New nitrogen mustards structurally related to (L)-carnitine

Article abstract of DOI:10.1016/S0968-0896(02)00458-3

Enantiopure nitrogen mustards which mimic (L)-carnitine framework are prepared by a multi-step synthesis from the (R)-di-tert-butyl malate and their antitumor properties evaluated.

Full text of DOI:10.1016/S0968-0896(02)00458-3

Bioorganic & Medicinal Chemistry 11 (2003) 325–334
New Nitrogen Mustards Structurally Related to (L)-Carnitine
Ludovic Faissat, Katja Martin, Claude Chavis, Jean-Louis Montero and Marc Lucas*
´ ´
ENSCM, Laboratoire de Chimie Biomoleculaire, UMR 5032, 8 Rue de l’Ecole Normale, 34296 Montpellier Cedex 05, France
Received 10 July 2002; accepted 19 September 2002
Abstract—Enantiopure nitrogen mustards which mimic (l)-carnitine framework are prepared by a multi-step synthesis from the
(R)-di-tert-butyl malate and their antitumor properties evaluated.
# 2002 Elsevier Science Ltd. All rights reserved.
The nitrogen mustards are among the earliest more
effective antitumor drugs used in human cancer chemo-
therapy.1 Actually, Mechlorethamine and Chlorambucil
are two of the most currently used anticancer agents,
and these cytotoxic drugs are believed to exert their
biological activity by inducing interstrand cross-links in
the major groove of DNA and this represents the most
toxic of all alkylation events.2,3 Numerous nitrogen
mustard structural modifications have been made dur-
ing the last 30 years in order to increase their cytotoxi-
city and their target affinity. In this respect, we designed
new nitrogen mustards structurally related to (l)-carni-
tine devoted to the specific tumor cells mitochondrial
DNA target. This idea was supported by the fact that
(l)-carnitine and some of its O-alkyl derivatives have
been demonstrated to play a crucial role in the transport
and the b-oxidation of long-chain fatty acids into
mitochondria4ꢀ6 and that a number of reports indicated
that mitochondria are potentially attractive targets for
multiple classes of DNA damaging agent.7ꢀ10 An
increase in the pool of mitochondria11 and a strong
expression of the carnitine transporter12 in cancer cells
provides an opportunity for tumor mitochondrial
DNA-selective alkylation.
groupor the nitrogen quaternarisation during the for-
mation of the requisite alkylating aziridinium cation will
give the necessary positive ion related to the trimethyl-
ammonium moiety of carnitine. The acyl groups are
introduced on the alcoholic function of mustard 1 in
order to enhance the lipophilicity of these molecules
(2a–e) and their cell penetration and because of the lack
of toxicity of some O-acyl carnitine derivatives when
orally administrated as human nutritional supplements
has been demonstrated.
Our retrosynthetic plan (Scheme 2) follows the route
proposed by Boots et al.13 for the preparation of (R,S)-
carnitine and its key stepis the regioselective ring-
We herein disclose the synthesis of the new structurally
modified carnitine analogues (R)-1 and (R)-2a–e
(Scheme 1), where the trimethylammonium moiety is
replaced by a bis(2-chloroethyl)amine, keeping the
required l-configuration to maintain as much as possi-
ble the molecular recognition properties of (l)-carnitine;
the in vivo protonation of the bis(2-chloroethyl)amino
Scheme 1.
*Corresponding author. Tel.:+33-4-6714-4342; fax:+33-4-6714-4344;
e-mail: lucas@univ-montp2.fr
Scheme 2.
0968-0896/03/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(02)00458-3

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L. Faissat et al. / Bioorg. Med. Chem. 11 (2003) 325–334
opening of the chiral epoxyester (R)-3 by a protected
bis(hydroxyethyl)amine used as a masked nitrogen
mustard moiety. The tert-butyl carboxylate moiety of 3
was chosen to prevent amide formation during the
epoxide ring-opening step. Moreover basic conditions
are no longer required during the last ester hydrolysis
step which could lead to a possible partial racemisation
of the target molecules. The enantiopure tert-butyl
epoxybutyrate (R)-3 was obtained by a multi-step
sequence from the di-tert-butyl malate (R)-4 (Scheme 3).
cleaner reaction and the best yields were obtained with
the sodium cyanoborohydride reduction of the cyclic
sulfate (R,R)-6. These results demonstrate that the
transformation of the cyclic sulfate and thioxocarbonate
of dialkyl tartrates into the corresponding malates can
be successfully applied to di-tert-butyl tartrate.
In addition, it is known that the regioselective Saito
reduction of a-hydroxy esters proceeds fairly well with
methyl,26 ethyl,27 isopropyl27 and benzyl28 esters, but to
the best of our knowledge, no attempts have been
reported in the literature with substrates containing a
bulkier tert-butyl ester. The Saito reduction conditions
(borane-methyl sulfide complex/sodium borohydride
cat.) gave cleanly the tert-butyl dihydroxy-3,4-butyrate
(R)-8 in 55% yield from (R)-4. Monotosylation of the
diol 8 produced the 4-tosyloxy derivative (R)-9 which
failed to afford the epoxide 3 when subjected to alkaline
conditions (potassium carbonate, methanol), but
instead gave the tert-butyl 4-hydroxy butenoate, a result
in accordance with earlier reports29ꢀ32 about the ability
of b,g-epoxy esters to isomerise easily into a,b-unsatu-
rated g-hydroxyesters. This problem was overcome by
converting the tosylate 9 into the bromohydrin (R)-10
(66% from 8) which finally gave successfully the desired
epoxide by using silver(I) oxide/DME (37%);32 the
epoxy ester (R)-3 displayed an optical rotation in good
accordance with the value reported by Liu et al.,33 who
recently described an efficient hydrolytic kinetic resolu-
tion of racemic epoxide 3 by using the (R,R)-salen
Co(III) Jacobsen catalyst.34
The direct esterification of (R)-malic acid was rather
disappointing and afforded the corresponding di-tert-
butyl malate in very poor yield;14ꢀ16 thus, the cyclic
sulfate (R,R)-6 and thioxocarbonate (R,R)-7 derived
from natural tartaric acid were selected as useful inter-
mediates known17ꢀ20 to give efficient access to the
unnatural (R)-dialkyl malates (Me, Et, Bun, Pri). The
(R,R)-di-tert-butyl tartrate 521 was converted by stan-
dard procedures22,23 into the cyclic sulfate (R,R)-6 and
thioxocarbonate (R,R)-7 already described by us24 in,
respectively, 82 and 60% yields (Scheme 3). We then
subjected 6 to a deoxygenation reaction23 using either
sodium borohydride or sodium cyanoborohydride fol-
lowed by a mild acid catalysed hydrolysis25 of the
resulting b-sulfate intermediate, in order to afford the
(R)-di-tert-butyl malate 4 in 56 and 80% yields, respec-
tively. Moreover, the less reactive cyclic thioxo-
carbonate 7 was also assessed for its suitability toward
two radical deoxygenation reactions using either tri-n-
butyltin hydride/AIBN17 or magnesium iodide,19 and
obtained the (R)-di-tert-butyl malate 4 in lower yields,
25 and 32%, respectively. It thus appeared that the
The silica gel-mediated ring opening of epoxide (R)-3
(Scheme 4) by the bis(tert-butyldiphenylsilyloxyethyl)-
amine24 gave the g-amino b-hydroxyester (R)-11 in 50%
yield. At this stage, different acyl groups (acetyl, pro-
Scheme 3. Reagents and conditions: (i), (ii) see ref 24; (iii) NaBH4,
DMA, rt, then concd H2SO4 (cat.), H2O (0.5–1.0 equiv), THF,
56%; (iv) NaBH3CN, THF, reflux, then concd H2SO4 (cat.), H2O
(0.5–1.0 equiv), THF, 80%; (v) HSnBu3, AIBN, benzene, reflux,
25%; (vi) MgI2, CH3CN, reflux, 32%; (vii) Me2S.BH3, NaBH4
(cat.), THF, then EtOH, PTSA, 55%; (viii) TsCl, Et3N, CH2Cl2,
0 ꢁC; (ix) LiBr, 2-butanone, reflux, 66% (two steps from 8); (x) Ag2O,
DME, reflux, 37%.
Scheme 4. Reagents and conditions: (i) bissilylated amine, SiO2,
CH3CN, reflux, 50%; (ii) anhydride or acid chloride, Et3N, DMAP
(cat.), CH2Cl2, reflux; 58–95% for 14a–c; PhCH2COOH or Chlor-
ambucil, 2,4,6-Cl3C6H2COCl, Et3N, CH2Cl2, rt for 14d (76%) and 14e
(52%); (iii) TBAF/THF/imidazole, rt; (iv) CH2Cl2, Et3N, DMAP,
PTSCl, rt, 34–51% (two steps from 14a–e); (v) aqueous HCl 2.4 M,
THF, rt, 75%; (vi) TFA, CH2Cl2, rt, 50–76%.

L. Faissat et al. / Bioorg. Med. Chem. 11 (2003) 325–334
327
pionyl, capryloyl, phenylacetyl and Chlorambucil-1-yl)
were introduced using appropriate anhydrides (mixed
anhydrides in the case of 14d–e) or acid chlorides
and afforded the expected O-acyl aminohydroxyesters
(R)-14a–e in yields ranging from 52 to 95%. It is worth
noting that reactions performed with capryloyl and
propionyl chlorides gave competitively a by-product (up
significant inhibitory activity, especially 2e with a IC50
of 193 mM close to that of Chlorambucil (IC50 202 mM).
A fairly good correlation appears between the anti-
tumor activity of compounds 2b–e and the lipophilicity
(log P)42 of their acyl chain; the presence of either a long
alkyl chain (C7H15, 2c) or a very lipophilic moiety
(Chlorambucil, 2e) proved to be effective in increasing
the cytotoxic activity by a factor upto 4 when com-
pared with the parent compound 1. These results may
account for the fact that these nitrogen mustards would
enter the cells by a diffusion rather than an active pro-
cess via a carnitine receptor. Moreover, the increased
lipophilicity brought by the tert-butyl ester function in
16c depleted its activity by comparison with activity of
the parent compound 2c pointing out the upmost
importance of the free acid function (as in Melphalan
and Chlorambucil structures).
1
to 15%) whose structure was assigned by H and 13C
nmr to a diastereomeric mixture of b-ketoesters 12
(formula shown for the propionyl moiety), presumably
resulting from a partial acylation of the alcoholic moi-
ety through the very reactive b-lactone dimer by-pro-
duct generated from ketene under the reaction
conditions used with acyl chlorides.35,36 On the other
hand, the same experimental conditions used with acetyl
chloride were unfavourable for the generation of dik-
etene37 itself from the very volatile ketene and this
explains the observed exclusive formation of the O-ace-
tyl aminohydroxyester (R)-14a and the lack of acetyl-
acetonate derivative 13 from (R)-11.
Nitrogen mustards 2c and 2e showed significant cyto-
toxicity against MCF7 cell line with IC50 values of 589
and 258 mM, respectively much better than Chlor-
ambucil which was found inactive in our conditions (1 h
drug exposure) compared with a recent reported IC50
value of 1.7 mM (24 h drug exposure).43 The two nitro-
gen mustard analogues 2c and 2e exhibited interesting
inhibitory activities with IC50 values of 723 and 753 mM,
respectively, against the HT29 cell line where both
Melphalan and Chlorambucil were inactive.
As substantial deacylation occurred when the desilyl-
ation of ethers (R)-14a–e was carried out with TBAF/
THF, the reaction was performed by using modified
conditions, that is TBAF/THF/imidazole38 which gave
the bis(2-hydroxyethyl)amine derivatives (R)-15a–e
which were prone to very fast acyl migration on stand-
ing. Therefore, they were immediately converted into
the corresponding dichlorides (R)-16a–e in 22–51%
yields by a classical procedure39 (p-toluenesulfonyl
chloride/DMAP). A final acidic hydrolysis step, gave
according to the experimental conditions, either the
fully deprotected nitrogen mustard (R)-1 (20% aq HCl/
anisole)40 (75% yield) or the O-acyl nitrogen mustards
(R)-2a–e (TFA/anisole)41 (50–87% yields).
Since the structural modification brought by the nitro-
gen mustard moiety of 2a–e to the trimethylammonium
core of natural carnitine seemed to preclude any recog-
nition of these molecules with the carnitine receptor, we
planned to evaluate the ability of the latter to act as a
carrier for two active principles, NaPa (sodium phenyl-
acetate)44ꢀ46 and Chlorambucil. Thus, the two ana-
logues 18 and 19 (Scheme 5) were prepared in order to
check the potential use of the natural l-carnitine for
prodrug development.
Biological Results
The in vitro antitumor activities of the nitrogen mus-
tards 2a–e were evaluated against the A375 human
melanoma, HT29 resistant type colon carcinoma and
MCF7 human breast carcinoma cells and were com-
pared with the cytotoxic activity of Melphalan and
Chlorambucil, chosen as references (Table 1). The parent
nitrogen mustard 1 containing a free hydroxylic function
showed a low cytotoxicity (IC50 870 mM) against A375
cell line whereas its O-acyl derivatives 2b–e exhibited a
Thus direct acylation47 of the commercially available
l-carnitine chloride 17 with the phenylacetyl and
Chlorambucil chlorides afforded the compounds 18 and
19 which unfortunately exhibited a very disappointing
lack of antitumoral activity against A375, HT29 and
MCF7 cell lines.
In summary, we have proposed a convenient multi-step
synthesis of new enantiopure nitrogen mustards which
mimic the (l)-carnitine framework. The broad anti-
tumor activities of the lipophilic compounds 2c,e
against A375, HT29 and MCF7 cell lines, appears pro-
mising for a future prospect in the design of new struc-
tural analogues. As nitrogen mustards are very
important anticancer drugs, our synthetic and biological
Table 1. In vitro cytotoxic activitiesa
Compound
Log Pb
A375
IC50 (mM)
HT29
IC50 (mM)
MCF7
IC50 (mM)
Melphalan
Chlorambucil
0.09
3.70
0.62
2.08
2.62
3.87
5.27
5.96
6.96
33
202
870
>1000
>1000
>1000
>1000
>1000
>1000
723
390
>1000
>1000
>1000
>1000
>1000
589
1
2a
2b
2d
2c
>1000
573
403
312
193
>1000
2e
16c
753
>1000
258
>1000
aThree independent experiments were realised for each compound.
bLog P values were calculated with ACD/log P calculator program.
Scheme 5. Reagents and conditions: (i) PhCH2COOH or Chlor-
ambucil, SOCl2, 60–80 ꢁC, 17.

328
L. Faissat et al. / Bioorg. Med. Chem. 11 (2003) 325–334
studies bring valuable breakthroughs for further work
in this area.
of the mixture concd H2SO4 (cat.)–H2O (120 mL), the
reaction was stirred for 3 h at rt, neutralized with a satd
soln of NaHCO3, extracted with diethyl ether, washed
with brine, dried over Na2SO4 and evaporated under
vacuum. Chromatography on silica gel as above affor-
Experimental
ded the title compound (1 g, 70%); Rf 0.7 (dichloro-
20
D
Elemental analyses were performed by the ‘Service de
Microanalyse de l’Ecole Superieure de Chimie de Mon-
tpellier’. 1H NMR spectra were determined on a Bruker
200 spectrometer (200 MHz frequency) and 13C NMR
spectra on a Bruker AC 400 spectrometer (100.6 MHz
frequency). Mass spectra were obtained with a JEOL
JMS-DX-300 by the FAB ionisation method (positive
mode) with p-nitrobenzyl alcohol (NBA) as the matrix;
Optical rotations were measured in a 0.1 dm cell on a
Perkin–Elmer 241 polarimeter. Petroleum spirit refers to
the fraction boiling in the range 40–65 ꢁC.
methane); ½aŠ + 7.0ꢁ Æ0.2 (c 5.0 chloroform) (lit.,15,16
no reported data); dH (200 MHz; CDCl3) 1.47 (9H, s, 4-
But), 1.50 (9H, s, 1-But), 2.65 (1H, d, J3 -2 5.7, J3 -3 16.5
0
0
Hz, 30-H), 2.76 (1H, d, J3-2 4.6, J3-3 16.5 Hz, 3-H), 3.23
0
0
(1H, d, JOH-2 5.7 Hz, OH) and 4.31 (1H, td, J2-3 5.7, J2-
OH 5.7, J2-3 4.6 Hz, 2-H); dC (100 MHz; CDCl3) 28.4 (3C,
CMe3), 28.5 (3C, CMe3), 40.3 (1C, 3-C), 67.9 (1C, 2-C),
81.8 (1C, 4-CMe3), 83.1 (1C, 1-CMe3), 170.2 (1C, 4-CO)
and 173.2 (1C, 1-CO); m/z (NBA) 247 [M+H]+, 269
[M+Na]+, 493 [2M+H]+, 515 [2M+Na]+ (Found: C,
58.4; H, 9.2. C12H22O5 requires C, 58.5; H, 9.0%).
(R)-tert-Butyl 3,4-dihydroxybutyrate 8. To the (R)-di-
tert-butyl malate 4 (5.15 g, 20.9 mmol) dissolved in
THF (130 cm3) was added borane dimethylsulfide (10
mol dmꢀ3 in THF) (2.3 cm3, 23.0 mmol). After 30 min
at rt sodium borohydride (39.7 mg, 1.0 mmol) was
added and the soln stirred until completion on tlc (4 h).
Addition of ethanol (50 cm3) and p-toluene sulfonic acid
(PTSA) (143 mg, 1.0 mmol) and stirring for 3 h was
followed by co-evaporation several times with benzene–
ethanol (1:1). Chromatography on silica gel with di-
chloromethane–methanol (9:1) as the eluent afforded
(R)-Di-tert-butyl malate 4 from the cyclic thioxocarbo-
nate 7.24 Method A. A mixture of 7 (0.2 g, 0.66 mmol),
tri-n-butyltin hydride (0.2 cm3, 0.72 mmol) and AIBN
(cat.) dissolved in benzene (1 cm3) were heated under
reflux under nitrogen for 1 h. To the soln cooled to rt
was added tert-butanol (10 cm3) and silica gel 60 (37–70
mm) (100 mg) then the solution was stirred for 30 min
and evaporated to dryness. The residue was chromato-
graphed on silica gel with dichloromethane–methanol
(99.5:0.5) as the eluent and the fractions were pooled,
evaporated and dissolved in acetonitrile, washed with
n-hexanes and concd under vacuum. Chromatography
on silica gel with dichloromethane–methanol (99.5:0.5)
as the eluent afforded the title compound (41 mg, 25%).
the title compound as an oil (2.2 g, 60%); Rf 0.42 [di-
20
D
chloromethane–methanol (95:5)]; ½aŠ + 20.7ꢁ Æ0.7 (c
1.5 chloroform); dH (200 MHz; CDCl3) 1.49 (9H, s,
But), 2.45 (1H, dd, J2-2 16.5, J2-3 4.8 Hz, 2-H), 2.51 (1H,
0
dd, J2 -2 16.5, J2 -3 7.7 Hz, 20-H); 3.52 (1H, dd, J4-4 11.3,
0
0
0
J4-3 6.2 Hz, 4-H), 3.68 (1H, dd, J4 -4 11.3, J4 -3 3.4 Hz, 40-
H) and 4.11 (1H, m, 3-H); dC (100 MHz; CDCl3) 27.3
(3C, CMe3), 37.5 (1C, 2-C), 64.7 (1C, 4-C), 67.5 (1C, 3-
C), 80.6 (1C, CMe3) and 171.2 (1C, CO); m/z (NBA) 177
[M+H]+, 199 [M+Na]+, 103 [M+HꢀOBut]+ (Found:
C, 54.7; H, 8.9. C8H16O4 requires C, 54.5; H, 9.1%).
0
0
Method B. A solution of thioxocarbonate 7 (50 mg,
0.16 mmol) and MgI2 (91.7 mg, 0.33 mmol) in acetoni-
trile (1.5 cm3) was refluxed for 45 min. To the reaction
was added at rt a satd soln of sodium bisulfide followed
by an extraction with dichloromethane. The organic
phase was washed with a satd solution of NaCl and dried
over Na2SO4 and concd under vacuum. Chromatography
as above afforded the title compound (13 mg, 32%).
(R)-tert-Butyl 3-hydroxy-4-bromobutyrate 10. To a stir-
red solution of p-toluenesulfonyl chloride (0.32 g, 1.7
mmol) in pyridine (1 cm3) was added at 0 ꢁC dropwise a
solution of (R)-tert-butyl 3,4-dihydroxybutyrateꢁ 8 (0.2
g, 1.1 mmol) in pyridine (1 cm3). After 24 h at 0 C, the
soln was concd under reduced pressure and extracted
with diethyl ether, washed with an aqueous soln of citric
acid (5%) and a satd soln of NaHCO3 until alkaline pH
and then with brine. The organic phase was dried over
Na2SO4 and concd under reduced pressure to give the
crude (R)-tert-butyl 3-hydroxy-4-tosyloxy butyrate 9
which was used further without purification. To the
crude tosylate dissolved in 2-butanone (5 cm3) was
added lithium bromide (395 mg, 4.5 mmol) and the soln
refluxed for 30 min. The mixture was diluted with di-
chloromethane and the soln washed with water and a
satd soln of NaCl and then dried over Na2SO4. After
evaporation of the solvents under reduced pressure the
crude product was chromatographed on silica gel with
petroleum ether–ethyl acetate (97:3) as the eluent to
From the cyclic sulfate 6.24. Method A. To the sulfate
(50 mg, 0.15 mmol) dissolved in DMA (2 cm3) was
added at 0 ꢁC sodium borohydride (5.8 mg, 0.15 mmol).
After 1 h at rt, the solution was evaporated to dryness.
THF (2 cm3) and a mixture of concd H2SO4 (cat.)–H2O
(2–3 mL) were added at 0 ꢁC. After 2 h at rt, a satd soln
of NaHCO3 was added until pH 8 and the aqueous
phase extracted with diethyl ether, the organic phase
washed with a saturated solution of NaCl, dried over
Na2SO4 and evaporated under vacuum. Chromato-
graphy on silica gel as above afforded the title compound
(21 mg, 56%).
Method B. To the sulfate (2 g, 6 mmol) dissolved in
THF (60 cm3) was added sodium cyanoborohydride
(tech., 85%) (0.91 g, 12.4 mmol). The soln was refluxed
for 15 h and after addition at 0 ꢁC of THF (40 cm3) and
afford the title compound as an oil (173 mg, 66%); Rf
0.53 [petroleum ether–ethyl acetate (85:15)]; ½aŠ
20
D
+

L. Faissat et al. / Bioorg. Med. Chem. 11 (2003) 325–334
329
15.2ꢁ Æ0.8 (c 1.4 chloroform); dH (200 MHz; CDCl3)
Preparation of O-acyl aminohydroxyesters 14d–e (mixed
anhydride method). To a cooled soln (0 ꢁC) of phenyl-
acetic acid or Chlorambucil (1.2 mmol) and triethyla-
mine (6.6 mmol) in dichloromethane (25 cm3) was
added dropwise 2,4,6-trichlorobenzoyl chloride (6.6
mmol). After 2 h at rt, hydroxyester 11 (5.5 mmol) in
dichloromethane (20 cm3) was added and the reaction
refluxed for 10 h, cooled and the organic phase washed
with a solution of NaHCO3 and brine then dried over
Na2SO4. After evaporation of the solvent, the crude oil
was chromatographed on silica gel with petroleum
ether–ethyl acetate (95:5) as the eluent to afford the
expected O-acyl hydroxyesters 14d–e.
1.48 (9H, s, But), 2.58 (1H, dd, J2-3 5.0, J2-2 16.7 Hz, 2-
0
H), 2.60 (1H, dd, J2 -3 7.1, J2 -2 16.7 Hz, 20-H), 3.45 (1H,
0
0
dd, J4 -3 5.5, J4 -4 10.4 Hz, 40-H), 3.52 (1H, dd, J4-3 5.14,
0
0
0
J4-4 10.4 Hz, 4-H) and 4.19 (1H, m, 3-H); dC (100 MHz;
CDCl3) 28.4 (3C, CMe3), 37.7 (1C, 4-C), 40.7 (1C, 2-C),
68.1 (1C, 3-c), 82.2 (1 C, CMe3) and 171.6 (1C, CO); m/
z (NBA) 239–241 [M+H]+ (Found: C, 40.0; H, 6.5.
C8H15BrO3 requires C, 40.2; H, 6.3%).
(R)-tert-Butyl 3,4-epoxybutyrate 3. A soln of (R)-tert-
butyl 3-hydroxy-4-bromobutyrate 10 (378 mg, 1.6
mmol) and Ag2O (375 mg, 1.6 mmol) in DME (5 cm3)
was refluxed for 12 h. The cooled soln was filtered and
concd under reduced pressure and the residue chroma-
tographed on silica gel with ethyl acetate (0–4%) in
petroleum ether as the eluent to give the title compound
(R)-tert-Butyl 3-acetyloxy-4-[N,N-bis(2-tert-butyldiphe-
nylsilyloxyethyl)amino]butyrate 14a. The title compound
was obtained according to the aforementioned proce-
as a colourless oil (92 mg, 37%); Rf 0.75 [petroleum
dure from acetic anhydride in 95% yield as an oil; Rf
20
D
20
D
ether–diethyl ether (85:15)]; ½aŠ + 5.2ꢁ Æ0.2 (c 5.3
0.76 [petroleum ether–diethyl ether (85:15)]; ½aŠ + 4.0ꢁ
20
chloroform), {lit.,33 ½aŠ + 4.7ꢁ (c 1.5 chloroform)}; dH
Æ0.7 (c 10.7 chloroform); dH (200 MHz; CDCl3) 1.04
(18H, s, 2ꢂSiBut), 1.41 (9H, s, CO2But), 1.95 (3H, s,
MeCO), 2.52–2.75 (4H, m, 2ꢂ2-H and 2ꢂ4-H), 2.72
(4H, t, JNCH CH O 6.4 Hz, 2ꢂCH2N), 3.64 (4H, t,
D
(200 MHz; CDCl3) 1.47 (9H, s, But), 2.40 (2H, m, 2ꢂ2-
H), 2.45–2.75 (2H, m, 2ꢂ4-H) and 3.15 (1H, m, 3-H); dC
(100 MHz; CDCl3) 27.8 (3C, CMe3), 39.0 (1C, 2-C),
46.4 (1C, 3-C), 48.0 (1C, 4-C), 80.9 (1C, CMe3) and
169.5 (1C, CO); m/z (NBA) 159 [M+H]+.
2
2
JOCH CH N 6.4 Hz, 2ꢂCH2O), 5.16 (1H, m, 3-H) and
2
7.38–27.67 (20H, m, aromatics); dC (100 MHz; CDCl3)
21.5 (1C, MeCO), 27.2 (6C, 2ꢂSiCMe3), 28.5 (3C,
CO2CMe3), 39.0 (1C, 2-C), 57.5 (2 C, 2ꢂCH2N), 58.5
(1C, 4-C), 62.9 (2C, 2ꢂCH2O), 69.9 (1C, 3-C), 81.0 (3C,
3ꢂCMe3), 128.1 (8C, Cm aromatics), 130.0 (4C, Cp aro-
matics), 134.1 (4C, Cq aromatics), 136.0 (8C, Co aro-
matics), 170.3 (1C, CO) and 170.5 (1C, CO); m/z (NBA)
782 [M+H]+ (Found: C, 70.8; H, 8.3. C46H63NO6Si2
requires C, 70.6; H, 8.1%).
(R)-tert-Butyl
3-hydroxy-4-[N,N-bis(2-tert-butyldiphe-
nylsilyloxyethyl)amino]butyrate 11. A stirred solution of
epoxyester 3 (100 mg, 0.63 mmol), bis(tert-butyldiphe-
nylsilyloxyethyl)amine (386 mg, 0.66 mmol) and silica
gel 60 (37–70 mm) (100 mg) in acetonitrile (10 cm3) was
refluxed for 12 h. After evaporation of the solvent under
reduced pressure, the residual oil was chromatographed
on silica gel with diethyl ether (0–5%) in petroleum
ether as the eluent to give the title compound as a col-
(R)-tert-Butyl 3-propionyloxy-4-[N,N-bis(2-tert-butyldi-
phenylsilyloxyethyl)amino]butyrate 14b. The title com-
pound was obtained according to the aforementioned
ourless oil (234 mg, 50%); Rf 0.62 [(petroleum ether–
20
D
ethyl acetate (85:15)]; ½aŠ ꢀ22.6ꢁ Æ0.7 (c 1.6 chloro-
form); dH (200 MHz; CDCl3) 0.97 (18H, s, 2ꢂSiBut),
procedure from propionic anhydride in 80% yield as an
20
D
1.40 (9H, s, CO2But), 2.25 (1H, dd, J2-3 5.5, J2-2 15.3
oil; Rf 0.62 [(petroleum ether–ethyl acetate (9:1)]; ½aŠ
0
Hz, 2-H), 2.39 (1H, dd, J2 -3 7.7, J2 -2 15.3 Hz, 20-H),
2.42 (1H, m, 40-H), 2.61 (1H, m, 4-H), 2.73 (4H, m,
2ꢂCH2N), 3.66 (4H, m, 2ꢂCH2O), 3.95 (1H, m, 3-H)
and 7.39–7.66 (20H, m, aromatics); dC (100 MHz;
CDCl3) 27.2 (6C, 2ꢂSiCMe3), 28.1 (3C, CO2CMe3),
41.1 (1C, 2-C), 57.0 (2C, 2ꢂCH2N), 61.2 (1C, 4-C), 62.5
(2C, 2ꢂCH2O), 66.0 (1C, 3-C), 81.0 (3C, 3ꢂCMe3),
128.1 (8C, Cm aromatics), 130.0 (4C, Cp aromatics),
134.1 (4C, Cq aromatics), 139.9 (8C, Co aromatics) and
171.5 (1C, CO); m/z (NBA) 740 [M+H]+ (Found: C,
71.1; H, 8.5. C44H61NO5Si2 requires C, 71.4; H, 8.3%).
+ 5.8ꢁ Æ0.2 (c 4.2 chloroform); dH (200 MHz; CDCl3)
1.02 (3H, t, J 7.5 Hz, Me), 1.03 (18H, s, 2ꢂSiBut), 1.39
(9H, s, CO2But), 2.20 (2H, q, J 7.5 Hz, CH2Me), 2.34
0
0
(1H, dd, J2-3 8.35, J2-2 15.6 Hz, 20-H), 2.49–2.73 (3H, m,
0
2-H et 2ꢂ4-H), 2.74 (4H, t, JNCH CH O 6.6 Hz, 2ꢂ
2
2
CH2N), 3.66 (4H, t, JOCH CH N 6.6 Hz, 2ꢂCH2O), 5.15
2
2
(1H, m, 3-H) and 7.30–7.60 (20H, m, aromatics); dC
(100 MHz; CDCl3) 9.5 (1C, Me), 27.3 (6C, 2ꢂSiCMe3),
28.1 (1C, COCH2), 28.5 (3C, CO2CMe3), 39.0 (1C, 2-C),
57.4 (2C, 2ꢂCH2N), 58.6 (1C, 4-C), 63.0 (2C,
2ꢂCH2O), 69.7 (1 C, 3-C), 81.0 (3C, 3ꢂCMe3), 128.1
(8C, Cm aromatics), 130.0 (4C, Cp aromatics), 134.1
(4C, Cq aromatics), 136.0 (8C, Co aromatics), 170.4 (1C,
CO) and 173.9 (1C, CO); m/z (NBA) 796 [M+H]+
(Found: C, 71.0; H, 8.4. C47H65NO6Si2 requires C, 70.9;
H, 8.2%).
General procedure for the preparation of O-acyl amino-
hydroxyesters 14a–c. To a cooled soln (0 ꢁC) of the
hydroxyester 11 (2.8 g, 3.8 mmol), triethylamine (1.06
cm3, 7.6 mmol) and DMAP (cat.) in dichloromethane
(55 cm3) was added dropwise anhydride (acetic, pro-
pionic, capryloyl) (5.7 mmol). The reaction was then
refluxed for 12 h, cooled and the organic phase washed
with a soln. of NaHCO3 and brine then dried over
Na2SO4. After evaporation of the solvent, the crude oil
was chromatographed on silica gel with ethyl acetate (0–
5%) in petroleum ether as the eluent to afford the
expected O-acyl hydroxyesters 14a–c.
(R)-tert-Butyl 3-capryloyloxy-4-[N,N-bis(2-tert-butyldi-
phenylsilyloxyethyl)amino]butyrate 14c. The title com-
pound was obtained according to the aforementioned
procedure from capryloyl anhydride in 85% yield as an
oil; Rf 0.44 [petroleum ether–ethyl acetate (95:5); ½aŠ
20
D
+ 3.7ꢁ Æ0.5 (c 1.9 chloroform); dH (200 MHz; CDCl3)
0.87 (3H, t, J 6.4 Hz, Me), 1.03 (18H, s, 2ꢂSiBut), 1.26

330
L. Faissat et al. / Bioorg. Med. Chem. 11 (2003) 325–334
(8H, m, 4ꢂCH2), 1.39 (9H, s, CO2But), 1.55 (2H, m,
CH2b), 2.19 (2H, t, J 7.7 Hz, CH2a), 2.29–2.81 (8H, m,
2ꢂ2-H, 2ꢂ4-H and 2ꢂCH2N), 3.63 (4H, t, JOCH CH N
(4C, Cq aromatics), 135.6 (8C, Co aromatics), 144.4 (1C, Cq
aromatic), 170.0 (1C, CO) and 172.6 (1C, CO); m/z
(NBA) 1025–1027 [M+H]+ (Found: C, 67.7; H, 7.6.
C58H78Cl2N2O6Si2 requires C, 67.9; H, 7.7%).
2
2
6.15 Hz, 2ꢂCH2O), 5.18 (1H, m, 3-H) and 7.38–7.63
(20H, m, aromatics); dC (100 MHz; CDCl3) 14.5 (1C,
Me), 23.0 (1C, CH2), 25.1 (1C, CH2b), 25.3 (1C, CH2),
27.0 (3C, SiCMe3), 27.2 (3C, SiCMe3), 28.5 (3C,
CO2CMe3), 29.3 (1C, CH2), 32.1 (1C, CH2), 34.9 (1C,
CH2a), 39.1 (1C, 2-C), 57.4 (2C, 2ꢂCH2N), 58.5 (1C, 4-
C), 62.9 (2C, 2ꢂCH2O), 69.6 (1C, 3-C), 80.9 (2C,
CMe3), 128.1 (8C, Cm aromatics), 130.1 (4C, Cp aro-
matics), 134.1 (4C, Cq aromatics), 136.0 (8C, Co aro-
matics), 170.4 (1C, CO) and 173.3 (1C, CO); m/z (NBA)
866 [M+H]+ (Found: C, 72.3; H, 8.9. C52H75NO6Si2
requires C, 72.1; H, 8.7%).
General procedure for the desilylation of silylated ethers
14a–e and for the chloration of bis(2-hydroxyethyl)amine
derivatives 15a–e. To a mixture of the disilylated ethers
14a–e (2.86 mmol) and imidazole (1.36 g, 20 mmol)
dissolved in THF (40 cm3), was added TBAF (1 mol
dmꢀ3 in THF) (17.2 cm3, 17.2 mmol) and the soln stir-
red at rt for 30 min then concd under reduced pressure.
The obtained crude material was quickly purified on
Florisil (60–100 mesh) with ethyl acetate (0–20%) in
petroleum ether as the eluent to afford the desired bis(2-
hydroxyethyl)amine derivatives 15a–e (characterised by
1H NMR) which are used immediately to avoid the easy
migration of the acyl groupfrom the 3-position to the
primary alcohols. To these later crude diols in di-
chloromethane (10 cm3) were added triethylamine (794
mL, 5.72 mmol), DMAP (419 mg, 3.43 mmol) and p-
toluenesulfonyl chloride (1.3 g, 6.86 mmol). The solu-
tion was stirred for 8 h at rt and concentrated under
reduced pressure to give a crude material which was
purified on Florisil (60–100 mesh) with ethyl acetate (0–
5%) in petroleum ether as the eluent to afford the
expected dichloro derivatives 16a–e.
(R)-tert-Butyl 3-phenylacetyloxy-4-[N,N-bis(2-tert-butyl-
diphenylsilyloxyethyl)amino]butyrate 14d. The title com-
pound was obtained according to the aforementioned
procedure from phenylacetic acid by the mixed anhy-
dride method in 76% yield as an oil; Rf 0.73 [petroleum
20
D
ether–ethyl acetate (9:1); ½aŠ +4.7ꢁ Æ0.2 (c 0.7 chloro-
form); dH (200 MHz; CDCl3) 1.05 (18H, s, 2ꢂSiBut),
1.37 (9H, s, CO2But), 2.37 (1H, dd, J2-3 8.35, J2-2 15.8
0
Hz, 20-H), 2.62 (3H, m, 2-H and 2ꢂ4-H), 2.69 (4H, m,
AA0BB0 system, JAB+JAB 12.5 Hz, 2ꢂCH2N), 3.52
0
(2H, s, COCH2), 3.62 (4H, m, AA0BB0 system, JBA
+
0
JBA 12.5 Hz, 2ꢂCH2O), 5.20 (1H, m, 3-H) and 7.19–
7.60 (25H, m, aromatics); dC (100 MHz; CDCl3) 27.2
(6C, 2ꢂSiCMe3), 28.4 (3C, CO2CMe3), 38.9 (1C, 2-C),
41.8 (1C, COCH2), 57.3 (2C, 2ꢂCH2N), 58.4 (1C, 4-C),
62.9 (2C, 2ꢂCH2O), 70.3 (1C, 3-C), 81.0 (3C, 3ꢂCMe3),
127.6 (2C, Cm aromatics), 128.1 (8C, Cm aromatics),
128.3 (2C, Cp aromatics), 129.6 (2C, Co aromatics),
130.0 (4C, Cp aromatics), 133.6 (1C, Cq aromatic), 134.1
(4C, Cq aromatics), 136.0 (8C, Co aromatics), 170.2 (1C,
CO) and 171.0 (1C, CO); m/z (NBA) 859 [M+H]+
(Found: C, 73.0; H, 8.2. C52H67NO6Si2 requires C, 72.8;
H, 7.9%).
(R)-tert-Butyl 3-acetyloxy-4-[N,N-bis(2-chloroethyl)ami-
no]butyrate 16a. The crude diol 15a was obtained
according to the aforementioned procedure as an oil; Rf
0.48 (ethyl acetate); dH (200 MHz; CDCl3) 1.46 (9H, s,
But), 2.10 (3H, s, MeCO), 2.49 (1H, dd, J2-3 6.9, J2-2
0
15.7 Hz, 2-H), 2.58 (1H, dd, J2 -3 5.9, J2 -2 15.7 Hz, 20-
H), 2.65–2.83 (6H, m, 2ꢂCH2N and 2ꢂ4-H), 3.63 (4H,
t, JOCH CH N 5.4 Hz, 2ꢂCH2O) and 5.36 (1H, m, 3-H);
0
0
2
2
the title compound was obtained according to the afore-
mentioned procedure in 51% yield (two steps) as an oil;
+
20
D
Rf 0.51 [petroleum ether–ethyl acetate (9/1)]; ½aŠ
0.9ꢁ Æ0.1 (c 21.7 chloroform); dH (200 MHz; CDCl3)
(R)-tert-Butyl 3-(4-{4-[bis(2-chloroethyl)amino]phenyl}-
butyryloxy)-4-[N,N-bis(2-tert-butyldiphenylsilyloxyethy-
l)amino]butyrate 14e. The title compound was obtained
according to the aforementioned procedure from
Chlorambucil by the mixed anhydride method in 52%
1.46 (9H, s, But), 2.06 (3H, s, MeCO), 2.49 (1H, dd, J2-3
0
0
0
7.2, J2-2 15.8 Hz, 2-H), 2.69 (1H, dd, J2 -3 5.6, J2 -2 15.8
Hz, 20-H), 2.76 (2H, d, J4-3 6.1 Hz, 2ꢂ4-H), 2.95 (4H, m,
AA0BB0 system, JAB + JAB’ 13.7 Hz, 2ꢂCH2N), 3.53
(4H, m, AA0BB0 system, JBA + JBA’ 13.7 Hz, 2ꢂCH2Cl)
0
yield as an oil; Rf 0.68 [petroleum ether–ethyl acetate
6.1ꢁ Æ0.2 (c 0.6 chloroform); dH
and 5.23 (1H, ddd, J3-2 7.2, J3-2 5.6, J3-4 6.1 Hz, 3-H);
20
D
(9:1); ½aŠ
+
(200 MHz; CDCl3) 1.02 (18H, s, 2ꢂSiBut), 1.38 (9H, s,
dC (100 MHz; CDCl3) 21.5 (1C, MeCO), 28.4 (3C,
CMe3), 38.5 (1C, 2-C), 42.3 (2C, 2ꢂCH2Cl), 57.6 (2C,
2ꢂCH2N), 57.8 (1C, 4-C), 69.5 (1C, 3-C), 81.5 (1C,
CMe3), 170.0 (1C, CO) and 170.6 (1C, CO); m/z (NBA)
342–344 [M+H]+ (Found: C, 50.2; H, 7.2.
C14H25Cl2NO4 requires C, 49.1; H, 7.4%).
CO2But), 1.83 (2H, m, CH2CH2CH2), 2.21 (2H, t,
0
JCH CH 7.2 Hz, COCH2), 2.34 (1H, dd, J2-3 8.35, J2-2
2
2
15.6 Hz, 20-H), 2.50 (2H, t, JCH CH 7.2 Hz, CH2Ph),
2
2
2.55–2.74 (7H, m, 2-H, 2ꢂ4-H and 2ꢂCH2N), 3.64 (10H,
m, 2ꢂNCH2CH2Cl and 2ꢂCH2O), 5.19 (1H, m, 3-H),
6.60 (2H, d, JCHCH 8.6 Hz, aromatics), 7.08 (2H, d,
JCHCH 8.6 Hz, aromatics) and 7.38–7.64 (20H, m, aro-
matics); dC (100 MHz; CDCl3) 26.7 (1C, CH2CH2CH2),
26.9 (6C, 2ꢂSiCMe3), 28.1 (3C, CO2CMe3), 33.7 (1C,
COCH2), 34.0 (1C, CH2Ph), 38.7 (1C, 2-C), 40.6 (2C,
2ꢂCH2Cl), 53.7 (2C, 2ꢂNCH2CH2Cl), 57.1 (2C,
2ꢂCH2N), 58.3 (1C, 4-C), 63.6 (2C, 2ꢂCH2O), 69.4
(1C, 3-C), 80.6 (3C, 3ꢂCMe3), 112.3 (2C, 2ꢂC aromatics),
127.8 (8C, Cm aromatics), 129.8 (2C, 2ꢂC aromatics),
129.9 (1C, Cq aromatic), 130.8 (4C, Cp aromatics), 134.9
(R)-tert-Butyl 3-propionyloxy-4-[N,N-bis(2-chloroethyl)-
amino]butyrate 16b. The crude diol 15b was obtained
according to the aforementioned procedure as an oil; Rf
0.46 [ethyl acetate–methanol (98:2)]; dH (200 MHz;
CDCl3) 1.13 (3H, t, JMe-CH 7.7 Hz, Me), 1.43 (9H, s,
2
But), 2.34 (2H, q, JCH -Me 7.7 Hz, CH2), 2.51 (2H, m,
2
2-H), 2.57–2.81 (6H, m, 2ꢂCH2N and 2ꢂ4-H), 3.60
(4H, m, AA0BB0 system, JAB+ JAB’ 10.3 Hz, 2ꢂCH2O)
and 5.33 (1H, m, 3-H). The title compound was obtained
according to the aforementioned procedure in 43%

L. Faissat et al. / Bioorg. Med. Chem. 11 (2003) 325–334
331
yield (two steps) as an oil; Rf 0.57 [petroleum ether–
COCH2), 42.3 (2C, 2ꢂCH2Cl), 57.4 (2C, 2ꢂCH2N),
57.6 (1C, 4-C), 70.0 (1C, 3-C), 81.5 (1C, CMe3), 127.5
(2C, Cm aromatics), 128.3 (1C, Cp aromatic), 129.7 (2C,
Co aromatics), 133.7 (1C, Cq aromatic), 169.9 (1C, CO)
and 170.5 (1C, CO); m/z (NBA) 418–420–422 [M+H]+
(Found: C, 57.2; H, 6.9. C20H29Cl2NO4 requires C, 57.4;
H, 7.0%).
20
D
ethyl acetate (85:15)]; ½aŠ + 3.8ꢁ Æ0.1 (c 9.3 chloro-
form); dH (200 MHz; CDCl3) 1.12 (3H, t, JMe-CH 7.6
2
Hz, Me), 1.43 (9H, s, But), 2.31 (2H, q, JCH -Me 7.6 Hz,
2
0
CH2CO), 2.47 (1H, dd, J2-3 7.2, J2-2 15.8 Hz, 2-H), 2.67
0
(1H, dd, J2 -3 5.4, J2 -2 15.8 Hz, 20-H), 2.75 (2H, d, J4-3
0
6.1 Hz, 2ꢂ4-H), 2.93 (4H, m, AA0BB0 system, JAB + JAB
0
13.8 Hz, 2ꢂCH2N), 3.50 (4H, m, AA0BB0 system, JBA
+
0
JBA 13.8 Hz, 2ꢂCH2Cl) and 5.20 (1H, m, 3-H); dC
(100 MHz; CDCl3) 9.5 (1C, Me), 28.1 (1C, COCH2), 28.4
(3C, CMe3), 38.5 (1C, 2-C), 42.3 (2C, 2ꢂCH2Cl), 57.6
(2C, 2ꢂCH2N), 57.7 (1C, 4-C), 69.4 (1C, 3-C), 81.4 (1C,
CMe3), 170.1 (1C, CO) and 174.0 (1C, CO); m/z (NBA)
356–358 [M+H]+, 378–380 [M+Na]+ (Found: C, 50.8;
H, 7.5. C15H27Cl2NO4 requires C, 50.6; H, 7.6%).
(R)-tert-Butyl 3-(4-{4-[bis(2-chloroethyl)amino]phenyl}-
butyryloxy-4-[N,N-bis(2-chloroethyl)amino]butyrate 16e.
The crude diol 15e was obtained according to the
aforementioned procedure as an oil; Rf 0.52 [ethyl ace-
tate–methanol (99:1)]; dHc (200 MHz; CDCl3) 1.45 (9H,
s, CO2But), 1.87 (2H, m, CH2CH2CH2), 2.32 (2H, t,
JCH CH 7.2 Hz, COCH2), 2.49–2.75 (10H, m, CH2Ph,
2
2
2ꢂ2-H, 2ꢂ4-H and 2ꢂCH2N), 3.65 (10H, m,
2ꢂNCH2CH2Cl and 2ꢂCH2O), 5.35 (1H, m, 3-H), 6.59
(2H, d, JCHCH 8.6 Hz, aromatics), 7.04 (2H, d, JCHCH
8.6 Hz, aromatics). The title compound was obtained
according to the aforementioned procedure in 23%
(R)-tert-Butyl 3-capryloyloxy-4-[N,N-bis(2-chloroethyl)-
amino]butyrate 16c. The crude diol 15c was obtained
according to the aforementioned procedure as an oil; Rf
0.54 [ethyl acetate–methanol (98:2)]; dH (200 MHz;
CDCl3) 0.86 (3H, m, Me), 1.26 (8H, m, 4ꢂCH2), 1.43
(9H, s, But), 1.60 (2H, m, CH2b), 2.31 (2H, t, J 7.5 Hz,
CH2a), 2.51 (2H, m, 2ꢂ2-H), 2.58–2.81 (6H, m,
2ꢂCH2N and 2ꢂ4-H), 3.60 (4H, t, JOCH CH N 5.2 Hz,
yield (two steps) as an oil; Rf 0.34 [petroleum ether–
20
ethyl acetate (9:1)]; ½aŠ + 2.5ꢁ Æ0.3 (c 3.7 chloroform);
D
dH(200 MHz; CDCl3) 1.42 (9H, s, CO2But), 1.88 (2H,
m, CH2CH2CH2), 2.29 (2H, t, JCH2CH2 7.2 Hz,
COCH2), 2.41–2.65 (4H, m, CH2Ph and 2ꢂ2-H), 2.73
(2H, d, J4-3 5.5 Hz, 2ꢂ4-H), 2.93 (4H, m, AA0BB0 sys-
2
2
2ꢂCH2O) and 5.34 (1H, m, 3-H). The title compound
was obtained according to the aforementioned proce-
dure in 34% yield (two steps) as an oil; Rf 0.37 [petro-
0
tem, JAB + JAB 13.8 Hz, 2ꢂCH2N), 3.50 (4H, m,
20
D
leum ether–ethyl acetate (9:1)]; ½aŠ + 3.8ꢁ Æ0.1 (c 8.2
AA0BB0 system, JBA + JBA 13.8 Hz, 2ꢂCH2Cl), 3.65
0
chloroform); dH (200 MHz; CDCl3) 0.91 (3H, m, Me),
1.25 (8H, m, 4ꢂCH2), 1.43 (9H, s, But), 1.58 (2H, m,
CH2b), 2.27 (2H, t, J 7.4 Hz, CH2a), 2.47 (1H, dd, J2-3
(8H, m, 2ꢂNCH2CH2Cl), 5.22 (1H, m, 3-H), 6.62 (2H,
d, JCHCH 8.6 Hz, aromatics), 7.06 (2H, d, JCHCH 8.6 Hz,
aromatics); dC (100 MHz; CDCl3) 26.6 (1C,
CH2CH2CH2), 28.0 (3 C, CMe3), 33.6 (1C, COCH2),
33.9 (1C, CH2Ph), 38.1 (1C, 2-C), 40.6 (2 C, 2ꢂCH2Cl),
41.8 (2C, 2ꢂCH2Cl), 53.6 (2C, 2ꢂNCH2CH2Cl), 57.1
(2C, 2ꢂCH2N), 57.3 (1C, 4-C), 69.0 (1C, 3-C), 81.0 (3C,
3ꢂCMe3), 112.2 (2C, 2ꢂCH aromatics), 127.0 (2C,
2ꢂCH aromatics), 129.7 (1C, Cq aromatic), 144.4 (1C,
Cq aromatic), 169.7 (1C, CO) and 172.7 (1C, CO); m/z
(NBA) 585–587–589 [M+H]+ (Found: C, 53.4; H, 7.1.
C26H40Cl4N2O4 requires C, 53.2; H, 6.9%).
0
0
0
7.0, J2-2 15.8 Hz, 2-H), 2.67 (1H, dd, J2 -3 5.5, J2 -2 15.8
Hz, 20-H), 2.74 (2H, d, J4-3 6.1 Hz, 2ꢂ4-H), 2.93 (4H, m,
AA0BB0 system, JAB + JAB 13.8 Hz, 2ꢂCH2N), 3.50
0
0
(4H, m, JBA + JBA 13.8 Hz, 2ꢂCH2Cl) and 5.24 (1H,
m, 3-H); dC (100 MHz; CDCl3) 13.0 (1C, Me), 22.7 (1C,
CH2), 23.9 (1C, CH2b), 27.0 (3 C, CMe3), 27.9 (1C,
CH2), 28.1 (1C, CH2), 30.6 (1C, CH2), 33.4 (1C, CH2a),
37.1 (1C, 2-C), 40.9 (2C, 2ꢂCH2Cl), 56.2 (2C,
2ꢂCH2N), 56.3 (1C, 4-C), 67.9 (1C, 3-C), 80.0 (1C,
CMe3), 168.6 (1C, CO) and 172.0 (1C, CO); m/z (NBA)
426–428 [M+H]+(Found: C, 56.0; H, 8.9.
C20H37Cl2NO4 requires C, 56.3; H, 8.7%).
(R)-3-Hydroxy-4-[N,N- bis(2 -chloroethyl)amino]butyric
acid 1. To the amino ester 16a (100 mg, 0.29 mmol)
dissolved in THF (0.3 cm3) was added anisole (31 mL,
0.29 mmol) and aqueous HCl (2.4 mol dmꢀ3) (3 cm3, 7.3
mmol) and the solution stirred at rt for 5 days. The
mixture was diluted with diethyl ether and the soln
washed with water. The resulting aqueous phase was
lyophilised and the obtained crude oil chromatographed
on silica gel (reversed phase C2) with THF–water (7:3)
(R)-tert-Butyl 3-phenylacetyloxy-4-[N,N-bis(2-chloroethyl)-
amino]butyrate 16d. The crude diol 15d was obtained
according to the aforementioned procedure as an oil; Rf
0.42 [ethyl acetate–methanol (99:1)]; dH (200 MHz;
CDCl3) 1.41 (9H, s, CO2But), 2.38–2.69 (8H, m, 2ꢂ2-H,
2ꢂ4-H and 2ꢂCH2N), 3.54 (4H, t, JOCH CH N 5.3 Hz,
2
2
2ꢂCH2O), 3.64 (2H, s, COCH2), 5.35 (1H, m, 3-H) and
7.34–7.48 (5H, m, aromatics). The title compound was
obtained according to the aforementioned procedure in
as the eluent to afford the title compound (54 mg, 76%);
Rf 0.63 [acetone–ethyl acetate–water (2:4:1)]; ½aŠ
20
D
ꢀ29.6ꢁ Æ0.2 (c 5.4 water); dH (200 MHz; D2O) 2.48
0 0
(1H, dd, J2-3 7.2, J2-2 16.5 Hz, 2-H), 2.59 (1H, dd, J2 -3
22% yield (two steps) as an oil; Rf 0.40 [petroleum
20
D
ether–ethyl acetate (9:1)]; ½aŠ + 1.2ꢁ Æ0.1 (c 2.2
5.1, J2 -2 16.5 Hz, 20-H), 3.31 (1H, dd, J4-3 10.3, J4-4 13.6
0
0
chloroform); dH (200 MHz; CDCl3) 1.41 (9H, s, But),
Hz, 4-H), 3.44 (1H, dd, J4 -3 2.7, J4 -4 13.6 Hz, 40-H),
0
0
0
0
2.47 (1H, dd, J2-3 7.2, J2-2 16.0 Hz, 2-H), 2.66 (1H, dd,
0
3.68 (4H, m, AA0BB0 system, JAB+JAB 11.2 Hz,
J2 -3 5.7, J2 -2 16.0 Hz, 20-H), 2.72 (2H, d, J4-3 6.0 Hz,
2ꢂCH2N), 3.89 (4H, m, AA0BB0 system, JBA+JBA 11.2
0
0
2ꢂ4-H), 2.86 (4H, m, AA0BB0 system, JAB+JAB 13.8
Hz, 2ꢂCH2Cl) and 4.41 (3H, m, 3-H); dC (100 MHz;
D2O) 37.5 (2C, 2ꢂCH2Cl), 39.5 (1C, 2-C), 55.7 (2C,
2ꢂCH2N), 58.0 (1C, 4-C), 62.2 (1C, 3-C) and 174.3 (1C,
CO); m/z (NBA) 244–246 [M+H]+ (Found: C, 39.6; H,
6.1. C8H15Cl2NO3 requires C, 39.4; H, 6.2%).
0
Hz, 2ꢂCH2N), 3.43 (4H, m, AA0BB0 system, JBA+JBA
0
13.8 Hz, 2ꢂCH2Cl), 3.60 (2H, s, COCH2), 5.23 (1H, m,
3-H) and 7.20–7.40 (5H, m, aromatics); dC (100 MHz;
CDCl3) 28.4 (3C, CMe3), 38.4 (1C, 2-C), 41.80 (1C,

332
L. Faissat et al. / Bioorg. Med. Chem. 11 (2003) 325–334
General procedure for the acidic hydrolysis of tert-butyl
diesters 16a–e
CH2a), 37.1 (1C, 2-C), 41.4 (2C, 2ꢂCH2Cl), 57.2 (2C,
2ꢂCH2N), 57.5 (1C, 4-C), 68.4 (1C, 3-C), 173.7 (1C,
CO) and 175.5 (1C, CO); m/z (NBA) 370–372 [M+H]+,
392–394 [M+Na]+ (Found: C, 51.8; H, 7.8.
C16H29Cl2NO4 requires C, 51.9; H, 7.9%).
To the amino esters 16a–e (0.24 mmol) dissolved in di-
chloromethane (1.5 cm3) were added anisole (26 mL,
0.24 mmol) and trifluoroacetic acid (185 mL, 2.41
mmol). The solution was stirred at rt for 10 h, concd
under reduced pressure, co-evaporated with cyclohex-
ane and the obtained crude residue was chromato-
graphed on silica gel with ethyl acetate as the eluent to
afford the corresponding amino acids 2a–e as colourless
oils.
(R)-3-Phenylacetyloxy-4-[N,N-bis(2-chloroethyl)amino]-
butyric acid 2d. The title compound was obtained
according to the aforementioned procedure in 71%
yield as an oil; Rf 0.57 [petroleum ether–ethyl acetate
+15.5ꢁ Æ0.4ꢁ (c 0.3 chloroform); dH
20
D
(400 MHz; CDCl3) 2.57 (1H, dd, J2-3 7.2, J2-2 16.4 Hz,
(4:6)]; ½aŠ
0
2-H), 2.65 (1H, dd, J2 -3 6.2, J2 -2 13.8 Hz, 40-H), 2.72
0
0
0
0
(R)-3-Acetyloxy-4-[N,N-bis(2-chloroethyl)amino]butyric
acid 2a. The title compound was obtained according
to the aforementioned procedure in 76% yield as an
(1H, dd, J2-3 6.0, J2-2 13.8 Hz, 4-H), 2.78 (1H, dd, J2 -3
0
5.4, J2 -2 16.4 Hz, 2-H), 2.82 (4H, m, AA0BB0 system,
JAB+JAB 13.5 Hz, 2ꢂCH2N), 3.38 (4H, m, AA0BB0
0
20
0
oil; Rf 0.64 [petroleum ether–ethyl acetate (4:6)]; ½aŠ
system, JBA+JBA 13.5 Hz, 2ꢂCH2Cl), 3.55 (2H,
D
20
D
ꢀ17.3Æ0.9ꢁ (c 1.1 water); ½aŠ +1.3ꢁ Æ0.2 (c 3.9
COCH2), 5.20 (1H, m, 3-H) and 7.17–7.28 (5H, m, aro-
matics); dC (100 MHz; CDCl3) 36.9 (1C, 2-C), 41.8 (1C,
COCH2), 42.2 (2C, 2ꢂCH2Cl), 57.3 (2C, 2ꢂCH2N),
57.4 (1C, 4-C), 69.5 (1C, 3-C), 127.6 (2C, Cm aromatics),
129.0 (1C, Cp aromatic), 129.6 (2C, Co aromatics), 134.0
(1C, Cq aromatic), 171.3 (1C, CO) and 175.9 (1C, CO);
m/z (NBA) 362–364 [M+H]+ (Found: C, 53.3; H, 6.0.
C16H21Cl2NO4 requires C, 53.0; H, 5.8%).
chloroform); dH (200 MHz; CDCl3) 2.03 (3H, s, Me),
0
2.65 (2H, dd, J2-3 6.4, J2-2 16.5 Hz, 2-H), 2.82 (2H,
0
dd, J2 -3 6.0, J2 -2 16.5 Hz, 20-H), 2.95 (2H, m, 2ꢂ4-
0
H), 3.09 (4H, m, AA0BB0 system, JAB+JAB 13.3 Hz,
0
2ꢂCH2N), 3.59 (4H, m, AA0BB0 system, JBA+JBA
0
13.3 Hz, 2ꢂCH2Cl) and 5.28 (1H, m, 3-H); dC
(100 MHz; D2O) 20.8 (1C, MeCO), 37.0 (1C, 2-C),
37.7 (2C, 2ꢂCH2Cl), 56.5 (1C, 4-C), 56.0 (2C,
2ꢂCH2N), 66.2 (1C, 3-C), 173.7 (2C, 1-C and
MeCO); m/z (NBA) 286–288 [M+H]+(Found: C,
42.2; H, 6.1. C10H17Cl2NO4 requires C, 42.0; H,
6.0%).
(R)-3-(4-{4-[Bis(2-chloroethyl)-amino]phenyl}butyryloxy-
4-[N,N-bis(2-chloroethyl)amino]butyric acid 2e. The title
compound was obtained according to the aforemen-
tioned procedure in 87% yield as an oil; Rf 0.48 [petro-
20
D
leum ether–ethyl acetate (4:6)]; ½aŠ + 6.9ꢁ Æ0.2ꢁ (c 0.8
(R)-3-Propionyloxy-4-[N,N-bis(2-chloroethyl)amino]buty-
ric acid 2b. The title compound was obtained according
to the aforementioned procedure in 67% yield as an
chloroform); dH (200 MHz; CDCl3) 1.81 (2H, m,
CH2CH2CH2), 2.27 (2H, t, JCH CH 7.4 Hz, COCH2),
2.47 (2H, t, JCH CH 7.4 Hz, CH22Ph), 2.62 (1H, dd, J2-3
2
2
2
20
D
0
0
0
oil; Rf 0.72 [petroleum ether–ethyl acetate (4:6)]; ½aŠ
6.1, J2-2 16.7 Hz, 2-H), 2.73 (1H, dd, J2 -3 6.5, J2 -2 16.7
Hz, 20-H), 3.04 (2H, d, J4-3 5.8 Hz, 2ꢂ4-H), 3.17 (4H, m,
2ꢂCH2N), 3.55 (4H, m, 2ꢂCH2Cl), 3.60 (8H, m,
2ꢂNCH2CH2Cl), 5.25 (1H, m, 3-H), 6.57 (2H, d, JCHCH
8.6 Hz, aromatics) and 7.00 (2H, d, JCHCH 8.6 Hz, aro-
matics); dC (100 MHz; CDCl3) 26.8 (1C, CH2CH2CH2),
33.9 (1C, COCH2), 34.2 (1C, CH2Ph), 37.0 (1C, 2-C),
40.3 (2C, 2ꢂCH2Cl), 40.9 (2C, 2ꢂCH2Cl), 54.1 (2C,
2ꢂNCH2CH2Cl), 56.8 (2C, 2ꢂCH2N), 57.3 (1C, 4-C),
67.7 (1C, 3-C), 112.8 (2C, 2ꢂCH aromatics), 130.1 (2C,
2ꢂCH aromatics), 130.9 (1C, Cq aromatic), 144.6 (1C,
Cq aromatic), 173.5 (1C, CO) and 174.1 (1C, CO); m/z
(NBA) 529 [M+H]+ (Found: C, 49.5; H, 5.8.
C22H32Cl4N2O4 requires C, 49.8; H, 6.1%).
+ 6.5ꢁ Æ0.7 (c 1.4 chloroform); dH (200 MHz; CDCl3)
1.11 (3H, t, JMe-CH 7.7 Hz, Me), 2.33 (2H, q, JCH -Me
0
2
2
7.7 Hz, COCH2), 2.66 (1H, dd, J2-3 6.4, J2-2 16.2 Hz, 2-
H), 2.81 (1H, dd, J2 -3 6.0, J2 -2 16.2 Hz, 20-H), 3.00 (2H,
0
0
d, J4-3 6.4 Hz, 2ꢂ4-H), 3.13 (4H, m, AA0BB0 system,
JAB+JAB 13.2 Hz, 2ꢂCH2N), 3.61 (4H, m, AA0BB0
0
0
system, JBA+JBA 13.2 Hz, 2ꢂH2Cl) and 5.28 (1H, m,
3-H); dC (100 MHz; CDCl3) 9.3 (1C, Me), 28.0 (1C,
COCH2), 37.0 (1C, 2-C), 41.3 (2C, 2ꢂCH2Cl), 57.1 (2C,
2ꢂCH2N), 57.4 (1C, 4-C), 68.4 (1C, 3-C), 171.8 (1C,
CO) and 174.4 (1C, CO); m/z (NBA) 300–302 [M+H]+,
322–324 [M+Na]+ (Found: C, 43.8; H, 6.3.
C11H19Cl2NO4 requires C, 44.0; H, 6.4%).
(R)-3-Capryloyloxy-4-[N,N-bis(2-chloroethyl)amino]bu-
tyric acid 2c. The title compound was obtained accord-
ing to the aforementioned procedure in 50% yield as an
(R)-(3-Carboxy-2-phenylacetoxypropyl)trimethylammo-
nium chloride 18. A mixture of phenyl acetic acid (506
mg, 3.7 mmol) and thionyl chloride (135 mL, 1.9 mmol)
was warmed for 40 min at 80 ꢁC. Then l-carnitine
chloride 17 (300 mg, 1.9 mmol) was added and the
reaction maintained at the same temperature for 45 min.
After concentration under vacuum, the crude residue
was chromatographed on silica gel (reverse-phase C2)
with water–THF (65:35) as the eluent to afford the title
compound as a colourless oil (426 mg, 72%); Rf 0.37
20
oil; Rf 0.73 [petroleum ether–ethyl acetate (4:6)]; ½aŠ
+
D
9.0ꢁ Æ0.4ꢁ (c 2.3 chloroform); dH (200 MHz; CDCl3)
0.81 (3H, t, J 7.0 Hz, Me), 1.21 (8H, m, 4ꢂCH2), 1.53
(2H, m, CH2b), 2.23 (2H, t, J 7.6 Hz, CH2a), 2.60 (1H,
0
0
dd, J2-3 6.4, J2-2 16.4 Hz, 2-H), 2.77 (1H, dd, J2 -3 5.4,
0
J2 -2 16.4 Hz, 20-H), 2.85 (2H, d, J4-3 6.4 Hz, 2ꢂ4-H),
3.00 (4H, m, AA0BB0 system, JAB+JAB 12.8 Hz,
0
2ꢂCH2N), 3.51 (4H, m, AA0BB0 system, JBA+JBA 12.8
[acetone–ethyl acetate—water–acetic acid (2:2:1:2.5)];
0
20
D
2.70 (1H, dd, J2-3 7.1, J2-2 17.0 Hz, 2-H), 2.79 (1H, dd,
Hz, 2ꢂCH2Cl) and 5.19 (1H, m, 3-H); dC (100 MHz;
CDCl3) 14.5 (1C, Me), 23.0 (2C, 2ꢂCH2), 25.2 (1C,
CH2b), 29.3 (1C, CH2), 32.0 (1C, CH2), 34.7 (1C,
½aŠ ꢀ29.5ꢁ Æ0.4ꢁ (c 2.8 water); dH (200 MHz; CD3OD)
0
J2 -3 5.4, J2 -2 17.0 Hz, 40-H), 2.93 (9H, s, 3ꢂNMe), 3.72
0
0

L. Faissat et al. / Bioorg. Med. Chem. 11 (2003) 325–334
333
(2H, s, COCH2), 3.67 (1H, m, 40-H), 3.83 (1H, dd, J4-3
0
8.51, J4-4 14.5 Hz, 4-H), 5.57 (1H, m, 3-H) and 7.21–
surface area was 50% when compared to untreated
cells.
7.37 (5H, m, aromatics); dC (100 MHz; CD3OD) 36.9
(1C, 2-C), 41.1 (1C, COCH2), 53.5 (3C, 3ꢂNCH3), 65.9
(1C, 3-C), 68.2 (1C, 4-C), 127.5 (2C, Cm aromatics),
128.8 (1C, Cp aromatic), 129.6 (2C, Co aromatics), 133.8
(1C, Cq aromatic), 171.0 (1C, CO) and 171.4 (1C,
CO2H); m/z (NBA) 280 [M]+(Found: C, 57.2; H, 7.1.
C15H22ClNO4 requires C, 57.0; H, 7.0%).
Acknowledgements
We are grateful to the Ministere de la Recherche et de la
Technologie and C.N.R.S for financial support of this
research. L. F. is pleased to acknowledge Mayoly
Spindler Company and ‘la Region Languedoc Roussil-
lon’ for providing a grant. We thank P. Cuq, A. Evrard
and L. Vian of the Laboratoire de Toxicologie du
Medicament (Universite de Montpellier I) for their
technical support during the biological testing.
(R) - [2 - (4 - {4 - [Bis(2 - chloroethyl)amino]phenyl}butyryl-
oxy)-3-carboxypropyl]trimethylammonium chloride 19.
A mixture of Chlorambucil (676 mg, 2.2 mmol) and
thionyl chloride (135 mL, 1.9 mmol) was warmed for 15
min at 60 ꢁC. Then l-carnitine chloride 17 (300 mg, 1.9
mmol) and trichloroacetic acid (450 mg) were added
and the reaction maintained at the same temperature for
2 h. After concn under vacuum, the crude residue was
chromatographed on silica gel (reverse-phase C2) with
water–THF (65:35) as the eluent to afford the title com-
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