Method for preparing activated esters
The present invention relates to the preparation of
activated esters of formula (I):

in which R is a (C1-C6) alkyl, aryl, heteroaryl,
cycloalkyl or heterocycloalkyl group, and Alk is a (C1-
C6)alkylene group. These activated esters can be used in
the preparation of conjugates, i.e. of antibodies to
which biologically active chemical compounds, such as
cytotoxic compounds, are attached by covalent bonding.
Further details on conjugate chemistry will be found,
for example, in Birch and Lennox, Monoclonal
Antibodies: Principles and Applications, Chap. 4,
Wiley-Liss, New York, N.Y. (1995).
[Prior art]
WO 2004/016801 describes activated esters comprising a
nitrosuccinimide unit. The preparations of these
compounds described in figures 1 to 6 are based on
reactions different than those envisioned in the
present invention.
J. Med. Chem. 2006, 49(14), 4392-4408 describes the
preparation of activated esters, in particular
N-succinimidyl-[4-methyl-4-(methyldithio)]pentanoate on
scheme 6, by means of reactions different than that
envisioned in the present invention.
Langmuir 2000, 16(1), 81-86 describes, on scheme 1, the
preparation of succinimidyl-3-(2-pyridyldithio)butyrate

(SPDB) by coupling of the corresponding acid with
N-hydroxysuccinimide.
US 6407263, US 5872261, US 5892057 and US 5942628
describe activated esters and the method for preparing
same.
Can. J. Chem. 1982, 60, 976 describes the preparation
of the dicyclohexylamine salt of N-hydroxysuccinimide
(P2) by reaction between dicyclohexylamine and
N-hydroxysuccinimide in acetone. This compound has the
CAS No. 82911-72-6.
Can. J. Chem. 1986, 64 (11),2097-2102; J. Chem. Soc,
Perkin Trans. 1 1985, 4, 765-8; Bull. Soc. Chem. Jpn
1986, 59(8), 2505-8; Coll. Czech. Chem. Comm. 1985,
50(12), 2925-2936 describe the preparation of
succinimide esters from dicyclohexylamine salts but
without using disuccinimidyl carbonate.
Tetrahedron Letters 1979, 49, 4745-4746 describes DSC
and its value in synthesis (see scheme 2).
Biochem. J. 1978, 173, 723-737 describes the
preparation of activated esters in the presence of
N-hydroxysuccinimide and of dicyclohexylcarbodiimide.
JACS 2003, 125(30), 8994-8995 is part of the technical
background.
[Brief description of the invention]
The invention relates to a method for preparing an
activated ester of formula (I):


in which R is a linear or branched (C1-C6) alkyl, aryl,
heteroaryl, cycloalkyl or heterocycloalkyl group, and
Alk is a linear or branched (C1-C6) alkylene group, said
method consisting in reacting the dicyclohexylamine
salt P1:

and disuccinimidyl carbonate (DSC) in a solvent in
which the dicyclohexylamine salt of
N-hydroxysuccinimide P2

precipitates.
The invention also relates to the products of formula
P1:

more particularly those of formula:


[Description of the invention]
definitions
• alkyl group: a linear or branched, saturated
aliphatic hydrocarbon-based group obtained by removing
a hydrogen atom from an alkane. Mention may in
particular be made of the following groups: methyl,
ethyl, propyl, butyl, pentyl, hexyl, 2-methylbutyl,
2-methylpentyl and 1-methylpentyl;
• alkylene group: a divalent group obtained by removing
two hydrogen atoms from an alkane. Mention may in
particular be made of the following groups: methylene
(-CH2-), ethylene (-CH2CH2-) , n-propylene (-CH2CH2CH2-)
and butylene (-CH2CH2CH2CH2-) ;
• cycloalkyl group: a cyclic alkyl group containing
from 3 to 10 carbon atoms involved in the cyclic
structure. Mention may in particular be made of the
following groups: cyclopropyl, cyclopentyl and
cyclohexyl;
• aryl group: an aromatic group containing from 6 to 10
carbon atoms. Mention may in particular be made of the
following groups: phenyl, naphthyl, indenyl and
fluorenyl;
• heteroaryl group: an aromatic group of 5 to 10 ring
members comprising, as atoms forming the ring, one or
more heteroatoms selected from 0, S or N;
• heterocycloalkyl group: a cycloalkyl group as defined
above, also comprising, as atom(s) forming the ring,
one or more heteroatoms selected from N, 0 or S.
The preparation is based on the reaction between the
dicyclohexylamine salt P1 and disuccinimidyl carbonate
(DSC) in a solvent in which the dicyclohexylamine salt
of N-hydroxysuccinimide P2 precipitates (scheme 1).


R is:
• a (C1-C6) alkyl group: for example a methyl, ethyl,
propyl, butyl or pentyl group, which is optionally
branched;
• a (C3-C7) cycloalkyl group: for example the
cyclopropyl group;
• an aryl group: for example the phenyl group;
• a heteroaryl group: for example the 2-pyridinyl
group
• a heterocycloalkyl group: for example the
piperidinyl group.
Alk is a (C1-C6)alkylene group, for example a propylene,
butylene or pentylene group, which is optionally
branched. It is more particularly the (CH2)n group, n
denoting an integer ranging from 1 to 6.
The function of the dicyclohexylamine is to promote the
reaction and to render insoluble the N-
hydroxysuccinimide which is released. This reaction has
the following advantages:
• ease of implementation: simple bringing into
contact, no heating, slow and controlled release of
CO2;
• since the compound P1 is in carboxylate form, it is
not necessary to add an additional base in order to
activate the reaction;
• the compound P2 which is released has only very low
solubility in the solvent used and it precipitates.

The majority of P2 can therefore be readily removed by
simple mechanical separation, for example filtration;
• the reaction makes it possible to readily obtain
the activated ester with a good yield and good
purity.
P2 is prepared by neutralization of the corresponding
acid with dicyclohexylamine. DSC is a commercial
product.
The solvent is advantageously a ketone, which exhibits
fewer toxicological problems than the solvents normally
used for this type of reaction (dichloromethane or
dimethylformamide). The ketone may, for example, be
acetone or methyl isobutyl ketone (MIBK). MIBK is
preferred because, since it is water-miscible (1.55%
w/w at 20°C), it allows aqueous washing of the product,
thus facilitating the removal of residual P2. It also
makes it possible to remove the residual water by
azeotropic distillation. Finally, MIBK is a good
solvent for the activated ester but not for the
compounds P2 and P1 and the DSC, which allows a slow and
controlled reaction between P1 and the DSC: the
reactants can thus be initially mixed in their entirety
without this posing a problem in terms of safety (rapid
reaction with uncontrolled release of CO2) .
The reaction is carried out at ambient temperature
(approximately 20°C). P2 can precipitate spontaneously
in certain solvents. In order to promote the
precipitation of P2, it is possible, after having
reacted P1 and the DSC, to cool the reaction mixture
(for example to a temperature close to 0°C).
This reaction makes it possible in particular to
prepare the following activated esters: N-succinimidyl-
3-(2-pyridyldithio)propionate (SPDP), N-succinimidyl-3-
(2-pyridyldithio)butyrate (SPDB) or N-succinimidyl-[4-

methyl-4-(methyldithio)]pentanoate from the
corresponding acid salts, namely, respectively:

[Examples]
Example 1: preparation of N-succinimidyl-[4-methyl-4-
(methyldithio)]pentanoate
The reaction is the following:

A suspension of the dicyclohexylamine salt of 4-methyl-
4-(methyldithio)pentanoic acid (23 g) and DSC (18.2 g,
1.1 eq.) in 161 ml of MIBK is stirred at approximately
20°C for 5 h. The suspension is then cooled to
approximately 0°C, stirred for 1 h at this temperature,
and then filtered.
The solid is washed with 2 x 23 ml of MIBK. The organic
phases are combined, and washed with 2 x 58 ml of a 6N
aqueous solution of HC1 and then with 92 ml of
demineralized water. The organic phase is then
concentrated to dryness under vacuum. The resulting
solid is solubilized in 230 ml of dichloromethane
(DCM), and the resulting solution is treated with 46 g
of silica and stirred for 10 min, and then the silica
is filtered off and washed with 2 x 69 ml of DCM. This
operation is repeated a second time. The organic phase
is then concentrated to approximately half the volume,
and then, at approximately 20°C, 391 ml of n-heptane
are added in approximately 30 min. The resulting white
suspension is stirred at this temperature for

approximately 1 h, cooled to approximately -10°C over
the course of approximately 1 h, and then stirred at
this temperature for approximately 1 h. The solid is
then filtered off, washed with 2 x 23 ml of n-heptane
cooled to approximately -10°C, and then dried under
vacuum at 40°C for 15 h. The 4-N-hydroxysuccinimidyl-
[4-methyl-4-(methyldithio)]pentanoate is isolated with
a yield of 70.6%. Its purity, determined by HPLC, is
99.65% (excluding solvents).
Example 2: preparation of N-succinimidyl-3-(2-
pyridyldithio)butyrate (SPDB)
The reaction is the following:

The dicyclohexylamine salt (40 g, 1 eq.) and the DSC
(28.7 g, 1.1 eq.) are suspended in 280 ml of MIBK. The
mixture is stirred for 4 h at 20±3°C. The suspension is
cooled to 0±3°C, left at this temperature for 30 min
and filtered, and the solid obtained is washed with
ice-cold MIBK (120 ml). The mother liquors are washed
with water (3 x 176 ml) and evaporated to dryness under
reduced pressure on a rotary evaporator with a bath at
50°C until an amount of MIBK of <2.5% is obtained. The
crude SPDB is obtained in the form of a yellow oil.
The SPDB (32.5 g) is then dissolved in ethanol (455 ml)
at 35+2°C. The solution obtained is cooled to 18±2°C:
the pure SPDB begins to crystallize. 90 ml of n-heptane
are added over the course of 10 min, the
crystallization intensifies. The mixture is cooled to
0+3°C and 820 ml of n-heptane are added over the course
of 20 min. The mixture is stirred for 1 h at 0±3°C. The
pure SPDB is isolated by filtration, washed with

2 x 90 ml of ice-cold n-heptane and dried in an oven
(30°C, 50 mbar). Yield: 84.8%, HPLC purity: 98.7%.

CLAIMS
1. A method for preparing an activated ester of
formula (I)

in which R is a linear or branched (C1-C6) alkyl,
aryl, heteroaryl, cycloalkyl or heterocycloalkyl
group, and Alk is a linear or branched (C1-
C6)alkylene group,
said method consisting in reacting the
dicyclohexylamine salt P1:

and disuccinimidyl carbonate (DSC) in a solvent in
which the dicyclohexylamine salt of N-
hydroxysuccinimide P2

precipitates.

2. The method as claimed in claim 1, in which R is a
methyl, ethyl, propyl, butyl or pentyl group,
which is optionally branched, or the 2-pyridinyl
group.
3. The method as claimed in claim 1 or 2, in which
Alk is a propylene, butylene or pentylene group,
which is optionally branched.
4. The method as claimed in claim 1 or 2, in which
Alk is the (CH2)n group, n denoting an integer
ranging from 1 to 6.
5. The method as claimed in one of claims 1 to 4, in
which the reaction is carried out in a ketone.
6. The method as claimed in claim 5, in which the
ketone is MIBK.
7. The method as claimed in any one of claims 1 to 6,
in which, after having reacted P1 and DSC, the
reaction mixture is cooled in order to promote the
precipitation of P2.
8. The method as claimed in one of claims 1 to 7, in
which P2 is removed by mechanical separation.
9. The method as claimed in claim 8, in which the
mechanical separation is filtration.
10. A product of formula P1:


in which R and Alk are as defined in one of claims
1 to 4.
11. The product as claimed in claim 10, of formula:

The invention relates to a method for preparing an activated
ester of the formula (I), where R is a (C1-C6) alkyl, aryl, heteroaryl, cycloalkyl,
or heterocycloalkyl group, and ALK is a (C1-C6) alkylene group, said method
consisting of reacting the dicyclohexylamine P1 salt and the disuccinimidyl
carbonate (DSC) in a solvent in which the dicyclohexylamine salt of the N-
hydroxysuccinimide P2 is precipitated. The invention also relates to products
of the formula P1.