Design of Biphenyl-Substituted Diarylpyrimidines with a Cyanomethyl Linker as HIV-1 NNRTIs via a Molecular Hybridization Strategy.

The key problems of human immunodeficiency virus (HIV) therapy are the rapid emergence of drug-resistant mutant strains and significant cumulative drug toxicities. Therefore, there is an urgent demand for new anti-HIV agents with low toxicity and broad-spectrum antiviral potency. A series of biphenyl-substituted diarylpyrimidines with a cyanomethyl linker were designed using a molecular hybridization strategy. The cell-based anti-HIV assay showed that most of the compounds exhibited moderate to good activities against wild-type HIV-1 and clinically relevant mutant strains with a more favorable toxicity, and the enzymatic assay showed they had nanomolar activity against reverse transcriptase (RT). Compound 10p exhibited the best activity against wild-type HIV-1 with an EC50 (50% HIV-1 replication inhibitory concentration) value of 0.027 µM, an acceptable CC50 (50% cytotoxic concentration) value of 36.4 µM, and selectivity index of 1361, with moderate activities against the single mutants (EC50: E138K, 0.17 µM; Y181C, 0.87 µM; K103N, 0.9 µM; L100I, 1.21 µM, respectively), and an IC50 value of 0.059 µM against the RT enzyme, which was six-fold higher than nevirapine (NVP). The preliminary structure-activity relationship (SAR) of these new compounds was concluded. The molecular modeling predicted the binding modes of the new compounds with RT, providing molecular insight for further drug design.


Introduction
Acquired immune deficiency syndrome (AIDS) is a serious public health problem worldwide caused by the human immunodeficiency virus (HIV) [1,2]. By the end of 2018, there were approximately 37.9 million people living with HIV, which poses a growing threat to the development of mankind [3]. Currently, there are over 30 drugs approved by the U.S. Food and Drug Administration (USA FDA) for AIDS treatment, targeting different steps of the HIV life cycle [4,5]. With the disclosure of highly active antiretroviral therapy (HAART) as the recommended treatment standard for HIV infection, the morbidity and mortality of HIV-infected patients were significantly reduced [6,7].
The nonnucleoside reverse transcriptase inhibitor (NNRTI), as the vital component of the HAART regimen, inhibits the transformation of the single-stranded viral RNA to double-stranded DNA (dsDNA). It binds to an allosteric hydrophobic pocket, also named the NNRTI-binding pocket (NNIBP), located 10 Å from the DNA catalytic active site of reverse transcriptase (RT). With the efforts of the past decades, six NNRTIs were approved by the FDA (Figure 1) [8]. Nevirapine (NVP), delavirdine (DLV), and efavirenz (EFV), the first-generation NNRTIs, bind to NNIBP in a rigid "butterfly-like" binding mode, commonly less active toward the mutant strains [9]. The second-generation NNRTIs, etravirine (ETR) and rilpivirine (RPV), bearing a diarylpyrimidine (DAPY) skeleton, typically have a U or horseshoe shape with torsional ("wiggling") and repositioning flexibility ("jiggling"), leading to their higher potency against wild-type (WT) and drug-resistant HIV infections [10]. However, adverse events, such as rash and central nervous system (CNS) disturbances and hypersensitivity reactions, were reported in the clinical utility of ETR [11,12]. For, RPV it was reported that a number of drug-resistant mutants lead to virologic failure, and the failure rate is even higher than ETR [13][14][15]. In addition, both ETR and RPV show high cytotoxicity (CC 50~6 µM), which limits long-term therapy of AIDS patients. For these reasons, the efficacy of DAPYs was compromised and the clinic therapeutic options were limited. Therefore, there is an urgent demand for new DAPYs with low toxicity and broad-spectrum antiviral potency.
The CN unit, a bioisostere of various functional groups including carbonyl, halogen, and hydroxyl groups, is widely used in drug discovery, especially in anti-HIV drugs, such as ETR, RPV, DOR, lersivine, dapivirine, and MV-150 [16]. The CN group with a short polarized linear triple bond can stretch into narrow clefts of drug targets, forming hydrogen bonding interactions, polar interactions, covalent interactions, or hydrophobic interactions with amino acids [16][17][18]. Over the past 20 years, our research group made considerable efforts to develop a variety of new DAPY analogues [19][20][21][22]. Incorporating a CN linkage between ring A and the central pyrimidine ring B was determined to be an effective strategy for developing novel DAPY analogues with promising antiviral potency. For instance, compound 1 was a new CN-DAPY with promising anti-HIV potency and selectivity (EC 50 = 1.8 nM, SI > 118595) ( Figure 2) [23]. The biphenyl-DAPYs were developed by our group with low nanomolar EC 50 values against the WT HIV-1 strain and several clinically resistant strains [24][25][26][27][28].
Compound 2 [29], a representative of biphenyl-DAPYs, showed excellent activities against WT HIV-1 (EC 50 = 1.0 ± 0.6 nM) and nanomolar EC 50 values toward several clinically important resistant mutant strains [27]. In the binding with RT, the biphenyl group was attributed to enhancing the π-π interactions with the hydrophobic cavity consisting of Y181, Y188, W229, and F227, and to stabilizing the "U" conformation with the RT enzyme. Inspired by these promising lead compounds, a molecular hybridization strategy was utilized to combine the cyanomethyl linker and biphenyl-DAPY derivatives for developing a new series of scaffolds as NNRTIs ( Figure 2).

Chemistry
The synthesis of target molecules is depicted in Scheme 1, leading to a mixture of enantiomers. The arylboronic acids 3a-n with different substituted 2-(4-bromophenyl)acetonitriles 4a-c via Suzuki-Miyaura cross-coupling reaction catalyzed by PdCl 2 provided the key intermediate biphenyl acetonitriles 5a-p in a mixed solvent of PEG 400 and water at room temperature [30]. The intermediate 4-((4-chloropyrimidin-2-yl)amino)benzonitrile 9 was generated from the commercially available thiouracil 6 through a three-step synthetic strategy according to our previously reported procedures [31,32]. Treatment of 9 with the corresponding biphenyl acetonitriles 5a-p in the presence of KOH in anhydrous DMSO at 50 • C for 4-8 h under a nitrogen atmosphere gave the title compounds 10a-p [33,34].
Firstly, three compounds (10a-c) with 2-F, 3-F, or H at R 1 were synthesized. Compound 10c with 3-F displayed an EC 50 value of 0.33 µM against the WT HIV-1 viral strain, better than that with H (10a) and 2-F (10b) substitutions. The compound 10b with 2-F showed somewhat lower activity than compound 10a without any substitution, indicating that 2-F substitution had a negative effect on the activity. Their cytotoxicities were decreased compared with the lead compound 2 (CC 50 = 2.1 µM) [27] and were better than the marketed drugs NVP, ETR, and RPV. Next, the 3-F group at R 1 was fixed and the effect of R 2 on the antiviral activity was explored. The electron-donating methyl or methoxy groups were introduced to afford compounds 10d-i. Unfortunately, this series showed decreased activity compared to the corresponding compound 10c with nonsubstitution at R 2 . The methyl substituted compounds (10d-f) displayed slightly higher activity than those of methoxy compounds (10g-i). For 10d-f, the attachment position of the methyl group exerted no apparent effect on HIV potency. For 10g-i, the activity followed the sequence 4'-methoxy > 2'-methoxy > 3'-methoxy. Their cytotoxicities were similar to 10c with the exception of compound 10i, which possessed a CC 50 value of 183 µM. Then, we replaced the electron-donating groups with the electron-withdrawing fluorine and chlorine groups. They exhibited better activity than those of the electron-donating methyl and methoxy groups. Similarly, the activity of the 4'-substituted compound (10l and 10o) was better than those of the 2'-and 3'-substituted analogues (10l > 10j > 10k, 10o > 10m > 10n). Finally, encouraged by the results, the electron-withdrawing cyano group was introduced to the 4'-position to obtain compound 10p. To our delight, compound 10p (EC 50 = 0.027 µM) exhibited the best activity against WT HIV-1, consistent with our previous research on biphenyl-DAPYs [27]. The activity of 10p was significantly improved by 12-fold compared with 10c. The cytotoxicity was also acceptable with a CC 50 value of 36.4 µM. The selectivity index (SI = 1361) was best in this series, which was better than the marketed drug ETR. Collectively, the above results indicated that the substitution of biphenyl could significantly influence their antiviral potency; 3-F at R 1 was beneficial to the activity; the electronic effect and position of the R 2 substitution were key factors for the antiviral activity and satisfied the following orders: CN > F ≈ Cl > CH 3 > OCH 3 , and 4'-position > 2'-positon > 3'-position, respectively.
The enzymatic assay against WT HIV-1 RT was conducted for most compounds ( Table 2). All of the target compounds displayed low micromolar RT inhibitory activity (IC 50 = 0.058-0.460 µM) superior or comparable to that of NVP (IC 50 = 0.332 µM) except 10b. Compound 10p (IC 50 = 0.059 µM) and 10j (IC 50 = 0.058 µM) showed great inhibitory activity toward HIV-1 RT, with values six-fold higher than that of NVP. Moreover, a good linear correlation (R 2 = 0.80) between the enzymatic RT inhibitory activities and antiviral activities in MT-4 cells was observed (Figure 3). These results suggested that these compounds could exhibit their anti-HIV-1 activity by targeting RT. Molecular modeling was performed to provide a deep understanding of the binding modes for explaining the SAR. Compounds 10l, 10o, and 10p with better antiviral activities were selected for docking into NNIBP. Considering the CH(CN) chiral center, we performed the docking study using the two enantiomers. As shown in Figure 4, all compounds could bind to the NNIBP in classical "U" conformation similar to other DAPYs. In general, both enantiomers could form the classic hydrogen-bonding interactions between the backbone of K101 and the N atom of pyrimidine and the NH linker. The biphenyl group could deeply insert into the hydrophobic pocket surrounded by the aromatic residues Y181, Y188, F227, and W229, forming the face-to-face π-π stacking interactions. R/S enantiomers displayed similar binding conformations with NNIBP. The cyanomethyl linker in both enantiomers could form three hydrogen bonds with amino-acid residues I180 and Y181 and a water-mediated hydrogen-bonding interaction with E138 (distance < 3.5 Å). For compound 10p, the 4'-CN was predicted to form an additional hydrogen bond with Y188 ( Figure 4A,B). For compound 10l, the 4'-F also formed a hydrogen bond with Y188 ( Figure 4C,D) while 4'-Cl of compound 10o formed a halogen bond ( Figure 4E,F). In the E138K RT ( Figure 4G), the binding mode was similar to the WT RT. However, in the Y188L RT ( Figure 4H), the compound 10p lost the hydrogen bond between 4'-CN and Y188 and decreased π-π stacking interactions. That might explain the higher antiviral activity against the E138K mutant than against the Y188L mutant. The binding modes of 10p with double mutant (F227L + V106A, K103N + Y181C) RT were also predicted (see Figure S1, Supplementary Materials). As previously mentioned, the "U" conformations of compounds located in the NNIBP were critical for the antiviral activity. However, in the double mutated enzymes generated by Bioluminate, the "U" conformations of compound 10p (both isomers) were lost probably due to the high steric hindrance caused by the site mutations. The 4-caynoaniline inserted into the entrance channel instead of the solvent-exposed region. The hydrogen-bonding interaction between the NH linker and K101 was missing. The π-π stacking interactions were reduced by Y181C and F227L mutations with decreased aromatic rings. This might explain the decrease in biological activity of compound 10p against the HIV-1 double mutants.

Apparatus, Materials, and Analytical Reagents
Chemical reagents and solvents were obtained commercially and were used without further purification. All the reactions were monitored by thin-layer chromatography (TLC) on the pre-coated silica gel G plates at 254 nm under an ultraviolet (UV) lamp using ethyl acetate/n-hexane as the eluent. Column chromatography was performed on glass column packed with silica gel (200-300 mesh) using ethyl acetate/n-hexane as the eluent. Melting points were measured on an SGW X-1 microscopic melting point apparatus. Proton nuclear magnetic resonance ( 1 H-NMR) and carbon nuclear magnetic resonance ( 13 C-NMR) spectra were recorded in acetone-d 6 at ambient temperature on a Bruker AV-400 MHz spectrometer, using tetramethylsilane (TMS) as the internal standard. High-resolution mass spectrometry (HRMS) was performed on a Bruker Compact instrument using electrospray ionization (ESI). The purities of the compounds were analyzed by HPLC (Agilent 1260) using a C 18 column (Eslipse XDB, 4.6 × 150 mm, 5 µm) with methanol/water as the mobile phase at a flow rate of 0.8 mL/min: (a) 0-12 min, 50%-95% MeOH; (b) 12-20 min, 95% MeOH; (c) 15-20 min, 60%-95% MeOH; (d) 20-25 min, 95%-50% MeOH; (e) 25-30 min, 50% MeOH. All final compounds exhibited purities greater than 95%. 1 H-NMR, 13 C-NMR, and HRMS spectra of the target compounds 10a-p are presented in Supplementary Materials.

General Procedure for Synthesis of Compounds 5a-p
A mixture of arylboronic acids 3a-n (5.5 mmol, 1.1 equiv.), different substituted 2-(4-bromophenyl) acetonitriles 4a-c (5.0 mmol, 1.0 equiv.), PdCl 2 (0.025 mmol, 0.005 equiv.), K 2 CO 3 (17.5 mmol, 3.5 equiv.), PEG 400 15 mL, and H 2 O 15 mL were added to a 100-mL round-bottom flask, and stirred at room temperature for the desired time until complete consumption of starting material as judged by TLC. Then, the reaction mixture was poured into water (100 mL) and extracted with ethyl acetate (30 mL × 3). The combined organic layers were washed with brine, dried over anhydrous Na 2 SO 4 , filtered, and removed by evaporation under reduced pressure to afford the crude products, which were further purified by column chromatography on silica gel eluting with EtOAc/petroleum ether to 5a-p as white solid.

General Procedure for Synthesis of Target Compounds 10a-p
Different substituted biphenylacetonitriles 5a-p (3.0 mmol, 1.0 equiv.) were dissolved in DMSO (10 mL) in the presence of KOH (15.0 mmol, 5.0 equiv.), followed by addition of the compound 9 (3.0 mmol, 1.0 equiv.). The reaction mixture was stirred at 50 • C for 4-8 h (monitored by TLC) under nitrogen atmosphere. Then the reaction was cooled to ambient temperature and poured into H 2 O (200 mL) yielding a precipitate. The precipitate was collected by filtration, and the residue was then purified via flash chromatography on silica gel, eluting with EtOAc/petroleum ether (1/3, v/v) to obtain final compounds 10a-p of 33%-84% yield. . HIV-1 RT was mutated and minimized using Schrödinger BioLuminate. The protein preparation followed our previous protocol [21,27].

Conclusions
In conclusion, we designed and synthesized 16 new biphenyl-DAPY compounds with a cyanomethyl linker based on a molecular hybridization strategy in order to extend chemical spaces of NNRTIs. Most of the compounds showed submicromolar activity against WT HIV-1 and mutant strains with a more favorable toxicity. The best compound 10p exhibited EC 50 values of 0.027, 0.17, 0.87, 0.90, and 1.21 µM against the WT, E138K, Y181C, K103N, and L100I variants, respectively. They also showed a good correlation with their enzymatic activity, indicating that they targeted RT in HIV-1-infected MT-4 cells. Molecular docking predicted the binding modes of these compounds with RT. These cyanomethyl-biphenyl-diarylpyrimidines could be potential lead templates for further optimization in the discovery of anti-HIV-1 drugs.
Author Contributions: Y.L. and S.H. are co-first authors; they performed molecule design and chemical synthesis, analyzed the NMR and mass spectra, and wrote the paper. Y.Y. analyzed data. C.Z. and F.-E.C. designed the project, provided guidance for optimization of the synthesis process, and they reviewed and edited the paper. C.P. and E.D.C. conceptualized and performed the biological assay. All authors read and agreed to the published version of the manuscript.