Positional linker effects in haptens for cocaine immunopharmacotherapy

Article abstract of DOI:10.1016/j.bmcl.2007.05.032

Cocaine use remains a serious problem, despite intensive efforts to curb abuse. Given the lack of effective pharmacotherapeutics for the treatment of cocaine addiction, research groups have targeted immunopharmacotherapy in which the drug user's immune sy

Full text of DOI:10.1016/j.bmcl.2007.05.032

Bioorganic & Medicinal Chemistry Letters 17 (2007) 4280–4283
Positional linker effects in haptens for cocaine
immunopharmacotherapy
a,b,
Akira Ino,a Tobin J. Dickersona,b, and Kim D. Janda
*
*
aDepartments of Chemistry and Immunology, and The Skaggs Institute for Chemical Biology,
10550 North Torry Pines Road, La Jolla, CA 92037, USA
bWorm Institute for Research and Medicine (WIRM), The Scripps Research Institute, 10550 North Torrey Pines Road,
La Jolla, CA 92037, USA
Received 21 April 2007; revised 8 May 2007; accepted 9 May 2007
Available online 16 May 2007
Abstract—Cocaine use remains a serious problem, despite intensive efforts to curb abuse. Given the lack of effective pharmacother-
apeutics for the treatment of cocaine addiction, research groups have targeted immunopharmacotherapy in which the drug user’s
immune system is trained to recognize and remove cocaine prior to entry into the central nervous system. Antibody cocaine esterases
and simple binders have been procured, however, rates and/or affinities still need improvement before clinical trials are warranted.
Herein, we report the synthesis and testing of two new haptens for the procurement of cocaine binding antibodies and cocaine ester-
ase catalytic antibodies. Central in the design of these haptens was the placement of the linker functionality distal from the antic-
ipated cocaine epitopes in an attempt to bury the hapten deep within an antibody combining site to gain possible entropic and
enthalpic advantages.
Ó 2007 Elsevier Ltd. All rights reserved.
Abuse of the psychostimulant cocaine (1), one of the
most addictive stimulants that directly affects the brain,
is a continuing medical, social, and political problem in
many areas of the world. According to the 2005
National Survey on Drug Use and Health (NSDUH),
an estimated 2.4 million people are current cocaine
users, an increase over the 2 million users in 2004.1 De-
spite intensive research efforts, there are no medications
with proven efficacy for the treatment of cocaine addic-
tion. Pharmacotherapies that are typically designed to
block the central neurochemical effects of cocaine have
had limited success, because their actions are non-
selective and thus often generate adverse side effects.
Furthermore, given the mechanism of action of cocaine
as a dopamine transport blocker, the inherent difficulty
in antagonizing an interaction such as this has led many
laboratories, including our own, to explore protein-
based therapeutics for the treatment of cocaine
addiction.
Recently, immunopharmacotherapy for cocaine abuse
has been explored as a possible alternative approach.2
This strategy is based on the synthesis of a cocaine vac-
cine that induces the production of anti-cocaine anti-
bodies. The antibodies bind the cocaine specifically to
inhibit the penetration into the CNS, thus this approach
can avoid the side effects of traditional pharmacother-
apy. In a series of investigations from our laboratory,
we have reported two effective haptens GNC (2)3 and
GND (3)4 that resemble the molecular structure of
cocaine (Fig. 1). Active immunization of rodents with
a GNC-keyhole limpet hemocyanin (KLH) conjugate
reduced the psychoactive effects of cocaine, and lowered
the cocaine levels in the CNS of treated rodents,2 while
immunization with GND–KLH resulted in a dramatic
suppression of the psychomotor stimulant effect of
cocaine.3
These, and other, previously reported haptens4 have
been designed with a linker moiety positioned at the
cocaine methyl ester functionality, primarily for ease
of preparation. However, placing the linker here also
may decrease efficient antibody binding as it may com-
promise the burying of key epitopes, that is, the benzoyl
and methyl esters within the antibody combining site.5
Herein, we report the synthesis of haptens AI1 (4) and
Keywords: Cocaine; Drugs of abuse; Immunopharmacotherapy; Vac-
cine; Transition state analog.
*
Corresponding authors. Tel.: +1 858 784 2516; fax: +1 858 784 2529
(K.D.J); e-mail: kdjanda@scripps.edu
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.05.032

A. Ino et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4280–4283
4281
Me
O
Me
TBSO
O
O
O
Me
Me
N
N
OH
N
N
OMe
X
X
a
TBSO
b
OMe
OMe
(1S)-6
O
O
O
O
O
O
O
O
6
7
O
Cocaine (1)
GNC (2) X=O
GND (3) X=NH
Scheme 1. Optical resolution of compound 6. Reagents and condi-
tions: (a) i—(1S)-(ꢀ)-camphanic chloride, Et3N, CH2Cl2, rt; ii—
diastereomer separation, 38%; (b) LiOH, quant.
O
Me
O
H
N
OMe
HO2C
N
O
zation. The stereochemistry was confirmed by single-
crystal X-ray analysis (Fig. 2).10 Meltzer et al. obtained
the (1R,10S) diastereomer and abandoned the isolation
of (1S,10S) diastereomer by this procedure in which
the protecting group of C-7 alcohol was a MOM group
instead of TBS.9 It is interesting to note that by chang-
ing the nature of the protecting group, a reversal in the
crystalline isomer was observed.
O
O
AI1 (4)
O
Me
H
N
OMe
HO2C
N
O
O
O
P
O
OH
AI2 (5)
To complete the synthesis of the desired hapten, the syn-
thetic route of Kozikowski et al. used for the synthesis
of racemic 7-hydroxylated cocaine was employed
(Scheme 2).11 Thus, Na–Hg reduction of (1S)-6 gave
(1S)-8 as a minor product along with major 2a-isomer.
Benzoylation and subsequent cleavage of the silyl ether
gave chiral (1S)-9 in good yield. Introduction of the lin-
ker moiety was achieved by activation with 4-nitro-
phenyl chloroformate, followed by reaction with
benzyl 6-aminohexanoate. Finally, the benzyl group
was removed to give the target hapten 4 which subse-
quently was coupled to the carrier proteins bovine serum
albumin (BSA) and keyhole limpet hemocyanin (KLH).
Coupling efficiency for the BSA immunoconjugate was
estimated to be 27:1 by MALDI-TOF analysis.
Figure 1. Structures of cocaine and haptens GNC, GND, AI1, and
AI2.
AI2 (5), designed as optimized haptens for immuno-
pharmacotherapy of cocaine abuse, and the ability of
antibodies induced from these haptens to bind cocaine.
We envisioned that by positioning the linker distal from
the native ester functionalities present in cocaine, an im-
proved immune response would be obtained by allowing
the cocaine molecule to be further sequestered within the
antibody binding site. This hypothesis is grounded upon
crystallographic analysis of a cocaine esterase6 and an
antibody-cocaine complex.5 Both GNC and GND have
a hexanoic acid linker for conjugation with a carrier
protein at the C-2 methyl ester moiety. Alternatively,
haptens 4 and 5 have the linker attached at the C-7 posi-
tion by means of a carbamate spacer. In essence, this
tact places the carrier protein a greater distance away
from the three anticipated epitopes of cocaine, that is,
the methyl ester, benzoate, and N-methyl amino moie-
ties. Although the importance of the two-carbon bridge
region for cocaine binding receptor topography has been
suggested,7 it is expected that antibodies induced from
haptens 4 and 5 will recognize cocaine better than anti-
bodies from GNC or GND.
The KLH conjugate was then used to immunize
129GIX+ mice followed by a booster injection after 1
week. Mice were then bled after an additional week
and antibody titers measured by ELISA using the 4-
BSA immunoconjugate. For comparison, the titer of a
comparable immunization of GNC–KLH also was mea-
sured. Both haptens resulted in robust immune
responses with measured titers of greater than 25,600.
In an alternative pharmacotherapeutic approach,
cocaine esterase catalytic antibodies have been envisioned
as a potentially powerful tool in treating cocaine addic-
tion.12 These antibodies would then overcome the requi-
site stoichiometry of cocaine binding antibodies, and
Although a large number of cocaine derivatives have
been synthesized, only a few chiral derivatives that pos-
sess substituents at the C-7 position have been reported.
We began the synthesis of hapten 4 from known racemic
7b-(tert-butyldimethylsilyloxy)-2b-(methoxycarbonyl)-
tropan-3-one (6), which can be prepared from 2,5-
dimethoxy-2,5-dihydrofuran.8 In our previous immuno-
pharmacotherapy programs, a critical feature of all
haptens has been the presence of the natural stereo-
chemistry. In order to obtain enantiopure 6, we per-
formed an optical resolution of the racemic compound
and achieved separation of the derived diastereomers,
following the method of Meltzer et al. (Scheme 1).9
Racemic 6 was reacted with (10S)-camphanic chloride
to give diastereomeric esters that could be separated
by column chromatography and subsequent recrystalli-
Figure 2. X-ray crystallographic structure of compound 7.

4282
A. Ino et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4280–4283
Me
TBSO
O
O
Me
TBSO
N
N
a
OMe
OH
b
OMe
OH
8
(1S)-6
O
O
Me
HO
Me
O
H
N
N
OMe
OMe
HO2C
N
c
d
O
O
O
O
O
AI1 (4)
9
O
Me
O
O
H
N
OMe
N
BSA
N
H
O
O
O
AI1-BSA conjugate (10)
Scheme 2. Synthesis of hapten AI1 immunoconjugate. Reagents and conditions: (a) Na–Hg, aq H2SO4, 9%; (b) i—BzCl, Et3N, DMAP; ii—TBAF,
84%; (c) i—4-nitrophenoxy chloroformate, Et3N, DMAP; ii—benzyl 6-aminohexanoate; iii—H2, Pd/C, 70%; (d) i—Sulfo-NHS, EDC; ii—BSA, PBS,
coupling efficiency = 27:1.
O
O
Me
O
Me
TBSO
H
N
N
OMe
OMe
a
BnO2C
N
b
c
(1S)-6
O
O
P
O
O
P
O
OBn
OBn
11
12
O
O
Me
O
Me
O
O
H
H
N
N
N
OMe
OMe
d
HO2C
N
BSA
N
H
O
O
O
O
O
O
P
P
OH
OH
A
AI2 (5)
I2-BSA conjugate (13)
Scheme 3. Synthesis of AI2 immunoconjugate. Reagents and conditions: (a) i—PhPOCl2, 1H-Tetrazole, i-Pr2NEt then BnOH; ii—TBAF, 89%, (b)
i—4-nitrophenoxy chloroformate, Et3N, DMAP; ii—benzyl 6-aminohexanoate, 59%; (c) H2, Pd/C, quant; (d) i—Sulfo-NHS, EDC; ii—BSA, PBS,
coupling efficiency = 7:1.
thus provide a mechanism to avoid saturation at larger
drug doses. However, much like previously developed
cocaine binding antibodies, the haptens used in these
studies have linkers positioned on either the cocaine
methyl ester or the tropane nitrogen atom.12 Antibodies
generated using this tact, while viable cocaine esterases,
still fall well short of the needed pharmacokinetic rates
for potential clinical utility. The most efficient cocaine
esterases are of bacterial origin and place the two
cocaine esters deep within the enzyme binding cleft. In
an attempt to model such interactions in an antibody
binding site, repositioning of the linker was needed.
Hence, we envisioned that by moving the linker distal
to the 30-transition state moiety, better presentation of
the hapten may be achieved, resulting in more efficient
catalysis.
and the KLH immunoconjugate injected into mice.
Again, good antibody titer was observed after immuni-
zation followed by a single booster injection, as mea-
sured by ELISA.
Using the sera from both AI1 and AI2 mice, competi-
tion studies were performed to assess antibody recogni-
tion of free cocaine. Unfortunately, these studies
revealed that both haptens bound cocaine rather poorly
(Kd,app P 400 lM) relative to GNC (Kd,app ꢁ 12.5 lM).
Further competition experiments demonstrated that the
antibodies resulting from immunization with either hap-
ten more efficiently bound benzoyl ecgonine
(Kd,app ꢁ 25 lM), the product of cocaine methyl ester
hydrolysis and primary cocaine metabolite. While this
compound is also psychoactive14 and thus could be
viewed as a target for immunotherapy, a viable vaccine
must be able to bind the parent drug prior to CNS entry
and any subsequent neurochemical effects. From a
mechanistic standpoint, the lability of this ester is
presumed to stem from neighboring-group participation
by the tropane nitrogen, as previously characterized
Our synthesis utilized the same enantiopure intermedi-
ate 6 as was used in the preparation of 4. Installation
of the benzyl phosphonate moiety,13 followed by TBS
deprotection and linker installation proceeded in accept-
able yield to give protected hapten 12, which was then
hydrogenated to yield the desired hapten 5 (Scheme 3).
This compound was then coupled to BSA and KLH,
with
cocaine,
but
not
pseudococaine
or
N-acylnorcocaine.15

A. Ino et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4280–4283
4283
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Acknowledgment
We gratefully thank The Skaggs Institute for Chemical
Biology and Shionogi & Co., Ltd. for financial support.
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