Review For reprint orders, please contact [email protected]

Arylsulfone-based HIV-1 non-nucleoside reverse transcriptase inhibitors HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTIs) represent one of the most significant classes of drugs for the treatment of AIDS/HIV infection. Over the past two decades several potent arylsulfone-based HIV-1 NNRTIs and related analogs have been developed. This review provides an essential overview of the structure–activity relationships of the arylsulfone-based HIV-1 NNRTIs. Furthermore, structural information useful for the design and development of new sulfur containing NNRTIs with enhanced antiretroviral activity against HIV-1 wild type and clinically relevant drug resistant HIV-1 mutant strains will be discussed.

AIDS was recognized in the early 1980s. Later, in 1983, HIV was identified as the etiological cause of HIV-1/AIDS pandemic [1,2]. The joint United Nations program on HIV/AIDS (UNAIDS) estimates that more than 34 million people are living with AIDS/HIV infection. In 2011, HIV virus newly infected an estimated 2.5 million people and AIDS caused 1.7 million deaths [201]. To date, the antiretroviral armamentarium is made up of 36 approved drug products falling into six classes, nucleoside and nucleotide reverse transcriptase inhibitors (NRTIs and NtRTIs, respectively), non-nucleoside RT inhibitors (NNRTIs), protease inhibitors, fusion inhibitors, entry inhibitors (CCR5 co-receptor antagonists) and HIV integrase strand transfer inhibitors. Agents in these classes have been used as single drug therapy or in multidrug combination products [202]. Highly active antiretroviral therapy (HAART) based on combination of at least two or three (preferably) anti­retroviral drugs from different drug classes proved to improve dramatically the prognosis of HIV-1 infection. The primary goal of HAART is to achieve a durable suppression of viral replication below the detectable levels (50 RNA copies/ ml) along with immune restoration of CD4+ cell count [3]. However, many HAART receiving patients with undetectable RNA copies still have low HIV levels in their blood and remain at risk of virologic rebound [4]. HAART produces an effective and prolonged reduction of morbidity and mortality in AIDS patients. However, HAART has proven to be unsuccessful in eradicating the infection [5]. The management of associated drug resistance and adverse effects remain unsolved problems of chronic long-term treatments [6].

The basic difficulty experienced with this viral infection is the ability of virus to mutate, leading to rapid drug resistance [7]. RT has a high error rate when transcribing RNA into proviral DNA since it has low proof­reading ability. These errors could be an opportunity for the viruses, improving their resistance to the antiviral drugs. The HIV infection and high mutation rates are major causes of various viral species, each with different mutations [8]. On the other hand, the development of resistance mechanisms is inevitable following the use of monotherapy; accordingly, the HAART consists of a combination of at least three antiviral drugs. It is difficult to generate genomic variances that make the virus resistant to treatment including three drugs with different target. Therefore, new potential drugs that display a broad spectrum of activity against clinically relevant HIV-1 mutant strains are needed. 3´-azido-2´,3´-dideoxythymidine (azidothymidine [AZT], also known as zidovudine) was the first drug licenced for the treatment of HIV-1. Various nucleoside analogs, didanosine, lamivudine, stavudine and abacavir, were used in patients who had become intolerant to AZT or in whom AZT treatment failed [9]. NRTIs were eventually joined by NNRTIs, which target at a specific binding site within the HIV-1 RT, distinct from the RT catalytic site [10,11]. Furthermore, NNRTIs are structurally very different from protease inhibitors [12– 14]. More recently, new agents targeting other phases of the HIV-1 life cycle have became available, gp41-mediated entry (fusion inhibitors) [15], early entry inhibitors (CCR5 coreceptor antagonists) [16,17] and HIV integrase strand transfer inhibitors [18,19]. Currently,

Valeria Famiglini1, Antonio Coluccia1, Andrea Brancale2 , Sveva Pelliccia1, Giuseppe La Regina1 & Romano Silvestri*1

10.4155/FMC.13.174 © 2013 Future Science Ltd

Future Med. Chem. (2013) 5(18), 2141–2156

ISSN 1756-8919

Istituto Pasteur – Fondazione Cenci Bolognetti, Dipartimento di Chimica e Tecnologie del Farmaco, Sapienza Università di Roma, Piazzale Aldo Moro 5, I-00185 Roma, Italy 2 Welsh School of Pharmacy, Cardiff University, King Edward VII Avenue, Cardiff, CF10 3NB, UK *Author for correspondence: Tel.: +39 06 4991 3800 Fax: +39 06 4969 3268 E-mail: [email protected] 1

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Review | Famiglini, Coluccia, Brancale, Pelliccia, La Regina & Silvestri Key Terms HIV-1: Belongs to a class of retroviruses. The genetic information is encoded by using RNA. During the replication cycle inside cells, HIV-1 uses an enzyme called reverse transcriptase to convert the RNA into DNA. Non-nucleoside reverse transcriptase inhibitors:

Class of antiretroviral drugs that bind to an allosteric pocket located close to the catalytic site in the p66 subunit.

Pyrrylarylsulfone: HIV-1

non-nucleoside reverse transcriptase inhibitors having the 1-benzensulfonyl-1H-pyrrole scaffold.

Indolylarylsulfones: HIV-1 non-nucleoside reverse transcriptase inhibitor having the 3-arylsulfonyl-1H-indole scaffold.

Benzenesulfonyl moiety:

Shift from position 3 to position 1 of the indole ring led to loss of activity, maybe due to the fact that these compounds interact with the non-nucleoside binding site differently from 3-benzenesulfonyl indoles.

there is a rich availability of anti­retroviral drugs for the treatment of initial HIV infection (S upplementary Table  1) [20] . Beside the recently approved etravirine and rilpivirine [21], many other new NNRTIs have been recently described that look promising in terms of their potency and resistance profile (e.g., MK-1439 [22] and Lersivirine [23]). RT is a heterodimeric macromolecule formed by two subunits, p66 and p51 [24,25]. Two domains were found in the p66 subunit, the N-terminal polymerase domain and the C-terminal RNase H domain. p66 holds the catalytic site that resembles a right hand with fingers, palm, thumb and connection subdomains [25,26]. The p51 subunit, even comprising the same domains of the p66 subunits with the exception of the RNase H domain, does not play a catalytic role and shows structural function only. RT converts RNA retroviral genome into proviral DNA in three steps [27]: RNA-dependent DNA polymerization, an RNA–DNA hybrid is generated by a cellular lysine transfer RNA primer (RNA reverse transcription);

n

Ribonuclease H degradation; RNA is degraded from RNA–DNA hybrids by ribonuclease RNase H, so the original template strand leaves a single-strand DNA;

n

DNA-dependent DNA polymerization, double-stranded DNA is formed.

n

The NNRTIs area class of noncompetitive allosteric inhibitors and bind to a hydrophobic pocket, the non-nucleoside binding site (NNBS), that is located close to the catalytic site in the p66 palm subdomain [28,29]. Diarylsulfones The story of HIV-1 arylsulfone-based inhibitors began in 1993 at the National Cancer Institute (MD, USA) where McMahon et al. screened a large collection of synthetic and natural products in search of new antiviral agents [30]. This study led to the selection of 2-nitrophenyl phenyl sulfone (1) as an optimal representative of the new diarylsulfone class that showed appreciable anti-HIV-1 activity (S upplementary F igure  1) . The preliminary screening led to the discovery of five hit compounds 1–5 with antiviral activities (EC50 values) ranging from 0.89 µM (2) to 11 µM (4). EC50 values represented 50% protection of infected MT-4 2142

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cells from the HIV-1IIIB-induced cytopathogenicity (the tetrazolium salt method). Despite 2,2´-dinitrophenylsulfone (2) being more potent than compound 1, the latter was expected to have greater in vivo bioavailability. Sulfone 1 selectively inhibited the HIV-1 RT, protected human CEM-SS lymphoblastoid cells at 1–5 µM concentrations and was not cytotoxic up to 100 µM. „„Pyrrylarylsulfones

Studies on diarylsulfone analogs led to the discovery of pyrrylarylsulfone (PAS) (6, 7) HIV-1 NNRTI class (Table 1). Both the 2-nitrophenyl and 2-ethoxycarbonyl-1H-pyrrole groups were key requirements of first generation PAS [31]. The development of PAS NNRTI class considerably advanced by replacing the 2-nitrobenzene group with a para-chloroanilino moiety to provide a new generation of amino-PAS (7) endowed with potent antiviral activity at micromolar concentrations [32,33,101,102]. Potent NNRTI agents’ analogs of 2-nitrophenyl phenyl sulfone with an amino group, such as PAS, were reported in during the period 1996–2001, namely compounds 8–12 [34–37]. Pyrrylarylsulfones were synthesized by arylsulfonation of pyrroles in the presence of potassium tert-butoxide and 18-crown-6 in tetrahydrofuran or via Clauson–Kaas reaction by heating with 2,5-dimethoxytetrahydrofuran in boiling glacial acetic acid [38]. Alkyl 1-(2-nitrophenylsulfonyl)1H-pyrrole-2-carboxylate were reduced to amino with iron powder in glacial acetic acid (Supplementary Figure 2). Pyrrolo[1,2-b][1,2,5] benzothiadiazepines The presence of the para-chloroaniline pharma­cophore was also of capital importance in improving the antiretroviral activity of pyrrolo[1,2-b][1,2,5]benzothiadiazepines (13; PBTDs) [39,40,102]. PBTD derivatives were active in HIV-1-infected cell cultures and inhibited the recombinant HIV-1 RT in enzyme assays, thus supporting that PBTDs are a novel class of NNRTIs structurally related to nevirapine. PBTD was obtained by intramolecular cyclization of ethyl 1-(2-aminophenylsulfonyl)1H-pyrrole-2-carboxylate by heating in the presence of 2-hydroxypyridine as bifunctional catalyst. Alkylation with the appropriate alkyl halide in the presence of potassium carbonate provided N10 alkylated PBTDs (Supplementary Figure 3) [39]. future science group

Arylsulfone-based HIV-1 non-nucleoside reverse transcriptase inhibitors

| Review

Table 1. Nitro-pyrrylarylsulfones, amino-pyrrylarylsulfones and structurally related HIV-1 non-nucleoside reverse transcriptase inhibitors.  NO2 SO2 N

NH2 Cl

O R

SO2

O R

N

O

O

Nitro-PAS (6)

Amino-PAS (7a–d)

O O N

SO2

NO2

Cl

SO2

NH

NH

Cl

CN SO2

SO2

SO2

NO2

NO2

NH2

NH2

NH

S

Cl 8 Inactive

9

Compound 7 (a) R = Me (b) R = Et (c) R = n-Pr (d) R = i-Pr

11

10

12

EC50† (µM) WT IIIB

SI‡

0.18 0.14 0.22 0.14

1666 >2140 500 >2140

MT-4 cells, tetrazolium salts method. CC50 /EC50 ratio. PAS: Pyrrylarylsulfone; SI: Selectivity index; WT: Wild type. † ‡

Pyrrolo[2,3-b][1,5] benzothiadiazepines & pyrrolo[3,2-b] [1,5]benzothiadiazepines The research project on HIV-1 NNRTIs based on a tricyclic scaffold led to the design pyrrolobenzothiadiazepines. The novel heterocyclic systems were synthesized by reaction of 2-amino­ thiophenol with pyrrole or 1-methylpyrrole in the presence of iodine and potassium iodide to afford a mixture of 2-(2-aminophenylsulfenyl)-1H-pyrrole (14a) and 3-(2-aminophenylsulfenyl)-1H-pyrrole (15a) (or the corresponding 1-methylpyrrole derivatives 14b and 15b). The amino compounds were treated with bis(trichloromethyl)carbonate (triphosgene) in the presence of triethyl­amine to provide 1H-pyrrolo[2,3-b][1,5]benzothiadiazepine (16a,b) and 1H-pyrrolo[3,2-b][1,5]benzothiadiazepine derivatives (17a,b) which were transformed into the corresponding dioxides 18 and 19 with hydrogen peroxide (Supplementary Figure 4) [41]. Structure–activity relationship (SAR) studies showed that the HIV-1 inhibition was correlated with the presence of the sulfone group in the 1H-pyrrolo[2,3-b][1,5]benzothiadiazepine series, whereas in the isomeric 1H-pyrrolo[3,2-b] [1,5]benzothiadiazepine the antiretroviral activity required the non-oxidized sulfur atom. future science group

Indolylarylsulfones In 1993, Merck reported L-737,126 (20) as a novel potent and selective HIV-1 RT inhibitor (HIV-1 wild type (WT) IIIB: EC50 = 1 nM; IC50 = 25 nM) [42,103,104]. Initially, the design of new indolylarylsulfones (IAS) was based on PASs’ structural features because of lack of SAR information. The phenyl ring of 20 was replaced by a para-chloroanilino moiety, the 3-benzenesulfonyl moiety was shifted from position 3 to position 1, and the carboxyamide was changed to ester and moved from position 2 to 3 [43] (Supplementary Figure 6). In general 1-benzenesulfonylindoles did not exhibit any significant antiretroviral activity, with the exception of derivatives bearing the para-chloroaniline pharmacophore that showed some anti­retroviral activity. Indoles bearing a carbethoxy group at the indole were inactive or weak inhibitors. Replacement of the 2-ester group with a carboxy­a mide function led to a notably increase of both potency and selectivity. „„Carboxyamides

& carboxhydrazides As inhibitors of HIV-1 WT, IAS derivatives 22 and 23 bearing only one methyl group at the 3-phenylsulfonyl moiety were less cytotoxic www.future-science.com

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Review | Famiglini, Coluccia, Brancale, Pelliccia, La Regina & Silvestri and more selective than 20. The 2,4- and 3,5-dimethyl derivatives 24 and 25 were also potent antiretroviral agents but were more cytotoxic that the mono-substituted counterparts. Both 5-chloro (25) with 5-bromo (26) derivatives were strong inhibitors in HIV-1 infected cells, whereas replacement of the halogen by acetyl (27) decreased the cytotoxicity (Table 2). A number of 2-carboxyhydrazides were also prepared, but unfortunately these compounds were remarkably less potent than corresponding carboxamides [43]. Monomethyl IAS derivatives 22 and 23 inhibited the Y181C mutant at submicromolar concentrations. The 2-carboxyamide sulfone derivatives turned out to be less cytotoxic and more potent than similar sulfur (compare 25 with 21, respectively). However, 22 and 23 were weak inhibitors of the HIV-1 K103N-Y181C double mutant (EC50 values) and efavirenz (EFV)resistant K103R-V179D-P225H triple mutant strains (EC90 values). Interestingly, derivative 25 showed strong inhibitory activity against these HIV-1 mutant strains; it was clearly superior Table 2. Indolylarylsulfones, carboxamides. R1 Cl

R2

SO2

X

NH2

N H

NH2

N H

O

O

IAS (21–27)

L-737, 126 (20) 21 R1 = 3,5-Me2, R2 = Cl, X = S 22 R1 = 2-Me, R2 = Cl, X = SO2 23 R1 = 3-Me, R2 = Cl, X = SO2

24 R1 = 2,4-Me2, R2 = Cl, X = SO2 25 R1 = 3,5-Me2, R2 = Cl, X = SO2 26 R1 = 3,5-Me2, R2 = Br, X = SO2 27 R1 = 3,5-Me2, R2 = COMe, X = SO2 20 R1 = H, R2 = Cl, X = SO2

Compound

EC50 (nM) WT IIIB

20 21 22 23 24 25 26

EFV

1 6 1 1 4 4 2 –

†

SI

‡

45,000 117 100,000 >100,000 9250 3750 9000 –

EC90 (nM)

Y181C

Y181C-K103N EFV-RES §

20 – 160 6 150 30 – 25

8000 – 10,000 7000 10,000 650 – 150

900 – 2600 260 5300 80 – 1800

MT-4 cells, tetrazolium salts method. CC50 /EC50 ratio. Efavirenz-resistant strain carrying K103R-V179D-P225H mutations, C8166 cells, p24 method. EFV-RES: Efavirenz resistant; IAS: Indolylarylsulfones; SI: Selectivity index; WT: Wild type. † ‡ §

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to 20 as inhibitor of the HIV-1 K103 mutated RT, and 22-fold more potent than EFV against the K103R-V179D-P225H triple mutant. SAR studies showed that the antiretroviral activity of 22–25 was correlated to the number and the position of the methyl groups bound to the 3-phenyl ring [43,105]. The 3,5-dimethyl groups at the 3-phenylsulfonyl moiety of the indole proved to be a key structural requirement for an effective inhibition of the HIV-1 mutant strains. Compound 25 was as active as 20 and EFV against the HIV-1 WT and Y181C mutant, more potent than 20 and slightly less potent than EFV against the double K103N-Y181C, and superior to 20 and EFV against the triple mutation. Compound 25 inhibited both the WT and Y181C RTs, but was less effective against the K103 RT (rRT). The double mutation creates a conformationaly different pocket than the single mutant, and then it is possible that a compound is active on double mutation and less on the single. The K103N mutation is the most frequently occurring HIV-1 mutation isolated from nevirapine (NVP)- or EFV-treated patients. Therefore, the impacts of either the single mutation or the double mutation on the high level resistance of the HIV-1 K103N-Y181C mutant were investigated. The required 3-arylthio intermediates were obtained by Atkinson reaction from appropriate 1H-indole-2-carboxylic acids and arylthiodisulfides in the presence of sodium hydride, and subsequent transformation of acids 28 in the corresponding methyl esters with (trimethylsilyl) diazomethane (Supplementary Figure 5) [44]. In an improved procedure, the esters of the indole were treated with N-(arylthio)succinimides in the presence of boron trifluoride diethyl etherate to furnish esters 29. The sulfur derivatives were oxidized to corresponding sulfones with 3-chloroperoxybenzoic acid. Finally, ester 30 gave amides on treatment with concentrated ammonium hydroxide in closed vessel, or hydrazides with hydrazine hydrate. „„N-(2-hydroxyethyl)carboxyamides & N-(2-hydroxyethyl)carboxyhydrazides Efforts at Merck Research Laboratories (NJ, USA) to improve the bioavailability of 20 resulted in the synthesis of bioisosteric analogs bearing nitrogen containing heterocyclic rings at position 2 of indole (31–33; S upplementary Figure  6) [45]. These compounds reached only partly the desired water solubility. Most interestingly, they showed improved activity against the HIV-1 mutant strains; for example, against future science group

Arylsulfone-based HIV-1 non-nucleoside reverse transcriptase inhibitors the K103N mutation compound 31 was 11-times more potent than 20. The binding mode of IAS derivatives was investigated by a series of molecular docking studies of 20 into the NNBSs of 14 HIV-1 RTs. A 3D quantitative SAR model was then developed from a training set of 70 IAS derivatives [43] and used to design new IASs bearing an hydroxyethylaminocarbonyl or 2-hydroxyethylhydrazinocarbonyl moiety at position 2 of the indole (Table 3) [46,47]. The structures of the training set were modeled starting from the structurally related diarylsulfone 739W9424 extracted from the corresponding complex with the HIV-1 RT (PDB code 1jlq) [48]. These compounds were also expected to be more soluble in water than the parent compound 20. SAR studies showed that against the HIV-1 WT, IAS derivatives bearing the 2-(2-hydroxyethyl)carboxyamide were superior to the corresponding 2-hydroxyethylcarboxyhydrazides. 3-[(3,5-dimethylphenyl)sulfonyl]-1H-indole-2N-(2-hydroxyethyl)carboxyamide (39) and the corresponding carboxyhydrazide 45 were the most potent derivatives within the two series with EC50 values = 1 nM. Compounds 34, 36, 39 and 45 were effective inhibitors of the HIV-1 Y181C mutant strain with EC50s ranging from 10 nM (39) to 50 nM (34). Compound 39 turned out more potent than 20 against Y181C and K103N-Y181C, respectively, and against the viral strain carrying the K103R-V179D-P225H mutations, 39 was more potent than 20 and EFV. As RT WT inhibitors, compounds 39 and 45 were superior to NVP and comparable to EFV. They also proved to be potent inhibitors of the HIV-1 K103N mutation being more potent than NVP and notably superior (13- and 24-fold, respectively) to EFV. Against the Y181I mutation, that is comparable with the Y181C in term of drug resistance, 39 and 45 were more active than NVP but slightly less potent than EFV. „„2-carboxyhydrazide

derivatives. The discovery that the N-(2-hydroxyethyl) carboxyhydrazide moiety was able to improve the antiviral activity against HIV-1 resistant mutants, prompted the design of new structurally correlated IASs. The previous 3D quantitative SAR model [48] was implemented expanding the training set composition from 70 to 101 molecules [49]. The use of this highly predictive model led to the design of new IAS compounds by simply modifying the substituents future science group

| Review

Table 3. 2-(2-hydroxyethyl) carboxyamides and 2-(2-hydroxyethyl) carboxyhydrazides. R1

Cl

SO2 N H

R1

Cl H N

N H

O

Compound

34 R1 = H-Me

– 2

†

SI

‡

– 50,000

OH

H N N H O

EC50† (nM) WT IIB

20

SO2

OH

EC90 (nM)

Y181C

Y181C-K103N

EFV-RES §

20 50

8000 6500

900 1200

35 R1 = 2-Me

16

3750

–

–

–

36 R1 = 3-Me

3

21,667

60

2600

600

37 R1 = 4-Me

4

835

–

–

–

38 R1 = 2,3-Me2

10

7100

–

–

–

39 R1 = 3,5-Me2

1

43,000

10

500

100

40 R1 = H

20

5000

–

–

–

41 R1 = 2 Me

70

1428

–

–

–

42 R1 = 3-Me

10

10,000

–

–

–

43 R1 = 4-Me2

40

>2500

–

–

–

44 R1 = 2,3-Me2

60

833

–

–

–

45 R1 = 3,5-Me2

1

12,000

–

–

–

NVP EVP

– –

– –

>30 µM 25

>30 µM 150

>30 µM 1800

MT-4 cells, tetrazolium salts method. CC50 /EC50 ratio. EFV-RES strain carrying K103R-V179D-P225H mutations, C8166 cells, p24 method. EFV-RES: Efavirenz resistant; NVP: Nevirapine; SI: Selectivity index; WT: Wild type. † ‡ §

at the 2-hydrazide position of compound 38. Combination of the structure-based alignment obtained by docking and the 3D quantitative SAR model estimation of the inhibitory values led to the design of new IAS derivatives 46–52 . Notably, compound 47 was shown to bind into the active site with the indole ring rotated by approximately 180° along the C3–C6 virtual axis as compared with 20 or 39. This different binding mode could break key interactions, thus lowering the anti-HIV-1 activity below the reference compounds 20 and 39. The binding mode of the other IASs was expected to be congruent with that of 39. Compounds 46 (EC50 = 2 nM) and 48 (EC50 = 3 nM) were the most potent inhibitors of the HIV-1 WT IIIB strain in MT-4 cells, and 46 showed the highest selectivity index (Table  4). Derivatives 46, 48 and 50–52 were effective against the HIV-1Y181C mutant strain, with inhibitory concentrations ranging from 10 nM (46) to 40 nM (48 & 51). Compound 46 (EC50 = 900nM) was the most active against www.future-science.com

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Review | Famiglini, Coluccia, Brancale, Pelliccia, La Regina & Silvestri the HIV-1K103N-Y181C double mutant. The most potent derivatives were evaluated against HIV-1 WTIIB and primary isolates HIV-112 (carrying K103N-V108I-M184V mutations) and HIV-AB1 (carrying L100I-V108I mutations) obtained from two HIV-1-Ab seropositive patients in failing HAART. In lymphocytes, compound 48 inhibited the primary isolates IIIB, 112 and AB1 with EC50 values in the subnano- (IIIB) or low nanomolar (112, AB1) range of concentration. In macrophages, compounds 46 and 50–51 inhibited the replication of IIIBBa-L strain at nanomolar concentration. Compound 46 was the most potent and selective inhibitor (EC50 = 2 nM, selectivity index [SI] >10,000). Against the HIV-1 K103N mutated RT, compounds 46 and 48 showed relative resistance (ID50_mutant /ID50_WT ) values 1.6- and 80-fold, respectively, whereas the same mutation showed a relative resistance >300-fold toward EFV [50]. Peptide derivatives A novel series IAS derivatives was synthesized by simple coupling of glycine/alanine units to the 2-carboxyamide function. The aim was remodeling of the indole-2-side chain (e.g., acetamido to urea or glycine to azaglycine) to form new chemical interactions with amino acid residues of the NNBS [51]. Against the HIV-1 WT IIIB strain, compounds 53, 55 and 57 were the most potent Table 4. 2-carboxyhydrazide derivatives. O 46 X =

N

O

49 X = NH-cyclohexyl 50 X = NHCONHEt

Cl

SO2

N H

Compound 46 47 48 49 50 51 52

EFV

47 X = NHMe H N X

48 X = NH-iso-Pr

51 X = NHCONHNH2 52 X = NHCOMe

O

EC50† (nM)

EC90‡ (nM)

WT IIIB

Y181C

Y181C-K103N

IIB

112

AB1

2 50 0.7 100 5 20 40 3

10 130 40 1200 20 40 20 10

900 >20,000 8600 >20,000 >20,000 9200 5700 200

10 20 0.8 800 10 20 30 7

30 40 6 1000 8 30 50 >20 µm

20 20 8 900 10 20 40 >20 µm

MT-4 cells, tetrazolium salts method. Primary isolates in lymphocytes antigen, p24 method. EFV: Efavirenz; WT: Wild type. † ‡

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derivatives within the series, with potencies comparable to those of 20 and EFV (Table 5). The presence of both the 3-(3,5-dimethylphenyl)­ sulfonyl group and short chains of simple amino acids at the 2-carboxamide certainly improved on the anti­retoviral activity of IAS derivatives. Interestingly, these peptide derivatives compare favorably with EFV as far as the activity against the triple mutant is considered. IAS derivatives 53– 62 were synthesized from 3-[(3,5-dimethylphenyl)sulfonyl]1H-indole-2-carboxylic acid and esters of glycine or alanine using benzotriazol-l-yl-oxy-tris(dimethylamino) phosphonium hexafluorophosphate and triethylamine as coupling reagents to obtain 63 or 64. Lithium hydroxide hydrolysis of the latter compounds afforded the corresponding acids 65 or 66 which were treated again with an ester of glycine or d,l-alanine to provide 67–70. Finally, reaction of the desired ester with ammonium hydroxide at 60°C, or with hydrazine hydrate at room-temperature, furnished the corresponding amides 53, 55, 57, 59 or 61 or hydrazides 54, 56, 58, 60 or 62 , respectively (S upplementary Figure 7). Coupling of natural and unnatural amino acids to the indole-2-carboxamide resulted in a new series of IAS HIV-1 NNRTI with inhibitory activities comparable to EFV in CEM cells (Table 6) [50]. Some new IASs inhibited the Coxsackie B4 virus at 2–9 µM concentrations. The antiviral potency was in general less pronounced in Vero than in HeLa cell cultures. Coxsackie B virus may be responsible of polymyositis and rhabdomyolysis in immunodepressed patients [52,53]. These agents have the potential to inhibit both the HIV-1 and Coxsackie B4 viruses. Superimposition of docked conformations of IASs 71 and 25 showed that the mutation of leucine to isoleucine does not affect the binding mode. Compound 71 showed quite matching binding modes in both HIV-1 RT and L100I mutated RT. „„ Di-halo-IASs

A major problem observed in >90% patients treated with EFV whose viral loads rebounded after an initial response to the drug, was the selection of the HIV-1 K103N mutation. Furthermore, additional double mutations (K103N-V108N, K103N-P225H) slowly appeared in many patients [54]. Moreover, the upregulation of P450 liver isoenzyme CYP3A4 caused an increase of the need for protease inhibitors to compensate the more rapid clearance [55]. DuPont Laboratories future science group

Arylsulfone-based HIV-1 non-nucleoside reverse transcriptase inhibitors (MS, USA) designed new anlogs of EFV bearing two halogen atoms at the quinazolinone ring that proved to inhibit HIV-1 WT and viral strains carrying the single mutation K103N and L100I at low nanomolar concentration. Quinazolinone derivatives possessing the 5,6-dihalogen substitution pattern proved to be more effective than their 5- and 6-monohalogenated counterparts against HIV-1 resistant mutant strains. This broader spectrum of activity was partly due to a weaker protein binding [56–58]. These findings prompted the design of new IAS derivatives bearing two halogen atoms at the indole ring (Table 7) [59] . The two halogen atoms at the positions 4 and 5 of the indole revealed to be an optimal substitution pattern for the di-halo IASs, similarly to some EFV’s analogs. Against the HIV-1 WT, the 4,5-difluoro-IAS 76 (EC50 = 1 nM) and 5-chloro-4-fluoro-IAS 81 (EC50 = 0.5 nM) were highly potent and selective. The most potent derivative 81 was as active as EFV in inhibiting of the HIV-1 Y181C (EC50 = 4 nM) and K103N-Y181C (EC50 = 300 nM) mutant strains. Compounds 76 and 81 were inhibitory to the 112 and the AB1 strains in lymphocytes and the IIIBBa-L strain in macrophages. Compounds 81 strongly inhibited the HIV-1 K103N, Y181I, and L100I mutated RTs. IAS derivatives showed different mechanisms of action, depending on the nature and position of their chemical substituents. In particular, di-halo-IASs were selective for the enzyme– substrate complex [60]. By detecting the activities

| Review

Table 5. Peptide derivatives. O 53–56 X =

Z Y Y

O Cl

SO2 N H

Compound Y

57–60 X =

H N X

N H

Z O

O

O 61, 62 X =

Z

N H

H N

O Z

O

EC50† (nM)

EC90‡ (nM)

WT IIIB†

Y181C

Y181C-103N

EFV-RES‡

53

H

NH2

6

30

800

100

54

H

NHNH2

10

50

2000

1000

55

Me

NH2

3

10

700

40

56

Me

NHNH2

30

140

4700

700

57

H

NH2

0.7

5

1200

100

58

H

NHNH2

60

80

2800

150

59

Me

NH2

14

30

2200

420

60

Me

NHNH2

80

160

3550

1000

61

H

NH2

120

230

5900

1900

62

H

NHNH2

180

330

>11,000

2300

EFV

–

–

4

25

150

1800

MT-4 cells, tetrazolium salts method. ‡ Efavirenz-resistant strain carrying K103R-V179D-P225H mutations, C8166 cells, p24 method. EFV-RES: Efavirenz resistant; WT: Wild type. †

against HIV-1 WT RT and enzyme carrying the resistance mutation K103N, L100I or Y181I, compound 81 proved to dissociate from the

Table 6. Natural and unnatural peptide derivatives. R1 = 5-Cl, 5-Br, 5-NO2, 5-Cl, 4-F

SO2

R1

Amino acid unit

NH N H

O

O

O

R2

R2 =

O

NH2 NH2

General formula

O

O

Compound

R1

NH2

NH2

NH2

R2

S

NH2 O

O

S

NH2 O

EC50† (nM) WT IIIB1 L1001

K103N

Y181C

53

5-Cl

CH2CONH2

1.9

8

29

74

55

5-Cl

CH2(Me)CONH2

1.6

9

14

96

71

5-Cl

CH2CH2CONH2

1.0

7

19

78

72

5-Cl

CH2CH2SO2NH2

1.4

1.2

40

230

73

5-Cl

CH2CH2(Me)SO2NH2

2.3

2.0

36

390

EFV

–

–

–

22

130

130

CEM cells, giant cell formation. EFV: Efavirenz; WT: Wild type. †

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Review | Famiglini, Coluccia, Brancale, Pelliccia, La Regina & Silvestri Table 7. Dihalo-indolylarylsulfones.

F3C Cl

F3C O

N H

O

N H

SO2

R

NH

R

NH2

O

N H

Quinazolinone analogs R = 5-Cl; R = 4,5-F2

EFV

Compound R

O

74–82

EC50† (nM)

EC50‡ (nM)

EC50 § (nM)

WT IIIB† Y181C K103N-Y181C IIB

112

AB1

IIIBBa-L

74

5,6-Cl2

10.0

1000

>20,000

nd

nd

nd

nd

75

5,7-Cl2

21.0

4200

>20,000

nd

nd

nd

nd

76

4,5-F2

1.0

50

1200

20

130

84

23

77

5,6-F2

20.0

40

>20,000

nd

nd

nd

nd

78

5,7-F2

7.0

1500

>20,000

nd

nd

nd

nd

79

4-Cl, 5-F 6-Cl, 5-F 5-Cl, 5-F 5-Cl, 6-F 5-Cl –

8.0 50.0 0.5 12.0 6.0 3.0

2200 700 4 50 50 10

>20,000 12,000 300 >20,000 800 200

nd nd 8 nd 200 7

nd nd 10 nd 300 >20,000

nd nd 12 nd 220 >20,000

nd nd 10 nd 115 nd

80 81 82 25

EFV

MT-4 cells, tetrazolium salts method. Primary isolates in lymphocytes antigen p24 method. § HIV-1 wild type (IIIBBa-L ) in macrophages, antigen p24 method. EFV: Efavirenz; nd: Not determined; WT: Wild type. † ‡

mutated enzyme almost tenfold slower than from the WT RT. The study suggested that by comparing with other IASs [60,61], the excellent activity of 81 may be correlated to its greater flexibility [62] allowing new stable dynamic interactions inside the NNBSs of the mutated viral RTs. Di-halo-IASs were synthesized as above starting from an appropriate ethyl indole2-carboxylate (83) [59] obtained by Fischer’s reaction [63] of the corresponding ethyl pyruvate phenylhydrazone (84) in PPA (S upplementary F igure  8) . By this procedure, isomeric ethyl 4,5-difluoro- (83c) and 5,6-difluoro-1H-indole2-carboxylates (83d) were obtained from ethyl pyruvate of 3,4-difluorophenylhydrazone after repeated column chromatography [64]. The phenylhydrazones were prepared starting from the corresponding aniline according to the method of Japp–Klingemann [65]. In order to simplify the above procedure and to improve yield, ethyl 5-chloro-6-fluoro(83g) and 5-chloro-4-fluoro- (83h) 1H-indole2-carboxylates were prepared by a different procedure [66]. The required intermediate ethyl pyruvate of 4-chloro-3-fluorophenylhydrazone was prepared by treatment of ethyl pyruvate of 3-f luorophenylhydrazone with 2148

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N-chlorosuccinimide. This three-step procedure allowed to prepare 83g and 83h from 3-fluoaniline with overall yield of 50%. However, since compound 83h was obtained in overall yield of 5% by this procedure [67], a more convenient synthesis was investigated (S upplementary Figure  9) [68]. N-pivaloyl 2-methyl-3-fluoroaniline (85) was treated with N-chlorosuccinimide to furnish N-(4-chloro3-fluoro-2-methylphenyl)pivalamide (86). The acid 87 was oxidized to nitro (88), which was transformed into ethyl 3-(3-chloro-2-fluoro6-nitrophenyl)-2-oxopropanoate (89) by reaction with diethyl oxalate and sodium ethoxide. Reduction of 89 and subsequent intramolecular cyclization of the intermediate amino derivative provided the ester 83 h. The latter six-steps procedure provided 83 h in 37% overall yield. In 2010 Idenix Pharmaceuticals (MA, USA) reported a further improvement of this synthetic procedure [67]. BOC-protected 4-chloro3-fluoroaniline 90 under went region selective iodination at C-2 (91). After deprotection, the resultant 2-iodoaniline 92 was treated with pyruvic acid in the presence of palladium II acetate and 1,4-diazabicyclo[2.2.2]octane to furnished 5-chloro-4-fluoro-indole-2-carboxylic acid (93) future science group

Arylsulfone-based HIV-1 non-nucleoside reverse transcriptase inhibitors which was subsequently transform end into the corresponding methyl ester 94 in 56% overall yield (Supplementary Figure 10). Several synthetic strategies for the preparation of intermediate compounds to easily achieve new sulfone HIV-1 NNRTIs were developed. For example, some phenylhydrazones undergo rapid degradation upon prolonged heating and high temperatures; a number of pyridinyl N-aryl hydrazones showed to decompose spontaneously even at room temperature and required storage at -20°C [Silvestri R, Unpublished Data]. Such hydrazones were prepared by an unexplored procedure including open vessel and cooling while heating microwave-assisted synthesis [69]. The compounds were obtained in 88–98% yields in 5 min, by reacting 4- and 2,4-(di)substituted phenylhydrazines bearing either electron-donating or with drawing groups, with 2-, 3- and 4-acetylpyridine. The method was successfully extended to other carbonyl compounds. Similarly, arylthioindole intermediates (97) were obtained in 90–98% yield in 4 min from 95 and 96 in closed vessel, independently on the nature and position of the substituents of the molecule (S upplementary Figure 11) [70].

| Review

strategy in the design of new HIV-1 NNRTIs [62]. Significant examples are diaryltriazine (Janssen [NJ, USA]) [71], dipyridodiazepinone BIRL 355 BS (Boehringer Ingelheim [CT, USA]) [72,73] and the pyrrolidin-1-ylsulfone (Merck [PA, USA]) [74] HIV-1 NNRTIs. The binding poses of IAS derivatives carrying an (hetero)cyclic moiety at the 2-carboxamide bridging group were predicted to resemble those of 53, 55 and 71 [50,51] establishing novel binding interactions in the solvent accessible cleft of the NNBS formed by residues R172, I180, V179 and E138:B, and T139:B. The observations prompted the design of new IAS derivatives characterized by a basic nitrogen atom and small hydrophobic groups at the 2-carboxamide side chain that could form striking interactions respectively with the E138:B acid function and hydrophobic residues in the NNRTI cleft [75]. These studies resulted in the synthesis of a new series of three-cycles IAS derivatives (98–105; Table  8) [75]. New IASs bearing a methylene/ ethylene spacer group were inhibitory to HIV-1 WT in CEM cells at nanomolar concentrations, whereas the substituents of the indole nucleus had a little effect on the antiretroviral activity. Against the HIV-1 L100I and K103N mutant strains, compounds 100–102 and 105 showed antiviral potency superior to that of EFV and were equipotent to ETV. Compounds 98, 100

„„Three-cycle

IAS The strategy to add a heterocyclic motif to the parent compound proved to be a successful Table 8. Three-cycle indolylarylsulfones.

SO2

R1

NR2

N H

Compound R1

O

R2

EC50† (nM)

EC50‡ (nM)

WT IIIB†

WT IIIB §

L1001§

K103N §

Y181C §

Clade A‡

98

5-Cl

CH2-pyrrolidinyl

3.3

nd

nd

nd

nd

0.1

99

5-Cl

CH2-pyperidinyl

1.3

nd

nd

nd

nd

nd

100

5-Cl

CH2-morpholinyl

1.9

3.2

8.0

11

23

0.1

101

5-Cl

CH2-phenyl

5.7

11

6.1

15

46

nd

102

5,7-F2

CH2-phenyl

5.7

16

7

11

54

nd

103

4-Cl, 5-F

CH2-phenyl

6.2

10

84

80

300

nd

104

6-Cl, 5-F

CH2CH2-phenyl

5.7

21

19

52

100

nd

105

5-Cl, 5-F

CH2CH2-pyrrolyl

6.5

44

11

15

67

2.1

EFV

–

–

nd

1.9

22

130

160

nd

CEM cells, giant cell formation. ‡ HIV-1 UG2373 clade in peripheral blood mononuclear cells. § MT-4 cells, tetrazolium salts method. EFV: Efavirenz; nd: Not determined; WT: Wild type. †

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and 105 proved to inhibit effectively different HIV-1 clades in peripheral blood mononuclear cells with EC50 values in the higher picomolar or lower nanomolar range of concentrations. Compounds 98 and 99 showed slower substratedependent dissociation rates from these HIV-1 mutated enzymes than from WT RT, suggesting that they can achieve more stable binding interactions into the mutated NNBS with respect to the WT form [76]. Mannich-base derivatives were obtained by heating at reflux in tert-butanol the carboxamide 106a or 106c with a secondary cyclic amine and 37% formaldehyde. The 5-bromoor 5-chloro-4-fluoro- derivatives were prepared by heating at reflux the corresponding carboxamide 106b or 106d with secondary amine in benzene for 3 h in the presence of paraformaldehyde using a Dean-Stark apparatus

EFV: EC50 = 980 nM). In peripheral blood mono­nuclear cells compound 107 also potently inhibited in the picomolar range various HIV-1 clades, independently of their coreceptor use. „„IAS

affinity against HIV-1 mutant RTs Antiretroviral agents [78–80] that were able to assume the horseshoe shape [81] binding mode proved to be uniformly active against the HIV-1 WT and drug resistant mutant strain. Docking experiments in mutated RTs (K103N, L100I, Y181C and Y188L) showed that IASs did not adopt the aforementioned horseshoe binding conformation but were still able to overcome several first and second generation NNRTIs limitations [50]. The loss of aromatic ring stacking interactions significantly reduces the binding energy of NNRTIs, including NVP and EFV, in the Y181C or Y188L mutation [82]. For IASs, the presence of the 3,5-dimethylphenyl moiety strengthened interaction with the highly conserved W229 and weakened contact with Y181 and Y188 while maintaining the binding mode observed for the WT [83]. The weaker binding of NNRTI in the K103N mutation, that is caused by a stabilization of the unbound state of the NNBS by an intramolecular H-bond between N103 and Y188 residues, is responsible of drug resistance [84]. Therefore, the observed effectiveness of ETV against the K103N RT should be correlated somehow to an interaction with N103/Y188 residues when approaching the RT

(S upplementary Figure 12).

A new series of IAS derivatives (for example, bearing nitrogen containing groups linked to the indole-2-carboxamide exhibited potent inhibitory activity against the HIV-1 WT (NL4-3 strain) and low cytotoxicity in MT-4 cells (Table 9) [77]. Compound 107 showed consistent inhibition of the HIV-1 Y181I, K103N and Y188L HIV-1 mutant strains, superior to EFV and comparable with AZT, and the HIV-1 IRLL98 multiresistant strain carrying the K101Q, Y181C and G190A mutations (HIV-1 IRLL98 mutant strain, 107: EC50 = 1 nM; 106–110)

Table 9. Three-cycle indolylarylsulfones (2) .

SO2

R1

NR2

N H

Compound R1

O

R2

EC50† (nM)

EC50‡ (nM)

WT†,§

Y181L§

K103N §

Y188L§

Clade A‡

106

5-Cl

CH2-pyridin-3-yl

2.2

8.8

37.8

44

nd

107

5-Cl

CH2-pyridin-4-yl

2.0

2.2

8.8

22

0.6–1.2

108

5-Cl

CH2CH2-pyridin-3-yl

2.1

6.3

27.3

504

nd

109

5-Cl

3-cyanophenyl

2.2

44

19.8

1386

nd

110

5-Cl

CH2-(4-cyanophenyl)

1.5

32.1

117.9

>7142

nd

AZT EFV

– –

– –

7.8 6.3

1.6 157.5

15.6 69.3

12 315

nd nd

HIV-1 NL4-3 wild-type strain. HIV-1 UG2373 clade in peripheral blood mononuclear cell. § MT-4 cells, tetrazolium salts method. AZT: Azidothymidine; EFV: Efavirenz; nd: Not determined; WT: Wild type. † ‡

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Arylsulfone-based HIV-1 non-nucleoside reverse transcriptase inhibitors

| Review

Table 10. Sulfonamides. R2

R3 N SO2

R1 N H

R3 N

R2

SO2

R1 NHR4

O

110–116

R2–R3

R4

111

Pyrrolidin-1-yl Piperidin-1-yl Pyrrolidin-1-yl Pyrrolidin-1-yl Pyrrolidin-1-yl Pyrrolidin-1-yl Pyrrolidin-1-yl

H H H H 2-Cl, 6-F-benzyl 2-idroxybenzyl 2-Me-imidazol-5-yl

112 113 114 115 116 117

O

117

Compound R1 5-Br 5-Br 5-Cl 5-CN 5-Br 5-Br 5-Br

R4

N H

IC50† (nM) WT RT

K103N RT Y181C RT

3.6 5.7 3.9 9.1 3.8 2.4 3.1

nd nd 1963 2300 20 31 42

nd nd 48 >10,000 16 10 25

IC50 versus isolated RT enzyme. nd: Not determined; WT: Wild type. †

entrance channel [85]. For IASs with inhibitory activities against K103N RT, it was postulated that they should interact with N103/Y188 residues in an ETV-like manner [85,86]. Finally, on Jorgensen’s studies [87], it was suggested that IASs and ETV featuring more plastic structures than NVP and EFV, are less affected by the L100I mutation. „„ Sulfonamides

Docking studies carried out at Merck and Co. Inc. research laboratories resulted in the replacement of the sulfone group in 20 to obtain sulfonamido derivatives (111–117; Table 10) [74]. Both secondary and tertiary indole sulfonamides exhibited potent inhibition of the HIV-1 WT RT, but only the tertiary cyclic sulfonamides 111 and 112 displayed a small shift between enzyme inhibition activity (WT RT) and cell based activity. The pyrrolidinosulfonamide and the halogens Br and Cl (113), and CN (114), were the most effective substituents at the position 3 and 5 of the indole, respectively. Consistent with previous results [42], 5-chloro 113 as well as 5-cyano 114 derivatives were well tolerated and provided potent enzymatic and cellular activity versus WT RT. However, despite potent activity versus WT enzyme and virus compounds, these compounds displayed only weak inhibition against the mutant K103N (111, 113, 114) and Y181C (111, 114) RT mutants. The indole3-pyrrolidine sulfonamide 111 was selected for further modification. The substituents at the position 2 of the indole were modulated to future science group

obtain effective inhibitors (115–117) in both enzymatic (WT, K103N and Y181C RTs) and cellular assays. Sulfonamides were obtained from the proper N-protected-indole 118 that was converted to sulfonyl chloride 119 with thionyl chloride (Supplementary Figure 13). The chlorine atom of 119 was replaced with an amine to provide 120. Subsequent one pot deprotection of indole N1 and aminolysis with ammonia of the 2-ester function (111–114) or treatment with a benzylamine (115, 116) led to the desired sulfonamides 111–116. Sulfonamide 117 was obtained by deprotecting of the corresponding indole 120 with tris-(2-aminoethyl)aminomethyl polystyrene-trisamine). The ester 121 was transform end into 122 by reduction to alcohol with lithium aluminum hydride and subsequent oxidation to carboxaldehyde 122 with manganese dioxide. Finally, 122 was cyclized to 117 on treatment with with pyruvic aldehyde in the presence of ammonium hydroxide. „„ Phosphinates IDX 899 (123) was

initially developed at Idenix Pharmaceuticals by bioisosteric replacement of the 3-sulfonyl bridging group of IAS NNRTI with a phosphinic acid methyl ester group. In 2009, Idenix Pharmaceuticals signed a license agreement granting GlaxoSmithKline pharmaceuticals (London, UK) exclusive worldwide rights to 123 [88]. The 3´-Z-cyanovinyl moiety was introduced in order to achieve broad spectrum of activity against HIV-1 NNRTI-resistant mutants, as it did for RPV [89]. Compound 123 showed potent www.future-science.com

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nucleoside reverse transcriptase inhibitors based on the 3-arylphosphonyl-1H-indole scaffold.

antiretroviral activity and barrier to resistance superior to EFV (Table 11) [90]. In breakthrough studies in MT-2 cells, 123 showed slower development of drug resistance than EFV (45 and 17 days, respectively) and selected different HIV-1 drug resistance mutations. The enantiomerically pure S-3-phosphoindole 123 inhibited HIV-1 WT RT (subtype B, BH10 strain) at an IC50 value of 340 nM and the K103N-Y181C mutant strain at an IC50 = 1610 nM. For compound (S)-124, IC50 values ranged from 370nM (HIV-1 WT RT) to 1410 nM (K103N-Y181C RT). In MT-4 cells, compounds 123 and 124 inhibited HIV-1 WT (subtype B, BH10 strain) reproduction with EC50 values of 0.2 nM (SI >18,000) and 0.3 nM (SI >22,0000), respectively [106]. Inspection of the 123/RT cocrystal structure clearly showed that the inhibitor assumed a ‘butterfly-like’ active conformation [90]. An exemplary procedure for the synthesis of phosphoindoles 123 and 124 is shown in S upplementary F igure  14 [106]. N1-proctected indole 125 was lithiated with n-BuLi followed by treatment with diethyl chlorophsosphite to give a P(III) intermediate which under acidic conditions yielded the H-phosphinate 126. Treatment of 126 with iodocinnamonitrile under palladium catalyzed coupling reaction provided 127. Transesterification from ethyl to methyl followed by lithium hydroxide hydrolysis of both carboxylate ester and N-phenylsulfonyl protecting groups provided 128. Chiral resolution of the enantiomers of 128, using a chiral base, followed by amide formation afforded the final compound 123 or 124.

Future perspective The success of HIV-1 NNRTIs as component of HAART prompted significant interest in the identification of new small non-nucleoside molecules acting at level of the NNBS of the RT. Effort in medicinal chemistry along with the greater availability of structural and crystallographic information resulted in the successful development of new potent NNRTIs as anti-HIV/AIDS agents. The approval of second generation NNRTIs ETV and RPV represents a shift in the treatment of AIDS/HIV infection. In particular, these new drugs showed a durable and uniform inhibition of HIV-1 WT and drug resistant mutant strains. They undoubtedly answered the question ‘is there a role for NNRTIs in the treatment of HIV infection?’ [91]. The primary goal of additional new antiretroviral drugs is to achieve a durable suppression of viral replication [4], hinder the progress of drug resistance and have the potential of drug compatibility and timely administration [11]. Beside the recently approved etravirine and rilpivirine, the aim of our future research project is the design and synthesis of new non-nucleoside reverse transcriptase inhibitors of both HIV-1 wild type and resistant mutant strains, which are characterized by a greater structural flexibility in order to provide an advantage in overcoming viral drug resistance. The strategy to add a heterocyclic motif to the parent compound proved to be a successful strategy in the design of new HIV-1 NNRTIs; so, the result suggested further exploration and optimization at the B region of IASs by new substitutions at the indole-2-carboxamide.

Table 11. Phosphinates. CN R

Cl

S

1) Bioisosteric replacement

O O

N H

NH2

Cl

O

O NH2

EC50† (nM) WT

EFV

O

N H O 123 R = H IDX 899 124 R = F

Compound

124

P

2) Introduction of the cyanovinyl arm

IAS R = H (25) IAS R = F (81)

123

R

Y181C

K103N

Y181C-K103N

BH10

IIIB

BH10

IIIB

BH10

IIIB

BH10

IIIB

0.2 0.3 0.6

0.5 1.0 1.5

1.5 1.8 1.6

2.1 2.8 6.9

0.1 0.3 41

nd nd nd

6.6 3.5 53

9.1 4.7 112

MT-4 cells. EFV: Efavirenz; IAS: Indolylarylsulfones; nd: Not determined; WT: Wild type. †

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Arylsulfone-based HIV-1 non-nucleoside reverse transcriptase inhibitors Supplementary data

Financial & competing interests disclosure

To view the supplementary data that accompany this paper please visit the journal website at: www.future-science.

This research is supported by funds from Istituto Pasteur – Fondazione Cenci Bolognetti, Sapienza Università di Roma, Roma, Italy. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.

com /doi /full /10.4155/FMC.13.174

Acknowledgements To M Artico on the occasion of his 80th birthday, whom the authors wish to thank for early development of arylsulfonebased HIV-1 non-nucleoside reverse transcriptase inhibitors.

| Review

Executive summary Pyrrylarylsulfones „„

Replacing of the 2-nitrobenzene group of 6 with a para-chloroanilino moiety provided new generation amino-pyrrylarylsulfone HIV-1 non-nucleoside reverse transcriptase inhibitors (7) with antiretroviral activity at submicromolar concentrations (7b: EC50 = 140 nM).

„„

The para-chloroaniline pharmacophore was also of capital importance in improving the antiretroviral activity of pyrrolo[1,2-b][1,2,5] benzothiadiazepines (13a : EC50 = 500 nM).

Indolylarylsulfones „„

For the indolylarylsulfones (IAS) class, the presence the 3-(3´,5´-dimethylphenyl) group revealed to be a key structural requirement for an effective inhibition of the HIV-1 mutant strains (25: WT IIIB EC50 = 4 nM, Y181C EC50 = 30 nM, K103R-V179D-P225H EC50 = 900 nM).

Hydroxyethylcarboxyamides „„

The 2-N-(2-hydroxyethyl)carboxamide group turned out to be an optimal group for the inhibition of the HIV-1 triple mutant (39 : EC50 = 100 nM; efavirenz (EFV): EC50 = 1800 nM).

Peptide derivatives Improvement of activity against HIV-1 resistant mutant strains was obtained by adding simple natural or unnatural amino acid units at indole-2-carboxamide. Di-halo-IAS „„

„„

IAS derivative 81 bearing the 5-chloro-4-fluoroindole substitution pattern was highly potent and selective against HIV-1 WT IIIB (EC50 = 0.5 nM) and as active as EFV as inhibitors of HIV-1 Y181C (EC50 = 4 nM) and K103N-Y181C (EC50 = 300 nM) mutant strains.

Three-cycle IAS „„

New IAS characterized by a third-cyclic nucleus linked to the 2-carboxamide by a short spacer group showed potent antiretroviral activity. Compound 107, characterized by the N-(4-pyridin-4-yl-methyl) group, inhibited uniformly the Y181C (EC50 = 2.2 nM), K103N (EC50 = 8.8 nM) and Y188L (EC50 = 22 nM) HIV-1 mutants and the multiresistant IRLL98 HIV-1 strain (EC50 = 1 nM) conferring resistance to nevirapine, DLV and EFV.

Oxidation of sulfur to sulfone group proved to be essential for antiviral potency of IAS. Phosphinates „„

„„

Replacement of the 3-sulfonyl bridging group of IAS with a phosphinic acid methyl ester group provided new highly potent HIV-1 non-nucleoside RT inhibitors.

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