Bioorganic & Medicinal Chemistry Letters 25 (2015) 3208–3212

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Sulfonamide bearing pyrazolylpyrazolines as potent inhibitors of carbonic anhydrase isoforms I, II, IX and XII Poonam Khloya a, Mariangela Ceruso b, Sita Ram a, Claudiu T. Supuran b,⇑, Pawan K. Sharma a,⇑ a

Department of Chemistry, Kurukshetra University, Kurukshetra 136119, India Università degli Studi di Firenze, Laboratorio di Chimica Bioinorganica, Rm 188, and Neurofarba Department, Sezione di Scienze Farmaceutiche, Via U. Schiff 6, I-50019 Sesto Fiorentino (Firenze), Italy b

a r t i c l e

i n f o

Article history: Received 23 January 2015 Revised 14 May 2015 Accepted 28 May 2015 Available online 14 June 2015 Keywords: Pyrazoles Carbonic anhydrase isoforms I, II, IX, XII Acetazolamide

a b s t r a c t A series of pyrazolylpyrazolines was designed, synthesized and evaluated for carbonic anhydrase (CA, EC 4.2.1.1) inhibitory activity against cytosolic human (h) isozymes hCA I and hCA II as well as transmembrane tumor associated isozymes, hCA IX and hCA XII. All the tested compounds exhibited an excellent CA activity profile against hCA I, hCA II and hCA XII when compared to the reference drug acetazolamide (AZA). Compounds 6d, 6f and 7a–7f have exhibited better inhibition profile against hCA XII (Ki = 0.47– 5.1 nM) as compared with AZA (Ki = 5.7 nM) especially, compounds 6a, 7a, 7c and 7d which were nearly 10-fold better than reference drug. Against hCA II, all of the tested compounds were better than the standard drug especially compounds 6c, 6d, 7c and 7d (Ki = 1.1–1.7 nM) were many fold better inhibitors than AZA (Ki = 12.1 nM). In addition, they acted as selective CA inhibitors of isoform hCA XII over the physiological isoform hCA I. Ó 2015 Elsevier Ltd. All rights reserved.

Cancer indeed is one of the major health issues bothering humankind. Although significant advances are increasingly being made in fight against cancer, development of new effective therapeutic approaches is still highly desirable. The ongoing research efforts in this field to diversify therapeutic interventions have led biologists and chemists to focus their attention toward the drugs which could target specific pathways of crucial importance in the growth and development of tumors.1 In the past few years, several new tumor cell targets have been identified which led to the emergence of carbonic anhydrase isozymes as promising druggable target. Carbonic anhydrases (CAs, EC 4.2.1.1) belong to the family of zinc metalloenzymes found in a diversity of organisms and primarily responsible for catalyzing simple but fundamental reaction, CO2 hydration into bicarbonate and protons. Different CA isoforms play vital roles in various crucial physiological processes such as respiration, calcification, acid balance, bone respiration etc.2 CA isoenzymes are among the most studied proteins that find applications as therapeutic agents such as antiglaucoma, antiobesity, antidiuretic, antiepileptic, antialtitude sickness, antipain, antiinfective, and antitumor agents or as diagnostic tools.3–7

⇑ Corresponding authors. Tel./fax: +39 055 4573005 (C.T.S.); tel.: +91 9416457355; fax: +91 1744 238277 (P.K.S.). E-mail addresses: claudiu.supuran@unifi.it (C.T. Supuran), [email protected] (P.K. Sharma). http://dx.doi.org/10.1016/j.bmcl.2015.05.096 0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.

Amongst 16 CA isoforms belonging to the a-class, hCA I and hCA II have been recognized as cytosolic isozymes while hCA IX and hCA XII are identified as tumor-associated, transmembrane carbonic anhydrase isozymes.8 hCA IX and hCA XII isoforms are mainly involved in the regulation of pH dynamics in solid tumors. Therefore, search for new heterocyclic lead compounds which could selectively inhibit the tumor associated hCA IX and hCA XII is increasingly gaining the attention of researchers with a view to develop new anticancer therapies, with such a sulfonamide based inhibitor, SLC-0111 entering in Phase I Clinical Trials this year.9,10 Aromatic or heterocyclic compounds containing primary sulfonamide group have been extensively studied as important scaffolds for the development of new carbonic anhydrase inhibitors (CAIs). Some members of this interesting class of sulfonamide derivatives such as acetazolamide (AZA), methazolamide (MZA), ethoxzolamide (EZA), pazopanib etc. are widely used CAIs as clinical drugs (Fig. 1). Literature survey indicates that pyrazole scaffold has been an important pharmacophore and privileged nucleus present in various novel CA inhibitors (1–4)11–14 as shown in Figure 1. Recently, we have reported 4-functionalized pyrazoles (5) as potent inhibitors of the tumor associated CA isozymes hCA IX and hCA XII over off-target cytosolic isozymes hCA I and hCA II.15 This promoted us to enhance the repertoire of 4-functionalized pyrazoles for evaluation as novel CAIs. Encouraged by the low nanomolar potencies of 4-functionalized pyrazoles15 as CAIs and as a part of our ongoing research

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O

O HN

N S

N N

N S

O N

N S

SO2NH2

SO2NH2 AZA

MZA

EZA

H2NO2S H2N

N

O CF3 N H

N

HO

OH

O

O

N

HN

H2N

1

HO

Ph

O N

R

Pazopanib

Celecoxib

N

N N

N

H3C

Ph

H2NO2S

N N

O

S

N N

SO2NH2

O N

N N

HN

R

NC

SO2NH2 2

OH

SO2NH2 4

3 Figure 1. Some CA inhibitors having pyrazole nucleus.

on heterocyclic compounds,16–24 of potential biological applications, we envisaged coupling the pyrazole and the pyrazoline moieties in one molecular frame. Here we report a small library of pyrazolylpyrazolines 6a–6f and 7a–7f and their CA evaluation against a panel of selective two cytosolic isozymes (hCA I and hCA II) as well as two transmembrane isozymes (hCA IX and hCA XII) (Fig. 2).

It is pertinent to mention here that, to the best of our knowledge, we have evaluated coupled pyrazole and pyrazoline moiety in one molecular frame for the first time for CA inhibition profile. The synthetic strategy employed to obtain the target compounds 6a–6f and 7a–7f is depicted in Scheme 1. Starting pyrazolone 9 was synthesized by the condensation of ethyl acetoacetate with 4-hydrazinobezenesulfonamide

H2NO2S

N N

R

CONH2/COOMe/CONHNH2 5 H2NO2S H2NO2S N N N N Cl H2NO2S

Cl O

CH3

Non Bulky group

N N

CH3 N N

Bulky Group

R R 6

Figure 2. Pyrazolylpyrazolines (6a–6f and 7a–7f) as CA inhibitors.

7

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hydrochloride (8).25 The pyrazolone 9 was then subjected to the Vilsmeier–Haack chloroformylation reaction using DMF/POCl3 to yield the corresponding 5-chloro-3-methyl-4-formylpyrazole with protected sulfonamide group (10). It is pertinent to mention here that under the Vilsmeier–Haack reaction conditions, the sulfonamide group of the benzenesulphonamide moiety also reacts with Vilsmeier–Haack reagent and gets converted into N-[(dimethylamino)methylidine]sulfonamide group. Having 5-chloro-3methyl-4-formylpyrazole (10) with protected sulfonamide group in hand, the dimethylaminomethyl group was removed under acidic conditions to afford the corresponding 5-chloro-3-methyl4-formylpyrazole (11) with free sulfonamide group. Chalcones 12 were prepared by using Claisen–Schmidt condensation reaction26 of 5-chloro-3-methyl-4-formylpyrazole (11) with appropriate acetophenones. The final compounds, pyrazolylpyrazolines 6 were obtained by the condensation of appropriate chalcones 12 with 4-hydrazinobenzenesulfonamide hydrochloride (8) in ethanol containing catalytic amount of glacial acetic acid while compounds 7 were obtained by treating chalcones with hydrazine hydrate in acetic acid solvent.

The structures of all the newly synthesized compounds (10, 11, 12a–12f, 6a–6f, and 7a–7f) were characterized by a rigorous analysis of their IR, 1H NMR and 13C NMR spectral data. IR spectrum of compound 10 showed a strong characteristic absorption band at 1674 cm 1 corresponding to carbonyl (C@O) stretching while the 1 H NMR spectrum of compound 10 showed a characteristic singlet at d 9.92 for formyl proton and was further supported by the appearance of a characteristic signal at d 184.6 in its 13C NMR. The IR spectrum of compound 11 showed a strong absorption band at 1682 cm 1 corresponding to carbonyl stretching, and two bands in the region 1342 cm 1 and 1157 cm 1 attributed to SO2 stretching. The 1H NMR spectrum of compound 11 showed a broad singlet, exchangeable in D2O, at d 7.56 attributed to free sulfonamide protons (SO2NH2). IR and 1H NMR spectrum of compound 12 clearly indicated the deprotection of sulfonamide group. The IR spectra of chalcones 12a–12f exhibited the characteristic absorption band for C@O stretching at 1651 cm 1 while the vinyl CH@CH appeared at 1288–1296 cm 1. The signals for vinyl protons in compounds 12a–12f were found to be merged with aromatic region in 1H NMR spectra. In general, the 1H NMR spectra of target pyrazolylpyrazolines 6a–6f showed characteristic ABX pattern of

HC NO2S N

H2NO2S

SO2NH2

N N

N N

(i) NHNH2.HCl

(ii)

Cl

O

CHO 10

9

8 H2NO2S (iii)

(iv)

N N

R

N N

H2NO2S

Cl

Cl

CHO

O 12

11 R R

N N

H2NO2S

Cl

(v)

N N

O

12

N

N

Cl H2NO2S

(vi)

H2NO2S 6 R

N N Cl

H2NO2S

6,7,12 R

7

a H

N N O

b CH3

c OCH3

d F

e Cl

f Br

Scheme 1. Synthesis of pyrazolylpyrazolines 6a–6f and 7a–7f. (i) ethyl acetoacetate, reflux; (ii) POCl3/DMF, 50–60 °C, stir 5–6 h; (iii) Conc. HCl, reflux; (iv) substituted acetophenones, ethanol-water, 24 h; (v) 4-hydrazinobenzenesulfonamide 8, ethanol reflux; (vi) hydrazine hydrate, acetic acid, reflux.

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three protons of pyrazoline including two methylene protons at C4 and one methine proton at C-5 pyrazoline. In compounds 6a–6f, methine proton (C5-H) of pyrazoline resonates at d 5.56–5.66 as a doublet of doublet with coupling constants of 12.6 Hz and 6.3 Hz. One of the methylene protons (C4-H) of pyrazoline appeared as a doublet of doublet around d 4.02 with coupling constants 18.0 Hz and 12.6 Hz in compounds 6a–6f. The other methylene proton (C4-H) could be found at d 3.36 merging in DMSO solvent peak in pyrazolylpyrazolines 6a–6f and 7a–7f. The pyrazolylpyrazolines 7a–7f followed same ABX pattern as that of compounds 6a–6f. The structures of the pyrazolylpyrazolines 6a–6f and 7a– 7f were further supported by their 13C NMR spectrum which displayed a signal at d 54.0 due to C-5 pyrazoline while signal for C-4 pyrazoline could be assigned at d 40.7, merging with DMSO signal. The other protons appeared as expected in their 1H NMR and 13C NMR spectra. Mass spectra of 6a–6f and 7a–7f showed the molecular ion peak at M+1 which tallies to their molecular formula. All the newly synthesized pyrazolylpyrazolines 6a–6f and 7a– 7f were screened for their CA inhibition prolife by stopped-flow, CO2 hydrase assay against hCA I, hCA II, hCA IX and hCA XII enzymes.27 The inhibition data of tested compounds are reported in Table 1. The data from Table 1 clearly indicate that pyrazolylpyrazolines 6a–6f and 7a–7f act as strong inhibitors of isoforms hCA I, hCA II and hCA XII. The following features could be extracted from the data given in Table 1 regarding the CAs inhibitory properties of pyrazolylpyrazoline derivatives:

(3)

(4)

(5) (1) The cytosolic isoform hCA I was in general significantly inhibited by both series of pyrazolylpyrazolines 6a–6f and 7a–7f. The most effective compounds against the hCA I were 7a, 6f, 7e, 7b, and 6b arranged in a descending order from the most effective to the least one with Ki 632.5 nM. However, all the pyrazolylpyrazolines 6a–6f and 7a–7f were found to be more potent than standard drug acetazolamide (AZA) with Ki value 250 nM. (2) All the synthesized analogs in both series of pyrazolylpyrazolines displayed excellent inhibitory potential in nanomolar and subnanomolar range with Ki values 0.17–10.9 nM against the most abundant isoform hCA II. Out of all the tested compounds, four compounds namely 7a

Table 1 Carbonic anhydrase activity of pyrazolylpyrazolines 6a–6f and 7a–7f using a stoppedflow, CO2 hydrase assay Compounds

R

6a 6b 6c 6d 6e 6f 7a 7b 7c 7d 7e 7f AZA

H CH3 OCH3 F Cl Br H CH3 OCH3 F Cl Br

Ki* (nM) hCA I

hCA II

hCA IX

hCA XII

245 32.5 185 143 320 20.7 20.4 26.7 85.6 1107 23.3 149 250

10.9 0.6 1.5 1.4 3.1 5.5 0.17 0.54 1.1 1.7 0.26 0.30 12.1

71.6 70.7 52.4 44.7 32.9 44.4 63.1 67.1 56.9 6.9 30.8 67.2 25

9.4 7.1 5.9 3.0 55.7 0.47 0.58 4.0 0.54 0.57 3.9 5.1 5.7

AZA = acetazolamide, reference compound, a standard sulfonamide CAI, is also provided for comparison. * Mean from 3 different assays, errors were in the range of ±5–10% of the reported value.

(6)

(Ki = 0.17 nM), 7b (Ki = 0.54 nM), 7e (Ki = 0.26 nM) and 7f (Ki = 0.30 nM) exhibited the inhibitory effect several fold superior than AZA (Ki = 12.1 nM) for hCA II while five compounds 6b, 6c, 6d, 7c and 7d were also found to be more potent than AZA exhibiting potency in nanomolar range with Ki 62 nM. All the tested compounds in both series of pyrazolylpyrazolines 6a–6f and 7a–7f weakly inhibited tumor associated isoform hCA IX except the compound 7d (R = F, Ki = 6.9 nM) when compared to the standard drug AZA (Ki = 25 nM). Tumor associated isoform hCA XII was strongly inhibited by all the synthesized pyrazolylpyrazoline analogs 6a–6f and 7a–7f, except 6e, in nano molar and subnanomolar range with Ki values 0.47–9.4 nM. Amongst all the tested compounds, three analogs in the series of N-acetyl-substituted pyrazolylpyrazolines (7a–7f) namely, 7a (R = H, Ki = 0.58 nM), 7c (R = OCH3, Ki = 0.54 nM) and 7d (R = F, Ki = 0.57 nM) displayed inhibitory potential approximately 10-fold superior than AZA (Ki = 5.7 nM). Indeed, one compound 6f (R = Br, Ki = 0.47 nM) in the series of N-benzenesulfonamide-substituted pyrazolylpyrazolines (6a–6f) was also found to be more effective exhibiting 12-fold better CA inhibition profile than the standard drug AZA. It can be concluded from the Table 1 that the trend of CA inhibitory potential against hCA XII was found to be 6f < 7c < 7d < 7a < 6d < 7e < 7b and 7f with Ki 65.1 nM from the most effective to the least effective compound. In general N-acetylsubstituted pyrazolylpyrazolines (7a–7f) fared better in inhibiting hCA XII as compared to N-benzenesulfonamide substituted pyrazolylpyrazolines (6a–6f) indicating that on a bulkier group at N-1 of pyrazoline is detrimental to inhibition potency against hCA XII. Selectivity ratio for inhibiting the tumor-associated isoforms (hCA IX and hCA XII) over the off-target cytosolic isoforms (hCA I and hCA II) by compounds 6a–6f and 7a–7f has also been presented in Table 2. It is evident that newly synthesized compounds 6a–6f and 7a–7f showed better selectivity ratio for tumor associated enzymes IX and XII with respect to hCA I (Table 2). The entire series of compounds 6a–6f and 7a–7f exhibited selectivity profile in the range of 160.4–0.32 for hCA IX and 1942.10–4.47 for hCA XII enzyme. Compound 7d being the most efficient inhibitor of both

Table 2 Selectivity ratios for the inhibition of the tumor-associated isozymes hCA IX and hCA XII over the cytosolic isozymes hCA I and hCA II for compounds Inhibitors

R

6a 6b 6c 6d 6e 6f 7a 7b 7c 7d 7e 7f AZA

H CH3 OCH3 F Cl Br H CH3 OCH3 F Cl Br

Selectivity ratio* hCA I/IX

hCA II/IX

hCA I/XII

hCA II/XII

3.42 0.45 3.53 3.19 9.72 0.46 0.32 0.39 1.50 160.40 0.75 2.21 10.0

0.15 0.01 0.02 0.03 0.09 0.12 0.01 0.01 0.02 0.24 0.01 0.01 0.4

26.06 4.47 31.35 47.66 5.74 44.04 35.17 6.67 158.51 1942.10 5.97 29.21 43.8

1.15 0.08 0.25 0.46 0.05 11.70 0.29 0.13 2.03 2.98 0.06 0.05 2.1

AZA = acetazolamide, reference compound, a standard sulfonamide CAI, is also provided for comparison. * The Ki ratios are indicative of isozyme selectivity: a weak selective inhibitor is characterized by a low ratio value.

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isoforms hCA XII and IX, was about forty-five fold more selective for hCA XII and sixteen-times more selective for hCA IX over physiological isoform hCA I. (7) A comparative study of N-benzenesulfonamide-substituted pyrazolylpyrazolines 6a–6f and N-acetyl-substituted pyrazolylpyrazolines 7a–7f revealed that in general the Nacetyl-substituted pyrazolylpyrazolines 7a–7f exhibited better inhibitory potency against hCA IX, XII and II when compared to the N-benzenesulfonamide-substituted pyrazolylpyrazolines (6a–6f). In the present report, twelve novel pyrazolylpyrazolines 6a–6f and 7a–7f were synthesized and screened for inhibition profile against four human carbonic anhydrase isoforms. Most of the tested compounds displayed excellent inhibitory potency in nanomolar range against hCA I, hCA II and hCA XII when compared to the reference drug acetazolamide. Some of the tested compounds in series of pyrazolylpyrazolines 6a–6f and 7a–7f namely, 6d, 6f, 7a, 7b, 7c, 7d, 7e, and 7f exhibited low nanomolar Ki values 65 nM for hCA XII while nine compounds, 6b, 6c, 6d, 7a, 7b, 7c, 7d, 7e and 7f were found to be more potent exhibiting Ki values 62 nM against hCA II. In addition, one compound 7d was more selective for tumor associated isoforms hCA IX and XII over cytosolic isoform hCA I. From the results it can be concluded that pyrazolylpyrazolines scaffold deserves to be investigated further as a novel scaffold for CAIs. Acknowledgements One of the authors (Poonam Khloya) is grateful to the Haryana State Council for Science and Technology (HSCST), Panchkula (Haryana), India and the other (Sita Ram) to the Council of Scientific and Industrial Research, New Delhi, India for the award of Senior Research Fellowships. The authors are thankful to Sophisticated Analytical Instrument Facility, Central Drug Research Institute, Lucknow for providing Mass spectra. Work from CTS lab was financed by an EU project of the FP7 programme (Metoxia). Supplementary data Supplementary data (biological assay, experimental procedure and spectroscopic characterization of compounds 6a–6f and 7a– 7f) associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.2015.05.096. References 1. Cairns, R.; Papandreou, I.; Denko, N. Mol. Cancer Res. 2006, 4, 61.

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