New synthesis of N1- And N2-substituted pyrazolo[4,3-b]pyridine-5-one derivatives as CB2 receptor ligands

Article abstract of DOI:10.1039/d0nj03400b

The derivatives of the 7-hydroxy-5-oxopyrazolo[4,3-b]pyridine-6-carboxamide scaffold have been reported recently as potent and selective agonists/inverse agonists of the cannabinoid type-2 receptor (CB2R), but the synthetic way adopted has not yet allowed the structure-activity relationship to be fully explored. Herein, we describe a novel synthetic approach based on the use of a 2-tetrahydropyranyl (THP)-protected precursor that opens the way to obtain either N1- or N2-substituted analogs endowed with agonist or inverse agonist activity, respectively, at the CB2 receptor.

Full text of DOI:10.1039/d0nj03400b

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New synthesis of N1- and N2-substituted pyrazolo[4,3-b]pyridine-
5-one derivatives as CB2 receptor ligands
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Claudia Mugnaini,a Antonella Brizzi,a Marco Paolino,a and Federico Corelli*,a
Received 00th January 20xx,
Accepted 00th January 20xx
Derivatives of 7-hydroxy-5-oxopyrazole[4,3-b]pyridine-6-carboxamide scaffold have been reported recently as potent and
selective agonists/inverse agonists of the cannabinoid type-2 receptor (CB2R), but the synthetic way adopted has not yet
allowed to explore fully the structure-activity relationship. Herein, we describe a novel synthetic approach, based on the
use of a 2-tetrahydropyranyl(THP)-protected precursor, that has open the way to obtain either N1- or N2-substituted
analogs endowed with agonist or inverse agonist activity, respectively, at CB2 receptor.
DOI: 10.1039/x0xx00000x
COR1022 and COR1023 exhibited a receptors selectivity index
(SI = KiCB1R/KiCB2R) of 167 and 62, respectively.[10]
Introduction
The main receptors of the endocannabinoid system that have
been identified so far are type-1 cannabinoid receptor (CB1R)
and type-2 cannabinoid receptor (CB2R).[1,2] During the last
decades, several pharmaceutical companies and academic
research laboratories have shown a particular interest in the
development of new selective agonists or inverse agonists of
cannabinoid receptors as potential therapeutic agents for
different conditions. In particular, CB2R agonists have been
proposed as therapy for diverse pain conditions, including
acute pain, chronic inflammation, and neuropathic pain, which
does not cause overt psychotropic effects as CB1R activation
does.[3,4] Furthermore, CB2R agonists could be helpful for the
treatment of neurodegenerative and neuroinflammatory
diseases, such as amyotrophic lateral sclerosis, Huntington’s
chorea and cerebellar ataxia.[5] Recently, several compounds
with excellent in vitro activities have been described, some of
which have passed the preclinical evaluation and have entered
clinical trials.[6,7] On the other hand, CB2R-specific inverse
agonists have been shown to modulate the migration of
immune cells in vivo and promise to lead to the identification
of immunomodulatory agents.[8]
Figure 1. General structure of 4-hydroxy-2-quinolone-3-
carboxamide derivatives 1 and 7-hydroxy-5-oxopyrazolo[4,3-
b]pyridine-6-carboxamide derivatives 2 along with structure of
representative compounds 2a and 2b.
Our research group has been working for several years on the
design and synthesis of new quinolone derivatives, with the
goal of identifying increasingly potent and selective CB2
ligands (e.g. compounds 1) endowed with improved
physicochemical properties.[9] As a recent development of this
research, it is worth mentioning the isosteric replacement-
based approach of the benzene ring of 1 with the pyrazole
nucleus, generating a new class of compounds, namely the 7-
hydroxy-5-oxopyrazolo[4,3-b]pyridine-6-carboxamide
derivatives (2), whose structure is shown in Figure 1. As far as
structure-activity relationships are concerned, the R1
substituent on the pyrazole ring is decisive for the functional
activity of the compounds, as the derivatives substituted at N1
activate the CB2 receptor, thereby acting as agonists, while the
isomers substituted at N2 show a different profile, being
inverse agonists or antagonists at the same receptor.[10]
Among the various synthesized molecules, compounds 2a and
2b (also referred to as COR1023 and COR1022, respectively)
(Figure 1) stand out as new cannabinoid ligands, displaying in
in vitro studies high binding affinity for the CB2R, with Ki values
of 0.18 ± 0.01 nM and 0.9 ± 0.1 nM, respectively. In addition,
Compounds 2 had been prepared through a multistep
synthesis,[10] which involved the early introduction of R1 on
the pyrazole ring and offered the possibility to mainly
investigate the influence of the R3 substituent, i.e. the amide
portion of the molecules, on biological activity. However, this
synthetic approach does not seem to be flexible enough if we
want to study the pharmacophoric role of the other
substituent R1, as the alkylation of the pyrazole nitrogen at an
early stage gives predominantly the N2-substituted isomer
and, not less importantly, chemical diversity on the pyrazole
moiety can only be obtained by starting a new synthesis every
time.
Based on these observations, the aim of this work was to
develop a more versatile synthesis of 7-hydroxy-5-oxopyrazole
[4,3-b]pyridine-6-carboxamide derivatives (2) that would allow
to explore the chemical space around the pyrazole nucleus by
varying the substituent R1, keeping the central core and the
amide functionality unchanged. Consequently, this work
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mail: federico.corelli@unisi.it

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involved the selection of a protecting group for the pyrazole In particular, we were looking for a PG that could tolerate all the
ring which can be easily inserted at the beginning and experimental conditions employed during the whole synthetic
View Article Online
DOI: 10.1039/D0NJ03400B
removed at an advanced stage of the synthetic sequence. This sequence and be easily removed at the end, so as to give us the
approach would offer the possibility of increasing the chemical possibility to introduce a variety of alkyl chains as replacement of
diversity at the level of this ring through the introduction of a the methyl group present in our prototypes 2a and 2b. The first PG
variety of R1 substituents, a result that cannot be pursued with we tried was the tert-butoxycarbonyl (BOC) group, which was
a chemical synthesis that starts from a pyrazole irreversibly inserted in position 2 of the pyrazole ring through the reaction of 3
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alkylated at the nitrogen atom (Scheme 1).
with di-tert-butyl dicarbonate (Boc2O) and Et3N in dry THF.
However, this reaction did not go to completion and the desired
product 4 was isolated in low yield (25%) after trituration with
petroleum ether.
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Scheme 1. Retrosynthetic analysis for the preparation of 7-
hydroxy-5-oxopyrazole[4,3-b]pyridine-6-carboxamide scaffold
variously functionalized at N1 and N2.
In a second attempt, we used ethyl vinyl ether to obtain
compound 5. The reaction was conducted in dry DCM with
ethyl vinyl ether and p-toluenesulfonic acid and 5 (racemic)
was obtained in high yield (94%) but it looked like a dark oil
which underwent rapid degradation.
Another PG we considered was the 2-methoxyethoxymethyl
(MEM) group, by alkylating compound 3 with MEM chloride
and K2CO3 in acetone, but the reaction did not lead to the
expected result, giving rise to a complex mixture that was not
possible to purify with the available purification methods and
therefore the formed compound 6 could not be isolated.
After these discouraging results, other routes that might prove
to be more practical were explored. Thus, the benzyl group
was considered as a possible PG for pyrazole. When 3 was
reacted with benzyl p-toluenesulfonate in the presence of
K2CO3 in acetone, compound 7 (racemic) was obtained as a
light yellow solid in good yield (66%) after chromatographic
purification. To test a further possibility, 3,4-dihydro-2H-pyran
(DHP) was taken into consideration. By treating 3 with DHP
and p-toluenesulfonic acid in DCM, the reaction was complete
in 2 h providing 8 as a colorless stable oil in 50% yield after
purification by flash column chromatography. Although the
alkylation reactions on the pyrazole nitrogen often gives rise to
mixtures of regioisomers,[10,11] the THP-protected compound
was obtained as a single isomer, which was assigned the
structure of N2-substituted isomer 8 on the basis of 2D-NOESY
experiments, that showed NOE correlations between H1
proton of the THP ring and the aromatic proton of the pyrazole
(Figures 2).
Results and Discussion
The first point we addressed was the selection of a suitable
protecting group (PG) for the pyrazole nitrogen of compound 3
(Scheme 2: for sake of simplicity, only the predominant N2-
regiosomers are shown).
Scheme 2. Selection of possible protected precursors.
In order to test which of the protected compounds 7 and 8
would be the most reliable for the whole synthetic sequence,
we designed simplified compounds which could serve as
models for the critical deprotection step. To this end, 7 and 8
were both reduced to the corresponding amines 9 and 10 and
then converted into the corresponding amides 11 and 12,
respectively, with p-toluoyl chloride in dry DCM (Scheme 3).
Reagents and conditions: i) Boc2O, Et3N, dry THF, RT,
overnight; ii) ethyl vinyl ether, p-toluenesulfonic acid, dry
DCM, r. t., 2 h; iii) MEM chloride, K2CO3, acetone, reflux,
overnight; iv) benzyl p-toluenesulfonate, K2CO3, acetone, r. t.,
5 h; v) 3,4-dihydro-2H-pyran, p-toluenesulfonic acid, DCM, r. t.,
2 h. For sake of simplicity, only the main N2-regiosomers are
shown.
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Scheme 3. Synthesis and evaluation of the deprotection procedure of the model compounds 11 and 12.
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Reagents and conditions: i) for 7: Fe0 powder, CaCl2, EtOH, H2O, 60 °C, 2 h; ii) for 8: 10% Pd/C, ammonium formate, EtOH, H2O,
50 °C, 1 h; iii) p-toluoyl chloride, Et3N, DMAP, dry DCM, r. t., 50 min; iv) p-toluenesulfonic acid, MeOH, r. t., overnight.
The
target
7-hydroxy-5-oxopyrazolo[4,3-b]pyridine-6-
carboxamide derivatives were prepared through a multistep
synthesis, starting with an N-alkylation reaction of compound
10 (Scheme 4). The insertion of an N-alkyl chain on the amine
in position 4, either by direct alkylation or reductive amination
procedure resulted unsuccessful, due to the exclusive or
predominant formation of the N,N-dialkylated product, likely
due to the higher nucleophilic reactivity of secondary amines
(generated in the reaction mixture by the first N-alkylation)
compared to primary ones. For the preparation of compounds 2a,b,
[10] this alkylation reaction was best performed in DMF as the
solvent and in the absence of any added base, because the initially
formed monoalkylated compound, being also the most basic
species present in solution, preferentially acted as scavenger for the
produced HX acid. As a result, protonation made the monoalkylated
compound less nucleophilic and allowed
a more selective
conversion of the primary amine into the corresponding secondary
amine, by reducing the possibility of formation of the dialkylated
product. On the other hand, the alkylation reaction of 10, protected
with the acid-sensitive THP group, was not possible in the absence
of a base. Thus, assuming that the use of a secondary alkyl
halide would avoid the formation of dialkylated compounds
for steric reasons, a modified procedure was adopted, using 2-
iodopropane as alkylating agent, propionitrile as solvent, and
K2CO3 as base to neutralize the hydrogen iodide which formed
during the reaction, and hence prevent the THP removal.
Under these conditions, compound 14 could be isolated in
satisfactory 64% yield, while tertiary alkylating agents - as
expected - proved to be completely unreactive. Accordingly, we put
aside the idea of making structural changes in position 4 and limited
ourselves to introduce an isopropyl chain on the nitrogen atom. By
reaction with methyl 3-chloro-3-oxopropionate and Et3N in dry
DCM, 14 was converted into the amide derivative 15, that was
subjected to Dieckmann condensation by reaction with NaH
and dry MeOH (catalytic amount) in dry THF. The bicyclic 4-
hydroxypyrazolo[4,3-b]pyridine-2-one derivative 16 thus
obtained was treated with 1-aminoadamantane in
toluene/THF as solvent to afford 17. Finally, the amide 17 was
treated with p-toluenesulfonic acid in MeOH to deprotect the
pyrazole nitrogen, leading to the compound 18 with a 67%
yield after purification by flash column chromatography
(Scheme 4).
Figure 2. 1H−1H NOESY spectra of A) compound 8 and B)
compound 12 registered in CDCl3.
As a result, 11 appeared as a white solid, insoluble in most
organic solvents, thus hampering the execution of
deprotection procedures, whereas compound 12 was easily
deprotected with p-toluenesulfonic acid in MeOH so as to get
13 in 93% yield and with no need of purification. In agreement
with what observed for compound 8, the presence of THP
group on the N2 was confirmed by 2D NOESY spectra also on
compound 12 (Figure 2). Accordingly, we selected the THP-
protected compound 10 as the starting point for the
preparation of the desired N1- and N2-substituted
compounds.
The deprotected product 18 was the starting point for studying
the substitution of the pyrazole ring with the introduction of a
variety of alkyl/aralkyl chains (Scheme 4 and Table 1).
Compounds 19-22 were prepared in good to excellent overall
yield (60-99%) starting from 18 by alkylation reactions
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performed by using the appropriate alkylating agent, K2CO3 as obtained in low yield, due to the formation ofVidewegArrtaicdleaOtniloinne
base, and acetone as solvent. To test a different alkylation products. In addition, all the alkylation reactions led to a
procedure, compounds 23 and 24 were obtained by phase- mixture of N1- and N2-substituted regioisomers, which were
transfer catalyzed reactions, using 50% NaOH/DCM and separated by flash column chromatography, with the
tetrabutylammonium bromide as catalyst. The reaction was exception of compound 21 for which only the regioisomer 21A
complete in 2-3 hours, but compounds 23 and 24 were was identified.
DOI: 10.1039/D0NJ03400B
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Scheme 4. Synthesis of the N1- and N2-substituted compounds.
Reagents and conditions: i) 2-iodopropane, K2CO3, propionitrile, 95 °C, 7-8 h; ii) methyl 3-chloro-3-oxopropionate, Et3N, dry
DCM, r. t., 2 h; iii) NaH, dry MeOH (cat), dry THF, 60 °C, 3 h; iv) 1-aminoadamantane, THF, toluene, 140 °C, 1 h; v) p-
toluenesulfonic acid, MeOH, r. t., overnight; vi) for 19-22: appropriate alkylating agent, K2CO3, acetone, r. t., 1-18 h; vii) for 23
and 24: alkylating agent, tetrabutylammonium bromide, 50% NaOH, DCM, r. t., 2-3 h.
Table 1. 7-Hydroxy-5-oxopyrazolo[4,3-b]pyridine-6-carboxamide derivatives.
Yield %
Compd
R1
A isomer
80
B isomer
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Me
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Figure 3. 1H NMR spectrum of compounds 19A (top) and 19B (bottom).
Interestingly, the 1H NMR spectra of the two isolated affected by the type of substituent present on the nitrogen
regioisomers show significant differences, as exemplified by atom.
the spectra of compounds 19A and 19B in Figure 3: a) the
derivatives of the A series (e.g. 19A in Figure 3) show a
splitting of the signals, that is not equally evident in the NMR
spectra of the B derivatives (e.g. 19B in Figure 3), and b) the
derivatives of A series show a downfield shift of the signals
compared to the chemical shift values for the same protons in
the regioisomers of the B series. These observations, together
with the comparison with spectral data of COR1022 and
COR1023,[10] allowed to assign the N1-substituted structure
to the compounds of the A series and the N2-substituted
structure to those of the B series.
Conclusions
In summary, with the aim to explore the chemical space
around
the
7-hydroxy-5-oxopyrazolo[4,3-b]pyridine-6-
carboxamide scaffold, keeping the central core and the amide
functionality unchanged, twelve new compounds have been
synthesized as potential CB2 ligands. The here described
alternative approach to the synthesis of 7-hydroxy-5-
oxopyrazolo[4,3-b]pyridine-6-carboxamide derivatives, despite
more lengthy for two more (protection-deprotection) steps,
has shown to be more versatile compared to the previously
reported synthesis. The protecting group approach allowed to
insert a variety of substituents on the pyrazole ring as last step
and hence to enhance the chemical diversity on this moiety, a
result that could not be easily achieved according to the
previously adopted multistep synthesis.
Scheme 5. Tautomeric equilibria that have been
hypothesized for the N1-substituted compounds.
On the other hand, this approach has limited the variety of
substituents to be installed at position 4, due to the widely
different reactivity between primary, secondary, and tertiary
alkylating agents. Last but not least, this synthesis allowed to
obtain in most cases either N1- or N2-substituted analogs, thus
opening the door to wider exploration of structure-activity
relationship for both CB2R agonists and inverse
agonists/antagonists with potential in diverse fields of
therapeutic relevance.
These findings have been tentatively explained by assuming
that: i) the compounds can give rise to tautomeric equilibria in
solution (Scheme 5), with tautomer A surmised to be the most
abundant although no attempt to compute the relative
stability of the putative tautomers has been made yet; ii) the
substituent at N1 favors tautomers B and C much more than
the substituent at N2 does; iii) the ratio between tautomers is
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mmol) were added. The reaction mixture was stiVrireewdAratictlerOonolimne
temperature for 5 h, then it was poured into H O (100 mL).
Experimental
DOI: 10.1039/D0NJ03400B
2
General. All commercially available reagents were used as
received unless otherwise specified. Solvents were treated
before use with suitable drying agents and used after
distillation in an atmosphere of N2. THF and toluene were
distilled from sodiun/benzophenone ketyl; DCM, acetonitrile,
and propionitrile from CaH2; MeOH from Mg/I2. Reactions
requiring anhydrous conditions were performed under N2.
Melting points were determined on a Gallenkamp apparatus
and are uncorrected. For flash chromatography 60 Merck
Kieselgel 60 (0.040-0.063 mm) was used. Thin layer
chromatography (TLC) was performed on silica gel plates
(Merck 60 F254) eluting with solvents indicated, visualized by a
254 nm UV lamp (λ = 254 nm) and stained with aqueous
potassium permanganate, Pancaldi and phosphomolybdic acid
The aqueous phase was extracted with EtOAc. The organic
layer was washed with brine, dried over anhydrous Na2SO4,
filtered and the solvent was removed under reduced pressure.
The colorless oily residue was purified by flash column
chromatography on silica gel, using PE:EtOAc 8:1 as eluent to
afford 7 (2.07 g; 66% yield) as a light yellow solid. Mp: 75.9-
79.7 °C. 1H NMR (400 MHz, CDCl3): δ 7.93 (s, 1H), 7.35 (m, 3H),
7.25 (m, 2H), 5.27 (s, 2H), 3.94 (s, 3H). MS (ESI): m/z 284
[M+Na]+ .
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Methyl
4-Nitro-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazole-3-
carboxylate (8).
A solution of 3,4-dihydro-2H-pyran (0.16 mL; 1.75 mmol) in
DCM (5 mL) was added dropwise to a suspension of methyl 4-
nitro-1H-pyrazole-3-carboxylate (3)[11] (0.20 g; 1.17 mmol)
and p-toluenesulfonic acid (0.021 g; 0.12 mmol) in DCM (15
mL), maintained at the temperature of 0 °C. The reaction
mixture was allowed to warm to room temperature and stirred
for further 2 h. After addition of Et2O, the solution was washed
with a saturated solution of NaHCO3 and brine, dried over
anhydrous Na2SO4 and filtered. Evaporation of the solvent
under reduced pressure gave a yellow oil which was purified
by flash column chromatography on silica gel, eluting with
EtOAc:PE 2:1 to provide 8 (0.15 g; 50% yield) as a colorless oil.
1H NMR (400 MHz, CDCl3): δ 8.31 (s, 1H), 5.35 (dd, J = 9.1 and
2.7 Hz, 1H), 4.02 (m, 1H), 3.92 (s, 3H), 3.67 (m, 1H), 2.15 (m,
2H), 2.00-1.78 (m, 2H), 1.75-1.47 (m, 2H). MS (ESI): m/z 278
[M+Na]+.
1
solutions. H NMR and 13C NMR spectra were recorded on a
Brucker Avance DPX-400 NMR spectrometer at 400 MHz (1H)
or 100 MHz (13C). Chemical shifts (δ) and coupling constants (J)
are reported in parts per million (ppm) and hertz (Hz),
respectively. Mass spectra were recorded with an LC-MSD 110
series AGILENT instrument, with electrospray interface and
with a 0.4 mL/min flow rate using a binary solvent system of
95:5 methanol/water. The UV detector was set at 254 nm and
the mass spectra were acquired either in positive or in
negative mode scanning over the mass range of 105-1500.
(tert-Butyl)
carboxylate (4).
3-Methoxycarbonyl-4-nitro-1H-pyrazole-1-
TEA (0.37 mL; 2.63 mmol) and Boc2O (0.57 g; 2.63 mmol) were
added to solution of methyl 4-nitro-1H-pyrazole-3-
a
carboxylate (3)[11] (0.30 g; 1.75 mmol) in dry THF (10 mL)
under a nitrogen atmosphere and the resulting solution was
stirred at room temperature for 16 h. The reaction mixture
was diluted with H2O (50 mL) and the aqueous phase was
extracted with EtOAc. The organic phase was dried over
anhydrous Na2SO4, filtered and evaporated under reduced
pressure. The oily residue was purified by trituration with PE to
give 4 as a pink solid (0.12 g; 25% yield). Mp: 98.6-100.0 °C. 1H
NMR (400 MHz, CDCl3): δ 8.70 (s, 1H), 3.94 (s, 3H), 1.61 (s, 9H).
MS (ESI): m/z 294 [M+Na]+.
Methyl 4-Amino-1-benzyl-1H-pyrazole-3-carboxylate (9).
Iron powder (1.71 g; 30.62 mmol), anhydrous CaCl2 (0.85 g;
7.66 mmol) and H2O (5 mL) were added successively to a
solution of methyl 1-benzyl-4-nitro-1H-pyrazole-3-carboxylate
(7) (2.00 g; 7.66 mmol) in EtOH (50 mL), warmed at 60 °C and
the reaction mixture was vigorously stirred at this temperature
for 2 h. After cooling to room temperature, the suspension
was filtered through a pad of celite, concentrated in vacuo and
brought to pH 9 with 5% Na2CO3 solution. The white
precipitate of iron hydroxide which formed was filtered out.
The aqueous phase was extracted with EtOAc, and the organic
layer was dried over anhydrous Na2SO4, filtered and
evaporated under reduced pressure. The oily residue was
purified by flash column chromatography on silica gel
(PE:EtOAc 1:2) and by trituration with PE to give 9 (1.01 g; 57%
Methyl
1-(1-Ethoxyethyl)-4-nitro-1H-pyrazole-3-carboxylate
(5).
Ethyl vinyl ether (0.32 mL; 3.33 mmol) was added to a
suspension of methyl 4-nitro-1H-pyrazole-3carboxylate (3)[11]
(0.30 g; 1.75 mmol) and p-toluenesulfonic acid (0.031 g; 0.18
mmol) in dry DCM (60 mL). The green solution which formed
was stirred at room temperature for 2 h. The reaction mixture
was diluted with H2O (50 mL) and extracted with DCM. The
organic phase was washed with NaHCO3 solution, then with
brine, dried over anhydrous Na2SO4, filtered and evaporated
under reduced pressure. The oily residue was purified by flash
column chromatography on silica gel (PE:EtOAc 2:1) to afford
1
yield) as a white solid. Mp: 110.1-113.2 °C. H NMR (400 MHz,
CDCl3): δ 7.33-7.23 (m, 4H), 7.18-7.12 (m, 1H), 6.87 (s, 1H),
5.17 (s, 2H), 4.08 (s, 2H), 3.85 (s, 3H). MS (ESI): m/z 254
[M+Na]+.
Methyl 4-Amino-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazole-3-
carboxylate (10).
To a solution of methyl 4-nitro-1-(tetrahydro-2H-pyran-2-yl)-
1H-pyrazole-3-carboxylate (8) (0.84 g; 3.29 mmol) in EtOH (10
mL) and H2O (1 mL), ammonium formate (2.06 g; 32.67 mmol)
and 10% Pd/C (0.042 g; 0.39 mmol) were added under a
nitrogen atmosphere. The reaction mixture was stirred at 50
°C for 1 h. After cooling to room temperature, the mixture was
filtered through a pad of celite and the filtrate was diluted
with H2O. The aqueous phase was extracted with EtOAc and
the organic layer was dried over anhydrous Na2SO4, filtered
1
0.40 g (94% yield) of 5 as an orange oil. H NMR (400 MHz,
CDCl3): δ 8.26 (s, 1H), 5.48 (q, J = 5.9 Hz, 1H), 3.91 (s, 3H), 3.43
(m, 2H), 1.60 (d, J = 6.0 Hz, 3H), 1.13 (t, J = 7.0 Hz, 3H). MS
(ESI): m/z 266 [M+Na]+.
Methyl 1-Benzyl-4-nitro-1H-pyrazole-3-carboxylate (7).
To a solution of methyl 4-nitro-1H-pyrazole-3-carboxylate
(3)[11] (2.06 g; 12.04 mmol) in acetone (150 mL), K2CO3 (1.84
g; 13.31 mmol) and benzyl p-toluenesulfonate (3.50 g; 13.34
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and evaporated under reduced pressure to give 10 (0.72 g; two further additions of K2CO3 (0.28 g; 2.02 mmol) and 2-
View Article Online
97% yield) as a yellow oil. 1H NMR (400 MHz, CDCl3): δ 7.04 (s, iodopropane (0.20 mL; 2.00 mmol) were made. Then the
DOI: 10.1039/D0NJ03400B
1H), 5.17 (dd, J = 9.0 and 2.8 Hz, 1H), 3.88 (m, 1H), 3.70 (s, 3H), mixture was concentrated under reduce pressure, diluted with
3.51 (m, 1H), 1.85 (m, 3H), 1.60-1.32 (m, 3H). MS (ESI): m/z H2O (30 mL), and extracted with EtOAc. The organic layer was
248 [M+Na]+.
washed with a saturated solution of Na2S2O3 and brine, dried
over anhydrous Na2SO4, filtered and evaporated. The oily
General procedure for the preparation of compounds 11 and residue was purified by flash column chromatography on silica
12. gel eluting with PE:EtOAc (2:1) to give the title product 14
1
10
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To a solution of amine 9 or 10 (1.78 mmol) in dry DCM (20 mL), (0.17 g, 64% yield) as a light yellow oil. H NMR (400 MHz,
Et3N (0.38 mL; 2.66 mmol) and a catalytic amount of DMAP CDCl3): δ 6.95 (s, 1H), 5.22 (dd, J = 8.9 and 3.3 Hz), 3.94 (m,
were added under a nitrogen atmosphere. Afterwards a 1H), 3.77 (s, 3H), 3.54 (m, 1H), 3.15 (m, 1H), 1.97-1.81 (m, 3H),
solution of p-toluoyl chloride (0.28 mL; 2.13 mmol) in dry DCM 1.60-1.41 (m, 3H), 1.08 (dd, J = 6.3 and 3.6 Hz). MS (ESI): m/z
(10 mL) was added dropwise and the reaction mixture was 290 [M+Na]+.
stirred at room temperature for 50 min. Then a saturated
solution of NaHCO3 (20 mL) was added and the mixture was Methyl
4-(N-Isopropyl-3-methoxy-3-oxopropanamido)-1-
stirred for further 15 min. The organic phase was washed (tetrahydro-2H-pyran-2-yl)-1H-pyrazole-3-carboxylate (15). A
successively with a saturated solution of NaHCO3, 1 N HCl, H2O solution of methyl 3-chloro-3-oxopropionate (1.00 mL; 9.31
and brine, dried over anhydrous Na2SO4 and filtered. Removal mmol) in dry DCM (5 mL) was added dropwise under a
of the solvent under reduced pressure left a residue which was nitrogen atmosphere to
a
solution of methyl 4-
purified by flash column chromatography on silica gel to yield (isopropylamino)-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazole-3-
the title products.
carboxylate (14) (1.66 g; 6.21 mmol) and Et3N (1.30 mL; 9.31
mmol) in dry DCM (35 mL). The reaction mixture was stirred at
Methyl 1-Benzyl-4-[(4-methylbenzoyl)amino]-1H-pyrazole-3- room temperature for 2 h and then was quenched with a
carboxylate (11). saturated solution of NaHCO3. The mixture was extracted with
Prepared from 9 according to the general procedure. Eluent: DCM and the organic phase was washed with 1 N HCl and H2O,
DCM:PE (2:1). White solid (0.48 g; yield 77%). Mp: 157.9-160.6 dried over anhydrous Na2SO4, filtered and evaporated. The
1
°C. H NMR (400 MHz, CDCl3): δ 9.86 (s, 1H), 8.26 (s, 1H), 7.75 orange oil obtained was purified by flash column
(d, J = 8.1 Hz, 2H), 7.39-7.16 (m, 7H), 5.30 (s, 2H), 3.96 (s, 3H), chromatography on silica gel, with PE:EtOAc (2:1) as eluent, to
1
2.35 (s, 3H). MS (ESI): m/z 372 [M+Na]+.
afford 15 (1.97 g; 86% yield) as a colorless oil. H NMR (400
MHz, CDCl3): δ 7.53 (s, 1H), 5.37 (d, J = 9.6 Hz, 1H), 4.91 (m,
Methyl 4-[(4-Methylbenzoyl)amino]-1-(tetrahydro-2H-pyran-2- 1H), 4.01 (m, 1H), 3.83 (s, 3H), 3.72-3.53 (m, 4H), 3.14 (m, 2H),
yl)-1H-pyrazole-3-carboxylate (12). 2.18-2.05 (m, 1H), 2.03-1.70 (m, 2H), 1.69-1.51 (m, 3H), 1.02
Prepared from 10 according to the general procedure. Eluent: (dd, J = 6.4 and 4.0 Hz, 3H), 0.81 (d, J = 6.8 Hz, 3H). MS (ESI):
PE:EtOAc (2:1). White solid (0.37 g; yield 60%. Mp: 139.4-140.5 m/z 390 [M+Na]+.
1
°C. H NMR (400 MHz, CDCl3): δ 9.83 (s, 1H), 8.51 (s, 1H), 7.78
(d, J = 8.2 Hz, 2H), 7.23 (d, J = 8.0 Hz, 2H), 5.38 (m, 1H), 4.02 Methyl 7-Hydroxy-4-isopropyl-5-oxo-2-(tetrahydro-2H-pyran-
(m, 1H), 3.95 (s, 3H), 3.64 (m, 1H), 2.36 (s, 3H), 2.15-1.89 (m, 2-yl)-4,5-dihydro-2H-pyrazolo[4,3-b]pyridine-6-carboxylate
3H), 1.79-1.48 (m, 3H). MS (ESI): m/z 366 [M+Na]+.
(16).
To a suspension of NaH (0.24 g; 10.0 mmol) in dry THF (50 mL),
Methyl 4-[(4-Methylbenzoyl)amino]-1H-pyrazole-3-carboxylate dry MeOH (0.066 mL) was added under
a nitrogen
(13). atmosphere. The reaction mixture was stirred at 60 °C for 10
p-Toluenesulfonic acid (0.009 g; 0.07 mmol) was added to a min, then a solution of methyl 4-(N-isopropyl-3-methoxy-3-
solution of methyl 4-[(4-methylbenzoyl)amino]-1-(tetrahydro- oxopropanamido)-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazole-3-
2H-pyran-2-yl)-1H-pyrazole-3-carboxylate (12) (0.050 g; 0.15 carboxylate (15) (1.21 g; 3.29 mmol) in dry THF (20 mL) was
mmol) in MeOH (10 mL) and the reaction mixture was stirred added dropwise. The suspension obtained was stirred at 60 °C
at room temperature for 16 h. Then, the solution was for 3 h. After cooling to room temperature, THF was removed
concentrated under reduced pressure and the residue under nitrogen atmosphere. The reaction mixture was brought
obtained was diluted into EtOAc. The organic layer was to pH 9 with 10% NaOH solution, diluted with H2O (10 mL) and
washed with a saturated solution of NaHCO3, dried over extracted with EtOAc. The aqueous phase was acidified with
anhydrous Na2SO4, filtered and evaporated to afford 13 (0.036 12 N HCl and then extracted with EtOAc. The organic layer was
g; 93% yield) as a white solid. Mp 229.3-230.4 °C. 1H NMR (400 washed with brine, dried over anhydrous Na2SO4 and filtered.
MHz, CDCl3): δ 9.75 (s, 1H), 8.55 (s, 1H), 7.78 (d, J = 8.0 Hz, 2H), Evaporation of the solvent under reduce pressure gave a
7.25 (d, J = 8.0 Hz, 2H), 6.13 (s, 1H), 4.04 (s, 3H), 2.37 (s, 3H). yellow oily residue, which was purified by flash column
MS (ESI): m/z 282 [M+Na]+.
chromatography on silica gel, eluting with PE:EtOAc (1:9) to
afford 16 (0.93 g; 84% yield) as a colorless oil. H NMR (400
1
Methyl 4-(Isopropylamino)-1-(tetrahydro-2H-pyran-2-yl)-1H- MHz, acetone-d6): δ 13.54 (br s, 1H), 8.00 (s, 1H), 5.54 (d, J =
pyrazole-3-carboxylate (14). 9.2 Hz, 1H), 5.08 (m, 1H), 3.95 (m, 1H), 3.83 (s, 3H), 3.67 (m,
To a solution of methyl 4-amino-1-(tetrahydro-2H-pyran-2-yl)- 1H), 2.24-2.07 (m, 1H), 2.06-1.88 (m, 2H), 1.79 -1.65 (m, 1H),
1H-pyrazole-3-carboxylate (10) (0.225 g, 1.00 mmol) in 1.59 (m, 2H), 1.36 (d, J = 9.2 Hz, 6H). MS (ESI): m/z 358
propionitrile (15 mL), K2CO3 (0.28 g, 2.02 mmol) and 2- [M+Na]+.
iodopropane (0.20 mL, 2.00 mmol) were added. The reaction
mixture was stirred at 95 °C for 7 h. During the first two hours,
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New Journal of Chemistry
oxo-4,5-dihydro-2H-pyrazolo[4,3-b]pyridine-6-carboxamide
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N-(Adamantan-1-yl)-7-hydroxy-4-isopropyl-5-oxo-2-
(tetrahydro-2H-pyran-2-yl)-4,5-dihydro-2H-pyrazolo[4,3-
b]pyridine-6-carboxamide (17).
View Article Online
(19B).
Prepared from 18 by reaction with dimethyl sulfate. Elution
DOI: 10.1039/D0NJ03400B
A
mixture of methyl 7-hydroxy-4-isopropyl-5-oxo-2- with PE:EtOAc (2:1) gave the first regioisomer 19A as a solid
(tetrahydro-2H-pyran-2-yl)-4,5-dihydro-2H-pyrazolo[4,3-
residue that was recrystallized from MeOH to afford a white
b]pyridine-6-carboxylate (16) (0.13 g; 0.39 mmol), 1- solid (mixture of tautomers in solution); yield, 80%. Mp 193.6-
aminoadamantane (0.11 g; 0.73 mmol), THF (1.5 mL), and 194.9 °C. H NMR (400 MHz, CDCl3): δ 18.92 and 18.30 (s, 1H
1
9
toluene (10 mL) was stirred at 140 °C for 1 h. During this time overall), 11.14 and 10.57 (s, 1H overall), 7.56 (s, 1H), 5.42 and
further toluene was added dropwise while the azeotropic 5.16 (m, 1H overall), 4.30 and 4.23 (s, 3H overall), 2.13 (m, 9H),
mixture MeOH/toluene was distilled out. Once the reaction 1.70 (m, 6H), 1.51 and 1.47 (d, J = 7.0 Hz, 6H overall). 13C NMR
was complete, the solvent was removed by a nitrogen flow. (100 MHz, CDCl3): δ 171.7, 165.7, 162.2, 124.1, 123.8, 122.2,
The residue was taken up into EtOAc (20 mL) and the organic 95.6, 52.6, 45.4, 41.5, 39.3, 36.4, 29.4, 19.8. MS (ESI): m/z 383
phase was washed with 1 N HCl, H2O and brine, dried over [M–H]–.
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anhydrous Na2SO4, filtered and evaporated under reduced Further elution with EtOAc furnished the second isomer 19B as
pressure. The oily residue was purified by flash column a white solid; yield, 17%. Mp 263.9-266.0 °C. 1H NMR (400
chromatography on silica gel, eluting with PE:EtOAc (2:1) to MHz, CDCl3): δ 17.70 (s, 1H), 10.30 (s, 1H), 7.37 (s, 1H), 5.32
furnish 17 (0.16 g; 90% yield) as a glassy white solid. Mp 122.1- (m, 1H), 4.03 (s, 3H), 2.08 (m, 9H), 1.65 (m, 6H), 1.36 (d, J = 7.0
124.9 °C. H NMR (400 MHz, CD3OD): δ 10.39 (s, 1H), 8.09 (s, Hz, 6H). 13C NMR (100 MHz, CDCl3): δ 171.6, 168.2, 162.9,
1
1H), 7.82 (s, 1H), 5.52 (dd, J = 9.6 and 2.4 Hz), 5.12 (m, 1H), 132.9, 125.6, 114.5, 96.7, 52.4, 45.4, 41.2, 40.9, 36.8, 30.0,
4.10-3.95 (m, 1H), 3.74-3.67 (m, 1H), 2.25-1.86 (m, 12H), 19.3. MS (ESI): m/z 407 [M+Na]+.
1.83.1.53 (m, 9H), 1.42 (d, J = 5.9 Hz, 6H). MS (ESI): m/z 931
[2M+Na]+.
N-(Adamantan-1-yl)-7-hydroxy-4-isopropyl-5-oxo-1-(prop-2-
yn-1-yl)-4,5-dihydro-1H-pyrazolo[4,3-b]pyridine-6-
carboxamide (20A) and N-(Adamantan-1-yl)-7-hydroxy-4-
isopropyl-5-oxo-2-(prop-2-yn-1-yl)-4,5-dihydro-2H-
N-(Adamantan-1-yl)-7-hydroxy-4-isopropyl-5-oxo-4,5-dihydro-
2H-pyrazolo[4,3-b]pyridine-6-carboxamide (18).
A solution of N-(adamantan-1-yl)-7-hydroxy-4-isopropyl-5-oxo- pyrazolo[4,3-b]pyridine-6-carboxamide (20B).
2-(tetrahydro-2H-pyran-2-yl)-4,5-dihydro-2H-pyrazolo[4,3-
b]pyridine-6-carboxamide (17) (0.82 g; 1.80 mmol) and p- with PE:EtOAc (5:1) afforded the first regioisomer 20A as a
Prepared from 18 by reaction with propargyl bromide. Elution
1
toluenesulfonic acid (0.11 g; 0.64 mmol) in MeOH (50 mL) was colorless oil (mixture of tautomers in solution); yield, 39%. H
stirred at room temperature overnight, then concentrated NMR (400 MHz, CDCl3): δ 18.83 and 18.39 (br s, 1H overall),
under reduce pressure to a small volume. EtOAc (20 mL) and a 11.01 and 10.48 (s, 1H overall), 7.59 (s, 1H), 5.51 and 5.34 (s,
saturated solution of NaHCO3 (5 mL) and H2O (10 mL) were 2H overall), 5.10 (m, 1H), 2.34 (s, 1H), 2.06 (m, 9H), 1.62 (m,
added to the solid residue obtained and the aqueous phase 6H), 1.45 and 1.41 (d, J = 7.0 Hz, 6H overall). 13C NMR (100
was extracted with EtOAc. The organic phase was washed with MHz, CDCl3): δ 171.6, 166.3, 162.2, 127.9, 125.6, 125.4, 95.8,
brine, dried over anhydrous Na2SO4 and filtered. Removal of 73.7, 73.4, 53.2, 52.8, 45.5, 41.4, 36.3, 29.4, 19.9. MS (ESI): m/z
the solvent under reduce pressure yielded a white solid 407 [M–H]–.
residue, which was purified by flash column chromatography Further elution with PE:EtOAc (4:1 to 1:1) gave 20B as a light
on silica gel, using DCM:MeOH (98:2) to obtain 18 (0.45 g; 67% yellow oil; yield, 60%. 1H NMR (400 MHz, CDCl3): δ 17.75 (br s,
yield) as a white glassy solid (mixture of tautomers in solution). 1H), 10.25 (s, 1H), 7.64 (s, 1H), 5.26 (m, 1H), 5.05 (s, 2H), 2.55
Mp 183.5-185.7 °C. H NMR (400 MHz, CDCl3): δ 18.93 and (s, 1H), 2.03 (m, 9H), 1.62 (m, 6H), 1.35 (d, J = 7.1 Hz, 6H). 13C
1
18.08 (s, 1H overall), 10.95 and 10.50 (s, 1H overall), 8.77 (br s, NMR (100 MHz, CDCl3): δ 171.2, 168.9, 162.8, 133.5, 125.5,
1H), 7.75 (s, 1H), 5.38 and 5.18 (m, 1H overall), 2.22-1.89 (m, 113.5, 97.1, 76.2, 75.6, 52.5, 45.6, 43.7, 41.5, 36.4, 29.4, 19.3.
9H), 1.78-1.60 (m, 6H), 1.49 and 1.44 (d, J = 6.8 Hz, 6H overall). MS (ESI): m/z 407 [M–H]–.
MS (ESI): m/z 371 [M+Na]+.
N-(Adamantan-1-yl)-1-(cyanomethyl)-7-hydroxy-4-isopropyl-5-
oxo-4,5-dihydro-1H-pyrazolo[4,3-b]pyridine-6-carboxamide
General procedure for the preparation of compounds 19-22.
To a solution of N-(adamantan-1-yl)-7-hydroxy-4-isopropyl-5- (21A).
oxo-4,5-dihydro-2H-pyrazolo[4,3-b]pyridine-6-carboxamide
Prepared from 18 using chloroacetonitrile as alkylating agent.
(18) (0.14 g; 0.38 mmol) in acetone (20 mL), maintained under Eluent: PE:EtOAc (3:1). Glassy white solid (mixture of
a nitrogen atmosphere, anhydrous K2CO3 (0.11 g; 0.80 mmol) tautomers in solution); yield, 58%. 1H NMR (400 MHz, CDCl3): δ
and the appropriate alkylating agent (0.80 mmol) were added. 18.78 and 18.52 (s, 1H overall), 10.91 and 10.47 (s, 1H overall),
The reaction mixture was stirred at room temperature for 2-16 7.65 (s, 1H), 5.67 and 5.49 (s, 2H overall), 5.33 and 5.13 (m, 1H
h. The mixture was concentrated under reduced pressure and overall), 2.07 (m, 9H), 1.65 (m, 6H), 1.46 and 1.42 (d, J = 7.0 Hz,
the solid residue was partitioned between H2O and EtOAc. The 6H overall). 13C NMR (100 MHz, CDCl3): δ 171.5, 171.4, 168.3,
aqueous phase was extracted with EtOA and the organic layer 166.6, 165.7, 162.2, 127.2, 126.9, 126.8, 122.0, 114.2, 113.9,
was washed with brine, dried over anhydrous Na2SO4, filtered 95.9, 93.0, 53.4, 53.1, 45.6, 41.5, 41.4, 39.3, 38.7, 36.3, 36.2,
and evaporated. The residue obtained was purified by flash 29.4, 20.0, 19.9. MS (ESI): m/z 432 [M+Na]+.
column chromatography on silica gel.
tert-Butyl
isopropyl-5-oxo-4,5-dihydro-1H-pyrazolo[4,3-b]pyridin-1-
4,5-dihydro-1H-pyrazolo[4,3-b]pyridine-6-carboxamide (19A) yl]acetate (22A) and tert-Butyl 2-[6-[(Adamantan-1-
and N-(adamantan-1-yl)-7-hydroxy-4-isopropyl-2-methyl-5-
2-[6-[(Adamantan-1-yl)carbamoyl]-7-hydroxy-4-
N-(Adamantan-1-yl)-7-hydroxy-4-isopropyl-1-methyl-5-oxo-
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1
yl)carbamoyl]-7-hydroxy-4-isopropyl-5-oxo-4,5-dihydro-2H-
pyrazolo[4,3-b]pyridin-2-yl]acetate (22B).
solution); yield, 12%. H NMR (400 MHz, CDCl3):Vδiew1A8rt.ic8le4Oanlninde
18.41 (s, 1H overall), 11.01 and 10.50 (sD, O1IH: 10o.v10e3r9a/lDl)0, N8J.043840(m0B,
Obtained from 18 by reaction with tert-butyl bromoacetate. 2H), 7.60 (s, 1H), 7.13 (m, 2H), 5.85 and 5.69 (s, 2H overall),
Elution with PE:EtOAc (4:1) afforded the first isomer 22A as a 5.37 and 5.12 (m, 1H overall), 2.06 and 1.92 (m, 9H overall),
1
colorless oil (mixture of tautomers in solution); yield, 18%. H 1.65-1.60 (m, 6H overall), 1.47 and 1.43 (d, J = 7.0 Hz, 6H,
NMR (400 MHz, CDCl3): δ 18.81 and 18.16 (s, 1H overall), 11.00 overall). MS (ESI): m/z 484 [M+Na]+.
and 10.47 (s, 1H overall), 7.58 (s, 1H), 5.34 (m, 1H), 5.27 and Further elution with EtOAc:PE (4:1) to EtOAc:MeOH (99:1)
5.14 (s, 2H overall), 2.06 (m, 9H), 1.64 (m, 6H), 1.48-1.38 (m, gave 24B as a yellow oil; yield, 16%. 1H NMR (400 MHz, CDCl3):
15H). MS (ESI): m/z 507 [M+Na]+. 13C NMR (100 MHz, CDCl3): δ δ 17.86 (s, 1H), 10.27 (s, 1H), 8.53 (m, 2H), 7.39 (s, 1H), 7.06
171.6, 167.1, 165.3, 162.3, 125.5, 125.1, 122.5, 96.0, 82.9, (m, 2H), 5.42 (s, 2H), 5.26 (m, 1H), 2.08 (m, 9H), 1.64 (m, 6H),
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18
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23
24
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53.7, 52.6, 45.6, 41.6, 36.3, 29.4, 28.0, 19.9.
1.33 (d, J = 7.1 Hz, 6H). MS (ESI): m/z 462 [M+H]+.
Further elution with PE:EtOAc (3:1) gave 22B as a white solid;
1
yield, 61%. Mp 199.1-200.0 °C H NMR (400 MHz, CDCl3): δ
17.72 (s, 1H), 10.28 (s, 1H), 7.50 (s, 1H), 5.28 (m, 1H), 4.90 (s,
2H), 2.24-1.99 (m, 9H), 1.71-1.57 (m, 6H), 1.49.1.27 (m, 15H).
MS (ESI): m/z 485 [M+H]+. 13C NMR (100 MHz, CDCl3): 171.4,
169.0, 165.8, 162.9, 133.6, 125.6, 115.6, 97.2, 83.5, 55.4, 52.5,
45.6, 41.6, 36.4, 29.4, 28.0, 19.3.
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
Authors acknowledge the partial support by MIUR Progetto
Dipartimenti di Eccellenza 2018-2022, grant n. L. 232/2016.
General procedure for the preparation of compounds 23 and
24.
A solution of N-(adamantan-1-yl)-7-hydroxy-4-isopropyl-5-oxo-
4,5-dihydro-2H-pyrazolo[4,3-b]pyridine-6-carboxamide
(18)
(0.14 g; 0.38 mmol) and the appropriate alkylating agent (0.40
mmol) in CH2Cl2 (5 mL) was added to a suspension of 50%
NaOH solution (3 mL) and tetrabutylammonium bromide (0.13 [1]
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as a colorless oil (mixture of tautomers in solution); yield, 12%. [5]
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PE:EtOAc (3:1 to 1:1). Light yellow oil; yield, 27%. 1H NMR (400 Benefits of Cannabinoid CB2 Receptor Agonists, Chem. Rev. 116
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