Antiprotozoal and antimicrobial activities of O-alkylated and formylated acylphloroglucinols

Article abstract of DOI:10.1016/j.bmc.2006.10.006

In the present article, we examined the antileishmanial, antimalarial, antibacterial, and antifungal activities of several newly synthesized O-alkylated phloroglucinol compounds (11-19) which are analogues of the naturally occurring antimalarial compound

Full text of DOI:10.1016/j.bmc.2006.10.006

Bioorganic & Medicinal Chemistry 15 (2007) 87–96
Antiprotozoal and antimicrobial activities of O-alkylated
and formylated acylphloroglucinols
Sandip B. Bharate,a Shabana I. Khan,b Nafees A. M. Yunus,a Siddheshwar K. Chauthe,a
Melissa R. Jacob,b Babu L. Tekwani,b Ikhlas A. Khanb and Inder Pal Singha,*
aDepartment of Natural Products, National Institute of Pharmaceutical Education and Research (NIPER) ,
Sector-67, S.A.S. Nagar, Punjab 160062, India
bNational Centre for Natural Products Research, School of Pharmacy, University of Mississippi, MS 38677, USA
Received 16 September 2006; revised 5 October 2006; accepted 6 October 2006
Available online 10 October 2006
Abstract—In the present article, we examined the antileishmanial, antimalarial, antibacterial, and antifungal activities of several
newly synthesized O-alkylated phloroglucinol compounds (11–19) which are analogues of the naturally occurring antimalarial com-
pound 1. Analogues 12 and 16 exhibited antileishmanial activity against, Leishmania donovani promastigotes with IC50s of 5.3 and
4.2 lg/mL, respectively. Naturally occurring monomeric formylated acylphloroglucinol compounds, grandinol (2), jensenone (3),
and their analogues (29–37), were also synthesized and evaluated for antileishmanial, antimalarial, antibacterial, and antifungal
activities. Amongst these, both grandinol and jensenone showed mild to moderate antibacterial, antifungal, and antileishmanial
activities. Jensenone (3) was effective against Candida albicans with an IC50 of 5.5 lg/mL but was ineffective against Cryptococcus
neoformans and methicillin-resistant Staphylococcus aureus. Among the analogues, 34 was the most active against C. albicans and C.
neoformans with IC50s of 2.0 and 2.5 lg/mL, respectively, and was fungicidal toward Candida albicans.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
Similarly, an increasing number of multidrug-resistant
microbial pathogens have become a serious problem
particularly during the last decade and provide the
impetus for the search and discovery of novel antibacte-
rial and antifungal agents active against these
pathogens.3
Parasitic diseases such as leishmaniasis and malaria have
a high mortality rate having a significant impact in
developing countries and affecting several hundred mil-
lions of people worldwide. Leishmaniasis is a tropical
disease caused by protozoal parasites of the genus Leish-
mania and it remains a significant health issue in large
part due to the lack of effective and affordable drugs
and increasing resistance against existing drugs.1 Present
drugs are either associated with side effects or are too
expensive for use by poor populations. Malaria is one
of the most important parasitic diseases in the world
and is a major global health problem affecting over
one hundred countries with disease prevalence escalat-
ing at an alarming rate, particularly in the last two dec-
ades. Rapid development of resistance by Plasmodium
falciparum to the conventional drugs such as chloro-
quine necessitates the search for new antimalarials.2
Naturally occurring phloroglucinol compounds have
shown diverse range of biological activities including
cancer chemopreventive, antimalarial, antibacterial,
HIV-RTase inhibitory, antifouling, etc.4 The O-preny-
lated phloroglucinol derivative 1 isolated from the light
petroleum ether extract of the aerial parts of Hypericum
calycinum has shown in vitro antimalarial activity with
an IC50 of 0.88 lg/mL against P. falciparum, while its
O-geranylated analogue showed antiviral and antimicro-
bial activities.5,6 As well, in a TLC bioassay, 1 demon-
strated antifungal activity against the plant pathogen
Cladosporium cucumerinum.5 The formylated acylphl-
oroglucinol compound grandinol (2), isolated from
leaves of Eucalyptus grandis, has shown germination
inhibitory activity, Epstein-Barr Virus inhibitory activi-
ty, and antibacterial activity against Bacillus subtilis and
Staphylococcus aureus (IC50s 5 and 25 lg/mL, respec-
tively).7 Another related acylphloroglucinol compound
Keywords: Antiprotozoal; Antimicrobial; Phloroglucinol; O-Alkylated
acylphloroglucinols; Grandinol; Jensenone.
NIPER Communication No. 386.
*
Corresponding author. Tel.: +91 172 2214683x2144; fax: +91 172
2214692; e-mail: ipsingh@niper.ac.in
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.10.006

88
S. B. Bharate et al. / Bioorg. Med. Chem. 15 (2007) 87–96
jensenone (3), isolated from Eucalyptus jensenii, has
shown antifeedant activity.8 Grandinol and jensenone
are assumed to be precursors of biologically active
phloroglucinols such as sideroxylonals, grandinal,
euglobals, and macrocarpals.4 The acylated phloroglu-
cinol compound, 1,3-diacetyl-2,4,6-trihydroxy benzene
(4), isolated from the bacterium Pseudomonas fluores-
cens, demonstrated antibacterial activity against
grandinol (2) and jensenone (3), of Eucalyptus spp.
along with several other synthetic analogues 29–37
were synthesized and evaluated for antimicrobial
and antiprotozoal activities.
2. Results and discussion
2.1. Chemistry
Streptococcus
pyogenes, Streptococcus
faecaelis,
Streptococcus mutans, Streptococcus sanguis, S. aureus,
Clostridium welchii, and Lactobacillus casei.9 Another
phloroacetophenone analogue, xanthoxylin (5), ob-
tained from Xanthoxylum piperitum exhibited antifungal
activity against C. albicans and two nonhuman patho-
genic fungi, Bortrytis cinera and Phomopsis perniciosa,
with ED50s of 45 and 410 lM on spore germination,
respectively, and a complete inhibition of germination
of B. cinera was observed at 550 lM.10 Multifidol (6)
and its glycoside 7 containing 2-methylbutyryl function-
ality isolated from 20% methanol fraction of the latex of
Jatropha multifida possessed immunomodulatory activi-
ty.11 Sessiliflorene (8) isolated from the hexane extract of
Melicope sessiliflora showed inhibitory activity against
the herpes simplex virus.12 Similarly caespitin (9) and
caespitate (10) isolated from Helichrysum caespitium
were found to possess antibacterial activity.13 Caespitate
(10), containing an acylated prenyl group on the phlor-
oglucinol nucleus, exhibited cytotoxic activity as well as
activity against pathogenic bacteria and fungi including
Mycobacterium tuberculosis.13 Acylphloroglucinols have
also been proposed as lead compounds for the treatment
of degenerative diseases because of their antioxidant
properties.14 The structures of bioactive monomeric
phloroglucinol compounds are shown in Figure 1.
2.1.1. Synthesis of O-alkylated acylphloroglucinols. A
number of O-alkyl phloroglucinols were designed and
synthesized based on the interesting biological activity
profile of the naturally occurring antimalarial O-preny-
lated phloroglucinol derivative, 1. Compounds were de-
signed by replacing in compound 1, the prenyl (with
allyl and isopentyl), methyl (with formyl, acetyl, and iso-
valeryl), and the isovaleryl side chain (with an acetyl or
isopentyl group) (Table 2).
As the key reaction involved in synthesis of O-alkyl-
ated phloroglucinols is O-alkylation, conditions were
optimized by varying bases, solvents, and tempera-
ture. The reported mono-allylation of phloroglucinol
via Zn catalyzed Williamson synthesis18 however,
did not produce satisfactory yield with various alkyl
halides. Several other conditions were attempted as
shown in Table 1. In most of the cases, results were
unsatisfactory, and although the expected product
was formed in most of these cases, yields were quite
low (2–20%); the expected product was obtained in
60–65% yield only with the use of sodium hydride
as a base and DMF as a solvent at room tempera-
ture. It is reported that these reactions give C-alkyl-
Based on the interesting biological activity profile of
monomeric acylphloroglucinols, and as a part of
our program to explore the naturally occurring phlor-
oglucinol compounds and their analogues for biolog-
ical potential,15–17 we have earlier found that
phloroglucinol-monoterpene adducts possessed good
antileishmanial activity.16 In this paper, we report
antiprotozoal and antimicrobial activities of mono-
meric phloroglucinol compounds. We have synthe-
sized several new O-alkylated phloroglucinol
compounds, 11–19, based on the antimalarial activity
of the naturally occurring O-prenylated analogue 1.
Naturally occurring formylated acylphloroglucinols,
Table 1. Conditions for O-alkylation of phloroglucinol (20)
Reagent/solvent
Reaction condition
Yieldsa (%)
K2CO3, acetone
K2CO3, acetone
NaH, DMF
rt, 10 h
50 ꢁC, 6 h
rt, 10 h
15–20
20–30
60–65
5–10
30
NaH, DMF
K2CO3, DMF
NaH, THF
60 ꢁC, 5 h
rt, 10 h
rt, 10 h
0
K2CO3, DMF
K2CO3, DMF
rt, 10 h
60 ꢁC, 10 h
5–10
5–10
a Isolated yields.
CH3
CHO
CHO
HO
O
O
HO
OH
O
HO
OH
O
HO
O
OCH3
HO
OH
O
H3C
OHC
OH
OH
OCH3
O
OH
OH
5
1
2
3
4
O
RO
O
OH
HO
OH
O
HO
OH
O
HO
OH
O
O
HO
O
OH
OH
OH
OH
6: R = H
7: R = β-D-Glu
8
9
10
Figure 1. Bioactive monomeric acylphloroglucinols.

S. B. Bharate et al. / Bioorg. Med. Chem. 15 (2007) 87–96
89
HO
OH
HO
R2
O
ated products via Claisen rearrangement at higher
temperature.19
R1
a
OH
OH
The rate of alkylation of phenolate ions is strongly
dependent on the solvent; polar aprotic solvents such
as dimethylformamide, dimethylsulfoxide, hexamethyl
phosphoric triamide (HMPA), etc., enhance the reactiv-
ity of phenolate ions leading to O-alkylation. In con-
trast, polar protic solvents such as water and alcohols
result in formation of C-alkylated product predominant-
ly via direct C-alkylation. If O-allylation reaction in po-
lar aprotic solvents such as DMF is performed at higher
temperatures, C-alkylated product is formed as a result
of Claisen rearrangement.19
22: R1 = Isopentyl, R2 = H
27: R1 = H, R2 = Isopentyl
20
CHO
+
AHO
O
HO
O
b
OHB
OH
28
19
Scheme 2. Reagents and conditions: (a) (CH3)2CHCH2CH2I, KOH/
MeOH:H2O (4:1), 70 ꢁC, 3 h, 60%; (b) POCl3, DMF, EtOAc, rt, 1 h,
70%.
Compound 19, which possesses a long alkyl side chain in
place of the acyl group, has been synthesized as shown
in Scheme 2. Treatment of phloroglucinol (20) with
isoamyl iodide in the presence of methanolic potassium
hydroxide resulted in formation of O-alkylated, 22,
C-alkylated, 27, and O/C-dialkylated phloroglucinol
compound, 28. Compound 28 further upon Vilsmeier–
Haack formylation yielded compound 19 in 70% yield.
Compounds, 11–18, have been synthesized by the strat-
egy shown in Scheme 1.
Treatment of phloroglucinol (20) with alkyl halide in the
presence of sodium hydride in DMF resulted in the for-
mation of O-alkylated phloroglucinols (21–22) in 60%
yield. O-Alkylated phloroglucinols (21 and 22) upon
Friedel-Crafts acylation with acyl chloride in the pres-
ence of titanium chloride resulted in the formation of
mono- (23–26) and diacylated (13–14 and 17–18)
phloroglucinols in 35–40% yield. Monoacyl phlorogluci-
nols (23–26) further upon Vilsmeier–Haack formylation
resulted in the formation of formylated acylphlorogluci-
nols (11–12 and 15–16) in 35–40% yield. Compound 16
has a structural similarity to that of naturally occurring
prenylated phloroglucinol compound 1, differing by
presence of O-isopentyl (reduced prenyl) and formyl
(in place of methyl) functionalities.
A typical pattern of chemical shift values for aromatic
1
hydroxyls was observed in the H NMR spectrum of
analogues 11–18. 1H NMR chemical shift for OHA
was downfield in analogues with diacyl functionality
compared with those possessing monoacyl functionality.
Hydroxyl protons, OHA and OHB, appeared downfield
in analogues with isovaleryl (12, 14, 16, and 18) com-
pared with analogues with acetyl (11, 13, 15, and 17)
functionality. The location of aromatic hydroxyls,
OHA and OHB was confirmed by 2-D NMR studies
viz. HMQC and HMBC. This observation may be use-
ful in structure elucidation of structurally similar natural
unknown O-alkylated acyl phloroglucinols.
2.1.2. Synthesis of formylated acylphloroglucinols. Natu-
rally occurring formylated phloroglucinol compounds,
grandinol (2) and jensenone (3), have been synthesized
along with several of their analogues 29–37 from com-
mercially available phloroglucinol (20) as described ear-
lier.15,16 Naturally occurring diformyl phloroglucinol
jensenone (3) has been synthesized by duff’s formylation
of phloroisovalerophenone.15 Compound 34 has been
synthesized by treatment of phloropropiophenone with
five equivalents each of dimethylformamide and phos-
phorous oxychloride. Compound 34 upon treatment
with prenyl bromide in the presence of sodium hydride
in dioxane, resulted in formation of C-prenylated
phloroglucinol compound 35 (Scheme 3).
The newly synthesized 34 has structural similarity with
sessiliflorene (8), which possesses antiviral activity
against the herpes simplex virus while analogue 35 is
similar to caespitin (9) and caespitate (10), which are
reported to possess antibacterial, antimycobacterial,
and cytotoxic activities.
All synthesized compounds were characterized by
NMR, MS, UV, and IR spectral data. All known acyl-
phloroglucinols were characterized by comparison of
Scheme 1. Reagents and conditions: (a) RX, NaH, DMF, 0 ꢁC, 10 h,
60–65%; (b) RCOCl, TiCl4, DCM, 0 ꢁC, 30 min, 35–40%; (c) POCl3,
DMF, EtOAc, rt, 1 h, 35–40%.

90
S. B. Bharate et al. / Bioorg. Med. Chem. 15 (2007) 87–96
CHO
comparable to that of the drug standard pentamidine.
HO
OH
O
HO
OH
O
Another interesting finding is that O-alkylated
phloroglucinols bearing formyl/isovaleryl moieties (12
and 16; IC50s 5.3 and 4.2 lg/mL, respectively) are
the most active against L. donovani promastigotes as
compared to compounds bearing formyl/acetyl (11
and 15), acetyl/acetyl (13 and 17) or isovaleryl/isovale-
ryl (14 and 18) functionalities. Similar results were
obtained in our previous work on phloroglucinol-ter-
pene adducts where natural as well as unnatural
euglobals with formyl and isovaleryl moieties pos-
sessed potent antileishmanial activity.16 Replacement
of the isovaleryl (16) functionality by isopentyl (19)
also resulted in a decrease in antileishmanial activity.
Among, formylated acylphloroglucinols (Table 3),
naturally occurring grandinol (2), jensenone (3), and
1,3-diacetyl-2,4,6-trihydroxy benzene (4) along with
analogues 31, 34, and 35 exhibited mild antileishma-
nial activities, while the remaining analogues were
inactive.
b
OH
OH
32
39
a
CHO
CHO
HO
OH
O
HO
OH
c
OH
OH
OH
34
OH
35
O
Scheme 3. Reagents and conditions: (a) POCl3/DMF (5 equiv each),
EtOAc, rt, 4 h, 40%; (b) POCl3/DMF (1 equiv each), EtOAc, rt,
30 min, 70%; (c) (CH3)2C@CH–CH2Br, NaH, dioxane, 60 ꢁC, 60%.
their spectroscopic data with literature values.7,8,15,16
The 13C NMR data and C, H, analyses have been taken
for all the final compounds.
2.2. Biological evaluation
None of these compounds (O-alkylated or formylated)
showed in vitro antimalarial activity against chloroquine
sensitive (D6) and chloroquine-resistant (W2) strains of
P. falciparum.21
All synthesized compounds were evaluated for in vitro
antileishmanial, antimalarial, antibacterial, antifungal,
and cytotoxic activities. Antileishmanial activity
against Leishmania donovani promastigotes was deter-
mined by an Alamar Blueꢂ assay.20 Most of the
O-alkylated acyl phloroglucinols showed moderate to
mild activities with IC50 values ranging from 4.2 to
40 lg/mL as depicted in Table 2. The most potent
antileishmanial activity was exhibited by analogues
12 and 16 (IC50 5.3 and 4.2 lg/mL, respectively),
which are structurally related to the naturally occur-
ring antimalarial compound 1. Analogue 12 differs
from 1 by the presence of allyl and formyl function-
alities in place of prenyl and methyl moieties, respec-
tively. Similarly, 16 possesses isopentyl and formyl
replacing prenyl and methyl of 1. The IC50 of 12 is
The antibacterial activity was tested against methicillin-
resistant S. aureus and Mycobacterium intracellulare.
Only naturally occurring formylated phloroglucinol
compounds, grandinol (2) and jensenone (3), exhibited
antibacterial activity against methicillin-resistant S.
aureus, while the rest of the analogues were inactive.
Ciprofloxacin was included as positive control for com-
parison (Table 3).
The antifungal activities were evaluated against a panel
of pathogenic fungi (C. albicans, Cryptococcus neofor-
mans, and Aspergillus fumigatus) associated with oppor-
Table 2. In vitro antileishmanial activity of O-alkylated acylphloroglucinols (11–19)c
a,b
Compound
Antileishmanial
activity against
Leishmania
Cytotoxicity IC50
R1
HO
R2
O
A
R
donovani
OHB
a,b
IC50
a,b
IC90
R
R1
R2
Vero cells
LLC-PK1 cells
11
12
–CH2CH@CH2
–CH2CH@CH2
–CH2CH@CH2
–CH2CH@CH2
–CH2CH2CH(CH3)2
–CH2CH2CH(CH3)2
–CH2CH2CH(CH3)2
–CH2CH2CH(CH3)2
–CH2CH2CH(CH3)2
CHO
CHO
COCH3
COCH3
COCH2CH(CH3)2
COCH3
na
5.3
na
14
na
nc
17.0
nc
nc
nc
nc
nc
nc
4.6
nt
nc
31
9.0
nc
13
14
na
COCH2CH(CH3)2
CHO
CHO
COCH2CH(CH3)2
COCH3
36
nc
nc
15
16
7.8
4.2
40
>40
>40
>40
>40
36
COCH2CH(CH3)2
COCH3
nc
nc
17
18
COCH3
COCH2CH(CH3)2
CHO
COCH2CH(CH3)2
CH2CH2CH(CH3)2
24
10
nc
19
11.5
nt
Pentamidine
Amphoterecin B
Doxorubicin
1.5
0.32
nt
4.9
0.9
nt
nt
nt
7.5
0.65
a IC50 and IC90, the concentration that affords 50% and 90% inhibition of leishmanial growth.
b Values expressed in terms of lg/mL; nc, not cytotoxic up to 23.8lg/mL; na, not active at 40 lg/mL.
c None of the compounds were active against bacteria/fungi; nt, not tested.

Table 3. In vitro antibacterial, antifungal, antileishmanial, and antimalarial activities of formylated acylphloroglucinols (2–3 and 29–37)
a,c,d
IC50
Entry
R1
HO
R2
OH
R3
OH
R1
R2
R3
Methicillin-resistant
Staphylococcus aureus
Candida
albicans
Cryptococcus
neoformans
Leishmania
donovani
Plasmodium
falciparum
D6/W2
Cytotoxicity
against
Vero cells
Cytotoxicity
against
LLC-PK1 cells
b,c
)
(IC50a/IC90
2
COCH2CH(CH3)2
COCH2CH3
COCH3
CHO
CHO
CHO
CHO
CHO
CHO
CH3
CH3
8
na
na
na
na
na
na
na
na
20
na
na
0.10
nt
10
5.0
15
na
na
na
na
2.0
na
5.5
na
na
nt
10
20
na
na
15
na
na
2.5
na
na
na
na
nt
21/39
na
na
na
na
8.0
nc*
nc*
14
7.0
nc*
nc*
9.5
11.0
10.0
11.0
nc
29
30
31
32
33
4
CH3
H
na
na
COCH2CH(CH3)2
COCH2CH3
COCH3
27/40
na
na
H
na
na
18.0
17.5
12.0
nc
H
COCH3
COCH3
CHO
CHO
CHO
CHO
CHO
H
27/>40
29/>40
16/>40
19/35
na
na
na
34
35
3
COC(CH3)@CHOH
COC(CH3)@CHOH
COCH2CH(CH3)2
CH2CH2CH(CH3)2
CH3
H
CH2CH@C(CH3)2
na
na
19
14.5
nc*
nc*
nc*
nt
CHO
CHO
CHO
nc*
nc*
nc*
nt
36
37
na
na
na
nt
Ciprofloxacin
Amphoterecin B
Pentamidine
Artemisinin
nt
0.3
nt
0.7
nt
0.32/ 0.9
1.5/ 4.9
nt
nt
nt
nt
nt
nt
nt
nt
nt
nt
nt
14.0/ 7.5
13.5/ 140
nt
nt
nt
Chloroquine
Doxorubicin
nt
nt
nt
nt
nt
nt
nt
nt
nt
nt
7.5
0.65
nc*, not cytotoxic up to 4.76 lg/mL; na, not active at 20 lg/mL (for MRS, Candida and Cryptococcus); nt, not tested.
a IC50, the concentration that affords 50% inhibition of bacterial/fungal/leishmanial growth.
b IC90, the concentration that affords 90% inhibition of leishmanial growth.
c Values expressed as lg/mL.
d None of the compounds showed activity against Mycobacterium intracellulare and Aspergillus fumigatus; nc, not cytotoxic up to 23.8 lg/mL.

92
S. B. Bharate et al. / Bioorg. Med. Chem. 15 (2007) 87–96
tunistic infections. Amphoterecin B was included as a
standard drug for comparison. None of the O-alkylated
acylphloroglucinols showed any antifungal activity to-
ward these pathogens.
4.1. General method for O-alkylation: synthesis of 21 and
22
To a suspension of sodium hydride (2 mmol, previous-
ly washed with hexane to remove fats) in anhydrous
DMF (25 mL), a solution of phloroglucinol (20,
1.0 mmol) in anhydrous DMF (30 mL) was added at
0 ꢁC and stirred at room temperature for 2 h. This
solution was cooled again at 0 ꢁC and alkyl halide
(1.2 mmol) was added slowly and the resulting mixture
was stirred at room temperature for 8–10 h. Reaction
mixture was diluted with water; extracted with ethyl
acetate; washed with brine solution and finally dried
over anhydrous sodium sulfate. Solvent evaporation
on vacuo rotavapor resulted in crude product, which
was purified by silica gel column chromatography
using hexane/EtOAc (70:30) to give O-alkylated
phloroglucinols, 21 and 22.
Among the formylated acylphloroglucinols, jensenone
(3) exhibited antifungal activity with an IC50 value
of 5.5 lg/mL against C. albicans, while it was inac-
tive against C. neoformans and A. fumigatus. Howev-
er, grandinol (2) exhibited comparatively weaker
antifungal activity with IC50 value of 10 lg/mL
against C. albicans and C. neoformans. Among syn-
thetic analogues, compound 34 showed improved
antifungal activity against C. albicans and C. neofor-
mans with an IC50 of 2.0 and 2.5 lg/mL, respective-
ly. Compounds 29 and 30 were moderately active
against C. albicans. None was active against
Aspergillus.
None of the acylphloroglucinols showed any toxicity to-
ward mammalian kidney fibroblasts (Vero) and kidney
epithelial (LLC-PK1) cells22 up to a concentration of
23.8 lg/mL, however, some of the formylated acylphl-
oroglucinols were moderately toxic with IC50s in the
range of 7–19 lg/mL.
4.1.1. 1-(2-Propenyloxy)-3,5-dihydroxybenzene (21).
Yield: 60%; Yellow oil. IR (neat): 3351, 2958, 1608,
1506, 1472, 1367, 1270, 1154, 1061 cmÀ1 1H NMR
.
(300 MHz, CDCl3): d 6.01 (s, 3H, 3· –Ar-H), 5.95 (m,
1H, –CH@CH2), 5.36 (d, J = 17.0 Hz, 1H, –CH@CH2),
5.25 (d, J = 10.4 Hz, 1H, –CH@CH2), 4.43 (d,
J = 5.2 Hz, 2H, –OCH2); CIMS: m/z 166.9 [M+1]+.
4.1.2. 1-(3-Methyl-butyloxy)-3,5-dihydroxybenzene (22).
Yield: 45%; dark brown viscous oil. IR (neat): 3356,
3. Conclusion
2957, 1607, 1471, 1506, 1471, 1366, 1153, 1050 cmÀ1
.
O-Alkylated acylphloroglucinols bearing formyl/iso-
valeryl functionality exhibited antileishmanial activity.
Amongst formylated acylphloroglucinols, naturally
occurring grandinol (2) and jensenone (3) exhibited
antileishmanial, antibacterial, and antifungal activities.
Antileishmanial activity of grandinol, jensenone, and
O-alkylated phloroglucinols is reported for the first
time. Antimicrobial and antileishmanial properties
associated with acylphloroglucinol compounds seem
to be dependent on the length of the acyl side chain.
Decrease in acyl chain length or replacement of acyl
with alkyl resulted in loss of activity.
1H NMR (300 MHz, CDCl3): d 5.99 (d, J = 1.7 Hz,
2H, 2 · Ar-H), 5.94 (d, J = 1.6 Hz, 1H, Ar-H), 3.83 (t,
J = 6.4 Hz, 2H, –OCH2–), 1.74 (sept, 1H, –CH–), 1.57
(q, J = 6.7, 13.4 Hz, 2H, –OCH2CH2–), 0.90 (d,
J = 6.3 Hz, 6H, 2· –CH3); CIMS: m/z 196.9 [M+1]+.
4.2. Synthesis of 1-(3-methyl-butyloxy)-4-(3-methyl-
butyl)-3,5-dihydroxybenzene (28)
A mixture of phloroglucinol (20, 10 g, 1 mmol), potas-
sium hydroxide (6.6 g, 1.5 mmol) and isoamyl iodide
(3 mL, 2 mmol) in methanol/water, 4:1 (100 mL) was
refluxed for 4 h. Reaction mixture was acidified with
dilute HCl; brine solution was added and extracted
with ethyl acetate. Ethyl acetate layer was washed
with brine solution and dried over anhydrous sodium
sulfate. Solvent evaporation on vacuo rotavapor
resulted in crude product which was purified by silica
gel column chromatography using hexane/EtOAc
(70:30) to afford 1-(3-methyl-butyloxy)-3,5-dihydroxy-
benzene (22) (2 g, 20%), 1-isopentyl 2,4,6-trihydroxy
benzene (27, 3.5 g, 35%) and 1-(3-methyl-butyloxy)-4-
(3-methylbutyl)-3,5-dihydroxy benzene (28) (300 mg,
5%).
4. Experimental
Melting points were recorded on capillary melting point
apparatus and are uncorrected. H NMR spectra were
1
recorded on 300 MHz Bruker FT-NMR (Avance
DPX300) spectrometer using tetramethylsilane as inter-
nal standard and the chemical shifts are reported in d
units. Mass spectra were recorded on either GCMS
(Shimadzu QP 5000 spectrometer) autosampler/direct
injection (EI/CI) or LCMS (APCI/ESI). All chromato-
graphic purifications were performed with silica gel
(60–120 mesh), whereas all TLC (silica gel) development
4.2.1. 1-Isopentyl-2,4,6-trihydroxybenzene (27). Yield:
35%; brown sticky solid. IR (neat): 3269, 2957,
2870, 1615, 1470, 1412, 1384, 1282, 1202, 1146,
was performed on silica gel coated (Merck Kiesel 60F254
,
0.2 mm thickness) sheets. All chemicals were purchased
from Sigma–Aldrich, SD fine chemicals, Lancaster, and
CDH. Solvents used for the chemical synthesis pur-
chased from commercial sources were of analytical grade
and were used without further purification unless other-
wise stated.
1
1112, 1025, 1013 cmÀ1. H NMR (300 MHz, CD3OD):
d 5.83 (s, 2H, 2 · Ar-H), 2.48 (t, J = 6.0 Hz, 2H, Ar-
CH2), 1.55 (m, 1H, –CH2CH–), 1.34 (m, 2H,
–CH2CH–), 0.93 (d, J = 6.6 Hz, 6H, 2· –CH3); CIMS:
m/z 196.9 [M+1]+.

S. B. Bharate et al. / Bioorg. Med. Chem. 15 (2007) 87–96
93
4.2.2.
1-(3-Methyl-butyloxy)-4-(3-methylbutyl)-3,5-
–CH3), 0.94 (d, J = 6.3 Hz, 6H, 2· –CH3); CIMS:
m/z 281 [M+1]+.
dihydroxybenzene (28). Yield: 5%; light brown solid;
mp 147–149 ꢁC. IR (KBr): 3410, 3328, 2952, 2871,
2376, 1628, 1609, 1532, 1442, 1406, 1366, 1206, 1153,
1112, 1018 cmÀ1. 1H NMR (300 MHz, CDCl3+CD3OD,
4:1): d 5.97 (s, 2H, 2 · Ar-H), 3.89 (t, J = 6.3 Hz, 2H,
–OCH2–), 2.53 (t, J = 8.1 Hz, 2H, Ar-CH2–), 1.78 (m,
2H, 2· –CH–), 1.62 (m, 2H, –OCH2CH2–CH), 1.40
(m, 2H, ArCH2-CH2–), 0.94 (d, J = 6.6 Hz, 6H, 2· –
CH3), 0.90 (d, J = 6.7 Hz, 6H, 2· –CH3); CIMS: 266.9
[M+1]+.
4.3.5. 1-(2-Propenyloxy)-2,4-diacetyl-3,5-dihydroxyben-
zene (13). Yield: 6%; white solid; mp 114–116 ꢁC; UV
(MeOH): kmax (loge) 267 nm (4.65). IR (neat): 3435,
3092, 1616 cmÀ1
.
1H NMR (300 MHz, CDCl3): d
16.26 (s, 1H, OHA), 14.80 (s, 1H, OHB), 6.05 (m, 1H,
CH@CH2), 5.90 (s, 1H, Ar-H), 5.45 (d, J = 17.0 Hz,
1H, –CH@CH2), 5.38 (d, J = 10.4 Hz, 1H, –CH@CH2),
4.64 (d, J = 5.7 Hz, 2H, –OCH2–), 2.72 (s, 3H,
–COCH3), 2.69 (s, 3H, –COCH3). 13C NMR (75 MHz,
CDCl3): d 204.4, 203.3, 171.9, 171.7, 166.0, 131.3,
119.7, 105.1, 104.3, 92.2, 70.2, 33.1, 33.0; CIMS: m/z
250.9 [M+1]+; analysis for C13H14O5 (250.2), calcd, C,
62.39; H, 5.64; found, C, 62.24; H, 5.79.
4.3. General method for Friedel-Crafts acylation:
synthesis of 23–26, 13–14 and 17–18
To the solution of O-alkylated phloroglucinols (21 and
22, 1 mmol) in DCM, titanium chloride and acyl chloride
(3 mmol) was added at 0 ꢁC and reaction mixture was stir-
red at room temperature for 30 min. The excess titanium
chloride was quenched with methanol (5 mL) and solvent
was removed from the reaction mixture under reduced
pressure. The crude reaction mixture was purified by silica
gel column chromatography using hexane/EtOAc (50:50)
as eluent to yield mono- (23–26) and di- (13–14 and 17–18)
acylphloroglucinols.
4.3.6. 1-(2-Propenyloxy)-2,4-di-(3-methyl-butyryl)-3,5-
dihydroxybenzene (14). Yield: 10%; yellow sticky solid;
UV (MeOH): kmax (loge) 276 nm (4.29). IR (neat):
2959, 1767, 1621, 1465, 1412, 1368, 1292, 1195, 1148,
1122 cmÀ1 1H NMR (300 MHz, CDCl3): d 16.50 (s,
.
1H, OHA), 14.94 (s, 1H, OHB), 6.09 (m, 1H,
CH@CH2), 5.85 (s, 1H, Ar-H), 5.49 (d, J = 17.3 Hz,
1H, CH@CH2), 5.41 (d, J = 10.4 Hz, 1H, CH@CH2),
4.61 (d, J = 5.5 Hz, 2H, –OCH2–), 2.98 (d,
J = 5.7 Hz, 2H, –COCH2), 2.87 (d, J = 5.95 Hz, 2H,
–COCH2), 2.23 (m, 2H, 2· –CH–), 0.99 (d,
J = 5.2 Hz, 6H, 2· –CH3), 0.96 (d, J = 5.9 Hz, 6H, 2·
–CH3); 13C NMR (75 MHz, CDCl3): d 207.3, 206.4,
172.5, 172.3, 166.1, 131.9, 120.4, 105.8, 104.8, 92.9,
70.7, 53.6, 53.5, 26.0, 25.4, 23.3, 23.2; CIMS: m/z
334.9 [M+1]+; analysis for C19H26O5 (334.4), calcd,
C, 68.24; H, 7.84; found, C, 68.35; H, 7.91.
4.3.1. 1-(2-Propenyloxy)-4-acetyl-3,5-dihydroxybenzene
(23). Yield: 35%; off white solid; mp 145–147 ꢁC. IR
(KBr): 3184, 2915, 1645, 1587, 1446, 1372, 1289, 1181,
1090, 1073, 1027 cmÀ1. H NMR (300 MHz, CDCl3): d
1
6.01 (m, 1H, –CH@CH2), 5.94 (s, 2H, 2 · Ar-H), 5.39
(d, J = 17.3 Hz, 1H, –CH@CH2), 5.31 (d, J = 10.7 Hz,
1H, –CH@CH2), 4.51 (d, J = 5.3 Hz, 2H, –OCH2), 2.70
(s, 3H, CH3); CIMS: m/z 208.9 [M+1]+.
4.3.2. 1-(2-Propenyloxy)-4-(3-methyl-butyryl)-3,5-dihydr-
oxybenzene (24). Yield: 20%; light brown viscous oil. IR
(neat): 3447, 2960, 2873, 1625, 1583, 1460, 1412, 1368,
1298, 1195, 1129,1045 cmÀ1. 1H NMR(300 MHz, CDCl3):
d 6.01 (m, 1H, –CH@CH2), 5.95 (s, 2H, 2· Ar-H), 5.41 (d,
J = 17.2 Hz, 1H, –CH@CH2), 5.30 (d, J = 10.9 Hz, 1H,
–CH@CH2), 4.49 (d, J = 5.2 Hz, 2H, –OCH2), 2.94 (d,
J = 6.8 Hz, 2H, –COCH2), 2.25 (m, 1H, –CH), 0.98 (d,
J = 6.4 Hz, 6H, 2· –CH3); CIMS: m/z 250.8 [M+1]+.
4.3.7. 1-(3-Methyl-butyloxy)-2,4-diacetyl-3,5-dihydroxy-
benzene (17). Yield: 12%; yellow solid; mp 69–71 ꢁC; UV
(MeOH): kmax (loge) 280 nm (4.14). IR (KBr): 2956,
2870, 1622, 1588, 1367, 1291, 1200, 1126 cmÀ1 1H
.
NMR (300 MHz, CDCl3): d 16.27 (s, 1H, OHA), 14.79
(s, 1H, OHB), 5.89 (s, 1H, Ar-H), 4.10 (t, J = 6.4 Hz,
2H, –OCH2–), 2.70 (s, 3H, –COCH3), 2.63 (s, 3H,
–COCH3), 1.86–1.73 (m, 3H, –CH2–CH–), 0.99 (d,
J = 6.1 Hz, 6H, 2· –CH3). 13C NMR (75 MHz, CDCl3):
d 204.9, 203.8, 172.6, 172.3, 167.2, 105.5, 104.8, 92.4,
68.7, 38.0, 33.5, 25.7, 23.0; CIMS: m/z 281 [M+1]+; anal-
ysis for C15H20O5 (280.3), calcd, C, 64.27; H, 7.19;
found, C, 64.13; H, 7.08.
4.3.3. 1-(3-Methyl-butyloxy)-4-(acetyl)-3,5-dihydroxy-
benzene (25). Yield: 40%; dark brown viscous oil. IR
(neat): 3374, 2956, 2863, 2361, 1619, 1588, 1429, 1367,
1286, 1194, 1128, 1050 cmÀ1 1H NMR (300 MHz,
.
CDCl3): d 5.91 (s, 2H, 2· Ar-H); 3.93 (t, J = 6.8 Hz,
2H, –OCH2), 2.66 (s, 3H, –COCH3), 1.77 (m, 1H,
–CH), 1.63 (m, 2H, –OCH2CH2), 0.98 (d, J = 6.8 Hz,
6H, 2· CH3); CIMS: m/z 239 [M+1]+.
4.3.8. 1-(3-Methyl-butyloxy)-2,4-di-(3-methyl-butyryl)-
3,5-dihydroxybenzene (18). Yield: 17%; yellow solid;
mp 65–67 ꢁC; UV (MeOH): kmax (loge) 271 nm (4.24).
IR (KBr): 3020, 2400, 1618, 1585, 1420 cmÀ1 1H
.
NMR (300 MHz, CDCl3): d 16.48 (s, 1H, OHA), 14.96
(s, 1H, OHB), 5.90 (s, 1H, Ar-H), 4.09 (t, J = 6.5 Hz,
2H, –OCH2–), 3.00 (d, J = 5.1 Hz, 2H, –COCH2–),
2.91 (d, J = 5.5 Hz, 2H, –COCH2–), 2.25 (m, 3H, 3·
–CH–), 0.98 (d, J = 6.0 Hz, 18H, 6· –CH3). 13C NMR
(75 MHz, CDCl3): d 207.3, 206.2, 172.6, 172.3, 166.8,
105.6, 104.8, 92.5, 68.5, 53.5, 38.1, 25.7, 25.4, 23.3,
22.9; CIMS: m/z 365 [M+1]+; analysis for C21H32O5
(364.5), calcd, C, 69.20; H, 8.85; found, C, 69.11; H,
8.77.
4.3.4.
1-(3-Methyl-butyloxy)-4-(3-methyl-butyryl)-3,5-
dihydroxybenzene (26). Yield: 35%; off white solid;
mp 168–172 ꢁC. IR (KBr): 3352, 2959, 2871, 1634,
1602, 1583, 1525, 1465, 1432, 1368, 1300, 1257,
1206, 1180, 1084, 1037, 1005 cmÀ1
.
1H NMR
(300 MHz, CDCl3): d 5.92 (s, 2H, 2· Ar-H), 3.98
(t, J = 6.7 Hz, 2H, –OCH2), 2.97 (d, J = 6.7 Hz, 2H,
–COCH2), 2.24 (m, 1H, –CH), 1.80 (m, 1H, –CH),
1.65 (m, 2H, –CH2), 0.98 (d, J = 6.4 Hz, 6H, 2·

94
S. B. Bharate et al. / Bioorg. Med. Chem. 15 (2007) 87–96
4.4. General method for Vilsmeier–Haack formylation:
synthesis of 11, 12, 15, 16 and 19
1H NMR (300 MHz, CDCl3): d 15.11 (s, 1H, OHB),
14.60 (s, 1H, OHA), 10.05 (s, 1H, CHO), 5.91 (s, 1H,
Ar-H), 4.09 (t, J = 6.1 Hz, 2H, –OCH2–), 2.99 (d,
J = 6.6 Hz, 2H, –COCH2–), 2.25 (m, 1H, –CH–), 1.79
(m, 1H, –CH–), 1.73 (m, 2H, –OCH2CH2–), 0.99 (d,
J = 4.0 Hz, 6H, 2· –CH3), 0.97 (d, J = 3.7 Hz, 6H, 2·
–CH3). 13C NMR (75 MHz, CDCl3): d 206.2, 191.7,
174.3, 169.7, 166.5, 104.4, 104.2, 92.5, 67.7, 52.6, 37.3,
25.1, 24.9, 22.7, 22.4; CIMS: m/z 308.8 [M+1]+; analysis
for C17H24O5 (308.3), calcd, C, 66.21; H, 7.84; found, C,
66.13; H, 7.87.
To the solution of 23–26 and 28 (1 mmol) in ethyl ace-
tate (15 mL) were added dimethyl formamide (1 mmol)
and phosphoryl chloride (1.1 mmol) at room tempera-
ture. Reaction mixture was further stirred for 30 min.
Reaction mixture was diluted with water; extracted with
ethyl acetate; washed with brine solution and finally
dried over Na2SO4. The crude product was purified by
column chromatography over silica gel using hexane/
EtOAc (70:30) as eluent to afford formylated acylphl-
oroglucinols (11–12, 15–16 and 19).
4.4.5. 1-(3-Methyl-butyloxy)-2-formyl-4-(3-methylbutyl)-
3,5-dihydroxybenzene (19). Yield: 70%; cream solid; mp
166–168 ꢁC; UV (MeOH): kmax (loge) 279 nm (4.19).
IR (KBr): 3175, 2957, 2872, 1650, 1592, 1505, 1470,
1443, 1385, 1322, 1286, 1265, 1210, 1146, 1124,
4.4.1. 1-(2-Propenyloxy)-4-acetyl-2-formyl-3,5-dihydr-
oxybenzene (11). Yield: 30%; cream white solid; mp
102–105 ꢁC; UV (MeOH): kmax (loge) 269 nm (4.43).
IR (KBr): 3705, 3563, 3092, 2929, 2592, 1741, 1612,
1082 cmÀ1 1H NMR (300 MHz, CDCl3): d 12.64 (s,
.
1277 cmÀ1
.
1H NMR (300 MHz, CDCl3): d 14.89 (s,
1H, OHA), 10.11 (s, 1H, CHO), 5.87 (s, 1H, Ar-H),
5.48 (br s, 1H, OHB), 3.99 (t, J = 6.4 Hz, 2H, –OCH2),
2.53 (t, J = 8.0 Hz, 2H, Ar-CH2), 1.82 (m, 2H, 2·
–CH), 1.71 (m, 4H, 2· –CH2), 0.97 (d, J = 4.0 Hz, 6H,
2· –CH3), 0.95 (d, J = 4.2 Hz, 6H, 2· –CH3). 13C
NMR (75 MHz, CDCl3): d 191.5, 164.2, 163.2, 161.4,
108.7, 105.3, 90.6, 66.6, 37.9, 37.5, 28.8, 25.0, 22.4,
22.3, 19.6; CIMS: m/z 294.9 [M+1]+; analysis for
C17H26O4 (294.4), calcd, C, 69.36; H, 8.90; found, C,
69.18; H, 9.02.
1H, OHB), 14.53 (s, 1H, OHA), 10.07 (s, 1H, CHO),
6.04 (m, 1H, –CH@CH2), 5.91 (s, 1H, Ar-H), 5.43 (d,
J = 17.4 Hz, 1H, –CH@CH2), 5.37 (d, J = 10.5 Hz, 1H,
–CH@CH2), 4.62 (d, J = 5.1 Hz, 2H, –OCH2–), 2.71
(s, 3H, –COCH3). 13C NMR (75 MHz, CDCl3): d
203.8, 191.7, 173.8, 169.8, 166.1, 131.1, 119.1, 104.3,
92.4, 69.6, 32.7; EIMS: m/z 236 [M]+; analysis for
C12H12O5 (236.2), calcd, C, 61.01; H, 5.12; found, C,
61.07; H, 5.01.
4.4.2. 1-(2-Propenyloxy)-4-(3-methyl-butyryl)-2-formyl-
3,5-dihydroxybenzene (12). Yield: 40%; light violet solid;
mp 96–98 ꢁC; UV (MeOH): kmax (loge) 270 nm (4.54).
IR (KBr): 3402, 3084, 2959, 2928, 2872, 1627,
4.5. Synthesis of 2,4,6-trihydroxy-3-(3-hydroxy-2-methyl-
acryloyl)-benzaldehyde (34)
To
a
solution of phloropropiophenone (1.0 g,
1579 cmÀ1
.
1H NMR (300 MHz, CDCl3): d 15.11 (s,
5.49 mmol) in ethyl acetate (10 mL), dimethylformam-
ide (2.12 mL, 27.45 mmol) and phosphoryl chloride
(2.56 mL, 27.45 mmol) was added and reaction mixture
were stirred at room temperature for 3 h. Water was
added to the reaction mixture and extracted with ethyl
acetate. Solvent was evaporated under reduced pressure
and crude product was purified by silica gel column
chromatography using hexane/EtOAc (70:30) as eluent
to afford 34. Yield: 60%; yellow solid; mp 149–151 ꢁC;
UV (CHCl3): kmax (loge) 276 nm (4.00). IR (KBr):
1H, OHB), 14.61 (s, 1H, OHA), 10.07 (s, 1H, CHO),
6.02 (m, 1H, –CH@CH2), 5.91 (s, 1H, Ar-H), 5.43 (d,
J = 17.2 Hz, 1H, –CH@CH2), 5.37 (d, J = 10.2 Hz, 1H,
–CH@CH2), 4.62 (d, J = 5.6 Hz, 2H, –OCH2–), 2.99
(d, J = 6.7 Hz, 2H, –COCH2–), 2.25 (m, 1H, –CH–),
0.99 (d, J = 6.3 Hz, 6H, 2· –CH3). 13C NMR
(75 MHz, CDCl3): d 206.3, 191.7, 174.2, 169.7, 165.9,
131.2, 119.1, 104.4, 92.5, 69.6, 52.7, 24.9, 22.7; EIMS:
277 [MÀ1]+; analysis for C15H18O5 (278.3), calcd, C,
64.74; H, 6.52; found, C, 64.70; H, 6.48.
3076, 2927, 1645, 1455, 1320, 1252, 1187, 1081 cmÀ1
.
1H NMR (CDCl3, 300 MHz): d 14.22 (s, 1H, OH),
12.37 (s, 1H, OH), 10.34 (s, 1H, CHO), 7.68 (s, 1H,
–CHOH), 6.28 (s, 1H, Ar-H), 2.00 (s, 3H, CH3). 13C
NMR (CDCl3): d 192.8, 182.6, 167.9, 167.4, 162.3,
152.4, 119.7, 106.5, 104.1, 94.5, 10.1; EIMS: m/z 220
(MÀH2O), 192, 163, 124, 69; analysis for C11H10O6
(238.2), calcd, C, 55.47; H, 4.23; found, C, 55.41; H,
4.16.
4.4.3.
1-(3-Methyl-butyloxy)-4-acetyl-2-formyl-3,5-
dihydroxybenzene (15). Yield: 30%; white solid; mp
130–132 ꢁC; UV (MeOH): kmax (loge) 269 nm (4.36).
IR (KBr): 3402, 3077, 2956, 2870, 1616, 1576 cmÀ1; H
1
NMR (300 MHz, CDCl3): d 14.88 (s, 1H, OHB), 14.52
(s, 1H, OHA), 10.02 (s, 1H, CHO), 5.91 (s, 1H, Ar-H),
4.10 (t, J = 6.7 Hz, 2H, –OCH2–), 2.70 (s, 3H,
–COCH3), 1.86 (m, 1H, –CH–), 1.74 (m, 2H,
–OCH2CH2–), 0.98 (d, J = 7 Hz, 6H, 2· –CH3). 13C
NMR (75 MHz, CDCl3): d 203.8, 191.7, 173.9, 169.8,
167.0, 104.3, 92.0, 67.7, 37.2, 32.6, 25.1, 22.4; CIMS:
m/z 266.9 [M+1]+; analysis for C14H18O5 (266.3), calcd,
C, 63.15; H, 6.81; found, C, 63.10; H, 6.71.
4.6. Synthesis of 2,4,6-trihydroxy-3-(3-hydroxy-2-methyl-
acryloyl)-5-(3-methyl-but-2-enyl)-benzaldehyde (35)
The mixture of 34 (0.2 g, 0.84 mmol) and sodium hy-
dride (0.06 g, 2.52 mmol) in dioxane (10 mL) was heated
at 60 ꢁC for 30 min. Prenyl bromide (0.145 mL,
1.26 mmol) was added and reaction mixture was stirred
further at 60 ꢁC for 2 h. Reaction mixture was allowed
to cool, filtered, and solvent evaporated under reduced
pressure and crude product was purified by silica gel
4.4.4. 1-(3-Methyl-butyloxy)-4-(3-methyl-butyryl)-2-for-
myl-3,5-dihydroxybenzene (16). Yield: 40%; off white sol-
id; mp 85–87 ꢁC; UV (MeOH): kmax (loge) 271 nm
(4.55). IR (KBr): 3077, 2958, 2863, 1621, 1494 cmÀ1
;

S. B. Bharate et al. / Bioorg. Med. Chem. 15 (2007) 87–96
95
column chromatography using hexane/EtOAc (80:20) as
4.9. Assay for in vitro antimicrobial activity
eluent to afford 35. Yield: 39%; white solid; mp 118–
120 ꢁC; UV (CHCl3): kmax (loge) 282 nm (4.03). IR
(KBr): 3564, 2925, 1645, 1444, 1297, 1270, 1188,
Susceptibility testing against C. albicans, C. neoformans,
methicillin-resistant S. aureus (MRS), and A. fumigatus
was performed using a modified version of the NCCLS
methods.23 Susceptibility testing against M. intracellu-
lare was done using the modified Alamar Blue procedure
of Franzblau et al.24 Samples (dissolved in DMSO) were
serially diluted using 0.9% saline and transferred in
duplicate to 96-well microplates. Microbial inocula were
prepared after comparison of the absorbance at 630 nm
of cell suspensions to the 0.5 McFarland standard and
diluting the suspensions in broth to afford recommended
inocula. Microbial inocula were added to the diluted
samples to achieve a final volume of 200 lL. Growth,
solvent, and media controls were included in each assay.
Plates were read at either 630 nm or 544ex/590em (M.
intracellulare) prior to and after incubation. Percent
growth was plotted versus test concentration to afford
the IC50.
1100 cmÀ1 1H NMR (CDCl3, 300 MHz): d 14.14 (s,
.
1H, OH), 12.61 (s, 1H, OH), 10.36 (s, 1H, CHO), 7.74
(s, 1H, @CHOH), 5.15 (t, J = 5.1 Hz, 1H, –CH2–
CH@C), 3.36 (d, J = 7.0 Hz, 2H, ArCH2-CH), 1.78 (s,
3H, CH3), 1.67 (s, 3H, CH3), 1.58 (s, 3H, CH3); 13C
NMR (75 MHz, CDCl3): d 193.2, 183.5, 166.6, 165.3,
160.2, 153.0, 133.3, 121.5, 119.9, 107.4, 106.7, 104.2,
26.3, 21.1, 18.3, 10.6; EIMS: m/z 288 [MÀH2O], 274,
245, 233, 193, 69; analysis for C16H18O6 (306.3), calcd,
C, 62.74; H, 5.92; found, C, 62.60; H, 5.80.
4.7. Assay for in vitro antileishmanial activity
Antileishmanial activity of the compounds was tested
in vitro against a culture of L. donovani promastigotes.
They were grown in RPMI 1640 medium supplemented
with 10% fetal calf serum (Gibco Chem. Co.) at 26 ꢁC.
A 3-day-old culture was diluted to 5 · 105 promasti-
gotes/mL. Drug dilutions were prepared directly in cell
suspension in 96-well plates. Plates were incubated at
26 ꢁC for 48 h and growth of leishmania promastigotes
was determined by Alamar blue assay as described earli-
er.20 Standard fluorescence was measured on a Fluostar
Galaxy plate reader (BMG Lab Technologies) at excita-
tion wavelength of 544 nm and emission wavelength of
590 nm. Pentamidine and Amphoterecin B were used
as the standard antileishmanial agents. IC50 and IC90 val-
ues were computed from dose-response curves generated
by plotting percent growth versus drug concentration.
4.10. Cytotoxicity assay
The in vitro cytotoxicity was determined against mam-
malian kidney fibroblasts (VERO) and kidney epithelial
(LLC-PK1) cells. The assay was performed in 96-well
tissue culture-treated plates as described earlier.22 Brief-
ly, cells were seeded to the wells of the plate (25,000
cells/well) and incubated for 24 h. Samples were added
and plates were again incubated for 48 h. The number
of viable cells was determined by neutral red assay.
IC50 values were determined from logarithmic graphs
of growth inhibition versus concentration. Doxorubicin
was used as a positive control, while DMSO was used as
vehicle control.
4.8. Assay for in vitro antimalarial activity
Acknowledgments
The assay is based on the determination of plasmodial
LDH activity. For the assay, a suspension of red blood
cells infected with D6 or W2 strains of P. falciparum
(200 lL, with 2% parasitemia and 2% hematocrit in
RPMI 1640 medium supplemented with 10% human ser-
um and 60 lg/mL Amikacin) was added to the wells of a
96well plate containing 10 lL of test samples diluted in
medium at various concentrations. The plate was placed
in a modular incubation chamber (Billups-Rothenberg,
CA) flushed with a gas mixture of 90% N2, 5% O2,
and 5% CO2, and incubated at 37 ꢁC, for 72 h. Parasitic
LDH activity was determined by using Malstatꢂ re-
agent (Flow Inc., Portland, OR) according to the proce-
dure of Makler and Hinrichs.21 Briefly, 20 lL of the
incubation mixture was mixed with 100 lL of the Mal-
statꢂ reagent and incubated at room temperature for
30 min. Twenty microliters of a 1:1 mixture of NBT/
PES (Sigma, St. Louis, MO) was then added and the
plate is further incubated in the dark for 1 h. The reac-
tion was then stopped by the addition of 100 lL of a 5%
acetic acid solution. The plate was read at 650 nm using
the EL-340 Biokinetics Reader (Bio-Tek Instruments,
Vermont). IC50 values were computed from the dose–re-
sponse curves. Artemisinin and chloroquine were includ-
ed in each assay as the drug controls. DMSO (0.25%)
was used as vehicle control.
I.P.S. is thankful to NIPER for start-up funds. S.B.B.,
N.A.M.Y., and S.K.C. are thankful to NIPER for fel-
lowship. USDA Agricultural Research Service Specific
Cooperative Agreement No. 58-6408-2-0009 is also
acknowledged for partial support of this work.
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