Molecular design, synthesis and biological evaluation of BP-O-DAPY and O-DAPY derivatives as non-nucleoside HIV-1 reverse transcriptase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2013.04.052

This paper reports the synthesis and antiviral evaluation of a series of non-nucleoside reverse transcriptase inhibitors (NNRTIs) that combine the peculiar structural features of diarylpyrimidine derivatives (DAPYs) and benzophenone derivatives (BPs). The DAPY derivatives bearing benzoyl or alkoxyl substitutes on the A-ring showed the inhibitory activity against wild-type HIV-1 at the cellular level within the range of EC₅₀ values from micromolar to nanomolar. Among these compounds, 1u exhibited the most potent anti-HIV-1 activity (EC₅₀ = 0.06 ± 0.01 μM, SI > 6260), which were about 1.8-fold more active than nevirapine (NVP) and delavirdine (DLV). In addition, the binding modes with HIV-1 RT and the preliminary SAR studies of these derivatives were also considered for further investigation.

Full text of DOI:10.1016/j.ejmech.2013.04.052

Accepted Manuscript
Molecular design, synthesis and biological evaluation of BP-O-DAPY and O-DAPY
derivatives as non-nucleoside HIV-1 reverse transcriptase inhibitors
Shiqiong Yang, Christophe Pannecouque, Dirk Daelemans, Xiao-Dong Ma, Yang Liu,
Fen-Er Chen, Erik De Clercq
PII:
S0223-5234(13)00284-5
10.1016/j.ejmech.2013.04.052
EJMECH 6162
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 14 December 2012
Revised Date: 24 April 2013
Accepted Date: 26 April 2013
Please cite this article as: S. Yang, C. Pannecouque, D. Daelemans, X.-D. Ma, Y. Liu, F.-E. Chen, E. De
Clercq, Molecular design, synthesis and biological evaluation of BP-O-DAPY and O-DAPY derivatives
as non-nucleoside HIV-1 reverse transcriptase inhibitors, European Journal of Medicinal Chemistry
(2013), doi: 10.1016/j.ejmech.2013.04.052.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
►Hybrid the special structural features of diarylpyrimidines and benzophenones
►Synthesize a series of BP-O-DAPY hybrids and O-DAPY derivatives as NNRTIs
► Evaluate of the synthesized compounds against HIV-1 and HIV-2
► Study the potential binding mode by molecular docking

ACCEPTED MANUSCRIPT
Molecular design, synthesis and biological evaluation of
BP-O-DAPY and O-DAPY derivatives as non-nucleoside
HIV-1 reverse transcriptase inhibitors
Shiqiong Yanga, Christophe Pannecouqueb, Dirk Daelemansb, Xiao-Dong Maa, Yang Liua,
Fen-Er Chena, and Erik De Clercqb
a Department of Chemistry, Fudan University, 220 Handan Road, Shanghai 200433,People’s Republic of China
b Rega Institute for Medical Research KU Leuven, 10 Minderbroedersstraat, B-3000 Leuven, Belgium
Abstract: This paper reports the synthesis and antiviral evaluation of a series of non-nucleoside
reverse transcriptase inhibitors (NNRTIs) that combine the peculiar structural features of
diarylpyrimidine derivatives (DAPYs) and benzophenone derivatives (BPs). The DAPY
derivatives bearing benzoyl or alkoxyl substitutes on the A-ring showed the inhibitory activity
against wild-type HIV-1 at the cellular level within the range of EC50 values from micromolar to
nanomolar. Among these compounds, 1u exhibited the most potent anti-HIV-1 activity (EC50
=
0.06 ± 0.01 ꢀM, SI > 6260), which were about 1.8-fold more active than nevirapine (NVP) and
delavirdine (DLV). In addition, the binding modes with HIV-1 RT and the preliminary SAR
studies of these derivatives were also considered for further investigation.
Keywords: HIV-1, NNRTIs, Diarylpyrimidine, Benzophenone, Molecular hybridization,
Antiviral activity
Abbreviations: NNRTIs, non-nucleoside reverse transcriptase inhibitors; DAPYs, diarylpyrimidine
derivatives; BPs, benzophenone derivatives; NVP, nevirapine; DLV, delavirdine; HIV-1 RT, Human
immunodeficiency virus type 1 reverse transcriptase; USFDA, United States Food and Drug
Administration; NNRTIs, non-nucleoside RT inhibitors; EFV, efavirenz; TMC125, etravirine; TMC278,
rilpivirine; NNIBP, non-nucleoside inhibitor binding pocket; wt, wild-type; BPs, benzophenone derivatives;
SI, selectivity index; AZT, zidovudine; ESI, electrospray ionization; TMS, tetramethylsilane ; MTT,
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide; CCID50, 50% cell culture infectious dose;
OD, optical density; CC50, 50% cytotoxic concentration; EC50, 50% effective concentration; Kd,
dissociation constant; IC50, 50% inhibitory concentration.
Corresponding author, Tel./fax: +86 21 65643811. E-mail address: rfchen@fudan.edu.cn (F.-E.
Chen).
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1. Introduction
Human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) is a key enzyme in the
HIV replicative cycle. It represents one of the main targets for the treatment of HIV/AIDS.
Nowadays, thirteen RT inhibitors have been approved by the United States Food and Drug
Administration (USFDA) for clinical use as anti-HIV drugs, among which the non-nucleoside RT
inhibitors (NNRTIs, Fig. 1) are nevirapine (NVP), delavirdine (DLV), efavirenz (EFV), etravirine
(TMC125) and rilpivirine (TMC278) [1-5]. The NNRTIs can directly inhibit HIV reverse
transcriptase enzyme by binding to an allosteric site, located around 10-15 Å away from the
catalytic site [6]. With the group of the NNRTIs, the emergence of RT mutations rapidly confers
resistance to the first-generation NNRTIs, such as the Y181C mutation associated with NVP
resistance and the K103N mutation associated with EFV resistance [7]. However, the
diarylpyrimidine (DAPY) analogues, represented by TMC125 and TMC278, have been identified
as highly potent second-generation NNRTIs [8]. It was found that NVP and DLV inhibited
K103N/Y181C RT mutants at a range of EC50 >10 ꢀM to EC50 >100 ꢀM in cell-based assays,
while TMC125 and TMC278 could inhibit K103N/Y181C RT mutants at an EC50 value of 5 nM
and 0.8 nM, respectively [9].
The crystal structures of HIV-1 RT/ DAPY complexes and/or molecular modeling studies have
revealed some important features of enzyme-ligand interaction, helping to maintain the antiviral
activity against a wide range of resistance mutations [10, 11]. Although DAPYs shared a similar
pharmacophore including hydrophobic center, hydrogen bond donor and acceptor as mentioned
for the first-generation NNRTIs (NEV䟍DLV and EFV), the binding conformation of DAPYs
resembled a horseshoe or “U” shape when bound in the non-nucleoside inhibitor binding pocket
(NNIBP) [9, 10]. For instance, in the molecule of TMC125, the ether and amino linkages of the
two cyanophenyl substituents provide sufficient flexibility to allow strong π-π stacking
interactions with Y181, Y188, F227 and W229 [10]. It was suggested that DAPY NNRTIs bind to
RT through torsional flexibility (“wiggling”) and repositioning flexibility (jiggling”) [9]. This
concept proposes a general strategy for designing flexible drugs to target a variety of resistant
mutants. Recently, some novel DAPY analogues [8, 12] and triazine derivatives [13] have also
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been investigated as NNRTIs, whereas these analogues did not show highly potent antiviral
activity against clinically relevant mutant strains.
In addition, numerous structurally different non-nucleoside compounds have also been
investigated for inhibitory effects against HIV replication. A class of novel NNRTIs,
benzophenone derivatives (BPs) was developed starting from a lead with moderate activity against
wild-type (wt) HIV and little or no activity against clinically relevant mutants in a high-throughput
screening [14, 15]. For example, GW678248 (Fig. 1) exhibited very low EC50 values in antiviral
assays: 0.5 nM against wild-type, 1 nM against K103N and 0.7 nM against Y181C, respectively
[14]. Upon binding of GW678248 to RT, the B-ring is placed at the top hydrophobic pocket that is
lined with the aromatic residues Y181, Y188, and W229, while the A-ring placed a “down”
position and interacting with V179 via the p-chloro substituent on the A-ring [15]. Additionally,
the interaction of the chloro substituent with the main-chain carbonyl of Y188 is also maintained
[15]. As shown by the structure modifications of BPs, the benzophenone group was essential for
maintaining high anti-HIV activity [15]. The RT-bound structure of BPs pointed to a number of
features that aided in the design flexible drugs to target a variety of resistant mutants.
In our present study, we tried to combine the special structural features of diarylpyrimidines
(DAPYs) and benzophenones (BPs) to develop new NNRTIs, which originated from the concept
of molecular hybridization [16, 17]. Twenty-one analogs (Fig. 2) were selected for synthesis and
evaluation of in vitro antiviral activity against wt HIV-1, double mutant HIV-1 (K103N/Y181C)
and HIV-2. The preliminary SAR studies and molecular docking analysis for further investigation
are also discussed.
2. Results and discussion
2.1. Chemistry
The synthesis of the target compounds (1a–u) is shown in Scheme 1. The key intermediates
4-(4-chloro-pyrimidin-2-ylamino)benzonitriles (2a–c) were synthesized according to our
previous reported method in 3 or 5 steps [18]. The 4-chloro-hydroxybenzophenone derivatives (3)
were prepared from 4-chloroanisole via Friedel-Crafts acylation [14], and the others are
commercial available. Treatment of 2a–c with appropriate hydroxybenzophenone or phenol
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derivatives (3) in the presence of K2CO3 in anhydrous DMF at 80 ºC under nitrogen atmosphere
afforded the desired compounds 1a–u. The yields varied from 52.2% to 98.4%.
In the reaction with hydroxybenzophenone derivatives (3), the use of 2b–c led to lower yields
than that of 2a, which could be due to the steric hindrance from the methyl group substituted at
the pyrimidine ring. Besides the hydroxybenzophenone derivatives (3) listed in Scheme 1, the
reaction was also tried by using 4-chloro-2-(2-chlorobenzoyl)phenol and 4-chloro-2-(2,
6-dichlorobenzoyl)phenol as reagents, whereas the reaction failed at 80 ºC and gave only
degradation unknown compounds. When carried out at lower temperature (25–50 ºC), the
reaction still did not work. Thus, we also introduced some phenol derivatives for comparison.
2.2. Biological activity
The antiviral activity and cytotoxicity of the synthesized compounds (1a–u) were tested in
MT-4 cells. Their capacity to inhibit the replication of wild-type (wt) HIV-1 strain IIIB, the double
mutant HIV-1 strain RES056 (K103N + Y181C) and the HIV-2 strain ROD was evaluated [19,
20]. The results, expressed as EC50, CC50, and SI (selectivity index) values are given in Table 1.
FDA-approved drugs, including the NNRTIs NEV, EFV, DLV, and the NRTI zidovudine (AZT)
were used as reference according to the same procedure. Additionally, the biological data of
TMC125 evaluated by the same laboratory [12], and the previously reported antiviral data of
GW678248 [21] and TMC278 [9] were also included in Table 1 for comparison.
In the antiviral assay, the majority of the target molecules showed moderate to good inhibitory
activity against wt HIV-1 within a range of EC50 values from 46.06 ± 13.98 ꢀM to 0.06 ± 0.01
ꢀM. Among these analogues, compound 1u displayed the highest potency with an EC50 value of
0.06 ± 0.01 ꢀM against HIV-1, and a selectivity index value > 6260, whereas it completely lost
potency against the double mutant type virus (K103N + Y181C) and HIV-2. In addition,
compound 1g displayed the inhibitory activity with an EC50 value of 0.26 ± 0.06 ꢀM against
HIV-1, 93.25 ꢀM against the double mutant type virus (K103N + Y181C) and 46.19 ꢀM against
HIV-2, respectively. Although compounds 1b, 1n and 1u were also potent against wide-type
HIV-1 within less than 1 ꢀM as NVP, DLV, and GW678248, these newly synthesized compounds
lacked inhibitory activities against the double mutant virus (K103N + Y181C) and HIV-2.
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In terms of SARs, it seemed that the changes in the A- and B-ring proved to have some
influence on the antiviral potency of the analogues. For example, compound 1b was about 2-fold
more active than compound 1c, which indicated 3, 5-dimethyl is more beneficial than 2-methyl as
the substituent on the B-ring. Nevertheless, it was unexpected that compound 1e with
para-position linked between A-ring and B-ring was 2 to 3-fold less active than compounds 1b and
1c with that linkage of ortho-position.
In addition, compounds 1b was 3-fold less active than compound 1g, while 58-fold more active
than compound 1f, and both 1g and 1f were not substituted by R5 at A-ring. It was found that
compound 1g (R4 = 2-MeO-Et-) was 177-fold more active against wt HIV-1 than compound 1f
(R4 = BnO-), which might imply the 2-MeO-Et- group was more favorable for the para-position
substitution on the A-ring of O-DAPYs than BnO- group. In general, the analogues 1a–e with
ortho- or para-position substituted benzoyl substituent as R5 displayed lower inhibitory activities
than the analogue 1g without such substitution.
Evaluation of the cytotoxicity showed that compounds 1a and 1d possess a CC50 value at low
micromolar concentration, which indicated that the introduction of the 4-Cl-4′-Me-BP and
3-OMe-BP in DAPYs was not as tolerable as the other benzophenones.
For the substitute R1 or R2 on the pyrimidine ring of BP-O-DAPYs, compounds 1h–l (R1 = H,
R2 = CH3) decreased the potency against wild type HIV-1 virus, when compared with the
analogues 1a–e (R1 = H, R2 = H) and 1o–s (R1 = CH3, R2 = H). As an example, 1l was 1.9-fold
and 1.4-fold less active against wt HIV-1 than 1e and 1s, respectively. In addition, compound 1u
(R1 = CH3, R2 = H) displayed the highest potency with an EC50 value of 0.06 ± 0.01 ꢀM against
HIV-1, which is 4.3-fold and 13.2-fold more active than compound 1g (R1 = H, R2 = H) and
compound 1n (R1 = H, R2 = CH3), respectively. Based on the results, further modifications on the
pyrimidine ring may be favorable for improving the biological activity.
To identify the binding target, 12 selected compounds were carried out an HIV-1 RT inhibitory
assay (Table 2), using poly(rA)/oligo(dT)16 as template and primer, NVP and EFV as reference [22,
23]. Although most of the tested compounds exhibited moderate inhibitory activity against wt
HIV-1 RT, the results confirmed the consistent SAR information as obtained in antiviral
evaluation in MT-4 cells. As an example, compound 1u still displayed the highest inhibitory
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activity against wt HIV-1 RT with IC50 value of 1.27 ± 0.02 ꢀM, which was higher than NVP
(IC50 = 2.74 ± 0.11 ꢀM), but lower than EFV (IC50 = 0.048 ± 0.000 ꢀM). These data suggest that
these synthesized compounds bind to HIV-1 RT and belong to HIV-1 NNRTIs.
2.3. Molecular Modeling Analysis
Molecular docking was used to study the potential binding mode of the synthesized compounds
in the non-nucleoside inhibitor-binding pocket (NNIBP) of wild-type and mutated HIV-1 RT,
respectively. The crystallographic structure of wt HIV-1 RT in complex with TMC125 (PDB ID
3MEC) [10] was used for the molecular docking of the representative compounds 1b, 1e, 1g and
1u in the binding site of wt HIV-1 RT, considering their structural diversity and potency. Among
the four compounds, 1b and 1e introduce benzoyl substituents on the A-ring, while 1g and 1u
have alkoxyl group at the para-postion of the A-ring. The docking simulations were carried out on
the basis of literature protocols [24-26], using the Base-Builder and Surflex-Dock programs
interfaced with SYBYL-1.2.
As shown in Fig. 3, compounds 1b, 1e, 1g and 1u are predicted to bind with NNIBP in a “U”
mode, which is similar to the binding mode of TMC125. Two typical hydrogen bonds indicated
the interaction between the nitrogen atom of pyrimidine ring, the NH linker and Lys 101, which is
crucial for the anti-HIV activity of DAPYs. The π-π interaction between these four compounds
and the aromatic amino acid residue Tyr181 was observed to be similar to that with TMC125. The
binding mode of compound 1b was not as expected and the direct-linked A-ring of 1b actually
moved downwards, while the other B-ring guided the interaction with Tyr 181. However,
compound 1e displayed the π-π interaction between the aromatic amino acid residues Tyr181 and
the direct-linked A-ring, which implied that the para-position of the benzophenone structure could
lead to less steric hindrance than that of the ortho-position. In addition, the orientation of the “U”
shape for these four compounds (1b, 1e, 1g and 1u) moved downward when comparing that for
TMC125 in purple. The linear nitrile group on the C-ring of the synthesized compounds was
positioned farther from the side chains of Phe227, Leu234 and His235 than that of TMC125. The
interaction with the residues Leu234 and Phe227 were not obviously observed with compounds 1b,
1e, 1g and 1u, and which may determine the loss of their anti-HIV-1 (wt) activities.
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To further explore the interaction of the synthesized compounds with mutated HIV-1 RT, the
crystallographic structure of mutant HIV-1 RT (K103N/Y181C) in complex with TMC278 (PDB
ID 3BGR) [9] was similarly used for docking the relatively active compounds 1b and 1g in the
NNIBP of HIV-1 RT (Fig. 4). The docking simulations clearly showed that the binding sites of
compounds 1b and 1g with enzyme were greatly different from that of TMC278 and there was
only one classical hydrogen bond between the nitrogen (N) in the pyrimdine ring of 1g with Lys
101. In addition, the crystallographic structure of mutated RT (K103N) with GW678248 (PDB ID
3DOK) [15] was also used for docked compounds 1b and 1d (Fig. 4) in the binding site. For 1d,
the important intramolecular hydrogen bond between Asn103 with NH group of the linker joining
the central pyrimidine and right phenyl ring still existed; however, there were no interactions (π-π,
van der Waals) with the key amide acid residues Leu234 and Tyr181.
Generally, the related binding features indicate that the benzoyl structure on the A-ring of
DAPYs may interact with the residues Tyr 181 and Tyr188 of wt RT enzyme as designed, whereas
most of the compounds lacked contact with Leu234 of both wt RT and mutant RT, and also failed
to interact with residues Cys181 and Asn 103 of the double-mutant strain RES056 (K103N +
Y181C) to some extent. Therefore, the predicted binding of these compounds with RT is
consistent with explaining their observed relatively higher activity against wild-type HIV-1, while
lacking activity against the double-mutant strain RES056.
2.4. Conclusion
In conclusion, a series of DAPY derivatives with benzoyl or alkoxyl group substitution on the
A-ring were synthesized and evaluated for their antiviral activity against HIV in MT-4 cells. Most
of the compounds showed the inhibitory activity against wild-type HIV-1 at a range of EC50
values from 46.06 ± 13.98 ꢀM to 0.06 ± 0.01 ꢀM. Compound 1u exhibited the most potent
activity with an EC50 value of 0.06 ± 0.01 ꢀM against HIV-1, and a selectivity index value of >
6260. However, these compounds lost the potency against the double mutant type virus (K103N +
Y181C) and HIV-2, except for compound 1g which displayed weak inhibitory activity with an
EC50 value of 93.25 ꢀM against the double mutant type virus (K103N + Y181C) and 46.19 ꢀM
against HIV-2, respectively. The HIV-1 reverse transcriptase assay of 12 synthesized compounds
also indicates that the target of these compounds is HIV-1 RT. In addition, we also carried out
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molecular docking to analyze the difference of the binding modes with both wt and mutant HIV-1
RT between the synthesized compounds and the highly potent TMC125 and TMC278,
respectively. Further structural modifications are needed for developing the compounds with
higher potency against mutated RT and lower cytotoxicity.
3. Experimental Section
3.1. Chemistry
All chemicals and solvents used were purchased from commercial sources, were of analytical grade and
were used without further purification. Melting points were measured on a SGW X-4 microscopic
melting-point apparatus and were uncorrected. Mass spectra were obtained on a Waters Quattro Micromass
1
instrument using electrospray ionization (ESI) techniques. H NMR and 13C NMR spectra on a Brucker AV
400 MHz spectrometer were recorded in DMSO-d6. Chemical shifts are listed in δ (ppm) units relative to the
internal standard tetramethylsilane (TMS). TLC analyses were run on silica gel 60 F254 plates (Merck)
using a variety of solvent systems and the spots were examined with UV light (254 nm). Column
chromatography was performed on silica gel (300~400 mesh) using hexane/ethyl acetate as eluents.
3.2. General Procedure for the Synthesis of 1a–u
To a solution of hydroxybenzophenone or phenol derivatives (3) (1 mmol) in anhydrous DMF (3 mL),
K2CO3 (2 mmol) was added at room temperature under N2. The mixture was stirred for 10 min and the
corresponding 2a–c (1 mmol) was added, respectively. The reaction mixture was heated at 80˚C for 5–15 h.
Then, the mixture was poured into ice-cold H2O (30 mL) and the resulting precipitate was filtered off. The
obtained solid was further washed with ice-cold water (10 mL). The crude products 1a–u were finally
purified by silica column chromatography eluting with hexane/ethyl acetate = 5 : 1 to 1 : 1 to yield the pure
compounds in 52.2% ~ 98.4%.
3.2.1. 4-{4-[4-Chloro-2-(4-methylbenzoyl)phenoxy]pyrimidin-2-ylamino}benzonitrile (1a).
Yield 91.0%; yellowish solid, mp 192.2–193.8 °C; 1H NMR (400 MHz, DMSO-d6) δ (ppm): 2.28 (s, 3H,
CH3), 6.36 (d, 1H, J = 5.6 Hz, CH), 7.18–7.81 (m, 11H, PhH), 8.30 (d, 1H, J = 5.6 Hz, CH), 9.99 (s, 1H,
NH); 13C NMR (100 MHz, DMSO-d6) δ (ppm): 21.65, 100.13, 103.16, 118.98 (2C), 119.90, 126.20, 129.53
(2C), 129.59 (2C), 130.23, 130.50, 132.33, 133.18 (2C), 133.78, 134.60, 144.84, 145.02, 148.84, 159.00,
160.61, 169.20, 192.56; MS (ESI+) m/z for C25H17ClN4O2 (M+H)+ calcd: 441.11, found: 441.16.
3.2.2. 4-{4-[4-Chloro-2-(3,5-dimethylbenzoyl)phenoxy]pyrimidin-2-ylamino}benzonitrile (1b).
1
Yield 79.4%; white solid, mp 179.7–181.0 °C; H NMR (400 MHz, DMSO-d6) δ (ppm): 2.11 (s, 6H,
2CH3), 6.35 (d, 1H, J = 5.6 Hz, CH), 7.15–7.82 (m, 10H, PhH), 8.29 (d, 1H, J = 5.6 Hz, CH), 10.09 (s, 1H,
NH); 13C NMR (100 MHz, DMSO-d6) δ (ppm): 20.32 (2C), 99.62, 102.68, 118.37 (2C), 119.34, 125.83,
127.01 (2C), 129.03, 130.03, 131.82, 132.65 (2C), 134.19, 135.12, 136.03, 137.77 (2C), 144.38, 148.32,
158.52, 160.09, 168.57, 192.86; MS (ESI+) m/z for C26H19ClN4O2 (M+H)+ calcd: 455.13, found: 455.24.
3.2.3. 4-{4-[4-Chloro-2-(2-methylbenzoyl)phenoxy]pyrimidin-2-ylamino}benzonitrile (1c).
1
Yield 93.4%; white solid, mp 173.3–174.0 °C; H NMR (400 MHz, DMSO-d6) δ (ppm): 2.18 (s, 3H,
CH3), 6.19 (d, 1H, J = 5.6 Hz, CH), 7.07–7.85 (m, 11H, PhH), 8.27 (d, 1H, J = 5.6 Hz, CH), 10.06 (s, 1H,
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NH); 13C NMR (100 MHz, DMSO-d6) δ (ppm): 19.74, 99.41, 102.66, 118.46 (2C), 119.41, 125.29, 125.96,
129.99, 130.19, 130.32, 131.20, 131.77, 132.64 (2C), 133.01, 134.49, 136.70, 137.49, 144.36, 148.83,
158.47, 159.95, 168.56, 194.36; MS (ESI+) m/z for C25H17ClN4O2 (M+H)+ calcd: 441.11, found: 441.21.
3.2.4. 4-{4-[2-Benzoyl-5-methoxyphenoxy]pyrimidin-2-ylamino}benzonitrile (1d).
1
Yield 92.9%; yellowish solid, mp 156.3–157.6 °C; H NMR (400 MHz, DMSO-d6) δ (ppm): 3.87 (s, 3H,
CH3), 6.28 (d, 1H, J = 5.6 Hz, CH), 7.03–7.67 (m, 12H, PhH), 8.27 (d, 1H, J = 5.6 Hz, CH), 9.99 (s, 1H,
NH); 13C NMR (100 MHz, DMSO-d6) δ (ppm): 56.06, 99.69, 102.65, 109.32, 111.61, 118.51 (2C), 119.53,
124.16, 128.34 (2C), 129.32 (2C), 132.45, 132.73 (2C), 132.97, 137.54, 144.53, 152.01, 158.68, 159.97,
163.05, 169.03, 193.41; MS (ESI+) m/z for C25H18N4O3 (M+H)+ calcd: 423.15, found: 423.27.
3.2.5. 4-{4-[4-(4-Hydroxybenzoyl)phenoxy]pyrimidin-2-ylamino}benzonitrile (1e).
Yield 96.0%; white solid, mp 244.9–245.5 °C; 1H NMR (400 MHz, DMSO-d6) δ (ppm): 6.68 (d, 1H, J =
5.6 Hz, CH), 6.91–7.82 (m, 12H, PhH), 8.49 (d, 1H, J = 5.6 Hz, CH), 10.16 (s, 1H, NH); 13C NMR (100
MHz, DMSO-d6) δ (ppm): 100.05, 102.61, 115.34 (2C), 118.47 (2C), 119.39, 122.02 (2C), 127.80, 131.12,
132.53 (2C), 132.68 (2C), 135.61, 144.45, 155.06, 158.96, 160.42, 162.14, 169.14, 193.46; MS (ESI+) m/z
for C24H16N4O3 (M-H)- calcd: 407.41, found: 407.65.
3.2.6. 4-{4-[4-Benzyloxyphenoxy]pyrimidin-2-ylamino}benzonitrile (1f).
1
Yield 96.6%; white solid, mp 191.3–192.0 °C; H NMR (400 MHz, DMSO-d6) δ (ppm): 5.17 (s, 2H,
CH2), 6.54 (d, 1H, J = 5.6 Hz, CH), 7.11–7.70 (m, 13H, PhH), 8.41 (d, 1H, J = 5.6 Hz, CH), 10.12 (s, 1H,
NH); 13C NMR (100 MHz, DMSO-d6) δ (ppm): 69.53, 99.58, 102.36, 115.67 (2C), 118.31 (2C), 119.43,
122.91 (2C), 127.69 (2C), 127.91, 128.47 (2C), 132.58 (2C), 137.01, 144.54, 145.69, 155.93, 158.97, 159.93,
169.76; MS (ESI+) m/z for C24H18N4O2 (M+H)+ calcd: 395.15, found: 395.24.
3.2.7. 4-{4-[4-(2-Methoxylethyl)phenoxy]pyrimidin-2-ylamino}benzonitrile (1g).
Yield 98.4%; white solid, mp 154.2–156.0 °C; 1H NMR (400 MHz, DMSO-d6) δ (ppm): 2.88 (t, 2H, J =
6.6 Hz, CH2), 3.29 (s, 3H, CH3), 3.62 (t, 2H, J = 6.6 Hz, CH2), 6.58 (d, 1H, J = 5.6 Hz, CH), 7.15–7.66 (m,
8H, PhH), 8.42 (d, 1H, J = 5.6 Hz, CH), 10.13 (s, 1H, NH); 13C NMR (100 MHz, DMSO-d6) δ (ppm): 34.63,
57.84, 72.67, 99.69, 102.36, 118.34 (2C), 119.45, 121.72 (2C), 130.12 (2C), 132.62 (2C), 136.74, 144.51,
150.52, 158.97, 160.05, 169.55; MS (ESI+) m/z for C20H18N4O2 (M+H)+ calcd: 347.15, found: 347.28.
3.2.8. 4-{6-Methyl-4-[4-chloro-2-(4-methylbenzoyl)phenoxy]pyrimidin-2-ylamino}benzonitrile (1h).
1
Yield 78.4%; white solid, mp 197.8–200.0 °C; H NMR (400 MHz, DMSO-d6) δ (ppm): 2.26 (s, 3H,
CH3), 2.29 (s, 3H, CH3), 6.23 (s, 1H, CH), 7.17–7.79 (m, 11H, PhH), 9.94 (s, 1H, NH); 13C NMR (100 MHz,
DMSO-d6) δ (ppm): 21.18, 23.46, 98.18, 102.51, 118.42 (2C), 119.47, 125.73, 128.99, 129.10 (2C), 129.74
(2C), 129.90, 131.79, 132.61 (2C), 133.39, 134.18, 144.52, 144.55, 148.53, 158.15, 169.21, 170.13, 192.13;
MS (ESI+) m/z for C26H19ClN4O2 (M+H)+ calcd: 455.92, found: 455.22.
3.2.9. 4-{6-Methyl-4-[4-chloro-2-(3,5-dimethylbenzoyl)phenoxy]pyrimidin-2-ylamino}benzonitrile (1i).
1
Yield 72.2%; white solid, mp 160.1–162.0 °C; H NMR (400 MHz, DMSO-d6) δ (ppm): 2.09 (s, 6H,
2CH3), 2.24 (s, 3H, CH3), 6.22 (s, 1H, CH), 7.14–7.79 (m, 10H, PhH), 10.02 (s, 1H, NH); 13C NMR (100
MHz, DMSO-d6) δ (ppm): 20.39 (2C), 23.48, 98.24, 102.55, 118.37 (2C), 119.49, 125.92, 127.10 (2C),
129.04, 130.00, 131.87, 132.69 (2C), 134.36, 135.18, 136.17, 137.89 (2C), 144.65, 148.63, 158.19, 169.15,
170.22, 193.03; MS (ESI+) m/z for C27H21ClN4O2 (M+H)+ calcd: 469.15, found: 469.27.
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3.2.10. 4-{6-Methyl-4-[4-chloro-2-(2-methylbenzoyl)phenoxy]pyrimidin-2-ylamino}benzonitrile (1j).
1
Yield 78.4%; yellowish solid, mp 153.6–155.2 °C; H NMR (400 MHz, DMSO-d6) δ (ppm): 2.18 (s, 3H,
CH3), 2.25 (s, 3H, CH3), 6.03 (s, 1H, CH), 7.07–7.84 (m, 11H, PhH), 9.99 (s, 1H, NH); 13C NMR (100 MHz,
DMSO-d6) δ (ppm): 19.67, 23.35, 97.86, 102.44, 118.32 (2C), 119.37, 125.19, 125.93, 129.85, 130.04,
130.15, 131.05, 131.60, 132.51 (2C), 132.87, 134.47, 136.74, 137.42, 144.49, 149.02, 158.03, 168.99,
169.82, 194.32; MS (ESI+) m/z for C26H19ClN4O2 (M+H)+ calcd: 455.92, found: 455.27.
3.2.11. 4-{6-Methyl-4-[2-benzoyl-5-methoxyphenoxy]pyrimidin-2-ylamino}benzonitrile (1k).
1
Yield 74.7%; yellowish solid, mp 169.2–170.8 °C; H NMR (400 MHz, DMSO-d6) δ (ppm): 2.25 (s, 3H,
CH3), 3.86 (s, 3H, CH3), 6.14 (s, 1H, CH), 6.99–7.66 (m, 12H, PhH), 9.93 (s, 1H, NH); 13C NMR (100 MHz,
DMSO-d6) δ (ppm): 23.45, 55.97, 102.41, 109.27, 111.37, 118.36 (2C), 119.50, 119.59, 124.21, 128.23 (2C),
129.26 (2C), 132.28, 132.60 (2C), 132.82, 137.53, 144.66, 152.14, 158.25, 162.94, 169.44, 169.80, 193.33;
MS (ESI+) m/z for C26H20N4O3 (M+H)+ calcd: 437.48, found: 437.29.
3.2.12. 4-{6-Methyl-4-[4-(4-hydroxybenzoyl)phenoxy]pyrimidin-2-ylamino}benzonitrile (1l).
1
Yield 83.5%; yellowish solid, mp 216.1–218.2 °C; H NMR (400 MHz, DMSO-d6) δ (ppm): 2.37 (s, 3H,
CH3), 6.52 (s, 1H), 6.91–7.78 (m, 12H, PhH), 10.08 (s, 1H, NH), 10.41 (br, 1H, OH); 13C NMR (100 MHz,
DMSO-d6) δ (ppm): 23.64, 98.57, 102.43, 115.07, 115.36, 118.38 (2C), 119.46, 122.00 (2C), 127.89, 128.84,
131.10, 132.07, 132.54, 132.62, 135.48, 144.64, 155.28, 158.58, 161.27, 162.14, 169.58, 170.38, 193.51; MS
(ESI+) m/z for C25H18N4O3 (M+H)+ calcd: 423.45, found: 423.27.
3.2.13. 4-{6-Methyl-4-[(4-benzyloxy)phenoxy]pyrimidin-2-ylamino}benzonitrile (1m).
1
Yield 74.9%; white solid, mp 111.6–112.0 °C; H NMR (400 MHz, DMSO-d6) δ (ppm): 2.35 (s, 3H,
CH3), 5.17 (s, 2H, CH2), 6.40 (s, 1H, CH), 7.10–7.67 (m, 13H, PhH), 10.07 (s, 1H, NH); 13C NMR (100
MHz, DMSO-d6) δ (ppm): 23.49, 69.51, 97.98, 102.13, 115.60 (2C), 118.16 (2C), 119.43, 122.87 (2C),
127.63 (2C), 127.87, 128.42 (2C), 132.53 (2C), 137.00, 144.67, 145.82, 155.82, 158.57, 169.72, 170.22; MS
(ESI+) m/z for C25H20N4O2 (M+H)+ calcd: 409.47, found: 409.23.
3.2.14. 4-{6-Methyl-4-[4-(2-methoxylethyl)phenoxy]pyrimidin-2-ylamino}benzonitrile (1n).
1
Yield 96.2%; white solid, mp 129.4–131.8 °C; H NMR (400 MHz, DMSO-d6) δ (ppm): 2.36 (s, 3H,
CH3), 2.88 (t, 2H, J = 6.6 Hz, CH2), 3.30 (s, 3H, CH3), 3.62 (t, 2H, J = 6.6 Hz, CH2), 6.45 (s, 1H, CH),
7.12–7.66 (m, 8H, PhH), 10.09 (s, 1H, NH); 13C NMR (100 MHz, DMSO-d6) δ (ppm): 23.48, 34.59, 57.78,
72.64, 98.11, 102.11, 118.17 (2C), 119.42, 121.66 (2C), 129.99 (2C), 132.52 (2C), 136.54, 144.63, 150.62,
158.53, 169.80, 169.98; MS (ESI+) m/z for C21H20N4O2 (M+H)+ calcd: 361.42, found: 361.24.
3.2.15. 4-{5-Methyl-4-[4-chloro-2-(4-methylbenzoyl)phenoxy]pyrimidin-2-ylamino}benzonitrile (1o).
1
Yield 74.0%; white solid, mp 183.6–185.5 °C; H NMR (400 MHz, DMSO-d6) δ (ppm): 1.73 (s, 3H,
CH3), 2.30 (s, 3H, CH3), 7.19–7.84 (m, 11H, PhH), 8.12 (1H, CH), 9.82 (s, 1H, NH); 13C NMR (100 MHz,
DMSO-d6) δ (ppm): 11.05, 21.21, 102.21, 108.69, 118.15 (2C), 119.68, 125.95, 129.08 (2C), 129.29, 129.24,
129.60 (2C), 130.11, 132.05, 132.64 (2C), 133.64, 134.17, 144.47, 148.75, 156.94, 159.08, 166.84, 192.39;
MS (ESI+) m/z for C26H19ClN4O2 (M+H)+ calcd: 455.92, found: 455.21.
3.2.16. 4-{5-Methyl-4-[4-chloro-2-(3,5-dimethylbenzoyl)phenoxy]pyrimidin-2-ylamino}benzonitrile (1p).
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1
Yield 52.2%; white solid, mp 196.7–197.3 °C; H NMR (400 MHz, DMSO-d6) δ (ppm): 1.74 (s, 3H,
CH3), 2.11 (s, 6H, 2CH3), 7.15–7.84 (m, 10H, PhH), 8.10 (1H, CH), 9.91(s, 1H, NH); 13C NMR (100 MHz,
DMSO-d6) δ (ppm): 10.98, 20.36 (2C), 102.17, 108.63, 118.00 (2C), 119.51, 126.15, 126.80 (2C), 129.27,
130.07, 132.01, 132.65 (2C), 134.11, 134.91, 136.39, 137.76 (2C), 144.72, 148.73, 156.94, 159.04, 166.65,
193.17; MS (ESI+) m/z for C27H21ClN4O2 (M+H)+ calcd: 469.95, found: 469.24.
3.2.17. 4-{5-Methyl -4-[4-chloro-2-(2-methylbenzoyl)phenoxy]pyrimidin-2-ylamino}benzonitrile (1q).
1
Yield 74.0%; white solid, mp 138.8–141.0 °C; H NMR (400 MHz, DMSO-d6) δ (ppm): 1.61 (s, 3H,
CH3), 2.08 (s, 3H, CH3), 7.11–7.85 (m, 11H, PhH), 8.09 (1H, CH), 9.88 (s, 1H, NH); 13C NMR (100 MHz,
DMSO-d6) δ (ppm): 10.83, 19.45, 102.15, 108.58, 118.04 (2C), 119.47, 125.29, 125.95, 129.85, 130.08,
130.38, 131.12, 131.66, 132.52 (2C), 133.15, 134.60, 137.04, 137.24, 144.63, 149.20, 156.90, 158.90,
166.67, 194.32; MS (ESI+) m/z for C26H19ClN4O2 (M+H)+ calcd: 455.92, found: 455.28.
3.2.18. 4-{5-Methyl-4-[2-benzoyl-5-methoxyphenoxy]pyrimidin-2-ylamino}benzonitrile (1r).
1
Yield 63.1%; white solid, mp 183.0–184.0 °C; H NMR (400 MHz, DMSO-d6) δ (ppm): 1.70 (s, 3H,
CH3), 3.87 (s, 3H, CH3), 7.02–7.54 (m, 12H, PhH), 8.09 (s, 1H, CH), 9.83 (s, 1H, NH); 13C NMR (100 MHz,
DMSO-d6) δ (ppm): 11.13, 56.03, 102.08, 108.72, 109.29, 111.78, 118.04 (2C), 119.59, 124.31, 128.28 (2C),
129.04 (2C), 132.61 (2C), 132.80, 137.93, 144.79, 152.28, 157.02, 158.83, 163.15, 165.44, 167.02, 193.66;
MS (ESI+) m/z for C26H20N4O3 (M+H)+ calcd: 437.48, found: 437.27.
3.2.19. 4-{5-Methyl-4-[4-(4-hydroxybenzoyl)phenoxy]pyrimidin-2-ylamino}benzonitrile (1s).
1
Yield 84.7%; white solid, mp >250 °C; H NMR (400 MHz, DMSO-d6) δ (ppm): 2.23 (s, 3H, CH3),
6.92–7.83 (m, 12H, PhH), 8.36 (s, 1H, CH), 9.98 (s, 1H, NH), 10.16 (s, 1H, OH); 13C NMR (100 MHz,
DMSO-d6) δ (ppm): 11.62, 101.92, 108.91, 115.20 (2C), 117.86 (2C), 119.34, 122.04 (2C), 127.75, 130.88
(2C), 132.36 (2C), 132.49 (2C), 135.32, 144.65, 155.37, 157.17, 159.34, 161.99, 167.21, 193.30; MS (ESI+)
m/z for C25H18N4O3 (M-H)- calcd: 421.43, found: 421.91.
3.2.20. 4-{5-Methyl-4-[(4-benzyloxy)phenoxy]pyrimidin-2-ylamino}benzonitrile (1t).
1
Yield 82.9%; white solid, mp 209.1–210.6 °C; H NMR (400 MHz, DMSO-d6) δ (ppm): 2.18 (s, 3H,
CH3), 5.16 (s, 2H, CH2), 7.10–7.58 (m, 13H, PhH), 8.26 (s, 1H, CH), 9.89 (s, 1H, NH); 13C NMR (100 MHz,
DMSO-d6) δ (ppm): 11.76, 69.60, 101.80, 108.69, 115.57 (2C), 117.89 (2C), 119.53, 123.15 (2C), 127.68
(2C), 127.95, 128.50 (2C), 132.48 (2C), 137.07, 144.84, 146.09, 155.82, 157.26, 158.87, 167.84; MS (ESI+)
m/z for C25H20N4O2 (M+H)+ calcd: 409.47, found: 409.21.
3.2.21. 4-{5-Methyl-4-[4-(2-methoxylethyl)phenoxy]pyrimidin-2-ylamino}benzonitrile (1u).
1
Yield 94.0%; white solid, mp 181.0–181.9 °C; H NMR (400 MHz, DMSO-d6) δ (ppm): 2.17 (s, 3H,
CH3), 3.29 (t, 2H, J = 6.6 Hz, CH2), 3.50 (s, 3H, CH3), 3.61 (t, 2H, J = 6.6 Hz, CH2), 7.10–7.55 (m, 8H,
PhH), 8.26 (s, 1H, CH), 9.90 (s, 1H, NH); 13C NMR (100 MHz, DMSO-d6) δ (ppm): 11.81, 34.74, 57.92,
72.79, 101.85, 108.72, 117.97 (2C), 119.61, 121.98 (2C), 130.01 (2C), 132.57 (2C), 136.57, 144.85, 150.95,
157.32, 159.09, 167.74; MS (ESI+) m/z for C21H20N4O2 (M+H)+ calcd: 361.42, found: 361.24.
3.3. Biological evaluation
Anti-HIV assays: The anti-HIV activity and cytotoxicity of the compounds 1a–u, were evaluated against
wild-type HIV-1 strain IIIB, a double RT mutant (K103N + Y181C) HIV-1 strain and HIV-2 strain ROD in
MT-4 cell cultures using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) method
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[19, 20]. Briefly, stock solutions (10 × final concentration) of test compounds were added in 25 µL volumes
to two series of triplicate wells so as to allow simultaneous evaluation of their effects on mock-and
HIV-infected cells at the beginning of each experiment. Serial 5-fold dilutions of test compounds were made
directly in flat-bottomed 96-well microtiter trays using a Biomek 3000 robot (Beckman instruments).
Untreated control HIV-and mock-infected cell samples were included for each sample. Virus stock (50 µL) at
100-300 CCID50 (50% cell culture infectious dose) or culture medium was added to either the virus-infected
or mock-infected wells of the microtiter tray. Mock-infected cells were used to evaluate the effect of test
compounds on uninfected cells in order to assess the cytotoxicity of the test compounds. Exponentially
growing MT-4 cells were centrifuged for 5 min at 220 g and the supernatant was discarded. The MT-4 cells
were resuspended at 6 × 105 cells/mL and 50 µL volumes were transferred to the microtiter tray wells. Five
days after infection, the viability of mock-and HIV-infected cells was examined spectrophotometrically by
the MTT assay.
The MTT assay is based on the reduction of yellow colored MTT (Acros Organics) by mitochondrial
dehydrogenase activity of metabolically active cells to a blue-purple formazan that can be measured
spectrophotometrically. The absorbances were read in an eight-channel computer-controlled photometer
(Infinite M1000, Tecan), at two wavelengths (540 and 690 nm). All data were calculated using the median
OD (optical density) values of tree wells. The 50% cytotoxic concentration (CC50) was defined as the
concentration of the test compound that reduced the absorbance (OD540) of the mock-infected control
sample by 50%. The concentration achieving 50% protection from the cytopathic effect of the virus in
infected cells was defined as the 50% effective concentration (EC50).
Reverse transcriptase assay: Recombinant wild type p66/p51 HIV-1 RT was expressed and purified as
the previously described procedure [22]. The RT assay is performed with the EnzCheck Reverse
Transcriptase Assay kit (Molecular Probes, Invitrogen), as described by the manufacturer. The assay is based
on the dsDNA quantitation reagent PicoGreen. This reagent shows a pronounced increase in fluorescence
signal upon binding to dsDNA or RNA-DNA heteroduplexes. Single-stranded nucleic acids generate only
minor fluorescence signal enhancement when a sufficiently high dye:base pair ratio is applied [23]. This
condition is met in the assay.
A poly(rA) template of approximately 350 bases long, and an oligo(dT)16 primer, are annealed in a molar
ratio of 1:1.2 (60 min, at room temperature). Fifty-two ng of the RNA/DNA is brought into each well of a
96-well plate in a volume of 20 µL polymerization buffer (60 mM Tris-HCl, 60 mM KCl, 8 mM MgCl2, 13
mM DTT, 100 µM dTTP, pH 8.1). Five µL of RT enzyme solution, diluted to a suitable concentration in
enzyme dilution buffer (50 mM Tris-HCl, 20% glycerol, 2 mM DTT, pH 7.6), is added. The reactions are
incubated at 25°C for 40 minutes and then stopped by the addition of EDTA (15 mM). Heteroduplexes are
then detected by addition of PicoGreen. Signals are read using an excitation wavelength of 490 nm and
emission detection at 523 nm using a spectrofluorometer (Safire2, Tecan). To test the activity of compounds
against RT, 1 µL of compound in DMSO is added to each well before the addition of RT enzyme solution.
Control wells without compound contain the same amount of DMSO. Results are expressed as relative
fluorescence i.e. the fluorescence signal of the reaction mix with compound divided by the signal of the same
reaction mix without compound.
3.4. Molecular simulation
Molecular modeling was carried out with the Tripos molecular modeling packages Sybyl-1.2. All the
molecules for docking were built using standard bond lengths and angles from Sybyl-1.2/base Builder and
were then optimized using the Tripos force field for 2000 generations two times or more, until the minimized
conformers of the ligand were the same. The flexible docking method, called Surflex-Dock, docks the ligand
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automatically into the ligand binding site of the receptor by using a protocol-based approach and an
empirically derived scoring function [26-28]. The protocol is a computational representation of a putative
ligand that binds to the intended binding site and is a unique and essential element of the docking algorithm.
The scoring function in Surflex-Dock, which contains hydrophobic, polar, repulsive, entropic, and salvation
terms, was trained to estimate the dissociation constant (Kd) expressed in –log(Kd)2. Prior to docking, the
protein was prepared by removing water molecules, the ligands (TMC125 and TMC278), and other
unnecessary small molecules from the crystal structure of the ligands–HIV-1 RT complex (PDB code:
3MEC [10] and 3BGR [9]); similarly with the ligand (GW678248), and other unnecessary small molecules
from the crystal structure of the with GW678248 (PDB code: 3DOK) [15]; simultaneously, polar hydrogen
atoms were added to the protein. Surflex-Dock default settings were used for other parameters, such as the
number of starting conformations per molecule (set to 0), the size to expand search grid (set to 8 Å), the
maximum number of rotatable bonds per molecule (set to 100), and the maximum number of poses per
ligand (set to 20). During the docking procedure, all of the single bonds in residue side chains inside the
defined RT binding pocket were regarded as rotatable or flexible, and the ligand was allowed to rotate on all
single bonds and move flexibly within the tentative binding pocket. The atomic charges were recalculated
using the Kollman all-atom approach for the protein and the Gasteiger-Hückel approach for the ligand. The
binding interaction energy was calculated to include van der Waals, electrostatic, and torsional energy terms
defined in the Tripos force field. The structure optimization was performed for 20000 generations using a
genetic algorithm, and the 20-best-scoring ligand–protein complexes were kept for further analyses. The
–log(Kd)2 values of the 20-best-scoring complexes, which represented the binding affinities of ligand with
RT, ranged over a wide scope of functional classes (10-2–10-9). Therefore, only the highest-scoring 3D
structural model of the ligand-bound RT was chosen to define the binding interaction [27, 28].
Acknowledgments
This work was supported in part by grants from the General Financial Grant from the China Postdoctoral
Science Foundation (No. 2012M511046) and the National Natural Science Foundation of China (No.
20872018 and No. 81172918). We would like to thank Jun Wang for MS and Dr. Wen-Xue Chen for
running the NMR spectra. We are indebted to Kristien Erven, Cindy Heens and Kris Uyttersprot for
evaluating the compounds for their antiviral activity.
Reference and note
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Figure captions
Table 1. Antiviral activity test of compounds 1a-u against HIV-1 and HIV-2 in MT-4 cells
Table 2. Activity of selected compounds against wt HIV-1 RT polymerization
Fig. 1. Structures of currently FDA-approved clinical NNRTIs and novel benzophenone NNRTI
derivatives (GW 678248 as the example)
Fig. 2. Strategy for designing BP-O-DAPY hybrids and O-DAPYs
Fig. 3. Predicted binding mode of the representative compounds in the non-nucleoside
inhibitor-binding pocket (NNIBP) of wild-type HIV-1 RT (PDB code: 3MEC[10]): a) compound
1b (green), b) compound 1e (brown), c) compound 1g (cyan), d) compound 1u (red); The docked
conformations of the representative compounds are shown in caped sticks. For comparison
purposes, the binding conformation of TMC125 is shown in purple stick representation. Possible
Hydrogen bonds are indicated with dashed lines in yellow. The figures are generated by the
software of SYBYL-1.2.
Fig. 4. Predicted binding mode of the representative compounds in the non-nucleoside inhibitor
binding pocket (NNIBP) of K103N/Y181C mutated HIV-1 RT (PDB code: 3BGR [9]): a)
compound 1b (green) and 1g (cyan), and that of K103N mutated HIV-1 RT (PDB code: 3DOK
[15]); b) compound 1b (green) and 1d (blue); The docked conformations of the representative
compounds are shown in caped sticks and superimposed with TMC278 in purple for comparison
in a), and GW678248 in purple for comparison in b), respectively. Possible Hydrogen bonds are
indicated with dashed lines in yellow. The figures are generated by the software of SYBYL-1.2.
Scheme 1. Synthesis of the target compounds 1a-u. Reagents and conditions: (a) K2CO3, DMF,
5–15 h, 80 °C.
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Table 1. Antiviral activity test of compounds 1a-u against HIV-1 and HIV-2 in MT-4 cells
EC50a (µM) HIV-1
EC50a (µM) HIV-2
Compound
CC50d(µM)
SIe
b
IIIB
RES056c
ROD
1a
> 1.71 ± 0.56
0.79 ± 0.13
1.36 ± 0.10
> 0.76
> 1.71 ± 0.56
> 28.81
> 28.81
> 0.76
> 1.71 ± 0.56
> 28.81
> 28.81
> 0.76
1.71 ± 0.56
28.81 ± 7.30
28.81 ± 1.81
0.76 ± 0.37
19.44 ± 8.86
> 316.91
< 1
36
1b
1c
21
1d
< 1
7
1e
2.72 ± 1.55
46.06 ± 13.98
0.26 ± 0.06
> 26.55
> 19.44
> 316.91
䏫 93.25
> 26.55
> 37.06
> 4.24
> 19.44
> 316.91
䏫 46.19
> 26.55
> 37.06
> 4.24
1f
> 7
359
< 1
< 1
< 1
< 1
䏪 6
6
1g
93.25 ± 46.94
26.55 ± 1.33
37.06 ± 7.12
4.24 ± 0.67
4.90 ± 0.81
32.00 ± 5.61
27.67 ± 3.47
13.79 ± 10.22
䏫 0.83
1h
1i
> 37.06
1j
> 4.24
1k
> 4.90
> 4.90
> 4.90
1l
䏫 5.18
> 32.00
> 27.67
> 13.79
> 0.83
> 32.00
> 27.67
> 13.79
> 0.83
1m
4.31 ± 1.04
0.79 ± 0.31
3.79 ± 0.08
1.48 ± 0.13
1.09 ± 0.37
䏫 3.51
1n
18
1o
< 1
9
1p
> 13.37
> 24.16
> 7.42
> 13.37
> 24.16
> 7.42
13.37 ± 5.34
24.16 ± 6.34
7.42 ± 3.75
> 295.90
1q
22
1r
䏪 2
> 82
29
1s
3.61 ± 1.19
> 170.47
>295.90
> 170.47
> 346.83
5.12 ± 5.14
0.51 ± 0.14
> 36.19
0.034 ± 0.005
0.001
> 295.90
> 170.47
> 346.83
> 15.02
> 6.34
1t
170.47 ± 71.31
> 346.83
1u
0.06 ± 0.01
0.11 ± 0.07
0.01 ± 0.0015
0.11 ± 0.07
0.002 ± 0.0004
0.0004
> 6260
> 132
> 727
> 338
12884
nd
NVP
> 15.02
EFV
> 6.34
DLV
> 36.19
> 27.79
nd
> 36.19
TMC125f [12]
TMC278 [9]
27.79 ± 11.54
nd
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GW678248f [21]
0.69 ± 0.43
0.0014 ± 0.0001
0.01 ± 0.00003
nd
> 386.8
> 93.55
> 563380
> 14445
AZT
0.01 ± 0.002
0.01 ± 0.001
a: effective concentration of compound required to protect the cells against viral cytopathogenicity by 50%.
b: wild-type HIV-1 strain.
c: HIV-1 mutated strain bearing both K103N and Y181C mutations.
d: cytotoxic concentration of compound that reduces the normal uninfected cell viability by 50%.
e: selectivity index: ratio CC50/EC50(HIV-1IIIB).
f: The data were evaluated in the same biological laboratory with the same essay.
nd: not determined.
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Table 2. Activity of selected compounds against wt HIV-1 RT polymerization
Compound
IC50 (µM)a
3.22 ± 0.35
3.45 ± 0.23
7.13 ± 0.24
30.96 ± 2.79
2.69 ± 0.61
16.90 ± 1.30
17.43 ± 2.33
11.64 ± 1.47
3.06 ± 0.21
5.23 ± 0.18
5.21 ± 0.00
1.27 ± 0.02
2.74 ± 0.11
0.048 ± 0.000
1b
1c
1e
1f
1g
1l
1m
1n
1p
1q
1s
1u
NVP
EFV
a: Compound concentration required to inhibit HIV-1 RT wt activity by 50%
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HN
O
H
O S
O
N
HN
F3C
N
N
Cl
O
N
N
N
N
N
N
H
O
H
O
Nevirapine
CN
Delavirdine
Efavirenz
CN
CN
CN
Cl
Cl
A
B
N
O
NH
SO2NH2
NC
O
HN
N
NH
N
O
O
Br
N
H
N
NH2
O-DAPYs
Etravirine (TMC125)
NH-DAPYs
Rilpivirine (TMC278)
GW 678248
Fig. 1. Structures of currently FDA-approved clinical NNRTIs and novel benzophenone
NNRTI derivatives (GW 678248 as the example)
20

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Fig. 2. Strategy for designing BP-O-DAPY hybrids and O-DAPY derivatives
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Fig. 3. Predicted binding mode of the representative compounds in the non-nucleoside
inhibitor-binding pocket (NNIBP) of wild-type HIV-1 RT (PDB code: 3MEC [10]): a) compound
1b (green), b) compound 1e (brown), c) compound 1g (cyan), d) compound 1u (red); The docked
conformations of the representative compounds are shown in caped sticks. For comparison
purposes, the binding conformation of TMC125 is shown in purple stick representation. Possible
Hydrogen bonds are indicated with dashed lines in yellow. The figures are generated by the
software of SYBYL-1.2.
22

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Fig. 4. Predicted binding mode of the representative compounds in the non-nucleoside
inhibitor binding pocket (NNIBP) of K103N/Y181C mutated HIV-1 RT (PDB code: 3BGR [9]): a)
compound 1b (green) and 1g (cyan), and that of K103N mutated HIV-1 RT (PDB code: 3DOK
[15]); b) compound 1b (green) and 1d (blue); The docked conformations of the representative
compounds are shown in caped sticks and superimposed with TMC278 in purple for comparison
in a), and GW678248 in purple for comparison in b), respectively. Possible Hydrogen bonds are
indicated with dashed lines in yellow. The figures are generated by the software of SYBYL-1.2.
23

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Scheme 1. Synthesis of the target compounds 1a–u. Reagents and conditions: (a) K2CO3,
DMF, 5–15 h, 80 °C.
24

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