Optimization of the antiviral potency and lipophilicity of halogenated 2,6-diarylpyridinamines as a novel class of HIV-1 NNRTIS

Article abstract of DOI:10.1002/cmdc.201400075

Nineteen new halogenated diarylpyridinamine (DAPA) analogues modified at the phenoxy C-ring were synthesized and evaluated for anti-HIV activity and certain drug-like properties. Ten compounds showed high anti-HIV activity (EC₅₀<10 nM). In particular, (E)-6-(2′′-bromo- 4′′-cyanovinyl-6′′-methoxy)phenoxy-N²- (4′-cyanophenyl)pyridin-2,3-diamine (8 c) displayed low-nanomolar antiviral potency (3-7 nM) against wild-type and drug-resistant viral strains bearing the E138K or K101E mutations, which are associated with resistance to rilvipirine (1 b). Compound 8 c exhibited much lower resistance fold changes (RFC: 1.1-2.1) than 1 b (RFC: 11.8-13.0). Compound 8 c also exhibited better metabolic stability (in vitro half-life) than 1 b in human liver microsomes, possessed low lipophilicity (clog D: 3.29; measured log P: 3.31), and had desirable lipophilic efficiency indices (LE>0.3, LLE>5, LELP<10). With balanced potency and drug-like properties, 8 c merits further development as an anti-HIV drug candidate. Taking the lead! Among 19 new halogenated diarylpyridinamine (DAPA) analogues, (E)-6-(2′′-bromo- 4′′-cyanovinyl-6′′-methoxy)phenoxy-N²- (4′-cyanophenyl)pyridin-2,3-diamine (8 c) displays low-nanomolar antiviral potency (3-7 nM) against wild-type HIV and E138K or K101E mutant strains; it also exhibits promising drug-like properties. Compound 8 c merits development as an anti-HIV drug candidate.

Full text of DOI:10.1002/cmdc.201400075

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DOI: 10.1002/cmdc.201400075
Optimization of the Antiviral Potency and Lipophilicity of
Halogenated 2,6-Diarylpyridinamines as a Novel Class of
HIV-1 NNRTIS
Zhi-Yuan Wu,[a] Na Liu,[a] Bingjie Qin,[a] Li Huang,[b] Fei Yu,[c] Keduo Qian,[d] Susan L. Morris-
Natschke,[d] Shibo Jiang,[c, e] Chin Ho Chen,[b] Kuo-Hsiung Lee,*[d, f] and Lan Xie*[a]
Nineteen new halogenated diarylpyridinamine (DAPA) ana-
logues modified at the phenoxy C-ring were synthesized and
evaluated for anti-HIV activity and certain drug-like properties.
Ten compounds showed high anti-HIV activity (EC50 <10 nm).
In particular, (E)-6-(2’’-bromo-4’’-cyanovinyl-6’’-methoxy)phe-
noxy-N2-(4’-cyanophenyl)pyridin-2,3-diamine (8c) displayed
low-nanomolar antiviral potency (3–7 nm) against wild-type
and drug-resistant viral strains bearing the E138K or K101E mu-
tations, which are associated with resistance to rilvipirine (1b).
Compound 8c exhibited much lower resistance fold changes
(RFC: 1.1–2.1) than 1b (RFC: 11.8–13.0). Compound 8c also ex-
hibited better metabolic stability (in vitro half-life) than 1b in
human liver microsomes, possessed low lipophilicity (clogD:
3.29; measured logP: 3.31), and had desirable lipophilic effi-
ciency indices (LE>0.3, LLE>5, LELP<10). With balanced po-
tency and drug-like properties, 8c merits further development
as an anti-HIV drug candidate.
Introduction
Non-nucleoside HIV-1 reverse transcriptase inhibitors (NNRTIs)
play an important role in antiretroviral therapy (ART)[1,2] and
prevention of HIV infection, owing to their advantages of high
potency and low toxicity. Five NNRTIs, including nevirapine, de-
lavirdine, efavirenz, etravirine (TMC125, 1a), and rilpivirine
(TMC278, 1b), have been approved by the US Food and Drug
Administration (FDA) and are currently available for the treat-
ment of AIDS and HIV infection. Diarylpyrimidine derivatives
(DAPYs) 1a and 1b (Figure 1) are new-generation NNRTI drugs
that possess extremely high potency against wild-type and
Figure 1. Next-generation NNRTI drugs TMC125 and TMC278.
a broad spectrum of mutated NNRTI-resistant strains.[3] It has
been reported that HIV-1 has a higher genetic barrier to evolve
resistance to 1a and 1b;[4] therefore, this compound type
might have a better chance to overcome the drawback of
rapid drug resistance observed with earlier NNRTI drugs. How-
ever, resistance mutations to the new-generation NNRTI drug
1b have also appeared in patients.[5,6] Therefore, efforts still
continue to develop additional new-generation NNRTI drugs
with high efficacy and different structural scaffolds that will
provide more clinical treatment options.
[a] Z.-Y. Wu, N. Liu, Dr. B. Qin, Prof. L. Xie
Beijing Institute of Pharmacology & Toxicology
27 Tai-Ping Road, Beijing, 100850 (China)
E-mail: lanxieshi@yahoo.com
[b] Dr. L. Huang, Prof. C. H. Chen
Duke University Medical Center, Box 2926
Surgical Oncology Research Facility, Durham, NC 27710 (USA)
[c] Dr. F. Yu, Prof. S. Jiang
Lindsley F. Kimball Research Institute
New York Blood Center, New York, NY 10065 (USA)
In our previous studies on novel NNRTI agents, we discov-
ered diarylpyridinamines (DAPAs)[7,8] with extremely high po-
tency against HIV-1 wild-type and drug-resistant viral strains.
As an example, DAPA lead compounds 2a and 2b (Figure 2)
exhibited sub-nanomolar potencies against HIV-1 wild-type
(EC50 0.63 and 0.71 nm) and RT multidrug-resistant (RTMDR)
viral strains (EC50 0.96 and 0.59 nm), which were better than or
similar to those of new-generation NNRTI drugs 1a (EC50 1.4
and 1.0 nm) and 1b (EC50 0.51 and 0.49 nm), respectively, in
the same assays. Although DAPA and DAPY analogues have
similar molecular flexibility and topology, DAPA compounds
have a new scaffold chemotype and can likely adopt confor-
[d] Dr. K. Qian, Dr. S. L. Morris-Natschke, Prof. K.-H. Lee
Natural Products Research Laboratories, Eshelman School of Pharmacy
University of North Carolina, Chapel Hill, NC 27599 (USA)
E-mail: khlee@unc.edu
[e] Prof. S. Jiang
Key Laboratory of Medical Molecular Virology of Ministries of Education
and Health, Shanghai Medical College
Fudan University, Shanghai 200032 (China)
[f] Prof. K.-H. Lee
Chinese Medicine Research and Development Center
China Medical University and Hospital, Taichung (Taiwan)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201400075: HPLC conditions and purity
data for target compounds.
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Results and Discussion
Chemistry
DAPAs are available at low cost, being readily synthesized from
commercial reagents via short and efficient routes. Modifica-
tions on the phenol ring (C-ring) were performed as shown in
Schemes 1 and 2 to produce new DAPA series 6a–n and 8a–e.
6-Chloro-2-(4-cyanophenyl)amino-3-nitropyridine (3), prepared
from commercially available 2,6-dichloro-3-nitropyridine and 4-
cyanophenol, as described in our previous publication,[8] was
coupled with various halogenated phenols in DMF in the pres-
ence of cesium carbonate or potassium carbonate under mi-
crowave irradiation at 90–1008C for 10–15 min to afford corre-
sponding halogenated 2,6-diaryl-3-nitropyridines 4a–e, 5a,b,
and 7a, respectively. The aldehyde group on the C-ring in 4a
was reduced with sodium borohydride to produce hydroxy-
Figure 2. Leads, new DAPAs, and optimization strategy.
mational changes (“wiggle”) as well as relocate and reorient
within the NNRTI binding pocket (“jiggle”),[9,10] leading to high
potency against drug-resistant viral replication. In our contin-
ued study, we now report the design and synthesis of a new
series of halogenated DAPA de-
rivatives, as well as an evaluation
of their anti-HIV activity and es-
sential drug-like properties. Our
aim was to develop potential
new drug candidates with good
in vitro antiviral potency and ac-
ceptable drug-like properties.
Based on prior SAR results,[11,12]
we know that the para-cyano-
phenyl moiety (A-ring), the cen-
tral pyridine (B-ring) with a 3-
amino group and the NH linker
between these two rings are
necessary pharmacophores of
DAPAs as HIV-1 NNRTIs. The
presence of the 3-amino group
on the central pyridine ring is es-
pecially important for interaction
with a key residue, K101, in the
Scheme 1. a) Cs2CO3 or K2CO3 in DMF, 90–1008C, 10–15 min, microwave; b) NaBH4, THF/CH3OH, RT, 30 min for 5c;
c) NH2OH·HCl, RT, 2 h for 5e; d) (EtO)2P(O)CH2CN, tBuOK, THF, 08C!RT, 1 h, for 5g; or CNCH2CONH2, ZnCl2, 1008C,
microwave, 10 min, for 5h; or CH3COCH3, NaOH, 2 h, RT, for 5i; or Ph3PCHCOOCH3, CHCl3, reflux, overnight, for 5j;
e) CH3COCl, Et3N, THF, RT, 30 min; f) Ac2O, reflux, overnight, for 5 f; g) 1. aq NaOH, RT, 36 h; 2. CDI, NH3·H2O, RT,
2 h; h) Na2S2O4, NH3·H2O, THF/H2O (v/v 1:1), RT, 3 h; i) Pd/C H2, EtOH, RT, 6 h, 65–78%; j) LiBH4, THF, CH3OH, 08C!
RT, 78%.
NNRTI binding site, which acts
as both hydrogen bond donor
and acceptor to form multiple
hydrogen bonds.[13] While the
known
pharmacophores
of
DAPAs were maintained in our
new series, current modifications focused on substituents X
and R on the phenoxy ring (C-ring; Figure 2) to extend our
knowledge of structure–activity (SAR) and structure–property
(SPR) relationships. Initially, we replaced the 2,6-dimethyl
groups, which are known major metabolic site(s) in drugs 1a
and 1b,[14] with halogens (X) in order to increase metabolic sta-
bility. We also introduced polar or ionizable groups or heteroa-
toms as para substituents (R) on the C-ring to investigate how
physicochemical properties affect drug-like properties. Herein
we report the synthesis of a new series of halogenated DAPAs,
their antiviral activity against HIV-1 wild-type and drug-resist-
ant strains, and certain in vitro drug-like properties, such as
logP values, metabolic stability, aqueous solubility, and lipophi-
licity.
methyl compound 5c, followed by esterification with acetyl
chloride to afford 5d. Treatment of 4a with hydroxylamine hy-
drochloride yielded oxime compound 5e, which, follow-
ing reflux in acetic anhydride overnight, was converted into
para-cyano compound 5 f. The formyl group in 4a was con-
densed with nucleophilic reagents, including diethyl cyano-
methylphosphonate [(EtO)2P(O)CH2CN], 2-cyanoacetamide
(CNCH2CONH2), Ph3PCHCOOCH3, or acetone, under basic condi-
tions to produce corresponding compounds 5g, 5h, 5i, or 5j,
respectively, containing various groups at the para position (R)
on the C-ring. Compound 5j yielded carboxamide compound
5k upon hydrolysis under basic conditions and treatment with
ammonia. Similarly to 5g, a series of para-cyanovinyl inter-
mediates 7b–e were prepared by condensation of the formyl
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(refer to PDB IDs 3MEC and
3BGR). Consequently, the pres-
ence of heteroatoms, polar, or
ionizable groups at the para po-
sition of the phenoxy ring might
impede insertion of the R moiety
in the tunnel and binding
pocket, in turn compromising
antiviral potency. Subsequently,
four additional halogenated
DAPA analogues, 8a–d, with
either para-cyano or para-cyano-
vinyl R groups on the C-ring,
were synthesized. The para-cya-
novinyl dibromo compound 8b
displayed the highest potency,
with a sub-nanomolar EC50 value
of 0.33 nm, more potent than
1b in the same assay. Dibromo
8a with a para-cyano R group
was also highly potent (EC50
0.99 nm) against NL4-3. Both 8a
Scheme 2. a) Cs2CO3 or K2CO3 in DMF, 90–1008C, 10–5 min, microwave; b) tBuOK, (EtO)2P(O)CH2CN, THF, 08C!RT,
1 h; c) Na2S2O4, NH3·H2O, THF/H2O (v/v 1:1), RT, 3 h.
group in 4b–e with diethyl cyanomethylphosphonate. Finally,
the 3-nitro group in series 5 and 7 compounds was reduced to
an amino group by using sodium hydrosulfite or catalytic hy-
drogenation with palladium on carbon (5–10%) to
and 8b were more potent than the corresponding difluoro 6 f
and 6g and similarly potent to dimethyl leads 2a and 2b. Fur-
thermore, para-cyanovinyl compound 8c with different X
obtain the corresponding halogenated series 6 and 8
Table 1. Anti-HIV activity of new DAPA series 6 and 8 analogues.[a]
diarylpyridinamines, respectively. Catalytic hydroge-
nation simultaneously reduced the double bond in
the para-substituents of 5i, 5j, 5g, and 5h, giving 6i,
6j, 6l, and 6m, respectively. The ester group in 6j
was further reduced with lithium borohydride to
afford 6n with a para-hydroxypropyl group on the C-
ring.
Compd
X
R
EC50 [nm][b]
CC50 [mm][c]
SI[d]
6a
6b
6c
6d
6e
6 f
6g
6h
6i
F
F
F
F
F
F
F
F
F
F
F
F
F
F
Br
Br
H
Br
CH2OH
CH2OAc
CH=NOH
CN
CH=CHCN
CH=C(CN)CONH2
CH2CH2COCH3
CH2CH2COOCH3
CH=CHCONH2
CH2CH2CN
CH2CH(CN)CONH2
CH2CH2CH2OH
CN
CH=CHCN
CH=CHCN
CH=CHCN
CH=CHCN
CN
6.78ꢀ1.18
7.43ꢀ1.1
57.1ꢀ9.2
290ꢀ46
19.2
>9.59
>27.2
1.58
>26.2
>9.39
11.0
15.8
>9.8
15.3
>24.6
>10.2
15.1
>25.2
13.2
2832
>1291
>476
544
>100
>1820
14667
43
>2103
155
>40
>3630
43
>525
13333
24909
6462
Evaluation of anti-HIV activity
261ꢀ55.1
5.16ꢀ1.57
0.75ꢀ0.18
370ꢀ59
The newly synthesized halogenated DAPA com-
pounds were first evaluated against wild-type HIV-
1 virus (NL4-3) infection of TZM-bl cells, and the data
are summarized in Table 1. In the fluoride DAPA
series 6, six analogues, 6a, 6b, 6 f, 6g, 6i, and 6l,
showed extremely high potency against HIV-1 infec-
tion with low-nanomolar EC50 values ranging from
0.75 to 7.43 nm. The para substituents R on the C-
ring in these six compounds were relatively nonpolar
(e.g., hydrogen, bromo, cyano, cyanovinyl); however,
a para R substituent with more polar groups or het-
eroatoms (see 6c, 6d, 6e, 6h, 6j, 6k, 6m, and 6n)
resulted in substantially lower antiviral activity (EC50
48–350 nm). These results indicate that 2,6-halogena-
tion on the C-ring is a beneficial approach to obtain
new DAPA derivatives with high antiviral activity, but
the identity of the para R group also affects potency.
These findings are consistent with our previous pos-
tulation that the “west wing” on the NNRTI binding
pocket might be a very hydrophobic narrow tunnel[6]
4.66ꢀ0.95
99ꢀ19
6j
6k
6l
614ꢀ118
2.81ꢀ0.59
350ꢀ52
6m
6n
8a
8b
8c
8d
8e
2a
2b
1b[e]
47.98ꢀ9.8
0.93ꢀ0.27
0.33ꢀ0.09
3.25ꢀ0.89
8.14ꢀ2.49
16.7ꢀ4.33
0.68ꢀ0.03
0.71ꢀ0.58
0.44ꢀ0.11
8.22
21.0
10.7
>20.6
8.98
Br/OMe
Cl
H/OMe
Me
1310
>1234
13206
13732
44091
Me
CH=CHCN
9.75
19.4
[a] Determined against HIV-1 NL3-4 (wild-type) virus in the TZM-bl cell line. [b] Con-
centration of compound that causes 50% inhibition of viral infection; values are the
meanꢀSD of at least three independent experiments. [c] Value that causes cytotoxici-
ty to 50% of cells; CytoTox-Glo cytotoxicity assays (Promega) were used, and values
were averaged from two independent tests. [d] Selectivity index: CC50/EC50 ratio.
[e] Reference drug used for comparison.
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groups, bromo and methoxy (Br/OMe), as well as dichloro 8d,
also exhibited high antiviral activity, with EC50 values of 3.25
and 8.14 nm, respectively, only slightly less potent than para-
cyanovinyl DAPAs 2b, 6g, and 8b. These results suggest that:
1) the favored order of halogenation (X) for antiviral activity is
dibromide > difluoride > monobromide > dichloride, and that
2) a para-cyanovinyl substituent (R) on the C-ring is preferable
over other substituents for enhancing antiviral potency. Non-
halogenated para-cyanovinyl DAPA 8e (X=H/MeO) remained
active, again indicating that other modifications of the X sub-
stituents could be acceptable, but was less potent (EC50
16.7 nm) than other para-cyanovinyl DAPAs.
Subsequently, potent compounds 6a, 6g, and 8a-c were se-
lected for further testing in parallel with 1b against wild-type
viral strain IIIB and NNRTI-resistant HIV-1 mutants, including RT-
multidrug-resistant (HIV-1RTMDR1), K101E, E138K, and A17
(Table 2). K101E and E138K are the most important mutations
associated with resistance against new-generation NNRTI drug
1b,[6] and A17 is an NNRTI-resistant strain with double muta-
tions of K103N and Y181C. More interestingly, we
shown in Table 3. Metabolic stability of active DAPAs was eval-
uated by a human liver microsome (HLM) incubation assay.
Compounds 6a, 6g, 8a, and 8c displayed similar or better
HLM stability (t1/2: 41–76 min) than 1b (t1/2: 39 min); however,
the most potent dibromo compound 8b appeared to be less
stable (t1/2: 21.87 min) than 1b in the same assay. Our experi-
mental data indicate that compounds 6g and 8a–c have logP
values of 3.30–3.55 (at pH 7.4) similar to dimethyl-DAPA 2b
and improved compared with 1b, implying decreased lipophi-
licity, even though their solubility was not notably enhanced
(0.50–8.42 mgmLꢁ1 at both pH 7.4 and 2.0). We postulated that
the presence of halogen and oxygen atoms with stronger elec-
tronegativity, i.e., fluoride (F 4.0), methoxy (O 3.5) and bromide
(Br 2.8), could contribute to the decreased lipophilicity. Next,
lipophilic efficiency indices (LE, LLE, and LELP) of these com-
pounds were predicted. Compliant with several desired thresh-
olds, such as LE>0.3, LLE>5, and LELP<10),[20] 6g and 8c
have potential for further development as drug candidates. In
contrast, compounds 6a, 8a,b, and 8d did not reach all de-
found that 8c displayed very similar potency against
Table 2. Antiviral activity of new DAPAs against mutated viral strains.
two wild-type (NL4-3 and III-B) and resistant viral
strains with RTMDR, K101E, or E138K mutations, re-
sulting in lower resistance fold change (RFC) values
(1.1–2.1) than those of 1b. The RFC value of 8c
against RT-resistant strain A17 was 18.6, similar to
that of 1b (18.4) and lower than those of other com-
pounds tested in the same assays. As listed in
Table 2, other halogenated DAPAs also exhibited
nearly single-digit nanomolar potency against drug-
resistant viral strains, except A17, thus indicating that
modification of the 2,6-substituents on the phenoxy
ring (C-ring) is acceptable and might be beneficial
against 1b-associated drug resistance.
Compd
EC50 [nm][a]
TZM-bl cells
NL4-3(WT) RTMDR[b] K101E[c]
MT-2 cells
E138K[c]
>29 (4.3)
IIIB(WT)
A17[d]
6a
6g
8a
8b
8c
1b
6.78
0.75
0.93
0.33
3.25
0.44
3.55 (0.52) 53 (7.82)
–
–
34.7 (46.3)
2.47 (2.6)
0.74 (2.2)
3.46 (1.1)
0.68 (1.5)
7.20 (9.6)
3.09 (3.3)
7.63 (23.1)
3.90 (1.2)
5.74 (13.0)
7.71 (10.3)
1.11 >500 (>450)
5.44 (5.8)
4.11 (12.4)
6.93 (2.1)
5.19 (11.8)
12.45
3.04
4.53
0.49
384 (30.8)
199.8 (65.7)
84.4 (18.6)
9.03 (18.4)
[a] Resistance fold change (RFC): each compound was tested in at least three inde-
pendent experiments. [b] HIV-1 RTMDR (obtained from the AIDS Research and Refer-
ence Reagent Program, Division of AIDS, NIAID, NIH), which contains mutations at RT
residues L74V, M41L, V106A, and T215Y, is resistant to AZT, ddI, nevirapine, and other
NNRTIs. [c] Mutated residues in NL4-3 NNRTI binding pocket confer resistance to 1b.
[d] Multi-NNRTI-resistant strain A17 from NIH with mutations at K103N and Y181C in
the viral RT.
In general, biological potency is often accompa-
nied by increased molecular lipophilicity. However,
high lipophilicity can result in undesirable ADME
properties and toxicity, which
hinder further drug develop-
ment. Therefore, molecular lipo-
Table 3. Drug-like properties and parameters of selected potent DAPAs.
philicity has recently been con-
sidered as a major factor in the
quality of a drug candidate. To
balance potency and lipophilicity
related to multiple drug-like
properties, the concepts of
ligand efficiency (LE), lipophilic
ligand efficiency (LLE),[15] and
ligand-efficiency-dependent lipo-
philicity (LELP)[16] have been ac-
cepted and applied[17–19] as use-
fully predictive tools to aid in de-
cision making. Accordingly, we
further assessed drug-like prop-
erties and predicted parameters
for these new active DAPAs in
parallel with 2b and 1b as
[b]
Compd
HLM[a]
t1/2 [min]
Aq.Sol. [mgmLꢁ1
pH 2.0
]
logP[c]
clogD[d]
Lipophilic Efficiency
tPSA[h]
pH 7.4
LE[e]
LLE[f]
LELP[g]
6a
6g
8a
8b
8c
8d
2b
1b
44.01
76.46
41.66
21.87
54.71
39.61
62.69
39.52
8.42
1.27
0.79
0.63
0.82
0.81
1.04
86.8
1.58
0.72
0.72
0.62
0.50
0.70
0.09
0.24
>5
3.56
3.81
4.13
5.15
3.29
4.39
4.52
3.62
0.45
0.43
0.46
0.45
0.39
0.38
0.43
0.45
4.61
5.31
4.87
4.33
5.20
3.70
4.63
5.66
7.95
8.84
9.04
11.50
8.49
11.49
10.46
7.87
83.43
107.22
107.22
107.22
116.45
107.22
107.22
96.36
3.48
3.30
3.55
3.31
>5
3.56
>5
[a] Human liver microsome incubation assay data from at least two experiments in parallel with propranolol as
a reference. [b] Aqueous solubility measured by HPLC in triplicate. [c] Values were measured at pH 7.4 by HPLC
method in triplicate. [d] Predicted by using ACD software (freeware in 2013). [e] Values were calculated by the
formula ꢁDG/HA(non-Hatom), in which normalizing binding energy DG=ꢁRTlnKd, presuming EC50 ꢂKd, HA=
heavy (non-hydrogen atom) count in a compound. [f] Calculated by pEC50ꢁclogD; pEC50 values (negative loga-
rithm of the molar effective concentration of compound that causes 50% inhibition of wild-type virus) were
converted from experimental data. [g] Defined as the ratio of clogD and LE. [h] Topological polar surface area
was predicted by using ChemDraw Ultra 12.0.
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phy (TLC) and preparative TLC plates used silica gel GF254 (200–
300 mesh) purchased from Qingdao Haiyang Chemical Company.
Medium-pressure column chromatography was performed using
a CombiFlash companion purification system. All reagents and sol-
vents were obtained from Beijing Chemical Works or Sigma–Al-
drich, Inc. NADPH, MgCl2, KH2PO4, K2HPO4, and reference com-
pound propranolol were purchased from Sigma–Aldrich. HPLC-
grade CH3CN for LC–MS analysis was purchased from VWR. Pooled
human liver microsomes (lot no. 28831) were purchased from BD
Biosciences (Woburn, MA, USA).
sired property indices, having either LLE values less than 5 or
LELP values greater than 10, as listed in Table 3. Moreover, mo-
lecular topological polar surface area (tPSA) values were calcu-
lated by using ChemDraw Ultra 12.0 as a prediction of oral bio-
availability. All compounds in Table 3 had calculated tPSA
values within the criterion of <140 ꢁ.[2,21] To balance favorable
activity against drug-resistant viral strains and reasonable
drug-like properties, compound 8c is considered as the most
promising drug candidate among the current DAPA com-
pounds. Its structure, with two different X groups (Br and
OMe), might be critical in influencing antiviral potency against
these drug-resistant viral strains, especially for E138K and
K101E, two major mutations associated with next-generation
NNRTI drugs 1a and 1b.
General coupling reaction procedure for preparation of 1,6-
diaryl-3-nitropyridines (4, 5a,b, 7a): A mixture of 6-chloro-N2-(4-
cyanophenyl)amino-3-nitropyridine (3, 1 equiv) and 2,4,6-trisubsti-
tuted phenol (1.2 equiv) in DMF (1–2 mL) in the presence of
Cs2CO3 or K2CO3 (3.6 equiv) was irradiated under microwave condi-
tions with stirring at 908C for 15 min or at 1008C for 10 min. The
mixture was poured into ice-water, adjusted to pH 2–3 with aq HCl
(5%), extracted with CH2Cl2 three times (30–50 mL), and dried over
anhydrous Na2SO4. After removal of organic solvent under reduced
pressure, crude product was purified by PTLC or flash column chro-
matography (gradual elution) to produce the corresponding prod-
uct.
Conclusions
Synthetic modifications of X and R substituents on the phen-
oxy ring (C-ring) of DAPA lead 2 led to a new series of halo-
genated DAPA analogues (6 and 8 series). Among them, com-
pound 8c was identified as a potential drug candidate with fa-
vorable physicochemical properties and good balance be-
tween potency and lipophilicity. Compound 8c exhibited high
anti-HIV potency against wild-type and resistant viral strains
with E138 or K101E mutation (EC50 4–7 nm), lower resistance
fold change values (RFC: 1.1–2.1) than those of DAPY drug 1b
(RFC: 11.8–13) in the same assays. It also exhibited desirable
lipophilicity (low clogD 3.29 and measured logP 3.31), moder-
ate metabolic stability (t1/2: 54.7 min) in HLM assay, and desira-
ble ligand lipophilic efficiency indices (LE>0.3, LLE>5, LELP<
10, and tPSA<140). The current modifications also revealed
new SAR and SPR findings. 1) Introduction of halogens (X) at
the 2- and/or 6-positions on the C-ring is an effective approach
to improve molecular drug-like properties without loss of po-
tency. 2) The presence of heteroatoms with stronger electrone-
gativity at the 2,6-positions on the C-ring might decrease mo-
lecular lipophilicity and improve ADME properties, despite
having less impact on aqueous solubility. 3) Dibromo DAPAs
appeared to have lower metabolic stability than corresponding
difluoro and dichloro compounds (see 8b, 6g, and 8d).
N2-(4’-Cyanophenyl))amino-6-(2’’,6’’-difluoro-4’’-formyl)phenoxy-
3-nitropyridine (4a): Starting with 6-chloro-N2-(4-cyanophenyl)ami-
no-3-nitropyridine (3) (274 mg, 1.0 mmol) and 2,6-difluoro-4-for-
mylphenol (190 mg, 1.2 mmol) in DMF (1–2 mL) in the presence of
Cs2CO3 (3.6 equiv) irradiated at 908C for 15 min to afford 4a as
1
a yellow solid (277 mg, 65%), mp: 217–2198C; H NMR (400 MHz,
[D6]DMSO): d=10.34 (1H, s, NH), 10.05 (1H, s, CHO), 8.73 (1H, d,
J=8.8 Hz, PyH-4), 7.92 (2H, d, J=8.8 Hz, ArH-3’,5’), 7.47 (2H, d, JF =
8.8 Hz, ArH-3’’,5’’), 7.35 (2H, d, J=8.8 Hz, ArH-2’,6’), 6.95 ppm (1H,
d, J=8.8 Hz, PyH-5); MS m/z (%): 394.9 [Mꢁ1, 100].
N2-(4’-Cyanophenyl))amino-6-(2’’,6’’-dibromo-4’’-formyl)phenoxy-
3-nitropyridine (4b): Starting with 3 (567 mg, 2.0 mmol), 2,6-dibro-
mo-4-formylphenol (693 mg, 2.48 mmol), and K2CO3 (3.6 equiv) at
1008C for 10 min to afford 4b as a yellow solid (518 mg, 50%),
1
mp: 227–2298C; H NMR (400 MHz, CDCl3): d=10.58 (1H, brs, NH),
10.03 (1H, s, CHO), 8.71 (1H, d, J=9.2 Hz, PyH-4), 8.18 (2H, s, ArH-
3’’,5’’), 7.25 (4H, s, ArH-2’,3’,5’,6’), 6.76 ppm (1H, d, J=9.2 Hz, PyH-
5); MS m/z (%): 517.0 [M+1, 20], 519.0 [M+3, 100], 521.1 [M+5,
40].
6-(2’’-Bromo-4’’-formyl-6’’-methoxy)phenoxy-N2-(4’-cyanopheny-
l))amino-3-nitropyridine (4c): Starting with 3 (274 mg, 1.0 mmol),
2-bromo-4-formyl-6-methoxyphenol (277 mg, 1.2 mmol), and
Cs2CO3 (3.6 equiv) at 908C for 15 min to afford 4c as a yellow solid
Experimental Section
1
(281 mg, 59%), mp: 222–2248C; H NMR (400 MHz, [D6]DMSO): d=
Chemistry
10.34 (1H, s, NH), 10.06 (1H, s, CHO), 8.69 (1H, d, J=8.8 Hz, PyH-4),
7.96 (1H, d, J=1.6 Hz, ArH-3’’), 7.73 (1H, d, J=1.6 Hz, ArH-5’’), 7.43
(2H, d, J=8.4 Hz, ArH-3’,5’), 7.35 (2H, d, J=8.8 Hz, ArH-2’,6’), 6.90
(1H, d, J=8.8 Hz, PyH-5), 3.82 ppm (3H, s, OMe); MS m/z (%): 467
[Mꢁ1, 100], 469 [M+1, 90].
N2-(4’-Cyanophenyl))amino-6-(2’’,6’’-dichloro-4’’-formyl)phenoxy-
3-nitropyridine (4d): Starting with 3 (274 mg, 1.0 mmol), 2,6-di-
chloro-4-formylphenol (225 mg, 1.2 mmol), and K2CO3 (3.6 equiv) at
1008C for 10 min to afford 4d as a yellow solid (233 mg, 55%),
mp: 232–2348C; 1H NMR (400 MHz, [D6]DMSO): d=10.34 (1H, s,
NH), 10.09 (1H, s, CHO), 8.75 (1H, d, J=8.8 Hz, PyH-4), 8.22 (2H, s,
ArH-3’’,5’’), 7.45 (2H, d, J=8.8 Hz, ArH-3’,5’), 7.32 (2H, d, J=8.8 Hz,
ArH-2’,6’), 6.98 ppm (1H, d, J=8.8 Hz, PyH-5); MS m/z (%): 427
[Mꢁ1, 80], 391.1 (100).
Melting points were measured with an RY-1 melting apparatus
without correction. H NMR spectra were measured on a JNM-ECA-
1
400 (400 MHz) spectrometer using tetramethylsilane (TMS) as inter-
nal standard. The solvent used was [D6]DMSO unless otherwise in-
dicated. Mass spectra were measured on an ABI PerkinElmer Sciex
API-150 mass spectrometer with electrospray ionization (ESI), and
the relative intensity of each ion peak is presented as a percentage.
The purities of target compounds were ꢃ95%, measured by HPLC,
performed on an Agilent 1200 HPLC system with UV detector and
Grace Alltima HP C18 column (100ꢂ2.1 mm, 3 mm), eluting with
a mixture of solvents A and B (condition 1: CH3CN/H2O 95:5, flow
rate 1.0 mLminꢁ1
; condition 2: MeOH/H2O 80:20, flow rate
0.8 mLminꢁ1, UV l 254 nm). Microwave reactions were performed
on a microwave reactor from Biotage, Inc. Thin-layer chromatogra-
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2014, 9, 1546 – 1555 1550

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N2-(4’-Cyanophenyl)amino-6-(4’’-formyl-2’’-methoxy)phenoxy-3-
nitropyridine (4e): Starting with 3 (274 mg, 1.0 mmol), 4-formyl-2-
methoxyphenol (182 mg,1.2 mmol), and Cs2CO3 (3.6 equiv) at 908C
for 15 min to afford 4e as a yellow solid (227 mg, 58), mp: 213–
8.8 Hz, ArH-3’,5’), 7.44 (2H, d, JH-F =8.8 Hz, ArH-3’’,5’’), 7.40 (2H, d,
J=8.8 Hz, ArH-2’,6’), 6.93 (1H, d, J=8.8 Hz, PyH-5), 5.17 ppm (2H,
s, CH2), 2.19 (3H, s, CH3); MS m/z (%): 439.6 [Mꢁ1, 100].
N2-(4’-Cyanophenyl))amino-6-[2’’,6’’-difluoro-4’’-(hydroxyimino)-
1
2158C; H NMR (400 MHz, [D6]DMSO): d=10.34 (1H, s, NH), 10.09
methyl]phenoxy-3-nitropyridine (5e): To
a solution of 4a
(1H, s, CHO), 8.65 (1H, d, J=8.8 Hz, PyH-4), 7.73 (1H, s, ArH-3’’),
7.67 (1H, d, J=8.0 Hz, ArH-5’’), 7.48 (1H, d, J=8.0 Hz, ArH-6’’), 7.39
(4H, s, ArH-2’,3’,5’,6’), 6.80 (1H, d, J=8.8 Hz, PyH-5), 3.78 ppm (3H,
s, OMe); MS m/z (%): 389.3 [Mꢁ1, 100].
N2-(4’-Cyanophenyl))amino-6-(2’’,6’’-difluoro)phenoxy-3-nitropyr-
idine (5a): Starting with 3 (2.74 g, 10 mmol), 2,6-difluorophenol
(1.56 g,12 mmol), and K2CO3 (3.6 equiv) at 1008C for 10 min to
afford pure 5a as a yellow solid (1.62 g, 44%) by flash column
chromatography (gradual elution: CH2Cl2/MeOH 0–3%), mp: 234–
2368C; 1H NMR (400 MHz, [D6]DMSO): d=10.64 (1H, s, NH), 8.66
(1H, d, J=8.8 Hz, PyH-4), 7.26–7.31 (5H, m, ArH), 7.11 (2H, m, ArH),
6.73 ppm (1H, d, J=8.8 Hz, PyH-5); MS m/z (%): 369.1 [M+1, 100].
(396 mg, 1.0 mmol) in THF (15 mL) was added hydroxylamine hy-
drochloride (175 mg, 25 mmol) in an ice bath. The mixture was
stirred for 2 h at room temperature and then poured into ice-
water, adjusted to pH 7–8 with 5% aq NaOH. The resulting solid
was collected and washed with H2O to neutral. The dried solid was
then purified by silica flash column (gradual elution: MeOH/CH2Cl2
0–1%) to afford 5e as a yellow solid (312 mg, 76%), mp: 240–
1
2428C; H NMR (400 MHz, [D6]DMSO): d=11.77 (1H, s, OH), 10.37
(1H, brs, NH), 8.72 (1H, d, J=8.8 Hz, PyH-4), 8.27 (1H, s, CH=), 7.57
(2H, d, JH-F =8.8 Hz, ArH-3’’,5’’), 7.44 (4H, m, ArH-2’,6’ and ArH-3’,5’),
6.94 ppm (1H, d, J=8.8 Hz, PyH-5); MS m/z (%): 410.2 [Mꢁ1, 100].
6-(4’’-Cyano-2’’,6’’-difluoro)phenoxy-N2-(4’-cyanophenyl))amino-
3-nitropyridine (5 f): 5e (150 mg, 0.36 mmol) in acetic anhydride
was held at reflux overnight and monitored with TLC (EtOAc/petro-
leum ether) until reaction was completed. After removal of most
acetic anhydride under reduced pressure, the residue was added
to H2O and stirred for ~1 h. The yellow solid produced was filtered,
washed with H2O to neutral, and dried. The crude product was pu-
rified by a silica flash column (gradual elution: CH2Cl2/petroleum
6-(4’’-Bromo-2’’,6’’-difluoro)phenoxy-N2-(4’-cyanophenyl)amino-
3-nitropyridine (5b): Starting with 3 (274 mg, 1.0 mmol), 4-bromo-
2,6-difluorophenol (251 mg, 1.2 mmol), and K2CO3 (3.6 equiv) at
1008C for 10 min to afford 5b as a yellow solid (290 mg, 70%),
mp: 213–2158C; 1H NMR (400 MHz, [D6]DMSO): d=10.43 (1H, s,
NH), 8.71 (1H, d, J=8.8 Hz, PyH-4), 7.80 (2H, d, J=8.8 Hz, ArH-
3’,5’), 7.55 (2H, d, JF =12 Hz, ArH-3’’,5’’), 7.39 (2H, d, J=8.8 Hz, ArH-
2’,6’), 6.92 ppm (1H, d, J=8.8 Hz, PyH-5); MS m/z (%): 445.2 [Mꢁ1,
98], 447.0 [M+1, 100].
1
ether) to afford solid 5 f (130 mg, 90%), mp: 282–2848C; H NMR
(400 MHz, [D6]DMSO): d=10.34 (1H, s, NH), 8.73 (1H, d, J=8.8 Hz,
ArH-4), 8.12 (2H, d, JF-H =8.0 Hz, ArH-3’’,5’’), 7.56 (2H, d, J=8.8 Hz,
ArH-3’,5’), 7.36 (2H, d, J=8.8 Hz, ArH-2’,6’), 6.95 ppm (1H, d, J=
8.8 Hz, ArH-5); MS m/z (%): 394 [M+1, 100].
N2-(4’-Cyanophenyl))amino-6-(2’’,4’’,6’’-tribromo)phenoxy-3-ni-
tropyridine (7a): Starting with 3 (2.75 mg, 10 mmol), 2,4,6-tribro-
mophenol (3.97 mg, 12 mmol), and K2CO3 (3.6 equiv) at 1008C for
10 min to afford 7a as a yellow solid (3.60 g, 67%), mp: 192–
1948C; 1H NMR (400 MHz, [D6]DMSO): d=10.57 (1H, s, NH), 8.68
(1H, d, J=8.8 Hz, PyH-4), 7.83 (2H, s, ArH-3’’,5’’), 7.38 (2H, d, J=
9.2 Hz, ArH-3’,5’), 7.26 (2H, d, J=9.2 Hz, ArH-2’,6’), 6.72 ppm (1H, d,
J=8.8 Hz, PyH-5); MS m/z (%): 567.2 [M+1, 20], 569.0 [M+3, 100],
571.1 [M+5, 90].
(E)-N2-(4’-Cyanophenyl))amino-6-(2’’,6’’-difluoro-4’’-(2-carbamoyl-
2-cyano)vinyl)phenoxy-3-nitropyridine (5h): A mixture of 4a
(396 mg, 1.0 mmol) in DMF (1.5 mL), ZnCl2 (27 mg, 0.2 mmol), and
2-cyanoacetamide (excess) was irradiated under microwave condi-
tions with stirring at 1008C for 10 min. The mixture was poured
into ice-water, adjusted to pH 2–3 with aq HCl (5%), extracted with
CH2Cl2 three times (30–50 mL), and dried over anhydrous Na2SO4.
After removal of solvent, crude product was purified by a silica
column (gradual elution: MeOH/CH2Cl2 0–5%) to afford 5h as
N2-(4’-Cyanophenyl))amino-6-(2’’,6’’-difluoro-4’’-hydroxymethyl)-
phenoxy-3-nitropyridine (5c): To
a solution of 4a (396 mg,
1.0 mmol) in THF (15 mL) and MeOH (5 mL) was added NaBH4
(191 mg, 5 mmol) slowly in an ice bath. The mixture was stirred for
another 30 min and then poured into ice-water, adjusted to pH 4–
5 with 5% aq HCl, extracted with EtOAc three times (40–60 mL),
and dried over anhydrous Na2SO4. After removal of organic solvent
in vacuo, crude product was purified by silica flash column (elu-
tion: CH2Cl2/MeOH 100:1) to afford pure 5c as a yellow solid
1
a yellow solid (415 mg, 90%), mp: 265–2678C; H NMR (400 MHz,
[D6]DMSO): d=10.36 (1H, brs, NH), 8.73 (1H, d, J=8.8 Hz, PyH-4),
8.29 (1H, s, CH=), 8.00 and 7.96 (each 1H, brs, NH2), 7.89 (2H, d, JH-
F =8.8 Hz, ArH-3’’,5’’), 7.50 (2H, d, J=8.8 Hz, ArH-3’,5’), 7.39 (2H, d,
J=8.8 Hz, ArH-2’,6’), 6.95 ppm (1H, d, J=8.8 Hz, PyH-5); MS m/z
(%): 461.4 [Mꢁ1, 25], 398.4 (100).
1
(276 mg, 70%), mp: 237–2398C; H NMR (400 MHz, [D6]DMSO): d=
(E)-N2-(4’-Cyanophenyl))amino-6-(2’’,6’’-difluoro-4’’-(3-oxobut-1-
en-1-yl)phenox-3-nitropyridine (5i): To a solution of 4a (396 mg,
1 mmol) in acetone (20 mL) was added aq NaOH (10%, 2 mL)
slowly in ice-water bath and stirred for 10 min. The mixture was
then stirred at room temperature for 1 h, poured into ice-water,
and pH was adjusted to 4–5 with 5% aq HCl. The solid was filtered,
washed with H2O, and purified on a silica gel column (gradient elu-
tion: EtOAc/petroleum ether 0–50%) to produce 5i as a yellow
10.38 (1H, brs, NH), 8.71 (1H, d, J=8.8 Hz, PyH-4), 7.52 (2H, d, J=
8.4 Hz, ArH-3’,5’), 7.40 (2H, d, J=8.4 Hz, ArH-2’,6’), 7.30 (2H, d, JH-
F =9.2 Hz, ArH-3’’,5’’), 6.92 (1H, d, J=8.8 Hz, PyH-5), 5.67 (1H, t, J=
5.6 Hz, OH), 4.60 ppm (2H, d, J=5.6 Hz, CH2); MS m/z (%): 397.0
[Mꢁ1, 100].
N2-(4’-Cyanophenyl))amino-6-(2’’,6’’-difluoro-4’’-acetoxymethyl)-
phenoxy-3-nitropyridine (5d): To
a solution of 5c (598 mg,
1
solid (226 mg, 52%), mp: 275–2778C; H NMR (400 MHz, CDCl3) d=
1 mmol) in THF (15 mL) was added dropwise acetyl chloride (2 mL)
and triethylamine (2 mL) simultaneously in an ice bath. Then the
mixture was stirred at room temperature for another 30 min,
poured into ice-water, adjusted to pH 4–5 with aq HCl, extracted
with EtOAc three times (40–60 mL), and dried over anhydrous
Na2SO4. After removal of solvent, crude product was purified by
a silica column eluted with CH2Cl2 to afford 5d as a yellow solid
10.33 (1H, brs, NH), 8.71 (1H, d, J=8.8 Hz, PyH-4), 7.76 (2H, JF-H
=
8.8 Hz, ArH-3’’,5’’), 7.68 (1H, d, J=16.4 Hz, ArCH=), 7.46 (2H, d, J=
8.8 Hz, ArH-3’,5’), 7.36 (2H, d, J=8.8 Hz, ArH-2’,6’), 7.03 (1H, d, J=
16.4 Hz, =CHCO), 6.92 (1H, d, J=8.8 Hz, PyH-5), 2.38 ppm (3H, s,
COCH3); MS m/z (%): 435.2 [Mꢁ1, 100].
(E)-N2-(4’-Cyanophenyl))amino-6-(2’’,6’’-difluoro-4’’-(2-carbome-
1
(412 mg, 94%), mp: 159–1618C; H NMR (400 MHz, [D6]DMSO): d=
thoxy)vinyl)phenoxy-3-nitropyridine (5j):
A
mixture of 4a
10.39 (1H, brs, NH), 8.72 (1H, d, J=8.8 Hz, PyH-4), 7.52 (2H, d, J=
(396 mg, 1 mmol) and Ph3PCHCOOCH3 (334 mg, 1 mmol) in CHCl3
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(20 mL) was heated at reflux under nitrogen overnight. After re-
moval of solvent, the residue was purified on a silica gel column
(elution: CH2Cl2) to give 5j as a yellow solid (380 mg, 83%), mp:
(E)-N2-(4’-Cyanophenyl))amino-6-(4’’-cyanovinyl-2’’,6’’-dichloro)-
phenoxy-3-nitropyridine (7d): Starting with 4d (389 mg) to
obtain 7d as yellow solid (339 mg, 88%), mp: 210–2128C; H NMR
1
1
263–2658C; H NMR (400 MHz, [D6]DMSO): d=10.34 (1H, brs, NH),
(400 MHz, [D6]DMSO): d=10.30 (1H, brs, NH), 8.72 (1H, d, J=
8.8 Hz, PyH-4), 8.02 (2H, s, ArH-3’’,5’’), 7.71 (1H, d, J=16.4 Hz,
ArCH=), 7.45 (2H, d, J=8.4 Hz, ArH-3’,5’), 7.33 (2H, d, J=8.4 Hz,
ArH-2’,6’), 6.94 (1H, d, J=8.8 Hz, PyH-5), 6.76 ppm (1H, d, J=
16.4 Hz, =CHCN); MS m/z (%): 450.3 [Mꢁ1, 100].
8.71 (1H, d, J=9.2 Hz, PyH-4), 7.84 (2H, JF-H =8.8 Hz, ArH-3’’,5’’),
7.73 (1H, d, J=16.0 Hz, ArCH=), 7.46 (2H, d, J=8.8 Hz, ArH-3’,5’),
7.38 (2H, d, J=8.8 Hz, ArH-2’,6’), 6.93 (1H, d, J=8.8 Hz, PyH-5),
6.90 (1H, d, J=16.4 Hz, =CHCO), 3.76 ppm (3H, s, OCH3); MS m/z
(%): 451.2 [Mꢁ1, 100].
(E)-N2-(4’-Cyanophenyl)amino-6-(4’’-cyanovinyl-2’’-methoxy)phe-
noxy-3-nitropyridine (7e): Starting with 4e (389 mg) to obtain 7e
(E)-N2-(4’-Cyanophenyl))amino-6-(2’’,6’’-difluoro-4’’-(2-carba-
1
moyl)vinyl)phenoxy-3-nitropyridine (5k):
A
solution of 5j
as yellow solid (343 mg, 83%), mp: 228–2308C; H NMR (400 MHz,
(452 mg, 1 mmol) in THF (50 mL) was added dropwise to aq NaOH
(10%, 1 mL) at room temperature and stirred for 36 h. The mixture
was poured into H2O and pH was adjusted to 2–3 with aq HCl. The
precipitated solid was collected and washed with H2O to neutral.
After drying, the solid was dissolved in CH2Cl2, N,N’-carbonyl diimi-
dazole (CDI, 194 mg, 1.2 mmol) was added in several portions, and
stirring continued for 2 h. The mixture was then added into ammo-
nia (30 mL, excess) and stirred for 2 h. The mixture was added to
H2O and extracted with CH2Cl2 three times. The combined organic
phase was washed with H2O, aq NaHCO3, and brine successively
and dried over Na2SO4. The solvent was removed to obtain crude
5k, used without purification in the next step.
[D6]DMSO): d=10.33 (1H, brs, NH), 8.63 (1H, d, J=9.2 Hz, PyH-4),
7.75 (1H, d, J=16.4 Hz, ArCH=), 7.59 (1H, d, J=1.6 Hz, ArH-3’’),
7.41 (4H, m, ArH), 7.33 (1H, d, J=8.4 and 1.6 Hz, ArH-5’’), 7.29 (1H,
d, J=8.4 Hz, ArH-6’’), 6.77 (1H, d, J=8.8 Hz, PyH-5), 6.64 (1H, d, J=
16.4 Hz, =CHCN), 3.73 ppm (3H, s, OCH3); MS m/z (%): 412.3 [Mꢁ1,
100].
General reduction procedures of nitro group to amine: Method
1 (reduction with sodium hydrosulfite, Na2S2O4): To a solution of
a 2,6-diaryl-3-nitropyridine (1 equiv, 5 or 7) in THF and H2O (v/v
1:1) was added NH3·H2O solution (25%, 0.5 mL) and sodium hydro-
sulfite (90% Na2S2O4, 10 equiv), successively, at room temperature
with stirring for 2 h monitored by TLC (CH2Cl2/MeOH 100:1) until
reaction was completed. The mixture was then poured into ice-
water and extracted with EtOAc three times (30–50 mL). After re-
moval of solvent in vacuo, the residue was purified on a silica gel
flash column (gradual elution: MeOH/CH2Cl2 0–1%) to obtain pure
corresponding diarylpyridinamines 6 (a–c, f–h, k) and 8, respective-
ly. Method 2 (catalytic hydrogenation, H2 on Pd/C): A solution of
a diarylnitrobenzene (1 equiv, 5) in a solvent mixture of anhydrous
EtOH and THF (15:15 mL) with hydrogen gas in the presence of
excess Pd/C (10%) under 15 psi was shaken until hydrogen was no
longer absorbed (~2 h) and also monitored by TLC (elution:
CH2Cl2/MeOH 100:1) until the reaction was completed. The catalyst
was filtered out from the solution and washed with EtOH several
times. After removal of solvent, the residue was purified by flash
column chromatography (gradual elution: MeOH/CH2Cl2, 0–1%) to
obtain required product 6 (d,e, i,j, or l,m).
General procedure for preparation of cyanovinyl compounds: To
a solution of diethylcyanomethylphosphonate [(EtO)2P(O)CH2CN,
266 mg, 1.5 mmol] in THF (15 mL) was added tBuOK (1.5 mmol) at
08C (ice-water bath) with stirring for 30 min and then for another
30 min at room temperature. The solution of aldehyde (4, 1 mmol)
in THF (15 mL) was quickly added dropwise into the above mixture
at room temperature and stirred until the reaction was completed
(1–48 h), as monitored by TLC. The mixture was poured into H2O,
extracted with EtOAc three times (40–60 mL), and dried over anhy-
drous Na2SO4. After removal of solvent under reduced pressure,
crude product was purified by a silica gel column using medium-
pressure system of CombiFlash companion (gradual elution:
CH2Cl2/petroleum ether) to give the corresponding cyanovinyl
product (5g, 7b–e).
(E)-N2-(4’-Cyanophenyl))amino-6-(4’’-cyanovinyl-2’’,6’’-difluoro)-
phenoxy-3-nitropyridine (5g): Starting with 4a (396 mg, 1 mmol)
to provide 5g as yellow solid (396 mg, 81%), mp: 262–2648C;
1H NMR (400 MHz, CDCl3): d=10.61 (1H, brs, NH), 8.68 (1H, d, J=
9.2 Hz, PyH-4), 7.40 (1H, d, J=16.4 Hz, ArCH=), 7.34–7.26 (4H, m,
ArH), 7.20 and 7.18 (each 1H, s, ArH), 6.95 (1H, d, J=8.8 Hz, PyH-
5), 5.98 ppm (1H, d, J=16.4 Hz, =CHCN); MS m/z (%): 418.2 [Mꢁ1,
100].
N2-(4’-Cyanophenyl)amino-6-(2’’,6’’-difluoro)phenoxypyridin-3-
amine (6a): Method 1: Starting with 5a (57 mg, 0.154 mmol) re-
duced by Na2S2O4 to afford 6a as a yellow solid (15 mg, 29%), mp:
161–1638C; 1H NMR (400 MHz, [D6]DMSO): d=8.35 (1H, s, NH),
7.38 (1H, m, ArH-4’), 7.33 (4H, m, ArH-2’,3’,5’,6’), 7.30 (2H, d, JF,H
=
12 Hz, ArH-3’’,5’’), 7.16 (1H, d, J=8.0 Hz, PyH-4), 6.56 (1H, d, J=
8.0 Hz, PyH-5), 4.92 ppm (2H, s, NH2); MS m/z (%): 339.2 [M+1,
100]; HPLC purity 95.18%.
(E)-N2-(4’-Cyanophenyl))amino-6-(4’’-cyanovinyl-2’’,6’’-dibromo)-
phenoxy-3-nitropyridine (7b): Starting with 4b (518 mg) to give
pure 7b as light-yellow solid (363 mg, 67%), mp: 224–2268C;
1H NMR (400 MHz, [D6]DMSO): d=10.31 (1H, s, NH), 8.72 (1H, d,
J=8.8 Hz, PyH-4), 8.17 (2H, s, ArH-3’’,5’’), 7.70 (1H, d, J=16.0 Hz,
CH=), 7.44 (2H, d, J=8.4 Hz, ArH-3’,5’), 7.33 (2H, d, J=8.4 Hz, ArH-
2’,6’), 6.94 (1H, d, J=8.8 Hz, PyH-5), 6.76 ppm (1H, d, J=16.0 Hz,
CH=); MS m/z (%): 538 [Mꢁ1, 30], 540 [M+1, 100], 542 [M+3, 20].
(E)-6-(2’’-Bromo-4’’-cyanovinyl-6’’-methoxy)phenoxy-N2-(4’-cyano-
phenyl))amino-3-nitropyridine (7c): Starting with 4c (469 mg) to
give 7c as yellow solid (418 mg, 85%), mp: 243–2458C; 1H NMR
(400 MHz, [D6]DMSO): d=10.32 (1H, br, NH), 8.67 (1H, d, J=8.8 Hz,
PyH-4), 7.72 (1H, d, J=16.4 Hz, ArCH=), 7.65 (2H, m, ArH-3’’,5’’),
7.44 and 7.35 (each 2H, d, J=8.8 Hz, ArH-2’,3’,5’,6’), 6.87 (1H, d, J=
8.8 Hz, PyH-5), 6.75 (1H, d, J=16.4 Hz, =CHCN), 3.76 ppm (3H, s,
OCH3); MS m/z (%): 490.2 [Mꢁ1, 100].
6-(4’’-Bromo-2’’,6’’-difluoro)phenoxy-N2-(4’-cyanophenyl)amino)-
pyridin-3-amine (6b): Method 1: Starting with 5b (447 mg) re-
duced byNa2S2O4 to produce 6b as a light-gray solid (218 mg,
1
52%), mp: 208–2108C; H NMR (400 MHz, [D6]DMSO): d=8.46 (1H,
s, NH), 7.75 (2H, d, JF-H =8.4 Hz, ArH-3’’,5’’), 7.39 (2H, d, J=8.8 Hz,
ArH-3’,5’), 7.31 (2H, d, J=8.8 Hz, ArH-2’,6’), 7.16 (1H, d, J=8.4 Hz,
PyH-4), 6.58 (1H, d, J=8.4 Hz, PyH-5), 4.96 ppm (2H, br, NH2); MS
m/z (%): 417 [M+1, 60], 415.3 [Mꢁ1, 70], 397 (90), 395.1 (100);
HPLC purity 95.41%.
N2-(4’-Cyanophenyl)amino-6-(2’’,6’’-difluoro-4’’-hydroxymethyl)-
phenoxypyridine-3-amine (6c): Starting with 5c (398 mg) reduced
by Na2S2O4 to give 6c as a gray solid (179 mg, 49%), mp: 210–
2128C; 1H NMR (400 MHz, [D6]DMSO): d=8.38 (1H, br, NH), 7.36
(4H, m, ArH on A-ring), 7.24 (2H, d, J=8.8 Hz, ArH-3’’,5’’), 7.15 (1H,
d, J=8.4 Hz, PyH-4), 6.55 (1H, d, J=8.4 Hz, PyH-5), 5.56 (1H, t, J=
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5.6 Hz, OH), 4.91 (2H, s, NH2), 4.57 ppm (2H, d, J=5.6 Hz, CH2); MS
m/z (%): 367.2 [Mꢁ1, 60], 320.1 (100); HPLC purity 95.22%.
(2H, JF-H =8.8 Hz, ArH-3’’,5’’), 7.14 (1H, d, J=8.0 Hz, PyH-4), 6.54
(1H, d, J=8.0 Hz, PyH-5), 4.90 (2H, brs, NH2), 3.60 (3H, s, OCH3),
2.93 and 2.72 ppm (each 2H, t, J=7.6 Hz, CH2); MS m/z (%): 423.3
[Mꢁ1, 40], 376.3 (100); HPLC purity 97.38%.
(E)-N2-(4’-Cyanophenyl)amino-6-(2’’,6’’-difluoro-4’’-(2-carbamoyl)-
vinyl)phenoxypyridin-3-amine (6k): Method 1: Starting with
crude 5k (452 mg, 1 mmol) reduced by Na2S2O4 to furnish 6k as
an off-white solid (123 mg, 32%), mp: 239–2418C; 1H NMR
(400 MHz, [D6]DMSO): d=8.43 (1H, br, NH), 7.56 (3H, m, ArH-3’’,5’’
and NH), 7.48 (1H, d, J=16.0 Hz, ArCH=), 7.32 (4H, m, ArH-
2’,3’,5’,6’), 7.22 (1H, br, NH), 7.16 (1H, d, J=8.0 Hz, PyH-4), 6.71
(1H, d, J=16.0 Hz, =CHCN), 6.58 (1H, d, J=8.0 Hz, PyH-5),
4.95 ppm (2H, br, NH2); MS m/z (%): 406.2 [Mꢁ1, 35], 386.2 (100);
HPLC purity 99.84%.
N2-(4’-Cyanophenyl)amino-6-(2’’,6’’-difluoro-4’’-acetoxymethyl)-
phenoxypyridin-3-amine (6d): Method 2. Starting with 5d
(440 mg) reduced by catalytic hydrogenation to furnish 6d as
a gray solid (209 mg, 51%), mp: 173–1758C; 1H NMR (400 MHz,
[D6]DMSO): d=8.40 (1H, s, NH), 7.36 (6H, m, ArH), 7.15 (1H, d, J=
8.0 Hz, PyH-4), 6.57 (1H, d, J=8.0 Hz, PyH-5), 5.14 (2H, s, CH2), 4.93
(2H, brs, NH2), 2.16 ppm (3H, s, CH3); MS m/z (%): 409.2 [Mꢁ1, 70],
329.3 (100); HPLC purity 95.11%.
N2-(4’-Cyanophenyl)amino-6-(2’’,6’’-difluoro-4’’-(hydroxyimino)-
methyl)phenoxypyridin-3-amine (6e): Starting with 5e (381 mg)
reduced by catalytic hydrogenation to afford 6e as a gray solid
1
(313 mg, 53%), mp: 214–2168C; H NMR (400 MHz, [D6]DMSO): d=
6-(4’’-(2-Cyanoethyl)-2’’,6’’-difluoro)phenoxy-N2-(4’-cyanophenyl)-
aminopyridin-3-amine (6l): Method 2. Starting with 5g
(396 mg,0.94 mmol) reduced by catalytic hydrogenation to afford
6l as an off-white solid (198 mg, 52%), mp: 202–2048C; 1H NMR
(400 MHz, [D6]DMSO): d=8.39 (1H, br, NH), 7.38 (2H, d, J=8.8 Hz,
ArH-3’,5’), 7.36 (2H, d, J=8.8 Hz, ArH-2’,6’), 7.30 (2H, JF-H =8.4 Hz,
ArH-3’’,5’’), 7.15 (1H, d, J=8.4 Hz, PyH-4), 6.55 (1H, d, J=8.0 Hz,
PyH-5), 4.91 (2H, brs, NH2), 2.96 and 2.89 ppm (each 2H, m, CH2);
MS m/z (%): 390.1 [Mꢁ1, 65], 370.0 (100); HPLC purity 95.25%.
N2-(4’-Cyanophenyl)amino-6-(4’’-(2-cyano)propylcarbamoyl-
2’’,6’’-difluoro)phenoxypyridin-3-amine (6m): Method 2: Starting
with 5h (462 mg) reduced by catalytic hydrogenation to produce
6m as a gray solid (233 mg, 60%), mp: 222–2248C; 1H NMR
(400 MHz, [D6]DMSO): d=8.40 (1H, br, NH), 7.83 and 7.59 (each
1H, br, CONH2), 7.42 (2H, d, J=8.8 Hz, ArH-3’,5’), 7.36 (2H, d, J=
8.8 Hz, ArH-2’,6’), 7.28 (2H, d, JF-H =9.2 Hz, ArH-3’’,5’’), 7.15 (1H, d,
J=8.0 Hz, PyH-4), 6.56 (1H, d, J=8.0 Hz, PyH-5), 4.95 (2H, brs,
NH2), 4.08 (1H, t, J=6.4 Hz, CH), 3.24 ppm (2H, m, CH2); MS m/z
(%): 433.5 [Mꢁ1, 10], 343.1 (100); HPLC purity 99.84%.
11.63 (1H, s, OH), 8.42 (1H, br, NH), 8.25 (1H, s, CH=N), 7.51 (2H, d,
J
H-F =9.2 Hz, ArH-3’’,5’’), 7.34 (4H, m, ArH-2’,6’,3’,5’), 7.16 (1H, d, J=
8.4 Hz, PyH-4), 6.58 (1H, d, J=8.4 Hz, PyH-5); 4.94 ppm (2H, s,
NH2); MS m/z (%): 380.1 [Mꢁ1, 20], 342.2 (100); HPLC purity
96.17%.
N2-(4’-Cyanophenyl)amino-6-(2’’,6’’-difluoro-4’’-cyano)phenoxy-
pyridin-3-amine (6 f): Method 1: Starting with 5 f (150 mg) in the
presence of NaHCO3 (328 mg, 3.9 mmol) reduced by Na2S2O4 to
give 6 f (85 mg, 60%), mp: 2658C (dec.), 1H NMR (400 MHz,
[D6]DMSO): d=8.46 (1H, s, NH), 8.08 (2H, d, JH-F =8.0 Hz, ArH-
3’’,5’’), 7.41 (2H, d, J=8.8 Hz, ArH-3’,5’), 7.26 (2H, d, J=8.8 Hz, ArH-
2’,6’), 7.16 (1H, d, J=8.4 Hz, ArH-4), 6.62(1H, d, J=8.4 Hz, ArH-5),
5.00 ppm (2H, s, NH2); MS m/z (%): 364 [M+1, 100]; HPLC purity
98.51% (CH3OH/H2O=70:30).
(E)-N2-(4’-Cyanophenyl)amino-6-(4’’-cyanovinyl-2’’,6’’-difluoro)-
phenoxypyridin-3-amine (6g): Method 1: Starting with 5g
(419 mg, 1 mmol) was reduced by Na2S2O4 to afford 6g as an off-
white solid (254 mg, 65%), mp: 210–2118C; 1H NMR (400 MHz,
CD3COCD3): d=7.80 (1H, br, NH), 7.62 (1H, d, J=16.8 Hz, ArCH=),
7.59 (2H, JF-H =8.8 Hz, ArH-3’’,5’’), 7.39 (2H, d, J=8.8 Hz, ArH-3’,5’),
7.29 (3H, m, ArH-2’,6’ and PyH-4), 6.57 (1H, d, J=8.0 Hz, PyH-5),
6.48 (1H, d, J=16.8 Hz, CH=), 4.39 ppm (2H, br, NH2); MS m/z (%):
387.9 [Mꢁ1, 25], 368.2 (100); HPLC purity 96.37%.
(E)-6-[4’’-(3-Amino-2-cyano-3-oxoprop-1-enyl)-2’’,6’’-difluoro]phe-
noxy-N2-(4’-cyanophenyl)pyridin-2,3-diamine (6h): Method 1:
Starting with of 5h (462 mg) reduced by Na2S2O4 to give 6h as
a gray solid (273 mg, 64%), mp: 227–2298C; 1H NMR (400 MHz,
[D6]DMSO): d=8.46 (1H, brs, NH), 8.28 (1H, s, CH=), 8.02 and 7.93
(each 1H, br, CONH2), 7.89 and 7.87 (each 1H, s, ArH-3’’,5’’), 7.35
(4H, m, ArH), 7.18 (1H, d, J=8.0 Hz, PyH-4), 6.63 (1H, d, J=8.0 Hz,
PyH-5), 5.02 ppm (2H, br, NH2); MS m/z (%): 431.1 [Mꢁ1, 20], 411.3
(100); HPLC purity 96.19%.
N2-(4’-Cyanophenyl)amino-6-(2’’,6’’-difluoro-4’’-(3-hydroxypro-
pyl)phenoxy)pyridin-3-amine (6n): The crude 6j prepared from
5j (452 mg, 1 mmol) was further reduced by LiBH4 (280 mg,
10 mmol) in ice bath with stirring for 30 min. The mixture was
poured into ice-water, adjusted pH to 4–5 with aq HCl, extracted
with EtOAc three times (40–60 mL), and dried over Na2SO4. After
removal of solvent, the residue was purified by a silica gel flash
column (gradual elution: MeOH/CH2Cl2, 0–1%) to give 6n as
a
light-gray solid (264 mg, 67%), mp: 160–1628C; 1H NMR
(400 MHz, [D6]DMSO): d=8.40 (1H, brs, NH), 7.36 (4H, s, ArH-
2’,3’,5’,6’), 7.15 (3H, m, ArH-3’’,5’’ and PyH-4), 6.54 (1H, d, J=8.4 Hz,
PyH-4), 6.54 (1H, d, J=8.0 Hz, PyH-5), 4.90 (2H, brs, NH2), 4.52 (1H,
brs, OH), 3.43 (2H, t, J=7.6 Hz, OCH2), 2.70 (2H, t, J=7.6 Hz,
ArCH2), 1.76 ppm (2H, m, CH2); MS m/z (%): 395 [Mꢁ1, 100]; HPLC
purity 96.78%.
N2-(4’-Cyanophenyl)amino-6-(2’’,6’’-difluoro-4’’-(3-oxobutyl)phe-
noxy)pyridin-3-amine (6i): Method 2: Starting with 5i (436 mg)
reduced by catalytic hydrogenation to furnish 6i as a gray solid
6-(4’’-Cyano-2’’,6’’-dibromo)phenoxy-N2-(4’-cyanophenyl)pyridin-
2,3-diamine (8a): Method 1: Starting with 7a (515 mg, 1 mmol)
reduced by Na2S2O4 to afford 8a as an off-white solid (309 mg,
1
(167 mg, 43%), mp: 142–1448C; H NMR (400 MHz, [D6]DMSO): d=
8.42 (1H, br, NH), 7.40 (2H, d, J=8.8 Hz, ArH-3’,5’), 7.35 (2H, d, J=
8.8 Hz, ArH-2’,6’), 7.14–7.19 (3H, m, ArH and PyH-4), 6.54 (1H, d,
J=8.0 Hz, PyH-5), 4.92 (2H, br, NH2), 4.85 (4H, m, CH2 ꢂ2);
2.14 ppm (3H, s, COCH3); MS m/z (%): 407.4 [Mꢁ1, 70], 360.3 (100);
HPLC purity 95.20%.
1
69%), mp: 218–2208C; H NMR (400 MHz, [D6]DMSO): d=8.46 (2H,
s, ArH-3’’,5’’), 8.40 (1H, brs, NH), 7.38 (2H, d, J=8.8 Hz, ArH-3’,5’),
7.26 (2H, d, J=8.8 Hz, ArH-2’,6’), 7.18 (1H, d, J=8.4 Hz, PyH-4),
6.59 (1H, d, J=8.4 Hz, PyH-5), 4.94 ppm (2H, s, NH2); MS m/z (%):
481.6 [Mꢁ1, 40], 483.7 [M+1, 100]; HPLC purity 99.85%.
(E)-N2-(4’-Cyanophenyl)amino-6-(4’’-cyanovinyl-2’’,6’’-dibromo)-
phenoxypyridin-3-diamine (8b): Method 1: Starting with 7b
(500 mg, 0.92 mmol) reduced by Na2S2O4 to produce 8b as a white
solid (200 mg, 43%), mp: 231–2338C; 1H NMR (400 MHz,
N2-(4’-Cyanophenyl)amino-6-(2’’,6’’-difluoro-4’’-propylcarbme-
thoxy)phenoxypyridin-3-amine (6j): Method 2: Starting with 5j
(452 mg) reduced by catalytic hydrogenation to produce 6j as
a gray solid (158 mg, 40%), mp: 203–2058C; 1H NMR (400 MHz,
[D6]DMSO): d=8.39 (1H, br, NH), 7.35 (4H, m, ArH-2’,3’,5’,6’), 7.22
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[D6]DMSO): d=8.39 (1H, brs, NH), 8.15 (2H, s, ArH-3’’,5’’), 7.70 (1H,
d, J=16.8 Hz, ArCH=), 7.30 (4H, brs, ArH-2’,3’,5’,6’), 7.16 (1H, d, J=
8.0 Hz, PyH-4), 6.71 (1H, d, J=16.8 Hz, =CHCN), 6.56 (1H, d, J=
8.0 Hz, PyH-5), 4.90 ppm (2H, s, NH2); MS m/z (%): 510.3 [M+1,
100], 512.2 [M+3, 50]; HPLC purity 97.11%.
concentrations for 50% inhibition (EC50) were calculated with Cal-
cuSyn software (version 2.0).[24]
Cytotoxicity assays: A CytoTox-Glo cytotoxicity assay (Promega) was
used to determine the cytotoxicity of the synthesized compounds.
Parallel to the anti-viral assays, TZM-bl cells were cultured in the
presence of various concentrations of the compounds for one day.
Percent of viable cells was determined by following the protocol
provided by the manufacturer. The 50% cytotoxic concentration
(CC50) was defined as the concentration that caused a 50% de-
crease in cell viability.
(E)-6-(2’’-Bromo-4’’-cyanovinyl-6’’-methoxy)phenoxy-N2-(4’-cyano-
phenyl)pyridin-2,3-diamine (8c): Method 1: Starting with 7c
(492 mg) reduced by Na2S2O4 to afford 8c as a white solid
1
(285 mg, 61%), 219–2208C; H NMR (400 MHz, [D6]DMSO): d=8.35
(1H, s, NH), 7.69 (1H, d, J=16.4 Hz, ArCH=), 7.65 (1H, s, ArH-3’’),
7.55 (1H, s, ArH-5’’), 7.31 (4H, brs, ArH-2’,3’,5’,6’), 7.13 (1H, d, J=
8.8 Hz, PyH-4), 6.68 (1H, d, J=16.4 Hz, =CHCN), 6.48 (1H, d, J=
8.8 Hz, PyH-5), 4.83 (2H, s, NH2), 3.75 ppm (3H, s, OCH3); MS m/z
(%): 460.3 [Mꢁ1, 100], 462.1 [M+1, 60]; HPLC purity 95.07%.
Aqueous solubility measurements: Solubility was measured separate-
ly at pH 7.4 and pH 2.0 by using an HPLC-UV method. Test com-
pounds were initially dissolved in DMSO at 10 mgmLꢁ1. This stock
solution (10 mL) was spiked into either pH 7.4 phosphate buffer
(1.0 mL) or 0.01m HCl (pH~2.0, 1 mL) with the final DMSO concen-
tration being 1%. The mixture was stirred for 4 h at RT, and then
concentrated at 3000 rpm for 10 min. The saturated supernatants
were transferred to other vials for analysis by HPLC-UV. Each
sample was performed in triplicate. For quantification, a model
1200 HPLC-UV (Agilent) system was used with an Agilent TC-C18
column (250ꢂ4.6 mm, 5 mm) and gradient elution of CH3CN in
H2O, starting with 0% of CH3CN, which was linearly increased up
to 70% over 10 min, then slowly increased up to 98% over 15 min.
The flow rate was 1.0 mLminꢁ1 and injection volume was 15 mL.
Aqueous concentration was determined by comparison of the
peak area of the saturated solution with a standard curve plotted
peak area versus known concentrations, which were prepared by
solutions of test compound in CH3CN at 50, 12.4, 3.125, 0.781, and
(E)-N2-(4’-Cyanophenyl)amino-6-(4’’-cyanovinyl-2’’,6’’-dichloro)-
phenoxypyridin-3-amine (8d): Method 1. Starting with 7d
(452 mg) reduced by Na2S2O4 to produce 8d as a white solid
1
(219 mg, 52%), mp: 216–2188C; H NMR (400 MHz, [D6]DMSO): d=
8.39 (1H, brs, NH), 8.00 (2H, s, ArH-3’’,5’’), 7.71 (1H, d, J=16.8 Hz,
ArCH=), 7.30 (2H, d, J=8.8 Hz, ArH-3’,5’), 7.27 (2H, d, J=8.8 Hz,
ArH-2’,6’), 7.17 (1H, d, J=8.4 Hz, PyH-4), 6.72 (1H, d, J=16.8 Hz, =
CHCN), 6.58 (1H, d, J=8.4 Hz, PyH-5), 4.91 ppm (2H, s, NH2); MS
m/z (%): 420.0 [Mꢁ1, 60], 422.3 [M+1, 100]; HPLC purity 96.11%.
(E)-N2-(4’-Cyanophenyl)amino-6-(2’’-methoxy-4’’-cyanovinyl)phe-
noxypyridin-3-amine (8e): Method 1. Starting with 7e (413 mg)
reduced by Na2S2O4 to furnish 8e as a white solid (198 mg, 52%),
1
82–848C; H NMR (400 MHz, [D6]DMSO): d=8.35 (1H, s, NH), 7.69
(1H, d, J=16.4 Hz, ArCH=), 7.53 (1H, s, ArH-3’’), 7.35 (4H, m, ArH-
2’,3’,5’,6’), 7.28 (1H, J=8.8 Hz, ArH-5’’), 7.13 (1H, d, J=8.8 Hz, PyH-
4), 7.10 (1H, J=8.8 Hz, ArH-6’’), 6.56 (1H, d, J=16.4 Hz, =CHCN),
6.43 (1H, d, J=8.8 Hz, PyH-5), 4.86 (2H, s, NH2), 3.74 ppm (3H, s,
OCH3); MS m/z (%): 382.2 [Mꢁ1, 100]; HPLC purity 96.24%.
0.195 mgmLꢁ1
.
LogP measurements: Using the above DMSO stock solution
(1 mgmLꢁ1), 20 mL of this solution were added into n-octane
(1 mL) and H2O (1 mL). The mixture was stirred at room tempera-
ture for 24 h and left to sit overnight. Each solution (~0.5 mL) was
transferred from two phases, respectively, into other vials for HPLC
analysis. The instrument and conditions were the same as those
for solubility determination. The logP value was calculated by the
peak area ratios in n-octane and in H2O.
Bioassays
Assays for measuring the inhibitory activity of compounds on HIV-1 in-
fection of TZM-bl cells: Inhibition of HIV-1 infection was measured
as a decrease in luciferase gene expression after a single round of
virus infection of TZM-bl cells, as described previously.[22] Briefly,
800 TCID50 of virus (NL4-3 or drug-resistant variants) was used to
infect TZM-bl cells in the presence of various concentrations of
compounds. One day after infection, the culture medium was re-
moved from each well, and 100 mL of Bright Glo reagent (Promega,
San Luis Obispo, CA, USA) was added to the cells for measurement
of luminescence using a Victor 2 luminometer. The effective con-
centration (EC50) against HIV-1 strains was defined as the concen-
tration that caused a 50% decrease in luciferase activity (relative
light units, RLU) relative to virus control wells.
Determination of predictive physicochemical properties:[20] Ligand ef-
ficiency (LE) values were calculated by normalizing binding free
energy of a ligand for the number of heavy atoms, as given by the
formula ꢁDG/HA(non-Hatom). Free binding energy calculation was car-
ried out as DG=ꢁRTlnKd, presuming EC50 ꢂKd, a temperature of
310 K, and given in kcal per heavy atom (non-hydrogen atom). The
pEC50 values, negative logarithm of the molar effective concentra-
tion of compound that causes 50% inhibition of wild-type virus,
were converted from experimental data. Lipophilic ligand efficiency
(LLE) was calculated by the formula pEC50ꢁclogD, in which clogD
values were calculated using ACD software (freeware in 2013).
LELP was defined as the ratio of clogD and LE. The topological
polar surface area (tPSA) was calculated by using ChemDraw Ultra
12.0.
HIV-1 infection assays using MT-2 cells: Inhibitory activity of com-
pounds against infection by HIV-1 IIIB and its variant A17, which is
resistant to multiple NNRTIs, was determined as previously de-
scribed.[23] Briefly, MT-2 cells (104 per well) were infected with an
HIV-1 strain (100 TCID50) in 200 mL RPMI 1640 medium containing
10% FBS in the presence or absence of a test compound at
graded concentrations overnight. The culture supernatants were
then removed, and fresh media containing no test compounds
were added. On the fourth day post-infection, 100 mL of culture su-
pernatants were collected from each well, mixed with equal vol-
umes of 5% Triton X-100, and assayed for p24 antigen, which was
quantitated by ELISA, and the percentage of inhibition of p24 pro-
duction was calculated as previously described.[23] The effective
Microsomal stability assays: Stock solutions of test compounds
(1 mgmLꢁ1) were prepared by dissolving the pure compound in
DMSO and storing at 48C. Before the assay, the stock solution was
diluted with CH3CN to 0.1 mm concentration. For measurement of
metabolic stability, all compounds were brought to a final concen-
tration of 1 mm with 0.1m potassium phosphate buffer at pH 7.4,
which contained 0.1 mgmLꢁ1 human liver microsomes and 5 mm
MgCl2. The incubation volumes were 300 mL, and reaction tempera-
ture was 378C. Reactions were started by adding 60 mL NADPH
(final concentration: 1.0 mm) and quenched by adding 600 mL ice-
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ChemMedChem 2014, 9, 1546 – 1555 1554

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Samples at the 0 min time point were prepared by adding 600 mL
ice-cold CH3CN first, followed by 60 mL NADPH. Incubation of all
samples was conducted in duplicate. After quenching, all samples
were centrifuged at 12000 rpm for 5 min at 08C. The supernatant
was collected, and 20 mL of the supernatant was directly injected
onto a Shimadzu LC–MS-2010 system with an ESI for further analy-
sis. The following controls were also conducted: 1) positive control
incubation containing liver microsomes, NADPH, and reference
compound propranolol; 2) negative control incubation omitting
NADPH; and 3) baseline control containing only liver microsomes
and NADPH. The peak heights of test compounds at various time
points were converted into log percentage remaining, and the
peak height values at initial time (0 min) served as 100%. The
slope of the linear regression from log percentage remaining
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vitro half-life (t1/2) values by the formula of in vitro t1/2 =0.693/k, re-
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This investigation was supported by grants 30930106 and
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(NSFC) awarded to L.X., and US NIH grants awarded to C.H.C.
(AI65310) and K.H.L. (AI33066).
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Keywords: anti-HIV
activity
·
antiviral
agents
·
diarylpyridinamines · drug-like properties · lipophilicity
Received: January 30, 2014
Revised: May 6, 2014
[1] G. R. Kaufmann, D. A. Cooper, Curr. Opin. Microbiol. 2000, 3, 508–514.
[2] S. Vella, L. Palmisano, Antiviral Res. 2000, 45, 1–7.
Published online on June 4, 2014
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ChemMedChem 2014, 9, 1546 – 1555 1555