Biomimetic synthesis and anti-HIV activity of dimeric phloroglucinols

Article abstract of DOI:10.1016/j.bmc.2010.01.023

Plants are an important source of a variety of bioactive compounds with different modes of action. Anti-HIV agents from plant sources can be useful in developing novel therapies for inhibiting HIV infection. Based on the reported anti-HIV activity of plant derived phloroglucinols, several new dimeric phloroglucinols were synthesized in the present study by varying substitution on aromatic ring and at methylene bridge. Some of the synthesized compounds have shown good HIV inhibitory activity in a human CD4+ T cell line (CEM-GFP) infected with HIV-1 NL_4.3 virus isolate. Structure-activity studies indicate that phenyl, 4-benzyloxy-1-phenyl and cyclohexyl substitution at methylene bridge gave compounds with better anti-HIV activity. Compounds 22 and 24 showed highest anti-HIV activity with an IC₅₀ of 0.28 μM and 2.71 μM, respectively, former was more active than the positive standard AZT in cell based assay.

Full text of DOI:10.1016/j.bmc.2010.01.023

Bioorganic & Medicinal Chemistry 18 (2010) 2029–2036
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Biomimetic synthesis and anti-HIV activity of dimeric phloroglucinols
Siddheshwar K. Chauthe a, Sandip B. Bharate a, Sudeep Sabde b, Debashis Mitra b,
,
*
Kamlesh K. Bhutani a, Inder P. Singh a,
*
a Department of Natural Products, National Institute of Pharmaceutical Education and Research (NIPER) , Sector-67, S.A.S. Nagar, Punjab 160 062, India
b National Center for Cell Science (NCCS), Pune University Campus, Ganeshkhind, Pune, Maharashtra 411 007, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Plants are an important source of a variety of bioactive compounds with different modes of action. Anti-
HIV agents from plant sources can be useful in developing novel therapies for inhibiting HIV infection.
Based on the reported anti-HIV activity of plant derived phloroglucinols, several new dimeric phloroglu-
cinols were synthesized in the present study by varying substitution on aromatic ring and at methylene
bridge. Some of the synthesized compounds have shown good HIV inhibitory activity in a human CD4+ T
cell line (CEM-GFP) infected with HIV-1 NL4.3 virus isolate. Structure–activity studies indicate that phe-
nyl, 4-benzyloxy-1-phenyl and cyclohexyl substitution at methylene bridge gave compounds with better
Received 3 November 2009
Revised 8 January 2010
Accepted 9 January 2010
Available online 15 January 2010
Keywords:
Phloroglucinol
Anti-HIV activity
CEM-GFP cells
Antivirals
anti-HIV activity. Compounds 22 and 24 showed highest anti-HIV activity with an IC50 of 0.28
lM and
2.71 M, respectively, former was more active than the positive standard AZT in cell based assay.
l
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
Donglei et al. have elaborately reviewed anti-HIV agents from
natural resources belonging to several classes including terpenoids,
coumarins, alkaloids, polyphenols, tannins and flavonoids.5 Earlier,
we reviewed natural products exhibiting anti-HIV activity.6 Anti-
HIV agents from these classes exhibit unique mechanism of action
pertaining to their physicochemical properties. These natural prod-
uct derived compounds can be used in conjunction with existing
anti-HIV agents and thus possibility of drug resistance could be
minimized in novel formulations both for therapeutic and preven-
tive use. Some of the natural products such as calanolides,7a calce-
olarioside B,7b macrocarpals,7c betulinic acid,7d and chicoric acid7e
have exhibited high anti-HIV activity. Calanolide A and a betulinic
acid derivative are in Phase II and IIb of clinical trials.7f Naturally
occurring phloroglucinol compounds have shown a diverse range
of biological activities including anti-HIV activity.8 These com-
pounds mainly occur in the genera Dryopteris, Aspidium, Myrtus,
Mallotus, Hypericum, Eucalyptus and Helichrysum.8 Dimeric
phloroglucinols comprise compounds having two phloroglucinol
units joined either through a methylene linkage or by the forma-
tion of a chroman ring. Dimeric phloroglucinol mallotojaponin
(1) isolated from Mallotus japonicus showed 90% inhibition of
Acquired immunodeficiency syndrome (AIDS) is a clinical syn-
drome caused by the infection with human immunodeficiency
virus-1 (HIV-1), which causes profound immunosuppression. It
has been a serious, life-threatening health problem since the first
case was identified in 1981 and is the most quickly spreading dis-
ease of the century.1 In 2008, an estimated 33.4 million people
lived with the disease worldwide, 2.7 million people were newly
infected with the deadly virus and it killed an estimated 2.0 million
people, including 280,000 children. In the last 25 years, HIV/AIDS
has claimed 25 million lives worldwide.2 Currently used HAART
therapy comprises of drugs that are either nucleoside/nucleotide
reverse transcriptase inhibitors or non-nucleoside reverse trans-
criptase inhibitor or protease inhibitors or supplemented with
the addition of a fusion inhibitor.3 This present therapy is able to
control the viral replication but fails to eradicate virus completely
from body. Recently, US FDA approved Maraviroc (CCR5 blocker)
and Raltegravir (HIV integrase inhibitor) for the treatment of
HIV-1 infection in combination with other antiretroviral agents.4
Most of the clinically used anti-HIV agents are nucleosides and
their toxicity, adverse effects and drug resistance limit their use.
Hence, there is an urgent need for newer anti-HIV agents with no-
vel mechanism of action.
HIV-RTase activity at concentration of 25
6,60-bieckol (2) obtained from Ecklonia cava showed inhibition of
HIV-1 induced syncytia formation (EC50 1.72 M), lytic effects
(EC50 1.23 M), and viral p24 antigen production (EC50
1.26
M).10 Macrocarpals A–E, which are phloroglucinol terpene
adducts showed HIV-RTase inhibitory activity, macrocarpal-B (3)
was the most active with an IC50 = 5.3
M.11 Polyisoprenylated
phloroglucinol derivative laxifloranone (4) showed inhibition of
cytopathic effect of in vitro HIV infection (EC50 = 0.62 g/mL and
l
g/mL.9 Phlorotannin
l
l
l
* Corresponding authors. Tel.: +91 2025708151 (D.M.); +91 172214683x2144
(I.P.S.).
l
E-mail addresses: dmitra@nccs.res.in (D. Mitra), ipsingh@niper.ac.in, ipsingh67
@yahoo.com (I.P. Singh).
l
y
NIPER Communication Number-465.
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.01.023

2030
S. K. Chauthe et al. / Bioorg. Med. Chem. 18 (2010) 2029–2036
IC50 = 6.6
l
g/mL),12 and phloroglucinol-
a
-pyrone derivative arza-
R
R
R
R
nol (5) inhibited HIV-1 replication in T cells (IC50 = 5 l
M).13 Struc-
HO
R
OH
HO
OH
R
HO
R
OH HO
OH
R
tures of these anti-HIV phloroglucinols are shown in Figure 1.
Biogenetically dimeric phloroglucinols can be proposed to be
formed by joining of two monomeric units via methylene linkage
as depicted in Figure 2. Methionine is presumed to be a source
for methylene unit.14
CH2
OH
OH
OH
OH
Figure 2. Proposed biogenesis of dimeric phloroglucinols.
The objective of the present study was to find new anti-HIV
compounds based on anti-HIV activity of phloroglucinol deriva-
tives and as a part of our continuing efforts to synthesize naturally
occurring phloroglucinol compounds and their analogues to ex-
plore their biological potential.15–17 Several dimers containing
phloroglucinol moiety were synthesized by changing length of acyl
functionality and varying substitution at methylene bridge. All the
synthesized compounds have been tested for their potential to in-
hibit the virus in HIV-1 infected T cell line and the active com-
pounds were further evaluated for reverse transcriptase
inhibitory activity.
formyl analogues methylene-bis-(3,5-di-isopentanoyl-2,4,6-trihy-
droxybenzene) (15)22
methylene-bis-(3,5-di-isobutanoyl-2,4,6-
,
trihydroxybenzene) (16)23,24 and methylene-bis-(3-formyl-5-iso-
pentanoyl-2,4,6-trihydroxybenzene) (32)25 from their respective
monomeric precursors 7–9 and 13 is depicted in Scheme 1. Several
new dimeric compounds were synthesized by varying the substitu-
tion on aromatic ring (changing acyl functionalities) and at meth-
ylene bridge (by using different aldehydes viz saturated, aromatic
and heteroaromatic) in a key condensation step as shown in
Scheme 1. Treatment of diacyl phloroglucinols 7 and 8 with ali-
phatic aldehydes viz formaldehyde, propionaldehyde, cyclohexane
carbaldehyde and dihydrocinnamaldehyde under neat conditions
and microwave irradiation (750 W) for 15 min resulted in forma-
tion of desired dimers 17–20 in 32–45% yield. Reaction of 7 and
2. Results and discussion
2.1. Synthesis of dimeric phloroglucinols
Based on the biogenetic pathway,14 our synthetic strategy in-
volved initial synthesis of monomeric units which can be further
dimerized in a key biomimetic step as depicted in Scheme 1. The
key monomeric precursors, 7–9 were synthesized from phloroglu-
cinol (6). The Friedel–Craft acylation of phloroglucinol (6) using
different acyl chlorides resulted in formation of mono- (10–12)
and diacetyl phloroglucinols (7–9) in 60% and 30% yield, respec-
tively.16,18 Synthesis of monomer 13 was carried out by treatment
of phloroisovalerophenone (11) with Vilsmeier–Haack reagent
(POCl3, DMF)19 in ethyl acetate resulting in formation of 3-formyl
phloroisovalerophenone (13)16 in 70% yield. After synthesis of
2,4-diacetyl phloroglucinol (7), the key step involved in the synthe-
sis of dimeric phloroglucinols is condensation of two monomeric
units with the use of proper linker molecule. Several experimental
conditions were attempted for condensation of two monomeric
units as shown in Table 1. Reaction of 7 with formaldehyde in pres-
ence of p-TsCl in chloroform at 50 °C resulted in formation of ex-
pected product 1420 in a yield of 35%. The reaction was not
complete in the absence of catalyst and product was obtained in
a yield of 10%. Higher yields were obtained when reaction was per-
formed under microwave irradiation (750 W, 10–15 min). Initially
when reaction was performed without catalyst under MW condi-
tions for 10 min, 35% product was obtained while reaction under
neat conditions (absence of solvent as well as catalyst) at 750 W
for 15 min resulted in formation of expected product in a yield of
38%. Synthesis of methylene-bis-(3,5-diacetyl-2,4,6-trihydroxy
benzene) (14)21 along with its isovaleryl isobutyryl and isovaleryl
8
with aromatic and heteroaromatic aldehydes under neat
conditions did not yield the desired products. However, the desired
dimers 21–31 were obtained in 42–50% yield in the presence of
catalyst p-TsCl and chloroform as solvent. Dimeric phloroglucinol
compounds 32–34 with formyl functionalities on aromatic ring
were synthesized by reaction of 13 with formaldehyde, pyridine-
2-carboxyaldehyde and 4-benzyloxybenzaldehyde and p-TsCl
catalyst in chloroform under microwave conditions for 5–10 min.
The structures of synthesized compounds are shown in Figure 3.
All compounds were purified by silica gel (# 60–120) column chro-
matography or chromatography using RP-C18 silica gel and charac-
terized by UV, IR, MS, 1H NMR and 13C NMR spectral data.
2.2. Assay for in vitro anti-HIV activity
Synthesized dimeric phloroglucinols were initially tested for
their cytotoxicity in MTT assay26 before testing for cell based
anti-HIV activity. The non-toxic concentrations of dimers were
used for further screening. The anti-HIV activity assay of com-
pounds was carried out in CEM-GFP T cells infected with HIV-1
NL4.3 virus. CEM-GFP is a human CD4+ reporter T cell line, which
expresses GFP (green fluorescent protein) upon HIV infection due
to transactivation by Tat protein of stably integrated long terminal
repeat regulated GFP gene.27 This cell line is used widely for deter-
mination of anti-HIV activity due to easy visualization of infected
cells.28 The results of cytotoxicity and anti-HIV activity are shown
in Table 2. Initially all the synthesized derivatives were tested for
CH3
CHO
OH
O
O
HO
O
O
HO
OH
OH O
HO
O
OHC
OH
H
i-Bu
OH
OH
4
3
1
HOOC
OH
O
OH
O
OH
HO
O
O
HO
OH
O
O
HO HO
HO
O
OH OH
O
O
OH
OH
HO
OH
HO
5
2
Figure 1. Anti-HIV phloroglucinol derivatives from plants.

S. K. Chauthe et al. / Bioorg. Med. Chem. 18 (2010) 2029–2036
2031
O
R
HO
R
OH
HO
OH
HO
R
OH
a
+
O
OH
OH
O
OH
HO
OH
6
7
8
10: R = CH3
: R = CH2CH(CH3)2
: R = CH(CH3)2
: R = CH3
b
11
12
: R = CH2CH(CH3)2
OHC
9: R = CH(CH3)2
OH O
13
R''
HO
R'
O
R''
OH HO
O
c
c
OH
R'
O
OH
OH O
R
Scheme 1. Reagents and conditions: (a) RCOCl, AlCl3, PhNO2, 1 h, 50 °C, 70%; (b) POCl3/DMF (1 equiv each), EtOAc, rt, 2 h, 70%; (c) RCHO, MW, 750 W, 15 min, 32–45%/RCHO,
p-TsCl, CHCl3, MW, 750 W, 15 min, 42–50%.
Table 1
tion in CEM-GFP cells. These seven compounds were analyzed fur-
ther for determination of CC50 and IC50 in order to look for a
potential lead molecule for further development. In order to find
mechanism of action, these seven active compounds were further
tested for reverse transcriptase inhibitory activity.
Condensation of two units of
conditions
7 using formaldehyde under different reaction
Reagent/solvent
Reaction condition
Yielda (%)
CHCl3, p-TsCl
CHCl3, p-TsCl
THF, p-TsCl
PhCH3, p-TsCl
CH3OH, KOH
CHCl3
CHCl3, p-TsCl
CHCl3
Neat
rt, 2 h
50 °C, 2 h
50 °C, 2 h
60 °C, 2 h
0
35
15
15
0
2.3. Structure–activity relationship (SAR)
60 °C, 2 h
We synthesized molecules with different aromatic and hetero-
aromatic substituents on linker methylene carbon and also chan-
ged two of the acyl groups on aromatic phloroglucinol moieties
with two formyl groups to study the changes in anti-HIV activity.
First, four different variations on phloroglucinol nucleus were
tried. Diisovaleryl, diisobutyryl, diacetyl and formyl-isovaleryl
groups were introduced onto the aromatic nuclei. All the four com-
pounds (14–16 and 32) were inactive in the anti-HIV assay. Next,
different alkyl, aromatic, heteroaromatic were introduced on the
linker methylene bridge. Results indicate that although analogue
50 °C, 2 h
10
25
35
38
MW, 750 W, 10 min
MW, 750 W, 10 min
MW, 750 W, 15 min
a
The yields are reported less than 50% as two equivalents of monomeric starting
material produce 1 equiv of dimeric product.
anti-HIV activity at highest non-toxic concentration. Seven out of
the 21 compounds showed 60% or more inhibition of HIV replica-
O
R
R
O
O
O
R
O
R
O
O
O
HO
R1
OH HO
OH
R1
HO
O
OH
HO
OH
HO
R1
OH
HO
O
OH
HO
HO
OH
R1
OH
O
OH
OH O
OH
OH O
O
OH
OH O
OH
OH O
14: R = R' = CH3
15
16
32
: R = R' = CH2CH(CH3)2
: R = R' = CH(CH3)2
: R = H, R' = CH2CH(CH3)2
17
19
18: R = R1 = CH3
20: R = R1 = CH2CH(CH3)2
R
O
R
O
R
O
R
O
R
O
R
R
O
R
O
O
HO
R1
OHHO
OH
R1
HO
OH
HO
OH
HO
OH
HO
O
HO
OH
R1
HO
OH
R1
OH
R1
R1
R1
R1
O
OH
O
OH
OH O
O
OH
O
OH
OH O
OH O
OH
O
O
Ph
: R = R1 = CH2CH(CH3)2
34: R = H; R1 = CH2CH(CH3)2
26: R = R1 = CH3
OH
: R = R1 = CH3
22
: R = R1 = CH2CH(CH3)2
29
: R = R1 = CH2CH(CH3)2
24
21
23: R = R1 = CH2CH(CH3)2
R
O
R
O
R
O
R
O
R
O
R
O
R
O
R
O
HO
R1
HO
OH
HO
R1
OH
HO
OH
HO
OH
R1
OH
R1
HO
HO
OH
N
OH
R1
HO
OH
R1
R1
R1
O
OH
O
OH
O
OH
OH O
OH O
OH
O
OH
O
OH O
N
O
N
O
25
: R = R1 = CH3
30: R = R1 = CH2CH(CH3)2
31
: R = R1 = CH2CH(CH3)2 28: R = R1 = CH2CH(CH3)2
33: R = H; R1 = CH2CH(CH3)2
27: R = R1 = CH3
Figure 3. Synthesized dimeric phloroglucinol compounds.

2032
S. K. Chauthe et al. / Bioorg. Med. Chem. 18 (2010) 2029–2036
Table 2
ents and linker methylene carbon substituted with bulky aromatic
group were found to be essential for anti-HIV activity.
Anti-HIV activity of phloroglucinol derivatives as analyzed by p24 antigen production
in HIV-1 NL4.3 infected CEM-GFP T cells due to treatment with the compounds
a
b
Code Highest non-toxic
% Inhibition
(p24 assay)
IC50
(lM)
CC50 (lM)
2.4. In vitro HIV-1 reverse transcriptase assay
concentration (lM)
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
AZT
0.23
0.17
0.09
2.17
1.87
9.73
0.71
14.31
0.74
1.45
5.75
35.78
15.06
9.07
7.39
3.75
3.44
2.77
4.10
15.91
7.46
5.00
0
0
0
ND
ND
ND
ND
ND
4.09 0.58
ND
ND
ND
ND
ND
ND
ND
58.93 6.34
ND
ND
Active analogues can inhibit the virus by targeting different
steps in virus life cycle or enzymes like, entry, reverse transcrip-
tase, integrase, protease and viral assembly. Seven dimeric
phloroglucinols showing activity in cell based assay were further
tested for reverse transcriptase inhibition as similar compounds
were earlier shown to be RT inhibitors. Active analogues 19, 22
and 24–28 were evaluated for in vitro reverse transcriptase activity
(Table 3) and Nevirapine a known reverse transcriptase inhibitor
was used as positive standard. Analogues 24 and 25 showed
87.75% and 80.46% inhibition of reverse transcriptase enzyme,
respectively, while 26 and 27 showed weak inhibition of reverse
transcriptase suggesting multiple modes of inhibition. The most
17.5 0.09
16 0.1
78.5 5.7
15 0.08
56 6.8
66.5 8.2
15.6 0.3
70.0 3.9
71.1 4.6
73 11.4
60.6 7.5
64.7 12.8
56.8 7.3
30.1 11.6
13.9 0.05
0
0.28 0.07
ND
2.71 0.49
3.15 0.77
ND
24.00 4.13
18.59 1.97 93.35 4.1
6.75 0.33
6.06 0.63
3.37 0.79
ND
ND
ND
ND
ND
ND
1.05 0.07
61.18 6.12
22.07 3.9
38.05 3.64
ND
ND
ND
ND
ND
ND
24.06 0.63
active compound 22 showed very weak activity at 20
lg/mL con-
centration indicating an alternative mechanism of viral inhibition.
3. Conclusion
26.9 0.02
0
89.75 7.07
Twenty-one dimeric phloroglucinols have been biomimetically
synthesized and screened for in vitro anti-HIV assay. Structure–
activity relationships for HIV-1 inhibition of this class of com-
pounds were elucidated. Compounds 19, 22 and 24–28 exhibited
moderate to good anti-HIV activity in HIV-1 NL4.3 infected CEM-
GFP T cells (Table 2). Seven dimeric phloroglucinols showing activ-
ities in initial assays were further screened for inhibition of reverse
transcriptase enzyme. Compound 22 which is diisovaleryl phloro-
glucinol dimer with phenyl substituent at methylene bridge was
found to be the most active in HIV-1 NL4.3 infected CEM-GFP T cells
a
IC50 = concentration of compound to achieve 50% inhibition of infected cells
(n = 3).
CC50 = concentration of compound indicating cytotoxicity against noninfected
cells (n = 3); ND = not determined.
b
25 was least toxic with CC50 of 93.35 lM, it exhibited weaker activ-
ity. Among the seven active compunds, 22 with phenyl group on
linker methylene and two diisovaleryl phloroglucinol moieties
was found to be most active having an IC50 of 0.28
lM. This com-
having an IC50 of 0.28
than AZT in the cell based assay. This compound showed a CC50
of 3.15 M indicating a good safety index for further analysis as
lM, thus showing better inhibitory activity
pound showed a CC50 of 3.15 M indicating a good safety index for
l
further analysis as a potential lead molecule. Activity and cytotox-
icity was diminished when phenyl moiety of 22 was substituted
with hydroxyl group at para position as observed in 23. Good activ-
ity was observed in 25–28 where phenyl moiety was replaced with
heteroaromatic moiety like quinoline-4-yl, furan-2-yl, 4-N, N-di-
methyl phenyl-1-yl, and pyridine-2-yl. Though these compounds
showed weaker activity compared to 22, but they have better
safety index. Compounds 14–16 and 32 which are devoid of substi-
tution on linker methylene carbon were inactive and highly toxic
to the cells. Replacing acyl functionality in 24 and 28 with formyl
group also resulted in loss of activity as observed in 34 and 33,
respectively. Dimeric phloroglucinols with directly attached aro-
matic ring or bulky alkyl substitution on linker methylene carbon
exhibited better anti-HIV activity as compared to simple aliphatic
substituents.
l
a potential lead molecule. Analogues 24 and 25 showed 87.75
and 80.46% inhibition of reverse transcriptase, respectively, while
the most active compound 22 showed very weak inhibition of
RTase suggesting an alternate mechanism of inhibition. It is also
possible that these compounds get modified inside the cell to be-
come potent RT inhibitor as is the case with AZT, which needs to
be phosphorylated to become active.29 It is suggested that few of
these dimeric phloroglucinol compounds, especially 22 could be
used as a lead molecule for further development of new anti-HIV
therapeutic molecule.
4. Experimental
SAR study established the basic pharmacophore responsible for
activity (Fig. 4). Dimeric phloroglucinols with four acyl substitu-
Melting points were recorded on capillary melting point appa-
ratus and are uncorrected. 1H NMR spectra were recorded on
300 MHz Bruker FT-NMR (Avance DPX300) spectrometer using tet-
Table 3
If R = H, loss of activity
Reverse transcriptase inhibitor activity due to treatment with the compounds
a
Code
Concentration
g/reactions)
% Reverse transcriptase
inhibition
EC50
(
lM)
O
R
R
O
(l
HO
R'
OHHO
R''
OH
R'
19
22
24
25
26
27
28
20
20
20
20
20
20
20
0.1
0
ND
ND
4.61 0.7
87.75 4.65
80.46 1.06
16.22 0.1
13.39 0.06
0
O
OH
OH O
1.035 0.012
0.47 0.0.04
ND
R'' = Phenyl functionality
(Ideal substituent)
R, R' = CH2CH(CH3)2
Isovaleryl functionality
(Ideal chain length)
ND
ND
Bulky aromatic or
heteroaromatic
substituents
Nevirapine
95.5 0.1
0.0036 0.0001
a
EC50 = concentration of compound to achieve 50% inhibition of reverse trans-
criptase (n = 3); ND = not determined.
Figure 4. Structure–activity relationship of dimeric phloroglucinols.

S. K. Chauthe et al. / Bioorg. Med. Chem. 18 (2010) 2029–2036
2033
ramethylsilane as internal standard and the chemical shifts are re-
ported in d units. Mass spectra were recorded on either GC–MS
(Shimadzu QP 5000 spectrometer) auto sampler/direct injection
(EI/CI) or LC–MS (APCI/ ESI). Domestic microwave oven (Whirlpool,
Sweden, Model: MT-243) was used to carry out microwave heated
reactions. All chromatographic purifications were performed with
silica gel (60–120 mesh), whereas all TLC (silica gel) development
was performed on silica gel coated (Merck Kiesel 60F254, 0.2 mm
thickness) sheets. All chemicals were purchased from Sigma-Al-
drich, SD fine chemicals, Lancaster and CDH. Solvents used for
the chemical synthesis purchased from commercial sources were
of analytical grade and were used without further purification un-
less otherwise stated.
1618, 1581, 1462, 1404, 1259, 1046 cmꢀ1
;
1H NMR (CDCl3,
300 MHz): d 17.73 (s, 1H, OH), 17.49 (s, 1H, OH), 16.25 (s, 2H,
2 ꢁ OH), 10.78 (s, 1H, OH), 9.95 (s, 1H, OH), 4.42 (t, J = 7.2 Hz,
1H), 2.76 (s, 6H), 2.73 (s, 6H), 2.31 (m, 2H), 0.88 (t, J = 7.1 Hz,
3H); 13C NMR (CDCl3, 75 MHz): d 205.7, 171.2, 166.4, 164.5,
107.5, 106.4, 104.2, 34.2, 33.0, 32.4, 24.3, 14.1; CIMS: m/z 461
[M+1]+, 251 [MꢀC10H10O5]. Anal. Calcd for C23H24O10 (460.1): C,
60.00; H, 5.25. Found: C, 59.89; H, 5.19.
4.1.5. 1-[3-Acetyl-5-[(3,5-diacetyl-2,4,6-trihydroxyphenyl)-2-
phenylethyl-methyl]-2,4,6-trihydroxyphenyl]ethanone (18)
Yield: 33%; cream colored solid; mp 188–190 °C; UV (CHCl3):
kmax (log
e
) 272 (4.42), 341 (3.78); IR (KBr): mmax 3204, 1615,
1578, 1473, 1417, 1363, 1320, 1264 1109 cmꢀ1
;
1H NMR (CDCl3,
4.1. General method for synthesis of dimeric phloroglucinols
14–20
300 MHz): d 17.86 (s, 1H, OH), 17.46 (s, 1H, OH), 16.25 (s, 2H,
2 ꢁ OH), 10.86 (s, 1H, OH), 9.90 (s, 1H, OH), 7.26–7.12 (m, 3H),
7.08 (d, J = 6.8 Hz, 2H), 4.53 (t, J = 7.1 Hz, 1H), 2.76 (s, 6H), 2.72
(s, 6H), 2.56 (m, 4H); 13C NMR (CDCl3, 75 MHz): d 205.7, 171.3,
169.4, 168.5, 166.4, 141.5, 129.0, 128.9, 126.6, 105.0, 104.7,
104.5, 35.7, 34.1, 32.9, 32.7, 29.9; CIMS: m/z 537 [M+1]+, 327
[MꢀC10H10O5]. Anal. Calcd for C29H28O10 (536.2): C, 64.92; H,
5.26. Found: C, 65.01; H, 5.18.
The mixture of diacyl phloroglucinol (7 or 8 1 mmol) and alde-
hyde (0.5 mmol) was irradiated with microwave radiations
(750 W) in domestic microwave oven for 10–15 min. On cooling,
the reaction mixture was diluted with chloroform. The resultant
mixture was washed with water and brine solution and finally
dried over anhydrous sodium sulfate. Solvent was removed under
vacuum and the crude product was purified by silica gel (# 60–
120) column chromatography using hexane–EtOAc as eluent.
4.1.6. 1-[3-Acetyl-5-[(3,5-diacetyl-2,4,6-trihydroxyphenyl)-
cyclohexyl-methyl]-2,4,6-trihydroxyphenyl]ethanone (19)
Yield: 35%; cream colored solid; mp 196–198 °C; UV (CHCl3):
4.1.1. Methylene-bis-(3,5-diacetyl-2,4,6-trihydroxy benzene)
(14)
kmax (log
e
) 276 (4.29), 341 (3.59); IR (KBr): mmax 3173, 2915,
2849, 1617, 1583, 1473, 1406, 1366, 1259, 1167, 1108 cmꢀ1
;
1H
Yield: 35%; cream colored solid; mp 278–280 °C; UV (CHCl3):
NMR (CDCl3, 300 MHz): d 17.78 (s, 1H, OH), 17.46, s, 1H, OH),
16.25 (s, 2H, 2 ꢁ OH), 10.86 (s, 1H, OH), 9.96 (s, 1H, OH), 4.17 (d,
J = 10.9 Hz, 1H), 2.76 (s, 6H), 2.72 (s, 6H), 1.64 (m, 1H), 1.26–1.10
(m, 6H), 0.92–0.70 (m, 4H); 13C NMR (CDCl3, 75 MHz): d 205.6,
205.2, 169.7, 167.4, 167.1, 165.7, 165.2, 106.4, 106.0, 105.2,
103.9, 36.7, 36.2, 33.8, 32.8, 32.4, 26.3, 26.0; CIMS: m/z 515
[M+1]+, 305 [MꢀC10H10O5]. Anal. Calcd for C27H30O10 (514.2): C,
63.03; H, 5.88. Found: C, 62.98; H, 5.72.
kmax (log
e
) 275 (4.34), 336 (3.88); IR (KBr): mmax 3201, 1616,
1594, 1477, 1424, 1370, 1268, 1181, 1132, 1112, 1022 cmꢀ1
;
1H
NMR (CDCl3, 300 MHz): d 17.38 (s, 2H, 2 ꢁ OH), 16.22 (s, 2H,
2 ꢁ OH), 10.23 (s, 2H, 2 ꢁ OH), 3.74 (s, 2H), 2.76 (s, 6H), 2.73 (s,
6H); 13C NMR (CDCl3, 75 MHz): d 205.8, 171.5, 169.1, 166.5,
106.6, 105.8, 104.9, 34.1, 33.0, 15.6; EIMS: m/z 432 [M]+, 223,
210, 195, 177, 85, 69, 67.
4.1.2. Methylene-bis-(3,5-di-isopentanoyl-2,4,6-
trihydroxybenzene) (15)
4.1.7. 1-[3-Isopentanoyl-5-[(3,5-diisopentanoyl-2,4,6-
trihydroxyphenyl)-phenyl-ethyl-methyl]-2,4,6-
Yield: 38%; yellow solid; mp 158–160 °C; UV (CHCl3): kmax (log
282 (4.44), 339 (3.97); IR (KBr): max 3201, 2959, 2872, 1622, 1545,
1470, 1427, 1367, 1302, 1204, 1170, 1131, 1075 cmꢀ1 1H NMR
(CDCl3, 300 MHz): d 17.64 (s, 2H, 2 ꢁ OH), 16.45 (s, 2H, 2 ꢁ OH),
10.32 (s, 2H, 2 ꢁ OH), 3.71 (s, 2H), 3.04 (d, J = 6.4 Hz, 8H), 2.27 (m,
4H), 1.00 (d, J = 6.4 Hz, 24H); 13C NMR (CDCl3, 75 MHz): d 208.3,
207.8, 171.3, 168.5, 165.5, 105.3, 105.1, 104.7, 53.7, 52.9, 25.6,
23.3, 16.0; CIMS: m/z 601 [M+1]+, 307 (MꢀC16H22O5), 295. Anal.
Calcd for C33H44O10 (600.3): C, 65.98; H, 7.38. Found: C, 65.81; H,
7.45.
e
)
trihydroxyphenyl]-3-methylbutan-1-one (20)
m
Yield: 32%; yellow solid; mp 138–140 °C; UV (CHCl3): kmax
;
(log
e
) 285 (4.55), 341 (4.05); IR (KBr): mmax 3202, 2958, 1615,
1580, 1469, 1392, 1367, 1299, 1201, 1128, 1054 cmꢀ1
;
1H NMR
(CDCl3, 300 MHz): d 18.13 (s, 1H, OH), 17.76 (s, 1H, OH), 16.46 (s,
2H, 2 ꢁ OH), 10.96 (s, 1H, OH), 10.03 (s, 1H, OH), 7.26–7.12 (m,
3H), 7.08 (d, J = 7.6 Hz, 2H), 4.54 (t, J = 7.1 Hz, 1H), 3.05 (d,
J = 6.5 Hz, 8H), 2.31 (m, 4H), 2.27 (m, 4H), 1.01 (d, J = 6.4 Hz,
24H); 13C NMR (CDCl3, 75 MHz): d 208.7, 208.0, 171.2, 169.7,
168.8, 166.2, 166.0, 141.7, 128.9, 126.5, 108.0, 105.7, 104.7, 53.9,
52.9, 35.8, 32.8, 30.3, 25.7, 23.3; CIMS: m/z 411 [MꢀC10H10O5].
Anal. Calcd for C41H52O10 (704.4): C, 69.86; H, 7.44. Found: C,
69.75; H, 7.53.
4.1.3. Methylene-bis-(3,5-di-isobutanoyl-2,4,6-
trihydroxybenzene) (16)
Yield: 34%; light orange solid; mp 200–202 °C; UV (CHCl3): kmax
(log
e
) 272 (4.33), 341 (3.73); IR (KBr): mmax 3188, 2970, 1618,
4.2. General method for synthesis of dimeric phloroglucinols
21–31
1467, 1382, 1273, 1211, 1160, 1065; 1H NMR (CDCl3, 300 MHz):
d 17.73 (s, 2H, 2 ꢁ OH), 16.51 (s, 2H, 2 ꢁ OH), 10.39 (s, 2H,
2 ꢁ OH), 4.04 (m, 4H), 3.76 (s, 2H), 1.22 (d, J = 5.9 Hz, 24H); 13C
NMR (CDCl3, 75 MHz): d 213.1, 212.8, 171.3, 168.8, 165.2, 105.3,
104.4, 103.8, 40.1, 39.7, 20.0, 19.6, 16.2; CIMS: m/z 545 [M+1]+,
279, 267. Anal. Calcd for C29H36O10 (544.2): C, 63.96; H, 6.66.
Found: C, 64.06; H, 6.78.
To the solution of diacyl phloroglucinols (7, 8 1 mmol) in chlo-
roform, p-TsCl and aldehyde (0.5 mmol) was added and the reac-
tion mixture was irradiated with microwave radiations (750 W)
in domestic microwave oven for 10–15 min. Remaining procedure
is same as given for 14–20.
4.1.4. 1-[3-Acetyl-5-[(3,5-diacetyl-2,4,6-trihydroxyphenyl)
ethylmethyl]-2,4,6-trihydroxyphenyl]ethanone (17)
Yield: 45%; yellow solid; mp 178–180 °C; UV (CHCl3): kmax
4.2.1. 1-[3-Acetyl-5-[(3,5-diacetyl-2,4,6-trihydroxyphenyl)-4-
hydroxyphenyl-methyl]-2,4,6-trihydroxyphenyl]ethanone (21)
Yield: 50%; light brown solid; mp 110–112 °C; UV (CHCl3): kmax
(log
e) 272 (4.27), 336 (3.50); IR (KBr): mmax 3437, 2928, 1697,
(log e) 272 (4.20), 339 (3.31); IR (KBr): mmax 3249, 2928, 1615,

2034
S. K. Chauthe et al. / Bioorg. Med. Chem. 18 (2010) 2029–2036
1511, 1425, 1366, 1266, 1111 cmꢀ1
;
1H NMR (CDCl3, 300 MHz): d
4.2.6. 1-[3-Acetyl-5-[(3,5-diacetyl-2,4,6-trihydroxyphenyl)-
furan-2-yl-methyl]-2,4,6-trihydroxyphenyl]ethanone (26)
Yield: 42%; light green solid; mp 143–145 °C; UV (CHCl3): kmax
17.63 (s, 2H, 2 ꢁ OH), 16.36 (s, 2H, 2 ꢁ OH), 10.18 (s, 2H,
2 ꢁ OH), 6.95 (d, J = 7.8 Hz, 2H), 6.76 (d, J = 7.9 Hz, 2H), 6.09 (s,
1H), 5.11 (br s, 1H), 2.78 (s, 6H), 2.72 (s, 6H); 13C NMR (CDCl3,
75 MHz): d 205.5, 205.3, 171.0, 168.6, 166.0, 154.2, 132.4, 127.8,
115.3, 106.1, 105.3, 104.3, 33.5, 32.4, 29.7; CIMS: m/z 315
[MꢀC10H10O5]. Anal. Calcd for C27H24O11 (524.1): C, 61.83; H,
4.61. Found: C, 61.91; H, 4.56.
(log
e
) 279 (4.34), 336 (3.80); IR (KBr): mmax 3216, 1618, 1580,
1412, 1364, 1261, 1185, 1114, 1020 cmꢀ1
;
1H NMR (CDCl3,
300 MHz): d 17.61 (s, 2H, 2 ꢁ OH), 16.34 (s, 2H, 2 ꢁ OH), 10.13 (s,
2H, 2 ꢁ OH), 7.31 (s, 1H), 6.32 (s, 1H), 6.00 (s, 2H), 2.76 (s, 6H),
2.73 (s, 6H); 13C NMR (CDCl3, 75 MHz): d 206.0, 205.6, 171.7,
168.9, 166.1, 151.0, 142.3, 110.8, 107.3, 105.7, 105.3, 104.8, 34.0,
33.9, 29.6; CIMS: m/z 499 [M+1]+, 289 [M-C10H10O5]. Anal. Calcd
for C25H22O11 (498.1): C, 60.24; H, 4.45. Found: C, 60.12; H, 4.57.
4.2.2. 1-[3-Isopentanoyl-5-[(3,5-diisopentanoyl-2,4,6-
trihydroxyphenyl)-phenyl-methyl]-2,4,6-trihydroxyphenyl]-3-
methylbutan-1-one (22)
4.2.7. 1-[3-Acetyl-5-[(3,5-diacetyl-2,4,6-trihydroxyphenyl)-4-
N,N-dimethyl-aminophenyl-methyl]-2,4,6-
trihydroxyphenyl]ethanone (27)
Yield: 43% yellow solid; mp 180–182 °C; UV (CHCl3): kmax
(log
e
) 272 (4.52), 340 (3.70); IR (KBr): mmax 2957, 1685, 1617,
1583, 1454, 1424, 1326, 1293, 1186, 1128, 1070, 1027 cmꢀ1
;
1H
Yield: 48%; white solid; mp 196–198 °C; UV (CHCl3): kmax (log
288 (4.44); IR (KBr): mmax 2930, 1621, 1596, 1509, 1424, 1364,
1271, 1183, 1107 cmꢀ1 1H NMR (pyridine-d5, 300 MHz): d 8.94
e)
NMR (CDCl3, 300 MHz): d 17.90 (s, 2H, 2 ꢁ OH), 16.55 (s, 2H,
2 ꢁ OH), 10.26 (s, 2H, 2 ꢁ OH), 7.30–7.23 (m, 3H), 7.10 (d,
J = 7.5 Hz, 2H), 6.16 (s, 1H), 3.06 (d, J = 16.3 Hz, 8H), 2.26 (m, 4H),
1.00 (d, J = 6.4 Hz, 24H); 13C NMR (CDCl3, 75 MHz): d 208.3,
171.5, 169.5, 166.2, 137.2, 128.9, 127.1, 126.9, 107.0, 105.8,
104.9, 53.9, 52.9, 30.2, 25.7, 23.3; CIMS: m/z 384 [MꢀC10H10O5].
Anal. Calcd for C39H48O10 (676.3): C, 69.21; H, 7.15. Found: C,
69.10; H, 7.28.
;
(d, J = 3.9 Hz, 1H), 8.24 (m, 4H), 7.08 (d, J = 8.4 Hz, 1H), 4.62 (s,
1H), 2.79 (s, 6H), 2.71 (s, 6H); 13C NMR (pyridine-d5, 75 MHz): d
205.1, 170.5, 131.3, 130.8, 128.4, 126.4, 118.4, 106.8, 34.0, 33.5,
33.3; EIMS: 559 [M]+, 348, 209. Anal. Calcd for C30H25NO10
(559.2): C, 64.40; H, 4.50; N, 2.50. Found: C, 64.51; H, 4.63; N, 2.39.
4.2.8. 1-[3-Isopentanoyl-5-[(3,5-diisopentanoyl-2,4,6-
trihydroxyphenyl)-pyridin-2-yl-methyl]-2,4,6-
trihydroxyphenyl]-3-methylbutan-1-one (28)
4.2.3. 1-[3-Isopentanoyl-5-[(3,5-diisopentanoyl-2,4,6-
trihydroxyphenyl)-4-hydroxyphenyl-methyl]-2,4,6-
trihydroxyphenyl]-3-methylbutan-1-one (23)
Yield: 46%; yellow solid; mp 190–192 °C; UV (CHCl3): kmax
Yield: 50%; brown oil; UV (CHCl3): kmax (log
(3.72); IR (Neat): mmax 3433, 2978, 1756, 1698, 1615, 1581, 1510,
1456, 1298, 1196, 1123, 1092 cmꢀ1 1H NMR (CDCl3, 300 MHz): d
e) 280 (4.51), 340
(log
e
) 282 (4.53), 340 (3.95); IR (KBr): mmax 3436, 2957, 2870,
1618, 1545, 1458, 1366, 1302, 1202, 1130, 1063 cmꢀ1
;
1H NMR
;
(CDCl3, 300 MHz): d 16.63 (s, 2H, 2 ꢁ OH), 8.42 (d, J = 5.1 Hz, 1H),
8.09 (t, J = 7.4 Hz, 1H), 7.89 (d, J = 8.1 Hz, 1H), 7.57 (t, J = 9.2 Hz,
1H), 7.07 (s, 1H), 3.15–2.84 (m, 8H), 2.21 (m, 4H), 0.97 (d,
J = 6.6 Hz, 24H); 13C NMR (CDCl3, 75 MHz): d 208.0, 206.9, 173.1,
170.8, 162.4, 144.2, 140.7, 127.4, 123.7, 106.6, 105.7, 104.8, 53.3,
34.3, 34.3, 26.0, 23.5, 23.3; CIMS: m/z 384 [MꢀC10H10O5]. Anal.
Calcd for C38H47NO10 (677.3): C, 67.34; H, 6.99; N, 2.07. Found:
C, 67.47; H, 6.89; N, 1.97.
17.87 (br s, 2H, 2 ꢁ OH), 16.53 (s, 2H, 2 ꢁ OH), 10.24 (br s, 2H,
2 ꢁ OH), 6.96 (d, J = 8.1 Hz, 2H), 6.77 (d, J = 8.4 Hz, 2H), 6.08 (s,
1H), 3.05 (d, J = 10.6 Hz, 8H), 2.27 (m, 4H), 0.99 (d, J = 6.7 Hz, 24
H); 13C NMR (CDCl3, 75 MHz): d 208.4, 171.5, 169.5, 166.2, 154.8,
128.8, 128.4, 114.6, 106.9, 105.8, 104.9, 52.6, 52.9, 25.8, 23.3,
14.7; CIMS: m/z 400 [MꢀC10H10O5]. Anal. Calcd for C39H48O11
(692.3): C, 67.61; H, 6.98. Found: C, 67.49; H, 7.07.
4.2.9. 1-[3-Isopentanoyl-5-[(3,5-diisopentanoyl-2,4,6-
trihydroxyphenyl)-furan-2-yl-methyl]-2,4,6-trihydroxyphenyl]-
3-methylbutan-1-one (29)
4.2.4. 1-[3-Isopentanoyl-5-[(3,5-diisopentanoyl-2,4,6-
trihydroxyphenyl)-4-benzyloxyphenyl-methyl]-2,4,6-
trihydroxyphenyl]-3-methylbutan-1-one (24)
Yield: 48%; yellow oil; UV (CHCl3): kmax (log e) 288 (4.54), 340
Yield: 42%; brown crystals; mp 152–154 °C; UV (CHCl3): kmax
(3.98); IR (Neat): mmax 3437, 2958, 1618, 1466, 1299, 1118,
(log
e
) 272 (3.24), 340 (3.72); IR (KBr): mmax 3253, 2965, 1750,
1046 cmꢀ1
;
1H NMR (CDCl3, 300 MHz): d 17.88 (s, 2H, 2 ꢁ OH),
1652, 1620, 1171, 1120, 1095 cmꢀ1
;
1H NMR (CDCl3, 300 MHz): d
16.56 (s, 2H, 2 ꢁ OH), 10.19 (s, 2H, 2 ꢁ OH), 7.32 (s, 1H), 6.33 (d,
J = 2.0 Hz, 1H), 6.01 (s, 1H), 5.80 (s, 1H), 3.03 (m, 8H), 2.26 (m,
4H), 1.00 (d, J = 3.2 Hz, 24H); 13C NMR (CDCl3, 75 MHz): d 208.4,
208.1, 171.7, 169.2, 165.8, 151.3, 142.2, 110.8, 107.3, 105.2,
104.8, 95.9, 53.8, 53.3, 52.9, 30.2, 29.9, 25.7, 23.3; CIMS: m/z 374
[MꢀC10H10O5]. Anal. Calcd for C37H46O11 (666.3): C, 66.65; H,
6.95. Found: C, 66.80; H, 7.08.
17.87 (br s, 2H, 2 ꢁ OH), 16.53 (s, 2H, 2 ꢁ OH), 10.23 (br s, 2H,
2 ꢁ OH), 7.43–7.35 (m, 5H), 6.99 (d, J = 8.7 Hz, 2H), 6.88 (d,
J = 8.7 Hz, 2H), 6.09 (s, 1H), 5.04 (s, 1H), 3.05 (d, J = 16.5 Hz, 8H),
2.27 (m, 4H), 0.98 (d, J = 6.7 Hz, 24 H); 13C NMR (CDCl3, 75 MHz):
d 208.3, 208.1, 171.5, 169.4, 166.1, 157.9, 137.5, 129.5, 129.1,
128.5, 128.2, 128.0, 115.1, 106.8, 105.7, 104.8, 70.5, 53.8, 52.9,
33.2, 25.7, 23.3; EIMS: m/z 489.13 [M-C16H22O5]; 295.13. Anal.
Calcd for C46H54O11 (782.9): C, 70.57; H, 6.95. Found: C, 70.52; H,
7.26.
4.2.10. 1-[3-Isopentanoyl-5-[(3,5-diisopentanoyl-2,4,6-
trihydroxyphenyl)-quinolin-4-yl-methyl]-2,4,6-
trihydroxyphenyl]-3-methylbutan-1-one (30)
4.2.5. 1-[3-Acetyl-5-[(3,5-diacetyl-2,4,6-trihydroxyphenyl)-
quinolin-4-yl-methyl]-2,4,6-trihydroxyphenyl]ethanone (25)
Yield: 48%; cream colored solid; mp 200–202 °C; UV (CHCl3):
kmax (log
e
) 276 (4.65), 340 (4.02); IR (KBr): mmax 2957, 2870,
2545, 1615, 1418, 1367, 1301, 1198, 1127 cmꢀ1
;
1H NMR (CDCl3,
Yield: 48%; white solid; mp 196–198 °C; UV (CHCl3): kmax (log
288 (4.44); IR (KBr): mmax 2930, 1621, 1596, 1509, 1424, 1364,
1271, 1183, 1107 cmꢀ1 1H NMR (pyridine-d5, 300 MHz): d 8.94
e)
300 MHz): d 16.49 (br s, 3H, 3 ꢁ OH), 16.16 (s, 2H, 2 ꢁ OH), 13.84
(br s, 1H, OH), 8.25 (s, 2H), 7.76–7.48 (m, 4H), 7.26 (s, 1H), 3.04
(br s, 8H), 2.29 (m, 4H), 1.01 (d, J = 4.4 Hz, 24H); 13C NMR (CDCl3,
75 MHz): d 207.1, 172.2, 170.4, 170.1, 140.1, 137.9, 127.7, 120.7,
10.5.1, 104.4, 103.9, 53.4, 34.1, 25.6, 23.4; CIMS: m/z 727 [M+1]+.
Anal. Calcd for C42H49NO10 (727.3): C, 69.31; H, 6.79; N, 1.92.
Found: C, 69.19; H, 6.65; N, 1.79.
;
(d, J = 3.9 Hz, 1H), 8.24 (m, 4H), 7.08 (d, J = 8.4 Hz, 1H), 4.62 (s,
1H), 2.79 (s, 6H), 2.71 (s, 6H); 13C NMR (pyridine-d5, 75 MHz): d
205.1, 170.5, 131.3, 130.8, 128.4, 126.4, 118.4, 106.8, 34.0, 33.5,
33.3; EIMS: 559 [M]+, 348, 209. Anal. Calcd for C30H25NO10
(559.2): C, 64.40; H, 4.50; N, 2.50. Found: C, 64.51; H, 4.63; N, 2.39.

S. K. Chauthe et al. / Bioorg. Med. Chem. 18 (2010) 2029–2036
2035
4.2.11. 1-[3-Isopentanoyl-5-[(3,5-diisopentanoyl-2,4,6-
trihydroxyphenyl)-3,4-methylenedioxyphenyl -methyl]-2,4,6-
trihydroxyphenyl]-3-methylbutan-1-one (31)
2H), 3.03 (d, 3.6 Hz 4H), 2.22–2.31 (m, 2H), 1.00 (d, J = 6.5 Hz,
12H); 13C NMR (CDCl3, 75 MHz): d 208.11, 194.0, 171.4, 169.4,
166.0, 158.0, 137.4, 129.1, 128.7, 128.5, 128.2, 128.0, 115.3,
107.2, 105.6, 104.3, 70.6, 53.9, 52.6, 32.5, 25.6, 23.3; CIMS: m/z
433.17, 238.1 [MꢀC12H13O5]. Anal. Calcd for C38H38O11 (670.70):
C, 68.05; H, 5.71. Found: C, 67.22; H, 6.05.
Yield: 45%; yellow crystalline solid; mp 172–174 °C; UV
(CHCl3): kmax
267 (2.94); IR (KBr): mmax 3201, 2958, 1614,
(log e)
1583, 1504, 1487, 1367, 1200, 1127, 1041 cmꢀ1 1H NMR (CDCl3,
;
300 MHz): d 17.9 (br s, 2H, 2 ꢁ OH), 16.56 (s, 2H, 2 ꢁ OH), 10.24
(br s, 2H, 2 ꢁ OH), 6.71 (d, J = 8.1 Hz, 1H), 6.58 (s, 1H), 6.55 (d,
J = 8.7 Hz, 1H), 6.07 (s, 1H), 5.94 (s, 1H), 3.04 (d, J = 14.4 Hz, 8H),
2.26 (m, 4H), 0.99 (d, J = 6.7 Hz, 24 H); 13C NMR (CDCl3, 75 MHz):
d 208.4, 208.1, 171.5, 169.3, 166.1, 148.5, 146.6, 131.4, 119.9,
108.4, 107.9, 106.7, 105.7, 104.8, 101.6, 53.8, 52.8, 33.6, 25.7,
23.3 Maldi Tof: m/z 720.47. Anal. Calcd for C40H48O12 (720.31): C,
66.65; H, 6.71; O, 26.64. Found: C, 66.52; H, 6.63.
4.4. Cell cytotoxicity assay using MTT
Cytotoxicity of potential candidates was assayed using MTT Kit
(Roche, Germany) in the CEM-GFP cell line, according to the man-
ufacturer’s protocol. Briefly, 2 ꢁ 104 cells/well were seeded in 96-
well plate; samples were then added into the wells at different
concentrations keeping untreated or vehicle treated wells as con-
trols. After 48–72 h incubation, 10 lL of MTT reagent (5 mg/mL)
4.3. General method for synthesis of dimeric phloroglucinols
32–34
were added in to the wells to allow the reaction. Formazan crystals
produced during the reaction were solubilized and color develop-
ment was read at 540 nm using microplate ELISA reader.26
To the solution of 3-formyl phloroisovalerophenone (13,
1 mmol) in chloroform, p-TsCl and aldehyde (0.5 mmol) was added
and the reaction mixture was irradiated with microwave radia-
tions (750 W) in domestic microwave oven for 10–15 min. On cool-
ing, the reaction mixture was diluted with chloroform. The
resultant mixture was washed with water and brine solution and
finally dried over anhydrous sodium sulfate. Solvent was removed
under vacuum and the crude product was purified by reverse
phase silica gel column chromatography using MeOH and water
as eluent.
4.5. Anti-HIV screening in CEM-GFP cells
Human CD4+ T cell line, CEM-GFP cells were infected with HIV-
1 NL4.3 virus at a multiplicity of infection (MOI) of 0.05 using the
standard protocol previously described from the laboratory.28
The infection was monitored by GFP visualization under a fluores-
cence microscope. The cells were then incubated with samples for
up to 9 days post infection. Preliminary screening was performed
by GFP quantitation by microfluorometry. Virus production was
assayed in the culture supernatant on day-7 post infection by
p24 antigen capture ELISA assay (Perkin–Elmer, USA).
4.3.1. Methylene-bis-(3-formyl-5-isopentanoyl-2,4,6-
trihydroxybenzene) (32)
Yield: 30%; cream colored solid; mp 228–230 °C; UV (CHCl3):
kmax (log
e
) 284 (4.33); IR (KBr): mmax 3373, 2921, 1621, 1474,
4.6. In vitro HIV-1 reverse transcriptase assay
1399, 1184, 1048 cmꢀ1
;
1H NMR (CDCl3, 300 MHz): d 17.65 (s,
2H, 2 ꢁ OH), 14.51 (s, 2H, 2 ꢁ OH), 10.15 (s, 2H, 2 ꢁ OH), 9.78 (s,
2H, 2 ꢁ CHO), 3.72 (s, 2H), 3.03 (d, J = 6.6 Hz, 4H), 2.26 (m, 2H),
1.01 (d, J = 6.6 Hz, 12H); 13C NMR (CDCl3, 75 MHz): d 207.6,
193.6, 171.2, 169.4, 165.5, 105.4, 105.1, 103.5, 52.6, 25.7, 23.3,
15.3; CIMS: m/z 489 [M+1]+. Anal. Calcd for C25H28O10 (488.2): C,
61.47; H, 5.78. Found: C, 61.61; H, 5.89.
The reverse transcriptase assay was performed with pure HIV-1
reverse transcriptase using the colorimetric enzyme immunoassay
kit obtained from Roche, Germany, following manufacturer’s pro-
tocol. Nevirapine, a potent non-nucleoside RT inhibitor was used
as a positive control.
Acknowledgment
4.3.2. 1-[3-Formyl-5-[(5-formyl-3-isopentanoyl-2,4,6-
trihydroxyphenyl)-pyridin-2-yl-methyl]-2,4,6-
trihydroxyphenyl]-3-methylbutan-1-one (33)
Authors thank Department of Biotechnology, Ministry of
Science and Technology, Government of India for the financial
support to this project (Grant No. BT/PR7020/Med/14/930/2005).
Yield: 44%; yellow solid; mp 148–149 °C; UV (CHCl3): kmax
(log
e
) 268 (3.40); IR (KBr): mmax; 3470, 2930, 1630, 1545, 1470,
1366, 1302, 1045 cmꢀ1
;
1H NMR (CD3OD + CDCl3, 300 MHz): 9.87
References and notes
(s, 2H), 8.25 (d, J = 5.3 Hz, 1H), 8.20 (t, J = 7.2 Hz, 1H), 7.64 (d,
J = 7.5 Hz, 1H), 7.59 (t, J = 9.0 Hz, 1H), 7.32 (s, 1H), 2.88 (d,
J = 5.4 Hz, 4H), 2.10 (m, 2H), 0.83 (d, J = 6.6 Hz, 12H); 13C NMR
(CD3OD + CDCl3, 75 MHz): d 205.5, 193.4, 190.6, 171.7, 169.8,
160.2, 145.5, 140.0, 126.2, 123.6, 106.2, 104.3, 101.9, 52.32,
29.46, 25.19, 22.37; CIMS: m/z 328.1 [MꢀC12H13O5]. Anal. Calcd
for C30H31NO10 (565.57): C, 67.71; H, 5.52; N, 2.48. Found: C,
67.47; H, 5.57; N, 2.43.
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