Fused heterocycles bearing bridgehead nitrogen as potent HIV-1 NNRTIs. Part 4: Design, synthesis and biological evaluation of novel imidazo[1,2-a]pyrazines

Article abstract of DOI:10.1016/j.ejmech.2015.02.022

Through a structure-guided core-refining approach, a series of novel imidazo[1,2-a]pyrazine derivatives were designed, synthesized and evaluated as HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTIs). Biological results of antiviral assay in MT

Full text of DOI:10.1016/j.ejmech.2015.02.022

Accepted Manuscript
Fused Heterocycles Bearing Bridgehead Nitrogen as Potent HIV-1 NNRTIs. Part 4:
Design, Synthesis and Biological evaluation of Novel Imidazo[1,2-a]pyrazines
Boshi Huang, Xin Liang, Cuicui Li, Wenmin Chen, Tao Liu, Xiao Li, Yueyue Sun, Lu
Fu, Huiqing Liu, Erik De Clercq, Christophe Pannecouque, Peng Zhan, Xinyong Liu
PII:
S0223-5234(15)00118-X
10.1016/j.ejmech.2015.02.022
EJMECH 7704
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 29 October 2014
Revised Date: 29 January 2015
Accepted Date: 13 February 2015
Please cite this article as: B. Huang, X. Liang, C. Li, W. Chen, T. Liu, X. Li, Y. Sun, L. Fu, H. Liu, E. De
Clercq, C. Pannecouque, P. Zhan, X. Liu, Fused Heterocycles Bearing Bridgehead Nitrogen as Potent
HIV-1 NNRTIs. Part 4: Design, Synthesis and Biological evaluation of Novel Imidazo[1,2-a]pyrazines,
European Journal of Medicinal Chemistry (2015), doi: 10.1016/j.ejmech.2015.02.022.
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Graphical abstract
Through structure-guided core-refining approach,
a
a
series of novel
imidazo[1,2-a]pyrazine derivatives were designed, synthesized and identified as
inhibitors of wild-type HIV-1 strain in this paper.
R2
CN
NH
R1
R1
N
O
N
N
4a R1=CH3 R2=CH3 EC50 = 0.26 M
5a R1=CH3 R2=CN EC50 = 0.32 M

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Fused Heterocycles Bearing Bridgehead Nitrogen as Potent HIV-1
NNRTIs. Part 4: Design, Synthesis and Biological evaluation of Novel
Imidazo[1,2-a]pyrazines
a
Boshi Huang a,1, Xin Liang a,1, Cuicui Li a, Wenmin Chen a, Tao Liu a, Xiao Li ,
a
b
c
c
Yueyue Sun a, Lu Fu , Huiqing Liu , Erik De Clercq , Christophe Pannecouque ,
Peng Zhan a,*, 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 Ji’nan, Shandong, PR China
b Institute of Pharmacology, School of Medicine, Shandong University, 44 West Culture Road,
250012 Ji’nan, Shandong, PR China
c
Rega Institute for Medical Research, KU Leuven Minderbroedersstraat 10, B-3000 Leuven,
Belgium
Abstract: Through a structure-guided core-refining approach, a series of novel
imidazo[1,2-a]pyrazine derivatives were designed, synthesized and evaluated as
HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTIs). Biological results of
antiviral assay in MT-4 cell cultures showed that 12 target compounds displayed
moderate activities against wild-type (wt) HIV-1 strain (IIIB) with EC50 values
ranging from 0.26 µM to 19 µM. Among them, 4a and 5a were found to be the two
most active analogues possessing EC50 values of 0.26 µM and 0.32 µM respectively,
comparable to delavirdine (DLV, EC50 = 0.54 µM) and nevirapine (NVP, EC50 = 0.31
µM) in a cell-based assay. Additionally, 9 compounds showed RT inhibitory activity
superior to that of NVP. Moreover, some predicted drug-like properties of
Corresponding authors. Tel: +86 531 88380270; fax: +86 531 88382731; E-mail address:
zhanpeng1982@sdu.edu.cn (Zhan P.); xinyongl@sdu.edu.cn (Liu X.Y.)
1. Huang B. and Liang X. contributed equally.
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representative compounds 4a and 5a, as well as the structure-activity relationship
(SAR) analysis were discussed in detail. The binding mode of compound 4a was
investigated by molecular simulation studies.
Keywords: HIV-1 RT, Imidazo[1,2-a]pyrazines, Core-refining, NNRTIs, Biological
activity, Molecular simulation.
1. Introduction
According to the newly released UNAIDS Gap report, there were approximately 35
million people living with HIV, 2.1 million new HIV infections and 1.5 million deaths
due to AIDS in 20131. In spite of the introduction of highly active antiretroviral
therapy (HAART) dramatically decreasing the morbidity and mortality caused by
HIV-1 infection, the AIDS prevalence remains one of the world’s most serious public
health problems. Owing to the excellent antiviral potency, high specificity and low
cytotoxicity, HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTIs) have
become inherent ingredients of HAART2. However, drug resistance due to reverse
transcriptase (RT) mutations inevitably emerge after long-term use of NNRTIs. Thus,
further development of novel inhibitors with efficient anti-HIV activity against both
wild-type (wt) and mutated HIV strains, is undoubtedly required for the successful
application of NNRTIs in drug combination regimens3, 4.
Among NNRTIs, diarylpyrimidine (DAPY) derivatives with remarkable activity
have attracted considerable attention over the past decade. As the most representative
series of second generation NNRTIs, DAPY derivatives culminated with the US
FDA’s approval of Etravirine (ETR, TMC125) and Rilpivirine (RPV, TMC278) for
their prominent potency against a large panel of HIV-1 mutant strains5 (Fig. 1).
However, most DAPY derivatives suffered from unsatisfactory pharmacokinetic
profiles. For instance, the clinical advancement of ETR and RPV was hindered
initially by poor aqueous solubility and bioavailability6. Besides, ETR proves to be an
inducer of cytochrome P450 3A4 enzyme (CYP3A4), while it is an inhibitor of CYP
2C9 and CYP 2C19 on the contrary7. Thus, a number of drug-drug interactions must
be considered when prescribing etravirine, which limits its clinical application.
Taking into account these drawbacks, efforts have been focused on exploring
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additional heterocyclic scaffolds as next-generation DAPY-like NNRTIs to improve
pharmacokinetic and druggable properties. For instance, pyrrolopyrimidines
RDEA427 and RDEA640 have been disclosed to exhibit remarkable antiviral potency
against wt HIV-1 (with EC50 values of 0.9 and 0.8 nM, respectively) and a broad
spectrum of HIV-1 mutants with low potential for CYP induction (Fig. 2) 8-10. In 2014,
two series of fused heterocyclic compounds bearing bridgehead nitrogen were
reported by our group as DAPY-like NNRTIs . Excellent anti-HIV-1 activities were
displayed by some compounds, such as pyrazolo[1,5-a] pyrimidine YT-5a (EC50= 70
nM)11 and 1,2,4-triazolo[1,5-a] pyrimidine LW-7c (EC50 = 50 nM)7 (Fig. 2). In
addition to furnishing highly potent NNRTIs, these bicyclic systems also provided
additional diversification points for structure-activity relationship (SAR) studies and
for improvement of the inhibitor’s overall pharmaceutical properties.
The advances in structural biology further gave insights into the important
interactions between the NNRTI-binding pocket (NNIBP) and the inhibitors, which
could provide new guidelines for improving the subsequent structure-based molecular
design of potent NNRTIs. Recently, another relatively unexplored open region termed
“entrance channel” in NNIBP extending into the solvent-exposed region, which was
surrounded by Leu100, Lys101, Glu138 and Val17912, has been initially identified by
Prof. W.L. Jorgensen’s group. Based on the detailed inspection of the binding mode
revealed by the crystallographic structure of ETR in its complex with RT, it was
found that the amino and bromo groups on the central pyrimidine ring of ETR exactly
pointed to the entrance channel. The amino group formed a hydrogen bond with
Glu138 while the bromo group developed electrostatic interaction with Val17913.
On the basis of this analysis, and in continuation of our pursuit of the exploitation
of novel biologically potent fused heterocycles as DAPY-like NNRTIs, we designed a
series of synthetically feasible imidazo[1,2-a]pyrazine14 NNRTIs via
a
structure-guided core-refining approach to exploit additional interactions with the
entrance channel region of NNIBP. Concretely, according to the bioisosterism
principle, we employed the privileged bridgehead nitrogen heterocycle
imidazo[1,2-a]pyrazine as the central ring of new NNRTIs, as shown in Figure 2.
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Meanwhile, the NH linker connecting the central ring with the right ring, which could
make a crucial hydrogen-bond interaction with the backbone of Lys101 especially in
the cases of the Lys101 mutant, was maintained in view of its paramount importance.
Besides, the diverse substituents (including the preponderant groups) on the left and
right rings were introduced. The overall molecular conformation of DAPYs which is
required for the antiviral potency should be retained. Additionally, compared to ETR,
the extra imidazole ring instead of the amino and bromo groups, was installed to
occupy the NNIBP entrance channel more effectively, which was expected to have
potentially enhanced interactions with the key residues inside the entrance channel.
Herein, to validate these hypotheses, we reported the synthesis, in vitro anti-HIV
potency, HIV-1 RT inhibitory activity and some predicted drug-like properties of
these novel imidazo[1,2-a]pyrazine derivatives. The SAR and molecular simulation
studies were also discussed in detail to gain further insight into this series of
analogues.
2. Results and discussion
2.1. Chemistry
The synthetic route of imidazo[1,2-a]pyrazine derivatives as target compounds is
depicted in Scheme 1. The intermediate 6,8-dibromoimidazo[1,2-a]pyrazine (2) was
prepared from commercially available 3,5-dibromopyrazin-2-amine (1) by cyclizing
with 2-chloroacetaldehyde15. Subsequently, different substituted phenols were
connected to the relatively active 8-position of the fused imidazo[1,2-a]pyrazine ring
by a nucleophilic substitution reaction in the presence of K2CO316, providing
intermediates 3-8. Finally, 3-8 underwent a Pd(OAc)2-catalyzed Buchwald–Hartwig
reaction17 with corresponding anilines to yield 18 title compounds (3a-d, 4a-d, 5a-d,
6a, 6b, 7a, 7b, 8a, 8b). Both physicochemical and spectral data of all the synthesized
compounds were found to be in full agreement with the proposed structures.
2.2. In vitro anti-HIV activity
The newly synthesized imidazo[1,2-a]pyrazine derivatives were tested for their
anti-HIV activity in MT-4 cell cultures infected with the wt HIV-1 (strain IIIB), the
double RT mutant (K103N+Y181C) HIV-1 strain (RES056), as well as HIV-2 (strain
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ROD), using the MTT method as previously described. The biological results are
presented as EC50 values, CC50 values and SI values as shown in Table 1. Zidovudine
(AZT), Didanosine (DDI), Lamivudine (3TC), Nevirapine (NVP), Delavirdine
mesylate (DLV), Efavirenz (EFV) and Etravirine (ETR) were used as reference drugs.
In total, 12 compounds displayed anti-HIV-1 (IIIB) activity in the micromolar
range (EC50 = 0.26 – 19 µM) with selectivity index (SI) values ranging from 5 to ≥
224. Among them, 4a and 5a were found to be the two most active analogues
possessing EC50 values of 0.26 µM and 0.32 µM respectively, which were much
lower than the reference drugs DDI (EC50 = 23 µM) and 3TC (EC50 = 2.2 µM), and
comparable to DLV (EC50 = 0.54 µM) and NVP (EC50 = 0.31 µM), while much higher
than for AZT (EC50 = 0.007 µM), EFV (EC50 = 0.006 µM) and ETR (EC50 = 0.004 µM)
against IIIB strain. Compounds 4a and 5a also exhibited high selectivity indexes (SI =
126, ≥ 224, respectively). Also, two compounds (7a, EC50 = 0.98 µM; 8a, EC50 = 0.87
µM) showed better potency than the control drugs DDI and 3TC. Unfortunately, none
of the new compounds was active against the double mutant HIV-1 strain RES056 at
subtoxic concentrations. Based on the anti-HIV-1 (IIIB) assay results, preliminary
SAR analysis was as follows.
First of all, to elucidate the SAR profiles of the substitutents R3, different goups
were introduced on the right ring of these imidazo[1,2-a]pyrazine analogues, while
keeping other positions unchanged. Comparing the EC50 and CC50 values of the 4a-d
sub-series characterized by a 2,4,6-trimethyl substituent on the left wing, we found
that compound 4a bearing a 4-cyano-substituent on the right ring possessed the best
activity and highest SI value (SI = 126). The sequences of potency and SI values in
this case indicated the same declining trend: 4-cyano > 4-nitro > 4- cyano
methylene > 4-chlorine. Compounds 3a and 5a also showed best potency and safety
profiles in the 3a-d and 5a-d sub-series, respectively. On the whole, 4-cyano-aniline
substitution was found to be the optimal functional group for R3 in these derivatives.
The results were in agreement with those of the DAPYs previously reported.
Next, the nature of substitutions (R1 and R2) on the left wing was explored.
Comparing the EC50 values of those analogues with same R3 substituents, compounds
4a-d, it was seen that a 2,4,6-trimethyl substituent on the left wing was most
favorable to the anti-HIV-1 (IIIB) activity, superior to 2,6-dimethyl-4-cyano group
and the others. Besides, by comparing compound 7a and 8a with 3a (EC50 = 6.5 µM),
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we can conclude that adding a bromine or chlorine atom at the 4-position of
2,6-dimethylphenoxy moiety was beneficial for the anti-HIV-1 potency. It was also
interesting to observe the “magic methyl phenomenon”: by comparing 3a to 4a, an
additional methyl group with its lipophilic surface well buried into a hydrophobic
pocket of NNIBP is anticipated to provide binding energy up to 1.5 kcal/mol, so give
18
up to a 10-fold improvement in binding constant Kd (or equivalent to EC50).
Certainly, a 25-fold increase for potency of 4a (EC50 = 0.26 µM) over 3a (EC50 = 6.5
µM) was observed . With a 2,4,6-trifluorine substituent on the left ring, 6a exhibited
the worst anti-HIV-1 profile in the 4-cyano-substituent (R3) sub-series. In short, the
decreased order of substituents on the left wing based on the antiviral activity was:
2,4,6-trimethyl (4a) > 2,6-dimethyl-4-cyano (5a) > 2,6-dimethyl-4-bromo (7a) ≈
2,6-dimethyl-4-chloro (8a) > 2,6-dimethyl (3a) >2,4,6-trifluoro (6a).
Moreover, in the 3a-d, 4a-d and 5a-d sub-series, it is noteworthy that the lowest
cytotoxicity was always found in the compounds bearing cyano methylene at the
R3-position (3c: CC50 = 156 µM; 4c: CC50 = 117 µM; 5c: CC50 = 282 µM),
Taken together, our preliminary SAR analysis of these novel
imidazo[1,2-a]pyrazine derivatives revealed the key structural fragments to maintain
high activity against wt HIV-1: (i) The optimal moiety for the right wing was a
4-cyano-aniline group; (ii) The best group for the left wing was
a
2,4,6-trimethyl-phenoxy group. (iii) Furthermore, our study revealed that the
cytotoxicity was directly related to the nature of the R3 substituent groups. Most
importantly, in spite of the fact that these imidazo[1,2-a]pyrazines did not exhibit
improved activity in comparison with ETR, they represented a novel class of
anti-HIV-1 agents with great potential for further optimization. It must be said, the
new imidazo[1,2-a]pyrazine derivatives can be readily derivatized, facilitating
extensive SAR studies in the future.
Moreover, all the target compounds were also screened for their inhibition against
HIV-2 (strain ROD) in MT-4 cells, but none was found effective at a subtoxic
concentration, indicating that the newly synthesized compounds were only specific
for HIV-1.
2.3. Inhibition of HIV-1 RT
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In order to further confirm the binding target, all the compounds were tested in
enzymatic assays against HIV-1 RT (WT), using poly [A] • oligo [dT]15 as
template/primer (RT kit, Roche). NVP and ETR were used as reference drugs in this
assay. As shown in Table 2, on the whole, most compounds displayed moderate RT
inhibitory activity. Among them, 9 compounds showed RT inhibitory activity superior
to that of NVP. In particular, compound 5c (IC50 = 0.17 µM) demonstrated the highest
potency compared to that of ETR (IC50 = 0.13 µM). Admittedly, the enzymatic
activity and SARs of these analogues exhibited some difference compared to their
anti-HIV (IIIB) results, especially for cyano methylene-containing compounds 3c, 4c
and 5c with the recovered RT inhibition. In summary, it’s reasonable that the newly
synthesized imidazo[1,2-a]pyrazine derivatives could specifically target HIV-1 RT,
and thus belonged to the class of the HIV-1 NNRTIs.
2.4. Molecular modeling analysis
Molecular modeling (docking) was performed using the Sybyl-X 1.1 software to
get deeper insight into the molecular bases of the inhibitory potency and to rationalize
SARs of these analogues. The most potent compound 4a in anti-HIV-1 (IIIB) in vitro
assay was selected to be docked into the NNIBP of HIV-1 RT (taken from the crystal
structure of the RT/ETR complex, PDB entry: 3MEC).
As shown in Figure 3, 4a was displayed in overlap with ETR by PyMOL version
1.5 (http://www.pymol.org/). 4a had nearly the same binding mode as etravirine.
Notable features of 4a in the NNIBP were summarized as follows: The
trimethylphenyl fitted into the aromatic-rich sub-pocket consisting of residues Tyr181,
Tyr188, Phe227 and Trp229, developing positive face-to-face π–π interactions with
the aromatic rings of Tyr181 and Tyr188. The right benzonitrile moiety extended to a
solvent exposure region, which was surrounded by residues His235 and Pro236.
Additionally, one hydrogen bond was formed between the NH linked to the 6-position
of the core ring and the backbone carbonyl of residue Lys101. Moreover, the fused
imidazole ring actually occupied the entrance channel effectively, giving rise to
favorable Van der Waals interaction. However, lacking one polar interaction (like
hydrogen bond interaction) between the central heterocyclic moiety and Glu138
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observed in ETR may be one reason for the inferior activity of 4a. On the whole, the
modeling analysis was able to support our original design hypothesis and of the
information provided above will be taken full advantage for further structural
optimization.
2.5. In silico prediction of physicochemical properties
Furthermore, some physicochemical properties of 4a, 5a (as representative
compounds) and RDEA427, LW-7c, ETR (as control), like LogP, molecular weight
(MW), topological polar surface area (TPSA), molecular volume (MV), were
determined using free on-line software (http://www.molinspiration.com/) for their
compliance with the Lipinski’s rule of five. The predicted results (Table 3) suggested
that compounds 4a, 5a and reference compounds conformed well to the Lipinski’s
rule of five. In addition, imidazo[1,2-a]pyrazine derivatives and ETR shared similar
properties. Physicochemical properties of 4a and 5a may not be mainly responsible
for their moderate activity against wt HIV-1. In short, considering all of the results,
compounds 4a and 5a have a better balance of moderate activity against wt HIV-1
and reasonable physicochemical properties, and thus considered as the two most
promising leads for further lead optimization.
3. Conclusions
Based on the crystallographic structures of reported DAPY (or DAPY-like)
analogues, a series of novel imidazo[1,2-a]pyrazines were designed via a
structure-guided core-refining approach, synthesized and evaluated for their activities
against wt HIV-1 (strain IIIB), K103N+Y181C double-mutated HIV-1 (strain
RES056) and HIV-2 (strain ROD) in MT-4 cells. Among them, 12 compounds
exhibited moderate anti-HIV-1 (IIIB) activity in micromolar range (EC50 = 0.26 – 19
µM). Especially, compounds 4a and 5a were identified as the two most active
analogues in inhibiting HIV-1 possessing EC50 values of 0.26 µM and 0.32 µM
respectively, which were much better than those of reference drugs DDI (EC50 = 23
µM) and 3TC (EC50 = 2.2 µM), comparable to DLV (EC50 = 0.54 µM) and NVP (EC50
= 0.31 µM). In addition, all the compounds were tested in enzymatic assays against
HIV-1 RT (WT), and 9 compounds showed RT inhibitory activity superior to that of
NVP. Furthermore, some physicochemical properties of representative compounds 4a
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and 5a were predicted, and the results suggested that 4a and 5a could be considered as
the two most promising leads for next-step optimization. Preliminary SARs based on
anti-HIV-1 activities were discussed in detail, and molecular modeling studies
performed supported our initial design hypothesis that the fused imidazole ring of new
compounds could occupy the entrance channel of HIV-1 RT effectively, but lacking
one polar interaction with Glu138 as observed in etravirine may be one reason for the
inferior activity of this series of derivatives. These valuable information and the initial
results immensely encourage us to further optimize this priviledged structure, which
will be reported in due course.
4. Experimental section
4.1. Chemistry
All melting points (mp) were determined on a micromelting point apparatus and are
uncorrected. Mass spectra were taken on a LC Autosampler Device: Standard
1
G1313A instrument by electrospray ionization. H NMR and 13C NMR spectra were
obtained on a Bruker AV-400 spectrometer (Bruker BioSpin, Switzerland) in the
indicated solvent DMSO-d6. Chemical shifts were expressed in δ units (ppm), using
TMS as an internal reference and J values were reported in hertz (Hz). TLC was
performed on Silica Gel GF254. Spots were visualized by irradiation with UV light (λ
254 nm). Flash column chromatography was performed on columns packed with
silica gel 60 (200–300 mesh). Solvents were of reagent grade, and if necessary, were
purified and dried by standard methods. The key reagents were purchased from
commercial suppliers and not further purified when used. Rotary evaporators served
in concentration of the reaction solutions under reduced pressure.
4.1.1.
General
procedure
for
the
synthesis
of
intermediates
6-bromo-8-substitutedphenoxy imidazo[1,2-a]pyrazines (3-8)
6,8-Dibromoimidazo[1,2-a]pyrazine (2): 2-chloroacetaldehyde (0.79 g, 4 mmol )
was slowly added to a freshly prepared solution of 3,5-dibromopyrazin-2-amine (1)
(0.51 g, 2 mmol) in 2-propanol (20 mL). The mixture was stirred at reflux under a
nitrogen atmosphere for 24 h. After cooling, CH2Cl2 (25 mL) and Et3N (1 mL) were
added to the reaction solution. Thereafter, the solvent was evaporated under reduced
pressure, and then the residue was washed with 11 mL mixed solution (water:
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2-propanol = 10:1) for 2 times, filtered and dried under vacuum to give
6,8-dibromoimidazo[1,2-a]pyrazine (2) as a brown solid, which was pure enough to
be used in the next step without further purification.
6-Bromo-8-substitutedphenoxy imidazo[1,2-a]pyrazines (3-8): A mixture of
appropriate substituted phenols (7 mmol) and potassium carbonate (1.93 g, 14 mmol)
in 30 mL DMF was stirred for 30 min at room temperature. Then,
6,8-dibromoimidazo[1,2-a]pyrazine (2) (1.94 g, 7 mmol) was added and the mixture
reacted overnight at room temperature. After the reaction was completed, 100 mL
H2O was added to the mixture. The resultant precipitate were filtered and dried in
vacuo to obtain the crude products of 6-bromo-8-substitutedphenoxy
imidazopyrazines (3-8), which could be used in the next step without further
purification.
4.1.2. General procedure for the synthesis of target compounds (3a-d, 4a-d, 5a-d, 6a,
6b, 7a, 7b, 8a, 8b)
Pd(OAc)2 (11.2 mg, 0.05 mmol) and Xantphos (28.9 mg, 0.05 mmol) were added
to 1,4-dioxane (5 mL) in a Schlenk-type flask. After stirring for 30 min at room
temperature, 6-bromo-8-substitutedphenoxy imidazo[1,2-a]pyrazines (3-8) (1 mmol),
different 4-substitued anilines (1.1 mmol) and Cs2CO3 (651.6 mg, 2 mmol) were
added to the mixture. The solution was allowed to stir at reflux under N2 protection,
and the reaction was followed by TLC until its completion. After cooling to room
temperature, the reaction solution was filtered and concentrated under reduced
pressure. The residue was purified by flash chromatography using proper eluant and
then recrystallization to afford the target compounds (3a-d, 4a-d, 5a-d, 6a, 6b, 7a, 7b,
8a, 8b).
4.1.2.1. 4-((8-(2,6-dimethylphenoxy)imidazo[1,2-a]pyrazin-6-yl)amino)benzonitrile
(3a)
1
White solid, yield: 51%. Decomposed at 187 °C. H NMR (400 MHz, DMSO-d6)
δ: 9.19 (s, 1H, NH), 8.18 (s, 1H, imidazole-H), 7.96 (s, 1H, pyrazine-H), 7.74 (s, 1H,
imidazole-H), 7.34 (d, J=8.80 Hz, 2H, PhH), 7.24-7.20 (m, 3H, OPh-H), 7.08 (d,
J=8.84 Hz, 2H, PhH), 2.11 (s, 6H, CH3×2). 13C NMR (100 MHz, DMSO-d6) δ:
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150.81, 150.09, 147.50, 137.65, 134.87, 133.22, 130.80, 130.18, 129.25, 126.25,
120.26, 117.22, 116.04, 102.28, 100.10, 16.48; ESI-MS: m/z 356.5 (M+1), 378.6
(M+23), C21H17N5O (355.14).
4.1.2.2.
8-(2,6-dimethylphenoxy)-N-(4-nitrophenyl)imidazo[1,2-a]pyrazin-6-amine
(3b)
Yellow solid, yield: 27%. mp: 259-261 °C. 1H NMR (400 MHz, DMSO-d6) δ: 9.62
(s, 1H, NH), 8.22 (s, 1H, imidazole-H), 8.03 (s, 1H, pyrazine-H), 7.82 (d, J=9.28 Hz,
2H, PhH), 7.77 (s, 1H, imidazole-H), 7.28-7.22 (m, 3H, OPh-H), 7.10 (d, J=9.28 Hz,
2H, PhH), 2.12 (s, 6H, CH3×2). 13C NMR (100 MHz, DMSO-d6) δ: 150.87, 150.12,
149.83, 138.78, 137.22, 135.02, 130.86, 130.28, 129.25, 126.23, 125.47, 117.40,
115.13, 103.15, 16.46; ESI-MS: m/z 376.5 (M+1), 398.4 (M+23), C20H17N5O3
(375.13).
4.1.2.3.
2-(4-((8-(2,6-dimethylphenoxy)imidazo[1,2-a]pyrazin-6-yl)amino)phenyl)acetonitrile
(3c)
White solid, yield: 31%. mp: 98-100 °C. 1H NMR (400 MHz, DMSO-d6) δ: 8.47 (s,
1H, NH), 8.11 (d, J=0.52 Hz, 1H, imidazole-H), 7.84 (s, 1H, pyrazine-H), 7.68 (d,
J=0.56 Hz, 1H, imidazole-H), 7.22-7.16 (m, 3H, OPh-H), 7.03-6.95 (m, 4H, PhH),
3.84 (s, 2H, CH2), 2.11 (s, 6H, CH3×2). 13C NMR (100 MHz, DMSO-d6) δ: 150.72,
150.12, 142.64, 139.44, 134.54, 130.85, 129.98, 129.14, 128.78, 126.06, 121.94,
120.03, 117.13, 116.77, 99.65, 22.02, 16.54. ESI-MS: m/z 370.4 (M+1), 392.4
(M+23), C22H19N5O (369.16).
4.1.2.4. N-(4-chlorophenyl)-8-(2,6-dimethylphenoxy)imidazo[1,2-a]pyrazin-6-amine
(3d)
1
White solid, yield: 21%. mp: 167-169 °C. H NMR (400 MHz, DMSO-d6) δ: 8.61
(s, 1H, NH), 8.12 (s, 1H, imidazole-H), 7.84 (s, 1H, pyrazine-H), 7.69 (s, 1H,
imidazole-H), 7.24-7.18 (m, 3H, OPh-H), 7.02-6.97 (m, 4H, PhH), 2.10 (s, 6H,
CH3×2). 13C NMR (100 MHz, DMSO-d6) δ: 150.72, 150.13, 142.16, 139.15, 134.61,
130.84, 129.95, 129.19, 128.61, 126.13, 122.96, 118.15, 116.84, 99.86, 16.52.
ESI-MS: m/z 365.4 (M+1), 367.4 (M+3), C20H17ClN4O (364.11).
11

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4.1.2.5. 4-((8-(mesityloxy)imidazo[1,2-a]pyrazin-6-yl)amino)benzonitrile (4a)
1
White solid, yield: 28%. mp: 163-165 °C. H NMR (400 MHz, DMSO-d6) δ: 9.20
(s, 1H, NH), 8.17 (s, 1H, imidazole-H), 7.98 (s, 1H, pyrazine-H), 7.73 (s, 1H,
imidazole-H), 7.35 (d, J=8.76 Hz, 2H, PhH), 7.10 (d, J=8.80 Hz, 2H, PhH), 7.03 (s,
2H, OPh-H), 2.32 (s, 3H, CH3), 2.06 (s, 6H, CH3×2). 13C NMR (100 MHz, DMSO-d6)
δ: 150.97, 148.84, 147.60, 137.55, 135.12, 134.81, 133.23, 130.36, 130.27, 129.68,
120.29, 117.20, 116.07, 102.39, 100.11, 20.82, 16.43. ESI-MS: m/z 370.5 (M+1),
392.5 (M+23), C22H19N5O (369.16).
4.1.2.6. 8-(mesityloxy)-N-(4-nitrophenyl)imidazo[1,2-a]pyrazin-6-amine (4b)
Yellow solid, yield: 55%. mp: 138-140 °C. 1H NMR (400 MHz, DMSO-d6) δ: 9.60
(s, 1H, NH), 8.21 (d, J=0.68 Hz, 1H, imidazole-H), 8.03 (s, 1H, pyrazine-H), 7.83 (dd,
J=7.52 Hz, J=1.84 Hz, 2H, PhH), 7.76 (d, J=0.68 Hz, 1H, imidazole-H), 7.10 (dd,
J=7.40 Hz, J=2.00 Hz, 2H, PhH), 7.06 (s, 2H, OPh-H), 2.34 (s, 3H, CH3), 2.07 (s, 6H,
CH3×2). 13C NMR (100 MHz, DMSO-d6) δ: 151.02, 149.85, 147.93, 138.76, 137.16,
135.17, 134.95, 130.46, 130.33, 129.70, 125.44, 117.38, 115.22, 103.10, 20.80, 16.39.
ESI-MS: m/z 390.4 (M+1), 412.5 (M+23), C21H19N5O3 (389.15).
4.1.2.7.
2-(4-((8-(mesityloxy)imidazo[1,2-a]pyrazin-6-yl)amino)phenyl)acetonitrile
(4c)
1
White solid, yield: 38%. mp: 185-187 °C. H NMR (400 MHz, DMSO-d6) δ: 8.50
(s, 1H, NH), 8.11 (s, 1H, imidazole-H), 7.84 (s, 1H, pyrazine-H), 7.68 (s, 1H,
imidazole-H), 7.05-6.96 (m, 6H, OPh-H, PhH), 3.87 (s, 2H, CH2), 2.32 (s, 3H, CH3),
2.06 (s, 6H, CH3×2). 13C NMR (100 MHz, DMSO-d6) δ: 150.86, 147.90, 142.58,
139.43, 135.01, 134.46, 130.40, 129.99, 129.62, 128.70, 121.90, 120.02, 117.20,
116.73, 99.43, 22.05, 20.84, 16.48. ESI-MS: m/z 384.4 (M+1), 406.6 (M+23),
C23H21N5O (383.17).
4.1.2.8. N-(4-chlorophenyl)-8-(mesityloxy)imidazo[1,2-a]pyrazin-6-amine (4d)
1
White solid, yield: 56%. mp: 105-107 °C. H NMR (400 MHz, DMSO-d6) δ: 8.59
(s, 1H, NH), 8.11 (d, J=0.72 Hz, 1H, imidazole-H), 7.84 (s, 1H, pyrazine-H), 7.68 (d,
J=0.72 Hz, 1H, imidazole-H), 7.04-6.98 (m, 6H, OPh-H, PhH), 2.31 (s, 3H, CH3),
2.05 (s, 6H, CH3×2). 13C NMR (100 MHz, DMSO-d6) δ: 150.88, 147.90, 142.19,
12

ACCEPTED MANUSCRIPT
139.12, 134.98, 134.53, 130.40, 130.04, 129.63, 128.61, 123.01, 118.29, 116.81,
99.83, 20.82, 16.46. ESI-MS: m/z 379.5 (M+1), 381.4 (M+23), C21H19ClN4O
(378.12).
4.1.2.9.
4-((6-((4-cyanophenyl)amino)imidazo[1,2-a]pyrazin-8-yl)oxy)-3,5-dimethylbenzonitr
ile (5a)
White solid, yield: 66%. decomposed at 184 °C. 1H NMR (400 MHz, DMSO-d6) δ:
9.19 (s, 1H, NH), 8.21 (d, J=0.84 Hz, 1H, imidazole-H), 8.03 (s, 1H, pyrazine-H),
7.79 (s, 2H, OPh-H), 7.76 (d, J=0.64 Hz, 1H, imidazole-H), 7.40 (d, J=8.84 Hz, 2H,
PhH), 7.04 (d, J=8.84 Hz, 2H, PhH), 2.16 (s, 6H, CH3×2). 13C NMR (100 MHz,
DMSO-d6) δ: 153.92, 150.14, 147.40, 137.24, 135.18, 133.26, 133.19, 129.97, 120.22,
119.01, 117.47, 115.97, 109.11, 103.20, 100.34, 16.20. ESI-MS: m/z 381.5 (M+1),
403.5 (M+23), C22H16N6O (380.14).
4.1.2.10.
3,5-dimethyl-4-((6-((4-nitrophenyl)amino)imidazo[1,2-a]pyrazin-8-yl)oxy)benzonitril
e (5b)
Yellow solid, yield: 30%. mp: 133-135 °C. 1H NMR (400 MHz, DMSO-d6) δ: 9.61
(s, 1H, NH), 8.24 (s, 1H, imidazole-H), 8.10 (s, 1H, pyrazine-H), 7.88 (d, J=9.28 Hz,
2H, PhH), 7.83 (s, 2H, OPh-H), 7.79 (s, 1H, imidazole-H), 7.05 (d, J=9.24 Hz, 2H,
PhH), 2.17 (s, 6H, CH3×2). 13C NMR (100 MHz, DMSO-d6) δ: 153.91, 150.20,
149.72, 138.90, 136.78, 135.33, 133.32, 133.19, 130.05, 125.53, 118.97, 117.66,
115.02, 109.13, 104.09, 16.19. ESI-MS: m/z 401.5 (M+1), 423.4 (M+23), C21H16N6O3
(400.13).
4.1.2.11.
4-((6-((4-(cyanomethyl)phenyl)amino)imidazo[1,2-a]pyrazin-8-yl)oxy)-3,5-dimethylb
enzonitrile (5c)
1
White solid, yield: 19%. mp: 174-176 °C. H NMR (400 MHz, DMSO-d6) δ: 8.48
(s, 1H, NH), 8.13 (d, J=0.44 Hz, 1H, imidazole-H), 7.91 (s, 1H, pyrazine-H), 7.76 (s,
2H, OPh-H), 7.70 (s, 1H, imidazole-H), 7.01 (m, 4H, PhH), 3.86 (s, 2H, CH2), 2.16 (s,
6H, CH3×2). 13C NMR (100 MHz, DMSO-d6) δ: 153.97, 150.06, 142.45, 139.12,
13

ACCEPTED MANUSCRIPT
134.86, 133.30, 133.06, 129.73, 128.82, 122.28, 119.94, 119.01, 117.24, 117.04,
109.02, 100.31, 22.08, 16.26. ESI-MS: m/z 395.4 (M+1), 417.5 (M+23), C23H18N6O
(394.15).
4.1.2.12.
4-((6-((4-chlorophenyl)amino)imidazo[1,2-a]pyrazin-8-yl)oxy)-3,5-dimethylbenzonitr
ile (5d)
1
White solid, yield: 20%. mp: 195-197 °C. H NMR (400 MHz, DMSO-d6) δ: 8.63
(s, 1H, NH), 8.15 (d, J=0.52 Hz, 1H, imidazole-H), 7.89 (s, 1H, pyrazine-H), 7.80 (s,
2H, OPh-H), 7.72 (d, J=0.52 Hz, 1H, imidazole-H), 7.04-6.95 (m, 4H, PhH), 2.15 (s,
6H, CH3×2). 13C NMR (100 MHz, DMSO-d6) δ: 154.01, 150.04, 141.95, 138.86,
134.93, 133.31, 133.14, 129.69, 128.65, 123.24, 119.05, 118.23, 117.10, 109.00,
100.53, 16.23. ESI-MS: m/z 390.4 (M+1), 392.4 (M+3), 412.4 (M+23), C21H16ClN5O
(389.10).
4.1.2.13.
4-((8-(2,4,6-trifluorophenoxy)imidazo[1,2-a]pyrazin-6-yl)amino)benzonitrile (6a)
White solid, yield: 19%. decomposed at 269 °C. 1H NMR (400 MHz, DMSO-d6) δ:
9.32 (s, 1H, NH), 8.24 (s, 1H, imidazole-H), 8.11 (s, 1H, pyrazine-H), 7.80 (s, 1H,
imidazole-H), 7.58 (t, JH-F=8.64 Hz, 2H, OPh-H), 7.50 (d, J=8.80 Hz, 2H, PhH), 7.13
(d, J=8.80 Hz, 2H, PhH). 13C NMR (100 MHz, DMSO-d6) δ: 154.38, 149.51, 147.24,
136.60, 135.63, 133.43, 129.59, 120.22, 117.74, 116.00, 104.25, 102.38 (t, JC-F=27.00
Hz, 3C, OPh-C), 100.60. ESI-MS: m/z 382.4 (M+1), 404.4 (M+23), C19H10F3N5O
(381.08).
4.1.2.14. N-(4-nitrophenyl)-8-(2,4,6-trifluorophenoxy)imidazo[1,2-a]pyrazin-6-amine
(6b)
Yellow solid, yield: 16%. mp: 268-270 °C. 1H NMR (400 MHz, DMSO-d6) δ: 9.71
(s, 1H, NH), 8.28 (s, 1H, imidazole-H), 8.18 (s, 1H, pyrazine-H), 7.97 (d, J=9.24 Hz,
2H, PhH), 7.83 (s, 1H, imidazole-H), 7.62 (t, JH-F=8.64 Hz, 2H, OPh-H), 7.15 (d,
J=9.28 Hz, 2H, PhH). 13C NMR (100 MHz, DMSO-d6) δ: 160.82, 158.37 (t,
JC-F=14.00 Hz, 2C, OPh-C), 155.61 (ddd, JC-F=248.00 Hz, JC-F=14.00 Hz, JC-F=6.00
Hz, 2C, OPh-C), 149.61, 139.18 (d, JC-F=5.00 Hz, 1C, Ph-C), 136.17, 135.76, 129.72,
14

ACCEPTED MANUSCRIPT
126.82, 125.64, 117.90, 115.07, 105.20, 102.38 (t, JC-F=28.00 Hz, 1C, OPh-C).
ESI-MS: m/z 402.4 (M+1), 424.4 (M+23), C18H10F3N5O3 (401.07).
4.1.2.15.
4-((8-(4-bromo-2,6-dimethylphenoxy)imidazo[1,2-a]pyrazin-6-yl)amino)benzonitrile
(7a)
1
Light yellow solid, yield: 29%. decomposed at 268 °C. H NMR (400 MHz,
DMSO-d6) δ: 9.20 (s, 1H, NH), 8.19 (d, J=0.60 Hz, 1H, imidazole-H), 7.99 (s, 1H,
pyrazine-H), 7.74 (d, J=0.60 Hz, 1H, imidazole-H), 7.48 (s, 2H, OPh-H), 7.38 (d,
13
J=8.80 Hz, 2H, PhH), 7.08 (d, J=8.84 Hz, 2H, PhH), 2.10 (s, 6H, CH3×2). C NMR
(100 MHz, DMSO-d6) δ: 150.43, 149.49, 147.45, 137.45, 135.00, 133.79, 133.22,
131.64, 130.08, 120.24, 118.33, 117.34, 116.12, 102.64, 100.26, 16.20. ESI-MS: m/z
436.4 (M+3), C21H16BrN5O (433.05).
4.1.2.16.
8-(4-bromo-2,6-dimethylphenoxy)-N-(4-nitrophenyl)imidazo[1,2-a]pyrazin-6-amine
(7b)
1
Yellow solid, yield: 32%. decomposed at 223 °C. H NMR (400 MHz, DMSO-d6)
δ: 9.60 (s, 1H, NH), 8.22 (d, J=0.80 Hz, 1H, imidazole-H), 8.05 (s, 1H, pyrazine-H),
7.89 (dd, J=7.40 Hz, J=2.00 Hz, 2H, PhH), 7.77 (d, J=0.76 Hz, 1H, imidazole-H),
7.51 (s, 2H, OPh-H), 7.09 (dd, J=7.36 Hz, J=2.00 Hz, 2H, PhH), 2.11 (s, 6H, CH3×2).
13C NMR (100 MHz, DMSO-d6) δ: 150.48, 149.72, 149.49, 138.87, 137.06, 135.15,
133.83, 131.65, 130.14, 125.51, 118.41, 117.51, 115.16, 103.37, 16.18. ESI-MS: m/z
454.3 (M+1), 456.3 (M+3), C20H16BrN5O3 (453.04).
4.1.2.17.
4-((8-(4-chloro-2,6-dimethylphenoxy)imidazo[1,2-a]pyrazin-6-yl)amino)benzonitrile
(8a)
1
Light yellow solid, yield: 33%, decomposed at 256 °C. H NMR (400 MHz,
DMSO-d6) δ: 9.19 (s, 1H, NH), 8.19 (d, J=0.84 Hz, 1H, imidazole-H), 8.00 (s, 1H,
pyrazine-H), 7.74 (d, J=0.68 Hz, 1H, imidazole-H), 7.38 (d, J=8.80 Hz, 2H, PhH),
7.35 (s, 2H, OPh-H), 7.08 (d, J=8.84 Hz, 2H, PhH), 2.10 (s, 6H, CH3×2). 13C NMR
(100 MHz, DMSO-d6) δ: 150.49, 148.94, 147.47, 137.41, 135.00, 133.37, 133.22,
15

ACCEPTED MANUSCRIPT
130.09, 130.00, 128.70, 120.25, 117.34, 116.08, 102.70, 100.23, 16.30; ESI-MS: m/z
390.3 (M+1), 412.4 (M+23), C21H16ClN5O (389.10).
4.1.2.18.
8-(4-chloro-2,6-dimethylphenoxy)-N-(4-nitrophenyl)imidazo[1,2-a]pyrazin-6-amine
(8b)
Yellow solid, yield: 40%, mp: 184-186 °C. 1H NMR (400 MHz, DMSO-d6) δ: 9.60
(s, 1H, NH), 8.22 (s, 1H, imidazole-H), 8.05 (s, 1H, pyrazine-H), 7.88 (d, J=9.32 Hz,
2H, PhH), 7.77 (s, 1H, imidazole-H), 7.37 (s, 2H, OPh-H), 7.09 (d, J=9.28 Hz, 2H,
PhH), 2.11 (s, 6H, CH3×2). 13C NMR (100 MHz, DMSO-d6) δ: 150.55, 149.75,
148.96, 138.66, 137.04, 135.15, 133.43, 130.16, 130.09, 128.72, 125.49, 117.51,
115.14, 103.45, 16.28; ESI-MS: m/z 410.4 (M+1), 432.4 (M+23), C20H16ClN5O3
(409.09).
4.2. Biological activity evaluation
4.2.1. In vitro anti-HIV assay
Evaluation of the antiviral activity of the newly synthesized compounds against
HIV-1 wt strain (IIIB), double mutant strain (RES056) and HIV-2 strain (ROD) was
performed using the MTT method as previously described19,20. 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 five-fold dilutions of
test compounds were made directly in flat-bottomed 96-well microtiter trays using a
Biomek 3000 robot (Beckman instruments, Fullerton, CA). Untreated control HIV-
and mock-infected cell samples were included for each sample.
HIV-1 wt strain (IIIB), HIV-1 double mutant strain (RES056) or HIV-2 strain
(ROD) stock (50 µL) at 100–300 CCID50 (50% cell culture infectious dose) or culture
medium was added to either the infected or mock-infected wells of the microtiter tray.
Mock-infected cells were used to evaluate the effect of test compound on uninfected
cells in order to assess its cytotoxicity. Exponentially growing MT-4 cells were
centrifuged for 5 min at 1000 rpm 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 method. The
16

ACCEPTED MANUSCRIPT
50% cytotoxic concentration (CC50) was defined as the concentration of the tested
compound that reduced the viability of the mock-infected MT-4 cells 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).
4.2.2. HIV-1 RT inhibition assay
Inhibition of HIV-1 RT was performed by using homopolymer template/primer
linked to a microtiter plate, biotin-dUTP and RT with detection by ELISA for
quantifying HIV-1 RT expression. The incorporated quantities of the biotin-dUTP
into the template represented the activity of HIV-1 RT. IC50 values corresponded to
the concentration of the imidazo[1,2-a]pyrazine derivatives required to inhibit
biotin-dUTP incorporation by 50%. The procedure for RT inhibition assay was
performed as depicted in details by the kit (Roche) protocol21. The percentage
inhibitory values were calculated by the following formula:
%Inhibition = [O.D. value without inhibitors (with RT) -O.D. value with RT and
inhibitors]/[O.D. value without inhibitors (with RT)- O.D. value without RT and
inhibitors].
Conflict of interest
The authors declare no conflict of interest.
Acknowledgments
The financial support from the Key Project of National Natural Science Foundation
of China (NSFC) for International Cooperation Program (No. 81420108027,
30910103908), National Natural Science Foundation of China (No. 81273354,
81102320), Research Fund for the Doctoral Program of Higher Education of China
(No. 20110131130005), Natural Science Foundation of Shandong Province
(ZR2009CM016) and KU Leuven (GOA 10/014) is gratefully acknowledged. We
thank K. Erven, K. Uyttersprot and C. Heens for technical assistance with the
anti-HIV assays.
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Figure captions
Fig. 1. Structures of etravirine and rilpivirine.
Fig. 2. Design of 6,8-disubstituted imidazo[1,2-a]pyrazine derivatives as HIV-1 NNRTIs via a
structure-guided core-refining approach.
Fig. 3. Predicted binding mode of 4a (orange) docked into wt RT (PDB code: 3MEC) in overlap
with ETR (yellow). Hydrogen bonds are indicated with dashed lines in red, and hydrogen atoms
are omitted for clarity.
Tables
Table 1. In vitro anti-HIV-1 activity and cytotoxicity of title compounds (3a-d, 4a-d, 5a-d, 6a, 6b,
7a, 7b, 8a, 8b).
R2
R3
R1
R1
N
O
N
NH
N
20

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Compd R1
CH3
R2
R3
EC50 a (µM)
RES056
CC50b (µM)
SIc
RES056
IIIB
IIIB
5
3a
3b
3c
3d
4a
4b
4c
4d
5a
5b
5c
5d
6a
6b
7a
7b
8a
H
H
H
H
CN
6.5 ± 2.6
>5.3
>32
ndd
32 ± 7.0
5.3 ± 0.77
156 ± 34
57 ± 65
33 ± 1.7
34 ± 3.7
117 ± 60
12 ± 6.2
≥73
<1
nd
CH3
CH3
CH3
NO2
<1
<1
<1
126
25
13
<1
≥224
13
15
<1
5
CH2CN >156
>156
>57
>33
>34
>117
>12
>73
>29
>282
nd
<1
<1
<1
<1
<1
<1
<orX1e
<1
<1
nd
Cl
>57
CH3 CH3 CN
CH3 CH3 NO2
0.26 ± 0.09
1.3 ± 0.16
CH3 CH3 CH2CN 9.2 ± 4.7
CH3 CH3 Cl
CH3 CN CN
CH3 CN NO2
>12
0.32 ± 0.11
2.3 ± 1.3
29 ± 5.3
282 ± 31
13 ± 7.1
38 ± 16
28 ± 2.8
28 ± 1.5
28 ± 6.1
34 ± 2.6
33 ± 3.0
CH3 CN CH2CN 19 ± 3.0
CH3 CN Cl
>13
F
F
F
F
CN
7.2 ± 1.7
>28
>38
>28
>28
>28
>34
>33
<1
<1
<1
<1
<1
<1
NO2
CN
<1
28
7
CH3 Br
CH3 Br
CH3 Cl
CH3 Cl
0.98 ± 0.18
4.0 ± 0.46
0.87 ± 0.18
4.6 ± 0.54
NO2
CN
39
7
8b
NO2
AZT
0.007 ± 0.002 0.01± 0.009 >94
>13144 >9149
DDI
3TC
NVP
DLV
EFV
23 ± 7.6
nd
>212
>87
>15
>36
>6.3
>9
nd
2.2 ± 0.82
0.31 ± 0.06
0.54 ± 0.51
nd
>39
nd
≥7.6
>36
48
>orX2
X1
>67
0.006 ± 0.002 0.16 ± 0.01
>1014
>1127
>41
>184
ETR
0.004± 0.0002 0.02 ± 0.003 >4.6
a
EC50: concentration of compound required to achieve 50% protection of MT-4 cells against
HIV-1-induced cytopathicity, as determined by the MTT method.
b
CC50: concentration required to reduce the viability of mock-infected cells by 50%, as
determined by the MTT method.
c SI: selectivity index (CC50/EC50).
d nd: not determined.
e X1: stands for ≥1 or <1.
Table 2. Inhibitory activity of target compounds, NVP and ETR against HIV-1 RT (WT)
Compd
3a
IC50a (µM)
0.82
Compd
5c
IC50a (µM)
0.17
3b
334
5d
5.5
3c
5.7
6a
0.47
3d
15
6b
11
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4a
4b
4c
4d
5a
5b
0.40
4.7
7a
7b
8a
0.41
4.1
0.36
5.9
1.1
8b
NVP
1.3
0.26
14
2.4
ETR
0.13
a
IC50: The inhibitory concentration of the tested compounds required to inhibit biotin
deoxyuridine triphosphate (biotin-dUTP) incorporation into the HIV-1 RT by 50%.
Table 3. Prediction of physicochemical propertiesa of 4a, 5a and reference compounds.
Compd
EC50
nViol natoms miLogP MW
nON nOHNH nrotb TPSA
MV
(µM)
Accepted
range
<500
Da
<10
<5
≤10
< 140
Å2
<5
4a
5a
0.26
0.32
0.0009
0.05
0.004
1
0
1
1
1
28
29
29
28
28
5.35
4.66
5.00
5.01
5.03
369.43
380.41
380.41
370.42
435.28
6
7
7
7
7
1
1
2
1
3
4
4
4
4
4
75.25
99.04
110.42
88.14
120.65
335.84
336.14
335.75
331.68
335.95
RDEA427
LW-7c
ETR
a nViol = number of violations; natoms = no. of atoms; miLogP = molinspiration predicted LogP;
MW= molecular weight; nON = no. of hydrogen bond acceptors; nOHNH = no. of hydrogen bond
donors; nrotb = no. of rotatable bonds; TPSA = topological polar surface area; MV = molar
volume.
Figures
22

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CN
CN
CN
NH
CN
NH
O
N
HN
N
N
Br
N
NH2
Etravirine (ETR, TMC125) Rilpivirine (RPV, TMC278)
Fig. 1. Structures of etravirine and rilpivirine
Fig. 2. Design of 6,8-disubstituted imidazo[1,2-a]pyrazine derivatives as HIV-1 NNRTIs via a
structure-guided core-refining approach.
23

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Fig. 3. Predicted binding mode of 4a (orange) docked into wt RT (PDB code: 3MEC) in overlap
with ETR (yellow). Hydrogen bonds are indicated with dashed lines in red, and hydrogen atoms
are omitted for clarity.
Schemes
Scheme 1. The synthetic route for imidazo[1,2-a]pyrazine derivatives. Reagents and conditions: (a)
ClCH2CHO, 2-propanol, reflux in N2 atmosphere, 24 h; (b) ArOH, K2CO3, DMF, r.t., overnight; (c)
ArNH2, Pd(OAc)2, Xanphos, Cs2CO3, 1,4-dioxane, reflux in N2 atmosphere.
24

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Highlights
·Novel imidazo[1,2-a]pyrazines were identified as HIV-1 inhibitors.
·4a and 5a exhibited anti-HIV-1 (IIIB) activity with EC50 values of 0.26 µM and 0.32
µM respectively.
·Some drug-like properties of 4a and 5a were predicted.
·Preliminary SARs and molecular simulation of these new analogues were detailed.

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Correspondingtocompound3a(HNM R)

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Correspondingtocompound3a(CNM R)

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Correspondingtocompound3a(M S)