Synthesis and QSAR of substituted 3-hydroxyanthranilic acid derivatives as inhibitors of 3-hydroxyanthranilic acid dioxygenase (3-HAO)

Article abstract of DOI:10.1016/S0223-5234(99)00220-2

Novel 4,5-, 4,6-disubstituted and 4,5,6-trisubstituted 3- hydroxyanthranilic acid derivatives were synthesized and their ability to reduce the production of the excitotoxin quinolinic acid (QUIN) by inhibition of brain 3-hydroxyanthranilic acid dioxygenase (3-HAO) was subsequently investigated. The potency of the compounds to inhibit 3-HAO was assayed in rat brain homogenate, while chemical stability of certain compounds was studied by HPLC. The data were used to generate quantitative structure- activity relationship (QSAR) models for potency of 3-HAO inhibition and compound stability. Compounds with longer half-lives were obtained when the difference between the HOMO and LUMO was increased, while electron withdrawing groups in the 4- and 5-positions increased the potency of 3-HAO inhibition. Selected compounds that showed high potency in vitro were also found to be efficacious inhibitors in vivo after cerebral administration in rats.

Full text of DOI:10.1016/S0223-5234(99)00220-2

Eur. J. Med. Chem. 34 (1999) 729−744
© 1999 Editions scientifiques et médicales Elevier SAS. All rights reserved
729
´
Original article
Synthesis and QSAR of substituted 3-hydroxyanthranilic acid derivatives
as inhibitors of 3-hydroxyanthranilic acid dioxygenase (3-HAO)
Mats Linderberga, Sven Hellberga, Susanna Björka, Birgitta Gotthammara, Thomas Högberga,
Kerstin Perssona, Robert Schwarczc, Johan Luthmanb, Rolf Johanssona*
aMedicinal Chemistry, Preclinical R& D, Astra Arcus, S-15185 Södertälje, Sweden
bCell Biology, Preclinical R& D, Astra Arcus, S-15185 Södertälje, Sweden
cMaryland Psychiatric Research Center, University of Maryland School of Medicine, Baltimore, MD 21228, USA
(Received 21 January 1999; revised 12 March 1999; accepted 18 March 1999)
Abstract – Novel 4,5-, 4,6-disubstituted and 4,5,6-trisubstituted 3-hydroxyanthranilic acid derivatives were synthesized and their ability to
reduce the production of the excitotoxin quinolinic acid (QUIN) by inhibition of brain 3-hydroxyanthranilic acid dioxygenase (3-HAO) was
subsequently investigated. The potency of the compounds to inhibit 3-HAO was assayed in rat brain homogenate, while chemical stability of
certain compounds was studied by HPLC. The data were used to generate quantitative structure-activity relationship (QSAR) models for
potency of 3-HAO inhibition and compound stability. Compounds with longer half-lives were obtained when the difference between the
HOMO and LUMO was increased, while electron withdrawing groups in the 4- and 5-positions increased the potency of 3-HAO inhibition.
Selected compounds that showed high potency in vitro were also found to be efficacious inhibitors in vivo after cerebral administration in rats.
´
© 1999 Editions scientifiques et médicales Elevier SAS
3-HAO / QSAR / hydroxyanthranilic acid / enzyme inhibitors / neuroprotection
1. Introduction
high degree of homology (94% similarity) between the
rat and the human 3-HAO amino acid sequence [11].
The cytosolic, non-haem ferrous (Fe2+) enzyme
3-hydroxyanthranilic acid dioxygenase (3-HAO; EC
1.13.11.6) plays an important role in the metabolic
transformation of L-tryptophan to nicotinamide in the
kynurenine pathway (figure 1). The enzyme oxidizes and
ring opens 3-hydroxyanthranilic acid (3-HANA) to pro-
duce α-amino-â-carboxymuconic acid ω-semialdehyde,
which subsequently, spontaneously cyclizes and forms
quinolinic acid (QUIN) [1]. 3-HAO seems mainly to be
localized to hepatic tissue, but it has also been demon-
strated that the enzyme is expressed both in the brain [2,
3] and in inflammatory cells [2, 4] where the production
of QUIN has been shown to be stimulated by certain
cytokines [5–9]. Biochemical and immunological analy-
sis in the rat suggest that the brain and liver 3-HAO are
identical proteins [10]. Moreover, there appears to be a
QUIN is an NMDA receptor agonist and an excito-
toxin [12] that has been reported to cause convul-
sions [13] or neurodegeneration [14] after intracerebral
administration. It has been shown that intrastriatal injec-
tions of QUIN in rats induce biochemical and morpho-
logical alterations similar to those observed in Hunting-
ton’s disease [15, 16]. Furthermore, increases in neuronal
Huntingtin immunoreactivity [17] have recently been
reported to occur after administration of QUIN into the
striatum of mice, providing further support for a role of
QUIN in the pathogenesis of Huntington’s disease. It is
also possible that QUIN is involved in epilepsy. For
example, the levels of QUIN are higher in epilepsy-prone
mice in a transgenic model of epilepsy than in the
corresponding wild type mice [18]. Moreover, increased
QUIN levels, or enhanced kynurenine pathway activity,
have been implicated in inflammatory diseases of the
central nervous system. For instance, QUIN has been
*Correspondence and reprints

730
Figure 1. The biotransformation pathway for tryptophan yielding the excitotoxin QUIN.
associated with neurological dysfunction occurring fol-
lowing various viral or bacterial infections [19–23].
To elucidate the possible role of 3-HAO-mediated
formation of QUIN in different neurological conditions,
inhibitors of the enzyme are valuable tools, and may also
find therapeutic utility. In this report, the synthesis of
several analogues of 3-HANA and their structure-activity
relationship as inhibitors of 3-HAO are presented. The
main objective was to develop inhibitors with improved
chemical and pharmacological properties compared to
previously described 4-halogenated 3-HANA ana-
logues [24]. A particular problem associated with
3-HANA analogues is the propensity of 3-HANA for
auto-oxidation leading to its degradation and the forma-
tion of oxidized cinnabarinic acid [25, 26]. Hence, the
stability of some of the synthesized compounds was
studied by HPLC. The potency of the compounds to
inhibit 3-HAO was assessed in rat brain tissue homoge-
nates, while the ability of selected compounds to inhibit
3-HAO in vivo was also studied following intracerebral
administration.
the 5-position is substituted, the 4-substituted halogens
can be obtained by a regio-selective reaction at the
4-position. Accordingly, compounds 8 and 10 were ob-
tained by direct bromination of the 5-substituted-2-
hydroxyanthranilic acids or esters (figures 2 and 3). To
confirm the identity of the structures, 5-bromo-3-
hydroxyanthranilic acid was chlorinated and shown to
give an identical product as formed by bromination of
4-chloro-3-hydroxyanthranilic acid. The identity of the
4,5-dichloro and 4,5-dibromo derivatives was confirmed
by comparison with the corresponding 4,6-disubstituted
compounds and by synthesis either from the 4- or
5-halogenated 3-HANA leading to identical products
(figure 2).
Compound 39 was synthesized by bromination of
5-hydroxy-2-methoxybenzoic acid, which after nitration
and reduction of the nitro group gave the desired product
2. Chemistry
A
series of 4,5-, 4,6-disubstituted and 4,5,6-
trisubstituted 3-hydroxyanthranilic acids were prepared.
The 4,5-dihalo substituted compounds were synthesized
from the known 4-chloro- and 4-bromo-3-hydroxy-
anthranilic acids. The strong directing effect of the amino
substituent gives access to derivatives 11 and 12 after
reaction with bromine and chlorine, respectively. When
Figure 2. i)
Cl2/HCl/HOAc.
Br2/HBr/HOAc,
ii)
Br2/HBr/HOAc
or

731
Figure 3. i) Br2/HOAc, ii) KOH/EtOH.
Figure 6. i) EtSNa/DMF, ii) HCl/MeOH, iii) HNO3/CH3NO2/
CH2Cl2, iv) KOH/EtOH, v) SnCl2xH2O/EtOH.
(figure 4). Compounds 6, 34 and 44 (figures 5 and 6) were
prepared by nitration of suitably substituted mono or
dihalogenated benzoic acid. The benzoic acids 32 and 42
were obtained by a palladium catalysed carbonylation of
the corresponding triflates. The directing effect of the
phenol gave the desired regio-selectivity for compounds
6 and 44. The more easily oxidized compounds 17, 25
and 36 (figure 7 and 8) were obtained after halogenation
of an intermediate without the strongly directing and
activating amino-function. To make the tri-substituted
compounds 21 and 29 and the 4,6-disubstituted 50 and 54
the carboxylic acid was introduced via ring closure of the
corresponding isonitrosoacetanilid followed by oxidative
ring-opening of the isatin-derivative (figure 9).
Figure 7. i) Cl2/CHCl3, ii) BnBr/K2CO3, iii) Cu(I)Cl/KBH4.
Figure 8. i) Br2/HOAc/NaOAc, ii) H2/PtS2.
Figure 4. i) Br2/HOAc, ii) NaNO3/La(NO3)3/HCl(aq.)/diethyl
ether, iii) H2/PtS2/EtOH.
Figure 9. i) CCl3CH(OH)2/NH2OH/DMF/H2O, ii) H2SO4/Dio-
xane or polyphosphoric acid, iii) NaOH/H2O2, iv) H2/Pd or
BBr3.
Figure 5. i) Pd(OAc)2/CO/MeOH/dppp, ii) KOH/MeOH, iii)
BBr3 or HBr, iv) HNO3/CH3NO2, v) Pd/H2.

732
Table I. Inhibition of 3-HAO and chemical stability of 3-HANA derivatives.
a
Compound
R4
R5
R6
IC50 nM
Stabilityb
38
55 [24, 32]
Cl
Br
F
H
H
H
OEt
Br
Me
Br
Cl
H
H
H
H
(CH2)2
Me
Me
H
H
H
H
H
H
H
H
H
Br
MeO
Cl
Ph
(CH2)2
Me
Cl
6
2
24
56
56 [24]
57 [24]
6
8
10
11
12
36
39
44
50
21
29
34
54
Br
Br
Br
Cl
Cl
Br
Br
Cl
Cl
Cl
Cl
Cl
Cl
0.3
2.3
0.26
0.3
5.8
120
10.1
11 000
200
8.2
4.4
7.8
21
90
0
89
79
0c
62
53
Me
aIn vitro inhibition of 3-HAO in rat cortex. bPercent remaining after 24 h in a PBS buffer at pH 7.5 and 37 °C. cPercent remaining after 24 h
in a PBS buffer at pH 7.5 and 50 °C.
3. QSAR
A quantitative structure-activity relationship was de-
Test compound concentrations resulting in a 50% inhibi-
tion of the enzyme (IC50) are reported.
Also, the ability of selected compounds to inhibit
cerebral 3-HAO in vivo was studied following intracere-
broventricular (i.c.v.) administration of the compounds in
rats. The de novo formation of QUIN in hippocampal
tissue after concomitant i.c.v. administration of the test
compounds and QUIN was measured by GC/MS.
veloped for the 16 compounds presented in table I. The σ
of the R1, R2 and R3 substituents, the π and MR for the
R4, R5 and R6 substituents were obtained from the
literature [27]. The pKa values for the CO2H, NH2 and
OH substituents and logP for the molecules were calcu-
lated using the Pallas software [28]. Semi-empirical cal-
culations using AM1 (Spartan) [29] provided values for
HOMO and LUMO energies, e-neg, hardness, heat of
formation, polarizability, surface area, molecular volume
and ovality [30] which were subsequently used as de-
scriptors in the QSAR calculations.
5. Results and discussion
A PLS analysis# (Codex, AP Scientific Service) of
potency resulted in a four component model, accounting
for 97% of the variance in pIC50 (cross-validated Q2 =
0.85) (figure 10). All the components in the model were
statistically significant according to cross-validation. The
two most important components accounted for 86% of
the variance. A plot of the PLS-weights for these com-
ponents indicated that the most important factors influ-
4. Pharmacology
The compounds were tested for their ability to inhibit
3-HAO in homogenates of rat brain tissue according to
the method of Foster et al. [18]. The production of
radioactive QUIN was measured after addition of
[1-14C]3-HANA to determine inhibition of the enzyme.
#
Descriptors used in the PLS analysis are available on the
Internet on request

733
Figure 10. Predicted vs. observed IC50 values for 16 3-HAO
inhibitors.
Figure 11. Plot of stability versus hardness.
encing the IC50 values are the size of the R6 substituent,
the lipophilicity of the R5 substituent and the pKa of the
phenol. This implies that small, electron-withdrawing
groups at R6 and lipophilic, electron-withdrawing groups
at R5 would increase the potency of the compounds.
Nine of the compounds were used to develop a QSAR
model for chemical stability. The PLS analysis resulted in
a two-component model, accounting for 94% of the
variance in stability. The single most important factor for
the model was the difference between the HOMO and
LUMO energies (hardness) of the molecules (figure 11).
The very labile compound 39, having the strongly
electron-donating 6-methoxy substituent could not be
accounted for in the model. This may indicate a different
mechanism for the degradation of this compound. The
increased instability observed for compounds with a
small difference in HOMO and LUMO-orbitals is in
agreement with the observation that dimerization has
been reported to be involved in the degradation of
4-bromo-3-hydroxyanthranilic acid [25, 26].
Several compounds that were found to be potent in
vitro inhibitors of 3-HAO were also efficacious in vivo
after intracerebral administration. There was a general
agreement between the in vitro and in vivo ability of the
3-HANA analogues to inhibit 3-HAO (tables I and II).
Hence, compounds which showed low nM IC50 values
were able to almost totally inhibit de novo production of
QUIN in vivo when given at doses that were equivalent to
or higher than those of 3-HANA, while 21, which showed
a high nM IC50 value in vitro, displayed no in vivo
activity. On the other hand, certain differences in potency
observed in vitro were not reflected in vivo. This may, at
least partially, depend on the inherent differences in
chemical stability of the compounds.
For example, the low stability of the very potent
compound 8 may have reduced its in vivo actions, but
distribution and local metabolism in vivo could also
contribute to the discrepancies seen.
Importantly, the finding that potency and stability
depend on separate structural features present possibili-
ties to further improve the characteristics of 3-HANA
analogues as useful inhibitors of 3-HAO.
Table II. In vivo effects of 3-HAO inhibitors on 3-HANA-induced
de novo production of QUIN after i.c.v. administration in rat.
Compound
Ratio dosea
% Inhibition
3-HANA:compound
55
8
36
1:1
1:1
1:0.3
1:1
1:30
1:30
1:30
1:30
1:0.3
1:30
96.9 ± 0.1
87.4 ± 2.0
67.2 ± 6.6
81.9 ± 1.4
95.2 ± 1.4
91.9 ± 1.9
0 ± 13
95.1 ± 1.2
69.6 ± 7.9
93.9 ± 0.6
44
21
29
34
a3-HANA was given in a dose of 10 nmol i.c.v., while the different
compounds were given in doses of either 3, 10 or 300 nmol i.c.v.
at the same time. The cerebral levels of QUIN at 2 h after
administration were determined in hippocampal tissue by GC/MS.
Data are the mean ± SEM of 5–7 determinations.

734
6. Experimental protocols
(DMSO-d6) δ 167.5, 156.5, 155.8, 131.3, 109.7, 104.5,
104.3, 64.6, 14.6, MS (TSP) m/z: 263/261 (M + 1).
6.1. Chemical methods
6.1.3. Methyl 4-bromo-5-ethoxy-3-hydroxybenzoate 3
A solution of 2 (115 mg, 0.44 mmol) in dry methanol
(10 mL) was saturated with hydrogen chloride at 0 °C
and the mixture was stirred overnight at room tempera-
ture. The solution was evaporated and the remainder was
dissolved in chloroform. The organic phase was washed
with sodium bicarbonate (sat.), water, brine and then
dried (Na2SO4) and evaporated to give the title com-
pound (94 mg, 79%). 1H NMR (CDCl3) δ 7.35 (d, J = 1.8
Hz, 1H), 7.14 (d, J = 1.8 Hz, 1H) 5.85 (br, 1H), 4.17 (q,
J = 7.0 Hz, 2H), 3.91 (s, 3H), 1.49 (t, J = 7.0 Hz, 3H),
13C NMR (CDCl3) δ 167.2, 156.5, 154.4, 130.8, 110.1,
106.0, 105.5, 65.4, 52.7, 14.7, MS (TSP) m/z: 277/275
(M + 1).
Melting points were determined on a Büchi SMP-20
1
apparatus. H and 13C NMR spectra were recorded at
ambient temperature on a Varian Unity 400 or Varian
Gemini 300 instrument. Chemical shifts are given in ppm
1
from internal standards. For H NMR and 13C NMR
spectra the internal references were tetramethylsilane (0.0
ppm), CDCl3 (δ 7.26 or δ 77.0 ppm), CD3OD (δ 3.38 or
δ 49.3 ppm) or DMSO-d6 (δ 2.49 or δ 39.5 ppm),
respectively. Coupling constants are given in Hertz, and
the splitting patterns are designated as follows: s, singlet;
d, doublet; dd, doublet of doublets; t, triplet; q, quartet
and br, broad. Mass spectra were obtained on an LKB
2091 (ELI, 70 eV or CI/CH4) or on a Finnigan-MAT TSQ
70 (thermospray) spectrometer. Elemental analyses were
performed by MIKRO KEMI AB, Uppsala, Sweden. The
values were within ± 0.4% of theoretical if not otherwise
indicated.
6.1.4. Methyl 4-bromo-5-ethoxy-3-hydroxy-2-nitrobenzo-
ate 4
A solution of 3 (89.7 mg, 0.326 mmol) in nitromethane
(1 mL) and dichloromethane (1 mL) was stirred with
nitric acid (90%, 15 µL) at 40 °C for 10 min. Ice was
added, and the layers were separated. The aqueous layer
was extracted with chloroform, and the combined organic
layer was washed with water and evaporated. Purification
of the remainder by flash chromatography (SiO2, toluene-
ethyl acetate 10:1) gave the title compound (37 mg,
35%). 1H NMR (CDCl3) δ 6.59 (s, 1H), 4.21 (q, J = 7.0
Hz, 2H) 3.90 (s, 3H), 1.47 (q, J = 7.0 Hz, 3H), 13C NMR
(CDCl3) δ 167.1, 162.2, 154.4, 132.2, 126.9, 105.4,
103.3, 66.6, 53.9, 14.6, MS (TSP) m/z: 339/337 (M +
NH4).
6.1.1. Ethyl 4-bromo-3,5-diethoxybenzoate 1
4-Bromo-3,5-dihydroxybenzoic acid [31] (4.68 g,
20 mmol), potassium carbonate (8.28 g) and diethyl sul-
fate (7.84 mL) in dry acetone (50 mL) under nitrogen
atmosphere were stirred at room temperature for 20 min
and then refluxed for 8 h. The salts were filtered off and
the solvent was evaporated, and the remainder was
dissolved in diethyl ether. The organic layer was washed
with water, sodium hydroxide (aq., 5%), ammonium
hydroxide (conc.), hydrochloric acid, water and finally
dried (Na2SO4). The organic phase was then evaporated
to give the title compound (2.63 g, 51%). 1H NMR
(CDCl3) δ 7.22 (s, 2H), 4.39 (q, J = 7.1 Hz, 2H) 4.18 (q,
J = 7.0 Hz, 4H), 1.5 (t, J = 7.0 Hz, 6H), 1.41 (t, J = 7.1
Hz, 3H), 13C NMR (CDCl3) δ 166.8, 157.0, 130.8, 107.9,
106.8, 65.4, 61.6, 14.7, 14.4.
6.1.5. 4-Bromo-5-ethoxy-3-hydroxy-2-nitrobenzoic acid 5
Methyl 4-bromo-5-ethoxy-3-hydroxy-2-nitrobenzoate
4 (30.2 mg, 0.094 mmol) was dissolved in ethanol
(1.2 mL) and potassium hydroxide (62 mg) in water
(0.54 mL) was added. The mixture was stirred at 40 °C
for 16 h and then cooled and acidified to pH 2 by the
addition of hydrochloric acid. The remainder was tritu-
rated with water, filtered and dried to give the title
compound (24.6 mg, 86%). 1H NMR (CD3OD) δ 6.82 (s,
1H), 4.74 (br, 2H) 4.00 (q, J = 7.0 Hz, 2H), 1.26 (t, J =
7.0 Hz, 3H), 13C NMR (CD3OD) δ 167.3, 159.7, 151.0,
135.3, 128.2, 106.9, 106.3, 67.4, 15.4, MS (EI, 70 eV)
m/z: 307/305 (M +, 99/100).
6.1.2. 4-Bromo-5-ethoxy-3-hydroxybenzoic acid 2
A solution of 1 (1.02 g, 3.2 mmol) in N,N-dimethyl-
formamide (5 mL) was added to sodium ethanethiolate
(4.53 g, 51.2 mmol) in N,N-dimethylformamide (25 mL)
under nitrogen atmosphere. The mixture was stirred at
100 °C for 8 h. After cooling, the mixture was poured into
water. The aqueous phase was acidified by the addition of
hydrochloric acid (1 M) and extracted with diethyl ether.
The organic phase was dried (Na2SO4) and evaporated
The remainder was purified by flash chromatography
(SiO2, toluene-acetic acid 5:1) to give the title compound
6.1.6. 4-Bromo-5-ethoxy-3-hydroxyanthranilic acid 6
A mixture of 5 (19.8 mg, 0.065 mmol) and SnCl2xH2O
(73 mg) in ethanol (2.0 mL) was heated at 70 °C for 5.5 h
under nitrogen atmosphere. After cooling, water (2.0 mL)
was added and the pH was adjusted to 7 by adding
1
(115 mg, 14%). H NMR (DMSO-d6) δ 7.13 (d, J = 1.8
Hz, 1H), 6.95 (d, J = 1.8 Hz, 1H) 4.08 (q, J = 7.0 Hz,
2H), 3.4 (br, 2H), 1.30 (t, J = 7.0Hz, 3H), 13C NMR

735
sodium bicarbonate (aq., 5%). The mixture was extracted
with ethyl acetate, and the organic phase was washed
with brine and dried (Na2SO4). The organic phase was
evaporated to give the title compound (8.4 mg, 47%). 1H
NMR (CD3OD) δ 6.93 (s, 1H), 4.80 (br, 4H) 3.89 (q, J =
7.0 Hz, 2H), 1.30 (t, J = 7.0 Hz, 3H), 13C NMR (CD3OD)
δ 171.7, 147.7, 145.1, 137.9, 109.5, 108.7, 107.2, 67.0,
15.7, MS (TSP) m/z: 278/276 (M + 1).
thin layer chromatography (SiO2, chloroform-methanol-
ammonium hydroxide 300:10:1, chloroform-methanol
1 000:1) to give the title compound (9 mg, 15%). H
NMR (DMSO- d6) δ 7.22 (s, 1H), 6.25 (br, 3H), 3.74 (s,
3H), 2.17 (s, 3H), MS (EI, 70 eV) m/z: 261/259 (M +,
85/86).
1
6.1.10. 4-Bromo-3-hydroxy-5-methylanthranilic acid 10
Methyl 4-bromo-3-hydroxy-5-methylanthranilate
9
6.1.7. 5-Bromo-3-hydroxyanthranilic acid 7
(14 mg, 0.05 mmol) was dissolved in ethanol (0.7 mL)
and flushed with argon. Potassium hydroxide (aq,
0.35 mL, 0.05 mmol) was added and the reaction was
stirred at 40 °C for 6 h. The mixture was acidified to pH
2 by the addition of hydrochloric acid (2 M). The solvent
was removed and the remainder was triturated with
ice-water. The crude product was purified by preparative
HPLC (Lichrosorb C18) using methanol-ammonium hy-
droxide (0.05 M) (40:60) as the eluent to give the title
compound (2 mg, 16%). 1H NMR (DMSO-d6) δ 7.23 (s,
1H), 3.35 (br, 2H), 2.13 (s, 2H), 2.05 (s, 3H), MS (EI, 70
eV) m/z: 247/245 (M +, 82/86). High resolution MS
C8H8BrNO3.
To 3-hydroxyanthranilic acid (5 g, 32.6 mmol) in ace-
tic acid (450 mL) was bromine (10.4 g, 65.2 mmol) in
acetic acid (50 mL) added drop-wise. The thick slurry
was stirred for 1 h at room temperature and was then
evaporated. The remainder was dissolved in methanol
(60 mL) and water was added. The precipitate was
filtered and dried to give the title compound (7.38 g,
1
98%), m.p.: 210–212 °C. H NMR (DMSO-d6) δ 10.20
(br, 1H), 7.30 (d, J = 2.4 Hz, 1H) 6.88 (d, J = 2.4 Hz,
1H), 13C NMR (DMSO-d6) δ 169.2, 146.4, 141.2, 123.2,
118.9, 110.9, 104.2, MS (EI, 70 eV) m/z: 233/231 (M +,
14/13).
6.1.8. 4,5-Dibromo-3-hydroxyanthranilic acid 8
5-Bromo-3-hydroxyanthranilic acid
7
(7.38 g,
6.1.11. 5-Bromo-4-chloro-3-hydroxyanthranilic acid 11
31.8 mmol) was dissolved in methanol (120 mL) and
dichloromethane (250 mL) saturated with hydrogen bro-
mide was added. More dichloromethane (300 mL) was
added and the mixture was stirred for 48 h whereafter the
solvent was evaporated. The residue (8.92 g) was dis-
solved in acetic acid (400 mL) and hydrobromic acid
(48%, 100 mL) and bromine (5.5 g, 34.4 mmol) in acetic
acid (50 mL) were added drop-wise for 20 min. The
mixture was heated to 70 °C and additional bromine
(5.50 g, 34.4 mmol) in acetic acid (50 mL) was added.
After 14 h at 90 °C the solvent and excess reagent were
evaporated. The remainder was crystallized from ethanol-
water to give the title compound (8.45 g, 85%), m.p.:
223–223.5 °C. 1H NMR (DMSO- d6) δ 9.3 (br, 1H), 7.56
(s, 1H), 6.0 (br, 1H), 5.3 (br, 1H). 13C NMR (DMSO-d6)
δ 168.9, 143.5, 142.5, 125.9, 117.9, 110.3, 107.2. MS (EI,
70 eV) m/z: 313/311/309 (M +, 51/100/50). Anal. for the
hydrochloride C7H5Br2NO3xHCl (C, H, N).
To 4-chloro-3-hydroxyanthranilic acid [23] (50 mg,
0.27 mmol) dissolved in acetic acid (2.0 mL) were hy-
drobromic acid (47%, 0.02 mL, 0.54 mmol) and then
bromine (86 mg, 0.54 mmol) in acetic acid (3 mL) added,
and the mixture was stirred at room temperature and
under nitrogen atmosphere for 2 d. The solvent was
evaporated and the remainder was crystallized from
ethanol (40%), to give the title compound (37 mg, 51%),
m.p.: 227–228 °C. 1H NMR (CD3OD) δ 7.72 (s, 1H), 5.0
(br, 4H), 13C NMR (CD3OD) δ 170.3, 143.5, 143.0,
127.2, 125.4, 111.0, 106.7. MS (TSP) m/z: 270/268/266
(M +, 28/100/80). Anal. C7H5BrClNO3 (C, H, N, O).
6.1.12. 4,5-Dichloro-3-hydroxyanthranilic acid 12
To
a
solution of 4-chloro-3-hydroxyanthranilic
acid [32] (150 mg, 0.80 mmol) in acetic acid (12 mL) and
under argon, were hydrochloric acid (12 M, 336 µL,
4.8 mmol) and then chlorine (1.76 mmol) in acetic acid
(1.78 mL) added drop-wise. The reaction was stirred for
20 h and then the precipitated material was filtered. The
crude material was recrystallized from ethanol (50%)
giving the title compound (14 mg, 8%). 1H NMR
(CD3OD) δ 7.57 (s, 1H), 4.98 (br, 4H), 13C NMR
(CD3OD) δ 170.7, 143.6, 143.4, 124.0, 118.7, 110.6,
109.5. MS (TSP) m/z: 224/222 (M +, 53/100). Anal.
C7H5Cl2NO3 (C, H, N).
6.1.9. Methyl 4-bromo-3-hydroxy-5-methylanthranilate 9
To a solution of methyl 3-hydroxy-5-methylanthra-
nilate hydrochloride (50 mg, 0.23 mmol) dissolved in
acetic acid (2.5 mL) under argon atmosphere was bro-
mine (36 mg, 0.23 mmol) in acetic acid (2.5 mL) added
drop-wise. After 3 h at room temperature, additional
bromine (18 mg, 0.11 mmol) in acetic acid (1 mL) was
added. The solvent and excess reagent were evaporated,
and the remainder was purified by repeated preparative

736
6.1.13. 6-Methoxy-7-nitrotetralin 13
mixture was stirred at room temperature for 24 h, the salts
were then filtered and the solvent was evaporated. The
remainder was dissolved in ethyl acetate, and the organic
phase was washed with brine, dried (MgSO4) and evapo-
rated. The crude material was purified by flash chroma-
tography (SiO2, chloroform-hexane 1:1) to give the title
compound (2.20 g, 69%), m.p.:80–82 °C. 1H NMR
(DMSO-d6) δ 7.73 (s, 1H), 7.48–7.37 (m, 5H), 5.06 (s,
2H), 2.79–2.75 (m, 4H), 1.79–1.69 (m, 4H). 13C NMR
(DMSO-d6) δ 144.8, 142.1, 135.8, 135.6, 129.6, 128.4,
123.4, 106.2, 105.5, 28.6, 27.6, 21.6, 21.4. MS (TSP)
m/z: 337/335 (M + NH4, 30/100). Anal. C17H16ClNO3
(C, H, N).
To a solution of 6-methoxytetralin (6.80 g, 41.9 mmol)
in dichloromethane (135 mL) under argon atmosphere
and at 0 °C was nitric acid (90%, 3.92 mL, 83.8 mmol)
added drop-wise. The mixture was stirred for 25 h when
iodomethane (7.85 mL, 126 mmol), tetra-n-butyl-
ammonium hydrogensulfate (28.4 g, 83.8 mmol) and so-
dium hydroxide (2 M, 8 mL, 16 mmol) were added and
the mixture was heated at reflux for 4 h. The organic layer
was collected, washed with brine, sodium hydroxide (2
M), brine, dried (MgSO4) and evaporated. The remainder
was purified by flash chromatography (SiO2, chloroform-
hexane 2:1) to give the title compound (2.62 g, 30%). 1H
NMR (CDCl3) δ 7.60 (s, 1H), 6.74 (s, 1H), 3.90 (s, 3H),
2.79 (m, 2H) 2.71 (m, 2H) 1.81–1.77 (m, 4H), 13C NMR
(CDCl3) δ 151.5, 145.5, 137.6, 130.0, 126.8, 114.2, 57.0,
30.5, 28.8, 23.3 23.1, MS (EI, 70 eV) m/z: 207 (M +, 59).
6.1.17. 7-Amino-6-benzyloxy-5-chlorotetralin 17
To a suspension of 16 (2.56 g, 8.33 mmol) in methanol
at 1 °C was copper(I) chloride (4.95 g, 25.0 mmol) and
potassium borohydride (3.25 g, 60.1 mmol) added
portion-wise for 8 h. The mixture was filtered and evapo-
rated. The remainder was dissolved in ethyl acetate and
the organic phase was washed with water, brine, dried
(Na2SO4) and evaporated. The crude material was puri-
fied by flash chromatography (SiO2, chloroform) to give
the title compound (1.82 g, 76%). 1H NMR (DMSO-d6) δ
7.55 (dd, J = 1.6 Hz, J = 8.1 Hz, 2H), 7.41–7.34 (m, 3H),
6.42 (s, 1H), 4.84 (s, 2H), 4.79 (s, 2H), 2.58–2.52 (m,
4H), 1.71–1.60 (m, 4H), 13C NMR (DMSO-d6) δ 140.1,
139.3, 137.3, 134.0, 128.3, 128.2, 128.1, 127.9, 127.0,
121.8, 114.0, 72.8, 29.0, 26.2, 22.7, 22.3, MS (TSP) m/z:
290/288 (M + 1, 27/100). Anal. C17H18ClNO (C, H, N).
6.1.14. 6-Hydroxy-7-nitrotetralin 14
To 13 (2.92 g, 14.1 mmol) dissolved in dichlo-
romethane (450 mL) at –65 °C was boron tribromide
(1 M, 28.2 mmol) in dichloromethane (28.2 mL) added
for 15 min under an atmosphere of argon. The solution
was stirred for 2 h when the temperature was gradually
increased to 0 °C. The organic phase was collected,
washed with sodium bicarbonate (sat., 200 mL), brine,
dried (MgSO4) and evaporated. The remainder was puri-
fied on a short column (SiO2, chloroform-hexane 2:1) to
give the title compound (2.10 g, 77%). 1H NMR (DMSO-
d6) δ 10.47 (br, 1H), 7.62 (s, 1H), 6.80 (s, 1H), 2.7–2.6
(m, 4H), 1.65–1.71 (m, 4H), 13C NMR (DMSO-d6) δ
150.0, 146.1, 134.0, 128.3, 124.7, 118.5, 28.9, 27.5, 22.3,
22.0, MS (EI, 70 eV) m/z: 193 (M +, 100).
6.1.18.
6-Benzyloxy-5-chloro-7-isonitrosoacetamino-
tetralin 18
To a solution of 17 (1.84 g, 6.41 mmol) in N,
N-dimethylformamide (80 mL) and water (8 mL) under
argon atmosphere, were hydrochloric acid (12 M,
0.53 mL) and chloral hydrate (1.17 g, 7.05 mmol) added.
The mixture was heated to 110 °C when hydroxylamine
hydrochloride (1.78 g, 25.6 mmol) in water (8 mL) was
added. The mixture was heated to 100 °C for 1 h and the
solvent was evaporated. The remainder was dissolved in
ethyl acetate, and the organic phase was washed with
water, brine, dried (Na2SO4) and evaporated. The crude
material was purified by flash chromatography (SiO2,
ethyl acetate-chloroform 1:5) to give the title compound
6.1.15. 5-Chloro-6-hydroxy-7-nitrotetralin 15
To a solution of 14 (2.44 g, 12.6 mmol) in chloroform
(290 mL) under argon atmosphere was chlorine (0.99 M,
25.6 mmol) in chloroform (25.6 mL) added. The mixture
was stirred at room temperature for 6 h when the solvent
was evaporated. The remainder was purified by flash
chromatography (SiO2, chloroform-hexane 1:1) to give
the title compound (2.29 g, 80%). 1H NMR (DMSO-d6) δ
10.62 (br, 1H), 7.71 (s, 1H), 2.75–2.69 (d, 4H), 1.77–1.64
(d, 4H), 13C NMR (DMSO-d6) δ 146.3, 143.6, 134.6,
129.7, 123.4, 122.8, 28.3, 27.9, 21.7, 21.6, MS (EI, 70
eV) m/z: 229/227 (M +, 32/100).
1
(1.35 g, 59%). H NMR (DMSO-d6) δ 12.33 and 9.72
6.1.16. 6-Benzyloxy-5-chloro-7-nitrotetralin 16
(E/Z) (2s, 1H), 9.20 and 8.28 (E/Z) (2s, 1H), 7.84 and
7.72 (E/Z) (2s, 1H), 7.60 (s, 1H), 7.57–7.37 (m, 5H), 4.90
and 4.88 (E/Z) (2s, 2H), 2.71–2.65 (m, 4H), 1.75–1.68
(m, 4H), 13C NMR (DMSO-d6) δ 160.3, 160.0, 143.4,
143.0, 136.6, 136.1, 134.4, 134.3, 131.2, 130.6, 129.8,
129.5, 128.8, 128.3, 128.2, 128.1, 127.0, 120.7, 120.3,
To a solution of 15 (2.28 g, 10.0 mmol) in dry N,
N-dimethylformamide (40 mL) under argon atmosphere,
were benzyl chloride (11.5 mL, 100 mmol), tetra-n-
butylammonium hydrogensulfate (95 mg, 0.25 mmol)
and potassium carbonate (41.5 g, 30.0 mmol) added. The

737
74.8, 74.3, 29.1, 26.7, 22.2, 22.0, MS (TSP) m/z: 361/359
(M + 1, 28/100).
(DMSO-d6) δ 169.7, 138.1, 136.4, 128.1, 123.3, 121.4,
115.2, 27.9, 26.9, 22.4, 22.2, MS (EI, 70 eV) m/z:
333/331 (M +, 21/65).
6.1.19. 9-Benzyloxy-8-chloro-1H-4,5,6,7-tetrahydro[e]
benzindole-2,3-dione 19
6.1.22. 4,5-Dimethyl-2-nitrophenol 22
To sulfuric acid (conc., 5 mL) at 60 °C, 18 (500 mg,
1.39 mmol) was added portion-wise for 1 h. The mixture
was poured on ice (50 mL) and extracted with ethyl
acetate (200 mL). The organic phase was dried (Na2SO4)
and evaporated. The remainder was dissolved in N,
N-dimethylformamide (3 mL) under argon atmosphere,
and benzyl bromide (0.16 mL, 1.39 mmol) and potassium
carbonate (192 mg, 1.39 mmol) were added. The mixture
was stirred at room temperature for 18 h. The salt was
filtered off and the solvent was evaporated. The remain-
der was purified by repeated flash chromatography (SiO2,
ethyl acetate-methanol 20:1, chloroform-methanol 50:1)
to give the title compound (56 mg, 12%). 1H NMR
(DMSO-d6) δ 11.40 (s, 1H), 7.58 (d, J = 7.3 Hz, 2H),
7.42–7.36 (m, 3H), 4.90 (s, 2H), 2.92 (t, J = 7.3 Hz, 2H),
2.63 (t, J = 6.0 Hz, 2H), 1.74–1.66 (m, 4H), 13C NMR
(DMSO-d6) δ 183.8, 159.4, 142.3, 137.3, 137.1, 136.2,
136.2, 129.9, 128.8, 128.2, 128.1, 114.7, 74.6, 26.7, 25.4,
21.7, 20.8, MS (EI, 70 eV) m/z: 343/341 (M +, 5/15).
To
a
solution of 3,4-dimethylphenol (20.0 g,
164 mmol) in dichloromethane (400 mL) under argon
atmosphere and at 2 °C was nitric acid (90%, 7.7 mL,
165 mmol) added drop-wise. The mixture was stirred for
90 min while the temperature was slowly increased to
ambient temperature. To the mixture was dichlo-
romethane (500 mL) added and the organic phase was
washed with brine, water, dried (MgSO4) and evaporated.
The remainder was purified by flash chromatography
(SiO2, ethyl acetate-hexane 1:1) to give the title com-
1
pound (10 g, 36%), m.p.: 82–83 °C. H NMR (DMSO-
d6) δ 11.5 (br, 1H), 7.71 (s, 1H), 6.92 (s, 1H), 2.22 (s,
3H), 2.17 (s, 3H), 13C NMR (DMSO-d6) δ 150.8, 146.3,
133.4, 128.0, 124.9, 119.7, 19.7, 18.1, MS (EI, 70 eV)
m/z: 167 (M +, 100).
6.1.23. 2-Chloro-3,4-dimethyl-6-nitrophenol 23
To a solution of 22 (7.0 g, 41.9 mmol) dissolved in
chloroform (300 mL) under argon atmosphere was chlo-
rine (0.99 M, 83.7 mmol) in chloroform (84.8 mL) added.
The mixture was stirred for 26 h and then the solvent and
excess reagents were evaporated. The remainder was
dissolved in dichloromethane (400 mL) and the organic
phase was washed with brine, dried (MgSO4) and evapo-
rated. The crude material was purified by flash chroma-
tography (SiO2, chloroform-hexane 1:1) to give the title
compound (6.47 g, 77%), m.p.: 62–63 °C. 1H NMR
(DMSO-d6) δ 11.5 (br, 1H), 7.80 (s, 1H), 2.34 (s, 3H),
2.27 (s, 3H). 13C NMR (DMSO-d6) δ 146.9, 143.8,
134.1, 128.8, 123.7, 122.9, 19.4, 17.4. MS (EI, 70 eV)
m/z: 203/201 (M +, 37/100).
6.1.20. 6-Amino-7-benzyloxy-5-carboxy-8-chlorotetralin
20
To a suspension of 19 (51 mg, 0.15 mmol) in sodium
hydroxide (0.68 M, 0.60 mmol) and water (0.46 mL) at
10 °C, was hydrogen peroxide (30%, 86 µL, 0.85 mmol)
in sodium hydroxide (0.68 M, 0.90 mmol) added. The
mixture was stirred for 3 h and then filtered. Water and
acetic acid were added and the mixture was extracted
with ethyl acetate (40 mL). The organic phase was
washed with brine, dried (Na2SO4) and evaporated to
give the title compound (35 mg, 70%). 1H NMR (DMSO-
d6) δ 7.56 (d, J = 7.0 Hz, 2H), 7.43–7.36 (m, 3H), 4.84
(s, 2H), 3.3 (br, 3H), 2.73 (m, 2H), 2.59 (m, 2H)
1.70–1.61 (m, 4H), 13C NMR (DMSO-d6) δ 169.3, 139.9,
139.5, 136.8, 133.1, 129.5, 128.3, 128.1, 122.3, 116.5,
73.1, 28.1, 26.8, 22.2, 22.0, MS (EI, 70 eV) m/z: 333/331
(M +, 7/18).
6.1.24. 2-Benzyloxy-3-chloro-4,5-dimethylnitrobenzene
24
To a solution of 23 (6.44 g, 31.9 mmol) in dry N,
N-dimethylformamide (105 mL) under argon atmo-
sphere, were benzyl bromide (4.17 mL, 35.1 mmol) and
potassium carbonate (13.2 g, 95.7 mmol) added. The
mixture was stirred at room temperature for 8 h and was
then filtered. The solvent was evaporated, and the remain-
der was purified by flash chromatography (SiO2,
chloroform-hexane 1:1) affording the title compound
6.1.21. 6-Amino-5-carboxy-8-chloro-7-hydroxytetralin
21
6-Amino-7-benzyloxy-5-carboxy-8-chlorotetralin 20
(33 mg, 0.10 mmol) in ethanol (3 mL) was hydrogenated
at room temperature and at atmospheric pressure for 2 h
with Pd/C (5%, 4 mg) as the catalyst. The mixture was
filtered, evaporated and dried to give the title compound
(21 mg, 87%), m.p.: 147 °C (dec.). 1H NMR (DMSO-d6)
δ 7.9 (br, 4H), 2.66 (t, J = 7 Hz, 2H), 2.54 (t, J = 7 Hz,
2H), 1.70–1.64 (m, 2H), 1.63–1.57 (m, 2H), 13C NMR
1
(8.44 g, 91%), m.p.: 74–75 °C. H NMR (DMSO-d6) δ
7.80 (s, 1H), 7.48–7.37 (m, 5H), 5.06 (s, 2H), 2.37 (s,
3H), 2.34 (s, 3H), 13C NMR (DMSO-d6) δ 145.2, 142.2,
142.1, 135.7, 134.7, 129.6, 128.4, 128.4, 123.6, 75.7,
19.7, 17.1. MS (EI, 70 eV) m/z: 293/291 (M +, 8/37).

738
6.1.25. 2-Benzyloxy-3-chloro-4,5-dimethylaniline 25
To a solution of 24 (3.00 g, 10.3 mmol) in methanol
(420 mL) at 2 °C, were copper(I) chloride (6.11 g,
30.8 mmol) and potassium borohydride (4.4 g,
81.6 mmol) added in portions. The mixture was filtered
and evaporated. The remainder was dissolved in ethyl
acetate, and the organic phase was washed with water,
dried (MgSO4) and evaporated. The crude material was
purified by flash chromatography (SiO2, chloroform) to
give the title compound (2.14 g, 79%), m.p.: 57–58 °C.
1H NMR (DMSO-d6) δ 7.54 (d, J = 6.5 Hz, 2H),
7.42–7.34 (m, 3H), 6.52 (s, 1H), 4.83 (s, 2H), 4.79 (s,
2H), 2.12 (s, 6H), 13C NMR (DMSO-d6) δ 140.0, 138.9,
137.3, 133.0, 128.3, 128.2, 127.9, 127.3, 121.2, 115.4,
72.7, 20.2, 15.4, MS (EI, 70 eV) m/z: 286/284 (M + 23,
42/100).
The remainder was purified by repeated flash chromatog-
raphy (SiO2, dichloromethane-methanol 50:1) to give the
title compound (13 mg, 2%). H NMR (DMSO-d6) δ
11.42 (s, 1H), 7.57 (d, J = 6.2 Hz, 2H), 7.42–7.35 (m,
3H), 4.92 (s, 2H), 2.44 (s, 3H), 2.22 (s, 3H), 13C NMR
(DMSO-d6) δ 184.3, 159.3, 141.9, 137.0, 137.0, 136.3,
135.4, 130.2, 128.8, 128.3, 128.2, 115.6, 74.6, 15.3, 14.2,
MS (EI, 70 eV) m/z: 317/315 (M +, 3/7).
1
6.1.28.
3-Benzyloxy-4-chloro-5,6-dimethylanthranilic
acid 28
To a suspension of 27 (13 mg, 0.04 mmol) in dioxane
(0.5 mL) and sodium hydroxide (0.68 M, 0.14 mmol) at
10 °C, was hydrogen peroxide (30%, 7 µL, 0.22 mmol)
dissolved in sodium hydroxide (0.41 mL) added in por-
tions. The mixture was concentrated in a stream of
nitrogen, when acetic acid (38 µL) was added. The
mixture was dissolved in ethyl acetate (3 mL) and the
organic phase was washed with brine, dried (MgSO4) and
6.1.26.
2-Benzyloxy-3-chloro-4,5-dimethylisonitroso-
acetanilide 26
1
evaporated to give the title compound (11 mg, 90%). H
To a solution of 25 (2.14 g, 8.19 mmol) in N,
N-dimethylformamide (60 mL) and water (2 mL) were
hydrochloric acid (conc., 0.68 mL, 8.19 mmol) and chlo-
ral hydrate (1.49 g, 9.00 mmol) added. The mixture was
heated to 105 °C and hydroxylamine hydrochloride
(2.28 g, 32.8 mmol) dissolved in water (4 mL) was
added. The mixture was stirred for 1 h and the solvent
was evaporated. The remainder was dissolved in ethyl
acetate, and the organic layer was washed with water,
dried (MgSO4) and evaporated. The crude material was
purified by flash chromatography (SiO2, chloroform-
ethyl acetate 10:1) affording the title compound (1.28 g,
NMR (DMSO-d6) δ 7.55 (d, J = 7.0 Hz, 2H), 7.43–7.35
(m, 3H), 4.82 (s, 2H), 2.21 (s, 3H), 2.19 (s, 3H), MS (EI,
70 eV) m/z: 307/305 (M +, 7/19).
6.1.29. 4-Chloro-5,6-dimethyl-3-hydroxyanthranilic acid
29
3-Benzyloxy-4-chloro-5,6-dimethylanthranilic acid 28
(10 mg, 0.03 mmol) in ethanol (1.5 mL) was hydrogen-
ated at ambient temperature and atmospheric pressure for
5 h with Pd/C (10%, 2 mg) as the catalyst. The mixture
was filtered and the solvent was evaporated. The remain-
der was purified by preparative HPLC (Lichrosorb-C18,
methanol-phosphate buffer, pH 3, 1:1). The fractions
containing the product were pooled and evaporated. To
the residue was ethyl acetate added and the organic phase
was washed with sodium carbonate (sat), dried and
1
47%). H NMR (DMSO-d6) δ 12.33 and 9.71 (E/Z) (2s,
1H), 9.20 and 8.27 (E/Z) (2s, 1H), 7.91 and 7.83 (2s, 1H),
7.60 (s, 1H), 7.65–7.36 (m, 5H), 4.90 (s, 1H), 4.87 (s,
1H), 2.26–2.23 (m, 6H), 13C NMR (DMSO-d6) δ 160.2,
160.0, 143.4, 143.1, 136.6, 136.1, 133.4, 133.3, 131.0,
130.4, 129.7, 129.4, 128.7, 128.4, 128.4, 128.3, 128.2,
128.1, 128.0, 127.4, 127.2, 121.5, 121.2, 74.7, 74.2, 20.3,
16.1, MS (EI, 70 eV) m/z: 334/332 (M +, 4/12).
1
evaporated to give the title compound (3 mg, 46%). H
NMR (DMSO-d6) δ 3.35 (br, 1H), 3.16 (s, 2H), 2.16 (s,
3H), 2.15 (s, 3H), 13C NMR (DMSO-d6) δ 169.9, 137.9,
135.2, 126.4, 123.5, 121.4, 117.3, 17.7, 16.0, MS (EI, 70
eV): m/z 217/215 (M +, 21/63).
6.1.27. 7-Benzyloxy-6-chloro-4,5-dimethylisatin 27
To sulfuric acid (conc., 6 mL) at 80 °C was 26
(700 mg, 2.10 mmol) added. The mixture was stirred at
80 °C for 10 min and then poured into ice-water
(200 mL). The mixture was extracted with ethyl acetate
(200 mL) and the organic phase was dried (MgSO4) and
evaporated. The remainder was dissolved in N,
N-dimethylformamide and benzyl bromide (0.28 mL,
2.30 mmol) and potassium carbonate (318 mg,
2.30 mmol) were added. The mixture was stirred at
ambient temperature for 30 h and filtered. Acetic acid
(1.5 mL) was added, and the solvents were evaporated.
6.1.30. 2,4-Dichloro-5-methoxy-3-methylphenyl trifluoro-
methanesulfonate 30
To a solution of 2,4-dichloro-5-methoxy-3-methyl-
phenol [33] (7.73 g, 37.3 mmol) in dichloromethane
(180 mL) were triethylamine (10.4 mL, 74.7 mmol) and
N,N-dimethylaminopyridine (10 mg, 0.08 mmol) added.
The solution was cooled to –78 °C and trifluoromethane-
sulfonic anhydride (9.4 mL, 74.7 mmol) was added drop-
wise. The mixture was then stirred for 20 min while the
temperature slowly reached 0 °C. To the solution was
added dichloromethane (200 mL) and the organic phase

739
was washed with water (150 mL), brine, dried (MgSO4)
and evaporated. The remainder was purified by flash
chromatography (SiO2, ethyl acetate-hexane 1:3) afford-
ing the title compound (12.3 g, 97%), m.p.: 58.5–59.0 °C.
1H NMR (DMSO-d6) δ 7.30 (s, 1H), 3.92 (s, 3H), 2.49 (s,
3H), 13C NMR (DMSO-d6) δ 154.2, 143.9, 137.0, 122.8,
118.1, 118.0 (q, J = 321Hz), 105.3, 57.2, 18.0, MS (EI,
70 eV) m/z: 340/338 (M +, 47/64).
6.1.33. 4,6-Dichloro-3-hydroxy-5-methyl-2-nitrobenzoic
acid 33
To a solution of 32 (90 mg, 0.41 mmol) in ni-
tromethane (9 mL) at 40 °C, was nitric acid (90%, 20 mL,
0.43 mmol) added. The mixture was stirred for 4 h, when
the solvent was removed by evaporation. The remainder
was purified by flash chromatography (SiO2, ethyl
acetate-acetic acid 30:1) to give the title compound
(79 mg, 72%), m.p.: 197–199 °C. 1H NMR (DMSO-d6) δ
2.45 (s, 3H). 13C NMR (DMSO-d6) δ 164.3, 147.1,
139.6, 136.3, 128.4, 126.5, 118.9, 18.6. MS (EI, 70 eV)
m/z: 267/265 (M +, 66/100).
6.1.31. Methyl 2,4-dichloro-5-methoxy-3-methylbenzoate
31
To a solution of 30 (7.60 g, 22.4 mmol) in dioxane
6.1.34. 4,6-Dichloro-3-hydroxy-5-methylanthranilic acid
34
(75 mL)
were
1,3-bis(diphenylphosphino)propane
(0.37 g, 0.90 mmol) and palladium acetate (0.20 g,
0.90 mmol) added. The mixture was flushed with carbon
monoxide, then triethylamine (6.90 mL, 49.4 mmol) and
methanol (23 mL) were added. The mixture was stirred at
70 °C and atmospheric pressure for 25 h and was then
filtered and evaporated. The remainder was dissolved in
diethyl ether and the organic phase was washed with
ammonium hydroxide (2 M, 150 mL), brine (150 mL),
dried (MgSO4) and evaporated. The crude material was
purified by flash chromatography (SiO2, ethyl acetate-
hexane 1:3) to give the title compound (4.18 g, 75%),
4,6-Dichloro-3-hydroxy-5-methyl-2-nitrobenzoic acid
33 (69 mg, 0.26 mmol) in acetic acid (10 mL) and hydro-
chloric acid (conc., 33 mL) was hydrogenated at atmo-
spheric pressure and room temperature for 2 h with Pd/C
(10%, 10 mg) as the catalyst. Methanol was added and
the mixture was filtered. The solvent was evaporated and
the remainder was purified by flash chromatography
(SiO2, ethyl acetate-acetic acid 45:1) affording the title
1
compound (55 mg, 90%), m.p.: 192 °C (dec.). H NMR
(DMSO-d6) δ 2.27 (s, 3H), 13C NMR (DMSO-d6) δ
167.4, 139.2, 136.0, 122.9, 121.3, 120.4, 116.9, 17.0, MS
(EI, 70 eV) m/z: 237/235 (M +, 45/79).
1
m.p.: 73.5–74 °C. H NMR (DMSO-d6) δ 7.33 (s, 1H),
3.88 (s, 3H), 3.86 (s, 3H), 2.45 (s, 3H), 13C NMR
(DMSO-d6) δ 165.7, 153.3, 136.1, 130.5, 125.2, 123.0,
110.8, 56.6, 52.7, 17.9, MS (EI, 70 eV) m/z: 250/248
(M +, 53/80).
6.1.35. 4,6-Dibromo-3-hydroxy-2-nitrobenzoic acid 35
To a cooled solution of 3-hydroxy-2-nitrobenzoic acid
(10.5 g, 0.057 mol) and sodium acetate (9.85 g, 0.57 mol)
in acetic acid (100 mL) was bromine (6.15 mL, 0.12 mol)
added drop-wise. The mixture was stirred at 60 °C for
68 h and then cooled and filtered. The solution was
evaporated, and the remainder was dissolved in ethyl
acetate. The organic phase was washed with dilute
hydrochloric acid, dried (MgSO4) and evaporated. The
crude product was purified by flash chromatography
(SiO2, toluene-acetic acid 5:1). The pure compound was
crystallized from methanol to give the title compound
(15.4 g, 79%), m.p.: 201–202 °C (dec.). 1H NMR
(DMSO-d6) δ 8.20 (s, 1H), 13C NMR (DMSO-d6) δ
164.4, 146.9, 139.1, 130.5, 116.2, 108.1, MS (EI, 70 eV)
m/z: 343/341/339 (M +, 46/98/49). Anal. C7H3Br2NO5
(C, H, N).
6.1.32. 2,4-Dichloro-5-hydroxy-3-methylbenzoic acid 32
To a solution of 31 (0.24 g, 0.96 mmol) in methanol
(30 mL) under argon atmosphere, was potassium hydrox-
ide (0.31 g, 4.8 mmol) added and the mixture was stirred
at 50 °C for 19 h. The solvent was evaporated, and
hydrobromic acid (48%, 30 mL) was added and the
mixture was stirred at 110 °C for 3 d. The acid was
evaporated, and the remainder was dissolved in ethyl
acetate (40 mL). The organic phase was extracted with
ammonium hydroxide (dil., 11 mL) and the pH of the
aqueous phase was lowered to 1. The aqueous phase was
extracted with ethyl acetate (40 mL), the organic phase
was washed with brine, dried (MgSO4) and evaporated to
give, after crystallization from diethyl ether, the title
compound (0.20 g, 94%), m.p.: 203.5–204.5 °C. 1H
NMR (DMSO-d6) δ 10.68 (br, 1H), 7.15 (s, 1H), 2.42 (s,
3H), 13C NMR (DMSO-d6) δ 166.7, 151.8, 135.9, 131.2,
123.7, 121.2, 114.4, 17.9, MS (EI, 70 eV) m/z: 222/220
(M +, 56/100).
6.1.36. 4,6-Dibromo-3-hydroxyanthranilic acid 36
4,6-Dibromo-3-hydroxy-2-nitrobenzoic
acid
35
(4.09 g, 12 mmol) in ethanol (150 mL) was hydrogenated
at atmospheric pressure and room temperature for 45 h,
with PtS2 (0.16 g, 0.62 mmol) as the catalyst. The mix-
ture was filtered and the solvent was removed by evapo-
ration. The remainder was purified by flash chromatog-

740
raphy (SiO2, toluene-acetic acid 5:1) affording, after
crystallization from methanol-water, the title compound
(2.51 g, 63%), m.p.: 162–164.5 °C. 1H NMR (DMSO-d6)
δ 6.96 (s, 1H), 13C NMR (DMSO-d6) δ 167.7, 140.6,
140.0, 121.8, 117.2, 112.5, 110.4. MS (EI, 70 eV) m/z:
313/311/309 (M +, 36/72/34). Anal. C7H5Br2NO3 (C, H,
N).
dichloromethane (10 mL) at –70 °C, was trifluro-
methanesulfonic anhydride (5.79 mL, 34.4 mmol) added.
The mixture was stirred for 20 min while the temperature
was increased to ambient temperature. Dichloromethane
(150 mL) was added and the organic phase was washed
with water (100 mL), brine (100 mL), dried (MgSO4) and
evaporated. The remainder was purified by flash chroma-
tography (SiO2, dichloromethane-hexane 1:2) affording
the title compound (6.37 g, 86%). 1H NMR (DMSO-d6) δ
7.99 (s, 1H), 7.42 (s, 1H), 3.92 (s, 3H), 13C NMR
(DMSO-d6) δ 154.6, 144.0, 130.9, 122.3, 118.0 (q, J =
321 Hz), 117.1, 108.2, 57.3, MS (EI, 70 eV) m/z:
328/326/324 (M +, 6/37/55).
6.1.37. 4-Bromo-5-hydroxy-2-methoxybenzoic acid 37
To
a
solution of 5-hydroxy-2-methoxybenzoic
acid [34] (1.24 g, 73.7 mmol) in acetic acid (100 mL)
was bromine (0.38 mL, 7.4 mmol) added drop-wise. The
mixture was stirred for 3 h and the solvent was then
evaporated. The remainder was purified by flash chroma-
tography (SiO2, toluene-acetic acid 10:1) to give the title
6.1.41. Methyl 2,4-dichloro-5-methoxybenzoate 41
1
compound (1.5 g, 83%). H NMR (CD3OD) δ 7.38 (s,
1H), 7.26 (s, 1H), 3.84 (s, 3H), 13C NMR (CD3OD) δ
To a solution of 40 (6.35 g, 19.5 mmol) in N,
N-dimethylformamide (65 mL) flushed with carbon mon-
oxide were 1,3-bis(diphenylphosphino)propane (314 mg,
0.76 mmol), palladium acetate (171 mg, 0.76 mmol), tri-
ethylamine (6.0 mL) and methanol (14.5 mL) added. The
reaction was stirred at 70 °C and atmospheric pressure for
5 h, the solvent was then evaporated. The remainder was
dissolved in diethyl ether (600 mL) and the organic phase
was washed with ammonium hydroxide (2 M, 300 mL),
brine (200 mL), dried (MgSO4) and evaporated. The
crude product was purified by flash chromatography
(SiO2, dichloromethane-hexane 2:3) affording the title
compound (1.65 g, 36%), m.p.: 88–89 °C. 1H NMR
(DMSO-d6) δ 7.73 (s, 1H), 7.49 (s, 1H), 3.90 (s, 3H),
3.86 (s, 3H), 13C NMR (DMSO-d6) δ 164.8, 153.4,
131.3, 129.6, 125.3, 123.2, 114.2, 56.7, 52.7, MS (EI, 70
eV) m/z: 238/236/234 (M +, 8/55/83).
169.0, 154.1, 149.6, 121.3, 119.6, 119.0, 116.6, 57.7.
6.1.38.
4-Bromo-3-hydroxy-6-methoxy-2-nitrobenzoic
acid 38
To a solution of sodium nitrate (361 mg, 4.25 mmol),
lanthanum nitrate hexahydrate (18 mg, 0.042 mmol) and
hydrochloric acid (12 M, 4 mL) in water (4 mL) at 0 °C,
was 37 (1.05 g, 4.25 mmol) in diethyl ether (20 mL)
added in portions. The mixture was stirred for 7 h, while
the solution reached ambient temperature. Dichlo-
romethane (90 mL) and water (20 mL) were added, and
the organic phase was collected, dried (MgSO4), filtered
and evaporated. The remainder was purified by flash
chromatography (SiO2, toluene-ethyl acetate-acetic acid
1
8:2:1) affording the title compound (600 mg, 48%). H
NMR (CD3OD) δ 7.57 (s, 1H), 3.85 (s, 3H), MS (EI, 70
eV) m/z 293/291 (M +, 21/19).
6.1.42. 2,4-Dichloro-5-hydroxybenzoic acid 42
6.1.39. 4-Bromo-3-hydroxy-6-methoxyanthranilic acid 39
4-Bromo-3-hydroxy-6-methoxy-2-nitrobenzoic acid
38 (52 mg, 0.18 mmol) in ethanol (7 mL) was hydrogen-
ated at atmospheric pressure and room temperature for
18 h with PtS2 as the catalyst. The solution was filtered
and the solvent was evaporated. The remainder was
purified by flash chromatography (SiO2, toluene-ethyl
acetate-acetic acid 8:2:1) to give the title compound
(30 mg, 64%). 1H NMR (CD3OD) δ 6.40 (s, 1H), 3.88 (s,
3H), 13C NMR (CD3OD) δ 170.8, 154.8, 145.6, 137.8,
115.5, 102.1, 101.6, 57.6, MS (EI, 70 eV): m/z 263/261
(M +, 79/80).
To a solution of 41 (600 mg, 2.55 mmol) in dichlo-
romethane (10 mL) at –70 °C and under argon atmo-
sphere was boron tribromide (0.72 mL, 7.66 mmol)
added. The mixture was stirred for 4 h while the tempera-
ture slowly reached ambient temperature. Dichloro-
methane (20 mL) and sodium hydroxide (2 M, 15 mL)
were added and stirring was continued for 30 min. The
aqueous phase was washed with dichloromethane
(10 mL) and was then acidified to pH 1 by the addition of
hydrochloric acid (12 M). The aqueous phase was ex-
tracted with ethyl acetate (40 mL), and the organic phase
washed with brine, dried (MgSO4) and evaporated to give
the title compound (350 mg, 66%), m.p.: 200.5–201.5 °C.
1H NMR (DMSO-d6) δ 10.8 (br, 1H) 7.56 (s, 1H), 7.38
(s, 1H), 13C NMR (DMSO-d6) δ 165.8, 152.0, 131.3,
130.3, 123.6, 121.6, 118.1, MS (EI, 70 eV) m/z:
210/208/206 (M +, 11/64/100).
6.1.40. 2,4-Dichloro-5-methoxyphenyl trifluoromethane-
sulfonate 40
To a solution of 2,4-dichloro-5-methoxyphenol [35]
(4.43 g, 22.9 mmol), triethylamine (6.40 mL, 45.9 mmol)
and N,N-dimethylaminopyridine (5 mg, 0.04 mmol) in

741
6.1.43. 4,6-Dichloro-3-hydroxy-2-nitrobenzoic acid 43
To a solution of 42 (280 mg, 1.35 mmol) in nitro-
methane (35 mL) at 45 °C was nitric acid (90%, 63 µL,
1.35 mmol) added. The mixture was stirred for 4 h and
then the solvent was evaporated. The remainder was
dissolved in ethyl acetate (100 mL), and the organic
phase was washed with water (5 mL), brine (5 mL), dried
(MgSO4) and evaporated. The crude product was purified
by flash chromatography (SiO2, ethyl acetate-acetic acid
50:1) affording the title compound (323 mg, 95%), m.p.:
perature for 3 h with Pd/C (10%, 180 mg) as the catalyst.
The mixture was filtered and the solvent was evaporated.
The remainder was dissolved in ethyl acetate and the
organic phase was washed with ammonium hydroxide (2
M), dried (MgSO4) and evaporated. The crude material
was crystallized from ethyl acetate-hexane affording the
title compound (5.05 g, 62%), m.p.: 65–66.5 °C. 1H
NMR (CDCl3) δ 7.49–7.31 (m, 5H), 6.96 (d, J = 2.1 Hz,
1H), 6.82 (d, J = 2.1 Hz, 1H), 3.97 (s, 2H), 3.86 (s, 3H),
13C NMR (CDCl3) δ 142.5, 141.4, 139.9, 138.4, 128.7,
127.9, 127.4, 126.8, 118.2, 112.9, 59.8, MS (EI, 70 eV)
m/z: 235/233 (M +, 24/48). Anal. C13H12ClNO (C, H, N).
1
186 °C (dec.). H NMR (DMSO-d6) δ 7.88 (s, 1H), 13C
NMR (DMSO-d6) δ 163.8, 147.2, 139.0, 133.1, 128.1,
126.3, 118.2, MS (EI, 70 eV) m/z: 253/251 (M +, 34/49).
6.1.47. 3-Chloro-2-methoxy-5-phenylisonitrosoacetani-
lide 47
6.1.44. 4,6-Dichloro-3-hydroxyanthranilic acid hydro-
chloride 44
To a stirred solution of chloralhydrate (1.08 g,
6.53 mmol) and sodium sulfate (5.0 g, 35.2 mmol) in
water (20 mL) was 46 (1.01 g, 4.33 mmol) dissolved in a
mixture of water (5 mL), N,N-dimethylformamide
(10 mL) and hydrochloric acid (conc., 0.43 mL) added.
The mixture was stirred at 95 °C for 90 min, when
hydroxylamine hydrochloride (1.36 g, 19.5 mmol) was
added, and the stirring was continued for another 22 h.
The mixture was cooled and evaporated and then ethyl
acetate was added. The organic phase was washed with
water, dried (MgSO4) and evaporated. The remainder was
purified by flash chromatography (SiO2, dichloromethane
→ ethyl acetate) to give the title compound (1.04 g,
79%). 1H NMR (DMSO-d6) δ 9.51 (s, 1H), 8.39 (s, 1H),
7.80 (s, 1H), 7.60 (d, 2H), 7.51–7.34 (m, 5H), 3.83 (s,
3H), 13C NMR (DMSO-d6) δ 160.4, 145.3, 143.6, 138.4,
137.3, 132.8, 129.0, 128.8, 127.9, 127.0, 126.6, 126.4,
123.3, 118.6, 60.8, MS (EI, 70 eV) m/z: 306/304 (M +,
6/20). Anal. C15H13ClN2O3 (C, H, N).
4,6-Dichloro-3-hydroxy-2-nitrobenzoic
acid
43
(223 mg, 0.88 mmol) dissolved in ethanol (50 mL) was
hydrogenated at atmospheric pressure and room tempera-
ture for 1 h with Pd/C (10%, 30 mg) as the catalyst. The
mixture was filtered and the solvent was evaporated. The
remainder was purified by flash chromatography (SiO2,
ethyl acetate-acetic acid 50:1) affording the free base. The
base was dissolved in tetrahydrofuran (0.6 mL) and
ethereal hydrogen chloride (3 M, 1 mL) was added to
give the title compound (32 mg, 14%), m.p.: 231 °C
1
(dec.). H NMR (DMSO-d6) δ 6.68 (s, 1H), 13C NMR
(DMSO-d6) δ 167.2, 140.3, 139.1, 122.5, 121.9, 116.2,
114.0, MS (EI, 70 eV) m/z: 223/221 (M +, 42/65). Anal.
C7H5Cl2NO3xHCl (C, H, N).
6.1.45. 3-Chloro-2-methoxy-5-phenylnitrobenzene 45
A solution of 2-chloro-6-nitro-4-phenylphenol [36]
(21.2 g, 0.08 mol), potassium carbonate (17.6 g,
0.13 mol) and iodomethane (13.5 mL, 0.22 mol) in dry N,
N-dimethylformamide (150 mL) was stirred at room
temperature under nitrogen atmosphere for 19 h, when
the solvent was evaporated. The remainder was purified
by flash chromatography (SiO2, hexane → ethyl acetate-
hexane 10:1) affording, after crystallization from ethyl
acetate-hexane, the title compound (22.2 g, 99%), m.p.:
76–77.5 °C. 1H NMR (CDCl3) δ 7.88 (d, J = 2.3 Hz, 1H),
7.81 (d, J = 2.3 Hz, 1H), 7.53–7.40 (m, 5H), 4.05 (s, 3H),
13C NMR (CDCl3) δ 148.8, 145.5, 138.1, 137.1, 132.8,
130.8, 129.4, 129.2, 128.7, 126.8, 121.8, 62.6, MS (EI,
70 eV) m/z: 265/263 (M +, 30/98). Anal. C13H10ClNO3
(C, H, N).
6.1.48. 6-Chloro-7-methoxy-4-phenylisatin 48
3-Chloro-2-methoxy-5-phenylisonitrosoacetanilide 47
(6.5 g, 21.3 mmol) and polyphosphoric acid (66 g) were
stirred at 80 °C for 3 h when ice, water and ethyl acetate
were added. The organic phase was washed with water,
dried (Na2SO4) and evaporated. The remainder was
purified by flash chromatography (SiO2, dichloromethane
→ ethyl acetate-hexane 1:1) to give the title compound
1
(5.06 g, 83%). H NMR (DMSO-d6) δ 11.54 (br s, 1H),
7.54–7.42 (m, 5H), 7.10 (s, 1H), 3.82 (s, 3H), 13C NMR
(DMSO-d6) δ 181.8, 159.3, 145.4, 139.8, 138.0, 135.6,
134.9, 128.9, 128.8, 128.1, 124.4, 114.5, 61.2, MS (EI,
70 eV) m/z: 289/287 (M +, 27/100). Anal. C15H10ClNO3,
(C, H, N).
6.1.46. 3-Chloro-2-methoxy-5-phenylaniline 46
3-Chloro-2-methoxy-5-phenylnitrobenzene 45 (9.1 g,
34.5 mmol) was hydrogenated in tetrahydrofuran
(100 mL), methanol (100 mL) and hydrochloric acid
(2 M, 34.5 mL) at atmospheric pressure and room tem-
6.1.49. 4-Chloro-3-methoxy-6-phenylanthranilic acid 49
To a solution of 48 (2.15 g, 7.47 mmol) in dioxane
(20 mL) and sodium hydroxide (0.68 M, 60 mL) at 0 °C

742
1
and under nitrogen atmosphere, was hydrogen peroxide
(30%, 4.0 mL, 39.2 mmol) in sodium hydroxide (0.68 M,
50 mL) added drop-wise. The solution was stirred at
ambient temperature for 6 h when acetic acid was added
to pH 5. The mixture was extracted with ethyl acetate, the
organic phase was dried (Na2SO4) and evaporated. The
remainder was purified by flash chromatography (SiO2,
ethyl acetate-hexane 1:2 → toluene-ethyl acetate-acetic
85%). H NMR (DMSO-d6) δ 9.36 (s, 1H), 7.90 (d, J =
1.6 Hz, 1H), 7.74 (s, 1H), 7.07 (d, J = 2.1 Hz, 1H), 3.75
(s, 3H), 2.25 (s, 3H), 13C NMR (DMSO-d6) δ 160.2,
143.6, 134.8, 132.1, 125.9, 125.5, 120.8, 60.7, 20.5,
MS (TSP) m/z: 262/260 (M + 18, 30/100). Anal.
C10H11ClN2O3 (C, H, N).
6.1.52. 6-Chloro-7-methoxy-4-methylisatin 52
1
3-Chloro-2-methoxy-5-methylisonitrosoacetanilide 51
(0.49 g, 2.0 mmol) and polyphosphoric acid (6.3 g) were
stirred at 80 °C for 3 h when ice was added. Ethyl acetate
(150 mL) was added and the organic phase was washed
with water (50 mL), dried (Na2SO4) and evaporated. The
remainder was purified by flash chromatography (SiO2,
ethyl acetate-hexane 1:2 → ethyl acetate) to give the title
acid 4:1:1) to give the title compound (1.37 g, 66%). H
NMR (DMSO-d6) δ 7.38–7.25 (m, 5H), 6.52 (s, 1H),
3.75 (s, 3H), 13C NMR (DMSO-d6) δ 169.3, 142.9,
141.6, 141.1, 139.0, 128.0, 127.9, 127.1, 117.5, 114.6,
59.4, MS (EI, 70 eV) m/z: 280/278 (M +, 15/47). Anal.
C14H12ClNO3 (C, H, N).
1
compound (0.37 g, 82%). H NMR (DMSO-d6) δ 11.41
6.1.50. 4-Chloro-3-hydroxy-6-phenylanthranilic acid 50
To 49 (110 mg, 0.40 mmol) dissolved in diethyl ether
was ethereal hydrogen chloride added. The solvent was
evaporated and dichloromethane (3 mL) was added. The
solution was cooled to –70 °C under nitrogen atmosphere
and boron tribromide (0.08 mL, 0.84 mmol) was added.
The solution was stirred for 40 min while the temperature
reached ambient temperature. The mixture was left over
night, and then sodium bicarbonate (sat, 15 mL) was
added and the aqueous phase was stirred with dichlo-
romethane for 1 h. Then the pH was lowered to 4 by the
addition of hydrochloric acid (2 M), and the aqueous
phase was extracted with dichloromethane, the organic
phase was dried (MgSO4) and evaporated. The crude
material was purified by flash chromatography (SiO2,
toluene-ethyl acetate-acetic acid 4:1:1) affording the title
compound (23 mg, 22%). 1H NMR (DMSO-d6) δ
7.33–7.22 (m, 5H), 6.52 (s, 1H), 13C NMR (DMSO-d6) δ
172.5, 143.6, 141.2, 141.1, 137.2, 129.6, 129.2, 128.0,
123.0, 119.8, 115.2, MS (EI, 70 eV) m/z: 265/263 (M +,
23/79).
(br, 1H), 7.00 (s, 1H), 3.74 (s, 3H), 2.38 (s, 3H), 13C
NMR (DMSO-d6) δ 183.8, 159.4, 144.5, 138.6, 136.0,
135.8, 125.1, 116.1, 61.1, 16.6, MS (TSP) m/z: 245/243
(M + 18, 37/100). Anal. C10H8ClNO3 (C, H, N).
6.1.53. 4-Chloro-3-methoxy-6-methylanthranilic acid 53
To a solution of 52 (320 mg, 1.4 mmol) in dioxane
(5 mL) at 5 °C and under nitrogen atmosphere was
hydrogen peroxide (30%, 0.73 mL, 7.2 mmol) in water
(10 mL) and sodium hydroxide (0.67 M, 26 mL) added
drop-wise. The mixture was stirred for 3 h while the
temperature slowly reached ambient temperature. Water
(25 mL) was added and the pH was adjusted to 5 by the
addition of acetic acid. The mixture was extracted with
ethyl acetate (120 mL) and the organic phase was dried
(Na2SO4) and evaporated. The remainder was purified by
flash chromatography (SiO2, ethyl acetate → ethyl
acetate-acetic acid 1 000:1) affording the title compound
1
(210 mg, 70%). H NMR (CD3OD) δ 6.45 (s, 1H), 3.76
(s, 3H), 2.37 (s, 3H), 13C NMR (CD3OD) δ 171.9, 146.4,
142.9, 138.3, 131.0, 120.3, 114.5, 60.2, 23.2, MS (TSP)
m/z: 218/216 (M + 1, 34/100). Anal. C9H10ClNO3 (C, H,
N).
6.1.51.
3-Chloro-2-methoxy-5-methylisonitrosoaceta-
nilide 51
To a stirred solution of chloral hydrate (0.99 g,
6.0 mmol) and sodium sulfate (3.4 g, 24 mmol) in water
(15 mL) was 3-chloro-2-methoxy-5-methylaniline [37]
(0.51 g, 3.0 mmol) in N,N-dimethylformamide (7 mL),
water (9 mL) and hydrochloric acid (conc., 0.37 mL)
added. The mixture was stirred at 90 °C for 25 min, when
hydroxylamine hydrochloride (1.24 g, 18 mmol) was
added and stirring was continued for another 18 h. The
mixture was cooled and ethyl acetate (100 mL) was
added. The organic phase was washed with water
(100 mL), dried (Na2SO4) and evaporated. The remainder
was purified by flash chromatography (SiO2, ethyl
acetate-hexane 1:1) affording the title compound (0.62 g,
6.1.54. 4-Chloro-3-hydroxy-6-methylanthranilic acid 54
To 53 (49 mg, 0.23 mmol) in diethyl ether was ethereal
hydrogen chloride (3 M) added. The solvent was evapo-
rated and dichloromethane (2 mL) was added. The solu-
tion was cooled to –65 °C under nitrogen atmosphere
when boron tribromide (0.1 mL, 1.12 mmol) was added.
The reaction was stirred for 43 h while the temperature
reached ambient temperature. Sodium bicarbonate (sat.,
15 mL) was added, and the pH of the aqueous phase was
adjusted to 2 by the addition of hydrochloric acid (2 M).
The aqueous phase was extracted with ethyl acetate, and
the organic phase was dried (Na2SO4) and evaporated.
The remainder was purified by flash chromatography

743
(SiO2, toluene-acetic acid 10:1 → 5:1) affording the title
cycle (12:12 h, lights on 6 a.m.). Food and tap water were
available ad libitum. The rats were anaesthetized with
enflurane (Efrane; Abbott) in a flow mixture of O2 and
N2O. They were thereafter placed in a stereotaxic frame.
The anaesthesia was maintained by free breathing into a
mask fitted over the nose of the rat of 3.5–5.0% of
enflurane maintained at a flow of 3 L/min of O2 and
7 L/min of N2O. Body temperature was kept at 37 °C
using a heating pad controlled via a rectal thermometer
(CMA/12). The skull was oriented with the horizontal
plane passing through bregma and lambda and through
the intraural line. The skin over the skull was opened and
a unilateral hole on the right side of the skull bone was
made by drilling with a 1 mm burr. A Hamilton microlitre
syringe (gauge 22S, 25 µL) was lowered 3.9 mm verti-
cally from the surface of the brain at AP 0 mm (bregma)
and L 1.0 mm. An intracerebroventricular (i.c.v.) injec-
tion of 3, 10, or 300 nmol of test compound together with
10 nmol 3-HANA dissolved in 10 µl 10 mM phosphate
buffer, containing 105 mM NaCl, 2.5 mM KCl, 1.18 mM
MgCl2, 1.26 mM CaCl2 and 0.1% ascorbic acid was
performed for 1 min. The syringe was then removed and
the skin was closed by wound clips. At various time-
points after the i.c.v. injection, the rats were decapitated,
and their brains rapidly removed and placed on an
ice-chilled petri-dish. The right hippocampus was dis-
sected out and stored at –70 °C until QUIN determina-
tions.
1
compound (22 mg, 47%). H NMR (CD3OD) δ 6.43 (s,
1H), 2.35 (s, 3H), 13C NMR (CD3OD) δ 172.2, 142.9,
139.9, 133.4, 123.8, 119.8, 113.8, 22.9, MS (TSP) m/z:
204/202 (M + 1, 28/100). Anal. C8H8ClNO3 (C, H, N).
6.2. Stability assay
The compounds were dissolved in PBS buffer at pH
7.5 and the solution was left in the dark for 24 h at 37 °C.
Aliquots were analysed by HPLC (Bondapak C18, 150 ×
3.9, phosphate buffer pH 3-acetonitrile-acetic acid
75:25:1) with UV detection (254 nm).
6.3. Pharmacological methods
6.3.1. In vitro measurement of 3-HAO inhibition
Male Sprague-Dawley rats (150–200 g) were anaesthe-
tized and perfused before decapitation. The cortex was
rapidly dissected out on ice and stored at –70 °C. To
prepare a homogenate, the cortex was thawed, minced
and sonicated in 9 volumes (w/v) of ice-cold distilled
water by 3 times 5 s bursts of sonication using a cell
disrupter (Branson sonifier). Aliquots of the crude homo-
genate were stored in vials (400 µL/vial) at –70 °C prior
to assay. To prepare cell-free homogenate, the vials were
thawed in an ice-bath, and to each vial, 600 µL 150 mM
(N-morpholino)-2-ethanesulfonic acid (MES)/NaOH
buffer (pH 6.5), was added. The vials were then vortexed
and centrifuged at 15 000 g for 10 min at 4 °C. 100 µL of
the supernatant was incubated at 37 °C under gently
shaking in a water bath for 1 h in a solution containing
FeSO4 (0.3 mM), ascorbic acid (aq, 0.1%) and [14C]3-
HANA (specific activity of 5.5 mCi/mmol, 5 µM, Astra
Arcus AB, final volume 200 µL). Test compounds were
added in a volume of 10 µL prior to the substrate. The
incubation was terminated by addition of HClO4 (aq.,
6%, 50 µL), the tubes cooled on ice and the precipitate
removed by centrifugation at 10 000 g for 4 min at 4 °C.
An aliquot (230 µL) of the supernatant was applied to a
Dowex 50W (200–400 mesh) cation-exchange column
(0.5 × 2 cm), which was thereafter washed with distilled
water (1 mL) to elute the [14C]-QUIN. Scintillation fluid
(10 mL, Beckman, Ultima Gold) was added to the eluate,
and the radioactivity was determined by liquid scintilla-
tion spectrophotometry.
For QUIN determination, the samples were homog-
enized in 10 volumes (w/w) 0.3 M formic acid with
[18O]4-quinolinic acid added as internal standard. After
filtration, the samples were applied to a pretreated anion-
exchanger column (0.5 mL Bio-Rad AG 1 × 8, 200–400
mesh, formate form). The column was washed with water
(4 mL) before use. QUIN and internal standard were
eluted with 3 mL 6.0 M formic acid. After evaporation,
the samples were esterified at 70 °C with hexafluoroiso-
propanol and trifluoroacetylimidazole for 1 h. The esters
were partitioned between water (0.4 mL) and n-heptane
(3 mL) by whirlimixing for 1 min followed by centrifu-
gation at 1 000 g for 5 min. The organic phase was
transferred to a new tube and washed with 0.4 mL of
water by whirlimixing. After a second centrifugation, the
water was discarded and the samples evaporated in a
stream of nitrogen at room temperature to about 5 mL.
Two µL of the final solution was assayed using gas
6.3.2. In vivo measurement of 3-HAO inhibition
chromatography coupled to a mass spectrometer
Male Sprague-Dawley rats (150–200 g) were acclima-
tized to the animal quarters for at least 7 d before
initiation of the experiments. The rats were housed five
animals per cage under controlled conditions of tempera-
ture (21 °C), relative humidity (55–65%) and light-dark
(GC/MS). The GC (Carlo Erba MFC 500) was operated
in splitless injection mode, and a capillary column
(NB-225, 25 m, i.d. 0.32 mm) was used. Helium was
employed as carrier gas. The oven was run from
70–135 °C, with a ramp of 20 °C/min. The MS (VG,

744
[12] Stone T.W., Pharmacol. Rev. 45 (1993) 309–379.
[13] Lapin I.P., J. Neuronal Trans. 42 (1978) 37–43.
TS-250) was adjusted to record m/e 467 (QUIN) and m/e
471 (internal standard) by negative chemical ionization.
Methane was used as reagent gas. The peak height ratio
(QUIN/internal standard) from standards with known
amounts of QUIN was calculated. The calibration curve
was plotted as peak height ratio against concentration of
QUIN in the standards. QUIN amount in the samples was
then calculated from the standard curve.
[14] Schwarcz R., Whetsell W.O. Jr., Mangano R.M., Science 219 (1983)
316–318.
[15] Beal M.F., Ferrante R.J., Swartz K.J., Kowall N.W., J. Neurosci. 11
(1991) 1649–1659.
[16] Huang Q., Zhou D., Sapp E., Aizawa H., Ge P., Bird E.D., Vonsattel
J.P., Di Figlia M., Neuroscience 65 (1995) 397–407.
[17] Tatter S.B., Galpern W.R., Hoogeveen A.T., Isacson O., Neuroreport
6 (1995) 1125–1129.
[18] Foster A.C., White B.J., Schwarcz R., J. Neurochem. 47 (1986)
22–30.
Acknowledgements
[19] Heyes M.P., Jordan E.K., Lee K., Saito K., Frank J.A., Snoy P.J.,
Markey S.P., Gravell M., Brain Res. 570 (1992) 237–250.
The authors are grateful for the analysis of QUIN by
Mr Göran Fredriksson, for surgical assistance by Ms Eva
Vänerman and to Mr Göran Stening for skillful technical
assistance. 4-Halogenated 3-HANA analogues were gen-
erously provided by Dr Barry Carpenter, Cornell Univer-
sity.
[20] Halperin J.J., Heyes M.P., Neurology 42 (1992) 43–50.
[21] Eastman C.L., Urbánska E., Löve A., Kristensson K., Schwarcz R.,
Exper. Neurol. 125 (1994) 119–124.
[22] Heyes M., Saito K., Major E.O., Milstien S., Markey S.P., Vickers
J.H., Brain 116 (1993) 1425–1450.
[23] Heyes M.P., Saito K., Milstien S., Schiff S.J., J. Neurol. Sciences
133 (1995) 112–118.
[24] Todd W.P., Carpenter B.K., Schwarcz R., Prep. Biochem. 19 (1989)
155–165.
References
[25] Manthey M.K., Pyne S.G., Truscott R.J.W., J. Org. Chem. 53 (1988)
1486–1488.
[1] Bokman A.H., Schweigert B.S., Arch. Biochem. Biophys. 33 (1951)
270–276.
[26] Manthey M.K., Pyne S.G., Truscott R.J.W., Biochim. Biophys. Acta
1034 (1990) 207–212.
[2] Roberts R.C., McCarthy K.E., Du F., Ottersen P., Okuno E.,
Schwarcz R., J. Neurosci. 15 (1995) 1150–1161.
[27] Hansch C., Leo A., Hoekman D., Exploring QSAR: Hydrophobic,
Electronic and Steric Constants, ACS, Washington DC, 1995.
[3] Heyes M.P., Chen C.Y., Major E.O., Saito K., Biochem. J. 326
(1997) 351–356.
[28] Pallas – CompDrug Chemistry Ltd.
[29] Dewar M.J.S., Zoenisch E.G., Healy E.F., Stewart J.J.P., J. Am.
Chem. Soc. 107 (1985) 3902–3909.
[4] Alberati-Giani D., Ricciardi-Castagnoli P., Köhler C., Cesura A.M.,
J. Neurochem. 66 (1996) 996–1004.
[30] Ovality is defined as ovality = A/4p(3V/4p) 2/3, where A is the
molecular surface area and V is the molecular volume.
[5] Saito K., Markey S.P., Heyes M.P., Brain Res. 546 (1991) 151–154.
[6] Saito K., Chen C.Y., Masana M., Crowley J.S., Markey S.P., Heyes
M.P., Biochem. J. 291 (1993) 11–14.
[31] Cannon J.R., Cresp T.M., Metcalf B.W., Sargent M.V., Vinciguerra
G., J. Chem. Soc. C (1971) 3495–3504.
[7] Saito K., Markey S.P., Heyes M.P., Neurosci. Lett. 178 (1994)
211–215.
[32] Melikian A., Boigegrain R., Kan J.P., Soubrie P., Eur. J. Med.
Chem./Chim. Ther. 25 (1990) 267–270.
[8] Heyes M.P., Saito K., Major E.O., Milstien S., Markey S.P., Vickers
J.H., Brain 116 (1993) 1425–1450.
[33] Calam C.T., Oxford A.E., J. Chem. Soc. (1939) 280–284.
[34] Schmidt U., Boekens H., Lieberknecht A., Griesser H., Tetrahedron
[9] Blight R.T., Cohen T.I., Saito K., Heyes M.P., Brain 118 (1995)
735–752.
Lett. 22 (1981) 4949–4952.
[35] Castelfranchi G., Perrotti E., Ann. Chim. 47 (1957) 1201–1214.
[10] Okuno E., Köhler C., Schwarcz R., J. Neurochem. 49 (1987)
771–780.
[36] Colbert J.C., Meigs W., Stuerke A.H., J. Amer. Chem. Soc. 56
(1934) 2128–2130.
[11] Malherbe P., Köhler C., Da Prada M., Lang G., Kiefer V., Schwarcz
R., Lahm H.W., Cesura A.M., J. Biol. Chem. 269 (1994)
13792–13797.
[37] Watanabe Y., Nakajima K., Seki T., Ozawa H., Chem. Pharm. Bull.
18 (1970) 2208–2215.