Synthesis and anti-leishmanial activity of heterocyclic betulin derivatives

Article abstract of DOI:10.1016/j.bmc.2010.01.003

Betulin, a naturally occurring abundant triterpene is converted in four steps to 3,28-di-O-acetyllupa-12,18-diene. When various 4-substituted urazoles were oxidized to the corresponding urazines with iodobenzene diacetate in the presence of 3,28-di-O-acetyllupa-12,18-diene, new heterocyclic betulin derivatives were produced. These betulin derivatives were examined in a microplate assay at 50 μM for their ability to inhibit the growth of Leishmania donovani axenic amastigotes, a species that causes the fatal visceral leishmaniasis. GI₅₀ (concentration for 50% growth inhibition) values of the most effective compounds were determined and their cytotoxicity on the human macrophage cell line THP-1 evaluated. The anti-leishmanial activity on L. donovani amastigotes growing in macrophages was also examined. The heterocycloadduct between 3,28-di-O-acetyllupa-12,18-diene and 4-methylurazine was the most effective derivative with an GI₅₀ = 8.9 μM against L. donovani amastigotes.

Full text of DOI:10.1016/j.bmc.2010.01.003

Bioorganic & Medicinal Chemistry 18 (2010) 1573–1582
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and anti-leishmanial activity of heterocyclic betulin derivatives
Sami Alakurtti a,b, Tuomo Heiska a, Alexandros Kiriazis a, Nina Sacerdoti-Sierra c,
Charles L. Jaffe c, Jari Yli-Kauhaluoma a,
*
a Division of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Helsinki, PO Box 56 (Viikinkaari 5 E), FI-00014 University of Helsinki, Finland
b VTT Technical Research Centre of Finland, VTT, PO Box 1000, FI-02044 VTT (Espoo), Finland
c Department of Microbiology and Molecular Genetics, IMRIC, Hebrew University-Hadassah Medical School, PO Box 12272, Jerusalem 91220, Israel
a r t i c l e i n f o
a b s t r a c t
Article history:
Betulin, a naturally occurring abundant triterpene is converted in four steps to 3,28-di-O-acetyllupa-
12,18-diene. When various 4-substituted urazoles were oxidized to the corresponding urazines with
iodobenzene diacetate in the presence of 3,28-di-O-acetyllupa-12,18-diene, new heterocyclic betulin
Received 21 October 2009
Revised 22 December 2009
Accepted 2 January 2010
Available online 7 January 2010
derivatives were produced. These betulin derivatives were examined in a microplate assay at 50 lM
for their ability to inhibit the growth of Leishmania donovani axenic amastigotes, a species that causes
the fatal visceral leishmaniasis. GI50 (concentration for 50% growth inhibition) values of the most effec-
tive compounds were determined and their cytotoxicity on the human macrophage cell line THP-1 eval-
uated. The anti-leishmanial activity on L. donovani amastigotes growing in macrophages was also
examined. The heterocycloadduct between 3,28-di-O-acetyllupa-12,18-diene and 4-methylurazine was
Keywords:
Antiprotozoal agents
Betulin
Cycloaddition
Leishmania sp.
Terpenoids
the most effective derivative with an GI50 = 8.9
l
M against L. donovani amastigotes.
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
against pentavalent antimony drugs has become a serious problem
and is present in approximately 65% of the patients with visceral
leishmaniasis in India.4
Leishmaniasis is a parasitic disease that affects millions of peo-
ple in developing countries and has been designated as a neglected
tropical disease by the World Health Organization. It is caused by
the protozoan parasite Leishmania and transmitted by sand flies
belonging to the genus Phlebotomus and Lutzomyia in the Old and
New World, respectively.1 Three main forms of leishmaniasis are
commonly observed: (1) visceral leishmaniasis, a fatal disease
where parasites invade the liver, spleen and bone marrow; (2)
mucocutaneous leishmaniasis, a highly disfiguring, chronic disease
of the nasopharynx and mucosal facial tissue and (3) cutaneous
leishmaniasis, a self-limiting disease resulting in scarring at the
site of skin lesion. First line drugs for treatment include pentava-
lent antimony compounds, pentamidine or amphotericin B. All
these drugs are administrated by intravenous injection and require
clinical supervision or hospitalization due the possibility of severe
side effects. Liposomal encapsulated amphotericin B shows lower
toxicity but is highly expensive. Several new drugs such as allopu-
rinol, primaquine, imipramine and 3-chloroimipramine are being
developed, however none of them are fully effective against Leish-
mania. Recently, miltefosine which can be administrated orally has
shown promising anti-leishmanial activity and is currently in
phase IV clinical studies, though the use of miltefosine in pregnant
women is limited due to teratogenic effects.2,3 Parasite resistance
Betulin (lup-20(29)-ene-3b,28-diol) 1 (Fig. 1) is a widespread
compound found in the plant kingdom. It can be isolated up to
30% dry weight from the outer bark of white-barked birches of
the genus Betula sp.5 This pentacyclic triterpene can be converted
to betulinic acid 2,6 which has shown anti-malarial,7 anti-inflam-
matory8 and especially cytotoxic activity against many tumor cell
lines by inducing apoptosis.9–11 Some betulin derivatives have also
shown remarkable anti-HIV activity with new mechanisms of
action.12,13
In a study by Chowdhury et al. dihydrobetulinic acid 3 was
found to inhibit growth of Leishmania donovani promastigotes
and amastigotes with an IC50 (50% inhibitory concentration) value
of 2.6 and 4.1 l
M, respectively.14 It acts by targeting DNA topoiso-
merase I and II preventing DNA cleavage and formation of en-
zyme–DNA complex, ultimately inducing apoptosis. Leishmanial
parasitic burden in golden hamsters was reduced by 92% after 6-
week treatment with dihydrobetulinic acid 3 (10 mg/kg body
weight). In a related study by Sauvain et al., a rare natural product,
betulinic aldehyde 4, obtained from Doliocarpus dentatus (Aubl.)
showed weak in vitro activity against Leishmania amazonensis
amastigotes with a survival index of 12% at 136
68
M.15 However, at these doses 4 also showed toxicity against
peritoneal macrophages with survival indices of 70% and 80%,
respectively. At a concentration of 34 M, aldehyde 4 was ineffec-
tive against L. amazonensis as well as non-toxic to macrophages. In
lM and 42% at
l
l
* Corresponding author. Tel. +358 9 191 59170; fax: +358 9 191 59556.
E-mail address: Jari.Yli-Kauhaluoma@helsinki.fi (J. Yli-Kauhaluoma).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.01.003

1574
S. Alakurtti et al. / Bioorg. Med. Chem. 18 (2010) 1573–1582
29
20
30
12
19
18
13
22
11
OH
OH
H
OH
17
1
14
28
O
O
O
10
8
3
5
7
HO
HO
HO
HO
6
4
3
2
1
Figure 1. Chemical structures of betulin 1, betulinic acid 2, dihydrobetulinic acid 3 and betulinic aldehyde 4.
another study, where anti-leishmanial activity of plethora of natu-
ral products was screened, betulinic acid 2 isolated in minute
quantities from Betula platyphylla var. japonica was found to be
weakly active against Leishmania major with an IC50 value of
not generally stable for long period of time, they were synthesized
in situ by oxidation of urazoles 15 with the hypervalent iodine re-
agent, (diacetoxyiodo)benzene.19 Of the two dienes 8a and 8b, only
the sterically least hindered 8a took part in the hetero Diels–Alder
reaction producing heterocyclic betulin derivatives 9a–9l.
88 l
M.16 It was also noted that in the triterpenes with ursane, ole-
anane or lupane skeletons, a carboxyl substituent was required for
anti-leishmanial activity.
Reactions of 4-phenyl- or 4-methyl-1,2,4-triazoline-3,5-dione
with a mixture of dienes 8a and 8b in toluene gave the correspond-
ing heterocycles 9m and 9n in 52% and 49% yields, respectively
(Scheme 2). Also in this case the only detected and isolated cyc-
loadducts were formed from the diene 8a. Treatment of the hetero-
cycles 9m and 9n with NaOH in a mixture of MeOH and
tetrahydrofuran (THF) (2:3) provided the diols 9o and 9p as the
hydrolysis products in 91% and 73% yields, respectively. The
[4+2] cycloaddition reactions between the mixture of dienes 8a/
8b and other less reactive dienophiles, such as maleic anhydride,
N-phenylmaleimide, diethyl azodicarboxylate and dimethyl acety-
lenedicarboxylate were unsuccessful.
In our previous work, the hydroxy groups of C-3 and C-28, and
the carbon–carbon double bond C-20–C-29 were used as a starting
point to prepare a library of 24 triterpenoid derivatives for anti-
leishmanial studies against L. donovani axenic amastigotes.17 Betu-
lonic acid was the most potent derivative giving a GI50 value of
14.6 l
M.17 To improve the aqueous solubility and convert our ini-
tial anti-leishmanial triterpenoids to more drug-like compounds
we embarked on the synthesis of their heterocyclic derivatives
and the evaluation of their leishmanicidal activity against L. dono-
vani axenic amastigotes.
Treatment of the dienes 8a/8b with NaOH in a mixture of MeOH
and THF (2:3) gave a mixture of 3,28-dihydroxylupa-12,18-diene
10a and 3,28-dihydroxylupa-18,21-diene 10b in 85% yield. The
subsequent acylation with various acyl chlorides in the presence
of 4-(dimethylamino)pyridine (DMAP) as a nucleophilic catalyst
and pyridine (py) in CH2Cl2 yielded a mixture of dienes 11a–11e.
Cycloaddition reactions of 4-methyl- or 4-phenyl-1,2,4-triazoline-
3,5-dione with a mixture of dienes 11a–11e provided the corre-
sponding heterocycles 9q–9v in moderate yields. When tert-bu-
tyl-substituted urazole 15l was oxidized to the corresponding 4-
substituted urazine 16 with (diacetoxyiodo)benzene in situ in the
presence of 11d, a new heterocycle 9w was obtained.
The dienophilic urazines 16 and their corresponding urazole
precursors 15 were synthesized as follows (Scheme 3): Reaction
of stoichiometric amount of ethyl hydrazinecarboxylate 12 and
isocyanates 13 in toluene yielded carbethoxysemicarbazides 14
in excellent 90–99% yields.20a,b If the substituent of carbethoxyse-
micarbazides was aromatic (14a–14i), the cyclization to the corre-
sponding urazoles 15a–15i was carried out by heating 14a–14i in
aqueous 4 M KOH solution for 90 min. Acidification of this basic
solution with aqueous HCl solution afforded urazoles 15a–15i in
2. Results and discussion
2.1. Chemistry
Betulin 1 was acetylated with excess of acetic anhydride to give
3,28-di-O-acetylbetulin 5 in 97% yield (Scheme 1). Treatment of 5
with hydrobromic acid (HBr) in the presence of acetic anhydride
and acetic acid in toluene caused the migration of the C-20–C-29
double bond of 5 to the position C-18–C-19, and thus afforded
3,28-di-O-acetyllup-18-ene 6 in 42% yield.18a,b The C-18–C-19 dou-
ble bond of 3,28-di-O-acetyllup-18-ene 6 was epoxidized with 3-
chloroperoxybenzoic acid (mCPBA) in chloroform to yield the oxi-
rane intermediate 7 in 65% yield. Treatment of 3,28-di-O-acetyl-
18,19-epoxylupane 7 with pyridine p-toluenesulfonate (PPTS) in
toluene gave a mixture (4:1) of conjugated dienes, 3,28-di-O-acet-
yllupa-12,18-diene 8a and 3,28-di-O-acetyllupa-18,21-diene 8b, in
68% yield.
Heterocycloadducts 9a–9l were obtained in moderate yields
from the reaction between a mixture of betulin-derived dienes
8a and 8b and urazines 16 (Scheme 2). Because urazines 16 are
c.
b.
a.
O
O
OH
O
O
O
O
O
HO
O
5
6
1
O
d.
O
O
O
+
O
O
O
O
O
O
O
O
O
8b
7
8a
Scheme 1. Reagents and conditions: (a) Ac2O (6 equiv), DMAP, py, CH2Cl2, rt, 17 h, 97%; (b) HBr, Ac2O, AcOH, PhMe, rt, 21 d, 42%; (c) mCPBA, Na2CO3, CHCl3, rt, 2 h, 65%; (d)
PPTS, Ac2O, PhMe, reflux, 3 h, 68%.

S. Alakurtti et al. / Bioorg. Med. Chem. 18 (2010) 1573–1582
1575
OR2
OH
11a, R2 = COEt, 107%
11b, R2 = COPr, 93%
11c, R2 = COi-Pr, 95%
11d, R2 = COcHex, 74%
11e, R2 = COPh, 74%
HO
R2O
10a
d.
11a1 - 11e1
+
+
OH
c.
OR2
O
HO
10b
O
O
R2O
11a2 - 11e2
O
8a
a.
R1
or b.
O
+
N
a.
O
or b.
N
N
H
O
c.
R1
N
O
O
O
O
O
O
O
N
O
N
O
H
9a - 9n
8b
9a, R1 = Bn, 62%
OR2
9b, R1 = 3-MeO-Ph, 47%
9c, R1 = 4-F-Ph, 44%
9d, R1 = 3-NO2-Ph, 60%
9e, R1 = 4-Cl-Ph, 38%
9f, R1 = 4-Ac-Ph, 30%
9g, R1 = 1-naphthyl, 53%
9h, R1 = indan-5-yl, 53%
9i, R1 = 1,3-dioxol-5-yl, 51%
9j, R1 = n-Bu, 28%
R2O
R1
N
O
9q - 9w
O
N
9q, R1 = Me, R2 = COEt, 54%
9r, R1 = Me, R2 = COPr, 52%
9s, R1 = Me, R2 = COi-Pr, 40%
N
H
OH
9t, R1 = Me, R2 = COcHex, 48%
9u, R1 = Me, R2 = COPh, 36%
9v, R1 = Ph, R2 = COPh, 39%
9w, R1 = t-Bu, R2 = COcHex, 16%
9k, R1 = Et, 36%
9l, R1 = H, 40%
9m, R1 = Ph, 52%
9n, R1 = Me, 49%
HO
9o, R1 = Ph, 91%
9p, R1 = Me, 73%
Scheme 2. Reagents and conditions: (a) 4-substituted urazole 15a–15l, PhI(OAc)2, CH2Cl2, THF, rt, 20 h; (b) 4-methyl- or 4-phenyl-1,2,4-triazoline-3,5-dione, CH2Cl2, THF, rt,
20 h; (c) NaOH, MeOH, THF, rt, 18 h, 10a + 10b 85%; (d) R2Cl, DMAP, py, CH2Cl2, 40 °C, 20 h.
O
previously described.21,22 Initial screening was carried out by
assessing the inhibition of amastigote growth at 50 M betulin
derivative. All compounds were tested at least twice in triplicate.
Complete medium both with and without dimethyl sulfoxide
(DMSO) was used as negative controls (0% inhibition of amastigote
growth). Amphotericin B was included as a positive control on each
O
O
NH2
a.
O=C=N-R1
R1
+
O
N
H
l
O
N
H
N
H
N
H
12
14a - 14l
13
R1
R1
plate and gave >90% inhibition at 1 lM. In some cases the GI50 was
N
O
O
N
O
O
d.
b.
or c.
also determined. Screening for activity on amastigotes growing in
macrophages was carried out as previously described using reti-
noic acid-treated human macrophage cell line THP-1 infected with
L. donovani expressing the luciferase gene (Ld:pSSU-int/LUC) at a
3:1 parasite: macrophage ratio.23 Compounds (at 50, 25 and
N
H
N
H
N
N
16
15a - 15l
Scheme 3. Reagents and conditions: (a) PhMe, rt, 2 h to 80 °C, 2 h, 90–99%; (b) (i)
4 M KOH in H2O, 70 °C, 90 min, (ii) 37% HCl 15a–15i, 37–88%; or (c) (i) Na, EtOH,
85 °C, 24 h, (ii) 1.25 M HCl in EtOH 15j–15l, 94–96%; (d) in situ PhI(OAc)2, CH2Cl2,
THF (for the substituents R1, see Scheme 3).
12.5 lM) to be tested were added for 48 h and the luminescence
determined after adding luciferase substrate and measuring in a
microplate reader. Cytotoxic effect of the compounds on THP-1
cells was assessed using the alamarBlue viability assay.
moderate to good yields (37–88%). If the substituent R1 was ali-
phatic (14j–14l), the cyclization reaction was carried out in a
refluxing mixture of 4% sodium ethoxide in ethanol for 24 h. Acid-
ification of the basic solution with HCl in ethanol afforded urazoles
15j–15l in excellent yields (94–96%).
2.2.1. Microplate assay at 50
amastigotes
lM against L. donovani axenic
Initial anti-leishmanial activity of heterocyclic compounds was
measured against L. donovani axenic amastigotes at 50 M on
l
2.2. Biological assays
microplate assay. The screening results of the first batch (com-
pounds 9a–9p; Scheme 3) showed that small R1 substituents at
the nitrogen atom (9k, R1 = Et and 9n, R1 = Me) gave the best
anti-leishmanial activity. Therefore, a second batch of heterocyclic
betulin derivatives 9q–9u was synthesized so that acyl groups at
The anti-leishmanial activity of heterocyclic betulin derivatives
9a–9w was screened using a fluorescent viability microplate assay
with L. donovani axenic amastigotes and alamarBlue (resazurin) as

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S. Alakurtti et al. / Bioorg. Med. Chem. 18 (2010) 1573–1582
R2 were altered and R1 group was kept as methyl. In addition, two
cycloadducts 9v and 9w with bulky R1 and R2 groups were
synthesized.
88%. Based on these results it seems that methyl group as R1 gives
the best anti-leishmanial activity.
Therefore, the effect to anti-leishmanial activity of different
(acyl) groups at R2 was studied, while keeping the R1 group as
methyl. Derivative 9p, with hydroxy groups as R2 had clearly de-
creased inhibition activity of 50% when compared to 9n with acetyl
groups. Dipropionate 9q showed good leishmanicidal activity, 93%
inhibition, but replacement of R2 with bulkier ester groups resulted
in weaker anti-leishmanial activity. Dibutyrate 9r and diisobuty-
rate 9s had weak anti-leishmanial activity of 26% and 20%, respec-
tively, but dicyclohexanoate 9t as well as dibenzoate 9u had
practically no activity.
When the R1 and R2 groups are bulky, such as phenyl and ben-
zoyl (R1, R2) in 9v and tert-butyl (R1) in 9w, practically no anti-
leishmanial activity was noticed (Table 1). When bulky acyl groups
in R2 were replaced with sterically less demanding acetyl group
and R1 was kept aromatic (compounds 9a–9i and 9m) anti-leish-
manial activity was improved. In general, different substituents
at the aromatic ring had only minor effect on leishmanicidal activ-
ity, and among these derivatives, 1-naphthyl derivative 9g and 1,3-
dioxol-5-yl derivative 9i showed the best inhibition activity at 58%
and 52%, respectively. Phenyl derivative 9o, with free hydroxy
groups had slightly improved inhibition activity of 48% when com-
pared to 9m (36%).
However, when the aromatic group R1 was replaced with less
hindered aliphatic groups and the R2 groups were kept as acetyl
(compounds 9j–9l), anti-leishmanial activity improved systemati-
cally. The n-butyl derivative 9j displayed moderate activity, 49%,
the ethyl derivative 9k showed 88% inhibition and finally, the
methyl derivative 9n displayed 98% inhibition. Derivative 9l with
a hydrogen atom as R1 displayed slightly decreased inhibition of
2.2.2. Determination of GI50 values, cytotoxicity and anti-
leishmanial activity on L. donovani amastigotes growing in THP-
1 cells
The most potent derivatives (9k, 9n, 9l and 9q) on microplate as-
say at 50 lM against L. donovani axenic amastigotes were selected
for further investigations, that is, determination of GI50 on axenic
amastigotes, cytotoxicity for macrophages and anti-leishmanial
activity against L. donovani amastigotes growing in THP-1 macro-
phages. The methyl derivative 9n had the best GI50 value = 8.9
while the other compounds showed slightly higher but very similar
GI50 values:9k30 M, 9l26 M and9q 25 M (Table1). Cytotoxicity
of the derivatives was evaluated at 50 M, 25 M and 12.5 M con-
lM,
l
l
l
Table 1
l
l
l
Anti-leishmanial activity of heterocyclic betulin derivatives at 50
lM
against L.
centrations using the human macrophage THP-1 cell line (Table 2).
Compound 9q was non-toxic at all test concentrations while 9k
and 9l only showed cytotoxicity at the highest concentration
(50 lM) examined 48% and 97% growth inhibition, respectively, of
THP-1 cell line. No significant cytotoxicity was observed at lower
concentrations. Compound 9n was cytotoxic at all concentrations
examined still inhibiting THP-1 growth, 39%, even at 12.5 lM.
donovani axenic amastigotes
R1
O
N
O
N
N
H
OR2
Finally, anti-leishmanial activity was tested on L. donovani
amastigotes growing in macrophages at concentrations, which
showed less than 40% cytotoxicity for the macrophage cell line
THP-1. The compound 9q showed activity on L. donovani amastig-
otes growing in macrophages similar to that seen with axenic
R2O
Compound
R1
R2
Average inhibitiona
(%)
GI50
b
(
l
M)
amastigotes at 50 lM, 83% and 93%, respectively. Even at 25 lM
concentration 9k, 9l and 9q still showed good activity inhibiting
amastigote growth in the macrophages 53%, 55% and 55%, respec-
tively. Compound 9q still displayed weak anti-parasitic activity,
9v
9w
9a
9b
9c
9d
9e
9f
Ph
tert-Bu
Bn
COPh
COcHex 4.8
0.0
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
25
44
48
30
30
45
58
23
52
3-MeO-Ph
4-F-Ph
3-NO2-Ph
4-Cl-Ph
4-Ac-Ph
1-Naphthyl
Indan-5-yl
1,3-Dioxol-
5-yl
18%, against amastigotes growing in macrophages at 12.5
lM.
Compound 9n showed good inhibition at 12.5 M (60%), however
l
even at this concentration it was also moderately cytotoxic (39%)
to macrophage cell line.
9g
9h
9i
3. Conclusion
9m
9o
9j
9k
9n
9l
9p
9q
9r
9s
9t
9u
Ph
Ph
n-Bu
Et
Me
H
Me
Me
Me
Me
Me
Me
Ac
H
Ac
Ac
Ac
Ac
H
36
48
49
88
98
88
50
93
26
19
A series of new heterocycles derived from the naturally occur-
ring and abundant triterpene betulin was synthesized. We found
30
8.9
26
Table 2
Cytotoxicity and anti-leishmanial activity of the most potent betulin derivatives on
macrophage cell line THP-1 and L. donovani amastigotes growing in THP-1 cells
COEt
COPr
COi-Pr
COcHex 5.5
COPh
25
Compound 50
l
M
25
lM
12.5 lM
Cytotoxic inhibition of THP-1 cell line growth (%)
9k
9n
9l
48
94
97
16
13
78
2.4
15
8.5
39
0.0
5.0
0.0
95
Positive
controlc
Negative
controld
9q
0.0
Inhibition of L. donovani amastigote growth (%) in the macrophage
cell line THP-1
9k
9n
9l
nta
nt
nt
53
nt
55
55
0.0
60
4.8
18
a
Determined at 50
l
M concentration of betulin derivative with the exception of
M due to their poor solubility.
GI50 = concentration for 50% growth inhibition.
compounds 9r and 9s that were used at 15
l
b
9q
83
c
Amphotericin B (1
Culture medium and DMSO.
l
M).
d
a
nt = not tested.

S. Alakurtti et al. / Bioorg. Med. Chem. 18 (2010) 1573–1582
1577
that small R1 substituents at the nitrogen atom of the triazolo
moiety as well as the least sterically hindered acyl groups at the
R2 positions in the betulin skeleton promoted anti-leishmanial
activity. Also triazolo ring had beneficial effect on anti-leishmanial
activity, as it was found out in our previous work that 3,28-di-O-
acetylbetulin 5 was totally inactive. The most effective derivative
against L. donovani amastigotes was the heterocycloadduct 9n with
1.02 (s, 3H), 1.68 (s, 3H), 2.38 (m, 1H), 3.18 (dd, J = 5.1, 10.8 Hz,
1H), 3.32 (d, J = 10.8 Hz, 1H), 3.79 (d, J = 10.8 Hz, 1H), 4.57 (s,
1H), 4.68 (s, 1H); 13C NMR (75 MHz, DMSO-d6): d = 14.7, 15.3,
15.9, 16.1, 18.3, 19.1, 20.8, 25.2, 27.0, 27.3, 27.9, 29.1, 29.7, 33.9,
34.2, 37.1, 37.3, 38.7, 38.8, 40.9, 42.7, 47.7, 47.7, 48.7, 50.4, 55.2,
~
60.5, 79.0, 109.7, 150.4; IR:
m
¼ 879, 1009, 1035, 1232, 1375,
1452, 1739, 2939, 3360; MS (EI, 70 eV) m/z: 442 [M]+; Anal. Calcd
a GI50 = 8.9
lM, however this compound showed some toxicity
for C30H50O2: C, 81.39; H, 11.38. Found: C, 81.40; H, 11.56.
(39%) for the macrophage cell line at approximately the same con-
centration (12.5
lM). Several additional derivatives with slightly
4.1.3. 3,28-Di-O-acetylbetulin (5)
higher GI50, ꢀ25
l
M, showed none or only limited toxicity for mac-
To a solution of 1 (15 g, 34 mmol), 4-(dimethylamino)pyridine
(0.41 g, 3.4 mmol), and pyridine (25 mL) in CH2Cl2 (150 mL) was
added acetic anhydride (19 mL, 200 mmol). The resulting mixture
was stirred at room temperature for 17 h. The reaction mixture
was then washed with 10% hydrochloric acid (200 mL), saturated
aqueous NaHCO3 solution (400 mL), water (100 mL) and dried with
anhydrous Na2SO4. Removal of the solvent in vacuo gave 5 as a
white solid (17 g, 97%): Rf = 0.6 (EtOAc/n-hexane 1:4); mp: 219–
220 °C; 1H NMR (300 MHz, CDCl3): d = 0.83 (s, 3H), 0.84 (s, 3H),
0.96 (s, 3H), 1.02 (s, 3H), 1.38 (s, 3H), 1.68 (s, 3H), 2.03 (s, 3H),
2.06 (s, 3H), 2.39–2.48 (m, 1H), 3.84 (d, J = 11.0 Hz, 1H), 4.24 (d,
J = 11.0 Hz, 1H), 4.46 (dd, J = 5.8, 10.2 Hz, 1H), 4.58 (s, 1H), 4.68
(s, 1H); 13C NMR (75 MHz, CDCl3): d = 14.6, 16.0, 16.1, 16.4, 18.1,
19.0, 20.7, 21.0, 21.3, 23.6, 25.1, 27.0, 27.9, 29.5, 29.7, 34.1, 34.5,
37.0, 37.5, 37.7, 38.3, 40.8, 42.6, 46.2, 47.6, 48.7, 50.2, 55.3, 62.7,
~
rophages at this concentration, and demonstrated good activity
against the intracellular parasites. New therapeutic agents for the
leishmaniases are urgently needed. Most existing drugs are toxic
or expensive and drug resistance by parasites causing fatal visceral
disease has led to discontinued use of first-line drugs in some
highly endemic regions of India. Heterocyclic betulin derivatives
show promising activity against L. donovani that causes fatal vis-
ceral disease. Further studies to develop more potent betulin deriv-
atives with leismanicidal properties but no toxicity for the human
host macrophages are underway.
4. Experimental section
4.1. Chemistry
80.8, 109.8, 150.1, 170.9, 171.6; IR:
m
¼ 1243, 1731, 2951; MS (EI,
70 eV) m/z: 526 [M]+; Anal. Calcd for C34H54O4: C, 77.52; H,
10.33. Found: C, 77.46; H, 10.36.
4.1.1. General procedures
Commercially available reagents were used without further
purification and all of the solvents were HPLC grade. Anhydrous
solvents were purchased from Sigma–Aldrich. All reactions in
anhydrous solvents were performed in oven dried glassware under
an inert atmosphere of anhydrous argon or nitrogen. Thin layer
chromatography (TLC) was performed on E. Merck Silica Gel 60
aluminium packed plates, with visualization accomplished by UV
illumination and staining with 5% H2SO4 in MeOH. The 1H NMR
spectra were measured on a Varian Mercury-VX 300 MHz or a Che-
magnetics CMX 400 MHz spectrometer with chemical shifts re-
ported as parts per million (in CDCl3 at 23 °C, solvent peak at
7.26 ppm as an internal standard or in DMSO-d6 at 23 °C, solvent
peak at 2.50 ppm as an internal standard). The 13C NMR spectra
were obtained on a Varian Mercury-VX 75 MHz or a Chemagnetics
CMX 100 MHz spectrometer with chemical shifts reported as parts
per million (in CDCl3 at 23 °C, solvent peak at 77.0 ppm as an inter-
nal standard or in DMSO-d6 at 23 °C, solvent peak at 39.50 ppm as
an internal standard). HPLC–MS and elemental analyses were per-
formed to determine purity of all tested compounds. Purity of all
tested compounds was >95%. Mass spectra were measured on a
Bruker Daltonik Esquire-HPLC spectrometer, with XTerra MS
4.1.4. 3,28-Di-O-acetyllup-18-ene (6)
To a solution of HBr (47%, 250 g), acetic anhydride (100 g) and
acetic acid (300 g) (resulting 14% HBr and 35% acetic acid) was
added 3,28-di-O-acetylbetulin
5 (17 g, 33 mmol) in toluene
(200 mL).17 The reaction mixture was allowed to stand for 3 weeks
at room temperature. The mixture was diluted with water
(400 mL) and aqueous phase was separated and extracted with tol-
uene (400 mL). Combined organic phases were washed with water
(300 mL), saturated aqueous NaHCO3 solution (600 mL) and dried
with anhydrous Na2SO4. Solvent was removed in vacuo and result-
ing crude product was purified by flash chromatography on silica
gel (hexane/EtOAc 8:1) to afford 3,28-di-O-acetyllup-18-ene 6
(7.4 g, 42%): Rf = 0.8 (EtOAc/n-hexane 1:4); mp: 202–203 °C; 1H
NMR (300 MHz, CDCl3): d = 0.83 (s, 3H), 0.84 (s, 3H), 0.88 (s, 3H),
0.91 (s, 3H), 0.97 (s, 3H), 1.00 (s, 3H), 1.05 (s, 3H), 2.03 (s, 3H),
2.04 (s, 3H), 2.24 (m, 2H), 2.41 (dd, J = 12.4 Hz, 1H), 3.13 (m, 1H),
4.00 (d, J = 4.4 Hz, 2H), 4.48 (dd, J = 6.2, 10.3 Hz, 1H); 13C NMR
(75 MHz, CDCl3): d = 15.5, 16.5, 16.5, 16.8, 18.1, 21.0, 21.3, 21.5,
21.6, 22.1, 23.7, 26.6, 27.9, 28.2, 28.2, 29.3, 32.3, 34.6, 34.9, 37.1,
37.8, 38.6, 40.8, 40.9, 43.2, 51.0, 52.1, 55.5, 66.9, 80.9, 134.0,
~
RP18 column (4.6 ꢁ 30 mm, 2.5
lm) or on a JEOL JMS-AX505 (To-
143.6, 171.0, 171.4; IR:
m
¼ 982, 1029, 1246, 1372, 1387, 1450,
1738, 2869, 2946, 2990; MS (EI, 70 eV) m/z: 526 [M]+; Anal. Calcd
kyo, Japan) spectrometer with direct input and electron ionization
(EI). Elemental analyses were performed by Robertson Microlit
Laboratories, Madison, NJ, USA. Melting points were obtained with
a Sanyo Gallenkamp apparatus without correction. The Fourier
transform infrared (FTIR) spectra were recorded on a Bruker Vertex
70 spectrometer with Pike MIRacle diamond crystal or with a Bru-
ker Equinox 55 spectrometer including IRScope II and diamond
anvil.
for C34H54O4: C, 77.52; H, 10.33. Found: C, 77.74; H, 10.61.
4.1.5. 3,28-Di-O-acetyl-18,19-epoxylupane (7)
To
a
solution of 3,28-di-O-acetyllup-18-ene
6
(4.91 g,
9.33 mmol) in CHCl3 (120 mL) was added Na2CO3 (4.94 g,
46.7 mmol) and m-chloroperoxybenzoic acid (mCPBA 70%, 3.69 g,
14.9 mmol), and the resulting mixture was stirred at room temper-
ature for 2 h.17 The reaction mixture was washed with water
(150 mL), saturated aqueous NaHSO3 solution (150 mL), saturated
aqueous NaHCO3 solution (150 mL) and dried with anhydrous
Na2SO4. Solvent was removed in vacuo, and the resulting crude
product was crystallized from EtOH (175 mL) to afford 3,28-di-O-
acetyl-18,19-epoxylupane 7 (3.31 g, 65%): Rf = 0.7 (EtOAc/n-hex-
ane 1:4); mp: 210–212 °C; 1H NMR (300 MHz, CDCl3): d = 0.81 (s,
3H), 0.86 (s, 3H), 1.00 (s, 3H), 1.02 (s, 3H), 1.03 (s, 3H), 1.04 (s,
4.1.2. Betulin (1)
Betulin 1 was isolated (95% purity) from the bark of birch (Bet-
ula sp.) by extraction and was obtained from UPM Kymmene
(Lappeenranta, Finland). The crude betulin was recrystallized from
2-propanol/H2O azeotrope to give 1 as a white solid:5 Rf = 0.2
(EtOAc/n-hexane 1:4); mp: 252–253 °C; 1H NMR (300 MHz,
CDCl3): d = 0.75 (s, 3H), 0.82 (s, 3H), 0.96 (s, 3H), 0.97 (s, 6H),

1578
S. Alakurtti et al. / Bioorg. Med. Chem. 18 (2010) 1573–1582
~
3H), 1.07 (s, 3H), 1.08 (s, 3H), 2.02 (s, 6H), 3.85 (d, J = 10.7 Hz, 1H),
4.45 (dd, J = 7.4, 8.8 Hz, 1H), 4.48 (d, J = 10.7 Hz, 1H); 13C NMR
(75 MHz, CDCl3): d = 16.4, 16.4, 16.7, 16.7, 18.1, 18.8, 19.9, 20.9,
21.3, 21.4, 22.6, 23.6, 23.7, 26.3, 26.6, 27.9, 28.5, 29.9, 34.1, 37.1,
37.7, 38.1, 38.5, 41.1, 43.1, 45.6, 51.3, 55.5, 66.7, 75.5, 77.7, 80.8,
~
542 [M]+; Anal. Calcd for C34H54O5: C, 75.23; H, 10.03. Found: C,
75.29; H, 10.29.
139.4, 148.2, 150.4, 170.8, 171.0; IR:
m
¼ 1028, 1241, 1441, 1736,
2940; MS (ESI) m/z: 714.4 [M+H]+; Anal. Calcd for C43H59N3O6: C,
72.34; H, 8.33; N, 5.89. Found: C, 70.62; H, 7.52; N, 5.05.
4.1.7.2. Heterocycloadduct (9b). Yield 47%: Rf = 0.6 (EtOAc/n-
hexane 1:2); mp: 122–123 °C; 1H NMR (300 MHz, CDCl3): d = 0.86
(s, 3H), 0.87 (s, 3H), 0.96 (s, 3H), 1.02 (s, 3H), 1.12 (s, 3H), 2.04 (s,
3H), 2.09 (s, 3H), 2.50 (m, 1H), 2.77 (m, 2H), 3.81 (s, 3H), 3.99 (q,
J = 14.4, 25.7 Hz, 2H), 4.49 (t, J = 8.0 Hz, 1H), 4.75 (t, J = 9.9 Hz, 1H),
6.88 (dd, J = 2.4, 8.4 Hz, 1H), 7.08 (m, 2H), 7.33 (t, J = 8.1 Hz, 1H);
13C NMR (75 MHz, CDCl3): d = 16.2, 16.5, 18.1, 19.2, 19.4, 20.3,
20.9, 21.2, 22.4, 23.0, 23.1, 26.0, 27.8, 29.2, 29.3, 33.2, 33.6, 33.8,
37.1, 37.8, 37.9, 38.4, 43.1, 43.4, 49.4, 52.8, 55.4, 55.6, 68.1, 73.2,
80.5, 111.3, 114.0, 118.1, 129.5, 132.6, 135.5, 139.4, 147.3, 149.4,
~
170.9, 171.3; IR:
m
¼ 982, 1244, 1732, 2940; MS (EI, 70 eV) m/z:
4.1.6. 3,28-Di-O-acetyllupa-12,18-diene (8a) and 3,28-di-O-
acetyllupa-18,21-diene (8b)
To a solution of 3,28-di-O-acetyl-18,19-epoxylupane 7 (2.00 g,
3.68 mmol) and p-toluenesulfonic acid (0.42 g, 2.21 mmol) in tolu-
ene (80 mL) was added acetic anhydride (0.560 mL, 5.90 mmol).17
The resulting mixture was refluxed for 4 h and then washed with
saturated aqueous NaHCO3 solution (150 mL), water (100 mL)
and dried with anhydrous Na2SO4. Solvent was removed in vacuo,
and the resulting crude product was purified by flash chromatogra-
phy on silica gel (hexane/EtOAc 12:1) and crystallized from EtOH
(20 mL) to give a mixture (4:1) of 3,28-di-O-acetyllupa-12,18-
diene 8a and 3,28-di-O-acetyllupa-18,21-diene 8b (1.31 g, 68%):
Rf = 0.6 (EtOAc/n-hexane 1:4); 8a: 1H NMR (300 MHz, CDCl3):
d = 2.03 (s, 3H), 2.05 (s, 3H), 2.30 (dd, J = 4.1, 9.6 Hz, 2H), 3.85 (q,
J = 11.0, 39.0 Hz, 2H), 4.50 (dd, J = 1.2, 7.4 Hz, 1H), 5.31 (t,
J = 3.8 Hz, 1H); 13C NMR (75 MHz, CDCl3): d = 16.1, 16.8, 17.6,
18.2, 21.1, 21.3, 21.4, 21.8, 22.6, 23.2, 23.6, 26.4, 27.5, 28.1, 28.4,
31.5, 34.2, 34.5, 36.8, 37.7, 38.7, 38.9, 44.1, 47.5, 51.4, 55.4, 66.1,
80.8, 122.9, 134.7, 136.6, 145.5, 171.0, 171.5.
169.9, 170.9, 171.0; IR:
m
¼ 1008, 1241, 1393, 1697, 2941; MS (ESI)
m/z: 730.4 [M+H]+; Anal. Calcd for C43H59N3O7: C, 70.75; H, 8.15;
N, 5.76. Found: C, 67.65; H, 7.20; N, 5.51.
4.1.7.3. Heterocycloadduct (9c). Yield 44%: Rf = 0.7 (EtOAc/n-
hexane 1:2); mp: 127–128 °C; 1H NMR (300 MHz, CDCl3):
d = 0.86 (s, 3H), 0.96 (s, 3H), 1.02 (s, 3H), 1.12 (s, 3H), 1.55 (s,
3H), 2.05 (s, 3H), 2.09 (s, 3H), 2.50 (m, 1H), 2.78 (m, 2H), 4.00 (q,
J = 13.2, 24.5 Hz, 2H), 4.50 (t, J = 8.1 Hz, 1H), 4.75 (t, J = 9.9 Hz,
1H), 7.12 (t, J = 8.4 Hz, 2H), 7.48 (m, 2H); 13C NMR (75 MHz, CDCl3):
= 16.2, 16.5, 18.1, 19.2, 19.4, 20.3, 20.9, 21.2, 22.3, 22.9, 23.1, 26.0,
27.8, 29.2, 33.2, 33.6, 33.8, 37.1, 37.8, 37.9, 38.0, 38.4, 43.1, 43.4,
49.4, 52.8, 55.6, 68.1, 73.2, 80.5, 115.6, 115.9, 127.5, 127.6, 127.6,
d
~
m
135.5, 139.4, 147.1, 149.2, 159.9, 170.8, 171.0; IR:
¼ 1028,
1241, 1512, 1696, 2942; MS (ESI) m/z: 718.4 [M+H]+; Anal. Calcd
for C42H56FN3O6: C, 70.27; H, 7.86; N, 5.85. Found: C, 68.82; H,
7.11; N, 5.08.
4.1.7. General procedure for the synthesis of
bis[acetyloxy]dihydro[1,2,4]triazolo[1,2-a]-pyridazine-1,3-
diones by generating urazine in situ (9a–9l) and (9w):
heterocycloadduct (9f)
4.1.7.4. Heterocycloadduct (9d). Yield 60%: Rf = 0.5 (EtOAc/n-
hexane 1:2); mp: 135–136 °C; 1H NMR (300 MHz, CDCl3):
d = 0.86 (s, 3H), 0.88 (s, 3H), 0.97 (s, 3H), 1.03 (s, 3H), 1.12 (s,
3H), 2.04 (s, 3H), 2.09 (s, 3H), 2.53 (m, 1H), 2.78 (m, 2H), 4.00 (q,
J = 14.4, 25.4 Hz, 2H), 4.50 (t, J = 8.3 Hz, 1H), 4.77 (t, J = 9.9 Hz,
1H), 7.61 (t, J = 8.3 Hz, 1H), 7.98 (dd, J = 1.2, 7.2 Hz, 1H), 8.18 (dd,
J = 2.1, 8.1 Hz, 1H), 8.53 (t, J = 2.1 Hz, 1H); 13C NMR (75 MHz,
CDCl3): d = 16.2, 16.5, 18.1, 19.1, 19.4, 20.3, 20.9, 21.2, 22.4, 22.9,
23.1, 26.0, 27.8, 29.2, 33.1, 33.5, 33.9, 37.1, 37.8, 37.9, 38.1, 38.4,
43.1, 43.4, 49.4, 53.0, 55.6, 68.1, 73.4, 80.4, 120.4, 122.0, 129.5,
131.0, 133.0, 135.3, 139.3, 146.3, 148.3, 148.4, 170.8, 171.0; IR:
~
Solution of p-acetyl-4-phenylurazole 15f (50 mg, 0.23 mmol)
and iodobenzene diacetate (74 mg, 0.23 mmol) in an anhydrous
THF/CH2Cl2 mixture (4 mL, 1:1) was stirred under inert atmo-
sphere for 15 min.19 To the formed red solution was added a mix-
ture (4:1) of 3,28-di-O-acetyllupa-12,18-diene 8a and 3,28-di-O-
acetyllupa-18,21-diene 8b (100 mg, 0.19 mmol) in THF/CH2Cl2
mixture (4 mL, 1:1). The reaction mixture was stirred at room tem-
perature for 24 h. Solvent was evaporated in vacuo, and purifica-
tion of crude product by flash chromatography on silica gel
(hexane/EtOAc 4:1?1:1) afforded 9f (43 mg, 30%): Rf = 0.3
(EtOAc/n-hexane 1:2); mp: 127–128 °C; 1H NMR (300 MHz,
CDCl3): d = 0.86 (s, 3H), 0.87 (s, 3H), 0.96 (s, 3H), 1.02 (s, 3H),
1.12 (s, 3H), 2.04 (s, 3H), 2.09 (s, 3H), 2.60 (s, 3H), 2.53 (m, 1H),
2.77 (m, 2H), 4.00 (q, J = 14.1, 25.1 Hz, 2H), 4.49 (t, J = 8.1 Hz,
1H), 4.76 (t, J = 10.1 Hz, 1H), 7.71 (d, J = 7.5 Hz, 2H), 8.02 (d,
J = 7.8 Hz, 2H); 13C NMR (75 MHz, CDCl3): d = 16.2, 16.5, 18.1,
19.2, 19.4, 20.3, 20.9, 21.2, 22.4, 23.0, 23.4, 26.0, 26.6, 27.8, 29.3,
33.1, 33.6, 33.9, 37.1, 37.8, 37.9, 38.0, 38.4, 43.1, 43.5, 49.4, 53.0,
55.6, 68.1, 73.4, 80.5, 125.1, 128.9, 135.5, 135.6, 136.1, 139.4,
~
m
¼ 749, 1030, 1244, 1351, 1535, 1701, 2945; MS (ESI) m/z: 745.4
[M+H]+; Anal. Calcd for C42H56N4O8: C, 67.72; H, 7.58; N, 7.52.
Found: C, 64.48; H, 7.41; N, 6.80.
4.1.7.5. Heterocycloadduct (9e). Yield 38%: Rf = 0.7 (EtOAc/n-
hexane 1:2); mp: 104–106 °C; 1H NMR (300 MHz, CDCl3):
d = 0.85 (s, 3H), 0.86 (s, 3H), 0.95 (s, 3H), 1.01 (s, 3H), 1.10 (s,
3H), 2.03 (s, 3H), 2.08 (s, 3H), 2.50 (m, 1H), 2.76 (m, 2H), 3.99 (q,
J = 11.3, 25,8 Hz, 2H), 4.48 (t, J = 8.5 Hz, 1H), 4.74 (t, J = 9.3 Hz,
1H), 7.26–7.59 (m, 5H); 13C NMR (75 MHz, CDCl3): d = 16.2, 16.5,
18.1, 19.2, 19.4, 20.3, 20.9, 21.2, 22.4, 23.0, 23.1, 26.0, 27.8, 29.3,
33.2, 33.6, 33.8, 37.1, 37.8, 37.9, 38.0, 38.4, 43.1, 43.4, 49.4, 52.9,
55.6, 68.1, 73.3, 80.5, 123.6, 125.7, 127.8, 129.7, 132.8, 134.4,
~
146.7, 148.8, 170.9, 171.0, 197.1; IR:
m
¼ 1029, 1242, 1391, 1699,
2941; MS (ESI) m/z: 742.5 [M+H]+; Anal. Calcd for C44H59N3O7: C,
71.23; H, 8.02; N, 5.66. Found: C, 70.43; H, 6.83; N, 5.04.
135.5, 139.4, 146.8, 148.9, 170.9, 171.0; IR:
m
¼ 1028, 1242, 1409,
1699, 1732, 2943; MS (ESI) m/z: 734.4 [M+H]+; Anal. Calcd for
C42H56ClN3O6: C, 68.69; H, 7.69; N, 5.72. Found: C, 66.46; H,
6.68; N, 4.48.
4.1.7.1. Heterocycloadduct (9a). Yield 62%: Rf = 0.6 (EtOAc/n-
hexane 1:2); mp: 114–115 °C; 1H NMR (300 MHz, CDCl3):
d = 0.86 (s, 3H), 0.88 (s, 3H), 0.91 (s, 3H), 0.99 (s, 3H), 1.05 (s,
3H), 2.05 (s, 3H), 2.07 (s, 3H), 2.41 (m, 1H), 2.76 (m, 2H), 3.95 (q,
J = 8.7, 19.5 Hz, 2H), 4.50 (t, J = 8.0 Hz, 1H), 4.64 (m, 1H), 4.65 (q,
J = 19.8, 34.2 Hz, 2H), 7.26–7.44 (m, 5H); 13C NMR (75 MHz, CDCl3):
4.1.7.6. Heterocycloadduct (9g). Yield 53%: Rf = 0.3 (EtOAc/n-
hexane 1:2); mp: 145–147 °C; 1H NMR (300 MHz, CDCl3):
d = 0.86 (s, 3H), 0.98 (s, 3H), 1.00 (s, 3H), 1.15 (s, 3H), 1.19 (s,
3H), 2.02 (s, 3H), 2.10 (s, 3H), 2.48 (m, 1H), 2.76 (m, 1H), 2.88
(m, 1H), 4.02 (q, J = 12.4, 23.9 Hz, 2H), 4.48 (t, J = 7.8 Hz, 1H),
d
= 16.2, 16.5, 18.1, 19.0, 19.1, 20.2, 20.9, 21.2, 22.1, 22.9, 23.1, 25.9,
27.8, 29.2, 33.3, 33.6, 37.2, 37.7, 37.8, 37.9, 38.3, 42.5, 43.1, 43.2,
49.4, 52.5, 55.6, 68.0, 72.9, 80.5, 127.7, 128.5, 128.6, 135.5, 136.2,

S. Alakurtti et al. / Bioorg. Med. Chem. 18 (2010) 1573–1582
1579
4.83 (q, J = 9.9, 20.7 Hz, 1H), 7.45–7.58 (m, 4H), 7.68 (dd, J = 7.2,
26.4 Hz, 1H), 7.91 (t, J = 7.5 Hz, 2H); 13C NMR (75 MHz, CDCl3):
d = 16.2, 16.6, 18,1, 19.4, 19.5, 20.3, 20.9, 21.2, 22.3, 23.1, 26.0,
27.9, 29.3, 33.2, 33.7, 37.1, 37.8, 37.9, 38.4, 43.2, 43.5, 49.5, 52.8,
53.0, 55.6, 67.6, 68.1, 73.2, 73.6, 80.5, 122.1, 125.3, 126.4, 127.1,
128.5, 129.9, 130.2, 134.5, 135.6, 139.5, 147.9, 148.1, 149.6,
~
d = 0.85 (s, 3H), 0.86 (s, 3H), 0.91 (s, 3H), 0.99 (s, 3H), 1.05 (s,
3H), 2.04 (s, 3H), 2.07 (s, 3H), 2.43 (m, 1H), 2.68 (m, 2H), 3.96 (q,
J = 17.4, 28.5 Hz, 2H), 4.49 (dd, J = 6.0, 10.2 Hz, 1H), 4.62 (t,
J = 10.0 Hz, 1H), 9,13 (br s, NH, 1H); 13C NMR (75 MHz, CDCl3):
d = 14.2, 16.2, 16.5, 18.1, 19.0, 19.3, 20.3, 20.9, 21.3, 22.2, 22.7,
23.2, 26.0, 27.9, 29.2, 33.1, 33.6, 37.1, 37.8, 38.0, 38.4, 43.0, 43.3,
49.4, 52.7, 55.7, 60.4, 68.1, 72.7, 80.6, 135.5, 139.2, 148.2, 150.3,
~
150.3, 170.9, 171.1; IR:
m
¼ 769, 1028, 1243, 1693, 2942; MS
(ESI) m/z: 750.4 [M+H]+; Anal. Calcd for C46H59N3O6: C, 73.67; H,
7.93; N, 5.60. Found: C, 70.84; H, 6.29; N, 4.29.
170.9, 171.1; IR:
m
¼ 1028, 1241, 1367, 1693, 2942; MS (ESI) m/z:
624.4 [M+H]+; Anal. Calcd for C36H53N3O6: C, 69.31; H, 8.56; N,
6.74. Found: C, 67.70; H, 8.03; N, 5.00.
4.1.7.7. Heterocycloadduct (9h). Yield 53%: Rf = 0.5 (EtOAc/n-
hexane 1:2); mp: 151–153 °C; 1H NMR (300 MHz, CDCl3):
d = 0.86 (s, 3H), 0.87 (s, 3H), 0.96 (s, 3H), 1.02 (s, 3H), 1.11 (s,
3H), 2.07 (s, 3H), 2.09 (s, 3H), 2.48 (m, 1H), 2.77 (m, 2H), 2.90 (q,
J = 7.2, 14.4 Hz, 4H), 3.99 (q, J = 13.8, 24.6 Hz, 2H), 4.49 (t,
J = 8.4 Hz, 1H), 4.74 (t, J = 9.9 Hz, 1H), 7.18–7.29 (m, 3H); 13C
NMR (75 MHz, CDCl3): d = 16.2, 16.5, 18.1, 19.2, 19.4, 20.3, 20.9,
21.2, 22.3, 23.0, 23.1, 25.5, 25.6, 26.0, 27.8, 29.3, 32.5, 32.8, 33.2,
33.6, 33.7, 37.1, 37.8, 37.9, 38.4, 43.1, 43.4, 49.4, 52.7, 55.6, 68.0,
73.1, 80.5, 122.1, 124.0, 124.5, 129.4, 135.5, 139.4, 144.1, 145.1,
~
4.1.7.12. Heterocycloadduct (9w). Yield 16%: Rf = 0.8 (EtOAc/n-
hexane 1:3); mp: 145–147 °C; 1H NMR (300 MHz, CDCl3):
d = 0.83 (s, 3H), 0.87 (s, 3H), 0.92 (s, 3H), 0.96 (s, 3H), 0.98 (s,
3H), 1.00 (s, 3H), 1.06 (s, 3H), 1.62 (s, 9H), 2.29 (m, 2H), 2.41 (m,
1H), 2.72 (m, 1H), 3.94 (dd, J = 3.6, 14.8 Hz, 2H), 4.48 (t,
J = 7.4 Hz, 1H), 4.60 (t, J = 9.3 Hz, 1H); 13C NMR (75 MHz, CDCl3):
d = 16.4, 16.5, 18.1, 19.4, 19.5, 20.4, 22.4, 23.2, 23.5, 25.3, 25.5,
25.7, 25.8, 25.9, 27.9, 28.7, 28.8, 28.9, 29.0, 29.1, 29.3, 33.1, 33.6,
33.8, 37.2, 37.3, 37.9, 38.0, 38.5, 43.3, 43.4, 43.6, 49.5, 52.4, 55.7,
57.6, 67.2, 73.2, 79.9, 135.4, 139.7, 149.1, 152.0, 175.7, 176.2; IR:
~
147.7, 149.8, 170.8, 171.0; IR:
m
¼ 1028, 1241, 1693, 1738, 2908;
MS (ESI) m/z: 740.5 [M+H]+; Anal. Calcd for C45H61N3O6: C, 73.04;
H, 8.31; N, 5.68. Found: C, 72.00; H, 8.11; N, 5.39.
m
¼ 1131, 1165, 1389, 1689, 1727, 2932; Anal. Calcd for
C50H77N3O6: C, 73.58; H, 9.51; N, 5.15. Found: C, 72.70; H, 9.55;
N, 4.52.
4.1.7.8. Heterocycloadduct (9i). Yield 51%: Rf = 0.3 (EtOAc/n-
hexane 1:2); mp: 117–119 °C; 1H NMR (300 MHz, CDCl3):
d = 0.86 (s, 3H), 0.87 (s, 3H), 0.96 (s, 3H), 1.02 (s, 3H), 1.11 (s,
3H), 2.04 (s, 3H), 2.09 (s, 3H), 2.48 (m, 1H), 2.77 (m, 2H), 3.99 (q,
J = 12.9, 23.7 Hz, 2H), 4.49 (t, J = 8.0 Hz, 1H), 4.73 (t, J = 9.9 Hz,
1H), 5.98 (s, 2H), 6.84 (d, J = 9.3 Hz, 1H), 6.93 (d, J = 8.2 Hz, 1H),
6.95 (s, 1H); 13C NMR (75 MHz, CDCl3): d = 16.2, 16.5, 18.1, 19.2,
19.4, 20.3, 20.9, 21.2, 22.3, 23.0, 23.1, 26.0, 27.8, 29.2, 29.3, 33.2,
33.6, 33.7, 37.1, 37.8, 37.9, 38.4, 43.1, 43.4, 49.4, 52.8, 55.6, 68.1,
73.2, 80.5, 101.5, 107.5, 108.1, 119.9, 125.0, 135.5, 139.4, 147.1,
~
4.1.8. General procedure for the synthesis of
bis[acetyloxy]dihydro[1,2,4]triazolo[1,2-a]-pyridazine-1,3-
diones from the commercially available urazines (9m and 9n)
and (9q–9v): heterocycloadduct (9n)
A mixture of 3,28-di-O-acetyllupa-12,18-diene 8a, 3,28-di-O-
acetyllupa-18,21-diene 8b (100 mg, 0.19 mmol together) [or a
mixture of 3,28-di-O-acyllupa-12,18-diene 11a1–11e1, 3,28-di-O-
acyllupa-18,21-diene 11a2–11e2 in case of compounds 9q–9v]
and 4-methyl-1,2,4-triazoline-3,5-dione (32 mg, 0.29 mmol) in
anhydrous toluene (5 mL) was stirred at room temperature for
22 h. Solvent was removed in vacuo, and the resulting crude prod-
uct was purified by flash chromatography on silica gel (hexane/
EtOAc 2:1) to afford adduct 9n (60 mg, 49%): Rf = 0.1 (EtOAc/n-hex-
ane 1:2); mp: 218 °C; 1H NMR (300 MHz, CDCl3): d = 0.83 (s, 3H),
0.84 (s, 3H), 0.90 (s, 3H), 0.99 (s, 3H), 1.03 (s, 3H), 2.02 (s, 3H),
2.05 (s, 3H), 2.42 (m, 1H), 2.69 (m, 2H), 3.01 (s, 3H), 3.94 (q,
J = 11.4, 24.0 Hz, 2H), 4.47 (t, J = 8.3, 1H), 4.63 (t, J = 10.1 Hz, 1H);
13C NMR (75 MHz, CDCl3): d = 16.2, 16.5, 18.1, 19.0, 19.2, 20.2,
20.9, 21.2, 22.2, 22.8, 23.1, 24.7, 24.8, 25.9, 27.8, 29.2, 33.2, 33.6,
37.1, 37.8, 37.9, 38.3, 43.0, 43.3, 49.4, 52.5, 55.6, 68.1, 72.7, 80.5,
~
147.5, 147.8, 149.6, 170.9, 171.0; IR:
m
¼ 1032, 1242, 1693, 2941;
MS (ESI) m/z: 744.4 [M+H]+; Anal. Calcd for C43H57N3O8: C, 69.42;
H, 7.72; N, 5.65. Found: C, 67.49; H, 6.55; N, 3.87.
4.1.7.9. Heterocycloadduct (9j). Yield 28%: Rf = 0.4 (EtOAc/n-
hexane 1:3); mp: 92–94 °C; 1H NMR (300 MHz, CDCl3): d = 0.84
(s, 3H), 0.86 (s, 3H), 0.92 (s, 3H), 1.00 (s, 3H), 1.05 (s, 3H), 2.03 (s,
3H), 2.06 (s, 3H), 2.41 (m, 1H), 2.73 (m, 2H), 3.49 (m, 2H), 3.95
(m, 2H), 4.47 (m, 1H), 4.65 (m, 1H); 13C NMR (75 MHz, CDCl3):
d = 13.6, 16.2, 16.5, 18.1, 19.1, 19.3, 19.9, 20.3, 20.9, 21.2, 22.2,
23.0, 23.2, 26.0, 27.9, 29.3, 30.1, 33.3, 33.5, 33.6, 37.2, 37.8, 37.8,
37.9, 38.4, 38.8, 43.1, 43.3, 49.4, 52.5, 55.7, 68.1, 72.8, 80.5,
~
135.6, 139.3, 148.5, 150.5, 170.8, 171.0; IR:
m
¼ 1029, 1223, 1691,
135.6, 139.5, 148.5, 150.6, 171.0, 171.8; IR:
m
¼ 1030, 1244, 1369,
1728, 2910; MS (ESI) m/z: 638.4 [M+H]+; Anal. Calcd for
C37H55N3O6: C, 69.67; H, 8.69; N, 6.59. Found: C, 66.36; H, 7.94;
N, 5.90.
1453, 1693, 1741, 2875, 2942; MS (ESI) m/z: 680.4 [M+H]+; Anal.
Calcd for C40H61N3O6: C, 70.66; H, 9.04; N, 6.18. Found: C, 69.56;
H, 8.91; N, 5.65.
4.1.8.1. Heterocycloadduct (9m). Yield 52%: Rf = 0.5 (EtOAc/n-
hexane 1:4); mp: 164 °C; 1H NMR (300 MHz, CDCl3): d = 0.85 (s,
3H), 0.87 (s, 3H), 0.96 (s, 3H), 1.02 (s, 3H), 1.11 (s, 3H), 2.04 (s,
3H), 2.09 (s, 3H), 2.50 (m, 1H), 2.78 (m, 2H), 3.94 (q, J = 10.8,
32.4 Hz, 2H), 4.49 (t, J = 8.4, 1H), 4.75 (t, J = 10.0 Hz, 1H), 7.32 (ttt,
J = 1.6, 7.4 Hz, 1H), 7.43 (t, J = 7.8 Hz, 2H), 7.50 (dd, J = 1.2, 8.4 Hz,
2H); 13C NMR (100 MHz, CDCl3): d = 14.2, 16.3, 16.6, 18.1, 19.2,
19.4, 20.3, 21.0, 21.0, 21.3, 22.4, 23.0, 23.2, 26.0, 27.9, 29.3, 33.2,
33.6, 33.8, 37.2, 37.8, 38.0, 38.4, 43.1, 43.4, 49.4, 52.8, 55.6, 60.4,
68.1, 73.2, 80.5, 125.8, 127.7, 128.9, 131.6, 135.5, 139.4, 147.3,
~
4.1.7.10. Heterocycloadduct (9k). Yield 36%: Rf = 0.3 (EtOAc/n-
hexane 1:3); mp: 197–202 °C; 1H NMR (300 MHz, CDCl3):
d = 0.83 (s, 3H), 0.84 (s, 3H), 0.91 (s, 3H), 0.99 (s, 3H), 1.04 (s,
3H), 2.02 (s, 3H), 2.05 (s, 3H), 2.44 (m, 1H), 2.72 (m, 2H), 3.53
(m, 2H), 4.92 (m, 2H), 4.47 (m, 1H), 4.63 (m, 1H); 13C NMR
(75 MHz, CDCl3): d = 13.3, 16.2, 16.5, 18.1, 19.1, 19.2, 20.2, 20.9,
21.2, 22.2, 22.9, 23.1, 25.9, 27.8, 29.3, 33.3, 33.4, 33.6, 33.9, 37.1,
37.8, 37.9, 38.4, 43.1, 43.3, 49.4, 52.4, 55.6, 68.0, 72.8, 80.5,
~
135.6, 139.4, 148.4, 150.6, 170.8, 171.0; IR:
m
¼ 758, 987, 1031,
1225, 1244, 1381, 1453, 1690, 1740, 2878, 2945; MS (ESI) m/z:
652.5 [M+H]+; Anal. Calcd for C38H57N3O6: C, 70.01; H, 8.81; N,
6.45. Found: C, 69.97; H, 8.74; N, 6.23.
149.5, 170.9, 171.1; IR:
m
¼ 756, 1028, 1241, 1391, 1694, 2941;
MS (ESI) m/z: 700.4 [M+H]+; Anal. Calcd for C42H57N3O6: C, 72.07;
H, 8.21; N, 6.00. Found: C, 71.85; H, 8.37; N, 5.96.
4.1.7.11. Heterocycloadduct (9l). Yield 40%: Rf = 0.2 (EtOAc/n-
hexane 1:1); mp: 119–120 °C; 1H NMR (300 MHz, CDCl3):
4.1.8.2. Heterocycloadduct (9q). Yield 54%: Rf = 0.3 (EtOAc/n-
hexane 1:3); mp: 133–139 °C; 1H NMR (300 MHz, CDCl3):

1580
S. Alakurtti et al. / Bioorg. Med. Chem. 18 (2010) 1573–1582
d = 0.82 (s, 3H), 0.85 (s, 3H), 0.90 (s, 3H), 0.99 (s, 3H), 1.03 (s, 3H),
1.12 (s, 3H), 2.40 (m, 6H), 2.71 (m, 2H), 3.01 (s, 3H), 3.97 (m, 2H),
4.48 (m, 1H), 4.63 (m, 1H); 13C NMR (75 MHz, CDCl3): d = 9.1, 9.2,
16.2, 16.5, 18.1, 19.0, 19.1, 20.2, 22.2, 22.8, 23.1, 24.7, 25.9, 27.5,
27.8, 27.9, 29.1, 33.1, 33.6, 33.6, 37.1, 37.7, 37.8, 37.9, 38.4, 43.1,
43.3, 49.4, 52.5, 55.6, 67.8, 72.7, 80.2, 135.5, 148.5, 150.5, 174.1,
~
1738, 2877, 2944; MS (ESI) m/z: 666.5 [M+H]+; Anal. Calcd for
C39H59N3O6: C, 70.34; H, 8.93; N, 6.31. Found: C, 69.42; H, 8.74;
N, 6.71.
3H), 2.61 (m, 1H), 2.81 (m, 2H), 4.29 (m, 2H), 4.80 (m, 2H), 7.52
(m, 11H), 8.05 (m, 4H); 13C NMR (75 MHz, CDCl3): d = 16.5, 16.5,
18.1, 19.3, 19.5, 20.4, 22.5, 23.0, 23.2, 25.9, 28.0, 28.6, 33.0, 33.6,
34.0, 37.2, 37.9, 38.0, 38.2, 38.4, 43.5, 43.6, 49.4, 52.8, 55.7, 67.6,
73.2, 81.1, 125.7, 127.7, 128.3, 128.4, 128.8, 129.5, 129.5, 129.9,
130.8, 131.6, 132.7, 133.2, 135.7, 139.4, 147.3, 149.4, 166.6; IR:
~
174.5; IR:
m
¼ 748, 1010, 1082, 1185, 1272, 1392, 1464, 1698,
m
¼ 711, 756, 969, 1026, 1069, 1110, 1273, 1392, 1415, 1452,
1503, 1601, 1702, 1758, 2875, 2944, 2963; Anal. Calcd for
C52H61N3O6: C, 75.79; H, 7.46; N, 5.10. Found: C, 75.61; H, 7.52;
N, 4.97.
4.1.8.3. Heterocycloadduct (9r). Yield 52%: Rf = 0.3 (EtOAc/n-
hexane 1:3); mp: 106–114 °C; 1H NMR (300 MHz, CDCl3):
d = 0.83 (s, 3H), 0.85 (s, 3H), 0.91 (s, 3H), 0.99 (s, 3H), 1.03 (s,
3H), 2.28 (m, 4H), 2.44 (m, 1H), 2.69 (m, 2H), 3.01 (s, 3H), 3.95
(m, 2H), 4.49 (m, 1H), 4.64 (m, 1H); 13C NMR (75 MHz, CDCl3):
d = 13.7, 16.3, 16.5, 18.1, 18.4, 18.5, 19.0, 19.2, 20.2, 22.2, 22.8,
23.2, 24.8, 25.9, 27.8, 29.1, 33.1, 33.6, 36.1, 36.6, 37.1, 37.7, 37.8,
37.9, 38.4, 43.1, 43.3, 49.4, 52.5, 55.6, 67.6, 72.7, 80.1, 135.5,
~
4.1.9. General procedure for the synthesis of 3-hydroxy-8a-
(hydroxymethyl)dihydro[1,2,4]-triazolo[1,2-a]pyridazine-1,3-
diones generating urazine in situ (9o and 9p):
heterocycloadduct (9o)
To a mixture of 9m (50 mg, 0.071 mmol) in a solution of MeOH/
THF 2:3 (1.4 mL) was added 1 M NaOH solution (0.30 mL), the and
reaction mixture was stirred at room temperature for 19 h. Water
(10 mL) was added and the precipitated product was filtered and
dried in vacuum oven to give 9o (40 mg, 91%): Rf = 0.5 (EtOAc/n-
hexane 1:1); mp: 154–156 °C; 1H NMR (300 MHz, CDCl3):
d = 0.79 (s, 3H), 0.94 (s, 3H), 0.98 (s, 3H), 0.99 (s, 3H), 1.12 (s,
3H), 2.56 (m, 1H), 2.71 (m, 2H), 3.20 (dd, J = 5.2, 10.7 Hz, 1H),
3.47 (d, J = 10.4 Hz, 1H), 3.61 (d, J = 10.7 Hz, 1H), 4.73 (t,
J = 10.0 Hz, 1H), 7.29–7.50 (m, 5H); 13C NMR (75 MHz, CDCl3):
d = 15.1, 16.5, 18.3, 19.2, 19.5, 20.3, 22.6, 23.0, 26.1, 26.9, 27.8,
28.4 32.6, 33.7, 34.3, 37.5, 37.8, 38.0, 38.5, 38.9, 43.5, 45.1, 49.6,
53.0, 55.6, 67.4, 73.5, 78.8, 125.8, 127.7, 128.9, 131.7, 134.8,
~
139.3, 148.6, 150.6, 173.3, 173.7; IR:
m
¼ 750, 987, 1182, 1258,
1394, 1467, 1700, 1737, 1796, 2877, 2945, 2967; MS (ESI) m/z:
694.5 [M+H]+; Anal. Calcd for C41H63N3O6: C, 70.96; H, 9.15; N,
6.05. Found: C, 69.89; H, 9.07; N, 6.06.
4.1.8.4. Heterocycloadduct (9s). Yield 52%: Rf = 0.3 (EtOAc/n-
hexane 1:3); mp: 134–142 °C; 1H NMR (300 MHz, CDCl3):
d = 0.82 (s, 3H), 0.85 (s, 3H), 0.90 (s, 3H), 0.99 (s, 3H), 1.03 (s,
3H), 1.14 (s, 3H), 1.16 (s, 3H), 2.51 (m, 2H), 2.67 (m, 2H), 3.01 (s,
3H), 3.95 (m, 2H), 4.46 (m, 1H), 4.63 (m, 1H); 13C NMR (75 MHz,
CDCl3): d = 16.3, 16.5, 18.0, 18.8, 19.0, 19.1, 19.2, 20.3, 22.2, 22.8,
23.1, 24.7, 25.9, 27.8, 28.8, 33.0, 33.5, 33.7, 34.0, 34.3, 37.1, 37.6,
37.9, 37.9, 38.4, 43.3, 49.4, 52.5, 55.6, 67.4, 72.7, 79.9, 135.5,
~
140.2, 147.4, 149.3; IR:
m
¼ 744, 1035, 1427, 1689, 2932, 3498;
MS (ESI) m/z: 616.4 [M+H]+; Anal. Calcd for C38H53N3O4: C, 74.11;
H, 8.67; N, 6.82. Found: C, 73.94; H, 8.39; N, 6.62.
139.5, 148.5, 150.5, 176.6, 177.1; IR:
m
¼ 749, 1158, 1192, 1391,
4.1.9.1. Heterocycloadduct (9p). Yield 73%: Rf = 0.1 (EtOAc/n-
hexane 1:1); mp: 130–131 °C; 1H NMR (300 MHz, CDCl3):
d = 0.79 (s, 3H), 0.90 (s, 3H), 0.93 (s, 3H), 0.95 (s, 3H), 0.98 (s,
3H), 1.00 (s, 3H), 1.02 (s, 3H), 1.05 (s, 3H), 2.52 (m, 1H), 2.65 (m,
2H), 3.03 (s, 3H), 3.20 (dd, J = 5.2, 10.4 Hz, 1H), 3.44 (d,
J = 10.4 Hz, 1H), 3.57 (d, J = 10.4 Hz, 1H), 4.63 (t, J = 9.6 Hz, 1H);
13C NMR (75 MHz, CDCl3): d = 15.1, 16.5, 18.3, 19.0, 19.4, 20.2, 22.4,
22.9, 24.7, 26.1, 26.9, 27.9, 28.3, 32.6, 33.6, 34.1, 37.5, 37.6, 37.9,
38.5, 38.8, 43.4, 45.1, 49.5, 52.7, 55.6, 67.3, 72.8, 78.8, 134.9,
~
1260, 1468, 1698, 1734, 1755, 2876, 2943, 2972; MS (ESI) m/z:
694.6 [M+H]+; Anal. Calcd for C41H63N3O6: C, 70.96; H, 9.15; N,
6.05. Found: C, 70.71; H, 9.19; N, 6.33.
4.1.8.5. Heterocycloadduct (9t). Yield 48%: Rf = 0.3 (EtOAc/n-
hexane 1:3); mp: 161–175 °C; 1H NMR (300 MHz, CDCl3):
d = 0.83 (s, 3H), 0.87 (s, 3H), 0.91 (s, 3H), 1.00 (s, 3H), 1.04 (s,
3H), 2.30 (m, 2H), 2.49 (m, 1H), 2.70 (m, 2H), 3.02 (s, 3H), 3.95
(m, 2H), 4.48 (m, 1H), 4.63 (m, 1H); 13C NMR (75 MHz, CDCl3):
d = 16.4, 16.5, 18.1, 19.1, 19.3, 20.3, 22.3, 22.9, 23.2, 24.8, 25.4,
25.5, 25.7, 25.8, 25.9, 27.9, 28.8, 28.9, 29.0, 29.2, 33.0, 33.6, 33.7,
37.1, 37.6, 37.9, 38.0, 38.4, 43.2, 43.3, 43.4, 43.5, 49.4, 52.7, 55.6,
67.2, 72.8, 79.8, 135.5, 139.4, 148.6, 150.6, 175.7, 176.1; IR:
~
140.1, 148.6, 150.5; IR:
m
¼ 728, 1046, 1468, 1678, 2939, 3438;
MS (ESI) m/z: 554.4 [M+H]+; Anal. Calcd for C33H51N3O4: C, 71.57;
H, 9.28; N, 7.59. Found: C, 64.03; H, 7.39; N, 5.77.
4.1.10. 3,28-Dihydroxylupa-12,18-diene (10a) and 3,28-
dihydroxylupa-18,21-diene (10b)
m
¼ 748, 1037, 1132, 1169, 1246, 1392, 1465, 1699, 1731, 2856,
2935; Anal. Calcd for C47H71N3O6: C, 72.93; H, 9.24; N, 5.43. Found:
A mixture of 3,28-di-O-acetyllupa-12,18-diene 8a and 3,28-di-
O-acetyllupa-18,21-diene 8b (9.45 g, 18.0 mmol), aqueous 2 M
NaOH (40 mL), MeOH (80 mL) and THF (120 mL) was stirred at
room temperature for 18 h. The reaction mixture was acidified
with aqueous 18% HCl solution (pH ꢀ5.5) and diluted with water
(900 mL). The formed crude product precipitate was filtered and
dried in vacuum oven for 3 h to give a mixture of 10a and 10b
(6.50 g, 85%, purity 72%) and it was used without further purifica-
tion in the next reaction step. Compound 10a: 1H NMR (300 MHz,
CDCl3): d = 0.79 (s, 3H), 1.00 (s, 3H), 1.26 (s, 3H), 2.28–2.39 (m, 1H;
m, 2H), 2.91 (m, 1H), 3.42 (m, 2H), 5.35 (m, 1H).
C, 72.68; H, 9.26; N, 5.18.
4.1.8.6. Heterocycloadduct (9u). Yield 36%: Rf = 0.3 (EtOAc/n-
hexane 1:3); mp: 175–183 °C; 1H NMR (300 MHz, CDCl3):
d = 0.92 (s, 3H), 0.96 (s, 3H), 1.03 (s, 3H), 1.06 (s, 3H), 1.09 (s,
3H), 2.56 (m, 1H), 2.71 (m,2H), 3.04 (s, 3H), 4.27 (m, 2H), 4.71
(m, 1H), 7.50 (m, 6H), 8.04 (m, 4H); 13C NMR (75 MHz, CDCl3):
d = 16.8, 16.8, 18.4, 19.4, 19.6, 20.7, 22.6, 23.2, 23.5, 25.1, 26.1,
28.3, 28.9, 33.9, 33.3, 34.1, 37.4, 37.9, 38.2, 38.5, 38.7, 43.8, 43.8,
49.7, 52.9, 56.0, 60.6, 67.9, 73.1, 81.4, 128.6, 128.7, 129.8, 129.8,
130.2, 131.1, 133.0, 133.5, 136.1, 139.6, 148.9, 150.9, 166.4,
~
166.9; IR:
m
¼ 711, 1110, 1273, 1452, 1466, 1697, 1718, 1755,
4.1.11. General procedure for the synthesis of mixture of lupa-
12,18-diene-3,28-diol and lupa-18,21-diene esters (11a–11e):
3,28-di-O-butyryllupa-12,18-diene (11b1) 3,28-di-O-
butyryllupa-18,21-diene (11b2)
2875, 2945; Anal. Calcd for C47H59N3O6: C, 74.08; H, 7.80; N,
5.51. Found: C, 72.98; H, 7.85; N, 5.45.
4.1.8.7. Heterocycloadduct (9v). Yield 39%: Rf = 0.7 (EtOAc/n-
hexane 1:3); mp: 192–204 °C; 1H NMR (300 MHz, CDCl3):
d = 0.94 (s, 3H), 0.98 (s, 3H), 1.04 (s, 3H), 1.09 (s, 3H), 1.17 (s,
Mixture of 3,28-dihydroxylupa-12,18-diene 10a and 3,28-
dihydroxylupa-18,21-diene 10b (1.50 g, 3.40 mmol), DMAP
(1.25 g, 10.2 mmol) and butyryl chloride (1.09 g, 10.2 mmol) in

S. Alakurtti et al. / Bioorg. Med. Chem. 18 (2010) 1573–1582
1581
anhydrous pyridine (35 mL) was heated with stirring at 40 °C for
20 h. EtOAc (250 mL) was added, and the formed precipitate was
filtered off. The filtrate was washed with aqueous HCl solution
(18%, 4 ꢁ 250 mL), dried with Na2SO4 and evaporated in vacuo to
provide mixture of 11b1 and 11b2 (1.84 g, 93%). Compound
11b1: 1H NMR (300 MHz, CDCl3): d = 0.87 (s, 3H), 0.97 (s, 3H),
2.30 (m, 2H), 2.90 (m, 1H), 3.85 (m, 2H), 4.52 (m, 1H), 5.31 (m, 1H).
4-butyl-1-carbethoxysemicarbazide 14j (3.94 g, 96%). It was used
without further purification in the next reaction step. Compound
14k, R1 = Et, 99%; 14l, R1 = t-Bu, 97%.
4.1.12.4. 4-Butylurazole (15j). Sodium metal (1.02 g, 44.3 mmol)
was reacted with EtOH (40 mL, 99.5%). After 1 h, 4-butyl-1-car-
bethoxysemicarbazide 14j (2.00 g, 9.84 mmol) was added and the
resulting brown reaction mixture was heated at 85 °C for 24 h.
The reaction mixture was acidified with 1.25 M HCl in EtOH
(50 mL). The formed precipitate was filtered off, and the filtrate
was evaporated to dryness in vacuo to give 4-butylurazole 15j
(1.44 g, 93%). Compound 15k, R1 = Et, 96%; 15l, R1 = t-Bu, 95%;
15m, R1 = H, urazole.
4.1.11.1. 3,28-Di-O-propionyllupa-12,18-diene (11a1) and 3,28-
di-O-propionyllupa-18,21-diene (11a2). Yield 88%. Compound
11a1: 1H NMR (300 MHz, CDCl3): d = 0.87 (s, 3H), 0.97 (s, 3H),
0.99 (s, 3H), 1.13 (s, 3H), 1.14 (s, 3H), 2.32 (m, 2H), 2.92 (m, 1H),
3.88 (m, 2H), 4.51 (m, 1H), 5.31 (s, 1H).
4.1.11.2. 3,28-Di-isobutyryllupa-12,18-diene (11c1) and 3,28-
di-O-isobutyryllupa-18,21-diene (11c2). Yield 95%. Compound
11c1: 1H NMR (300 MHz, CDCl3): d = 1.14 (s, 3H), 1.17 (s, 3H),
2.37 (m, 2H), 2.55 (m, 2H), 2.90 (m, 1H), 3.84 (m, 2H), 4.50 (m,
1H), 5.31 (m, 1H).
4.2. Biology
4.2.1. Parasite and cell culture
L. donovani (MHOM/SD/1962/1S-Cl2d) was used in all bioas-
says. Axenic amastigotes were grown at 37 °C in a 5% CO2 incuba-
tor as described20 in complete RPMI 1640 containing 20% fetal calf
serum, pH 5.5. Stably transfected L. donovani promastigotes
expressing the firefly luciferase gene (Ld:pSSU-int/LUC) were cul-
tured in Medium-199 adjusted to pH 6.8 and supplemented with
4.1.11.3. 3,28-Di-O-cyclohexanoyllupa-12,18-diene 11d1 and
3,28-di-O-cyclohexanoyllupa-18,21-diene (11d2). Yield 74%.
Compound (11d1): 1H NMR (300 MHz, CDCl3): d = 0.86 (s, 3H)
0.88 (s, 3H), 0.97 (s, 3H), 0.99 (s, 3H), 1.26 (s, 3H), 2.32 (m, 4H),
2.86 (m, 1H), 3.87 (m, 2H), 4.49 (m, 1H), 5.36 (m, 1H).
L
-glutamine (2 mM), adenosine (100
1 ꢁ BME vitamin mix, 10% fetal calf serum, penicillin G (100 IU),
streptomycin (100 g/mL) and hygromycin B (100 g/mL). The hu-
lM), folic acid (23 lM),
l
l
4.1.11.4. 3,28-Di-O-benzoyllupa-12,18-diene (11e1) and 3,28-
di-O-benzoyllupa-18,21-diene (11e2). Yield 74%. Compound
11e1: 1H NMR (300 MHz, CDCl3): d = 0.95 (s, 3H), 1.03 (s, 3H),
2.37 (m, 2H), 2.95 (m, 1H), 4.11 (m, 2H), 5.74 (m, 1H), 5.37 (m,
1H), 7.49 (m, 6H), 8.16 (m, 4H).
man leukemia monocyte cell line (THP-1) was cultured in com-
plete RPMI-1640 supplemented with antibiotics (100 IU penicillin
G and 100 mg/mL streptomycin), 2 mM
serum (10% v/v).
L-glutamine and fetal calf
4.2.2. Axenic amastigote viability assay
4.1.12. General procedures for the synthesis of N-substituted
1,2,4-triazolidine-3,5-diones (15)18
Screening of the compounds for leishmanicidal activity was car-
ried out using the alamarBlue (AbD Serotec, Oxford, UK) viability
assay similar to that reported for leishmanial promastigotes.20
Standardization and optimization of the assay for axenic amastig-
otes have been described elsewhere.21 Compounds to be assayed
were diluted to twice the final concentration in the complete
amastigote medium, containing 1% DMSO, and were aliquoted in
4.1.12.1. Aryl isocyanates (13a–13i): p-Acetyl-4-phenyl-1-car-
bethoxysemicarbazide (14f). To a solution of ethyl hydrazine-
carboxylate 12 (275 mg, 2.64 mmol) in anhydrous PhMe (5 mL)
was added dropwise 4-acetylphenyl isocyanate 13f (425 mg,
2.64 mmol) in anhydrous PhMe (5 mL). The reaction mixture was
stirred at room temperature for 2 h and then at 80 °C for 2 h. The
formed precipitate was filtered and dried in vacuum oven to give
p-acetyl-4-phenyl-1-carbethoxysemicarbazide 14f (630 mg, 90%).
It was used without further purification in the next reaction step.
14a, R1 = Bn;18b 14b, R1 = 3-MeO-Ph;18b 14c, R1 = 4-F-Ph;18b 14d,
R1 = 3-NO2-Ph;18b 14e, R1 = 3-Cl-Ph, 92%; 14g, R1 = 1-naphthyl;18b
14h, R1 = indan-5-yl;18b 14i, R1 = 1,3-dioxol-5-yl.18b
triplicate (125 lL/well) into 96-well flat-bottom plates (Nunc, Ros-
kilde, Denmark). Amastigotes (5.0 ꢁ 105 cells/mL; 125
lL/well)
were added to each well and incubated for 24 h at 37 °C in a 5%
CO2 incubator. The alamarBlue viability indicator was added
(25 lL/well) and the plates incubated for an additional 24 h at
which time the fluorescence (kex = 544 nm; kem = 590 nm) was
measured in a microplate reader (Fluoroskan Ascent FL, Finland).
Complete medium both with and without DMSO was used as neg-
ative controls (0% inhibition of amastigote growth). Amphotericin
B (Sigma–Aldrich, St. Louis MO), a drug used to treat VL, was in-
cluded as a positive control on each plate and gave >90% inhibition
4.1.12.2. p-Acetyl-4-phenylurazole (15f). p-Acetyl-4-phenyl-1-
carbethoxysemicarbazide 14f (300 mg, 1.13 mmol) was reacted
with aqueous 4 M KOH solution (0.570 mL, 2.26 mmol) at 70 °C
for 1.5 h. Remaining precipitate was filtered off, and filtrate was
cooled to room temperature and acidified with concentrated HCl
(10 drops). Formed precipitate was filtered and dried in desiccator
to give p-acetyl-4-phenylurazole 15f (162 mg, 65%). Compound
15a, R1 = Bn18b; 15b, R1 = 3-MeO-Ph, 88%; 15c, R1 = 4-F-Ph, 73%;
15d, R1 = 3-NO2-Ph, 37%; 15e, R1 = 3-Cl-Ph, 68%; 15g, R1 = 1-naph-
thyl18b; 15h, R1 = indan-5-yl, 92%; 15i, R1 = 1,3-dioxol-5-yl, 37%.
of parasite growth at 1 lM.
4.2.3. Screening on infected macrophages
THP-1 cells in the log-phase of growth were differentiated by
incubation for 3 days in complete RPMI-1640 containing 1 lM
retinoic acid (Sigma–Aldrich, St. Louis, MO).22 Excess retinoic
acid was removed by washing the cells three times with RPMI-
1640 (250g, 100, 4 °C) and the treated macrophages suspended
in complete RPMI-1640 and transferred to 75 mL tissue culture
flasks (Costar Brand, NUNC™, Denmark). Stationary-phase
Ld:pSSU-int/LUC promastigotes were added to the treated mac-
rophages (3:1 parasite/macrophage ratio) and incubated in a
5% CO2 incubator for 16 h at 37 °C to allow for infection and dif-
ferentiation of the Leishmania into intracellular amastigotes. Any
remaining extracellular parasites were removed by centrifuga-
tion 4 - 5 times (210g, 80, 4 °C). This was validated by phase
4.1.12.3. Alkyl isocyanates (13j– 13l): 4-butyl-1-carbethoxyse-
micarbazide (14j). To a solution of ethyl hydrazinecarboxylate 12
(2.10 g, 20.2 mmol) in anhydrous PhMe (70 mL) was added drop-
wise butyl isocyanate 13j (2.00 g, 20.2 mmol) in anhydrous PhMe
(5 mL). The reaction mixture was stirred at room temperature for
2 h and then at 80 °C for 2 h. The reaction mixture was cooled to
room temperature, and the formed precipitate was filtered,
washed with PhMe (60 mL) and dried in vacuum oven to provide

1582
S. Alakurtti et al. / Bioorg. Med. Chem. 18 (2010) 1573–1582
microscopy. Infected THP-1 cells in complete RPMI-1640 were
lL/well) in triplicate
into opaque 96-well flat bottom plates (Costar Brand, NUNC™,
Denmark). Test substances diluted in complete RPMI-1640 con-
taining 1% DMSO (10 lM, 50 lL/well) were added to the infected
Supplementary data
counted and aliquoted (1 ꢁ 105 cells in 50
Supplementary data (1H and 13C NMR spectra of compounds
9a–9w) associated with this article can be found, in the online ver-
sion, at doi:10.1016/j.bmc.2010.01.003.
cells. The cultures were incubated for 48 h (37 °C, 5% CO2). Cells
were lysed by the addition of Steady-GloÒ Luciferase Assay sub-
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luted in the complete medium containing 1% DMSO (10
lM)
and aliquoted in triplicate (125 L/well) into 96-well flat-bottom
l
plates. THP-1 macrophages in complete RPMI-1640 were added
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l
L/well) to the plates and incubated for
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lL)
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The financial support from the Finnish Funding Agency for
Technology and Innovation (Tekes), Foundation for Research of
Natural Resources in Finland, Marjatta ja Eino Kollin Säätiö, Acad-
emy of Finland (Grant 108376) and the projects by the European
Commission (Contract No LSHB-CT-2004-503467 and EU-KBBE-
227239) are gratefully acknowledged. C.L.J. holds the Michael
and Penny Feiwel Chair in Dermatology and is grateful to the
American Friends of Hebrew University for financial support of this
project. We thank Adjunct Professor Salme Koskimies for valuable
discussions.
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