Synthesis of an immunogenic template for the generation of catalytic antibodies for (-)-cocaine hydrolysis

Full text of DOI:10.1021/jo960522q

5686
J . Org. Chem. 1996, 61, 5686-5689
O
Syn th esis of a n Im m u n ogen ic Tem p la te for
th e Gen er a tion of Ca ta lytic An tibod ies for
(-)-Coca in e Hyd r olysis
O
O
H
N
CH3
N
O
O
O
N
O
O
P
O
Ph
OH
Clifford E. Berkman, Gail E. Underiner, and
J ohn R. Cashman*
1
O
CH3
N
Seattle Biomedical Research Institute, 4 Nickerson Street,
Suite 200 Seattle, Washington 98109
H
N
OH
O
O
O
P
Received March 18, 1996
Ph
OH
2
In tr od u ction
F igu r e 1.
Abuse of (-)-cocaine has become a major public health
problem in the United States. The relative risk to
individuals and the cost to society is difficult to estimate
but the burden to the United States is large in terms of
economic and personal loss. In fact, (-)-cocaine abuse
is one of the most common causes for emergency room
visits in United States hospitals, especially in the inner
cities.
Sch em e 1
O
O
CH3
N
CH3
N
H
N(CH2)5CO2CH3
OH
OH
OH
4
3
The pharmacotherapies to combat (-)-cocaine abuse
that have been attempted thus far have not been suc-
cessful. Consequently, there has been a great interest
in developing novel therapeutic strategies, particularly
those derived from immunological sources for combating
health problems arising from (-)-cocaine abuse.1,2 Lan-
dry and co-workers attempted to procure catalytic anti-
bodies specific for (-)-cocaine hydrolysis, and although
antibodies were isolated, their activity was reportedly
quite low.1 In that study, the ester hapten 1 was coupled
to a carrier protein and used for immunization (Figure
1). Although 1 possessed the C3 phosphonate functional-
ity commonly used for mimicking the transition state of
benzoyl ester hydrolysis, it is possible that the ester
linkage at the C2 position was metabolically prone to
hydrolysis in vivo and thus decreased its immunogenic
lifetime. As a consequence of a decreased residence time
of the hapten in the immunized animal, the potential for
an appropriate immune response was possibly reduced.
For example, it is known that aliphatic C2-esters of (-)-
cocaine are extensively hydrolyzed by serum and hepatic
microsomal esterases from animals and humans.3 There-
fore, to circumvent such potential shortcomings, we
designed and synthesized an analogous hapten (2) with
a more metabolically stable linkage at the C2 position
(i.e., an amide) in order to procure antibodies with more
catalytic activity for (-)-cocaine hydrolysis. This report
will describe the successful synthesis of phosphonate 2.
The novel strategy utilized in our study was to avoid the
difficulties associated with (-)-cocaine’s unique chemistry
by synthesizing the phosphonate 2 through an N-8-amino
protection strategy.
O
CH3
N
H
N(CH2)5CO2CH3
O
O
P
Ph
OCH3
5
acceptable synthetic results. Therefore, the initial strat-
egy for the synthesis of 2 envisioned a straightforward
amidation of ecgonine (3) followed by phosphonylation
(Scheme 1). Although treatment of ecgonine (3) with
oxalyl chloride followed by reaction with methyl 6-ami-
nocaproate generated amide 4, attempts at efficient
phosphonylation of 4 under various conditions were
unsuccessful.
The bridgehead N-8 nitrogen, in addition to steric
hindrance induced by the alkyl amide at C2, may
contribute to the lack of reactivity toward phosphon-
ylating reagents by possible electronic interactions. It
was anticipated that protection of the nitrogen would
circumvent the first of these problems (Scheme 2). This
type of strategy has been recently employed in the
preparation of the tropane alkaloid ferruginine.6 There-
fore, N-norcocaine (6) was fully hydrolyzed to N-norecgo-
nine in an analogous fashion to (-)-cocaine being hydro-
lyzed to ecgonine7 and subsequently N-protected with
benzyl chloroformate (CBZ-Cl) to give the hydroxy acid
7. Coupling of 7 with methyl 6-aminocaproate in the
presence of DCC provided the alcohol 8. Phosphonylation
of 8 was carried out by the method described by Zhao
and Landry8 (i.e., tetrazole catalysis) to afford the phen-
ylphosphonate 9 in an acceptable overall yield of 23%
from N-norcocaine. In contrast to the 15 h phosphon-
ylation protocol for secondary alcohols by Zhao and
Landry,8 it is noteworthy that a decrease of the reaction
time for phosphonylation to 1 h increased the yield of 9
Resu lts a n d Discu ssion
The preparation of (-)-cocaine analogues, where either
the C2 or C3 functional groups were altered, has gener-
ally relied on simple and efficient functional group
transformations.4,5 Such modifications have not required
special consideration of the bridgehead nitrogen for
(4) Kozikowski, A. P.; Xiang, L.; Tanaka, J .; Bergmann, J . S.;
J ohnson, K. M. Med. Chem. Res. 1991, 1, 312.
(5) Lewin, A. H.; Gao, Y.; Abraham, P.; Boja, J . W.; Kuhar, M. J .;
Carroll, F. I. J . Med. Chem. 1992, 35, 135.
(6) Hernandez, A. S; Thaler, A.; Castells, J .; Rapoport, H. J . Org.
Chem. 1996, 61, 314.
(7) Kozikowski, A.; Saiah, M. K. E.; J ohnson, K. M.; Bergmann, J .
S. J . Med. Chem. 1995, 38, 3086.
(8) Zhao, K.; Landry, D. W. Tetrahedron Lett. 1993, 49, 363.
(1) Landry, D. W.; Zhao, K.; Yang, G. X. Q.; Glickman, M.; Geor-
giadis, T. M. Science 1993, 259, 1899.
(2) Rocio, M.; Carrera, A.; Ashley, J . A.; Parsons, L. H.; Wirsching,
P.; Koob, G. F.; J anda, K. D. Nature 1995, 378, 727.
(3) Brzezinski, M. R.; Abraham, T. L.; Stone, C. L.; Dean, R. A.;
Bosron, W. F. Biochem. Phamacol. 1994, 48, 1747.
S0022-3263(96)00522-1 CCC: $12.00 © 1996 American Chemical Society

Notes
J . Org. Chem., Vol. 61, No. 16, 1996 5687
Sch em e 2
O
O
CBZ N
HN
H
O
N(CH2)5CO2CH3
O
CBZ N
CBZ N
OCH3
H
O
O
1. PhP(O)Cl2
DEA, tetrazole
2. MeOH
DCC
OH
1. 1.0 N HCl
2. CBZ-Cl
O
O
N(CH2)5CO2CH3
OH
P
H2N(CH2)5CO2CH3
Ph
OCH3
OH
Ph
9
7
8
6
Sch em e 3
O
O
CBZ N
CBZ N
H
N(CH2)5CO2CH3
O-CBZ
OH
8
6
Na2CO3
MeOH
DCC
H2N(CH2)5CO2CH3
1. 1.0 N HCl
2. CBZ-Cl 3 equiv
O-CBZ
11
10
Sch em e 4
O
O
CBZ N
H
PATH A
N(CH2)5CO2CH3
H2N(CH2)5CO2CH3
O
O
P
P
O
(CH3O)2SO2
CBZ N
Ph
OH
13
O
7
PhP(O)Cl2
DEA
9
O
O
P
CBZ N
12
DCC
H2N(CH2)5CO2CH3
Ph
OH
PATH B
MeOH
O
O
Ph
OCH3
14
Sch em e 5
from 25% to nearly 60%. Although 9 existed as a mixture
of isomers (presumably diastereomeric at phosphorus)
identified by 31P and 1H NMR, mass spectral and
elemental analysis of 9 were consistent with the expected
product. Separation of these isomers was not necessary
because the ultimate synthesis of 15 or 2 (i.e., after
dealkylation) would result in the loss of chirality at
phosphorus.
O
CH3
N
H
N(CH2)5CO2R
5
9
1. H2/Pd
2. CH3I
TMSBr
O
O
P
Ph
OH
15 R = CH3
R = H
LiOH
2
It is important to note that the reaction of N-norecgo-
nine with excess CBZ-Cl (Scheme 3) led to the bis-N,O-
CBZ-protected carboxylic acid 10. Although unstable, the
bis-N,O-CBZ-protected carboxylic acid 10 was success-
fully coupled with methyl 6-aminocaproate to form amide
11. Additionally, we found that amide 11 could be
selectively O-deprotected to give alcohol 8.
Deprotection of 9 via catalytic hydrogenolysis followed
by methylation afforded the N-methyl product 5. Dealky-
lation of the methyl phosphonate 5 was readily ac-
complished with trimethylsilyl bromide10 (TMSBr) to
form the phosphonic acid 15. The hydrolysis of the
methyl ester of 15 was achieved with LiOH to give the
final hapten 2 as the dilithium salt. Although combus-
tion analysis of 2 as the dilithium salt gave accurate
results for hydrogen and nitrogen, an acceptable result
for carbon was not feasible due to interference with
lithium. However, 1H and 31P NMR as well as mass
spectral analysis were consistent with the desired prod-
uct.
In conclusion, synthetic difficulties in phosphonylation
of the C3 position of ecgonine-related materials were
circumvented by protection of the N-8 bridgehead nitro-
gen. In addition, use of the UV-active CBZ protecting
group facilitated chromatographic isolation of the ecgo-
nine derivatives described herein. A parallel synthetic
strategy utilizing the favorable proximity and configura-
tions of the C2 carboxylic acid and the C3 hydroxyl of
N-norecgonine to form a reactive cyclic carboxylic-phos-
phonic mixed anhydride was explored. It was discovered
that both routes were viable and gave similar overall
yields. The synthesized hapten 2 has now been coupled
to an immunogenic protein, and results regarding the
An alternative route to 9 was developed taking advan-
tage of the favorable proximity and configuration of the
C2 carboxylic acid and C3 alcohol. As shown in Scheme
4, this reaction sequence involved a cyclic mixed anhy-
dride. The precedent for this synthetic approach is based
on the regioselective formation of amides from carbon-
phosphorus mixed anhydrides when reacted with amines.9
Thus, reaction of the hydroxy acid 7 with phenylphos-
phonic dichloride followed by ring-opening amidation
with methyl 6-aminocaproate provided the amide phos-
phonate 13 in good yield (Scheme 4, path A) and
subsequent methylation afforded the methyl phosphonate
9. As expected, the opposite regioselectivity was observed
when the cyclic intermediate was intercepted with metha-
nol to produce the corresponding methyl phosphonate 14
(Scheme 4, path B). Coupling of 14 with methy 6-ami-
nocaproate in the presence of DCC also provided 9.
To obtain the desired final product 2 from 9, a sequence
of simple transformations was executed (Scheme 5).
(9) J ackson, A. G.; Kenner, G. W.; Moore, G. A.; Ramage, R.; Thorpe,
W. D. Tetrahedron Lett. 1976, 40, 3627.
(10) Mckenna, C. E.; Schmidhauser, J . J . Chem. Soc., Chem.
Commun. 1979, 739.

5688 J . Org. Chem., Vol. 61, No. 16, 1996
Notes
preparative TLC (hexane:ethyl acetate, 1:2 v:v; Rf ) 0.12) to give
a colorless oil (0.0125 g, 82% yeild). Spectral data were identical
to that described above.
procurement of catalytic antibodies utilizing hapten 2 will
be forthcoming.
3â-((Ben zyloxyca r bon yl)oxy)-8-(ben zyloxyca r bon yl)-8-
a za b icyclo[3.2.1]oct a n e-2â-ca r b oxylic Acid N-(5-Ca r -
bom eth oxyp en tyl)a m id e (11). N-Norcocaine (6) (0.289 g, 1.0
mmol) was dissolved in 0.8 N HCl (10 mL) and refluxed
overnight to generate N-norecgonine. The solution was allowed
to cool to room temperature and extracted with diethyl ether
(20 mL) to remove benzoic acid. The aqueous layer was treated
with Na2CO3 (solid) to adjust the pH to 10. Benzyl chloroformate
(0.428 mL, 3.0 mmol) was added to the aqueous solution and
stirred at room temperature for 3 h. Ethyl acetate (15 mL) was
added to the reaction mixture followed by the addition of HCl
(concd) to adjust the pH to 2. The aqueous layer was extracted
twice with ethyl acetate. The organic layers were combined,
dried over Na2SO4, and concentrated in vacuo to give a yellow
oil. The crude mixture was purified by flash silica gel chroma-
tography (hexane:ethyl acetate, 2:1, v:v; Rf ) 0.15) to give 10 as
a pale yellow oil. The bis-O,N-CBZ-carboxylic acid 10 was
observed to decompose readily and was therefore used im-
mediately in the subsequent reaction. The carboxylic acid 10
(0.054 g, 0.123 mmol) was dissolved in CH2Cl2 (1.5 mL) at room
temperature, followed by the addition of TEA (0.021 mL, 0.147
mmol) and methyl 6-aminocaproate (0.027 g, 0.147 mmol). DCC
was dissolved in CH2Cl2 (0.5 mL), added to the reaction mixture,
and allowed to stir overnight. The reaction was filtered twice,
concentrated to an oil, and purified by flash chromatography
(hexane:ethyl acetate, 1:1 v:v; Rf ) 0.12) to give the product as
a clear oil (0.022 g, 31% yeild). 1H NMR (CDCl3): δ 1.24 q, 2H,
J ) 7.2; 1.34 q, 2H, J ) 7.3; 1.53 m, 2H; 1.68 d, 2H, J ) 7.3;
1.88-2.02 m, 3H; 2.23 t, 2H, J ) 7.5; 2.36 dt, 1H, J ) 2.9, 12.2;
2.85 d, 1H, J ) 6.8; 3.05-3.14 m, 2H; 3.63 s, 1H; 4.52 s, 1H;
4.65 d, 1H, J ) 6.6; 4.99-5.14 m, 5H; 5.83 s, 1H; 7.27-7.34 m,
10H. MS (FAB): m/ z 567 (M + H).
Exp er im en ta l Section
Gen er a l P r ocd u r es. All solvents used in reactions were
distilled prior to use. All other reagents were used as supplied
unless otherwise stated. Ecgonine and N-norcocaine were
obtained from NIDA. All chromatography was performed with
silica gel unless specified otherwise. 1H and 31P NMR spectra
were recorded on a Varian VXR 300. 1H NMR chemical shifts
are relative to TMS (δ ) 0.00 ppm) or CDCl3 (δ ) 7.24 ppm).
31P NMR chemical shifts are relative to H3PO4 (δ ) 0.00 ppm).
3â-Hyd r oxy-8-m et h yl-8-a za b icyclo[3.2.1]oct a n e-2â-ca r -
boxylic Acid N-(5-Ca r bom eth oxyp en tyl)a m id e (4). To a
stirred suspension of ecgonine‚HCl (1.0 g, 4.51 mmol) in CH2-
Cl2 (40 mL) at 0 °C was added pyridine (0.365 mL, 4.51 mmol)
followed by oxalyl chloride (1.57 mL, 18.04 mmol). The reaction
was allowed to warm to room temperature for 3 h, at which time
the solution became pale pink in color. The solvent was
evaporated and dried overnight in vacuo at room temperature
to give the crude acid chloride of ecgonine. A solution of methyl
6-aminocaproate (1.23 g, 6.76 mmol) and pyridine (1.46 mL,
18.04 mmol) in CH2Cl2 was added to the crude ecgonine acid
chloride at 0 °C and the reaction was allowed to stir for 1 h.
The mixture was treated with Na2CO3 (10% w/v) and extracted
twice with CH2Cl2. The organic layers were combined and dried
over MgSO4, and the solvent was evaporated in vacuo overnight
to remove excess pyridine. The crude mixture was purified by
flash silica chromatography (MeOH:CHCl3, 25:75; Rf ) 0.15) to
give the product as a pale yellow semisolid (0.788 g, 56% yield).
1H NMR (CDCl3): δ 1.31-1.39 m, 2H; 1.48-1.55 m, 3H; 1.58-
1.65 m, 2H; 1.76 dd, 1H, J ) 4.1, 19.2; 1.86 t, 1H, J ) 9.3; 2.13
t, 2H, J ) 7.4; 2.33 s, 3H; 2.56 d, 1H, J ) 16.8; 3.22 s, 1H; 3.28
q, 2H, J ) 6.9; 3.64 s, 3H; 3.68 d, 1H, J ) 5.3; 5.71 s, 1H br;
6.23 s, 1H. MS (FAB): m/ z 313 (M + H).
8-(Ben zyloxyca r bon yl)-3â-(m eth oxyp h en ylp h osp h in yl)-
8-a za b icyclo[3.2.1]oct a n e-2â-ca r b oxylic Acid N-(5-Ca r -
bom eth oxyp en tyl)a m id e (9). F r om 8. Tetrazole (0.0021 g
0.03 mmol) and 8 (0.141 g, 0.326 mmol) were dissolved in
benzene and stirred under Ar(g) at 4 °C. N,N-Diisopropylethy-
lamine (0.13 mL, 0.730 mmol) was added via syringe followed
by the addition of phenylphosphonic dichloride (0.048 mL, 0.338
mmol). The reaction was allowed to warm to room temperature
for 1 h, followed by the dropwise addition of methanol (0.040
mL, 0.905 mmol), and the mixture was allowed to stir an
additional 30 min. The solvent was removed in vacuo, and the
product was purified by flash chromatography (MeOH:ethyl
acetate 5:95 v:v, Rf ) 0.2) to give a colorless oil (0.110 g, 58%
yield). 1H NMR (CDCl3): δ 1.14-1.74 m, 8H; 1.80-2.05 m, 3H;
2.14-2.31 m, 2H; 2.55 ddt, 1H, J ) 48.0, 12.1, 2.6; 2.77 ddd,
1H, J ) 48.6, 6.5, 2.0; 2.91-3.10 m, 2H; 3.61-3.68 m, 6H; 4.37-
4.45 m, 1H; 4.60 d, 1H, J ) 6.1; 4.64-4.82 m, 1H; 5.06 d, 2H, J
) 5.4; 5.89 dt, 1H, J ) 73.0, 5.3; 7.25-7.33 m, 5H; 7.37-7.58
m, 3H; 7.74 dd, 2H, J ) 13.6, 6.9. 31P NMR: δ 20.67, 21.01.
MS (FAB): m/ z 587 (M + H) Anal. Calcd for C16H19NO5: C,
60.62; H, 6.84; N, 4.88. Found: C, 60.95; H, 6.77; N, 4.81. F r om
12, P a th A. A benzene (3 mL) solution of N-CBZ-norecgonine
(7) (100 mg, 0.33 mmol), diisopropylamine (0.23 mL, 1.32 mmol),
and tetrazole (2 mg, 0.03 mmol) under Ar(g) was cooled to 5 °C
and treated with phenylphosphonic dichloride (0.047 mL, 0.33
mmol). The ice bath was removed, and the reaction was stirred
for 1 h. Methyl 6-aminocaproate hydrochloride (60 mg, 0.33
mmol) was added and stirring was continued for 1 h. Saturated
ammonium chloride solution (10 mL) and dichloromethane (30
mL) were added. The organic layer was separated, dried over
sodium sulfate, and evaporated to give 13 as a pale yellow oil.
The crude phosphonic acid 13 was taken up in dichloromethane
and treated with sodium carbonate (100 mg) and dimethyl
sulfate (0.032 mL, 0.50 mmol). After the solution was stirred
overnight at room temperature, solvent was removed under
vacuum and the residue was purified by flash chromatography
(CH2Cl2:methanol 95:5 v:v) to give 9 (46 mg, a 24% yield) as a
pale yellow oil. The spectral data for this product were identical
to that descibed above. F r om 12, P a th B. In situ formation of
12 from 7 was identical to the procedure described above from
12, path A with the exception that all amounts were doubled.
The synthetic intermediate 12 was then treated with MeOH
(0.066 mL, 1.5 mmol) to form 14. The solvent was removed in
8-(Ben zyloxyca r b on yl)-3â-h yd r oxy-8-a za b icyclo[3.2.1]-
octa n e-2â-ca r boxylic Acid (7). N-Norcocaine (6) (0.5 g, 1.73
mmol) was dissolved in 0.8 N HCl (10 mL) and refluxed
overnight to generate N-norecgonine. The solution was allowed
to cool to room temperature and extracted with diethyl ether
(20 mL) to remove benzoic acid. The aqueous layer was treated
with Na2CO3 (solid) to adjust the pH to 10. Benzyl chloroformate
(0.271 mL, 1.90 mmol) was added to the aqueous solution and
stirred at room temperature for 3 h. Ethyl acetate (15 mL) was
added to the reaction mixture followed by the addition of HCl
(concd) to adjust the pH to 2. The aqueous layer was extracted
once with ethyl acetate and twice with CH2Cl2. The organic
layers were combined, dried over Na2SO4, and concentrated in
vacuo to a yellow oil. The crude mixture was purified by flash
silica gel chromatography (MeOH:CH2Cl2 10:90, v:v; Rf ) 0.25)
to give 7 as a pale yellow oil (0.235 g, 44% yield). 1H NMR
(CDCl3): δ 1.64-1.67 m, 2H; 1.88-1.97 m, 2H; 1.99-2.09 m,
2H; 2.96 s, 1H; 4.04-4.08 m, 1H; 4.44 s, 1H; 4.86 s, 1H; 5.12 s,
2H; 7.33 s, 5H.
8-(Ben zyloxyca r b on yl)-3â-h yd r oxy-8-a za b icyclo[3.2.1]-
oct a n e-2â-ca r b oxylic Acid N-(5-Ca r b om et h oxyp en t yl)-
a m id e (8). F r om 7. N-CBZ-norecgonine (7) (0.249 g, 0.86 mmol)
was dissolved in CH2Cl2 (8 mL) at room temperature followed
by the addition of DCC (0.185 g, 0.898 mmol). The mixture was
stirred for 10 min, after which a white precipitate formed.
6-Aminocaproic acid methyl ester hydrochloride (0.178 g, 0.980
mmol) along with TEA (0.137 mL, 0.980 mmol) was dissolved
in CH2Cl2 (2 mL) and added to the above reaction mixture. The
mixture was allowed to stir overnight at room temperature, after
which it was filtered twice. The filtrate was concentrated in
vacuo and purified by flash silica gel chromatography (ethyl
acetate; Rf ) 0.3) to give the desired product as a colorless oil
(0.281 g, 80% yield). 1H NMR (CDCl3): δ 1.23-1.33 m, 2H;
1.39-1.41 m, 2H; 1.57-1.74 m, 5H; 1.89-2.04 m, 3H; 2.29 t,
2H J ) 7.4; 2.74 s, 1H; 3.15-3.17 m, 2H; 3.66 s, 3H; 4.11-4.13
m, 1H; 4.49 s, 1H; 4.65 s, 1H; 5.11 s, 2H; 7.33 s, 5H. F r om 11.
Amide 11 (0.020 g, 0.035 mmol) was dissolved in MeOH (2 mL)
at room temperature, Na2CO3 (0.015 g, 0.142 mmol) was added,
and the reaction was allowed to stir until complete consumption
of starting material was observed by TLC (3 h). The solvent
was evaporated in vacuo, and the product was purified by

Notes
J . Org. Chem., Vol. 61, No. 16, 1996 5689
vacuo, acidified with 10% HCl (10 mL), extracted twice with CH2-
Cl2, dried over sodium sulfate, concentrated, and purified by
flash silica chromatography (MeOH:CH2Cl2, 5:95; Rf ) 0.2) to
give 14 (0.061 g, 0.133 mmol) in 20% yield. Compound 14 was
dissolved in CH2Cl2 (3 mL) followed by the addition of DCC
(0.030 g, 0.146 mmol) and allowed to stir at room temperature
for 15 min. A solution of 6-aminocaproic acid methyl ester
hydrochloride (0.029 g, 1.59 mmol) and TEA (0.022 mL, 1.59
mmol) in CH2Cl2 was then added, and the mixture was allowed
to stir overnight. The reaction mixture was filtered, concen-
trated in vacuo, and purified by silica chromatography (MeOH:
ethyl acetate 5:95 v:v, Rf ) 0.2) to give the methyl phosphonate
9 as a colorless oil. The spectral data for this product was
identical to that described above.
8-Meth yl-3â-(m eth oxyp h en ylp h osp h in yl)-8-a za bicyclo-
[3.2.1]octa n e-2â-ca r boxylic Acid N-(5-Ca r bom eth oxyp en -
tyl)a m id e (5). To a stirred solution of 9 (0.300 g, 0.512 mmol)
in MeOH (15 mL) was added 10% Pd/C (0.060 g). The flask was
then charged with H2(g) (balloon pressure) and stirred for 2 h.
The solution was filtered and the solvent removed in vacuo to
give the N-desmethyl intermediate as a pale yellow oil (0.222 g,
0.491 mmol). This material was dissolved in acetonitrile fol-
lowed by the addition of Na2CO3 (0.052 g, 0.491 mmol) and
methyl iodide (0.037 mL, 0.598 mmol). The reaction mixture
was allowed to stir at room temperature for 2 h and then filtered.
The filtrate was concentrated in vacuo, and the product was
purified by column chromatography (neutral aluminum oxide,
MeOH:CH2Cl2 5:95 v:v, Rf ) 0.5). 1H NMR (CDCl3): δ 1.30-
1.40 m, 2H; 1.45-1.69 m, 5H; 1.78 d, 2H, J ) 16.7; 1.88 d, J )
7.4; 1.98-2.17 m, 2H; 2.20 s, 3H; 2.23-2.32 m, 2H; 2.72 dd, 1H,
J ) 74.6,7.7; 3.13-3.31 m, 4H; 3.63 s, 3H; 3.69 dd 3H, J ) 34.2,
11.3; 4.70 dm, J ) 73.3; 7.39-7.54 m, 3H; 7.86 ddd, 2H, J )
49.5, 13.4, 8.0. 31P NMR: δ 20.12, 21.01. MS (FAB): m/ z 467
(M + H). Anal. Calcd for C23H35N2O6P: C, 59.22; H, 7.56; N,
6.00. Found: C, 59.15; H, 7.75; N, 6.20.
mmol) via syringe. The reaction was allowed to stir at room
temperature for 1 h, after which trimethylsilyl bromide (0.007
mL, 0.052 mmol) was again added via syringe and the reaction
was allowed to stir for an additional 1 h. Water was added
(0.050 mL), and the solvent was removed in vacuo. The product
was purified by column chromatography (C-18 solid phase
extraction column, MeOH:NH4OH (30%) 100:1 v:v) to give the
product as a white solid (0.010 g, 52%). 1H NMR (CD3OD): δ
1.24-1.64 m, 7H; 1.90 s, 1H; 1.97-2.05 m, 2H; 2.20-2.28 m,
2H; 2.29-2.35 m, 2H; 2.75 s, 3H; 3.06 s, 1H; 3.10-3.19 m, 2H;
3.62 s, 3H; 3.82 s, 1H; 4.03 s, 1H; 4.65-4.80 m, 1H; 7.39-7.46
m, 3H; 7.75 dd, 2H, J ) 13.0, 7.8. 31P NMR: δ 18.53. MS
(FAB): m/ z 453 (M + H).
3â-(Hyd r oxyp h en ylp h osp h in yl)-8-m eth yl-8-a za bicyclo-
[3.2.1]oct a n e-2â-ca r b oxylic Acid N-(5-Ca r b oxyp en t yl)-
a m id e (2) Dilith iu m Sa lt. To a methanolic solution (0.665 mL)
of 15 (0.040 g, 0.088 mmol) was added LiOH (1.0 N, 0.265 mL).
The reaction was allowed to stir for 15 h at room temperature.
The solvent was evaporated in vacuo, and the product was
purified by column chromatography (C-18 solid phase extraction
column, MeOH) to give the product as a white solid (0.023 g,
60% yield). 1H NMR (CD3OD): δ 1.24-1.34 m, 2H; 1.40-1.59
m, 4H; 1.81-1.90 m, 3H; 2.03-2.23 m, 3H; 2.09 t, 2H, J ) 7.4;
2.55 s, 3H; 2.87 d, 1H, J ) 5.0; 3.08-3.26 m, 2H; 3.56 s, 1H;
3.79 s, 1H; 4.57-4.62 m, 1H; 7.32-7.39 m, 3H; 7.19 dd, 2H, J )
12.6,7.7. 31P NMR (CD3OD): δ 18.86. MS (FAB): m/ z 451 (M
+ H + 2Li), 445 (M + H + Li), 439 (M + H). Anal. Calcd for
C
21H29Li2N2O6P: C, 56.01; H, 6.49; N, 6.22. Found: C, not
available; H, 6.40; N, 6.22.
Ack n ow led gm en t. This project was financially sup-
ported (Grant DA08531) by the National Institutes on
Drug Abuse. Donations of rare chemicals from the
National Institutes on Drug Abuse are also gratefully
acknowledged. Thanks are due to Dr. J . J . Kiddle for
helpful discussions.
3â-(Hyd r oxyp h en ylp h osp h in yl)-8-m eth yl-8-a za bicyclo-
[3.2.1]octa n e-2â-ca r boxylic Acid N-(5-Ca r bom eth oxyp en -
tyl)-a m id e (15). To a stirred solution of 5 (0.020 g, 0.043 mmol)
under Ar(g) was added trimethylsilyl bromide (0.007 mL, 0.052
J O960522Q