The synthesis of haptens and their use for the development of monoclonal antibodies for treating methamphetamine abuse

Article abstract of DOI:10.1021/jm901134w

In addition to addiction, the repeated use of (+)-methamphetamine [(+)-METH], (+)-amphetamine [(+)-AMP], or (±)-3,4- methylenedioxymethamphetamine ((±)-MDMA, commonly called ecstasy) can lead to life-threatening medical problems including cardiovascular i

Full text of DOI:10.1021/jm901134w

J. Med. Chem. 2009, 52, 7301–7309 7301
DOI: 10.1021/jm901134w
The Synthesis of Haptens and Their Use for the Development of Monoclonal Antibodies for Treating
Methamphetamine Abuse
F. Ivy Carroll,*,† Philip Abraham,† Paul K. Gong,† Ramakrishna R. Pidaparthi,† Bruce E. Blough,† Yingni Che,‡
Amber Hampton,‡ Melinda Gunnell,‡ Jackson O. Lay Jr.,§ Eric C. Peterson,‡ and S. Michael Owens‡
†Center for Organic and Medicinal Chemistry, Research Triangle Institute, P.O. Box 12194, Research Triangle Park, North Carolina 27709,
‡Department of Pharmacology and Toxicology, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205,
and §Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, Arkansas 72701
Received July 30, 2009
In addition to addiction, the repeated use of (þ)-methamphetamine [(þ)-METH], (þ)-amphetamine
[(þ)-AMP], or (()-3,4-methylenedioxymethamphetamine ((()-MDMA, commonly called ecstasy) can
lead to life-threatening medical problems including cardiovascular injury, severe depression, and psycho-
sis. Currently, there are no specific pharmacotherapies to treat these medical problems. In this study, we
report the design and synthesis of two haptens, (S)-(þ)-3-(9-carboxynonyloxy)methamphetamine
(3a, (þ)-METH MO10) and (S)-(þ)-3-(5-carboxypentyloxy)methamphetamine (3b, (þ)-METH MO6),
and their use in generating high affinity (low KD value) monoclonal antibodies (mAbs) against (þ)-
METH, (þ)-AMP, and/or (þ)-MDMA. On the basis of results from the determination of mAb KD values
and ligand specificity, the mAbs generated from hapten 3a showed the greatest promise for generating
active and passive immunotherapies for treating overdose or addiction from (þ)-METH-like stimulants.
The abuse of (S)-(þ)-methamphetamine [1a-(þ)-METH]a
and other amphetamine-like stimulants continues to be a
major health problem worldwide.1-3 Indeed, a study by
RAND Corporation estimates the economic cost of (þ)-
METH use in the United States in 2005 was $623.4 billion.4
This comprehensive estimate includes the economic burden of
addiction, premature death, drug treatment, and many other
aspects of the drug’s impact on Americans. The 2008 National
Survey on Drug Use and Health estimates that over 12 million
individuals, aged 12 and older, had used (þ)-METH in their
lifetime, that 850000 had used (þ)-METH during the past
year, and that 314000 individuals had used (þ)-METH during
the last month, which defines them as current users.5 Accord-
ing to the 2005 Drug Abuse Warning Network (DAWN)
report, there were 108905 methamphetamine-related emer-
gency departments (ED) visits in 2005.6 If (þ)-(S)-ampheta-
mine [1b, (þ)-AMP] was included, there were 138950 ED
visits.6
antibodies (mAbs) can rapidly remove the drug from its sites
of action in critical tissues like the brain and heart, suggesting
that immunotherapy could provide an important new medical
strategy for addressing (þ)-METH-induced adverse health
effects in humans while others suggest antibody catalyzed
inactivation of METH could be a possible therapeutic ap-
proach.7 (S)-(þ)-Amphetamine [1b, (þ)-AMP], which is a
major metabolite of (þ)-METH and a drug of abuse, and (þ)-
methylenedioxymethamphetamine, the stimulant-inducing
chemical in the racemic mixture of (()-methylenedioxy-
methamphetamine (2, (()-MDMA, commonly referred
to as ecstasy) are two other widely abused and dangerous
stimulants. The potency and stimulant effects of these (þ)-
3,4-METH-like compounds are influenced by the drug’s
stereochemistry, with the (þ)- or (S)-isomers producing
significantly more psychomimetic effects, stereotyped be-
havior, locomotor activity,8 and increased production of
reactive oxygen species in mice.9 Given the medical im-
portance of all three of these structurally related (þ)- or
(S)-isomers, it could be medically advantageous to have a
single mAb that could be used in the treatment of medical
problems resulting from (þ)-METH, (þ)-AMP, and/or
(þ)-MDMA. Indeed, we recently reported the immunolo-
gical design strategy, discovery, and development of an
mAb antibody named anti-(þ)-METH mAb4G9 that ex-
hibited a novel optimal combination of high affinity and
drug-class specificity for (þ)-METH, (þ)-AMP, and (þ)-
MDMA without significant cross-reactivity against other
METH-like ligands, over-the-counter medications, or
endogenous neurotransmitters.10
At present, there are no specific pharmacotherapies for
managing adverse (þ)-METH-induced effects like acute over-
dose and chronic addiction. Preclinical studies in rats show
that systemic administration of anti-(þ)-METH monoclonal
*To whom correspondence should be addressed. Phone: 919 541-
6679. Fax: 919 541-8868. E-mail: fic@rti.org.
a Abbreviations: (þ)-METH, (þ)-methamphetamine; mAb, monoclo-
nal antibody(ies); (þ)-AMP, (þ)-amphetamine; (þ)-MDMA, (þ)-3,4-
methylenedioxymethamphetamine; (-)-METH, (-)-methamphetamine;
(-)-AMP, (-)-amphetamine; (-)-MDMA, (-)-3,4-methylenedioxy-
methamphetamine; (()-MDMA, (()-3,4-methylenedioxymethampheta-
mine; BSA, bovine serum albumin; c-BSA, cationized bovine serum
albumin; RIA, radioimmunoassay; DAWN, Drug Abuse Warning Net-
work; ED, emergency department; OVA, ovalbumin; ELISA, enzyme-
linked immunosorbent assay; MALDI, matrix-assisted laser desorption/
ionization; MS, mass spectrometry.
In this study, we report the synthesis of the hapten (S)-(þ)-
3-(9-carboxynonyloxy)methamphetamine (3a, designated
(þ)-METH MO10) used to prepare the mAb4G9 along with
r
2009 American Chemical Society
Published on Web 10/29/2009
pubs.acs.org/jmc

7302 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22
Carroll et al.
comparisons with the synthesis of (S)-(þ)-3-(5-carboxypen-
tyloxy)methamphetamine (3b, designated (þ)-METH MO6),
an earlier prototypic hapten. We also present the development
and characterization of new anti-(þ)-METH mAbs resulting
from immunizations with these haptens covalently bound to
three different carrier proteins. The mAbs generated from
immunization with (þ)-METH MO10 hapten had signifi-
cantly lower KD valuesthanthe mAbs generated fromthe (þ)-
METH MO6 hapten. Furthermore, the mAbs generated
against the (þ)-METH MO10 hapten coupled to ovalbumin
showed the richest diversity of specificity and higher affinity
for all three medically important (þ)-METH-like stimulants,
(þ)-METH, (þ)-AMP, and (þ)-MDMA (see below).
Scheme 1a
Chemistry
Thesynthesisofthehaptens3aand 3b isshowninScheme1.
(S,S)-3-Methoxyphenyl-2-propyl-R-methylbenzylamine (5)
was synthesized by modification of a method reported for
other similar compounds.11 Thus, reductive alkylation of m-
methoxyphenylacetone (4) with (S)-(-)-R-methylbenzyla-
mine using triacetoxyborohydride in ethylene dichloride gave
a mixture of (R,S)- and (S,S)-5. Recrystallization of the
hydrochloride salts of the mixture from an isopropyl alcohol
and ethyl ether mixture gave pure (S,S)-5, which was con-
verted to the N-formyl intermediate 6 using a formic acid/
acetic anhydride mixture. O-Demethylation of 6 using bro-
mine tribromide in methylene chloride afforded the phenol 7.
The sodium salt of the phenol prepared using sodium hydride
in dimethylformamide was alkylated with methyl 10-bromo-
decanoate or methyl 6-bromohexanoate to give 8a and 8b,
respectively. Reduction of 8a and 8b using diborane in tetra-
hydrofuran provided the N-methyl compounds 9a and 9b.
Subjection of 9a and 9b to transfer hydrogenation using
ammonium formate and 5% palladium on carbon catalyst
in methanol yielded 10a and 10b. Hydrolysis of 10a and 10b
using 6N hydrochloride acid afforded the desired haptens 3a
and 3b.
a Reagents: (a) Na(OAc)3BH3, (CH2Cl)2; (b) ethenal HCl; (c) recrys-
tallization from (CH3)2CHOH and Et2O; (d) HCO2H, (CH3CO)2O,
toluene; (e) BBr3, CH2Cl2; (f) NaH, Br(CH2)9CO2CH3 or Br-
(CH2)5CO2CH3, DMF; (g) BH3, THF; (h) HCO2NH4, 5% Pd/C,
CH3OH; (i) 6N HCl.
Scheme 2a
The hapten/protein antigens were synthesized as shown in
Scheme 2 using a carbodiimide coupling procedure.12 Briefly,
a solution of the hapten 3a or 3b was combined with a solution
of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI)
hydrochloride to give the activated intermediate 12a or 12b,
which was not isolated. Addition of a solution of bovine
serum albumin(BSA), cationizedBSA(c-BSA), orovalbumin
(OVA) to the solution of 12a or 12b provided the desired
hapten-protein antigen 13a or 13b, which was separated
from unconjugated haptens and other starting materials by
dialysis against multiple changes of distilled water followed
by phosphate-buffered saline (pH 7.35). The degree of
incorporation of the haptens onto the OVA and BSA carrier
proteins was determined by matrix-assisted laser des-
orption/ionization (MALDI) mass spectrometry (MS) analy-
sis. The degree of incorporation of haptens onto the
c-BSA carrier proteins was not determined because these
antigens were generated before MS analysis of proteins was
a Reagents: (a) EDCI, DMF of 0.1 M buffer; (b), BSA, OVA, or
cationized BSA in buffer; (c) dialysis against water then PBS buffer.
available to this project. Parts A and B of Figure 1 show the
MALDI mass spectra obtained from BSA and MO10-mod-
ified BSA, respectively. The observed mass for BSA by
MALDIMSwas inexcellent agreementwiththe value derived
from the known amino acid sequence. The mass difference
between the two spectra divided by the mass added by each
hapten (317 Da) indicated an average incorporation of 5.6
haptens. A similar MALDI MS analysis of OVA and the
MO10-modified OVA indicated an incorporation of 4-5
haptens.

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Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22 7303
Figure 1. (A) MALDI mass spectrum of BSA. (The measured m/z is taken as the correct mass to minimize error in mass assignment). (B)
MALDI mass spectrum of METH MO10-modified BSA. The number of modifying groups (5.6) was determined by dividing the mass increase
upon modification by the mass added by each modifying group (317 Da).
Results and Discussion
Because (þ)-AMP does not possess the N-methyl group
present in (þ)-METH and (þ)-MDMA, we hypothesized that
a longer linker group between the hapten (like MO10) and
backbone structure would lead to improved immune recogni-
tion and higher affinity antibodies for (þ)-METH while
maintaining cross-reactivity and affinity with (þ)-MDMA
as well as (þ)-AMP. While our primary goal was to discover
haptens that would generate mAbs with high affinity for (þ)-
METH, we also hoped to discover mAbs with a broader
specificity for all three of these drugs of abuse. We hypothe-
sized that this broader specificity for (þ)-METH-like drugs
would improve the likelihood of producing a single therapeu-
tic antibody that could be used to treat medical problems
The overall goal of this study was to design and develop a
hapten that would stimulate immune cell production of mAbs
with high affinity for (þ)-METH as well as (þ)-AMP and/or
(þ)-MDMA. Because all three structures have in common an
(S)-aminopropyl substituent on an aromatic ring that helps make
the molecules pharmacologically and immunologically distinct, it
was decided to leave this part of the structure alone and to attach
the linker moiety of the haptens to the aromatic ring at a site that
was distal to the (S)-aminopropyl structures. The linker was
connected to the aromatic ring through an oxygen atom at the
meta position to improve the probability of MDMA recognition,
which has oxygen atoms at both the meta and para positions.

7304 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22
Carroll et al.
Table 1. Haptens, Antigens, and Immunochemical Specifications of Anti-(þ)-METH Monoclonal Antibodies
(þ)-METH-like hapten and
monoclonal
antibody
IgG isotype, light
chain
(þ)-METH KD
(þ)-AMP KD (nM) or KI
(þ)-MDMA KI
antigen
(nM)
(nM)a
(nM)b
MO6 bound to c-BSA
mAb9B11
mAb10E1
mAb7H11
mAb5C2
mAb9C4
mAb1C1
IgG1, λ
IgG1, λ
IgG1, κ
IgG1, κ
IgG2a, κ
IgG1, κ
110
91
52
39
77
58
>1000
>5000
>5000
>5000
>5000
>5000
24
24
30
28
45
22
MO10 bound to OVA
mAb4G9
mAb2F11
mAb1A12
mAb6C1
mAb10D1
IgG2b, κ
IgG2a, κ
IgG2a, κ
IgG1, κ
IgG1, κ
16
13
13
47
34
50
49
68
65
5
>1000
47
51
52
69
MO10 bound to BSA
mAb7F9
mAb9C10
IgG1, κ
IgG1, κ
9
38
>1000
>1000
14
27
a Only four mAbs (all from immunization with a MO10-OVA antigen) had high affinity for (þ)-AMP, and these KD values were determined in a
homologous immunoassay with [3H]-AMP as the radioligand and inhibition with unlabeled (þ)-AMP. If we determined in screening assays that the
mAb would have a very high KD value >1000-5000 nM, we conducted a heterologous immunoassay with [3H]-METH as the radioligand and inhibition
with a high range of unlabeled (þ)-AMP doses to obtain an approximate KI value for (þ)-AMP binding. b [3H]-(þ)-MDMA was unavailable, so we
determined a KI value for binding to each mAb using [3H]-METH as the radioligand and inhibition with unlabeled (þ)-MDMA.
(e.g., addiction, overdose) resulting from any of these drugs.
By analogy, we attempted to produce a “designer antibody”
to treat the medical problems caused by these “designer
drugs.” We also reasoned that the future medical applications
for a broader specificity antibody would be greater because
hospital pharmacies would only have to stock one medication
for the treatment of medical problems resulting from (þ)-
METH, (þ)-MDMA, and (þ)-AMP.
protein has less uniform properties than the other carrier
proteins, with varying amounts of activation within lots or
batches. However, by the time synthesis of the MO10 antigens
was started, the mass spectrometry technology to directly
determinethe number of haptensper protein moleculebecame
available to us. These mass spectrometry studies showed that
about five haptens per protein molecule (either OVA or BSA)
were sufficient to produce high affinity anti-(þ)-METH anti-
bodies. We also found that hapten incorporation rates on
OVA and BSA of less than five per protein molecule led to
inconsistent results or no antigenic response (results not
shown). While the c-BSA proved a good antigenic protein,
wechosetouse OVA and BSA antigensbecausetheseproteins
were small enough to allow direct mass spectrometric analysis
and direct determination of hapten epitope densities. This
proved valuable in optimizing various outcomes. In general,
this allows the user to optimize the hapten to protein ratios
(i.e., number of (þ)-METH-like antigenic epitopes) in syn-
thetic reactions and will also prove valuable in future studies
of the relationship between epitope density and antidrug
response in vaccinated animals.
The current studies provide a comprehensive analysis of the
importance of hapten length and its relationship to affinity
and specificity for very small haptens. Previous studies from
our laboratory have shown that five unique mAbs generated
against five different METH-like haptens (including MO6
and MO10) can produce mAbs against (þ)-METH-like hap-
tens that range in KD value from 250-10 nM.10 These five
mAbs resulted from immunizing mice with (þ)-METH
coupled to protein antigens through progressively longer
linker groups of 4, 6, and 10 atoms connected to the aromatic
ring at the para or meta positions.
Our hypothesis was supported by the finding that immuni-
zations with antigens containing an MO10 hapten epitope
produced significantly greater affinities for (þ)-METH (as
judged by lower KD values for (þ)-METH) than did immu-
nization with the MO6-containing hapten epitope (p < 0.05
using a Student’s t test; Table 1 and Figures 2 and 3). It should
be noted that we designed our immunization schedules to
include the minimum antigen dose and long periods between
boost (up to 2 months) to favor the likelihood that we would
generate high affinity anti-(þ)-METH mAbs. We also
screened for anti-(þ)-METH mAbs with a minimum amount
of hapten protein conjugate to favor the discovery of high
affinity antibodies. In practice, only antibodies of the highest
affinity can stay bound when the hapten dose is minimal.
However, on many occasions, we also discovered low affinity
antibodies but only kept the mAbs with KD values for (þ)-
METH of approximately 100 nM or less. We chose this cutoff
point after considering the outcomes of a wide range of
pharmacological and behavioral studies in rats from our
laboratory using various anti-(þ)-METH mAbs. From these
observations, we hypothesize that mAbs with KD values of
g100 nM will not be clinically useful, and KD values of at least
e10-30 nM will be needed for the treatment of medical
problems caused by addiction.13,14
For the current studies, we redetermined the (þ)-METH
KD values for mAb9B11 and mAb4G9 (from the previous
studies), along with 11 other never before reported anti-(þ)-
METH mAbs, using a significantly improved radioimmu-
noassay (RIA) for determination of KD and KI values. This
improved RIA method does not require a second dilution or
incubation step to separate the drug (þ)-METH mAb com-
plex from the unbound drug. These procedural steps in an
Another critical factor in the discovery process was the
number of haptens bound to the antigen. In our earlier studies
with the MO6 haptens attached to c-BSA carrier proteins, we
did not have the analytical capabilities to directly determine
the number of haptens per c-BSA molecule. Furthermore, we
later found MALDI MS analysis (or any other method) was
not dependable with hapten-c-BSA, which is slightly larger in
molecular size than BSA. More importantly, the c-BSA

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Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22 7305
RIA often result in a less than optimal estimation of the KD
values for ligand binding. Indeed, mAb9B11 and mAb4G9
KD values for (þ)-METH in our previously reported RIA
were 41 and 34 nM,10 respectively, but in the current study,
they are 110 and 16 nM with improved reproducibility.
Importantly, four mAbs generated from MO10, bound to
the OVA, have KD or Ki values of 13-47, 47-51, and 52-
69 nM for (þ)-METH, (þ)-AMP, and (þ)-MDMA, respec-
tively. In contrast, six mAbs generated from (þ)-METH MO6
bound to c-BSA antigen, and two mAbs generated from
MO10, bound to BSA antigen, sometimes had very low KD
values for (þ)-METH and (þ)-MDMA binding but always
possessed KD values of >1000 nM for (þ)-AMP. One mAb
(mAb1A12) generated from the MO10, bound to OVA anti-
gen, also had a KD value of >1000 nM for (þ)-AMP. We are
now in the process of testing the best of these antibodies as
medications in preclinical studies in rodents. It will be of
particular interest to determine if the mAb4G9, with its broad
specificity for all three (þ)-METH-like drugs, is actually
therapeutically better than mAb7F9 (generated from the
(þ)-METH MO10-BSA antigen). In our experience, thera-
peutic efficacy of mAbs is best judged by the ability of the
mAbs to (1) block and/or reduce brain concentration of
(þ)METH, (2) provide prolonged redistribution of
(þ)METH out of the brain and into the bloodstream, and
(3) the ability of the mAbs to block or significantly reduce
(þ)METH-induced behavioral effects in preclinical animal
models like the rat. MAb7F9 had the highest affinity for (þ)-
METH of any of the antibodies (KD = 9 nM) in this report
but no significant binding to (þ)-AMP. Also worthy of noting
is the very high affinity binding of mAb1A12 for (þ)-MDMA
(KI = 5 nM).
Over the course of more than 10years of studies of anti-(þ)-
METH mAbs, we have used a range of protein antigens
including OVA, c-BSA, BSA, keyhole limpet hemocyanin,
thymoglobulin, and immunoglobulin. We have also used five
different adjuvants: Freund’s Complete, Freund’s Incom-
plete, RIBI, TiterMax, and Alum. The adjuvant does not
appear to be the major factor in generating a broad specificity
mAbs (i.e., cross-reactivity with (þ)-METH, (þ)-AMP, and
(þ)-MDMA), but it does significantly affect the ability to
generate high affinity antibodies. Freund’s Complete adju-
vant consistently proved to be the best of the five adjuvants. In
addition, the use of haptens derived from (þ)-isomers has
never generated mAbs that significantly cross-react with (-)-
isomers of METH-like ligands (results not shown). Our
studies also suggest that attachment of the hapten spacer
arm linkage at the meta portion of the (þ)-METH aromatic
ring structure appears superior to a para position linkage
because it is the only hapten to ever generate mAbs with
significant cross reactivity with (þ)-AMP (Table 1 and ref 7).
Another important factor to emerge from this research pro-
gram is the suggestion that the combination of MO10 linked
to OVA is best for generating mAbs with simultaneously high
affinity (i.e., low KD values) for (þ)-METH, (þ)-AMP, and
(þ)-MDMA (Table 1). On the basis of the results of these
studies, the ability of a single (þ)-METH-like hapten to
generate high affinity for (þ)-METH and (þ)-MDMA ap-
pears less dependent on choosing the optimal combination of
hapten and carrier protein.
Figure 2. Representative RIA plots for the determination of anti--
(þ)-METH mAb4G9 KD values for (þ)-METH (upper) and (þ)-
AMP (middle), and KI values for (þ)-MDMA (lower). Similar RIA
inhibition curves were determined in duplicate or triplicate for all 13
mAbs listed in Table 1. After a mathematical correction for the
contribution of [3H]-METH or [3H]-AMP binding, the final average
K
D and KI value was calculated.
Figure 3. Individual (open circles) and average (solid bar) KD
values for (þ)-METH binding to all 13 monoclonal antibodies
generated for these studies. Side-by-side circles indicate that two
different antibodies had the same apparent KD value. The KD values
for (þ)-METH binding to antibodies generated against (þ)-METH
MO10 haptens (n = 7) were significantly lower (p < 0.05; t test)
than the KD values for the antibodies generated against (þ)-METH
M06 haptens (n = 6).
In summary, haptens were designed and developed to
maximize the potential for a single mAb to specifically
recognize the common molecular features of (þ)-METH,
(þ)-AMP, and (þ)-MDMA. Four mAbs have been

7306 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22
Carroll et al.
developed from the MO10 hapten bound to OVA, which have
simultaneously low KD and KI values for (þ)-METH, (þ)-
AMP, and (þ)-MDMA. The high affinity Abs generated in
this study have the potential to block or reduce the stimulant-
induced medical effects in patients addicted to (or abusing)
these drugs. In addition, the mAbs derived from hapten 3a
have the potential to provide an acute treatment for life-
threatening stimulant overdose resulting from (þ)-AMP, (þ)-
METH, or (þ)-MDMA by removing the drugs from their
sites of action in the central nervous, heart, and other critical
organs, thereby reducing METH-induced psychosis, neuro-
toxicity, and cardiovascular effects. Finally, these studies also
suggest MO10 or a similar hapten will be the best for use in
developing active vaccines. These studies are currently in
progress in our laboratories.
15% NH4OH (200 mL). The organic layer was separated, and
the aqueous layer was extracted with CH2Cl2 (3 ꢀ 100 mL). The
combined organic layers were washed with saturated brine
solution (150 mL), dried (Na2SO4), and concentrated to give
9.24 g (99%) of 5 as a colorless oil. 1H NMR (CDCl3) δ
7.27-7.44 (m, 4H), 7.20-7.27 (m, 1H), 7.16 (t, J = 7.9 Hz,
1H), 6.72 (dd, J = 7.9, 2.3 Hz, 1H), 6.68 (d, J = 7.5 Hz, 1H), 6.62
(at, J = 2.3 Hz, 1H), 3.92 (q, J = 6.4 Hz, 1H), 3.76 (s, 3H), 2.85
(dd, J = 12.8, 5.3 Hz, 1H), 2.78 (dq, J = 6.4, 5.3 Hz, 1H), 2.46
(dd, J = 12.8, 7.2 Hz, 1H), 1.42 (bs, 1H, NH), 1.30 (d, J = 6.4
Hz, 3H), 0.92 (d, J = 6.4 Hz, 3H). 13C NMR (300 MHz, CDCl3)
δ 159.5, 146.2, 141.2, 129.1, 128.4, 126.8, 126.5, 121.8, 115.0,
111.3, 55.3, 55.1, 51.8, 42.7, 24.5, 21.2.
The free base dissolved in Et2O (100 mL) was treated with
HCl solution in Et2O (2.0 M, 16.0 mL, 10.0 equiv) at ice-cold
conditions. Addition of Et2O (200 mL) yielded 8.48 g (92%) of a
white, amorphous solid. Recrystallization of the solid using hot
isopropyl alcohol (725 mL) followed by the slow addition of
Experimental Section
Et2O (600 mL) at room temperature yielded 5.5 g (65%) of
1
Nuclear magnetic resonance (1H NMR and 13C NMR) spectra
were recorded on a 300 MHz (Bruker AVANCE 300) spectro-
meter. Chemical shift data for the proton resonances were
reported in parts per million (ppm) relative to internal standard.
Optical rotations were measured on an AutoPol III polarimeter,
purchased from Rudolf Research. Elemental analyses were per-
formed by Atlantic Microlab, Norcross, GA. Purity of com-
pounds (>95%) was established by elemental analyses.
Analytical thin-layer chromatography (TLC) was carried out
on plates precoated with silica gel GHLF (250 μM thickness).
TLC visualization was accomplished with a UV lamp or in an
iodine chamber. All moisture-sensitive reactions were performed
under a positive pressure of nitrogen maintained by a direct line
from a nitrogen source. Anhydroussolvents were purchasedfrom
Aldrich Chemical Co.
5 HCl as a white crystalline solid: mp 219-221 °C. H NMR
3
(CDCl3) δ 10.32 (bs, 1H), 9.84 (bs, 1H), 7.69 (ad, J = 7.2 Hz,
2H), 7.48 (tt, J = 7.2, 1.9 Hz, 2H), 7.41 (tt, J = 7.2, 1.5 Hz, 1H),
7.15 (t, J = 7.9 Hz, 1H), 6.75 (dd, J = 7.9, 1.9 Hz, 1H), 6.59 (d,
J = 7.5 Hz, 1H), 6.51 (at, J = 1.9 Hz, 1H), 4.39 (ap, J = 6.8 Hz,
1H), 3.73 (s, 3H), 3.40 (dd, J = 12.8, 4.1 Hz, 1H), 3.05 (bs, 1H),
2.85 (dd, J = 12.8, 9.8 Hz, 1H), 1.96 (d, J = 6.8 Hz, 3H), 1.43 (d,
J = 6.8 Hz, 3H). 13C NMR (CDCl3) δ 159.8, 138.1, 136.5, 129.7,
129.5, 129.3, 127.9, 121.4, 114.6, 112.7, 55.9, 55.2, 53.9, 38.1,
21.2, 17.9. Anal. (C18H24ClNO) C, H.
(S,S)-N-Formyl-N-r-methylbenzyl-3-methoxyamphetamine (6).
To a stirred solution of 24 mL of formic acid (36.6 g, 0.795 mol)
at 0 °C, 40 mL of acetic anhydride (58.3 g, 0.572 mol) in toluene
(100 mL) was added dropwise. After 30 min, the amine 5 (10.2 g,
0.038 mol, obtained from the hydrochloride salt) in a minimum
volume of formic acid was added. The reaction mixture was
heated under reflux for 16 h and allowed to stir at RT for 8 h.
The acidic solution was basified with 40 mL of 30% NH4OH
(aq) solution and extracted with 3 ꢀ 100 mL of CH2Cl2. The
extracts were washed with brine (100 mL) and dried (MgSO4).
Concentration of the extracts gave an oil. The oil was purified
by flash chromatography (silica gel, 200 g ISCO column) to give
10.3 g (92%) of 6 as a clear, colorless thick oil. 1H NMR (CDCl3)
δ 8.41 (s, 1H), 8.40 (s, 1H), 7.27- 7.50 (m, 5H), 7.07 (t, J = 7.9Hz,
1H), 7.07(t, J= 7.9 Hz, 1H), 6.69 (d, J= 1.9 Hz, 1H), 6.66 (d, J=
1.9 Hz, 1H), 6.41 (d, J = 7.5 Hz, 1H), 6.30 (at, J = 1.9 Hz, 1H),
6.26 (d, J = 7.5 Hz, 1H), 6.09 (at, J =1.9Hz, 1H), 5.94 (q, J = 7.2
Hz, 1H), 4.65 (q, J = 7.2 Hz, 1H), 3.70 (s, 3H), 3.68 (s, 3H),
3.34-3.53 (m, 1H), 3.19-3.36 (m, 1H), 3.00 (dd, J = 13.2, 9.8 Hz,
1H), 2.61 (dd, J = 13.2, 4.9 Hz, 1H), 2.38-2.54 (m, 2H), 1.58 (d,
J = 7.2 Hz, 3H), 1.54 (d, J = 7.2 Hz, 3H), 1.31 (d, J = 6.8 Hz,
3H), 1.25 (d, J = 6.8 Hz, 3H). 13C NMR (CDCl3) δ 162.5, 161.4,
159.55, 159.48, 140.9, 140.2, 139.8, 139.6, 129.3, 129.1, 128.6,
128.4, 128.1, 127.9, 127.7, 127.2, 121.5, 121.3, 114.3, 114.2, 112.1,
111.9, 56.9, 55.1, 53.3, 51.1, 49.7, 44.7, 40.4, 21.8, 19.7, 17.3, 16.4.
LCMS (APCI) m/z 298 (M þ 1).
(S)-3-(9-Carboxynonyloxy)methamphetamine (3a) Hydro-
chloride. Compound 10a (420 mg, 1.1 mmol) was heated under
reflux in 9 mL of 6N HCl for 8 h. The cooled solution was
concentrated to a tan residue that was recrystallized 3ꢀ from a
CH3OH and Et2O mixture to yield 260 mg (65%) of 3a as its
white powdery HCl salt: mp 124-127 °C; [R]25 þ0.30 (c 1.0,
D
CH3OH). 1H NMR (CD3OD) δ 7.25 (t, J = 7.9 Hz, 1H),
6.75-6.90 (m, 3H), 3.97 (t, J = 6.4 Hz, 3H), 3.40-3.55 (m,
1H), 3.11 (dd, J = 13.2, 4.9 Hz, 1H), 2.73 (dd, J = 13.2, 9.0 Hz,
1H), 2.72 (s, 3H), 2.28 (t, J = 7.2 Hz, 2H), 1.77 (ap, J = 7.2 Hz,
2H), 1.60 (ap, J = 7.2 Hz, 2H), 1.42-1.54 (m, 2H), 1.28-1.43
(m, 9H), 1.25 (d, J = 6.8 Hz, 3H). 13C NMR (300 MHz,
CD3OD) δ 161.5, 138.8, 131.4, 122.8, 117.2, 114.7, 69.3, 58.1,
40.3, 35.0, 30.9, 30.5, 30.4, 30.3, 30.2, 27.1, 26.1, 15.9. LCMS
(APCI) m/z 336.7 (M þ 1)þ. Anal. (C20H34ClNO3 0.25H2O) C,
3
H, N.
(S)-3-(5-carboxypentyloxy)methamphetamine Hydrochloride
(3b). Compound 3b was prepared by a procedure analogous to
that described for 3a to afford 59% of 3b as the hydrochloride
1
salt: mp 73-76 °C; [R]25 þ0.64 (c 1.10, CH3OH). H NMR
D
(CD3OD) δ 7.24 (t, J = 7.9 Hz, 1H), 6.76-6.91 (m, 3H), 3.97 (t,
J = 6.4 Hz, 3H), 3.39-3.59 (m, 1H), 3.35 (s, 3H), 3.16 (dd, J =
13.2, 4.9 Hz, 1H), 2.75 (dd, J = 13.2, 9.0 Hz, 1H), 2.72 (s, 3H),
2.32 (t, J = 7.2 Hz, 2H), 1.78 (ap, J = 7.2 Hz, 2H), 1.67 (ap, J =
(S,S)-N-Formyl-N-r-methylbenzyl-3-hydroxyamphetamine (7).
To a stirred cold (0 °C) solution of 6 (2.10 g, 0.007 mol) in
60 mL of CH2Cl2 under N2 atmosphere was added dropwise
1.33 mL (3.54 g, 0.014 mol) of BBr3. The reaction mixture was
allowed to stir at 25 °C for 8 h, 100 mL of water was carefully
added to quench excess BBr3, and the resulting mixture was
extracted with 3 ꢀ 100 mL of CH2Cl2 and dried (Na2SO4). The
resulting oil was subjected to silica gel chromatography using
hexanes/CH2Cl2/MeOH (4:8:1) as the eluent to give 1.96 g (98%)
of pure 7 as a white powder: mp 195-198 °C; Rf 0.43 (1:1,
hexanes/EtOAc). 1H NMR (CDCl3) δ 8.36 (s, 1H), 8.32 (s, 1H),
7.29-7.46 (m, 4H), 7.26 (at, J = 6.8 Hz, 1H), 7.01 (t, J = 7.2 Hz,
1H), 6.99(t, J= 7.2 Hz, 1H), 6.72 (d, J= 1.9 Hz, 1H), 6.69 (d, J=
1.9 Hz, 1H), 6.55 (at, J = 1.9 Hz, 1H), 6.26 (at, J = 1.9 Hz, 1H),
6.24 (d, J = 7.5 Hz, 1H), 6.11 (d, J = 6.8 Hz, 1H), 5.89 (q, J = 7.2
7.2 Hz, 2H), 1.42-1.58 (m, 2H), 1.25 (d, J = 6.8 Hz, 3H). 13
C
NMR (300 MHz, CD3OD) δ 131.4, 123.0, 117.1, 114.8, 69.2,
58.2, 40.6, 35.3, 31.4, 30.5, 27.2, 26.3, 16.2. LCMS (ESI) m/z
280.3 (M þ 1)þ, m/z 278.6 (M - 1)þ. Anal. (C16H26ClNO3) C,
H, N.
(S,S)-N-r-Methylbenzyl-3-methoxyamphetamine (5) Hydro-
chloride. To a solution of m-methoxyphenylacetone (5.0 g,
0.030 mol) and (S)-(-)-R-methylbenzylamine (3.81 g, 0.031
mol) in ethylene dichloride (200 mL) at room temperature was
added sodium triacetoxyborohydride (10.0 g, 0.047 mol) in four
equal portions over a 10 min interval. After stirring overnight at
ambient temperature, the reaction mixture was quenched with

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22 7307
Hz, 1H), 4.56 (q, J = 7.2 Hz, 1H), 3.42-3.63 (m, 1H), 3.23-3.42
(m, 1H), 2.93 (dd, J = 13.2, 9.4 Hz, 1H), 2.61 (dd, J = 13.2, 5.3
Hz, 1H), 2.25-2.46 (m, 2H), 1.54 (d, J = 6.4 Hz, 3H), 1.52 (d, J =
Hz, 1H), 6.62-6.83 (m, 3H), 3.90 (t, J = 6.4 Hz, 2H), 3.63 (s,
3H), 2.76 (sixtet, J = 6.4 Hz, 1H), 2.60 (dq, J = 13.2, 6.4 Hz,
2H), 2.35 (s, 3H), 2.27 (t, J = 7.2 Hz, 2H), 1.74 (p, J = 6.4 Hz,
2H), 1.59 (p, J = 7.2 Hz, 2H), 1.18-1.50 (m, 10H), 1.03 (d, J =
6.4 Hz, 3H). 13C NMR (CDCl3) δ 174.1, 159.1, 140.9, 129.2,
121.3, 121.4, 115.5, 112.0, 67.7, 56.2, 51.3, 43.4, 34.0, 33.8, 29.21,
29.20, 29.04, 28.99, 25.9, 25.8, 19.6.
6.4 Hz, 3H), 1.30 (d, J = 6.8 Hz, 1H), 1.21 (d, J = 6.8 Hz, 1H). 13
C
NMR (300 MHz, CDCl3) δ 162.9, 162.1, 156.61, 156.58, 140.5,
139.8, 139.3, 139.2, 129.4, 129.3, 128.65, 128.57, 128.5, 128.0,
127.91, 127.86, 127.0, 120.4, 116.2, 116.1, 113.7, 113.4, 57.5,
53.8, 51.2, 50.2, 44.2, 40.1, 21.6, 19.8, 17.2, 16.3. Anal.
(S,S)-N-Formyl-N-r-methylbenzyl-3-(50-carbomethoxypenty-
loxy)amphetamine (8b). Compound 8b was prepared by a pro-
cedure analogous to that described for 8a to afford 76% of 8b.
1H NMR (300 MHz, CDCl3) δ 8.41 (s, 1H), 8.40 (s, 1H),
7.22-7.50 (m, 5H), 7.06 (t, J = 7.9 Hz, 1H), 7.05 (t, J = 7.9
Hz, 1H), 6.67 (d, J = 1.9 Hz, 1H), 6.64 (d, J = 1.9 Hz, 1H), 6.39
(d, J = 7.5 Hz, 1H), 6.28 (bs, 1H), 6.24 (d, J = 7.5 Hz, 1H), 6.08
(bs, 1H), 5.93 (q, J = 7.2 Hz, 1H), 4.64 (q, J = 7.2 Hz, 1H), 3.82
(q, J = 6.4 Hz, 1H), 3.68 (s, 3H), 3.35-3.54 (m, 1H), 3.19-3.35
(m, 1H), 2.98 (dd, J = 12.8, 9.8 Hz, 1H), 2.60 (dd, J = 12.8, 4.9
Hz, 1H), 2.42-2.50 (m, 2H), 2.33 (t, J = 7.2 Hz, 3H), 1.64-1.83
(m, 4H), 1.58 (d, J = 6.8 Hz, 3H), 1.54 (d, J = 6.8 Hz, 3H),
1.41-1.52 (m, 2H), 1.31 (d, J = 6.8 Hz, 3H), 1.25 (d, J = 6.8 Hz,
3H). 13C NMR (300 MHz, CDCl3) δ 173.5, 162.4, 161.4, 158.94,
158.88, 140.8, 140.2, 139.7, 139.5, 129.2, 129.0, 128.5, 128.4,
128.0, 127.8, 127.6, 127.1, 121.3, 121.0, 114.8, 114.7, 112.6,
112.4, 67.9, 60.1, 56.8, 53.2, 51.1, 49.7, 44.6, 40.4, 34.1, 28.9,
28.8, 25.5, 24.6, 21.7, 19.7, 17.2, 16.3.
(S,S)-N-Methyl-N-r-methylbenzyl-3-(50-carbomethoxypentyl-
oxy)amphetamine (9b). Compound 9b was prepared by a proce-
dure analogous to that described for 9a to afford 58% of 9b. 1H
NMR (300 MHz, CDCl3) δ 7.24-7.44 (m, 4H), 7.15 (m, 1H),
7.10 (t, J = 7.9 Hz, 1H), 6.67 (d, J = 1.9 Hz, 1H), 6.60 (d, J = 7.9
Hz, 1H), 6.52(at, J = 1.9 Hz, 1H), 3.78-3.96 (m, 2H), 3.55-3.75
(m, 3H), 2.96-3.12 (m, 1H), 2.58 (dd, J = 12.8, 4.5 Hz, 1H), 2.31
(dd, J = 12.8, 9.8 Hz, 1H), 2.24 (s, 3H), 1.69-1.84 (m, 2H),
1.54-1.69 (m, 2H), 1.37-1.54 (m, 4H), 1.32 (d, J = 6.8 Hz, 3H),
0.90 (d, J = 6.8 Hz, 3H). 13C NMR (300 MHz, CDCl3) δ 129.35,
128.72, 127.68, 127.07, 121.95, 115.52, 112.22, 67.83,
62.56, 56.22, 51.90, 38.36, 34.41, 32.77, 29.42, 26.11, 25.14,
22.41, 16.02.
(C18H21NO2 0.25H2O) C, H, N.
3
(S,S)-N-Formyl-N-r-methylbenzyl-3-(90-carbomethoxynony-
loxy)amphetamine (8a). A solution of 1.97 (0.007 mol) of phenol
7 in 3 mL of DMF was carefully added to a suspension of 300 mg
of 60% NaH (washed with hexanes in 5 mL of DMF) followed
by the addition of 2 g of methyl-10-bromodecanoate in 4 mL of
DMF. After 16 h of stirring, the reaction mixture was quenched
with water and extracted with 4 ꢀ 200 mL of CH2Cl2 and dried
(Na2SO4). The oil obtained on concentration was purified using
an 80 g silica gel ISCO column with gradient 0-10% of polar B:
hexanes/CH2Cl2/MeOH (4:8:1) in a polar CH2Cl2 collecting 25
mL fraction. Concentration of the product fraction gave 1.98 g
(63%) of pure 8a as an oil. 1H NMR (CDCl3) δ 8.40 (s, 1H), 8.37
(s, 1H), 7.25-7.47 (m, 5H), 7.054 (t, J = 7.9 Hz, 1H), 7.045 (t,
J = 7.9 Hz, 1H), 6.68 (d, J = 1.9 Hz, 1H), 6.65 (d, J = 1.9 Hz,
1H), 6.39 (d, J = 7.5 Hz, 1H), 6.29 (at, J = 1.9 Hz, 1H), 6.23 (d,
J = 7.5 Hz, 1H), 6.08 (at, J = 1.9 Hz, 1H), 5.93 (q, J = 7.2 Hz,
1H), 4.64 (q, J = 7.2 Hz, 1H), 3.80 (qd, J = 13.2, 6.4 Hz, 2H),
3.66 (s, 3H), 3.36-3.55 (m, 1H), 3.18-3.36 (m, 1H), 2.98 (dd,
J = 13.2, 9.8 Hz, 1H), 2.61 (dd, J = 13.2, 5.3 Hz, 1H), 2.36-2.53
(m, 2H), 2.31 (t, J = 7.5 Hz, 2H), 1.68-1.82 (m, 2H), 1.50-1.68
(m, 2H), 1.57 (d, J = 6.8 Hz, 3H), 1.54 (d, J = 7.2 Hz, 3H),
1.39-1.50 (m, 2H), 1.28-1.39 (m, 13H), 1.25 (d, J = 7.2 Hz,
3H). 13C NMR (CDCl3) δ 174.1, 162.4, 161.4, 159.05, 158.98,
140.8, 140.1, 139.7, 139.5, 129.2, 129.0, 128.5, 128.4, 128.0,
127.9, 127.8, 127.7, 127.6, 127.15, 127.07, 121.2, 121.0, 114.9,
114.7, 112.7, 112.5, 67.73, 67.7, 56.9, 53.3, 51.3, 51.1, 49.7, 44.7,
40.4, 34.0, 29.23, 29.21, 29.18, 29.16, 29.1, 29.0, 25.9, 24.8, 21.7,
19.7, 17.2, 16.3. Anal. (C29H41NO4 H2O) C, H, N.
3
(S,S)-N-Methyl-N-r-methylbenzyl-3-(90-carbomethoxynonyloxy)-
amphetamine (9a). To 1.90 g (0.004 mol) of 8a in 5 mL of anhydrous
THF was added 10 mL of 1 M BH3 in THF solution. The reaction
mixture was stirred for 1 h, carefully quenched with 1 mL MeOH,
10 mL of 1 M HCl, and basified with 20 mL of 15% NH4OH
(aq) solution. The aqueous layer was extracted with 3 ꢀ 50 mL of
CH2Cl2. The oil obtained on concentration was subjected to chro-
matographyusinga120gsilicagelISCOcolumnwithhexanes/Et2O/
TEA (10:9:1) as the eluent collecting 25 mL fractions. The fractions
containing the first major product were concentrated to give 489 mg
(S)-3-(5-Carbomethoxypentyloxy)methamphetamine Hydro-
chloride (10b). Compound 10b was prepared by a procedure
analogous to that described for 10a to afford 67% of 10b. H
1
NMR (CDCl3) δ 7.20 (t, J = 7.5 Hz, 1H), 6.67-6.81 (m, 3H),
3.94 (t, J = 6.4 Hz, 3H), 3.67 (s, 3H), 2.73-2.87 (m, 1H), 2.68
(dd, J = 13.2, 6.8 Hz, 1H), 2.58 (dd, J = 13.2, 6.4 Hz, 1H), 2.39
(s, 3H), 2.35 (t, J = 7.2 Hz, 2H), 1.80 (ap, J = 7.2 Hz, 2H), 1.71
(ap, J = 7.2 Hz, 2H), 1.40-1.62 (m, 3H), 1.06 (d, J = 6.0 Hz,
3H). 13C NMR (300 MHz, CDCl3) δ 174.4, 159.7, 138.0, 129.7,
121.4, 115.4, 113.0, 67.6, 56.8, 51.4, 39.9, 33.9, 30.5, 28.9, 25.6,
24.6, 16.1.
General Synthesis of Hapten-Protein Antigens. In prelimin-
ary studies, each hapten, (þ)-METH MO6 or (þ)-METH
MO10, was covalently bound to at least 2-3 different protein
antigens, cationized bovine serum albumin (c-BSA), ovalbumin
(OVA), or bovine serum albumin (BSA), and then used to
immunize groups of mice (see below) to test for the serum
anti-(þ)-METH immune response. The individual mouse and
hapten-protein antigen combination that yielded the highest
anti-(þ)-METH IgG titers from each group of immunizations
was chosen for production of mAbs.
A. From c-BSA. To generate the antigens for immunization
with the c-BSA carrier protein, each hapten was conjugated to
Imject SuperCarrier Immune Modulator (c-BSA; Thermo Fish-
er, Rockford, IL), following the manufacturer’s protocol. To
separate the hapten/c-BSA conjugates from uncoupled hapten
and other synthesis reagents, the antigens were passed through a
PD-10 Sephadex G-25 column (GE Healthcare Bio-Sciences
Corp., Piscataway, NJ) equilibrated with phosphate-buffered
saline. Absorbance of the protein eluent was monitored at
280 nM to determine when to collect the conjugated product
as it eluted from the column. Once collected, the final antigen
1
(26%) of 9a as an oil. H NMR (CDCl3) δ 7.18-7.34 (m, 4H),
7.09-7.18 (m, 1H), 7.03 (t, J = 7.9 Hz, 1H), 6.60 (dd, J = 7.5, 1.9
Hz, 1H), 6.52 (d, J = 7.5 Hz, 1H), 6.45 (s, 1H), 3.69-3.87 (m, 2H),
3.56 (s, 3H), 3.50-3.68 (m, 1H), 2.89-3.07 (m, 1H), 2.82 (dd, J =
12.8, 4.5 Hz, 1H), 2.17 (s, 3H), 2.15-2.30 (m, 2H), 1.44-1.74 (m,
5H), 1.09-1.44 (m, 13H), 0.82 (d, J = 6.4 Hz, 3H). 13C NMR
(CDCl3) δ174.3, 159.0, 146.4, 142.6, 128.9, 128.3, 127.3, 126.6, 121.4,
115.1, 111.8, 67.8, 62.7, 62.1, 55.8, 51.4, 38.0, 34.1, 32.3, 29.8, 29.3,
29.14, 29.08, 26.0, 24.9, 21.9, 15.5.
(S)-3-(9-Carbomethoxynonyloxy)methamphetamine (10a). A
mixture of 489 mg (1.08 mmol) of 9a in 40 mL of CH3OH
containing 2.7 g of HCO2NH4 and 350 mg of 5% Pd/C was
refluxed for 2 h. The cooled reaction solution was filtered
through a Celite pad and concentrated. The resulting residue
was basified in 100 mL of Et2O using 1 mL of TEA. This mixture
was gravity filtered (to remove excess formate salts), and the
filtrate was concentrated to an oil. The oil was dissolved in a
minimum amount of CH2Cl2 (about 1-2 mL) and was subjected
to column chromatography using a 120-g silica gel ISCO
column using 5% B/A to 100% B/A gradient elution over
30 min (A = hexanes/Et2O/TEA (10:9:1); B = 10% MeOH/
CH2Cl2). Product fractions were concentrated to give 378 mg
(91%) of 10a as an oil. 1H NMR (CDCl3) δ 7.16 (dt, J = 7.2, 1.5

7308 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22
Carroll et al.
concentrations were determined by adding 8.3 μL of antigen
solution to 250 μL of Coomassie Plus protein assay reagent
(Pierce Chemical Company, Rockford, IL) and comparing the
color development against a BSA standard curve. A microtiter
plate reader with detection at 590 nM was used to detect the
color change. The final products were stored at -20 °C until
needed.
titer was reached. Serum samples were taken via tail bleed
periodically to measure anti-(þ)-METH IgG response by a
enzyme-linked immunosorbant assay (ELISA) similar to the
method described by Peterson et al.10
After sufficient anti-(þ)-METH IgG titers were achieved
(typically after 2-4 months of immunization), hybridomas were
produced using previously reported methods.16 The hybridoma
fusion partner for mouse B cells was cell line P3 ꢀ 63Ag8.653
(American Type Culture Collection, Manassas, VA). Once
hybridomas were produced, initial screening for potential anti--
(þ)-METH mAbs was conducted by an ELISA using 96-well
microtiter plates coated with the original hapten (either MO6 or
MO10) conjugated to a different protein carrier (thyroglobulin,
Sigma Chemical Company). This procedure avoided selecting
carrier protein-reactive antibodies (e.g., anti-OVA antibodies
for mice immunized with a MO10-OVA antigen). To ensure that
only mouse IgG isotypes were selected during these processes,
anti-IgG constant region antibodies were used for the ELISA.
The IgG isotype and light chain identity were determined using a
mouse antibody isotyping kit (Boehringer Mannheim, Indiana-
polis, IN). Once potential anti-(þ)-METH secreting hybridoma
cell lines were discovered in a specific well of a microtiter plate,
the cells from this location were repeatedly subcloned to ensure
the monoclonality of the cell line. The supernatant from these
cell lines were also rescreened by a [3H]-(þ)-METH radioimmu-
noassay (RIA) with (þ)-METH, (þ)-AMP, and (þ)-MDMA as
the inhibitors to confirm that the cell line was secreting IgG
antibodies against (þ)-METH-like compounds. This RIA
method is described in an upcoming section.
Production and Purification. Monoclonal antibodies were
produced in either a Cell-Pharm System 2500 hollow fiber
bioreactor16,17 (Unisyn Technologies, Inc., Hopkinton, MA)
or in a Biostat B 10-L bioreactor (Sartorius Corp., Edgewood,
NY).10 All antibodies were harvested and stored at -80 °C until
purification. mAb were purified either by affinity chromatog-
raphy using Protein-G Sepharose (Amersham Biosciences, Pis-
cataway, NJ) as described by Peterson et al.,10 ion exchange
chromatography using SP Sepharose (Amersham Biosciences,
Piscataway, NJ) as described in Hardin et al.,18 or a combina-
tion of the two methods. Following purification, mAbs were
concentrated and buffer exchanged into 15 mM sodium phos-
phate containing 150 mM sodium chloride (pH 6.5-7.5), as
described in McMillan et al.19
Determination of Monoclonal Antibody KD and KI Values for
(þ)-METH, (þ)-AMP, and (þ)-MDMA. A 100 μL aliquot of
(þ)-[20,60-3H(n)]methamphetamine ([3H]-(þ)-METH; specific
activity = 18 Ci/mmol; ∼50000 decays/min) in RIA buffer
(0.05 M Tris; 0.9% NaCl; 2% BSA; 0.2% NaN3; 0.05% Tween
20, adjusted to pH 7.6) was added to 50 mm ꢀ 14 mm
polypropylene test tubes. The [3H]-(þ)-METH radioligand
was synthesized at the Research Triangle Institute (Research
Triangle Park, NC) and was a gift from the National Institute on
Drug Abuse (Bethesda, MD). The mAbs (in 100 μL) were
diluted in RIA buffer to a concentration that would bind
∼15-22% of the ∼50000 decays/min of the [3H]-(þ)-METH
in each tube. Each tube also received 10 μL of unlabeled (þ)-
METH (or (þ)-MDMA for determining the KI value for (þ)-
MDMA) at an appropriate range of (þ)-METH concentrations
above and below the expected KD (or KI) value for each mAb.
[3H]-(þ)-MDMA was unavailable, so we determined a KI value
for binding to each mAb using [3H]-METH as the radioligand
and inhibition with unlabeled (þ)-MDMA. Next, 20 μL of
Pierce MagnaBind Protein G Magnetic Beads was added to
each tube to allow easy separation of bound (to mAb) and free
[3H]-(þ)-METH at the end of the assay. The nonspecific binding
tubes had all of the same reagents except that a saturating dose
(10 μM) of (þ)-METH was added to inhibit all specific antibody
binding. The tubes were vortexed, centrifuged at 1000 rpm for
30 s, and incubated overnight with gentle shaking at 4 °C. The
next day tubes were centrifuged at 1000 rpm for 30 s and placed
B. From BSA and OVA. To generate the antigens for im-
munization with OVA or BSA carrier proteins, we used a
carbodiimide procedure similar to the method of Owens
et al.15 All reagents used in hapten-protein conjugation were
obtained from Sigma Chemical Company (St. Louis, MO)
unless otherwise specified. For the synthesis of MO10 antigens
using OVA or BSA, the starting hapten/carrier protein molar
ratio was 60:1. For comparison the synthesis of MO6 antigens
using c-BSA, the starting hapten/carrier protein molar ratio was
90:1. These ratios of hapten to carrier protein were optimized in
preliminary experiments. Each hapten was dissolved in 1 mL of
DMF or 0.1 M 2-(N-morphalino)ethanesulfonic acid buffer (pH
4.5) to conduct the synthesis. The reaction mixture was allowed
to stir overnight at 25 °C protected from light. The final product
was dialyzed against 4 L of deionized water (with at least 2-3
changes of water) for 24 h at 4 °C then against 4 L of pH 7.35
phosphate-buffered saline (with at least 2-3 changes) for 24 h at
4 °C to yield the hapten/protein antigens. Because of the protein
modification resulting from the addition of haptens to the
protein surface, the antigens often partially precipitated. After
dialysis, the soluble and insoluble fractions of the antigenic
preparations were stored at -20 °C until needed for immuniza-
tion.
Mass Spectrometry Analysis of (þ)-MO6-BSA, (þ)-MO6-
OVA, (þ)-METH MO10-BSA, and (þ)-METH MO10-OVA.
Because the analysis of (þ)-MO6 and (þ)-MO10 antigens were
similar, we will only discuss (þ)-MO10 antigens. Hapten to
carrier protein incorporation rates for the (þ)-MO6-cBSA or
(þ)-M010-cBSA antigens were not determined, as discussed
earlier. Mass spectra were obtained of (þ)-METH MO10-
OVA and (þ)-METH MO10-BSA using a Bruker Reflex III
MALDI TOF mass spectrometer. The mass spectrometer was
operated in the linear mode with an acceleration voltage of
approximately 25 kV. Pulsed ion extraction with a delay of
700 ns was used. The instrument was calibrated using a com-
mercial reference material from the manufacturer (Bruker Pro-
tein Standard II) for high mass (10-100 kDa) range calibration.
Ion suppression was used up to m/z 10 KDa to enhance the high
mass ions. After synthesis of the (þ)-METH MO10-OVA or
(þ)-METH MO10-BSA conjugate, a small aliquot of the solu-
ble antigen in the supernatant fraction, along with the native
OVA or BSA protein, was analyzed using dihydroxy benzoic
acid (approximately 10 mg/mL in 0.1% TFA/ACN 2:1) as the
MALDI matrix. Small volumes of dialyzed samples were mixed
1:1 with the matrix solution, and 1 μL was spotted on the target.
For samples that had not previously been dialyzed, a standard
desalting Zip Tip procedure was used to cleanup and concen-
trate the samples. However, the protein dialysis procedure
against deionized H2O consistently produced better results.
Immunization, Screening, and Hybridoma Generation. Female
BALB/c mice (Charles River Laboratories, Wilmington, MA)
were used for all immunizations, as previously described by
Peterson et al.10 For production of the (þ)-METH MO6 and
(þ)-METH MO10 mAbs, all mice were initially immunized
subcutaneously in the hindquarters with 10-100 μg of antigen
emulsified in either Hunter’s TiterMax adjuvant (for mAb9B11
and mAb10E1) or Freund’s Complete Adjuvant (for all others).
This initial immunization was followed by a boost with
10-50 μg of antigen emulsified in TiterMax or Freund’s In-
complete Adjuvant (or Freund’s Complete Adjuvant for the
mice used to produce mAb1A2, mAb1C1, mAb10D1, mAb7F9,
and mAb9C10; see Table 1) 3-6 weeks later. Subsequent boost
followed at 6 week intervals until a high anti-(þ)-METH IgG

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22 7309
in a magnetic separating rack for 6 min. The supernatant fluid
was slowly aspirated, and the magnetic bead pellet containing
mAb was resuspended in the same test tube with 2 mL scintilla-
tion fluid (Ecoscint A, National Diagnostics, Atlanta, GA). The
tubes were placed into an empty 20 mL scintillation vial carrier
and the mAb bound concentration of [3H]-METH in each tube
was determined by liquid scintillation spectrometry. An IC50
value for inhibition of [3H]-(þ)-METH for each mAb was
determined after fitting a logistic curve to the data points using
Origin graphing and data analysis software (OriginLab Cor-
poration, Northampton, MA). KD (for (þ)-METH or KI (þ)-
MDMA values for (þ)-METH binding to each mAb) were
determined after correction for the binding of [3H]-(þ)-METH
by the method of Akera and Cheng.20
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A similar homogeneous assay was conducted using [3H]-(þ)-
AMP with inhibition using (þ)-AMP for determination of KD
values for (þ)-AMP binding to the mAbs. If we determined in
initial screening assays that the mAb would have a very high KD
value (>1000-5000 nM) for (þ)-AMP, we conducted a hetero-
logous immunoassay with [3H]-METH as the radioligand and
inhibition with a high range of unlabeled (þ)-AMP doses to
obtain an approximate KI value for (þ)-AMP binding.
Representative binding curves for mAb4G9 for (þ)-METH,
(þ)-AMP, and (þ)-MDMA are shown in Figure 2. Each of the
determinations was made with duplicate (for (þ)-METH and
(þ)-AMP) or triplicate (for (þ)-MDMA) RIA dose-response
curves, and the average value was reported in Table 1.
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H. P.; Owens, S. M. Development and preclinical testing of a high-
affinity single-chain antibody against (þ)-methamphetamine.
J. Pharmacol. Exp. Ther. 2008, 325, 124–133.
(14) Gentry, W. B.; Ruedi-Bettschen, D.; Owens, S. M. Development of
active and passive human vaccines to treat methamphetamine
addiction. Hum. Vaccines 2009, 5, 206–213.
(15) Davis, M. T.; Preston, J. F. A simple modified carbodiimide
method for conjugation of small-molecular-weight compounds to
immunoglobulin G with minimal protein crosslinking. Anal. Bio-
chem. 1981, 116, 402–407.
(16) Valentine, J. L.; Arnold, L. W.; Owens, S. M. Anti-phencyclidine
monoclonal Fab fragments markedly alter phencyclidine pharma-
cokinetics in rats. J. Pharmacol. Exp. Ther. 1994, 269, 1079–1085.
(17) Valentine, J. L.; Owens, S. M. Antiphencyclidine monoclonal
antibody therapy significantly changes phencyclidine concentra-
tions in brain and other tissues in rats. J. Pharmacol. Exp. Ther.
1996, 278, 717–724.
(18) Hardin, J. S.; Wessinger, W. D.; Proksch, J. W.; Owens, S. M.
Pharmacodynamics of a monoclonal antiphencyclidine Fab with
broad selectivity for phencyclidine-like drugs. J. Pharmacol. Exp.
Ther. 1998, 285, 1113–1122.
Acknowledgment. This research was supported by the
National Institute on Drug Abuse under projects DA05477,
DA-1-881, DA11560, DA14361, and U01 DA23900.
Supporting Information Available: Elemental analysis of
compounds 5, 7, 8a, 3a, and 3b. This material is available free
of charge via the Internet at http://pubs.acs.org.
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