Synthesis and characterization of novel classes of PDE10A inhibitors - 1H-1,3-benzodiazoles and imidazo[1,2-a]pyrimidines

Article abstract of DOI:10.1016/j.ejmech.2018.05.043

New compounds containing [1,2,4]triazolo [1,5-a]pyridine (I), pyrazolo [1,5-a]pyridine (II), 1H-1,3-benzodiazole (III) and imidazo [1,2-a]pyrimidine (IV) backbones were designed and synthesized for PDE10A interaction. Among these compounds, 1H-1,3-benzodiazoles and imidazo [1,2-a]pyrimidines showed the highest affinity for PDE10A enzyme as well as good metabolic stability. Both classes of compounds were identified as selective and potent PDE10A enzyme inhibitors.

Full text of DOI:10.1016/j.ejmech.2018.05.043

Accepted Manuscript
Synthesis and characterization of novel classes of PDE10A inhibitors - 1H-1,3-
benzodiazoles and imidazo[1,2-a]pyrimidines
Rafał Moszczyński-Pętkowski, Jakub Majer, Małgorzata Borkowska, Łukasz Bojarski,
Sylwia Janowska, Mikołaj Matłoka, Filip Stefaniak, Damian Smuga, Katarzyna
Bazydło, Krzysztof Dubiel, Maciej Wieczorek
PII:
S0223-5234(18)30456-2
10.1016/j.ejmech.2018.05.043
EJMECH 10454
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 15 February 2018
Revised Date: 24 May 2018
Accepted Date: 25 May 2018
Please cite this article as: Rafał. Moszczyński-Pętkowski, J. Majer, Mał. Borkowska, Ł. Bojarski,
S. Janowska, Mikoł. Matłoka, F. Stefaniak, D. Smuga, K. Bazydło, K. Dubiel, M. Wieczorek,
Synthesis and characterization of novel classes of PDE10A inhibitors - 1H-1,3-benzodiazoles
and imidazo[1,2-a]pyrimidines, European Journal of Medicinal Chemistry (2018), doi: 10.1016/
j.ejmech.2018.05.043.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Synthesis and characterization of novel classes of PDE10A inhibitors - 1H-1,3-benzodiazoles
and imidazo[1,2-a]pyrimidines
Rafał Moszczyński-Pętkowskia,*, Jakub Majera, Małgorzata Borkowskaa, Łukasz Bojarskib, Sylwia
Janowskab, Mikołaj Matłokab, Filip Stefaniaka, Damian Smugaa, Katarzyna Bazydłob, Krzysztof
Dubiela, Maciej Wieczorekb
aCelon Pharma S.A., Medicinal Chemistry Department, Mokra 41a, Kiełpin, 05-092 Łomianki, Poland
b Celon Pharma S.A., Innovative Drugs Research and Development Department, Mokra 41a, Kiełpin,
05-092 Łomianki, Poland
Keywords:
PDE10A
1H-1,3-benzodiazoles
Imidazo[1,2-a]pyrimidines
Abstract:
New compounds containing [1,2,4]triazolo[1,5-a]pyridine (I), pyrazolo[1,5-a]pyridine (II), 1H-1,3-
benzodiazole (III) and imidazo[1,2-a]pyrimidine (IV) backbones were designed and synthesized for
PDE10A interaction. Among these compounds, 1H-1,3-benzodiazoles and imidazo[1,2-a]pyrimidines
showed the highest affinity for PDE10A enzyme as well as good metabolic stability. Both classes of
compounds were identified as selective and potent PDE10A enzyme inhibitors.
1. Introduction
Phosphodiesterase 10A (PDE10A) is an enzyme from the phosphodiesterases family with highly
specific expression in the striatum [1], the part of basal ganglia having various functions involved
in control of motor movement, cognitive processes, emotions and learning [2]. PDE10A
hydrolyzes both cAMP and cGMP in the striatal neurons and regulates throughout stratonigral
(direct) and stratopallidal (indirect) pathways in the brain. Activation of these pathways has
opposite output effects leading to activation or inhibition of thalamus circuits [3]. In the
stratopallidal pathway, inhibition of the PDE10A leads to diminishing of the D2 activation
signalling in contrast to the stratonigral pathway, where inhibition of the PDE10A result in
mimicking the D1 signalling [4]. Balance between these two circuits plays essential function in
controlling motor and executive functions [5]. Therefore cyclic nucleotides level modulation in this
neurons can be advantageous in several disorders connected to the basal ganglia system,
including psychotic, neurological and cognitive functions disorders, for example such as
psychosis, including psychosis in schizophrenia, Huntington’s disease, Parkinson’s disease,
addictions and obsessive-compulsive disorders [5,6,7 ].
In recent years numerous companies have reported PDE10A inhibitors from different chemical
groups [8-10]. Among them Lundbeck in a series of patent applications [11-14] presented novel
classes of compounds as potential antipsychotic agents, where these derivatives are described to
have PDE10A potencies of <50 nM and potent in vivo activity measured by the reversal of PCP
induced hyperactivity. These compounds are certainly selectivity pocket binders, with crucial
interactions with Tyr 683 and Gln 716 within the PDE10A enzyme [15]. Their structure was derived
from benzodiazole derivative (Fig.1) which was selected on the basis of HTS-screening [16]. Further
optimization of benzodiazole HTS hit led to compound LuAE90074, which incorporated 4-
phenylimidazole moiety as most crucial fragment for PDE10A interaction (IC50 = 67 nM). (Fig.1). The
important feature of molecules developed by Lundbeck within this chemical class was the presence of
5,7-dimethyl-[1,2,4]triazolo[1,5-a]pyrimidine and 5,8-dimethyl-[1,2,4]triazolo[1,5-a]pyrazine moieties
(HET1) which were responsible for selectivity of obtained compounds toward PDE10A and were
optimized in terms of metabolic stability [16].
*Corresponding author. Tel.:+48 882149529 E.mail address: rafal.moszczynski@celonpharma.com

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Subsequently, in the period between 2012-2013 Bristol Myers Squibb published a series of patent
applications concerning the compounds of similar structure and significant potency toward PDE10A
[17,18]. The BMS’s compounds incorporated initially a cyclopropane ring instead of the 4-
phenylimidazole moiety in LuAE90074 [17]. During further development cyclopropane was replaced
with a bicyclic scaffold of imidazo[1,2-a]pyridine, while keeping the critical nitrogen N(1) atom in the
central bicyclic core responsible for interaction with Tyr683 [18] (e.g., the most potent, Example 1a,
IC50 = 37 nM) (Fig.1). Since the initial publication of the structures by BMS in patent application [18]
there was neither literature available about the further development of these compounds nor
biological data concerning their in vitro and in vivo activity.
Figure 1. Structure of PDE10A inhibitors (Lundbeck LuAE90074 and BMS’s Example 1a [18])
Thus in the frame of our research, considering Example 1a [18] as model compound, four different
heterocyclic bicyclic scaffolds, including [1,2,4]triazolo[1,5-a]pyridine (I), pyrazolo[1,5-a]pyridine (II),
1H-1,3-benzodiazole (III) and phenylimidazo[1,2-a]pyrimidine (IV) have been designed (Fig.2). We
planned also to make use of three different HET1 moieties (HET1-1: 5,8-dimethyl-[1,2,4]triazolo[1,5-
a]pyrazine, HET1-2: 5,7-dimethyl-[1,2,4]triazolo[1,5-a]pyrimidine and HET1-3: 4-methylquinazoline) to
check the influence of these motifs on the potency, selectivity over other PDE’s and metabolic
stability. As for HET2 moiety we decided to check the influence of different fragments (e.g. 5, 6, 10-
membered rings) as well as different substituents on PDE10A inhibition activity. In this paper we
report the synthesis and biological evaluation of novel 1H-1,3-benzodiazole and imidazo[1,2-
a]pyrimidine derivatives.
2. Results and discussion
2.1.Chemistry

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Figure 2. Bioisosteric modification of the imidazo[1,2-a]pyridine core
The chemistry employed to prepare the [1,2,4]triazolo[1,5-a]pyridines (I) is outlined in Scheme 1.
For all synthesized compounds in this series the first three steps were common. In the first step,
isonicotinic acid 1 was converted into methyl ester 2 [19] which was treated with amino 2,4,6-
trimethylbenzene-1-sulfonate [20] to give pyridinium salt 3. The subsequent condensation of 3 with
corresponding benzonitrile or pyrimidinecarbonitrile in refluxing ethanol afforded key intermediate,
methyl 2-aryl[1,2,4]triazolo[1,5-a]pyridine-7-carboxylate 4 [21]. Depending on the HET1 moiety, the
further synthetic routes for final compounds 7-12 were different. For compounds 9, 10 and 12, the 2-
aryl[1,2,4]triazolo[1,5-a]pyridine-7-carboxylate 4 was hydrolyzed to corresponding carboxylic acid 8a-
b [22], which was converted to final Compound 9 or 10 by refluxing with 1-amino-4,6-dimethyl-1H-
pyrimidin-2-ylidene-ammonium diphenylphosphinate [23] in POCl3. The products were isolated in
yields ranging from 10% (9) to 20% (10). For compound 7, the 2-aryl[1,2,4]triazolo[1,5-a]pyridine-7-
carboxylate 4 was reduced by lithium aluminium hydride in THF to give corresponding primary alcohol
5 which was converted to aldehyde 6 via Swern Oxidation. Finally, the aldehyde 6 was reacted with
1-amino-2-imino-3,6-dimethyl-2,3-dihydro-1-pyrazinium diphenylphosphinate [23] in DMF to give
Compound 7 in 23% yield.
For Compound 12, the carboxylic acid 8a was coupled with 1-(2-aminophenyl)ethanone in the
presence of HATU/DIPEA in DMF. The obtained amide intermediate 11 was then converted to 12 with
78% yield by heating with ammonia solution in ethanol at 130oC in a sealed vessel.

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Scheme 1. Syntheses of new [1,2,4]triazolo[1,5-a]pyridine analogues. Reagents and conditions: (i)
H2SO4, MeOH, reflux, 72h, 92%; (ii) Mes(SO2)-ONH2, DCM, 0->25oC, 18h, 72%; (iii) benzonitrile (for
7, 9, 12) or 2-pyrimidinecarbonitrile (for 10), EtOH, reflux, 18h, 36-88%; (iv) THF/MeOH, LiOH*H2O,
RT, 48h, 64%; (v) 1-amino-4,6-dimethyl-1H-pyrimidin-2-ylidene-ammonium diphenylphosphinate,
POCl3, reflux, 3h, 11- 20%; (vi) LiAlH4, THF, -40->10oC, 2,5h, 42%; (vii) (COCl)2, DMSO, TEA, DCM, -
78oC
->
RT,
2,5h,
87%;
(viii)
1-amino-2-imino-3,6-dimethyl-2,3-dihydro-1-pyrazinium
diphenylphosphinate, Na2S2O5, DMF, 50-80oC, 24h, 23%; (ix) 1-(2-aminophenyl)ethanone, HATU,
DIPEA, DMF, 60oC, 18h, 36%; (x) NH3/MeOH, 130oC, 18h, 78%.
The syntheses of compounds 17 and 18, containing pyrazolo[1,5-a]pyridine scaffold are outlined in
Scheme 2. In the first step, condensation of ethyl benzoate 13 with 5-cyano-2-methylpyridine via
intermediate 14 [24] and subsequent reaction with 2,4,6-trimethylbenzene-1-sulfonate led to key
intermediate, 2-phenylpyrazolo[1,5-a]pyridine-6-carbonitrile 15 [24]. The nitrile 15 was converted to
corresponding aldehyde 16 via reduction with DIBALH in DCM. The aldehyde 16 was then reacted
with 1-amino-4,6-dimethyl-1H-pyrimidin-2-ylidene-ammonium diphenylphosphinate or 1-amino-2-
imino-3,6-dimethyl-2,3-dihydro-1-pyrazinium diphenylphosphinate [23] in DMF to give final
compounds, 17 and 18, respectively.

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Scheme 2. Syntheses of new pyrazolo[1,5-a]pyridine analogues. Reagents and conditions: (i) 5-
cyano-2-methylpyridine, LiHMDS, THF, -5oC->RT, 19h, 93%; (ii) Mes(SO2)-ONH2, DIPEA, DCM, 0oC-
>RT, 20h, 54%; (iii) DIBALH, DCM, -78oC->RT, 4h, 33%; (iv) 1-amino-4,6-dimethyl-1H-pyrimidin-2-
ylidene-ammonium diphenylphosphinate, Na2S2O5, DMF, 80oC, 22h, 7%; (v) 1-amino-2-imino-3,6-
dimethyl-2,3-dihydro-1-pyrazinium diphenylphosphinate, Na2S2O5, DMF, 100oC, 24h, 26%.
Syntheses of novel 1H-1,3-benzodiazole analogues are depicted in Scheme 3. The main substrate,
ethyl 3,4-diaminobenzoate 19 was reacted with carboxylic acid or aldehyde containing HET2 moiety
which can be aryl, heteroaryl or benzyl. The reactions of carboxylic acids of general formula
HET2COOH were carried out either in polyposphoric acid or phosphorus oxychloride [25]. Generally
reaction with POCl3 provided better yields of 1H-1,3-benzodiazole esters 20a-iv. The yields,
depending on HET2 substituents varied between 13-85%. The reaction of aldehydes of general
fomula HET2CHO were performed either at RT with sodium metabisulfite in DMF (instead of heating
in EtOH-water, [26-28]) or in refluxing toluene [29] with catalytic amount of PTSA. The 1H-1,3-
benzodiazole esters 20a-iv were obtained in yields up to 88%. Intermediates 20a-iv were
subsequently hydrolyzed to corresponding carboxylic acids 21a-iv with good yields [30-31]. The acids
21a-iv were converted to final derivatives 22-24 and 26-53 via reactions with iminium salts [23] using
coupling agents (HATU or EDCI-HOBt) in DMF. The N(3) and N(1) methylated derivatives were
obtained from compound 22 via methylation with MeI in basic conditions in DMF. The final
compounds 25a and 25b were separated via column chromatography.

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Scheme 3. Syntheses of new 1H-1,3-benzodiazole analogues. Reagents and conditions: (i) HET2-
COOH, PPA, 140oC, 2h, 46-62% or (ii) HET2-CHO, Na2S2O5, DMF, RT, 20h, 25-38% or (iii) HET2-
CHO, PTSA, toluene, reflux, 20h, 9-88% or (iv) HET2-COOH, POCl3, 120oC, 3h, 4-88%; (v) MeOH,
NaOH aq., 60oC, 20h, 53-96%; (vi) 1-amino-4,6-dimethyl-1H-pyrimidin-2-ylidene-ammonium
diphenylphosphinate, HOBt, EDCI, TEA, DMF, RT, 48h, AcOH, 100oC, 24h, 30% or (vii) 1-amino-4,6-
dimethyl-1H-pyrimidin-2-ylidene-ammonium diphenylphosphinate, POCl3, reflux, 3h, 18%; (viii) 1-
amino-2-imino-3,6-dimethyl-2,3-dihydro-1-pyrazinium diphenylphosphinate, HATU, DIPEA, DMF, RT,
48h, 16-74%; (ix) MeI, K2CO3, DMF, 24h, 48%; (x) MeI, K2CO3, DMF, 24h, 56%.
The synthesis of compound 58 (Scheme 4) possessing 4-methylquinazoline as HET1 moiety was
performed using 3,4-dinitrobenzoic acid 54 as starting material, which was converted into acid
chloride and subsequently reacted with 1-(2-aminophenyl)ethanone giving amide intermediate 55
which was converted to quinazoline derivative 56 with high yield by heating with ammonia solution in
methanol at 140oC in an autoclave [32]. The dinitro compound 56 was then reduced with hydrogen on
palladium and obtained diamine 57 was directly reacted with benzaldehyde in the presence of sodium
bisulfite in DMF to give final derivative 58 in 24% yield.

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Scheme 4. Synthesis of compound 58. Reagents and conditions: (i) (COCl)2, DCM/DMF, RT, 2h, 1-
(2-aminophenyl)ethanone, TEA, THF, 18h, 74%; (ii) NH3/MeOH, 140oC, 5h, 93%; (iii) Pd/H2,
AcOEt/EtOH, RT, 24h, not isolated; (iv) PhCHO, Na2S2O5, DMF, 100oC, 20 h, 24%.
Syntheses of novel imidazo[1,2-a]pyrimidine analogues are depicted in Scheme 5. The main
substrate, 2-amino-4-methylpyrimidine 59 was reacted in refluxing ethanol [33] with corresponding
bromoacetophenone to give 7-methyl-2-arylo-imidazo[1,2-a]pyrimidine compounds 60a-j containing
substituted or unsubstituted HET2 group in yields up to 90%. In the next step, the derivatives 60a-j
were converted in the presence of an excess of DMA-DMF acetal in DMF to corresponding
ethenamines 61a-j in yields up to 90% [34,35]. The ethenamines 61 were subsequently reacted with
sodium periodate in water-THF mixture to give corresponding aldehydes 62a-j in yields up to 67%
[36-37]. In the last step the aldehydes 62 were reacted either with pyrazinium salt in toluene or 2-
propanol, giving the final compounds 63-65 in yields up to 42% or with iminium salt delivering final
products 66-75 in yields up to 24%.

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Scheme 5. Syntheses of new imidazo[1,2-a]pyrimidine analogues. Reagents and conditions: (i) HET2-
COCH2Br, EtOH, 80oC, 5h, 33-90% (ii) DMA-DMF, DMF, 140oC, 20h, 89%; (iii) NaIO4, THF/H2O 0oC,
5h, 28-67%; (iv) 1-amino-2-imino-3,6-dimethyl-2,3-dihydro-1-pyrazinium diphenylphosphinate,
toIuene/IPA,
80oC,
1,5
h,
5-42%;
(v)
1-amino-4,6-dimethylpyrimidin-2(1H)-iminium
diphenylphosphinate DMF, 50-80oC, 4d, 1-24%.
The synthesis of compound 82 possessing imidazo[1,2-a]pyrimidine core and 4-methylquinazoline
as HET1 moiety is outlined in Scheme 6. In the first step ethyl vinyl ether 76 was reacted with ethyl
oxalyl chloride, affording oxoester 77 [38]. The ester 77 was further condensed with guanidinium
chloride giving aminopyrimidine derivative 78 [39]. Subsequent cyclization of 78 with
bromoacetophenone afforded imidazo[1,2-a]pyrimidine ester 79 which was further hydrolyzed to
corresponding carboxylic acid 80. Coupling of 80 with 1-(2-aminophenyl)ethanone and subsequent
cyclization of amide intermediate 81 in methanolic ammonia solution afforded final compound 82 in
73% yield.

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Scheme 6. Synthesis of compound 82 .Reagents and conditions: (i) ethyl oxalyl chloride, 10oC, 24h,
25%; (ii) guanidinium chloride, propanenitrile, TEA, propionitrile, 100oC, 24h, 27%; (iii) 2-bromo-1-
phenylethanone, toluene, 110oC, 6h, 38%; (iv) LiOH*H2O, THF/MeOH, RT, 18h, 42%; (v) 1-(2-
aminophenyl)ethanone, HATU, DIPEA, DMF, RT, 24h, 49%; (vi) NH3/MeOH, 100oC, 18h, 73%.
2.2. Inhibition of PDE10A activity
The possible binding mode of compounds containing bicyclic scaffolds (I-IV) was examined by a
manual docking placement procedure into the PDE10A protein (PDB 3HQY) using AutoDockVina
[40]. On the basis of virtual docking we assumed that nitrogen N(1) in the central bicyclic core is
responsible for crucial hydrogen-bond interaction with Tyr683 in the PDE10A structure and functions
as hydrogen bond acceptor (substitution of nitrogen N(1) by methyl group suppresses PDE10A
inhibitory activity for compound 25b) while the HET1 motif might occupy the clamp region and make π-
stacking interaction with Phe719 (Fig 3). Besides the nitrogen N(1) we also found the influence of
substituent in the HET2 moiety on inhibitory activity.

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Figure 3. 3D representation of possible binding mode of Example 1a by BMS (yellow), Compound 26
(green) and Compound 66 (cyan) in the binding site of PDE10A enzyme (PDB ID 3HQY)
A library of compounds comprising four mentioned bicyclic scaffolds and three HET1 moieties has
been prepared. The scaffolds I-IV comprising four different bicyclic systems were chosen using
“scaffold hopping” approach. Although the carbon-nitrogen swap in the central bicyclic core might
seem obvious, however the obtained in vitro data indicated strong relationship between scaffold type
and activity toward PDE10A (Tables 1-4). The obtained results showed that scaffolds possessing less
basic nitrogen N(1) had lesser PDE10A inhibition potency.
The compounds (Tables 1 and 2) containing scaffolds such as [1,2,4]triazolo[1,5-a]pyridine (I) and
pyrazolo[1,5-a]pyridine (II) were the least potent of all tested molecules. Their IC50 were in the range
between 268 nM (Compound 7) and >5000 nM (Compound 10). Replacing the phenyl group (HET2) in
compound 7 by a pyrimidin-2-yl group in compound 10 resulted in total loss of potency. On the other
hand, a slightly increasing trend for PDE10A inhibition (IC50 = 268 nM) was observed for compound 7,
which incorporated another HET1 moiety (5,8-dimethyl-[1,2,4]triazolo[1,5-a]pyrazine instead of 5,7-
dimethyl-[1,2,4]triazolo[1,5-a]pyrimidine in 9).
As for the second tested scaffold, pyrazolo[1,5-a]pyridine (II, Table 2) the obtained IC50 values were
also in the range of 500-800 nM (Compounds 17 and 18).
Assuming the binding mode depicted on Fig 3. the basicity of N(1) nitrogen in both scaffolds is weak
(pKa 1,6-2,3) thus the potency of the compounds might be expected to be low (in micromolar range).
Table 1
The PDE 10A inhibition activity of [1,2,4]triazolo[1,5-a]pyridine (I) analogues

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Compound#
HET1
HET2
Phenyl
Phenyl
IC50 (nM)
268
381
>5000
884
7
9
10
12
HET1-2
HET1-1
HET1-1
HET1-3
Pyrimidin-2-yl
Phenyl
Table 2
The PDE 10A inhibition activity of pyrazolo[1,5-a]pyridine (II) analogues
Compound#
HET1
HET1-1
HET1-2
HET2
Phenyl
Phenyl
IC50 (nM)
770
17
18
581
For the more basic scaffold, 1H-1,3-benzodiazole (IV), the IC50 values for the first synthesized
compounds, 22 and 26, were 47nM and 27nM, respectively - the strong increasing trend for PDE10A
inhibition was observed – the obtained values were 10-fold greater than for previous compounds
incorporating scaffolds I and II (Table 3). The potency for Compound 26 (possessing HET1-2 = 5,8-
dimethyl-[1,2,4]triazolo[1,5-a]pyrazine) was increased 2-fold than for Compound 22. Since we
observed this trend in the case of previous scaffolds (I-II), we decided to develop a library of
compounds which possessed only 5,8-dimethyl-[1,2,4]triazolo[1,5-a]pyrazine (HET1-2) as HET1

ACCEPTED MANUSCRIPT
moiety. Simultaneously, we decided to check the influence of different HET2 moieties on inhibitory
potency toward PDE10A. The best results (Table 3) were obtained when HET2 was an unsubstituted
phenyl. Replacing the phenyl group by another aryl such as thiazole, imidazole, oxazole, pyridine,
pyrimidine or pyrazine resulted in slightly lesser potency (compounds 30, 31, 33, 42, 48). Only in
case when HET2 = thiophene or furan (compounds 40 and 41) potency was retained (IC50 = 67 nM
and 28 nM, respectively). In the case of pyrazine we observed a significant loss in potency (>1000
nM, 48). We noted that substitution of the HET2 moiety (when HET2 = phenyl or pyridine) at the C-2
position led to increase in potency toward PDE10A. Especially methoxy group or fluorine proved to be
the most favorable (compounds 27 and 32, IC50 = 37 nM and 19,5 nM, respectively). The same trend
can be observed for pyridine substituent. Compound 46, having pyridin-3-yl group was nearly 3-fold
less potent (IC50 = 166 nM) than 30 which possessed pyridin-2-yl group (IC50 = 63 nM). Bulkier
groups at C-2 position of the phenyl ring, such as trifluoromethoxy or trifluoromethyl (compounds 36
and 37) contributed to a lesser inhibitory activity (IC50 = 280 nM and >1000 nM, respectively). In the
case where HET2 was substituted by fluorine or methoxyl at pyridine C-3 position (49 and 50) the
potency was retained (IC50 = 69 and 40 nM, respectively), but methoxy group at position C-4
contributed to lesser potency (IC50 = 280 nM for Compound 51).
Both addition of a –CH2- linker between the center scaffold and HET2 moiety (compound 34) as well
as replacing the HET2 group by a bulkier substituent (bicyclic, such as quinoxalin-2-yl, 35) resulted in
significant decreasing of potency (IC50 > 1000 nM). Also replacing HET1 with 4-methylquinazoline
(HET1-3) while maintaining phenyl group as HET2 resulted in decreasing of inhibition activity toward
PDE10A (58, IC50 = 315 nM).
We also have observed that after the methylation of nitrogen N(3) in the center scaffold (thus blocking
the possibility of formation of tautomeric forms within the 1H-1,3-benzodiazole core) the activity
toward PDE10A significantly decreased (to 794 nM for compound 25a whereas for compound 26 IC50
was 27 nM). When the critical nitrogen N(1) was methylated (compound 25b) the inhibitory activity
was completely suppressed. This observation seems to confirm the critical role of nitrogen N(1) in this
series of compounds as for the interaction with the PDE10A enzyme.
Table 3
The PDE 10A inhibition activity of 1H-1,3-benzodiazole (III) analogues
Compound#
R1
H
H
H
HET1
HET2
Phenyl
Pyridin-2-yl
Pyridin-4-yl
Phenyl
Phenyl
Phenyl
2-Fluorophenyl
3-Fluorophenyl
4-Fluorophenyl
Pyridin-2-yl
IC50 (nM)
47
279
953
794
inactive
27
37
172
54
63
84
19,5
280
>1000
>1000
280
22
23
24
25a
25b
26
27
28
29
30
31
32
33
34
35
36
HET1-1
HET1-1
HET1-1
HET1-1
HET1-1
HET1-2
HET1-2
HET1-2
HET1-2
HET1-2
HET1-2
HET1-2
HET1-2
HET1-2
HET1-2
HET1-2
N(3)CH3
N(1)CH3
H
H
H
H
H
H
H
H
H
H
H
1,3-Thiazol-2-yl
2-Methoxyphenyl
1-Methyl-1H-imidazol-2-yl
Benzyl
Quinoxalin-2-yl
2-(Trifluoromethoxy)phenyl

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37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
58
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
HET1-2
HET1-2
HET1-2
HET1-2
HET1-2
HET1-2
HET1-2
HET1-2
HET1-2
HET1-2
HET1-2
HET1-2
HET1-2
HET1-2
HET1-2
HET1-2
HET1-2
HET1-3
2-(Trifluoromethyl)phenyl
>1000
382
242
67
2-Cyanophenyl
2-Methylphenyl
Furan-2-yl
Thiophen-2-yl
28
1,3-Oxazol-4-yl
1,3-Thiazol-4-yl
1,3-Thiazol-5-yl
3-Bromophenyl
Pyridin-3-yl
>300
268
135
146
166
161
>1000
69
5-Chlorothiophen-2-yl
Pyrazin-2-yl
3-Fluoropyridin-2-yl
3-Methoxypyridin-2-yl
4-Methoxypyridin-2-yl
4-Methyl-1,3-thiazol-2-yl
5-Methyl-1,3-thiazol-2-yl
Phenyl
40
>280
117
198
315
For the next scaffold, imidazo[1,2-a]pyrimidine (IV) the inhibition activity (Table 4) was within the
same range as for 1H-1,3-benzodiazole analogues (Table 3). The obtained IC50 values for the most
active compounds (66 and 67; 35 nM and 52 nM, respectively) were consistent with those observed
for Compounds 22 and 32. The compound with HET2 = pyridine-2-yl (69) was twice less potent (IC50 =
115 nM) than its 1H-1,3-benzodiazole counterpart (30, IC50 = 63 nM). In this series of compounds we
also have observed that the substitution especially at C-2 position in the phenyl ring of HET2 moiety is
critical for retaining high potency.
Table 4
The PDE 10A inhibition activity of imidazo[1,2-a]pyrimidine (IV) analogues
Compound#
HET1
HET2
Phenyl
Pyridin-2-yl
1,3-Thiazol-2-yl
Phenyl
2-Methoxyphenyl
1,3-Thiazol-2-yl
Pyridin-2-yl
4-Fluorophenyl
2-Chloro-4-
fluorophenyl
4-Bromophenyl
2,5-
IC50 (nM)
96
63
64
65
66
67
68
69
70
71
HET1-1
HET1-1
HET1-1
HET1-2
HET1-2
HET1-2
HET1-2
HET1-2
HET1-2
280
458
35
52
135
115
76
>1000
>1000
>280
72
73
HET1-2
HET1-2
Dimethoxyphenyl
74
75
HET1-2
HET1-2
107
145
3-Chlorophenyl
2-Bromophenyl
Phenyl
82
HET1-3
163

ACCEPTED MANUSCRIPT
2.3. Metabolic stability and selectivity
During ADMET studies on drug discovery microsomal stability tests are primary assays for compound
selection for further development. Microsomal stability assays are “gold standard” for investigation of
First Phase metabolism, measurement in vitro intrinsic clearance or identifying formed metabolites
with good correlation to hepatic stability approach [41]. On the base of microsomal stability it is
possible to rank order compounds undergoing enzymatic reactions. Microsomal stability assessment
is rapid, reproducible in high throughput analyses and cost reducing approach in comparison to
hepatic stability [42]. Moreover, the liver microsomal in vitro T1/2 approach can be a suitable way to
measure in vitro CLint, which can be scaled up to the in vivo situation and used in the prediction of
human clearance [43].
The set of compounds, possessing the 1H-1,3-benzodiazole (III) and imidazo[1,2-a]pyrimidine (IV)
scaffold were selected for metabolic stability study. The metabolic stability was assessed (Table 5) by
measuring the intrinsic clearance in human and rat liver microsomes (HLM Cl and RLM Cl). The HET1
fragment had critical importance as for the stability of the given compound. The 4-methylquinazoline
moiety (HET1-3) proved to be very unstable (Cl > 450 µl x min-1 x mg -1, Compound 58), whereas
compounds possessing 5,8-dimethyl-[1,2,4]triazolo[1,5-a]pyrazine (HET1-2) moiety had mostly
optimal clearance value ranging from 0,7 to 34,4 µl x min-1 x mg -1 (Table 5).
Table 5
Metabolic stability of selected compounds
Cl int (µl x min-1 x mg -1)
Compound#
Microsome type
26
26
30
40
49
50
50
58
66
67
70
71
72
74
75
RLM
HLM
RLM
RLM
RLM
RLM
HLM
RLM
RLM
RLM
RLM
RLM
RLM
RLM
RLM
27.4
34.4
11.1
26.3
1.8
5.4
0.7
>450
5.6
9.1
3.1
11.5
15.0
18.9
1.8
RLM – rat liver microsomes, 1st phase, HLM – human liver microsomes, 1st phase
The model compound, BMS’s Example 1a ([18],Fig. 1) is significantly less metabolically stable, when
compared to its imidazo[1,2-a]pyrimidine analogue 66. The comparison of metabolic stability on
various types of microsomes between BMS’s Example 1a and its closest analogue is depicted in
Table 6.
Table 6
Metabolic stability of Example 1a and compound 66 in various types of microsomes
Cl int (µl x min-1 x mg -1)
Compound#
RLM
68.86
5.6
MLM*
28.9
0.6
HLM
11.6
1.8
DLM
16.3
7.4
Example 1a
Compound 66
RLM – rat liver microsomes, 1st phase, HLM – human liver microsomes, 1st phase, MLM* – mouse liver
microsomes, 1st and 2nd phase, DLM – dog liver microsomes, 1st phase

ACCEPTED MANUSCRIPT
The newly obtained compound 66 is nearly 10-fold more stable on HLM than its analogue (the Clint
value in HLM for Example 1a was 11,6 µl x min-1 x mg -1 whereas for compound 66 Cl value
decreased to 1,8 µl x min-1 x mg -1).
The most active compounds from the 1H-1,3-benzodiazole (III) and imidazo[1,2-a]pyrimidine (IV)
series (with IC50 values < 200 nM) were also screened for PDE selectivity. The selectivity screen was
performed using an enzymatic activity luminescent assay PDE-Glo. The compounds were screened
against PDE1C and PDE9A. The preliminary selectivity assessment revealed no activity against
them. For all twenty-five tested compounds (22, 26, 27, 29, 30, 31, 32, 40, 41, 44, 45, 46, 47, 49, 50,
52, 53, 63, 66, 67, 68, 69, 70 ,74, 75) IC50 values for PDE1C and PDE9A inhibitory activity were
greater than 10 µM. Additionally, Compounds 26 and 66 as representatives of both leading series
were screened against representative members of all other PDE families at 100 nM (Table 7). Both
compounds proved to be very selective toward PDE10A.
Table 7
Selectivity screen against representative members of all other PDE families at 100 nM. Results are
expressed as % inhibion of PDE enzyme
% inhibition
Enzyme
PDE1C
PDE2A
PDE3B
PDE4B1
PDE5A1
Compound 26
Compound 66
16%
8%
-3%
3%
5%
9%
7%
4%
6%
18%
26%
-1%
-3%
98%
9%
6%
PDE6C
PDE7A
PDE8A1
PDE9A2
PDE10A1
PDE11A
-3%
8%
-4%
-7%
99%
8%
3. Conclusions
We have identified two new classes of compounds who proved to be potent and stable PDE10A
inhibitors. Overall 34 1H-1,3-benzodiazole and 14 imidazo[1,2-a]pyrimidine derivatives were
synthesized and screened for their in vitro activity. SAR analysis indicated that the substitution of the
HET2 moiety in C-2 position of the phenyl ring resulted in high potency of the compounds. Hovewer,
the addition of a linker between the central bicyclic core and HET2 motif as well as replacement of 5-
or 6- membered ring by bulkier substituent (10-membered, such as quinoxalinyl group) as HET2 group
resulted in decreased potency probably due to spatial limitations in the active binding pocket. We also
noticed that nitrogen N(1) in central bicyclic core plays important role for PDE10A interaction – in the
1H-1,3-benzodiazole series the inhibitory activity may be suppressed in case when the N(1) is
methylated.
Both obtained series of inhibitors possess similar metabolic stability (Clint in the range 0,7-34,4 µl x
min-1 x mg -1) and high selectivity toward PDE10A. The compound 66 from the imidazo[1,2-
a]pyrimidine series proved to be equally potent and 10-fold more stable than model compound
(Example 1a) developed by BMS. Further work on these interesting classes of PDE10A inhibitors will
be disclosed in due course.
4. Experimental section
4.1.Chemistry: general procedures
Chemicals (with purity at least 95%) were purchased from ABCR, Acros, Alfa Aesar, Combi-Blocks,
Fluorochem, Fluka, Merck and Sigma Aldrich and used without further purification. Solvents were

ACCEPTED MANUSCRIPT
purified according to standard procedures, if required. Air or moisture sensitive reactions were carried
out under argon atmosphere. All compounds were routinely checked by thin-layer chromatography
(TLC). TLC was performed on silica gel coated plates (Kieselgel F254) and visualized using UV. Flash
1
chromatography was performed using Merck silica gel 60 (230-400 mesh ASTM). H NMR spectra
were performed on a Varian Inova 300 NMR spectrometer or Bruker DRX 500 NMR spectrometer
1
with H being observed at 300 MHz or 500 MHz, respectively. 13C NMR spectra were taken on a
Varian Inova 300 NMR spectrometer with 13C being observed at 75 MHz. Due to the poor solubility of
some obtained final compounds a standard characterization by 13C NMR was omitted. Chemical shifts
1
for H and 13C NMR spectra were reported in δ (ppm) using tetramethylsilane as internal standard.
The abbreviations for multiplicity of signals were as follows: s (singlet), d (doublet), t (triplet), m
(multiplet), dd (doublet of doublets), dt (doublet of triplet), q (quartet). Coupling constants (J) were
expressed in Hertz. Mass spectra (Atmospheric Pressure Ionization Electrospray, API-ES) were
obtained on Agilent 6130 LC/MSD spectrometer.
4.1.1. 1-Amino-4-(methoxycarbonyl)pyridin-1-ium 2,4,6-trimethylbenzene-1-sulfonate (3)
Amino-2,4,6-trimethylbenzene-1-sulfonate [20] (5,46 g, 25,4 mmol, 1 eq.) was dissolved in dry DCM
(30 mL) and cooled under argon atmosphere to 0oC, then methyl isonicotinate (3,83 g, 27,9 mmol, 1,1
eq.) was added dropwise within 1 min. The whole was slowly (without removal of the cooling bath)
warmed to RT (about 3 h) and further stirring at RT under inert gas was continued overnight (18h).
The solvent was evaporated, Et2O (100 mL) was added to the solid residue and the whole was
refluxed for 30 min, then cooled to 0oC, filtered, washed with Et2O and dried under vacuum. 6,43 g of
3 were obtained as a solid (yield 72%). API-ES: Calculated for C7H9N2O2 (pyridinium cation) [M+H]-
m/z 154.07, found 154.1; calculated for C9H11O3S (2,4,6-trimethylbenzenesulfonate anion) [M-H]- m/z
198.03, found 198.1.
4.1.2. {2-Phenyl-[1,2,4]triazolo[1,5-a]pyridin-7-yl}methanol (5)
A solution of methyl 2-phenyl-[1,2,4]triazolo[1,5-a]pyridine-7-carboxylate 4 [21] (2,71 g, 10,7 mmol, 1
eq.) in dry THF (70 mL) was cooled under argon atmosphere to -40oC, then LiAlH4 (0,83 g, 2 eq.) was
added in small portions during 10 min. After addition of LiAlH4 the whole was stirred at -40oC for 30
min, then the reaction mixture was slowly warmed initially to 0oC (within 1 h) and stirred at this
temperature for 30 min. Next, the whole was warmed to 10oC and stirred for further 2h. Water (15 mL)
was cautiously added to destroy the excess of LiAlH4, then AcOEt (50 ml) was added and the whole
was filtered through celite. Water phase was extracted with AcOEt (3 x 50 mL), combined extracts
were dried with Na2SO4 and concentrated. The obtained crude product (2,07 g) was purified by flash
chromatography (eluent CHCl3/MeOH 0-2%). 1,01 g of pure 5 were obtained as a solid (yield 42%).
1H NMR (300 MHz, DMSO-d6): δ 8.91 (d, J = 7.0 Hz, 1H), 8.20 (dd, J = 7.4, 1.9 Hz, 2H), 7.71 (s, 1H),
7.60 – 7.35 (m, 3H), 7.12 (dd, J = 7.0, 1.3 Hz, 1H), 5.62 (t, J = 5.8 Hz, 1H), 4.65 (d, J = 5.7 Hz, 2H).
4.1.3. 2-Phenyl-[1,2,4]triazolo[1,5-a]pyridine-7-carbaldehyde (6)
To a cooled (-78oC) solution of oxalyl chloride (2,01 g, 15,5 mmol, 3,5 eq.) in dry DCM (45 mL),
DMSO (4,86 g, 62,2 mmol, 14 eq.) was added under argon atmosphere. The mixture was vigorously
stirred for 5 min. at -78oC, then suspension of 5 (1,0 g, 4,44 mmol, 1 eq.) in DCM (35 mL) was added
and the whole was stirred for 15 min. TEA (3,23 g, 32 mmol, 7,2 eq.) was added and the reaction
mixture was slowly (1h) warmed to RT, then stirred at RT for further 1h. Water (80 mL) and DCM (50
mL) were added and water phase was extracted with DCM (2 x 30 mL). Combined organic extracts
were washed successively with water (1 x 50 mL), 1% aq. H2SO4 (1 x 50 mL), water (1 x 50 mL),
saturated aq. Na2CO3 (1 x 50 mL), water ( 1 x 50 mL), brine (1 x 50 mL), dried with Na2SO4 and
evaporated. The obtained crude product (1,02 g) was purified by flash chromatography (eluent
CHCl3/MeOH 0-1%). 0,86 g of 6 were obtained as a solid (yield 87%). 1H NMR (300 MHz, DMSO-d6):
δ 10.15 (s, 1H), 9.25 – 8.97 (m, 1H), 8.56 (s, 1H), 8.39 – 8.03 (m, 2H), 7.76 – 7.17 (m, 4H).
4.1.4. 7-{5,8-Dimethyl-[1,2,4]triazolo[1,5-a]pyrazin-2-yl}-2-phenyl-[1,2,4]triazolo[1,5-a]pyridine (7)
To a solution of 6 (0,31 g, 1,41 mmol 1,3 eq.) in DMF (40 mL) 1-amino-2-imino-3,6-dimethyl-2,3-
dihydro-1-pyrazinium diphenylphosphinate [23] (0,38 g, 1,09 mmol, 1 eq.) were added and the whole
was stirred under inert gas at 80oC for 4h. 0,27 g (1,41 mmol, 1,3 eq.) of Na2S2O5 were added and
the reaction mixture was heated under air atmosphere at 100oC overnight (18h). The solvent was
evaporated and the residue was purified by flash chromatography (eluent heptane/AcOEt 0-100%).
86 mg of 7 were obtained as a solid (yield 23%). 1H NMR (300 MHz, CDCl3): δ 8.68 (ddd, J = 8.0, 4.4,

ACCEPTED MANUSCRIPT
0.9 Hz, 2H), 8.36 – 8.22 (m, 2H), 7.98 (dd, J = 7.1, 1.7 Hz, 1H), 7.89 (d, J = 0.9 Hz, 1H), 7.57 – 7.37
(m, 3H), 2.96 (s, 3H), 2.78 (s, 3H). 13C NMR (75 MHz, CDCl3): δ 165.0, 161.2, 151.7, 149.9, 146.8,
132.0, 130.5, 130.2, 130.1, 129.9, 128.6, 128.1, 127.2, 114.6, 112.5, 20.5, 14.3.
4.1.5. 7-{5,7-Dimethyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-yl}-2-phenyl-[1,2,4]triazolo[1,5-a]pyridine (9)
The mixture of 8a [22] (0,16 g, 0,67 mmol,1 eq.) and 1-amino-4,6-dimethyl-1H-pyrimidin-2-ylidene-
ammonium diphenylphosphinate [23] (0,28 g, 0,80 mmol, 1,2 eq.) in POCl3 (7 mL) was heated to
reflux under argon atmosphere for 3h. The reaction mixture was poured on crushed ice with excess of
solid Na2CO3, and CHCl3 (30 mL) was added. The water phase was extracted with CHCl3 (5 x 20 mL),
the extracts were dried (Na2SO4) and concentrated. The crude product was chromatographed on a
preparative plate (PLC Kieselgel 60 F254, 2 mm) eluting with CHCl3/MeOH/TEA (97:2:1). 29 mg of 9
1
were obtained (yield 10%). H NMR (300 MHz, DMSO-d6): δ 9.09 (d, J = 7.1 Hz, 1H), 8.44 (s, 1H),
8.27 – 8.19 (m, 2H), 7.88 (d, J = 7.0 Hz, 1H), 7.63 – 7.50 (m, 3H), 7.20 (s, 1H), 2.81 (s, 3H), 2.60 (s,
3H).
4.1.6. 2-(7-{5,7-Dimethyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-yl}-[1,2,4]triazolo[1,5-a]pyridin-2-
yl)pyrimidine (10)
Synthesized from 8b [22] following the procedure described for 9. Purified on a preparative plate (PLC
1
Kieselgel 60 F254, 2 mm) eluting with CHCl3/MeOH/TEA (97:2:1). Solid, 42 mg (yield 20%). H NMR
(300 MHz, DMSO-d6): δ 9.21 (d, J = 7.1 Hz, 1H), 9.07 (d, J = 4.9 Hz, 2H), 8.58 (s, 1H), 8.01 (dd, J =
7.1, 1.7 Hz, 1H), 7.70 (t, J = 4.9 Hz, 1H), 7.28 (s, 1H), 2.87 (s, 3H), 2.65 (s, 3H).
4.1.7. N-(2-Acetylphenyl)-2-phenyl-[1,2,4]triazolo[1,5-a]pyridine-7-carboxamide (11)
To a solution of 8a [22] (0,30 g, 1,25 mmol, 1 eq.) in DMF (30 mL) DIPEA (0,81 g, 6,27 mmol, 5 eq.)
and HATU (1,19 g, 3,14 mmol, 2,5 eq.) were added and the whole was stirred at RT for 10 min. 1-(2-
Aminophenyl)ethanone (0,34 g, 2,51 mmol, 2 eq.) was added and the reaction mixture was heated to
60oC for 18h. The solvent was evaporated and the residue was purified by flash chromatography
(eluent heptane/AcOEt 0-50%). 0,16 g of 11 were obtained as a solid (yield 36%). 1H NMR (300 MHz,
DMSO-d6): δ 12.40 (s, 1H), 9.18 (d, J = 6.9 Hz, 1H), 8.55 (d, J = 7.5 Hz, 1H), 8.36 (d, J = 0.9 Hz, 1H),
8.25 (dd, J = 7.4, 2.2 Hz, 2H), 8.12 (dd, J = 8.0, 1.4 Hz, 1H), 7.76 – 7.67 (m, 1H), 7.65 – 7.52 (m, 4H),
7.33 (dd, J = 11.0, 4.2 Hz, 1H), 2.71 (s, 3H).
4.1.8. 4-Methyl-2-{2-phenyl-[1,2,4]triazolo[1,5-a]pyridin-7-yl}quinazoline (12)
To a solution of ammonia in MeOH (1,2M, 17 ml) in a pressure tube 0,15 g (0,42 mmol) of 11 were
added and the whole was heated with vigorous stirring to 100oC for 18h. The precipitated crude
product was washed with hot MeOH, filtered and dried under vacuum. 0,11 g of 12 were obtained as
1
a solid (yield 78%). H NMR (300 MHz, CDCl3): δ 8.95 (d, J = 0.7 Hz, 1H), 8.62 (d, J = 6.5 Hz, 1H),
8.25 (ddd, J = 9.5, 7.3, 1.7 Hz, 3H), 8.04 (dd, J = 7.9, 4.4 Hz, 2H), 7.94 – 7.76 (m, 1H), 7.59 (dd, J =
11.2, 4.0 Hz, 1H), 7.53 – 7.32 (m, 3H), 2.98 (s, 3H). 13C NMR (75 MHz, CDCl3): δ 168.8, 164.6, 156.8,
151.7, 149.8, 140.2, 133.9, 130.2, 129.1, 128.6, 127.8, 127.5, 127.1, 124.9, 123.2, 115.4, 113.5, 21.8.
4.1.9. 2-Phenylpyrazolo[1,5-a]pyridine-6-carbaldehyde (16)
A solution of 15 [24] (0,22 g, 1 mmol, 1 eq.) in dry DCM (20 mL) was cooled to -78oC under argon
atmosphere, then DIBALH (1M solution in hexane, 1,50 ml, 1,5 eq.) was added dropwise. The whole
was stirred at -78oC for 2h, then slowly warmed to RT (2h). The reaction was quenched by addition of
saturated aqueous solution of NH4Cl (40 mL). Water phase was extracted with DCM (5 x 30 mL),
extracts were dried with Na2SO4, and concentrated. The crude product was purified by flash
chromatography (eluent heptane/AcOEt 0-30%). 73 mg of 16 were obtained as a solid (yield 33%). 1H
NMR (300 MHz, CDCl3): δ 9.91 (s, 1H), 8.92 (d, J = 1.0 Hz, 1H), 8.04 – 7.93 (m, 2H), 7.60 – 7.54 (m,
2H), 7.54 – 7.38 (m, 3H), 6.91 (d, J = 0.7 Hz, 1H).
4.1.10. 6-{5,7-Dimethyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-yl}-2-phenylpyrazolo[1,5-a]pyridine (17)
Synthesized from 16 (94 mg, 0,41 mmol, 1 eq.) and 1-amino-4,6-dimethyl-1H-pyrimidin-2-ylidene-
ammonium diphenylphosphinate [23] (145 mg, 0,41 mmol, 1 eq.) following the procedure described
for 7. Purified on a preparative plate (PLC Kieselgel 60 F254, 2 mm) eluting with CHCl3/MeOH/TEA
(97:2:1). Solid, 10 mg (yield 7%). 1H NMR (300 MHz, CDCl3): δ 9.44 (s, 1H), 8.10 – 7.90 (m, 3H), 7.63
(d, J = 9.2 Hz, 1H), 7.43 (ddd, J = 20.0, 13.8, 6.2 Hz, 3H), 6.86 (d, J = 7.8 Hz, 2H), 2.84 (s, 3H), 2.67
(s, 3H).

ACCEPTED MANUSCRIPT
4.1.11. 6-{5,8-Dimethyl-[1,2,4]triazolo[1,5-a]pyrazin-2-yl}-2-phenylpyrazolo[1,5-a]pyridine (18)
Synthesized from 16 (0,17 g, 0,77 mmol, 1 eq.) and 1-amino-2-imino-3,6-dimethyl-2,3-dihydro-1-
pyrazinium diphenylphosphinate [23] (0,30 g, 0,84 mmol, 1.1 eq.) following the procedure described
for 7. Purified by flash chromatography (eluent heptane/AcOEt 0-30%). Solid, 69 mg (yield 26%). 1H
NMR (300 MHz, CDCl3): δ 9.50 (s, 1H), 8.08 – 7.91 (m, 3H), 7.85 (s, 1H), 7.60 (d, J = 9.0 Hz, 1H),
7.43 (dt, J = 25.1, 7.2 Hz, 3H), 6.84 (s, 1H), 2.93 (s, 3H), 2.76 (s, 3H).
4.1.12. General procedure I – synthesis of 1H-1,3-benzodiazole esters with PPA (intermediates 20a-
c)
The mixture of ethyl 3,4-diaminobenzoate 19 (1 eq., 27.7 mmol) and appropriate benzoic acid (1
o
eq.,27.7 mmol) and 20 mL of polyphosphoric acid was heated at 140 C for 3 hours. Warm reaction
mixture was poured onto ice covered with excess of solid NaHCO3 and then 60 mL of AcOEt were
added. Aqueous phase was extracted with AcOEt (4 x 70 mL). Extracts were dried over Na2SO4 and
concentrated. The crude product was purified by chromatography on silica gel (eluent heptane/AcOEt,
0-60% or CHCl3/MeOH 0-5%).
4.1.12.1. Ethyl 2-phenyl-1H-1,3-benzodiazole-6-carboxylate (20a)
Synthesized from 19 (5 g, 27,2 mmol, 1 eq.) and benzoic acid (3,32 g, 27,2 mmol, 1 eq.). Solid, 4,5 g
1
(yield 62%). H NMR (500 MHz, CDCl3): δ 8.37 (s, 1H), 8.12 (dd, J = 7.2, 2.0 Hz, 2H), 7.98 (dd, J =
8.5, 1.3 Hz, 1H), 7.63 (d, J = 8.4 Hz, 1H), 7.52 – 7.37 (m, 3H), 4.40 (q, J = 7.1 Hz, 2H), 1.40 (t, J = 7.1
Hz, 3H). API-ES: m/z 267.1 [M+H]+, 555.1 [2M+Na]+.
4.1.12.2. Ethyl 2-(pyridin-2-yl)-1H-1,3-benzodiazole-6-carboxylate (20b)
Synthesized from 19 (3 g, 16,3 mmol, 1 eq.) and picolinic acid (2,41 g, 19,6 mmol, 1,1 eq.). Solid, 2,0
g (yield 46%). API-ES: m/z 290.1 [M+Na]+, 266.1 [M-H]-.
4.1.12.3. Ethyl 2-(pyridin-4-yl)-1H-1,3-benzodiazole-6-carboxylate (20c)
Synthesized from 19 (3 g, 16,3 mmol, 1 eq.) and isonicotinic acid (2,43 g, 19,6 mmol, 1,1 eq.). Solid,
2,22 g (yield 50%). API-ES: m/z 290.1 [M+Na]+, 266.1 [M-H]-.
4.1.13. General procedure II – synthesis of 1H-1,3-benzodiazole esters with sodium metabisulfite
(intermediates 20d-g)
The mixture of ethyl 3,4-diaminobenzoate 19 (1 eq., 13.6 mmol), appropriate carboxyaldehyde (2,2
eq., 29.9 mmol) and Na2S2O5 (2,2 eq., 29.9 mmol) in DMF (50 mL) was stirred at RT under argon
atmosphere for 20h, then the solvent was evaporated. Water (100 mL) and AcOEt (50 mL) were
added and the phases were separated. Water phase was extracted with AcOEt (5 x 30 mL). Organic
extracts were dried (Na2SO4), concentrated and chromatographed on silica gel (eluent
heptane/AcOEt, 0-50% or CHCl3/MeOH, 0-5%).
4.1.13.1. Ethyl 2-(2-fluorophenyl)-1H-1,3-benzodiazole-6-carboxylate (20d)
Synthesized from 19 (1,06 g, 5,76 mmol, 1 eq.) and 2-fluorobenzaldehyde (1,61 g, 12,7 mmol, 2,2
1
eq.). Solid, 0,62 g (yield 38%). H NMR (300 MHz, DMSO-d6): δ 12.91 (s, 1H), 8.27 (t, J = 6.8 Hz,
2H), 7.88 (d, J = 8.4 Hz, 1H), 7.73 (d, J = 7.5 Hz, 1H), 7.62 (dd, J = 14.5, 6.3 Hz, 1H), 7.55 – 7.33 (m,
2H), 4.34 (q, J = 7.1 Hz, 2H), 1.36 (t, J = 7.1 Hz, 3H).
4.1.13.2. Ethyl 2-(3-fluorophenyl)-1H-1,3-benzodiazole-6-carboxylate (20e)
Synthesized from 19 (1,09 g, 5,93 mmol, 1 eq.) and 3-fluorobenzaldehyde (1,62 g, 12,7 mmol, 2,2
eq.). Solid, 0,58 g (yield 35%).1H NMR (300 MHz, DMSO-d6): δ 8.23 (s, 1H), 8.06 (d, J = 8.0 Hz, 1H),
7.99 (dd, J = 9.7, 1.9 Hz, 1H), 7.88 (dd, J = 8.5, 1.5 Hz, 1H), 7.73 (s, 1H), 7.71 – 7.60 (m, 1H), 7.41
(td, J = 8.5, 2.3 Hz, 1H), 4.35 (q, J = 7.1 Hz, 2H), 1.48 – 1.27 (m, 3H). API-ES: m/z 283.1 [M-H]-.
4.1.13.3. Ethyl 2-(4-fluorophenyl)-1H-1,3-benzodiazole-6-carboxylate (20f)

ACCEPTED MANUSCRIPT
Synthesized from 19 (1,08 g, 5,87 mmol, 1 eq.) and 4-fluorobenzaldehyde (1,59 g, 12,7 mmol, 2,2
eq.). Solid, 0,45 g (yield 27%).1H NMR (300 MHz, DMSO-d6): δ 13.29 (d, J = 8.4 Hz, 1H), 8.25 (dd, J
= 8.9, 5.4 Hz, 2H), 8.12 (s, 1H), 7.93 – 7.80 (m, 1H), 7.75 (d, J = 8.7 Hz, 1H), 7.63 (d, J = 8.4 Hz, 1H),
7.45 (t, J = 7.9 Hz, 2H), 4.34 (q, J = 7.0 Hz, 2H), 1.36 (t, J = 7.1 Hz, 3H). API-ES: m/z 283.1 [M-H]-.
4.1.13.4. Ethyl 2-(2-methoxyphenyl)-1H-1,3-benzodiazole-6-carboxylate (20g)
Synthesized from 19 (1,0 g, 5,44 mmol, 1 eq.) and o-anisaldehyde (1,63 g, 12 mmol, 2,2 eq.). Solid,
0,41 g (yield 25%). 1H NMR (300 MHz, CDCl3): δ 8.59 (dt, J = 15.6, 4.7 Hz, 1H), 8.03 – 7.96 (m, 1H),
7.54 – 7.41 (m, 2H), 7.17 (ddd, J = 7.8, 7.4, 1.0 Hz, 1H), 7.09 (d, J = 8.4 Hz, 1H), 4.45 – 4.33 (m, 2H),
4.16 – 4.06 (m, 3H), 1.36 (t, J = 7.1 Hz, 3H).
4.1.14. General procedure III - synthesis of 1H-1,3-benzodiazole esters with PTSA (intermediates
20h-k)
The mixture of ethyl 3,4-diaminobenzoate 19 (1 eq., 13.6 mmol), appropriate carboxyaldehyde (1,5
eq., 20.4 mmol) and p-toluenesulphonic acid (0,2 eq., 2.72 mmol) in toluene (150 mL) was heated at
reflux with Dean-Stark apparatus for 6h. After completion of the reaction (TLC control) the whole was
concentrated and chromatographed on silica gel (heptane/ethyl acetate, gradient 0-60% or
CHCl3/MeOH 0-5%).
4.1.14.1. Ethyl 2-(1,3-thiazol-2-yl)-1H-1,3-benzodiazole-6-carboxylate (20h)
Synthesized from 19 (5,0 g, 27,7 mmol, 1 eq.) and 2-thiazolecarboxaldehyde (3,56 g, 30,5 mmol, 1,1
eq.). Solid, 7,4 g (yield 88%). 1H NMR (300 MHz, CDCl3): δ 8.88 (d, J = 0.9 Hz, 1H), 8.01 (d, J = 3.2
Hz, 1H), 7.91 (d, J = 1.8 Hz, 1H), 7.83 (dd, J = 8.4, 1.9 Hz, 1H), 7.50 (dd, J = 3.2, 1.0 Hz, 1H), 6.75 (d,
J = 8.4 Hz, 1H), 4.75 (s, 2H), 4.34 (q, J = 7.1 Hz, 2H), 1.39 (dd, J = 8.7, 5.6 Hz, 3H). API-ES: m/z
272.1 [M-H]-.
4.1.14.2. Ethyl 2-(1-methyl-1H-imidazol-2-yl)-1H-1,3-benzodiazole-6-carboxylate (20i)
Synthesized from 19 (2,52 g, 13,7 mmol, 1 eq.) and 1-methyl-1H-imidazole-2-carboxaldehyde (2,31 g,
20,6 mmol, 1,5 eq.). Solid, 0,33 g (yield 9%). 1H NMR (300 MHz, DMSO-d6): δ 13.35 (s, 1H), 8.16 (s,
1H), 7.87 (d, J = 8.6 Hz, 1H), 7.81 – 7.65 (m, 1H), 7.49 (s, 1H), 7.23 – 7.17 (m, 1H), 4.34 (q, J = 7.1
Hz, 2H), 4.20 (s, 3H), 1.36 (t, J = 7.1 Hz, 3H). API-ES: m/z 269.1 [M-H]-.
4.1.14.3. Ethyl 2-(1,3-thiazol-4-yl)-1H-1,3-benzodiazole-6-carboxylate (20j)
Synthesized from 19 (3,37 g, 18,7 mmol, 1 eq.) and thiazole-4-carboxaldehyde (2,16 g, 18,7 mmol, 1
eq.). Solid, 1,72 g (yield 32%). 1H NMR (300 MHz, CDCl3): δ 8.96 (d, J = 2.0 Hz, 1H), 8.42 (d, J = 2.0
Hz, 1H), 8.02 (dd, J = 8.5, 1.6 Hz, 1H), 7.67 (s, 1H), 4.43 (q, J = 7.1 Hz, 2H), 1.44 (t, J = 7.1 Hz, 3H).
4.1.14.4. Ethyl 2-(1,3-thiazol-5-yl)-1H-1,3-benzodiazole-6-carboxylate (20k)
Synthesized from 19 (3,37 g, 18,7 mmol, 1 eq.) and thiazole-5-carboxaldehyde (2,2 g, 18,7 mmol, 1
eq.). Solid, 4,0 g (yield 70%). 1H NMR (300 MHz, CDCl3): δ 8.95 (s, 1H), 8.50 (s, 1H), 8.46 – 8.15 (m,
1H), 7.99 (d, J = 8.5 Hz, 1H), 7.63 (d, J = 74.1 Hz, 1H), 4.42 (q, J = 7.1 Hz, 2H), 1.44 (t, J = 7.1 Hz,
3H).
4.1.15. General procedure IV – synthesis of 1H-1,3-benzodiazole esters with POCl3 (compounds, 20l-
20iv)
The mixture of ethyl 3,4-diaminobenzoate 19 (1 eq., 7.34 mmol) and appropriate benzoic acid (1 eq.,
7.34 mmol) was put under argon atmosphere and 15 mL of POCl3 were added. The whole was
heated at reflux for 3 hours. The mixture was cooled to room temperature, poured on ice and
neutralized with 6M NaOH (80 mL), then brought to pH ca. 9 by addition of 20 g of solid NaHCO3. 100
mL of CHCl3 were added and phases were separated. Aqueous phase was extracted with CHCl3 (2 x
50 mL). Combined organic phases were purified by chromatography on silica gel (CHCl3/MeOH,
gradient 0-5%).
4.1.15.1. Ethyl 2-benzyl-1H-1,3-benzodiazole-6-carboxylate (20l)

ACCEPTED MANUSCRIPT
Synthesized from 19 (1,32 g, 7,34 mmol, 1 eq.) and benzeneacetic acid (1,0 g, 7,34 mmol, 1 eq.).
Solid, 1,75 g (yield 85%). 1H NMR (300 MHz, CDCl3) δ 8.21 (dd, J = 1.5, 0.6 Hz, 1H), 7.92 (dd, J =
8.5, 1.6 Hz, 1H), 7.46 (dd, J = 8.5, 0.6 Hz, 1H), 7.37 – 7.28 (m, 1H), 7.25 – 7.13 (m, 5H), 4.35 (q, J =
7.1 Hz, 2H), 4.18 (s, 2H), 1.37 (t, J = 7.1 Hz, 3H). API-ES: m/z 269.1 [M-H]-.
4.1.15.2. Ethyl 2-(quinoxalin-2-yl)-1H-1,3-benzodiazole-6-carboxylate (20m)
Synthesized from 19 (2,54 g, 14,1 mmol, 1 eq.) and 2-quinoxalinecarboxylic acid (2,5 g, 14,1 mmol, 1
eq.). Solid, 3,01 g (yield 67%). API-ES: m/z 317.1 [M-H]-.
4.1.15.3. Ethyl 2-[2-(trifluoromethoxy)phenyl]-1H-1,3-benzodiazole-6-carboxylate (20n)
Synthesized from 19 (1,75 g, 9,7 mmol, 1 eq.) and 2-(trifluoromethoxy)benzoic acid (2,0 g, 9,7 mmol,
1 eq.). Solid, 2,52 g (yield 74%). 1H NMR (300 MHz, CDCl3): δ 8.70 (s, 1H), 8.18 (d, J = 1.3 Hz, 1H),
8.05 – 7.96 (m, 1H), 7.93 (dd, J = 7.7, 1.7 Hz, 1H), 7.62 – 7.51 (m, 2H), 7.49 – 7.40 (m, 1H), 7.32 (dt,
J = 8.3, 7.6 Hz, 1H), 4.39 (q, J = 7.1 Hz, 2H), 1.34 (t, J = 7.1 Hz, 3H). API-ES: m/z 351.1 [M+H]+,
373.1 [M+Na]+, 723.1 [2M+Na]+, 349.1 [M-H]-.
4.1.15.4. Ethyl 2-[2-(trifluoromethyl)phenyl]-1H-1,3-benzodiazole-6-carboxylate (20o)
Synthesized from 19 (2,79 g, 15,5 mmol, 1 eq.) and 2-trifluoromethylbenzoic acid (3,0 g, 15,5 mmol, 1
eq.). Solid, 1,45 g (yield 28%). API-ES: m/z 333.2 [M-H]-.
4.1.15.5. Ethyl 2-(2-cyanophenyl)-1H-1,3-benzodiazole-6-carboxylate (20p)
Synthesized from 19 (6,12 g, 34 mmol, 1 eq.) and o-cyanobenzoic acid (5 g, 34 mmol, 1 eq.). Solid,
0,4 g (yield 4%). 1H NMR (300 MHz, DMSO-d6): δ 13.46 (s, 1H), 8.28 (s, 1H), 8.10 (dd, J = 13.4, 7.6
Hz, 2H), 7.93 (t, J = 7.7 Hz, 2H), 7.75 (t, J = 7.2 Hz, 2H), 4.35 (q, J = 7.1 Hz, 2H), 1.37 (t, J = 7.1 Hz,
3H).
4.1.15.6. Ethyl 2-(2-methylphenyl)-1H-1,3-benzodiazole-6-carboxylate (20q)
Synthesized from 19 (6,62 g, 36,7 mmol, 1 eq.) and 2-methylbenzoic acid (5 g, 36,7 mmol, 1
eq.).Solid, 9,7 g (yield 80%). 1H NMR (300 MHz, CDCl3): δ 8.36 (dd, J = 1.6, 0.7 Hz, 1H), 8.02 (dd, J
= 8.5, 1.6 Hz, 1H), 7.70 (dd, J = 8.5, 0.7 Hz, 1H), 7.68 – 7.63 (m, 1H), 7.44 – 7.29 (m, 3H), 4.41 (qd, J
= 7.2, 2.8 Hz, 2H), 2.58 (s, 3H), 1.40 (t, J = 7.1 Hz, 3H). API-ES: m/z 281.1 [M+H]+, 303.1 [M+Na]+,
583.1 [2M+Na]+, 279.1 [M-H]-, 315.1 [M+Cl]-.
4.1.15.7. Ethyl 2-(furan-2-yl)-1H-1,3-benzodiazole-6-carboxylate (20r)
Synthesized from 19 (4,28 g, 23,3 mmol, 1 eq.) and 2-furancarboxylic acid (3,02 g, 25,6 mmol, 1,1
eq.). Solid, 1,52 g (yield 25%). API-ES: m/z 257.1 [M+H]+, 297.1 [M+Na]+, 535.1 [2M+Na]+, 255.1
[M-H]-.
4.1.15.8. Ethyl 2-(thiophen-2-yl)-1H-1,3-benzodiazole-6-carboxylate (20s)
Synthesized from 19 (2,11 g, 11,7 mmol, 1 eq.) and 2-thiophenecarboxylic acid (1,5 g, 11,7 mmol, 1
eq.). Solid, 0,52 g (yield 16%). 1H NMR (300 MHz, CDCl3): δ 10.26 (s, 1H), 8.26 (s, 1H), 7.92 (d, J =
7.6 Hz, 1H), 7.83 (s, 1H), 7.56 (d, J = 7.8 Hz, 1H), 7.36 (d, J = 4.4 Hz, 1H), 6.98 (s, 1H), 4.37 (q, J =
7.0 Hz, 2H), 1.37 (t, J = 7.1 Hz, 3H). API-ES: m/z 273.1 [M+H]+, 295.1 [M+Na]+, 567.1 [2M+Na]+,
271.1 [M-H]-.
4.1.15.9. Ethyl 2-(1,3-oxazol-4-yl)-1H-1,3-benzodiazole-6-carboxylate (20t)
Synthesized from 19 (2,32 g, 12,8 mmol, 1 eq.) and oxazole-4-carboxylic acid (1,5 g, 13,3 mmol, 1,03
1
eq.). Solid, 0,79 g (yield 24%). H NMR (300 MHz, DMSO-d6): δ 13.31 (s, 1H), 8.94 (d, J = 3.3 Hz,
1H), 8.71 (d, J = 3.5 Hz, 1H), 8.18 (d, J = 30.9 Hz, 1H), 7.86 (t, J = 8.9 Hz, 1H), 7.65 (dd, J = 34.8, 8.5
Hz, 1H), 4.34 (q, J = 7.1 Hz, 2H), 1.35 (t, J = 7.1 Hz, 3H). API-ES: m/z 280.1 [M+Na]+, 537.1
[2M+Na]+, 256.1 [M-H]-.
4.1.15.10. Ethyl 2-(3-bromophenyl)-1H-1,3-benzodiazole-6-carboxylate (20u)

ACCEPTED MANUSCRIPT
Synthesized from 19 (4,93 g, 27,4 mmol, 1 eq.) and 3-bromobenzoic acid (5,5 g, 27,4 mmol, 1 eq.).
Solid, 2,16 g (yield 19%). API-ES: m/z 345.1, 347.1 [M+H]+, 343.1, 345.1 [M-H]-.
4.1.15.11. Ethyl 2-(pyridin-3-yl)-1H-1,3-benzodiazole-6-carboxylate (20v)
Synthesized from 19 (4,37 g, 24,2 mmol, 1 eq.) and niacin (3,0 g, 24,2 mmol, 1 eq.). Solid, 1,01 g
1
(yield 15%). H NMR (300 MHz, CDCl3): δ 9.35 (d, J = 1.7 Hz, 1H), 8.62 (dt, J = 13.0, 6.5 Hz, 1H),
8.54 – 8.41 (m, 1H), 8.27 (s, 1H), 7.91 (dd, J = 8.5, 1.5 Hz, 1H), 7.56 (d, J = 8.2 Hz, 1H), 7.39 (dd, J =
8.0, 4.9 Hz, 1H), 4.37 (q, J = 7.1 Hz, 2H), 1.34 (t, J = 7.1 Hz, 3H). API-ES: m/z 268.1 [M+H]+.
4.1.15.12. Ethyl 2-(5-chlorothiophen-2-yl)-1H-1,3-benzodiazole-6-carboxylate (20w)
Synthesized from 19 (3,26 g, 18,1 mmol, 1 eq.) and 5-chloro-2-thiophenecarboxylic acid (3,0 g, 18,1
mmol, 1 eq.). Solid, 0,86 g (yield 13%). API-ES: m/z 307.1 [M+H]+, 305.1 [M-H]-.
4.1.15.13. Ethyl 2-(pyrazin-2-yl)-1H-1,3-benzodiazole-6-carboxylate (20x)
Synthesized from 19 (3,56 g, 19,7 mmol, 1 eq.) and 2-pyrazinecarboxylic acid (2,5 g, 19,7 mmol, 1
eq.). Solid, 1,75 g (yield 32%). API-ES: m/z 269.1 [M+H]+, 267.1 [M-H]-.
4.1.15.14. Ethyl 2-(3-fluoropyridin-2-yl)-1H-1,3-benzodiazole-6-carboxylate (20y)
Synthesized from 19 (3,75 g, 20,8 mmol, 1 eq.) and 3-fluoropicolinic acid (3,0 g, 20,8 mmol, 1 eq.).
Solid, 1,30 g (yield 18%). API-ES: m/z 284.1 [M-H]-, 593.1 [2M+Na]+.
4.1.15.15. Ethyl 2-(3-methoxypyridin-2-yl)-1H-1,3-benzodiazole-6-carboxylate (20z)
Synthesized from 19 (1,21 g, 6,58 mmol, 1,05 eq.) and 3-methoxy-2-pyridinecarboxylic acid (1,0 g,
6,27 mmol, 1 eq.). Solid, 1,30 g (yield 18%). API-ES: m/z 298.1 [M+H]+, 320.1 [M+Na]+, 617.1
[2M+Na]+, 296.1 [M-H]-.
4.1.15.16. Ethyl 2-(4-methoxypyridin-2-yl)-1H-1,3-benzodiazole-6-carboxylate (20ii)
Synthesized from 19 (3,46 g, 19,2 mmol, 1 eq.) and 4-methoxypicolinic acid (3,0 g, 19,2 mmol, 1 eq.).
Solid, 1,82 g (yield 31%). 1H NMR (300 MHz, CDCl3): δ 10.18 (s, 1H), 8.43 – 8.39 (m, 1H), 8.32 –
8.28 (m, 1H), 8.05 – 7.98 (m, 1H), 7.87 (d, J = 2.4 Hz, 1H), 7.82 (t, J = 3.3 Hz, 1H), 6.99 (dd, J = 5.7,
2.6 Hz, 1H), 4.33 (q, J = 7.1 Hz, 2H), 3.95 (s, 3H), 1.36 (t, J = 7.1 Hz, 3H). API-ES: m/z 320.1
[M+Na]+, 332.0 [M+Cl]-.
4.1.15.17. Ethyl 2-(4-methyl-1,3-thiazol-2-yl)-1H-1,3-benzodiazole-6-carboxylate (20iii)
Synthesized from 19 (3,63 g, 20,1 mmol, 1 eq.) and 4-methyl-1,3-thiazole-2-carboxylic acid (3,0 g,
20,1 mmol, 1 eq.). Solid, 3,63 g (yield 62%). 1H NMR (300 MHz, CDCl3): δ 10.14 (s, 1H), 8.74 – 8.67
(m, 1H), 8.31 (d, J = 1.8 Hz, 1H), 8.09 – 8.02 (m, 1H), 7.28 – 7.26 (m, 1H), 7.20 (t, J = 0.8 Hz, 1H),
4.34 (q, J = 7.1 Hz, 2H), 2.64 (s, 3H), 1.37 (t, J = 7.1 Hz, 3H). API-ES: m/z 286.0 [M-Hl]-.
4.1.15.18. Ethyl 2-(5-methyl-1,3-thiazol-2-yl)-1H-1,3-benzodiazole-6-carboxylate (20iv)
Synthesized from 19 (1,26 g, 6,98 mmol, 1 eq.) and 5-methyl-1,3-thiazole-2-carboxylic acid (1,0 g,
6,98 mmol, 1 eq.). Solid, 1,33 g (yield 66%). API-ES: m/z 310.1 [M+Na]+, 286.1 [M-H]-.
4.1.16. General procedure V – synthesis of 1H-1,3-benzodiazole carboxylic acids (21a-iv)
To the solution of 1H-1,3-benzodiazole ester 20a-20iv (1 eq., 5.63 mmol) in MeOH (20 mL) 1.35 g
(33,75 mmol, 6 eq.) of NaOH dissolved in 15 mL of water were added. The whole was heated to 60
oC for 6 hours. The reaction mixture was concentrated to about 30 percent of initial volume, cooled to
o
0 C and 6M hydrochloric acid was added adjusting the pH to 3. Precipitated abundant solid was
filtered, washed with Et2O and dried under vacuum. The products were used in the next step without
further purification.
4.1.16.1. 2-Phenyl-1H-1,3-benzodiazole-6-carboxylic acid hydrochloride (21a)
Synthesized from 20a (1,5 g, 5,63 mmol). Solid, 1,14 g (yield 74%). API-ES: m/z 237.1 [M-H]-.

ACCEPTED MANUSCRIPT
4.1.16.2. 2-(Pyridin-2-yl)-1H-1,3-benzodiazole-6-carboxylic acid hydrochloride (21b)
Synthesized from 20b (2 g, 7,48 mmol). Solid, 1,50 g (yield 73%). API-ES: m/z 238.1 [M-H]-.
4.1.16.3. 2-(Pyridin-4-yl)-1H-1,3-benzodiazole-6-carboxylic acid hydrochloride (21c)
Synthesized from 20c (2,2 g, 8,31 mmol). Solid, 2,2 g (yield 96%). API-ES: m/z 238.1 [M-H]-.
4.1.16.4. 2-(2-Fluorophenyl)-1H-1,3-benzodiazole-6-carboxylic acid hydrochloride (21d)
Synthesized from 20d (0,62 g, 2,18 mmol). Solid, 0,58 g (yield 90%). API-ES: m/z 255.1 [M-H]-.
4.1.16.5. 2-(3-Fluorophenyl)-1H-1,3-benzodiazole-6-carboxylic acid hydrochloride (21e)
Synthesized from 20e (0,58 g, 2,04 mmol). Solid, 0,59 g (yield 91%). API-ES: m/z 255.1 [M-H]-.
4.1.16.6. 2-(4-Fluorophenyl)-1H-1,3-benzodiazole-6-carboxylic acid hydrochloride (21f)
Synthesized from 20f (0,45 g, 1,58 mmol). Solid, 0,40 g (yield 88%). API-ES: m/z 255.1 [M-H]-.
4.1.16.7. 2-(2-Methoxyphenyl)-1H-1,3-benzodiazole-6-carboxylic acid hydrochloride (21g)
Synthesized from 20g (0,40 g, 1,36 mmol). Solid, 0,33 g (yield 80%). API-ES: m/z 267.1 [M-H]-.
4.1.16.8. 2-(1,3-Thiazol-2-yl)-1H-1,3-benzodiazole-6-carboxylic acid hydrochloride (21h)
Synthesized from 20h (0,82 g, 3,0 mmol). Solid, 0,39 g (yield 46%). API-ES: m/z 244.1 [M-H]-.
4.1.16.9. 2-(1-Methyl-1H-imidazol-2-yl)-1H-1,3-benzodiazole-6-carboxylic acid hydrochloride (21i)
Synthesized from 20i (1,65 g, 6,1 mmol). Solid, 1,42 g (yield 84%). API-ES: m/z 241.1 [M-H]-.
4.1.16.10. 2-(1,3-Thiazol-4-yl)-1H-1,3-benzodiazole-6-carboxylic acid hydrochloride (21j)
Synthesized from 20j (1,72 g, 5,03 mmol). Solid, 1,54 g (yield 87 %). API-ES: m/z 244.1 [M-H]-.
4.1.16.11. 2-(1,3-Thiazol-5-yl)-1H-1,3-benzodiazole-6-carboxylic acid hydrochloride (21k)
Synthesized from 20k (4,0 g, 11,7 mmol). Solid, 3,27 g (yield 79 %). API-ES: m/z 244.1 [M-H]-.
4.1.16.12. 2-Benzyl-1H-1,3-benzodiazole-6-carboxylic acid (21l)
Synthesized from 20l (1,75 g, 6,24 mmol). Solid, 1,37 g (yield 76 %). API-ES: m/z 251.1 [M-H]-.
4.1.16.13. 2-(Quinoxalin-2-yl)-1H-1,3-benzodiazole-6-carboxylic acid hydrochloride (21m)
Synthesized from 20m (5,0 g, 15,7 mmol). Solid, 1,50 g (yield 29 %). API-ES: m/z 289.1 [M-H]-.
4.1.16.14. 2-[2-(Trifluoromethoxy)phenyl]-1H-1,3-benzodiazole-6-carboxylic acid hydrochloride (21n)
Synthesized from 20n (2,52 g, 5,76 mmol). Solid, 1,23 g (yield 48 %). API-ES: m/z 321.1 [M-H]-.
4.1.16.15. 2-[2-(Trifluoromethyl)phenyl]-1H-1,3-benzodiazole-6-carboxylic acid hydrochloride (21o)
Synthesized from 20o (1,45 g, 4,34 mmol). Solid, 0,81 g (yield 54 %). API-ES: m/z 305.1 [M-H]-.
4.1.16.16. 2-(2-Cyanophenyl)-1H-1,3-benzodiazole-6-carboxylic acid hydrochloride (21p)
Synthesized from 20p (0,40 g, 1,37 mmol). Solid, 0,38 g (yield 94 %). API-ES: m/z 262.1 [M-H]-.
4.1.16.17. 2-(2-Methylphenyl)-1H-1,3-benzodiazole-6-carboxylic acid hydrochloride (21q)
Synthesized from 20q (9,70g, 27,7 mmol). Solid, 7,22 g (yield 72 %). API-ES: m/z 251.1 [M-H]-.
4.1.16.18. 2-(Furan-2-yl)-1H-1,3-benzodiazole-6-carboxylic acid hydrochloride (21r)
Synthesized from 20r (1,52 g, 5,93 mmol). Solid, 1,55 g (yield 99 %). API-ES: m/z 227.1 [M-H]-.

ACCEPTED MANUSCRIPT
4.1.16.19. 2-(Thiophen-2-yl)-1H-1,3-benzodiazole-6-carboxylic acid hydrochloride (21s)
Synthesized from 20s (0,50 g, 1,84 mmol). Solid, 0,40 g (yield 78 %). API-ES: m/z 243.1 [M-H]-.
4.1.16.20. 2-(1,3-Oxazol-4-yl)-1H-1,3-benzodiazole-6-carboxylic acid hydrochloride (21t)
Synthesized from 20t (0,79 g, 3,07 mmol). Solid, 0,59 g (yield 72 %). API-ES: m/z 228.1 [M-H]-.
4.1.16.21. 2-(3-Bromophenyl)-1H-1,3-benzodiazole-6-carboxylic acid hydrochloride (21u)
Synthesized from 20u (2,16 g, 6,26 mmol). Solid, 1,98 g (yield 90 %). API-ES: m/z 315.1, 317.1 [M-
H]-.
4.1.16.22. 2-(Pyridin-3-yl)-1H-1,3-benzodiazole-6-carboxylic acid hydrochloride (21v)
Synthesized from 20v (1,01 g, 3,76 mmol). Solid, 0,90 g (yield 87 %). API-ES: m/z 238.1 [M-H]-.
4.1.16.23. 2-(5-Chlorothiophen-2-yl)-1H-1,3-benzodiazole-6-carboxylic acid hydrochloride (21w)
Synthesized from 20w (0,86 g, 2,25 mmol). Solid, 0,81 g (yield 92 %). API-ES: m/z 277.1 [M-H]-.
4.1.16.24. 2-(Pyrazin-2-yl)-1H-1,3-benzodiazole-6-carboxylic acid hydrochloride (21x)
Synthesized from 20x (5,0 g, 18,6 mmol). Solid, 0,90 g (yield 17 %). API-ES: m/z 239.1 [M-H]-.
4.1.16.25. 2-(3-Fluoropyridin-2-yl)-1H-1,3-benzodiazole-6-carboxylic acid hydrochloride (21y)
Synthesized from 20y (1,30 g, 3,65 mmol). Solid, 1,20 g (yield 90 %). API-ES: m/z 256.1 [M-H]-.
4.1.16.26. 2-(3-Methoxypyridin-2-yl)-1H-1,3-benzodiazole-6-carboxylic acid hydrochloride (21z)
Synthesized from 20z (0,65 g, 2,19 mmol). Solid, 0,31 g (yield 47 %). API-ES: m/z 268.1 [M-H]-.
4.1.16.27. 2-(4-Methoxypyridin-2-yl)-1H-1,3-benzodiazole-6-carboxylic acid hydrochloride (21ii)
Synthesized from 20ii (2,0 g, 6,73 mmol). Solid, 1,01 g (yield 49 %). API-ES: m/z 268.1 [M-H]-.
4.1.16.28. 2-(4-Methyl-1,3-thiazol-2-yl)-1H-1,3-benzodiazole-6-carboxylic acid hydrochloride (21iii)
Synthesized from 20iii (3,60 g, 12,5 mmol). Solid, 1,80 g (yield 48 %). API-ES: m/z 258.1 [M-H]-.
4.1.16.29. 2-(5-Methyl-1,3-thiazol-2-yl)-1H-1,3-benzodiazole-6-carboxylic acid hydrochloride (21iv)
Synthesized from 20iv (1,33 g, 3,70 mmol). Solid, 0,96 g (yield 70 %). API-ES: m/z 258.1 [M-H]-.
4.1.17. 6-{5,7-Dimethyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-yl}-2-phenyl-1H-1,3-benzodiazole (22)
To a solution of 2-phenyl-1H-1,3-benzodiazole-5-carboxylic acid 21a (0.30 g, 1.26 mmol, 1 eq.) in dry
DMF (15 mL) were added successively HOBt (0.17 g, 1.26 mmol, 1 eq.), TEA (0.53 mL, 3.78 mmol, 3
eq.), 1-amino-4,6-dimethyl-1H-pyrimidin-2-ylidene-ammonium diphenylphosphinate ([23], 0.45 g, 1.26
mmol, 1 eq.) and EDCI (0.29 g, 1.51 mmol, 1,2 eq.). The whole was put under argon and stirred at
room temperature for 48 hours. The solvent was evaporated and to the obtained residue 15 mL of ice-
cold AcOH were added and the mixture was heated at 100 °C for 24 hours. The mixture was
concentrated to about ¼ volume, then saturated aqueous NaHCO3 solution (50 mL) and 50 mL of
CHCl3 were added. Aqueous phase was separated and extracted with CHCl3 (6 x 40 mL). Combined
organic phases were dried over Na2SO4 and concentrated. The crude product was purified by
chromatography on silica gel (CHCl3/MeOH, gradient 0-10%). 0.13 g of compound 22 were obtained
1
as a solid (yield 30%). H NMR (500 MHz, DMSO-d6): δ 13.10 (s, 1H), 8.42 (s, 1H), 8.22 (d, J = 7.2
Hz, 2H), 8.13 (d, J = 8.2 Hz, 1H), 7.75 (s, 1H), 7.59 (t, J = 7.4 Hz, 2H), 7.57 – 7.50 (m, 1H), 7.12 (d, J
= 0.6 Hz, 1H), 2.80 (s, 3H), 2.59 (s, 3H). 13C NMR (75 MHz, DMSO-d6): δ 146.6, 130.1, 129.8, 128.9,
126.5, 110.5, 24.4, 16.5. API-ES: m/z 341.1 [M+H]+.
4.1.18. 6-{5,7-Dimethyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-yl}-2-(pyridin-2-yl)-1H-1,3-benzodiazole (23)

ACCEPTED MANUSCRIPT
To a mixture of 2-(pyridin-2-yl)-1H-1,3-benzodiazole-6-carboxylic acid (21b) (0,3 g, 1,25 mmol, 1 eq.)
and 1-amino-4,6-dimethyl-1H-pyrimidin-2-ylidene-ammonium diphenylphosphinate ([23], 0,4 g, 1,14
mmol, 0,9 eq.) POCl3 (7 mL) was added and the whole was refluxed for 3 hours. The reaction mixture
was cooled to RT and cautiously poured onto ice mixed with excess of solid Na2CO3. Aqueous phase
(alkaline) was extracted with chloroform (5 x 50 mL), dried with Na2SO4 and concentrated. The crude
product was chromatographed on silica gel (chloroform/methanol 93:7). 70 mg of 23 was obtained as
1
a solid (yield 18%). H NMR (500 MHz, DMSO-d6): δ 13.36 (d, J = 6.1 Hz, 1H), 8.78 (t, J = 4.4 Hz,
1H), 8.43 (d, J = 0.9 Hz, 1H), 8.38 (dd, J = 7.7, 6.6 Hz, 1H), 8.18 (dd, J = 8.4, 1.5 Hz, 1H), 8.13 (dd, J
= 8.5, 1.6 Hz, 1H), 8.04 (td, J = 7.8, 1.6 Hz, 1H), 7.84 (d, J = 8.5 Hz, 1H), 7.68 (d, J = 8.4 Hz, 1H),
7.57 (tdd, J = 7.3, 2.8, 1.7 Hz, 1H), 7.18 – 7.13 (m, 1H), 2.81 (s, 3H), 2.60 (s, 3H). 13C NMR (75 MHz,
DMSO-d6): δ 164.6, 156.0, 152.7, 149.9, 148.6, 147.1, 146.0, 138.1, 135.6, 126.0, 125.4, 122.1,
121.8 (d, J = 44.2 Hz), 120.0, 112.8, 111.1, 25.0, 17.1.
4.1.19. 6-{5,7-Dimethyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-yl}-2-(pyridin-4-yl)-1H-1,3-benzodiazole (24)
Synthesized from 21c
following the procedure described for 22. Purified on silica gel
1
(chloroform/methanol 0-7%). 90 mg, solid, yield 21%. H NMR (500 MHz, DMSO-d6): δ 13.67 (s, br,
1H), 8.79 (dd, J = 11.3, 6.1 Hz, 3H), 8.31 (s, 1H), 8.18 (s, 1H), 8.16 – 8.08 (m, 2H), 7.18 (s, 1H), 2.82
(s, 3H), 2.61 (s, 3H).
4.1.20.
5-{5,7-Dimethyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-yl}-1-methyl-2-phenyl-1H-1,3-benzodiazole
(25a) and
6-{5,7-Dimethyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-yl}-1-methyl-2-phenyl-1H-1,3-benzodiazole (25b)
To a solution of 6-{5,7-Dimethyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-yl}-2-phenyl-1H-1,3-benzodiazole
(22, 80 mg, 0.23 mmol, 1 eq.) in dry DMF (3.5 mL) K2CO3 (65 mg, 0.47 mmol, 2 eq.) was added. The
whole was put under argon and after 10 minutes MeI (0.03 mL, 0.47 mmol, 2 eq.) was added. The
reaction was carried out for 24 hours at room temperature. To the mixture water (10 mL) and AcOEt
(20 mL) were added. Aqueous phase was separated and extracted with ethyl acetate (4 x 20 mL).
Combined extracts were dried with Na2SO4 and concentrated. The residue was chromatographed on
a preparative plate (PLC Kieselgel 60 F254, 2 mm) eluting with CHCl3/MeOH (95:5). 47 mg of the
compound 25a and 40 mg of the compound 25b were obtained as solids (yields 48 and 56%,
respectively).
1
Compound 25a: H NMR (300 MHz, DMSO-d6): δ 8.51 (d, J = 1.0 Hz, 1H), 8.22 (dd, J = 8.4, 1.5 Hz,
1H), 7.90 (dd, J = 7.7, 1.8 Hz, 2H), 7.79 (d, J = 8.4 Hz, 1H), 7.64 – 7.57 (m, 3H), 7.17 (s, 1H), 3.94 (s,
3H), 2.82 (s, 3H), 2.61 (d, J = 6.8 Hz, 3H).
Compound 25b: 1H NMR (300 MHz, DMSO-d6): δ 8.42 (d, J = 1.0 Hz, 1H), 8.18 (dd, J = 8.4, 1.5 Hz,
1H), 7.89 (dt, J = 4.3, 2.3 Hz, 2H), 7.82 (d, J = 8.4 Hz, 1H), 7.65 – 7.57 (m, 3H), 7.18 (d, J = 0.8 Hz,
1H), 3.99 (s, 3H), 2.82 (s, 3H), 2.61 (s, 3H).
4.1.21. 6-{5,8-Dimethyl-[1,2,4]triazolo[1,5-a]pyrazin-2-yl}-2-phenyl-1H-1,3-benzodiazole (26)
To the solution of 2-phenyl-1H-1,3-benzodiazole-5-carboxylic acid 21a (1,7 g, 6.2 mmol, 1 eq.) in dry
DMF (90 mL) DIPEA (4.0 g, 31 mmol, 5 eq.) and HATU (2.47 g, 6.5 mmol, 1,05 eq.) were added.
After addition of HATU, the whole was put under argon and stirred at room temperature for 5 minutes.
1-Amino-2-imino-3,6-dimethyl-2,3-dihydro-1-pyrazinium diphenylphosphinate ([23], 2.09 g, 6.2 mmol,
1 eq.) was added and stirring was continued for 72h. The solvent was removed under reduced
pressure, and the dark oily residue was chromatographed on silica gel (chloroform/methanol, gradient
1
0-5%). 1.57 g of 26 were obtained as a solid (yield 74%). H NMR (300 MHz, DMSO-d6): δ 13.09 (s,
1H), 8.41 (s, 1H), 8.26 – 8.16 (m, 2H), 8.12 (dd, J = 8.4, 1.4 Hz, 1H), 7.92 (s, 1H), 7.72 (d, J = 8.4 Hz,
1H), 7.56 (dt, J = 8.2, 6.6 Hz, 3H), 2.79 (s, 3H), 2.70 (s, 3H). 13C NMR (75 MHz, DMSO-d6): δ 163.5,
152.8, 148.0, 146.3, 130.5, 129.6, 129.2, 129.0, 128.7, 126.7, 124.2, 121.6, 20.3, 14.0. API-ES: m/z
339.1 [M-H]-, 341.1 [M+H]+, 375.1 [M+Cl]-, 703 [2M+Na]+.
4.1.22. 6-{5,8-Dimethyl-[1,2,4]triazolo[1,5-a]pyrazin-2-yl}-2-(2-fluorophenyl)-1H-1,3-benzodiazole (27)
Synthesized from 21d (0,28 g, 1.09 mmol) following the procedure described for 26. Purified on silica
1
gel (chloroform/methanol 0-5%), solid 0.19g (yield 50%). H NMR (300 MHz, DMSO-d6): δ 12.82 (s,
1H), 8.50 (s, 1H), 8.35 – 8.23 (m, 1H), 8.16 (dd, J = 8.4, 1.5 Hz, 1H), 7.95 (s, 1H), 7.78 (d, J = 8.4 Hz,

ACCEPTED MANUSCRIPT
1H), 7.68 – 7.54 (m, 1H), 7.46 (ddd, J = 15.0, 9.4, 4.8 Hz, 2H), 2.78 (s, 3H), 2.72 (s, 3H). 13C NMR (75
MHz, DMSO-d6): δ 164.1, 161.8, 158.5, 148.6 , 146.9, 132.9 (d, J = 8.7 Hz), 131.0, 130.8, 130.0,
125.8, 125.0, 122.3, 118.4 (d, J = 11.4 Hz), 117.2 (d, J = 21.5 Hz), 20.9, 14.7. API-ES: m/z 357 [M-
H]-, 359.1 [M+H]+, 381.1 [M+Na]+, 739.1 [2M+Na]+.
4.1.23. 6-{5,8-Dimethyl-[1,2,4]triazolo[1,5-a]pyrazin-2-yl}-2-(3-fluorophenyl)-1H-1,3-benzodiazole (28)
Synthesized from 21e (0,29 g, 1,1 mmol) following the procedure described for 26. Purified on silica
gel (chloroform/methanol 0-5%), solid 0,16 g (yield 38%). 1H NMR (300 MHz, DMSO-d6): δ 13.22 (s,
1H), 8.45 (s, 1H), 8.17 (dd, J = 8.4, 1.5 Hz, 1H), 8.06 (d, J = 7.8 Hz, 1H), 7.99 (t, J = 4.1 Hz, 2H), 7.76
(d, J = 8.1 Hz, 1H), 7.65 (td, J = 8.1, 6.2 Hz, 1H), 7.39 (td, J = 8.5, 2.3 Hz, 1H), 2.84 (s, 3H), 2.75 (s,
3H). 13C NMR (75 MHz, DMSO-d6): δ 164.7, 164.0, 161.5, 152.2, 148.7, 146.9, 132.8, 131.9 (d, J =
8.6 Hz), 130.8, 130.1, 125.1, 123.4, 122.5, 117.7, 117.5, 113.8 (d, J = 23.7 Hz), 20.9, 14.6. API-ES:
m/z 357 [M-H]-, 359.1 [M+H]+ ,739 [2M+Na]+.
4.1.24. 6-{5,8-Dimethyl-[1,2,4]triazolo[1,5-a]pyrazin-2-yl}-2-(4-fluorophenyl)-1H-1,3-benzodiazole (29)
Synthesized from 21f (0,23 g, 0.89 mmol) following the procedure described for 26. Purified on silica
gel (chloroform/methanol 0-5%), solid 80 mg (yield 25%). 1H NMR (300 MHz, DMSO-d6): δ 8.46 (dd,
J = 8.9, 5.2 Hz, 3H), 8.29 (dd, J = 8.6, 1.4 Hz, 1H), 7.94 (d, J = 0.9 Hz, 1H), 7.87 (d, J = 8.5 Hz, 1H),
7.52 (t, J = 8.8 Hz, 2H), 2.79 (s, 3H), 2.71 (s, 3H). API-ES: m/z 357 [M-H]-.
4.1.25. 6-{5,8-Dimethyl-[1,2,4]triazolo[1,5-a]pyrazin-2-yl}-2-(pyridin-2-yl)-1H-1,3-benzodiazole (30)
Synthesized from 21b (0,27 g, 1.13 mmol) following the procedure described for 23. Purified on silica
1
gel (chloroform/methanol 0-5%), solid 0,1 g (yield 26%). H NMR (300 MHz, DMSO-d6): δ 13.38 (s,
1H), 8.77 (s, 1H), 8.42 (s, 1H), 8.37 (d, J = 7.8 Hz, 1H), 8.23 – 8.09 (m, 1H), 8.03 (dd, J = 10.7, 4.7
Hz, 1H), 7.96 (s, 1H), 7.83 (d, J = 8.5 Hz, 1H), 7.67 (d, J = 8.4 Hz, 1H), 7.57 (dd, J = 8.6, 3.7 Hz, 1H),
2.81 (s, 3H), 2.72 (s, 3H). 13C NMR (75 MHz, DMSO-d6): δ 164.0, 153.0, 149.5, 148.5, 146.9, 146.2,
138.3, 137.3, 135.8, 130.8, 130.1, 125.6, 124.8, 123.2, 122.2, 118.7, 113.2, 21.0, 14.7. API-ES: m/z
340 [M-H]-, 342 [M+H]+, 364 [M+Na]+, 705 [2M+Na]+.
4.1.26. 6-{5,8-Dimethyl-[1,2,4]triazolo[1,5-a]pyrazin-2-yl}-2-(1,3-thiazol-2-yl)-1H-1,3-benzodiazole (31)
Synthesized from 21h (1,5 g, 5.32 mmol) following the procedure described for 26. Purified on silica
gel (chloroform/methanol 0-5%), solid 0,98 g (yield 53%). 1H NMR (300 MHz, DMSO-d6): δ 13.64 (s,
1H), 8.42 (s, 1H), 8.17 (d, J = 8.6 Hz, 1H), 8.13 (d, J = 3.2 Hz, 1H), 8.03 (d, J = 3.2 Hz, 1H), 7.96 (s,
1H), 7.77 (s, 1H), 2.81 (s, 3H), 2.77 (s, 3H). 13C NMR (75 MHz, DMSO-d6): δ 163.2, 158.3, 148.1,
147.4, 146.3, 144.2, 130.2, 129.4, 125.1, 123.3, 122.3, 20.3, 14.1. API-ES: m/z 346 [M-H]-, 370
[M+Na]+, 717 [2M+Na]+.
4.1.27.
(32)
6-{5,8-Dimethyl-[1,2,4]triazolo[1,5-a]pyrazin-2-yl}-2-(2-methoxyphenyl)-1H-1,3-benzodiazole
Synthesized from 21g (0,33 g, 0.9 mmol) following the procedure described for 23. Purified on silica
1
gel (ethyl acetate/methanol 0-30%), solid 0,11 g (yield 28%). H NMR (300 MHz, DMSO-d6): δ 10.75
(s, 1H), 8.66 – 8.56 (m, 1H), 8.51 (s, 1H), 8.29 (d, J = 8.3 Hz, 1H), 7.83 (s, 1H), 7.51 – 7.38 (m, 1H),
7.27 (s, 1H), 7.17 (t, J = 7.5 Hz, 1H), 7.07 (d, J = 8.3 Hz, 1H), 4.10 (s, 3H), 2.95 (s, 3H), 2.78 (s, 3H).
API-ES: m/z 369 [M-H]-, 371 [M+H]+, 393 [M+Na]+.
4.1.28.
6-{5,8-Dimethyl-[1,2,4]triazolo[1,5-a]pyrazin-2-yl}-2-(1-methyl-1H-imidazol-2-yl)-1H-1,3-
benzodiazole (33)
Synthesized from 21i (0,30 g, 1,24 mmol) following the procedure described for 23. Purified on silica
1
gel (chloroform/methanol 0-2%), solid 0,16 g (yield 39%). H NMR (300 MHz, DMSO-d6): δ 8.63 (s,
1H), 8.31 (d, J = 8.5 Hz, 1H), 7.86 (s, 1H), 7.82 (s, 1H), 7.37 (s, 1H), 7.16 (d, J = 9.1 Hz, 2H), 4.29 (s,
3H), 2.97 (s, 3H), 2.83 (s, 3H). 13C NMR (75 MHz, DMSO-d6): δ 149.1, 138.0, 130.8, 129.3, 128.6,
124.9, 35.8, 20.3, 14.5. API-ES: m/z 343.1 [M-H]-, 367.1 [M+Na]+.
4.1.29. 2-Benzyl-6-{5,8-Dimethyl-[1,2,4]triazolo[1,5-a]pyrazin-2-yl}-1H-1,3-benzodiazole (34)

ACCEPTED MANUSCRIPT
Synthesized from 21l (0,3 g, 1.18 mmol) following the procedure described for 26. Purified on silica
1
gel (chloroform/methanol 0-5%), solid 0,18 g (yield 42%). H NMR (300 MHz, DMSO-d6): δ 8.42 (s,
1H), 8.19 (dd, J = 8.4, 1.6 Hz, 1H), 7.80 (d, J = 1.0 Hz, 1H), 7.64 (s, 1H), 7.34 – 7.30 (m, 3H), 7.25
(tdd, J = 8.5, 3.5, 2.2 Hz, 1H), 4.25 (s, 2H), 2.91 (s, 3H), 2.77 (s, 3H). 13C NMR (75 MHz, DMSO-d6): δ
164.9, 155.6, 148.9, 146.9, 136.5, 130.7 , 129.8, 128.9, 127.3, 124.4, 122.2, 35.8, 20.4, 14.5. API-
ES: m/z 353.1 [M-H]-, 389.1 [M+Cl]-, 355.1 [M+H]+, 731.1 [2M+Na]+.
4.1.30. 2-(6-{5,8-Dimethyl-[1,2,4]triazolo[1,5-a]pyrazin-2-yl}-1H-1,3-benzodiazol-2-yl)quinoxaline (35)
Synthesized from 21m (0,6 g, 2,07 mmol) following the procedure described for 26. Purified on silica
1
gel (chloroform/methanol 0-5%), solid 0,44 g (yield 54%). H NMR (300 MHz, DMSO-d6): δ 9.82 (s,
1H), 8.52 (s, 1H), 8.30 – 8.11 (m, 3H), 7.97 (d, J = 15.3 Hz, 4H), 2.84 (s, 3H), 2.75 (s, 3H). API-ES:
m/z 391.1 [M-H]-, 415.1 [M+Na]+, 807.1 [2M+Na]+.
4.1.31.
6-{5,8-Dimethyl-[1,2,4]triazolo[1,5-a]pyrazin-2-yl}-2-[2-(trifluoromethoxy)phenyl]-1H-1,3-
benzodiazole (36)
Synthesized from 21n (0,39 g, 1,09 mmol) following the procedure described for 26. Purified on silica
1
gel (chloroform/methanol 0-4%), solid 0,24 g (yield 52%). H NMR (300 MHz, DMSO-d6): δ 8.52 (s,
1H), 8.18 (d, J = 6.9 Hz, 2H), 7.95 (s, 1H), 7.77 (d, J = 13.9 Hz, 1H), 7.68 (dt, J = 22.1, 6.5 Hz, 3H),
2.82 (s, 3H), 2.72 (s, 3H). 13C NMR (75 MHz, DMSO-d6): δ 163.3, 148.8, 147.9, 146.2, 145.6, 131.7
(d, J = 19.9 Hz), 130.1, 129.3, 128.1, 124.1, 122.1, 118.3, 20.1, 13.9. API-ES: m/z 423.1 [M-H]-,
425.1 [M+H]+, 447.1 [M+Na]+, 871.1 [2M+Na]+.
4.1.32.
6-{5,8-Dimethyl-[1,2,4]triazolo[1,5-a]pyrazin-2-yl}-2-[2-(trifluoromethyl)phenyl]-1H-1,3-
benzodiazole (37)
Synthesized from 21o (0,29 g, 0,95 mmol) following the procedure described for 26. Purified on silica
1
gel (chloroform/methanol 0-5%), solid 0,22 g (yield 50%). H NMR (300 MHz, DMSO-d6): δ 13.06 (s,
1H), 8.48 (s, 1H), 8.20 (d, J = 8.6 Hz, 1H), 8.05 – 7.94 (m, 2H), 7.94 – 7.68 (m, 4H), 2.84 (s, 3H), 2.75
(s, 3H). 13C NMR (75 MHz, DMSO-d6): δ 164.1, 148.7, 147.0, 133.2, 132.9, 131.2, 130.9, 130.3 (d, J
= 31.7 Hz), 129.9, 129.6, 128.8, 127.3, 126.2, 20.9, 14.7. API-ES: m/z 407.1 [M-H]-, 409.1 [M+H]+,
431.1 [M+Na]+, 839.1 [2M+Na]+.
4.1.33. 2-(6-{5,8-Dimethyl-[1,2,4]triazolo[1,5-a]pyrazin-2-yl}-1H-1,3-benzodiazol-2yl)benzonitrile (38)
Synthesized from 21p (0,37 g, 1,41 mmol) following the procedure described for 26. Purified on silica
gel (chloroform/methanol 0-6%), solid 0,18 g (yield 35%). 1H NMR (300 MHz, DMSO-d6): δ 13.39 (s,
1H), 8.56 (s, 1H), 8.26 (d, J = 8.7 Hz, 1H), 8.20 – 8.07 (m, 2H), 8.05 (d, J = 1.0 Hz, 1H), 7.97 (td, J =
7.7, 1.3 Hz, 1H), 7.92 – 7.81 (m, 1H), 7.77 (td, J = 7.7, 1.2 Hz, 1H), 2.87 (s, 3H), 2.79 (s, 3H). API-
ES: m/z 364.1 [M-H]-, 400.1 [M+Cl]-.
4.1.34.
6-{5,8-Dimethyl-[1,2,4]triazolo[1,5-a]pyrazin-2-yl}-2-(2-methylphenyl)-1H-1,3-benzodiazole
(39)
Synthesized from 21q (0,5 g, 1,98 mmol) following the procedure described for 26. Purified on silica
1
gel (chloroform/methanol 0-6%), solid 0,44 g (yield 62%). H NMR (300 MHz, DMSO-d6): δ 12.87 (s,
1H), 8.46 (d, J = 38.6 Hz, 1H), 8.15 (d, J = 7.2 Hz, 1H), 7.96 (s, 1H), 7.76 (dd, J = 19.5, 17.8 Hz, 2H),
7.51 – 7.29 (m, 3H), 2.81 (s, 3H), 2.72 (s, 3H), 2.63 (s, 3H). 13C NMR (75 MHz, DMSO-d6): δ 163.3,
153.5, 147.8, 146.1, 137.0, 136.1, 131.2, 130.0, 129.4 (dd, J = 17.3, 7.8 Hz), 125.9, 120.7, 20.9, 20.1,
13.8. API-ES: m/z 353.1 [M-H]-, 355.1 [M+H]+.
4.1.35. 6-{5,8-Dimethyl-[1,2,4]triazolo[1,5-a]pyrazin-2-yl}-2-(furan-2-yl)-1H-1,3-benzodiazole (40)
Synthesized from 21r (1,56 g, 6,84 mmol) following the procedure described for 26. Purified on silica
gel (chloroform/methanol 0-5%), solid 0,83 g (yield 37 %). 1H NMR (300 MHz, DMSO-d6): δ 13.16 (d,
J = 12.0 Hz, 1H), 8.41 (d, J = 35.9 Hz, 1H), 8.15 (ddd, J = 10.2, 8.5, 1.5 Hz, 1H), 8.01 (dd, J = 8.3, 4.1
Hz, 2H), 7.70 (dd, J = 36.7, 8.5 Hz, 1H), 7.28 (d, J = 3.4 Hz, 1H), 6.78 (dt, J = 5.1, 2.5 Hz, 1H), 2.83
(s, 3H), 2.74 (s, 3H). 13C NMR (75 MHz, DMSO-d6): δ 163.4 (d, J = 12.8 Hz), 148.0, 146.2, 145.4,

ACCEPTED MANUSCRIPT
145.0 (d, J = 15.0 Hz), 134.5, 130.1, 129.4, 124.4, 124.0, 122.1, 121.3, 119.0, 117.4, 112.5, 111.7,
111.2, 110.1, 20.2, 14.0. API-ES: m/z 329.1 [M-H]-, 365.1 [M+Cl]-, 331.1 [M+H]+, 683.1 [2M+Na]+.
4.1.36. 6-{5,8-Dimethyl-[1,2,4]triazolo[1,5-a]pyrazin-2-yl}-2-(thiophen-2-yl)-1H-1,3-benzodiazole (41)
Synthesized from 21s (0,4 g, 1,64 mmol) following the procedure described for 26. Purified on silica
gel (chloroform/methanol 0-6%), solid 0,28 g (yield 49 %). 1H NMR (300 MHz, DMSO-d6): δ 13.17 (d,
J = 17.3 Hz, 1H), 8.39 (d, J = 33.9 Hz, 1H), 8.13 (t, J = 9.0 Hz, 1H), 7.97 (s, 1H), 7.89 (d, J = 3.1 Hz,
1H), 7.80 (d, J = 4.8 Hz, 1H), 7.69 (dd, J = 31.6, 8.3 Hz, 1H), 7.33 – 7.19 (m, 1H), 2.82 (s, 3H), 2.73
(s, 3H). 13C NMR (75 MHz, DMSO-d6): δ 148.7, 146.9, 133.9, 130.8, 130.1, 129.1, 127.9, 124.7,
122.8, 121.9, 119.5, 117.9, 112.2, 110.5, 20.9, 14.7. API-ES: m/z 345.1 [M-H]-, 381.1 [M+Cl]-, 347.1
[M+H]+, 715.1 [2M+Na]+.
4.1.37.
6-{5,8-Dimethyl-[1,2,4]triazolo[1,5-a]pyrazin-2-yl}-2-(1,3-oxazol-4-yl)-1H-1,3-benzodiazole
(42)
Synthesized from 21t (0,59 g, 2,57 mmol) following the procedure described for 26. Purified on silica
gel (eluent: chloroform), solid 0,20 g (yield 23 %). 1H NMR (300 MHz, DMSO-d6): δ 13.22 (s, 1H), 8.93
(s, 1H), 8.72 (dd, J = 5.4, 1.0 Hz, 1H), 8.45 (d, J = 21.2 Hz, 1H), 8.23 – 8.11 (m, 1H), 8.01 (s, 1H),
7.72 (dd, J = 32.7, 8.6 Hz, 1H), 2.84 (s, 3H), 2.75 (s, 3H). API-ES: m/z 330.1 [M-H]-, 366.1 [M+Cl]-.
4.1.38.
6-{5,8-Dimethyl-[1,2,4]triazolo[1,5-a]pyrazin-2-yl}-2-(1,3-thiazol-4-yl)-1H-1,3-benzodiazole
(43)
Synthesized from 21j (0,50 g, 2,04 mmol) following the procedure described for 26. Purified on silica
1
gel (chloroform/methanol 0-6%), solid 0,46 g (yield 65 %). H NMR (300 MHz, CDCl3): δ 8.88 (d, J =
2.0 Hz, 1H), 8.39 (m, 2H), 8.28 (s, 1H), 8.19 (dd, J = 8.5, 1.6 Hz, 1H), 7.76 (s, 2H), 2.85 (s, 3H), 2.72
(s, 3H). 13C NMR (75 MHz, DMSO-d6): δ 163.4, 155.7, 148.5, 147.9, 146.6, 146.2, 130.1, 129.3,
124.2, 121.6, 120.2, 20.2, 14.0. API-ES: m/z 348.1 [M+H]+, 370.1 [M+Na]+, 382.1 [M+Cl]-.
4.1.39.
6-{5,8-Dimethyl-[1,2,4]triazolo[1,5-a]pyrazin-2-yl}-2-(1,3-thiazol-5-yl)-1H-1,3-benzodiazole
(44)
Synthesized from 21k (0,50 g, 2,04 mmol) following the procedure described for 26. Purified on silica
1
gel (chloroform/methanol 0-6%), solid 0,44 g (yield 63 %). H NMR (300 MHz, DMSO-d6): δ 9.27 (s,
1H), 8.63 (s, 1H), 8.45 (s, 1H), 8.18 (dd, J = 8.3, 1.6 Hz, 1H), 8.03 (s, 1H), 7.75 (d, J = 8.4 Hz, 1H),
2.87 (s, 3H), 2.78 (s, 3H). API-ES: m/z 346.1 [M-H]-, 382.1 [M+Cl]- .
4.1.40.
2-(3-bromophenyl)-6-{5,8-Dimethyl-[1,2,4]triazolo[1,5-a]pyrazin-2-yl}-1H-1,3-benzodiazole
(45)
Synthesized from 21u (2,48 g, 6,26 mmol) following the procedure described for 26. Purified on silica
gel (chloroform/methanol 0-6%), solid 0,91 g (yield 27 %). 1H NMR (300 MHz, DMSO-d6): δ 8.42 (d, J
= 15.6 Hz, 1H), 8.20 (s, 1H), 7.99 (s, 1H), 7.73 (s, 2H), 7.49 (s, 3H), 2.83 (s, 3H), 2.74 (s, 3H). API-
ES: m/z 417.1 [M-H]-, 453.1 [M+Cl]- , 873.1 [2M+Na]+.
4.1.41. 6-{5,8-Dimethyl-[1,2,4]triazolo[1,5-a]pyrazin-2-yl}-2-(pyridin-3-yl)-1H-1,3-benzodiazole (46)
Synthesized from 21v (0,40 g, 1,67 mmol) following the procedure described for 26. Purified on silica
gel (ethyl acetate/methanol 0-30%), solid 0,18 g (yield 31 %). 1H NMR (300 MHz, DMSO-d6): δ 13.23
(s, 1H), 9.30 (d, J = 1.5 Hz, 1H), 8.64 (dd, J = 4.7, 1.4 Hz, 1H), 8.37 (dd, J = 47.5, 12.8 Hz, 2H), 8.09
(d, J = 8.1 Hz, 1H), 7.90 (s, 1H), 7.70 (d, J = 25.7 Hz, 1H), 7.55 (dd, J = 7.8, 4.8 Hz, 1H), 2.76 (s, 3H),
2.66 (s, 3H). 13C NMR (75 MHz, DMSO-d6): δ 150.9, 148.1, 147.7, 146.3, 134.0, 130.2, 129.5, 125.9,
124.1, 20.3, 14.1. API-ES: m/z 340.1 [M-H]-, 370.1 [M+Cl]-.
4.1.42.
2-(5-Chlorothiophen-2-yl)-6-{5,8-dimethyl-[1,2,4]triazolo[1,5-a]pyrazin-2-yl}-1H-1,3-
benzodiazole (47)
Synthesized from 21w (0,40 g, 1,15 mmol) following the procedure described for 26. Purified on silica
1
gel (chloroform/methanol 0-6%), solid 0,18 g (yield 42 %). H NMR (300 MHz, DMSO-d6): δ 8.28 (s,
1H), 8.05 (dd, J = 8.4, 1.4 Hz, 1H), 7.90 (s, 1H), 7.73 – 7.53 (m, 2H), 7.21 (d, J = 4.0 Hz, 1H), 2.75 (s,

ACCEPTED MANUSCRIPT
3H), 2.66 (s, 3H). 13C NMR (75 MHz, DMSO-d6): δ 163.4, 148.1, 147.6, 146.3, 132.5, 131.1, 130.2,
129.5, 128.4, 126.8, 124.5, 121.9, 20.3, 14.1. API-ES: m/z 379.1 [M-H]-, 415.1 [M+Cl]-.
4.1.43. 6-{5,8-Dimethyl-[1,2,4]triazolo[1,5-a]pyrazin-2-yl}-2-(pyrazin-2-yl)-1H-1,3-benzodiazole (48)
Synthesized from 21y (0,90 g, 3,00 mmol) following the procedure described for 26. Purified on silica
gel (chloroform/methanol 0-6%), solid 0,42 g (yield 40 %). 1H NMR (300 MHz, DMSO-d6): δ 13.56 (s,
1H), 9.52 (s, 1H), 8.82 (d, J = 9.0 Hz, 2H), 8.49 (d, J = 38.9 Hz, 1H), 8.18 (d, J = 8.8 Hz, 1H), 7.97 (s,
1H), 7.93 – 7.64 (m, 1H), 2.83 (s, 3H), 2.75 (s, 3H). API-ES: m/z 341.1 [M-H]-, 377.1 [M+Cl]-, 707.1
[2M+Na]+.
4.1.44. 6-{5,8-Dimethyl-[1,2,4]triazolo[1,5-a]pyrazin-2-yl}-2-(3-fluoropyridin-2-yl)-1H-1,3-benzodiazole
(49)
Synthesized from 21x (0,80 g, 2,33 mmol) following the procedure described for 26. Purified on silica
gel (chloroform/methanol 0-6%), solid 0,41 g (yield 49 %). 1H NMR (300 MHz, DMSO-d6): δ 13.38 (s,
1H), 8.65 (s, 1H), 8.52 (d, J = 35.4 Hz, 1H), 8.19 (dd, J = 16.4, 8.2 Hz, 1H), 7.99 (t, J = 9.7 Hz, 2H),
7.89 (d, J = 8.5 Hz, 1H), 7.71 (d, J = 8.9 Hz, 1H), 2.83 (s, 3H), 2.74 (s, 3H). API-ES: m/z 358.1 [M-H]-,
394.1 [M+Cl]-, 382.1 [M+Na]+, 741.1 [2M+Na]+.
4.1.45.
6-{5,8-Dimethyl-[1,2,4]triazolo[1,5-a]pyrazin-2-yl}-2-(3-methoxypyridin-2-yl)-1H-1,3-
benzodiazole (50)
Synthesized from 21z (0,31 g, 1,15 mmol) following the procedure described for 26. Purified on silica
gel (chloroform/methanol 0-5%), solid 70 mg (yield 16 %). 1H NMR (300 MHz, DMSO-d6): δ 13.22 (s,
br, 1H), 8.53 (s, 1H), 8.38 (d, J = 4.2 Hz, 1H), 8.17 (d, J = 8.5 Hz, 1H), 8.00 (s, 1H), 7.75 (d, J = 8.3
Hz, 2H), 7.57 (dd, J = 8.4, 4.4 Hz, 1H), 4.01 (s, 3H), 2.84 (s, 3H), 2.75 (s, 3H). 13C NMR (75 MHz,
DMSO-d6): δ 155.3, 151.4, 148.7, 147.1, 141.7, 137.5, 130.9, 130.1, 126.5, 121.5, 56.7, 21.0, 14.7.
API-ES: m/z 372.1 [M+H]+, 394.1 [M+Na]+, 765.1 [2M+Na]+.
4.1.46.
6-{5,8-Dimethyl-[1,2,4]triazolo[1,5-a]pyrazin-2-yl}-2-(4-methoxypyridin-2-yl)-1H-1,3-
benzodiazole (51)
Synthesized from 21ii (0,40 g, 1,49 mmol) following the procedure described for 26. Purified on silica
gel (chloroform/methanol 0-3%), solid 0,11 g (yield 19 %). 1H NMR (300 MHz, DMSO-d6): δ 13.37 (s,
1H), 8.58 (d, J = 5.7 Hz, 1H), 8.50 (d, J = 31.9 Hz, 1H), 8.25 – 8.12 (m, 1H), 8.00 (s, 1H), 7.88 (d, J =
2.3 Hz, 1H), 7.77 (dd, J = 48.4, 8.3 Hz, 1H), 7.14 (dd, J = 5.7, 2.6 Hz, 1H), 3.98 (s, 3H), 2.84 (s, 3H),
2.75 (s, 3H). API-ES: m/z 372.1 [M+H]+, 394.1 [M+Na]+, 765.1 [2M+Na]+.
4.1.47.
6-{5,8-Dimethyl-[1,2,4]triazolo[1,5-a]pyrazin-2-yl}-2-(4-methyl-1,3-thiazol-2-yl)-1H-1,3-
benzodiazole (52)
Synthesized from 21iii (1,0 g, 2,78 mmol) following the procedure described for 26. Purified on silica
gel (chloroform/methanol 0-6%), solid 0,66 g (yield 65 %). 1H NMR (300 MHz, DMSO-d6): δ 13.62 (s,
1H), 8.41 (d, J = 39.1 Hz, 1H), 8.17 (dd, J = 16.5, 8.6 Hz, 1H), 7.96 (s, 1H), 7.71 (dd, J = 49.9, 8.5 Hz,
1H), 7.56 (s, 1H), 2.81 (s, 3H), 2.72 (s, 3H), 2.52 (s, 3H). API-ES: m/z 360.1 [M-H]-, 745.1 [2M+Na]+.
4.1.48.
6-{5,8-Dimethyl-[1,2,4]triazolo[1,5-a]pyrazin-2-yl}-2-(5-methyl-1,3-thiazol-2-yl)-1H-1,3-
benzodiazole (53)
Synthesized from 21iv (0,50 g, 1,74 mmol) following the procedure described for 26. Purified on silica
gel (chloroform/methanol 0-5%), solid 0,29 g (yield 46 %). 1H NMR (300 MHz, DMSO-d6): δ 8.44 (d, J
= 36.9 Hz, 1H), 8.16 (d, J = 9.0 Hz, 1H), 7.99 (s, 1H), 7.82 (s, 1H), 7.67 (m, 1H), 2.84 (s, 3H), 2.75 (s,
3H), 2.58 (s, 3H). 13C NMR (75 MHz, DMSO-d6): δ 163.2, 156.3, 148.1, 147.5, 145.0, 142.2, 137.3,
130.2, 129.4, 121.6, 119.5, 110.7, 20.2, 14.0, 11.7. API-ES: m/z 360.1 [M-H]-, 384.1 [M+Na]+, 745.1
[2M+Na]+.
4.1.49. N-(2-acetylphenyl)-3,4-dinitrobenzamide (55)
To a solution of 3,4-dinitrobenzoic acid (54, 1,92 g, 8.88 mmol, 1,2 eq.) in dry DCM (50 ml) 0,5 ml of
dry DMF were added at RT and oxalyl chloride (1,15 mL, 13,3 mmol, 1,8 eq.) was slowly added