Structure-activity relationships: Analogues of the dicaffeoylquinic and dicaffeoyltartaric acids as potent inhibitors of human immunodeficiency virus type 1 integrase and replication

Article abstract of DOI:10.1021/jm9804735

The dicaffeoylquinic acids (DCQAs) and dicaffeoyltartaric acids (DCTAs) are potent and selective inhibitors of human immunodeficiency virus type 1 (HIV-1) integrase. They also inhibit HIV-1 replication at nontoxic concentrations. Since integrase is an excellent target for anti-HIV therapy, structure-activity relationships were employed to synthesize compounds with: (1) improved potency against HIV-1 integrase, (2) improved anti-HIV effect in tissue culture, and (3) increased selectivity as indicated by low cellular toxicity. Thirty-four analogues of the DCTAs and DCQAs were synthesized and tested for cell toxicity, anti-HIV activity, and inhibition of HIV-1 integrase. Seventeen of the 34 analogues had potent activity against HIV-1 integrase ranging from 0.07 to > 10 μM. Seventeen analogues that were synthesized or purchased had no inhibitory activity against integrase at concentrations of 25 μM. Of the biologically active analogues, 7 of the 17 inhibited HIV replication at nontoxic concentrations. The most potent compounds were D-chicoric acid, meso-chicoric acid, bis(3,4- dihydroxydihydrocinnamoyl)-L-tartaric acid, digalloyl-L-tartaric acid, bis(3,4-dihydroxybenzoyl)-L-tartaric acid, dicaffeoylglyceric acid, and bis(3,4-dihydroxyphenylacetyl)-L-tartaric acid. Anti-HIV activity of the active compounds in tissue culture ranged from 35 to 0.66 μM. Structure- activity relationships demonstrated that biscatechol moieties were absolutely required for inhibition of integrase, while at least one free carboxyl group was required for anti-HIV activity. These data demonstrate that analogues of the DCTAs and the DCQAs can be synthesized which have improved activity against HIV integrase.

Full text of DOI:10.1021/jm9804735

J . Med. Chem. 1999, 42, 497-509
497
Str u ctu r e-Activity Rela tion sh ip s: An a logu es of th e Dica ffeoylqu in ic a n d
Dica ffeoylta r ta r ic Acid s a s P oten t In h ibitor s of Hu m a n Im m u n od eficien cy
Vir u s Typ e 1 In tegr a se a n d Rep lica tion †
Peter J . King,‡ Guoxiang Ma,∇ Wenfang Miao,∇ Qi J ia,∇ Brenda R. McDougall,§ Manfred G. Reinecke,∇
Chris Cornell,‡ J ean Kuan,§ Tracey R. Kim,§ and W. Edward Robinson, J r.*,‡,§
Departments of Microbiology and Molecular Genetics and of Pathology, University of California, Irvine, California 92697-4800,
and Department of Chemistry, Texas Christian University, Fort Worth, Texas
Received August 13, 1998
The dicaffeoylquinic acids (DCQAs) and dicaffeoyltartaric acids (DCTAs) are potent and selective
inhibitors of human immunodeficiency virus type 1 (HIV-1) integrase. They also inhibit HIV-1
replication at nontoxic concentrations. Since integrase is an excellent target for anti-HIV
therapy, structure-activity relationships were employed to synthesize compounds with: (1)
improved potency against HIV-1 integrase, (2) improved anti-HIV effect in tissue culture, and
(3) increased selectivity as indicated by low cellular toxicity. Thirty-four analogues of the DCTAs
and DCQAs were synthesized and tested for cell toxicity, anti-HIV activity, and inhibition of
HIV-1 integrase. Seventeen of the 34 analogues had potent activity against HIV-1 integrase
ranging from 0.07 to >10 µM. Seventeen analogues that were synthesized or purchased had
no inhibitory activity against integrase at concentrations of 25 µM. Of the biologically active
analogues, 7 of the 17 inhibited HIV replication at nontoxic concentrations. The most potent
compounds were D-chicoric acid, meso-chicoric acid, bis(3,4-dihydroxydihydrocinnamoyl)-L-
tartaric acid, digalloyl-L-tartaric acid, bis(3,4-dihydroxybenzoyl)-L-tartaric acid, dicaffeoylglyceric
acid, and bis(3,4-dihydroxyphenylacetyl)-L-tartaric acid. Anti-HIV activity of the active
compounds in tissue culture ranged from 35 to 0.66 µM. Structure-activity relationships
demonstrated that biscatechol moieties were absolutely required for inhibition of integrase,
while at least one free carboxyl group was required for anti-HIV activity. These data
demonstrate that analogues of the DCTAs and the DCQAs can be synthesized which have
improved activity against HIV integrase.
In tr od u ction
222 µM. Therefore, L-DCTA was the most selective and
potent of the previously reported inhibitors of HIV-1 IN
(reviewed in ref 6).
The human immunodeficiency virus type 1 (HIV-1)
integrase (IN) is absolutely required for stable infec-
tion.1-5 Therefore, it represents a potentially important
enzymatic target for antiviral therapy. Despite reports
of numerous inhibitors of HIV IN, to date, there has
been little success developing inhibitors of IN that block
HIV replication at nontoxic concentrations (reviewed in
ref 6). One exception has been the dicaffeoylquinic acids
(DCQAs) and the dicaffeoyltartaric acids (DCTAs). This
class of inhibitors is potently active against HIV IN in
biochemical assays and has moderate anti-HIV activity
in tissue culture. The lead compound in this series was
L-DCTA (L-chicoric acid) (1) with a fifty percent inhibi-
tory activity (IC50) against HIV-1 IN of 0.3 µM.7,8 This
same compound blocked HIV-1 infection by 50%, the
fifty percent effective dose (ED50), at 4 µM.7,8 The
concentration of this molecule that inhibited cellular
replication by 5%, the five percent lethal dose (LD5), was
The utility of the DCQAs and DCTAs as a class was
highlighted in several recent publications. It has been
shown that these compounds are highly selective for
HIV-1 IN when compared to their activity against other
HIV proteins and metalloenzymes.9 Neamati et al.
recently used the structure of the DCQAs10 and the
DCTAs11 to generate a three-part or four-part pharma-
cophore. When these pharmacophores were used to
search a chemical structure database, they returned a
high “hit-rate” for inhibitors of HIV-1 IN,10,11 although
none of the compounds identified blocked HIV replica-
tion.10,11
IN can mediate several in vitro activities including
3′-end processing, strand transfer, and disintegration
(reviewed in ref 12). Although a number of investigators
have utilized the 3′-end processing and strand transfer
reactions to screen compounds for anti-HIV activity,13-18
the disintegration reaction offers several advantages.
First, steady-state kinetic analyses can be performed
using the dumbbell substrate in the disintegration
reaction.19,20 Second, disintegration is not simply the
reversal of integration but may also measure other
properties of IN including single-stranded DNA repair.21
Third, for the DCTAs and DCQAs, disintegration seems
less susceptible to nonspecific inhibition by dicaffeoyl-
† Taken in part from the dissertations of Qi J ia (1996) and Wenfang
Miao (1998) submitted to Texas Christian University in partial
fulfillment of the requirements of the Ph.D. degree. Preliminary
report: Ma, G. X.; Reinecke, M. G.; J ia, Q.; Miao, W.; Robinson, W.
E., J r. Chicoric Acid Analogues as Potential Anti-HIV Integrase
Inhibitors. Abst. Natl. Mtg. Am. Chem. Soc. 1998, 215, MED#28.
* Corresponding author: W. Edward Robinson, J r., M.D., Ph.D.
Phone: 949-824-3431. Fax: 949-824-2505. E-mail: ewrobins@uci.edu.
‡ Department of Microbiology and Molecular Genetics.
§ Department of Pathology.
∇ Department of Chemistry.
10.1021/jm9804735 CCC: $18.00 © 1999 American Chemical Society
Published on Web 01/27/1999

498 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 3
King et al.
Sch em e 1a
Sch em e 2a
a
See footnote to Scheme 1.
Sch em e 3a
a
Abbreviations: OM ) O-methylcarbonyl ) -OCOOMe; DPM
) diphenylmethyl ) -CHPh2; CAF ) caffeoyl ) -COCHdCH[3,4-
(OH)2-phenyl]; BMC ) bis(methoxycarbonylcaffeoyl) ) -COCHd
CH[3,4-(OM)2-phenyl]. Procedures: [A] ClCOOMe; [B] Na2CO3; [C]
Ph2CHN2; [D] HOAc; [E] RCOCl; [F] RCOCl/R3N; [G] L-tartaric
acid; [H] (COCl)2; [I] SOCl2.
benzoic acid derivatives.8 And finally, the catalytic core
alone, rather than the whole IN molecule, can perform
disintegration;22,23 thus, inhibitors of disintegration are
more likely to act against the IN core than other regions
of the molecule.7,8 We have previously shown that the
DCTAs and DCQAs act on the catalytic core.8
In an effort to develop more potent and selective
inhibitors of HIV-1 IN, analogues of the DCTAs and
DCQAs were synthesized. The length of the side chains,
the spatial arrangement of the phenolic hydroxyl groups,
the size and structure of the central molecular core
structure, and the requirement of one or more free
carboxyl groups were all studied. The effects of these
changes were assayed against HIV-1 IN in the disinte-
gration reaction as well as against HIV-1 replication and
cell growth in tissue culture.
a
See footnote to Scheme 1.
neous anti-HIV and HIV IN inhibitory activities, a
series of DCTA and DCQA analogues was prepared and
their biological activities determined.
L- (1), D- (2)-, and meso- (3) DCTAs were synthesized
by acylation of the bis(diphenylmethyl) tartrates 1a , 2a ,
and 3a with the protected caffeoyl chloride 35a to give
the fully blocked compounds 20, 2b, and 3b, from which
the phenol and carboxyl blocking groups were sequen-
tially removed (21, 2c, and 3c) (Scheme 1). [This
synthesis, without experimental detail, was alluded to
in ref 7 but misrepresented in a recent paper (Synth.
Commun. 1998, 737-740), as involving condensation of
the bisdiphenyl ester (sic) of tartaric acid and the 3,4-
cyclocarbonate of caffeoyl chloride.]
To determine the importance of a free carboxyl group
for bioactivity, a series of alkane (4, 12, 14) and
cyclohexane (5, 6, 9, 10, 16, 18) dicaffeoyl esters lacking
this group were prepared by direct acylation of the
appropriate diol with 35a to give the protected com-
pounds 4a , 5a , 6a , 7, 8, 11, 13, 15, and 17, from which
Resu lts
Syn t h et ic Goa ls a n d Met h od ology. Both the
DCQAs and the DCTAs are potent and selective inhibi-
tors of both HIV-1 IN and HIV-1 replication in tissue
culture.7-9 Caffeic acid, quinic acid, chlorogenic acid (5-
caffeoylquinic acid), and CAPE (caffeic acid phenethyl
ester) show no such anti-HIV activity, consistent with
the “biscatechol hypothesis”,15,18,24,25 although the last
of these is reported to have weak anti-IN activity.14
Using these compounds as “lead structures”, structure-
activity relationship (SAR) studies were initiated. To
probe the structural features responsible for simulta-

HIV IN Inhibitors: SAR of DCQA and DCTA Analogues
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 3 499
Sch em e 4a
Sch em e 6a
a
See footnote to Scheme 1.
Sch em e 5a
a
See footnote to Scheme 1.
Sch em e 7a
a
See footnote to Scheme 1.
the phenol blocking groups were removed (Scheme 2).
Two compounds with a single carboxyl group, dicaf-
feoylglyceric acid (19) and 3,5-dicaffeoylbenzoic acid
(34), also were prepared by acylation with 35a and
deprotection via 19a and 34a -c (Scheme 3).
To determine the effect on bioactivity of the group
linking the catechol and tartaric acid moieties of 1,
analogues were prepared with the CHdCH replaced by
two (22), one (28), or zero (26) methylene groups. Direct
acylation of L-tartaric acid with the acid chlorides of the
phenol-protected acids 22a , 28a , and 26a gave 22b, 28b,
and 26b from which the phenol blocking groups were
removed (Scheme 4).
The significance for bioactivity of the number and
position of the phenol groups was determined in both
the cinnamic acid series (Scheme 5) and benzoic acid
series (Scheme 6). Analogues with one (23, 24) or three
(25) phenolic hydroxyl groups as well as the resorcinol
(27), 2,3-catechol (31), and hydroquinone (32) analogues
were synthesized by acylation of L-tartaric acid (Scheme
5) or its diester 1a (Scheme 6) with the acid chlorides
of the phenol-protected acids 23a , 24a , 25a , 27a , 31c,
and 32c followed by removal of the appropriate blocking
groups. The preparation of acids 31c and 32c required
initial blocking of the carboxyl groups as the diphenyl-
methyl esters so that the ortho phenol group could be
protected (Scheme 7). Finally, several analogues {29,
a
See footnote to Scheme 1.
30 (Scheme 5) and 33 (Scheme 6)} lacking phenol groups
were made by direct acylation of L-tartaric acid.
Act ivit y a ga in st H IV-1 IN (In it ia l Scr een in g).
Each compound was screened for inhibitory activity
against HIV-1 IN in the disintegration assay.21 The
results of this screen are illustrated in Figure 1. The
data were quantitated and are presented in Tables 1
and 2. Active analogues were defined as those com-
pounds which inhibited HIV-1 IN by greater than 65%
in the disintegration assay at 25 µM. Seventeen ana-
logues met this criterion; they are listed in Table 1.
Another 17 new analogues were inactive, as defined by

500 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 3
King et al.
Ta ble 1. Anti-HIV Activity of Biologically Active DCQA and
DCTA Analoguesa
% inhibn
of IN at
25 µMd
b
c
formula
wt
LD5
ED50
compd
(in µM)
(µM)
IC50e (µM)
1
2
3
4
5
6
9
10
12
14
16
18
19
22
25
26
28
31
34
474
474
474
386
440
440
440
440
400
414
440
440
430
478
454
422
450
422
478
516
516
516
516
602
264
115
373
194
170
20.5 >45
9.1 >34.1
36.4 >68.2
117.5 >350
121
63.6 >136
22.7 >90.9
145
>263
2.7
90
70
19
100
145
290
116
145
372
4.2
6.3
4.2
96.9
100
97.5
83.4
85.2
90.3
80.7
73.3
81.2
67.6
80.2
81.7
100
94.5
100
97.2
100
100
NT
0.18 (0.04)f-h
0.07 (0.01)
0.08 (0.01)
>10
1.03 (0.47)
1.01 (0.21)
1.8 (0.2)
1.63 (0.39)
>10
>10
>10
8.36 (0.36)
0.52 (0.06)
2.38 (0.67)
0.97 (0.00)
0.43 (0.05)
0.88 (0.40)
1.07 (0.29)
6.9 (1.79)h
1.6 (0.16)h
1.3 (0.46)g,h
1.4 (0.6)h
0.6 (0.06)h
0.8 (0.45)g,h
>388
>341
>242
F igu r e 1. Representative screen of several DCTA and DCQA
analogues in the disintegration assay. Compounds were
diluted to 500 µM; 1 µL was added to 19 µL of disintegration
reaction mixture containing 1.5 pmol of HIV-1 IN and 0.1 pmol
of radiolabeled and annealed oligonucleotide primers. Reac-
tions proceeded at 37 °C for 60 min. Products were separated
on a 15% denaturing acrylamide gel. Compounds: lane 1,
substrate alone; lane 2, substrate and enzyme; lane 3, sub-
strate, enzyme, and 0.7% ethanol; lane 4, quinic acid; lane 5,
chlorogenic acid; lane 6, tartaric acid; lane 7, 12; lane 8, 14;
lane 9, 1; lane 10, 2; lane 11, 3; lane 12, 22; lane 13, 23; lane
14, 24; lane 15, 25; lane 16, 26; lane 17, 27; lane 18, 28; lane
19, 29. Dashed long arrow indicates product; solid, short arrow
indicates substrate.
2.3
8.4
0.66
0.95
35
>38
>75
4
2
12
4
1,5-DCQA
3,5-DCQA
3,4-DCQA
4,5-DCQA
1-MO-3,5-
DCQA
NT
NT
NT
NT
7
NT
a
Analogues of L-DCTA or the DCQAs were synthesized and
screened for activity against HIV IN. Only active analogues
(inhibition of enzyme by greater than 65% at 25 µM) are shown.
less than or equal to 65% inhibition of HIV-1 IN in the
disintegration assay at 25 µM (Table 2).
b
An ti-IN Activities of Biologica lly Active Com -
p ou n d s. The IC50 values for all of the biologically active
compounds were determined using the disintegration
assay. One representative assay is illustrated in Figure
2. The IC50 values for five representative compounds
ranging from an inactive compound, chlorogenic acid,
to the most potent inhibitor (25) are illustrated in Figure
3. The most potent compounds were 2 and 3 which had
IC50 values of approximately 70-80 nM. The DCTAs,
L-DCTA (1), D-DCTA (2), and m-DCTA (3), were the
most potent group of compounds against HIV-1 IN in
the disintegration assay. The least active compound that
also demonstrated anti-HIV activity in tissue culture
was 22 with an IC50 of 2.4 µM. With the exception of 2
and 3, which were more potent than the lead compounds
1 and the DCQAs, all were of similar potency to 1 and
the DCQAs.
The five percent lethal dose (LD5) is indicated. This is the
concentration of analogue that inhibits growth of cells by 5%. This
represents a truly nontoxic dose and is within 1 SD of cell control
wells lacking any inhibitor. c The fifty percent effective dose (ED50
)
is the concentration of compound that inhibits HIV-induced cell
d
death by 50%. Each compound was tested for inhibition of HIV
IN (whole molecule) in the disintegration reaction21 at a concen-
tration of 25 µM. Any compound that inhibited the reaction by
65% or greater was selected for further testing. e IC50 of triplicate
reactions performed against wild-type HIVNL4-3 IN. Analogues
were tested in 0.5 log dilutions from 0.033 to 10 µM; 1.5 pmol of
HIS-tagged whole HIVNL4-3 IN was tested for inhibitory activity
in the disintegration assay using the method of Chow et al.21
Reactions were allowed to proceed for 60 min at 37 °C. Products
were separated on a 15% polyacrylamide sequencing gel and
quantitated using a PhosphorImager. Numbers in parentheses are
1 SD. f Inhibitory activity for 1 differs slightly from that published
by us previously (0.3 µM) but falls within the original standard
deviation for the assays. The source of IN in these experiments
was different from that published previously. Furthermore, IC50
results shown in this table were performed in 0.5 log dilutions
rather than 1.0 log dilutions in the reactions reported previously.
Either may play a role in the slightly different (50% lower) IC50
Toxicity a n d An ti-HIV Activities. Next, all of the
analogues were tested for cell toxicity and anti-HIV
activity in tissue culture. Although the toxicities of these
compounds varied widely, from a low of 7.4 µM to a high
of 1389 µM, none of the compounds defined as biologi-
cally inactive (less than or equal to 65% inhibition of
HIV-1 IN at 25 µM) blocked HIV-1 replication in tissue
culture (Table 2). The biologically active compounds, on
the other hand, contained compounds with anti-HIV
activity and those without detectable activity. As shown
in Table 1, cell toxicity for these compounds varied from
a low of 2.7 µM to a high of 373 µM. Eleven compounds
failed to block HIV-1 replication at nontoxic concentra-
tions, while 7 compounds were potent inhibitors of
HIV-1 replication (Table 1). The most active compound,
25, had an ED50 of 660 nM. The least active compound,
28, had an ED50 of 35 µM. The other six compounds had
ED50 values ranging from 1 to 8 µM. One representative
cell toxicity and anti-HIV activity experiment for each
h
reported herein. g Reference 7. Reference 8. Numbers in bold are
for new compounds with anti-HIV activity at nontoxic concentra-
tions.
of the seven new, active compounds is illustrated in
Figure 4.
Activities a ga in st a n L-Ch icor ic Acid -Resista n t
Molecu la r Clon e of HIV-1. We have previously de-
scribed
a molecular clone of HIV-1, called HIV-
1NL4-3clone1-D4, that is resistant to the antiviral activity
of 1.26 Resistance results from a single amino acid
change in IN. To determine whether analogues of 1 were
also inhibiting HIV replication through inhibition of IN,
the anti-HIV activity of 22, 25, 26, and 28 was tested
against HIV-1NL4-3clone1-D4. The results are illustrated
in Table 3. The mutation in IN confers cross-resistance
to all four analogues; therefore, all four are acting to
inhibit HIV replication, at least in large part, through
inhibition of IN.

HIV IN Inhibitors: SAR of DCQA and DCTA Analogues
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 3 501
Ta ble 2. Anti-HIV Activity of Biologically Inactive DCQA and
DCTA Analoguesa
% inhibn
b
c
formula
wt
LD5
ED50
of IN at
compd
(in µM)
(in µM)
25 µMd
quinic acid
caffeic acid
L-tartaric acid
chlorogenic acid
7
8
11
13
15
17
20
21
23
24
27
29
30
32
33
192
180
150
354
672
672
632
646
672
672
1038
806
442
442
422
410
530
422
430
907
1389
>1000
249
>1823
>1389
>1000
>497
>14.8
>29.6
>55.4
>23.3
>14.8
>44.6
>48.2
>37.2
>283
>227
>119
>245
>178
>238
>59.2
18.7
0
0
59.7
-1.4
30.6
16.8
6.3
19.0
39.7
16.7
22.8
23.7
29.9
25.9
7.4
7.4
7.4
26.9
12.4
8.9
11.9
24.1
16.1
566
114
>119
>245
89
0
20
28
>238
28.9
a
Analogues of L-DCTA or the DCQAs were synthesized and
screened for activity against HIV IN. Only inactive analogues
(inhibition of enzyme by less than 65% at 25 µM) are shown. See
F igu r e 3. IC50 analysis of five representative analogues of
the DCTAs and DCQAs in the disintegration assay. 1, the lead
compound, as well as 25, the most potent inhibitor of IN, 28,
a strong inhibitor, 31, a weak inhibitor, and chlorogenic acid,
an inactive compound, were diluted and tested in the HIV
disintegration assay. Each point represents the mean of
triplicate assays; error bars are 1 SD.
b
Table 1 for active analogues. The five percent lethal dose (LD5)
is indicated. This is the concentration of analogue that inhibits
growth of cells by 5%. This represents a truly nontoxic dose and
is within 1 SD of cell control wells lacking any inhibitor. c The fifty
percent effective dose (ED50) is the concentration of compound that
d
inhibits HIV-induced cell death by 50%. Each compound was
tested for inhibition of HIV IN (whole molecule) in the disintegra-
tion reaction21 at a concentration of 25 µM.
replication in tissue culture? and (3) selective for inhibi-
tion of HIV-1 at nontoxic concentrations?
The first compounds in the series altered the central
core by lengthening or shortening the aliphatic central
core domain. Compounds 4, 12, and 14 vary the length
of the core from 2 to 4 carbons but lack the free
carboxylic acid(s) of the DCQAs and 1. All three
compounds weakly inhibited HIV-1 IN (IC50 > 10 µM)
but had no activity in tissue culture. Blocking the
phenolic OH on the biscatechols of these compounds (11
and 13) resulted in loss of anti-IN activity. Having a
cyclohexanediol central core (5, 6, 9, 10, 16, and 18)
increased the potency of the compounds against IN but
did not result in compounds that could interfere with
HIV-1 replication in tissue culture. Interestingly, sev-
eral of these compounds, 5, 6, 9, and 10, were as active
against HIV-1 IN as the DCQAs in biochemical assays
(IC50 of 1-2 µM) but did not inhibit HIV-1 replication.
These data suggest that a free carboxylic acid or
additional, free hydroxyl groups on the central core may
be necessary for uptake of the compounds by the cell or
into the virion. Blocking the phenolic groups of the
dicaffeoylcyclohexanediols (7, 8, 15, and 17) abolished
activity.
F igu r e 2. Representative assay for determining IC50 of active
analogues. 31 was tested in the disintegration assay: lane 1,
0.1 pmol of substrate alone; lanes 2-24, 1.5 pmol of enzyme
plus 0.1 pmol of substrate; lanes 2-4, enzyme and substrate
alone. The remaining lanes contained in addition to enzyme
and substrate: lane 5, 25 µM L-tartaric acid; lane 6, 25 µM 1;
lanes 7-9, 0.03 µM 31; lanes 10-12, 0.1 µM 31; lanes 13-15,
0.3 µM 31; lanes 16-18, 1.0 µM 31; lanes 19-21, 3.0 µM 31;
lanes 22-24, 10.0 µM 31. Reactions were allowed to proceed
at 37 °C for 60 min.
Previous data on the DCQA8 had indicated that
substitution patterns around the quinic acid core had
little effect on anti-HIV-1 activity and IN inhibition. The
same was not true with the dicaffeoylcyclohexanediols;
although both the 1,4-substitution (5, 6) and the 1,3-
substitution (9, 10) resulted in an IC50 of approximately
1-2 µM, the 1,2-substitution (16, 18) resulted in
compounds with IC50 values of greater than 10 µM. Both
cis- (6, 10, and 18) and trans- (5, 9, and 16) substituted
rings were equally potent inhibitors of HIV-1 IN. The
same was not true for the D- (2) and meso- (3) isomers
of L-DCTA, which inhibited HIV-1 IN at submicromolar
Discu ssion
Structure-activity relationships for IN inhibitors
have demonstrated that biscatechols are active inhibi-
tors of IN in biochemical assays, if not HIV in tissue
culture7-11,14,15,17,18,24,27-29 (reviewed in ref 6). However,
the DCQAs and DCTAs, while biscatechols, are unique
in their selectivity against HIV-1 IN and are also potent
inhibitors in tissue culture.7-9 Thus, it is possible to ask
which structural components are (1) necessary for
inhibition of IN? (2) necessary for inhibition of HIV-1

502 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 3
King et al.
F igu r e 4. Cell toxicity and anti-HIV effects of seven novel integrase inhibitors. Representative experiments for each novel analogue
of the DCTAs and the DCQAs that also inhibited HIV-1 IN are illustrated: (A) 2; (B) 3; (C) 19; (D) 22; (E) 25; (F) 26; (G) 28.
Closed circles are cell toxicity, and open circles are anti-HIV effect. Error bars are 1 SD of assays performed in triplicate.
concentrations with IC50 values of 0.07 and 0.08 µM,
respectively. L-DCTA (1), on the other hand, was 2-3
times less potent. The addition of large blocking groups
to the free carboxylic acids on L-DCTA (21) or the
blocking of both phenolic hydrogens and the free car-
boxylic acid (20) resulted in loss of activity against
HIV-1 IN. Although this was expected for 20, the loss
of activity when blocking the L-DCTA carboxyl groups
(21) was unexpected in view of the HIV IN inhibition
by the other biscatechols discussed above. This suggests
that the presence of large groups on the central core
(such as the diphenylmethylesters on 21) may interfere
with the ability of the compounds to fit in the active
site of IN. Only one carboxylic acid was required for both
anti-HIV IN activity and antiviral activity for the
DCTAs, as 19 was a potent inhibitor of both disintegra-
tion and viral replication.
We had previously demonstrated that two caffeic acid
groups were necessary for both potent IN inhibition and
anti-HIV activity in tissue culture.7,8 Several isomers

HIV IN Inhibitors: SAR of DCQA and DCTA Analogues
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 3 503
IN protein from avian sarcoma virus.31 The close
proximity of the mutant amino acid to the likely drug-
binding site supports the hypothesis that the anti-HIV-1
activity of this class of compounds, at least in part, is
directed toward the IN protein. Studies on the anti-HIV
activity of this class of compounds against the drug-
resistant clone demonstrates that the four compounds
tested (22, 25, 26, and 28) were all less active against
the drug-resistant virus. This data demonstrates that,
like the parent molecule 1, all are likely mediating their
anti-HIV activity in tissue culture through inhibition
of IN.
Several groups have recently identified compounds
that are potent inhibitors of HIV IN in vitro and HIV
replication in tissue culture.7,8,32,33 New lead compounds
described herein would include both meso- (3) and D-
(2) DCTA, as well as dicaffeoylglyceric acid (19), di-
galloyl-L-tartaric acid (25), bis(3,4-dihydroxybenzoyl)-
L-tartaric acid (26), and bis(3,4-dihydroxyphenylacetyl)-
L-tartaric acid (28). Improvement in the selectivity of
25 and, likely, cell uptake of 28 may significantly
enhance their use as lead compounds in the search for
potent and selective inhibitors of HIV-1 IN. These new
successes in analogue synthesis, coupled with improved
crystallographic information on the IN core,31,34,35 in-
dicate that, through diligent SAR studies, clinically
useful inhibitors of IN may be “just over the horizon”.
Ta ble 3. Anti-HIV Activity of L-Chicoric Acid Analogues
against an L-Chicoric Acid-Resistant Molecular Clone of HIV,
HIV-1NL4-3clone1-D4
ED50 (in µM)
a
b
compd
HIVNL4-3
HIVNL4-3clone1-D4
fold change
1
22
25
26
28
0.78
7.8
0.29
0.82
15.6
>61.25
31.25
31.25
>52.5
>62.5
>78
4
107
>64
>4
a
The anti-HIV activity of each compound was tested against
the infectious molecular clone of HIV-1, HIVNL4-3. The ED50 is
expressed as the mean of triplicate infections. Methods were the
same as for uncloned HIV although the multiplicity of infection is
b
between 0.1 and 1 for the clones. The anti-HIV activity of each
compound was measured against an L-chicoric acid-resistant
26
molecular clone of HIV-1NL4-3, HIV-1NL4-3clone1-D4
.
The results
are expressed as the mean of triplicate infections. The multiplicity
of infection was the same as for HIV-1NL4-3
.
or analogues were also active, however, including gallic
acid (3,4,5-trihydroxybenzoyl) (25), which was signifi-
cantly more toxic to dividing cells than were the other
compounds (LD5 of 2.7 µM) indicating that the third
phenolic hydroxyl group decreases specificity against IN.
The activity of the 3,4-dihydroxyphenylacetyl (28), 3,4-
dihydroxybenzoyl (26), and 3,4-dihydroxydihydro-
cinnamoyl (22) analogues indicates that the length (0,
1, or 2 carbons) or hybridization (sp2 or sp3) of the linker
group separating the catechol from the central core is
not critical. The 3,4-biscatechol moiety was absolutely
required, however, since the 3,5- (27), 2,3- (31), and 2,5-
(32) dihydroxyphenyl isomers were inactive. This indi-
cates that the anti-IN and anti-HIV activity of the
biscatechols is not simply due to their antioxidant or
metal-chelating properties. Blocking the phenolic hy-
droxyls (30) or reducing the number of hydroxyls from
two to one (23 and 24) or none (29) also abolished
activity as did replacement of the OH with fluorine (33).
Without exception, compounds that inhibited HIV-1
IN by less than 65% at 25 µM failed to inhibit HIV-1
replication. Indeed, only two compounds that inhibited
HIV-1 IN by more than 90% at 25 µM (6) failed to inhibit
HIV-1 replication in tissue culture. Therefore, for this
class of compounds using the disintegration assay,
inhibition of IN by >90% at a concentration of 25 µM
would seem to be a reasonable “cutoff” value to screen
for inhibitors of HIV-1 IN. We have identified two
compounds that are more potent than 1, the most potent
inhibitor of HIV-1 IN reported previously. These two
compounds, 2 and 3, were as active or more active in
tissue culture than either 1 or the DCQAs.
Exp er im en ta l Section
Ch em ica l Syn th eses. Gen er a l. All melting points were
measured on a Mel-Temp apparatus and are corrected. Ele-
mental analyses were performed by M-H-W laboratories of
Phoenix, AZ. MS analyses were obtained on a HP-5989A
instrument at 70 eV in the EI mode with GC sampling unless
otherwise noted as DIP (direct insertion probe). Selected peaks
are reported as m/z (rel. int.) including all (except isotope
peaks) with m/z >100 and rel. int. >25. HRFABMS were
performed at the Washington University Resource for Bio-
medical and Bio-Organic Mass Spectrometry, St. Louis, MO.
NMR spectra were obtained on a Varian XL-300 instrument
at 299.936 (1H) or 75.427 (13C) unless otherwise noted in the
indicated solvent (D ) DMSO-d6, C ) CDCl3, M ) CD3OD)
and are reported in order as: ppm downfield from TMS at δ
) 0, multiplicity (s, d, dd, m, bs), observed couplings J in Hz,
and relative number of H’s. APT spectra results are expressed
as d ) C or CH2, u ) CH or CH3. HPLC used a C-18 10-µm,
250-mm × 4.6-mm analytical column or a C18 10-µm, 250- ×
22-mm preparative column eluted with either methanol-water
or acetonitrile-water mixtures containing 1% HOAc and UV
detection at 254 nm. The following general synthetic proce-
dures refer to both Schemes 1-7 and the preparation of the
specific compounds to follow. No attempt was made to optimize
yields.
One question surrounding the use of this class of
compounds as lead molecules in the search for inhibitors
of HIV IN has been the mechanism of their anti-HIV
activity. Previous work by Mahmood et al.30 had sug-
gested that one DCQA blocked HIV-1 gp120 from
binding to its cellular receptor, CD4. Therefore, it
seemed plausible that their mechanism of anti-HIV-1
activity was at the level of virus binding to its receptor.
However, recent work has demonstrated that the DC-
TAs and DCQAs are selective inhibitors of HIV-1 IN.9
Furthermore, the isolation of an L-DCTA-resistant
HIV-1 molecular clone mapped the site of resistance to
a single amino acid change in HIV-1 IN.26 A likely
L-DCTA-binding site was identified by cocrystallization
of a structurally similar compound and an analogous
P r oced u r e A, Ca r boxym eth yla tion of P h en ols. A solu-
tion of the phenolic acid in 2.2 equiv of 1 N NaOH was cooled
to 0 °C, and 3.5 equiv of methyl chloroformate was added
dropwise with stirring. The precipitate was collected by
filtration, washed with DI water, air-dried, and recrystallized
to give the blocked phenolic acid.
P r oced u r e B, Rem ova l of Ca r boxym eth yl Gr ou p s. The
carboxymethylated phenol was dissolved in THF and hydro-
lyzed with 2% Na2CO3 with enough MeOH for homogeneity
at room temperature, under N2 for 5-7 h. The reaction mixture
was acidified to pH ) 1-2 with 10 N HCl and extracted with
ether, the ether evaporated, and the residue purified by
chromatography on silica gel, if necessary.
P r oced u r e C, F or m a tion of Dip h en ylm eth yl Ester s. A
solution of 1.25 equiv of diphenyldiazomethane per COOH
group in chloroform was added to the carboxylic acid in
MeOH-CHCl3. The mixture was stirred at room temperature

504 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 3
King et al.
until the red color disappeared and washed successively with
1 N HCl, saturated NaHCO3, and H2O. The organic layer was
dried with Na2SO4 and evaporated on a Rotovap to afford the
diphenylmethyl ester which was recrystallized if necessary.
P r oced u r e D, Hyd r olysis of Dip h en ylm eth yl Ester s.
The diphenylmethyl esters were deprotected in refluxing 70%
acetic acid under N2 for 4 h. The solvent was removed by
lyophilization and the residue purified by chromatography on
Sephadex LH-20.
P r oced u r e E, Dir ect Acyla tion of Alcoh ol Gr ou p s. The
alcohol was reacted with a slight excess of the acid chloride
without solvent in an oil bath at 130-140 °C for 15 min and
the residue chromatographed on silica gel.
P r oced u r e F , Solu tion Acyla tion of Alcoh ol Gr ou p s.
The acyl chloride was reacted with the alcohol in anhydrous
benzene with pyridine or triethylamine as a catalyst for 2.5 h
at room temperature. The reaction mixture was successively
washed with 1 N HCl, saturated NaHCO3, and water and the
organic layer evaporated to give the ester which was purified
on a silica gel column.
P r oced u r e G, Dir ect Acyla tion of L-Ta r ta r ic Acid . A
modification of Scarpati’s method36 involves heating L-tartaric
acid with an excess of an acyl chloride in an oil bath at 135-
160 °C for 10-30 min followed by hydrolysis of the intermedi-
ate anhydride with 80% HOAc on a steam bath for 30 min.
The residue from removal of the solvent below 40 °C under
reduced pressure was partitioned between water and ether and
the latter dried and evaporated to give a crude product which
was purified by chromatography on silica gel or Sephadex LH-
20.
153.1, 152.9, 144.3, 143.9, 142.7, 138.8, 138.7, 133.0, 128.63,
128.57, 128.2 (×2), 127.3, 127.0 (×2), 123.50, 122.6, 117.5, 79.1,
71.1, 56.0 (×2). Removal of the methoxycarbonyl groups by
tr a te (2c), mp ) 134-136 °C dec; 1H NMR (M) 7.49 (d, 15.8,
2H), 6.90 (s, 2H), 6.80-7.33 (m, 20H), 6.08 (s, 2H), 6.07 (d,
15.8, 2H); 13C NMR (M) 167.4d, 166.8d, 150.0d, 148.7u, 146.8d,
140.6d, 140.5d, 129.6u (×2), 129.1u (×2), 128.1u, 127.9u,
127.4d, 123.6u, 116.5u, 115.3u, 113.2u, 80.4u, 72.5u. Removal
of the diphenylmethyl groups by procedure D gave D-DCTA
(2), whose 1H and 13C NMR agreed with the literature.38
procedure B gave bis(d ip h en ylm eth yl) d ica ffeoyl-
D-ta r -
Dica ffeoyl-m eso-ta r ta r ic Acid (m eso-Ch icor ic Acid ,
m eso-DCTA) (3). meso-Tartaric acid was converted by pro-
cedure C to bis(d ip h en ylm eth yl) m eso-ta r tr a te (3a ) as a
1
white powder, mp ) 108-109 °C; H NMR (C) 7.18-7.27 (m,
20H), 6.82 (s, 2H), 4.72 (s,2H); 13C NMR (C) 170.2, 139.0, 138.7,
128.52, 128.45, 128.23, 128.18, 128.0, 127.2, 79.4, 73.1; DIP/
MS 315 (2), 183 (37), 167 (100), 165 (47); DIP/MS 315 (2), 183
(35), 167 (100), 165 (48). Reaction of 3a with 35a via procedure
F gave bis(d ip h en ylm eth yl) bis[bis(m eth oxyca r bon yl)-
1
ca ffeoyl]-m eso-ta r tr a te (3b), mp ) 72-74 °C; H NMR (C)
7.59 (d, 16.0, 2H), 7.21-7.38 (m, 20H), 6.88 (s, 2H), 6.29 (d,
16.0, 2H), 6.02 (s, 2H), 3.91 (s, 6H), 3.90 (s, 6H); 13C NMR (C)
164.9, 164.8, 153.0, 152.9, 144.6, 143.9, 142.7, 139.01, 138.98,
133.01, 128.6, 128.5, 128.27, 128.1, 127.3, 127.1 (×2), 123.5,
122.6, 117.8, 79.6, 71.6, 55.9 (×2). Removal of the methoxy-
carbonyl groups by procedure B gave bis(d ip h en ylm eth yl)
d ica ffeoyl-m eso-ta r tr a te (3c), mp ) 142-144 °C dec; 1H
NMR (M) 7.55 (d, 16.0, 2H), 6.82 (s, 2H), 6.77-7.25 (m, 20H),
6.20 (d, 16.0, 2H), 6.01 (s, 2H); 13C NMR (M) 167.4d, 166.8d,
150.1d, 148.9u, 146.8d, 140.8 (×2)d, 129.6u, 129.5u, 129.2u,
129.1u, 128.2u, 128.1u, 127.3d, 123.5u, 116.5u, 115.4u, 113.4u,
80.8u, 72.8u. Removal of the diphenylmethyl groups by
procedure D gave m eso-DCTA (3) whose 1H and 13C NMR
agreed with the literature except that C-1 and C-2 of the
tartaric acid portion were 3.5 ppm upfield from that of an
alleged meso-chicoric acid from Equisetum arvense.39 Since the
latter was isolated by chromatography with 1% NH3 in MeOH,
it may have been the ammonium salt. Dissociation of a
carboxyl group deshields both the carbonyl and R-carbon
resonances by 3-4 ppm.38 This hypothesis was confirmed since
the 13C NMR of the ammonium salt of 3 matched that of the
“meso-chicoric acid” isolated from Equisetum which therefore
was of the corresponding ammonium salt.
P r oced u r e H, F or m a tion of Acyl Ch lor id es w ith Ox-
a lyl Ch lor id e. A solution of the acid in excess oxalyl chloride
was stirred with a 25-fold excess of oxalyl chloride at room
temperature for 1 h, the excess reagent removed on a Rotovap,
and the acid chloride used immediately without purification.
P r oced u r e I, F or m a tion of Acyl Ch lor id es w ith Th io-
n yl Ch lor id e. A solution of the acid in excess thionyl chloride
was heated (NaOH trap) in an oil bath at 80-90 °C until HCl
evolution ceased. Removal of the thionyl chloride on a Rotovap
gave the acid chloride used immediately without purification.
Sp ecific Syn t h eses. Dica ffeoyl-L-t a r t a r ic Acid (L-
Ch icor ic Acid , L-DCTA) (1). L-Tartaric acid was converted
by procedure C to bis(d ip h en ylm eth yl) L-ta r tr a te (1a ) as
a white powder, mp ) 107-108 °C; 1H NMR (C) 7.24-7.32
(m, 20H), 6.98 (s, 2H), 4.75 (s, 2H); 13C NMR (C) 170.7, 139.0,
138.9, 128.6, 128.5, 128.4, 128.1, 127.6, 126.9, 79.2, 72.3; DIP/
MS 315 (2), 183 (33), 167 (100), 165 (43). Reaction of 1a with
35a via procedure F gave bis(d ip h en ylm eth yl) bis[bis-
(m eth oxyca r bon yl)ca ffeoyl]-L-ta r tr a te (20), mp ) 62-64
1,2-Dica ffeoyleth a n ed iol (4). Ethylene glycol reacted with
35a by procedure E to give 1,2-bis[bis(m eth oxyca r bon yl)-
ca ffeoyl]eth a n ed iol (4a ), mp ) 88-90 °C; 1H NMR (C) 7.66
(d, 16.0, 2H), 7.48 (d, 1.9, 2H), 7.45 (dd, 1.9, 8.4, 2H), 7.32 (d,
8.4, 2H), 6.44 (d, 16.1, 2H), 4.48 (s, 4H), 3.91 (s, 12H); 13C NMR
(C) 166.2, 153.07, 152.92, 143.6, 143.2, 142.7, 133.4, 126.7,
123.5, 122.4, 119.1, 62.5, 55.9 (×2); DIP/MS 618 (1), 233 (61),
189 (100), 145 (26), 117 (32). Hydrolysis of 4a by procedure B
gave 4, mp ) 239-240 °C; 1H NMR (M) 7.57 (d, 15.9, 2H),
7.04 (d, 1.9, 2H), 6.93 (dd, 2.0, 8.2, 2H), 6.75 (d, 8.2, 2H), 6.28
(d, 16.0, 2H), 4.43 (s, 4H); 13C NMR (M) 169.1, 150.4, 147.6,
147.1, 127.3, 123.2, 116.6, 115.0, 114.4, 63.5. Anal. (C20H18O8)
C, H.
1
°C; H NMR (C) 7.50 (d, 16.0, 2H), 7.10-7.38 (m, 20H), 6.94
(s, 2H), 6.16 (d, 16.0, 2H), 6.04 (s, 2H), 3.93 (s, 6H), 3.92 (s,
6H); 13C NMR (C) 164.8 (×2), 153.1, 152.9, 144.3, 143.9, 142.7,
138.8, 138.7, 133.0, 128.63, 128.57, 128.2 (×2), 127.3, 127.0
(×2), 123.50, 122.6, 117.5, 79.1, 71.1, 56.0 (×2). Removal of
the methoxycarbonyl groups by procedure B gave bis(d i-
p h en ylm eth yl) d ica ffeoyl-L-ta r tr a te (21), mp ) 135-136
°C dec; 1H NMR (M) 7.47 (d, 15.8, 2H), 6.91 (s, 2H), 6.79-
7.36 (m, 20H), 6.10 (s, 2H), 6.07 (d, 15.8, 2H); 13C NMR (M)
167.4d, 166.8d, 150.2d, 148.8u, 146.9d, 140.7d (×2), 129.7u
(×2), 129.2u (×2), 128.2u, 128.0u, 127.5d, 123.6u, 116.6u,
115.4u, 113.3u, 80.3u, 72.5u. Removal of the diphenylmethyl
groups by procedure D gave L-DCTA (1), whose 1H and 13C
NMR agreed with the literature.37
1,4-tr a n s- a n d 1,4-cis-Dica ffeoylcycloh exa n ed iols (5
a n d 6). A mixture of cis- and trans-1,4-cyclohexanediols
(Aldrich C10,120-6) reacted with 35a by procedure E to give
a stereoisomeric mixture of 1,4-bis[bis(methoxycarbonyl)caf-
feoyl]cyclohexanediols which was separated with benzene:
acetone on a silica gel column. The first isomer to elute was
identified as the 1,4-trans-isomer because the diaxial carbinol
protons are, as expected,40 at higher field (4.98 ppm) than those
of the axial-equatorial carbinol protons (5.02 ppm) of the cis-
isomer. 1,4-tr a n s-Bis[bis(m eth oxycar bon yl)caffeoyl]cyclo-
h exa n ed iol (5a ), mp ) 174-177 °C; 1H NMR (C) 7.62 (d, 16.0,
2H), 7.47 (s, 2H), 7.43 (d, 8.5, 2H), 7.32 (d, 8.5, 2H), 6.40 (d,
16.0, 2H), 4.98 (bs, 2H), 3.92 (s, 12H), 2.08 (bd, 4H), 1.67 (m,
4H); 13C NMR (C) 165.9, 153.1, 152.9, 143.5, 142.7, 142.4,
133.5, 126.6, 123.5, 122.3, 120.0, 71.2, 55.9 (×2), 28.0. The
second isomer to elute was 1,4-cis-b is[b is(m et h oxyca r -
Dica ffeoyl-D-ta r ta r ic Acid (D-Ch icor ic Acid , D-DCTA)
(2). D-Tartaric acid was converted by procedure C to bis-
(d ip h en ylm eth yl) D-ta r tr a te (2a ) as a white powder, mp )
108-110 °C; 1H NMR (C) 7.28-7.34 (m, 20H), 6.99 (s, 2H),
4.76 (s, 2H); 13C NMR (C) 170.7, 139.0, 138.9, 128.6, 128.5,
128.4, 128.1, 127.6, 126.9, 79.2, 72.3; DIP/MS 315 (2), 183 (37),
167 (100), 165 (47). Reaction of 2a with 35a via procedure F
gave bis(d ip h en ylm eth yl) bis[bis(m eth oxyca r bon yl)ca f-
feoyl]-D-ta r tr a te (2b), mp ) 61-63 °C; 1H NMR (C) 7.50 (d,
16.0, 2H), 7.09-7.37 (m, 20H), 6.94 (s, 2H), 6.16 (d, 16.0, 2H),
6.04 (s, 2H), 3.93 (s, 6H), 3.92 (s, 6H); 13C NMR (C) 164.8 (×2),
1
bon yl)ca ffeoyl]cycloh exa n ed iol (6a ), mp ) 60-64 °C; H

HIV IN Inhibitors: SAR of DCQA and DCTA Analogues
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 3 505
NMR (C) 7.64 (d, 16.0, 2H), 7.48 (d, 1.9, 2H), 7.44 (dd, 1.9,
8.5, 2H), 7.32 (d, 8.5, 2H), 6.43 (d, 16.0, 2H), 5.02 (m, 2H),
3.91 (s, 12H), 1.90-2.10 (m, 4H), 1.67-1.85 (m, 4H); 13C NMR
(C) 165.8, 153.1, 153.0, 143.5, 142.7, 142.4, 133.6, 126.6, 123.5,
122.3, 120.1, 70.4, 55.9 (×2), 27.4. Hydrolysis of 5a by
6.89 (dd, 2.0, 8.2, 2H), 6.62 (d, 8.1, 2H), 6.16 (d, 15.8, 2H), 4.14
(bs, 4H), 1.73 (bs, 4H); 13C NMR (D) 166.8, 152.9, 147.2, 145.8,
122.8, 121.9, 115.6, 113.5, 111.4, 63.0, 25.0. Anal. (C22H22O8‚
0.8H2O) C, H.
1,2-tr a n s-Dica ffeoylcycloh exa n ed iol (16). 1,2-trans-
Cyclohexanediol reacted with 35a by procedure E to give
1,2-t r a n s-b is [b is (m e t h o x y c a r b o n y l)c a ffe o y l]c y c lo -
h exa n ed iol (15), mp ) 46-47 °C; 1H NMR (C) 7.58 (d, 16.0,
2H), 7.43 (d, 2.0, 2H), 7.39 (dd, 2.0, 8.5, 2H), 7.29 (d, 8.5, 2H),
6.34 (d, 15.9, 2H), 5.02 (m, 2H), 3.91 (s, 6H), 3.90 (s, 6H), 2.16
(bd, 2H), 1.78 (m, 2H), 1.42-1.53 (m, 4H); 13C NMR (C) 165.8,
153.1, 152.9, 143.6, 142.8, 142.6, 133.5, 126.7, 123.4, 122.3,
119.6, 74.1, 55.9 (×2), 30.3, 23.5. Hydrolysis of 15 by procedure
1
procedure B gave 5, mp ) 294-296 °C; H NMR (M) 7.53 (d,
15.8, 2H), 6.98 (d, 2.0, 2H), 6.84 (dd, 8.1, 2.0, 2H), 6.65 (d, 8.1,
2H), 6.12 (d, 15.8, 2H), 4.92 (bs, 2H), 2.07 (m, 4H), 1.76 (m,
4H); 13C NMR (M) 169.5, 156.4, 149.5, 148.2, 124.3, 123.8,
117.0, 113.3, 112.3, 72.1, 29.2. Anal. (C24H24O8) C, H. Hydroly-
1
sis of 6a by procedure B gave 6, mp ) 262 °C; H NMR (M)
7.56 (d, 15.8, 2H), 7.04 (s, 2H), 6.91 (d, 8.1, 2H), 6.73 (d, 8.1,
2H), 6.22 (d, 15.8, 2H), 4.96 (bs, 2H), 1.92 (m, 4H), 1.84 (m,
4H); 13C NMR (M) 169.0, 153.0, 148.2, 147.5, 126.0, 123.4,
116.8, 114.3, 114.0, 71.3, 28.5. Anal. (C24H24O8) C, H.
1
B gave 16, mp ) 99 °C; H NMR (D) 7.44 (d, 16.0, 2H), 7.01
(s, 2H), 6.96 (d, 8.4, 2H), 6.73 (d, 8.4, 2H), 6.19 (d, 15.8, 2H),
4.91 (d, 6.4, 2H), 2.01 (bd, 10.9, 2H), 1.46 (m, 2H), 1.41-1.47
(m, 4H); 13C NMR (D) 165.8, 148.3, 145.4, 145.3, 125.3, 121.2,
115.6, 114.8, 113.7, 73.0, 29.8, 22.9. Anal. (C24H24O8) C, H.
1,3-tr a n s- a n d 1,3-cis-Dica ffeoylcycloh exa n ed iols (9
a n d 10). A mixture of cis- and trans-1,3-cyclohexanediols
(Aldrich C10,110-9) reacted with 35a by procedure E to give
a stereoisomeric mixture of 1,3-bis[bis(methoxycarbonyl)caf-
feoyl]cyclohexanediols which was separated with benzene:
acetone on a silica gel column. The first isomer to elute was
identified as the 1,3-trans-isomer because the axial-equatorial
carbinol protons are at lower field (5.28 ppm) than those of
the diaxial carbinol protons (4.93 ppm) of the cis-isomer (cf.
5a and 6a ). 1,3-tr a n s-Bis[bis(m eth oxyca r bon yl)ca ffeoyl]-
1,2-cis-Dica ffeoylcycloh exa n ed iol (18). 1,2-cis-Cyclohex-
anediol reacted with 35a by procedure E to give 1,2-cis-bis-
[bis(m eth oxyca r bon yl)ca ffeoyl]cycloh exa n ed iol (17), mp
) 60.5-63 °C; 1H NMR (C) 7.61 (d, 16.0, 2H), 7.45 (d, 1.9, 2H),
7.41 (dd, 8.5, 1.9, 2H), 7.33 (d, 9.1, 2H), 6.41 (d, 15.9, 2H), 5.21
(d, 7.0, 2H), 3.91 (s, 12H), 1.97 (m, 2H), 1.73 (m, 4H), 1.52 (m,
2H); 13C NMR (C) 165.7, 153.1, 152.9, 143.6, 142.7, 142.7,
133.6, 126.7, 123.5, 122.3, 119.9, 71.5, 55.9 (×2), 27.8, 21.8.
Hydrolysis of 17 by procedure B gave 18, mp ) 105-106 °C;
1H NMR (D) 7.47 (d, 16.0, 2H), 7.06 (s, 2H) 6.99 (dd, 8.2, ca. 2,
2H), 6.76 (d, 8.1, 2H), 6.27 (d, 16.0, 2H), 5.10 (d, 6.4, 2H), 1.86
(m, 2H), 1.69 (m, 4H) 1.47 (m, 2H); 13C NMR (D) 165.8, 148.3,
145.4, 145.1, 125.4, 121.4, 115.6, 114.7, 114.0, 70.4, 27.3, 21.3.
Anal. (C24H24O8‚0.33H2O) C, H.
1
cycloh exa n ed iol (7), mp ) 56-57 °C; H NMR (C) 7.62 (d,
16.0, 2H), 7.47 (d, 1.9, 2H), 7.44 (dd, 2.0, 8.5, 2H), 7.32 (d, 8.4,
2H), 6.41 (d, 16.0, 2H), 5.28 (bs, 2H), 3.93 (s, 6H), 3.92 (s, 6H),
1.99 (m, 2H), 1.68-1.90 (m, 6H); 13C NMR (C) 165.7, 153.1,
153.0, 143.6, 142.7, 142.5, 133.6, 126.6, 123.5, 122.3, 120.0,
70.3, 55.9 (×2), 35.7, 30.3, 19.3; DIP/MS 672 (1), 279 (29), 252
(33), 238 (26), 235 (53), 221 (25), 208 (54), 194 (25), 191 (40),
189 (92), 81 (100). The second isomer to elute was 1,3-cis-bis-
[bis(m eth oxyca r bon yl)ca ffeoyl]cycloh exa n ed iol (8), mp
) 59-61 °C; 1H NMR (C) 7.61 (d, 15.9, 2H), 7.45 (d, 1.9, 2H),
7.40 (dd, 1.9, 8.5, 2H), 7.30 (d, 8.4, 2H), 6.37 (d, 16.0, 2H), 4.93
(m, 2H), 3.91 (s, 12H), 2.38 (d, 11.8, 1H), 1.60-2.06 (m, 5H),
1.26-1.48 (m, 2H); 13C NMR (C) 165.6, 153.1, 152.9, 143.6,
142.7, 142.5, 133.5, 126.5, 123.5, 122.3, 119.9, 70.8, 55.9 (×2),
37.1, 30.8, 20.0; DIP/MS 672 (1), 538 (29), 252 (38), 235 (34),
221 (26), 208 (78), 194 (26), 191 (40), 189 (100). Hydrolysis of
7 by procedure B gave 9, mp ) 105-106 °C; 1H NMR (M) 7.55
(d, 15.8, 2H), 7.03 (s, 2H) 6.93 (d, 8.2, 2H), 6.75 (d, 8.2, 2H),
6.24 (d, 15.8, 2H), 5.21 (m, 2H), 1.68-1.98 (m, 8H); 13C NMR
(M) 168.7, 151.0, 147.4, 147.2, 127.0, 123.2, 116.6, 114.8, 114.7,
71.3, 36.8, 31.3, 20.5. Anal. (C24H24O8‚0.5H2O) C, H. Hydrolysis
Dica ffeoyl-L-glycer ic Acid (19). Calcium L-glycerate di-
hydrate (Aldrich 37241-2) was dried to constant weight and
reacted with 35a by procedure E to give bis[bis(m eth oxy-
1
ca r bon yl)ca ffeoyl]glycer ic a cid (19a ), gum; H NMR (C)
7.73 (d, 16.0, 1H), 7.66 (d, 16.0, 1H), 7.3-7.5 (m, 6H), 6.51 (d,
16.0, 1H), 6.44 (d, 16.0, 1H), 5.56 (m, 1H), 4.6-4.8 (m, 2H),
3.92 (s, 12H); 13C NMR (C) 171.9, 165.9, 165.4, 153.1, 153.0,
152.9 (×2), 144.6 (×2), 143.83 (×2), 143.79, 142.7, 133.2, 133.1,
126.90, 126.85, 123.5 (×2), 122.6, 122.5, 118.5, 117.9, 70.2,
62.9, 56.0 (×4). Hydrolysis of 19a by procedure B but with a
1
reaction time of only 10 min gave 19, mp ) 234-236 °C; H
NMR (D) 7.54 (d, 15.9, 1H), 7.51 (d, 15.9, 1H), 7.0-7.1 (m,
4H), 6.78 (dd, 1.6, 8.1, 2H), 6.37 (d, 15.9, 1H), 6.29 (d, 15.9,
1H), 5.36 (m, 1H), 4.56 (m, 2H); 13C NMR (D) 168.5, 166.0,
165.6, 148.6, 148.5, 146.4, 146.0 (×2), 145.5, 125.2 (×2), 121.5,
121.4, 115.7 (×2), 115.0, 114.9, 113.0, 112.8, 70.2, 62.4. Anal.
(C21H18O10) C, H.
1
of 8 by procedure B gave 10, mp ) 108 °C; H NMR (M) 7.53
(d, 15.7, 2H), 7.00 (s, 2H) 6.85 (d, 7.9, 2H), 6.69 (d, 7.9, 2H),
6.18 (d, 15.7, 2H), 4.93 (bs, 2H), 2.29 (m, 1H), 1.67-1.96 (m,
5H) 1.38 (m, 2H); 13C NMR (M) 168.8, 152.4, 148.0, 147.5,
126.3, 123.2, 116.7, 114.5, 114.1, 71.6, 37.8, 31.8, 20.6. Anal.
(C24H24O8) C, H.
Bis(3,4-d ih yd r oxyd ih yd r ocin n a m oyl)-L-ta r ta r ic Acid
(22). 3,4-Dihydroxydihydrocinnamic acid was reacted by pro-
cedure A to give a mixture of 3,4-d im eth oxyca r bon yld ih y-
d r ocin n a m ic a cid (22a ) and its anhydride which were
separated by silica gel column chromatography and the latter
hydrolyzed by 80% HOAC solution on a steam bath to give
22a , gum; 1H NMR (C) 7.16 (d, 7.2, 1H), 7.14 (s, 1H), 7.10 (d,
7.2, 1H), 3.90 (s, 6H), 2.97 (t, 8.0, 2H), 2.69 (t, 8.1, 2H); 13C
NMR (C) 177.5, 153.4 (×2), 142.2, 140.8, 139.4, 126.7, 123.0,
122.9, 55.8 (×2), 35.0, 29.9. Reaction of 22a by procedure I
gave the acid chloride which was subjected to procedure G to
give b is(3,4-d im et h oxyca r b on yld ih yd r ocin n a m oyl)-L-
ta r ta r ic a cid (22b), gum; 1H NMR (C) 7.18 (d, 8.1, 2H), 7.10
(dd, 8.1, 1.9, 2H), 7.09 (d, 1.9, 2H), 5.55 (s, 2H), 3.92 (s, 6H),
3.91 (s, 6H), 2.98 (m, 4H), 2.81 (m, 4H); 13C NMR (C) 171.0,
167.1, 154.7, 153.7, 142.0, 140.5, 139.3, 127.0, 123.2, 123.0,
70.3, 56.1, 56.0, 34.6, 30.1. Deprotection of 22b by procedure
B and preparative HPLC gave 22 as a gum which on analytical
HPLC gave a single peak with a retention time of 2.5 min (70%
MeOH-H2O + 1% HOAc) or 3.1 min (45% MeCN-H2O + 1%
1,3-Dica ffeoylp r op a n ed iol (12). 1,3-Propanediol reacted
with 35a by procedure E to give 1,3-b is[b is(m et h oxyca r -
bon yl)ca ffeoyl]p r op a n ed iol (11), gum; 1H NMR (C) 7.63 (d,
2H, 16.1), 7.47 (d, 2H, 1.9), 7.41 (d, 2H, 2.0, 8.5), 7.30 (d, 2H,
8.4), 6.41 (d, 2H, 16.1),4.35 (t, 4H, 6.2), 3.92 (s, 6H), 3.91 (s,
6H), 2.12 (t, 2H, 6.2); 13C NMR (C) 166.3, 153.1, 152.9, 143.6,
142.8, 142.7, 133.4, 126.6, 123.5, 122.3, 119.4, 61.4, 55.9 (×2),
28.1. Hydrolysis of 11 by procedure B gave 12, mp ) 210-
1
212 °C; H NMR (D) 7.47 (d, 15.9, 2H), 7.02 (d, 1.6, 2H), 6.92
(dd, 1.7, 8.2, 2H), 6.67 (d, 8.1, 2H), 6.22 (d, 15.9, 2H), 4.21 (t,
6.3, 4H), 2.01 (t, 6.3, 2H); 13C NMR (D) 166.6, 151.0, 146.7,
145.7, 124.0, 121.4, 116.0, 114.7, 112.3, 60.6, 27.8. Anal.
(C21H20O8) C, H.
1,4-Dica ffeoylbu ta n ed iol (14). 1,4-Butanediol reacted
with 35a by procedure E gave 1,4-bis[bis(m eth oxyca r bon -
yl)ca ffeoyl]bu ta n ed iol (13), mp ) 143-144 °C; 1H NMR (C)
7.62 (d, 16.0, 2H), 7.47 (d, 1.9, 2H), 7.42 (dd, 8.5, 1.9, 2H), 7.31
(d, 8.4, 2H), 6.41 (d, 15.9, 2H), 4.27 (t, 5.0, 4H), 3.91 (s, 6H),
3.90 (s, 6H), 1.84 (bs, 4H); 13C NMR (C) 166.5, 153.1, 152.9,
143.4, 142.7, 142.6, 135.5, 126.6, 123.5, 122.3, 119.6, 64.2, 55.9
(×2), 25.4. Hydrolysis of 13 by procedure B gave 14, mp )
241-242 °C; 1H NMR (D) 7.45 (d, 15.7, 2H), 6.99 (d, 2.0, 2H),
1
HOAc); H NMR (D) 6.66 (bs, 2H), 6.54 (d, 8.0, 2H), 6.35 (bd,
7.5, 2H), 5.38 (s, 2H), 2.75 (m, 4H), 2.49 (m, 4H); 13C NMR (D)
172.2, 170.8, 145.5, 143.5, 131.4, 118.5, 115.6, 115.5, 74.8, 35.3,
29.5; HRFABMS calcd m/z for C22H22O12Na 501.1009, found
501.1006.

506 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 3
King et al.
Bis(3-h yd r oxycin n a m oyl)-L-ta r ta r ic Acid (23). 3-Hy-
droxycinnamic acid was reacted by procedure A to give
3-m eth oxyca r bon ylcin n a m ic a cid (23a ), mp ) 158-160
Bis(3,5-d ih yd r oxyben zoyl)-L-ta r ta r ic Acid (27). 3,5-
Dihydroxybenzoic acid was reacted by procedure A to give 3,5-
d im eth oxyca r bon ylben zoic a cid (27a ), mp ) 155-156 °C;
1H NMR (C) 7.84 (d, 2.3, 2H), 7.38 (d, 2.3, 1H), 3.94 (s, 6H);
13C NMR (C) 169.8, 153.5, 151.4, 131.6, 120.5, 119.8, 55.8.
Procedure I converted 27a to the acid chloride which reacted
by procedure G to give bis(3,5-d im eth oxyca r bon ylben zoyl)-
L-ta r ta r ic a cid (27b), gum; 1H NMR (D) 7.79 (d, 2.1, 4H),
7.67 (m, 2H), 5.89 (s, 2H), 3.86 (s, 12H); 13C NMR (D) 166.8,
162.9, 152.9, 151.3, 130.8, 120.8, 120.0, 71.8, 55.7. Deprotection
of 27b by procedure B and preparative HPLC gave 27, mp )
1
°C; HNMR (C) 7.75 (d, 16.0, 1H), 7.44 (m, 2H), 7.38 (s, 1H),
7.24 (m, 1H), 6.46 (d, 16.0, 1H), 3.93 (s, 3H); 13C NMR (C)
171.3, 151.5, 145.5, 135.8, 130.2, 130.1, 126.1, 123.2, 120.5,
118.7, 55.6. Procedure I converted 23a to the acid chloride
which reacted by procedure G to give b is(3-m et h oxyca r -
bon ylcin n a m oyl)-L-ta r ta r ic a cid (23b), mp ) 108-110
°C: 1H NMR (D) 7.7-7.8 (m, 6H), 7.49 (t, 7.6, 1H), 7.32 (1H,
d, 6.6), 6.79 (d, 16.2, 1H), 5.66 (s, 1H), 3.85 (s, 3H); 13C NMR
(D) 167.6, 165.0, 153.4, 151.1, 144.4, 135.4, 130.1, 126.4, 123.4,
120.9, 118.4, 71.2, 55.4. Deprotection of 23b by procedure B
1
193-195 °C; H NMR (D) 9.78 (s, 4H), 6.87 (s, 4H), 6.50 (s,
2H), 5.77 (s, 2H). 13C NMR (D) 167.0, 164.7, 158.5, 130.0, 107.8,
107.3, 71.2. Anal. (C18H14O12‚3H2O) C, H.
1
and preparative HPLC gave 23, mp ) 115-116 °C; H NMR
(D) 7.57 (d, 15.9, 2H), 7.20 (t, 7.5, 2H), 7.12 (d, 7.1, 2H), 7.05
(1H, s, 2H), 6.84 (d, 7.5, 2H), 6.53 (d, 15.9, 2H), 5.57 (s, 2H);
13C NMR (D) 168.4, 165.3, 157.7, 145.1, 135.0, 129.8, 119.1,
117.7, 117.5, 114.7, 72.0. Anal. (C22H18O10‚0.5H2O) C, H.
Bis(3,4-d ih yd r oxyp h en yla cetyl)-L-ta r ta r ic Acid (28).
3,4-Dihydroxyphenylacetic acid was reacted by procedure A
to give 3,4-d im eth oxyca r bon ylp h en yla cetic a cid (28a ), oil;
1H NMR (C) 7.28-7.17 (m, 3H), 3.89 (s, 6H), 3.63 (s, 2H); 13
C
NMR (C) 175.7, 153.2 (×2), 142.1, 141.5, 132.5, 127.9, 124.0,
123.0, 55.8 (×2), 40.1. Reaction of 28a by procedure H gave
the acid chloride which was subjected to procedure G to give
bis(3,4-d im eth oxyca r bon ylp h en yla cetyl)-L-ta r ta r ic a cid
(28b), gum; 1H NMR (M) 7.35-7.20 (m, 3H), 5.72 (s, H-2), 3.85
(s, 6H), 3.80 (s, 2H); 13C NMR (M) 171.3, 168.9, 154.74, 154.69,
143.6, 143.0, 134.2, 129.1, 125.3, 124.1, 72.7, 56.4 (×2), 40.3.
Deprotection of 28b by procedure B and preparative HPLC
gave 28 as a gum. Anal. (C20H18O12‚H2O) Calcd: C, 51.3; H,
4.3. Found: C, 51.5; H, 4.8. Analytical HPLC gave a single
major peak with a retention time of 2.72 min (10:1 MeCN:
Bis(4-h yd r oxycin n a m oyl)-L-ta r ta r ic Acid (24). 4-Hy-
droxycinnamic acid was reacted by procedure A to give
4-m eth oxyca r bon ylcin n a m ic a cid (24a ), mp ) 201-203
1
°C; H NMR (D) 7.78 (d, 8.6, 2H), 7.62 (d, 16.1, 1H), 7.30 (d,
8.6, 2H), 6.55 (d, 16.1, 1H), 3.85 (s, 3H); 13C NMR (D) 167.5,
153.3, 152.0, 142.8, 132.3, 129.6, 121.8, 119.6, 55.6. Procedure
H
converted 24a to the acid chloride which reacted by
procedure G to give bis(4-m eth oxyca r bon ylcin n a m oyl)-L-
ta r ta r ic a cid (24b), mp ) 157-160 °C; 1H NMR (D) 7.84 (d,
8.6, 2H), 7.72 (d, 16.0, 1H), 7.31 (d, 8.6, 2H), 6.73 (d, 16.0, 1H),
5.68 (s, 1H), 3.85 (s, 3H); 13C NMR (D) 168.0, 165.2, 153.2,
152.2, 144.3, 131.8, 129.9, 121.8, 117.7, 71.7, 55.5. Deprotection
of 24b by procedure B and preparative HPLC gave 24, mp )
137-140 °C; 1H NMR (M) 7.73 (d, 15.9, 2H), 7.51 (d, 8.7, 2H),
6.82 (d, 8.7, 2H), 6.44 (d, 15.9, 2H), 5.81 (s, 2H); 13C NMR (M)
169.6, 167.7, 161.6, 148.1, 131.5, 127.0, 116.9, 113.7, 72.5.
Anal. (C22H18O10) C, H.
Diga lloyl-L-ta r ta r ic Acid (25). Gallic acid (3,4,5-trihy-
droxybenzoic acid) was reacted by procedure A to give 3,4,5-
tr im eth oxyca r bon ylben zoic a cid (25a ), mp ) 143-144 °C;
1H NMR (C) 7.99 (s, 2H), 3.94 (s, 6H), 3.93 (s, 3H); 13C NMR
(C) 169.1, 152.6, 151.6, 143.8, 139.1, 127.8, 122.6, 56.4, 56.2.
Procedure I converted 25a to the acid chloride which reacted
with bis(diphenylmethyl) L-tartrate (1a ) according to proce-
dure F to give bis(d ip h en ylm eth yl) bis(3,4,5-tr im eth oxy-
ca r bon ylben zoyl)-L-ta r tr a te (25b), gum; 1H NMR (C) 7.62
(s, 4H), 7.30-7.36 (m, 20H), 6.96 (s, 2H), 6.08 (s, 2H), 3.94 (s,
6H), 3.92 (s, 12H); 13C NMR (C) 164.0, 162.5, 152.4, 151.5,
143.6, 138.4, 138.1, 128.6, 128.54, 128.50, 128.3, 128.2, 127.5,
126.8, 126.5, 122.3, 79.3, 71.6, 56.4, 56.1. Sequential depro-
tection of 25b by procedures D and B and preparative HPLC
gave 25 as a gum which on analytical HPLC gave a single
dominant peak with a retention time of 1.7 min (10% MeOH-
water + 1% HOAc) or 4.8 min (10% MeCN-H2O + 1% HOAc);
1H NMR (D) 7.01 (s, 4H), 5.70 (s, 2H); 13C NMR (D) 167.3,
164.8, 117.8, 108.8, 145.5, 139.1, 70.9; HRFABMS calcd m/z
for C18H14O14Na 477.0281, found 477.0276.
1
H2O + 1% HOAc) or 1.76 min (5:1 MeOH:H2O); H NMR (M)
6.72-6.68 (m, 4H), 6.59 (dd, 8.0, 1.9, 2H), 5.65 (s, 2H), 3.59
(s, 4H); 13C NMR (M) 172.6, 169.1, 146.2, 145.5, 126.2, 121.9,
117.6, 116.3, 72.4, 40.6.
Dicin n a m oyl-L-t a r t a r ic Acid (29). L-Tartaric Acid was
treated with cinnamoyl chloride according to procedure G to
1
give 29, mp ) 90-94 °C; H and 13C NMR values agree with
literature;41 1H NMR (D) 7.80 (bs, 4H), 7.77 (d, 16.1, 2H), 7.47
(m, 6H), 6.78 (d, 16.1, 2H), 5.79 (s, 2H); 13C NMR (D) 167.5,
165.3, 146.4, 133.7, 130.9, 129.0, 128.7, 116.7, 71.1; DIP/MS
392 (10) (M - H2O), 148 (32), 147 (38), 131 (100), 103 (60).
Anal. (C22H18O8‚1.25H2O) C, H.
Bis(3,4-d im eth oxycin n a m oyl)-L-ta r ta r ic Acid (30). The
acid chloride of 3,4-dimethoxycinnamic acid prepared via
procedure I was reacted with L-tartaric acid according to
1
procedure G to give 30, mp ) 204-206 °C; H NMR (M) 7.70
(d, 15.9, 2H), 7.15 (m, 4H), 6.93 (d, 8.2, 2H), 5.86 (s, 2H), 3.83
(s, 6H), 3.82 (s, 6H); 13C NMR (M) 170.3, 167.6, 152.9, 150.6,
147.8, 128.4, 124.4, 115.1, 112.5, 111.3, 72.9, 56.4 (×2); DIP-
MS: 512 (1) (M - H2O), 209 (13), 208 (100), 193 (24), 191 (34).
Anal. (C26H26O12) C, H.
Bis(2,3-d ih yd r oxyben zoyl)-L-ta r ta r ic Acid (31). 2,3-
Dihydroxybenzoic acid treated by procedure C gave d ip h en -
ylm eth yl 2,3-d ih yd r oxyben zoa te (31a ), gum; 1H NMR (C)
10.81 (s, 1H), 7.57 (dd, 8.1, 1.7, 1H), 7.31-7.44 (m, 11H), 7.11
(s, 1H), 6.84 (t, 8.0, 1H), 5.68 (s, 1H); 13C NMR (C) 169.4, 149.1,
145.1, 139.5, 128.7, 128.3, 127.1, 120.6, 120.0, 119.3, 112.5,
78.1. Application of procedure A to 31a gave d ip h en ylm eth yl
2,3-d im eth oxyca r bon ylben zoa te (31b), mp ) 88-89 °C; 1H
NMR (C) 8.00 (dd, 8.0, 1.7, 1H), 7.50 (dd, 8.0, 1.7, 1H), 7.25-
7.40 (m, 11H), 7.10 (s, 1H), 3.90 (s, 3H), 3.60 (s, 3H); 13C NMR
(C) 163.1, 153.2, 152.7, 143.9, 142.7, 139.6, 129.5, 128.6, 128.1,
127.5, 127.4, 126.5, 125.3, 78.3, 56.0, 55.7. Hydrolysis of 31b
according to procedure D gave 2,3-d im eth oxyca r bon ylben -
zoic a cid (31c), mp ) 107-110 °C; 1H NMR (C) 8.00 (dd, 7.9,
1.7, 1H), 7.77 (dd, 7.9, 1.7, 1H), 7.38 (t, 8.0, 1H), 3.92 (s, 6H);
13C NMR (C) 169.1, 153.1, 152.7, 143.8, 143.1, 129.8, 128.2,
126.5, 124.1, 56.0 (×2). Procedure I converted 31c to the acid
chloride which was reacted with 1a by procedure F to give bis-
(d ip h en ylm eth yl) bis(2,3-d im eth oxyca r bon ylben zoyl)-L-
ta r tr a te (31d ), mp ) 187-189 °C; 1H NMR (C) 7.52 (dd, 7.9,
1.4, 2H), 7.48 (dd, 1H, 7.8, 1.4), 6.9-7.4 (m, 22H), 6.88 (s, 2H),
6.11 (s, 2H), 3.93 (s, 6H), 3.80 (s, 6H); 13C NMR (C) 164.2 161.8
153.1 152.4 143.8 143.3 138.64 138.60 129.5 128.5 128.4 128.3
128.2 128.1 127.2 127.0 126.3 126.3 123.0 79.5 71.5 56.0 (×2).
Bis(3,4-d ih yd r oxyben zoyl)-L-ta r ta r ic Acid (26). 3,4-
Dihydroxybenzoic acid (Aldrich 10,980-0) was reacted by
procedure A to give 3,4-d im eth oxyca r bon ylben zoic a cid
1
(26a ) as an off-white solid, mp ) 168-169 °C; H NMR (C +
M) 8.00 (dd, 8.6, 2.2, 1H), 7.99 (s, 1H), 7.39 (d, 8.6, 1H), 3.93
(s, 6H); 13C NMR (C + M) 167.0, 153.3, 153.0, 146.1, 142.3,
129.7, 128.8, 125.0, 123.1, 56.1 (×2). Procedure I converted
26a to the acid chloride which reacted by procedure G to give
bis(3,4-d im eth oxyca r bon ylben zoyl)-L-ta r ta r ic a cid (26b),
1
gum; H NMR (C + M) 8.04 (dd, 8.4, 1.9, 2H), 8.07 (bs, 2H),
7.46 (d, 8.4, 2H), 6.00 (s, 2H), 5.78 (bs, 2H), 3.93 (s, 3H), 3.92
(s, 3H); 13C NMR (C + M) 167.5, 163.5, 152.7, 152.4, 146.3,
142.0, 128.5, 127.4, 124.6, 123.0, 71.8, 55.6 (×2). Deprotection
of 26b by procedure B and preparative HPLC gave 26 as a
white solid, mp ) 175-176 °C; 1H NMR (D) 9.97 (s, 2H), 9.56
(s, 2H), 7.41 (d, 2.5, 2H), 7.39 (d, 8.1, 2H), 6.87 (dd, 8.1, 2.5,
1H), 5.73 (d, 2.7, 2H); 13C NMR (D) 167.4, 164.5, 151.1, 145.1,
122.3, 119.1, 116.4, 115.4, 71.0. Anal. (C18H14O12‚1.5H2O) C,
H.

HIV IN Inhibitors: SAR of DCQA and DCTA Analogues
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 3 507
77.7. Hydrolysis of 34c by procedure D gave 3,5-d ica ffeoyl-
Hydrolysis of 31d by procedure D gave bis(2,3-d im eth oxy-
ca r bon ylben zoyl)-L-ta r ta r ic a cid (31e), gum; 1H NMR (C)
9.91 (s, 2H), 7.98 (dd, 7.9, 1.4, 2H), 7.50 (dd, 7.9, 1.4, 2H), 7.36
(t, 7.9, 2H), 5.94 (s, 2H), 3.90 (s, 6H), 3.81 (s, 6H); 13C NMR
(C) 169.9, 162.2, 153.1, 152.7, 143.8, 143.0, 129.7, 128.3, 126.8,
123.3, 71.3, 56.1, 56.0. Deprotection of 31e by procedure B gave
bis(2,3-d ih yd r oxyben zoyl)-L-ta r ta r ic a cid (31), mp ) 202
°C; 1H NMR (D) 7.26 (dd, 7.9, 1.3, 2H), 7.08 (dd, 7.9, 1.3, 2H),
6.81 (t, 7.9, 2H), 5.86 (s, 2H); 13C NMR (D) 167.2, 167.1, 149.1,
146.2, 121.0, 119.7, 119.1, 112.8, 71.8. Anal. (C18H14O12) C, H.
1
ben zoic a cid (34), mp ) 170-172 °C; H NMR (D) 9.82 (bs,
2H), 9.33 (bs, 2H), 7.76 (d, 15.7, 2H), 7.67 (d, 2.2, 2H), 7.44 (t,
2.2, 1H), 7.19 (d, 1.9, 2H), 7.14 (dd, 8.3, 2.0, 2H), 6.84 (d, 8.1),
6.53 (d, 15.9, 2H); 13C NMR (D) 165.8, 164.9, 151.0, 149.0,
147.7, 145.6, 132.8, 125.2, 122.1, 120.4, 120.0, 115.8, 115.1,
112.3. Anal. (C25H18O10‚1.6H2O) C, H.
3,4-Dim eth oxyca r bon ylca ffeic Acid (35). Caffeic acid
was treated according to procedure A to give 35, mp )142-
143 °C (lit.43 mp 141.5-143 °C); 1H NMR (M) 7.63 (d, 16.0,
1H), 7.58 (d, 2.0, 1H), 7.53 (d, 8.4, 1H), 7.34 (d, 8.5, 1H), 6.48
(d, 16.0, 1H), 3.870 (s, 3H), 3.866 (s, 3H); 13C NMR (M) 169.8,
154.6, 154.4, 145.0, 144.2, 144.0, 135.1, 127.8, 124.7, 123.5,
121.1, 56.5 (×2); DIP/MS 296 (75), 252 (100), 221 (79), 193
(55). The acid chloride was made according to procedure H or
I to give 3,4-d im eth oxyca r bon ylca ffeoyl ch lor id e (35a ),
MS 314 (22), 235 (100), 191 (80), which was used immediately.
Biologica l Assa ys. 1. Cells a n d Vir u s. MT-2 cells are a
CD4+ T-lymphoblastoid cell line that is completely lysed by
T-cell tropic isolates of HIV-1. HIVNL4-3 and HIVNL4-3clone1-D4
were obtained as described.26 Plasmids were transfected into
HeLa cells with Lipofectin (Gibco/BRL); the cells were washed
and then cultured with H9 cells, a CD4+ T-lymphoblastoid cell
line that supports chronic production of HIV. Virus was
obtained when the culture became 100% positive for HIV
antigens by immunofluorescence. HIVLAI is the LAI isolate of
HIV-1. HIVLAI was grown in H9 cells. Cells were grown in
RPMI-1640 containing 25 mM 4-(2-hydroxyethyl)-1-piper-
azineethanesulfonic acid (HEPES) and supplemented with
11.5% fetal bovine serum and 2 mM L-glutamine. All HIVs
were obtained from H9 cell culture supernatant clarified of
cells by low-speed centrifugation followed by filtration through
0.45-µm nitrocellulose filters.
2. Cell Toxicity Assa ys. Cell toxicity assay was performed
as reported, previously.7,8,44 Briefly, lyophilized compounds
were solubilized in either H2O or 95% ethanol, diluted 1:5 in
growth medium, filter-sterilized, and further 2-fold serially
diluted from 1:10 to 1:1280 in triplicate wells of a microtiter
plate. To each 50 µL of diluted compound, 50 µL of growth
medium was added followed by 100 µL of MT-2 cell suspension
(2 × 105 cells). Cells were incubated with drug for 48 or 72 h
at 37 °C and then harvested for cell viability in a neutral red
dye assay as described previously.32 The lethal dose was
defined as 5% inhibition of MT-2 cell growth in 48 h (LD5).
Several of the compounds were not available in sufficient
quantity or demonstrated a solubility profile that precluded
determination of a true LD5. For these compounds, cell toxicity
is defined as greater than the maximum concentration of
compound tested. The LD5 is a better measure of toxicity than
LD50 as 5% inhibition of cell growth is within 1 SD of the cell
controls. Thus, this is a truly nontoxic concentration of
compound.
Bis(2,5-d ih yd r oxyben zoyl)-L-ta r ta r ic Acid (32). 2,5-
Dihydroxybenzoic acid treated by procedure C gave d ip h en -
ylm eth yl 2,5-d ih yd r oxyben zoa te (32a ), mp ) 140-141 °C;
1H NMR (C) 10.30 (s, 1H), 7.47 (d, 3.1, 1H), 7.30-7.39 (m,
10H), 7.09 (s, 1H), 7.00 (dd, 8.9, 3.1, 1H), 6.87 (d, 8.9, 1H); 13
C
NMR (C) 168.8, 156.1, 147.7, 139.5, 128.7, 128.3, 127.1, 124.3,
118.6, 114.7, 112.3, 78.0. Application of procedure A to 32a
gave d ip h en ylm et h yl 2,5-d im et h oxyca r b on ylb en zoa t e
(32b), gum; 1H NMR (C) 7.90 (d, 2.9, 1H), 7.45-7.25 (m, 11H),
7.21 (d, 8.8, 1H), 7.10 (s, 1H), 3.92 (s, 3H), 3.62 (s, 3H); 13C
NMR (C) 162.7, 153.7 (×2), 148.6, 148.3, 139.5, 128.5, 128.1,
127.4, 126.7, 126.5, 124.6, 124.4, 78.3, 55.7, 55.5. Hydrolysis
of 32b according to procedure D gave 2,5-d im eth oxyca r bon -
1
ylben zoic a cid (32c), mp ) 150-152 °C; H NMR (C) 7.95
(d, 3.0, 1H), 7.46 (dd, 8.7, 3.0, 1H), 7.25 (d, 8.7, 1H), 3.94 (s,
6H); 13C NMR (C) 168.5, 153.73, 153.68, 148.8, 148.6, 127.5,
125.0, 124.7, 123.1, 55.8, 55.7. Procedure I converted 32c to
the acid chloride which was reacted with 1a by procedure F
to give bis(d ip h en ylm eth yl) bis(2,5-d im eth oxyca r bon yl-
ben zoyl)-L-ta r tr a te (32d ), gum; 1H NMR (C) 7.51 (d, 2.9, 2H),
7.43 (dd, 8.8, 2.9, 2H), 7.20 (d, 2H, 8.8), 7.00-7.36 (m, 20H),
6.88 (s, 2H), 6.14 (s, 2H), 3.89 (s, 6H), 3.76 (s, 6H); 13C NMR
(C) 164.0, 161.7, 153.5, 153.4, 148.8, 148.4, 138.9, 128.6,
128.52, 128.46, 128.2, 128.1, 127.6, 127.3, 127.2, 126.9, 124.52,
124.47, 79.4, 71.4, 55.7, 55.6. Hydrolysis of 32d by procedure
D gave bis(2,5-dim eth oxycar bon ylben zoyl)-L-tar tar ic acid
1
(32e), gum; H NMR (D) 7.84 (d, 2H, 2.9), 7.72 (dd, 8.8, 3.0,
2H), 7.53 (d, 8.8, 2H), 5.87 (s, 2H), 3.87 (s, 6H), 3.79 (s, 6H);
13C NMR (D) 166.7, 161.7, 153.3, 153.0, 148.4, 148.2, 128.5,
125.6, 124.0, 122.5, 71.6. Deprotection of 32e by procedure B
gave bis(2,5-d ih yd r oxyben zoyl)-L-ta r ta r ic a cid (32), mp
1
) 138.5-140 °C; H NMR (D) 9.35 (s, 2H), 7.18 (d, 3.0, 2H),
7.03 (dd, 9.0, 3.0, 2H), 6.86 (d, 9.0, 2H), 5.85 (s, 2H); 13C NMR
(D) 167.0 (×2), (in 20% CD3OD splits into 167.2/166.6) 153.1,
149.5, 124.4, 118.4, 114.3, 111.9, 71.5. Anal. (C18H14O12) C, H.
Bis(3,4-d iflu or oben zoyl)-L-ta r ta r ic Acid (33). The acid
chloride of 3,4-difluorobenzoic acid prepared via procedure I
was reacted with L-tartaric acid according to procedure G to
give 33, mp 200-202 °C; 1H NMR (M) 7.95 (m, 4H), 7.45 (ddd,
ca. 8.8 each, 2H), 5.98 (s, 2H); 13C NMR (M) 168.9, 164.7, 155.2
(dd, 255.4, 12.8), 151.4 (dd, 249.0, 13.2), 128.4 (dd, 7.8, 3.6),
127.6 (m), 120.0 (d, 18.6), 119.0 (d, 18.4), 73.4. Anal. (C26H26O12)
C, H.
3. An ti-HIV Assa y. Anti-HIV assays were performed as
described.7,8,44 On the basis of cell toxicity data, compounds
were diluted in growth medium such that a final 1:4 dilution
of the sample would result in a concentration of sample equal
to the LD5. The compounds were then 2-fold serially diluted
in triplicate. To each 50 µL of diluted compound, 50 µL of
HIVLAI was added, and the virus-compound mixture was
incubated for 1 h at 37 °C. Next, 100 µL of MT-2 cell
suspension (2 × 105 cells) was added to each well, and cells
were incubated for 72 h at 37 °C. Final multiplicity of infection
(MOI) was 1-5. Cells were harvested to quantitate cytopathic
effect using a neutral red dye assay as described.44 The
antiviral concentration reported is the concentration of sample
necessary to protect 50% of MT-2 cells from viral-induced cell
death; this is referred to as the ED50. Inhibition of virus-
induced cell death has correlated well with virus replication
as measured by synthesis of antigens, reverse transcriptase
release, and production of infectious progeny virions.45
3,5-Dica ffeoylben zoic Acid (34). 3,5-Dihydroxybenzoic
acid treated by procedure C gave d ip h en ylm eth yl 3,5-
d ih yd r oxyben zoa te (34a ), mp ) 129-131 °C; 1H NMR (M)
7.25-7.42 (m, 10H), 7.12 (d, 2, 2H), 7.00 (s, 1H), 6.58 (s, 1H);
13C NMR (M) 167.0, 159.8, 141.8, 133.1, 129.5, 128.9, 127.8,
108.9, 108.6, 78.9; DIP/MS 320 (19), 167 (100), 152 (31).
Reaction of 34a and 35a according to procedure F gave
d ip h en ylm eth yl 3,5-bis(3,4-d im eth oxyca r bon ylca ffeoyl)-
ben zoa te (34b), mp ) 62-64 °C; 1H NMR (C) 7.84 (d, 2.2,
2H), 7.82 (d, 2.2, 2H), 7.25-7.53 (m, 17H), 7.13 (s, 1H), 6.58
(d, 16.0, 2H), 3.93 (s, 6H), 3.92 (s, 6H); 13C NMR (C) 164.2.
163.9, 153.0, 152.9, 151.1, 145.1, 144.1, 142.8, 139.8, 133.0,
132.4, 128.6, 128.1, 127.2, 127.0, 123.7, 122.6, 120.5, 120.4,
118.1, 78.1, 56.0 (×2); DIP/MS 437 (13), 436 (47), 253 (32),
167 (73), 166 (100), 165 (79). Hydrolysis of 34b according to
procedure B gave d ip h en ylm eth yl 3,5-d ica ffeoylben zoa te
1
(34c), mp ) 179-181 °C dec; H NMR (D) 7.87 (d, 2.1, 2H),
7.79 (d, 15.7, 2H), 7.22-7.59 (m, 13H), 7.14 (dd, 8.4, 1.7, 2H),
7.11 (s, 1H), 6.86 (d, 8.0, 2H), 6.55 (d, 15.8, 2H); 13C NMR (D)
164.9, 163.4, 151.3, 149.7, 148.0, 145.9, 140.2, 131.4, 128.6,
127.9, 126.53, 126.46, 125.0, 122.2, 120.1, 115.8, 114.9, 112.0,
4. Disin tegr a tion Assa y. The disintegration activities of
IN in the presence and absence of inhibitors was assayed in
vitro as modified from Chow et al.21 The following oligonucleo-
tides (GenoSys, Inc.) were used as DNA substrates: T1 (16-

508 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 3
King et al.
(9) McDougall, B.; King, P. J .; Wu, B. W.; Hostomsky, Z.; Reinecke,
M. G.; Robinson, W. E., J r. Dicaffeoylquinic and dicaffeoyltartaric
acids are selective inhibitors of human immunodeficiency virus
type 1 integrase. Antimicrob. Agents Chemother. 1998, 42, 140-
146.
mer), 5′-CAGCAACGCAAGCTTG-3′; T3 (30-mer), 5′-GTC-
GACCTGCAGCCCAAGCTTGCGTTGCTG-3′; V2 (21-mer), 5′-
ACTGCTAGAGATTTTCCACAT-3′; V1/T2 (33-mer), 5′-
ATGTGGAAAATCTCTAGCAGGCTGCAGGTCGAC-3′.
The oligonucleotides were gel-purified. Oligonucleotide T1
was labeled at the 5′-end using T4 polynucleotide kinase and
[γ-32P]ATP (3000 Ci/mmol; Amersham). The substrate for
assaying disintegration activity, the Y-oligomer, was prepared
by annealing the labeled T1 strand with oligonucleotides T3,
V2, and V1/T2.21 In a 20-µL volume, the DNA substrate (0.1
pmol) was incubated with 1.5 pmol of recombinant IN for 60
min at 37 °C in a buffer containing a final concentration of 20
mM HEPES (pH 7.5), 10 mM DTT, 0.05% Nonidet P-40, and
10 mM MnCl2. To each 19 µL of reaction mixture, 1 µL of
inhibitor at various concentrations in solvent or solvent alone
was added. The reaction was stopped by the addition of EDTA
to a final 18 mM concentration. Reaction products were heated
at 90 °C for 3 min before analysis by electrophoresis on a 15%
polyacrylamide gel with 7 M urea in Tris-borate-EDTA
buffer. All reactions were performed at enzyme excess, and
reactions were stopped within the linear range of the reac-
tion.21 Although it has been suggested by one group that the
inhibitory effects of biscatechols are metal ion-dependent,46,47
all reactions were performed in the presence of MnCl2 rather
than MgCl2. Our findings, in collaboration with our collabora-
tor Prof. Samson Chow, indicate that the DCTAs and DCQAs
inhibit HIV IN whether Mg2+ or Mn2+ is the source of divalent
cation, and recent work from our groups suggests that metal
ion is not required at all (Zhu et al., unpublished results).
These data are more consistent with the inhibitory activity of
biscatechols against avian sarcoma virus IN.46 All compounds
were first tested at 25 µM. For active compounds, IC50 analysis
was determined from a median effect plot using CalcuSyn
software (Biosoft, Cambridge, U.K.) on 0.5 log dilutions of
inhibitor in triplicate experiments.
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deVreese, K.; Este, J . A.; Reymen, D.; Pallansch, L. A.; Lackman-
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Ack n ow led gm en t. This work was supported in part
by grants from the National Institutes of Health
(1RO1AI-41360, W.E.R.) and a NIH training grant
(5T32-GM07311, P.J .K.). The authors are indebted to
S. Chow for his gift of the HIV-1 IN expression vector
and his advice on the purification of biologically active
HIV-1 IN.
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