Synthesis and biological evaluation of cyclic nitrogen mustards based on carnitine framework

Article abstract of DOI:10.1016/j.ejmech.2010.06.003

Two series of cyclic nitrogen mustards structurally related to l-carnitine have been prepared. The cytotoxic activity of these compounds was evaluated by using Chlorambucil as a reference. In accordance with earlier report, the cytotoxicity is in direct correlation with the lipophilicity of the introduced alkyl chains. Among the cyclic nitrogen mustards synthesized, the most cytotoxic compounds were the one acylated with a palmitoyl side chain, which showed activities comparable to that of Chlorambucil.

Full text of DOI:10.1016/j.ejmech.2010.06.003

European Journal of Medicinal Chemistry 45 (2010) 4140e4148
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and biological evaluation of cyclic nitrogen mustards based
on carnitine framework
*
Simon Leiris, Marc Lucas, Arnaud Dupuy d’Angeac, Alain Morère
Institut des Biomolécules Max Mousseron (IBMM), UMR 5247, CNRS, Université Montpellier 2 et 1, Bâtiment de Recherche Max Mousseron, Ecole Nationale Supérieure
de Chimie de Montpellier, 8 Rue de l’Ecole Normale, F-34296 Montpellier Cedex 05, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Two series of cyclic nitrogen mustards structurally related to L-carnitine have been prepared. The
Received 13 March 2010
Received in revised form
28 May 2010
Accepted 2 June 2010
Available online 9 June 2010
cytotoxic activity of these compounds was evaluated by using Chlorambucil as a reference. In accordance
with earlier report, the cytotoxicity is in direct correlation with the lipophilicity of the introduced alkyl
chains. Among the cyclic nitrogen mustards synthesized, the most cytotoxic compounds were the one
acylated with a palmitoyl side chain, which showed activities comparable to that of Chlorambucil.
Ó 2010 Elsevier Masson SAS. All rights reserved.
Keywords:
Cyclic nitrogen mustard
L-Carnitine
Mitochondria
1. Introduction
activities on MCF-7, H460 and A375 cell lines comparable to that of
chlorambucil. As cyclic analogues of
very effective mimics of -carnitine, we designed and prepared new
cyclic nitrogen mustards based on -carnitine specifically aimed at
tumor cell mitochondrial targeting. Indeed, the over-expression of
the -carnitine transporter in cancer cells provides an opportunity
L-carnitine were shown to be
Nitrogen mustards like Chlorambucil are commonly used as
anticancer agents. These cytotoxic compounds are believed to exert
their biological activity by inducing interstrand cross-links in the
major groove of DNA. This linkage represents the most toxic of all
alkylation events [1,2]. During the past years, many nitrogen
mustard structural modifications have been envisioned to increase
their cytotoxicity and especially their specificity towards tumor
cells [3]. A number of reports indicate that mitochondrial DNA are
over-expressed in tumor cells making them an attractive loci for
selective tumor cell targeting using multiple classes of DNA
damaging agents [4e6]. These findings have led to the mitochon-
drial DNA being targeted for antitumoral treatment [7e10]. We
L
L
L
to selectively alkylate mitochondrial DNA of tumor cells [12,13].
2. Targets design
We herein report the synthesis of two series of racemic cyclic
nitrogen mustards 1aed and 2aed (Fig. 1) structurally related to
L-carnitine, where the original trimethylammonium moiety is
replaced by a bis(2-chloroethyl)amine. We believed that the in vivo
protonation of the bis(2-chloroethyl)amino group or the nitrogen
quaternarisation during the formation of the requisite alkylating
aziridinium cation will give a positive ion corresponding to the
trimethylammonium moiety of carnitine.
The first series of nitrogen mustards 1aed was designed on the
1,2- and 2,3-trans relative configurations of the tetramethy-
lammonium and the hydroxyl group along with the hydroxyl and
the carboxylic acid moiety of cyclic carnitine. Another series of
mustards (2aed), which differ from 1aed by different relative
configuration was also designed and prepared. Biological studies
would shed more light on the effect of this configuration on the
cytotoxic activity of these compounds. As in our previous study,
acyl groups with various saturated carbon chains were introduced
on these mustards to study the impact of chain length on
hypothesized that
L-carnitine, an essential cofactor that transfers
long chain fatty acids to the mitochondria for
b-oxidation, could be
considered as an appropriate vector to target these organelles. Our
work is based on previous research carried out in our laboratory
concerning nitrogen mustard derivatives of
L-carnitine, for which
the trimethylammonium moiety of -carnitine was replaced by the
L
appropriate bis(2-chloroethyl) amine group of nitrogen mustard
[11]. The alcohol function of the mustard-containing carnitine was
acylated by alkyl chains of various lengths (acetyl, propionyl,
palmitoyl.). The acylated mustards showed high cytotoxic
* Corresponding author. Tel.: þ33 467 14 43 45; fax: þ33 467 14 43 44.
E-mail address: alain.morere@univ-montp2.fr (A. Morère).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.06.003

S. Leiris et al. / European Journal of Medicinal Chemistry 45 (2010) 4140e4148
4141
silica gel-mediated epoxide opening of 4 (Scheme 2) did not give
any reaction. This problem was overcome by using lithium triflate
LiOTf, well known to favour the regioselective opening of epoxides
[16]. In this way the g-amino-b-hydroxyester 9 was obtained in 75%
yield. At this stage, different acyl groups (acetyl, capryloyl and
palmitoyl) were introduced using appropriate anhydrides or acid
chlorides. The expected O-acyl aminohydroxyesters 10aec were
obtained in yields ranging from 78 to 97%.
The cleavage of the silyl ethers 10aec was performed by using
TBAF and imidazole in THF (Scheme 2) [11,17]. However, the
obtained bis(2-hydroxyethyl)amine derivatives were prone to very
fast acyl migration or degradation on standing and appeared to be
really sensitive to silica gel acidity. These difficulties were circum-
vented by flash chromatography on florisil and immediate
conversion of the dihydroxyl compounds to their corresponding
dichloro derivatives 11aec. The chlorination of diols was performed
using p-TsCl in presence of TEA and DMAP [11,18,19], leading to
chlorinated compounds 11aec in quite good yields (42e65% over
two steps) for such derivatives (Scheme 2.). The obtained dichloro-
compounds, unstable and sensitive to silica gel, were quickly
purified by chromatography on a florisil column. The final acidic
hydrolysis step, gave, according to the experimental conditions,
either the fully deprotected nitrogen mustard 2d (20% aq HCl/
anisole) [11,20,21] (41% yield) or the O-acyl nitrogen mustards 2aec
(TFA/anisole) [11,22e25] (62e79% yields) (Scheme 2.).
Fig. 1. Synthetic targets 1aed and 2aed.
cytotoxicity. We anticipate that the activity should increase with
the lipophilicity of these molecules by improving their cell
penetration capacity.
The soft reaction conditions leading to regioselective opening of
cis-epoxide 4 were unsuccessful on the trans-epoxide 3. Stronger
conditions were required to obtain
g-amino-b-hydroxyester 12
3. Synthetic studies
finally in 25% yield (Scheme 3). Acylation of 12 by the procedure
described above led to compounds 13aec. These were then
converted to the corresponding mustards 14aec by silyl cleavage
followed by chlorination in good yields (42e65% over two steps)
(Scheme 3). The low yields associated with the synthesis of 12 led
us to consider the opening of 3 by using a less sterically hindered
nucleophile, such as NaN3 [26]. This reaction was not regioselective,
Our retrosynthetic approach (Fig. 2) follows the synthetic
pathway reported for the preparation of cyclic
L-carnitine
analogues [12,13]. The key steps are the regioselective openings of
the trans-epoxide 3 and cis-epoxide 4 which allow the preparation
of the two series of targeted molecules. Trans- and cis-epoxides (3
and 4) were prepared by a multi-step sequence from methyl ester 7,
synthesized from 3-bromocyclohexene following the synthetic
pathway reported by Hutchison et al. (Scheme 1) [12,13]. The
nucleophilic substitution of 3-bromocyclohexene by KCN in pres-
ence of 18-crown-6 gave nitrile 6 in good yield (75%) [13,14]. The
corresponding methyl ester 7 was obtained in almost quantitative
yield by the treatment of 6 with methanolic HCl [14,15]. According
to Davies and Whitham’s procedure, the tert-butyl ester 5 was then
prepared by acidic hydrolysis of 7 with APTS, leading quantitatively
to carboxylic acid 8, followed by treatment with isobutylene in
catalytic presence of H2SO4 [14]. The desired cis and trans-epoxides
3 and 4 were prepared in 3:2 ratio by the usual epoxidation
procedure using m-CPBA in CH2Cl2. The two stereoisomers were
successfully purified and separated by chromatography on silica gel
with a global yield of 71%.
since a 3:2 mixture of expected
g-azido-b-hydroxyester 15 and its
regioisomer -azido- -hydroxyester was obtained. Purified 15 was
b
g
acylated to afford 16aec in yields ranging from 75 to 98%. Azides
16aec were reduced with H2 and Pd/C to give the corresponding
amines which were immediately used for the next step due to their
unstable nature. The bis(2-hydroxyethyl)amine derivatives were
obtained by reaction with ethylene oxide in a sealed tube [27e32].
The obtained unstable diols were then converted to their corre-
sponding chloro derivatives by the conditions described above.
After purification on florisil the chloro derivatives 14 aec were
obtained in 39e58% yields from azides 16aec. Fully deprotected
nitrogen mustard 1d (20% aq HCl/anisole) (47% yield) or the O-acyl
nitrogen mustards 1aec (TFA/anisole) (65e75% yields) were
successfully obtained by the same procedure used for the prepa-
ration and isolation of compounds 2aed.
The nitrogen mustards 2aed were obtained by regioselective
ring-opening of the cis-epoxide 4 with bis(tert-butyldiphenylsily-
loxyethyl)-amine, prepared previously in our laboratory [11]. The
4. Biological studies and discussion
The cytotoxicities of the prepared nitrogen mustards 1aed and
2aed were evaluated on two tumor cell lines: A375 (human
melanoma) and H460 (adenocarcinoma) (Table 1 and Fig. 3). O-acyl
derivatives 1bec and 2bec exhibited significant cytotoxic activity,
especially 1c and 2c with CC50 respectively of 164
210 M (H460) and 310 M (A375)/298 M (H460), which are
comparable to that of Chlorambucil (CC50 ¼ 127 M for A375 and
M for H460). A fairly good correlation appears between
mM (A375)/
m
m
m
m
CC50 ¼ 104
m
the antitumor activity of compounds 1aec and 2aec and the lip-
ophilicity of their alkyl chains; the presence of either a long alkyl
chain (C7H15, 1b or 2b) or a very lipophilic moiety (C15H31, 1c or 2c)
proved to be effective in improving the cytotoxic activity (the
Fig. 2. Retrosynthetic analysis.

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S. Leiris et al. / European Journal of Medicinal Chemistry 45 (2010) 4140e4148
Scheme 1. Synthesis of epoxides 3 and 4. Reagents and conditions: (a) KCN, 18-crown-6, CH2Cl2, r.t., 4 days, 75%; (b) HCl, MeOH, reflux, 2 h, 95%; (c) APTS, 9:1 dioxane/H2O, reflux,
overnight, 100%; (d) Isobutylene, H2SO4, CH2Cl2, r.t., 12 h, 90%; (e) m-CPBA, CH2Cl2, 25 ꢀC, 12 h, 4 (28%) and 3 (42%).
cytotoxic activity increased by a factor of four) when compared
with acetylated products 1a and 2a. These results could suggest
that the nitrogen mustards are entering the cells by passive diffu-
sion as it was proposed previously [11]. However, it seems that
compounds 1 of the “trans” series (with stereochemistry corre-
6. Experimental
6.1. Chemistry
Chemical and solvents were of reagent grade or distilled using
the procedures described in “D.D. Perrin et W.L.F. Amarego,
“Purification of Laboratory Chemicals”, Pergamon Press, 3rd edition,
London, 1988”. Anhydrous grade solvents were purchased from
Aldrich and VWR. All reactions involving air or moisture sensitive
reagents or intermediates were performed under an argon atmo-
sphere. Chromatographic separations were performed on Carlo
sponding to the -carnitine) are more potent than compounds 2 of
L
the “cis” series. Moreover, cyclic nitrogen mustard with free
hydroxyl 1d, which displays the same relative configuration as
cyclic carnitine, showed relative good cytotoxic activities for H460
cell line (CC50 304 mM) compared to its stereoisomer 2d which was
not significantly cytotoxic.
Therefore, these results could suggest an active transport into
Erba (Silica Gel 60A, 35e70 mm) or C18 reverse phase silica gel.
the cell possibly by
hypothesis, further experiments with labelled compounds 1 and 2
are planned and will be published in due course.
L
-carnitine transporters. In order to verify this
Elemental analyses were carried out by the service de microanal-
yses du CNRS, division de Vernaison (France). TLC was performed
on Merck 60 F354 (art. 5554) brand silica gel plates that were
visualized by irradiation (254 nm) or by staining with p-anisalde-
hyde stain. 1H and 13C NMR spectra were obtained using a Bruker
DRX-400 (400 MHz) instrument. Chemical shifts were reported in
5. Conclusion
parts per million (ppm, d
) referenced to the residual 1H resonance
In conclusion we synthesized two series of cyclic nitrogen
of the solvents (CDCl3, 7.26 ppm). 13C spectra were referenced to the
residual 13C resonance of the solvent (CDCl3, 77 ppm). Samples
recorded in D2O were referenced to either H2O (1H NMR) or
dioxane (13C NMR). Splitting patterns were designated as follow: s,
singlet; br, broad; d, doublet; dd, doublet of doublets; t, triplet; q,
quartet; m, multiplet. Elemental analyses were carried out by the
Service de Microanalyses du CNRS, Division de Vernaison (France).
Mass spectra were recorded by electrospray on a Waters micromass
ZQ spectrometer.
mustards structurally related to
L-carnitine. Biological studies
identified the best cytotoxic candidate to be 1c which exhibits
better cytotoxic activity than the most potent acyclic nitrogen
mustard carnitine analogue we previously reported [11]. Moreover,
products 1aed (“trans” series), presenting the same relative
configuration than L-carnitine, showed better cytotoxicities than
compounds 2 (“cis” series). In order to determine which transport
pathways are involved, labelled compounds are now under
development.
Scheme 2. Synthesis of mustards 2aed Reagents and conditions: (a) NH(CH2CH2OTBDPS)2, LiOTf, CH3CN, reflux, 2 days, 75%; (b) Acyl chloride or anhydride, TEA, DMAP, CH2Cl2, reflux,
12 h, 78e97%; (c) (i) TBAF, Imidazole, THF, r.t., 30 min; (ii) p-TsCl, TEA, CH2Cl2, r.t., 12 h, 42e65% over 2 steps; (d) TFA, anisole, CH2Cl2, r.t., 8 h, 62e79%; (e) HCl, THF, r.t., 5 days, 41%.

S. Leiris et al. / European Journal of Medicinal Chemistry 45 (2010) 4140e4148
4143
Scheme 3. Synthesis of mustards 1aed. Reagents and conditions: (a) NH(CH2CH2OTBDPS)2, LiOTf, CH3CN, reflux, 2 days, 25%; (b) Acyl chloride or anhydride, TEA, DMAP, CH2Cl2,
reflux, 12 h, 74e96%; (c) (i) TBAF, Imidazole, THF, r.t., 30 min; (ii) p-TsCl, TEA, CH2Cl2, r.t., 12 h, 42e65% over 2 steps; (d) NaN3, NH4Cl, MeOH, reflux, 12 h, 51%; (e) Acyl chloride or
anhydride, TEA, DMAP, CH2Cl2, reflux, 12 h, 75e98%; (f) (i) H2, Pd/C, THF, r.t., 6 h (ii) Ethylene oxide, EtOH, sealed tube, r.t., 24 h (iii) p-TsCl, TEA, DMAP, CH2Cl2, r.t., 12 h, 39e58% over
3 steps; (g) TFA, anisole, CH2Cl2, r.t., 8 h, 65e75%.; (h) HCl, THF, r.t., 5 days, 47%.
6.1.1. Cyclohex-2-enecarbonitrile (6)
6.1.2. Methyl cyclohex-2-enecarboxylate (7)
Compound 6 was prepared according to the method of Hutch-
ison et al. [13]. To a solution containing 14.0 g (87.0 mmol) of
3-bromocyclohexene in 50 mL of CH2Cl2 were added 22.0 g
(348 mmol) of KCN and 300 mg (1.10 mmol) of 18-crown-6. The
reaction mixture was stirred at 25 ꢀC for 4 days. The unreacted KCN
was filtered, washed with CH2Cl2 and excess solvent was removed
under diminished pressure to afford a crude oil. The residue was
purified by chromatography on a silica gel column. Step gradient
elution with petroleum ether / 95:5 petroleum ether/ethyl
acetate gave 6 as a colourless oil: yield 6.97 g (75%); silica gel TLC Rf
0.84 (9:1 petroleum ether/ethyl acetate); bp 84 ꢀC/20 mm Hg ([33]
Compound 7 was prepared according to the method of Hutch-
ison et al. [13]. Anhydrous hydrogen chloride was bubbled into
a refluxing solution containing 5.00 g (46.0 mmol) of 6 in 40 mL of
MeOH for 1 h. The reaction mixture was heated at reflux for an
additional hour and stirred at room temperature for 12 h. The
reaction mixture was poured into ice and extracted with diethyl
ether. The combined organic phase was washed with 5% NaHCO3,
dried (Na2SO4), filtered and concentrated under diminished pres-
sure to afford 7 as a colourless oil which was used without further
purification in the next step: yield 6.10 g (95%); silica gel TLC Rf 0.78
(9:1 petroleum ether/ethyl acetate); 1H NMR (CDCl3)
d 1.41e2.24
bp 89 ꢀC/23 mm Hg); 1H NMR (CDCl3)
d
1.40e2.24 (m, 6H), 3.21 (m,
(m, 6H), 2.92 (m, 1H), 3.72 (s, 3H) and 5.60e5.79 (m, 2H); MS (ESI):
1H) and 5.61e5.80 (m, 2H); MS (ESI): m/e 108 (M þ H)þ.
m/e 141 (M þ H)þ and 163 (M þ Na)þ.
6.1.3. Cyclohex-2-enecarboxylate (8)
Table 1
Human cell line cytotoxicity activities (CC50) in
m
M of compounds 1aed and 2aed
To a solution containing 2.00 g (14.0 mmol) of 7 in 75 mL of 9:1
dioxane/H2O were added 500 mg (2.86 mmol) of APTS. The reaction
mixture was warmed to reflux and stirred overnight. The reaction
was quenched with H2O and the mixture was extracted with
diethyl ether. The combined organic phase was washed with brine
and satd aq NaHCO 3. The aqueous phase was acidified until pH ¼ 3
with 2 N HCl and the mixture extracted with diethyl ether. This
combined organic phase was dried (Na2SO4), filtered and concen-
trated under diminished pressure to give 8 as a colourless oil: yield
1.76 g (100%); silica gel TLC Rf 0.21 (9:1 petroleum ether/ethyl
determined by MTT assay.a
Compound
A375
H460
1a
2a
1b
2b
1c
2c
1d
2d
>1000
>1000
>1000
>1000
295 ꢁ 19
322 ꢁ 21
164 ꢁ 17
311 ꢁ 25
857 ꢁ 27
>1000
414 ꢁ 12
>1000
215 ꢁ 13
298 ꢁ 15
304 ꢁ 17
>1000
acetate); 1H NMR (CDCl3)
5.61e5.79 (m, 2H) and 12.1 (br. s, 1H); Anal. Calcd for C7H10O2: C,
d 1.41e2.24 (m, 6H), 3.15 (m, 1H),
Chlorambucil
127 ꢁ 9
104 ꢁ 11
a
Cell type: A375 (human melanoma); H460 (human adenocarcinoma).

4144
S. Leiris et al. / European Journal of Medicinal Chemistry 45 (2010) 4140e4148
Fig. 3. Viabilities of A375 and H460 cell lines exposed to 1ae2a ( ), 1be2b ( ), 1ce2c ( ) and 1de2d ( ) for 24 h. Chlorambucil (ꢃ) was chosen as a reference. All experiments
were triplicated.
66.65; H, 7.99; Found C, 66.69; H, 7.82; MS (ESI): m/e 127 (M þ H)þ,
149 (M þ Na)þ and 125 (M ꢂ H)ꢂ.
and brine, dried (Na2SO4), filtered and concentrated under diminished
pressure to give a crude oil. The residue was purified by chromatog-
raphy on a silica gel column. Step gradient elution with petroleum
ether / 97:3 petroleum ether/ethyl acetate gave 3 as a colourless oil:
yield 648 mg (42%); silica gel TLC Rf 0.52 (4:1 petroleum ether/diethyl
ether). Further elution gave 4 as a colourless oil: yield 432 mg (28%);
silica gel TLC Rf 0.49 (4:1 petroleum ether/diethyl ether).
6.1.4. tert-Butyl cyclohex-2-enecarboxylate (5)
To a cooled (ꢂ78 ꢀC) solution containing 2.50 g (20.0 mmol) of 8
in 100 mL of CH2Cl2 was bubbled w10 mL of isobutylene. The
mixture was warmed up to room temperature and 100 mL of H2SO4
were added. The reaction mixture was stirred at room temperature
for 12 h. Excess isobutylene was removed and the organic phase
was washed with satd aq NaHCO3 and brine, dried (Na2SO4),
filtered and concentrated under diminished pressure to give 5 as an
oil: yield 3.28 g (90%); silica gel TLC Rf 0.87 (9:1 petroleum ether/
Compound 3: 1H NMR (CDCl3)
d 1.41 (s, 9H), 1.42e1.96 (m, 6H),
2.71 (m, 1H), 3.12 (m, 1H) and 3.31 (d, 1H, J ¼ 3.8 Hz); 13C NMR
(CDCl3) d 17.2, 24.2, 24.3, 28.3, 41.9, 52.6, 52.8, 81.4 and 173.2; Anal.
Calcd for C11H18O3: C, 66.64; H, 9.15; Found C, 66.67; H, 9.20; MS
(ESI): m/e 199 (M þ H)þ and 221 (M þ Na)þ.
ethyl acetate); 1H NMR (CDCl3)
d
1.41 (s, 9H), 1.41e2.24 (m, 6H),
Compound 4: 1H NMR (CDCl3)
d 1.41 (s, 9H), 1.4e1.96 (m, 6H),
2.23 (m, 1H) and 5.62e5.81 (m, 2H); Anal. Calcd for C11H18O2: C,
2.66 (m, 1H), 3.12 (m, 1H) and 3.36 (t, 1H, J ¼ 3.4 Hz); 13C NMR
72.49; H, 9.95; Found C, 72.44; H, 9.91; MS (ESI): m/e 183 (M þ H)þ.
(CDCl3) d 19.3, 21.7, 23.8, 28.5, 42.1, 52.6, 52.8, 81.4 and 173.2; Anal.
Calcd for C11H18O3: C, 66.64; H, 9.15; Found C, 66.68; H, 9.17; MS
6.1.5. tert-Butyl trans-2,3-epoxycyclohexanecarboxylate (3) and
tert-butyl cis-2,3-epoxycyclohexanecarboxylate (4)
(ESI): m/e 199 (M þ H)þ and 221 (M þ Na)þ.
To a cooled (0 ꢀC) solution containing 1.40 g (7.70 mmol) of 5 in
50 mL of CH2Cl2 was added dropwise a solution containing 2.80 g
(16.0 mmol) of m-CPBA in 120 mL of CH2Cl2. The reaction mixture was
stirred at 25ꢀC for 12 h. The organic phase was washed with 5% Na2CO3
6.1.6. tert-Butyl 2-hydroxy-3-[N,N-bis(2-tert-
butyldiphenylsilyloxyethyl)amino] cyclohexanecarboxylate (9)
To a solution containing 540 mg (2.73 mmol) of 4 in 10 mL of
CH3CN was added 722 mg (4.63 mmol) of lithium triflate LiOTf. The

S. Leiris et al. / European Journal of Medicinal Chemistry 45 (2010) 4140e4148
4145
reaction mixture was stirred at room temperature for 2 h and 2.35 g
(4 mmol) of NH(CH2CH2OTBDPS)2 were added. The mixture was
warmed to reflux and stirred for 2 days. Excess solvent was
removed under diminished pressure and the residue was taken up
in 20 mL of CH2Cl2. The organic phase was washed with water,
dried (Na2SO4), filtered and concentrated under diminished pres-
sure to afford a crude white solid. The residue was purified by
chromatography on a silica gel column. Step gradient elution with
petroleum ether/ 9:1 petroleum ether/diethyl ether gave 9 as
a colourless oil: yield 1.61 g (75%); silica gel TLC Rf 0.71 (85:15
cooling the organic phase was washed with satd aq NaHCO3 and
brine, dried (Na2SO4), filtered and concentrated under diminished
pressure to afford a crude residue. The residue was purified by
chromatography on a silica gel column. Step gradient elution with
petroleum ether / 95:5 petroleum ether/ethyl acetate gave O-acyl
hydroxyesters 10aec, 13aec and 16aec.
6.1.9.1. tert-Butyl
2-acetyloxy-3-[N,N-bis(2-tert-butyldiphenylsily-
loxyethyl)amino] cyclohexanecarboxylate (10a). Yield 97%; silica gel
TLC Rf 0.65 (9:1 petroleum ether/ethyl acetate); 1H NMR (CDCl3)
hexane/ethyl acetate); 1H NMR (CDCl3)
d
0.91 (s, 18H), 1.41 (s, 9H),
d 0.91 (s 18H),1.41 (s, 9H),1.40e1.92 (m, 6H),1.81(s, 3H), 2.61 (m, 4H),
1.38e1.91 (m, 6H), 2.61 (m, 4H), 2.72 (m, 1H), 2.86 (m, 1H), 3.30 (br.
2.72 (m, 1H), 2.86 (m, 1H), 3.47 (m, 4H), 4.84 (dd, 1H, J ¼ 4.8 Hz and
s, 1H), 3.47 (m, 4H), 3.72 (m, 1H) and 7.21e7.55 (m, 20H); 13C NMR
J ¼ 8.5 Hz) and 7.21e7.55 (m, 20H); 13C NMR (CDCl3)
d 19.1, 21.4, 26.4,
(CDCl3)
d
19.1, 22.2, 26.4, 27.3, 28.2, 43.6, 53.5, 58.5, 63.4, 65.0, 81.0,
21.2, 26.8, 28.1, 43.6, 53.5, 58.9, 63.4, 72.1, 81.0, 127.5e137.4 (20C),
170.1 and 172.3; Anal. Calcd for C49H67NO6Si2: C, 71.58; H, 8.21; N,
1.70; Found C, 71.64; H, 8.26; N, 1.66; MS (ESI): m/e 825 (M þ H)þ.
127.5e137.4 (20C) and 172.9; Anal. Calcd for C47H65NO5Si2: C, 72.35;
H, 8.40; N, 1.80; Found C, 72.32; H, 8.37; N, 1.78; MS (ESI): m/e 780
(M þ H)þ.
6.1.9.2. tert-Butyl 2-capryloyloxy-3-[N,N-bis(2-tert-butyldiphenylsi-
lyloxyethyl)amino] cyclohexanecarboxylate (10b). Yield 78%; silica
gel TLC Rf 0.78 (9:1 petroleum ether/ethyl acetate); 1H NMR (CDCl3)
6.1.7. tert-Butyl 2-hydroxy-3-[N,N-bis(2-tert-
butyldiphenylsilyloxyethyl)amino] cyclohexanecarboxylate (12)
To a solution containing 150 mg (0.75 mmol) of 3 in 2 mL of
CH3CN were added 472 mg (3.03 mmol) of lithium triflate LiOTf.
The reaction mixture was stirred at room temperature for 2 h and
880 mg (1.51 mmol) of NH(CH2CH2OTBDPS)2 were added. The
mixture was warmed to reflux and stirred for 2 days. Excess solvent
was removed under diminished pressure and the residue was taken
up in 20 mL of CH2Cl2. The organic phase was washed with water,
dried (Na2SO4), filtered and concentrated under diminished pres-
sure to afford a crude white solid. The residue was purified by
chromatography on silica gel column. Step gradient elution with
petroleum ether/ 9:1 petroleum ether/diethyl ether gave 12 as
a colourless oil: yield 145 mg (25%); silica gel TLC Rf 0.75 (85:15
d
0.81 (t, 3H, J ¼ 6.7 Hz), 0.91 (s, 18H), 1.10e1.71 (m, 10H), 1.41 (s,
9H),1.40e1.92 (m, 6H), 2.08 (t, 2H, J ¼ 6.6 Hz), 2.64 (m, 4H), 2.81 (m,
1H), 2.96 (m, 1H), 3.46 (m, 4H), 4.87 (dd, 1H, J ¼ 4.6 Hz and
J ¼ 7.9 Hz) and 7.21e7.55 (m, 20H); 13C NMR (CDCl3)
d 19.1, 21.2,
21.4, 25.6, 26.4, 27.5e30.7 (5C), 27.1, 33.6, 42.0, 52.3, 57.8, 62.2, 70.9,
79.0, 126.1e137.4 (20C), 171.1 and 171.9; Anal. Calcd for
C55H79NO6Si2: C, 72.88; H, 8.79; N, 1.55; Found C, 72.98; H, 8.85; N,
1.52; MS (ESI): m/e 906 (M þ H)þ.
6.1.9.3. tert-Butyl 2-palmitoyloxy-3-[N,N-bis(2-tert-butyldiphenylsi-
lyloxyethyl)amino] cyclohexanecarboxylate (10c). Yield 85%; silica
gel TLC Rf 0.95 (9:1 petroleum ether/ethyl acetate); 1H NMR (CDCl3)
hexane/ethyl acetate); 1H NMR (CDCl3)
d
0.91 (s, 18H), 1.41 (s, 9H),
d
0.81 (t, 3H, J ¼ 6.9 Hz), 0.91 (s, 18H), 1.10e1.71 (m, 26H), 1.41 (s,
1.40e1.92 (m, 6H), 2.59 (m, 4H), 2.70 (m, 1H), 2.89 (m, 1H), 3.31 (br.
9H),1.40e1.92 (m, 6H), 2.08 (t, 2H, J ¼ 6.5 Hz), 2.64 (m, 4H), 2.81 (m,
1H), 2.96 (m, 1H), 3.46 (m, 4H), 4.85 (dd, 1H, J ¼ 4.6 Hz and
s, 1H), 3.47 (m, 4H). 3.78 (m, 1H); 7.2e7.55 (m, 20H); 13C NMR
(CDCl3)
d
19.1, 22.2, 26.4, 27.2, 28.5, 44.2, 53.0, 58.9, 63.4, 65.0, 81.2,
J ¼ 7.8 Hz), and 7.21e7.55 (m, 20H); 13C NMR (CDCl3)
d 19.1, 21.2,
127.5e137.4 (20C) and 172.9; Anal. Calcd for C47H65NO5Si2: C, 72.35;
H, 8.40; N, 1.80; Found C, 72.37; H, 8.35; N, 1.83; MS (ESI): m/e 780
(M þ H)þ.
21.4, 25.6, 26.4, 27.5e30.7 (13C), 27.1, 33.6, 42.0, 52.3, 57.8, 62.2,
70.9, 79.0, 126.1e137.4 (20C), 171.1 and 172.0; Anal. Calcd for
C63H95NO6Si2: C, 74.29; H, 9.40; N, 1.38 Found C, 74.36; H, 9.45; N,
1.34; MS (ESI): m/e 1018 (M þ H)þ.
6.1.8. tert-Butyl 3-azido-2-hydroxycyclohexanecarboxylate (15)
To a solution containing 950 mg (4.80 mmol) of 3 in 20 mL of
MeOH were added 530 mg (8.70 mmol) of NaN3 and 382 mg
(7.21 mmol) ofNH4Cl. The reaction mixturewaswarmedto refluxand
stirred for12 h. The reactionmixturewascooledto room temperature
and excess solvent was removed under diminished pressure to give
a crude oil. The residue was taken up in 20 mL of H2O and extracted
with ethyl acetate. The combined organic phase was washed with
brine, dried (Na2SO4), filtered and concentratedunder vacuum to give
a 3:2 mixture of 17 and its isomer. The residue was purified by
chromatography on silica gel column. Step gradient elution with
petroleum ether / 4:1 petroleum ether/ethyl acetate gave 17 as an
oil: yield 590 mg (51%); silica gel TLC Rf 0.32 (4:1 petroleum ether/
6.1.9.4. tert-Butyl 2-acetyloxy-3-[N,N-bis(2-tert-butyldiphenylsily-
loxyethyl)amino] cyclohexanecarboxylate (13a). Yield 96%; silica gel
TLC Rf 0.61 (9:1 petroleum ether/ethyl acetate); 1H NMR (CDCl3)
d
0.91 (s, 18H), 1.41 (s, 9H), 1.40e1.92 (m, 6H), 1.83 (s, 3H), 2.19 (m,
1H), 2.43 (m, 1H), 2.50e2.62 (m, 4H), 3.47 (m, 4H), 4.77 (t, 1H,
J ¼ 9.8 Hz) and 7.21e7.55 (m, 20H); 13C NMR (CDCl3)
d 19.1, 21.2,
21.4, 25.6, 26.9, 28.1, 43.6, 53.5, 58.9, 63.4, 72.1, 81.0, 127.5e137.4,
170.1 and 172.3; Anal. Calcd for C49H67NO6Si2: C, 71.58; H, 8.21; N,
1.70; Found C, 71.63; H, 8.23; N, 1.72; MS (ESI): m/e 826 (M þ H)þ.
6.1.9.5. tert-Butyl 2-capryloyloxy-3-[N,N-bis(2-tert-butyldiphenylsi-
lyloxyethyl)amino] cyclohexanecarboxylate (13b). Yield 74%; silica
gel TLC Rf 0.75 (9:1 petroleum ether/ethyl acetate); 1H NMR (CDCl3)
ethyl acetate); 1H NMR (CDCl3)
(m, 1H); 3.19 (m, 1H), 3.28 (br. s, 1H) and 3.59 (m, 1H); 13C NMR
(CDCl3) 23.5, 27.7, 28.2, 30.1, 50.6, 65.2, 74.5, 81.4 and 173.2; Anal.
d 1.41 (s, 9H), 1.40e1.92 (m, 6H), 2.23
d
0.78 (t, 3H, J ¼ 6.6 Hz), 0.91 (s, 18H), 1.10e1.71 (m, 10H), 1.41 (s,
d
9H),1.40e1.92 (m, 6H), 2.08 (t, 2H, J ¼ 6.6 Hz), 2.20 (m, 1H), 2.42 (m,
1H), 2.49e2.61 (m, 4H), 3.46 (m, 4H), 4.91 (t, 1H, J ¼ 10.1 Hz) and
Calcd for C11H19N3O3: C, 54.76; H, 7.94; N, 17.41; Found C, 54.71; H,
7.89; N, 17.46; MS (ESI): m/e 242 (M þ H)þ and 264 (M þ Na)þ.
7.21e7.55 (m, 20H); 13C NMR (CDCl3)
d 19.1, 21.2, 21.4, 25.6, 26.4,
27.5e30.7 (5C), 27.0, 33.6, 42.1, 52.3, 57.8, 62.2, 70.9, 79.0,
126.0e137.4 (20C), 171.1 and 171.9; Anal. Calcd for C55H79NO6Si2: C,
72.88; H, 8.79; N, 1.55; Found C, 72.97; H, 8.84; N, 1.52; MS (ESI):
m/e 906 (M þ H)þ.
6.1.9. General procedure for the preparation of O-acyl
aminohydroxyesters (10aec, 13aec, 16aec)
To a solution containing 9, 12 or 15 (1 eq.), triethylamine (2 eq.)
and DMAP (cat.) in 55 mL of CH2Cl2 was added dropwise anhydride
(acetic, palmitoic) or acid chloride (capryloyl, lauroyl) (1.2 eq.). The
reaction mixture was warmed to reflux and stirred for 12 h. After
6.1.9.6. tert-Butyl 2-palmitoyloxy-3-[N,N-bis(2-tert-butyldiphenylsi-
lyloxyethyl)amino] cyclohexanecarboxylate (13c). Yield 80%; silica

4146
S. Leiris et al. / European Journal of Medicinal Chemistry 45 (2010) 4140e4148
gel TLC Rf 0.92 (9:1 petroleum ether/ethyl acetate); 1H NMR (CDCl3)
their instabilities by using the procedure reported herein thereafter
in Section 6.1.12.
d
0.81 (t, 3H, J ¼ 6.9 Hz), 0.91 (s, 18H), 1.10e1.71 (m, 26H), 1.41 (s,
9H), 1.43e1.90 (m, 6H), 2.08 (t, 2H, J ¼ 6.5 Hz), 2.15 (m, 1H), 2.39 (m,
1H), 2.42e2.61 (m, 4H), 3.46 (m, 4H), 4.92 (t, 1H, J ¼ 10.3 Hz) and
6.1.12. General procedure for the synthesis of chlorinated products
(11aec and 14aec)
7.21e7.55 (m, 20H); 13C NMR (CDCl3)
d 19.0, 21.1, 21.3, 25.6, 26.4,
27.5e30.7 (13C), 27.2, 33.6, 42.1, 52.3, 57.8, 62.2, 70.9, 79.1,
126.2e137.4 (20C), 171.1 and 172.5; Anal. Calcd for C63H95NO6Si2: C,
74.29; H, 9.40; N, 1.38; Found C, 74.36; H, 9.45; N, 1.36; MS (ESI):
m/e 1018 (M þ H)þ.
To a solution containing 0.25 mmol of diol in 5 mL of CH2Cl2
were added 79.4
mL (0.57 mmol) of triethylamine, 41.9 mg
(0.686 mmol) of DMAP and 130 mg (0.686 mmol) of p-TsCl. The
reaction mixture was stirred for 12 h at room temperature and
excess solvent was removed under diminished pressure to afford
a crude oil. The residue was purified by flash chromatography on
a florisil column. Step gradient elution with petroleum ether / 2:3
petroleum ether/ethyl acetate gave 11aec and 14aec.
6.1.9.7. tert-Butyl
2-acetyloxy-3-azidocyclohexanecarboxylate
(16a). Yield 98%; silica gel TLC Rf 0.49 (9:1 petroleum ether/ethyl
acetate); 1H NMR (CDCl3)
d 1.41 (s, 9H), 1.40e1.91 (m, 6H), 2.23 (m,
1H), 3.19 (m, 1H) and 5.03 (t, 1H, J ¼ 10.1 Hz); 13C NMR (CDCl3)
d
19.4, 21.3, 26.4, 28.2, 50.6, 65.1, 72.2, 81.4, 171.2 and 173.2; Anal.
6.1.12.1. tert-Butyl
cyclohexanecarboxylate (11a). Yield 42%; silica gel TLC Rf 0.23 (9:1
petroleum ether/ethyl acetate); 1H NMR (CDCl3)
1.41 (s, 9H),
2-acetyloxy-3-[N,N-bis(2-chloroethyl)amino]
Calcd for C13H21N3O4: C, 55.11; H, 7.47; N, 14.83; Found C, 55.23; H,
7.52; N, 14.81; MS (ESI): m/e 284 (M þ H)þ.
d
1.40e1.92 (m, 6H), 1.81 (s, 3H), 2.97 (m, 1H), 3.15e3.37 (m, 4H), 3.51
(m, 1H), 3.71 (m, 4H) and 4.90 (dd, 1H, J ¼ 5.1 Hz and J ¼ 8.2 Hz);
Anal. Calcd for C17H29Cl2NO4: C, 53.41; H, 7.65; N, 3.66; Found C,
53.50; H, 7.68; N, 3.62; MS (ESI): m/e 382 (M þ H)þ.
6.1.9.8. tert-Butyl
(16b). Yield 75%; silica gel TLC Rf 0.61 (9:1 petroleum ether/ethyl
acetate); 1H NMR (CDCl3)
2-capryloyloxy-3-azidocyclohexanecarboxylate
d
0.83 (t, 3H, J ¼ 7.0 Hz), 1.10e1.71 (m,
10H), 1.41 (s, 9H), 1.40e1.92 (m, 6H), 2.05 (t, 2H, J ¼ 6.5 Hz), 2.26 (m,
1H), 3.15 (m, 1H) and 5.09 (t, 1H, J ¼ 9.9 Hz, 1H); 13C NMR (CDCl3)
6.1.12.2. tert-Butyl 2-capryloyloxy-3-[N,N-bis(2-chloroethyl)amino]
d
19.2, 21.2, 22.1, 26.4, 27.5e30.7 (5C), 28.1, 50.6, 65.1, 72.3, 81.1,
cyclohexane carboxylate (11b). Yield 51%; silica gel TLC Rf 0.38 (9:1
170.9 and 173.1; Anal. Calcd for C19H33N3O4: C, 62.10; H, 9.05; N,
petroleum ether/ethyl acetate); 1H NMR (CDCl3)
d 0.81 (t, 3H,
11.43; Found C, 62.19; H, 9.09; N, 11.40; MS (ESI): m/e 368 (M þ H)þ.
J ¼ 6.9 Hz), 1.10e1.71 (m, 10H), 1.41 (s, 9H), 1.40e1.92 (m, 6H), 2.15
(t, 2H, J ¼ 7.2 Hz), 2.99 (m, 1H), 3.11e3.32 (m, 4H), 3.51 (m, 1H), 3.72
(m, 4H) and 4.91 (dd, 1H, J ¼ 5.7 Hz and J ¼ 10.6 Hz); Anal. Calcd for
C23H41Cl2NO4: C, 59.22; H, 8.86; N, 3.00; Found C, 59.28; H, 8.93; N,
2.98; MS (ESI): m/e 466 (M þ H)þ.
6.1.9.9. tert-Butyl
(16c). Yield 86%; silica gel TLC Rf 0.89 (9:1 petroleum ether/ethyl
acetate); 1H NMR (CDCl3)
0.81 (t, 3H, J ¼ 7.4 Hz), 1.09e1.71 (m,
2-palmitoyloxy-3-azidocyclohexanecarboxylate
d
26H),1.41 (s, 9H),1.38e1.92 (m, 6H), 2.06 (t, 2H, J ¼ 6.9 Hz), 2.27 (m,
1H), 3.10 (m,1H) and 5.15 (t,1H, J ¼ 10.1 Hz); 13C NMR (CDCl3)
d
19.1,
6.1.12.3. tert-Butyl
amino]cyclohexane carboxylate (11c). Yield 65%; silica gel TLC Rf
0.78 (9:1 petroleum ether/ethyl acetate); 1H NMR (CDCl3)
0.81 (t,
2-palmitoyloxy-3-[N,N-bis(2-chloroxyethyl)
21.2, 22.3, 26.4, 27.5e30.7 (13C), 28.2, 50.6, 65.1, 72.3, 81.1, 171.1 and
173.2; Anal. Calcd for C27H49N3O4: C, 67.60; H, 10.30; N, 8.76; Found
C, 67.72; H, 10.35; N, 8.73; MS (ESI): m/e 480 (M þ H)þ.
d
3H, J ¼ 6.8 Hz), 1.10e1.71 (m, 26H), 1.41 (s, 9H), 1.40e1.92 (m, 6H),
2.22 (t, 2H, J ¼ 7.0 Hz), 2.97 (m, 1H), 3.13e3.35 (m, 4H), 3.56 (m, 1H),
3.72 (m, 4H) and 4.87 (dd, 1H, J ¼ 5.3 Hz and J ¼ 10.2 Hz); Anal.
Calcd for C31H57Cl2NO4: C, 64.34; H, 9.93; N, 2.42; Found C, 64.41;
H, 9.97; N, 2.41; MS (ESI): m/e 578 (M þ H)þ.
6.1.10. General procedure for the reduction of the azides 16aec and
for the preparation of the corresponding bis(2-hydroxyethyl)amines
using ethylene oxide
To a solution containing 0.71 mmol of 16aec in 8 mL of THF
was added Pd/C (0.150 mmol) and the reaction mixture was
purged with hydrogen and stirred for 6 h under hydrogen
atmosphere. The reaction mixture was filtered through a celite
pad which was washed with ethyl acetate. Excess solvent was
removed under diminished pressure to give a crude oil. The
residue was taken up in EtOH (2 mL) under an ethylene oxide
atmosphere in a sealed tube. The reaction mixture was stirred at
room temperature for 24 h and excess solvent was removed
under diminished pressure to afford a crude oil. The residue was
purified by flash chromatography on a florisil column. Step
gradient elution with petroleum ether / 2:3 petroleum ether/
ethyl acetate gave expected diols which were immediately chlo-
rinated due to their instabilities by using the procedure reported
thereafter in Section 6.1.12.
6.1.12.4. tert-Butyl
2-acetyloxy-3-[N,N-bis(2-chloroethyl)amino]
cyclohexanecarboxylate (14a). Yield 39% from 16a and 42% from
13a; silica gel TLC Rf 0.25 (9:1 petroleum ether/ethyl acetate); 1H
NMR (CDCl3)
d 1.41 (s, 9H), 1.40e1.92 (m, 6H), 1.81 (s, 3H), 2.28 (m,
1H), 2.39 (m, 1H), 2.62e2.74 (m, 4H), 3.37 (m, 4H) and 5.02 (t, 1H,
J ¼ 9.5 Hz); Anal. Calcd for C17H29Cl2NO4: C, 53.41; H, 7.65; N, 3.66;
Found C, 53.52; H, 7.70; N, 3.63; MS (ESI): m/e 382 (M þ H)þ.
6.1.12.5. tert-Butyl 2-capryloyloxy-3-[N,N-bis(2-chloroethyl)amino]
cyclohexane carboxylate (14b). Yield 49% from 16b and 61% from
13b; silica gel TLC Rf 0.64 (9:1 petroleum ether/ethyl acetate); 1H
NMR (CDCl3)
d
0.82 (t, 3H, J ¼ 6.9 Hz), 1.10e1.69 (m, 10H), 1.41 (s,
9H), 1.40e1.95 (m, 6H), 2.11 (t, 2H, J ¼ 6.4 Hz), 2.25 (m, 1H), 2.39 (m,
1H), 2.59e2.71 (m, 4H), 3.41 (m, 4H), 5.05 (t, 1H, J ¼ 10.2 Hz); Anal.
Calcd for C23H41Cl2NO4: C, 59.22; H, 8.86; N, 3.00; Found C, 59.35;
H, 8.91; N, 2.96; MS (ESI): m/e 466 (M þ H)þ.
6.1.11. General procedure for the cleavage of silylated ethers (10aec
and 13aec)
To a solution containing 0.286 mmol of 10aec or 13aec in 4 mL
of THF were added 136 mg (2.00 mmol) of imidazole and 1.72 mL
(1.72 mmol) of TBAF (1 M in THF). The reaction mixture was stirred
at room temperature for 30 min and excess solvent was removed
under diminished pressure to afford a crude oil. The residue was
purified by flash chromatography on a florisil column. Step gradient
elution with petroleum ether / 2:3 petroleum ether/ethyl acetate
gave expected diols which were immediately chlorinated due to
6.1.12.6. tert-Butyl 2-palmitoyloxy-3-[N,N-bis(2-chloroethyl)amino]
cyclohexane carboxylate (14c). Yield 58% from 16c and 65% from
13c; silica gel TLC Rf 0.78 (9:1 petroleum ether/ethyl acetate); 1H
NMR (CDCl3)
d
0.81 (t, 3H, J ¼ 7.1 Hz), 1.10e1.72 (m, 26H), 1.41 (s,
9H), 1.40e1.89 (m, 6H), 2.15 (t, 2H, J ¼ 6.0 Hz), 2.28 (m, 1H), 2.49 (m,
1H), 2.68e2.75 (m, 4H), 3.39 (m, 4H) and 5.10 (t, 1H, J ¼ 10.2 Hz);
Anal. Calcd for C31H57Cl2NO4: C, 64.34; H, 9.93; N, 2.42; Found C,
64.35; H, 9.98; N, 2.40; MS (ESI): m/e 578 (M þ H)þ.

S. Leiris et al. / European Journal of Medicinal Chemistry 45 (2010) 4140e4148
4147
6.1.13. General procedure for the synthesis of carboxylic acids (2aec
and 1aec)
4H) and 5.1 (t, 1H, J ¼ 10.2 Hz); 13C NMR (CDCl3)
d
15.2, 17.5e29.2
(16C), 34.4, 38.0, 43.4, 53.2, 56.4, 69.7, 169.4 and 173.2; Anal. Calcd
for C27H49Cl2NO4: C, 62.05; H, 9.45; N, 2.68; Found C, 61.89; H, 9.50;
N, 2.64; MS (ESI): m/e 520 (M ꢂ H)ꢂ.
To a solution containing 0.12 mmol of 11aec or 14aec in 1 mL of
CH2Cl2 were added 13.1
mL (0.12 mmol) of anisole and 92.5 mL
(1.20 mmol) of TFA. The reaction mixture was stirred at room
temperature for 8 h and excess solvent was removed under
diminished pressure. The residue was coevaporated with 20 mL of
cyclohexane and purified by flash chromatography on a silica gel
column. Elution with ethyl acetate gave 2aec and 1aec as colour-
less oils.
6.1.14. General procedure for the preparation of 1d and 2d
To a solution containing 0.15 mmol of 11a or 14a in 150
mL of THF
were added 16.0 L (0.15 mmol) of anisole and 0.3 mL (3.7 mmol) of
m
aqueous HCl (2.4 mol Lꢂ1). The reaction mixture was stirred at
room temperature for 5 days. The solution was diluted with 5 mL of
diethyl ether and the organic phase was washed with water. The
aqueous phase was lyophilised to afford a crude oil. The residue was
purified by chromatography on a reverse phase silica gel column
(C18). Elution with water gave 2d or 1d respectively.
6.1.13.1. 2-Acetyloxy-3-[N,N-bis(2-chloroethyl)amino]cyclo-
hexanecarboxylic acid (2a). Yield 62%; silica gel TLC Rf 0.35 (2:3
petroleum ether/ethyl acetate); 1H NMR (CDCl3)
d 1.38e1.89 (m,
6H), 1.83 (s, 3H), 3.21 (m, 1H), 3.15e3.37 (m, 4H), 3.45 (m, 1H), 3.71
(m, 4H) and 4.90 (dd,1H, J ¼ 5.1 Hz and J ¼ 8.6 Hz); 13C NMR (CDCl3)
6.1.14.1. 2-Hydroxy-3-[N,N-bis(2-chloroethyl)amino]cyclo-
d
19.2, 21.2, 22.5, 26.5, 37.1, 41.2, 53.5, 56.1, 72.1, 170.1 and 173.7;
hexanecarboxylic acid (1d). Yield 47%; silica gel TLC Rf 0.82 (2:3:1
Anal. Calcd for C13H21Cl2NO4: C, 47.86; H, 6.49; N, 4.29; Found C,
acetone/ethyl acetate/water); 1H NMR (D2O)
d 1.41e1.88 (m, 6H),
47.82; H, 6.54; N, 4.25; MS (ESI): m/e 325 (M ꢂ H)ꢂ.
2.45 (m, 1H), 2.52 (m, 1H), 2.69e2.74 (m, 4H), 3.44 (m, 4H) and 4.15
(t, 1H, J ¼ 9.5 Hz); 13C NMR (D2O)
d 17.5, 21.2, 29.0, 36.9, 38.7, 51.8,
6.1.13.2. 2-Capryloyloxy-3-[N,N-bis(2-chloroethyl)amino]cyclo-
55.2, 63.1 and 171.9; Anal. Calcd for C11H19Cl2NO3: C, 46.49; H, 6.74;
N, 4.93; Found C, 46.42; H, 6.81; N, 4.90; MS (ESI): m/e 283
(M ꢂ H)ꢂ.
hexanecarboxylic acid (2b). Yield 71%; silica gel TLC Rf 0.59 (2:3
petroleum ether/ethyl acetate); 1H NMR (CDCl3)
d 0.81 (t, 3H,
J ¼ 6.9 Hz), 1.10e1.71 (m, 10H), 1.41e1.93 (m, 6H), 2.15 (t, 2H,
J ¼ 7.2 Hz), 3.22 (m, 1H), 3.21e3.39 (m, 4H), 3.42 (m, 1H), 3.72 (m,
4H) and 4.96 (dd, 1H, J ¼ 5.7 Hz and J ¼ 10.1 Hz); 13C NMR (CDCl3)
6.1.14.2. 2-Hydroxy-3-[N,N-bis(2-chloroethyl)amino]cyclo-
hexanecarboxylic acid (2d). Yield 41%; silica gel TLC Rf 0.78 (2:3:1
d
14.2, 19.2, 20.3, 22.7, 22.9, 24.5, 26.7, 27.2, 29.8, 33.2, 37.8, 41.8,
acetoneeethyl acetate/water); 1H NMR (D2O)
d 1.40e1.89 (m, 6H),
55.3, 56.1, 71.4, 169.2 and 173.9; Anal. Calcd for C19H33Cl2NO4: C,
55.61; H, 8.11; N, 3.41; Found C, 55.53; H, 8.15; N, 3.38; MS (ESI):
m/e 409 (M ꢂ H)ꢂ.
2.45 (m, 1H), 2.52 (m,1H), 3.21e3.40 (m, 4H), 3.72 (m, 4H), 4.21 (dd,
1H, J ¼ 5.2 Hz and J ¼ 9.8 Hz); 13C NMR (D2O)
d 17.5, 21.9, 29.4, 37.1,
38.2, 53.6, 55.9, 62.2 and 172.6; Anal. Calcd for C11H19Cl2NO3: C,
46.49; H, 6.74; N, 4.93; Found C, 46.38; H, 6.77; N, 4.92; MS (ESI):
m/e 283 (M ꢂ H)ꢂ.
6.1.13.3. 2-Palmitoyloxy-3-[N,N-bis(2-chloroxyethyl)amino]cyclo-
hexanecarboxylic acid (2c). Yield 79%; silica gel TLC Rf 0.82 (2:3
petroleum ether/ethyl acetate); 1H NMR (CDCl3)
d 0.81 (t, 3H,
6.2. Biological procedure
J ¼ 6.8 Hz), 1.10e1.72 (m, 26H), 1.42e1.89 (m, 6H), 2.19 (t, 2H,
J ¼ 6.9 Hz), 3.18 (m, 1H), 3.13e3.35 (m, 4H), 3.51 (m, 1H), 3.72 (m,
4H) and 4.87 (dd, 1H, J ¼ 5.3 Hz and J ¼ 10.1 Hz); 13C NMR (CDCl3)
Cytotoxicity assays for the nitrogen mustards 1aed and 2aed
were carried out in triplicate on two different cell lines (all wild
type) obtained from the American Type Culture Collection
(Bethesda, MD, USA). Human melanoma cells (A375) grown in
DMEM (Dulbecco’s Modified Eagle’s Medium) and human large cell
carcinoma of the lung (NCI-H460) grown in RPMI 1640 (Roswell
Park Memorial Institute), both supplemented with 10% fetal calf
serum, were selected. Cells were seeded at a density of 3 ꢃ 104 cells/
well in a 96-well plate and left to adhere for 24 h at 37 ꢀC in the
presence of 5% CO2 for attachment. Culture media containing
increasing concentrations (0.1e1000 mM) of test compound was
prepared and the cells exposed to it for 24 h. For the viability
staining, a neutral red solution (33 mg/L) was added for 4 h and the
cells were stained (15 min) with a mixture glacial acetic acid-
eethanol [1:50 (v/v)]. Absorbances were read at 540 nm. The
cytotoxic activity of the drugs was expressed as CC50, the concen-
tration at which the proportion of death cells was 50% when
compared to untreated cells. Chlorambucil was chosen as reference.
d
14.7, 17.0e29.5 (16C), 34.0, 37.8, 42.5, 53.2, 56.7, 70.3, 169.3 and
173.4; Anal. Calcd for C27H49Cl2NO4: C, 62.05; H, 9.45; N, 2.68;
Found C, 61.97; H, 9.49; N, 2.64; MS (ESI): m/e 520 (M ꢂ H)ꢂ.
6.1.13.4. 2-Acetyloxy-3-[N,N-bis(2-chloroethyl)amino]cyclo-
hexanecarboxylic acid (1a). Yield 65%; silica gel TLC Rf 0.29 (2:3
petroleum ether/ethyl acetate); 1H NMR (CDCl3)
d 1.41e1.90 (m,
6H), 1.81 (s, 3H), 2.46 (m, 1H), 2.51 (m, 1H), 2.62e2.74 (m, 4H), 3.37
(m, 4H) and 5.02 (t, 1H, J ¼ 9.5 Hz); 13C NMR (CDCl3)
d 19.0 21.5,
22.5, 27.3, 37.8, 42.4, 54.5, 57.2, 71.8, 169.0 and 172.5; Anal. Calcd for
C13H21Cl2NO4: C, 47.86; H, 6.49; N, 4.29; Found C, 47.79; H, 6.53; N,
4.31; MS (ESI): m/e 325 (M ꢂ H)ꢂ.
6.1.13.5. 2-Capryloyloxy-3-[N,N-bis(2-chloroethyl)amino]cyclo-
hexanecarboxylic acid (1b). Yield 67%; silica gel TLC Rf 0.59 (2:3
petroleum ether/ethyl acetate); 1H NMR (CDCl3)
d 0.82 (t, 3H,
J ¼ 6.9 Hz), 1.10e1.71 (m, 10H), 1.41e1.93 (m, 6H), 2.11 (t, 2H,
J ¼ 6.4 Hz), 2.48 (m, 1H), 2.54 (m, 1H), 2.59e2.71 (m, 4H), 3.42 (m,
Acknowledgements
4H) and 5.05 (t, 1H, J ¼ 10.0 Hz); 13C NMR (CDCl3)
d 15.5, 17.8e31.2
(8C), 33.4, 38.2, 41.7, 55.4, 56.3, 71.8, 169.4 and 173.1; Anal. Calcd for
C19H33Cl2NO4: C, 55.61; H, 8.11; N, 3.41; Found C, 55.56; H, 8.15; N,
3.39; MS (ESI): m/e 409 (M ꢂ H)ꢂ.
We are grateful to the Ministère de la Recherche et de la Tech-
nologie for financial support of this research. We thank the team of
M. Garcia (IRCM, U896 INSERM) for their technical support during
the biological testing.
6.1.13.6. 2-Palmitoyloxy-3-[N,N-bis(2-chloroethyl)amino]cyclo-
hexanecarboxylic acid (1c). Yield 75%; silica gel TLC Rf 0.76 (2:3
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d 0.81 (t, 3H,
J ¼ 7.1 Hz), 1.10e1.72 (m, 26H), 1.40e1.93 (m, 6H), 2.15 (t, 2H,
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