Development of novel linkers to conjugate pharmacophores to a carrier antibody

Article abstract of DOI:10.1016/j.bmcl.2012.05.040

We have developed modified maleimide novel linkers with improved chemical stability that could potentially be used in conjugating various pharmacophores such as oligo nucleotides, peptides, and proteins to antibodies to afford novel biologics with well-de

Full text of DOI:10.1016/j.bmcl.2012.05.040

Bioorganic & Medicinal Chemistry Letters 22 (2012) 4249–4253
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Development of novel linkers to conjugate pharmacophores
to a carrier antibody
⇑
Moorthy S. S. Palanki , Abhijit Bhat, Rodney W. Lappe, Bin Liu, Bryan Oates, John Rizzo,
Nebojsa Stankovic, Curt Bradshaw
CovX Research, Pfizer Worldwide Research and Development, 9381 Judicial Drive, Suite 200, San Diego, CA 92121, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
We have developed modified maleimide novel linkers with improved chemical stability that could poten-
tially be used in conjugating various pharmacophores such as oligo nucleotides, peptides, and proteins to
antibodies to afford novel biologics with well-defined therapeutic benefits and improved pharmacoki-
netic properties. These linkers expand the array of tools available for bioconjugation of pharmacophores
to antibodies.
Received 11 April 2012
Accepted 8 May 2012
Available online 17 May 2012
Keywords:
Linkers
Ó 2012 Elsevier Ltd. All rights reserved.
Bioconjugation
Synthesis of novel linkers
Protein based drugs have enabled targeted modulation of bio-
logically relevant targets not easily druggable by small molecules.
There is a growing interest in developing chemical, biochemical,
and genetic tools for doing ‘medicinal chemistry’ with proteins in
order to optimize their pharmaceutical properties. One of the areas
of focus is developing new bioconjugation chemistries that enable
modulation of specific residues on a protein with improved phar-
macokinetic and pharmacodynamic properties. Many of these pro-
teins have been modified chemically for various purposes such as;
proteins attached to polyethylene glycol as novel therapeutics;
proteins attached to chemical probes for studying cellular func-
tion; proteins with photoreactive group to study the protein bind-
ing site; and proteins with artificial cofactors to impart novel
activity.1 There is an ongoing need for additional techniques to
selectively modify proteins to further develop conjugates as thera-
peutics, probes or sensors.
If a protein has solvent accessible cysteine thiol, one can take
advantage of the reactivity of thiol to selectively modify the func-
tional group. The thiol group is a more reactive nucleophile than
amine towards certain electrophiles, such as maleimide under
physiological conditions. Maleimide offers several advantages
including high selectivity towards thiol under neutral and acidic
conditions in the presence of an amine,2 ease of synthesis and rea-
sonable aqueous solubility. Thiols in proteins and peptides react
with maleimides in a Michael reaction with very high efficiency
to form succinimide rings. In the absence of an accessible surface
thiol functional group, a widely used protein modification is the
O
O
COOH
HN R2
H2O
+
N
R2
R1
HN R2
COOH
R1
S
R1
S
O
S
O
2
1
3
Figure 1. Hydrolysis of an amide bond.
replacement of a suitable surface accessible amino acid with a
cysteine. The cysteine can be introduced relatively easily in most
of the proteins and conjugated to maleimide with relative ease.
However, after the maleimide conjugation with a thiol, the result-
ing succinimide ring is susceptible to ring-opening hydrolysis in
aqueous buffers, or a retro-Michael reaction followed by reaction
with other thiols (such as albumin or glutathione) in vivo. These
liabilities could influence drug stability as well as PK/PD profile
of the molecules. While the implications of succinamide ring-open-
ing in vivo are not well understood, it can present a problem in
drug product stability. In this publication we disclose modified
maleimide analogs with improved chemical stability, while main-
taining the efficiency of maleimide reactions towards thiol nucleo-
philes in Michael reaction. We also disclose the synthesis of new
linkers with the modified maleimide ring that would be suitable
for conjugating proteins to an antibody.3
The succinimide ring amide bond undergoes hydrolysis to form
acid products at either of the two carbonyl groups even under
neutral conditions (Fig. 1).4–6
It is known that the amide bond’s stability towards hydrolysis is
⇑
Corresponding author. Tel.: +1 858 964 2053.
E-mail address: moorthy.palanki@pfizer.com (M.S.S. Palanki).
affected by the succinimide ring’s substituents.7 The stability of
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.05.040

4250
M. S. S. Palanki et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4249–4253
molecule from undergoing a retro-Michael reaction. These modifi-
cations were selected to provide improved stability to the succini-
mide ring without affecting the efficiency of the Michael reaction
with a thiol.
The stability of these glutathione adducts (8–11) was studied at
pH 7.5 at 40 °C in a formulation buffer (100 mM Histidine, 200 mM
Glycine, and 200 mM Sucrose buffer). The materials were analyzed
for the formation of succinimide ring-opened acids as well as retro-
Michael products. The incubation mixture did not show any frag-
ments as a result of retro-Michael reaction or peptide hydrolysis.
The maleimide analog 8 underwent complete hydrolysis of the suc-
cinimide ring in 72 h, and was the least stable among the four fol-
lowed by the itaconimide analog 11. While dimethyl maleimide
analog was the most stable, the formation of the product 10 was
the least efficient (69% after 16 h at room temperature). It appears
that by simply substituting a methyl group on the succinimide ring,
the stability can be improved without sacrificing the conjugation
efficiency.
Next we examined the effect of groups substituted on the nitro-
gen of the succinimide ring and their role in facilitating a water
molecule to attack either carbonyl groups of the succinimide ring.
We evaluated four different connectors as shown below using the
model compounds 12–15 (Fig. 3). We used glutathione as a model
peptide in all four compounds. Methyl maleimide ring was chosen
due to its improved stability and comparable reactivity as malei-
mide towards thiol for further development. Three different linkers
were connected to the nitrogen of the methyl maleimide ring.
These nitrogen atom substituents were selected because these
modifications were of interest for incorporation in the linkers
(16–19) to be used for bioconjugation applications. These linkers
(16–19) have a maleimide ring on one end and a propanoyl azeti-
din-2-one group on the other end.12
Figure 2. Stability studies of succinimide groups at pH 7.5 (100 mM Histidine,
200 mM Glycine, and 200 mM Sucrose buffer), 40 °C. The incubation mixture was
analyzed using LC–MS.
methyl and dimethyl succinimide rings towards hydrolysis was
studied under basic conditions, although there was no example of
thiol substituted succinimide rings and their stability.8,9 Subse-
quently, it was recognized that thiol-substituted succinimide rings
undergo ring opening hydrolysis to form mixture of acids under
neutral conditions.10 The substitution of a methyl group on the suc-
cinimide ring improves the amide bond stability towards hydroly-
sis. We undertook the first systematic study to examine the
stability of thio-maleimide ring in neutral buffer, with the aim of
developing more stable succinimide linkers suitable for bioconju-
gations. These new linkers offer a potential advantage for conjugat-
ing various small molecules, oligonucleotides, peptides and
proteins to antibodies with improved stability in buffered solutions.
The preliminary hydrolysis studies were carried out on glutathi-
one conjugated N-phenyl succinimide compounds as shown in Fig-
ure 2. The syntheses of these compounds were outlined in Scheme 1.
Appropriately substituted N-phenyl succinimides (4–7)11 were
treated with reduced glutathione to give compounds 8–11. Com-
pound 9 has one methyl group on a carbon adjacent to a carbonyl
group, and 10 has methyl groups on each carbon. Compound 11
has a methylene bridge between glutathione and succinimide ring.
While compounds 8–10 can potentially undergo retro-Michael
reaction, the extra methylene group in 11 would slow down this
In recent publications we have demonstrated successfully that
one could combine a carrier monoclonal antibody such as aldolase
antibody with favorable pharmacokinetic properties and a small
molecule or peptide with therapeutic benefit to generate CovX
bodies that have desirable pharmacokinetic properties of the
antibody and therapeutic effect of peptides.13–15 The aldolase anti-
body has a lysine at position 93 of the heavy chain and located
deep in the hydrophobic binding pocket on each of the two Fab
arms.16 We have developed phenylpropanoylazetidin-2-one to
COOH
HN
O
O
N
H
SH
COOH
O
R1
S
HN
H2N
O
O
O
COOH
R1
R2
N
H
N
N
DMSO
R2
O
r.t.
H2N
O
Overnight
COOH
8
, R1 = R2 = H, 96%
4, R1 = R2 = H
9
, R1 = H, R2 = CH3, 98%
5
, R1 = H, R2 = CH3
10, R1 = R2 = CH3, 69%
6, R1 = R2 = CH3
COOH
O
HN
O
N
O
N
H
O
S
N
O
H2N
COOH
7
11, 93%
O
Scheme 1. Reagents and Conditions: (a) reduced glutathione (1.2 equiv), DMSO, rt, overnight.

M. S. S. Palanki et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4249–4253
4251
O
O
R
O
COOH
2
N
HN
O
O
O
N
O
O
N
Glutathione
O
12, R = H
c
a
O
13, R = CH3
O
O
O
O
O
O
N
O
N
b
O
30
29
COOH
Glutathione
N
O
2
O
18
O
HO
d
O
14
O
N
Glutathione
H
COOH
N
O
14
O
2
O
Scheme 3. Reagents and conditions: (a) 22, DIC, HOBT, 80 °C; (b) HCl, CH3CN, rt; (c)
28, HBTU, DIEA, DMF, rt; (d) glutathione, DMSO, rt.
15
COOH
HN
O
S
O
The synthesis of compounds 12 and 13 and linkers 16 and 17
are shown in Scheme 2. Nitrophenyl acid chloride 26 was prepared
from 25 as described.18 An amide proton in azetidin-2-one was
deprotanated with n-butyl lithium in tetrahydrofuran and reacted
with the acid chloride 26 to give 27. The nitro group in 27 was re-
duced to an amine using palladium on carbon in methanol at 35 °C
to give 28. Maleimide acids (20 and 21) were activated with N-hy-
droxy succinimide and reacted with PEG amine 22.19 The tertiary
butyl group was cleaved using 15% trifluroacetic acid in dichloro-
methane to give acids 23 and 24, respectively. The acids (23 and
24) were treated with glutathione to give glutathione adducts 12
and 13. The acids 23 and 24 were activated with HBTU and treated
with amine 28 to give linkers 16 and 17, respectively.
N
H
Glutathione
H2N
COOH
Figure 3. Structures of glutathione-maleimide linker.
react selectively with Lys-93 located deep in the hydrophobic
pocket and not with any other lysine on the surface of the antibody
or protein.17 We propose linkers 16–19 which would be useful for
conjugating either peptides or proteins to the aldolase antibody.
O
O
O
O
b
a
HO
Cl
N
25
26
27
NO2
NO2
NO2
c
R
R
O
O
O
O
N
N
N
O
NH2
O
N
O
O
N
28
R
NH2
O
d
O
O
N
+
O
f
23
24
O
O
, R = H
O
O
HO
O
e
O
, R = CH3,
N
O
N
22
tBu-O
20,
R = H
21
, R = CH3
O
O
HO
g
O
16
17
, R = H
, R = CH3,
12
, R = H
13
, R = CH3,
Scheme 2. Reagents and conditions: (a) SOCl2, toluene, rt; (b) azetidin-2-one, n-BuLi, THF, À78 °C to rt; (c) H2, 1 N HCl, Pd/C, methanol, 35 °C; (d) N-hydroxysuccinimide,
diisopropylcarbodiimide, THF, rt; (e) 15% TFA in CH2Cl2, rt; (f) 28, HBTU, DIEA, DMF, rt; (g) glutathione, DMSO, rt.

4252
M. S. S. Palanki et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4249–4253
Figure 4. Hydrolytic stability studies of succinamide ring in a formulation buffer (100 mM Histidine, 200 mM Glycine, and 200 mM Sucrose) at pH 7.5, 40 °C; 7.5, 4 °C; and
6.5, 40 °C . The formation of the succinimide ring-opened acid products was measured using LC–MS.
treated with glutathione in DMSO to afford 15 in quantitative yield.
The acid 33 was coupled to 28 using HBTU in the presence of HOBT
in DMF to afford linker 19.
O
O
O
a
F
F
N
H2N
O
F
F
F
OH
b
The ring stability of glutathione adducts 12–15 was studied
under the same conditions as described above. Figure 3 shows
the result of succinimide ring stability studies at neutral pH. When
the phenyl ring in 8 and 9 was replaced with an aliphatic chain as
shown in 12 and 13 we saw improved stability of the succinimide
ring. While the stability improvement from compound 8 to 12 was
modest, the stability improvement from 9 to 13 was much more
pronounced. These improvements in stability were anticipated
since electron withdrawing groups such as phenyl would facilitate
the hydrolysis of an amide bond in succinimide. We noticed further
improvement in stability when propionyl group was replaced with
more hydrophobic alkyl chain (14) and cyclohexyl chain (15).
We also examined the stability of compounds 12-15 at lower
temperature, 4 °C at pH 7.5 and at lower pH 6.5 at 40 °C (Fig. 4).
By lowering the temperature, from 40 to 4 °C, the stability im-
proved approximately 15-fold. Similarly, by lowering the pH from
7.5 to 6.5, the stability of the ring increased approximately twofold.
The studies showing the improved stability in the succinimide
ring would help to develop appropriate buffer systems for admin-
istering the compound as well as storage conditions. We also
examined the stability of the succinimide ring in different buffers
at the same pH and at the same temperature and observed the type
of buffer has effect on the stability of the succinimide ring.
In summary, we have developed four different maleimide link-
ers (16–19). The methyl maleimide group in linkers 17–19 offer
improved stability towards hydrolysis without compromising the
reaction efficiency towards thiols (data not shown). These linkers
have the required aqueous solubility to dissolve in buffered solu-
tions for conjugation. These linkers have an azetidin-2-one group
that selectively reacts with lysine-93 in aldolase antibody and
modified maleimide groups to react selectively with a thiol group
in a pharmacophore. These new linkers expand the list of available
linkers in the bioconjugation field that could be used to conjugate
small molecules/oligonucleotides/proteins/peptides to an anti-
body. We have successfully used these linkers to conjugate both
peptides and proteins to the carrier antibody and evaluated their
activity both in vitro and in vivo. The application of these linkers
in conjugating a protein therapeutic to an antibody for generating
novel therapeutics will be disclosed in future.
O
31
32
O
O
c
e
N
OH
N
O
15
O
d
O
O
33
f
O
O
N
O
N
O
N
O
O
O
N
19
O
Scheme 4. Reagents and conditions: (a) 29, DMF, rt; (b) DIPEA, DMF, rt; (c) 22,
NMM, THF, rt; (d) 50% TFA, CH2Cl2, rt; (e) glutathione, DMSO, rt; (f) 28, HBTU, HOBT,
NMM, DMF, rt.
The synthesis of glutathione analog of linker 14 and the synthe-
sis of linker 18 were started from citraconic anhydride (29) as
shown in Scheme 3. A solution of citraconic anhydride, 29, PEG
amine 22 in DMF was stirred at room temperature for 2 h. Diiso-
propylcarbodiimide and HOBT were added and the reaction was
heated at 80 °C for 2 h that resulted in higher yield of an ester
intermediate compared to acetic acid approach.20 The ester inter-
mediate was treated with 50% 6 N HCl in acetonitrile to afford
30. The resulting acid 30 was treated with glutathione in DMSO
to give glutathione adduct 14. Acid 30 was coupled to amine 28
using HBTU and DIEA to give 18.
The synthesis of 15 and 19 are shown in Scheme 4. A solution of
citraconic anhydride (29) and trans-4-aminomethyl cyclohexane
carboxylic acid (31) in dimethylformamide was stirred at room
temperature for 6 h. The reaction solution was cooled to 0 °C, DI-
PEA followed by pentafluorophenyl trifluoroacetate in DMF were
added. The reaction mixture was warmed to room temperature
and stirred for another 16 h under N2 to give 32. Compound 32
was treated with PEG amine 22 in the presence of N-methyl mor-
pholine to give a crude intermediate which was treated with 50%
trifluoroacetic acid in dichloromethane to afford the acid interme-
diate 33 as white solid after HPLC purification. The acid 33 was
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