Design and synthesis of N1-aryl-benzimidazoles 2-substituted as novel HIV-1 non-nucleoside reverse...

Accepted Manuscript

Design and synthesis of N 1-aryl-benzimidazoles 2-substituted as novel HIV-1non-nucleoside reverse transcriptase inhibitors

Anna-Maria Monforte, Stefania Ferro, Laura De Luca, Giuseppa Lo Surdo,Francesca Morreale, Christophe Pannecouque, Jan Balzarini, Alba Chimirri

PII: S0968-0896(13)01053-5DOI: http://dx.doi.org/10.1016/j.bmc.2013.12.045Reference: BMC 11307

To appear in: Bioorganic & Medicinal Chemistry

Received Date: 21 October 2013Revised Date: 11 December 2013Accepted Date: 17 December 2013

Please cite this article as: Monforte, A-M., Ferro, S., Luca, L.D., Surdo, G.L., Morreale, F., Pannecouque, C.,Balzarini, J., Chimirri, A., Design and synthesis of N 1-aryl-benzimidazoles 2-substituted as novel HIV-1 non-nucleoside reverse transcriptase inhibitors, Bioorganic & Medicinal Chemistry (2013), doi: http://dx.doi.org/10.1016/j.bmc.2013.12.045

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Graphical Abstract

Design and synthesis of N1-aryl-benzimidazoles

2-substituted as novel HIV-1 non-nucleoside reverse

transcriptase inhibitors

Anna-Maria Monfortea,*

, Stefania Ferroa, Laura De Luca

a, Giuseppa Lo Surdo

a, Francesca Morreale

a,

Christophe Pannecouqueb, Jan Balzarini

b and Alba Chimirri

a

aDipartimento di Scienze del Farmaco e Prodotti per la Salute, Università di Messina,

Viale Annunziata, I-98168 Messina, Italy bRega Institute for Medical Research, Katholieke Universiteit Leuven, Minderbroedersstraat 10, B-3000

Leuven, Belgium

N

NRX

Me

Me

SNH

OR'

Leave this area blank for abstract info.

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Bioorganic & Medicinal Chemistry journal homepage: www.e lsevier .com

Design and synthesis of N1-aryl-benzimidazoles 2-substituted as novel HIV-1 non-

nucleoside reverse transcriptase inhibitors

Anna-Maria Monfortea,*

, Stefania Ferroa, Laura De Luca

a, Giuseppa Lo Surdo

a, Francesca Morreale

a,

Christophe Pannecouqueb, Jan Balzarini

b and Alba Chimirri

a

aDipartimento di Scienze del Farmaco e Prodotti per la Salute, Università di Messina, Viale Annunziata, I-98168 Messina, Italy bRega Institute for Medical Research, Katholieke Universiteit Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium

1. Introduction

Human immunodeficiency virus type 1 reverse transcriptase

(HIV-1 RT) is a heterodimer of p66 (66 kDa) and p51 (51 kDa)

subunits, targeted by almost half of the approved anti-AIDS

drugs.

Particularly, Non-Nucleoside Reverse Transcriptase Inhibitors

(NNRTIs) have gained an important place in clinical use based

on their unique antiviral potency with generally low toxicity and

favourable pharmacokinetic properties.1

More than 50 structurally diverse classes of compounds have

been identified as NNRTIs, which specifically suppress HIV-1

replication.2-6

NNRTIs bind a specific and allosteric site in the RT which is a

flexible pocket formed upon the binding with the inhibitor

located about 10Å from the polymerase active site. Although

NNRTIs belong to different and diverse chemical families, they

share a common and unique mechanism of action: their

interaction with HIV-1 reverse transcriptase induces

conformational changes that inhibit the catalytic activities of the

enzyme.

Nevertheless, to date only five molecules have been approved

for clinical use: nevirapine, delavirdine, efavirenz, etravirine and

more recently rilpivirine.2, 7, 8

Like other types of anti-HIV drugs,

the therapeutic efficacy of these inhibitors has been limited by

the emergence of drug-resistant mutants as well as the severe side

effects, therefore identification of new NNRTIs active against

relevant mutant strains (such as Y188C, Y181C, K103N, and

L100I) and with improved pharmacokinetic profile is a constant

goal for the development of new drugs able to combat the

growing AIDS pandemic.1

Fortunately, the structural diversity of NNRTIs and the flexibility

of the binding pocket in RT provided a wide space for novel lead

discovery, and the pharmacophore similarity of NNRTIs gave

valuable hints for lead discovery and optimization.

In our previous papers 9-15

we reported structure-based molecular

modeling and docking approaches which led to the rational

discovery of a series of novel N1-substituted 6-chloro-1,3-

dihydro-2H-benzimidazol-2-ones and their 2-thione analogues

(1) as potent HIV-1 non-nucleoside reverse transcriptase

inhibitors. Structure–activity relationships (SAR) studies

highlighted that 3,5-dimethylphenyl moiety at N-1 of

benzimidazolone system showed very low toxicity and potent

antiretroviral activity similar to that of efavirenz and higher than

nevirapine against both wilde type and some mutant strains of

HIV-1 RT (Fig 1).

Considering compounds 1 as a starting point for lead

optimization strategy and taking into account potent NNRTIs

especially active against key mutants resistant to the clinically

used NNRTIs, we herein report the rational design, SAR of new

N1-aryl-2-arylthioacetamido-benzimidazoles with the aim of

improving the activity of this class of compounds as well as their

ART ICLE INFO AB ST R ACT

Article history:

Received

Received in revised form

Accepted

Available online

A series of novel N1-aryl-2-arylthioacetamido-benzimidazoles were synthesized and evaluated

as inhibitors of human immunodeficiency virus type-1 (HIV-1). Some of them proved to be

effective in inhibiting HIV-1 replication at submicromolar and nanomolar concentration acting

as HIV-1 non-nucleoside RT inhibitors (NNRTIs), with low cytotoxicity. The preliminary

structure-activity relationship (SAR) of these new derivatives was discussed and rationalized by

docking studies.

2009 Elsevier Ltd. All rights reserved.

Keywords:

HIV-1 reverse transcriptase

NNRTIs

N1-aryl-benzimidazoles 2-substituted

synthesis

*Corresponding author:Phone 00390906766477. Fax

00390906766402. e-mail: [email protected]

mailto:[email protected]

selective index. Finally, docking results are herein reported to

further clarify their mode of binding.

NH

NCl

Y

X

Me

Me

X=CH2, SO

2

Y= O, S1

Figure 1. Structure of N1-substituted 6-chloro-1,3-dihydro-2H-benzimidazol-

2-ones and 2-thione analogues

2. Results and discussion

2.1 Rational design

Numerous compounds which present a thioacetamide linker

have been reported as promising NNRT inhibitors. Particularly

some sulfanyltriazole/tetrazole derivatives demonstrated high

potency in inhibiting HIV-1 proliferation at nanomolar

concentration (Fig. 2).3, 16-21

It has been shown that a suitable

combination of substitution patterns both on the aryl linked to the

tetrazole/triazole core and the anilide moiety, lead to the

identification of compounds which maintain the same intrinsic

activity on the wild-type HIV-1 enzyme and the clinically

relevant mutant strain (K103N/Y181C). Currently, one of these

derivatives (RDEA-806) is being considered for further clinical

trials for the treatment of HIV infection (Fig.2).

1, 22-24

N

NN

SBrNH

O

Cl

OH O

N

NN

X SNH

O

R'

R''

R

RDEA-806Tetr(tri)azole-based NNRTIs

Figure. 2

On these bases, as extension of our ongoing efforts towards

the development and identification of new molecules with anti-

HIV activity, we designed and planned the synthesis of new 2-

[1-(3,5-dimethylbenzyl)-1H-benzimidazol-2-yl]sulfanyl-N-

phenylacetamides and 2-(1-[(3,5-dimethylphenyl)sulfonyl]-1H-

benzimidazol-2-ylsulfanyl)-N-phenylacetamides (2-8) (Fig.3) as

new potential NNRTIs.

The new synthesized 2-arylthioacetamidobenzimidazole

derivatives N1-substituted (2-8) keep in their structure the

benzimidazole system of derivatives 1 and the 3,5-

dimethylphenyl substituent which had proved to improve the

anti-HIV activity and the selectivity index (SI) of the previously

studied molecules.

Four subsets (a-d) of compounds were designed and

synthesized containing H or Cl at C6 atom of benzimidazole, a

methylene or a sulfonyl spacer between the heterocyclic ring and

3,5-dimethylphenylic moiety. Different substituents have been

inserted at orto or/and para position of the arylthioacetamide

group with the aim to study their influence on anti-HIV and anti-

RT activity.

N

NRX

Me

Me

SNH

OR'

2-8

Figure 3. Structure of N1-aryl-benzimidazoles 2-substituted

2.2. Chemistry

The synthesis of new benzimidazole derivatives was achieved

following the reaction sequence straightforward depicted in

Scheme 1.

The appropriate 2-nitroanilines were N-substituted by

treatment with 3,5-dimethyl-benzylbromides or 3,5-dimethyl-

benzensulphonylchloride in the presence of sodium hydride to

give the desired products 9a-d in high yields. The obtained nitro-

intermediates 9a-d were reduced by reflux with Zn dust in

ethanol and acid medium for concentrated hydrochloric acid.

The ring closure reaction of aminoderivatives 10a-d was

easily accomplished via the reaction with

thiocarbonyldiimidazole (TCDI) that rapidly and quantitatively

reacts with both the amino groups of reactants, affording

compounds 1a-d.

Compounds 18-24 were synthesized by condensing suitable

anilines 11-17 with chloroacetyl chloride, at room temperature, in

dichloromethane and using diisopropylethylamine as a base. In

the last step of the synthetic pathway, reaction of N1-aryl-

thiobenzimidazoles 1a-d with intermediates 18-24 in

dimethylformamide, followed by crystallization from ethanol or

by column flash-chromatography on silica gel, afforded

compounds 2(a–d)-8(a–d) in excellent yield. Both analytical and

spectral data (1H NMR) of all synthesized compounds are in full

agreement with the proposed structures.

2.3 Biological activity

All the obtained derivatives were tested in MT-4 cells for

inhibition of HIV-1 (strain IIIB) and HIV-2 (strain ROD).

Compound-induced cytotoxicity was also measured in MT-4

cells in parallel with antiviral activity.

The compounds were also tested for their ability to inhibit the

enzymatic activity of HIV-1 RT as well as against a significant

NNRTIs resistant strain (RES056) containing (K103N/Y181C)

double mutations. The results from the cell-based and enzimatic

assays are summarised in Table 1 and compared to nevirapine

and efavirenz as reference drugs.

NH

NR

S

X

Me

Me

NH2

NO2

R

NO2

R NH

X

Me

Me

NH2

R NH

X

Me

Me

N

NRX

Me

Me

SNH

O

R'

R''

NH

Cl

O

R'

R''

NH2

R'

R''

2a-d - 8a-d

1a-d

R= H, Cl 9a-d 10a-d

18-24

a b

c

d

e

11-17

Compd R X R’ R’’ Compd R X R’ R’’

1a H CH2 - - 5a H CH2 Cl CH3

1b * Cl CH2 - - 5b Cl CH2 Cl CH3

1c

H SO2 - - 5c

H SO2 Cl CH3

1d # Cl SO2 - - 5d Cl SO2 Cl CH3

2a H CH2 Cl H 6a H CH2 Cl COOCH3

2b Cl CH2 Cl H 6b Cl CH2 Cl COOCH3

2c H SO2 Cl H 6c H SO2 Cl COOCH3

2d Cl SO2 Cl H 6d Cl SO2 Cl COOCH3

3a H CH2 Br H 7a H CH2 Cl SO2CH3

3b Cl CH2 Br H 7b Cl CH2 Cl SO2CH3

3c H SO2 Br H 7c H SO2 Cl SO2CH3

3d Cl SO2 Br H 7d Cl SO2 Cl SO2CH3

4a H CH2 NO2 H 8a H CH2 Cl SO2NH2

4b Cl CH2 NO2 H 8b Cl CH2 Cl SO2NH2

4c H SO2 NO2 H 8c H SO2 Cl SO2NH2

4d Cl SO2 NO2 H 8d Cl SO2 Cl SO2NH2

Scheme 1: reagents and conditions: (a) DMF, NaH, rt, 2-6 h (b) Zn/HCl, EtOH, 80°C, 1h; (c) TCDI, pirydine, rt, 1h; (d) K2CO3, DMF,

rt, 1,5h; (e) chloroacetyl chloride, DIPEA, CH2Cl2, rt, 1h .* See ref. (10) , # see

ref.(12) .

Table 1. Anti-RT and anti-HIV-1 activities, cytotoxicity and selectivity index in MT-4 cells

Compd. IC50 (µM)a EC50 (µM)

b CC50 (µM)

c SI

d

1a 27.24±6.66 1.3±0.95 5.74±1.6 4

1b 0.046 ±0.006 0.076± 0.03 0.305±0.018 4

1c 1.35±0.4

0.09± 0.07 40.95± 5.62

455

1d 1.76±0.31

0.05± 0.01 93.07±55.17 1861

2a 42.13±4.1 1.12±0.66 111.06±42.06 99

2b >255 19.59± 2.33 141.49±11.67 7

2c 51.93±7,38 0.088± 0.11 107.07±16.06 1216

2d 18.64±3.68 1.77±0.99 99.3±19.5 56

3a 15.02±5.63 1.12±0.04 ≥143.7

≥128

3b >233 32.49±10.42. 174.46±44.63 5

3c 0.18±0.018 0.06±0.02 ≥235.64

342.60

≥3927

3d 63.5±35.82. 3.86.±0.24 144.9 ±38.04 37

4a 3.91± 2.19

>

1.05±0.13 193.54±12.43 184.33

4b >249.49 ≥25.99 130.73±15.14 ≤5

4c 100.00±29.98 0.04 ±0.01 ≥26.08 ≥652

4d

4d

37.04±1.58 1.3±0.21. >235.4 >182

5a 178.93±124 1.53±0.31 49.15±3.22 32.12

5b >247.70 >79.27 79.27±45.08 <1

5c 0.55±0.079 0.04±0.01 5.4±0.3 135

5d 46.41±15.19 1.57±0.56 >234 >149

6a 40.30±1.17 6.03±3.9 190.72±28.74 32

6b >227.07 >104.83 104.83±27.53 <1

6c 162.17 0.11±0.07 144.99±40.68 1318

6d 37.56±5.55 4.82±1.03 ≥130.05 ≥27

7a 1.93±1.94 0.54±0.31 106.2±32.21 196

7b 70.55±8.53 >88.75 88.75 <1

7c 0.12±0.035 0.04±0.01 >221.59 >5540

7d 2.61±0.5 0.5±0.3 3.59±0.79 7

8a 1.02±0.19 0.21±0.09 111.17±27.06 530

8b 86.64±11.41 ≥22.57 ≥18.56 <1

8c 18.51±11.37 0.02±0.01 25.57±1.68 1279

8d 3.04±0.76 0.62±.0.2 23.38±4.89 38

Nevirapine 2.55± 0.93 0.19±0.06 > 15.02 >79

Efavirenz 0.032±0.009 0.006±0.0001 > 6.34 >1056

a Concentration required to inhibit by 50% the in vitro RNA-dependent DNA polymerase activity of recombinant RT. b Effective concentration required to

reduce HIV-1-induced cytopathic effect by 50% in MT-4 cells. c Cytotoxic concentration required to reduce MT-4 cell viability by 50%. d Selectivity index: ratio

CC50/EC50

The corresponding thiocarbonylbenzimidazolones (1a-d) are

reported for comparative purposes. The experimental results

indicated that most of the newly synthesized compounds showed

inhibitory activity against HIV-1 RT and prevented the

cytopathic effects of HIV-1 IIIB at micromolar or nanomolar

concentrations and in some cases had very low toxicity against

MT-4 cells, thus resulting in high selectivity indices (3c, SI≥3927

and 7c, SI>5540) (Table 1). None of the compounds was active

against HIV-2 and RES056 strain.

The SAR of benzimidazole and arylthioacetamide moieties

was probed and the effect of the linker between heterocyclic ring

and dimethylphenyl group is described.

The results of this study show that all derivatives of the series

a, c and d are particularly interesting with EC50 values ranging

from 0.02 to 6.03 µM. Only derivatives b show low potency and

all of them (2b-8b) display in their structure the contemporary

presence of a chlorine atom at C6 and a methylene bridge.

This clearly demonstrates the great influence of these

chemical features on the anti-HIV activity. In fact, the

substitution of Cl with H and/or the CH2 with SO2 led to the

identification of promising new NNRTIs with submicromolar to

nanomolar activities.

In particular derivatives 2c-8c proved to be highly potent as

their activities are superior to the clinically approved NNRTI

nevirapine.

Similarly, looking at the RT enzymatic inhibition the best

results were generally obtained for molecules 6-unsubstituted and

containing a sulfonyl-linker (i.e. 3c, 5c and 7c).

Our previous molecular modeling studies suggested that the

greatest potency of arylsulfonyl derivatives might be due to the

electronic characteristics of the sulfonyl groups able to make

closer intermolecular contacts with the NNIBP. 12

Furthermore it is interesting to note how the anti-HIV activity

depends also on the nature of the substituents on the phenyl ring

linked to the thioacetamide group. The introduction of SO2CH3 in

derivatives 7 or SO2NH2 in 8 at C-4 position, generally resulted

in increased potency compared to the unsubstituted analogues (1-

4) whereas CH3 (5) and COOCH3 moiety (6) negatively

influenced the anti-HIV activity.

2.4 Docking studies

In order to explore the possible binding mode of the

synthesized compounds, several analogues were docked into the

non-nucleoside binding site of HIV-1 RT wt (PDB code: 3DLG) 25

using AutoDock Vina. 26

As regards 3DLG, the originally crystallized inhibitor

(GW69564, Fig. 4) shows a good structural similarity compared

to our newly synthesized compounds and the interactions formed

are discussed below.

F

O O

O

NH

SNH

2

F

F

F

O O

Cl

GW69564

Figure 4

GW69564 forms a panel of interactions including: a)

hydrophobic contacts between the 3,5-disubstituted phenyl group

and residues Tyr181, Tyr188 and Trp229; b) H-bond interaction

between the carbonyl oxygen of amide group, and the NH of

Lys103 backbone; c) the phenyl group bearing the 2-methyl and

4-sulfonamide substituents is located toward a solvent accessible

region that forms one entrance to the NNRTI pocket (Fig. 5A). 25

When the crystallographic position is reproduced by AutoDock

Vina, the computed binding energy is -14 kcal/mol.

The most active derivative of our series (7c; IC50 = 0.12 M)

shows a binding mode similar to compound GW69564 (Fig. 5B).

The calculated binding energy for the docking pose of 7c is -13

kcal/mol. The 3,5-dimethyl substituted phelyl ring forms a

interaction with Tyr188 and contacts Tyr181 and Trp229; the

carbonyl oxygen of amide group forms an hydrogen bond with

Lys103 backbone; the benzimidazole portion occupies the same

region of the 4-Cl phenyl portion of GW69564.

On the other hand compound 7b (IC50 = 70.55 M) with a

chlorine substituent on the benzimidazole ring and a methylene

linker rather than a sulfonyl moiety shows a different orientation

of the aromatic rings and a distorted conformation of the

thioacetamide linker which does not allow the H-bond formation

with Lys103 (Fig 5C). This is likely to be determined by the

steric hindrance of chlorine atom on the benzimidazole nucleus,

and explains the significant drop in activity compared to

derivative 7c, as well as the general trend in this series, where we

observe a lower activity if the benzimidazole ring is chlorine

substituted.

Accordingly, binding energy of 7b is decreased (-12.0

kcal/mol). The presence of the chlorine atom makes the bicyclic

system unsuitable to fit the same region of 4-Cl phenyl ring of

GW69564, and determines a different binding mode, where the

benzimidazole moiety forms a stacking interaction with

Tyr188. Furthermore the thioacetamide linker position differs

from that of GW69564 and 7c, and H-bond formation with

Lys103 is hampered by a distance of 6.87 Å between the oxygen

of amide group of 7b and the NH of Lys103 backbone.

Figure 5. Binding mode of compounds GW69564 (A), 7c (B), 7b (C). Superposition of the docking conformation of 7c (in gray), 7b (in green) and the

crystallographic ligand GW69564 (in magenta) in the active site. This figure was produced by Pymol.27

3. Conclusion

In summary we have identified a novel family of potent anti-

HIV agents through the screening of a small library of 2-

arylthioacetamidebenzimidazoles. SAR studies suggest that some

chemical features can be considered critical pharmacophore

elements for anti-HIV activity and RT inhibition. In fact, some

derivatives proved to be effective in inhibiting HIV-1 RT wt and

HIV-1 replication at nanomolar concentration. Among these

compounds, 3c and 7c were identified as the most promising

candidates showing potent activity and no toxicity. These

findings and in particular the high selectivity of these small

molecules would constitute a good starting point for future

design and development of a focused chemical library to evaluate

and gain insight into the requirements for new NNRTIs with

improved activities against mutants, better barrier to resistance

and favorable tolerability for HIV-1 infection treatment.4

4. Experimental section

4.1 Chemistry. Melting points were determined on a BUCHI

Melting Point B-545 apparatus and are uncorrected. Elemental

analyses (C, H, N) were carried out on a C. Erba Model 1106

ElementalAnalyzer and the results were within ± 0.4% of the

theoretical values and purity of tested compounds was >95%.

Merck silica gel 60 F254 plates were used for TLC; column

chromatography was performed on Merck silica gel 60 (230-400

mesh) and Flash Chromatography (FC) on Biotage SP1 EXP. 1H

NMR spectra were measured with a Varian Gemini-300

spectrometer in CDCl3 with TMS as internal standard or in

DMSO-d6. Coupling constants (J) are reported in hertz and

chemical shifts are expressed in (ppm).

General procedures for the synthesis of N-substituted-2-

nitroanilines and N-(2-nitrophenyl)-benzenesulfonamides

(9a-d)

A mixture of anhydrous sodium hydride (5 mmol) and 2-

nitroaniline (138 mg, 1 mmol) or 5-chloro-2-nitroaniline (173

mg, 1 mmol) in DMF (5 mL) was stirred for 10 min at 0°C and

then 3,5-dimethylbenzyl bromide (597 mg, 3 mmol) or 3,5-

dimethylbenzensulphonyl chloride (614 mg, 3 mmol) was added.

When the reaction was completed (2-6h) a saturated NaHCO3

aqueous solution was added. The mixture was extracted with

dichloromethane (3 x 10 mL) and dried over Na2SO4. After

removal of the solvent under reduced pressure, the residue was

triturated by treatment with diethyl ether and crystallized from

ethanol.

N-(3,5-dimethylbenzyl)-2-nitroaniline (9a)

Mp: 79-81 °C, yield 34%.1H NMR (CDCl3): 2.33 (s, 6H,

CH3), 4.48 (d, J= 5.3, 2H),6.66-7.44 (m, 6H, ArH ), 8.22 (d, J=

8.3, 1H, ArH) 8.41 (bs, 1H, NH). Anal. Calcd for C15H16N2O2: C:

70.29; H: 6.29; N:10.93; Found C: 70.53; H: 6.12; N: 10.71.

5-Chloro-N-(3,5-dimethylbenzyl)-2-nitroaniline (9b)

See reference 10.

N-(2-nitrophenyl)-3,5-dimethylbenzenesulfonamide (9c)

Mp: 156-158 °C, yield 34%. 1H NMR (CDCl3): 2.35 (s, 6H,

CH3), 7.14-8.15 (m, 6H, ArH ), 9.89 (bs, 1H, NH). Anal. Calcd

for C14H14N2O4S: C: 54.88; H: 4.61; N: 9.14; Found: C: 55.07; H:

4.83; N: 9.35.

N-(5-Chloro-2-nitrophenyl)-3,5-dimethylbenzene

sulfonamide (9d)

See reference 12.

General procedures for the synthesis of N1-(substituted-

benzyl)-2-amino-anilines and N-(2-aminophenyl)-benzene

sulfonamides (10a-d)

The mixture of N-substituted-2-nitroanilines (1 mmol) or N-

(2-nitrophenyl)-benzenesulfonamides (1 mmol) in HCl conc. (5

mL) and EtOH (7 mL) was stirred vigorously, then zinc dust

(2.18 g, 33 mmol) was added in several portions at room

temperature. When the addition was completed the reaction was

refluxed for 1h. The resulting mixture was cooled, made alkaline

with NaOH 2N aqueous solution and then extracted with ethyl

acetate (3 x 10 mL). The extracted was washed with water, dried

over Na2SO4 and evaporated. The residue was crystallized from

ethanol or purified by column fash chromatography using

cyclohexane/AcOEt as eluent.

2-Amino-1-(3,5-dimethylbenzyl)-aniline (10a)

Mp: 145-147 °C, yield 61%. 1H NMR (DMSO-d6): 2.22 (s,

6H, CH3), 3.90 (bs, 2H, NH2), 5.00 (bs, 1H, NH), 4.17 (s, 2H,

CH2), 6.32-6.96 (m, 6H, ArH ). Anal. Calcd for C15H18N2: C:

79.61; H: 8.02; N: 12.38; Found: C: 79.48; H: 8.23; N: 12.55.

2-Amino-5-chloro-1-(3,5-dimethylbenzyl)-aniline (10b)

See reference 10.

N-(2-Aminophenyl)-3,5-dimethylbenzenesulfonamide (10c)

Mp: 164-166°C, yield 40%. 1H NMR (DMSO-d6): 2.29 (s,

6H, CH3), 4.92 (bs, 2H, NH2), 6.38 (t, J= 7.7, 1H, ArH), 6.63 (m,

2H, ArH ), 6.88 (t, J= 7.7, 1H, ArH), 7.28 (s, 2H, ArH), 9.14 (bs,

1H, NH). Anal. Calcd for C14H16N2O2S: C: 60.85; H: 5.84; N:

10.14; Found: C: 60.96; H: 6.04; N: 10.40.

N-(2-Aminophenyl-5-chloro)-3,5-dimethylbenzene

sulfonamide (10d)

See reference 12.

General procedures for the synthesis of 1-(3,5-

dimethylbenzyl)-1,3-dihydro-2H-benzimidazol-2-ones and 1-

(3,5-dimethylphenylsulfonyl)-1,3-dihydro-2H-benzimidazol-

2-ones (1a-d).

To a solution of N1-(substituted-benzyl)-2-amino-anilines

(10a-b) (1 mmol) or N-(2-aminophenyl)-benzenesulfonamides

(10c-d) (1 mmol) in pyridine (10 mL) 1,1’-

thiocarbonyldiimidazole (250 mg, 1.4 mmol) was added at room

temperature and the resulting mixture was maintained under

stirring for 1h. After this time, distilled water was added to

quench the reaction and the precipitate was filtered off to give the

desired products after cooling.

1-(3,5-Dimethylbenzyl)-1,3-dihydro-2H-benzimidazole-2-

thione (1a)


6H, CH3), 5.42 (s, 2H, CH2), 6.89-7.22 (m, 7H, ArH ), 12.88 (bs,

1H, NH). Anal. Calcd for C16H16N2S: C: 71.61; H: 6.01; N:

10.44; Found: C: 71.49; H: 6.23; N: 10.58.

6-Chloro-1-(3,5-dimethylbenzyl)-1,3-dihydro-2H-

benzimidazole-2-thione (1b)

See reference 10.

1-(3,5-Dimethylphenylsulfonyl)-1,3-dihydro-2H-

benzimidazole-2-thione (1c)


CH3), 7.13-7.17 (m, 1H, ArH), 7.28-7.33 (m, 3H, ArH), 7.78 (s,

2H, ArH), 8.15-8.18 (m, 1H, ArH ), 10.31 (bs, 1H, NH). Anal.

Calcd for C15H14N2O2S2: C: 56.58; H: 4.43; N: 8.80; Found: C:

56.42; H: 4.59; N: 8.69.

6-Chloro-1-(3,5-dimethylphenylsulfonyl)-1,3-dihydro-2H-

benzimidazole-2-thione (1d)

See reference 12.

General procedures for the synthesis of 2-chloro-N-

phenylacetamides (18-24).

N,N-Diisopropylethylamine (175 µL, 1mmol) and then

chloroacetyl chloride (78 µL, 1mmol) were added dropwise to a

solution of suitable substituted anilines (11-17) (1 mmol) in

dichloromethane (5 mL). The mixture was stirred for 1 h at room

temperature. Successively, the reaction was quenched with a

saturated NaHCO3 aqueous solution. The reaction mixture was

extracted with ethyl acetate (3 x 10 mL), dried over Na2SO4 and

evaporated under reduced pressure. The residue was crystallized

from ethanol.

N-(2-Chlorophenyl)-2-chloroacetamide (18)


CH2), 7.10 (t, J= 7.6, 1H, ArH), 7.30 (t, J= 7.6, 1H, ArH), 7.40

(d, J= 7.6, 1H, ArH), 8.36 (d, J= 8.2, 1H, ArH), 8.93 (bs, 1H,

NH). Anal. Calcd for C8H7Cl2NO: C: 52.87; H: 4.05; N: 8.04;

Found: C: 52.66; H: 4.23; N: 8.16.

N-(2-Bromophenyl)-2-chloroacetamide (19)


CH2), 7.04 (t, J= 7.5, 1H, ArH), 7.35 (t, J= 7.5, 1H, ArH), 7.57


NH). Anal. Calcd for C8H7BrClNO: C: 38.67; H: 2.84; N: 5.64;

Found: C: 38.51; H: 2.79; N: 5.79.

N-(2-Nitrophenyl)-2-chloroacetamide (20)

Mp: 90-92°C, yield 79%. 1H NMR (CDCl3): 4.27 (s, 2H,

CH2), 7.28 (t, J= 8.2, 1H, ArH), 7.71 (t, J= 8.8, 1H, ArH), 8.27


NH). Anal. Calcd for C8H7ClN2O3: C: 44.77; H: 3.29; N: 13.05;

Found: C: 44.58; H: 3.43; N: 12.96.

2-Chloro-N-(2-chloro-4-methylphenyl)acetamide (21)


CH3), 4.23 (s, 2H, CH2), 7.11 (d, J= 8.3, 1H, ArH), 7.23 (s, 1H,

ArH), 8.21 (d, J= 8.8, 1H, ArH), 8.84 (bs, 1H, NH). Anal. Calcd

for C9H9Cl2NO: C: 49.57; H: 4.16; N: 6.42; Found: C: 49.72; H:

4.28; N: 6.56.

Methyl-3-chloro-4-[(4-chloroacetyl)amino]benzoate (22)

Mp: 118-120°C, yield 75%. 1H NMR (CDCl3): 3.94 (s, 3H,

CH3), 4.28 (s, 2H, CH2), 8.00 (d, J= 8.8, 1H, ArH), 8.11 (s, 1H,


for C10H9Cl2NO3: C: 45.83; H: 3.46; N: 5.34; Found: C: 45.92;

H: 3.68; N: 5.56.

2-Chloro-N-[2-chloro-4-(methylsulfonyl)phenyl]acetamide

(23)

Mp: 206-208°C, yield 66%. 1H NMR (DMSO-d6): 3.26 (s,

3H, CH3), 4.45 (s, 2H, CH2), 7.88 (d, J= 8.5, 1H, ArH), 8.04 (s,

1H, ArH), 8.12 (d, J= 8.5, 1H, ArH), 10.11 (bs, 1H, NH). Anal.

Calcd for C9H9Cl2NO3S: C: 38.31; H: 3.22; N: 4.96; Found: C:

38.42; H: 3.38; N: 5.26.

2-Chloro-N-(2-chloro-4-sulfamoylphenyl)acetamide (24)


2H, CH2), 7.48 (bs, 2H, NH2), 7.76 (d, J= 8.5, 1H, ArH), 7.89 (s,


Calcd for C8H8Cl2NO3S: C: 33.94; H: 2.85; N: 9,89; Found: C:

33.78; H: 2.68; N: 9.95.

General procedures for the synthesis of 2-[1-(3,5-

dimethylbenzyl)-1H-benzimidazol-2-yl]sulfanyl-N-

phenylacetamides and 2-(1-[(3,5-dimethylphenyl)sulfonyl]-

1H-benzimidazol-2-ylsulfanyl)-N-phenylacetamides (2a-d

8a-d)

A solution of 1-(3,5-dimethylbenzyl)-1,3-dihydro-2H-

benzimidazol-2-one (1a-b) (1 mmol) or 1-(3,5-

dimethylphenylsulfonyl)-1,3-dihydro-2H-benzimidazol-2-one

(1c-d) (1 mmol) in DMF (3 mL), anhydrous potassium carbonate

(138 mg, 1mmol) and the appropriate 2-chloro-N-

phenylacetamide (1mmol) was stirred at room temperature for

1h. The reaction was quenched by the addition of saturated

NaHCO3 aqueous solution and the mixture was extracted with

ethyl acetate (3 x 10 mL). After removal of the solvent under

reduced pressure, the residue was crystallized by treatment with

ethanol or purified by column fash chromatography using

cyclohexane/AcOEt as eluent.

2-[1-(3,5-Dimethylbenzyl)-1H-benzimidazol-2-

yl]sulfanyl-N-2-chlorophenylacetamide (2a).

Mp: 134-136 °C, yield 72%, Rf: 0,51 (cyclohexane/EtOAc=

8:2). 1H NMR (CDCl3): 2.24 (s, 6H, CH3), 4.18 (s, 2H, CH2),

5.20 (s, 2H, CH2), 6.78 (s, 2H, ArH), 6.91 (s, 1H, ArH), 6.97-

7.03 (m, 1H, ArH), 7.19-7.31 (m, 5H, ArH), 7.71 (d, J= 8.0, 1H,


for C4H22ClN3OS: C: 66.12; H: 5.09; N: 9.64; Found: C: 66.35;

H: 5.22; N: 9.81.

2-[6-Chloro-1-(3,5-dimethylbenzyl)-1H-benzimidazol-2-

yl]sulfanyl-N-2-chlorophenylacetamide (2b).



5.15 (s, 2H, CH2), 6.75 (s, 2H, ArH), 6.93 (s, 1H, ArH), 6.98-

7.04 (m, 1H, ArH), 7.20-7.32 (m, 4H, ArH), 7.60 (d, J= 7.4, 1H,


for C24H21Cl 2N3OS: C: 61.28; H: 4.50; N: 8.93; Found: C: 61.43;

H: 4.77; N: 9.01.

2-(1-[(3,5-Dimethylphenyl)sulfonyl]-1H-benzimidazol-2-

ylsulfanyl)-N-2-chlorophenylacetamide (2c).



7.00 (t, J= 7.7, 1H, ArH), 7.18 (s, 1H, ArH), 7.23-7.28 (m, 3H,

ArH), 7.33-7.35 (m, 2H, ArH), 7.65 (s, 2H, ArH), 7.96-7.99 (m,


Calcd for C23H20ClN3O3S2: C: 56.84; H: 4.15; N: 8.65; Found: C:

57.05; H: 4.33; N: 8.81.

2-(6-Chloro-1-[(3,5-dimethylphenyl)sulfonyl]-1H-

benzimidazol-2-ylsulfanyl)-N-2-chlorophenylacetamide (2d).



7.00 (t, J= 7.7, 1H, ArH), 7.20-7.33 (m, 4H, ArH), 7.53 (d, J=

8.8, 1H, ArH), 7.64 (s, 2H, ArH), 7.98 (s, 1H, ArH), 8.34 (d, J=

8.8, 1H, ArH), 9.51 (bs, 1H, NH). Anal. Calcd for

C23H19Cl2N3O3S2: C: 53.08; H: 3.68; N: 8.07; Found: C: 53.23;

H: 3.81; N: 8.37.


yl]sulfanyl-N-2-bromophenylacetamide (3a).



5.20 (s, 2H, CH2), 6.79 (s, 2H, ArH), 6.91-6.96 (m, 2H, ArH),

7.20-7.30 (m, 4H, ArH), 7.48 (d, J= 8.0, 1H, ArH), 7.73 (d, J=

8.0, 1H, ArH), 8.21 (d, J= 8.0, 1H, ArH), 10.29 (bs, 1H, NH).

Anal. Calcd for C24H22BrN3OS: C: 60.00; H: 4.62; N: 8.75;

Found: C: 60.26; H: 4.98; N: 8.86.


yl]sulfanyl-N-2-bromophenylacetamide (3b).



5.16 (s, 2H, CH2), 6.76 (s, 2H, ArH), 6.93-6.95 (m, 2H, ArH),

7.20-7.30 (m, 3H, ArH), 7.48 (d, J= 8.0, 1H, ArH), 7.62 (d, J=


Anal. Calcd for C24H22BrN3OS: C: 55.99; H: 4.11; N: 8.16;

Found: C: 56.13; H: 4.23; N: 8.31.


ylsulfanyl)-N-2-bromophenylacetamide (3c).



6.83-6.89 (m, 1H, ArH), 7.10 (s, 1H, ArH), 7.19-7.27 (m, 4H,

ArH), 7.34 (d, J= 7.5, 1H, ArH), 7.57 (s, 2H, ArH), 7.87-7.90 (m,


Calcd for C23H20BrN3O3S2: C: 52.08; H: 3.80; N: 7.92; Found: C:

52.25; H: 4.03; N: 8.08.


benzimidazol-2-ylsulfanyl)-N-2-bromophenylacetamide (3d).



6.96 (t, J= 7.7, 1H, ArH), 7.22-7.33 (m, 3H, ArH), 7.44 (d, J=

9.4, 1H, ArH), 7.55 (d, J= 8.9, 1H, ArH), 7.65 (s, 2H, ArH), 8.00

(s, 1H, ArH), 8.26 (d, J= 6.6, 1H, ArH), 9.26 (bs, 1H, NH). Anal.

Calcd for C23H19BrClN3O3S2: C: 48.90; H: 3.39; N: 7.44; Found:

C: 49.13; H: 3.53; N: 7.67.


yl]sulfanyl-N-2-nitrophenylacetamide (4a).



5.23 (s, 2H, CH2), 6.79 (s, 2H, ArH), 6.90 (s, 1H, ArH), 7.15-

7.25 (m, 4H, ArH), 7.59 (t, J= 8.5, 1H, ArH), 7.82 (d, J= 7.5, 1H,

ArH), 8.09 (d, J= 8.5, 1H, ArH), 8.57 (d, J= 8.5, 1H, ArH), 11.64

(bs, 1H, NH). Anal. Calcd for C24H22N4O3S: C: 64.56; H: 4.97;

N: 12.55; Found: C: 64.77; H: 5.08; N: 12.69.


yl]sulfanyl-N-2-nitrophenylacetamide (4b).



5.21 (s, 2H, CH2), 6.79 (s, 2H, ArH), 6.95 (s, 1H, ArH), 7.18-

7.23 (m, 3H, ArH), 7.63 (t, J= 7.2, 1H, ArH), 7.74 (d, J= 9.2, 1H,


(bs, 1H, NH). Anal. Calcd for C24H21ClN4O3S: C: 59.93; H: 4.40;

N: 11.65; Found: C: 64.77; H: 5.08; N: 12.69.


ylsulfanyl)-N-2-nitrophenylacetamide (4c).



5.23 (s, 2H, CH2), 7.16-7.30 (m, 5H, ArH), 7.61-7.89 (m, 4H,


(bs, 1H, NH). Anal. Calcd for C23H20N4O5S2: C: 55.63; H: 4.06;

N: 11.28; Found: C: 56.79; H: 4.21; N: 11.49.


benzimidazol-2-ylsulfanyl)-N-2-nitrophenylacetamide (4d).



7.20-7.31 (m, 3H, ArH), 7.65-7.69 (m, 3H, ArH), 7.94 (s, 1H,


(bs, 1H, NH). Anal. Calcd for C23H19ClN4O5S2: C: 52.02; H:

3.61; N: 10.55; Found: C: 52.31; H: 3.86; N: 10.76.


yl]sulfanyl-N-2-chloro-4-methylphenylacetamide (5a).



4.16 (s, 2H, CH2), 5.19 (s, 2H, CH2), 6.78 (s, 2H, ArH), 6.91 (s,

1H, ArH), 7.03 (d, J= 8.5, 1H, ArH), 7.11 (s, 1H, ArH), 7.19-

7.27 (m, 3H, ArH), 7.70 (d, J= 7.5, 1H, ArH), 8.17 (d, J= 8.5,

1H, ArH), 10.53 (bs, 1H, NH). Anal. Calcd for C25H24ClN3OS:

C: 66.73; H: 5.38; N: 9.34; Found: C: 66.92; H: 5.59; N: 9.60.


yl]sulfanyl-N-2-chloro-4-methylphenylacetamide (5b).



4.16 (s, 2H, CH2), 5.17 (s, 2H, CH2), 6.77 (s, 2H, ArH), 6.95 (s,

1H, ArH), 7.06 (d, J= 8.2, 1H, ArH), 7.13 (s, 1H, ArH), 7.22-

7.28 (m, 3H, ArH), 7.61 (d, J= 7.7, 1H, ArH), 8.19 (d, J= 8.7,

1H, ArH), 10.36 (bs, 1H, NH). Anal. Calcd for C25H23Cl2N3OS:

C: 61.98; H: 4.79; N: 8.67; Found: C: 62.32; H: 5.01; N: 8.80.


ylsulfanyl)-N-2-chloro-4-methylphenylacetamide (5c).



4.15 (s, 2H, CH2), 7.05 (d, J= 8.3, 2H, ArH), 7.18 (s, 1H, ArH),

7.33-7.36 (m, 2H, ArH), 7.61-7.65 (m, 3H, ArH), 7.97 (d, J= 6.6,



57.98; H: 4.71; N: 8.69


benzimidazol-2-ylsulfanyl)-N-2-chloro-4-

methylphenylacetamide (5d).



4.13 (s, 2H, CH2), 7.05 (d, J= 8.7, 2H, ArH), 7.22 (s, 1H, ArH),

7.28-7.33 (m, 2H, ArH), 7.53 (d, J= 8.2, 1H, ArH), 7.65 (s, 2H,

ArH), 7.99 (s, 1H, ArH), 8.18 (d, J= 8.2, 1H, ArH), 9.48 (bs, 1H,

NH). Anal. Calcd for C24H21Cl2N3O3S2: C: 53.93; H: 3.96; N:

7.86; Found: C: 54.28; H: 4.09; N: 7.99.

Methyl-3-chloro-4-[([1-(3,5-dimethylphenyl)-1H-

benzimidazol-2-yl]sulfanylacetyl)amino] benzoate (6a).



4.19 (s, 2H, CH2), 5.20 (s, 2H, CH2), 6.79 (s, 2H, ArH), 6.91 (s,

1H, ArH), 7.23-7.26 (m, 3H, ArH), 7.70 (d, J= 7.4, 1H, ArH),

7.90 (d, J= 9.1, 1H, ArH), 7.99 (s, 1H, ArH), 8.50 (d, J= 8.5, 1H,

ArH), 9.48 (bs, 1H, NH). Anal. Calcd for C26H24ClN3O3S: C:

63.21; H: 4.90; N: 8.51; Found: C: 63.55; H: 5.26; N: 8.84.

Methyl-3-chloro-4-[([6-chloro-1-(3,5-dimethylphenyl)-1H-

benzimidazol-2-yl]sulfanylacetyl)amino] benzoate (6b).



4.19 (s, 2H, CH2), 5.17 (s, 2H, CH2), 6.77 (s, 2H, ArH), 6.95 (s,

1H, ArH), 7.24-7.28 (m, 3H, ArH), 7.61 (d, J= 8.8, 1H, ArH),

7.93 (d, J= 8.8, 1H, ArH), 8.01 (s, 1H, ArH), 8.52 (d, J= 8.3, 1H,

ArH), 10.70 (bs, 1H, NH). Anal. Calcd for C26H23Cl2N3O3S: C:

59.09; H: 4.39; N: 7.95; Found: C: 59.23; H: 4.50; N: 8.12.

Methyl-3-chloro-4-[(1-[(3,5-dimethylphenyl)sulfonyl]-

1H-benzimidazol-2-ylsulfanyl)acetyl]aminobenzoate (6c).



4.17 (s, 2H, CH2), 7.17 (s, 1H, ArH), 7.33-7.38 (m, 2H, ArH),

7.60-7.63 (m, 3H, ArH), 7.89-7.98 (m, 3H, ArH), 8.50 (d, J= 8.5,



55.43; H: 4.31; N: 7.94.

Methyl-3-chloro-4-[(6-chloro-1-[(3,5-

dimethylphenyl)sulfonyl]-1H-benzimidazol-2-

ylsulfanyl)acetyl]aminobenzoate (6d).


8:2). 1H NMR (DMSO-d6): 2.32 (s, 6H, CH3), 3.85 (s, 3H, CH3),

4.45 (s, 2H, CH2), 7.40 (d, J= 8.3, 2H, ArH), 7.57 (d, J= 8.9, 1H,

ArH), 7.82 (s, 2H, ArH), 7.90 (d, J= 5.0, 2H, ArH), 7.97 (s, 1H,


for C25H21Cl2N3O5S2: C: 51.91; H: 3.66; N: 7.26; Found: C:

52.13; H: 3.81; N: 7.45.


yl]sulfanyl-N-[2-chloro-4-(methylsulfonyl)phenyl]-acetamide

(7a).



4.21 (s, 2H, CH2), 5.22 (s, 2H, CH2), 6.81 (s, 2H, ArH), 6.94 (s,

1H, ArH), 7.30 (s, 3H, ArH), 7.71 (d, J= 7.7, 1H, ArH), 7.82 (d,

J= 8.8, 1H, ArH), 7.92 (s, 1H, ArH), 8.66 (d, J= 8.8, 1H, ArH),

11.14 (bs, 1H, NH). Anal. Calcd for C25H24ClN3O3S2: C: 58.41;

H: 4.71; N: 8.17; Found: C: 58.63; H: 4.92; N: 8.46.



(7b).



4.19 (s, 2H, CH2), 5.17 (s, 2H, CH2), 6.77 (s, 2H, ArH), 6.96 (s,

1H, ArH), 7.25 (s, 2H, ArH), 7.61 (d, J= 8.8, 1H, ArH), 7.83 (d,

J= 8.4, 1H, ArH), 7.93 (s, 1H, ArH), 8.66 (d, J= 8.4, 1H, ArH),

10.93 (bs, 1H, NH). Anal. Calcd for C25H23Cl2N3O3S2: C: 58.41;

H: 4.71; N: 8.17; Found: C: 58.63; H: 4.92; N: 8.46.



(7c).



4.17 (s, 2H, CH2), 7.21 (s, 1H, ArH), 7.34-7.39 (m, 2H, ArH),

7.59-7.65 (m, 3H, ArH), 7.78-7.86 (m, 2H, ArH), 7.97 (d, J= 7.4,



51.41; H: 4.22; N: 7.56.


benzimidazol-2-yl]sulfanyl-N-[2-chloro-4-

(methylsulfonyl)phenyl]-acetamide (7d).

Mp: 218-220 °C, yield 80%, Rf: 0,33 (cyclohexane /EtOAc=


4.43 (s, 2H, CH2), 7.37-7.46 (m, 2H, ArH), 7.57 (d, J= 8.2, 1H,

ArH), 7.72 (d, J= 8.8, 1H, ArH), 7.81 (s, 2H, ArH), 7.87 (d, J=


Anal. Calcd for C24H21Cl2N3O5S3: C: 48.16; H: 3.54; N: 7.02;

Found: C: 48.31; H: 3.73; N: 7.29.


yl]sulfanyl-N-(2-chloro-4-sulfamoylphenyl)-acetamide (8a).



5.11 (s, 2H, CH2), 6.36 (s, 2H, ArH), 6.70 (s, 2H, ArH), 6.82 (s,

1H, ArH), 7.15-7.19 (m, 3H, 1ArH and NH2), 7.58 (d, J= 7.7,

1H, ArH), 7.69 (d, J= 8.7, 1H, ArH), 7.80 (s, 1H, ArH), 8.44 (d,

J= 8.7, 1H, ArH), 10.89 (bs, 1H, NH). Anal. Calcd for

C24H23ClN4O3S2: C: 55.97; H: 4.50; N: 10.88; Found: C: 56.23;

H: 4.71; N: 11.09.


yl]sulfanyl-N-(2-chloro-4-sulfamoylphenyl)-acetamide (8b).



5.35 (s, 2H, CH2), 6.80 (s, 2H, ArH), 6.90 (s, 1H, ArH), 7.20 (d,

J= 8.5, 1H, ArH), 7.57 (d, J= 8.5, 1H, ArH), 7.66-7.73 (m, 4H,

2ArH and NH2), 7.73 (s, 1H, ArH), 8.11 (d, J= 8.5, 1H, ArH),


H: 4.04; N: 10.20; Found: C: 52.64; H: 4.31; N: 10.49.

2-(1-[(3,5-Dimethylbenzyl)sulfonyl]-1H-benzimidazol-2-

ylsulfanyl)-N-(2-chloro-4-sulfamoylphenyl)-acetamide (8c).



7.31-7.37 (m, 3H, ArH), 7.44 (bs, 2H, NH2), 7.50-7.62 (m, 1H,

ArH), 7.75 (t, J= 8, 3H, ArH), 7.84-7.90 (m, 2H, ArH), 8.01 (d,

J= 8.5, 1H, ArH), 10.89 (bs, 1H, NH). Anal. Calcd for

C23H21ClN4O5S3: C: 48.89; H: 3.75; N: 9.91; Found: C: 49.12; H:

3.91; N: 10.07.

2-(6-Chloro-1-[(3,5-dimethylbenzyl)sulfonyl]-1H-

benzimidazol-2-ylsulfanyl)-N-(2-chloro-4-sulfamoylphenyl)-

acetamide (8d).



6.31 (s, 2H, ArH), 7.17 (s, 1H, ArH), 7.27 (s, 1H, ArH), 7.44 (d,

J= 8.7, 1H, ArH), 7.56 (bs, 2H, NH2), 7.72 (d, J= 8.7, 1H, ArH),

7.78 (s, 1H, ArH), 7.90 (s, 1H, ArH), 8.45 (d, J= 9.3, 1H, ArH),


H: 3.36; N: 9.35; Found: C: 46.25; H: 3.59; N: 9.71.

4.2 Docking studies. In order to explore the possible binding

mode of the designed compounds, several representive analogues

were docked into the non-nucleoside binding site of HIV-1 RT

wt (PDB code: 3DLG 25

) using AutoDock Vina. 26

Ligands were built using Discovery Studio 2.5.5 28

and

submitted for ligand minimization using CHARMm forcefield

with Smart Minimizer algorithm. AutoDockTools 1.5.4 29

was

used to further process ligands ensuring that its atoms were

assigned the correct AutoDock atom types, adding Gasteiger

charges, merging non-polar hydrogens, detecting aromatic

carbons if any, and setting up the 'torsion tree'. Using ADT polar

hydrogen atoms were added to the protein and its nonpolar

hydrogen atoms were merged. A grid box with a dimension of 20

x 20 x 20 points was defined around the crystallized ligand to

cover the entire non-nucleoside binding site and accommodate

ligands to move freely.

Docking validation has been carried out removing the ligand

GW69564 from the crystal structure and then attempting to

reproduce the X-ray orientation and conformation. The best

docking pose found for GW69564 agreed well with its

experimental binding mode, with a root-mean square deviation

(RMSD) of 0.55 Å.

4.3 Anti-HIV activity assays

In vitro anti-HIV assay. The methodology of the anti-HIV

assays has been previously described. 22

Briefly, MT-4 cells were

infected with HIV-1 (IIIB) at ~ 100-times the CCID50 (50% cell

culture infective dose) per ml of cell suspension. 100 l of the

infected cell suspension was then transferred to microtiter plate

wells, mixed with 100 l of the appropriate dilutions of the test

compounds, and further incubated at 37 °C. After 5 days (MT-4)

incubation, the number of viable MT-4 cells was determined. The

50% effective concentration (EC50) was defined as the

concentration of compound required to reduce the virus-induced

cytopathicity by 50%.

Reverse transcriptase assay. Recombinant wild type p66/p51

HIV-1 RT was expressed and purified as described by. 1 The RT

assay is performed with the EnzCheck Reverse Transcriptase

Assay kit (Molecular Probes, Invitrogen), as described by the

manufacturer. The assay is based on the dsDNA quantitation

reagent PicoGreen. This reagent shows a pronounced increase in

fluorescence signal upon binding to dsDNA or RNA-DNA

heteroduplexes. Single-stranded nucleic acids generate only

minor fluorescence signal enhancement when a sufficiently high

dye:base pair ratio is applied. 24

This condition is met in the

assay.

A poly(rA) template of approximately 350 bases long, and an

oligo(dT)16 primer, are annealed in a molar ratio of 1:1.2 (60

min. at room temperature). Fifty-two ng of the RNA/DNA is

brought into each well of a 96-well plate in a volume of 20 µl

polymerization buffer (60 mM Tris-HCl, 60 mM KCl, 8 mM

MgCl2, 13 mM DTT, 100 µM dTTP, pH 8.1). Five µl of RT

enzyme solution, diluted to a suitable concentration in enzyme

dilution buffer (50 mM Tris-HCl, 20% glycerol, 2 mM DTT,

pH 7.6), is added. The reactions are incubated at 25°C for 40

minutes and then stopped by the addition of EDTA (15 mM fc).

Heteroduplexes are then detected by addition of PicoGreen.

Signals are read using an excitation wavelength of 490 nm and

emission detection at 523 nm using a spectrofluorometer (Safire

2, Tecan). To test the activity of compounds against RT, 1 µl of

compound in DMSO is added to each well before the addition of

RT enzyme solution. Control wells without compound contain

the same amount of DMSO. Results are expressed as relative

fluorescence i.e. the fluorescence signal of the reaction mix with

compound divided by the signal of the same reaction mix without

compound.

Acknowledgment

Financial support for this research by the Fondo Ateneo di

ricerca (2008, Messina, Italy).

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