2,4,5-Trisubstituted thiazole derivatives as HIV-1 NNRTIs effective on both wild-type and mutant HIV-1 reverse transcriptase: Optimization of the substitution of positions 4 and 5

Article abstract of DOI:10.1016/j.ejmech.2016.07.047

In our previous work, novel 2,4,5-trisubstituted thiazole derivatives (TSTs) were synthesized, and their activities were evaluated against HIV-1 reverse transcriptase. Some interesting results were obtained, which led us to a new discovery regarding these TSTs. In the present study, 21 new 2,4,5-trisubstituted thiazole derivatives were rationally designed and synthesized as HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTIs) in accordance with our previous study. Among the synthesized target compounds, compounds 14, 16, 17, and 19 showed more potent inhibitory activities against HIV-1 with an IC₅₀value of 0.010?μM. Compounds 4, 9, 10, 11, 13 and 16 were further tested on nine NNRTI-resistant HIV-1 strains, and all of these compounds exhibited inhibitory effects. A molecular docking study was conducted, and the results showed a consistent and stable binding mode for the typical compounds. These results have provided deeper insights and SAR of these types of NNRTIs.

Full text of DOI:10.1016/j.ejmech.2016.07.047

Accepted Manuscript
2,4,5-Trisubstituted thiazole derivatives as HIV-1 NNRTIs effective on both wild-type
and mutant HIV-1 reverse transcriptase: Optimization of the substitution of positions 4
and 5
Zhongliang Xu, Jiamei Guo, Ying Yang, Mengdi Zhang, Mingyu Ba, Zhenzhong Li,
Yingli Cao, Ricai He, Miao Yu, Hua Zhou, Xiaoxi Li, Xiaoshan Huang, Ying Guo,
Changbin Guo
PII:
S0223-5234(16)30608-0
10.1016/j.ejmech.2016.07.047
EJMECH 8765
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 7 March 2016
Revised Date: 19 July 2016
Accepted Date: 20 July 2016
Please cite this article as: Z. Xu, J. Guo, Y. Yang, M. Zhang, M. Ba, Z. Li, Y. Cao, R. He, M. Yu, H.
Zhou, X. Li, X. Huang, Y. Guo, C. Guo, 2,4,5-Trisubstituted thiazole derivatives as HIV-1 NNRTIs
effective on both wild-type and mutant HIV-1 reverse transcriptase: Optimization of the substitution of
positions 4 and 5, European Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.07.047.
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2,4,5-Trisubstituted Thiazole Derivatives as HIV-1 NNRTIs Effective
on Both Wild-Type and Mutant HIV-1 Reverse Transcriptase:
Optimization of the Substitution of Positions 4 and 5
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Zhongliang Xua,1, Jiamei Guob,1, Ying Yangb,1, Mengdi Zhanga, Mingyu Bab, Zhenzhong Lia, Yingli Caob,
Ricai Hea, Miao Yub, Hua Zhoua, Xiaoxi Lia, Xiaoshan Huanga, Ying Guob,*, Changbin Guoa,*
a Department of Chemistry, Capital Normal University, Beijing, 100048, China.
b State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese
Academy of Medical Sciences and Peking Union Medical College, Beijing, 100050, China
Corresponding Authors
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Phone, +86-10-68902249; E-mail, guocb@cnu.edu.cn (G.CB.). Phone, +86-10-63161716; E-mail, yingguo6@imm.ac.cn
(G.Y.).
1 Authors Zhongliang Xu, Jiamei Guo, and Ying Yang contributed equally.
List of abbreviations: RT, reverse transcriptase; NNRTIs, non-nucleoside reverse transcriptase inhibitors; TSTs,
2,4,5-trisubstituted thiazole derivatives; DMSO, dimethyl sulfoxide; VSVG, vesicular stomatitis virus G protein.
Highlights:
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1. Twenty-one novel 2,4,5-trisubstituted thiazoles were designed and synthesized.
2. Reasonable design and combination of optimal substituents resulted in increased activity.
3. SAR exploration focused on positions 4 and 5 of the thiazole ring.
4. Some compounds showed inhibition against WT and some mutant viruses.
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Keywords: HIV-1, non-nucleoside reverse transcriptase inhibitors, 2,4,5-trisubstituted thiazole derivatives,
structure optimization, structure activity relationship.
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ABSTRACT: In our previous work, novel 2,4,5-trisubstituted thiazole derivatives (TSTs) were
synthesized, and their activities were evaluated against HIV-1 reverse transcriptase. Some interesting
results were obtained, which led us to a new discovery regarding these TSTs. In the present study, 21 new
2,4,5-trisubstituted thiazole derivatives were rationally designed and synthesized as HIV-1 non-nucleoside
reverse transcriptase inhibitors (NNRTIs) in accordance with our previous study. Among the synthesized
target compounds, compounds 14, 16, 17, and 19 showed more potent inhibitory activity against HIV-1
NNRT with an IC50 value of 0.010 ꢀM. Compounds 4, 9, 10, 11, 13 and 16 were further tested on nine
NNRTI-resistant HIV-1 strains, and all of these compounds exhibited inhibitory effects. A molecular
docking study was conducted, and the results showed a consistent and stable binding mode for the typical
compounds. These results have provided deeper insights and SAR of these types of NNRTIs.
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Introduction
Highly active antiretroviral therapy (HAART) consists of three drugs to sufficiently control HIV
infection and restore human immune system functions. Although HAART has significantly improved the
treatment of HIV/AIDS [1-4], the emergence of resistance to antiretroviral agents and serious side effects
cause widespread failures in the treatment of HIV/AIDS. Therefore, the discovery and development of
new drugs should target potent efficacy against resistant viruses and safety properties.
HIV reverse transcriptase (RT) is an enzyme that is essential for viral replication; this enzyme
converts the viral single-stranded RNA genome into a double-stranded DNA [5]. RT is a multifunctional
enzyme that possesses three activities: RNA-dependent DNA polymerase, DNA-dependent DNA
polymerase and ribonuclease H (RNase H) activities [6]. There are two classes of RT inhibitors, namely,
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nucleoside reverse transcriptase inhibitors (NRTIs) and non-nucleoside reverse transcriptase inhibitors
(NNRTIs), which block DNA polymerase activity and prevent synthesis of the double-stranded DNA [7,8].
NNRTIs are crucial components in HAART due to their unique antiviral efficacy, high specificity and low
toxicity characteristics.[9-11] Five NNRTIs have currently been approved by the FDA and have become
the cornerstone of HIV/AIDS treatment. Nevertheless, drug resistance is still the main reason for clinical
treatment failures [12-14]. NNRTIs can effectively inhibit wild-type HIV replication, but the long-term
use of these NNRTIs has led to the generation of resistant viruses, such as RT-K103N and RT-Y181C,
which are the most prevalent mutations in clinical HIV-1 isolates and have high-level resistance to current
NNRTIs [15]. Therefore, innovative NNRTIs with a higher genetic barrier against clinically relevant
mutant strains are still needed [16,17].
In our previous work, 2,4,5-trisubstituted thiazole derivatives (TSTs) were first reported as a novel class
of NNRTIs against both wild-type and resistant HIV-1, and the IC50 of the most active of these compounds
is 0.046 ꢀM [18]. The activity apparently increased when compound 1 transformed to compound 2 (Figure
1), which led us to hypothesize that the pseudo 5-membered ring formed by the hydrogen bond between
‘-OH’ and ‘-OCH3’ may be beneficial to the activity. 3D-QSAR contour maps of the active compounds
also suggested that increasing the volume of the side chain at position 2 of the thiazole ring may increase
the activities of the TSTs [18]. Based on the above SAR, compound 3 was designed and synthesized,
which was transformed from compound 2. The activity of 3 against HIV-1 RT increased, and thus, 3 was
used as the lead compound in this study. The 2,3-dihydrobenzofuran-5-yl-methylamino on the side chain
at position 2 of the thiazole ring was retained, and the substituents at positions 4 and 5 of the thiazole ring
were further optimized.
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Figure 1. Structures and anti-HIV activities of compounds 1, 2, and 3.
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Results and discussion
The main synthetic route for the target compounds, which is described in reference 15, is shown in
Scheme 1. Appropriate benzaldehydes and acetophenones were condensed to provide chalcones, which
were then hydrogenated, brominated, and condensed with an appropriate sulfur source to afford
2-aminothiazoles. The 2-aminothiazoles were condensed with appropriate benzaldehydes to afford the
Schiff bases. Reduction of the Schiff bases afforded the target compounds.
Scheme 1. Synthesis of 3-23.
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Reagents and conditions: (a) 10% NaOH, EtOH, rt, 5-10 h, 70-90% yield; (b) H2, Pd/C, EA, rt, 3-4 h, 80-95% yield; (d) Br2,
AlCl3, CHCl3, 0°C, 5-10 h; (e) thiourea, CH3COONa, EtOH, 80°C, 10-15 h, 70-80% yield; (f)
2,3-dihydrobenzofuran-5-carbaldehyde, p-toluenesulfonic acid, toluene, 130°C, 10-24 h, 29-80% yield.
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The structures of compounds 3-23 and their activities against wild-type HIV are shown in Table 1.
According to our previous study [18], we first retained the benzene ring as the substituent at position 4 of
the thiazole ring and changed the substituents at position 5. Comparing compound 3 with compounds 4, 5
and 6, the activity substantially increased when “-OCH3” was introduced at the ortho position of the
phenyl ring at position 5, slightly increased when introduced at the meta position and almost disappeared
when introduced at the para position, thus suggesting that the ortho position is the optimal position for
substitution. The IC50 values of compounds 7 and 8 also supported this deduction. The activities of these
compounds revealed that the introduction of an electron-donating group, ‘-OCH3’, on the ortho position of
the benzene ring at position 5 of the thiazole scaffold may be beneficial. Then, considering the extensively
used strategy to introduce halogen atoms and perform multi-substitutions in a drug structure to yield
enhanced activity, compounds 9 and 10 were synthesized and exhibited potent IC50 values, suggesting that
disubstitution on the benzene ring at this position led to a marked increase in activity.
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In our previous study, compounds with the substituents “2-F”, “2-Cl”, “2,4-F2” and “2,4-Cl2” on
position 4 showed potent inhibitory activities. However, only four compounds with the above-mentioned
substituents were obtained in the previous study, and all exhibited good activity, a finding that indicated
tremendous potential for higher activity, but further investigation was needed to confirm the effect of
certain substituents. According to the results described above, we selected ‘2-OCH3’, ‘2,6-Cl2’, and
‘3,5-F2’ as the optimal substituents on the benzene ring at position 5 to explore the influence of the
mentioned substituents at position 4.
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As shown in Table 1, the comparisons of compound 11 with compound 17 and compound 13 with
compound 19 revealed that most compounds with a 2-F-phenyl at position 4 exhibited higher activities
than the corresponding compounds with a 2,4-F2-phenyl at the same position. Furthermore, the IC50 values
of compounds 14 to 16, which have a “-Cl” at the ortho position, and of compounds 20 to 22, which have
a 2,4-Cl2, also exhibited the same phenomenon. Additionally, the activityof compound 23 (IC50=7.32 ꢀM),
which was disubstituted at the meta position, decreased more than 100-fold compared with that of
compound 17 (IC50=0.01 ꢀM). These results suggested that when position 5 was occupied by the same
substituent, the inhibitory activities may be primarily provided by the ortho substituent at position 4 and
that the atom located on the meta position may have some negative contribution to the activity. Moreover,
compounds 9 to 22 showed obviously increased activities (0.088 to 0.010 ꢀM) compared with the lead
compound 3 (0.239 ꢀM) and the previous best compound (compound 24 in ref. 15, 0.046 ꢀM). This
finding not only demonstrates that the strategy used successfully yielded enhanced activity by the
combination of the optimal substituents on positions 4 and 5 but also provides strong evidence supporting
the hypothesis that increasing the bulk of the side chain at position 2 of the thiazole ring is beneficial to
the activity of the TSTs. The best compounds 14, 16, 17, and 19 (0.010 ꢀM) highlight the significance of
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this investigation, which represents a successful optimization of a reasonable drug design.
Table 1. Chemical structures and cell-based antiviral activities of compounds 3-23a against wild-type HIV-1
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IC50b(ꢀM)
Compd
R1
R2
3
H
H
0.239
0.072
0.136
>10
4
H
2-OCH3
3-OCH3
4-OCH3
4-F
5
H
6
H
7
H
5.670
>10
8
H
4-Cl
9
H
2,6-Cl2
3,5-F2
2-OCH3
2,6-Cl2
3,5-F2
2-OCH3
0.062
0.037
0.045
0.021
0.014
0.010
10
11
12
13
14
H
2,4-F2
2,4-F2
2,4-F2
2-Cl
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23
2-Cl
2-Cl
2-F
2,6-Cl2
0.022
0.010
0.010
0.032
0.010
0.088
0.071
0.068
7.320
3,5-F2
2-OCH3
2,6-Cl2
3,5-F2
2-F
2-F
2,4-Cl2
2,4-Cl2
2,4-Cl2
3,5-F2
2-OCH3
2,6-Cl2
3,5-F2
2-OCH3
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a All tested compounds had no cytotoxicity at a final concentration of 10 ꢀM.
b Inhibitory concentration 50% (IC50, ꢀM) was calculated from the dose-infectivity curves.
In our previous study, we have proved that the 2,4,5-trisubstituted thiazole derivatives’ inhibitory
activity on HIV-1 replication is due to the reverse transcription blockage by Time-of-Addition (TOA)
assay results [18]. In this paper, we tested the four most potent compounds on reverse transcriptase
activity by RT enzyme-based assay. As shown in table 2, compounds 14, 16, 17 and 19 inhibited RT
RNA-dependent DNA polymerase activities with IC50 values from 0.014 to 0.064 ꢀM; and NVP, a NNRTI
targeting RNA-dependent DNA polymerase, was used as positive control with IC50 as 2.1 ꢀM. This
indicated that the primary target of 2,4,5-trisubstituted thiazole derivatives for their anti-HIV activities is
RT RNA-dependent DNA polymerase, i.e., they are NNRTIs.
Table 2. Antiviral activities of compound 14, 16, 17 and 19 against wild-type HIV-1
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IC50 (ꢀM)
Compd
RNA-dependent DNA polymerase a
0.014 ± 0.0028
VSVG/HIV-1 b
0.01
14
0.025 ± 0.0057
0.01
16
0.020 ± 0.0085
0.01
17
0.064 ± 0.00071
0.01
19
2.1 ± 0.087
0.031
NVP
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a. Enzyme-based antiviral assay (Average ± SD, n = 2)
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b. Cell-based antiviral assay
c. NVP: Nevirapine
Resistance
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Potent activity against resistant HIV-1 is an important feature for the discovery of novel NNRTIs.
Therefore, six compounds, 4, 9, 10, 11, 13, and 16, were selected for evaluation of their activities against
NNRTI-resistant HIV-1 replication. Nine NNRTI-resistant HIV-1 recombinant virus models were used:
HIVRT-Y181C, HIVRT-K103N, HIVRT-L100I/RT-K103N, HIVRT-Y188L, HIVRT-K103N/RT-P225H, HIVRT-K103N/RT-G190A and
HIVRT-K103N/RT-V108I, HIVRT-K103N,Y181C and HIVRT-K103N,Y188L. Compound 11 exhibited high activity against seven
of the nine resistant strains, with IC50 values ranging from 2.9- to 23.5-fold greater than the values against
wild-type HIV, which is better than that of the positive control drugs nevirapine (90- to 2152-fold) and
efavirenz (5.4- to 2394-fold). However, when the viruses carried a mutation at RT-Try188, such as
HIVRT-Y188L or HIVRT-K103N/Y188L, the viruses exhibited severe resistance to this series of compounds (Table
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3). The docking result showed that the Y188 residue may have a π-π stacking interaction with the phenyl
ring at the 5 position of the thiazole. We hypothesize that this interaction plays a key role in the mutual
effect between the compounds and the enzyme.
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Table 3. Inhibitory effects of 4, 9, 10, 11, 13, 16 and the references NVP and EFV on wild-type and NNRTI-resistant
HIV-1 replication, as determined through a cell-based antiviral assay
Compd
4
9
10
11
13
16
NVP
IC50
EFV
IC50
IC50
(µM)
IC50
(µM)
IC50
(µM)
IC50
(µM)
IC50
(µM)
IC50
(µM)
(µM)
(µM)
Resistance
0.072
0.0618
0.0372
0.0447
0.0139
0.010
0.031
0.00104
a
VSVG/HIVwt
0.88
(12.2)*
0.512
(8.8)
1.53
(24.8)
0.39
1.41
(37.9)
5.56
0.29
(6.5)
0.13
(2.9)
0.87
(19.5)
>10
0.2
(14)
1.1
0.08
(7.8)
0.4
16.1
(519)
66.7
0.0588
(56)
VSVG/HIVRT-
K103N
0.00565
(5.4)
VSVG/HIVRT-
(6.3)
5.05
(149.5)
1.17
(79)
(39)
(2152)
5.0
Y181C
5.31
2.49
VSVG/HIVRT-
NTb
NT
(73.8)
>10
(81.7)
>10
(31.5)
>10
(161)
>10
(2394)
0.48
L100I,K103N
7
1.4
VSVG/HIVRT-
(>223.7) (>161.8) (>268.8) (>223.7)
(504)
(136)
(>322)
(461.5)
Y188L
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4.98
(69.2)
0.58
>10
(>161.8)
0.56
5.03
(135.2)
2
1.05
(23.5)
0.49
5.48
(177)
>10
0.16
(154)
VSVG/HIVRT
NT
NT
-
K103N,P225H
0.056
VSVG/HIVRT-
NT
NT
(8.1)
1.53
(9.1)
(53.8)
10.4
(11.0)
0.76
(>322)
2.8
(538)
K103N,G190A
3.0
3.9
0.3
0.083
VSVG/HIVRT-
(21.3)
9.56
(48.5)
>10
(279.6)
>10
(17.0)
0.45
(218)
(29)
(90)
(79.8)
0.0587
(56.4)
13.8
K103N,V108I
>10
VSVG/HIVRT-
NT
NT
(132.8)
>10
(>161.8) (>268.8)
(10.1)
>10
(>322)
>10
K103N,Y181C
>10
>10
VSVG/HIVRT-
NT
NT
(>223.7) (>161.8) (>268.8) (>223.7)
(>322)
(13269.2)
K103N,Y188L
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a. VSV-G: vesicular stomatitis virus G protein
b. NT: not tested
* Mean change (fold) in IC50 compared to wild type.
Docking studying
Compounds 16, 17 and 19, which exhibited the highest activity against the WT HIV-1, were used as
representatives to conduct the docking experiments in the WT HIV-1 RT non-nucleoside binding site
(NNBS) and to study the binding mode of the TSTs. To take the flexibility of the experimental NNBS into
account, the structural data of six RTs (1c1b, 1c1c, 1ikx, 2b5j, 3lak, and 2rki) were selected. The Surflex
program was used to dock the compounds into all six RTs. The results indicated that the three compounds
almost aligned in all cases, and a nearly identical binding mode for the three compounds with all RTs was
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observed: (i) the two nitrogen atoms on the thiazole ring and on the side chain of position 2 formed two
hydrogen bonds with the K103 and K101 amino groups, respectively; (ii) the side chain at the 2 position
of the thiazole occupied a pocket formed primarily by the side chains of F227, L234, H235, P225, P226
and P236 and fit in the pocket well, which may explain why the compounds with the
2,3-dihydrobenzofuran on the side chain exhibited higher activities than the corresponding compounds
with 4-methoxyphenyl on the side chain; and (iii) the phenyl ring at the 5 position of the thiazole has a π-π
stacking interaction with Y181 or Y188. These possible reactions may explain the decrease in the IC50 of
the above six compounds, 4, 9, 10, 11, 13 and 16, against Y188 mutants. Moreover, to explain the
improved activity of 11 against other mutants, we can speculate that both 2-OCH3 on position 5 and 2,4-F2
on position 4 may exhibit good interactions with the surrounding residues, a speculation that might require
additional investigation to provide deeper insights.
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Figure 2. Binding conformation of 16 (magenta), 17 (white) and 19 (orange) into the NNBS of HIV-1WT RT (PDB id:2rki).
Hydrogen bonds are shown as dotted yellow lines.
Conclusion
Based on a previous study, 21 novel 2,4,5-trisubstituted thiazole derivatives (3-23) were designed and
synthesized. Their anti-HIV activity and the SAR analysis provided us with a deeper understanding of the
mechanism of the TSTs. Among the synthesized derivatives, 19 compounds exhibited potent inhibition of
HIV-1 replication with submicromolar IC50 values against wild-type strains. Compounds, 4, 9, 10, 11, 13
and 16 were also evaluated for their activities against nine HIV-1 RT mutants, and compound 11 exhibited
high activity against seven of the nine resistant strains. RT enzymatic assay results illustrated that
2,4,5-trisubstituted thiazole derivatives are RT RNA-dependent DNA polymerase inhibitors. The docking
study revealed the most likely mechanism of interaction between this type of compound and the HIV-1 RT,
and the result suggested a possible steady conformation of the binding mode.
Experimental
Chemistry. The starting materials and other reagents were purchased from commercial suppliers and
were used as received without further purification unless otherwise indicated. The melting points were
measured using a WRS-1B digital melting point apparatus from Shanghai Measuring Instruments
1
Equipment Co., Ltd. H NMR and 13C NMR spectra were measured on a VARIAN Mercury 600 MHz
1
spectrometer using TMS as an internal standard. H NMR and 13C NMR spectra were obtained in
DMSO-d6 or CDCl3 solutions as indicated (reported in ppm). The mass spectra were obtained using liquid
chromatography mass spectrometry (LC-MS) on a Bruker APEXIIFT-ICR mass spectrometer with an ESI
interface. Thin-layer chromatography (TLC) was performed using silica gel GF254. The hydrogenation
reactions were conducted using a GCD-500 high-purity hydrogen generator and a BLT-2000
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medium-pressure hydrogenation apparatus produced by Beijing Jiaweikechuang Company. The boiling
range for petroleum ether is 60-90°C.
General procedure for the preparation of derivatives of 3-23. The 4,5-disubstituted thiazol-2-amine
obtained from the previous step, a substituted aryl aldehyde (1 equivalent) and a catalytic amount of
p-toluenesulfonic acid were added to a 50 mL round-bottom flask, and the mixture was refluxed in toluene
for 24 h. Then, the solvent was removed, and sodium borohydride (5 equivalents) in ethanol was added.
The mixture was stirred for 2-3 h and monitored by TLC. After the solvent was removed and the mixture
was extracted, washed and concentrated, column chromatography was performed for further purification.
5-Benzyl-N-((2,3-dihydrobenzofuran-5-yl)methyl)-4-phenylthiazol-2-amine (3) was obtained as a slight
yellow solid in 76.0% yield. 1H NMR (DMSO-d6, 600 MHz) δ 3.12 (t, J = 8.4 Hz, 2H, CH2), 4.05 (s, 2H,
CH2), 4.30 (d, J = 5.4 Hz, 2H, CH2), 4.47 (t, J = 8.4 Hz, 2H, CH2), 6.68 (d, J = 8.4 Hz, 1H, ArH), 7.05 (d,
J = 8.4 Hz, 1H, ArH), 7.19 (dd, J = 17.4, 6.6 Hz, 4H, ArH), 7.29 (t, J = 7.2 Hz, 3H, ArH), 7.38 (t, J = 7.8
Hz, 2H, ArH), 7.57-7.52 (m, 2H, ArH), 7.84 (t, J = 6.0 Hz, 1H, NH); 13C NMR (151 MHz, CDCl3) δ
159.70, 140.54, 135.26, 129.70, 128.52, 128.25, 127.70, 127.51, 126.49, 124.54, 109.17, 77.21, 77.00,
76.79, 71.33, 49.59, 32.93, 29.64; ESI-MS m/z: 477 ([M + H]+; m.p. 125.3-126.4°C.
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N-((2,3-Dihydrobenzofuran-5-yl)methyl)-5-(2-methoxybenzyl)-4-phenylthiazol-2-amine
(4)
was
1
obtained as a white solid in 55.1% yield. H NMR (DMSO-d6, 600 MHz) δ 3.14 (t, J= 8.4 Hz, 2H,
CH2CH2), 3.75 (s, 3H, OCH3), 3.98 (s, 2H, ArCH2), 4.31 (d, J= 6.0 Hz, 2H, ArCH2N), 4.49 (t, J= 8.4 Hz,
2H, CH2CH2), 6.70 (d, J= 7.8 Hz, 1H, ArH), 6.88 (t, J= 7.2 Hz, 1H, ArH), 6.98 (d, J= 8.4 Hz, 1H, ArH),
7.07 (d, J= 7.2 Hz, 2H, ArH), 7.22 (m, 2H, ArH), 7.30 (t, J= 7.2 Hz, 1H, ArH), 7.39 (t, J= 7.2 Hz, 2H,
ArH), 7.56 (d, J= 7.2 Hz, 2H, ArH), 7.80 (t, J= 6.0 Hz, 1H, NH); 13C NMR (151 MHz, CDCl3) δ 166.91,
159.64, 156.99, 147.40, 135.61, 129.91, 129.36, 128.95, 128.50, 128.21, 127.70, 127.43, 127.29, 124.54,
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120.54, 119.76, 110.13, 109.11, 77.24, 77.02, 76.81, 71.33, 55.17, 49.56, 29.66, 27.25; IR (KBr, cm-1):
3206(νN-H), 3098, 2966, 2891, 1582, 1491, 1461, 1430, 1331, 1244, 1167, 1103, 975, 818, 755, 700;
ESI-HRMS: Calcd. for C26H24N2O2S: m/z 429.1637 [M + H]+, found: 429.1597; m.p. 128.5-129.5°C.
219
220
221
222
223
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225
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N-((2,3-Dihydrobenzofuran-5-yl)methyl)-5-(3-methoxybenzyl)-4-phenylthiazol-2-amine
(5)
was
obtained as slight yellow solid in 75.0% yield. 1H NMR (DMSO-d6, 600 MHz) δ 3.12 (t, J = 8.4 Hz, 2H,
CH2), 3.68 (s, 3H, OCH3), 4.05 (s, 2H, CH2), 4.33-4.28 (d, J = 5.4 Hz, 2H, CH2), 4.47 (t, J = 8.4 Hz, 2H,
CH2), 6.68 (d, J = 8.4 Hz, 1H, ArH), 6.84-6.69 (m, 3H, ArH), 7.05 (d, J = 8.4 Hz, 1H, ArH), 7.22-7.19 (m,
1H, ArH), 7.29 (dd, J = 8.4, 6.6 Hz, 1H, ArH), 7.38 (td, J = 7.8, 2.4 Hz, 2H, ArH), 7.60-7.42 (m, 3H, ArH),
7.85 (t, J = 5.4 Hz, 1H, NH); 13C NMR (151 MHz, CDCl3) δ 159.74, 159.16, 142.16, 140.76, 135.27,
133.21, 129.82, 128.37, 127.62, 124.53, 120.56, 115.92, 114.50, 113.98, 113.62, 111.80, 109.16, 77.22,
77.01, 76.80, 71.34, 55.41, 55.17, 49.57, 33.53, 32.91, 29.64; ESI-MS m/z: 429 [M + H]+; m.p.
115.8-117.5°C.
228
229
230
231
232
233
234
N-((2,3-Dihydrobenzofuran-5-yl)methyl)-5-(4-methoxybenzyl)-4-phenylthiazol-2-amine
(6)
was
1
obtained as a slight yellow solid in 45.5% yield. H NMR (DMSO-d6, 600 MHz) δ 3.70 (s, 3H, OCH3),
3.73 (s, 3H, OCH3), 4.02 (s, 2H, CH2), 4.36 (d, J=6.0 Hz, 2H, CH2), 6.76-6.79 (m, 1H, ArH), 6.89-6.93
(m, 4H, ArH), 7.17-7.21 (m, 4H, ArH), 7.27 (t, J=7.2, 2H, ArH), 7.44-7.47 (m, 2H, ArH), 7.89 (t, J=6.0
Hz, 1H, NH); 13C NMR (151 MHz, CDCl3) δ 167.61, 159.81, 159.03, 146.91, 140.57, 139.56, 129.62,
128.56, 128.17, 127.69, 126.67, 129.56, 119.67, 118.43, 113.71, 113.07, 112.76, 55.22, 55.18, 49.66,
32.88; ESI-MS m/z 417 [M + H]+
.
235
236
N-((2,3-Dihydrobenzofuran-5-yl)methyl)-5-(4-fluorobenzyl)-4-phenylthiazol-2-amine (7) was obtained
as a white solid in 42% yield. 1H NMR (600 MHz, DMSO-d6) δ 3.14 (t, J = 8.7 Hz, 2H, CH2), 4.07 (s, 2H,
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CH2), 4.36-4.29 (m, 2H, CH2), 4.49 (td, J = 8.7, 1.7 Hz, 2H, CH2), 6.70 (dd, J = 8.2, 1.6 Hz, 1H, ArH),
7.07 (d, J = 8.2 Hz, 1H, ArH), 7.12 (td, J = 8.7, 1.7 Hz, 2H, ArH), 7.24-7.19 (m, 3H, ArH), 7.33-7.28 (m,
1H, ArH), 7.40 (td, J = 7.7, 1.7 Hz, 2H, ArH), 7.58-7.53 (m, 2H, ArH), 7.90-7.85 (t, J = 5.4 Hz, 1H, NH2);
13C NMR (151 MHz, CDCl3) δ 167.10, 162.41, 160.79, 159.71, 147.09, 136.09, 134.88, 129.70, 128.41,
127.68, 127.50, 124.51, 119.56, 115.44, 115.30, 109.16, 77.23, 77.01, 76.80, 71.34, 49.59, 32.13, 29.64;
IR(KBr, cm-1): 3309, 1552, 1508, 1490, 1352, 1223, 1104, 831, 694; m.p. 153-154.5°C.
243
244
245
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248
249
5-(4-Chlorobenzyl)-N-((2,3-dihydrobenzofuran-5-yl)methyl)-4-phenylthiazol-2-amine (8) was obtained
1
as a slight yellow solid in 76.0% yield. H NMR (DMSO-d6, 600 MHz) δ 3.14 (t, J = 8.4 Hz, 2H, CH2),
4.07 (s, 2H, CH2), 4.33 (dd, J = 15.6, 5.4 Hz, 2H, CH2), 4.49 (t, J = 8.4 Hz, 2H, CH2), 6.70 (d, J = 8.4 Hz,
1H, ArH), 7.07 (d, J = 8.4 Hz, 1H, ArH), 7.24-7.18 (m, 3H, ArH), 7.33-7.28 (m, 1H, ArH), 7.42-7.33 (m,
4H, ArH), 7.54 (d, J = 8.4 Hz, 2H, ArH), 7.90 (t, J = 5.4 Hz, 1H, NH); 13C NMR (151 MHz, CDCl3) δ
167.12, 159.69, 147.81, 139.01, 135.15, 132.27, 129.58, 128.68, 128.39, 127.64, 127.48, 124.48, 119.07,
109.16, 77.23, 77.01, 76.80, 71.33, 49.55, 32.28, 29.65; ESI-MS m/z: 433 [M + H]+; m.p. 137.2-137.8°C.
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254
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5-(2,6-Dichlorobenzyl)-N-((2,3-dihydrobenzofuran-5-yl)methyl)-4-phenylthiazol-2-amine
(9)
was
obtained as a slight yellow in 76.0% yield. 1H NMR (DMSO-d6, 600 MHz) δ 3.13 (t, J = 8.4 Hz, 2H, CH2),
4.29 (d, J = 5.4 Hz, 2H, CH2), 4.36 (s, 2H, CH2), 4.48 (t, J = 8.4 Hz, 2H, CH2), 6.68 (d, J = 7.8 Hz, 1H,
ArH), 7.04 (d, J = 7.8 Hz, 1H, ArH), 7.19 (s, 1H, ArH), 7.30 (t, J = 7.8 Hz, 1H, ArH), 7.34 (t, J = 7.8 Hz,
1H, ArH), 7.44 (t, J = 6.6 Hz, 4H, ArH), 7.64 (d, J = 7.8 Hz, 2H, ArH), 7.76 (t, J = 5.4 Hz, 1H, NH); 13C
NMR (151 MHz, CDCl3) δ 166.62, 159.66, 147.66, 136.59, 135.73, 135.54, 129.74, 128.70, 128.45,
128.25, 127.70, 127.43, 124.55, 117.61, 109.14, 77.22, 77.01, 76.80, 71.32, 49.61, 29.63, 29.30; ESI-MS
m/z: 467 [M + H]+; m.p. 147.2-148.9°C.
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5-(3,5-Difluorobenzyl)-N-((2,3-dihydrobenzofuran-5-yl)methyl)-4-phenylthiazol-2-amine (10) was
obtained as a slight yellow solid in 79% yield. mp:158.4-159.5℃, 1H NMR (DMSO-d6, 600 MHz) δ 3.15
(t, J = 8.4 Hz, 2H, -O-CH2-CH2-), 4.11 (s, 2H, CH2), 4.34 (dd, J = 15.6, 6.0 Hz, 2H, -NH-CH2-), 4.49 (t,
J = 8.4 Hz, 2H, -O-CH2-CH2-), 6.70 (d, J = 8.4 Hz, 1H, ArH), 6.88 (d, J = 6.6 Hz, 2H, ArH), 7.09 (t, J =
9.0 Hz, 2H, ArH), 7.24 (d, J = 11.4 Hz, 1H, ArH), 7.32 (t, J = 7.2 Hz, 1H, ArH), 7.40 (t, J = 7.8 Hz, 2H,
ArH), 7.53 (d, J = 7.2 Hz, 2H, ArH), 7.94 (t, J = 5.4 Hz, 1H, -NH-CH2-); 13C NMR (151 MHz, CDCl3) δ
167.37, 163.95, 162.30, 159.71, 148.52, 144.55, 134.99, 129.57, 128.40, 127.71, 127.50, 124.46, 117.44,
110.99, 109.17, 102.18, 102.01, 101.84, 77.23, 77.01, 76.80, 71.34, 49.53, 32.52, 29.64; ESI-MS m/z: 435
[M + H]+.
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276
4-(2,4-Difluorophenyl)-N-((2,3-dihydrobenzofuran-5-yl)methyl)-5-(2-methoxybenzyl)thiazol-2-amine
(11) was obtained as a slight yellow solid in 72% yield. 1H NMR(DMSO-d6, 600 MHz) δ 3.13 (t, J = 8.4
Hz, 2H, -O-CH2-CH2-), 3.66 (d, J = 8.4 Hz, 3H, OCH3), 3.72 (s, 2H, CH2), 4.26 (d, J = 5.4 Hz, 2H,
-NH-CH2-), 4.48 (t, J = 8.4 Hz, 2H, -O-CH2-CH2-), 6.67 (d, J = 8.4 Hz, 1H, ArH), 6.83 (t, J = 7.2 Hz, 1H,
ArH), 6.91 (d, J = 8.4 Hz, 1H, ArH), 6.98 (d, J = 7.2 Hz, 1H, ArH), 7.03 (d, J = 8.4 Hz, 1H, ArH),
7.23-7.10 (m, 3H, ArH), 7.30 (t, J = 9.6 Hz, 1H, ArH), 7.47 (dd, J = 15.6, 7.2 Hz, 1H, ArH), 7.81 (t, J =
13
5.4 Hz, 1H, -NH-CH2-); C NMR (151 MHz, CDCl3) δ 167.32, 163.40, 161.75, 160.93, 159.70, 159.27,
156.89, 140.35, 132.61, 129.71, 128.59, 127.82, 127.67, 127.50, 124.51, 122.89, 111.14, 110.18, 109.14,
104.28, 104.11, 103.94, 77.22, 77.01, 76.80, 71.34, 55.09, 49.55, 29.63, 27.19; ESI-MS m/z: 465 [M + H]+;
m.p. 126.8-127.7°C.
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5-(2,6-Dichlorobenzyl)-4-(2,4-difluorophenyl)-N-((2,3-dihydrobenzofuran-5-yl)methyl)thiazol-2-amine
(12) was obtained as a slight yellow solid in 70.0% yield. 1H NMR(DMSO-d6, 600 MHz) δ 3.19-3.07 (m,
2H, -O-CH2-CH2-), 4.07 (s, 2H, CH2), 4.26 (d, J = 5.4 Hz, 2H, -NH-CH2-), 4.53-4.45 (m, 2H,
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-O-CH2-CH2-), 6.68 (d, J = 8.4 Hz, 1H, ArH), 7.03 (t, J = 9.6 Hz, 1H, ArH), 7.16 (dd, J = 22.2, 13.8 Hz,
2H, ArH), 7.28 (t, J = 8.4 Hz, 1H, ArH), 7.34 (t, J = 9.6 Hz, 1H, ArH), 7.43 (d, J = 8.4 Hz, 2H, ArH), 7.55
(dd, J = 15.6, 7.8 Hz, 1H, ArH), 7.85 (t, J =5.4 Hz, 1H, -NH-CH2-); 13C NMR (151 MHz, CDCl3) δ
167.08, 161.80, 159.77, 141.00, 136.18, 135.44, 132.63, 129.47, 128.48, 128.33, 128.02, 127.73, 127.55,
124.58, 120.06, 111.38, 109.22, 104.27, 104.10, 103.93, 77.23, 77.01, 76.80, 71.36, 49.63, 29.63, 29.06;
ESI-MS m/z: 503 [M + H]+; m.p. 143.8-145.1°C.
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5-(3,5-Difluorobenzyl)-4-(2,4-difluorophenyl)-N-((2,3-dihydrobenzofuran-5-yl)methyl)thiazol-2-amine
(13) was obtained as a slight yellow solid in 76.0% yield. 1H NMR(DMSO-d6, 600 MHz) δ 3.12 (t, J = 8.4
Hz, 2H, -O-CH2-CH2-), 3.83 (s, 2H, CH2), 4.27 (d, J = 5.4 Hz, 2H, -NH-CH2-), 4.47 (t, J = 8.4 Hz, 2H,
-O-CH2-CH2-), 6.68 (d, J = 7.8 Hz, 1H, ArH), 6.79 (d, J = 7.8 Hz, 2H, ArH), 7.04 (d, J = 7.2 Hz, 2H, ArH),
7.13 (t, J = 8.4 Hz, 1H, ArH), 7.19 (s, 1H, ArH), 7.30 (t, J = 9.6 Hz, 1H, ArH), 7.48 (dd, J = 15.6, 7.8 Hz,
1H, ArH), 7.95 (t, J = 5.4 Hz, 1H, -NH-CH2-); 13C NMR (151 MHz, CDCl3) δ 167.64, 163.82, 163.57,
162.17, 161.91, 160.62, 159.79, 159.04, 143.99, 141.37, 132.55, 129.29, 127.61, 124.45, 120.83, 119.2,
111.54, 111.13, 109.23, 104.41, 104.23, 104.07, 102.21, 102.05, 101.88, 77.21, 77.00, 76.79, 71.35, 49.54,
32.62, 29.62; ESI-MS m/z: 471 [M + H]+; m.p. 112.7-114.4°C.
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301
4-(2-Chlorophenyl)-N-((2,3-dihydrobenzofuran-5-yl)methyl)-5-(2-methoxybenzyl)thiazol-2-amine (14)
1
was obtained as a white solid in 55.0% yield. H NMR(DMSO-d6, 600 MHz) δ 3.13 (t, J = 8.7 Hz, 2H,
CH2), 3.66 (s, 2H, CH2), 3.69 (d, J = 1.1 Hz, 3H, OCH3), 4.26 (d, J = 5.6 Hz, 2H, CH2), 4.49 (td, J = 8.8,
1.3 Hz, 2H, CH2), 6.68 (d, J = 8.1 Hz, 1H, ArH), 6.82 (td, J = 7.4, 1.1 Hz, 1H, ArH), 6.91 (d, J = 8.2 Hz,
1H, ArH), 6.95 (dd, J = 7.4, 1.5 Hz, 1H, ArH), 7.05 (dd, J = 8.2, 1.7 Hz, 1H, ArH), 7.17 (td, J = 7.8, 1.6
Hz, 1H, ArH), 7.22 (s, 1H, ArH), 7.39 (m, 3H, ArH), 7.53 (m, 1H, ArH), 7.79 (s, 1H, NH); 13C NMR (151
MHz, CDCl3) δ 167.26, 159.60, 156.86, 144.62, 134.86, 134.22, 132.00, 129.84, 129.65, 129.18, 128.71,
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127.70, 127.39, 126.40, 124.58, 122.12, 120.42, 110.12, 109.04, 71.32, 55.07, 49.50, 29.65, 27.15;
IR(KBr, cm-1): 3088(νC-H), 2965, 2887(νC-H), 1579, 1484(νAr), 1327(δC-H), 1244(νC-O), 1098(νC-O), 1020,
819, 752(δC-H); ESI-MS m/z: 462 [M + H]+; m.p. 129.4-131.1°C.
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4-(2-Chlorophenyl)-5-(2,6-dichlorobenzyl)-N-((2,3-dihydrobenzofuran-5-yl)methyl)thiazol-2-amine (15)
1
was obtained as a white solid in 34% yield. H NMR (DMSO-d6, 600 MHz) δ 3.12 (t, J = 8.4 Hz, 2H,
CH2), 4.02 (s, 2H, CH2), 4.25 (d, J = 6.0 Hz, 2H, CH2), 4.48 (t, J = 8.4 Hz, 2H, CH2), 6.67 (d, J = 8.4 Hz,
1H, ArH), 7.05-7.01 (m, 1H, ArH), 7.20 (s, 1H, ArH), 7.27 (t, J =7.8Hz, 1H, ArH), 7.47-7.39 (m, 5H,
ArH), 7.56-7.53 (m, 1H, ArH), 7.81 (t, J = 5.4 Hz, 1H, NH); 13C NMR (151 MHz, CDCl3) δ 166.85,
159.72, 145.17, 136.22, 135.48, 134.64, 134.03, 132.04, 129.66, 129.30, 128.34, 127.77, 127.50, 126.49,
124.65, 119.53, 109.18, 77.23, 77.02, 76.81, 71.35, 49.65, 29.65, 29.22; IR(KBr, cm-1): 3422, 3200, 3093,
2931, 1587, 1560, 1492, 1434, 1249, 983, 939, 762; ESI-MS m/z 501[M+1]+; m.p. 179-181°C.
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321
4-(2-Chlorophenyl)-5-(3,5-difluorobenzyl)-N-((2,3-dihydrobenzofuran-5-yl)methyl)thiazol-2-amine (16)
1
was obtained as a yellow solid in 51.3% yield. H NMR(DMSO-d6, 600 MHz) δ 3.14 (t, J = 8.7 Hz, 2H,
CH2), 3.79 (s, 2H, CH2), 4.28 (d, J = 5.6 Hz, 2H, CH2), 4.49 (t, J = 8.7 Hz, 2H, CH2), 6.69 (d, J = 8.1 Hz,
1H, ArH), 6.77 (m, 2H, ArH), 7.09-6.99 (m, 2H, ArH), 7.23 (d, J = 1.7 Hz, 1H, ArH), 7.41 (m, J = 9.5, 7.8,
6.4, 2.0 Hz, 3H, ArH), 7.54 (dt, J = 6.7, 1.4 Hz, 1H, ArH), 7.95 (d, J = 6.4 Hz, 1H, NH); 13C NMR (151
MHz, CDCl3) δ 163.77, 162.13, 159.74, 144.07, 134.03, 131.77, 129.82, 129.60, 129.39, 127.73, 127.52,
126.67, 124.57, 111.13, 109.17, 102.11, 101.94, 101.77, 71.35, 49.56, 32.57, 29.63; IR(KBr, cm-1):
3094(νC-H), 2943(νC-H), 1624, 1579, 1484(νAr), 1316(νC-N), 1115(νC-N), 886, 757(δC-H); ESI-MS m/z: 468
[M + H]+; m.p. 89.9-92.2°C.
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N-((2,3-Dihydrobenzofuran-5-yl)methyl)-4-(2-fluorophenyl)-5-(2-methoxybenzyl)thiazol-2-amine (17)
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was obtained as a slight yellow solid in 63.0% yield. H NMR(DMSO-d6, 600 MHz) δ 3.12 (td, J = 8.8,
2.7 Hz, 2H), 3.65 (d, J = 9.4 Hz, 3H), 3.72 (d, J = 3.3 Hz, 2H), 4.25 (dd, J = 8.3, 5.8 Hz, 2H), 4.47 (td, J =
8.7, 1.9 Hz, 2H), 6.67 (dd, J = 8.1, 3.4 Hz, 1H), 6.81 (t, J = 7.4 Hz, 1H), 6.88 (dd, J = 14.2, 8.4 Hz, 1H),
6.95 (m, 1H), 7.05-7.01 (m, 1H), 7.08 (d, J = 2.5 Hz, 1H), 7.00-7.13 (m, 1H), 7.24 (q, J = 8.8, 7.5 Hz, 2H),
13
7.44-7.31 (m, 2H), 7.68 (ddt, J = 28.4, 5.9, 3.4 Hz, 1H), 7.81 (dt, J = 32.8, 5.8 Hz, 1H); C NMR (151
MHz, CDCl3) δ 167.29, 160.79, 159.71, 159.15, 156.93, 156.07, 141.38, 132.29, 131.82, 131.05, 130.33,
129.83, 128.83, 127.75, 127.51, 124.59, 123.94, 123.47, 122.87, 121.52, 120.47, 115.86, 111.84, 110.18,
109.16, 71.36, 55.39, 55.11, 49.60, 29.66, 27.22, 27.00; IR(KBr, cm-1): 3188(νC-H), 2926(νC-H), 1585,
1490(νAr), 1244(νC-O), 1115(νC-O), 1026, 819, 763(δC-H); ESI-MS m/z: 446 [M + H]+; m.p. 96.1-98.0°C.
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5-(2,6-Dichlorobenzyl)-N-((2,3-dihydrobenzofuran-5-yl)methyl)-4-(2-fluorophenyl)thiazol-2-amine (18)
1
was obtained as a white solid in 41% yield. H NMR (DMSO-d6, 600 MHz) δ 3.13 (t, J = 8.4 Hz, 2H,
CH2), 4.09 (s, 2H, CH2), 4.27 (d, J = 6.0 Hz, 2H, CH2), 4.48 (t, J = 8.4 Hz, 2H), 6.68 (d, J = 8.4 Hz, 1H,
ArH), 7.05 – 7.01 (m, 1H, ArH), 7.20 – 7.17 (m, 1H, ArH), 7.32 – 7.26 (m, 3H, ArH), 7.51 (td, J = 7.8, 1.8
Hz, 1H, ArH), 7.83 (t, J = 6.0 Hz, 1H, NH); IR(KBr, cm-1): 3192, 3088, 2926, 1537, 1489, 1433, 1331,
1248, 1218, 980, 929, 775, 752; ESI-MS m/z 485 [M+H]+; m.p. 140-141.5°C.
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5-(3,5-Difluorobenzyl)-N-((2,3-dihydrobenzofuran-5-yl)methyl)-4-(2-fluorophenyl)thiazol-2-amine (19)
1
was obtained as a white solid in 43% yield. H NMR (600 MHz, DMSO-d6) δ 7.96 (t, J = 5.8 Hz, 1H),
7.48-7.39 (m, 2H), 7.30-7.23 (m, 2H), 7.22 (s, 1H), 7.09-7.02 (m, 2H), 6.81 (h, J = 4.6 Hz, 2H), 6.70 (d, J
= 8.0 Hz, 1H), 4.49 (t, J = 8.7 Hz, 2H), 4.30 (d, J = 5.7 Hz, 2H), 3.86 (s, 2H), 3.14 (t, J = 8.7 Hz, 2H); 13C
NMR (151 MHz, CDCl3) δ 167.70, 163.82, 162.18, 160.54, 159.79, 158.90, 144.24, 131.73, 129.92,
129.38, 127.71, 127.56, 124.52, 124.22, 122.84, 120.74, 116.05, 115.90, 111.19, 109.22, 102.16, 101.99,
101.82, 77.23, 77.02, 76.81, 71.37, 49.59, 32.73, 29.65; IR(KBr, cm-1): 3201, 3096, 2931, 1590, 1492,
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1459, 1326, 1114, 987, 764; m.p. 104.5-106°C.
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4-(2,4-Dichlorophenyl)-N-((2,3-dihydrobenzofuran-5-yl)methyl)-5-(2-methoxybenzyl)thiazol-2-amine
1
(20) was obtained as a white solid in 80.0% yield. H NMR (DMSO-d6, 600 MHz) δ 3.11 (t, J = 8.4 Hz,
2H, -O-CH2-CH2-), 3.64 (s, 2H, CH2), 3.67 (s, 3H, OCH3), 4.23 (d, J = 5.4 Hz, 2H, -NH-CH2-), 4.46 (t, J
= 8.4 Hz, 2H, -O-CH2-CH2-), 6.66 (d, J = 8.4 Hz, 1H, ArH), 6.81 (t, J = 7.8 Hz, 1H, ArH), 6.89 (d, J = 8.4
Hz, 1H, ArH), 6.97 – 6.93 (m, 1H, ArH), 7.02 (d, J = 8.4 Hz, 1H, ArH), 7.18 – 7.13 (m, 1H, ArH), 7.19 (s,
1H, ArH), 7.38 (d, J = 8.4 Hz, 1H, ArH), 7.46 (dd, J = 8.4, 2.4 Hz, 1H, ArH), 7.67 (d, J = 2.4 Hz, 1H,
ArH), 7.80 (t, J = 5.4 Hz, 1H, -NH-CH2-); 13C NMR (151 MHz, CDCl3) δ 167.19, 159.67, 156.83, 143.48,
135.01, 134.28, 133.46, 132.73, 129.57, 128.44, 127.82, 127.56, 126.74, 124.52, 122.73, 120.44, 110.15,
109.11, 77.22, 77.01, 76.80, 71.33, 55.06, 49.53, 29.65, 27.20; ESI-MS m/z: 497 [M + H]+; m.p.
147.7-148.5°C.
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5-(2,6-Dichlorobenzyl)-4-(2,4-dichlorophenyl)-N-((2,3-dihydrobenzofuran-5-yl)methyl)thiazol-2-amine
(21) was obtained as a white solid in 29% yield. 1H NMR (DMSO-d6, 600 MHz) δ 3.12 (t, J = 8.7 Hz, 2H,
CH2), 4.02 (s, 2H, CH2), 4.25 (d, J = 5.7 Hz, 2H, CH2), 4.48 (t, J = 8.7 Hz, 2H, CH2), 6.67 (d, J = 8.1 Hz,
1H, ArH), 7.02 (dd, J = 8.1, 1.8 Hz, 1H, ArH), 7.19 (d, J = 1.8 Hz, 1H, ArH), 7.28 (dd, J = 8.4, 7.7 Hz, 1H,
ArH), 7.43 (d, J = 8.0 Hz, 2H, ArH), 7.51-7.45 (m, 2H, ArH), 7.71 (d, J = 1.9 Hz, 1H, ArH), 7.86 (t, J =
5.7 Hz, 1H, NH); 13C NMR (151 MHz, CDCl3) δ 166.86, 135.41, 132.78, 129.88, 129.52, 129.34, 128.50,
128.33, 127.79, 127.56, 126.89, 124.65, 120.02, 109.22, 77.20, 76.99, 76.78, 71.35, 49.66, 29.76, 29.70,
29.37; IR(KBr, cm-1): 2980, 2927, 1474, 1253, 1056, 1026, 966, 848; ESI-MS m/z: 536 [M+3]+; m.p.
172.5-173.5°C.
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4-(2,4-Dichlorophenyl)-5-(3,5-difluorobenzyl)-N-((2,3-dihydrobenzofuran-5-yl)methyl)thiazol-2-amine
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(22) was obtained as a yellow solid in 53.6% yield. 1H NMR (600 MHz, DMSO-d6) δ 7.97 (t, J = 5.7 Hz,
1H, ArH), 7.70 (d, J = 2.1 Hz, 1H, ArH), 7.47 (dd, J = 8.3, 2.1 Hz, 1H, ArH), 7.43 (d, J = 8.3 Hz, 1H,
ArH), 7.25-7.20 (m, 1H, ArH), 7.08-7.00 (m, 2H, ArH), 6.78 (h, J = 4.4 Hz, 2H, ArH), 6.69 (d, J = 8.1 Hz,
1H,NH), 4.49 (t, J = 8.7 Hz, 2H,OCH2CH2), 4.28 (d, J = 5.7 Hz, 2H,CH2), 3.79 (s, 2H,CH2), 3.14 (t, J =
8.7 Hz, 2H,OCH2CH2); 13C NMR (151 MHz, CDCl3) δ 167.39, 159.62, 144.77, 140.34, 139.55, 134.53,
134.10, 132.68, 131.89, 130.38, 129.92, 129.38, 128.42, 128.29, 128.14, 127.67, 127.45, 126.63, 124.53,
122.41, 109.08, 71.32, 49.50, 33.21, 32.96, 29.64; ESI-MS m/z: 503.1 [M + H]+; m.p. 125.8-127.1°C.
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4-(3,5-Difluorophenyl)-N-((2,3-dihydrobenzofuran-5-yl)methyl)-5-(2-methoxybenzyl)thiazol-2-amine
(23) was obtained as a slight yellow solid in 78.0% yield. 1H NMR(DMSO-d6, 600 MHz) δ 3.15 (t, J = 8.4
Hz, 2H, -O-CH2-CH2-), 3.76 (s, 3H, OCH3), 4.04 (s, 2H, CH2), 4.32 (d, J = 5.4 Hz, 2H, -NH-CH2-), 4.50
(t, J = 8.4 Hz, 2H, -O-CH2-CH2-), 6.70 (d, J = 8.4 Hz, 1H, ArH), 6.90 (t, J = 7.2 Hz, 1H, ArH), 7.01 (d, J
= 8.4 Hz, 1H, ArH), 7.11-7.05 (m, 2H, ArH), 7.18 (t, J = 9.0 Hz, 1H, ArH), 7.24 (dd, J = 13.8, 6.6 Hz, 4H,
ArH), 7.91 (t, J = 5.4 Hz, 1H, -NH-CH2-); 13C NMR (151 MHz, CDCl3) δ 166.68, 163.64, 162.00, 159.74,
156.93, 129.54, 129.35, 128.07, 127.73, 127.53, 124.57, 121.93, 120.63, 111.29, 110.30, 109.18, 102.74,
102.57, 102.40, 77.23, 77.02, 76.80, 71.35, 55.15, 49.50, 29.62, 27.26; ESI-MS m/z: 465[M + H]+;
m.p.129.3-131.0℃.
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Biology.
Cells and Plasmids. HEK 293T cells were obtained from the National Platform of Experimental Cell
Resources for Sci-tech (Beijing, China). The pNL4-3.luc.R-E- plasmid was obtained from the National
Institute of Health AIDS Research and Reference Reagent Program. VSVG plasmid was kindly provided
by Dr. Lijun Rong (University of Illinois at Chicago). The pNL4-3 plasmids with RT mutations were
constructed as described previously [19].
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Anti-HIV Activity Assay by Pseudotyped Viruses. Vesicular stomatitis virus glycoprotein (VSVG)
plasmid and env-deficient HIV vector (pNL4-3.luc.R-E- or pNL4-3.luc.R-E-RT-mutant) [20,21]. were
co-transfected into HEK 293T cells using the Ca3(PO4)2 method [22]. The supernatant containing
pseudotyped virions was collected and filtered through 0.22 ꢀm filters 48 h post-transfection. The
harvested viral solution was quantified using p24 concentrations using an ELISA kit (ZeptoMetrix, Cat.:
0801111) and diluted to 0.2 ng p24/mL.
HEK 293T cells were seeded on 24-well plates at a density of 6×104 cells/well one day prior to
infection. The compounds were added to the target cells 15 min prior to infection. Cells were infected
using VSV-G/HIVwt or VSV-G/HIVmut virus calculated to 0.2 ng p24/mL. Forty-eight hours post-infection,
293T cells were lysed, and the luciferase activity of the cell lysate was measured using a Sirius
luminometer (Berthold Detection System) according to the manufacturer’s instructions.Cytotoxicity.
HEK 293T cells were seeded at a density of 1×104 cells/well in 96-well plates. The next day, serially
diluted compounds were incubated with 293T cells and further cultured for 48 hours. The cytotoxicity was
measured using a cell proliferation assay kit (Promega) according to the manufacturer’s instructions.
RT RNA-Dependent DNA Polymerase Activity Detection. HIV-1 reverse transcriptase
RNA-dependent DNA polymerase activity was detected as our previously described [23]. The tested
compound was added into 60 ꢀL reaction system containing 11.7 ng/L poly(rA), 11.7 ng/L oligo(dT)15,
2.8 ꢀM dTTP, 800 nM Digoxigenin-11-dUTP, 40 nM Biotin-11-dUTP, 50 mM Tris-HCl (pH7.8), 290 mM
KCl, 30 mM MgCl2 and 10 mM DTT. The reaction was initiated by the addition of 0.226 ng/L RT
followed by 1 h incubation at 37℃. Then the mixture was transferred into a streptavidin-coated plate
(Roche) to incubate at 37℃ for 1 h. The supernatant was then removed and the plate was washed with
PBS. Anti-DIG-POD was added to the plate and incubated at 37℃ for 1 h. Washed the plate and added
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TMB (3,3,5,5-tetramethylbenzidine). Read OD450 values by a microplate reader (Molecular Devices).
Computational
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Docking and 3D-QSAR. All of the reported calculations were performed on an HP Z800 workstation with
Red Hat Enterprise Linux 5 operating system using the Tripos Sybyl-X 1.2 (Tripos Inc., St Louis, MO, USA)
molecular modeling package. The parameters in the study were set to default values except for those
specifically mentioned.
Acknowledgments
This work was supported by the National Natural Basic Research Program of China (No. 81273561 &
81473256), the National Science and Technology Major Project (No. 2015ZX09102-023-004) and the
central level, scientific research institutes for basic R & D special fund business (No. 2015CX16).
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Appendix A. Supplementary data
Supplementary data related to this article can be found at XXXX
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