Biomimetic synthesis, antimicrobial, antileishmanial and antimalarial activities of euglobals and their analogues

Article abstract of DOI:10.1016/j.bmc.2005.10.027

In the present communication, naturally occurring phloroglucinol- monoterpene adducts, euglobals G1-G4 (3b/a and 4a/b) and 16 new analogues (13a/b-18a/b and 19-22) were synthesized by biomimetic approach. These synthetic compounds differ from natural euglobals in the nature of monoterpene and acyl functionality. All of these compounds were evaluated for their antibacterial, antifungal, antileishmanial and antimalarial activities. Analogue 17b possessed good antibacterial activity against methicillin-resistant Staphylococcus aureus, while analogues 19-22 possessed potent antifungal activity against Candida glabrata with IC₅₀s ranging from 1.5 to 2.5 μg/mL. Euglobals along with all synthesized analogues exhibited antileishmanial activity. Amongst these, euglobal G2 (3a), G3 (4a) and analogues 13a and 14a showed potent antileishmanial activity with IC₅₀s ranging from 2.8 to 3.9 μg/mL. Analogue 16a possessed antimalarial activity against chloroquine sensitive D6 clone of Plasmodium falciparum. None of the compounds showed toxicity against mammalian kidney fibroblasts (vero cells) upto the concentration of 4.76 μg/ml.

Full text of DOI:10.1016/j.bmc.2005.10.027

Bioorganic & Medicinal Chemistry 14 (2006) 1750–1760
Biomimetic synthesis, antimicrobial, antileishmanial
and antimalarial activities of euglobals and their analogues
Sandip B. Bharate,a Kamlesh K. Bhutani,a Shabana I. Khan,b Babu L. Tekwani,b
Melissa R. Jacob,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 2005; revised 14 October 2005; accepted 15 October 2005
Available online 7 November 2005
Abstract—In the present communication, naturally occurring phloroglucinol–monoterpene adducts, euglobals G1–G4 (3b/a and 4a/
b) and 16 new analogues (13a/b–18a/b and 19–22) were synthesized by biomimetic approach. These synthetic compounds differ from
natural euglobals in the nature of monoterpene and acyl functionality. All of these compounds were evaluated for their antibacterial,
antifungal, antileishmanial and antimalarial activities. Analogue 17b possessed good antibacterial activity against methicillin-resis-
tant Staphylococcus aureus, while analogues 19–22 possessed potent antifungal activity against Candida glabrata with IC50s ranging
from 1.5 to 2.5 lg/mL. Euglobals along with all synthesized analogues exhibited antileishmanial activity. Amongst these, euglobal
G2 (3a), G3 (4a) and analogues 13a and 14a showed potent antileishmanial activity with IC50s ranging from 2.8 to 3.9 lg/mL. Ana-
logue 16a possessed antimalarial activity against chloroquine sensitive D6 clone of Plasmodium falciparum. None of the compounds
showed toxicity against mammalian kidney fibroblasts (vero cells) upto the concentration of 4.76 lg/ml.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
ed; it is characterized by its effect on the internal organs,
particularly the liver, the spleen and the bone marrow.1
Parasitic diseases such as leishmaniasis, malaria and try-
panosomiasis have a significant impact in developing
countries and affect millions of people and are cause
of high mortality rate. Leishmaniasis is a group of trop-
ical diseases caused by a number of species of protozoan
parasites belonging to the genus Leishmania. This ail-
ment affects around 12 million people in 80 countries
and it is estimated that there are about two to three mil-
lion new cases each year. It is also considered that pres-
ently there exists a population of 350 million people
under risk of infection.1 Moreover, a person with HIV
infection whose immune system is suppressed is at high
risk of leishmaniasis. Visceral leishmaniasis is the most
severe clinical form of the disease caused by parasite
Leishmania donovani and it can be fatal when not treat-
Leishmaniasis remains a significant health issue in large
part of the world due to the lack of effective and afford-
able drugs and increasing resistance against existing
drugs. The available chemotherapeutic agents for treat-
ment of leishmaniasis include antimony compounds
(e.g., sodium stibogluconate and meglumine antimonite)
and amphotericin B. However, the antimonials cause
serious side effects that include pain at the site of injec-
tion, stiff joints, gastrointestinal problems, cardiotoxic-
ity and, in some cases, hepatic and renal insufficiency.
These drugs also require lengthy treatments and their
cost is rather high. Amphotericin B is also associated
with a number of side effects, including the alteration
of the renal function in approximately 80% of treated
individuals. Amongst the natural products, different
classes of secondary metabolites have been reported to
possess antileishmanial activity.2 These include qui-
nones, alkaloids (quinoline, isoquinoline, indole, and
steroidal), terpenes and phenolics (chalcones, flavonoids
and lignans). Although several phloroglucinols have
been reported to possess antiparasitic activities such as
Keywords: Euglobals; Biomimetic synthesis; Antimicrobial; Anti-
leshmanial; antimalarial.
*
Corresponding author. Tel.: +91 172 2214682; fax: +91 172
2214692; e-mail: ipsingh@niper.ac.in
NIPER communication number 363.
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.10.027

S. B. Bharate et al. / Bioorg. Med. Chem. 14 (2006) 1750–1760
1751
antimalarial robustadials,3 to our knowledge none of the
phloroglucinol derivatives have been evaluated for
antileishmanial activity.
Naturally occurring phloroglucinol compounds possess
a number of biological activities such as antibacterial,
antimalarial, antiviral and antifouling, etc.6 This class
of compounds is of wide occurrence in myrtaceae fami-
ly, particularly in Eucalyptus spp. and also in Hypericum
spp. Phloroglucinol compounds of Eucalyptus spp. in-
clude monomeric compounds such as grandinol (1),
jensenone (2), phloroglucinol-monoterpene adducts
such as euglobals and phloroglucinol–sesquiterpene ad-
ducts such as macrocarpals. Grandinol and jensenone
are proposed to be the key precursors in the biogenesis
of many bioactive phloroglucinol molecules found in
Eucalyptus.6 These include Epstein–Barr virus inhibitory
euglobals7 (3a, 3b, 4a, 4b), antimalarial robustadials3
(5a, 5b), antibacterial and anti-HIV macrocarpals8,9 (6)
and antifouling sideroxylonals (7) and grandinal10 (8).
Apart from antimalarial robustadials, a number of other
acyl phloroglucinols (robustaol3 (9), sarothralen11 (10),
japonicine12 (11) and O-prenylated phloroglucinol
derivative13 (12)) have also been reported to possess
antimalarial activity (Fig. 1).
There are a number of antileishmanial drugs available
but to date none of them has been demonstrated to be
fully effective against Leishmania parasites. Therefore,
there is an urgent need to search for new classes of
antileishmanial agents that are fully effective against
Leishmania parasites.2
Malaria is another most important parasitic disease in
tropical areas. Four parasitic species of the genus Plas-
modium infect human beings, but two cause majority of
the infections. Nearly all malaria deaths and a large
proportion of morbidity are caused by Plasmodium fal-
ciparum. More than a third of the worldÕs population
(about two billion people) lives in malaria-endemic
areas. Majority of deaths caused by falciparum malaria
occur in sub-Saharan Africa, primarily among children
younger than 5 years and pregnant women living in re-
mote rural areas with limited access to health services.4
The estimates for annual malaria mortality range from
0.5 to 3.0 million people. This increase in mortality can
be attributed to spread of Plasmodium falciparum that
is resistant to antimalarial drugs. In last few decades,
resistance to several antimalarials became widely dis-
seminated, while the cost of effective treatment is pro-
hibitive for the large majority of the populations in
these areas. Thus, rapid development of resistance by
Plasmodium falciparum to the conventional drugs like
chloroquine necessitates search for new antimalarial
drugs.4 Similarly, increasing number of multidrug-resis-
tant microbial pathogens have become a serious prob-
lem particularly during the last decade and provide
impetus for the search and discovery of novel antibac-
terial and antifungal agents active against these
pathogens.5
Euglobals constitute an important group of bioactive
phloroglucinol compounds. Structurally euglobals differ
from each other in the nature of terpenoid moiety, with
nine different terpenes implicated in the formation of
about thirty naturally known euglobals.14
The euglobals G1 (3b), G2 (3a), G3 (4a) and G4 (4b)
have a pinane skeleton as the terpenic component.
Euglobals G1 and G2 show a-pinene fused to dihydro-
pyran ring, while Euglobal G3 and G4 have b-pinene
joined to pyran ring to form spiro compounds. These
have been isolated from leaves of Eucalyptus grandis
and have structural similarity with antimalarial robusta-
dials. Euglobals G3 (4a) and G4 (4b) differ from
robustadials (5a and 5b) in the location of isobutyl
chain, present either on aromatic ring or the pyran ring.
R1
O
R1
O
CHO
CHO
HO
OH
OH
HO
O
HO
R2
O
HO
R2
O
H
OHC
OHC
H
H
OH
OH
O
OH
O
OH
3a: R1 = H, R2 = COCH2CH(CH3)2 4a: R1 = H, R2 = COCH2CH(CH3)2
3b: R1 = COCH2CH(CH3)2, R2 = H 4b: R1 = COCH2CH(CH3)2, R2 = H
5a: 4α
5b: 4β
6
CHO
CHO
HO
OH
HO
OH
CHO
OH
CHO
OH
CH3
CHO
OH
HO
O
HO
O
OH
HO
OCH3
O
HO
O
O
CHO
CHO
OH
OH
OHC
OH
8
7
9
O
CH3
HO
OH
HO
OH
O
OH
O
O
OH
HO
OH
O
O
O
O
O
OH
OH
O
10
11
12
Figure 1. Bioactive phloroglucinol compounds.

1752
S. B. Bharate et al. / Bioorg. Med. Chem. 14 (2006) 1750–1760
CHO
HO
O
O
Euglobal G1(3b) and G2 (3a)
Euglobal G3 (4a) and G4 (4b)
CHO
OH
HO
O
OH
CHO
HO
O
O
OH
1
OH
Figure 2. Proposed biogenesis of euglobals G1–G2 (3b/a) and G3–G4 (4a/b).
Biogenetically, the robustadials and euglobals have been
proposed to be formed by Diels–Alder type cycloaddi-
tion of O-quinone methides derived from oxidation of
phloroglucinol derivatives jensenone (2) or grandinol
(1) with various mono- or sesqui-terpenes as depicted
in Figure 2.14
aluminium chloride resulted in formation of acyl
phloroglucinols (24 and 25) in 70% yield. Acyl phlorog-
lucinols (24 and 25) upon Vilsmeier–Haack formylation
using phosphorus oxychloride and dimethylformamide
resulted in the formation of formylated acyl phloroglu-
cinols (26 and 27), which upon methylation with iodom-
ethane in the presence of potassium hydroxide resulted
in formation of C-methylated products, 1 and 28 (over-
all yield from 23, 25%).15,16
Based on the interesting biological activity profile of
these phloroglucinol–terpene adducts, we planned to
synthesize natural and unnatural euglobals by biomi-
metic approach. Some of the structural analogues of
euglobals were designed and synthesized for evaluation
as antimicrobial, antileishmanial and antimalarial
agents. These included adducts with monoterpenes not
encountered in natural euglobals.
In order to synthesize diformylated phloroglucinol–ter-
pene adducts (19–22), the key precursor 31 was essen-
tial and has been synthesized from 23 in three steps.
Formylation of 23 with Vilsmeier–Haack reagent
resulted in the formation of 29, which upon deoxygen-
ation with sodium cyanoborohydride led to formation
of 2,4,6-trihydroxytoluene, 30. The conversion of 30
to 31 was found to be challenging since the introduc-
tion of one formyl group onto an aromatic ring deac-
tivates the phloroglucinol nucleus for second
formylation. A number of reactions and different
experimental conditions (POCl3/DMF, CH(OC2H5)3/
DCM) were tried to achieve diformylation of 30 to
obtain 31 but in all cases monoformylated product
was predominating. The most exciting and striking re-
sults were obtained with modified Duff reaction.
Refluxing of 30 with two equivalents of hexamethyl-
ene tetramine in trifluoroacetic acid for 20 h at 70 ꢁC
resulted in exclusive formation of diformylated prod-
uct 31 in 40% yield.16
2. Results and discussion
2.1. Synthesis of euglobals and their analogues
We have synthesized naturally occurring phloroglucin-
ol–terpene adducts, euglobals G1–G4 (3b/a and 4a/b)
and analogues (13a/b–18a/b and 19–22) by biomimetic
approach in four steps starting from commercially avail-
able phloroglucinol (23). Our synthetic strategy involves
initial synthesis of the key precursors required for syn-
thesis of target phloroglucinol–terpene adducts. Synthe-
sis of key precursors, 1, 28, and 31 is shown in Scheme 1.
The Friedel–Crafts acylation of 23 in the presence of
CHO
CHO
HO
OH
HO
OH
HO
OH
HO
OH
a
b
c
R
R
R
CH3
OH
OH
OH
O
OH
O
O
24: R = CH2CH(CH3)2
25: R = CH3
23
26: R = CH2CH(CH3)2
27: R = CH3
1: R = CH2CH(CH3)2
28: R = CH3
b
CHO
HO
OH
HO
OH
HO
OH
d
e
CHO
CH3
OHC
CH3
OH
OH
OH
29
30
31
Scheme 1. Reagents and conditions: (a) RCOCl, AlCl3, 50 ꢁC, 70%; (b) POCl3, DMF, rt, 70%; (c) CH3I, KOH, MeOH, 70 ꢁC, 50%; (d) NaBH3CN,
THF, pH 4, rt, 15 h, 60%; (e) (CH2)6N4, TFA, 70 ꢁC, 43%.

S. B. Bharate et al. / Bioorg. Med. Chem. 14 (2006) 1750–1760
1753
R1
O
3a : R1 = H, R2 = CH2CH(CH3)2
3b : R1 = CH2CH(CH3)2, R2 = H
15a : R1 = H, R2 = CH3
15b : R1 = CH3, R2 = H
19 : R1 = H, R2 = H
10
O
4
5
AHO
6
7
4a
3
R2
2
α-Pinene
8
9
1
H
O
OHB
R1
O
4a : R1 = H, R2 = CH2CH(CH3)2
4b : R1 = CH2CH(CH3)2, R2 = H
16a : R1 = H, R2 = CH3
1
O
1'
8
6'
AHO
5'
2'
2
7
6
4'
R1
O
3'
R2
O
3
β-Pinene
16b : R1 = CH3, R2 = H
5
4
AHO
R2
OH
20 : R1 = H, R2 = H
OHB
a
CH3
R1
O
O
OHB
13a : R1 = H, R2 = CH2CH(CH3)2
13b : R1 = CH2CH(CH3)2, R2 = H
17a : R1 = H, R2 = CH3
17b : R1 = CH3, R2 = H
21 : R1 = H, R2 = H
H
3
8
H
2
9
4
1
: R1 = H, R2 = CH2CH(CH3)2
28 : R1 = H, R2 = CH3
31 : R1 = H, R2 = H
AHO
O
5
1a
H
2a
1
6
R2
3-Carene
9a
7
H
H
O
OHB
R1
O
6'
14a : R1 = H, R2 = CH2CH(CH3)2
14b : R1 = CH2CH(CH3)2, R2 = H
18a : R1 = H, R2 = CH3
18b : R1 = CH3, R2 = H
22 : R1 = H, R2 = H
1'
3
1
O
8
5'
4'
AHO
7
6
2'
2
3'
Camphene
R2
5
4
O
OHB
Scheme 2. Reagents and conditions: (a) PhNO2, DDQ, 50 ꢁC, 1 h.
After synthesis of the precursors, the key step involved
in the synthesis of phloroglucinol–terpene adducts is
the oxidative activation of compounds, 1, 28, and
31 with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone
(DDQ) in nitromethane leading to the generation of
corresponding O-quinone methides which further
undergo intermolecular cycloaddition with monoter-
penes such as a-pinene, b-pinene, (+)-3-carene and
(ꢀ)-camphene (Scheme 2). Several strategies have been
employed to generate O-quinone methides for cycload-
dition with unactivated alkenes. These include conven-
tional17 as well as electrochemical synthesis18
performed on the surface of PTFE-fibre coated elec-
trode and with the use of metallated 2-alkenyl sulfoxi-
mines.19 We synthesized four natural euglobals and 16
new analogues using biomimetic approach and the
structures were established by extensive spectral data
Diels–Alder reaction of 1 and its acetyl analogue 28
with terpenes resulted in the formation of a pair of
regioisomers which differ from each other in the loca-
tion of formyl and acyl moieties on the aromatic ring.
These regioisomers were separated by preparative TLC
(hexane/EtOAc = 98: 2). The separated regioisomeric
pairs, 13a/13b and 14a/14b, were characterized by 2D
NMR experiments in which the position of isovaleryl
and formyl group was confirmed by HMBC
correlations.
When the separation was attempted on reverse-phase
high performance liquid chromatography coupled with
mass spectrometry (RPHPLC–MS), it has been no-
ticed that, the isomer possessing acyl moiety b- to
the pyran oxygen gets eluted earlier as compared to
the other isomer. We also observed a typical pattern
of chemical shift values for formyl group and the
hydroxyl group between formyl and acyl in 1H
1
viz., H NMR, MS and IR. The point of attachment
of terpene moiety onto the pyran ring has been deter-
mined by the DEPT analysis and location of formyl
and acyl functionalities was established by extensive
2D NMR spectral data, viz., HSQC and HMBC.
1
NMR spectrum as shown in Table 1. H NMR spec-
tra of euglobals (3a, 4a) and analogues (13a–18a) in
which formyl group is b- to the pyran oxygen showed
peaks ranging from d 14.30 to d 14.47 for OHA and
peaks ranging from d 9.93 to d 10.06 for formyl
group, while regioisomers (3b, 4b, 13b–18b) of these,
in which acyl functionality is at b-position to the pyr-
an oxygen showed peaks ranging from d 13.10 to
13.45 and d 10.17 to 10.20 for OHA and formyl
group, respectively. The chemical shifts for OHB are
fairly identical in all the regioisomers ranging from d
15.10 to 15.45. These observations can be generalized
as this has been the case for almost 30 naturally
occurring14 as well as 16 synthetic compounds report-
ed in this paper. This observation makes the structure
elucidation of this class of compounds simpler without
the need of 2D NMR experiments.
Diels–Alder reaction of 1 with b-pinene in the presence
of DDQ in different solvents viz., dioxane, tetrahydro-
furan, dimethyl formamide, dimethyl sulfoxide and
methanol has been attempted, however, none of these
yielded the expected products. Products were obtained
only with nitromethane as a solvent. Reactions were
also performed under microwave conditions. Reaction
of
1 with b-pinene under microwave conditions
(750 W, 1 min) using nitromethane as a solvent resulted
in the formation of 4a and 4b in a yield of 15%. How-
ever, same reaction when performed using conventional
heating, products 4a and 4b were formed in a yield of
30%.

1754
S. B. Bharate et al. / Bioorg. Med. Chem. 14 (2006) 1750–1760
Table 1. Parameters to distinguish pair of regioisomers
1H NMR (d values)b
a
Entry
RP-LCMS tR
CHO
OHA
OHB
3a/3b
4a/4b
10.54/8.42
12.60/10.08
11.63/8.97
13.56/10.85
6.59/5.68
7.72/6.46
6.75/5.76
7.66/6.17
9.93/10.20
10.01/10.18
10.06/10.19
10.00/10.20
9.94/10.20
14.36/13.14
14.43/13.18
14.47/13.21
14.38/13.15
14.28/13.16
14.35/13.20
14.39/13.22
14.30/13.19
15.34/15.43
15.37/15.37
15.40/15.36
15.36/15.38
15.13/15.20
15.13/15.31
15.15/15.17
15.12/15.44
13a/13b
14a/14b
15a/15b
16a/16b
17a/17b
18a/18b
10.02/10.17
10.06/10.19
10.01/10.19
a Retention time in minutes.
b 1H NMR chemical shift values for formyl and hydroxyl groups (OHA and OHB) on aromatic ring.
In general, the Diels–Alder reaction between unsymmet-
rical dienophile and unsymmetrical diene takes place in
two ways to give two isomeric products. However, for-
mation of one of the isomer, is strongly favoured
depending upon the electronic nature of substituent. In
case of [4 + 2] Diels–Alder reaction between O-quinone
methide and 3-carene, presence of the methyl group
onto the double bond results in the formation of isomer
13a-I rather than 13a-II. Similarly in case of Diels–Alder
reaction of O-quinone methide with camphene, presence
of the ring residues onto double bond favours the for-
mation of 1,2-adduct (14a-I) rather than 1,3-adduct
(14a-II).20a
trum should have been present. Absence of any signal
around d 4.0 ruled out the possibility of 14a-II. This
was also confirmed by the DEPT experiment which
showed C-2 as singlet indicating its quaternary nature.
In 14a-II, C-2 should have been a triplet (Fig. 4).
It has been reported that in Diels–Alder reaction, cis-
product is the main product at moderate temperature,
but the proportion of trans-product increases with rise
in temperature.20a As we carried out Diels–Alder reac-
tions at 50 ꢁC, the cis-products are expected to be
formed in Diels–Alder reactions of a-pinene and 3-car-
ene. This is supported by our previous work20b as well
as cis stereochemistry reported for all naturally occur-
ring monoterpene adducts.14c
The point of attachment of the terpene moiety onto the
chroman ring has been identified on the basis of 1H
NMR chemical shifts. In the case of Diels–Alder reac-
tion of 1 with 3-carene, two possibilities arise with re-
spect to orientation of terpene moiety. Structures 13a-I
and 13a-II as shown in Figure 3 can be formed. In
The essential oil of Eucalyptus is a rich source of mono-
terpenes which mainly include 1,8-cineole (50–90%), a-
pinene (15-50%) and a-terpeneol (1–8%) in most of the
species but the percentage of these varies from species
to species. Apart from these major monoterpenes, euca-
lyptus oil also contains a number of other terpenoids
such as limonene, 3-carene, camphene, and citronellal,
citronellol in minor amounts. 3-Carene occurs in Euca-
lyptus radiata (2.7%), Eucalyptus dives (0.6%), Eucalyp-
tus bakeri (0.32%), Eucalyptus smithi (0.2%),
Eucalyptus polybractea (0.09%) and also in Eucalyptus
globulus. Camphene occurs in Eucalyptus gigantangion
(2.2%) and also in Eucalyptus polybractea (0.065%),
Eucalyptus smithi (0.053%) and Eucalyptus bakeri
(0.012%).21 As euglobals are proposed to be formed
biogenetically from Diels–Alder cycloaddition between
1
13a-II, triplet for 2a-H around d 4.0 in the H NMR
spectrum should have been present. Absence of any sig-
nal around d 4.0 ruled out the possibility of 13a-II. This
was confirmed by DEPT experiments which showed C-
2a as a singlet at d 76.9 indicating quaternary nature
of this oxycarbon. In 13a-II, this oxycarbon should have
been a doublet.
Similarly in the case of Diels–Alder reaction of 1 with
camphene, the alternative structure 14a-II has been ru-
led out based on H NMR data. In 14a-II, singlet for
1
1
methylenoxy protons around d 4.0 in H NMR spec-
CHO
CHO
CHO
CHO
AHO
O
O
AHO
O
O
AHO
O
O
AHO
O
O
OHB
OHB
OHB
OHB
CHO
CHO
CHO
CHO
H
AHO
O
AHO
O
O
2
AHO
O
O
AHO
O
2
2a
2a
H
O
OHB
OHB
OHB
O
OHB
14a-I
13a-I
13a-II
14a-II
Figure 3. Diels–Alder cycloaddition of grandinol (1) with 3-carene.
Figure 4. Diels–Alder cycloaddition of grandinol (1) with camphene.

S. B. Bharate et al. / Bioorg. Med. Chem. 14 (2006) 1750–1760
1755
O-quinone methides generated from phloroglucinol
derivatives, grandinol or jensenone and monoterpenes,
the possibility of 13a/b and 14a/b being discovered at
some stage from Eucalyptus containing these terpenes
(3-carene and camphene) in a major amount is not ruled
out, and hence these may be hitherto undiscovered nat-
ural products. Compounds 13a/b and 14a/b are structur-
ally related to natural euglobals, however these have
new skeleton that have not been reported so far, so we
propose to call these unnatural euglobals as S-euglobals
(S-for synthetic).
Diformylated euglobal analogues, 19–22, exhibited most
potent antifungal activity against C. glabrata with IC50s
of 2.5, 1.5, 1.5 and 2.5 lg/mL, respectively, while
amongst acyl-formyl euglobal analogues, only 16a and
3a exhibited antifungal activity against C. glabrata with
IC50s of 3.0 and 8.0 lg/ml, respectively. Analogues 20
and 21 also exhibited activity against C. krusei at much
higher concentrations. Amphotericin B was included, as
a standard drug, for comparison.
Antileishmanial activity against L. donovani promasti-
gotes was determined by the Alamar Blue assay.24 All
the compounds exhibited antileishmanial activity with
IC50 and IC90 values ranging from 3.6 to 24 lg/ml and
7.8 to 40 lg/ml, respectively, as shown in Table 3. Two
important structure-activity correlations were observed
from the results. The compounds possessing an isovale-
ryl functionality on the aromatic ring (3a/b and 4a/b and
13a/b–14a/b) showed activity with IC50s ranging from
3.6 to 14 lg/mL compared to analogues with an acetyl
functionality (15a/b–18a/b) which showed IC50s ranging
from 4.3 to 18 lg/mL. Another interesting difference in
activity was noticed between the pair of regioisomers.
The regioisomers in which formyl functionality is at b-
to the pyran oxygen (3a–4a and 13a–18a) showed better
antileishmanial activity than those in which acyl func-
tionality is at the b-position to pyran oxygen (3b–4b
and 13b–18b). Thus, compounds (3a, 4a, 13a and 14a)
comprising a combination of these two favorable struc-
tural requirements (presence of isovaleryl moiety and
formyl b- to the pyran oxygen) are the most potent with
IC50s ranging from 2.8 to 3.9 lg/mL and IC90s ranging
from 7.8 to 24 lg/ml. Another important aspect is the
position of double bond in monoterpene prior to cyclo-
addition. Terpenoids (a-pinene and 3-carene) containing
2.2. Biological evaluation
Susceptibility of S. aureus and methicillin-resistant S.
aureus to test compounds was determined according to
the procedure as described by the National Committee
for Clinical Laboratory Standards (NCCLS).22 Suscep-
tibility of M. intracellulare was done using the modified
Alamar Blue procedure of Franzblau et al.23 As shown
in Table 2, euglobal G4 (4b) and analogues 14b and
17b exhibited good antibacterial activity against methi-
cillin-resistant S. aureus with IC50s of 10, 15 and 3 lg/
mL, respectively, while none of the compounds pos-
sessed activity against M. intracellulare (data not
shown).Ciprofloxacin was used as positive control for
comparison.
The antifungal activities against pathogenic fungi asso-
ciated with opportunistic infections (Candida albicans,
Candida glabrata, Candida krusei, Cryptococcus neofor-
mans and Aspergillus fumigatus) were also evaluated.
Most of the compounds were active against C. glabrata,
a few against C. krusei, while none showed activity
against C. albicans, C. neoformans and A. fumigatus.
Table 2. In vitro antibacterial and antifungal activities of euglobals
and their analogues
Table 3. In vitro antileishmanial activities of euglobals and their
analogues
a,b
Entry
IC50
Antibacterial activity
Methicillin-resistant
a
Entry
IC50a(lg/mL)
IC90 (lg/mL)
Antifungal activity
3a
3b
3.6
7.1
3.9
12
24
29
S. aureus
4a
4b
24
32
C. glabrata C. krusei
3a
4a
NA
NA
10
8
16
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
15
13a
13b
2.8
6.2
3.6
14
7.8
27
4b
13a
NA
20
14a
14b
15
33
NA
15
14b
15b
NA
10
15a
15b
9.4
17
30
34
NA
NA
NA
3
16a
17a
3
15
16a
16b
16
16
>40
34
17b
18a
NA
3.5
2.5
1.5
1.5
2.5
0.20
—
17a
17b
9.5
14
30
33
NA
NA
NA
NA
NA
—
19
20
18a
18b
4.3
18
20
37
21
22
10
19
20
24
21
>40
40
NA
0.70
—
Amphotericin B
Ciprofloxacin
21
22
19
14
36
34
0.10
Pentamidine
Amphoterecin B
0.9
0.35
3.0
0.8
NA, not active.
a IC50: The concentration that affords 50% inhibition of bacterial/
fungal growth.
b Values expressed in terms of lg/mL.
a IC50 and IC90, sample concentrations that kill 50% and 90% cells
compared to solvent controls.

1756
S. B. Bharate et al. / Bioorg. Med. Chem. 14 (2006) 1750–1760
endocyclic double bond lead to the formation of adducts
with xanthan skeleton (e.g., 3a/b) in which terpenoid is
attached in a linear fashion. Adducts formed by terpenes
containing exocyclic double bond (b-pinene and camph-
ene) contain spiroskeleton. Euglobals with xanthan skel-
eton 3a/b showed a comparatively higher activity with
spiro-euglobals 4a/b. Phenolic compounds, such as chal-
cones, aurones and hydroxynaphthoquinones, are
reported to affect ultrastructure and function of the par-
asite mitochondria, thus exerting their effect on parasite
respiratory chain.2b Euglobals contain both phenolic
and formyl groups and may be acting through a similar
mechanism.
antileishmanial activity compared to adducts formed
from monoterpenes with exocyclic double bond (b-pi-
nene and camphene). Euglobal analogues bearing a dif-
ormyl functionality possessed potent antifungal activity,
while other analogues bearing an acyl–formyl function-
ality possessed weaker or were devoid of antifungal
activity, indicating the role of diformyl functionality
for antifungal activity. Thus, it can be concluded that
euglobal analogues bearing long chain acyl and formyl
functionalities at d and b- to the pyran oxygen, respec-
tively, provide an interesting lead in our search for
newer antileishmanial agents, while diformyl euglobal
analogues are the promising leads for antifungal drug
discovery.
Since, phloroglucinol class of compounds have never
been evaluated for their antileishmanial activity, inter-
esting activity profile of natural, as well as synthetic
phloroglucinol-monoterpene adducts, opens up a new
class of antileishmanial compounds.
4. Experimental
Melting points were recorded with a capillary melting
point apparatus and are uncorrected. H NMR spectra
1
In vitro antimalarial activity was evaluated against chlo-
roquine sensitive (D6) and chloroquine resistant (W2)
clones of P. falciparum. Analogue 16a exhibited antima-
larial activity against D6 clone of P. falciparum with an
IC50 of 2.3 lg/mL. Rest of the analogues were inactive.
Determination of in vitro antimalarial activity was
based on the determination of plasmodial LDH
activity.25
are recorded on a 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). Elemental
analyses were recorded on Elementar Vario EL spec-
trometer. The HPLC analysis was carried out on Phe-
nomenex C18 column (250 · 4.6 mm) connected to a
Shimadzu (USA manufacturing Inc) HPLC system
consisting of a model LC-10AT VP fitted with 20 ll
injection loop and a model SPD-M10A VP photodiode
array detector. The analysis was carried out using
MeOH/water/acetic acid—100:5:3 as a mobile phase
with the flow rate of 1.7 ml/min. Class-VP software
(Shimadzu) was used for data collection. All chromato-
graphic purifications were performed with silica gel
(60–120 mesh), whereas all TLC (silica gel) develop-
ment 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 purchased from commercial sources were of
analytical grade and were used without further purifi-
cation unless otherwise stated.
None of the compounds were found to have any cyto-
toxic effects towards mammalian kidney fibroblasts
(vero cells) up to a concentration of 4.76 lg/ml (data
not shown).26,27
More importantly, from this work euglobals have
emerged as a promising new class of antileishmanial
compounds and further work to synthesize and evaluate
more compounds from this class using monocyclic and
bicyclic monoterpenes is in progress.
3. Conclusion
In conclusion, euglobals G1–G4 (3b/a–4a/b), S-euglo-
bals (13a/b–14a/b) and other structural analogues were
synthesized in four steps from phloroglucinol. The S-
euglobals were designed and synthesized so as to incor-
porate those bicyclic monoterpenes not encountered in
natural euglobals. Also compounds were designed and
synthesized by varying the acyl and formyl functional-
ities. Euglobals and their analogues possessed in vitro
antileishmanial activity against L. donovani promasti-
gotes. Amongst these, 3a, 4a, 13a and 14a showed the
most potent activity. The presence of isovaleryl moiety
and formyl functionality b- to the pyran oxygen seems
to be necessary for potent activity, as analogues with
acetyl functionality and acyl moiety b- to the pyran oxy-
gen showed a comparatively weaker activity. Decrease
in the acyl chain length from isovaleryl to acetyl to
formyl resulted in decrease in antileishmanial activity.
Adducts formed by Diels–Alder cycloaddition of O-qui-
none methide with monoterpenes containing an endocy-
clic double bond (a-pinene and 3-carene) showed better
4.1. General method for Friedel–Crafts acylation:
synthesis of 24–25
A solution of phloroglucinol (23, 1 mmol) and anhy-
drous aluminium chloride (3.17 g, 3 mmol) in carbon
disulfide (10 mL) was stirred at room temperature for
20 min. Nitrobenzene (15 mL) was added and the tem-
perature of the reaction mixture was allowed to increase
up to 50 ꢁC. Acyl chloride (3 mmol) was added and the
reaction mixture was stirred further for 30 min. Nitro-
benzene was removed from the reaction mixture under
reduced pressure and crude reaction mixture was
purified by silica gel column chromatography using hex-
ane/EtOAc (50:50) as eluent to yield acyl phlorogluci-
nols (24–25). Compounds were characterized by
comparing their spectral data and melting point with lit-
erature values.15

S. B. Bharate et al. / Bioorg. Med. Chem. 14 (2006) 1750–1760
1757
4.2. General method for Vilsmeier–Haack formylation:
synthesis of 29, 26 and 27
4.5.1. 3,5-Diformyl-2,4,6-trihydroxy toluene (31). Yield:
43%; light yellow solid; mp 188–190 ꢁC; 1H NMR
(300 MHz, CDCl3+CD3OD): d 10.09 (s, 2H), 2.01 (S,
To the solution of phloroglucinol or acyl phlorogluci-
nols (23–25) (1 mmol) in ethyl acetate (15 mL) were add-
ed dimethylformamide (1 mmol) and phosphoryl
chloride (1.1 mmol) at room temperature. The reaction
mixture was further stirred for 30 min. Water was added
to the reaction mixture and extracted with ethyl acetate.
Ethyl acetate layer was washed with brine solution and
dried over Na2SO4 and concentrated to afford the crude
product. Column chromatography over silica gel using
hexane/EtOAc (70:30) as eluent provided formyl phlor-
oglucinol (29) or formyl acyl phloroglucinols (26–27).
Compounds were characterized by comparing their
spectral data and melting point with literature
values.15,28
3H); IR (KBr) mmax: 3119, 1622, 1257, 1182 cmꢀ1
CIMS: m/z 197 [M+1]+.
;
4.6. General method for Diels–Alder cycloaddition: syn-
thesis of 3a/b–4a/b, 13a/b–18a/b and 19–22
To the solution of 4, 28 or 31 (1 mmol) and DDQ
(1 mmol) in nitromethane (10 mL) was added monoter-
pene (1 mmol) and the reaction mixture was stirred at
50 ꢁC for 2 h. The solvent was evaporated on rotavapor
and the crude product was purified by silica gel (# 60–
120) column chromatography to get a viscous liquid as
a mixture of two isomers. These isomers were separated
by preparative thin layer chromatography (hexane/
EtOAc = 98:2) to afford 3a/b and 4a/b and 13a/b–18a/
b. In case of reaction of monoterpenes with 31, a single
product was obtained and was purified by column chro-
matography on silica gel using hexane/EtOAc (95:5) to
afford 19–22. Euglobals G1–G4 (3b/a and 4a/b) were
characterized by comparing their spectral data with lit-
erature values.14
4.3. General method for methylation: synthesis of 1 and 28
To the solution of formylated acyl phloroglucinols (26
and 27, 1 mmol) and potassium hydroxide (2 mmol) in
methanol (10 mL) was added methyl iodide (5 mmol).
The reaction mixture was refluxed for 2 h. Solvent was
evaporated and the crude product was purified by silica
gel column chromatography using hexane/EtOAc
(80:20) as eluent to afford 1, 28. Compounds were char-
acterized by comparing their spectral data and melting
point with literature values.15
4.6.1. 5,7-Dihydroxy-1,1,2a-trimethyl-6-(3-methyl-butan-
oyl)-1,1a,2,2a,8,8a,9,9a-octahydro-3-oxa-cyclopropa[b]-
anthracene-4-carbaldehyde (13a). Yield: 19%; brownish
yellow oil; 1H NMR (300 MHz, CDCl3): d 15.40 (s,
1H, OHB), 14.47 (s, 1H, OHA), 10.06 (s, 1H, CHO),
2.99 (d, J = 6.6 Hz, 2H), 2.58 (dd, J = 5.8, 16.7 Hz,
1H), 2.39–2.24 (m, 3H), 2.16 (m, 1H), 2.04–1.88 (m,
1H), 1.76–1.48 (m, 3H), 1.41–1.28 (m, 2H), 1.26 (s,
3H), 1.21 and 1.00 (s, 3H each), 0.98 (d, J = 7.7 Hz,
6H); 13C NMR (75 MHz, CDCl3): d 206.4, 191.9,
172.4, 168.2, 162.4, 104.2, 103.6, 99.0, 76.9, 52.7, 32.2,
31.5, 29.7, 28.6, 25.1, 24.8, 23.2, 22.8, 19.1, 16.5; IR
(neat) mmax: 3620, 3020, 2400, 1619, 1424, 1216,
1046 cmꢀ1; CIMS: m/z 387 [M+1]+, 251 [MꢀC10H16];
analysis for C23H30O5 (386.2), calcd: C, 71.48; H, 7.82;
found: C, 71.37; H, 7.98.
4.4. General method for deoxygenation: synthesis of 30
To the solution of 1-formyl-2,4,6-trihydroxy benzene
(29, 1 mmol) in tetrahydrofuran (20 mL) was added
methyl orange (1–2 drops) followed by addition of
sodium cyanoborohydride (3 mmol). The pH of the
reaction mixture was maintained at 4.0 throughout
the reaction by addition of 1 N HCl. The reaction mix-
ture was stirred at room temperature for 12 h and then
extracted with ethyl acetate. Ethyl acetate layer was
washed with brine solution and finally dried over
Na2SO4. Solvent was evaporated to get the crude prod-
uct and the column chromatography over silica using
hexane/EtOAc (50:50) as eluent afforded 2,4,6-trihy-
droxy toluene (30). The compound was characterized
by comparing its spectral data and melting point with
literature values.29
4.6.2. 5,7-Dihydroxy-1,1,2a-trimethyl-4-(3-methyl-butan-
oyl)-1,1a,2,2a,8,8a,9,9a-octahydro-3-oxa-cyclopropa[b]-
anthracene-6-carbaldehyde (13b). Yield: 17%; yellow
1
sticky solid; H NMR (300 MHz, CDCl3): d 15.36 (s,
1H, OHB), 13.21 (s, 1H, OHA), 10.19 (s, 1H, CHO),
2.97 (d, J = 9.8 Hz, 2H), 2.63 (dd, J = 5.8, 16.8 Hz,
1H), 2.42–2.20 (m, 4H), 2.01 (m, 1H), 1.78–1.68 (m,
2H), 1.57–1.53 (m, 1H), 1.44–1.25 (m, 2H), 1.26 (s,
3H), 1.24 and 1.02 (s, 3H each), 0.98 (d, J = 7.0 Hz,
6H); 13C NMR (75 MHz, CDCl3): d 206.0, 192.4,
169.9, 168.0, 163.5, 104.5, 98.0, 76.6, 53.1, 32.4, 31.0,
29.7, 28.6, 25.3, 24.8, 23.3, 23.0, 22.9, 22.7, 19.0, 16.7;
IR (neat) mmax: 3401, 2926, 2856, 1613, 1447, 1419,
1313, 1192, 1156 cmꢀ1; CIMS: m/z 387 [M+1]+, 251
[MꢀC10H16]; analysis for C23H30O5 (386.2), calcd, C,
71.48; H, 7.82; found, C, 71.21; H, 7.55.
4.5. General method for Duff formylation: synthesis of 31
A mixture of 2,4,6-trihydroxy toluene (30, 1 mmol)
and hexamethylene tetramine (2 mmol) in anhydrous
trifluoroacetic acid (5 mL) was refluxed for 20 h. The
reaction mixture was cooled to room temperature
and then poured into 4 N HCl (10 mL) and stirred
for 10 min. The product was extracted with ethyl ace-
tate. The combined ethyl acetate layer was washed
with 4 N HCl, brine solution and then dried over
Na2SO4. The solvent evaporated under reduced pres-
sure and the crude product was purified by silica gel
column chromatography using hexane/EtOAc (90:10)
as eluent to afford 3,5-diformyl-2,4,6-trihydroxy tolu-
ene (31).
4.6.3.
3,4-Dihydro-5,7-dihydroxy-30,30-dimethyl-6-(3-
methyl-butanoyl)-spiro-[2H-1-benzopyran-2,20-bicyclo-
[2.2.1]heptane]-8-carboxaldehyde (14a). Yield: 14%;
yellow oil; 1H NMR (300 MHz, CDCl3): d 15.36 (s,

1758
S. B. Bharate et al. / Bioorg. Med. Chem. 14 (2006) 1750–1760
1H, OHB), 14.38 (s, 1H, OHA), 10.00 (s, 1H, CHO), 2.97
(d, J = 6.7 Hz, 2H), 2.73–2.53 (m, 1H), 2.42–2.32 (m,
1H), 2.31–2.20 (m, 2H), 2.18–2.08 (m, 1H), 2.06–1.98
(m, 1H), 1.96–1.73 (m, 2H), 1.72–1.53 (m, 1H), 1.51–
1.41 (m, 1H), 1.38–1.31 (m , 1H), 1.26 and 1.07 (s, 3H
each), 0.98 (d, J = 6.6 Hz, 6H); 13C NMR (CDCl3,
75 MHz): d 206.4, 191.6, 171.4, 168.2, 162.1, 104.5,
103.3, 101.5, 90.1, 52.7, 49.4, 45.1, 34.4, 29.7, 25.2,
25.1, 23.7, 22.8, 22.5, 22.3, 16.8; IR (neat) mmax: 3373,
4.6.7. 6-Acetyl-3,4-dihydro-5,7-dihydroxy-60,60-dimethyl-
spiro-[2H-1-benzopyran-2,20-bicyclo[3.1.1]-heptane]-8-
carboxaldehyde (16a). Yield: 12%; brown solid; mp 107–
1
109 ꢁC; H NMR (300 MHz, CDCl3): d 15.13 (s, 1H,
OHB), 14.35 (s, 1H, OHA), 10.02 (s, 1H, CHO), 2.70
(s, 3H), 2.55 (t, J = 6.5 Hz, 2H), 2.26 (m, 1H), 2.16 (t,
J = 4.5 Hz, 1H), 2.04–1.95 (m, 5H), 1.91–1.81 (m, 2H),
1.62 (d, J = 10 Hz, 1H), 1.29 and 1.02 (s, 3H each);
13C NMR (75 MHz, CDCl3): d 203.8, 191.7, 170.8,
168.4, 162.1, 104.3, 103.3, 101.0, 85.0, 49.6, 40.6, 38.2,
32.6, 31.8, 28.5, 27.5, 26.5, 24.7, 23.2, 15.3; IR (KBr)
2928, 1621, 1425, 1293, 1216, 1178, 1124, 1038 cmꢀ1
;
CIMS: m/z 387 [M+1]+, 251 [MꢀC10H16]; analysis for
C23H30O5 (386.2) calcd: C, 71.48; H, 7.82; found: C,
71.21; H, 7.62.
m
max: 3568, 2923, 1621, 1444, 1295, 1203, 1105 cmꢀ1
;
CIMS: m/z 345 [M+1]+, 209 [MꢀC10H16]; analysis for
C20H24O5 (344.2) calcd: C, 69.75; H, 7.02; found; C,
69.40; H, 6.92.
4.6.4. 3,4-Dihydro-5,7-dihydroxy-30,30-dimethyl-8-(3-meth-
yl-butanoyl)-spiro-[2H-1-benzopyran-2,20-bicyclo[2.2.1]hep-
tane]-6-carboxaldehyde (14b). Yield: 13%; yellow oil; H
4.6.8. 8-Acetyl-3,4-dihydro-5,7-dihydroxy-60,60-dimethyl-
spiro-[2H-1-benzopyran-2,20-bicyclo[3.1.1]heptane]-6-car-
boxaldehyde (16b). Yield: 10%; cream colored solid; mp
1
NMR (300 MHz, CDCl3): d 15.38 (s, 1H, OHB), 13.15
(s, 1H, OHA), 10.20 (s, 1H, CHO), 3.06–2.95 (m, 2H),
2.70–2.58 (m, 1H), 2.53–2.38 (m, 1H), 2.40–2.22 (m,
2H), 2.12–1.93 (m, 2H), 1.92–1.72 (m, 3H), 1.70–1.58
(m, 2H), 1.55–1.45 (m, 1H), 1.44–1.36 (m, 1H), 1.26
(s, 3H), 1.06 (s, 3H), 0.98 (d, J = 4.3 Hz, 6H); 13C
NMR (CDCl3, 75 MHz): d 205.4, 192.5, 169.7, 166.8,
164.0, 105.0, 104.3, 101.2, 93.0, 52.9, 50.1, 46.3, 35.0,
29.7, 27.2, 24.4, 23.5, 23.4, 22.8, 22.6, 22.5, 16.5; IR
(neat) mmax: 3620, 3026, 2400, 1602, 1522, 1476, 1423,
1323, 1214, 1046 cmꢀ1; CIMS: m/z 387 [M+1]+, 251
[MꢀC10H16]; analysis for C23H30O5 (386.2) calcd: C,
71.48; H, 7.82; found: C, 71.10; H, 7.52.
1
137–140 ꢁC; H NMR (300 MHz, CDCl3): d 15.31 (s,
1H, OHB), 13.20 (s, 1H, OHA), 10.17 (s, 1H, CHO),
2.65 (s, 3H), 2.57 (m, 2H), 2.28 (m, 1H), 2.19 (t,
J = 4.7 Hz, 1H), 2.06–1.88 (m, 7H), 1.57 (d,
J = 6.6 Hz, 1H), 1.29 and 1.03 (s, 3H each); 13C NMR
(75 MHz, CDCl3): d 203.3, 192.4, 170.0, 167.2, 163.5,
104.7, 104.1, 100.3, 86.8, 48.9, 40.4, 38.3, 33.0, 31.3,
28.9, 27.5, 27.0, 24.7, 23.3, 15.2; IR (KBr) mmax: 3568,
2915, 2869, 1630, 1425, 1320, 1172, 1146 cmꢀ1; CIMS:
m/z 345 [M+1]+, 209 [MꢀC10H16]; analysis for
C20H24O5 (344.2) calcd: C, 69.75; H, 7.02; found: C,
68.87; H, 6.74.
4.6.5. 7-Acetyl-2,3,4,4a,9,9a-hexahydro-6,8-dihydroxy-
3,3,4a-trimethyl-2,4-methano-1H-xanthene-5-carboxal-
4.6.9. 6-Acetyl-5,7-dihydroxy-1,1,2a-trimethyl-1,1a,2,
2a,8,8a,9,9a-octahydro-3-oxa-cyclopropa[b]anthracene-4-
1
dehyde (15a). Yield: 14%; reddish brown viscous oil; H
1
carbaldehyde (17a). Yield: 16%; yellow sticky solid; H
NMR (300 MHz, CDCl3): d 15.13 (s, 1H, OHB), 14.28
(s, 1H, OHA), 9.94 (s, 1H, CHO), 2.71 (s, 3H), 2.72–2.68
(m, 2H), 2.48–2.41 (dd, J = 6.5, 15.9 Hz, 1H), 2.25 (t,
J = 5.4 Hz, 1H), 2.19–2.06 (m, 2H), 1.98 (m, 1H), 1.90
(m, 1H), 1.47 (s, 3H), 1.32–1.22 (m, 1H), 1.31 and 1.10
(s, 3H each); 13C NMR (75 MHz, CDCl3): d 203.9,
191.7, 170.9, 168.4, 164.6, 103.8, 103.6, 100.8, 87.7, 54.9,
44.2, 40.6, 34.5, 32.7, 32.0, 28.8, 28.2, 27.9, 22.7, 19.7;
IR (neat) mmax: 3401, 2922, 2849, 1625, 1449, 1377, 1299,
1205, 1119, 1020 cmꢀ1; CIMS: m/z 345 [M+1]+, 209
[MꢀC10H16]; analysis for C20H24O5 (344.2) calcd: C,
69.75; H, 7.02; found: C, 69.24; H, 7.42.
NMR (300 MHz, CDCl3): d 15.15 (s, 1H, OHB), 14.39
(s, 1H, OHA), 10.06 (s, 1H, CHO), 2.71 (s, 3H), 2.71–
2.69 (m, 1H), 2.58 (dd, J = 5.6, 16.7 Hz, 1H), 2.39–
2.28 (m, 3H), 2.04 (br s, 1H), 1.73 (m ,2H), 1.42–1.21
(m, 1H), 1.21 (s, 3H), 1.01 and 0.95 (s, 3H each); 13C
NMR (75 MHz, CDCl3): d 203.9, 192.0, 172.0, 168.4,
162.6, 104.2, 103.7, 98.9, 77.2, 32.7, 32.2, 31.5, 30.2,
28.6, 24.8, 23.1, 22.8, 19.1, 16.5; IR (KBr) mmax: 3403,
2924, 1624, 1443, 1281, 1204, 1108, 1021 cmꢀ1; CIMS:
m/z 345 [M+1]+, 209 [MꢀC10H16]; analysis for
C20H24O5 (344.2) calcd: C, 69.75; H, 7.02; found: C,
69.51; H, 6.93.
4.6.6. 5-Acetyl-2,3,4,4a,9,9a-hexahydro-6,8-dihydroxy-
3,3,4a-trimethyl-2,4-methano-1H-xanthene-7-carboxal-
4.6.10. 4-Acetyl-5,7-dihydroxy-1,1,2a-trimethyl-1,1a,2,
2a,8,8a,9,9a-octahydro-3-oxa-cyclopropa[b] anthracene-
6-carbaldehyde (17b). Yield: 15%; yellow sticky solid;
1H NMR (300 MHz, CDCl3): d 15.17 (s, 1H, OHB),
13.22 (s, 1H, OHA), 10.19 (s, 1H, CHO), 2.71 (s, 3H),
2.70–2.66 (m, 1H), 2.62 (dd, J = 5.6, 16.4 Hz, 1H),
2.41–2.31 (m, 3H), 2.02 (br s, 1H), 1.78–1.68 (m ,2H),
1.44–1.24 (m, 1H), 1.24 (s, 3H), 1.02 and 0.96 (s, 3H
each); 13C NMR (75 MHz, CDCl3): d 203.5, 192.3,
169.7, 168.4, 163.8, 104.4, 98.1, 77.2, 33.1, 32.4, 31.0,
1
dehyde (15b). Yield: 12%; yellow sticky solid; H NMR
(300 MHz, CDCl3): d 15.20 (s, 1H, OHB), 13.16 (s,
1H, OHA), 10.20 (s, 1H, CHO), 2.80–2.60 (m, 2H),
2.56 (s, 3H), 2.40 (dd, J = 5.9, 15.3 Hz, 1H), 2.29 (t,
J = 5.4 Hz, 1H), 2.15 (m, 2H), 2.02 (m, 1H), 1.90 (m,
1H), 1.51 (s, 3H), 1.32–1.24 (m, 1H), 1.31 and 1.10 (s,
3H each); 13C NMR (75 MHz, CDCl3): d 203.4, 192.3,
170.1, 167.2, 166.3, 104.5, 103.9, 100.6, 89.2, 55.3,
44.1, 40.3, 33.8, 32.2, 31.8, 29.2, 28.1, 27.5, 22.7, 19.8;
IR (neat) mmax: 3402, 2924, 2849, 2325, 1628, 1559,
1447, 1252, 1049, 1021 cmꢀ1; CIMS: m/z 345 [M+1]+,
209 [MꢀC10H16]; analysis for C20H24O5 (344.2) calcd:
C, 69.75; H, 7.02; found: C, 69.51; H, 6.89.
29.7, 28.5, 25.0, 23.0, 22.6, 18.9, 16.6; IR (Neat) mmax
:
3401, 2924, 2856, 1618, 1454, 1312, 1200, 1112,
1020 cmꢀ1; CIMS: m/z 345 [M+1]+, 209 [MꢀC10H16];
analysis for C20H24O5 (344.2) calcd: C, 69.75; H, 7.02;
found: C, 69.62; H, 6.90.

S. B. Bharate et al. / Bioorg. Med. Chem. 14 (2006) 1750–1760
1759
4.6.11. 6-Acetyl-3,4-dihydro-5,7-dihydroxy-30,30-dimethyl-spi-
analysis for C19H22O5 (330.1), calcd; C, 69.07; H, 6.71;
found: C, 68.96; H, 6.59.
ro-[2H-1-benzopyran-2,20-bicyclo[2.2.1]heptane]-8-carboxalde-
hyde (18a). Yield: 16%; yellow oil; H NMR (300 MHz,
1
CDCl3): d 15.12 (s, 1H, OHB), 14.30 (s, 1H, OHA),
10.01 (s, 1H, CHO), 2.70 (s, 3H), 2.66 (dd, J = 5.6,
16.7 Hz, 1H), 2.50–2.26 (m, 1H), 2.24 (d, J = 4.6 Hz,
1H), 2.10–2.01 (m, 2H), 1.89 (m, 1H), 1.75–1.60 (m,
3H), 1.50–1.24 (m, 2H), 1.12 and 1.02 (s, 3H each);
13C NMR (CDCl3, 75 MHz): d 203.9, 191.9, 170.9,
169.2, 162.4, 104.6, 103.3, 101.9, 90.2, 49.4, 45.2, 34.4,
4.6.15. 5,7-Dihydroxy-1,1,2a-trimethyl-1,1a,2,2a,8,8a,
9,9a-octahydro-3-oxa-cyclopropa[b]anthracene-4,6-dicar-
1
baldehyde (21). Yield: 20%; yellow brown oil; H NMR
(300 MHz, CDCl3): d 13.47 and 13.24 (s, 1H each,
OHA and OHB), 10.16 and 10.08 (s, 1H each, CHO),
2.57 (dd, J = 5.8, 16.6 Hz, 1H), 2.36–2.28 (m, 2H),
2.04–1.95 (m, 2H), 1.74–1.56 (m, 3H), 1.39–1.25 (m,
1H), 1.23 (s, 3H), 1.01 and 0.96 (s, 3H each); 13C
NMR (75 MHz, CDCl3): d 192.1, 191.6, 169.9, 168.1,
164.1, 104.3, 103.9, 98.6, 79.1, 32.2, 31.3, 29.7, 28.6,
32.9, 29.7, 25.2, 23.6, 22.5, 22.3, 16.67; IR (Neat) mmax
:
;
3435, 2963, 1628, 1446, 1372, 1296, 1198, 1107 cmꢀ1
CIMS: m/z 345 [M+1]+, 209 [MꢀC10H16]; analysis for
C20H24O5 (344.2) calcd: C, 69.75; H, 7.02; found: C,
69.61; H, 7.00.
27.6, 24.9, 22.7, 22.6, 19.0, 16.4; IR (Neat) mmax
:
3343, 3019, 2930, 2401, 1632, 1521, 1426, 1216,
1046 cmꢀ1; CIMS: m/z 331 [M+1]+, 195 [MꢀC10H16];
analysis for C19H22O5 (330.1) calcd: C, 69.07; H,
6.71; found: C, 69.01; H, 6.62.
4.6.12. 8-Acetyl-3,4-dihydro-5,7-dihydroxy-30,30-dimethyl-
spiro-[2H-1-benzopyran-2,20-bicyclo[2.2.1]heptane]-6-carbox-
aldehyde (18b). Yield: 15%; yellow solid; mp 135–138 ꢁC;
1H NMR (300 MHz, CDCl3): d 15.44 (s, 1H, OHB),
13.19 (s, 1H, OHA), 10.19 (s, 1H, CHO), 2.71 (s, 3H),
2.66 (m, 1H), 2.48 (m, 1H), 2.26 (d, J = 4.6 Hz, 1H),
2.09–1.99 (m, 2H), 1.90–1.87 (m, 2H), 1.86–1.81 (m,
1H), 1.66–1.61 (m, 2H), 1.51–1.46 (m, 1H), 1.43–1.37
(m, 1H), 1.10 and 1.06 (s, 3H each); 13C NMR (CDCl3,
75 MHz): d 203.1, 192.4, 170.0, 167.2, 164.1, 104.7,
104.2, 101.5, 93.1, 50.1, 46.8, 35.0, 33.2, 29.7, 27.0,
23.5, 23.3, 22.9, 22.5, 16.4; IR (KBr) mmax: 3568, 2939,
2881, 1627, 1421, 1376, 1317, 1142 cmꢀ1; CIMS: m/z
345 [M+1]+, 209 [MꢀC10H16]; analysis for C20H24O5
(344.2) calcd: C, 69.75; H, 7.02; found: C, 69.83; H, 6.91.
4.6.16. 3,4-Dihydro-5,7-dihydroxy-30,30-dimethyl-spiro-
[2H-1-benzopyran-2,20-bicyclo[2.2.1]heptane]-6,8-dicar-
boxaldehyde (22). Yield: 21%; yellow solid; mp 107–
109 ꢁC, 1H NMR (300 MHz, CDCl3): d 13.38 and
13.22 (s, 1H each, OHA and OHB), 10.15 and 10.03 (s,
1H each), 2.72–2.57 (m, 2H), 2.46–2.30 (m, 1H), 2.25
(m, 1H), 2.20–2.09 (m, 1H), 2.05–1.95 (m, 1H), 1.96–
1.73 (m, 2H), 1.72–1.51 (m, 3H), 1.56–1.38 (m, 1H),
1.38–1.22 (m, 1H), 1.12 and 1.02 (s, 3H each); 13C
NMR (CDCl3, 75 MHz): d 192.2, 191.4, 168.8, 168.1,
163.8, 104.7, 103.6, 101.1, 90.7, 49.4, 45.2, 34.4, 29.7,
27.2, 25.2, 23.6, 22.5, 22.5, 16.3; IR (KBr) mmax: 3742,
2941, 1640, 1440, 1377, 1303, 1169, 1071 cmꢀ1; CIMS:
m/z 331 [M+1]+,195 [MꢀC10H16]; analysis for C19H22
O5 (330.1) calcd: C, 69.07; H, 6.71; found: C, 68.87;
H, 6.51.
4.6.13. 2,3,4,4a,9,9a-Hexahydro-6,8-dihydroxy-3,3,4a-
trimethyl-2,4-methano-1H-xanthene-5,7-dicarboxalde-
hyde (19). Yield: 17%; brown viscous oil, 1H NMR
(300 MHz, CDCl3): d 13.36 and 13.20 (s, 1H each,
OHA and OHB), 10.15 and 9.95 (s, 1H each, CHO),
2.71 (m, 1H), 2.66 (m, 1H), 2.45 (dd, J = 5.6,
15.7 Hz, 1H), 2.26 (t, J = 4.5 Hz, 1H), 2.15 (m, 2H),
1.90 (m, 1H), 1.80 (d, J = 15.0 Hz, 1H),1.48 (s, 3H),
1.32–1.22 (m, 1H), 1.31 and 1.10 (s, 3H each); 13C
NMR (75 MHz, CDCl3): d 192.0, 191.7, 168.6, 168.1,
166.0, 103.7, 100.4, 88.3, 54.7, 43.9, 40.5, 34.2, 31.7,
28.6, 28.0, 27.8, 22.6, 19.4; IR (Neat) mmax: 3402,
2925, 1632, 1558, 1446, 1306, 1255, 1181 cmꢀ1; CIMS:
m/z 331 [M+1]+, 195 [MꢀC10H16]; analysis for
C19H22O5 (330.1) calcd: C, 69.07; H, 6.71; found: C,
69.00; H, 6.67.
4.7. Assay for in vitro antimicrobial activity
Antibacterial and antifungal activities of all the synthe-
sized natural and unnatural euglobals against pathogen-
ic bacteria and fungi were evaluated in the similar way
as described earlier.30
4.8. Assay for in vitro antileishmanial activity
Antileishmanial activity was tested in vitro against a
culture of L. donovani promastigotes as described ear-
lier.30 Pentamidine and amphotericin B were used as
the standard antileishmanial agents. IC50 and IC90 val-
ues were computed from dose–response curves gener-
ated by plotting the percent growth versus drug
concentration.
4.6.14. 3,4-Dihydro-5,7-dihydroxy-60,60-dimethyl-spiro-
[2H-1-benzopyran-2,20-bicyclo[3.1.1]heptane]-6,8-dicar-
boxaldehyde (20). Yield: 25%, light brown solid; mp
1
109–111 ꢁC; H NMR (300 MHz, CDCl3): d 13.42 and
4.9. Assay for in vitro antimalarial activity
13.21 (s, 1H each, OHA and OHB), 10.13 and 10.02 (s,
1H each, CHO), 2.55 (t, J = 6.7 Hz, 2H), 2.26 (m, 1H),
2.16 (t, J = 6.0 Hz, 1H), 2.05–1.96 (m, 5H), 1.91–1.84
(m, 2H), 1.60 (d, J = 10 Hz, 1H), 1.30 and 1.02 (s, 3H
each); 13C NMR (CDCl3, 75 MHz): d 191.9, 191.5,
168.6, 168.1, 163.5, 104.4, 103.5, 100.6, 85.5, 49.6,
40.5, 38.2, 31.5, 28.5, 27.4, 26.4, 24.6, 23.2, 14.7; IR
(KBr) mmax: 3401, 2923, 1637, 1547, 1444, 1306, 1181,
1018 cmꢀ1; CIMS: m/z 331 [M+1]+, 195 [MꢀC10H16];
In vitro antimalarial activity was determined against
chloroquine sensitive and resistant (D6 and W2) clones
of P. falciparum as described.30
4.10. Assay for in vitro cytotoxicity to vero cells
Cytotoxicity was determined against mammalian kidney
fibroblasts (vero cells) as described earlier.30

1760
S. B. Bharate et al. / Bioorg. Med. Chem. 14 (2006) 1750–1760
16. Bharate, S. B.; Chauthe, S. K.; Bhutani, K. K.; Singh, I. P.
Aust. J. Chem. 2005, 58, 551.
Acknowledgments
17. Chiba, K.; Arakawa, T.; Tada, M. Chem. Commun. 1996,
1763.
18. Chiba, K.; Arakawa, T.; Tada, M. J. Chem. Soc., Perkin
Trans. 1 1998, 2939.
S.B.B. is thankful to Director, NIPER, for the fellow-
ship. Financial assistance from NIPER for synthesis of
compounds is gratefully acknowledged.
19. Reggelin, M.; Gerlach, M.; Vogt, M. Eur. J. Med. Chem.
1999, 1011.
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