Bioorganic & Medicinal Chemistry 21 (2013) 1396–1403

Contents lists available at SciVerse ScienceDirect

Bioorganic & Medicinal Chemistry

journal homepage: www.elsevier .com/locate /bmc

Carbonic anhydrase inhibitors: Benzenesulfonamides incorporatingcyanoacrylamide moieties are low nanomolar/subnanomolarinhibitors of the tumor-associated isoforms IX and XII

0968-0896/$ - see front matter � 2012 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.bmc.2012.12.004

⇑ Corresponding authors. Tel.: + 966 50 706 9896 (A.M.A.); tel.: +39 055 4573005; fax: +39 055 4573385 (C.T.S.).

E-mail addresses: alafeefy@hotmail.com (A.M. Alafeefy), claudiu.supuran@unifi.it (C.T. Supuran).

Ahmed M. Alafeefy a,⇑, Semra Isik b, Hatem A. Abdel-Aziz c,g, Abdelkader E. Ashour d, Daniela Vullo b,Nabila A. Al-Jaber e, Claudiu T. Supuran b,f,⇑a Department of Pharmaceutical Chemistry, College of Pharmacy, Salman Bin Abdulaziz University, PO Box 173, Alkharj 11942, Saudi Arabiab Università degli Studi di Firenze, Polo Scientifico, Laboratorio di Chimica Bioinorganica, Rm. 188, Via della Lastruccia 3, 50019 Sesto Fiorentino (Florence), Italyc Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, PO Box 2457, Riyadh 11451, Saudi Arabiad Department of Pharmacology and Toxicology, College of Pharmacy, King Saud University, PO Box 2457, Riyadh 11451, Saudi Arabiae Women Students-Medical Studies and Sciences Sections, Chemistry Department, College of Science, King Saud University, PO Box 22452, Riyadh 11495, Saudi Arabiaf Università degli Studi di Firenze, Polo Scientifico, Dipartimento di Scienze Farmaceutiche, 50019 Sesto Fiorentino (Florence), Italyg Department of Applied Organic Chemistry, National Research Centre, Dokki, Cairo 12622, Egypt

a r t i c l e i n f o

Article history:Available online 2 January 2013

Keywords:SulfonamideCarbonic anhydraseCyanoacrylamideIsoform-selective inhibitor

a b s t r a c t

A series of benzenesulfonamides incorporating cyanoacrylamide moieties (tyrphostine analogues)have been obtained by reaction of sulfanilamide with ethylcyanoacetate followed by condensationwith aromatic/heterocyclic aldehydes, isothiocyanates or diazonium salts. The new compounds havebeen investigated as inhibitors of the metalloenzyme carbonic anhydrase (CA, EC 4. 2.1.1), and morespecifically against the cytosolic human (h) isoforms hCA I and II, as well as the transmembrane,tumor-associated ones CA IX and XII, which are validated antitumor targets. Most of the new ben-zenesulfonamides were low nanomolar or subnanomolar CA IX/XII inhibitors whereas they were lesseffective as inhibitors of CA I and II. The structure–activity relationship for this class of effective CAinhibitors is also discussed. Generally, electron donating groups in the starting aldehyde reagentfavored CA IX and XII inhibition, whereas halogeno, methoxy and dimethylamino moieties led to verypotent CA XII inhibitors.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

Unsubstituted aromatic sulfonamides were known to inhibitthe metaloenzyme carbonic anhydrase (CA, EC 4.2.1.1) since thebeginning of research in this field, in the 40s.1 Starting with the1950-s, potent CA inhibitors (CAIs) belonging to the heterocyclicsulfonamide class (such as acetazolamide AAZ) have been devel-oped that led to the benzothiadiazine and high-ceiling diuretics(hydrochlorothiazide HCT, furosemide FUR, etc.), widely useddrugs even nowadays, as well as to the systemic antiglaucomadrugs (acetazolamide AAZ, methazolamide MZA, ethoxzolamideEZA and dichlorophenamide DCP, to mention only the first gener-ation such compounds).2–5

HN

NNSH2NO

O SO

AAZ

NSSH2NO

O N N

O

MZA

S NH

HN

SO

O

H2N OO

Cl

HCT

S

NSO

ONH2

OEZA

O

H2NCl Cl

DCP

OHO

SO

H2NO

Cl

NH O

FUR

Mann and Keilin reported already in 1940 that sulfanilamide SAacts as a potent and specific CAI,1 and this prompted much re-search in the field of designing such inhibitors based on the ben-zenesulfonamide scaffold.6 However, most of the first generationdrugs mentioned above, as well as the second generationsulfonamide CAIs, dorzolamide DZA and brinzolamide BRZ, wereheterocyclic sulfonamides. The interest in aromatic, benzenesul-fonamide-based compounds has reemerged only recently, by our

A. M. Alafeefy et al. / Bioorg. Med. Chem. 21 (2013) 1396–1403 1397

report that 1,3,5-triazinyl-substituted benzenesulfonamides act ashighly potent, and isoform-selective CAIs targeting two of the mostinvestigated mammalian CAs,7 that is, the tumor-associated iso-forms CA IX and XII, which have been shown to be anticancer drugtargets.8–12

S SS

O O

OO

NH2

HN C2H5DZA

N S SS

O O

OO

NH2

HN C2H5BRZ

H3CO

Indeed, some ureido-substituted benzenesulfonamides devel-oped in one of our laboratories using the 1,3,5-triazinyl derivatives

mentioned above as lead molecules, were then used in the proof-of-concept study to show that CA IX/XII inhibition has profoundantitumor effects in vivo, both for the inhibition of the growth ofprimary tumors and metastases.13–15 Some of these derivatives(as well as the corresponding sulfamates) are being evaluated inpreclinical models as potentially new classes of anticancer drugs/diagnostic tools for imaging hypoxic tumors.8a

Based on this interesting recent data, we report here a new classof highly effective CAIs incorporating the classical benzenesulfon-amide scaffold as well as cyanoacrylamide moieties substitutedwith aromatic groups, a scaffold never considered earlier fordesigning CAIs. The new class of CAIs described here showed excel-lent activity against the tumor-associated human (h) isoforms hCAIX and XII, as well as some selectivity for inhibiting the transmem-brane isoforms versus the cytosolic, offtarget ones CA I and II.

2. Chemistry

The key intermediate in the preparation of the new CAIs reportedhere was 2-cyano-N-(4-sulfamoylphenyl)acetamide (1), which wasprepared by the reaction of sulfanilamide with ethyl cyanoacetateaccording to the reported literature procedure.16 The cyanoacetylderivative 1 was then reacted with a series of aromatic/heterocyclicaldehydes in refluxing ethanol, in the presence of a catalytic amountof piperidine, to afford the corresponding acrylamides 2–18 and thechromene 19, respectively (Scheme 1). The rationale behind thedrug design reported here resides in the fact that it has been shownearlier2–5 by means of X-ray crystallography of adducts of variousCA isoforms with sulfonamide inhibitors, that the most importantcontribution for the isoform selectivity is assured by the ‘tail’of the inhibitor (Fig. 1), which can participate in favorable/non-favorable interactions with amino acid residues towards the middlepart of the active site or towards its edge.

Thus, we have explored here scaffolds/tails not investigated ear-lier as CAIs, which incorporate the classical benzenesulfonamidegroup which will bind to the Zn(II) ion from the enzyme active site,to which the rigid substituted acrylamido scaffold has been at-tached by exploiting the condensation reaction of the active meth-ylene (NC–CH2–CONH–) from compound 1 with aromatic/heterocyclic aldehydes. In this way a rather high degree of chemi-cal diversity may be achieved easily, and this type of scaffold hasnot been investigated earlier in the drug design of CAIs.2–5

The new compounds 2–18 were characterized by routine spec-tral data which confirmed their structures (see Section 5 for de-tails). For example, the IR spectra of compounds 2–18 showedthe appearance of NH and NH2 absorption band in the region of3500–3250 cm�1 and the band of nitrile function around2225 cm�1 in addition to the carbonyl band at 1700–1650 cm�1.In their 1H NMR spectra the @CH proton appeared in the regionof 8.15–8.20 ppm in addition to the D2O exchangeable signals ofNH and NH2 (at around 10.50 and 7.50 ppm), respectively.

On the other hand, compound 1 couples readily with diazotizedaniline in ethanol, in the presence of sodium acetate at 0–5 �C, toafford the hydrazone 20 (Scheme 2). Furthermore, derivative 1was reacted with phenyl isothiocyanate in dimethylformamide,in the presence of potassium hydroxide, yielding the thioacetani-lide 21 (Scheme 2). Treatment of the latter compound with thehydrazonoyl chloride in refluxing ethanol and in the presence ofa catalytic amount of triethylamine afforded the corresponding1,3,4-thiadiazole derivatives 22 (Scheme 2). The IR spectrum ofcompound 22 revealed the appearance of two carbonyl absorptionbands around 1685 cm�1 in addition to a nitrile absorption band ataround 2225 cm�1. The mass spectrum of compound 22 showed apeak corresponding to its molecular ion. These results indicate thatthe reaction of the thioacetanilide 3 with hydrazonyl chloride pro-ceeded, via loss of hydrogen chloride to form a non-isolable inter-mediate which undergoes cyclization by elimination of an anilinemolecule to form the 1,3,4-thiadiazole derivatives 22.17–19

3. CA inhibition

Inhibition data of compounds 1–22 reported here and acetazol-amide AAZ (as a standard drug), against the cytosolic CA isozymesh (h = human) hCA I and II, as well as the tumor-associated, targetisoforms hCA IX and XII, are shown in Table 1.20

The following structure–activity relationship (SAR) can be ob-served from data of Table 1:

(i) The cytosolic isoforms hCA I was moderately inhibited bymost sulfonamides reported here, with inhibition constantsin the range of 18.7–116 nM. Two compounds, 16 and 17,were on the other hand low nanomolar hCA I inhibitors(KIs of 5.3–6.1 nM). Both of them incorporate a five-mem-bered heterocyclic ring (furan and thiophene, respectively)in contrast to all other derivatives which are derivatives ofaromatic, substituted benzaldehydes. Thus, the smallerfive-membered ring is more favorable than the six-mem-bered one in generating potent hCA I inhibitors. On the otherhand, the quite bulky derivative 22, incorporating a highlysubstituted 1,3,4-thiadiazole with bulky (phenyl) substitu-ents in position 3, was one of the least effective hCA I inhib-itors reported here. It should be however stressed that allthese compounds are better hCA I inhibitors than the clini-cally used drug acetazolamide, AAZ, which is a weak hCA Iinihibitor (KI of 250 nM). For the compounds incorporatingsubstituted benzene rings, 2–14, the hCA I inhibition wasmodestly influenced by the substitution pattern, as most ofthem showed rather similar inhibition power, with KIs vary-ing between 25.6–71.0 nM (Table 1). The ortho-methoxygroup, present in 5, led to the least effective hCA I inhibitor,probably due to a steric hindrance caused by the orthosubstituent.

(ii) Compound 1, the starting material from which the otherderivatives were obtained already behaved as a strong hCAII inhibitor (KI of 6.1 nM). As this is an ubiquitous, house-keeping isoform, this may be not a valuable property if com-pounds targeting other isoforms (e.g., the tumor-associatedones hCA IX and XII) should be obtained. Thus, it is interest-ing to note that some of the substitution patterns present inthe new derivatives reported here led to less effective hCA IIinhibitors compared to the parent compound 1. Among suchcompounds are 2, 3, 8, 10, 13–15, and 18, which showedinhibition constants in the range of 14.3–61.0 nM. Theremaining ones were highly effective as hCA II inhibitors,with KIs varying between 2.7–10.9 nM (the same activityrange as acetazolamide AAZ, the standard drug, see Table

S

NH

O

CN

H2N

OO

S

NH

O

CN

H2N

OO

R

S

NH

OH2N

OO

O

NH

R-CHO

Cl Cl

Cl O2N

OMe

MeO MeO

MeO

MeO

MeO

MeO

OMe

MeO

OMe

OMe

MeO

MeO

HO

MeO

HO

HO

HO NMe

Me

O S

O

O

1

2-18 19

R =

2-OH-C6H4-CHO

2 3 4 5 6 7 8

9 10 11 12 13 14

15 16 17 18

Scheme 1. Preparation of compounds 2–19 by condensation of cyanoacetamido-sulfanilamide 1 with aldehydes.

O

NH

H

N

O

O

O

Zn2+

R

R'

His119His96His94

Glu106

Hydrophilicpart of active site

Hydrophobic partof active siteThr199

ZBG

SCAFFOLD

TAIL

Figure 1. Drug design strategy based on the ‘tail-approach’: the zinc-binding group(ZBG) of the inhibitor molecule directly coordinates the Zn(II) ion from the enzymeactive site (also coordinated by three His residues), being in this case a SO2NH�

moiety. The scaffold of the inhibitor is tailored in such a way as to interact bothwith the hydrophilic and the hydrophobic halves of the active site. The tails (one ormore of them; two are shown in this schematic representation) of the cyano-acrylamide type have never been investigated up until now for the design of CAIs.

1398 A. M. Alafeefy et al. / Bioorg. Med. Chem. 21 (2013) 1396–1403

1). It is thus clear that small modifications in the scaffold ofthe aldehyde from which the CAIs were obtained, leads toimportant differences in the hCA II inhibitory potency (e.g.,compare the regiomers 7, 8 and 9, which differ in activityby a factor of 6, comparing the most active with the leastactive one).

(iii) The tumor associated target isoform hCA IX was highly inhib-ited by most sulfonamides reported here, generally in the lownanomolar to subnanomolar range (Table 1). Thus, several

new compounds, such as 3, 5, 7 and 10, were less effectiveas hCA IX inhibitors, with KIs in the range of 21.2–35.6 nM.They incorporate 2,4-dichlorophenyl-, 2-methoxyphenyl,3,4,5-trimethoxy and 3,4-dimethoxyphenyl moieties in theirmolecules. The remaining sulfonamides reported here showedexcellent hCA IX inhibitory power, with KIs in the range of0.65–12.8 nM (being much more effective than AAZ, whichhas a KI of 25 nM). The most effective, subnanomolar hCA IXinhibitors (compounds 6, 8, 12 and 13) incorporated the fol-lowing substitution patterns: 4-methoxyphenyl-, 2,4,5-trime-thoxyphenyl-, 3,4-dihydroxyphenyl- and 4-hydroxyphenyl. Itmay be observed again (as for hCA II, discussed above) that forsome of the most effective and the most ineffective hCA IXinhibitors there are only small structural differences betweenthe scaffolds, (residing in the nature of groups substituting themoiety originating from the starting aldehyde reagent). Thus,the fine-tuning of the inhibitory activity is dependent of verysmall structural changes in the scaffold of these sulfonamidederivatives.

(iv) The second tumor-associated isoform, hCA XII, was highlyinhibited by the compounds investigated here, with mostof them being subnanomolar (derivatives 1–9, 13, 14 and22) or low nanomolar (compounds 10–12, 15–18 and 20)CAIs. The less effective hCA XII inhibitors were 19 and 21(KIs of 11.7–24.3 nM) which were anyhow quite effectiveCAIs. Thus, a rather large number of substitution patternsfrom these derivatives lead to highly effective hCA XII inhib-itors. Further studies (X-ray crystallography) are currentlybeing done in order to clarify the exact mechanism of actionand structure–activity correlation for this class of effectivesulfonamide CAIs.

S

NH

OCN

H2N

OO

S

NH

OCN

H2N

OO

NNH

S

NH

OCN

H2N

OO

SH

Ph-N2Cl 1- Ph-NCS

HNPh

S

NH

OCN

H2N

OO

SNN Ph

Ph

MeO

Me

ON NHCl

Ph

2021

1

22

2- HCl

Scheme 2. Preparation of compounds 20–22.

Table 1Inhibition data of the newly synthesized compounds 1–22 against isoforms hCA I, II,IX and XII, by a stopped-flow CO2 hydrase assay20

Compound KIa (nM)

hCA I hCA II hCA IX hCA XII

1 52.4 6.1 5.2 0.672 41.3 61.0 8.9 0.863 47.1 32.9 35.6 0.724 40.0 5.4 0.93 0.665 71.0 6.9 32.6 0.606 39.6 9.0 0.86 0.577 38.8 7.5 20.5 0.598 42.4 38.2 0.96 0.679 25.6 6.7 7.8 0.53

10 31.3 18.6 21.2 2.311 32.9 6.0 5.4 5.212 52.4 4.2 0.65 2.313 58.9 33.8 0.67 0.6514 47.3 19.4 3.4 0.9315 18.7 31.3 4.9 2.416 6.1 5.5 4.3 6.317 5.3 2.7 3.7 6.418 24.7 14.3 9.5 8.919 32.9 10.9 12.8 24.320 34.0 8.5 9.5 3.521 67.6 6.5 5.8 11.722 116 7.9 0.33 0.64AAZ 250 12 25 5.7

Inhibition with acetazolamide AAZ, a standard sulfonamide CAI, is also provided forcomparison.

a Mean from 3 different assays. Errors were in the range of ±10% of the reportedvalues (data not shown).

Table 2Selectivity ratios for the inhibition of hCA IX over hCA II and hCA XII over hCA II forthe compounds 1–22 reported in the paper (and acetazolamide AAZ as standard drug)

Compound Selectivity ratio

hCA IX/hCA II hCA XII/hCA II

1 1.17 9.102 6.85 70.93 0.92 45.74 5.80 8.185 0.21 11.56 10.4 15.87 0.36 12.78 39.8 57.09 0.85 12.6

10 0.87 8.0811 1.11 1.1512 6.46 1.8213 50.4 52.014 5.70 20.815 6.38 13.016 1.28 0.8717 0.73 0.4218 1.50 1.6119 0.85 0.4420 0.89 2.4221 1.12 0.5522 23.9 12.3AAZ 0.48 2.10

A. M. Alafeefy et al. / Bioorg. Med. Chem. 21 (2013) 1396–1403 1399

(v) Selectivity for inhibiting the tumor-associated isoforms (hCAIX and XII) over the widespread cytosolic ones (hCA I and II) isa critical issue when designing CAIs.2,3 As seen from data ofTable 2, where these ratios are provided, many of the com-pounds reported here showed relatively strong inhibitoryproperties against all four CAs investigated (e.g., compounds16 and 17 showed highly similar inhibitory propertiesagainst hCA I, II, IX and XII, with KIs in the range of 2.7–6.4 nM, Tables 1 and 2). This is not highly desirable whenonly the tumor-associated isoforms would be targeted. How-ever, some of the compounds reported here do show indeedsuch an inhibition profile. For example 6 has a selectivityratio for inhibiting hCA IX over hCA II of 10.5, and forinhibiting hCA IX over hCA I of 46. These parameters are even

better for the selective inhibition of hCA XII over hCA I and II(hCA XII/II selectivity ratio of 15.8). Compound 13 is evenmore selective (than 6) as an inhibitor of the tumor-ssociatedover the cytosolic isoforms, with a selectivity ratio for inhib-iting hCA IX over hCA II of 50.4, and for inhibiting hCA IX overhCA I of 87.9. The same parameters for inhibiting hCA XII overhCA II are of 52.0, and for inhibiting hCA XII over hCA I of 90.6,respectively. Thus, the new class of sulfonamides reportedhere may indeed lead to isoform-selective CAIs targetingthe tumor-associated CAs.

4. Conclusions

We report a series of benzenesulfonamides incorporatingcyanoacrylamide moieties (tyrphostine analogues), which havebeen obtained by reaction of sulfanilamide with ethylcyanoacetate,

1400 A. M. Alafeefy et al. / Bioorg. Med. Chem. 21 (2013) 1396–1403

followed by condensation of the active methylene from the cyano-acetamide group with aromatic/heterocyclic aldehydes, diazoniumsalts or isothiocyanates. Most of the new benzenesulfonamides ob-tained and evaluated in this study were low nanomolar or subn-anomolar CA IX/XII inhibitors whereas they were less effective asinhibitors of CA I and II, but substantially inhibited also those iso-forms. Some CA IX/XII-selective inhibitors were also detected inthis study. Further studies should be done to explore in detailthe exact mechanism of action and to broaden the structure–activ-ity relationship for this interesting class of sulfonamide CAIs.

5. Experimental protocols

5.1. General

Melting points (�C, uncorrected) were determined in open cap-illaries on a Gallenkamp melting point apparatus (Sanyo Gallenk-amp, Southborough, UK) and were uncorrected. Precoated silicagel plates (silica gel 0.25 mm, 60G F254; Merck, Germany) wereused for thin layer chromatography, dichloromethane/methanol(9.5:0.5) mixture was used as a developing solvent system andthe spots were visualized by ultraviolet light and/or iodine. Infrared spectra were recorded in KBr discs using IR-470 Shimadzuspectrometer (Shimadzu, Tokyo, Japan). 1H NMR spectra were re-corded on Bruker AC-300 Ultra Shield NMR spectrometer (Bruker,Flawil, Switzerland, d ppm) at 300 MHz for 1H and 75 MHz for13C, using TMS as internal standard and peak multiplicities are de-signed as follows: s, singlet; d, doublet; t, triplet; m, multiplet.Electron Impact Mass Spectra were recorded on a Shimadzu GC–MS-QP 5000 instrument (Shimadzu, Tokyo, Japan). Elemental anal-yses were performed, on Carlo Erba 1108 Elemental Analyzer(Heraeus, Hanau, Germany), at the Microanalytical Unit, Facultyof Science, Cairo University, Cairo, Egypt, and the found resultswere within ±0.4% of the theoretical values.

5.2. Chemistry

5.2.1. 2-Cyano-N-(4-sulfamoylphenyl)acetamide (1)This compound was prepared according to reported literature

procedures.19 It had the same physico-chemical parameters (1H,13C NMR spectra, as well as MS, as reported in Ref. 19, data notshown).

5.2.2. Synthesis of compounds 2–19To a mixture of 2-cyano-N-(4-sulfamoylphenyl)acetamide

(0.239 g, 1 mmol) and the appropriate aldehyde (1 mmol) in etha-nol (25 ml), few drops of piperidine were added. The reaction mix-ture was refluxed for 4 h, and then cooled to room temperature.The precipitate was filtered, dried and washed with ethanol. Thecrude product was recrystallized from EtOH/DMF to afford com-pounds 2–19, respectively, in 45–80% yield.

5.2.3. 3-(2-Chlorophenyl)-2-cyano-N-(4-sulfamoylphenyl)acrylamide (2)

Compound 2: Yield: 81%, mp: 221–23 �C. IR, m (cm�1): 3412,3331, 3253 (SO2NH2 and NH), 3045 (Ar-CH), 2941 (CH), 2225(CN), 1668 (C@O). 1H NMR (DMSOd6): d 7.01–7.23 (m, 4H, Ar-H),7.42 (s, 2H, SO2NH2), 7.73 (d, J = 7.5 Hz, 2H, H-3 and H-5, PhSO2),7.85 (d, J = 7.5 Hz, 2H, H-2 and H-6, PhSO2), 8.33 (s, 1H, @CH),10.46 (s, 1H, N–H, exchange). 13C NMR: d 106.50 (C-CN), 116.43(CN), 122.03, 126.90, 127.82, 128.92, 129.64, 131.41, 133.24,135.30, 139.15, 146.0, 153.42 (Ar-C), 164.34 (C@O). MS m/z (Rel.Int.): 363 (M++2, 17), 361 (M+, 5.7). Anal. (C16H12ClN3O3S) C, H, N.

5.2.4. 2-Cyano-3-(2,4-dichlorophenyl)-N-(4-sulfamoylphenyl)acrylamide (3)

Compound 3: Yield: 80%, mp: 235–37 �C. IR, m (cm�1): 3410,3328, 3257 (SO2NH2 and NH), 3049 (Ar-CH), 2944 (CH), 2222(CN), 1666 (C@O). 1H NMR (DMSOd6): d 7.01 (d, 1H, J = 6.4 Hz,Ar-H), 7.13 (d, 1H, J = 6.4 Hz, Ar-H), 7.25(s, 1H, Ar-H), 7.87 (d,J = 7.5 Hz, 2H, H-3 and H-5, PhSO2), 7.91 (d, J = 7.5 Hz, 2H, H-2and H-6, PhSO2), 8.21 (s, 2H, SO2NH2), 8.33 (s, 1H, @CH), 10.46(s, 1H, N–H, exchange). 13C NMR: d 106.50 (C-CN), 116.43 (CN),122.03, 126.90, 127.82, 128.92, 129.64, 131.41, 132.35, 133.24,135.30, 139.15, 153.42 (Ar-C), 164.34 (C@O). MS m/z (Rel. Int.):398 (M++2, 26), 396 (M+, 8.5). Anal. (C16H11Cl2N3O3S) C, H, N.

5.2.5. 2-Cyano-3-(4-nitrophenyl)-N-(4-sulfamoylphenyl)acrylamide (4)

Compound 4: Yield: 49%, mp: 218–220 �C. IR, m (cm�1): 3415,3324, 3257 (SO2NH2 and NH), 3075 (Ar-CH), 2981 (CH), 2230(CN), 1684 (C@O). 1H NMR (DMSOd6): d 7.59 (d, J = 7.5 Hz, 2H, H-2 and H-6, PhNO2), 7.66 (s, 2H, SO2NH2), 7.88 (d, J = 7.6 Hz, 2H,H-3 and H-5, PhSO2), 7.92 (d, J = 7.5 Hz, 2H, H-2 and H-6, PhSO2),8.12 (d, J = 8.0 Hz, 2H, H-3 and H-5, PhNO2), 8.31 (s, 1H, @CH),10.27 (s, 1H, N–H, exchange). 13C NMR: d 106.3 (C-CN), 115.5,116.4 (CN), 121.4, 127.5, 127.64, 129.3, 135.7, 139.5, 153.8, 158.4(Ar-C), 164.6 (C@O). MS m/z (Rel. Int.): 374 (M++2, 20). Anal.(C16H12N4O5S) C, H, N.

5.2.6. 2-Cyano-3-(2-methoxyphenyl)-N-(4-sulfamoylphenyl)acrylamide (5)

Compound 5: Yield: 52%, mp: 208–10 �C. IR, m (cm�1): 3412,3331, 3253 (SO2NH2 and NH), 3067 (Ar-CH), 2984 (CH), 2257(CN), 1680 (C@O). 1H NMR (DMSOd6): d 3.73 (s, 3H, OCH3), 7.05–7.22 (m, 4H, Ar-H), 7.45 (s, 2H, SO2NH2), 7.77 (d, J = 7.5 Hz, 2H,H-3 and H-5, PhSO2), 7.92 (d, J = 7.5 Hz, 2H, H-2 and H-6, PhSO2),8.35 (s, 1H, @CH), 10.40 (s, 1H, N–H, exchange). 13C NMR: d 56.3(OCH3), 106.5 (C-CN), 114.7, 115.4, 116.7 (CN), 120.8, 121.6,127.5, 129.2, 135.7, 139.5, 153.9, 157.5 (Ar-C), 164.0 (C@O). MSm/z (Rel. Int.): 357 (M+, 36). Anal. (C17H15N3O4S) C, H, N.

5.2.7. 2-Cyano-3-(4-methoxyphenyl)-N-(4-sulfamoylphenyl)acrylamide (6)

Compound 6: Yield: 47%, mp: 215–17 �C. IR, m (cm�1): 3414,3329, 3254 (SO2NH2 and NH), 3068 (Ar-CH), 2987 (CH), 2231(CN), 1683 (C@O). 1H NMR (DMSOd6): d 3.74 (s, 3H, OCH3), 7.12(d, J = 7.5 Hz, 2H, H-2 and H-6, PhOCH3), 7.41 (s, 2H, SO2NH2),7.68 (d, J = 7.6 Hz, 2H, H-3 and H-5, PhSO2), 7.79 (d, J = 7.5 Hz,2H, H-2 and H-6, PhSO2), 8.02 (d, J = 8.0 Hz, 2H, H-3 and H-5,PhOCH3), 8.31 (s, 1H, @CH), 10.46 (s, 1H, N–H, exchange). 13CNMR: d 56.3 (OCH3), 106.7 (C-CN), 115.2, 116.5 (CN), 121.3,127.6, 129.3, 135.7, 139.5, 153.9, 158.5 (Ar-C), 164.3 (C@O). MSm/z (Rel. Int.): 357 (M+, 36). Anal. (C17H15N3O4S) C, H, N.

5.2.8. 2-Cyano-N-(4-sulfamoylphenyl)-3-(3,4,5-trimethoxyphenyl)acrylamide (7)

Compound 7: Yield: 50%, mp: 225–27 �C. IR, m (cm�1): 3398,3324, 3250 (SO2NH2 and NH), 3073 (Ar-CH), 2955 (CH), 2233(CN), 1689 (C@O). 1H NMR (DMSOd6): d 3.74 (s, 9H, 3OCH3), 6.27(s, 2H, H-2 and H-6, Ar-H), 7.41 (s, 2H, SO2NH2), 7.82 (d,J = 8.0 Hz, 2H, H-3 and H-5, HNPhSO2), 7.90 (d, J = 7.6 Hz, 2H, H-2and H-6, HNPhSO2), 8.32 (s, 1H, @CH), 10.48 (s, 1H, N–H, ex-change). 13C NMR: d 56.5 (2OCH3), 56.6 (OCH3), 102.9, 106.7 (C-CN), 111.4, 113.2, 116.2 (CN), 121.4, 127.3, 135.6, 139.8, 143.5,150.2, 151.6, 153.9 (Ar-C), 165.0 (C@O). MS m/z (Rel. Int.): 417(M+, 37). Anal. (C19H19N3O6S) C, H, N.

A. M. Alafeefy et al. / Bioorg. Med. Chem. 21 (2013) 1396–1403 1401

5.2.9. 2-Cyano-N-(4-sulfamoylphenyl)-3-(2,4,5-trimethoxyphenyl)acrylamide (8)

Compound 8: Yield: 46%, mp: 226–28 �C. IR, m (cm�1): 3414,3330, 3253 (SO2NH2 and NH), 3075 (Ar-CH), 2956 (CH), 2236(CN), 1685 (C@O). 1H NMR (DMSOd6): d 3.74 (s, 3H, OCH3), 3.80(s, 3H, OCH3), 3.89 (s, 3H, OCH3), 6.19 (s, 1H, Ar-H), 6.60 (s, 1H,Ar-H), 7.41 (s, 2H, SO2NH2), 7.76 (d, J = 8.0 Hz, 2H, H-3 and H-5,HNPhSO2), 7.89 (d, J = 7.6 Hz, 2H, H-2 and H-6, HNPhSO2), 8.32 (s,1H, @CH), 10.48 (s, 1H, N–H, exchange). 13C NMR: d 56.33(2OCH3), 56.65 (OCH3), 101.90, 106.37 (C-CN), 109.14, 113.20,116.23 (CN), 121.40, 127.35, 135.65, 139.58, 143.52, 150.22,151.46, 153.99 (Ar-C), 165.0 (C@O). MS m/z (Rel. Int.): 417 (M+,67). Anal. (C19H19N3O6S) C, H, N.

5.2.10. 2-Cyano-N-(4-sulfamoylphenyl)-3-(2,4,6-trimethoxyphenyl)acrylamide (9)

Compound 9: Yield: 43%, mp: 226–28 �C. IR, m (cm�1): 3399,3324, 3253 (SO2NH2 and NH), 3074 (Ar-CH), 2953 (CH), 2236(CN), 1686 (C@O). 1H NMR (DMSOd6): d 3.72 (s, 9H, 3OCH3), 5.97(s, 2H, Ar-H), 7.81 (s, 2H, SO2NH2), 7.88 (d, J = 8.0 Hz, 2H, H-3 andH-5, HNPhSO2), 7.93 (d, J = 7.6 Hz, 2H, H-2 and H-6, HNPhSO2),8.33 (s, 1H, @CH), 10.27(s, 1H, N–H, exchange). 13C NMR: d 56.2(2OCH3), 56.4 (OCH3), 102.5, 106.3 (C-CN), 112.1, 113.6, 116.3(CN), 121.0, 127.4, 135.8, 139.8, 143.6, 150.5, 151.4, 154.0 (Ar-C),165.3 (C@O). MS m/z (Rel. Int.): 417 (M+, 60). Anal. (C19H19N3O6S)C, H, N.

5.2.11. 2-Cyano-3-(3,4-dimethoxyphenyl)-N-(4-sulfamoylphenyl)acrylamide (10)

Compound 10: Yield: 46%, mp: 205–07 �C. IR, m (cm�1): 3421,3342, 3255 (SO2NH2 and NH), 3075 (Ar-CH), 2953 (CH), 2243(CN), 1682 (C@O). 1H NMR (DMSOd6): d 3.81 (s, 3H, OCH3), 3.86(s, 3H, OCH3), 6.78 (d, J = 7.0 Hz, 1H, Ar-H), 6.96 (d, J = 7.0 Hz, 1H,Ar-H), 7.41 (s, 2H, SO2NH2), 7.77 (d, J = 8.5 Hz, 2H, H-3 and H-5,HNPhSO2), 7.99 (d, J = 7.5 Hz, 2H, H-2 and H-6, HNPhSO2), 8.22 (s,1H, @CH), 10.43 (s, 1H, N–H, exchange). 13C NMR: d 56.33(2OCH3), 56.45 (OCH3), 107.08 (C-CN), 112.20, 116.23 (CN),117.0, 120.14, 122.10, 127.65, 128.87, 135.95, 139.14, 148.75,149.54, 153.96 (Ar-C), 165.10 (C@O). MS m/z (Rel. Int.): 387 (M+,65). Anal. (C18H17N3O5S) C, H, N.

5.2.12. 2-Cyano-3-(4-hydroxy-3-methoxyphenyl)-N-(4-sulfamoyl-phenyl)acrylamide (11)

Compound 11: Yield: 48%, mp: 211–13 �C. IR, m (cm�1): 3429,3345, 3234 (SO2NH2 and NH), 3044 (Ar-CH), 2935 (CH), 2222(CN), 1677 (C@O). 1H NMR (DMSOd6): d 3.65 (s, 3H, OCH3), 6.46(d, J = 7.0 Hz, 1H, Ar-H), 6.60 (s, 1H, Ar-H), 6.65 (d, J = 7.0 Hz, 1H,Ar-H), 7.80 (s, 2H, SO2NH2), 7.90 (d, J = 8.5 Hz, 2H, H-3 and H-5,HNPhSO2), 7.94 (d, J = 7.5 Hz, 2H, H-2 and H-6, HNPhSO2), 8.18 (s,1H, @CH), 8.56 (s, 1H, OH, exchange.), 10.36 (s, 1H, N–H, exchange).13C NMR: d 56.5 (OCH3), 106.6 (C-CN), 114.0, 116.4 (CN), 118.1,121.1, 122.3, 127.6, 128.8, 135.8, 139.3, 146.7, 147.7, 153.6 (Ar-C), 164.2 (C@O). MS m/z (Rel. Int.): 373 (M+, 35). Anal.(C17H15N3O5S) C, H, N.

5.2.13. 2-Cyano-3-(3,4-dihydroxyphenyl)-N-(4-sulfamoylphenyl)acrylamide (12)

Compound 12: Yield: 41%, mp: 235–37 �C. IR, m (cm�1): 3428(2OH), 3348, 3239 (SO2NH2 and NH), 3046 (Ar-CH), 2939 (CH),2223 (CN), 1677 (C@O). 1H NMR (DMSOd6): d 6.58 (d, J = 7.0 Hz,1H, Ar-H), 7.09 (s, 1H, Ar-H), 7.41 (s, 2H, SO2NH2), 7.60 (d,J = 8.5 Hz, 1H, Ar-H), 7.77 (d, J = 8.5 Hz, 2H, H-3 and H-5, HNPhSO2),7.99 (d, J = 7.5 Hz, 2H, H-2 and H-6, HNPhSO2), 8.18 (s, 1H, @CH),9.98 (s, 2H, 2OH), 10.39 (s, 1H, N–H, exchange). 13C NMR: d106.32 (C-CN), 113.22, 116.24 (CN), 117.11, 120.13, 122.04,127.46, 129.88, 135.85, 139.37, 146.75, 147.74, 153.76 (Ar-C),

164.21 (C@O). MS m/z (Rel. Int.): 359 (M+, 65). Anal. (C16H13N3O5S)C, H, N.

5.2.14. 2-Cyano-3-(4-hydroxyphenyl)-N-(4-sulfamoylphenyl)acrylamide (13)

Compound 13: Yield: 42%, mp: 219–21 �C. IR, m (cm�1): 3426(OH), 3345, 3241 (SO2NH2 and NH), 3041 (Ar-CH), 2938 (CH),2227 (CN), 1672 (C@O). 1H NMR (DMSOd6): d 6.68 (d, J = 7.0 Hz,2H, Ar-H), 7.11 (d, J = 6.6 Hz, 2H, Ar-H), 7.60 (s, 2H, SO2NH2), 7.87(d, J = 8.5 Hz, 2H, H-3 and H-5, HNPhSO2), 7.92 (d, J = 7.5 Hz, 2H,H-2 and H-6, HNPhSO2), 8.25 (s, 1H, @CH), 8.54 (s, 1H, OH),10.30 (s, 1H, N–H, exchange). 13C NMR: d 106.3 (C-CN), 115.5,116.4 (CN), 122.4, 127.0, 127.6, 134.5, 138.7, 153.4, 156.7 (Ar-C),164.2 (C@O). MS m/z (Rel. Int.): 359 (M+, 65). Anal. (C16H13N3O4S)C, H, N.

5.2.15. 2-Cyano-3-(4-(dimethylamino)phenyl)-N-(4-sulfamoyl-phenyl)acrylamide (14)

Compound 14: Yield: 49%, mp: 212–14 �C. IR, m (cm�1): 3305,3211 (SO2NH2 and NH), 3071 (Ar-CH), 2958 (CH), 2222 (CN), 1674(C@O). 1H NMR (DMSOd6): d 6.56 (d, J = 7.0 Hz, 2H, Ar-H), 7.12 (d,J = 6.6 Hz, 2H, Ar-H), 7.66 (s, 2H, SO2NH2), 7.89 (d, J = 8.5 Hz, 2H,H-3 and H-5, HNPhSO2), 7.93 (d, J = 7.5 Hz, 2H, H-2 and H-6,HNPhSO2), 8.34 (s, 1H, @CH), 10.30 (s, 1H, N–H, exchange). 13CNMR: d 41.5 (2CH3), 107.1 (C-CN), 114.9, 116.4 (CN), 122.4, 125.0,127.2, 127.5, 135.5, 139.3, 149.0, 153.4, (Ar-C), 163.8 (C@O). MSm/z (Rel. Int.): 370 (M+, 36). Anal. (C18H18N4O3S) C, H, N.

5.2.16. 2-Cyano-5-phenyl-N-(4-sulfamoylphenyl)penta-2,4-dienamide (15)

Compound 15: Yield: 72%, mp: 187–89 �C. IR, m (cm�1): 3352,3318, 3261 (NH2 and NH), 3075 (Ar-CH), 2229 (CN), 1685 (C@O).1H NMR (DMSOd6): d 6.66 (d, J = 7.0 Hz, 1H, C@H), 6.90 (m, 1H,C@H), 7.12 (m, 1H, Ar-H), 7.21 (m, 2H, Ar-H), 7.36 (d, J = 7.0 Hz,2H, C@H), 7.53 (s, 2H, SO2NH2), 7.90 (d, J = 7.0 Hz, 2H, Ar-H), 7.92(d, J = 7.0 Hz, 2H, Ar-H), 8.0 (d, J = 7.0 Hz, 1H, C@H), 8.45 (s, 1H,N–H, exchange). 13C NMR: d 93.0 (C-CN), 115.3, 116.2 (C„N),120.1, 121.2, 125.0, 125.9, 127.2, 128.0, 128.5, 131.26, 133.8,135.1, 135.3, 139.5 (Ar-C), 163.5 (C@O). MS m/z (Rel. Int.): 353(M+, 5.5). Anal. (C18H15N3O3S) C, H, N.

5.2.17. 2-Cyano-3-(furan-2-yl)-N-(4-sulfamoylphenyl)acrylamide (16)

Compound 16: Yield: 46%, mp: 201–203 �C. IR, m (cm�1): 3305,3211 (SO2NH2 and NH), 3071 (Ar-CH), 2958 (CH), 2222 (CN),1674 (C@O). 1H NMR (DMSOd6): d 6.62 (t, J = 6.3 Hz, 1H, Ar-H),7.10 (d, J = 6.9 Hz, 1H, Ar-H), 7.80 (d, J = 6.5 Hz, 1H, Ar-H), 7.86 (s,2H, SO2NH2), 7.90 (d, J = 8.5 Hz, 2H, H-3 and H-5, HNPhSO2), 7.94(d, J = 7.5 Hz, 2H, H-2 and H-6, HNPhSO2), 8.33 (s, 1H, @CH),10.23 (s, 1H, N–H, exchange). 13C NMR: d 111.1 (C-CN), 112.5,114.0, 116.4 (CN), 121.5, 127.5, 135.7, 139.4, 147.0, 152.0, 154.7,(Ar-C), 164.2 (C@O). MS m/z (Rel. Int.): 317 (M+, 34). Anal.(C14H11N3O4S) C, H, N.

5.2.18. Cyano-N-(4-sulfamoylphenyl)-3-(thiophen-2-yl)acrylamide (17)

Compound 17: Yield: 51%, mp: 186–88 �C. IR, m (cm�1): 3305,3211 (SO2NH2 and NH), 3071 (Ar-CH), 2958 (CH), 2222 (CN),1674 (C@O). 1H NMR (DMSOd6): d 7.32 (t, J = 6.3 Hz, 1H, Ar-H),7.60 (d, J = 6.9 Hz, 1H, Ar-H), 7.63 (d, J = 6.5 Hz, 1H, Ar-H), 7.85 (s,2H, SO2NH2), 7.91 (d, J = 8.5 Hz, 2H, H-3 and H-5, HNPhSO2), 7.95(d, J = 7.5 Hz, 2H, H-2 and H-6, HNPhSO2), 8.32 (s, 1H, @CH),10.20 (s, 1H, N–H, exchange). 13C NMR: d 112.0 (C-CN), 116.4(CN), 121.5, 127.5, 128.6, 130.8, 135.7, 139.4, 147.0, 152.0, 154.7,(Ar-C), 164.2 (C@O). MS m/z (Rel. Int.): 333 (M+, 26). Anal.(C14H11N3O3S2) C, H, N.

1402 A. M. Alafeefy et al. / Bioorg. Med. Chem. 21 (2013) 1396–1403

5.2.19. (3-(Benzo[d][1,3]dioxol-5-yl)-2-cyano-N-(4-sulfamoyl-phenyl)acrylamide (18)

Compound 18: Yield: 50%, mp: 195–97 �C. IR, m (cm�1): 3305,3211 (SO2NH2 and NH), 3071 (Ar-CH), 2958 (CH), 2222 (CN),1674 (C@O). 1H NMR (DMSOd6): d 5.92 (s, 2H, CH2), 6.60 (d,J = 6.7 Hz, 1H, Ar-H), 6.77 (d, J = 6.5 Hz, 1H, Ar-H), 6.90 (s, 1H, Ar-H), 7.85 (s, 2H, SO2NH2), 7.91 (d, J = 8.5 Hz, 2H, H-3 and H-5,HNPhSO2), 7.96 (d, J = 7.5 Hz, 2H, H-2 and H-6, HNPhSO2), 8.34 (s,1H, @CH), 10.20 (s, 1H, N–H, exchange). 13C NMR: d 100.9 (CH2),107.3 (C-CN), 112.1, 115.0, 116.1 (CN), 122.0, 127.6, 128.5, 135.4,139.6, 147.8, 149.0, 152.8 (Ar-C), 163.5 (C@O). MS m/z (Rel. Int.):371 (M+, 41). Anal. (C17H13N3O5S) C, H, N.

5.2.20. 2-Imino-N-(4-sulfamoylphenyl)-2H-chromene-3-carboxamide (19)

Compound 19: Yield: 44%, mp: 222–24 �C. IR, m (cm�1): 3305,3211 (SO2NH2 and NH), 3071 (Ar-CH), 2958 (CH), 2222 (CN),1674 (C@O). 1H NMR (DMSOd6): d 5.70 (bs, 1H, =NH, D2Oexchangeable), 6.67 (d, J = 6.4 Hz, 1H, Ar-H), 6.89 (t, J = 6.2 Hz, 1H,Ar-H), 6.94 (t, J = 6.3 Hz, 1H, Ar-H), 7.14 (d, J = 6.5 Hz, 1H, Ar-H),7.87 (s, 2H, SO2NH2), 7.92 (d, J = 8.5 Hz, 2H, H-3 and H-5,HNPhSO2), 7.95 (d, J = 7.5 Hz, 2H, H-2 and H-6, HNPhSO2), 8.35 (s,1H, @CH), 10.21 (s, 1H, N–H, exchange). 13C NMR: d 115.7 (CH2),116.3, 119.7, 121.5, 127.3, 127.8, 128.4, 129.2, 135.5, 139.8, 153.9(Ar-C), 163.1, 164.5. MS m/z (Rel. Int.): 343 (M+, 41). Anal.(C16H13N3O4S) C, H, N.

5.2.21. 2-Oxo-N’-phenyl-2-(4-sulfamoylphenylamino)acetohydrazonoyl cyanide (20)

To a stirred solution of 2-cyano-N-(4-sulfamoylphenyl)acetam-ide (0.239 g, 1 mmol) in ethanol (50 ml) sodium acetate trihydrate(0.13 g, 1 mmol) was added. After stirring for 15 min, the mixturewas chilled at 0 �C and treated with cold solution of aniline(0.93 g, 1 mmol) in 6 M hydrochloric acid (1.5 ml) with sodium ni-trite solution (0.07 g, 1 mmol) in water (3 mL). The addition of thediazonium salt was stirred for an additional 2 h at 0–5 �C and thenleft for 8 h in a refrigerator (4 �C). The resulting solid was collectedby filtration, washed thoroughly with water and dried. The crudeproduct was crystallized from ethanol to give hydrazone 20 in55% yield.

Compound 20: Yield: 55%, mp: 226–28 �C. IR, m (cm�1): 3385,3230, 3190 (SO2NH2 and NH), 3040 (Ar-CH), 2225 (CN), 1679(C@O). 1H NMR (CH3OD): d 6.58 (d, J = 6.4 Hz, 2H, ArH), 6.70 (t,J = 6.2 Hz, 1H, ArH), 7.11 (t, J = 6.8 Hz, 2H, ArH), 7.36 (s, 2H,SO2NH2), 7.78 (d, J = 8.0 Hz, 2H, H-2 and H-6, NHPhSO2NH2), 7.91(d, J = 8.0 Hz, 2H, H-3 and H-5, NHPhSO2NH2), 8.35 (s, 1H, N–H, ex-change.), 10.48 (s, 1H, CON–H, exchange). 13C NMR: d 107.13,114.25, 116.10, 119.0, 122.4, 128.1, 129.7, 135.7, 137.25, 140.0,143.9 (Ar-C), 162.7 (C@O). MS m/z (Rel. Int.): 343 (M+, 20). Anal.(C15H13N5O3S) C, H, N.

5.2.22. 2-Cyano-3-mercapto-3-(phenylamino)-N-(4-sulfamoyl-phenyl)acrylamide (21)

To a stirred solution of KOH (0.56 g 10 mmol) in DMF (20 mL),compound 1 (2.39 g, 10 mmol) was added. After stirring for30 min phenyl isothiocyanate (1.35 g, 10 mmol) was added to theresulting mixture. Stirring was continued for 6 h and then pouredover crushed ice containing HCl. The solid product so formed wasfiltered off, washed with water, dried and finally crystallizationfrom EtOH to afford compound 21 as pale yellow solid in 41% yield.

Compound 21: Yield: 41%, mp: 196–98 �C. IR, m (cm�1): 3353,3330, 3264 (SO2NH2 and NH), 3062 (Ar-CH), 2221 (CN), 1682(C@O). 1H NMR (DMSOd6): d 5.06 (s, 1H, NH, exchange.), 6.75–7.05 (m, 5H, Ar-H), 7.33 (s, 2H, SO2NH2), 7.76 (d, J = 8.0 Hz, 2H,H-2 and H-6, NHPhSO2NH2), 7.90 (d, J = 8.0 Hz, 2H, H-3 and H-5,NHPhSO2NH2), 10.45 (s, 1H, N–H, exchange.), 11.51 (s, 1H, S-H, ex-

change). 13C NMR: d 86.43 (C-CN), 115.38, 116.75 (CN), 119.10,122.25, 127.95, 130.26, 136.14, 139.85, 144.56 (Ar-C), 160.54(@CH), 163.57 (C@O). MS m/z (Rel. Int.): 374 (M+, 5.5). Anal.(C16H14N4O3S2) C, H, N.

5.2.23. 2-(5-Acetyl-3-phenyl-1,3,4-thiadiazol-2(3H)-ylidene)-2-cyano-N-(4-sulfamoylphenyl)acetamide (22)

To a solution of thioanilide 21 (0.37 g, 1 mmol) dissolved in eth-anol (20 mL), (Z)-2-oxo-N’-phenylpropanehydrazonoyl chloride(22) (0196 g, 1 mmol), and of triethylamine (0.5 mL) were added.The mixture was refluxed for 6 h, then allowed to cool. The formedsolid was filtered off, washed with ethanol and recrystallized fromEtOH/DMF to afford 1,3,4-thiadiazole derivative 22 in 46% yield.

Compound 22: Yield: 46%, mp: 204–06 �C. IR, m (cm�1): 3305,3211 (SO2NH2 and NH), 3071 (Ar-CH), 2958 (CH), 2222 (CN),1674 (C@O). 1H NMR (DMSOd6): d 2.27 (s, 3H, CH3), 6.46 (d,J = 6.5 Hz, 2H, Ar-H), 6.63 (t, J = 6.5 Hz, 1H, Ar-H), 6.98 (t, J = 6.7 Hz, 2H, Ar-H), 7.88 (s, 2H, SO2NH2), 7.91 (d, J = 8.5 Hz, 2H, H-3 andH-5, HNPhSO2), 7.94 (d, J = 7.5 Hz, 2H, H-2 and H-6, HNPhSO2),10.23 (s, 1H, N–H, exchange). 13C NMR: d 24.0 (CH3), 87.6 (C-CN),116.0, 116.7 (CN), 119.3, 119.7, 121.3, 127.5, 129.3, 135.6, 139.7,146.5, 154.6, 161.2 (Ar-C), 163.1, 184.7 (C@O). MS m/z (Rel. Int.):441 (M+, 21). Anal. (C19H15N5O4S2) C, H, N.

5.3. CA inhibition

An Applied Photophysics stopped-flow instrument has beenused for assaying the CA catalysed CO2 hydration activity.20 Phenolred (at a concentration of 0.2 mM) has been used as indicator,working at the absorbance maximum of 557 nm, with 20 mMHepes (pH 7.4) as buffer, and 20 mM Na2SO4 (for maintaining con-stant the ionic strength), following the initial rates of the CA-cata-lyzed CO2 hydration reaction for a period of 10–100 s.20 The CO2

concentrations ranged from 1.7 to 17 mM for the determinationof the kinetic parameters and inhibition constants. For each inhib-itor at least six traces of the initial 5–10% of the reaction have beenused for determining the initial velocity. The uncatalyzed rateswere determined in the same manner and subtracted from the to-tal observed rates. Stock solutions of inhibitor (10 mM) were pre-pared in distilled-deionized water and dilutions up to 0.001 nMwere done thereafter with the assay buffer. Inhibitor and enzymesolutions were preincubated together for 15 min at room temper-ature prior to assay, in order to allow for the formation of the E–Icomplex. The inhibition constants were obtained by non-linearleast-squares methods using PRISM 3, as reported earlier,10–15

and represent the mean from at least three different determina-tions. All CA isoforms were recombinant ones obtained in houseas reported earlier.10–15 The concentration of the enzymes in theassays was of 8.3 nM for hCA I, of 5.7 nM for hCA II, of 6.9 nM forhCA IX and of 7.5 nM for hCA XII, respectively.

Acknowledgments

This research was financed by a 7th FP EU project (Metoxia) andby a grant from the National Plan of Science, Technology and Inno-vation (Grant No. 10-MED1188-02 & Grant No. 11-MED-1874-2),King Saud University, Riyadh, by the Graduate Studies and Scien-tific Research Agency, Salman bin Abdulaziz University, Grant No.2.H.33, Alkharj, Saudi Arabia. Semra Isik was supported by a grantfrom the Council of Higher Education of Turkey.

Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.bmc.2012.12.004.

A. M. Alafeefy et al. / Bioorg. Med. Chem. 21 (2013) 1396–1403 1403

References and notes

1. Mann, T.; Keilin, D. Nature 1940, 146, 164.2. (a) Alterio, V.; Di Fiore, A.; D’Ambrosio, K.; Supuran, C. T.; De Simone, G. Chem.

Rev. 2012, 112, 4421; (b) Supuran, C. T. Nat. Rev. Drug Disc. 2008, 7, 168; (c)Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2012, 27, 759.

3. (a) Fisher, S. Z.; Aggarwal, M.; Kovalevsky, A. Y.; Silverman, D. N.; McKenna, R. J.Am. Chem. Soc. 2012, 134, 14726; (b) Supuran, C. T. Bioorg. Med. Chem. Lett.2010, 20, 3467; (c) Pastorekova, S.; Parkkila, S.; Pastorek, J.; Supuran, C. T. J.Enzyme Inhib. Med. Chem. 2004, 19, 199; (d) Clare, B. W.; Supuran, C. T. Eur. J.Med. Chem. 1999, 34, 463.

4. (a) Jain, A.; Whitesides, G. M.; Alexander, R. S.; Christianson, D. W. J. Med. Chem.1994, 37, 2100; (b) Graham, S. L.; Shepard, K. L.; Anderson, P. S.; Baldwin, J. J.;Best, D. B.; Christy, M. E.; Freedman, M. B.; Gautheron, P.; Habecker, C. N.;Hoffman, J. M.; Lyle, P. A.; Michelson, S. R.; Ponticello, G. S.; Robb, C. M.;Schwam, H.; Smith, A. M.; Smith, R. L.; Sondey, J. M.; Strohmaier, K. M.; Sugrue,M. F.; Varga, S. L. J. Med. Chem. 1989, 32, 2548; (c) Baranauskiene, L.; Hilvo, M.;Matuliene, J.; Golovenko, D.; Manakova, E.; Dudutiene, V.; Michailoviene, V.;Torresan, J.; Jachno, J.; Parkkila, S.; Maresca, A.; Supuran, C. T.; Grazulis, S.;Matulis, D. J. Enzyme Inhib. Med. Chem. 2010, 25, 863.

5. (a) Supuran, C. T. Future Med. Chem. 2011, 3, 1165; (b) Supuran, C. T.;Scozzafava, A.; Casini, A. Med. Res. Rev. 2003, 23, 146; (c) Wilkinson, B. L.;Bornaghi, L. F.; Houston, T. A.; Innocenti, A.; Supuran, C. T.; Poulsen, S. A. J. Med.Chem. 2006, 49, 6539.

6. (a) Supuran, C. T.; Scozzafava, A.; Casini, A. Development of SulfonamideCarbonic Anhydrase Inhibitors (CAIs). In Carbonic Anhydrase—Its Inhibitors andActivators; Supuran, C. T., Scozzafava, A., Conway, J., Eds.; CRC Press: Boca Raton(FL, USA), 2004; pp 67–147; (b) Owa, T.; Nagasu, T. Expert Opin. Ther. Pat. 2000,10, 1725; (c) Carta, F.; Scozzafava, A.; Supuran, C. T. Expert Opin. Ther. Pat. 2012,22, 747; (d) Aggarwal, M.; McKenna, R. Expert Opin. Ther. Pat. 2012, 22, 903.

7. (a) Carta, F.; Garaj, V.; Maresca, A.; Wagner, J.; Avvaru, B. S.; Robbins, A. H.;Scozzafava, A.; McKenna, R.; Supuran, C. T. Bioorg. Med. Chem. 2011, 19, 3105;(b) Supuran, C. T.; Scozzafava, A. J. Enzyme Inhib. 1997, 12, 37.

8. a) Neri, D.; Supuran, C. T. Nat. Rev. Drug Disc. 2011, 10, 767; (b) Winum, J. Y.;Maresca, A.; Carta, F.; Scozzafava, A.; Supuran, C. T. Chem. Commun. 2012, 48,8177.

9. Ahlskog, J. K.; Schliemann, C.; Mårlind, J.; Qureshi, U.; Ammar, A.; Pedleym, R.B.; Neri, D. Bioorg. Med. Chem. Lett. 2009, 19, 4851.

10. (a) Hen, N.; Bialer, M.; Yagen, B.; Maresca, A.; Aggarwal, M.; Robbins, A. H.;McKenna, R.; Scozzafava, A.; Supuran, C. T. J. Med. Chem. 2011, 54, 3977; (b)Bootorabi, F.; Jänis, J.; Hytönen, V. P.; Valjakka, J.; Kuuslahti, M.; Vullo, D.;Niemelä, O.; Supuran, C. T.; Parkkila, S. J. Enzyme Inhib. Med. Chem. 2011, 26,862.

11. (a) Swietach, P.; Wigfield, S.; Supuran, C. T.; Harris, A. L.; Vaughan-Jones, R. D.BJU Int. 2008, 101, 22; (b) Ebbesen, P.; Pettersen, E. O.; Gorr, T. A.; Jobst, G.;Williams, K.; Kienninger, J.; Wenger, R. H.; Pastorekova, S.; Dubois, L.; Lambin,P.; Wouters, B. G.; Supuran, C. T.; Poellinger, L.; Ratcliffe, P.; Kanopka, A.;Görlach, A.; Gasmann, M.; Harris, A. L.; Maxwell, P.; Scozzafava, A. J. EnzymeInhib. Med. Chem. 2009, 24, 1.

12. Alterio, V.; Hilvo, M.; Di Fiore, A.; Supuran, C. T.; Pan, P.; Parkkila, S.; Scaloni, A.;Pastorek, J.; Pastorekova, S.; Pedone, C.; Scozzafava, A.; Monti, S. M.; DeSimone, G. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 16233.

13. (a) Pacchiano, F.; Carta, F.; McDonald, P. C.; Lou, Y.; Vullo, D.; Scozzafava, A.;Dedhar, S.; Supuran, C. T. J. Med. Chem. 1896, 2011, 54; (b) Lou, Y.; McDonald, P.C.; Oloumi, A.; Chia, S. K.; Ostlund, C.; Ahmadi, A.; Kyle, A.; Auf dem Keller, U.;Leung, S.; Huntsman, D. G.; Clarke, B.; Sutherland, B.W.; Waterhouse, D.; Bally,M. B.; Roskelley, C. D.; Overall, C. M.; Minchinton, A.; Pacchiano, F.; Carta, F.;Scozzafava, A.; Touisni, N.; Winum, J. Y.; Supuran, C. T.; Dedhar, S. Cancer Res.2011, 71, 3364.; (c) Pacchiano, F.; Aggarwal, M.; Avvaru, B. S.; Robbins, A. H.;Scozzafava, A.; McKenna, R.; Supuran, C. T. Chem. Commun. 2010, 46, 8371.

14. (a) Winum, J. Y.; Carta, F.; Ward, C.; Mullen, P.; Harrison, D.; Langdon, S. P.;Cecchi, A.; Scozzafava, A.; Kunkler, I.; Supuran, C. T. Bioorg. Med. Chem. Lett.2012, 22, 4681; (b) Liu, F.; Martin-Mingot, A.; Lecornué, F.; Jouannetaud, M. P.;Maresca, A.; Thibaudeau, S.; Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2012, 27,886.

15. Gieling, R. G.; Babur, M.; Mamnani, L.; Burrows, N.; Telfer, B. A.; Williams, S. R.;Davies, K. E.; Carta, F.; Winum, J. Y.; Scozzafava, A.; Supuran, C. T.; Williams, K.J. J. Med. Chem. 2012, 55, 5591.

16. Trivedi, J. M.; Mehta, C. M., J. Indian Chem. Soc. 1975, LII, 708.17. Dawood, K. M.; Farag, A. M.; Abdel-Aziz, H. A. J. Chem. Res. 2005, 278.18. Hamdy, N. A.; Abdel-Aziz, H. A.; Farag, A. M.; Fakhr, I. M. I. Monatsh. Chem.

2007, 138, 1001.19. Nikolay, Y. G.; Behrooz, H. Y.; Ferdinand, B.; Oliver, C. K. Tetrahedron 2004, 60,

8633.20. Khalifah, R. J. J. Biol. Chem. 1971, 246, 2561.