A Scaffold-Hopping Strategy toward the Identification of Inhibitors of Cyclin G Associated Kinase

Article abstract of DOI:10.1002/cmdc.201800690

We recently reported the discovery of isothiazolo[4,3-b]pyridine-based inhibitors of cyclin G associated kinase (GAK) displaying low nanomolar binding affinity for GAK and demonstrating broad-spectrum antiviral activity. To come up with novel core structures that act as GAK inhibitors, a scaffold-hopping approach was applied starting from two different isothiazolo[4,3-b]pyridines. In total, 13 novel 5,6- and 6,6-fused bicyclic heteroaromatic scaffolds were synthesized. Four of them displayed GAK affinity with K_d values in the low micromolar range that can serve as chemical starting points for the discovery of GAK inhibitors based on a different scaffold.

Full text of DOI:10.1002/cmdc.201800690

Accepted Article
Title: A scaffold hopping strategy towards the identification of inhibitors
of cyclin G associated kinase
Authors: Randy Wouters, Junjun Tian, Piet Herdewijn, and Steven De
Jonghe
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To be cited as: ChemMedChem 10.1002/cmdc.201800690
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10.1002/cmdc.201800690
ChemMedChem
FULL PAPER
A scaffold hopping strategy towards the identification of
inhibitors of cyclin G associated kinase
Randy Wouters,[a] Junjun Tian,[a] Piet Herdewijn,[a] and Steven De Jonghe*,§,[a]
[a]
Mr. R. Wouters (0000-0003-2878-6984), Dr. J. Tian, Prof. P. Herdewijn (0000-0003-3589-8503), Dr. S. De Jonghe (0000-0002-3872-6558)
Medicinal Chemistry, Rega Institute for Medical Research, KU Leuven, Herestraat 49 – box 1041, 3000 Leuven, Belgium
E-mail: steven.dejonghe@kuleuven.be
§
Present affiliation: Laboratory of Virology and Chemotherapy, Rega Institute for Medical Research, KU Leuven, Herestraat 49 - box 1043, 3000 Leuven,
Belgium
Abstract: We recently reported the discovery of isothiazolo[4,3-
b]pyridine based inhibitors of cyclin G associated kinase (GAK)
displaying low nanomolar binding affinity for GAK and demonstrating
broad-spectrum antiviral activity. In order to come up with novel core
structures that act as GAK inhibitors, a scaffold hopping approach was
applied starting from two different isothiazolo[4,3-b]pyridines. In total,
13 novel 5,6- and 6,6-fused bicyclic heteroaromatic scaffolds were
synthesized. Four of them displayed GAK affinity with Kd values in the
low micromolar range that can serve as chemical starting points for
the discovery of GAK inhibitors based on a different scaffold.
trafficking. This phosphorylation of APs enhances their binding to
cargo proteins and helps the recruitment of clathrin to the
membrane to form a clathrin coated vesicle (CCV).[2],[8]–[11] In
analogy to auxilin I, GAK contains a C-terminal J-domain that acts
as a cochaperone with the heat shock cognate 71 Kda protein
(HSC70) to uncoat CCVs in order to recycle clathrin back to the
cell surface.[2] Depletion of GAK inhibits CME by inhibiting the
recruitment of clathrin and clathrin adaptors.[2],[12] Furthermore, we
previously discovered that GAK’s involvement in CME is required
in both early and late stages of the lifecycle of distinct viruses,
such as dengue virus (DENV), Ebola virus (EBOV), chikungunya
virus (CHIKV), and hepatitis C virus (HCV). [10],[13],[14]
Recently, we reported the discovery of potent GAK inhibitors
based on an isothiazolo[4,3-b]pyridine scaffold, of which
compounds 1, 2 and 3 are the most promising representatives
(Figure 1). These compounds display potent GAK affinity (Kd
values in the range of 8-80 nM), are selective and are also
endowed with broad-spectrum in vitro antiviral activity against
HCV, DENV, EBOV and CHIKV.[15],[16]
Previous explorations of the structure-activity relationship (SAR)
of isothiazolo[4,3-b]pyridines as GAK inhibitors focused on
modifications of the functional groups at positions 3 and 6 of the
scaffold.[15]–[17] In this manuscript, an alternative approach based
on scaffold hopping was followed. Scaffold hopping is a widely
applied medicinal chemistry strategy in which the core skeleton
(the scaffold) of lead molecules is subjected to bioisosteric
replacements, leading to the discovery of structurally novel
compounds. There are different reasons to search for biologically
active compounds based on alternative scaffolds. Examples
include the development of analogues with improved activity
and/or selectivity, altered physicochemical and ADMET
properties, as well as obtaining molecules with a favourable
intellectual property situation.[18]
Introduction
Cyclin G associated kinase (GAK), also known as auxilin 2, is a
cellular serine/threonine protein kinase first identified in 1997.[1] It
is highly homologous to auxilin I, with the notable exception of the
presence of an N-terminal kinase domain on GAK.[2] In contrast
with auxilin I, GAK is ubiquitously expressed while auxilin I is
solely expressed in neurons. Many biological effects have been
attributed to GAK as a result of its abundance. It has been
demonstrated that siRNA-mediated GAK knockdown results in
mitotic arrest during metaphase and multipolar spindle
formation.[3] Due to this mitotic involvement, GAK inhibition
represents a possible drug target for the treatment of BXW7-
deficient tumours, such as cholangiocarcinoma.[4] GAK was also
identified as a potential target for the inhibition of prostate cancer
growth in cells expressing androgen receptor splice variants.[5]
Furthermore, GAK was found to be involved in osteosarcoma,
which is the most frequent primary malignant bone tumour among
children. In both osteosarcoma cell lines and tissue samples an
overexpression of GAK is seen, compared to human osteoblasts.
Its knockdown by siRNA decreased cell proliferation in both drug-
sensitive and multidrug-resistant osteosarcoma cell lines,
suggesting osteosarcoma growth and proliferation to be GAK
dependent.[6]
In Parkinson’s disease, the leucine rich repeat kinase 2 (LRRK2)
is a major contributor to the illness. It is implicated in vesicle
trafficking and was found to interact with GAK, where it plays a
role in the dysfunction of LRRK2.[7]
Figure 1. Previously reported isothiazolo[4,3-b]pyridines as GAK inhibitors.
GAK is a key regulator of clathrin-mediated endocytocis (CME) as
it is one of the kinases known to phosphorylate Thr144 and
Thr156 of the μ-subunits of adaptor protein complex (AP) 1 and
AP2, respectively. While GAK-dependent phosphorylation of AP2
is important for its endocytic activity, GAK’s phosphorylation of
AP1 is implicated in trans-golgi network (TGN) to lysosome
A scaffold hopping exercise starts with an active compound and
ends up with a novel chemotype by solely modifying the central
core structure of the molecule. Two isothiazolo[4,3-b]pyridines
1
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10.1002/cmdc.201800690
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with a different substitution pattern were selected as the starting
point for the scaffold hopping exercise (Figure 2). Compound 4[16]
bears a 3,4-dimethoxyphenyl residue at position 6, instead of its
3,4,5-trimethoxy (compound 2)
or
3-methoxy-4-amino
(compound 2) counterparts, and displays a Kd value of 52 nM for
GAK. Despite the 5-fold lower GAK affinity of compound 4
compared to compounds 1 and 2, it was selected as a reference
compound because of the easy access and low cost of large
amounts of 3,4-dimethoxyphenylboronic acid. For some of the
scaffolds (such as the pyrazolo[1,5-a]pyrimidine, pyrrolo[3,2-
b]pyridine, pyrazolo[4,3-b]pyridine and thieno[3,2-b]pyridine), the
insertion of a morpholine residue via either a nucleophilic aromatic
substitution, a palladium-catalyzed Buchwald reaction or a CuI/L-
proline mediated amination was cumbersome. In contrast, the
introduction of an aryl group via palladium-catalyzed Suzuki
cross-coupling reaction proceeded smoothly. Therefore,
compound 5, characterized by the presence of a phenyl moiety at
position 3 (instead of the morpholine residue of compound 4) was
used for comparison purposes.[19] Although, GAK affinity of
compound 5 (Kd of 0.77 µM) is 15-fold lower than the
corresponding 3-morpholino analogue 4, the straightforward
synthesis of different phenyl-substituted scaffold analogues
justifies its use as a reference compound. As the substitution
pattern is kept intact, the influence of scaffold modification on
GAK affinity can be easily studied.
Scheme 1. Synthesis of isothiazolo[3,4-b]pyridine. Reagents and conditions: a)
3,4-dimethoxyphenyl B(OH)2, K2CO3, Pd(PPh3)4, H2O, dioxane, 90°C (63%); b)
NaN3, DMF, 70°C (83%); c) PPh3, pyridine, rt; d) 80% aq CH3COOH, reflux
(95%); e) P2S5, ethanol, reflux (71%); f) 30% aq. H2O2, MeOH, 0°C (83%); g)
NaNO2, HBr, CuBr, H2O, 0°C to rt (25%); h) morpholine, EtOH, reflux (40%).
Synthesis of isothiazolo[3,4-b]pyrazine
Applying the same aqueous Suzuki conditions that successfully
yielded compound 7 (Scheme 1) to the synthesis of compound 16
resulted merely in the formation of different unidentified products.
However, the regioselective introduction of
a
3,4-
dimethoxyphenyl residue on 3,5-dichloropyrazine-2-carbonitrile
15 was possible via an anhydrous Suzuki reaction using
tripotassium phosphate as a base affording compound 16
(Scheme 2). The chlorine at position 3 was then exchanged by
azide affording compound 17. The azido moiety was
subsequently converted to an amino group via Staudinger
reduction with concomitant acidic hydrolysis of the
iminophosphorane intermediate 18 yielding pyrazine 19.
Thionation of its cyano moiety yielding thioamide 20 was achieved
with Lawesson's reagent (LR) rather than P2S5, as the work-up
with LR was less labour intensive and consistently led to higher
yields. The thioamide 20 was then submitted to an oxidative ring
closure furnishing the isothiazolo[3,4-b]pyrazine 21. Applying the
typical Sandmeyer reaction conditions (as in Scheme 1) to
convert the amino group into a bromine was unsuccessful, as a
complex mixture of unidentified products was formed. An
alternative Sandmeyer procedure using tert-butyl nitrite (t-
BuONO) and CuBr2 in dry acetonitrile yielded the desired product
22.[22] Finally, nucleophilic substitution with morpholine yielded the
title compound 23 in good yield.
Figure 2. Reference GAK inhibitors for scaffold hopping.
Chemistry
Synthesis of isothiazolo[3,4-b]pyridine
A
palladium-catalyzed Suzuki coupling between 2,6-
dichloronicotinonitrile 6 and 3,4-dimethoxyphenylboronic acid
yielded compound 7 regioselectively (Scheme 1).[20] Nucleophilic
displacement of the remaining chlorine at position 2 by ammonia
in either water or methanol only led to the recovery of starting
material. In contrast, the reaction with sodium azide proceeded
smoothly, affording compound 8 in high yield. Staudinger
reduction by treatment of compound 8 with triphenylphosphine
formed the iminophosphorane intermediate 9, which was
subsequently submitted to an acidic hydrolysis yielding the 2-
amino pyridine derivative 10.[21] The cyano group of compound 10
was converted to its corresponding thioamide 11 using
phosphorus pentasulfide. An oxidative ring closure using
hydrogen peroxide in methanol allowed to construct the
isothiazole moiety, furnishing isothiazolo[3,4-b]pyridine 12. A
Sandmeyer reaction using sodium nitrite, hydrogen bromide and
CuBr yielded 3-bromo-6-(3,4-dimethoxyphenyl)isothiazolo[3,4-
b]pyridine 13. Finally, a nucleophilic aromatic substitution with
morpholine afforded the desired isothiazolo[3,4-b]pyridine 14.
Scheme 2. Synthesis of isothiazolo[3,4-b]pyrazine. Reagents and conditions:
a) 3,4-dimethoxyphenyl- B(OH)2, K3PO4, Pd(PPh3)4, dioxane, 90°C (74%); b)
NaN3, DMF, 70°C (83%); c) PPh3, pyridine, rt; d) 80% aq CH3COOH, reflux
(95%); d) LR, ethanol, reflux (83%); f) 30% aq. H2O2, MeOH, 0°C (67%); g) t-
BuONO, CuBr2, CH3CN, rt (32%); h) morpholine, EtOH, reflux (64%).
Synthesis of isothiazolo[3,4-d]pyrimidine
2
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A Suzuki coupling on 4-amino-2-chloropyrimidine-5-carbonitrile
24 using the aforementioned reaction circumstances (Schemes 1
and 2) only resulted in the formation of side products. The
presence of a free amino group is known to hamper Suzuki
reactions and a common practice to circumvent this is by
protecting the amino group.[23] However, additional protecting and
deprotecting sequences are time consuming and lead to overall
lower reaction yields. Therefore, alternative reaction conditions
were explored. Performing the Suzuki reaction using tripotassium
phosphate as a base, Pd(OAc)2 as a catalyst and 1,1′-bis(di-tert-
Synthesis of pyrrolo[3,2-b]pyridine
In analogy to the Lemgruber-Batcho synthesis of indoles, the
synthesis of this scaffold started by reacting 5-bromo-2-methyl-3-
nitropyridine 34 with N,N-dimethylformamide dimethyl acetal
(DMF-DMA) to give enamine 35 (Scheme 5).[26] Reductive
cyclization of enamine 35 using iron in acetic acid led to the
formation of pyrrolo[3,2-b]pyridine 36. Electrophilic iodination
using N-iodosuccinimide (NIS) in THF yielded the 3-iodo
congener 37 in low yield. Suzuki reaction using 37 as substrate,
with the classical reaction conditions (Scheme 1) did not yield the
desired product and only starting material was recovered.
Protection of the pyrrole nitrogen prior to the palladium-catalyzed
cross-coupling reaction turned out to be necessary. The pyrrole
nitrogen was protected by reaction of 37 with di-tert-butyl
carbonate (Boc2O), yielding the Boc-protected derivative 38.
Suzuki coupling of 38 with phenylboronic acid proceeded with
concomitant cleavage of the Boc group, yielding compound 39.
Reprotection with Boc (compound 40) and subsequent Suzuki
coupling furnished compound 41. Remarkably, during this last
Suzuki reaction, no Boc deprotection was observed, despite using
the same reaction conditions as in step (e). Finally, acidic
hydrolysis using a 4N HCl solution in dioxane yielded pyrrolo[3,2-
b]pyridine 42 in moderate yield.
butylphosphino)ferrocene (D-t-BPF) as
a ligand provided
compound 25 efficiently (Scheme 3).[23] The subsequent steps are
similar as in Schemes 1 and 2. Briefly, thioamide 26 was
accessible via thionation of the cyano group of compound 25. The
isothiazolo moiety was then constructed via an oxidative ring
closure
Conversion of the amino group to a bromine 28, followed by the
introduction of morpholine moiety, gave access to
yielding
3-amino-isothiazolo[3,4-d]pyrimidine
27.
a
isothiazolo[3,4-d]pyrimidine 29.
Scheme 3. Synthesis of isothiazolo[3,4-d]pyrimidine. Reagents and conditions:
a) 3,4-dimethoxyphenyl- B(OH)2, K3PO4, Pd(OAc)2, D-t-BPF, dioxane (53%); b)
LR, ethanol, reflux (60%); c) 30% aq. H2O2, MeOH, 0°C (84%); d) t-BuONO,
CuBr2, CH3CN, rt (49%); e) morpholine, EtOH, reflux (65%).
Scheme 5. Synthesis of pyrrolo[3,2-b]pyridine. Reagents and conditions. a)
DMF-DMA, DMF, 90°C (98%); b) Fe, CH3COOH, rt (36%); c) NIS, THF, rt
(37%); d) Boc2O, TEA, DMAP, THF, rt (95%); e) phenyl-B(OH)2 (70%) or 3,4-
dimethoxyphenyl-B(OH)2 (15%), K2CO3, Pd(PPh3)4, H2O, dioxane; f) 4N HCl in
dioxane, reflux (47%).
Synthesis of pyrazolo[1,5-a]pyrimidine
Condensation of 4-bromo-1H-pyrazol-3-amine 30 and 2-
chloromalonaldehyde under acidic conditions afforded 3-bromo-
6-chloropyrazolo[1,5-a]pyrimidine 31 (Scheme 4).[24] Two
consecutive Suzuki reactions allowed to introduce both aromatic
substituents regioselectively, as bromine is favoured over chlorine
during the oxidative addition step.[25] The first Suzuki coupling
yielded selectively the 3-phenyl-pyrazolo[1,5-a]pyrimidine 32. A
subsequent Suzuki coupling under identical reaction conditions,
except for using 3,4-dimethoxyphenylboronic acid, yielded the
desired pyrazolo[1,5-a]pyrimidine 33 in good yield.
Synthesis of pyrazolo[4,3-b]pyridine
Suzuki coupling between 5-bromo-3-fluoropicolinonitrile 43 and
3,4-dimethoxyphenylboronic acid proceeded smoothly yielding
compound 44 in excellent yield (Scheme 6). An aromatic
nucleophilic substitution with hydrazine and consecutive ring
closure gave access to the pyrazolo[4,3-b]pyridine scaffold 45.[27]
Several attempts to convert the amino group into a bromine via
Sandmeyer reactions (using the conditions from Schemes 1 or 2)
resulted in unidentified side products. Instead, a diazotation
reaction using sodium nitrite, para-toluenesulphonic acid (p-
TsOH) and potassium iodide was used[28], yielding the 3-iodo
pyrazolo[4,3-b]pyridine analogue 46 in low yield. Finally,
introduction of a phenyl moiety via a Suzuki coupling yielded the
title compound 47 in moderate yield. Noteworthy, unlike in
Scheme 5, the Suzuki coupling of pyrazolo[4,3-b]pyridine 46 did
not require Boc protection of its pyrrole-like nitrogen for the
reaction to be successful.
Scheme 4. Synthesis of pyrazolo[1,5-a]pyrimidine. Reagents and conditions:
a) 2-chloromalonaldehyde, MeOH, CH3COOH, 70°C (57%); b) phenyl-B(OH)2,
K2CO3, Pd(PPh3)4, H2O, dioxane (56%); c) 3.4-dimethoxyphenyl-B(OH)2, K2CO3,
Pd(PPh3)4, H2O, dioxane (27%).
3
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Scheme 6. Synthesis of pyrazolo[4,3-b]pyridine. Reagents and conditions: a)
3,4-dimethoxyphenyl- B(OH)2, K2CO3, Pd(PPh3)4, H2O, dioxane (86%); b)
NH2NH2.H2O, n-BuOH, 121°C (83%); c) p-TsOH, KI, NaNO2, CH3CN, rt (28%);
d) phenyl-B(OH)2, K2CO3, Pd(PPh3)4, H2O, dioxane, MW, 120°C (23%).
Synthesis of thieno[3,2-b]pyridine
Scheme 8. Synthesis of thieno[3,2-b]pyridine. Reagents and conditions. a) ethyl
2-mercaptoacetate, KOtBu, DMF (54%); b) 3,4-dimethoxyphenyl-B(OH)2 (76%)
or phenyl-B(OH)2 (42%), K3PO4, Pd(PPh3)4, dioxane, 110°C; c) t-BuONO, CuBr2,
CH3CN (47%); d) NaOH, EtOH, rt (89%); e) Cu, quinoline, 200°C (48%).
The synthesis started from 3-amino-thiophene 51, that was
formed by reaction of thiophene-3-carboxylic acid 48 with
diphenyl phosphoryl azide (DPPA) resulting in the acyl azide
intermediate 49 (Scheme 7). This was subsequently submitted to
a Curtius rearrangement by addition of dry t-BuOH yielding a Boc-
protected 3-amino-thiophene 50.[24] The use of dry t-BuOH was
essential to prevent the formation of thiophene-3-carbamoylic
acid. Deprotection of Boc using a 4N HCl solution in dioxane
yielded 3-amino-thiophene hydrochloride 51, which turned out to
be UV-sensitive and needed to be protected from light. Reaction
of 51 with 2-chloromalonaldehyde did not result in the formation
of the desired thieno[3,2-b]pyridine scaffold 52a, as mass spectral
analysis showed 2-chloro-N1,N3-di(thiophen-3-yl)propane-1,3-
diimine 52b to be the main product. To avoid this side reaction a
different synthetic route was chosen starting from the pyridine
analogue 53 (Scheme 8).
Synthesis of [1,2,3]triazolo[4,5-b]pyridine
Nucleophilic displacement of the chlorine at position 2 of 2,5-
dichloro-3-nitropyridine 60 by aniline furnished substitution
product 61 (Scheme 9).[31] Catalytic reduction of the nitro group
with Raney nickel under a hydrogen atmosphere yielded the 3-
amino-pyridine 62. Formation of the triazole ring was achieved by
a diazotation reaction using sodium nitrite in acetic acid with
concomitant cyclisation yielding the [1,2,3]triazolo[4,5-b]pyridine
scaffold 63. Finally, a Suzuki coupling afforded the target
compound 64.
Scheme 9. Synthesis of [1,2,3]triazolo[4,5-b]pyridine. Reagents and conditions:
a) aniline, DIPEA, dioxane, 80°C (85%°; b) Raney nickel, H2, THF, rt; c) NaNO2,
acetic acid, CH2Cl2, water (62%); d) 3,4-dimethoxyphenyl-B(OH)2, Cs2CO3,
Pd(PPh3)4, H2O, DMF (78%).
Scheme 7. Attempted synthesis of thieno[3,2-b]pyridine. Reagents and
conditions: a) DPPA, DIPEA, DMF, rt; b) t-BuOH, reflux (61%); c) 4N HCl in
dioxane, reflux (100%); d) 2-chloromalonaldehyde, CH3COOH/MeOH (2:3),
70°C.
Synthesis of imidazo[4,5-b]pyridine
Initial attempts for the synthesis of the imidazo[4,5-b]pyridine
scaffold followed a similar procedure as in Scheme 9. Reaction of
compound 62 with triethyl orthoformate allowed to build up the
imidazole ring and afforded 6-chloro-3-phenyl-imidazo[4,5-
b]pyridine. Unfortunately, despite exploring different reaction
conditions, the subsequent Suzuki coupling never yielded the
desired compound. Therefore, an alternative synthetic route was
developed in which the 3,4-dimethoxyphenyl residue was
introduced early on in the synthetic sequence.[32] As the Suzuki
coupling on the brominated pyridine analogue was consistently
higher yielding, 5-bromo-2-chloro-3-nitropyridine 65 was selected
as starting material. Introduction of an anilino moiety at position 2
by nucleophilic aromatic substitution afforded compound 66
(Scheme 10). Suzuki coupling formed the addition product 67 in
good yield. For the reduction of the nitro group, we opted for zinc
in acetic acid as this proceeded faster and cleaner than catalytic
hydrogenation. The amino intermediate 68 was used as such
without further purification for the formation of the imidazole
moiety. Reaction of 68 with triethyl orthoformate afforded
imidazo[4,5-b]pyridine 69 in low yield.
Reaction of pyridine 53 with ethyl 2-mercaptoacetate and
potassium tert-butoxide (KOtBu) as a strong base afforded
thieno[3,2-b]pyridine 54.[29] Introduction of a 3,4-dimethoxyphenyl
residue at position 6 of the scaffold required harsher reaction
conditions compared to previous scaffolds. Higher temperature
(110 °C) and a longer reaction time (~3 days) allowed to isolate
the desired compound 55 in moderate yield. A classical
Sandmeyer reaction using conditions from Scheme 2, followed by
a Suzuki coupling, yielded product 57. Hydrolysis of the ester
using NaOH in EtOH yielded the carboxylic acid 58 as a pink
precipitate. Refluxing compound 58 in a concentrated HCl
solution was not able to achieve decarboxylation of the carboxylic
acid group. Instead, only harsh conditions of metallic copper in
quinoline at 200°C yielded the title compound 59, albeit in low
yield.[30]
4
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Scheme 10. Synthesis of imidazo[4,5-b]pyridine. Reagents and conditions: a)
aniline, DIPEA, NMP, 120°C (78%); b) 3,4-dimethoxyphenyl-B(OH)2, K2CO3,
Pd(PPh3)4, H2O, dioxane (78%); c) Zn, CH3COOH; d) CH(OEt)3, 130°C (35%).
Scheme 12. Synthesis of pyrido[3,2-d]pyrimidine. Reagents and conditions: a)
Fe, CH3COOH, rt (56%); b) 3,4-dimethoxyphenyl-B(OH)2, K2CO3, Pd(PPh3)4,
H2O, dioxane, 90°C (98%); c) formamide, 160°C, µW (74%); d) 6N HCl, reflux
(66%); e) POCl3, 1,2,4-triazole, DIPEA, CH3CN; f) morpholine, dioxane, reflux
(25%).
Synthesis of quinazoline
Condensation of methyl 2-amino-4-bromobenzoate 70 with
triethyl orthoformate as a one-carbon fragment and ammonium
acetate as nitrogen source allowed the isolation of 7-
bromoquinazolin-4(3H)-one 71 (Scheme 11).[33] The tautomeric
hydroxyl group of the lactam moiety was converted into a chlorine
by treatment with phosphorus oxychloride, yielding a 4-chloro-
quinazoline analogue 72.[34] A nucleophilic aromatic substitution
with morpholine yielded the 4-morpholino-quinazoline 73.
Coupling of 73 with 3,4-dimethoxyphenylboronic acid using the
aforementioned conditions for the Suzuki reaction furnished
quinazoline 74 in good yield.
Synthesis of pyrido[2,3-d]pyrimidine
Starting from pyridine analogue 10 (synthesized in Scheme 1),
the construction of the pyrimidine moiety and the introduction of
morpholine was performed in analogy to Scheme 12, yielding the
title compound 84 in moderate yield (Scheme 13).
Scheme 13. Synthesis of pyrido[2,3-d]pyrimidine. Reagents and conditions: a)
formamide, 160°C, µW (46%); b) HCl 6N, reflux (60%); c) POCl3, 1,2,4-triazole,
DIPEA, CH3CN; d) morpholine, dioxane, 60 °C (19%).
Scheme 11. Synthesis of quinazoline. Reagents and conditions a) NH4OAc,
CH(OEt)3, reflux (60%); b) POCl3, reflux; c) morpholine, rt (57%); d) 3,4-
dimethoxyphenyl-B(OH)2, K2CO3, Pd(PPh3)4, H2O, dioxane, 90°C (75%).
Synthesis of pteridine
Synthesis of pyrido[3,2-d]pyrimidine
Heating of compound 19 (available from Scheme 3) in formamide
(as in Schemes 12 and 13) did not allow to form the pyrimidine
moiety and only starting material was recovered. Alternatively,
attempts to build up the pyrimidine ring using concentrated formic
acid and concentrated sulphuric acid under microwave
irradiation,[22],[36] solely led to the hydrolysis of the cyano group
affording carboxamide 85 as a bright yellow precipitate (Scheme
14). The subsequent ring closure by treatment with formamide
afforded pteridin-4(3H)-one 86. Chlorination with phosphorus
oxychloride yielded the 4-chloro-pteridine 87, that was sufficiently
stable to be purified by silica gel flash chromatography. Finally,
introduction of morpholine yielded the target compound 88.
First, the nitro group of commercially available 3-nitro-5-
bromopyridine-2-carbonitrile 53 was reduced by treatment with
iron under acidic conditions, generating the desired aniline 75 as
the major product (Scheme 12). However, due to the acid
hydrolysis of the cyano group, a substantial amount (12%) of 3-
amino-5-bromopicolinamide was also formed. These two
compounds were easily separated by silica gel flash
chromatography. Introduction of a 3,4-dimethoxyphenyl moiety on
75 via a Suzuki coupling yielded compound 76. Construction of
the pyrimidine moiety using triethyl orthoformate, as performed
before for the synthesis of the quinazoline analogue, did not result
in the isolation of the desired product. In contrast, heating of
compound 76 in formamide (which functions both as a reagent
and solvent) under microwave conditions furnished pyrido[3,2-
d]pyrimidine 77 as a brown precipitate in excellent yield and the
product was used as such without further purification.[35] Acid
hydrolysis of the 4-amino group of 77 afforded pyrido[3,2-
d]pyrimidin-4(3H)-one 78. Phosphorus oxychloride mediated
chlorination of compound 78 turned out to be cumbersome as no
product was retrieved, even though starting material appeared to
be fully consumed. Instead of chloride, 1,2,4-triazole was
introduced as a leaving group successfully yielding intermediate
79. Subsequent substitution with morpholine yielded the title
compound 80 in moderate yield.
Scheme 14. Synthesis of pteridine. Reagents and conditions: a) HCOOH,
H2SO4, 120°C, µW (78%); b) formamide, 160°C, µW (71%); c) POCl3, toluene,
reflux, rt (55%); d) morpholine, dioxane, reflux (97%).
Biological evaluation and SAR
All compounds were tested for GAK binding affinity using the
KINOMEscan™ platform, that quantitatively measures the ability
of a compound to compete with an immobilized active-site-
5
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10.1002/cmdc.201800690
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directed ligand.[37] In an initial series of compounds, the focus was
on structural modification of the pyridine moiety of the
isothiazolo[4,3-b]pyridine scaffold. Shuffling the position of the
nitrogen yielded isothiazolo[3,4-b]pyridine 14, displaying a 75-
fold drop in GAK affinity, when compared to the parent
isothiazolo[4,3-b]pyridine scaffold. Insertion of an additional
nitrogen atom in the pyridine moiety of compound 14 yielded a
pyrazine and pyrimidine analogue. The isothiazolo[3,4-
d]pyrimidine 29 (Kd = 3.2 µM) is equally active as GAK ligand as
Table 2. SAR of the isothiazolo moiety of the isothiazolo[4,3-b]pyridine scaffold
GAK
Compound#
Name scaffold
Scaffold
affinity
isothiazolo[3,4-b]pyridine 14 (Kd
=
3.9 µM), whereas
Kd (µM)a
isothiazolo[3,4-b]pyrazine 23 (Kd = 0.36 µM) is 10-fold more
potent than isothiazolo[3,4-b]pyridine 14, but still shows a 10-fold
decreased GAK affinity when compared to the original
isothiazolo[4,3-b]pyridine skeleton. Overall, the SAR of the
pyridine moiety points towards an essential role of the nitrogen at
position 4 of the scaffold.
5
isothiazolo[4,3-b]pyridine
pyrazolo[1,5-a]pyrimidine
0.77
0.69
33
42
47
pyrrolo[3,2-b]pyridine
pyrazolo[4,3-b]pyridine
4
Table 1. SAR of the pyridine moiety of the isothiazolo[4,3-b]pyridine scaffold.
1.7
59
64
69
thieno[3,2-b]pyridine
[1,2,3]triazolo[4,5-b]pyridine
imidazo[4,5-b]pyridine
26
GAK
>30
0.14
Compound#
Name scaffold
Scaffold
affinity
Kd (µM)a
4
isothiazolo[4,3-b]pyridine
isothiazolo[3,4-b]pyridine
0.052
3.9
aKd = dissociation constant. Values represent the average of two independent
experiments.
14
Finally, the 6-5 bicyclic isothiazolo[4,3-b]pyridine core was
replaced by a number of 6-6 bicyclic structures (Table 3). Keeping
the pyridine moiety of isothiazolo[4,3-b]pyridine intact and
replacing the isothiazole moiety by pyrimidine afforded the
pyrido[3,2-d]pyrimidine 78, which is endowed with a Kd value of
7.3 µM, and hence displays a 140-fold drop in GAK affinity when
compared to the original isothiazolo[4,3-b]pyridine skeleton.
Switching the position of the nitrogen in the pyridine ring (yielding
pyrido[2,3-d]pyrimidine 84) or insertion of an additional nitrogen
(affording pteridine 88) afforded scaffolds that were completely
lacking GAK affinity (Kd of 20 µM or more). On the other hand,
quinazoline 74 shows only a 10-fold drop in GAK affinity (Kd =
0.62 µM).
23
29
isothiazolo[3,4-b]pyrazine
isothiazolo[3,4-d]pyrimidine
0.36
3.2
aKd = dissociation constant. Values represent the average of two independent
experiments.
To further evaluate the SAR with respect to GAK binding, the
pyridine moiety was kept intact and the isothiazolo moiety was
replaced by a number of five-membered heteroaromatic rings
(Table 2). As mentioned before in the introduction, introducing
morpholine turned out to be problematic and therefore, the 3-
phenyl-isothiazolo[4,3-b]pyridine 5 (Kd of 0.77 µM) was selected
as a reference compound. Neither the pyrrolo[3,2-b]pyridine 42,
pyrazolo[4,3-b]pyridine
47,
thieno[3,2-b]pyridine
59
or
[1,2,3]triazolo[4,5-b]pyridine 64 were able to reach the GAK
affinity value of the reference compound 5. In contrast,
pyrazolo[1,5-a]pyrimidine 33 and imidazo[4,5-b]pyridine 69 were
the only scaffolds with a similar GAK affinity as isothiazolo[4,3-
b]pyridine 5.
6
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2.45 GHz with continuous irradiation power from 0 to 300 W utilizing the
standard absorbance level (300 W maximum power). The reactions were
carried out in 10 mL glass tubes, sealed with an aluminum/Teflon crimp
top, which can be exposed to 250 °C and 20 bar internal pressure. The
temperature was measured with an IR sensor on the outer surface of the
Table 3. SAR of 6-6 bicyclic scaffolds
process vial. After the irradiation period, the reaction vessel was cooled
1
rapidly (60–120 s) to ambient temperature by gas jet cooling. H and 13
C
NMR spectra were recorded on a Bruker Avance 300 MHz instrument (1H
NMR, 300 MHz; 13C NMR, 75 MHz), 500 MHz instrument (1H NMR, 500
MHz; 13C NMR, 125 MHz) or a 600 MHz instrument (1H NMR, 600 MHz;
13C NMR, 150 MHz), using tetramethylsilane as internal standard for 1H
NMR spectra and DMSO-d6 (39.5 ppm) or CDCl3 (77.2 ppm) for 13C NMR
spectra. Abbreviations used are s = singlet, d = doublet, t = triplet, q =
quartet, m = multiplet, b = broad. Coupling constants are expressed in Hz.
High resolution mass spectra were acquired on a quadrupole orthogonal
acceleration time-of-flight mass spectrometer (Synapt G2 HDMS, Waters,
Milford, MA). Samples were infused at 3uL/min and spectra were obtained
in positive or negative ionization mode with a resolution of 15000 (FWHM)
using leucine enkephalin as lock mass. Precoated aluminum sheets (Fluka
silica gel/TLC-cards, 254 nm) were used for TLC. Column chromatography
was performed on silica gel 0.060 – 0.200 mm, 60 Å (Acros Organics).
Purity of final compounds was verified to be >95% by HPLC analysis.
HPLC conditions to asses purity were as follows: Shimadzu HPLC
equipped with a LC-20AT pump, DGU-20A5 degasser, and a SPD-20A
UV-VIS detector; Symmetry C18 column (5 μm, 4.6 mm × 150 mm);
gradient elution of H2O/CH3CN from 95/5 or 70/30 to 5/95 over 25 minutes;
GAK
Compound#
Name scaffold
isothiazolo[4,3-b]pyridine
quinazoline
Scaffold
affinity
Kd (µM)a
4
0.052
0.62
74
78
84
88
pyrido[3,2-d]pyrimidine
pyrido[2,3-d]pyrimidine
pteridine
7.3
20
25
flow rate
1 mL/min; wavelength, UV 254 nm. Preparative HPLC
aKd = dissociation constant. Values represent the average of two independent
experiments.
purifications were performed using a Phenomenex Gemini 110A column
(C18, 10 µM, 21.2 mm x 250 mm).
Chemistry
Conclusion
2-Chloro-6-(3,4-dimethoxyphenyl)nicotinonitrile (7)
To a solution of 2,6-dichloronicotinonitrile 6 (2.0 g, 11.56 mmol) in dioxane
(40 mL) and water (10 mL) were added 3,4-dimethoxyphenyl boronic acid
(2.5 g, 13.87 mmol), potassium carbonate (3.2 g, 23.12 mmol), and
Pd(PPh3)4 (134 mg, 0,12 mmol). The reaction mixture was stirred at 90 °C
for 3 hours. The volatiles were evaporated and the crude residue was
purified by silica gel flash column chromatography (using a mixture of
dichloromethane/methanol 99:1) yielding the title compound (2.7 g, 63%).
1H NMR (300 MHz, DMSO) δ 8.46 (d, J = 8.2 Hz, 1H), 8.21 (d, J = 8.3 Hz,
1H), 7.79 (dd, J = 8.5, 2.1 Hz, 1H), 7.69 (d, J = 2.0 Hz, 1H), 7.12 (d, J =
8.6 Hz, 1H), 3.87 (s, 3H), 3.85 (s, 3H) ppm; 13C NMR (75 MHz, DMSO) δ
159.71, 151.89, 150.95, 149.21, 144.37, 127.95, 121.18, 118.56, 115.65,
111.93, 110.33, 106.55, 55.79 ppm; HRMS m/z [M+H]+ calcd for
C14H11Cl1N2O2: 275.0582, found: 275.0589.
A number of 5,6- and 6,6- fused bicyclic heteroaromatic scaffolds
that bear essential structural features for GAK affinity and that are
based on the original isothiazolo[4,3-b]pyridine scaffolds 4 and 5
were synthesized. Four of the 13 novel scaffolds (isothiazolo[3,4-
b]pyrazine 23, pyrazolo[1,5-a]pyrimidine 33 imidazo[4,5-
b]pyridine 69 and quinazoline 74) displayed GAK affinity with Kd
values under 1 µM. Although these compounds are not potent
enough to serve as chemical tools to decipher GAK biology in a
cellular context, they can serve as starting point for medicinal
chemists to further improve the GAK binding affinity by
modification of the decoration pattern.
2-Azido-6-(3,4-dimethoxyphenyl)nicotinonitrile (8)
Moreover, this manuscript also describes the synthesis of new
scaffolds that are underrepresented in organic/medicinal
chemistry. Examples include the isothiazolo[3,4-b]pyridine (206
known compounds according to SciFinder), isothiazolo[3,4-
b]pyrazine (25 known derivatives according to SciFinder) and
isothiazolo[3,4-d]pyrimidine (80 known analogues in SciFinder).
To a solution of 2-chloro-6-(3,4-dimethoxyphenyl)nicotinonitrile 7 (2.0 g,
7.80 mmol) in DMF (25 mL) was added NaN3 (812 mg, 12.48 mmol). The
mixture was stirred at 70°C for 4 hours upon which a bright yellow
precipitate formed. The mixture was allowed to cool to room temperature,
the precipitate was filtered off and carefully washed with water to yield the
title compound (1.8 g, 83%).
1H NMR (300 MHz, DMSO) δ 8.78 – 8.55 (m, 1H), 7.96 – 7.65 (m, 3H),
7.47 – 7.19 (m, 1H), 3.91 (s, 3H), 3.88 (s, 3H) ppm; HRMS m/z [M+H]+
calcd for C14H11N5O: 282.0985, found: 282.0985.
2-Amino-6-(3,4-dimethoxyphenyl)nicotinonitrile (10)
To a solution of 2-azido-6-(3,4-dimethoxyphenyl)nicotinonitrile 8 (1.5 g,
5.33 mmol) in pyridine was added triphenyl phosphine (2.2 g, 8.53 mmol)
and the mixture was stirred at room temperature overnight. After
disappearance of the starting material the volatiles were evaporated and
the crude residue was dissolved in an 80% solution of acetic acid in water
and refluxed for 1 hour. The volatiles were evaporated in vacuo and the
crude residue was purified by silica gel flash column chromatography
(eluting with a gradient of dichloromethane/ethyl acetate in a ratio of 99:1
to 95:5) to yield the title compound (1.92 g, 95%).
Experimental section
For all reactions, analytical grade solvents were used. All moisture-
sensitive reactions were carried out in oven-dried glassware (125 °C). All
microwave irradiation experiments were carried out in a dedicated CEM-
Discover monomode microwave apparatus, operating at a frequency of
7
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1H NMR (300 MHz, DMSO) δ 7.87 (d, J = 8.1 Hz, 1H), 7.67 (d, J = 1.8 Hz,
1H), 7.62 (dd, J = 8.5, 1.8 Hz, 1H), 7.19 (d, J = 8.1 Hz, 1H), 7.01 (d, J =
8.5 Hz, 1H), 6.89 (bs, 2H), 3.84 (s, 3H), 3.81 (s, 3H) ppm; 13C NMR (75
MHz, DMSO) δ 170.11, 159.71, 158.91, 150.82, 148.89, 142.83, 130.25,
120.31, 117.67, 111.55, 110.41, 108.16, 87.08, 55.65 ppm; HRMS m/z
[M+H]+ calcd for C14H13N3O2:256.1080, found: 256.1085.
2-Amino-6-(3,4-dimethoxyphenyl)pyridine-3-carbothioamide (11)
To a solution of 2-amino-6-(3,4-dimethoxyphenyl)nicotinonitrile 10 (500
mg, 1.96 mmol) in ethanol (25 mL) was added P2S5 (1.74 g, 7.84 mmol)
and the mixture was stirred at reflux overnight. After disappearance of the
starting material, the volatiles were evaporated and the crude residue was
purified by silica gel flash column chromatography (eluting with a mixture
of dichloromethane/acetone in a ratio of 90:10) to yield the title compound
(400 mg, 71%).
gel flash column chromatography (eluting with
dichloromethane/ethyl acetate in a ratio of 99:1) yielding the title
compound (2.34 g, 74%).
a
mixture of
1H NMR (300 MHz, DMSO) δ 9.48 (s, 1H), 7.91 (dd, J = 8.5, 2.1 Hz, 1H),
7.75 (d, J = 2.1 Hz, 1H), 7.18 (d, J = 8.6 Hz, 1H), 3.88 (s, 3H), 3.88 (s, 3H)
ppm; HRMS m/z [M+H]+ calcd for C13H10Cl1N3O2: 276.0534, found:
276.0528.
3-Azido-5-(3,4-dimethoxyphenyl)pyrazine-2-carbonitrile (17)
To a solution of 3-chloro-5-(3,4-dimethoxyphenyl)pyrazine-2-carbonitrile
16 (2.34 g, 8.45 mmol) in DMSO (50 mL) was added NaN3 (604 mg, 9.30
mmol). The mixture was stirred at 70°C overnight. After disappearance of
the starting material the mixture was allowed to cool and water was added.
The formed precipitate was filtered off and washed with water to yield the
title compound (2.10 g, 83%).
1H NMR (300 MHz, DMSO) δ 9.70 (bs, 1H), 9.43 (bs, 1H), 7.75 – 7.57 (m,
3H), 7.22 – 7.12 (m, 3H), 7.04 (d, J = 8.4 Hz, 1H), 3.85 (s, 3H), 3.82 (s,
3H) ppm; 13C NMR (75 MHz, DMSO) δ 198.24, 157.11, 156.33, 150.28,
148.87, 136.13, 130.94, 119.72, 115.66, 111.70, 110.22, 107.65, 55.69;
HRMS m/z [M+H]+ calcd for C14H15N3O2S1: 290.0958, found: 290.0959.
6-(3,4-Dimethoxyphenyl)isothiazolo[3,4-b]pyridin-3-amine (12)
HRMS m/z [M+Na]+ calcd for C13H10N6O2Na: 305.0758, found: 305.0755.
3-Amino-5-(3,4-dimethoxyphenyl)pyrazine-2-carbonitrile (19)
To a solution of 3-azido-5-(3,4-dimethoxyphenyl)pyrazine-2-carbonitrile 17
(2.07 g, 7.00 mmol) in pyridine (30 mL) was added triphenyl phosphine
(2.99 g, 11.40 mmol) and the mixture was stirred at room temperature
overnight. After disappearance of the starting material, the volatiles were
evaporated and the crude residue was dissolved in an 80% solution of
acetic acid in water (20 mL) and refluxed for 1 hour. The volatiles were
evaporated and the crude residue was purified by silica gel flash column
chromatography (eluting with a gradient of dichloromethane/acetone in a
ratio of 95:5) to yield the title compound (1.72 g, 95%).
1H NMR (300 MHz, CDCl3) δ 8.47 (s, 1H), 7.63 – 7.56 (m, 2H), 7.01 – 6.93
(m, 1H), 5.27 (bs, 2H), 3.99 (s, 3H), 3.97 (s, 3H) ppm 13C NMR (126 MHz,
DMSO) δ 156.66, 153.39, 151.90, 149.48, 131.23, 127.86, 121.35, 117.03,
112.21, 110.74, 108.93, 56.11 ppm; HRMS m/z [M+H]+ calcd for
C13H12N4O2: 257.1033, found: 257.1032.
3-Amino-5-(3,4-dimethoxyphenyl)pyrazine-2-carbothioamide (20)
To a solution of 3-amino-5-(3,4-dimethoxyphenyl)pyrazine-2-carbonitrile
19 (700 mg, 2.73 mmol) in ethanol (20 mL) was added Lawesson reagent
(2.21 g, 5.46 mmol) and the mixture was refluxed overnight. After
disappearance of the starting material the volatiles were evaporated in
vacuo. The residue was dissolved in water and extracted three times with
dichloromethane. The combined organic layers were evaporated under
reduced pressure and the crude residue was purified by silica gel silica gel
flash column chromatography (using a mixture of dichloromethane/ethyl
acetate in a ratio of 99:1 as mobile phase) yielding the title compound (660
mg, 83%).
To
a
solution
of
2-amino-6-(3,4-dimethoxyphenyl)pyridine-3-
carbothioamide 11 (300 mg, 1.04 mmol) in methanol (5 mL) at 0°C was
added dropwise a 30% aqueous H2O2 solution (354 µL, 4.15 mmol) and
the mixture was stirred overnight at room temperature. After
disappearance of the starting material, the mixture was cooled to 0°C and
the precipitate was filtered off and washed with cold methanol to yield the
title compound (250 mg, 83%).
1H NMR (300 MHz, DMSO) δ 8.29 (d, J = 8.6 Hz, 1H), 8.13 (bs, 2H), 7.80
(s, 1H), 7.75 (d, J = 8.3 Hz, 1H), 7.50 (d, J = 8.8 Hz, 1H), 7.08 (d, J = 8.3
Hz, 1H), 3.88 (s, 3H), 3.84 (s, 3H) ppm; 13C NMR (75 MHz, DMSO) δ
173.76, 164.69, 159.85, 150.93, 149.03, 131.91, 131.03, 120.78, 111.65,
110.94, 110.50, 109.72, 55.73, 55.68 ppm; HRMS m/z [M+H]+ calcd for
C14H13N3O2S1: 288.0801, found: 288.0802.
3-Bromo-6-(3,4-dimethoxyphenyl)isothiazolo[3,4-b]pyridine (13)
A solution of 6-(3,4-dimethoxyphenyl)isothiazolo[3,4-b]pyridin-3-amine 12
(200 mg, 0.70 mmol) in HBr (5 mL) was stirred for 10 min at room
temperature, then CuBr was added at once (200 g, 1.40 mmol). The
resulting mixture was cooled to 0 °C and solution of sodium nitrite (146 g,
2.10 mmol) in H2O (5 mL) was added dropwise over a period of 15 min.
The reaction mixture was stirred for 4 h at 0 °C followed by overnight at
room temperature. After disappearance of the starting material, the
mixture was cooled to 0 °C and carefully neutralized using Na2CO3. The
neutralised mixture was washed with ethyl acetate 3 times, organic phases
were collected and concentrated in vacuo. The crude residue was purified
by silica gel flash column chromatography (eluting with a mixture of
hexane/acetone in a ratio of 80:20) yielding the title compound (60 mg,
25%).
1H NMR (300 MHz, CDCl3) δ 8.47 (s, 1H), 7.65 – 7.55 (m, 2H), 7.02 – 6.93
(m, 1H), 3.99 (s, 3H), 3.97 (s, 3H) ppm; 13C NMR (126 MHz, DMSO) δ
192.85, 155.01, 153.30, 151.59, 149.49, 128.39, 127.92, 125.29, 120.96,
112.19, 110.61, 56.10, 56.08 ppm.
6-(3,4-Dimethoxyphenyl)isothiazolo[3,4-b]pyrazin-3-amine (21)
To
a
solution
of
3-amino-5-(3,4-dimethoxyphenyl)pyrazine-2-
HRMS m/z [M+H]+ calcd for C14H11Br1N2O2S1: 350.9798, found: 350.9801
4-(6-(3,4-Dimethoxyphenyl)isothiazolo[3,4-b]pyridin-3-yl)morpholine
(14)
carbothioamide 20 (600 mg, 2.07 mmol) in methanol (5 mL) at 0°C was
added dropwise a 30% aqueous H2O2 solution (703 µL, 8.27 mmol). The
mixture was stirred overnight at room temperature. After disappearance of
the starting material the volatiles were evaporated under reduced pressure
and the crude residue was purified by silica gel flash column
chromatography (eluting with a gradient of dichloromethane/acetone in a
ratio of 9:1 to 8:2) to yield the title compound (400 mg, 67%).
1H NMR (300 MHz, DMSO) δ 8.95 (s, 1H), 8.24 (s, 2H), 7.89 (d, J = 8.4
Hz, 1H), 7.82 (s, 1H), 7.13 (d, J = 8.5 Hz, 1H), 3.90 (s, 3H), 3.86 (s, 3H)
ppm; HRMS m/z [M+H]+ calcd for C13H12N4O2S1: 289.0754, found:
289.0767
3-Bromo-6-(3,4-dimethoxyphenyl)isothiazolo[3,4-b]pyrazine (22)
To a solution of CuBr2 (170 mg, 0,76) and tert-butylnitrite (93 mg, 0.90
mmol) in dry acetonitrile (5 mL) under argon atmosphere at 0°C was added
6-(3,4-dimethoxyphenyl)isothiazolo[3,4-b]pyrazin-3-amine 21 (200 mg,
0.67 mmol) and the mixture was stirred at 0°C for 5 hours. After
disappearance of the starting material, volatiles were evaporated and the
crude residue was purified by silica gel flash column chromatography
(eluting with a mixture of dichloromethane/ethyl acetate in a ratio of 99:1)
yielding the title compound (77 mg, 32%).
To
a
solution of 3-bromo-6-(3,4-dimethoxyphenyl)isothiazolo[3,4-
b]pyridine 13 (50 mg, 0.14 mmol) in ethanol (5 mL) was added morpholine
(37 mg, 0.43 mmol). The reaction was stirred at reflux for 4 days. After
disappearance of the starting material volatiles were evaporated in vacuo
and the crude residue was purified by silica gel flash column
chromatography (eluting with a mixture of dichloromethane/methanol in a
ratio of 97:3) to yield the title compound (20 mg, 40%).
1H NMR (300 MHz, DMSO) δ 8.41 (d, J = 9.1 Hz, 1H), 7.84 (d, J = 1.9 Hz,
1H), 7.79 (dd, J = 8.4, 2.0 Hz, 1H), 7.58 (d, J = 9.2 Hz, 1H), 7.09 (d, J =
8.5 Hz, 1H), 3.98 – 3.81 (m, 10H), 3.54 (m, 4H) ppm; 13C NMR (75 MHz,
DMSO) δ 175.75, 166.13, 159.37, 151.12, 149.11, 132.68, 130.71, 120.92,
112.38, 111.68, 110.37, 109.96, 65.38, 55.76, 55.71, 50.66 ppm; HRMS
m/z [M+H]+ calcd for C18H19N3O3S1: 358.1220, found: 358.1219.
3-Chloro-5-(3,4-dimethoxyphenyl)pyrazine-2-carbonitrile (16)
To a solution 3,5-dichloropyrazine-2-carbonitrile 15 (2.0 g, 11.50 mmol) in
dioxane (50 mL) were added 3,4 dimethoxyphenylboronic acid (2.51 g,
13.80 mmol), tripotassium phosphate (4.88 g, 22.99 mmol), and Pd(PPh3)4
(132 mg, 0,12 mmol). The reaction mixture was stirred at 90 °C overnight.
The volatiles were evaporated and the crude residue was purified by silica
1H NMR (300 MHz, CDCl3) δ 9.27 (s, 1H), 7.97 (s, 1H), 7.79 (d, J = 8.4 Hz,
1H), 7.01 (d, J = 8.4 Hz, 1H), 4.02 (s, 3H), 3.98 (s, 3H) ppm.
8
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4-(6-(3,4-Dimethoxyphenyl)isothiazolo[3,4-b]pyrazin-3-yl)morpholine
(23)
evaporated and the crude residue was purified by silica gel flash column
chromatography (eluting with a mixture of dichloromethane/acetone in a
ratio of 95:5) yielding the title compound (60 mg, 49%).
To
a
solution of 3-bromo-6-(3,4-dimethoxyphenyl)isothiazolo[3,4-
b]pyrazine 22 (77 mg, 0.22 mmol) in ethanol (5 mL) was added morpholine
(57 mg, 0.66 mmol) and the reaction was stirred at reflux overnight. After
disappearance of the starting material, volatiles were evaporated in vacuo
and the crude residue was purified by silica gel flash column
chromatography (eluting with a mixture of dichloromethane/methanol in a
ratio of 98:2) to yield the title compound (48 mg, 64%).
1H NMR (300 MHz, CDCl3) δ 9.34 (s, 1H), 8.34 (d, J = 8.0 Hz, 1H), 8.22
(s, 1H), 7.00 (d, J = 8.3 Hz, 1H), 4.04 (s, 3H), 3.99 (s, 3H) ppm; HRMS m/z
[M+H]+ calcd for C13H10Br1N3O2S1: 351.9750, found: 351.9746.
4-(6-(3,4-Dimethoxyphenyl)isothiazolo[3,4-d]pyrimidin-3-
yl)morpholine (29)
To
a
solution of 3-bromo-6-(3,4-dimethoxyphenyl)isothiazolo[3,4-
1H NMR (300 MHz, CDCl3) δ 8.80 (s, 1H), 7.96 (d, J = 2.0 Hz, 1H), 7.73
(dd, J = 8.4, 2.0 Hz, 1H), 6.99 (d, J = 8.5 Hz, 1H), 4.03 (s, 3H), 3.98 (s,
3H), 3.97 – 3.96 (m, 8H) ppm; 13C NMR (75 MHz, CDCl3) δ 173.06, 160.31,
154.08, 152.07, 150.02, 136.81, 129.26, 125.65, 121.33, 111.10, 110.81,
66.40, 56.48, 56.34, 50.05 ppm; HRMS m/z [M+H]+ calcd for
C17H18N4O3S1: 359.1172, found: 359.1161.
d]pyrimidine 28 (50 mg, 0.14 mmol) in ethanol (5 mL) was added
morpholine (37 mg, 0.43 mmol). The reaction was stirred at reflux
overnight. After disappearance of the starting material, volatiles were
evaporated in vacuo and the crude residue was purified by silica gel flash
column
chromatography
(eluting
with
a
mixture
of
dichloromethane/methanol in a ratio of 95:5) to yield the title compound
(33 mg, 65%).
4-Amino-2-(3,4-dimethoxyphenyl)pyrimidine-5-carbonitrile (25)
A solution of 4-amino-2-chloropyrimidine-5-carbonitrile 24 (3.00 g, 19.50
mmol), tripotassium phosphate (8.28 g, 39 mmol) and 3,4-
dimethoxyphenylboronic acid (3.67 g, 29.25 mmol) in dry dioxane (30 mL)
was degassed and refilled with nitrogen gas two times. Pd(OAc)2 (219 mg,
0.98 mmol) and D-t-BPF (501 mg, 0.98 mmol) were added and the mixture
was degassed and refilled with nitrogen gas two times and stirred at reflux
overnight. After disappearance of the starting material, the mixture was
allowed to cool to room temperature and the solid was filtered off and
thoroughly washed with ethyl acetate. The filtrate was concentrated in
vacuo and the crude residue was purified by silica gel flash column
chromatography (eluting with a mixture of dichloromethane/ethyl acetate
in a ratio of 95:5) to yield the title compound (2.76 g, 53%).
1H NMR (300 MHz, CDCl3) δ 9.33 (s, 1H), 8.27 – 8.21 (m, 1H), 8.20 – 8.15
(m, 1H), 7.02 – 6.91 (m, 1H), 4.04 – 3.92 (m, 10H), 3.69 (d, J = 5.2 Hz,
4H); 13C NMR (126 MHz, CDCl3) δ 164.64, 158.16, 153.06, 153.01, 149.38,
125.62, 123.05, 111.19, 111.07, 65.31, 56.20, 56.09, 50.89 ppm; HRMS
m/z [M+H]+ calcd for C17H18N4O3S1: 359.1172, found: 359.1168.
3-Bromo-6-chloropyrazolo[1,5-a]pyrimidine (31)
To a solution of 4-bromo-1H-pyrazol-3-amine 30 (1.0 g, 6.17 mmol) in
methanol (18 mL) and acetic acid (12 mL) was added 2-
chloromalonaldehyde (690 mg, 6.48 mmol), and the mixture was stirred at
70°C for 2 hours. After disappearance of the starting material, the mixture
was cooled to 0°C and the formed precipitate was filtered off and washed
with methanol to yield the title compound (820 mg, 57%).
1H NMR (300 MHz, DMSO) δ 9.62 (d, J = 2.2 Hz, 1H), 8.69 (d, J = 2.2 Hz,
1H), 8.42 (s, 1H) ppm; 13C NMR (75 MHz, DMSO) δ 150.27, 145.38,
143.30, 134.90, 117.44, 84.18 ppm; HRMS m/z [M+H]+ calcd for
C6H3Br1Cl1N3: 231.9272, found: 231.9273.
1H NMR (300 MHz, DMSO) δ 8.68 (s, 1H), 7.97 (d, J = 8.4 Hz, 1H), 7.91 –
7.80 (m, 3H), 7.09 (d, J = 8.5 Hz, 1H), 3.84 (s, 3H), 3.83 (s, 3H) ppm; 13
C
NMR (126 MHz, DMSO) δ 164.65, 162.55, 161.47, 151.98, 148.55, 128.98,
122.15, 116.06, 111.33, 111.10, 86.80, 55.68, 55.49 ppm; HRMS m/z
[M+H]+ calcd for C13H12N4O2: 257.1033, found: 257.1038.
6-Chloro-3-phenylpyrazolo[1,5-a]pyrimidine (32)
4-Amino-2-(3,4-dimethoxyphenyl)pyrimidine-5-carbothioamide (26)
To a solution 4-amino-2-(3,4-dimethoxyphenyl)pyrimidine-5-carbonitrile
25 (2.00 g, 7.80 mmol) in ethanol (30 mL) was added Lawesson’s reagent
(6.31 g, 15.60 mmol). The mixture was refluxed overnight. After
disappearance of the starting material volatiles were evaporated in vacuo
and the residue was dissolved in water and extracted three times with
dichloromethane. The combined organic layers were evaporated under
reduced pressure and the crude residue was purified by silica gel flash
column chromatography (using a gradient of dichloromethane/acetone in
a ratio of 85:15 to 75:15 as mobile phase) yielding the title compound (1.36
g, 60%).
To a solution of 3-bromo-6-chloropyrazolo[1,5-a]pyrimidine 31 (400 mg,
1.72 mmol) in dioxane (15 mL) and water (5 mL) were added phenyl
boronic acid (254 g, 2.08 mmol), potassium carbonate (476 mg, 3.44
mmol), and Pd(PPh3)4 (20 mg, 0,02 mmol). The reaction mixture was
degassed and refilled with nitrogen gas and stirred at 90 °C overnight. After
disappearance of the starting material, volatiles were evaporated and the
crude residue was purified by silica gel flash column chromatography
(using a mixture of heptane/acetone in a ratio of 90:10) to yield the title
compound (220 mg, 56%).
1H NMR (300 MHz, DMSO) δ 9.60 (d, J = 2.3 Hz, 1H), 8.80 (s, 1H), 8.72
(d, J = 2.3 Hz, 1H), 8.14 – 8.07 (m, 2H), 7.50 – 7.41 (m, 2H), 7.33 – 7.22
(m, 1H) ppm; HRMS m/z [M+H]+ calcd for C12H8Cl1N3: 230.0479, found:
230.0471.
1H NMR (300 MHz, DMSO) δ 9.83 (s, 1H), 9.58 (s, 1H), 8.44 (s, 1H), 8.00
– 7.89 (m, 4H), 7.07 (d, J = 8.5 Hz, 1H), 3.83 (s, 6H) ppm; 13C NMR (126
MHz, DMSO) δ 195.84, 163.00, 161.06, 153.48, 151.46, 148.56, 129.69,
121.53, 112.96, 111.36, 110.90, 55.68, 55.53 ppm.
6-(3,4-Dimethoxyphenyl)-3-phenylpyrazolo[1,5-a]pyrimidine (33)
To a solution of 6-chloro-3-phenylpyrazolo[1,5-a]pyrimidine 32 (100 mg,
0.44 mmol) in dioxane (4 mL) and water (1 mL) were added 3,4-
dimethoxyphenyl boronic acid (96 mg, 0.53 mmol), potassium carbonate
(121 mg, 0.88 mmol), and Pd(PPh3)4 (4 mg, 0,003 mmol). The reaction
mixture was degassed and refilled with nitrogen gas and stirred at 90 °C
overnight. After disappearance of the starting material, volatiles were
evaporated in vacuo and the crude residue was purified by silica gel flash
column chromatography (using a mixture of dichloromethane/acetone in a
ratio of 90:10) yielding the title compound (40 mg, 27%).
6-(3,4-Dimethoxyphenyl)isothiazolo[3,4-d]pyrimidin-3-amine (27)
To
a
solution of 4-amino-2-(3,4-dimethoxyphenyl)pyrimidine-5-
carbothioamide 26 (500 mg, 1.72 mmol) in methanol (5 mL) at 0°C was
added dropwise a 30% aqueous H2O2 solution (585 µL, 6.89 mmol). The
mixture was stirred overnight at room temperature. After disappearance of
the starting material, the volatiles were evaporated in vacuo and the crude
residue was purified by silica gel flash column chromatography (eluting
with
a gradient of dichloromethane/acetone 8:2) yielding the title
compound (418 mg, 84%).
1H NMR (300 MHz, DMSO) δ 9.38 (s, 1H), 8.87 (s, 2H), 8.09 (d, J = 8.4
Hz, 1H), 8.00 (s, 1H), 7.09 (d, J = 8.6 Hz, 1H), 3.87 (s, 3H), 3.84 (s, 3H)
ppm; 13C NMR (126 MHz, DMSO) δ 178.21, 165.37, 163.19, 156.33,
151.90, 149.04, 130.51, 122.50, 111.75, 111.50, 108.97, 56.05, 55.91
ppm; HRMS m/z [M+H]+ calcd for C13H12N4O2S1: 289.0754, found:
289.0747.
3-Bromo-6-(3,4-dimethoxyphenyl)isothiazolo[3,4-d]pyrimidine (28)
To a stirred solution of CuBr2 (87 mg, 0.39) and tert-butylnitrite (46 mg,
0.45 mmol) in dry acetonitrile (10 mL) under argon atmosphere at 0°C was
added 6-(3,4-dimethoxyphenyl)isothiazolo[3,4-d]pyrimidin-3-amine 27
(100 mg, 0.35 mmol) and the mixture was stirred at room temperature
overnight. After disappearance of the starting material, volatiles were
1H NMR (300 MHz, DMSO) δ 9.51 (d, J = 2.3 Hz, 1H), 9.07 (d, J = 2.3 Hz,
1H), 8.78 (s, 1H), 8.22 – 8.15 (m, 2H), 7.50 – 7.40 (m, 4H), 7.30 – 7.23 (m,
1H), 7.10 (d, J = 8.4 Hz, 1H), 3.90 (s, 3H), 3.81 (d, J = 7.7 Hz, 3H) ppm;
13C NMR (75 MHz, DMSO) δ 150.23, 149.84, 149.66, 143.45, 143.37,
132.68, 132.36, 129.14, 126.46, 126.32, 126.08, 122.11, 119.63, 112.80,
110.98, 109.34, 56.23, 56.11 ppm; HRMS m/z [M+H]+ calcd for
C20H17N3O2: 332.1393, found: 332.1403
(E)-2-(5-Bromo-3-nitropyridin-2-yl)-N,N-dimethylethen-1-amine (35)
To a solution of 5-bromo-2-methyl-3-nitropyridine 34 (1.00 g, 4.61 mmol)
in dry DMF (10 mL) was added DMF-DMA (1.01 g, 9.22 mmol) dropwise.
The mixture stirred at 90°C for 4 hours, upon which a dark red colour
formed. After disappearance of the starting material, volatiles were
9
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10.1002/cmdc.201800690
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evaporated in vacuo yielding the title compound (1.23 g, 98%). The crude
residue was used without further purification in the following reaction.
HRMS m/z [M+H]+ calcd for C9H10Br1N3O2: 272.0030, found: 272.0032
6-Bromo-1H-pyrrolo[3,2-b]pyridine (36)
ppm; HRMS m/z [M+H]+ calcd for C14H9Br1N2O2: 316.9921, found:
316.9917.
tert-Butyl
6-(3,4-dimethoxyphenyl)-3-phenyl-1H-pyrrolo[3,2-
b]pyridine-1-carboxylate (41)
A suspension of iron powder (1.13 g, 20.26 mmol) in glacial acetic acid
(20mL) was stirred at 0°C for 10 minutes. Subsequently, a solution of (E)-
2-(5-bromo-3-nitropyridin-2-yl)-N,N-dimethylethen-1-amine 35 (1.00 g,
3.68 mmol) in glacial acetic acid (10 mL) was added dropwise and the
mixture was stirred at room temperature for 3 hours. After disappearance
of the starting material, ethyl acetate was added to the mixture and it was
filtrated using a paper filter. The filtered cake was washed thoroughly with
ethyl acetate and the filtrate was evaporated in vacuo. The crude residue
was purified by silica gel flash column chromatography (eluting with a
mixture of dichloromethane/ethyl acetate in a ratio of 90:10) to yield the
title compound (660 mg, 91%).
To a solution of tert-butyl 6-bromo-3-phenyl-1H-pyrrolo[3,2-b]pyridine-1-
carboxylate 40 (65 mg, 0.17 mmol) in dioxane (4 mL) and water (1 mL)
were added 3,4-dimethoxyphenylboronic acid (38 g, 0.21 mmol),
potassium carbonate (47 mg, 0.34 mmol), and Pd(PPh3)4 (2 mg, 0.002
mmol). The reaction mixture was degassed and refilled with nitrogen gas
and stirred at 90 °C overnight. The volatiles were evaporated in vacuo and
the crude residue was purified by silica gel flash column chromatography
(using a mixture of dichloromethane/ethyl acetate in a ratio of 90:10) to
yield the title compound (11 mg, 15%).
1H NMR (300 MHz, DMSO) δ 8.93 (d, J = 1.9 Hz, 1H), 8.54 (d, J = 1.7 Hz,
1H), 8.40 (s, 1H), 8.36 – 8.29 (m, 2H), 7.51 – 7.44 (m, 2H), 7.38 – 7.28 (m,
3H), 7.11 (d, J = 8.3 Hz, 1H), 3.88 (s, 3H), 3.83 (s, 3H), 1.70 (s, 9H) ppm;
13C NMR (75 MHz, DMSO) δ 149.50, 149.07, 148.77, 144.93, 144.22,
132.16, 131.74, 130.50, 129.15, 128.59, 127.31, 127.14, 126.00, 119.68,
119.62, 119.42, 112.61, 110.74, 85.10, 55.76, 55.73, 27.72 ppm.
6-(3,4-Dimethoxyphenyl)-3-phenyl-1H-pyrrolo[3,2-b]pyridine (42)
1H NMR (300 MHz, DMSO) δ 11.46 (bs, 1H), 8.37 (d, J = 2.1 Hz, 1H), 8.03
– 8.00 (m, 1H), 7.69 – 7.66 (m, 1H), 6.60 – 6.57 (m, 1H) ppm; HRMS m/z
[M+H]+ calcd for C7H5Br1N2: 196.9709, found: 196.9709.
6-Bromo-3-iodo-1H-pyrrolo[3,2-b]pyridine (37)
To a solution of 6-bromo-1H-pyrrolo[3,2-b]pyridine 36 in dry THF (15 mL)
was added N-iodosuccinimide at once and the mixture was stirred at room
temperature for 3 hours. After disappearance of the starting material,
volatiles were evaporated and the crude residue was purified by silica gel
To
a
solution of tert-butyl 6-(3,4-dimethoxyphenyl)-3-phenyl-1H-
pyrrolo[3,2-b]pyridine-1-carboxylate 41 (11 mg, 0.026 mmol) in methanol
(2 mL) was added a 4N solution of HCl (2 mL) in dioxane and the mixture
was stirred at 70°C for 4 hours. After disappearance of the starting material,
volatiles were evaporated in vacuo and the crude residue was dissolved in
water and washed three times with dichloromethane. Organic fractions
were collected and concentrated in vacuo to yield the title compound (4
mg, 47%).
1H NMR (300 MHz, DMSO) δ 12.97 (s, 1H), 8.84 – 8.78 (m, 1H), 8.70 (s,
1H), 8.53 – 8.44 (m, 1H), 7.92 (d, J = 7.4 Hz, 2H), 7.57 – 7.34 (m, 5H),
7.14 (d, J = 8.4 Hz, 1H), 3.91 (s, 3H), 3.84 (s, 3H) ppm; 13C NMR (75 MHz,
DMSO) δ 149.54, 149.47, 135.55, 133.46, 132.76, 131.62, 129.91, 128.97,
128.61, 127.52, 127.09, 123.88, 119.92, 112.62, 112.42, 111.26, 55.98,
55.84 ppm; HRMS m/z [M+H]+ calcd for C21H18N2O2: 331.1441, found:
331.1442.
flash column chromatography (eluting with
a
gradient of
dichloromethane/ethyl acetate in a ratio of 99.5:0.5 to 99:1) yielding the
title compound (400 mg, 37%).
1H NMR (300 MHz, DMSO) δ 8.43 (d, J = 2.0 Hz, 1H), 8.07 (d, J = 2.0 Hz,
1H), 7.85 (s, 1H) ppm; 13C NMR (75 MHz, DMSO) δ 143.72, 134.62,
129.17, 121.50, 113.07 ppm; HRMS m/z [M+H]+ calcd for C7H4Br1I1N2:
322.8678, found: 322.8670
tert-Butyl
6-bromo-3-iodo-1H-pyrrolo[3,2-b]pyridine-1-carboxylate
(38)
A mixture of 6-bromo-3-iodo-1H-pyrrolo[3,2-b]pyridine 37 (200 mg, 0.60
mmol), DMAP (8 mg, 0.06 mmol), TEA (80 mg, 0.8 mmol) and di-tert-butyl
dicarbonate (158 mg, 0.72 mmol) was stirred in dry THF (6 mL) for 5 hours
at room temperature. After disappearance of the starting material, volatiles
were evaporated in vacuo and the crude residue was purified using silica
5-(3,4-Dimethoxyphenyl)-3-fluoropicolinonitrile (44)
To a solution 5-bromo-3-fluoropicolinonitrile 43 (5.00 g, 24.86 mmol) in
dioxane (40 mL) and water (10 mL) were added 3,4-
dimethoxyphenylboronic acid (5.432 g, 29.85 mmol), potassium carbonate
(6.87 g, 49.72 mmol), and Pd(PPh3)4 (287 mg, 0.248 mmol). The reaction
mixture was degassed and refilled with nitrogen gas and stirred at 90 °C
overnight. After disappearance of the starting material, volatiles were
evaporated in vacuo and the crude residue was purified by silica gel flash
column chromatography (using a mixture of dichloromethane/ethyl acetate
in a ratio of 99.5:0.5) to yield the title compound (5.54 g, 86%).
1H NMR (300 MHz, DMSO) δ 9.05 (s, 1H), 8.58 – 8.42 (m, 1H), 7.54 – 7.47
(m, 2H), 7.13 (d, J = 8.4 Hz, 1H), 3.88 (s, 3H), 3.84 (s, 3H) ppm; 13C NMR
(126 MHz, DMSO) δ 162.61, 160.50, 150.70, 149.43, 145.35, 141.96,
126.22, 121.78, 121.64, 120.65, 118.51, 113.97, 112.24, 110.94, 55.88,
55.75 ppm. HRMS m/z [M+H]+ calcd for C14H11F1N2O2: 259.0877,
found:259.0881.
6-(3,4-Dimethoxyphenyl)-1H-pyrazolo[4,3-b]pyridin-3-amine (45)
To a solution of 5-(3,4-dimethoxyphenyl)-3-fluoropicolinonitrile 44 (3.00 g,
11.61 mmol) in n-butanol (30 mL) was added hydrazine hydrate (2.91 g,
58.08 mmol) and the mixture was stirred at 121°C overnight. After
disappearance of the starting material, volatiles were evaporated in vacuo
and the crude residue was purified by silica gel flash column
chromatography (eluting with a mixture of dichloromethane/methanol in a
ratio of 95:5) to yield the title compound (2.6 g, 83%).
gel flash column chromatography (eluting with
a
mixture of
heptane/acetone in a ratio of 9:1) to yield the title compound (240 mg,
95%).
1H NMR (300 MHz, DMSO) δ 8.66 (d, J = 2.0 Hz, 1H), 8.45 (d, J = 2.0 Hz,
1H), 8.20 (s, 1H), 1.63 (s, 9H) ppm; HRMS m/z [M+H]+ calcd for
C8H4Br1I1N2O2: 366.8576, found: 366.8572.
6-Bromo-3-phenyl-1H-pyrrolo[3,2-b]pyridine (39)
To a solution of tert-butyl 6-bromo-3-iodo-1H-pyrrolo[3,2-b]pyridine-1-
carboxylate 38 (200 mg, 0.57 mmol) in dioxane (4 mL) and water (1 mL)
were added phenylboronic acid (83 g, 0.68 mmol), potassium carbonate
(158 mg, 1.14 mmol), and Pd(PPh3)4 (7 mg, 0,006 mmol). The reaction
mixture was degassed and refilled with nitrogen gas and stirred at 90 °C
overnight. After disappearance of the starting material, volatiles were
evaporated in vacuo and the crude residue was purified by silica gel flash
column chromatography (using a mixture of heptane/acetone in a ratio of
70:30) to yield the title compound (109 mg, 70%).
1H NMR (300 MHz, DMSO) δ 11.72 (bs, 1H), 8.51 (d, J = 2.1 Hz, 1H), 8.25
– 8.18 (m, 3H), 8.08 (d, J = 2.1 Hz, 1H), 7.43 – 7.37 (m, 2H), 7.25 – 7.18
(m, 1H) ppm; HRMS m/z [M+H]+ calcd for C13H9Br1N2:273.0022, found:
273.0019
tert-Butyl 6-bromo-3-phenyl-1H-pyrrolo[3,2-b]pyridine-1-carboxylate
(40)
A mixture of 6-bromo-3-iodo-1H-pyrrolo[3,2-b]pyridine 39 (70 mg, 0.26
mmol), DMAP (3 mg, 0.03 mmol), TEA (35 mg, 0.35 mmol) and di-tert-
butyl dicarbonate (68 mg, 0.31 mmol) were stirred in dry THF (5 mL) for 5
hours. After disappearance of the starting material, volatiles were
evaporated and the crude residue was purified by silica gel flash column
chromatography (eluting with a mixture of heptane/acetone in a ratio of
9:1) to yield the title compound (65 mg, 67%).
1H NMR (300 MHz, DMSO) δ 11.66 (bs, 1H), 8.59 (s, 1H), 7.84 (s, 1H),
7.38 – 7.24 (m, 2H), 7.08 (d, J = 8.4 Hz, 1H), 5.39 (bs, 2H), 3.88 (s, 3H),
3.82 (s, 3H) ppm.
6-(3,4-Dimethoxyphenyl)-3-iodo-1H-pyrazolo[4,3-b]pyridine (46)
A
solution of 6-(3,4-dimethoxyphenyl)-1H-pyrazolo[4,3-b]pyridin-3-
fluoropicolinonitrile 45 (1.00 g, 3.70 mmol) and p-toluenesulfonic acid (2.11
g, 11.1 mmol) in acetonitrile was stirred for 10 minutes. Subsequently a
solution of sodium nitrite (640 mg, 9.30 mmol) and potassium Iodide (1.54
g, 9.30 mmol) in water (10 mL) was added dropwise over 30 minutes and
1H NMR (300 MHz, DMSO) δ 8.72 (s, 1H), 8.54 (s, 1H), 8.43 (s, 1H), 8.25
(d, J = 7.5 Hz, 2H), 7.52 – 7.43 (m, 2H), 7.39 – 7.30 (m, 1H), 1.68 (s, 9H)
the mixture was stirred at room temperature for
5 hours. After
10
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10.1002/cmdc.201800690
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disappearance of the starting material, volatiles were evaporated in vacuo
and the crude residue was purified by silica gel flash column
chromatography (eluting with a mixture of dichloromethane/methanol in a
ratio of 95:5) to yield the title compound (400 mg, 28%).
1H NMR (300 MHz, DMSO) δ 9.19 – 9.11 (m, 1H), 8.89 – 8.85 (m, 1H),
7.68 – 7.61 (m, 2H), 7.56 (s, 1H), 7.22 – 7.14 (m, J = 7.7 Hz, 2H), 3.96 (s,
3H), 3.89 (s, 3H) ppm.
6-(3,4-Dimethoxyphenyl)-3-phenyl-1H-pyrazolo[4,3-b]pyridine (47)
To a solution 6-(3,4-dimethoxyphenyl)-3-iodo-1H-pyrazolo[4,3-b]pyridine
46 (100 mg, 0.26 mmol) in dioxane (4 mL) and water (1 mL) were added
3,4-dimethoxyphenylboronic acid (38 mg, 0.31 mmol), potassium
carbonate (72 mg, 0.52 mmol), and Pd(PPh3)4 (60 mg, 0.0.052 mmol). The
reaction mixture was stirred in a microwave at 120°C for 1 hour. After
disappearance of the starting material, volatiles were evaporated in vacuo
and the crude residue was purified by silica gel flash column
chromatography (using a mixture of dichloromethane/methanol in a ratio
of 98:2) to yield the title compound (20 mg, 23%).
1H NMR (300 MHz, DMSO) δ 13.46 (s, 1H), 8.95 (s, 1H), 8.54 (d, J = 7.3
Hz, 2H), 8.20 (s, 1H), 7.60 – 7.33 (m, 5H), 7.12 (d, J = 8.4 Hz, 1H), 3.90
(s, 3H), 3.83 (s, 3H) ppm; 13C NMR (75 MHz, DMSO) δ 149.53, 149.33,
145.10, 141.99, 137.58, 134.53, 133.30, 132.87, 130.41, 128.70, 128.05,
126.47, 120.00, 115.41, 112.69, 111.52, 55.93, 55.87 ppm; HRMS m/z
[M+H]+ calcd for C20H17N3O2: 332.1393, found: 332.1396.
material, volatiles were evaporated in vacuo and the crude residue was
purified by silica gel flash column chromatography (eluting with a mixture
of heptane/ethyl acetate in a ratio of 80:20) to yield the title compound
(3.04 g, 76%).
1H NMR (300 MHz, DMSO) δ 9.00 (d, J = 2.0 Hz, 1H), 8.66 (d, J = 2.0
Hz, 1H), 7.45 – 7.36 (m, 2H), 7.10 (d, J = 8.1 Hz, 1H), 6.89 (bs, 2H), 4.31
(q, J = 7.1 Hz, 2H), 3.89 (s, 3H), 3.82 (s, 3H), 1.32 (t, J = 7.1 Hz, 3H)
ppm 13C NMR (126 MHz, DMSO) δ 164.11, 147.57, 145.02, 134.92,
134.15, 133.21, 119.17, 118.62, 112.24, 110.49, 60.42, 55.69, 14.48
ppm; HRMS m/z [M+H]+ calcd for C18H18N2O4S1: 359.1060, found:
359.1054.
Ethyl
3-bromo-6-(3,4-dimethoxyphenyl)thieno[3,2-b]pyridine-2-
carboxylate (56)
To a stirred solution of CuBr2 (1.72 g, 7.67) and tert-butyl nitrite (789 mg,
7.66 mmol) in dry acetonitrile (25 mL) under argon atmosphere at 0°C was
added ethyl 3-amino-6-(3,4-dimethoxyphenyl)thieno[3,2-b]pyridine-2-
carboxylate 55 (2.5 g, 6.98 mmol) and the mixture was stirred at 0°C for 6
hours and then at room temperature overnight. After disappearance of the
starting material, volatiles were evaporated in vacuo and the crude residue
was purified by silica gel flash column chromatography (eluting with a
mixture of heptane/ethyl acetate in a ratio of 70:30) yielding the title
compound (1.38 g, 47%).
1H NMR (300 MHz, DMSO) δ 9.20 (s, 1H), 8.88 (s, 1H), 7.48 – 7.35 (m,
2H), 7.12 (d, J = 8.1 Hz, 1H), 4.41 (q, J = 7.0 Hz, 2H), 3.89 (s, 3H), 3.83
(s, 3H), 1.37 (t, J = 7.0 Hz, 3H) ppm; HRMS m/z [M+H]+ calcd for
C18H16Br1N1O4S1: 422.0056, found: 422.0052.
tert-Butyl thiophen-3-ylcarbamate (50)
To a solution of thiophene-3-carboxylic acid 48 (2.00 g, 15.61 mmol) in dry
dimethylformamide (25 mL) at 0 °C was added diphenylphosphoryl azide
(4.30 g, 15.61 mmol) and the mixture was stirred at room temperature for
2 hours. After disappearance of the starting material, tert-butanol was
added (15 mL, 156.10 mmol) and the mixture was stirred at reflux for 17
hours. After disappearance of the intermediate 49, the volatiles were
evaporated and the crude residue was purified by silica gel flash column
chromatography (using a mixture of heptane/acetone in a ratio of 9:1) to
yield the title compound (1.91 g, 61%).
1H NMR (300 MHz, DMSO) δ 9.62 (bs, 1H), 7.42 – 7.34 (m, 1H), 7.19 –
7.12 (m, 1H), 7.02 – 6.95 (m, 1H), 1.47 (s, 9H) ppm; 13C NMR (75 MHz,
DMSO) δ 152.89, 137.48, 124.71, 121.26, 105.90, 28.25 ppm; HRMS m/z
[M+H]+ calcd for C5H5N1O2S1: 144.0114, found: 144.0112.
Ethyl
6-(3,4-dimethoxyphenyl)-3-phenylthieno[3,2-b]pyridine-2-
carboxylate (57)
To a solution ethyl 3-bromo-6-(3,4-dimethoxyphenyl)thieno[3,2-b]pyridine-
2-carboxylate 56 (1.00 g, 2.40 mmol) in dioxane (25 mL) were added
phenylboronic acid (340 mg, 2.80 mmol), tripotassium phosphate (1.02 g,
4.80 mmol), and Pd(PPh3)4 (280 mg, 0.24 mmol). The reaction mixture
was stirred at reflux overnight. After disappearance of the starting material,
volatiles were evaporated in vacuo and the crude residue was purified by
silica gel flash column chromatography (eluting with a mixture of
heptane/acetone in a ratio of 80:20) yielding the title compound (422 mg,
42%).
3-Aminothiophene hydrochloride (51)
1H NMR (600 MHz, DMSO) δ 9.08 (d, J = 2.1 Hz, 1H), 8.87 (d, J = 2.1 Hz,
1H), 7.52 – 7.45 (m, 5H), 7.43 (d, J = 2.0 Hz, 1H), 7.40 (dd, J = 8.3, 2.1
Hz, 1H), 7.11 (d, J = 8.4 Hz, 1H), 4.21 (q, J = 7.1 Hz, 2H), 3.88 (s, 3H),
3.82 (s, 3H), 1.14 (t, J = 7.1 Hz, 3H) ppm; 13C NMR (151 MHz, DMSO) δ
161.90, 152.13, 149.46, 149.43, 147.58, 142.51, 134.65, 133.54, 133.33,
131.34, 130.60, 129.12, 128.12, 128.02, 127.45, 119.75, 112.43, 110.90,
61.49, 55.78, 55.70, 13.80 ppm; HRMS m/z [M+H]+ calcd for
C24H21N1O4S1: 420.1264, found: 420.1261.
tert-Butyl thiophen-3-ylcarbamate 50 (500 mg, 2.50 mmol) was dissolved
in a 4N solution of HCl in dioxane (10 mL) and the mixture was protected
from light using aluminium foil and stirred at reflux for 3 hours. After
disappearance of the starting material, volatiles were evaporated and the
crude residue (340 mg, 100%) was protected from light and used as such
without further purification in the following reaction.
1H NMR (300 MHz, DMSO) δ 10.46 (bs, 2H), 7.76 – 7.63 (m, 1H), 7.63 –
7.51 (m, 1H), 7.15 – 7.10 (m, 1H) ppm; 13C NMR (75 MHz, DMSO) δ
129.43, 128.02, 123.32, 118.50 ppm; HRMS m/z [M+H]+ calcd for
C4H5N1S1: 100.0215, found: 100.0227.
6-(3,4-Dimethoxyphenyl)-3-phenylthieno[3,2-b]pyridine-2-carboxylic
acid (58)
To
a solution of ethyl 6-(3,4-dimethoxyphenyl)-3-phenylthieno[3,2-
Ethyl 3-amino-6-bromothieno[3,2-b]pyridine-2-carboxylate (54)
To a solution of 5-bromo-3-nitropyridine-2-carbonitrile 53 (2.00 g, 21.93
mmol) and ethyl 2-mercaptoacetate (3.95 g, 32.89 mmol) in
dimethylformamide (50 mL) was added potassium tert-butoxide (4.92,
43.86 mmol) portion wise. The mixture turned red and was stirred at room
temperature for 3 hours. After disappearance of the starting material the
volatiles were evaporated in vacuo and the crude residue was purified by
silica gel flash column chromatography (eluting with a mixture of heptane
/ethyl acetate in a ratio of 80:20) to yield the title compound (4.24 g, 54%).
1H NMR (300 MHz, DMSO) δ 8.89 – 8.60 (m, 2H), 6.90 (bs, 2H), 4.30 (q,
J = 7.0 Hz, 2H), 1.31 (t, J = 6.8 Hz, 3H) ppm; 13C NMR (126 MHz, DMSO)
δ 164.47, 147.92, 147.88, 145.40, 135.30, 134.49, 119.52, 98.47, 60.78,
14.85 ppm; HRMS m/z [M+H]+ calcd for C10H9Br1N2O2S1: 300.9641, found:
300.9638.
b]pyridine-2-carboxylate 57 (200 mg, 0.48 mmol) in ethanol (10 mL) was
added a solution of NaOH (192 mg, 4.8 mmol) in water (2 mL) and the
mixture was stirred at room temperature for 4 hours. After disappearance
of the starting material, the mixture was neutralised using a 6 N HCl
solution in water, upon which a precipitate formed. The precipitate was
filtered off and washed with water to yield the title compound (168 mg,
89%).
HRMS m/z [M+H]+ calcd for C22H17N1O4S1: 392.0951, found: 392.0941
6-(3,4-Dimethoxyphenyl)-3-phenylthieno[3,2-b]pyridine (59)
To a solution of 6-(3,4-dimethoxyphenyl)-3-phenylthieno[3,2-b]pyridine-2-
carboxylic acid 58 (100 mg, 0.25 mmol) in quinoline (1 mL) was added
copper (11 mg, 0.18 mmol) and the reaction was stirred at 200°C overnight.
After disappearance of the starting material, the mixture was diluted with
water and washed three times with dichloromethane. Organic phases were
collected and concentrated in vacuo and the crude residue was purified by
silica gel flash column chromatography (eluting with a mixture of
heptane/acetone 80:20) to yield the title compound (42 mg, 48%).
1H NMR (300 MHz, DMSO) δ 9.10 (d, J = 2.1 Hz, 1H), 8.84 (d, J = 2.1 Hz,
1H), 8.37 (s, 1H), 8.19 – 8.08 (m, 2H), 7.54 – 7.36 (m, 5H), 7.10 (d, J = 8.4
Hz, 1H), 3.89 (s, 3H), 3.82 (s, 3H) ppm; 13C NMR (75 MHz, DMSO) δ
151.54, 149.47, 149.15, 146.00, 135.21, 134.51, 134.32, 131.19, 129.70,
Ethyl
3-amino-6-(3,4-dimethoxyphenyl)thieno[3,2-b]pyridine-2-
carboxylate (55)
To a solution ethyl 3-amino-6-bromothieno[3,2-b]pyridine-2-carboxylate
54 (3.50 g, 11.62 mmol) in dioxane (50 mL) were added 3,4-
dimethoxyphenylboronic acid (2.55 g, 13.95 mmol), potassium phosphate
(4.90 g, 23.24 mmol), and Pd(PPh3)4 (671 mg, 0,58 mmol). The reaction
mixture was stirred at reflux for 2 days. After disappearance of the starting
11
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201800690
ChemMedChem
FULL PAPER
128.57, 128.50, 128.23, 127.70, 119.49, 112.50, 110.87, 55.80, 55.74
ppm; HRMS m/z [M+H]+ calcd for C21H17N1O2S1: 348.1053, found:
348.1053.
1H NMR (300 MHz, CDCl3) δ 10.04 (s, 1H), 8.63 (s, 1H), 8.48 (d, J = 2.1
Hz, 1H), 7.59 (d, J = 8.0 Hz, 2H), 7.39 (t, J = 7.8 Hz, 2H), 7.27 – 7.13 (m,
1H) ppm; 13C NMR (126 MHz, DMSO) δ 155.29, 148.38, 138.04, 136.94,
129.23, 128.70, 124.59, 123.14, 106.20 ppm.
5-Chloro-3-nitro-N-phenylpyridin-2-amine (61)
To a mixture of 2,5-dichloro-3-nitropyridine 60 (1.93 g, 10 mmol) and
aniline (2.8 mL, 30 mmol) in 1,4-dioxane (100 mL) was added DIPEA (0.52
mL, 35 mmol) and the reaction was stirred at 80 oC overnight. After
disappearance of the starting material, the reaction was cooled to room
temperature and diluted with ethyl acetate, washed with water, dried over
Na2SO4, and concentrated in vacuo. The crude residue was purified by
silica gel flash column chromatography (eluting with a gradient of ethyl
acetate/Methanol in a ratio of 100:0 to 95:5) to yield the title compound
(2.12 g) in 85% yield.
5-(3,4-Dimethoxyphenyl)-3-nitro-N-phenylpyridin-2-amine (67)
To a solution of 5-bromo-3-nitro-N-phenylpyridin-2-amine 66 (600 mg, 2.0
mmol), in dioxane (16 mL) and water (4 mL) were added (3,4-
dimethoxyphenyl)boronic acid (408 mg, 2.2 mmol), sodium carbonate (912
mg, 8.6 mmol) and Pd(PPh3)4 (120 mg, 0.1 mmol). The reaction mixture
was degassed and refilled with nitrogen gas and stirred at 80 °C overnight.
After disappearance of the starting material, volatiles were evaporated in
vacuo and the crude residue was dissolved in ethyl acetate and washed
with water. Collected organic phases were dried over Na2SO4,
concentrated in vacuo and the crude residue was purified using silica gel
flash column chromatography (eluting with a mixture of Pentane/Ethyl
acetate in a ratio of 75:25) to yield the title compound (360 mg, 78%).
6-(3,4-Dimethoxyphenyl)-3-phenyl-3H-imidazo[4,5-b]pyridine (69)
To a solution of 5-(3,4-dimethoxyphenyl)-3-nitro-N-phenylpyridin-2-amine
68 (360 mg, 1 mmol) in glacial acetic acid (5 mL), was added zinc (2.2 g,
34 mmol) and the mixture was stirred at reflux for 3 hours. After
disappearance of the starting material, the reaction was allowed to cool to
room temperature and filtrated through celite, washed with acetic acid and
concentrated in vacuo. The crude residue was subsequently suspended in
triethyl orthoformate (10 mL) and stirred at 130oC for 2 h. After
disappearance of the starting material the volatiles were evaporated in
vacuo and the crude residue was dissolved in ethyl acetate, washed with
water, saturated sodium bicarbonate and brine. Collected organic phases
were dried over MgSO4 and concentrated in vacuo. The crude residue was
purified using silica gel flash column chromatography (eluting with a
mixture of ethyl acetate/pentane in a ratio of 6:4) to give the title compound
which was further purified by crystallisation from methanol (116 mg, 35%).
1H NMR (300 MHz, CDCl3) δ 8.68 (d, J = 2.0 Hz, 1H), 8.38 (s, 1H), 8.29
(d, J = 2.1 Hz, 1H), 7.84 – 7.77 (m, 2H), 7.65 – 7.56 (m, 2H), 7.51 – 7.43
(m, 1H), 7.20 (dd, J = 8.2, 2.1 Hz, 1H), 7.15 (d, J = 2.1 Hz, 1H), 7.01 (d, J
= 8.3 Hz, 1H), 3.98 (s, 3H), 3.95 (s, 3H); 13C NMR (75 MHz, CDCl3) δ
149.77, 149.27, 146.24, 144.29, 143.76, 136.36, 133.16, 131.70, 130.01,
128.07, 126.43, 123.64, 120.08, 112.17, 111.21, 56.24; HRMS: [M + H]+
calcd for C20H17N3O2, 392.1393; found, 392.1393.
1H NMR (300 MHz, CDCl3) δ 10.05 (s, 1H), 8.51 (d, J = 2.5 Hz, 1H), 8.42
(d, J = 2.5 Hz, 1H), 7.65 – 7.56 (m, 2H), 7.47 – 7.35 (m, 2H), 7.28 – 7.16
(m, 1H).
5-Chloro-N2-phenylpyridine-2,3-diamine (62)
A mixture of 5-chloro-3-nitro-N-phenylpyridin-2-amine 61 (0.5 g, 2 mmol)
and Raney nickel (0.1 g) in THF (20 mL) was degassed and filled with
hydrogen gas. The reaction was allowed to stir at room temperature for
two hours. After disappearance of the starting material the mixture was
filtered, concentrated in vacuo and used in the following reaction without
further purification.
6-Chloro-3-phenyl-3H-[1,2,3]triazolo[4,5-b]pyridine (63)
To a solution of 5-chloro-N2-phenylpyridine-2,3-diamine 62 in a mixture of
glacial acetic acid (0.56 mL, 10 mmol), water (0.5 mL) and
dichloromethane (0.5 mL) at 0°C was added sodium nitrite (0.18 g, 2.6
mmol) dropwise. After 20 minutes of stirring, the mixture was diluted with
dichloromethane, washed with water and dried over Na2SO4.. Organic
phases were collected and concentrated in vacuo. The crude residue was
purified by silica gel flash column chromatography (eluting with gradient of
pentane/ethyl acetate in a ratio of 100:0 to 60:40) to yield the title
compound (0.28 g, 62 %)
1H NMR (300 MHz, CDCl3) δ 8.66 (d, J = 2.2 Hz, 1H), 8.39 (d, J = 2.2 Hz,
1H), 8.26 – 8.19 (m, 2H), 7.63 – 7.54 (m, 2H), 7.50 – 7.43 (m, 1H). 13C
NMR (75 MHz, CDCl3) δ 149.95, 143.72, 138.23, 136.19, 129.61, 128.60,
128.16, 127.71, 121.36.
6-(3,4-Dimethoxyphenyl)-3-phenyl-3H-[1,2,3]triazolo[4,5-b]pyridine
(64)
7-Bromoquinazolin-4-ol (71)
To a solution of 6-chloro-3-phenyl-3H-[1,2,3]triazolo[4,5-b]pyridine 63 (120
mg, 0.52 mmol) in dimethylformamide (8 mL) and water (2 mL) were added
3,4-dimethoxyphenyl boronic acid (227 mg, 1.24 mmol), Cesium
carbonate (542 mg, 1.61 mmol) and Pd(PPh3)4 (60 mg, 0.052 mmol). The
reaction mixture was degassed and refilled with nitrogen gas and stirred
at 100 °C overnight. After disappearance of the starting material, volatiles
were evaporated and the crude residue was dissolved in ethyl acetate and
washed with water. Collected organic phases were dried over Na2SO4,
concentrated in vacuo and the crude residue was purified using silica gel
flash column chromatography (eluting with a gradient of pentane/ethyl
acetate in a ratio of 100:0 to 70:30) to yield the title compound (134 mg,
78%).
1H NMR (300 MHz, CDCl3) δ 8.96 (d, J = 2.1 Hz, 1H), 8.51 (d, J = 2.1 Hz,
1H), 8.36 – 8.28 (m, 2H), 7.66 – 7.56 (m, 2H), 7.52 – 7.43 (m, 1H), 7.20
(dd, J = 8.2, 2.1 Hz, 1H), 7.14 (d, J = 2.1 Hz, 1H), 7.01 (d, J = 8.3 Hz, 1H),
3.99 (s, 3H), 3.95 (s, 3H); 13C NMR (75 MHz, CDCl3) δ 150.50, 149.85,
149.72, 144.63, 138.34, 136.65, 134.26, 130.16, 129.62, 128.34, 125.75,
121.41, 120.25, 112.11, 111.00, 56.21, 56.17; HRMS: [M + H]+ calcd for
C19H16N4O2: 333.1346; found, 333.1345.
To a solution of methyl 2-amino-4-bromobenzoate 70 (1.00 g, 4.35 mmol)
in triethyl orthoformate (20 mL) was added ammonium acetate (419 mg,
5.43 mmol) and the mixture was stirred at reflux overnight. After
disappearance of the starting material the volatiles were evaporated in
vacuo and the crude residue was dissolved in water. The formed
precipitate was filtered off, yielding the title compound (600 mg, 60%).
1H NMR (300 MHz, DMSO) δ 12.38 (bs, 1H), 8.14 (s, 1H), 8.03 (d, J = 8.5
Hz, 1H), 7.89 (d, J = 1.8 Hz, 1H), 7.69 (dd, J = 8.5, 1.8 Hz, 1H).
7-Bromo-4-chloroquinazoline (72)
7-Bromoquinazolin-4-ol 71 (200 mg, 0,88 mmol) was suspended in
phosphorus oxychloride (3,0 mL) and stirred at 125°C overnight. After
disappearance of the starting material, the solvent was evaporated in
vacuo and the crude residue was used as such without further purification
in the following reaction.
4-(7-Bromoquinazolin-4-yl)morpholine (73)
To a solution of crude residue containing 7-bromo-4-chloroquinazoline 72
in dioxane was added morpholine (958 µL, 11 mmol) and the mixture was
stirred at room temperature for 3 hours. After disappearance of the starting
material volatiles were evaporated in vacuo and the crude residue was
purified by silica gel flash column chromatography (eluting with a mixture
of dichloromethane/methanol 95:5) yielding the title compound (148 mg,
57%).
5-Bromo-3-nitro-N-phenylpyridin-2-amine (66)
To a solution of 5-Bromo-2-chloro-3-nitropyridine 65 (2.37g, 10 mmol) in
NMP (5 mL) was added aniline (1.4 mL, 15 mmol) and DIPEA (5 mL, 30
mmol) and the mixture was stirred at 120°C for 1.5 h. After disappearance
of the starting material, the reaction was allowed to cool to room
temperature, diluted with water and washed with ethyl acetate. Organic
phases were collected, washed with brine, dried over Na2SO4 and
concentrated under reduced pressure. The crude residue was purified by
silica gel flash column chromatography (eluting with a mixture of
pentane/ethyl acetate in a ratio of 90:10) to yield the title compound (2.29
g, 78%).
1H NMR (300 MHz, DMSO) δ 8.83 (s, 1H), 8.15 – 8.03 (m, 2H), 7.82 (dd,
J = 9.0, 1.9 Hz, 1H), 4.21 – 4.00 (m, 4H), 3.91 – 3.72 (m, 5H).
4-(7-(3,4-Dimethoxyphenyl)quinazolin-4-yl)morpholine (74)
To a solution of 4-(7-bromoquinazolin-4-yl)morpholine 73 (100 mg, 0.34
mmol) in dioxane (4 mL) and water (1 mL) were added 3,4
dimethoxyphenyl boronic acid (74 mg, 0.41 mmol), potassium carbonate
(94 mg, 0.68 mmol), and Pd(PPh3)4 (12 mg, 0,01 mmol). The reaction
mixture was degassed, refilled with nitrogen and stirred at 90 °C overnight.
12
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10.1002/cmdc.201800690
ChemMedChem
FULL PAPER
The volatiles were evaporated in vacuo and the crude residue was purified
by silica gel flash column chromatography (eluting with a mixture of
dichloromethane/methanol in a ratio of 99:1) yielding the title compound
(90 mg, 75%).
1H NMR (300 MHz, DMSO) δ 8.66 (s, 1H), 8.10 – 8.03 (m, 2H), 7.90 – 7.83
(m, 1H), 7.43 – 7.37 (m, 2H), 7.11 (d, J = 8.9 Hz, 1H), 3.90 (s, 3H), 3.83
(s, 3H), 3.81 – 3.73 (m, 8H); 13C NMR (75 MHz, DMSO) δ 163.68, 154.18,
152.06, 149.59, 149.38, 144.17, 131.28, 125.90, 124.57, 124.41, 119.72,
114.52, 112.38, 110.80, 66.17, 55.78, 55.75, 49.81; HRMS m/z [M+H]+
calcd for C20H21N3O3: 352.16555, found 352.1660.
1 hour. After disappearance of the starting material, the volatiles were
evaporated in vacuo and the crude residue was purified by silica gel flash
column
chromatography
(eluting
with
a
mixture
of
dichloromethane/methanol in a ratio of 95:5) yielding the title compound
(26 mg 25%).
1H NMR (300 MHz, DMSO) δ 9.18 (d, J = 2.3 Hz, 1H), 8.56 (s, 1H), 8.36
(d, J = 2.3 Hz, 1H), 7.52 – 7.47 (m, 2H), 7.12 (d, J = 9.0 Hz, 1H), 4.49 –
4.39 (m, 4H), 3.90 (s, 3H), 3.83 (s, 3H), 3.81 – 3.76 (m, 4H) ppm; 13C NMR
(75 MHz, DMSO) δ 158.58, 154.97, 149.99, 149.54, 147.24, 145.83,
138.64, 131.77, 131.35, 128.17, 120.06, 112.44, 110.92, 66.52, 55.86,
55.77, 47.80 ppm; HRMS m/z [M+H]+ calcd for C19H20N4O3: 353.1608,
353.1601.
7-(3,4-Dimethoxyphenyl)pyrido[2,3-d]pyrimidin-4-amine (81)
2-Amino-6-(3,4-dimethoxyphenyl)nicotinonitrile 10 (400 mg) was
suspended in formamide (4 mL) and heated in a microwave for 10 minutes
at 160 °C. The reaction was allowed to cool to room temperature and the
precipitate was filtered off and washed with water to yield the title
compound (200 mg, 46%).
1H NMR (300 MHz, DMSO) δ 8.69 (d, J = 8.6 Hz, 1H), 8.52 (s, 1H), 8.15
(d, J = 8.7 Hz, 1H), 8.02 (bs, 1H), 7.93 – 7.83 (m, 2H), 7.13 (d, J = 9.0 Hz,
1H), 3.90 (s, 3H), 3.86 (s, 3H) ppm; 13C NMR (75 MHz, DMSO) δ 163.01,
161.23, 159.06, 158.68, 151.15, 149.14, 134.21, 130.56, 120.86, 117.87,
111.78, 110.56, 107.50, 55.75, 55.72 ppm; HRMS m/z [M+H]+ calcd for
C15H14N4O2:283.1189, found: 283.1189.
7-(3,4-Dimethoxyphenyl)pyrido[2,3-d]pyrimidin-4-ol (82)
7-(3,4-Dimethoxyphenyl)pyrido[2,3-d]pyrimidin-4-amine 81 (200 mg, 0.71
mmol) was suspended in a 6 N HCl solution (5 mL) and stirred at reflux for
2 hours. The reaction was allowed to cool to room temperature and the
precipitate was filtered off and washed with water to yield the title
compound (120 mg, 60%).
3-Amino-5-bromopyridine-2-carbonitrile (75)
To a stirred solution of iron powder (4.900 g, 87.7 mmol) in 50 mL of acetic
acid at room temperature, was added 5-bromo-3-nitropyridine-2-
carbonitrile 53 (10 g, , 43.9 mmol) at once and the reaction was stirred for
3 hours. After completion, ethyl acetate was added to the mixture and the
precipitate was filtered off. The filtered cake was washed thoroughly with
ethyl acetate and the filtrate was evaporated in vacuo. Crude was purified
by silica gel flash column chromatography (eluting with dichloromethane)
to yield the title compound (4.845 g 56%).
1H NMR (300 MHz, DMSO) δ 8.08 – 7.66 (m, 1H), 7.55 – 7.26 (m, 1H),
6.58 (bs, 2H); HRMS m/z [M+H]+ calcd for C6H4BrN3: 197.96618, found
197.9656.
3-Amino-5-(3,4-dimethoxyphenyl)picolinonitrile (76)
To a solution 3-amino-5-bromopyridine-2-carbonitrile 75 (1.0 g, 5.05
mmol) in dioxane (40 mL) and water (10 mL) were added 3,4
dimethoxyphenylboronic acid (1.11 g, 6.06 mmol), potassium carbonate
(1.38 g, 10.1 mmol), and Pd(PPh3)4 (58 mg, 0,05 mmol). The reaction
mixture was stirred at 90 °C overnight. The volatiles were evaporated and
the crude residue was purified by silica gel flash column chromatography
(eluting with a mixture of dichloromethane/acetone in a ratio of 90:10)
yielding the title compound (1.27 g, 98%).
1H NMR (300 MHz, DMSO) δ 8.22 (d, J = 1.9 Hz, 1H), 7.42 (d, J = 1.9 Hz,
1H), 7.24 – 7.20 (m, 2H), 7.12 – 7.05 (m, 1H), 3.85 (s, 3H), 3.81 (s, 3H);
13C NMR (75 MHz, DMSO) δ 149.87, 149.34, 148.80, 139.62, 137.72,
128.63, 119.65, 117.34, 113.03, 112.34, 110.51, 55.76 ppm.
7-(3,4-Dimethoxyphenyl)pyrido[3,2-d]pyrimidin-4-amine (77)
3-Amino-5-(3,4-dimethoxyphenyl)picolinonitrile 76 (200 mg) was
suspended in formamide (4 mL) and heated in a microwave for 10 minutes
at 160 °C. The reaction was allowed to cool to room temperature and the
formed precipitate was filtered off and washed with water to yield the title
compound (164 mg, 74 %).
1H NMR (300 MHz, DMSO) δ 8.69 (s, 1H), 8.53 (d, J = 8.4 Hz, 1H), 8.21
(d, J = 8.4 Hz, 1H), 7.91 – 7.80 (m, 2H), 7.15 (d, J = 8.3 Hz, 1H), 3.89 (s,
3H), 3.86 (s, 3H) ppm; 13C NMR (75 MHz, DMSO) δ 161.38, 160.93,
151.50, 149.99, 149.18, 136.81, 132.19, 131.67, 131.55, 129.58, 128.99,
128.83, 121.09, 119.34, 111.94, 110.62, 55.83 ppm; HRMS m/z [M+H]+
calcd for C15H13N3O3: 284.1030, found: 284.1025.
7-(3,4-Dimethoxyphenyl)-4-(1H-1,2,4-triazol-1-yl)pyrido[2,3-
d]pyrimidine (83)
A solution of phosphorus oxychloride (268 mg, 1.73 mmol), triazole (242
mg, 3.5 mmol) and DIPEA (136 mg, 1.05 mmol) in acetonitrile (5 mL) was
stirred for 20 minutes at room temperature when 7-(3,4-
dimethoxyphenyl)pyrido[3,2-d]pyrimidin-4-ol 82 (100 mg, 0.35 mmol) was
added. The mixture was stirred for 48 hours at room temperature. After
disappearance of the starting material, the precipitate was filtered of and
the filtrate was washed with a 10% solution of HCl. Organic phases were
collected and concentrated in vacuo. The crude residue was used as such
in the following reaction.
1H NMR (300 MHz, DMSO) δ 9.12 (d, J = 2.1 Hz, 1H), 8.45 (s, 1H), 8.30
(d, J = 2.1 Hz, 1H), 7.93 (bs, 2H), 7.50 – 7.39 (m, 2H), 7.10 (d, J = 8.1 Hz,
1H), 3.90 (s, 3H), 3.83 (s, 3H); 13C NMR (75 MHz, DMSO) δ 161.91,
156.56, 149.87, 149.51, 147.32, 144.88, 139.76, 131.01, 129.79, 128.67,
120.14, 112.39, 111.11, 55.88, 55.75 ppm; HRMS m/z [M+H]+ calcd for
C15H14N4O2: 283.1189, found: 383.1188.
7-(3,4-Dimethoxyphenyl)pyrido[3,2-d]pyrimidin-4-ol (78)
7-(3,4-Dimethoxyphenyl)pyrido[3,2-d]pyrimidin-4-amine 77 (120 mg, 0.43
mmol) was suspended in a 6 N HCl solution (5 mL) and stirred at reflux for
1 hour. The reaction was allowed to cool to room temperature and the
formed precipitate was filtered off to yield the title compound (85 mg, 66%).
HRMS m/z [M+H]+ calcd for C15H13N3O3:284.1030, found: 284.1029.
7-(3,4-Dimethoxyphenyl)-4-(1H-1,2,4-triazol-1-yl)pyrido[3,2-
d]pyrimidine (79)
A solution of phosphorus oxychloride (230 mg, 1.50 mmol), triazole (207
mg, 3.0 mmol) and DIPEA (116 mg, 0.90 mmol) in acetonitrile (5 mL) was
stirred for 20 minutes at room temperature when 7-(3,4-
dimethoxyphenyl)pyrido[3,2-d]pyrimidin-4-ol 78 (85 mg, 0.30 mmol) was
added and the mixture was stirred for 48 hours at room temperature. After
disappearance of the starting material, the precipitate was filtered off and
the filtrate was washed with a 10% solution of HCl. Organic phases were
collected and concentrated in vacuo. The crude residue was used as such
without further purification in the following reaction.
4-(7-(3,4-Dimethoxyphenyl)pyrido[2,3-d]pyrimidin-4-yl)morpholine
(84)
To
a
solution
of
7-(3,4-dimethoxyphenyl)-4-(1H-1,2,4-triazol-1-
yl)pyrido[2,3-d]pyrimidine 83 (0.35 mmol) in dioxane (5 mL) was added
morpholine (152 mg, 1.75 mmol) and the mixture was stirred at reflux for
1 hour. The volatiles were evaporated in vacuo and the crude residue was
purified by silica gel flash column chromatography (eluting with a mixture
of dichloromethane/methanol in a ratio of 95:5) yielding the title compound
(20 mg, 19%).
1H NMR (300 MHz, DMSO) δ 8.71 (s, 1H), 8.43 (d, J = 8.8 Hz, 1H), 8.05
(d, J = 8.7 Hz, 1H), 7.92 – 7.80 (m, 2H), 7.13 (d, J = 8.2 Hz, 1H), 3.90 (s,
3H), 3.86 (s, 3H), 3.85 – 3.75 (m, 8H) ppm; 13C NMR (75 MHz, DMSO) δ
163.99, 160.90, 159.98, 157.06, 151.36, 149.19, 135.87, 130.21, 121.00,
117.33, 111.83, 110.49, 108.67, 66.13, 55.77, 55.72, 49.48 ppm; HRMS
m/z [M+H]+ calcd for C19H20N4O3:353.1608, found:353.1597.
3-Amino-5-(3,4-dimethoxyphenyl)pyrazine-2-carboxamide (85)
To a solution of 3-amino-5-(3,4-dimethoxyphenyl)pyrazine-2-carbonitrile
19 (500 mg, 1.95 mmol) in concentrated formic acid (3 mL) was added
concentrated sulphuric acid (140 µL). The mixture was heated in a
microwave for 20 minutes at 100°C. After disappearance of the starting
material the mixture was allowed to cool and the formed bright yellow
4-(7-(3,4-Dimethoxyphenyl)pyrido[3,2-d]pyrimidin-4-yl)morpholine
(80)
To
a
solution
of
7-(3,4-dimethoxyphenyl)-4-(1H-1,2,4-triazol-1-
yl)pyrido[3,2-d]pyrimidine 79 (0.35 mmol) in dioxane (5 mL) was added
morpholine (152 mg, 1.75 mmol) and the mixture was stirred at reflux for
13
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10.1002/cmdc.201800690
ChemMedChem
FULL PAPER
precipitate was filtered off and washed with water to yield the title
compound (417 mg, 78%).
Acknowledgements
1H NMR (300 MHz, CDCl3) δ 8.26 (s, 1H), 7.63 (d, J = 2.0 Hz, 1H), 7.58
(dd, J = 8.4, 2.1 Hz, 1H), 6.97 (d, J = 8.4 Hz, 1H), 4.00 (s, 3H), 3.96 (s, 3H)
ppm; 13C NMR (75 MHz, DMSO) δ 168.73, 154.58, 152.77, 150.98, 149.10,
128.35, 127.88, 123.66, 120.34, 111.83, 110.23, 55.73 ppm; HRMS m/z
[M+H]+ calcd for C13H14N4O3: 275.1139, found: 275.1164.
R.W. is the recipient of a doctoral fellowship from the Agency for
Innovation by Science and Technology in Flanders (IWT.141103).
This work was supported by award number W81XWH-16-1-0691
from the Department of Defense (DoD), Congressionally Directed
Medical Research Programs (CDMRP) to P.H.
7-(3,4-Dimethoxyphenyl)pteridin-4(3H)-one (86)
3-Amino-5-(3,4-dimethoxyphenyl)pyrazine-2-carbonitrile 85 (400 mg, 1.56
mmol) was suspended in formamide (10 mL) and heated in a microwave
at 160°C for 20 minutes. The reaction was allowed to cool to room
temperature, the precipitate was filtered off and washed with water to yield
the title compound (315 mg, 71%).
HRMS m/z [M+H]+ calcd for C14H12N4O3: 285.0982, found: 285.0982.
4-Chloro-7-(3,4-dimethoxyphenyl)pteridine (87)
To a solution of 7-(3,4-dimethoxyphenyl)pteridin-4(3H)-one 86 (300 mg,
1.05 mmol) in dry toluene were added DIPEA (407 mg, 3.15 mmol) and
phosphorus oxychloride (483 mg, 3.15 mmol) and the mixture was stirred
at reflux overnight. After disappearance of the starting material, the
volatiles were evaporated and the crude residue was purified by silica gel
Keywords: cyclin G associated kinase • medicinal chemistry •
nitrogen heterocycles • scaffold hopping • isothiazolo[4,3-
b]pyridine
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15
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Entry for the Table of Contents
Starting from a cyclin G associated kinase (GAK) inhibitor based on an isothiazolo[4,3-b]pyridine skeleton, a scaffold hopping
strategy was applied, resulting in the synthesis of 13 novel scaffolds. Four of these scaffolds (isothiazolo[3,4-b]pyrazine, pyrazolo[1,5-
a]pyrimidine, imidazo[4,5-b]pyridine and quinazoline) display potent GAK affinity with Kd values of less than 1 µM. These
heterocycles can be used as starting points for the discovery of novel GAK inhibitors based on a different chemotype.
16
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