An immunotherapeutic program for the treatment of nicotine addiction: Hapten design and synthesis

Article abstract of DOI:10.1021/jo001442w

People continue to smoke and use tobacco products despite well-established hazardous consequences. The most contributing factor is the addictive nature of nicotine. There is no highly effective treatment for the problem of nicotine dependence. Immunotherapy offers an alternative to conventional approaches. The chemistry necessary for a comprehensive immunopharmacological program is presented. Haptens for the generation of antibodies specific for naturally occurring (S)-nicotine, (S)- and (R)-nornicotine, and the metabolite (S)-cotinine were prepared with high optical purity. Preliminary data for antinicotine antibodies are reported.

Full text of DOI:10.1021/jo001442w

J . Org. Chem. 2001, 66, 4115-4121
4115
An Im m u n oth er a p eu tic P r ogr a m for th e Tr ea tm en t of Nicotin e
Ad d iction : Ha p ten Design a n d Syn th esis
Shigeki Isomura, Peter Wirsching,* and Kim D. J anda*
The Scripps Research Institute, Department of Chemistry BCC-582, 10550 North Torrey Pines Road,
La J olla, California 92037
kdjanda@scripps.edu
Received October 6, 2000
People continue to smoke and use tobacco products despite well-established hazardous consequences.
The most contributing factor is the addictive nature of nicotine. There is no highly effective treatment
for the problem of nicotine dependence. Immunotherapy offers an alternative to conventional
approaches. The chemistry necessary for a comprehensive immunopharmacological program is
presented. Haptens for the generation of antibodies specific for naturally occurring (S)-nicotine,
(S)- and (R)-nornicotine, and the metabolite (S)-cotinine were prepared with high optical purity.
Preliminary data for antinicotine antibodies are reported.
Nicotine is the most widely used addictive drug in the
world. As an alkaloid [(S)-(-)-1-methyl-2-(3-pyridyl)-
pyrrolidine] derived from tobacco leaves, nicotine is
available in several forms such as cigarettes, cigars, pipe
tobacco, and chewing tobacco. Hence, the drug is inti-
mately linked with cigarette smoking, the leading pre-
ventable cause of death in the United States.1 Smoking
contributes to coronary heart disease, stroke, vascular
disease, peptic ulcers, chronic lung diseases and lung
cancer, and fetal brain damage and morbidity. Although
the dangers of smoking are well-known, people continue
to smoke.
A great deal of evidence supports the view that people
continue to smoke because of the addictive effects of
nicotine.2 However, since nicotine is legally and widely
accessible there is relatively little stigma associated with
its use, unlike cocaine, heroin, and other illicit drugs.
Although a large percentage of addicted smokers have
expressed a desire to stop smoking and most who quit
do so without treatment, less than 5% of unaided at-
tempts lead to successful long-term abstinence.3 Simi-
larly, the two most popular therapies, nicotine gum and
transdermal nicotine patches used to slowly wean the
user off the drug, have afforded inadequate long-term
success rates of <20%.4 Perhaps because relatively little
is known about the specific neuropharmacologic mecha-
nisms underlying nicotine addiction and the response to
smoking cessation treatment, no highly effective therapy
has been developed.
There is a need to develop a treatment approach to
nicotine addiction which does not depend solely on
unaided compliance or on the administration of nicotine
itself for rehabilitation. One alternative might rely on
immunological reagents and the immune system. Re-
cently, we demonstrated the efficacy of immunological
strategies with regard to the cocaine abuse problem.5 The
immune-mediated binding of nicotine would impede its
passage into the central nervous system and would result
in a suppression of its characteristic actions. In particu-
lar, we believe the use of both active immunization
(immunoconjugate vaccine) and passive immunization
(monoclonal antibodies, mAbs) protocols would provide
the most beneficial therapy. Herein, we describe the
design and synthesis of haptens necessary to establish
an immunopharmacologic arsenal to combat nicotine
dependence. Significantly, we have developed a compre-
hensive program aimed at nicotine as the primary target,
as well as the secondary targets nornicotine and cotinine.
Resu lts a n d Discu ssion
In the past 30 years, a number of reports appeared in
the literature which described nicotine haptens and
immunoconjugates.6 One of these haptens was recently
used in vaccination studies with rats.7 Our general
approach to the design of haptens for naturally occurring
ligands has been to retain the significant structural and
(5) (a) Carrera, M. R. A.; Ashley, J . A.; Wirsching, P.; Koob, G. F.;
J anda, K. D. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 1988-1992. (b)
Carrera, M. R. A.; Ashley, J . A.; Zhou, B.; Wirsching, P.; Koob, G. F,
J anda, K. D. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 6202-6206. (c)
Carrera, M. Rocio A.; Ashley, J . A.; Parsons, L. H.; Wirsching, P.; Koob,
G. F.; J anda, K. D. Nature 1995, 378, 727-730.
(6) (a) Hieda, Y.; Keyler, D. E.; VanDeVoort, J . T.; Kane, J . K.; Ross,
C. A.; Raphael, D. E.; Niedbala, R. S.; Pentel, P. R. J . Pharmacol. Exp.
Ther. 1997, 283, 1076-1081. (b) Abad, A.; Manclu´s, J . J .; March, C.;
Montoya, A. Anal. Chem. 1993, 65, 3227-3231. (c) Bjercke, R. J .; Cook,
G.; Rychlik, N.; Gjika, H. B.; Van Vunakis, H.; Langone, J . J . J .
Immunol. Methods 1986, 90, 203-213. (d) Castro, A.; McKennis, H.,
J r.; Monji, N.; Bowman, E. R. Biochem. Arch. 1985, 1, 205-214. (e)
Castro, A.; Monji, N.; Hacer, A.; Bowman, E. R.; McKennis, H., J r.
Biochem. Arch. 1985, 1, 173-183. (f) Langone, J . J .; Van Vunakis, H.
Methods Enzymol. 1982, 84, 628-640. (g) Matsushita, H.; Noguchi,
M.; Tamaki, E. Biochem. Biophys. Res. Commun. 1974, 57, 1006-1010.
(h) Langone, J . J .; Gjika, H. B.; Van Vunakis, H. Biochemistry 1973,
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(1) (a) Nelson, D. E.; Kirkendall, R. S.; Lawton, R. L.; Chrismon, J .
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Rep. 1994, 43, 1-8. (b) US Dep. Health Hum. Serv. Reducing the health
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General; Public Health Services: Rockville, MD, 1989.
(2) (a) Benowitz, N. L. Annu. Rev. Pharmacol. Toxicol. 1996, 36,
597-613. (b) Rose, J . E. Annu. Rev. Med. 1996, 47, 493-507.
(3) (a) Hughes, J . R.; Gulliver, S. B.; Fenwick, J . W. et al. Health
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Psychosoc. Oncol. 1988, 6, 217-234.
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(b) Hughes, J . R. In Integrating Behavior Theories with Medication in
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10.1021/jo001442w CCC: $20.00 © 2001 American Chemical Society
Published on Web 05/12/2001

4116 J . Org. Chem., Vol. 66, No. 12, 2001
Isomura et al.
Nornicotine 2b was then obtained with the (S)-(-)
stereochemistry in g98% ee via a classical resolution of
the racemic 8.9 In the process, the (R)-isomer 2c was also
procured with a similar purity. The reaction with linker
14 proceeded with a good yield of monoalkylation. We
anticipated that, after purification of the intermediate
benzyl ester, hydrogenation would afford isolated NIC
ready for use. However, somewhat surprisingly, ∼25%
of the ring-opened, 2′-C-N cleavage product 15 was also
produced, even at 1 atm of hydrogen and short reaction
times, which necessitated purification of the final hapten.
Benzylic amines are generally more resistant to hydro-
genolysis than benzyl esters and acidic media is often
necessary to facilitate the reaction.10 In this case, the
benzylic-like C-N bond adjacent to the pyridyl ring was
sufficiently labile to compete with hydrogenolysis of the
benzyl ester C-O bond. Although there was no byproduct
in the conversion of 8 to nornicotine, the reaction condi-
tions were different and incorporated triethylamine.
Since we omitted a base in the reaction of 9 in order to
avoid salt formation of the NIC hapten, perhaps the more
acidic environment, especially after benzyl ester hydro-
genolysis, activated the C-N bond toward cleavage.
F igu r e 1. Tobacco components and metabolites under discus-
sion.
stereochemical features of the target compound unaltered
upon conversion into a hapten.5b Furthermore, the linker
required for coupling to a carrier protein should mimic a
functional group of the ligand and be of a sufficient length
and with a position of attachment to allow appropriate
presentation of all important recognition elements. In
this way, enhanced antibody titers in vivo, as well as
affinity and specificity of isolated mAbs, are obtained that
benefit immunopharmacotherapy.
For optimum results, the design and synthesis of
nicotine-like haptens demands attention to stereochem-
istry, acid/base properties, and incorporation of a suitable
linker moiety. (S)-(-)-Nicotine 1 as well as structurally
related compounds, nornicotine 2 present in tobacco and
also a metabolite and cotinine 3 the major primary
metabolite of nicotine, are included in the development
of our program. N-Methylpyrrolidine 4, a minor tobacco
component, is only important with regard to the elicita-
tion and selection of premium antinicotine specificities
in order to afford the best therapy (Figure 1).
Implementation of our strategy with regard to nicotine
requires a hapten with the natural (S)-configuration.
Although an immune response might be expected against
both antipodes of a racemic hapten, stereospecificity and
serum titers should be enhanced using the (S)-antipode
alone. This has been shown for instance in the case of
propranolol8 and in work from our laboratory (unpub-
lished results and see below). The hapten should also
contain an alkyl linker of appropriate length at the
position of the methyl group on the pyrrolidine nitrogen.
With the above approach, all of the characteristics of
nicotine remain intact for display to the immune system,
namely stereochemistry, two unaltered ring structures,
correct charge at physiological pH, and conservation of
the important pyrrolidyl methyl group mimicked as part
of the linker.
Consequently, hapten 10 (code: NIC) was synthesized
with the (S)-configuration corresponding to natural nico-
tine (Figure 2). To this end, the formation of 5-bro-
momyosmine 7 occurred via the base-mediated con-
densation of ethyl 5-bromonicotinate 6 with N-vinyl-
pyrrolidinone, followed by in situ acid-catalyzed hydroly-
sis of the â-keto-N-vinyllactam, decarboxylation, and
cyclization.9 The isolated imine was then reduced with
sodium borohydride to afford 5-bromonornicotine 8.
All previously reported nicotine haptens contained
short linkers, at either the pyridyl or pyrrolidyl ring
carbons, and were racemic in nature. The linker used
here, ∼12 Å in length and with an internal amide bond
we recently found useful for hapten immune responses,
should yield complete and high affinity recognition of all
features of the nicotine molecule.
In the secondary phase of our program, anti-nornico-
tine and anti-cotinine mAbs provide a potentially ben-
eficial adjunct to nicotine therapy. Nornicotine 2 occurs
in tobacco as a mixture of (R)- and (S)-enantiomeric
forms. Although present as only 2% of total alkaloids,
the compound is pharmacologically active in the central
nervous system similar to nicotine.11,12 Both optical
isomers of nornicotine have similar binding affinities to
the nicotinic acetylcholine receptor, but there is evidence
that the enantiomers differ in their behavioral and
pharmacological activities.12 Nornicotine also accounts for
∼8% of metabolized nicotine in both humans and rats
and has a plasma half-life of about 8 h which is longer
than the average 2-h half-life of nicotine.13 Most signifi-
cant, animal behavioral studies of rats demonstrate that
(9) J acob, P., III. J . Org. Chem. 1982, 47, 4165-4167.
(10) Rylander, P. N. Hydrogenation Methods; Academic Press: San
Diego, 1985; pp. 163-165.
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N. L. J . Chromatogr. 1993, 619, 49-61. (b) Saitoh, F.; Noma, M.;
Kawashima, N. Phytochemistry 1985, 24, 477-480.
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Dwoskin, P. L. Pharmacol. Biochem. Behav. 1997, 58, 1083-1087. (b)
Crooks, P. A.; Dwoskin, L. P. Biochem. Pharmacol. 1996, 54, 743-
753. (c) Dwoskin, L. P.; Teng, L.; Buxton, S. T.; Ravard, A.; Deo, N.;
Crooks, P. A. Eur. J . Pharmacol. 1995, 276, 195-199. (d) Dwoskin, L.
P.; Buxton, S. T.; J ewell, A. L.; Crooks, P. A. J . Neurochem. 1993, 60,
2167-2174. (e) Copeland, J . R.; Adem, A.; J acob, P., III; Nordberg, A.
Naunyn-Schmiedeberg’s Arch. Pharmacol. 1991, 343, 123-127. (f)
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Exp. Ther. 1988, 244, 807-813.
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loids: Absorption, Distribution, Metabolism, and Excretion; Gorrod,
J . W., Wahren, J ., Eds.; Chapman Hall: London, 1993; pp. 147-179.
(b) Kyerematen, G. A.; Morgan, M.; Chattopadhyay, B.; DeBethizy, J .
D.; Vesell, E. S. Clin. Pharmacol. Ther. 1990, 48, 641-651. (c) Cundy,
K. C.; Crooks, P. A. J . Chromatogr. B Biomed. Sci. Appl. 1984, 306,
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Chem. 1962, 237, 541-545.
(7) (a) Pentel, P. R.; Malin, D. H.; Ennifar, S.; Hieda, Y.; Keyler, D.
E.; Lake, J . R.; Milstein, J . R.; Basham, L. E.; Coy, R. T.; Moon, J . W.
D.; Naso, R.; Fattom, A. Pharmacol. Biochem. Behav. 1999, 65, 191-
198. (b) Hieda, Y.; Keyler, D. E.; VanDeVoort, J . T.; Niedbala, R. S.;
Raphael, D. E.; Ross, C. A.; Pentel, P. R. Psychopharmacology 1999,
143, 150-157.
(8) Spector, S. US Patent 4026879, 1977.

Hapten Design and Synthesis
J . Org. Chem., Vol. 66, No. 12, 2001 4117
F igu r e 2. Synthesis of the (S)-nicotine hapten: (a) ethanol, concentrated H2SO4, reflux; (b) (i) NaH, THF, 1-vinyl-2-pyrrolidinone,
reflux, (ii) aq HCl, reflux, (iii) aq NaOH; (c) NaBH4; (d) (i) resolution (MTPA salts), (ii) H2, Pd/C, NEt3, EtOH; (e) 14, DIEA,
acetonitrile; (f) H2, Pd/C, MeOH; (g) HBTU, N-methylmorpholine, DMF; (h) NaI, acetonitrile.
flexible linker will afford a substantial fraction of anti-
bodies that recognize only the pyridyl and pyrrolidyl rings
and not the alkyl-chain region. Consequently, binding
should be more specific for nornicotine rather than the
nicotine structure bearing a methylated nitrogen.
Cotinine 3 accounts for 70-80% of metabolized nicotine
and has a long half-life of 16-20 h.15 In addition, cotinine
levels can average 15-fold higher than nicotine during
smoking or nicotine replacement therapy.15a Apparently,
cotinine also (1) has an effect on nicotinic cholinergic
receptors in vitro, (2) influences release of neurotrans-
mitters, (3) inhibits androgen biosynthesis, and (4) pos-
F igu r e 3. Synthesis of the racemic and (S)- and (R)-norni-
cotine haptens: (a) 20, DIEA, acetonitrile; (b) H2, Pd/C, MeOH;
sibly contributes to the lower blood pressure of smokers
during nonsmoking intervals.16 Notably, cotinine is re-
ported to effect the pharmacology of nicotine withdrawal
and could promote cardiovascular and endocrine effects
and withdrawal symptoms after nicotine abstinence.17
Hence, inclusion of cotinine as a target is warranted in
an immunotherapeutic approach to the nicotine problem.
(c) benzyl alcohol, p-TsOH, cyclohexane; (d) NaI, acetonitrile.
nornicotine effects locomotor activity and is self-admin-
istered.14
At first, the hapten 16a (code: NOC) was synthesized
starting from rac-nornicotine 2a (Figure 3). However,
preliminary tests indicated the absence of a viable
immune response against the hapten in accord with
previous hypotheses. Therefore, separate (S)-NOC and
(R)-NOC haptens were prepared in order to obtain
antibodies specific for each isomer of nornicotine.
Hence, linker 20 was introduced onto the nornicotine
framework using reaction chemistry similar to that
employed for the preparation of 9. Hydrogenation of
16a -c afforded NOC haptens which required purifica-
tion, again due to the formation of a ring-opened product
analogous to 15. Although the NOC hapten is structur-
ally analogous to NIC, the “tuning” of the linker length
should be critical. We anticipate that the shorter and less
For cotinine-specific antibodies, the (S)-hapten 24
(code: COT) was synthesized starting from 2b (Figure
4). The central transformation was the Wenkert oxidation
of the amine 21 using conditions similar to those in the
(15) (a) J acob, P., III.; Benowitz, N. L. Clin. Pharmacol. Ther. 1994,
56, 483-493. (b) J acob, P., III.; Benowitz, N. L.; Shulgin, A. T.
Pharmacol. Biochem. Behav. 1988, 30, 249-253. (c) Benowitz, N. L.;
Kuyt, F.; J acob, P., III; J ones, R. T.; Osman, A. L. Clin. Pharmacol.
Ther. 1983, 34, 604-611. (d) Benowitz, N. L.; Hall, S. M.; Herning, R.
I. N. Engl. J . Med. 1983, 309, 139-142.
(16) (a) Dwoskin, L. P.; Teng, L.; Buxton, S. T.; Crooks, P. A. J .
Pharmacol. Exp. Ther. 1999, 288, 905-911. (b) Benowitz, N. L.; Sharp,
D. S. Circulation 1989, 80, 1309-1312. (c) Yeh, J .; Barbieri, R. L.;
Friedman, A. J . J . Steroid Biochem. 1989, 33, 627-630. (d) Fuxe, K.;
Everitt, B. J .; Hokfelt, T. Pharmacol. Biochem. Behav. 1979, 10, 671-
677.
(14) (a) Bardo, M. T.; Green, T. A.; Crooks, P. A.; Dwoskin, L. P.
Psychopharmacology 1999, 146, 290-296. (b) Dwoskin, L. P.; Crooks,
P. A.; Teng, L.; Green, T. A.; Bardo, M. T. Psychopharmacology 1999,
145, 442-451.
(17) Keenan, R. M.; Hatsukami, D. K.; Pentel, P. R.; Thompson, T.
N.; Grillo, M. A. Clin. Pharmacol. Ther. 1994, 55, 581-590.

4118 J . Org. Chem., Vol. 66, No. 12, 2001
Isomura et al.
F igu r e 4. Synthesis of the (S)-cotinine hapten: (a) 27, DIEA, acetonitrile; (b) Hg(OAc)2, aq EDTA, pH 9, dioxane, reflux; (c) (i)
TFA, CH2Cl2, (ii) 29, HBTU, N-methylmorpholine, DMF; (d) H2, Pd/C, MeOH; (e) di-tert-butyldicarbonate, MeOH; (f) (i)
methanesulfonyl chloride, NEt3, CH2Cl2, (ii) NaI, acetonitrile; (g) benzyl alcohol, DMAP, CH2Cl2.
Ta ble 1. Da ta for Som e An tin icotin e MAbs Der ived fr om
literature for the conversion of nicotine to cotinine.18 The
stereochemical integrity should not be affected, since no
racemization occurs in this reaction.
NIC-KLH
NIC mAb
isotype (IgG)
Kd (M x 107)
specificitya (%)
3G2
κγ1
κγ1
κγ2a
κγ1
κγ1
κγ1
3.0
2.7
1.6
2.5
3.4
2.0
0.30, nd
0.15, nd
0.10, nd
0.20, nd
0.65, nd
0.055, nd
Modification of the linker chemistry by using 27 was
necessary to provide a compound that was stable during
the basic conditions of the oxidation. The benzyl ester-
containing linker in 9 decomposed and gave products that
could not be purified. Interestingly, unlike in the NIC
synthesis, the hydrogenation to produce COT was nearly
quantitative. There was no cleavage of the 5′-C-N bond
likely due to deactivation by the pyrrolidone ring and the
absence of a basic nitrogen as in 9. Notably, the final
linker in 24 differs from that in 10 by only one bond
length while maintaining the desirable internal amide
bond.
Coupling of 10 to keyhole limpet hemocyanin (KLH)
and bovine serum albumin (BSA) afforded the conjugates
NIC-KLH for immunization and NIC-BSA for screening.
Our preliminary studies indicated that NIC-KLH could
be used to immunize mice in a very consistent fashion
and thereby produce both a high quality, reproducible
immune response, as well as mAbs with excellent affinity
and specificity for nicotine (Table 1). We anticipate
similar results for NOC-KLH and COT-KLH.
The Kd values for nicotine binding surpassed all
previously reported examples except those of Langone et
al. that were ∼10-fold better.6c However, the same hapten
employed by these workers was used by others and
afforded mAbs that bound nicotine ∼100-fold less tightly.6b
Hence, the characteristics of this hapten, or its immu-
noconjugates, might not be conducive for achieving
consistent titers which would be a drawback for thera-
peutic purposes. Although our antinicotine mAbs had
lower affinities, the specificity for nicotine relative to
nornicotine and cotinine was improved by severalfold
compared to the Langone mAbs which lends support to
aspects of our hapten design. Consequently, 2, 3, and 4
6C12
13A3
1B10
5E8
9D9
a
Determined by enzyme-linked immunosorbent assay (ELISA)
using NIC-BSA; percent cross-reactivity (2b, 3) relative to the
amount of free nicotine that produces 50% decrease in mAb
binding; nd ) not detectable (no change in ELISA reading at 1000-
fold molar excess of 3 compared to nicotine).
should not compete with nicotine for antibody binding
sites and therefore would not pose a problem during
treatment.
While both a nicotine vaccine and an antinicotine mAb
are part of our therapeutic strategy, we believe passive
immunization protocols would be most practical with
regard to 2 and 3. Certainly, the binding and blockade
of nicotine is of paramount importance for therapy.
However, the additional treatment with a “cocktail” of
mAbs specific for nornicotine and cotinine could have
important ramifications with regard to reducing rein-
forcement and relapse potential, especially for some
individuals. The haptens described provide the essential
elements in a program of immunopharmacotherapy that
offers a new avenue in the challenging battle against
nicotine addiction.
Exp er im en ta l Section
Gen er a l Meth od s. 1H and 13C NMR spectra were measured
at 400 MHz on a Brucker AMX-400 spectrometer. Chemical
sifts (ppm) were reported relative to internal CDCl3 (1H, 7.26
ppm and 13C, 77.0 ppm) and CD3OD (1H, 3.30 ppm and 13C,
49.2 ppm). HRMS spectra were recorded using electrospray
ionization (ESI) or MALDI techniques. Glassware and solvents
were dried by standard methods. Flash chromatography was
performed on silica gel 60 (230-400 mesh) and thin-layer
chromatography on glass plates coated with a 0.02 mm layer
(18) Wenkert, E.; Angell, E. C. Synth. Commun. 1988, 18, 1331-
1337.

Hapten Design and Synthesis
J . Org. Chem., Vol. 66, No. 12, 2001 4119
of silica gel 60 F-254. The rac-nornicotine 2a and other
chemical reagents and solvents were from Aldrich Chem. Co.,
unless otherwise noted, and used without further purification.
Eth yl 5-Br om on icotin a te 6. A solution of 5-bromonicotinic
acid 5 (20.6 g, 102 mmol) in a mixture of ethanol (400 mL)
and concentrated sulfuric acid (5 mL) was stirred at reflux
under nitrogen for 18 h. The ethanol was removed and
evaporated and the resulting white residue was dissolved in
water. The aqueous solution was made basic to pH 8 with sat.
sodium bicarbonate and extracted with ether. The organic
layer was washed with brine, dried over Na2SO4, and evapo-
was dissolved in EtOAc (25 mL) and treated with a solution
of (+)-MTPA (3.9 g, 16.7 mmol) in EtOAc (13 mL) with stirring.
After the solution was left standing for 15 min, the crystallized
product was collected by filtration. Three recrystallizations
from boiling acetonitrile yielded 4.0 g (52%) of colorless needles
[(S)-5-bromonornicotine (+)-MTPA salt].
(S)-Nor n icotin e 2b. A suspension of (S)-5-bromonornico-
tine (+)-MTPA salt (2.39 g, 5.17 mmol) in ether (120 mL) was
vigorously shaken with 1 M KOH (50 mL) in a separatory
funnel. The ether layer was washed with 1 M KOH (50 mL),
dried over anhydrous K2CO3, and evaporated. The residual oil
was dissolved in EtOH (50 mL) containing Et3N (1.25 mL) and
hydrogenated at 1 atm with 10% Pd/C (500 mg). After 1 h,
the mixture was filtered through Celite and the filter cake
washed with EtOH. The filtrate was poured into 1 M K2CO3
(125 mL) which was then extracted with two 125 mL portions
of CH2Cl2. After washing with brine (50 mL), the combined
extracts were dried over anhydrous K2CO3 and evaporated.
The residue was purified by chromatography on silica gel
eluting with CH2Cl2/MeOH (7:1) to give (S)-2b as a pale yellow
1
rated to give 6 as a pale yellow oil (22 g, 94% yield). H NMR
(CDCl3) δ: 9.08 (d, J ) 1.8 Hz, 1H), 8.79 (d, J ) 2.4 Hz, 1H),
8.38 (dd, J ) 1.8, 2.4 Hz, 1H), 4.38 (q, J ) 7.0 Hz, 2H), 1.38 (t,
J ) 7.0 Hz, 3H). 13C NMR (CDCl3) δ: 163.9, 154.3, 148.7, 139.3,
127.5, 120.5, 61.8, 14.1. HRMS (MALDI-FTMS): calcd for
C8H9BrNO2 (MH+) 229.9817, found 229.9816.
3-Br om o-5-(4, 5-d ih yd r o-3H-p yr r ol-2-yl)-p yr id in e (5-
Br om om yosm in e) 7. Sodium hydride (2.4 g, 60 mmol, 60%
dispersion in oil) in a three-neck flask was washed with three
20 mL portions of hexane. The flask was fitted with a reflux
condenser, flushed with nitrogen, and charged with THF (85
mL). A solution of 6 (10.6 g, 46.1 mmol) and 1-vinyl-2-
pyrrolidinone (5.5 g, 49.5 mmol) in THF (15 mL) was added
in one portion. The mixture was stirred and refluxed for 1 h
and then cooled to room temperature. A solution of concen-
trated HCl (8 mL) in water (12 mL) was added, and the THF
was removed on a rotary evaporator. Additional concentrated
HCl (12 mL) and water (24 mL) were added, and the mixture
was heated at reflux overnight. In an ice-cooled bath, the
solution was made basic with concentrated aqueous NaOH,
which resulted in precipitation of the crude product, and then
it was extracted with CH2Cl2. The CH2Cl2 layer was washed
with water and brine and evaporated. The residue was purified
by chromatography on silica gel eluting with CH2Cl2/acetone
1
oil (621 mg, 81% yield). H NMR (CDCl3) δ: 8.54 (d, J ) 2.1
Hz, 1H), 8.42 (dd, J ) 1.8, 5.0 Hz, 1H), 7.67 (dt, J ) 1.8, 7.9
Hz, 1H), 7.19 (ddd, J ) 0.6, 4.7, 7.9 Hz, 1H), 4.11 (t, J ) 7.6
Hz, 1H), 3.15 (ddd, J ) 5.6, 7.6, 10.0 Hz, 1H), 3.03-2.97 (m,
1H), 2.21-2.14 (m, 1H), 1.95-1.77 (m, 2H), 1.67-1.57 (m, 1H).
13C NMR (CDCl3) δ: 148.5, 148.1, 140.0, 134.0, 123.2, 59.9,
46.8, 34.2, 25.4. HRMS (MALDI-FTMS): calcd for C9H13N2
(MH+) 149.1079, found 149.1074. [R]23 ) -35.8° (c ) 2.184,
D
MeOH).
The (R)-2c isomer was prepared in a similar fashion from
(R)-5-bromonornicotine (-)-MTPA salt. [R]23 ) + 37.5° (c )
D
3.806, MeOH).
1H NMR of a diastereomeric derivative was used to deter-
mine the enantiomeric excess of each isomer. A small amount
of (S)-, (R)-, or rac-nornicotine (∼10 mg) in CH2Cl2 (50 µL) was
added Et3N (30 µL, 0.215 mmol) and a solution of (S)-R-
methoxy-R-(trifluoromethyl)-phenylacetyl chloride (800 µL, 0.1
M in CH2Cl2). The reaction mixture was mixed briefly and then
evaporated. The residue was partitioned between EtOAc and
water, and the EtOAc layer was washed with brine, dried over
Na2SO4, and evaporated. The starting material and byproducts
in the residue were removed by chromatography on silica gel,
eluting with CH2Cl2/MeOH (50:1) to give the amide derivative
as a colorless oil (TLC g 90% pure). The 2′-H proton shifted
from 4.11 to 5.21 ppm for the (S)-nornicotine derivative and
to 5.17 ppm for the (R)-nornicotine derivative. As indicated
by the racemic sample, the patterns were different and clearly
distinguishable. Neither diastereomer showed the presence of
the other. To obtain a threshold of detection, each isomer was
mixed with 1 mol % of the other isomer. Both samples showed
a trace signal above noise at the expected location. Hence, it
was possible to assign a lower limit of 98% ee to both
(S)-nornicotine and (R)-nornicotine. (S)-Nornicotine deriva-
tive (2′-H proton) δ: 5.21 (dd, J ) 3.8, 6.2 Hz, 1H). HRMS
(MALDI-FTMS): calcd for C19H20F3N2O2 (MH+) 365.1471,
found 365.1474. (R)-Nornicotine derivative (2′-H proton) δ:
5.17 (dd, J ) 6.8, 7.9 Hz, 1H). HRMS (MALDI-FTMS): calcd
for C19H20F3N2O2 (MH+) 365.1471, found 365.1483.
1
(19:1) to 7 as a pale yellow solid (7.4 g, 72% yield). H NMR
(CDCl3) δ: 8.82 (d, J ) 1.8 Hz, 1H), 8.65 (d, J ) 2.4 Hz, 1H),
8.29 (t, J ) 2.1 Hz, 1H), 4.06-4.01 (m, 2H), 2.91-2.85 (m, 2H),
2.07-1.98 (m, 2H). 13C NMR (CDCl3) δ: 169.7, 152.0, 146.9,
137.0, 131.5, 120.8, 61.6, 34.7, 22.4. MS (ESI): C9H9BrN2 (Fw
224/226), m/z 225/227 (MH+).
3-Br om o-5-(2-p yr r olid in yl)-p yr id in e (5-Br om on or n i-
cotin e) 8. Sodium borohydride (4.5 g, 119 mmol) was added
portionwise over 10 min with vigorous stirring to a solution
of 7 (12.1 g, 53.8 mmol) in 125 mL of 80:20 methanol/acetic
acid cooled to -40 °C with a dry ice-acetone bath. During the
course of the addition, the temperature rose to -20 °C. After
warming to room temperature, most of the solvent was
removed with a rotary evaporator. Water (300 mL) was added
and the solution was made basic with NaOH and extracted
with CH2Cl2 (2 × 90 mL). The combined extracts were washed
with brine, dried over K2CO3, and evaporated. The residue was
purified by chromatography on silica gel eluting with EtOAc/
MeOH (1:1) to give racemic 8 as a pale yellow oil (9.9 g, 81%).
1H NMR (CDCl3) δ: 8.49 (d, J ) 2.1 Hz, 1H), 8.45 (d, J ) 1.8
Hz, 1H), 7.87 (t, J ) 1.8 Hz, 1H), 4.14 (t, J ) 7.6 Hz, 1H), 3.13
(ddd, J ) 5.3, 7.6, 10.0 Hz, 1H), 3.02 (ddd, J ) 6.8, 7.9, 10.0
Hz, 1H), 2.24-2.16 (m, 1H), 1.93-1.77 (m, 2H), 1.64-1.55 (m,
1H). 13C NMR (CDCl3) δ: 149.0, 146.6, 142.7, 136.6, 120.7,
N-[1-Oxo-6-[(2S)-2-(3-p yr id in yl)-1-p yr r olid in yl]h exyl]-
â-a la n in e P h en ylm eth yl Ester 9. A solution of 14 (136 mg,
0.338 mmol) in acetonitrile (500 µL) was added to a mixture
of 2b (52.1 mg, 0.338 mmol) and diisopropylethylamine (DIEA)
(117 µL, 0.676 mmol) in acetonitrile (850 µL) with stirring at
room temperature. After 18 h, the mixture was evaporated and
the residue was purified by chromatography on silica gel
eluting with CH2Cl2/MeOH (19:1) to give product 9 as a pale
59.1, 46.9, 34.5, 25.4. HRMS (MALDI-FTMS): calcd for C9H12
-
BrN2 (MH+) 227.0184, found 227.0175.
Resolu tion of Ra cem ic 5-Br om on or n icotin e 8 via th e
r-Meth oxy-r-(tr iflu or om eth yl) P h en yla ceta te (MTP A)
Sa lts. A solution of (-)-MTPA (3.9 g, 16.7 mmol) in EtOAc
(13 mL) was added to a solution of 8 (7.57 g, 33.3 mmol) in
EtOAc (52 mL) with stirring. The mixture was allowed to stand
at room temperature for 15 min, after which time the crystal-
line product was collected by filtration to give (R)-isomer
enriched crystals. Three recrystallizations from boiling aceto-
nitrile yielded 4.1 g (54%) of colorless needles [(R)-5-bromonor-
nicotine (-)-MTPA salt]. The filtrate was extracted with 1 N
sulfuric acid (2 times). The acid extracts were combined,
washed with ether, made basic with NaOH, and extracted with
CH2Cl2. The organic layer was evaporated and purified on a
silica gel column to give an (S)-isomer enriched oil. The oil
1
yellow oil (72 mg, 48% yield). H NMR (CDCl3) δ: 8.53 (br s,
1H), 8.46 (br d, 1H), 7.67 (br d, 1H), 7.36-7.31 (m, 5H), 7.23
(dd, J ) 4.7, 7.9 Hz, 1H), 6.07 (br s, 1H), 5.12 (s, 2H), 3.50 (q,
J ) 5.9, 2H), 3.31 (dt, J ) 2.1, 8.2 Hz, 1H), 3.23 (t, J ) 8.2 Hz,
1H), 2.57 (t, J ) 5.9 Hz, 2H), 2.43 (dt, J ) 8.2, 11.7 Hz, 1H),
2.20-2.13 (m, 2H), 2.05 (t, J ) 8.2 Hz, 2H), 2.07-2.01 (m, 1H),
1.97-1.85 (m, 1H), 1.85-1.76 (m, 1H), 1.69-1.60 (m, 1H),
1.53-1.45 (m, 2H), 1.43-1.36 (m, 2H), 1.32-1.13 (m, 2H). 13
C
NMR (CDCl3) δ: 172.9, 172.5, 149.4, 148.4, 135.6, 134.9, 128.6,

4120 J . Org. Chem., Vol. 66, No. 12, 2001
Isomura et al.
128.4, 128.2, 123.5, 67.6, 66.5, 54.0, 53.5, 36.6, 35.1, 34.7, 34.1,
(2S)-2-(3-P yr id in yl)-1-p yr r olid in ebu ta n oic Acid P h e-
n ylm eth yl Ester 16b. Linker 20 (198 mg, 0.650 mmol), (S)-
nornicotine 2b (96.3 mg, 0.650 mmol), DIEA (226 µL, 1.30
28.4, 26.9, 25.4, 22.6. HRMS (MALDI-FTMS): calcd for
C
25H34N3O3 (MH+) 424.2595, found 424.2607. [R]23 ) -65.3°
D
(c ) 1.432, MeOH).
mmol). Pale yellow oil (124 mg, 58% yield). [R]23 ) -88.7° (c
D
N-[1-Oxo-6-[(2S)-2-(3-p yr id in yl)-1-p yr r olid in yl]h exyl]-
â-a la n in e 10 (NIC). The benzylester 9 (51.4 mg, 0.122 mmol)
in MeOH (2 mL) was hydrogenated with 10% Pd/C (9.3 mg)
at 40 psi. After 90 min, the mixture was filtered through Celite
and the filter cake was washed with MeOH. The filtrate was
evaporated and the residue was purified by chromatography
on silica gel eluting with CH2Cl2/MeOH (3:2) to give hapten
) 1.076, MeOH).
(2R)-2-(3-P yr id in yl)-1-p yr r olid in ebu ta n oic Acid P h e-
n ylm eth yl Ester 16c. Linker 20 (198 mg, 0.650 mmol), (R)-
nornicotine 2c (96.3 mg, 0.650 mmol), DIEA (226 µL, 1.30
mmol). Pale yellow oil (131 mg, 62% yield). [R]23 ) +87.5° (c
D
) 1.072, MeOH).
ra c-2-(3-P yr idin yl)-1-pyr r olidin ebu tan oic Acid 17a (r a c-
NOC). The benzylester 16a (58.3 mg, 0.180 mmol) in MeOH
(1.5 mL) was hydrogenated with 10% Pd/C (15 mg) using a
balloon technique. After 75 min, the reaction mixture was
filtered through Celite and the filter cake was washed with
MeOH. The filtrate was evaporated, and the residue was
purified by chromatography on silica gel eluting with CH2Cl2/
MeOH (3:2) to give 17a as a colorless oil (26 mg, 62% yield).
1H NMR (CD3OD) δ: 8.60 (d, J ) 1.8 Hz, 1H), 8.53 (dd, J )
1.8, 5.0 Hz, 1H), 7.98 (dt, J ) 2.1, 7.9 Hz, 1H), 7.48 (ddd, J )
0.9, 5.0, 7.9 Hz, 1H), 3.94 (dd, J ) 7.6, 9.4 Hz, 1H), 3.71-3.65
(m, 1H), 2.85-2.69 (m, 3H), 2.43-2.38 (m, 1H), 2.33-2.26 (m,
1H), 2.17-1.98 (m, 4H), 1.83-1.71 (m, 2H). 13C NMR (CD3-
OD) δ: 179.7, 150.7, 150.6, 138.0, 136.1, 125.9, 69.5, 55.3, 54.6,
35.8, 34.4, 24.0, 23.3. HRMS (MALDI-FTMS): calcd for
1
10 as a pale yellow oil (26 mg, 66% yield). H NMR (CD3OD)
δ: 8.53 (s, 1H), 8.45 (d, J ) 3.5 Hz 1H), 7.93-7.90 (m, 1H),
7.44 (dd, J ) 5.0, 7.9 Hz, 1H), 3.55 (t, J ) 8.8 Hz, 1H), 3.49-
3.43 (m, 1H), 3.38 (t, J ) 6.8 Hz, 2H), 2.58-2.51 (m, 1H), 2.50-
2.43 (m, 1H), 2.37 (t, J ) 6.8 Hz, 2H), 2.32-2.24 (m, 2H), 2.11
(t, J ) 7.3 Hz, 2H), 2.07-1.90 (m, 2H), 1.86-1.76 (m, 1H),
1.55-1.45 (m, 4H), 1.34-1.17 (m, 2H). 13C NMR (CD3OD) δ:
179.1, 176.0, 150.2, 149.7, 139.3, 138.0, 125.7, 69.5, 55.4, 54.8,
37.6, 37.6, 37.1, 35.2, 28.8, 28.0, 26.8, 23.5. HRMS (MALDI-
FTMS): calcd for C18H28N3O3 (MH+) 334.2131, found 334.2139.
[R]23 ) -71.0° (c ) 0.534, MeOH).
D
N-(1-Oxo-6-br om oh exyl)-â-alan in e 13. A mixture of 6-bro-
mohexanoic acid 11 (2.20 g, 11.4 mmol) and HBTU (Interna-
tional Peptides) (12.5 mmol) in DMF (15 mL) was stirred at 0
°C for 10 min. To the reaction mixture was added a solution
of â-alanine benzyl ester p-TsOH salt 12 (Sigma) (4.00 g, 11.4
mmol) and N-methyl morpholine (3.75 mL) in DMF (10 mL)
at 0 °C with stirring. After 24 h at room temperature, the
reaction mixture was poured into water and extracted with
EtOAc. The EtOAc layer was washed with brine, dried over
Na2SO4, and evaporated. The residue was purified by chro-
matography on silica gel eluting with hexane/EtOAc (1:1) to
C
13H19N2O2 (MH+) 235.1441, found 235.1440.
(2S)-2-(3-P yr id in yl)-1-p yr r olid in eb u t a n oic Acid 17b
[(S)-NOC]. The benzylester 16b (120 mg, 0.370 mmol), 10%
Pd/C (30 mg), reaction time 20 min. Colorless oil (63 mg, 73%
yield). [R]23 ) -15.7° (c ) 1.202, MeOH).
D
(2R)-2-(3-P yr id in yl)-1-p yr r olid in eb u t a n oic Acid 17c
[(R)-NOC]. The benzylester 16c (95.0 mg, 0.293 mmol), 10%
Pd/C (25 mg), reaction time 20 min. Colorless oil (41 mg, 59%
1
13 as a pale yellow oil (3.4 g, 84% yield). H NMR (CDCl3) δ:
yield). [R]23 ) +13° (c ) 0.822, MeOH).
D
7.37-7.34 (m, 5H), 6.02 (br s, 1H), 5.14 (s, 2H), 3.52 (q, J )
5.9 Hz, 2H), 3.39 (t, J ) 6.8 Hz, 2H), 2.59 (t, J ) 5.9 Hz, 2H),
2.13 (t, J ) 7.6 Hz, 2H), 1.89-1.82 (m, 2H), 1.67-1.58 (m, 2H),
1.47-1.40 (m, 2H). 13C NMR (CDCl3) δ: 172.6, 172.4, 135.5,
128.5, 128.3, 128.2, 66.5, 36.2, 34.7, 34.0, 33.6, 32.3, 27.6, 24.6.
HRMS (MALDI-FTMS): calcd for C16H22BrNO3Na (MNa+)
378.0681, found 378.0693.
Ben zyl 4-Br om obu ta n oa te 19. A solution of 4-bromobu-
tanoic acid 18 (25 g, 0.15 mol), benzyl alcohol (21 g, 0.194 mol),
and p-TsOH hydrate (1.3 g, 6.87 mmol) in cyclohexane (225
mL) was heated to reflux. The water was azeotropically
removed with the aid of a Dean-Stark trap. After 1 hr, the
stoichiometric amount of water was collected and the solution
refluxed an additional hr. After cooling to room temperature,
the solution was washed with sat. sodium bicarbonate and
brine and dried over Na2SO4 and evaporated. The residue was
distilled (bp 120-125 °C, 2 mm Hg) to give 19 as a clear,
colorless liquid (30 g, 78% yield).1H NMR (CDCl3) δ: 7.40-
7.32 (m, 5H), 5.13 (s, 2H), 3.46 (t, J ) 6.5 Hz, 2H), 2.56 (t, J
) 7.1 Hz, 2H), 2.23-2.16 (m, 2H). 13C NMR (CDCl3) δ: 172.3,
135.7, 128.5, 128.3, 128.2, 66.4, 32.7, 32.4, 27.7.
N-(1-Oxo-6-iod oh exyl)-â-a la n in e 14. A mixture of 13 (1.00
g, 2.81 mmol) and sodium iodide (2.10 g, 14.1 mmol) in
acetonitrile (6 mL) was vigorously stirred at room temperature
1
for 18 h. The progress of the reaction was followed by H NMR.
After completion, the mixture was evaporated and the residue
was partitioned between EtOAc and water. The EtOAc layer
was washed with brine, dried over Na2SO4, and evaporated.
The residue was purified by chromatography on silica gel
eluting with hexane/EtOAc (1:1) to give 14 as a colorless oil
(1.11 g, 98% yield). 1H NMR (CDCl3) δ: 7.38-7.32 (m, 5H),
6.08 (br s, 1H), 5.13 (s, 2H), 3.51 (q, J ) 6.2 Hz, 2H), 3.16 (t,
J ) 7.0 Hz, 2H), 2.58 (t, J ) 6.2 Hz, 2H), 2.12 (t, J ) 7.6 Hz,
2H), 1.84-1.76 (m, 2H), 1.64-1.56 (m, 2H), 1.42-1.34 (m, 2H).
13C NMR (CDCl3) δ: 172.6, 172.5, 135.6, 128.6, 128.4, 128.2,
66.5, 36.2, 34.7, 34.0, 33.0, 29.9, 24.4, 6.7. HRMS (MALDI-
FTMS): calcd for C16H22INO3Na (MNa+) 426.0537, found
426.0553.
Ben zyl 4-Iod obu ta n oa te 20. A mixture of 19 (500 mg, 1.95
mmol) and sodium iodide (1.46 g, 9.75 mmol) in acetonitrile
(4 mL) was vigorously stirred at room temperature for 18 h.
1
The progress of the reaction was followed by H NMR. After
completion, the reaction mixture was evaporated and the
residue was partitioned between EtOAc and water. The EtOAc
layer was washed with brine, dried over Na2SO4, and evapo-
rated. The residue was purified by chromatography on silica
gel eluting with hexane/CH2Cl2 (2:1) to give 20 as a colorless
oil (466 mg, 79% yield). 1H NMR (CDCl3) δ: 7.39-7.34 (m,
5H), 5.14 (s, 2H), 3.23 (t, J ) 6.8 Hz, 2H), 2.51 (t, J ) 7.1 Hz,
2H), 2.19-2.12 (m, 2H). 13C NMR (CDCl3) δ: 172.0, 135.7,
ra c-2-(3-P yr id in yl)-1-p yr r olid in ebu ta n oic Acid P h e-
n ylm eth yl Ester 16a . A solution of 20 (103 mg, 0.338 mmol)
in acetonitrile (500 µL) was added to a solution of rac-
nornicotine (50.0 mg, 0.338 mmol) and DIEA (117 µL, 0.676
mmol) in CH3CN (850 µL) with stirring at room temperature.
After 18 h, the reaction mixture was evaporated and the
residue was purified by chromatography on silica gel, eluting
with CH2Cl2/MeOH (19:1) to give 16a as a pale yellow oil (65
128.5, 128.2, 128.1, 66.3, 34.7, 28.3, 5.4. MS (GCMS) C11H13
-
IO2 (Fw 304), m/z 304 (M+).
[6-[(2S)-2-(3-P yr id in yl)-1-p yr r olid in yl]h exyl]-ca r ba m -
ic Acid 1,1-Dim eth yleth yl Ester 21. A solution of 27 (425
mg, 1.30 mmol) in acetonitrile (2 mL) was added to a mixture
of 2b (200 mg, 1.35 mmol) and DIEA (470 µL, 2.70 mmol) in
acetonitrile (4 mL) with stirring at room temperature. After
15 h, the reaction mixture was evaporated and the residue
was purified by chromatography on silica gel eluting with CH2-
Cl2/MeOH (30:1) to give 21 as a pale yellow oil (266 mg, 59%
1
mg, 59% yield). H NMR (CD3OD) δ: 8.46 (br s, 1H), 8.38 (d,
J ) 4.7 Hz, 1H), 7.82-7.79 (m, 1H), 7.35-7.29 (m, 6H), 5.06
(d, J ) 12.3 Hz, 1H), 5.00 (d, J ) 12.3 Hz, 1H), 3.36-3.28 (m,
2H), 2.44 (dt, J ) 8.2, 12.0 Hz, 1H), 2.32 (dt, J ) 2.1, 7.0 Hz,
2H), 2.24-2.11 (m, 3H), 1.96-1.81 (m, 2H), 1.80-1.69 (m, 2H),
1.67-1.57 (m, 1H). 13C NMR (CD3OD) δ: 174.9, 149.9, 149.0,
141.7, 137.8, 137.6, 129.7, 129.4, 129.4, 125.5, 68.9, 67.3, 54.7,
54.5, 36.2, 32.9, 25.0, 23.7. HRMS (MALDI-FTMS): calcd for
1
yield). H NMR (CD3OD) δ: 8.49 (s, 1H), 8.41 (d, J ) 3.8 Hz,
1H), 7.85 (dt, J ) 1.8, 7.9 Hz, 1H), 7.40 (dd, J ) 5.0, 7.9 Hz,
1H), 3.36-3.29 (m, 2H), 2.96 (t, J ) 7.0 Hz, 2H), 2.44 (dt, J )
8.5, 12.0 Hz, 1H), 2.29-2.07 (m, 2H), 2.13 (m, 1H), 1.96-1.85
(m, 2H), 1.72-1.63 (m, 1H), 1.46-1.35 (m, 13H), 1.30-1.18
C
20H25N2O2 (MH+) 325.1911, found 325.1900.

Hapten Design and Synthesis
J . Org. Chem., Vol. 66, No. 12, 2001 4121
1.97-1.89 (m, 1H), 1.46-1.35 (m, 4H), 1.30-1.17 (m, 4H). 13
(m, 4H). 13C NMR (CD3OD) δ: 158.7, 149.9, 149.0, 141.7, 137.7,
125.5, 79.9, 69.2, 55.7, 54.9, 41.4, 36.0, 31.1, 29.8, 29.0, 28.4,
27.9, 23.6. HRMS (MALDI-FTMS): calcd for C20H34N3O2
C
NMR (CD3OD) δ: 179.7, 178.1, 175.5, 150.3, 149.4, 139.0,
136.9, 126.0, 61.4, 42.0, 40.4, 33.0, 31.3, 30.3, 29.2, 27.9, 27.5.
HRMS (MALDI-FTMS): calcd for C19H27N3O4Na (MNa+)
384.1894, found 384.1898. [R]23D ) -14.2° (c ) 1.186, MeOH).
(6-Hyd r oxyh exyl)-ca r ba m ic Acid 1,1-Dim eth yleth yl
Ester 26. A mixture of 6-aminohexanol 25 (2.0 g 17.1 mmol)
and di-tert-butyldicarbonate (4.1 g, 18.8 mmol) in MeOH (40
mL) was stirred at room temperature for 90 min. The reaction
mixture was evaporated, and the residue was purified by
chromatography on silica gel eluting with CH2Cl2/MeOH (49:
1) to give 26 as a pale yellow oil (3.7 g, 99%). 1H NMR (CDCl3)
δ: 4.63 (br s, 1H), 3.58 (t, J ) 6.5 Hz, 2H), 3.07 (q, J ) 6.5 Hz,
2H), 2,12 (br s, 1H), 1.56-1.49 (m, 2H), 1.48-1.40 (m, 11H),
1.37-1.27 (m, 4H). 13C NMR (CDCl3) δ: 156.0, 79.0, 62.4, 40.3,
32.5, 29.9, 28.3, 26.3, 25.2. MS (ESI): C11H23NO3 (Fw 217),
m/z 240 (MNa+).
(MH+) 348.2646, found 348.2630. [R]23 ) -74.2° (c ) 0.96,
D
MeOH).
[6-[(5S)-2-Oxo-5-(3-p yr id in yl)-1-p yr r olid in yl]h exyl]-
ca r ba m ic Acid 1,1-Dim eth yleth yl Ester 22. A solution of
21 (265 mg, 0.764 mmol) in dioxane (30 mL) was added to a
mixture of Hg(OAc)2 (1.22 g, 3.82 mmol) and aqueous EDTA
(7.66 mL, 5 mmol/10 mL, pH 9.0) in water (39 mL), and the
mixture was refluxed with stirring for 2 h. After cooling, the
mixture was filtered through Celite and the filter cake was
washed with a small amount of dioxane. The filtrate was
extracted with CH2Cl2, and the organic layer was washed with
brine, dried over Na2SO4, and evaporated. The residue was
purified by chromatography on silica gel eluting with CH2Cl2/
MeOH (25:1) to give 22 as a pale yellow oil (95 mg, 34% yield).
1H NMR (CD3OD) δ: 8.52 (dd, J ) 1.5, 5.0 Hz, 1H), 8.50 (d, J
) 1.5 Hz, 1H), 7.78 (dt, J ) 2.1, 7.9 Hz, 1H), 7.49 (ddd, J )
0.9, 5.0, 7.9 Hz, 1H), 6.54 (br s, 1H), 4.88-4.85 (m, 1H), 3.61-
3.53 (m, 1H), 3.00-2.95 (m, 2H), 2.64-2.48 (m, 4H), 1.95-
1.89 (m, 1H), 1.42-1.35 (m, 13H), 1.28-1.19 (m, 4H). 13C NMR
(CD3OD) δ: 178.2, 158.7, 150.3, 149.4, 139.0, 136.9, 126.0, 79.9,
61.5, 42.1, 41.4, 31.3, 30.9, 29.3, 29.0, 27.9, 27.6, 27.5. HRMS
(MALDI-FTMS): calcd for C20H31N3O3Na (MNa+) 384.2258,
(6-Iod oh exyl)-ca r ba m ic Acid 1,1-Dim eth yleth yl Ester
27. To a mixture of 26 (1.55 g, 7.13 mmol) and Et3N (1.5 mL,
10.7 mmol) in CH2Cl2 (20 mL) in an ice-cooled bath was added
mesyl chloride (607 µL, 7.8 mmol) with stirring. After 2 h at
room temperature, the reaction mixture was washed with
water and brine, dried over Na2SO4, and evaporated. The
residue was dissolved in acetonitrile (30 mL), and then sodium
iodide (5.35 g, 35.7 mmol) was added at room temperature with
stirring. After 18 h, the mixture was evaporated and the
residue was partitioned between EtOAc and water. The
organic layer was washed with brine, dried over Na2SO4, and
evaporated. The residue was purified by chromatography on
silica gel eluting with hexane/EtOAc (10:1) and to give 27 as
found 384.2273. [R]23 ) -16.4° (c ) 1.834, MeOH).
D
4-Oxo-4-[[6-[(5S)-2-oxo-5-(3-p yr id in yl)-1-p yr r olid in yl]]-
h exyl]a m in o]-bu ta n oic Acid P h en ylm eth yl Ester 23. A
solution of 22 (91.7 mg, 0.254 mmol) in CH2Cl2 (500 µL) was
treated with TFA (400 µL) at 0 °C. After completion, the
mixture was evaporated with toluene (3 times). HBTU (111
mg, 0.292 mmol) was added to a solution of 29 (58.1 mg, 0.279
mmol) in DMF (500 µL) at 0 °C with stirring. After 10 min, a
solution of the deprotected amine residue and N-methylmor-
pholine (167 µL, 1.52 mmol) in DMF (300 µL) was added at 0
°C with stirring. After 18 h at room temperature, the mixture
was partitioned between EtOAc and water. The EtOAc layer
was washed with brine, dried over Na2SO4, and evaporated.
The residue was purified by chromatography on silica gel
eluting with CH2Cl2/MeOH (35:1) to give 23 as a pale yellow
oil (79 mg, 69% yield). 1H NMR (CD3OD) δ: 8.52 (dd, J ) 1.5,
4.7 Hz, 1H), 8.49 (d, J ) 1.5 Hz, 1H), 7.77 (dt, J ) 1.8, 7.9 Hz,
1H), 7.48 (dd, J ) 4.7, 7.9, Hz, 1H), 7.34-7.28 (m, 5H), 5.10
(s, 2H), 4.86-4.83 (m, 1H), 3.56 (dt, J ) 7.6, 13.8 Hz, 1H),
3.09 (dt, J ) 2.1, 7.0 Hz, 2H), 2.65 (t, J ) 6.8 Hz, 2H), 2.63-
2.51 (m, 4H), 2.47 (t, J ) 6.8 Hz, 2H), 1.96-1.88 (m, 1H), 1.42-
1.35 (m, 4H), 1.27-1.16 (m, 4H). 13C NMR (CD3OD) δ: 178.1,
174.2, 150.3, 149.4, 139.0, 137.8, 136.9, 129.7, 129.3, 129.3,
126.0, 67.5, 61.4, 42.0, 40.3, 31.6, 31.3, 30.7, 30.3, 29.2, 27.9,
27.5. HRMS (MALDI-FTMS): calcd for C26H33N3O4Na (MNa+)
474.2363, found 474.2384. [R]23D ) -12.6° (c ) 1.598, MeOH).
4-Oxo-4-[[6-[(5S)-2-oxo-5-(3-p yr id in yl)-1-p yr r olid in yl]]-
h exyl]a m in o]-bu ta n oic Acid 24 (COT). The benzyl ester 23
(77.0 mg, 0.171 mmol) in MeOH (2 mL) was hydrogenated with
10% Pd/C (15 mg) using a balloon technique. After 40 min,
Pd/C was removed by filtration washing with MeOH. The
filtrate was evaporated, and the residue was purified by
chromatography on silica gel eluting with CH2Cl2/MeOH (3:
1
a colorless oil (1.8 g, 77% yield). H NMR (CDCl3) δ: 4.55 (br
s, 1H), 3.16 (t, J ) 6.8 Hz, 2H), 3.08 (q, J ) 6.5 Hz, 2H) 1.80
(m, 2H), 1.48-1.35 (m, 13H), 1.34-1.28 (m, 2H). 13C NMR
(CDCl3) δ: 155.9, 79.0, 40.4, 33.3, 30.1, 29.8, 28.3, 25.6, 6.9.
MS (ESI): C11H22INO2 (Fw 327), m/z 350 (MNa+).
Su ccin ic Acid Mon oben zyl Ester 29. A mixture of
succinic anhydride 28 (1.00 g, 10.0 mmol), benzyl alcohol (860
µL, 8.30 mmol), and DMAP (1.02 g, 8.30 mmol) in CH2Cl2 (30
mL) was stirred at room temperature for 18 h. After this time,
a solution of 5% Na2CO3 was poured into the reaction mixture
and the layers separated. The aqueous layer was acidified with
1 M HCl and extracted with EtOAc. The EtOAc layer was
washed with brine, dried over Na2SO4, and evaporated to give
1
29 as a colorless oil (1.63 g, 94%). H NMR (CDCl3) δ: 7.37-
7.34 (m, 5H), 5.15 (s, 2H), 2.74-2.66 (m, 4H). 13C NMR
(CDCl3) δ: 178.4, 172.0, 135.6, 128.5, 128.2, 128.1, 66.6, 28.9,
28.8. HRMS (MALDI-FTMS): calcd for C11H12O4Na (MNa+)
231.0628, found 231.0627.
Ack n ow led gm en t. This work was supported by
funding from the Skaggs Institute for Chemical Biology.
K. D. J . is an Arthur C. Cope Scholar. The text
appearing on the cover was reproduced from The Merck
Index, 12th Edition. Copyright 1996 by Merck & Co.,
Whitehouse Station, NJ , USA. All rights reserved.
Su p p or tin g In for m a tion Ava ila ble: Copies of the 1H
NMR and 13C NMR spectra for all prepared compounds. This
material is available free of charge via the Internet at
http://pubs.acs.org.
1
1) to give 24 as a pale yellow oil (59 mg, 96% yield). H NMR
(CD3OD) δ: 8.51 (br s, 2H), 7.79 (dt, J ) 1.8, 7.9 Hz, 1H), 7.49
(dd, J ) 5.0, 7.9 Hz, 1H), 4.88-4.85 (m, 1H), 3.57 (dt, J ) 7.6,
14.1 Hz, 1H), 3.11 (dt, J ) 1.2, 7.0 Hz, 2H), 2.63-2.4 (m, 8H),
J O001442W