Synthesis of diverse analogues of Oenostacin and their antibacterial activities

Article abstract of DOI:10.1016/j.bmc.2006.09.034

Several diverse analogues of Oenostacin, a naturally occurring potent antibacterial phenolic acid derivative, have been synthesized. A small library with more than forty analogues having different aromatic rings and varied side chains has been achieved through solution phase synthesis. Some of these analogues, that is, 22, 23 and 42, possessed potent antibacterial activities against Staphylococcus epidermidis and Staphylococcus aureus having EC₅₀ ranging from 0.49 to 0.67 μM as compared to Oenostacin (EC₅₀ = 0.12 μM).

Full text of DOI:10.1016/j.bmc.2006.09.034

Bioorganic & Medicinal Chemistry 15 (2007) 518–525
Synthesis of diverse analogues of Oenostacin and their
antibacterial activitiesq
Vandana Srivastava, Mahendra P. Darokar, Atiya Fatima, J. K. Kumar,
Chinmay Chowdhury, Hari Om Saxena, Gaurav R. Dwivedi, Kunal Shrivastava,
Vivek Gupta, S. K. Chattopadhyay, Suaib Luqman, M. M. Gupta, Arvind S. Negi*
and Suman P. S. Khanuja
Central Institute of Medicinal and Aromatic Plants (CIMAP), PO CIMAP, Kukrail Picnic Spot Road, Lucknow 226 015, India
Received 21 August 2006; revised 14 September 2006; accepted 16 September 2006
Available online 10 October 2006
Abstract—Several diverse analogues of Oenostacin, a naturally occurring potent antibacterial phenolic acid derivative, have been
synthesized. A small library with more than forty analogues having different aromatic rings and varied side chains has been achieved
through solution phase synthesis. Some of these analogues, that is, 22, 23 and 42, possessed potent antibacterial activities against
Staphylococcus epidermidis and Staphylococcus aureus having EC50 ranging from 0.49 to 0.67 lM as compared to Oenostacin
(EC50 = 0.12 lM).
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
man pathogen and is the predominant cause of
infections associated with indwelling medical devices, as
well as the primary cause of many nosocomial infections.7
Multidrug-resistant gram-positive bacteria have contin-
ued to pose challenges to medicinal research community.1
Oenothera biennis (Oenagraceae) commonly known as
‘Evening Primarose’ is cultivated in Indian gardens.2 A
number of compounds have been isolated from its aerial
parts.3 A potent antibacterial compound Oenostacin4
was isolated during the systematic investigation of its
roots along with several other compounds5 in the recent
past at this institute. Being a phenolic acid derivative with
an aliphatic ester chain Oenostacin, has shown potent
antibacterial activity against Staphylococcus aureus and
Staphylococcus epidermidis. It is known that S. aureus,
one of the most successful opportunistic human Gram-
positive pathogens, is responsible for postoperative
wound infections, bacteraemia, pneumonia, osteomyeli-
tis, mastitis, acute endocarditis, and deep abscesses in
various organs.6 In contrast to S. aureus, infections
caused by S. epidermidis are less acute in nature. Howev-
er, S. epidermidis is now recognized as an important hu-
In view of promising antibacterial activities4 of Oenost-
acin 1, and also in continuation to our research interest
on the synthesis of biologically active gallic acid deriva-
tives,8 we became curious to study the efficacy of various
analogues of 1. Also, this could give us some possible
antibacterial lead compounds of pharmacological spec-
trum of Oenostacin analogues and related compounds
to evaluate their biological activities against S. epidermi-
dis and S. aureus. Some of the synthesized compounds
exhibited very good antibacterial activities as given in
Figure 1 with their EC50 values in parentheses.
2. Results and discussion
2.1. Chemistry
Different analogues were synthesized as described in
Schemes 1–5. In Scheme 1, orcinol 2, used as starting
material, underwent Reimer–Tiemann reaction with
CHCl3–aqueous alkali system to yield the aldehyde 4,
which upon methylation with dimethyl sulfate in ace-
tone gave 2,6-dimethoxy-4-methyl benzaldehyde (5).
Keywords: Oenostacin; Antibacterial; Analogue synthesis; Phenolic
acids.
q
CIMAP Communication No. 2006-29J.
Corresponding author. Tel.: +91 522 2717529x327; fax: +91 522
2342666; e-mail: arvindcimap@rediffmail.com
*
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.09.034

V. Srivastava et al. / Bioorg. Med. Chem. 15 (2007) 518–525
519
OH
OH
OH
3
O
1'
3'
5'
OH
2
1
CHO
OH
OMe
4
OH
COOH
HO
H3C
HOOC
6
22: (0.67μM)
1: (EC50=0.12μM)
4:(1.64μM)
O
O
OH
OH
OH O
HO
HO
OH
HO
HO
OH
OH
OH
23: (0.99μM)
26: (1.04μM)
42:( 0.63μM)
Figure 1. Oenostacin and some of the active analogues against S. epidermidis.
O
OMe
OMe
OH
OH
4
OH
CHO
OH
CHO
OMe
v
ii
i
OMe
vii
OH
H3C
H3C
H3C
H3C
6
5
2
iii
vi
iv
O
OH
OMe
OMe
3
O
OMe
OH
OH
OMe
H3C
CH3
MeO
H3C
OMe
H3C
8
7
viii
O
OMeO
OH
OMe
H3C
9
Scheme 1. Reagents and conditions: (i) CHCl3–aq KOH (30%), reflux, 4 h, 14%; (ii) Me2SO4, K2CO3, dry acetone, reflux, 3 h, 87%; (iii) Me2SO4,
K2CO3, acetone, reflux, 3 h, 93%; (iv) DMF, POCl3, 0 ꢁC for 30 min then rt for 3 h, 62%; (v) DMSO, NaOH, (Ph3P+@CHCOOC2H5)BrÀ, rt,
overnight, 32%; (vi) 1% ethanolic KOH, acetone, rt, 50–60 h, 62%; (vii) BBr3, DCM, À78 ꢁC for 1 h then rt, 42%; (viii) glutaric anhydride, DCM,
AlCl3, rt, overnight, 24%.
Due to poor yields in this approach, the reaction pathway
was modified through methylation of orcinol followed by
Vilsmeier–Haack formylation9 using dimethylformam-
ide and phosphorus oxychloride (DMF–POCl3) to ob-
tain 5 in good yield (62%). Wittig reaction of 5 with
triphenyl phosphonium ethyl acetate bromide in DMSO
and sodium hydroxide at room temperature yielded the
corresponding cinnamic acid derivative 6. Later, it was
demethylated by stirring with boron tribromide in dichlo-
romethane to get 8 in 42% yield.
dichloromethane gave a mixture of products keto (12)
and enol lactone (13), which were carefully separated
by silica gel column chromatography. Compound 12
was demethylated with anhydrous aluminium chloride
in dichloromethane affording deprotected products 14,
15 and 16 in 2:3:1 ratio. The keto acid 12 on reacting
with sodium borohydride in trifluoroacetic acid (TFA)
yielded a saturated C5 fatty acid chain containing com-
pound 17 in 42% yield.11 The fully protected ester 11, on
Vilsmeier–Haack reaction with DMF and POCl3, yield-
ed the aldehyde 18, where the aldehydic group was unex-
pectedly introduced in the ring between one of the
methoxyl and carboxylic ester groups. The methoxy
groups of 18 were deprotected with boron tribromide
in dichloromethane to furnish fully deprotected product
21, which on borohydride reduction in methanol yielded
corresponding alcohol 22. On the other hand, com-
pound 18 was selectively demethylated with AlCl3–
CH2Cl2 system12 to get mono demethylated ester 19,
which on further hydrolysis yielded acid 20.
Aldehyde 5 on stirring overnight with acetone at room
temperature in 1% alcoholic KOH yielded an aldol
product 7. However, compound 3 underwent Friedel-
Craft’s acylation with glutaric anhydride in dichloro-
methane and aluminium chloride at room temperature
to give 9 having some similarity with the C5 side chain
of Oenostacin 1.10
In Scheme 2, the starting compound, 3,5-dihydroxy ben-
zoic acid (10) was first methylated with dimethyl sulfate
in acetone to obtain trimethylated product 11 in 93%
yield. Interestingly, compound 11 on Friedel-Crafts
acylation with glutaric anhydride in presence of anhy-
drous aluminium chloride at room temperature in
In Schemes 1 and 2, for the attachment of the required
C5 aliphatic chain at the aromatic ring, as in Oenostacin
1, we attempted Friedel-Crafts reaction on 3 and 11 with
glutaric anhydride using anhydrous aluminium chloride

520
V. Srivastava et al. / Bioorg. Med. Chem. 15 (2007) 518–525
O
OMe
13
OMe
O
OH
10
O
OMe
O
3'
3
4
5
OH
i
2
1'
ii
5'
OH
+
1
MeO
COOH
1
5
MeO
COOMe
HOOC
MeOOC
OMe
11
v
12
iv
OMe
17
O
iii
OMe
OH
OH
vi
MeO
COOMe
OMe
O
O
MeOOC
OMe
HOOC
OH
CHO
18
OH
CHO
21
viii
MeO
COOH
14
+
vii
OH
OH
O
COOH
OH
MeOOC
19
OMe
CHO
MeO
COOH
HOOC
22
OH
CH2OH
15
ix
+
OH
OMe
O
COOH
HOOC
20
OMe
CHO
HO
COOMe
16
Scheme 2. Reagents and conditions: (i) Me2SO4, K2CO3, acetone, reflux, 3 h, 92%; (ii) glutaric anhydride, DCM, AlCl3, rt, overnight, 12: 45%, 13:
18%; (iii) AlCl3, DCM, rt,overnight 78% 14/15/16 = 2:3:1; (iv) TFA, NaBH4, 0 ꢁC for 4 h then rt, 42%; (v) DMF, POCl3, 0 ꢁC for 30 min then rt for
4 h, 42%; (vi) BBr3, DCM, À78 ꢁC for 30 min then RT overnight, 24%; (vii) NaBH4, MeOH, rt, 40 min, 79%; (viii) AlCl3-DCM, rt, overnight, 78%;
(ix) 5% NaOH in MeOH/water (3:1), 50 ꢁC, 1 h, 52%.
O
O
OH
26
O
OMeO
25
OMe
24
OH
23
HO
HO
MeO
MeO
OH
OH
MeO
MeO
HO
HO
ii
i
iii
Scheme 3. Reagents and conditions: (i) Me2SO4, K2CO3, acetone, reflux, 3 h, 89%; (ii) glutaric anhydride, DCM, AlCl3, rt, overnight, 22%; (iii) BBr3,
DCM, À78 ꢁC for 1 h then rt, 58%.
O
MeO
MeO
R
MeO
MeO
COOH
ii
HO
HO
COOH
i
O
OMe
OMe
28
OH
27
29: R= -CH2CH2CH2COOC2H5,
30: R= -CH2-CH=CH-COOC2H5
iii
MeO
R-O
COOH
MeO
COOMe
vi
MeO
COOMe
MeO
MeO
COOMe
iv
v
R-O
HO
OMe
OMe
OMe
32
OMe
31
35: R= -CH2CH2CH2COOH,
36: R= -CH2-CH=CH-COOH
33: R= -CH2CH2CH2COOC2H5,
34: R= -CH2-CH=CH-COOC2H5
Scheme 4. Reagents and conditions: (i) Me2SO4, 30% aqueous KOH, 10 ꢁC for 1 h then reflux for 3 h, 62%; (ii) ethyl bromo butyrate/ethyl bromo
crotonate, K2CO3, acetone, reflux, 4 h, 53%; (iii) Me2SO4, acetone, K2CO3, reflux, 4 h, 89%; (iv) AlCl3, DCM, rt, 4 h, 69%; (v) ethyl bromoesters,
acetone, K2CO3, reflux, 3–4 h; 33: 64%, 34: 71%; (vi) 5% KOH in MeOH/water (3:1), 50 ꢁC, 1 h, 35: 61%, 36: 67%.
in CH2Cl2 at room temperature. In the above cases, we
expected the attachment of the keto acid unit between
both the methoxyl groups (i.e., at C4 position). Indeed,
in case of 3 the attachment of the side chain was as
expected to give 9 but surprisingly in 11 it was at C2-
position (i.e., ortho to COOMe group), leading to a

V. Srivastava et al. / Bioorg. Med. Chem. 15 (2007) 518–525
521
R1
R1
R1
MeO
MeO
CHO
HO
HO
COOH
MeO
MeO
COOH
ii
i
R2
R2
R2
41: R1= OH, R2=H;
42: R1= H, R2=OH
37: R1= OMe, R2=H;
38: R1= H, R2=OMe
39: R1= OMe, R2=H;
40: R1= H, R2=OMe
Scheme 5. Reagents and conditions: (i) DMSO, KOH, (Ph3P+@CH-COOC2H5)BrÀ, overnight, 39: 48%, 40: 54%; (ii) BBr3, DCM, À78 ꢁC for 30 min
then rt, 41: 49%, 42: 62%.
mixture of products 12 and 13. All these structural
assignments have been unambiguously confirmed by
various NMR experiments and mass spectral data.
er being more upfield indicates the side-chain acid was
possibly cyclised at enol site to form a six-membered lac-
tone ring. Further evidence to the proposed lactone moi-
ety in 13 came from IR (a strong band at 1768 cmÀ1
,
Different 1D/2D-NMR experiments on 12 and 13 have
confirmed the side-chain attachment at C2 position on
typical for a lactone carbonyl) and Mass spectral data.
ESI-MS showed mass peaks at 279.2 (M+H) and
301.1 (M+Na) indicating the unambiguous assignment
of 13 as a lactone.
1
the aromatic ring. H NMR of 13 revealed two distinct
singlet resonances in the aromatic region at d 6.89 and
7.12 (ppm) due to two non-identical protons indicating
the possible attachment of side chain at C2 position
only. All the carbon resonances of 13C NMR coupled
with DEPT editing experiments are well in agreement
with the proposed structure for 13. Also, the appearance
of only one long range correlation, in the 1H–13C inverse
correlated HMBC experiment, between a carbonyl car-
bon of acid group attached to benzene ring of 13 and
one of its aromatic protons attached to C6 position con-
firmed the side-chain attachment unambiguously at C2
position of aromatic ring. Similarly, a two-bond correla-
tion between C3 and a proton attached to C4 (as well as
two such similar correlations between C5 and protons
attached to C4 and C6, respectively) further supported
the proposed side-chain attachment of 13. All the
important long-range correlations of 13 are shown in
Figure 2. A similar long-range HMBC correlation found
in compound 12 confirms the same type of attachment
as proposed in 13.
In case of compound 9 (Scheme 1) the 1H NMR showed
a singlet resonance at d 6.28 ppm integrating for two
protons indicating the side-chain attachment at C4 posi-
tion, that is, between two aromatic methoxyl groups
(whereas in 12 and 13 two distinct singlet resonances ap-
peared indicating their non-identical nature). Further
confirmation of the structure proposed to compound 9
came from the 1H–13C inverse correlated HMBC
experiment.
In Scheme 3, the phenolic hydroxy groups of pyrogallol
23 were protected through methylation with dimethyl
sulfate as described earlier. Subsequent Friedel-Crafts
reaction with glutaric anhydride yielded the keto deriv-
ative 25, which was further demethylated with boron
tribromide to furnish the phenolic keto acid 26.
Several derivatives of gallic acid were also synthesized,
as shown in Scheme 4, in which both ester chain deriv-
atives (29 and 30) and phenolic ether derivatives (33–
36) were prepared. In this approach, gallic acid was
methylated with dimethyl sulfate in aqueous alkali to
get trimethoxy benzoic acid 28, which on reaction with
ethyl bromo butyrate and ethyl bromo crotonate yielded
29 and 30, respectively. On the other hand, gallic acid on
methylation with dimethyl sulfate in acetone–K2CO3
yielded the fully protected ester 31, which on selective
demethylation with AlCl3–CH2Cl2 system12 yielded phe-
nolic ester 32. Compound 32 reacted with bromo esters
In 13C NMR of 13, the two resonances at d 167.2 and
175.3 ppm are due to two carbonyl functions. The form-
O
OCH3
2
1'
O
2'
3
4
5'
3'
4'
COOH
H3CO
5
1
6
Figure 2. 1H–13C HMBC correlations of 13.
Some more analogues
COOH
COOH
COOH
COOH
MeO
HO
HO
43
OMe
45
OMe
46
OH
44
O
COOH
AcO
HO
HO
COOH
CH3
AcO
OMe
OAc
OH
49
47
48
Figure 3. Some more analogues of Oenostacin.

522
V. Srivastava et al. / Bioorg. Med. Chem. 15 (2007) 518–525
(ethyl bromo butyrate/ethyl bromo crotonate) to afford
the desired products 33 and 34, respectively. Basic
hydrolysis treatment on esters 33 and 34 afforded the
acids 35 and 36, respectively.
an essential requisite for showing activity, while their
protection renders it inactive. When an aldehyde group
was introduced in the aromatic ring, antibacterial activ-
ity was increased. On reducing aldehyde to correspond-
ing alcohol, bioactivity was further enhanced. The
position of the phenolic hydroxyl in the ring has no ef-
fect on biological activity. Replacement of methyl group
in the ring with a carboxylic group had no significant
impact on the bioactivity. Increasing the side-chain
length from C1 to C3 did not show much effect on the
biological activity. Several analogues possessed very
good activity but none of the analogues was found as ac-
tive as Oenostacin 1.
In Scheme 5, 2,3,4-trimethoxy benzaldehyde (37) and
3,4,5-trimethoxy benzaldehyde (38) underwent Wittig
reaction using the similar reaction conditions as de-
scribed earlier in Scheme 1 to furnish the product
2,3,4-trimethoxy cinnamic acid (39) and 3,4,5-trimeth-
oxy cinnamic acid (40), respectively. Compounds 39
and 40 were demethylated in boron tribromide–CH2Cl2
system to get corresponding trihydroxy cinnamic acid
derivatives 41 and 42, respectively.
In conclusion, inspired by fascinating chemical structure
and potent antibacterial activity of Oenostacin 1, we
developed a new family of non-natural analogues. The
present study provided some insight into the essential
structural features of Oenostacin 1. The above studies
will be helpful for further lead optimization of
Oenostacin.
Besides the above derivatives, a few more compounds as
shown in Figure 3 (few are commercial samples and oth-
,
ers are their derivatives)14a b were used for biological
activity studies to establish structure and activity rela-
tionship of Oenostacin 1.
2.2. Biological evaluation
All the analogues were screened for in vitro antibacterial
activity against S. epidermidis and S. aureus following
the method described by Petersdorf et al.13 Compounds
having EC50 values beyond 5 lM concentrations were
considered inactive. The EC50 values were means of
three experiments in triplicate. Only active analogues
have been shown in Table 1. Amongst these 22, 23
and 42 possessed higher level of antibacterial activity
against both the strains and hence are best out of all
the analogues of Oenostacin 1. Some of the analogues
4, 21, 26 and 49 possessed moderate level of activity,
while others 2, 19, 20 and 44 showed low level of anti-
bacterial activities. However, none of these analogues
was found equipotent to Oenostacin 1.
4. Experimental
4.1. Materials and methods
Melting points (mp) were determined on a JSGW melt-
ing point apparatus and are uncorrected. FT-IR spectra
were recorded in KBr on Perkin-Elmer AC-1 spectrome-
ter. H NMR and 13C NMR spectra were recorded at
1
300 MHz and 75 MHz, respectively, on a Bruker Avance
DRX-300 spectrometer. Chemical shifts are given in d
values, downfield from the TMS as internal standard.
All the 1H and a few 13C spectra are reported. Coupling
constant (J) values are given in Hz. Electrospray mass
spectra were recorded on API-3000, LC-Ms/Ms (Applied
Biosystem) after dissolving the compounds in acetoni-
trile. FAB mass was done on a JEOL SX 102/DA-6000
Mass spectrometer using argon as the FAB gas and
m-Nitrobenzyl alcohol as the matrix. All the solvents
and reagents were of LR/AR grade. Dry solvents were
prepared as per standard methods. Reactions were mon-
itored on Merck aluminium thin layer chromatography
(TLC, UV254nm) plates. Visualization was accomplished
either on UV chamber (254 nm and 320 nm) or by spray-
ing TLC plates with 2% ceric sulfate in 10% aqueous
sulfuric acid solution and charring them at higher tem-
peratures (100–120 ꢁC). Column chromatography was
carried out on silica gel (60–120 mesh, Merck chemicals).
Elemental analysis was carried out in Heraus CHN
analyzer.
3. Summary and conclusion
More than forty analogues have been synthesized and
evaluated for antibacterial activities. From these studies,
it revealed that the presence of a free phenolic group is
Table 1. Antibacterial activity of some of the active analogues of
Oenostacin against S. epidermidis and S. aureus
S. No.
Compound
S. epidermidis
EC50 (lM)
S. aureus
EC50(lM)
1.
2.
1, Oenostacin
2
0.12
4.03
1.64
2.38
2.55
1.37
0.67
0.99
1.04
0.63
2.78
1.48
0.12
4.03
3.
4
1.64
4.76
4.
19
20
21
22
23
26
42
44
49
4.2. Syntheses
5.
Inactive
Inactive
0.67
6.
4.2.1. General procedure for the synthesis of compounds 5
and 18
7.
8.
0.49
2.08
9.
4.2.1.1. Synthesis of 4-methyl, 2,6-dimethoxy benzal-
dehyde (5). In a 25 ml round-bottomed-flask 3,5 dime-
thoxy toluene 3 (100 mg, 0.65 mmol) was taken in dry
DMF (0.1 ml, 1.29 mmol). The reaction flask was kept
in an ice-bath (0–10 ꢁC). To this stirred reaction mix-
ture, phosphorus oxychloride (0.1 ml, 1.07 mmol) was
10.
11.
12.
1.26
2.78
2.96
*Analogues possessing antibacterial activity higher than 5 lM con-
sidered being inactive.

V. Srivastava et al. / Bioorg. Med. Chem. 15 (2007) 518–525
523
added dropwise. The reaction mixture was further kept
for 30 min in the cooling bath and then heated at
80 ꢁC for 3 h. On completion, the reaction mixture was
slowly poured into ice-cold water and then it was made
alkaline with 10% aqueous NaOH to precipitate the de-
sired aldehyde. It was filtered and recrystallized from
chloroform–hexane (1:3 v/v) to get 5 as creamish white
solid (62% yield).
Yield = 62%; oil, 1H NMR (300 MHz, CDCl3): d 2.44 (s,
6H, 2· CH3), 3.83 (s, 6H, 2· OCH3), 3.89 (s, 6H,
2· OCH3), 6.37–6.40 (d, 4H, aromatic, J = 9Hz), 7.23–
7.28 (d, 2H, 2· @CHCO, J = 16.2 Hz), 7.91–7.97 (d,
2H, 2· CH@, J = 16.2 Hz); Electrospray Mass (CH3CN):
383.4 [M+H]+, 405.3 [M+Na]+, 421.2 [M+K]+, 765.5
[2M+H]+, 803.6 [2M+K]+; Elemental analysis Calcd for
C23H26O5: C, 72.23; H, 6.85. Obsd: C, 72.02; H, 6.93.
1
Compound 22: Yield = 79%; oil, H NMR (300 MHz,
4.2.2. General procedure for the synthesis of compounds 6,
39 and 40. All the Wittig salts were prepared by taking
the corresponding alkyl halide with triphenyl phosphine
in dry toluene under refluxing condition for 1–2 h.
acetone d6): d 2.77 (s, 2H, CH2OH), 6.57 (d, 1H, aromatic,
J = 1.87 Hz), 6.63 (d, 1H, aromatic, J = 1.88 Hz), 8.82 (s,
1H, exchangeable, phenolic OH), 9.13 (s, 1H, exchange-
able, phenolic OH); Electrospray Mass (CH3CN): 184.2
[M]+, 369.0 [2M+H]+.
4.2.2.1. Synthesis of 3-(2,6-dimethoxy-4-methylphe-
nyl)-acrylic acid (6). In a 25 ml round-bottomed-flask
4-methyl, 2, 6-dihydroxy benzaldehyde 5 (300 mg,
1.66 mmol), dry DMSO (2 ml) and sodium hydroxide
(250 mg, 6.25 mmol) were stirred at room temperature.
After 20 min of stirring, Wittig salt was added and the
reaction mixture was further stirred overnight (ꢀ16 h).
Later the reaction mixture was poured into water,
acidified with 10% dil HCl and extracted with ethyl
acetate. The organic layer was washed with water and
dried over anhydrous sodium sulfate. The solvent was
evaporated get a crude residue. Further purification
was done on a silica gel column eluting with chloro-
form–methanol to get the desired compound 6 as white
crystalline solid.
4.2.4. General procedure for the synthesis of compounds 9,
12, 13 and 25
4.2.4.1. Synthesis of 5-(2,6-Dimethoxy-4-methyl-phen-
yl)-5-oxo-pentanoic acid (9). In a 25 ml round-bottomed-
flask glutaric anhydride (506 mg, 3.84 mmol) was stirred
with dichloromethane (5 ml.) and anhydrous aluminium
chloride (506 mg, 3.79 mmol). It was stirred for 20 min
and then to this 1,3-dimethoxy 5-methyl benzene 3
(500 mg, 3.29 mmol) was added in portions. The reac-
tion mixture was stirred overnight (18 h) at room tem-
perature. To this 5% dil HCl (5 ml) was added and
extracted with dichloromethane (3· 25 ml). Organic
layer was washed with water, dried over anhydrous sodi-
um sulfate and evaporated to dryness. The residue thus
obtained was purified through silica column and eluted
with chloroform–acetone. Compound 9 was obtained
at 3% acetone–CHCl3 (v/v) as an oil.
Compound 6: Yield = 32%; mp = 164–165 ꢁC, IR (KBr,
cmÀ1): 2952, 2595, 1654, 1508; 1H NMR (300 MHz,
CDCl3): d 2.38 (s, 3H, CH3), 3.81 (s, 3H, OCH3), 3.89
(s, 3H, OCH3), 6.28 (d, 1H, aromatic, J = 2.17 Hz),
6.32 (d, 1H, aromatic, J = 2.10 Hz), 6.56–6.61 (d, 1H,
@CH-CO, J = 16.05 Hz), 7.88–7.93 (d, 1H, CH@,
J = 16.02 Hz); 13C NMR (75 MHz, CDCl3): d 21.48,
55.48, 55.54, 96.59, 108.11, 115.53, 119.15, 139.68,
141.85, 161.44, 161.68, 171.25; Electrospray Mass
(CH3CN): 223.1 [M+H]+; Elemental analysis Calcd for
C12H14O4: C, 64.85; H, 6.35. Obsd: C, 65.12; H, 6.23.
1
Yield = 32%; oil, H NMR (300 MHz, CDCl3): d 1.88-
1.97 (quintet, 2H, CH2CH2COOH, J = 7.23 Hz), 2.27
(s, 3H, CH3), 2.30–2.36 (t, 2H, CH2COOH), 2.69–2.74
(t, 2H, –CO–CH2–, J = 7.04 Hz), 3.69 (s, 3H, 2· OCH3),
6.28 (s, 2H, aromatic protons); Electrospray Mass
(CH3CN): 289.1 [M+Na]+; Elemental analysis Calcd for
C14H18O5: C, 63.16; H, 6.77. Obsd: C, 62.72; H, 7.13.
Compound 39: Yield = 48%; mp = 162–164 ꢁC, IR (KBr,
cmÀ1): 2570, 1694, 1619, 1498, 1590; 1H NMR
(300 MHz, CDCl3): d 3.88 (s, 3H, OCH3), 3.91 (s, 3H,
OCH3), 3.94 (s, 3H, OCH3), 6.41–6.46 (d, 1H, @CHCO,
J = 16.08 Hz), 6.69–6.72 (d, 1H, aromatic, J = 8.79 Hz),
7.28–7.31 (d, 1H, aromatic, J = 8.79 Hz), 7.95–8.01 (d,
1H, CH@, J = 16.08 Hz); Electrospray Mass (CH3CN):
239.1 [M+H]+, 261.2 [M+Na]+; Elemental analysis Calcd
for C12H14O5: C, 60.50; H, 5.92. Obsd: C, 60.72; H, 6.23.
4.2.4.2. Synthesis of 2-(4-Carboxy butyryl)-3,5-dime-
thoxy-benzoic acid methyl ester (12) and 2-(4-Carboxy-1-
hydroxy-but-1-enyl)-3,5-dimethoxy-benzoic acid (13). In
a
25 ml round-bottomed-flask glutaric anhydride
(175 mg, 1.54 mmol) was stirred with dichloromethane
(5 ml) and anhydrous aluminium chloride (210 mg,
1.57 mmol). It was stirred for 20 min and then to this
3,5-dimethoxy benzoic acid methyl ester 11 (250 mg,
1.28 mmol) was added in portions. The reaction mixture
was stirred overnight (16–18 h) at room temperature. To
this 5% dil HCl (5 ml) was added and extracted with
dichloromethane (3· 25 ml). Organic layer was washed
with water, dried over anhydrous sodium sulfate and
evaporated to dryness. The residue thus obtained was
purified through silica column and eluted with chloro-
form–acetone. Compound 12 was obtained at 1% ace-
tone–CHCl3 and 13 was obtained at 2% acetone–
CHCl3 to get both crystalline solids.
4.2.3. Synthesis of 1,5 Di [(2,6-dimethoxy, 4-methyl
phenyl)]-penta 1,4-dien, 3-one (7). In a 100 ml round-bot-
tomed-flask 2,6-dimethoxy, 4-methyl benzaldehyde (5,
100 mg, 0.56 mmol) was taken in ethanol (2 ml). To this
stirred solution sodium hydroxide (4 mg, 0.1 mmol) and
acetone (20 mg, 0.34 mmol) were added and further stir-
red at room temperature for 72 h. On completion of the
reaction a yellow solid mass was precipitated in the
reaction mixture. It was filtered, washed with alcohol
and recrystallized with chloroform–acetone to get 7 as
an oil.
Compound 12: Yield = 45%; mp = 107–109 ꢁC, IR
1
(KBr, cmÀ1): 2954, 2843, 1689, 1712, 1508; H NMR

524
V. Srivastava et al. / Bioorg. Med. Chem. 15 (2007) 518–525
(300 MHz, CDCl3): d 2.03–2.10 (distorted quintet, 2H,
CH2–CH2COOH), 2.49–2.54 (t, 2H, CH2COOH,
J = 7.08 Hz), 2.84–2.89 (t, 2H, CH2 benzylic, J =
6.81 Hz), 3.78 (s, 3H, COOCH3), 3.84 (s, 6H,
2· OCH3), 6.63 (s, 1H, aromatic proton), 7.02 (s, 1H,
aromatic proton); 13C NMR (75 MHz, CDCl3): d 19.1,
33.3, 43.19, 52.58, 55.99, 56.41, 103.45, 106.51, 127.08,
130.32, 157.78, 161.36, 166.69, 178.81, 204.34; Electro-
spray Mass (CH3CN): 311.2 [M+H]+, 333.3 [M+Na]+,
349.1 [M+K]+; Elemental analysis Calcd for C15H18O7:
C, 58.06; H, 5.85. Obsd: C, 57.86; H, 6.03.
NMR(75 MHz, CDCl3): d 25.2, 26.07, 30.11, 34.36,
52.29, 55.79, 56.10, 102.56, 105.69, 125.54, 132.12,
158.64, 159.31, 168.83, 179.97; Electrospray Mass
(CH3CN): 297.1 [M+H]+; Elemental analysis Calcd for
C15H20O6: C, 60.80; H, 6.80. Obsd: C, 61.02; H, 6.63.
4.2.6. General procedure for the synthesis of compounds
14, 15, 16 and 19
4.2.6.1. Synthesis of 2-(4-carboxy-butyryl)-3,5-dime-
thoxy-benzoic acid (14), 2-(4-carboxy-butyryl)-3-hy-
droxy,5-methoxy-benzoic acid (15) and 2-(4-carboxy-
butyryl)-5-hydroxy, 3-methoxy benzoic acid (16). In a
25 ml round-bottomed-flask 12 (200 mg, 0.64 mmol)
was taken in dry dichloromethane (10 ml). To this stir-
ring solution anhydrous aluminium chloride (800 mg,
5.99 mmol) was added and stirred overnight at room
temperature. On completion of reaction dil HCl was
added dropwise to it and stirred for 10 min. It was
extracted with dichloromethane and washed with water.
The organic layer was dried over anhydrous sodium sul-
fate and evaporated to get a residue. The residue thus
obtained was purified through a column of silica gel to
get 14, 15 and 16 at 2% methanol–CHCl3 one by one
in the ratio 2:3:1, respectively.
Compound 13: Yield = 18%; mp = 148–152 ꢁC, IR(KBr,
1
cmÀ1): 3215, 2950, 1719, 1653, 1605, 1507; H NMR
(300 MHz, pyridine d5): d 2.57–2.62 (t, 2H, CH2-COOH
of lactone ring, J = 7.2 Hz), 2.73–2.80 (m, 2H,
CH2CH2COOH of lactone ring, J = 7.3 Hz), 3.87 (s,
3H, OCH3), 3.93 (s, 3H, OCH3), 5.78–5.83 (t, 1H,
@CH–CH2 lactone ring), 6.69 (d, 1H, aromatic proton,
J = 1.64 Hz), 6.71 (d, 1H, aromatic proton, J =
1.66 Hz); 13C NMR (75 MHz, pyridine d5): d 22.73,
34.79, 49.84, 56.23, 56.30, 99.58, 105.89, 110.37, 121.71,
127.91, 145.34, 156.29, 163.32, 167.23, 175.28; Electro-
spray Mass (CH3CN): 279.2 [M+H]+, 301.1 [M+Na]+,
317.0 [M+K]+. Elemental analysis Calcd for C14H14O6:
C, 60.43; H, 5.04. Obsd: C, 60.03; H, 5.26.
Compound 14: Yield = 23%; 1H NMR (300 MHz,
CDCl3): d 1.84–1.94 (quintet, 2H, CH2CH2COOH,
J = 7.16 Hz), 2.32–2.37 (t, 2H, CH2COOH, J =
7.33 Hz), 2.76–2.81 (t, 2H, CH2CO–, J = 7.0 Hz), 3.73
(s, 3H, OCH3), 3.75(s, 3H, OCH3), 6.61 (d, 1H, aromatic,
J = 1.0 Hz), 6.83 (d, 1H, aromatic, J = 1.0 Hz); Electro-
spray Mass (CH3CN): 297.1 [M+H]+; Elemental analysis
Calcd for C14H16O7: C, 56.76; H, 5.40. Obsd: C, 57.02;
H, 5.88.
Compound 25: Yield = 22%; oil, IR (neat, cmÀ1): 2940,
1735, 1654, 1508, 1631; H NMR (300 MHz, CDCl3):
1
d 2.03–2.13 (m, 2H,CH2CH2COOH), 2.49–2.53 (t, 2H,
CH2COOH, J = 7.00 Hz), 3.02–3.06 (t, 2H, –COCH2-
CH2, J = 7.08 Hz), 3.89 (s, 3H, OCH3), 3.93 (s, 3H,
OCH3), 3.96 (s, 3H, OCH3), 6.48–6.51(d, 1H, aromatic,
J = 9.03 Hz), 7.52–7.55 (d, 1H, aromatic, J = 9.03 Hz);
Electrospray Mass (CH3CN): 291.2 [M+K]+.
Compound 15: Yield = 38%; 1H NMR (300 MHz,
CDCl3): d 1.86–1.93 (quintet, 2H, CH2CH2COOH,
J = 7.16 Hz), 2.28–2.33 (t, 2H, CH2COOH, J =
7.37 Hz), 2.78–2.83 (t, 2H, CH2CO–, J = 7.03 Hz), 3.72
(s, 3H, OCH3), 6.39 (s, 1H, aromatic), 6.65 (s, 1H, aro-
matic); Electrospray Mass (CH3CN): 283.1 [M+H]+; Ele-
mental analysis Calcd for C13H14O7: C, 55.32; H, 4.96.
Obsd: C, 54.89; H, 5.36.
4.2.5. Synthesis of 2-(4-carboxy-butyl)-3,5-dimethoxy-
benzoic acid methyl ester (17). In a 25 ml round-bottom-
med-flask 2-(4-carboxy butyryl)-3,5-dimethoxy-benzoic
acid methyl ester 12 (100 mg, 0.34 mmol) was taken in
2 ml trifluoroacetic acid (TFA). The reaction mixture
was kept in an ice-bath (0–10 ꢁC). To this stirred reac-
tion mixture, sodium borohydride (40 mg, 1.05 mmol)
was added in portions to avoid excessive heat. It was
stirred for 4 h at this temperature and then at room tem-
perature for an hour. The reaction mixture was slowly
poured into ice and acidified (5% HCl). It was then
extracted with ethyl acetate (3· 25 ml) and washed with
water. The organic layer was dried over anhydrous sodi-
um sulfate and evaporated to dryness. The residue thus
obtained was purified through column chromatography
over silica gel and eluted with chloroform–acetone and
chloroform–methanol. The desired product 17 was ob-
tained at 5% acetone–CHCl3 as a white crystalline solid.
Compound 16: Yield = 12%; 1H NMR (300 MHz,
CDCl3): d 1.99–2.04 (broad triplet, 2H, CH2CH2COOH),
2.39–2.44 (t, 2H, CH2COOH), 2.74–2.79 (t, 2H, CH2CO–),
3.83 (s, 3H, OCH3), 3.91(s, 3H, –COOCH3), 6.52 (s,
1H, aromatic), 6.68 (s, 1H, aromatic); Electrospray Mass
(CH3CN): 297.1 [M+H]+; Elemental analysis Calcd for
C14H16O7: C, 56.76; H, 5.40. Obsd: C, 56.44; H, 5.89.
4.2.6.2. Synthesis of 2-formyl, 3-methoxy, 4-hydroxy
benzoic acid methyl ester (19). In a 25 ml round-bot-
tomed-flask 2-formyl, 3,5-dimethoxy-benzoic acid meth-
yl ester (18, 100 mg, 0.45 mmol) was taken in dry
dichloromethane (6 ml). To this stirring solution anhy-
drous aluminium chloride (200 mg, 1.6 mmol) was add-
ed and stirred overnight at room temperature. On
completion of reaction dil HCl was added dropwise to
it and stirred for 10 min. It was extracted with dichloro-
methane and washed with water. The organic layer was
dried over anhydrous sodium sulfate and evaporated to
Yield = 42%; mp = 125–130 ꢁC, IR (KBr, cmÀ1): 3420,
2948, 1773, 1713, 1503, 1620; 1H NMR (300 MHz,
CDCl3): d 1.73–1.81 (m, 2H, CH2CH2COOH), 1.85–
1.95 (m, 2H, CH2CH2CH2COOH), 2.57–2.62 (t, 2H,
CH2COOH, J = 7.45 Hz), 3.00–3.05 (t, 2H, benzylic,
J = 7.62 Hz), 4.00 (s, 3H, OCH3), 4.01 (s, 3H, OCH3),
4.08 (s, 3H, COOCH3), 6.77 (d, 1H, Aromatic,
J = 2.38 Hz), 7.08 (d, 1H, aromatic, J = 2.46 Hz); 13C

V. Srivastava et al. / Bioorg. Med. Chem. 15 (2007) 518–525
525
get a residue. It was passed through a small column of
silica gel to get 19 at hexane/CHCl3 (1:1 v/v) as creamish
white solid.
project work was supported from Department of Bio-
technology (DBT) Govt. of India.
4.2.7. General procedure of the synthesis of compounds 8,
21, 26, 41 and 42
References and notes
4.2.7.1. Synthesis of 3-(3,4,5-trihydoxyphenyl)-acrylic
acid (42). In a 25 ml round-bottomed-flask 3,4,5-tri-
methoxy cinnamic acid 40 (50 mg, 0.21 mmol) was taken
in dry dichloromethane (5 ml). The reaction mixture was
kept in acetone bath and cooled to À78 ꢁC with liquid
nitrogen. To the chilled reaction mixture boron tribro-
mide (0.12 ml, 12 mmol) was added dropwise and fur-
ther stirred for 2 h at this temperature. Then the
reaction mixture was stirred overnight at room temper-
ature (16 h). On completion, the reaction mixture was
poured into water and dil HCl (10%, 5 ml) was added
to it. It was extracted with chloroform, organic layer
was washed with water, dried over anhydrous sodium
sulfate and evaporated. The residue thus obtained was
recrystallized with CHCl3–MeOH (3:1 v/v) to get desired
demethylated product 42 as light brown crystalline solid.
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B. J. F. J. Am. Oil Chem. Soc. 1984, 61, 540–543; (c)
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(b) Shukla, Y. N.; Santha Kumar, T. R.; Srivastava, A.;
Khanuja, S. P. S.; Gupta, V.; Kumar, S. US patent no.
6,365,197, 2002.
Yield = 62%; mp = 180–182 ꢁC, IR (KBr, cmÀ1): 3277,
1702, 1641, 1613, 1539; H NMR (300 MHz, CD3OD):
1
d 6.14–6.19 (d, 1H, @CHCO, J = 15.81 Hz), 6.59 (s,
2H, aromatic), 7.41–7.46 (d, 1H, CH = , J = 15.81 Hz);
Electrospray Mass (CH3CN): 197.0 [M+H]+, 415.2
[2M+Na]+; Elemental analysis Calcd for C9H8O5: C,
55.11; H, 4.11; Obsd: C, 54.92; H, 4.23.
5. (a) Shukla, Y. N.; Srivastava, A.; Kumar, S. Ind. J. Chem.
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4.3. Biological evaluation
4.3.1. Antibacterial bioassay
4.3.1.1. Estimation of minimum inhibitory concentra-
tion (MIC). Twofold serial dilution technique was used
to assess the minimal inhibitory concentration (MIC)
of a test compound against the bacterial strains. In a ser-
ies of eight tubes serial dilutions was made. In first tube,
2 ml of nutrient broth was taken and in subsequent tube
1 ml of broth was taken after that, test compound of
known concentration was added in first tube and mix
properly. From the first tube 1 ml of broth containing
antibiotic was taken and added to the second tube and
mixed properly. This was repeated until the seventh
tube. 1 ml of mixture was expelled out from the last
tube. Only broth culture was used as a control. To each
of this tube, 10 ll of properly diluted log phase culture
of test organism with a titre of 104 cfu/ml was added.
The tubes were incubated at 37 ꢁC and examined by tur-
bidity measurement. The MIC was the lowest concentra-
tion of test compound inhibiting the development of
visible growth.
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14. (a) For methylation: Gilman, H.; Blat, A. H.. In Book,
Organic Synthesis; John Wiley: London, 1932; Vol. I, pp
537–538; (b) For acetylation: Furniss, B. S.; Hannaford,
A. J.; Smith, P. W. G.; Tatchel, A. R. In Book, Vogel’s
Text Book of practical organic chemistry; Addison-Wesley
Longman, Inc.: Reading, MA, 1998; p 1248.
Acknowledgments
V. S. and H. O. S. are grateful to C.S.I.R. and U.G.C.,
New Delhi for their fellowships, respectively. The