Optimization of 2,4-diarylanilines as non-nucleoside HIV-1 reverse transcriptase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2012.02.055

The current optimization of 2,4-diarylaniline analogs (DAANs) on the central phenyl ring provided a series of new active DAAN derivatives 9a-9e, indicating an accessible modification approach that could improve anti-HIV potency against wild-type and resis

Full text of DOI:10.1016/j.bmcl.2012.02.055

Bioorganic & Medicinal Chemistry Letters 22 (2012) 2376–2379
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Optimization of 2,4-diarylanilines as non-nucleoside HIV-1 reverse
transcriptase inhibitors
Lian-Qi Sun a, Bingjie Qin a, Li Huang b, Keduo Qian c, Chin-Ho Chen b, Kuo-Hsiung Lee c,d, Lan Xie a,
⇑
a Beijing Institute of Pharmacology & Toxicology, 27 Tai-Ping Road, Beijing 100850, China
b Duke University Medical Center, Box 2926, Surgical Oncology Research Facility, Durham, NC 27710, USA
c Natural Products Research Laboratories, UNC Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC 27599, USA
d Chinese Medicine Research and Development Center, China Medical University and Hospital, Taichung, Taiwan
a r t i c l e i n f o
a b s t r a c t
Article history:
The current optimization of 2,4-diarylaniline analogs (DAANs) on the central phenyl ring provided a ser-
ies of new active DAAN derivatives 9a–9e, indicating an accessible modification approach that could
improve anti-HIV potency against wild-type and resistant strains, aqueous solubility, and metabolic sta-
bility. A new compound 9e not only exhibited extremely high potency against wild-type virus (EC50
0.53 nM) and several resistant viral strains (EC50 0.36–3.9 nM), but also showed desirable aqueous solu-
bility and metabolic stability, which were comparable or better than those of the anti-HIV-1 drug
TMC278 (2). Thus, new compound 9e might be a potential drug candidate for further development of
novel next-generation NNRTIs.
Received 23 December 2011
Revised 16 February 2012
Accepted 17 February 2012
Available online 24 February 2012
Keywords:
Diarylaniline
NNRTIs
Lead optimization
Anti-HIV agents
Ó 2012 Elsevier Ltd. All rights reserved.
Since the first case of acquired immunodeficiency syndrome
(AIDS) was reported by the US in 1981, the scientific progress in
HIV/AIDS research has been extraordinary, especially in the
development of antiretroviral therapy (ART) that has proven to
be life-saving to millions of people. Recent scientific evidence has
broad spectrum of mutated viral strains. There is also a higher
genetic barrier8,9 to delay the emergence of drug-resistance toward
etravirine and rilpivirine.10 Although both drugs are poorly water
soluble over a wide pH range, 2 has the advantage of better
bioavailability when compared to 1, resulting in a once-daily oral
dosing. The success of etravirine and rilpivirine greatly encouraged
more research to explore additional novel next-generation NNRTI
agents with new scaffolds, high potencies, improved resistant pro-
files, and better pharmacokinetic profiles than 1 and 2 for more
efficacious therapy and potential AIDS prevention.
In prior studies toward the design and synthesis of novel next-
generation NNRTIs, we discovered a series of new diarylanilines
(DAANs, Fig 2) with nano- to subnano-molar anti-HIV potencies
against wild-type and multi-RT-resistant viral strains,11,12 as
exemplified by compound 3 with higher potency against wild-type
(EC50 0.38 nM) and RT multi-resistant viral strains (EC50 0.87 nM)
than 1 in the same assays. Previous SAR results also defined the
key pharmacophores of DAANs as NNRTIs: (1) a para-cyanoaniline
moiety (A-ring), (2) a crucial amino group on the central phenyl
ring (B-ring) ortho to the A-ring position, (3) a trisubstituted phen-
oxy ring (C-ring) with a para-linear hydrophobic substituent, and
(4) similar molecular flexibility to 1 and 2. Their high potency
and straightforward synthesis prompted us to develop DAANs as
new anti-AIDS drug candidates. For orally anti-HIV drug candi-
dates, aqueous solubility is one of the most critical physicochemi-
cal properties to be considered in the process of lead optimization,
because low aqueous solubility would limit molecular absorption
demonstrated that ART is also effective at preventing infection1,2
;
thus it offers an unprecedented opportunity to control the AIDS
pandemic. Therefore, the discovery and development of novel
highly potent anti-HIV drugs is imperative.
Among current anti-HIV drugs, non-nucleoside reverse trans-
criptase inhibitors (NNRTIs) are used in combination with other
drugs as key components in the highly active antiretroviral therapy
(HAART).3,4 NNRTIs target an allosteric binding pocket on HIV-1
reverse transcriptase (RT) in a noncompetitive manner to cause
distortion of the three-dimensional structure of the enzyme to in-
hibit RT catalytic function.5 Currently, five NNRTIs drugs have been
approved by FDA and are marketed (Fig 1) with the advantages of
high potency and low toxicity. Despite the clinical high efficiency,
the first-generation NNRTIs drugs (nevirapine, delavirdine, efavi-
renz) are limited in clinical use due to rapid emergence of drug-
resistance. However, the second-generation NNRTI drugs etravi-
rine (TMC125, 1)6 and rilpivirine (TMC278, 2)7 greatly overcome
this deficiency. Both 1 and 2 possess potent antiviral activity with
subnano- or low nano-molar EC50 values against wild-type and a
⇑
Corresponding author.
E-mail address: lanxieshi@yahoo.com (L. Xie).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2012.02.055

L.-Q. Sun et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2376–2379
2377
DAANs was associated with antiviral potency by creating a small
electrostatic interaction with the positively charged amino acid
K172 on the NNRTI binding site.11 Thus, we proposed that other
polar or ionizable groups, similar to the nitro group, on the B-ring
might provide comparable or additional interaction point(s) with
the NNRTI binding site via a ‘salt bridge’ and/or H-bonds to en-
hance antiviral potency. Furthermore, the presence of R1 substitu-
ent(s) to serve as a H-bond acceptor or donor is expected to be
favorable for improving aqueous solubility. Therefore, a series of
new DAAN derivatives with different R1 groups on the central phe-
nyl ring are reported herein, including chemical synthesis, antiviral
activity, aqueous solubility, and metabolic stability in vitro.
HN
H
N
O
N
N
N
F3C
O
HN
S
O
Cl
N
N
N
O
O
N
H
O
N
H
Nevirapine
Delavirdine
Efavirenz
CN
CN
O
CN
NH
CN
As shown in Scheme 1, the nitration of 2,4-dichlorobenzoic acid,
a commercially available and inexpensive reagent, was performed
with concentrated HNO3 in H2SO4 at low temperature (0–5 °C) to
produce 2,4-dichloro-5-nitrobenzoic acid (4) with a 91% yield.
Compound 4 was esterified with MeOH in the presence of H2SO4
to afford methyl 2,4-dichloro-5-nitrobenzoate (5). Coupling of 5
and 4-aminobenzo-nitrile was performed in DMF in the presence
of excess cesium carbonate at 100 °C for about 10 h to provide
methyl 2-chloro-4-(4-cyanophenyl amino)-5-nitrobenzoate (6),
which was characterized by an NH signal at d 9.76 ppm in the 1H
NMR spectrum, ascribable to the chelated nitro group and consis-
tent with previous results that the chlorine ortho to the nitro group
has higher reactivity for nucleophilic substitution with an aromatic
amine. Next, intermediate 6 was reacted with 4-hydroxy-3,5-
dimethylbenzaldehyde under microwave irradiation in DMF in
the presence of potassium carbonate with stirring at 190 °C for
about 15 min to afford 7 with a three-phenyl ring skeleton in a
67% yield. Subsequently, the aldehyde group in 7 was converted
into a cyanovinyl moiety by condensation with diethyl cyanometh-
yl phosphonate in the presence of potassium tert-butoxide to af-
ford compound 8a. Then, the ester group in 8a was hydrolyzed
under basic conditions to afford the corresponding benzoic acid
compound 8b. The carboxylic acid group was then converted into
an amide or N-methyl amide by treatment with SOCl2, followed
with ammonia or methylamine to afford corresponding com-
pounds 8c and 8d respectively. In addition, the ester group in 8a
was reduced with LiBH4 to provide compound 8e with a hydroxy-
methyl substituent. Finally, the nitro group on the central ring in
compounds 8a–8e was reduced by using sodium hydrosulfite
N
HN
N
NH
N
Br
N
NH2
2
1
Rilpivirine (TMC278)
EC50 0.52 nM
Eravirine (TMC125)
EC50 1.5 nM
Figure 1. Current marketed NNRTIs.
R2
C
CN
A
O
NH
NH2
B
R1
DAANs
3
R
1 = NO2, R2 = (E)-CH=CHCN
EC50 0.38 nM (wild-type), 0.87 nM (RTMDR)
Figure 2. 2,4-Diarylaniline leads.
and bioavailability in vivo. On the other hand, most small organic
molecules generally have poor water solubility. Therefore, our cur-
rent optimization of DAANs must generate active DAANs with
desirable molecular aqueous solubility. Our early results indicated
that the nitro group (R1) on the central phenyl ring (B-ring) of
CN
CN
Cl
Cl
Cl
Cl
Cl
Cl
a
b
NH2
c
Cl
NH
HOOC
NO2
H3COOC
NO2
HOOC
H3COOC
NO2
6
4
5
CN
CN
CHO
CHO
CN
CN
CN
OH
d
k
e
NH
O
O
NH
O
NH
H3COOC
NO2
R1
NO2
R1
NH2
7
8a-e
9a-e
1
R
a
b
c
d
e
COOCH3
f
COOH
g
h
CONH2
CONHCH3
CH2OH
j
Scheme 1. Reagent and conditions: (a) HNO3/H2SO4, 0–5 °C to rt, 2 h; (b) H2SO4/MeOH, reflux, 2 h; (c) Cs2CO3/DMF, 100 °C, 10 h; (d) K2CO3/DMF, 190 °C, microwave, 10–
15 min; (e) (EtO)2P(O)CH2CN, t-BuOK/THF, 0 °C to rt, 3 h; (f) THF/MeOH, aq NaOH, 0.5 h; (g) (i) CH2Cl2/SOCl2, reflux, 3 h; (ii) ammonia in THF, 0 °C, 0.5 h; (h) (i) SOCl2/CH2Cl2,
reflux, 3 h; (ii) methylamine in THF, 0 °C, 0.5 h; (j) LiBH4, THF/MeOH, 1 h; (k) Na2S2O4, ammonia, THF/H2O (1:1, v/v), rt, 2 h.

2378
L.-Q. Sun et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2376–2379
Table 1
a
Anti-HIV activities of 9a–9e in cellular assay and aqueous solubility
H
N
O
CN
NC
R1
NH2
b
c
R1
EC50 (nM)
CC50
(
lM)
SId
Aqueous solubility (lg/mL)
pH 7.4
pH 2.0
9a
9b
9c
9d
COOCH3
COOH
CONH2
CONHCH3
CH2OH
NO2
2.74 0.71
230 38
>22.8
14.9
>23.6
>9.2
16.3
>47.1
19.4
>8465
65
1.30
117
1.75
4.40
2.10
8.63
21.0
0.29
156
0.87 0.28
5.72 0.94
0.53 0.13
0.38 0.07
0.52 0.14
>27126
>1606
30754
>123947
37305
0.63
1.32
3.23
0.29
0.24
9e
3e
2 (TMC278)
a
b
c
HIV-1 NL3-4 (wild-type) virus in TZM-bl cell lines.
Concentration of compound that causes 50% inhibition of viral replication and presented as mean standard deviation (SD) in at least triplicate.
Concentration of compound that causes cytotoxicity to 50% of cells.
d
e
Selectivity index (SI) is the ratio of CC50/EC50
.
Compound and activity data were published previously.12
Table 2
Data for 9c and 9e against resistant viral strains and metabolic stability
a
EC50 (nM)
Human liver microsomeb
t1/2 min CL mL/min/mg
Rat liver microsomeb
t1/2 min CL mL/min/mg
HIV-1RTMDR1
K101E
E138 K
9c
9e
3
0.31 0.10
0.36 0.10
0.87 0.33
0.49 0.10
8.8 3.3
3.4 1.05
10.8 3.53
5.7 1.4
9.0 2.6
3.9 0.90
11.3 3.76
5.2 1.6
35
30
47
34
0.20
0.23
0.15
0.20
122
84
139
120
0.057
0.083
0.050
0.057
2
a
Experiments were performed at least in triplicate and data presented as mean SD.
Data from at least two experiments and presented as means.
b
dehydrate to produce corresponding target compounds 9a–9e, a
series of 5-substituted 2-(4-cyanophenylamino)-4-(4-cyanovinyl-
2,6-dimethylphenoxy)anilines.13
liver microsome (RLM and HLM, respectively) assays in vitro,14,15
respectively, to predict their metabolic stability in vivo. The two
data sets in Table 2 indicated a similar metabolic stability pattern.
Compounds 9c and 9e had moderate stability with half-lives of 35
and 30 min in HLM and 122 and 84 min in RLM assays, respectively.
Compound 9c had a longer maintaining time (was more stable) than
9e in both assays, and both compounds showed comparable meta-
bolic stability to that of 2 (t1/2 34 min) in the HLM assay.
In conclusion, our optimization focused on the central phenyl
ring of DAANs provided a series of new active compounds 9a–9e
and also verified our previous hypothesis that the R1 substitution
could optimize the antiviral potency and drug-like properties. This
study resulted in the discovery of a new compound 9e that not only
exhibited extremely high potency against wild-type virus (EC50
0.53 nM) and several resistant viral strains (EC50 0.36–3.9 nM),
but also showed desirable aqueous solubility and metabolic stabil-
ity, which were better than those of drug 2. Thus, compound 9e
could serve as a potential drug candidate for further development.
New compounds 9a–9e were first tested against wild-type HIV-
1 NL4-3 infection of TZM-bl cells in parallel with TMC278 and 3. The
data are summarized in Table 1. Compounds 9c (R1 = CONH2) and 9e
(R1 = CH2OH) exhibited extremely high potency with EC50 values of
0.87 and 0.53 nM, respectively, and both showed high selective in-
dexes (SIs) of >27,126, comparable to those of new drug 2 (TMC278)
and 3 in the same assay. Compounds 9a (R1 = COOCH3) and 9d
(R1 = CONHCH3) were also potent with EC50 values of 2.7 and
5.7 nM, respectively, and SI values of >8464 and >1606, respectively.
On the other hand, 9b with a carboxylic acid group (R1 = COOH) was
significantly less potent (EC50 230 nM). Thus, the current results,
even with the limited data set, demonstrated that the R1 substituent
on the central phenyl ring was modifiable and could greatly affect
antiviral potency. The aqueous solubility of new compounds
9a–9e at pH 7.4 and pH 2.0 were measured by a HPLC/UV method
in parallel with 2 and 3, and the data are also shown in Table 1.
Compared to 2 and 3, all new 9 series compounds had improved
aqueous solubility at pH 7.4 and were generally more soluble at
pH 2.0, likely due to the presence of a free amino group. The most
active new compound 9e (R1 = CH2OH) showed better aqueous
Acknowledgments
This investigation was supported by grants 30930106 and
81120108022 from the Natural Science Foundation of China (NSFC)
awarded to L. Xie and U.S. NIH grants awarded to C. H. Chen
(AI65310) and K. H. Lee (AI33066).
solubility (3.23 lg/mL at pH 7.4 and 21.0 lg/mL at pH 2.0) than
the remaining four compounds in the series. Subsequently, the most
active compounds 9c and 9e were selected for testing against NNRTI
resistant HIV-1 mutants RT-multi-drug-resistant (RTMDR), K101E,
and E138K. As shown in Table 2, new compounds 9c and 9e exhib-
ited improved antiviral activity compared to 3 against the drug
resistant viral strains, and a little better or comparable to those of
2. Compounds 9c and 9e were further evaluated in rat and human
References and notes
1. Fauci, A. S. Science 2011, 333, 13.
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3. Kaufmann, G. R.; Cooper, D. A. Curr. Opin. Microbiol. 2000, 3, 508.
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5. Andries, K.; Azijn, H.; Thielemans, T. Antimicrob. Agents Chemother. 2004, 48,
4680.
6. Pecora Fulco, P.; McNicholl, I. R. Pharmacotherapy 2009, 29, 281.
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S. H.; Arnold, E. J. Mol. Biol. 2009, 385, 693.
J = 8.8 Hz, ArH-30,50), 7.47 (2H, s, ArH-30,50), 7.57 (1H, d, J = 16.8 Hz, CH@),
7.61 (1H, s, ArH-3), 8.20 (1H, s, NH); MS m/z (%) 424.2 (M+1, 100); purity
(HPLC) 98.2%. 9d: yield 31%, white solid, mp 112–114 °C; 1H NMR (CDCl3) d
ppm 2.16 (6H, s, CH3 ꢀ 2), 3.07 (3H, d, NCH3), 5.80 (1H, s, NH), 5.83 (1H, d,
J = 16.8 Hz, @CH), 6.18 (1H, s, ArH-6), 6.65 (2H, d, J = 8.8 Hz, ArH-20,60), 7.21
(2H, s, ArH-300,500), 7.32 (1H, d, J = 16.8 Hz, CH@), 7.41 (2H, d, J = 8.8 Hz, ArH-
30,50), 7.81 (1H, s, ArH-3); MS m/z (%) 438.4 (M+1, 100); HPLC-purity 100.0%.
9e: yield 81%, white solid, mp 186–188 °C; 1H NMR (CDCl3) d ppm 2.13 (6H,
s, CH3 ꢀ 2), 4.87 (2H, s, CH2), 5.50 (1H, s, NH), 5.79 (1H, d, J = 16.8 Hz, CH@),
6.03 (1H, s, ArH-6), 6.55 (2H, d, J = 8.8 Hz, ArH-20,60), 6.94 (1H, s, ArH-3),
7.17 (2H, s, ArH-300,500), 7.30 (1H, d, J = 16.8 Hz, CH@), 7.40 (2H, d, J = 8.8 Hz,
ArH-30,50); MS m/z (%) 411.3 (M+1, 100); HPLC-purity 99.9%.
9. Vingerhoets, J.; Azijn, H.; Fransen, E.; De Baere, I.; Smeulders, L.; Jochmans, D.;
Andries, K.; Pauwels, R.; de Bethune, M. P. J. Virol. 2005, 79, 12773.
10. Pecora Fulco, P.; McNicholl, I. R. Pharmacotherapy 2009, 29, 281.
11. Qin, B. J.; Jiang, X. K.; Lu, H.; Tian, X. T.; Barbault, F.; Huang, L.; Qian, K.; Chen, C.
H.; Huang, R.; Jiang, S.; Lee, K. H.; Xie, L. J. Med. Chem. 2010, 53, 4906.
12. Tian, X. T.; Qin, B. J.; Wu, Z. Y.; Wang, X. F.; Lu, H.; MorrisNatschke, S. L.; Chen,
C. H.; Jiang, S.; Lee, K. H.; Xie, L. J. Med. Chem. 2010, 53, 8287.
13. Synthetic procedure for 4-substituted 1,5-diarylbenzene-1,2-diamines (9a–
9e). To a solution of a diaryl-nitrobenzene (8, 1 equiv) in THF and water
(30 mL, v/v 1:1) was added ammonia aqueous solution (25%) and sodium
hydrosulfite (10 equiv) successively with stirring at room temperature for
2 h. The reaction was monitored by TLC (CH2Cl2/MeOH 60:1) until
completed. The mixture was poured into ice-water and extracted with
EtOAc three times. After removal of organic solvent under reduced pressure,
crude product (9) was purified by a flash silica gel column chromatograph
(eluent: CH2Cl2/MeOH = 30/1) with the CombiFlash Flash chromatography
system, ISCO company, Inc. to obtain pure target compounds 9a–9e
respectively. HPLC analyses for purities of 9a–9e were performed on an
Agilent 1200 HPLC system with UV detector and a Grace Alltima HP C18
14. Microsomal stability assay. Stock solutions of test compounds (1 mg/mL) were
prepared by dissolving the pure compound in DMSO and stored at 4 °C. Before
assay, the stock solution was diluted with ACN to 0.1 mM concentration. For
measurement of metabolic stability, all test compounds were brought to a final
concentration of 1
contained 0.1 mg/mL human liver microsomes and 5 mM MgCl2. The
incubation volumes were 300 L, and reaction temperature was 37 °C.
Reactions were started by adding 60 L of NADPH (final concentration of
1.0 mM) and quenched by adding 600 L of ice-cold ACN to stop the reaction at
5, 15, 30, 60 min time points. Samples at 0 min time point were prepared by
adding 600 L ice-cold ACN first, followed by 60 L NADPH. Incubations of all
lM with 0.1 M potassium phosphate buffer at pH 7.4, which
l
l
l
l
l
samples were conducted in duplicate. After quenching, all samples were
centrifuged at 12,000 rpm for 5 min at 0 °C. The supernatant was collected, and
column (100 ꢀ 2.1 mm, 3
l
m) eluting with a mixture of solvents A and B in
20 lL of the supernatant was directly injected onto a Shimadzu LC-MS-2010
two conditions: (1) acetonitrile (ACN)/water 70:30, flow rate 1.0 mL/min; (2)
MeOH/water 70:30, flow rate 0.8 mL/min. The samples were detected under
system with an electrospray ionization source (ESI) for further analysis. The
following controls were also conducted: (1) positive control incubation
containing liver microsomes, NADPH, and reference compound; (2) negative
control incubation omitting NADPH; and (3) baseline control containing only
liver microsomes and NADPH. The peak heights of test compounds at different
time points were converted to percentage of remaining, and the peak height
values at initial time (0 min) served as 100%. The slope of the linear regression
from log percentage remaining versus incubation time relationships (ꢁk) was
used to calculate in vitro half-life (t1/2) value by the formula of in vitro t1/
2 = 0.693/k, regarded as first-order kinetics. Conversion to in vitro CLint (in units
of ml/min/mg protein) was calculated by the formula15: CLint = (0.693/in vitro
t1/2) ꢀ (ml incubation/mg microsomes).
UV wavelength at 254 nm and an injection volume of 3 lL. Compounds: 9a,
yield 64%, white solid, mp 240–242 °C; 1H NMR (CDCl3) d ppm 2.16 (6H, s,
CH3 ꢀ 2), 3.93 (3H, s, OCH3), 5.78 (1H, s, NH), 5.80 (1H, d, J = 16.8 Hz, @CH),
6.20 (1H, s, ArH-6), 6.67 (2H, d, J = 8.8 Hz, ArH), 7.17 (2H, s, ArH), 7.31 (1H,
d, J = 16.8 Hz, CH@), 7.42 (2H, d, J = 8.8 Hz, ArH), 7.45 (1H, s, ArH-3); MS m/z
(%) 439.3 (M+1, 100); HPLC-purity 96.1%. 9b: yield 35%, brown solid, mp
226–228 °C. 1H NMR (CDCl3)
d
ppm 2.19 (6H, s, CH3 ꢀ 2), 5.84 (1H, d,
J = 16.8 Hz, @CH), 6.04 (1H, s, NH), 6.27 (1H, s, ArH-6), 6.75 (2H, d, J = 8.8 Hz,
ArH-20,60), 7.22 (2H, s, ArH-300,500), 7.32 (1H, d, J = 16.8 Hz, CH@), 7.44 (2H, d,
J = 8.8 Hz, ArH-30,50), 7.72 (1H, s, ArH-3); MS m/z (%) 423.2 (M-1, 100); HPLC
purity 100.0%. 9c: yield 63%, white solid, mp 290–292 °C; 1H NMR (DMSO-
d6) d ppm 2.10 (6H, s, CH3 ꢀ 2), 4.75 (2H, s, NH2), 6.01 (1H, s, ArH-6), 6.39
(1H, d, J = 16.8 Hz, @CH), 6.63 (2H, d, J = 8.8 Hz, ArH-20,60), 7.45 (2H, d,
15. Obach, R. S.; Baxter, J. G.; Liston, T. E.; Silber, B. M.; Jones, B. C.; MacIntyre, F.;
Rance, D. J.; Wastall, P. J. Pharmacol. Exp. Ther. 1997, 283, 46.