European Journal of Medicinal Chemistry 76 (2014) 531e538
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Discovery of nitropyridine derivatives as potent HIV-1 non-nucleoside reverse transcriptase inhibitors via a structure-based core refining approach Jun Wang a, Peng Zhan a, *, Zhenyu Li a, Huiqing Liu c, Erik De Clercq b, Christophe Pannecouque b, Xinyong Liu a, * a Department of Medicinal Chemistry, Key Laboratory of Chemical Biology (Ministry of Education), School of Pharmaceutical Sciences, Shandong University, 44, West Culture Road, 250012 Jinan, Shandong, PR China b Rega Institute for Medical Research, KU Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium c Institute of Pharmacology, School of Medicine, Shandong University, 44 West Culture Road, 250012 Ji’nan, Shandong, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 October 2013 Received in revised form 13 February 2014 Accepted 17 February 2014 Available online 19 February 2014
As a continuation of our efforts to discover and develop back-up analogs of DAPYs, novel substituted nitropyridine derivatives were designed via a structure-based core refining approach, synthesized and evaluated for their in vitro HIV-1 activity in MT-4 cells. Preliminary biological evaluation indicated that most of the compounds exhibited marked inhibitory activity against wild-type HIV-1 IIIB. Most notably, the compound 7b was identified as the most promising candidate in inhibiting HIV-1 replication with an EC50 value of 0.056 mM and a selective index (SI) of 1251, which were much better than those of NVP (EC50 ¼ 0.23 mM) and DLV (EC50 ¼ 0.51 mM). Some other compounds, 7k, 7c, 7j and 7e, were also endowed with a favorable anti-HIV-1 potency (EC50 ¼ 0.034, 0.11, 0.11 and 0.16 mM, respectively). Some antivirally active compounds also showed moderate inhibitory activity against RT. Preliminary structureeactivity relationships (SARs) and molecular modeling of these new analogs provide valuable avenues for future molecular optimization. Ó 2014 Elsevier Masson SAS. All rights reserved.
Keywords: HIV-1 Reverse transcriptase NNRTIs Drug design Organic synthesis Pyridine Bioactivity
1. Introduction Human immunodeficiency virus (HIV) is the causative agent of acquired immunodeficiency syndrome (AIDS). From the data of AIDS Epidemic Update reported by World Health Organization, 1.7 million patients died from AIDS and related diseases, and 34.0 million peoples were living with AIDS in 2011 [1]. Currently, there is no effective vaccine against HIV, but the generally adopted highly active antiretroviral therapy (HAART) has significantly reduced the morbidity and mortality of HIV-infected people. HIV type 1 reverse transcriptase (HIV-1 RT) as a key enzyme in the HIV replicative cycle represents one of the main targets for the treatment of HIV/ AIDS [2]. Among RT inhibitors, non-nucleoside RT inhibitors (NNRTIs) can directly inhibit RT by binding to an allosteric site, termed as the NNRTIs binding pocket (NNIBP), which is located
* Corresponding authors. E-mail addresses: [email protected] (P. Zhan), [email protected] (X. Liu). http://dx.doi.org/10.1016/j.ejmech.2014.02.047 0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.
about 10 A away from the RT catalytic site [3]. HIV-1 NNRTIs is a key component of HAART regimens for the long-term management of HIV infection because of their unique antiviral potency, high specificity and low toxicity. However, during the clinical use of the first generation NNRTIs (nevirapine, delavirdine and efavirenz), significant resistance has been generated by the emergence of mutant viral strains. Therefore, there is still need for discovery of novel HIV-1 NNRTIs with an improved potency against the wild and clinically observed mutant virus strains [4e9]. The diarylpyrimidine (DAPY) family, represented by the newly approved anti-HIV-1 drugs etravirine (TMC125) and rilpivirine (TMC278), has been regarded as promising HIV-1 NNRTIs with robust anti-HIV-1 potency against both the wild type (wt) and drug-resistant strains carrying multiple mutations [10]. Over the past few years, considerable efforts have been devoted to the structural modification of DAPYs to discover more potent back-up congeners. As determined by X-ray crystal structures of DAPYs-RT complexes and molecular simulation, DAPYs resembled a horseshoe or “U”
532
J. Wang et al. / European Journal of Medicinal Chemistry 76 (2014) 531e538
shape when bound in the NNIBP in contrast to the typical butterflylike binding shape of the first-generation NNRTIs (NVP, DLV and EFV). The relative flexibility of the six-membered heterocycle core might allow DAPYs to better adapt to the plasticity and changes of the NNIBP, which appear to be critical for maintaining potency against wt and a wide range of drug-resistant mutant HIV-1 RTs [11,12]. As shown in Fig. 1, the DAPYs also shared a similar pharmacophore as mentioned for the first-generation NNRTIs, including a hydrophobic center able to participate in pep stacking interactions (C ring) as well as hydrogen bond donor and acceptor interactions. The six-membered heterocyclic moiety not only stays in the center of the NNIBP, anchoring the functional groups for optimal engagement with the residues around NNIBP, but also serves as a critical hydrogen bond acceptor (the pyrimidine nitrogen at 1-position) to generate hydrogen bonding with the a-amino of LYS101. Besides, the NH linker connecting the central B ring and the right A wing can function as a hydrogen bond donor able to interact with the backbone carbonyl of LYS101 [13]. These conclusions provided critical insights for further structural studies or scaffold refining towards improving their activity and drug resistance profiles. Scaffold refining via the replacement of the central core in bioactive molecules combined with introducing privileged substituents is a common practice in contemporary medicinal chemistry to obtain intellectual property and novel hits, as well as to improve synthetic accessibility. To discover highly potent DAPY derivatives that are easier to synthesize or to avoid existing patent property, further modifications were focused on the replacement of the pyrimidine core with an array of aromatic scaffolds [14,15]. In this regard, previous investigations in our laboratories using structure-based drug design and isosterism resulted in the discovery of a novel series of potent DAPYs back-up analogs, such as pyridazine derivatives, with proved to be highly effective in inhibiting HIV-1 replication at double-digit nanomolar concentrations acting as RT inhibitors [16e18]. According to the molecular modeling and structureeactivity relationship (SAR) results, the central pyrimidine ring of DAPYs is relatively tolerant. As an extension of these investigations and with the aim to generate novel NNRTIs with desirable potency and properties, we designed and synthesized novel nitropyridine derivatives in which the pyrimidine ring of the original DAPY scaffold was replaced with a synthetically accessible nitropyridine structural motif to adhere to the optimal pharmacophore moieties, while the NH linker connecting the central ring and the right wing, and the constant substitutions of A and C ring were maintained in view of their paramount importance in the previous series. The general structure of such a new scaffold is illustrated in Fig. 1. Herein, we will further report the synthesis, anti-HIV evaluation against wt HIV-1 and HIV-2 ROD, as well as against a doublemutated HIV-1 strain RES056 (K103N þ Y181C). In addition, preliminary SAR data and molecular modeling results of these new
CN
CN
C
A
compounds are also discussed to give further insight in their probable binding modes in the allosteric binding site. 2. Chemistry The synthesis of the newly designed nitropyridines compounds was accomplished using an expeditious straight-forward route described in Scheme 1. Chlorination of commercially available picolinic acid (1) with SOCl2 followed by esterification with MeOH resulted in the formation of methyl 4-chloropicolinate (2). The latter was further converted to 4-chloropicolinamide (3) by amidation using saturated ammonia. Then, 4-chloropyridin-2-amine (4) was generated from intermediate 3 using standard procedures of Hofmann rearrangement. Further, nitration reaction of compound 4 in the presence of HNO3/H2SO4 formed the key intermediate 4-chloro5-nitropyridin-2-amine (5) in 41% yield according to the methods previously reported [19,20]. Compound 5 was subjected to treat with substituted phenols or substituted anilines to obtain compounds 6 [21], which was converted into the final nitropyridine derivatives 7 with various 4-substituted phenyl bromides using K2CO3 as a base and Pd(OAc)2 and Xantphos as catalysts, in good yields [22,23]. The synthesized compounds were characterized by physicochemical and spectral means. The MS and NMR spectral data are in full agreement with the proposed structures. 3. Results and discussion 3.1. Anti-HIV activity evaluation All of the newly synthesized nitropyridine derivatives were evaluated for their anti-HIV activity and cytotoxicity in MT-4 cells infected by wt HIV-1 strain IIIB and the double mutant strain RES056 (K103N þ Y181C) along with HIV-2 (ROD). The results, expressed as CC50 (cytotoxicity), EC50 (anti-HIV activity) and SI (selectivity index, given by the CC50/EC50 ratio), are summarized in Table 1 together with those of nevirapine (NVP), delaviridine (DLV), efavirenz (EFV) and zidovudine (azidothymidine, AZT) as the reference drugs. As listed in Table 1, the majority of these compounds showed high activity against wt HIV-1(IIIB) with EC50 values in the range of 0.034e30.17 mM, except for the two compounds, 7m and 7q. Half of them are of low toxicity in MT-4 cells with CC50 values more than 125 mM. Encouragingly, compound 7b (EC50 ¼ 0.056 mM, SI ¼ 1251), compound 7k (EC50 ¼ 0.034 mM, SI ¼ 691), compound 7c (EC50 ¼ 0.11 mM, SI ¼ 339) and compound 7h (EC50 ¼ 0.17 mM, SI ¼ 97) were more active than reference drugs NVP and DLV against wt HIV-1(IIIB), but were still inferior to the clinically used EFV and AZT. It was also observed that some compounds, 7a and 7r, had high anti-HIV-1 potency (EC50 ¼ 0.58 and 0.72 mM, SI ¼ 214 and 200, respectively). These promising results demonstrated that the
CN R1
CN
R
C O Br
N B
Scaffold refining
NH HN
N
NH
N
NH2
N
Etravirine (TMC125, ETV)
Rilpivirine (TMC278)
Lead compounds
R2
A R3
X
NH B
O2N
N
X = O, NH
nd in bo ma en do g o n dr tio Hy tera n i
Newly designed nitropyridine derivatives
Fig. 1. Design of nitropyridine derivatives as back-up DAPYs via a scaffold refining approach.
J. Wang et al. / European Journal of Medicinal Chemistry 76 (2014) 531e538
Cl
Cl
533
Cl Cl
OH
N
i
ii
O
N
O 2
R1
v
R3
R2
X
NH2 N
O2N
N
NH2 4
3
R1
R2
NH2
iv
N
O2N
O
O
1
NH2
N
iii
vi
R
R3 X
NH N
O2N
6
5
7
R1, R2, R3 = H, F, Cl, Br, CH3, OCH3, CN, NO3 R = CN, CH3, NO2 X = O, NH
Scheme 1. Reagents and conditions. i: (a) SOCl2; (b) MeOH; ii: NH3 (aq); iii: Br2, NaOH(aq); iv: HNO3/H2SO4; v: Substituted phenols, K2CO3, DMF; or substituted anilines, K2CO3, DMF; vi: Pd(OAc)2, Xantphos, Cs2CO3, dioxane.
isosteric replacement of pyridine for pyrimidine in the central B-ring of DAPY compounds was gratifying and afforded a series of potent nitropyridine NNRTIs. Based on the results of antiviral assay (Table 1), several important structural features for conferring optimum HIV inhibition and the cytotoxicity were investigated and summarized as follows: (a) The biological assay results clearly indicated that the activities of the compounds where X is O are generally better than the compounds where X is NH. For instance, the active sequence of the compounds with the same substitutions in A and C ring was as follows: 7a > 7m, 7b > 7n, 7j > 7o. (b) Next we turned our attention to SAR of the R substituent at the para position of the A-ring. Like the DAPY analogs TMC125 and TMC278, the compound 7c (EC50 ¼ 0.11 mM, CC50 ¼ 36.06 mM) containing a cyano group as the R substituent was found to be more active than the analogs containing NO2 (7r, EC50 ¼ 0.72 mM, CC50 ¼ 141.44 mM) and CH3 (7q, EC50 > 206.87 mM, CC50 ¼ 206.87 mM). These results in conjunction with previous studies suggested that the paracyano moiety would be a favorable structural moiety for antiHIV activity. Besides, it is noteworthy that the cytotoxicity of these congeners increased in the same order (7c > 7r > 7q). After determining that the O atom was the favorable linker connecting the central ring and the left wing and a nitrile was the optimal substituent at the R-position on the right wing, we then focused our attention on the SAR of the left C wing.
analogs, indicating that perhaps the R1 substituents may have some unfavorable aspects regarding cytotoxicity. (d) The nature of the substituents at the R2/R3 position of the C-ring essentially influences the anti-HIV-1 activity. The R2/R3-methyl derivative 7a showed activity in the low micromolecular range with EC50 value (0.58 mM) comparable to that of the R2/R3chlorine derivative 7g (EC50 ¼ 0.62 mM), much lower than that of the methoxy counterpart 7f (EC50 ¼ 4.28 mM). Even so, compound 7f was a compelling molecule with the highest CC50 value (318.78 mM) of all the tested compounds. (e) In the case of 2,4,6-trihalogen-phenoxy derivatives (7h, 7j, 7l), we found that their antiviral potency was in the order 2,4,6-tribromo > 2,4,6-trichloro > 2,4,6-trifluoro. Compared with their congeners, significantly increased cytotoxicities were observed. Compound 7l characterized by the absence of the 2,4,6-trifluoro substitution at the phenoxy showed the highest toxicity among all the nitropyridine derivatives. With the aim to define the resistance profiles of these compounds, they were also assayed against the double mutant strain RES056 (K103N þ Y181C), the often encountered clinical strain in the treatment of AIDS with HIV-1 NNRTIs (results shown in Table 1). Unfortunately, all the compounds lost their activity against the double mutant strain RES056. Furthermore, none of the derivatives tested showed inhibition of HIV-2 (ROD) replication in MT-4 cells, which might suggest that this new series of nitropyridine derivatives was specific for HIV-1. 3.2. Inhibition of HIV-1 RT
(c) As highlighted in Table 1, the anti-HIV activity is strongly dependent on the nature of the substituents at the R1 position of the C-ring. In the case of 2,6-dimethylphenoxy analogs (7ae7e), a clear order of R1-substituent for anti-HIV activity was observed by direct comparison: CH3 (7b) > CN (7c) > Cl (7e) > Br (7d) > H (7a). This conclusion is in agreement with 2,6-dichlorophenoxy analogs (Cl > H > NO2) and 2,6-dibromophenoxy analogs (CH3 > Br). This set of results seemed to indicate that R1 substituents with appropriate bulk are beneficial for potency. The negative impact on the potency was greatly exacerbated with the replacement of the H in 7g by NO2 (7i). It was also observed that, compound 7a with the lowest potency in the 2,6dimethylphenoxy series demonstrated the lowest cytotoxicity (CC50 ¼ 125.85 mM), and likewise, this will also be the case for compound 7h among three 2,6-dichlorophenoxy
With aim to verify their binding target, three selected title compounds (7b, 7c, and 7k) were tested in enzyme assays against highly purified recombinant HIV-1 RT using poly (rC)-oligo (dG) as template primer. IC50 values corresponded to the concentration of the substituted nitropyridine derivatives required to inhibit biotindUTP incorporation into the HIV-1 RT by 50%. The three compounds exhibited moderate inhibition of enzymatic activity with IC50 values of 6.9 mM, 8.5 mM and 10.4 mM, respectively, which was slightly higher than that of TMC125 (6.5 mM) (Table 2). It suggests that the newly synthesized nitropyridine derivatives bind to HIV-1 RT and belong to the genuine NNRTIs. What needs to be stressed is that the anti-RT potency of compound 7k was slightly weaker than that of 7b and 7c even though compound 7k was more potent against HIV-1 replication (Table 1). The discrepancy in potency between anti-HIV-1 replication and
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J. Wang et al. / European Journal of Medicinal Chemistry 76 (2014) 531e538
Table 1 HIV-1 wt (IIIB) inhibition effect and cytotoxicity of the title compounds.
Compds
X
R
R1
R2
R3
HIV-1 IIIB
7a 7b 7c 7d 7e 7f 7g 7h 7i 7j 7k 7l 7m 7n 7o 7p 7q 7r NVP DLV EFV AZT a b c
O O O O O O O O O O O O NH NH NH NH O O
CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CH3 NO2
H CH3 CN Br Cl H H Cl NO2 Br CH3 F H CH3 CH3 Br CN CN
CH3 CH3 CH3 CH3 CH3 OCH3 Cl Cl Cl Br Br F CH3 CH3 Br Br CH3 CH3
CH3 CH3 CH3 CH3 CH3 OCH3 Cl Cl Cl Br Br F CH3 CH3 Br F CH3 CH3
RES056 SI
EC50(mM)
0.58 0.056 0.11 0.27 0.16 4.28 0.62 0.17 16.8 0.11 0.034 0.72 >12.78 30.17 3.85 3.05 >206.87 0.72 0.23 0.51 0.0057 0.0075
214 1251 339 45 70 74 44 97 2 65 691 5 71 >1112 >12,473
>125.84 >69.86 >36.06 >12.26 >11.06 >318.78 >27.69 >16.24 >26.51 >7.49 >23.55 >3.54 >12.78 >262.36 >166.52 >149.25 >206.87 >141.44 4.09 >36.19 0.32 0.016
c
CC50(mM)b
ROD
EC50(mM)
a
a
SI
EC50(mM)
149.25 >206.87 >141.44
c
a
0.012
SI
c
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Discovery of nitropyridine derivatives as potent HIV-1 non-nucleoside reverse transcriptase inhibitors via a structure-based core refining approach Jun Wang a, Peng Zhan a, *, Zhenyu Li a, Huiqing Liu c, Erik De Clercq b, Christophe Pannecouque b, Xinyong Liu a, * a Department of Medicinal Chemistry, Key Laboratory of Chemical Biology (Ministry of Education), School of Pharmaceutical Sciences, Shandong University, 44, West Culture Road, 250012 Jinan, Shandong, PR China b Rega Institute for Medical Research, KU Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium c Institute of Pharmacology, School of Medicine, Shandong University, 44 West Culture Road, 250012 Ji’nan, Shandong, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 October 2013 Received in revised form 13 February 2014 Accepted 17 February 2014 Available online 19 February 2014
As a continuation of our efforts to discover and develop back-up analogs of DAPYs, novel substituted nitropyridine derivatives were designed via a structure-based core refining approach, synthesized and evaluated for their in vitro HIV-1 activity in MT-4 cells. Preliminary biological evaluation indicated that most of the compounds exhibited marked inhibitory activity against wild-type HIV-1 IIIB. Most notably, the compound 7b was identified as the most promising candidate in inhibiting HIV-1 replication with an EC50 value of 0.056 mM and a selective index (SI) of 1251, which were much better than those of NVP (EC50 ¼ 0.23 mM) and DLV (EC50 ¼ 0.51 mM). Some other compounds, 7k, 7c, 7j and 7e, were also endowed with a favorable anti-HIV-1 potency (EC50 ¼ 0.034, 0.11, 0.11 and 0.16 mM, respectively). Some antivirally active compounds also showed moderate inhibitory activity against RT. Preliminary structureeactivity relationships (SARs) and molecular modeling of these new analogs provide valuable avenues for future molecular optimization. Ó 2014 Elsevier Masson SAS. All rights reserved.
Keywords: HIV-1 Reverse transcriptase NNRTIs Drug design Organic synthesis Pyridine Bioactivity
1. Introduction Human immunodeficiency virus (HIV) is the causative agent of acquired immunodeficiency syndrome (AIDS). From the data of AIDS Epidemic Update reported by World Health Organization, 1.7 million patients died from AIDS and related diseases, and 34.0 million peoples were living with AIDS in 2011 [1]. Currently, there is no effective vaccine against HIV, but the generally adopted highly active antiretroviral therapy (HAART) has significantly reduced the morbidity and mortality of HIV-infected people. HIV type 1 reverse transcriptase (HIV-1 RT) as a key enzyme in the HIV replicative cycle represents one of the main targets for the treatment of HIV/ AIDS [2]. Among RT inhibitors, non-nucleoside RT inhibitors (NNRTIs) can directly inhibit RT by binding to an allosteric site, termed as the NNRTIs binding pocket (NNIBP), which is located
* Corresponding authors. E-mail addresses: [email protected] (P. Zhan), [email protected] (X. Liu). http://dx.doi.org/10.1016/j.ejmech.2014.02.047 0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.
about 10 A away from the RT catalytic site [3]. HIV-1 NNRTIs is a key component of HAART regimens for the long-term management of HIV infection because of their unique antiviral potency, high specificity and low toxicity. However, during the clinical use of the first generation NNRTIs (nevirapine, delavirdine and efavirenz), significant resistance has been generated by the emergence of mutant viral strains. Therefore, there is still need for discovery of novel HIV-1 NNRTIs with an improved potency against the wild and clinically observed mutant virus strains [4e9]. The diarylpyrimidine (DAPY) family, represented by the newly approved anti-HIV-1 drugs etravirine (TMC125) and rilpivirine (TMC278), has been regarded as promising HIV-1 NNRTIs with robust anti-HIV-1 potency against both the wild type (wt) and drug-resistant strains carrying multiple mutations [10]. Over the past few years, considerable efforts have been devoted to the structural modification of DAPYs to discover more potent back-up congeners. As determined by X-ray crystal structures of DAPYs-RT complexes and molecular simulation, DAPYs resembled a horseshoe or “U”
532
J. Wang et al. / European Journal of Medicinal Chemistry 76 (2014) 531e538
shape when bound in the NNIBP in contrast to the typical butterflylike binding shape of the first-generation NNRTIs (NVP, DLV and EFV). The relative flexibility of the six-membered heterocycle core might allow DAPYs to better adapt to the plasticity and changes of the NNIBP, which appear to be critical for maintaining potency against wt and a wide range of drug-resistant mutant HIV-1 RTs [11,12]. As shown in Fig. 1, the DAPYs also shared a similar pharmacophore as mentioned for the first-generation NNRTIs, including a hydrophobic center able to participate in pep stacking interactions (C ring) as well as hydrogen bond donor and acceptor interactions. The six-membered heterocyclic moiety not only stays in the center of the NNIBP, anchoring the functional groups for optimal engagement with the residues around NNIBP, but also serves as a critical hydrogen bond acceptor (the pyrimidine nitrogen at 1-position) to generate hydrogen bonding with the a-amino of LYS101. Besides, the NH linker connecting the central B ring and the right A wing can function as a hydrogen bond donor able to interact with the backbone carbonyl of LYS101 [13]. These conclusions provided critical insights for further structural studies or scaffold refining towards improving their activity and drug resistance profiles. Scaffold refining via the replacement of the central core in bioactive molecules combined with introducing privileged substituents is a common practice in contemporary medicinal chemistry to obtain intellectual property and novel hits, as well as to improve synthetic accessibility. To discover highly potent DAPY derivatives that are easier to synthesize or to avoid existing patent property, further modifications were focused on the replacement of the pyrimidine core with an array of aromatic scaffolds [14,15]. In this regard, previous investigations in our laboratories using structure-based drug design and isosterism resulted in the discovery of a novel series of potent DAPYs back-up analogs, such as pyridazine derivatives, with proved to be highly effective in inhibiting HIV-1 replication at double-digit nanomolar concentrations acting as RT inhibitors [16e18]. According to the molecular modeling and structureeactivity relationship (SAR) results, the central pyrimidine ring of DAPYs is relatively tolerant. As an extension of these investigations and with the aim to generate novel NNRTIs with desirable potency and properties, we designed and synthesized novel nitropyridine derivatives in which the pyrimidine ring of the original DAPY scaffold was replaced with a synthetically accessible nitropyridine structural motif to adhere to the optimal pharmacophore moieties, while the NH linker connecting the central ring and the right wing, and the constant substitutions of A and C ring were maintained in view of their paramount importance in the previous series. The general structure of such a new scaffold is illustrated in Fig. 1. Herein, we will further report the synthesis, anti-HIV evaluation against wt HIV-1 and HIV-2 ROD, as well as against a doublemutated HIV-1 strain RES056 (K103N þ Y181C). In addition, preliminary SAR data and molecular modeling results of these new
CN
CN
C
A
compounds are also discussed to give further insight in their probable binding modes in the allosteric binding site. 2. Chemistry The synthesis of the newly designed nitropyridines compounds was accomplished using an expeditious straight-forward route described in Scheme 1. Chlorination of commercially available picolinic acid (1) with SOCl2 followed by esterification with MeOH resulted in the formation of methyl 4-chloropicolinate (2). The latter was further converted to 4-chloropicolinamide (3) by amidation using saturated ammonia. Then, 4-chloropyridin-2-amine (4) was generated from intermediate 3 using standard procedures of Hofmann rearrangement. Further, nitration reaction of compound 4 in the presence of HNO3/H2SO4 formed the key intermediate 4-chloro5-nitropyridin-2-amine (5) in 41% yield according to the methods previously reported [19,20]. Compound 5 was subjected to treat with substituted phenols or substituted anilines to obtain compounds 6 [21], which was converted into the final nitropyridine derivatives 7 with various 4-substituted phenyl bromides using K2CO3 as a base and Pd(OAc)2 and Xantphos as catalysts, in good yields [22,23]. The synthesized compounds were characterized by physicochemical and spectral means. The MS and NMR spectral data are in full agreement with the proposed structures. 3. Results and discussion 3.1. Anti-HIV activity evaluation All of the newly synthesized nitropyridine derivatives were evaluated for their anti-HIV activity and cytotoxicity in MT-4 cells infected by wt HIV-1 strain IIIB and the double mutant strain RES056 (K103N þ Y181C) along with HIV-2 (ROD). The results, expressed as CC50 (cytotoxicity), EC50 (anti-HIV activity) and SI (selectivity index, given by the CC50/EC50 ratio), are summarized in Table 1 together with those of nevirapine (NVP), delaviridine (DLV), efavirenz (EFV) and zidovudine (azidothymidine, AZT) as the reference drugs. As listed in Table 1, the majority of these compounds showed high activity against wt HIV-1(IIIB) with EC50 values in the range of 0.034e30.17 mM, except for the two compounds, 7m and 7q. Half of them are of low toxicity in MT-4 cells with CC50 values more than 125 mM. Encouragingly, compound 7b (EC50 ¼ 0.056 mM, SI ¼ 1251), compound 7k (EC50 ¼ 0.034 mM, SI ¼ 691), compound 7c (EC50 ¼ 0.11 mM, SI ¼ 339) and compound 7h (EC50 ¼ 0.17 mM, SI ¼ 97) were more active than reference drugs NVP and DLV against wt HIV-1(IIIB), but were still inferior to the clinically used EFV and AZT. It was also observed that some compounds, 7a and 7r, had high anti-HIV-1 potency (EC50 ¼ 0.58 and 0.72 mM, SI ¼ 214 and 200, respectively). These promising results demonstrated that the
CN R1
CN
R
C O Br
N B
Scaffold refining
NH HN
N
NH
N
NH2
N
Etravirine (TMC125, ETV)
Rilpivirine (TMC278)
Lead compounds
R2
A R3
X
NH B
O2N
N
X = O, NH
nd in bo ma en do g o n dr tio Hy tera n i
Newly designed nitropyridine derivatives
Fig. 1. Design of nitropyridine derivatives as back-up DAPYs via a scaffold refining approach.
J. Wang et al. / European Journal of Medicinal Chemistry 76 (2014) 531e538
Cl
Cl
533
Cl Cl
OH
N
i
ii
O
N
O 2
R1
v
R3
R2
X
NH2 N
O2N
N
NH2 4
3
R1
R2
NH2
iv
N
O2N
O
O
1
NH2
N
iii
vi
R
R3 X
NH N
O2N
6
5
7
R1, R2, R3 = H, F, Cl, Br, CH3, OCH3, CN, NO3 R = CN, CH3, NO2 X = O, NH
Scheme 1. Reagents and conditions. i: (a) SOCl2; (b) MeOH; ii: NH3 (aq); iii: Br2, NaOH(aq); iv: HNO3/H2SO4; v: Substituted phenols, K2CO3, DMF; or substituted anilines, K2CO3, DMF; vi: Pd(OAc)2, Xantphos, Cs2CO3, dioxane.
isosteric replacement of pyridine for pyrimidine in the central B-ring of DAPY compounds was gratifying and afforded a series of potent nitropyridine NNRTIs. Based on the results of antiviral assay (Table 1), several important structural features for conferring optimum HIV inhibition and the cytotoxicity were investigated and summarized as follows: (a) The biological assay results clearly indicated that the activities of the compounds where X is O are generally better than the compounds where X is NH. For instance, the active sequence of the compounds with the same substitutions in A and C ring was as follows: 7a > 7m, 7b > 7n, 7j > 7o. (b) Next we turned our attention to SAR of the R substituent at the para position of the A-ring. Like the DAPY analogs TMC125 and TMC278, the compound 7c (EC50 ¼ 0.11 mM, CC50 ¼ 36.06 mM) containing a cyano group as the R substituent was found to be more active than the analogs containing NO2 (7r, EC50 ¼ 0.72 mM, CC50 ¼ 141.44 mM) and CH3 (7q, EC50 > 206.87 mM, CC50 ¼ 206.87 mM). These results in conjunction with previous studies suggested that the paracyano moiety would be a favorable structural moiety for antiHIV activity. Besides, it is noteworthy that the cytotoxicity of these congeners increased in the same order (7c > 7r > 7q). After determining that the O atom was the favorable linker connecting the central ring and the left wing and a nitrile was the optimal substituent at the R-position on the right wing, we then focused our attention on the SAR of the left C wing.
analogs, indicating that perhaps the R1 substituents may have some unfavorable aspects regarding cytotoxicity. (d) The nature of the substituents at the R2/R3 position of the C-ring essentially influences the anti-HIV-1 activity. The R2/R3-methyl derivative 7a showed activity in the low micromolecular range with EC50 value (0.58 mM) comparable to that of the R2/R3chlorine derivative 7g (EC50 ¼ 0.62 mM), much lower than that of the methoxy counterpart 7f (EC50 ¼ 4.28 mM). Even so, compound 7f was a compelling molecule with the highest CC50 value (318.78 mM) of all the tested compounds. (e) In the case of 2,4,6-trihalogen-phenoxy derivatives (7h, 7j, 7l), we found that their antiviral potency was in the order 2,4,6-tribromo > 2,4,6-trichloro > 2,4,6-trifluoro. Compared with their congeners, significantly increased cytotoxicities were observed. Compound 7l characterized by the absence of the 2,4,6-trifluoro substitution at the phenoxy showed the highest toxicity among all the nitropyridine derivatives. With the aim to define the resistance profiles of these compounds, they were also assayed against the double mutant strain RES056 (K103N þ Y181C), the often encountered clinical strain in the treatment of AIDS with HIV-1 NNRTIs (results shown in Table 1). Unfortunately, all the compounds lost their activity against the double mutant strain RES056. Furthermore, none of the derivatives tested showed inhibition of HIV-2 (ROD) replication in MT-4 cells, which might suggest that this new series of nitropyridine derivatives was specific for HIV-1. 3.2. Inhibition of HIV-1 RT
(c) As highlighted in Table 1, the anti-HIV activity is strongly dependent on the nature of the substituents at the R1 position of the C-ring. In the case of 2,6-dimethylphenoxy analogs (7ae7e), a clear order of R1-substituent for anti-HIV activity was observed by direct comparison: CH3 (7b) > CN (7c) > Cl (7e) > Br (7d) > H (7a). This conclusion is in agreement with 2,6-dichlorophenoxy analogs (Cl > H > NO2) and 2,6-dibromophenoxy analogs (CH3 > Br). This set of results seemed to indicate that R1 substituents with appropriate bulk are beneficial for potency. The negative impact on the potency was greatly exacerbated with the replacement of the H in 7g by NO2 (7i). It was also observed that, compound 7a with the lowest potency in the 2,6dimethylphenoxy series demonstrated the lowest cytotoxicity (CC50 ¼ 125.85 mM), and likewise, this will also be the case for compound 7h among three 2,6-dichlorophenoxy
With aim to verify their binding target, three selected title compounds (7b, 7c, and 7k) were tested in enzyme assays against highly purified recombinant HIV-1 RT using poly (rC)-oligo (dG) as template primer. IC50 values corresponded to the concentration of the substituted nitropyridine derivatives required to inhibit biotindUTP incorporation into the HIV-1 RT by 50%. The three compounds exhibited moderate inhibition of enzymatic activity with IC50 values of 6.9 mM, 8.5 mM and 10.4 mM, respectively, which was slightly higher than that of TMC125 (6.5 mM) (Table 2). It suggests that the newly synthesized nitropyridine derivatives bind to HIV-1 RT and belong to the genuine NNRTIs. What needs to be stressed is that the anti-RT potency of compound 7k was slightly weaker than that of 7b and 7c even though compound 7k was more potent against HIV-1 replication (Table 1). The discrepancy in potency between anti-HIV-1 replication and
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Table 1 HIV-1 wt (IIIB) inhibition effect and cytotoxicity of the title compounds.
Compds
X
R
R1
R2
R3
HIV-1 IIIB
7a 7b 7c 7d 7e 7f 7g 7h 7i 7j 7k 7l 7m 7n 7o 7p 7q 7r NVP DLV EFV AZT a b c
O O O O O O O O O O O O NH NH NH NH O O
CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CN CH3 NO2
H CH3 CN Br Cl H H Cl NO2 Br CH3 F H CH3 CH3 Br CN CN
CH3 CH3 CH3 CH3 CH3 OCH3 Cl Cl Cl Br Br F CH3 CH3 Br Br CH3 CH3
CH3 CH3 CH3 CH3 CH3 OCH3 Cl Cl Cl Br Br F CH3 CH3 Br F CH3 CH3
RES056 SI
EC50(mM)
0.58 0.056 0.11 0.27 0.16 4.28 0.62 0.17 16.8 0.11 0.034 0.72 >12.78 30.17 3.85 3.05 >206.87 0.72 0.23 0.51 0.0057 0.0075
214 1251 339 45 70 74 44 97 2 65 691 5 71 >1112 >12,473
>125.84 >69.86 >36.06 >12.26 >11.06 >318.78 >27.69 >16.24 >26.51 >7.49 >23.55 >3.54 >12.78 >262.36 >166.52 >149.25 >206.87 >141.44 4.09 >36.19 0.32 0.016
c
CC50(mM)b
ROD
EC50(mM)
a
a
SI
EC50(mM)
149.25 >206.87 >141.44
c
a
0.012
SI
c
