Carbonic anhydrase inhibitors: Synthesis, molecular docking, cytotoxic and inhibition of the human carbonic anhydrase isoforms I, II, IX, XII with novel benzenesulfonamides incorporating pyrrole, pyrrolopyrimidine and fused pyrrolopyrimidine moieties

Accepted Manuscript

Carbonic anhydrase inhibitors: Synthesis, molecular docking, cytotoxic and in-

hibition of the human carbonic anhydrase isoforms I, II, IX, XII with novel

benzenesulfonamides incorporating pyrrole, pyrrolopyrimidine and fused pyr-

rolopyrimidine moieties

Mostafa M. Ghorab, Mansour S. Alsaid, Mariangela Ceruso, Yassin M. Nissan,

Claudiu T. Supuran

PII:

S0968-0896(14)00349-6

DOI:

http://dx.doi.org/10.1016/j.bmc.2014.05.009

BMC 11570

Reference:

Bioorganic & Medicinal Chemistry

To appear in:

Received Date:

Revised Date:

Accepted Date:

27 March 2014

5 May 2014

6 May 2014

Please cite this article as: Ghorab, M.M., Alsaid, M.S., Ceruso, M., Nissan, Y.M., Supuran, C.T., Carbonic anhydrase

inhibitors: Synthesis, molecular docking, cytotoxic and inhibition of the human carbonic anhydrase isoforms I, II,

IX, XII with novel benzenesulfonamides incorporating pyrrole, pyrrolopyrimidine and fused pyrrolopyrimidine

moieties, Bioorganic & Medicinal Chemistry (2014), doi: http://dx.doi.org/10.1016/j.bmc.2014.05.009

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Carbonic anhydrase inhibitors: Synthesis, molecular docking, cytotoxic and inhibition of

the human carbonic anhydrase isoforms I, II, IX, XII with novel benzenesulfonamides

incorporating pyrrole, pyrrolopyrimidine and fused pyrrolopyrimidine moieties.

Mostafa M. Ghorab*^a,b, Mansour S. Alsaid^a, Mariangela Ceruso ^c, Yassin M. Nissan^dand

Claudiu T. Supuran*^c,e

^aPharmacognosy Department, College of Pharmacy, King Saud University, P. O. Box 2457,

Riyadh 11451, Saudi Arabia

^bDrug Radiation Research Department, National Center for Radiation Research & Technology,

Atomic Energy Authority, Cairo, Egypt.

^cUniversità degli Studi di Firenze, Laboratorio di Chimica Bioinorganica, Rm. 188, Via della

Lastruccia 3, I-50019 Sesto Fiorentino (Firenze), Italy.

^dPharmaceutical Chemistry Department, Faculty of Pharmacy, Cairo University, Kasr Al- Aini

st., P.O.Box 11562, Cairo, Egypt.

^eUniversità degli Studi di Firenze, Polo Scientifico, Dipartimento Neurofaba; Sezione di

Scienze Farmaceutiche e Nutraceutiche, Via Ugo Schiff 6, 50019 Sesto Fiorentino (Firenze),

Italy.

*Corresponding authors: M.M. Ghorab, Professor of Applied Organic Chemistry,

Pharmacognosy Department, College of Pharmacy, King Saud University, P.O. Box 2457,

Riyadh 11451, Saudi Arabia. Tel.: +966 534292860; Fax: +966 01 4670560. E-mail address:

mmsghorab@yahoo.com; and C.T. Supuran: Tel./Fax: +39-055-4573005;

E-mail: claudiu.supuran@unifi.it.

1

Abstract

series

A

of

novel

pyrroles,

pyrrolopyrimidines,

pyrazolopyrrolopyrimidine,

triazolopyrrolopyrimidines, tetrazolopyrrolopyrimidine, triazinopyrrolopyrimidines and

pyrrolopyrimidotriazepines bearing the biologically active benzenesulfonamide moiety were

synthesized by using pyrrole-o-amino-carbonitrile as key intermediate. All the synthesized

compounds were evaluated for their in vitro carbonic anhydrase (CA, EC 4.2.1.1) inhibitory

effects against the human (h) isoforms hCA I, II, IX and XII. Among the tested derivatives,

compounds 16, 18 and 20-24 showed potent activity as inhibitors for the tumor associated

transmembrane isoforms (hCA IX and XII) in the nanomlar and subnanomolar range, with

high selectivity. All compounds underwent cytotoxic activity assays on human breast cancer

cell line (MCF-7) showing effective activity, comparable to that of the clinically used drug

Doxorubicin.

Key words: Pyrrolopyrimidines, sulfonamide, cytotoxic activity, carbonic anhydrase

inhibitors, molecular docking.

2

1. Introduction

Several pyrrole and fused pyrrole derivatives have been reported as biologically active

compounds with variant activities^1-6especially anticancer activity.^7-10In addition, sulfonamides

constitute an important class of drugs with several types of pharmacological activities,

including antibacterial^11,12, antiviral ¹³, anti-inflammatory ¹⁴and anticancer activity.^15,16On the

other hand, pyrimidines and its fused derivatives play an essential role in several biological

17-23

processes.

Also, the potential use of aromatic/ heterocyclic sulfonamides as carbonic

24-29

anlydrase (CA, EC 4.2.1.1) inhibitors

has been only recently explored to date for the

treatment/imaging of hypoxic tumors, considered the second cause of death for the time

being.^30,31

Fifteen isoforms of human carbonic anhydrase have been discovered, among them the

transmembrane tumor associated isoforms (hCA IX and hCA XII) are over expressed in

several types of tumors. Many sulfonamide derivatives have been investigated for their CA

inhibition activity seeking for selective hCA IX and hCA XII inhibitors since the lack of

selectivity is the major challenge for the wide use of chemotherapeutic agents in cancer

therapy.^24-29Additionally, 5,7-diphenyl-pyrrolo[2,3-d]pyrimidines were described as potent

cytotoxic agents.^32-34

In the lights of the previous facts and based on our previous work in the field of design and

synthesis of sulfonamide based molecules as cytotoxic agents ^35-38, we herein report the results

achieved from the design and synthesis of novel pyrrole, pyrrolo[2,3-d]pyrimidine and fused

pyrrolopyrimidine derivatives carrying a biologically active benzenesulfonamide moiety. All

the newly synthesized compounds were subjected to cytotoxic screening against the breast

cancer cell line MCF-7. To explore these derivatives prospective mechanism of action,

assessment of their in vitro enzyme-inhibitory capacity against hCA I, II, IX and XII was

performed and compared with the well known CAI acetazolamide (AZA).³⁹Finally, molecular

docking of all compounds in the active site of hCA XII was carried out in an attempt to

speculate the possible binding mode of these molecules in the active site of their target

enzyme.

2. Results and Discussion

2.1. Chemistry

The aim of this work was to design, synthesis some novel pyrrole, pyrrolopyrimidine and

fused pyrrolopyrimidine derivatives with the biologically active benzenesulfonamide moiety

3

and evaluate their carbonic anhydrase inhibition as well as their cytotoxic activity. Thus,

interaction of sulfanilamide 1 with 2-bromo-1-(4-bromophenyl) ethanone 2 furnished the

corresponding 4-(2-oxo-2-(bromophenyl) ethylamino) sulfonamide 3. The reactivity of

compound 3 towards the active methylene compound, namely, malononitrile, was investigated.

When compound 3 was refluxed with malononitrile in ethanol containing sodium ethoxide, the

pyrrole derivative 5 was produced (Scheme 1). The formation of compound 5 was assumed to

proceed via condensation of 3 with malononitrile to give the intermediate 4 followed by

intramolecular cyclization to give pyrrole derivative 5. Refluxing of compound 5 with

triethylorthoformate neat or in presence of acetic anhydride gave compound 6. When

compound 6 was refluxed with hydrazine hydrate in absolute ethanol the corresponding N-

amino-imino derivative 8 was obtained through the formation of intermediate 7. Interaction of

compound 8 with malononitrile in ethanol in presence of sodium ethoxide afforded the

pyrazolo derivative 9 in good yield. On the other hand, 1, 2, 4-triazole derivatives 10-12 were

obtained directly by heating under reflux of 8 with diethyl oxalate or diethylmalonate and/or

ethylacetoacetate. Interaction of compound 8 with an equimolar ratio of ethyl chloroformate

gave the corresponding 1,2,4-triazole derivative 14 through the formation of intermediate 13

followed by loss 1 mole of ethanol. Also, when compound 8 was subjected to the diazotization

reaction condition, the tetrazolo derivative 15 was formed. Reaction of compound 8 with ethyl

chloroacetate in refluxing sodium ethoxide yielded the triazino derivative 16 rather than its

isomeric structure 17 (Scheme 2). Structure 16 was suggested rather than structure 17, based

on the assumption that the reaction basic condition allowed proceeding through formation of

sodium salt on the less basic NH and elimination of sodium chloride followed by cyclization.

In addition the IR spectrum of the isolated product showed band at 1654 cm^-1which was at less

1

frequency than that expected for structure 17. Further evidence was the H-NMR spectrum

which showed a singlet at 4.7 ppm for the methylene protons.

Treatment of compound 8 with oxalyl chloride in refluxing dry benzene afforded the

corresponding dioxotriazine derivative 18. In contrast, in our hands when the N-amino-imino 8

was allowed to react with acetylacetone the corresponding triazepino derivative 19 was

obtained. On the other hand, the reaction of 8 with ethoxymethylenemalononitrile or ethyl

ethoxymethylenecyanoacetate yielded the corresponding triazepine aminocarbonitrile 20 and

aminoester 21, respectively.

4

Conversely, condensation of the key compound 8 with isatin in refluxing ethanol gave the

corresponding cyclocondensation product indeno-1,2,4-triazine derivative 22 (Scheme 3).

Also, it was condensed with N-acetyl isatin to give the condensation product 23 in good yield.

It is noteworthy that when the reaction was carried out with 8 and benzoylacetone, the product

was identified as the azomethine compound 24 which could be cyclized to the corresponding

triazepine 25 by heating in refluxing phosphoryl chloride (Scheme 3).

2.2. Carbonic anhydrase inhibition

The CA inhibitory ability of all the synthesized compounds was measured against the

cytosolic isoforms hCA I and II as well as the membrane associated isoforms hCA IX and XII

using stopped flow assay method and the results are displayed in Table 1. The well known

carbonic anhydrase inhibitor acetazolamide (AZA) was used as the reference drug for this

assay.³⁹Data obtained from the CA inhibition assay led to the following inferences.

(i) The cytosolic isoform hCA I was moderately inhibited by the sulfonamide derivatives with

k_ivalues in the range of 21.6- 498 nM. The most active compounds against hCA I were 8, 14,

23, 19, 11, 25 and 15 arranged in a descending order from the most active to the least with k_i

values less than 30nM. However, all the synthesized compounds were more active than AZA

except compound 22 with k_ivalue 498 nM.

(ii) With regards to the profiling assay against hCA II, all compounds were active with k_i

values in the range of 3.2-123.7 nM. Compounds 6, 8, 24, 14, 5, 10, 9, 16, 20, 15, 23, 25, 21

and 22 were the most active compounds arranged in a deseeding order from the most active to

the least with k_ivalues less than 9 nM being more active than AZA while compounds 11, 12,

18 and 19 were less active than AZA.

(iii) The tumor associated target isoform hCA IX was highly inhibited by sulfonamides 9, 14,

8, 10, 22, 15, 24, 11, 21, 23, 25, 6 and 5 (arranged in a descending manner with respect to

inhibition efficiency) showing k_ivalues lower than that of AZA. On the other hand, only three

compounds 16, 18 and 19 were less active than AZA.

(iv) Finally, excellent results were displayed by eight of the novel sulfonamides (22, 5, 16, 23,

21, 20, 18 and 24 arranged in a desending order) in the profiling assay performed against the

transmembrane, tumor associated isozyme hCA XII where they were potent in a subnanomolar

concentration being 6 to 8.5 fold more active than AZA against the target CA isoform.

Moreover, compounds 19 and 25 showed k_ivalues of 5.4 and 3.4 nM, respectively yet still

more active than AZA. The other synthesized derivatives were less active than AZA.

5

Selectivity of the newly synthesized compounds towards carbonic anhydrase isoforms received

great importance in order to reduce the unwanted side effects of using such candidates as

anticancer agents as the cystolic isoforms hCA I and hCA II inhibition will lead to potential

diueresis. Selectivity index (SI) was calculated for each compound as hCA I/ hCA XII and

hCA II/ hCA XII to measure their selectivity towards the transmembranal tumor associated

isoform hCA XII. Compounds 5, 6, 18 and 20-22 showed high selectivity for hCA XII than

hCA I especially compound 22 with SI value more than 691. On the other hand, compound 18

showed high selectivity for hCA XII than hCA II with SI value more than 356.

2.3. In vitro cytotoxic activity

The newly synthesized compounds were evaluated for their in vitro cytotoxic activity against

human breast cancer cell line (MCF7). Doxorubicin which is one of the most effective

anticancer agents was used as the reference drug in this study. The relationship between

surviving fraction and drug concentration was plotted to obtain the survival curve of breast

cancer cell line (MCF7). The response parameter calculated was the IC₅₀value, which

corresponds to the concentration required for 50% inhibition of cell viability. (Table 2) shows

the in vitro cytotoxic activity of the synthesized compounds, where all compounds exhibited

significant activity compared to doxorubicin as reference drug. All the synthesized compounds

showed promising cytotoxic activity especially compounds 5-10, 14, 15, 19 and 21- 25 with

better activity than Doxorubicin as positive control, while compounds 16 and 20 showed

comparable activity to that of doxorubicin with IC₅₀value of 8.11 µM for each. On the other

hand, compounds 11, 12 and 18 were slightly less active than doxorubicin.

The strategic starting pyrrole derivative 5 showed better activity than doxorubicin with IC₅₀

value of 7.56 µM. Upon reaction of the amino group to form the imino derivative 6 the activity

remained the same with no change. Cyclization of 6 to the imino-amino pyrrolopyrimidine

derivative 8 had a good effect on activity as it increased to reach 7.29 µM. Adding another

five-member ring to the pyrrolopyrimidine derivative 8 as in compounds 9-12, 14 and 15 did

not improve the activity with IC₅₀values of 8.94, 7.29, 7.56, 8.94, 7.56 and 7.84 µM for

compounds 9-12, 14 and 15, respectively. The most active compound of this series was the

triazolopyrrolopyrimidine derivative with an ethyl ester substituent 10 with IC₅₀value of 7.29

µM. The other cylization was performed to get an additional six member ring as in compounds

16 and 18 which did not also improve the activity with IC₅₀values of 8.11 and 8.66 µM,

respectively. On our attempt to study the effect of other cyclization, seven member ring

6

derivatives were also synthesized and investigated for cytotoxic activity represented by

compounds 19, 20, 21 and 25. The IC₅₀values for these derivatives were 7.84, 8.11, 7.84 and

7.29 µM, respectively. The most active compound of this series was compound 25 with IC₅₀

value of 7.29 µM. The final cyclization attempt was performed to get the benzopyrrolotriazine

derivative 22 which showed IC₅₀value of 7.84 µM.

Moreover, two hydrazone derivatives 23 and 24 were evaluated for their cytotoxic activity

with IC₅₀values of 7.56 and 7.84, respectively.

In the light of the previous biological results, we can retrieve that: the pyrrolo derivative 5

exhibited both good CA inhibitory activity and cytotoxic activity with good selectivity for

inhibiting hCA XII over the other isoforms, whereas the formation of the Schiff's base

derivative 6 has decreased the hCA inhibition activity. Cyclization to pyrrolopyrmidine

derivative 8 also tends to decrease the activity on hCA IX and XII. However, the fused

triazinopyrrolopyrimidine derivatives 16, 18 and 24 showed potent activity towards hCA XII,

in the subnanomolar range, with cytotoxic activity comparable to that of doxorubicin. Fusion

of the pyrrolopyrimidine moiety with five membered rings such as pyrazole, triazole and

tetrazole, in compounds 9, 10-12, 14 and 15, respectively, tends to decrease activity towards

hCA XII while still preserving good cytotoxic activity. On the other hand, fusion of

pyrrolopyrimidine with triazepine moiety as in compounds 19, 20, 21 and 25 was better for

hCA XII inhibitory activity, especially for 20 and 21, which showed a subnanomolar range of

activity, with cytotoxic activity comparable to doxorubicin. Fusion of the pyrrolopyrimidine

scaffold with triazinoindole or substitution with an indole moiety as in compounds 23 and 22,

respectively, again was beneficial to hCA inhibitory activity with even better cytotoxic activity

than doxorubicin.

2.4. Molecular Docking Results

It was of interest to perform molecular docking for the newly synthesized compounds, in order

to rationalize their promising biological activity. The protein data bank file (PDB ID: 1JD0)

was selected for this purpose. The file contains hCA XII co-crystallized with acetazolamide.

Docking on the active site of hCA XII was performed for all synthesized sulfonamide

derivatives 5, 6, 8-12, 14-16 and 18-25. Acetazolamide interacts with the active site of hCA

XII by two hydrogen bonds between Thr199 and the SO₂group, one hydrogen bond between

Thr200 and N3 of the thiadiazole ring, a water mediated hydrogen bond between Pro201 and

N4 of the thiadiazole ring, and finally a metal complex between the Zn(II) ion, His94, His96

7

and His119 and the SO₂NH₂group of AZA (Figure 1). All synthesized compounds were

docked to the active site of hCA XII with good energy scores (S) supporting the observed

activity of these sulfonamides as CA XII inhibitors. Energy scores (S) and amino acid

interactions of the newly synthesized compounds were listed in Table 3.

Docking scores for the newly synthesized compounds fall in the range of (-12.7252 to

-52.7571 Kcal/mol). The best docking score was assigned to compound 16 (-52.7571

Kcal/mol). On the other hand, all of the newly synthesized compounds were capable of forming

a metal complex with the Zn ion through His 94, His 96 and His 119 in a similar manner to the

co-crystallized ligand (AZA). It was observed from the amino acid interactions of the newly

synthesized compounds that interactions with Thr 199 were achieved only by compounds 5, 16,

18 and 20-24 in a very comparable way to that of AZA while the rest of compounds giving

lower activity in the hCA XII inhibition assay failed to show interaction with this amino acid in

the docking study.

In a further look to the docking results side by side to CA XII inhibition results, we

can observe that the most active compounds (5, 16, 18 and 20-24) have shown excellent

docking scores (-41.3027, -52.7571, -51.5937, -49.5079, -46.6627, -41.5627, -40.2157 and -

44.9134, respectively) in addition to their interactions with Thr 199 by at least one hydrogen

bond which can give us a simulating illustration of their receptor interaction with active site of

CA XII. Amino acid interactions of the most selective compounds (16 and 18) are illustrated in

Figures 2 and 3, respectively.

3. Conclusions

A

series

of

pyrroles,

pyrrolopyrimidines,

pyrazolopyrrolopyrimidine,

triazolopyrrolopyrimidines, tetrazolopyrrolopyrimidine, triazinopyrrolopyrimidines and

pyrrolopyrimidotriazepines bearing the biologically active benzenesulfonamide moiety were

designed and synthesized as potential CAIs. The carbonic anhydrase inhibitory activity of

these compounds was assessed against the human cytosolic isoforms hCA I and hCA II as well

as the transmembrane isoforms hCA IX and hCA XII which are over expressed in various

types of tumors and hence are valid targets for antitumor agents. The tested sulphonamides

showed variable degrees of activities against all four evaluated hCA isoforms some being more

potent than the well known CAI AZA. All of the newly synthesized compounds were more

potent than AZA against the tumor associated isoform hCA IX except 18 and 19. Most of the

synthesized compounds were more potent than AZA against the tumor associated isoform hCA

XII particularly, 5, 16, 18, 20-24 exhibiting subnanomolar K_ivalues. These new sulfonamides

8

also displayed high selectivity towards the tumor associated isoform hCA XII as displayed by

their hCA I/XII and hCA II/XII SI. Docking of all compounds was performed with hCA XII,

and enzyme-ligand binding poses obtained displayed interactions in a fashion comparable to

that of AZA especially for compounds 5, 16, 18, 20-24. Cytotoxic activity of the newly

synthesized compounds showed better or comparable results to the reference drug

Doxorubicin. These results merit further investigation of these new sulfonamides as potential

antitumor agents.

4. Experimental

4.1. Chemistry

Melting points (^oC, uncorrected) were determined in open capillaries on a Gallenkemp melting

point apparatus (Sanyo Gallenkemp, Southborough, UK) and were uncorrected. Pre coated

silica gel plates (silica gel 0.25 mm, 60 G F254; Merck, Germany) were used for thin layer

chromatography, dichloromethane/methanol (9.5:0.5) mixture was used as a developing

solvent system and the spots were visualized by ultraviolet light and/or iodine. Infra-red

spectra were recorded in KBr discs using IR-470 Shimadzu spectrometer (Shimadzu, Tokyo,

Japan). NMR spectra (in DMSO-d₆) were recorded on Bruker AC-500 Ultra Shield NMR

spectrometer (Bruker, Flawil, Switzerland, δ ppm) at 500 MHz using TMS as internal Standard

and peak multiplicities are designed as follows: s, singlet; d, doublet; t, triplet; m, multiplet.

Elemental analyses were performed on Carlo Erba 1108 Elemental Analyzer (Heraeus, Hanau,

Germany).

4.1.1. 4-(2-(4-Bromophenyl)-2-oxoethylamino)benzenesulfonamide 3

A mixture of sulfanilamide 1 (1.72 g, 0.01 mol.) and 2-bromo-1-(4-bromophenyl)ethanone 2

(2.77 g, 0.01 mol.) was refluxed in N,N’-dimethylformamide (20 mL) in the presence of

catalytic amount of triethylamine for 6 h. The solid obtained was filtered off and recrystallized

o

from ethanol to give 3. Yield 89%, m.p. 232.6 C, IR:υ_max./cm^-13358, 3255 (NH, NH₂), 3100

(CH arom.), 2970, 2863 (CH aliph.), 1685 (C=O), 1381, 1157 (SO₂).¹H-NMR (DMSO-d_6,

D₂O) δ: 4.7 (s, 2H, CH₂), 6.6 (s, 1H, NH, D₂O exchangeable), 6.7, 7.5 (2d, 4H, Ar-H, AB

system, J=7.1 Hz), 7.8, 8.0 (2d, 4H, Ar-H, AB system, J=6.9 Hz).¹³C-NMR (DMSO-d₆) δ :

49.4, 111.4 (2), 127.1, 127.7, 129.9 (2), 130.7 (2), 131.8 (2), 133.9, 150.8, 195.3.Anal. Calcd.

for C₁₄H₁₃BrN₂O₃S (369.23): C, 45.54; H, 3.55; N, 7.59. Found: C, 45.54; H, 3.31; N, 7.24.

4.1.2. 4-(2-Amino-4-(4-bromophenyl)-3-cyano-1H-pyrrol-1- yl)benzenesulfonamide 5

9

A mixture of compound 3 (3.69 g, 0.01 mol.) and malononitrile (0.66 g, 0.01 mol.) in ethanol

(20 mL) containing sodium ethoxide (0.5 g) was refluxed for 8 h. The reaction mixture was

cooled and acidified with dil. HCl. The solid obtained was filtered off and recrystallized from

dioxane to give 5. Yield 78%, m.p. 221.2 ^oC, IR:υ_max./cm^-13419, 3344, 3238 (NH₂), 3095 (CH

arom.), 2187 (C≡N), 1342, 1176 (SO₂).¹H-NMR (DMSO-d_6,D₂O) δ: 6.1 (s, 2H, NH₂, D₂O

exchangeable), 7.0 (s, 1H, CH pyrrole), 7.5- 7.9 (m, 10H, Ar-H+SO₂NH₂).¹³C-NMR (DMSO-

d₆) δ : 70.5, 113.5, 117.5, 119.5, 121.3 (2), 125.2, 127.3, 131.2 (2), 131.6 (2), 132.3 (2), 139.5,

142.9, 148.5. Anal. Calcd. for C₁₇H₁₃BrN₄O₂S (417.28): C, 48.93; H, 3.14; N, 13.43. Found: C,

48.71; H, 3.50; N, 13.16.

4.1.3. Ethyl N-4- (4-bromophenyl)-3-cyano-1-(4-sulfamoylphenyl)-1H-pyrrol-2-yl)-form-

imidate (6)

A mixture of compound 5 (4.17 g, 0.01 mol.) and triethylorthoformate (20 mL) was refluxed

for 6 h. The reaction mixture was cooled and then poured onto ice/water. The formed residue

was recrystallized from ethanol to give 6. Yield 78%, m.p. 120.2 ^oC, IR:υ_max./cm^-13151, 3136

1

(NH₂), 2987, 2865 (CH aliph.), 2206 (CN), 1629 (C=N), 1354, 1155 (SO₂). H-NMR

(DMSO-d_6,D₂O) δ: 1.0 (t, 3H, CH₃), 4.3 (q, 2H, CH₂), 7.6- 7.9 (m, 8H, Ar-H), 8.1 (s, 1H, CH

13

pyrrole), 8.5 (s, 2H, SO₂NH₂, D₂O exchangeable), 8.7 (s, 1H, N=CH). C-NMR (DMSO-d₆)

δ: 13.6, 63.6, 79.1, 116.8, 117.4, 120.3, 122.8 (2), 125.4, 127.7, 127.8 (2), 131.5 (2), 131.8 (2),

138.5, 140.6, 145.3, 161.9. Anal. Calcd. for C₂₀H₁₇BrN₄O₃S (473.34): C, 50.75; H, 3.62; N,

11.84. Found: C, 50.48; H, 3.91; N, 11.54.

4.1.4. 4-(3-Amino-5-(4-bromophenyl)-4-imino-3,4-dihydropyrrolo[2,3-d]pyrimidin-7-yl)b-

enzenesulfonamide (8)

A mixture compound 6 (4.73 g, 0.01 mol) and hydrazine hydrate (1 g, 0.02 mol) was stirred

in ethanol at room temperature for 1h, the solid formed was filtered, and recrystallized from

ethanol to give 8. Yield % 88, m.p. 184.5^oC; IR (KBr, cm^-1): 3331, 3296, 3230, 3132 (NH,

1

NH₂), 1637 (C=N), 1328, 1165 (SO₂). H-NMR (DMSO-d₆) δ: 5.6 [s, 2H, NH₂, D₂O-

exchangeable], 7.3- 7.8 [m, 9H, Ar-H + NH], 7.9 [s, 1H, CH pyrimidine], 8.0 [s, 2H, SO₂NH₂,

D₂O-exchangeable], 8.1 [s, 1H, CH pyrrole]. ¹³C-NMR (DMSO-d₆) δ: 103.8, 119.8, 120.1,

121.1 (2), 124.0, 126.7, 130.8 (2), 131.1 (2), 132.9 (2), 139.6, 141.9, 142.7, 147.6, 153.0. Anal.

Calcd for C₁₈H₁₅BrN₆O₂S (459.32): C, 47.07; H, 3.29; N, 18.30. Found: C, 47.29; H, 3.56; N,

18.66.

10

4.1.5. 4-(8-Amino-9-cyano-1-(4-bromophenyl)-3H-pyrazolo[1,5-c]-pyrrolo[3,2-e]pyrimi-

din-3-yl)benzenesulfonamide (9)

A mixture of 8 (0.459 g, 0.01 mol) and malononitrile (0.079 g, 0.0012 mol) in ethanol (20

mL) containing sodium ethoxids (0.023 g, 0.01 mole) was refluxed for 6h. The reaction

mixture was cooled and acidified with diluted HCl. The obtained residue was filtered and

recrystallized from dioxane to give 9. Yield % 73, m.p. 237.8 ^oC; IR (KBr, cm^-1): 3386, 3350,

3210 (NH₂), 2198 (C≡N), 1591 (C=N), 1325, 1161 (SO₂). ¹H-NMR (DMSO-d₆) δ: 4.2 [s, 2H,

NH₂, D₂O-exchangeable], 7.2- 8.1 [m, 10H, Ar-H + SO₂NH₂], 8.1 [s, 1H, CH pyrrole], 8.6 [s,

1H, CH pyrimidine]. ¹³C-NMR (DMSO-d₆) δ: 101.4, 109.6, 116.5, 120.5, 125.4 (2), 126.8,

128.9 (2), 129.3, 130.7 (2), 131.5, 131.9 (2), 132.2, 139.1, 140.6, 142.2, 150.0, 154.4. Anal.

Calcd. for C₂₁H₁₄BrN₇O₂S (508.35): C, 49.62; H, 2.78; N, 19.29. Found: C, 49.31; H, 2.48; N,

19.55.

4.1.6. Ethyl 9-(4-bromophenyl)-7-(4-sulfamoylphenyl)-7H-pyrrolo[3,2-e][1,2,4]triazolo-

[1,5-c]pyrimidine-2-carboxylate (10)

A mixture of 8 (4.59 g, 0.01 mol) and diethyloxalate (5 mL) was refluxed for 20h. The

reaction mixture was then concentrated at reduced presure and left to cool. The solid product

formed was filtered off and recrystallized from ethanol to give 10. Yield % 82, m.p. 309.2 ^oC;

IR (KBr, cm^-1): 3340, 3255 (NH₂), 3076 (CH arom.), 2926, 2861 (CH aliph.), 1728 (C=O),

1622 (C=N), 1336, 1163 (SO₂). ¹H-NMR (DMSO-d₆) δ: 1.3 [t, 3H, CH₃], 4.3 [q, 2H, CH₂], 7.4

[s, 1H, CH pyrrole], 7.5- 8.1 [m, 10H, Ar-H + SO₂NH₂], 9.8 [s, 1H, CH pyrimidine]. ¹³C-NMR

(DMSO-d₆) δ: 14.0, 62.6, 112.7, 119.6, 124.5 (2), 124.8, 126.9 (2), 129.4, 130.6 (2), 133.6 (2),

137.3, 138.1, 142.8, 143.2, 153.7, 155.2, 157.3, 161.3. Anal. Calcd for C₂₂H₁₇BrN₆O₄S

(541.38): C, 48.81; H, 3.17; N, 15.52. Found: C, 48.58; H, 3.51; N, 15.19.

4.1.7. Ethyl 2-(9-(4-bromophenyl)-7-(4-sulfamoylphenyl)-7H-pyrrolo[3,2-e][1,2,4]triaz-

olo[1,5-c]pyrimidin-2-yl)acetate (11)

A suspension of compound 8 (4.59 g, 0.01 mol) and diethylmalonate (10 mL) was heated

under refluxed for 12h. The obtained product was filtered and recrystallized from dioxane to

o

give 11. Yield % 59, m.p. 268.3 C; IR (KBr, cm^-1): 3348, 3234 (NH₂), 3066 (CH arom.),

2987, 2876 (CH aliph.), 1716 (C=O), 1625 (C=N), 1363, 1157 (SO₂). ¹H-NMR (DMSO-d₆) δ:

1.2 [t, 3H, CH₃ethyl], 4.0 [s, 2H, CH₂], 4.1 [q, 2H, CH₂ethyl], 7.5 [s, 1H, CH pyrrole], 7.6-

8.3 [m, 10H, Ar-H + SO₂NH₂], 9.2 [s, 1H, CH pyrimidine]. ¹³C-NMR (DMSO-d₆) δ: 14.0,

38.9, 60.8, 103.6, 117.0, 124.6 (2), 124.8, 126.8 (2), 129.2, 131.3 (2), 131.7 (2), 136.2, 139.2,

11

141.3, 142.7, 149.0, 152.9, 160.9, 168.6. Anal. Calcd for C₂₃H₁₉BrN₆O₄S (555.40): C, 49.74;

H, 3.45; N, 15.13. Found: C, 49.39; H, 3.12; N, 15.48.

4.1.8. 4-(2-Acetylmethyl-9-(4-bromophenyl)-7H-pyrrolo[3,2-e][1,2,4]triazolo[1,5-c]pyri-

midin-7-yl)benzenesulfonamide (12)

A mixture of 8 (4.59 g, 0.01 mol) and ethylacetoacetate (2.60 g, 0.02 mol) was heated at

220^oC in an oil bath for 4h. After cooling the product was collected by filteration and

recrystallized from ethanol to give 12. Yield % 75, m.p. 320.0 ^oC; IR (KBr, cm^-1): 3390, 3282

(NH₂), 3074 (CH arom.), 2961, 2846 (CH aliph.), 1710 (C=O), 1624 (C=N), 1336, 1163 (SO₂).

¹H-NMR (DMSO-d₆) δ: 2.1 [s, 3H, COCH₃], 4.1 [s, 2H, CH₂], 7.5 [s, 1H, CH pyrrole], 7.6- 8.2

13

[m, 10H, Ar-H + SO₂NH₂], 9.4 [s, 1H, CH pyrimidine]. C-NMR (DMSO-d₆) δ: 30.9, 49.1,

103.4, 117.0, 119.9 (2), 124.0, 124.4 (2), 126.7, 129.2 (2), 131.2 (2), 135.8, 139.2, 142.5,

145.6, 149.3, 152.8, 163.5, 205.3. Anal. Calcd for C₂₂H₁₇BrN₆O₃S (525.38): C, 50.29; H, 3.26;

N, 16.00. Found: C, 50.56; H, 3.48; N, 16.33.

4.1.9. 4-(2-Carboethoxy-9-(4-bromophenyl)-7H-pyrrolo[3,2-e][1,2,4]triazolo[1,5-c]py-

rimidin-7-yl)benzenesulfonamide (14)

A mixture of 8 (4.59 g, 0.01 mol) and ethylchloroformate (1.4 g, 0.015 mole) in benzene (20

mL) was refluxed for 8h. The reaction mixture was then filtered off and recrystallized from

ethanol to give 14.

o

Yield % 82, m.p. 229.2 C; IR (KBr, cm^-1): 3241, 3374 (NH₂), 3100 (CH arom.), 2966, 2817

(CH aliph.), 1732, 1645 (2C=O), 1593 (C=N), 1373, 1159 (SO₂). ¹H-NMR (DMSO-d₆) δ: 1.1

[t, 3H, CH₃], 4.0 [q, 2H, CH₂], 7.0 [s, 1H, CH pyrrole], 7.4- 8.1 [m, 10H, Ar-H + SO₂NH₂], 8.7

[s, 1H, CH pyrimidine]. ¹³C-NMR (DMSO-d₆) δ: 14.0, 59.7, 100.0, 118.6, 120.8, 121.3 (2),

124.7, 126.5, 130.7 (2), 130.8 (2), 132.0 (2), 138.1, 143.2, 145.1, 147.5, 152.3, 163.2, 170.3.

Anal. Calcd for C₂₂H₁₇BrN₆O₅S (557.38): C, 47.41; H, 3.07; N, 15.08. Found: C, 47.09; H,

3.36; N, 15.39.

4.1.10. 9-(4-Bromophenyl)-7H-pyrrolo[3,2-e][1,2,3,4]tetrazolo[1,5-c]pyrimidin-7-yl)ben-

zenesulfonamide (15)

To a cold solution of 8 (4.59 g, 0.01 mol) in acetic acid (20 mL) was added an ice-cold

solution of sodium nitrite (2.07 g; 0.03 mol) in H₂O (10 mL) with stirring during 5 min. Strring

was then continued for 3h. The reaction mixture was poured onto ice/ water and the solid

formed was filtered off and recrystallized from dioxene to give 15. Yield % 78, m.p. 278.3

12

1

^oC; IR (KBr, cm^-1): 3325, 3265 (NH₂), 3100 (CH arom.), 1591 (C=N), 1384, 1163 (SO₂). H-

NMR (DMSO-d₆) δ: 6.8 [s, 1H, CH pyrrole], 7.4- 8.3 [m, 10H, Ar-H + SO₂NH₂], 8.6 [s, 1H,

CH pyrimidine]. ¹³C-NMR (DMSO-d₆) δ: 106.3, 119.7, 123.2 (2), 126.3, 127.1 (2), 130.3,

131.8 (2), 132.4 (2), 136.7, 139.4, 142.2, 146.5, 155.8, 158.4. Anal. Calcd for C₁₈H₁₂BrN₇O₂S

(470.3): C, 45.97; H, 2.57; N, 20.85. Found: C, 45.62; H, 2.80; N, 20.54.

4.1.11. 4-(2-Oxo-10-(4-bromophenyl)-3,4-dihydropyrrolo[2,3-d]pyrimido [3,4-e][1,2,4]-

triazin-8-yl)benzenesulfonamide (16)

A mixture of 8 (4.59 g, 0.01 mol) and ethyl chloroacetate (1.2 g, 0.0 mole) was refluxed in

ethanol (30 mL) containing sodim ethoxide (0.23 g, 0.01 mol) for 22h, the reaction mixture

was then cooled and poured onto ice water, the solid separated was recrystallized from dioxane

to give 16. Yield % 75, m.p. 292.7 ^oC; IR (KBr, cm^-1): 3323, 3296, 3110 (NH, NH₂), 3100 (CH

arom.), 1654 (C=O), 1593 (C=N), 1327, 1161 (SO₂). ¹H-NMR (DMSO-d₆) δ: 4.7 [s, 2H, CH₂],

13

6.9 [s, 1H, CH pyrrole], 7.4- 8.1 [m, 10H, Ar-H + SO₂NH₂], 8.7 [s, 1H, CH pyrimidine]. C-

NMR (DMSO-d₆) δ: 55.9, 100.0, 118.6, 120.9, 121.3 (2), 124.8, 126.9, 129.3 (2), 131.3 (2),

132.0 (2), 138.1, 143.2, 147.5, 152.3, 160.9, 171.0. Anal. Calcd for C₂₀H₁₅BrN₆O₃S (499.34):

C, 48.11; H, 3.03; N, 16.83. Found: C, 48.46; H, 3.29; N, 16.48.

4.1.12. 4-(2,3-Dioxo-10-(4-bromophenyl)-3,4-dihydropyrrolo[2,3-d]pyrimido[3,4-e][1,2,-

4]triazin-8-yl)benzenesulfonamide (18)

To a solution of 8 (4.59 g, 0.01 mol) in dry benzene (20 mL), oxalyl chloride (1.26 g, 0.01

mol) was added. The reaction mixture was heated under reflux for 12h. The solid formed was

collected by filteration and recrystallized from dimethylformamide-ethanol (1: 2) to give 18.

Yield % 74, m.p. > 350; IR (KBr, cm^-1): 3439, 3376 (NH, NH₂), 3086 (CH arom.), 1745, 1737

(2C=O), 1627 (C=N), 1371, 1172 (SO₂). ¹H-NMR (DMSO-d₆) δ: 7.2 [s, 1H, CH pyrrole], 7.3-

8.4 [m, 10H, Ar-H + SO₂NH₂], 9.2 [s, 1H, CH pyrimidine], 9.7 [s, 1H, NH, D₂O-

13

exchangeable]. C-NMR (DMSO-d₆) δ: 102.5, 119.7, 123.6, 124.7 (2), 125.5, 126.5, 129.3

(2), 130.6 (2), 135.1 (2), 136.8, 141.7, 148.3, 157.3, 160.8, 168.8, 172.9. Anal. Calcd for

C₂₀H₁₃BrN₆O₄S (513.32): C, 46.80; H, 2.55; N, 16.37. Found: C, 46.57; H, 2.72; N, 16.06.

4.1.13. p-{3-(p-Bromophenyl)-11,13-dimethyl-5.7.9.10.14-pentazatricyclo[7.5.0.0^2,6]tetra-

deca-1(14),2(6),3,7,10-pentaen-5-yl}benzenesulfonamide (19)

A mixture of 8 (4.59 g, 0.01 mol) and acetylacetone (1.0g, 0.01 mol) in ethanol (30 mL) was

refluxed for 4h. After cooling the solid formed was collected and recrystallized from dioxane

13

o

to give 19. Yield % 54, m.p. 293.0 C; IR (KBr, cm^-1): 3392, 3284 (NH₂), 3078 (CH arom.),

1

2976, 2836 (CH aliph.), 1624 (C=N), 1334, 1165 (SO₂). H-NMR (DMSO-d₆) δ: 1.0 [s, 3H,

CH₃], 1.9 [s, 3H, CH₃], 7.4 [s, 1H, CH pyrrole], 7.5- 8.3 [m, 10H, Ar-H + SO₂NH₂], 8.4 [s, 1H,

13

CH triazepine], 9.4 [s, 1H, CH pyrimidine]. C-NMR (DMSO-d₆) δ: 19.3, 29.0, 103.5, 114.2,

117.0, 120.0, 124.0 (2), 124.5, 126.8, 129.2 (2), 131.3 (2), 131.7 (2), 135.9, 141.3, 142.5,

148.9, 154.6, 158.8, 163.6. Anal. Calcd for C₂₃H₁₉BrN₆O₂S (523.41): C, 52.78; H, 3.66; N,

16.06. Found: C, 52.46; H, 3.39; N, 16.37.

4.1.14. p-{13-Amino-3-(p-bromophenyl)-12-cyano-5, 7, 9, 10, 14-pentazatricyclo-[7.5.-0-

.0^2,6]-tetradeca-1(14),2(6),3,7,10,12-hexaen-5-yl}benzenesulfonamide (20)

A mixture of 8 (4.59 g, 0.01 mol) and ethoxymethylenemalononitrile (1.22 g, 0.01 mol) in

absolute ethanol (20 mL) was refluxed for 8h. The precipitate formed after cooling was filtered

off and recrystallized from acetic acid to give 20. Yield % 67, m.p. 356.8 ^oC; IR (KBr, cm^-1):

1

3400, 3265, 3130 (NH₂), 3066 (CH arom.), 2193 (C≡N), 1618 (C=N), 1371, 1165 (SO₂). H-

NMR (DMSO-d₆) δ: 6.7 [s, 2H, NH₂, D₂O-exchangeable], 7.5 [s, 1H, CH pyrrole], 7.6- 8.4 [m,

10H, Ar-H + SO₂NH₂], 8.5 [s, 1H, CH triazepine], 9.4 [s, 1H, CH pyrimidine]. ¹³C-NMR

(DMSO-d₆) δ: 73.9, 103.9, 117.0, 119.9, 124.4, 124.7 (2), 126.7, 127.1, 129.0 (2), 130.6 (2),

131.9 (2), 136.2, 139.2, 148.2, 154.1, 166.8, 176.9, 178.3. Anal. Calcd for C₂₂H₁₅BrN₆O₂S

(535.38): C, 49.36; H, 2.82; N, 20.93. Found: C, 49.08; H, 2.49; N, 21.29.

4.1.15. p-{13-Amino-3-(p-bromophenyl)-12-ethoxycarbonyl- 5, 7, 9, 10, 14- pentazatricy-

clo [7.5.0.0^2,6]tetradeca-1(14),2(6),3,7,10,12-hexaen-5-yl}benzenesulfonamide (21)

A mixture of 8 (4.59 g, 0.01 mol) and ethyl ethoxymethylenecyanoacetate (1.6 g, 0.01 mol)

in absolute ethanol (30 mL) was refluxed for 8h. After cooling the solid precipitate was

o

filtered off and recrystallized from dioxane to give 21. Yield % 90, m.p. 368.7 C; IR (KBr,

cm^-1): 3400, 3267, 3130 (NH₂), 3082 (CH arom.), 2976, 2863 (CH aliph.), 1733 (C=O), 1620

1

(C=N), 1383, 1165 (SO₂). H-NMR (DMSO-d₆) δ: 1.0 [t, 3H, CH₃], 4.4 [q, 2H, CH₂], 7.5 [s,

1H, CH pyrrole], 7.8- 8.4 [m, 10H, Ar-H + SO₂NH₂], 8.5 [s, 1H, CH triazepine], 9.5 [s, 1H,

CH pyrimidine]. ¹³C-NMR (DMSO-d₆) δ: 18.5, 56.0, 93.0, 104.0, 117.0, 119.9, 123.8 (2),

124.4, 126.7, 129.0 (2), 130.6 (2), 131.5 (2), 136.2, 139.2, 148.2, 154.1, 161.6, 165.8, 167.2,

172.1. Anal. Calcd for C₂₄H₂₀BrN₇O₄S (582.43): C, 49.49; H, 3.46; N, 16.83. Found: C, 49.12;

H, 3.81; N, 16.54.

4.1.16. p-{5-(p-Bromophenyl)-2, 7, 9, 11, 12, 20- hexazapentacyclo[11.7.0.0^3,11.0^4,8.0^14,19]-

icosa-1(20), 2, 4(8), 5, 9, 12, 14(19), 15, 17-nonaen-7-yl}benzenesulfonamide (22)

14

A mixture of 8 (4.59 g, 0.01 mol) and isatin (1.5 g, 0.01 mol) in absolute ethanol (20 mL)

was refluxed for 12h. The product which was separated during reflux was filtered off and

o

recrystallized from acetic acid to give 22. Yield % 82, m.p. 342.4 C; IR (KBr, cm^-1): 3354,

3290 (NH₂), 3061 (CH arom.), 1629 (C=N), 1330, 1157 (SO₂). ¹H-NMR (DMSO-d₆) δ: 7.3 [s,

13

1H, CH pyrrole], 7.4- 8.1 [m, 14H, Ar-H + SO₂NH₂], 9.4 [s, 1H, CH pyrimidine]. C-NMR

(DMSO-d₆) δ: 107.6, 119.6, 120.0, 120.4 (2), 122.1, 123.1, 124.7, 126.3, 126.9, 129.1 (2),

130.6 (2), 131.4, 131.9 (2), 132.9, 133.2, 142.7, 145.6, 148.2, 162.6, 164.2, 166.7, 170.1. Anal.

Calcd for C₂₆H₁₆BrN₇O₂S (570.42): C, 54.75; H, 2.83; N, 17.19. Found: C, 54.45; H, 2.43; N,

17.51.

4.1.17 4-(3-(1-Acetyl-2-oxoindolin-3-ylideneamino)-5-(4-bromophenyl)-4-imino-3,4-

dihydropyrrolo[2,3-d]pyrimidin-7-yl)benzenesulfonamide (23)

A mixture of 8 (4.59 g, 0.01 mol) and N-acetylisatin (1.75 g, 0.01 mol) in absolute ethanol

(30 mL) was refluxed for 10h. After cooling the product was filtered off and recrystallized

from dioxane to give 23.

Yield % 77, m.p. 340.3 ^oC; IR (KBr, cm^-1): 3298, 3210 (NH, NH₂), 1691, 1683 (2C=O), 1585

1

(C=N), 1338, 1161 (SO₂). H-NMR (DMSO-d₆) δ: 2.1 [s, 3H, COCH₃], 6.6 [s, 1H, CH

pyrrole], 7.2- 8.6 [m, 14H, Ar-H + SO₂NH₂], 9.4 [s, 1H, CH pyrimidine], 10.5 [s, 1H, NH,

D₂O-exchangeable]. ¹³C-NMR (DMSO-d₆) δ: 18.5, 110.1, 117.3, 118.1, 120.1, 120.8, 122.9

(2), 123.2, 124.5, 124.8, 127.0 (2), 130.1, 130.7 (2), 131.6, 131.9 (2), 132.3, 138.2, 141.5,

143.1, 144.0, 160.0, 164.3, 167.8, 173.8. Anal. Calcd for C₂₈H₂₀BrN₇O₄S (630.47): C, 53.34;

H, 3.20; N, 15.55. Found: C, 53.10; H, 3.57; N, 15.21.

4.1.18 4-(5-(4-Bromophenyl)-4-imino-3-(4-oxo-4-phenylbutan-2-ylidene-amino)-3,4-

dihydropyrrolo[2,3-d]pyrimidin-7-yl)benzenesulfonamide (24)

A mixture of 8 (4.59 g, 0.01 mol) and benzoylacetone (1.62 g, 0.01 mol) in absolute ethanol

(30 mL) was refluxed for 15h. The solvent was evaporated under reduced pressure and the

solid formed was collected and recrystallized from dioxane to give 24. Yield % 86, m.p. 249.4

^oC; IR (KBr, cm^-1): 3327, 3286 (NH, NH₂), 3084 (CH arom.), 1653 (C=O), 1591 (C=N), 1328,

1

1161 (SO₂). H-NMR (DMSO-d₆) δ: 0.9 [s, 3H, CH₃], 2.8 [s, 2H, CH₂], 6.6 [s, 1H, CH

pyrrole], 7.5- 8.3 [m, 15H, Ar-H + SO₂NH₂], 9.2 [s, 1H, CH pyrimidine], 9.6 [s, 1H, NH, D₂O-

exchangeable]. ¹³C-NMR (DMSO-d₆) δ: 18.6, 102.6, 109.0, 118.9, 120.9, 121.0 (2), 124.6,

125.0, 125.3 (2), 126.8 (2), 129.3 (2), 130.7 (2), 131.4 (2), 131.7, 138.5, 142.9, 144.5, 147.5,

15

152.5, 164.3, 165.8, 193.2. Anal. Calcd for C₂₈H₂₃BrN₆O₃S (603.49): C, 55.73; H, 3.84; N,

13.93. Found: C, 55.46; H, 3.50; N, 13.66.

4.1.19. p-{3-(p-Bromophenyl)-11-methyl-13-phenyl-5, 7, 9, 10, 14-pentazatricyclo-

[7.5.0.0^2,6] tetradeca-1(14),2(6), 3, 7, 10,12- hexaen-5-yl}benzenesulfonamide (25)

A solution of 24 (0.603 g, 0.001 mol) in POCl₃(20 mL) was heated under reflux for 6h. After

cooling the reaction mixture was poured onto a mixture of ice water and neutralized with

ammonium hydroxide solution. The solid formed was collected and recrystallized from ethanol

to afford 25. Yield % 69, m.p. 219.4 ^oC; IR (KBr, cm^-1): 3460, 3331 (NH₂), 3061 (CH arom.),

1

2970, 2926 (CH aliph.), 1627 (C=N), 1338, 1161 (SO₂). H-NMR (DMSO-d₆) δ: 0.9 [s, 3H,

CH₃], 5.9 [s, 1H, CH triazipene], 6.8 [s, 1H, CH pyrrole], 7.1- 8.2 [m, 15H, Ar-H + SO₂NH₂],

13

9.4 [s, 1H, CH pyrimidine]. C-NMR (DMSO-d₆) δ: 19.1, 108.9, 112.7, 119.6, 121.4, 122.6

(2), 124.7, 127.0, 127.4 (2), 128.0 (2), 128.6, 128.8 (2), 131.4 (2), 132.0 (2), 132.7, 134.2,

140.1, 143.2, 152.7, 157.4, 165.6, 168.8. Anal. Calcd for C₂₈H₂₁BrN₆O₂S (585.47): C, 57.44;

H, 3.62; N, 14.35. Found: C, 57.70; H, 3.29; N, 14.03.

4.2. Carbonic anhydrase inhibition assay

An Applied Photophysics stopped flow instrument has been used for assaying the CA catalysed

CO₂hydration activity. Phenol red (at a concentration of 0.2 mM) has been used as indicator,

working at the absorbance maximum of 557 nm, with 20 mM Hepes (pH 7.5) as buffer, and 20

mM Na₂SO₄(for maintaining constant the ionic strength), following the initial rates of the CA-

catalyzed CO₂hydration reaction for a period of 10–100 s. The CO₂concentrations ranged from

1.7 to 17 mM for the determination of the kinetic parameters and inhibition constants. For each

inhibitor at least six traces of the initial 5-10% of the reaction have been used for determining the

initial velocity. The uncatalyzed rates were determined in the same manner and subtracted from

the total observed rates. Stock solutions of inhibitor (0.1 mM) were prepared in distilled-ionized

water and dilutions up to 0.01 nM were done thereafter with the assay buffer. Inhibitor and

enzyme solutions were pre-incubated together for 15 min at room temperature or 4°C prior to

assay, in order to allow for the formation of the E-I complex. Data from Table 1 were obtained

after 15 minutes incubation of enzyme and inhibitor.⁴⁰The inhibition constants were obtained by

41

non-linear least squares methods using PRISM 3, as reported earlier and represent the mean

from three different determinations. All CA isoforms were recombinant ones obtained in-house

as reported earlier. ^{40, 42-47}

4.3. In vitro cytotoxic activity

16

Human tumor breast cell line (MCF-7) was used in this study. The cytotoxic activity was

measured in vitro for the newly synthesized compounds using the Sulfo-Rhodamine-B stain

(SRB) assay using the method of Skehan et al.⁴⁸The in vitro anticancer screening was done by

the pharmacology unit at the National Cancer Institute, Cairo University. Cells were plated in 96-

multiwell plate (10⁴cells/ well) for 24 h before treatment with the compound(s) to allow

attachment of cell to the wall of the plate. Test compounds were dissolved in dimethylsulfoxide.

Different concentrations of the compound under test (10, 25, 50, and 100 µM) were added to the

cell monolayer. Triplicate wells were prepared for each individual concentration. Monolayer

cells were incubated with the compound(s) for 48 h at 37 ^oC and in atmosphere of 5% CO₂. After

48 h, cells were fixed, washed and stained for 30 min with 0.4% (wt/vol) SRB dissolved in 1%

acetic acid. Excess unbound dye was removed by four washes with 1% acetic acid and attached

stain was recovered with Tris-EDTA buffer. Color intensity was measured in an ELISA reader.

The relation between surviving fraction and drug concentration is plotted to get the survival

curve for breast tumor cell line after the specified time. The molar concentration required for

50% inhibition of cell viability (IC₅₀) was calculated and compared to the reference drug

Doxorubicin (CAS, 25316-40-9). The surviving fractions were expressed as means ± standard

error.

4.4. Molecular Docking Study

All the molecular modeling studies were carried out on an Intel Pentium 1.6 GHz processor, 512

MB memory with Windows XP operating system using Molecular Operating Environment

(MOE, 10.2008) software. All the minimizations were performed with MOE until a RMSD

gradient of 0.05 Kcalmol^-1Å^-1with MMFF94X force field and the partial charges were

automatically calculated. The X-ray crystallographic structure of CA isozyme XII complexed

with acetazolamide (PDB ID: 1JD0) was obtained from the protein data bank. The enzymes were

prepared for docking studies where: (i) The co-crystallized ligand molecule (AZA) was removed

from the enzyme active site. (ii) Hydrogen atoms were added to the structure with their standard

geometry. (iii) MOE Alpha Site Finder was used for the active sites search in the enzyme

structure and dummy atoms were created from the obtained alpha spheres. (iv) The obtained

model was then used in predicting the ligand enzymes interactions at the active site. (v) Docking

protocol was verified by re-docking of the co-crystallized ligand (acetazolamide) in the vicinity

of the active site of the enzyme with energy score (S) = -36.3568 Kcal/ mol and root mean

standard deviation (RMSD) = 1.1976.

Acknowledgment

17

The authors would like to extend their sincere appreciation to the Deanship of Scientific

Research at King Saud University for its funding of this research through the Research Group

Project no. RGP-VPP-302. Research from CTS group was financed by two EU projects of FP7,

Metoxia and Dynano.

References

1- Mohamed, M.; Kamel, R.; Abd El-hameed, R. Med. Chem. Res. 2013, 22, 2244.

2- Hassan, S. M.; El-Maghraby, A. A.; Abdel Aal, M. M.; Bashandy, M. S. Phosphorus,

Sulfur, and Silicon and the Related Elements. 2009, 184, 291.

3- Hilmy, K. M.; Khalifa, M. M.; Hawata, M. A.; Keshk, R. M.; el-Torgman, A. A. Eur. J.

Med. Chem. 2010, 45, 5243.

4- Esteves, M. A.; Ortet, O.; Capelo, A.; Supuran, C. T.; Marques, S. M.; Santos, M. A.

Bioorg. Med. Chem. Lett. 2010, 20, 3623.

5- Evers, D. L.; Breitenbach, J. M.; Borysko, K. Z.; Townsend, L. B.; Drach, J. C.

Antimicrob. Agents Chemother. 2002, 46, 2470.

6- Boger, D. L.; Boyce, C. W.; Labroli, M. A.; Sehon, C. A.; Jin, Q. J. Am. Chem. Soc. 1998,

121, 54.

7- Ghorab, M. M.; Heiba, H. I.; Khalil, A. I.; Abou El Ella, D. A.; Noaman, E. Phosphorus,

Sulfur, and Silicon and the Related Elements. 2007, 183, 90.

8- Clark, M. P.; George, K. M.; Bookland, R. G.; Chen, J.; Laughlin, S. K.; Thakur, K. D.;

Lee, W.; Davis, J. R.; Cabrera, E. J.; Brugel, T. A.; VanRens, J. C.; Laufersweiler, M. J.;

Maier, J. A.; Sabat, M. P.; Golebiowski, A.; Easwaran, V.; Webster, M. E.; De, B.; Zhang,

G. Bioorg. Med. Chem. Lett. 2007, 17, 1250.

9- Merighi, S.; Mirandola, P.; Varani, K.; Gessi, S.; Leung, E.; Baraldi, P. G.; Tabrizi, M. A.;

Borea, P. A. Pharmacol. Ther. 2003, 100, 31.

10- Abou El Ella, D. A.; Ghorab, M. M.; Noaman, E.; Heiba, H. I.; Khalil, A. I. Bioorg. Med.

Chem. 2008, 16, 2391.

11- Turk, S. R.; Shipman, C., Jr.; Nassiri, R.; Genzlinger, G.; Krawczyk, S. H.; Townsend, L.

B.; Drach, J. C. Antimicrob. Agents Chemother. 1987, 31, 544.

12- Evers, D. L.; Breitenbach, J. M.; Borysko, K. Z.; Townsend, L. B.; Drach, J. C.

Antimicrob. Agents Chemother. 2002, 46, 2470.

13- El-Sabbagh, O. I. Arch. Pharm. 2013, 346, 733.

14- Gangapuram, M.; Mazzio, E.; Eyunni, S.; Soliman, K. F.; Redda, K. K. Arch. Pharm.

2014, 2, DOI: 10.1002/ardp.201300379.

15- Basanagouda, M.; Shivashankar, K.; Kulkarni, M. V.; Rasal, V. P.; Patel, H.; Mutha, S. S.;

Mohite, A. A. Eur. J. Med. Chem. 2010, 45, 1151.

16- Patrick, G. L.: Antibacterial agents. In An Introduction to Medicinal Chemistry. Part 2, 3

rd. ed.; Oxford university press: 2005, New York, USA 379.

17- Nasr, M. N.; Gineinah, M. M. Arch. Pharm. 2002, 335, 289.

18- Lu, X.; Chen, Y.; Guo, Y.; Liu, Z.; Shi, Y.; Xu, Y.; Wang, X.; Zhang, Z.; Liu, J. Bioorg.

Med. Chem. 2007, 15, 7399.

19- Mohamed, M. S.; Awad, S. M.; Sayed, A. I. Molecules. 2010, 15, 1882.

20- Huang, T.; Tu, H.; Aibibu, Z.; Hou, C.; Zhang, A. ARKIVOC, 2011, (ii), 1.

21- Sondhi, S. M.; Jain, S.; Dinodia, M.; Shukla, R.; Raghubir, R. Bioorg. Med. Chem. 2007,

15, 3334.

22- Nofal, Z. M.; Fahmy, H. H.; Zarea, E. S.; El-Eraky, W. Acta. Pol. Pharm. 2011, 68, 507.

18

23- El-Gazzar, A. R.; Hussein, H. A.; Hafez, H. N. Acta. Pharm. 2007, 57, 395.

24- Winum, J. Y.; Maresca, A.; Carta, F.; Scozzafava, A.; Supuran, C. T. Chem. Commun.

2012, 48, 8177.

25- a) Supuran, C. T.; Scozzafava A. Expert. Opin. Ther. Pat. 2000, 10, 575; b) Supuran, C.T.

Future Med. Chem. 2011, 3, 1165; c) Chohan, Z.H.; Supuran, C.T.; Scozzafava, A. J.

Enzyme Inhib. Med. Chem. 2005, 20, 303.

26- a) Supuran, C. T. Bioorg. Med. Chem. Lett. 2010, 20, 3467; b) Supuran, C.T. J. Enzyme

Inhib. Med. Chem. 2012, 27, 759.

27- Supuran, C. T. Nat. Rev. Drug Discov. 2008, 7, 168.

28- Neri, D.; Supuran, C. T. Nat. Rev. Drug Discov. 2011, 10, 767.

29- Supuran, C. T.; Scozzafava, A. Bioorg. Med. Chem. 2007, 15, 4336.

30- Jemal, A.; Bray, F.; Center, M. M.; Ferlay, J.; Ward, E.; Forman, D. CA Cancer J. Clin.

2011, 61, 69.

31- Thun, M. J.; DeLancey, J. O.; Center, M. M.; Jemal, A.; Ward E. M. Carcinogenesis.

2010, 31, 100.

32- a) Ghorab, M. M.; Ragab, F. A.; Heiba, H. I.; Youssef, H. A.; El-Gazzar, M. G. Bioorg.

Med. Chem. Lett. 2010, 20, 6316; b) Aggarwal, M.; Boone, C.D.; Kondeti, B.; McKenna,

R. J. Enzyme Inhib. Med. Chem. 2013, 28, 267.

33- Ghorab, M. M.; Ragab, F. A.; Alqasoumi, S. I.; Alafeefy, A. M.; Aboulmagd, S. A. Eur. J.

Med. Chem. 2010, 45, 171.

34- Ghorab, M. M.; Ragab, F. A.; Noaman, E.; Heiba, H. I.; Aboulmagd, S. A.

Arzneimittelforschung. 2009, 59, 96.

35- Bashandy, M. S.; Alsaid, M. S.; Arafa, R. K.; Ghorab, M. M. J. Enzyme Inhib. Med.

Chem. 2013, DOI:10.3109/14756366.2013.833197.

36- Al-Dosari, M. S.; Ghorab, M. M.; Alsaid, M. S.; Nissan, Y. M.; Ahmed, A. B. Eur. J.

Med. Chem. 2013, 69, 373-83

37- Al-Dosari, M. S.; Ghorab, M. M.; Al-Said, M. S.; Nissan, Y. M. Chem. Pharm. Bull. 2013,

61, 50.

38- Ghorab, M. M.; Al-Said, M. S.; Nissan, Y. M. Life Sci. J. 2013, 10, 2170.

39- Yusuf, M.; Khan, R. A.; Khan, M.; Ahmed, B. Chem. Biol. Drug Des. 2013, 81, 666.

40- Tars, K.; Vullo, D.; Kazaks, A.; Leitans, J.; Lends, A.; Grandane, A.; Zalubovskis, R.;

Scozzafava, A.; Supuran, C. T. J. Med. Chem. 2013, 56, 293.

41- Alterio, V.; Di Fiore, A.; D'Ambrosio, K.; Supuran, C. T.; De Simone, G. Chem. Rev.

2012, 112, 4421.

42- Maresca, A.; Temperini, C.; Vu, H.; Pham, N. B.; Poulsen, S. A.; Scozzafava, A.; Quinn,

R. J.; Supuran, C. T. J. Am. Chem. Soc. 2009, 131, 3057.

43- Touisni, N.; Maresca, A.; McDonald, P. C.; Lou, Y.; Scozzafava, A.; Dedhar, S.; Winum,

J. Y.; Supuran, C. T. J. Med. Chem. 2011, 54, 8271.

44- a) Carta, F.; Maresca, A.; Scozzafava, A.; Supuran, C. T. Bioorg. Med. Chem. Lett. 2012,

22, 267; b) Maresca, A.; Carta, F.; Vullo, D.; Supuran, C.T. J. Enzyme Inhib. Med. Chem.

2013, 28, 407.

45- a) Maresca, A.; Temperini, C.; Pochet, L.; Masereel, B.; Scozzafava, A.; Supuran, C. T. J.

Med. Chem. 2010, 53, 335; b) Fabrizi, F.; Mincione, F.; Somma, T.; Scozzafava, G.;

Galassi, F.; Masini, E.; Impagnatiello, F.; Supuran, C.T. J. Enzyme Inhib. Med. Chem.

2012, 27, 138; c) Borras, J.; Scozzafava, A.; Menabuoni, L.; Mincione, F.; Briganti, F.;

Mincione, G.: Supuran, C.T. Bioorg. Med. Chem. 1999, 7, 2397.

46- a) Davis, R. A.; Vullo, D.; Maresca, A.; Supuran, C. T.; Poulsen, S. A. Bioorg. Med.

Chem. 2013, 21, 1539; b) De Simone, G.; Alterio, V.; Supuran, C.T. Expert Opin. Drug

19

Discov. 2013, 8, 793; c) Supuran, C.T.; Vullo, D.; Manole, G.; Casini, A.; Scozzafava, A.

Curr. Med. Chem. – Cardiovasc. Hematol. Agents, 2004, 2, 49.

47- a) Kazancioglu, E. A.; Guney, M.; Senturk, M.; Supuran, C. T. J. Enzyme Inhib. Med.

Chem. 2012, 27, 880; ; b) Supuran, C.T.; Scozzafava, A.; Casini, A. Med. Res. Rev. 2003,

23, 146; c) Supuran, C.T. J. Enzyme Inhib. Med. Chem. 2013, 28, 229; d) Supuran, C.T.;

Mincione, F.; Scozzafava, A.; Briganti, F.; Mincione, G.; Ilies, M.A. Eur. J. Med. Chem.

1998, 33, 247.

48- Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMahon, J.; Vistica, D.; Warren, J. T.;

Bokesch, H.; Kenney, S.; Boyd, M. R. J. Natl. Cancer Inst. 1990, 82, 1107.

20

Table 1. hCA I, II, IX and XII inhibition data of compounds 5-25 and acetazolamide as

standard.

Compound

K_i* (nM)

SI**

hCA IX

hCA I

84.7

159.6

21.6

32.4

66

hCA II

hCA XII hCA I/XII hCA II/XII

4.6

3.2

6.3

3.9

0.74

31.6

114.46

5.05

6.22

0.12

5

6

3.8

4.9

2.7

2.4

2.7

3

211

70.8

365

184

79.2

9.3

0.10

0.46

0.01

0.03

0.01

0.02

0.04

0.28

0.30

15.39

356.36

9.19

3.44

3.61

3.75

3.83

2.98

1.06

2.12

8

9

4.7

0.18

10

11

12

14

15

16

18

19

20

21

22

23

24

25

AZA

27.6

33.2

23.2

28.4

94.1

69.1

25.4

80.4

126

24.5

123.7

4.5

0.15

3.1

2.6

2.8

11.7

325

49.6

3.1

3

0.42

2.49

5.3

9.2

3.09

4.9

0.76

0.912

5.4

123.82

75.77

4.70

64.3

18.2

5.2

0.9

89.33

151.81

691.67

30.99

17.34

8.29

7.3

0.83

0.72

0.81

0.94

3.4

498

8.5

2.7

3.1

2.8

3.6

25.0

25.1

16.3

28.2

250

5.6

3.9

7.1

12.1

5.7

43.86

AZA (Acetazolamide), a well known CAI, was used as a standard for comparison.

* K_ipresented is the mean from 3 different assays; errors are in the range of ± 5-10% of the

reported values (data not shown).

** SI (Selectivity index) is a ratio between the K_ivalues observed for two hCA isoforms; low

value index is indicative of weak selectivity.

21

Table 2

In vitro anticancer screening of the synthesized compounds against the human breast cancer

cell line MCF7.

Compound

Compound concentration (µM)

25µM 50µM

Surviving fraction (Mean ± S.E.)^*

IC₅₀(µM)

10µM

100µM

0.314±0.032 0.309±0.016 0.251±0.023 0.266±0.032

0.327±0.121 0.273±0.043 0.233±0.011 0.255±0.020

0.340±0.090 0.294±0.021 0.246±0.110 0.256±0.002

0.330±0.211 0.309±0.016 0.227±0.110 0.268±0.132

0.347±0.218 0.292±0.100 0.213±0.152 0.221±0.101

0.305±0.113 0.281±0.100 0.274±0.109 0.309±0.001

0.426±0.100 0.410±0.121 0.348±0.133 0.335±0.104

0.400±0.011 0.399±0.110 0.365±0.021 0.350±0.002

0.361±0.003 0.311±0.011 0.232±0.021 0.216±0.011

0.348±0.110 0.294±0.021 0.254±0.100 0.216±0.111

0.393±0.014 0.358±0.001 0.337±0.111 0.362±0.001

0.436±0.021 0.366±0.136 0.350±0.011 0.358±0.011

0.380±0.002 0.285±0.018 0.332±0.006 0.375±0.001

0.382±0.021 0.380±0.031 0.401±0.058 0.379±0.110

0.372±0.001 0.377±0.012 0.353±0.032 0.391±0.011

0.347±0.111 0.338±0.035 0.315±0.012 0.381±0.008

0.342±0.022 0.287±0.018 0.259±0.002 0.326±0.001

0.381±0.001 0.333±0.008 0.251±0.001 0.301±0.039

0.303±0.002 0.306±0.006 0.277±0.011 0.280±0.011

8.02

7.56

7.29

7.56

7.29

8.94

7.56

7.84

8.11

8.66

7.84

8.11

7.84

7.56

7.84

7.29

Doxorubicin

5

6

8

9

10

11

12

14

15

16

18

19

20

21

22

23

24

25

* Each value is the mean of three values ± Standard Error.

22

Table 3. Docking results of the synthesized sulfonamide derivatives with CA XII using MOE

software version 2008.10.

Compound

S

Amino

acids

Thr199

His94

Interacting Type of interaction Length

(Kcal/mol)

-41.3027

groups

SO₂

(A^o)

2.41

3.00

1.49

2.06

2.03

2.04

2.73

2.90

H-bond (acceptor)

H-bond (donor)

5

SO₂

NH₂

SO₂NH₂

SO₂

Metal complex (Zn)

H-bond (acceptor)

Solvent (H₂O)

His96

His119

His199

Thr91

Gln92

CN

Solvent (H₂O)

-19.9791

-13.0846

-23.6440

-12.7252

-15.3873

-13.2802

-35.6833

-35.7456

His94

His 6

His119

SO₂NH₂

Metal complex (Zn)

2.06

2.03

2.04

6

His94

His96

His119

SO₂NH₂

Metal complex (Zn)

2.06

2.03

2.04

8

His94

His96

His119

SO₂NH₂

Metal complex (Zn)

2.06

2.03

2.04

9

His94

His96

His119

SO₂NH₂

Metal complex (Zn)

2.06

2.03

2.04

10

11

12

14

15

His94

His96

His119

SO₂NH₂

Metal complex (Zn)

2.06

2.03

2.04

His94

His96

His119

SO₂NH₂

Metal complex (Zn)

2.06

2.03

2.04

His94

His96

His119

SO₂NH₂

Metal complex (Zn)

2.06

2.03

2.04

His94

SO₂NH₂

Metal complex (Zn)

2.06

23

His96

His119

SO₂NH₂

Metal complex (Zn)

2.03

2.04

-52.7571

Thr199

His94

His96

His119

Thr200

Asn62

SO₂

SO₂NH₂

SO₂

H-bond (acceptor)

Metal complex (Zn)

H-bond (acceptor)

Solvent (H₂O)

3.57

2.06

2.03

2.04

2.93

3.44

2.42

16

NH

C=O

Solvent (H₂O)

-51.5937

Thr199

His94

His96

His119

His96

His119

Thr91

Gln92

SO₂

NH₂

SO₂NH₂

NH₂

SO₂

C=O

H-bond (acceptor)

Metal complex (Zn)

H-bond (acceptor)

Solvent (H₂O)

2.64

2.93

1.35

2.06

2.03

2.04

2.34

3.10

2.47

2.58

18

-32.2959

-49.5079

His94

His96

His119

SO₂NH₂

Metal complex (Zn)

2.06

2.03

2.04

19

20

Thr199

His94

His96

SO₂

H-bond (acceptor)

Metal complex (Zn)

2.87

2.06

2.03

2.04

SO₂NH₂

His119

-46.6627

-41.5627

Thr199

His94

His96

His119

SO₂

NH₂

SO₂NH₂

H-bond (acceptor)

H-bond (donor)

Metal complex (Zn)

2.76

1.68

2.06

2.03

2.04

21

22

Thr199

His119

His94

His96

His119

SO₂

SO₂NH₂

H-bond (acceptor)

Metal complex (Zn)

3.70

3.20

2.06

2.03

2.04

24

-40.2157

-44.9134

-36.0029

Thr199

His94

His96

SO₂

H-bond (acceptor)

Metal complex (Zn)

3.70

2.06

2.03

2.04

23

42

25

SO₂NH₂

His119

Thr199

His94

His96

SO₂

H-bond (acceptor)

Metal complex (Zn)

3.66

2.06

2.03

2.04

SO₂NH₂

His119

His94

His96

His119

SO₂NH₂

Metal complex (Zn)

2.06

2.03

2.04

25

Br

NH₂

HN

CN

O

NC

CN

DMF

TEA

Br

C

+

N

NaOEt

NH

Br

SO₂NH₂

SO2NH2

2

1

3

Br

SO₂NH₂

4

CN

CH(OC H )

2

5 3

OEt

N

NH₂

N

Br

SO₂NH₂

NH₂

SO2NH2

6

NC

5

N

EtOH

N₂H₄.H₂O

Br

NC

CN

N

HN

NaOEt

H

N

C

NH2

NH₂

N

SO₂NH₂

Br

R

N

9

N

EtO

OEt

O

SO₂NH₂

i)

8

O

N

7

N

O

ii)

OEt

O

OEt

O

iii)

OEt

SO2NH2

10; R= COOEt

11; R= CH₂COOEt

12

; CH₂COCH₃

Scheme 1: Synthesis of pyrrole and pyrrolopyrimidine derivatives 5- 12.

26

Br

O

Br

O

N

NH

N

SO2NH2

O

15

SO₂NH₂

Cl

SO₂NH₂

O

Cl

18

19

O

E

t

O

H

O

H

h

NaNO₂

Br

P

Br

O

y

r

D

O

OEt

HN

N

NH

O

N

O

Cl

O

8

N

NaOEt

Excess

N

O

Br

SO₂NH₂

N

NH

13

16

N

- EtOH

O

Br

N

O

N

O

N

SO₂NH₂

17

SO₂NH₂

14

Scheme 2: Synthesis of triazolo, tetrazolo, triazino, and triaziepenopyrrolopyrimidine derivatives.

27

O

CN

H2N

Br

H₂N

Br

N

O

H

CN

N

O

H

CN

O

N

O

N

8

EtOH

SO₂NH₂

SO2NH2

O

20

21

O

N

O

N

O

H

O

EtOH

N

O

Br

N

Br

N

Br

O

HN

N

HN

N

P

O

C

l

3

SO2NH2

22

SO₂NH₂

Br

SO₂NH₂

23

24

N

25

SO₂NH₂

Scheme 3

: Synthesis of triaziepeno, triazinopyrrolopyrimidine and pyrrolopyrimidine derivatives

28

Figure 1. 2D interaction of AZA with the active site amino acids of CA XII: (S) = -36.3568

Kcal/ mol (RMSD) = 1.1976.

Figure 2. 2D enzyme-ligand interaction of compound 16 with CA XII: energy score (S) = -

52.7571 Kcal/ mol.

29

Article Doi

Carbonic anhydrase inhibitors: Synthesis, molecular docking, cytotoxic and inhibition of the human carbonic anhydrase isoforms I, II, IX, XII with novel benzenesulfonamides incorporating pyrrole, pyrrolopyrimidine and fused pyrrolopyrimidine moieties

DOI: 10.1016/j.bmc.2014.05.009

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Authors:

Article abstract of DOI:10.1016/j.bmc.2014.05.009

Full text of DOI:10.1016/j.bmc.2014.05.009

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