Faculty Bibliography

This paper describes an extended structure-activity relationships study of aminotetralin-piperazine-based hybrid molecules developed earlier for dopamine D2/D3 receptors. Various analogues as positional isomers have been developed where location of the phenolic hydroxyl group has been varied on the aromatic ring. Between two catechol derivatives, compound 6e with a two methylene linker length exhibited higher affinity and selectivity for D3 over D2 receptors over compound 6f with four methylene linkers (D2/D3 = 50.6 vs 7.51 for 6e and 6f, respectively). In general, the (-)-isomer was more potent than the (+)-isomeric counterpart. Binding results indicated highest selectivity for D3 receptors in compound (-)- 10 ( K i = 0.35 nM; D2/D3 = 71). In the 5-hydroxy series, highest selectivity for D3 receptors was exhibited by compound (-)- 25 ( K i = 0.82 nM; D2/D3 = 31.5). Most potent compounds exhibited binding and functional affinities at the sub-nanomolar level for the D3 receptor. Binding assays were carried out with HEK-293 cells expressing either D2 or D3 receptors by using tritiated spiperone as radioligand for competition studies to evaluate inhibition constants ( K i). A functional assay of selected compounds for stimulating GTPgammaS binding was carried out with CHO cells expressing human D2 receptors and AtT-20 cells expressing human D3 receptors. The functional assay results indicated partial to full agonist characteristics of test compounds. Compound (-)- 25 was selected further for in vivo study to evaluate its potency in producing contralateral rotations in rats with unilateral lesion in the nigrostriatal region induced by neurotoxin 6-OHDA, a Parkinsonian animal model. Compound (-)- 25 at 5 micromol/kg exhibited rotational activity that lasted beyond 12 h, whereas at a 1 micromol/kg dose the rotations lasted beyond 8 h

Deficiency in dopaminergic activity has been linked to a depressed state in pharmacological and clinical studies. Current pharmacological treatment for depression primarily involves modulation of serotonergic and noradrenergic systems but not dopaminergic neurotransmission. Available pharmacotherapy for depression has a number of drawbacks as a significant number of people are either refractory or develop tolerance to the antidepressant agents resulting in relapse. Furthermore, the slow onset of action of current therapies often poses a challenge for effective treatment. In our effort to develop novel molecules impacting all three above mentioned monoamine systems, we discovered structurally unique pyran derivatives with various profiles in inhibiting monoamine transporters. One of our lead molecules, D-161 exhibited triple monoamine transporter inhibitory activity with the highest affinity for norepinephrine transporter (NET) followed by its affinity for serotonin transporter (SERT) and dopamine transporter (DAT). D-161 exhibited potent activity in reducing immobility significantly in the rat forced swim test as well as in the mouse tail suspension test. Moreover, results from locomotor activity tests indicated that the reduction of immobility by D-161 was not due to motor activation as no significant motor activation was observed when the rats were subjected to the same doses of drug under the same conditions as in the forced swim test. These results suggest that the novel asymmetric pyran derivative D-161 with unique molecular structure exhibiting triple monoamine transporter inhibitory activity could possess potent antidepressant activity

In the current report, we extend the SAR study on our hybrid structure 7-{[2-(4-phenyl-piperazin-1-yl)ethyl]propylamino}-5,6,7,8-tetrahydronaphth alen-2-ol further to include heterocyclic bioisosteric analogues. Binding assays were carried out with HEK-293 cells expressing either D2 or D3 receptors with tritiated spiperone to evaluate inhibition constants (Ki). Functional activity of selected compounds in stimulating GTPgammaS binding was assessed with CHO cells expressing human D2 receptors and AtT-20 cells expressing human D3 receptors. The highest binding affinity and selectivity for D3 receptors were exhibited by (-)-34 (Ki=0.92 nM and D2/D3=253). In the functional GTPgammaS binding assay, (-)-34 exhibited full agonist activity with picomolar affinity for D3 receptor with high selectivity (EC50=0.08 nM and D2/D3=248). In the in vivo rotational study, (-)-34 exhibited potent rotational activity in 6-OH-DA unilaterally lesioned rats with long duration of action, which indicates its potential application in neuroprotective treatment of Parkinson's disease

A series of structurally constrained analogues based on hybrid compounds containing octahydrobenzo[g or f]quinoline moieties were designed, synthesized, and characterized for their binding to dopamine D2 and D3 receptors expressed in HEK-293 cells. Among the newly developed constrained molecules, trans-octahydrobenzo[f]quinolin-7-ol (8) exhibited the highest affinity for D2 and D3 receptors, the (-)-isomer being the eutomer. Interestingly, this hybrid constrained version 8 showed significant affinity over the corresponding nonhybrid version 1 (representing a constrained version of the aminotetralin structure only) when assayed under same conditions (K(i) of 49.1 and 14.9 nM for 8 vs 380 and 96.0 nM for 1 at D2 and D3, respectively). Similar results were found with other lead hybrid compounds, indicating a contribution of the piperazine moiety in the observed enhanced affinity. On the basis of the data of new lead constrained derivatives and other lead hybrid derivatives developed by us, a unique pharmacophore model was proposed consisting of three pharmacophoric centers, two with aromatic/hydrophobic and one with cationic features

Our earlier effort to develop constrained analogues of flexible piperidine derivatives for monoamine transporters led to the development of a series of 3,6-disubstituted piperidine derivatives, and a series of 4,8-disubstituted 1,4-diazabicyclo[3.3.1]nonane derivatives. In further structure-activity relationship (SAR) studies on these constrained derivatives, several novel analogues were developed where an exocyclic hydroxyl group was introduced on the N-alkyl-aryl side chain. All synthesized derivatives were tested for their affinities for the dopamine transporter (DAT), serotonin (5-HT) transporter (SERT), and norepinephrine transporter (NET) in the brain by measuring their potency in inhibiting the uptake of [(3)H]DA, [(3)H]5-HT, and [(3)H]NE, respectively. Compounds were also tested for their binding potency at the DAT by their ability to inhibit binding of [(3)H]WIN 35,428. The results indicated that position of the hydroxyl group on the N-alkyl side chain is important along with the length of the side chain. In general, hydroxyl derivatives derived from more constrained bicyclic diamines exhibited greater selectivity for interaction with DAT compared to the corresponding 3,6-disubstituted diamines. In the current series of molecules, compound 11b with N-propyl side chain with the hydroxyl group attached in the benzylic position was the most potent and selective for DAT (K(i)=8.63nM; SERT/DAT=172 and NET/DAT=48.4)

The widely abused psychostimulant cocaine is thought to elicit its reinforcing effects primarily via inhibition of the neuronal dopamine transporter (DAT). However, not all DAT inhibitors share cocaine's behavioral profile, despite similar or greater affinity for the DAT. This may be due to differential molecular interactions with the DAT. Our previous work using transporter mutants with altered conformational equilibrium (W84L and D313N) indicated that benztropine and GBR12909 interact with the DAT in a different manner than cocaine. Here, we expand upon these previous findings, studying a number of structurally different DAT inhibitors for their ability to inhibit [(3)H]CFT binding to wild-type, W84L and D313N transporters. We systematically tested structural intermediates between cocaine and benztropine, structural hybrids of benztropine and GBR12909 and a number of other structurally heterologous inhibitors. Derivatives of the stimulant desoxypipradrol (2-benzhydrylpiperidine) exhibited a cocaine-like binding profile with respect to mutation, whereas compounds possessing the diphenylmethoxy moiety of benztropine and GBR12909 were dissimilar to cocaine-like compounds. In tests with specific isomers of cocaine and tropane analogues, compounds with 3alpha stereochemistry tended to exhibit benztropine-like binding, whereas those with 3beta stereochemistry were more cocaine-like. Our results point to the importance of specific molecular features--most notably the presence of a diphenylmethoxy moiety--in determining a compound's binding profile. This study furthers the concept of using DAT mutants to differentiate cocaine-like inhibitors from atypical inhibitors in vitro. Further studies of the molecular features that define inhibitor-transporter interaction could lead to the development of DAT inhibitors with differential clinical utility

The use of heterologous expression systems for studying dopamine (DA) transporter (DAT) function has provided important information corroborating and complementing in situ obtained knowledge. Preliminary experiments with human embryonic kidney cells (HEK293) heterologously expressing varying amounts of DAT suggested fluctuations in the potency of cocaine in inhibiting DA uptake and led to the present systematic assessment of the impact of the density of DAT on its function. Transiently expressing intact HEK293 cells, transfected with increasing amounts of DAT cDNA, displayed increasing levels of surface DAT, binding of the cocaine analog [(3)H]2beta-carbomethoxy-3beta-(4-fluorophenyl)tropane ([(3)H]CFT), and uptake of [(3)H]DA, [(3)H]N-methyl-4-phenylpyridinium ([(3)H]MPP(+)), [(3)H]norepinephrine, and [(3)H]serotonin. However, the amount of DAT cDNA and the DAT expression level required to produce 50% of maximal activity was threefold higher for CFT binding than for DA uptake. Increased DAT expression was accompanied by weakened potency in inhibiting [(3)H]DA uptake for cocaine, CFT, benztropine, and its analog JHW025, GBR 12909 and mazindol; their potency in inhibiting [(3)H]CFT binding was unaffected. Inhibition of uptake by the substrates DA, m-tyramine, d-amphetamine, or MPP(+) was also unaffected. Increasing DAT in stably expressing HEK293 cells by stimulation of gene expression with sodium butyrate also decreased the uptake inhibitory potency of a number of the above blockers without affecting the interaction between substrates and DAT. The present results prompt discussion of models explaining how factors regulating DAT expression at the plasma membrane can regulate DAT function and pharmacology.