volume of paw and the percentage of edema inhibition for each rat group for different compounds is summarized in Table 2.3. 75

Pyrazole derivatives: Synthesis and biological evaluation Table 2.3. In vivo anti-inflammator y activity of pyrazolylpyrazolines 31 and 32 Compound a b Volume of edema (ml) and %AI c 1h 2h 3h 4h Control 0.68±0.02 0.95±0.03 1.86±0.03 1.94±0.02 Indomethacin 0.20±0.02* 0.21±0.02** 0.40±0.02** 0.48±0.20** (71) (78) (78) (75) 31a 0.30±0.07 0.31±0.05** 0.61±0.11** 1.21±0.11* (44) (67) (67) (38) 31c 0.24±0.03* 0.25±0.07** 0.41±0.09** 1.27±0.11* (65) (74) (78) (35) 31d 0.22±0.18* 0.27±0.07** 0.43±0.05** 1.30±0.15* (68) (72) (77) (33) 31e 0.29±0.04 0. 30±0.2** 0.53±0.10** 1.37±0.13 (57) (68) (72) (29) 31g 0.22±0.04* 0.32±0.02** 0.41±0.01** 1.31±0.01* (68) (66) (78) (32) 31h 0.55±0.02 0. 80±0. 2 1.21±0.20** 1.49±0.14 (19) (16) (35) (23) 31i 0. 23±0.03* (66) 0. 54±0. 1 (43) 0.56±0.03** (70) 1.54±0.13 (21) 31k 0.32±0.09 0.33±0.1* 0.63±0.13** 1.30±0.12* (53) (65) (66) (33) 31l 0.30±0.13 0.35±0. 04 0.58±0.04** 1.26±0.17* (44) (63) (69) (35) 32a 0.33±0.04 0.75±0.11 0.89±0.08** 1.35±0.20 (51) (21) (52) (34) 32c 0.46±0.02 0.92±0.26 1.2 ±0.10** 1.60±0.14 (32) (3) (35) (18) 32d 0.21±0.02* 0.28±0.14** 0.89±0.08** 1.25±0.27* (69) (71) (52) (36) 32e 0.33±0.04 0.75±0. 11 1.24±0.07** 1.08±0.16** (51) (21) (33) (44) 32g 0.24±0.03* 0.28±0.09** 0.54±0.07** 0.92±0.07** (65) (71) (71) (53) 32h 0.61±0.13 0.90±0.18 1.05±0.15** 1.37±0.13 (10) (5) (44) (29) 32i 0.57±0.17 0.91±0.28 1.22±0.08** 1.29±0.22* (16) (4) (34) (34) 32k 32l 0.34±0.01 (50) 0.40±.05 (41) 0.79±0.11 (17) 0.42±0.06 (56) 1.58±0.18 (15) 0.49±0.19** (74) 1.60±0.20 (18) 1.61±0.17 (17) * Significantly different compared to respective control values, P < 0.05. ** Significantly different compared to respective control values, P < 0.01. a Dose levels: test compounds (50 mg/kg body wt.), Indomethacin (10 mg/kg body wt.). b Values are expressed as mean ± SEM (no. of animals = 6) and analyzed by AVA. c Values in parentheses (percentage anti-inflammatory activity, AI%). Amongst eighteen compounds tested, two compounds (31c and 31g) showed identical AI activity as that of the standard drug indomethacin (78% inhibition) 3h after carrageenan injection while five compounds (31d, 31e, 31i, 32g, 32l) showed 76

excellent AI activity ( 70% inhibtion) after 3h and three compounds (31a, 31k, 31l) showed significant activity with 66-69% inhibition after 3 h. one of the compound was able to maintain its AI activity after 4h suggesting that these compounds get easily metabolized in the system. In general, unsubstituted pyrazolylpyrazolines (31) showed better AI activity as compared to -benzenesulfonamido-substituted pyrazolylpyrazolines (32) indicating that addition of another benzenesulfonamide group on pyrazoline -1 is detrimental to AI activity. This effect may probably be attributed to the steric bulk or the electronic effects associated with the electron withdrawing behavior of the benzenesulfonamide moiety. Within individual series (31 and 32), compounds containing methoxy substituent in general showed better AI activity followed by fluoro and. This effect was more pronounced when two methoxy (31g and 32g) substituents were present together. More or less same type of observation was noted in case of compounds containing fluoro substituents (31l and 117,118 32l). These results were in accordance with our earlier observations that the compounds with methoxy (or) and fluoro substituents show higher activity. 2.5.3.2.2. In vitro antibacterial activity All newly synthesized pyrazolylpyrazolines (31a-31l and 32a-32l) were evaluated for in vitro antibacterial activity. Results revealed that in general, all the tested compounds possessed variable antibacterial activity against Gram-positive bacteria (S. aureus, B. subtilis). owever, none of them was found to be effective against any Gram-negative bacteria (E. coli, P. aeruginosa). n the basis of zone of inhibition against the test bacterium, four compounds 31a, 31d, 31f and 31k were found to be most effective against S. aureus showing the maximum zone of inhibition of >19 mm, and four compounds 31g, 32f, 32h and 32i producing >19 mm zone of inhibition against B. subtilis (Table 2.4) when compared with standard drug ciprofloxacin which showed the zone of inhibition 27.6 mm against S. aureus and 26.3 mm against B. subtilis. Besides these compounds, 31c and 32h showed moderate antibacterial activity against S. aureus with zone of inhibition >18.0 mm. Besides 31g, 32f, 32h and 32i, compounds 31d and 31h showed zone of inhibition >18.0 mm against B. subtilis. Five compounds namely 32c, 32e, 32g, 32j and 32k didn t show any activity against S. aureus. owever, in terms of MIC, none of the compounds was found to possess appreciable antibacterial activity. Amongst all the compounds, the 77

Pyrazole derivatives: Synthesis and biological evaluation MIC ranged between 64 and 256 µg/ml against Gram-positive bacteria (Table 2.4). A comparison between the two series of compounds (31 and 32) indicates that in general, unsubstituted pyrazolylpyrazolines (31a-31l) showed better activity as compared to -benzenesulfonamido-substituted pyrazolylpyrazolines (32a-32l) in terms of zone of growth inhibition as well as MIC, a trend similar to that observed in t he case of anti-inflammatory activity. Within the individual series, no correlation between the antibacterial activity with respect to the substituent on phenyl ring was observed. Table 2.4. In vitro antibacterial activity and MIC of pyrazolylpyrazolines 31 and 32 Compound a Diameter of growth of inhibition zone Minimum inhibi tory (mm) b concentration (MIC) (μg/ml) S. aureus B. subtil is E. co li P. aeruginosa S. aureus B. subtilis Ciprofloxacin 27.6 26.3 25.0 25.3 5 5 31a 19.3 17.6 64 64 31b 12.3 16.3 > 256 128 31c 18.6 16.6 64 128 31d 19.6 18.6 64 64 31e 17.6 15.0 128 128 31f 19.6 16.0 64 128 31g 16.3 19.3 128 64 31h 16.3 18.6 128 64 31i 14.3 16.3 256 128 31j 16.6 15.3 128 128 31k 19.6 16.3 64 128 31l 16.3 16.0 128 128 32a 16.0 16.6 128 128 32b 16.3 15.6 128 128 32c 16.0 128 32d 15.6 15.3 128 128 32e 15.6 128 32f 16.0 19.6 128 32g 16.3 128 32h 18.6 19.3 64 64 32i 15.6 19.3 128 64 32j 14.0 256 32k 16.3 128 78

32l 16.3 15.6 128 128 a Concentration 4.0 mg/ml. b Values, including diameter of the well (8 mm), are means of three replicates. o activity. 2.5.3.2.3. In vitro antifungal activity The antifungal activity of all targeted pyrazolylpyrazolines (31a-31l and 32a- 32l) was evaluated in comparison with the standard drug fluconazole (75.3% & 74.6% against A. niger and A. flavus respectively). It was noted that while none of the compounds showed activity comparable to the standard drug fluconazole, all the compounds displayed moderate to good activity in the range 59% 44% against both the fungi. otably, none of the compounds was devoid of antifungal activity (Scheme 2.5). ere, no correlation was observed between the two series (31 and 32) as well as within individual series. Table 2.5. In vitro antifungal activity of pyrazolylpyrazolines 31 and 32 through poisoned food method Compound a Mycelial growth inhibiti on (%) A. nige r A. flavus Fluconazole 75.3 74.6 31a 52.5 53.3 31b 50.0 51.1 31c 45.5 44.4 31d 47.7 51.1 31e 52.5 51.1 31f 58.8 62.5 31g 50.0 47.7 31h 51.1 50.0 31i 51.1 48.8 31j 50.0 51.1 31k 55.5 58.8 31l 48.8 50.0 32a 56.6 52.5 32b 52.2 50.0 32c 48.8 50.0 32d 57.7 58.8 32e 55.5 58.8 79

Pyrazole derivatives: Synthesis and biological evaluation 32f 48.8 52.5 32g 48.8 51.1 32h 44.4 48.8 32i 45.5 48.8 32j 50.0 52.5 32k 44.4 44.4 32l 44.4 45.5 a Concentration 4.0 mg/ml 2.5.3.3. Thiazolylhydrazinomethylidenepyrazoles (48a-48o) 2.5.3.3.1. In vivo anti-inflammatory activity Increa se in the volume of paw and the percentage of edema inhibition for each rat a nd each group is summarized in Table 2.6. Table 2.6. In vivo anti-inflammator y activity of thiazolylhydrazinomethylidene- pyrazoles 48 a Compound Volume of ed ema (ml) and %AI c 1h 2h 3h 4h Control 0.99±0.12 1.98±0.23 1.73±0.22 1.77±0.33 Indomethacin 0. 04±0.02* 0. 19±0.03* 0. 26±0.02* 0. 17±0.03* (95.95) (90.40) (84.97) (90.39) 48a 0. 23±0.08* 1.44±0.12 1.210±0.19 1. 90±0.18 (76.76) (27.27) (30.05) (-7.34) 48b 0. 12±0.02* 1.37±0.24 1.378±0.16 1. 29±0.03 (87.87) (30.80) (20.35) (27.11) 48c 0. 74±0.2* 0. 67±0.14* 0. 79±0.16* 0. 45±0.18* (25.25) (66.16) (54.33) (74.57) 48d 0. 28±0.07* 0. 24±0.09* 0. 20±0.15* 0. 16±0.08* (71.71) (87.87) (88.43) (90.96) 48e 0. 16±0.04* 1.04±0.1* 1.09±0.32 0. 67±0.10* (83.83) (47.47) (36.99) (62.14) 48f 0. 31±0.03* 0. 39±0.05* 0. 29±0.07* 0. 15±0.04* (68.68) (80.30) (83.23) (91.52) 48g 0. 28±0.03* 0. 41±0.05* 0. 37±0.04* 0. 28±0.02* (71.71) (79.29) (78.61) (84.18) 48h 0. 21±0.07* 0. 27±0.06* 0. 32±0.07* 0. 12±0.04* (78.78) (86.36) (81.50) (93.22) 48i 0. 26±0.12* 1.03±0.18* 0. 48±0.04* 1.4±0.25 (73.73) (47.97) (72.25) (20.90) 48j 0. 30±0.04* 0. 32±0.03* 0. 28±0.08* 0. 19±0.06* (69.69) (84.84) (83.81) (89.26) 48k 0. 32±0.07* 0. 28±0.04* 1.56±0.18 1. 70±0.27 (67.67) (85.85) (9.82) (3.95) 48l 48m 0. 36±0.08 * (63.63) 0.24±0.08* (75.75) 1.55±0.16 (43) 1.03±0.23* (47.97) 1.44±0.14 (16. 76) 1.24±0.23 (28.32) 1. 46±0.13 (17.51) 1.26±0.02 (28.81) 48n 0.21±0.02* (78.78) 0.22±0.05* (88.88) 0.22±0.06* (87.28) 0.11±0.16* (95.95) 80

48o 0.98±0.1 (1.01) 1.38±0.25 (30.30) 1.414±0.10 (18.26) *Significantly different compared to respective control values, P < 0.01. a Dose levels: test compounds (50 mg/kg body wt.), Indomethacin (10 mg/kg body wt.). b Values are expressed as mean ± SEM (no. of animals = 6) and analyzed by AVA. c Values in parentheses (percentage anti-inflammatory activity, AI%). 1.19±0.22 (32.76) Amongst fifteen compounds (48a-48o) tested, four compounds (48d, 48f, 48h and 48n) possessed AI activity surpassing that of the standard drug indomethacin 4h after carrageenan injection while two other compounds 48g and 48j possessed excellent AI activity comparable to the standard drug. Compound 48c showed moderate AI activity with 54% inhibition after 3h but good activity with 74% inhibition after 4h. It suggests that these seven compounds do not get easily metabolized in the system, maintaining AI activity for a long period of time. Amongst rest of the compounds, many of them showed fair activity after 1h and 2h but failed to maintain its AI activity after 3h. A careful observation showed that the compounds containing methyl, methoxy, fluoro or chloro substituent on pyrazole displayed better AI activity as compared to unsubstituted pyrazole but no correlation could be drawn with respect to the substituent on coumarin ring. owever, a close look indicated that a combination of methoxy or chloro substituent on pyrazole ring and chloro substituent on coumarin ring exhibited pronounced AI activity. These results were nearly in accordance with our earlier observations 117,118 that the compounds with methoxy and/or chloro substituents show higher activity. When we compared the AI results of these compounds with phenyl analogues of our earlier study, 85 we found that in general, coumarin analogues containing methyl, methoxy, fluoro or chloro substituent on pyrazole (48d, 48e, 48g, 48h, 48j, 48k, 48m, 48n) displayed better AI activity as compared to phenyl analogues but loss of AI activity was found in coumarin analogues with unsubstituted pyrazole (48a, 48b). The comparison of AI results with starting materials, pyrazole-4-carbaldehyde thiosemicarbazones (50a-50e) displayed an increase in activity by construction of thiazole ring in compounds containing methyl, methoxy or fluoro substituent on pyrazole (48d, 48g, 48j) while decrease in activity in those containing no or chloro substituent on pyrazole (48a, 48m). 81

Pyrazole derivatives: Synthesis and biological evaluation 2.5.3.3.2. Docking analysis of the most active (48n) and the least active (48a)compound of thiazolylhydrazinomethylidenepyrazoles series with CX-1 & CX-2 enzymes active sites The docking experiments were carried out using the GLD docking 119 program. For docking studies, the atomic co-ordinates of CX-1 (PDB-ID: 2YE), and CX-2 (PDB-ID: 4CX) were downloaded from the Brookhaven Protein Data Bank (www.rcsb.org) and prepared using Discovery Studio 2.1(DS2.1) software 120 package where bond orders were assigned, water and other residues except bound ligands were deleted and finally, the protein was backbone constrained minimized using Charmm force field implemented in DS2.1. The bound conformation of cocrystallized ligands were used as controls in order to define the active site in CX-1 and CX-2 respectively, and optimized 3D-structures of new ligands were docked within 10 Å radius by running 20 genetic algorithm (GA) steps for each. The docked poses of geometry optimized ligands were ranked using the GoldScore (GS), to find the most optimal binding pose of each ligand. In the GLD program, the default parameters of population size (100); selection-pressure (1.1); number of operations (10,000); number of islands (1); niche size (2); and operator weights for migrate (0), mutate (100) and crossover (100) were applied. Finally, the top five binding poses of each ligand were analyzed to select the best binding pose of each ligand in order to untangle the essential parameters in terms of direct- (-bonds) and indirect (hydrophobic) interactions governing binding disparities among the series of these compounds. 2.5.3.3.2.1. Docking studies Although, only crystallographic data can fully clarify the binding mode of chemical compounds, the docking studies can be utilized to gain an insight about the structure activity relationships and to elucidate the essential structural requirements for molecules acting on the same receptor/enzyme. 121 Molecular docking studies were performed using the automated GLD docking program 119 on the most active (48n) and the least active (48a) compound of the series in the anti-inflammatory studies along with the control drug indomethacin to gain an insight about the probable nature of their binding at the CX-1 and CX-2 active site using crystal structure data obtained from the RCSB Protein Data Bank (PDB). 82

2.5.3.3.2..2. Docking to CX-2 active site Initially indomethacin was docked at the active site of the CX-2 protein cothe correct binding crystallized with the indomethacin (PDB ID: 4CX) 1222 to ensure mode prediction of the docking program. The comparative binding site analysis of CX-2 protein ligand docked complex and CX-2 X-ray crystal structure revealed that there is no significant change in the binding mode of indomethacin both in terms of indomethacin conformation or in the binding amino acid residues, thus suggesting the correct binding prediction of docking program (Figure 2.1). Figure 2. 1. Comparison of conformation for ndomethacin in the co-crystallized (pink colored carbon) and docked (cyan colored carbon) binding pose of CX-2 The binding site analysis of indomethacin showed that it penetrates deep into the cycloxygenase active site (Figure 2.2A). The benzoyl oxygen interacts with Ser530 amino acid residue whichh is the acetylation site of aspirin in both CX-1 and CX-2. 123 The carboxylate group of indomethacin is positioned towards the mouth of the active site near Arg120 and Tyr355 and the methoxy group of indole points towards the secondary pocket of the CX-2 active site. Additional close contacts with Ser353, Ala527 and Val523 were also seen (Figure 2.2A). The binding site analysis of the most active compound 48n showed that the benzenesulfonamide group is positioned in the vicinity of secondary pocket of CX-2 isozyme, surrounded by is90, Tyr355, Arg513, Ser353, Gln192 and Leu352 (Figure 2.2A and 2.2B). This secondary pocket is the major difference in the active site of CX-1 & CX-2 isoforms as similar pocket is found in CX-1 but is inaccessible due to the presence of bulkier Ile523 in CX-1 whichh is Val523 in CX-2. 122,124 The two oxygens of the S 2 2 group are inserted into the secondary pocket region, showing close contact 83