Substituted 3-(4-(1,3,5-triazin-2-yl)-phenyl)-2-aminopropanoic acids as novel tryptophan hydroxylase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2009.07.005

Tryptophan hydroxylase (TPH) is a key enzyme in the synthesis of serotonin. As a neurotransmitter, serotonin plays important physiological roles both peripherally and centrally. Here we describe the discovery of substituted triazines as a novel class of tryptophan hydroxylase inhibitors. This class of TPH inhibitors can selectively reduce serotonin levels in murine intestine after oral administration without affecting levels in the brain. These TPH 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:10.1016/j.bmcl.2009.07.005

Bioorganic & Medicinal Chemistry Letters 19 (2009) 5229–5232
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Substituted 3-(4-(1,3,5-triazin-2-yl)-phenyl)-2-aminopropanoic acids
as novel tryptophan hydroxylase inhibitors
a
a
a
a
Haihong Jin a, , Giovanni Cianchetta , Arokiasamy Devasagayaraj , Kunjian Gu , Brett Marinelli ,
Lakshman Samala a, Sheldon Scott a, Terry Stouch a, Ashok Tunoori a, Ying Wang a, Yi Zang a,
Chengmin Zhang a, S. David Kimball a, Alan J. Main a, Zhi-Ming Ding b, Weimei Sun b, Qi Yang b,
Xiang-Qing Yu b, David R. Powell b, Alan Wilson b, Qingyun Liu b, Zhi-Cai Shi a
*
a Lexicon Pharmaceuticals, 350 Carter Road, Princeton, NJ 08540, United States
b Lexicon Pharmaceuticals, 8800 Technology Forest Place, The Woodlands, TX 77381, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Tryptophan hydroxylase (TPH) is a key enzyme in the synthesis of serotonin. As a neurotransmitter, sero-
tonin plays important physiological roles both peripherally and centrally. Here we describe the discovery
of substituted triazines as a novel class of tryptophan hydroxylase inhibitors. This class of TPH inhibitors
can selectively reduce serotonin levels in murine intestine after oral administration without affecting lev-
els in the brain. These TPH inhibitors may provide novel treatments for gastrointestinal disorders asso-
ciated with dysregulation of the serotonergic system, such as chemotherapy-induced emesis and
irritable bowel syndrome.
Received 15 March 2009
Revised 23 June 2009
Accepted 2 July 2009
Available online 8 July 2009
Keywords:
5-HT
TPH1
Ó 2009 Elsevier Ltd. All rights reserved.
Tryptophan hydroxylase
Gastrointestinal disorders
Serotonin (5-hydroxytryptamine, 5-HT) is an important sub-
stance involved in the modulation of behavior and control of gas-
trointestinal (GI) function. The rate-limiting enzyme of serotonin
synthesis in the GI tract is tryptophan hydroxylase type I (TPH1).
Serotonin is known to markedly influence bowel motility by acti-
vating at least five receptor types, namely 5-HT 1–4 and 7.1 Irrita-
ble bowel syndrome (IBS) is a functional gastrointestinal disorder
characterized by abdominal pain, diarrhea, and constipation. The
role of 5-HT in the pathophysiology of IBS has been investigated.2
In IBS patients with diarrhea-predominant symptoms (IBS-D), the
number of enterochromaffin cells is significantly enhanced3 and
the plasma 5-HT levels following a meal are significantly higher.4
Furthermore, 5HT-3 antagonists are proven to be effective in the
treatment of IBS-D.5 These findings suggest that modulation of 5-
HT in the gut via inhibition of TPH1 is a potential treatment for
functional GI disorders.
essential for the TPH1 in vitro potency and cannot be replaced. On
the other hand, the pyrazine core (e.g. compound 2) can be re-
placed by pyrimidine (compound 1) or triazine. In this Letter, we
focus on the discovery of substituted 3-(4-(1,3,5-triazin-2-yl)-phe-
nyl)-2-aminopropanoic acids as novel TPH1 inhibitors.
During the optimization of hit compound 2, different heterocy-
clic replacements for the pyrazine moiety were investigated with
regard to their inhibition of TPH1. Our results showed that the pyr-
azine core is sensitive to change but could be effectively replaced
by 2-amino-triazine. Additional triazine derived analogs ( 6a–l,
7a–m) were prepared to investigate SAR. The general synthetic
route is outlined in Scheme 1. Nucleophiles 3, such as amines or
alcohols, reacted with a variety of dichloro triazines 4 through
nucleophilic aromatic substitution to form intermediate chlorides
represented by 5. Suzuki coupling of 5 with L-phenylalanine–boro-
nic acid using bis(triphenylphosphine) palladium(II) dichloride as
the catalyst and sodium carbonate as the base gave the target com-
pounds 6 and 7 which were generally purified by preparative HPLC
using a MeOH/water/TFA solvent system.
We recently disclosed the discovery and structure–activity rela-
tionship (SAR) of small molecule TPH inhibitors, exemplified by 1
(Fig. 1).6 These molecules can selectively reduce peripheral seroto-
nin levels without affecting brain serotonin levels after oral admin-
istration. We also disclosed the key SAR for these TPH1 inhibitors.
In vitro inhibitory potency (IC50) was obtained using a continu-
ous fluorescence assay which measured inhibition of purified, re-
For example, we demonstrated that the
L
-phenylalanine moiety is
combinant human TPH1.7 Cellular potency (EC50
)
was
determined by measuring the inhibition of serotonin biosynthesis
in the rat mastocytoma cell line RBL-2H3.8 Compounds were
examined in both the TPH1 fluorescence assay and RBL cellular as-
* Corresponding author. Tel.: +1 609 466 5593; fax: +1 609 466 3562.
E-mail address: hjin@lexpharma.com (H. Jin).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.07.005

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H. Jin et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5229–5232
O
O
F
OH
OH
N
N
NH2
O
NH2
CF3
N
N
N
H
NH2
1
2
Figure 1. Chemical structures of compounds 1 and 2.
say. As shown in Table 1, introduction of the 2-amino triazine re-
sulted in compound 6a having an IC50 of 0.5 M against TPH1,
which is slightly more potent than the HTS hit, pyrazine 2
(IC50 = 0.67 M). The effect of varying substitutions on the distal
sis by introducing a bulky fused ring system. Adamantane analog
6l proved highly potent (IC50 = 0.019 M) and demonstrated an
l
l
additional threefold improvement in cell potency compared to 6k.
As illustrated in Table 2, we continued to look at sizeable hydro-
phobic substitutions on the left part of the molecule. To this end,
we decided to introduce a naphthalene moiety. Not surprisingly,
naphthalene analog 7a (Table 2) showed a good potency of
l
phenyl ring was evaluated. A fourfold in vitro potency increase
was observed when either electron withdrawing groups, such as
a trifluoromethyl group (6b), or electron donating groups, such
as methyl groups (6c) were substituted on the phenyl ring. How-
ever, these changes did not improve the cell potency. Replacement
of the phenyl ring with cyclohexyl gave analog 6d which was equi-
potent to the substituted phenyl derivatives. Using the published
apo-structure of human TPH1, a homology model was constructed
which suggested that the binding pocket could accommodate lar-
ger groups on the left side of the molecule. Biphenyl analogs were
designed to test the hypothesis. Compared to 6a, the para-biphenyl
0.024
at the benzylic position. The (R)-2-methyl amino isomer 7b
(IC50 = 0.026 M) demonstrated much better cellular potency than
lM against TPH1. We next investigated the stereochemistry
l
(S) isomer 7c but maintained similar primary potency. Further-
more, we evaluated modulating the nature of the linker of 7b. As
shown in Table 2, 7d (IC50 = 0.055 lM, EC50 = 1.7 lM) with a gem-
inal dimethyl group exhibited a twofold decrease in enzyme po-
tency and a fivefold decrease in cell potency compared with 7b.
6e (IC50 = 0.06
fold in the biochemical assay. Moving the distal phenyl ring to
the ortho-position resulted in 6f (IC50 = 0.007 M, EC50 = 2.2 M),
which further improved the primary potency. meta-Biphenyl 6g
(IC50 = 0.028 M, EC50 = 1.4 M) improved primary potency by
twofold compared with 6e. Interestingly, these biphenyl analogs
improved the cellular potency more than sixfold compared with
the corresponding phenyl analogs. Electronic effects on the biphe-
nyl system were also investigated. Introduction of electron donat-
l
M, EC50 = 1.8
l
M) increased the activity by eight-
Trifluoromethyl substituted alkoxy compound 7e (IC50 = 0.32
EC50 = 9.6 M) and trifluoromethyl substituted amine 7f
(IC50 = 0.33 M, EC50 = 6.3 M) both proved detrimental to the en-
zyme and cellular potencies. N-methylation of 7b resulted in 7g
(IC50 = 0.05 M, EC50 = 0.48 M), which showed a twofold decrease
in primary potency and had little effect on cell potency. Changing
the -methyl group to ethyl, isobutyl, and isopropyl (7h, 7i, and 7j,
respectively) resulted in loss of cell potency. Efforts toward modi-
fying the triazine substitution are also summarized in Table 2.
Morpholino triazine 7k, 2-N-methyl triazine 7l and des-amino tri-
azine 7m all had reduced potencies.
An X-ray crystal structure of 7b co-crystallized with TPH1 was
obtained (Fig. 2) after our SAR exploration on the triazine series.9
This crystal structure revealed that the carboxylate group of com-
pound 7b interacted with Arg257 through a charge–charge interac-
tion and accepted two hydrogen bonds: one from the backbone
nitrogen of Thr265 and another one from the hydroxyl group on
lM,
l
l
l
l
l
l
l
l
l
a
ing groups, such as
EC50 = 2.3 M) retained potency while electron withdrawing
groups (e.g., 6j, IC50 = 0.16 M, EC50 = 4.1 M) led to decreased
a methyl group (6i, IC50 = 0.014 lM,
l
l
l
in vitro potency. Phenoxyphenyl analog 6h displayed fourfold re-
duced in vitro potency relative to 6g. The addition of a benzylic
a-methyl group as in 6k (IC50 = 0.024 lM, EC50 = 2.5 lM) increased
in vitro potency by more than twofold compared with the corre-
sponding unsubstituted analog 6e. We further tested our hypothe-
R2
R1
R3
X
N
Y
Cl
Cl
N
Cl
a
R2
X
+
R3
R1
N
N
N
N
Y
3
4
5
4-1 Y=NH2
b
4-2
4-3
Y=H
Y=NHCH3
O
4-4
N
O
Y=
OH
R2
R1
R3
X
N
Y
NH2
N
N
6a-l
7a-m
Scheme 1. General procedure for the synthesis of triazine analogs. Reagents and conditions: (a) 1,4-dioxane, K2CO3, reflux, 95%; (b)
(PPh3)2PdCl2, 1 M Na2CO3, acetonitrile, microwave, 150 °C, 10 min, 92%; all compounds were characterized by 1H NMR, LC/MS, and HPLC.
L-phenylalanine-boronic acid,

H. Jin et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5229–5232
5231
Table 1
Table 2
In vitro inhibitory and cellular potencies of 6a–l
In vitro inhibitory and cellular potencies of 7a–m
O
O
OH
OH
R2
R1
H
H
R2
N
N
NH2
X
N
Y
NH2
R1
N
N
N
N
NH2
R1
R2
X
Y
TPH1 IC50
(lM)
RBL EC50 (lM)
Example
R2
R1
H
TPH1IC50
0.5
(lM)
RBLEC50
>11
(lM)
7a
7b
7c
7d
7e
7f
H
H
H
H
CH3
CF3
CF3
CH3 (R)
CH2CH3
NH
NH
NH
NH
O
NH
NCH3
NH
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
0.024
0.026
0.044
0.055
0.32
0.33
0.05
0.15
2.1
0.3
2.8
1.7
9.6
6.3
0.48
2.3
*
CH3(R)
CH3(S)
CH3
H
H
6a
*
6b
6c
H
H
0.12
0.10
36
7g
7h
H
H
F3C
*
7i
H
H
H
NH
NH
NH
NH2
NH2
0.12
0.25
15.6
>11
5.7
*
>11
7j
*
N
O
6d
6e
H
H
0.12
0.06
>11
1.8
7k
H
>40
*
7l
7m
H
H
CH3 (R)
CH3
NH
NH
NHCH3
H
0.61
0.19
10.2
16.2
*
the side chain of Ser336. The amino group of the amino acid moiety
in compound 7b formed three hydrogen-bond interactions: one
with the carbonyl oxygen of Thr265 and two with water molecules.
The phenyl ring in 7b made contacts with Pro268 and His272
through hydrophobic interactions. The triazine ring formed an
6f
H
0.007
2.2
edge-to-face
p–p interaction with Phe313, being placed slightly
more than 4 Å away from the ring plane.
*
6g
6h
H
H
0.038
0.15
1.4
3.4
Furthermore, one of the nitrogen atoms of the triazine ring was
within hydrogen bond distance from one of the water molecules
that coordinates the iron atom. Another nitrogen atom formed a
hydrogen bond with a bridging water molecule, and the 2-amino
group established an additional hydrogen-bond interaction with
another water molecule.
O
*
*
The X-ray structure showed that the naphthalene moiety was
sandwiched between the Tyr235 side chain (p–p-edge-to-face
stacking) and Pro268 (hydrophobic interaction) and restricted ac-
cess to the pocket that is normally occupied by the pterin cofactor.
6i
6j
H
H
0.014
0.16
2.3
4.1
*
F3C
*
6k
6l
CH3
CH3
0.024
0.019
2.5
*
0.82
Figure 2. Crystal structure of TPH1 and 7b.

5232
H. Jin et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5229–5232
The pharmacokinetic profile of compound 7b was examined in
gastrointestinal diseases caused by dysregulation of the serotoner-
gic pathway in the periphery.
two groups of C57-albino male mice. One group received an intra-
venous injection of 1 mg/kg, and another was orally dosed at
10 mg/kg. A low plasma clearance (CL = 0.5 L/h/kg) and a relatively
small volume of distribution (VSS = 0.5 L/kg) were observed follow-
ing the intravenous injection. Following oral gavage dosing, the
Acknowledgements
The authors thank Ken Carson, Joseph Barbosa for their helpful
discussions and Jay Mitchell for assistance in manuscript
preparation.
compound was poorly absorbed with
a
Cmax = 0.5 lM,
AUC = 1.6 M h and an oral bioavailability of only 5%.
l
Compound 7b was examined in vivo by administering the com-
pound to mice by intraperitoneal injection (ip) and oral gavage (po)
at a dose level of 100 mg/kg twice a day (200 mg/kg/day) for five
days.8 Levels of 5-HT in the blood and intestine were then mea-
sured. We observed a 17% decrease of 5-HT levels in the blood
and a 38% decrease of 5-HT levels in duodenum following oral
administration. There were a 26% decrease in blood 5-HT and a
37% decrease in duodenum 5-HT following IP administration. In a
follow-up study, the compound was given at 100 mg/kg, twice a
day by IP for eight days. A 50% decrease in blood 5-HT and a 57%
decrease in duodenum 5-HT was observed in this case. Brain 5-
HT levels, however, were not significantly altered.
In summary, we have described another novel class of potent
TPH inhibitors. TPH inhibitors, such as 7b, can selectively inhibit
peripheral 5-HT synthesis without effects on the central nervous
system because of very poor systemic exposure and the inability
to cross the bloodÀbrain barrier. This novel class of peripheral
TPH inhibitors may provide potential treatments for a variety of
References and notes
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been deposited at Protein Data Bank file name: 3HF6.