Tricyclononene carboxamide derivatives as novel anti-HIV-1 agents

Article abstract of DOI:10.1016/j.ejmech.2010.05.070

By modifying the chemical structure of anti-orthopoxvirus compound ST-246, we designed and synthesized a series of tricyclononene carboxamide derivatives and tested their anti-HIV-1 activity and cytotoxicity. We found that benzoimidazol-containing compound 7g was highly effective in inhibiting HIV-1 R5 infection with an IC₅₀ value of 0.41 μM and a selectivity index of 292, but it exhibited no significant inhibitory activity on HIV-1 reverse transcriptase, integrase and protease. CoMFA was used to analyze structure-activity relationships with good predictive power (r² = 0.921; q² = 0.582). Moreover, the CoMFA model showed that the length of the molecule, the amide, and the amine moieties all played crucial roles in anti-HIV activity. These results suggest that 7g may serve as a lead for the development of novel anti-HIV-1 therapies. A series of tricyclononene carboxamide derivatives based on anti-orthopoxvirus compound ST-246 were synthesized and characterized as novel anti-HIV-1 agents.

Full text of DOI:10.1016/j.ejmech.2010.05.070

European Journal of Medicinal Chemistry 45 (2010) 4096e4103
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Tricyclononene carboxamide derivatives as novel anti-HIV-1 agents
,
a,
*
Ming-xin Dong a, Jian Zhang c, Xu-qing Peng a, Hong Lu b, Liu-hong Yun d, Shibo Jiang b , Qiu-yun Dai
**
a Beijing Institute of Biotechnology, Beijing 100071, PR China
b Lindsley F. Kimball Research Institute, New York Blood Center, 310 East 67th Street, New York, NY 10065, USA
c Department of Pathophysiology, Key Laboratory of Cell Differentiation and Apoptosis of the Chinese Ministry of Education, Shanghai Jiao-Tong University School of Medicine
(SJTU-SM), Shanghai 200025, PR China
d Bejing Institute of Toxicology and Pharmacology, Beijing 100850, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
By modifying the chemical structure of anti-orthopoxvirus compound ST-246, we designed and
synthesized a series of tricyclononene carboxamide derivatives and tested their anti-HIV-1 activity and
cytotoxicity. We found that benzoimidazol-containing compound 7g was highly effective in inhibiting
Received 26 January 2010
Received in revised form
19 May 2010
Accepted 29 May 2010
Available online 9 June 2010
HIV-1 R5 infection with an IC50 value of 0.41
mM and a selectivity index of 292, but it exhibited no
significant inhibitory activity on HIV-1 reverse transcriptase, integrase and protease. CoMFA was used to
analyze structureeactivity relationships with good predictive power (r2 ¼ 0.921; q2 ¼ 0.582). Moreover,
the CoMFA model showed that the length of the molecule, the amide, and the amine moieties all played
crucial roles in anti-HIV activity. These results suggest that 7g may serve as a lead for the development of
novel anti-HIV-1 therapies.
Keywords:
HIV-1
Inhibitor
Synthesis
Ó 2010 Elsevier Masson SAS. All rights reserved.
Tricyclononene carboxamide derivatives
anti-HIV-1 activity
QSAR
1. Introduction
resistant viruses in a growing number of patients has compromised
HAART efficacy and limits therapeutic options [7]. Thus, it is still an
Since the 1980s, acquired immune deficiency syndrome (AIDS)
has become a global epidemic disease. By the end of 2008, more
than 33.2 million people had been infected worldwide, and 25
million people had died [1]. Currently, there are more than 25 drugs
approved for the treatment of human immunodeficiency virus
(HIV) infection. They belong to 6 classes of inhibitors and target
four steps of the HIV life cycle: fusion/entry, reverse transcription,
integration and proteolytic maturation [2]. The combination of
reverse transcriptase inhibitors (RTIs) and protease inhibitors (PIs),
commonly known as Highly Active Antiretroviral Therapy (HAART)
[3], has significantly decreased morbidity and mortality among
patients infected with HIV-1, transforming HIV/AIDS into
a manageable chronic illness. However, HIV-1 can acquire resis-
tance against all currently available antiviral drugs, including RTIs
and PIs, the fusion inhibitor Enfuvirtide (T-20), and the CCR5
antagonist, selzentry [4e6]. Moreover, development of multidrug-
arduous task to discover and develop new anti-HIV drugs with
improved potency and resistance profiles.
Based on the chemical structure of the anti-orthopoxvirus
compound ST-246 [8], a series of tricyclononene carboxamide
derivatives were designed, synthesized and evaluated for their in
vitro antiviral activity against HIV-1 R5 strain Bal infection in
CEMX174 5.25M7 cells. While a majority of these compounds
moderately inhibited HIV-1 infection, the benzoimidazol-contain-
ing compound 7g exhibited the most potent anti-HIV-1 activity
with an IC50 (the concentration causing 50% inhibition) of 0.42
mM
and a selectivity index (SI) of 292. These results suggest that
compound 7g can serve as an attractive lead for development of
novel anti-HIV-1 drugs.
2. Results and discussion
2.1. Chemistry
The synthesis of the designed compounds are outlined in
Schemes 1e3. The commercially available methyl 4-methyl-
benzoate 1 was selected as the starting material for the synthesis of
* Corresponding author. Tel.: þ86 10 66948897; fax: þ86 10 63833521.
** Corresponding author. Tel.: þ1 212 570 3058; fax: þ1 212 570 3099.
E-mail addresses: sjinag@nybloodcenter.org (S. Jiang), qy_dai@yahoo.com
(Q.-y. Dai).
compounds 7aei. Compound
1
reacted with NBS (1-
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.05.070

M.-x. Dong et al. / European Journal of Medicinal Chemistry 45 (2010) 4096e4103
4097
Scheme 1. Synthesis of compound 7aei.
bromopyrrolidine-2,5-dione) in refluxing tetrachloromethane to
give methyl 4-(bromomethyl)benzoate 2 in 90% yield (as shown in
Scheme 1). In the presence of DIEA (Diisopropyl ethylenediamine),
compound 2 coupled with 3aei to provide the corresponding
products 4aei. The hydrazides 5aei were prepared by treating the
esters 4aei with hydrazine hydrate in refluxing ethanol for 0.5 h.
The key intermediate 6 could be generated in excellent yield by
treating cycloheptatriene with maleic anhydride in boiling xylene
as reported in the synthesis of ST-246 [8]. Finally, the synthesis of
the target compounds 7aei were accomplished via the condensa-
tion of equimolar amounts of hydrazides 5aei and intermediate 6
in refluxing ethanol [8]. The side reaction of anhydride 6 with
ethanol is suppressed because the aromatic hydrazide production is
more stable than ester and water form in the condensation process
as well.
For the synthesis of compounds 12aei (Scheme 2), esters 8a,
b and 9dee were chosen as the corresponding starting materials.
Intermediates 10a and 10cee could be prepared easily through
a one-step reaction, while compound 10b was obtained in a four-
step procedure according to a known literature procedure [9,10].
Then, target compounds 12aei could be synthesized by the
procedures described for the synthesis of 7aei.
As shown in Scheme 3, heating equimolar amounts of starting
materials 13aec and intermediate 6 in ethanol at reflux afforded
Scheme 2. Synthesis of compound 12aee.

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M.-x. Dong et al. / European Journal of Medicinal Chemistry 45 (2010) 4096e4103
Scheme 3. Synthesis of compound 14c and 15aec.
the target compound 14c and intermediates 14a, b. The latter
coupled with the corresponding amides to obtain target
compounds 15a, b.
a minor change of benzoimidazol with methyl benzoimidazol (7h)
would result in significant increase in cytotoxicity
(CC50 ¼ 1.02 M), while still maintaining antiviral activity. Taken
a
m
together, block C is very sensitive to the bulk and tropism of the
substitution in this region such that substituents are either too big
or too small will affect its anti-HIV activity.
2.2. Biological evaluation
In addition, the soft linkers in block B might contribute to the
decreased antiviral activity, such as 12cee, 14c and 15b. This
suggests that the rigid benzamide structure is a key structural
feature maintaining anti-HIV-1 activity.
Compound 7g was also tested for inhibitory activity on HIV-1
reverse transcriptase, integrase and protease. The results showed
that it exhibited no significant inhibitory activity against these
The inhibitory activities of compounds on HIV-1 R5 strain Bal
infection in CEMX174 5.25M7 cells were determined by luciferase
assay [11,12], and the in vitro cytotoxicity of compounds on
CEMX174 5.25M7 cells was measured by XTT assay [13]. The IC50
and CC50 values were calculated using the CalcuSyn program [14].
The results are summarized in Table 1.
ST-246, an otherwise active compound against multiple ortho-
poxviruses [8,15], showed no antiviral activity against HIV-1 Bal
(Table 1). However, its derivatives by replacement of the tri-
fluoromethyl group of ST-246, most of which are neonatal
compounds, did exhibit HIV-1 inhibitory activities. Compound 7g,
which contains benzoimidazol instead of the trifluoromethyl group
of compound ST-246, exhibited the most potent antiviral activity
enzymes at the concentration as high as 100 mM, while the corre-
sponding inhibitors e Nevirapine, ABPS, and Indinavir, were highly
effective in inhibiting the activities of reverse transcriptase, inte-
grase and protease, respectively (Table 2). These results suggest
that the compound 7g does not target these enzymes. Since the
compound 7g exhibited potent antiviral activity against HIV-1 R5
strain (Bal), this compound may inhibit HIV-1 infection by targeting
the HIV-1 coreceptor CCR5.
(IC50 ¼ 0.41
m
M) and the lowest cytotoxicity (CC50 ¼ 119.6
mM),
values which are comparable to those of TAK-779, a CCR5 antago-
nist under development [16,17].
2.3. CoMFA analysis
In contrast, compounds 7aed, 7i, 12a, and 12b, which contain
small hydrophilic or hydrophobic groups, were much less active
than compound 7g. On the other hand, the block C (Fig. 1) should
remain medium size because the compounds with bigger sized
block C, such as the compounds 7e and 7f (Scheme 1), exhibited
much lower antiviral activities (Table 1). It was unexpected that
To correlate the structure of title compound with the inhibitory
activity on HIV-1 replication, a 3D QSAR analysis was performed by
use of a CoMFA module following the standard procedure [18,19].
The thirteen compounds with explicit IC50 values were constructed
by Sketch module in SYBYL. Compound 7g was taken as a template
with few rotatable bonds and high potency. CoMFA was applied to
generate a predictive model, which may identify the physico-
chemical features of this kind of compound and provide useful
information for molecular design. All compounds were fit to the
common backbone for alignment, and the resultant alignments of
the training set molecules are shown in Fig. 2. The IC50 values of the
13 structurally related compounds were subjected to CoMFA
analysis. The best model was obtained with the optimum compo-
nent 4, which was indicated by the cross-validation test. The cross-
validation q2 value was 0.582, and a non-cross-validated r2 value
was 0.921, with SEE (standard error of estimation) ¼ 0.282 and
F ¼ 23.229. The contributions of steric and electrostatic fields were
59.3% and 40.7% by PLS analysis [20], respectively. This indicates
that steric factors play a more important role than electronic factors
on the activity of these kinds of compounds.
Table 1
Anti-HIV activities and cytotoxicities of tricyclononene carboxamide derivatives.
code
IC50
(m
M)
CC50
(
m
M)
SI (CC50/IC50)
ST-246
7a
7b
7c
7d
>200
NDa
ND
27.84 ꢀ 4.87
32.71 ꢀ 3.86
57.03 ꢀ 13.34
25.49 ꢀ 3.75
>165.45
82.81 ꢀ 2.24
13.26 ꢀ 2.50
13.18 ꢀ 1.87
57.21 ꢀ 4.94
17.08 ꢀ 2.16
>165.45
2.01
2.48
1.00
1.51
7e
7f
7g
7h
7i
0.41 ꢀ 0.16
1.68 ꢀ 0.38
31.45 ꢀ 1.55
7.8 ꢀ 0.02
119.59 ꢀ 5.04
1.02 ꢀ 0.27
29.69 ꢀ 6.17
3.54 ꢀ 1.02
>185.40
>117.96
>181.20
ND
84.00 ꢀ 9.79
44.03 ꢀ 12.85
>221.36
7.88 ꢀ 0.36
291.68
0.61
0.94
12a
12b
12c
12d
12e
14c
15a
15b
TAK-779b
0.45
>185.40
>117.96
154.8 ꢀ 21.6
89.40 ꢀ 2.26
30.42 ꢀ 2.05
5.70 ꢀ 1.90
>221.36
>1.17
The predicted pIC50 and experimental data of the compounds
are listed in Table 3. Most of the compounds were predicted
correctly. Fig. 3 illustrates the contour maps of the field contribu-
tions determined by different properties as well as the template
compound 7g. The contour maps indicate how the change of the
molecule affects the activity. The green contours indicate where the
bulky group substitutions improve the activity, while yellow
2.76
7.72
0.05 ꢀ 0.004
157.6
a
ND ¼ Not done.
b
TAK-779 is a CCR5 antagonist [16,17].

M.-x. Dong et al. / European Journal of Medicinal Chemistry 45 (2010) 4096e4103
4099
Fig. 1. The structure of ST-246 and its derivative, compound 7g.
contours depict regions where steric bulk would not be tolerated.
The blue and red polyhedrons denote the favorable sites for posi-
tively and negatively charged groups, respectively. It can be
observed that most contour maps tend to focus at the right side of
the molecular structures.
predictive power (r2 ¼ 0.921; q2 ¼ 0.582). Based on the success of
this highly predictive QSAR model and CoMFA contours, further
design, synthesis, biological evaluation are continuing in our
laboratory.
Since most of the diverse substitutions occurred at the right side
of the compounds, the disproportion of the field distribution was
generated from the limited structural changes on the left side.
Within the CoMFA contour map, as shown in Fig. 3, several red
polyhedrons, which indicate regions favorable to negative charge,
are located on the upper middle of the molecule, nearby or pointing
to the carbonyl group in the amide moiety. The amide moiety is one
of the important structural characteristics of these kinds of
compounds and exists in every active compound, including TAK-
799. The red polyhedrons here show that electro-negative carbonyl
oxygen has a favorable distribution for this activity. The green
polyhedrons located in the lower right of the molecule indicate that
the bulky group substitutions enhance the activity. The aza-indole
ring indicated here gives a high potency for compound 7a. The blue
polyhedrons in the vicinity of the N atom show that the electro-
positive groups have an advantage with respect to affinity.
However, the amine moiety is another important characteristic of
these compounds and can also be found in TAK-799. For
compounds 12d and 12e, the benzene ring in the right side is
connected to the amide positioned near the yellow polyhedrons,
but it is not long enough to extend to the green contour map region.
Consequently, the activities of these kinds of compounds are rela-
tively weak. In sum, the length of the molecule, the amide moieties
and the amine moiety all play crucial roles in the affinity of the
studied molecules binding to the target.
4. Experimental section
4.1. Chemistry
All chemicals and reagents used in current study were of
analytical grade. Thin-layer chromatography (TLC) was performed
on silica gel GF254 plates. Silica gel for column chromatography was
obtained from Qingdao Haiyang Chemical Company. Melting points
were obtained with an RY-1 melting point apparatus without
correction. All 1H NMR and 13C NMR spectra were measured on
a JNM-ECA-400 (400 MHz) spectrometer using tetramethylsilane
(TMS) as an internal standard. Mass spectra (MS) were measured
on either a Micromass ZabSpec or an ABI PerkineElmer Sciex API-
3000 mass spectrometer with electrospray ionization.
4.1.1. The synthesis of intermediate 2
To a stirred solution of methyl 4-methylbenzoate (3.07 g,
20 mmol) in tetrachloromethane (30 mL) was added NBS (4.14 g,
23 mmol). The mixture was heated under reflux for 5 h. After
filtration, the organic layer was evaporated in vacuo to give 4.22 g
the aimed product as yellow oil, yield: 90%.
4.1.2. General procedure for the preparation of intermediates 4aei
To the solution of intermediate 2 (4.22 g, 18 mmol) in CH2Cl2
(35 mL) were added the corresponding starting materials 3aei
(20 mmol) and DIEA (3.1 mL, 20 mmol), and the reaction mixture
was stirred at room temperature overnight. Then, the mixture was
washed with water (20 mL) and hydrochloric acid solution (2 M,
20 mL), dried (Na2SO4), filtered and evaporated in vacuo to afford
the crude products 4aei, which were used directly in the next step
without further purification.
3. Conclusion
In summary, a series of tricyclononene carboxamide derivatives
were synthesized and tested for their inhibitory activity against
HIV-1 Bal infection in CEMX174 5.25M7 cells. Since the benzoimi-
dazol-containing compound 7g showed submicromolar anti-HIV-1
activity and low cytotoxicity toward the same cell line, it could act
as a promising lead compound for the future optimization of CCR5
receptor inhibitor. The CoMFA model was employed to compre-
hensively elucidate the structureeactivity relationship with good
Table 2
Inhibition of the compound 7g on the activities of HIV-1 reverse transcriptase,
intergrase and protease.
Compound
IC50 (mM) for inhibiting the activity of
Reverse transcriptase
Protease
Intergrase
Nevirapine
Indinavir
ABPS
13.20
ND
ND
ND
<0.01
ND
ND
ND
0.42
>228
7g
>457
>114
*Nevirapine, Indinavir, ABPS are inhibitors of HIV-1 reverse transcriptase, protease,
and intergrase, respectively. ND ¼ not done.
Fig. 2. Common substructure alignment.

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M.-x. Dong et al. / European Journal of Medicinal Chemistry 45 (2010) 4096e4103
Table 3
4.1.4.2. N-(3,3a,4,4a,5,5a,6,6a-octahydro-1,3-dioxo-4,6-ethenocyclo-
prop[f]isoindol-2 (1H)-yl)-4-((3-chlorophenylamino)methyl)benza-
mide (7b). White crystals, yield: 91%; mp: 175e177 ꢁC; 1H NMR
Experimental pIC50 and the corresponding predicted values for the training
compounds of the CoMFA model.
Code
7a
7b
7c
7d
7e
7g
7h
(400 MHz, CDCl3):
d
8.11(s, 1H, NH), 7.76 (d, J ¼ 7.6 Hz, 2H, Ar-H),
pIC50
Predicted pIC50
4.082
4.018
4.878
4.713
4.880
4.829
4.243
4.355
4.768
4.885
6.854
6.352
5.775
6.315
7.38 (d, J ¼ 8.4 Hz, 2H, Ar-H), 7.06 (t, J ¼ 8.4 Hz, 1H, Ar-H), 6.70 (d,
J ¼ 8.0 Hz, 1H, Ar-H), 6.60 (s, 1H, Ar-H), 6.47 (d, J ¼ 8.0 Hz, 1H, Ar-H),
5.83 (t, 2H, CH]CH), 4.36 (s, 2H, Ar-CH2), 3.45 (m, 2H, 2 ꢂ COCH),
3.13 (m, 2H, 2 ꢂ CHeCH]CH), 1.13 (m, 2H, 2 ꢂ CHeCH2), 0.32 (m,
Code
7i
12a
12d
12e
14c
15a
pIC50
Predicted pIC50
4.502
4.477
5.108
5.017
3.810
3.857
4.049
4.020
4.517
4.650
5.244
5.221
2H, CH2); 13C NMR (100 MHz, CDCl3):
d 174.86, 164.72, 148.59,
144.05, 135.05, 130.28, 129.71, 128.11, 127.80, 127.40, 117.96, 112.79,
111.31, 47.71, 43.80, 33.39, 9.59, 4.42; MS (Turbo Spray) m/z calcd for
C25H22ClN3O3 (M þ 1): 448.1, found: 448.2.
4.1.3. General procedure for the preparation of intermediates 5aei
and 11aee
4.1.4.3. N-(3,3a,4,4a,5,5a,6,6a-octahydro-1,3-dioxo-4,6-ethenocyclo-
prop[f]isoindol-2 (1H)-yl)-4-((isobutyl(phenyl)amino)methyl)benza-
mide (7c). White crystals, yield: 87%; mp: 158e159 ꢁC; 1H NMR
To the solution of intermediates 4aei or 10aee (10 mmol) in
ethanol (10 mL) was added 85% hydrazine hydrate (2 mL, 30 mmol).
The mixture was heated under reflux for 1 h. DM water (20 mL) was
added to the reaction mixture, and the resulting product was
extracted with CH2Cl2 (25 mL) three times. The combined organic
layers were washed with DM water, dried (Na2SO4), filtered and
evaporated in vacuo to give crude products 5aei or 11aee, which
would be used directly in the next step without further purification.
(400 MHz, CDCl3):
d
7.93 (s, 1H, CONH), 7.74 (d, J ¼ 7.6 Hz, 2H, Ar-
H), 7.25 (m, 2H, Ar-H), 7.17 (m, 2H, Ar-H), 6.63 (m, 3H, Ar-H), 5.83
(t, 2H, CH]CH), 4.62 (s, 2H, Ar-CH2), 3.45 (m, 2H, 2 ꢂ COCH), 3.23
(d, J ¼ 7.2 Hz, 2H, NeCH2), 3.13 (m, 2H, 2 ꢂ CHeCH]CH), 2.15 (m,
1H, CH(CH3)2), 1.13 (d, J ¼ 3.6 Hz, 2H, 2 ꢂ CHeCH2), 0.96 (d,
J ¼ 6.8 Hz, 6H, 2 ꢂ CH3), 0.31 (m, 2H, CH2); 13C NMR (100 MHz):
d
174.92, 164.78, 148.20, 144.52, 129.14, 128.41, 127.97, 127.76,
4.1.4. General procedure for the target compounds 7aei, 12aee and
compounds 14aec
126.81, 116.32, 112.39, 59.91, 55.22, 43.74, 33.33, 27.34, 20.50, 9.55,
4.40; MS (Turbo Spray) m/z calcd for C29H31N3O3 (M þ 1): 470.2,
found: 469.8.
To a solution of intermediates 5aei and compound 6 (Yield:
85.4%; mp: 94e96 ꢁC) in ethanol (5 mL) were added two drops of
DIEA. The resulting mixture was heated under reflux for 6 h. Upon
cooling to room temperature, the organic mixture was evaporated
under reduced pressure to give a white residue. The residue was
purified by column chromatography to give the target compounds
7aei. A similar procedure was used to synthesize 12aee and 14aec.
4.1.4.4. N-(3,3a,4,4a,5,5a,6,6a-octahydro-1,3-dioxo-4,6-ethenocyclo-
prop[f]isoindol-2 (1H)-yl)-4-((enthyl(phenyl)amino)methyl)benza-
mide (7d). White crystals, yield: 90%; mp: 155e156 ꢁC; 1H NMR
(400 MHz, DMSO-d6):
d 11.03 (s, CONH), 7.83 (m, 2 H, Ar-H), 7.34
(m, 2H, Ar-H),7.10 (m, 2H, Ar-H), 6.62 (m, 3H, Ar-H), 5.79 (m, 2H,
CH]CH), 4.59 (s, 2H, Ar-CH2), 3.48 (m, 2H, CH2CH3), 3.28 (m, 4H,
2 ꢂ COCH, 2 ꢂ CHeCH]CH), 1.15 (m, 2H, 2 ꢂ CHeCH2), 1.13 (m, 3H,
4.1.4.1. N-(3,3a,4,4a,5,5a,6,6a-octahydro-1,3-dioxo-4,6-ethenocyclo-
prop[f]isoindol-2
mide (7a). White crystals, Yield 76%; mp 123e124 ꢁC; 1H NMR
(400 MHz, CDCl3):
(1H)-yl)-4-(4-methylpiperazin-1-methyl)benza-
CH3), 0.27 (m, 2H, CH2); 13C NMR (100 MHz, CDCl3):
d 175.00,
164.78, 147.98, 144.85, 129.25, 128.01, 127.72, 126.68, 116.36, 112.09,
53.79, 45.33, 43.72, 33.31, 12.09, 9.53, 4.39; MS (Turbo Spray) m/z
calcd for C27H27N3O3 (M þ 1): 442.2, found: 442.3.
d
7.79 (d, J ¼ 8.4 Hz, 2H, Ar-H), 7.38 (d, J ¼ 8.4 Hz,
2H, Ar-H), 5.84 (t, J ¼ 3.6 Hz, 2H, CH]CH), 3.53 (s, 2H, Ar-CH2), 3.46
(m, 2H, 2 ꢂ COCH), 3.14 (m, 2H, 2 ꢂ CHeCH]CH), 2.50 (m, 8H,
piperazine-H), 2.34 (s, 3H, CH3), 1.14 (d, J ¼ 4 Hz, 2H, 2 ꢂ CHeCH2),
4.1.4.5. N-(3,3a,4,4a,5,5a,6,6a-octahydro-1,3-dioxo-4,6-ethenocyclo-
prop[f]isoindol-2 (1H)-yl)-4-((benzyl(phenyl)amino)methyl)benza-
mide (7e). White crystals, yield: 64%; mp: 162e163 ꢁC; 1H NMR
0.32 (m, 2H, CH2); 13C NMR (100 MHz, DMSO-d6):
d 174.88, 164.33,
143.28, 128.80, 127.66, 127.53, 127.24, 61.38, 54.47, 52.22, 45.36,
43.26, 42.90, 33.00, 32.71, 9.18, 4.09; MS (FAB, Glycerin) m/z calcd
for C24H28N4O3 (M þ 1): 421.2, found: 421.3.
(400 Hz, CDCl3):
d 8.13 (s, 1H, CONH), 7.76 (m, 2H, Ar-H), 7.31 (m,
7H, Ar-H), 7.17 (m, 2H, Ar-H), 6.71 (m, 3H, Ar-H), 5.83 (t, 2H, CH]
CH), 4.64 (d, 4H, 2 ꢂ Ar-CH2), 3.45 (m, 2H, 2 ꢂ COCH), 3.13 (m, 2H,
2 ꢂ CHeCH]CH), 1.12 (m, 2H, 2 ꢂ CHeCH2), 0.31 (m, 2H, CH2); 13
C
NMR (100 MHz, CDCl3):
d 174.85, 164.76, 148.68, 144.26, 138.10,
129.29, 128.66, 128.41, 128.07, 127.80, 127.04, 126.92, 126.68, 117.20,
112.53, 54.40, 54.08, 43.76, 33.35, 9.57, 4.41; MS (Turbo Spray) m/z
calcd for C32H29N3O3 (M þ 1): 504.2, found: 504.4.
4.1.4.6. N-(3,3a,4,4a,5,5a,6,6a-octahydro-1,3-dioxo-4,6-ethenocyclo-
prop[f]isoindol-2 (1H)-yl)-4-((2-oxo-3-(pyridin-4-yl)imidazolidin-1-
yl)methyl)benzamide (7f). White crystals, yield: 32%; mp: >250 ꢁC;
1H NMR (400 MHz, DMSO-d6):
d 11.08 (br, 1H, CONH), 8.40 (d,
J ¼ 5.2 Hz, 2H, pyridine-H), 7.90 (d, J ¼ 7.6 Hz, 2H, Ar-H), 7.58 (d,
J ¼ 6.4 Hz, 2H, Ar-H), 7.46 (d, J ¼ 8 Hz, 2H, pyridine-H), 5.80 (t, 2H,
CH]CH), 4.50 (s, 2H, Ar-CH2), 3.85 (dd, J1 ¼ 2.4 Hz, J2 ¼ 6.0 Hz, 2H,
imidazolidin-CH2), 3.44 (dd, J1 ¼ 8.4 Hz, J2 ¼ 8.0 Hz, 2H, imidazo-
lidin-CH2), 3.29 (m, 4H, 2 ꢂ COCH, 2 ꢂ CHeCH]CH), 1.12 (m, 2H,
2 ꢂ CHeCH2), 0.26 (m, 2H, CH2); 13C NMR (100 MHz, CD3OD):
Fig. 3. Compound 7g shown together with the contour map of CoMFA steric and
electrostatic fields. Sterically favored areas (contribution level of 80%) and disfavored
areas (contribution level of 20%) are represented by green polyhedron and yellow
polyhedron, respectively. Blue and red polyhedrons depict the favorable site for
positively (80%) and negatively (20%) charged groups, respectively.
d
176.90, 158.42, 149.97, 149.44, 143.03, 129.41, 129.28, 129.11,
128.92, 112.77, 112.63, 45.01, 42.76, 42.27, 39.07, 34.77, 10.44, 4.74;
MS (Turbo Spray) m/z calcd for C27H25N5O4 (M þ 1): 484.2, found:
484.3.

M.-x. Dong et al. / European Journal of Medicinal Chemistry 45 (2010) 4096e4103
4101
4.1.4.7. N-(3,3a,4,4a,5,5a,6,6a-octahydro-1,3-dioxo-4,6-ethenocyclo-
prop[f]isoindol-2 (1H)-yl)-4-((1H-benzo[d]imidazol-1-yl)methyl)
benzamide (7g). White crystals, yield 60%; mp: 272e274 ꢁC; 1H
NMR (400 MHz, DMSO-d6): 11.05 (s, 1H, CONH), 8.42 (s, H, imi-
7.17 (t, J ¼ 7.6 Hz, 1H, Ar-H), 7.06 (t, J ¼ 6.8 Hz, 2H, Ar-H), 5.73 (t, 2H,
CH]CH), 4.54 (d, J ¼ 12.8 Hz, 1H, piperidine-CH), 3.24 (m, 4H,
2 ꢂ COCH, 2 ꢂ CHeCH]CH), 3.02 (m, 1H, piperidine-H), 2.89 (m,
1H, piperidine-H), 2.55 (m, 2H, piperidine-H), 2.17 (s, 3H, CH3), 2.11
(s, 3H, CH3), 1.86 (d, J ¼ 11.6 Hz, 1H, piperidine-H), 1.68 (d,
J ¼ 12.8 Hz, 1H, piperidine-H), 1.50 (d, J ¼ 10 Hz, 1H, piperidine-H),
1.39 (d, J ¼ 8.8 Hz, 1H, piperidine-H), 1.14 (m, 2H, 2 ꢂ CHeCH2), 0.25
d
dazol-2-H), 7.85 (m, 2H, Ar-H), 7.67 (m, 1H, Ar-H), 7.42 (m, 3H, Ar-
H), 7.20 (m, 2H, Ar-H), 5.78 (t, 2H, CH]CH), 5.60 (s, 2H, Ar-CH2),
3.32(m, 4H, 2 ꢂ COCH, 2 ꢂ CHeCH]CH), 1.07 (m, 2H, 2 ꢂ CHeCH2),
0.26 (m, 2H, CH2); 13C NMR (100 MHz, CD3OD):
d
176.60, 144.59,
(m, 2H, CH2); 13C NMR (100 MHz, CD3OD):
d 174.74, 169.76, 135.02,
143.18, 142.19, 129.28, 128.64, 128.25, 124.50, 123.81, 111.66, 44.73,
34.47, 10.18, 4.53; MS (FAB, Glycerin) m/z calcd for C26H22N4O3
(M þ 1): 439.2, found: 439.2(M þ 1).
132.92, 132.79, 127.91, 126.88, 126.84, 126.61, 44.61, 42.95, 39.56,
39.44, 32.75, 28.00, 27.41, 17.32, 17.00, 8.45, 2.74; MS (Turbo Spray)
m/z calcd for C26H29N3O4 (M þ 1): 448.2, found: 448.1.
4.1.4.8. N-(3,3a,4,4a,5,5a,6,6a-octahydro-1,3-dioxo-4,6-ethenocyclo-
prop[f]isoindol-2 (1H)-yl)-4-((2-methyl-1H-benzo[d]imidazol-1-yl)
methyl)benzamide (7h). White crystals, yield: 74%; mp: 261e262 ꢁC;
4.1.4.13. N-(3,3a,4,4a,5,5a,6,6a-octahydro-1,3-dioxo-4,6-ethanocy-
cloprop[f]isoindol-2 (1H)-yl)-N-(2-amino-2-oxo-1-phenylethyl)ben-
zamide (12d). White crystals, yield: 45%; mp: 223e225 ꢁC; 1H NMR
1H NMR (400 MHz, CDCl3):
d
10.62 (s, 1H, CONH), 7.88 (d, J ¼ 8.0 Hz,
(400 MHz, DMSO-d6):
d
11.00 (s, 1H, CONHN), 9.02 (d, J ¼ 8.4 Hz, 1H,
2H, Ar-H), 7.68 (d, J ¼ 7.6 Hz, 1H, Ar-H), 7.23 (m, 3H, Ar-H), 6.94 (d,
J ¼ 7.6 Hz, 2H, Ar-H), 5.68 (t, 2H, CH]CH), 5.35(s, 2H, Ar-CH2), 3.43
(m, 2H, 2 ꢂ COCH), 3.12 (m, 2H, 2 ꢂ CHeCH]CH), 2.35 (s, 3H, CH3),
1.11 (d, J ¼ 4.4 Hz, 2H, 2 ꢂ CHeCH2), 0.28 (m, 2H, CH2); 13C NMR
CONH), 7.94 (m, 2H, Ar-H), 7.54 (m, 3H, Ar-H), 7.45 (m, 2H, Ar-H),
7.37 (m, 3H, Ar-H), 5.95 (d, J ¼ 8.0 Hz, 1H, Ar-CH), 5.72 (t, 2H, CH]
CH), 3.23 (m, 4H, 2 ꢂ COCH, 2 ꢂ CHeCH]CH), 1.07 (m, 2H,
2 ꢂ CHeCH2), 0.23 (m, 2H, CH2); 13C NMR (100 MHz, CDCl3):
(100 MHz, CD3OD):
d
174.82, 164.12, 151.89, 142.34, 141.65, 135.28,
d 174.29, 168.74, 167.05, 136.53, 133.14, 131.90, 128.85, 128.43,
130.12, 128.21, 127.52, 127.22, 126.78, 121.71, 121.42, 118.33, 109.94,
45.96, 43.26, 42.89, 32.98, 32.69, 13.57, 9.16, 4.07; MS (Turbo Spray)
m/z calcd for C27H24N4O3 (M þ 1): 453.2, found: 453.1.
127.59, 127.25, 55.45, 43.65, 33.20, 9.46, 4.27; MS (FAB, Glycerin) m/
z calcd for C26H23N3O4 (M þ 1): 442.2, found: 442.2.
4.1.4.14. N-(3,3a,4,4a,5,5a,6,6a-octahydro-1,3-dioxo-4,6-ethanocy-
cloprop[f]isoindol-2 (1H)-yl)-N-(1-amino-1-oxo-3-phenylpropan-2-
yl)benzamide (12e). White crystals, yield: 88%; mp: 108e109 ꢁC; 1H
4.1.4.9. N-(3,3a,4,4a,5,5a,6,6a-octahydro-1,3-dioxo-4,6-ethenocyclo-
prop[f]isoindol-2
(7i). White crystals, yield: 85%; mp: 189e191 ꢁC; 1H NMR (400 MHz,
CDCl3):
(1H)-yl)-4-((4-fluorophenoxy)methyl)benzamide
NMR (400 MHz, DMSO-d6):
d10.82 (s, 1H, CONHN), 8.68 (d,
d
8.03(s, 1H, CONH), 7.83 (d, J ¼ 8.4 Hz, 2H, Ar-H), 7.48 (d,
J ¼ 8.4 Hz, 1H, CONH), 7.78 (m, 2H, Ar-H), 7.45(m, 8H, Ar-H), 5.75 (t,
2H, CH]CH), 4.88 (t, J ¼ 8.4 Hz, 1H, COCHN), 3.19 (m, 4H, 2 ꢂ COCH,
Ar-CH2, 2 ꢂ CHeCH]CH), 1.16 (m, 2H, 2 ꢂ CHeCH2), 0.26 (m, 2H,
J ¼ 8.0 Hz, 2H, Ar-H), 6.98 (m, 2H, Ar-H), 6.89 (m, 2H, Ar-H), 5.84 (t, 2H,
CH]CH), 5.07(s, 2H, Ar-CH2), 3.46 (m, 2H, 2 ꢂ COCH), 3.14 (m, 2H,
2 ꢂ CHeCH]CH), 1.13 (m, 2H, 2 ꢂ CHeCH2), 0.31 (m, 2H, CH2); 13
C
CH2); 13C NMR (100 MHz, CDCl3):
d 174.60, 174.37, 169.58, 168.23,
NMR(100 MHz, CD3OD):
d
176.71,174.19,159.75,157.37,155.98,143.62,
135.81, 133.18, 133.03, 129.48, 128.68, 128.60, 127.67, 127.09, 52.78,
43.67, 36.78, 36.69, 33.27, 18.40, 9.51, 4.35; MS (FAB, Glycerin) m/z
calcd for C27H25N3O4 (M þ 1): 456.2, found: 456.2.
129.34, 128.90, 128.71, 128.25, 128.02, 116.96, 116.87, 116.66, 116.41,
70.53, 46.94, 44.79, 35.84, 34.77, 34.57, 10.23, 10.06, 4.55, 3.08; MS
(Turbo Spray) m/z calcd for C25H21FN2O4 (M þ 1): 433.1, found: 433.4.
4.1.4.15. N-(3,3a,4,4a,5,5a,6,6a-octahydro-1,3-dioxo-4,6-ethanocy-
4.1.4.10. N-(3,3a,4,4a,5,5a,6,6a-octahydro-1,3-dioxo-4,6-ethanocy-
cloprop[f]isoindol-2 (1H)-yl)-4-(2,5-dimethyl-1H-pyrrol-1-yl)benza-
mide (12a). White crystals, yield: 86%; mp: 224e226 ꢁC; 1H NMR
cloprop[f]isoindol-2
(1H)-yl)-N0-benzyl-N0-methylpropane-1,3-
diamine (14c). Yellow oil, yield: 95%; 1H NMR (400 MHz, CDCl3):
d
7.31(m, 5H, Ar-H), 5.66 (t, J ¼ 1.6 Hz, 2H, CH]CH), 3.45 (m, 6H,
(400 MHz, CDCl3):
d
7.95 (m, 2H, Ar-H), 7.31 (m, 2H, Ar-H), 5.93 (s,
2 ꢂ COCH, Ar-CH2, CONeCH2), 2.93 (m, 2H, 2 ꢂ CHeCH]CH), 2.33
(t, J ¼ 7.2 Hz, 2H, CH2N(CH3)), 2.16 (s, 3H, CH3), 1.67 (m, 2H,
CH2(CH2)2), 1.08 (m, 2H, 2 ꢂ CHeCH2), 0.27 (m, 2H, CH2); 13C NMR
2H, pyrrole-CH]CH), 5.86 (t, J ¼ 4.0 Hz, 2H, CH]CH), 3.48 (m, 2H,
2 ꢂ COCH), 3.17 (m, 2H, 2 ꢂ CHeCH]CH), 2.03 (s, 6H, 2 ꢂ CH3), 1.16
(m, 2H, 2 ꢂ CHeCH2), 0.34 (m, 2H, CH2); 13C NMR (100 MHz,
(100 MHz, CDCl3):
d 178.52, 129.06, 128.20, 127.51, 127.08, 62.11,
CD3OD):
d
176.39, 166.82, 143.95, 129.46, 129.17, 128.81, 128.45,
54.38, 45.09, 41.65, 36.46, 33.33, 25.23, 9.78, 4.65; MS (Turbo Spray)
107.15, 44.53, 34.30, 12.64, 9.95, 4.27; MS (Turbo Spray) m/z calcd
for C24H23N3O3 (M þ 1): 402.2, found: 402.1.
m/z calcd for C22H26N2O2 (M þ 1): 351.2, found: 351.3.
4.1.5. General procedure for the preparation of target compounds
15aeb
To the solution of intermediate 14a or 14b (1 mmol) in DMF
(Dimethylformamide, 10 mL) were added the corresponding amide
(0.1 g, 1.1 mmol) and DIEA (0.23 mL, 1.5 mmol). The solution was
chilled to 0e5 ꢁC under an atmosphere of nitrogen. HBTU (2-(1H-
4.1.4.11. N-(3,3a,4,4a,5,5a,6,6a-octahydro-1,3-dioxo-4,6-ethenocy-
cloprop[f]isoindol-2 (1H)-yl)-4-(3-isopropyl-5-methyl-4H-1,2,4-tri-
azol-4-yl)benzamide (12b). White crystals, yield: 77%; mp:
276e277 ꢁC; 1H NMR (400 MHz, CDCl3):
d 8.69 (s, 1H, CONH), 8.13
(d, J ¼ 8.0 Hz, 2H, Ar-H), 7.35 (d, J ¼ 8.0 Hz, 2H, Ar-H), 5.86 (t,
J ¼ 4.0 Hz, 2H, CH]CH), 3.48 (m, 2H, 2 ꢂ COCH), 3.17 (m, 2H,
2 ꢂ CHeCH]CH), 2.78 (m, 1H, CH(CH3)2), 2.24 (s, 3H, CH3), 1.24 (d,
J ¼ 6.8 Hz, 6H, 2 ꢂ CH3),1.17 (m, 2H, 2 ꢂ CHeCH2),0.33 (m, 2H, CH2);
Benzotriazole-1-yl)-1,1,3,3-tetramethyluronium
hexa-
fluorophosphate, 0.57 g, 1.5 mmol) was added and the solution
stirred for 10 min, then warmed to room temperature and stirred an
additional 2.5 h. Water (50 mL) was added to the reaction mixture to
precipitate the crude products. The crude products were purified by
column chromatography to give the target compounds 15a, b.
13C NMR (100 MHz, DMSO-d6):
d 184.26, 172.99, 167.65, 159.91,
147.00, 141.58, 141.18, 138.90, 137.35, 137.06, 136.78, 69.22, 52.44,
42.50, 34.13, 30.5, 23.56, 20.09, 18.68, 13.61; MS (Turbo Spray) m/z
calcd for C24H25N5O3 (M þ 1): 432.2, found: 432.2.
4.1.5.1. N-(3,3a,4,4a,5,5a,6,6-octahydro-1,3-dioxo-4,6-ethanocyclo-
4.1.4.12. N-(3,3a,4,4a,5,5a,6,6a-octahydro-1,3-dioxo-4,6-ethanocy-
cloprop[f]isoindol-2 (1H)-yl)-N-(1-(2,6-dimethylbenzoyl)piperidine-
4-yl)carboxamide (12c). White crystals, yield: 91%; mp:
prop[f]isoindol-2 (1H)-yl)-4-(formanilide)benzyl (15a). White crys-
tals, yield: 86%; mp: 224e226 ꢁC; 1H NMR (400 MHz, CDCl3):
d 8.01
(s, 1H, CONH), 7.78 (d, J ¼ 8.0 Hz, 2H, Ar-H), 7.64 (d, J ¼ 8.0 Hz, 2H,
185e187 ꢁC; 1H NMR (400 MHz, DMSO-d6):
d 10.53 (s, 1H, CONH),
Ar-H), 7.36 (m, 4H, Ar-H), 7.15 (m, 1H, Ar-H), 5.62 (t, 2H, CH]CH),

4102
M.-x. Dong et al. / European Journal of Medicinal Chemistry 45 (2010) 4096e4103
4.59 (s, 2H, Ar-CH2), 3.37 (t, J ¼ 1.6 Hz, 2H, 2 ꢂ COCH), 2.99 (m, 2H,
Medical Sciences). The increase of fluorescence was detected at
490 nm, using 355 nm excitation wavelength in BMG POLARstar
Galaxy Fluorescence Plate Readers at 37 ꢁC.
2 ꢂ CHeCH]CH),1.09 (m, 2H, 2 ꢂ CHeCH2), 0.33 (m, 2H, CH2); 13
C
NMR (100 MHz, CDCl3):
d 178.16, 165.42, 139.43, 137.96, 134.25,
128.91, 128.54, 127.57, 127.27, 124.43, 120.27, 45.19, 41.51, 33.39,
9.374, 4.61; MS (Turbo Spray) m/z calcd for C25H22N2O3 (M þ 1):
399.2, found: 399.2.
The inhibitory activity of the compound 7g on HIV-1 integrase
activity was measured using the procedure described by Guo et al.
[23]. An intergrase inhibitor, achyranthes bidentata polysaccharide
sulfate (ABPS, MW: 4000 Da) was included as a control. Briefly,
wells of a 96 well Covalink-NH plate (NUNC) were coated with
donor substrate (50P-ACC CTT TTA GTC AGT GTG GAA AAT CTC TAG
CAGT-30; 30-GAA AAT CAG TCA CAC CTT TTA GAG ATC GTCA-50),
followed by three washes with PBS. The reaction mixture
(20 mmol/L TriseHCl, pH7.8, 7.5 mmol/L MgCl2, 20 mmol/L DTT,
0.1% NP40, and 25 mmol/L MOPS, pH7.2), the compound 7g or ABPS
at indicated concentrations of compounds and the HIV-1 intergrase
4.1.5.2. N-(3,3a,4,4a,5,5a,6,6a-octahydro-1,3-dioxo-4,6-ethanocyclo-
prop[f]isoindol-2 (1H)-yl)-2-((1H-benzo[d]imidazol -1-yl) meth-
anone) ethyl (15b). White crystals, yield: 64%; mp: 164e166 ꢁC; 1H
NMR (400 MHz, CDCl3):
d 8.35 (s, 1H, imidazol-H), 8.22 (m, 1H, Ar-
H), 7.80 (m, 1H, Ar-H), 7.42 (m, 2H, Ar-H), 5.66 (m, 2H, CH]CH),
3.96 (t, J ¼ 7.2 Hz, 2H, NeCH2), 3.40 (m, 2H, 2 ꢂ COCH), 3.27 (t,
J ¼ 7.2 Hz, 2H, COCH2), 3.00 (d, J ¼ 1.6 Hz, 2H, 2 ꢂ CHeCH]CH), 1.10
(m, 2H, 2 ꢂ CHeCH2), 0.28 (m,1H, CH2); 13C NMR (100 MHz, DMSO-
were added in a final 100 m
L volume, co-incubated at 37 ꢁC for 1 h.
d6):
d
177.89, 171.73, 169.06, 143.48, 143.24, 141.86, 131.15, 127.24,
Then the target biotin linked DNA (50-TGA CCA AGG GCT AAT TCA
CT-30-biotin; biotin-30-ACT GGT TCC CGA TTA AGT GA-50) was
added, followed by an incubation at 37 ꢁC for 1 h. After washing
three times with PBS contained 0.05% Tween 20, and an incubation
with 1% BSA at 37 ꢁC for 40 min, the alkaline phosphatase reaction
system was used to detected the absorbance at 405 nm.
125.31, 124.63, 121.71, 119.93, 115.15, 44.61, 44.55, 33.46, 33.05,
32.90, 31.64, 9.37, 4.32; MS (Turbo Spray) m/z calcd for C21H19N3O3
(M þ 1): 362.1, found: 362.3.
4.2. Biological evaluation
The inhibitory activity of the compound 7g on HIV-1 reverse
transcriptase inhibitor was tested as previously described [24].
Nevirapine (MW: 266.3 Da), an HIV-1 reverse transcriptase inhib-
itor, was used as a reference compound. Briefly, Poly A was
immobilized via its 50-terminal phosphate to Covalink-NH micro-
4.2.1. Reagents
HIV-1 Bal strain (R5) was obtained from the NIH AIDS Research
and Reference ReagentProgram. LymphoidcelllineCEMx174 5.25M7,
kindly provided by C. Cheng-Mayer, is stably transduced with an HIV-
1 long terminal repeat (LTR)-green fluorescent protein (GFP) reporter
and luciferase reporter construct. These cells, expressing CD4 and
both coreceptors, CXCR4 and CCR5 [11], were maintained in RPMI
1640 medium supplemented with 10% fetal bovine serum (FBS), 1
of puromycin/mL, and 200 g of G418/mL. HIV-1 intergrase was
expressed in E. coli transformed with F185K/C280S IN1-288
recombinant plasmid as described by Jenkins TM et al. [21].
titer plates. The reaction mixture (60
(50 mmol/L TriseHCl (pH 8.3), 6 mmol/L MgCl2, 100 mmol/L KCl,
5 mmol/L DTT (DL-dithiothreitol), 0.13 mg/ml BSA, 0.31 g/mL oligo
(dT)15, 0.26 M biotin-11-dUTP, 0.52 M dTTP), compound 7g or
Nevirapine at grade concentrations, and 0.04 L of the HIV-1
mL) contained reaction buffer
m
mg
m
m
m
m
reverse transcriptase (Calbiochem). After incubation at 37 ꢁC for
1 h, the plate was washed with a wash buffer (10 mmol/L TriseHCl
(pH 7.5), 0.15 mol/L NaCl, 1 mmol/L EDTA, 0.01% Tween 20). After
4.2.2. Determination of the inhibitory activity of the compounds
against HIV-1 Bal infection in CEMx174 5.25M7 cells
The inhibitory activities of compounds on infection by HIV-1 R5
strain Bal in CEMx174 5.25M7 cells were determined as previously
100 mL of 1% BSA and SA-HRPO (streptavidin-horseradish peroxi-
dase) (Pharmacia) in wash buffer without Tween 20 were added to
each well and incubated for 30 min at 37 ꢁC, followed by three
washes with wash buffer. Then, 100 mL of TMB (tetramethylbenzi-
described [11,12]. Briefly, 50
m
l of a compound at graded concen-
dine) solution at 100 ng/mL was added before an incubation for
15 min at room temperature. The reaction was stopped by addition
of 50 mL 2 M H2SO4 and the absorbance at 450 nm was measured by
EMax Precision Microplate ELISA Reader (Molecuar Devices).
The percent inhibition and IC50 value of the compounds for
inhibiting the activities of the HIV-1 enzymes were calculated using
CalcuSyn program as described above.
tration in triplicate was incubated with an equal volume of the HIV-
1 Bal at an MOI of 0.01 at 37 ꢁC for 30 min, followed by addition of
100
m
l of CEMx174 5.25M7 cells (5 ꢂ 105/mL). After incubation at
37 ꢁC overnight, the culture medium was replaced with fresh
medium without CAP. On day 3 post-infection, the cells were har-
vested and lysed for analysis of luciferase activity, using a luciferase
assay kit (Promega, Madison, WI) and a luminometer (Ultra 386;
Tecan, Durham, NC) according to the manufacturer’s instructions.
The percent inhibition of luciferase activity and the concentrations
for 50% inhibition (IC50) were calculated using a computer program,
designated CalcuSyn, kindly provided by Dr. T. C. Chou
(SloaneKettering Cancer Center, New York) [14].
4.2.4. Assessment of in vitro cytotoxicity
The in vitro cytotoxicity of compounds on CEMx174 5.25M7 cells
was measured by XTT assays as previously described [13]. Briefly,
100 mL of the test compound at graded concentration was added to
equal volumes of cells (5 ꢂ 105/mL) in wells of 96-well plates. After
incubation at 37 ꢁC for 4 days, 50
mL of XTT solution (1 mg/mL)
4.2.3. Determination of the inhibitory activity of the compound 7g
against HIV-1 protease, integrase and reverse transcriptase
The inhibitory activity of the compound 7g on HIV-1 protease
activity was detected using a previously reported method [22] with
modification. Indinavir (MW 613.8 Da), the HIV-1 protease inhib-
containing 0.02 mM of phenazine methosulfate (PMS) was added.
After 4 h, the absorbance at 450 nm was measured with an ELISA
reader. The CC50 (concentration for 50% cytotoxicity) values were
calculated using the CalcuSyn computer program [14].
itor, was included as a control. Briefly, 1
the graded concentrations or Indinavir at 10 nM was added into
200 L reaction buffer (0.1 mol/L CH3COONa, pH5.0, 0.9 mol/L NaCl,
1 mmol/L DTT, 1 mmol/L EDTA, and 5 mol/L fluorescent peptide
substrate from Molecular Probes) in wells of a microtiter plate.
After preincubated for 20 min at 37 ꢁC, the reaction was initiated by
the addition of the purified recombinant HIV-1 protease (provided
by the Institute of Medicinal Biotechnology at Chinese Academy of
m
L of the compound 7g at
4.3. CoMFA analysis
m
All computational studies were performed in the molecular
modeling package SYBYL8.0 on a Dell Precision 7400 workstation.
Systematic conformation search was adopted to find the lowest
energy of each compound. Partial atomic charges were assigned to
each atom using the GasteigereeHückel method. All structures of
compounds were optimized using a conjugate gradient minimizer
m

M.-x. Dong et al. / European Journal of Medicinal Chemistry 45 (2010) 4096e4103
4103
[6] R. Delgado, Enferm. Infecc. Microbiol. Clin. 11 (2008) 28e33.
[7] P. Yeni, J. Hepatol. 44 (2006) 100e103.
and the standard Tripos force field with a distance-dependent
dielectric function. The minimization was terminated when the
energy gradient convergence criterion of 0.001 kcal/mol was ach-
ieved or when the 1000-step minimization cycle limit was exceeded.
The CoMFA descriptors, steric and electrostatic field energies, were
calculated using the SYBYL default parameters: 2Aꢁ grid points
[8] R.B. Thomas, R.R. Susan, O. Elizabeth, J.B. Christopher, C.P. Daniel, S.C. Marc,
R. Gerry, T. Sanjeev, W.H. John, O.B. Robert, R.K. Earl, A.K. Kathy, D.C. Dai,
G. Yang, H. Dennis, J. Robert, J. Med. Chem. 50 (2007) 1442e1444.
[9] B.C. Jordan, B.D. Clive, PCT Int. Appl (WO). Pfizer Ltd., 2007, 066201.
[10] K. Liu, H. Lu, L. Hou, Z. Qi, C. Teixeira, F. Barbault, B.T. Fan, S.W. Liu, S.B. Jiang,
L. Xie, J. Med. Chem. 51 (2008) 7843e7854.
[11] H. Lu, Q. Zhao, G. Wallace, S. Liu, Y. He, R. Shattock, A.R. Neurath, S.B. Jiang,
AIDS. Res. Hum. Retroviruses 22 (2006) 411e418.
[12] Y.X. He, J.W. Cheng, J.J. Li, Z. Qi, H. Lu, M.X. Dong, S.B. Jiang, Q.Y. Dai, J. Virol. 82
(2008) 6349e6355.
spacing, sp3 carbon probe atom with þ1 charge and a minimum
s
(column filtering) of 2.0 kcal/mol, and an energy cutoff of 30.0 kcal/
mol [25].
[13] A.K. Debnath, L. Radigan, S.B. Jiang, J. Med. Chem. 42 (1999) 3203e3209.
[14] T.C. Chou, M.P. Hayball, Calusyn: Windows Software for Dose Effect Analysis.
BIOSOFT, Ferguson, MO 63125, USA, 1991.
Acknowledgements
[15] G. Yang, D.C. Pevear, M.H. Davies, M.S. Collett, T. Bailey, S. Rippen, L. Barone,
C. Burns, G. Rhodes, S. Tohan, J.W. Huggins, R.O. Baker, R.L. Mark Buller,
E. Touchette, K. Waller, J. Schriewer, J. Neyts, E. Declercq, K. Jones, D. Hruby,
R. Jordans, J. Virol. 79 (2005) 13139e13149.
[16] M. Baba, O. Nishimura, N. Kanzaki, M. Okamoto, H. Sawada, Y. Iizawa,
M. Shiraishi, Y. Aramaki, K. Okonogi, Y. Ogawa, K. Meguro, M. Fujino, Proc.
Natl. Acad. Sci. U.S.A 96 (1999) 5698e5703.
[17] T. Ikemoto, A. Nishiguchi, H. Mitsudera, M. Wakimasu, K. Tomimatsu, Tetra-
hedron 57 (2001) 1525e1529.
[18] R.D. Cramer, D.E. Patterson, J.D. Bunce, J. Am. Chem. Soc. 110 (1988)
5959e5967.
[19] G.F. Yang, X.Q. Huang, Curr. Pharm. Des. 12 (2006) 4601e4611.
[20] T.A. Halgren, J. Am. Chem. Soc. 112 (1990) 4710e4723.
[21] D.J. Hazuda, J.C. Hastings, A.L. Wolfe, et al., Nucleic Acid Res. 22 (1994)
1121e1122.
[22] B. Dong, T. Zhang, P.Z. Tao, Chin. J. AIDS STD. 12 (2006) 402e405.
[23] Z.M. Guo, H.S. Chen, Chin. J. Exp. Clin. Virol. 16 (2002) 119e123.
[24] Y.X. Han, J.N. Li, L.X. Fu, J.D. Jiang, Chin. J. Microbiol. Immunol. 24 (2004)
748e750.
We thank Dr. Zonggen Peng from the Institute of Medicinal
Biotechnology at Chinese Academy of Medical Sciences, for the tests
of inhibitory activities of HIV-1 protease, reverse transcriptase and
integrase. This work was supported by the China Specific Project for
Developing New Drugs during the Eleventh Five-Year Plan Period
(NO. 2009ZX09103-628, 2009ZX09503-015) awarded to Q.Y.D and
by NIH grant AI 46221 awarded to S.J.
References
[1] UNAIDS. Geneva, Switzerland; 2008.
[2] W.C. Greene, Z. Debyser, Y. Ikeda, E.O. Freed, E. Stephens, W. Yonemoto, R.
W. Buckheit, J.A. Esté, T. Cihlar, Antivir. Res. 80 (2008) 251e265.
[3] J. Cohen, Science 296 (2002) 2320e2324.
[4] Y.X. He, J.W. Cheng, H. Lu, J.J. Li, J. Hu, Z. Qi, Z.H. Liu, S.B. Jiang, Q.Y. Dai, Proc.
Natl. Acad. Sci. U.S.A 105 (2008) 16332e16337.
[25] P. Shah, A. Mittal, P.V. Bharatam, Eur. J. Med. Chem. 43 (2008) 2784e2791.
[5] C.S. Adamson, E.O. Freed, Drug Discov. Today 13 (2008) 424e432.