3-Methyl-α-himachalene is confirmed, and the relative stereochemistry defined, by synthesis as the sex pheromone of the sandfly Lutzomyia longipalpis from Jacobina, Brazil

Article abstract of DOI:10.1039/a900242a

The structure of the sex pheromone produced by the male sandfly Lutzomyia longipalpis, from the Jacobina region of Brazil, previously proposed tentatively as the novel homosesquiterpene 3-methyl-α-himachalene is confirmed by synthesis and biological activ

Full text of DOI:10.1039/a900242a

3-Methyl-a-himachalene is confirmed, and the relative stereochemistry
defined, by synthesis as the sex pheromone of the sandfly Lutzomyia longipalpis
from Jacobina, Brazil
J. Gordon C. Hamilton,a Antony M. Hooper,b Kenji Mori,c John A. Pickett*b and Satoshi Sanoc
a School of Life Sciences, University of Keele, Keele, Staffordshire, UK ST5 5BG
b IACR-Rothamsted, Harpenden, Hertfordshire, UK AL5 2JQ. E-mail: john.pickett@bbsrc.ac.uk
c Department of Chemistry, Science University of Tokyo, Kagurazaka 1-3, Shinjuku-ku, Tokyo 162-8601, Japan
Received (in Cambridge, UK) 7th January 1999, Accepted 11th January 1999
The structure of the sex pheromone produced by the male
sandfly Lutzomyia longipalpis, from the Jacobina region of
Brazil, previously proposed tentatively as the novel homo-
sesquiterpene 3-methyl-a-himachalene is confirmed by syn-
thesis and biological activity; the relative stereochemistry is
defined as 1RS,3RS,7RS by comparing the natural product
with the four synthetic diastereoisomers.
Co-injection of natural product with the diastereoisomers,
using high-resolution capillary GC on columns of different
polarity (Table 1) gave peak enhancement only with diastereo-
isomer 1c. In the case of chiral GC, only one enantiomer of 1c
enhanced the natural product peak. The 1H NMR results
reported,6 although incomplete, showed good agreement with
only those for structure 1c. The mass spectrum for the natural
product6 was almost identical with that for 1c, with greater
differences from those for 1a, 1b and 1d. All had a base peak at
m/z 94, different from that for a-himachalene (m/z 93), which
was suggested as likely when the earlier tentative structural
prediction was made.6 Bioassays¶ involving attraction of
female L. longipalpis and conducted in a Y-tube olfactometer
showed that only diastereoisomer 1c and the natural pheromone
extract were active and both showed high statistical sig-
nificance. This was in agreement with the chromatographic and
spectral data, giving the sex pheromone structure as
(1RS,3RS,7RS)-3-methyl-a-himachalene, i.e. (1RS,3RS,7RS)-
2-methylene-3,6,6,9-tetramethylbicyclo[5.4.0]undec-8-ene
(1c). The cis stereochemistry of the natural product is analogous
The sandfly Lutzomyia longipalpis (Lutz and Neiva) (Diptera:
Psychodidae) is the vector of the protozoan parasite Leishmania
chagasi (Cunha and Chagas) (Kinetoplastida: Trypanosomati-
dae), the causative agent of visceral leishmaniasis in the New
World. Male L. longipalpis release a sex pheromone from
glands on the tergites of the abdomen1 which is highly attractive
to females.2 The sex pheromone gland of L. longipalpis from
Jacobina (Bahia State) in northeastern Brazil produces several
compounds but only the principal volatile component is
responsible for the attraction of females.3 It was proposed,
mostly on the basis of mass spectrometry of the natural product
and of products of microchemical reactions, that the pheromone
comprised the novel homosesquiterpene 3-methyl-a-himache-
lene.4 The sex pheromones of other sympatric and allopatric
populations of L. longipalpis are different;5 for example, that
from the Lapinha region of Brazil (Minas Gerais State) has been
tentatively proposed as another novel sesquiterpene, 9-me-
thylgermacrene-B.6 The purpose of this work was to test the
proposed structure for the Jacobina L. longipalpis sex pher-
omone by synthesis of 3-methyl-a-himachalene 1.
i
ii,iii
OH
TBDMSO
TBDMSO
CN
2
3
4
iv,v
vi,vii
The synthetic route adopted followed the intramolecular
Diels–Alder methodology employed by Wenkert and Naemura7
for a-himachalene and is described in Scheme 1. Diene 7
comprised a mixture of E/Z isomers (77: 23–90 : 10) by 1H
NMR spectroscopy. The subsequent cyclisation of the keto
enediene 8 in xylene under reflux gave a mixture of cyclic
products, which could be separated by medium pressure liquid
chromatography into enantiomeric pairs of diastereoisomers
9a–d.† Final conversion to the diastereoisomeric 3-methyl-a-
himachalenes 1a–d‡ was achieved quantitatively using the
Tebbe reagent. Assignment of structures 9a–d was by correla-
tion and two dimensional NOE spectroscopy (Fig. 1). Com-
pounds 9a and 9c were designated as cis-fused by the ring
junction protons showing a NOE to each other. The relative
stereochemistry of the methyl group could be determined for 9c
by a NOE to the ring junction proton of C-1, a signal not seen
for 9a. The relative stereochemistries of the C-3 methyl group of
trans-fused 9b and 9d were also elucidated by NOE experi-
ments. A NOE was observed between the C-1 proton of 9b and
one of the C-6 methyl groups, while the other C-6 methyl group
showed a NOE to the C-3 methyl, which therefore lies on the
opposite face to the C-1 proton (Fig. 1). The structures of 1a–d
were inferred from 9a–d and in comparison with NMR data for
natural a-himachalene.4 Mass spectra were obtained by GC-
MS.§
HO
CO2Me
TBDMSO
CO2Et
6
5
viii,ix
x–xiii
CO2Me
O
7
8
xiv
O
xv
1a–d
9a–d
Scheme 1 Reagents and conditions: i, NaH (1.0 equiv.), TBDMSCl (1.1
equiv.), THF, 94%; ii, TsCl, Py, CHCl3; iii, NaCN, DMSO, heat, 94% (2
steps); iv, DIBALH, CH2Cl2, H3O+, 67%; v, CH3CN(PPh3)CO2Et,
benzene, reflux, quant.; vi, Mg, MeOH, 88%; vii, aq. HF, Me3CN, 97%;
viii, Swern oxidation; ix, Ph3PNCHCMeNCH2, THF, reflux, 56% (2 steps);
x, LiAlH4, Et2O, 92%; xi, PCC, MS 4A, CH2Cl2; xii, CH2NCHMgBr, THF,
70% (2 steps); xiii, Dess–Martin oxidation, 86%; xiv, xylene, reflux, 62%;
xv, Tebbe reagent, quant.
Chem. Commun., 1999, 355–356
355

δ 2.87
δ 1.06
Notes and references
H
NOE
δ 2.42
O
H
H
H
† Selected data for 9a: Rf 0.64; dH 0.70 (s, 3H, 6-CH3), 1.01 (s, 3H, 6-CH3),
1.06 (d, J 6.5, 3-CH3), 1.71 (br s, 3H, 9-CH3), 2.42 (m, 1H, 1-H), 2.68 (br
s, 1H, 7-H), 2.87 (m, 1H, 3-H), 5.47 (br s, 1H, 8-H). For 9b: Rf 0.64; dH 0.80
(s, 3H, 6-CH3), 0.96 (s, 3H, 6-CH3), 1.06 (d, J 6.5, 3-CH3), 1.69 (br s,
9-CH3), 1.81 (m, 1H, 7-H), 2.67 (m, 1H, 1-H), 2.59 (m, 1H, 3-H), 5.30 (br
s, 1H, 8-H). For 9c: Rf 0.61; dH 0.85 (s, 3H, 6-CH3), 0.97 (d, J 6.5, 3-CH3),
1.07 (s, 3H, 6-CH3), 1.71 (br s, 9-CH3), 2.14 (br s, 1H, 7-H), 2.66 (m, 1H,
3-H), 2.77 (m, 1H, 1-H), 5.57 (br s, 1H, 8-H). For 9d: Rf 0.61; dH 0.77 (s,
3H, 6-CH3), 1.01 (s, 3H, 6-CH3), 1.06 (d, J 6.5, 3-CH3), 1.69 (br s, 9-CH3),
2.06 (m, 1H, 7-H), 2.49 (m, 1H, 1-H), 2.65 (m, 1H, 3-H), 5.32 (br s, 1H,
8-H). Rf values in hexane–EtOAc 10:1.
‡ Selected data for 1a: dH 0.81 (s, 3H, 6-CH3), 0.94 (s, 3H, 6-CH3), 1.10 (d,
J 7, 3-CH3), 1.70 (br s, 3H, 9-CH3), 4.82 (s, 1H, 2-CHaCHb), 4.86 (s, 1H,
2-CHaCHb), 5.49 (br s, 1H, 8-H). For 1b: dH 0.71 (s, 3H, 6-CH3), 0.94 (s,
3H, 6-CH3), 1.06 (d, J = 7, 3-CH3), 1.69 (br s, 9-CH3), 4.76 (s, 1H,
2-CHaCHb), 4.86 (s, 1H, 2-CHaCHb), 5.30 (br s, 1H, 8-H). For 1c: dH 0.96
(s, 3H, 6-CH3), 1.00 (s, 3H, 6-CH3), 1.01 (d, J 7, 3-CH3), 1.68 (br s, 9-CH3),
4.76 (s, 1H, 2-CHaCHb), 4.81 (s, 1H, 2-CHaCHb), 5.51 (br s, 1H, 8-H). For
1d: dH 0.70 (s, 3H, 6-CH3), 0.94 (s, 3H, 6-CH3), 0.97 (d, J 7, 3-CH3), 1.67
(br s, 9-CH3), 4.75 (s, 1H, 2-CHaCHb), 4.79 (s, 1H, 2-CHaCHb), 5.31 (br s,
1H, 8-H).
Me
Me
NOE
Me
Me
H
δ 1.71
H
δ 5.47
O
δ 2.68
9a
NOE
δ 2.66
δ 0.97
δ 2.77
O
NOE
H
H
H
H
Me
H
δ 1.71
Me
H
Me
O
δ 5.57
δ 2.14
Me
9c
§ GC–MS: 0.32 mm id 3 50 m HP-1(a siloxane 3 0.52 mm film thickness,
30 to 250 °C at 5 °C min21; EI at 70 EV, 250 °C (VG-Autospec, Fisons
Instruments), elution order 1d, 1c, 1b, 1a. Selected data for 1a: m/z 94
(100%), 79 (55), 121 (54), 41 (52), 93 (51), 91 (41), 107 (40), 105 (35), 69
(35), 55 (34), 119 (32), 81 (29), 95 (24), 77 (24), 148 (23), 149 (19), 133
(18), 65 (18), 203 (16), 161 (16), 175 (15), 53 (15), 39 (15), 218 (11, M+).
For 1b: m/z 94 (100%), 121 (87), 105 (61), 91 (58), 79 (57), 55 (56), 41 (53),
148 (51), 107 (50), 136 (47), 93 (46), 69 (43), 175 (35), 119 (33), 39 (31),
42 (28), 77 (28), 133 (27), 95 (23), 161 (22), 218 (21), 81 (21), 92 (20), 27
(20), 67 (19), 123 (18), 162 (18), 131 (17), 148 (17), 147 (16), 120 (15), 43
(15). For 1c: m/z 94 (100%), 41 (69), 93 (63), 107 (61), 79 (59), 121 (58),
175 (53), 91 (46), 105 (45), 69 (44), 55 (43), 119 (40), 203 (38), 81 (36), 77
(32), 133 (26), 67 (26), 95 (25), 149 (24), 148 (23), 147 (21), 39 (21), 43
(20), 29 (20), 109 (20), 43 (20), 162 (19), 27 (19), 218 (18, M+), 123 (17),
161 (16), 136 (15), 67 (15). For 1d: m/z 94 (100%), 121 (65), 79 (50), 93
(49), 107 (45), 41 (44), 91 (40), 136 (39), 105 (38), 119 (38), 148 (36), 69
(32), 55 (31), 175 (30), 81 (27), 77 (26), 133 (25), 149 (24), 218 (23 M+),
95 (22), 161 (18), 147 (17), 67 (16), 109 (15), 53 (15).
¶ Virgin female sandflies were removed from larval rearing pots within 10
h after eclosion to ensure that they were unmated. They were provided with
a saturated sugar solution on cotton wool and subsequently maintained for
5–6 days in Barraud cages (18 3 18 3 18 cm). Bioassays were conducted
in a glass (9 mm internal diameter) Y-tube olfactometer. Zero grade air was
passed (2 ml min21) through two charcoal filters into the test and control
arms (10 cm long). The olfactometer was connected to the air supply by
Teflon tubing. A filter paper disk (1.5 cm diameter) was inserted into the
Teflon tubing at the connection with the olfactometer test and control arms.
During bioassays pheromone extracts or synthetic chemicals in hexane were
placed on one of the filter paper disks. Hexane in the same quantity as for
the test arm was placed on the other filter paper disk. The female sandfly
was introduced into the third arm (10 cm long) of the olfactometer and its
response observed for 5 min. Female attraction to 1c and extract prepared
from 5–7 day old male L. longipalpis was highly significant (c2 = 6.2 3
1027 and P = 1.4 3 10226, respectively). Females were not significantly
attracted to 1a, 1b or 1d, nor to a natural a-himachalene sample at the same
levels.
δ 2.59
δ 1.06
δ 2.67
O
H
NOE
H
H
H
Me
δ 1.69
Me
H
H
δ 0.96
δ 0.80
δ 1.81
Me
H
δ 5.30
O
NOE
NOE
9b
Me
NOE
δ 2.65
δ 1.06
O
δ 2.49
H
H
H
δ 1.69
H
δ 5.32
δ 2.06
9d
Fig. 1 Assignment of the relative stereochemistry of 9a–d.
Table 1 Retention times of all isomers of 3-methyl-a-himachalene 1a–d by
GC and peaks enhanced on coinjection with natural material of L.
longipalpis from Jacobina
Retention time/min
Compound
HP-5 (a siloxane)a
16.67
Chiral GC (b-cyclodextrin)b
1a
1b
1c
1d
37.01
No chiral separation
35.15
1 R. P. Lane, A. Phillips, D. H. Molyneux, G. Procter and R. D. Ward, Ann.
Trop. Med. Parasitol., 1985, 79, 225
2 I. E. Morton and R. D. Ward, Med. Vet. Entomol., 1989, 3, 219
3 J. G. C. Hamilton, M. J. Dougherty and R. D. Ward, J. Chem. Ecol., 1994,
20, 141; M. J. Dougherty, P. Guerin and R. D. Ward, Physiol. Entomol.,
1995, 20, 23
16.03
36.35
32.50
33.01c
32.58
33.36
14.73c
14.71
4 J. G. C. Hamilton, G. W. Dawson and J. A. Pickett, J. Chem. Ecol., 1996,
22, 2331
a 0.32 mm id 3 30 m 3 25 mm film thickness, 40 to 150 °C at 5 °C min21
b 0.25 mm id 3 30 m 3 25 mm film thickness, 40 to 180 °C at 3 °C min21
c Peaks enhanced on coinjection with natural material.
.
.
5 J. G. C. Hamilton and R. D. Ward, Parassitologia, 1991, 33 (Suppl 1),
283; J. G. C. Hamilton, R. D. Ward, M. J. Dougherty, C. Ponce, E. Ponce,
H. Noyes and R. Zeledon, Ann. Trop. Med. Parasitol., 1996, 90, 533
6 J. G. C. Hamilton, G. W. Dawson and J. A. Pickett, J. Chem. Ecol., 1996,
22, 1477
to that of natural a-himachalene, with absolute stereochemistry
1R,7R.8 The absolute stereochemistry of the pheromone is now
under further investigation, although the bioassay results
suggest no interference with biological activity by the sample
containing both enantiomers. Attempts are being made to scale-
up the synthesis for field and pest control studies.
7 E. Wenkert and K. Naemura, Synth. Commun., 1973, 3, 45
8 T. C. Joseph and S. Dev, Tetrahedron, 1968, 24, 3841
Communication 9/00242A
356
Chem. Commun., 1999, 355–356