Pyrrolo[3,2-c]quinoline derivatives: A new class of kynurenine-3-hydroxylase inhibitors

Article abstract of DOI:10.1016/S0014-827X(99)00009-9

A series of pyrrolo[3,2-c]quinoline derivatives were synthesised and evaluated as inhibitors of selected enzymes of the kynurenine pathway. 7-Chloro-3-methyl-1H-pyrrolo[3,2-c]quinoline-4-carboxylic acid (7a) was found to be a relatively potent and selecti

Full text of DOI:10.1016/S0014-827X(99)00009-9

Il Farmaco 54 (1999) 152–160
Pyrrolo[3,2-c]quinoline derivatives: a new class of
kynurenine-3-hydroxylase inhibitors
Franco Heidempergher, Paolo Pevarello *, Antonio Pillan, Vittorio Pinciroli,
Arturo Della Torre, Carmela Speciale, Marina Marconi, Massimo Cini,
Salvatore Toma, Felicita Greco, Mario Varasi
Pharmacia & Upjohn, Chemistry Department, Viale Pasteur 10, I-20014 Ner6iano, Milan, Italy
Received 17 June 1998; accepted 30 December 1998
Abstract
A series of pyrrolo[3,2-c]quinoline derivatives were synthesised and evaluated as inhibitors of selected enzymes of the
kynurenine pathway. 7-Chloro-3-methyl-1H-pyrrolo[3,2-c]quinoline-4-carboxylic acid (7a) was found to be a relatively potent and
selective inhibitor of kynurenine-3-hydroxylase (KYN-3-OHase). A molecular modelling study showed a good superimposition of
7a with PNU-156561 and kynurenine the natural substrate of KYN-3-OHase. © 1999 Elsevier Science S.A. All rights reserved.
Keywords: Kynurenine; Hydroxylase; Enzyme inhibition
1. Introduction
disease and other neurodegenerative diseases where ex-
citotoxic mechanisms have been implicated [9].
With the aim of obtaining new KYN-3-OHase in-
hibitors we prepared and evaluated in vitro a series of
pyrrolo[3,2-c]quinoline derivatives (7a–c, 8, 9, 10) and
a correlated tetrahydroquinoline analogue 11. A molec-
ular modelling study was performed in order to identify
pharmacophoric features of these compounds by com-
paring the representative compound of this series (7a)
with PNU-156561, a potent benzoylalanine analogue
inhibitor [10] and with KYN, the natural substrate of
KYN-3-OHase.
It is well known that through the kynurenine (KYN)
pathway tryptophan metabolism gives rise to the for-
mation of both quinolinic acid (QUIN) and kynurenic
acid (KYNA) [1].
Kynurenine aminotransferase (KAT) is the enzyme
responsible for the synthesis of KYNA from KYN.
Kynurenine-3-hydroxylase (KYN-3-OHase) provides 3-
hydroxy-kynurenine (3-OH-KYN), which is further me-
tabolized to QUIN [2] (Fig. 1).
KYNA is endowed with neuroprotective properties
[3], whereas QUIN is a relatively potent neurotoxin [4]
which has been implicated in the pathogenesis of a
variety of neurological disorders [5]. The enzymes re-
sponsible for the synthesis of both KYNA and QUIN
are attractive pharmacological targets for shifting
kynurenine metabolism towards enhanced KYNA
synthesis.
Inhibitors of KYN-3-OHase, if devoid of KAT in-
hibitory activity, may play a role in the neuronal pro-
tection by increasing KYNA levels in the brain [6–8].
The strategy aimed at favourably altering KYNA/
QUIN balance may also be a useful tool in Parkinson’s
2. Results
2.1. Chemistry
The synthesis of the pyrrolo[3,2-c]quinoline deriva-
tives (7a–c, 8) was performed according to the route
shown (Scheme 1). The Reissert reaction [11] on the
acetylated intermediates 3a–c and 4 and subsequent
acid hydrolysis of 5a–c and 6 with 48% HBr afforded
the tricyclic compounds 7a–c and 8 (Scheme 1).
Compound 9 was obtained by alkylating 7a with MeI
in the presence of NaOH and by acid hydrolysis of the
* Corresponding author.
0014-827X/99/$ - see front matter © 1999 Elsevier Science S.A. All rights reserved.
PII: S0014-827X(99)00009-9

F. Heidempergher et al. / Il Farmaco 54 (1999) 152–160
153
Fig. 2. 1-NH vs. 5-NH tautomerism in compound 7a.
intermediate methyl ester with 23% HCl in AcOH
(Scheme 2).
Compound 10 was obtained by dehalogenation of 7a
under reductive conditions with ammonium formate
and 10% Pd/C (Scheme 3). Starting from 7a, an attempt
to reduce the double bond of the pyrrole ring with
H2/PtO2 failed, and compound 11 was obtained as the
product of partial reduction and dehalogenation of the
quinoline moiety (Scheme 3).
7-Chloro-quinolinic acid (12) was synthesized follow-
ing a known procedure [12], and compound 13 was
prepared starting from 14 [13] through N-alkylation of
the N-tosyl intermediate following a procedure previ-
ously described for analogues [14] (Scheme 4).
A nuclear Overhauser effect (NOE) NMR study was
performed on 7a (Fig. 2), the representative compound
of this series. Theoretically, two tautomers that differ in
the position of the mobile proton (either on N-1 or on
N-5) are possible for this molecule.
Only one is observed in DMSO-d6 solution and NOE
data are in agreement with a structure bearing the
exchangeable proton on N-1. In fact, upon irradiation
of the NH proton NOE enhancements are observed for
H-2 and H-9 signals. Similarly, irradiation of H-9 and
H-2 enhances the NH-1 signal.
Fig. 1. Kynurenine pathway. IDO, indoleamine 2,3-dioxygenase;
TDO, tryptophan pyrrolase; KYN-3-OHase, kynurenine-3-hydroxy-
lase; KAT, kynurenine aminotransferase; KYNase, kynureninase/3-
hydroxykynureninase; 3-HAO, 3-hydroxy anthranilic acid oxygenase;
QUIN, quinolinic acid; KYNA, kynurenic acid.
Scheme 1.

154
F. Heidempergher et al. / Il Farmaco 54 (1999) 152–160
Scheme 2. (i) MeI, KOH, DMSO; (ii) 23% HCl/AcOH.
Scheme 4. (i) p-TsNCO, CH3CN; (ii) MeI, K2CO3; (iii) 37% HCl/
AcOH.
3. Discussion
Compound 7a showed a remarkable selectivity to-
wards other enzymes (KAT and KYNase). The affinity
of 7a for a related microbial enzyme, para-hydroxy-
benzoate-hydroxylase (PHBH), was negligible thus re-
inforcing its specificity for KYN-3-OHase.
Although 7a is considerably less active (micromolar
versus low micromolar [10] or nanomolar [16] range)
than previously reported inhibitors, its rigid structure
makes it an interesting tool, particularly useful for
modelling studies.
In vitro inhibitory activity on KYN-3-OHase of the
compounds was evaluated in rat liver tissue (Table 1).
Among the compounds tested, 7-chloro-3-methyl-1H-
pyrrolo[3,2-c]quinoline-4-carboxylic acid (7a) was
found to be a micromolar inhibitor of this enzyme. The
importance of the carboxy group is apparent from the
complete inactivity of 1a (Scheme 1). All the chemical
modifications performed on the 7a scaffold did not
afford any improvement in potency (7b–11). The na-
ture of the substitution at position 3 (Me versus Ph; 7a
versus 8) did not dramatically influence the inhibitory
potency (24.0 versus 34.5 mM), thus indicating that a
quite large lipophilic pocket may exist in that region.
The influence of the substitution pattern on the aro-
matic ring was not completely aligned with that ob-
served for the benzoylalanine class [10]: while a chlorine
in the formal 4% position of the benzoylalanine scaffold
(corresponding to position 7 in 7a) was clearly benefi-
cial to activity, a fluorine replacement in the same
position led to an inactive compound (7c). In the
benzoylalanine series and in another closely related
series [15] a fluorine and a chlorine on the aromatic ring
were, by and large, interchangeable. This may reflect a
subtle difference in orientation of the phenyl rings in
the catalytic site between the series. It might however
be anticipated that a chlorine substitution at position 6
would increase activity in this series. Furthermore, ei-
ther the complete deletion of the pyrrole moiety (12) or
its deletion while maintaining the amino functionality
(13), led to completely inactive compounds.
Fig. 3. Superimposition of compound 7a (grey) and KYN (black).
Fig. 4. Superimposition of compound 7a (grey) and PNU-156561
(black).
Scheme 3.

F. Heidempergher et al. / Il Farmaco 54 (1999) 152–160
155
Table 1
Physicochemical data of intermediates 3a–c, 4 and 5a–c, 6
Comp.
X
Y
R
Yield (%)
M.p. (°C)
Formula
C14H11ClN2O
C14H11ClN2O
C14H11FN2O
C19H13ClN2O
C22H16ClN3O2
C22H16ClN3O2
C22H16FN3O2
C27H18ClN3O2
3a
3b
3c
4
5a
5b
5c
6
Cl
H
F
Cl
Cl
H
F
H
Cl
H
H
H
Cl
H
H
Me
Me
Me
Ph
Me
Me
Me
Ph
66
63
65
83
60
77
63
64
159–160.5
210–213
150–152
149–150
196–201
190 dec.
85 dec. (amorphous)
90 dec. (amorphous)
Cl
A molecular modelling study was then performed to
mimic a mechanism for KYN-3-OHase inhibition by
7a. In this study PNU-156561, a potent KYN-3-OHase
inhibitor, and KYN, the natural substrate of KYN-3-
OHase, were superimposed on the rigid compound 7a.
Although a substrate and an inhibitor do not necessar-
ily share the same interactions at a catalytic site, in this
case, due to the remarkable similarity of PNU-156561
and KYN structures, it is reasonable that the inhibitor
and the substrate act by the same mechanism (see Table
2 for KYN-3-OHase inhibition of compounds 7–13).
The conformational analysis of KYN showed that,
among the several possible conformations, those lead-
ing to an intramolecular hydrogen bond between the
carbonyl moiety and the aromatic amino group were
preferred. This conclusion was supported by a confor-
mational search and by a dynamic simulation study
performed either in vacuum or in water, and by the
semi-empirical quantum mechanics AM1 method [17].
The same conformation was found for the X-ray crys-
tallographic structure of N-acetylkynurenine (ref. code
ACKYNU0001) reported in the Cambridge Structural
Database (CSD) (release 5.12). Such an intramolecular
hydrogen bond is energetically and geometrically so
favourable that it is reasonable to assume it to be
maintained in the active site of the enzyme. The stable
conformations of PNU-156561 and KYN were super-
imposed on compound 7a using the carboxylic group
and the phenyl ring as common pharmacophore fea-
tures. The most stable conformation of PNU-156561
and KYN showed the best overlapping (see Figs. 3 and
4). The analysis of the superimposed compounds sug-
gested that the aromatic NH2 of kynurenine and the
pyrrolo NH of 7a, pointing in the same direction, may
give a hydrogen bond with the same residue of the
enzyme. However, this hydrogen bond does not seem
essential for a strong inhibitory activity since it is not
present in PNU-156561.
In conclusion, the synthesis and biological in vitro
evaluation of a series of pyrrolo[3,2-c]quinolines were
presented. The parent compound structure (7a) was
attributed by NOE analysis. Pharmacophoric features
of this series have been defined by a molecular mod-
elling study of the parent compound (see Table 3 for
biological data for 7a).
4. Experimental
4.1. Chemistry
Melting points were determined in open glass capil-
1
laries on a Buchi apparatus and are not corrected. H
NMR spectra were registered on a VXR-200 or VXR-
400S instrument at 27°C. The reference signal was the
solvent (DMSO-d6, 2.49 ppm). Pre-saturation delay was
set at 7.0 s in the stationary 1D NOE experiment.
Positive ion fast bombardment (FAB), field desorption
(FD) and electronic impact (EI) mass spectra (MS) were
obtained on a Varian MAT 311-A and on a Finnigan

156
F. Heidempergher et al. / Il Farmaco 54 (1999) 152–160
Table 2
TSQ 70 instrument. Elemental analyses were performed
for new compounds by a Carlo Erba 1106 instrument.
Where elemental analyses are indicated, the results were
within 0.4% of the theoretical values (see Table 4).
Yields refer to the purified products and are not opti-
mized. Column chromatography was carried out using
silica gel 60, 230–400 mesh (Carlo Erba). Starting
materials that were not commercially available were
prepared according to procedures already published. In
particular, intermediates 1c and 2 were prepared fol-
lowing the procedure described for 1a and 1b [18].
KYN-3-OHase inhibition of compounds 7–13
Comp.
X
Y
H
R
R1
KYN-3-OH-inhibition IC50
(mM)/C or % enzyme inhibition
(100 mM) rat liver tissue
7a
7b
7c
8
9
10
Cl
H
F
Cl
Cl
H
Me
H
H
H
H
24.093.1
61.496.5
23@100
34.591.8
53.393.2
97911
Cl Me
4.1.1. General procedure for the synthesis of intermedi-
ates 3a–c, 4
H
H
H
H
Me
Ph
Me Me
Me
A solution of the appropriate pyrrolo[3,2-c]quinoline
1a–c, 2 (31 mmol) in an excess of acetic anhydride (35
ml) was refluxed for 4 h under nitrogen atmosphere.
The cooled mixture was cautiously poured into crushed
ice and allowed to stand overnight. The crude solid so
obtained was collected by filtration and crystallized
from anhydrous ethanol.
H
1a
5@100
Yields and analytical data of intermediates 3a–c, 4
are reported in Table 1.
11
100912
4.1.2. General procedure for the preparation of carboni-
trile intermediates 5a–c, 6
The procedure is reported for the preparation of
1-acetyl-5-benzoyl-7-chloro-3-phenyl-4,5-dihydro-1H-
pyrrolo[3,2-c]quinoline-4-carbonitrile (6). Benzoyl chlo-
ride (5.64 ml, 48.6 mmol) was dropped at room
temperature (r.t.) into a vigorously stirred mixture of 4
(7.79 g, 24.3 mmol) and potassium cyanide (4.73 g, 72.9
mmol) in methylene chloride (80 ml) and water (30 ml)
and left for 2 h. The mixture was stirred for 20 h, then
diluted with water. The aqueous layer was extracted
with methylene chloride; the combined organic layers
were washed with brine, dried over anhydrous sodium
sulfate and evaporated to dryness. The residue was
purified by silica gel flash-chromatography (diisopropyl
ether as eluant) to give 5.55 g of the desired product as
a beige solid.
12
13
14.4@100
1.5@100
PNU-156561
0.3390.03
0.2090.02
(rat brain tissue)
1H NMR (200 MHz, DMSO): l 2.79 (s, 3H,
CH3CO), 6.65 (s, 1H, H-4), 6.86 (d, J=2.2 Hz, 1H,
H-6), 7.27 (dd, J=8.8, 2.2 Hz, 1H, H-8), 7.3–7.7 (m,
10H, Ph×2), 7.79 (d, J=8.8 Hz, 1H, H-9), 8.06 (s,
1H, H-2). MS (EI) m/z (rel. int.) 451 (M +, 4.5), 346
’
(7.3), 304 (16.4), 278 (17.3), 105 (100).
Intermediates 5a–c, 6 were prepared similarly. Yields
and analytical data are reported in Table 1. The H
NMR spectra and MS data are consistent with the
assigned structures.
xylic acid (7a). A solution of 5a (4.9 g, 12.6 mmol) in
acetic acid (35 ml) and 48% hydrobromic acid (35 ml)
was heated at 100°C for 1 h. The cooled solution was
evaporated to dryness and the residue crystallized from
ethanol/water 1:1 to obtain a beige solid (2.06 g, 63%
yield), m.p. 300°C dec.
1
4.1.3. General procedure for the synthesis of compounds
7a–c, 8
1
Anal. (C13H9ClN2O2) C, H, Cl, N. H NMR (400
This procedure is illustrated for the preparation of
7-chloro-3-methyl-1H-pyrrolo[3,2-c]quinoline-4-carbo-
MHz, DMSO): l 2.35 (d, J=0.8 Hz, 3H, CH3), 7.48
(m, 1H, H-2), 7.72 (dd, J=8.8, 2.1 Hz, 1H, H-8), 8.12

F. Heidempergher et al. / Il Farmaco 54 (1999) 152–160
157
Table 3
Biological data for compound 7a
KYN 3-OHase inhibition IC50 (mM)
KAT enzyme inhibition (%)
KYNase enzyme inhibition (%)
PHBH IC50 (mM)
\1000
Liver
Brain
24.093.1
8.191.2
24@100 mM
25@100 mM
(d, J=2.1 Hz, 1H, H-6), 8.44 (d, J=8.8 Hz, 1H, H-9),
12.7 (bs, 1H, H-1). FAB-MS m/z 259 (M−H)−.
The following compounds were also prepared by the
general procedure described above.
line-4-carboxylic acid methyl ester as a beige solid (0.32
g, 78% yield), m.p. 165°C dec. Anal. (C15H13ClN2O2) C,
H, Cl, N.
The solution of 7-chloro-1,3-dimethyl-1H-pyrrolo-
[3,2-c]quinoline-4-carboxylic acid methyl ester (0.32 g,
1.1 mmol) in acetic acid (5 ml) and 23% HCl (5 ml)
was refluxed for 16 h. The mixture was evaporated to
dryness, taken up with water and basified with 2 N
sodium hydroxide. The basic solution was extracted
with ethyl acetate, acidified with 2 N hydrochloric acid
and the light yellow solid collected by filtration (0.27 g,
90% yield), m.p. 195°C dec. Anal. (C14H11ClN2O2) C,
H, Cl, N. 1H NMR (200 MHz, DMSO): l 2.30 (d,
J=0.9 Hz, 3H, CH3), 4.23 (s, 3H, NCH3), 7.39 (m, 1H,
H-2), 7.69 (dd, J=9.0, 2.2 Hz, 1H, H-8), 8.14 (d,
J=2.3 Hz, 1H, H-6), 8.57 (d, J=9.0 Hz, 1H, H-9).
8-Chloro-3-methyl-1H-pyrrolo[3,2-c]quinoline-4-
carboxylic acid (7b): dark yellow solid, 53% yield, m.p.
230°C dec. Anal. (C13H9ClN2O2) C, H, Cl, N. 1H NMR
(200 MHz, DMSO): l 2.31 (d, J=1.0 Hz, 3H, CH3),
7.44 (m, 1H, H-2), 7.62 (dd, J=8.9, 2.3 Hz, 1H, H-7),
8.05 (d, J=8.9 Hz, 1H, H-6), 8.50 (d, J=2.3 Hz, 1H,
H-9), 12.5 (bs, 1H, H-1). MS (EI) m/z (rel. int.) 260
(M +, 10), 216 (84.5), 215 (100).
’
7-Fluoro-3-methyl-1H-pyrrolo[3,2-c]quinoline-4-
carboxylic acid hydrate (7c): yellow solid, 72% yield,
m.p. \300°C dec. Anal. (C13H9FN2O2 · H2O) C, H, N.
1H NMR (200 MHz, DMSO): l 2.31 (d, J=0.9 Hz,
3H, CH3), 7.41 (m, 1H, H-2), 7.58 (ddd, J=8.8, 8.8,
2.6 Hz, 1H, H-8), 7.80 (dd, J=10.5, 2.6 Hz, 1H, H-6),
8.45 (dd, J=8.8, 6.0 Hz, 1H, H-9), 12.6 (bs, 1H, H-1).
FAB-MS m/z 243 (M−H)−.
’
FAB-MS m/z 275 (M+H) .
4.1.5. 3-Methyl-1H-pyrrolo[3,2-c]quinoline-4-
carboxylic acid hydrate (10)
7-Chloro-3-phenyl-1H-pyrrolo[3,2-c]quinoline-4-
Ammonium formate (2.3 g, 36 mmol) and 10% Pd/C
(0.2 g) were added to a solution of 7a (0.8 g, 3 mmol)
in ethanol (30 ml) and the mixture was refluxed for 2 h,
cooled, and filtered. After evaporation, the residue was
taken up with water and 1 N sodium hydroxide was
added until complete dissolution. The solution was
acidified with 2 N hydrochloric acid, and the precipitate
was collected by filtration and washed with water to
give a yellow solid (0.57 g, 76% yield), m.p. 230°C dec.
Anal. (C13H10N2O2 · H2O) C, H, N. 1H NMR (200
MHz, DMSO): l 2.38 (d, J=1.1 Hz, 3H, CH3), 7.48
(m, 1H, H-2), 7.70 (m, 2H, H-8 and H-7), 8.13 (m, 1H,
H-6), 8.44 (m, 1H, H-9), 12.7 (bs, 1H, H-1). MS (EI)
carboxylic acid (8): yellow solid, 57% yield, m.p.
1
\300°C. Anal. (C18H11ClN2O2) C, H, Cl, N. H NMR
(200 MHz, DMSO): l 7.2–7.5 (m, 5H, Ph), 7.73 (dd,
J=8.8, 2.0 Hz, 1H, H-8), 7.75 (s, 1H, H-2), 8.12 (d,
J=2.0 Hz, 1H, H-6), 8.50 (d, J=8.8 Hz, 1H, H-9),
13.0 (bs, 1H, H-1), 13.4 (bs, 1H, COOH). FD MS m/z
322 (M +).
’
4.1.4. 7-Chloro-1,3-dimethyl-1H-pyrrolo[3,2-c]-
quinoline-4-carboxylic acid (9)
Potassium hydroxide (0.47 g, 8.4 mmol) was added to
a solution of 7a (0.37 g, 1.4 mmol) in anhydrous
dimethylsulfoxide (6 ml). The mixture was stirred at r.t.
for 1 h, then methyl iodide (0.35 ml, 5.6 mmol) was
added dropwise and the mixture stirred at r.t. for 3 h.
The solution was poured into cold water and extracted
with ethyl acetate. The organic layer was washed with
brine, dried over anhydrous sodium sulfate, filtered and
evaporated to dryness. The residue was purified by
silica gel flash-chromatography (ethyl acetate as eluant)
to give 7-chloro-1,3-dimethyl-1H-pyrrolo[3,2-c]quino-
m/z (rel. int.) 226 (M +,19), 182 (71.8), 181 (100).
’
4.1.6. 3-Methyl-6,7,8,9-tetrahydro-1H-pyrrolo-
[3,2-c]quinoline-4-carboxylic acid hydrate (11)
The solution of 7a (0.1 g, 0.38 mmol) in acetic acid
(20 ml) and 0.1 M HCl (3.8 ml, 0.38 mmol) added to
0.02 g PtO2 was hydrogenated at 3 atm for 18 h at r.t.
The mixture was filtered, evaporated and the crude
product was purified by silica gel flash-chromatography

158
F. Heidempergher et al. / Il Farmaco 54 (1999) 152–160
Table 4
Elemental analyses
Comp.
Formula
Elemental analysis [Calc. (found)]
C
H
N
Cl
3a
3b
3c
4
5a
5b
5c
6
C
14H11ClN2O
64.99 (64.91)
64.99 (64.83)
69.41 (69.21)
71.14 (71.04)
67.77 (67.48)
67.77 (67.53)
70.77 (70.43)
71.76 (71.46)
59.89 (59.50)
59.89 (59.53)
59.54 (59.23)
4.29 (4.30)
4.29 (4.07)
4.58 (4.62)
4.08 (4.11)
4.13 (4.48)
4.13 (4.49)
4.32 (4.42)
4.01 (4.24)
3.48 (3.61)
3.48 (3.63)
4.23 (4.19)
10.83 (10.80)
10.83 (10.61)
11.57 (11.54)
8.74 (8.70)
10.78 (10.59)
10.78 (10.49)
11.25 (10.95)
9.30 (8.99)
13.71 (13.69)
13.71 (13.59)
C14H11ClN2O
C14H11FN2O
C19H13ClN2O
C22H16ClN3O2
C22H16ClN3O2
C22H16FN3O2
C27H18ClN3O2
C13H9ClN2O2
C13H9ClN2O2
C13H9FN2O2 · H2O
11.05 (11.06)
9.09 (9.24)
9.09 (8.82)
7.85 (7.53)
13.60 (13.65)
13.60 (13.67)
7a
7b
7c
10.74 (10.58)
10.74 (10.48)
10.68 (10.44)
H2O (K.F.) Calc. 6.87% Found: 7.10%
8
9
10
C18H11ClN2O2
C14H11ClN2O2
C13H10N2O2 · H2O
66.98 (66.67)
61.21 (60.89)
63.92 (63.58)
3.44 (3.51)
4.04 (4.03)
4.95 (4.92)
8.68 (8.45)
10.20 (10.04)
11.43 (11.23)
10.99 (10.66)
12.91 (13.15)
H2O (K.F.) Calc. 7.38% Found: 7.42%
62.88 (62.67) 6.49 (6.43)
H2O (K.F.) Calc. 7.26% Found: 7.20%
51.87 (52.13) 4.35 (4.36)
H2O (K.F.) Calc. 7.10% Found: 6.84%
11
13
C13H14N2O2 · H2O
C11H9ClN2O2 · H2O
11.28 (11.15)
11.00 (10.89)
13.92 (13.67)
(chloroform/methanol/30%
80:20:2 as eluant) to give a beige solid (0.042 g, 45%
ammonium
hydroxide
a light yellow solid was utilized without further purifi-
cation.
yield), m.p. 230°C dec. Anal. (C13H14N2O2 · H2O) C, H,
N. H NMR (200 MHz, DMSO): l 1.7–2.0 (m, 4H,
CH2-7 and CH2-8), 2.37 (d, J=1.0 Hz, 3H, CH3),
A solution of 7-chloro-4-methyl-(toluene-4-sulfonyl)-
amino-quinoline-2-carboxylic acid methyl ester (0.2 g,
0.49 mmol) in hydrochloric acid (2 ml) and acetic acid
(2 ml) was refluxed for 7 h. The solution was evapo-
rated to dryness and the residue was taken up with
water. The insoluble white solid was collected by filtra-
tion (0.11 g, 88% yield), m.p. 280.5–285°C. Anal.
(C11H9ClN2O2 · H2O) C, H, Cl, N. 1H NMR (200 MHz,
DMSO): l 3.17 (d, J=4.8 Hz, 3H, CH3), 7.20 (s, 1H,
H-3), 7.83 (dd, J=9.1, 2.2 Hz, 1H, H-6), 8.29 (d,
J=2.2 Hz, 1H, H-8), 8.45 (d, J=9.1 Hz, 1H, H-5),
9.80 (q, J=4.8 Hz, 1H, NH). MS (EI) m/z (rel. int.)
1
2.7–3.1 (m, 4H, CH2-6 and CH2-9), 7.40 (m, 1H, H-2),
+
’
11.9 (bs, 1H, H-1). MS (EI) m/z (rel. int.) 230 (M
,
39.2), 186 (59.2), 185 (100).
4.1.7. 7-Chloro-4-methylamino-quinoline-2-carboxylic
acid hydrate (13)
p-Tosyl-isocyanate (1.92 g, 12.6 mmol) was added to
a solution of 7-chloro-4-quinolone-2-carboxylic acid
methyl ester (14) (3 g, 12.6 mmol) in acetonitrile (50 ml)
and the mixture was refluxed for 13 h. After evapora-
tion the residue was ground with ethyl acetate to afford
crude 7-chloro-4-(toluene-4-sulfonylimino)-1,4-dihydro-
quinoline-2-carboxylic acid methyl ester (0.7 g, 14%
yield) as a light yellow solid that was utilized without
further purification. Methyl iodide (0.16 ml, 2.56 mmol)
and potassium carbonate (0.27 g, 1.95 mmol) were
added to a solution of 7-chloro-4-(toluene-4-sulfonyl-
imino)-1,4-dihydro-quinoline-2-carboxylic acid methyl
ester (0.25 g, 0.64 mmol) in acetonitrile (5 ml). The
mixture was refluxed for 24 h, then evaporated to
dryness. The residue was taken up with water and
extracted with ethyl acetate. The organic layer was
washed with brine, dried over anhydrous sodium sul-
fate, and evaporated to dryness after filtration. The
crude 7-chloro-4-methyl-(toluene-4-sulfonyl)-amino-quino-
line-2-carboxylic acid methyl ester (0.2 g, 77% yield) as
236 (M +, 17), 192 (100).
’
4.2. Biological testing
4.2.1. Kynurenine-3-hydroxylase assay in the rat li6er
The efficacy of the compounds of the invention in the
inhibition of the enzyme kynurenine-3-hydroxylase was
evaluated in rat liver mitochondrial extract according
to the method of Erickson et al. [19] with minor
modifications.
This assay is based on the enzymatic synthesis of
tritiated water during the hydroxylation reaction.
Radio-labelled water was quantified following selective
adsorption of the isotopic substrate and its metabolite
with activated charcoal.
The assay for kynurenine 3-hydroxylase activity was
carried out at 37°C for 30 min. The reaction mixture

F. Heidempergher et al. / Il Farmaco 54 (1999) 152–160
159
(total volume 30 ml) constituted 44 mg of suspended
extract, 100 mM Tris–Cl− buffer pH 8.1, 10 mM
EDTA, 100 mM KCl, 0.8 mM NADPH, 0.025 mM
4.2.4. Kynurenine aminotransferase (KAT) assay
KAT activity was quantified in the rat following the
forebrain homogenates by the conversion of
L
-
L
-kynurenine, 0.3 mCi
L
-(3,5-3H)kynurenine (10 Ci/
kynurenine to kynurenic acic according to the method
by Okuno et al. [21] with minor modifications. Brain
tissue was diluted 1:10 in ice-cold 50 mM pyridoxal-5-
phosphate, 10 mM 2-mercaptoethanol in 5 mM Tris–
HCl pH 8.0, with a Polytron homogenizer (setting 7,
twice for 15 s). The reaction mixture consisted of 80 ml
of tissue homogenates, 20 ml of inhibitor and 1000 ml
substrate-cofactor solution containing 80 mM pyri-
doxal-5-phosphate, 1 mM a-ketoglutaric acid, 50 mM
mmol) and 3 ml of different concentrations of inhibitor
solutions. After the incubation, the reaction was termi-
nated by the addition of 300 ml of 7.5% (wt./vol.)
activated charcoal, and centrifuged for 7 min.
A 75 ml aliquot of supernatant was transferred to an
optiplate and 200 ml of liquid scintillation added. The
optiplates were vortexed and the radioactivity counted
in a scintillation counter.
L
-kynurenine in 150 mM Tris–HCl pH 8.0 (final con-
centration). The reaction was terminated after 60 min
of incubation at 37°C by addition of 20 ml of 50%
trichloroacetic acid. After centrifugation at 10 000 rpm
for 15 min, the supernatants were directly injected into
the HPLC system and kynurenic acid was quantified.
4.2.2. Kynurenine-3-hydroxylase assay in the rat brain
The inhibition of the enzyme kynurenine-3-hydroxy-
lase has been evaluated in rat brain homogenate by the
conversion of
L-kynurenine to L-3-hydroxykynurenine
according to Carpenedo et al. [7].
Brain was homogenized in ice-cold 0.32 M sucrose
and centrifuged at 12 000×g for 30 min at 4°C. The
pellet was washed three times with 0.32 M sucrose by
centrifugation and suspended in 0.14 M KCl in 20 mM
K-phosphate buffer at pH 7 (1 g brain in 2 ml buffer).
The reaction mixture contained 75 ml of suspended
homogenate, 100 ml of 2 mM MgCl2, 0.4 mM NADPH,
4.2.5. p-Hydroxybenzoate hydroxylase (PHBH) assay
The activity of the enzyme PHBH from Pseudomonas
fluorescens was measured by the conversion of p-hy-
droxybenzoic acid to 3,4-dihydroxybenzoic acid. The
enzyme was incubated in the presence of 3 mM FAD,
150 mM NADPH, 25 mM substrate and increasing
concentrations of inhibitors (final volume 200 mM) at
30°C. The reaction was terminated after 15 min by
adding 200 ml of 1 M HClO4. After centrifugation,
supernatants were directly used for the measurement of
3,4-dihydroxybenzoic acid. The concentration of 3,4-di-
hydroxybenzoic acid was measured on a HPLC system
coupled to a coulometric detection (det 1+0.05 det
2+0.2 V) with a reversed phase (C 18, 3 mm) column.
The mobile phase contained water/acetonitrile/tri-
fluoroacetic acid (95:5:0.05). The flow rate was 1 ml/min.
50 mM L-kynurenine in 50 mM K-phosphate buffer pH
7.5 and 25 ml of different concentrations of the tested
compound. The reaction was terminated by addition of
200 ml of 1 M HClO4 after 1 h incubation at 37°C.
L
-3-Hydroxykynurenine formed was quantified by
HPLC with coulometric detection at working voltage of
+0.2 V. The column was a 10 cm C 18 reversed phase
(3 mm particulate). The mobile phase consisted of 950
ml distilled water, 20 ml acetonitrile, 9 ml triethyl-
amine, 5.9 ml phosphoric acid, 100 mg sodium EDTA
and 1.5 g heptanesulfonic acid. The flow rate was 1
ml/min.
4.3. Molecular modelling
The three-dimensional structures of the compounds
reported in Figs. 2–4 were built from available struc-
tural fragments of the INSIGHTII version 97.2 (MSI,
San Diego, CA). The built structures were energy mini-
mized by DISCOVER (version 2.9) with molecular
mechanics using the gradient method and cvff.frc force
fields. The minimization was carried out until the rms
4.2.3. Kynureninase assay in the rat li6er
The inhibition of the enzyme kynureninase has been
evaluated in rat liver according to the method of
Takikawa et al. [20] with minor modifications, by the
conversion of
L-kynurenine to anthranilic acid. Liver
tissue (2 g) was homogenized in 10 ml of ice-cold 0.14
M KCl/0.02 M potassium phosphate buffer, pH 7.0
with a Polytron homogenizer (setting 7, twice for 15
min).
˚
of the gradients was B0.001 kcal/(mol A). The struc-
tures were then submitted to a complete torsion angle
search with increments of 30°, for the bonds free to
rotate, discarding the conformations above 5 kcal/mol.
The obtained conformations were minimized by molec-
ular mechanics as described above. The conformations
of KYN showing an intramolecular hydrogen bond
between the carbonylic moiety and the aromatic amino
group, leading to a formal 6-atom ring, were at a
relative lower energy. This was also the only allowed
conformation in the AM1 semi-empirical method (MO-
PAC program, version 6.0). Indeed, geometry minimiza-
The reaction mixture contained 100 ml of ho-
mogenate, 40 ml of inhibitor and 60 ml of a substrate-co-
factor solution containing 50 ml pyridoxal-5-phosphate,
10 mM -kynurenine in 100 mM Tris–HCl pH 8.0 (final
L
concentration). The reaction at 37°C was started by
adding the substrate and was terminated after 30 min
by adding 0.2 ml of 10% trichloroacetic acid. After
centrifugation at 10 000 rpm for 15 min the superna-
tants were directly injected into the HPLC apparatus.

160
F. Heidempergher et al. / Il Farmaco 54 (1999) 152–160
Schwarcz, Multiple ways to modulate the content of quinolate
and kynurenate in the central nervous system, Adv. Exp. Med.
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Protection against quinolinic acid-mediated excitotoxicity in ni-
grostriatal dopaminergic neurons by endogenous kynurenic acid,
Neuroscience 78 (1997) 967–975.
[10] A. Giordani, L. Corti, M. Cini, R. Bormetti, M. Marconi, O.
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tion of different kynurenine conformations always led
to this intramolecular hydrogen bond conformation. A
high-energy conformation of kynurenine was submitted
to a dynamic simulation at 300 K, either in vacuum or
˚
in water (in this case a 15 A layer of water molecules
was used). In both cases after nearly 5 ps, a conversion
to the most stable conformation was observed. The
compounds remained in this conformation for the rest
of the dynamic simulation (100 ps). The superimposi-
tion of PNU-156561 and KYN on compound 7a
˚
[11] F.D. Popp, Developments in the chemistry of Reissert com-
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tively, using the carboxylic group and the phenyl ring
for the matching process.
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