Development of diarylpyrimidine derivatives (& other heterocycles) as HIV-1 and WT RT inhibitors

Abstract

Reverse transcriptase (RT) is an enzyme encoded by the genetic material of retroviruses. Viruses such as HIV and hepatitis B employ an enzyme reverse transcriptase (RT) to generate complementary DNA from the RNA template during reverse transcription. Thus, viruses replicate their genomes and proliferate within the host genome. In particular, researchers are concerned about the pathogenic viruses that cause numerous diseases through this mechanism. The retroviruses that cause diseases in humans include human immunodeficiency virus (HIV), which causes AIDS, and human T-cell lymphotropic virus I (HTLV-1), which causes leukemia. HIV has been the most devastating health problem for decades. The number of recorded HIV cases was found to be approximately 39 million worldwide in 2022. Acquired immune deficiency syndrome (AIDS), most devastating disease caused by HIV-1 needs potent antiretroviral therapy for treatment. Among the effective treatments for AIDS, NNRTIs are key drugs in highly active antiretroviral therapy (HAART). Heterocyclic small molecules play an important role in drug discovery for treatment of HIV-1 infection. Particularly, diarylpyrimidines class of drugs have shown promising activity. In this review, anti-HIV-1 activity and RT inhibitory activity of heterocycle small molecules focusing mostly on diarylpyrimidines was discussed. Furthermore, structure–activity relationship was discussed emphasizing most potent molecules.

Heterocyclic molecules, in particular diarylpyrimidine and diaryltriazines derivatives possessed excellent RT inhibitory and anti-HIV properties. Hence, molecules are suitable for design of potent anti-HIV drug molecules.

1. Introduction

Reverse transcriptases were discovered by Howard Temin in Rous sarcoma virions¹ and independently isolated by David Baltimore from two RNA viruses, murine leukemia virus and Rous sarcoma virus, in 1970.² Viruses such as HIV and hepatitis B employ an enzyme reverse transcriptase (Fig. 1) to generate complementary DNA from the RNA template during reverse transcription. Thus, viruses replicate their genomes and proliferate within the host genome. Eukaryotic cells use RT to extend telomeres to the ends of their linear chromosomes. To date, only a few reverse transcriptases have been well studied, including HIV-1 reverse transcriptase from human immunodeficiency virus type 1,³ M-MLV reverse transcriptase from the Moloney murine leukemia virus, AMV reverse transcriptase from the avian myeloblastosis virus,⁴ and telomerase reverse transcriptase that maintains the telomeres of eukaryotic chromosomes.⁵

Fig. 1. Crystallographic structure of HIV-1 reverse transcriptase, active sites of polymerase and nuclease are highlighted [reproduced with permission from ref. 6].

Viruses encode reverse transcriptase enzymes and use them for reverse transcription as a step in the replication process. HIV infects humans with the use of the RT enzyme, wherein new viruses are generated by coping with its genetic material using the RT enzyme.⁷ Acquired immune deficiency virus (AIDS) has become a serious problem because of its special invasion mechanism and characteristics of easy mutation. As HIV uses RT for replication, it is considered as an important target for HIV therapy.⁸ Hence, inhibition of RT is an efficient approach for HIV treatment. There are two main classes of RT inhibitors: nucleoside reverse transcription inhibitors (NRTIs) and non-nucleoside reverse transcription inhibitors (NNRTIs).^9,10 Heterocyclic small molecules are an important class of molecules in drug design. Both NRTIs and NNRTIs contain an excellent group of molecules for HIV treatment. NNRTIs have received considerable attention because of their unique antiviral properties, low cytotoxicity, and favorable pharmacokinetic properties.¹⁰ NNRTIs disrupt the normal functioning of the HIV-1 RT enzyme by binding to the NNRTI binding pocket. The US Food and Drug Administration approved first-generation NNRTIs such as nevirapine, delavirdine, and efavirenz. Second-generation NNRTIs approved by the FDA include etravirine, rilpivirine and doravirine (Table 1).^11,12 However, adverse side effects, in addition to the high mutation rate of HIV-1 RT, limit the clinical use of NNRTIs.^13–15

Table 1. Structures of FDA-approved NNRTIs [reproduced from ref. 16].

Structure of drug molecule	Type	Structure of drug molecule	Type
Delavirdine	NNRTI	Nevirapine	NNRTI
Rilpivirine	NNRTI	Efavirenz	NNRTI
Doravirine	NNRTI	Etravirine	NNRTI

In this context, research in drug discovery needs more stringency to identify efficient RT inhibitors. Small molecule heterocycles find their way in designing promising RT inhibitors. Recent literature witnesses the role of heterocyclic molecules in discovery of RT inhibitors. In this review, the literature pertaining to inhibition of RTs was systematically reviewed. The review is classified based on type of heterocyclic molecule. Also, emphasis was made regarding most potent RT inhibitors. The review might be helpful in bringing out potent heterocycle-based RT inhibitors.

2. Diarylpyrimidine/pyrimidine derivatives

Among the FDA approved NNRTIs, diarylpyrimidine derivatives are a successful class of anti-HIV drugs.^17,18 Based on previously reported diarylpyrimidine derivatives with NH linker exhibiting inhibitory activity in nanomolar range against WT-HIV RT,¹⁹ X. Chen et al. have come up with design of stereoisomers containing diarylpyrimidine moiety.²⁰ The synthesized compounds were tested for inhibitory activity against HIV-1 (III_B) strains where in most of the compounds rendered potent activity; in particular (S)-isomers of compounds 1 and 2 (Fig. 2) showed most potent activity with EC₅₀ values of 1.6 nM. However, compound 1 was found to possess excellent cytotoxicity in addition to inhibitory activity. Inhibitory activity of synthesized molecules against a panel of clinically relevant HIV-1 mutant strains and WT HIV-1 RT was found to be promising. Again compound 1 among potent inhibitors demonstrated the strongest activity (EC₅₀ = 0.0067–8.48 μM) against panel of HIV-1 mutant strains. In case of inhibitory activity against HIV-1 RT, compound 1 and 2 showed the most potent activity with IC₅₀ values of 5.5 nM and 9.5 nM, respectively. Structure–activity relationship (SAR) indicates that derivatives with electron-donating groups excelled in the inhibitory activity. Especially, (S)-isomers presented superior activity over (R)-isomers. This fact indicates that in addition to general structure of a molecule, its stereochemistry also plays a pivotal role in the inhibitory activity. Besides this, the presence of substituent at 3-position of phenyl ring rendered the strongest activity. Further, at a dosage of 2 g kg⁻¹, the compound 1 was found to be well tolerable, possessing significant cardiovascular safety.

Fig. 2. Structure of potent WT HIV-1 RT inhibitors [reproduced from ref. 20 with permission from Elsevier, copyright 2020].

A small variation in the structural design has been made to diarylpyrimidine moiety to obtain 5-substituted diarylpyrimidine derivatives by P. Gao et al.²¹ The study was inspired by the fact that the introduction of fused-core can fully occupy the tolerant region II and enhance the activity of the compound.^22,23 The designed molecules were subjected to inhibitory activity against clinically resistant WT HIV-1 (III_B) strains where almost all the molecules (except for 3,5-dimethylphenyl and 1-naphthyl substituted derivatives) exhibited potent activity (EC₅₀ = 0.0025–0.93 μM). Among these, compounds 3 and 4 (Fig. 3) demonstrated the strongest activity against HIV-1 induced cytotoxicity with EC₅₀ values of 2.5 nM. In case of inhibitory activity against resistant WT HIV-1 (III_B) strains, compounds 3 and 4 showed activity in the rage of 2.5–41.3 nM and 2.5–112 nM, respectively. However, compound 4 was found to be comparatively more cytotoxic than compound 3. Based on these facts, compound 3 was selected for inhibition against HIV-1 RT enzyme wherein compound 3 presented moderate activity with IC₅₀ value of 0.57 μM. Furthermore, in docking analysis of compound 3, binding mode resembled etravirine (ETV) as a horse shoe conformation; while benzene ring is stretched into tolerable region II site. SAR reveals that introduction of non-benzenoid aromatic rings on to phenyl ring enhanced the activity. In addition, position of linkage of substituent to phenyl ring affects the activity significantly.

Fig. 3. Illustration of 5-phenyl substituted diarylpyrimidine derivatives [reproduced from ref. 21 with permission from Elsevier, copyright 2021].

Replacement of dimethylphenyl moiety with difluorophenyl moiety resulted in enhanced anti-HIV activity. Considering this, L. Ding et al. designed a series of difluoro-biphenyl-diarylpyrimidine derivatives (with alkylation of linker) in view of blocking potential metabolic sensitive sites and maintain the high antiviral potency.²⁴ Initially, N-alkyl-substituted derivatives were screened for anti-HIV-1 (III_B) in MT-4 cells. Most of them rendered potent antiviral activity. Surprisingly, unalkylated derivative 5 (Fig. 4) possessing methyl group at pyrimidine 5-position demonstrated the strongest activity (EC₅₀ = 1 nM) with low cytotoxicity and great selectivity. Excellent inhibitory activity has been exhibited by some of the compounds against a panel of HIV-1 mutant strains. Compound 5 proved to be the most potent molecule with remarkable EC₅₀ value (EC₅₀ = 5.4–16.3 nM) coupled with high selectivity against all strains except Y188L strain. Most potent antiviral molecules were chosen for inhibitory activity against WT HIV-1 RT wherein noteworthy activity was noticed. Among them, compound 5 elicited inhibitory activity (IC₅₀ = 13 nM) very close to reference compounds. Besides, compound 5 was well tolerated at a dose of 2 g kg⁻¹ and exhibited enhanced half-life (t_1/2 = 46.1 min). In brief SAR indicates N-alkylation improves antiviral activity. Having said that, keeping NH-linker unsubstituted but methylation of pyrimidine 5-position showed comparatively better activity.

Fig. 4. Structure of difluoro-biphenyl diarylpyrimidine derivative [reproduced from ref. 24 with permission from American Chemical Society, copyright 2021].

To improve inhibitory activity against K103N and E138K strains, S. Han et al. have come up with molecular hybridization of pyrimidine with benzene to obtain diarylbenzopyrimidine analogs,²⁵ which is an extension of their previous research on diarylbenzopyrimidine derivatives.²⁶ The synthesized molecules exhibited excellent-to-moderate inhibitory activity against HIV-1 III_B and resistant mutant strains. Of those, compound 6 (Fig. 5) exhibited promising HIV-1 III_B activity (EC₅₀ = 10.6 nM) and inhibitory activity against mutant strains E138K (EC₅₀ = 17.7 nM) and K103N (EC₅₀ = 10.2 nM). Some of the evaluated derivatives possessed inhibitory properties better than compound 6. However, compound 6 showed superior drug-likeliness properties; based on drug-likeliness properties, compound 6 was chosen for microsome stability and rat pharmacokinetic (PK) profile wherein moderate clearance and favorable half-lives (t_1/2 = 3.95 at 5 mg kg⁻¹ dosage) were observed. Further optimization of molecules by changing substituent on fused-benzene ring and phenyl ring resulted in a series of derivatives. Evaluation of these molecules showed interesting facts. Irrespective of substitution on phenyl ring, 6-fluoro and 7-chlorobenzopyrimidine derivatives demonstrated potent activity. Further improved activity was observed for derivatives without any substitution on fused-benzene ring. Of these molecules, compound 7 with substituents methyl and nitro moieties elicited the strongest activity. Structure of compound 7 is obtained by removing 6-chloro group on fused benzene ring of compound 6. Inhibitory activity of compound 7 against HIV-1 III_B was found to be 3.4 nM as EC₅₀ value which was better than ETR and close to efavirenz (EFV). In case of E138K and K103N, EC₅₀ values were found to be 4.3 nM and 3.6 nM, respectively. The selected compounds presented excellent inhibitory activity (IC₅₀ = 7–17.7 nM) against WT HIV-1 RT.

Fig. 5. Structure of diaryl benzopyrimidine derivatives with potent anti-HIV activity [reproduced from ref. 25 with permission from American Chemical Society, copyright 2021].

A privileged structure, fused dihydrothiophene-pyrimidine of previously synthesized diaryl-dihydrothiophene-pyrimidine derivatives²⁷ was retained and a series of diaryl-dihydrothiophene-pyrimidine derivatives and other heterocycle-fused pyrimidine derivatives were prepared with various linkers.²⁸ The motive of extension of the research was to design a molecule that could show inhibitory activity against double resistant mutant strains which previously designed molecules could not. Initially, derivatives with various linkers were prepared and evaluated wherein derivatives piperidine linker exhibited potent antiviral activity. Although, various heterocycle-fused pyrimidine derivatives with piperidine linker displayed potent antiviral properties, compounds 8 and 9 (Fig. 6) with fused-dihydrofuran were found to be exceptionally potent inhibitors, eliciting EC₅₀ values of 0.9–7.0 nM against resistant strains and the activity was approximately 1.3 to 3.6-fold higher compared to ETV. In addition, inhibitory activity of compounds 8 (F227L + V106A: EC₅₀ = 19.0 nM) and 9 (F227L + V106A: EC₅₀ = 10.5 nM) against double mutant strain was comparable to ETV. Also, compound 8 exhibited a very low toxicity and high selectivity index. In case of inhibition of WT HIV-1 RT, the best activity (IC₅₀ = 0.051 nM) was noticed. Furthermore, an efficient PK values were showed by compound 8 with good half-life (t_1/2, po = 11.1 h) and better oral bioavailability (F = 30.96%).

Fig. 6. Structure of furo-pyrimidine derivatives as remarkable anti-HIV-1 agents [reproduced from ref. 28 with permission from American Chemical Society, copyright 2019].

Interesting findings encouraged Y. Sun et al. to continue research²⁹ to bring out a more potent and less cytotoxic fused-thiophene-pyrimidine derivatives.³⁰ The aim was to design drug molecules that enhance potency against mutant strains. Among the evaluated molecules against HIV-1 III_B, few analogs exhibited potent activity. In particular, compounds 10 and 11 (Fig. 7) displayed the strongest inhibitory activity with EC₅₀ values of 7 nM and 8 nM, respectively. The activity was found to be superior to that of standard NVP. Alongside this, compound 11 possessed low cytotoxicity (CC₅₀ > 217.5 μM) and high selectivity index. On contrary, most potent antiviral molecules 10 and 11 demonstrated much lower WT HIV-1 RT inhibitory activity with IC₅₀ values of 0.961 μM and 0.299 μM, respectively. Above facts reveal inconsistence between antiviral properties and anti-HIV-1 RT enzyme inhibitory activity. In case of inhibition of a panel HIV-1 mutant strains, most of the evaluated compounds elicited moderate activity. Interestingly the compounds 10 (EC₅₀ = 0.007–0.544 μM) and 11 (EC₅₀ = 0.007–0.877 μM) turned out as the most potent against mutant strains also. While, these compounds showed lower activity against double mutant strains compared to reference. SAR indicates that the derivatives with 1-benzylpiperazine motif presented comparatively potent activity than derivatives with 1-phenylpiperazine and 1-phenylpiperidine-3-amine moieties. In addition, attaching SO₂NH₂ substituent was essential for high antiviral potency.

Fig. 7. Fused-thiophene-pyrimidine analogs with excellent antiviral properties [reproduced from ref. 30 with permission from Elsevier, copyright 2021].

In case of biphenyl-diarylpyrimidine derivatives, change in planarity of molecule might contribute to enhanced antiviral activity. But no such effects were observed with conformational restriction of biphenyl unit on antiviral activity.³¹ In this context, Y. Sang et al. introduced halogens or methyl groups to obtain a series of biphenyl-thiophene-diarylpyrimidine derivatives and to study antiviral properties.³² Moderate-to-remarkable inhibitory activity was exhibited by synthesized molecules. Especially, compounds 12 and 13 (Fig. 8) bestowed excellent inhibitory activity against HIV-1 (III_B) with EC₅₀ values of 14 nM and 17 nM, respectively. Although possessing lower activity, compound 13 rendered low cytotoxicity (CC₅₀ = 264.19 μM) and a very high selectivity index (SI = 18 564). Selected compounds were screened for inhibitory activity against drug-resistant mutant strains wherein compound 12 presented the most potent activity (EC₅₀ = 0.02–0.13 μM); while compound 13 exhibited abated activity. Similarly, compound 13 was found to be slightly inferior to reference with respect to inhibitory activity against WT HIV-1 RT. Whereas, compound 12 witnessed HIV-1 RT inhibitory activity (IC₅₀ = 27 nM) as good as reference compounds. Observation of activity and structure of evaluated molecules reveals that introduction of fluoro groups on central and terminal phenyl rings of biphenyl moiety, slight increase in antiviral activity was evident. Further, replacement of fluoro groups with methyl groups enhanced the activity. Particularly, position of methyl attachment as 2,2 or 2,3 resulted in the strongest activity. As expected, the presence of dimethyl groups might have changed dihedral angle and modulated the conformation of biphenyl moiety so that the molecules possess suitable 3-D orientation to fit into NNRTI's binding pocket.

Fig. 8. Fused-thiophene-pyrimidine derivatives with restricted biphenyl moieties [reproduced from ref. 32 with permission from Elsevier, copyright 2019].

A series of benzopyrimidine derivatives were designed and synthesized by modifying them by introducing a privileged pharmacophore,^33,34 thioacetamide as one of the linkers.³⁵ Also, sulfur-containing compounds were clinically being used against HIV infections.³⁶ Initially synthesized diaryl benzopyrimidine derivatives without thioacetamide linker showed nanomolar anti-HIV properties. Particularly, compound 14 (Fig. 9) exhibited the most potent HIV-1 III_B (EC₅₀ = 0.0127 μM), K103N (EC₅₀ = 0.0104 μM), HIV-1 WT RT inhibitory activity (EC₅₀ = 0.55 μM). While cytotoxicity and selectivity indices were not favorable. Insertion of thioacetamide linker resulted in another series of molecules with lower anti-HIV properties. However, further optimization of thioacetamide series by oxidation of sulfur to sulfone had positive impact on antiviral activity. Among various molecules designed, compound 15 embarked with strongest HIV-1 III_B inhibitory activity (EC₅₀ = 0.0249 μM), mutant strain, K103N (EC₅₀ = 0.0104 μM), HIV-1 WT RT inhibitory activity (EC₅₀ = 0.32 μM). Gratifyingly, compound 15 showed low cytotoxicity (CC₅₀ > 221 μM) and the best selectivity indices. Replacement of –CN group on phenyl ring attached to acetamide linker with other electron-withdrawing groups or replacement of dimethyl groups on other phenyl ring resulted in decreased anti-HIV properties.

Fig. 9. Diaryl benzopyrimidine derivatives possessing noteworthy anti-HIV-1 activity [reproduced from ref. 35 with permission from Elsevier, copyright 2019].

Diarylpyrimidine derivatives with sulfonylacetanilide linker were reported to be potent anti-HIV-1 agents.³⁷ Presence of sulfonylacetanilide moiety has been shown to exhibit hydrogen bonding with main chain of residues K101 and K103.³⁸ Considering these, Y. Sang et al. envisaged synthesis of diarylpyrimidine derivatives with modified sulfonylacetamide linker.³⁹ After structural optimization, synthesized molecules were tested for HIV-1 (WT) wherein compounds 16 and 17 (Fig. 10) elicited the most potent activity with EC₅₀ values of 7 nM and 6 nM, respectively. Additionally, a very high selectivity indices with low cytotoxicity were noticed. Furthermore, a remarkable inhibitory profile (EC₅₀ = 0.006–0.026 μM) was observed against a panel of HIV-1 single mutant strains. In case of the mutant strains, K103N (EC₅₀ = 0.006 μM) and E138K (EC₅₀ = 0.026 μM), noteworthy inhibitory activities were reported. Also, the compound 17 showed potent inhibitory activity against double mutant strains. Unfortunately, compound 16 exhibited reduced potency against mutant strains. In consistent with the strong inhibitory values of compound 17 against mutant strains, low HIV-1 RT inhibitory activity (IC₅₀ = 0.142 μM) was observed. It was evident from SAR that regardless of substitution on phenyl ring, introduction of alkyl groups in between sulfonylacetamide linker and aryl group resulted in reduced activity. This confirms that sulfonylacetanilide is optimized linker for the better activity. Retaining sulfonylacetanilide structure, joining a methyl group to acetamide methylene group decreased the activity.

Fig. 10. Structure of diarylpyrimidines derivative with sulfonylacetanilide linker [reproduced from ref. 39 with permission from Elsevier, copyright 2020].

As piperidine-linked aminopyrimidine derivatives could improve water solubility and bioavailability,^40,41 indazole was connected to piperidine to obtain a series of indazolyl-substituted piperidin-4-yl-aminopyrimidines.⁴² The hybrid molecules were tested for inhibitory potency against HIV-1 III_B wherein most of the compounds displayed moderate activity; while compounds 18 and 19 (Fig. 11) expressed the strongest activity with EC₅₀ values of 8.6 nM and 6.4 nM, respectively. Further, against mutant strains, excellent inhibitory potencies were noticed for compounds 18 and 19. In particular, compound 19 demonstrated the best activity against K103N (EC₅₀ = 77 nM) and E138K (EC₅₀ = 57 nM) with high selectivity index of 2500. However, inhibitory properties of compounds 18 and 19 were found to be inferior compared to reference, ETR. Unfortunately, almost all hybrid molecules rendered low inhibitory potency against double mutant strains. Besides this, in molecular modeling analysis of compound 19 with crystal structure of WT HIV-1 RT, favorable interactions with various amino acid residues were noticed that include a key hydrogen bond between NH hydrogen and Lys101, indazolyl NH and main chain of Lys103, and extensive interactions with Trp229, Thr181 and Phe227. SAR reveals that derivative featuring 2,6-dimethyl-4-cyanophenyl wing showed strong potency, while removal of 4-CN group could not reduce the potency much. However, the change in position of dimethyl groups rendered abated anti-HIV-1 activity.

Fig. 11. Structure of indazole-appended piperidine containing diarylpyrimidines [reproduced from ref. 42 with permission from Elsevier, copyright 2019].

In addition to diary moieties as appendants/chemotypes for pyrimidine, cyclohexyl group was classified as chemotype, subtype 4 that witnessed excellent anti-HIV-1 properties. Derivatives with cyclohexylmethyl moiety conferred high biochemical inhibition against HIV-1 RT polymerase and RNase H.⁴³ Triggered by above facts, Tang J. et al. designed and prepared a series of cyclohexylmethyl-hydroxypyrimidine-one derivatives.⁴⁴ Initially, synthesized molecules were screened for inhibition of RT-associated RNase H and RT polymerase. 2-Fluoro-3-chlorobenzyl substituted derivative 20 (Fig. 12) demonstrated most potent RNase inhibitory activity with IC₅₀ value of 1.1 μM. While inhibition of RT polymerase by compound 20 was not significant. Removal of other series of molecules devoid of N-hydroxy group of pyrimidinone ring resulted in poor RNase inhibitory activity, but enhanced RT polymerase inhibitory activity and nanomolar inhibition of virus replication. Among those, isopropyl-pyrimidinone analog 21 presented remarkable RT polymerase inhibitory activity (IC₅₀ = 0.085 μM). With the IC₅₀ value of 5.2 nM, compound 21 displayed excellent MAGI antiviral activity. While derivatives with substitution on benzyl ring turned out to have comparatively lower inhibitory activity.

Fig. 12. Cyclohexylmethyl-substituted pyrimidinone analog with excellent anti-HIV-1 activity [reproduced from ref. 44 with permission from Elsevier, copyright 2017].

Literature witnesses that dihydro-alkyl-oxy-benzyl-oxo-pyrimidines (S-DABOs) possess potency against cross-resistance.^45,46 Pyrazole-containing S-DABOs exhibited strong WT HIV-1 inhibitory activity.⁴⁷ Considering above facts, Y. M. Li et al. designed and synthesized a series of 2-mercaptopyrimidinones⁴⁸ introducing 1,3-benzodioxole moiety. Inhibitory activity of synthesized molecules against HIV-1 reveals moderate-to-potent activity. Especially, compound 22 (Fig. 13) bestowed the strongest activity (IC₅₀ = 0.06 μM) with low cytotoxicity (CC₅₀ = 96.23 μM). Among the potent compounds screened for inhibitory activity against HIV-1 resistant strains, again compound 22 turned out to be an excellent inhibitor (IC₅₀ = 0.445–0.645 μM). While, poor inhibitory potency was observed against two strains, HIV-1_A17 and HIV-1_K103N. Furthermore, the selected compounds demonstrated remarkable inhibitory against HIV-1 clinical isolates. The inhibitory activity of the compounds was far better than reference ETR. Especially, compound 22 displayed extraordinary inhibitory potency (IC₅₀ = 0.064–0.103 μM). Additionally, compound 22 expressed noteworthy inhibitory activity against RT RNA dependent DNA polymerase (IC₅₀ = 0.518 μM) and RT DNA dependent DNA polymerase (IC₅₀ = 0.072 μM). The presence of pyrazole resulted in moderate anti-HIV-1 activity.

Fig. 13. Structure of 1,3-benzodioxole-containing pyrimidinone derivative [reproduced from ref. 48 with permission from Elsevier, copyright 2020].

Dihydropyrimidinone moiety was hybridized with pharmacologically active group isatin to get dihydropyrimidinone-isatin hybrid molecules.⁴⁹ Isatin has been reported to possess good activity against RT enzyme as well as other targets.⁵⁰ All the synthesized hybrid molecules were tested for RT inhibition wherein compound 23 and 24 (Fig. 14) elicited the strongest inhibitory activity with IC₅₀ values of 64 μM and 67 μM, respectively. While, corresponding % RT inhibition of compounds 23 and 24 were found to be 95.96 and 94.63, respectively. RT inhibitory properties of compounds 23 and 24 were stronger than that of rilpivirine. Isatin derivatives without substitution at pyrimidinone 4-position possessed low inhibitory activity. Also, substituents larger than ethyl group showed diminished activity. In addition, derivatives with triazoleamine at isatin 3-position exhibited deteriorated activity.

Fig. 14. Structure of isatin-appended pyrimidinone analog with RT inhibitory activity [reproduced from ref. 49 with permission from Elsevier, copyright 2017].

In continuation of their previous work on oxathiadiazole derivatives as NNRTIs,^51,52 V. K. Singh et al. synthesized a series of diarylpyrimidine derivatives performed TOPKAT analysis and molecular dynamics simulations.⁵³ Drug-likeliness of the most the designed compounds were found within the range of Lipinski's rule and expected to behave as NNRTIs against WT HIV-1. Molecular modeling studies of compounds reveal H-bond interactions with Lys103 in the NNRTI binding pocket. Introduction of nitro groups at position-5 of both aryl rings (compound 25, Fig. 15) led to formation of stabilized complex of the compound 25 with HIV RT by having H-bond interaction with amino acids His235 and Trp229. Furthermore, piecewise linear potential (PLP) functions of synthesized compounds with target protein, HIV RT revealed higher PLP scores and stronger binding; particularly, the compound 25 showed the highest score, 109.74. Also, favorable ligand internal energy and significant dock scores were evident for the designed molecules. TOPKAT analysis indicated that the in silico toxicity values such as DTP (development toxicity potential), LD₅₀, EC₅₀, of all compounds were found to be less than reference drug ETV. All the above studies confirm that the compound 25 may be a potent NNRTI.

Fig. 15. Structure of diarylpyrimidine derivative with potent in silico NNRTI activity [reproduced from ref. 53 with permission from Taylor & Francis, copyright 2020].

3. Indole derivatives

P. Gao et al. envisaged design and synthesis of a series of indolylarylsulfone derivatives,⁵⁴ considering the fact that indolylarylsulfone derivatives form a potent class of NNRTIs⁵⁵ and reported to exhibit inhibitory activity against WT HIV in nanomolar range.⁵⁶ The synthesized molecules were screened for inhibition of WT HIV-1 RT wherein most the compounds showed only mild activity. However, a few molecules rendered significant activity, notably, compound 26 (Fig. 16) embarked the strongest activity (IC₅₀ = 9.74 μM). In case of mutant strain Y181C, good inhibitory activity (IC₅₀ = 2.36 μM) with mild cytotoxicity (CC₅₀ = 20 μM) were exhibited by the compound 26. Additionally, selected compounds were evaluated for WT HIV RT in nucleotide incorporation assay wherein poor inhibitory activity was noticed against WT BH10 strain; while mild activity (IC₅₀ = 18.2 μM) was observed against Y181C RT. SAR reveals that indolyl derivatives possessing acrylamide sulfonamide groups showed comparatively higher anti-HIV-1 activity than that of ethylene warheads. Furthermore, cytotoxicity was reduced for derivatives with six-member ring-substitution.

Fig. 16. Structure of indolylsulfonamide with acrylsulfonamide group [reproduced from ref. 54 with permission from Elsevier, copyright 2020].

As indole-2-carboxamide derivatives endowed with remarkable anti-HIV-1 activity, and positive results obtained through chemical modifications at indole-2-position,^57,58 T. Zhao et al. prepared a series of hybrid molecules based on 5-chloroindole, 3-arylsufonyl and 2-carboxamide groups.⁵⁹ Moderate-to-extraordinary HIV-1 III_B inhibitory activity was witnessed by evaluated molecules. Of those, compounds 27–29 (Fig. 17) demonstrated explicit inhibitory activity with EC₅₀ values of 0.0047 μM, 0.0043 μM, and 0.0098 μM, respectively. Whereas, only the compound 27 could exhibit an excellent inhibitory potency (IC₅₀ = 7.6 μM) against RES056 strain. All the three compounds showed low cytotoxicity. Inhibitory potency of both the compounds 27 and 28 were better than reference drugs, ETR and EFV. In addition, few compounds were chosen for inhibition of HIV-1 mutant strains, wherein compound 29 turned out to be the most potent molecule (EC₅₀ = 0.011–0.31 μM). Additionally, compound 29 was also a remarkable inhibitor (EC₅₀ = 0.094 μM) of double mutant strain, F227L + V106A. In case of inhibition of HIV-1 WT RT, the compound 28 elicited noteworthy activity (IC₅₀ = 0.61 μM). Brief SAR analysis reveals that regardless of size of cyclic secondary amine appended to carboxamide fragment, connecting small groups such as methy sulfonyl or acetamidyl groups resulted in the most potent activity. In most of the cases, absence of cyclic secondary amine paved to low activity. Also, stereochemistry at carboxyamide N-atom did not play a pivotal role in inhibitory activity.

Fig. 17. 5-Chloroindole-carboxamide derivatives with nanomolar anti-HIV-1 activity [reproduced from ref. 59 with permission from Elsevier, copyright 2019].

Similar research was carried out by researcher X. Li et al. on anti-HIV-1 activity of indolylarylsulfones wherein indole 5-position was substituted with either Br or Cl and carboxamide fragment was appended to N-substituted alkylpiperidine moiety.⁶⁰ Almost all synthesized molecules presented moderate-to-excellent inhibitory activity against HIV-1 III_B strain. Among the potent inhibitors, compounds 30 and 31 (Fig. 18) displayed the most potent activity with EC₅₀ values of 6 nM and 9 nM, respectively. In case of inhibition of mutant strains (expect Y188L), the compound 30 demonstrated a good inhibitory profile (EC₅₀ = 0.017–0.22 μM). While compound 31 could show slightly reduced activity profile (EC₅₀ = 0.043–0.27 μM). However, among the selected compounds for HIV-1 RT inhibition, compound 31 was limited to moderate activity (IC₅₀ = 7.0 μM). When it comes to SAR, halogen substitution at 5-position and methylene group between carboxamide group and piperidine ring did not affect the anti-HIV-1 activity significantly. However, connecting substituted (such as 4-Cl, 3-Cl, 4-CF₃, 4-NO₂, 3-F, and 3-CN) phenyl moieties to carboxamide group led to low potency.

Fig. 18. Piperidine-containing arylindolylsulfone derivatives [reproduced from ref. 60 with permission from Elsevier, copyright 2016].

In view of promising pharmacological activity phosphorylated indoles,^61,62 for instance, phosphoindole derivative, fosdevirine is considered as HIV NNRTI.⁶³ In addition to these facts, considering replacement of carboxamide at indole 2-position could improve anti-HIV-1 activity,⁶⁴ El-Hussieny et al. designed and synthesized a series of thiophenyl-indole hybrids.⁶⁵ Evaluation of synthesized compounds against HIV-1 RT reveals the most promising inhibitory activity (IC₅₀ = 2.93–24.90 nM). Most of the compounds were found to be as good as efavirenz. Of those, the strongest activity has been exhibited by compounds 32 and 33 (Fig. 19) with IC₅₀ values of 2.93 nM and 3.54 nM, respectively. As of SAR, replacement of formyl group with thione group led to threefold decreased activity. While replacement of formyl group with methyl acrylate increased the activity significantly. Especially E-isomer (compound 32) showed approximately twofold higher activity compared to its Z-isomer. Similarly, in case of other derivatives also, E-isomers exhibited superior activity over Z-isomers.

Fig. 19. Thiophene-containing indole derivative with the promising HIV-1 RT inhibitory activity [reproduced from ref. 65 with permission from Elsevier, copyright 2019].

4. Miscellaneous heterocycle derivatives

Quinoline is a part of most of the nitrogen-containing pharmaceutically active molecules.^66,67 Hence, Makarasen et al. have come up with design and synthesis of amino-oxy-diarylquinoline derivatives as potent NNRTIs.⁶⁸ The synthesized molecules were evaluated for percentage inhibitory activity against HIV-1 RT wherein moderate inhibitory percentages were noticed. Among the evaluated molecules, compound 34 and 35 (Fig. 20) turned out to be potent molecules with inhibitory percentages, 34.02 and 39.71, respectively. Molecular modeling studies of the compounds 34 and 35 with HIV-1 RT demonstrates good binding energies of −13.18 kcal mol⁻¹ and −12.56 kcal mol⁻¹, respectively. Additionally, analyzed molecules bound to HIV-1 RT with three hydrogen bonds to amino acid residues Lys101, and His235.

Fig. 20. Diarylquinoline derivatives possessing good HIV-1 RT inhibitory percentages [reproduced from ref. 68 with permission from Thieme, copyright 2019].

Studies indicate that chloroaquiline moiety conjugated with ribonucleoside displayed potent HIV-1 replication inhibitory activity.^69,70 Furthermore, 4-oxoquinoline moiety turned out to be a lead compound with low toxicity by bioisosteric substitution of the carboxylic acid by an amide group.⁷¹ Based on these, da S. M. Forezi et al. envisaged design and synthesis of 4-oxoquinoline ribonucleoside derivatives.⁷² Potent-to-mild HIV-1 RT inhibitory effects were noticed for the evaluated molecules. Among those, few derivatives expressed remarkable activity; particularly, compounds 36 and 37 (Fig. 21) elicited the strongest inhibitory activity with IC₅₀ value of 1.4 μM and 1.6 μM, respectively. These compounds 36 and 37 exhibited very low cytotoxicity values of 1486 μM and 1394 μM, respectively as CC₅₀ values. SAR analysis indicates benzoylated ribonucleoside derivatives presented enhanced activity compared to non-benzoylated analogs. Among non-benzoylated derivatives, replacement of aryl moiety with cyclohexyl ring rendered poor activity. In case of benzoylated analogs, derivatives without substitution/CH₃ substitution at quinoline 6-position exhibited the strongest activity. While, the presence of electron-withdrawing groups such as Cl or CF₃ displayed lowered activity. Molecular modeling studies reveal that the potent compound 36 bound to the allosteric site of the HIV-1 RT through favorable interactions with Try181, Leu100, Phe227, and Trp229. Additionally, compound 36 formed van der Waals interactions and hydrophobic interactions with important amino acid residues.

Fig. 21. Structure of quinolinone-ribonucleoside derivatives with HIV-1 RT inhibitory activity [reproduced from ref. 72 with permission from Elsevier, copyright 2020].

Structures of FDA approved NNRTIs reveal that cyclopropylacetylene and trifluoromethyl groups are pivotal pharmacophores in EFV, while 4-cyano aniline group is essential for pharmacological activities of ETV and RPV.^73,74 Hence, K. Jin et al. made use of molecular hybridization using structural features of FDA-approved molecules and synthesized a series of dihydroquinazoline derivatives.⁷⁵ Of the hybrid molecules designed, few molecules could exhibit excellent inhibitory potency against HIV-1 III_B. Among those, compound 38 (Fig. 22) witnessed extraordinary inhibitory activity (EC₅₀ = 0.84 nM). The inhibitory activity of compound 38 was found to be twofold stronger than reference drug EFV. Also, noteworthy inhibitory activity was observed against mutant strains E138K and RES056 with EC₅₀ values of 3.5 nM and 0.066 μM, respectively. Again, inhibitory activity against E138K was approximately twofold stronger compared to ETV. Furthermore, among compounds chosen for inhibitory activity against WT HIV-1 RT, the strongest activity (IC₅₀ = 1.1 nM) was observed for the compound 39, better than both NVP and EFV. On contrary, compound 38 exhibited slightly lowest activity (IC₅₀ = 0.01 nM) among the evaluated molecules. SAR of evaluated molecules implies that the presence of nitrile group at 4-positin of phenyl ring resulted in superior activity compared to derivatives with either Br, or CF₃. Molecular modeling analysis reveals that van der Waals interaction with residues E138 and V179.

Fig. 22. Quinazoline derivative with extraordinary anti-HIV-1 activity [reproduced from ref. 75 with permission from Elsevier, copyright 2019].

Diaryl-thiazolidinone containing heteroaryl substituents demonstrated anti-HIV activity and are considered as potent NNRTIs.^76,77 Also, considering N-(hetero)arylacetamide tail as a promising moiety in some NNRTIs,⁷⁸ Buemi et al. envisaged design and synthesis of a series of thiadiazole-thiadiazolidinone derivatives.⁷⁹ Newly synthesized molecules failed to render potent anti-HIV-1 activity. Moderate-to-mild inhibitory activity was observed for the evaluated molecules when compared to reference drug NVP. In the assessment of anti-HIV-1 inhibitory activity, poor activity was noticed. While, in case of HIV-1 RT, moderate activity has exhibited by a few synthesized molecules. Out of those, compound 40 (Fig. 23) expressed the highest activity (IC₅₀ = 11.83 μM). SAR suggests that the connecting 2-Cl, or 3-Cl phenyl moieties to thiazolidinone ring showed enhanced activity. Furthermore, docking analysis of compound 40 revealed interaction with residues of Tyr181, Tyr188 and Trp229 of hydrophobic pocket. Additionally, a hydrogen bond was established with Lys103.

Fig. 23. Structure of thiazole-thiazolidinone derivative with moderate anti-HIV-1 activity [reproduced from ref. 79 with permission from Elsevier, copyright 2020].

Heterobicyclic compounds possessing thiazole and pyrazole are an important class of heterocycles.^80,81 For instance, thiazole derivatives display a wide range of antiviral properties.⁸² Inspired by these facts, Kasralikar et al. planned to design and synthesize pyrazolothiazole derivatives.⁸³ Initially, the synthesized molecules were analyzed by molecular modeling studies wherein few molecules showed favorable interactions that were screened for anti-HIV-1 inhibitory activity. Of those, compounds 41 and 42 (Fig. 24) turned out to be the strongest inhibitors of HIV-1 III_B (41: EC₅₀ = 0.74 μg ml⁻¹; 42: EC₅₀ = 1.2 μg ml⁻¹) and ADA5 (41: EC₅₀ = 1.08 μg ml⁻¹; 42: EC₅₀ = 0.34 μg ml⁻¹). Both the compounds 41 and 42 exhibited low cytotoxicity values. Besides this, selected compounds were tested for inhibitory activity against percentage inhibition of HIV-RT wherein compounds 41 (90.57%) and 42 (89.80%) displayed almost similar inhibitory activity exhibited by NVP. Docking studies of potent molecules described π–π interaction in to hydrophobic binding pocket with aromatic side chain of Trp229 residue in addition to existence of π-cation interaction with Lys101 residue. These interactions were found to be responsible for enhanced potency against RT. Brief SAR suggests that combination of either electron-donating group-substituted phenyl rings or electron-withdrawing group-substituted phenyl rings elicited higher activity compared to derivatives containing mixed substituents.

Fig. 24. Structure of pyrazolothiazoles as potent anti-HIV-1 agents [reproduced from ref. 83 with permission from Elsevier, copyright 2019].

Fused pyrazolo-pyridine derivatives were designed and synthesized by Sanjay Kumar et al.,⁸⁴ inspired by anti-HIV-1 activity of previously reported fused pyrazolo-pyridine analogs.^85,86 Drug-likeliness of synthesized molecules was assessed by using in silico QED and ADMET approach. In this assessment, all the compounds displayed QED value greater than 0.5 indicating drug-likeliness of designed molecules. ADMET analysis revealed the compounds that cross blood brain barrier (BBB) are permeable for Caco-2 cells. Besides, the designed compounds were found to be substrate for CYP450 3A4 and are also found to be noncarcinogenic and nonmutagenic. Then, the compounds were investigated for inhibitory activity against HIV-1 strains wherein some of the compounds showed poor activity. While, a few molecules expressed moderate-to-mild activity; in particular compound 43 (Fig. 25) demonstrated the highest activity against HIV-1_VB59 (IC₅₀ = 3.67 μM) and HIV-1_UG070 (IC₅₀ = 2.79 μM) strains. Additionally, potent inhibitory activity was displayed by compound 43 against HIV-1 primary isolates, HIV-1_92/BR/018 (IC₅₀ = 7.42 μM), HIV-1_NARI-DR (IC₅₀ = 2.53 μM), and HIV-1_N119 (IC₅₀ = 3.24 μM). Few compounds were chosen for inhibition of HIV-1 RT wherein moderate activity (IC₅₀ = 30.80 μM) was presented by compound 43. SAR of evaluated molecules infers that the derivatives possessing phenyl rings substituted with electron-withdrawing groups resulted in deteriorated activity. While, the presence of methyl groups, particularly at 3- and 5-positions led to the highest activity. Furthermore, molecular modeling studies indicate interaction of the compound 43 occupies hydrophobic pocket containing residues such as Tyr181, Tyr188, Phe227, and Trp229. Additional interaction was noticed between the pyridinone ring and residues Y181 and Y188.

Fig. 25. Structure of pyrazolo-pyridine with moderate anti-HIV-1 activity [reproduced from ref. 84 with permission from Elsevier, copyright 2019].

Diaryltriazines (DATAs) and DAPYs share structural similarities.^87,88 Hence, DATAs may be a class of an efficient NNRTIs. Also, DATAs exhibit seahorse conformation as a binding conformation within allosteric non-nucleosidic binding pocket.⁸⁹ Triggered by these observations, K. Jin et al. designed biphenyl-substituted diaryltriazines.⁹⁰ Against WT HIV-1 III_B strain, excellent-to-moderate activity has been exhibited by the synthesized molecules. Approximately, half of the evaluated derivatives demonstrated remarkable activity. Of those, compounds 44 and 45 (Fig. 26) showed the promising inhibitory activity with IC₅₀ values of 3.4 nmol L⁻¹ and 2.6 nmol L⁻¹, respectively. The compound 45 was found to be as good as reference drugs, ETR and ETV. Few potent molecules were chosen for inhibition of mutant strains of HIV-1.

Fig. 26. Structure of diaryltriazine derivatives with promising anti-HIV-1 activity.⁹⁰.

In consistent with HIV-1 III_B inhibitory activity, both the compounds 44 (EC₅₀ = 3.6–23 nmol L⁻¹) and 45 (EC₅₀ = 2.3– 10 nmol L⁻¹) turned out to be the strongest inhibitors of single mutant strains. Besides this, the compound 44 was also an outstanding inhibitor of double mutant strains,RES056 (EC₅₀ = 34 nmol L⁻¹) and F227L + V106A (EC₅₀ = 17 nmol L⁻¹). While, compound 45 displayed good potency against RES056 (EC₅₀ = 60 nmol L⁻¹) but mild potency against F227L + V106A. On contrary, the strongest WT HIV-1 RT inhibitory was exhibited by the compounds 46 (EC₅₀ = 0.035 μg mL⁻¹) and 47 (EC₅₀ = 0.033 μg mL⁻¹). While, the compounds 44 and 45 elicited reduced inhibitory activity. SAR reveals that derivatives with NH-linker rendered comparatively stronger activity than derivatives with O-linker. Whereas, compounds with cyclic secondary amine, especially, hydroxypyrrolidine-containing derivatives excelled in anti-HIV-1 activity.

Discussion and conclusion

A retrovirus, HIV-1 is one of the causative strains responsible for AIDS in humans.⁹¹ AIDS is condition of progressive failure of immune system that allows life-threatening opportunistic infections and cancer to occur.⁹² Reverse transcriptase is the enzyme used by HIV-1 for its replication process in host cells.⁹³ Hence, inhibition of RT is one of the efficient approaches of HIV-1 control. Furthermore, HIV-1 has undergone mutation giving rise to various single mutant strains such as Y181C, L1001, K103N, Y188L, E138K and double mutant strains, RES056 and F227L + V106A. Research has been going on in design and development of medicinal drugs for treatment of HIV-1 and its mutant strains. In this arena, small molecule heterocycles play a pivotal role in inhibition of HIV-1 strains. Among the various heterocycles, diarylpyrimidines were found to be the most promising scaffolds with the remarkable anti-HIV-1 activity. Hence, a comprehensive review was planned about inhibitory activity of diarylpyrimidines in addition to some other important heterocyclic molecules. Besides, describing anti-HIV-1 activity of most potent molecules, structure–activity relationship was established among a set of molecules. In case of diarylpyrimidine derivatives, the presence of 4-cyanophenylamine moiety on pyrimidine ring along with substituted benzeneamine ring or a substituted biphenyl amine or substituted 4-acrylnitrile benzeneamine moieties resulted in the promising anti-HIV-1 activity. While, slight reduction in the activity was evident for the benzene ring-fused pyrimidine derivatives. Whereas, tetrahydrofuran-fused pyrimidine connecting to piperidine amine linker rendered increased activity. Furthermore, almost similar inhibitory potency was noticed for thiophene-fused pyrimidine derivatives. Using sulfonylacetamide as linker in pyrimidine derivatives bestowed the finest activity. Insertion of indazolyl group could not improve the activity significantly. Replacement of aryl group with a cyclohexyl methyl group might not be beneficial as this process exhibited decreased activity. Monoaryl pyrimidine derivatives were not found to be as good as diarylpyrimidine analogs.

Among other heterocyclic derivatives, simple diarylquinolines which are quinoline analogs of diarylpyrimidines demonstrated reduced activity. Whereas, quinazoline-containing scaffolds possessing arylamine and cyclopropylacetylene displayed extraordinary anti-HIV-1 properties. Derivatives containing thiazole or thiazolidinone or pyrazolo-thiazole rings did not fetch significant inhibitory properties. Besides, structural analogs of diaryl pyrimidines, diaryl triazine derivatives were turned out to be successful NNRTIs with almost similar anti-HIV-1 activity compared to DAPYs. There are certain limitations in this review. Cytotoxicity of the prepared molecules is very essential in deciding efficacy of therapeutic activity. However, the complete cytotoxic activity was not provided in the manuscript because of unavailability of the data for some research articles. In addition, complete molecular docking studies was not included in the manuscript because of lack of relevant research data.

Future perspectives

Based on structure–activity relationship of the most potent molecules, it is possible to design a template molecule that would possibly demonstrate the strongest anti-HIV-1 activity with low cytotoxicity, and excellent pharmacokinetics. The core pharmacophore may be pyrimidine or a triazine that should be appended with a substituted 4-cyanobyphenyl moiety and/4-cyanophenyl moiety. In addition, insertion of sulphonylacetamide linker may enhance the activity. Researchers should focus on the above-mentioned structural features in order to design an efficient NNTRI. Also, AI could also contribute much to drug design and discovery of RT inhibitors. Researchers should focus on potential ability of AI in drug design of RT inhibitors.

Data availability

No primary research results, software or code have been included and no new data were generated or analyzed as part of this review.

Conflicts of interest

There is no conflict of interest.

Biography

Atukuri Dorababu.

Dr. Atukuri Dorababu obtained his M.Sc (2010) and Ph.D (2017) in Chemistry from Karnatak University, Dharwad. He worked as Research Associate in Syngene, Bengaluru for a short period, later, he worked as a Lecturer for five years (2013–17) in Govt. Pre-University College, Belgaum. Meanwhile, he has been appointed as Assistant Professor in SRMPP GFGC, Huvinahadagali in 2017 and is presently working there in the same position. He has around 36 publications in various reputed international journals. His research focused on drug discovery, antimicrobial activity, anticancer activity, natural product extraction and their pharmacological activity.