Synthesis and anti-HIV-1 activity evaluation for novel 3a,6a-dihydro-1H-pyrrolo[3,4-c]pyrazole-4,6-dione derivatives

Article abstract of DOI:10.3390/molecules21091198

The search for new molecular constructs that resemble the critical two-metal binding pharmacophore and the halo-substituted phenyl functionality required for HIV-1 integrase (IN) inhibition represents a vibrant area of research within drug discovery. As reported herein, we have modified our recently disclosed 1-[2-(4-fluorophenyl)ethyl]-pyrrole-2,5-dione scaffolds to design 35 novel compounds with improved biological activities against HIV-1. These new compounds show single-digit micromolar antiviral potencies against HIV-1 and low toxicity. Among of them, compound 9g and 15i had potent anti-HIV-1 activities (EC₅₀ < 5 μM) and excellent therapeutic index (TI, CC₅₀ /EC₅₀ > 100). These two compounds have potential as lead compounds for further optimization into clinical anti-HIV-1 agents.

Full text of DOI:10.3390/molecules21091198

molecules
Article
Synthesis and Anti-HIV-1 Activity Evaluation for Novel
3a,6a-Dihydro-1H-pyrrolo[3,4-c]pyrazole-4,6-dione
Derivatives
Guan-Nan Liu 1,2, Rong-Hua Luo 3, Yu Zhou 2, Xing-Jie Zhang 3, Jian Li 2, Liu-Meng Yang 3,
Yong-Tang Zheng 3,* and Hong Liu 2,
*
1
College of Life Sciences, China Jiliang University, Hangzhou 310018, Zhejiang, China; gnliu@cjlu.edu.cn
CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica,
Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 201203, China;
zhouyu@mail.shcnc.ac.cn (Y.Z.); jianl@mail.shcnc.ac.cn (J.L.)
Key Laboratory of Animal Models and Human Disease Mechanisms of Chinese Academy of Sciences and
Yunnan Province, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, Yunnan,
China; luorh@mail.kiz.ac.cn (R.-H.L.); kmhz123@163.com (X.-J.Z.); lmyang@mail.kiz.ac.cn (L.-M.Y.)
Correspondence: zhengyt@mail.kiz.ac.cn (Y.-T.Z.); hliu@mail.shcnc.ac.cn (H.L.);
2
3
*
Tel.: +86-871-6519-5684 (Y.-T.Z.); +86-21-5080-7042 (H.L.)
Academic Editor: Diego Muñoz-Torrero
Received: 9 August 2016; Accepted: 30 August 2016; Published: 8 September 2016
Abstract: The search for new molecular constructs that resemble the critical two-metal binding
pharmacophore and the halo-substituted phenyl functionality required for HIV-1 integrase (IN)
inhibition represents a vibrant area of research within drug discovery. As reported herein, we have
modified our recently disclosed 1-[2-(4-fluorophenyl)ethyl]-pyrrole-2,5-dione scaffolds to design
35 novel compounds with improved biological activities against HIV-1. These new compounds
show single-digit micromolar antiviral potencies against HIV-1 and low toxicity. Among of them,
compound 9g and 15i had potent anti-HIV-1 activities (EC50 < 5 µM) and excellent therapeutic
index (TI, CC50/EC50 > 100). These two compounds have potential as lead compounds for further
optimization into clinical anti-HIV-1 agents.
Keywords: anti-HIV-1; integrase; synthesis; SAR; AIDS
1. Introduction
Human immunodeficiency virus type 1 (HIV-1) is the main causative agent of acquired
immunodeficiency syndrome (AIDS), which remains a serious public health problem throughout
the world [
mediates insertion of the double-stranded DNA provirus into the host genome [
the final step before irreversible and productive HIV-1 infection of the target cell [
was identified as an attractive target nearly 20 years ago [ ], the first generation drugs targeting
IN (raltegravir, RAL, and elvitegravir, ELV) were only recently approved by the Food and Drug
Administration (FDA) [ ] as part of combination antiretroviral therapy (Figure 1). Unfortunately,
1
]. HIV-1 integrase (IN) is a virally encoded enzyme essential for virus replication, which
]. Integration is
]. Although it
2
3
4
1
5,6
continuous mutation of the viral genome leads to multi-drug resistant viral strains that are no longer
susceptible to the current therapy [7,8].
As a result there has been a surge of research driving force to address these limitations with
significant contributions from both academia and industry [2,9,10]. The current studies have been
focused on the identification of new chemical classes that are able to retain the intrinsic potency and
structural elements of the bidentate metal-binding pharmacophore (red color) which is essential for
strand transfer inhibition and the halo-substituted phenyl rings (blue color) which interact with
Molecules 2016, 21, 1198; doi:10.3390/molecules21091198
www.mdpi.com/journal/molecules

Molecules 2016, 21, 1198
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the penultimate cytosine base near the 3’-end of the viral DNA to prevent the insertion of the
viral DNA into the host genome [11 12]. These efforts provide several distinct structural classes.
Some second-generation drug targeting IN, such as dolutegravir (DTG, ) [13], which has been
approved for clinical use, and GSK364735 ( ) [14 15] is in phase II clinical trials (Figure 1). Although
they display superior characteristics to RAL and EVG, the cross-resistance has still been observed and
the subsequent secondary mutations diminish the inhibitory activity [16 18]. Therefore, intensified
,
2
3
,
–
search for IN inhibitors with novel chemical features to overcome the resistance and more investigation
of the structure-activity relationship are urgently needed.
N
O
O
O
R
N
NH
N
4
H
R=H, OH, NHAr
F
O
F
F
N
N
N
N
OH
Raltegravir (RAL, 1)
Dolutegravir (DTG, 2)
NH
O
O
OH
OH
O
O
5
H
N
O
EC =1.65μM
N
50
NH
N
F
TI=56.3
N
O
F
F
O
N
O
OEt
OH
O Me
F
N
O
O
O
H
N
N
GSK 364735 (3)
OH
F
OH
6
Phase II
O
OH
OH
N
N
F
Figure 1. HIV-1 integrase inhibitors.
Recently, 1-hydroxy-1,8-naphthyridin-2H-one-3-carboxamides
inhibit wild-type IN in biochemical assays and show good antiviral efficacies in single-round infection
assays of HIV-1 infectivity [19 20] (Figure 1). Importantly, members of these series retain good
antiviral potency against a set of mutants resistant to in these latter assays. A considerable
feature of is that the carbonyl oxygen of the 2’,4’-difluorobenzyl amide group (blue color), which
4 have been reported to potently
,
1
4
binds to the penultimate base adjacent to the 3’-end, may not be an obligatory component of the
metal-chelating triad (red color). As a consequence, there may be greater flexibility in this region
of the molecule than is found with other inhibitors, such as
halobenzyl amide participates in Mg2+ chelation. This flexibility is reminiscent of what is seen
with , where the flexibility of the haloamide component is thought to contribute to the ability of
to maintain efficacy against certain forms of IN that are resistant to . Simultaneously, we reported
that 7-hydroxy-1,3-dioxo-2,3-dihydro-1H-pyrrolo[3,4-c]pyridine-4-carboxylates such as , show good
anti-HIV activities [21] (Figure 1). In the case of , the binding function is served by a 4’-fluorophenyl
1, where the carbonyl oxygen of the
2
2
1
5
5
ethyl group, which is appended at the 1-position of the pyrrole-2,5-dione nucleus. Previous work has
shown that the nature and pattern of halogen phenyl substitution can significantly affect the potency
of IN inhibitors [22]. In the discovery of compound
5, we found that a 4’-fluorophenyl moiety, which
is also present in and , was superior to the other halophenyl rings we tested. As a continuation
1
3
of our research in this field, we further modified the 1-[2-(4-fluorophenyl)ethyl]-pyrrole-2,5-dione
nucleus by incorporating a new 2-(2,3-dihydroxyphenyl)-4,5-dihydropyrazole at the 3- and 4-positions
to form a new component of the metal-chelating triad (red color) independent of the halogen phenyl
functionality (blue color). Additionally, we noted that an extra aryl group in compound
4 can increase

Molecules 2016, 21, 1198
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their inhibitory potency [20]. Therefore, we have designed compound
6
which has two 4’-fluorophenyl
moieties and a novel bidentate metal-binding pharmacophore (Figure 1). Subsequently, we prepared
a series of analogues
and redesign.
6–16 using an iterative process of design, synthesis, biological evaluation,
2. Results and Discussion
2.1. Chemical Synthesis
As outlined in Scheme 1, target compounds 6–16 were successfully synthesized from commercially
available reagents. In a similar reported methodology, the treatment of maleic anhydride with the
corresponding phenylethylamines in acetic anhydride in the presence of sodium acetate gave the
intermediate 1-phenylethyl-pyrrole-2,5-diones M1 [23]. Another intermediaries M2 was obtained
from aryl-aldehydes by treatment of the corresponding phenylhydrazines in ethanol without further
purification. Subsequently, M1 were reacted with M2 in refluxing ethanol via 1,3-dipolar cycloaddition
reaction yielding the target compounds
1H-NMR and MS.
6–
16
[
24]. All the target compounds were characterized by
O
O
Phenylethylamines
a
N
O
R1
M1
c
O
O
O
R
R
H
N
N
R
NH2
NH
R2
N
N
+
N
O
b
O
R2
R2
R1
6—16
M2
Scheme 1. Synthetic route of compounds 6–16. Reagents and conditions: (a) NaOAc, (Ac)2O, reflux,
30 min; (b) AcOH, EtOH, room temperature, 8 h; (c) EtOH, reflux, 12–20 h.
2.2. Anti-HIV-1 Evaluation
Target compounds 6–16 were evaluated for their inhibitory activity against HIV-1 replication in
acutely infected C8166 cells in vitro according to the previously described method [25 26], and AZT
,
was used as a positive control. The assay results of the target compounds are presented in Tables 1
and 2, expressed as CC50, EC50 and therapeutic index (TI). Several of these new compounds show
single-digit micromolar antiviral potencies against HIV-1 and low toxicity for cultured cells, which in
two cases (9g and 15i), results in therapeutic index (TI, CC50/EC50) of greater than 100.
Using
6 as a starting point, the initial series of compounds was designed to examine the role
of aromatic functionality of the R-substitution on the nucleus (Table 1). We initiated our work by
replacing the 2-, and 3-hydroxy on the phenyl group. Despite of the improved anti-HIV-1 potencies
of analogs without the 2-hydroxy (7a and 7b, EC50 values of 5.37 and 9.21
cytotoxicities were increased, revealing the essentiality of the hydroxy on the 2-position. The removal
of the 3-hydroxy gave the compound with a better anti-HIV-1 potency and a comparable cytotoxicity,
µM, respectively), the
8
suggesting the possibility of replacing the hydroxy on the 3-position of the phenyl group. Thus, we
carefully continued our structural optimization effort by introducing some other groups into the 3- and
other positions of the phenyl group and remaining the hydroxy on the 2-position. Although moving
of the hydroxy from 3-position of the phenyl group to 4-position (9a) slightly decreased the potency
against HIV-1 relative to
6. The fluorine substituent was tolerated well, both in positions 3 and 5 of
the phenyl ring (compounds 9b and 9c), with the EC50 values of 5.74
µ
M and 4.21 M, respectively.
µ
When the fluorine atom in position 3 was replaced by a chlorine atom (9d) or a nitro group (9e),
both the activities and TI values decreased. Interestingly, the introduction of the electron-donating

Molecules 2016, 21, 1198
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groups to the 3-position (giving 9f and 9g) enhanced the potencies, particularly when utilizing the
methoxy group.
Table 1. Anti-HIV-1 activity of compounds 6–14 in vitro a.
O
R
N
N
N
F
O
6—14
F
c
b
TI d
Compd.
R
EC50 (µM)
CC50 (µM)
6
7a
7b
8
9a
9b
9c
9d
9e
2,3-OHPh
2-OMe,3-OHPh
Ph
>431.92
39.14
26.39
5.37
9.21
10.11
35.31
5.74
4.21
20.42
7.55
4.90
3.98
8.52
4.73
115.87
41.94
11.13
7.11
>16.37
7.29
16.03
>44.25
4.91
24.58
37.70
16.23
6.56
30.73
>105.26
2.51
39.85
>3.51
2.15
>36.56
31.29
2.00
147.64
>447.35
173.48
141.01
158.78
331.51
49.53
150.52
>418.88
21.36
188.62
>407.19
90.29
>406.96
222.45
9.03
2-OHPh
2,4-OHPh
2-OH,3-FPh
2-OH,5-FPh
2-OH,3-ClPh
2-OH,3-NO2Ph
2-OH,3-MePh
2-OH,3-OMePh
2-OH,4-OMePh
2-OH,5-OMePh
2-OH,3-OEtPh
2-OH,3-OMe,5-NO2Ph
2,3-OMePh
benzo[1,3]dioxol-4-yl
2-OH-naphthalen-1-yl
thiazol-2-yl
9f
9g
10a
10b
10c
11
12a
12b
13
14a
14b
AZT e
4.50
158.41
149.85
3779
35.55
26.16
0.0085
4.46
5.73
444,588
pyridin-2-yl
a
Values are means of two separate experiments; b CC50 (50% cytotoxic concentration), concentration of drug
that causes 50% reduction in total C8166 cell number; c EC50 (50% effective concentration), concentration of
drug that reduces syncytia formation by 50%; d In vitro therapeutic index (CC50 value/EC50 value) and e AZT
was used as a positive control.
We were gratified to discover that the 2-hydroxy-3methoxyphenyl substituted derivative 9g was
nearly eight times more active than its parent 2,3-dihydroxyphenyl substituted counterpart
values of 3.98 and 26.39 M, respectively) with a TI of greater than 100. The shift of the methoxy group
from position 3 to 4 and 5 gave the compounds 10a and 10b with slightly decreased anti-HIV-1 activities
(EC50 values of 8.52 and 4.73 M, respectively), while replacing the methoxy group on position 3 by
ethoxy (compound 10c) or adding a nitro group to position 5 of the phenyl group (compound 11
6 (EC50
µ
µ
)
had negative impacts on anti-HIV-1 potency too. Unfortunately, the attempt to replace the hydroxy
group in position 2 by electron-donating groups (compounds 12a and 12b) led to the decreased
potencies. We sequentially continued our structural optimization effort by introduction some other
aryl groups into the R group. Good biological activity was observed when 2-hydroxy-naphthalen-1-yl
was introduced into the molecule (13, EC50 = 4.50 µM), while the introduction of the thiazol-2-yl group
led to a decrease in the activity by an order of magnitude (compound 14a), as well as its substitution
with pyridin-2-yl group (compound 14b).
Encouraged by the improved potency of compound 9g, the diversities of R1 and R2 substituents
were further investigated (Table 2). We prepared an additional series of analogues 15 in which several
substituents were introduced at the R2-position. Historically, a halo-substituted moiety in this position
has been optimal, but little structural variation was tolerated in this series.

Molecules 2016, 21, 1198
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Both the shift of the fluorine atom from the 4-position to the 2-, and 3-position (15a and 15b),
or the replacement of the fluorine atom on 4-position by chlorine atom (15c) led to a potency decrease,
even with the use of the 2,4-difluoro phenyl group as in
2 and 4 (15d). In general, the derivatives with
electron-withdrawing groups also provided poor activities against HIV-1 with EC50 values >30 µM
(15e–15g).
15e
4-fluorine atom on the phenyl group the resulting compound 15i showed comparable potency to 9g
(EC50 = 4.10 M, TI > 103.09), while the introduction of the 4-methoxy (15j) had a negative impact
on HIV-1 inhibition. Finally, a diversity of R1 substituents has been screened (series 16), which led
a slightly decrease of the antiviral activities, with EC50 values of more than 10 M. An exception was
–15, as well as the unsubstituted derivative 15h. Luckily, when the methyl group replaced the
µ
µ
the 2-fluoro substituted derivative 16a, which provided the best potency (EC50 = 2.52 µM) among all
the series, but also the highest cytotoxicity.
Table 2. Anti-HIV activity of compounds 15–16 in vitro a.
O
O
OH
N
N
N
O
R2
R1
15—16
c
b
TI d
Compd.
R1
R2
EC50 (µM)
CC50 (µM)
15a
15b
15c
15d
15e
15f
15g
15h
15i
4-F
4-F
4-F
4-F
4-F
4-F
4-F
4-F
4-F
4-F
2-F
3-F
4-Cl
H
3-F
2-F
4-Cl
128.89
>418.88
166.33
>403.67
>379.17
>372.44
46.02
>435.77
>422.83
>408.61
19.58
143.35
70.13
>435.73
219.37
3779
12.08
112.85
25.47
65.92
32.10
65.34
30.43
28.63
4.10
10.67
>3.71
6.52
>6.12
>11.81
>5.70
1.51
>15.22
>103.09
>19.34
7.78
11.01
6.73
2,4-F
4-CF3
4-SO2Me
4-SO2NH2
H
4-Me
4-OMe
4-F
15j
21.12
2.52
16a
16b
16c
16d
16e
AZT e
4-F
4-F
4-F
4-F
13.02
10.43
18.87
87.15
0.0085
>23.09
2.52
444,588
4-OMe
a
Values are means of two separate experiments; b CC50 (50% cytotoxic concentration), concentration of drug
that causes 50% reduction in total C8166 cell number; c EC50 (50% effective concentration), concentration of
drug that reduces syncytia formation by 50%; d In vitro therapeutic index (CC50 value/EC50 value) and e AZT
was used as a positive control.
3. Experimental Section
3.1. Chemistry
3.1.1. General Information
All commercially available compounds and solvents were used without further purification.
1
All target products were characterized by their H-NMR and MS spectra. 1H-NMR spectra were
recorded in deuterochloroform (CDCl3) on a Bruker AMX-300 NMR spectrometer (Bruker Cor.,
Zurich, Switzerland). Chemical shifts were reported in parts per million (ppm, δ) downfield from
tetramethylsilane. Proton coupling patterns are described as singlet (s), doublet (d), triplet (t), quartet
(q), multiplet (m), and broad (br). Low- and high-resolution mass spectra (LRMS and HRMS) were

Molecules 2016, 21, 1198
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measured on Finnigan MAT 95 and LCQ-DE-CA mass spectrometers (Thermo Scientific Inc., Waltham,
MA, USA).
3.1.2. General Procedure for the Preparation of M1
In a 100 mL three-necked flask provided with a stirrer, a reflux condenser, and a dropping funnel
were placed maleic anhydride (0.98 g, 10 mmol) and diethyl ether (25 mL). The maleic anhydride
dissolved upon stirring, at which point a solution of the corresponding phenylethylamines (10 mmol,
1 equiv) in diethyl ether (5 mL) was run through the dropping funnel. The resulting thick suspension
was stirred at room temperature for 1 h and was then cooled in an ice bath, filtered and dried.
Subsequently, the residue was added to a flask containing a solution of anhydrous sodium acetate
(0.33 g, 4 mmol) in acetic anhydride (5 mL) and stirred under reflux for 30 min. The reaction mixture
was then cooled to room temperature in a cold water bath and was then poured into an ice-water
mixture (30 mL). The precipitated product was recovered by filtration, washed three times with
portions 10 mL of ice-cold water, and dried as white to yellow solids. The physical data of M1 have
been reported recently [21].
3.1.3. General Procedure for the Preparation of M2
To a solution of aryl-aldehydes (5.0 mmol) and the corresponding phenylhydrazines (5.0 mmol)
in ethanol (25 mL), was added one drop of acetic acid. The reaction mixture was stirred at room
temperature for 8 h. After removing of the solvent, the residue was dissolved in CH2Cl2 (DCM) (20 mL),
washed by water (10 mL), dried over Na2SO4, filtered and concentrated to give the hydrazones M2
that were used without further purification.
3.1.4. General Procedure for the Preparation of Target Compounds 6–16
A solution of hydrazone M2 (0.40 mmol) and M1 (0.40 mmol) in EtOH was heated at reflux
for 12–20 h. After cooling, the solvents were removed. The residue was subjected to a column
chromatography on silica gel with petroleum ether (PE)/EtOAc (4/1, v/v) as eluent to afford the target
compounds 6–16.
3-(2,3-Dihydroxyphenyl)-1-(4-fluorophenyl)-5-[2-(4-fluorophenyl)ethyl]-3a,6a-dihydro-1H-pyrrolo[3,4-
c]pyrazole-4,6-dione (6 δ 7.44–7.38 (m, 3H), 7.13–6.99 (m, 5H), 6.93
): Pale yellow solid; 1H-NMR (CDCl3)
(t, J = 7.5 Hz, 1H), 6.83 (t, J = 8.4 Hz, 2H), 4.99–4.88 (m, 2H), 3.83 (t, J = 6.9 Hz, 2H), 2.94 (t, J = 7.2 Hz,
2H) ppm; MS (EI, m/z) 463 [M]+; HRMS (EI) m/z calcd C25H19F2N3O4 [M]+ 463.1344, found 463.1341.
1-(4-Fluorophenyl)-5-[2-(4-fluorophenyl)ethyl]-3-(3-hydroxy-2-methoxyphenyl)-3a,6a-dihydro-1H-pyrrolo[3,4-
c]pyrazole-4,6-dione (7a): Pale yellow solid; 1H-NMR (CDCl3)
δ 7.54 (d, J = 8.7 Hz, 1H), 7.50–7.45 (m,
2H), 7.06–7.00 (m, 4H), 6.95 (t, J = 8.1 Hz, 2H), 6.47–6.43 (m, 2H), 5.30 (d, J = 10.5 Hz, 1H), 4.89 (d,
J = 10.8 Hz, 1H), 3.88 (s, 3H), 3.76–3.71 (m, 2H), 2.85 (t, J = 6.9 Hz, 2H) ppm; MS (ESI, m/z) 478 [M + H]+;
HRMS (ESI) m/z calcd C26H21F2N3O4Na [M + Na]+ 500.1398, found 500.1400.
1-(4-Fluorophenyl)-5-[2-(4-fluorophenyl)ethyl]-3-phenyl-3a,6a-dihydro-1H-pyrrolo[3,4-c]pyrazole-4,6-dione
(
7b): Pale yellow solid; 1H-NMR (CDCl3)
δ
8.02–7.98 (m, 1H), 7.54–7.43 (m, 5H), 7.09–7.00 (m, 4H),
6.84 (t, J = 8.7 Hz, 2H), 5.05 (d, J = 7.5 Hz, 1H), 4.82 (d, J = 7.8 Hz, 1H), 3.81–3.76 (t, J = 6.9 Hz, 2H), 2.87
(t, J = 8.1 Hz, 2H) ppm; MS (ESI, m/z) 430 [M
430.1367, found 430.1369.
−
H]+; HRMS (ESI) m/z calcd C25H18F2N3O2 [M
−
H]+
1-(4-Fluorophenyl)-5-[2-(4-fluorophenyl)ethyl]-3-(2-hydroxyphenyl)-3a,6a-dihydro-1H-pyrrolo[3,4-c]pyrazole-
4,6-dione ( 7.84–7.81 (m, 1H), 7.44–7.33 (m, 3H), 7.11–6.98 (m,
): Pale yellow solid; 1H-NMR (CDCl3)
8
δ
6H), 6.84 (t, J = 8.4 Hz, 2H), 4.98–4.89 (m, 2H), 3.81 (t, J = 6.9 Hz, 2H), 2.88 (t, J = 7.2 Hz, 2H) ppm; MS
(ESI, m/z) 446 [M − H]+; HRMS (ESI) m/z calcd C25H18F2N3O3 [M − H]+ 446.1316, found 446.1327.

Molecules 2016, 21, 1198
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3-(2,4-Dihydroxyphenyl)-1-(4-fluorophenyl)-5-[2-(4-fluorophenyl)ethyl]-3a,6a-dihydro-1H-pyrrolo[3,4-c]pyrazole-
1
4,6-dione (9a): Pale yellow solid; H-NMR (CDCl3)
δ 7.72 (d, J = 8.4 Hz, 1H), 7.42–7.38 (m, 2H),
7.11–7.00 (m, 4H), 6.88–6.82 (m, 2H), 6.55–6.50 (m, 2H), 4.89–4.88 (m, 2H), 3.86 (t, J = 6.9 Hz, 2H), 2.87
(t, J = 7.5 Hz, 2H) ppm; MS (EI, m/z) 463 [M]+; HRMS (EI) m/z calcd C25H19F2N3O4 [M]+ 463.1344,
found 463.1331.
1-(4-Fluorophenyl)-5-[2-(4-fluorophenyl)ethyl]-3-(3-fluoro-2-hydroxyphenyl)-3a,6a-dihydro-1H-pyrrolo[3,4-
c]pyrazole-4,6-dione (9b): Pale yellow solid; 1H-NMR (CDCl3)
δ
7.60 (d, J = 8.9 Hz, 1H), 7.45–7.40 (m,
2H), 7.09–6.95 (m, 6H), 6.84 (t, J = 8.7 Hz, 4H), 5.0 (d, J = 10.5 Hz, 1H), 4.91 (d, J = 11.1 Hz, 1H), 3.83
(t, J = 7.2 Hz, 2H), 2.89 (t, J = 6.9 Hz, 2H) ppm; MS (ESI, m/z) 464 [M
C25H17F3N3O3 [M − H]+ 464.1222, found 464.1217.
−
H]+; HRMS (ESI) m/z calcd
1-(4-Fluorophenyl)-5-[2-(4-fluorophenyl)ethyl]-3-(5-fluoro-2-hydroxyphenyl)-3a,6a-dihydro-1H-pyrrolo[3,4-
c]pyrazole-4,6-dione (9c): Pale yellow solid; 1H-NMR (CDCl3)
7.69-7.65 (m, 1H), 7.22–7.19 (m, 1H),
7.06–6.92 (m, 7H), 6.87–6.81 (m, 2H), 5.41 (d, J = 10.5 Hz, 1H), 4.76 (d, J = 9.9 Hz, 1H), 3.75 (t, J = 7.5 Hz,
δ
2H), 2.86 (t, J = 6.9 Hz, 2H) ppm; MS (ESI, m/z) 464 [M
−
H]+; HRMS (ESI) m/z calcd C25H17F3N3O3
[M − H]+ 464.1222, found 464.1228.
3-(3-Chloro-2-hydroxyphenyl)-1-(4-fluorophenyl)-5-[2-(4-fluorophenyl)ethyl]-3a,6a-dihydro-1H-pyrrolo[3,4-
c]pyrazole-4,6-dione (9d): Pale yellow solid; 1H-NMR (CDCl3)
7.78–7.75 (m, 1H), 7.46–7.41 (m,
δ
3H), 7.12–6.94 (m, 6H), 6.84 (t, J = 8.7 Hz, 2H), 5.02 (d, J = 10.8 Hz, 1H), 4.91 (d, J = 10.8 Hz, 1H),
3.83 (t, J = 6.9 Hz, 2H), 2.89 (t, J = 6.9 Hz, 2H) ppm; MS (EI, m/z) 481 [M]+; HRMS (EI) m/z calcd
C25H18ClF2N3O3 [M]+ 481.1005, found 481.1016.
1-(4-Fluorophenyl)-5-[2-(4-fluorophenyl)ethyl]-3-(2-hydroxy-3-nitrophenyl)-3a,6a-dihydro-1H-pyrrolo[3,4-
c]pyrazole-4,6-dione (9e): Pale yellow solid; 1H-NMR (CDCl3)
δ
8.13–8.11 (m, 1H), 8.03–7.99 (m, 1H),
7.51–7.47 (m, 2H), 7.11–6.99 (m, 5H), 6.86 (t, J = 9.0 Hz, 4H), 5.24 (d, J = 11.1 Hz, 1H), 5.05 (d, J = 10.8 Hz,
1H), 3.79 (t, J = 6.9 Hz, 2H), 2.88 (t, J = 6.9 Hz, 2H) ppm; MS (ESI, m/z) 491 [M
calcd C25H17F2N4O5 [M − H]+ 491.1167, found 491.1171.
−
H]+; HRMS (ESI) m/z
1-(4-Fluoro-phenyl)-5-[2-(4-fluoro-phenyl)-ethyl]-3-(2-hydroxy-3-methyl-phenyl)-3a,6a-dihydro-1H-pyrrolo[3,4-
c]pyrazole-4,6-dione (9f): Pale yellow solid; 1H-NMR (300 MHz, CDCl3)
7.69–7.66 (m, 1H), 7.46–7.41
(m, 2H), 7.25–7.22 (m, 1H), 7.11–6.99 (m, 4H), 6.93 (t, J = 7.5 Hz, 1H), 6.83 (t, J = 8.7 Hz, 2H), 4.98–4.90
δ
(m, 2H), 3.81 (t, J = 7.2 Hz, 2H), 2.87 (t, J = 7.5 Hz, 2H), 2.35 (s, 3H) ppm; MS (ESI, m/z) 460 [M
−
H]+;
HRMS (ESI) m/z calcd C26H20F2N3O3 [M − H]+ 460.1473, found 460.1476.
1-(4-Fluorophenyl)-5-[2-(4-fluorophenyl)ethyl]-3-(2-hydroxy-3-methoxyphenyl)-3a,6a-dihydro-1H-pyrrolo[3,4-
c]pyrazole-4,6-dione (9g): Pale yellow solid; 1H-NMR (CDCl3)
7.44–7.40 (m, 3H), 7.11–6.96 (m, 6H),
δ
6.88–6.84 (m, 2H), 4.96–4.94 (m, 2H), 3.96 (s, 3H), 3.79 (t, J = 7.5 Hz, 2H), 2.87 (t, J = 7.2 Hz, 2H) ppm;
MS (EI, m/z) 477 [M]+; HRMS (EI) m/z calcd C26H21F2N3O4 [M]+ 477.1500, found 477.1495.
1-(4-Fluorophenyl)-5-[2-(4-fluorophenyl)ethyl]-3-(2-hydroxy-4-methoxyphenyl)-3a,6a-dihydro-1H-pyrrolo[3,4-
c]pyrazole-4,6-dione (10a): Pale yellow solid; 1H-NMR (CDCl3)
δ
7.75–7.72 (m, 1H), 7.42–7.37 (m, 2H),
7.10–6.99 (m, 4H), 6.87–6.82 (m, 2H), 6.59–6.56 (m, 2H), 4.92–4.84 (m, 2H), 3.86–3.78 (m, 5H), 2.87
(t, J = 7.5 Hz, 2H) ppm; MS (ESI, m/z) 476 [M
476.1422, found 476.1427.
−
H]+; HRMS (ESI) m/z calcd C26H20F2N3O4 [M
−
H]+
3-(2,3-Dihydroxyphenyl)-1-(4-fluorophenyl)-5-[2-(4-fluorophenyl)ethyl]-3a,6a-dihydro-1H-pyrrolo[3,4-
c]pyrazole-4,6-dione (10b): Pale yellow solid; 1H-NMR (CDCl3)
7.44–7.39 (m, 3H), 7.09–6.96 (m, 6H),
6.84–6.81 (m, 2H), 4.99 (d, J = 11.1 Hz, 1H), 4.90 (d, J = 10.8 Hz, 1H), 3.86–3.79 (m, 5H), 2.87 (t, J = 7.5 Hz,
δ
2H) ppm; MS (ESI, m/z) 476[M
found 476.1406.
−
H]+; HRMS (ESI) m/z calcd C26H20F2N3O4 [M
−
H]+ 476.1422,

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1-(4-Fluorophenyl)-5-[2-(4-fluorophenyl)ethyl]-3-(3-ethoxy-2-hydroxyphenyl)-3a,6a-dihydro-1H-pyrrolo[3,4-
c]pyrazole-4,6-dione (10c): Pale yellow solid; 1H-NMR (CDCl3)
7.46–7.39 (m, 3H), 7.08–6.93 (m, 6H),
δ
6.83 (t, J = 8.4 Hz, 2H), 4.98–4.91 (m, 2H), 4.16 (q, 2H), 3.82–3.77 (m, 2H), 2.93–2.83 (m, 2H), 1.51
(t, J = 6.9 Hz, 3H) ppm; MS (EI, m/z) 491 [M]+; HRMS (EI) m/z calcd C27H23F2N3O4 [M]+ 491.1657,
found 491.1654.
1-(4-Fluorophenyl)-5-[2-(4-fluorophenyl)ethyl]-3-(2-hydroxy-3-methoxy-5-nitrophenyl)-3a,6a-dihydro-1H-
pyrrolo[3,4-c]pyrazole-4,6-dione (11): Pale yellow solid; 1H-NMR (CDCl3)
δ 8.52 (d, J = 2.7 Hz, 1H), 7.84
(d, J = 2.7 Hz, 1H), 7.45–7.40 (m, 2H), 7.11 (t, J = 8.1 Hz, 2H), 7.03–6.98 (m, 2H), 6.81 (t, J = 8.4 Hz, 2H),
5.08 (d, J = 10.8 Hz, 1H), 4.98 (d, J = 11.1 Hz, 1H), 4.05 (s, 3H), 3.85 (t, J = 7.8 Hz, 2H), 2.90 (t, J = 6.9 Hz,
2H) ppm; MS (ESI, m/z) 521 [M
found 521.1281.
−
H]+; HRMS (ESI) m/z calcd C26H19F2N4O6 [M
−
H]+; 521.1273,
3-(2,3-Dimethoxyphenyl)-1-(4-fluorophenyl)-5-[2-(4-fluorophenyl)ethyl]-3a,6a-dihydro-1H-pyrrolo[3,4-
c]pyrazole-4,6-dione (12a): Pale yellow solid; 1H-NMR (CDCl3)
7.52–7.47 (m, 2H), 7.26–7.23 (m, 1H),
7.12–6.98 (m, 6H), 6.90 (t, J = 8.7 Hz, 2H), 5.23 (d, J = 10.8 Hz, 1H), 4.97 (d, J = 10.8 Hz, 1H), 3.99 (s, 3H),
δ
3.91 (s, 3H), 3.77–3.71 (m, 2H), 2.85 (t, J = 7.2 Hz, 2H) ppm; MS (ESI, m/z) 490 [M
−
H]+; HRMS (ESI)
m/z calcd C27H22F2N3O4 [M − H]+ 490.1578, found 490.1586.
3-Benzo[1,3]dioxol-4-yl-1-(4-fluorophenyl)-5-[2-(4-fluorophenyl)ethyl]-3a,6a-dihydro-1H-pyrrolo[3,4-
c]pyrazole-4,6-dione (12b): Pale yellow solid; 1H-NMR (CDCl3)
7.53–7.49 (m, 2H), 7.36–7.34 (m, 1H),
7.08–7.01 (m, 4H), 6.92–6.85 (m, 4H), 6.09 (s, 2H), 5.02–4.94 (m, 2H), 3.75 (t, J = 6.9 Hz, 2H), 2.85
δ
(t, J = 7.5 Hz, 2H) ppm; MS (ESI, m/z) 474 [M
474.1265, found 474.1259.
−
H]+; HRMS (ESI) m/z calcd C26H18F2N3O4 [M
−
H]+
1-(4-Fluorophenyl)-5-[2-(4-fluorophenyl)ethyl]-3-(2-hydroxynaphthalen-1-yl)-3a,6a-dihydro-1H-pyrrolo[3,4-
c]pyrazole-4,6-dione (13): Pale yellow solid; 1H-NMR (CDCl3)
7.97 (d, J = 2.1 Hz, 1H), 7.83–7.75 (m,
δ
2H), 7.517.39 (m, 4H), 7.176.99 (m, 5H), 6.87 (t, J = 8.7 Hz, 2H), 5.55 (d, J = 10.8 Hz, 1H), 5.00 (d,
J = 10.5 Hz, 1H), 3.69 (t, J = 7.2 Hz, 2H), 2.80 (t, J = 7.5 Hz, 2H) ppm; MS (EI, m/z) 497 [M]+; HRMS (EI)
m/z calcd C29H21F2N3O3 [M]+ 497.1551, found 497.1553.
1-(4-Fluorophenyl)-5-[2-(4-fluorophenyl)ethyl]-3-thiazol-2-yl-3a,6a-dihydro-1H-pyrrolo[3,4-c]pyrazole-4,6-dione
(
14a): Yellow solid; 1H-NMR (CDCl3)
δ
7.97 (d, J = 3.0 Hz, 1H), 7.54–7.50 (m, 2H), 7.40 (d, J = 3.0 Hz,
1H), 7.18–7.14 (m, 1H), 7.04–6.97 (m, 3H), 6.85 (t, J = 8.4 Hz, 2H), 5.14–5.03 (m, 2H), 3.81 (t, J = 6.9 Hz,
2H), 2.88 (t, J = 6.9 Hz, 2H) ppm; MS (ESI, m/z) 437 [M
[M − H]+ 437.0884, found 437.0880.
−
H]+; HRMS (ESI) m/z calcd C22H15F2N4O2S
1-(4-Fluorophenyl)-5-[2-(4-fluorophenyl)ethyl]-3-pyridin-2-yl-3a,6a-dihydro-1H-pyrrolo[3,4-c]pyrazole-4,6-dione
1
(
14b): Pale yellow solid; H-NMR (CDCl3)
δ
8.75 (m, 1H), 7.98–7.96 (m, 1H), 7.81–7.78 (m, 1H),
7.34–7.32 (m, 1H), 7.21–7.15 (m, 3H), 7.08–6.96 (m, 5H), 5.53 (d, J = 10.8 Hz, 1H), 5.14 (d, J = 10.8 Hz,
1H), 3.75–3.70 (m, 2H), 2.89-2.84 (m, 2H) ppm; MS (ESI, m/z) 431 [M
−
H]+; HRMS (ESI) m/z calcd
C24H17F2N4O2 [M − H]+ 431.1320, found 431.1318.
1-(3-Fluorophenyl)-5-[2-(4-fluorophenyl)ethyl]-3-(2-hydroxy-3-methoxyphenyl)-3a,6a-dihydro-1H-pyrrolo[3,4-
c]pyrazole-4,6-dione (15a): Pale yellow solid; 1H-NMR (CDCl3)
7.48–7.45 (m, 1H), 7.34–7.13 (m, 3H),
δ
7.04–6.94 (m, 4H), 6.87–6.81 (m, 2H), 6.78–6.72 (m, 1H), 5.03 (d, J = 10.8 Hz, 1H), 4.94 (d, J = 10.5 Hz,
1H), 3.96 (s, 3H), 3.81 (t, J = 7.5 Hz, 2H), 2.87 (t, J = 7.2 Hz, 2H) ppm; MS (ESI, m/z) 477 [M]+; HRMS
(ESI) m/z calcd C26H21F2N3O4 [M]+ 477.1500, found 477.1489.
1-(2-Fluorophenyl)-5-[2-(4-fluorophenyl)ethyl]-3-(2-hydroxy-3-methoxyphenyl)-3a,6a-dihydro-1H-pyrrolo[3,4-
c]pyrazole-4,6-dione (15b): Pale yellow solid; 1H-NMR (CDCl3)
δ
7.52–7.49 (m, 1H), 7.28–7.26 (m, 1H),
7.19–7.12 (m, 3H), 6.98–6.90 (m, 4H), 6.81 (t, J = 8.4 Hz, 2H), 5.41–5.37 (m, 1H), 4.83 (d, J = 10.5 Hz, 1H),
3.96 (s, 3H), 3.75 (t, J = 6.9 Hz, 2H), 2.81 (t, J = 7.2 Hz, 2H) ppm; MS (ESI, m/z) 476 [M
(ESI) m/z calcd C26H20F2N3O4 [M − H]+ 476.1422, found 476.1419.
−
H]+; HRMS

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1-(4-Chlorophenyl)-5-[2-(4-fluorophenyl)ethyl]-3-(2-hydroxy-3-methoxyphenyl)-3a,6a-dihydro-1H-pyrrolo[3,4-
c]pyrazole-4,6-dione (15c): Pale yellow solid; 1H-NMR (CDCl3)
7.47–7.30 (m, 4H), 7.18–7.13 (m, 1H),
7.03–6.95 (m, 4H), 6.86–6.81 (m, 2H), 5.00 (d, J = 10.7 Hz, 1H), 4.93 (d, J = 10.7 Hz, 1H), 3.95 (s, 3H), 3.80
δ
(t, J = 6.6 Hz, 2H), 2.87 (t, J = 6.9 Hz, 2H) ppm; MS (ESI, m/z) 492 [M
−
H]+; HRMS (ESI) m/z calcd
C26H19ClFN3O4 [M − H]+ 492.1126, found 492.1120.
1-(2,4-Difluorophenyl)-5-[2-(4-fluorophenyl)ethyl]-3-(2-hydroxy-3-methoxyphenyl)-3a,6a-dihydro-1H-pyrrolo
[3,4-c]pyrazole-4,6-dione (15d): Pale yellow solid; 1H-NMR (CDCl3)
7.51–7.48 (m, 1H), 7.23–7.15 (m,
1H), 7.05–6.78 (m, 8H), 5.25 (d, J = 7.8 Hz, 1H), 4.82 (d, J = 10.5 Hz, 1H), 3.95 (s, 3H), 3.75 (t, J = 7.2 Hz,
δ
2H), 2.81 (t, J = 7.5 Hz, 2H) ppm; MS (ESI, m/z) 494 [M
−
H]+; HRMS (ESI) m/z calcd C26H19F3N3O4
[M − H]+ 494.1328, found 494.1332.
5-[2-(4-Fluorophenyl)-ethyl]-3-(2-hydroxy-3-methoxyphenyl)-1-(4-trifluoromethylphenyl)-3a,6a-dihydro-1H-
1
pyrrolo[3,4-c]pyrazole-4,6-dione (15e): Pale yellow solid; H-NMR (CDCl3)
δ 7.63 (d, J = 9.0 Hz, 2H),
7.53–7.46 (m, 3H), 7.03-6.98 (m, 4H), 6.83 (t, J = 8.7 Hz, 2H), 5.11 (d, J = 10.8 Hz, 1H), 4.97 (d, J = 10.5 Hz,
2H), 3.97 (s, 3H), 3.82 (t, J = 7.2 Hz, 2H), 2.87 (t, J = 6.9 Hz, 2H) ppm; MS (ESI, m/z) 526 [M
−
H]+;
HRMS (ESI) m/z calcd C27H20F4N3O4 [M − H]+ 526.1390, found 526.1401.
5-[2-(4-Fluorophenyl)ethyl]-3-(2-hydroxy-3-methoxyphenyl)-1-(4-methanesulfonylphenyl)-3a,6a-dihydro-1H-
1
pyrrolo[3,4-c]pyrazole-4,6-dione (15f): Pale yellow solid; H-NMR (CDCl3)
δ 7.89 (d, J = 8.7 Hz, 2H),
7.54–7.46 (m, 3H), 7.00–6.96 (m, 4H), 6.84–6.78 (m, 2H), 5.19–5.16 (m, 1H), 5.04–5.00 (m, 1H), 3.96
(s, 3H), 3.81 (t, J = 7.8 Hz, 2H), 3.11 (s, 3H), 2.86 (t, J = 6.9 Hz, 2H) ppm; MS (ESI, m/z) 536 [M
HRMS (ESI) m/z calcd C27H23FN3O6S [M − H]+ 536.1292, found 536.1293.
−
H]+;
4-[5-[2-(4-Fluorophenyl)ethyl]-3-(2-hydroxy-3-methoxyphenyl)-4,6-dioxo-4,5,6,6a-tetrahydro-3aH-pyrrolo[3,4-
c]pyrazol-1-yl]-benzenesulfonamide (15g): Pale yellow solid; 1H-NMR (CDCl3) δ 7.92 (d, J = 9.0 Hz, 2H),
7.53–7.46 (m, 3H), 7.02–6.96 (m, 4H), 6.85–6.79 (m, 2H), 5.16–4.99 (m, 2H), 4.75 (br, 2H, NH2), 3.97
(s, 3H), 3.83 (t, J = 6.9 Hz, 2H), 2.87 (t, J = 7.2 Hz, 2H) ppm; MS (ESI, m/z) 537 [M
m/z calcd C26H22FN4O6S [M − H]+ 537.1244, found 537.1252.
−
H]+; HRMS (ESI)
5-[2-(4-Fluorophenyl)ethyl]-3-(2-hydroxy-3-methoxyphenyl)-1-phenyl-3a,6a-dihydro-1H-pyrrolo[3,4-c]pyrazole-
1
4,6-dione (15h): Pale yellow solid; H-NMR (CDCl3)
δ 7.48–7.45 (m, 3H), 7.38 (t, J = 7.8 Hz, 2H),
7.06–6.95 (m, 5H), 6.84 (t, J = 8.7 Hz, 2H), 5.05 (d, J = 10.5 Hz, 1H), 4.91 (d, J = 10.5 Hz, 1H), 3.96 (s, 3H),
3.80 (t, J = 7.5 Hz, 2H), 2.87 (t, J = 7.2 Hz, 2H) ppm; MS (ESI, m/z) 460 [M
−
H]+; HRMS (ESI) m/z calcd
C26H22FN3O4Na [M + Na]+ 482.1492, found 482.1505.
5-[2-(4-Fluorophenyl)ethyl]-3-(2-hydroxy-3-methoxyphenyl)-1-(4-methylphenyl)-3a,6a-dihydro-1H-pyrrolo[3,4-
c]pyrazole-4,6-dione (15i): Pale yellow solid; 1H-NMR (CDCl3)
7.48–7.45 (m, 1H), 7.36 (d, J = 8.4 Hz,
δ
2H), 7.19 (d, J = 8.4 Hz, 2H), 7.05–6.95 (m, 4H), 6.85 (t, J = 9.0 Hz, 2H), 5.01 (d, J = 11.8 Hz, 1H), 4.91
(d, J = 10.5 Hz, 1H), 3.96 (s, 3H), 3.80 (t, J = 7.2 Hz, 2H), 2.87 (t, J = 7.5 Hz, 2H), 2.34 (s, 3H) ppm; MS
(ESI, m/z) 472 [M − H]+; HRMS (ESI) m/z calcd C27H23FN3O4 [M − H]+ 472.1673, found 472.1680.
5-[2-(4-Fluorophenyl)-ethyl]-3-(2-hydroxy-3-methoxyphenyl)-1-(4-methoxyphenyl)-3a,6a-dihydro-1H-pyrrolo
1
[3,4-c]pyrazole-4,6-dione (15j): Pale yellow solid; H-NMR (CDCl3)
δ 7.43–7.39 (m, 3H), 7.02–6.85
(m, 8H), 4.93–4.92 (m, 2H), 3.96 (s, 3H), 3.83–3.78 (m, 5H), 2.87 (t, J = 7.5 Hz, 2H) ppm; MS (EI, m/z) 489
[M]+; HRMS (EI) m/z calcd C27H24FN3O5 [M]+ 489.1700, found 489.1693.
1-(4-Fluorophenyl)-5-[2-(2-fluorophenyl)ethyl]-3-(2-hydroxy-3-methoxyphenyl)-3a,6a-dihydro-1H-pyrrolo[3,4-
c]pyrazole-4,6-dione (16a): Pale yellow solid; 1H-NMR (CDCl3)
δ 7.46–7.39 (m, 3H), 7.09–6.90 (m, 8H),
4.98–4.89 (m, 2H), 3.96 (s, 3H), 3.89–3.85 (m, 2H), 2.97–2.92 (m, 2H) ppm; MS (ESI, m/z) 476 [M
−
H]+;
HRMS (ESI) m/z calcd C26H20F2N3O4 [M − H]+ 476.1422, found 476.1416.
1-(4-Fluorophenyl)-5-[2-(3-fluorophenyl)ethyl]-3-(2-hydroxy-3-methoxyphenyl)-3a,6a-dihydro-1H-pyrrolo[3,4-
c]pyrazole-4,6-dione (16b): Pale yellow solid; 1H-NMR (CDCl3)
7.49–7.40 (m, 3H), 7.10–6.96 (m, 5H),
δ

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6.85–6.47 (m, 3H), 4.99–4.92 (m, 2H), 3.96 (s, 3H), 3.83 (t, J = 6.6 Hz, 2H), 2.91 (t, J = 7.5 Hz, 2H) ppm;
MS (ESI, m/z) 476 [M
H]+; HRMS (ESI) m/z calcd C26H20F2N3O4 [M H]+ 476.1422, found 476.1424.
−
−
5-[2-(4-Chlorophenyl)-ethyl]-1-(4-fluorophenyl)-3-(2-hydroxy-3-methoxyphenyl)-3a,6a-dihydro-1H-pyrrolo[3,4-
c]pyrazole-4,6-dione (16c): Pale yellow solid; 1H-NMR (CDCl3)
7.42–7.38 (m, 2H), 7.28–7.27 (m, 2H),
7.13–7.03 (m, 5H), 6.98–6.94 (m, 2H), 4.99–4.91 (m, 2H), 3.94 (s, 3H), 3.80 (t, J = 7.2 Hz, 2H), 2.87–2.83
δ
(m, 2H) ppm; MS (ESI, m/z) 492 [M
found 492.1117.
−
H]+ ; HRMS (ESI) m/z calcd C26H20FClN3O4 [M
−
H]+ 492.1126,
1-(4-Fluorophenyl)-3-(2-hydroxy-3-methoxyphenyl)-5-phenethyl-3a,6a-dihydro-1H-pyrrolo[3,4-c]pyrazole-
1
4,6-dione (16d): Pale yellow solid; H-NMR (CDCl3)
δ 7.49–7.41 (m, 3H), 7.16 (d, J = 6.6 Hz, 2H),
7.11–7.04 (m, 5H), 6.98–6.96 (m, 2H), 4.98–4.90 (m, 2H), 3.96 (s, 3H), 3.84 (t, J = 7.2 Hz, 2H), 2.90
(t, J = 8.1 Hz, 2H) ppm; MS (ESI, m/z) 458 [M
458.1516, found 458.1517.
−
H]+; HRMS (ESI) m/z calcd C26H21FN3O4 [M
−
H]+
1-(4-Fluorophenyl)-3-(2-hydroxy-3-methoxyphenyl)-5-[2-(4-methoxyphenyl)ethyl]-3a,6a-dihydro-1H-pyrrolo
[3,4-c]pyrazole-4,6-dione (16e): Pale yellow solid; 1H-NMR (CDCl3)
7.49–7.41 (m, 3H), 7.10–7.07 (m,
2H), 6.98–6.95 (m, 4H), 6.68 (d, J = 8.7 Hz, 2H), 4.97–4.89 (m, 2H), 3.96 (s, 3H), 3.80 (t, J = 6.3 Hz,
δ
2H), 3.65 (s, 3H), 2.83 (t, J = 7.5 Hz, 2H) ppm; MS (ESI, m/z) 488 [M
−
H]+; HRMS (ESI) m/z calcd
C27H23FN3O5 [M − H]+ 488.1622, found 488.1623.
The 1H-NMR spectra of all new compounds 6–16 are provided in the Supplementary Materials.
3.2. Biological Evaluation
3.2.1. Cytotoxicty Assay
The cytotoxicty assay was performed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazoliumbromide
(MTT) method. Briefly, 100
µ
L 4
×
104 C8166 cells were seeded each well with 100
µ
L◦of different
concentrations of compounds in a 96-well plate. The plate was incubated 72 h at 37 C◦, 5% CO2
incubator. MTT (20 µL, 5 mg/mL) was added to each well and the plate was incubated at 37 C for 4 h.
Supernatant (100 µL) was discarded and 20% SDS-50% DMF (100 µL) was added. The plates were read
at 570/630 nm by a Bio-Tek Elx 800 reader (Bio-Tek Instruments, Inc., Winooski, VT, USA). The 50%
cytotoxic concentration (CC50) was calculated.
3.2.2. Syncytia Inhibition Assay
The syncytia inhibition assay was applied by counting syncytia which were formed by HIV-1IIIB
infected C8166 cells. Briefly, HIV-1IIIB supernatant and C8166 cells were seeded in a 96-well plate with
different concentration of compounds. The plate was incubated in a 37 ◦C, 5% CO2 incubator for 72 h.
Syncytia were counting under an inverted microscope and 50% effective concentration (EC50) was
calculated. Therapeutic index (TI) was calculated by the ratio of CC50/EC50.
4. Conclusions
In summary, we have designed and synthesized a series of 3a,6a-dihydro-1H-pyrrolo[3,4-
c]pyrazole-4,6-dione derivatives
novel compounds show single-digit micromolar antiviral potency against HIV-1. Among them, 9g
and 15i exhibited the best overall absolute performance (EC50 < 5 M, TI > 100), approximately 5- to
6–16 that exhibited anti-HIV-1 activity. As expected, most of the
µ
10- fold enhancement compared to the starting compound 6. The structural class of agents presented
herein may represent an attractive platform for developing anti-HIV-1 drugs. Further SAR studies and
lead optimization are warranted to select clinically viable compound for development.
Supplementary Materials: The 1H-NMR spectral charts of
6–16 can be accessed at: http://www.mdpi.com/1420-
3049/21/9/1198/s1.

Molecules 2016, 21, 1198
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Acknowledgments: We gratefully acknowledge financial support from the Zhejiang Provincial Natural
Science Foundation (LQ14B020004), the National Natural Science Foundation of China (81220108025, 81302633,
2147220 and 91229204), SA-SIBS scholarship program, the National Health and Family Planning Commission
(2012ZX09304-011, 2013ZX09401003-005, 2013ZX09507001, 2013ZX09507-002 and 2014ZX09507002-001), Shanghai
Science and Technology Development Fund (15DZ2291600), the Thousand Talents Program in China, the
National Natural Science Foundation of China (81102483), the National Science and Technology Major Project
(2009ZX09501-029), Yunnan Province (2007BC006) and the Knowledge Innovation Program of the Chinese
Academy of Sciences (KFJ-EW-STS-026).
Author Contributions: G-N.L. and R.-H.L. conceived and performed the experiments; H.L. and Y.-T.Z. designed
the experiments; J.L. and X.-J.Z. analyzed the data; G-N.L., Y.Z. and L.-M.Y. discussed the results; G-N.L. wrote
the paper.
Conflicts of Interest: The authors declare no conflict of interest.
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