An integrated in silico 3D model-driven discovery of a novel, potent, and selective amidosulfonamide 5-HT1A agonist (PRX-00023) for the treatment of anxiety and depression

3116

J. Med. Chem. 2006, 49, 3116-3135

An Integrated in Silico 3D Model-Driven Discovery of a Novel, Potent, and Selective

Amidosulfonamide 5-HT_1AAgonist (PRX-00023) for the Treatment of Anxiety and Depression

Oren M. Becker,^†,* Dale S. Dhanoa,^‡,*^,§Yael Marantz,^†Dongli Chen,^‡Sharon Shacham,^†Srinivasa Cheruku,^‡

Alexander Heifetz,^†Pradyumna Mohanty,^‡Merav Fichman,^†Anurag Sharadendu,^‡Raphael Nudelman,^†Michael Kauffman,^‡and

Silvia Noiman^†

Predix Pharmaceuticals Ltd., 3 Hayetzira Street, Ramat Gan 52521, Israel, and Predix Pharmaceuticals Inc., 4 Maguire Road,

Lexington, Massachusetts 02421

ReceiVed August 31, 2005

We report the discovery of a novel, potent, and selective amidosulfonamide nonazapirone 5-HT_1Aagonist

for the treatment of anxiety and depression, which is now in Phase III clinical trials for generalized anxiety

disorder (GAD). The discovery of 20m (PRX-00023), N-{3-[4-(4-cyclohexylmethanesulfonylaminobutyl)-

piperazin-1-yl]phenyl}acetamide, and its backup compounds, followed a new paradigm, driving the entire

discovery process with in silico methods and seamlessly integrating computational chemistry with medicinal

chemistry, which led to a very rapid discovery timeline. The program reached clinical trials within less than

2 years from initiation, spending less than 6 months in lead optimization with only 31 compounds synthesized.

In this paper we detail the entire discovery process, which started with modeling the 3D structure of 5-HT_1A

using the PREDICT methodology, and then performing in silico screening on that structure leading to the

discovery of a 1 nM lead compound (8). The lead compound was optimized following a strategy devised

based on in silico 3D models and realized through an in silico-driven optimization process, rapidly overcoming

selectivity issues (affinity to 5-HT_1Avs R1-adrenergic receptor) and potential cardiovascular issues (hERG

binding), leading to a clinical compound. Finally we report key in vivo preclinical and Phase I clinical data

for 20m tolerability, pharmacokinetics, and pharmacodynamics and show that these favorable results are a

direct outcome of the properties that were ascribed to the compound during the rational structure-based

discovery process. We believe that this is one of the first examples for a Phase III drug candidate that was

discovered and optimized, from start to finish, using in silico model-based methods as the primary tool.

Introduction

depression are the most prevalent mental illnesses. In the US

alone an estimated four million adults suffer from GAD and

nearly 19 million adults are affected by depressive disorders.

Since GAD and depression are often present together, most

patients suffering from these disorders are treated with the same

drugs.¹⁴The most commonly used therapies are selective

serotonin reuptake inhibitors (SSRIs) and the more recently

developed serotonin noradrenaline reuptake inhibitors (SNRIs).

These drugs are thought to exert their effects by increasing

neurotransmitter availability and transmission. Another class of

drugs used for the short-term relief of GAD is benzodiazepines.

These sedating agents are controlled substances because of their

addictive properties and can be lethal when used in combination

with alcohol.

Serotonin (5-hydroxytryptamine or 5-HT) is involved in a

variety of physiological functions such as vasoconstriction,¹

thermoregulation, sexual behavior, pain, appetite, and sleep.^2-4

In addition, 5-HT-based mechanisms are implicated in neuro-

psychiatric disturbances such as depression, anxiety, and

psychosis.^5,6The numerous and varied actions of 5-HT are

mediated through 14 receptor subtypes.^7,8One of the most

actively studied 5-HT receptor subtypes, 5-HT_1A, is a 46 kDa,

421 amino acid protein coded on human chromosome 5 by an

intronless gene. 5-HT_1Areceptors are expressed in the CNS with

highest density in the dorsal and median raphe nuclei as well

as in the hippocampus. High density is also seen in the frontal

cortex, entorhinal cortex, amygdale, and septum.

Importantly, most of the drugs used for anxiety and depression

suffer from troublesome side effects. SSRIs, which make up

60% of the worldwide antidepressant and antianxiety market,

are frequently associated with sexual dysfunction, appetite

disturbances, and sleep disorders. Because SSRIs and SNRIs

can increase 5-HT levels in the brain, they can indirectly

sitmulate all serotonergic receptors, some of which are believed

to lead to adverse side effects associated with these drugs. The

use of benzodiazepines is associated with addiction, dependency,

and cognitive impairment.

5-HT_1Aagonists and partial agonists have been implicated

in and have demonstrated effectiveness in the treatment of

anxiety and depression in the clinic.^9-13Partial agonists of the

5-HT_1Areceptor mediate antidepressant activity through an

increase in serotonergic neurotransmission. Although the mech-

anism of action is not yet fully understood, there is substantial

evidence that the physiological and behavioral responses are

achieved following desensitization of the 5-HT_1Areceptor-

mediated response.¹³

According to the U.S. National Institute of Mental Health,

anxiety (in particular generalized anxiety disorder, or GAD) and

Another therapeutic alternative for treatment of anxiety and

depression is 5-HT_1Aagonists. Azipirones comprise the major

class of 5-HT_1Aagonists of which buspirone¹⁵1 (Buspar) is

the only FDA approved 5-HT_1Aselective agonist currently on

the US market for anxiety (Buspar package insert).¹⁶Buspirone

has shown efficacy in randomized controlled trials of GAD for

* To whom correspondence should be addressed: Phone: +972-3-612-

8590, Fax: +972-3-612-8528, e-mail: becker@predixpharm.com.

^†Predix Pharmaceuticals Ltd., Israel.

^‡Predix Pharmaceuticals Inc., Lexington, MA.

^§Current address: P.O. Box 265, Del Mar, CA 92014.

Published on Web 05/09/2006

SelectiVe 5-HT_1AAgonist

Journal of Medicinal Chemistry, 2006, Vol. 49, No. 11 3117

Chart 1. Azapirones, 5-HT_1AAgonists, Developed for the Treatment of Anxiety and Depression: Buspirone 1, the Only Marketed

Azapirone, Gepirone 2, Zalospirone 3, Ipsapirone 4, Tandospirone 5, and Sunepitron 6

Table 1. Properties of Azapirones and Their Selectivity Profiles

5-HT_1A

K_i[nM]

R₁

K_i[nM]

D2

K_i[nM]

compound

5-HT_1A/R₁ratio

5-HT_1A/D2 ratio

dizziness, nausea, headache

human half-life (dosing)

buspirone

ipsapirone

sunepitron

gepiron ER

20

6

92

13

367

229

35

18

38

0.4

185

13

110

0.7

18

yes

2 h (3× daily)

6 h (1× daily)

2400

58

4

yes

which it was approved.^17-19Unlike the benzodiazepines, bus-

pirone is not addictive, even with long-term use. Buspirone also

seems to have less pronounced side effects than benzodiazepines

and SSRIs/SNRIs and no withdrawal effects, even when the

drug is discontinued quickly. Sexual dysfunction, very prominent

in depressed and anxious patients, and a major side effect of

other psychotropic agents, is not associated with 5-HT_1Aagonist

use. Buspirone has been reported to show modest activity in

reducing SSRI-induced sexual side effects.^20,21

elimination half-life [t_1/2] ) 2-3 h)^22,23and to a three-times

daily dosing (Table 1). This short half-life is due to the rapid

formation of the pyrimidinepiperazin metabolite 7, which is a

potent R₂-adrenergic receptor antagonist.²⁷Even with an

extended release formulation the elimination half-life for

gepirone ER is only about 6 h.²⁸

The main disadvantages of buspirone are side effects such

as nausea, headache, nervousness, restlessness, dizziness, light

headedness, and fatigue. To overcome these side effects,

buspirone has to be gradually titrated for several days or weeks

until reaching the therapeutic dose, taking weeks for the drug

to be fully effective. For these reasons, buspirone is initiated at

5 mg three times daily (tid) or 7.5 mg twice daily (bid) and

then escalated gradually to the clinically effective dose of 10

to 20 mg tid. Another limitation of buspirone is its short

elimination time in the body (∼2 h),^22,23necessitating its three-

times a day (tid) dosing and making it less appealing compared

to the once a day dosing of SSRIs.

Furthermore, it was found that most of these azapirones share

a similar side effect profile consisting of dizziness, lighthead-

edness, fatigue, nausea, nervousness/restlessness, and in some

cases also sleep disorders. Close inspection of the pharmaco-

logical profiles of different azapirones suggests that the poor

tolerability of this class of drugs may be due to lack of

selectivity. While the primary target for these drugs is the

5-HT_1Areceptor, many of them show high affinity also to R₁

adrenergic receptors and to dopamine D2 receptors (Table 1).

This lack of selectivity, together with the potent R₂adrenergic

antagonism effect of the azapirones’ primary metabolite, 7,

suggests that the side effects of azapirones may be related to

R-adrenergic activities (blood pressure) or dopaminergic activi-

ties (nausea, restlessness).

Hence, there is still a medical need for new antianxiety and

antidepressant drugs with improved efficacy for partial/non-

responding and noncompliant patients. These new drugs should

have a faster onset of action compared to that of SSRIs, SNRIs,

and azapirones (two to four weeks), allow once a day dosing,

and have a reduced side effects profile. It should not have the

same degree of dizziness, lightheadedness, nervousness/restless-

ness, and fatigue characteristic of azapirones, the sexual

dysfunction, sleep disorders, and other side effects associated

with SSRIs, nor the addiction, sedation, and dependence which

accompany the use of benzodiazepines. The experience gained

with buspirone and other azapirones suggest that a nonazapirone

5-HT_1Aagonist has the potential of fulfilling this desirable

The introduction of buspirone prompted attempts by many

pharmaceutical companies to develop new azapirone drugs,

structurally similar 5-HT_1Aagonists, with a superior side effect

profile and metabolic stability that will allow them to be dosed

once daily. Among these new azapirones are drugs such as

gepirone 2, zalospirone 3, ipsapirone 4, tandospirone 5, and

sunepitron 6 (Chart 1). Extended release gepirone²⁴and ipsa-

pirone²⁵have shown activity in major depressive disorder

studies. Tandospirone improved depressed mood, anxiety, and

agitation in a pilot study of patients with dementia.²⁶However,

despite their apparent efficacy, most of these drugs have failed

to reach the market due to poor tolerability. Only exceptions

are tandospirone, which is marketed only in Japan, and gepirone

that is still under active development in an extended release

(ER) formulation.

Like buspirone, the other azapirones also suffer from rapid

metabolism leading to a very short elimination half-life (serum

3118 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 11

Becker et al.

profile provided it is highly selective and metabolically stable.

The selectivity should ensure a better side effect profile and

should allow reaching the full therapeutic dose on day 1 to

enhance onset of action.

Several classes of compounds such as aminotetralins, aryl-

oxyalkylamines, ergolines, indolylamines, and arylpiperazines

have been reported as 5-HT_1Areceptor ligands.^29-36Most

interesting is a series of studies by Lopez-Rodriguez and

collaborators^37-44who using extensive SAR analysis together

with 3D-QSAR models (e.g., CoMFA) have characterized a

range of arylpiperarzines, mainly focusing on bicyclohydantoin

or diketopiperazine analogues. These studies have shown that

the pharmacophore part of these molecules is their pyrimi-

dinepiperazine or arylpiperazine part while the rest of the

molecule is nonpharmacophoric in nature. The same studies

also showed that most of these compounds exhibit high

levels of undesirable affinity to R₁adrenergic receptor, and that

it is not trivial to engineer potent compounds that are selective

to one or the other, although examples of selective 5-HT_1A

agonists have been reported. In general, the lack of an X-ray

crystallography structure for the 5-HT_1Areceptor has so far

prevented the application of structure-based approaches to the

discovery of selective 5-HT_1Aagonists, although homology-

based models, which use the structure of rhodopsin as a

template, have been recently used to rationalize some of the

data.^42,44

Figure 1. A PREDICT 3D model of the 5-HT_1Areceptor binding site

with a bound 5-HT molecule. Key interactions between the amino group

of 5-HT and Asp 116, and between the 5-HT hydroxyl and Ser 199

are highlighted.

To overcome this lack of structural information, which in

general has hampered the application of structure based drug

discovery to GPCRs, we have developed PREDICT. PREDICT

is a unique nonhomology method for modeling the 3D structure

of membrane-embedded GPCRs.^45,46The resulting 3D structures

have been successfully validated for multiple GPCR drug targets

and together with in silico screening methodologies have

repeatedly yielded novel potent lead candidates.^46,47

We herein report on the discovery and development of a novel

class of potent, selective, and orally bioavailable amidosulfon-

amides as 5-HT_1Aagonists for the treatment of anxiety,

depression, and possibly also attention deficit hyperactivity

disorder (ADHD). The discovery was made by applying a

structure-based and in silico-driven approach to the discovery

and optimization of this class of compounds. The whole

discovery process, from in silico screening through lead

optimization, preclinical, and into clinical studies, was very

rapid, requiring the synthesis of only 31 compounds and taking

less than 2 years from program initiation to Phase I clinical

trial. This compound is now in Phase III clinical trials in GAD

after successfully completed phase II clinical trials, showing

efficacy and tolerability in patients.

Figure 2. Enrichment graph for in silico screening of 34 known 5-HT_1A

partial agonists embedded in a 10 000-compound random drug-like

library with similar physiochemical properties after docking into the

PREDICT 3D model of the 5-HT_1Areceptor. Library compounds are

ranked along the x axis according to their docking score (best scorers

on the left, worst scorers near the 100% mark). Enrichment at 50% is

15-fold better than with random screening (dashed line).

It was previously shown that this 3D model also explains

the binding of the azapirones buspirone 1 and sunepitron 6 in

the 5-HT_1Abinding pocket.⁴⁷In both cases, residue Asp116 is

interacting with the piperazine amine and residue Ser199

interacts with the azapirones’ imide moiety. In addition, the

model shows that residue Phe362 from TM6 stacks against these

compounds’ pyrimidine rings and that residue Asn386 from

TM7 also contributes to this interaction, in agreement with

experimental data.⁵⁰

To further validate the model, as well as to calibrate the

docking protocol, a virtual ‘enrichment’ experiment was carried

out. Docking a diverse set of 34 known 5-HT_1Aligands

embedded in a random 10 000 drug-like compound library, with

average properties similar to those of the 5-HT_1Aligands,⁴⁶

yielded an enrichment factor 15-fold better than random (at the

point where 50% of the compounds were identified) when

ranked by the initial DOCK score (Figure 2). Furthermore, 90%

of the known ligands were ranked among the top 10% of the

DOCK-score ranked library.⁴⁵

In Silico Screening. A virtual screening library of 40 000

compounds was extracted from Predix’s 2 100 000 compound

library. In silico docking and scoring of these 40 000 compounds

against the 5-HT_1APREDICT model led to the selection of 78

virtual hits. These 78 compounds were purchased from the

respective vendors and experimentally tested in vitro for binding

Results and Discussion

Modeling. PREDICT is a GPCR modeling methodology that

combines the properties of the protein sequence with those of

its membrane environment, without relying on the X-ray

structure of rhodopsin as a template. The PREDICT algorithm

and methodology were reported in detail elsewhere.^45,46Using

PREDICT, a 3D model of the 5-HT_1Areceptor was generated

from its amino acid sequence (Swissprot entry 5-HT_1A_human).

This model was then virtually complexed with serotonin (5-

HT) to form a 3D model of the receptor-ligand complex. A

binding pocket was easily identified in the extracellular portion

of the TM domain, allowing the amine moiety of 5-HT to

interact with residue Asp116 in TM3 and its hydroxyl moiety

to interact with residue Ser199 in TM5 (Figure 1), both in good

agreement with experimental data.^48-49

SelectiVe 5-HT_1AAgonist

Journal of Medicinal Chemistry, 2006, Vol. 49, No. 11 3119

Figure 3. 3D models generated by PREDICT of compound 8 docked into the binding pocket of (a) the 5-HT_1Areceptor and (b) the R₁adrenergic

receptor. Highlighted are interactions that are formed between the compound and key residues in the binding pocket. The region that is present in

the 5-HT_1Apocket and is missing in the R₁pocket is highlighted by a dotted square.

to the 5-HT_1Areceptor using a radioligand displacement assay.

Compounds with experimentally validated binding affinities

lower than 5 µM are defined as actual hits. The in vitro 5-HT_1A

binding assays confirmed that16 of the 78 compounds tested

were hits with Ki < 5µM, reflecting a 21% hit rate, 9 of which

had a K_i< 1 µM (K_ivalues of 1.0, 2.3, 43, 79, 139, 160, 193,

301, and 688 nM, respectively). These 16 compounds represent

five distinct chemical scaffolds and 87% of them (14 of 16

compounds) were found to be novel chemical entities, not

covered by any patent or publication (other than the supplier’s

catalog). The best hit was a novel 1.0 nM compound 8, which

was selected as the lead compound for further optimization.

R₁(K_i) 6 nM) and R_2a(K_i) 12 nM), as well as for 5-HT₇(K_i

) 30 nM), 5-HT_2B(K_i) 53 nM), 5-HT transporter (K_i) 200

nM), 5-HT_2A(K_i) 490 nM) and dopamine receptor D₂(K_i)

67 nM). Compound 8 was also tested for cardiovascular safety

by assessing its potency for inhibition of the hERG

(human ether-a-go-go related gene) K⁺channel in a patch-

clamp assay, yielding an IC₅₀of >5000 nM which is considered

safe.

Structure-Based in Silico-Driven Optimization. The above

battery of in vitro and in vivo assays has shown that the lead

compound 8, which was obtained directly from the in silico

screening process, is a novel, potent partial-agonist of 5-HT_1A

;

it is, however, suboptimal in both its pharmacokinetic properties

and its selectivity profile. Clearly, both properties had to be

optimized. Recalling that the poor tolerability of most azapirones

stems from their poor selectivity toward R-adrenergic receptors,

we targeted a 5-HT_1A/R₁selectivity ratio >200 as the highest

priority for this program.

Lead Compound. Arylpiperazinylsulfonamide, 8, possessed

high binding affinity (K_i) 1.0 nM) for the 5-HT_1Areceptor

and was tested as a partial agonist in an in vitro cell-based assay

(Gi linked to GTP-γ-S), showing 65% activity relative to 5-HT

with an EC50 of 21 nM. This functionality compared favorably

with buspirone, 1, the only 5-HT_1Apartial agonist approved as

a drug, which has a 5-HT_1Abinding K_iof 20 nM, and shows

50% activity relative to 5-HT with an EC50 of ∼80 nM in a

cell-based assay. It also compares well with gepirone, which

has a 5-HT_1Abinding K_iof 13 nM and shows 80% activity

relative to 5-HT with an EC50 of ∼288 nM.

Step 1: Introducing Selectivity for 5-HT_1Aover R₁-

Adrenergic. The introduction of selectivity toward R-adrenergic

receptors while maintaining affinity to 5-HT_1Ais not an easy

task, as has been demonstrated in the extensive research that

has been carried out on this topic over the years. Since the R₁

adrenergic receptor is also a GPCR, we used PREDICT to model

the 3D structure of the R₁receptor (Swissprot entry

AD1AA_human) complexed with compound 8, to obtain a

structure-based strategy for introducing selectivity. Figure 3

shows compound 8 docked into the two binding pockets of the

5-HT_1Aand the R₁adrenergic receptors. According to the 3D

model of the 5-HT_1Abinding pocket (Figure 3a), the aryl moiety

of 8 interacts with Phe362 from TM6, the piperazine moiety

interacts with Asp116 from TM3, and residue Ser199 interacts

with the sulfonamide moiety. This binding mode is very similar

to the binding modes or buspirone 1 and of sunepitron 6, which

were discussed above. Interestingly, residue Asn386 from TM7,

which contributed to the binding of buspirone, appears not to

participate in the binding of 8.

Compound 8 pharmacokinetic (PK) properties in rats are a

1-2 h serum half-life po and 2% oral bioavailability. Al-

though these PK characteristics are suboptimal for a once a day

dosing drug, they are no worse than buspirone 1, which in rats

has a 1 h serum half-life po and as little as 1% oral bioavail-

ability, and therefore represent a good starting point for lead

optimization.

The main downside of compound 8 was its selectivity profile,

which like many other 5-HT_1Aagonists was not selective

enough. Testing 8 in vitro against a panel of 50 G-protein

coupled receptors showed rather high binding affinities for some

of the other receptors, most notably for the adrenergic receptors

The 3D model of 8 docked into the binding pocket of the

PREDICT generated R₁adrenergic receptor model (Figure 3b)

is also in agreement with its high binding affinity. The aryl

3120 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 11

Becker et al.

Figure 4. 3D models generated by PREDICT of 20h, a m-^cPrCONH piperidinesulfonyl analogue of 8, in the binding pockets of (a) 5-HT_1Aand

(b) R₁receptors. Highlighted are the distances between the piperazine amine atom and the proteins’ aspartic acid (Asp116 in 5-HT_1Aand Asp106

in R1), indicating that this interaction is maintained in the 5-HT_1Astructure but lost in the R₁structure.

Chart 2. Strategy for Optimization of the Lead Compound 8

moiety of 8 interacts with Phe290 from TM6, the piperazine

moiety interacts with Asp106 from TM3, and residues Ser188

and Ser192 interact with the sulfonamide moiety. In addition

the 2-OMe group seems to interact with Asn318 from TM7,

and the phenyl ring that is attached to the sulfonamide interacts

with residue Phe193 from TM5, an interaction that is not

observed in the 5-HT_1Abinding site. This binding mode is in

general agreement with putative binding modes suggested in a

recently published ligand-supported homology model of R₁,

which has yielded very good in silico screening results.⁵¹

O-aryl, and esters as well as N-alkyl substituents was not favored

because of the potential propensity of these appendages for

metabolic degradation. Hence the strategy that was pursued was

to introduce sulfonamides and amides at the meta-position for

their metabolic stability. Both groups have been shown in the

past to be tolerated at the meta-position of other 5-HT_1A

ligands.^41,42Such amides and sulfonamides groups would

potentially have an added value of increasing the compound’s

solubility and improving its metabolic stability, thus favor-

ably affecting its oral bioavailability and improving the com-

pound’s PK profile. A computational analysis of a series of

alkyl-amides and alkyl- sulfonamides docked into the 5-HT_1A

binding site suggested that the maximal size of the alkyl group

that can be accommodated at that position was isopropyl or

cyclopropyl.

Figure 4 depicts the binding of a m-^cPrCONH virtual analogue

of 8 in the binding pockets of 5-HT_1Aand R₁receptors. As can

be seen, this larger moiety at the meta-position does not prevent

the compound from docking well into the 5-HT_1Abinding site,

maintaining all the original interactions (e.g., the piperazine-

aspartic acid interaction) as well as adding the putative interac-

tion with Asn386 (Figure 4a). On the other hand, according to

the 3D model, this bulkier group interferes with binding to R₁,

increasing the distance between the piperazine and the aspartic

acid to more than 5 Å, effectively preventing this salt-bridge

from forming (Figure 4b). Hence the strategy that was selected

is depicted in Chart 2. The length of the alkyl linker was not

selected for optimization because it has been previously

established that maximal affinity for both 5-HT_1Aand R₁

receptors is reached with a three-carbon or four-carbon linker.^40-41

Naturally, even with a well defined strategy, there are multiple

compounds that can be proposed for synthesis. To limit the

Namely, in these 3D models the arylpiperazine (or the

pyrimidinepiperazine) group is embedded deep within the

binding pocket in a way that is similar for both 5-HT_1Aand R₁

receptors, supporting the hypothesis that this part of the molecule

contains the pharmacophore.⁴³Accordingly, the nonpharma-

cophoric parts of the 5-HT_1Aligands, e.g., the imide group of

buspirone 1 or the sulfonamide group of 8, are oriented toward

the top of the binding pocket and are less tightly bound.

These two 3D structures were used to outline an optimization

strategy for 8 leading to a 5-HT_1Aover R₁selectivity. Comparing

the two binding sites, one could identify a region near the aryl

group that was not occupied by 8 when docked in the 5-HT_1A

receptor and was missing altogether in the 3D model of R₁.

This region is primarily hydrophobic with the exception of

residue Asn386, which also contributes to it. According to these

3D structures, replacing the original o-OMe group with a bulkier

moiety at the meta-position would lead to a more selective

compound since this added moiety should bind well to the

5-HT_1Apocket but will not fit into the R₁site. This strategy is

in agreement with similar conclusions that were reached using

CoMFA analysis³⁸and Artificial Neural Networks analysis⁴²

of a series of bicyclohydantoin-phenylpiperazines with affinity

to 5-HT_1Aand R₁. However, it is of course essential that this

group that is expected to reduce the affinity to R₁will not

decrease the activity of the new potential compounds to the

target protein, 5-HT_1A. Since this additional moiety would bind

in a tight region of the 5-HT_1Asite, its flexibility should be

limited in order to minimize the entropic penalty which would

reduce its affinity to 5-HT_1A. Furthermore, if the new group

would have hydrogen donors or acceptors that could interact

with Asn386, that added interaction could serve to compensate

for the entropic loss as well. The incorporation of O-alkyl,

SelectiVe 5-HT_1AAgonist

Journal of Medicinal Chemistry, 2006, Vol. 49, No. 11 3121

Scheme 1^a

^aReagents: (i). NH(C₂H₄Cl)₂, Na₂CO₃, C₆H₅Cl, 40-45%; (ii). Et₃N, CH₂Cl₂, 80-96%; (iii) 11, Et₃N, THF, 70-76%; (iv) TFA, CH₂Cl₂, 90%; (v)

SnCl₂, MeOH, HCl, 75%.

Table 2. Optimization of Selectivity for 5-HT_1Aover R-Adrenergic

number of compounds synthesized, and hence to speed up the

Receptors

optimization process, each proposed compound was computa-

tionally analyzed before making a decision whether to synthesize

that compound or not. The computational analysis included in

silico evaluation of the affinity to 5-HT_1A, evaluation of

selectivity vs R1, and prediction of several ADME properties.

In particular, an estimate of the compound’s affinity to the target

was obtained by docking each proposed compound to the

5-HT_1Areceptor model and using the resulting binding poses

for calculating a predicted 5-HT_1AK_ivalue using a 2D/3D

QSAR-based model (in general, pK_ipredictions were accurate

to within 0.5 log units). Selectivity was assessed by docking

each proposed compounds to the R₁-adrenergic receptor model

and to a 3D model of the hERG channel (see below). ADME-

related properties included brain penetration predictions, and

other physicochemical parameters such as rotatable bonds, polar

surface area, cLogP, and more. The details of these prediction

methodologies will be presented elsewhere. These predicted

profiles were instrumental in prioritizing and deprioritizing

compounds for synthesis, resulting in actual synthesis of only

10% on the proposed compounds.

compd

R

5-HT_1AK_i(nM)^a

R₁, %

6 nM

98

51

47

22

0

12

15

<10

0

R₂, %^b

8

9

2-OMe

1.0

40

46

141

47

6.8

426

56

12 nM

90

21

14

12

43

31

12

H

20a

20b

20c

20d

20e

20f

20g

20h

20i

3-MeSO₂NH

3-EtSO₂NH

3-ⁱPrSO₂NH

3-MeCONH

3-EtCONH

3-ⁿPrCONH

3-ⁱPrCONH

3-^cPrCONH

3-^tBuCONH

78

22

1360

<10

10

11

^aK_iis the binding affinity determined using h5-HT_1Areceptor expressed

in HEK-293 cells using [³H]8-OH-DPAT as the radioligand (mean value;

n ) 2). ^b% displacement of the radioligand at 1 µM from the R₁and R₂

adrenergic receptors in rat cerebral cortex cells using [³H]prazosin and

[³H]RX 821002, respectively (mean value; n ) 2).

The selected optimization strategy necessitated the insertion

of a pivotal link such as an amino group via which alkyl, aryl,

esters, alkylsulfonyl, and acyl groups can be introduced. The

synthesis shown in Scheme 1 was developed to allow rapid and

efficient preparation as well as scale-up of various compounds

shown in Tables 2 and 3. Nitrophenylpiperazine 11 as an

common intermediate was prepared by refluxing 10 with bis-

(2-chloroethyl)amine chloride salt and Na₂CO₃in chlorobenzene

(Scheme 1). The intermediate 18 was synthesized by two

different methods (Scheme 1). In method 1, 18 was directly

prepared by treating 11 with 13 which was obtained by

disulfonylation of aminobutanol 12. Compounds 20a-j and 20p

were synthesized with method 1. In method 2, tosylation

products 15 of N-boc-aminobutanol 14 were used as alkylating

reagent which reacted with 11 to give boc-protected compounds

16. Deprotection of compounds 16 by TFA yielded TFA salt

16 which was treated with different sulfonyl chlorides to afford

intermediates 18. Compounds 20k-o and 20q were synthesized

by method 2. The nitro intermediates 18 were reduced by SnCl₂

in methanol to give corresponding amino compounds 19 with

good yield. The final compounds 20a-q were obtained by

acylation or sulfonylation of intermediates 19 with correspond-

ing acyl chloride and sulfonyl chloride.

Table 2 shows that the resulting structure-activity relation-

ships (SAR) of the sulfonamides 8, 9, 20a-i agree well with

the structure-based hypotheses that were laid as the foundation

of the optimization strategy. Comparison of the lead compound

8 with the corresponding unsubstituted phenyl analogue 9

showed that presence of the o-OMe in 8 affects a 40-fold

increase in its 5-HT_1Abinding affinity with a lesser effect on

the activity for both R-adrenergic receptors R₁and R₂.

Introducing the alkyl-amides and alkyl-sulfonamides groups

at the meta-position significantly reduced the affinity of the

compounds to R₁, moving them from the low nM range (>98%

at 1 µM) to the µM range (<50% at 1 µM) for all the

compounds 20a-i in these series. In general the amide series

showed greater selectivity toward R₁with all amide com-

pounds exhibiting R₁K_ivalues greater than 3500 nM (<22%

at 1 µM), while the sulfonamide compounds exhibited R₁

binding affinities in the vicinity of 1000 nM (∼50% at 1 µM).

3122 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 11

Becker et al.

Figure 5. 3D model of the pore forming region (S5-P-S6) of the hERG K⁺channel with astemizole bound to it (IC₅₀) 0.9 nM). (a) Top-view

and side-view of the hERG tetramer, showing the backbone of each monomer and a space-filling representation of the bound compound. (b) A

detailed view of astemizole docked within the hERG channel pore: Phe656 residues are flanking the terminal methoxy-phenyl group of astemizole,

Tyr652 residues are within interaction distance from the p-fluorophenyl group, and the Ser660 residues are at interaction distance from the terminal

methoxy group.

Selectivity toward R₂was in general commensurate with the

selectivity toward R₁.

selectivity goal, exhibiting a 5-HT_1A/R₁selectivity ratio >500

(R₁K_i> 3500 nM) and showing partial agonist activity with

an EC₅₀) 20 nM in an in vitro cell-based assay, similar to that

of the lead compound 8. Compound 20d also demonstrated

excellent half-life both in human liver microsomal assay (hLM

t_1/2) 39 min) and in rat pharmacokinetic evaluation (t_1/2) 3.3

h, oral adminstration), with a rat oral bioavailability of 15%,

showing a significant improvement in oral bioavailability

compared to the 2% of the lead compound 8. The observed

improvement in oral bioavilability and metabolic stability during

the optimization was another validation of the strategy discussed

above.

The SAR also suggests that alkyl groups ranging in size from

Me to Pr were tolerated within the 5-HT_1Abinding pocket. On

the other hand, in agreement with the computational prediction,

the bulkier ^tBu group 20i does not fit in that pocket as reflected

by its reduced potency (K_i) 1360 nM). In fact, compound 20i

showed 200-fold less 5-HT_1Abinding affinity than the most

active analogue 20d (K_i) 6.8 nM).

As expected, the best 5-HT_1AK_ivalues were obtained for

analogues which are conformationally the most constrained,

c

MeCONH 20d (K_i) 6.8 nM), PrCONH 20h (K_i) 22 nM),

However, when 20d was examined for its cardiovascular

safety there was an unpleasant surprise. While the lead

compound 8 exhibited very poor binding to the hERG K⁺

channel (IC₅₀> 5000 nM), the optimized compound 20d has

become a more potent hERG blocker, inhibiting the channel

(when evaluated in a patch-clamp assay) with a IC₅₀of 300

nM. Since the hERG K⁺channel is a major regulatory molecular

mechanism underlying QT prolongation syndrome,⁵²which is

linked to a predisposition of ventricular arrhythmia that can lead

to ventricular fibrillation and sudden death,⁵³hERG blocking

is considered a serious hazard which has led to removal from

the market of drugs such as aztemizole and cisapride.

Step 2: Reducing Affinity to hERG. The high affinity of

20d to the hERG channel had to be reduced at least 10-fold for

a derivative of this compound to reach development candidate

status. As in the case of the selectivity profile, we applied a

structure-based approach to alleviate the hERG affinity problem.

Since there is no experimentally determined X-ray structure of

and MeSO₂NH 20a (K_i) 46 nM). Accordingly, higher K_ivalues

were measured for conformationally less constrained analogues,

such as EtCONH 20e (K_i) 426 nM) and EtSO₂NH 20b (K_i)

141 nM). The effect of entropy can be seen when comparing

the propyl series of amide derivatives, 20f, 20g, and 20h substi-

n

i

c

tuted with Pr, Pr, and Pr, respectively, at the meta-position

to the phenyl of piperazine which showed high to moderate

binding affinity depending upon the size and volume of the

appendage. The ⁿPr and the ⁱPr analogues (K_i) 56 nM and 78

nM, respectively) exhibited 2-3-fold less 5-HT_1Abinding affin-

ity than the conformationally restricted ^cPr derivative 20h which

demonstrated 5-HT_1AK_iof 22 nM and virtually no affinity for

adrenergic receptors. It is not clear why the flexible ⁿPr analogue

i

20f has a lower K_ithan the corresponding Pr compound 20g;

this may be attributed to assay variability or its ability to

maximize hydrophobic interactions in that region of the site.

The optimized Me-amide analogue 20d not only has high

5-HT_1Aaffinity (K_i) 6.8 nM) it also surpassed the target

SelectiVe 5-HT_1AAgonist

Journal of Medicinal Chemistry, 2006, Vol. 49, No. 11 3123

Figure 6. A comparison of the hERG binding modes of compounds 8 and 20d. Sown are a detailed 3D view of the binding of compounds 8 in

the hERG pore, as well as two schematic views of the binding of compounds 8 and 20d next to each other. The four main interaction regions are

highlighted in all views: An aromatic region formed by the four Tyr652 residues, a K⁺pocket, an aromatic region formed by the four Phe656

residues, and a polar region formed by four Ser660 residues (shown only schematically). The greater hERG affinity of 20d is probably due to its

interaction with the Ser660 region. This interaction is not formed by compound 8.

the hERG channel, the first step was to model its 3D structure

based on the structure of a bacterial channel. The hERG K⁺

channel is composed of four identical subunits that coassemble

to form a tetrameric structure. The basic secondary structure of

each subunit is identical to that of other voltage-gated K⁺

channels subunits. Each monomer consists of six transmembrane

segments (S1-S6), with large intracellular amino and carboxy-

termini. The pore domain is formed by the S5-P-S6 segments,

where P is a reentrant pore-forming linker that contains a helical

K⁺selectivity filter.⁵⁴Because the closed KcsA ion channel

(PDB code 1BL8)⁵⁵that was used as a template for modeling

the hERG channel (procedure described in the Experimental

Section) has only two transmembrane domains, which are

equivalent to the S5 and S6 domains of voltage-gated channels

such as the hERG, the resulting 3D model is restricted to that

channel forming portion of the tetramer. The channel pore

structure was refined using a molecular-dynamics-based virtual

complexation process with astemizole, a drug that was found

to be an extremely potent hERG-channel blocker (IC₅₀) 0.9

nM) causing QT-prolongation (astemizole was removed from

the market in 1998 due to its potential hazards). Figure 5 depicts

the 3D model of the hERG channel with astemizole bound to

it (for more details, see Kalid, O et al., manuscript in prepara-

tion).

interacts with Tyr652, the piperazine amine is located in the

"K⁺pocket", and the aryl group is sandwiched between two

Phe656 residues. The only difference is that while the m-

NHCOMe moiety of 20d extends further and interacts also with

residues Ser660, this additional interaction does not occur in

the binding of 8, which as an o-OMe moiety that points in a

different direction. Interestingly, the potent hERG binder

astemizole also has a moiety (p-OMe) that interacts with Ser660

(see Figure 5).

The structural analysis of the hERG binding mode of 20d

suggests that removing the H-bond capable group from the meta-

position of 20d should reduce hERG binding. However, this

would also annul all of the previous optimization efforts.

Namely, this analysis shows that the optimization strategy of

Chart 2, which led to an efficient optimization of affinity,

selectivity, and pharmacokinetics, is also the source of the

increased affinity to the hERG channel. Since this was not a

promising path, the conclusion was that the effort to reduce

hERG affinity should focus on the other side of the compound,

on the p-toluenesulfonyl region of 20d. On the basis of the 3D

model, the p-toluenesulfonyl group contributes to the hERG

binding affinity of compound 20d via interactions with the

aromatic Tyr562 residues. Replacing the aromatic p-toluene (p-

tol) substituent of 20d with a nonaromatic hydrophobic group

may offer an alternative strategy to reducing the hERG binding

affinity. It should be noted that replacing the aromatic group

by a nonaromatic group was not expected to remove hERG

binding altogether, but it was hoped that it will reduce it enough

to obtain the required selectivity window for the drug.

To develop an optimization strategy that would reduce hERG

binding without compromising the affinity, selectivity, and PK

goals that were already achieved in compound 20d, it was

necessary to understand why compound 20d had 10-fold greater

affinity to the hERG channel compared to the lead compound

8. Figure 6 compares the binding modes of compounds 8 and

20d in the hERG channel. As expected, both compounds adopt

a similar binding-mode such that most of the interactions are

identical for both compounds: the p-toluenesulfonyl group

Fortunately, the structural analysis of the 5-HT_1Amodel also

suggested that this modification should not have a significant

effect on binding to the primary target. According to the 3D

structure of 5-HT_1A(Figure 3a), there are no major aromatic

3124 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 11

Table 3. Optimization of hERG Binding

Becker et al.

compd

R

R₂

R₃

5-HT_1A: K_i(nM)^a

R₁%^b

6 nM

22

39

<10

47

10

5

37

27

R₂%^b

hERG: IC₅₀nM (%)^c

8

2-OMe

4-Me-Ph

4-F-Ph

^chexyl

H

1

6.8

18

40

24

5.1

8.9

9.2

1.3

9.7

12 nM

43

>5000 (11%)

20d

20j

20k

20l

20m

20n

20o

20p

20q

3-MeCONH

3-^cPrCONH

3-MeCONH

300 (73%)

<10

0

ⁱPr

CH₂-^chexyl

ⁱBu

∼3800 (21%)

∼1500 (41%)

∼3000 (25%)

∼4000 (20%)

∼2200 (31%)

<10

0

CH₂-^chexyl

ⁱBu

Me

47

16

^aK_iis the binding affinity determined using h5-HT_1Areceptor expressed in HEK-293 cells using [³H]8-OH-DPAT as the radioligand (mean value; n )

2). ^b% displacement of the radioligand at 1 µM from the hR₁and hR₂adrenergic receptors in rat cerebral cortex cells using [³H]prazosin and [³H]RX

821002 as the radioligands, respectively (mean value; n ) 2). ^cEstimated IC₅₀(in nM) for inhibition of the hERG K⁺current in a patch-clamp assay, based

on measuring the % inhibition at 1 µM (mean value, n ) 2, given in parentheses).

to be a weak hERG K⁺channel blocker with low inhibitory

Chart 3. Strategy for Reducing hERG Affinity of 20d

potency of only 20% at 1 µM (estimated IC₅₀3800 nM). Since

the cyclopropyl amide 20h was found to be the most active

(5-HT_1AK_i22 nM) among all three propyl amide analogues

(20f, 20g, and 20h) examined, a cyclopropyl amide compound

20n was also designed and synthesized in which the terminal

p-toluenesulfonamide group was replaced with an optimal

cyclohexylmethyl moiety to improve target activity and selectiv-

ity. However, although the cyclopropyl amide 20n showed good

5-HT_1Abinding affinity (K_i) 8.9 nM) and even better selectivity

residues that interact with the p-toluenesulfonyl group, which

than 20m, its hERG K⁺channel blocking affinity was too high,

is the nonpharmacophoric part of our compounds, suggesting

that such a modification should not adversely impact binding

41% at 1 µM (estimated IC₅₀1500 nM) not quite making the

hERG selectivity target.

to the 5-HT_1Atarget. Finally, this modification was also not

Replacement of the p-toluene (4-Me-Ph) sulfonyl with

expected to significantly increase the compound’s affinity to

metabolically more stable 4-fluorophenylsulfonyl moiety yielded

R₁, since in the 3D structure of R₁one finds an aromatic Phe

20j which has a nearly 3-fold reduced 5-HT_1Abinding affinity

residue in interaction with the p-toluene moiety (Figure 3b).

(K_i) 18 nM) compared to 20m, and was not pursued further.

Hence, the structure-based strategy that was selected for

reducing the hERG affinity of 20d without affecting 5-HT_1A

affinity and R₁selectivity was to replace the p-toluene group

with alicyclic and cyclic carbocycles such as isopropyl, isobutyl,

Compound 20m with 5-HT_1AK_iof 5.1 nM behaved as a full

agonist in an in vitro cell-based functional assay with an EC₅₀

of 20 nM and showed excellent selectivity and pharmacokinetic

profiles. Hence, 20m was selected as the clinical candidate, and

this compound is now in Phase III clinical trials for generalized

anxiety disorder (GAD). A detailed report of the properties of

cyclohexyl, and cyclohexylmethyl. Chart 3 describes this

strategy, which led to the synthesis of compounds 20k-q.

Table 3, which reports the SAR results for compounds

20m and its analogues is given in the following section.

20j-q, shows that the structure-based strategy of Chart 3 was

Note, that during the entire optimization process we did not

change the length of the linker connecting the arylpiperazine

to the sulfonamide. The reason is that a detailed in silico analysis

of various linker lengths suggested that the four-carbon linker

is already the optimal spacer. This is in full agreement with

thorough SAR analysis on other arylpiperazine compounds

which have demonstrated that the maximum affinity for both

5-HT_1Aand R₁receptors is reached with n ) 3 or 4. Reduction

to n ) 2 or 1 leads to inactive or poorly active compounds.⁴³

Nonetheless, the computational analysis has suggested that

making the linker more rigid may be beneficial. In particular it

was suggested that replacing the linear n-butyl-sulfonamide

linker with a 4-methylpiperidine-sulfonyl moiety would result

in compounds that dock well into the 5-HT_1Abinding site and

thus will maintain, or even improve, the high affinity of

corresponding linear compound. On the basis of this analysis,

we prepared compounds 21a-c. Compounds 21a-c in Scheme

proven to be very successful. All compounds in this series

maintained excellent binding to the 5-HT_1Areceptor as well as

their selectivity toward R₁while significantly reducing hERG

binding by factors ranging from 5-fold to more than 10-fold,

reducing hERG affinity from an IC₅₀of 300 nM in 20d to IC₅₀

values ranging from 1500 nM (20n) to 4000 nM (20p).

The cyclohexyl replacement of p-tol on the sulfonamide gave

20k that had reduced hERG activity and slightly diminished

binding affinity for 5-HT_1A. This compound, which is the closest

analogue of the original p-tol in this series, also had lower

affinity toward the R₁and R₂receptors, confirming the estimated

role of the aromatic residue Phe193 in the binding of 20d to

R1. The 5-HT_1Aactivity was improved by nearly 2-fold when

reducing the size of the lipophilic cyclohexyl ring by replacing

it with an isopropyl group to give compound 20l (5-HT_1AK_i

24 nM). The lower K_iof 20k and 20l may be attributable to the

shorter length and/or size of the cyohexyl and isopropyl groups

on the sulfonamide moiety. The size and the conformational

constraint of the sulfonamides was optimized by incorporating

cyclohexylmethylsulfonyl to yield 20m which displayed im-

proved 5-HT_1Aactivity (K_i5.1 nM) while maintaining good

selectivity. In hERG patch-clamp assay, 20m was determined

SelectiVe 5-HT_1AAgonist

Journal of Medicinal Chemistry, 2006, Vol. 49, No. 11 3125

Scheme 2^a

^aReagents: (i). Et₃N, CH₂Cl₂, TsCl, 90%; (ii) 11, Et₃N, THF, 92%; (iii) TFA, CH₂Cl₂,; (iv) R₂SO₂Cl, Et₃N, CH₂Cl₂, 90%; (v) SnCl₂, MeOH, HCl, 95%;

(vi) CH₃COCl, Et₃N, CH₂Cl₂, 91%.

Table 4. Effect of Rigidifying the Linker

Table 5. Selectivity of 20m or Serotonin Receptors

receptor

K_i

tissue

5-HT_1A

5-HT_1B

5-HT_1D

5-HT_2A

5-HT_2B

5-HT_2C

5-HT₃

5.1-11 nM

17 nM

human cDNA

rat cerebral cortex

bovine

human cDNA

human

mouse

human

human cDNA

human

>2 µM*

>5 µM*

700 nM*

5-HT_1A

hERG

compd

R₂

linker^aK_i(nM)^bR₁, %^cR₂, %^c

IC₅₀(nM)^d

20m CH₂-^chexyl linear

21a

20l

21b

20j

21c

5.1

10

24

112

18

47

<10

39

0

∼3800 (21%)

CH₂-^chexyl rigid

13 ∼2000 (35%)

5-HT_4e

5-HT_5A

5-HT₆

ⁱPr

linear

rigid

linear

rigid

<10

ⁱPr

<10

4-F-Ph

5-HT₇

human cDNA

13

49

^a*, Estimated based on % displacement at 1 µM.

^a“linear” ) n-butyl-sulfonamide ((CH₂)₄NH); “rigid” ) 4-methylpip-

eridinesulfonyl . ^bK_iis the binding affinity determined using h5-HT_1A

receptor expressed in HEK-293 cells using [³H]8-OH-DPAT as the

radioligand (mean value; n ) 2). ^c% displacement of the radioligand at 1

µM from the hR₁and hR₂adrenergic receptors in rat cerebral cortex cells

using [³H]prazosin and [³H]RX 821002 as the radioligands, respectively

(mean value; n ) 2). ^dEstimated IC₅₀(in nM) for inhibition of the hERG

K⁺current in a patch-clamp assay, based on measuring the % inhibition at

1 µM (mean value, n ) 2, given in parentheses).

200-fold 5-HT_1A/hERG selectivity target (5-HT_1AK_i) 10 nM).

However, the pharmacokinetic properties of 21a in rats showed

that its methylpiperidine linker is being rapidly metabolized

resulting in a very short 0.75 h serum half-life po, which is

much shorter than the serum half-life of the linear linker

compound 20m.

2 were synthesized by the same procedure as in method 2 of

Scheme 1.

Table 4 shows the results for compounds 21a-c in compari-

son to their linear counterparts, compounds 20m, 20l, and 20j,

respectively. The results show that two of the three rigid-linker

compounds, 21a and 21c, maintained a 5-HT_1Aaffinity similar

to that of their linear counterparts, in agreement with the

computational prediction. Only compound 21b was 4-fold less

active compared to its linear linker counterpart 20l, which in

itself was the least active compound of the three linear

compounds. Furthermore, rigidifying the linker also did not

affect the affinity of the compounds to the R₁and R₂receptors,

with the exception of 21a which became even more selective

toward R₁relative to its linear counterpart 20m. When tested

for hERG binding compound 21a showed 35% inhibition of

the ion current at 1 µM (IC₅₀∼ 2000 nM), which is within the

Clinical Candidate. In Vitro Pharmacology. The develop-

ment candidate 20m (PRX-00023) demonstrates high affinity

binding for the human and rat 5-HT_1Areceptors (K_is of

5.1-11 nM and 17 nM, respectively) with more than a 400-

fold difference in affinities for the 5-HT_1Areceptor compared

with all other 5-HT receptors (K_i> 2 µM, except for the 5-HT₇

with a K_i) 700 nM [Table 5]). 20m was also devoid of binding

to >50 additional proteins tested (K_i> 2 µM), including other

neurotransmitters, other GPCRs, ion channels and transporters,

including R- and â-adrenergics, dopamine, histamine, angio-

tensin II, benzodiazepines, bradykinins, endothelins, GABA,

3126 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 11

Becker et al.

Table 6. Pharmacological and Pharmacokinetic Data for Selected Compounds

assay

20m

20o

20q

20p

buspirone, 1

20 nM

80 nM

367 nM

13 nM

In Vitro Pharmacology^a

9.2 nM

60 nM

>5000 nM

3000 nM

>90 min

5-HT_1AK_i

5.1-11 nM

20 nM

1600 nM

>2000 nM

3800 nM

28 min

65 min

92%

none

9.7 nM

32 nM

27%^b

1.3 nM

3.6 nM

37%^b

EC₅₀

R₁K_i

D₂K_i

hERG K_i

hLM T_1/2

hS9 T_1/2

serum prot binding

Cyp inhib

2200 nM

>45 min

4000 nM

12 min

65 min

57%

none

In Vivo Pharmacokinetics^c

50%

3.5 h

350 ng/mL

>80%

2.4 h

600 ng/mL

ND

rat F%

11%

<1%

rat T_1/2po

rat C_maxpo

dog F%

dog T_1/2po

dog C_maxpo

rat MTD po

dog MTD po

2-3.5 h

24 ng/mL

16%

1 h

4 ng/mL

9%

1.0 h

146 ng/mL

<200 mg/kg (LD₅₀)

<600 mg/kg

1.1 h

174 ng/mL

600 mg/kg^d

600 mg/kg

^aFor all in vitro pharmacology data shown is the mean value (n g 2). ^b% displacement of the radioligand from the R-1 adrenergic receptor at 10 µM.

^cRat and dog pharmacokinetics determined following intravenous administration (1 mg/kg) and oral administration (3 mg/kg) (mean value, n g 3). ^dLD₁₀

could not be determined.

galanin, cholecystokinin, chemokines, melanocortins, muscar-

inics, neurokinins, neurotensin, orphanin, opioids, somatostatin,

sigmas, vasoactive intestinal peptide, vasopressin, neuropeptides,

and ion channels including Na⁺, K⁺, and Cl^-, as well as

transporters such as 5-HT, norepinephrine (NE), and dopamine

(CEREP and MDS Pharma Services binding profiles; data not

shown). The only receptor for which 20m has significant affinity

is the guinea pig sigma receptor (K_i) 100 nM). As reported

above, in hERG patch-clamp assay, 20m was determined to be

a weak hERG K⁺channel blocker with an estimated IC₅₀of

3800 nM. Overall these data demonstrate that 20m is a very

selective ligand for the 5-HT_1Areceptor. Compound 20m was

tested in functional cell-based assay, demonstrating that it has

an EC₅₀of 20 nM and behaves as a full agonist.

20m demonstrates a half-life (t_1/2) of 28 min in a human liver

microsome (HLM) stability assay, which is three times greater

than that of buspirone (t_1/2) 9 min), and a t_1/2of 65 min in a

human liver S9 fraction stability assay (without phase 2

cofactors), with 75% of the compound remaining after 15 min.

20m does not inhibit cytochrome P450 isoforms (CYP) 1A2,

2C9, 2C19, 2D6, and 3A4. The major metabolites of 20m were

determined to be its mono-hydroxylated and deacetylated

derivatives with 85% of 20m remaining after 4 h in human

hepatocytes. In a human serum protein binding assay, 20m was

found to be 92% protein bound at 2 µM and 10 µM, with the

majority of this binding due to an interaction with R₁-acid

glycoprotein.

pharmacokinetic profile of 20m shows 16% oral bioavailability,

a serum t_1/2of 1.1 h po, and a C_maxlevel of 174 ( 141 ng/mL

(3 mg/kg, po). This pharmacokinetics profile is far superior to

that of buspirone 1 (Table 6). In rats buspirone exhibits less

than 1% oral bioavailability and a 1 h serum t_1/2, reaching barely

detectable maximal drug levels of 4 ng/mL (3 mg/kg, po).

Buspirone does not have a significantly improved PK profile

in dogs, in which the oral bioavailability is 9%, serum t_1/2

remains 1 h, although C_maxlevels reach 146 ( 10 ng/mL

(3 mg/kg, po).

Namely, 20m in vitro shows a half-life approximately 3 times

longer than buspirone in human liver microsomes and human

S9 liver fraction. In vivo, the serum t_1/2is more than 3 times

longer in rats and is similar to buspirone in dogs. Oral

bioavailability of 20m is superior to buspirone in both rats and

dogs. Since buspirone requires three times daily (tid) dosing,

these data suggest that 20m may require only a single daily

dose, a highly desirable property for any drug.

Backup Compounds. After identifying 20m as the early

development candidate, we continued to explore the strategy

depicted in Chart 3 for the discovery of backup candidates. In

compound 20o the cyclohexylmethyl moiety of 20m was

replaced with an acyclic mimic, the isobutyl group. The isobutyl

analogue 20o showed excellent over all drug candidate profile

On the basis of the binding affinity, selectivity and functional

potency, HLM stability, and other in vitro pharmacology

parameters, 20m was selected for further evaluation for efficacy

in appropriate animal models, PK behavior in rats and dogs,

and general safety. Some key properties of 20m are listed in

Table 6.

In Vivo Pharmacokinetics. A pharmacokinetic analysis of

20m in both rats and dogs showed that the compound has a

favorable PK profile. In rats 20m shows 11% oral bioavailability

with a serum t_1/2of 2-3.5 h when administrated po, attaining

a C_maxlevel of 24 ( 13 ng/mL (3 mg/kg, po). 20m shows

significant brain penetration, achieving a brain:serum concentra-

tion ratio of approximately 0.5 in the rat at 1 h following either

intravenous or oral administration and reaching brain concentra-

tion approximately equivalent to that of buspirone. In dogs the

with high 5-HT_1Abinding affinity (K_i) 9.2 nM) and higher

selectivity than 20m (Tables 3 and 5). The agonist activity EC50

of 20o in 5-HT_1Afunctional assay was determined to be 60 nM

and its hERG binding IC₅₀∼ 3000 nM, well within the target

selectivity range. The half-life of 20o in HLM assay was

determined to be the longest of all compounds in the series with

t_1/2of >90 min which is attributable to its reduced lipophilicy

and fewer number of sites available for enzymatic oxidative

cleavage than that in 20m. This metabolic stability was also

observed in the cases of the N-Me analogue 20q of compound

20o, which was found to have a half-life >45 min in the HLM

assay. Compound 20o is only 57% protein bound and it does

not inhibit CYPs 1A2, 2C9, 2C19, 2D6, and 3A4. Compound

SelectiVe 5-HT_1AAgonist

Journal of Medicinal Chemistry, 2006, Vol. 49, No. 11 3127

20o also has a favorable PK profile, even better than that of

20m. In rats compound 20o shows 50% oral bioavailability,

serum t_1/2of 3.5 h po and C_maxlevels of 350 ng/mL, which are

7-fold higher than the corresponding levels of 20m. In dogs

compound 20o shows >80% oral bioavailability, serum t_1/2of

2.4 h po, and C_maxlevels of 600 ng/mL, which are 4-fold higher

than the corresponding levels of 20m. Compound 20o, was

selected as a backup to 20m.

N-Methyl analogues, 20p and 20q of 20m and 20o, respec-

tively, were designed to assess the effect of N-Me vs N-H of

the sulfonamide on the biological activity. N-Me derivative 20p

of the cyclohexylmethylsulfonamide 20m exhibited 4-fold

increase in the 5-HT_1Abinding affinity and slightly reduced

selectivity than that of 20m while the N-Me analogue 20q of

the isobutylsulfonamide 20o was nearly equipotent in binding

affinity and less selective than 20o for the target 5-HT_1A

receptor. Both N-methylated compounds 20p and 20q exhibited

excellent agonist activity with EC₅₀values of 3.6 nM and 32

nM, higher in both cases than their corresponding NH analogues

20m and 20o, respectively. The hERG inhibitory activity of

20p and 20q also met the target selectivity goal, estimated as

4000 nM, 2200 nM, 20% and 31% inhibition at 1 µM in the

patch-clamp assay, respectively. However, when tested in the

HLM assay, it was found that both compounds were metabolized

significantly faster than their corresponding N-H analogues

(Table 6). Within the cylohexylmethyl series 20p has a HLM

t_1/2of 12 min compared to 28 min for 20m, and within the

isobutyl series 20q has a HLM t_1/2of >45 min compared to

>90 min for 20o. Overall, both compounds 20p and 20q have

favorable profiles and could in principle also be considered as

backup candidates.

Figure 7. Effect of 20m on Delta T (°C) in the stress-induced

hyperthermia test. Seen are the increase in rectal temperature in vehicle-

treated animals (stress-induced hyperthermia) and the blocking effect

of buspirone (20 mg/kg; P < 0.05 vs vehicle-treated group) and 20m

(20 mg/kg; P < 0.01 vs vehicle-treated group). ANOVA revealed

significant effect of Treatment (p ) 0.0009). Follow-up Fisher’s PLSD

for paired comparison showed that buspirone (20 mg/kg) significantly

decreased (p ) 0.024) stress-induced hyperthermia. Similarly, 20m in

20 mg/kg dose resulted in significant decrease (p ) 0.0067). 20m at 1

and 3 mg/kg did not show significant effects (p ) 0.618 and p ) 0.2911,

respectively).

In Vivo Efficacy. Compound 20m was tested for anxiolytic

efficacy in mice using the stress-induced hyperthermia model

following oral administration of 1, 3, and 20 mg/kg doses of

the compound. In addition, two doses of buspirone (3 and 20

mg/kg, ip) were included. The test is based upon the observation

that an anxiogenic or stressful event will cause a rise in core

body temperature in mammals.⁵⁶Anxiolytics such as benzo-

diazepine receptor agonists (e.g., diazepam, chlordiazepoxide,

alprazolam) and 5-HT_1Areceptor agonists (e.g., buspirone)

decrease the hyperthermic response to stress.⁵⁷

One hour after dosing, two rectal temperature measurements

were obtained, basal temperature and, 10 min later, the body

temperature due to the stress of the first rectal measurement.

The difference (Delta T) in the two measurements reflects a

stress-induced anxiogenic response. The procedure also mea-

sures the intrinsic activity of drugs on the basal body temper-

ature, and Delta T is relatively independent from the intrinsic

temperature effects of drugs.

Figure 8. Effect of 20m on basal rectal temperature (°C) in the stress-

induced hyperthermia test. *p < 0.05, **p < 0.01 vs vehicle-treated

group (water). ANOVA revealed a significant effect of treatment (p )

0.0109). Follow-up Fisher’s PLSD for paired comparison showed that

20m (20 mg/kg) significantly decreased (p ) 0.0023) basal rectal

temperature. Similarly, buspirone in 20 mg/kg dose resulted in

significant decrease (p ) 0.044). Lower dose of 20m (1 and 3 mg/kg)

did not show significant effects (p ) 0.808 and p ) 0.326, respectively).

hyperprolactinemia effects at high doses). The maximum

tolerated dose (MTD) of 20m with a single oral administration

was determined to be 300 and 600 mg/kg in rat and dog,

respectively. The MTD values of 20m exceed those of bus-

pirone, which has an LD50 of 196 mg/kg in rats and 586

mg/kg in dogs (Buspar package insert).

Both 20m and buspirone exerted a dose-dependent anxiolytic-

like effect with a significant decrease in the hyperthermic

response to stress (Delta T) at the 20 mg/kg dose (Figure 7). A

significant reduction in basal rectal temperature was also seen

at the high dose for both compounds (Figure 8).

Additional studies of 20m have been performed using other

anxiolytic assays such as the elevated plus maze and in animal

models of attention-deficit hyperactivity disorder (ADHD). 20m

demonstrated significant efficacy in these studies, the results

of which will be presented elsewhere.

Preclinical Safety. 20m was subjected to a full range of

preclinical safety and toxicology studies in two species. Both

single and repeated dose studies indicated that the compound

was well tolerated. As found with other 5-HT_1Aagonists,

increased prolactin levels were observed (in rats, this leads to

Clinical Development. On the basis of the desirable phar-

macology, pharmacokinetics, animal efficacy, and toxicology

results, 20m was selected for clinical development for the

treatment of anxiety and depression. Preclinical PK suggested

that in humans 20m could be dosed once- or twice-daily, and

the negligible cross reactivity to other receptors, which was

introduced into the compound through the rational structure-

based optimization process, suggested that in humans 20m

should be well tolerated at efficacious doses.

To date, 20m has completed three Phase I clinical trials in

84 healthy volunteers to evaluate the tolerability and pharma-

cokinetics of this drug at doses of 10 to 150 mg. In addition, a

Phase II study has completed and the first of at least two pivotal

Phase III clinical trials in patients with generalized anxiety

disorder has been initiated.

3128 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 11

Becker et al.

A detailed review of 20m clinical results is not within the

scope of the present paper. Here we will only briefly report

some key results which are important for comparison with the

discovery effort and the preclinical data. 20m was well tolerated

at the expected efficacious doses while showing a desirable PK

profile with a terminal half-life >10 h, superior to any azapirone

tested so far, and supporting once-daily dosing. In a single-

dose study, 20m AUCs at all doses (10, 30, 40, and 60 mg)

were substantially greater than buspirone AUC after a 30 mg

buspirone dose. After single and multiple dosing, transient

increases in serum prolactin levels were observed in response

to 20m at doses greater than 30 mg. Mean increases from

baseline were approximately comparable to those seen with

buspirone 30 mg, but the intrasubject variability was less with

20m treatment.

the human Phase I studies, with good absorption and a terminal

half-life >10 h supporting a once daily dosing. Namely, 20m

shows promise as a once-daily alternative to other 5-HT_1A

agonists such as buspirone (Buspar), which require three times

daily (tid) dosing.

•Pharmacodynamics: 20m is a potent full agonist of 5-HT_1A

with EC₅₀levels of 20 nM in cell-based assays. The drug showed

activity in animal models of anxiety and ADHD. While

discussion of 20m human efficacy is not within the scope of

this paper, we have reported response of a known surrogate

marker for 5-HT_1Aefficacy (transient prolactin levels) both in

the in vivo animal studies and in healthy volunteers in the Phase

I studies. The efficacy of 20m was suggested in the Phase II

study that was completed, leading to the current ongoing Phase

III study in generalized anxiety disorder.

The clinical experience with healthy volunteers receiving 20m

revealed no unexpected adverse events associated with treat-

ment. 20m was well tolerated in healthy volunteers in single

doses up to 40 mg when administered in the fasting state.

Headache and dizziness were observed in some subjects given

a single dose of 60 mg after an overnight fast.

20m was also well tolerated when titrated up to doses of 150

mg daily in the multiple-dose studies when subjects initiated

dosing at a dose level below 60 mg. There were no clinically

meaningful changes observed in 12-lead electrocardiogram

(ECG) intervals, clinical laboratory results, or vital signs.

As described above, the lead optimization process that led

from the lead compound 8 to the clinical candidate 20m and

its backup candidate 20o was in silico-driven, adopting a

structure-based approach which relied on in silico generated 3D

models of the target receptors. The total number of compounds

synthesized in this project was only 31 compounds, representing

approximately 10% of the number of virtual compounds that

were computationally evaluated during the optimization process.

The whole optimization process took less than 6 months from

start to end, engaging only three medicinal chemists working

together with two to three computational chemists fully dedi-

cated to the project. This 1:1 ratio of medicinal to computational

chemists is a key aspect of the drug discovery paradigm

developed at Predix. This team structure is essential for enabling

a thorough in silico analysis of each suggested compound, which

leads to focused prioritization of compound synthesis that in

turn translates to a more efficient drug discovery process. The

discovery and optimization processes detailed here have cul-

minated in a safe and tolerable drug candidate with a desirable

PK profile that is undergoing a Phase III study in GAD, all

within 30 months.

Conclusion

In conclusion, using an in silico model-driven and structure-

based approach we have discovered a novel series of potent

and selective acylaminophenylpiperazinyl sulfonamide-based

5-HT_1Aagonists that exhibit the desired oral bioavailability,

safety, and efficacy in preclinical animal studies. On the basis

of in vitro and in vivo potency, efficacy, and pharmacokinetic

characteristics, 20m, N-(3-(4-(4-(cyclohexylmethylsulfonamido)-

butyl)piperazin-1-yl)phenyl)acetamide, was chosen as the clini-

cal development candidate and 20o, N-(3-(4-(4-(2-methyl-

propylsulfonamido)butyl)piperazin-1-yl)phenyl)acetamide, was

selected as the back-up candidate for the treatment of anxiety,

depression, and ADHD.

In Phase I human clinical trials, 20m (once daily and 28 day

multiple doses up to 60 mg) was well tolerated showing an

excellent side-effect profile with no effect on cardiovascular or

electrocardiographic parameters, as expected from a selective

5-HT_1Areceptor agonist.

It is important to note that the favorable clinical properties

of 20m that were seen in the Phase I clinical studiess

tolerability, pharmacokinetics, and pharmacodynamics-can be

directly attributed to the structure-based optimization process

described in this paper:

•Tolerability: 20m is the product of a structure-based in

silico-driven optimization process that rationally and efficiently

minimized cross-reactivity to other receptors including R₁-

adrenergic receptor (R₁-AR), R₂-AR, and the hERG K⁺channel,

leading to a potent, selective, full agonist of 5-HT_1A. This in

vitro selectivity profile correlates well with the favorable clinical

safety and tolerability profile of 20m, as observed in 84 healthy

volunteers in three Phase I studies.

Experimental Section

GPCR Modeling. The PREDICT algorithm^45-47searches through

the receptors’ conformational space for the most stable 3D

structure(s) of the transmembrane (TM) domain of the GPCR

protein within the membrane environment. To ensure that the final

model represents the most stable conformation, the method

simultaneously optimizes several thousand alternative conformations

of the receptor. Following two successive application of the

PREDICT algorithm (coarse modeling followed by fine modeling),

the resulting model is subjected to molecular dynamics refinement

using CHARMM⁵⁸and the CHARMM22 force field.⁵⁹Multiple

constraints are applied during the simulations to ensure that the

model does not deviate significantly from the initial PREDICT

model. These refinement dynamics introduce helical kinks and relax

the side chain conformations. Finally, a protein-ligand complex

is carefully constructed through molecular dynamics.

The 3D model of the 5-HT_1Areceptor and the R₁receptor was

modeled by the PREDICT program, following the procedure that

was reported in detail in refs 45-47. Briefly, following two

successive application of the PREDICT algorithm (coarse modeling

followed by fine modeling), the resulting model was subjected to

molecular dynamics refinement using CHARMM⁵⁸and the

CHARMM22 force field.⁵⁹Multiple constraints were applied during

the simulations to ensure that the model does not deviate signifi-

cantly from the initial PREDICT model. Finally, a protein-ligand

complex is carefully constructed through molecular dynamics.

Screening Library. The screening library consists of virtual 2D

representations of approximately 2 100 000 drug-like compounds,

obtained from electronic catalogs of more than 20 vendors

worldwide. The library is updated quarterly, so that at any given

•Pharmacokinetics: The pharmacokinetic profile of the

target compound was one of the most important parameters in

the optimization process, leading to 20m which shows a

preclinical PK profile with a serum half-life approximately 3

times longer than, and oral bioavailability superior to, buspirone.

This preclinical profile translated to an excellent PK profile in

SelectiVe 5-HT_1AAgonist

Journal of Medicinal Chemistry, 2006, Vol. 49, No. 11 3129

time, it represents compounds that can be purchased on short notice.

The multiple sources of the library ensure its diversity, allowing

us to explore broad chemical spaces.

t (triplet), q (quartet), m (multiplet), and sb (singlet broad). Flash

chromatography was performed in columns of various diameters

with Merck silica gel (230-400 mesh) by elution with solvent

systems indicated in the parentheses. Positive electrospray ionization

(ESI) mass spectra were determined on a Finnigan LAD00416 mass

spectrometer. Elemental analyses were carried out by Atlantic

Microlab, Inc., and were within (0.4% of the theoretical value.

Melting points were performed on a Mettler-Toledo FP62 instru-

ment. The instrument uses optical detection when determining the

sample melting point.

1-(3-Nitro-phenyl)-piperazine (11). A mixture of 3-nitro-

phenylamine (10) (13.8 g, 0.1 mol), bis-(2-chloro-ethyl)-amine

hydrochloride salt (17.5 g, 0.1 mol), and potassium carbonate (48.3

g, 0.35 mol) in chlorobenzene (300 mL) was refluxed for 48 h.

The mixture was poured into water (300 mL). The organic layer

was separated, and the water phase was extracted with dichloro-

methane (3 × 150 mL). The combined organic phases was dried

over sodium carbonate, filtered, concentrated under pressure, and

purified with flash chromatography on silica gel (eluent 2% MeOH

in CH₂Cl₂) to give 9.3 g of the titled compound 11 (45% yield).

¹H NMR (CDCl₃): δ 7.71 (s, 1H), 7.65 (d, J ) 8.0 Hz, 1H), 7.39-

7.35 (m, 1H), 7.19 (d, J ) 8.4 Hz, 1H), 3.25-3.23 (m, 4H), 3.07-

3.04 (m, 4H), 1.68 (s, 1H); MS (ESI): m/z 208 (M + H)⁺.

Toluene-4-sulfonic Acid 4-(Toluene-4-sulfonylamino)-butyl

Ester (13a). To an ice-cooled solution of 4-amino-butan-1-ol (12a)

(1.0 g, 11.0 mmol) in anhydrous methylene chloride (25 mL) under

N₂was added Et₃N (6.1 mL, 44.0 mmol), followed by tosyl chloride

(4.6 g, 24.2 mmol). The mixture was stirred at 0 °C for 3 h and

then quenched with cold aqueous saturated NaHCO₃solution (25

mL), and the organic phase was washed with water (3 × 25 mL),

dried over Na₂SO₄, and filtered. Solvent was removed under reduced

pressure to give a brownish-red residue. The crude material was

used for next step without further purification. MS (ESI): m/z 398

(M + H)⁺.

4-Methyl-N-{4-[4-(3-nitro-phenyl)-piperazin-1-yl]-butyl}-ben-

zenesulfonamide (18a). A mixture of 1-(3-nitro-phenyl)-piperazine

(11) (2.07 g, 10 mmol), 13a (3.93 g, 10 mmol), and triethylamine

(2.02 g, 20 mmol) in THF (100 mL) was stirred under nitrogen at

ambient temperature for 48 h. The reaction mixture was diluted

with dichloromethane, washed with 10% aqueous sodium carbonate

solution (2 × 100 mL) and water (2 × 100 mL), and dried over

sodium sulfate. After removal of sodium sulfate, solvent was

evaporated under reduced pressure. The residue was purified by

flash chromatography on silica gel (eluent 2% MeOH in CH₂Cl₂)

to give 3.3 g (76% yield) of the title compound as a white solid.

¹H NMR (CDCl₃): δ 7.74 7.66 (m, 4H), 7.40 (t, J ) 8.4 Hz, 1H),

7.29 (d, J ) 7.6 Hz, 2H), 7.22-7.20 (m, 1H), 3.34-3.2 (m, 4H),

2.99 (t, J ) 4.4 Hz, 1H), 2.63-2.61 (m, 4H), 2.42 (s, 3H), 2.39 (t,

J ) 5.6 Hz, 2H), 1.61-1.59 (m, 4H); MS (ESI): m/z 433 (M +

H)⁺.

Library Preparation. The virtual compound library is processed

prior to docking. This step includes 2D to 3D conversion (using

CONCORD),⁶⁰assignment of Gasteiger atom types,⁶¹assignment

of atomic charges, identification of multiple anchors for docking

(using HYPERION software from Predix), generating multiple

conformations for these anchors (using CONFORT),⁶²and more.

Approximately 10% of the full library (on the order of 150 000

compounds) is selected for screening, according to the character

of the binding site (charges, polar, or hydrophobic), and to the

desired range of molecular weights, compound diversity, drug-like

properties, etc.

In Silico Screening. The screening library was docked into the

binding site using the DOCK4.0.^63,64Docking for each compound

(often represented by five or more different anchor conformations)

is repeated 10 times. The specific docking parameters are fine-

tuned for each target separately, by optimizing the docking of a

small set of known binders (if available) relative to a large drug-

like random compound library.

Scoring and Selection. A sequential application of several

scoring tools and selection criteria is used, until a list of fewer than

100 virtual hits is reached (Marantz et al, manuscript in preparation).

First, an automated binding-mode analysis (using the Predix

program BMA) is performed on all docked conformations to ensure

proper docking. Ten additional 3D scores are calculated for the

top 10% of the library based on the best DOCK scores that pass

BMA (using DOCK,⁶⁴CSCORE,⁶⁵and CHARMM⁵⁸). Specific

score cutoff values or combination cutoff values are used to further

narrow the list of virtual hits. Finally, the remaining compounds in

the hit list are clustered, with only the best-scored representative-

(s) of the similarity cluster selected for in vitro testing.

Modeling the hERG Ion Channel. The S5-S6 region of the

hERG channel was modeled using the structure of the closed KcsA

ion channel (PDB code 1BL8),⁵⁵which only two transmembrane

domains, as a template. The sequence alignment between the pore

regions of KcsA (residues 43-119) and hERG (residues 572-668)

was based on the partial alignment published by Doyle et al.⁵⁵and

previously utilized by Mitcheson et al.⁵⁴Initially, all KcsA residues

without identical hERG counterparts were manually mutated to fit

the hERG sequence using the Sybyl Biopolymer module (version

6.8; Tripos Inc.: St. Louis, MO). This process distorted the structure

of some proline residues, which were subsequently fixed with the

“Fix Proline” function in the same Sybyl module. The structure

was then energy-minimized with a fixed backbone constraint using

the Tripos force field and the Powell minimization algorithm set

to terminate at an energy gradient of 0.01 kcal/(mol‚Å²). Following

minimization, all rotamers were optimized using the “rotamer

optimization” function in the InsightII Biopolymer module (Release

2000.1; Accelrys Inc., San Diego, CA).

The channel pore structure was further refined using a molecular-

dynamics-based virtual complexation process with astemizole

(Hismanal), a drug that was found to be an extremely potent hERG-

channel blocker (IC₅₀) 0.9 nM) causing QT-prolongation (astemi-

zole was removed from the market in 1998 due to its potential

hazards). Astemizole was manually docked into the channel pore

region of the hERG model using InsightII. Molecular dynamics

simulations were performed using CHARMm⁵⁸and the CFF force

field,⁶⁶following a procedure described in detail elsewhere, where

a detailed analysis of the model can also be found (Kalid O. et al.,

manuscript in preparation).

Chemistry. All reagents used were obtained from commercially

available sources. Solvents used in reaction were purchased from

Sigma-Aldrich and Alfa Aesar while solvents for chromatography

were obtained from VWR. ¹H nuclear magnetic resonance (NMR)

spectra were recorded at 400 MHz on a Varian AS400 spectrometer

and referenced to the NMR solvent. Chemical shifts were reported

in parts per million (ppm, δ) relative to an internal standard

(tetramethylsilane) with coupling constants given in hertz (Hz, J).

¹H NMR multiplicity data are denoted by s (singlet), d (doublet),

N-(3-{4-[4-(Toluene-4-sulfonylamino)-butyl]-piperazin-1-yl}-

phenyl)-acetamide (9). This compound (as a colorless oil-like

liquid) was prepared by using 1-phenyl-piperazine and 12a in the

same manner as compound 18a, described above, and was purified

by silica gel chromatography (eluent 1% MeOH in CH₂Cl₂) (97%

1

yield); H NMR (CDCl₃): δ 7.96 (d, J ) 8.0 Hz, 2H), 7.60 (d, J

) 7.6 Hz, 1H), 7.46-7.42 (m, 3H), 7.04 (d, J ) 8.0 Hz, 1H), 6.90-

6.86 (m, 1H), 3.35-3.30 (m, 4H), 3.06-3.02 (m, 2H), 2.70-2.66

(m, 4H), 2.46 (s, 3H), 2.42 (t, J ) 5.4 Hz, 2H), 2.28 (s, 3H), 1.62-

1.66 (m, 4H); MS (ESI): m/z 445 (M + H)⁺. Anal. (C₂₁H₂₉N₃O₂S)

C, H, N.

N-{4-[4-(3-Amino-phenyl)-piperazin-1-yl]-butyl}-4-methyl-

benzenesulfonamide (19a). To a solution of tin chloride (3.5 g,

18.5 mmol) in methanol (50 mL) and concentrated hydrochloric

acid (10 mL) was added a solution of 18a (800 mg, 1.85 mmol) in

methanol (50 mL) in one portion at -10°C. The reaction mixture

was allowed to warm to ambient temperature and stirred until the

reaction was completed as determined by TLC. The reaction was

quenched by adding 10% sodium carbonate solution (200 mL) and

extracted with dichloromethane (3 × 200 mL). The combined

extracts were dried over sodium sulfate and evaporated under

3130 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 11

Becker et al.

reduced pressure. The residue was separated by flash chromatog-

raphy on silica gel (eluent 2% MeOH in CH₂Cl₂) to give the titled

compound (670 mg, 90% yield). H NMR (CDCl₃): δ 7.75 (d, J

) 8.4 Hz, 2H), 7.29 (d, J ) 8.4 Hz, 2H), 7.09 (t, J ) 8.0 Hz, 1H),

6.41 (d, J ) 8.0 Hz, 1H), 6.30 (s, 1H), 6.28 (d, J ) 8.0 Hz, 1H),

3.65 (sb, 2H), 3.25-3.22 (m, 4H), 3.00 (t, J ) 5.6 Hz, 2H), 2.63-

2.61 (m, 4H), 2.45 (s, 3H), 2.41 (t, J ) 5.6 Hz, 2H), 1.64-1.60

(m, 4H); MS (ESI): m/z 403 (M + H)⁺.

N-(3-{4-[4-(Toluene-4-sulfonylamino)-butyl]-piperazin-1-yl}-

phenyl)-butyramide (20f). This compound (as a white solid) was

prepared by using 19a and butyryl chloride in the same manner as

compound 20a, described above, and was purified by silica gel

1

chromatography (eluent 1% MeOH in CH₂Cl₂) (91% yield); H

NMR (CDCl₃): δ 7.70 (d, J ) 8.0 Hz, 2H), 7.36 (s, 1H), 7.27-

7.16 (m, 4H), 6.87 (d, J ) 7.6 Hz, 1H), 6.66 (d, J ) 8.4 Hz, 1H),

3.24-3.22 (m, 4H), 2.97-2.95 (m, 1H), 2.58-2.56 (m, 4H), 2.40

(s, 3H), 2.37-2.32 (m, 4H), 1.79-1.73 (m, 2H), 1.58-1.57 (m,

4H), 1.01 (t, J ) 7.6 Hz, 3H). MS (ESI): m/z 473 (M + H)⁺.

Anal. (C₂₅H₃₆N₄O₃S) C, H, N.

N-{4-[4-(3-Methanesulfonylamino-phenyl)-piperazin-1-yl]-but-

yl}-4-methyl-benzenesulfonamide (20a). To a mixture of com-

pound 19a (31 mg, 0.077 mmol) and triethylamine (0.5 mL) in

dichloromethane (5 mL) was added methanesulfonyl chloride (0.008

mL, 0.10 mmol) at 0 °C. The mixture was stirred at ambient

temperature for 30 min and concentrated on vacuum to dryness.

The resulting residue was diluted with dichloromethane, washed

with aqueous sodium carbonate solution and water, dried over

sodium sulfate, filtered, and concentrated. Purification by silica gel

chromatography (eluent 1% MeOH in CH₂Cl₂) gave 36 mg (97%)

N-(3-{4-[4-(Toluene-4-sulfonylamino)-butyl]-piperazin-1-yl}-

phenyl)-isobutyramide (20g). This compound (as a white solid)

was prepared by using 19a and isobutyryl chloride in the same

manner as compound 20a, described above, and was purified by

silica gel chromatography (eluent 1% MeOH in CH₂Cl₂) (92%

1

yield); H NMR (CDCl₃): δ 7.71 (d, 2H, J ) 8.4 Hz), 7.44 (m,

1H), 7.25-7.12 (m, 3H), 6.84-6.82 (m, 1H), 6.68-6.65 (m, 1H),

3.27-3.26 (m, 4H), 2.98-2.95 (m, 1H), 2.61-2.58 (m, 4H),

2.553-2.49 (m, 1H), 2.41 (s, 3H), 2.40-2.38 (m, 2H), 1.60-1.58

(m, 4H), 1.27 (s, 3H), 1.26 (s, 3H). MS (ESI): m/z 473 (M + H)⁺.

Anal. (C₂₅H₃₆N₄O₃S) C, H, N.

1

of the desired product 14 as a white solid. H NMR (CDCl₃): δ

7.71 (d, J ) 8.0 Hz, 2H), 7.27 (d, J ) 8.0 Hz, 2H), 7.24-7.20 (m,

1H), 6.81 (s, 1H), 6.74 (d, J ) 7.6 Hz, 1H), 6.67 (d, J ) 7.4 Hz,

1H), 3.28-3.22 (m, 4H), 3.01 (s, 3H), 2.96 (t, J ) 5.6 Hz, 2H),

2.62-2.58 (m, 4H), 2.41 (s, 3H), 2.37 (t, J ) 6.0 Hz, 2H), 1.61-

1.57 (m, 4H); MS (ESI): m/z 481 (M + H)⁺. Anal. (C₂₂H₃₂N₄O₄S₂)

C, H, N.

Cyclopropanecarboxylic Acid (3-{4-[4-(Toluene-4-sulfonyl-

amino)-butyl]-piperazin-1-yl}-phenyl)-amide (20h). This com-

pound (as a white solid) was prepared by using 19a and cyclopro-

panecarbonyl chloride in the same manner as compound 20a,

described above, and was purified by silica gel chromatography

N-{4-[4-(3-Ethanesulfonylamino-phenyl)-piperazin-1-yl]-but-

yl}-4-methyl-benzenesulfonamide (20b). This compound (as a

white solid) was prepared by using 19a and ethanesulfonyl chloride

in the same manner as compound 20a, described above, and was

purified by silica gel chromatography (eluent 1% MeOH in CH₂-

1

(eluent 1% MeOH in CH₂Cl₂) (75% yield); H NMR (CDCl₃): δ

7.70 (d, J ) 8.0 Hz, 2H), 7.42-7.40 (m, 2H), 7.26-7.16 (m, 2H),

6.83 (d, J ) 7.6 Hz, 1H), 6.65 (d, J ) 8.0 Hz, 1H), 3. 24-3.22

(m, 4H), 2.96 (t, J ) 5.2 Hz, 2H), 2.59-2.567 (m, 4H), 2.40 (s,

3H), 2.37 (m, 2H), 1.60-1.48 (m, 5H), 1.09-1.07 (m, 2H), 0.86-

0.83 (m, 2H). MS (ESI): m/z 471 (M + H)⁺. Anal. (C₂₅H₃₄N₄O₃S)

C, H, N.

1

Cl₂) (98% yield); H NMR (CDCl₃): 7.72 (d, J ) 8.0 Hz, 2H),

7.29 (d, J ) 8.0 Hz, 2H), 7.24-7.20 (m, 1H), 6.85-6.81 (m, 1H),

6.74 (d, J ) 7.6 Hz, 1H), 6.64 (d, J ) 7.4 Hz, 1H), 3.32-3.28 (m,

4H), 3.16-3.12 (m, 2H), 2.98-2.86 (m, 2H), 2.69-2.66 (m, 4H),

2.48-2.44 (m, 2H), 2.42 (s, 3H), 1.64-1.60 (m, 4H), 1.39-1.35

(m, 3H); MS (ESI): m/z 495 (M + H)⁺. Anal. (C₂₃H₃₄N₄O₄S₂) C,

H, N.

N-(3-(4-(4-(Tosylamino)butyl)piperazin-1-yl)phenyl)pivala-

mide (20i). This compound (as a white solid) was prepared by using

19a and pivaloyl chloride in the same manner as compound 20a,

described above, and was purified by silica gel chromatography

4-Methyl-N-(4-{4-[3-(propane-2-sulfonylamino)-phenyl]-pip-

erazin-1-yl}-butyl)-benzenesulfonamide (20c). This compound (as

a white solid) was prepared by using 19a and propane-2-sul-

fonyl chloride in the same manner as compound 20a, described

above, and was purified by silica gel chromatography (eluent 1%

MeOH in CH₂Cl₂) (93% yield); ¹H NMR (CDCl₃): δ 7.72 (d, J )

8.0 Hz, 2H), 7.26 (d, J ) 8.0 Hz, 2H), 7.20-7.16 (m, 1H), 6.85-

6.84 (m, 1H), 6.71-6.67 (m, 2H), 3.36-3.30 (m, 1H), 3.24-3.22

(m, 4H), 2.96 (t, J ) 5.4 Hz, 2H), 2.59-2.56 (m, 4H), 2.41 (s,

3H), 2.36 (t, J ) 5.2 Hz, 2H), 1.58-1.56 (m, 4H), 1.38 (d, J ) 6.8

Hz, 6H); MS (ESI): m/z 509 (M + H)⁺. Anal. (C₂₄H₃₆N₄O₄S₂) C,

H, N.

1

(eluent 1% MeOH in CH₂Cl₂) (85% yield); H NMR (CDCl₃): δ

7.71 (d, J ) 7.6 Hz, 2H), 7.44-7.35 (m, 1H), 7.29-7.26 (m, 3H),

7.19 (t, J ) 8.4 Hz, 1H), 6.82 (d, J ) 8.0 Hz, 1H), 6.67 (d, J ) 8.4

Hz, 1H), 3.25 (m, 4H), 2.96 (t, J ) 5.2 Hz, 2H), 2.60-2.56 (m,

4H), 2.40 (s, 3H), 2.36 (t, J ) 5.2 Hz, 2H), 1.59-1.58 (m, 4H),

1.32 (s, 9H); MS (ESI): m/z 487 (M + H)⁺. Anal. (C₂₆H₃₈N₄O₃S)

C, H, N.

4-Fluoro-benzenesulfonic Acid 4-(4-Fluoro-benzenesulfon-

ylamino)-butyl Ester (13b). This compound was prepared by using

12a and 4-fluorobenzene-1-sulfonyl chloride in the same manner

as compound 13a, described above, and was used for next step

without further purification. MS (ESI): m/z 406 (M + H⁺).

4-Fluoro-N-{4-[4-(3-nitro-phenyl)-piperazin-1-yl]-butyl}-ben-

zenesulfonamide (18b). This compound was prepared by using

11 and 13b in the same manner as compound 18a, described above,

and was purified by silica gel chromatography (eluent 2% MeOH

in CH₂Cl₂) (75% yield). ¹H NMR (CDCl₃): δ 7.85 (m, 2H), 7.75-

7.63 (m, 2H), 7.22-7.06 (m, 4H), 3.39-3.37 (m, 4H), 2.97-2.95

(m, 2H), 2.45-2.43 (m, 4H), 2.05-2.03 (m, 1H), 1.61-1.63 (m,

4H); MS (ESI): m/z 437 (M + H)⁺.

N-(3-{4-[4-(Toluene-4-sulfonylamino)-butyl]-piperazin-1-yl}-

phenyl)-acetamide (20d). This compound (as a white solid) was

prepared by 19a and acetyl chloride in the same manner as

compound 20a, described above, and was purified by silica gel

1

chromatography (eluent 1% MeOH in CH₂Cl₂) (97% yield); H

NMR (CDCl₃): δ 7.96 (d, J ) 8.0 Hz, 2H), 7.60 (d, J ) 7.6 Hz,

1H), 7.46-7.42 (m, 3H), 7.04 (d, J ) 8.0 Hz, 1H), 6.90-6.86 (m,

1H), 3.35-3.30 (m, 4H), 3.06-3.02 (m, 2H), 2.70-2.66 (m, 4H),

2.46 (s, 3H), 2.42 (t, J ) 5.4 Hz, 2H), 2.28 (s, 3H), 1.62-1.66 (m,

4H); MS (ESI): m/z 445 (M + H)⁺. Anal. (C₂₃H₃₂N₄O₃S) C, H,

N.

4-Fluoro-N-{4-[4-(3-amino-phenyl)-piperazin-1-yl]-butyl}-

benzenesulfonamide (19b). This compound was prepared by

reducing 18b in the same manner as compound 19a, described

above, and was purified by silica gel chromatography (eluent 2%

N-(3-{4-[4-(Toluene-4-sulfonylamino)-butyl]-piperazin-1-yl}-

phenyl)-propionamide (20e). This compound (as a white solid)

was prepared by using 19a and propionyl chloride in the same

manner as compound 20a, described above, and was purified by

silica gel chromatography (eluent 1% MeOH in CH₂Cl₂) (97%

1

MeOH in CH₂Cl₂) (yield 87%); H NMR (CDCl₃): δ 7.95 (m,

2H), 7.45-6.95 (m, 4H), 6.51-6.19 (m, 2H), 3.95-3.45 (m, 4H)

3.12-2.83 (m, 4H), 2.65-2.31 (m, 7H), 1.61 (m, 4H); MS (ESI):

m/z 407 (M + H)⁺.

1

yield); H NMR (CDCl₃): δ 8.30 (s, 1H), 7.72 (d, J ) 8.0 Hz,

2H), 7.40 (s, 1H), 7.32 (m, 3H), 6.84 (d, J ) 7.6 Hz, 1H), 6.62 (d,

J ) 7.4 Hz, 1H), 3.24-3.22 (m, 4H), 2.94 (t, J ) 5.6 Hz, 2H),

2.64-2.60 (m, 4H), 2.40 (s, 3H), 2.38 (m, 4H), 1.59(m, 4H), 1.24

(t, J ) 6.8 Hz, 3H); MS (ESI): m/z 459 (M + H)⁺. Anal.

(C₂₄H₃₄N₄O₃S) C, H, N.

N-(3-{4-[4-(4-Fluoro-benzenesulfonylamino)-butyl]-piperazin-

1-yl}-phenyl)-acetamide(20j). This compound (as a white solid)

was prepared by using 19b and acetyl chloride in the same manner

as compound 20a, described above, and was purified by silica gel

1

chromatography (eluent 1% MeOH in CH₂Cl₂) (95% yield); H

SelectiVe 5-HT_1AAgonist

Journal of Medicinal Chemistry, 2006, Vol. 49, No. 11 3131

1

NMR (CDCl₃): δ 7.45-7.16 (m, 4H), 6.81 (d, J ) 8.9 Hz, 2H),

6.65 (d, J ) 8.9 Hz 2H), 3.48 (s, 1H), 3.28 (m, 4H), 2.95 (m, 2H),

2.62 (m, 5H), 2.35 (m, 2H), 2.08 (s, 3H), 1.65 (m, 4H); MS (ESI):

m/z 449 (M + H)⁺. Anal. (C₂₂H₂₉FN₄O₃S) C, H, N.

chromatography (eluent 1% MeOH in CH₂Cl₂) (61% yield); H

NMR (CDCl₃): δ 7.76 (t, J ) 2.4 Hz, 1H), 7.71 (t, J ) 1.6 Hz,

1H), 7.40 (t, J ) 7.7 Hz, 1H), 7.22 (d, J ) 2.2 Hz, 1H), 3.49 (t, J

) 5.6 Hz, 4H), 3.21 (t, J ) 5.2 Hz, 2H), 2.88 (m, 4H), 2.57 (t, J

) 5.2 Hz, 4H), 2.45 (t, J ) 5.6 Hz, 2H), 1.96-1.20 (m, 14H); MS

(ESI): m/z 424 (M + H)⁺.

Cyclohexanesulfonic Acid {4-{4-(3-Amino-phenyl)-piperazin-

1-yl]-butyl}-amide (19d). This compound was prepared by reduc-

tion of 18d in the same manner as compound 19a, described above,

and was purified by silica gel chromatography (eluent 2% MeOH

Cyclohexyl-methanesulfonic Acid 4-(Cyclohexylmethane-

sulfonyl-methyl-amino)-butyl Ester(13c). This compound was

prepared by using 4-(methylamino)butan-1-ol (12b) and cyclo-

hexylmethanesulfonyl chloride in the same manner as compound

13a, described above, and was used for next step without further

1

purification. H NMR (CDCl₃): δ 4.23 (m, 4H), 3.29 (t, J ) 6.7

1

Hz, 4H), 3.00-2.61 (m, 8H), 2.27 (s, 3H), 1.99-1.45 (m, 18H);

in CH₂Cl₂) (75% yield); H NMR (CDCl₃): δ 7.34 (m, 1H), 7.11

MS (ESI): m/z 424 (M + H)⁺.

(m, 1H), 6.35 (m, 2H), 3.27 (t, J ) 5.6 Hz, 4H), 3.02 (t, J ) 5.2

Hz, 2H), 2.79 (m, 4H), 2.60 (t, J ) 5.2 Hz, 4H), 2.49 (t, J ) 5.6

Hz, 2H), 1.99-1.12 (m, 14H); MS (ESI): m/z 394 (M + H)⁺.

N-(3-(4-(4-(Cyclohexylsulfonylamino)butyl)piperazin-1-yl)-

phenyl)acetamide (20k). This compound was prepared by using

19d and acetyl chloride in the same manner as compound 20a,

described above, and was purified by silica gel chromatography

C-Cyclohexyl-N-methyl-N-{4-[4-(3-nitro-phenyl)-piperazin-

1-yl]-butyl}-methanesulfonamide (18c). This compound was

prepared by using 11 and 13c in the same manner as compound

18a, described above, and was purified by silica gel chromatography

1

(eluent 1% MeOH in CH₂Cl₂) (71% yield). H NMR (CDCl₃): δ

7.35 (s, 1H), 7.23 (t, J ) 7.3 Hz, 1H), 6.94 (d, J ) 6.9 Hz, 1H),

6.81 (d, J ) 7.4 Hz, 1H), 3.87 (m, 2H), 3.68 (m, 2H), 3.25-3.03

(m, 14H), 2.17 (s, 3H), 1.96-1.34 (m, 11H); MS (ESI): m/z 453

(M + H)⁺.

1

(eluent 1% MeOH in CH₂Cl₂) (95% yield); H NMR (CDCl₃): δ

7.36 (t, J ) 1.6 Hz, 1H), 7.28 (t, J ) 1.6 Hz,1H), 6.91 (d, J ) 7.7

Hz, 1H), 6.73 (d, J ) 2.2 Hz, 1H), 3.28 (t, J ) 5.6 Hz, 4H), 3.18

(t, J ) 5.2 Hz, 2H), 2.86 (m, 1H), 2.66 (t, J ) 5.2 Hz, 4H), 2.49

(t, J ) 5.6 Hz, 2H), 2.21 (s, 3H), 1.99-1.12 (m, 14H); MS (ESI):

m/z 437 (M + H)⁺. Anal. (C₂₂H36N₄O₃S) C, H, N.

N-{4-[4-(3-Amino-phenyl)-piperazin-1-yl]-butyl}-C-cyclohexyl-

N-methyl-methanesulfonamide (19c). This compound was pre-

pared by the reduction of 18c in the same manner as compound

19a, described above, and was purified by silica gel chromatography

Propane-2-sulfonic Acid {4-[4-(3-Nitro-phenyl)-piperazin-1-

yl]-butyl}-amide(18e). This compound was prepared with 4-[4-

(3-nitro-phenyl)-piperazin-1-yl]-butylamine trifluoroacetic acid salt-

(17a) and propane-2-sulfonyl chloride in the same manner as

compound 20a, described above, and was purified by silica gel

1

(eluent 2% MeOH in CH₂Cl₂) (yield 76%); H NMR (CDCl₃): δ

7.28 (s, 1H), 7.23 (t, J ) 7.1 Hz, 1H), 6.94 (d, J ) 7.0 Hz 1H),

6.30 (d, J ) 7.2 Hz, 1H), 3.85 (sb, 2H), 3.63 (m, 2H), 3.28-3.05

(m, 16H), 2.17 (s, 3H), 1.99-1.39 (m, 11H); MS (ESI): m/z 423

(M + H)⁺.

1

chromatography (eluent 1% MeOH in CH₂Cl₂) (63% yield); H

NMR (CDCl₃): δ 7.81-7.69(m, 2H), 7.44-7.22 (m, 2H), 6.60 (sb,

1H), 3.44-3.33 (m, 4H), 3.24-3.13 (m, 3H), 2.75-2.64 (m, 4H),

2.58-2.50 (m, 2H), 1.77-1.70 (m, 4H), 1.40 (d, J ) 6.8 Hz, 6H);

MS (ESI): m/z 385 (M + H)⁺.

N-(3-{4-[4-(Cyclohexylmethanesulfonyl-methyl-amino)-butyl]-

piperazin-1-yl}-phenyl)-acetamide (20p). This compound (as a

white solid) was prepared by 19c and acetyl chloride in the same

manner as compound 20a, described above, and was purified by

silica gel chromatography (eluent 1% MeOH in CH₂Cl₂) (83%

Propane-2-sulfonic Acid {4-[4-(3-Amino-phenyl)-piperazin-

1-yl]-butyl}-amide (19e). This compound was prepared by reduc-

tion of 18e in the same manner as compound 19a, described above,

and was purified by silica gel chromatography (eluent 2% MeOH

in CH₂Cl₂) (70% yield); ¹H NMR (CDCl₃): δ 7.06-7.02 (m, 1H),

6.36 (d, J ) 7.6 Hz, 1H), 6.26-6.21 (m, 2H), 3.60 (sb, 2H), 3.22-

3.01 (m, 7H), 2.63-2.61 (m, 4H), 2.50-2.44 (m, 2H), 1.70-1.64

(m, 4H), 1.34 (d, J ) 6.8 Hz, 6H); MS (ESI): m/z 355 (M + H)⁺.

N-(3-{4-[4-(Propane-2-sulfonylamino)-butyl]-piperazin-1-yl}-

phenyl)-acetamide (20l). This compound was prepared with 19e

and acetyl chloride in the same manner as compound 20a, described

above, and was purified by silica gel chromatography (eluent 1%

1

yield); H NMR (CDCl₃): δ 7.37 (s, 1H), 7.15 (t, J ) 7.4 Hz,

1H), 6.98 (d, J ) 7.2 Hz, 1H), 6.71 (d, J ) 7.3 Hz, 1H),), 3.27 (t,

J ) 6.3 Hz, 4H), 3.15 (t, J ) 6.2 Hz, 2H), 2.89 (d, J ) 6.7 Hz,

2H), 2.46 (t, J ) 6.3 Hz, 4H), 2.45 (t, J ) 6.3 Hz, 2H), 2.23 (s,

3H), 2.19 (s, 3H), 1.99 (t, J ) 6.4 Hz, 2H), 1.68 (m, 7H), 1.33-

1.02 (m, 6H); MS (ESI): m/z 465 (M + H)⁺.Anal. (C₂₄H₄₀N₄O₃S)

C, H, N.

Toluene-4-sulfonic Acid 4-tert-Butoxycarbonylamino-butyl

Ester (15a). This compound was prepared with (4-hydroxy-butyl)-

carbamic acid tert-butyl ester and tosyl chloride in the same manner

as compound 13a, described above, and was used for next step

without further purification. MS (ESI): m/z 344 (M + H)⁺.

{4-[4-(3-Nitro-phenyl)-piperazin-1-yl]-butyl}-carbamic Acid

tert-Butyl Ester (16a). This compound was prepared by alkylation

of 11 with 15a in the same manner as compound 18a, described

above, and was purified by silica gel chromatography (eluent 1%

MeOH in CH₂Cl₂) (63% yield); ¹H NMR (CDCl₃): δ 7.78 (s, 1H),

7.67 (d, J ) 8.0 Hz, 1H), 7.43-7.40 (m, 1H), 7.21 (d, J ) 7.6 Hz,

1H), 5.21 (sb, 1H), 3.39-3.21 (m, 4H), 2.69-2.67 (m, 4H), 2.24-

2.20 (m, 2H), 2.19-2.17 (m, 2H), 1.78-1.42 (m, 4H), 1.44 (s,

9H); MS (ESI): m/z 379 (M + H)⁺.

1

MeOH in CH₂Cl₂) (91% yield); H NMR (CDCl₃): δ 7.32-7.21

(m, 2H), 6.92 (d, J ) 7.6 Hz, 1H), 6.71 (d, J ) 8.0 Hz, 2H), 3.29-

3.11 (m, 7H), 2.67-2.66 (m, 4H), 2.50-2.48 (m, 2H), 2.22

(s, 3H), 1.77-1.66 (m, 4H), 1.39 (d, J ) 6.8 Hz, 6H); MS (ESI):

m/z 397 (M + H)⁺. Anal. (C₁₉H₃₂N₄O₃S) C, H, N.

C-Cyclohexyl-N-{4-[4-(3-nitro-phenyl)-piperazin-1-yl]-butyl}-

methanesulfonamide (18f). This compound was prepared with

4-[4-(3-nitro-phenyl)-piperazin-1-yl]-butylamine trifluoroacetic acid

salt (17a) and cyclohexyl-methanesulfonyl chloride in the same

manner as compound 20a, described above, and was purified by

silica gel chromatography (eluent 2% MeOH in CH₂Cl₂) (88%

yield); ¹H NMR (CDCl₃): δ 7.45 (s, 1H), 7.22-7.20 (m, 1H), 6.97

(d, J ) 8.0 Hz, 1H), 6.75 (d, J ) 7.6 Hz, 1H), 3.87-3.84 (m, 2H),

3.65-3.63 (m, 2H), 3.28-3.05 (m, 14H), 1.99-1.39 (m, 12H);

MS (ESI): m/z 439 (M + H)⁺.

N-{4-[4-(3-Amino-phenyl)-piperazin-1-yl]-butyl}-C-cyclohexyl-

methanesulfonamide (19f). This compound was prepared by the

reduction of 18f in the same manner as compound 18a, described

above, and was purified by silica gel chromatography (eluent 2%

MeOH in CH₂Cl₂) (76% yield); ¹H NMR (CDCl₃): δ 7.50 (s, 1H),

7.13-7.10 (m, 1H), 6.89 (d, J ) 8.0 Hz, 1H), 6.69 (d, J ) 7.6 Hz,

1H), 3.85 (sb, 2H), 3.65-3.63 (m, 2H), 3.35-3.02 (m, 16H), 2.01-

1.44 (m, 12H); MS (ESI): m/z 409 (M + H)⁺.

4-[4-(3-Nitro-phenyl)-piperazin-1-yl]-butylamine Trifluoro-

acetic Acid Salt (17a). To a solution of 16a (14.0 g, 0.037 mol) in

dichloromethane (100 mL) at 0°C trifluoroacetic acid (10 mL, 0.13

mol) was added. The mixture was stirred for additional 2 h. Solvent

was evaporated under reduced pressure. The residue was washed

with diethyl ether (100 mL). Precipitate was dried to obtain the

1

desired product 17a(16.85 g, 90% yield); H NMR (CD₃OD): δ

10.05 (sb, 1H), 7.79-7.42 (m, 4H), 4.07-3.48 (m, 4H), 3.25 (sb,

3H), 2.85-2.81 (m, 4H), 2.19-2.15 (m, 2H), 1.99-1.42 (m, 6H);

MS (ESI): m/z 279 (M + H)⁺.

Cyclohexanesulfonic Acid {4-{4-(3-Nitro-phenyl)-piperazin-

1-yl]-butyl}-amide (18d). This compound was prepared with 4-[4-

(3-nitro-phenyl)-piperazin-1-yl]-butylamine trifluoroacetic acid salt

(17a) and cyclohexanesulfonyl chloride in the same manner as

compound 20a, described above, and was purified by silica gel

N-{3-[4-(4-Cyclohexylmethanesulfonylamino-butyl)-piperazin-

1-yl]-phenyl}-acetamide (20m). This compound was prepared by

3132 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 11

Becker et al.

1

using 19f and acetyl chloride in the same manner as compound

20a, described above, and was purified by silica gel chromatography

(eluent 1% MeOH in CH₂Cl₂) (89% yield); mp 200.4 °C; ¹H NMR

(CDCl₃): δ 7.44 (s, 1H), 7.30 (s, 1H), 7.22-7.20 (m, 1H), 6.93

(d, J ) 7.6 Hz, 1H), 6.70 (sb, 1H), 6.68 (d, J ) 8.0 Hz, 1H), 3.28-

7.25 (m, 4H), 3.15 (t, J ) 5.2 Hz, 2H), 2.89 (d, J ) 6.0 Hz, 2H),

2.64-2.62 (m, 4H), 2.45 (t, J ) 5.6 Hz, 2H), 2.19 (s, 3H), 2.01-

1.97 (m, 2H), 1.68 (m, 7H), 1.33-1.02 (m, 6H); MS (ESI): m/z

451 (M + H)⁺. Anal. (C₂₃H₃₈N₄O₃S) C, H, N.

in CH₂Cl₂) (75% yield); H NMR (CDCl₃): δ 7.26 (s, 1H), 7.12

(t, J ) 7.3 Hz, 1H), 6.96 (d, J ) 7.4 Hz, 1H), 6.77 (d, J ) 7.2 Hz,

1H), 3.89 (m, 2H), 3.65 (m, 2H), 3.29-3.02 (m, 14H), 2.17 (s,

3H), 1.97-1.34 (m, 7H); MS (ESI): m/z 413 (M + H)⁺.

2-Methyl-propane-1-sulfonic Acid {4-[4-(3-Amino-phenyl)-

piperazin-1-yl]-butyl}-methyl-amide (19h). This compound was

prepared by the reduction of 18h in the same manner as compound

19a, described above, and was purified by silica gel chromatography

1

(eluent 2% MeOH in CH₂Cl₂) (73% yield); H NMR (CDCl₃): δ

Cyclopropanecarboxylic Acid {3-[4-(4-Cyclohexylmethane-

sulfonylamino-butyl)-piperazin-1-yl]-phenyl}-amide (20n). This

compound was prepared by using 19f and cyclopropanecarbonyl

chloride in the same manner as compound 20a, described above,

and was purified by silica gel chromatography (eluent 1% MeOH

in CH₂Cl₂) (89% yield); ¹H NMR (CDCl₃): δ 7.41-7.17 (m, 2H),

6.82 (d, J ) 7.6 Hz, 1H), 6.65 (d, J ) 8.0 Hz, 1H), 3.26-3.22 (m,

4H), 3.07-3.03 (m, 4H), 2.89-2.85 (m, 4H), 2.64-2.60 (m, 4H),

2.43-2.40 (m, 2H) 1.95-1.49 (m, 13H), 1.38-0.91 (m, 5H); MS

(APCI): m/z 477 (M + H)⁺. Anal. (C₂₅H₄₀N₄O₃S) C, H, N.

2-Methyl-propane-1-sulfonic Acid {4-[4-(3-Nitro-phenyl)-pip-

erazin-1-yl]-butyl}-amide (18g). This compound was prepared

with 4-[4-(3-nitro-phenyl)-piperazin-1-yl]-butylamine trifluoroacetic

acid salt (17a) and 2-methyl-propane-1-sulfonyl chloride in the same

manner as compound 20a, described above, and was used for next

step without further purification; MS (ESI): m/z 399 (M + H)⁺.

2-Methyl-propane-1-sulfonic Acid {4-[4-(3-Amino-phenyl)-

piperazin-1-yl]-butyl}-amide (19g). This compound was prepared

by reducing 18g in the same manner as compound 19a, described

above, and was purified by silica gel chromatography (eluent 2%

MeOH in CH₂Cl₂) (79% yield); ¹H NMR (CDCl₃): δ 7.05-7.01-

(m, 1H), 6.37-6.34(m, 1H), 6.25-6.20(m, 2H), 3.21-3.18 (m, 4H),

3.08 (t, J ) 5.6 Hz, 2H), 2.85 (d, J ) 6.4 Hz, 2H), 2.61-2.85 (m,

4H), 2.42 (t, J ) 6.4 Hz, 2H), 2.27-2.16 (m, 1H), 1.68-1.61 (m,

4H), 1.08 (d, J ) 7.2 Hz, 6H); MS (ESI): m/z 369 (M + H)⁺.

N-(3-{4-[4-(2-Methyl-propane-1-sulfonylamino)-butyl]-piper-

azin-1-yl}-phenyl)-acetamide (20o). This compound was prepared

with 19g and acetyl chloride in the same manner as compound 20a,

described above, and was purified by silica gel chromatography

7.27 (s, 1H), 7.13 (t, J ) 7.5 Hz, 1H), 6.95 (d, J ) 7.4 Hz, 1H),

6.74 (d, J ) 7.5 Hz, 1H), 3.95 (sb, 2H), 3.21-3.18 (m, 4H), 3.08

(t, J ) 6.3 Hz, 2H), 2.85 (d, J ) 6.0 Hz, 2H), 2.61-2.85 (m, 4H),

2.42 (t, J ) 6.3 Hz, 2H), 2.29 (s, 3H), 2.27-2.16 (m, 1H), 1.68-

1.61 (m, 4H), 1.08 (d, J ) 6.4 Hz, 6H); MS (ESI): m/z 383 (M +

H)⁺.

N-[3-(4-{4-[Methyl-(2-methyl-propane-1-sulfonyl)-amino]-

butyl}-piperazin-1-yl)-phenyl]-acetamide (20q). This compound

was prepared with 19h and acetyl chloride in the same manner as

compound 20a, described above, and was purified by silica gel

1

chromatography (eluent 1% MeOH in CH₂Cl₂) (91% yield); H

NMR (CDCl₃): δ 7.47 (s, 1H), 7.23 (m, 1H), 6.98 (d, J ) 7.4 Hz,

1H), 6.73 (d, J ) 8 Hz, 1H),), 3.21-3.18 (m, 4H), 3.12 (t, J ) 6.7

Hz, 2H), 2.82 (d, J ) 6.3 Hz, 2H), 2.63-2.60 (m, 4H), 2.45 (t, J

) 6.2 Hz, 2H),2.31 (s, 3H), 2.27-2.20 (m, 1H), 1.68-1.61 (m,

4H), 1.08 (d, J ) 6.4 Hz, 6H); MS (ESI): m/z 425 (M + H)⁺.

Anal. (C₂₁H₃₆N₄O₃S) C, H, N.

4-(Toluene-4-sulfonyloxymethyl)-piperidine-1-carboxylic Acid

tert-Butyl Ester (23). This compound was prepared with 4-hy-

droxymethyl-piperidine-1-carboxylic acid tert-butyl ester (22) and

tosyl chloride in the same manner as compound 13a, described

above, and was used for next step without further purification. MS

(ESI): m/z 370 (M + H)⁺.

4-[4-(3-Nitro-phenyl)-piperazin-1-ylmethyl]-piperidine-1-car-

boxylic Acid tert-butyl Ester (24). This compound was prepared

with 23 and 11 in the same manner as compound 18a, described

above, and was purified by silica gel chromatography (eluent 1%

1

MeOH in CH₂Cl₂) (92% yield); H NMR (CDCl₃): δ 7.30-6.69

(m, 4 H), 3.80-3.61 (m, 4 H), 3.21-3.19 (m, 4 H), 2.51-2.49

(m, 4 H), 2.23 (d, J ) 7.6 Hz, 2 H), 1.80-1.59 (m, 5 H), 1.39 (s,

9 H); MS (ESI): m/z 405 (M + H)⁺.

1

(eluent 1% MeOH in CH₂Cl₂) (93% yield); H NMR (CDCl₃): δ

7.21 (s, 1H), 7.04-7.00 (m, 1H), 6.87 (d, J ) 7.2 Hz, 1H), 6.66

(d, J ) 8.4 Hz, 1H), 3.21-3.18 (m, 4H), 3.08 (t, J ) 6.0 Hz, 2H),

2.85 (d, J ) 6.4 Hz, 2H), 2.61-2.59 (m, 4H), 2.42 (t, J ) 6.0 Hz,

2H), 2.27-2.20 (m, 1H), 1.68-1.61 (m, 4H), 1.08 (d, J ) 6.8 Hz,

6H); MS (ESI): m/z 411 (M + H)⁺. Anal. (C₂₀H₃₄N₄O₃S) C, H,

N.

1-(3-Nitro-phenyl)-4-piperidin-4-ylmethyl-piperazine Tri-

fluoroacetic Acid Salt (25). This compound was prepared with

24 in the same manner as compound 17a, described above, and

was used for next step without further purification; MS (ESI): m/z

305 (M + H)⁺.

Toluene-4-sulfonic Acid 4-(tert-Butoxycarbonyl-methyl-amino)-

butyl Ester (15b). This compound was prepared with tert-butyl

4-hydroxybutylmethylcarbamate and tosyl chloride in the same

manner as compound 13a, described above, and was used for next

step without further purification. MS (ESI): m/z 358 (M + H)⁺.

Methyl-{4-[4-(3-nitro-phenyl)-piperazin-1-yl]-butyl}-carbam-

ic Acid tert-Butyl Ester(16b). This compound was prepared with

11 and 15b in the same manner as compound 18a, described above,

and was purified by silica gel chromatography (eluent 1% MeOH

1-(1-Cyclohexylmethanesulfonyl-piperidin-4-ylmethyl)-4-(3-

nitrophenyl)-piperizine (26a). This compound was prepared with

25 and cyclohexyl-methanesulfonyl chloride in the same manner

as compound 20a, described above, and was purified by silica gel

1

chromatography (eluent 1% MeOH in CH₂Cl₂) (90% yield); H

NMR (CDCl₃): δ 7.30-6.66 (m, 4 H), 3.90-3.52 (m, 2 H), 3.22-

3.19 (m, 4 H), 3.02 (d, J ) 6.4 Hz, 2 H), 2.51-2.49 (m, 4 H), 2.21

(d, J ) 7.6 Hz, 2 H), 1.98-1.22 (m, 16 H); MS (ESI): m/z 465

(M + H)⁺.

1

in CH₂Cl₂) (51% yield); H NMR (CDCl₃): δ 7.75 (s, 1H), 7.63

3-[4-[(1-Cyclohexylmethanesulfonyl-piperidin-4-ylmethyl)-

piperazin-1-yl]-phenylamine (27a). This compound was prepared

by the reduction of 26a in the same manner as compound 19a,

described above, and was purified by silica gel chromatography

(m, 1H), 7.34 (m, 1H), 7.19 (m, 1H), 3.47 (m, 4H), 2.68 (m, 4H),

2.37 (s, 3H), 2.23 (m, 2H), 2.17 (m, 2H), 1.88-1.45 (m, 4H), 1.44

(s, 9H); MS (ESI): m/z 393 (M + H)⁺.

Methyl-{4-[4-(3-nitro-phenyl)-piperazin-1-yl]-butyl}-amine Tri-

fluoroacetic Acid Salt (17b). This compound was prepared with

16b in the same manner as compound 17a, described above, and

was used for next step without further purification; ¹H NMR (CD₃-

OD): δ 7.75 (s, 1H), 7.53 (m, 1H), 7.25 (m, 1H), 7.09 (m, 1H),

4.56 (sb, 3H), 3.48 (m, 4H), 2.78 (m, 4H), 2.27 (s, 3H), 2.13 (m,

2H), 1.99-1.45 (m, 6H); MS (ESI): m/z 293 (M + H)⁺.

2-Methyl-propane-1-sulfonic Acid Methyl-{4-[4-(3-nitro-

phenyl)-piperazin-1-yl]-butyl}-amide (18h). This compound was

prepared with methyl-{4-[4-(3-nitro-phenyl)-piperazin-1-yl]-butyl}-

amine trifluoroacetic acid salt (17b)and 2-methyl-propane-1-sulfonyl

chloride in the same manner as compound 20a, described above,

and was purified by silica gel chromatography (eluent 1% MeOH

1

(eluent 2% MeOH in CH₂Cl₂) (95% yield); H NMR (CDCl₃): δ

7.79-7.77 (m, 1 H), 7.30-7.39 (m, 3 H), 3.88-3.85 (m, 2 H),

3.61 (br s, 2 H), 3.22-3.19 (m, 4 H), 3.02 (d, J ) 6.4 Hz, 2 H),

2.51-2.48 (m, 4 H), 2.20 (d, J ) 7.6 Hz, 2 H), 1.90-1.20 (m, 16

H); MS (ESI): m/z 435 (M + H)⁺.

N-{3-[4-(1-Cyclohexylmethanesulfonyl-piperidin-4-ylmethyl)-

piperizine-1-yl]-phenyl}-acetamide (21a). This compound was

prepared with 27a and acetyl chloride in the same manner as

compound 20a, described above, and was purified by silica gel

1

chromatography (eluent 1% MeOH in CH₂Cl₂) (91% yield); H

NMR (CDCl₃): δ 7.30-6.60 (m, 4 H), 3.87-3.50 (m, 4 H), 3.21-

3.19 (m, 4 H), 3.02 (d, J ) 6.4 Hz, 2 H), 2.52-2.50 (m, 4 H), 2.22

SelectiVe 5-HT_1AAgonist

Journal of Medicinal Chemistry, 2006, Vol. 49, No. 11 3133

(d, J ) 7.6 Hz, 2 H), 2.15 (s, 3 H), 2.00-1.20 (m, 16 H); MS

(ESI): m/z 477 (M + H)⁺. Anal. (C₂₅H₄₀N₄O₃S) C, H, N.

1-(3-Nitro-phenyl)-4-[1-(propane-2-sulfonyl)-piperidin-4-yl-

methyl)-4-piperizine (26b). This compound was prepared with 25

and propane-2-sulfonyl chloride in the same manner as compound

20a, described above, and was purified by silica gel chromatography

cases where >50% displacement was observed, specific K_ivalues

were determined (n ) 2). 67 binding sites were examined, including

other 5-HT receptors, biogenic amine receptors, other GPCRs, ion

channels, and transporters. The majority of assays were performed

using a human recombinant receptor. Where a human assay system

was unavailable, receptor binding was performed in tissue from

mouse, rat, guinea pig, chicken, or bovine source.

1

(eluent 1% MeOH in CH₂Cl₂) (96% yield); H NMR (CDCl₃): δ

7.30-6.61 (m, 4 H), 3.86-3.20 (m, 4 H), 3.23-3.19 (m, 4 H),

3.25-3.23 (m, 1 H), 2.53-2.51 (m, 4 H), 2.21 (d, J ) 7.6 Hz, 2

H), 1.88-1.41 (m, 5 H), 1.39 (d, J ) 6.4 Hz, 6H); MS (ESI): m/z

411.3 (M + H)⁺.

[³⁵S]GTPγS Binding Studies. Studies were performed by MDS

Pharma Services (Taiwan, Republic of China). Assays were

performed in triplicates (n ) 3) and nonspecific binding was

determined in the presence of 100 µM GTPγS.

3-{4-[(1-(Propane-2-sulfonyl)-piperidin-4-ylmethyl]-piperazin-

1-yl}-phenylamine (27b). This compound was prepared by the

reduction of 26b in the same manner as compound 19a, described

above, and was purified by silica gel chromatography (eluent 2%

hERG Binding Assay. The ability to block the hERG (K⁺)

channel was evaluated in HEK-293 cells stably expressing hERG

(human ether-a-go-go related gene) by the whole-cell patch clamp

method. The degree of inhibition (%) was obtained by measuring

the tail current amplitude before and after drug perfusion at 1 µM

(n ) 2).

HLM Microsomes. The metabolic stability of compounds in

the presence of human liver microsomes was evaluated at 5 µM,

measured by LC/MS following 0, 15, 30, 45, 60, and 90 min of

incubation with human liver microsomes (1.0 mg protein/mL) at

37°C.

1

MeOH in CH₂Cl₂) (95% yield); H NMR (CDCl₃): δ 7.78-7.77

(m, 1 H), 7.31-6.61 (m,3 H), 3.86-3.20 (m, 4 H), 3.23-3.19 (m,

4 H), 3.24-3.22 (m, 1 H), 2.53-2.50 (m, 4 H), 2.21 (d, J ) 7.6

Hz, 2 H), 1.87-1.40 (m, 5 H), 1.39 (d, J ) 6.4 Hz, 6H); MS

(ESI): m/z 381 (M + H)⁺.

N-(3-{4-[1-(Propane-2-sulfonyl)-piperidin-4-ylmethyl]-piper-

izine-1-yl}-phenyl)-acetamide (21b). This compound was prepared

with 27b and acetyl chloride in the same manner as compound

20a, described above, and was purified by silica gel chromatography

LC/MS/MS metabolite identification was performed on the 30

min time point samples.

1

(eluent 1% MeOH in CH₂Cl₂) (92% yield); H NMR (CDCl₃): δ

S9 Assay. The metabolic stability was evaluated in the presence

of human liver S9 fraction (with 2 mM NADPH) at a concentration

of 5 µM, measured by LC/MS following 0, 15, 30, 45, 60, and 90

min of incubation with pooled human liver S9 fraction (0.25-10

mg protein/mL) at 37 °C.

7.32-6.60 (m, 4 H), 3.88-3.22 (m, 4 H), 3.22-3.19 (m, 4 H),

3.25-3.22 (m, 1 H), 2.54-2.52 (m, 4 H), 2.21 (d, J ) 7.6 Hz, 2

H), 2.16 (s, 3 H), 1.87-1.41 (m, 5 H), 1.38 (d, J ) 6.4 Hz, 6H);

MS (ESI): m/z 423 (M + H)⁺. Anal. (C₂₁H₃₄N₄O₃S) C, H, N.

1-[1-(4-Fluoro-benzenesulfonyl)-piperidin-4-ylmethyl]-4-(3-ni-

tro-phenyl)-piperazine (26c). This compound was prepared with

25 and 4-fluoro-benzenesulfonyl chloride in the same manner as

compound 20a, described above, and was purified by silica gel

Cytochrome P450 Isoenzyme Inhibition. The inhibition of

human recombinant cytochrome P450 enzyme activity (isoenzymes

1A2, 2C9, 2C19, 2D6, and 3A4) was determined in this assay.

Enzyme activity was measured using fluorogenic substrates

{3-cyano-7-ethoxycoumarin, 7-methoxy-4-trifluoromethylcoumarin,

3-[2-(N,N-diethyl-N-methylamino)ethyl]-7-methoxy-4-methyl-

coumarin, 7-benzyloxyquinoline, and 7-benzyloxy-4-(trifluorometh-

yl)-coumarin} in the absence and presence of the compound. The

isoenzyme incubations were performed in the presence of an

NADPH regenerating system (glucose-6-phosphate/glucose-6-

phosphate dehydrogenase) at approximately 20 °C for 30 min over

a range of 10 concentrations (at 1:4 serial dilutions from 30 mM).

The IC₅₀values were calculated using a standard 4 parameter curve

fit.

1

chromatography (eluent 1% MeOH in CH₂Cl₂) (95% yield); H

NMR (CDCl₃): δ 7.79-7.75 (m, 2H), 7.27-7.18 (m, 3 H), 6.40-

6.38 (m, 3 H), 3.22-3.20 (m, 4 H), 2.52-2.49 (m, 4 H), 2.20 (d,

J ) 7.6 Hz, 2 H), 1.88-1.39 (m, 5 H); MS (ESI): m/z 463 (M +

H)⁺.

3-{4-[(4-Fluoro-benzenesulfonyl)-piperidin-4-ylmethyl]-pip-

erazin-1-yl}-phenylamine (27c). This compound was prepared by

the reduction of 26c in the same manner as compound 19a,

described above, and was purified by silica gel chromatography

1

(eluent 2% MeOH in CH₂Cl₂) (97% yield); H NMR (CDCl₃): δ

7.81-7.99 (m, 2H), 7.24-7.00 (m, 3 H), 6.39-6.20 (m, 3 H),

3.81-3.79 (m, 2 H), 3.60 (br s, 2 H), 3.22-3.20 (m, 4 H), 2.52-

2.49 (m. 4 H), 2.25-2.22 (m, 2 H), 2.20 (d, J ) 7.6 Hz, 2 H),

1.88-1.39 (m, 5 H); MS (ESI): m/z 433 (M + H)⁺.

Human Hepatocytes Stability Assay. The metabolic stability

in the presence of human hepatocytes was evaluated at a concentra-

tion of 5 µM, measured by LC/MS following 0, 30, 60, 90, 120,

and 180 min of incubation with human cryopreserved hepatocytes

at 37 °C.

In Vivo Studies. Drugs. 20m (Lot # DC-006-022-L2,

C₂₃H₃₈N₄O₃S₂‚HCl, doses expressed in mg of salt), buspirone (3,

10, and 20 mg/kg, Sigma, Lot # 101H0402). All compounds were

dissolved in sterile injectable water, which served as the vehicle

control. All solutions were prepared on the day of the experiments.

20m was given to animals orally (po) in a volume of 10 mL/kg

body weight; buspirone was given to animals intraperitoneally (ip).

Test compounds were administered to the animals one hr before

testing.

N-(3-{4-[4-(Fluoro-benzenesulfonyl)-cycloheyl]-piperazin-1-

yl)-acetamide (21c). This compound was prepared with 27c and

acetyl chloride in the same manner as compound 20a, described

above, and was purified by silica gel chromatography (eluent 1%

1

MeOH in CH₂Cl₂) (94% yield); H NMR (CDCl₃): δ 7.85-7.83

(m, 2H), 7.47-7.46 (m, 1 H), 7.38-7.22 (m, 3 H), 6.95 (d, J ) 8

Hz, 1 H), 6.78 (d, J ) 7.6 Hz, 1 H), 3.78-3.60 (m, 4 H), 3.20 (m,

4 H), 2.52-2.49 (m, 4 H), 2.22 (d, J ) 7.6 Hz, 2 H), 2,21 (s, 3 H),

1.70-1.52 (m, 5 H); MS (ESI): m/z 475.2 (M + H)⁺. Anal.

(C₂₄H₃₁FN₄O₃S) C, H, N.

In Vitro Pharmacology. Screening Radioligand Binding. The

selected compounds were purchased from the respective vendors

and experimentally tested in vitro for binding to the 5-HT_1Areceptor

using a radioligand displacement assay performed by CEREP (Paris,

France). Compounds were initially tested at a 10 µM concentration

in duplicates. Hits showing more than 50% inhibition at 10 µM

were validated by a full concentration dose response curve,

measured between 10^-10and 10^-4M. Compounds with experi-

mentally validated binding affinities lower than 5 µM are defined

as actual hits.

Radioligand Binding Studies. All radioligand binding studies

were performed by CEREP (Paris, France) or MDS Pharma Services

(Taiwan, Republic of China). All studies were initially carried out

at 1 µM in duplicates, unless otherwise indicated in the results. In

Animals. Male 129S6/SvEvTac mice from Taconic, German-

town, NY, were used in the stress-induced hyperthermia test. Mice

were 6 weeks of age upon arrival and were group housed in

polycarbonate cages with filter tops for 7 days prior to testing. A

12-h light/dark cycle (lights on 7:00 a.m.) was maintained in the

temperature and humidity controlled animal room (20-23 °C and

30%-70%, respectively). Chow and water were provided ad libitum.

In each test, animals were randomly assigned across treatment

groups and balanced by cage number. Ten animals were used in

each treatment group. Mice were handled once daily for 2

consecutive days prior to testing.

Stressed Induced Hyperthermia (SIH). The study was carried

out during the daytime portion of the light cycle. The test involves

two measures of rectal temperature repeated in the same animal

3134 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 11

Becker et al.

(5) Baumgarten, H. G., Go¨thert, M., Eds. Serotoninergic Neurons and

5-HT Receptors in the CNS: Handbook of Experimental Pharma-

cology, Vol. 129; Springer-Verlag: Berlin, 1997.

(6) Martin, G. R., Eglen, R. M., Hoyer, D., Hamblin, M. W., Yocca, F.,

Eds. AdVances in Serotonin Receptor Research: Molecular Biology,

Signal Transmission, and Therapeutics; Annals of the New York

Academy of Sciences: New York, 1998.

(7) Hoyer, D.; Hannon, J. P.; Martin, G. R. Molecular,Pharmacological

and Functional Diversity of 5-HT Receptors. Pharmacol. Biochem.

BehaV. 2002, 71, 533-554.

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The structure and signalling properties of 5-HT receptors: an endless

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within a 10 min period. On the day prior to testing, animals were

brought to the experimental room 1 h prior to scheduled lights out

and singly housed overnight with food and water ad libitum. On

the morning of the experiment, animals were first injected with

treatment compound or vehicle. After 1 h posttreatment, the animal

was removed from the holding cage and held in a supine position

and had rectal temperature measured by using a rectal probe

attached to a PhysiTemp thermometer (Fisher Scientific). For each

animal tested, the rectal probe was cleaned with an alcohol pad

and lubricated with sterile K-Y jelly and slowly inserted into the

animal’s rectum at a length of approximately 3-5 mm. The probe

remained within the animal’s rectum for approximately 5 s or until

body temperature reached stability, and the baseline rectal temper-

ature (T1) was recorded. The animal was immediately placed back

to the holding cage, and after a 10-min interval a second rectal

temperature (T2) was taken using the same procedure as T1. The

animal was then returned to the home cage and at the completion

of the experiment returned to the colony room.

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All data were analyzed by comparing the groups treated with

the test substance to the vehicle control or reference treated groups.

Statistical analysis was performed by ANOVA followed by Fisher’s

post-hoc test where appropriate. Statistical significance was estab-

lished at a p value of <0.05. Data are represented as the means

and standard error of the mean (SEM).

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Rat and Dog PK Studies. The pharmacokinetic properties were

investigated in male Sprague-Dawley rats following both intra-

venous and oral administration. Six animals (three per group) were

fasted overnight. On day 1, Group 1 animals were administered

the compound intravenously at 1 mg/kg in a dose volume of 0.5

mL/kg. Group 2 animals were administered the compound at 3 mg/

kg in a dose volume of 1.5 mL/kg. Serial blood collections were

performed at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, 12, and 24 h following

intravenous dosing, and at 0.25, 0.5, 1, 2, 4, 6, 8, 12, and 24

following oral administration from the cannulated jugular vein (or

via retroorbital bleed or tail vein depending upon cannula patency).

On day 2, all animals were dose as described above followed by

blood and brain collection at 1 our post dose. On days 1 and 2 all

animals were observed for clinical signs. Presence of the compound,

in both blood and brain, was measured by LC/MS.

The pharmacokinetic properties were investigated in male beagle

dogs following both intravenous and oral administration. Three

animals were fasted overnight and on day 1 were administered the

compound intravenously at a dose level of 1 mg/kg in a dose volume

of 1 mL/kg. On day 2 the compound was administered orally at a

dose level of 3 mg/kg in a dose volume of 5 mL/kg. Serial blood

collections were performed at 5, 15, 30 min and 1, 2, 4, 6, 8, 12,

and 24 h following intravenous administration; and 15 and 30 min

and 1, 2, 4, 6, 8, 12, and 24 following oral administration from a

cephalic or jugular vein. On days 1 and 2, animals were observed

for clinical signs. Plasma levels was measured by LC/MS.

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Acknowledgment. We thank our clinical drug development

operations team members, Christine Wang, Alexandra Petrusich,

and Steve Potter. We also thank Kimberley Gannon, Stephen

Donahue, Joseph Rutkowski, and Adam Muzikant for significant

assistance in preparing this manuscript.

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JM0508641

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An integrated in silico 3D model-driven discovery of a novel, potent, and selective amidosulfonamide 5-HT1A agonist (PRX-00023) for the treatment of anxiety and depression

DOI: 10.1021/jm0508641

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