5-Hydroxyindole-2-carboxylic acid amides: Novel histamine-3 receptor inverse agonists for the treatment of obesity

Article abstract of DOI:10.1021/jm900409x

Obesity is a major risk factor in the development of conditions such as hypertension, hyperglycemia, dyslipidemia, coronary artery disease, and cancer. Several pieces of evidence across different species, including primates, underscore the implication of the histamine 3 receptor (H3R) in the regulation of food intake and body weight and the potential therapeutic effect of H3R inverse agonists. A pharmacophore model, based on public information and validated by previous investigations, was used to design several potential scaffolds. Out of these scaffolds, the 5-hydroxyindole-2-carboxylic acid amide appeared to be of great potential as a novel series of H3R inverse agonist. Extensive structure-activity relationships revealed the interconnectivity of microsomal clearance and hERG (human ether-a-go-go-related gene) affinity with lipophilicity, artificial membrane permeation, and basicity. This effort led to the identification of compounds reversing the (R)-R-methylhistamine-induced water intake increase in Wistar rats and, further, reducing food intake in diet-induced obese Sprague-Dawley rats. Of these, the biochemical, pharmacokinetic, and pharmacodynamic characteristics of (4,4-difluoropiperidin- 1-yl)[1-isopropyl-5-(1-isopropylpiperidin-4-yloxy)-1H-indol-2-yl]-methanone 36 are detailed.

Full text of DOI:10.1021/jm900409x

J. Med. Chem. 2009, 52, 3855–3868
3855
5-Hydroxyindole-2-carboxylic Acid Amides: Novel Histamine-3 Receptor Inverse Agonists for
the Treatment of Obesity
Pascale David Pierson, Alec Fettes, Christian Freichel, Silvia Gatti-McArthur, Cornelia Hertel, Jo¨rg Huwyler, Peter Mohr,
Toshito Nakagawa, Matthias Nettekoven, Jean-Marc Plancher,* Susanne Raab, Hans Richter, Olivier Roche,
Rosa Mar´ıa Rodr´ıguez Sarmiento, Monique Schmitt, Franz Schuler, Tadakatsu Takahashi, Sven Taylor, Christoph Ullmer, and
Ruby Wiegand
F. Hoffmann-La Roche Ltd., Pharmaceuticals DiVision, CH-4070 Basel, Switzerland
ReceiVed February 6, 2009
Obesity is a major risk factor in the development of conditions such as hypertension, hyperglycemia,
dyslipidemia, coronary artery disease, and cancer. Several pieces of evidence across different species, including
primates, underscore the implication of the histamine 3 receptor (H3R) in the regulation of food intake and
body weight and the potential therapeutic effect of H3R inverse agonists. A pharmacophore model, based
on public information and validated by previous investigations, was used to design several potential scaffolds.
Out of these scaffolds, the 5-hydroxyindole-2-carboxylic acid amide appeared to be of great potential as a
novel series of H3R inverse agonist. Extensive structure-activity relationships revealed the interconnectivity
of microsomal clearance and hERG (human ether-a-go-go-related gene) affinity with lipophilicity, artificial
membrane permeation, and basicity. This effort led to the identification of compounds reversing the (R)-
R-methylhistamine-induced water intake increase in Wistar rats and, further, reducing food intake in diet-
induced obese Sprague-Dawley rats. Of these, the biochemical, pharmacokinetic, and pharmacodynamic
characteristics of (4,4-difluoropiperidin-1-yl)[1-isopropyl-5-(1-isopropylpiperidin-4-yloxy)-1H-indol-2-yl]-
methanone 36 are detailed.
Introduction
side effects, the perception the patient has of his own disease,
and additionally the discrepancy between the patient’s expecta-
tion and the achievable weight loss under medication (typically
5% within 1 year) are hampering compliance.7 Although a
change of lifestyle remains an excellent therapy, there exists a
large medical need for safe and efficient treatment of obesity,
stimulating research in this field.2
In the past 2 decades, obesity has became epidemic and is
nowadays recognized as a significant health care burden.
Already in 1997, the World Health Organisation (WHOa)
officially declared human obesity as one of the most significant
health problems facing mankind.1 This declaration followed the
massive expansion in the prevalence of obesity in almost all
societies.2a,3a According to the WHO body mass index (BMI)
classification, more than 120 million people are today considered
obese. Obesity is a major risk factor for the development of
conditions such as hypertension, hyperglycemia, dyslipidemia,
coronary artery disease, and cancer.3,4 As a consequence, the
total cost in the U.S. for all obesity-related health problems
exceeds $200 billion. Sadly, 400 000 people die each year
prematurely in the U.S. from obesity-related complications.5
Increasingly sedentary lifestyles and high-fat, energy rich diets
are the two main causes for the global rise in the prevalence of
obesity. Several drugs have reached the market, among them
the most prominent being sibutramine (a nonselective norepi-
nephrine/serotonine/dopamine reuptake inhibitor) and orlistat
(a lipase inhibitor).2 The obesity market is expected to increase
to over U.S. $1.3 billion by 2010.6 However, their potential
Histamine 3 Receptor Subtype and Its Pharmacology.
Belonging to the G-protein-coupled receptor (GPCR) type A
superfamily, the histamine receptor family consists of four
subtypes.8 The histamine H1 and H2 receptors (H1R and H2R)
have already been extensively studied, particularly for their
involvement in the modulation of allergic response and gastric
acid secretion, respectively, leading to the identification of very
useful medicines.9 The H4 receptor (H4R) was discovered more
recently,10,11 and its potential utility is still being investigated.12
The histamine 3 receptor (H3R), discovered in 1983,13 cloned
in 1999 (human receptor),14 and consisting of 445 amino acids,
shares limited similarity with its H1R, H2R, and H4R congeners
and is predominantly expressed in the central nervous system
(CNS). As a presynaptic autoreceptor, H3R is negatively coupled
to adenylcyclase and regulate neurotransmitter releases. Its
blockade increases the synthesis and the release of histamine,13
which in turn can bind to and activate central H1R. It has been
proposed that stimulation of H1R can reduce food intake and
increase wakefulness.15,16d H3R has also been shown to act as
a heteroreceptor, regulating the release of other neurotransmitters
such as acetylcholine, dopamine, norepinephrine, and sero-
tonin,16d known for their important roles in modulating vigilance,
attention, and cognitive function.
* To whom correspondence should be addressed. Phone: +41 61 68 86
725. Fax: +41 61 68 88 714. E-mail: jean-marc.plancher@roche.com.
a Abbreviations: aq, aqueous; b.i.d., bis in die (twice a day); BMI, body
mass index; BSS, behavioral satiety sequence; CNS, central nervous system;
DCM, dichloromethane; DIO, diet induced obesity; h, human; DMF, N,N-
dimethylformamide; H1R, histamine-1 receptor subtype; H2R, histamine-2
receptor subtype; H3R, histamine-3 receptor subtype; H4R, histamine-4
receptor subtype; hERG, human ether-a-go-go-related gene; HFD, high-fat
diet; HPLC, high pressure liquid chromatography; ip, intraperitonal; LYSA,
lyophilization solubility assay; MNT, micrometerucleus test; OGTT, oral
glucose tolerance test; po, per os (oral); RAMH, (R)-R-methylhistamine;
SD, Sprague-Dawley; TBTU, 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetram-
ethyluronium tetrafluoroborate; THF, tetrahydrofuran; U.S., United States
of America; WHO, World Health Organization.
The H3R is a constitutively active receptor,17 and ligands have
been described as falling into three functional classes: agonists,
inverse agonists, and neutral antagonists.
10.1021/jm900409x CCC: $40.75  2009 American Chemical Society
Published on Web 05/20/2009

3856 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 13
Pierson et al.
Scheme 1. Some Representative H3R Antagonists/Inverse Agonists: 1-6 Used as Preclinical Tools and 7-9 Claimed To Be in Clinical
Trialsa
a Colored circles highlight the conserved pharmacophore requirements.
Over 20 isoforms of the human H3R have been identified so
far, varying in length, N and C termini, sequence of the third
intracellular loop, and deletions of transmembrane domains,
although pharmacological relevance of these isoforms remain
elusive.18
As a consequence of this complex and rich pharmacology,
H3R antagonists/inverse agonists are raising interest in two main
fields: cognitive diseases/sleep disorders16,19 (e.g., Alzheimer,
depression, anxiety, schizophrenia, and narcolepsy) and meta-
bolic diseases (e.g., obesity, dyslipidemia).16,20
Historically, the first H3R antagonists/inverse agonists were
derived directly from the endogenous ligand histamine and
therefore contained the imidazole core. This pharmacophore was
perceived to be responsible for two majors drawbacks, namely,
significant binding discrepancies across species (e.g., rat vs
human H3R binding properties)21 and undesired affinity for the
cytochromes P450.22 A breakthrough came with the replacement
of the offending imidazole by a tertiary amine. Several non-
imidazole-based compounds unleashed the rich H3R pharmacol-
ogy in both the cognition/sleep and food intake arenas but
revealed also some of the undesired properties and limitations
linked to the H3R pharmacophore or its different chemical series
(see Scheme 1).23 Representative of the innovative piperidine-
3-propyloxy motif, 1 (JNJ-5207852) is flanked by a second basic
nitrogen.24 1 is a tight and selective H3R binder with clear in
vivo efficacy in rodent arousal models and interestingly lacks
the appetite-suppressant effect.24
loxybiphenyl, 4 (A-349821), was very potent in rodent model
of cognitive enhancement, yet did not show any effect in an
obesity model, even at higher dose.23,28 This distinct pharmacol-
ogy between obesity and cognitive/arousal models, also reported
above for 1, remains to be explained.26 Eventually, development
of 4 was terminated because of an inadequate safety margin
with respect to a prolonged duration of cardiac action potential.23
5 (ABT-239)29 showed broad efficacy in preclinical models of
cognitive deficits;29c however, its safety ratio toward cardio-
vascular risk was not sufficient for development, despite a more
favorable brain/blood ratio compared to 4.30 Again, 5 was
claimed to be ineffective in a rodent model of obesity (at 30
mg/kg).26 Finally, 6 (NNC-1202) brings an additional piece of
evidence by successfully reducing average calorie intake in
rhesus monkeys.31 However, neither application of this com-
pound in cognitive models nor any clinical developments have
been reported.
Three representatives compounds currently in clinical trials
are listed in Scheme 1, two of which are being evaluated in
phase II for narcolepsy (7, BF 2.649;32 8, GSK18925433).
Interestingly, only 9 (OBE101), which has already been
marketed to treat vestibular vertigo, went successfully into phase
II with obesity as indication.34 Surprisingly, affinity of 9 for
the rat H3R was reported to be only in the micromolar range35
(see Scheme 1). To the best of our knowledge, up to 10 H3R
antagonists/inverse agonists were tested in clinical trials.36
Capitalizing as well on the propyloxy motif, 2 (A-331440)
demonstrates sustained efficacy in chronic obesity models,
mainly by reducing adipose tissues mass, and a modest effect
in the cognition enhancing model.25,26 However, 2 was reported
to be genotoxic in the in vitro micronucleus test (MNT).27
Fortunately, some of its fluorinated congeners, e.g., 3 (A-
423579), were devoid of any in vitro genotoxicity while
retaining efficacy in obesity models.27 Another aminopropy-
Chemistry
Prior to the synthesis of the H3R ligands, we describe here
first the preparation of some noncommercially available pip-
eridine and piperazine derivatives.
Piperidines 12 and 15 were prepared by a double Michael
addition on ethyl acrylate, followed by a Dieckmann condensa-
tion and subsequent decarboxylation and reduction of the ketone

5-Hydroxyindole-2-carboxylic Acid Amides
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 13 3857
Scheme 2. Preparation of 1-Cyclopropyl- and
To facilitate the SAR exploration of the indole ring, several
syntheses were developed, most of them from commercially
available 2,5-disubstituted indoles. Incorporation of the mor-
pholine into the 5-hydroxyindole-2-carboxylic acid, followed
by a Mitsunobu reaction to vary the 5-substituent, proved to be
the most expeditious approach, although the yields of the
Mitsunobu reaction suffered from using standardized, unopti-
mized reaction conditions (see Scheme 3).
1-tert-Butylpiperidin-4-ol 12 and 15 and 1-Cyclobutylpiperazine
17a
The use of 5- and 6-hydroxy-1H-indole-2-carboxylic acid
ethyl esters allowed the exploration of the amide functionality
(see Scheme 4). Whereas the former building block is com-
mercially available, the 6-hydroxy isomer was prepared accord-
ing to a known procedure.38
Derivatization of the indole nitrogen was achieved mainly
by stoichiometric deprotonation using sodium hydride, followed
by quenching with the appropriate electrophile (see Scheme 5,
compounds 33-40). An Ullmann coupling was instrumental for
the incorporation of an N-aryl substituent (41).39
Since the 2,2,2-trifluoroethyl substituent on the indole nitrogen
was found to be of particular interest, the reaction sequence
was altered to allow for a late stage derivatization of the amide
moiety. The Mitsunobu/N-alkylation/amidation sequence turned
out to be favorable as far as the overall yield was concerned
(see Scheme 6).
Introduction of the piperidine, protected as its carbamate,
allowed the late modulation of the N-piperidine substituent (see
Scheme 6). Whereas both cyclobutanone and oxetanone are
commercially available and readily undergo reductive amination,
leading to compounds 45 and 46, respectively, cyclopropanone
a Reagents and conditions: (a) ethyl acrylate 2.0 equiv, room temp, 4
days, 54%; (b) NaH 1.5 equiv, ethanol 1.0 equiv, THF, reflux, 2 days; (c)
aq HCl 18%, reflux, 5 h, then NaOH 1 N; (d) NaBH4 0.75 equiv, ethanol,
room temp, then aq NaOH 28%; (e) ethyl acrylate 3.5 equiv, reflux, 6 days,
14%; (f) cyclobutanone 1.0 equiv, sodium triacetoxy borohydride 1.0 equiv,
DCM, room temp, 16 h, 97%; (g) 4 M HCl in dioxane, room temp, 16 h,
96%.
(see Scheme 2).37 Steric bulk of the tert-butylamine dramatically
decreases the reactivity of the mono-Michael adduct, leading
to a modest yield despite a prolonged reaction time (14%, 6
days). N-Cyclobutylpiperidine 17 was prepared by reductive
amination followed by acidic cleavage of the carbamate
protecting group.
Scheme 3. Preparation of Disubstituted Indoles from 5-Hydroxyindole-2-carboxylic Acida
a Reagents and conditions: (a) morpholine 1.15 equiv, TBTU 1.15 equiv, i-Pr2NEt 5.0 equiv, DMF, room temp, 16 h, 86%; (b) R1OH 1.3 equiv,
azodicarbonyldipiperidine 2.0 equiv, tri-n-butylphosphine 2.0 equiv, THF, 0 °C to room temp, 16 h.
Scheme 4. Exploration of the 2,5- and 2,6-Positionsa
a Reagents and conditions: (a) 1-isopropylpiperidin-4-ol 1.2 equiv, triphenylphosphine 1.2 equiv, di-tert-butylazodicarboxylate 1.2 equiv, THF, room
temp, 2 days, 67% (pos-5) 14% (pos-6); (b) LiOH·H2O 1.1 equiv, methanol, THF, water; quant (pos-5), 99% (pos-6); (c) HNR2 1.2 equiv, TBTU 1.2 equiv,
i-Pr2NEt 6.0 equiv, DMF, room temp, 16 h.

3858 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 13
Scheme 5. Exploration of the N-Indole Substituenta
Pierson et al.
a Reagents and conditions: (a) 25, 2,2,2-trifluoroethyl trifluoromethanesulfonate 1.2 equiv, NaH 1.2 equiv, DMF, 70 °C, 24 h, 74%; (b) 31, isopropyl
methanesulfonate 2.0 equiv, cesium carbonate 2.0 equiv, acetonitrile, 95 °C, 22 h, 47%; (c) 30, 2,2,2-trifluoroethyl trifluoromethanesulfonate 1.2 equiv, NaH
1.2 equiv, DMF, 70°C, 24 h, 88%; (d) 30, 2-bromopropane 1.1 equiv, NaH 1.2 equiv, DMF, 70 °C, 18 h, 59%; (e) 30, acetyl chloride 2.5 equiv, NaH 1.2
equiv, DMF, room temp, 24 h, 77%; (f) 30, methanesulfonoyl chloride 2.5 equiv, sodium hydride 1.2 equiv, DMF, room temp, 2 days, 9%; (g) 30,
4-fluorobenzenesulfonyl chloride 1.2 equiv, NaH 1.1 equiv, DMF, 60 °C, 20 h, 60%; (h) 30, bromoacetonitrile 1.1 equiv, NaH 1.1 equiv, DMF, 70 °C, 5 h,
38%; (i) 30, 3,4-dichlorophenylboronic acid 3.0 equiv, copper acetate 2.0 equiv, pyridine 4.0 equiv, DCM, room temp, 3 days, 81%.
Scheme 6. Early Stage Incorporation of the 2,2,2-Trifluoroethyl Residue on the Indole Nitrogen and Variation of the N-Piperidine
Substituenta
a (a) 1-Isopropylpiperidin-4-ol 1.2 equiv, triphenylphosphine 1.2 equiv, di-tert-butylazodicarboxylate 1.2 equiv, THF, room temp, 2 days, 67%; (b) 2,2,2-
trifluoroethyl trifluoromethanesulfonate 1.2 equiv, NaH 1.1 equiv, DMF, 70 °C, 16 h, quant; (c) LiOH ·H2O 1.2 equiv, THF, water, methanol, reflux, 16 h,
quant.; (d) dioxothiomorpholine 1.2 equiv, TBTU 1.2 equiv, i-Pr2NEt 5.0 equiv, DMF, room temp, 24 h, 92%; (e) N-Boc-4-hydroxypiperidine 1.04 equiv,
PPh3 1.25 equiv, diisopropylazodicarboxylate 1.22 equiv, DCM, room temp, 3 days, 58%; (f) LiOH ·H2O 1.1 equiv, THF, MeOH, water, 80 °C, 16 h, 96%;
(g) morpholine 1.5 equiv, t-BuCOCl 1.2 equiv, 4-methylmorpholine 1.2 equiv, 0 °C, 4.5 h, 75%; (h) 2,2,2-trifluoroethyl trifluoromethanesulfonate 1.2 equiv,
NaH 1.1 equiv, DMF, 70°C, 20 h, 65%; (i) trifluoroacetic acid 10 equiv, DCM, room temp, 1 h, 92%; (j) (1-ethoxycyclopropoxy)trimethylsilane 1.5 equiv,
sodium cyanoborohydride 1.5 equiv, acetic acid 3.0 equiv, THF, 55 °C, 20 h, 45%; (k) oxetanone 2.0 equiv, sodium triacetoxyborohydride 2.0 equiv, acetic
acid 3.0 equiv, THF, 65°C, 2 h, 60%; (l) cyclobutanone 1.5 equiv, sodium cyanoborohydride 1.5 equiv, acetic acid 3.0 equiv, THF, 55 °C, 16 h, 59%.
is not available because of its large preference for the sp3
hybridization state. However, the problem was circumvented
by the use of the known (1-ethoxycyclopropyl)trimethylsilane
as synthetic equivalent.40
5-Aminoindoles were derived from the commercially avail-
able 5-nitroindolecarboxylic acid ethyl ester (see Scheme 7).
Reduction of the nitro group using hydrogen over platinium
oxide did not interfere with the indole nucleus, and the
piperidinyl group was introduced using a reductive amination.
After the incorporation of the amide moiety, the aniline was
selectively alkylated, leading to compound 50.
The Fisher indole synthesis41 was found to be a convenient
strategy to introduce from the beginning a carboxylic acid
functionality at the 5-position, later converted to the N-
cyclobutylpiperidine amide (see Scheme 8). The 1- and 2-posi-
tions of the indole could subsequently be further manipulated.

5-Hydroxyindole-2-carboxylic Acid Amides
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 13 3859
Scheme 7. Modulation of the N-Piperidine Substituenta
a (a) PtO2 ·H2O 0.11 equiv, H2, THF, room temp, 2 h, 98%; (b) 1-isopropyl-4-piperidone 1.0 equiv, Ti(O-i-Pr)4 1.2 equiv, NaBH4 0.6 equiv, methanol,
room temp, 16 h, 85%; (c) LiOH ·H2O 1.1 equiv, THF, methanol, water, 75 °C, 2 h, quant; (d) morpholine 1.2 equiv, TBTU 1.2 equiv, i-Pr2NEt 6.0 equiv,
DMF, room temp, 2 h, 78%; (e) potassium carbonate 1.25 equiv, iodoethane 2.5 equiv, dimethylacetamide, 60 °C, 2 h, 77%.
Scheme 8. Fisher Indole Synthesis for the Preparation of 2,5-Bis-aminocarbonylindolesa
a (a) Ethyl pyruvate 1.2 equiv, ethanol, reflux, 18 h, 82%; (b) polyphosphoric acid, 165 °C, 20 min, 44%; (c) 17 1.25 equiv, TBTU 1.5 equiv, i-Pr2NEt,
DMF, room temp, 1.5 h, 63%; (d) LiOH 1.25 equiv, THF, water, 75 °C, 2 h, quant; (e) morpholine 1.25 equiv, i-Pr2NEt 5.0 equiv, TBTU 1.25 equiv, DMF,
room temp, 3.5 h, 73%; (f) 2,2,2,-trifluoroethyl-trifluoromethanesulfonate 1.1 equiv, NaH 1.1 equiv, THF, 75 °C, 2 days, 76%.
Scheme 9. Preparation of 5-Hydroxy-3-carboxamideindolea
a (a) NaOH 5.0 equiv, THF, water, reflux, 2 days, 75%; (b) morpholine 1.2 equiv, TBTU 1.2 equiv, i-Pr2NEt 5.0 equiv, DMF, room temp, 2 h, 20%; (c)
1-isopropylpiperidin-4-ol 1.3 equiv, azodicarbonyldipiperidine 2.0 equiv, tri-n-butylphosphine 2.0 equiv, THF, room temp, 3 days, 22%.
To explore the indole scaffold systematically, the 3,5- and
2,6-disubstituted indoles were also prepared and evaluated, in
addition to the favorable 2,5-disubstituted and 1,2,5-trisubstituted
derivatives. The 3,5-derivatives were assembled in a straight-
forward manner by saponification42 of the commercially avail-
able ethyl 5-hydroxy-2-methylindole-3-carboxylate and subse-
quent standard amidation/Mitsunobu process (see Scheme 9).
Because of the lack of useful commercially available materi-
als, the 2,6-disubstituted indoles were accessed by the
Hemtsberger-Knittel reaction41 between ethyl azidoacetate and
4-benzyloxy-3-fluorobenzaldehyde, which led mainly to the
desired regioisomer 56 (see Scheme 10). Again, the electron-
rich indole double bond proved to be quite stable toward
hydrogenation, making the benzyl cleavage a high yield process.
in Scheme 1 and Figure 1) acting as a main anchor, most
probably through salt bridge formation with Asp3.32. At a
distance of 4-5 Å, equivalent to four bonds in linear arrange-
ment, an electron rich entity, typically an ether or a carbonyl
function (red circle in Scheme 1 and Figure 1), is often observed.
Optionally, a second basic nitrogen (e.g., 1) can be present at
a distance of 10-11 Å from the first one, most probably
interacting with Glu5.46 (see Figure 1). As this glutamate
residue is only present in H3R and H4R, this favorable interaction
may also increase potency and selectivity over H1R and H2R.
Alternatively, a second electron rich entity (e.g., 4, 8), probably
interacting with Thr6.52, appears to be useful. Interestingly, this
threonine residue is only present in H3R. Between these two
electron rich entities, several scaffolds such as naphthalene,44
quinoline,45 and indole were investigated and these demonstrated
similar in vitro profiles. However, the naphthalene scaffold
resulted in a rather large amphiphilic vector,45,46 raising concerns
about toxicological liabilities such as phospholipidosis. More-
over, our naphthalene series did not lead to efficacious
compounds in a rodent model of obesity, after oral application.
Results and Discussion
Lead Identification. The publication of dozens of potent H3R
binders has facilitated the construction of an accurate pharma-
cophore model.44 It is very much conserved across chemical
series and is characterized by a basic sp3 nitrogen (blue circle

3860 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 13
Scheme 10. Preparation of 6-Hydroxy-2-carboxamideindolea
Pierson et al.
a (a) Benzyl bromide 1.1 equiv, potassium carbonate 1.2 equiv, DMF, 55 °C, 2 h, quant; (b) ethyl azidoacetate 4.6 equiv, NaOMe 4.1 equiv, toluol,
methanol 0 °C, 3 h, then p-xylene, reflux, 2 h, 35%; (c) Pd/C 10% 0.03 equiv, ethyl acetate, H2, room temp, 2 h, 74%; (d) 1-isopropyl-3-piperidinol 1.3
equiv, azodicarbonyldipiperidine 2.0 equiv, tri-n-butylphosphine 2.0 equiv, THF, room temp, 22 h, 48%; (e) LiOH ·H2O 1.1 equiv THF, water, reflux, 1 h,
quant; (f) morpholine 1.2 equiv, TBTU 1.2 equiv, i-Pr2NEt 6.0 equiv, room temp, 17 h, 68%.
Figure 1. Description of a unique pharmacophore model. Various cores were docked to this pharmacophore semiautomatically and selected based
on fitting and chemical tractability. On the basis of this, the 5-oxy-indole-2-carboxylic acid amide series was selected.
On the other hand, some of our H3R ligands based on the
quinoline core were associated with in vitro phototoxicity (data
not shown). Both amphiphilic vector and in vitro phototoxicity
are only surrogate markers of potential toxicity. However, the
perspective to fail late, i.e., in toxicological studies, motivated
us to identify alternatives. Characterized by lower lipophilicity
compared to naphthalenes (clogP ) 2.13 vs 3.32 for the core)
and a distinct electronic density compared to quinolines, the
indole central core additionally held the promise of an easy
access to a third attachment point on the indole nitrogen, so far
poorly explored, and an excellent chemical tractability.
First Attempts. Popularized by others, since it excellently
fits the pharmacophore requirements with respect to the distance
between the basic nitrogen and a first electron rich entity,16c
the N-propoxypiperidine was first introduced together with a
morpholine amide at the 2-position. To our delight, 20 was
found to be an inverse agonist (hH3R EC50 ) 54 nM) with a
high degree of selectivity toward the related H1R, H2R, and H4R
isoforms (6%, 3%, and 4% inhibition at 3 µM, respectively),
securing the 5-oxyindole-2-carboxylic acid amide as a selective
scaffold. Expecting a gain in entropy by restricting free
rotation,29a the linear n-propyl chain was replaced by a more
rigid piperidine. Indeed, more than 1 log-unit affinity improve-
ment was measured for 25, both in rat and in human receptor
(hH3R Ki ) 5 nM and hH3R Ki ) 7 nM; see Table 1). Actually,
species discrepancies minimally affected binding affinities for
all the compounds described herein (below 10-fold difference;
see Table 1).
crosomes preparation (12 and <1 (µL/min)/mg protein, respec-
tively) warranted evaluation of its in vivo efficacy.
An acute model of obesity did not seem appropriate to
characterize H3R antagonists/inverse agonists. Therefore, we
turned our attention toward the widely used dipsogenia assay.
Thus, first-line in vivo screening was based on the antagonist/
inverse agonist reversion of the water-intake induced by the
H3R selective agonist (R)-R-methylhistamine RAMH in rat (for
assay setup, see Figure 2).47 To our delight, 25 was also able
to block the RAMH-induce water intake (inhibition was 91%
at 30 mg/kg ip; see Figure 2).
Further profiling included inhibition of the human ether-a-
go-go-related gene (hERG) channel, a marker for potential
cardiac arrhythmia, which was a show-stopper in some competi-
tor projects (vide supra). 25 showed only a modest inhibition
of the hERG channel (28% at 10 µM).
However, oral application of 25 at a dose of 30 mg/kg b.i.d.
for 3 days in a DIO rat did not induce any significant reduction
in food intake or body weight. Although the compound was
able to permeate the CNS (brain to plasma ratio of 0.3; brain
exposure at 2 h postdosing at 30 mg/kg ip, 2228 ng/g),
pharmacokinetic analysis revealed a fast metabolism (Clint
)
50 (mL/min)/kg) and a short half-life (t1/2 ) 1 h). We
hypothesized that a longer half-life was required to cover the
whole active period of the animal (i.e., 12 h during the dark
phase) and started seeking compounds with such characteristics.
It is worth mentioning that the dipsogenia assay, lasting only
2 h, does not have the same requirement for a prolonged half-
Additionally, subnanomolar functional activity for 25 and
good to excellent metabolic stability in rat and human mi-

5-Hydroxyindole-2-carboxylic Acid Amides
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 13 3861
Table 1. Structure-Activity Relationship of Selected Compoundsd
log D at
pH 7.4
hH3R Ki
rH3R Ki
hH3R EC50
(nM)a
Cl micros Human
((µL/min)/mg)
Cl micros Rat
((µL/min)/mg)
dipso inhib
(% at mg/kg ip)
compd
pKa
(nM)a
(nM)a
20
21
22
25
26
27
28
29
30
31
32
33
35
36
37
38
39
40
41
42
45
46
47
49
50
51
53
55
58
10.0
7.7
10.0
10.0
9.7
9.9
9.8
10.0
9.7
nt
9.7
9.7
9.7
9.9
nt
0.32
1.95
0.03
0.09
1.75
0.59
-0.15
1.23
1.2
71
2
91
2
54
4
<1
5
10
<1
10
5
12
<1
5
6
66 at 10
nt
347
13
12
55
20
5
4
4
6
nt
5
7
<1
<1
9
91 at 30
76 at 10
70 at 30
83 at 30
86 at 10
58 at 1
nt
6
53
37
14
3
9
8
1
2
3
17
9
nt
8
13
8
12
nt
10
20
16
104
4
2
3
7
4
nt
7
6
nt
1
0.31
1.1
2.08
1.67
nt
33
3
95
1
23
4
60 at 10
87 at 10c
49 at 3
80 at 3
nt
<1
3
7
1
4
2
5
5
4
13
3
5
7
<1
<1
<1
<1
<1
1
9.6
nt
9.8
nt
1.31
3.04
1.13
3.38
0.35
2.09
1.83
2.79
-0.7
nt
0.04
1.97
-0.12
0.61
4
4
81 at 3c
nt
2
4
11
2
3
2
nt
1
0.3
5
2
nt
9.6
9.1
6.4
7.5
9.9
nt
11
1
4
nt
0.2
13
0.4
28
nt
7
5
nt
nt
2
4
8
28
228
143
9
nt
23
2
64
19b
2
7
86
366
9
nt
2
11
5
nt
<1
10
<1
6
nt
44
nt
5
99 at 30
nt
nt
nt
15
16
15
18
nt
6.8
9.8
9.5
3
405
112
93 at 10c
nt
nt
a Ki and EC50 were calculated from dose-response curves. b Percentage inhibition measured at 3 µM. c Per os application. Water intake increase in
absence of RAMH stimulation. d nt: not tested.
Figure 2. Dipsogenia as the acute pharmacological model. Principle is the reversal of H3R-induced short-term water intake using an H3R antagonist/
inverse agonist. The H3R inverse agonist is applied ip at t ) 0, and water intake is recorded automatically (cf. Experimental Section) during 1 h
(green bar) and compared with vehicle treated group (gray bar). The compound-treated groups do not exhibit significant modification of their water
intake. Then RAMH, a H3R selective agonist, is applied ip and water intake is again recorded for 1 h (light-blue bar). Percentage inhibition is
calculated for the antagonist/inverse agonist-treated groups (dark-blue bars) relative to the group treated solely with RAMH (light-blue bar). A
significant 91% inhibition is observed for compound 25 at a dose of 30 mg/kg ip.
life and that discrepancies between this first line assay and the
longer 3-day food intake experiment were therefore not
unexpected.
Replacement of the 5-oxy by the 5-amino group impacted
the binding slightly negatively but did not prevent 49 from fully
blocking the RAMH-induce water intake (inhibition of 99% at

3862 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 13
Pierson et al.
30 mg/kg ip). However, 49 showed a high blood-to-plasma ratio
(68-fold), presumably due to active accumulation in erythro-
cytes. Risk of blood toxicity excluded this compound from
further development. Interestingly, this finding was highly
compound specific; minor modifications (e.g., replacement of
the morpholine ring by a pyrrolidine) prevented this accumula-
tion (data not shown). On the other hand, N-alkylation of the
5-amino residue abolished any hH3R binding affinity (see
compound 50, 19% inhibition at 3 µM on hH3R, Table 1).
Exchange of the Attachment Point on the Indole Ring.
With 25 as a reference compound, the effect of the attachment
point on the indole was studied. Shifting the N-isopropyl-4-
piperidineoxy from the 5- to the 6-position (indole numbering;
see compound 32, Table 1) weakened the binding on human
receptor from 5 to 33 nM. Introduction of fluorine to block the
reactive 5-indole position further decreased the affinity to H3R
and, counterintuitively, reduced the stability in human mi-
crosomes preparation as well (Clh.micros ) 1 (µL/min)/mg protein
for 32 to 6 (µL/min)/mg protein for 58; see Table 1).
Moving the carbonylmorpholine on the indole from the 2- to
the 3-position did not affect the affinity but had a dramatic impact
on the efficacy, although 55 was still classified as an inverse agonist
(hH3R EC50 ) 405 nM). All these observations were in agreement
with the pharmacophore model proposed above.
Figure 3. Rat microsomal clearance is negatively correlated with the
measured pKa (two subseries in blue and magenta).
an extended half-life (t1/2 ) 11 h). A food intake reduction of
17% at 10 mg/kg po b.i.d. was recorded (brain exposure 2 h
postdosing at 10 mg/kg po, 1274 ng/g). hERG affinity was
improved to 42% inhibition at 10 µM. N-2,2,2-trifluoroethylin-
dole 35 led to undesired side effects in the IRWIN-type safety
assay and was discarded,48 whereas replacement of the difluo-
ropiperidine by a morpholine did not exhibit any side effects at
the same dose (30 mg/kg ip). In line with a short half-life (t1/2
) 1.8 h) and despite an inhibition of 87% at 10 mg/kg ip (brain
exposure 2 h postdosing at 10 mg/kg ip, 435 ng/g) in the
dispogenia assay, 33 was not active in the subchronic obesity
model at 10 mg/kg po b.i.d. N-Indole substitution proved to be
very tolerant with respect to both size and electronic properties.
However, large N-substituents hampered the selectivity toward
H2R (cf. compounds 39 and 41, >30% inhibition at 3 µM) and
were therefore no longer pursued.
Variations on the Basic Side Chain: Consequences on
Microsomal Clearance and hERG Affinity. Replacement of
the isopropyl group by cyclobutyl modestly reduced the pKa
(9.1 for 45 vs 9.7 for 33), although the stronger electron
withdrawing cyclopropyl dramatically reduced the pKa (e.g., for
21, pKa ) 7.7; for 47, pKa ) 7.5). Similarly, the potent binder
46 (hH3R Ki ) 23 nM) was identified with a pKa as low as 6.4,
the lowest value within this series. In contrast, electron-donating
N-tert-butyl increased the basicity significantly without large
alteration of the affinity (compound 22: pKa ) 10.0; hH3R Ki
) 4 nM). Thus, within a pKa range spanning more than 3 log
units, no correlation could be drawn between pKa and hH3R Ki,
although it is assumed that within the receptor binding pocket
these H3R inverse agonists are protonated, forming a salt bridge
with Asp 3.32.
Clearance for 46 and 47, particularly in rat microsomal
preparations, was poor (228 and 143 (µL/min)/mg protein,
respectively). Although only loose correlation was observed
between microsomal clearance and log D, it turned out that
microsomal clearance was negatively and almost linearly
correlated with the pKa (two subseries in blue and magenta;
see Figure 3). Introduction of local polarity (e.g., compound
46) or increased s-bond character (e.g., compounds 21 and 47)
does not appear to improve metabolic stability. These data
suggest that metabolic stability is exclusively dictated by the
pKa for this series.
Encouraged by this observation to introduce more drastic
changes on the basic side chain, the 4-piperidineoxy moiety was
replaced by a N-carbonylpiperazine. Although 53 was not
optimal as far as microsomal stability was concerned, inhibition
in the dipsogenia assay was excellent (93% at 10 mg/kg po).
The in vivo intrinsic clearance was acceptable (Clint ) 24 (mL/
min)/kg), and 53 also penetrated the brain (brain/plasma of 0.3);
Investigations around the Amide. Lipophilic azepane 26
is extensively metabolized in vitro. Evaluation of the morpholine
replacements, maintaining the local hydrophilicity (e.g., carbo-
nyl-4-methoxypiperidine 27 or carbonylpyrrolidyl-3-ol 28), did
not help in improving the metabolic stability compared to 25.
Therefore, we attempted to shield the amide using polyfluori-
nated rings. Such an approach could lead to a dramatic reduction
of the aqueous solubility, hampering further development.
However, driven by the basic amine, solubility was in general
not an issue within this series. Thus, carbonyl-4,4′-difluoropi-
peridine 30 was soluble (>540 mg/L in LYSA assay), potent
on the H3R receptor (hH3R Ki ) 2 nM), and metabolically stable
in vitro (Clh.micros ) 5 (µL/min)/mg protein; Clr.micros ) 9 (µL/
min)/mg protein; see Table 1). Its pharmacokinetic profile
combined an acceptable intrinsic clearance (Clint ) 18 (mL/
min)/kg), a perfect bioavailability (∼100%), and an extended
half-life (t1/2 ) 4 h). More interestingly, this compound was
active in our 3-day obesity model at 10 mg/kg b.i.d. after ip
application (12% reduction in food intake), despite a reduced
brain exposure compared to 25 (brain exposure 2 h postdosing
at 10 mg/kg ip, 1615 ng/g), strengthening our working hypoth-
esis concerning the importance of the half-life for efficacy (vide
supra). Further, po application did not alter the efficacy or the
exposure during the 3-day obesity model (10% reduction in food
intake; brain exposure 2 h postdosing at 10 mg/kg po, 1692
ng/g), ruling out an obvious taste aversion effect on food intake.
However, 73% inhibition of the hERG channel at 10 µM was
considered as being suboptimal for this indication.
N-Indole Substituant as a Third Attachment Point. Acy-
lation of N-indole generated compounds that were slightly
chemically unstable in basic aqueous media (compound 37).
However, sulfonylation (e.g., compound 38) provided molecules
with an excellent overall profile. Most importantly, the half-
life of 38 reached 6 h and, to our delight, was found to be highly
efficacious in our 3-day obesity model (14% reduction of food
intake at 10 mg/kg po b.i.d.). However, hERG affinity was still
of concern with 64% inhibition at 10 µM. The isopropyl
derivative 36 exhibited an excellent in vitro profile as well.
Intrinsic clearance of 38 and 36 was identical, but the larger
volume of distribution (Vss ) 9.2 L ·kg-1) for the latter led to

5-Hydroxyindole-2-carboxylic Acid Amides
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 13 3863
Figure 4. Correlation of experiment vs calculated hERG pIC50, using Roche automatic SAR analyzer (ROSARA). The model is relying solely on
two descriptors, the percentage of membrane-bound compound in the parallel artificial membrane assay (PAMPA), and log D and is independent
of pKa. Red squares represent compounds used to develop the model (training set). Blue squares represent compounds used to validate it (validation
set). The line of identity is represented in green, with a regression coefficient of 0.736 (based on the training set).
Figure 5. Chronic dosing with compound 36 at 5, 10, and 20 mg/kg b.i.d. po in a diet-induced obesity rat model. Dose dependent reduction in
body weight was observed. The body weight development for the 10 mg/kg group and the pair-fed group was comparable. The rebound effect
observed after stopping treatment was less marked with the treated groups than with the pair-fed group.
however, the low protein binding (52% in rat) and the low Vss
(1.5 L/kg) shortened the half-life. Therefore, inefficacy of 53
in the subchronic food intake model at 10 mg/kg po b.i.d. was
somewhat expected. hERG inhibition of 53 was reduced (33%
at 10 µM).
descriptors: the percentage of membrane-bound compound in
the parallel artificial membrane assay (PAMPA)51 and log D.
As some small molecule hERG inhibitors are assumed to interact
with the hERG channel through the membrane section, this
result made a lot of sense. Translation of hERG single point
values measured at 10 µM into pIC50 values significantly
improved the regression coefficient to 0.736. Interestingly,
although the pKa in the training set spanned more than 3 log
units, no correlation with hERG affinity was observed. The
model has been validated with two new compounds from the
same series (compounds 34 and 40, blue squares; see Figure 4)
and then routinely applied not to select but to prioritize
candidates at an early stage (before in vivo experiments). The
independence of pKa and hERG inhibition, together with the
improved metabolic stability for compounds having pKa > 9
(vide supra), biased us toward the selection of rather basic
ligands.
Such a data set suggested keeping a rather basic nitrogen (i.e.,
pKa ≈ 9-10) and replacing the morpholine by difluoropiperidine
for optimal metabolic stability in vitro. Concerning hERG, a
basic nitrogen is often considered as the usual culprit, although
definitely not the only one.49 To gain a better understanding of
the main driver for hERG inhibition, we applied the Roche
automatic SAR analyzer (ROSARA).50 This proprietary soft-
ware, based on the use of partial least squares analysis, is
designed to study potential correlation between a defined
compound property (i.e., hERG inhibition in this case) and
numerous descriptors. These descriptors can be both measured
(e.g., LYSA solubility, log D, hH3R Ki, pKa, etc.) or calculated
(e.g., clogP, PSA, etc.). In our case, ROSARA was able to
generate a valuable hERG model by selecting only two in vitro
Characterization of Compound 36. Among those com-
pounds, 36 attracted our attention for its striking combination

3864 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 13
Pierson et al.
Detailed Description. 5-(1-Isopropylpiperidin-4-yloxy)-1H-
indole-2-carboxylic Acid Ethyl Ester (23). To a cold (ice-water)
solution of 5-hydroxyindole-2-carboxylic acid ethyl ester (20 g, 97
mmol) and triphenylphosphine (30.68 g, 117 mmol, 1.2 equiv) in
tetrahydrofuran (500 mL) was added dropwise a solution of di-
tert-butyl azodicarboxylate (26.93 g, 117 mmol, 1.2 equiv) in
tetrahydrofuran (100 mL). The mixture was stirred for 2 days at
room temperature and concentrated in vacuo. Purification was
performed by column chromatography on silica gel using a gradient
of dichloromethane/methanol/aqueous ammonia (19:1:0 to 95:5:
0.25) as eluant to afford, after evaporation, a colorless oil (40.26
g). This oil was dissolved in ethyl acetate and concentrated in vacuo
to ∼50 mL. Methyl tert-butyl ether was added, and the mixture
was stirred at ∼0-3 °C (ice bath) and then filtered and dried in
vacuo to afford the desired product as a white solid (21.53 g, 67%).
1H NMR (400 MHz, CDCl3) δ 8.77 (s broad, 1H); 7.30 (d, J ) 8
Hz, 1H); 7.15-7.10 (mult, 2H); 7.00 (dd, J ) 8 Hz, 2 Hz, 1H);
4.40 (quad, J ) 7 Hz, 2H); 4.26 (hept, J ) 4 Hz, 1H); 2.87-2.76
(mult, 2H); 2.75 (hept, J ) 8 Hz, 1H); 2.42-2.33 (mult, 2H);
2.08-1.98 (mult, 2H); 1.87-1.77 (mult, 2H); 1.41 (t, J ) 7 Hz,
3H); 1.06 (d, J ) 6 Hz, 6H) ppm. MS (EI) m/e: 331.1 (M + H)+.
5-(1-Isopropylpiperidin-4-yloxy)-1H-indole-2-carboxylic Acid,
Hydrochloride Salt, with Lithium Chloride. To a solution of 5-(1-
isopropylpiperidin-4-yloxy)-1H-indole-2-carboxylic acid ethyl ester
(23, 10.0 g, 30 mmol) in a mixture of tetrahydrofuran (130 mL)
and methanol (27 mL) was added a solution of lithium hydroxide
monohydrate (1.40 g, 33 mmol, 1.1 equiv) in water (65 mL). The
mixture was stirred for 1 h at reflux and concentrated in vacuo.
The pH of the resulting suspension was adjusted to pH 2 with
hydrochloric acid (2 N). The solution was dried in vacuo to afford
the desired product as a brown solid (11.6 g, quant.). This solid
of potent H3R binding (hH3R Ki ) 7 nM; rH3R Ki ) 5 nM),
low clearance in vivo (intrinsic clearance in rat, 10 (µL/min)/
mg protein), extended half-life (t1/2 ) 11 h in rat), rather low
hERG inhibition (42% hERG inhibition at 10 µM), and
ultimately excellent performance in the 3-day obesity model
(17% reduction in food intake at 10 mg/kg po b.i.d.).
To evaluate whether food intake is decreased in a specific
manner or by disruption of normal feeding behavior in a
nonspecific manner (e.g., by inducing sedation, hyperactivity,
or any other motor effects that could interfere with food intake),
the effect of 36 was recorded on the different components of
the behavioral satiety sequence (BSS).52 Compound 36 was
tested at three different doses (10, 20, 30 mg/kg po) and
compared to the vehicle treated group. At all tested doses,
administration of 36 did not cause a disruption of the behavioral
pattern of rats, providing confidence that reduction of food intake
was achieved by enhancing natural satiety in a specific manner.
To test 36 in a disease relevant model, male Sprague-Dawley
rats were fed with a high fat diet (HFD, 43% of energy as fat,
19% w/w fat) 10 weeks prior to the treatment period and during
dosing. Chronic application of 36 (5, 10, and 20 mg/kg b.i.d.
po) for 17 days in this diet induced obesity rat model (DIO)
led to a dose-dependent slower progression of body weight in
all groups (-4%, -10%, -13%, respectively), statistically
significant for the 10 and 20 mg/kg groups (cf. Figure 5). A
pair-fed group (a group of animals receiving exactly the amount
of food eaten by the 10 mg/kg group) was incorporated into
the study. As expected, the body weight gains for the 10 mg/
kg group (orange line) and the pair-fed group (green line) were
comparable. The body weight was still recorded for 13 days
after withdrawal of the treatment. Interestingly, the rebound
effect observed after stopping treatment was less marked with
the treated groups than with the pair-fed group.
1
was used in the next step without further purification. H NMR
(300 MHz, DMSO-d6) δ 11.67 (s, 1H); 7.35 (d, J ) 9 Hz, 1H);
7.23 (broad s, 1H); 6.97 (d, J ) 1 Hz, 1H); 7.00-6.90 (mult, 1H);
4.85-4.75 (mult, 1H); 3.46 (hept, J ) 7 Hz, 1H); 3.25-3.15 (mult,
4H); 2.30-2.20 (mult, 2H); 2.10-2.00 (mult, 2H); 1.31 (d, J ) 7
Hz, 6H) ppm. MS (EI) m/e: 301.2 (M-H)-.
Conclusion
(4,4-Difluoropiperidin-1-yl)[5-(1-isopropyl-piperidin-4-yloxy)-
1H-indol-2-yl]methanone (30). To a solution of 5-(1-isopropylpi-
peridin-4-yloxy)-1H-indole-2-carboxylic acid, hydrochloride salt,
with lithium chloride (5.0 g, 13 mmol) and 2-(1H-benzotriazole-
1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (5.3 g, 15.7
mmol, 1.2 equiv) in N,N-dimethylformamide (75 mL) were added
ethyldiisopropylamine (11.5 mL, 65.6 mmol, 5.0 equiv) and 4,4′-
difluoropiperidine hydrochloride (2.48 g, 15.7 mmol, 1.2 equiv).
The mixture was stirred overnight at room temperature and
partitioned between saturated aqueous sodium hydrogenocarbonate
solution (300 mL) and ethyl acetate (300 mL). The aqueous layer
was extracted with ethyl acetate, and the combined organic phases
were washed with water and brine, dried over sodium sulfate,
filtered, and concentrated to ∼40 mL. Methyl tert-butyl ether (40
mL) was added with stirring, and the precipitate was filtered, washed
with cold (5-10 °C) methyl tert-butyl ether, and dried in vacuo to
afford the desired product as a white solid (4.95 g, 93%). 1H NMR
(300 MHz, CDCl3) δ 8.97 (s, 1H); 7.32 (d, J ) 9 Hz, 1H); 7.11 (d,
J ) 2 Hz, 1H); 6.99 (dd, J ) 9 Hz, 2 Hz, 1H); 6.69 (d, J ) 2 Hz,
1H); 4.24 (hept, J ) 4 Hz, 1H); 4.08-3.95 (mult, 4H); 3.87-3.70
(mult, 3H); 2.42-2.35 (mult, 2H); 2.19-1.97 (mult, 6H); 1.92-1.75
(mult, 2H); 1.06 (d, J ) 7 Hz, 6H) ppm. MS (EI) m/e: 406.2 (M
+ H)+. Anal. (C22H29N3O2) C, H, N.
(4,4-Difluoropiperidin-1-yl)[1-isopropyl-5-(1-isopropylpiperi-
din-4-yloxy)-1H-indol-2-yl]methanone (36). To a solution of (4,4-
difluoropiperidin-1-yl)[5-(1-isopropylpiperidin-4-yloxy)-1H-indol-
2-yl]methanone (30, 1.5 g, 3.7 mmol) in N,N-dimethylformamide
(18 mL) was added sodium hydride (50% w/w dispersion in oil,
178 mg, 4.1 mmol, 1.1 equiv). The mixture was stirred 50 min at
70 °C before the addition of 2-bromopropane (0.55 mL, 4.4 mmol,
1.2 equiv). The mixture was stirred 18 h at 70 °C and partitioned
between ethyl acetate and water. The aqueous layer was extracted
with ethyl acetate, and the combined organic phases were washed
with water, dried over sodium sulfate, filtered, and concentrated in
A series of 5-hydroxyindole-2-carboxylic acid amide deriva-
tives was identified as a novel class of H3R inverse agonist.53
By reliance on a pharmacophore model built on public informa-
tion, the indole scaffold, in addition to other scaffolds,43-45 was
identified as a promising chemical entry point. Following SAR
development, extensive multidimensional optimization, and
careful evaluation of the underlying drivers of microsomal
clearance and hERG affinity, 36 proved to combine the best
overall characteristics for further profiling. In an indirect acute
pharmacodynamic model, 36 reversed dose-dependently the
water intake induced by an H3R selective agonist (RAMH).
Furthermore, in a 17-day diet-induce obesity (DIO) rat model,
36 demonstrated a robust reduction in body weight gain after
po application and showed a reduced rebound effect compared
to a per-fed group. On the basis of its efficacy, metabolic
stability, and safety profile, compound 36 was selected for
further in depth evaluation.
Experimental Section
Chemistry. General. Proton NMR spectra were obtained on
Bruker 300 or 400 MHz instrument with chemical shifts (δ in ppm)
reported relative to tetramethylsilane as an internal standard. NMR
abbreviations are as follows: s, singlet; d, doublet; t, triplet; quad,
quadruplet; quint, quintuplet; sext, sextuplet; hept, heptuplet; mult,
multiplet.
Elemental analyses were performed by Solvias AG (Matten-
strasse, Postfach, CH-4002 Basel, Switzerland). Column chroma-
tography was carried out on silica gel 60 (32-60 mesh, 60 Å) or
on prepacked columns (Isolute Flash Si). Mass spectra were
recorded on SSQ 7000 (Finnigan-MAT) for electron impact
ionization.

5-Hydroxyindole-2-carboxylic Acid Amides
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 13 3865
vacuo. Purification was performed by column chromatography on
silica gel using a gradient of dichloromethane/methanol (1:0 then
19:1) as eluant to afford, after evaporation, the desired product as
a white solid (984 mg, 59%). 1H NMR (400 MHz, CDCl3) δ 7.41
(d, J ) 9 Hz, 1H); 7.09 (dd, J ) 2 Hz, 1H); 6.92 (dd, J ) 9 Hz,
2 Hz, 1H); 6.40(s, 1H); 4.73 (hept J ) 7 Hz, 1H); 4.30-4.20 (mult,
1H); 3.87-3.78 (mult, 4H); 2.85-2.75 (mult, 2H); 2.74 (hept, J )
7 Hz, 1H); 2.38 (broad t, J ) 9 Hz, 2H); 2.08-1.95 (mult, 6H);
1.87-1.75 (mult, 2H); 1.62 (d, J ) 7 Hz, 6H); 1.05 (d, J ) 7 Hz,
6H) ppm. MS (EI) m/e: 448.2 (M + H)+. Anal. (C25H35F2N3O2) C,
H, N.
Torrance, CA). Quantitative detection was achieved on a SCIEX
2000 instrument (MDS Sciex, Concord, ON, Canada) using electron
spray ionization. Concentrations were determined by ratio of test
compound and internal standard peaks and given as a percentage
of the concentration measured at the first time point (substrate
depletion). Intrinsic clearance (CLint, in (µL/min)/mg microsomal
protein) is the rate constant of the first-order decay of the test
compound, normalized for the protein concentration in the incubation.
In Vivo Assays. Animals. Male Wistar rats around 350 g body
weight (11 and 13 weeks of age) were used for the dispogenia
studies. For the food intake studies male Sprague-Dawley (SD)
rats were put on a high fat diet (HFD, KLIBA 2157, 43% of energy
as fat, 19% w/w fat) for 2 weeks until use. For the chronic
experiments 100 male DIO SD rats were used. All animals were
obtained from Iffa Credo/Charles River, France. Animals were put
on HFD (KLIBA 2157, 43% of energy as fat, 19% w/w fat) at 3
weeks of age for 10 weeks and on a reversed light cycle (9.30 a.m.
lights off; 9.30 p.m. lights on). During the growing period, body
weight was measured at the beginning of the high fat feeding and
before the stratification. Responders to HFD were separated from
nonresponder animals using the following criteria: the difference
between average body weight of high responders and the body
weight of rats fed standard chow diet was at least 2 times the
standard deviation of the average body weight of the standard chow
diet group.
Dipsogenia Assay. Rats (n ) 10 animals/group) were tested for
the inhibition of the (R)-R-methylhistamine (RAMH) induced short-
term water intake in a dipsogenia model. Each rat was injected po
or ip with either vehicle (citric acid buffer pH 3.3) or H3R inverse
agonists, and water intake was monitored automatically (TSE
system) for 1 h. Afterward 10 mg/kg RAMH or 0.9% NaCl was
administered ip and water intake was again monitored for 1 h.
Three-Day Food Intake. A subchronic, 3-day model for food
intake in male SD rat on HFD was used to assess efficacy of H3R
inverse agonists. Animals were put on a reversed light cycle (9.30
a.m. lights off; 9.30 p.m. lights on) for 2 weeks, for adaptation.
Access to tap water and food was ad libitum. Food intake and body
weight were monitored daily 1 week prior to compound testing,
and animals were stratified into homogeneous groups according to
food intake and body weight. The compound was applied b.i.d. po
at different doses 1 h prior light cycle change and 6 h later, and
food intake was monitored daily for 3 consecutive days.
In Vitro Assays. H1R, H2R, H3R, and H4R Radioligand
Binding Assays. For the rat H3R and human H1R, H3R, and H4R
binding assays, membranes were prepared from recombinant CHO
cells stably expressing the respective histamine receptor. Membranes
for the H2R binding assay were purchased at Euroscreen, Belgium.
For H3 receptor competition binding (human and rat), membrane
preparations were incubated with [3H]-(R)-R-methylhistamine (1.0
nM for human H3R, 2.0 nM for rat H3R) in the presence or absence
of increasing concentrations (10 concentrations over a 4.5 log-unit
range) of ligands for 120 min at 25 °C in a final volume of 0.2 mL
of 50 mM Tris-HCl, 5 mM MgCl2, pH 7.4, buffer, gently shaking.
Nonspecific binding was defined with 5 µM (R)-(-)-R-methylhis-
tamine. Cloned human histamine H1 receptor radioligand binding
assays were performed using 1.5 nM [3H]pyrilamine for 30 min in
50 mM Tris-HCl, 2 mM MgCl2, 20 mM NaCl, and 250 mM
sucrose, pH 7.4. Nonspecific binding was defined with 10 µM
pyrilamine. Cloned human H2 receptor binding assays were
performed using 13 nM [3H]tiotidine for 60 min in 50 mM Tris-
HCl, 500 mM NaCl, pH 7.4. Nonspecific binding was defined with
100 µM tiotidine. Cloned H4 receptor binding was performed using
25 nM [3H]histamine for 60 min in 50 mM Tris-HCl, 5 mM EDTA,
250 mM sucrose, pH 7.4. Nonspecific binding was defined with
100 µM histamine. All binding reactions were terminated by
vacuum filtration onto polyethylenimine (0.3% and 0.5% for H4
receptor binding only) presoaked GF/B filter plates (Packard)
followed by three brief washes with 2 mL of ice-cold binding buffer
with the exception that the binding buffer for the H1 and H3
receptors contained 500 mM NaCl. Liquid scintillation counting
was used to determine bound radiolabel. IC50 values and Hill slopes
were determined by a four parameter logistic model using Activi-
tyBase (ID Business Solution, Ltd.). pKi values were determined
by the generalized Cheng-Prusoff equation (Cheng and Prusoff,
1973).
H3R GTPγS-Binding Assay. This assay was performed to
determine the activity of compounds at human H3 receptors using
membranes from CHO cells (Euroscreen, Belgium). After thawing,
the membranes were suspended in 20 mM HEPES-NaOH buffer
(pH7.4) containing 1 mM MgCl2, 100 mM NaCl, 45 µg/mL saponin
and 10 µM GDP. Membrane suspension and wheat germ agglutinin
SPA beads (Amersham) were mixed (beads, 13 mg/mL; mem-
branes, 150 µg protein/mL). GTPγ35S binding was performed in
96-well microplates in a total volume of 180 µL with 30 µg of
membrane proteins and 0.28 nM GTPγ35S. Nonspecific binding was
measured in the presence of 10 µM cold GTPγS. Plates were sealed
and agitated (350 rpm) at room temperature for 2 h. The beads
were then settled by centrifugation (1000 rpm, 10 min), and the
plate was counted in a Top count using quench correction.
Rat and Human Microsomal Clearances. Human or rat
microsome incubations were conducted by an automated procedure
implemented on a Genesis workstation (Tecan, Switzerland).
Compounds (2 µM) were incubated in microsomes at 0.5 mg
protein/mL in a 50 mM potassium phosphate buffer, pH 7.4, at 37
°C. Cofactor (NADPH) was produced by a generating system
(glucose 6-phosphate, 3.2 mM; ꢀ-NADP, 2.6 mM; MgCl2, 6.5 mM).
Addition of the NADPH generating system to the prewarmed
microsomes containing the test compound started the reaction.
Aliquots (50 µL) were taken at six defined time points within 30
min and transferred into 100 µL of methanol containing an internal
standard. Concentration of each compound was analyzed by
LC-MS/MS using a Synergy-4-Polar RP 18 column (Phenomenex,
Chronic DIO SD Rat Model. To evaluate the long-term efficacy
of H3R inverse agonists on body weight development, food intake,
body fat, and plasma parameters, a chronic DIO SD rat model was
used. After separation of responders from nonresponders to the
HFD, responders were adapted to their housing for 2 weeks for
adaptation. In parallel, animals were monitored for food intake and
body weight daily. Animals were stratified into homogeneous
treatment groups according to their food intake, body weight, and
body fat. In order to assess possible effects on body weight and fat
depots beyond a reduction of food intake, a pair-fed group was
run in parallel to the 10 mg/kg dose group, given the exact amount
of food of the treatment group with a 24 h delay. Any improvement
of the metabolic status would therefore be distinguishable from the
effect of body weight reduction.
Acknowledgment. The authors thank Dr. Holger Fischer
for his help during the use of Roche structure-activity relation-
ship analyzer (ROSARA) and Dr. Liudmila Polonchuk for the
measurement of the hERG channel inhibitions.
Supporting Information Available: Preparation details of all
compounds described in Table 1 and their intermediates, together
with spectroscopic data; table of elemental analysis data of target
compounds; description of the pKa measurement, lipophilicity
determination (log D), lyophilization solubility assay (LYSA),
hERG inhibition assay, rat pharmacokinetic measurement, and
behavioral satiety sequence (BSS) method. This material is available
free of charge via the Internet at http://pubs.acs.org.

3866 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 13
Pierson et al.
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