Non-Nucleoside Reverse Transcriptase Inhibitors: the NNRTI Boom

Antiviral Chemistry & Chemotherapy 10:285–314

Review Non-nucleoside reverse transcriptase inhibitors: the NNRTI boom

Ole S Pedersen and Erik B Pedersen*

Department of Chemistry, University of Southern Denmark, Odense University, DK-5230 Odense M, Denmark

*Corresponding author: Tel: +45 6550 2555; Fax: +45 6615 8780; E-mail: [email protected]

Non-nucleoside reverse transcriptase inhibitors approved NNRTI drugs and focuses on the recent (NNRTIs) are promising drugs for the treatment of efforts being made to produce second genera- HIV when used in combination with other anti- tion inhibitors that circumvent this resistance HIV drugs such as nucleoside reverse transcriptase problem. (RT) inhibitors and protease inhibitors. The first generation of NNRTIs have, however, suffered Keywords: HIV-1; non-nucleoside reverse from the rapid development of resistance. This transcriptase inhibitors; HEPT; nevirapine; review discusses the properties of the FDA- delavirdine; efavirenz; trovirdine

Introduction

The HIV epidemic is still a major concern. Virtually every interest in NNRTIs we intend to review this class of com- country in the world has seen new infections in 1998, and pounds, especially the second generation of NNRTIs, using the epidemic is out of control in many places according to a chemical approach. This review will focus on the recent the World Health Organization (WHO) and the Joint and most interesting published results. The NNRTIs syn- United Nations Programme on HIV/AIDS (UNAIDS). thesized before 1996 are covered by reviews by Artico The introduction of highly active antiretroviral therapy (1996) and Tucker et al. (1996). A review focusing on the (HAART), which has been used mainly in North America role of NNRTIs in the therapy of HIV-1 infections has and Western Europe, has reduced the number of deaths been published by De Clercq (1998a). caused by AIDS. However, because new infections continue to occur and infected people are kept alive with HAART Non-nucleoside reverse transcriptase and other combinations of anti-HIV drugs, the number of inhibitors people living with HIV has increased in North America and Western Europe. Combinations of anti-HIV drugs NNRTIs are bound in a hydrophobic pocket proximal to often contain a non-nucleoside reverse transcriptase (RT) the catalytic site of RT in HIV-1 (Tantillo et al.,1994). X- inhibitor (NNRTI), a nucleoside RT inhibitor (NRTI) and ray crystallographic studies of NNRTIs in complex with a protease inhibitor. This review will concentrate on the RT (Ren et al., 1995; Ding et al., 1995) have shown that the NNRTIs, of which the first, nevirapine (Viramune, NNRTIs maintain a very similar conformational ‘butterfly- Boehringer Ingelheim), was approved as a drug for the like’ shape and appear to function as π-electron donors to treatment of HIV-1 infection by the US Food and Drug aromatic side-chain residues surrounding the binding Administration (FDA) in 1996. Nevirapine was followed pocket (Kroeger et al., 1995; De Clercq, 1998b). by delavirdine mesylate (Rescriptor, Pharmacia & Upjohn) The major problem in the development of new and efavirenz (Sustiva, DuPont), approved for the treat- NNRTIs is the rapid emergence of resistant strains of ment of HIV-1 infection by the FDA in 1997 and 1998, HIV-1 in cell culture and patients. In patients receiving respectively. A further three compounds, MKC-442 monotherapy with nevirapine, drug resistance developed (Triangle Pharmaceuticals), Calanolide A (Sarawak rapidly owing to mutations in the RT; particularly signifi- MediChem Pharmaceuticals) and AG 1549 (Agouron cant is the Y181C mutation (Cywin et al.,1998). Cross- Pharmaceuticals) are in clinical trials according to the resistance is also a contributing factor and Pharmaceutical Research and Manufacturers of America pyridinone-resistant strains containing the Y181C, K103N (PhRMA) 1998 survey report. Because of the increasing or both mutations (Nunberg et al., 1991) have been found

Figure 1. The structure of nevirapine mutant strains of HIV-1 have to be considered in the development of new NNRTI drug candidates. These aspects O include metabolic stability, clearance rates, the ability to H3C 5 H N cross the blood–brain barrier and protein binding. Protein binding is a complex issue; a high protein binding could 4 reduce the metabolism of the drug, the clearance rates and 11 maintain high concentration of the drug in the blood. 2 N N N However, too strong protein binding may reduce the con- 10 1 centration of the free drug available for inhibitory action. Some of the currently most interesting subclasses of NNRTIs are described below. Tables showing the antiviral activity and activity against purified RT are presented. The to confer resistance to both TIBO R82150 (Pauwels et al., presentation of each drug begins with the subclass with an 1990) and nevirapine (Merluzzi et al., 1990). It has been FDA approved drug, followed by a subclass with a drug in shown that NNRTIs rapidly select for resistant mutant clinical trials according to the PhRMA 1998 survey report, HIV-1 strains when selection pressure is applied by drugs and ends with a description of some additional characteris- in vitro (Kleim et al., 1995; Nunberg et al., 1991) or in tic subclasses. monotherapy (Cywin et al., 1998; Miller et al., 1998). Cross-resistance has been observed between many Nevirapine NNRTIs in development (Miller et al., 1998). Mutations Nevirapine (Viramune; Boehringer Ingelheim) (Figure 1) commonly selected for by NNRTIs occur at amino acid was approved for the treatment of HIV in combination positions 98 to 108, 179 to 190 and 230 to 236 (Miller et with nucleoside analogues in 1996 in the USA, and in 1998 al., 1998). Cross-resistance is one of the obstacles that has in the European Union. to be overcome for the next generation of NNRTIs, new Nevirapine has a good potency against wild-type RT, drugs that have a resistance profile that differs from the with a 50% inhibitory concentration of 84 nM (Hargrave resistance profile of the already known drugs. et al.,1991), good metabolic stability, good bioavailability Many aspects beside selectivity and activity against (Cywin et al. 1998) and crosses the blood–brain barrier eas-

Table 1. The activity of some nevirapine analogues against wild-type RT and two mutant strains of HIV-1 RT

O H3C N

N N N R1 R2

IC50 (µM) HIV-1 RT*

Compound R1 R2 WT† Y181C Y188L Reference

1ClEt0.08 0.21 ND‡Proudfoot et al. (1995a) 2 N-pyrrolyl Et 0.09 0.21 ND Proudfoot et al. (1995a) 3 2-furanyl Et 0.11 0.16 ND Proudfoot et al. (1995a) 4 3-furanyl Et 0.04 0.11 ND Proudfoot et al. (1995a) 5 2-pyrrolyl Et 0.07 0.07 ND Proudfoot et al. (1995a) 6 3-pyrrolyl Et 0.033 0.050 ND Kelly et al. (1997) 7 3-pyrrolyl c-Pr 0.05 0.06 ND Proudfoot et al. (1995a) 8 4-pyrazolyl Et 0.02 0.06 ND Proudfoot et al. (1995a) 9 4-pyrazolyl c-Pr 0.06 0.05 ND Proudfoot et al. (1995a)

10 4-NH2-phenyl Et 0.04 0.12 ND Proudfoot et al. (1995a) 11 indol-3-yl Et 0.028 0.028 0.090 Kelly et al. (1997) 12 5-azaindol-3-yl Et 0.044 0.098 0.384 Kelly et al. (1997)

*Inhibitor concentration to give 50% inhibition of incorporation of [3H]dGTP into a [(poly)rC•(oligo)dG] template. †Wild-type HIV-1RT. ‡ND, Not determined.

Table 2. The activity of some nevirapine analogues against wild-type RT and two mutant strains of HIV-1 RT

H3C O N R2

N N N R1

IC50 (µM) HIV-1 RT*

Compound R1 R2 WT† Y181C Y188L Reference 13 Cl (3-ureidophenyl)ethyl 0.06 0.08 0.23 Klunder et al. (1998) 14 Cl (3-anilinyl)ethyl 0.12 0.23 0.25 Klunder et al. (1998) 15 Cl (4-(2-aminopyridyl))ethyl 0.02 0.07 0.16 Klunder et al. (1998) 16 F (4-pyridyl)ethyl 0.05 0.08 0.25 Klunder et al. (1998) 17 I (4-pyridyl)ethyl 0.09 0.18 0.43 Klunder et al. (1998) 18 Cl (4-pyridyl)ethyl 0.08 0.12 1.85 Klunder et al. (1998) 19 Cl (phenylthio)methyl 0.02 0.01 0.03 Cywin et al. (1998) 20 Cl (phenyloxy)methyl 0.11 0.06 0.64 Cywin et al. (1998) 21 Cl (2-methyl-4-pyridinyl-oxy)methyl 0.03 0.03 0.23 Cywin et al. (1998) 22 Cl phenylethyl 0.05 0.15 0.96 Cywin et al. (1998) 23 Cl (3-(aminocarbonyl)phenyloxy)methyl 0.13 0.28 0.34 Cywin et al. (1998) 24 Cl (3-aminophenyloxy)methyl 0.05 0.10 0.37 Cywin et al. (1998)

*Inhibitor concentration to give 50% inhibition of incorporation of [3H]dGTP into a [(poly)rC•(oligo)dG] template. †Wild-type HIV-1 RT. ily (Glynn & Yazdanian, 1998). Like all current NNRTIs, HIV activity against both wild-type RT and a broad spec- nevirapine selects for mutations in the RT, the most com- trum of mutant RTs (Klunder et al.,1998). The aim in the mon resistant strain of HIV-1 being characterized by the synthesis of these derivatives has been to achieve multiple Y181C mutation. Mutations appear rapidly in response to interaction points with the enzyme or to achieve interac- treatment with NNRTIs administered as monotherapy, tion between the compound and some more conserved and the focus of researchers working with nevirapine ana- residues of RT. Some of these residues include the catalyt- logues has been to develop drug candidates that retain ic aspartic acid residues, Asp-110, Asp-185 and Asp-186. activity against both wild-type HIV-1 and known Mutation of these residues results in inactive RT enzymes NNRTI-resistant HIV-1 strains. (Larder et al.,1987). Further research has investigated the The substitution pattern of the dipyridodiazepinone linker between the aryl substituent and the dipyridodi- ring system in the search for new derivatives was changed azepine system (Cywin et al.,1998). The preferred substi- from C-4 and N-11 to N-5 and N-11, as this appears to be tution at the 8 position was with an aryloxymethyl or an optimum for activity against both wild-type RT and arylthiomethyl, the arylthiomethyl derivatives being the Y181C RT (Proudfoot et al., 1995a). Molecular modelling most active compounds (Table 2). The aryloxymethyl com- of the X-ray crystal structure of nevirapine bound to wild- pounds though, were metabolically more stable, less toxic type RT has revealed a lipophilic cavity proximal to the C- and yet possessed good activity, and are thus the choice for 4 position, which allows for placement of an aryl group at further development rather than the thio analogues (Cywin this position. Although the placement of an amino group et al.,1998). in the para position of this aryl group produced derivatives Several derivatives of nevirapine with changes in the conferring activity against the Y181C mutant enzyme, the ring system have been synthesized and tested. Some of activity was not strong (Kelly et al., 1995). Adding an aro- these are the dipyrido[2,3-b:2′,3′-c]diazepinones matic substituent at position 2 afforded several analogues (Proudfoot et al., 1995b), pyridazinobenzodiazepines with activity against both wild-type RT and Y181C (Heinisch et al., 1997a; Barth et al., 1996) and pyridoben- mutant RT (Proudfoot et al., 1995a) and some have also zodiazepines (Hargrave et al.,1991).However,none of shown activity against the Y188L mutant RT (Kelly et al., these have shown an improved anti-HIV potency. 1997) (Table 1). Changing the position of the aromatic Dibenzoxapinones and pyridobenzoxazepinones (Tables substituent from position 2 to position 8, and introducing 3 and 4) are analogues of nevirapine that have shown activ- an ethyl linker, have afforded new analogues with anti- ity against HIV-1 RT in the nanomolar range (Klunder et

Antiviral Chemistry & Chemotherapy 10:6 287 OS Pedersen & EB Pedersen

Table 3. The anti-HIV-1 RT activity of some Table 4. The anti-HIV-1 RT activity of some dibenz[b,f][1,4]oxazepin-11(10H)-ones pyrido[2,3-b][1,5]benzoxazepin-5(6H)-ones

R1 O R1 O N 9 1 N 7 4 2 8 8 3 R R 2 O 3 3 7 R R3 6 4 2 9 O N 2 1 Compound R1 R2 R3 IC50 (nM)*

Compound R1 R2 R3 IC50 (nM)* 1CH3 7-CH3 2-NH2 30

2CH2CH3 9-CH3 2-NH2 60 8CH2CH3 8,9-(CH3)2 3-NH2 71

3CH3 7,9-(CH3)2 2-NH2 20 9 CH(CH3)2 8,9-(CH3)2 3-NH2 45

4CH2CH3 7,9-(CH3)2 2-NH2 43 10 CH3 7,9-(CH3)2 3-NH2 46

5CH3 7,9-(CH3)2 H5711 CH2CH3 7,9-(CH3)2 3-NH2 27

6CH3 7,9-(CH3)2 2-CN 50 12 CH2CH3 7,9-(CH3)2 H29

7CH3 7,9-(CH3)2 2-OH 63 13 CH2CH3 7-NO2, 9-CH3 H31 14 CH CH 7-CN, 9-CH H19 *Inhibitor concentration to give 50% inhibition of incorporation of 2 3 3 [3H]dGTP into a [(poly)rC•(oligo)dG] template. 15 CH3 7-CN, 9-CH3 H55

Data from Klunder et al. (1992). 16 CH3 7-NO2, 9-CH3 H23

17 CH3 7-CO2CH3, 9-CH3 H22 al., 1992). As a class, oxazepinones are less potent HIV-RT *Inhibitor concentration to give 50% inhibition of incorporation of [3H]dGTP into a [(poly)rC•(oligo)dG] template. inhibitors than the diazepinones. This problem has been Data from Klunder et al. (1992). partly solved by substituent optimization. However, solubility is a problem for this class of compounds because oxazepinones have the disadvantage of being less soluble for clinical evaluation (Romero et al.,1994). The structures than diazepinones. Replacing a phenyl ring with a pyrimi- of delavirdine mesylate (U90152S) and atevirdine mesylate dine ring in the dibenzoxazepinones may increase the sol- (U87201E) are shown in Figure 2. ubility, but is often accompanied by a decrease in the Later work has led to new analogues, the (alky- inhibitory effect on RT (Klunder et al., 1992). lamino)piperidine BHAPs (AAP-BHAP). This has produced compounds with activity against RT containing the BHAP Y181C and P236L mutations. The proline to leucine Bis(heteroaryl)piperazine (BHAP) RT inhibitors form a mutation at position 236 appears in RT after several HIV- class of compounds, discovered by the Upjohn laboratories 1 passages in vitro in the presence of increased concentra- (delavirdine, atevirdine; Pharmacia & Upjohn) (Romero et tions of atevirdine or delavirdine (Romero et al.,1996). al., 1991). This subclass of NNRTIs contain delavirdine Some AAP-BHAPs have, in a structure–activity relation- mesylate (rescriptor), which was given FDA approval in ship programme, demonstrated excellent activity against April 1997 for therapeutic use against HIV-1. Atevirdine the wild-type virus (Romero et al., 1996) (Table 5). mesylate is another compound of this class that was chosen Delavirdine has been found to block the replication of 25

Figure 2. The structures of delavirdine mesylate and atevirdine mesylate

HN HN H3CO H3CSO2-NH

N N N N N N N N H H O .CH3SO3H O .CH3SO3H

Delavirdine mesylate Atevirdine mesylate U90152S U87201E DLV ATV

Table 5. The in vitro inhibition of recombinant HIV-1IIIB RT mutants

HN R3

N R4 N N N H O R1

IC50 (µM)*

Compound R1 R2 R3 R4 WT† P236L Y181C Atevirdine mesylate 2.3 >60 >60 Delavirdine mesylate 0.26 18.0 8.32 Nevirapine 3.1 0.32 >60 1 mesylate Me i-Pr H H 0.23 0.74 0.80

2Mei-Pr H (OCH2CH2)OH 0.3 0.41 0.77

3Mei-Pr CH3SO2HN H 0.5 1.5 1.1

4Mei-Pr CH3NHCONH H 0.18 NT‡ 1.6 5 c-Pr i-Pr H H 0.70 0.59 2.31

6Mei-Pr H HOCH2 0.17 0.37 0.49

*IC50 (µM), concentration that inhibited the RNA-dependent DNA polymerase activity of RT by 50% using a rA.dT template:primer. †Wild-type HIV-1 RT. ‡NT, Not tested. Data from Romero et al. (1996). primary HIV-1 isolates in peripheral blood lymphocytes replaced C-6 (Romero et al.,1996). Substituting an ethyl (PBL), including variants that are highly resistant to 3′- for a methyl group on the aminopiperidine spacer azido-3′-deoxythymidine (zidovudine) and 2′,3′-dideoxyi- enhanced the activity against Y181C and P236L mutant nosine (didanosine), with a mean 50% effective dose (ED50) RTs (Romero et al.,1996). Some of the most active com- of 0.066±0.137 µM (Dueweke et al., 1993). The mutations pounds in this series are presented in Table 6. Y181C and K103N conferred some resistance to delavir- Replacing the 3-alkylamino in both BHAPs and AAP- dine, as for many other NNRTIs. However, delavirdine has BHAPs with alkoxy groups has also been investigated been shown to inhibit the Y181C mutant RT with the (Genin et al.,1996). Incorporation of a 3-alkoxy sub- rA.dT template:primer at a 50% inhibitory concentration stituent has been shown to be beneficial on the metabolic

(IC50) of 8.3 µM, whereas nevirapine and L-697,661 failed stability, evaluated in the presence of hepatic microsomal to achieve 50% inhibition at 60 µM. This, together with cytochrome P450 in vitro,thus providing analogues with good oral bioavailability and good serum drug levels in ani- similar or greater metabolic stability than delavirdine. mals, made the mesylate salt of delavirdine a candidate for Some of these compounds possessed good activity against clinical trials (Dueweke et al., 1993). wild-type and P236L RT enzymes, but the activity against These compounds did not, however, possess the desired Y181C RT was diminished (Genin et al., 1996). pharmacokinetic profile, primarily due to high clearance rates. This afforded a new structure–activity relationship Benzoxazinones programme focusing on compounds with increased activi- Benzoxazinones are the third class of compounds from which ty against mutant RT (P236L and Y181C) with the appro- a non-nucleoside inhibitor has been approved by the FDA. priate pharmaceutical properties. These included alteration Efavirenz (DMP-266; DuPont) (Table 7) was approved in of the 3-pyridine substituent because N-dealkylation of the September 1998 by the FDA for once-daily dosing to be used pyridine 3-alkylamino substituent is a predominant path- in combination with other anti-HIV drugs in both adult and way for metabolism of the BHAPs (Romero et al.,1996). paediatric patients. Work included varying the lipophilicity and introducing Efavirenz is a very potent inhibitor against a wide range polar water solubilizing groups. Because delavirdine also of mutant HIV-1 RTs. Its activity against some of these are underwent hydroxylation at C-6 of the pyridine ring, this presented in Table 8. In cell culture efavirenz selects for a position was targeted for blocking. This was done by syn- double mutation (L100I plus L103A) following 10 serial thesizing compounds containing a halogen at C-6 and one passages at increasing concentrations (Young et al., 1995a). compound containing a pyridazine ring where nitrogen However, no single RT substitution has yielded a mutant

Antiviral Chemistry & Chemotherapy 10:6 289 OS Pedersen & EB Pedersen

Table 6. Antiviral activities of selected compounds against resistant viruses

HN 1. R = Et, Y = t-Bu and X = H CH3SO2HN 2. R = n-Pr, Y = t-Bu and X = H

N N N X 3. R = Et, Y = Et and X = H N H R 4. R = Et, Y = i-Pr and X = F O 5. R = Et, Y = t-Bu and X = F

Y H3C N HN H 6. Z = CO, Y = i-Pr and X = H N N Z 7. Z = CO, Y = t-Bu and X = H N N N X 8. Z = SO2, Y = t-Bu and X = H N Et H 9. Z = CO, Y = t-Bu and X = F O 10. Z = SO2, Y = t-Bu and X = F

EC90 (µM)*

Compound HIV-1MF (P236L)† HIV-1IIIB (L100I, M230L)† HIV-1IIIB (Y181C)‡ Delavirdine mesylate >10 >10 5.2 1 0.19 0.08 0.03 2 0.32 0.11 0.03 3 0.31 0.08 0.18 4 0.3–1.0 0.11 0.14 5 0.22 0.12 ≤0.03 6 0.24 0.11 0.05–0.1 7 0.16 0.16 ~0.05–0.10 8 0.34 0.14 <0.03 9 0.16 0.14 0.13 10 0.29 0.29 0.15

*EC90, concentration of drug that inhibited p24 production in the antiviral assays by 90%. †Delavirdine was used for the selection of BHAP-resistant MF and IIIB HIV-1 variants. ‡L-697,661 was used for the selection of the resistant IIIB HIV-1 variant. Data from Romero et al. (1996).

Figure 3. The structure of 1-[(2-hydroxyethoxy)methyl]- virus for which the EC95 for inhibition by efavirenz has 6-(phenylthio)thymine (HEPT) and the benzyl been >1.5 µM. Most mutants are inhibited by efavirenz at analogue MKC-442 EC95 values of 50 nM or less (Table 8). The lowest concentration of efavirenz that has yielded evidence of cyto- O O toxicity, both in primary cells and in T cells, is 80 µM, giving a selectivity index of approximately 80000 (Young et HN 5 3 HN al., 1995a).

1 O 6 N S O N HEPT derivatives 1-[(2-Hydroxyethoxy)methyl]-6-(phenylthio)thymine, O O HEPT (Figure 3), is the lead compound of this class of HO NNRTI and was described in 1989 (Miyasaka et al.,1989). HEPT was synthesized as an acyclonucleoside and was expected to be a member of the nucleoside class of RT HEPT MKC-442 inhibitors, which act as competitive inhibitors by mimick- ing the natural substrate for the enzyme (Tantillo et al.,

Table 7. The activity of selected benzoxazinones against HIV-1 RT and against a strain of HIV-1 with a double mutation in RT

X R Cl O

N O H

Configuration X R IC50 (nM)* WT IC50 (nM)† A17 RT ClC95 (nM)‡

(–) Efavirenz CF3 2856

(–) CF3 8.6 69 12

(+/–) CF3 25 480 6

(+/–) CClF2 12 350 ND§

CH 3 (+/–) CF N 43 1950 100 3 CH 3

*Inhibitor concentration to give 50% inhibition[(poly)rC•(oligo)dG] in wild-type RT. †Inhibitor concentration to give 50% inhibition[(poly)rC•(oligo)dG] in A17RT, which is a mutant with double mutation: K103N and Y181C. ‡Concentration of inhibitor that reduced the spread of infection by at least 95% in MT-4 cells. HIV-1 p24 accumulation was directly correlated with virus spread. §ND, not determined. Data from Young et al. (1995a).

1994). However, in antiviral testing it was found only to Phase III clinical trials. The importance of the substitution inhibit HIV-1 and not HIV-2. From this it was deduced pattern of the thiophenyl ring in the 6 position and the sub- that HEPT was different in its mechanism of inhibition of stituent at the 5 position were among the first characteris- HIV than the known nucleoside inhibitors zidovudine, tics to be studied (Tanaka et al., 1992a). Substituting the stavudine and didanosine. methyl group in the 5 position of HEPT with ethyl or iso- Studies of the structure–activity relationship of HEPT propyl gave a marked increase in potency (Tanaka et al., analogues have led to the synthesis of MKC-442 (I-EBU; 1992a), whereas substitution with a hydrogen atom resulted Triangle Pharmaceuticals) (Figure 3), which has entered in loss of activity. Also, the substitution pattern of the 6-

Table 8. Inhibition of wild-type and mutant HIV-1 infection in cell culture by efavirenz*

EC (nM)‡ EC (nM)‡ Virus or amino 95 Virus or amino 95 acid substitution† Efavirenz L-697,661 acid substitution† Efavirenz L-697,661 Wild-type IIIB 1.5 100 V108I 3 400 Wild-type MN 3 50 V179D 3 400 Wild-type RFII 3 50 Y181C 6 >3000.0 A98G 12 800 Y188L 1500 >3000.0 L100I 100 200 K101D+K103N 1500 >3000.0 K101E 25 800 K101D+L100I 1500 >3000.0 K103N 100 800 K103N+Y181C 400 >3000.0 V106A 12 100 L100I+K103N§ 25000 1500

*Comparative values are presented for efavirenz and the unrelated pyridinone L-697,661. †Each mutant virus expressed the noted amino acid substitution at the indicated RT residue.

‡The EC95 was defined as the concentration of test compound that inhibited virus expression by at least 95% relative to virus expression in untreated control cultures. Assays were performed in MT-4 human T-lymphoid cells. §This variant was selected by passage of the HIV-1 IIIB strain in MT-4 cells in increasing concentrations of efavirenz. Data from Young et al. (1995b).

Antiviral Chemistry & Chemotherapy 10:6 291 OS Pedersen & EB Pedersen

Table 9. Inhibition of HIV-1 replication in MT-4 cells by HEPT derivatives

R HN 1

O N R2

Compound R1 R2 R3 EC50 (µM) CC50 (µM) SI

HEPT Me SPh HOCH2CH2OCH2 7.0* 740 106

E-HEPU-dM Et SPh(3,5-di-Me) HOCH2CH2OCH2 0.013* 149 11500

I-HEPU-dM i-Pr SPh(3,5-di-Me) HOCH2CH2OCH2 0.0027* 128 47400

E-HEBU-dM Et CH2Ph(3,5-di-Me) HOCH2CH2OCH2 0.013† 281 22000

I-HEBU-dM i-Pr CH2Ph(3,5-di-Me) HOCH2CH2OCH2 0.0027† 221 82000

E-BPU Et SPh PhCH2OCH2 0.0059‡ 34 5800

E-EBU-dM Et CH2Ph(3,5-di-Me) EtOCH2 0.0016† 207 130000

E-EBU Et CH2Ph EtOCH2 0.041† 245 6000

I-EBU-dM i-Pr CH2Ph(3,5-di-Me) EtOCH2 0.0006† 43 72000

MKC-442 i-Pr CH2Ph EtOCH2 0.0042† 186 44000

1EtCH2Ph(3,5-di-Me) EtSCH2 0.004§ 68 17000

2 i-Pr CH2Ph EtSCH2 0.006§ 37 6200

3EtCH2Ph MeSCH2 0.002§ 32 16000

4 i-Pr SePh(3-Me) EtOCH2 0.0081¶ 53 6540

5 i-Pr SePh(3,5-di-Me) EtOCH2 0.0047¶ >200 42600

Values may not be directly comparable due to differences in assay conditions. Data from: *Tanaka et al. (1992a); †Tanaka et al. (1995); ‡Tanaka et al. (1991), two separate experiments. The values was determined by the MTT method; §Danel et al. (1996).

EC50 was determined by the p24 antigen method and the MTT method was used for CC50. The values are expressed as the mean of three independent determinations; ¶Kim et al. (1996). The values were determined in CEM-SS cells and are the mean of two independent determinations in duplicate.

(phenylthio) moiety was investigated and substitution at the functionalities that are introduced into the N-1 linker 2 or 4 position was ineffective, whereas substitution at the 3 between the pyrimidyl moiety and different aromatic rings, position with methyl, ethyl or a fluoro substituent increased for example, phenyl, 2-furyl, 2-thienyl, 2-benzofuranyl and the potency. Introduction of a methyl group or a chlorine at so on (Pontikis et al.,1997). Some 1-substituted (ethyl- both the 3 and 5 position was even more effective (Tanaka thio)methyl and (methylthio)methyl analogues have shown et al., 1992a). Removal of the hydroxyl group in the (2- anti-HIV-1 activity comparable to MKC-442 and although hydroxyethoxy)methyl side chain also improved the poten- they have higher cytotoxicity, they still have high selectivity cy (Tanaka et al., 1992b). Changing the phenylthio moiety indices (6200 to 17000) (Danel et al.,1996). Derivatives with benzyl analogues yielded even more potent inhibitors with a selenyl in place of sulphur in the (phenylthio) moiety of HIV-1 with selectivity indices of up to 130000 (Tanaka have also been prepared (Kim et al., 1996) and some with et al., 1995). After studies on pharmacokinetics and follow- activities and selectivities that are comparable to those ing toxicology tests, MKC-442 was selected as the candi- obtained for MKC-442. date for clinical trials for AIDS chemotherapy (Tanaka et Some HEPT derivatives are highly bound to human al., 1995). The oral bioavailability of MKC-442 in rats was serum proteins and have been tested for antiviral activity in 18.4% and the 50% lethal dose in rats was >2000 mg/kg the presence of varying percentages of human serum (HS) (Tanaka et al.,1995). After molecular modelling studies on in place of foetal bovine serum (FBS) (Baba et al.,1993).

HEPT derivatives (Hopkins et al.,1996), an analogue of MKC-442 (I-EBU) showed EC50 values of 0.014 µM (in MKC-442 with naphtyl in place of phenyl was synthesized, 10% FBS), 0.018 µM (in 10% HS), 0.026 µM (in 30% HS) but it was 10-fold less active than MKC-442 and more toxic and 0.063 µM (in 50% HS). Some 2-thio analogues of (Danel et al., 1997). Many derivatives with variations in the HEPT have also been prepared and although the anti-HIV N-1 substituent have been synthesized. Among these are activity has been good, they all bound with high affinity to some with more bulky groups, such as E-BPU (Tanaka et HS proteins, some with only 0.3 to 0.7% of the total com- al., 1991) (Table 9). Some have amines, amides and alkene pound remaining unbound to proteins (Baba et al.,1993).

Figure 4. The basic structure of dihydroalkoxybenzyloxopyrimidines (DABOs) and dihydrothioalkylbenzyloxopyrimidines (S-DABOs)

O O 5′ R 5′ 4 1 R1 HN 4′ 4 4′ 3 5 HN 3 5 2 6 3′ 2 6 R2 1 R2 1 3′ O N 2 S N ′ 2′ DABOs S-DABOs

R1 : H, Me, Et or i-Pr

R2 : alkyl or cycloalkyl

Dihydroalkoxybenzyloxopyrimidines and thio best activities are achieved with bulky substituents for R2 analogues (Figure 4), such as cyclohexyl, cyclopentyl or sec-butyl The dihydroalkoxybenzyloxopyrimidines (DABOs) and (Artico et al.,1993). A methyl substituent in the 3′ position their thio analogues (S-DABOs) are closely related to the increases the activity, whereas an additional methyl sub- HEPT derivatives. DABO derivatives are characterized by stituent in the 5′ position does not always increase the an alkoxy substituent, the S-DABOs by a thioalkyl group, potency. A compound with methyl in the 5 position, 3′,5′- at C-2 in place of the N-1 substituent in the HEPT deriv- dimethyl substituted in the phenyl moiety and a sec-butyl atives (Figure 4). in the alkoxy group at C-2 had a selectivity of >416, and Some 6-benzylpyrimidines were synthesized as dihy- has been one of the most active among the first DABO drofolate reductase inhibitors and because of their structur- compounds (Massa et al., 1995). al similarities with HEPT they were tested, and some were Replacing the side chain oxygen in the 2 position with found to be active, against HIV-1 (Botta et al.,1992). sulphur led to the S-DABO compounds, which showed Unlike the HEPT derivatives, the DABOs are active with increased anti-HIV-1 activity (Mai et al.,1995). Alkylation hydrogen in the 5 position comparable with compounds at N-3 and replacing C=O with C=S in the 4 position of the having a methyl group in the 5 position (Artico et al., pyrimidine ring led to compounds devoid of activity, where- 1993). Substituting the 5 position with an ethyl or iso- as substituting the 6-benzyl group with 1-naphtylmethyl propyl does not increase the anti-HIV activity, however the enhanced the activity of the S-DABO compounds (Mai et size of the C-2 alkoxy group seems to be important. The al., 1997). Some highly active anti-HIV-1 compounds with

Table 10. Anti-HIV-1 activities of selected compounds of the S-DABO class

R1 HN

R 3 R2 S N

R1 R2 R3 IC50 (µM)* ED50 (µM)† CD50 (µM)‡ SI§

Et phenyl allyl ND 1.5¶ >100 >67 H 1-naphtyl sec-butyl ND 0.33** >300 >909

Me phenyl MeSCH2 28.8±10.0†† 3.4±1.1 >100 32±10

Et phenyl MeSCH2 12.0±2.3†† 0.8±0.2 >100 146±38 i-Pr phenyl MeSCH2 5.6±2.4†† <0.001±0.000 >100 100000±0 H2,6-di-F-phenyl sec-butyl 0.05‡‡ 0.04 >200 5000 Me 2,6-di-F-phenyl i-propyl 0.05‡‡ 0.05 >200 4000

*Concentration required to inhibit recombinant RT by 50%. †Concentration to prevent the spread of HIV-1 in cell culture by 50%. ‡Concentration of compound that prevented proliferation of host cells by 50%.

§Selectivity index, CD50/ED50. ¶Danel et al. (1998). **Mai et al. (1997). ††Sudbeck et al. (1998), (rA.dT template:primer). ‡‡Mai et al. (1999), (rC.dG template:primer).

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Figure 5. The structure of 2,3-dihydro-7H-thiazolo Figure 6. The structure of AG 1549, 5-(3,5-dichloro- [3,2-a]pyrimidin-7-ones, which can be considered a phenylthio)-4-isopropyl-1-(4-pyridyl)methyl-1H- hybrid between S-DABOs and HEPT imidazol-2-yl-methyl carbamate

O Cl N 2 Cl R1 c R1: Et, i-Pr N 6 b 5 R : Ph, 3,5-Me C H , 1-Naphtyl a R 2 2 6 3 2 O N N S 4 R3 : Me, Et, CH2CH2OH S 1 O OR3 N NH2 a (methylthiomethyl)thio substituent at C-2 of the pyrimidine ring (Table 10) have been designed based on the struc- selected from a set of derivatives of an initial lead com- ture of the NNRTI binding pocket of RT (Sudbeck et al., pound (Fujiwara et al., 1998). AG 1549 is a highly substi-

1998). Molecular modelling supporting a hypothesis of tuted imidazole (Figure 6), and had an IC50 value of 0.45 beneficial π-stacking interaction between Tyr-188 of the µM in a standard RT assay with poly(rA) and oligo(dT). NNRTI binding pocket in RT and the DABOs with 2,6- Some of the beneficial characteristics for AG 1549 are a dihalogenation in the benzyl group, has led to highly potent higher concentration in lymph nodes than in plasma after inhibitors of HIV-1 (Mai et al.,1999). Some activities of S- oral administration to rats and an in vitro selectivity index DABO compounds are presented in Table 10. of 8000 (Fujiwara et al.,1998). The activities of AG 1549, Bicyclic derivatives that can be considered as hybrids nevirapine, delavirdine and loviride were compared against between S-DABOs and HEPT analogues have also been HIV-1 mutant clones (Table 11). synthesized (Danel et al., 1998) (Figure 5). Mutant clones with single amino acid substitutions at These compounds showed only moderate anti-HIV residues 100, 103, 106, 181, 188, 190, 227 and 236 showed activity with the lowest EC50 value of 0.7 µM for the less than 10-fold reduced sensitivities compared to the compound with R1=ethyl, R2=3,5-Me2C6H3CH2 and wild-type strain, whereas the clone with mutation L234I

R3=ethyl. Some analogues with the thiazole ring fused to was 22-fold less sensitive to AG 1549 (Fujiwara et al., the C-2 and N-3 bond of the S-DABO pyrimidine ring 1998) and was more sensitive to nevirapine and loviride was also synthesized, but these were either too toxic to the than the wild-type HIV strain. The double mutant strain host cells or devoid of antiviral activity (Danel et al., with the V106A plus Y181C mutation was still sensitive to 1998). AG 1549, whereas the double mutant strain V106A plus F227L was only somewhat sensitive. Imidazoles AG 1549 was tested for in vitro selection of mutant In a screening programme seeking NNRTIs that would strains of HIV-1 and two clones were found and sequenced. inhibit HIV-1 strains that are resistant to known NNRTIs In both case more than one amino acid substitution muta- or to zidovudine, AG 1549 (formerly S1153; Agouron tion was present. One strain contained the mutations V106A

Pharmaceuticals and Shionogi Research Laboratories) was plus F227L and the EC50 value for the clone was 740 ng/ml

Figure 7. The structures of some TIBO derivatives

S S S HN HN HN N N N H H H CH3 CH3 CH3 N N N Cl Cl TIBO R82150 TIBO R82913 TIBO R86183 (9-Cl-TIBO) (8-Cl-TIBO)

Table 11. Sensitivities of molecular clones of HIV with RT gene mutations to AG 1549 (S1153) and other anti-HIV-agents

EC50 (µM)* Virus mutation AG 1549 Nevirapine Delavirdine Loviride Wild-type (strain NL342) 0.0069 0.025 0.011 0.018 L100I 0.0021 0.052 >0.905 0.015 K103N 0.0069 1.13 0.832 0.399 Y106A 0.0031 >1.88 0.271 0.285 Y181C 0.0093 >1.88 >0.905 >1.42 Y188C 0.0010 >1.88 0.058 0.655 G190A 0.0075 >1.88 0.0024 0.6712 F227C 0.0053 0.33 0.017 0.16 F227L 0.0010 0.11 0.0014 0.065 L234I 0.015 0.0013 0.024 0.0013 P236L 0.0024 0.079 >0.905 0.0080 V106A+Y181C 0.0055 >1.88 >0.905 >1.42 V106A+F227L 0.266 >1.88 0.067 >1.42

*Each value represents the mean of at least three experiments, each of which was performed in duplicate. Data from Fujiwara et al. (1998).

(1.6 µM). Another clone containing K103T, V106A plus found that a 3-methyl-2-butenyl substituent on N-6

L234I had an EC50 value of 37 ng/ml (0.08 µM) in cell cul- (Kukla et al., 1991a), a methyl substituent at position 5 and ture using the MTT assay (Fujiwara et al., 1998). a thione in position 2, gave a very potent inhibitor of HIV- Many NNRTIs bind to serum proteins, and AG 1549 1, particularly if the 5 position had the S-configuration has the greatest affinity for human albumin. In the presence (R82150) (Pauwels et al., 1990) (Figure 8). Additionally, a of 50 mg/ml of HS albumin and 1 mg/ml alpha-1-acid gly- chloro substituent in position 9 gave a compound coprotein, AG 1549 showed a ninefold reduction in its activ- (R82913) that had a 10-fold increase in activity, but also an ity compared with the activity without albumin. Taking this increase in cytotoxicity (Pauwels et al.,1990). Changing into account, AG 1549 may still have a sixfold greater antivi- the chloro substituent from position 9 to position 8 afford- ral activity than nevirapine in human plasma (Fujiwara et al., ed a compound (R86183) even more potent (Ho et al., 1998). AG 1549 is now undergoing clinical testing. 1995). Compound R82913 has been subject to clinical Phase I testing. TIBO In the imidazole part of the structure it has been found Since the TIBO class ( Janssen Research Foundation) was that replacement of the carbonyl oxygen with sulphur or introduced as non-nucleoside inhibitors of HIV in vitro selenium causes an increase in the activity. A number of (Pauwels et al., 1990), a lot of work concerning struc- variations in this position have been tested, including S- ture–activity relationships has been done using this class of phenylthiourea and O-methylated urea, and all have been compounds (Figure 7). inactive. The proton on nitrogen in position 1 appeared to In the early work of optimizing the TIBO class, it was be necessary, especially for the urea compounds, and this group may be involved in direct hydrogen bonding to the Figure 8. General structure of TIBO derivatives enzyme. Replacing the nitrogen in position 3 with a carbon resulted in loss of activity. Most changes in the imidazole X structure resulted in less active compounds or a total loss of X = O, S or Se activity (Kukla et al., 1991b). HN 2 Substitutions on the diazepine ring have also been inves- N 4 tigated (Breslin et al., 1995). The 5-mono-methyl-substi- CH3 tuted analogues, which were the original substitutions in the 9 N early lead compounds, and 7-mono-methyl-substituted 8 7 analogues were comparable, being the most active in the series. The 4,5,7-unsubstituted analogue and the 4-mono- methyl-substituted analogues were less active. Substituting larger and more bulky groups such as isopropyl and phenyl

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Table 12. Anti-HIV activities of some TIBO derivatives

HN N

R1 N R 3 R2

Compound X R1 R2 R3 EC50(µM)* CC50(µM)† SI‡

R78305 O (+)-(S)CH3 allyl H 70§ 674 10

R82150 S (+)-(S)CH3 3,3-dimethylallyl H 0.028§ >870 >31071

R82913 S (+)-(S)CH3 3,3-dimethylallyl 9-Cl 0.0015§ 31 20667

1S(±)-CH3 3,3-dimethylallyl H 0.097¶ ND** ND

2Se(+)-(S)CH3 3,3-dimethylallyl 9-Cl 0.018¶ ND ND

3S(+)-(S)CH3 3,3-dimethylallyl 8-SCH3 0.0050†† ND ND

4S(+)-(S)CH3 3,3-dimethylallyl 8-F 0.0058†† ND ND

5S(+)-(S)CH3 3,3-dimethylallyl 9-F 0.0250†† ND ND

R86775 S (+)-(S)CH3 3,3-dimethylallyl 8-Br 0.003‡‡ 53 18000

R84674 S (+)-(S)CH3 3,3-dimethylallyl 8-CH3 0.0014‡‡ 80 5700

*EC50 is the 50% inhibitory concentration for cytopathicity by HIV-1 in MT-4 cells. †CC50 is the 50% cytotoxic dose in mock-infected MT-4 cells. ‡The ratio CC50/EC50 is the selectivity index (SI). §Data from Pauwels et al. (1990). ¶Data from Kukla et al. (1991b). **ND, not determined. ††Data from Ho et al. (1995). ‡‡Data from Pauwels et al. (1994). produced no advantage except for compounds substituted (Ahgren et al., 1995; Cantrell et al.,1996). The lead com- with isopropyl in position 4 and with an oxo group in posi- pound of this series was LY73497 (Figure 9). Optimization tion 2, which were more active than the 4-methyl ana- of this lead gave N-[2-(2-pyridyl)ethyl]-N-[2-(5-bro- logues. Some (4 plus 5, 4 plus 7, 5 plus 6, 5 plus 7, 6 plus 7 mopyridyl)]thiourea hydrochloride (LY300046:HCl) (tro- and 7 plus 8) disubstituted or ring fused analogues were also virdine; Medivir), which has been selected for clinical trials. prepared (Breslin et al., 1995). The 5,7-di-Me (trans) ana- Extensive structure–activity relationship studies of the logue was slightly better than the 5-mono substituted ana- PETT compounds have been made. For this purpose the logue, being the most promising with an EC50 value of structure of PETT is considered as a product of four parts 0.042 µM in MT-4 cells. From this, the R,R (trans) and S,S (Figure 10). (trans) 5,7-dimethyl analogues were prepared for the 2-oxo In part 1 of the structure, the phenethyl moiety, the sub- and 2-thio analogues. These compounds have a different stitution patterns have been investigated. Meta and partic- structure–activity relationship to the 5-methyl analogues. ularly ortho substitution is preferred over para substitution The 2-oxo compounds were slightly more active than the (Bell et al.,1995). Both mono substitution (Vig et al., thio analogues and a chloro substituent at the 9 position 1998), di and tri substitution (Bell et al., 1995) have given resulted in a significant loss in activity. very active compounds. The characteristics of the sub- Substitution in the aromatic part of the TIBO structure stituents have also been investigated (Bell et al.,1995). has also been investigated (Ho et al.,1995). Substituents in Both electron donating and electron withdrawing of small the 8 position gave a large improvement in activity compared groups like fluoro, chloro, azido and methoxy substituents with the parent compound, whereas substituents at the 9 were comparable with good activity. The chain length of an position tended to have little effect on activity and 10 sub- alkoxy substituent was investigated, and the ethoxy substituents decreased the activity (Ho et al., 1995) (Table 12). stituent gave the maximum activity, whereas the more ster- ically demanding propoxy and isopropoxy substituents PETT decreased the activity. Compounds where the phenyl group The phenethylthiazolylthiourea (PETT) compounds were was replaced with 2-pyridyl, as in trovirdine, gave the most derived from a systematic disassemblage of the molecular active compounds in a series with different heterocycles. structure of the known TIBO HIV-1 RT inhibitors Active compounds have also been prepared with saturated

Figure 9. The structure of trovirdine and the lead Figure 10. Phenethylthiazolethiourea (PETT), compound LY73497 separated into four parts for discussion of structure–activity

S N

S S N N N H H S N N LY 73497 H H

Br S 123 4

N N N N H H .HCl pounds (Figure 11). LY 300046 HCl The activity of new NNRTIs against strains of HIV (trovirdine) resistant to known NNRTIs is becoming more important, and the PETT compounds show good activity against Trovirdine has an ED in HIV-1-infected MT-4 cells of 0.020 M, and 50 µ some of the known resistant strains of HIV. Some of the an IC50 value against wild-type HIV-1 RT of 0.015 µM and IC50 values against mutant HIV-1 RTs of 0.43 µM (Ile-100) and 2.50 µM (Cys-181) PETT derivatives showing good activity against single (Cantrell et al., 1996). mutations (Ile-100 and Cys-181) were tested in assays against two double mutant HIV-1 strains. Five of the test- heterocycles such as 1-piperidinyl and 1-piperazinyl (Mao ed compounds showed ED50 values below 10 µM against et al., 1998). one of the two double mutants (Ile-100 plus Asn-103 or In part 2 of the molecule the ethyl linker was optimal for Ile-100 plus Cys-181) (Cantrell et al., 1996). These five activity. Methyl substitution in the benzylic position compounds are listed in Table 13. enhanced activity, whereas a methyl in the phenylethyl The PETT compound trovirdine has shown to readily position diminished activity (Bell et al., 1995). A cyclo- penetrate the blood–brain barrier, as the concentration in propyl linker with cis configuration, has been shown to be brain tissue parallelled those in plasma of male Fischer 344 an advantage for the urea-PETT analogues (Sahlberg et rats (Ahgren et al.,1995). The in vitro protein binding of al., 1998). trovirdine was 88.7% in rat plasma and 95.5% in human For part 3 of the molecule the N,N-unsubstituted- thiourea was most active (Bell et al., 1995). Methyl substitution on nitrogen adjacent to the phenethyl side chain Figure 11. The structure of PETT derivatives and trovirdine with the proposed internal hydrogen completely eliminated activity, whereas methyl substitution bond on the nitrogen adjacent to the thiazole ring only decreased the activity slightly. The reason for the difference in activi- S ty for N-methyl substitution may be owing to an internal (H,Me) R hydrogen bond (Figure 11). N N Compounds with a urea part in place of the thiourea H have also been prepared. They give lower activity but may S N possess better pharmacological properties than the thiourea compounds (Sahlberg et al., 1998). PETT derivatives In part 4, the thiazole moiety, the most optimum compounds have been achieved by replacing the thiazole with a

5-bromopyrid-2-yl. In the thiazole series several 4-substi- S tuted derivatives, including small alkyl, cyano, trifluoromethyl and ethoxycarbonyl substituted compounds were HN N N quite potent inhibitors (Bell et al., 1995). In a series where H the thiazole was replaced with diazinyl it seemed that the N nitrogen in the 2 position was essential because the 4-pyri- dazinyl derivate was devoid of activity (Heinisch et al.,

1997b). This seems to be in accordance with the proposed Br internal hydrogen bond in anti-HIV active PETT com- Trovirdine

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Table 13. Five compounds tested with activity against double mutant HIV-1 RT in MT-4 cells

R R2 R1 4 S

N N N H H R5

ED50 (µM)*

R1 R2 R3 R4 R5 WT† Ile-100 Cys-181 Ile-100/Asn-103 Ile-100/Cys-181 Cl F MeO H MeO 0.002 0.009 0.003 1.0 1.0 Br F H H EtO 0.013 0.09 0.007 1.6 1.0

Br F CN Me2NH 0.013 0.15 0.020 5.5 7.5 Br F EtO H F 0.006 0.10 0.006 10 1.2 Br CN EtO H F 0.004 0.04 0.005 >10 3.0

*Concentration of inhibitor that inhibited the infection of HIV-1IIIB in MT-4 cells, assayed with the XTT method after 5 or 6 days. †Wild-type HIV-1 RT. Data from Cantrell et al. (1996).

Table 14. Inhibition of HIV-1 by urea-PETT compounds

R 1 R R4 2 O R2 R4 O R1 N N N H H N N N H H R3 R3

1–67–13

ED50 (µM)*

Compound R1 R2 R3 R4 WT WT† Clone 118‡ Clone 90§ 1HFF Cl 0.03 0.85 32 >32

2 NMe2 FF Br 0.5 NT¶ >25 >25 3 OMe F OMe Cl 0.011 0.09 17 3 4 OEt F OMe Cl 0.13 NT 14 >27 5 COMe F OMe Cl 0.2 NT >27 >27 6 OEt Cl F Br 0.07 NT 15 8 7HFF Cl 0.01 0.01 NT NT 8 OEt Cl F Cl 0.016 0.06 0.1 NT 9 OEt Cl F CN 0.01 0.02 0.27 0.53 10 OEt F F Cl 0.01 0.03 0.75 0.27 11 OMe F OMe Cl 0.012 0.1 1.6 2.7 12 OEt F Cl Cl 0.01 0.1 0.1 0.1 13 OEt F OMe Br 0.025 0.4 NT NT Trovirdine 0.02 5 0.8 >5 9-Chloro-TIBO 0.25 NT >22 124 Nevirapine 0.15 0.12 0.62 22 L-697,661 0.065 NT 0.85 >11

*Concentration of compound that reduced the CPE of HIV-1IIIB in MT-4 cells by 50% in an XTT assay. †Tested in the presence of 15% human AB serum. ‡Clone 118 contains a L100I mutation. §Clone 90 contains a Y181C mutation. ¶NT, Not tested. Data from Sahlberg et al. (1998).

Table 15. The anti-HIV activity and inhibition of HIV-1 Table 16. Pyrimidine thioethers inhibition against RT of selected alkenyldiarylmethanes (ADAM) HIV-1 wild-type and a delavirdine resistant strain compared to delavirdine

H3COOC COOCH3 Cl

H3CO OCH3 N

X X H2N N SR

R EC90 (µM)*

Compound R HIV-1IIIB HIV-1MF†

RXIC50*EC50†CC50‡ TI§ 1 2-naphtyl 0.22 2.76 2 CH=CHphenyl 0.15 5.05 (CH2)4CH3 Br 0.38 9.2 138 15 3 CH=CHCONMe2 0.09 0.74 CH2CH2N3 Br 94 1.1 >316 >278 4 CH=CHCONEt2 0.02 0.11 CH2CH2N3 Cl 2.0 0.27 41.8 155 5 3-methylphenyl 0.11 1.76 (CH2)3COOCH3 Cl 0.3 0.013 31.6 2430 6 3-methoxyphenyl 0.27 3.21 *Inhibitory activity (µM) versus HIV-1 RT with rC.dG as the template 7 3-bromophenyl 0.35 4.52 primer. Delavirdine‡ 0.03 53.58 †50% inhibitory concentration (µM) for CPE of HIV-1RF in CEM-SS cells. ‡Cytotoxic concentration (µM) for mock-infected CEM cells. *Concentration of drug that inhibited p24 production by 90% in infected MT-4 cells. §Therapeutic index, CC50 divided by EC50. Data from Cushman et al. (1998a). †Laboratory derived delavirdine-resistant variant of HIV-1. ‡Reference compound. Data from Nugent et al. (1998). plasma at a concentration of 2 µg/ml. Because of its simple chemical synthesis, excellent antiviral activity, satisfactory potent compound of this class, with an EC50 value of 13 pharmacokinetic profile and acceptable toxicity, trovirdine nM and a selectivity index of 2430 (Cushman et al., entered Phase I clinical trials (Ahgren et al., 1995), but was 1998a,b). later withdrawn. Bromo- and chloro substituents are preferred over iodo- and unsubstituted derivatives (Table 15). The length of the Urea-PETT alkenyl substituent is also important and it appears that a The urea-PETT compounds may have better toxicological six atom chain is optimal for anti-HIV-1 activity and phamacokinetic properties than the PETT com- (Cushman et al., 1998a). pounds. It was found that the urea-PETT compounds maintain their antiviral activity in cell culture in the pres- Pyrimidine thioethers ence of added HS much better than the thiourea com- Pyrimidine thioethers (Pharmacia & Upjohn) as inhibitors pounds (Sahlberg et al., 1998). This point urged the of HIV-1 RT were first reported by Althaus et al. (1996). researchers at Medivir AB to make a structure–activity This class of RT inhibitors is primarily characterised by its research (SAR) programme in which different urea-PETT potent activity against the P236L mutant, which renders analogues were synthesized and evaluated (Table 14). This HIV-1 resistant to delavirdine (Nugent et al.,1998). The gave ethyl linked (1–6) and conformational restricted lead compound of this class was 6-chloro-2-benzylthio-4- cyclopropyl analogues (7–13). The cyclopropyl compounds pyrimidinamine, and several derivatives were investigated were in general more potent than the ethyl linked com- by Nugent et al. (1998). Exchange of the chloro moiety in pounds, especially against mutant HIV strains. the pyrimidine ring with trifluoromethyl gave a compound only a little less potent, whereas exchange with other elec- Alkenyldiarylmethanes tron-withdrawing groups was not so good. In the place of Alkenyldiarylmethanes (ADAMs) were introduced as a R (Table 16) the enzyme can accommodate a large, but flat, new class of NNRTIs in 1995 (Cushman et al., 1995, hydrophobic group. This could be an aryl group or an 1996). This afforded the first lead compound 3′,3′′-dibromo- alkenyl group. A secondary amide attached to an alkenyl 4′,4′′-dimethoxy-5′,5′′-bis(methoxycarbonyl)-1,1-diphenyl- has significantly less activity compared to a tertiary amide.

1-heptene, with an EC50 value of 9.2 µM and a selectivity This is thought to be caused by a hydrogen bonding pro- index of 15. Further work has lead to methyl 3′,3′′- ton, or for steric reasons, in the lipophilic pocket of the dichloro-4′,4′′-dimethoxy-5′,5′′-bis(methoxycarbonyl)-6,6- enzyme. Substitution of the sulphur with oxygen or nitro- diphenylhexenoate (ADAM II) as the currently most gen decreased or removed the activity. Oxidation of the sul-

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Table 17. Antiviral activity of furo[2,3-c]pyridine pyrimidine thioethers against in vitro-selected NNRTI-resistant HIV-1 variants

S N Cl N

CH3 N

NH2

EC90 (µM)* DLVR MF L-697,661R IIIB DLVR IIIB R88703R IIIB Compound IIIB (WT) (P236L)† (Y181C)† (L100I)† (Y181C)† (–)-(S)(PNU-142721) 0.001 0.008 1.1 0.07 0.17 (+)-(R) ND‡ >0.01 >3.0 ND ND Racemate 0.002 0.01 2.4 0.06 0.17 Delavirdine 0.05 >10 5.2 >10 1.0

*EC90 concentration of drug that inhibited p24 antigen production by 90% in infected MT-4 cells. †Primary resistance conferring mutation at the designated codon of HIV-1 RT. ‡ND, Not determined. Data from Wishka et al. (1998a). phur diminished the activity, and removal of the methylene c]pyridinethiopyrimidine class of compounds. These are linker resulted in compounds devoid of activity. Table 16 characterized by the aralkyl group and a methyl substituent shows some of the compounds with activity against wild- at the methylene linker. This methyl substitution introduces type and the delavirdine-resistant HIV-1 (P236L) mutant a stereocentre at the carbon in the methylene linker (Table (Nugent et al., 1998). 17). Both the (R) and (S) enantiomers proved to be Further study within the pyrimidine thioethers for compounds with broad activity against several NNRTI-resistant Figure 12. Structure of other bicyclic TTD analogues variants of HIV-1 have led to the furo[2,3- O O

Table 18. Antiviral activity of some TTDs S R1 N O O 2′ 7 S X S N 1 O 2N 3′ 6 S (a) R2 5 4 N O O O R N S R1 N Compound X R EC50 (µM)* CC50 (µM)† H3C N QM96625 H 2-Cl-benzyl 0.1 >119 N O QM96521 H CH CN 0.9 502.7 2 (b) R2 QM96537 H CH2CCH 1.0 376 QM96539 Cl CH2CN 0.09 340 O O Br CH2CN 0.09 68.6 QM96639 F CH CN 0.05 93.6 S S R1 2 N Cl *Compound concentration required to achieve 50% protection of N MT-4 cells from HIV-1IIIB induced cytopathogenecity, as determined O by the MTT method. †Compound concentration dosage required to reduce the viability of R2 (c) mock-infected cells by 50%, as determined by the MTT method. All data represent mean values of at least two separate experiments. (a) Thieno[2,3-e][1,2,4]thiadiazine,(b) 6-methylpyrazolo[4,3- Data from Arranz et al. (1998). c][1,2,4]thiadiazine, (c) 6-chlorothieno[3,2-e][1,2,4]thiadiazine.

Figure 13. Structure of carboxanilide UC84 with the istry of these thiopyrimidines. PNU-142721, the (S)-enan- separation into four parts marked tiomer, and the racemate were found to inhibit the enzyme

with IC50 values in the submicromolar range, whereas the (R)-enantiomer was inactive. In cell culture PNU-142721 O CH3 O CH3 was very potent against HIV-1 wild-type and several strains H N of HIV-1 resistant to known NNRTIs (Table 17). S O CH3 Another compound in this series, PNU-109886, with a O methyl substituent in the furo[2,3-c]pyridine moiety has Cl ab c d displayed increased potency against virus containing the Y181C mutation. However, it is characterized by a less favourable pharmacokinetic profile than PNU-142721 extremely potent inhibitors of wild-type HIV-1 RT with (Wishka et al., 1998b). PNU-142721 possess high antivi-

IC50 values (rA.dT) from 20 to 85 nM, and of the mutant ral activity against a broad spectrum of HIV-1 variants and

RT P236L that is resistant to delavirdine, with IC50 values a favourable pharmacokinetic profile, including penetra- from 22 to 25 nM (Wishka et al., 1998a). Interestingly, tion of the blood–brain barrier in the rat. The levels of activity against Y181C RT,which is observed to occur when PNU-142721 found in the brain in an experiment with HIV-1 cultures are under pressure from many known rats was 75% of the simultaneous plasma concentrations NNRTIs, was found to be dependent on the stereochem- (Wishka et al., 1998a).

Figure 14. Structure of some active derivatives of UC 84

O CH3 O CH3 N H S O CH3 O Cl ab c d H H3C N UC 38 O H3C S H O H3C N CH 1 O 3 H3C S H CH H3C 3 N CH NO 2 O 2 CH3 H3C S H C 3 H O CH N 3 3 O H3C S H3C H O N O 4 H C 3 O CH3 S H3C H O N O 5 H3C O S CH H 3 N O CH3

6 H3C O O CH S 3 CH H CH 3 N O 3

7 H C 3 O O CH3 S CH H O 3 N CH3 8 H C 3 O O CH3 S CH3

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Table 19. Antiviral activity of oxathiin carboxanilide derivatives against wild-type and mutant HIV-1 strains in CEM cells

EC50 (µM)*

Compound IIIB L100I V106A G138K Y181C CC50 (µM)† UC 84 0.042 ≥112 >56 ≥56 42 25 UC 38 0.03 2.2 2.2 1.3 2.0 40 1 0.01 2 2 0.3 2.0 19 2 0.03 0.67 1.4 0.2 1.1 >317 3 0.1 1.3 1.7 0.33 1.8 20 4 0.1 1.8 2.1 1.7 1.8 36 5 0.6 2.2 1.6 1.8 1.6 14 6 0.6 0.91 1.2 0.2 1.8 30 7 0.9 2.1 1.2 2 2.0 30 8 0.9 1.9 1.0 0.35 1.5 2.3 Nevirapine 0.03 0.1 8.6 8.6 0.37 >75 TIBO R82913 0.03 0.9 6.2 2 5.3 >62

*Compound concentration required to reduce HIV-1-induced giant cell formation in CEM cell cultures by 50%. †Compound concentration required to reduce viability of the host cells by 50%. Data from Balzarini et al. (1995).

TTD resulted in less active compounds. Systems that have been 1,1,3-Trioxo-2H,4H-thieno[3,4-e][1,2,4]thiadiazine (T- investigated are thieno[2,3-e], 6-methylpyrazolo[3,4-e] TD) is a new class of NNRTI (Witvrouw et al., 1998). The and 6-chlorothieno[3,2-e] fused TTD analogues (Arranz basic requirements for the structure of TTDs is N-4 alkyl et al., 1998) (Figure 12). and N-2 benzyl groups (Table 18). Two compounds with a 2-picolyl in place of the N-2 benzyl were less active, where- Carboxanilides and thiocarboxanilides as a 3-picolyl derivative and a 2-picolyl derivative with a Oxathiin carboxanilide, UC 84 (NSC 615985), was origi- benzyl group at N-4 was more potent than the benzyl ana- nally synthesized as a potential fungicide by chemists at the logues (Arranz et al., 1998). Halogens have been substitut- Uniroyal Chemical Company. In a screening programme ed at the N-2 benzyl moiety, with optimum results by the National Cancer Institute, UC 84 exhibited anti- achieved by one ortho- or metha substituent because a 3- HIV activity. UC 84 completely protected CEM-SS cells fluorobenzyl derivative was more potent than the 3,5- infected with HIV-1 from the cytopathic effect of HIV difluorobenzyl analogue (Witvrouw et al., 1998). The best with an EC50 value ranging from 0.1 µM to 1.0 µM, results concerning the N-4 alkyl substituent have so far depending on the infectivity condition (Bader et al.,1991). been with cyanomethyl, propargyl and benzyl. Substituents UC 84 served as a lead (Balzarini et al., 1995) and consists of methyl, ethyl, propyl, allyl and cyanoethyl at N-4 pro- of an oxathiin moiety (part a), a carboxamide group (part b) duce less active compounds (Arranz et al., 1998; Witvrouw and a 2-chlorobenzoic acid (part c) that is esterified with et al., 1998). an isopropyl group (part d) (Figure 13). Other bicyclic systems, where changes in the thiophene A series of derivatives was synthesized (Balzarini et al., ring of the thieno[3,4-e][1,2,4]thiadiazine have been tried, 1995) with the following changes: part a, the oxathiin part,

Figure 15. Structure and substituents of UC 10, UC 82, UC 781 and UC 040

R =N and Z = O (UC 10) O

Cl S

N R R = and Z = S (UC 82) H O and Z = O (UC 781) Z CH3

R = and Z = S (UC 040) O

Table 20. Anti-HIV activities of UC 10, UC 82, UC 781 thus be very interesting in a more protective manner as an and UC 040 agent in retrovirucidal formulations.

Compound EC (µM)* CC (µM)† TI‡ 50 50 TDA (thiadiazole derivatives) UC 10 0.008 20 2500 The basic structure of the TDA derivatives is presented in UC 82 0.008 16 2000 Table 22. A derivative (RD3-2356) with a 2-chloro sub- UC 781 0.008 >500 62500 UC 040 0.016 10 625 stituent in the phenyl moiety enhanced the activity compared with a derivative containing an unsubstituted phenyl *Concentration required to inhibit the spread of HIV-1 by 50% moiety (RD3-2105) (Hanasaki et al.,1995). A 4-chloro (XTT method), for paticular cell lines and virus isolates; see Buckheit et al. (1997). substituent has the opposite effect, producing compounds †50% cytotoxic concentration. with decreased or lacking activity (Ijichi et al.,1996). ‡Therapeutic index. Data from Buckheit et al. (1997). Investigating the R2 and R3 alkyl substituents revealed that the most potent congener of the TDA derivatives is 4- (2,6-dichlorophenyl)-1,2,5-thiadiazol-3-yl N-methyl-N- was replaced by various alkoxy groups; part b was modified propylcarbamate (RD4-2024), whereas the from C=O to C=S, which in most cases increased the activ- monoalkylcarbamate derivative, RD3-2380, was without ity; part c was mostly unmodified; and part d was modified inhibitory effect (Ijichi et al.,1996). The easy synthesis of with a variety of substituents. The eight most interesting TDA derivatives made them potential drug candidates for compounds in this series, UC 38 being the most potent, are HIV-1 infection (Hanasaki et al., 1995). presented in Figure 14, and test results against HIV wild- Sequence analysis of mutants resistant to TDA deriv- type and four mutant strains are presented in Table 19. atives has revealed a point mutation at position 181 Further work with the thio analogues of this series have (Tyr→ Cys) (Ijichi et al., 1996). When anti-HIV-1 led to three new highly active compounds related to UC 10 assays were carried out in the presence of 50% HS to (Figure 15 and Table 20), with the compound UC 781 as a evaluate TDA under more physiological conditions, the very potent inhibitor (Buckheit et al., 1997). inhibitory effects on HIV-1 replication showed a UC 781 shows excellent inhibitory activity against sev- 10–200-fold decrease. The degree of reduction depends eral mutant HIV-1 strains, including the L100I, V106A, on the lipophility, more lipophilic leading to less E138K and Y181C mutations (Table 21). In a test it has inhibitory effect. The TDA derivatives were bound to selected for the mutations Y181C, V108I and K101E in serum protein by more than 90% under these conditions order of appearance (Buckheit et al., 1997). UC 781 was (Ijichi et al., 1996). shown to be a rapid tight-binding inhibitor of HIV-1 RT (Barnard et al., 1997). In addition, UC 781 has been found Diarylsulphones to readily penetrate isolated HIV-1 particles, and treatment The basic structure of diarylsulphones consists of two sub- of isolated HIV-1 virions with UC 781 result in rapid inac- stituted phenyl groups linked by a sulphone group. In a tivation of the virus (Borkow et al., 1997). UC 781 may screening of 54 diarylsulphones and related derivatives, 2-

Table 21. Activities of UC compounds against viruses resistant to HIV-1-specific inhibition

EC ( M)* Inhibitor to which isolate 50 µ is resistant (mutation) UC 10 UC 040 UC 781 UC 82 Nevirapine

HIV-1IIIB (control) 0.2 0.2 0.01 0.008 0.01 Oxathiin caboxanilide (L100I) 2.0 >2 0.5 0.3 0.1 UC 10-Costatolide (K103N) 7.3 >2 0.9 1.0 ND† Thiazolobenzimidazole (V108I) 1.2 0.9 0.1 0.06 0.3 TIBO (A98G/V108I) 1.1 1.2 0.3 0.2 0.6 Calanolide A (T139I) 0.3 0.4 0.04 0.03 0.01 Diphenylsulphone (Y181C) 1.1 >2 0.9 0.2 5.9 Pyridionone (Y181C/L103N) >100 >2 >2 >2 >38 Costatolide (Y188H) 1.6 1.0 0.1 0.07 ND HEPT (P236L) 0.2 0.4 0.02 0.02 0.02

*Concentration required to inhibit the spread of HIV-1IIIB in CEM-SS cells by 50% (XTT method). †ND, Not determined. Data from Buckheit et al. (1997).

Antiviral Chemistry & Chemotherapy 10:6 303 OS Pedersen & EB Pedersen

Table 22. Structure-activity relationsship of the TDA derivatives

N R3 O R1 O

N N S

Compound R1 R2 R3 EC50 (µM)* CC50 (µM)† RD3-2105 – Me Me 29 210 RD3-2356 2-Cl Me Me 0.44 162 RD3-2107 4-Cl Me Me 353 353 RD3-2236 2,3-dichloro Me Me 0.94 127 RD3-2219 2,4-dichloro Me Me 8.3 77 RD3-2218 3,4-dichloro Me Me 315 315 RD3-2233 2,5-dichloro Me Me 0.37 111 RD3-2220 2,6-dichloro Me Me 0.20 190 RD4-2025 2,6-dichloro Me Et 0.037 29 RD4-2024 2,6-dichloro Me Pr 0.013 28 RD3-2102 2,6-dichloro Me Bu 0.020 23 RD4-2031 2,6-dichloro Me Hex 0.12 19

RD3-2222 2,6-dichloro C5H10 0.96 221 RD3-2101 2,6-dichloro Et Et 0.25 314 RD4-2023 2,6-dichloro Et Bu 0.33 44 RD3-2380 2,6-dichloro H Bu 139 139

*50% Effective concentration, required to inhibit HIV-1-induced CPE by 50% in MT-4 cells (MTT method). †50% Cytotoxic concentration, required to reduce cell viability by 50% in mock-infected MT-4 cells. Data from Ijichi et al., (1996). nitrophenyl phenyl sulphone (NPPS) was identified as a 1996). Several of the most active compounds in a series RT inhibitor of the non-nucleoside class (McMahon et al., studied by Buckheit et al. (1996) had a chloro and a methy- 1993). The most active compounds are those with an lamine on the ring not containing the nitro group (Table ortho-nitro group (McMahon et al., 1993; Buckheit et al., 23). Compounds where methyl replaced chloro were only

Figure 16. The structure of calanolide A and some anti-HIV active analogues

O O O O

O O O O O O O O O O O O

OH OH OH OAc

(+) Calanolide A (+) Calanolide B (–) Calanolide B 12-Acetoxycalanolide

ED50 0.1 µM ED50 0.4 µM (Costatolide) ED50 2.7 µM

IC50 20 µM IC50 15 µM ED50 0.3 µM IC50 13 µM

IC50 5.8 µM

EC50 is the concentration of inhibitor needed to prevent the spread of HIV by 50%. IC50 is the concentration of the inhibitor which reduce the viability of host cells by 50%.

Table 23. Antiviral HIV-1 activity and RT inhibition of some diarylsulphones

R1 OO NO2

R2 S

Compound (NSC) R1 R2 R3 R4 EC50 (µM)* IC50 (µM)†

665527 -NH(CH2)3-ClH0.3 >200 667948 -NH(CH2)3-MeH0.5 33 667951 NHMe H H Me 0.4 18.4 667952 NHMe H H Cl 0.08 61 671291 H Me NHMe H 0.2 32.7 671292 H Cl NHMe H 0.1 >200

*Concentration of compound required to inhibit the spread of HIV-1 in CEM-SS cells by 50% (XTT method). †Concentration of compound required to inhibit HIV-1 RT by 50% using a rC.dG template primer. Data from Buckheit et al. (1996). slightly less active (Buckheit et al., 1996) and reduction of from Sarawak in Malaysia, showed anti-HIV activity in an the sulphone linker also reduces the antiviral activity initial test (Kashman et al., 1992). From this extract some (McMahon et al., 1993). Compound NSC 667952 was the coumarins were isolated and identified. The most active of most active in the series, retaining activity against the drug- these compounds, in descending order, were (+)calanolide resistant virus isolate and having a sensitivity profile against A, (+)calanolide B and the ester derivative 12-acetoxy- the L100I mutation (Buckheit et al., 1996). The diarylsul- calanolide (Sarawak MediChem Pharmaceuticals) (Figure phones have recently been investigated as HIV-1 integrase 16). In a search for calanolide A in other and more abun- inhibitors. Some have been found to be interesting enough dant sources, several species of callophyllum were tested for further investigation (Neamati et al., 1997). (Fuller et al., 1994). In the latex from C. teysmannii miq. var. inophylloide,a compound identified as the (–)calanolide Calanolide A B or costatolide was isolated and tested. This costatolide,

A 1:1 CH2Cl2:MeOH extract of fruits and twigs from though a known compound not previously identified as Callophyllum lanigerum var. austrocoriaceum,a rainforest tree having antiviral activity, showed anti-HIV activity compa-

Figure 17. The structure of the two pyridinones L-697,639 and L-697,661

Cl H3C

N N H H N N O O Cl CH3 N O N O H H

L-697, 639 L-697, 661

HIV-1 RT HIV-1IIIB*

IC50† CIC95‡

L-697,639 20 nM 25–50 nM L-697,661 19 nM 25–50 nM

*Assays using MT-4 cell culture preinfected with HIV-1IIIB at a low multiplicity. †Concentration that caused 50% inhibition of HIV RT. ‡Concentration that inhibited the spread of HIV-1 infection by ≥95%. Data from Goldman et al. (1991).

Antiviral Chemistry & Chemotherapy 10:6 305 OS Pedersen & EB Pedersen

Table 24. Anti-HIV-1 and anti-HIV-1 RT activity of Pyridinones some pyridyl- and phenyl analogues of the 2- pyridinone class The pyridinones L-697,639 and L-697,661 (Merck Research Laboratories; Figure 17) were reported in 1991 3′ MeO X (Goldman et al., 1991). Compound L-697,661, with 95% 4′ clearance concentration (ClC95) values in the nanomolar H R N 5′ range, has been clinically tested and good antiviral activity Y 6′ was observed. However, antiviral activity was of short dura- tion owing to rapid onset of viral resistance (Hoffman et al., N O H 1992). When new drug candidates are tested in HIV antiviral and RT enzymatic assays they are often compared XY RIC(nM)* ClC (nM)† 50 95 to L-697,661. NCH4′-Me, 5′-Et 19 13 Structural modifications on the 2-pyridone moiety have CH CH 4′,5′-Me2 813not been successful (Hoffman et al., 1992; Saari et al., NCH4,5 -Me 950 ′ ′ 2 1992). These include N-methylation, O-methylation, dele- CH N 4 ,5 -Me 70 100 ′ ′ 2 tion of one or both alkyl groups and substituting 5-ethyl CH CH 265 400 with 5-methyl. Some structure–activity studies have also NCH 680 800 been done replacing the benzoxazole by a pyridyl or a *Concentration of compound that inhibit HIV-1 RT by 50% using phenyl (Wai et al., 1993) (Table 24). It was found that rC.dG template primer. †Concentration of compound that inhibits the spread of HIV-1 maximum enzyme inhibition was obtained with com- infection in MT-4 cells, by ≥95% using a p24 core antigen ELISA pounds containing a 2′-methoxy group on either the assay. Data from Wai et al. (1993). phenyl or pyridyl ring. In the phenyl subclass several substituents were tried in the ortho position. Ethoxy- and rable to (+)calanolide A (Fuller et al., 1994). nitro substituents were comparable with the methoxy sub- A lot of structure–activity studies concerning (+)calano- stituent and some substituents (CN, F, Cl, MeS, Me) gave lide A and (–)calanolide B have been performed (Galinis et only modestly active compounds. Substituents such as CF3, al., 1996; Zembower et al., 1997) and despite increased NH2 and MeOCH2 were detrimental to activity. There is knowledge of the structure–activity relationship for this no significant difference between the 2-methoxyphenyl class of NNRTI, no new compounds more potent than the and 2-methoxy-3-pyridyl derivatives. In the pyridyl sub- lead compounds have been found. Calanolide A is present- class the position of the pyridine nitrogen was found to be ly in clinical testing. important, with the position of nitrogen at 3′ as the most

Table 25. Inhibition of HIV-1 RT and viral infection spread by selected 2-pyridinone analogues together with the rates of acid hydrolysis

N X R

O N O H

Compound X R IC50 (nM)* ClC95 (nM) T1/2 (min)‡

L-697,661 NH 4,7-Cl2 20±4 50–100 17±4

1CH2 H23±1 50–100 218±18

2CH2 4,7-Cl2 14±3 50 29±1 3NHH 210±20 400–800 35±7 4OH 191±30 ND¶12±0.2

5O4,7-Cl2 88±16 200 11±0.2

6S4,7-Cl2 11±1 25–50 34±7

*The concentration that produced 50% inhibition of RT using a rC.dG template primer, and stated as the mean of at least three determinations ±SEM. †The cell culture inhibitor concentration required to inhibit by > 95% the spread of HIV-1 in susceptible cell culture, stated as a range of values if multiple determinations were made. An assay using MT-4 cells and HIV-1IIIB strain was used. ‡Determination were carried out in 90% ethanol, 10% 1 M HCl to ensure compound solubility and to maintain a final HCl concentration of 0.1 M. ¶ND, not determined. Data from Hoffman et al. (1993).

Table 26. Anti-HIV-1 RT activity and cytotoxicity of some α-APA analogues

O NH Cl 2

N H R1 Cl

Compound Isomer R1 R2 EC50 (nM)* CC50 (µM)† SI‡

R 15345 (±) OCH3 H 610 15 25

R 18893 (±) NO2 H8880 909

R 87231 (+) NO2 H 1700 82 48

R 87232 (–) NO2 H3366 2000

R 88703 (±) C(O)CH3 H26 130 5000

R 88976 (+) C(O)CH3 H 6500 120 18

R 88977 (–) C(O)CH3 H1952 2737

R 89439 (±) C(O)CH3 CH3 13 710 54615

R 90384 (+) C(O)CH3 CH3 2100 270 129

R 90385 (–) C(O)CH3 CH3 5 420 84000 L-697,661 22 320 14545 AZT 0.5 3.5 7000

*Concentration required for prevention of spread of HIV-1 in cell culture by 50% (MTT procedure). †Fifty percent toxicity concentration in MT-4 cells (MTT procedure).

‡Selectivity index is the ratio of CC50 to EC50. Data from Pauwels et al. (1993). Figure 18. Other NNRTIs

H C 3 NH2 O N N N O Cl S Cl N OO S O O CH3 O OO 123

NH2

H3C F

Cl N S F N N OO N N OO FF F S F 456

N O N N H3C H NH N O Cl S SiO O O SiO N O O O O NH2 N NH2 H NH O OSi 2 OOSi S S OO O 789O

Antiviral Chemistry & Chemotherapy 10:6 307 OS Pedersen & EB Pedersen

Figure 19. Other NNRTIs

H H N S N S

CH3 CH3 H3C S N CH3 O N H

S O O O O N CH H C CH3 H2C 3 3 O 10 11 12 S-2720 HBY 097 Thiazoloisoindol-5-one, C10H7 derivative

MeO

MeO NCN MeO O

MeO OMe Cl

OMe 13 14 U-78036 favourable. Small lipophilic groups at 4′- and 5′-positions ysis than the other derivatives. Compounds with an ether, are required for optimum activity and analogues with larg- or a thioether linker were also prepared and tested (Table er alkyl groups are less potent. Methyl substitution at the 25). The ether linker decreased the activity, whereas the 3′- and 6′-positions was detrimental. In this series of com- analogue of L-697,661 with a thioether linker was more pounds 3-[[(5-ethyl-2-methoxy-6-methyl-3-pyridyl)- inhibitory to HIV-1 RT than L-697,661 and had an methyl]amino]-5-ethyl-6-methylpyridin-2(1H)-one (first improved stability in an acid environment. It was not as entry in Table 24) was selected for clinical evaluation due stable as the unsubstituted counterpart with an ethyl link- to the best oral availability in rats and monkeys. er (Compound 1 in Table 25). A structure–activity relationship study of 2-pyridinones The early candidate of the pyridinones, L-697,661, was with a hydrolytically more stable ethyl linker in place of the very tightly bound (99.6%) to human plasma and com- amino-methylene linker was made (Hoffman et al., 1993) pound 1 was found to be 4% unbound to human plasma although the original lead had shown less potential with an protein, which suggested a 10-fold higher free drug level in ethyl linker. As observed in earlier studies, a change in the vivo.Compound 1 has been studied clinically in HIV-pos- length or a reduction in the flexibility decreased the itive patients. Resistance to compound 1 has been observed inhibitory potency. The effect of thiocarbonyl modification in cell culture experiments with virus from clinical isolates of the pyridinones was minimal in enzyme inhibition, but obtained from patients who developed resistance to L- they were less effective in the prevention of virus spread in 697,661 (Hoffman et al.,1993). cell culture, which could reflect poorer cell penetration of these pyridinethione analogues. Many of the compounds α-APA (α-anilinophenylacetamide) with a benzoxazole as in L-697,661 and an ethylene linker A screening programme of 2000 compounds belonging to were highly active, particularly those unsubstituted, substi- different classes led to the discovery of the lead α-APA tuted in the 4 or 7 position or disubstituted in the 4 and 7 derivative R 15345 with an EC50 of 0.6 µM in MT-4 cells position. The number of potent candidates was reduced by (Table 26). Through evaluation of structurally related investigating the susceptibility of hydrolysis in acidic envi- compounds, R 18893 with a nitro group in place of the ronments. This revealed one compound, the unsubstituted methoxy substituent in the ortho position of the aniline analogue 1 in Table 25, 10-fold more stable to acid hydrol- moiety was identified as a more potent inhibitor, with an

4 EC50 value of 88 nM in MT-4 cells (Pauwels et al., 1993). nanomolar range and a selectivity index of 10 (Kleim et At this stage it appeared as the antiviral activity was stere- al., 1993). HBY 097, another quinoxaline, was withdrawn ospecific as the (–) isomer was 50-fold more potent (EC50 after finishing Phase IIa clinical testing. HBY 097 showed

33 nM) compared with the (+) isomer (EC50 1700 nM). activity against HIV-1 in PBL (mean of 41 clinical iso-

Further optimization was made by replacing the nitro lates) with an EC50 of 0.002 µM and with a selectivity substituent with an acetyl group, R88703 and by adding a index of 12500 (Kleim et al., 1995). Strains of HIV-1 gen- methyl substituent in the para position of the acetyl group erated under high selective pressure of HBY 097 revealed on the aniline moiety. This led to the most potent a G190E mutation which is characteristic for the quinox- inhibitor of this series, 90385, with a selectivity index of alines (Kleim et al.,1995). RT with glutamic acid in posi- 80000 (Pauwels et al., 1993). The compound R 89439 (SI tion 190 has shown a dramatic decrease in enzymatic

54,615) was further evaluated against some mutant activity compared to the HIV-1MN RT (wild-type) (Kleim strains of HIV-RT and RT mutants containing a Y181C et al.,1993). Compound 12 [(R)-9b-(1-naphtyl)-2,3- or Y188C mutation showed resistance to α-APA deriva- dihydrothiazolo[2,3-a]isoindol-5(9bH)-one] is a member tives, whereas RT containing a I100L mutation was still of the thiazoloisoindol-5-ones, which has shown an EC50 sensitive to R 89439. A relatively easy chemical synthesis value of 0.046 µM in MT-2 cells, but the compound was of R 89439, good oral availability and favourable pharma- only moderately orally available in rats (Mertens et al., cokinetics made R 89439 (loviride) a valid candidate 1993). Also, the quinoline U-78036 (13) has shown activ- (Pauwels et al. 1993). The α-APA project and further ity against HIV-1 RT (Althaus et al., 1993). A tetrahy- development of loviride has been stopped by Janssen dronaphtalene lignan derivative (14) has shown a 50% Pharmaceuticals. effective inhibition concentration in antiviral assays of 0.15 to 0.8 µM and a selectivity index of 70–400 (Hiroto Other compounds et al.,1997). A lot of compounds and classes of compounds have been found to have activity against HIV-1 RT have been syn- Future perspectives for NNRTIs thesized. Some of these are presented in Figures 18 and 19. Compound 1 was the most active of the 5-H-pyrrolo[1,2- NNRTIs appear to be very promising therapies in the b][1,2,5]benzothiadiazepines (PBTDs) with an EC50 value treatment of HIV, when used in combination with other of 0.5 M and a selectivity index of >600 (Artico et al., anti-HIV drugs such as nucleoside RT inhibitors and pro- 1996). Compound 2, a pyrrolobenzoxazepinone with an tease inhibitors. NNRTIs have the advantage that they are

IC50 value in an enzymatic assay (rC.dG) of 250 nM com- able to cross the blood–brain barrier and in this way can pared to nevirapine (IC50 500 nM), was the most potent have an antiviral effect against HIV-1 infections in reser- NNRTI in a series by Campiani et al. (1996). Further work voirs that are out of reach for many other anti-HIV drugs, with sulphones and NPPS as a lead afforded the pyrryl aryl including most of the nucleoside RT inhibitors. sulphones (PAS). Two of these compounds, 3 and 4, had The first generation of NNRTIs, nevirapine and similar anti-HIV-1 activities with an EC50 of 0.14 µM in delavirdine, have suffered from a rapid development of MT-4 cells and a selectivity index of >2140. This is a high- resistance. To overcome this problem, the NNRTIs have er EC50 value and selectivity index than for zidovudine been used in high doses and in combination with two or (Silvestri et al., 1997). three other anti-HIV drugs in HAART. The problem of With TBZ (5) (Chimirri et al., 1997) as the lead struc- resistant strains of HIV may be reduced with the second ture, a 2-aryl-substituted benzimidazole 6 with an EC50 generation of NNRTIs because RT will need two or more value of 0.20 Μ in an antiviral assay was synthesized (Roth mutations for HIV-1 to be resistant to these drugs. Also, et al., 1997). the uptake of the second generation of NNRTIs in the A sulphone compound (7) was found to inhibit viral blood and cells may be increased and thereby increase the spread in MT-4 cells with a ClC95 of 3 nM (Williams et al., concentration that can effect the inhibition of RT.The per- 1993). spectives of the new generation of NNRTI inhibitors are Derivatives of TSAO-T (8) are also inhibitors of HIV-1 thus promising with an expected slower development of RT and are unique in that they interfere at the interface resistance combined with their ability to cross the between the p51 and p66 subunits of RT. One 1,2,3-triazole- blood–brain barrier. TSAO analogue, compound 9, is reported to have a selectivity index of 1470 in MT-4 cells (Velázquez et al., 1998). References Quinoxaline derivatives have also been an important class of NNRTIs. S-2720 has shown activity in a cell Ahgren C, Backro K, Bell FW, Cantrell AS, Clemens M, Colacino spread assay with a 50% effective concentration in the JM, Deeter JB, Engelhardt JA, Hogberg M, Jaskunas SR, Johansson

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Received 26 March 1999; accepted 20 July 1999