Ivermectin-derived leishmanicidal compounds

Article abstract of DOI:10.1016/j.bmc.2008.12.003

In the present study a family of macrocyclic and acyclic analogues as well as seco-analogues of avermectins were prepared from commercial Ivermectin (IVM) and their antileishmanial activity assayed against axenic promastigote and intracellular amastigote

Full text of DOI:10.1016/j.bmc.2008.12.003

Bioorganic & Medicinal Chemistry 17 (2009) 496–502
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Ivermectin-derived leishmanicidal compounds
Anderson Rouge dos Santos a, Camila Alves Bandeira Falcão b, Michelle Frazão Muzitano b,c
,
Carlos Roland Kaiser a, Bartira Rossi-Bergmann b, Jean-Pierre Férézou d,
*
a Instituto de Química, Universidade Federal do Rio de Janeiro, Ilha do Fundão, CT, Bloco A, CEP 21941-909, Rio de Janeiro, RJ, Brazil
b Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Ilha do Fundão, CCS, Bloco H, CEP 21941-590, Rio de Janeiro, RJ, Brazil
c Centro de Biociências e Biotecnologia, Universidade Estadual do Norte Fluminense, CEP 28013-602, Campos dos Goytacazes, RJ, Brazil
d École Polytechnique, DCSO, UMR CNRS 7652, 91128 Palaiseau cedex, France
a r t i c l e i n f o
a b s t r a c t
Article history:
In the present study a family of macrocyclic and acyclic analogues as well as seco-analogues of avermec-
tins were prepared from commercial Ivermectin (IVM) and their antileishmanial activity assayed against
axenic promastigote and intracellular amastigote forms of Leishmania amazonensis. Contrarily to the filar-
icidal activity, the leishmanicidal potentiality of avermectin analogues does not appear to depend on the
Received 23 September 2008
Revised 28 November 2008
Accepted 2 December 2008
Available online 8 December 2008
integrity of the non-conjugated
D D
3,4-hexahydrobenzofuran moiety. Conjugated 2,3-IVM or its corre-
sponding conjugated secoester show higher anti-leishmania activity than the parent compound. Surpris-
ingly, the diglycosylated northern sub-unit exhibits the same anti-amastigote potentiality as the
southern hexahydrobenzofuran. As expected for compounds derived from the widely used Ivermectin
antibiotic, little toxicity has been noticed for most of the novel analogues prepared.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Ivermectin
Avermectins
Seco-analogues
Ozonolysis
Leishmania
Leishmanicidal activity
Promastigotes
Amastigotes
1. Introduction
industrial processes.5 Aiming at developing new orally effective
drugs against leishmaniasis, we focused our efforts on Ivermectin
Leishmaniases constitute a group of endemic tropical diseases
provoked by protozoan parasites of the genus Leishmania, transmit-
ted by the phlebotomine sandfly. About 12 million people are in-
fected worldwide, particularly in the developing countries and 350
million people are at risk.1 Depending upon involved parasite spe-
cies, leishmaniasis occurs either as cutaneous/muco-cutaneous
(CL) or the most severe visceral form (VL, or Kala-azar), that is often
fatal when untreated. No vaccine yet exists and conventional che-
motherapy with pentavalent antimonials, pentamidine or ampho-
tericin-B exhibit limitations such as parenteral administration,
long course of treatment, toxic side effects, high treatment cost
and/or drug resistance. Despite recent advances in the disease
knowledge and promising drug discovery programs,2 therapy of
leishmaniasis still represents a major health problem,3 with increas-
ing drug resistance constituting a major concern for the future.4
The use of known pharmaceutical drugs (old drugs) for new
therapeutic applications is an interesting cost-effective strategy
with several obvious advantages such as low development cost
due to known pharmaco-toxicological profile and established
1, a well known antiparasitic semi-synthetic analogue of the aver-
mectin family of macrolides, widely used for its potent anti-
helminthic properties against livestock parasitic diseases and
human filariasis.6
Apart from two early studies demonstrating the in vitro and
in vivo activities of Ivermectin against Leishmania donovani, a caus-
ative agent of VL in the Old World,7 and the in vitro inhibitory con-
centration of 100 lg/ml against the promastigote insect form of
Leishmania major, a causative agent of CL also in the Old World,8
nothing is reportedly known on the leishmanicidal properties of
the avermectin family of compounds. We thus proposed to inves-
tigate the effect of Ivermectin and several of its analogues or
seco-analogues on Leishmania amazonensis, a causative agent of
CL in South America. Besides its oral activity, another decisive
argument for undertaking such a study starting from commercial
Ivermectin is the existence of improved efficient industrial fermen-
tation/hydrogenation processes as well as its recent availability as
a generic drug that makes these molecules cost-effective starting
materials for the development of more efficient or differently pro-
filed therapeutic drugs.
Therefore, several semi-synthetic Ivermectin analogues were
tested against L. amazonensis promastigotes and intracellular
* Corresponding author. Tel.: +33 1 69 33 59 79; fax: +33 1 69 33 59 72.
E-mail address: ferezou@dcso.polytechnique.fr (J.-P. Férézou).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.12.003

A. R. d. Santos et al. / Bioorg. Med. Chem. 17 (2009) 496–502
497
amastigotes and the main results of this comparative study are
presented here.
diol 10 and the isomeric 1,4-pentanediol derivatives 11a and
11b were subsequently prepared by ozonolysis of subunit 7 in
MeOH at ꢀ78 °C followed by NaBH4 reduction and chromato-
graphic separation. Respective configurations of the newly cre-
ated stereogenic center at C-14 in 11a and 11b have not been
assigned.
2. Results and discussion
2.1. Chemistry
The presence of the intact non-conjugated
been claimed to be important for the antiparasitic activity of aver-
mectins.12 Therefore, for bioassay comparisons, conjugated D2,3
D
3,4-double bond has
The structures of all the Ivermectin analogues tested in the
present study are depicted in Figure 1. For clarity, these com-
pounds have been gathered in three sub-families of derivatives:
those where the integrity of the Ivermectin C1–C25 carbon skele-
ton of the natural avermectins is preserved (1–6), those corre-
sponding to the northern sub-unit after selective oxidative
scission of the C10–C11 double bond (7–11) and finally those cor-
responding to the C1–C9 southern counter part (12 and 13).
A number of these Ivermectin-derived analogues were prepared
according to methodologies already described. IVM–monosaccha-
ride 2 and IVM–aglycone 3 were obtained upon hydrolysis condi-
tions reported by Mrozik et al.9 The physical data were identical
to those already reported.10 According to a methodology already
applied by us to open avermectin macrolides,11 refluxing Ivermec-
tin in anhydrous ethanol in the presence of Ti(OEt)4 afforded IVM
ethyl secoester 4 (68% yield).
Selective ozonolysis of C10–C11 double bond of Ivermectin
secoester 4 has been carried out as previously described, afford-
ing the northern alcohol 7 and the corresponding 5-OTBS C1–
C10 southern alcohol which was subsequently deprotected un-
der acidic conditions to afford the desired southern diol 12 in
only a 12% yield probably due to the lability of the final com-
pound. No attempt has been made to optimize this step. Spiro-
-
Ivermectin 5, and the corresponding unprecedented conjugated
secoester 6 were synthesized (Scheme 1). According to known pro-
cedure,13
D
2,3-(4S)-Ivermectin 5 was prepared upon treatment of
commercial Ivermectin 1 with substoichiometric amount of DBU
until total consumption of 1, monitored by the disappearance of
the 174 ppm 13C NMR signal in crude reaction mixture aliquots.
The resulting conjugated
D
2,3-Ivermectin ethyl secoester 6 was ob-
tained by refluxing 5 in anhydrous ethanol in the presence of
Ti(OEt)4 for five days under inert atmosphere. The reaction was
slower than in the case of 1, probably due to the lower reactivity
of the conjugated carboxyl group. Interestingly, this process pro-
vides one single (4S) epimer in more than 98% of diastereomeric
excess , a result quite different from the approximately 20:80 ratio
of (4S)/(4R) isomers obtained when inverting the conjugation—
transesterification sequence.
The novel conjugated
D
2,3-(4S) southern alcohol 13 was subse-
quently prepared by selective ozonolysis of conjugated secoester 6
followed by NaBH4 reduction as above. During the whole sequence,
no protection of the 5-OH group is required, probably as a result of
the greater stability of the hexahydrobenzofuran unit resulting
from the conjugation of the double bond. The route used here to
Figure 1. Tested analogues and seco-analogues derived from IVM.

498
A. R. d. Santos et al. / Bioorg. Med. Chem. 17 (2009) 496–502
H
O
O
(L-ole)2
O
O
O
(L-ole)2
O
O
O
OH
OEt
HO
OH
OEt
(b)
(a)
(c)
O
O
OH
1
IVM
7 +
OH
89%
53%
O
3
H
93%
4
OH
O
O
H
13 58%
OH
OH
5
6
Scheme 1. Preparation of conjugated
D
2,3-Ivermectin analogues: (a) DBU (0.4 equiv), THF, 85 °C, 15 h; (b) ethanol (10 equiv), Ti(OEt)4 (1.5 equiv), 90 °C, 5 days; (c) (i) O3, 3:1
CH2Cl2/EtOH, –78 °C; (ii) NaBH4 (4.0 equiv), MeOH, 0 °C, 40 min.
Table 1
obtain 13 represents a flexible alternative to the one previously
described by Hanessian et al.14
Finally, the northern C11-C25 monosaccharide 8 as well as the
corresponding aglycone 9 was prepared by controlled hydrolysis
of northern disaccharide 7 using slightly modified conditions in
comparison with those used for intact Ivermectin (Scheme 2).
Antileishmanial and cytotoxic activities of IVM analogues compared with reference
drugs
Compound
IC50
(
l
M)a
Macrophages
Promastigotes
Amastigotes
Selectivity
indexb
IVM analogues with intact carbon skeleton
1 (IVM)
7.0 1.5
7.7 1.7
8.3 1.6
10.7 1.6
13.8 1.5
2.8 1.7
5.0 1.3
29.7 1.2
32.0 1.6
6.5 1.2
3.6 1.3
4.6 1.2
72.4 1.5
>90
>90
42.9 1.6
65.5 2.1
27.1 1.6
14.5
>3.0
>2.8
0.8
18.2
5.8
2.2. Biological activities
2
3
4
5
6
The antileishmanial activities of the compounds were deter-
mined in vitro against both the insect promastigote and the in-
tra-macrophage amastigote forms of L. amazonensis in three
independent experiments. Pentostam and Amphotericin B were
used as reference drugs for anti-promastigote and anti-amastigote
activities, respectively. Two controls were used because Pentostam
is ineffective against promastigotes. The mean IC50 (the concentra-
tion required to induce half of the maximum inhibition) values are
summarized in Table 1. High IC50 on macrophages reflects undesir-
able cytotoxicity to mammalian cells, and was determined by the
release of the cytoplasmic enzyme lactate dehydrogenase (LDH).15
As can be seen in Table 1, compounds 1–7 possessing intact
Northern-derived IVM analogues
7
8
9
10
11a
11b
17.1 1.5
65.7 1.2
>90
>90
>90
13.2 1.2
41.5 1.3
30.5 1.4
54.5 1.4
>90
55.0 1.4
32.1 1.6
>90
>90
86.2 1.1
>90
4.2
0.8
>3.0
1.7
<1.0
—
>90
>90
Southern-derived IVM analogues
12
13
>90
>90
38.6 1.2
13.8 1.2
>90
>90
2.3
6.5
Ivermectin backbone, particularly the conjugated
D
2,3-conjugated
Reference drugs
Pentostam
Amphotericin B
—
17.9 1.2
—
>90
—
>5.0
—
secoester 6, were very active against axenic cultures of promastig-
otes, indicating a direct leishmanicidal action. Noteworthy, com-
pound 6 was more potent than the control drug Amphotericin B.
The activity of Ivermectin 1 against L. amazonensis is in agreement
with the antileishmanial activity previously described against L.
donovani and L. major promastigotes.7,8 Compounds 8–13 did not
4.4 1.3
a
Values are means and standard deviations of triplicate samples.
The selectivity index was calculated as macrophage value/Amastigote value.
b
With respect to the cytotoxicity against the macrophage host
show significant anti-promastigote activity (IC50 > 60 lM).
cells, secoester 4 and 6, together with the northern analogues 7
and 8, revealed more toxic than the parent IVM 1, whilst most of
the other Ivermectin-derived analogues exhibited lower toxicity
We also assessed the activity of the compounds against the
amastigote parasite forms that would require transmembrane
transport to reach the macrophage phagolysosome where amastig-
otes grow. Whereas compounds 2–3, 8–10 and 12 had intermedi-
ate activity, IVM 1 and compounds 4–7 as well as 13 were shown
to be more capable at inhibiting parasite growth than the control
drug Pentostam. It is worth noting that both northern sub-units
(IC50 P 60 lM).
3. Discussion–conclusion
For commercial Ivermectin 1, the results obtained are consis-
tent with previous observations reported in the literature (see
above).
Dealing with anti-promastigote activities (Fig. 2), it clearly ap-
pears that the integrity of the Ivermectin skeleton is required.
The skeleton can be either under the macrolactone form as in
IVM 1 itself, monosaccharide 2, aglycone 3 or D2,3 -conjugated
macrolide 5, or under the corresponding secoester form as in 4
9
and 10 were active against intracellular amastigotes
(IC50 = 30.5 M and 54.5 M, respectively) but not against axenic
promastigotes (IC50 > 90 M).
l
l
l
or 6. The later
D
2,3-conjugated secoester appeared to be the most
active anti-promastigote analogue prepared from 1, showing an
in vitro activity of the same order as that of the very potent control
drug Amphotericin B. Interestingly, contrarily to the anti-amasti-
gote case (see below), neither the northern nor southern dissected
analogues demonstrate important activities. Particularly striking is
the fact that conjugation of the C3–C4 double bond resulted in an
increase of the anti-promastigote activity, a tendency rather in
Scheme 2. Controlled hydrolysis of the northern disaccharide 7.

A. R. d. Santos et al. / Bioorg. Med. Chem. 17 (2009) 496–502
499
100.0
80.0
60.0
40.0
20.0
0.0
>90.0
>90.0
>90.0
>90.0 >90.0
>90.0
65.68
17.11
13.8
10.7
8.3
7.7
7
4.4
2.8
Figure 2. Comparative anti-promastigote activity of the different IVM analogues.
contradiction with that observed for the antihelminthic activity of
avermectins for which the native double bond has been demon-
strated to be necessary.12
The reason why 7, 12 and 13 are active against the intracellular
amastigotes but not the promastigotes that are more readily acces-
sible to drug does not appear obvious and requires more thorough
investigations.
Concerning the anti-amastigote activity of the IVM analogues
on infected macrophages, the results obtained show some specific
differences in comparison with the results observed above for the
promastigotes, as illustrated in Figure 3. Here again, IVM analogues
In a first consideration, as illustrated by the differences ob-
served in the activity of 1, 2 and 3, the necessity of the presence
of the diglycosylated side chain in the amastigote case may reflect
polarity or recognition requirements for the macrophage internal-
ization of the substances. The importance of such glycosyl substit-
uents is reminiscent of the results of a recent study on the anti-
amastigote activity of the two flavonoids quercetin and quercetin
3-rhamnoside (quercitrin).16 The latter glycoside is more active
than the aglycone, probably due to a better ability for intra-macro-
phage uptake of the glycoside form. Furthermore, in the case of
quercitrin, the known affinity of rhamnose for macrophage mem-
branes may be, at least, partially responsible for the higher activity
observed.17
with non-fragmented skeleton show greater activity.
D
2,3-Conju-
gated macrolide 5 or its corresponding
D
2,3-secoester 6 are almost
equally active and significantly more efficient than standard Pento-
stam. However, for the anti-amastigote efficiency, contrarily to the
anti-prosmastigote case, the presence of the polar disaccharide
side chain is required (compare 1 with 2 and 3 in both cases). Fur-
thermore, the diglycosylated northern analogue 7 as well as the
southern moiety 12, and particularly the conjugated
D
2,3-southern
analogue 13, exhibit relevant anti-amastigote activities, which was
not the case for promastigotes. These differences may account for
different mechanisms of action, but also for an increased stability
The possibility that oleandrosyl residues, which are formal 2-
deoxy analogues of rhamnose, present in compounds 1, 4–6 and
also in the northern moiety 7 favored the binding of these mole-
of the conjugated
parison with the native
D
2,3-analogues under the test conditions in com-
D
3,4-isomers.
100
80
>90.0 >90.0
60
54.5
41.5
38.6
40
32
30.5
29.7
17.9
13.8
20
13.2
6.5
5
4.6
3.6
0
Figure 3. Comparative anti-amastigote activity of the different IVM analogues.

500
A. R. d. Santos et al. / Bioorg. Med. Chem. 17 (2009) 496–502
cules to the macrophage membrane through lectin-related recep-
tors,18 should also be considered. Such recognition would explain
the increased activity of these glycosylated molecules against the
intracellular parasites.
with 107 GFP-transfected fluorescent promastigotes for 4 h at
37 °C. Cell monolayers were washed to remove free parasites
and cultured for a further 72 h with varying concentrations
of the test compounds (0, 10, 30 and 90 lM) in 0.5% DMSO.
Taking into account both anti-promastigote and anti-amasti-
gote activities observed during the present study, compounds
1–7 revealed the most promising. However, considering the cyto-
Controls were 0.5% DMSO alone or Pentostam (Welcome).
The fluorescence intensity of the infected cell monolayers
was measured as for the antipromastigote activity. Maximum
and minimum inhibitory activities were fluorescence units of
uninfected macrophages and infected cells without drugs,
respectively.
toxic profile to macrophages culture, only
D
3,4-macrolides 1–3 as
well as
D
2,3-conjugated macrolide 5 deserve further attention
due their higher selectivity against the parasite.
Oral drugs are highly needed for both cutaneous and visceral
leishmaniasis due to the obvious drawbacks of intramuscular and
intravenous conventional chemotherapy. Miltefosine, the only oral
drug currently licensed for treating antimony-resistant visceral
leishmaniasis in India also poses limitations due to its teratogenic-
ity and a narrow therapeutic window.4 Due to the oral effective-
ness of Ivermectin in other infections, and the absence of acid-
sensitive groups in the in vitro-active 7, 12 and 13 analogues, it
is expected that they also serve for oral administration. Although
the majority of drug targets is conserved amongst the various
Leishmania species as apparently is the Ivermectin target in L.
donovani and L. major,7,8 the activity of those analogues against L.
donovani remains to be confirmed.
4.1.4. Cytotoxicity to macrophages
Mouse peritoneal macrophages were plated at 2 ꢁ 106 cells/
well in 24-well culture plates and incubated for 48 h at 37 °C in
1 mL of DMEM culture medium containing 5% HIFCS and different
concentrations of the test compounds (0, 10, 30 and 90 lM) in 0.5%
DMSO. The release of the cytoplasmic enzyme lactate dehydroge-
nase (LDH) into the culture medium was measured using an assay
kit (Doles Reagentes, Brazil).15 Maximum and minimum release
values were cells cultured with 2% Triton X-100 or 0.5% DMSO,
respectively.
4.1.5. Data analysis
In conclusion, the present study confirms and complements
preliminary studies on the antileishmanial properties of Ivermec-
tin, a well-known semi-synthetic macrolide widely used since
more than 20 years by the oral route for its antihelmintic proper-
ties. Our attempts to prepare analogues with improved antipara-
sitic activity were successful, leading to some molecules more
active against L. amazonensis than the parental compound. More
interestingly, the present preliminary SAR observations clearly
establish a different profile than for antihelminthic activities for
which the non-conjugated southern hexahydrobenzofuran is
claimed to be an essential structural feature for optimal activity.
Furthermore, different specific structural requirements are demon-
strated for anti-promastigote or anti-amastigote activity, a result
that, in the perspective of a future pharmacological application,
may serve to design more optimized molecules against the tar-
geted parasite.
The data were analyzed by one-way analysis of variance fol-
lowed by a Tukey posttest.
4.2. Chemistry
4.2.1. General
Optical rotations were determined on a JASCO instrument,
model DIP-370. Mass spectra were obtained by electron-spray
on a Micromass ZQ 4000 spectrometer by direct introduction.
High-resolution, high mass-accuracy measurements were per-
formed either on a Micromass Autospec mass spectrometer with
EBE configuration using 70 eV electron ionisation (EI) at the
Instituto de Química, Campinas, Brazil or on a Micromass ZAB-
SpecTOF spectrometer at the Centre Régional de Mesures Phy-
siques de l’Ouest, CRMPO, Rennes, France, using electron spray
ionisation (ESI) and methanol as solvent (4 kV acceleration,
60 °C source temperature). Infrared spectra were obtained in
potassium bromide on a Nicolet-Nexus 670 model instrument
(wavelengths are given in wavenumbers). 1H NMR spectra were
recorded on a Bruker DRX 300 or Bruker DRX 400 instruments.
The chemical shifts (d) are expressed in parts per million
(ppm) downfield from tetramethylsilane (TMS). Coupling con-
stants (J) are given in Hertz (Hz). 13C NMR spectra were recorded
on a Bruker AC 200 at 50.32 MHz or on a Bruker DRX 300 instru-
ment at 75.47 MHz. The chemical shifts are expressed in parts
per million (ppm), and are referenced to residual chloroform
(d = 77.0 ppm).
4. Experimental
4.1. Biological assays
4.1.1. Obtention of L. amazonensis-GFP
L. amazonensis (Josefa strain) transfected with the green fluores-
cence reporter protein (GFP), was periodically isolated as amastig-
otes from experimentally infected mice and maintained as
promastigotes in DMEM culture medium (Sigma Aldrich) supple-
mented with 10% heat inactivated foetal calf serum (HIFCS) at
27 °C as previously described.15 Transfected promastigotes were
Commercial Ivermectin {[
a
]
D
= +34.8 (c, 1.00, CHCl3)} used
throughout this work was a mixture of 22,23-dihydroavermectin
B1a and B1b, in a ratio greater than 97:3 (HPLC analysis) and is
therefore described throughout this paper as the B1a constituent.
This material was kept under high vacuum for several hours before
use. Most of the reagents (Aldrich) were used without treatment.
Titanium(IV) ethylate [Ti(OEt)4], was prepared before use by
refluxing [Ti(Oi-Pr)4] in anhydrous ethanol, followed by distillation
under reduced pressure (150–170 °C, 650–1300 Pa), and stored un-
der nitrogen. Ozone was obtained from a BMT 802 ozone generator
(BMT Messtechnik GMBH, Berlin, Germany) and the enriched
ozone stream was controlled using a flow rotameter. All the sol-
vents were distilled before use.
periodically selected in 150 lg/mL of geneticin antibiotic.
4.1.2. Anti-promastigote activity
Promastigotes of GFP-transfected L. amazonensis were cultured
at 5 ꢁ 105 cells/ml in 0.2 mL of DMEM culture medium containing
5% HIFCS, 0.5% DMSO plus the drugs at 0, 10, 30 and 90 lM for 72 h
at 27 °C in 96-well culture plates. Amphotericin B was used as a
reference drug. At the end of culture time, the fluorescence inten-
sity of the cultures was measured using a plate-reader fluorometer
(Bio-Tek) at 435 nm excitation and 538 nm emission.15
4.1.3. Anti-amastigote activity
For clarity, avermectins/Ivermectin carbon and proton
numbering has been used for all compounds throughout the
paper.
Mouse peritoneal macrophages were plated at 2 ꢁ 106 cells/
well of 24-well culture plates for adherence and then infected

A. R. d. Santos et al. / Bioorg. Med. Chem. 17 (2009) 496–502
501
4.2.2. Southern diol (12)
15.1 (Me-14), 12.3 (Me-26), 11.9 (Me-27) ppm. ES MS: m/z
(%) = 898 (100) [M++Na], 569 (11), 307 (22). HRMS (ESI, MeOH):
m/z calcd for C48H74O14 [M+] 874.5079; found 874.5063.
A solution of 5-O-(tert-butyldimethylsilyl) southern alcohol
(800 mg, 2.0 mmol) resulting from ozonolysis of 5-O-(tert-butyldi-
methylsilyl) IVM as previously described,11 was stirred in MeOH
(6.0 mL) under nitrogen at room temperature for 90 min in the
presence of PTSA (1% w/v). After total substrate consumption, the
mixture was diluted by AcOEt (50 mL) and washed with saturated
aqueous NaHCO3 (20 mL) and brine (20 mL). The organic layer was
dried with anhydrous MgSO4, filtered and concentrated under re-
duced pressure to give, after purification by flash chromatography
on silica gel (hexanes/AcOEt mixtures from 1:0 to 0:1), 71 mg
(12%) of southern alcohol 12 as colourless oil.
4.2.4.
An oven-dried 25-mL flask was charged under nitrogen with
2,3-(4S)-Ivermectin
(2.47 g, 2.82 mmol), ethanol (1.65 mL,
D
2,3-(4S)-Ivermectin ethyl secoester 6
D
5
29.0 mmol; 10.0 equiv) and Ti(OEt)4 (0.89 mL, 4.25 mmol,
1.5 equiv). The reaction mixture was stirred under reflux for five
days. After cooling to room temperature, the mixture was diluted
with diethyl ether (200 mL) and washed with aqueous 0.5 N HCl
(3 ꢁ 100 mL). The aqueous layer was washed with diethyl ether
(100 mL), and the combined organic fractions were washed with
brine (2 ꢁ 150 mL), dried with anhydrous MgSO4, filtered and con-
centrated under reduced pressure to give, after purification by sil-
ica gel flash chromatography (hexanes/AcOEt mixtures from 4:1 to
1H NMR (200 MHz; CDCl3): d = 5.67 (dddd, 1H, J = 2.2, 6.1 Hz, H-
9), 5.41 (dt, 1H, J = 1.8, 4.3 Hz, H-3), 4.65 (br d, 1H, J = 14.0 Hz, HA-
8a), 4.47 (br d, 1H, J = 14.0 Hz, HB-8a), 4.41 (br d, 1H, J = 4.9 Hz, H-
5), 4.20 (q, 2H, J = 7.1 Hz, COOCH2CH3), 4.19 (d, 1H, J = 4.9 Hz, H-6),
4.11 (br d, 2H, J = 6.1 Hz, H2-10), 3.41 (app. dt, 1H, J = 2.0, 4.3 Hz, H-
2), 2.91 (br s, 3H, HO-5. HO-7 and HO-10), 1.84 (br s, 3H, Me-4),
1.29 (t, 3H, J = 7.1 Hz, COOCH2CH3) ppm. 13C NMR (50.32 MHz,
CDCl3): d = 173.0 (C-1), 145.0 (C-8), 138.9 (C-4), 120.3 (C-9),
117.3 (C-3), 82.7 (C-6), 78.2 (C-7), 68.4 (C-8a), 67.9 (C-5), 61.5
(COOCH2CH3), 60.0 (C-10), 47.4 (C-2), 19.2 (Me-4), 14.1
(COOCH2CH3) ppm. HRMS (ESI, MeOH): m/z calcd for C14H20O6
[M+] 284.1260; found 284.1263.
1:9), 0.62 g (25%) of recovered 5 and 1.37 g (53%) of
mectin ethyl secoester 6 as a pale-yellow foam.
D
2,3-(4S)-Iver-
[a
]
D = +58.3 (c, 1.06, CHCl3). IR (KBr):
m
= 3473, 3000–2850,
1694, 1456, 1382, 1150–1000, 986 cmꢀ1
.
1H NMR (400 MHz;
CDCl3): d = 6.63 (d, 1H, J = 1.8 Hz, H-3), 6.33 (app dt, 1H, J = 2.4,
10.0 Hz, H-9), 5.97–5.84 (m, 2H, H-10 e H-11), 5.44 (br t, 1H,
J = 6.8 Hz, H-15), 5.28 (d, 1H, J = 3.2 Hz, H-100), 4.72 (d, 1H,
J = 2.8 Hz, H-10), 4.66 (dd, 1H, J = 2.4, 14.0 Hz, HA-8a), 4.54 (dd,
1H, J = 2.4, 14.0 Hz, HB-8a), 4.22 (m, 2H, COOCH2CH3), 4.07 (m,
1H, H-19), 4.02 (d, 1H, J = 2.4 Hz, H-6), 3.78–3.45 (m, 5H, H-17,
H-30, H-50, H-300 and H-500), 3.63 (d, 1H, J = 8.4 Hz, H-13), 3.55 (dd,
1H, J = 2.4, 9.6 Hz, H-5), 3.42 (s, 3H, OMe), 3.37 (s, 3H, OMe),
3.17–3.08 (m, 3H, H-40, H-400 and H-25), 2.63 (ddddd, 1H, J = 1.8,
7.1, 9.6 Hz, H-4), 2.45 (m, 1H, H-12), 2.33–2.23 (m, 3H, H2-16
and Heq-200), 2.09 (app ddd, 1H, J = 1.0, 4.8, 12.0 Hz, Heq-20), 2.00–
1.87 (m, 3H, including Heq-20 and Heq-18), 1.64 (br d, 1H,
J = 10.1 Hz), 1.54 (br s, 3H, Me-14), 1.53–1.45 (m, 6H, H2-22, H2-
23, H2-27), 1.34–1.22 (m, 9H, including Me-50 and Me-500), 1.32
(t, 3H, J = 7.2 Hz, COOCH2CH3), 1.12 (d, 3H, J = 6.0 Hz, Me-12),
1.11 (app q, 1H, J = 11.6 Hz, Hax-18), 0.92–0.86 (m, 6H, Me-4 e
Me-27), 0.80 (d, 3H, J = 6.8 Hz, Me-24), 0.78 (d, 3H, J = 6.0 Hz,
Me-26) ppm. 13C NMR (50.32 MHz, CDCl3): d = 167.6 (C-1), 144.5
(C-8), 140.4 (2C, C-3 e C-11), 133.8 (C-14), 128.7 (C-2), 127.2 (C-
10), 125.3 (C-15), 123.0 (C-9), 99.0 (C-100), 97.2 (C-21), 93.3 (C-10),
86.6 (C-13), 83.8 (C-6), 81.8 (C-40), 79.3 (C-30), 78.3 (C-300), 77.9
(C-5), 77.2 (C-25), 76.2 (C-400), 71.9 (C-7), 68.2 (2C, C-8a e C-500),
67.6 (C-17), 66.7 (C-50), 64.7 (C-19), 61.1 (COOCH2CH3), 56.3
(OMe), 56.1 (OMe), 45.0 (C-20), 40.6 (C-18), 38.3 (C-12), 35.8 (C-
22), 35.4 (C-26), 34.8 (C-20), 34.5 (C-200), 34.1 (C-16), 33.1 (C-4),
31.3 (C-24), 28.1 (C-23), 27.4 (C-27), 18.0 (Me-12), 17.6 (Me-500),
17.3 (Me-50), 16.7 (2C, Me-4 and Me-24), 14.0 (COOCH2CH3), 12.4
(Me-26), 11.4 (Me-27) ppm. ES MS: m/z (%) = 944 (100) [M++Na],
926 (6), 403 (7), 291 (12). HRMS (ESI, MeOH); m/z calcd for
C50H80O15 [M+] 920.5497; found 920.5466.
4.2.3.
D
2,3-(4S)-Ivermectin (5)
Ivermectin 1 (5.36 g, 6.12 mmol) and DBU (0.37 mL, 2.47 mmol,
0.4 equiv) were dissolved in 30 mL anhydrous THF and the solution
stirred at 85 °C for 15 h before cooling to room temperature. The
reaction mixture was diluted with diethyl ether (150 mL) and
washed with aqueous 0.5 N HCl (2 ꢁ 100 mL). The aqueous layer
was washed with diethyl ether (2 ꢁ 100 mL), and the combined or-
ganic extracts were washed with brine (2 ꢁ 100 mL), dried with
anhydrous MgSO4, filtered and concentrated under reduced pres-
sure to give, after purification by silica gel flash chromatography
(hexanes/AcOEt mixtures from 4:1 to 1:4), 4.78 g (89%) of D2,3
(4S)-Ivermectin 5 as a pale-yellow foam.
-
[
a]
D = +206.0 (c, 0.73, CHCl3). IR (KBr):
m
= 3500, 3000–2850,
1699, 1456, 1383, 1200–1000, 983 cmꢀ1
.
1H NMR (400 MHz;
CDCl3): d = 6.18 (dt, 1H, J = 2.4, 10.8 Hz, H-9), 6.15 (d, 1H,
J = 1.6 Hz, H-3), 5.78 (dd, 1H, J = 10.0, 14.8 Hz, H-11), 5.69 (dd,
1H, J = 10.8, 14.8 Hz, H-10), 5.41 (d, 1H, J = 3.6 Hz, H-100), 5.39–
5.29 (m, 1H, H-19), 4.94 (br d, 1H, J = 10.8 Hz, H-15), 4.80 (s, 1H,
HO-7), 4.76 (d, 1H, J = 3.2 Hz, H-10), 4.59 (dd, 1H, J = 2.4, 14.0 Hz,
HA-8a), 4.51 (dd, 1H, J = 2.4, 14.0 Hz, HB-8a), 4.05 (d, 1H,
J = 2.0 Hz, H-6), 3.93 (br s, 1H, H-13), 3.88–3.55 (m, 5H, H-5, H-30,
H-50, H-300 and H-500), 3.53–3.43 (m, 1H, H-17), 3.47 (s, 3H, OMe),
3.44 (s, 3H, OMe), 3.25–3.20 (app d, 1H, H-25), 3.25 (t, 1H,
J = 9.2 Hz, H-40 or H-400), 3.17 (t, 1H, J = 9.2 Hz, H-400 or H-40), 2.61
(br s, 1H, HO-400), 2.56–2.43 (m, 2H, H-4 and H-12), 2.36–2.18
(m, 4H, H2-16, Heq-20 and Heq-200), 1.95 (dd, 1H, J = 4.3, 12.4 Hz,
Heq-20), 1.87 (br d, 1H, J = 12.0 Hz, Heq-18), 1.70 (br s, 1H, HO-5),
1.68 (br d, 1H), 1.60–1.35 (m, 10H, including H2-22, H2-23 and
H2-27), 1.46 (br s, 3H, Me-14), 1.30–1.20 (m, 9H, Me-50, Me-500
and Me-4), 1.16 (d, 3H, J = 6.8 Hz, Me-12), 0.93 (t, 3H, J = 7.6 Hz,
Me-27), 0.84 (d, 3H, J = 6.4 Hz, Me-24), 0.79 (d, 3H, J = 5.2 Hz,
Me-26), 0.71 (q, 1H, J = 12.0 Hz, Hax-18) ppm. 13C NMR
(50.32 MHz, CDCl3): d = 168.8 (C-1), 138.9 (C-8), 138.6 (C-3),
138.0 (C-11), 134.7 (C-14), 129.7 (C-2), 125.4 (C-10), 122.7 (C-9),
118.0 (C-15), 98.4 (C-100), 97.3 (C-21), 94.7 (C-10), 82.9 (C-6), 81.8
(C-13), 80.3 (C-40), 79.2 (C-30), 78.4 (C-5), 78.1 (C-300), 76.6 (C-25),
75.9 (C-400), 72.0 (C-8a), 68.9 (C-19), 68.1 (C-7), 67.8 (C-500), 67.1
(2C, C-50 and C-17), 56.5 (OMe), 56.3 (OMe), 40.3 (C-20), 39.6 (C-
12), 36.8 (C-18), 35.7 (C-22), 35.3 (C-26), 34.4 (C-20), 34.3 (C-16),
34.1 (C-200), 33.1 (C-4), 31.1 (C-24), 27.9 (C-23), 27.2 (C-27), 20.1
(Me-12), 18.3 (Me-50), 17.6 (Me-500), 17.3 (Me-24), 16.8 (Me-4),
4.2.5.
A trace amount of Sudan Red 7B was added to a magnetically
stirred solution of
2,3-(4S)-Ivermectin ethyl secoester 6 (1.28 g,
D
2,3-(4S) Southern diol (13)
D
1.39 mmol) in 38.5 mL of 3:1 CH2Cl2/EtOH mixture. The mixture
was cooled to ꢀ78 °C under nitrogen and then ozone was bubbled
until TLC monitoring showed complete consumption of the start-
ing material (TLC monitoring was done after a rapid nitrogen
purge, independently of the solution colour fading). The reaction
mixture was then purged with nitrogen, added with NaBH4
(211 mg, 5.58 mmol, 4.0 equiv) in 9.6 mL of methanol, allowed to
warm to 0 °C and maintained at this temperature for 40 min. The
stirred solution was diluted with water at 0 °C and aqueous 1.0 N
HCl was added dropwise down to pH 4.0. The phases were sepa-
rated and the aqueous layer was extracted with chloroform
(2 ꢁ 50 mL), saturated with NaCl and re-extracted with 9:1

502
A. R. d. Santos et al. / Bioorg. Med. Chem. 17 (2009) 496–502
CHCl3/MeOH mixture (2 ꢁ 50 mL). The combined organic extracts
were washed with brine (2 ꢁ 100 mL), dried with anhydrous
MgSO4, filtered and concentrated under reduced pressure. The
crude product was purified by silica gel column chromatography
(4:1 hexanes/AcOEt) to give, in order of elution, 874 mg (93%) of
known northern alcohol 7 as a white powder and 230 mg (58%)
4.2.7. Northern aglycone (9)
Northern alcohol 7 (400 mg, 0.595 mmol) was added to a solu-
tion of 2% H2SO4 in 1:9 H2O/i-PrOH mixture (40 mL) and stirred at
50 °C for 24 h. Subsequent chloroform extraction as above fur-
nished a crude product which gave 90 mg (40%) of northern alco-
hol aglycone 9 as a white powder after chromatography (CH2Cl2/
AcOEt mixtures from 1:0 to 2:3).
of
D
2,3-(4S) southern diol 13 as a colourless oil.
IR (KBr):
= 3407, 3000–2850, 1695, 1456, 1372, 1200–1000 cmꢀ1
m
.
[a]
D = +59.4 (c, 0.96, AcOEt). IR (KBr):
m = 3390, 3000–2850,
1H NMR (400 MHz; CDCl3): d = 6.63 (d, 1H, J = 2.0 Hz, H-3), 5.94
(dddd, 1H, J = 2.5, 5.8, 6.8 Hz, H-9), 4.95 (br s, 1H, HO-7), 4.60 (app.
ddt, 1H, J = 1.3, 2.5, 13.7 Hz, HA-8a), 4.43 (app. ddt, 1H, J = 1.3, 2.5,
13.7 Hz, HB-8a), 4.24 (q, 2H, J = 7.2 Hz, COOCH2CH3), 4.12 (ddt, 1H,
J = 1.3, 6.8, 13.5 Hz, HA-10), 4.09 (ddt, 1H, J = 1.3, 5.8, 13.5 Hz, HB-
10), 4.03 (d, 1H, J = 2.4 Hz, H-6), 3.56 (dd, 1H, J = 2.4, 9.4 Hz, H-5),
2.59 (ddddd, 1H, J = 2.0, 7.3, 9.4 Hz, H-4), 2.40 (br s, 2H, HO-5 and
HO-10), 1.33 (t, 3H, J = 7.2 Hz, COOCH2CH3), 1.26 (d, 3H, J = 7.3 Hz,
Me-4) ppm. 13C NMR (50.32 MHz, CDCl3): d = 167.5 (C-1), 144.4 (C-
3), 143.6 (C-8), 128.9 (C-2), 122.8 (C-9), 83.4 (C-6), 78.3 (C-7), 72.3
(C-5), 67.9 (C-8a), 61.3 (COOCH2CH3), 60.3 (C-10), 33.4 (C-4), 16.8
(Me-4), 14.1 (COOCH2CH3) ppm. HRMS (ESI, MeOH): m/z calcd for
C14H20O6 [M+] 284.1260; found 284.1258.
1460, 1383, 1150–1000, 984 cmꢀ1 1H NMR (300 MHz; CDCl3):
.
d = 5.33 (br t, 1H, J = 6.8 Hz, H-15), 4.00 (m, 1H, H-19), 3.80 (d,
1H, J = 8.9 Hz, H-13), 3.70-3.15 (m, 5H, including H2-11, H-17),
3.07 (br d, 1H, H25), 2.20 (m, 2H, H2-16), 2.00-1.75 (m, 3H, includ-
ing Heq-18 and Heq-20), 1.56 (br s, 3H, Me-14), 1.65–1.35 (m, 6H,
H2-22, H2-23, H2-27), 1.35–1.15 (m, 4H), 1.02 (q, 1H, J = 11.6 Hz,
Hax-18), 0.82 (t, 3H, J = 7.2 Hz, Me-27), 0.78–0.60 (m, 9H, Me-12,
Me-24, Me-26) ppm. 13C NMR (50.32 MHz, CDCl3): d = 137.8 (C-
14), 124.2 (C-15), 97.3 (C-21), 84.6 (C-13), 77.2 (C-25), 68.0 (C-
11), 67.6 (C-17), 64.8 (C-19), 44.8 (C-20), 40.2 (C-18), 37.0 (C-12),
35.7 (C-22), 35.4 (C-26), 33.9 (C-16), 31.2 (C-24), 28.0 (C-23),
27.3 (C-27), 17.4 (Me-24), 13.8 (Me-12), 12.4 (Me-26), 11.7 (Me-
27), 11.2 (Me-14) ppm. HRMS (ESI, MeOH); m/z calcd for
C22H40O5 [M+] 384.2876; found 384.2876.
4.2.6. Northern monosaccharide (8)
Northern alcohol 7 (400 mg, 0.595 mmol) was added to a solu-
tion of 2% H2SO4 in 1:9 H2O/i-PrOH mixture (40 mL) and stirred at
rt for 24 h. Then chloroform (50 mL) was added, and the solution
transferred into a separatory funnel, washed with aqueous NaHCO3
solution (3 ꢁ 30 mL). The aqueous layer was re-extracted with
chloroform (2 ꢁ 30 mL), and the combined organic extracts were
washed with brine (50 mL), dried with anhydrous MgSO4, filtered
and concentrated under reduced pressure. The crude product was
purified by silica gel column chromatography (CH2Cl2/AcOEt mix-
tures from 1:0 to 1:1) to give 314 mg (90%) of northern alcohol
monosaccharide 8 as a white powder.
Acknowledgments
Michelle F. Muzitano thanks CAPES (Brazil) and FAPERJ (Brazil)
for fellowships. Anderson Rouge dos Santos thanks CNPq (Brazil)
for doctoral fellowship.
References and notes
1. World Health Organization. http://www.who.int/leishmaniasis/en/ (2008);
http://www.who.int/tdr/diseases/leish/default.htm (2008).
2. Croft, S. L.; Coombs, G. H. Trends Parasitol. 2003, 19, 502.
3. Desjeux, P. Comp. Immun. Microbiol. Infect. Dis. 2004, 27, 305.
4. Croft, S. L.; Sundar, S.; Fairlamb, A. H. Clin. Microbiol. Rev. 2006, 19, 111.
5. For an example: Christensen, D. Sci. News 2003, 162, 296.
6. Omura, S. Int. J. Antimicrob. Agent 2008, 31, 91.
7. Rifaat, L. K.; Mohammad, M. A.; Jawdat, S. Z. Jpn. J. Med. Sci. Biol. 1989, 42, 51.
8. Rasheid, K. A.; Morsy, T. A. J. Egypt Soc. Parasitol. 1998, 28, 207.
9. Mrozik, H.; Eskola, P.; Arison, B. H.; Albers-Schönberg, G.; Fisher, M. H. J. Org.
Chem. 1982, 47, 489.
[a
]
D = ꢀ16.7 (c, 0.73 , AcOEt). IR (KBr):
m = 3406, 3000–2850,
1458, 1383, 1150–1000, 987 cmꢀ1 1H NMR (300 MHz; CDCl3):
.
d = 5.52 (br t, 1H, J = 6.8 Hz, H-15), 4.82 (d, 1H, J = 3.0 Hz, H-10),
4.08 (m, 1H, H-19), 3.80 (d, 1H, J = 9.8 Hz, H-13), 3.75–3.30 (m,
5H, H2-11, H-17, H-30, H-50), 3.40 (s, 3H, OMe), 3.17–3.07 (m, 2H,
H-40, H25), 2.57 (br s, 1H, HO-11), 2.53 (br s, 1H, HO-40), 2.28 (br
t, 2H, J = 6.8 Hz, H2-16), 2.14 (dd, 1H, J = 4.9, 13.2 Hz, Heq-20),
2.02–1.87 (m, 3H, including Heq-18 and Heq-20), 1.63 (br d, 1H,
J = 7.6 Hz), 1.55 (br s, 3H, Me-14), 1.58–1.43 (m, 6H, H2-22, H2-
23, H2-27), 1.37–1.22 (m, 4H), 1.29 (br d, 3H, J = 6.0 Hz. Me-50),
1.13 (q, 1H, J = 11.7 Hz, Hax-18), 0.88 (t, 3H, J = 7.2 Hz, Me-27),
0.84–0.73 (m, 9H, Me-12, Me-24, Me-26) ppm. 13C NMR
(75.47 MHz, CDCl3): d = 132.8 (C-14), 128.1 (C-15), 97.2 (C-21),
92.8 (C-10), 85.5 (C-13), 78.5 (C-30), 77.3 (C-25), 76.1 (C-40), 68.0
(C-50), 67.6 (C-17), 66.6 (C-11), 64.8 (C-19), 56.6 (OMe-30), 45.0
(C-20), 40.6 (C-18), 36.8 (C-12), 35.8 (C-22), 35.4 (C-26), 34.1
(C20), 34.0 (C-16), 31.3 (C-24), 28.1 (C-23), 27.5 (C-27), 18.0 (Me-
50), 17.4 (Me-24), 14.1 (Me-12), 12.5 (Me-26), 11.4 (Me-27), 10.9
(Me-14) ppm. HRMS (ESI, MeOH); m/z calcd for C29H50O7 [M+–
18] 510.3557; found 510.3560.
10. Chabala, J. C.; Mrozik, H.; Tolman, R. L.; Eskola, P.; Lusi, A.; Peterson, L. H.;
Woods, M. F.; Fisher, M. H. J. Med. Chem. 1980, 23, 1134.
11. Santos, A. R.; Ferreira, M. L. G.; Kaiser, C. R.; Férézou, J.-P. Eur. J. Org. Chem. 2005,
3348.
12. For example, against mites, conjugated avermectin B1a exhibited only one-
fifth of the natural product activity: Pivnichny, J. V.; Arison, B. H.; Preiser, F. A.;
Shim, J.-S. K.; Mrozik, H. J. Agric. Food Chem. 1988, 36, 826.
13. Hanessian, H.; Dubé, D.; Hodges, P. J. J. Am. Chem. Soc. 1987, 109, 7063.
14. Hanessian, H.; Ugolini, A.; Hodges, P. J.; Dubé, D. Tetrahedron Lett. 1985, 27,
2699.
15. Boeck, P.; Falcão, C. B.; Leal, P. V.; Cechinel-Filho, V.; Torres-Santos, E. C.; Yunes,
R. A.; Rossi-Bergmann, B. Bioorg. Med. Chem. 2006, 16, 1538.
16. Muzitano, M. F.; Tinoco, L. W.; Guette, C.; Kaiser, C. R.; Rossi-Bergmann, B.;
Costa, S. S. Phytochemistry 2006, 67, 2071.
17. Bozue, J. A.; Parthasarathy, N.; Phillips, L. R.; Cote, C. K.; Fellows, P. F.;
Mendelson, I.; Shafferman, A.; Friedlander, A. M. Microb. Pathog. 2005, 38, 1.
18. Monteiro, G. C. T. S.; Hirata, R.; Andrade, A. F. B.; Mattos-Guaraldi, A. L.; Nagao,
P. E. Int. J. Mol. Med. 2004, 13, 175.