Synthesis and dopamine transporter binding of 2β-isopropyl ester analogs of cocaine

Article abstract of DOI:10.1016/S0223-5234(01)01314-9

A series of 2β-isopropyl ester analogs of cocaine (7-11) was synthesised and evaluated in an in vitro dopamine transporter (DAT) binding assays. Ecgonine HCl (5) was obtained from (-)-cocaine (1) by hydrolysis using 1 N HCl. Acid catalysed esterification of 5 using 2-propanol and HCl gas afforded 2β-isopropyl ecgonine (6). Compounds 7-9 were obtained via esterification of the 3β-hydroxyl group of 6 using the appropriate acid chloride. Compound 10 was obtained via selective hydrolysis and re-esterification of 7 using 2-propanol and HCl gas. Compound 11 was obtained by reduction of 9 using H₂/Pd-C. Compounds 7, 10 and 11 showed high binding affinity to the DAT (as indicated from the inhibition of the binding of [³H]WIN 35,428 (3)) with IC₅₀ values (mean±S.E.M.) 208.5±9.5, 47.43±1.79 and 11.25±3.37 nM, respectively). Compound 7 is comparatively as active as cocaine, 10 is ca. fivefold more active than cocaine and 11 is ca. 20-fold more active than cocaine and even twice more active than the radioligand 3. Compound 11, like its methyl ester analog (2′ aminococaine), exhibited the highest affinity to the DAT. These results, along with previous results, emphasise the importance of a hydrogen-bond donor group at the 2′-position of cocaine and its isopropyl ester analogs to enhance binding affinity to the DAT.

Full text of DOI:10.1016/S0223-5234(01)01314-9

Eur. J. Med. Chem. 37 (2002) 171–176
Short communication
Synthesis and dopamine transporter binding of 2b-isopropyl ester
analogs of cocaine
Tarek F. El-Moselhy a,b,*, Kwasi S. Avor a, Garo P. Basmadjian a
a Department of Medicinal Chemistry and Pharmaceutics, College of Pharmacy, Uni6ersity of Oklahoma Health Sciences Centre, Oklahoma City,
OK 73190, USA
b Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Tanta Uni6ersity, Tanta 31527, Egypt
Received 5 July 2001; received in revised form 7 November 2001; accepted 7 November 2001
Abstract
A series of 2b-isopropyl ester analogs of cocaine (7–11) was synthesised and evaluated in an in vitro dopamine transporter
(DAT) binding assays. Ecgonine HCl (5) was obtained from (−)-cocaine (1) by hydrolysis using 1 N HCl. Acid catalysed
esterification of 5 using 2-propanol and HCl gas afforded 2b-isopropyl ecgonine (6). Compounds 7–9 were obtained via
esterification of the 3b-hydroxyl group of 6 using the appropriate acid chloride. Compound 10 was obtained via selective
hydrolysis and re-esterification of 7 using 2-propanol and HCl gas. Compound 11 was obtained by reduction of 9 using H2/PdꢀC.
Compounds 7, 10 and 11 showed high binding affinity to the DAT (as indicated from the inhibition of the binding of [3H]WIN
35,428 (3)) with IC50 values (mean9S.E.M.) 208.599.5, 47.4391.79 and 11.2593.37 nM, respectively). Compound 7 is
comparatively as active as cocaine, 10 is ca. fivefold more active than cocaine and 11 is ca. 20-fold more active than cocaine and
even twice more active than the radioligand 3. Compound 11, like its methyl ester analog (2% aminococaine), exhibited the highest
affinity to the DAT. These results, along with previous results, emphasise the importance of a hydrogen-bond donor group at the
´
2%-position of cocaine and its isopropyl ester analogs to enhance binding affinity to the DAT. © 2002 Published by Editions
scientifiques et me´dicales Elsevier SAS.
Keywords: Cocaine analogs; Isopropyl cocaine analogs; Dopamine transporter binding assays
1. Introduction
The current view, termed, ‘the dopamine hypothesis’
is that behaviours associated with cocaine addiction
result, to a large extent, not from a direct message
elicited by the binding of (−)-cocaine but rather from
the accumulation of DA in the synapse and its action at
one or more of the D1–D5 dopamine receptors [11–14].
In experiments involving knockout mice genetically
lacking the DA transporter, cocaine had no stimulant
effect [15]. This finding directly confirms the crucial role
of dopamine transporter (DAT) in cocaine action [15].
It has been reported that modification of substituents
at the 2b-position has led to improved binding affinities
and transporter selectivities in the phenyltropanes [16–
19]. The compounds of this investigation have been
prepared on the basis of the observation that replace-
ment of the methyl ester in the 2b-position by an
Cocaine (1), a naturally occurring local anesthetic
first isolated from Coca leaves (Erythroxylum coca) has
been recognised to be a potent central nervous stimu-
lant and binds to specific sites in the mammalian brain
[1–3]. The behavioural effects of cocaine are characte-
rised by reinforcement of responding and drug seeking
[4], drug discrimination ability [5] and locomotor acti-
vity stimulation [6,7]. While cocaine inhibits the neu-
ronal uptake of dopamine (DA) [8], serotonin (5-HT)
[9] and norepinephrine (NE) [10], the rewarding proper-
ties of cocaine clearly require activation of the do-
paminergic system.
* Correspondence and reprints.
E-mail address: mona63@hotmail.com (T.F. El-Moselhy).
´
0223-5234/02/$ - see front matter © 2002 Published by Editions scientifiques et me´dicales Elsevier SAS.
PII: S0223-5234(01)01314-9

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isopropyl moiety resulted in enhancement of binding
selectivity for DAT compared to NE and 5-HT bin-
dings [18,20]. Compounds 7, 10 and 11 were prepared
in view of high binding affinities exhibited by 2%-ace-
toxy-, 2%-hydroxy- and 2%-amino-cocaines (IC50
(mean9S.E.M.) were 6991, 2594 and 1892 nM,
respectively) [20], which were in the range of the 3-
phenyltropane WIN-35,428 (3) (IC50; mean9S.E.M.,
2494 nM), which is unusual for the benzoyl ester class
of tropanes [20–23].
Systematic evaluation of substitution at 4%-position
has resulted in the identification of high affinity ligand
selection for 5-HT transporter [24]. In contrast, few
publications have described the effects of substitution
at the 2%-position [21–23,25–27]. For these reasons, we
began a limited characterisation of the chemical nature
at 2%-substituents of 2b-isopropyl ester analogs of co-
caine on binding affinities to the DAT. In contrast to
most substituted cocaines, certain substituents at the
2%-position increase significantly the binding potency of
2b-isopropyl ester analogs of cocaine for DAT.
The structures of cocaine, WIN-35,428 and the pre-
pared compounds are shown in Fig. 1.
chloride in presence of benzene and Et3N, Fig. 2.
Compound 9 was prepared using the same method after
preparing the acid chloride from the corresponding acid
using SOCl2, Fig. 3. Compound 11 was prepared, ac-
cording to Fig. 3, by reduction of 9 using H2/PdꢀC,
while 10 was obtained from 7 by selective hydrolysis
using 2-propanol saturated with HCl gas (Fig. 4).
Preparation of salicyloyl chloride using SOCl2 or
oxalyl chloride activated DMF [28] was not feasible due
to the intramolecular hydrogen bonding found in sali-
cylic acid and the possibility of the formation of b-lac-
tone and/or acid anhydride. So, 10 was prepared by
adapting a procedure that was reported by our group
to prepare 2%-acetoxy- and 2%-hydroxy-cocaines
[21,22,29]. This procedure depends on selective hydroly-
sis and re-esterification of the 2%-acetoxy group of 7.
The 2%-acetoxy group and 2b-isopropyl ester of 7 were
hydrolysed, while re-esterification of the 2-carboxylic
group took place in a condition of acid catalysed
esterification (2-propanol, HCl gas and reflux
overnight). The 3b-benzoyl ester was not affected.
Compound 11 was prepared by the reduction of 9 using
H2/PdꢀC, because of the same reasons that have been
mentioned in the preparation of 10.
2. Results and discussion
2.2. Dopamine transporter binding assays
2.1. Chemistry
The IC50 values of 2b-isopropyl ester analogs of
cocaine 7–11, for inhibiting 4 nM of the radioligand
[3H]WIN 35,428 (3) to the DAT, are listed in Table 1.
The prepared compounds comprised 2%-substituents of
different nature such as: (1) a bulky group, 8; (2)
electron-donating groups, 7 and 8; (3) an electron-with-
drawing group, 9 and; (4) hydrogen-bond donor
groups, 10 and 11.
The intermediate compound 2b-isopropyl ecgonine
(6) was synthesised, according to Fig. 2, via hydrolysis
of cocaine with aqueous 1 N HCl, followed by acid
catalysed esterification using 2-propanol and HCl gas.
Compounds 7 and 8 were prepared by esterification of
3b-hydroxyl group of 6 using the appropriate acid
Fig. 1. Structures of the investigated compounds.

T.F. El-Moselhy et al. / European Journal of Medicinal Chemistry 37 (2002) 171–176
173
Fig. 2. Preparation of 5–8.
hydrogen-bond with the serine residue at the acceptor
site of DAT [21].
It is apparent from Table 1 that compounds 8 and 9
have low binding affinities, while 7, 10 and 11 showed
high binding affinities with IC50 values less than that of
cocaine. The IC50 values (mean9S.E.M.) for 7, 10 and
11 were 208.599.5, 47.4391.79 and 11.2593.37 nM,
respectively. So, compound 7 has IC50 close to that of
cocaine (249937 nM), 10 is ca. fivefold more active
than cocaine and 11 is ca. 20-fold more active than
cocaine and even twice more active than the radioli-
gand [3H]WIN 35,428 (3). These results are in accor-
dance with our previously published ones that showed
high binding affinities exhibited by 2%-acetoxy-, 2%-hy-
droxy- and 2%-amino-cocaines (IC50; mean9S.E.M.;
7091, 2594 and 1892 nM, respectively) [21–23].
These results emphasise that 2b-isopropyl ester and
2%-substituents are optimal and a hydrogen-bond donor
group is crucial in enhancing binding affinity to the
DAT.
3. Experimental
Melting points were determined on a Thomas
Hoover capillary melting point apparatus and are un-
corrected. Elemental analyses were performed by Mid-
west Microlab LTD, Indianapolis, IN, USA and were
90.4% of the calculated values. NMR spectra were
recorded on a Varian XL-300 spectrometer. All organic
reagents were obtained from Aldrich Co. and were used
without further purification. Silica gel (200–400 mesh,
,
60 A) used for column chromatography was obtained
from Aldrich and silica gel chromatographic sheets with
a fluorescent indicator used for thin layer chromatogra-
phy (TLC) were obtained from Eastman Kodak Co.,
Rochester, NY. [3H]WIN-35,428 was obtained
from Dupont-New England Nuclear, Boston, MA.
It was postulated that the hydroxyl group, and simi-
larly the amino group, may engage in an intermolecular

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T.F. El-Moselhy et al. / European Journal of Medicinal Chemistry 37 (2002) 171–176
Table 1
DAT binding affinities of compounds 7–11
Compound
IC50 (nm) a
Cocaine (1)
WIN 35,428 (2)
249937
2494
7
8
9
10
11
208.599.5
35,9859206
1881.59506
47.4291.79
11.2593.37
a IC50 values are mean9S.E.M. of two experiments performed in
triplicates.
200 mL of 2-propanol, the mixture was saturated with
dry HCl gas and refluxed overnight to obtain a solu-
tion. After cooling, the mixture was saturated again
with HCl gas while maintained cool in ice. The reaction
mixture was stirred at room temperature (r.t.) for 24 h.
2-Propanol was evaporated under vacuum. The remain-
ing solution was neutralised with 20% NH4OH and
extracted with CHCl3 (3×40 mL). The combined or-
ganic extracts were dried over MgSO4 (anhyd), concen-
trated under vacuum, passed through a silica gel plug
and eluted with EtOAc–petroleum ether (50:50) to
1
obtain an oil (4.19 g, 82% yield). H-NMR (CDCl3, l):
5.19–5.11 (m, 1H, CH(CH3)2); 3.82 (s, br, 2H, C-3,
OH); 3.59–3.58 (m, 1H, C-1); 3.17–3.16 (m, 1H, C-5);
2.72 (m, 1H, C-2); 2.2 (s, 3H, NꢀCH3); 2.11–1.81 (m,
4H, C-4, C-7); 1.56–1.52 (m, 2H, C-6); 1.30–1.28 (d,
6H, CH(CH3)2).
Fig. 3. Preparation of 9 and 11.
Homogenisation of the striata was performed using a
Polytron Homogenizer, Kinematic Kriens-Luzern,
Switzerland. Centrifugation of the membrane ho-
mogenate was carried out using a Dupont Sorvel RC-5
superspeed refrigerated centrifuge (rotor SS-34). Filtra-
tion of the bound-membrane was carried out using a
Brandel Cell Harvester. Counting of the activity of the
receptor-bound ligand was performed using a Beckman
LS 1701, liquid scintillation counter.
3.2. 3i-[(2%-Acetoxybenzoyl)oxy]-1R-(exo,exo)-8-
methyl-8-azabicyclo[3.2.1]octane-2-carboxylic acid
isopropyl ester (7)
Ecgonine isopropyl ester free-base (6) (0.30 g, 1.32
mmol) was dissolved in 15 mL dry C6H6, to this was
added 1 mL (10 mmol) Et3N. To the stirred solution
was added acetylsalicyloyl chloride (0.39 g, 1.98 mmol)
under dry N2. The reaction mixture was stirred at
40 °C overnight. The reaction was stopped and the
benzene layer was washed with 10 mL water and 5%
aq. Na2CO3 solution (3×5 mL) and dried over MgSO4
(anhyd), and the solvent was removed under vacuum to
give an oil. The oil was purified over silica gel column
3.1. 3i-Hydroxy-1R-(exo,exo)-8-methyl-8-azabicyclo-
[3.2.1]octane-2-carboxylic acid isopropyl ester (6)
To ecgonine HCl (5) (5.0 g, 22.5 mmol) was added
Fig. 4. Preparation of 10.

T.F. El-Moselhy et al. / European Journal of Medicinal Chemistry 37 (2002) 171–176
175
(EtOAc–petroleum ether, 50:50) to afford 0.35 g (69%
yield) of the clear oil. The oil was converted to the
tartrate salt, which was recrystallised from EtOH–
Et2O. M.p. 87–90 °C. Anal. C25H33NO12: 1H-NMR
(D2O, l): 7.87–7.83 (d, 1H, C(6%)H); 7.58–7.52 (m, 1H,
C(4%)H); 7.29–7.24 (m, 1H, C(5%)H); 7.08–7.05 (d, 1H,
C(3%)H); 5.40–5.30 (m, 1H, C(3)H); 4.80–4.70 (m, 1H,
CH(CH3)2); 4.03–4.02 (m, 1H, C(1)H); 3.89 (m, 1H,
C(5)H); 3.41–3.38 (m, 1H, C(2)H); 2.68 (s, 3H, NCH3);
2.27–2.23 (m, 4H, C(4,7)H); 2.19 (s, 3H, CH3COO);
2.02–1.98 (m, 2H, C(6)H); 0.95–0.93 (d, 3H, CHCH3);
0.62–0.60 (d, 3H, CHCH3).
and eluted with EtOAc to afford 0.45 g (90.66% yield)
of the clear oil. The oil was converted to the tartrate
salt and recrystallised from isopropyl alcohol. M.p.
1
159–161 °C. Anal. C23H30N2O12·H2O: H-NMR (D2O,
l): 7.88–7.85 (d, 1H, C(3%)H); 7.70–7.62 (m, 3H,
C(4%,5%,6%)H); 5.48–5.44 (m, 1H, C(3)H); 4.80–4.76 (m,
1H, CH(CH3)2); 4.05–4.03 (m, 1H, C(1)H); 3.90 (m,
1H, C(5)H); 3.32–3.29 (m, 1H, C(2)H); 2.68 (s, 3H,
NCH3); 2.30–2.28 (m, 4H, C(4,7)H; 2.05–2.00 (m, 2H,
C(6)H); 0.98–0.96 (d, 3H, CHCH3); 0.59–0.57 (d, 3H,
CHCH3).
3.3. 3i-[(2%-Methoxybenzoyl)oxy]-1R-(exo,exo)-8-
methyl-8-azabicyclo[3.2.1]octane-2-carboxylic acid
isopropyl ester (8)
3.5. 3i-[(2%-Hydroxybenzoyl)oxy]-1R-(exo,exo)-8-
methyl-8-azabicyclo[3.2.1]octane-2-carboxylic acid
isopropyl ester (10)
Ecgonine isopropyl ester free-base (6) (0.30 g, 1.32
mmol) was dissolved in 15 mL dry C6H6, to this was
added 1 mL (10 mmol) Et3N. To the stirred solution
was added anisoyl chloride (0.34 g, 1.98 mmol) under
dry N2. The reaction mixture was stirred at 40 °C
overnight. The reaction was stopped and the C6H6 layer
was washed with 10 mL water and 5% aq. Na2CO3
solution (3×5 mL) and dried over MgSO4 (anhyd) and
the solvent was removed under vacuum to give an oil.
The oil was purified over silica gel column (EtOAc–
petroleum ether, 50:50) to afford 0.34 g (72% yield) of
the clear oil. The oil was converted to the tartrate salt
and recrystallised from isopropyl alcohol. M.p. 184–
2%-Acetoxyisopropyl cocaine free-base (7) (0.1 g; 0.26
mmol) was dissolved in 10 mL isopropyl alcohol and
dry HCl gas was passed. The resulting solution was
refluxed with stirring for 48 h. It was then cooled and
the solvent was evaporated under vacuum to obtain a
white solid, which was recrystallised from MeOH–Et2O
to afford 0.84 g (85% yield) of the desired compound.
1
M.p. 165–167 °C. Anal. C19H25NO5: H-NMR (D2O,
l): 7.66–7.63 (d, 1H, C(6%)H); 7.42–7.35 (t, 1H,
C(4%)H); 6.86–6.76 (m, 2H, C(3%,5%)H); 5.44–5.36 (m,
1H, C(3)H); 4.81–4.76 (m, 1H, CH(CH3)2); 4.05–4.03
(m, 1H, C(1)H); 3.92 (m, 1H, C(5)H); 3.49–3.47 (m,
1H, C(2)H); 2.71 (s, 3H, NCH3); 2.31–2.25 (m, 4H,
C(4, 7)H; 2.05–2.02 (m, 2H, C(6)H); 0.93–0.91 (d, 3H,
CHCH3); 0.64–0.62 (d, 3H, CHCH3).
1
186 °C. Anal. C24H33NO11: H-NMR (D2O, l): 7.67–
7.64 (d, 1H, C(6%)H); 7.46–7.43 (m, 1H, C(4%)H);
7.02–6.99 (d, 1H, C(3%)H); 6.90–6.85 (m, 1H, C(5%)H);
5.38–5.18 (m, 1H, C(3)H); 4.74–4.61 (m, 1H,
CH(CH3)2); 4.02–4.00 (m, 1H, C(1)H); 3.88 (m, 1H,
C(5)H); 3.72 (s, 3H, ArOCH3); 3.44–3.41 (m, 1H,
C(2)H); 2.68 (s, 3H, NCH3); 2.24–2.20 (m, 4H, C(4,
7)H; 2.03–2.00 (m, 2H, C(6)H); 0.92–0.90 (d, 3H,
CHCH3); 0.56–0.54 (d, 3H, CHCH3).
3.6. 3i-[(2%-Aminobenzoyl)oxy]-1R-(exo,exo)-8-
methyl-8-azabicyclo[3.2.1]octane-2-carboxylic acid
isopropyl ester (11)
To a 100 mL vacuumed sealed round-bottomed flask
were added, 0.3 g (0.79 mmol) of 9, 30 mg of Pd on
activated carbon (Pd content 10%) and 40 mL cyclo-
hexane. A hydrogen balloon was attached to the flask
while it was under vacuum to allow hydrogen to be
sucked into the flask. The mixture was allowed to stir
for 24 h. The crude product was filtered over celite and
the solvent evaporated to obtain 0.26 g (95% yield) of a
pure oil. The oil was converted to the tartrate salt and
recrystallised from MeOH–Et2O. M.p. 172–174 °C.
3.4. 3i-[(2%-Nitrobenzoyl)oxy]-1R-(exo,exo)-8-
methyl-8-azabicyclo[3.2.1]octane-2-carboxylic acid
isopropyl ester (9)
2-Nitrobenzoic acid (0.33 g, 1.98 mmol) and 10 mL
thionyl chloride were refluxed for 3 h. The solution was
cooled and evaporated under vacuum to remove excess
thionyl chloride. The residue obtained was taken in 5
mL dry C6H6 and added to ecgonine isopropyl ester
free-base (6) (0.30 g, 1.32 mmol) and 1 mL (10 mmol)
Et3N in 15 mL of dry C6H6. The mixture was stirred at
r.t. for 6 h. The reaction was stopped and the organic
layer was washed with 10 mL water and 5% aq.
Na2CO3 solution (3×5 mL) and dried over MgSO4
(anhyd) and the solvent was removed under vacuum to
give an oil. The oil was purified over silica gel column
1
Anal. C23H32N2O10: H-NMR (D2O, l): 7.62–7.59 (d,
1H, C(6%)H); 7.24–7.18 (t, 1H, C(4%)H); 6.71–6.68 (d,
1H, C(3%)H); 6.59–6.54 (t, 1H, C(5%)H); 5.32–5.26 (m,
1H, C(3)H); 4.78–4.74 (m, 1H, CH(CH3)2); 4.01 (m,
1H, C(1)H); 3.90 (m, 1H, C(5)H); 3.42–3.39 (m, 1H,
C(2)H); 2.69 (s, 3H, NCH3); 2.32–2.22 (m, 4H,
C(4,7)H; 2.03–2.00 (m, 2H, C(6)H); 0.94–0.92 (d, 3H,
CHCH3); 0.66–0.64 (d, 3H, CHCH3).

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3.7. Dopamine transporter binding assays
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The binding was performed in rat striatal tissue using
the method of Reith [30].
1. Materials: [3H]WIN-35,428 was obtained from Du-
pont-New England Nuclear, Boston, MA.
2. Preparation of test substances: stock solutions of the
test substances were prepared freshly by dissolving
them in the incubation buffer (phosphate buffer pH
7.4).
3. Phosphate buffer: obtained by mixing 35 mM
NaH2PO4 and 17.5 mM Na2HPO4 to obtain pH 7.4.
4. Membrane preparation: Whole brains from male
Sprague–Dawley rats weighing from 350 to 400 g
(Sasco Inc., Wilmington, MA) were rapidly harvested
after decapitation with a guillotine. The striata were
isolated and homogenised using Polytron Homoge-
nizer (setting six for 15 s) in ice-cold 0.32 M sucrose
solution (1.5 mL/100 mg of tissue). The Homogenizer
and blade were then rinsed with twice the volume of
0.32 M sucrose solution and added to the ho-
mogenate. The combined mixture was centrifuged at
3300 rpm for 10 min at 4 °C. The resulting superna-
tant was subsequently centrifuged at 13,800 rpm for
20 min at 4 °C to obtain a pellet (P2), which was
homogenised in ice-cold 35 mM phosphate buffer.
Each assay tube contained 130 mL buffer or buffer
plus 10 mL of unlabeled test compound (1×10−10
100×10−6 M), [3H]WIN-35,428 in the same buffer
(20 mL, 4 nM) and 50 mL of membranes (4 mg mL−1
–
)
to a total volume of 200 mL. (−)-Cocaine (100 mM)
was used for non-specific binding. Assays performed
in triplicates were incubated for 2 h in an ice bath and
terminated by rapid filtration through Whatman
GF/B glass fiber filters presoaked for 30 min in 0.05%
polylysine solution. Membranes were rapidly washed
three times with ice-cold buffer. Filters containing
membrane-bound radioligand were added to vials
containing 10 mL of scintillation fluid (Ecolume,
Costa Mesa, CA), stored overnight and counted for
5 min on a Beckman LS 1701, liquid scintillation
counter.
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5. Calculation of IC50: IC50 values were determined from
competition curves of 12 points using the curve-fitting
program EBDA (Biosoft software, Ferguson, MO).
Mean values and standard errors were calculated
from two to three assays for each test compound.
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Acknowledgements
This work is supported by a grant from the National
Institute on Drug Abuse (DA 08587).
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