Anne Blow and Mikael Bols, Department of Chemistry, University of Aarhus, Langelandsgade 140, DK-8000 Aarhus, Denmark Steffen

Sinning and Ove Wiborg, Lab. of Molecular Neurobiology, Psychiatric Hospital in Aarhus, Skovagervej 2, DK-8240 Risskov, Denmark

Synthesis of Cocaine Analogues by Multicomponent Grignard Reactions

Introduction
Cocaine abuse is a growing problem in the US and in Western Europe, but at present no medical therapy is available for the treatment of cocaine addiction. It is suggested that it is possible to design cocaine analogues that could be useful in abuse treatment.1 This has resulted in synthesis of numerous cocaine analogues though yet without the desired biological effect.2 Combinatorial chemistry is an efficient way to generate many compounds in few reactions. Even though very useful in drug discovery combinatorial chemistry has not yet been used for synthesis of cocaine analogues. In the present study small libraries of cocaine analogues were synthesized by multicomponent Grignard reactions followed by 2 dimensional screening for biological activity.
N O OCH3 O O R-cocaine WIN 35065-2 Analogues N O OCH3 N O OCH3 R
C. Normal dopamine reuptake B. Stimulated

Presynaptic Neuron

Postsynaptic Neuron

A. Rest state

Cocaine is a stimulant of the central nervous system. When a nerve terminal in the resting state (A) is stimulated (B), dopamine ( ) is released and diffuses across the synaptic cleft D. Cocaine inhibition of reuptake where a dopamine receptor ( ) is stimulated. The stimulating action of dopamine ends with its reuptake by dopamine transporters ( ), into the presynaptic neuron (C). Cocaine ( ) binds to and blocks the transporter function, flooding the synapse with excess dopamine (D), which prolongs the signalling at key brain synapses. Though, a potential drug candidate is desired to inhibit cocaine binding without affecting the normal dopamine reuptake.

The multicomponent Grignard reactions were carried out on R-anhydroecgonine methyl ester (1), which was prepared from R-cocaine in three steps. First the ester functionalities were hydrolysed by aqueous HCl followed by dehydration with POCl3. The last step was esterification using MeOH and acid.3 Grignard reagents were freshly prepared from 5 alkyl- or aryl bromides before addition of 2 equivalents to the ,-unsaturated ester 1. By quenching the reaction at -78C using anhydrous TFA we mainly obtained the desired products 2 and only small amounts of the epimer with a 2-equatorial carbomethoxy group.
N O OCH3 O
R-cocaine 1M aq HCl reflux 15h

Results

Library 6 Library 5
N O OCH3

Library 7
N O OCH3

Library 8
N O OCH 3

Library 9
N O OCH3

Library 10
N O OCH3

Library 4

O OCH3

O OCH3

O OCH3

O OCH3

O OCH3

OH

POCl3 reflux 1h

OH

MeOH rt 2h

OCH3 1. RMgBr, Et2O, o

4h -40 C 2. TFA in Et2O, -78 C
o

OCH3 R

Library 3

O OCH 3

O OCH3
N

O OCH3

O OCH3

O OCH3

OH 1 2

Lib rar y8

Lib rar y9

Lib rar y8

Lib rar y1 0

Lib rar y9

Lib rar y1 0

In this way 25 different cocaine analogues were synthesized in libraries (1-10) containing 5 compounds each according to the matrix to the right (Each library contain the compounds of a row or column). 24 of the compounds are new but the last compound (WIN35065-2) is known to bind to the dopamine transporter and included in the library to have a positive control in the screening procedure. The libraries were synthesized in two dimensions both horizontally (Library 1-5) and vertically (Library 6-10) in order to facilitate identification of a possible lead compound, since such a compound must be active in both dimensions. The 10 libraries were screened for binding affinity to the human Dopamine reuptake Dopamine transporter binding dopamine, serotonin, and norepinephrine transporters and for monoamine reuptake. Here only results concerning the dopamine 700 700 transporter will be presented. In the diagrams to the left a high column 600 600 indicates high affinity (low Ki) and a low column low affinity (high Ki). As 500 500 seen in the diagrams, the compounds that bind to the dopamine 400 400 300 300 transporter do also inhibit dopamine reuptake. The positive control 200 200 came up and two new high affinity compounds were identified. These 100 100 and three other compounds were resynthesized individually and 0 0 evaluated biologically (see below).
Library 2
N O OCH3
N O

OCH 3

OCH3

OCH3

OCH3

Library 1

OCH3

OCH3

OCH3

OCH3

OCH3

Positive control

High affinity

High affinity

100000/Ki(nM)

100000/Ki(nM)

Library 5 Library 4 Library 3

Library 5 Library 4 Library 3

Library 2

Library 2

Lib rar y6

Lib rar y7

Lib rar y6

Lib rar y7

Library 1

Library 1

The results show that an aromatic group in the 3 position is necessary to obtain high binding affinity. If an alkyl group is present in that position the affinity decreases dramatically. But the aromatic group can be too bulky, since the presence of a t-butyl phenyl substituent totally destroys binding and uptake affinity.

O OCH3

O OCH3

O OCH3

O OCH3

O OCH3

Ki (uptake)

Ki (binding)

13500 nM 39000 nM

Ki (binding)

Ki (uptake)

20 nM 25 nM

Ki (uptake)

Ki (binding)

85 nM 140 nM

Ki (uptake)

Ki (binding)

2300 nM 6700 nM

Ki (binding)

Ki (uptake)

5300 nM 18000 nM

25 cocaine analogues were synthesized using solution-phase combinatorial chemistry. This was done by a 1,4-conjugate addition of a multicomponent Grignard reagent to an ,-unsaturated ester. By screening for biological activity in 2 dimensions two new high affinity compounds were identified and resynthesized.

Conclusion

1. Carroll, F.I.; Lewin, A.H.; Boja, J.W.; Kuhar, M.J. J. Med. Chem. 1992, 35, 969-981 2. Singh, S. Chem. Rev. 2000, 100, 925-1024 3. Zheng, Q-H.; Mulholland, G.K. Nucl. Med. Biol. 1996, 23, 981-986

References