Design and synthesis of novel inhibitors of human kynurenine aminotransferase-I

Article abstract of DOI:10.1016/j.bmcl.2011.12.138

Herein we report 6-ethoxy-6-oxo-5-(2-phenylhydrazono) hexanoic acid and 3-(2-carboxyethyl)-1Hindole-2-carboxylic acid derivatives as synthetically accessible leads for human kynurenine aminotransferase-I (KAT-I) inhibitors. In total, 12 compounds were synthesized and their biological activities were determined using the HPLC-UV based KAT-I inhibition assay. Of the 12 compounds synthesized, 10 were found to inhibit human KAT-I and the most active compound was found to be 5-(2-(4-chlorophenyl) hydrazono)-6-ethoxy-6-oxohexanoic acid (9a) with an IC50 of 19.8 lM.

Full text of DOI:10.1016/j.bmcl.2011.12.138

Bioorganic & Medicinal Chemistry Letters 22 (2012) 1579–1581
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design and synthesis of novel inhibitors of human kynurenine
aminotransferase-I
Fady N. Akladios a, , Naveed A. Nadvi a,b, , Joohong Park b, Jane R. Hanrahan a, Vimal Kapoor c,
Mark D. Gorrell b, W. Bret Church a,
⇑
a Faculty of Pharmacy A15, The University of Sydney, Sydney NSW 2006, Australia
b Centenary Institute, Sydney Medical School, The University of Sydney, Sydney NSW 2006, Australia
c School of Medicine and Pharmacology, The University of Western Australia, Perth WA 6009, Australia
a r t i c l e i n f o
a b s t r a c t
Article history:
Herein we report 6-ethoxy-6-oxo-5-(2-phenylhydrazono) hexanoic acid and 3-(2-carboxyethyl)-1H-
indole-2-carboxylic acid derivatives as synthetically accessible leads for human kynurenine aminotrans-
ferase-I (KAT-I) inhibitors. In total, 12 compounds were synthesized and their biological activities were
determined using the HPLC–UV based KAT-I inhibition assay. Of the 12 compounds synthesized, 10 were
found to inhibit human KAT-I and the most active compound was found to be 5-(2-(4-chlorophenyl)
Received 27 October 2011
Revised 28 December 2011
Accepted 29 December 2011
Available online 10 January 2012
hydrazono)-6-ethoxy-6-oxohexanoic acid (9a) with an IC50 of 19.8 lM.
Keywords:
Schizophrenia
Ó 2012 Elsevier Ltd. All rights reserved.
Kynurenic acid
Kynurenine
Kynurenine aminotransferase-I
Phenyl hydrazones
Indole propionic acid
Schizophrenia is a complex neuropsychiatric disorder which is
caused by a combination of numerous genetic and environmental
factors leading to neurochemical disturbances.1 Several hypothe-
ses have been proposed with the aim of understanding the etiology
of such disturbances. Deficit in glutaminergic neurotransmission
has been linked to the disease, especially after the original observa-
tion of reduced glutamate levels in the cerebrospinal fluid (CSF) of
schizophrenic patients.2 Glutaminergic hypofunction provided a
better explanation of neurochemical disturbances in schizophrenia
due to studies implicating Kynurenic acid (KA). Kynurenic acid is
5-phosphate (PLP) as cofactor.6 Four different KAT orthologs (KAT
I–IV) are expressed in mammalian brain and their relative impor-
tance appears to vary with species, tissue and developmental stage.
All KAT orthologs are capable of catalyzing the same transamina-
tion reaction of Kyn to KA using different
a-ketoacids as amino
group acceptor.7 Compensatory upregulation of KAT-I and KAT-III
expression has been found to occur in transgenic kat2À/À mice,
demonstrating the importance of these two homologs.8
KAT-I, also known as glutamine transaminase K9 is inhibited by
competing substrates like tryptophan and phenylalanine which are
present in the human body under physiological conditions.10,11
These observations suggest that the major functional moieties
facilitating the binding of competitive inhibitors are the hydropho-
bic aromatic ring and the carboxylic acid. Studies on six different
indole molecules which were structurally similar to tryptophan
have been reported (Fig. 1, structures 1–6).12 Owing to the lack
the only known endogenous antagonist of N-methyl D-aspartate
subtype of glutamate receptor in brain.3 Investigations into the
association of KA with schizophrenia revealed high concentrations
of KA in postmortem prefrontal cortex4 and in the CSF5 of schizo-
phrenics when compared with normal brain samples.
Human kynurenine aminotransferases (KATs) are members of
the aminotransferase family which catalyse the transamination
of kynurenine (Kyn) into Kynurenic acid (KA) utilizing pyridoxal
of
can be transaminated by human KAT-I (hKAT-I). Indole-3-propi-
onic acid (6) was reported to have the lowest IC50 of 140 20
M.12
a-amino and a-keto groups, none of the molecules 1, 4, 5, 6
l
The crystal structures of hKAT-I in complex with some compet-
itive inhibitors have been solved. The first crystal structure of
Abbreviations: CSF, cerebrospinal fluid; KA, kynurenic acid; Kyn, kynurenine;
PLP, pyridoxal 5-phosphate; KAT, kynurenine aminotransferase; hKAT-I, human
KAT-I.
hKAT-I revealed was in complex with L-phenylalanine (PDB ID:
⇑
Corresponding author. Tel.: +61 2 9036 6569; fax: +61 2 9351 4391.
E-mail address: bret.church@sydney.edu.au (W. Bret Church).
1W7L).13 Later, the crystal structure of hKAT-I in complex with in-
dole-3-acetic acid (IAC) was reported (PDB ID: 3FVU)12 and the
These authors contributed equally to this work.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.12.138

1580
F. N. Akladios et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1579–1581
O
O
O
OH
OH
OH
O
NH2
N
H
N
H
N
H
(1) Indole-3-acetic acid
(3) Indole-3-pyruvic acid
(2) Tryptophan
O
O
O
OH
OH
OH
OH
N
H
N
H
N
H
(4) Indole-3-lactic acid
(6) Indole-3-propionic acid
(5) Indole-3-acrylic acid
Figure 1. Chemical structures of six indole derivatives (1–6), reported to inhibit hKAT-I.12
binding interactions of IAC inside the substrate binding site of
hKAT-I were identified (Fig. 2). The indole ring of IAC is inserted
into a hydrophobic pocket defined by several residues, including
Tyr63, His279, Phe278, Tyr101, and Phe125, while the carboxylic
end forms a salt bridge with the guanido group Arg398.
In search for new leads for hKAT-I inhibitors, we now report
6-ethoxy-6-oxo-5-(2-phenylhydrazono) hexanoic acid and 3-(2-
carboxyethyl)-1H-indole-2-carboxylic acid derivatives as novel
synthetically accessible inhibitors of KAT-I. The investigation of
their inhibitory actions has provided much insight into the under-
standing of hKAT-I inhibition and afforded new lead compounds
for the development of more potent KAT-I inhibitors. The interac-
tions of 3-chlorophenylhydrazone hexanoic acid derivative (9f) in-
side the substrate binding site were predicted and superimposed
on IAC (Fig. 2).
The diazonium salts (7a–g) were prepared from commercially
available 3 or 4-substituted anilines using sodium nitrite and
hydrochloric acid. A Japp-Klingmann condensation of the diazo-
nium salts with the enolate anion of ethyl 4-oxocyclopentane car-
boxylate and subsequent hydrolysis afforded the phenylhydrazono
hexanoic acid derivatives (9a–g) in moderate to good yields.14,15
Indoles (10a,b,d,e) were prepared via Fischer indole cyclisation in
the presence of sulfuric acid as catalyst.16 However cyclisation of
4-nitrophenylhydrazono hexanoic acid (9c) using sulfuric acid
was unsuccessful, with only esterification occurring yielding
diethyl 2-(2-(4-nitrophenyl)hydrazono)hexanedioate. Therefore
the diester product of 9c was treated with p-toluene sulfonic acid
in benzene to effect the indolisation yielding ethyl 3-(3-ethoxy-3-
oxopropyl)-5-nitro-1H-indole-2-carboxylate (10c).17 Hydrolysis in
ethanolic sodium hydroxide at reflux gave the corresponding
dicarboxylic acids (11a–e) (Scheme 1).
The hKAT-I inhibition assays for compounds (9a–g) and (11a–e)
were performed as previously described.11,18 The normalised KA
values were used in the statistical analysis using non-linear regres-
sion to obtain the IC50 of the inhibitors. Table 1 shows the IC50 val-
ues of the synthesised molecules, and Figure S1 shows dose
response inhibition curves of four representative inhibitors.
The substituent on the phenyl or indole ring can be seen to have
a significant effect on the potency of the inhibitor. The presence of
strongly electron withdrawing groups such as a nitro substituent
(9c) on the phenyl ring of the phenylhydrazone hexanoic acid ser-
ies of compounds led to an increase in the IC50 (179
lM) compared
to the methyl substituted compound (9d) (IC50 33
lM). On the
other hand the presence of hydrophobic groups and especially hal-
ogens results in increased potency of inhibition with a subsequent
decrease in IC50. The 3- and 4-chloro substituted compounds, 9f
and 9a, respectively, both had IC50 values of 20
ogen containing phenylhydrazone hexanoic acid series, the 4-iodo
substituted compound (9g) was the weakest with IC50 of 34 M.
The most potent indole based inhibitor we found (11b), was
lM. Of the four hal-
l
Figure 2. Stereo view of the interaction of the ligand 9f with hKAT-1 superimposed on indole acetic acid (IAC). The polypeptide and cofactor within 3.50 Å of the IAC were
chosen for display. 9f is shown with carbons in cerise, and IAC in grey. The PDB ID of the structure used is 3FVU12 and the figure was created with MOLSCRIPT.19

F. N. Akladios et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1579–1581
1581
Scheme 1. Reagents and conditions: (a) NaNO2/HCl, À5 °C, 1 h; (b) ethyl 2-oxocyclopentane carboxylate/4 M KOH, 0 °C; (c) H2SO4/ethanol, reflux overnight; (d) p-toluene
sulfonic acid, benzene, reflux overnight (for compound 10c); (e) 2 M NaOH/ethanol, reflux, 24 h.
Table 1
to Alaina Ammit, Noeris Salam, David Hibbs, Lenka Munoz and
IC50 values (lM) of the synthesised inhibitors of hKAT-I
Nick West for their support. This work was supported by The Re-
becca L. Cooper Medical Research Foundation (WBC, MDG) and Sel-
by Research Award (WBC), an Australian Postgraduate Award
(NAN) and University of Sydney Postgraduate Award(JP). The
authors wish to thank members of their respective research groups
for a supportive environment and their collegiality.
Compounds
X
Y
IC50 SD (
20
21 15
179 43
lM)
9a
9b
9c
9d
9e
9f
9g
11a
11b
11c
11d
11e
Cl
Br
NO2
CH3
OCH3
H
I
Cl
Br
H
H
H
H
H
Cl
H
H
H
H
H
H
8
33
43
6
4
20 14
34 20
293 29
271 51
666 74
>1000
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.12.138.
NO2
CH3
OCH3
>6000
References and notes
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determined to have IC50 of 271 51 lM, which is over 10 fold
weaker than the most potent phenylhydrazone compounds (9a
and 9f).
In this work, we have described the synthetic methodology to
produce a series of phenylhydrazone hexanoic acid and indole
based compounds as potential new classes of KAT-I inhibitors.
The synthesized compounds were assayed for their inhibitory
activity against recombinant human KAT-I enzyme and were found
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to have IC50 values ranging from 20 to 6000 lM. Interestingly, the
phenylhydrazone compounds were found to be more potent as
KAT-I inhibitors than the indole based compounds. It is notable
that to date the most potent KAT-I inhibitor reported is indole-3-
propionic acid (6) with an IC50 140 200 lM, making the 3- and
4-chloro substituted phenylhydrazone hexanoic acids, approxi-
mately seven fold more potent than indole-3-propionic acid.
Therefore, our approach has led to the identification of a new class
of potent KAT-I inhibitors.
17. Johnson, G.; Malone, T. C.; Yuen, P.-W. Substituted 2-carboxylindoles having
pharmaceutical activity, US Patent 5,145,845, 1992.
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Acknowledgments
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The authors thank Bruce Tattam for technical assistance with
mass spectrometry measurements. The authors are also grateful