Synthesis of 2-amino-4H-thiazolo[5,4-b]indole and characterization of its colored conversion products with protein tyrosine phosphatase inhibitory activity

Article abstract of DOI:10.1002/jhet.5570380303

In DMSO-solution 2-amino-4H-thiazolo[5,4-b]indole is converted into a complex mixture of colored products. The three major conversion end-products, of which two are inhibitors of protein tyrosine phosphatases (PTPs), were isolated by chromatographic methods and their structures characterized by spectroscopic analysis, including NMR and MS combined with computer assisted structure elucidation, and, finally, confirmed by independent chemical synthesis. Synthesis of 2-amino-4H-thiazolo[5,4-b]indole as well as its N-acetyl derivatives prepared from either oxindole or 2-bromo-1-(2-nitro-phenyl)ethanone is described.

Full text of DOI:10.1002/jhet.5570380303

May-Jun 2001
Synthesis of 2-Amino-4H-thiazolo[5,4-b]indole and
Characterization of its Colored Conversion Products with Protein
Tyrosine Phosphatase Inhibitory Activity
569
£
$
##
Jens Breinholt*, Claus Bekker Jeppesen , Sven Branner , Carl Erik Olsen , Niels Peter
#
Hundahl Møller , Bjarne Hilmer Nielsen** and Henrik Sune Andersen***
MedChem. Research I*** and IV*;
Novo Nordisk A/S, Novo Nordisk Park, DK-2760 Maaloev, Denmark
$
£ §
Protein Biochemistry; New Lead Discovery
#
Signal Transduction,
Novo Nordisk A/S, Novo Allé, DK-2880 Bagsvaerd, Denmark
Chemistry Department, The Royal Veterinary and Agricultural University, DK-1871 Frederiksberg C, Denmark
**Department of Organic Chemistry, The Technical University of Denmark, DK-2800 Lyngby, Denmark
##
§
Present address: Maxygen, Agern Allé, DK-2970, Hoersholm, Denmark
Received November 16, 2000
In DMSO-solution 2-amino-4H-thiazolo[5,4-b]indole is converted into a complex mixture of colored
products. The three major conversion end-products, of which two are inhibitors of protein tyrosine phos-
phatases (PTPs), were isolated by chromatographic methods and their structures characterized by spectro-
scopic analysis, including NMR and MS combined with computer assisted structure elucidation, and, finally,
confirmed by independent chemical synthesis. Synthesis of 2-amino-4H-thiazolo[5,4-b]indole as well as its
N-acetyl derivatives prepared from either oxindole or 2-bromo-1-(2-nitro-phenyl)ethanone is described.
J. Heterocyclic Chem., 38, 569 (2001).
Introduction.
Protein-tyrosine phosphatases (PTPs) are critically
involved in the regulation of intracellular signaling path-
ways that control cellular metabolism, differentiation and
mitogenicity (reviewed in [1]). Inhibition of PTPs that
negatively control protein tyrosine kinases (PTKs) would
potentially lead to enhanced (duration and perhaps ampli-
tude) signaling [2]. Therefore, PTPs are considered attrac-
tive therapeutic targets in different disease states where
improved PTK signaling is warranted, including type 2
diabetes [3]. Although it has been known for more than a
decade that certain PTPs are negatively regulating the
insulin receptor signaling pathway, the exact identity of
these enzymes remained elusive. Based on in vitro studies,
several PTPs have been proposed as negative regulators of
insulin signaling, including PTPα [4-8], PTP-LAR [9],
and PTP1B [10-12]. Importantly, recent studies on PTP1B
knockout mice provided significant support for the view
that PTP1B is a key regulator of insulin signaling. The lack
of PTP1B gave rise to both increased insulin sensitivity
and resistance to diet-induced obesity, thus pointing to this
phosphatase as a relevant therapeutic target in type 2 dia-
betes and obesity [13].
determination of the acetate and for separate biological
testing of single compounds, 1, 2, and 3 were synthesized.
Surprisingly, when testing for inhibitory activity against
PTP1B neither of the compounds 1, 2, and 3 displayed any
activity. In contrast, colored products formed during storage
of 1 in DMSO-solution inhibited PTP1B efficiently.
We here report the isolation, structural characterization
and biological activity of the major conversion products
derived from compound 1. Furthermore, we describe the
independent synthesis of the major conversion product to
confirm the spectroscopy derived structure, and, finally we
describe the synthesis of compounds 1, 2 and 3.
Results.
Preparation and Purification of Conversion Products.
We therefore initiated high throughput screening (HTS)
of the Novo Nordisk compound library using PTP1B and a
Automated HTS revealed that a solution of the aminoth-
iazole 1, contaminated with minor amounts of its N-acetyl
derivative 3, the synthesis of which is described below,
exhibited relatively potent PTP1B inhibitory activity. The
surprising observation that freshly prepared solutions of 1
and 3 were unable to inhibit PTP1B prompted us to further
investigate the chemical stability of 1 in solution.
Immediately after preparation, solutions of 1 in DMSO
appeared colorless and inactive against PTP1B, but the
33
P-phosphorylated peptide as substrate. Several inhibitors
were identified and one of these seemed to be 2-amino-4H-
thiazolo[5,4-b]indole, 1 since a sample containing this com-
pound relatively potently inhibited PTP1B. However, NMR
and MS analysis of the original sample revealed the pres-
ence of two components, namely the expected compound, 1
and an acetyl-derivative thereof, represented by either
formula 2 or 3 (Scheme 1). For unambiguous structure

570
J. Breinholt, C. B. Jeppesen, S. Branner, C. E. Olsen,
N. P. H. Møller , B. H. Nielsen and H. S. Andersen
Vol. 38
progress of a chemical reaction at room temperature was
evident from the appearance of a red color with a concomi-
tant increase in the PTP inhibitory activity. Increasing the
storage temperature accelerated this development and a
TFA salt. Liberation of the corresponding free base
resulted in a colour change from red to blue (see UV-VIS
data in experimental section). For reasons of solubility and
spectral quality the NMR analysis were performed on a
solution of the TFA-salt in DMSO-d added approximately
solution with K = 100 µM improved to 6 µM after 3 days
6
ic
1
one additional equivalent of TFA. H-NMR, including
at 37° (or 1.4 µM by heating at 80° for one hour). The reac-
tion mixture obtained by heating a DMSO-solution of 1 at
80° for sixteen hours was dominated by three major, col-
ored components (Figure 1), which were separated and
isolated by reversed phase HPLC as described in the
experimental section.
1
1
H, H-COSY, established the presence of eight aromatic
protons in two separated four-proton spin systems.
1
Additional H-resonances at δ 12.68 (1H, sharp) and δ
+
9.30 (2H, broad) were attributed to isolated NH and NH
2
13
groups, respectively. The C-NMR spectrum exhibited
Figure 1. HPLC trace detected at 300 nm of a fresh DMSO-solution of 1 (dotted line) and the reaction mixture obtained after heating at 80 ° for 16 hours
(solid line). The retention times for 1, 4, 5 and 6 are 1.64, 1.44, 2.10, and 2.25 minutes, respectively. The insert shows the UV-spectra of 1 (dotted line)
and 5 (solid line). Chromatographic details are given in Experimental.
Structural Characterization.
seventeen individual signals corresponding to aromatic or
1
13
carbonyl carbon atoms. H, C-HSQC data served to
assign carbon atoms directly coupled to protons and the
Structure and Biochemical Activity of Yellow Conversion
Product.
1
13
H, C-HMBC experiment to extend the carbon frame-
1
13
NMR analysis including single- and multiple-bond
H, C-correlation experiments (HSQC and HMBC)
work by observation of long-range H, C-couplings to
1
13
quaternary carbon atoms. High-resolution FAB-MS (m/z
+
established the structure of the yellow product as indole-
2,3-dione, 4 (isatin). The identity was supported by EI-
MS, exhibiting a molecular ion peak at m/z 147 and,
finally, confirmed by spectral and chromatographic com-
parison with an authentic sample of isatin. Isatin was
found to be unable to inhibit PTPs.
287.0945 [M+H] ) in conjunction with the NMR estab-
lished contents of protons and carbon atoms, implied the
molecular composition of the protonated species to be
1
15
C H N O. In conjunction with H, N-connectivities
17 11
15
4
15
and N-chemical shifts obtained by the N-versions of
1
15
the heteronuclear shift correlation experiments ( H, N-
HSQC and -HMBC) the substructures and fragments pre-
sented in Figure 2 were established. Key, diagnostic
H, C- and H, N-HMBC correlations are depicted by
arrows, and nitrogen atoms are labelled with their recorded
Structure and Biochemical Activity of the Red Conversion
Product.
1
13
1
15
The HPLC-purification scheme (as outlined in the
experimental section) using an acidic mobile phase
afforded the major conversion product as a red, amorphous
15
d
N-chemical shifts, except for one (N ) which exhibited
no detectable direct or long-range couplings.

May-Jun 2001
Synthesis of 2-Amino-4H-thiazolo[5,4-b]indole
571
Computer-generation of all structures conforming to the
established molecular formula, substructures and frag-
ments by the structure generation program NMRSAMS
(Version 2.0, Spectrum Research, LLC, Madison,
Wisconsin, USA) resulted in hundreds of possible struc-
tures if no further constraints on the constitution were
included. However, the number of candidate structures
could be reduced significantly by taking into account a few
additional spectroscopy-derived restrictions. First, the low
field chemical shift for the carbonyl carbon atom (δ
C
181.8) excluded an amide. Second, the chemical shift of
a
N (δ 146.2) excluded a double bond heteroaromatic
N
nitrogen. Third, lack of absorptions in the IR spectrum in
-1
the 2100-2400 cm range excluded the presence of a
nitrile, and, finally, a strong NOE observed between the
c
protons (δ 9.30) on the basic nitrogen (N ) and the aro-
matic proton (δ 8.36) attached to substructure A (Figure 2)
demonstrated their close proximity in space. By including
these restrictions structures 5, 5a, 5b and 5c (Scheme 2)
were found to be the only plausible structural candidates.
Of note independent chemical synthesis (see below) con-
firmed the structure 5 for the major conversion product.
The purified conversion product 5 exhibited K values
ic
against PTP1B and PTPα of 2.6 and 1.0 µM, respectively,
and appears to account for the major part of the increase in
enzyme inhibition observed by heat treatment of com-
pound 1 in DMSO-solution. Compound 5 was also found
to inhibit PTPβ, PTPε, CD45, LAR and SHP1 in the 1-5
µM range, indicating that 5 is a general inhibitor of PTPs.
Structure and Biochemical Activity of the Blue Conversion
Product.
The minor conversion product, obtained as a blue solid,
exhibited NMR characteristics very similar to those
recorded for 5, except for the lack of signals corresponding
to protons attached to a basic nitrogen and the occurrence
of an additional six-proton singlet signal at δ 3.18 corre-
H
1
13
sponding to two equivalent methyl groups. H, C-HSQC
1
13
and H, C-HMBC experiments furnished evidence for
substructures A and B (Figure 2) identical to those of 5.
The chemical shift for the carbon atoms corresponding to
the two equivalent methyl groups (δ 31.8) suggested their
C
geminal attachment to a sulphur atom. Additionally, a
Figure 2. Fragments and NMR-derived substructures A and B of major
1
13
1
15
1
15
conversion product with key, diagnostic long-range H, C- and H, N-
long-range correlation observed in the H, N-HMBC
spectrum from the methyl groups to a nitrogen atom res-
onating at 145 ppm demonstrated that the sulphur atom is
attached to a nitrogen atom, all in accordance with the
presence of a S,S-dimethylsulfilimine group.
d
HMBC correlations depicted by arrows. Nitrogen atoms (except for N )
15
are labeled with their N-chemical shifts.
The presence of a basic nitrogen with double bond char-
acter is in accordance with the relatively low-field chemical
The HRFAB-MS spectrum exhibited a molecular ion
+
c
13
peak at m/z 347.0979 M in correspondence with the molec-
shift for the basic nitrogen, N (96.2 ppm), and the C-res-
onance at 148.8 ppm attributable to the carbon atom
ular composition C H N OS, leading to 6 as the proposed
19 15
4
c
structure of the blue compound (Scheme 3). Expected NOE-
interaction was observed between the S-methyl groups and
an aromatic proton (δ 8.81) attached to substructure A. The
presence of a DMSO-derived S,S-dimethylsulfilimine group
attached to N . In comparison, the guanidinium carbon atom
in diphenyl guanidine was in DMSO-d observed to res-
6
onate between δ 147 (free base) and 155 (excess TFA), and
C
15
the N-chemical shift for the imino-nitrogen recorded in a
H, N-HSQC experiment under acidic conditions (excess
1
15
was further substantiated by using DMSO-d as the solvent
6
during the conversion of 1 to 6. In this case the isolated blue
product 6 exhibited in its FAB mass spectrum a molecular
ion peak at m/z 353 corresponding to C H D N OS.
TFA to obtain sharp NH-resonances) was δ 82.8.
N
The low-field chemical shift for C-7a (δ 162.8) is
C
c
explained by delocalization of the positive charge at N involv-
19
9 6 4
Compound 6 inhibited PTP1B with K = 2.6 µM.
ing a resonance structure carrying the positive charge on C-7a.
ic

572
J. Breinholt, C. B. Jeppesen, S. Branner, C. E. Olsen,
N. P. H. Møller , B. H. Nielsen and H. S. Andersen
Vol. 38
Reaction Mechanism and Independent Synthesis.
hydrolysed directly to 4 (isatin) perhaps via a cyanoimine.
Formation of the bis(cyanoimino) intermediate m/z 311
To rationalize the formation of 5 and 6 from 1 the time
course of the reaction was investigated further. Thus, a
solution of 1 in DMSO was heated at 80° and samples
were analyzed by LC-MS over a period of 26 hours. In
parallel an identical experiment, except for using DMSO-
+
([M+H] , C H N ) 10 proceeds via elimination of
18 11
6
hydrogen sulfide perhaps in a way similar to that described
for the formation of aryl aminocyans [14]. Cyclisation of
10 yields compound 11, containing the pentacyclic ring
system pyrimido[1,6-a: 5,4-b']diindole [15], which is
hydrolyzed to 5 or reacts with DMSO under the formation
of water and a S,S-dimethylsulfilimine species 12 which is
finally hydrolysed to 6. Heating a HPLC purified sample
of 5 at 80° in DMSO does not convert it to 6, demonstrat-
ing that 6 is not derived from 5 but from an earlier inter-
mediate. Additional evidence for the proposed routes to 5
and 6 outlined in Scheme 3 was obtained by synthesis of
the postulated intermediate 10 from indole and cyanogen
azide as described by Gompper et al. [16]. Heating a solu-
tion of 10 in DMSO at 80° transformed the compound into
a mixture of which 5 and 6 are major and minor compo-
d as the solvent, was conducted allowing identification of
6
intermediates and products carrying DMSO-derived
methyl groups. In addition to the starting material 1 (m/z
+
190, [C H N S+H] ) and end-products isatin, 4 (m/z 148,
9
7 3
+
+
[C H NO +H] ), 5 (m/z 287, [C H N O+H] ) and 6
(m/z 347 [C H N OS] and 353 [C H D N OS] ),
8
5
2
17 10 4
+
+
19 15
4
19 9 6 4
four major intermediate components were detected. Three
exhibited m/z 311 and one m/z 371 and 377 for the non-
deuterated and deuterated species, respectively. The time
dependent concentration profiles of individual species
were established by extracting selective ion traces corre-
sponding to the masses of (pseudo) molecular ions of each
component as shown in Figure 3. Initially a rapid disap-
pearance of 1 and a concomitant build-up of the m/z 311
and m/z 371/377 intermediates are observed. The interme-
diates are then slowly disappearing in concert with forma-
tion of the products 4, 5 and 6. Based on these findings we
suggest that 4, 5 and 6 are formed from 1 according to
Scheme 3. In brief, the aminothiazole moiety in 1 is
believed to ring open which results in a thiourea-substi-
tuted indole 7. The indole is either dimerized in an oxida-
tive manner to compound 8 liberating one equivalent of
water or forms a thiourea-substituted oxindole 9, which is
nents, respectively. Isatin (4) was not detected in
this mix-
ture, which supports its formation via a separate route not
involving compound 10.
Synthesis of 1, 2 and 3.
To unequivocally identify the acetate derivative of 1 present
in the HTS-sample routes offering the possibility for a regios-
elective introduction of the N-acetyl group in N-(4H-thia-
zolo[5,4-b]indole-2-yl)acetamide, 2 and 1-(2-aminothiazolo-
[5,4-b]indol-4-yl)ethanone, 3 were explored as outlined in
Scheme 4. The routes should provide the possibility of having
Figure 3. Relative concentration profiles of starting material 1 (m/z 190, ◆), products 4 (m/z 148, ◆), 5 (m/z 287, +), and 6 (m/z 353,
◊) and intermediates
(m/z 311, ×, ◆, ◆ and 377, ◆) observed by LC-MS during heating of 1 in DMSO-d at 80° over a period of 26 hours.
6

May-Jun 2001
Synthesis of 2-Amino-4H-thiazolo[5,4-b]indole
573
either the 2-aminothiazole ring (route A) or the indole ring
system (route B) formed prior to the final ring closure afford-
ing the thiazolo[5,4-b]indole ring system, which would allow
for a regioselective introduction of the acetyl group.
2-Amino-4H-thiazolo[5,4-b]indoles are to our knowledge
unprecedented within the chemical literature. Potts and
Marshall [17] have described the synthesis of 2-phenyl-4H-
thiazolo[5,4-b]indole wherein the thiazole ring was formed
prior to the final ring closure. Using a slightly modified pro-
cedure, compound 1 and 2 were synthesized. In brief, treat-
ment of 2-bromo-1-(2-nitrophenyl)ethanone, 13, as shown in
Scheme 5, with N-acetyl thiourea afforded the thiazole com-
pound 14, which was also obtained via treatment of 13 with
thiourea followed by acetylation with acetic acid anhydride of
the intermediate 2-amino-4-(2-nitrophenyl)thiazole, 15.
Deoxygenation of aromatic nitro compounds with triethyl
phosphite [18] affords products expected from the corre-
sponding nitrenes. In the present case, electrophilic attack of
the nitrene intermediate on the unsubstituted 5-thiazole posi-
tion afforded the desired N-(4H-thiazolo[5,4-b]indol-2-
yl)acetamide, 2. Deacetylation using hydrochloric acid in
ethanol afforded 2-amino-4H-thiazolo[5,4-b]indole, 1, which
proved identical with the major component in the HTS-sam-
ple based on NMR, HPLC and MS data, whereas compound
2 exhibited NMR and HPLC properties different from those
recorded for the minor component in the HTS-sample.
1-(2-Aminothiazolo[5,4-b]indol-4-yl)ethanone, 3 was
synthesized using route B (Scheme 4) from oxindole, 16

574
J. Breinholt, C. B. Jeppesen, S. Branner, C. E. Olsen,
N. P. H. Møller , B. H. Nielsen and H. S. Andersen
Vol. 38
Scheme 6
HMBC) were obtained in DMSO-d or chloroform-d at 300 K on
which offers the possibility of introducing the acetyl group
at the indole nitrogen atom prior to the thiazole formation
as shown in Scheme 6. Fused 2-aminothiazoles are most
conveniently synthesized by condensing an α-haloketone
with thiourea [19] which suggests 1-acetyl-2-chloro-1,2-
dihydroindol-3-one [20], 19 as a key intermediate synthe-
sized in three steps as outlined in Scheme 6. Oxindole, 16
was chloroformylated using phosphorus oxychloride in
DMF followed by acetylation of the indole nitrogen atom
with acetic acid anhydride which afforded 1-acetyl-2-
chloro-1H-indol-3-carbaldehyde, 18. Baeyer-Villiger oxi-
dation of the formyl group using m-chloroperbenzoic acid
(m-CPBA) yielded the acetylindole, 19 which underwent
cyclisation when treated with thiourea in ethanol affording
the desired 1-(2-aminothiazolo[5,4-b]indol-4-yl)ethanone,
3 exhibiting NMR and HPLC characteristics identical with
those of the minor component in the HTS-sample.
6
Bruker DRX400, DRX300 or DRX200 instruments equipped
1
15
with 5 mm selective inverse z-gradient probes. H, N-HSQC
1
(optimized for J = 101 Hz) and HMBC (two individual exper-
NH
n
iments optimized for J = 2.5 and 7 Hz, respectively) experi-
NH
ments were performed on a Bruker AMX2-400 instrument
1
equipped with a 5 mm broadband inverse z-gradient probe. H
13
and C chemical shifts (δ-values) are reported in ppm downfield
15
from tetramethylsilane. N chemical shifts are reported relative
to nitromethane at 379.5 ppm. LC-MS analysis was performed on
a HP1100 MSD equipped with an electro spray interface. EI mass
spectra, at 70 eV ionisation potential, and high-resolution fast
atom bombardment (HR-FAB) mass spectra were recorded on a
JEOL AX505W instrument. Microanalyses were carried out on a
Perkin-Elmer Model 240 elemental analyzer. Merck Kieselgel 60
F
on glass - or aluminium plates were used for tlc monitoring,
254
detection by UV (254 nm).
Synthesis of N-(4H-thiazolo[5,4-b]indol-2-yl)acetamide 2.
N-[4-(2-Nitrophenyl)-thiazol-2-yl]acetamide (14).
A mixture of 1-acetyl-2-thiourea (9.7 g, 0.082 mole) and
2-bromo-1-(2-nitrophenyl)ethanone (10.0 g, 0.041 mole) in
ethanol (150 mL) was heated at reflux temperature for 20 min-
utes. After cooling to room temperature the precipitate was fil-
tered off, washed with ethanol (2 × 50 mL) and dried in vacuo at
EXPERIMENTAL
Melting points were determined in open capillary tubes with a
BÜCHI 535 melting point apparatus and are uncorrected. The
H -, C - and H, C-heteronuclear nmr spectra (HSQC and
1
13
1
13
1
50°, which afforded 14 in 62 % yield (6.7 g), mp 216-218°; H

May-Jun 2001
Synthesis of 2-Amino-4H-thiazolo[5,4-b]indole
575
Table 1
1
13
H and C NMR data (δ, ppm) for Compounds 5 and 6 in DMSO-d
6
Red compound, 5
H
Blue compound, 6
H
1
13 15
1
13 15
Position [a]
C/
N
C/ N [b]
1
2
3
4
4a
5
6
7
7a
7b
8
8.10 (d, J = 7.8 Hz)
125.8
129.1
137.2
117.2
145.6
146
8.00 (d, J = 7.5 Hz)
125.1
128.3
137.1
119.4
147.1
n.o. [c]
n.o. [c]
n.o. [c]
162.3
117.5
124.3
122.3
137.0
113.7
151.8
110
7.69 (t, J = 7.8 and 7.6 Hz)
8.00 (t, J = 7.6 and 8.3 Hz)
8.36 (d, J = 8.3 Hz)
7.59 (t, J = 7.5 and 7.5 Hz)
7.92 (t, J = 7.5 and 8.2 Hz)
8.81 (d, J = 8.2 Hz)
148.8
n.o. [c]
162.8
117.2
124.2
122.7
137.4
113.9
151.9
111
121.7
122.2
181.8
123.9
96
8.12 (d, J = 8.0 Hz)
8.17 (d, J = 7.9 Hz)
9
7.38 (t, J = 8.0 and 7.7 Hz)
7.88 (t, J = 7.7 and 8.2 Hz)
7.58 (d, J = 8.2 Hz)
7.33 (t, J = 7.9 and 7.5 Hz)
7.83 (t, J = 7.5 and 8.2 Hz)
7.52 (d, J = 8.2 Hz)
10
11
11a
12
12a
12b
13
13a
12.68 (s)
12.40 (s)
120.7
n.o. [c]
183.2
123.7
145
NH
9.30 (bs)
2
S(Me)
3.18 (s)
31.8
2
13
[a] Atom numbering as indicated in Scheme 2; [b] C-Chemical shifts extracted from HSQC and HMBC spectra;
[c] Not observed. Carbon or nitrogen atoms for which no direct- or long range correlations were observed.
Anal. Calcd. for C H N O S: C, 48.86; H, 3.19; N, 18.99.
Found: C, 49.26; H, 3.22; N, 18.69.
Table 2
values (µM) of HTS-hit and Compounds 1-3 and Conversion
9
7 3 2
K
ic
Products 4-6 as Inhibitors of two Different Protein-Tyrosine
Phosphatases, PTP1B and PTPα
N-[4-(2-Nitrophenyl)thiazol-2-yl]acetamide (14).
A mixture of 15 (15.0 g, 0.07 mole) and sodium acetate (12.8 g,
0.16 mole) in acetic acid anhydride (200 mL) was stirred at room
temperature for 18 hours. The precipitate was isolated by filtration,
washed with water (3 × 80 mL), heptane (2 × 100 mL) and dried in
vacuo at 50° for 18 hours, which afforded 14 in 73 % yield (13.0 g),
Compound
HTS HIT
PTP1B
50
PTPα
30
1
2
3
4
5
6
75-300
>1000
>400
100-160
>1000
>400
1
mp 217-218°; H nmr (DMSO-d ): δ 2.16 (s, 3H), 7.51 (s, 1H), 7.60
6
> 1000
2.6
> 1000
1
(dt, 1H), 7.73 (dt, 1H), 7.78 (dd, 1H), 7.89 (dd, 1H), 12.17 (s, 1H).
Anal. Calcd. for C H N O S: C, 50.18; H, 3.45; N, 15.96.
11
9 3 3
2.6-16
1.7-6.5
Found: C, 50.16; H, 3.43; N, 15.77.
N-(4H-Thiazolo[5,4-b]indol-2-yl)acetamide (2).
nmr (DMSO-d ): δ 2.15 (s, 3H), 7.50 (s, 1H), 7.59 (dt, 1H), 7.72
A mixture of compound 14 (9.3 g, 0.035 mole), triethyl phos-
phite (20 mL) and xylene (70 mL) was heated at reflux tempera-
ture for 18 hours. After cooling the precipitate was isolated by fil-
tration, washed with diethyl ether (3 × 20 m) and dried in vacuo
6
(dt, 1H), 7.80 (dd, 1H), 7.89 (dd, 1H), 12.2 (bs, 1H).
Anal. Calcd. for C H N O S: C, 50.18; H, 3.45; N, 15.96.
11
9 3 3
Found: C, 50.28; H, 3.28; N, 15.53.
1
at 50°, which afforded 2 in 61 % yield (5.0 g), mp > 250°; H nmr
2-Amino-4-(2-nitrophenyl)thiazole (15).
(DMSO-d ): δ 2.16 (s, 3H), 7.11 (t, 1H), 7.28 (t, 1H), 7.48 (d,
6
1H), 7.74 (d, 1H), 11,41 (s, 1H), 12.15 (bs, 1H, NHCOMe).
A mixture of thiourea (12.8 g, 0.17 mole) and 2-bromo-1-(2-
nitrophenyl)ethanone (20.5 g, 0.084 mole) in ethanol (150 mL)
was heated at reflux temperature for 30 minutes. After cooling to
room temperature the volatiles were evaporated in vacuo. The
residue was dissolved in water (150 mL), the pH was adjusted to
7 by addition of 8 N sodium hydroxide and extracted with ethyl
acetate (2 × 200 mL). The combined extracts were washed with
Anal. Calcd. for C H N OS: C, 57.13; H, 3.92; N, 18.17.
11
9 3
Found: C, 57.50; H, 3.93; N, 18.13.
Synthesis of 2-Amino-4H-thiazolo[5,4-b]indole 1.
2-Amino-4H-thiazolo[5,4-b]indole Hydrochloride (1).
To a mixture of compound 2 (3.5 g, 0.015 mole) in ethanol (50
mL) was added concentrated hydrochloric acid (17.5 mL). The
reaction mixture was refluxed for 6 hours and stirred at room
temperature over night. The precipitate was isolated by filtration,
washed with ethanol (2 × 20 mL) and dried in vacuo at 50°,
brine (2 × 100 mL), dried (MgSO ), filtered and the solvent evap-
4
orated in vacuo, which afforded 15 in 88 % yield (16.3 g), mp
1
105-107°; H nmr (chloroform-d): δ 5.05 (bs, 2H), 6.67 (s, 1H),
7.44 (dt, 1H), 7.58 (dt, 1H), 7.67 (dd, 1H), 7.73 (dd, 1H).

576
J. Breinholt, C. B. Jeppesen, S. Branner, C. E. Olsen,
N. P. H. Møller , B. H. Nielsen and H. S. Andersen
Vol. 38
1
were as follows. Appropriately diluted inhibitors (4 different con-
centrations: diluted 1, 3, 9 and 27 fold in DMSO) were added to
microtiter plate wells containing different concentrations of the sub-
strate, p-nitrophenyl phosphate (usual range: 0.31 to 20 mM - final
assay concentration). The buffer used was 100 mM sodium acetate
pH 5.5, 50 mM sodium chloride, 0.1 % (w/v) bovine serum albu-
min, 5 mM DTT (dithiothreitol) and 10 % DMSO from the inhibitor
solution (total volume 100 µL). The reactions were started by addi-
tion of the enzyme to the wells and were carried out at 25° for 1
hour. The reactions were stopped by addition of NaOH. The
enzyme activity was determined by measurement of the absorbance
at 405 nm with appropriate corrections for absorbance of the com-
pounds and p-nitrophenyl phosphate. The data were analyzed using
nonlinear regression hyperbolic fit to classical Michaelis Menten
which afforded 1 in 59 % yield (2.0 g), mp 248-250°; H nmr
(DMSO-d ): δ 7.14 (t, 1H), 7.21 (t, 1H), 7.59 (d, 1H), 7.81 (d,
6
1H), 9.48 (bs, 2H, NH ), 11.45 (s, 1H, NH).
2
Anal. Calcd. for C H N S, 1×HCl, 1×H O: C, 44.36; H, 4.14;
9
7
3
2
N, 17.24. Found: C, 44.60; H, 4.25; N, 17.07.
Synthesis of 1-(2-Aminothiazolo[5,4-b]indol-4-yl)ethanone 3.
1-(2-Aminothiazolo[5,4-b]indol-4-yl)ethanone (3).
A mixture of thiourea (2.8 g, 36.3 mmoles), compound 19 [19]
(3.8 g, 18 mmoles) and iodine (4.6 g, 18 mmoles) in absolute
ethanol (100 mL) was heated at reflux temperature for 1 hour. After
cooling to room temperature the volatiles were evaporated in vacuo.
The residue was suspended in a mixture of water (100 mL) and
ethyl acetate (100 mL) and stirred for 30 minutes. The solid was fil-
tered off, suspended in ethyl acetate (25 mL) and stirred at reflux
temperature for 5 minutes. This was repeated with ethanol (40 mL)
instead of ethyl acetate. The solid was filtered off, suspended in a
mixture of water (20 mL) and diethyl ether (20 mL) and stirred at
room temperature for 15 minutes, filtered off and dried in vacuo at
50° for 18 hours, which afforded 3 in 8 % yield (0.35 g), mp > 250°
enzyme kinetic models. Inhibition is expressed as K values in µM,
ic
assuming mixed type inhibition.
Acknowledgment.
We wish to acknowledge the contributions of Anette L. P.
Sørensen for skilled synthesis of the target compounds, Lise-
Lotte N. Schmidt for determination of inhibitor constants and
Anne Mette Jacobsen for skilled assistance in HPLC purification.
+
Decomp; MS (EI) m/z 231 (M , 50), 189 (100), 162 (75), 144 (18),
129 (30), 102 (32); H nmr (DMSO-d ): δ 2.77 (bs, 3H), 7.18 (bs,
2H, NH ), 7.33 (m, 2H), 7.65 (m, 1H), 8.03 (bs, 1H, Ar-H5).
1
6
2
Anal. Calcd. for C H N OS: C, 57.13; H, 3.92; N, 18.17.
REFERENCES AND NOTES
11
9 3
Found: C, 56.70; H, 3.90; N, 18.00.
*** To whom all correspondence should be addressed.
[1] T. R. Burke, Jr. and Z. Y. Zhang, Biopolymers (Peptide
Sci-Ence), 47, 225 (1998).
[2] J. L. Evans and B. Jallal, Exp. Opin. Invest. Drugs, 8, 139
(1999).
[3] T. Hunter, Phil. Trans. R. Soc. Lond. B, 353, 583 (1998).
[4] L. N. Cong, H. Chen, Y. H. Li, C. H. Lin, J. Sap, and M. J.
Quon, Biochem. Biophys. Res. Commun., 255, 200 (1999).
[5] L. N. Cong, H. Chen, Y. H. Li, C. H. Lin, J. Sap, and M. J.
Quon, Diabetes, 47 (Suppl. 1), 178 (1998).
[6] R. Lammers, N. P. H. Møller and A. Ullrich, Biochem.
Biophys. Res. Commun., 242, 32 (1998).
[7] N. P. H. Møller, K. B. Møller, R. Lammers, A.
Kharitonenkov, E. Hoppe, F. C. Wiberg, I. Sures, and A. Ullrich, J.
Biol. Chem., 270, 23126 (1995).
[8] K. K. Jacob, J. Sap, and F. M. Stanley, J. Biol. Chem., 273,
4800 (1998).
[9] B. J. Goldstein, J. Cell. Biochem., 48, 33 (1992).
[10] K. A. Kenner, E. Anyanwu, J. M. Olefsky, and J. Kusari, J.
Biol. Chem., 271, 19810 (1996).
[11] J. C. H. Byon, A. B. Kusari, and J. Kusari, Mol. Cell.
Biochem., 182, 101 (1998).
[12] L. Seely, P. A. Staubs, D. R. Reichart, P. Berhanu, K. L.
Milarski, R. A. Saltiel, J. Kusari, and J. M. Olefsky, Diabetes, 45,
1379 (1996).
[13] M. Elchebly, P. Payette, E. Michaliszyn, W. Cromlish, S.
Collins, A. L. Loy, D. Normandin, A. Cheng, J. Himms-Hagen, C. C.
Chan, C. Ramachandran, M. J. Gresser, M. L. Tremblay, and B. P.
Kennedy, Science, 283, 1544 (1999).
[14] G. Crank, and M. I. H. Makin, J. Chem. Soc. Chem.
Commun., 53 (1984).
[15] The ring system pyrimido[1,6-a:5,4-b']diindole has to our
knowledge only been described once by A. N. Grinev et al. with a
different substituents pattern; A. N. Grinev, N. N. Suvorov, S. Yu.
Ryabova, G. N. Kurilo, K. F. Turchin and V. S. Velezheva, Chem.
Heterocycl. Comp. (Engl. Transl.), 16, 1193 (1979).
[16] R. Gompper, K. Hartmann, R. Kellner and K. Polborn,
Preparation and Isolation of Isatin (4), 6-Imino-12H-pyrim-
ido[1,6-a:5,4-b']diindole-13-one (5) and S,S-Dimethyl-13-oxo-
12H-pyrimido[1,6-a:5,4-b']diindole-6-imino Sulfonium (6).
2-Amino-4H-thiazolo[5,4-b]indole, 1 (100 mg) dissolved in
DMSO (3.0 mL) was heated in a sealed vial at 80° for 16 hours. The
components of the resulting dark solution were (in five portions)
separated by preparative reversed phase HPLC (Column: YMC
RP18 (15 µm), 250 × 20 mm; Gradient: 10 → 50 % CH CN (0.05 %
3
TFA) (50 min), 50 → 100 % CH CN (0.05 % TFA) (15 minutes),
3
flow rate 15 mL/minute; Detection: UV 210/550 nm). Fractions
were analyzed by reversed phase HPLC using a Nucleosil 100 (3
µm, 60 × 4 mm) column eluted with a linear gradient from H O (0.1
2
% H PO ) to CH CN over a period of 6 minutes at a flow rate of 2.7
3
4
3
mL/minute, and employing diode array detection at 200-600 nm.
Appropriate pooling of fractions yielded isatin, 4 (25 mg), the red
compound 5 (25 mg), and the blue compound 6 (4 mg), all obtained
1
13
15
as amorphous solids. 5: H- , C - and N nmr: see table 1.
HRFAB-MS: Calcd. for C H N O 287.0942; Found 287.0945
17 10
4
+
+
+
([M+H] ). EI-MS: 286 (100, M ), 258 (28, [M-CO] ), 182 (5), 156
(6), 143 (7), 129 (6), 103 (10), 76 (5). ir (potassium bromide): 3398,
-1
3126, 1681, 1640, 1614, 1572, 1203 and 1129 cm . uv (λ max nm
(log ε)) (MeOH + TFA): 265 (4.20), 313 (3.87), 415 (3.65), and 539
(3.36); (MeOH + NaOH): 281 (4.26), 333 (3.85), 450 (2.98), 605
1
13
15
(3.51), and 640 (3.47). 6: H -, C- and N nmr: see table 1;
HRFAB-MS: Calcd. for C H N OS 347.0966: Found 347.0979
19 15
4
+
(M ); ir (potassium bromide): 3436, 1691, 1605, 1200, and 1123
-1
cm ; uv (λ max nm (log ε)) (MeOH + TFA): 277 (4.33), 328 (3.79),
444 (3.22), and 576 (3.40); (MeOH + NaOH): 249 (4.23), 282
(4.27), 352 (3.85), 458 (3.53) and 750 (3.01).
Enzyme Kinetics.
Determination of Inhibitor Constants, K .
ic
The enzyme reactions were carried out using standard conditions
essentially as described by Burke et al. [21]. The assay conditions

May-Jun 2001
Synthesis of 2-Amino-4H-thiazolo[5,4-b]indole
577
Angew. Chem. Int. Ed. Engl., 34, 464 (1995).
[17] K. T. Potts, and J. L. Marshall, J. Org. Chem., 41, 129
(1976).
[18] R. A. Abramovitch, J. Court, and E. P. Kyba, Tetrahedron
Lett., 4059 (1972).
Heterocyclic Chemistry, Vol 3, I. Shinkai ed, Pergamon Press,
Oxford, 1996, pp 373-474.
[20] A. S. Bourlot, E. Desarbre, and J. Y. Mérour, Synthesis,
411 (1994).
[21] T. R. Burke, B. Ye, X. J. Yan, S. M. Wang, Z. C. Jia, L.
Chen, Z. Y. Zhang, and D. Barford, Biochemistry, 35, 15989 (1996).
[19] A. Dondoni and P. Merino, in Comprehensive