Novel 1-[2-(Diarylmethoxy)ethyl]-2-methyl-5 ... - ACS Publications

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J. Med. Chem. 2005, 48, 4378-4388

Novel 1-[2-(Diarylmethoxy)ethyl]-2-methyl-5-nitroimidazoles as HIV-1 Non-Nucleoside Reverse Transcriptase Inhibitors. A Structure-Activity Relationship Investigation Gabriella De Martino,† Giuseppe La Regina,† Alessandra Di Pasquali,† Rino Ragno,# Alberto Bergamini,‡ Chiara Ciaprini,‡ Anna Sinistro,‡ Giovanni Maga,| Emmanuele Crespan,| Marino Artico,† and Romano Silvestri*,† Istituto Pasteur - Fondazione Cenci Bolognetti, Dipartimento di Studi Farmaceutici, Universita` di Roma “La Sapienza”, Piazzale Aldo Moro 5, I-00185 Roma, Italy, Dipartimento di Studi di Chimica e Tecnologia delle Sostanze Biologicamente Attive, Universita` di Roma “La Sapienza”, Piazzale Aldo Moro 5, I-00185 Roma, Italy, Dipartimento di Sanita` Pubblica e Biologia Cellulare, Universita` di Roma “Tor Vergata”, Via Tor Vergata 135, I-00133 Roma, Italy, and Istituto di Genetica Molecolare - CNR, via Abbiategrasso 207, I-27100 Pavia, Italy Received March 25, 2005

1-[2-(Diarylmethoxy)ethyl]-2-methyl-5-nitroimidazoles (DAMNIs) is a novel family of HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTIs) active at submicromolar concentration. Replacement of one phenyl ring of 1-[2-(diphenylmethoxy)ethyl]-2-methyl-5-nitroimidazole (4) with heterocyclic rings, such as 2-thienyl or 3-pyridinyl, led to novel DAMNIs with increased activity. In HIV-1 WT cell-based assay the racemic 1-{2-[R-(thiophen-2-yl)phenylmethoxy]ethyl}2-methyl-5-nitroimidazole (7) (EC50 ) 0.03 µM) proved 5 times more active than compound 4. Docking experiments showed that the introduction of a chiral center would not affect the binding of both (R)-7 and (S)-7. The internal scoring function of the Autodock program calculated the same inhibition constant (Ki ) 7.9 nM) for the two enantiomers. Compounds 7 (ID50 ) 8.25 µM) were found more active than efavirenz (ID50 ) 25 µM) against the viral RT carrying the K103N mutation, suggesting for these compounds a potential use in efavirenz based antiAIDS regimens. Introduction

Chart 1. Approved NNRTIs

Acquired immune deficiency syndrome (AIDS)/ infection caused by human immunodeficiency virus (HIV) is unceasingly widespreading. According to WHO/ UNAIDS estimates 45 million people will become HIV infected between 2002 and 2010.1 Anti-AIDS drugs fall into three main categories, the nucleoside reverse transcriptase inhibitors (NRTIs), the non-nucleoside reverse transcriptase inhibitors (NNRTIs), and the protease inhibitors (PIs). Recently the viral entry inhibitor enfuvirtide has been licensed for the treatment of HIV infection.2 Effective anti-AIDS treatments are usually obtained combining the drugs in highly active anti-retroviral therapies (HAARTs), which are able to reduce the incidence of AIDS-infections and deaths. Despite their efficacy, HAARTs cannot suppress completely the viral infection,3 thus forcing to long-term or permanent treatments. These conditions clearly promote the emergence of drug resistant variants, cross-resistance inside the same category and unwanted metabolic side effects.3 The NNRTIs act through the allosteric inhibition of the viral reverse transcriptase (RT) and then are endowed with low cytotoxity.4 Nevertheless this real advantage is vanished by the broad cross-resistance * Corresponding author: phone +390649913800, fax +3906491491, e-mail: [email protected] † Istituto Pasteur - Fondazione Cenci Bolognetti, Dipartimento di Studi Farmaceutici, Universita` di Roma “La Sapienza”. # Dipartimento di Studi di Chimica e Tecnologia delle Sostanze Biologicamente Attive, Universita` di Roma “La Sapienza”. ‡ Dipartimento di Sanita ` Pubblica e Biologia Cellulare, Universita` di Roma “Tor Vergata”. | Istituto di Genetica Molecolare-CNR.

displayed by all approved NNRTIs nevirapine (1), delavirdine (2), and efavirenz (3) (Chart 1).5,6 To overcome these difficulties novel anti-HIV agents are searched with obstinacy either by improvements of the existing drug classes (NRTIs, NNRTIs, and PIs) or by discovery of agents targeting new mechanism of action (integrase inhibitors, Rnase-H inhibitors, and viral entry inhibitors).7-9 Our decennial commitment in the field of anti-AIDS agents10 led to the discovery of 1-[2-(diarylmethoxy)ethyl]-2-methyl-5-nitroimidazoles (DAMNIs, e.g. 4 (RS1478)) (Chart 2), a novel family of NNRTIs active at submicromolar concentration.11 SAR studies showed that the diarylmethane moiety strongly influenced the antiviral activity of DAMNIs, leading to different results

10.1021/jm050273a CCC: $30.25 © 2005 American Chemical Society Published on Web 05/27/2005

Novel DAMNIs as HIV NNRTIs

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 13 4379

Chart 2. Reference and Novel Derivativesa

a X ) S, O; Y ) H, phenyl, 1-naphthyl, cyclohexyl; Z ) S, SO , NH, N-benzyl, O; R -R ) H, CH , Cl, F; R ) H, Cl; R ,R ) H, NO ; 2 1 3 3 4 5 6 2 R7 ) H, CH3.

Scheme 1a

a

Reagents. a: NaBH4; b: 1-(2-hydroxyethyl)-2-methyl-5-nitroimidazole, PTSA, Dean-Stark trap.

depending on nature and position of atoms/groups introduced.11b Inside the RT non-nucleoside binding site (NNBS), DAMNIs assume a typical “butterfly-like” conformation, the diphenylmethane moiety mimicking the wings of the Scha¨fer’s 3D model,12 and the 1-(2methyl-5-nitroimidazolyl)ethane portion the indolyl ring of BHAPs. Since the introduction of different substituents on the phenyl ring of DAMNIs did not increase strongly the antiviral potency,11b we planned to replace one of the phenyl rings with a thiophene nucleus. Docking experiments on a thiophene DAMNI analogue indeed supported the design of novel derivatives. Autodock internal scoring functions predicted the inhibition constant (Ki) of the thiophene DAMNI in the low nanomolar range. Our project was also encouraged by some reports. Sowell13 et al. synthesized a series of 3-arylsulfonylthiophenes as novel antiviral/antitumor agents, with 3-(2-nitrophenylsulfonyl)-2-aminothiophene (5) as the most active compound within the series (CC50 ) >351 µM and EC50 ) 13.3 µM). Uckun14 et al. prepared N-[2(5-bromopyridyl)]-N′-[2-(2-thiophene)ethyl]-thiourea (HI443, 6) by replacing the pyridyl ring of trovirdine with a 2-thiophenyl nucleus. This compound showed high selectivity index and low cytotoxicity. The above findings prompted us to synthesize the 1-{2-[aryl-(R-thiophen-2-yl)methoxy]ethyl}-2-methyl-5nitroimidazoles (7-17) here reported. To further elucidate SAR analysis, some DAMNI derivatives were also prepared by replacing one aryl with furan (18) or pyridine (19) or by substituting the oxygen of the side chain with NH, S or SO2 (20-37). Finally derivatives

38-44 were also synthesized to complete the SAR evaluation. Chemistry Aryl-(thiophen-2-yl)-methanones15 45-55 were prepared by reacting proper aroyl chlorides with thiophene or 2-chlorothiophene in the presence of anydrous aluminum chloride in dichloromethane at room temperature. Sodium borohydride reduction of ketones 45-55 furnished the corresponding aryl-(thiophen-2-yl)-methanols16 56-66. These compounds were reacted with 1-(2hydroxyethyl)-2-methyl-5-nitroimidazole in the presence of para-toluenesulfonic acid in boiling benzene using a Dean-Stark trap to afford 1-{2-[aryl-(R-thiophen-2-yl)methoxy]ethyl}-2-methyl-5-nitroimidazoles (7-17). Similarly was prepared 1-{2-[phenyl-(R-furan-2-yl)methoxy]ethyl}-2-methyl-5-nitroimidazole (18) using (furan-2-yl)phenylmethanol (Scheme 1). Phenyl-(pyridin-3-yl)methanone17a (67) was prepared following the method of Bochis et al.17b by reacting pyridin-3-carbonyl chloride with benzene in the presence of anhydrous aluminum chloride. Sodium borohydride reduction of 67 gave phenyl-(pyridin-3-yl)methanol (68) which was transformed into R-(pyridin-3-yl)phenylmethyl chloride hydrochloride (69) with thionyl chloride. Reaction of 69 with 1-(2-hydroxyethyl)-2-methyl-5-nitroimidazole in the presence of triethylamine led to 1-{2-[R-(pyridin-3yl)phenylmethoxy]ethyl}-2-methyl-5-nitroimidazole (19) (Scheme 2). Reaction of proper arylphenylbromomethanes11b or benzyl chloride with 2-(2-methyl-5-nitroimidazol-1-yl)ethanethiol in the presence of potassium carbonate in

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Scheme 2a

a Reagents. a: thionyl chloride; b: benzene, anydrous aluminum chloride; c: NaBH4; d: thionyl chloride; e: 1-(2-hydroxyethyl)-2methyl-5-nitroimidazole, triethylamine.

Scheme 3a

a Reagents. a: 2-(2-methyl-5-nitroimidazol-1-yl)ethanethiol, K2CO3; b: 2-(2-methyl-5-nitroimidazol-yl)ethylamine dihydrochloride, K2CO3.

boiling acetone furnished derivatives 20-25 and 27 (Scheme 3). Sulfur derivative 20 was oxidized to the corresponding sulfone 26 with MCPBA. Derivatives 2836 were obtained by reacting the arylphenylbromomethanes or benzyl chloride with 2-(2-methyl-5-nitroimidazol-yl)ethylamine dihydrochloride18 in the presence of potassium carbonate in DMF at 100 °C. Reaction of 2-(diphenylmethoxy)ethanol19 with 2-methyl-4(5)-nitroimidazole in the presence of diethyl azodicarboxylate and triphenylphosphine according to the Mitsunobu reaction20 afforded a mixture of 1-[2-(diphenylmethoxy)ethyl]-2-methyl-5-nitroimidazole (4)11a and the related isomer 1-[2-(diphenylmethoxy)ethyl]-2methyl-4-nitroimidazole (38). In a similar way were prepared 1-[2-(diphenylmethoxy)ethyl]-5-nitroimidazole (41), 1-[2-(diphenylmethoxy)ethyl]-4-nitroimidazole (42), 1-[2-(phenylmethoxy)ethyl]-2-methyl-5-nitroimidazole (43), and 1-[2-(phenylmethoxy)ethyl]-2-methyl-4-nitroimidazole (44), using 4(5)-nitroimidazole and 2-(diphenylmethoxy)ethanol or 2-(phenylmethoxy)ethanol, respectively (Scheme 4). SAR Evaluation Test derivatives were evaluated for cytotoxicity and anti-retroviral activity against HIV-1 WT strain. The efficacy of test compounds against HIV-1 WT was evaluated in MT-4 cells by means of an MTT assay (EC50 values). Selected compounds were tested in vitro against RT WT and a panel of RTs containing the single amino acid mutation L100I, K103N, Y181I, V179D, and Y188L (ID50 values). Among eleven newly synthesized 1-{2-[R-(thiophen2-yl)arylmethoxy]ethyl}-2-methyl-5-nitroimidazoles (7-

De Martino et al.

Scheme 4a

a Reagents. a: 2-methyl-4(5)-nitroimidazole, diethyl azodicarboxylate, triphenylphosphine; b: 4(5)-nitroimidazole, diethyl azodicarboxylate, triphenylphosphine.

Table 1. Cytotoxicity, Anti-HIV-1 Activity, and Selectivity Index of Compounds 7-19

compd

X

R1

R2

R3

R4

CC50a

EC50b

SIc

7 8 9 10 11 12 13 14 15 16 17 18 19 4d

S S S S S S S S S S S O -

H CH3 H Cl H H F H H F H H -

H H H H Cl H H F H F H H -

H H CH3 H H Cl H H F H H H -

H H H H H H H H H H Cl H -

>100 92 >100 >100 72 >100 >100 >100 >100 >100 >100 84 >100 >100

0.03 0.45 0.3 1.9 0.08 58 0.12 0.14 3.1 0.06 1.2 0.1 0.08 0.2

>3333 204 >333 52.6 900 >1.7 >833 >714 >32.3 >1667 8.33 840 >1250 >500

a Data are mean values of two experiments performed in triplicate. b Compound dose (µM) required to reduce the viability of mock-infected cells by 50%, as determined by MTT method. c Compound dose (µM) required to achieve 50% protection of MT-4 cells from HIV-1 induced cytopathogenicity, as determined by MTT method. d Selectivity Index, CC50:EC50 ratio. d Literature11a

17), seven derivatives (7-9, 11, 13, 14, and 16) were endowed with antiviral activities at submicromolar concentrations, three compounds (10, 15, and 17) were active at micromolar concentration, and only one (12) was poorly active (Table 1). With the only exception of compound 12, the inhibitory activities ranged from 0.03 µM (compound 7) to 3.1 µM (compound 15). 1-{2-[R(Furan-2-yl)arylmethoxy]ethyl}-2-methyl-5-nitroimidazole (18) and 1-{2-[R-(pyridin-3-yl)arylmethoxy]ethyl}2-methyl-5-nitroimidazole (19) were also active at submicromolar concentrations, showing EC50s ) 0.1 and 0.08 µM, respectively. Among compounds 7-19, 10 derivatives (7, 9, 10, 12-17, and 19) were found poor cytotoxic showing CC50 greater or equal to 100 µM. The



Table 2. Cytotoxicity, Anti-HIV-1 Activity, and Selectivity Index of Compounds 20-44

compd

Y

Z

R1

R2

R3

R4

R5

R6

CC50a

EC50b

SIc

20 21 22 23 24 25 26 27 28d 29 30 31d 32 33d 34d 35d 36d 37 38 39e 40 41 42 43 44 4d

phenyl phenyl phenyl phenyl phenyl phenyl phenyl H phenyl phenyl phenyl phenyl phenyl phenyl phenyl phenyl H H phenyl 2-naphthyl cyclohexyl phenyl phenyl H H -

S S S S S S SO2 S NH NH NH NH NH NH NH NH NH N-benzyl O O O O O O O -

H Cl F H H H H H H Cl F H H H H H H H H H H H H H H -

H H H CH3 Cl F H H H H H CH3 Cl F H H H H H H H H H H H -

H H H H H H H H H H H H H H CH3 F H H H H H H H H H -

NO2 NO2 NO2 NO2 NO2 NO2 NO2 NO2 NO2 NO2 NO2 NO2 NO2 NO2 NO2 NO2 NO2 NO2 H NO2 NO2 NO2 H NO2 H -

H H H H H H H H H H H H H H H H H H NO2 H H H NO2 H NO2 -

CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 H H CH3 CH3 -

>100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100

1.2 12 5 3 64 80 5.2 60 12 90 50 65 2.1 1.8 44 92 >100 84 67 >100 0.3 >100 78 4.2 22 0.2

>83.5 >8.3 >20 >33.3 >1.6 >1.2 >19.2 >1.7 >8.3 >1.1 >2 >1.5 >47.6 >55.6 >2.3 >1.1 >1.2 >1.5 >333.3 >1.3 >2.38 >4.5 >500

a Data are mean values of two experiments performed in triplicate. b Compound dose (µM) required to reduce the viability of mockinfected cells by 50%, as determined by MTT method. c Compound dose (µM) required to achieve 50% protection of MT-4 cells from HIV-1 induced cytopathogenicity, as determined by MTT method. d Selectivity Index: CC50:EC50 ratio. d Tested as dihydrochloride. e Literature10a,b

selectivity indexes of derivatives 7-19 ranged from 2275 >3333 (7) to >1.7 (12). 1-{2-[R-(Thiophen-2-yl)phenymethoxy]ethyl}-2-methyl-5-nitroimidazole (7, EC50 ) 0.03 µM for the racemic compound) was 6.7 times more potent than the parent compound 4 (EC50 ) 0.2 µM), and it was also the most potent derivative within the series. In general the introduction of chlorine or fluorine atoms or a methyl group on the phenyl ring of 7 decreased the antiviral activity in the order ortho > meta > para (compare 8, 10, and 13 with 11, 14, and with 9, 12, and 15). With the exception of derivatives 8 and 11, the cytotoxitity >100 µM. Among ortho-substituted derivatives, the fluoro compound 13 (EC50 ) 0.12 µM) was found 3.7 and 15.8 times more potent than 8 (EC50 ) 0.45 µM) and 10 (EC50 ) 1.9 µM), respectively. Interestingly, the introduction of a fluorine atom at meta position of 13 did not decrease the antiviral activity as it did for the monosubstituted derivatives (compare 13 with 16). Introduction of a chlorine atom at position 2 of the thiophene ring of 7 led to a 40 times abatement of the antiviral activity (compare 7 with 17). Replacement of the 2-thiophenyl nucleus for a 2-furanyl or a 3-pyridinyl ring led to derivatives 18 and 19 endowed with 2- or 2.5-fold superior activity than 4, but 3.3 and 2.7 times less active than 7, respectively. Although scarcely cytotoxic, sulfur and amino derivatives 20-39 weakly inhibited the HIV multiplication

(Table 2). Replacement of the ethereal oxygen of 4 with a sulfur atom or an amino group resulted in 6 and 60 times abatement of the inhibitory activity, respectively (compare 4 with 20, EC50 ) 1.2 µM, and 28, EC50 ) 12 µM). Oxidation of sulfur to sulfone further reduced 4.3 times the activity of 20 (compare 20 with 26). Introduction of substituents on the phenyl ring did not affect remarkably the antiviral activity of derivatives 21-25 and 29-35. In the sulfur series, only the meta-methyl derivative 23 (EC50 ) 3 µM) retained partially the antiviral activity of 20, being 2.5 times less potent. In the amino series, the meta-chloro- and the meta-fluoroderivatives (32 and 33) were about 5.7 and 6.7 times more potent than 28. Finally we confirmed the essential role of the second (hetero)aryl group for the antiviral activity of DAMNIs (compare 27 with 20, 36, 37 with 28, and 39, 40, 43, 44 with 4). Morover the presence of the methyl and nitro groups at position 2 and 5 of the imidazole ring, respectively, were also crucial for inhibition the RT of HIV-1. In fact, 1-[2-(diphenylmethoxy)ethyl]-2-methyl4-nitroimidazole (38), 1-[2-(diphenylmethoxy)ethyl]-5nitroimidazole (41), and 1-[2-(diphenylmethoxy)ethyl]4-nitroimidazole (42) were drammatically less active than the 4. The SAR evaluations of the DAMNI family are summarized in Chart 3. Derivatives 7 and 11 were tested against RT WT and a panel RTs containing the single amino acid mutation (L100I, K103N, Y181I, V179D, and Y188L) responsible

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Molecular Docking of Compound 7

Chart 3. SARs on the DAMNI Family

Table 3. HIV-1 RT Inhibitory Activity of Compounds 7, 11, 20, and 28 against Wild-Type and Mutant Enzymes Carrying Single Amino Acid Substitutionsa compd 7 11 20 28 NVP (1)d EFV (3)e

wt L100I K103N wt ID50 EC50 ID50b ID50

Y181I ID50

0.04 0.37 0.1 1.17 2.3 10.64 8 16.5 0.25 0.4 0.004 0.08

>50 6.25 n.t. n.t. 36 0.4

5.5 3.2 n.t.c n.t. 9 0.2

8.25 5.1 n.t. n.t. 7 25

De Martino et al.

V179D Y188L ID50 ID50 1.9 10.8 n.t. n.t. 0.3 0.09

30.2 15.1 n.t. n.t. 18 0.1

a Data represent mean values of at least three separate experiments. b Compound dose (Ki, µM) required to inhibit by 50% the RT activity of the indicated strain. c n.t., not tested. d Literature.10e e Literature.10k

for resistance to NNRTIs. The esperimental results are shown in Table 3. Compound 7 showed a profile of inhibition comparable or slightly less potent to that of nevirapine (1) against all the tested RTs. Compound 11 was found more potent than 1 against the L100I, K103N, and Y181I RTs, but less potent than 3 against all the tested RTs. Both compounds 7 and 11 were generally less active than efavirenz (3), with the notable exception of the K103N mutation for which 7 and 11 were 3- and 5-times more potent.

Because of the very well-known isosteric interchange between benzene ring and thiophene nucleus, the binding mode of designed compound 7 was investigated as possible new DAMNI lead compound. Either the R or S absolute configurations were modeled starting from the available model structure of 411b and optimized using a standard molecular dynamics protocol. Autodock program was used to dock (R)-7 and (S)-7 into the RT nonnucleoside binding pocket (NNBS). Analogously as previously reported the NNBS was obtained using the coordinates of HIV-1 RT/TNK-651 (70)21 taken from the Protein Brookheaven database (PDB entry code 1rt2). The docking experiments were carried out employing the Lamarckian genetic algorithm with local search (GA-LS) hybrid formalism of the docking program Autodock 3.0.522 that predicts the bound conformations of flexible ligands to macromolecular targets. Primarily, the docking of 70 into the RT was performed to test the reliability of the docking formalism for our purposes. Second similar docking experiment was conducted on lead compound 4 as test for our previous findings11b and for comparison purposes. Autodock was able to reproduce the experimental binding conformation of 70 within a minimal root-meansquare deviation (RMSD ) 0.99 Å) and confirmed our previous findings about the binding mode of compound 4 (see Experimental Section). About the docking experiments on the designed derivative 7, interestingly it seems that the introduction of a chiral center would not affect the binding of both (R)-7 and (S)-7. Either R or S absolute configurations of 7 seems to bind into the NNBS in similar way, in fact autodock internal scoring function calculated the same inhibition constant for the two enantiomers (Ki ) 7.9 nM). A closer investigation of the (R)-7 and (S)-7 binding mode reveals that the thiophene rings are not overlapped each other, but are cross superimposed with the phenyl rings confirming the full benzene - thiophene interchange (Figure 1).

Figure 1. Superimposition of compound 7 as found from Autodock. In yellow is reported the (R) absolute configuration and in magenta the (S) configuration. A 3 Å core of NNBS is also displayed.



Moreover as a consequence of the thiophene ring inversion a positional switch between the nitro and the methyl groups of the imidazole moiety occurs. This is in agreement with the dual binding mode observed for compound 4.10b Ligand/receptors interactions also resemble those already described for 4, the phenyl ring of (R)-7 (thiophene for (S)-7) makes favorable interactions with a mild hydrophobic pocket formed by the side chain of Val106, the R-CH2 of Gly190, and β-CH2 of Tyr181; the thiophene ring of (R)-7 (benzene for (S)-7) makes favorable interactions with a bigger hydrophobic and aromatic rich pocket formed by the side chains of Tyr181, Tyr188, Phe227, Trp229, and Leu234. On the other side of the molecule the basic nitrogen of imidazole ring of (R)-7 makes an hydrogen bond with the NH of Lys103 (distance Nimidazole‚‚‚NHLys103 ) 2.5 Å) while the same interaction occurs for (S)-7 by the mean of the nitro group (average distance NO2‚‚‚NHLys103 ) 2.9 Å).

indicator at 254 nm) and silica gel TLC cards from Fluka (silica gel-precoated aluminum cards with fluorescent indicator at 254 nm) were used for thin-layer chromatography (TLC). Developed plates were visualized by a Spectroline ENF 260C/F UV apparatus. Organic solutions were dried over anhydrous sodium sulfate. Concentration and evaporation of the solvent after reaction or extraction were carried out on a rotary evaporator (Bu¨chi Rotavapor) operating at reduced pressure. Elemental analyses were found to be within (0.4% of the theoretical values. (Furan-2-yl)-phenylmethanol, (cyclohexyl)phenylmethanone, phenylmethoxyethanol, and 1-(2-hydroxyethyl)-2-methyl-5-nitroimidazole were obtained from commercial sources. General Procedure for the Synthesis of DAMNIs 7-18. Example. 1-{2-[R-(Thiophen-2-yl)phenylmethoxy]ethyl}2-methyl-5-nitroimidazole (7). A solution phenyl-(thiophen2-yl)-methanol (56, 1.9 g, 0.01 mol), 1-(2-hydroxyethyl)-2methyl-5-nitroimidazole (0.51 g, 0.003 mol), and para-toluenesulfonic acid (0.10 g,) in benzene (80 mL) was refluxed under nitrogen atmosphere with azeotropic removal of the water using a Dean-Stark trap. After evaporation of the solvent, the crude residue was treated with sodium hydrogen carbonate solution and extracted with ethyl acetate. The organic solution was shaken with brine, dried, and evaporated in vacuo to leave a dark oil which was purified through a silica gel column chromatography (chloroform as eluent) to give 7 (0.55 g, 53%), mp 90-92 °C (from cyclohexane). Anal. (C17H17N3O3S (343.41)) C, H, N, S. 1-{2-[R-(Thiophen-2-yl)-(2-methylphenyl)methoxy]ethyl}-2-methyl-5-nitroimidazole (8). Was prepared as 7 using alcohol 57. Yield 82%, oily material. 1H NMR (CDCl3): δ 2.18 (s, 3H), 2.56 (s, 3H), 3.77 (m, 2H), 4.53 (t, J ) 4.8 Hz, 2H), 5.63 (s, 1H), 6.61 (m, 1H), 6.85 (dd, J ) 1.9 and 5.0 Hz, 1H), 7.08-7.25 (m, 5H), 7.95 ppm (s, 1H). Anal. (C18H19N3O3S (357.43)) C, H, N, S. 1-{2-[R-(Thiophen-2-yl)-(4-methylphenyl)methoxy]ethyl}-2-methyl-5-nitroimidazole (9). Was prepared as 7 using alcohol 58. Yield 50%, oily material. Anal. (C18H19N3O3S (357.43)) C, H, N, S. 1-{2-[R-(Thiophen-2-yl)-(2-chlorophenyl)methoxy]ethyl}2-methyl-5-nitroimidazole (10). Was prepared as 7 using alcohol 59. yield 80%, mp 45-47 °C (from cyclohexane). Anal. (C17H16ClN3O3S (377.84)) C, H, Cl, N, S. 1-{2-[R-(Thiophen-2-yl)-(3-chlorophenyl)methoxy]ethyl}2-methyl-5-nitroimidazole (11). Was prepared as 7 using alcohol 60. Yield 60%, oily material. Anal. (C17H16ClN3O3S (377.84)) C, H, N, Cl, S. 1-{2-[R-(Thiophen-2-yl)-(4-chlorophenyl)methoxy]ethyl}2-methyl-5-nitroimidazole (12). Was prepared as 7 using alcohol 61. Yield 66%, mp 75-77 °C (from cyclohexane). Anal. (C17H16ClN3O3S (377.84)) C, H, N, Cl, S. 1-{2-[R-(Thiophen-2-yl)-(2-fluorophenyl)methoxy]ethyl}2-methyl-5-nitroimidazole (13). Was prepared as 7 using alcohol 62. Yield 74%, oily material. Anal. (C17H16FN3O3S (361.39)) C, H, N, F, S. 1-{2-[R-(Thiophen-2-yl)-(3-fluorophenyl)methoxy]ethyl}2-methyl-5-nitroimidazole (14). Was prepared as 7 using alcohol 63. Yield 42%, oily material. Anal. (C17H16FN3O3S (361.39)) C, H, N, F, S. 1-{2-[R-(Thiophen-2-yl)-(4-fluorophenyl)methoxy]ethyl}2-methyl-5-nitroimidazole (15). Was prepared as 7 using alcohol 64. Yield 78.4%, mp 81-85 °C (from cyclohexane). Anal. (C17H16FN3O3S (361.39)) C, H, N, F, S. 1-{2-[R-(Thiophen-2-yl)-(2,3-difluorophenyl)methoxy]ethyl}-2-methyl-5-nitroimidazole (16). Was prepared as 7 using alcohol 65. Yield 71%, oily material. Anal. (C17H15F2N3O3S (379.38)) C, H, N, F, S. 1-{2-[R-(5-Chlorothiophen-2-yl)phenylmethoxy]ethyl}2-methyl-5-nitroimidazole (17). Was prepared as 7 using alcohol 66. Yield 82%, mp 109-112 °C (from cyclohexane). Anal. (C17H16ClN3O3S (377.84)) C, H, N, Cl, S. 1-{2-[R-(Furan-2-yl)phenylmethoxy]ethyl}-2-methyl-5nitroimidazole (18). Was prepared as 7 using the (furan-2-

Conclusions DAMNIs are a novel family of NNRT agents initially designed taking account the “butterfly-like” conformation as a determinant requisite for antiretroviral activity. Although not very potent, DAMNIs were found active with compound 4 showing EC50 ) 0.2 µM, CC50 > 100 µM, and SI > 500. Replacement of one phenyl group with heterocyclic rings led to novel DAMNIs with increased activity. Compounds 7 and 19, with 2-thienyl and 3-pyridinyl rings, were found 6.7 and 2.5 times more active than compound 4. Introduction of halogens, chlorine, and fluorine in the phenyl ring of 7 did not improve the antiviral activity, thus proving that sterical impediments produced by substituents are deleterious for binding of DAMNIs at the NNBS of the RT. Efavirenz is the a first line NNRTI which was found highly effective when used in combination with two NRTIs in protease-sparing regimen. However the majority of patients (>90%) treated with efavirenz, whose viral loads rebounded after an initial response to the drug, selected the K103N mutation. Furthermore, after the emergence of the K103N mutation, additional double mutations (K103N-V108N, K103N-P225H) slowly appear in many patients.24 These observations highlighted the dramatic need of novel NNRTI effective against HIV-1 RT containing either single or double K103N mutations, when used in combination with other antiretroviral agents. Compounds 7 and 11 were found more active than efavirenz against the viral RT carrying the K103N mutation, suggesting for these compounds a potential use in efavirenz based anti-AIDS regimens. These findings are of great stimulus to develop novel DAMNI derivatives. Experimental Section Chemistry. Melting points were determined on a Bu¨chi 510 apparatus and are uncorrected. Infrared spectra were run on Perkin-Elmer 1310 and SpectrumOne spectrophotometers. Band position and absorption ranges are given in cm-1. Proton nuclear magnetic resonance spectra were recorded on Bruker A300 (200 MHz) Fourier transform spectrometer in the indicated solvent. Chemical shifts are expressed in δ units (ppm) from tetramethylsilane. Chromatographic columns were packed with alumina (Merck, 70-230 mesh) and silica gel (Merck, 70-230 mesh). Aluminum oxide TLC cards from Fluka (aluminum oxide-precoated aluminum cards with fluorescent

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yl)-phenylmethanol. Yield 7.5%, yellow oil. Anal. (C17H17N3O4 (327.38)) C, H, N. 1-{2-[R-(Cyclohexyl)phenylmethoxy]ethyl}-2-methyl-5nitroimidazole (40). Was prepared as 7 using the (cyclohexyl)-phenylmethanol. Yield 46%, yellow oil. Anal. (C19H25N3O3 (343.43)) C, H, N. 1-{2-[R-(Pyridin-3-yl)phenylmethoxy]ethyl}-2-methyl5-nitroimidazole (19). A solution of thionyl chloride (1.27 g, 0.78 mL, 0.011 mol) in chloroform (5 mL) was added dropwise to a an ice-cooled mixture of phenyl-(pyridin-3-yl)methanol (68) (1.65 g, 0.0089 mol) in the same solvent (18.1 mL). The reaction was heated at 50 °C for 5 h. The R-(pyridin-3-yl)phenylmethyl chloride hydrochloride (70) was precipitated with anhydrous dry diethyl ether, filtered, and used without purification. A mixture of 69, triethylamine (0.63 g, 0.88 mL, 0.0075 mol), and 1-(2-hydroxyethyl)-2-methyl-5-nitroimidazole (0.33 g, 0.0019 mol) in anhydrous N,N-dimethylformamide was stirred under dry nitrogen atmosphere at 50 °C overnight. After cooling, the reaction mixture was diluted with water and extracted with ethyl acetate. The organic layer was separated, washed with brine, and dried. Evaporation of the solvent gave a residue which was purified by passing through an alumina column (chloroform as eluent) to afford 19, (0.06 g, 9.3%) as a yellow oil. Anal. (C18H18N4O3 (338.36)) C, H, N. 1-[2-(Diphenylmethylthio]ethyl]-2-methyl-5-nitroimidazole (20). A mixture of dipheylbromomethane (0.13 g, 0.0005 mol), 2-(2-methyl-5-nitroimidazol-1-yl)ethanethiol (0.10 g, 0.0005 mol), and potassium carbonate (0.22 g, 0.0016 mol) in acetone (6 mL) was stirred at reflux for 4 days. After 24, 48, and 72 h, diphenylbromomethane (0.13 g, 0.0005 mol) and potassium carbonate (0.218 g, 0.0016 mol) were added. After cooling, the reaction was poured on ice-water, stirred for 15 min, and extracted with ethyl acetate. The organic layer was washed with brine, dried, and evaporated in vacuo to give a the crude product which was purified through a silica gel column cromatography (chloroform as eluent) to afford 20 (0.042 g, yield 24%), mp 124-126 °C (from cyclohexane). Anal. (C19H19N3O2S (353.44)) C, H, N, S. 1-{2-[R-(2-Chlorophenyl)phenylmethylthio]ethyl}-2methyl-5-nitroimidazole (21). Was prepared as 20 using (2chlorophenyl)phenylbromomethane. Yield 56%, oily material. Anal. (C19H18ClN3O2S (387.88)) C, H, Cl, N, S. 1-{2-[R-(2-Fluorophenyl)phenylmethylthio]ethyl}-2methyl-5-nitroimidazole (22). Was prepared as 20 using (2fluorophenyl)phenylbromomethane. Yield 30%, mp 93-95 °C (from n-hexane). Anal. Calcd. (C19H18FN3O2S (371.43)) C, H, F, N, S. 1-{2-[R-(3-Methylphenyl)phenylmethylthio]ethyl}-2methyl-5-nitroimidazole (23). Was prepared as 20 using (3methylphenyl)phenylbromomethane. Yield 30%, mp 113-115 °C (from n-hexane). Anal. (C20H21N3O2S (367.46)) C, H, N, S. 1-{2-[R-(3-Chlorophenyl)phenylmethylthio]ethyl}-2methyl-5-nitroimidazole (24). Was prepared as 20 using (3chlorophenyl)phenylbromomethane. Yield 58%, mp 81-83 °C (from ligroin). Anal. (C19H18ClN3O2S (387.88)) C, H, Cl, N, S. 1-{2-[R-(3-Fluorophenyl)phenylmethylthio]ethyl}-2methyl-5-nitroimidazole (25). Was prepared as 20 using (3fluorophenyl)phenylbromomethane. Yield 57%, mp 92-93 °C (from n-hexane). Anal. (C19H18FN3O2S (371.43)) C, H, F, N, S. 1-[2-(Diphenylmethylsulfonyl]ethyl]-2-methyl-5-nitroimidazole (26). 3-Chloroperbenzoic acid (0.048 g, 0.00028 mol) was added while stirring to an ice-cooled solution of 1-[2(diphenylmethylthio]ethyl]-2-methyl-5-nitroimidazole (20, 0.10 g, 0.00028 mol). The reaction was stirred at 25 °C for 30 min and then was poured on ice-water and extracted with ethyl acetate. The organic layer was washed with brine and dried. Evaporation of the solvent gave a residue which was purified through a silica gel column chromatography (ethyl acetate as eluent) to afford 26, (0.11 g, yield 100%), mp 189-191 °C (from benzene). Anal. (C19H19N3O4S (385.88)) C, H, N, S. 1-[2-(Phenylmethylthio]ethyl]-2-methyl-5-nitroimidazole (27). A mixture of benzyl chloride (0.33 g, 0.36 mL, 0.0026 mol), 2-(2-methyl-5-nitroimidazol-1-yl)etanethiol (0.45 g, 0.0026 mol), potassium carbonate (1.02 g, 0.0074 mol), and

acetone (28 mL) was refluxed for 48 h. Benzyl chloride (0.33 g, 0.36 mL, 0.0026 mol) and potassium carbonate (1.00 g, 0.0074 mol) were added at half time. After cooling, the reaction was diluted with water and extracted with ethyl acetate. The organic layer was washed with brine, dried, and evaporated. The remaining oil was purified through a silica gel column chromatography (ethyl acetate as eluent) to furnish 27 (0.19 g, yield 42%) as a yellow oil. Anal. (C13H15N3O2S (277.34)) C, H, N, S. 1-[2-(Diphenylmethylamino]ethyl]-2-methyl-5-nitroimidazole (28). To a suspension of 2-(2-methyl-5-nitroimidazol-yl)ethylamine dihydrochloride23 (0.64 g, 0.003 mol) and triethylamine (0.76 g, 1.045 mL, 0.0075 mol) in dry DMF (10 mL) was added a solution of diphenylbromomethane (0.62 g, 0.0025 mol) in the same solvent (2 mL). Reaction was stirred at 100 °C overnight under dry nitrogen atmosphere. After cooling, the mixture was diluted with water and extracted with ethyl acetate. The organic layer was washed with brine, dried, and evaporated in vacuo. The residue was purified by passing at first through a silica gel column chromatography (ethyl acetate as eluent) and then through an alumina column chromatography (chloroform as eluent) to afford 28 (0.11 g, yield 13%), mp 145 °C (dihydrochloride, triturated with diethyl ether). Anal. (C19H20N4O2‚2HCl (409.31)) C, H, Cl, N. 1-{2-[R-(2-Chlorophenyl)phenylmethylamino]ethyl}-2methyl-5-nitroimidazole (29). Was prepared as 28 using (2chlorophenyl)phenylbromomethane. Yield 18%, mp 96-98 °C (from n-hexane). Anal. (C19H19ClN4O2 (370.83)) C, H, Cl, N. 1-{2-[R-(2-Fluorophenyl)phenylmethylamino]ethyl}-2methyl-5-nitroimidazole (30). Was prepared as 28 using (2fluorophenyl)phenylbromomethane. Yield 23%, mp 72-73 °C (from n-hexane). Anal. (C19H19FN4O2 (354.38)) C, H, F, N. 1-{2-[R-(3-Methylphenyl)phenylmethylamino]ethyl}-2methyl-5-nitroimidazole (31). Was prepared as 28 using (3methylphenyl)phenylbromomethane. Yield 10%, mp 147 °C (dihydrochloride, from triturated with diethyl ether). Anal. (C20H22N4O2‚2HCl (423.34)) C, H, Cl, N. 1-{2-[R-(3-Chlorophenyl)phenylmethylamino]ethyl}-2methyl-5-nitroimidazole (32). Was prepared as 28 using (3chlorophenyl)phenylbromomethane. Yield 36%, mp 106-110 °C (from n-hexane). Anal. (C19H19ClN4O2 (370.83)) C, H, Cl, N. 1-{2-[R-(3-Fluorophenyl)phenylmethylamino]ethyl}-2methyl-5-nitroimidazole (33). Was prepared as 28 using (3fluorophenyl)phenylbromomethane. Yield 23%, mp 226-228 °C (dihydrochloride, triturated with diethyl ether). Anal. (C19H19FN4O2‚2HCl (427.30)) C, H, Cl, F, N. 1-{2-[R-(4-Methylphenyl)phenylmethylamino]ethyl}-2methyl-5-nitroimidazole (34). Was prepared as 28 using (4methylphenyl)phenylbromomethane. Yield 3%, mp 162-163 °C (dihydrochloride, triturated with diethyl ether). Anal. (C20H22N4O2‚2HCl (423.34)) C, H, Cl, N. 1-{2-[R-(4-Fluorophenyl)phenylmethylamino]ethyl}-2methyl-5-nitroimidazole (35). Was prepared as 28 using (4fluorophenyl)phenylbromomethane. Yield 10%, mp 220-222 °C (dihydrochloride, triturated with diethyl ether). Anal. (C19H19FN4O2‚2HCl (427.30)) C, H, Cl, F, N. 1-[2-(Phenylmethylamino)ethyl]-2-methyl-5-nitroimidazole (36). To a solution of 2-(2-methyl-5-nitroimidazol-yl)ethylamine dihydrochloride (0.5 g, 0.002 mol), and triethylamine (0.21 g, 1.045 mL, 0.0075 mol) in dry DMF (8 mL) was added a solution of benzyl chloride in the same solvent (1.6 mL). The reaction was stirred overnight at room temperature under nitrogen atmosphere and then diluted with water and extracted with ethyl acetate. The organic layer was washed with brine, dried, and evaporated in vacuo. The crude product was purified through a silica gel column chromatography (chloroform-ethanol 95:5 as eluent) to afford 36, (0.14 g, yield 29%), mp 107-110 °C (dihydrochloride, triturated with diethyl ether). Anal. (C13H16N4O2‚2HCl (333.21)) C, H, Cl, N. 1-{2-[(Diphenylmethyl)amino]ethyl}-2-methyl-5-nitroimidazole (37). To a suspension of 36 (0.5 g, 0.002 mol) and triethylamine (0.22 g, 0.3 mL, 0.002 mol) in dry DMF (8 mL) was added a solution of benzyl chloride (0.25 g, 0.23 mL, 0.002



mol) in the same solvent (1.6 mL). The mixture was stirred for 12 h at room temperature. Benzyl chloride (0.25 g, 0.23 mL, 0.002 mol) and triethylamine (0.212 g, 0.3 mL, 0.002 mol) were added, and the reaction was kept for additional 2 h. After dilution with water, the reaction mixture extracted with ethyl acetate. Organic extracts were combined, washed with brine, and dried. Removal of the solvent gave a residue which was purified through a silica gel column chromatography (chloroform as eluent) to afford 37 (0.06 g, yield 9%), mp 198-200 °C (dihydrochloride, triturated with diethyl ether). Anal. (C20H22N4O2‚2HCl (423.34)) C, H, N. 1-[2-(Diphenylmethoxy)ethyl]-2-methyl-4-nitroimidazole (38). A solution of diethyl azodicarboxylate (0.63 g, 0.56 mL, 0.0036 mol) in dry THF (10 mL) was added dropwise to a mixture of 2-(diphenylmethoxy)ethanol19 (0.75 g, 0.0033 mol), 2-methyl-5-nitroimidazole (0.46 g, 0.0036 mol), and anhydrous triphenylphosphine (0.95 g, 0.0036 mol) in the same solvent (20 mL). The reaction was stirred under dry nitrogen at roomtemperature overnight. Evaporation of the solvent gave a residue which was diluted with water and extracted with ethyl acetate. The organic layer was washed with brine, dried, and evaporated in vacuo. The crude residue was purified at first through a silica gel column chromatography (chloroformethanol 95:5 as eluent) and then through an alumina column (chloroform as eluent). First eluates gave to give 1-[2-(diphenylmethoxy)ethyl]-2-methyl-5-nitroimidazole (4)10a (0.33 g, 34%). Further elution with the same solvent afforded 38 (0.20 g, yield 20%), mp 112-114 °C (from cyclohexane). Anal. (C19H19N3O3 (337.38)) C, H, N. 1-[2-(Diphenylmethoxy)ethyl]-5-nitroimidazole (41) and 1-[2-(Diphenylmethoxy)ethyl]-4-nitroimidazole (42). Were prepared as 4 and 38 using 4(5)-nitroimidazole. The mixture of isomers was separated through a silica gel column chromatography (two repeated passages, chloroform-ethanol 95:5 and ethyl acetate-petroleum ether 1:1). First eluates gave 42, yield 33%, mp 126-127 °C (from toluene-cyclohexane). Further elution with the same solvent afforded 41, yield 22%, mp 110112 °C (from toluene-cyclohexane). Anal. (C18H17N3O3 (323.35)) C, H, N. 1-[2-(Phenylmethoxy)ethyl]-5-nitroimidazole (43) and 1-[2-(Phenylmethoxy)ethyl]-4-nitroimidazole (44). Were prepared as 4 and 38 using 2-(phenylmethoxy)ethanol. The mixture of isomers was separated through a silica gel column chromatography (chloroform-ethanol 95:5 as eluent). First eluates gave 43, yield 22%, mp 60-62 °C (from cyclohexane). Anal. (C13H15N3O3 (261.28)) C, H, N. Further elution with the same solvent afforded 44, yield 30%, mp 88-90 °C. Anal. (C13H15N3O3 (261.28)) C, H, N. General Procedure for the Synthesis of Methanones 45-55. Example. Phenyl-(thiophen-2-yl)methanone (45). A solution of benzoyl chloride (1.84 g, 0.013 mol) in dichloromethane (18 mL) was added dropwise to an ice-cooled suspension of anhydrous aluminum chloride (1.9 g, 0.013 mol) and thiophene (1.09 g, 0.013 mol) in the same solvent (18 mL). The reaction was sirred at room temperature for 2 h. After quenching on crushed ice the mixture was made acid with 37% HCl (1.3 mL) extracted with chloroform. The organic layer was washed with brine, dried, and evaporated to dryness. The crude product was purified through a silica gel column chromatography (dichloromethane petroleum ether 1:1 as eluent) to afford pure 45 (2.4 g, 90%) as yellow oil.15a (2-Methylphenyl)-(thiophen-2-yl)methanone (46). Was prepared as 45 in yield 80% using 2-methylbenzoyl chloride.15b (4-Methylphenyl)-(thiophen-2-yl)methanone (47). Was prepared as 45 in yield 90% using 4-methylbenzoyl chloride.15c (2-Chlorophenyl)-(thiophen-2-yl)methanone (48). Was prepared as 45 in yield 90% using 2-chlorobenzoyl chloride.15e (3-Chlorophenyl)-(thiophen-2-yl)methanone (49). Was prepared as 45 in yield 90% using 3-chlorobenzoyl chloride.15c (4-Chlorophenyl)-(thiophen-2-yl)methanone (50). Was prepared as 45 in yield 86% using 4-chlorobenzoyl chloride.15e (2-Fluorophenyl)-(thiophen-2-yl)methanone (51). Was prepared as 45 in yield 82% using 2-fluorobenzoyl chloride.15e

(3-Fluorophenyl)-(thiophen-2-yl)methanone (52). Was prepared as 45 using 2-fluorobenzoyl chloride. Yield 97%, yellow oil. Anal. (C11H7FOS (206,23)) C, H, F, S. (4-Fluorophenyl)-(thiophen-2-yl)methanone (53). Was prepared as 45 in yield 84% using 4-fluorobenzoyl chloride.15f (2,3-Difluorophenyl)-(thiophen-2-yl)methanone (54). Was prepared as 45 using 2,3-difluorobenzoyl chloride. Yield 26%, brown oil. Anal. (C11H6F2OS (224.22)) C, H, F, S. Phenyl-(5-chlorothiophen-2-yl)methanone (55). Was prepared as 45 in yield 86% using 2-chlorothiophene.15g General Procedure for the Synthesis of Methanols 56-66. Example. Phenyl-(thiophen-2-yl)methanol (56). Sodium borohydride (3.80 g, 0.01 mol) was added to a solution of phenyl-(thiophen-2-yl)methanone (45) (1.50 g, 0.008 mol) in THF (16 mL) containing 0.5 mL of water. The mixture was refluxed for 2 h. After cooling, the mixture was carefully diluted with water, concentrated to a small volume, and extracted with ethyl acetate. The organic layer was washed with brine, dried, and evaporated to afford 56, (1.46 g, yield 96%), mp 49-51 °C (from cyclohexane).16a (2-Methylphenyl)-(thiophen-2-yl)methanol (57). Was prepared as 56 by reduction of the methanone 46. Yield 90%, yellow oil. Anal. (C12H12OS (204.28)) C, H, S. (4-Mehylphenyl)-(thiophen-2-yl)methanol (58). Was prepared as 56 in yield 94% by reduction of the methanone 47.16b (2-Chlorophenyl)-(thiophen-2-yl)methanol (59). Was prepared as 56 in yield 92% by reduction of the methanone 48.16c (3-Chlorophenyl)-(thiophen-2-yl)methanol (60). Was prepared as 56 by reduction of the methanone 49. Yield 94%, brown oil. Anal. (C11H9ClOS (224.70)) C, H, Cl, O, S. (4-Chlorophenyl)-(thiophen-2-yl)methanol (61). Was prepared as 56 in yield 92% by reduction of the methanone 50.16c (2-Fluorophenyl)-(thiophen-2-yl)methanol (62). Was prepared as 56 by reduction of the methanone 51. Yield 88%, yellow oily material. Anal. (C11H9FOS (208.25)) C, H, F, S. (3-Fluorophenyl)-(thiophen-2-yl)methanol (63). Was prepared as 56 by reduction of the methanone 52. Yield 92%, yellow oily material. Anal. (C11H8F2OS (226.24)) C, H, F, S. (4-Fluorophenyl)-(thiophen-2-yl)methanol (64). Was prepared as 56 by reduction of the methanone 53.16d (2,3-Difluorophenyl)-(thiophen-2-yl)methanol (65). Was prepared as 56 by reduction of the methanone 54. Yield 89%, brown oil. Anal. (C11H8F2OS (226.24)) C, H, F, O, S. Phenyl-(5-chlorothiophen-2-yl)methanol (66). Was prepared as 56 by reduction of the methanone 55, yield 100%. Phenyl-(pyridin-3-yl)methanone (67). A suspension of nicotinic acid (3.00 g, 0.024 mol) in thionyl chloride (17 mL) was heated at 85 °C under stirring for 90 min. The excess of thionyl chloride was evaporated in vacuo, and the crude residue was treated with benzene (21 mL) and portionwise while stirring with anhydrous aluminum chloride (12.11 g, 0.091 mol) at 0 °C. The mixture was heated at reflux for 6 h, cooled, poured on ice containing 37% HCl (5 mL), and extracted with chloroform. The organic solution was washed with 1 N NaOH (50 mL) and with water (50 mL) and then dried and evaporated in vacuo. The residue was passed through a silica gel column chromatography (chloroform as eluent) to give 67 (1.75 g, yield 40%) as oily material.17a Phenyl-(pyridin-3-yl)methanol (68). Was prepared as 56 in yield 43% by reduction of the methanone 67.17c 2-(2-Methyl-5-nitroimidazol-1-yl)ethanthiol. A solution of 1-(2-hydroxyethyl)-2-methyl-5-nitroimidazole mesylate (2.00 g, 0.008 mol) and NaHS (0.58 g, 0.008 mol) in dry DMF was stirred under nitrogen stream at room temperature for 48 h. The reaction was poured on ice-water, stirred for additional 15 min, and extracted with ethyl acetate. The organic layer was washed with brine and dried. After removing of the solvent, the crude product was purified through a silica gel column chromatography (ethyl acetate-ethanol 1:1 as eluent) to give 2-(2-methyl-5-nitro-imidazol-1-yl)ethanthiol, (0.41 g, yield 30%), mp 115-118 °C (from ethanol).25

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Figure 2. A: Superimposition of experimental bound conformation of 70 (black) and that predicted by Autodock (grey). B: Previous model for 4 (black) compared to that found by Autodock (grey). Computational Studies. All molecular modeling calculations and manipulations were performed using the software packages Macromodel 7.1,26 Autodock 3.0.5,27 running on Silicon Graphics O2 R10000 and Octane R10000, IBM-compatible AMD Athlon XP 3.0 GHz workstations. For any minimization the all-atom Amber force field27 was adopted as implemented in the Macromodel package. The geometry of the NNBS of the wt RT strain was taken from the structure of HIV-1 RT/70 complex filed in the Brookhaven Protein Data Bank28 (entry code 1rt2). All the residues within 20 Å from any ligand’s atom (70) were used to define the NNBS. The reference compound 4 modeled as previously reported11b was used to build the two enantiomeric configurations of 7. The starting conformations for docking studies were obtained using molecular dynamics with simulated annealing as implemented in Macromodel version 7.1 and conducted as following: either (R)-7 or (S)-7 was energy minimized to a low gradient. The nonbonded cutoff distances were set to 20 Å for both van der Waals and electrostatic interactions. An initial random velocity to all atoms corresponding to 300 K was applied. Three subsequent molecular dynamic runs were then performed. The first was carried out for 10 ps with a 1.5 fs time-step at a constant temperature of 300 K for equilibration purposes. The next molecular dynamic was carried out for 20 ps, during which the system is coupled to a 150 deg thermal bath with a time constant of 5 ps. The time constant represents approximately the half-life for equilibration with the bath; consequently the second molecular dynamic command caused the molecule to slowly cool to approximately 150 K. The third and last dynamic cooled the molecule to 50 K over 20 ps. A final energy minimization was then carried out for 250 iterations using conjugate gradient. The minimizations and the molecular dynamics were in all cases performed in aqueous solution. The atom charges automatically assigned by the batchmin module were retained for the docking calculations.29 For the docking procedure the program Autodock was used to explore the binding conformation of either (R)-7 or (S)-7. For the docking a grid spacing of 0.375 Å and 60 × 80 × 60 number of points was used. The grid was centered on the mass center of the experimental bound 70 coordinates. The GA-LS method was adopted using the default settings. Amber united atom charges were assigned to the protein using the program ADT (AutoDock Tools).30 Autodock generated 250 possible binding conformations for each docking. To validate the use of the Autodock program, the docking studies were performed on the reference compound 70 and for comparison purposes also on the previously reported 4. Autodock successfully reproduced the experimental binding conformations of the reference drug 70 with acceptable root-mean-

square deviation (RMSD) of atom coordinates in either (RMSD ) 1.01 Å) and confirmed our previous findings about 4. In Figure 2A is reported the superimposition of experimental bound conformation of 70 and that predicted by Autodock; in Figure 2B our previous model for 4 compared to that found by Autodock (RMSD ) 1.2 Å). Cell Based Antiviral Assay Procedures. Viruses. A laboratory lymphocyte-tropic strain of HIV (HTLV-IIIB also called HIV-IIIB) was used to infect MT-4 cells. The strain is available through the AIDS Research and Reference Reagent Program (NIH, Bethesda, MD). HIV Titration. Titration to determine the infectivity of laboratory viral strains was performed in C8166 cells (HIVIIIB)31 The titer of virus stocks, expressed as 50% tissue culture infectious dose (TCID50) was determined as previously described.32 Anti-HIV Assays. The activity of test compounds against multiplication of HIV-1 in MT-4 cells was based on inhibition of virus-induced cytopathogenicity. Briefly, 150 µL of culture medium containing 2 × 104 cells were added to each well flatbottom microtiter trays containing 50 µL of culture medium with or without various concentrations of test compounds. Then 50 µL of HIV suspension (100 TCID50) was added. After 5 days incubation at 37 °C, the viral activity of compounds was evaluated by quantifying the HIV induced cytopathogenicity by means of the 3-(4,5-dimethylthiazol-1-yl)-2,5diphenyltetrazolium bromide (MTT) method.33 Protection (%) from the viral induced cytopathogenicity in HIV-infected cells as mesured by the MTT assay was calculated as follows: (ODT)HIV - (ODC)HIV: (ODC)mock - (ODC)HIV, whereby (ODT)HIV is the optical density measured with a given concentration of cysteamine in HIV infected cells, (ODC)HIV is the optical density measured for the control untreated HIV-infected cells, (ODC)mock is the optical density measured for the control untreated mock-infected cells. The optical density at 540/690 nm was measured using a plate reader. Cytotoxicity. The cytotoxicity of test compounds was evaluated in parallel with their antiviral activity and was based on the viability of mock-infected cells, as monitored by the MTT method. The 50% effective dose (ED50) and 50% cytotoxic dose (TD50) values were calculated from pooled values in the effective dynamic range of the antiviral activity and cytotoxicity assays (5-95%) using the median effect equation as previously described.34 The selectivity index (SI) was calculated ad TD50:ED50 ratio. Enzymatic Assay Procedures. Chemicals. [3H]dTTP (40 Ci/mmol) was from Amersham and unlabeled dNTP's from Boehringer. Whatman was the supplier of the GF/C filters. All other reagents were of analytical grade and purchased from Merck or Fluka.



Nucleic Acid Substrates. The homopolymer poly(rA) (Pharmacia) was mixed at weight ratios in nucleotides of 10: 1, to the oligomer oligo(dT)12-18 (Pharmacia) in 20 mM TrisHCl (pH 8.0), containing 20 mM KCl and 1 mM EDTA, heated at 65 °C for 5 min and then slowly cooled at room temperature. Expression and Purification of Recombinant HIV-1 RT Forms. The coexpression vectors pUC12N/p66(His)/ p51with the wild-type or the mutant forms of HIV-1 RT p66 (Boyer et al., 1994a) were kindly provided by Dr. S. H. Hughes (NCI-Frederick Cancer Research and Development Center). Proteins were expressed in E. coli and purified as described.35 HIV-1 RT RNA-Dependent DNA Polymerase Activity Assay. RNA-dependent DNA polymerase activity was assayed as follows: a final volume of 25 µL contained reaction buffer (50 mM Tris-HCl pH 7.5, 1 mM DTT, 0.2 mg/mL BSA, 4% glycerol), 10 mM MgCl2, 0.5 µg of poly(rA)/oligo(dT)10:1 (0.3 µM 3′-OH ends), 10 µM [3H]-dTTP (1Ci/mmol) and 2-4 nM RT. Reactions were incubated at 37 °C for the indicated time. Aliquots (20 µL) were then spotted on glass fiber filters GF/C which were immediately immersed in 5% ice-cold TCA. Filters were washed twice in 5% ice-cold TCA and once in ethanol for 5 min, dried, and acid-precipitable radioactivity was quantitated by scintillation counting. Inhibition Assays. Reactions were performed under the conditions described for the HIV-1 RT RNA-dependent DNA polymerase activity assay. Incorporation of radioactive dTTP into poly(rA)/oligo(dT) at different substrate (nucleic acid or dTTP) concentrations was monitored in the presence of increasing fixed amounts of inhibitor. Data were then plotted according to Lineweaver-Burke and Dixon. For Ki determination, an interval of inhibitor concentrations between 0.2 Ki and 5 Ki was used.

Acknowledgment. We thank the Universita` degli Studi di Roma “La Sapienza”, Finanziamento progetti di ricerca di Ateneo (ex quota 60%) - Anno 2002. We also acknowledge the finacial support of the Italian MIUR (cofin 2000) and the Italian Ministero della Salute - Istituto Superiore di Sanita` - Fourth National Research Program on AIDS (grants no. 40C.8 and no. 40D.46). Supporting Information Available: Spectroscopic data of new compounds 7-38, 40-44, 52, 54, 57, 60, 62, 63, and 65 and elemental analyses data of 7-38 and 40-44 are available free of charge via the Internet at http://pubs.acs.org.

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