4-Acyl-1-(4-aminoalkoxyphenyl)-2-ketopiperazines as a novel class of non-brain-penetrant histamine H3 receptor antagonists

Article abstract of DOI:10.1021/jm0708228

A series of ketopiperazines were prepared and evaluated for their activity as histamine H₃ antagonists. From investigation of the tertiary basic center in the aminopropyloxyphenyl template, the 2(R)-methylpyrrolidine was identified as the most

Full text of DOI:10.1021/jm0708228

6706
J. Med. Chem. 2007, 50, 6706–6717
4-Acyl-1-(4-aminoalkoxyphenyl)-2-ketopiperazines as a Novel Class of Non-Brain-Penetrant
Histamine H3 Receptor Antagonists
Panayiotis A. Procopiou,*,† Rachael A. Ancliff,† Mark J. Bamford,‡ Christopher Browning,§ Helen Connor,§ Susannah Davies,‡
Yvonne C. Fogden,† Simon T. Hodgson,† Duncan S. Holmes,† Brian E. Looker,† Karen M. L. Morriss,† Christopher A. Parr,‡
Elizabeth A. Pickup,# Sanjeet S. Sehmi,‡ Gemma V. White,† Clarissa J. Watts,# David M. Wilson,‡ and Michael D. Woodrow†
Departments of Chemistry, Pharmacology, Drug Metabolism and Pharmacokinetics, GlaxoSmithKline Medicines Research Centre, Gunnels
Wood Road, SteVenage, Hertfordshire, SG1 2NY, U.K., and GlaxoSmithKline New Frontiers Science Park South, Third AVenue,
Harlow, Essex, CM19 5AW, U.K.
ReceiVed July 9, 2007
A series of ketopiperazines were prepared and evaluated for their activity as histamine H3 antagonists. From
investigation of the tertiary basic center in the aminopropyloxyphenyl template, the 2(R)-methylpyrrolidine
was identified as the most potent amine. In the more rigid piperidineoxyphenyl template the N-cyclobutyl
group was the most potent amine. The 4-fluorobenzyol, 4-cyanobenzoyl, and 2,4-difluorobenzoyl groups
provided good pharmacokinetic profiles for the various amides. The PSA and log D values of these compounds
suggested low brain penetration. The compounds had very high selectivity over other receptors and did not
inhibit hepatic cytochrome P450, indicating low drug-drug interaction potential. Compound 22i was identified
as the best compound of this series based on its overall profile of high potency, selectivity, low brain
penetration, lack of CYP450 inhibition, high oral bioavailability, and pharmacokinetic properties.
Allergic rhinitis, also known as “hay fever”, affects at least
10–25% of the world’s population and has shown a steady
increase in prevalence during the past 40 years.1 There are two
types of allergic rhinitis: seasonal and perennial. The symptoms
of seasonal allergic rhinitis include at the early stage nasal
itching, irritation, and sneezing, and at the late stage rhinorrhea
and nasal congestion. The symptoms of perennial allergic rhinitis
are similar; however, nasal congestion may be more pronounced.
Either type of allergic rhinitis may also cause other symptoms
such as irritation of the throat and/or eyes, epiphora, and edema
around the eyes.2 Allergic rhinitis and other allergic conditions
are associated with the release of histamine from various cell
types but particularly mast cells. The physiological effects of
histamine are mediated by four major G-protein-coupled recep-
tors, termed H1, H2, H3, and H4, which differ in their expression,
signal transduction, and histamine-binding characteristics.3 H1
receptors are widely distributed throughout the central nervous
system (CNS) and periphery and play a critical role in regulating
inflammatory responses and CNS activity, such as wakefulness.
H1 antagonists, also known as H1 blockers or antihistamines,
are the most commonly used first-line medications for allergic
rhinitis.4 H2 receptors regulate gastric acid secretion, and H2
antagonists are used clinically to treat excess acid production
and gastric ulceration.5 The third receptor subtype (H3) is a
presynaptic autoreceptor that controls the synthesis and release
of histamine as well as other neurotransmitters such as
acetylcholine, dopamine, GABA, glutamate, 5-HT, and nora-
drenaline.6 Consequently many applications have been proposed
for H3 receptor–ligands, particularly in the CNS, where centrally
acting H3 antagonists may provide novel therapies for neuro-
logical disorders such as epilepsy, Parkinson’s disease, Alzhe-
imer’s disease, attention-deficit hyperactivity disorder, sleep
disturbances, cognition, schizophrenia, and obesity.7,8 The more
recently identified fourth receptor subtype (H4) appears to be
restricted to cells of the immune and inflammatory systems; a
physiological role for this receptor remains to be identified.9
H1 receptor antagonists are effective in treating all the
symptoms of allergic rhinitis, apart from the nasal congestion.
Hence, they are often used in combination with R-adrenergic
agonist decongestants such as pseudoephedrine. However, the
use of R-adrenergic agonists is limited because of their potential
to produce hypertension, agitation, and insomnia.
Histamine H3 receptors are expressed widely on both CNS
and peripheral nerve endings and mediate the inhibition of
neurotransmitter release.6,10 Activation of H3 receptor by
histamine modulates outflow to resistance and capacitance
vessels, causing vasodilatation in rats,11 guinea pigs,12 and cats.13
In vitro electrical stimulation of peripheral sympathetic nerves
in isolated human saphenous vein14,15 and porcine nasal
mucosa16 results in an increase in noradrenaline release and
smooth muscle contraction, which can be inhibited by histamine
H3 receptor agonists such as (R)-R-methylhistamine. In addition,
activation of H3 receptors in isolated human nasal turbinate
mucosa inhibits sympathetic vasoconstriction.17 The same report
also described high distribution of H3 receptors in the human
nasal mucosa. It is thought that activation of the H3 receptor
on the presynaptic terminals of sympathetic neurones reduces
noradrenaline release, and this may contribute, together with
the activation of the postsynaptic H1 receptors, to the nasal
blockage caused by histamine release. Consistent with this
hypothesis, combination treatment of H1 and H3 antagonists has
been shown to reverse the effects of mast cell activation on
nasal airway resistance and nasal cavity volume in a cat model
of nasal congestion in vivo.18,19 Further evidence for the
contribution of H3 receptors to histamine-induced blockage of
the nasal airway in normal healthy human volunteers was
provided recently by acoustic rhinometry.20 Scientists at the
* To whom correspondence should be addressed. Phone: (+44) 1438
762883. Fax: (+44) 1438 768302. E-mail: pan.a.procopiou@gsk.com.
†
Medicinal Chemistry, Respiratory CEDD, GlaxoSmithKline Medicines
Research Centre, Stevenage.
‡
Medicinal Chemistry, Neurology CEDD, GlaxoSmithKline, Harlow.
Asthma Rhinitis Biology, GlaxoSmithKline Medicines Research Centre,
§
Stevenage.
#
Department of Drug Metabolism and Pharmacokinetics, GlaxoSmith-
Kline Medicines Research Centre, Stevenage.
10.1021/jm0708228 CCC: $37.00
2007 American Chemical Society
Published on Web 12/06/2007

Ketopiperazines as Antagonists
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 26 6707
Chart 1
compounds 1. Optimization of the properties of 1 would require
variation of the acyl group of the amide, and the basic center.
Chemistry
The synthesis of analogues of 1 is outlined in Scheme 1.
Treatment of 4-benzyloxyaniline 2 with chloroacetyl chloride,
followed by reaction with ethanolamine gave amide 3. The
amino group of 3 was converted to the BOC derivative 4. The
hydroxyl group was reacted with mesyl chloride to the meth-
anesulfonate 5 and then cyclized with sodium hydride to the
protected ketopiperazine 6. Hydrogenolysis of 6 gave the phenol
7, which was reacted with 1-bromo-3-chloropropane to give the
chloride 8. Reaction of 8 with piperidine, pyrrolidine, homopi-
peridine, 2(R)-methylpyrrolidine, or 2(S)-methylpyrrolidine gave
the corresponding amines 9–13. Cleavage of the BOC protecting
group provided the free amines 14–18, which were acylated
with a variety of acids to give the corresponding amides 19–23.
In addition to these molecules possessing the flexible amino-
propyloxy chain, analogues where the chain is fixed in a ring
were also prepared in the hope of improving oral absorption.
The synthesis of the more rigid analogues is outlined in Scheme
2. Reaction of 1-iodo-4-fluorobenzene (24) with 1-BOC-4-
hydroxypiperidine (25) in the presence of sodium hydride in
1-methyl-2-pyrrolidinone gave 26, which was then reacted with
4-benzyloxycarbonylpiperazin-2-one (27) in the presence of
N,N′-dimethylethylenediamine and copper(I) iodide to give 28.53
The BOC protecting group was removed selectively under acid
conditions to give the piperidine 29, which was reductively
alkylated to tertiary amines 30–32 using sodium triacetoxy-
borohydride and the appropriate ketone (acetone, cyclobutanone,
or cyclopentanone). The protected amines were hydrogenolyzed
to give the secondary amines 33–35 and finally acylated with a
number of acids to provide the corresponding amides 36–38.
All carboxylic acids used were commercially available except
for 41, which was prepared from the commercially available
acid chloride 39 and azetidine, followed by ester hydrolysis of
the resulting amide 40 (Scheme 3).
Schering-Plough Research Institute have published the first dual
H1H3 receptor antagonist based on the first-generation H1
antagonist chlorpheniramine and a 4-substituted imidazole.21 In
principle there are two ways of targeting dual H1H3 pharmacol-
ogy, either by using a combination of two individual selective
antagonists or by identifying a molecule that exhibits antagonism
at both receptors. We have examined both strategies, and in
this paper we are reporting our efforts in identifying an H3
antagonist suitable for oral administration for the potential
treatment of allergic rhinitis.
Several compounds are known to be potent histamine H3
receptor antagonists. Most of the early H3 antagonists were
imidazole derivatives, such as thioperamide, ciproxifan, or
clobenpropit (Chart 1). The design of H3 receptor antagonists
devoid of an imidazole ring, as a means of providing compounds
with improved pharmacokinetic properties and avoiding the
possibility of drug-drug interactions, leads to analogues of
ciproxifan where the imidazole ring was replaced by piperi-
dine.22 During the past 3 years a large number of reports were
published by both pharmaceutical research groups in industry
and academia describing various classes of H3 receptor antago-
nists including the Johnson and Johnson,23–31 Hoffmann-La
Roche,32 Novo Nordisk,33,34 Eli Lilly,35 Schering Plough,36,37
Abbott,38,39 and the universities of Parma,40,41 Krakow,42
Amsterdam,43 and Lodz;44 however, all these compounds were
designed for the treatment of CNS diseases.
The target profile for this project was a nonimidazole, human
H3 antagonist (pKi g 9), selectivity over H1 (>100-fold to ensure
that the H1 effects from the fixed dose combination are entirely
due to the admixed H1 antagonist), low brain penetration to
avoid CNS H3 effects (low lipophilicity, high polar surface area,
rat brain/blood ratio of less than 0.5), high oral bioavailability
(molecular weight less than 500, rat F > 30%, dog F > 30%),
low clearance (rat and dog Cl < 1/3 liver blood flow), moderate
half-life (rat t1/2 ) 2 h, dog t1/2 ) 4 h), no CYP450 liability
(IC50 >10 µM on human isoforms), and no hERG liability (IC50
> 30-fold the predicted human free Cmax). A basic amine connected
through an alkoxy chain to a lipophilic group is a feature shared
by several classes of nonimidazole H3 antagonists,22,26–30,45–51
and that we wished to retain in our analogues. The lipophilic
group is often a biaryl moiety,45,46,48–52 which it was our
intention to replace with a group of higher polar surface area
in order to keep our H3 antagonists out of the brain, thus
avoiding any off-target activity. We envisaged replacing one
of the aryl rings of the biphenyl group with an N-linked
ketopiperazine, which would be a planar ring like the replaced
aryl ring, and acylating the remaining nitrogen to provide target
Results and Discussion
Amides 19–23 and 36–38 were evaluated in vitro in mem-
branes from CHO cells transfected with cloned human histamine
H3 receptors for their ability to reduce histamine stimulated
GTP-γ-S binding as determined by scintillation proximity
detection. Selected compounds were also assessed for their
ability to bind to native H3 receptor preparations from rat or
guinea pig cortex. The affinity of compounds was also evaluated
at recombinant human histamine H1 receptor and adrenergic R1A
and R1B receptors in intact CHO cells by means of plate-based
calcium imaging. Inhibition of agonist-induced cellular calcium
mobilization was monitored using the calcium sensing dye Fluo-
4AMa in a FLIPR instrument.54 Antihistamines are regarded
aAbbreviations: Fluo-4AM, 4-(6-acetoxymethoxy-2,7-difluoro-3-oxo-9-
xanthenyl)-4′-methyl-2,2′-(ethylenedioxy)dianiline-N,N,N′,N′-tetraacetic acid
tetrakis(acetoxymethyl) ester; GTP-γ-S, guanosine 5′-[γ-thio]triphosphate;
TBTU ) (O-benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluo-
roborate.

6708 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 26
Procopiou et al.
Scheme 1a
a Reagents and conditions: (a) chloroacetyl chloride, K2CO3, THF; (b) ethanolamine 60°C; (c) BOC2O, (d) MsCl, Et3N, DCM; (e) NaH; (f) H2, 10%
Pd/C, EtOH; (g) 1-bromo-3-chloropropane, K2CO3, 2-butanone; (h) amine piperidine, pyrrolidine, homopiperidine, 2(R)-methylpyrrolidine, or 2(S)-
methylpyrrolidine; (i) TFA, DCM; (j) acylation, RCO2H, Et3N, TBTU, DMF; RCOCl, Et3N, DCM; or RCO2H, HOBT, resin bound DCC, Et3N.
as a class of compounds with a potential for inducing electro-
physiological effects that may occasionally trigger life-threaten-
ing cardiac arrhythmias in humans.55 The binding affinity of
the test compounds for the human ether-a-go-go gene (hERG)
was assessed in 3H-dofetilide radioligand competition assay in
CHO cell membranes, and the data are summarized in Table 1.
The optimal cyclic amine groups reported from various H3
antagonists of different chemical series were piperidine and
pyrrolidine. In the present study piperidines 19a-j (see Chart
2 for structure information) were found to be potent antagonists
with pKi values ranging from 8.4 to 9.2.
Heteroaromatic amides such 19k and 19l and alkyl amides
such as 19m and 19n were found to possess pKi values in the
range 8.1–8.6. The para-regioisomeric sulfone 19f was equi-
potent to the meta isomer 19g. The p-azetidine amide 19j and
toluamide 19b were the most potent analogues from this
subgroup; however, 19b might be metabolically unstable,
whereas the molecular weight of the 19j parent was >500, and
it had a measured log D of -0.95 and possessed too many
hydrogen bond acceptors, making it an unlikely candidate for
good oral absorption.56,57 The next best compounds were the
fluoride and nitrile (19d and 19e). The fluoride 19d was selective
for H3 against H1, hERG, R1A, and R1B. The selectivity of 19d
for H3 over H1 receptors was confirmed in the guinea pig ileum
(H3, 8.4, n ) 2; H1, <6.0, n ) 2). Several second-generation
H1 antagonists are substrates and modulators of hepatic cyto-
chrome P450, in particular the subtype CYP3A4. It is essential
to avoid any drug-drug interactions that might lead to CYP450
activation and reduced safety in the clinic. The inhibitory effects
of selected antagonists for the CYP450 system are summarized
in Table 2. Clearly 19d had no activity against 1A2, 2C19, 2C9,
2D6, and 3A4 (IC50 >100 µM). Furthermore, 19d had low brain/
plasma ratio (0.3 and 0.4) (Table 3), making this compound
suitable for progressing to in vivo pharmacokinetic studies.
Finally the pharmacokinetic properties of 19d were investigated
in rat and dog, and the data are summarized in Table 4,
indicating high oral bioavailability in both species. The pyrro-
lidine analogues 20 had lower potencies than the piperidine
analogues 19 except for 20d and 20h. The latter compound had
potency below the cutoff value of 9 and was therefore rejected.
The former compound was further tested in the guinea pig ileum
where it showed selectivity for H3 over H1 receptors (H3, 8.9,
n ) 2; H1, <6.0, n ) 2). Its pharmacokinetic properties however,
were disappointing with rat oral bioavailability of 12% (Table
4). The homopiperidine analogues 21 had lower in vitro
potencies than the piperidine analogues 19. The introduction
of a methyl group at the C2 position of a pyrrolidine ring was
reported by the Abbott group to impart high in vitro potency at
human and rat H3 receptors.51,58 We have examined both the
(R)- and (S)- enantiomers (22 and 23) and confirmed the higher
potency of 2(R)-methylpyrrolidines 22. The analogue 22d was
a potent H3 antagonist and selective for H3 against H1, hERG,
R
1A, and R1B. Racemic 22d was tested in the guinea pig ileum
where it showed selectivity over H1 receptors (H3, 8.7; H1, <6.6;
n ) 2). Analogue 22e was also a potent H3 antagonist and
selective for H3 against H1, hERG, R1A, and R1B. Its pharma-
cokinetic properties, however, were disappointing with oral
bioavailability of 7% (Table 4). The low bioavailability might
be due to a combination of its low log D value of -0.59 and
high PSA of 77 Å2.59 Analogues 22h and 22i had identical H3
potency and almost identical selectivity profile. In addition, 22i
had a low brain/blood ratio (0.2 and 0.3, Table 3). The

Ketopiperazines as Antagonists
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 26 6709
Scheme 2a
a Reagents and conditions: (a) NaH, 1-methyl-2-pyrrolidinone, 81°C; (b) CuI, K3PO4, N,N′-dimethylethylenediamine, dioxane, 100°C; (c) TFA,
dichloromethane; (d) NaOH; (e) acetone, cyclobutanone, or cyclopentanone, AcOH, NaB(OAc)3H, DCM; (f) H2, Pd/C, EtOH; (g) acylation, RCO2H, Et3N,
TBTU, DMF.
Scheme 3a
potent than the analogues 19 or had pKi below 9, apart from
36e, which had a low log D value of -0.64. The cyclobutylpi-
peridines 37 were either equipotent or more potent than the
analogues 19. The most potent analogue (pKi 9.5) was the
azetidine amide (37j), which as expected from its high PSA
(73 Å2) had a low brain/blood ratio of 0.1; however, it had high
molecular weight (>500), high number of hydrogen bond
acceptors, and a low log D value of -0.29, properties that
predict low oral bioavailabity, and this was confirmed in rats
(8%). The sulfone 37f had high molecular weight, high number
of H-bond acceptors, and low log D value, properties similar
to those of the amide 37j, and not unexpectedly low oral
exposure as measured by the AUC (67 h ·ng/mL). From the
remaining compounds 37 that had potencies higher than their
a Reagents and conditions: (a) azetidine hydrochloride, Et3N, DCM; (b)
2 M NaOH, MeOH, H2O.
analogues of structure 19, the chloride 37c had a log D of 1.52
and a brain/blood ratio of 0.4. The fluoride 37d was a potent
H3 antagonist with good selectivity for H3 against H1, hERG,
R1a, and R1b and an acceptable log D value of 0.90. The
selectivity for H3 against H1 was confirmed in other species
such as rat (H3, 8.0, H1, <5.3) and guinea pig cortex (H3, 8.3,
H1, <5.6). The rat bioavailability of 35%, and half-life of 2 h,
makes this compound one of the preferred ones in this subgroup.
The nitrile 37e, however, had a selectivity profile similar to that
of 37d against H1, hERG, R1A, and R1B. It had an acceptable
log D of 0.38, a low brain/blood ratio of 0.2, good rat and dog
bioavailability (42% and 69%, respectively), low or no activity
against CYP450 (Table 2), and no time-dependent inhibition
of CYP450. The pharmacokinetic half-life of 37e was, however,
low (1.3 and 1.4 h in rats and 2.3 h in the dog). Finally, the
cyclopentylpiperidine analogues 38 had no advantage over the
cyclobutyl analogues 37 and were not investigated any further.
pharmacokinetic properties of 22i were investigated in rat and
dog, and the data are summarized in Table 4, indicating high
oral bioavailability in both species (48% for the rat and 100%
for the dog).
The Abbott group has recently published on some very
lipophilic H3 antagonists, analogues of 22i possessing a
naphthalene core substituted with a 4-cyanophenyl ring, in order
to enhance CNS penetration.60 This is exactly the reverse of
our own strategy, where we have used a polar core with a
ketopiperazine ring and an amide linker in order to keep out of
the CNS. The SAR observed with both series is remarkably
similar and fits the typical H3 pharmacophore of a basic amine
connected through an alkoxy group to a core.
It is expected that molecules with fewer rotatable bonds have
better pharmacokinetic properties, and for this reason analogues
having the aminopropyloxy chain locked in a piperidine ring
were prepared. The isopropyl piperidines 36 were generally less
The above studies identified 22i and 37d as the two
compounds fulfilling the selection criteria set at the outset of
this project. Further selectivity for 22i was examined versus a

6710 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 26
Procopiou et al.
Table 1. Antagonist Potency of Target Compounds at the Human H3 Receptor, Determined by a Functional GTPγ[S] Assay, H1, hERG, R1A, R1B, log D
and PSA
compd
H3 pKi ( SEM (n)a
H1 pKi ( SEM (n)
hERG pIC50 (n)
R1A pKi (n)
R1B pKi (n)
log D
PSA (Å2)
19a·HCl
8.4 ( 0.2 (5)
9.1 ( 0.1 (5)
8.7 ( 0.1 (3)
8.7 ( 0.1 (13)
8.8 ( 0.1 (4)
8.8 ( 0.1 (3)
8.6 ( 0.1 (4)
8.5 ( 0.1 (6)
8.7 ( 0.0 (4)
8.6 ( 0.1 (4)
9.2 ( 0.1 (5)
8.1 ( 0.1 (4)
8.4 ( 0.1 (6)
8.5 ( 0.1 (6)
8.6 ( 0.0 (9)
9.1 ( 0.1 (4)
8.2 ( 0.1 (4)
8.7 ( 0.2 (6)
8.4 ( 0.1 (4)
8.4 ( 0.1 (4)
8.5 ( 0.1 (4)
8.3 ( 0.1 (4)
8.1 ( 0.0 (4)
9.2 ( 0.1 (8)
9.3 ( 0.1 (8)
9.2 ( 0.1 (8)
9.2 ( 0.1 (15)
8.5 ( 0.1 (4)
8.8 ( 0.1 (4)
8.3 ( 0.0 (4)
8.5 ( 0.1 (4)
8.6 ( 0.1 (3)
8.4 ( 0.1 (3)
9.1 ( 0.4 (6)
8.7 ( 0.1 (4)
8.4 ( 0.1 (4)
8.5 ( 0.2 (6)
9.0 ( 0.1 (6)
9.2 ( 0.1 (6)
9.2 ( 0.1 (8)
9.0 ( 0.1 (8)
8.9 ( 0.1 (4)
9.1 ( 0.2 (4)
8.8 ( 0.1 (6)
9.5 ( 0.0 (4)
8.5 ( 0.2 (4)
8.6 ( 0.0 (4)
9.1 ( 0.1 (4)
9.1 ( 0.1 (4)
8.0 ( 0.1 (4)
8.6 ( 0.1 (4)
8.4 ( 0.1 (4)
5.9 ( 0.1 (3)
5.7 ( 0.1 (3)
5.7 ( 0.1 (4)
<5.7 (13)
<5.7 (4)
5.7 ( 0.1 (3)
<5.7 (4)
<5.5 (7)
<5.7 (4)
<5.7 (4)
<5.6 (7)
<5.6 (4)
<5.5 (9)
<5.7 (6)
<5.6 (4)
<5.6 (4)
<5.6 (4)
<5.7 (6)
<5.7 (4)
<5.6 (4)
<5.6 (4)
<5.6 (4)
<5.6 (4)
<5.7 (8)
<5.6 (9)
<5.6 (9)
<5.6 (15)
<5.7 (4)
<5.7 (4)
<5.7 (4)
<5.7 (4)
<5.5 (2)
<5.6 (2)
<5.5 (6)
<5.6 (4)
<5.6 (4)
<5.6 (6)
<5.7 (6)
<5.7 (5)
<5.6 (6)
<5.6 (8)
<5.6 (4)
<5.8 (4)
<5.6 (6)
<5.6 (4)
<5.8 (4)
<5.6 (6)
<5.6 (4)
<5.6 (6)
<5.6 (6)
<5.6 (6)
<5.6 (6)
4.2 (1)
<5.6 (4)
<5.6 (4)
NDb
<5.6 (4)
<5.8(4)
NDb
-0.09
0.48
53
53
53
53
53
77
87
87
53
53
73
66
83
53
53
53
77
53
53
53
77
53
53
53
77
53
53
53
77
53
53
53
53
77
53
53
53
53
53
53
77
87
53
53
73
53
53
77
87
87
53
53
19b·HCl
4.5 (1)
19c·HCl
19d
NDb
NDb
<4.5 (1)
<5.6 (4)
<5.8 (4)
0.11
0.12
-0.38
-1.19
-1.22
0.20
19d·HCO2H
4.3 (1)
NDb
NDb
19e·HCl
NDb
NDb
NDb
19f
<4.2 (2)
<4.2 (1)
4.4 (1)
<4.2 (1)
<4.2 (1)
<4.2 (1)
<4.2 (1)
<4.2 (2)
NDb
5.2 (1)
<4.8 (1)
5.1 (1)
<4.8 (1)
4.9 (1)
4.9(1)
4.9 (1)
4.3 (1)
4.7 (1)
<4.2 (1)
6.3 (1)
5.8 (2)
4.6 (1)
<4.2 (1)
4.9 (1)
NDb
NDb
19g·HCO2H
19h·HCO2H
19i·HCO2H
19j·HCO2H
19k. HCO2H
19l·HCO2H
19m·HCl
NDb
NDb
NDb
NDb
NDb
NDb
0.10
NDb
NDb
-0.95
-1.60
-1.10
-0.02
NDb
NDb
NDb
NDb
NDb
<5.4 (4)
<5.6 (4)
19n·HCl
NDb
NDb
20d·HCO2H
20e·HCO2H
20h
NDb
NDb
0.14
NDb
NDb
-1.07
-0.38
-0.45
-0.47
-0.39
0.24
NDb
NDb
20i
NDb
NDb
21d·HCO2H
21e·HCO2H
21h·HCO2H
21i·HCO2H
22d·HCO2H
22e·HCO2H
22h·HCO2H
22i·HCO2H
23d·HCO2H
23e·HCO2H
23h·HCO2H
23i·HCO2H
36c·HCl
NDb
NDb
NDb
NDb
NDb
NDb
NDb
NDb
0.15
<5.8 (1)
<5.7 (3)
<5 (1)
<5.7 (3)
NDb
<5.8(2)
<6.1 (2)
<5.8 (2)
<6.1 (2)
NDb
-0.13
-0.59
-0.02
-0.08
-0.18
-0.60
0.09
NDb
NDb
NDb
NDb
4.5 (1)
NDb
NDb
-0.13
NDb
NDb
NDb
NDb
36d·HCl
NDb
NDb
NDb
NDb
36e·HCO2H
36h·HCO2H
36i·HCO2H
37a·HCl
<4.2 (1)
<4.2 (1)
<4.2 (1)
<4.2 (2)
<4.2 (1)
4.5 (1)
<4.5 (2)
5.03 (1)
<4.2 (1)
<4.2 (1)
<4.2 (2)
<4.2 (1)
5.4 (1)
NDb
NDb
-0.64
0.06
-0.05
0.73
1.30
1.51
0.90
0.38
-0.38
0.99
0.95
-0.29
1.52
0.88
0.37
NDb
NDb
NDb
NDb
<5.4 (4)
<5.4 (2)
<5.4 (4)
<5.5 (2)
<5.7(2)
NDb
<5.6 (4)
<5.7 (2)
<5.6(4)
<5.5 (2)
<6.1(2)
NDb
37b·HCl
37c·HCl
37d·HCl
37e·HCO2H
37f·HCO2H
37h·HCO2H
37i·HCO2H
37j·HCO2H
38c·HCO2H
38d·HCO2H
38e·HCO2H
38f·HCO2H
38g·HCO2H
38h·HCO2H
38i·HCO2H
<5.7 (2)
<6.1 (2)
NDb
NDb
NDb
NDb
NDb
NDb
4.7 (1)
NDb
NDb
<4.2 (1)
<4.2 (1)
<4.2 (1)
5.1 (1)
NDb
NDb
NDb
NDb
-0.46
-0.41
1.03
NDb
NDb
NDb
NDb
4.6 (1)
NDb
NDb
0.96
a Negative log of the antagonist dissociation constant obtained from concentration-response curves of antagonist inhibition of a response to a fixed
concentration of agonist in functional assays and calculated by application of a modified form of the Cheng-Prusoff equation.62 b ND ) not determined.
panel of 50 other monoamine and hormone receptors, ion
channels, and neurotransmitter uptake sites and found to be
inactive in all apart from PDE4B with pIC50 of 6.15. Moreover,
human serum albumin binding (HSA) for 22i was 36%.
Compound 22i was chosen as a candidate for further progres-
sion. Compound 37d, being a piperidinyloxy restrained ana-
logue, was retained as a backup compound.
absence of hepatic CYP450 inhibition indicated low drug-drug
interaction potential. Optimization of the pharmacokinetic
properties of the new antagonists was achieved by variation of
the amide group. The 4-fluorobenzoyl, 4-cyanobenzoyl, and 2,4-
difluorobenzoyl groups provided better pharmacokinetic profiles
for the various amides. The optimal overall profile of compound
22i (high potency, very good selectivity, low brain penetration,
lack of CYP450 liability, high oral bioavailability) merits further
investigation for the potential use in the treatment of allergic
rhinitis.
Conclusion
A novel series of potent ketopiperazine human H3 receptor
antagonists have been synthesized. The most potent ketopip-
erazine template was the 2(R)-methylpyrrolidine (22). Other
potent templates include the piperidine (19) and the constrained
cyclobutylpiperidine (37). The new compounds had very high
selectivity over other receptors and low brain penetration. The
Experimental Section
Organic solutions were dried over anhydrous Na2SO4 or MgSO4.
TLC was performed on Merck 0.25 mm Kieselgel 60 F254 plates.
Products were visualized under UV light and/or by staining with

Ketopiperazines as Antagonists
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 26 6711
Chart 2
Table 2. In Vitro IC50 Concentration for 19d, 19e, 22i, and 37e against Human Cytochrome P450 Enzymes Expressed in E. coli, Assessed by the Effect
on the O-Dealkylase Metabolic Rate for Specific Profluorescent Probe Substratesa
enzyme isoform and substrate
compd
1A2, ER (µM)
2C19, BMC (µM)
2C9, FCA (µM)
2D6, MMMC (µM)
3A4, DEF (µM)
3A4, 7BQ (µM)
3A4, PPR (µM)
19d
19e
22i
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
77
>100
41
100
ND
>100
>100
100
ND
100
>100
ND
>100
>100
ND
37e
a ER ) ethoxyresorufin, incubated at 0.5 µM; BMC ) 3-butyryl-7-methoxycoumarin, incubated at 10 µM; FCA ) 7-methoxy-4-trifluoromehylcoumarin-
3-acetic acid, incubated at 50 µM; MMMC ) 4-methylaminomethyl-7-methoxycoumarin, incubated at 10 µM; DEF ) diethoxyfluorescein, incubated at 1
µM; 7BQ ) 7-benzloxyquinolone, incubated at 10 µM; PPR ) 7-{3-(4-phenylpiperazin-1-ylmethyl)benzyl} resorufin, incubated at 2 µM; ND ) not determined.
Table 3. Rat Brain/Blood Ratio Determined at 5 min after a 1 mg/kg
Intravenous Dose of 19d, 22i, 37c, 37e, and 37j
0.1% formic acid in water (solvent A) and 0.1% formic acid in
acetonitrile (solvent B), using an appropriate elution gradient over
15 min at a flow rate of 20 mL min-1 and detecting at 200-320
nm at room temperature. Mass spectra were recorded on Micromass
ZMD mass spectrometer using electrospray positive and negative
modes, alternate scans. The software used was MassLynx 3.5 with
OpenLynx and FractionLynx options. 1H NMR spectra were
recorded at 400 MHz unless otherwise stated. The chemical shifts
are expressed in ppm relative to tetramethylsilane. High-resolution
positive ion mass spectra were acquired on a Micromass Q-Tof 2
hybrid quadrupole time-of-flight mass spectrometer. Chiral HPLC
was conducted on Chiralpak AD (25 cm × 0.46 cm) column, eluting
with ethanol-heptane-TFA (25:75:0.1) at a flow rate of 1 mL
min-1 for 60 min at room temperature, detecting at 215 nm. Optical
rotations were measured with an Optical Activity AA100 digital
polarimeter.
19d
19m
22i
37c
37e
37j
0.4
0.3
0.1
0.2
0.3
0.4
0.2
0.1
aqueous KMnO4 solution. LCMS analysis was conducted on a
Supelcosil LCABZ+PLUS column (3.3 cm × 4.6 mm id), eluting
with 0.1% formic acid and 0.01 M ammonium acetate in water
(solvent A) and 0.05% formic acid and 5% water in acetonitrile
(solvent B), using the following elution gradient 0.0-0.7 min 0%
B, 0.7-4.2 min 100% B, 4.2–5.3 min 0% B, 5.3–5.5 min 0% B at
a flow rate of 3 mL min-1. The mass spectra were recorded on a
Fisons VG Platform spectrometer using electrospray positive and
negative mode (ES+ve and ES-ve). This method was the default,
unless otherwise stated. In an alternative method (method B), LCMS
was conducted on a Waters Atlantis column (50 mm × 4.6 mm
id), eluting with 0.05% formic acid in water (solvent A) and 0.05%
formic acid in acetonitrile (solvent B), using the following elution
N2-(2-Hydroxyethyl)-N1-{4-[(phenylmethyl)oxy]phenyl}glycina-
mide (3). 4-(Benzyloxy)aniline hydrochloride (2) (10 g, 42.5 mmol)
was stirred in dry tetrahydrofuran (100 mL) and cooled in an ice/
water bath to ∼5 °C. A solution of potassium carbonate (16.15 g,
117 mmol) in water (60 mL) was added to the above mixture,
followed by the dropwise addition of chloroacetyl chloride (4.22
mL, 53 mmol) over 30 min The mixture was allowed to warm to
room temperature and the organic phase separated. The organic
phase was cooled to ∼5 °C in an ice/water bath and 2-aminoethanol
(9.0 g, 150 mmol) added. The mixture was allowed to warm to
room temperature and heated at 60 °C for 2 h, left overnight at
room temperature, and heated at 60 °C for a further 2 h. The reaction
mixture was partitioned between EtOAc and water. The organic
phase was separated, washed with water and brine, dried, and
concentrated. The residue was recrystallized from chloroform to
give 3 (8.1 g, 64%). ES+ve m/z 301 (M + H)+; 1H NMR δ
(CDCl3) 9.17 (1H, s), 7.50 (2H, d, J ) 9 Hz), 7.45–7.30 (5H, m),
6.95 (2H, d, J ) 9 Hz), 5.05 (2H, s), 3.76 (2H, t, J ) 5 Hz), 3.42
(2H, s), 2.85 (2H, t, J ) 5 Hz).
gradient 0.0–4.0 min 0-100% B at a flow rate of 1 mL min-1
.
The mass spectra were recorded on a Waters ZQ platform using
electrospray positive and negative mode (ES+ve and ES-ve). In
yet another method (method C), LCMS was conducted on a Waters
Atlantis column (50 mm × 4.6 mm id), eluting with 0.05% formic
acid in water (solvent A) and 0.05% formic acid in acetonitrile
(solvent B), using the following elution gradient 0.0–1.5 min
0-100% B at a flow rate of 1 mL min-1. The mass spectra were
recorded on a Waters ZQ platform using electrospray positive and
negative mode (ES+ve and ES-ve). Column chromatography was
performed on Merck Kieselgel 60 (article 9385) or Biotage
prepacked silica gel cartridges containing KP-Sil run on a flash
12i chromatography module or on Flashmaster 2. The Flashmaster
2 is an automated multiuser flash chromatography system, available
from Argonaut Technologies Ltd., which utilizes disposable, normal
phase, SPE cartridges (2-100 g). Mass-directed autopreparative
HPLC (MDAP) was conducted on a Waters FractionLynx system
comprising a Waters 600 pump with extended pump heads, Waters
2700 autosampler, Waters 996 diode array, and Gilson 202 fraction
collector on a 10 cm × 2.54 cm id ABZ+ column, eluting with
1,1-Dimethylethyl (2-Hydroxyethyl)[2-oxo-2-({4-[(phenyl-
methyl)oxy]phenyl}amino)ethyl]carbamate (4). Bis(1,1-dimeth-
ylethyl) dicarbonate (14.5 g, 67.5 mmol) and 4-dimethylaminopy-
ridine (150 mg, 1.2 mmol) were added to a solution of 3 (8.1 g, 27
mmol) in DCM (200 mL). The mixture was stirred for 30 min,

6712 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 26
Procopiou et al.
Table 4. Pharmacokinetic Parameters for Compounds 19d, 20d, 22e, 22i, 37d, 37e, and 37f
dose (mg/kg)
(route)
AUC
(h·ng/mL)
compd
19d
MW
species
Cmax (ng/mL)
tmax (h)
Cl ((mL/min)/kg
V (L/kg)
t1/2 (h)
F (%)
439.6
rat
rat
rat
dog
dog
rat
rat
rat
rat
rat
rat
rat
rat
rat
dog
dog
dog
dog
rat
rat
rat
rat
rat
3.0 (po)
3.0 (po)
1.0 (iv)
1.0 (iv)
2.0 (po)
3.0 (po)
1.0 (iv)
1.0 (iv)
3.0 (po)
1.0 (iv)
3.0 (po)
1.0 (iv)
3.0 (po)
1.0 (iv)
2.0 (po)
1.0 (iv)
2.0 (po)
1.0 (iv)
2.9 (po)
1.0 (iv)
1.0 (iv)
3.0 (po)
1.0 (iv)
1.0 (iv)
2.0 (po)
1.0 (iv)
3.0 (po)
118
155
300
530
400
40.0
342
386
52.0
729
46.0
291
109
235
734
414
734
414
91.0
337
262
149
305
350
294
419
10.0
3.00
2.00
0.08
0.08
0.75
7.00
0.08
0.08
7.00
0.08
4.00
0.08
2.00
0.08
4.00
0.08
4.00
0.08
2.00
0.08
0.08
1.00
0.08
0.08
1.00
0.08
4.00
614
805
437
1570
3090
355
536
557
441
1190
375
682
753
529
4800
2160
4800
2160
760
837
680
1150
577
601
1540
1100
67
47
61
37
9.0
2.4
2.9
1.5
4.3
19d·HCO2H
20d·HCO2H
485.6
471.5
86
12
31
30
2.0
1.9
1.3
1.2
22e·HCO2H
22i·HCO2H
492.6
503.5
7
13
24
31
6.5
6.5
1.6
2.1
2.0
2.3
2.3
1.8
1.3
1.1
4.3
4.3
48
100
100
35
37d·HCl
451.6
504.6
18
23
2.5
2.9
2.0
2.0
37e·HCO2H
42
69
29
27
2.1
2.2
1.3
1.4
rat
dog
dog
rat
15
2.3
2.3
37f·HCO2H
557.7
and aqueous saturated sodium bicarbonate was added (150 mL).
The organic phase was separated, dried, and evaporated. The residue
was dissolved in methanol (150 mL), and potassium carbonate (14.5
g, 105 mmol) was added. The mixture was heated on a steam bath
for 15 min, allowed to cool to room temperature, and left standing
for 18 h. The reaction mixture was evaporated and partitioned
between DCM and water. The organic phase was separated, washed
with brine, dried, and evaporated. The residue was preabsorbed
onto flash silica and purified by flash chromatography (100 g silica
cartridge, from 0% to 100% EtOAc-cyclohexane gradient over
30 min) to give 4 (5.0 g, 46%). ES+ve m/z 401 (M + H)+.
To a solution of 7 (5.0 g, 17 mmol) in 2-butanone (200 mL) was
added potassium carbonate (4.7 g, 34 mmol) followed by 1-bromo-
3-chloropropane (4.0 g, 25.5 mmol). The mixture was heated at 90
°C under nitrogen for 72 h and cooled to room temperature, and
water (200 mL) was added. The organic phase was separated, and
the aqueous layer was extracted twice with DCM. The organic
phases were combined, dried, and evaporated. The residue was
purified by flash chromatography (100 g silica cartridge, from 0%
to 100% EtOAc-cyclohexane gradient over 60 min) to give a
mixture of 8 and 8′ (6.2 g, 9:1). ES+ve m/z 369/371 (chloride)
and 413/415 (bromide) (M + H)+; 1H NMR δ (CDCl3) 7.19 (2H,
d, J ) 9 Hz), 6.94 (2H, d, J ) 9 Hz), 4.25 (2H, s), 4.12 (2H, t, J
) 6 Hz), 3.80–3.74 (2H, m), 3.74 (1.8H, t, J ) 6 Hz), 3.71–3.67
(2H, m), 3.60 (0.2H, t, J ) 7 Hz), 2.28–2.21 (2H, m), 1.50 (9H, s).
1,1-Dimethylethyl3-Oxo-4-(4-{[3-(1-piperidinyl)propyl]oxy}phenyl)-
1-piperazinecarboxylate (9). To a solution of 8 and 8′ (1.28 g,
3.45 mmol) in acetone (50 mL) was added sodium iodide (0.517
g, 3.45 mmol) and piperidine (2 mL). The resultant mixture was
heated to 60 °C and stirred for 16 h and then at room temperature
for 5 days. More sodium iodide (0.517 g, 3.45 mmol) and piperidine
(2 mL) were added, and the reaction mixture was stirred at 60 °C
for a further 5 h. The reaction mixture was allowed to cool to room
temperature and concentrated in vacuo. The residue was partitioned
between EtOAc and saturated sodium bicarbonate solution. The
aqueous portion was extracted with EtOAc, and the combined
organic solutions were washed with water, brine, dried (Na2SO4),
and evaporated. The residue was purified by Biotage column
chromatography (90 g cartridge) and eluted with DCM–EtOH-
aqueous NH3 (150:8:1) to give 9 (345 mg, 25%). ES+ve m/z 418
(M + H)+; 1H NMR δ (CDCl3) 7.17 (2H, d, J ) 9 Hz), 6.92 (2H,
d, J ) 9 Hz), 4.24 (2H, s), 4.01 (2H, d, J ) 6 Hz), 3.80–3.75 (2H,
m), 3.72–3.66 (2H, m), 2.54–2.40 (6H, m), 2.04–1.95 (2H, m),
1.65–1.57 (4H, m), 1.50 (9H, s), 1.5–1.40 (2H, m).
1,1-Dimethylethyl 3-Oxo-4-{4-[(phenylmethyl)oxy]phenyl}-
1-piperazinecarboxylate (6). Methanesulfonyl chloride (2.5 mL,
32 mmol) was added to a solution of 4 (4.99 g, 9.93 mmol) in dry
DCM (50 mL) and triethylamine (20 mL) under nitrogen. The
mixture was stirred for 30 min and then partitioned between
dichloromethane and aqueous saturated sodium bicarbonate. The
organic layer was separated and concentrated in vacuo. The residue
was dissolved in dry DMF (20 mL), and sodium hydride (60% oil
dispersion, 0.6 g, 15 mmol) was added. The mixture was stirred
for 30 min, quenched with aqueous saturated sodium bicarbonate,
and extracted with EtOAc. The organic phases were combined,
dried, and evaporated. The residue was purified by flash chroma-
tography(100gsilicacartridge,from0%to100%EtOAc-cyclohexane
gradient over 40 min) to give 6 (4.1 g). ES+ve m/z 383 (M +
1
H)+; H NMR δ (CDCl3) 7.45–7.32 (5H, m), 7.19 (2H, d, J ) 9
Hz), 7.01 (2H, d, J ) 9 Hz), 5.08 (2H, s), 4.25 (2H, s), 3.80–3.75
(2H, m), 3.71–3.68 (2H, m), 1.50 (9H, s).
1,1-Dimethylethyl 4-(4-Hydroxyphenyl)-3-oxo-1-piperazin-
ecarboxylate (7). A solution of 6 (3.1 g, 8.1 mmol) in EtOH (25
mL) and EtOAc (25 mL) was hydrogenated over 10% Pd/C (1 g)
for 2 h. The catalyst was removed by filtration through Celite and
washed with EtOH. The combined filtrate and washings were
concentrated to give 7 (1.92 g, 81%). ES+ve m/z 293 (M + H)+;
1H NMR δ (CDCl3) 6.98 (2H, d, J ) 9 Hz), 6.64 (2H, d, J ) 9
Hz), 4.26 (2H, s), 3.80–3.75 (2H, m), 3.68–3.64 (2H, m), 1.51
(9H, s).
1-(4-{[3-(1-Piperidinyl)propyl]oxy}phenyl)-2-piperazinone Tri-
fluoroacetate (14). A solution of 9 (0.345 g, 0.826 mmol) in
DCM-trifluoroacetic acid (20 mL, 4:1) was stirred at room
temperature for 45 min. The solvents were then removed in vacuo
to leave 14 (0.745 g). ES+ve m/z 318 (M + H)+; 1H NMR δ
(CD3OD) 7.26 (2H, d, J ) 9 Hz), 7.01 (2H, d, J ) 9 Hz), 4.12
(2H, t, J ) 6 Hz), 3.99 (2H, s), 3.93–3.88 (2H, m), 3.69–3.64 (2H,
1,1-Dimethylethyl 4-{4-[(3-Chloropropyl)oxy]phenyl}-3-oxo-
1-piperazinecarboxylate (8) and 1,1-Dimethylethyl 4-{4-[(3-
Bromopropyl)oxy]phenyl}-3-oxo-1-piperazinecarboxylate (8′).

Ketopiperazines as Antagonists
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 26 6713
m), 3.63–3.55 (2H, m), 3.33–3.27 (2H, m), 3.00–2.92 (2H, m),
2.27–2.18 (2H, m), 2.01–1.93 (2H, m), 1.90–1.70 (3H, m), 1.6–1.5
(1H, m).
H)+; 1H NMR δ (CD3OD) 7.42–7.28 (5H, m), 7.19 (2H, d, J ) 8
Hz), 6.98 (2H, d, J ) 8 Hz), 5.18 (2H, s), 4.51–4.43 (1H, m), 4.25
(2H, m), 3.90–3.80 (2H, m), 3.75–3.68 (2H, m), 3.10–3.02 (2H,
m), 2.75–2.67 (2H, m), 2.05–1.95 (2H, m), 1.70–1.60 (2H, m).
Phenylmethyl 4-(4-{[1-(1-Methylethyl)-4-piperidinyl]oxy}phe-
nyl)-3-oxo-1-piperazinecarboxylate (30). A suspension of 29 (545
mg, 1.33 mmol) in acetone (10 mL), DCM (5 mL), and AcOH (1
mL) was treated portionwise with sodium triacetoxyborohydride
(0.57 g, 2.66 mmol), and the mixture was stirred at room
temperature under nitrogen overnight. A further portion of sodium
triacetoxyborohydride (0.57 g, 2.66 mmol) was added, and the
mixture was stirred for another day. The reaction mixture was
partitioned between EtOAc and aqueous NaHCO3. The organic
solution was washed with NaHCO3 and brine, dried, and concen-
1-(4-{[3-(Hexahydro-1H-azepin-1-yl)propyl]oxy}phenyl)-2-
piperazinone (16). A solution of 11 (0.9 g, 2 mmol) in DCM (10
mL) was treated with a mixture of trifluoroacetic acid-DCM (10
mL, 2:5). The reaction mixture was stirred at room temperature
for 18 h. The solvent was removed in vacuo. The residue was
dissolved in methanol, and the solution was applied to an SCX-2
cartridge (70 g). The cartridge was washed with methanol, and the
product eluted with 10% 0.880 NH3 in methanol. The appropriate
ammoniacal fractions were combined and concentrated to give 16
1
(0.67 g, 97%). ES+ve m/z 332 (M + H)+; H NMR δ (CDCl3)
7.18 (2H, d, J ) 9 Hz), 6.93 (2H, d, J ) 9 Hz), 4.01 (2H, t, J )
6 Hz), 3.70 (2H, s), 3.65 (2H, t, J ) 5 Hz), 3.21 (2H, t, J ) 5 Hz),
2.68–2.62 (6H, m), 2.08–1.90 (2H, m), 1.80–1.58 (8H, m).
1-[4-({3-[(2R)-2-Methyl-1-pyrrolidinyl]propyl}oxy)phenyl]-2-
piperazinone (17). 2-(R)-Methylpyrrolidine was prepared according
to the method of Ku, Cowart, and Sharma.61 ES+ve m/z 318 (M
1
trated to give 30 (576 mg, 96%). ES+ve m/z 452 (M + H)+, H
NMR δ (CDCl3) 7.40–7.32 (5H, m), 7.15 (2H, d, J ) 9 Hz), 6.92
(2H, d, J ) 9 Hz), 5.20 (2H, s), 4.32 (2H, s), 4.32–4.27 (1H, m),
3.85 (2H, br t, J ) 6 Hz), 3.74–3.67 (2H, m), 2.83–2.74 (3H, m),
2.47–2.38 (2H, m), 2.08–2.00 (2H, m), 1.90–1.78 (2H, m), 1.08
(6H, d, J ) 6 Hz).
1
+ H)+; H NMR δ (CDCl3) 7.18 (2H, d, J ) 9 Hz), 6.92 (2H, d,
J ) 9 Hz), 4.05–4.00 (2H, m), 3.75 (2H, s), 3.65 (2H, t, J ) 5
Hz), 3.24–3.15 (3H, m), 3.01–2.94 (1H, m), 2.33–1.65 (9H, m),
1.48–1.38 (1H, m), 1.10 (3H, d J ) 6 Hz).
1-(4-{[1-(1-Methylethyl)-4-piperidinyl]oxy}phenyl)-2-piperazi-
none (33). A solution of 30 (1.168 g, 2.586 mmol) in EtOH (20
mL) and EtOAc (5 mL) was hydrogenated over 10% Pd/C (200
mg) for 2 h. More catalyst (200 mg) was added, and the mixture
was stirred under hydrogen for a further 1.5 h. The catalyst was
removed by filtration, and the filtrate was hydrogenated over a fresh
batch of catalyst (200 mg) overnight. The catalyst was removed
by filtration through a Celite cartridge (10 g) and washed with
EtOH. The combined filtrate and washings were concentrated to
1,1-Dimethylethyl 4-[(4-Iodophenyl)oxy]-1-piperidinecarbox-
ylate (26). Sodium hydride (60% oil dispersion, 2.04 g, 52 mmol)
was added to a solution of N-BOC-4-hydroxypiperidine (25) (10
g, 50 mmol) in 1-methyl-2-pyrrolidinone (50 mL) under nitrogen
at room temperature. 1-Iodo-4-fluorobenzene (11.1 g, 50 mmol)
was added at room temperature, and then the mixture was heated
to 81 °C overnight. The mixture was allowed to cool to room
temperature and partitioned between water and EtOAc. The aqueous
phase was extracted with more EtOAc, and the combined organic
solutions were washed with brine, dried, and concentrated. The
residue was dissolved in DCM and purified by chromatography on
Flashmaster II, eluting with EtOAc-cyclohexane (from 0% to 100%
over 60 min) to give 26 (11.6 g, 57%). ES+ve m/z 304 (MH -
BOC)+; 1H NMR δ (CDCl3) 7.56 (2H, d, J ) 9 Hz), 6.70 (2H, d,
J ) 9 Hz), 4.46–4.40 (1H, m), 3.72–3.64 (2H, m), 3.38–3.30 (2H,
m), 1.95–1.86 (2H, m), 1.79–1.69 (2H, m), 1.58 (9H, s).
Phenylmethyl 4-{4-[(1-{[(1,1-Dimethylethyl)oxy]carbonyl}-4-
piperidinyl)oxy]phenyl}-3-oxo-1-piperazinecarboxylate (28). A
mixture of 26 (10.9 g, 27 mmol), 4-benzyloxycarbonylpiperazin-
2-one (27) (7.6 g, 32 mmol), tripotassium phosphate (6.79 g, 32
mmol), N,N′-dimethylethylenediamine (0.29 mL, 2.7 mmol), and
copper(I) iodide (257 mg, 1.35 mmol) in dioxane (200 mL) was
heated under nitrogen to 100 °C for 24 h. The mixture was allowed
to cool to room temperature and partitioned between water and
EtOAc. The aqueous solution was extracted with more EtOAc,
and the combined organic solutions were washed with brine, dried,
and concentrated. The residue was dissolved in DCM and purified
bychromatographyonBiotage(400g),elutingwithEtOAc-cyclohexane
(2:3 to 1:1) to give 28 (7.36 g, 53%). 1H NMR δ (CDCl3) 7.40–7.33
(5H, m), 7.17 (2H, d, J ) 9 Hz), 6.93 (2H, d, J ) 9 Hz), 5.20 (2H,
s), 4.40–4.43 (1H, m), 4.33 (2H, s), 3.89–3.84 (2H, m), 3.74–3.64
(4H, m), 3.39–3.31 (2H, m), 1.95–1.87 (2H, m), 1.80–1.70 (2H,
m), 1.48 (9H, s).
1
give 33 (732 mg, 89%). ES+ve m/z 318 (M + H)+; H NMR δ
(CD3OD) 7.19 (2H, d, J ) 9 Hz), 6.98 (2H, d, J ) 9 Hz), 4.44–4.37
(1H, m), 3.65 (2H, br t, J ) 5 Hz), 3.55–3.51 (2H, m), 3.15 (2H,
t, J ) 5 Hz), 2.88–2.73 (3H, m), 2.54–2.46 (2H, m), 2.08–1.99
(2H, m), 1.84–1.74 (2H, m), 1.10 (6H, d, J ) 6.5 Hz).
Generic Method for Acylation of 14 in Array Format
Using Resin Bound DCC. Polymer bound DCC (Nova; 1.8 mmol/
g, 853 mg, 1.5 mmol) was suspended in enough DCM to mobilize
on stirring in Radley’s tube on a carousel reaction station. The
carboxylic acid (a, e) (0.77 mmol, 2 equiv) was added to the resin
after 10 min and diluted with DMF to form a solution. The reaction
mixture was then treated with N-hydroxybenztriazole monohydrate
(104 mg, 0.77 mmol, 2 equiv), followed by a mixture of 14 (150
mg, 0.38 mmol) and triethylamine (0.1 mL, 0.8 mmol) in DCM
and stirred at room temperature for 3 days. The resin was removed
by filtration and washed with DCM, and the filtrate was acidified
with acetic acid. The solution was then applied to a 10 g SCX ion
exchange cartridge, eluting sequentially with MeOH, water and 10%
aqueous ammonia in MeOH. The ammoniacal fractions were
evaporated, and the residue was dissolved in DCM and purified by
chromatography on silica, eluting with (5% aqueous ammonia in
MeOH)-DCM (1:19). Appropriate fractions were combined and
evaporated under reduced pressure to give the product, which was
then dissolved in MeOH and treated with 1 equiv of 1 M HCl in
diethyl ether. The solvent was removed by evaporation, and the
residue was triturated in diethyl ether to give the hydrochloride
salts of the products.
Generic Method for the Acylation of 14 Using Acid
Chlorides. A solution of 14 (130 mg, 0.41 mmol) in DCM (5 mL)
was treated with diethylaminomethyl polystyrene (3.2 mmol/g, 194
mg, 0.62 mmol) and the appropriate acid chloride (a, m, n) (0.62
mmol) and stirred for 30 min. The mixture was diluted with MeOH
and passed down a SCX-2 cartridge, eluting with MeOH and then
with 10% aqueous ammonia in MeOH. The ammoniacal fractions
were combined and evaporated under reduced pressure to give the
product, which was then dissolved in MeOH and treated with 1
equiv of 1 M HCl in diethyl ether. The solvent was removed by
evaporation, and the residue was triturated in diethyl ether to give
the hydrochloride salts of the products.
Phenylmethyl 3-Oxo-4-[4-(4-piperidinyloxy)phenyl]-1-pipera-
zinecarboxylate (29). A solution of 28 (7.413 g, 145 mmol) in
DCM-TFA (4:1, 200 mL) was stirred at room temperature for 1 h
and then concentrated under reduced pressure. The residue was
diluted with water and basified with NaOH to give a white
suspension. The solid was collected by filtration, and the aqueous
filtrate was extracted with EtOAc. The organic solution was washed
with water and brine, dried, and evaporated to give 29 (812 mg).
The aqueous solution was basified with more NaOH to pH 13 and
extracted with EtOAc. The organic solution was washed with water
and brine, dried, and evaporated to give additional 29 (2.7 g). The
solid (3.5 g) was dissolved in a mixture of DCM and MeOH (80
mL) and applied to an aminopropyl cartridge (70 g), eluting with
MeOH. The appropriate fractions were combined and concentrated
to give a further quantity of 29 (2.28 g). ES+ve m/z 410 (M +
Generic Method for Acylation of Ketopiperazines 14–18
and 33–35 in Array Format Using TBTU. A solution of the
appropriate amine (0.1 mmol) in DMF (0.5 mL) and triethylamine

6714 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 26
Procopiou et al.
(0.14 mL, 1 mmol) was treated with the appropriate carboxylic
acid (0.11 mmol) and finally (O-benzotriazol-1-yl)-N,N,N′,N′-
tetramethyluronium tetrafluoroborate (TBTU) (42 mg, 0.13 mmol).
The mixture was stirred overnight at room temperature and then
applied to a 10 g SCX-2 cartridge, eluting with MeOH, followed
by 10% aqueous ammonia in MeOH. The ammoniacal fractions
were combined and evaporated under reduced pressure to give the
free base of the amides 19–23 and 36–38 in Table 1. All the
analogues in Table 1 shown as formate salts were the product of a
further purification by MDAP. The appropriate fractions possessing
the expected mass ion were evaporated under reduced pressure or
under a constant stream of nitrogen gas.
The arylamides in Table 1 were obtained as mixtures of rotamers
that produced broad NMR spectra. A selection of NMR solutions
was heated, and the spectra of these compounds were recorded at
elevated temperatures as indicated. The spectra of these compounds
sharpened up, confirming the presence of rotamers.
phosphate buffer (740 µL) in a deep-well microtiter plate. Buffer
saturated octanol (160 µL) was added (via a Beckman Biomek 2000
robot). The plate was sealed with a silicon rubber mat and then
inverted, 3 sets of 50 inversions each set. The plate was centrifuged
at 300g for 15 min to separate the two phases. The sample
concentration in both phases was quantified by HPLC and log D
calculated using the following equation:
log D ) log[HPLC sample peak area octanol phase/octanol phase
sample injection volume (1µL)]/[HPLC sample peak area aqueous
phase/aqueous phase sample injection volume (25µL)]
Human Serum Albumin Binding Assay. The HSA binding of
test compounds was determined using HPLC on chemically bonded
protein stationary phases according to the method of Valko.62
Histamine H3 Functional Antagonist Assay. Membranes
derived from a CHO cell line stably transfected with human H3
receptor (5 µg membranes/well) were adhered to wheat germ
agglutinin polystyrene LeadSeeker (WGA PS LS) scintillation
proximity assay (SPA) beads (250 µg/well) in assay buffer and
GDP (16.6 µM). Assay buffer contained N-2-hydroxyethylpipera-
zine-N′-2-ethanesulfonic acid (HEPES) (20 mM), MgCl2 (10 mM),
100 mM NaCl, and the pH was adjusted to 7.4 using 5 M KOH.
After 1 h of precoupling at room temperature, the bead/membrane
suspension (30 µL/well) was dispensed into a white Greiner
polypropylene 384-well plate containing stock solution of the test
compounds in DMSO (10 mM, 0.5 µL) or 0.5 µL of DMSO as a
control. [35S]-GTPγS (Amersham, radioactivity concentration 37
MBq mL-1; specific activity ) 1160 Ci mmol-1) made up in assay
buffer containing histamine (at a concentration that results in the
final assay concentration of histamine being EC80) was then added
(15 µL/well, 0.4 nM final radioligand concentration), and the assay
plates were sealed and centrifuged at 1000 rpm for 1 min. The
plates were left to equilibrate and to adapt in the dark between 2
and 6 h before reading on a ViewLux luminescence imager using
a 613/55 filter for 5 min plate-1. Data were analyzed using a four-
parameter logistical equation to determine the antagonist IC50.
Antagonist dissociation constants (Ki) were derived from the IC50
using a generalized form of the Cheng-Prusoff equation and
expressed as the negative log of the Ki (pKi).63,64
Histamine H1 and Adrenergic r1A and r1B Receptor Se-
lectivity Assays. CHO cells stably expressing the human recom-
binant histamine H1 receptor or Rat1 fibroblast cells stably
expressing the adrenergic R1A or R1B receptors were seeded into
black-walled clear-bottom 384-well plates at a density of 10 000
cells/well. The following day the cells were washed and incubated
with Tyrodes buffer (NaCl 145 mM, KCl 2.5 mM, HEPES 10 mM,
glucose 10 mM, MgCl2 1.2 mM, CaCl2 1.5 mM, probenecid 2.5
mM, pH adjusted to 7.40 with 1 M NaOH) containing Fluo-4AM
(10 µM) and brilliant black (500 µM) for 60 min at 37 °C. An
amount of 10 µL of antagonist was added (concentration range 30
µM to 30 pM, 2% v/v DMSO), and the cells were incubated for
30 min at 37 °C to allow equilibration of the test compound with
the receptor. An amount of 10 µL of histamine (H1) or phenyle-
phrine (R1a and R1b), at a concentration approximately equivalent
to an EC80 on the agonist concentration–response curve, was added
to each well using the fluorescence imaging plate reader (FLIPR;
Molecular Devices, Sunnyvale, CA), and fluorescence emissions
were monitored for 1 min. Agonist responses were measured as
the peak stimulated fluorescence expressed as a percentage of the
basal fluorescence, and antagonist affinity was determined as
described in the previous section.
4-[(4-Fluorophenyl)carbonyl]-1-(4-{[3-(1-piperidinyl)propyl]-
oxy}phenyl)-2-piperazinone Formate (19d). ES+ve m/z 440 (M
1
+ H)+; H NMR δ (CD3OD) 8.38 (1H, s), 7.60 (2H, dd, J ) 9,6
Hz), 7.26 (2H, d, J ) 9 Hz), 7.24 (2H, t, J ) 9 Hz), 6.99 (2H, d,
J ) 9 Hz), 4.51–4.30 (2H, m), 4.11 (2H, t, J ) 6 Hz), 4.09–3.82
(2H, m), 3.82–3.74 (2H, m), 3.64–3.06 (6H, s), 2.28–2.18 (2H, m),
1.96–1.81 (4H, m), 1.78–1.60 (2H, m); HRMS ES+ve m/z found
440.2354, calcd for C25H31FN3O3 440.2349.
4-[(2,4-Difluorophenyl)carbonyl]-1-[4-({3-[(2R)-2-methyl-1-
pyrrolidinyl]propyl}oxy)phenyl]-2-piperazinone formate (22i).
[R]2D2 –26 (c 0.88 in MeOH); ES+ve m/z 458 (M + H)+; chiral
1
HPLC tR ) 42.2 min, 100%; H NMR δ (CD3OD) 8.40 (1H, s),
7.64–7.50 (1H, m), 7.27 (2H, d, J ) 9 Hz), 7.19–7.10 (2H, m),
7.01 (2H, d, J ) 9 Hz), 4.49 (1.2H, s), 4.16 (0.8H, s), 4.13 (2.8H,
m), 3.85–3.64 (4.2H, m), 3.60–3.48 (2H, m), 3.27–3.13 (2H, m),
2.39–2.28 (1H, m), 2.28–2.16 (2H, m), 2.16–2.03 (2H, m),
1.82–1.71 (1H, m), 1.45 (3H, d, J ) 6.5 Hz); HRMS ES+ve m/z
found 458.2250, calcd for C25H30N3O3F2 458.2255.
1-{4-[(1-Cyclobutyl-4-piperidinyl)oxy]phenyl}-4-[(4-fluorophe-
nyl)carbonyl]-2-piperazinone Hydrochloride (37d). ES+ve m/z
452 (M + H)+; 1H NMR δ (CD3OD) 7.60 (2H, dd, J ) 9, 6 Hz),
7.28 (2H, d, J ) 9 Hz), 7.23 (2H, t, J ) 9 Hz), 7.06 (2H, d, J )
9 Hz), 4.83–4.76 (0.5H, m), 4.70–4.50 (0.5H, m), 4.45–4.30 (2H,
m), 4.30–3.70 (5H, m), 3.70–3.30 (2H, m), 3.20–2.90 (2H, m),
2.40–2.18 (6H, m), 2.15–2.00 (2H, m), 1.95–1.78 (2H, m). When
the solution was heated to 55 °C, the spectrum sharpened: 4.77–4.63
(1H, m), 4.37 (2H, s), 4.00–3.90 (2H, m), 3.83–3.67 (3H, m).
HRMS ES+ve m/z found 452.2353, calcd for C26H31FN3O3
452.2349.
Methyl 4-(Azetidin-1-ylcarbonyl)benzoate (40). Methyl 4-chlo-
rocarbonylbenzoate (39) (4.98 g, 25.1 mmol) was stirred with
triethylamine (8.7 mL, 63 mmol) in dry DCM (50 mL) at 13 °C.
Azetidine hydrochloride (2.8 g, 30.2 mmol) was added portionwise
over 5 min. Cooling in ice was required to keep the temperature
below 20 °C. After being stirred at room temperature under nitrogen
for 3.5 h, the solution was diluted with DCM and partitioned with
aqueous sodium bicarbonate solution. The cloudy organic solution
was then washed with dilute hydrochloric acid, water, and brine
and dried. The solution was evaporated to give 40 (3.97 g, 72%):
1
ES+ve m/z 220 (M + H)+; H NMR δ (DMSO-d6) 8.00 (2H, d,
J ) 8 Hz), 7.73 (2H, d, J ) 8 Hz), 4.28 (2H, t, J ) 7.5 Hz), 4.05
(2H, t, J ) 7.5 Hz), 3.87 (3H, s), 2.31–2.20 (2H, m).
4-(Azetidin-1-ylcarbonyl)benzoic Acid (41). A 2 M NaOH
solution (10.8 mL, 21.6 mmol) was added to a solution of 40 (2.38
g, 10.8 mmol) in MeOH (24 mL) and stirred for 0.5 h. The mixture
was acidified with 2 M hydrochloric acid and extracted with ethyl
acetate. The aqueous phase was extracted with more ethyl acetate,
and the combined organic solutions were washed with water and
brine, dried, and evaporated under reduced pressure to give 41 (1.92
g, 86%): ES+ve m/z 206 (M + H)+; 1H NMR δ (DMSO-d6) 13.21
(1H, br s), 7.99 (2H, d, J ) 8 Hz), 7.71 (2H, d, J ) 8 Hz), 4.28
(2H, t, J ) 8 Hz), 4.05 (2H, t, J ) 8 Hz), 2.31–2.21 (2H, m).
Determination of log D. A stock solution of the test compounds
in DMSO (10 mM, 10 µL) was added to octanol saturated pH 7.4
Guinea Pig and Rat Cortex Histamine Receptor Radio-
ligand Binding Assays. Membranes prepared from rat and guinea
pig cerebral cortex (7 mg of wet weight tissue in 0.4 mL) were
incubated with 50 µL of radioligand (H1, [3H]mepyramine 0.5–1
nM; H3, [3H]-R-R-methylhistamine 1–2 nM final assay concentra-
tion) and increasing concentrations of antagonist (50 µM to 0.5
nM and 1% v/v DMSO final assay concentration) for 2 h at room
temperature. Samples were then filtered through Whatman GF/C
filter paper using a Brandell cell harvester and washed three times
using distilled water at 4 °C. Bound radioligand was determined

Ketopiperazines as Antagonists
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 26 6715
using standard liquid scintillation spectrometry. Antagonist inhibi-
tion curves were analyzed using nonliner regression to obtain an
IC50 as described above and converted to antagonist dissociation
constants (Ki) using the method of Cheng and Prusoff.63
generated and noncompartmental PK analysis used to generate
estimates of half-life, clearance, volume of distribution, and oral
bioavailability.
Acknowledgment. We thank Bill J. Leavens for collecting
the HRMS data, Sean M. Lynn for checking the NMR spectra,
Pat J. McDonough for the log D determinations, James E.
Rowedder for H3 screening, Stephen J. Brough for H1 screening,
Vicky A. Holland and Sharon I. Butler for R screening, Phil
Green for hERG screening, and Klara L. Valko for the HSA
binding assay.
Isolated Guinea Pig Ileum Longitudinal Smooth Muscle
H1 and H3 Functional Assays. Isolated guinea pig ileum longi-
tudinal smooth muscle was prepared for the measurement of
isometric contractions as described elsewhere.65 For H1 receptors,
cumulative concentration–response curves for direct smooth muscle
contraction in response to histamine were constructed in the absence
and presence of test compound. For H3 receptors, tissues were
electrically stimulated (0.1 Hz, 0.5 ms pulse width, submaximal
voltage) and cumulative concentration–response curves for inhibi-
tion of electrical field stimulation by (R)-R-methylhistamine were
constructed in the absence and presence of test compound.
Antagonist pA2 values were calculated as described previously.66
3H-Dofetilide Radioligand Binding Assay for hERG. hERG-
expressing CHO-K1 membranes (2 µg membranes/well) were
adhered to wheat germ agglutinin-coated SPAPEI imaging beads
(120 µg/well) in assay buffer containing HEPES (25 mM) and
MgCl2 (1.2 mM), and pH was adjusted to 7.4 using 5 M KOH.
The final potassium concentration in the assay was 13 mM. After
1 h of mixing at 4 °C, the bead/membrane suspension (20 µL/
well) was dispensed into a white Nunc polypropylene 384-well plate
containing 0.5 µL of test compound in DMSO. Tritiated dofetilide,
made up in assay buffer containing 0.2% pluronic, was then added
(20 µL/well, 5 nM final radioligand concentration), and the assay
plates were sealed and centrifuged at 1000 rpm for 1 min The plates
were left to equilibrate and to adapt in the dark for 4 h before
reading on a ViewLux luminescence imager. Single-shot data were
generated from a single concentration of 12.5 µM. pIC50 data were
generated from an 11-point inhibition curve (top assay concentration
of 60 µM and a 1:3 step dilution), a 4- parameter curvefit being
applied.
In Vitro Inhibition of CYP450 System. Test compounds were
incubated using a concentration range from 0.1 to 100 µM with
human CYP expressed in E. coli or Gentest microsomes. The
compounds were preincubated at 37 °C for 5 or 10 min with the
CYP enzyme isoforms, in the presence of 50 mM phosphate buffer
(pH 7.4) and fluorescent probes for each CYP tested. The
incubations were started by addition of a cofactor solution contain-
ing NADP, glucose 6-phosphate and glucose 6-phosphate dehy-
drogenase. The plates were incubated at 37 °C for 10 min reading
the fluorescence signal on a plate reader throughout. The fluores-
cence data were then used to calculate the IC50 values for each
compound and each CYP.
CNS Penetration. Compounds were dosed intravenously at a
nominal dose level of 1 mL kg-1 to male CD Sprague-Dawley
rats. The test compounds were formulated as solutions in
DMSO-PEG 200-water (5:45:50). Blood samples were taken
under terminal anesthesia with isoflurane at 5 min postdose, and
the brains were also removed for assessment of brain penetration.
Blood samples were taken directly into heparinised tubes and
prepared for analysis using protein precipitation, and brain samples
were prepared using extraction of drug from brain by homogeniza-
tion and subsequent protein precipitation. The concentration of
parent drug in blood and brain extracts was determined by
quantitative LCMS/MS analysis using compound-specific mass
transitions.
Pharmacokinetics. Pharmacokinetics were determined in male
CD Sprague-Dawley rats and male Beagle dogs following single
oral and single intravenous administration. In the rat, doses of 1
and 3 mg kg-1 were used for iv and po, respectively. In the dog,
doses of 1 and 2 mg kg-1 were used for iv and po, respectively.
For all doses the compounds were formulated as solutions in
DMSO-PEG 200-water (5:45:50). Profiles were obtained by
taking serial and terminal blood samples over a range of time points
up to 7, 12, or 24 h postdose. Blood samples were taken directly
into heparinized tubes, prepared by protein precipitation, and
subjected to quantitative analysis by LCMS/MS using compound-
specific mass transitions. Drug concentration-time profiles were
Supporting Information Available: NMR data for 10–13, 15,
18, 19a-n, 20d-i, 21d-i, 22d-h, 23d-i, 31, 32, 34, 35, 36c-i,
37a-j, 38c-i, LCMS retention times and purity of target com-
pounds, and LCMS traces for 19d, 22i, and 37d. This material is
available free of charge via the Internet at http://pubs.acs.org.
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