Facile synthesis of maleimide bifunctional linkers

Article abstract of DOI:10.1016/S0040-4039(02)00192-2

The synthesis of maleimide containing derivatives capable of serving as linkers for the conjugation of proteins is described. These compounds differ from previously reported linkers by having multiple sites available for drug attachment. Compounds 3a, 3b,

Full text of DOI:10.1016/S0040-4039(02)00192-2

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 43 (2002) 1987–1990
Facile synthesis of maleimide bifunctional linkers
H. Dalton King,* Gene M. Dubowchik and Michael A. Walker*
Bristol Myers Squibb, Pharmaceutical Research Institute, 5 Research Parkway, Wallingford, CT 06492, USA
Received 29 November 2001; revised 23 January 2002; accepted 25 January 2002
Abstract—The synthesis of maleimide containing derivatives capable of serving as linkers for the conjugation of proteins is
described. These compounds differ from previously reported linkers by having multiple sites available for drug attachment.
Compounds 3a, 3b, and 3d were synthesized from the corresponding amino alcohols by the addition of two equivalents of t-butyl
bromoacetate to a series of amino alcohols followed by the introduction of the maleimide under Mitsunobu reaction conditions
and ester hydrolysis. © 2002 Elsevier Science Ltd. All rights reserved.
Maleimide linkers1 such as the readily available 1 and 2
have become very useful in the area of bioconjugate
chemistry. These linkers allow the conjugation of
sulfhydryl containing molecules such as peptides having
cysteine residues, that react with the maleimide func-
tionality, to a second molecule via an amide or ester
bond (Fig. 1). This allows the construction of various
moieties including peptide-conjugate haptens, immobi-
lized antibodies or enzymes, immuno-conjugates or
-toxins, and immunodiagnostic agents.2
Maleimide is a very good linker since it reacts exclu-
sively with cysteine SH groups over other nucleophilic
amino acids.3 Because cysteine is usually present in only
discrete numbers, this results in nearly homogenous
conjugates where the stoichiometry and site of attach-
ment are predictable. The activity and physical proper-
ties of the protein are thus preserved by reducing
deleterious conjugation and by maintaining the native
pI.
In an effort to synthesize immunoconjugates with
improved activity4 we have designed the maleimide
bifunctional linker 3. Our primary interest was develop-
O
O
O
O
N
N
OH
O
O
O
OH
Z
NH
O-tBu
Br
N
O-tBu
1
2
Z
NH
O
O
NH2
•HCl
KHCO3,
O-tBu
DMF
N
O
ligand
X
S
4
O
5
N
protein
( )n
N
N
OH
O
H2N
O-tBu
( )n
N
O
O
H2, 10% Pd-C
HOAc
1. Maleic anhydride
O
O
O
O
O
X
2. TMS-Cl
Et3N
OH
ligand
O-tBu
•HOAc
ACN
6
3
Protein-conjugate
(X = NH, O)
O
O
N
O-tBu
N
OH
Figure 1. Maleimide linkers and protein conjugate.
p-TsOH
N
N
O
O
O
O
O
O
O-tBu
OH
3a
•TsOH
7a
* Corresponding authors. Tel.: 203-677-6685 (H.D.K.); fax: 203-677-
7702; tel.: 203-677-6686 (M.A.W.); e-mail: dalton.king@bms.com;
michael.a.walker@bms.com
Scheme 1. Standard synthesis of 3a (Z=benzyloxycarbonyl).
0040-4039/02/$ - see front matter © 2002 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(02)00192-2

1988
H. D. King et al. / Tetrahedron Letters 43 (2002) 1987–1990
which was then deprotected to 6. Reaction with maleic
anhydride followed by dehydration with TMS-Cl/tri-
ethylamine yielded the maleimide 7a. Removal of the
t-butyl esters was accomplished with p-toluenesul-
phonic acid (Scheme 1).7 Alternatively, we employed
our method for synthesizing N-alkyl maleimides,
namely the Mitsunobu reaction.8 Thus, selective protec-
tion is not required since the maleimide is introduced
via displacement of a hydroxyl group onto an amino
alcohol (Scheme 2). This allows the synthesis to be
accomplished in just three steps starting from the
appropriate amino alcohol.
Amino alcohols 8a–d were readily alkylated with two
equivalents of tert-butyl bromoacetate to provide the
corresponding bis-esters 9a–d in good yield. Our
modified Mitsunobu conditions (method A), wherein
0.5 equiv. of neopentyl alcohol is used as a non-reacting
ligand for phosphorous, gave favorable yields for 7a–b
and 7d.9 When unmodified conditions (method B) were
used the yields were lower and occasionally, depending
on the addition order and reagent stoichiometry, no
product was obtained.
Unexpectedly, the Mitsunobu reaction was not success-
ful for 9c,10 rather, a side reaction appeared to be
interfering. Instead of the expected product (not
shown), 11 and 12 were isolated from the crude reac-
tion mixture in modest yield (ꢀ30% combined yield).
The by-products, 11 and 12, can arise from a Stevens
rearrangement of the putative zwitterion 10 shown in
Scheme 3. In the absence of maleimide and neopentyl
alcohol, the reaction of 9c with Ph3P and DEAD11
gives 12 exclusively.12
Scheme 2. Modified synthesis of series 3.
ing a linker which would increase drug loading onto a
MAb in the presence of a limited number of available
cysteine conjugation sites without causing aggregation.
Linker 3 offers the following advantages over 1 and 2
by: (i) the presence of a water solubilizing amine func-
tionality; (ii) the ability to couple two components at a
time to the MAb (cf. protein-conjugate, Fig. 1) and (iii)
a straight-forward synthesis that can be modified, if
necessary, to optimize the linker length. A MAb such as
BR96, which generates eight thiol groups on mild DTT
reduction would yield a conjugate with a possible mole
ratio of 16 drugs per MAb when used with linker 3.5
The synthesis of 3a–b,d was completed by acid medi-
ated removal of the tert-butyl ester groups with p-
toluenesulphonic acid.13 The resulting linkers can be
coupled directly to amines using DCC. As an example,
3a was coupled to the protected dipeptide amide 1314 to
give 14 in good yield (Scheme 4). Compound 14 may be
further elaborated to couple an NH2- or OH-containing
cytotoxic drug 15.14 A full description of this chemistry
will be the subject of a future publication.7
The synthesis of 3a using standard literature methods
requires manipulation of protecting groups on the
diaminoalkane, alkylation, and installation of the
maleimide via condensation of maleic acid.6 Thus, 4
was dialkylated with tert-butyl bromoacetate to give 5,
In conclusion, we have demonstrated a facile three-step
synthesis of a unique set of maleimide linkers 3a–b,d15
CO2-tBu
Ph3P/DEAD
-
CO2-tBu
CO2-tBu
+
N
N
CO2-tBu
HO
9c
10
CO2-tBu
CO2-tBu
N
N
CO2-tBu
11
12
Scheme 3.

H. D. King et al. / Tetrahedron Letters 43 (2002) 1987–1990
1989
Toru, T.; Ueno, Y. J. Org. Chem. 1997, 62, 2652 and
references cited therein.
7. Experimental details for this synthesis are reported in
Dubowchik et al., submitted for publication.
8. (a) Walker, M. A. Tetrahedron Lett. 1994, 35, 665; (b)
Walker, M. A. J. Org. Chem. 1995, 60, 5352.
3a
OH
L-Phe-L-Lys(Mtr)
N
H
NHS
DCC
DME
13
O
9. Representative synthetic procedure for 7a: In a 1000 mL
3-neck round bottomed flask, equipped with two drop-
ping funnels, Ph3P (27.0 g, 0.103 mol) was dissolved in
700 mL of THF and cooled to −78°C. DEAD (16.22 mL,
0.103 mol) was added dropwise via a dropping funnel
and the resulting light yellow solution stirred 5 min.
Compound 9a (29.7 g, 0.103 mol) was dissolved in 100
mL of THF and added dropwise over 15 min using the
second dropping funnel. Neopentyl alcohol (4.54 g, 0.052
mol) was added in one portion followed by maleimide (10
g, 0.103 mol). The flask was removed from the cooling
bath and allowed to stir overnight. The reaction mixture
was concentrated to 1/4 the original volume by rotary
evaporation and purified by flash column chromatogra-
phy with 6:1 hexanes/EtOAc on SiO2. Pure 7a (25 g, 66%
yield) was isolated as a solid mp 64–66°C. 1H NMR
(CDCl3) l 1.37 (s, 9), 2.85 (t, 2, J=6.4), 3.36 (s, 4), 3.54
(t, 2, J=6.5), 6.62 (s, 2). 13C NMR (CDCl3) l 28.08,
35.68, 51.42, 55.34, 80.92, 134.03, 170.35, 170.82. 7b:
OH
N
L-Phe-L-Lys(Mtr)-N
H
N
O
O
O
OH
L-Phe-L-Lys(Mtr)-N
H
14
Ab
S
O
O
O
N
L-Phe-L-Lys-N
NH-Drug
N
H
O
O
O
NH-Drug
O
L-Phe-L-Lys-N
H
O
Scheme 4.
1
Solid mp 51–52°C. H NMR (CDCl3) l 1.33 (s, 9), 1.62
(p, 2, J=7.1), 2.61 (t, 2, J=7.0), 3.29 (s, 3), 3.48 (t, 2,
J=7.1), 6.57 (s, 2). 13C NMR (CDCl3) l 27.74, 35.82,
51.28, 55.50, 80.78, 134.01, 134.92, 170.41, 170.69. 7d:
which, utilizing standard disconnections, require five–
six steps to accomplish. Our synthesis utilizes modified
Mitsunobu reaction conditions to install a maleimide
moiety in one step onto appropriately substituted
amino alcohols. Linkers 3a–b,d may be coupled to
cytotoxic drugs and used to construct MAb conjugates.
1
Oil. H NMR (CDCl3): l 1.04 (m, 6), 1.22 (s, 18), 1.30
(m, 2), 2.42 (dd, 2, J=7.27), 3.17 (s, 4), 3.26 (t, 2, J=7.2),
6.45 (s, 2). 13C NMR (CDCl3): l 26.49, 26.55, 27.69,
27.99, 28.33, 37.64, 53.83, 55.72, 80.61, 133.86, 170.54,
170.66.
10. We have found that the Mitsunobu procedure is difficult
for amino alcohols which have an amine group which is
optimally positioned for cyclization and/or has a higher
pKa.
References
11. Abbreviations: DEAD (diethylazodicarboxylate), DIAD
(diisopropylazodicarboxylate), Mtr (methoxytrityl), DCC
(dicyclohexylcarbodiimide).
12. For a more detailed discussion of this side reaction see:
Walker, M. A. Tetrahedron Lett. 1996, 37, 8133.
13. p-Toluenesulphonic acid (TsOH) was found to be supe-
rior to trifluoroacetic acid in this procedure and also
provides the products as solid p-tosylate salts, making
them convenient to handle and purify.
14. (a) Dubowchik, G. M.; Radia, S. Tetrahedron Lett. 1997,
38, 5257; (b) Dubowchik, G. M.; Firestone, R. A. Bioorg.
Med. Chem. Lett. 1998, 8, 3341; (c) Dubowchik, G. M.;
Mosure, K.; Knipe, J. O.; Firestone, R. A. Bioorg. Med.
Chem. Lett. 1998, 8, 3347; (d) De Groot, F. M. H.; de
Bart, A. C. W.; Verheijen, J. H.; Scheeren, H. W. J. Med.
Chem. 1999, 42, 5277.
1. Wong, S. S. Chemistry of Protein Conjugation and Cross-
linking; CRC Press: Boca Raton, 1991 and references
cited therein.
2. For reviews and recent examples, see: (a) Dubowchik, G.
M.; Walker, M. A. Pharmacol. Ther. 1999, 83, 67–123;
(b) Pai, L. H.; Pastan, I. J. Am. Med. Assoc. 1993, 269,
78; (c) Rusiecki, V. K.; Warne, S. A. Bioorg. Med. Chem.
Lett. 1993, 3, 707; (d) Janda, K. D.; Ashley, J. A.; Jones,
T. M.; McLeod, D. A.; Schloeder, D. M.; Weinhouse, M.
I. J. Am. Chem. Soc. 1990, 112, 8886; (e) Pietersz, G. A.
Bioconjugate Chem. 1990, 1, 89; (f) Maggio, E. T.
Enzyme-Immunoassays; CRC Press: Boca Raton, 1980.
3. Maleimide reacts approximately 1000-times faster with
thiols than with amines at neutral pH and below. At this
pH the amino groups of lysine and arginine are mostly
protonated.
15. Analytical data 3a: 1H NMR (DMSO-d6) l 2.25 (s, 3),
3.51 (m, 2), 3.80 (m, 2), 4.21 (s, 4), 7.01 (s, 2), 7.10 (d, 2,
J=8.0), 7.50 (d, 2, J=8.0), 11.09 (br s, 2). 13C NMR
(DMSO-d6): l 20.86, 32.28, 52.54, 53.66, 125.58, 128.39,
134.96, 138.47, 144.65, 167.44, 170.84. MS (HR ESI)
calcd for C10H12N2O6·H+ (MH+): 257.0774. Found:
257.0779. 3b: 1H NMR (DMSO-d6): l 1.88 (m, 2), 2.28 (s,
3), 3.20 (m, 2), 3.42 (m, 2), 4.12 (s, 4), 7.04 (s, 2), 7.11 (d,
2, J=8.0), 7.48 (d, 2, J=8.0). 13C NMR (DMSO-d6): l
20.75, 23.55, 34.23, 53.68, 54.21, 125.46, 128.07, 134.58,
4. Trail, P. A.; Willner, D.; Hellstro¨m, K. E. Drug Develop.
Res. 1995, 34, 196.
5. (a) Willner, D.; Trail, P. A.; Hofstead, S. J.; King, H. D.;
Lasch, S. J.; Braslawsky, G. R.; Greenfield, R. S.;
Kaneko, T.; Firestone, R. A. Bioconjugate Chem. 1993, 4,
521; (b) King, H. D.; Yurgaitis, D.; Willner, D.; Fire-
stone, R. A.; Yang, M. B.; Lasch, S. J.; Hellstrom, K. E.;
Trail, P. A. Bioconjugate Chem. 1999, 10, 279.
6. For a recent example, see: Reddy, P. Y.; Kondo, S.;

1990
H. D. King et al. / Tetrahedron Letters 43 (2002) 1987–1990
J=8.0), 7.48 (d, 2, J=8.0). 13C NMR (DMSO-d6): l
137.71, 145.49, 167.69, 171.01. Anal. calcd for
20.75, 22.98, 25.29, 25.58, 27.72, 36.89, 54.11, 55.94,
125.46, 128.06, 134.45, 137.72, 145.48, 167.63, 171.08. MS
(HR ESI) calcd for C14H20N2O9·H+ (MH+): 313.1400.
Found: 313.1408.
C11H14N2O6·C7H8SO3: C, 48.86; H, 5.01; N, 6.33. Found:
C, 48.66; H, 5.07; N, 6.21. 3d: H NMR (DMSO-d6): l
1.08 (m, 4), 1.45 (m, 2), 1.59 (m, 2), 2.28 (s, 3), 3.18 (m,
2), 3.37 (t, 2, J=6.9), 4.14 (s, 4), 7.01 (s, 2), 7.11 (d, 2,
1