Synthesis and HIV-1 inhibitory activities of dicaffeoyl and digalloyl esters of quinic acid derivatives

Article abstract of DOI:10.2174/092986713804999349

Twenty analogues of the anti-HIV-1 integrase (IN) inhibitors dicaffeoylquinic acids (DCQAs) were prepared. Their IC₅₀ values for 3'-end processing and strand transfer against recombinant HIV-1IN were determined in vitro, and their cell toxiciti

Full text of DOI:10.2174/092986713804999349

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7
24
Current Medicinal Chemistry, 2013, 20, 724-733
Synthesis and HIV-1 Inhibitory Activities of Dicaffeoyl and Digalloyl Esters of Quinic
Acid Derivatives
1
2
1
1
1
2
C.O.R. Junior , S.C. Verde , C.A.M. Rezende , W. Caneschi , M.R.C. Couri , B.R. McDougall ,
2
,1
W.E. Robinson, Jr. and M.V. de Almeida*
1
Departamento de Química, Instituto de Ciências Exatas, Universidade Federal de Juiz de Fora, Juiz de Fora, MG, Brazil;
Department of Pathology and Laboratory Medicine, University of California, Irvine, CA, USA 92697
2
Abstract: Twenty analogues of the anti-HIV-1 integrase (IN) inhibitors dicaffeoylquinic acids (DCQAs) were prepared. Their IC50 values
for 3’-end processing and strand transfer against recombinant HIV-1IN were determined in vitro, and their cell toxicities and EC50 against
HIV-1 were measured in cells (ex vivo). Acetylated or benzylated and/or with cyclohexylidene group compounds exhibited no inhibition
of integration in biochemical assays or viral replication in HIV-infected cells, with the exception of 16 and 36. Removal of these groups,
however, correlated with potent inhibition. Compounds 19, 31, and 38, all digalloyls, exhibited the most robust inhibitory performance in
biochemical assays as well as in cell culture and less toxicity than other molecules in the current study.
Keywords: Integrase inhibitor, HIV-1, dicaffeoyl, digalloyl, second generation inhibitor, esters, quinic acid, cyclitol, anti-HIV agents,
catechol.
OCaf
INTRODUCTION
HO
2
C
CO
2
H
2
HO C
OH
Human immunodeficiency virus (HIV-1) is the etiologic agent
of acquired immune deficiency syndrome (AIDS), a serious global
public health problem. HIV integrase (IN) is one of three enzymes
required by HIV to infect a host cell. The other two, reverse tran-
scriptase and protease, have traditionally been exploited as antiviral
targets [1]. IN inhibition is relatively novel, and only two U.S.
FDA-approved inhibitors, raltegravir (RGV) and elvitegravir
CaflO
OCaf
OH
OCaf
3
,5-dicaffeoylquinic acid (1)
L-(-)-dicaffeoyltartaric
acid (2)
CO
2
H
HO
HO
2
CO H
OH
OH
(
EVG), exist. IN catalyzes the insertion of the HIV cDNA into the
HO
2
C
OH
HO
host cell DNA, which is essential for the production of progeny
viruses. Therapeutic agents that can inhibit this process are effec-
tive anti-HIV agents [1-3].
OH
OH
Caffeic acid (3)
OH
Gallic acid (4)
Quinic acid (5)
The dicaffeoylquinic acids (DCQAs, for example, compound 1)
and dicaffeoyltartaric acids (DCTAs, for example, compound 2)
Fig. (1) are potent and selective inhibitors of IN. They also inhibit
HIV-1 replication at nontoxic concentrations [4]. Other derivatives
of caffeic acid (3) and gallic acid (4) condensates with different
classes of substrates, specifically carbohydrates and cyclohex-
anepolyols, also show inhibition of IN [1-9]. It has been proposed
that the catechol moiety plays a major role in the inhibition by these
compounds by chelation with divalent metals Mg (II) and Mn (II) in
the catalytic site of IN [9-11].
Fig. (1). Structure of the 3,5-dicaffeoylquinic, L-(-)-dicaffeoyltartaric, caf-
feic, gallic and quinic acids.
hydroxyl groups followed by reaction with oxalyl chloride [18]. The
spectroscopic data of 6 and 9 are reported in the literature [18, 19].
The acid chloride 10 was prepared by perbenzylation of gallic acid
followed by basic hydrolysis of the ester group and reaction with
oxalyl chloride (Scheme 1) [20, 21].
O
O
OH
a, b
Cl
The quinic acid (5) Fig. (1) is a carboxylated cyclohexanepolyol
that is widespread in the plant kingdom, where it is free or in the
form of various esters with caffeic acid (3) and gallic acid (4) Fig.
HO
AcO
OH
OAc
(1). It is widely used as an optically-active synthetic precursor in
6
3
multistep chemical synthesis [12]. Barco and co-workers [12] pub-
lished a review that reports the different modifications on all carbon
atoms from quinic acid. Among all applications, quinic acid is
widely used to obtain carbasugar derivatives [13-17].
O
O
O
HO
HO
RO
RO
OH
a or c
OH
Cl
b
In this context, we describe the synthesis of dicaffeoyl and di-
galloyl esters of quinic acid derivatives and their anti-HIV and anti-
IN activities.
RO
RO
OH
OR
OR
4
7 R= Ac
9
R= Ac
8
R= Bn
10 R= Bn
RESULTS
Synthesis
Scheme 1. a: Ac
2
O, py, 0°C-r.t.; b: (COCl) , toluene, 0°C-r.t.; c: i) NaH,
2
DMF, r.t. for 30 min. then BnBr, 100°C; ii) NaOH, acetone, r.t.; iii) HCl.
The isomers 11 and 12 were obtained from quinic acid (5) in
four steps in 10% and 57% yield, respectively, as previously re-
ported [22]. These compounds were condensed with 6 or 9 in the
presence of pyridine followed by hydrolysis of the acetyl and cy-
clohexylidene groups using hydrochloric acid in THF to furnish the
diesters 17-20 (Scheme 2).
The acyl chlorides 6 and 9 were prepared from caffeic acid (3)
and gallic acid (4), respectively, by acetylation of phenolic
*
Address correspondence to this author at the Departamento de Química, Instituto de
Ciências Exatas, Universidade Federal de Juiz de Fora, Juiz de Fora, MG, Brazil; Tel:
55 32 21023310; Fax: +55 32 21023310; E-mail: mauro.almeida@ufjf.edu.br
+
1
875-533X/13 $58.00+.00
© 2013 Bentham Science Publishers

Synthesis and HIV-1 Inhibitory Activities
Current Medicinal Chemistry, 2013, Vol. 20, No. 5
725
OR1
O
OH
3
1
OR2
HO
R O
O
O
a
b
1
O
2
5
R O
OH
OH
OH
OH
1
2
1
1
13 R = 3',4'-di-O-acetylcaffeoyl
1
17 R = caffeoyl
19 R = galloyl
HOOC
OH
4 steps
ref. 21
1
2
OH
5 R = 3',4',5'-tri-O-acetylgalloyl
OH
3
OR1
5
5
OR2
1
HO
R O
a
b
1
2
O
O
O
O
R O
OH
OH
1
2
12
14 R = 3',4'-di-O-acetylcaffeoyl
1
18 R = caffeoyl
1
2
6 R = 3',4',5'-tri-O-acetylgalloyl
20 R = galloyl
-
1
Scheme 2. a: 6 or 9, py, toluene, 0°C-r.t.; b: HCl (1 mol.L ), THF, r.t.
The compounds 21 and 22 were obtained by benzylation of
diols 11 and 12, respectively, by treatment with benzyl bromide
using phase transfer conditions, followed by the hydrolysis of the
cyclohexylidene group with trifluoroacetic acid (TFA) to give 23
and 24 (Scheme 3). These diols were subsequently condensed with
end of the oligonucleotide substrate, and the strand transfer (ST)
substrate, which quantifies the covalent joining of the processed
oligonucleotide into another oligonucleotide. The latter two of these
reactions, 3’-EP and ST, quantify what is, in effect, the integration
reaction. Following incubation, the reaction products were resolved
on a urea-PAGE gel, which was subsequently exposed to phos-
phorimager analysis. The inhibition of percent product conversion
was used to calculate the 50% inhibitory concentration (IC50) Fig.
(2). Epimers 13 and 14 exhibited no inhibition of integration in
biochemical assays or viral replication in HIV-infected cells. When
the compounds were deacetylated and the cyclohexylidene group
hydrolyzed to yield 17 and 18, both inhibited in vitro integration at
low to submicromolar concentrations (Table 1) and displayed mild
selectivity for the 3’-EP and ST reactions. Compound 17 exhibited
modest inhibition of integration in HIV-infected cells.
6
or 10 in the presence of pyridine leading to 25-28. Then the com-
pounds 25 and 26 were treated with palladium (0) in methanol lead-
ing to compounds 29 and 30, [23] respectively, while 27 and 28
were hydrogenated in ethyl acetate and presence of palladium 10%
leading to compounds 31 and 32, respectively (Scheme 3).
The quinic acid (5) was transformed into the ꢀꢁlactone 33 by
reaction with cyclohexanone in the presence of Amberlite IR120
(
Scheme 4) [24]. The ꢀ-lactone was reduced using lithium alumin-
ium hydride (LAH) in THF to give the triol 34 in 90% yield. The
primary and secondary hydroxyl groups of 34 were condensed with
6
or 9 in the presence of pyridine to give compounds 35 and 36,
Compounds 15 and 16 possess acetylated digalloyl and cyclo-
hexylidene side chains linked to cyclitol cores. The acetylated 15
did not exhibit any inhibitory activity during biochemical or cellular
integration assays. Compound 16, despite its acetylated hydroxyl
groups, inhibited integration in vitro at low micromolar concentra-
tions with clear selectivity towards the ST reaction. The removal of
the cyclohexylidene and acetyl groups from 15 and 16 to produce
the compounds 19 and 20 again correlated with a dramatic increase
in anti-IN activity. Compound 20 exhibited modest inhibition of
integration in vitro and slight ST selectivity, but did not prevent
integration in infected cells. Compound 19, potently inhibited all
reactions of integrase in vitro at submicromolar concentrations.
Compound 19 was also active in cellular assays and inhibited inte-
gration at low micromolar concentrations with low cytotoxicity.
which were treated with hydrochloric acid in THF leading to di-
esters 37 and 38 (Scheme 4).
Acetylated Caffeoyl, Galloyl Moieties and Cyclohexylidene
Groups Abolish Inhibitory Efficacy
To evaluate the hypothesis that caffeoyl or galloyl side chains
linked to quinic acid-derived cyclohexanepolyols could inhibit inte-
gration, compounds 13-16 and 35-36 were incubated in the pres-
ence of purified, recombinant IN and radiolabeled oligonucleotide
substrates. Integrase activities can be measured using several sub-
strates, including the disintegration substrate, which quantifies the
reversal of integration, the 3’-end processing (3’-EP) substrate,
which quantifies the removal of two oligonucleotides from the 3’
OH
OBn
OBn
OBn
OH
a
b
c
d for 25
e for 27
HO
BnO
O
O
OR1
HO
OR2
BnO
BnO
OH
O
O
OR1
OR2
OH
2
1
29 R = caffeoyl
11
21
23
25 R = 3',4'-di-O-acetylcaffeoyl
2
2
1
31 R = galloyl
7 R = 3',4',5'-tri-O-benzylgalloyl
OH
OBn
HO
BnO
OBn
OBn
OH
d for 26
e for 28
a
b
c
O
O
O
BnO
BnO
HO
O
OH
R
1
O
2
R O
OR1
OR2
OH
1
2
12
22
24
26 R = 3',4'-di-O-acetylcaffeoyl
2
30 R = caffeoyl
1
2
8 R = 3',4',5'-tri-O-benzylgalloyl
32 R = galloyl
Scheme 3. a: BnBr, n-Bu
reflux for 25 and 26; e: H
4
NBr, CH
2
Cl
2
, NaOH 50% (m/v), r.t.; b: CF
3
COOH, H
2
O, CH
2
Cl , reflux; c: 6 or 10, py, toluene, 0°C-50°C; d: Pd/C (10%), MeOH,
2
2
, Pd/C (10%), EtOAc, r.t. for 27 and 28.

7
26 Current Medicinal Chemistry, 2013, Vol. 20, No. 5
Junior et al.
OH
5
O
O
OH
O
O
OH
HOOC
O
OH
a
O
b
d
1
OH
3
OH
OH
OH
5
33
34
c
OR1
OR1
OR2
OR2
OH
O
OH
OH
OH
O
2
1
3
3
7 R = caffeoyl
35 R = 3',4'-di-O-acetylcaffeoyl
2
1
8 R = galloyl
36 R = 3',4',5'-tri-O-acetylgalloyl
-
1
Scheme 4. a: i) cyclohexanone, toluene, DMF, reflux for 50 h; ii) Amberlite IR 120, reflux; b: LiAlH , THF, r.t.; c: 6 or 9, py, toluene, r.t.; d: HCl (1 mol.L ),
4
THF, r.t.
a
Table 1.
Biochemical and Antiviral Activities in ꢀM of Active Compounds 16 – 38
Anti-HIVLAI
Activity c
Disinteg. IC50
3’-EP
IC50 (SD)
ST
IC50 (SD)
Cell Toxicity
Cell Toxicity
CT50 (SD)
e
Compound
b
b
b
d
d
TI
(
SD)
CT
5
(SD)
EC50 (SD)
f
RGV
35.2 (9.2)
>100
2.50 (0.64)
2.82 (0.13)
1.71 (0.06)
20.6 (1.16)
0.71 (0.16)
2.69 (0.18)
0.33 (0.01)
34.3 (1.47)
3.78 (3.3)
5.39 (0.49)
0.71 (0.16)
3.33 (0.19)
>100
0.11 (0.01)
0.24 (0.06)
1.68 (0.75)
4.5 (0.55)
0.004 (0.002)
0.001 (0.0001)
0.41(0.13)
>10
41.2 (0.8)
8.68(1.62)
>80.6
291 (15)
24.7(2.63)
>80.6
72.750
24.700
>196
g
EVG
,5-DCQA
h
3
5.53 (0.24)
>100
i
1
1
1
1
2
2
3
3
3
3
3
3
2
3
6
7
8
9
0
9
0
1
2
6
7
8
9
0
6.6 (2.7)
31.3 (3.3)
352 (117)
261 (20)
na
7.29 (0.47)
24 (0.7)
0.67 (0.9)
149 (9.8)
>264
142 (47.5)
161 (9.5)
69.3 (4.5)
39 (11.3)
111 (42.5)
34.6 (3.7)
82.6 (7.6)
33.6 (2.5)
51.3 (0.65)
56.2 (7.2)
>207.2
2.47
na
1.58 (0.34)
0.24 (0.05)
15.1 (1.84)
3.67 (0.48)
1.83 (0.25)
0.46 (0.01)
3.89 (0.41)
30.5 (1.34)
3.99 (0.84)
3.2 (0.14)
0.79 (0.05)
71 (11.4)
52.2 (7.9)
62.3 (7.9)
1.21 (0.8)
11.4 (0.6)
>100
2.73 (0.37)
>34.5
182 (3.6)
136 (9.3)
5415 (6202)
268 (7.0)
196 (6.6)
205 (8.0)
75.2 (1.6)
487 (137)
>207.2
66
na
>264
na
>66.2
na
3.19 (0.5)
>69
61.4
na
>61
na
23.3 (3.2)
11.4 (0.65)
52.2 (7.9)
62.3 (7.9)
6.82 (0.63)
2.79 (0.17)
3.78 (3.3)
5.39 (0.49)
56.1 (18.3)
12.2 (1.1)
>264
8.66
>17
na
3.67 (0.48)
1.83 (0.25)
111 (42.5)
34.6 (3.7)
5415 (6202)
268 (7.0)
>66.2
na
a
b
c
Values represent the mean of triplicate assays; parenthetical values are 1 standard deviation (SD). IC50, the concentration of compound required to inhibit 50% of enzymatic activity. EC50, the concentration of
d
compound required to inhibit 50% of HIV-induced cytopathic effect. CT
and the concentration of compound is nontoxic. TI, therapeutic index, ratio of the inhibitor concentration that induces 50% toxicity-related cell death (CT50) to the effective dose of a compound (EC50). RGV and
EVG exhibit increased potency in vivo due integrase higher-order multimer formation. 3,5-DCQA: 3,5-dicaffeoylquinic acid. na, not applicable, TI unable to be calculated for this compound. Disin-
5
, the concentration of compound required to inhibit cellular replication by 5% relative to an untreated control, i.e., cells are 95% viable
e
f
g
h
teg.=disintegration reaction, 3’-EP=3’-EP reaction, ST=ST reaction.
Compounds 35 and 36 contain acetylated 3,5-dicaffeoyl or di-
galloyl, respectively, linkages to the cyclohexylidene modified
core, which possesses a hydroxymethylene group (pseudosugar)
instead of carboxylic group. Though 35 does not exhibit biochemi-
cal or antiviral efficacy, 36 selectively inhibits the ST reaction and
is slightly active in cellular assays. This ST-specificity is mimicked
by compound 16, suggesting that digalloyl groups, even when ace-
tylated, are sufficient to inhibit IN.
involving compounds 13 and 14, neither compound 25 nor 26,
which possessed acetylated caffeoyl groups, exhibited any inhibi-
tion of integration. Likewise, the introduction of benzylated digal-
loyl residues, compounds 27 and 28 did not produce inhibitory
activity. Removal of the benzyl and acetyl groups, however, corre-
lated with potent inhibition.
Compounds 29 and 30 both exhibited low micromolar inhibi-
tion of the 3’-EP and ST reactions. Neither compound inhibited
viral replication in HIV-infected cells. Compounds 31 and 32 also
inhibited integration in biochemical assays at low micromolar con-
centrations. Indeed, 31 inhibited both 3’-EP and ST reactions at
submicromolar concentrations and exhibited low micromolar po-
Benzylated Caffeoyl, Galloyl Moieties Reduce Anti-IN and An-
tiviral Efficacy
To investigate the hypothesis that acetylated dicaffeoyl or ben-
zylated galloyl residues in the presence of a benzylated cyclitol core
would inhibit integration activity, compounds 25-28 were employed
in biochemical and cellular assays. Similar to previous experiments
tency in anti-HIV assays, yet exhibited a CT
the compound’s EC50
5
25-fold higher than
.

Synthesis and HIV-1 Inhibitory Activities
Current Medicinal Chemistry, 2013, Vol. 20, No. 5
727
3
2
Fig. (2). Compound 17 inhibits HIV-IN mediated strand transfer catalysis. 100 nM HIV IN was incubated for one hour at 37ꢀC in presence of 0.1 pmol P-
labelled oligonucleotide end-processed substrate homologous to the viral long-terminal repeat region, reaction buffer, and decreasing ꢁM concentrations of the
inhibitor, compound 17. Reactions were stopped with the addition of 18 mM EDTA and formamide. Products were resolved via denaturing urea-PAGE. Phos-
phorimager analysis was used to quantify percent-product conversion, and the IC50 was obtained with CalcuSyn software (Biosoft, Cambridge, UK). STP:
Strand transfer products. Arrow: Strand transfer substrate. S: Reaction performed in the absence of IN. DMSO: Reactions performed in the presence of inhibi-
tor solvent, 100% dimethylsulfoxide. LTA: Denotes addition of 25 ꢁM L-tartaric acid, an inactive analogue of L-chicoric acid [25]. LCA: Denotes addition of
2
5 ꢁM L-chicoric acid [25]. 100-0.3: Denotes ꢁM concentration of compound 17 in which reactions were incubated.
3
,5 Digalloyl, But Not Dicaffeoyl, Linkages Inhibit Integration
sistance to inhibition by 31. These data indicate that cyclitol-based
compounds are effective at prohibiting integration by highly drug-
resistant IN variants.
Of those compounds that inhibited integration in vitro, 17-20,
9-32, and 36-38, only five, 17, 19, 31, 37, and 38 prevented HIV-
2
induced cytopathic effect in cells. Both 17 and 37 have caffeoyl or
caffeoyloxymethyl moieties linked to the 3 and 5 carbon atoms of
the core molecule. The 3,5-linkage proved critical to caffeoyl-based
compounds’ inhibition of HIV replication, as no other arrangement
of caffeoyl pharmacophores around the cyclitol core was effective
in cellular assays. Compounds involving the arrangement of gal-
loyls were more pronounced in their antiviral performance and
exhibited less cytotoxicity. Compounds 19 and 38 are comprised of
galloyl or galloyloximethyl groups linked to the 3 and 5 cyclitol
carbon atoms and 31 contains 1,2-linkages of the same. These com-
pounds inhibit integration, in vitro and ex vivo, at submicromolar
and low micromolar concentrations, respectively. Compounds that
consisted of galloyls linked to other carbon atoms, for example 32,
did not possess inhibitory activity in cellular assays.
DISCUSSION AND CONCLUSION
Initial studies employing quinic acid as the inhibitory molecule
for integration concluded that the compound was ineffective at
prohibiting integrase activity [1, 25]. Similar results have been ob-
tained for chlorogenic acid [10, 25]. While ineffective alone, both
of these compounds, when used as scaffolds for hydroxylated-
phenyl moiety attachment, potently inhibit integration in biochemi-
cal assays [3, 25, 26]. Indeed, DCQAs and DCQA-like compounds
comprised the bulk of early structure-activity-relationship (SAR)
studies aimed at discovering potential anti-IN leads [10].
Multiple SAR studies have determined that the inhibitory ac-
tivities of DCQAs depend upon a quinic acid core possessing at
least one free carboxyl and a bisphenol [25]. Complete acetylation,
methylation, or benzylation of the caffeoyl hydroxyls abolishes
anti-IN as well as antiviral activity [1, 26]. Partial acetylation or
methylation of the hydroxyls restores minimal inhibitory behavior
in biochemical contexts, though such compounds remain inactive in
cellular assays [3]. Stereoisomers of DCQAs perform similarly;
Raltegravir-Resistant IN Remains Susceptible to Compounds
1
7, 19, and 31
To compare the novel cyclitols with current IN inhibitors, both
RGV and EVG were investigated for their inhibition of IN catalysis
and viral replication. RGV was approved by the FDA in 2007, and
EVG is currently enrolled in extended Phase III clinical studies.
Both inhibitors are selective for the ST reaction and exhibit low-
nanomolar efficacy in vivo. Accordingly, neither RGV nor EVG
was capable of inhibiting the disintegration reaction at attractive
concentrations (Table 1). In addition, RGV and EVG exhibited
micromolar potency for the inhibition of the 3’-EP reaction. Only in
the ST reaction, as well as in vivo, was nanomolar potency observed
for either drug.
[
26] the same is not true for more recent inhibitory molecules [27].
Examination of the ester linkages revealed that substitution with
diamides does not reduce potency, [3, 26] though increasing the
length of the catechol’s poly-carbon chain linker correlates with a
precipitous decline of inhibitory efficacy [3]. Finally, cyclohex-
anediol cores, a category that includes all compounds in the current
study, improve the inhibitory performance of biscatechols at the
expense of antiviral potency [28].
Similar to previous reports, acetylation or benzylation of the
biscatechol groups abolished inhibitory activity for all compounds
analyzed (Table I), with the exception of 16 and 36. Also similar to
previous findings, [4, 26] the removal of these groups restored po-
tency. The compounds 17-20 and 31 exerted anti-IN activity. This
inhibitory activity persisted in the absence of a carboxylated quinic
acid, previously identified as essential for inhibition [25, 26]. In-
deed, a singular result of this study is the demonstration that IN
catalytic inhibition does not require the presence of hydroxylated
quinic acid. The carboxyl group of the quinic acid and the diacid
moeties of such compounds as LCA that exhibit low micromolar
potency have been predicted, through modeling studies, [29] to
Due to their persistent efficacy in biochemical and cellular as-
says, compounds 17, 19, and 31 were examined for their inhibition
of integration performed by IN that exhibited mutations commonly
identified as RGV-resistant (Table 2). These IN mutations included
Q148H, N155H, and the double mutant G140S+Q148H. Similar to
previous biochemical assays, each IN mutant was examined for its
inhibitor susceptibility in the disintegration, 3’-EP, and ST reac-
tions. Q148H, G140S+Q148H, and N155H IN mutants were not
resistant to the inhibitory effects of 17. Q148H was slightly resis-
tant to 19 in the 3’-EPST assay, and N155H was slightly resistant to
inhibition by 19 in ST assay. No IN mutant exhibited >10-fold re-

7
28 Current Medicinal Chemistry, 2013, Vol. 20, No. 5
Junior et al.
a
Table 2.
Biochemical Activities in ꢀM of Compounds 17, 19, and 31 Against Drug-Resistant IN
Disinteg.
IC50 (SD)
3’-EP
IC50 (SD)
ST
IC50 (SD)
Inhibitory
Compound
IN Mutant
Reference
Q148H
7.29 (0.47)
0.64 (0.14)
0.63 (0.17)
1.24 (0.24)
0.7 (0.05)
0.71 (0.16)
0.91 (0.08)
0.67 (0.9)
17
17
17
17
19
19
19
19
31
31
31
31
†
5.64 (1.56)
†
†
G140SQ148H
N155H
6.28 (1.29)
1.25 (0.31)
0.33 (0.01)
2.31 (0.5)
1.75 (0.5)
0.32 (0.04)
0.24 (0.05)
Reference
Q148H
†
0.16 (0.02)
0.26 (0.02)
0.26 (0.02)
1.21 (0.8)
0.84 (0.17)
1.37 (0.61)
†
†
G140SQ148H
N155H
1.13 (0.11)
0.25 (0.04)
0.71 (0.16)
0.99 (0.14)
5.75 (3.36)
0.46 (0.01)
Reference
Q148H
†
0.73 (0.04)
0.94 (0.07)
1.03 (0.15)
70.1 (13.3)
3.04 (0.96)
†
†
G140SQ148H
N155H
2.72 (0.17)
1.29 (0.29)
1.24 (0.56)
a
Values represent the mean of triplicate assays; parenthetical values are 1 standard deviation (SD). †Assays performed in the presence of 350 nM IN due to diminished catalytic activity of IN mutants. 350 nM
reference IN micromolar 3’-EP IC50(SD) for compounds 17, 19, and 31 are, respectively, 5.22 (0.54), 0.88 (0.14), and 2.95 (0.64). 350 nM reference IN ST IC50(SD) for compounds 17, 19, and 31 are, respec-
tively, 1.76 (0.12), 4.06 (0.78), and 16.02 (7.99). Disinteg.=disintegration reaction, 3’-EP=3’-EP reaction, ST=ST reaction.
interfere with IN’s requisite divalent metal positioning. All of the
While many inhibitory assays are performed in the presence of
2
+
free-catechol compounds that exhibited potency (17-20, 31, 37, and
Mg , the biochemical assays completed herein were performed in
2
+
3
8), while absent carboxyls, do possess consecutive hydroxyl
the presence of Mn as the requisite metal cofactor. Initial descrip-
tions of the anti-IN potency of DCQA compound were based upon
groups in the 1,2 (17-20), 3,5 (31) or 1,3,4 (37, 38) positions. It is
possible that these hydroxyl groups perturb active site architecture,
either by hydrogen bonding to the active site residues or to the diva-
lent metals themselves, [11, 30] of an order sufficient to disrupt
catalysis.
2
+
biochemical assays performed in the presence of Mn [1, 25].
2
+
Hence, Mn was utilized herein as an internal control. Reports
describe increased potency of anti-IN compounds, such as DCQAs
2
+
or RGV, in the presence of Mg [30-32]. However, IN catalysis is
2
+
exquisitely sensitive to reaction conditions in the presence of Mg
Compounds 19, 31, and 38 exhibited the most robust inhibitory
performance in biochemical assays as well as in cell culture. In-
deed, these compounds were the only molecules evaluated that
prevented viral replication at concentrations substantially lower
than those at which they exerted minimal toxicity. While TIs of 66
are unsuitable for actual therapeutic application, these values do
render these molecules attractive as scaffolds for further modifica-
tion. Galloyl-based biscatechols generally present with greater inhi-
bition of integration as well as greater cellular toxicity [3, 6, 10,
and consequently exhibits lower specific activity [32, 33]. Thus,
diminished enzymatic activity reduces the concentration of anti-IN
compounds necessary for prescribed inhibition, which can account
2
+
for the enhanced “potency” of drugs in the presence of Mg . IN is
2
+
far more catalytically robust in Mn and thus requires more inhibi-
tor present to elicit inhibition [33]. Those compounds that exhibited
the greatest potency in vitro were also the most potent in vivo,
which provided a useful comparison to the generated biochemical
data. These data indicate that the selection of metal ion by IN does
not alter the relative inhibitory profile of the experimental com-
pounds described herein.
2
7]. Compounds 19, 31, and 38 exhibited less toxicity than other
molecules in the current study, perhaps due to the lack of the car-
boxylated cyclohexanepolyol core.
The data contained herein continue to refine our understanding
of the essential DCQA and digalloylquinic acid (DGQA) moieties
involved in inhibition of HIV IN. The previous finding that a free
carboxylic acid moiety is necessary for DCQA and DGQA-based
inhibition of HIV-IN is no longer supported. Furthermore, these
data confirm the robust activity of DGQAs and present three com-
pounds, 19, 31, and 38 that retain both potent inhibitory profiles
and attractive TIs. Finally, the DCQA 17 and DGQAs 19 and 31
effectively inhibited IN mutants resistant to two IN inhibitors, RGV
and EVG. Combined, these data present attractive scaffolds for
future SAR evaluation and chemical modification.
Galloyl linkages have also been described as reducing IN selec-
tivity, [1] a feature consistent with the results obtained in this study.
Compounds 19, 31, and 38 prevent IN biochemical activities at
submicromolar concentrations; however, they exert little or no se-
lectivity for any of the three reactions IN is capable of performing.
This selectivity could perhaps be restored with the addition of a
third galloyl group, as trigallic acids linked to a glucose scaffold
strongly and selectively inhibit HIV-IN [27]. Additionally, galloyl
groups linked to pyrimidine cores, which resemble the molecular
geometry of 19 and 31, also inhibit IN [28]. Combined, these data
suggest that galloyl-modified molecular scaffolds present an attrac-
tive base for future SAR studies.
EXPERIMENTAL SECTION
Chemical Syntheses. General
The anti-IN activity of compounds 16 and 36 was surprising, as
the compounds not only possess acetylated digalloyl groups, but a
cyclohexane group bound to the 3,4 or 1,2 hydroxyls, respectively.
Modification of the cyclitol core does not typically result in favor-
able performance in inhibitory studies, [3, 5, 25] though highly-
negatively charged linkages, such as trifluorocarbons, can enhance
anti-IN activity [28]. The acetylation of the galloyls would render
the hydrogen-bonding potential, and thus binding affinity, of the
compound within the IN active site relatively inert. Therefore, an-
other mechanism must exist.
The solvents were pretreated, when necessary, according to the
appropriate standard procedures before being used. The compounds
were purified by column chromatography on silica gel (70-230
2 4
mesh ASTM) with visualization under UV light and H SO char-
ring. All reaction mixtures were stirred magnetically. The hydro-
genolysis reactions were performed on an A16CA 4L hydrogena-
tion apparatus. The melting points were recorded on a MQAPF-

Synthesis and HIV-1 Inhibitory Activities
Current Medicinal Chemistry, 2013, Vol. 20, No. 5
729
Microquímica. NMR spectra were obtained with a BRUKER
AVANCER DRX/300 spectrometer using tetramethylsilane as the
internal standard. IR spectra were recorded on a BOMEM-FTIR
MB-120 spectrometer. Mass spectra were recorded on a KRATOS
MS-80 spectrometer. Optical rotations were measured on a
Bellingham Stanley ADP410 polarimeter.
(1R,2S,3R,5S)-3,5-Di-O-caffeoyl-1,2,3,5-cyclohexanetetrol
(17): 70% (0.8 g; 1.8 mmol); White solid; mp 117-119°C; [ꢀ]
D
: -
1
10.0 (c 0.2; CH
3
OH); H NMR (300 MHz, CD
3
OD): ꢀ (ppm) 1.64
(m, 4H), 4.63 (dd, J 9.0 and 2.8 Hz, 1H), 5.04 (m, 1H), 6.05 (m,
1H), 6.15 (m, 1H), 6.25 (d, J 15.9 Hz, 1H), 6.27 (d, J 15.9 Hz, 1H),
6.82-7.16 (m, 6H), 7.54 (d, J 15.9 Hz, 1H), 7.57 (d, J 15.9 Hz, 1H);
1
3
C NMR (75 MHz, CD
3
OD): ꢀ (ppm) 35.0, 36.4, 68.1, 69.2, 72.1,
10Na [M
General Procedure to Prepare 13-16
73.7, 115.3-148.8, 166.8, 167.3; HRMS calcd for C24H O
24
+
+
Na ] 495.1267, found 495.1268.
1R,2S,3R,5R)-3,5-Di-O-caffeoyl-1,2,3,5-cyclohexanetetrol
18): 55% (0.7 g; 1.4 mmol); White solid; mp 148-150°C; [ꢀ]
To a solution of 11 or 12 (1.14 g, 5 mmol) in pyridine (10 mL)
was added at 0°C a solution of 6 or 9 (20 mmol) in toluene (40
mL). The reaction mixture was stirred for 24 h at room temperature.
(
(
D
: -
OD): ꢀ (ppm) 1.90-
1
-1
10.0 (c 0.2; CH
3
OH); H NMR (300 MHz, CD
3
Then the mixture was acidified with HCl (4 mol.L ) until pH 3 and
extracted with EtOAc (3 x 100 mL). The combined organic extracts
2
1
6
.04 (m, 4H), 3.82 (m, 1H), 3.94 (m, 1H), 5.06 (m, 1H), 5.20 (m,
H), 6.22 (d, J 15.7 Hz, 1H), 6.26 (d, J 15.9 Hz, 1H), 6.73-7.04 (m,
2 4
were dried over Na SO , filtered, and the solvents were removed
1
3
H), 7.52 (d, J 15.9 Hz, 1H), 7.54 (d, J 15.9 Hz, 1H); C NMR (75
under reduced pressure. The residue was purified by silica gel col-
umn chromatography (CH Cl / MeOH) leading to compounds 13-
6.
MHz, CD
3
OD): ꢀ (ppm) 31.6, 34.8, 68.4, 69.4, 70.3, 73.1, 115.0-
2
2
+
1
4
49.8, 168.2, 168.8; HRMS calcd for C24
95.1267, found 495.1288.
24
H O10Na [M + Na ]
1
(
1R,2S,3R,5S)-1,2-O-Cyclohexylidene-3,5-di-O-(3’,4’-di-O-
(
1R,2S,3R,5S)-3,5-Di-O-galloyl-1,2,3,5-cyclohexanetetrol
(19): 45% (0.5 g; 1.1 mmol); Colorless oil; [ꢀ] : +266.7 (c 0.24;
OD): ꢀ (ppm) 1.20-2.48 (m,
H), 3.77 (dd, J 9.1 and 2.7 Hz, 1H), 4.18 (m, 1H), 5.27-5.36 (m,
acetyl)-caffeoyl-1,2,3,5-cyclohexanetetrol (13): 57% (2.1 g; 2.9
1
D
mmol); White solid; mp 87-89°C; [ꢀ]
NMR (300 MHz, CDCl ): ꢀ (ppm) 2.31 (s, 12 H), 1.20-2.60 (m, 14
H), 4.16 (t, J 5.9 Hz, 1H), 4.49 (s, 1H), 5.17 (m, 1H), 5.31 (m, 1H),
D 2 2
: -40.0 (c 0.2; CH Cl ); H
1
CH
4
2
3
OH); H NMR (300 MHz, CD
3
3
1
3
3
H), 7.03 (s, 2H), 7.09 (s, 2H); C NMR (75 MHz, CD OD):
6
7
2
1
.28 (t, J 16.0 Hz, 2H), 7.20-7.40 (m, 6H), 7.61 (d, J 16.0 Hz, 1H),
13
ꢀ (ppm) 35.8, 36.8, 68.3, 69.8, 72.4, 74.1, 109.9-146.1, 166.2,
3
.65 (d, J 16.0 Hz, 1H); C NMR (75 MHz, CDCl ): ꢀ (ppm) 20.5,
1
66.7.
(1R,2S,3R,5R)-3,5-Di-O-galloyl-1,2,3,5-cyclohexanetetrol
0.6, 23.7-32.8, 34.7, 37.5, 66.8, 72.0, 72.5, 75.8, 110.0, 119.1,
22.6-143.2, 143.5, 165.5, 167.9, 168.0; HRMS calcd for
+
C
38
H
40
O
14Na [M + Na ] 743.2316, found 743.2337.
(20): 50% (0.6 g; 1.3 mmol); White solid; mp 138-140°C; [ꢀ]
D
: -
OD): ꢀ (ppm)
.26-1.89 (m, 4H), 3.89-3.97 (m, 2H), 5.22-5.30 (m, 2H), 7.01-7.06
1
7
1
(
2.7 (c 0.11; CH
3
OH); H NMR (300 MHz, CD
3
(
1R,2S,3R,5R)-1,2-O-Cyclohexylidene-3,5-di-O-(3’,4’-di-O-
acetyl)-caffeoyl- 1,2,3,5-cyclohexanete-trol (14): 56% (2.0 g; 2.8
mmol); White solid; mp 79-81°C; [ꢀ] : +30 (c 0.2; CH Cl ); (300
MHz, CDCl ): ꢀ (ppm) 2.31 (s, 12H), 1.39-2.20 (m, 14H), 4.12 (t,
J 5.0 Hz, 1H), 4.42 (m, 1H), 5.23 (s, 1H), 5.52 (s, 1H), 6.38 (d, J
1
3
m, 4H); C NMR (75 MHz, CD
3
OD): ꢀ (ppm) 35.2, 38.0, 64.5,
D
2
2
6
C
8.6, 71.7, 74.0, 110.2-146.7, 167.3, 168.0; HRMS calcd for
3
+
20
H
20
O
12Na [M + Na ] 475.0852, found 475.0862.
1
1
2
1
5.8 Hz, 1H), 6.41 (d, J 15.8 Hz, 1H), 7.20-7.45 (m, 6H), 7.64 (d, J
13
Preparation of 21 and 22
3
5.8 Hz, 2H); C NMR: (75 MHz, CDCl ): ꢀ (ppm) 20.8, 20.9,
3.9-32.1, 35.3, 38.3, 67.5, 70.6, 72.6, 75.3, 110.1, 119.2, 119.6,
To a solution of 11 or 12 (2.28 g, 10 mmol) in dichloromethane
40 mL) was added tetrabutylammonium bromide (0.65 g, 2 mmol),
22.9-143.7, 143.8, 165.8, 166.0, 168.2, 168.3; ESI MS: m/z 759.1
(
+
(
M+K ).
sodium hydroxide 50 % v/v (20 mL) and benzyl bromide (4.79 mL,
40 mmol) at room temperature. The reaction mixture was stirred for
96 hours at room temperature and then was extracted with di-
chloromethane (2 x 100 mL). The combined organic extracts were
(
1R,2S,3R,5S)-1,2-O-Cyclohexylidene-3,5-di-O-(3’,4’,5’-tri-
O-acetyl)-galloyl-1,2,3,5-cyclohexanete-trol (15): 50% (2.0 g; 2.5
1
mmol); White solid; mp 89-91°C; [ꢀ]
NMR: (300 MHz, CDCl ): ꢀ (ppm) 2.29 (s, 18H), 1.50-2.66 (m,
4H), 4.19 (t, J 5.3 Hz, 1H), 4.49 (s, 1H), 5.29 (s, 1H), 5.42 (s, 1H),
.75 (s, 2H), 7.81 (s, 2H); C NMR: (75 MHz, CDCl
0.3, 20.7, 23.8-33.1, 34.9, 37.7, 68.1, 72.7, 73.3, 75.9, 110.4,
22.3-143.6, 163.4, 166.5, 167.8; HRMS calcd for C38 18Na [M
D 2 2
: -40.0 (c 0.2; CH Cl ); H
3
2 4
dried over Na SO , filtered, and the solvents were removed under
1
7
2
1
reduced pressure. The residue was purified by silica gel column
chromatography (EtOAc / hexane) to give the corresponding diben-
zyl ethers.
1
3
3
): ꢀ (ppm)
40
H O
(
1R,2S,3R,5S)-1,2-O-Cyclohexylidene-3,5-di-O-benzyl-
+
+
Na ] 807.2112, found 807.2126.
1R,2S,3R,5R)-1,2-O-Cyclohexylidene-3,5-di-O-(3’,4’,5’-tri-
O-acetyl)-galloyl-1,2,3,5-cyclohexanete-trol (16): 45% (1.8 g; 2.3
1
,2,3,5-cyclohexanetetrol (21): 90% (3.7 g; 9.0 mmol); Colorless
1
(
D 2 2 3
oil; [ꢀ] : +50.0 (c 0.16; CH Cl ); H NMR (300 MHz, CDCl ):
ꢀ (ppm) 0.80-1.70 (m, 2H), 2.30-2.50 (m, 2H), 3.50 (m, 1H), 3.72
(m, 1H), 4.02 (t, J 6.6 Hz, 1H), 4.38 (m, 1H), 4.66 (s, 2H), 4.68 (d,
1
mmol); White solid; mp 76-78°C; [ꢀ]
NMR: (300 MHz, CDCl ): ꢀ (ppm) 2.29 (s, 18H), 1.50-2.23 (m,
4H), 4.17 (t, J 5.5 Hz, 1H), 4.42 (m, 1H), 5.37 (m, 1H), 5.54 (m,
D 2 2
: -10.0 (c 0.2; CH Cl ); H
1
3
3
J 12.3 Hz, 1H), 4.76 (d, J 12.3 Hz, 1H), 7.29-7.33 (m, 10 H);
C
1
1
2
1
3
NMR (75 MHz, CDCl ): ꢀ (ppm) 23.9-34.9, 35.4, 38.1, 70.9, 71.5,
1
3
H), 7.77-7.81 (m, 4H); C NMR: (75 MHz, CDCl
3
): ꢀ (ppm)
72.0, 73.7, 79.8, 109.3, 127.7-138.8; HRMS calcd for C26
H
32
O
4
Na
+
0.3, 20.8, 23.9-35.2, 38.3, 38.4, 68.6, 71.8, 72.6, 75.4, 110.2,
22.5-143.7, 163.6, 166.5, 167.7, 167.8.
[M + Na ] 431.2198, found 431.2199.
(
1R,2S,3R,5R)-1,2-O-Cyclohexylidene-3,5-di-O-benzyl-
1
,2,3,5-cyclohexane-tetrol (22): 53% (2.2 g; 5.3 mmol); Colorless
General Procedure to Prepare 17-20
1
oil; [ꢀ]
D 2 2 3
: +29.2 (c, 0.48; CH Cl ); H NMR (300 MHz, CDCl ):
ꢀ
(ppm) 1.19-2.01 (m, 14H), 3.73 (m, 1H), 3.97 (m, 1H), 4.05 (m,
To a solution of 13, 14, 15 or 16 (2.5 mmol) in THF (25 mL)
1
3
-1
1H), 4.28 (m, 1H), 4.40-4.67 (m, 4H), 7.30 (m, 10H); C NMR (75
was added an aqueous solution of 1 mol.L HCl (90 mL). The reac-
tion mixture was stirred for 10 days at room temperature, then was
saturated with solid NaCl and the aqueous phase extracted with
EtOAc (3 x 100 mL). The combined organic extracts were dried
MHz, CDCl
3
): ꢀ (ppm) 23.9-33.3, 35.5, 38.2, 70.1, 71.1, 73.1, 75.0,
+
7
4
7.6, 109.3, 127.5-138.8; HRMS calcd for C26
32 4
H O Na [M + Na ]
31.2198, found 431.2201.
over Na
duced pressure. The residue was purified by silica gel column
chromatography (CH Cl /MeOH) leading to compounds 17-20.
2 4
SO , filtered, and the solvents were removed under re-
Preparation of 23 and 24
To a solution of 21 or 22 (2.05 g, 5 mmol) in dichloromethane
20 mL) was added TFA (2.5 mL) and water (2.0 mL). The reaction
2
2
(

7
30 Current Medicinal Chemistry, 2013, Vol. 20, No. 5
Junior et al.
mixture was stirred for 96 hours under reflux. After this time the
mixture was cooling, water was added (60 mL) and extracted with
dichloromethane (3 x 100 mL). The combined organic extracts
(1R,2S,3R,5R)-1,2-Di-O-(3’,4’,5’-tri-O-benzyl)-galloyl-3,5-
di-O-benzyl-1,2,3,5-cyclohexanetetrol (28): 65% (1.9 g; 1.6
1
mmol); Colorless oil; [ꢀ]
MHz, CDCl ): ꢀ (ppm) 1.61 (m, 2H), 2.26-2.52 (m, 2H), 3.95-3.99
(m, 1H), 4.56-5.13 (m, 16H), 5.46 (td, J 10.9 and 3.1 Hz, 1H), 5.63
D 2 2
: -30.8 (c 0.13; CH Cl ); H NMR (300
were dried over Na
2
SO
4
, filtered, and the solvents were removed
3
under reduced pressure. The residue was purified by silica gel col-
umn chromatography (EtOAc/hexane) giving 23 and 24, respec-
tively.
1
3
(t, J 3.1 Hz, 1H), 7.21-7.31 (m, 44H); C NMR (75 MHz, CDCl
3
):
ꢀ (ppm) 29.9, 32.7, 69.7-75.2, 109.1-152.8, 165.3; ESI MS: m/z
+
1
196.5 (M+Na ).
(
1R,2S,3R,5S)-3,5-Di-O-benzyl-1,2,3,5-cyclohexanetetrol
23): 72% (1.2 g; 3.6 mmol); Colorless oil; [ꢀ] : +140 (c 0.1;
): ꢀ (ppm) 1.21-1.42 (m, 2H),
(
D
Preparation of 29 and 30
1
2 2 3
CH Cl ); H NMR (300 MHz, CDCl
To a solution of 25 or 27 (0.13 g, 0.15 mmol) in methanol (10
mL) was added Pd/C 10% (0.15 g). The reaction mixture was re-
fluxed for 96 hours. After completion of the reaction, the resulting
mixture was filtered and concentrated under reduced pressure. The
2
3
1
1
ꢀ
.33-2.41 (m, 1H), 2.54-2.60 (m, 1H), 2.62 (s, 1H), 2.91 (s, 1H),
.52 (dd, J 9.2 and 3.1 Hz, 1H), 3.59-3.67 (m, 1H), 3.79-3.86 (m,
H), 4.14 (m, 1H), 4.44 (d, J 11.2 Hz, 1H), 4.55 (s, 2H), 4.70 (d, J
1
3
3
1.2 Hz, 1H), 7.25-7.30 (m, 10H); C NMR (75 MHz, CDCl ):
residue was purified by silica gel column chromatography (CH
2
Cl
2
(ppm) 35.2, 36.1, 68.5, 71.0, 71.3, 71.9, 75.3, 76.4, 127.8-138.7;
/
MeOH) leading to compounds 29 and 30, respectively.
1R,2S,3R,5S)-1,2-Di-O-caffeoyl-1,2,3,5-cyclohexanetetrol
D
29): 60% (0.09 mmol; 0.043 g); White solid; mp 144-146°C; [ꢀ] :
Anal. calcd. for C20
H, 7.58.
24 4 2
H O .H O: C, 69.38; H, 7.57. Found: C, 69.40;
(
(
(
1R,2S,3R,5R)-3,5-Di-O-benzyl-1,2,3,5-cyclohexanetetrol
24): 79% (1.3 g; 4.0 mmol); Colorless oil; [ꢀ] : +77.8 (c 0.18;
): ꢀ (ppm) 1.48-1.57 (m, 1H),
1
1
1
1
81.8 (c 0.1; CD
3
OD); H NMR (300 MHz, C
5 5
D N): ꢀ (ppm) 0.88-
(
D
1
.69 (m, 4H), 4.06 (m, 2H), 4.59 (m, 1H), 5.58 (m, 1H), 6.72 (t, J
2 2 3
CH Cl ); H NMR (300 MHz, CDCl
1
3
5.9 Hz, 2H), 6.76-7.00 (m, 6H), 7.47 (t, J 15.9 Hz, 2H); C (75
1
1
4
7
7
.69-1.75 (m, 1H), 2.15-2.29 (m, 2H), 3.60 (dd, J 9.2 and 3.1 Hz,
H), 3.81 (dt, J 9.5 and 4.0 Hz, 1H), 3.88 (m, 1H), 4.04 (m, 1H),
.47 (s, 2H), 4.62 (d, J 11.9 Hz, 1H), 4.68 (d, J 11.9 Hz, 1H), 7.26-
MHz, C
5
D
5
N): ꢀ (ppm) 38.3, 42.5, 65.4, 66.3, 69.5, 70.6, 114.9-
+
1
4
47.4, 168.1, 168.5; HRMS calcd for C24
95.1267, found 495.1280.
24
H O10Na [M + Na ]
1
3
3
.35 (m, 10H); C NMR (75 MHz, CDCl ): ꢀ (ppm) 32.8, 34.0,
0.3, 70.8, 72.1, 74.4, 74.7, 75.2, 127.6-137.7; Anal. calcd. for
.H O: C, 69.38; H, 7.57. Found: C, 69.40; H, 7.01.
(
1R,2S,3R,5R)-1,2-Di-O-caffeoyl-1,2,3,5-cyclohexanetetrol
C
20
H
24
O
4
2
(
-
30): 60% (0.09 mmol; 0.043 g); White solid; mp 133-135°C; [ꢀ] :
D
1
19.6 (c 0.1; CD
3 5 5
OD); H NMR (300 MHz, C D N): ꢀ (ppm) 1.17-
General Procedure to Prepare 25-28
2
.01 (m, 4H), 2.64 (s, 2H), 4.03 (m, 2H), 5.13-5.28 (m, 2H), 6.10
To a solution of 23 or 24 (0.82 g, 2.5 mmol) in pyridine (7 mL)
was added a solution of 6 or 10 (10 mmol) in toluene (30 mL) at
(d, J 15.9 Hz, 1H), 6.22 (d, J 16.0 Hz, 1H), 6.61-6.96 (m, 6H), 7.37
13
(d, J 15.9 Hz, 1H), 7.48 (d, J 16.0 Hz, 1H); C NMR (75 MHz,
0
°C. The reaction mixture was stirred for 48 h at 50°C. The mixture
C D N): ꢀ (ppm) 36.4, 38.0, 65.2, 66.7, 69.6, 72.4, 114.9-147.7
5 5
-1
+
was acidified with HCl (4 mol.L ) until pH 3 and extracted with
EtOAc (3 x 100 mL). The combined organic extracts were dried
168.3, 168.4; HRMS calcd for C24
found 495.1270.
24
H O10Na [M + Na ] 495.1267,
2 4
over Na SO , filtered, and the solvents were removed under re-
duced pressure. The residue was purified by silica gel column
chromatography (CH Cl /MeOH) leading to compounds 25-28.
Preparation of 31 and 32
2
2
A solution of 27 or 28 in EtOAc (20 mL) was hydrogenated un-
der pressure of 4 psi in presence of Pd/C 10% (0.30 g). The suspen-
sion was stirred for 6 h at room temperature, the catalyst was re-
moved by filtration, washed with methanol (10 mL) and concen-
trated under reduced pressure. The residue was purified by silica gel
(
1R,2S,3R,5S)-1,2-Di-O-(3’,4’-di-O-acetyl)-caffeoyl-3,5-di-O-
benzyl-1,2,3,5-cyclohexanetetrol (25): 71% (1.5 g; 1.8 mmol);
1
White solid; mp 72-74°C; [ꢀ]
300 MHz, CDCl ): ꢀ (ppm) 1.10-1.85 (m, 2H), 2.28-2.31 (m,
2H), 2.57 (m, 2H), 3.76-3.94 (m, 2H), 4.56-4.71 (m, 4H), 5.13 (dd,
D 2 2
: -206.0 (c 0.32; CH Cl ); H NMR
(
1
3
column chromatography (CH
2 2
Cl / MeOH) leading to compounds
3
1 and 32, respectively.
J 9.4 and 2.8 Hz, 1H), 5.64 (m, 1H), 6.34 (d, J 16.0 Hz, 1H), 6.37
(
d, J 16.0 Hz, 1H), 7.25-7.38 (m, 16H), 7.54 (d, J 16.0 Hz, 1H),
(1R,2S,3R,5S)-1,2-Di-O-galloyl-1,2,3,5-cyclohexanetetrol
(31): 93% (0.6 g; 1.4 mmol); White solid; mp 179-181°C; [ꢀ]
1
3
7
3
1
8
.56 (d, J 16.0 Hz, 1H); C NMR (75 MHz, CDCl
3
): ꢀ (ppm) 20.8,
D
: -
N): ꢀ (ppm)
2.12-2.14 (m, 2H), 2.52-2.81 (m, 2H), 4.55 (m, 1H), 4.70 (m, 1H),
1
4.5, 36.2, 69.4, 70.8, 71.4, 72.1, 74.1, 75.2, 119.0-143.8, 165.4,
3 5 5
270.6 (c 0.23; CH OH); H NMR (300 MHz, C D
+
65.8, 168.1, 168.2; HRMS calcd for C46
43.2629, found 843.2611.
44
H O14Na [M + Na ]
1
3
5.77 (dd, J 8.5 and 2.4 Hz, 1H), 6.29 (m, 1H), 7.90 (s, 4H);
C
NMR (75 MHz, C N): ꢀ (ppm) 30.6, 38.8, 65.6, 67.8, 70.2, 77.9,
10.7-150.7, 166.9, 167.8; HRMS calcd for C20
451.0877, found 451.0894.
5 5
D
(
1R,2S,3R,5R)-1,2-Di-O-(3’,4’-di-O-acetyl)-caffeoyl-3,5-di-O-
+
1
19
H O12 [M - H ]
benzyl-1,2,3,5-cyclohexanetetrol (26): White solid; 75% (1.5 g;
1
1
.9 mmol); mp 65-67°C; [ꢀ]
D 2 2
: +82.4 (c 0.17; CH Cl ); H NMR
): ꢀ (ppm) 2.29 (m, 16H), 3.88-3.95 (m, 2H),
(
300 MHz, CDCl
3
(1R,2S,3R,5R)-1,2-Di-O-galloyl-1,2,3,5-cyclohexanetetrol
4
2
.50-4.71 (m, 4H), 5.30-5.42 (td, J 11.0 and 2.8 Hz, 1H), 5.50 (t, J
(32): 95% (0.6 g; 1.4 mmol); White solid; mp 174-176°C; [ꢀ]
D
:
1
.8 Hz, 1H), 6.32 (d, J 16.0 Hz, 1H), 6.42 (d, J 16.0 Hz, 1H), 7.34
+136.4 (c 0.15; CH
3
OH); H NMR (300 MHz, C
5
D
5
N): ꢀ (ppm)
1
3
(
m, 16H), 7.61 (t, J 16.0 Hz, 2H); C NMR (75 MHz, CDCl
3
):
2.28-2.70 (m, 4H), 4.68 (m, 1H), 4.86 (m, 1H), 6.15 (m, 1H), 6.24
13
ꢀ
(ppm) 20.8, 32.3, 32.6, 68.9, 69.3, 70.9, 71.8, 71.9, 74.2, 119.0-
5 5
(m, 1H), 7.87 (s, 2H), 7.94 (s, 2H); C NMR (75 MHz, C D N):
1
[
43.9, 165.6, 165.8, 168.1, 168.2; HRMS calcd for C46
H
44
O
14Na
ꢀ (ppm) 37.7, 39.2, 64.8, 68.2, 70.1 72.9, 110.7-148.2, 167.0, 167.2;
+
+
M + Na ] 843.2629, found 843.2640.
1R,2S,3R,5S)-1,2-Di-O-(3’,4’,5’-tri-O-benzyl)-galloyl-3,5-di-
O-benzyl-1,2,3,5-cyclohexanetetrol (27): 70% (2.1 g; 1.8 mmol);
19
HRMS calcd for C20H O12 [M - H ] 451.0877, found 451.0899.
(
Preparation of 33
1
To a solution of 5 (10.91 g, 56.8 mmol) in toluene (40 mL) and
DMF (40 mL) was added cyclohexanone (36.0 mL, 237.0 mmol).
The reaction mixture was coupled to the dean-stark and refluxed for
Colorless oil; [ꢀ]
D
: -200.0 (c 0.16; CH
): ꢀ (ppm) 1.60-1.90 (m, 2H), 2.33-2.60 (m, 2H), 3.70 (m,
H), 3.74 (m, 1H), 4.48-5.04 (m, 16H), 5.31 (dd, J 9.0 and 2.6 Hz,
2 2
Cl ); H NMR (300 MHz,
CDCl
3
1
1
Cl
1
3
5
0 hours. Then was added amberlite IR 120 (14.0 g) and refluxed
H), 5.77 (m, 1H), 7.22-7.35 (m, 44H); C NMR (75 MHz, CD-
): ꢀ (ppm) 34.4, 35.9, 69.8-75.3, 109.3-152,8, 164.9, 165.3; A-
12: C, 77.80; H, 5.58. Found: C, 77.44; H,
for another 18 hours. The solution was filtered and neutralized with
solution of sodium bicarbonate (5% m/v). The mixture was ex-
tracted with ethyl ether (3 x 200 mL). The combined organic ex-
3
nal. calcd. for C76
.91.
68
H O
5

Synthesis and HIV-1 Inhibitory Activities
Current Medicinal Chemistry, 2013, Vol. 20, No. 5
731
tracts were dried over Na
2
SO
4
, filtered, and concentrated under
mixture was stirred for 10 days at room temperature and after com-
pletion of the reaction, was saturated with solid NaCl. The aqueous
phase was extracted with EtOAc (3 x 100 mL) and the combined
reduced pressure. The residue was purified by silica gel column
chromatography (EtOAc / hexane) leading to compound 33 in 50%
yield (7.2 g, 28.4 mmol).
2 4
organic extracts dried over Na SO , filtered and concentrated under
reduced pressure. The residue was purified by silica gel column
chromatography (CH Cl /MeOH) leading to compounds 37 and 38.
(
1R,3R,4R,5R)-3,4-O-Cyclohexylidene-1,3,4,5-cyclohexane
2
2
tetrol-1,5-carbolactone (33): White solid; mp 141-143°C; [ꢀ]
D
: -
): ꢀ (ppm) 1.21-
1
3
1
2
6
5.2 (c 1.18; CHCl
3
); H NMR (300 MHz, CDCl
3
(1R,2R,3R,5R)-3-O-caffeoyl-5-C-(caffeoyloxymethyl)-1,2,3,5-
.90 (m, 10H), 2.18 (dd, J 14.7 and 3.1 Hz, 1H), 2.28-2.41 (m, 2H),
.65 (d, J 11.9 Hz, 1H), 3.27 (s, 1H), 4.28-4.32 (m, 1H), 4.48 (td, J
cyclohexanetetrol (37): 58% (0.3 g; 0.6 mmol); White solid; mp
1
120-122°C; [ꢀ]
CD
(m, 1H), 5.43 (m, 1H), 6.31 (t, J 15.6 Hz, 2H), 6.85-7.18 (m, 6H),
D
= -38.5 (c 0.10; MeOH); H NMR (300 MHz,
1
3
.2 and 2.8 Hz, 1H), 4.74 (dd, J 6.2 and 2.8 Hz, 1H); C NMR (75
3
OD): ꢀ (ppm) 1.29 (m, 4H), 3.70 (m, 1H), 4.06 (s, 2H), 4.20
MHz, CDCl
10.7, 179.2; Anal. calcd. for C13
calcd. for C13 : C, 61.40; H, 7.14. Found: C, 61.20; H, 7.08.
3
): ꢀ (ppm) 23.6-34.5, 37.0, 38.4, 71.2, 71.7, 71.8, 76.0,
1
3
1
H
18
O
5
: C, 61.40; H, 7.14. Anal.
7.54 (d, J 15.6 Hz, 1H), 7.57 (d, J 15.6 Hz, 1H); C NMR (75
MHz, CD OD): ꢀ (ppm) 37.4, 39.1, 71.1, 72.0, 73.9, 74.3, 115.2-
49.0, 167.6; HRMS calcd for C25
found 525.1375.
H
18
O
5
3
+
1
26
H O11Na [M + Na ] 525.1373,
Preparation of 34
To a solution of 33 (2.3 g, 10 mmol) in dry THF (50 mL) was
(
1R,2R,3R,5R)-3-O-galloyl-5-C-(galloyloxymethyl)-1,2,3,5-
added LiAlH (0.8 g, 20.0 mmol). The reaction mixture was stirred
for 5 h at room temperature. After the completion of the reaction
the excess of LiAlH was destroyed with EtOAc. The mixture was
filtered and the filtrate extracted with water (100 mL) and EtOAc (3
4
cyclohexanetetrol (38): White solid; 54% (0.3 g; 0.5 mmol); mp
1
1
51-153°C; [ꢀ]
CD OD): ꢀ (ppm) 2.20-2.60 (m, 4H), 4.67 (m, 1H), 4.85 (m, 1H),
.13 (s, 2H), 7.86 (s, 2H), 7.92 (s, 2H); C NMR (75 MHz,
D
= -19.0 (c 0.21; MeOH); H NMR (300 MHz,
4
3
1
3
6
2 4
x 100 mL). The combined organic extracts were dried over Na SO ,
CD
3
OD): ꢀ (ppm) 38.2, 40.1, 71.7, 72.2, 74.1, 74.9, 110.7-148.2,
+
filtered, and concentrated under reduced pressure. The residue was
1
4
67.6; HRMS calcd for C21
81.1003.
21
H O13 [M - H ] 481.0982, found
chromatographed on silica gel column to afford 34 in 90% yield
(2.3 g; 9.0 mmol).
(
1R,2R,3R,5R)-1,2-O-Cyclohexylidene-5-C-(hydroxymethyl)-
,2,3,5-cyclohexanetetrol (34): White solid; mp 91-93°C; [ꢀ] = -
): ꢀ (ppm) 1.43-
.26 (m, 14H), 3.43 (d, J 19.7 Hz, 1H), 3.45 (d, J 19.7 Hz, 1H),
BIOLOGICAL ASSAYS
Oligonucleotides
1
6
2
3
D
1
3 3
8.3 (c 1.3; CHCl ); H NMR (300 MHz, CDCl
1
3
All oligonucleotides employed throughout this study were syn-
thesized by Integrated DNA Technologies and gel-purified and
desalted prior to use. The disintegration substrate, a self-annealing
oligonucleotide that is homologous to the HIV long terminal repeat
regions (LTR), is dBY-1: 5’- TGCTAGTTCTAGCAGGCCC
TTGGGCCGGCGCTTGCGCC-3’ [34]. The 3’-end processing/
strand transfer substrate (V1/V2) consists of an annealed pair, V1:
.95 (t, J 5.9 Hz, 1H), 4.02-4.09 (m, 1H), 4.45 (m, 1H); C NMR
(
300 MHz, CDCl
3
): ꢀ (ppm) 23.8-34.8, 38.2, 38.4, 69.3, 70.2, 72.8,
+
7
2
4.0, 76.8, 110.0; HRMS calcd for C13
81.1365, found 281.1367.
22 5
H O Na [M + Na ]
Preparation of 35 and 36
To a solution of 34 (0.5 g, 2.0 mmol) in pyridine (5 mL) was
added a solution of 6 or 9 (6 mmol) in toluene (30 mL) at 0°C. The
reaction mixture was stirred for 24 h at room temperature. Then the
5
’- ATGTGGAAAATCTCTAGCAGT-3’, and V2: 5’- ACTGCTA
GAGATTTTCCACAT-3’ [31]. The strand transfer substrate con-
sists of U5V1P: 5’- ATGTGGAAAATCTCTAGCA-3’ annealed to
V2 in order to generate U5V1P/V2, a pre-processed form of V1V2
homologous to the end-processed LTR (2) [31]. This substrate is
used to separate the effect of compounds upon 3’-end processing
from strand transfer catalysis. Oligonucleotides used for site-
directed mutagenesis include: G140S (+): 5’-G ATCAAGCAG-
GAATTTAGCATTCCCTACAATC-3; G140S(-): 5’-GATTGTA
GGGAATGCTAAATTCCTGCTTGATC-3’; Q148H(+):5’-CAAT
CCCCAAAGTCATGGA GTAATAGAATC-3’; Q148H(-):5’-GAT
TCTATTACTCCATGACTTTGGGGATTG-3’; N155H(+): 5’-
GTAATAGAATCTATGCATAAAGAATTAAAG-3’; N155H(-):
5’-CTTTAATTCTTTATGCATAGATTCTATTAC-3’. Underlined
sequence denotes mutated codon.
-1
mixture was acidified with HCl (4 mol.L ) until pH 3 and extracted
with EtOAc (3 x 100 mL). The combined organic extracts were
2 4
dried over Na SO , filtered and concentrated under reduced pres-
sure. The residue was purified by silica gel column chromatography
CH Cl /MeOH) leading to 35 and 36, respectively.
(
2
2
(
1R,2R,3R,5R)-1,2-O-cyclohexylidene-3-O-(3’,4’-di-O-acetyl)-
caffeoyl-5-C-((3’,4’-di-O-acetyl)-caffeoyloxymethyl)-1,2,3,5-cyclo
hexanetetrol (35): 70% (1.1 g; 1.4 mmol); White solid; mp 69-
1
7
ꢀ
4
6
1°C; [ꢀ]
D
= +26.0 (c 0.31; CH
2
Cl
2
); H NMR (300 MHz, CDCl
3
):
(ppm) 1.60-1.80 (m, 12H), 2.31 (m, 12H), 4.09-4.20 (m, 3H),
.54 (m, 1H), 5.45 (m, 1H), 6.41 (t, J 16.3 Hz, 2H), 7.20-7.41 (m,
H), 7.64 (d, J 16.3 Hz, 1H), 7.65 (d, J 16.3 Hz, 1H); 13C NMR (75
MHz, CDCl
3
): ꢀ (ppm) 20.8-34.9, 36.2, 37.9, 70.9, 71.4, 71.7, 73.9,
7
6.4, 110.6, 118.8-143.8, 165.8, 166.6, 168.2, 168.3; HRMS calcd
Generation of Recombinant IN Genes
+
for C39
H
42
O
15Na [M + Na ] 773.2421, found 773.2448.
Mutated IN genes were produced by performing the Quick-
Change XL site-directed mutagenesis protocol (Stratagene, La
Jolla, CA 92037) upon the stable, infectious molecular clone of
HIV, HIVNL4-3 (GenBank Accession #M19921). Mutation success
was confirmed by fluorophore-labeled dideoxynucleotide DNA
sequencing (GeneWiz, La Jolla, CA 92037) and restriction digest.
By utilizing engineered silent restriction sites, the mutated IN gene
was subsequently cloned into and replaced the reference IN gene in
a recombinant protein expression vector [35] pT7.7, which encodes
an amino-terminal six-histidine tag.
(
1R,2R,3R,5R)-1,2-O-cyclohexylidene-3-O-(3’,4’,5’-tri-O-ace
tyl)-galloyl-5-C-((3’,4’,5’-tri-O-acetyl)-galloyloxymethyl)-1,2,3,5-
cyclohexanetetrol (36): 50% (0.8 g; 1.0 mmol); White solid; mp 73-
1
7
ꢀ
4
2
6
1
5°C; [ꢀ]
D
= +22.2 (c 0.18; CH
2
Cl
2
); H NMR (300 MHz, CDCl
3
):
(ppm) 1.23-2.10 (m, 14H), 2.27-2.29 (m, 18H), 3.50 (m, 1H),
.19-4.31 (m, 3H), 4.51 (s, 1H), 5.54 (m, 1H), 7.80 (s, 2H), 7.81 (s,
1
3
3
H); C NMR (75 MHz, CDCl ): ꢀ (ppm) 20.4-34.9, 36.3, 37.9,
4.6, 71.6, 72.8, 73.9, 76.4, 110.7, 122.6-143.7, 163.6, 164.4,
66.6, 166.9.
Preparation of 37 and 38
Generation of Recombinant IN
To a solution of 35 or 36 (1.0 mmol) in THF (10 mL) was
Recombinant IN protein was expressed from BL21 DE3 pLysS
Escherichia coli (Stratagene, La Jolla, CA 92037) and purified
-1
added an aqueous solution of HCl (1 mol.L ) (35 mL). The reaction

7
32 Current Medicinal Chemistry, 2013, Vol. 20, No. 5
Junior et al.
following bacterial lysis [36]. Briefly, transformed bacterial cul-
tures were induced to express IN protein by the addition of 1 mM
isopropyl ꢀ-D-1-thiogalactopyranoside (IPTG). Following harvest
by low-speed centrifugation, cells were lysed by freeze-thaw, ultra-
sonication, and digestion by the endogenous lysozyme encoded by
the pLysS plasmid. The resultant mixture was clarified by
centrifugation, and the precipitate was washed with 100 mM NaCl.
The suspension was again centrifuged, and the precipitate was
slowly stirred in a buffer containing 1 mM NaCl, and 5 mM
CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesul
fonate). Each of the washing steps occurred at 5˚C under reducing
conditions (5 mM ꢀ-mercaptoethanol) in a buffer containing 20
mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)
at pH 7.5. Recombinant IN containing an amino-terminal 6-
MT-2 cells to the cytostatic and cytotoxic effects of compounds 13-
38 was determined in a similar fashion as described, previously
[38]. The concentration of compound at which 95% of the cells are
5
viable, CT , is a nontoxic concentration of the compound.
CONFLICT OF INTEREST
The authors confirm that this article content has no conflicts of
interest.
ACKNOWLEDGEMENTS
This paper is dedicated to Dr. Jeannine Cleophax. FAPEMIG,
CNPq, and CAPES for fellowships and financial supports. This
work was supported in part by the California Center for Antiviral
Drug Discovery (University of California MRPI#143226 to WER).
2
+
histidine tag was purified via Ni -column affinity chromatography
as previously described (10) [37]. IN purity was confirmed via
sodium-dodecyl-sulfate polyacrylamide gel-electrophoresis (SDS-
PAGE) followed by Coomassie Blue staining; IN resolved at a MW
of ꢁ 32 kDa. Highly-purified fractions containing IN were dialyzed
into storage buffer (20 mM HEPES pH 7.5, 300 mM NaCl, 5 mM
CHAPS, 10% glycerol, and 5 mM dithiothreitol) and stored at -
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32
IN was incubated for 1 h at 37˚C in the presence of 0.1 pmol -P
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[
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7
1
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the addition of loading buffer (98% deionized formamide, 10 mM
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mented with 11.5% fetal bovine serum (Atlas, Fort Collins, CO).
HIVLAI was obtained from H9 cell lysates clarified of cells by low-
speed centrifugation followed by filtration through 0.45 ꢂm filters.
[
12]
Fifty Percent Effective Concentration and Cell Toxicity Analy-
sis (EC50 and CT50/CT
5
)
[13]
Susceptibility of HIVLAI to compounds 13-38 was determined
in MT-2 cells as previously described [38]. Triplicate samples of a
compound were diluted serially 128-fold in 96-well culture plates.
HIVLAI was pre-incubated with each compound (13-38) for 1 h prior
to the addition of MT-2 cells at a multiplicity of infection (MOI)>1.
Upon observation of HIV-induced cytopathic effect, cells were
transferred to poly-L-lysine coated plates and stained with Finter’s
neutral red dye. Plates were then incubated at 37˚C for 1 h. Follow-
ing incubation, cells were washed 3 times with PBS and lysed in
acidified methanol. Viability was subsequently quantified on a mi-
crocolorimeter by A540. The percentage of viable cells was calcu-
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Received: June 25, 2012
Revised: November 20, 2012
Accepted: November 21, 2012