Synthesis, antimalarial and antileishmanial activity of novel 13-benzyl-15,16-bisnorlabdane derivatives

Article abstract of DOI:10.3184/174751913X13794472538261

Twelve 13-benzyl-15,16-bisnorlabdanes in which the C-13 and C-14 substituents are varied have been prepared from the naturally occurring labdane diterpene (+)-manool. These synthesised compounds were evaluated for antimalarial activity, in vitro as hemozo

Full text of DOI:10.3184/174751913X13794472538261

JOURNAL OF CHEMICAL RESEARCH 2013
NOVEMBER, 657–661
RESEARCH PAPER 657
Synthesis, antimalarial and antileishmanial activity of novel 13-benzyl-15,16-
bisnorlabdane derivatives
a
b
c
d
Franklin J. Salazar , Alberto Quintero , Neira Gamboa de Domínguez , Juan L. Concepción ,
María E. Acosta , Eleonora Tropper and José E. Villamizar *
c
a
a,b
aCentro de Química, Instituto Venezolano de Investigaciones Científicas (IVIC), Caracas 1020-A, Venezuela
bDepartamento de Química Medicinal, Instituto Venezolano de Investigaciones Científicas (IVIC), Caracas 1020-A, Venezuela
cUnidad de Bioquimica, Facultad de Farmacia, Universidad Central de Venezuela (UCV), Caracas 1051, Venezuela
dLaboratorio de Enzimología de Parásitos, Facultad de Ciencias, Universidad de Los Andes (ULA), Mérida 5101, Venezuela
Twelve 13-benzyl-15,16-bisnorlabdanes in which the C-13 and C-14 substituents are varied have been prepared from the naturally
occurring labdane diterpene (+)-manool. These synthesised compounds were evaluated for antimalarial activity, in vitro as
hemozoin formation inhibitors and in vivo against Plasmodium berghei. These derivatives were also assayed for antileishmanial
activity against Leishmania mexicana.
Keywords: bisnorlabdanes, hemozoin formation inhibitors, antimalarial, antileishmanial
Malaria and leishmaniasis are protozoan diseases with a
large global distribution and which have a high mortality rate
affecting millions of people every year. Globally, an estimated
as therapeutic agents to their natural counterparts. However,
half the drugs on the market are direct descendants of natural
10
products. Labdanes diterpenoids are among the most common
11–15
3.3 billion people were at risk of malaria in 2011, and estimates
types of bicyclic diterpenes isolated from plants and sponges
by the World Health Organization (WHO) have indicated that
approximately 80% of cases and 90% of deaths occur in Africa,
with children less than five years of age and pregnant women
and they have shown a large spectrum of interesting biological
activity such as cytotoxic, antifungal, anti-inflammatory,
antiparasitic and analgesic activity. Several natural labdane-
16
1
most severely affected. The Plasmodium falciparum parasite is
type diterpenes have shown good activity against chloroquine
responsible for most of the fatal cases of malaria. Leishmaniasis
threatens about 350 million people in 98 countries or territories
aroundtheworld, about2 millionestimatednewcasesoccurring
each year, and 12 million people are believed to be currently
sensitive (compounds 1–6, IC =24.0–54.0 µM) and resistant
5
0
(
compounds 8–13, IC =5.0–39.9 µM) Plasmodium falciparum
50
17–19
strain (3D7 and FcB1, respectively)
donovani (compounds 7, 14 and 15, IC =5.7–60.0 µM).
and against Leishmania
2
0–21
2
5
0
infected. Malarial parasites have developed unacceptable
levels of resistance towards existing treatment regimens3–8
In this context, we previously reported the synthesis of
optically active labdane-related natural products showing
antimalarial activity, which act by inhibiting the β-hematin
formation and/or the globin proteolysis. Some of them showed
a significant inhibition of one process or a moderate inhibition
of both.22 In continuation of this work, aimed at discovering
compounds with antiparasitic activity, we report the preparation
and, antimalarial and antileishmanial activity of twelve novel
related-labdane 13-benzyl-15,16-bisnorlabdanes 21a–23d in
which the C-13 and C-14 substituents are varied.
and drugs currently used against leishmaniasis show high
toxicities, producing clinical resistance and requiring long-
9
term treatment. Therefore, there is an urgent need for a rapid
search and discovery of new antimalarial and antileishmanial
agents with a novel structural backbone.
Plants have traditionally been an excellent and reliable source
for the discovery and development of new drugs. Although
severalsyntheticdrugspossessstructureswithlittleresemblance
to those of natural products, these compounds are often superior
R
1
O
OH
O
CHO
O
CHO
O
OHC
O
OHC
R
O
O
R
2
O
R
3
1
2
3
4 R = CHO
6
7
R
R
1
1
= R
= OCH
2
= H; R
3
= CO
2
CH
3
5
2 5 2
R = CH(OC H )
3
; R = OH; R
2
3
= CH
3
3
H CO
O
O
O
CO CH
2 3
O
CO
2
CH
3
HO
O
O
O
O
R
1
HO
HO
R
2
R
3 2
H CO C
8
9
1
R
R
0 R
1
1
= OH; R
= R = OH
= R = H
2
= H
11
12 R = H
14
15
2
13 R = OH
1
2
Scheme 1
*
Correspondent. E-mail: jvillami@ivic.gob.ve
JCR1302067_FINAL.indd 657
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658 JOURNAL OF CHEMICAL RESEARCH 2013
5
'
R1
N
14
1
'
3
'
R2
1
3
OH
O
R
2
0
11
1
7
1
i
ii
iv
10
8
3
6
1
8
19
1
6
17
1
8 R= CH
2
OH
21a-21d
iii
v
19 R= CHO
0 R= CH Br
R1
2
2
R
vi
R2
1
2
a: R = R = H
1
2
b: R = OH; R = H
1
2
c: R = OCH
3
; R = H
1
2
d: R = OCH
2
O = R
2
2
2a-22d R= CN
3a-23d R= CH
vii
+
-
2
NH
3
Cl
Scheme 2 (i) KMnO , Bu NBr, CHCl , 0 °C, 12 h; (ii) refs. 23,24; (iii) refs. 23,24; (iv) K CO , CH OH, γ-bicyclohomofarnesal
4
4
3
2
3
3
1
2
1
2
1
9, (R ,R )-benzylacetonitrile, rt, 4 h; (v) refs. 23,24; (vi) NaH, THF, (R ,R )-benzylacetonitrile, rt → reflux, 90 min →
bromide 20, rt → reflux, 3 h; (vii) LiAlH , THF, 0 °C → reflux, 6 h → Et O•HCl, 0 °C, 1 h.
4
2
Results and discussion
(H-12) with the aromatic protons (H-2’ and H-6’). Compound
2
0 was coupled with appropriate benzylacetonitrile in the
1
3-Benzyl-15,16-bisnorlabdanes 21a–23d were obtained from
presence of NaH and THF for 5 h at reflux temperature to
afford an inseparable mixture (1:1, calculated from H NMR
integrals) of 13-α and 13-β-benzyl-15,16-bisnorlabda-8(17)-en-
naturally occurring and commercially available (+)-manool
1
16, a labdane diterpene commonly used as semi-synthetic
2
3
precursor for the synthesis of others labdane diterpenes.
γ-Bicyclohomofarnesal 19 and the bromide 20 were prepared
by removing the side chain of the starting material 16 using
a procedure reported by Villamizar et al.
key intermediaries in the preparation of derivatives 21a–23d.
Compound 16 was oxidised according to a slightly modified
method of Costa et al. to afford ketone 17 in 91% yield.
γ-Bicyclohomofarnesal 19 was condensed with the appropriate
benzylacetonitrile in the presence of K CO and CH OH at room
14-nitrile derivatives 22a–d. The nitrile group of compounds
22a–d was reduced with LiAlH in THF refluxing for 6 h, then
4
2
4–25
the respective amine chloride derivatives 23a–d were formed
by treating the crude product with a saturated solution of
Et O•HCl at 0 °C.
and used as
2
2
6
The antimalarial and antileishmanial activity of compounds
21a–23d are presented in Table 1. A possible relationship
between the structure and the inhibition of β-haematin
formation (IHF) of compounds containing nitrile double bond
conjugation 23a–d suggested that removing the conjugated
double bond tended to improve inhibition and removal of the
conjugated double bond and converting the nitrile to an amino
group significantly enhanced inhibition. Among the compounds
2
3
3
temperature for 4 h to obtain 13-benzyl-15,16-bisnorlabda-
(17),12(13)-dien-14-nitrile derivatives 21a–d (Scheme 2). The
8
new alkene (C12–C13) which was formed on compounds 21a–d
with an exclusive -Z configuration, was confirmed by NOESY
through observing the spatial interaction of the olefinic proton
Table 1 Antimalarial and antileishmanial evaluation of bisnorlabdanes 21a–23d
In vitro antimalarial
In vivo antimalarial
Antileishmanial
PI/%h IC /µMi
Compound
IHF/%a
IC /mM
b
P/%c
NDj
ND
ND
S/%d
ST/daye
5
0
50
2
2
2
2
2
2
2
2
2
2
2
2
1a
1b
1c
1d
2a
2b
2c
2d
3a
3b
3c
<40
<40
<40
<40
<40
>25
−
−
−
−
−
−
−
0.09
0.74
2.31
72.27
0
>100
>100
84.26
26.3
>25
>25
>25
>25
>25
6.12
>25
3.47
0.22
2.36
3.87
0.48
−
ND
−
2.40±0.89
11.15±2.21
2.40±1.14
18.16±5.67
5.80±3.27
10.60±3.84
3.40±1.67
10.57±4.19
0.95±0.75
46.60±1.94
94.85
76.07
94.85
61.03
87.55
77.25
92.70
77.32
97.96
−
8.75±3.59
10.40±3.20
23.80±4.32
9.20±4.32
9.40±2.30
11.33±3.93
8.50±1.73
8.00±4.30
28.00±1.34
8.60±1.42
>100
49.04±1.29
69.37±0.69
46.90±0.59
88.69±0.74
88.53±1.06
73.00±4.57
78.12±1.03
88.03±0.77
−
0
0
0
>100
>100
>100
m
k
8.58
13.26
5.87
69.24
−
48.92
k
52.30
38.66
70.38
−
3d
f
CQ
g
NC
−
a
b
c
IHF, Inhibition of β-haematin formation, at 25 mM. IC , Concentration required to inhibit β-haematin formation by 50%. P, Parasitemia,
5
0
d
e
at fourth day post-infection in infected mice with Plasmodium berghei. S, Parasitemia suppression, at fourth day post-infection. ST,
f
g
h
Survival time of infected mice. CQ, Chloroquine. NC, Negative control, infected mice and only treated with saline-Tween solution. PI,
i
Promastigote inhibition, incubation by 54 h with drugs at 20 µM. IC , Concentration required to inhibit L. mexicana promastigote by 50%.
ND, Not determined. P>0.05 compared to IHF obtained with CQ. P>0.05 compared to survival time of infected mice and treated with CQ.
5
0
j
k
m
JCR1302067_FINAL.indd 658
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JOURNAL OF CHEMICAL RESEARCH 2013 659
that were tested, 22c, 23c and 23d presented good inhibition
and, 23a and 23b inhibited the β-haematin formation as much
as chloroquine (P>0.05). Special attention must be paid to
125.48 (C-2′, C-6′), 116.74 (C-14), 115.51 (C-13), 107.75 (C-17), 56.70 (C-
9), 55.44 (C-5), 42.01 (C-3), 39.70 (C-10), 39.15 (C-1), 37.98 (C-7), 33.59
(
2
[
C-4, C-18), 27.33 (C-11), 24.19 (C-6), 21.71 (C-19), 19.31 (C-2), 14.41 (C-
–1
0). IR (KBr) ν /cm : 3081, 2933, 2210, 1638. MS (ESI): m/z= 334.10
2
3b, which exhibited an IC50 twice as potent as chloroquine.
On in vivo antimalarial assay, compounds 22a, 22c, 23a and
3c gave good parasitemia suppression on the fourth day post-
max
+
M+H] . Anal. Calcd for C H N (333.25): C, 86.43; H, 9.37; N, 4.20.
24 31
Found: C, 86.23; H, 9.31; N, 4.16%.
3-(4-Hydroxy)benzyl-15,16-bisnorlabda-8(17),12(13)-dien-14-nitrile
2
1
infection compared to the non-treated mice (87.55–94.85%).
However, only compound 22c increased the mean survival time
of animals as significantly as chloroquine (P>0.05).
2
D
4
(
1
21b): Yield: 75%; white solid, m.p. 156–158 °C. [α] –3.9 (c 4.1, CHCl3).
H NMR (CDCl , 300 MHz): δ 0.74, 0.81 and 0.87 (s, 3H each, CH ),
3
3
4
.54 and 4.86 (s, 1H each, H-17), 6.66 (t, 1H, J=7.0 Hz, H-12), 6.82 (d,
On in vitro antileishmanial evaluation, compounds 21d, 23a
and 23c (IC =26.3–48.92 µM) were found to be moderately
13
2H, J=8.2 Hz, H-2′, H-6′), 7.32 (d, 2H, J=8.2 Hz, H-3′, H-5′). C NMR
5
0
(
CDCl , 75.45 MHz): δ 156.43 (C-4′), 148.15 (C-8), 146.36 (C-12), 126.92
3
active. Compounds 21c, 23b and 23d showed weak activities
IC =52.30–84.26 µM) and compounds 21a, 21b and 22a–d
(C-2′, C-6′), 125.80(C-1′), 116.96 (C-14), 115.79 (C-3′, C-5′), 114.73 (C-13),
(
5
0
107.68 (C-17), 56.72 (C-9), 55.40 (C-5), 41.98 (C-3), 39.66 (C-10), 39.10
were found to be completely inactive (IC >100 µM). Possible
5
0
(C-1), 37.96 (C-7), 33.56 (C-4, C-18), 27.16 (C-11), 24.17 (C-6), 21.65
SAR suggests that the decline in activities of compounds 22a–d
may be due to the removal of the conjugated double bond linked
to the nitrile and aromatic groups. Compound 21d having a
nitrile group, conjugated double bond and 3,4-methylendioxy
substituent on the aromatic ring was found to be the most active
compound in the series.
–1
(
2
C-19), 19.29 (C-2), 14.42 (C-20). IR (KBr) ν /cm : 3421, 3080, 2933,
max
+
218, 1641. MS (ESI): m/z=350.09 [M+H] . Anal. Calcd for C H NO
24 31
(349.24): C, 82.47; H, 8.94; N, 4.01. Found: C, 82.26; H, 8.87; N, 3.53%.
1
3-(4-Methoxy)benzyl-15,16-bisnorlabda-8(17),12(13)-dien-14-nitrile
24
(
1
21c): Yield: 85%; white solid, m.p. 100–102 °C. [α]D –4.7 (c 5.9, CHCl3).
H NMR (CDCl , 300 MHz): δ 0.75, 0.81 and 0.87 (s, 3H each, CH ), 3.79
3
3
This study reports the synthesis of novel 13-benzyl-15,16-
bisnorlabda-8(17),12(13)-dien-14-nitrile 21a–d, 13-benzyl-
(
s, 3H, Ar–OCH ), 4.55 and 4.86 (s, 1H each, H-17), 6.66 (t, 1H, J=7.0 Hz,
3
H-12), 6.86 (d, 2H, J=8.6 Hz, H-3′, H-5′), 7.40 (d, 2H, J=8.9 Hz, H-2′,
13
15,16-bisnorlabda-8(17)-en-14-nitrile 22a–d and 13-benzyl-
H-6′). C NMR (CDCl , 75.45 MHz): δ 159.95 (C-4′), 148.17 (C-8),
3
15,16-bisnorlabda-8(17)-en-14-amine 23a–d derivatives.
146.06 (C-12), 126.74 (C-2′, C-6′), 126.03 (C-1′), 116.94 (C-14), 114.89 (C-
Several of the bisnorlabdanes which had been prepared
exhibited in vitro and in vivo significant antimalarial activity
and some displayed an in vitro moderate antileishmanial
activity. Compound 22c is promising and provides a useful
model for further structural and biological optimisation to
obtain new antimalarial agents.
13), 114.19 (C-3′, C-5′), 107.71 (C-17), 56.75 (C-9), 55.43 (C-5), 55.34 (Ar–
OCH ), 42.02 (C-3), 39.69 (C-10), 39.13 (C-1), 37.99 (C-7), 33.58 (C-4,
3
C-18), 27.17 (C-11), 24.19 (C-6), 21.70 (C-19), 19.31(C-2), 14.39 (C-20). IR
–1
+
(KBr) νmax/cm : 3081, 2938, 2214, 1642. MS (ESI): m/z=364.18 [M+H] .
Anal. Calcd for C H NO (363.26): C, 82.60; H, 9.15; N, 3.85. Found: C,
25 33
82.41; H, 9.08; N, 3.49%.
1
3-(3,4-Methylendioxy)benzyl-15,16-bisnorlabda-8(17),12(13)-dien-
24
Experimental
14-nitrile (21d): Yield: 90%; white solid, m.p. 21–123 °C. [α]
c 6.4, CHCl ). H NMR (CDCl , 300 MHz): δ 0.75, 0.81 and 0.87 (s, 3H
D
–10.4
1
(
Melting points were measured with a Kofler hot-stage apparatus
and were uncorrected. NMR spectra were recorded with Bruker
Avance-300 and Avance-500 spectrometers. IR spectra were recorded
using a Nicolet Magna 560 FT-IR spectrometer. Mass Spectra (MS)
were obtained on a TSQ Quantum mass spectrometer. Elemental
analyses were performed using a Fisons EA-1108 instrument. Optical
3
3
each, CH ), 4.53 and 4.86 (s, 1H each, H-17), 5.95 (s, 2H, OCH O), 6.63
3
2
(
t, 1H, J=7.0 Hz, H-12), 6.77 (d, 1H, J=7.9 Hz, H-5′), 6.92 (d, 1H, J= 1.6,
13
H-2′), 6.98 (dd, 1H, J=8.2, 1.6 Hz, H-6′). C NMR (CDCl , 75.45 MHz):
3
δ 148.24 (C-3′), 148.15 (C-8), 148.07 (C-4′), 146.66 (C-12), 127.70 (C-1′),
119.91 (C-6′), 116.80 (C-14), 114.99 (C-13), 108.41 (C-5′), 107.71 (C-2′),
1
05.50 (C-17), 101.44 (OCH O), 56.75 (C-9), 55.45 (C-5), 42.03 (C-3),
rotations were obtained for CHCl solutions on a Perkin-Elmer 341
2
3
3
9.72 (C-10), 39.15 (C-1), 38.01 (C-7), 33.62 (C-4, C-18), 27.20 (C-11),
polarimeter, and their concentrations are expressed in g/100 mL.
Manool resin was purchased from Westchem Industries, Ltd. and
–1
24.22 (C-6), 21.74 (C-19), 19.34 (C-2), 14.43 (C-20). IR (KBr) ν /cm :
max
+
2
4
3081, 2931, 2218, 1634. MS (ESI): m/z=378.10 [M+H] . Anal. Calcd for
purified to obtain (+)-manool 16, [α] +28 (c 1.5, CHCl ). THF and
D
3
C H NO (377.24): C, 79.54; H, 8.28; N, 3.71. Found: C, 79.32; H, 8.20;
CH OH were purified before use. All other solvents and reagents
25 31
2
3
N, 3.62%.
were obtained from commercial suppliers and used without further
purification. Merck silica gel (70–230 mesh ASTM) was used for
column chromatography. TLC was performed on Analtech silica gel 60
G254 and the spots were observed either by exposure to iodine or by
Synthesis of 22a–d; general procedure
A solution of benzylnitrile, 4-hydroxybenzylnitrile, 4-methoxy-
benzylnitrile or 3,4-methylendioxybenzylnitrile (2.0047 mmol) in
THF (2.0 mL) was added to a suspension of NaH (6.6824 mmol) in THF
UV light. All organic extracts were dried over MgSO and evaporated
4
under reduced pressure below 60 °C.
(6.0 mL) was added at room temperature. The reaction mixture was
Synthesis of 21a–d; general procedure
stirred and refluxed for 90 minutes. Then, a solution of the bromide
20 (435 mg, 1.4534 mmol) in THF (2.0 mL) was added and the reaction
was stirred and refluxed for an additional 3 h. Finally, the reaction
was diluted with water, neutralised with HCl (2 M) and extracted with
A
mixture of aldehyde 19 (50.0 mg, 0.2133 mmol) and
benzylnitrile, 4-hydroxybenzylnitrile, 4-methoxybenzylnitrile or
,4-methylendioxybenzylnitrile (0.2133 mmol) in methanol (0.50 mL)
was added to a suspension of K CO (147.4 mg; 1.0666 mmol) in
3
diethyl ether. The organic extract was dried over MgSO , evaporated
2
3
4
methanol (1.50 mL) under nitrogen at room temperature, the reaction
mixture was stirred for 4 h. The reaction was diluted with water,
neutralised with HCl (2 M) and extracted with diethyl ether. The
and the crude product was chromatographed over silica gel using
mixtures of hexane and ethyl acetate.
13-Benzyl-15,16-bisnorlabda-8(17)-en-14-nitrile (22a): Yield: 93%;
1
organic extract was dried over MgSO , evaporated and the crude
white solid; H NMR (CDCl , 300 MHz): δ 0.64–0.66, 0.78–0.79 and
4
3
product was chromatographed over silica gel using mixtures of hexane
and ethyl acetate.
0.85 (s, 3H each, CH ), 3.73 (m, 1H, H-13), 4.33–4.40 and 4.78–4.79 (s, 1H
3
13
each, H-17), 7.33 (m, 5H, H-2′, H-3′, H-4′, H-5′, H-6′). C NMR (CDCl ,
3
1
3-Benzyl-15,16-bisnorlabda-8(17),12(13)-dien-14-nitrile (21a): Yield:
75.45 MHz): δ 148.23–147.83 (C-8), 136.03–135.92 (C-1′), 128.93 (C-3′,
C-5′), 127.93–127.87 (C-4′), 127.21–127.10 (C-2′, C-6′), 120.98–120.75
(C-14), 106.60–106.23 (C-17), 56.57–56.16 (C-9), 55.53–55.47 (C-5),
42.06 (C-3), 39.76–39.69 (C-13), 38.99–38.93 (C-10), 38.21–38.17 (C-
1), 37.82–37.59 (C-7), 35.03–34.88 (C-12), 33.56–33.51 (C-4, C-18),
24.34 (C-6), 21.65 (C-19), 21.30–21.18 (C-11), 19.30–19.27 (C-2), 14.38–
2
4
1
8
(
(
0%; white solid, m.p. 80–82 °C. [α] +4.1 (c 6.8, CHCl ). H NMR
CDCl , 300 MHz): δ 0.77, 0.82 and 0.88 (s, 3H each, CH ), 4.56 and 4.87
s, 1H each, H-17), 6.80 (t, 1H, J=7.0 Hz, H-11), 7.34 (m, 3H, H-3′, H-4′,
H-5′), 7.47 (m, 2H, H-2′, H-6′). C NMR (CDCl , 75.45 MHz): δ 148.38
D
3
3
3
13
3
(
C-8), 148.15 (C-12), 133.38 (C-1′), 128.83 (C-3′, C-5′), 128.64 (C-4′),
JCR1302067_FINAL.indd 659
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6
60 JOURNAL OF CHEMICAL RESEARCH 2013
–
1
1
4.35 (C-20). IR (KBr) ν /cm : 3076, 2929, 2235, 1641. MS (ESI):
13-(4-Hydroxy)benzyl-15,16-bisnorlabda-8(17)-en-14-amine (23b):
max
+
1
m/z=336.13 [M+H] . Anal. Calcd for C H N (335.26): C, 85.91; H, 9.91;
Yield: 83%; white solid; H NMR (CDCl , 300 MHz): δ 0.56–0.59,
2
4
33
3
N, 4.17. Found: C, 85.70; H, 9.84; N, 4.04%.
0.77–0.78, 0.84–0.86 (s, 3H each, CH ), 2.74 (m, 1H, H-13), 3.08 (m, 1H,
3
1
3-(4-Hydroxy)benzyl-15,16-bisnorlabda-8(17)-en-14-nitrile (22b):
H-14), 4.14–4.49, 4.69–4.82 (d, 1H each, J=2.2–2.2 Hz, H-17), 6.80–6.83
(d, 1H, J=13.8 Hz, H-3′, H-5′), 7.07–7.10 (d, 1H, J=14.1 Hz, H-2′, H-6′).
1
Yield: 85%; white solid; H NMR (CDCl , 300 MHz): δ 0.62–0.65,
.77–0.78 and 0.84 (s, 3H each, CH ), 3.66 (m, 1H, H-13), 4.31–4.39 and
3
13
0
C NMR (CDCl , 75.45 MHz): δ 158.13–158.07 (C-4′), 149.96–149.56
3
3
4
.76–4.78 (s, 1H each, H-17), 5.25 (bs, 1H, Ar–OH), 6.79–6.81 (d, 2H,
(C-8), 131.90–131.62 (C-2′, C-6′), 130.16–130.02 (C-1′), 116.92–116.88
(C-3′, C-5′), 107.05–106.92 (C-17), 58.46–57.75 (C-9), 56.93–56.82 (C-5),
45.56–45.15 (C-14), 43.32–43.27 (C-3), 40.72–40.68 (C-10), 40.17–39.98
(C-1), 39.41–39.34 (C-7), 34.47–34.43 (C-12), 34.33–34.09 (C-13), 34.06–
33.94 (C4, C18), 25.60–25.53 (C-6), 22.45 (C-19), 22.16–22.11 (C-11),
13
J=8.9 Hz, H-3′, H-5′), 7.13–7.15 (d, 2H, J=8.8 Hz, H-2′, H-6′). C NMR
CDCl , 75.45 MHz): δ 155.40–155.34 (C-4′), 148.28–147.91 (C-8),
(
3
1
1
(
(
28.56–128.44 (C-2′, C-6′), 128.01–127.89 (C-1′), 121.35–121.11 (C-14),
15.83 (C-3′, C-5′), 106.60–106.26 (C-17), 56.57–56.19 (C-9), 55.55–55.50
C-5), 42.08 (C-3), 39.79–39.72 (C-13), 39.02–38.98 (C-10), 38.23–38.19
C-1), 37.01–36.82 (C-7), 35.00–34.87 (C-12), 33.55 (C-4, C-18), 24.36 (C-
), 21.67 (C-19), 21.23–21.14 (C-11), 19.30 (C-2), 14.37 (C-20). IR (KBr)
–1
20.38–20.30 (C-2), 14.86 (C-20). IR (KBr) ν /cm : 3277, 2927, 1957.
max
+
MS (ESI): m/z=356.41 [M–Cl] . Anal. Calcd for C H ClNO (391.26):
2
4
38
6
C, 73.53; H, 9.77; N, 3.57. Found: C, 73.31; H, 9.71; N, 3.37%.
–1
+
νmax/cm : 3342, 3068, 2932, 2253, 1640. MS (ESI): m/z=352.16 [M+H] .
Anal. Calcd for C H NO (351.26): C, 82.00; H, 9.46; N, 3.98. Found: C,
13-(4-Methoxy)benzyl-15,16-bisnorlabda-8(17)-en-14-amine (23c):
1
Yield: 98%; white solid; H NMR (CDCl , 300 MHz): δ 0.56–0.59, 0.77–
2
4
33
3
81.81; H, 9.41; N, 3.86%.
0.78, 0.84–0.86 (s, 3H each, CH ), 2.74 (m, 1H, H-13), 3.05 (m, 1H, H-14),
3
1
3-(4-Methoxy)benzyl-15,16-bisnorlabda-8(17))-en-14-nitrile (22c):
3.80 (s, 3H, Ar–OCH ), 4.14–4.49, 4.69–4.82 (s, 1H each, H-17), 6.95–
3
1
Yield: 94%; white solid; H NMR (CDCl , 300 MHz): δ 0.62–0.65, 0.77
and 0.84 (s, 3H each, CH ), 3.67 (m, 1H, H-13), 4.31–4.40 and 4.76–4.78
6.98 (dd, 2H, J=8.9, 2.2 Hz, H-3′, H-5′), 7.28–7.31 (dd, 2H, J=8.9, 2.2 Hz,
3
13
H-2′, H-6′). C NMR (CDCl , 75.45 MHz): 160.20–160.09 (C-4′), 149.61–
3
3
(
7
s, 1H each, H-17), 6.85–6.88 (dd, 2H, J=8.7, 1.5 Hz, H-3′, H-5′), 7.19–
149.51 (C-8), 134.90–134.79 (C2′, C6′), 128.98–128.91 (C1′), 115.26 (C3′,
C5′), 106.99–106.90 (C17), 57.69–56.98 (C-9), 55.92–55.82 (C-5), 55.10
13
.22 (dd, 2H, J=8.7, 1.5 Hz, H-2′, H-6′). C NMR (CDCl , 75.45 MHz):
3
δ 159.20–159.15 (C-4′), 148.24–147.89 (C-8), 128.34–128.22 (C-2′, C-6′),
28.03–127.92 (C-1′), 121.29–121.05 (C-14), 114.31 (C-3′, C-5′), 106.61–
06.26 (C-17), 56.56–56.18 (C-9), 55.52–55.47 (C-5), 55.29 (Ar–OCH3),
(Ar–OCH ), 45.57–45.15 (C-14), 43.32–43.29 (C-3), 40.71–40.68 (C-10),
3
1
1
40.17–39.98 (C-1), 39.42–39.34 (C-7), 34.49–34.47 (C-12), 34.33–34.06
(C-13), 34.09–33.93 (C-4, C-18), 25.60–25.53 (C-6), 22.47 (C-9), 22.13–
–1
4
3
2.07 (C-3), 39.78–39.69 (C-13), 38.99–38.95 (C-10), 38.21–38.18 (C-1),
6.98–36.79 (C-7), 35.03–34.90 (C-12), 33.56–33.52 (C-4, C-18), 24.33
22.00 (C-11), 20.41–20.30 (C-2), 14.85 (C-20). IR (KBr) ν /cm : 2940,
max
+
1998. MS (ESI): m/z=370.43 [M–Cl] . Anal. Calcd for C H ClNO
25
40
(
(
C-6), 21.66 (C-19), 21.22–21.13 (C-11), 19.31 (C-2), 14.38 (C-20). IR
KBr) max/cm : 3081, 2934, 2235, 1642. MS (ESI): m/z=366.19 [M+H] .
(405.28): C, 73.95; H, 9.93; N, 3.45. Found: C, 73.71; H, 9.84; N, 3.27%.
–1
+
13-(3,4-Methylendioxy)benzyl-15,16-bisnorlabda-8(17)-en-14-amine
1
Anal. Calcd for C H NO (365.27): C, 82.14; H, 9.65; N, 3.83. Found: C,
(23d): Yield: 98%; white solid; H NMR (CDCl , 300 MHz): δ 0.57–0.61,
25
35
3
81.94; H, 9.56; N, 3.79%.
0.78, 0.84–0.86 (s, 3H each, CH ), 2.78 (m, 1H, H-13), 3.07 (m, 1H, H-14),
3
1
3-(3,4-Methylendioxy)benzyl-15,16-bisnorlabda-8(17)-en-14-nitrile
4.18–4.50 (d, 1H, J=1.3–1.6 Hz, H-17), 4.72–4.82 (d, 1H, J=2.2–2.6 Hz,
1
13
(
22d): Yield: 98%; white solid; H NMR (CDCl , 300 MHz): δ 0.63–0.65,
H-17), 5.95–5.96 (s, 2H, OCH O), 6.78 (m, 3H, H-2′, H-5′, H-6′). C NMR
3
2
0
.77–0.78 and 0.85 (s, 3H each, CH ), 3.63 (m, 1H, H-13), 4.33–4.41 and
(CDCl , 75.45 MHz): δ 149.91 (C-3′), 149.61 (C-8), 148.66–148.59 (C-4′),
3
3
4
.77–4.80 (s, 1H each, H-17), 5.95 (s, 2H, OCH O), 6.76 (m, 3H, H-2′,
135.17–134.88 (C-1′), 122.81–122.64 (C-6′), 109.54 (C-5′), 108.69–108.63
2
13
H-5′, H-6′). C NMR (CDCl , 75.45 MHz): δ 148.26–148.13 (C-3′), 147.89
(C-2′), 106.99 (C-17), 102.55 (OCH O), 58.43–57.76 (C-9), 56.92 (C-5),
3
2
(
1
1
4
C-8), 147.33–147.28 (C-4′), 129.75–129.61 (C-1′), 121.07–120.84 (C-
4), 120.69–120.53 (C-6′), 108.51 (C-5′), 107.64–107.59 (C-2′), 106.64–
45.71 (C-14), 43.31 (C-3), 40.71 (C-10), 40.20 (C-1), 39.40–39.34 (C-7),
34.48–34.43 (C-12), 34.23–34.09 (C-13), 33.84 (C-4, C-18), 25.60 (C-
6), 22.44 (C-9), 22.17–22.12 (C-11), 20.39–20.32 (C-2), 14.88 (C-20).
06.27 (C-17), 101.32 (OCH O), 56.59–56.19 (C-9), 55.56–55.50 (C-5),
2
–1
+
2.09 (C-3), 39.79–39.72 (C-13), 39.03–38.99 (C-10), 38.23 (C-1), 37.50–
IR (KBr) νmax/cm : 2926, 1953. MS (ESI): m/z=384.14 [M–Cl] . Anal.
37.30 (C-7), 35.07–34.96 (C-12), 33.57 (C-4, C-18), 24.36 (C-6), 21.67 (C-
Calcd for C H ClNO (419.26): C, 71.49; H, 9.12; N, 3.33. Found: C,
2
5
38
2
1
9), 21.26–21.18 (C-11), 19.32 (C-2), 14.40–14.37 (C-20). IR (KBr) νmax/
71.30; H, 9.07; N, 3.25%.
–1
+
cm : 3079, 2937, 2239, 1641. MS (ESI): m/z=380.15 [M+H] . Anal.
Calcd for C H NO (379.25): C, 79.11; H, 8.76; N, 3.69. Found: C, 78.90;
Antimalarial evaluation
2
5
33
2
In vitro inhibition of β-haematin formation: The β-haematin formation
assay was performed according to a slightly modified method of
Baelmans et al.26 Briefly, 50 μL of a fresh solution of haematin
H, 8.70; N, 3.60%.
Synthesis of 23a–d; general procedure
A solution of compound 22a, 22b, 22c or 22d (0.1639 mmol) in dry
THF (1.0 mL) was added to a suspension of LiAlH (0.6557 mmol) in
dry THF (2.0 mL) under nitrogen and with stirring at 0 °C. The reaction
mixture was then refluxed for 6 h. Then water was added, neutralised
with HCl (2 M) and extracted with ethyl acetate. A saturated solution of
–1
chloride (5.2 mg mL , 4 mM) in dimethyl sulfoxide (DMSO), was
distributed in 96-well micro plates. The tested compounds 21a–23d
(50 μL, 0.1–100 mM) in DMSO, positive control CQ (50 μL, 0.1–
100 mM) in water, 50 µL of the solvents as negative controls (water
and DMSO) and 100 µL of acetate buffer (0.2 M, pH 4.4), were added
in triplicate to test wells. The final concentration of CQ and tested
compounds ranged from 0.025 to 25 mM/well. Plates were incubated
at 37 °C for 48 h to allow completion of the reaction and centrifuged
at 4000 RPM for 15 min. After discarding the supernatant, the pellet
was washed twice with DMSO (200 µL) and finally dissolved in NaOH
(200 µL, 0.2 N). The solubilised aggregates were further diluted 1:2
with NaOH (0.1 N) and the absorbance recorded at 405 nm by using
Microplate Reader (BIORAD-550).
4
Et O•HCl at 0 °C was added dropwise to a solution of crude product in
2
ether (0.5 mL).
1
3-Benzyl-15,16-bisnorlabda-8(17)-en-14-amine (23a): Yield: 87%;
1
white solid; H NMR (CDCl , 300 MHz): δ 0.54–0.58, 0.77–0.78 and
3
0.84–0.86 (s, 3H each, CH ), 2.84 (m, 1H, H-13), 3.11 (m, 1H, H-14),
3
4.10–4.50 (d, 1H, J=1.6–1.9 Hz, H-17), 4.68–4.82 (d, 1H, J=2.6–2.5 Hz,
13
H-17), 7.34 (m, 5H, H-2′, H-3′, H-4′, H-5′, H-6′). C NMR (CDCl3,
5.45 MHz): δ 149.87–149.55 (C-8), 141.61–141.37 (C-1′), 130.20–130.17
C-3′, C-5′), 129.21–129.07 (C-4′), 128.83–128.73 (C-2′, C-6′), 106.99 (C-
7), 58.45–57.79 (C-9), 56.90–56.81 (C-5), 46.04–45.88 (C-14), 43.30–
3.24 (C-3), 40.72–40.68 (C-10), 40.16–39.95 (C-1), 39.39–39.32 (C-7),
7
(
1
4
In vivo antimalarial assay: The compounds were also tested in a
malaria murine model according to Peters and Robinson.27 Male albino
mice of Hygiene National Institute (HNI) strain, weighing 18–23 g were
maintained on a commercial pellet diet and housed under conditions
approved by the Ethics Committee of the Faculty of Pharmacy, Central
University of Venezuela. Plasmodium berghei (ANKA strain), a rodent
malarial parasite, was used for infection. Briefly, mice (n=6) were
3
2
4.47–34.42 (C-12), 34.19–34.09 (C-13), 34.05–33.82 (C-4, C-18), 25.59–
5.52 (C-6), 22.42 (C-19), 22.19–22.10 (C-11), 20.37–20.28 (C-2), 14.84
–1
+
(C-20). IR (KBr) νmax/cm : 2929, 1982. MS (ESI): m/z=340.20 [M–Cl] .
Anal. Calcd for C H ClN (375.27): C, 76.66; H, 10.19; N, 3.73. Found: C,
2
4
38
6
76.45; H, 10.12; N, 3.60%.
infected by IP passage of 10 infected erythrocytes diluted in phosphate
JCR1302067_FINAL.indd 660
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JOURNAL OF CHEMICAL RESEARCH 2013 661
buffered saline solution (PBS 10 mM, pH 7.4, 0.1 mL). The compounds
were dissolved in DMSO (0.1 M), diluted with Saline-Tween 20 solutions
References
1
World Health Organization, World malaria report 2012, WHO Press,
Switzerland, Geneva, 2012. pp. 88.
(
2%). Two hours after infection, each compound was administered
–1
–1
2
World Health Organization, Research priorities for Chagas disease,
human African Trypanosomiasis and Leishmaniasis (technical report
series, No. 975), WHO Press, Switzerland, Geneva, 2012. pp. 100.
World Health Organization, Global report on antimalarial drug efficacy
and drug resistance: 2000–2010, WHO Press, Switzerland, Geneva, 2010.
pp. 115.
once IP (20 mg kg day ) for four consecutive days. On day 4, the
parasitemia was monitored by microscopic examination of Giemsa-
–1
–1
stained smears. Chloroquine (20 mg kg day ) was used as a positive
control. The results were expressed as the percentage of parasitemia at
the fourth day post-infection and were compared to the values reported
by non-treated Plasmodium berghei infected mice. The survival time
beyond the control group (without drug treatment) was recorded. Data
were statistically analysed using unpaired t-tests for specific group
comparisons; assuming 95% confidence according GraphPad Prism 5.0.
3
4
5
6
7
U. Farooq and R.J. Mahajan, Vect. Borne. Dis., 2004, 41, 45.
C. Amaratunga, S. Sreng, S. Suon et al., Lancet Infect. Dis., 2012, 12, 851.
A.P. Phyo, S. Nkhoma, K. Stepniewska et al., The Lancet, 2012, 379, 1960.
Q. Cheng, D.E. Kyle and M.L. Gatton, Int. J. Parasitol. Drugs Drug
Resist., 2012, 2, 249.
8
9
10
A.M. Dondorp, F. Nosten, P. Yi et al., N. Engl. J. Med., 2009, 361, 455.
H.W. Murray, Am. J. Trop. Med. Hyg., 2004, 71, 787.
D. Kinghorn, J.N. Pan and C.F. Heebyung, J. Nat. Prod., 2011, 74, 1539.
Antileishmanial evaluation
Promastigotes of the L. mexicana strain AZV were maintained at 28 °C
on a Schneider medium supplemented with 20% foetal bovine serum
11 J.R. Hanson, Nat. Prod. Rep., 2012, 29, 890.
1
1
14
2
3
J.R. Hanson, Nat. Prod. Rep., 2011, 28, 1755.
J.R. Hanson, Nat. Prod. Rep., 2009, 26, 1156.
J.R. Hanson, Nat. Prod. Rep., 2007, 24, 1332.
(
FBS). Hamster footpad lesions were used as sources of amastigotes
for isolation and maintenance of the parasites. Briefly, L. mexicana
15 I.S. Marcos, L. Castañeda, P. Basabe, D. Díez and J.G. Urones, Mini-Rev.
Org. Chem., 2012, 9, 54.
16 I. Chinou, Curr. Med. Chem., 2005, 12, 1295.
promastigotes were seeded in 96-well micro plates to a final density
6
–1
of 10 promastigote mL . One row was left with medium and without
compounds (negative controls). Bisnorlabdane derivatives 21a–23d in
triplicate, were added to the culture at different concentrations (20, 40,
17
G. Duker-Eshun, J.W. Jaroszewski, W.A. Asomaning et al., Planta Med.,
002, 68, 642.
18 J. Asili, M. Lambert, H.L. Ziegler et al., J. Nat. Prod., 2004, 67, 631.
2
1
2
9
M. Kenmogne, E. Prost, D. Harakat et al., Phytochemistry, 2006, 67, 433.
6
0, 80 and 120 µM). After incubation for 54 h at 28 °C, the remaining
0 Z. Cheikh-Ali, T. Okpekon, F. Roblot et al., Phytochemistry Lett., 2011, 4,
parasites were counted in a Neubauer’s chamber and compared with
the controls.
2
40.
21 V. Samoylenko, D.C. Dunbar, M.A. Gafur et al., Phytother. Res., 2008, 22,
570.
J.E. Villamizar, J.P. Pittelaud, J.R. Rodrigues et al., Nat. Prod. Res., 2009,
3, 891.
23 F.J. Salazar and J.E. Villamizar, J. Chem. Res., 2013, 37, 63.
1
2
2
This research was supported by grants from FONACIT
2
(2007000960 and 2011000899). The authors thank the mass
24
25
26
27
28
J.E. Villamizar, J. Fuentes, F. Salazar, E. Tropper and R. Alonso, J. Nat.
Prod., 2003, 66, 1623.
J.E. Villamizar, F.J. Salazar, J. Fuentes, E. Tropper and R. Alonso, J.
Chem. Res., 2003, 504.
spectrometry (Ana Angarita and Matilde Gómez), NMR (Sara
Pekerar, Alberto Fuentes and Ligia Llovera) and elemental
analyses (Eleinne Severino) laboratories of Scientific Research
Venezuelan Institute (IVIC) for providing spectral data.
M. Costa, R. Tavares, W.B. Motherwell and M.J. Curto, Tetrahedron Lett.,
1
994, 35, 8839.
R. Baelmans, E. Deharo, E. Muñoz, M. Sauvain and H. Gisnburg, Exp.
Parasitol., 2000, 96, 243.
Received 15 July 2013; accepted 2 September 2013
Paper 1302067 doi:10.3184/174751913X13794472538261
Published online: 11 November 2013
W. Peters and B.L. Robinson, Handbook of animal models of infection, eds
O. Zack and M. Sande, Academic Press, San Diego, CA, 1999. Malaria, pp.
757–773.
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