Modulation of peripheral serotonin levels by novel tryptophan hydroxylase inhibitors for the potential treatment of functional gastrointestinal disorders

Article abstract of DOI:10.1021/jm800338j

The discovery of a novel class of peripheral tryptophan hydroxylase (TPH) inhibitors is described. This class of TPH inhibitors exhibits excellent potency in in vitro biochemical and cell-based assays, and it selectively reduces serotonin levels in the murine intestine after oral administration without affecting levels in the brain. These TPH1 inhibitors may provide novel treatments for gastrointestinal disorders associated with dysregulation of the serotonergic system, such as chemotherapy-induced emesis and irritable bowel syndrome.

Full text of DOI:10.1021/jm800338j

3684
J. Med. Chem. 2008, 51, 3684–3687
90% of the body’s 5-HT is synthesized and stored, 5-HT plays
a general prokinetic role and is an important mediator of
sensation (e.g., nausea and satiety) between the GI tract and
the brain.10–12 Dysregulation of the peripheral 5-HT signaling
system is involved in the etiology of several conditions such as
functional GI disorders, chemotherapy-induced emesis, and heart
valve damage.13–16 The large number of pharmaceutical agents
that block or stimulate 5-HT receptors is indicative of the wide
range of medical disorders that have been associated with 5-HT
dysregulation.17 One example, p-chlorophenylalanine (pCPA),
a weak inhibitor of TPH, has proven effective in treating
chemotherapy-induced emesis, as well as diarrhea, in carcinoid
tumor patients. Unfortunately, administration of pCPA has been
linked to the onset of depression and other alterations of CNS
function in patients.18–20
Modulation of Peripheral Serotonin Levels by
Novel Tryptophan Hydroxylase Inhibitors for
the Potential Treatment of Functional
Gastrointestinal Disorders
Zhi-Cai Shi,*,†,§ Arokiasamy Devasagayaraj,†,§
Kunjian Gu,†,§ Haihong Jin,†,§ Brett Marinelli,†,§
Lakshman Samala,†,§ Sheldon Scott,†,§ Terry Stouch,†,#
Ashok Tunoori,†,§ Ying Wang,†,§ Yi Zang,§
Chengmin Zhang,†,§ S. David Kimball,†,§ Alan J. Main,†,§
Weimei Sun,†, Qi Yang,‡, Amr Nouraldeen,‡,¶
Xiang-Qing Yu,‡,¶ Eric Buxton,‡, Shiv Patel,‡,
Nghi Nguyen,‡, Jon Swaffield,‡, David R. Powell,‡,
Alan Wilson,‡,¶ and Qingyun Liu‡,
In contrast to known serotonin reduction treatments, disrup-
tion of the mouse TPH1 gene results in loss of most peripheral
5-HT while leaving neuronal levels unaffected.8,21 We therefore
hypothesized that TPH inhibitors could be developed to
selectively modulate only peripheral serotonin levels, thereby
avoiding CNS-related side effects. Such selectivity can be
achieved by developing (1) a TPH1 inhibitor that is selective
against TPH2, (2) a TPH inhibitor that does not penetrate the
blood-brain barrier, (3) a TPH inhibitor that acts locally in the
GI tract, or (4) a combination of these approaches.
Herein, we report the discovery of a novel class of tryptophan
hydroxylase inhibitors that selectively reduce peripheral sero-
tonin levels in animal models without affecting central serotonin
levels. Compounds within that class were optimized to act
locally in the intestine with minimal systemic exposure after
oral administration and do not penetrate the blood-brain barrier.
When administered orally to mice, these compounds were able
to reduce 5-HT levels in the GI tract without affecting brain
5-HT levels.
Several biological assays were developed to determine the
inhibitory effect of compounds against TPH in vitro and in vivo.
In vitro biochemical assays for human, mouse, and rat TPH1
and human TPH2, PAH, and TH were used to measure
inhibition of enzyme activity and the selectivity among TPH1,
TPH2, PAH, and TH. Cell based assays using rat mastocytoma
cells (RBL) or human carcinoid cells (BON) were developed
to measure inhibition of serotonin biosynthesis in whole cells.
In addition, an in vivo tissue 5-HT assay was developed to
determine the effect of compounds on 5-HT levels in various
tissues after oral administration of TPH inhibitors.21
Discovery of the compounds began with high-throughput
screening of a library of approximately 200,000 compounds
using a radiometric enzyme assay. Several classes of hits were
identified. Among them, phenylalanine derivative 1 was con-
firmed as a TPH1 inhibitor with an IC50 of 0.67 µM. The
S-isomer 1a was found to be the active form, with an IC50 of
0.24 µM against TPH1. p-Ethynylphenylalanine (pEPA) and
pCPA were tested in the same in vitro assays, side by side with
1a, and have an IC50 of 30 and 92 µM, respectively (Scheme
1). Compared to pCPA and pEPA, 1a shares the same
phenylalanine moiety but is about 380-fold more potent than
pCPA and 120-fold more potent than pEPA. It was envisioned
that 1a could be further optimized not only to improve in vitro
and in vivo potency but also to improve physicochemical
properties to achieve local action in the GI tract and to ensure
that no penetration of the blood-brain barrier occurred.
Lexicon Pharmaceuticals, 350 Carter Road,
Princeton, New Jersey 08540, and Lexicon Pharmaceuticals,
8800 Technology Forest Place, The Woodlands, Texas 77381
ReceiVed March 25, 2008
Abstract: The discovery of a novel class of peripheral tryptophan
hydroxylase (TPH) inhibitors is described. This class of TPH inhibitors
exhibits excellent potency in in vitro biochemical and cell-based assays,
and it selectively reduces serotonin levels in the murine intestine after
oral administration without affecting levels in the brain. These TPH1
inhibitors may provide novel treatments for gastrointestinal disorders
associated with dysregulation of the serotonergic system, such as
chemotherapy-induced emesis and irritable bowel syndrome.
Serotonin (5-hydroxytryptamine, 5-HTa) is a neurotransmitter
that modulates central and peripheral functions through action
on neurons, smooth muscle, and other cell types. The rate-
limiting step in serotonin biosynthesis is the hydroxylation of
tryptophan, which is catalyzed by tryptophan hydroxylase
(TPH). The resulting 5-hydroxytryptophan is then decarboxy-
lated by an amino acid decarboxylase to 5-HT.1–4 Together with
phenylalanine hydroxylase (PAH) and tyrosine hydroxylase
(TH), TPH belongs to the pterin-dependent aromatic amino acid
hydroxylase family.1–4 Two vertebrate isoforms of TPH, TPH1
and TPH2, have been described.5–7 TPH1 is primarily expressed
in the pineal gland and non-neuronal tissues, such as entero-
chromaffin (EC) cells of the gastrointestinal (GI) tract.5,8,9 TPH2
(the dominant form in the brain) is expressed exclusively in
neuronal cells, such as dorsal raphe or myenteric plexus cells.5,8,9
5-HT is involved in the control and modulation of multiple
physiological and psychological processes. In the central nervous
system (CNS), 5-HT regulates mood, appetite, and other
behavioral functions. In the GI system, where approximately
* To whom correspondence should be addressed. Phone: 609 466-5576.
Fax: 609 466-6079. E-mail: zshi@lexpharma.com.
†
Lexicon Pharmaceuticals, NJ.
Department of Medicinal Chemistry.
Department of Computational Chemistry.
Department of Pharmaceutical Discovery.
Lexicon Pharmaceuticals, TX.
Department of Metabolic Diseases.
§
#
‡
¶
Department of Drug Metabolism, Pharmacokinetics, and Toxicology.
a Abbreviations: 5-HT, 5-hydroxytryptamine; TPH, tryptophan hydroxy-
lase; PAH, phenylalanine hydroxylase; TH, tyrosine hydroxylase; EC,
enterochromaffin; pCPA, p-chlorophenylalanine; GI, gastrointestinal; CNS,
central nervous system; RBL, rat mastocytoma cell; BON, human carcinoid
cell; pEPA, p-ethynylphenylalanine.
10.1021/jm800338j CCC: $40.75
2008 American Chemical Society
Published on Web 06/17/2008

Letters
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 13 3685
Table 1. In Vitro Potency of Compounds 9a–l
Scheme 1. Chemical Structures of 1a, pCPA, and pEPA
compd
R
TPH1 IC50 (µM)
9a
9b
9c
9d
9e
9f
9g
9h
9i
cyclohexyl
3-furanyl
4-cyanophenyl
2-pyridyl
3-quinolinyl
2-tetrahydronaphthyl
2-naphthyl
3-cyclopentyloxy-4-methoxyphenyl
3-(9-ethyl-9H-carbazolyl)
3,4-dimethoxyphenyl
4′-biphenyl
0.24
0.96
0.11
6.15
0.19
Scheme 2. Synthesis of TPH1 Inhibitors
0.046
0.013
0.045
0.031
0.069
0.044
0.040
9j
9k
9l
2′-methyl-ortho-biphenyl
Table 2. In Vitro Potency of L-Phe Analogues
Analogues were designed and synthesized to develop
structure-activity relationships of the lead molecules and to
improve potency and physicochemical properties. Scheme 2
outlines the general synthetic route for the preparation of these
analogues.
Intermediates 4 and 7 were made through reductive amination
of 2-amino-5-bromopyrazine 3 with various aldehydes 2 or
nucleophilic displacement of pyrimidine or triazine dichloride
6 with nucleophiles such as amines, thiols, or alcohols. Halides
4 and 7 were then coupled with 8 using microwave-assisted
Suzuki coupling to give 9 and 10. Compounds 9 and 10 were
purified by preparative HPLC using a MeOH/H2O/TFA system.
This finding was further confirmed by a solid-state crystal
structure of TPH1 protein with inhibitor 21 (LP-533401)21
(Figure 1A and Figure 2). As observed upon the binding of
ligands to the phenylalanine hydroxylases,22 this protein un-
dergoes substantial conformational change, with the mobile
loops “closing” on the ligand relative to the “partially closed”
form seen for the apo structure of tryptophan hydroxylase.3 Also,
Arg 287 moves to form a salt bridge with the ligands’
carboxylate groups. A web of hydrogen bonds ties the amino
portion of the amino acid to residues of the binding pocket
directly and via a bridging water molecule.
Once the SARs at the 2 and 5 positions of the pyrazine were
developed, our efforts turned to the modification of the pyrazine
core. A variety of heterocycles were studied. Several examples
are given in Table 3. The results suggest that the pyrazine core
is sensitive to alteration but can be replaced with a 2-amino-
4,6-disubstitued triazine (10g) or a 2-amino-4,6-disubstitued
pyrimidine (10h). The SAR that led to compounds with activity
in cells and in vivo were derived from the 2-amino-4,6-
disubstituted pyrimidine class.
During the development of SAR and optimization of lead
compounds, potent TPH1 inhibitors in biochemical assays were
tested in cell-based assays. One significant finding based on the
cell potency of these TPH1 inhibitors was that introduction of
an R-trifluoromethylalkoxy linker at the 4 position of the
2-amino-4,6-disubstituted pyrimidine analogues greatly im-
proved the TPH inhibitory activity in cells. Table 4 summarizes
the comparison of the R-methylamino linker (10i), R-methyl-
The initial SAR of the 2,5-disubstituted pyrazine class
revealed that a lipophilic substitutent at the 2 position of 2,5-
pyrazine was preferred. Arylmethylamino substituted analogues
generally had good in vitro potencies against TPH1. Introduction
of polarity at the 2 position decreased in vitro activity (Table
1).
The SAR also suggested that a 4′-phenylalanine moiety is
essential to the in vitro potency against TPH and cannot be
substituted by common acid isosteres. Replacement of phenyl-
alanine with phenylglycine, tetrahydroisoquinoline carboxylic
acid, or N-methyl-L-phenylalanine, as well as its corresponding
amino alcohol or R-aminotetrazole, all resulted in significant
loss of activity (11-15; chemical structures and data are in
Supporting Information).
Compared to 10a, analogues with various changes on the
L-phenylalanine moiety (10b, 10c, 16, and 17, Table 2) all
showed significantly decreased in vitro activity against TPH1.
Furthermore, replacing the phenyl group of the phenylalanine
moiety with pyridine, piperidine, or pyrazole (10d-f) signifi-
cantly reduces in vitro activity against TPH1. These data suggest
that the 4′-phenylalanine moiety is essential to the potency.

3686 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 13
Letters
Table 4. R-CF3 Alkoxy Linker Improves Cell Potency
compd
X/Y
TPH1 IC50 (µM)
RBL EC50 (µM)
10i
10j
10k
NH/CH3 (R)
O/CH3 (R)
O/CF3 (RS)
0.032
0.38
0.18
1.7
11
0.51
Table 5. In Vitro Potency of TPH1 Inhibitors
TPH1 IC50 RBL EC50 BON EC50
compd
A/X
(µM)
(µM)
(µM)
10l
Ph/3′-Me-Ph
Ph/2′-furanyl
Ph/3′-CF3-pyrazole
Thiophene/2′-furanyl
0.070
0.024
0.029
0.054
0.056
0.048
0.070
0.063
0.56
0.80
0.75
1.19
10m
10n
10o
Figure 1. TPH1 crystal structures.
The crystal structure of TPH1 with another inhibitor 9l further
revealed the binding modes of the lipophilic moiety of the TPH1
inhibitors within the protein’s active site (Figure 1B,C). The
pendent 2′-substituent on the pyrazine core is generally tolerant
of substitution, with lipophilic substituents being preferred.
However, the solid-state structures of 21 and 9l showed that
the orientations of the aryl substituent differ significantly.
For both ligands, this lipophilic moiety occupies the outermost
regions of the pterin cofactor binding site. In the case of 21, it
stretches across this site to make nonspecific interactions with
the outside of the protein. However, 9l binds with one ring
nestled into a shallow sub-binding pocket. It has enhanced
binding to TPH1 compared to the para-biaryl substitution pattern
(21). This is also in agreement with the in vitro TPH1 potency
of these two compounds, where the ortho-biaryl analogue 9l is
about 10 times more potent than the para-biaryl analogue (21).
This information was further applied to our optimization strategy
and the design of new analogues.
With the understanding of SAR, the binding mode in the
TPH1 active site, and the contribution of the R-trifluoromethyl-
alkoxy linker to the improved cell potency, we further optimized
the lead compounds by introducing a variety of ortho-biaryl
R-trifluoromethylalkoxy substituents at the 4 position of 2-amino-
4,6-disubstitued pyrimidine (Table 5). These compounds showed
excellent potencies in in vitro biochemical assays and good
cellular potencies.
Figure 2. Chemical structures of 21 and 22.
alkoxy linker (10j), and R-trifluoromethylalkoxy linker (10k).
The data showed that the R-CF3 alkoxy analogue (10k) has
decreased activity in the in vitro enzyme assay but has improved
activity in the cell-based assay. The ratio EC50/IC50 is about 3,
which is significantly lower than that for the R-methylalkoxy
analogue (ratio of 29).
Although the exact reason for the improved cell potency of
these R-CF3 alkoxy analogues is unknown, this finding is
consistent across a wide variety of structural variations in the
pendent aryl substituent and sets the stage for the in vivo
optimization of these compounds.
Table 3. Replacement of Pyrazine Core
compd
R
“core”
TPH1 IC50 (µM)
Compounds with good in vitro potencies were evaluated in
in vitro ADME assays and in vivo pharmacokinetic assays. In
general, these compounds have poor systemic exposure and do
not penetrate the blood-brain barrier in mouse pharmacokinetic
studies. For example, 21 showed a low plasma clearance (3.33
( 0.56 (mL/min)/kg) and a small volume of distribution (0.73
( 0.07 L/kg) following intravenous injection (1 mpk). After
oral administration, 21 was poorly absorbed with a low oral
bioavailability (7.6 ( 1.6%). The compound level in the brain
is negligible following oral administration, indicating no
penetration of the blood-brain barrier (see detailed 21 PK
results in Supporting Information).
9g
A
A
B
B
B
C
C
D
E
F
E
G
H
E
I
0.013
0.024
0.045
>10
10g
9h
18
19
9i
20
10h
0.97
0.031
>10
0.024
J
To test the in vivo efficacy of the compound, 21 was given
to mice orally at 30 or 90 mg/kg, twice a day, for a total of six

Letters
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 13 3687
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doses. Compared to the vehicle control group, 5-HT levels in
the small intestine (jejunum and ileum) were significantly lower
in the 21-dosed animals, averaging a 55% and 70% reduction
at 30 and 90 mg/kg, respectively. 5-HT levels in the colon were
also reduced, averaging a 24% and 36% reduction at 30 and 90
mg/kg, respectively. In contrast, no significant change in brain
5-HT levels was observed at either dose.21
Compound 22 (LP-615819),21 the ethyl ester prodrug of 21,
was also tested in the mouse side by side with a known TPH
inhibitor (pCPA) to compare their in vivo effects. Compound
22 was given orally at 45 mg/kg, while pCPA was given at 90
mg/kg, twice daily. After 3 days of dosing, both compounds
reduced 5-HT levels in the intestine, with 22 showing greater
efficacy in the jejunum. More importantly, pCPA, as expected,
significantly lowered 5-HT levels in the brain while 22 showed
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intestinal tract in a dose-dependent fashion with a 88% reduction
in the jejunum at 90 mg/kg. Most importantly, the compound
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In summary, we describe here a novel class of potent TPH
inhibitors. We show that these TPH inhibitors, such as 21 and
22, can selectively inhibit peripheral 5-HT synthesis without
affecting CNS 5-HT synthesis, because of the very poor
systemic exposure and their inability to cross the blood-brain
barrier. This novel class of peripheral TPH inhibitors may
provide potential treatment for a variety of gastrointestinal
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Supporting Information Available: Experimental details, ana-
lytical data of the compounds, in vitro potency data of 11-15, and
PK of LP-533401. This material is available free of charge via the
Internet at http://pubs.acs.org.
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E.; Brommage, R.; Cullinan, E.; Platt, K.; Wilson, A.; Powell, D.;
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