Design, synthesis, and preclinical evaluations of novel 4-substituted 1,5-diarylanilines as potent HIV-1 non-nucleoside reverse transcriptase inhibitor (NNRTI) drug candidates

Article abstract of DOI:10.1021/jm3007678

Twenty-one new 4-substituted diarylaniline compounds (DAANs) (series 13, 14, and 15) were designed, synthesized, and evaluated against wildtype and drug resistant HIV-1 viral strains. As a result, approximately a dozen new DAANs showed high potency with l

Full text of DOI:10.1021/jm3007678

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Article
Design, Synthesis, and Preclinical Evaluations of Novel 4-
Substituted 1,5-Diarylanilines as Potent HIV-1 Non-nucleoside
Reverse Transcriptase Inhibitior (NNRTI) Drug Candidates
Lian-Qi Sun, Lei Zhu, Keduo Qian, Bingjie Qin, Li Huang, Chin-Ho Chen, Kuo-Hsiung Lee, and Lan Xie
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm3007678 • Publication Date (Web): 02 Aug 2012
Downloaded from http://pubs.acs.org on August 3, 2012
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Design, Synthesis, and Preclinical Evaluations of Novel
4-Substituted 1,5-Diarylanilines as Potent HIV-1
Non-Nucleoside Reverse Transcriptase Inhibitor (NNRTI)
Drug Candidates
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Lian-Qi Sun,† Lei Zhu,‡ Keduo Qian,║ Bingjie Qin,† Li Huang,‡
Chin Ho Chen,‡ Kuo-Hsiung Lee║,┴ and Lan Xie†*
†Beijing Institute of Pharmacology & Toxicology, 27 Tai-Ping Road, Beijing, 100850,
China
‡Duke University Medical Center, Box 2926, Surgical Oncology Research Facility,
Durham, NC 27710, USA
║Natural Products Research Laboratories, UNC Eshelman School of Pharmacy,
University of North Carolina, Chapel Hill, NC 27599-7568, USA
┴ Chinese Medicine Research and Development Center, China Medical University and
Hospital, Taichung, Taiwan
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Abstract
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Twenty-one new 4-substituted diarylaniline compounds (DAANs) (Scheme 2, series 13,
14, and 15) were designed, synthesized, and evaluated against wild-type and drug
resistant HIV-1 viral strains. As a result, approximately a dozen new DAANs showed
high potency with low nano- to sub-nanomolar EC50 values ranging from 0.2 to 10 nM.
The three most promising compounds 14e, 14h, and 15h exhibited high potency against
wild-type and drug-resistant viral strains with EC50 values at the sub-nanomolar level
(0.29-0.87 nM), and were comparable to or more potent than the new NNRTI drug
riplivirine (2) in the same assays. Drug-like physicochemical property assessments
revealed that the most active DAANs (EC50 <10 nM) have better aqueous solubility
(>1-90 ꢀg/mL at pH 7.4 and pH 2) and metabolic stability in vitro than 2, as well as
desirable log P values (<5) and polar surface area (PSA) (<140 Å2). These promising
results warrant further development of this novel compound class as potential potent
anti-AIDS clinical trial candidates.
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INTRODUCTION
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Since the first case of acquired immunodeficiency syndrome (AIDS) was reported by the
U. S. 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 demonstrated that ART
is also effective at preventing infection,1,2 thus offering an unprecedented opportunity to
control the AIDS pandemic. Therefore, the discovery and development of novel highly
potent anti-HIV drugs remains imperative. Among current anti-HIV drugs,
non-nucleoside reverse transcriptase inhibitors (NNRTIs) are used in combination as key
components in the highly active antiretroviral therapy (HAART).3,4 Currently, five
NNRTI drugs have been approved by the US FDA for AIDS therapy, including three
first-generation compounds, nevirapine, delavirdine, and efavirenz, and two
second-generation compounds, eravirine (1)5 and rilpivirine (2)6 (Figure 1). NNRTI
drugs have the advantages of high potency and low toxicity, but rapid viral
drug-resistance to the first-generation NNRTI drugs has limited their clinical uses.
Fortunately, the new NNRTI drugs 1 and2 overcome the deficiency of early NNRTI
drugs. Both 1 and 2 possess extremely potent antiviral activity with low nano- or
sub-nanomolar EC50 values against wild-type and a broad spectrum of mutated NNRTI
resistant strains. There is also a higher genetic barrier for HIV-1 to evolve resistance to
the two new NNRTIs.7 , 8 Drugs 1 and 2 belong to the same structural family,
diarylpyrimidines (DAPYs). Rilpivirine is three-fold more potent than etravirine and is
used in a low oral dose (25 mg/tablet) once-daily. These successes greatly encourage
continuing research to discover and develop additional novel next-generation NNRTI
drugs with diverse molecular scaffolds for more efficacious therapy and potential AIDS
prevention.
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During our prior studies,9 10 we discovered a series of diarylaniline (DAAN)
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compounds with low nanomolar anti-HIV potency against wild-type and HIV-1
RT-resistant viral strains, as well as a new chemo-type scaffold and simplified synthesis.
As an example, lead 3 (Figure 1) exhibited sub-nanomolar EC50 values against wild-type
(EC50 0.63 nM) and RT multi-resistant (0.9 nM) viral strains, which were comparable to
those of rilpivirine (0.52 nM and 0.49 nM, respectively) in the same assays. However,
further physical chemistry studies revealed that 3 possessed poor aqueous solubility
(0.29 ꢀg/mL) and low permeability (Paap < 2 × 10-6 cm/s) in a Caco-2 assay, which
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might limit molecular absorption and cause low oral bioavailability in vivo. Therefore,
further lead optimization of DAANs is necessary to surmount these deficiencies.
Our further optimization strategy is shown in Figure 2. Based on prior SAR
results, the two known pharmacophores of DAANs, the para-cyanoaniline moiety
(A-ring) and the crucial amino group (NH2) on the central phenyl ring (B-ring) ortho to
the NH group linking the B and A rings were maintained. With regard to the structural
difference between drugs 1 and 2, 11 one modifiable point appeared to be the
para-substituent (R2) on the trisubstituted phenoxy ring (C-ring). We postulated that a
hydrophobic, flexible, and more linear substituent at this position might insert into the
narrow tunnel on the NNRTI binding site and interact with the reserved W229 to
enhance antiviral potency against wild-type and resistant viral strains. While early
molecular modeling results on DAANs supported the importance of the NH2 on the
central phenyl (ring B) for high anti-HIV activity, the results also revealed that the nitro
group (R1) opposite the amino group on the central phenyl ring (B-ring) created a small
electrostatic interaction with the positively charged amino acid K172 on the NNRTI
binding site. 9 Thus, R1 presents another opportunistic position that we hypothesized
could lead to improvement in molecular physicochemical properties and related
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drug-like properties without loss of anti-HIV potency. Accordingly, a series of new
DAAN analogues with different R1 substituents on the B-ring, either ionizable, polar
groups or H-bond donors and acceptors, were synthesized and evaluated. Herein, we
report the synthesis, anti-HIV activity, physicochemical properties, and some drug-like
properties in vitro of new 4-substituted 1,5-diarylaniline analogues (DAANs). Their
structure-activity relationship (SAR) and structure-property relationship (SPR)
correlations are also discussed.
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Figure 1. Next-generation NNRTI drugs and DAAN lead 3.
Figure 2. Leads DAANs, known SAR, and further optimization strategy
Chemistry. We designed a short synthetic route (see Schemes 1 and 2) that
would allow the rapid and simultaneous optimization of the two variable points (i.e., R1
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and R2) to produce three (13, 14, 15) new series of DAAN compounds. Our approach
was to first prepare building blocks 7–9 (Scheme 1). 2,4-Dichloro-5-trifloromethyl-
nitrobenzene (4) is commercially available. 2,4-dichloro-5-nitrobenzenesulfonamide (5)
was prepared from 2,4-dichloronitrobenzene by treatment with chlorosulfuric acid,
followed by addition of ammonia hydroxide solution. Nitration of 2,4-dichlorobenzoic
acid with concentrated HNO3 and H2SO4, followed by esterification with methanol in
the presence of H2SO4 produced methyl 2,4-dichloro-5- nitrobenzoate (6). Subsequently,
coupling of 4, 5, or 6 with 4-cyanoaniline in DMF in the presence of excess cesium
carbonate provided the corresponding intermediate 4-substituted 5-chloro-N-(4
-cyanophenyl)-2- nitroanilines 7–9, respectively. Consistent with our previous results,
the 4-cyanoaniline moiety preferred to attack the chloride at the ortho-position to the
nitro group.9 The 1H NMR signals of the linker NH in 7–9 were shifted down-field to δ
9.76–9.84 ppm due to a neighboring chelating effect with the adjacent nitro group.
As shown in Scheme 2, intermediates 7–9 were subsequently coupled with
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4-substituted 3,5-dimethylphenols in DMF in the presence of potassium carbonate,
either under microwave irradiation at 190 °C for 10–15 min or by traditional heating at
130 °C for 5 h, to afford corresponding 2,4-diarylnitrobenzene compounds 10a-c and
11c-d with the required three-phenyl ring scaffold. Furthermore, Aldol condensation of
10a with acetone generated compound 11a. Wittig-Honer reaction of aldehyde 10a-10c
with diethyl cyanomethyl phosphonate generated corresponding cyanovinyl compounds
12a-12c. Compound 12c was hydrolyzed under basic condition to yield acid 12d.
Compound 12d was treated with thionyl chloride and the resulting acid chloride was
treated with: (i) ammonia or (ii) methylamine or (iii) hydrazine to generate
corresponding compounds 12e or 12f or 12g respectively. The reduction of the ester
group in 10c and 12c with lithium borohydride (LiBH4) produced hydroxymethyl
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compounds 11e and 12h, respectively. The preparation of intermediate 12i was reported
previously.10 Finally, the nitro group on the central ring in 11a and 12 was treated with
the mild reducing reagent sodium hydrosulfite to produce corresponding target DAAN
compounds 13a and series 14 (a-f, h). Catalytic hydrogenation of 12 with hydrogen
(30-40 p.s.i.) in the presence of Pd/C (5-10%) in anhydrous ethanol afforded the new
series 15 DAAN compounds, in which the nitro group on the central ring and the double
bond of the para-cyanovinyl group on the phenoxy moiety (C-ring) were reduced. By
using the same catalytic hydrogenation, 5-substituted 2,4-diaryl-nitrobenzenes 11a and
11c-e were converted target series 13 (b-e). To confirm the importance of the NH2 on the
B-ring for anti-HIV activity, 1,6-diaryl-benzoimidazole compounds 16a and 16b were
prepared by cyclization of 14f and 15f, respectively, with triethyl orthoformate.12. The
successful synthesis of 16a and 16b further validated the structures of intermediates 7–9
as shown and described above.
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Scheme 1 a) ClSO3H, 0-140 °C; b) NH4OH/THF, 0 °C, 0.5 h; c) HNO3/H2SO4, 2h; d) H2SO4/MeOH,
reflux, 2 h; e) t-BuOK/DMF, r.t. 1 h.
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R1
O
O2N
R1
O
H2N
HN
O2N
HN
R1
Cl
O2N
HN
R1
O
HN
a
b
i
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CHO
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CN
14a-f, h
CN
CN
CHO
12
7-9
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10a R1 = CF3
CN
CN
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12a R1= CF3
12b R1= SO2NH2
14a, 15a R1= CF3
10b R1 = SO2NH2
10c R1 = COOCH3
14b, 15b R1= SO2NH2
14c, 15c R1 = COOCH3
14d, 15d R1 = COOH
14e, 15e R1 = CONH2
14f, 15f R1 = CONHMe
15g R1= CONHNH2
14h, 15h R1 = CH2OH
15i R1= NH2
12c R1= COOCH3
12d R1 = COOH
12e R1= CONH2
12f R1= CONHMe
12g R1= CONHNH2
12h R1= CH2OH
12i R1= NO2
a
HO
R3
c
d
g or h
h
e
f
O2N
HN
R1
O
H2N
HN
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O
i or j
k
j
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N
CONHMe
CN
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CN
R2
H2N
HN
R1
O
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11
13a R1 = CF3, R2 = CH=CHCOMe
13b R1 = CF3, R2 = CH2CH2COMe
13c R1 = CF3, R2 = CN
13d R1 = CF3, R2 = NH2
13e R1 = CH2OH, R2 = CH2OH
k
O
11a R1= CF3, R3 = CH=CHCOMe
11c R1= CF3, R3 = CN
11d R1= CF3, R3 = NO2
11e R1= CH2OH, R3 = CH2OH
CN
R2
CN
16a R2 = (E)-CH=CHCN
16b R2 = -CH2CH2CN
15a-i
CN
Scheme 2. a) K2CO3/DMF, 190 °C, MW, 15 min or traditional heating at 130 °C, 5 h; b)
(EtO)2P(O)CH2CN, t-BuOK/THF, 0 °C – r.t., 3 h; c) aq NaOH/THF/MeOH, r.t., 4 h; d)
(i) SOCl2/CH2Cl2, reflux, 3 h; (ii) NH4OH/THF, 0 °C, 0.5 h; e) (i) SOCl2/CH2Cl2, reflux,
3 h; (ii) NH2CH3 in THF, 0 °C, 0.5 h; f) (i) SOCl2/CH2Cl2, reflux, 3 h; (ii) N2H4·H2O, r.t,
1 h; g) CH3COCH3, MeOH/aq NaOH (10%), 0 °C – rt, 1 h; h) LiBH4, THF/MeOH, 1 h;
i) Na2S2O4/NH4OH, THF/H2O (v/v 1:1), r.t.; j) H2/Pd-C, EtOH, 2-4 h; k) CH(OEt)3, HCl
(1N), C2H5OC2H5/DMF, r.t. 3 h.
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RESULTS AND DISCUSSION
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Initially, we evaluated the 4-trifluoromethyl DAAN compounds (13a-d, 14a, and 15a) in
which the nitro group (R1) on the B-ring of lead 3 had been replaced with a strong
electron-withdrawing trifluoromethyl group, and various para-substituents (R2) were
present on the C-ring. All six compounds exhibited high potency against wild-type
HIV-1 replication with low nanomolar EC50 values ranging from 1.3 to 13.8 nM and
selective indexes (SI) between 789 and 6454 (Table 1). These results showed that the R1
substituent on the central phenyl ring could be modified without losing high anti-HIV
activity. However, the introduced trifluoromethyl group resulted in log P values greater
than 5 and did not improve aqueous solubility (< 1 ꢀg/mL) over a broad pH range
(2.0-7.4) (Table 1). Consequently, R1 on the B-ring was changed to other substituents in
an attempt to achieve potential drug candidates with more desirable drug-like properties
and high anti-HIV potency. Newly synthesized series 14 and 15 DAANs with various R1
groups on the B-ring and either a para-cyanovinyl or para-cyanoethyl R2 group,
respectively, on the C-ring were evaluated against wild-type HIV-1 replication. Except
for 14b, 15b, 14d, and 15d, the compounds in these two series exhibited very high
antiviral potency with low to sub-nanomolar EC50 values (0.39–10 nM). Among them,
the two most potent compounds 14h and 15h, with a 4-hydroxymethyl group (R1),
exhibited sub-nanomolar EC50 values of 0.53 nM and 0.39 nM, respectively, which were
comparable with or slight better than that of 2 in the same assay. Three other pairs of
analogues, 14c and 15c with a 4-ester (COOCH3), 14e and 15e with 4-amide (CONH2),
and14f and 15f with 4-methylamide (CONHCH3), also displayed high potency with low
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nanomolar EC50 values ranging from 0.87 to 5.72 nM, regardless of whether R2 on the
C-ring was para-cyanovinyl (14) or para-cyanoethyl (15). In contrast, the presence of
4-sulfamoyl (SO2NH2, 14b & 15b) or 4-carboxylic acid (COOH, 14d & 15d) on the
B-ring resulted in substantially reduced antiviral activity (EC50 94-380 nM), suggesting
that a very polar substituent at the 4-position might impede the interaction between the
inhibitor(s) and the NNRTI binding site. In a comparison of 4-amide-DAAN 15e,
4-amino-DAAN 15i, and 4-hydrazine-DAAN 15g, compound 15e (EC50 1.39 nM) was 5
and 13-fold more potent than 15i (7.30 nM) and 15g (EC50 19.1), respectively, indicating
that the distance between the amino group in the R1 substituent and the central phenyl
ring might influence the antiviral activity. Therefore, we postulated that an
electron-withdrawing R1 group with appropriate volume and length on the B-ring might
be necessary to provide additional interaction(s) within the NNRTI binding pocket for
maintaining or enhancing antiviral potency. However, when the two neighboring amino
groups on the central phenyl ring of the active compound 14f and 15f were cyclized,
both products 16a and 16b lost antiviral potency (EC50 > 220 nM). This result supported
our previous hypothesis that an amino group on the central B-ring ortho to the
aniline moiety is crucial for achieving high potency. Finally, DAAN 13e, with two
hydroxymethyl groups (R1 and R2), exhibited 20-fold lower activity (EC50 10.0 nM) than
the most active compounds 14h and 15h, in which R2 was para-cyanovinyl or
para-cyanoethyl, respectively. This finding is consistent with our previous SAR
results,10 that a hydrophobic and linear para-R2 on the C-ring is favored for antiviral
activity.
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Considering them to be next-generation NNRTIs, we chose new DAANs with
EC50 values less than 5 nM to test against three NNRTI resistant HIV-1 mutants
RT-multi-drug-resistant (MRTDR), K101E, and E138K. It is reported that K101E and
E138K are two of the mutations that confer resistance to new drugs 1 and 2.13
Interestingly, the newly synthesized active DAANs exhibited potent activity against the
drug resistant viral strains with low nanomolar EC50 values. The most active compounds
14h and 15h were slightly more potent than 2 and three- to four-fold more potent than 3
against these mutated viral strains, with the notable exception that 15h was now more
resistant against the RTMDR strain.
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To evaluate their potential as drug candidates, five pairs of new active DAANs
(EC50 < 10 nM) were further assessed for their physicochemical and drug-like properties
as shown in Table 4. We first measured their aqueous solubility by using an HPLC/UV
method, in parallel with 2 and 3. All tested DAANs, except 14a and 15a, showed
improved solubility at both pH 7.4 and pH 2.0 compared with 3, and were generally
more soluble in acidic than neutral conditions, because of the presence of a free amine
group. Notably, the most active compound 15h had aqueous solubility of 89.8 ꢀg/mL at
pH 2.0 and 18.8 ꢀg/mL at pH 7.4, comparable to and better than 2 (86 ꢀg/mL at pH 2.0
and 0.24 ꢀg/mL at pH 7.4) in the same assay (lit. 20 ng/mL at pH 7.0).14 In addition,
15c, 15f, 14h, and 13e also showed moderate solubility ranging from 20.9-44.7 ꢀg/mL
under acidic conditions, which would enhance molecular absorbability in the stomach. A
comparison of the 14 and 15 series showed that the 15 series compounds were generally
more soluble than the corresponding 14 series compounds, suggesting that the greater
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flexibility of the cyanoethyl group (R2) on the C-ring might contribute to better aqueous
solubility. Additionally, 15 series compounds had slightly or significantly lower melting
points than corresponding 14 series compounds. Next, we assessed the lipophilicity of
the active DAANs by measuring their octanol/water (aqueous buffer pH 7.4) partition
coefficients by an HPLC/UV method. The log P values of 14c/15c, 14e/15e, 14f/15f,
and 14h/15h ranged from 2.65–4.15 and, consistent with aqueous solubility, were also
improved compared with 14c/15c, 2, and 3. The pattern was also similar to the predicted
clog D values. Subsequently, molecular PSA (polar surface area) values were calculated
by using DS software (3.0) as a prediction of oral bioavailability. The values for all
compounds fell in a desirable range of 88.13-138 (a criterion <140 Å2).15 Furthermore,
metabolic stability of these active DAANs was evaluated by a human liver microsome
incubation assay, in parallel with 2. Most of the active DAANs displayed better stability
than 2 (t1/2 34.2 min); however, the most potent 4-hydroxymethyl DAANs 14h and 15h
appeared to be less stable (t1/2 30.2 and 20.9 min, respectively) and had higher clearance
(CL 0.23 and 0.33, respectively) than 2 and others in the same assay.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
CONCLUSION
Based on our current optimization strategy, 21 new DAAN analogues (series 13, 14, and
15) were synthesized by modifying R1 on the central phenyl ring (B-ring) and R2 on the
phenoxy ring (C-ring). Seventeen of the new target compounds showed significant
anti-HIV activity (EC50 < 20 nM). The most promising compounds 14e, 14h, and 15h
exhibited high potency against wild-type and drug-resistant viral strains with EC50
values at a subnanomolar level (0.29-0.87 nM), and were comparable or more potent
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than new NNRTI drug 2 in the same assays. The modifications of the R1 substituent on
the central B-ring provided the following new structure-activity relationship (SAR)
correlations for DAANs: (1) R1 is modifiable to improve molecular drug-like properties
without loss of potency. (2) R1 as H-bond donor and/or acceptor is favorable for
anti-HIV potency and drug-like properties, and (3) the presence of a too polar R1, such
as COOH or SO2NH2, greatly decreases potency. In addition, current results also
supported our previous SAR: (1) a favorable R2 substituent on the C-ring should be
hydrophobic, flexible and more linear, such as either a para-cyanovinyl or
para-cyanoethyl; (2) the amino (NH2) group on the central ring is crucial for achieving
high potency (see 16a and 16b). The assessment of drug-like physicochemical properties
for four pairs of active DAANs (EC50 < 6 nM) indicated that introducing a suitable R1
on the central phenyl ring can indeed improve molecular physicochemical properties,
such as aqueous solubility, log P values, PSA, and metabolic stability, which will benefit
drug ADME properties. The current results hold great promise for the identification of
potential new drug candidates with high potency and desirable drug-like properties.
Further studies on pharmacokinetic properties in vivo are ongoing and will be reported
later.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
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Table 1. Structures, antiviral activity, and physicochemical properties of 4-trifluoro-
methyl diarylanilines 13a-d, 14a, and 15a
H2N
CF3
NC
N
H
O
R2
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Aqueous
solubility
pH 7.4 pH 2.0
NL4-3 (wt) in TZM-bl cells
Log P
R2
EC50
CC50
(ꢀM)b
>8.6
SIc
(nM)a
--
--
CH=CHCOCH3 10.9 2.58
>789
1620
13a
13b
--
0.16
0.28
>5
CH2CH2COCH3 6.85 1.73
11.1
CN
3.79 0.83
13.8 4.37
13.3
15.8
3509
1145
13c
13d
0.26
--
0.96
--
>5
--
NH2
0.24
0.24
0.42
>5
CH=CHCN
CH2CH2CN
9.38 2.45
15.0
1599
14a
15a
0.33
0.29
>5
>5
1.13 0.31 > 7.1
0.63 0.13 >24.4
> 6454
>38730
37305
3
2
0.29
0.52 0.14
19.4
a
Concentration of compound that causes 50% inhibition of viral replication, presented
b
as mean standard deviation (SD), performed at least in triplicate. XTT assay was
c
used to determine the CC50 value that causes cytotoxicity to 50% cells. SI (Selectivity
index) is the ratio of CC50/EC50.
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Table 2. Structures and antiviral activity of diarylanilines 14-16 and 13ea
H2N
R1
N
R1
5
HN
O
N
O
6
7
8
R2
CN
R2
13-15
16
CN
9
NL4-3 (wt)
CC50(ꢀM)c
>21.8
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
R1
R2
EC50(nM)b
SId
SO2NH2
SO2NH2
COOCH3
COOCH3
COOH
CH=CHCN
CH2CH2CN
CH=CHCN
CH2CH2CN
CH=CHCN
CH2CH2CN
CH=CHCN
CH2CH2CN
>233
14b
15b
14c
15c
14d
15d
14e
15e
14f
15f
15g
14h
15h
15i
13e
16a
16b
3
93.7 ± 24.0
380 67
2.74 0.71
4.32 1.54
230 38
11.1
>22.8
>22.7
14.9
29.2
>8321
>5255
65
COOH
96.0 21
0.87 0.28
1.39 0.33
5.72 0.94
2.73 0.41
19.1 5.68
0.53 0.13
0.39 0.11
7.30 1.26
10.0 2.06
>220
14.3
149
CONH2
CONH2
>23.6 >27126
>9.4
>9.2
>9.1
>22.7
16.3
>6762
>1608
>3330
>1188
30755
61026
>3906
1590
__
CONHCH3 CH=CHCN
CONHCH3 CH2CH2CN
CONHNH2 CH2CH2CN
CH2OH
CH2OH
NH2
CH=CHCN
CH2CH2CN
CH2CH2CN
CH2OH
>23.8
>25
CH2OH
15.9
CONHCH3 CH=CHCN
CONHCH3 CH2CH2CN
>890
>890
>220
__
NO2
CH=CHCN
0.63 0.13
0.52 0.14
>24.4 >38730
19.4 37305
2
a Against HIV-1 NL3-4 (wild-type) virus in TZM-bl cell lines. b Concentration of
compound that causes 50% inhibition of viral replication, presented as mean standard
deviation (SD), and performed at least in triplicate. c XTT assay was used to determine
the CC50 value that causes cytotoxicity to 50% cells. d SI (Selectivity index) is the ratio
of CC50/EC50.
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Table 3. Antiviral activity against wild-type and mutated viral strains of selected active
5
6
DAANs
7
8
9
H2N
R1
O
NC
N
H
R2
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
EC50 (nM)a (Fold Change)b
NL4-3 RTMDRc
R1
R2
K101Ed
E138Kd
CONH2
CH2OH
CH=CHCN
CH=CHCN
CH2CH2CN
0.87 1.8 0.5 (2.1) 8.3 3.3 (9.5) 9.0 2.6 (10.3)
0.53 0.4 0.1 (0.7) 3.4 1.0 (6.4) 3.9 0.9 (7.4)
4.32 4.1 0.7 (0.9) 14 4.3 (3.2) 7.5 2.7 (1.7)
2.73 8.0 4.3 (2.9) 14 4.8 (5.1) 10 3.9 (3.7)
0.39 1.8 0.5 (4.6) 3.4 1.3 (8.7) 2.9 1.2 (7.4)
0.63 0.9 0.3 (1.4) 11 3.6 (17.5) 12 3.9 (19.0)
0.52 0.5 0.1 (0.9) 5.7 1.4(11.0) 5.2 1.6 (10.0)
14e
14h
15c
15f
15h
3
COOCH3
CONHCH3 CH2CH2CN
CH2OH
NO2
CH2CH2CN
CH=CHCN
2
a Experiments performed at least in triplicate and data presented as means SD. b
resistant fold change. c HIV-1 RTMDR (obtained from AIDS Research and Reference
Reagent Program, Division of AIDS, NIAID, NIH), which contains mutations in RT
amino acid residues L74, M41L, V106A, and T215Y, is resistant to AZT, ddI, nevrapine,
and other noncucleoside RT inhibitors. d mutated amino acids in the HIV-RT
non-nucleoside binding pocket (NNBP) confer resistance to 2.
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Table 4. Structures and drug-like properties of active DAANs 14 and 15 (EC50 <10 nM)
14 R2 = -CH=CHCN, 15 R2 = -CH2CH2CN
HLM c
Aqueous solubility
(ꢀg/mL) a
EC50
(nM)
R1
logP a clogD b PSAb
10
11
12
13
14
15
16
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59
60
Mp °C
t1/2
CL
pH 2.0
pH 7.4
min mL/min/mg
224-6
172-4
CF3
CF3
9.38
1.13
2.74
4.32
0.87
1.39
0.42
0.33
1.75
21.8
2.09
9.11
8.63
44.7
20.9
89.8
0.29
86.8
0.24
0.24
1.30
1.21
0.63
0.06
1.32
8.28
3.23
18.8
0.29
>5
>5
7.09 94.86 45.0 0.15
14a
15a
14c
15c
14e
6.49 94.86 55.7
0.12
0.02
0.04
0.20
0.16
0.15
0.12
0.23
0.33
0.15
0.20
CO2CH3
CO2CH3
CONH2
240-2 4.15
236-8 4.15
290-2 3.53
274-6 2.65
112-4 4.12
110-2 2.65
186-8 4.14
115-7 3.68
5.06 121.16 283
4.46 121.16 186
4.38 137.94 35.0
3.79 137.94 42.0
3.93 123.96 44.8
3.33 123.96 55.7
3.55 115.09 30.2
2.95 115.09 20.9
5.88 140.67 47.8
3.62 97.41 34.2
15e CONH2
14f CONHCH3 5.72
CONHCH3 2.73
15f
14h
15h
3
CH2OH
CH2OH
NO2
0.53
0.39
0.63
0.52
252-4
0.24d 246-8
>5
>5
2
a Measured at pH 7.4 in triplicate. A general solubility guideline for human oral
absorption is <10 ꢀg/mL low; 10-60 ꢀg/mL moderate, >60 ꢀg/mL high.16
b Predicted by using software ACD or DS 3.0, respectively. c Human liver microsome
incubation assay, data from at least two experiments in parallel. d 20 ng/mL at pH 7.0
reported in reference 14.
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EXPERIMENTAL SECTION
4
5
6
7
8
Chemistry. Melting points were measured with an RY-1 melting apparatus
without correction. The proton nuclear magnetic resonance (1H NMR) spectra were
measured on a JNM-ECA-400 (400 MHz) spectrometer using tetramethylsilane (TMS)
as the internal standard. The solvent used was CDCl3 unless otherwise indicated. Mass
spectra were measured on an ABI Perkin-Elmer Sciex API-150 mass spectrometer with
electrospray ionization, and the relative intensity of each ion peak is presented as percent
(%). The purities of target compounds were ≥95%, measured by HPLC analyses, which
were performed on an Agilent 1100 HPLC system with a UV detector and a Grace
Alltima HP C18 column (100 × 2.1 mm, 3 ꢀm) eluting with a mixture of solvents A and
B (condition 1: acetonitrile/water 70:30, flow rate 1.0 mL/min; condition 2:
MeOH/water 70:30, flow rate 0.8 mL/min; UV 254 nm). The microwave reactions were
performed on a microwave reactor from Biotage, Inc. Thin-layer chromatography (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 chemical reagents
and solvents were obtained from Beijing Chemical Works or Sigma-Aldrich, Inc.
NADPH, MgCl2, KH2PO4, K2HPO4 and reference compound propranolol were
purchased from Sigma-Aldrich. HPLC-grade acetonitrile for LC-MS analysis was
purchased from VWR. Pooled human liver microsomes (Lot No# 28831) were
purchased from BD biosciences (Woburn, MA).
9
10
11
12
13
14
15
16
17
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2,4-Dichloro-5-nitrobenzene-1-sulfonyl Chloride. Chlorosulfonic acid (30
mL) cooled to 0 °C was added dropwise into 2,4-dichloro-nitrobenzene (9.6 g, 50 mmol)
with stirring. Then the solution was allowed to warm to rt, followed by heating at 140 °C
for 2 h. After cooling to rt, ice-water was added dropwise to the mixture. A solid
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precipitated, and was filtered and washed with cold water. The collected crude product
was re-crystallized from EtOAc to give 10.6 g of pure sulfonyl chloride in 73% yield,
7
8
1
white solid, mp 116-118 °C; H NMR (DMSO-d6) δppm 7.95 (1H, s, ArH-3), 8.44 (1H,
9
s, ArH-5).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
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47
48
49
50
51
52
53
54
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60
2,4-Dichloro-5-nitrobenzenesulfonamide (5). A solution of the above
sulfonyl chloride (290 mg, 1 mmol) in THF was added to aqueous ammonia solution
(25%, 1 mL, excess) at 0 °C (ice-bath) and stirred at the same temperature for an
additional 0.5 h. The mixture was then poured into ice-water and let stand overnight.
The precipitated white solid was filtered and washed with cold water to provide 207 mg
of 5, 77% yield, white powder, mp 176-178 °C; 1H NMR (DMSO-d6) δ ppm 8.02 (2H, s,
SO2NH2), 8.27 (1H, s, ArH-3), 8.58 (1H, s, ArH-5).
2, 4-Dichloro-5-nitrobenzoic Acid. 2, 4-Dichlorobenzoic acid (191 mg, 1
mmol) was dissolved completely in H2SO4 (98%, 5 mL) with a mechanical stirrer at
0 °C. Then the solution was added dropwise to HNO3 (con., 0.5 mL) with stirring, and
the temperature was kept below 5 °C. The mixture was stirred at rt for 2 h with
monitoring by TLC to reaction finish. The mixture was slowly poured into ice-water
with stirring and let stand overnight. The precipitated solid was collected and washed
with cold water to neutral pH. After drying, 215 mg of 2,4-dichloro-5-nitrobenzoic acid
was obtained in 91% yield, white powder, mp 148-150 °C; 1H NMR (DMSO-d6) δ ppm
8.15 (1H, s, ArH-3), 8.50 (1H, s, ArH-5).
Methyl 2,4-Dichloro-5-nitrobenzoate (6). A solution of
2,4-dichloro-5-nitrobenzoic acid (235 mg, 1 mmol) in MeOH (10 mL) in the presence of
H2SO4 (conc., 2 mL) was refluxed for 2 h. The mixture was poured into ice-water and let
stand overnight. The precipitated white solid was collected, and washed with cold water
to neutral. After drying, 215 mg of 6 was obtained in 86% yield, white powder, mp
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5
46-48 °C; 1H NMR δ ppm 3.98 (3H, s, CH3), 7.71 (1H, s, ArH-3), 8.49 (1H, s, ArH-5).
General Preparation of 4-Substituted
6
7
8
9
5-Chloro-N1-(4-cyanophenyl)-2-nitroanilines (7-9). To a solution of 5-substituted
2,4-dichloro-nitrobenzene (1 equiv) and para-cyanoaniline (1.1 equiv) in DMF (3 mL)
was slowly added potassium tert-butoxide (2 equiv) at 0 °C (ice-water bath) with stirring
and then for an additional 1 h at room temperature. The reaction was monitored by TLC
until completion. The mixture was poured into ice-water and pH adjusted to 6 with aq
HCl (5%). The solid crude product was collected, washed with cold water, and purified
by CombiFlash medium-pressure column chromatography system.
10
11
12
13
14
15
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23
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58
59
60
5-Chloro-N1-(4-cyanophenyl)-4-trifluoromethyl-2-nitroanilines (7).
Starting with 260 mg (1 mmol) of commercially available
2,4-dichloro-5-nitrobenzotrifluoride (4) and 130 mg (1.1 mmol) of para-cyanoaniline to
afford 281 mg of 7, yield 82% yield, yellow solid, mp 180-182 °C; 1H NMR δ ppm 7.40
(1H, s, ArH-6), 7.41 (2H, d, J = 8.4 Hz, ArH-2’,6’), 7.78 (2H, d, J = 8.4 Hz, ArH-3’,5’),
8.61 (1H, s, ArH-3), 9.78 (1H, s, NH); MS m/z (%) 359.2 (M+NH4, 100).
5-Chloro-N1-(4-cyanophenyl)-4-sulfonamide-2-nitroanilines (8). Yield
33%, starting with 271 mg (1 mmol) of 5 and 130 mg (1.1 mmol) of para-cyanoaniline
to afford 116 mg of 8, yellow solid, mp 236-238 °C; 1H NMR δ ppm 5.09 (2H, s, NH2),
7.39 (1H, s, ArH-6), 7.41 (2H, d, J = 8.8 Hz, ArH-2’,6’), 7.79 (2H, d, J = 8.8 Hz,
ArH-3’,5’), 8.99 (1H, s, ArH-3), 9.84 (1H, s, NH); MS m/z (%) 375.1 (M+Na, 100).
5-Chloro-N1-(4-cyanophenyl)-4-methoxycarbonyl-2-nitroanilines (9).
Yield 57%, starting with 250 mg (1 mmol) of 6 and 130 mg (1.1 mmol) of
para-cyanoaniline to afford 189 mg of 9, yellow solid, mp 198-202 °C; 1H NMR δ ppm
3.95 (3H, s, CH3), 7.38 (2H, d, J = 8.4 Hz, ArH-2’,6’), 7.42 (1H, s, ArH-6), 7.77 (2H, d,
J = 8.4 Hz, ArH-3’,5’), 8.92 (1H, s, ArH-3), 9.76 (1H, s, NH); MS m/z (%) 354.3 (M+Na,
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100).
5
6
General Coupling Reaction of Diphenylether (under microwave
7
8
irradiation) for 10a, 10c, 11c, and 11d. A mixture of 4-substituted
9
N1-(4-cyanophenyl)-5-chloro-2-nitroaniline (1 equiv, 7 or 9) and para-substituted
2,6-dimethylphenol (1.2 equiv) in DMF (3 mL) in the presence of anhydrous potassium
carbonate (2 equiv) was irradiated under microwave conditions with stirring at 190 °C
for 15 min. The mixture was poured into ice-water, adjusted to neutral pH with aq HCl
(5%), and extracted with EtOAc three times. After removal of organic solvent under
reduced pressure, crude product was purified by PTLC or flash column chromatography
(gradual eluant: petroleum ether/CH2Cl2).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
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59
60
N1-(4’-Cyanophenyl)-5-(2”,6”-dimethyl-4”-formylphenoxy)-4-trifluoro
methyl-2-nitroaniline (10a). Starting with 171 mg (0.5 mmol) of 7 and 90 mg (0.6
mmol) of 2,6-dimethyl-para-formylphenol to afford 182 mg of 10a, yield 80%, yellow
1
solid, mp 192-194 °C; H NMR δ ppm 2.21 (6H, s, 2 × CH3), 6.24 (1H, s, ArH-6), 7.08
(2H, d, J = 8.4 Hz, ArH-2’,6’), 7.51 (2H, d, J = 8.4 Hz, ArH-3’,5’), 7.67 (2H, s,
ArH-3”,5”), 8.68 (1H, s, ArH-3), 9.92 (1H, s, NH), 9.97 (1H, s, CHO); MS m/z (%)
456.4 (M+1, 100).
N1-(4’-Cyanophenyl)-5-(2”,6”-dimethyl-4”-formylphenoxy)-4-methoxy
carbonyl-2-nitroaniline (10c). Starting with 166 mg (0.5 mmol) of 9 and 90 mg (0.6
mmol) of 2,6-dimethyl-para-formylphenol to afford 150 mg of 10c, yield 67%, yellow
1
solid, mp 220-222 °C; H NMR δ ppm 2.22 (6H, s, 2 × CH3), 3.97 (3H, s, OCH3), 6.23
(1H, s, ArH-6), 7.06 (2H, d, J = 8.8 Hz, ArH-2’,6’), 7.48 (2H, d, J = 8.8 Hz, ArH-3’,5’),
7.65 (2H, s, ArH-3”,5”), 9.02 (1H, s, ArH-3), 9.88 (1H, s, NH), 9.96 (1H, s, CHO); MS
m/z (%) 446.1 (M+1, 100).
N1-(4’-Cyanophenyl)-5-(4”-cyano-2”,6”-dimethylphenoxy)-4-trifluoro
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methyl-2-nitroaniline (11c). Starting with 171 mg (0.5 mmol) of 7 and 88 mg (0.6
mmol) of para-cyano-2,6-dimethylphenol to afford 150 mg of 11c, yield 66%, yellow
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solid, mp 182-184 °C; H NMR δ ppm 2.17 (6H, s, 2 × CH3), 6.21 (1H, s, ArH-6), 7.09
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(2H, d, J = 8.4 Hz, ArH-2’,6’), 7.44 (2H, s, ArH-3”,5”), 7.56 (2H, d, J = 8.4 Hz,
ArH-3’,5’), 8.67 (1H, s, ArH-3), 9.92 (1H, s, NH); MS m/z (%) 453.2 (M+1, 100).
N1-(4’-Cyanophenyl)-5-(2”,6”-dimethyl-4”-nitrophenoxy)-4-trifluorom
ethyl-2-nitroaniline (11d). Starting with 171 mg (0.5 mmol) of 7 and 100 mg (0.6
mmol) of 2,6-dimethyl-para-nitrophenol to afford 215 mg of 11d, yield 91%, yellow
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solid, mp 240-242 °C; H NMR δ ppm 2.24 (6H, s, 2 × CH3), 6.22 (1H, s, ArH-6), 7.10
(2H, d, J = 8.8 Hz, ArH-2’,6’), 7.55 (2H, d, J = 8.8 Hz, ArH-3’,5’), 8.02 (2H, s,
ArH-3”,5”), 8.69 (1H, s, ArH-3), 9.92 (1H, s, NH); MS m/z (%) 473.3 (M+1, 100).
N1-(4’-Cyanophenyl)-5-(2”,6”-dimethyl-4”-formylphenoxy)-4-sulfamo
yl-2-nitroaniline (10b). A mixture of 353 mg (1 mmol) of 8 and 180 mg (1.2 mmol) of
2,6-dimethyl-para-formylphenol in DMF in the presence of anhydrous potassium
carbonate (2 equiv) was heated at 130 °C for 5 h. The mixture was poured into ice-water,
adjusted to neutral pH with aq HCl (5%), and extracted with EtOAc three times. After
removal of organic solvent under reduced pressure, crude product was purified by PTLC
or medium-pressure column (gradual eluant: petroleum ether/CH2Cl2) to afford 310 mg
of 10b, yield 67%, yellow solid, mp 178-180 °C; 1H NMR (DMSO-d6) δ ppm 2.26 (6H,
s, 2 × CH3), 6.35 (1H, s, ArH-6), 7.49 (2H, d, J = 8.8 Hz, ArH-2’,6’), 7.56 (2H, s,
ArH-3”,5”), 7.82 (2H, d, J = 8.8 Hz, ArH-3’,5’), 8.24 (1H, s, ArH-3), 9.76 (1H, s, NH),
9.88 (1H, s, -CHO); MS m/z (%) 465.2 (M-1, 100).
N1-(4’-Cyanophenyl)-5-(2”,6”-dimethyl-4”-(3-oxobut-1-enyl)phenoxy)-
4-trifluoromethyl-2-nitroaniline (11a). To a solution of 10a (228 mg, 0.5 mmol) in
acetone (10 mL) and MeOH (3 mL) was added 10% aq NaOH (2 mL) slowly at 0 °C
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(ice-water bath) with stirring for about 10 min and then for additional 1 h at room
temperature. After the reaction was completed, the mixture was poured into ice-water,
and the pH adjusted with 5% aq. HCl to neutral. The solid crude product was collected,
washed with water, and purified on a flash silica gel column (eluent: CH2Cl2/MeOH =
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100/1) to afford 138 mg of 11a, 56% yield, yellow solid, mp 190-192 °C. H NMR δ
ppm 2.14 (6H, s, 2 × CH3), 2.42 (3H, s, COCH3), 6.27 (1H, s, ArH-6), 6.68 (1H, s, J =
16.4 Hz, =CH), 7.09 (2H, d, J = 8.8 Hz, ArH-2’,6’), 7.30 (2H, s, ArH-3”,5”), 7.43 (1H, s,
J = 16.4 Hz, CH=), 7.51 (2H, d, J = 8.8 Hz, ArH-3’,5’), 8.66 (1H, s, ArH-3), 9.89 (1H, s,
NH); MS m/z (%) 496.5 (M+1, 100).
N1-(4’-Cyanophenyl)-5-(2”,6”-dimethyl-4”-hydroxymethylphenoxy)-4-
hydroxymethyl-2-nitroaniline (11e). To a solution of LiBH4 (5 mmol) in THF (5
mL) was added dropwise 223 mg (0.5 mmol) of 10c in THF (5 mL) at r.t. The mixture
was stirred for 15 min and then 1-2 mL MeOH was added, and stirring continued for 1 h.
The mixture was poured into ice-water, pH was adjusted to 2-3 with 5% aq HCl, and the
resulting solution extracted with EtOAc three times. After removal of organic solvent in
vacuo, crude product was purified by PTLC or medium-pressure column (eluent:
1
PE/EtOAc = 3/1) to afford 178 mg of 11e, 85% yield, yellow solid, mp 216-218 °C; H
NMR δ ppm 2.13 (6H, s, 2 × CH3), 4.66 and 4.88 (each 2H, d, CH2O), 6.28 (1H, s,
ArH-6), 7.03 (2H, d, J = 8.8 Hz, ArH-2’,6’), 7.12 (2H, s, ArH-3”,5”), 7.47 (2H, d, J =
8.8 Hz, ArH-3’,5’), 8.38 (1H, s, ArH-3), 9.70 (1H, s, NH); MS m/z (%) 442.4 (M+Na,
100).
General
Preparation
of
4-Substituted
N1-(4’-Cyanophenyl)-5-(4”-cyanovinyl-2”,6”-dimethylphenoxy)-2-nitroanilin
e (12a-12c). To a solution of diethylcyanomethyl phosphonate [(EtO)2P(O)CH2CN,
266 mg, 1.5 mmol] in THF (15 mL) was added t-BuOK (168 mg, 1.5 mmol) at 0 °C
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(ice-water bath) with stirring for 30 min. The solution of an aldehyde (10a, 10b, or 10c,
1 mmol) in THF (15 mL) was added dropwise into the above mixture at rt and stirring
continued for about 3 h until the reaction was completed. The mixture was poured into
water and extracted with EtOAc three times. After removal of solvent under reduced
pressure, the crude product was purified by a silica gel column using the
medium-pressure system of CombiFlash companion (gradual eluant: MeOH / CH2Cl2,
0-4.5%).
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N1-(4’-Cyanophenyl)-5-(4”-cyanovinyl-2”,6”-dimethylphenoxy)-4-trifl
uoromethyl-2-nitroaniline (12a). Starting with 455 mg (1 mmol) of 10a and 266 mg
(1.5 mmol) of (EtO)2P(O)CH2CN to afford 416 mg of pure 12a, 87% yield, yellow solid,
mp 248-250 °C; 1H NMR δ ppm 2.15 (6H, s, 2 × CH3), 5.85 (1H, d, J = 16.4 Hz, =CH),
6.27 (1H, s, ArH-6), 7.10 (2H, d, J = 8.8 Hz, ArH-2’,6’), 7.21 (2H, s, ArH-3”,5”), 7.32
(1H, d, J = 16.4 Hz, CH=), 7.52 (2H, d, J = 8.8 Hz, ArH-3’,5’), 8.67 (1H, s, ArH-3),
9.91 (1H, s, NH); MS m/z (%) 501.1 (M+Na, 100).
N1-(4’-Cyanophenyl)-5-(4”-cyanovinyl-2”,6”-dimethylphenoxy)-4-sulf
amoyl-2-nitroaniline (12b). Starting with 10b (466 mg, 1 mmol) and 266 mg (1.5
mmol) of (EtO)2P(O)CH2CN to afford 218 mg of 12b, yield 45%, yellow solid, mp
198-200 °C; 1H NMR (DMSO-d6) δ ppm 2.16 (6H, s, 2 × CH3), 6.38 (1H, d, J = 16.4 Hz,
=CH), 6.54 (1H, s, ArH-6), 7.32 (2H, s, ArH-3”,5”), 7.51 (2H, d, J = 8.8 Hz, ArH-2’,6’),
7.52 (1H, d, J = 16.4 Hz, =CHCN), 7.85 (2H, d, J = 8.8 Hz, ArH-3’,5’), 8.25 (1H, s,
ArH-3), 9.79 (1H, s, NH); MS m/z (%) 488 (M-1, 100).
N1-(4’-Cyanophenyl)-5-(4”-cyanovinyl-2”,6”-dimethylphenoxy)-4-met
hoxycarbonyl-2-nitroaniline (12c). Starting with 445 mg (1 mmol) of 10c and 266
mg (1.5 mmol) of (EtO)2P(O)CH2CN to afford 412 mg of 12c, 88% yield, yellow solid,
mp 260-262 °C; 1H NMR δ ppm 2.16 (6H, s, 2 × CH3), 3.96 (3H, s, OCH3), 5.84 (1H, d,
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J = 16.4 Hz, =CH), 6.26 (1H, s, ArH-6), 7.08 (2H, d, J = 8.8 Hz, ArH-2’,6’), 7.20 (2H, s,
ArH-3”,5”), 7.32 (1H, d, J = 16.4 Hz, CH=), 7.50 (2H, d, J = 8.8 Hz, ArH-3’,5’), 9.00
(1H, s, ArH-3), 9.87 (1H, s, NH); MS m/z (%) 469.4 (M+1, 100).
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N1-(4’-Cyanophenyl)-5-(4”-cyanovinyl-2”,6”-dimethylphenoxy)-4-carb
oxy-2-nitroaniline (12d). To a solution of 12c (234 mg, 0.5 mmol) in THF (15 mL)
and MeOH (10 mL) was slowly added NaOH aqueous solution (80 mg NaOH in 4 mL
of water) at rt with stirring for 4 h. The mixture was poured into ice-water, pH adjusted
to 2–3 with 5% aq HCl, and solid was collected and washed with cold water to neutral.
The crude product was purified on a silica gel column using a CombiFlash system
(gradual eluant: MeOH/CH2Cl2, 0-4.5%) to afford 204 mg of pure 12d in 90% yield,
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yellow solid, mp 262-265 °C; H NMR (DMSO-d6) δ ppm 2.06 (6H, s, 2 × CH3), 5.95
(1H, s, ArH-6), 6.44 (1H, d, J = 16.4 Hz, =CH), 7.22 ( 2H, d, J = 8.8 Hz, ArH-2’,6’),
7.47 (2H, s, ArH-3”,5”), 7.58 (1H, d, J = 16.4 Hz, CH=), 7.65 (2H, d, J = 8.8 Hz,
ArH-3’,5’), 8.72 (1H, s, ArH-3), 9.79(1H, s, NH), 13.21 (1H, s, COOH); MS m/z (%)
453.4 (M-1, 100).
Preparation of Amidated Compounds (12e-g). To a solution of 12d (0.5
mmol) in CH2Cl2 (5 mL) was added dropwise SOCl2 (0.5 mL) at rt and then heated at
reflux for 3 h. After removal of solvent, the obtained brown solid was dissolved in dry
THF (5 mL), followed by addition of an amine aqueous solution (1 mL, excess, NH3,
methylamine, or hydrazine) with stirring for an additional 30 min at 0 °C (ice-bath) until
the reaction was completed. The mixture was poured into ice-water and pH was adjusted
to neutral with 5% aq. HCl. The solid was collected, washed with cold water, and dried.
The crude product was purified on a flash silica gel column (gradual eluant:
MeOH/CH2Cl2, 0-4.5%) using a CombiFlash syetem.
N1-(4’-Cyanophenyl)-5-(4”-cyanovinyl-2”,6”-dimethylphenoxy)-4-
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carbamoyl-2-nitroaniline (12e). Starting with 227 mg of 12d (0.5 mmol) in CH2Cl2
(5 mL), SOCl2 (0.5 mL) was added dropwise to obtain acyl chloride (brown solid),
which was dissolved in dry THF (5 mL) and added to aqueous ammonia solution (1 mL)
with stirring for 30 min at 0 °C (ice-bath) until the reaction was completed. After general
work-up as described above, 198 mg of 12e was obtained in 87% yield, yellow solid, mp
278-280 °C; 1H NMR δ ppm 2.16 (6H, s, 2 × CH3), 5.88 (1H, d, J = 16.4 Hz, =CH), 6.29
(1H, s, ArH-6), 7.08 (2H, d, J = 8.8 Hz, ArH-2’,6’), 7.24 (2H, s, ArH-3”,5”), 7.33 (1H, d,
J = 16.4 Hz, CH=), 7.50 (2H, d, J = 8.8 Hz, ArH-3’,5’), 9.30 (1H, s, ArH-3), 9.82 (1H, s,
NH); MS m/z (%) 454.3 (M+1, 100).
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N1-(4’-Cyanophenyl)-5-(4”-cyanovinyl-2”,6”-dimethylphenoxy)-4-met
hylcarbamoyl-2-nitroaniline (12f). Starting with 12d (227 mg, 0.5 mmol) with
SOCl2 (0.5 mL), followed by dry THF (5 mL) and aqueous methylamine solution
(25-30%, 1 mL) at ice-bath with stirring for 30 min. After the same work-up as above,
140 mg of pure 12f was obtained in 60% yield, yellow solid, mp 218-220 °C; 1H NMR δ
ppm 2.17 (6H, s, 2 × CH3), 3.08 (3H, d, CH3), 5.88 (1H, d, J = 16.4Hz, =CH), 6.28 (1H,
s, ArH-6), 7.07 (2H, d, J = 8.8 Hz, ArH-2’,6’), 7.25 (2H, s, ArH-3”,5”), 7.34 (1H, d, J =
16.4Hz, CH=), 7.49 (2H, d, J = 8.8 Hz, ArH-3’,5’), 7.50 (1H, NMe), 9.29 (1H, s, ArH-3),
9.78 (1H, s, NH); MS m/z (%) 468.3 (M+1, 100).
N1-(4’-Cyanophenyl)-5-(4”-cyanovinyl-2”,6”-dimethylphenoxy)-4-hyd
razide-2-nitroaniline (12g). Starting with 12d (140 mg, 0.30 mmol) with SOCl2 (0.50
mL), followed by dry THF (3 mL) and dropped slowly into hydrazine hydrate aqueous
solution (85%, 1 mL) at rt with stirring for 1 h. After the same work-up as above, crude
product was purified on a flash silica gel column (gradual eluant: MeOH/CH2Cl2,
0-4.5%) to afford 116 mg of pure 12g in 80% yield, yellow solid; 1H NMR δ ppm 2.13
(6H, s, 2×CH3) 5.82 (1H, d, J = 16.4 Hz, =CHCN), 6.30 (1H, s, ArH-6), 6.98 (2H, d, J =
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8.8 Hz, ArH-2’,6’), 7.21 (2H, s, ArH-3”, 5”), 7.33 (1H, d, J = 16.4 Hz, =CHAr), 7.53
(2H, d, J = 8.8 Hz, ArH-3’,5’), 9.20 (H, s, ArH-3), 9.81 (1H, s, NH); MS m/z (%) 469
(M + 1, 100).
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N1-(4’-Cyanophenyl)-5-(4”-cyanovinyl-2”,6”-dimethylphenoxy)-4-hyd
roxymethyl-2-nitroaniline (12h). The preparation was the same as that of 11e.
Starting with 234 mg (0.5 mmol) of 12c, reduction with LiBH4 afforded 100 mg of pure
12h in 91% yield, yellow solid, mp 232-234 °C; 1H NMR δ ppm 2.15 (6H, s, 2 × CH3),
4.89 (2H, s, OCH2), 5.85 (1H, d, J = 16.4 Hz, =CH), 6.27 (1H, s, ArH-6), 7.05 (2H, d, J
= 8.8 Hz, ArH-2’,6’), 7.21 (2H, s, ArH-3”,5”), 7.28 (1H, d, J = 16.4 Hz, CH=), 7.47 (2H,
d, J = 8.8 Hz, ArH-3’,5’), 8.41 (1H, s, ArH-3), 9.70 (1H, s, NH); MS m/z (%) 439.3
(M-1, 100).
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General Preparation of 4-Substituted 1,5-Diarylbenzene-1,2-diamines
(13a, 14a-f, and 14h). To a solution of a diaryl-nitrobenzene (1 equiv) in THF and
water (30 mL, v/v 1:1) was added aqueous ammonia solution (25%) and sodium
hydrosulfite (10 equiv) successively. The mixture was stirred at rt for 2 h monitored by
TLC (CH2Cl2/MeOH 60:1) until reaction was completed. The mixture was poured into
ice-water and extracted with EtOAc several times. After removal of organic solvent
under reduced pressure, crude product was purified on a flash column chromatograph
(gradual eluant: MeOH/CH2Cl2, 0-4.5%) with a CombiFlash separation system to obtain
pure target compound.
(E)-N1-(4’-Cyanophenyl)-5-(2”,6”-dimethyl-4”-(3-oxobut-1-enyl)pheno
xy)-4-trifluoromethylbenzene-1,2-diamine (13a). Starting with 495 mg (1 mmol)
of 11a to afford 316 mg of 13a, yield 68%, brown solid, mp > 300 °C; 1H NMR δ ppm
2.14 (6H, s, 2 × CH3), 2.37 (3H, s, COCH3), 5.71 (1H, s, NH), 6.21 (1H, s, ArH-6), 6.64
(2H, d, J = 8.8 Hz, ArH-2’,6’), 6.65 (1H, s, J = 16.4 Hz, =CH), 7.16 (1H, s, ArH-3), 7.27
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(2H, s, ArH-3”,5”), 7.42 (2H, d, J = 8.8 Hz, ArH-3’,5’), 7.43 (1H, s, J = 16.4Hz, CH=);
MS m/z (%) 466.2 (M+1, 100); HPLC purity 97.9%.
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(E)-N1-(4’-Cyanophenyl)-5-(4”-cyanovinyl-2”,6”-dimethylphenoxy)-4-t
rifluoromethylbenzene-1,2-diamine (14a). Starting with 478 mg (1 mmol) of 12a
to afford 112 mg of 14a, yield 25%, white solid, mp 224-226 °C; 1H NMR δ ppm 2.15
(6H, s, 2 × CH3), 5.68 (1H, s, NH), 5.80 (1H, d, J = 16.8 Hz, =CH), 6.18 (1H, s, ArH-6),
6.63 (2H, d, J = 8.8 Hz, ArH-2’,6’), 7.16 (1H, s, ArH-3), 7.18 (2H, s, ArH-3”,5”), 7.27
(1H, d, J = 16.8 Hz, CH=), 7.43 (2H, d, J = 8.8 Hz, ArH-3’,5’); MS m/z (%) 449 (M+1,
100); HPLC purity 97.3%.
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(E)-N1-(4’-Cyanophenyl)-5-(4”-cyanovinyl-2”,6”-dimethylphenoxy)-4-
sulfamoylbenzene-1,2-diamine (14b). Starting with 489 mg (1 mmol) of 12b to
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afford 184 mg of 14b in 40% yield, brown solid, mp 135-137 °C; H NMR (DMSO-d6)
δ ppm 2.14 (6H, s, 2 × CH3), 4.59 (2H, s, NH2), 6.37 (1H, d, J = 16.8 Hz, =CH), 6.78
(1H, s, ArH-6), 6.93 (1H, d, J = 16.8 Hz, CH=), 6.95 (2H, d, J = 8.8 Hz, ArH-2’,6’),
7.30 (2H, s, ArH-3”,5”), 7.61 (2H, d, J = 8.8 Hz, ArH-3’,5’), 8.20 (1H, s, ArH-3); MS
m/z (%) 460.3 (M+1,100); HPLC purity 95.8%.
(E)-N1-(4’-Cyanophenyl)-5-(4”-cyanovinyl-2”,6”-dimethylphenoxy)-4-
methoxycarbonylbenzene-1,2-diamine (14c). Starting with 468 mg (1 mmol) of
12c to afford 280 mg of 14c, yield 64%, brown solid, mp 240-242 °C. 1H NMR δ ppm
2.16 (6H, s, 2 × CH3), 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-2’,6’), 7.17 (2H, s, ArH-3”,5”),
7.31 (1H, d, J = 16.8 Hz, CH=), 7.42 (2H, d, J = 8.8 Hz, ArH-3’,5’), 7.45 (1H, s, ArH-3);
MS m/z (%) 439.3 (M+1, 100); HPLC purity 96.6%.
(E)- 4-Carboxy-N1-(4’-cyanophenyl)-5-(4”-cyanovinyl-2”,6”-dimethyl
phenoxy)benzene-1,2-diamine (14d). Starting with 454 mg (1 mmol) of 12d to
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afford 148 mg of 14d, yield 35%, brown solid, mp 226-228 °C; 1H NMR δ ppm 2.19
(6H, s, 2 × CH3), 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-2’,6’), 7.22 (2H, s, ArH-3”,5”), 7.32 (1H, d, J = 16.8 Hz,
CH=), 7.44 (2H, d, J = 8.8 Hz, ArH-3’,5’), 7.72 (1H, s, ArH-3); MS m/z (%) 423.2 (M-1,
100); HPLC purity 95.6%.
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(E)-4-Carbamoyl-N1-(4’-cyanophenyl)-5-(4”-cyanovinyl-2”,6”-dimethy
lphenoxy)benzene-1,2-diamine (14e). Starting with 453 mg (1 mmol) of 12e to
1
afford 266 mg of 14e, yield 63%, white solid, mp 290-292 °C; H NMR (DMSO-d6) δ
ppm 2.10 (6H, s, 2 × CH3), 4.76 (2H, s, NH2), 6.00 (1H, s, ArH-6), 6.39 (1H, d, J = 16.4
Hz, =CH), 6.63 (2H, d, J = 8.8 Hz, ArH-2’,6’), 7.47 (2H, s, ArH-3”,5”), 7.47 (2H, d, J =
8.8 Hz, ArH-3’,5’), 7.56 (1H, d, J = 16.4 Hz, CH=), 7.60 (1H, s, ArH-3), 8.20 (1H, s,
NH); MS m/z (%) 424.2 (M+1, 100); HPLC purity 98.2%.
(E)-N1-(4’-Cyanophenyl)-5-(4”-cyanovinyl-2”,6”-dimethylphenoxy)-4-
methylcarbamoylbenzene-1,2-diamine (14f). Starting with 467 mg (1 mmol) of
1
12f to afford 135 mg of 14f, yield 31%, brown solid, mp 112-114 °C; H NMR δ ppm
2.16 (6H, s, 2 × CH3), 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-2’,6’), 7.21 (2H, s, ArH-3”,5”),
7.32 (1H, d, J = 16.8 Hz, CH=), 7.41 (2H, d, J = 8.8 Hz, ArH-3’,5’), 7.81 (1H, s, ArH-3);
MS m/z (%) 438.4 (M+1, 100); HPLC purity 100.0%.
(E)-N1-(4’-Cyanophenyl)-5-(4”-cyanovinyl-2”,6”-dimethylphenoxy)-4-
hydroxymethylbenzene-1,2-diamine (14h). Starting with 440 mg (1 mmol) of 12h
to afford 332 mg of 14h, yield 81%, brown solid, mp 186-188 °C. 1H NMR δ ppm 2.13
(6H, s, 2 × CH3), 4.87 (2H, s, ArCH2O), 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-2’,6’), 6.94 (1H, s, ArH-3),
7.17 (2H, s, ArH-3”,5”), 7.30 (1H, d, J = 16.8 Hz, CH=), 7.40 (2H, d, J = 8.8 Hz,
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