Molecular Hybridization-Inspired Optimization of Diarylbenzopyrimidines as HIV-1 Nonnucleoside Reverse Transcriptase Inhibitors with Improved Activity against K103N and E138K Mutants and Pharmacokinetic Profiles

Article abstract of DOI:10.1021/acsinfecdis.9b00229

Molecular hybridization is a powerful strategy in drug discovery. A series of novel diarylbenzopyrimidine (DABP) analogues were developed by the hybridization of FDA-approved drugs etravirine (ETR) and efavirenz (EFV) as potential HIV-1 nonnucleoside reverse transcriptase inhibitors (NNRTIs). Substituent modifications resulted in the identification of new DABPs with the combination of the strengths of the two drugs, especially compound 12d, which showed promising activity toward the EFV-resistant K103N mutant. 12d also had a favorable pharmacokinetic (PK) profile with liver microsome clearances of 14.4 μL/min/mg (human) and 33.2 μL/min/mg (rat) and an oral bioavailability of 15.5% in rat. However, its activity against the E138K mutant was still unsatisfactory; E138K is the most prevalent NNRTI resistance-associated mutant in ETR treatment. Further optimizations resulted in a highly potent compound (12z) with no substituents on the phenyl ring and a 2-methyl-6-nitro substitution pattern on the 4-cyanovinyl-2,6-disubstitued phenyl motif. The antiviral activity of this compound was much higher than those of ETR and EFV against the WT, E138K, and K103N variants (EC₅₀ = 3.4, 4.3, and 3.6 nM, respectively), and the cytotoxicity was decreased while the selectivity index (SI) was increased. In particular, this compound exhibited acceptable intrinsic liver microsome stability (human, 34.5 μL/min/mg; rat, 33.2 μL/min/mg) and maintained the good PK profile of its parent compound EFV and showed an oral bioavailability of 16.5% in rat. Molecular docking and structure-activity relationship (SAR) analysis provided further insights into the binding of the DABPs with HIV-1 reverse transcriptase and provided a deeper understanding of the key structural features responsible for their interactions.

Full text of DOI:10.1021/acsinfecdis.9b00229

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Article
Molecular Hybridization-Inspired Optimization of Diarylbenzopyrimidines
as HIV-1 Nonnucleoside Reverse Transcriptase Inhibitors with Improved
Activity against K103N, E138K Mutants, and Pharmacokinetic Profiles
Sheng Han, Yali Sang, Yan Wu, Yuan Tao, Christophe
Pannecouque, Erik De Clercq, Chunlin Zhuang, and Fener Chen
ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.9b00229 • Publication Date (Web): 10 Oct 2019
Downloaded from pubs.acs.org on October 13, 2019
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ACS Infectious Diseases
Molecular Hybridization-Inspired Optimization of Diarylbenzopyrimidines as
HIV-1 Nonnucleoside Reverse Transcriptase Inhibitors with Improved Activity
against K103N, E138K Mutants, and Pharmacokinetic Profiles
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Sheng Han,†,‡ Yali Sang,†,‡ Yan Wu,†,‡ Yuan Tao,†,‡ Christophe Pannecouque,§ Erik De Clercq,§ Chunlin
Zhuang,*,†,‡ and Fen-Er Chen *,†,‡
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†Engineering Center of Catalysis and Synthesis for Chiral Molecules, Department of Chemistry, Fudan
University, Shanghai 200433, People's Republic of China
‡Shanghai Engineering Center of Industrial Asymmetric Catalysis for Chiral Drugs, Shanghai 200433, People's
Republic of China
§Rega Institute for Medical Research, KU Leuven, Herestraat 49, B-3000 Leuven, Belgium
Corresponding Authors: *(F.C.). E-mail: rfchen@fudan.edu.cn.; (C.Z.): zclnathan@163.com.
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Molecular hybridization is a powerful strategy in drug discovery. A series of novel diarylbenzopyrimidine (DABP)
analogues were developed by the hybridization of FDA-approved drugs etravirine (ETR) and efavirenz (EFV) as
potential HIV-1 nonnucleoside reverse transcriptase inhibitors (NNRTIs). Substituent modifications resulted in
the identification of new DABPs with the combination of the strengths of the two drugs, especially compound
12d, which showed promising activity toward the EFV-resistant K103N mutant. 12d also had a favorable
pharmacokinetic (PK) profile with liver microsome clearances of 14.4 µL/min/mg (human) and 33.2 µL/min/mg
(rat) and an oral bioavailability of 15.5% in rat. However, its activity against the E138K mutant was still
unsatisfactory; E138K is the most prevalent NNRTI resistance-associated mutant in ETR treatment. Further
optimizations resulted in a highly potent compound (12z) with no substituents on the phenyl ring and a 2-methyl-
6-nitro substitution pattern on the 4-cyanovinyl-2,6-disubstitued phenyl motif. The antiviral activity of this
compound was much higher than those of ETR and EFV against the WT, E138K and K103N variants (EC50 =
3.4, 4.3 and 3.6 nM, respectively), and the cytotoxicity was decreased while the selectivity index (SI) was
increased. In particular, this compound exhibited acceptable intrinsic liver microsome stability (human, 34.5
µL/min/mg; rat, 33.2 µL/min/mg) and maintained the good PK profile of its parent compound EFV and showed
an oral bioavailability of 16.5% in rat. Molecular docking and structure−activity relationship (SAR) analysis
provided further insights into the binding of the DABPs with HIV-1 reverse transcriptase and provided a deeper
understanding of the key structural features responsible for their interactions.
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Keywords: Molecular Hybridization; Diarylbenzopyrimidines; HIV-1; Reverse Transcriptase; NNRTI; PK
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ACS Infectious Diseases
According to the 2017-WHO report, approximately 36.9 million people were infected with human
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immunodeficiency virus (HIV) worldwide, 1.8 million people were newly infected with HIV, and 0.94 million
people died.1 Acquired immune deficiency syndrome (AIDS), one of the most devastating infectious diseases that
has not been eradicated, still requires potent antiretroviral therapy agents to reduce the death toll related to human
immunodeficiency virus type 1 (HIV-1), the etiological agent of AIDS.
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Currently, therapies against AIDS are based on several classes of anti-HIV drugs that target different steps
of the viral life cycle:2 fusion inhibitors (FIs), the coreceptor antagonist (maraviroc), integrase strand transfer
inhibitors (INSTIs), protease inhibitors (PIs), and nucleoside and nonnucleotide reverse transcriptase inhibitors
(NRTIs and NNRTIs). NNRTIs are key drugs in highly active antiretroviral therapy (HAART).3 However, the
effectiveness of NNRTIs has been significantly reduced because of the rapid development of resistance in a
growing number of mutant viral strains.4-7 Efavirenz (EFV, 1) is an early generation NNRTI. In the Gilead 934
study, 84% of EFV resistance possessed the K103N mutation.8 It is reported to be ineffective against the K103N
variant.9-11 Therefore, the K103N was the most prevalent NNRTI resistance-associated mutation (RAM) in EFV
treatments.12-14
CN
CN
CN
CN
NH
O
O
O
N
NH
F3C
NH
HN
N
N
Br
Cl
N
NH2
1 (Efavirenz)
2 (Etravirine)
3 (Rilpivirne)
Figure 1. The chemical structures of efavirenz, etravirine and rilpivirine
Diarylpyrimidine (DAPY) analogues are one of the most potent families of NNRTIs developed thus far.
Etravirine (ETR, 2) and rilpivirine (RPV, 3), representative DAPYs approved by the FDA, exhibit high anti-viral
activity against wild-type (WT) and the K103N mutation.15 This phenomenon may be caused by the fact that
DAPY inhibitors bind to the nonnucleoside reverse transcriptase inhibitor binding pocket (NNIBP) to form a
flexible “horseshoe” conformation, minimizing the loss of binding stabilization.16-19 However, E138K, the most
prevalent NNRTI RAM, is recognized to be the reason for the failure of RPV or ETR treatments.20-26 About 77%
patients failed with RPV treatments due to the E138K mutation (NCT00543725).12 Besides, the oral
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bioavailability or PK properties is another important aspect to limit the efficacy of NNRTIs.27-29 The absolute oral
bioavailability of ETR in human is still unknown probably due to its bad solubility and lack of an intravenous
formulation.30-32 Thus, finding novel NNRTIs to overcome drug resistance and improved PK profiles is of great
importance.
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enhance hydrophobic interactions
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CN
CN
CN
O
CN
NH
A
B
N
optimization 1
R2
N
R1
optimization
HN
O
O
H
N
F3C
C
H- bond with E138(K)
N
SAR
NH
D
N
Br
Molecualr
Hybridation
X
Cl
NH2
optimization 2
Figure 2. The optimization strategy of DABPs
In our previous work, we reported that several DABP hybrids based on ETR and DPC083 showed promising
HIV-1 inhibitory activities with low cytotoxicities and high selectivity indexes (SI).33 In this study, we used a
molecular hybridization strategy on the basis of ETR and EFV to address the drawbacks of two generations of
NNRTIs, improve the potency of DABPs against the K103N and E138K mutants, expand the SARs and improve
the PK profiles. These structural optimizations focused on three important factors. (1) Conversion of the ether
linkage between rings B and C to an amino group to allow the formation of additional water-mediated hydrogen
bonds with the carbonyl of E138. (2) Insertion of a vinyl group between the cyano group and ring B to extend the
π−π stacking interactions with the highly conserved amino acid W229 around the hydrophobic tunnel in RT. (3)
Various substituents with different sizes and electronic properties were introduced on ring B and ring D.
RESULTS AND DISCUSSION
Chemistry
The synthetic route to the desired quinazoline diamines is outlined in Scheme 1. Briefly, the first set of key
intermediates (6a-f) were synthesized via a well-established two-step procedure.34-38 In the meantime, ortho-nitro
benzoic acids 7a-f were catalytically hydrogenated to obtain anthranilic acids 8a-f, which were subsequently
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treated with urea to afford quinazolinediones 9a-f.33, 39 9a-f were then reacted with phosphorus oxychloride in the
presence of N,N-diisopropylethylamine, leading to the formation of 2,4-dichloroquinazolines 10a-f.40 Without
purification, key intermediates (10a-f) were immediately reacted with aromatic amines 6a-f under Pd catalysis38,
41, 42 to yield 4-substituted-2-chloroquinazolines 11a-z.43-47 Finally, 11a-z were reacted with 4-aminobenzonitriles
to give target quinazoline diamines 12a-z.48
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Scheme 1. Synthesis of quinazoline diamines 12a-z
6a
6b
6c
6d
6e
6f
R1 = R2 = Cl
R1 = R2 = F
R1= Me, R2= F
R1= Me, R2= NO2
R1= R2 = Me
R1= F, R2 = Cl
R1
R1
R1
a
b
H2N
R2
H2N
R2
H2N
R2
71-88%
56-85%
I
CN
4a-f
5a-f
6a-f
10a
10b
10c
10d
10e
10f
X= H
X= 6-F
X= 6-Cl
X= 6-methoxy
X= 7-Cl
O
O
OH
Cl
5
4
c
d
e
3
N
6
X
OH
NO2
OH
NH2
N
X
X
X
7
N
OH
N
1
Cl
2
X= 6-NO2
8
7a-f
8a-f
9a-f
10a-f
N
Cl
N
NH
X
X
N
N
f
g
R1
R1
CN
HN
R2
HN
R2
CN
CN
11a-z
12a-z
Reagents and conditions: (a) NaHCO3, iodine chloride, methanol, dichloromethane, room temperature, 6 h; (b)
acrylonitrile, 10% Pd/C wet, P(o-Tol)3, sodium acetate, tetrabutylammonium bromide, N,N-dimethylacetamide,
140 oC, 12 h; (c) 10% Pd/C wet, hydrogen atmosphere (balloon), ethanol, reflux, 24 h; (d) urea, 180 oC, 3 h; (e)
POCl3, N,N-diisopropylethylamine, reflux, 6 h; (f) (E)-3-(4-amino-3,5-disubstituted)acrylonitrile, palladium
acetate, DavePhos, K3PO4, N,N-dimethylacetamide, 140 oC, 12 h; (g) 4-aminobenzonitrile, n-butanol, reflux, 6-8
h.
Molecular hybridization of ETR and EFV leads to promising antiviral activity with a good combination of
their strengths.
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Based on the modeling study, the chlorophenyl group of EFV was introduced as ring D by molecular
hybridization (Figure 2). Four novel 6-chloride quinazoline diamines (12a-d) with different substituents on ring
B were synthesized. They maintained the activities of the parent compounds (EFV and ETR) against WT HIV-1.
The activity of compound 12a against K103N was ~5-fold greater than that of EFV. Next, we replaced the
chloride on ring B with a fluoride, as it might improve the potency and other properties, such as conformational
restriction, lipophilicity and metabolic stability.49 Compound 12b, with two fluoride substituents on ring B,
showed the best antiviral potency against WT HIV and moderate cytotoxicity, but it was not the most active
compound in this series against the K103N mutant strain. Replacement of one fluoride with a methyl group, which
should improve the conformation, on ring B (compound 12c) significantly improved activity against the K103N
mutant virus, and the activities against WT HIV and the E138K were maintained. Then, a nitro group was
introduced as it is considered to be a versatile and unique functional group with various therapeutic effects.50-52
When compared with 12c and EFV, compound 12d had similar potencies against WT HIV, the E138K and K103N
mutants.
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Table 1. Activity of 12a-d against HIV-1 IIIB, E138K, K103N strains and cytotoxicity in MT-4 cells.a
EC50b (nM)
E138K
SId
IIIB
CC50c (nM)
Compound
12a
X
R1
-Cl
R2
-Cl
-F
HIV-1 IIIB
13.6 ± 9.1
9.6 ± 2.8
10.3 ± 3.1
10.6 ± 10.0
3.9 ± 1.4
3.2 ± 1.4
1
K103N
29.4 ± 0.2
42.3 ± 10.5
12.1 ± 3.5
10.2 ± 0.8
3.5 ± 0.9
147.4 ± 85.9
1
6-Cl
6-Cl
6-Cl
6-Cl
41.6 ± 16.8
13.1 ± 0.2
11 ± 0.2
5264 ± 1348
12435 ± 5079
9200 ± 1514
3954 ± 1154
>4620
390
12b
-F
1301
890
12c
-CH3
-F
12d
-CH3 -NO2
17.7 ± 2.7
10.9 ± 7.4
5.4 ± 1.3
6
375
ETR
EFV
>1165
>2000
3989
> 40196
e
>3980
RPV
aAll data represent the mean values for at least three independent experiments.
bEC50: The effective concentration required to protect MT-4 cells against virus-induced cytopathicity by 50%.
cCC50: The cytotoxic concentration of the compound that reduces the normal uninfected MT-4 cell viability by
50%.
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dSI: selectivity index, ratio of CC50/EC50 (WT).
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eThe data were obtained from the same laboratory (Prof. Erik De Clercq, Rega Institute for Medical Research,
KU Leuven, Belgium) with the same method.53
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Table 2. Rat PK profile and liver microsome stability of 12d a
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Rat PK profile
Liver microsome
stability
Parameter
AUCall (ng*h/mL)
AUCinf (ng*h/mL)
MRTinf (h)
5.0 mg/kg (p.o.)
10.0 mg/kg (p.o.)
1183 ± 75
1.0 mg/kg (i.v.)
983 ± 131
991 ± 131
3.10 ± 0.21
4.02 ± 0.37
—
the clearance
756 ± 129
768 ± 129
6.61 ± 0.70
3.95 ± 1.00
1.0 ± 0.0
rate of rat
1198 ± 71
30.6
14.4
(μL/min/mg)
7.45 ± 1.78
3.15 ± 0.40
1.33 ± 0.58
133 ± 100
t1/2 (h)
the clearance
rate of human
(μL/min/mg)
Tmax (h)
Cmax (ng/mL)
F (%)
128 ± 74
—
15.5 ± 2.6
12.1 ± 0.7
—
aPK parameters (mean ± SD, n = 3).
Next, we examined the drug-like properties of the new DABP hybrids, and compound 12d was selected for
its promising antiviral potency against both WT and mutant strains. The liver microsome stability and rat PK
profile of compound 12d were examined. The intrinsic rat and human microsome clearances of 12d were 30.6
and 14.4 μL/min/mg proteins, respectively (Table 2), showing the property of moderate clearance (classification:
human, low < 8.6 μL/min/mg, high > 47.0 μL/min/mg; rat, low < 13.2 μL/min/mg, high > 71.9 μL/min/mg).54
The PK results showed that compound 12d was absorbed with short Tmax values (1 h and 1.33 h), favorable half-
lives (3.95 h and 3.15 h), and mean residence times (MRTs) of 6.61 h and 7.45 h when orally administered at two
doses of 5 mg/kg and 10 mg/kg, respectively. The Cmax values were 128 and 133 ng/mL. Moreover, the oral
bioavailabilities (F) were calculated to be acceptable values of 15.5% and 12.1%, respectively, which were
comparable to that of EFV (16%).55 A single-dose toxicity test of compound 12d was carried out in rats. After
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intragastric administration of 12d at a dose of 183 mg/kg body weight, no mortality was observed in the rats, and
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Further optimizations indicate an SAR of DABP hybrids and provide derivatives with much higher
antiviral activities against WT, E138K and K103N variants and improved PK.
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Encouraged by the results of 6-chloride hybrids 12a-d, we further replaced the chloride with different
functional groups (e.g., fluorine, methoxy, hydroxy, and nitro groups) to establish the SAR of the DABP hybrids.
This region of E138 was tolerant of substitutions, and these distinct substitution patterns could potentially affect
the mutant variant (Figure 2).
First, 6-fluorine quinazoline diamines 12e-i were synthesized. They showed significantly improved
potencies against the E138K and K103N mutant strains when compared with their corresponding chlorinated
analogues (12a-d). Compound 12f had better or comparable activities against WT HIV-1, E138K and K103N
mutant strains (EC50 = 4.6, 4.6, and 5.0 nM, respectively) relative to those of ETR and EFV, and it showed a high
SI value (SI = 3924).
In addition to fluorine and chlorine, electron-donating groups (e.g., methoxy and hydroxy groups) were
introduced to afford compounds 12j-l. Unfortunately, although the cytotoxicity was much lower, this series
showed decreased activities toward the WT HIV-1, E138K and K103N mutant strains. Then, the nitro group was
similarly introduced at the C6 position of ring D, affording compound 12n. Compound 12n showed a 2-fold
decrease in activity against the HIV-1 WT strain (EC50 = 9.6 nM) compared with its corresponding 6-fluorinated
analogue 12g (EC50 = 5.3 nM), and its activity was comparable to that of its corresponding 6-chloride analogue
12b (EC50 = 9.6 nM). Remarkably, the activity of 12n against the E138K mutant strain was greater (EC50 value
of 5.8 nM) than those of 12b (EC50 = 13.1 nM) and ETR (EC50 = 10.9 nM). Moreover, introducing a nitro group
into a drug molecule should be careful due to associated toxicity issues.50 Surprisingly, the cytotoxicity of 12n
was determined, and it showed the highest CC50 and SI values (CC50 >216 μM, SI= 22498) of all compound in
this series. Compound 12n showed moderate activity against the K103N mutant strain, and its EC50 was higher
than that of EFV.
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Table 3. Activity of 12e-z against HIV-1 IIIB, E138K, K103N strains and cytotoxicity in MT-4 cells.a
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EC50b (nM)
SId
IIIB
CC50c (nM)
Compound
12e
X
6-F
R1
-Cl
-F
R2
-Cl
-Cl
-F
HIV-1 IIIB
8.4 ± 9.2
4.6 ± 4.6
5.0 ± 0.9
3.9 ± 1.1
2.6 ± 0.6
10.5 ± 6.0
19.1 ± 14.1
9.0 ± 4.6
161 ± 108
9.6± 3.6
8.7 ± 3.7
11.8 ± 8.2
7.8 ± 2.8
9.9 ± 5.9
5.0 ± 3.1
15.9 ± 5.5
10.3 ± 4.1
3.7± 0.9
2.5 ± 1.1
4.7 ± 1.7
10.5 ± 5.5
3.4 ± 0.9
3.9 ± 1.4
3.2 ± 1.4
1
E138K
17.9 ± 2.1
4.6 ± 1.3
5.3
K103N
10.1 ± 2.3
5.0 ± 0.2
29.6 ± 4.1
9.8 ± 0.9
4.7 ± 0.2
11.0 ± 0.7
27.9 ± 5.1
24.6 ± 12.5
131.4 ± 2.5
38.8 ± 14.9
11.2 ± 1.0
7.8 ± 1.3
7.2 ± 2.2
9.2 ± 0.9
19.6 ± 3.7
45.5 ± 4.0
10.8 ± 1.7
4.6 ± 0.2
5.2 ± 0.2
11.3 ± 1.9
5.0 ± 0.5
3.6 ± 0.9
3.5 ± 0.9
147.4 ± 85.9
1
3373 ± 1394
17623 ± 1757
8407 ± 1532
7574 ± 1265
5070 ± 1036
94347 ± 58197
7319 ± 1066
12273 ± 6598
>283750
405
12f
6-F
3924
1730
1909
1925
9000
383
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12g
6-F
-F
12h
12i
6-F
-CH3
-F
8.7 ± 0.2
6.0 ± 1.5
28.0 ± 12.5
10.0 ± 4.3
19.6 ± 10.3
76.0 ± 5.9
5.8 ± 1.5
14.2 ± 2.6
10.3 ± 2.3
6.1 ± 0.2
12.1 ± 0.2
15.6 ± 5.2
32.7 ± 2.4
24.7 ± 13.7
6.6 ± 0.9
4.8 ± 1.1
8.7 ± 8.3
7.1 ± 1.0
4.3 ± 1.3
10.9 ± 7.4
5.4 ± 1.3
6
6-F
-CH3 -NO2
-CH3 -CH3
12j
6-OCH3
6-OCH3
6-OCH3
6-OH
6-NO2
7-Cl
7-Cl
7-Cl
7-Cl
7-Cl
7-Cl
-H
12k
12l
-Cl
-F
-Cl
-F
1355
>1763
22498
456
12m
12n
12o
-F
-F
-F
-F
216838 ± 39782
4009 ± 757
52920 ± 20792
144257 ± 65189
9108 ± 1100
9227 ± 2762
9105 ± 1348
25238 ± 9163
6436 ± 2729
7918 ± 1332
9891 ± 3932
6854 ± 488
6138 ± 1897
>4620
-Cl
-F
-Cl
-Cl
-F
12p
12q
12r
4520
18510
920
-F
-CH3
-F
12s
-CH3 -NO2
-CH3 -NH2
-CH3 -CH3
1888
569
12t
12u
12v
2457
1781
3100
2096
657
-H
-Cl
-F
-Cl
-Cl
-F
12w
12x
-H
-H
-F
12y
-H
-CH3
-F
12z
-H
-CH3 -NO2
1827
>1165
>2000
3989
ETR
EFV
RPV
> 40196
>3980
aAll data represent the mean values for at least three independent experiments.
bEC50: The effective concentration required to protect MT-4 cells against virus-induced cytopathicity by 50%.
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cCC50: The cytotoxic concentration of the compound that reduces the normal uninfected MT-4 cell viability by
50%.
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6
dSI: selectivity index, ratio of CC50/EC50 (WT).
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9
Third, moving the chloride to the C7 position placed it still close to residue E138, and the pocket might be
tolerant of an additional substituent (Figure 2). On the basis of the results of the 6-substituted analogues, an
electron-withdrawing chloride was introduced at the C7 position, yielding compounds 12o-s. In addition to good
potency against WT HIV-1, all compounds in this series exhibited low nanomolar potencies against E138K and
K103N mutant strains (EC50 values ranging from 6.1 to 15.6 nM and 7.2 to 19.6 nM, respectively). Remarkably,
compound 12q showed better or comparable activity (EC50= 6.1 and 7.2 nM, respectively) against the E138K and
K103N mutant strains relative to those of ETR and EFV as well as high CC50 (=144 μM) and SI (18510) values.
The reduction of the nitro group of 12s afforded compound 12t, which showed 2 to 3-fold lower potency against
the WT, E138K and K103N mutant strains.
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Finally, we removed the substituent on ring D to further elucidate the SAR. Compounds 12u-z were
synthesized on the basis of representative substituents on ring D in the 12a-t series. Fortunately, all the compounds
exhibited excellent potency against WT HIV-1 (EC50 = 2.5–10.5 nM), the E138K mutant strain (EC50 = 4.3–8.7
nM) and the K103N mutant strain (EC50 = 3.6–11.3 nM), except for 12u. Compound 12u, with a 2,6-dimethyl
substitution pattern, had activity comparable to those of corresponding compound 12j against WT, E138K and
K103N mutant strains (EC50 = 10.3 nM, 24.7 and 10.8 nM, respectively). When changing the chloride or fluoride
at the ortho position of ring B (12v-y), the activity was improved, and activities better than or comparable to those
of the drugs ETR or EFV against E138K and K103N were observed. Considering the promising activities of 12i,
12n and 12s, the 2-methyl-6-fluoro motif was then replaced by a 2-methyl-6-nitro pattern on ring B to afford
compound 12z. This compound showed a great combination of the strength of the two drugs (ETR and EFV).
Compound 12z was 2.5-fold more potent than ETR against E138K and was 41-fold more potent than EFV against
K103N. The EC50 value of 12z against WT HIV-1 was 3.4 nM relative to 3.9 nM for ETR and 3.2 nM for EFV.
As they showed the best anti-E138K and K103N activities, compounds 12d, 12f, 12n, 12q, 12w and 12z
were selected for further evaluation against the HIV-1 mutant RT enzyme (Table 4). They exhibited activities
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better or comparable to those of ETR and EFV at the enzymatic level, with IC50 values of 7.0 to 18.1 nM, showing
a good correlation (R2 = 0.95) with their antiviral activities in MT-4 cells (Figure S2). The time-of-addition
experiment was also performed to support that RT is the main and exclusive target of these compounds. 56 The
results clearly indicated our compounds acting as the NNRTI-type mode of action and better than the reference
compounds NVP and ETR (Figure S3).
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Table 4. The activity of selected compounds against WT HIV-1 RT enzyme.a
Compound
IC50 (nM)
12d
12f
12n
12q
12w
12z
ETR
EFV
17.7 ± 0.8
11.3 ± 0
18.1 ± 0.9
12.4 ± 1.5
7.0 ± 0.3
13.7 ± 1.6
11 ± 0
12.7 ± 9.5
aIC50: inhibitory concentration of test compound required to inhibit WT HIV-1 RT polymerase activity by 50%.
Compounds 12n and 12z were then selected for PK studies based on their structural features (containing a
nitro group), marked antiviral activities against WT and mutant strains, and/or high SI. Unfortunately, the oral
bioavailability (F) of compound 12n was calculated to be ~ 2% at 5 mg/kg (see the Supporting Information Table
S1). This result may be attributable to the nitro group at the 6-position of the quinazoline core exhibiting an
electron-withdrawing effect and making the π-electron system more electron deficient, leading to its metabolic
instability57 and poor bioavailability. Fortunately, the oral bioavailability (F) of 12z, with no substituents on ring
D, was significantly improved to 16.5% at a dose of 5 mg/kg in rats. The intrinsic rat and human microsome
clearances of 12z were 33.2 and 34.5 μL/min/mg proteins, respectively (Table 5). The PK study and safety
assessment of 12z showed that it was absorbed with mean residence times (MRTs) of 11.8 h and 11.4 h at these
two doses. The Cmax of 12z was 39.9 ng/mL at a dose of 5 mg/kg. A single-dose toxicity test of compound 12z in
rats showed no mortality, and there was no abnormal body weight decrease in the animals in the week following
an intragastrical dose at 293 mg/kg body weight (Figure S1). The above results indicate that compound 12z could
be an orally bioavailable candidate for treating human HIV-1 infection.
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Table 5. Rat PK profile and liver microsome stability of 12z a
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3
4
5
6
7
8
9
Rat PK profile
5.0 mg/kg (p.o.)
39.9 ± 12.5
—
Liver microsome
stability
Parameter
AUCall (ng*h/mL)
AUCinf (ng*h/mL)
MRTinf (h)
1.0 mg/kg (i.v.)
48.4 ± 9.8
50.4 ± 9.7
0.65 ± 0.15
3.26 ± 1.37
—
the clearance
rate of rat
33.2
34.5
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(μL/min/mg)
11.8 ± 4.7
—
t1/2 (h)
the clearance
rate of human
(μL/min/mg)
Tmax (h)
13.3 ± 11.0
4.16 ± 0.31
16.5 ± 5.2
Cmax (ng/mL)
F (%)
—
—
aPK parameters (mean ± SD, n = 3).
Molecular modeling analysis provides further insights into the binding of the DABPs to HIV-1 RT.
Compounds 12d, 12n and 12z were selected for the molecular modeling study to predict their binding modes
with RT and further clarify their activities. The E138K or K103N mutated enzyme was generated and its energy
was minimized using BioLuminate. The docking study was performed by Glide package using default parameters.
After docking study, Desmond molecular dynamics simulations was run using the docking poses with the best
Glidescore. The predicted results were then selected after 3 ns simulations with a suitable RMSD (~3 Å, see
supporting information). They showed several common features (Figure 3). (1) Consistent with previous
publications,58 the NH linkage of ring B and C formed a water-mediated hydrogen bonding interaction with the
carbonyl of E138. (2) The cyanovinyl group enhanced the interactions with Y181, Y188, F227, and W229 in the
modified hydrophobic tunnel.58 (3) The NH linker connecting the central pyridine ring and the cyano-phenyl ring
and the nitrogen of the pyrimidine formed hydrogen bonds with the backbone of K101.59, 60 The distance between
the 6-chloride substituent of compound 12d and E138/K138 was very short (Figure 3A, 3D and 3G). Compound
12n, with a 6-nitro group, formed an additional water-mediated hydrogen bond with the carbonyl of E138 (Figure
3B). The orientation of the new K138 was different due to the existence of the adjacent K101, making the space
between the residue and ring D more tolerated than it was in WT RT (Figure 3D, 3E and 3F). The water-mediated
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hydrogen bond was still observed in the mutant strain. The binding mode of 12n with RT explained its higher
activity against the E138K mutant than against the WT strain. Compound 12z had a binding mode similar to that
of 12n (Figure 3C and 3F). The 2-methyl-6-nitro and 2,6-difluro groups restricted the conformation of ring B,
making it more orientated to the new N103, thus resulting in a water-mediated hydrogen bond with the NH of
amide (the distance between nitro/ fluorine and hydrogen was 4-5 Å, Figure 3G, 3H and 3I).
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Figure 3 Predicted binding modes of 12d, 12n and 12z with the HIV-1 WT and E138K mutant RT crystal
structure. (A) WT with 12d (yellow); (B) WT with 12n (yellow); (C) WT with 12z (yellow); (D) E138K mutant
RT with 12d; (E) E138K mutant RT with 12n; (F) E138K mutant RT with 12z; (G) K103N mutant RT with 12d;
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(H) K103N mutant RT with 12n; (I) K103N mutant RT with 12z. The carbons of the compounds are depicted in
yellow. Residues involved in interactions are shown as green or gray sticks. Mutated residues are depicted as cyan
sticks. Hydrogen bonds are depicted as yellow dashed lines.
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Conclusion
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9
A series of novel quinazoline-2,4-diamine derivatives were designed by molecular hybridization and synthesized.
SAR studies led to the discovery of a number of HIV-1 inhibitors with nanomolar potencies that addressed the
drawbacks of ETR and EFV against two of the most prevalent NNRTI RAMs and PK profiles. The results of
biological evaluations highlight the promise of DABP compounds as potential drug candidates. (1) The new
DABPs represent a novel class of NNRTI showing potent inhibition of the HIV-1 WT strain (HIV-1 IIIB) not
only in cells but also with the free RT enzyme. (2) They are also active against HIV-1 E138K and K103N mutants
in the low nanomolar concentration range, acceptable SI values and low cytotoxicities as compared to ETR and
EFV. (3) Molecular modeling studies provided further insights into the allosteric binding of DABPs to HIV-1
WT and mutant RT. (4) They possess acceptable metabolic stabilities and good oral bioavailability for the
eventual clinical treatment of human HIV-1 infection. The best compound 12z exhibited EC50 values of 3.4, 4.3
and 3.6 nM against the WT, E138K and K103N variants, had acceptable intrinsic liver microsome stability
(human, 34.5 µL/min/mg; rat, 33.2 µL/min/mg), and showed an oral bioavailability of 16.5% in rat. The
compound could be a potential lead for further optimization in anti-RT drug discovery.
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Experimental section
1. Chemistry
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Chemical reagents and solvents, purchased from commercial sources, were of analytical grade and used without
further purification. All air-sensitive reactions were under a nitrogen atmosphere. All the reactions were
monitored by TLC on the pre-coated silica gel G plates at 254 nm under a UV lamp using ethyl acetate/n-hexane
as the eluent. Column chromatography was performed on glass column packed with silica gel (200-300 mesh)
using ethyl acetate/n-hexane as the eluent. Melting points were measured on a SGW X-1 microscopic melting
point apparatus. 1H NMR and 13C NMR spectra were recorded on a Bruker AV400 MHz spectrometer in DMSO-
d6. Chemical shifts were reported in δ (ppm) units relative to the internal standard tetramethylsilane (TMS). Mass
spectra and HRMS were obtained on a Waters Quattro Micromass instrument and Brukersolari X-70 FT-MS
instrument, respectively, using electrospray ionization (ESI) techniques. The purities of the target compounds
were ≥ 95%, measured by LC-MS, performed on an Waters 2695 system with UV detector and Synergi 4 μm
Hydro-RP 80 Å column (250×4.6 mm), eluting with a mixture of water (A) and methanol (B) (VA:VB=50:50 to
10:90) over 30 min with a flow rate of 1.0 mL/min and detection at 254 nm.
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1.1 General procedure to prepare the intermediates of 6a-f:
A mixture of various kinds of aniline (50 mmol, 1.0 eq), sodium bicarbonate (12.6 g, 150 mmol, 3.0 eq) and
methanol (70 mL) was stirred at room temperature. A solution of iodine chloride (8.93 g, 55 mmol, 1.1 eq) in
dichloromethane (70 mL) was added to the mixture dropwise in 20 minutes, then the solution was stirred at room
temperature for 6 h until GC-MS showed that the reaction was completed. The resulting mixture was filtered,
then the filtrate was concentrated. The residue was diluted with ethyl acetate, washed with an aqueous sodium
thiosulfate solution, dried with anhydrous sodium sulfate, filtered and concentrated to afford the crude 5a-f. The
crude product was used in the next step without further purification.
The crude 5a-f (50 mmol, 1.0 eq), acrylonitrile (5.30 g, 100 mmol, 2.0 eq)，P(o-tol)3 (1.52 g, 5 mmol, 0.1 eq),
tetrabutylammonium bromide (16.1 g, 50 mmol, 1.0 eq), 10% Pd/C wet (2.7 g, 0.05 eq), sodium acetate (4.93 g,
60 mmol, 1.2 eq) and N, N-dimethylacetamide (200 mL) was heated at 140 °C under nitrogen atmosphere for 12
h until GC-MS showed the reaction was completed. The resulting mixture was filtered, diluted with ethyl acetate
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and washed with brine. Then, it was dried over anhydrous sodium sulfate, concentrated and purified by
chromatography to obtain a white or pale yellow solid 6a-f of 42-75% yield in two steps.
1.2 General procedure to prepare the intermediates of 9a-f:
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3
4
5
6
7
8
9
Commercially available 2-nitrobenzoic acid 7a-f (177 mmol, 1.0 eq), 10% wet Pd/C (1.5 g, 0.05 eq) and ethanol
(300 mL) were refluxed under the atmosphere of hydrogen balloon reduction for 24 h. After TLC showed the
reaction was completed, the result mixture was filtered and concentrated to afford the crude reduction product
8a-f.
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A flask containing substituted anthranilic acids 8a-f (177 mmol, 1.0 eq) and urea (159 g, 2.65 mol, 15.0 eq) was
heated at 180 °C. After 3 h, the reaction mixture was cooled to 100 °C and an equal volume of water was added.
The obtained suspension was left to stir for another 30 min, after which it was cooled to room temperature. The
precipitate was filtered, washed with fresh water and dried in vacuum. Quinazoline-2,4-diones 9a-f were white
or pale yellow solids of 55-78% yield in two steps.
1.3
General procedure to prepare the intermediates of 11a~z:
Quinazoline-2,4-diones 9a-f (13 mmol, 1.0 eq), N,N-diisopropylethylamine (3.37 g, 26 mmol, 2.0 eq) and
phosphorus oxychloride (19.9 g, 130 mmol, 10 eq) were refluxed for 6 h to form 2,4-dichloroquinazolines. The
cooling mixtures were poured into ice-water before being stirred at room temperature. The resulting precipitates
were filtered, washed with fresh cold water and dried in vacuum to give products 10a-f, which were hygroscopic
and unstable in air and were used immediately.
(E)-3-(4-amino-3,5-disubstituted)acrylonitrile (6b-e, 10 mmol, 1.0 eq), the crude 2,4-dichloroquinazoline 10a-f
(13 mmol, 1.3 eq), palladium acetate (112 mg, 0.5 mmol, 0.05 eq), DavePhos (394 mg, 1 mmol, 0.1 eq), K3PO4
(6.37 g, 30 mmol, 3.0 eq) in N,N-dimethylacetamide (80 mL) were heated at 140 °C for 12 h under nitrogen
atmosphere. 4-Substituted-2-chloroquinazoline was purified by chromatography to obtain a white or pale yellow
solid (11b~d, 11g~j, 11l~n, 11q~u, 11u and 11x~z) of 21-43% yield in two steps.
(E)-3-(4-amino-3,5-disubstituted)acrylonitrile (6a and 6f, 10 mmol, 1.0 eq), the crude 2,4-dichloroquinazoline
10a-f (13 mmol, 1.3 eq), K3PO4 (6.37 g, 30 mmol, 3.0 eq) in N,N-dimethylacetamide (80 mL) were heated at 140
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°C for 12 h under nitrogen atmosphere. The 4-substituted-2-chloroquinazoline was purified by chromatography
1
2
3
to obtain a white or pale yellow solid (11a, 11e~f, 11k, 11o~p, and 11v~w) of 15-30% yield in two steps.
4
5
6
1.4
General procedure to prepare 12a~z:
7
8
9
4-Substituted-2-chloroquinazoline 11a~z (2 mmol, 1.0 eq), 4-aminobenzonitrile (4 mmol, 472 mg, 2.0 eq) in n-
butanol (5 mL) were refluxed for 6-8 h. The resulting precipitate was filtered off, washed with dichloromethane
(5 mL ×3) and dried in vacuum to obtain 12a~z as a white or pale yellow solid of 37-55% yield.
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1.5
2,6-difluoro-4-iodoaniline (5b). Synthesized following general procedure 1.1. Yield 83%, black solid. 1H
NMR (400 MHz, CDCl3) δ 7.21 – 7.08 (m, 2H, ArH), 3.76 (s, 2H, NH2).
1.6
solid. 1H NMR (400 MHz, CDCl3) δ 7.20 – 7.14 (m, 2H, ArH), 3.66 (s, 2H, NH2), 2.14 (s, 3H, CH3).
4-iodo-2,6-dimethylaniline (5e). Synthesized following general procedure 1.1. Yield 88%, black solid. 1H
2-fluoro-4-iodo-6-methylaniline (5c). Synthesized following general procedure 1.1. Yield 71%, black
1.7
NMR (400 MHz, CDCl3) δ 7.24 (s, 2H, ArH), 3.57 (s, 2H, NH2), 2.13 (s, 6H, CH3×2).
1.8 (E)-3-(4-amino-3,5-dichlorophenyl)acrylonitrile (6a). Synthesized following general procedure 1.1.
Yield 56%, white solid. 1H NMR (400 MHz, DMSO-d6) δ 7.62 (s, 2H, ArH), 7.42 (d, J = 16.6 Hz, 1H, olefinic
H), 6.26 (d, J = 16.6 Hz, 1H, olefinic H), 6.18 (s, 2H, NH2). 13C NMR (101 MHz, DMSO-d6) δ 148.94, 143.94,
128.19, 123.19, 119.72, 118.45, 93.52.
1.9
(E)-3-(4-amino-3,5-difluorophenyl)acrylonitrile (6b). Synthesized following general procedure 1.1.
Yield 65%, white solid. 1H NMR (400 MHz, DMSO-d6) δ 7.42 (d, J = 16.5 Hz, 1H, olefinic H), 7.36 – 7.26 (m,
2H, ArH), 6.21 (d, J = 16.5 Hz, 1H, olefinic H), 5.96 (s, 2H, NH2). 13C NMR (101 MHz, DMSO-d6) δ 152.10 (d,
JC-F = 10.0 Hz), 149.84 – 149.62 (m), 129.29 (t, JC-F = 16.9 Hz), 120.41 (t, JC-F = 8.9 Hz), 119.71, 111.35 (dd,
JC-F = 14.5, 7.2 Hz), 93.33.
1.10 (E)-3-(4-amino-3-fluoro-5-methylphenyl)acrylonitrile (6c). Synthesized following general procedure 1.1.
Yield 85%, pale yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 7.38 (d, J = 16.5 Hz, 1H, olefinic H), 7.28 (d,
JH-F = 12.3 Hz, 1H, ArH), 7.11 (s, 1H, ArH), 6.07 (d, J = 16.5 Hz, 1H, olefinic H), 5.63 (s, 2H, NH2), 2.12 (s, 3H,
CH3). 13C NMR (101 MHz, DMSO-d6) δ 150.45 (d, JC-F = 237.7 Hz), 150.71 (d, JC-F = 2.6 Hz), 138.37 (d, JC-F
=
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12.6 Hz), 127.00, 123.82 (d, JC-F = 4.8 Hz), 121.47 (d, JC-F = 7.8 Hz), 120.22, 111.93 (d, JC-F = 19.1 Hz), 91.18,
17.65 (d, JC-F = 3.0 Hz).
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1.11 (E)-3-(4-amino-3-methyl-5-nitrophenyl)acrylonitrile (6d). Synthesized following general procedure 1.1.
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Yield 59%, yellow solid. H NMR (400 MHz, DMSO-d6) δ 8.12 (s, 1H, ArH), 7.70 (s, 1H, ArH), 7.59 (s, 2H,
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NH2), 7.51 (d, J = 16.6 Hz, 1H, olefinic H), 6.22 (d, J = 16.6 Hz, 1H, olefinic H), 2.22 (s, 3H, CH3). 13C NMR
(101 MHz, DMSO-d6) δ 149.57, 146.63, 133.18, 130.75, 127.74, 125.77, 121.37, 119.73, 93.69, 18.50.
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1.12
(E)-3-(4-amino-3,5-dimethylphenyl)acrylonitrile (6e). Synthesized following general procedure 1.1.
Yield 69%, pale yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 7.31 (d, J = 16.4 Hz, 1H, olefinic H), 7.13 (s,
2H, ArH), 5.92 (d, J = 16.5 Hz, 1H, olefinic H), 5.31 (s, 2H, NH2), 2.08 (s, 6H, CH3×2). 13C NMR (101 MHz,
DMSO-d6) δ 151.63, 148.54, 128.61, 121.68, 120.90, 120.76, 88.81, 18.18.
1.13 (E)-3-(4-amino-3-chloro-5-fluorophenyl)acrylonitrile (6f). Synthesized following general procedure 1.1.
Yield 78%, red-brown solid. 1H NMR (400 MHz, DMSO-d6) δ 7.49 – 7.38 (m, 3H, ArH and olefinic H), 6.21 (d,
J = 16.5 Hz, 1H, olefinic H), 6.11 (s, 2H, NH2). 13C NMR (101 MHz, DMSO-d6) δ 151.74, 149.38 (d, JC-F = 2.6
Hz), 137.07 (d, JC-F = 16.0 Hz), 126.08 (d, JC-F = 1.7 Hz), 121.87 (d, JC-F = 8.2 Hz), 119.73, 118.26 (d, JC-F = 6.5
Hz), 112.92 (d, JC-F = 19.5 Hz), 93.34.
1.14 6-fluoroquinazoline-2,4-diol (9b). Synthesized following general procedure 1.2. Yield 86%, white solid.
1H NMR (400 MHz, DMSO-d6) δ 11.38 (s, 1H, OH), 11.17 (s, 1H, OH), 7.70 – 7.37 (m, 2H, ArH), 7.22 – 7.14
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(m, 1H, ArH). C NMR (101 MHz, CDCl3) δ 162.56 (d, JC-F = 2.6 Hz), 157.75 (d, JC-F = 239.7 Hz), 150.51,
137.99, 123.33 (d, JC-F = 24.3 Hz), 118.02 (d, JC-F = 7.8 Hz), 115.84 (d, JC-F = 7.4 Hz), 112.40 (d, JC-F = 23.8 Hz).
1.15 6-methoxyquinazoline-2,4-diol (9d). Synthesized following general procedure 1.2. Yield 68%, off-white
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solid. H NMR (400 MHz, DMSO-d6) δ 11.24 (s, 1H, OH), 10.99 (s, 1H, OH), 7.32 (d, J = 2.7 Hz, 1H, ArH),
7.29 – 7.24 (m, 1H, ArH), 7.11 (d, J = 8.8 Hz, 1H, ArH), 3.78 (s, 3H, CH3). 13C NMR (101 MHz, DMSO-d6) δ
163.18, 155.07, 150.54, 135.43, 124.24, 117.39, 115.30, 108.51, 55.99.
1.16 (E)-3-(4-((2-chloroquinazolin-4-yl)amino)-3,5-dimethylphenyl)acrylonitrile (11u).
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Synthesized following general procedure 1.3. Yield 23%, white solid. H NMR (400 MHz, DMSO-d6) δ 10.15
(s, 1H, NH), 8.54 (d, J = 8.1 Hz, 1H, ArH), 7.89 (t, J = 7.6 Hz, 1H, ArH), 7.72 (d, J = 8.2 Hz, 1H, ArH), 7.68 –
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7.58 (m, 2H, ArH and olefinic H), 7.51 (s, 2H, ArH), 6.47 (d, J = 16.8 Hz, 1H, olefinic H), 2.18 (s, 6H, CH3×2).
13C NMR (101 MHz, DMSO-d6) δ 160.38, 156.74, 150.62, 150.12, 137.73, 136.50, 134.14, 132.56, 127.43,
126.80, 126.66, 123.42, 118.85, 113.17, 109.49, 96.74, 18.00. LCMS (ESI, M+1): 335.2.
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1.17 (E)-4-((6-chloro-4-((2,6-dichloro-4-(2-cyanovinyl)phenyl)amino)quinazolin-2-yl)amino)
benzonitrile
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(12a). Synthesized following general procedure 1.4. Yield 41%, white solid, mp >325 °C (from n-butanol). H
NMR (400 MHz, DMSO-d6) δ 11.61 (s, 1H, NH), 10.76 (s, 1H, NH), 8.84 (s, 1H, ArH), 8.07 (s, 2H, ArH), 7.96
(d, J = 8.6 Hz, 1H, ArH), 7.82 – 7.69 (m, 2H, ArH and olefinic H), 7.53 (s, 4H), 6.81 (d, J = 16.8 Hz, 1H, olefinic
H). 13C NMR (101 MHz, DMSO-d6) δ 160.18, 153.38, 147.77, 142.99, 136.15, 135.04, 134.80, 133.15, 129.23,
128.31, 124.17, 120.72, 119.44, 118.63, 111.85, 105.25, 101.11. HRMS calcd for C24H13Cl3N6 [M+H]+: 491.0340,
found: 491.0338. HPLC analysis: retention time = 21.15 min; peak area, 96.42%.
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1.18 (E)-4-((6-chloro-4-((4-(2-cyanovinyl)-2,6-difluorophenyl)amino)quinazolin-2-yl)amino)
benzonitrile(12b). Synthesized following general procedure 1.4. Yield 42%, yellow solid, mp 285-289 °C (from
n-butanol). 1H NMR (400 MHz, DMSO-d6) δ 11.25 (s, 1H, NH), 10.68 (s, 1H, NH), 8.82 (s, 1H, ArH), 7.98 –
7.90 (m, 1H, ArH), 7.80 – 7.70 (m, 4H, ArH and olefinic H), 7.62 (s, 4H, ArH), 6.75 (d, J = 16.6 Hz, 1H, olefinic
H). 13C NMR (101 MHz, DMSO-d6) δ 160.33, 159.60 (d, JC-F = 5.4 Hz), 157.12 (d, JC-F = 5.3 Hz), 153.62, 148.36,
143.07, 136.06, 135.46 (t, JC-F = 10.2 Hz), 133.25, 129.15, 124.25, 120.85, 119.50, 118.61, 116.86 (t, JC-F = 15.4
Hz), 112.06 (d, JC-F = 23.4 Hz), 105.24 (t, JC-F = 7.8 Hz), 100.64. HRMS calcd for C24H13ClF2N6 [M+H]+:
459.0931, found: 459.0928. HPLC analysis: retention time = 20.49 min; peak area, 98.30%.
1.19 (E)-4-((6-chloro-4-((4-(2-cyanovinyl)-2-fluoro-6-methylphenyl)amino)quinazolin-2-
yl)amino)benzonitrile(12c). Synthesized following general procedure 1.4. Yield 52%, yellow solid, mp 278-282
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°C (from n-butanol). H NMR (400 MHz, DMSO-d6) δ 10.98 (s, 1H, NH), 10.56 (s, 1H, NH), 8.81 – 8.74 (m,
1H, NH), 8.04 – 7.52 (m, 9H, ArH and olefinic H), 6.36 (d, 14.2 Hz, 1H, olefinic H), 2.31 (s, 3H, CH3). 13C NMR
(101 MHz, DMSO-d6) δ 160.29, 157.06, 149.47 (d, JC-F = 2.5 Hz), 143.22, 139.19, 135.79, 134.84, 133.44 (d,
JC-F = 11.7 Hz), 133.17, 129.01, 126.65 (d, JC-F = 2.1 Hz), 124.20, 120.54, 119.51, 118.96, 112.63 (d, JC-F = 22.0
Hz), 112.17, 99.10, 18.12 (d, JC-F = 2.3 Hz). HRMS calcd for C25H16ClFN6 [M+H]+: 455.1182, found: 455.1168.
HPLC analysis: retention time = 19.25 min; peak area, 95.01%.
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1.20 (E)-4-((6-chloro-4-((4-(2-cyanovinyl)-2-methyl-6-nitrophenyl)amino)quinazolin-2-
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yl)amino)benzonitrile(12d). Synthesized following general procedure 1.4. Yield 51%, yellow solid, mp 324-326
°C (from n-butanol). 1H NMR (400 MHz, DMSO-d6) δ11.56 (s, 1H, NH), 10.72 (s, 1H, NH), 8.84 (s, 1H, ArH),
8.31 (s, 1H, ArH), 8.14 (s, 1H, ArH), 7.99 – 7.70 (m, 3H, ArH and olefinic H), 7.62 – 7.42 (m, 4H, ArH), 6.79
(d, J = 16.5 Hz, 1H, olefinic H), 2.37 (s, 3H, CH3). 13C NMR (101 MHz, DMSO-d6) δ 160.29, 153.01, 148.34,
147.66, 142.75, 140.23, 136.22, 134.54, 134.32, 133.18, 131.50, 129.34, 124.37, 123.18, 122.50, 120.88, 119.41,
118.73, 112.11, 105.46, 100.66, 18.45. HRMS calcd for C25H16ClN7O2 [M+H]+: 482.1127, found: 482.1125.
HPLC analysis: retention time = 19.08 min; peak area, 95.05%.
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1.21 (E)-4-((4-((2,6-dichloro-4-(2-cyanovinyl)phenyl)amino)-6-fluoroquinazolin-2-yl)amino)
benzonitrile
(12e). Synthesized following general procedure 1.4. Yield 49%, pale yellow solid, mp >325 °C (from n-butanol).
1H NMR (400 MHz, DMSO-d6) δ 11.28 (s, 1H, NH), 10.53 (s, 1H, NH), 8.51 (d, J = 8.9 Hz, 1H, ArH), 8.15 –
7.99 (m, 2H, ArH), 7.97 – 7.48 (m, 7H, ArH and olefinic H), 6.80 (d, J = 16.8 Hz, 1H, olefinic H). 13C NMR (101
MHz, DMSO-d6) δ 160.34 (d, JC-F = 3.2 Hz), 158.53 (d, JC-F = 244.2 Hz), 151.73, 147.22, 141.85, 135.87, 134.23,
133.13, 132.86, 132.70, 127.84, 125.06 (d, JC-F = 25.1 Hz), 122.34 (d, JC-F = 8.4 Hz), 120.64, 118.78, 118.11,
110.68 (d, JC-F = 8.9 Hz), 109.93 (d, JC-F = 24.3 Hz), 105.41, 100.77, 40.15, 39.94, 39.73, 39.52, 39.31, 39.10,
38.89. HRMS calcd for C24H13Cl2FN6 [M+H]+: 475.0636, found: 475.0630. HPLC analysis: retention time =
17.83 min; peak area, 95.10%.
1.22 (E)-4-((4-((2-chloro-4-(2-cyanovinyl)-6-fluorophenyl)amino)-6-fluoroquinazolin-2-
yl)amino)benzonitrile(12f). Synthesized following general procedure 1.4. Yield 45%, white solid, mp 297-299
°C (from n-butanol). 1H NMR (400 MHz, DMSO-d6) δ 11.53 (s, 1H, NH), 10.78 (s, 1H, NH), 8.64 (s, 1H, ArH),
7.93 – 7.85 (m, 3H, ArH), 7.82 – 7.72 (m, 2H, ArH and olefinic H), 7.55 (dd, J = 20.5, 8.3 Hz, 4H, ArH), 6.78
(d, J = 16.8 Hz, 1H, olefinic H). 13C NMR (101 MHz, DMSO-d6) δ 160.89 (d, JC-F = 3.1 Hz), 160.09 (d, JC-F
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5.5 Hz), 157.63 (d, JC-F = 11.7 Hz), 152.76, 148.05, 142.73, 136.19 (d, JC-F = 9.1 Hz), 133.99, 133.96, 133.22,
125.78, 125.75, 125.26 (d, JC-F = 23.9 Hz), 123.47, 121.02, 119.39, 118.59, 114.53 (d, JC-F = 21.7 Hz), 111.41 (d,
JC-F = 8.7 Hz), 110.14 (d, JC-F = 26.6 Hz), 105.54, 100.96. HRMS calcd for C24H13ClF2N6 [M+H]+: 459.0931,
found: 459.0932. HPLC analysis: retention time = 17.58 min; peak area, 98.35%.
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1.23 (E)-4-((4-((4-(2-cyanovinyl)-2,6-difluorophenyl)amino)-6-fluoroquinazolin-2-yl)amino)
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benzonitrile(12g). Synthesized following general procedure 1.4. Yield 48%, pale yellow solid, mp 293-299 °C
(from n-butanol). 1H NMR (400 MHz, DMSO-d6) δ 10.98 (s, 1H, NH), 10.52 (s, 1H, NH), 8.51 (d, J = 9.0 Hz,
1H, ArH), 8.14 – 7.69 (m, 6H, ArH and olefinic H), 7.69 – 7.58 (m, 3H, ArH), 6.74 (d, J = 16.6 Hz, 1H, olefinic
H). 13C NMR (101 MHz, DMSO-d6) δ 160.64, 159.88, 159.69, 157.46, 157.19 (d, JC-F = 4.8 Hz), 153.66, 148.38,
143.50, 133.47 (d, JC-F = 16.5 Hz), 133.22, 124.94, 124.68, 123.84, 120.51, 119.61, 118.62, 112.05 (d, JC-F = 24.3
Hz), 111.72, 109.61 (d, JC-F = 24.5 Hz), 106.58, 104.77, 104.68, 100.56. HRMS calcd for C24H13F3N6 [M+H]+:
443.1227, found: 443.1218. HPLC analysis: retention time = 16.99 min; peak area, 95.05%.
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1.24 (E)-4-((4-((4-(2-cyanovinyl)-2-fluoro-6-methylphenyl)amino)-6-fluoroquinazolin-2-
yl)amino)benzonitrile(12h). Synthesized following general procedure 1.4. Yield 47%, pale yellow solid, mp 284-
288 °C (from n-butanol). 1H NMR (400 MHz, DMSO-d6) δ 11.60 (s, 1H, NH), 10.91 (s, 1H, NH), 8.78 (d, J =
9.0 Hz, 1H, ArH), 8.12 – 7.67 (m, 5H, ArH and olefinic H), 7.59 – 7.51 (m, 3H, ArH), 7.50 – 7.44 (m, 2H, ArH),
6.67 (d, J = 16.6 Hz, 1H, olefinic H), 2.32 (s, 3H, CH3). 13C NMR (101 MHz, DMSO-d6) δ 160.99 (d, JC-F = 3.2
Hz), 160.23, 158.171 (d, JC-F = 248.9 Hz), 157.80, 152.07, 149.39, 142.29, 139.14, 135.17 (d, JC-F = 8.7 Hz),
133.22, 126.66, 126.31 (d, JC-F = 13.4 Hz), 125.22 (d, JC-F = 24.3 Hz), 122.16 (d, JC-F = 6.2 Hz), 121.11, 119.10
(d, JC-F = 36.8 Hz), 112.63 (d, JC-F = 21.7 Hz), 111.58 (d, JC-F = 8.7 Hz), 110.73 (d, JC-F = 25.1 Hz), 105.92, 99.27,
18.11 (d, JC-F = 2.2 Hz). HRMS calcd for C25H16F2N6 [M+H]+: 439.1477, found: 439.1470. HPLC analysis:
retention time = 15.42 min; peak area, 95.05%.
1.25 (E)-4-((4-((4-(2-cyanovinyl)-2-methyl-6-nitrophenyl)amino)-6-fluoroquinazolin-2-
yl)amino)benzonitrile(12i). Synthesized following general procedure 1.4. Yield 47%, yellow solid, mp 294-298
°C (from n-butanol). 1H NMR (400 MHz, DMSO-d6) δ 11.73 (s, 1H, NH), 10.78 (s, 1H, NH), 8.66 (d, J = 8.8 Hz,
1H, ArH), 8.31 (s, 1H, ArH), 8.14 (s, 1H, ArH), 7.90 – 7.44 (m, 7H, ArH and olefinic H), 6.80 (d, J = 16.6 Hz,
1H, olefinic H), 2.38 (s, 3H, CH3). 13C NMR (101 MHz, DMSO-d6) δ 160.80, 158.92 (d, JC-F = 242.1 Hz), 152.42,
148.31, 147.69, 142.57, 140.22, 134.56, 134.42, 133.60, 133.37, 133.19, 131.35, 122.50, 121.01, 119.36, 118.72,
111.57, 105.64, 100.72, 18.44. HRMS calcd for C25H16FN7O2 [M+H]+: 466.1416, found: 466.1421. HPLC
analysis: retention time = 15.48 min; peak area, 95.01%.
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1.26
(E)-4-((4-((4-(2-cyanovinyl)-2,6-dimethylphenyl)amino)-6-methoxyquinazolin-2-yl)amino) benzonitrile
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(12j). Synthesized following general procedure 1.4. Yield 46%, pale yellow solid, mp 303-308 °C (from n-
butanol). 1H NMR (400 MHz, DMSO-d6) δ 11.45 (s, 1H, NH), 10.77 (s, 1H, NH), 8.32 (s, 1H, ArH), 7.75 (d, J =
16.8 Hz, 1H, olefinic H), 7.69 – 7.54 (m, 4H, ArH), 7.42 (dd, J = 22.6, 8.5 Hz, 4H, ArH), 6.58 (d, J = 16.8 Hz,
1H, olefinic H), 3.96 (s, 3H, OCH3), 2.23 (s, 6H, CH3×2). 13C NMR (101 MHz, CDCl3) δ 160.58, 157.22, 150.98,
150.57, 142.68, 138.11, 136.84, 133.71, 133.17, 128.94, 128.06, 126.40, 120.81, 120.28, 119.32, 111.33, 106.11,
105.35, 97.64, 56.86, 18.41. HRMS calcd for C27H22N6O [M+H]+: 447.1928, found: 447.1940. HPLC analysis:
retention time = 12.95 min; peak area, 97.30%.
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1.27 (E)-4-((4-((2,6-dichloro-4-(2-cyanovinyl)phenyl)amino)-6-methoxyquinazolin-2-yl)amino)
benzonitrile(12k). Synthesized following general procedure 1.4. Yield 47%, yellow solid, mp 156-159 °C (from
n-butanol). 1H NMR (400 MHz, DMSO-d6) δ 11.95 (s, 1H, NH), 10.90 (s, 1H, NH), 8.33 (s, 1H, ArH), 8.15 (s,
2H, ArH), 7.69 (dd, J = 31.4, 8.5 Hz, 2H, ArH), 7.59 (d, J = 12.1 Hz, 1H, olefinic H), 7.49 (dd, J = 38.1, 8.4 Hz,
4H, ArH), 6.26 (d, J = 12.1 Hz, 1H, olefinic H), 3.96 (s, 3H, OCH3). 13C NMR (101 MHz, DMSO-d6) δ 160.86,
157.37, 150.98, 145.96, 142.39, 136.35, 136.12, 134.80, 134.65, 133.44, 133.17, 129.09, 127.27, 121.28, 120.74,
119.21, 117.53, 111.02, 105.72, 100.23, 56.90. HRMS calcd for C25H16Cl2N6O [M+H]+: 487.0835, found:
487.0837. HPLC analysis: retention time = 17.97 min; peak area, 95.03%.
1.28 (E)-4-((4-((4-(2-cyanovinyl)-2,6-difluorophenyl)amino)-6-methoxyquinazolin-2-yl)amino)
benzonitrile(12l). Synthesized following general procedure 1.4. Yield 53%, pale yellow solid, mp 301-305 °C
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(from n-butanol). H NMR (400 MHz, DMSO-d6) δ 11.67 (s, 1H, NH), 10.85 (s, 1H, NH), 8.31 (s, 1H, ArH),
7.79 – 7.67 (m, 4H, ArH and olefinic H), 7.66 – 7.47 (m, 5H, ArH), 6.76 (d, J = 16.6 Hz, 1H, olefinic H), 3.94 (s,
3H, OCH3). 13C NMR (101 MHz, DMSO-d6) δ 161.02, 159.58 (d, JC-F = 5.0 Hz), 157.29, 157.10 (d, JC-F = 5.5
Hz), 151.15, 148.29, 142.33, 135.74 (t, JC-F = 9.0 Hz), 133.28, 127.27, 121.17, 119.33, 118.58, 116.63 (t, JC-F
=
17.7 Hz), 112.08 (d, JC-F = 23.8 Hz), 111.38, 105.88, 105.71, 100.78, 56.90. HRMS calcd for C25H16F2N6O
[M+H]+: 455.1426, found: 455.1417. HPLC analysis: retention time = 13.89 min; peak area, 96.94%.
1.29 (E)-4-((4-((4-(2-cyanovinyl)-2,6-difluorophenyl)amino)-6-hydroxyquinazolin-2-yl)amino)
benzonitrile(12m). Compound 12m was synthesized via a known demethylation procedure47, 61 of 12l, not from
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1
intermediate 11m. Yield 41%, yellow solid, mp >325 °C (from n-butanol). H NMR (400 MHz, DMSO-d6) δ
9.79 (s, 1H, NH), 9.69 (s, 1H, NH), 9.54 (s, 1H, OH), 7.87 – 7.59 (m, 6H, ArH and olefinic H), 7.59 – 7.45 (m,
3H, ArH), 7.41 – 7.32 (m, 1H, ArH), 6.70 (d, J = 16.5 Hz, 1H, olefinic H). 13C NMR (101 MHz, DMSO-d6) δ
160.07, 159.63, 157.59, 154.18, 153.87, 148.61, 145.62, 134.42, 133.11, 126.66, 125.78, 120.15, 118.73, 112.47,
111.94 (d, JC-F = 23.4 Hz), 110.01, 106.44, 102.29, 100.06. HRMS calcd for C24H14F2N6O [M+H]+: 441.1270,
found: 441.1265. HPLC analysis: retention time = 10.23 min; peak area, 95.04%.
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1.30 (E)-4-((4-((4-(2-cyanovinyl)-2,6-difluorophenyl)amino)-6-nitroquinazolin-2-yl)amino) benzonitrile(12n).
Synthesized following general procedure 1.4. Yield 47%, yellow solid, mp 320-325 °C (from n-butanol). 1H NMR
(400 MHz, DMSO-d6) δ 10.96 (s, 1H, NH), 10.42 (s, 1H, NH), 9.55 (s, 1H, ArH), 8.51 (d, J = 8.9 Hz, 1H, ArH),
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7.84 – 7.66 (m, 6H, ArH and olefinic H), 7.65 – 7.55 (m, 2H, ArH), 6.74 (d, J = 16.6 Hz, 1H, olefinic H). C
NMR (101 MHz, DMSO-d6) δ 161.27, 159.69 (d, JC-F = 5.2 Hz), 157.21 (d, JC-F = 5.5 Hz), 148.44, 144.11, 142.69,
135.08 (t, JC-F = 9.6 Hz), 133.92, 133.17, 128.58, 125.88, 122.02, 120.18, 119.70, 118.66, 117.31 (t, JC-F = 16.2
Hz), 112.07 (d, JC-F = 23.8 Hz), 110.71, 104.35, 100.44. HRMS calcd for C24H13F2N7O2 [M+Na]+: 492.0991,
found: 492.0995. HPLC analysis: retention time = 20.72 min; peak area, 98.63%.
1.31 (E)-4-((7-chloro-4-((2,6-dichloro-4-(2-cyanovinyl)phenyl)amino)quinazolin-2-yl)amino)
benzonitrile(12o). Synthesized following general procedure 1.4. Yield 55%, pale yellow solid, mp >325 °C (from
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n-butanol). H NMR (400 MHz, DMSO-d6) δ 11.02 (s, 1H, NH), 10.37 (s, 1H, NH), 8.54 (d, J = 8.7 Hz, 1H,
ArH), 8.05 (s, 2H, ArH), 7.81 – 7.47 (m, 7H, ArH and olefinic H), 6.79 (d, J = 16.6 Hz, 1H, olefinic H). 13C NMR
(101 MHz, DMSO-d6) δ 160.70, 153.40, 147.74, 142.76, 140.82, 136.20, 135.03, 134.82, 133.13, 128.29, 127.05,
125.78, 120.97, 120.40, 119.37, 118.61, 109.54, 105.51, 101.15. HRMS calcd for C24H13Cl3N6 [M+H]+: 491.0340,
found: 491.0333. HPLC analysis: retention time = 15.42 min; peak area, 95.03%.
1.32 (E)-4-((7-chloro-4-((2-chloro-4-(2-cyanovinyl)-6-fluorophenyl)amino)quinazolin-2-
yl)amino)benzonitrile(12p). Synthesized following general procedure 1.4. Yield 40%, white solid, mp 304-306
°C (from n-butanol). 1H NMR (400 MHz, DMSO-d6) δ 11.24 (s, 1H, NH), 10.69 (s, 1H, NH), 8.67 (d, J = 8.8 Hz,
1H, ArH), 7.91 (s, 1H, ArH), 7.87 (d, J = 10.4 Hz, 1H, ArH), 7.79 – 7.71 (m, 2H, ArH and olefinic H), 7.68 –
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7.50 (m, 5H, ArH), 6.77 (d, J = 16.8 Hz, 1H, olefinic H). C NMR (101 MHz, DMSO-d6) δ 160.81, 160.16,
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157.65, 153.89, 148.08, 140.58, 136.05 (d, JC-F = 9.1 Hz), 134.08 (d, JC-F = 2.9 Hz), 133.17, 126.97, 125.65 (d,
JC-F = 21.6 Hz), 120.88, 119.46, 118.61, 114.50 (d, JC-F = 21.8 Hz), 109.82, 100.88. HRMS calcd for
C24H13Cl2FN6 [M+H]+: 475.0636, found: 475.0632. HPLC analysis: retention time = 20.95 min; peak area,
95.10%.
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1.33 (E)-4-((7-chloro-4-((4-(2-cyanovinyl)-2,6-difluorophenyl)amino)quinazolin-2-yl)amino)
benzonitrile(12q). Synthesized following general procedure 1.4. Yield 49%, white solid, mp 318-322 °C (from n-
butanol). 1H NMR (400 MHz, DMSO-d6) δ 10.59 (s, 1H, NH), 10.21 (s, 1H, NH), 8.50 (d, J = 8.7 Hz, 1H, ArH),
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7.81 – 7.68 (m, 4H, ArH and olefinic H), 7.66 – 7.54 (m, 5H, ArH), 6.73 (d, J = 16.6 Hz, 1H, olefinic H). C
NMR (101 MHz, DMSO-d6) δ 160.79, 159.68 (d, JC-F = 5.7 Hz), 157.20 (d, JC-F = 5.5 Hz), 154.23, 148.36, 143.23,
140.41, 135.40 (t, JC-F = 9.0 Hz), 133.44 (d, JC-F = 19.0 Hz), 133.21, 126.93, 125.37, 120.83, 119.53, 118.61,
116.99 (t, JC-F = 16.7 Hz), 112.04 (d, JC-F = 23.8 Hz), 110.07, 105.11, 100.60. HRMS calcd for C24H13ClF2N6
[M+H]+: 459.0931, found: 459.0932. HPLC analysis: retention time = 25.58 min; peak area, 98.28%.
1.34 (E)-4-((7-chloro-4-((4-(2-cyanovinyl)-2-fluoro-6-methylphenyl)amino)quinazolin-2-
yl)amino)benzonitrile(12r). Synthesized following general procedure 1.4. Yield 43%, white solid, mp 248-251
°C (from n-butanol). 1H NMR (400 MHz, DMSO-d6) δ 11.37 (s, 1H, NH), 10.82 (s, 1H, NH), 8.77 (d, J = 8.8 Hz,
1H, ArH), 7.79 – 7.61 (m, 4H, ArH and olefinic H), 7.58 – 7.50 (m, 5H, ArH), 6.66 (d, J = 16.6 Hz, 1H, olefinic
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H), 2.30 (s, 3H, CH3). C NMR (101 MHz, DMSO-d6) δ 160.89, 158.25 (d, JC-F = 247.5 Hz), 153.20, 149.45,
142.69, 140.63, 139.21, 135.03 (d, JC-F = 8.7 Hz), 133.18, 127.29, 126.65, 126.52, 125.66, 120.99, 119.75, 119.16
(d, JC-F = 43.8 Hz), 112.63 (d, JC-F = 21.7 Hz), 109.89, 105.59, 99.19, 18.11 (d, JC-F = 2.0 Hz). HRMS calcd for
C25H16ClFN6 [M+H]+: 455.1182, found: 455.1182. HPLC analysis: retention time = 23.80 min; peak area, 95.19%.
1.35 (E)-4-((7-chloro-4-((4-(2-cyanovinyl)-2-methyl-6-nitrophenyl)amino)quinazolin-2-
yl)amino)benzonitrile(12s). Synthesized following general procedure 1.4. Yield 49%, yellow solid, mp 287-289
°C (from n-butanol). 1H NMR (400 MHz, DMSO-d6) δ 11.38 (s, 1H, NH), 10.62 (s, 1H, NH), 8.64 (d, J = 8.7 Hz,
1H, ArH), 8.29 (s, 1H, ArH), 8.12 (s, 1H, ArH), 7.83 (d, J = 16.6 Hz, 1H, olefinic H), 7.75 (s, 1H, ArH), 7.62 (d,
J = 8.8 Hz, 1H, ArH), 7.53 (s, 4H, ArH), 6.77 (d, J = 16.6 Hz, 1H, olefinic H), 2.35 (s, 3H, CH3). 13C NMR (101
MHz, DMSO-d6) δ 160.66, 153.76, 148.37, 147.73, 140.51, 140.23, 134.48, 134.20, 133.15, 127.01, 125.50,
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122.48, 120.79, 119.48, 118.75, 110.04, 105.22, 100.59, 18.46. HRMS calcd for C25H16ClN7O2 [M+H]+:
482.1127, found: 482.1122. HPLC analysis: retention time = 12.57 min; peak area, 95.01%.
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1.36 (E)-4-((4-((2-amino-4-(2-cyanovinyl)-6-methylphenyl)amino)-7-chloroquinazolin-2-
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yl)amino)benzonitrile(12t). Compound 12t was synthesized from reduction of 12s, not from intermediate 11t.
Compound 12s (482 mg, 1 mmol, 1.0 eq), zinc powder (327 mg, 5 mmol, 5.0 eq), NH4Cl (535 mg, 10 mmol,
10.0eq) and ethanol/water (10 mL, v/v=1:1) was heated at 60 °C for 2 h, then the resulting mixture was filtered.
The filtrate was diluted with ethyl acetate, and was washed with water. The organic layer was dried with
anhydrous Na2SO4, concentrated. The residue was purified by a silica gel chromatography column. Yield 46%,
yellow solid, mp 210-213 °C (from n-butanol). 1H NMR (400 MHz, DMSO-d6) δ 9.72 (s, 1H, NH), 9.47 (s, 1H,
NH), 8.40 (d, J = 8.6 Hz, 1H, ArH), 7.84 (d, J = 7.3 Hz, 2H, ArH), 7.60 (d, J = 16.6 Hz, 1H, olefinic H), 7.53 (s,
1H, ArH), 7.42 (d, J = 7.9 Hz, 2H, ArH), 7.36 (d, J = 8.1 Hz, 1H, ArH), 6.90 (s, 2H, ArH), 6.32 (d, J = 16.6 Hz,
1H, olefinic H), 5.21 (s, 2H, NH2), 2.09 (s, 3H, CH3). 13C NMR (101 MHz, DMSO-d6) δ 160.62, 157.76, 152.93,
151.72, 146.64, 146.05, 138.17, 137.64, 133.27, 132.89, 126.42, 125.22, 124.56, 122.92, 120.19, 119.57, 118.84,
117.18, 112.56, 111.20, 101.98, 96.12, 18.60. HRMS calcd for C25H18ClN7 [M+H]+: 452.1385, found: 452.1381.
HPLC analysis: retention time = 20.82 min; peak area, 95.41%.
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1.37
(E)-4-((4-((4-(2-cyanovinyl)-2,6-dimethylphenyl)amino)quinazolin-2-yl)amino) benzonitrile (12u).
Synthesized following general procedure 1.4. Yield 38%, white solid, mp 280-284 °C (from n-butanol).1H NMR
(400 MHz, DMSO-d6) δ 11.33 (s, 1H, NH), 10.85 (s, 1H, NH), 8.67 (d, J = 7.9 Hz, 1H, ArH), 7.95 (t, J = 7.5 Hz,
1H, ArH), 7.77 – 7.65 (m, 2H, ArH and olefinic H), 7.64 – 7.55 (m, 3H, ArH), 7.42 (dd, J = 19.3, 8.1 Hz, 4H,
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ArH), 6.57 (d, J = 16.6 Hz, 1H, olefinic H), 2.21 (s, 6H, CH3×2). C NMR (101 MHz, DMSO-d6) δ 160.49,
151.53, 150.05, 142.03, 137.54, 136.33, 135.88, 133.24, 132.66, 127.54, 125.26, 124.62, 120.01, 118.79, 110.23,
105.07, 97.17, 17.86. HRMS calcd for C26H20N6 [M+H]+: 417.1822, found: 417.1813. HPLC analysis: retention
time = 15.03 min; peak area, 95.69%.
1.38
(E)-4-((4-((2,6-dichloro-4-(2-cyanovinyl)phenyl)amino)quinazolin-2-yl)amino)
benzonitrile(12v).
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Synthesized following general procedure 1.4. Yield 52%, white solid, mp >325 °C (from n-butanol). H NMR
(400 MHz, DMSO-d6) δ 11.73 (s, 1H, NH), 10.92 (s, 1H, NH), 8.67 (d, J = 8.2 Hz, 1H, ArH), 8.09 (s, 2H, ArH),
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7.99 (t, J = 7.7 Hz, 1H, ArH), 7.81 – 7.71 (m, 2H, ArH and olefinic H), 7.63 (t, J = 7.6 Hz, 1H, ArH), 7.51 (dd, J
= 33.7, 8.4 Hz, 4H, ArH), 6.83 (d, J = 16.6 Hz, 1H, olefinic H). 13C NMR (101 MHz, DMSO-d6) δ 161.23, 147.73,
142.38, 136.78, 136.33, 134.93, 134.78, 133.21, 128.32, 125.97, 125.07, 121.07, 119.29, 118.61, 110.46, 101.22.
HRMS calcd for C24H14Cl2N6 [M+H]+: 457.0730, found: 457.0729. HPLC analysis: retention time = 12.57 min;
peak area, 99.09%.
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1.39 (E)-4-((4-((2-chloro-4-(2-cyanovinyl)-6-fluorophenyl)amino)quinazolin-2-yl)amino) benzonitrile(12w).
Synthesized following general procedure 1.4. Yield 51%, white solid, mp 304-308 °C (from n-butanol). 1H NMR
(400 MHz, DMSO-d6) δ 11.63 (s, 1H, NH), 10.94 (s, 1H, NH), 8.69 (d, J = 8.2 Hz, 1H, NH), 7.97 (t, J = 7.8 Hz,
1H, ArH)), 7.93 (s, 1H, ArH)), 7.89 (d, J = 10.3 Hz, 1H, ArH)), 7.78 – 7.72 (m, 2H, ArH and olefinic H), 7.63 –
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7.45 (m, 5H, ArH)), 6.79 (d, J = 16.6 Hz, 1H, olefinic H). C NMR (101 MHz, DMSO-d6) δ 161.45, 160.06,
157.57, 152.34, 148.04, 142.30, 136.80, 136.26, 134.03 (d, JC-F = 2.7 Hz), 133.25, 125.86 (d, JC-F = 22.3 Hz),
125.18, 121.27, 118.94 (d, JC-F = 70.9 Hz), 114.54 (d, JC-F = 21.7 Hz), 110.62, 101.04. HRMS calcd for
C24H14ClFN6 [M+H]+: 441.1025, found: 441.1018. HPLC analysis: retention time = 12.20 min; peak area, 97.85%.
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(E)-4-((4-((4-(2-cyanovinyl)-2,6-difluorophenyl)amino)quinazolin-2-yl)amino)
benzonitrile(12x).
Synthesized following general procedure 1.4. Yield 39%, white solid, mp 306-312 °C (from n-butanol). 1H NMR
(400 MHz, DMSO-d6) δ 11.74 (s, 1H, NH), 11.06 (s, 1H, NH), 8.76 (d, J = 7.9 Hz, 1H, ArH), 7.98 (t, J = 1.8 Hz,
1H, ArH), 7.84 – 7.71 (m, 4H, ArH and olefinic H), 7.70 – 7.57 (m, 3H, ArH), 7.55 – 7.47 (m, 2H, ArH), 6.78
(d, J = 16.6 Hz, 1H, olefinic H). 13C NMR (101 MHz, DMSO-d6) δ 161.60, 159.59 (d, JC-F = 5.2 Hz), 157.10 (d,
JC-F = 5.2 Hz), 152.11, 148.27, 142.02, 136.89, 135.87 (t, JC-F = 9.9 Hz), 133.31, 126.03, 125.42, 121.49, 119.43,
119.26, 118.57, 116.53 (t, JC-F = 17.0 Hz), 112.07 (d, JC-F = 23.4 Hz), 110.74, 106.30, 100.86. HRMS calcd for
C24H14F2N6 [M+H]+: 425.1321, found: 425.1314. HPLC analysis: retention time = 15.80 min; peak area, 95.49%.
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(E)-4-((4-((4-(2-cyanovinyl)-2-fluoro-6-methylphenyl)amino)quinazolin-2-yl)amino) benzonitrile(12y).
Synthesized following general procedure 1.4. Yield 50%, white solid, mp 286-292 °C (from n-butanol). 1H NMR
(400 MHz, DMSO-d6) δ 11.33 (s, 1H, NH), 10.83 (s, 1H, NH), 8.69 (d, J = 7.9 Hz, 1H, ArH), 8.08 – 7.47 (m,
10H, ArH and olefinic H), 6.66 (d, J = 16.8 Hz, 1H, olefinic H), 2.31 (s, 3H, CH3). 13C NMR (101 MHz, DMSO-d6)
δ 161.38, 152.30, 149.43, 142.50, 139.24, 139.09, 136.49, 135.12 (d, JC-F = 9.7 Hz), 133.48 (d, JC-F = 17.8 Hz),
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133.22, 126.64, 125.79, 125.08, 121.07, 119.15 (d, JC-F = 40.9 Hz), 112.63 (d, JC-F = 17.7 Hz), 110.79, 99.24,
18.04 (d, JC-F = 1.7 Hz). HRMS calcd for C25H17FN6 [M+H]+: 421.1571, found: 421.1568. HPLC analysis:
retention time = 14.87 min; peak area, 95.04%.
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(E)-4-((4-((4-(2-cyanovinyl)-2-methyl-6-nitrophenyl)amino)quinazolin-2-yl)amino) benzonitrile(12z).
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Synthesized following general procedure 1.4. Yield 53%, yellow solid, mp 285-288 °C (from n-butanol). 1H NMR
(400 MHz, DMSO-d6) δ 11.51 (s, 1H, NH), 10.88 (s, 1H, NH), 8.67 (d, J = 7.9 Hz, 1H, ArH), 8.31 (s, 1H, ArH),
8.14 (s, 1H, ArH), 8.02 – 7.91 (m, 1H, ArH), 7.84 (d, J = 16.6 Hz, 1H, olefinic H), 7.71 (d, J = 8.2 Hz, 1H, ArH),
7.62 (t, J = 7.3 Hz, 1H, ArH), 7.57 – 7.32(m, 4H, ArH), 6.79 (d, J = 16.8 Hz, 1H, olefinic H), 2.36 (s, 3H, CH3).
13C NMR (101 MHz, DMSO-d6) δ 161.35, 152.15, 148.32, 147.76, 140.27, 136.71, 134.56, 133.21, 131.50,
125.95, 125.25, 122.49, 121.16, 119.30, 118.72, 110.81, 110.01, 105.97, 100.76, 18.40. HRMS calcd for
C25H17N7O2 [M+H]+: 448.1516, found: 448.1506. HPLC analysis: retention time = 10.55 min; peak area, 98.89%.
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2. In Vitro Anti-HIV Assay.
Evaluation of the antiviral activity of the compounds against HIV in MT-4 cells was performed using the MTT
assay as previously described.62, 63 All used NNRTI-resistant strains were a gift of Prof. Jan Balzarini from Rega
Institute for Medical Research, KU Leuven. All used strains were analyzed and reported earlier.64 65, 66 Prior to
use in our assays, every freshly grown virus stock is the subject of a genotypic analysis. Besides the genotypic
analysis every stock is subject to a phenotypic analysis using a panel of relevant reference compounds in MT-4
cells. In the case of NNRTI-resistant strains the reference compounds used are NVP, EFV and ETR. Stock
solutions (10 x final concentration) of test compounds were added in 25 µl volumes to two series of triplicate
wells so as to allow simultaneous evaluation of their effects on mock- and HIV-infected cells at the beginning of
each experiment. Serial 5-fold dilutions of test compounds were made directly in flat-bottomed 96-well microtiter
trays using a Biomek 3000 robot (Beckman instruments, Fullerton, CA). Untreated HIV- and mock-infected cell
samples were included as controls. HIV stock (50 µl) at 100-300 CCID50 (50 % cell culture infectious doses) and
culture medium (final 200 μL volume per well) were added to either the infected or mock-infected wells of the
microtiter tray. Mock-infected cells were used to evaluate the effects of test compound on uninfected cells in
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order to assess the cytotoxicity of the test compounds. Exponentially growing MT-4 cells were centrifuged for 5
minutes at 220 g and the supernatant was discarded. The MT-4 cells were resuspended at 6 x 105 cells/ml and 50
µl volumes were transferred to the microtiter tray wells. Five days after infection, the viability of mock-and HIV-
infected cells was examined spectrophotometrically using the MTT assay. The MTT assay is based on the
reduction of yellow colored 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Acros
Organics) by mitochondrial dehydrogenase activity in metabolically active cells to a blue-purple formazan that
can be measured spectrophotometrically. The absorbances were read in an eight-channel computer-controlled
photometer (Infinite M1000, Tecan), at two wavelengths (540 and 690 nm). All data were calculated using the
median absorbance value of three wells. The 50% cytotoxic concentration (CC50) was defined as the concentration
of the test compound that reduced the absorbance (OD540) of the mock-infected control sample by 50%. The
concentration achieving 50% protection against the cytopathic effect of the virus in infected cells was defined as
the 50% effective concentration (EC50).
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3. Reverse transcriptase assay.
Recombinant wild type p66/p51 HIV-1 RT was expressed and purified as described.67 The RT assay was
performed with the EnzCheck Reverse Transcriptase Assay kit (Molecular Probes, Invitrogen), as described by
the Manufacturer. The assay is based on the dsDNA quantitation reagent PicoGreen. This reagent shows a
pronounced increase in fluorescence signal upon binding to dsDNA or RNA-DNA heteroduplexes. Single-
stranded nucleic acids generate only minor fluorescence signal enhancement when a sufficiently high dye:base
pair ratio is applied.68 This condition is met in the assay. A poly(rA) template of approximately 350 bases long,
and an oligo(dT)16 primer, are annealed in a molar ratio of 1:1.2 (60 min at room temperature). Fifty-two ng of
the RNA/DNA is brought into each well of a 96-well plate in a volume of 20 µl polymerization buffer (60 mM
Tris-HCl, 60 mM KCl, 8 mM MgCl2, 13 mM DTT, 100 µM dTTP, pH 8.1). Five µl of RT enzyme solution,
diluted to a suitable concentration in enzyme dilution buffer (50 mM Tris-HCl, 20% glycerol, 2 mM DTT, pH
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