Indolizine derivatives as HIV-1 VIF-elonginc interaction inhibitors

Article abstract of DOI:10.1111/cbdd.12119

Compound 1 (VEC-5) was identified as a potent small-molecular HIV-1 viron infectivity factor inhibitor that targets the viron infectivity factor-ElonginC interaction. A structure-activity relationship study was carried out to develop compounds with improved efficacy against HIV-1 and 49 indolizine derivatives of three categories were designed and synthesized. We found that five compounds exhibited promising anti-HIV-1 activity, and the most active compound 2g had an IC₅₀ value of 11.0 μm. These results provide new information to develop highly potent small-molecule HIV-1 viron infectivity factor inhibitors.

Full text of DOI:10.1111/cbdd.12119

Chem Biol Drug Des 2013; 81: 730–741
Research Article
Indolizine Derivatives as HIV-1 VIF–ElonginC
Interaction Inhibitors
Wenlin Huang1, Tao Zuo2, Xiao Luo1, Hongwei
Jin1, Zhenming Liu1, Zhenjun Yang1, Xianghui
Yu2,3, Liangren Zhang1,* and Lihe Zhang1
these binding interactions within the A3G–VIF–E3 complex
presents new opportunities for therapeutic intervention.
Recently, several small molecules (RN-18 and IMB-26/35)
have been identified as VIF inhibitors by cell-based screen-
ing from chemical libraries (9,10) (Figure 1). These com-
pounds were shown to reduce the capacity of VIF to
downregulate A3G. IMB-26/35 was shown to specifically
bind to A3G and inhibit the interaction of VIF–A3G, thereby
stabilize A3G expression, while RN-18 was shown to inhi-
bit VIF–A3G interaction through reduction of VIF in the
host cells. Optimization of RN-18 generated new inhibitors
with improved activities (11).
1State Key Laboratory of Natural and Biomimetic Drugs,
School of Pharmaceutical Sciences, Peking University,
Beijing, 100191, China
2National Engineering Laboratory for AIDS Vaccine,
College of Life Science, Jilin University, Changchun,
130012, China
3Key Laboratory for Molecular Enzymology and
Engineering of the Ministry of Education, College of Life
Science, Jilin University, Changchun, 130012, China
*Corresponding author: Liangren Zhang,
liangren@bjmu.edu.cn
To find novel HIV-1 VIF inhibitors, our research was
focused on identifying small-molecular inhibitors targeting
the VIF–ElonginC interaction. We previously constructed a
three-dimensional VIF–ElonginB/C homology model (12),
then a virtual screening was performed and 15 com-
pounds with high-docking scores were selected for biolog-
ical evaluation (13). Compound 1 (VEC-5) was identified as
potent HIV-1 VIF inhibitor with an IC50 of 36.1 lM and a
CC50 of 167.4 lM. Further biological investigation revealed
that compound 1 protects A3G from VIF-mediated degra-
dation and drastically inhibits VIF function through blocking
interaction between VIF and ElonginC (13). To the best of
our knowledge, compound 1 was the first HIV-1 VIF inhibi-
tor that targets the VIF–ElonginC interaction.
Compound 1 (VEC-5) was identified as a potent small-
molecular HIV-1 viron infectivity factor inhibitor that
targets the viron infectivity factor–ElonginC interaction.
A structure–activity relationship study was carried out
to develop compounds with improved efficacy against
HIV-1 and 49 indolizine derivatives of three categories
were designed and synthesized. We found that five
compounds exhibited promising anti-HIV-1 activity,
and the most active compound 2g had an IC50 value of
11.0 lM. These results provide new information to
develop highly potent small-molecule HIV-1 viron infec-
tivity factor inhibitors.
Key words: anti-HIV-1, indolizine derivatives, structure–activity
relationship, VEC-5, VIF–ElonginC interaction inhibition
Indolizine derivatives have been comprehensively applied in
drug discovery due to their particular structures (14–17). As
compound 1 contains the unique indolizine core structure
and the 1, 3, 7-substituents are amenable to chemical
modification, we carried out a structure–activity relationship
investigation of compound 1 with the goal to search more
potent indolizine-based inhibitors of HIV-1 VIF. The modifi-
cation of compound 1 was focused on the three substituted
regions (Moieties A, B and C shown in Figure 2) of the ind-
olizine core structure, and 49 derivatives of three categories
were designed and synthesized. The information obtained
will be helpful for the finding of new anti-HIV agents.
Received
6 July 2012, revised 19 December 2012 and
accepted for publication 6 February 2013
The apolipoprotein B mRNA-editing enzyme catalytic poly-
peptide 3G (APOBEC3G, A3G) is a ‘host restriction factor’
for human immunodeficiency virus type 1 (HIV-1) (1,2).
However, HIV-1 viron infectivity factor (VIF) neutralizes A3G
by forming an E3 ubiquitinligase with Cullin5, ElonginB and
ElonginC that targets A3G for degradation by the ubiquitin
–proteasome pathway. VIF associates with the Cullin5-
ElonginB–ElonginC complex by binding directly to Elong-
inC via its SOCS-box motif and to Cullin5 via hydrophobic
residues within a zinc-binding region formed by a con-
served HCCH motif. The HIV-1 VIF-Cullin5–ElonginB/C
complex is then able to ubiquitnate the A3G factor by its
N-terminal domain binding (3–8). Therefore, preventing
Experimental Section
Chemistry
All commercial chemicals and solvents were purchased
from commercial suppliers of analytical grade and used
730
ª 2013 John Wiley & Sons A/S. doi: 10.1111/cbdd.12119

HIV-1 VIF-ElonginC Interaction Inhibitors
Figure 1: Structures of HIV-1 viron infectivity factor (VIF) inhibitors.
Figure 2: Structures of indolizine derivatives.
without further purification. 1H and 13C NMR spectra were
recorded on a Bruker Avance III 400 spectrometer. Chemi-
cal shifts were reported as values from an internal tetra-
methylsilane standard. High-resolution mass spectra
(HRMS) were recorded on a Bruker Apex IV FTMS. LC/MS
analyses were performed on an Agilent 1200-6110 instru-
ments. Elemental analyses were performed on a Vario EL
III instrument and were Æ0.3 of theoretical. Melting points
were determined on a MelTemp apparatus and were
uncorrected.
conditions to afford the corresponding carboxylic acid 12,
which was then condensed with various amines to afford
compounds 6. Compound 6h was further reacted with
compounds 13a, 13b and 13c to produce 6j, 6k and 6l,
respectively, then removal of the tert-butyloxycarbonyl
group of 6l and subsequent incorporation of the biotin
group generated compound 6n. The procedures and
spectral data of representative compounds are as follows:
spectral data of other compounds are available as Sup-
porting information.
The general route for the synthesis of indolizine com-
pounds is shown in Scheme 1. a-Bromoketone 7 was
reacted with 4-substituted pyridine 8 to afford the pyridini-
um salt 9. Then the pyridinium salt 9 was reacted with
ethyl propiolate under basic conditions to afford 1, 3, 7-tri-
substituted indolizine compounds. Compounds 1, 2, 4, 5,
10a were all synthesized by this route.
Representative procedure for the synthesis of
indolizine derivatives
Ethyl 3-(2-naphthoyl)-7-benzoylindolizine-1-
carboxylate (1)
A mixture of phenyl (pyridin-4-yl) methanone (500 mg,
2 mmol) and 2-bromo-1- (naphthalene-2-yl)ethanone
(360 mg, 2 mmol) in EtOAc (20 mL) was heated to reflux
over night. The crude product precipitated, and the pre-
cipitate was filtered off and washed with EtOAc to give
pyridinium salt as off-white solid. The crude product was
used for the next reaction without further purification or
characterization.
The general route for the synthesis of compounds 3 and 6
is shown in Scheme 2. The 7-benzyl ester of compound
10a was selectively hydrolysed to afford the corresponding
7-carboxy indolizine compound 11, which was then con-
densed with various amines to afford compounds 3. The
ester group of compound 1 was hydrolysed under basic
Chem Biol Drug Des 2013; 81: 730–741
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Huang et al.
O
Ar
Br
N
O
Ar
N
a
b
N
Br
Ar
O
R
R
R
CO2C2H5
10
7
8
9
Scheme 1: (a) Ethyl acetate, reflux, over night; (b) K2CO3, CH3CN, 0 °C, 30 min, then ethyl propiolate, r.t, 72 h.
O
O
O
b
a
N
R2
N
N
N
HO
R1
BnO2C
CO2C2H5
CO2C2H5
11
CO2C2H5
O
O
10a
3
O
O
N
O
c
d
N
N
R3
CO2C2H5
OH
O
O
O
O
O
1
12
6
N
N
CF3
O
N
N
R4
O
O
CF3
O
O
Biotin-NHS
e
6n
e
N
NH
R4
H2N
O
O
N
O
N
O
N
H
O
O
O
O
O
O
N
O
O
H
6h
13a: R4 =∗
13b: R4 = ∗
CH3
6j: R4 = ∗
CH3
O
N3
2
O
6k: R4
6l: R4
=
=
∗
N3
2
O
13c: R4 = ∗
NHBoc
O
∗
2
NHBoc
2
f
O
6m: R4 = ∗
NH2
2
Scheme 2: (a) (1) LiOH-H2O, tetrahydrofuran (THF)/C2H5OH/H2O, r.t, 2 h; or (2) tetrabutylammonium fluoride (TBAF), THF, r.t, over night;
(b) 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), HOBt, DIPEA, R1NHR2, THF, r.t, over night; or (COCl)2, dimethyl
formamide (DMF), then R1NR2, K2CO3, THF, r.t, over night; (c) NaOH, THF/CH3OH/H2O, reflux, 5 h; (d) (1) SOCl2, reflux, 2 h, then R3H,
K2CO3, acetone, 0 °C -r.t, over night; (2) R3H, EDC, HOBt, DIPEA, DMF, r.t, over night; or (3) N-hydroxy succinimide (NHS), EDC, DMF,
r.t, over night; then R3H, TEA, DCM, r.t, over night; (e) TEA, DCM, r.t, over night; (f) TFA, DCM, r.t, 24 h.
The pyridinium salt (430 mg, 1 mmol) was suspended in
CH3CN (10 mL) at 0 °C, then potassium carbonate
(276 mg, 2 mmol) was added portionwise to maintain the
temperature at 0 °C. After 30 min, ethyl propiolate
(108 mg, 1.1 mmol) was added dropwise at 0 °C, and the
resulting mixture was stirred at room temperature for
3 days. The solvent was evaporated under reduced pres-
sure, and the residue was diluted with water, extracted
three times with CH2Cl2. The organic layers were com-
bined, washed with brine, dried over Na2SO4 and evapo-
rated. The residue was purified with silica gel
chromatography (petroleum ether:EtOAc = 6:1) to give the
target compound 1 476.5 mg (53.3% over two steps) as a
yellow solid; Mp: 154–156 °C; 1H NMR (400 MHz, CDCl3)
d 10.02 (dd, J = 7.3 Hz, 0.9 Hz, 1H), 8.84–8.76 (m, 1H),
8.36 (s, 1H), 8.02 (s, 1H), 8.00 (s, 1H), 7.97–7.92 (m, 3H),
7.92–7.90 (m, 1H), 7.90–7.88 (m, 1H), 7.69–7.53 (m, 6H),
4.34 (q, J = 7.1 Hz, 2H), 1.31 (t, J = 7.1 Hz, 3H); 13C
NMR (100 MHz, CDCl3) d 194.2, 185.8, 163.9, 138.1,
136.9, 135.2, 135.0, 133.3, 132.6, 130.4, 130.2, 129.5,
129.4, 129.1, 128.9, 128.8, 128.4, 128.1, 127.2, 125.5,
124.16, 123.0, 114.8, 109.7, 61.2, 14.3; ESI-MS m/z:
448.0 [M + H]+; Anal. calcd. for C29H21NO4: C 77.85, H
4.79, N 2.99, Found: C 77.84, H 4.73, N 3.13.
Ethyl 3-(2-naphthoyl)-7-isonicotinoylindolizine-1-
carboxylate (2g)
Start with di(pyridin-4-yl) methanone and 2-bromo-1-
(naphthalene-2-yl)ethanone, follow the same procedure as
732
Chem Biol Drug Des 2013; 81: 730–741

HIV-1 VIF-ElonginC Interaction Inhibitors
in the synthesis of 1, purify with silica gel chromatography
(petroleum ether:EtOAc = 4:1) to afford 2g. Yield: 15.6%
over two steps; Light yellow solid; Mp: 134–136 °C; 1H
NMR (400 MHz, CDCl3) d 10.00 (d, J = 6.5 Hz, 1H), 8.88
(s, 2H), 8.80 (s, 1H), 8.35 (s, 1H), 8.02–7.89 (m, 5H), 7.62
–7.54 (m, 5H), 4.34 (q, J = 6.1 Hz, 2H), 1.31 (t,
J = 6.1 Hz, 3H); 13C NMR (100 MHz, CDCl3) d 192.8,
186.1, 163.6, 150.8, 143.6, 137.6, 136.6, 135.3, 132.9,
132.6, 130.44, 130.41, 129.5, 129.3, 128.8, 128.5, 128.1,
127.2, 125.4, 124.5, 123.7, 122. 9, 113.8, 110.4, 60.8,
14.5; ESI-MS m/z: 448.9 [M + H]+; HRMS (ESI-TOF+) m/z
Calcd. for C28H20N2O4 [M + H]+: 449.1501; Found
449.1486.
(petroleum ether:EtOAc = 5:1) to afford 5f. Yield: 31.0%
over two steps; Yellow solid; Mp: 136–138 °C; 1H NMR
(400 MHz, CDCl3) d 10.04 (d, J = 7.3 Hz, 1H), 8.77 (s,
1H), 8.57 (s, 1H), 7.88 (d, J = 7.7 Hz, 2H), 7.74 (s, 1H),
7.65 (t, J = 7.3 Hz, 1H), 7.55 (t, J = 7.7 Hz, 3H), 7.42 (s,
1H), 6.65 (s, 1H), 4.38 (q, J = 7.0 Hz, 2H), 1.36 (t,
J = 7.0 Hz, 3H); 13C NMR (100 MHz, CDCl3) d 194.3,
171.5, 163.9, 153.3, 146.4, 137.8, 136.8, 134.9, 133.2,
130.12, 129.1, 128.8, 128.1, 122.84, 122.82, 118.6,
114.6, 112.6, 110.1, 60.6, 14.6; ESI-MS m/z: 388.1
[M + H]+, 797.1 [2M + Na]+; Anal. Calcd. for C23H17NO5:
C, 71.31; H, 4.42; N, 3.62; Found: C, 71.23; H, 4.58; N,
3.59.
7-Benzyl 1-ethyl 3-(2-naphthoyl)indolizine-1,7-
dicarboxylate (10a)
Representative procedure for the synthesis of
compounds 3
Start with benzyl isonicotinate and 2-bromo-1- (naphtha-
lene-2-yl)ethanone, follow the same procedure as in the
synthesis of 1, purify with silica gel chromatography (petro-
leum ether:EtOAc = 6:1) to afford 10a. Yield: 58.6% over
Ethyl 3-(2-naphthoyl)-7-(piperidine-1-carbonyl)
indolizine-1-carboxylate (3a)
Synthesis of acid 11 (procedure A): LiOH-H2O (42 mg,
2 mmol) was added to a solution of ester 10a (477 mg,
1 mmol) in tetrahydrofuran (THF) (6 mL), methanol (6 mL)
and H2O (2 mL). The mixture was stirred at room tempera-
ture for 2 h, then the solvent was evaporated under
reduced pressure and 1 N HCl solution was added. The
precipitated solid was filtered, washed with water and
dried to afford the corresponding carboxylic acid com-
pound 11 almost quantitatively, which was used for the
next reaction without further purification.
1
two steps; Light yellow solid; H NMR (400 MHz, CDCl3) d
9.97 (d, J = 7.4 Hz, 1H), 9.14–9.10 (m, 1H), 8.35 (s, 1H),
8.00 (d, J = 8.5 Hz, 2H), 7.97–7.91 (m, 3H), 7.67 (dd,
J = 7.3 Hz, 1.8 Hz, 1H), 7.65–7.57 (m, 2H), 7.51 (dd,
J = 8.0 Hz, 1.4 Hz, 2H), 7.46–7.38 (m, 3H), 5.45 (s, 2H),
4.40 (q, J = 7.1 Hz, 2H), 1.39 (t, J = 7.1 Hz, 3H); 13C
NMR (100 MHz, CDCl3) d 186.1, 164.8, 163.8, 138.4,
136.8, 135.7, 135.2, 132.6, 130.3, 129.5, 129.4, 128.9,
128.8, 128.7, 128.7, 128.6, 128.3, 128.1, 127.1, 125.5,
124.1, 122.3, 114.4, 109.6, 67.7, 60.7, 14.6; ESI-MS m/z:
478.0 [M + H]+.
Synthesis of acid 11 (procedure B): 1 M tetrabutylammoni-
um fluoride (TBAF) solution in THF (3 mL, 3 mmol) was
added to a solution of ester 10a (477 mg, 1 mmol) in THF
(10 mL). The mixture was stirred at room temperature over
night. The solvent was evaporated under reduced pres-
sure, and the residue was dissolved in CH2Cl2. The
organic solution was washed with 1 N HCl solution and
brine, successively, dried over Na2SO4. The solvent was
concentrated, and the residue was purified with silica gel
chromatography (CH2Cl2/CH3OH = 20:1) to afford acid 11
325 mg (84.0%).
Ethyl 3-(2-naphthoyl)-7-benzylindolizine-1-
carboxylate (4a)
Start with 4-benzylpyridine and 2-bromo-1- (naphthalene-
2-yl)ethanone, follow the same procedure as in the synthe-
sis of 1, purify with silica gel chromatography (petroleum
ether:EtOAc = 6:1) to afford 4a. Yield: 44.8% over two
steps; off-white solid; 1H NMR (400 MHz, CDCl3) d 9.90
(dd, J = 7.2 Hz, 0.6 Hz, 1H), 8.31 (s, 1H), 8.25 (d,
J = 0.9 Hz, 1H), 8.01–7.96 (m, 2H), 7.96–7.89 (m, 2H),
7.86 (s, 1H), 7.64–7.56 (m, 2H), 7.39–7.33 (m, 2H), 7.30–
7.25 (m, 3H), 6.95 (dd, J = 7.2 Hz, 1.9 Hz, 1H), 4.35 (q,
J = 7.2 Hz, 2H), 4.13 (s, 2H), 1.35 (t, J = 7.2 Hz, 3H); 13C
NMR (100 MHz, CDCl3) d 185.5, 164.4, 142.5, 140.5,
139.1, 137.4, 135.0, 132.7, 129.9, 129.5, 129.4, 129.3,
129.0, 128.5, 128.0, 127.9, 127.0, 126.9, 125.7, 122.7,
118.5, 117.2, 106.0, 60.2, 42.0, 14.7; ESI-MS m/z: 434.1
Acid 11 (194 mg, 0.5 mmol) was dissolved in THF
(10 mL), catalytic amount of dimethyl formamide (DMF)
was added and then oxalyl chloride (127 mg, 1 mmol)
was added dropwise and the mixture was stirred at room
temperature for 2 h. The solvent was concentrated, and
the residue was diluted with THF and was used for the
next reaction without further purification.
[M + H]+
.
Piperidine (85 mg, 1 mmol) was dissolved in THF (10 mL),
K2CO3 (69 mg, 0.5 mmol) was added and then the crude
acyl chloride solution in THF was added dropwise at 0 °C.
The reaction mixture was stirred at room temperature over
night, the solvent was evaporated under reduced pressure
and the residue was dissolved in CH2Cl2. The organic
solution was washed with brine, dried over Na2SO4. The
Ethyl 7-benzoyl-3-(furan-2-carbonyl)indolizine-1-
carboxylate (5f)
Start with phenyl(pyridin-4-yl) methanone and 2-bromo-1-
(furan-2-yl)ethanone, follow the same procedure as in the
synthesis of 1, purify with silica gel chromatography
Chem Biol Drug Des 2013; 81: 730–741
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Huang et al.
solvent was concentrated, and the residue was purified
with silica gel chromatography (petroleum ether:
EtOAc = 3:1) to afford compound 3a 135.7 mg (59.8%)
as a yellow solid; Mp: 168–170 °C; 1H NMR (400 MHz,
CDCl3) d 9.99 (d, J = 7.2 Hz, 1H), 8.46 (s, 1H), 8.33 (s,
1H), 7.99 (d, J = 8.4 Hz, 2H), 7.96–7.89 (m, 3H), 7.66–
7.56 (m, 2H), 7.14 (dd, J = 7.2, 1.7 Hz, 1H), 4.38 (q,
J = 7.2 Hz, 2H), 3.76 (s, 2H), 3.46 (s, 2H), 1.81–1.61 (br,
6H), 1.38 (t, J = 7.2 Hz, 3H); 13C NMR (100 MHz, CDCl3)
d 185.8, 167.8, 164.0, 138.9, 137.0, 135.3, 135.1, 132.6,
130.1, 129.4, 128.6, 128.2, 128.1, 127.1, 125.6, 123.4,
117.9, 114.3, 107.9, 60.5, 24.7, 14.8; ESI-MS m/z: 455.1
[M + H]+; HRMS (ESI-TOF+) m/z: Calcd for C28H26N2O4:
454.1893; Found: 909.3858 [2M + H]+, 931.3677
[2M + Na]+.
Representative procedure for the synthesis of
compounds 6
3-(2-Naphthoyl)-7-benzoyl-N-ethylindolizine-1-
carboxamide (6a)
Ten per cent NaOH solution (5 mL) was added to the
solution of 1 (448 mg, 1 mmol) in THF (10 mL) and
CH3OH (5 mL). The mixture was heated to reflux for 5 h
and then the solvent was evaporated under reduced pres-
sure, and 2 N HCl solution was added. The precipitate
was filtered, washed with water and dried to afford the
corresponding carboxylic acid 12 almost quantitatively,
which was used for the next reaction without further
purification.
The corresponding carboxylic acid 12 (210 mg, 0.5 mmol)
was dissolved in the mixture of DMF (10 mL) and THF
(10 mL) and then 1-(3-dimethylaminopropyl)-3-ethylcarbodii-
mide hydrochloride (EDC) (144 mg, 0.75 mmol), 1-hydrox-
ybenzotriazole (101 mg, 0.75 mmol), N-ethyldiisopropylamine
(129 mg, 1 mmol) and ethanamine (27 mg, 0.6 mmol) were
added. The reaction mixture was stirred at room temperature
over night. After the solvent was removed, the residue was
extracted with CH2Cl2. The combined organic extracts were
washed with brine, dried over Na2SO4. The solvent was con-
centrated, and the residue was purified with silica gel chroma-
tography (CH2Cl2/CH3OH = 50:1) to afford the target
compound 200 mg (89.6%) as a yellow solid; Mp: 252–
(S)-Ethyl 3-(2-naphthoyl)-7-(1-methoxy-1-
oxopropan-2-ylcarbamoyl)indolizine-1-carboxylate
(3e)
Start with 3-(2-naphthoyl)-1-(ethoxycarbonyl)indolizine-7-
carboxylic acid and (S)-methyl 2-amino propanoate, follow
the same procedure as in the synthesis of 3a, purify with
silica gel chromatography (petroleum ether:EtOAc = 5:1) to
afford 3e. Yield: 60.1%; off-white solid; Mp: 197–199 °C;
1H NMR (400 MHz, CDCl3) d 9.93 (d, J = 7.3 Hz, 1H),
8.77 (s, 1H), 8.33 (s, 1H), 8.01–7.96 (m, 2H), 7.92 (t,
J = 8.3 Hz, 2H), 7.88 (s, 1H), 7.66–7.55 (m, 2H), 7.51 (dd,
J = 7.3, 1.8 Hz, 1H), 7.14 (d, J = 7.3 Hz, 1H), 4.86 (t,
J = 7.2 Hz, 1H), 4.37 (q, J = 7.2 Hz, 2H), 3.83 (s, 3H),
1.60 (d, J = 7.2 Hz, 3H), 1.38 (t, J = 7.1 Hz, 3H); 13C
NMR (100 MHz, CDCl3) d 185.9, 173.5, 164.8, 163.9,
138.6, 136.9, 135.2, 132.6, 132.3, 130.2, 129.5, 129.2,
128.7, 128.3, 128.0, 127.1, 125.5, 123.8, 118.1, 113.6,
108.6, 60.6, 52.9, 49.0, 18.6, 14.7; ESI-MS m/z: 473.2
[M + H]+; Anal. Calcd. for C27H24N2O6: C, 68.63; H, 5.12;
N, 5.93; Found: C, 68.59; H, 4.99; N, 5.90.
1
254 °C; H NMR (400 MHz, CDCl3) d 9.89 (s, 1H), 8.88 (s,
1H), 8.23 (s, 1H), 7.96–7.77 (m, 6H), 7.66–7.41 (m, 7H), 6.34
(s, 1H), 3.4 (q, J = 7.2 Hz, 2H), 1.17 (t, J = 7.2 Hz, 3H); 13C
NMR (100 MHz, CDCl3) d 194.4, 185.7, 163.7, 137.7, 137.0,
136.8, 134.9, 134.3, 133.1, 132.5, 130.0, 129.9, 129.2,
128.8, 128.6, 128.5, 128.1, 127.9, 127.0, 125.4, 124.6,
123.6, 123.3, 114.7, 112.8, 34.5, 15.2; ESI-MS m/z: 447.2
[M + H]+; HRMS (ESI-TOF+) m/z: Calcd for C29H22N2O3:
446.1630; Found: 893.3333 [2M + H]+; m/z,915.3153
[2M + Na]+.
Ethyl 3-(2-naphthoyl)-7-(benzylcarbamoyl)
indolizine-1-carboxylate (3j)
3-(2-Naphthoyl)-N-(2-(2-(2-aminoethoxy)ethoxy)
ethyl)-7-benzoylindolizine-1-carboxamide (6h)
The corresponding carboxylic acid 12 (419 mg, 1.0 mmol),
N-hydroxy succinimide (NHS) (138 mg, 1.2 mmol), and
EDC (228 mg, 1.2 mmol) were dissolved in DMF (30 mL).
The reaction mixture was stirred at room temperature for
8 h. After the solvent was removed, the residue was
extracted with CH2Cl2. The combined organic extracts
were washed with brine, dried over Na2SO4. The solvent
was concentrated, and the residue was purified with silica
gel chromatography (petroleum ether:EtOAc = 3:1) to
afford the NHS activated carboxylic acid compound
450 mg (87.0%) as a yellow solid.
Start with 3-(2-naphthoyl) -1-(ethoxycarbonyl)indolizine-7-
carboxylic acid and phenylmethanamine, follow the same
procedure as in the synthesis of 3a, purify with silica gel
chromatography (CH2Cl2/CH3OH = 50:1) to afford 3j.
Yield: 18.9%; off-white solid; Mp: 147–149 °C; 1H NMR
(400 MHz, CDCl3) d 9.90 (d, J = 7.3 Hz, 1H), 8.73 (s, 1H),
8.30 (s, 1H), 7.98–7.83 (m, 5H), 7.65–7.54 (m, 2H), 7.51
(d, J = 7.3 Hz, 1H), 7.42–7.33 (m, 4H), 7.31 (d,
J = 6.8 Hz, 1H), 6.95 (t, J = 5.4 Hz, 1H), 4.68 (d,
J = 5.6 Hz, 2H), 4.32 (q, J = 7.1 Hz, 2H), 1.34 (t,
J = 7.1 Hz, 3H); 13C NMR (100 MHz, CDCl3) d 185.9,
165.2, 164.0, 138.7, 137.9, 136.9, 135.2, 132.9, 132.6,
130.2, 129.4, 129.2, 129.1, 128.6, 128.2, 128.0, 128.0,
128.1, 125.5, 123.7, 117.7, 113.8, 108.4, 60.6, 44.6,
14.6; ESI-MS m/z: 477.0 [M + H]+, 953.9 [2M + H]+; Anal.
Calcd. for C30H24N2O4: C, 75.63; H, 5.17; N, 5.74; Found:
C, 75.61; H, 5.08; N, 5.88.
The NHS activated carboxylic acid compound (0.35 g,
0.68 mmol), triethylamine (0.14 g, 1.36 mmol) were dis-
solved in CH2Cl2 (200 mL) and then 2,2′-(ethane-1,2-diyl-
bis(oxy))diethanamine (0.6 g, 4.08 mmol) in CH2Cl2
734
Chem Biol Drug Des 2013; 81: 730–741

HIV-1 VIF-ElonginC Interaction Inhibitors
(30 mL) was added. The reaction mixture was stirred at
room temperature for 12 h. Then the reaction mixture was
washed with saturated NaHCO3 and brine, successively,
dried over Na2SO4. The solvent was concentrated, and
the residue was purified with silica gel chromatography
(CH2Cl2/CH3OH = 50:1) to afford 6h 290 mg (77.7%) as a
CDCl3) d 11.16 (s, 1H), 9.90 (d, J = 7.3 Hz, 1H), 8.94 (s,
1H), 8.28 (s, 1H), 8.20 (s, 1H), 8.01 (d, J = 8.3 Hz, 1H),
7.96 (s, 1H), 7.93–7.76 (m, 5H), 7.63–7.44 (m, 7H), 6.75
(d, J = 8.2 Hz, 1H), 6.21–6.68 (m, 3H), 4.36 (s, 1H), 4.13
(s, 2H), 3.78 (s, 2H), 3.66 (d, J = 3.9 Hz, 1H), 3.43–3.61
(m, 15H), 3.37–3.42 (m, 2H), 3.28 (s, 2H), 3.09 (dd,
J = 7.4 Hz, 4.1 Hz, 2H), 2.97 (s, 1H), 2.74 (s, 1H), 2.60
(s, 1H), 2.03 (s, 2H), 1.50–1.46 (m, 4H), 1.41 (d,
J = 6.7 Hz, 2H); ESI-MS m/z: 1157.3 [M + Na]+; HRMS
(ESI-TOF+): m/z [M + H]+ Calcd for C58H61F3N8O11S:
1135.4211, Found: 1135.4175.
yellow solid; 1H NMR (400 MHz, CDCl3)
d 9.98 (d,
J = 7.3 Hz, 1H), 8.99 (s, 1H), 8.36 (s, 1H), 8.03–7.88 (m,
7H), 7.86 (s, 1H), 7.65–7.52 (m, 7H), 7.26–7.20 (m, 1H),
3.64–3.59 (m, 4H), 3.58–3.54 (m, 2H), 3.51–3.48 (m, 2H),
3.29 (t, J = 4.5 Hz, 2H), 2.55 (t, J = 4.5 Hz, 2H); 13C
NMR (100 MHz, CDCl3) d 194.3, 185.6, 163.9, 137.7,
136.9, 136.7, 134.8, 134.2, 133.0, 132.4, 130.0, 129.3,
128.7, 128.4, 128.0, 127.8, 126.9, 125.4, 125.3, 123.4,
123.3, 114.5, 112.7, 77.6, 77.2, 76.9, 72.4, 70.4, 70.3,
61.4, 39.4; ESI-MS m/z: 550.1 [M + H]+; HRMS (ESI-
TOF+): m/z [M + H]+ Calcd for C33H31N3O5: 550.2342,
Found: 550.2339.
Biological assay
Preliminary screening for VIF inhibitors
A total of 293T cells were seeded into 24-well plates in
DMEM supplemented with 10% PBS and incubated over-
night at 37 °C. Then cells were transfected with pEYFP-
A3G plus HxB2VIF-cmyc or a control plasmid VR1012
using Lipofectamine2000 (Invitrogen, Grand Island, NY,
USA) as recommended by the manufacturer. Cells were
then cultured with different samples (final concentration at
50 lM) or dimethyl sulfoxide (DMSO). Cells were harvest at
48-h post-transfection. YFP fluorescence was monitored
using a Fluoroskan Ascent FL plate reader (Thermo Scien-
tific, Waltham, MA, USA) with excitation at 485 nm and
emission at 538 nm. YFP expression transfected with
pEYFP-A3G plus VR1012 and treated with DMSO was set
to 100%.
Compound 6l
Amine 6h (40 mg, 0.073 mmol), NHS activated carboxylic
acid 13c (42 mg, 0.073 mmol) and triethylamine (15 mg,
0.15 mmol) were dissolved in CH2Cl2. The reaction mix-
ture was stirred at room temperature for 12 h. The solvent
was diluted with CH2Cl2, washed with saturated NaHCO3
and brine, successively, dried over Na2SO4. The solvent
was concentrated, and the residue was purified with silica
gel chromatography (CH2Cl2/CH3OH = 50:1) to afford 6l
37 mg (50.0%) as a yellow solid; 1H NMR (400 MHz,
CDCl3) d 9.89 (d, J = 7.3 Hz, 1H), 8.94 (s, 1H), 8.25 (s,
1H), 8.04 (d, J = 4.4 Hz, 1H), 7.96 (d, J = 8.2 Hz, 1H),
7.90 (s, 1H), 7.88–7.81 (m, 5H), 7.79 (d, J = 7.9 Hz, 1H),
7.57 (t, J = 7.3 Hz, 1H), 7.53–7.42 (m, 5H), 7.29 (s, 1H),
6.73 (d, J = 8.2 Hz, 1H), 6.54 (s, 1H), 4.98 (s, 1H), 4.13–
4.08 (m, 2H), 3.77 (d, J = 4.2 Hz, 2H), 3.59–3.45 (m,
15H), 3.43–3.35 (m, 4H), 3.20 (s, 2H), 1.34 (s, 9H); ESI-
MS m/z: 1032.3 [M + Na]+.
Antiviral assay
Exponentially growing H9 cells were incubated with wild-
type HIV-1 virus at 37 °C for 3 h. After removal of the
inocula and three extensive washings, cells were seeded
into 96-well plates at a density of 1 9 104/well in 100 L of
RPMI 1640. A 100-lL aliquot of compound solutions
(ranging from 0 to 200 lM) was added, and the cells were
incubated for 7 days at 37 °C under 5% CO2. At day 7,
p24 levels in each wells were evaluated using a p24 ELISA
kit (Perklin Elmer, Norwalk, CT, USA). The IC50 (concentra-
tion inhibiting HIV-1 by 50%) value was determined by
plotting the p24 antigen contents against the drug con-
centration. To test the cytotoxicity of the compounds,
exponentially growing uninfected H9 cells were identically
seeded and cultured to that in the HIV antiviral assay.
At day 7, cytotoxicity was evaluated with a 3-(4, 5-dim-
ethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide colori-
metric assay (MTT assay). CC50 represent the compound
concentration that decreases the cell viability by 50%.
Compound 6n
Boc-protected amine 6l (33 mg, 0.033 mmol) was dis-
solved in CH2Cl2 (30 mL), trifluoroacetic acid (0.1 mL) was
added, and the reaction mixture was stirred at room tem-
perature over night. The solvent was diluted with CH2Cl2,
washed with saturated NaHCO3 and brine, successively,
dried over Na2SO4. The solvent was concentrated to
afford amine 6m, which was used for the next reaction
without further purification.
Amine 6m, Biotin-NHS (23 mg, 0.067 mmol), and triethyl-
amine (three drops) were dissolved in CH2Cl2, the reaction
mixture was stirred at room temperature for 24 h. The sol-
vent was diluted with CH2Cl2, washed with saturated
brine, dried over Na2SO4. The solvent was concentrated,
and the residue was purified with silica gel chromatogra-
phy (CH2Cl2/CH3OH = 15:1) to afford 6n 30 mg (80.1%
over two steps) as a yellow solid; 1H NMR (400 MHz,
Results and Discussion
Design of indolizine derivatives
The phenyl ring of moiety A was firstly modified by introduc-
ing hydrophobic and hydrophilic substitutes to determine
Chem Biol Drug Des 2013; 81: 730–741
735

Huang et al.
the effect of substitutes on activity and then it was replaced
by heterocycles and nonaromatic groups. The carbonyl
group of moiety A was also probed with more flexible linkers
to determine the subtle modifications of this region on activ-
ity. The naphthalene ring of moiety B was replaced with vari-
ous aromatic rings especially substituted phenyl rings to
probe its role on activity. The ethoxycarbonyl group of moi-
ety C was modified by introducing hydrophilic groups to
improve solubilities of compounds, and it was found that
appropriate modification was tolerable. Based on the above
information, a photophore was introduced to the ester
group, which generated three compounds as potential
photoaffinity labelling probes (18–21).
Moiety A modification
To investigate the effect of the left side benzoyl group on
anti-HIV-1 activity, a series of compounds with modifica-
tions of moiety A were performed based on two structural
elements, that is phenyl ring and carbonyl group.
To examine the effect of the phenyl group, the modifica-
tion began with the replacement of the phenyl ring by vari-
ous aromatic rings. It was found that introduction of
hydrophobic substituent (2a, 2b) to the phenyl ring
decreased solubility of compounds. A significant decrease
of activity was observed in the preliminary assay when a
hydrophilic group (2c, 2e) was introduced. Replacing the
phenyl ring using different heterocycles gave variable
results. The 2-pyridine and furan analogues (2f, 2h) exhib-
ited decreased solubilities, while the 4-pyridine analogue
2g exhibited the most potent activity in the preliminary
assay. Further anti-HIV-1 activity evaluation revealed that
compound 2g was the most potent HIV-1 VIF–ElonginC
interaction inhibitor obtained so far with an IC50 of 11.0 lM
and low cytotoxicity (137.3 lM), and resulting in a selectiv-
ity index of 12.5.
Synthesis of indolizine derivatives
For the synthesis of indolizine derivatives, the key step
was to construct the indolizine core structure. Among vari-
ous approaches (22–24), 1, 3-dipolar cycloaddition reac-
tion of pyridinium with electron-deficient alkynes provides
an efficient methodology for synthesizing indolizine motifs,
and it was used in this study to get the target 1, 3, 7-tri-
substituted compounds.
The next modification to moiety A was the replacement of
the phenyl ring with nonaromatic groups. The phenyl ring
was firstly replaced by heterocyclic aliphatics. The prelimin-
ary assay showed that activity decreased in the presence of
hydrophilic groups (3c, 3d), while the other compounds
showed decreased solubilities (3a, 3b). Then, amino acid
residues and various amine groups were chosen to replace
the phenyl ring; the preliminary assay showed that com-
pounds 3g and 3l resulted in improved potency. However,
further anti-HIV-1 activity assay revealed that compound 3l
showed onefold loss of inhibitory activity, while high toxicity
was observed for compound 3g. Taking all the above
results into account, the unsubstituted aromatic ring in
moiety A was essential to the activity, and it was retained in
subsequent modifications.
Compounds 3 were obtained by the reaction of 7-carboxyl
indolizine with various amines. The 7-carboxyl indolizine
compound was obtained by the hydrolysis of 7-benzyl
ester group, and condition of hydrolysis was optimized.
Complex products were obtained when the reaction was
conducted under typical catalytic hydrogenation condi-
tions. Then, TBAF was adopted as deprotecting reagent
according to the facile method described in literature (25),
and the benzyl ester group was removed successfully.
Through further investigation, it was found that the benzyl
ester group can also be hydrolysed selectively under basic
conditions using LiOH as the base.
The ester group of compound 1 was very stable, and it
was hydrolysed under strong alkaline conditions. The
resulted carboxylic acid 12 reacted with amines to afford
compounds 6. The photophore and the three potent
photoaffinity labelling probes were synthesized in a simi-
lar way as the methods described in the literatures
(26,27).
Finally, the modification of moiety A was carried out on the
carbonyl group as it was considered as linker to combine
the aromatic ring and the indolizine core structure. It was
found that replacement of the carbonyl group with more
flexible linker such as methene, hydroxymethyl and amino
groups resulted in a decreased activity in the preliminary
assay, suggesting that the carbonyl group of moiety A is
necessary for maintaining activity.
Biological activities and SARs
Most of the designed compounds were firstly assessed
at a single concentration of 50 lM for the effect of inhibit-
ing VIF-mediated A3G degradation using the preliminary
screening assay. The values were presented as the nor-
malized intensity relative to the value in the HxB2VIF-cmyc
or untreated pEYFP-A3G group as shown in Table 1.
Some of the designed compounds were not assessed in
the preliminary screening assay due to their poor solubili-
ties. Then nine compounds were further evaluated for
their antiviral activities, and the results were shown in
Table 2.
Moiety B modification
The significance of the naphthoyl group for anti-HIV activity
was examined and five analogues (5a–5e) with modifica-
tions of moiety B by replacing the naphthoyl group with
substituted benzoyl group were obtained via two-dimen-
sional similarity searching. These compounds all contained
hydrophobic group substituted phenyl rings and biological
evaluation indicated that they showed reduced activities in
the preliminary assay.
736
Chem Biol Drug Des 2013; 81: 730–741

HIV-1 VIF-ElonginC Interaction Inhibitors
Table 1: Chemical structures of compounds and their preliminary activities.
O
N
O
N
F
CO2C2H5
O
CO C H
2 5
O
2
O
O
NO2
NH2
N
N
CO2C2H5
O
CO C H
2 2 5
O
O
O
HO
BnO
N
N
CO C H
CO C H
2 2 5
O
O
2
2
5
O
O
N
N
N
N
O
CO2Et
CO Et
2
O
O
O
O
N
N
N
CO2Et
CO2C2H5
O
O
O
O
HN
N
O
N
N
N
CO2C2H5
CO2C2H5
O
O
O
O
OH
N
N
H
N
N
CO2C2H5
CO2C2H5
H3CO2C
O
O
O
O
SCH3
N
N
H
N
H
N
CO2C2H5
CO2C2H5
H3CO2C
O
H3CO2C
O
O
O
N
N
H
N
HN
CO C H
2 5
2
CO2C2H5
O
H3CO2C
O
O
O
N
N
H
N
H
N
O
CO Et
CO2Et
2
O
O
Chem Biol Drug Des 2013; 81: 730–741
737

Huang et al.
Table 1: continued
O
O
N
N
N
N
H
H
N
N
CO2Et
CO2Et
O
O
O
O
N
N
CO2C2H5
O
CO2C2H2
OH
O
N
N
N
H
CO2C2H5
CO2C2H5
O
NO2
Br
O
O
N
N
CO2C2H5
CO2C2H5
O
O
Cl
O
O
OCH3
N
Cl
N
CO2C2H5
O
CO2C2H5
O
O
O
O
N
N
CO2Et
CO2C2H5
O
O
F
O
O
NH2
N
N
CO2C2H5
CO2C2H5
O
O
OH
O
N
OH
O
CHO
N
CO2C2H5
CO2Et
O
O
OH
OH
OH
O
O
COOH
N
N
CO2C2H5
CO2C2H5
O
O
738
Chem Biol Drug Des 2013; 81: 730–741

HIV-1 VIF-ElonginC Interaction Inhibitors
Table 1: continued
O
O
O
N
N
CO2CH3
O
N
H
O
O
NHC2H5
O
O
O
N
N
O
N
O
N
H
O
H
H3CO2C
O
O
O
NH
N
N
HN
O
H
N
OH
O
O
N
H
O
O
O
O
O
O
O
O
O
O
N
N
HO
H2N
O
N
O
N
O
O
H
H
O
N
H
N
O
O
O
N
O
O
O
N
CF3
O
N
H3CO
O
H
N
O
N
O
O
H
O
N
N
N3
O
CF3
O
O
N
O
H
N
O
O
N
O
H
O
O
S
H
N
H
O
N
N
HN
H
O
NH
CF3
O
O
O
O
N
O
H
N
O
O
N
O
H
O
aCompounds were tested in triplicate, and the data were presented as the mean value.
bNot detected due to poor solubility.
To enrich structural diversity, the naphthalene ring of moi-
ety B was further replaced by other aromatic rings and
hydrophilic group substituted phenyl rings. It was found
that large aryl groups such as a-naphthyl and biphenyl-4-
yl resulted in reduced solubilities, and compounds with
hydrophilic groups were devoid of activity in the preliminary
Chem Biol Drug Des 2013; 81: 730–741
739

Huang et al.
Table 2: Anti-HIV-1 activity of the selected compounds.
Compounda
IC50 (lM)b
CC50 (lM)c
SId
Compound
IC50 (lM)
CC50 (lM)
SI
1
36.1
11.0
51.8
82.0
40.7
167.4
137.3
43.3
123.9
137.4
4.6
12.5
0.84
1.5
3.4
6h
6i
n.d.e
56.5
14.9
27.4
34.6
13.6
125.4
174.6
168.5
160.1
2g
3g
3l
2.2
11.7
6.1
6j
6k
6n
6f
4.6
aCompounds were tested in triplicate.
bThe concentration that affords 50% inhibition of p24 production in the H9 cells.
c50% cell toxicity concentration determined by MTT assay.
dSelectivity index calculated as CC50/IC50
eNot detected.
.
assay. As a result, it was concluded that the naphthoyl
group was essential for conserving activity of these com-
pounds.
mined. Compound 2g provided the highest potency with
IC50 of 11.0 lM and SI of 12.5, which is the most active
HIV-1 VIF inhibitor targeting the VIF–ElonginC interaction
reported to date. The modification of the ester group gave
us promising clues for further improvement of the
inhibitor’s activity. The three potential photoaffinity labelling
probes might be useful for the identification of molecular
targets of compound 1.
Moiety C modification
The moiety C was modified to probe the importance of
the ethoxycarbonyl moiety as well as to improve the solu-
bility of the compounds by introducing hydrophilic groups.
The ester group was firstly replaced by various amide
groups as the later is a common bioisostere of ester group
and shows high stability. In this case, different amine
groups were introduced to examine the effect of steric
hindrance on anti-HIV-1 activity. It was found that the
introduction of a short-chain amine group reduced activity,
while the introduction of a long amine chain (6f) restores
activity to an IC50 of 40.7 lM. Then long hydrophilic chains
were introduced to the ester group to improve the solubil-
ity of the compound. Unfortunately, the resulted com-
pounds 6g and 6h lost activities in the preliminary assay,
and 6h showed high cytotoxicity with a CC50 of 13.6 lM.
We assume that the cytotoxicity may be caused by the
terminal hydrophilic group and then acyl group was intro-
duced which resulted in compounds 6i, 6j, 6k and 6n. It
was found that these four compounds all showed
decreased in cytotoxicity and restored the anti-HIV-1 activ-
ity to the level of compound 1. The results showed that
modification of the ester group was tolerable.
Acknowledgments
This work was supported by the National Natural Science
Foundation of China (20972009) and the Doctoral Fund of
Ministry of Education of China (20090001120049).
Conflict of Interest
All authors declare no conflict of interest.
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Additional Supporting Information may be found in the
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Appendix S1. Similarity search method. Detailed synthetic
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