Synthesis and properties of methyl 5-(1′R,2′S)-(2- octadecylcycloprop-1-yl)pentanoate and other ω-19 chiral cyclopropane fatty acids and esters related to mycobacterial mycolic acids

Article abstract of DOI:10.1016/j.chemphyslip.2003.09.001

A 23-26-carbon chain length range of ω-19 (1′R,2′S) cyclopropane fatty acids, related to mycobacterial mycolic acids, has been prepared. The key cyclopropyl intermediate, (1′R,2′S)-(Z)-1-formyl- 2-octadecylcyclopropane, underwent Wittig chemistry with var

Full text of DOI:10.1016/j.chemphyslip.2003.09.001

Chemistry and Physics of Lipids 127 (2004) 35–46
Synthesis and properties of methyl 5-(1ꢀR,2ꢀS)-
(2-octadecylcycloprop-1-yl)pentanoate and other
␻-19 chiral cyclopropane fatty acids and esters
related to mycobacterial mycolic acids
Geoffrey D. Coxona, Juma’a R. Al Dulayymib, Caroline Morehousec,
Patrick J. Brennanc, Gurdyal S. Besrad, Mark S. Bairdb, David E. Minnikind,∗
a
Department of Chemistry, University of Newcastle, Newcastle upon Tyne NE1 7RU, UK
b
Department of Chemistry, University of Wales, Bangor, Gwynedd LL57 1BE, UK
c
Department of Microbiology, Colorado State University, Fort Collins, CO 80523-1677, USA
School of Biosciences, The University of Birmingham, Edgbaston, Birmingham B15 2TT, UK
d
Received 24 June 2003; received in revised form 20 August 2003; accepted 4 September 2003
Abstract
A 23–26-carbon chain length range of ␻-19 (1ꢀR,2ꢀS) cyclopropane fatty acids, related to mycobacterial mycolic acids, has been
prepared. The key cyclopropyl intermediate, (1ꢀR,2ꢀS)-(Z)-1-formyl-2-octadecylcyclopropane, underwent Wittig chemistry with
various reagents to provide vinylic precursors, which were selectively reduced to the corresponding saturated ␻-19 cyclopropane
fatty acids or esters. The 24-carbon ␻-19 cyclopropane ester was made by chain elongation of the 23-carbon ester. Saturated
and unsaturated chiral cyclopropane acids and esters were assayed, using wall extracts of Mycobacterium smegmatis; the
incorporation of 14C-acetate was used to measure inhibition or stimulation of mycolic acid synthesis. Minor inhibition (2–3%)
was shown by the 23- and 24-carbon saturated esters; all the other compounds were stimulants. The most effective (38–55%)
stimulators of mycolate synthesis were the unsaturated esters with 23- and 26-carbons and the saturated and unsaturated 25-carbon
acids.
© 2003 Elsevier Ireland Ltd. All rights reserved.
Keywords: Fatty acids; Mycolic acids; Chiral cyclopropane; Biosynthetic inhibitors
1. Introduction
It was estimated in 1991 that almost one-third of the
world’s population, or 1.7 billion people was infected.
In recent years, tuberculosis infections among certain
population groups have increased dramatically. This
is partly due to a decline in living standards of these
groups and to the increasing resistance of various
strains to present antibiotic treatment. In addition
to this is the coexistence of the disease within HIV
Tuberculosis remains the leading cause of death
from infectious diseases (Enarson and Murray, 1994).
∗
Corresponding author. Tel.: +44-121-414-8126;
fax: +44-121-414-5925.
E-mail address: d.e.minnikin@bham.ac.uk (D.E. Minnikin).
0009-3084/$ – see front matter © 2003 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.chemphyslip.2003.09.001

36
G.D. Coxon et al. / Chemistry and Physics of Lipids 127 (2004) 35–46
infected patients. Because of the ability of HIV to de-
stroy the immune system this has emerged as the most
significant risk factor for progression of dormant TB
infection to the clinical disease. It has been estimated
that from the beginning of the HIV pandemic until
mid-1993, more than 5 million people worldwide
had dual HIV and TB infection. This has highlighted
the need for a better understanding of the nature
of the disease and its control (Enarson and Murray,
1994).
Me(CH2)17
(CH2)nCO2Me
cis
4
n = 3,4
(CH2)14OH
OAc
Me(CH2)19
(CH2)14
5
CO2Me
(CH2)11
Me(CH2)19
(CH2)14
Mycobacterium tuberculosis is the causative agent
of tuberculosis and its pathogenicity depends on the
presence of a number of very unusual lipid molecules.
The high molecular weight 2-alkyl-3-hydroxy mycolic
acids (e.g. 1) are key structural components of M. tu-
berculosis (Minnikin, 1982, 1993) being essential for
the integrity of the cell envelope. Mycolic acids show a
wide variety of structural variations, including a range
of homologues and functional units, such as methoxy
and keto groups (Watanabe et al., 2001, 2002). All
mycobacteria have so-called ␣-mycolates, which have
no oxygen functions in addition to the 3-hydroxy
acid unit. Structure 1 represents the ␣-mycolic acids
from M. tuberculosis. Investigations into mycolic acid
biosynthesis in M. tuberculosis by Takayama and
Qureshi (1984) indicated that (Z)-tetracos-5-enoic
acid (2) is a key initial intermediate. This acid 2
was shown to stimulate ␣-mycolic acid synthesis in
extracts of the environmental agent, Mycobacterium
smegmatis (Besra et al., 1993a; Wheeler et al., 1993a).
(Z)-Tetracos-5-enoic acid (2) was considered to be
formed by a ꢀ5-desaturase from tetracosanoate and
this was strongly supported by the inhibition of my-
colate synthesis by the cyclopropene analogue 3 of 2
(Besra et al., 1993b; Wheeler et al., 1993b). Fatty acid
desaturases had previously be shown to be inhibited
by cyclopropene fatty acid derivatives (Johnson et al.,
1967).
cis
cis
(CH2)23CH3
6
Racemic cis-cyclopropane acids 4, related to
␣-mycolates have been synthesised and shown to
be weak inhibitors of ␣-mycolate synthesis in M.
smegmatis (Hartmann, 1993; Hartmann et al., 1994;
Coxon et al., 2003a). Recently, parallel routes to
chiral long-chain cyclopropane fatty acids, such as
lactobacillic acid, have been developed (Coxon et al.,
1999, 2003b). One of these routes has been used
to synthesise a so-called meromycolate portion 5
(Al Dulayymi et al., 2000) and a derivative of an
␣-mycolic acid enantiomer 6 (Al Dulayymi et al.,
2003). This report details the synthesis of the key
chiral intermediate 10 (Scheme 1) and its conversion
into the chiral cis-cyclopropane fatty acids and esters
(11, 12, 16, 17, 18–25) (Schemes 1 and 2), which
were tested against mycolate-synthesising extracts
of M. smegmatis. The synthesis of some of these
long-chain cyclopropane esters has been outlined in a
preliminary communication (Coxon et al., 1999).
2. Experimental
2.1. Materials and methods
1H and 13C NMR spectra were recorded on Bucker
AC 200, AC 250 CP/MAS or JEOL ␭500 NMR spec-
trometers in CDCl3. IR spectra were recorded using
a Nicolet FTIR PCIR or Perkin-Elmer 1600 FTIR
Infrared spectrophotometer as liquid films. Electron
impact (EI) mass spectra (MS) were recorded using
either a Kratos MS 80RF of Micromass Auto Spec
M spectrometer. Gas chromatography (GC) analysis
was carried out on a Perkin-Elmer 8410 gas chro-
matograph. Optical rotations were achieved using
OH
CO2H
(CH2)11
Me(CH2)19
(CH2)14
cis
cis
(CH2)23CH3
1
Me(CH2)17
(CH2)3CO2H
Me(CH2)17
(CH2)3CO2Me
2
3

G.D. Coxon et al. / Chemistry and Physics of Lipids 127 (2004) 35–46
37
_
O
O
O
+
H
H
ZnEt2/ ICH2Cl
91 %
Me(CH2)18PPh3Br
H
H
O
O
O
nBuLi
77 %
H
O
Me(CH2)16
8
Me(CH2)17
7
MeOH/ TFA
65 %
O
H
HO
HO
Me(CH2)16
MeO2C
H
NaIO4/ H2O
74 %
H
Ph3P=CHCO2Me
53 %
H
H
H
H
Me(CH2)16
Me(CH2)16
11
10
9
Hydrazine hydrate
CH3CO2H/ CuSO4
NaIO4/ H2O 600 C
53%
H
H
TsO
NC
MeO2C
H
HO
TsCl/ pyridine
91 %
LiAlH4
H
H
H
Me(CH2)16
Me(CH2)16
Me(CH2)16
89 %
14
12
13
82 %
NaCN
DOWEX H+/
H
H
H
HO2C
MeO2C
NaOH
H
MeOH
H
H
Me(CH2)16
Me(CH2)16
Me(CH2)16
82 %
90 %
17
16
15
Scheme 1.
DOWEX H+
89 %
H
MeO2C
H
HO2C
H
H
MeO2C
H
H
Me(CH2)16
Me(CH2)16
Me(CH2)16
MeOH
19
21
20
Hydrazine hydrate
CH3CO2H/ CuSO4
NaIO4/ H2O
DOWEX H+/ MeOH
74 %
600C 90 %
O
_
_
+
+
H
H
HO2C
MeO2C(CH2)3PPh3Br
HO2C
H
H
HO2C(CH2)4PPh3Br
H
H
H
Me(CH2)16
Me(CH2)16
Me(CH2)16
Na(Me3Si)2N
86 %
Na(Me3Si)2N
58 %
18
10
22
Hydrazine hydrate
CH3CO2H/ CuSO4
DOWEX H+/ MeOH
MeO2C
84 %
600C
NaIO4/ H2O
85 %
H
H
H
MeO2C
DOWEX H+
MeOH 70 %
HO2C
H
H
H
Me(CH2)16
Me(CH2)16
Me(CH2)16
25
23
24
Scheme 2.

38
G.D. Coxon et al. / Chemistry and Physics of Lipids 127 (2004) 35–46
Found M+: 380.3648. Calculated for C25H48O2:
380.3654.
an Optical Activity POLAAR 2001 automatic po-
larimeter. Melting points (uncorrected) were carried
out on a Kofler hot stage apparatus. Thin layer chro-
matography was performed using glass backed plates
precoated with silica gel F254 of layer thickness
0.25 ml and Fluka aluminium backed TLC cards with
fluorescent indicator 254 nm with a silica gel thick-
ness of 0.2 mm. Flash column chromatography was
performed at medium pressure using Fluka 60738 sil-
ica gel 60. Starting materials and chemical reagents
were all purchased from Aldrich, Fluka or Lancaster
Synthesis. Organic solutions were dried over mag-
nesium sulfate. Petrol refers to petroleum ether bp
40–60 ◦C.
2.2.2. (1R,2S,4ꢀS)-1-(2ꢀ,2ꢀ-Dimethyl-1ꢀ,3ꢀ-dioxolan-
4ꢀ-yl)-2-octadecylcyclopropane (8)
Diethyl zinc (67.0 ml, 73.6 mmol) followed by
chloroiodomethane (26.0 g, 147.2 mmol) was added
to compound 7 (14.0 g, 36.8 mmol) stirred in anhy-
drous 1,2-dichloroethane (100 ml) at −30 ◦C under
nitrogen. The solution was warmed to 0 ◦C and
stirred for 1 h whilst being monitored by TLC, then
quenched with saturated aq. ammonium chloride
solution (40 ml), the organic phase extracted with
chloroform (2 × 60 ml) and the combined organic
phases washed with water (3 × 50 ml) and dried. The
organic phase was concentrated under vacuum and
the impure compound was purified by flash chro-
matography, using petrol and diethyl ether (5:1), as
eluent to yield the product 8 (13.2 g, 91%) as a pale
yellow residue. Mp: 36–38 ◦C; [α]D24 = +6.8◦ (c:
0.46 mg ml−1, CHCl3); νmax (film): 3031, 2945, 2826,
1227, 929 cm−1; δH: 4.07 (br, q, CH, 1H, J = 5.2 Hz),
3.61 (m, 2 × CH, 2H), 1.44 (s, Me, 3H), 1.35 (s, Me,
3H), 1.25 (s, C16H32, 32H), 1.15 (m, CH2, 2H), 0.87
(br, t, 3 × CH, CH3, 6H, J = 6.0 Hz), 0.24 (m, CH,
1H); δC: 108.3, 78.2, 69.6, 31.4, 30.7, 29.1, 28.5,
28.3, 27.5, 27.1, 26.9, 26.4, 23.2, 22.7, 17.5, 16.4,
9.8. Found M+: 394.3794. Calculated for C26H50O2:
394.3810.
2.2. Synthesis
2.2.1. (1Z,4ꢀS)-1-(2ꢀ,2ꢀ-Dimethyl-1ꢀ,3ꢀ-dioxolan-
4ꢀ-yl)-1-icosene (7)
n-Nonadecyltriphenylphosphonium bromide (Al
Dulayymi et al., 2000) (36.0 g, 59.2 mmol) was sus-
pended in anhydrous tetrahydrofuran (THF; 200 ml)
and stirred under nitrogen at −78 ◦C. n-Butyllithium
(77.0 ml, 86.0 mmol) was added and the reaction
warmed to ambient temperature for 10 min before be-
ing recooled to −78 ◦C, when 2,3-O-isopropylidene-
d-glyceraldehyde (7.0 g, 53.8 mmol) in THF (10 ml)
was added. The reaction was stirred between −10
and 0 ◦C for 2 h, until no starting material remained
by TLC. Saturated aq. ammonium chloride solu-
tion (20 ml) was then added. The organic phase
was extracted with dichloromethane (3 × 50 ml),
washed with water (3 × 50 ml), dried and con-
centrated under vacuum. The crude residue was
purified by column chromatography, using petrol
and diethyl ether (5:2), to yield the product 7
(15.7 g, 77%) as a white solid. Mp: 35–37 ◦C;
[α]2D4 = +67.5◦ (c: 16.5 mg ml−1, CHCl3); νmax
(film): 3030, 2975, 2945, 2832, 1620, 1043, 9134,
780 cm−1; δH: 5.65 (d, CH, 1H, J = 10.0 Hz),
5.41 (t, CH, 1H, J = 8.6 Hz), 4.85 (br, q, CH, 1H,
J = 8.1 Hz), 4.05 (dd, CH, 1H, J = 8.0, 6.1 Hz), 3.52
(t, CH, 1H, J = 8.1 Hz), 2.08 (m, CH2, 2H), 1.43
(s, Me, 3H), 1.41 (s, Me, 3H), 1.26 (s, C16H32,
32H), 0.89 (br, t, CH3, 3H, J = 5.6 Hz); δC: 135.2
(CH), 126.8 (CH), 109.2 (OCO), 66.8 (CO), 67.2
(CO), 31.9, 31.8, 31.4, 31.0, 30.8, 30.6, 30.5,
30.1, 29.8, 29.7, 29.2, 27.4, 26.8, 26.0, 22.7, 14.1.
2.2.3. (1R,2S,1ꢀS)-1-(1ꢀ,2ꢀ-Dihydroxyeth-1ꢀ-yl)-
2-octadecylcyclopropane (9)
Trifluoroacetic acid (TFA) (80 ml, 80%) in
methanol (20 ml, 20%) was added to a solution of 8
(11.0 g, 27.9 mmol) in methanol (100 ml) at 0 ◦C. The
solution was stirred for 2 h, the TFA and methanol
were removed under vacuum and the residue was pu-
rified by column chromatography, eluting with petrol
and diethyl ether (1:1), to yield 9 (6.2 g, 65%) as
a white solid. Mp: 32–34 ◦C; [α]D24 = +22.4◦ (c:
1.05 mg ml−1, CHCl3); νmax (film): 3428 (br), 3056,
2989, 2970, 1252 cm−1; δH: 3.76 (m, CH, 1H), 3.50
(m, CH, 1H), 3.32 (m, CH, 1H), 2.06 (br, s, 2 × OH,
2H), 1.25 (s, C16H32, 32H), 0.92 (m, CH2, 2H), 0.89
(br, t, CH3, 3H, J = 6.1 Hz), 0.87 (m, 3 × CH, 3H),
0.11 (m, CH, 1H); δC: 73.3 (COH), 67.1 (COH),
31.9, 30.0, 29.7, 29.6, 29.1, 27.8, 22.8, 20.4, 19.8,

G.D. Coxon et al. / Chemistry and Physics of Lipids 127 (2004) 35–46
39
2.2.6. (1R,2S)-1-(2ꢀ-Methoxycarbonyleth-1ꢀ-yl)-2-
octadecylcyclopropane (12)
19.6, 19.4, 19.1, 18.4, 16.8, 15.7, 14.1. Found M+:
354.3496. Calculated for C23H46O2: 354.3497.
Sodium periodate (90 mg, 9.3 mmol) in water
(10 ml) was added dropwise via a dropping funnel over
2 h to a stirred mixture of 11 (350 mg, 0.93 mmol),
hydrazine hydrate (0.65 ml), ethanoic acid (0.5 ml)
and saturated aq. copper sulfate solution (0.5 ml) in
isopropanol (20 ml). The reaction was stirred for 3
days before being quenched with saturated aq. am-
monium chloride (20 ml). The organic phase was
extracted with dichloromethane (2 × 50 ml), washed
with water (2 × 20 ml) and dried. The organic phase
was reduced under vacuum and the residue purified
by column chromatography, eluting with petrol and
ethyl acetate (8:1), to yield 12 (150 mg, 42%) as a
low melting solid. Mp: 35–36 ◦C; [α]D24 = +34.7◦
(c: 0.37 mg ml−1, CHCl3); νmax (film): 3052, 2980,
2971, 1745, 1205 cm−1; δH: 3.61 (s, Me, 3H), 2.27 (t,
CH2, 2H, J = 7. 2 Hz), 1.82 (m, CH2, 2H), 1.34 (m,
CH2, 2H), 1.28 (s, C16H32, 32H), 0.92 (br, t, CH3,
3H, J = 5.6 Hz), 0.72 (m, 3×CH, 3H), −0.32 (q, CH,
1H, J = 4.6 Hz); δC: 51.5 (OMe), 34.7, 32.0, 30.2,
29.7, 29.4, 29.2, 29.1, 29.0, 28.9, 28.7, 28.6, 24.1,
22.7, 16.0, 15.3, 14.1, 10.8. Found M+: 380.3656.
Calculated for C25H48O2: 380.3654.
2.2.4. (1R,2S)-1-Formyl-2-octadecylcyclopropane
(10)
Sodium periodate (4.5 g, 225 mmol) in distilled
water (100 ml) was added to a solution of 9 (6.0 g,
17.5 mmol) in methanol (200 ml) cooled to 0 ◦C. The
mixture was stirred for 20 min until TLC indicated the
completion of the reaction and then quenched with
saturated aq. ammonium chloride (20 ml). The organic
phase was extracted with ethyl acetate (2 × 50 ml),
dried, concentrated under vacuum and purified by col-
umn chromatography using petrol and diethyl ether
(2:1), to yield the product 10 (4.2 g, 74%) as a pale
yellow residue. Mp: 33–34 ◦C; [α]D24 = +15.6◦ (c:
0.73 mg ml−1, CHCl3); νmax (film): 3071, 2986, 2970,
1648, 1239 cm−1; δH: 9.35 (d, CHO, 1H, J = 5.6 Hz),
1.49 (m, CH2, 2H), 1.35 (s, C16H32, 4 × CH, 36H),
0.86 (br, t, CH3, 3H, J = 6.0 Hz); δC: 179.7 (CHO),
68.4, 32.8, 30.1, 29.6, 29.2, 29.0, 26.9, 24.1, 22.9,
27.4, 23.4, 23.1, 22.8, 17.8, 14.4, 14.0. Found M+:
322.3239. Calculated for C22H42O: 322.3235.
2.2.5. (1ꢀE,1R,2S)-1-(3ꢀ-Methoxycarbonyleth-2ꢀ-
en-1ꢀ-yl)-2-octadecylcyclopropane (11)
2.2.7. (1R,2S)-1-(3ꢀ-Hydroxypropyl)-2-
Carbomethoxymethylenetriphenylphosphorane (400
mg, 3.1 mmol) was added to a solution of 10 (1.0 g,
3.1 mmol) in methanol (50 ml) and stirred at ambient
temperature overnight. The reaction was quenched
with saturated aq. ammonium chloride (10 ml),
the organic phase extracted with dichloromethane
(2 × 30 ml), the combined organic phases washed
with water (2 × 30 ml) and dried. The crude prod-
uct was purified by flash chromatography, using
petrol and diethyl ether (5:2), to yield 11 as a white
solid (62 mg, 53%). Mp: 41–42 ◦C; [α]D24 = +34.7◦
(c: 0.37 mg ml−1, CHCl3); νmax (film): 3053, 2981,
2947, 1652 cm−1; δH: 6.71 (dd, CH, 1H, J = 15.3,
10.5 Hz), 5.90 (d, CH, 1H, 15.3 Hz), 3.70 (s, MeO,
3H), 1.57 (m, CH2, 2H), 1.21 (s, C16H32, 3 × CH,
35H), 0.82 (br, t, CH3, 3H, J = 6.0 Hz), 0.51 (m, CH,
1H); δC: 170.3 (COOMe), 133.8 (CH), 131.9 (CH),
53.8, 51.2, 31.9, 31.7, 30.9, 29.7, 29.3, 29.2, 25.3,
22.7, 22.0, 21.8, 20.3, 19.5, 16.7, 15.7, 15.0, 14.1.
Found M+: 378.3507. Calculated for C25H46O2:
378.3497.
octadecylcyclopropane (13)
Lithium aluminium hydride (260 mg, 6.3 mmol)
in anhydrous THF (30 ml) was added dropwise over
15 min to 12 (1.1 g, 2.89 mmol) in THF (100 ml).
The solution was stirred at reflux temperature for 1 h
before being quenched with 10% aq. sodium sulfite
solution (20 ml), allowed to cool, filtered through
magnesium sulfate and reduced under vacuum. The
residue was purified by column chromatography,
eluting with petrol and diethyl ether (2:1), to yield
13 (950 mg, 94%) as a white solid. Mp: 33–34 ◦C;
[α]2D4 = +11.6◦ (c: 0.22 mg ml−1, CHCl3); νmax
(film): 3412 (br), 3062, 2980, 2954, 1208 cm−1; δH:
3.44 (t, CH2, 2H, J = 6.6 Hz), 2.85 (s, OH, 1H),
1.62 (m, CH2, 2H), 1.42 (m, 2 × CH2, 4H), 1.08
(s, C16H32, 32H), 0.76 (br, t, CH3, 3H, J = 5.6 Hz),
0.48 (m, 3 × CH, 3H), −0.41 (m, CH, 1H); δC:
62.9 (COH), 33.2, 31.8, 30.1, 29.6, 29.3, 28.6, 25.2,
24.9, 24.3, 24.1, 23.4, 23.0, 22.6, 15.8, 15.4, 14.0,
10.9. Found M+: 352.3701. Calculated for C24H48O:
352.3705.

40
G.D. Coxon et al. / Chemistry and Physics of Lipids 127 (2004) 35–46
2.2.8. (1R,2S)-2-Octadecyl-1-[[(3ꢀ-tosyl)oxy]-
propyl]cyclopropane (14)
2.2.10. (1R,2S)-1-(3ꢀ-Carboxypropyl)-2-
octadecylcyclopropane (16)
To a stirred solution of 13 (900 mg, 2.55 mmol)
in pyridine (50 ml) at 0 ◦C: was added tosyl chlo-
ride (730 mg, 3.83 eq.). The mixture was allowed
to warm to ambient temperature and stirred for
2 h until TLC analysis showed no starting ma-
terial. Saturated aq. ammonium chloride (20 ml)
was added to the solution and the organic phase
extracted with chloroform (3 × 30 ml). The com-
bined organic phases were dried and reduced un-
der vacuum. The crude mixture was purified by
column chromatography, eluting with petrol and
diethyl ether (5:2), to yield 14 (800 mg, 62%) as
a white solid. Mp: 39–41 ◦C; [α]D24 = +8.7◦ (c:
1.3 mg ml−1, CHCl3); νmax (film): 3110, 3092, 3062,
2998, 2978, 1410, 1380, 1252 cm−1; δH: 7.64 (m,
2 × CH, 2H), 7.19 (m, 2 × CH, 2H), 3.95 (t, CH2,
2H, J = 7.4 Hz), 2.25 (s, Me, 3H), 1.59 (t, CH2,
2H, J = 7.4 Hz), 1.48 (m, 2 × CH2, 4H), 1.10 (m,
C16H32, 32H), 0.89 (br, t, CH3, 3H, J = 5.6 Hz), 0.46
(m, 3 × CH, 3H), −0.32 (m, CH, 1H); δC: 144.8,
129.7, 127.9, 70.9 (COTs), 31.9, 30.1, 29.7, 28.6,
24.5, 22.7, 21.6, 15.7, 14.9, 10.9; MS: no parent ion
found.
To a stirred solution of 15 (400 mg, 1.12 mmol)
in ethanol (20 ml) was added sodium hydroxide
(600 mg, 8.7 mmol). The mixture was stirred at re-
flux for 3 h before being cooled to 0 ◦C, and wa-
ter (10 ml) added. The organic phase was extracted
with dichloromethane (2 × 30 ml), dried and re-
duced under vacuum. Purification by column chro-
matography, eluting with petrol and diethyl ether
(2:1), gave the product as a white solid 16 (310 mg,
73%). Mp: 31–32 ◦C; [α]D24 = 9.4◦ (c: 1.7 mg ml−1
,
CHCl3); νmax (film): 3820 (br), 3051, 2994, 2891,
1412 cm−1; δH: 2.20 (t, CH2, 2H, J = 7.3 Hz), 1.52
(p, CH2, 2H, 7.3 Hz), 1.42 (m, 2 × CH2, 2H), 1.08
(s, C17H34, 34H), 0.86 (br, t, CH3, 3H, J = 5.6 Hz),
0.43 (m, 3 × CH, 3H), −0.48 (m, CH, 1H); δC: 179.2
(COOH), 33.7, 31.9, 30.2, 29.7, 29.4, 28.7, 28.1,
27.2, 26.4, 25.2, 23.8, 22.9, 22.7, 15.7, 15.2, 14.1,
10.9. Found M+: 380.3642. Calculated for C25H48O2:
380.3654.
2.2.11. (1R,2S)-1-(3ꢀ-Methoxycarbonylprop-1ꢀ-yl)-2-
octadecylcyclopropane (17)
To a stirred solution of 16 (200 mg, 0.52 mmol)
in dry methanol (30 ml) was added DOWEX®
50WX2-100 ion-exchange resin (0.2 g). The solution
was stirred at reflux for 3 h, allowed to cool to ambi-
ent temperature and filtered. The filtrate was reduced
under vacuum and purified by column chromatogra-
phy, using petrol and diethyl ether (5:2), to yield 17
as a pale yellow solid (120 mg, 54%). Mp: 29–30 ◦C;
[α]2D4 = +8.7◦ (c: 2.12 mg ml−1, CHCl3); νmax (film):
3060, 2996, 2894, 1410, 1230 cm−1; δH 3.47 (s, OMe,
3H), 2.16 (t, CH2, 2H, J = 7.4 Hz), 1.57 (p, CH2, 2H,
J = 7.4 Hz), 1.44 (m, 2 × CH2, 2H), 1.11 (s, C16H32,
CH2, 34H), 0.85 (br, t, CH3, 3H, J = 5.6 Hz), −0.41
(m, 3 × CH, 3H), −0.41 (m, CH, 1H); δC: 176.2
(COOMe), 51.4 (OMe), 34.0, 31.9, 30.2, 29.7, 29.4,
28.7, 28.2, 27.2, 26.4, 25.5, 23.8, 23.7, 23.5, 23.2,
23.0, 22.9, 22.7, 15.7, 15.3, 14.1, 10.9. Found M+:
394.3805. Calculated for C26H50O2: 394.3810.
2.2.9. (1R,2S)-1-(3ꢀ-Cyanopropyl)-2-
octadecylcyclopropane (15)
To a slurry of sodium cyanide (73 mg, 1.52 mmol)
in DMSO (10 ml), stirred at 90 ◦C, was added to
14 (700 mg, 1.38 mmol). The solution was warmed
to 120 ◦C and stirred for 2 h before being cooled
to 0 ◦C. Water (10 ml) was added, the organic
phase was extracted with chloroform (3 × 20 ml),
dried and reduced under vacuum. The crude mix-
ture was purified by column chromatography, elut-
ing with petrol and diethyl ether (2:1), to yield 15
(480 mg, 84%) as a white solid. Mp: 43–44 ◦C;
[α]2D4
=
112◦ (c: 2.24 mg ml−1, CHCl3); νmax
(film): 3054, 3898, 2010, 1410, 1251 cm−1; δH:
2.18 (t, CH2, 2H, J = 6.7 Hz), 1.57 (t, CH2, 2H,
J = 6.5 Hz), 1.32 (m, 2 × CH2, 4H), 1.08 (s, C16H32,
32H), 0.61 (br, t, CH3, 3H, J = 5.6 Hz), 0.46 (m,
3 × CH, 3H), −0.42 (m, CH, 1H); δC: 31.9, 30.1,
29.7, 29.4, 28.7, 27.6, 26.8, 26.5, 26.2, 26.0, 25.3,
25.0, 24.3, 22.5, 21.7, 16.9, 15.7, 14.7, 14.2, 10.9.
Found M+: 361.3706. Calculated for C25H47N:
361.3708.
2.2.12. (1ꢀZ,1R,2S)-1-(4ꢀ-Carboxybut-2ꢀ-en-1ꢀ-yl)-2-
octadecylcyclopropane (18)
Sodium bis(trimethylsilyl) amide (3.1 ml, 3.6 mmol)
was added to a suspension of carbomethoxypropyl
triphenylphosphonium bromide (1.51 g, 3.41 mmol)

G.D. Coxon et al. / Chemistry and Physics of Lipids 127 (2004) 35–46
41
in anhydrous THF (20 ml), stirring under nitrogen at
ambient temperature. The mixture was stirred for a
further 10 min, cooled to −78 ◦C and 10 (100 mg,
1.0 eq., 3.1 mmol) in THF (20 ml) added. The re-
action was stirred for 2 h at ambient temperature
before being quenched with saturated aq. ammonium
chloride (20 ml). The organic phase was extracted
with dichloromethane (30 ml), washed with water
(2 × 50 ml), dried and reduced under vacuum. The
crude product was purified by column chromatogra-
phy, eluting with petrol and diethyl ether (5:2), to
yield 18 as a white low melting solid (62 mg, 86%).
2 h to a stirred mixture of 18 (20 mg, 0.051 mmol),
hydrazine hydrate (0.65 ml), ethanoic acid (0.5 ml)
and saturated aq. copper sulfate solution (0.5 ml)
dissolved in isopropanol (20 ml). The reaction was
stirred for 3 days before being quenched with satu-
rated aq. ammonium chloride (10 ml). The organic
phase was extracted with dichloromethane (2×50 ml),
washed with water (2 × 20 ml), dried and reduced
under vacuum. The residue was purified by column
chromatography, eluting with petrol and ethyl acetate
(8:1), to yield 20 (18 mg, 90%) as a white low melting
solid. Mp: 36–38 ◦C; [α]D24 = +1.6◦ (c: 0.5 mg ml−1
,
Mp: 38–40 ◦C; [α]D24 = +21.9◦ (c: 0.43 mg ml−1
,
CHCl3); νmax (film): 3510 (br), 3071, 2985, 2953,
2916, 1719 cm−1; δH: 2.29 (t, CH2, 2H, J = 7.2 Hz),
1.61 (m, CH2, 2H), 1.48 (3 × CH2, 6H), 1.18 (s,
C16H32, 32H), 0.91 (br, t, CH3, 3H, J = 5.6 Hz), 0.58
(m, 3 × CH, 3H), −0.34 (m, CH, 1H); δC: 180.3
(COOH), 34.2, 32.0, 30.2, 29.7, 29.4, 28.7, 28.3,
24.7, 24.2, 23.5, 22.7, 15.8, 15.5, 14.1, 10.9. Found
M+: 394.3793. Calculated for C26H50O2: 394.3810.
CHCl3); νmax (film): 3400 (br), 3058, 3035, 3035,
3015, 2990, 2920, 1687 cm−1; δH: 5.38 (dt, CH,
1H, J = 10.8, 6.5 Hz), 5.12 (t, CH, 1H, J = 10.8 Hz),
2.46 (m, 2 × CH2, 4H), 1.52 (br, m, CH2, 2H), 1.22
(s, C16H32, 32H), 0.84 (br, t, CH3, 3H, J = 5.6 Hz),
0.83 (m, 3 × CH, 3H), 0.12 (m, CH, 1H); δC: 179.2
(COOH), 131.8 (CH), 127.3 (CH), 34.4, 32.2, 29.9,
29.8, 29.7, 29.6, 25.3, 25.1, 24.9, 24.7, 24.6, 23.1,
22.9, 22.6, 18.9, 15.4, 14.4, 14.3, 14.2. Found M+:
392.3812. Calculated for C26H48O2: 392.3810.
2.2.15. (1R,2S)-1-(4ꢀ-Methoxycarbonylbut-1ꢀ-yl)-2-
octadecylcyclopropane (21)
DOWEX® 50WX2-100 ion-exchange resin (0.2 g)
was added to a stirred solution of 20 (10 mg,
0.025 mmol) in dry methanol (15 ml), stirred at re-
flux for 4 h, allowed to cool to ambient temperature
and filtered. The filtrate was reduced under vacuum
and purified by column chromatography, eluting with
petrol and diethyl ether (5:2), to yield a white solid,
21 (9 mg, 89%). Mp: 39–40 ◦C; [α]D24 = +7.1◦ (c:
0.7 mg ml−1, CHCl3); νmax (film): 2953, 2920, 2851,
1742 cm−1; δH: 3.61 (s, Me, 3H), 2.21 (t, CH2, 2H,
J = 7.2 Hz), 1.45–1.24 (m, 3 × CH2, 6H), 1.18 (m,
C16H32, CH2, 34H), 0.89 (t, CH3, 3H, J = 5.6 Hz),
0.56 (m, 3 × CH, 3H), −0.34 (m, CH, 1H); δC: 185.0
(COOMe), 52.1 (OMe), 34.1, 31.9, 30.2, 29.7, 29.4,
28.7, 24.5, 24.2, 24.1, 23.5, 23.4, 23.0, 22.8, 22.6,
19.4, 19.3, 16.1, 12.2, 10.1. Found M+: 408.3955.
Calculated for C27H52O2: 408.3967.
2.2.13. (1ꢀZ,1R,2S)-1-(4ꢀ-Methoxycarbonylbut-2ꢀ-
en-1ꢀ-yl)-2-octadecylcyclopropane (19)
To a stirred solution of 18 (30 mg, 0.076 mmol)
in dry methanol (15 ml) was added DOWEX®
50WX2-100 ion-exchange resin (0.1 g). The solution
was stirred at reflux for 2 h, allowed to cool to ambi-
ent temperature and filtered. The filtrate was reduced
under vacuum and purified by column chromatogra-
phy, using petrol and diethyl ether (5:2), to yield 19 as
a white solid (23 mg, 74%). Mp: 38–40 ◦C; [α]D24
=
+9.2◦ (c: 0.06 mg ml−1, CHCl3); νmax (film): 3129,
3052, 2996, 2991, 2983, 1730 cm−1; δH: 5.38 (dt, CH,
1H, J = 10.6, 6.6 Hz), 5.12 (t, CH, 1H, J = 10.6 Hz),
3.65 (s, OMe, 3H), 2.21 (m, 2 × CH2, 4H), 1.42 (m,
CH2, 2H), 1.10 (s, C16H32, 32H), 0.91 (br, t, CH3,
3 × CH, 6H), 0.06 (m, CH, 1H); δC: 131.3 (CH),
127.4 (CH), 51.5 (OMe), 34.2, 31.9, 29.7, 29.6, 29.5,
29.4, 23.2, 22.7, 18.7, 16.0, 15.6, 14.5, 14.1. Found
M+: 408.3766. Calculated for C27H50O2: 408.3767.
2.2.16. (1ꢀZ,1R,2S)-1-(5ꢀ-Carboxypent-1ꢀ-yl)-2-
octadecylcyclopropane (22)
To a stirred suspension of 4-carboxybutyltriphenyl-
phosphonium bromide (0.38 g, 3.41 mmol) in an-
hydrous THF (40 ml), under an atmosphere of
nitrogen at ambient temperature was added sodium
bis(trimethylsilyl) amide (3.1 ml, 2.0 eq., 6.2 mmol).
2.2.14. (1R,2S)-1-(4ꢀ-Carboxybut-1ꢀ-yl)-2-
octadecylcyclopropane (20)
Sodium periodate (1.2 g, 245 mmol) in water
(10 ml) was added dropwise via a dropping funnel over

42
G.D. Coxon et al. / Chemistry and Physics of Lipids 127 (2004) 35–46
The mixture was stirred for a further 10 min cooled
to −78 ◦C and 10 dissolved in THF (10 ml) (100 mg,
3.1 mmol) was added. The reaction was stirred for 3 h
at ambient temperature before being quenched with
saturated aq. ammonium chloride (30 ml). The organic
phase was extracted with dichloromethane (30 ml),
washed with water (2 × 40 ml), dried and reduced
under vacuum. The crude product was then purified
by column chromatography, eluting with petrol and
diethyl ether (5:2), to yield 22 as a white low melting
solid (72 mg, 58%). Mp: 42–43 ◦C; [α]D24 = +56.2◦
(c: 0.8 mg ml−1, CHCl3); νmax (film): 3400–3100 (br),
3057, 3013, 2988, 2953, 2920, 1705 cm−1; δH: 6.53
(dd, CH, 1H, J = 15.6, 10.2 Hz), 6.19 (d, CH, 1H,
J = 15.6 Hz), 2.41 (t, CH2, 2H, J = 7.5 Hz), 2.21 (p,
CH2, 2H, J = 7.5 Hz), 1.50 (t, CH2, 2H, J = 8.4 Hz),
1.34 (m, CH2, 2H), 1.22 (s, C16H32, 32H), 0.89 (br, t,
CH3, 3H, J = 5.6 Hz), 0.88 (m, 3 × CH, 3H), 0.57 (q,
CH, 1H, J = 5.9 Hz); δC: 207.1 (COOH), 150.7 (CH),
130.1 (CH), 32.1, 31.1, 29.8, 29.7, 29.6, 29.5, 29.3,
27.2, 27.1, 23.1, 22.8, 22.3, 20.0, 16.2, 15.7,14.2,
14.1; MS: no parent ion found.
22 (20 mg, 0.05 mmol), hydrazine hydrate (0.65 ml),
ethanoic acid (0.5 ml) and saturated aq. copper sulfate
solution (0.5 ml) in isopropanol (40 ml). The reaction
was stirred for 2 days before being quenched with
saturated aq. ammonium chloride (25 ml). The mix-
ture was extracted with dichloromethane (2 × 50 ml),
washed with water (2 × 20 ml) and dried. The organic
phase was reduced under vacuum and the residue pu-
rified by column chromatography, eluting with petrol
and ethyl acetate (8:1), to yield 24 (17 mg, 85%) as
a pale yellow solid. Mp: 41–43 ◦C; [α]D24 = +2.9◦
(c: 0.3 mg ml−1, CHCl3); νmax (film): 3450–3120 (br),
3068, 2986, 2953, 2916, 1733 cm−1; δH: 2.25 (m,
CH2, 2H), 1.58 (m, 2 × CH2, 4H), s, (C16H36, C3H6,
38H), 0.92 (br, t, CH3, 3H, J = 5.6 Hz), 0.57 (m, 3 ×
CH, 3H), −0.31 (m, CH, 1H); δC: 178.0 (COOH),
31.9, 30.2, 29.7, 29.4, 28.8, 28.5, 25.3, 24.8, 24.6,
24.2, 23.8, 22.7, 15.8, 15.6, 14.2, 10.9; MS: no parent
ion found.
2.2.19. (1R,2S)-1-(5ꢀ-Methoxycarbonypent-1ꢀ-yl)-2-
octadecylcyclopropane (25)
To a stirred solution of 24 (10 mg, 0.025 mmol)
in dry methanol (15 ml) was added DOWEX®
50WX2-100 ion-exchange resin (0.3 g), stirred at
reflux for 5 h, allowed to cool to ambient temper-
ature and filtered and the filtrate evaporated. The
residue was purified by column chromatography, us-
ing petrol and diethyl ether (5:2) to yield 25 (7 mg,
70%) as a white solid. Mp: 43–44 ◦C; [α]D24 = +1.6◦
(c: 0.06 mg ml−1, CHCl3); νmax (film): 3058, 2988,
2822, 2853, 1743 cm−1; δH: 60 (s, Me, 3H), 2.21
(t, CH2, 2H, J = 7.2 Hz), 1.25 (m, 2 × CH2, 4H),
1.20 (m, C16H32, C3H6, 38H), 0.88 (br, t, CH3, 3H,
5.6 Hz), 0.52 (m, 3 × CH, 3H), −0.33 (m, CH, 1H);
δC: 179.4 (COOMe), 51.4 (OMe), 34.2, 31.9, 30.2,
29.7, 29.4, 29.2, 28.7, 28.5, 25.0, 22.7, 15.8, 15.7,
15.6, 14.1, 10.9. Found M+: 422.4137. Calculated for
C28H54O2: 422. 4123.
2.2.17. (1ꢀZ,1R,2S)-1-(5ꢀ-Methoxycarbonylpent-2ꢀ-
en-1ꢀ-yl)-2-octadecylcyclopropane (23)
To a stirred solution of 22 (30 mg, 0.074 mmol)
in dry methanol (15 ml) was added DOWEX®
50WX2-100 ion-exchange resin (0.3 g). The solution
was stirred at reflux for 2 h, cooled to ambient tem-
perature and filtered. The filtrate was reduced under
vacuum and purified by chromatography, eluting with
petrol and diethyl ether (5:2), to yield a white low
melting solid 23 (26 mg, 84%). Mp: 43–45 ◦C; [α]D24
=
+7.8◦ (c: 0.1 mg ml−1, CHCl3); νmax (film): 3067,
3023, 2987, 1756, 1270 cm−1; δH: 5.22 (dt, CH, 1H,
J = 15.2, 10.1 Hz), 4.99 (t, CH, 1H, J = 15.0 Hz), 3.49
(s, OMe, 3H), 2.24 (t, CH2, 2H, J = 7.5 Hz), 1.52–1.41
(m, 2 × CH2, 4H), 1.19 (s, C16H32, CH2, 34H), 0.89
(br, t, CH3, 3×CH, 6H, J = 5.6 Hz), 0.01 (m, CH, 1H);
δC: 179.4 (COOMe), 130.9 (CH), 128.5 (CH), 51.5
(OMe), 33.5, 31.9, 29.7, 29.6, 29.4, 26.9, 25.0, 24.3,
24.2, 24.0, 23.7, 23.4, 22.7, 18.6, 14.2, 14.1. Found
M+: 418.3813. Calculated for C28H52O2 418.3810.
2.3. Biological testing
M. smegmatis mc2 cells were used to prepare a
cell-wall fraction, with mycolate synthesising ability
(Wheeler et al., 1993a,b). The “P60” wall fraction
was obtained from broken cells by Percoll-density
gradient centrifugation. For each low water assay,
performed in triplicate, a mixture of P60 (130 ␮l),
2.2.18. (1R,2S)-1-(5ꢀ-Carboxypent-1ꢀ-yl)-2-
octadecylcyclopropane (24)
Sodium periodate (1.2 g, 0.5 mmol) in water (15 ml)
was added dropwise over 2 h to a stirred mixture of

G.D. Coxon et al. / Chemistry and Physics of Lipids 127 (2004) 35–46
43
hexane (900 ␮l) and inhibitor/stimulator (200 ␮g ml−1
in hexane) solution (100 ␮l) concentration was pre
incubated at 37 ◦C for 30 min. Uniformly labelled
1 ␮Ci 14C-acetate (2.11 GBq mmol−1) (10 ␮l) and a
pH 5.0 buffer, consisting of di-potassium hydrogen
orthophosphate 0.5 M, and potassium hydrogen car-
bonate 0.1 M (14 ␮l) were then added to give a total
volume of 1154 ␮l; each assay was incubated for
1 h. The reaction was quenched with aqueous tetra-
butylammonium hydroxide (15%, 900 ␮l), and the
hexane evaporated. After heating at 100 ◦C for 18 h,
dichloromethane (2 ml) and iodomethane (200 ␮l)
were added, and each assay agitated for 45 min. The
aqueous phase was separated and the organic phase
washed with hydrochloric acid (1 M, 1.0 ml) and wa-
ter (2 × 1 ml). The organic phase was evaporated
and the residue extracted with ether (2 × 1 ml). My-
colic and fatty acid methyl esters were isolated from
this extract using preparative TLC with petrol and
acetone (95:5) as eluent and diphenyl hexatriene for
Table 1
Effect on the incorporation of 14C-acetate into mycolic acids, in extracts of M. smegmatis, of long-chain cyclopropane compounds (tested
at 20 ␮g/1154 ␮l)
Structure number
Control
Cyclopropane compound
None
Counts per min (cpm)
75755
% Inhibition
0
Chain length
–
CO2Me
RacC24
11
88633
104876
73995
−17
−38
+2
5
24
23
23
(
(
)
17
17
37
1
)
CO2Me
12
(
)
17
CO2Me
CO2Me
17
18
19
78027
106041
85232
+3
−40
−13
2
24
25
25
(
(
(
)
)
17
17
CO2H
5
20
CO2Me
)
17
17
20
21
22
117562
78586
91161
−55
−4
17
5
25
25
26
(
(
)
CO2H
)
17
CO2Me
−20
7
(
)
17
CO2H
23
24
25
106889
83942
88815
−41
−11
−18
13
20
5
26
26
26
(
)
17
CO2Me
CO2H
(
)
17
)
17
CO2Me
(

44
G.D. Coxon et al. / Chemistry and Physics of Lipids 127 (2004) 35–46
visualisation under long wave UV light. The radioac-
tivity of the methyl esters was then determined by
scintillation counting. Control assays were carried out
in the absence of inhibitor/stimulator to provide base-
line information against which to assess inhibitory or
stimulatory behaviour (Table 1).
ysed to the acid 16. Acid catalysed esterification con-
verted the acid 16 to the desired ester 17 (Scheme 1).
The phosphonium salt prepared from methyl
4-bromobutyrate, reacted with the aldehyde 10, in
the presence of sodium bis-trimethylsilyl amide to
give the acid 18 with only the Z diasteromer formed
as indicated by NMR (Scheme 2). It seemed likely
that the excess base used to ensure generation of the
yield had hydrolysed the ester to the acid. A similar
reaction using 4-carboxybutyl triphenylphosphonium
bromide afforded the acid 22 again with only the Z
diasteroisomer indicated by NMR (Scheme 2). Se-
lective reduction of the vinylic acids acid 18 and 22
gave the saturated acids 20 and 24, respectively. The
unsaturated (18, 22) and saturated (20, 24) cyclo-
propane acids were converted into the corresponding
unsaturated (19, 23) and saturated (21, 25) esters,
with DOWEX® 50WX2-100 H+ resin in methanol
for 4 h (Scheme 2).
The (1R,2S) long-chain ␻-19 cyclopropane acids
(18, 20, 22, 24) and esters (11, 12, 17, 19, 21, 23, 25)
and the racemic C24 methyl ester analogue (Hartmann
et al., 1994) were assayed for inhibition of mycolic
acid biosynthesis, using a cell-wall preparation from
M. smegmatis (Wheeler et al., 1993a,b) (Table 1). The
assay essentially measures elongation of endogenous
fatty acid precursors associated with the cell wall
extracts, it being unlikely that enzymes for de novo
mycolate synthesis are present in high concentrations
in these preparations. Inhibition by these long-chain
compounds probably reflects binding to the essential
ꢀ5-desaturase, which appears to initiate mycolate
synthesis; this was dramatically shown for the cyclo-
propene analogue 3 (Wheeler et al., 1993a). Stimu-
lation may be a result of direct incorporation of the
long-chain cyclopropane compounds, but extensive
studies would be necessary to establish this.
3. Results and discussion
The key chiral intermediate 10 was prepared from
2,3-O-isopropylidene-d-glyceraldehyde
(Jackson,
1988) as outlined in Scheme 1. Nonadecyl triph-
enylphosphonium bromide (Al Dulayymi et al.,
2000) reacted, in a Wittig reaction, with 2,3-O-
isopropylidene-d-glyceraldehyde to give the Z alkene
7, in 100% de as indicated by 1H and 13C NMR.
Enantioselective formation of the cyclopropane
ring, according to Morikawa et al. (1994) and
Zhao et al. (1995), was achieved, using a modified
Simmons–Smith reaction (Furakawa et al., 1966;
Nishimura et al., 1969). This involved the stereoselec-
tive addition of diethyl zinc and chloroiodomethane
to the alkene 7 to yield the cis-cyclopropane 8. De-
protection of the isopropylidene ring of 8 with TFA
in methanol gave the diol 9 and oxidative cleav-
age with sodium(meta)periodate then gave the key
formylcyclopropane intermediate 10 (Scheme 1). It
was expected that Wittig chemistry on 10, using the
appropriate phosphonium salts would enable the addi-
tion of 3-, 4-, 5- and 6-carbon carboxy side chains to
provide the corresponding chiral cyclopropane acids.
This approach was successful for the synthesis of
three of the acids or esters (12, 21, 25), as outlined
in Schemes 1 and 2. It was found necessary to pre-
pare the remaining acid 17 by chain extension of 12
(Scheme 1).
Reaction of the aldehyde 10 with carbomethoxy-
methylenetriphenylphosphorane yielded the E-alkene
11 (Scheme 1). Reduction of the alkene 11, using
hydrazine hydrate, sodium(meta)periodate, copper(II)
sulfate and ethanoic acid at 60 ◦C for 24 h yielded the
saturated ester 12. The phosphonium salt, prepared
from ethyl 3-bromopropanoate, failed to react with 10
to provide a route to the ester 17 (Scheme 1). There-
fore, reduction of 12 to the alcohol 13 was followed
by tosylation to give 14. Nucleophlic substitution with
cyanide on 14 gave the nitrile 15, which was hydrol-
Preliminary studies had indicated that racemic
cis-5, 6-methanomethyloctadecanoate (RacC24) was
a marginal inhibitor of mycolic acid biosynthesis in
M. smegmatis. (Hartmann, 1993; Hartmann et al.,
1994). In the present study, RacC24 actually stimu-
lated mycolate synthesis but the (1R,2S) enantiomer
17 showed small inhibition. Considering the saturated
analogues first, the 23-carbon chain length analogue
12 was also a weak inhibitor. However, the 25- and
26-carbon analogues 21 and 25 showed significant
stimulation of mycolate biosynthesis. The saturated

G.D. Coxon et al. / Chemistry and Physics of Lipids 127 (2004) 35–46
45
acid analogues 20 and 24 appeared to stimulate
biosynthesis although the 25-carbon acid 20 showed
a larger stimulation than the 26-carbon analogue 24.
This was also the case with the olefinic acids 18
and 22. In the case of the vinyl esters, these were
also found to be stimulating, with the 25-carbon ana-
logue 19 showing less stimulation than the 23-carbon
analogue 11 and the 26-carbon analogue 23 which
showed similar stimulation values. The C24 acid 16
was not tested for mycolic acid inhibition.
esters (19, 21) but the reverse was found for the C26
acids (22, 24) and esters (23, 25). It may be surmised
that the C26 esters are more readily hydrolysed by li-
pases due to their even numbered chain lengths being
more biocompatible.
The diverse effects on mycolic acid synthesis
shown by these long-chain ␻-19 cyclopropane acids
and esters indicate that specific interactions are taking
place with key biosynthetic enzymes. These enzymes
seem to be catholic in their tastes, perhaps demon-
strating that recognition is governed, largely by the
␻-19 cis-cyclopropane part of the molecule. These re-
sults support the hypothesis (Takayama and Qureshi,
1984; Wheeler et al., 1993a,b) that long-chain acids
with ␻-19 cis unsaturations have importance in the
biosynthesis of mycolic acids.
Considering the four saturated cyclopropane esters
(12, 17, 21, 25), it appears that the C23 and C24 com-
pounds (12, 17) are able to interact with the active site
of possibly a key ꢀ5-desaturase and cause mild inhi-
bition of mycolate synthesis. The extra carbon atom in
the C25 ester 21 seems to disallow such interactions,
giving mild stimulation; the addition of a further car-
bon in the C26 ester 25 reinforces this trend. The in-
creased inhibition of the C24 (1R,2S) form 17 over the
racemic form (RacC24) suggests that this enantiomer
has enhanced affinity for an enzymic active site.
The presence of an additional double bond does not
abolish stimulation of mycolate synthesis (Table 1).
The increased rigidity conferred by the trans unsatu-
ration in the C23 ester 11 negates the inhibitory effect
seen in the saturated analogue 12. Some interesting
trends were discernable for the C25 and C26 com-
pounds. The unsaturated methyl esters (19, 23) were
notably more stimulatory than the corresponding sat-
urated esters (21, 25). However, the behaviour of the
unsaturated (18, 22) and saturated acids (20, 24) was
not so well distinguished. This possibly reflects the
differential effect of any lipases needed to hydrolyse
the esters, the unsaturated esters (19, 23) apparently
being better substrates than the saturated analogues
(21, 25). In view of the relatively high stimulation of
mycolate synthesis by some of the unsaturated ana-
logues, such as compounds 11, 18 and 23, it would be
of interest to know if their constituent double bonds
are preserved through into intact mycolates. It is inter-
esting that the most potent stimulators 11, 18, 20 and
23 (Table 1) have either odd chain lengths (11, 18, 20)
or a double bond (23), suggesting that these substrates
may not bind well to a desaturase active site but are
capable of being elongated. Concerning different ac-
tivities of cyclopropane acids and the corresponding
esters, the only point of interest was that C25 acids
(18, 20) were stronger mycolate stimulators than their
Acknowledgements
D.E.M. and G.S.B. (Lister Institute Jenner Research
Fellow) received support from the UK Medical Re-
search Council Cooperative (G9901077) and Project
(G9901075) grants. The European Social Fund is
thanked for partial support of G.D.C. D.E.M., C.M.,
G.S.B. and P.J.B. had support from a Cooperative
Agreement AI-3807 from the National Cooperative
Drug Discovery Groups for the treatment of Oppor-
tunist Infections, NIAID and NIH (USA).
References
Al Dulayymi, J.R., Baird, M.S., Roberts, E., 2000. The synthesis
of single enantiomer of a meromycolic acid. Tetrahedron Lett.
41, 7107–7110.
Al Dulayymi, J.R., Baird M.S., Roberts E., 2003. The synthesis of a
single enantiomer of a major ␣-mycolic acid of Mycobacterium
tuberculosis. Chem. Commun. 228–229.
Besra, G.S., Minnikin, D.E., Wheeler, P.R., Ratledge, C., 1993a.
Synthesis of methyl (Z)-tetracos-5-enoate and both enantiomers
of ethyl (E)-6-methyltetracos-4-enoate; possible intermediates
in the biosynthesis of mycolic acids in mycobacteria. Chem.
Phys. Lipids 66, 23–34.
Besra, G.S., Minnikin, D.E., Simpson, M.J., Baird, M.S., Wheeler,
P.R., Ratledge, C., 1993b. Inhibition of mycolic acid biosyn-
thesis in a cell-wall preparation from Mycobacterium smeg-
matis by methyl 3-(2-octadecylcyclopropen-1-yl) butanoate, a
structural analogue of a key precursor. Chem. Phys. Lipids 66,
35–40.

46
G.D. Coxon et al. / Chemistry and Physics of Lipids 127 (2004) 35–46
Coxon, G.D., Knobl, S., Roberts, E., Baird, M.S., Al-Dulayymi,
J.R., Besra, G.S., Brennan, P.J., Minnikin, D.E., 1999. The
synthesis of both enantiomers of lactobacillic acid and mycolic
acid analogues. Tetrahedron Lett. 40, 6689–6692.
Coxon, G.D., Douglas, J.D., Minnikin, D.E., 2003a. Facile
synthesis of (Z)-tetracos-5-enoic acid and racemic cis-4-(2-
octadecylcyclopropane-1-yl)-butanoic acid. Chem. Phys. Lipids
126, 49–53.
Coxon, G.D., Al-Dulayymi, J.R., Baird, M.S., Knobl, S., Roberts,
E., Minnikin, D.E., 2003b. The synthesis of (11R,12S)-
lactobacillic acid and its enantiomer. Tetrahedron Asym. 14,
1211–1222.
Enarson, D.A., Murray, J.F., 1994. Global epidemiology of
tuberculosis. In: Bloom, B.R. (Ed.), Tuberculosis: Pathogenesis,
Protection and Control. American Society for Microbiology,
Washington, DC, pp. 3–11.
Furakawa, J., Kawabata, N., Nishimura, J., 1966. A novel route
to cyclopropanes from olefins. Tetrahedron Lett. 7, 3353–
3354.
Hartmann, S., 1993. Studies on the structure and biosynthesis
of mycobacterial lipids. Ph.D. thesis, University of Newcastle
upon Tyne, UK.
Hartmann, S., Minnikin, D.E., Römming, H.-J., Baird, M.S.,
Ratledge, C., Wheeler, P.R., 1994. Synthesis of methyl
3-(2-octadecylcyclopropen-1-yl)propanoate and methyl 3-(2-
octadecylcyclopropen-1-yl)pentanoate and cyclopropane fatty
acids as possible inhibitors of mycolic acid biosynthesis. Chem.
Phys. Lipids 71, 99–108.
The Biology of the Mycobacteria, vol. 1. Academic Press,
London, pp. 95–184.
Minnikin, D.E., 1993. Mycolic acids. In: Mukherjee, K.D., Weber,
N. (Eds.), CRC Handbook of Chromatography: Analysis of
Lipids. CRC Press, Boca Raton, FL, pp. 339–348.
Morikawa, T., Sasaki, H., Hanai, R., Shibuya, A., Taguchi, T., 1994.
Synthesis of optically active cis- and trans-1,2-disubstituted
cyclopropane derivatives by the Simmons–Smith reaction
of allyl alcohol derivatives derived from (R)-2,3-O-isopro-
pylideneglyceraldehyde. J. Org. Chem. 59, 97–103.
Nishimura, J., Kawabata, N., Furukawa, J., 1969. A novel synthesis
of methylcyclopropanes. Tetrahedron 25, 2647–2656.
Takayama, K., Qureshi, N., 1984. Structure and synthesis of lipids.
In: Kubica, G.P., Wayne, L.G. (Eds.), The Mycobacteria: A
Source Book, Part A. Marcel Dekker, New York, pp. 315–344.
Watanabe, M., Aoyagi, Y., Ridell, M., Minnikin, D.E., 2001.
Separation and characterization of individual mycolic acids in
representative mycobacteria. Microbiology 147, 1825–1837.
Watanabe, M., Aoyagi, Y., Mitome, H., Fujita, T., Naoki, H.,
Ridell, M., Minnikin, D.E., 2002. Location of functional groups
in mycobacterial meromycolate chains the recognition of new
structural principles in mycolic acids. Microbiology 148, 1881–
1902.
Wheeler, P.R., Besra, G.S., Minnikin, D.E., Ratledge, C., 1993a.
Stimulation of mycolic acid biosynthesis by incorporation
of cis-tetracos-5-enoic acid in a cell-wall preparation from
Mycobacterium smegmatis. Biochim. Biophys. Acta 1167, 182–
188.
Jackson, D.Y., 1988. An improved preparation of (+)-2,3-O-
isopropylidene-d-glyceraldehyde. Synth. Commun. 18, 337–
341.
Johnson, A.R., Pearson, J.A., Shenstone, F.S., Fogerty, A.C.,
1967. Inhibition of the desaturation of stearic to oleic acid by
cyclopropene fatty acids. Nature 214, 1244–1245.
Minnikin, D.E., 1982. Lipids: complex lipids, their chemistry,
biosynthesis and roles. In: Ratledge, C., Stanford, J.L. (Eds.),
Wheeler, P.R., Besra, G.S., Minnikin, D.E., Ratledge, C.,
1993b. Inhibition of mycolic acid biosynthesis in a cell-wall
preparation from Mycobacterium smegmatis by methyl 3-(2-
octadecylcyclopropen-1-yl) butanoate, a structural analogue of
a key precursor. Lett. Appl. Microbiol. 17, 33–36.
Zhao, Y., Yang, T., Lee, M., Lee, D., Newton, G.M., Chu,
C.K., 1995. Asymmetric synthesis of (1ꢀS,2ꢀR)-cyclopropyl
carboxylic nucleosides. J. Org. Chem. 60, 5236–5242.