Pyrazolo[3,4-: D] pyrimidines as sigma-1 receptor ligands for the treatment of pain. Part 2: Introduction of cyclic substituents in position 4

Article abstract of DOI:10.1039/c7md00078b

The replacement of acylamino by cyclic substituents in the position 4 of the pyrazolo[3,4-d]pyrimidine scaffold, led to highly active sigma-1 receptor (σ₁R) ligands. Phenyl or pyrazolyl groups were the best in terms of affinity for the σ₁R and the 4-(1-methylpyrazol-5-yl) derivative, 12f, was the most selective. Compound 12f is also one of the best σ₁R ligands ever described in terms of lipophilic ligand efficiency, which translates into a good physicochemical and ADMET profile. In addition, 12f was identified as an antagonist of the σ₁R in view of its potent antinociceptive profile in several pain models in mice.

Full text of DOI:10.1039/c7md00078b

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Corbera, D. Martinez, M. Bordas, C. Sicre, R. Pascual, M. J. Pretel, A. P. Marín, A. Montero, A. Dordal, I.
ALVAREZ and C. Almansa, Med. Chem. Commun., 2017, DOI: 10.1039/C7MD00078B.
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Pyrazolo[3,4ꢀd]pyrimidines as Sigmaꢀ1 Receptor
Ligands for the Treatment of Pain. Part 2:
Introduction of Cyclic Substituents in Position 4†
José Luis Díaz§, Jordi Corbera§, Daniel Martínez§, Magda Bordas§, Cristina Sicre#, Rosalia Pascual§,
Mª José Pretel§, Ana Paz Marín§, Ana Montero§, Albert Dordal§, Inés Alvarez§, and Carmen Almansa§*
§Drug Discovery and Preclinical Development, Esteve, Baldiri Reixach, 4ꢀ8, 08028 Barcelona, Spain. #Galchimia,
Cebreiro s/n 15823 O Pino A Coruña, Spain.
Corresponding author information: Carmen Almansa. Drug Discovery and Preclinical Development,
Esteve, Baldiri Reixach, 4ꢀ8, 08028 Barcelona, Spain. Tel: 0034934466000, email: calmansa@esteve.es.
Electronic Supplementary Information (ESI) available: Analytical and characterization data for all the
compounds. See DOI: 10.1039/x0xx00000x
†The authors declare no competing interests.
1

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Abstract
The replacement of acylamino by cyclic substituents in the 4 position of the pyrazolo[3,4ꢀ
d]pyrimidine scaffold, led to highly active sigmaꢀ1 receptor (σ1R) ligands. Phenyl or pyrazolyl groups
were the best in terms of affinity for the σ1R and the 4ꢀ(1ꢀmethylpyrazolꢀ5ꢀyl) derivative, 12f, was the
most selective. Compound 12f is also one of the best σ1R ligands ever described in terms of lipophilic
ligand efficiency, which translates into a good physicochemical and ADMET profile. In addition, 12f
was identified as an antagonist of the σ1R in view of its potent antinociceptive profile in several pain
models in mice.
Key words: sigmaꢀ1 receptor, antagonist, pyrazolo[3,4ꢀd]pyrimidine, antinociceptive, pain.
2

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Introduction
After the discovery of the σ receptor (σR) four decades ago and the subsequent identification of two
differentiated subtypes (σ1 and σ2),1 several σ1R ligands were identified and studied for treating different
central nervous system (CNS)ꢀrelated pathologies, such as schizophrenia and depression.2 More
recently, the σ1R has been revealed to act as an intracellular chaperone3 implicated in the modulation of
the activity of different molecular targets involved in transmission and amplification (e.g., central
sensitization) of nociceptive messages, supporting a role for σ1R antagonists in the treatment of pain.4
However, the finding of σ1R antagonists with adequate drugꢀlike properties and good absorption,
distribution, metabolism, excretion and toxicity (ADMET) profiles is not evident.5 First, as described in
the preceding article,6 the establishment of the functional behavior of σ1R ligands is not obvious, due to
the unique characteristics of the protein, and often relies on in vivo testing. Second, it is well known that
compounds with optimal lipophilicity have increased chances of success in development,7 but the σ1R
binding site is highly lipophilic,8 as can be inferred from the crystalline structure recently published9 and
the most accepted pharmacophores, such as Glennon’s10 and Laggner’s models.11 This lies together with
the observation that the majority of the described σ1R ligands are highly lipophilic and challenges the
discovery of ligands with optimal drugꢀlike properties. Third, the overlap of the σ1R requirements with
those of other receptor systems (e.g., monoaminergic receptors and transporters) and proteins
responsible for offꢀtarget effects (e.g., hERG channel), complicates the finding of selective agents. Thus,
although designing compounds active on the σ1R may be relatively simple, it is much more challenging
to find selective drugꢀlike compounds.
In the preceding article, the design and synthesis of a series of 4ꢀacylaminopyrazolo[3,4ꢀd]pyrimidine
derivatives as σ1R ligands is described.6 This series was designed based on Glennon pharmacophore and
taking into account the structureꢀactivity relationship (SAR) study that led to the selection of Eꢀ52862
(1), currently in phase II clinical trials for the treatment of different pain conditions..12 Several 4ꢀ
acylaminopyrazolo[3,4ꢀd]pyrimidine derivatives, represented by 2 (Figure 1), showed interesting in
3

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vitro and ADMET profiles with the potential drawback of a certain unstability at acidic pH values.
Although this would not preclude a potential development of this type of compounds, a search for more
stable alternatives was undertaken. In this work, the replacement of the 4ꢀacylamino group by cyclic
substituents is described. This modification led to stable and highly active σ1R ligands, showing as well
good physicochemical and ADMET profiles.
Results and Discussion
Chemistry
The synthesis of the final compounds was effected from the commercially available 4ꢀchloro (3) and
4ꢀamino (6) pyrazolo[3,4ꢀd]pyrimidine derivatives (Scheme 1).13 Reaction of 3 with 1ꢀ(2ꢀ
hydroxyethyl)piperidine (4) under Mitsunobu conditions afforded the 1ꢀalkylated chloropyrimidine 5 in
good yield, while reaction of 6 with 1ꢀ(2ꢀchloroethyl)piperidine (7) provided 8. As reported in the
preceding article for the 4ꢀacylaminopyrazolo[3,4ꢀd]pyrimidine derivatives, the N1ꢀsustituted pyrazole
was the major regioisomer.6
Derivatives 9, in which the heterocycle is linked to the central scaffold by a nitrogen atom (Table 1),
were prepared from 5 or 8 using different conditions depending on the type of reagent used. Thus, the
saturated derivatives 9aꢀe were prepared by nucleophilic substitution of 5 with the corresponding amines
under mild basic conditions (Method A), the pyrrolidone 9f was prepared from 5 using Buchwaldꢀ
Hartwig conditions (Method B), the imidazole (9g) and benzimidazole (9h) derivatives were obtained
via nucleophilic substitution using a strong base (ButOK, Method C) and finally, the dimethylpyrrole 9i
was prepared by reaction of 8 with hexaneꢀ2,5ꢀdione (Method D).
Derivatives in which the cyclic group is linked to the central scaffold by a carbon atom (Tables 2ꢀ4)
were prepared from 5 using arylboronic derivatives (acids or esters, Method E, 10, 11aꢀg, 12aꢀk) or
aryltributylstannanes (Method F, 11hꢀj, 12lꢀm) under palladium catalysis and microwave irradiation.
Suzuki or Stille coupling were selected depending on availability and price of the required reagents and
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afforded the desired compounds with moderate to good yields, which decreased on steric hindrance
increase of the metalated reagents. General conditions used are depicted in Scheme 1, but in the case of
12f the more reactive catalyst PdCl2(dppf)·CH2Cl2 was used for the coupling, as described in the
experimental part.
SAR
In the previous article,6 where the structureꢀactivity relationship (SAR) study of 4ꢀacylamino
pyrazolo[3,4ꢀd]pyrimidine derivatives is described, the 2ꢀpiperidinylethyl substituent was shown to be
preferred in position 1. Thus, this group was selected for exploring the replacement of the amide moiety
in position 4 by cyclic derivatives, a change expected to improve the chemical stability of 2 at acidic pH
values.
All the compounds synthesized were evaluated in primary σ1R binding assay using [3H]ꢀ(+)ꢀ
pentazocine14 as radioligand (Ki values are given in Tables 1ꢀ3). Compounds having a Ki for the σ1R
lower than 500 nM were evaluated against the σ2R at 1 ꢀM using [3H]ꢀdiꢀoꢀtolylguanidine15 as
radioligand. As discussed previously,6 selectivity for the σ1R vs the σ2R is desired in view of the
differentiated pharmacological properties of the two receptors.
The SAR study was initiated by the introduction of heterocycles linked to the pyrazolo[3,4ꢀ
d]pyrimidine scaffold by a nitrogen atom (Table 1). The saturated derivatives piperidine (9a),
difluoropiperidine (9b), 3,3ꢀdimethylpyrrolidine (9c) and 3ꢀphenylpyrrolidine (9d) were shown to be
less active and selective than the parent pyvalamide 2. This result cannot be attributed to steric factors,
since as indicated in Figure 2A, compounds 2 and 9a showed a good overlap and fitted the positive
ionizable feature and two (HYD2 and HYD4) out of the four hydrophobic features of the Laggner11 σ1R
pharmacophore. The introduction of polar substituents (9e,f) was more detrimental, matching the results
obtained in the SAR study of the 4ꢀacylamino derivatives. The low activity of the imidazolꢀ1ꢀyl (9g) and
dimethylpyrrolꢀ1ꢀyl (9i) derivatives was recovered by 9h. As shown in Figure 2B, the benzimidazole
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group of 9h was able to cover the HYD4 hydrophobic feature, while the imidazole ring matched quite
well the carbonyl region of the acylamino derivatives.
Next, the introduction of Cꢀlinked aryl and heteroaryl derivatives was explored. As shown in Table 2,
different types of phenyl substituents were tolerated by the σ1R, the less active derivatives being the two
compounds containing a hydrogen acceptor moiety (OMe, 10f; CN, 10g) in position 4. However, none
of the phenyl derivatives showed the desired selectivity for the σ2R. The introduction of nitrogen atoms
in the phenyl ring was subsequently explored (Table 3), but it was detrimental. Compound 11f, with a
substituent in position 2, was the most active among the pyridyl derivatives, but yet less potent than the
phenyl analogues.
Finally, five membered heterocycles were introduced in position 4 (Table 4). The isothiazole (12a),
oxazole (12l) and thiazole (12m) derivatives did not improve the pyridine analogues, while the
intermediate affinity of pyrazole 12b was impaired on alkylation (12cꢀd). The 2,5ꢀdimethyloxazolꢀ3ꢀyl
derivative 12e provided an improvement in σ1R affinity, while the 1ꢀmethylpyrazolꢀ5ꢀyl derivative 12f
was even more active, affording as well an improvement in selectivity vs the σ2R. Introducing a
trifluoromethyl substituent on the pyrazole ring of 12f provided a decrease in activity (12g), as did
changing to the imidazole isomers (12i,j). The 1ꢀmethylpyrrolꢀ2ꢀyl (12h) and 3ꢀchlorothiophene (12k)
recovered affinity, but less selectivity was observed again.
Chemical stability was assayed for all the compounds having a Ki value for the σ1R below 100 nM
and none of them showed any sign of degradation in solution at pH 2 or 7.4, indicating that, as expected,
the replacement of amide groups by cyclic derivatives provided an improvement in stability.
The previous results show that the introduction of cyclic substituents in position 4 provides a decrease
in selectivity for the σ2R in relation to the amide analogues discussed in the previous paper. The higher
selectivity of compound 12f could be explained by the concurrence of two factors: its increased polarity
(cLogP 1.3) and the presence of the pyrazole nitrogen atom, which matches quite well the carbonyl
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group of 2 (Figure 2C) and indicates that the σ2R is less tolerant to polar groups in this region than the
σ1R.
Being the most selective compound, 12f was further profiled in vitro and failed to show any
significant affinity (inhibition at 10 ꢁM < 50%) in a panel of 65 receptors17 and no significant inhibition
(below 50 % at 1 µM) in recombinant human cytochrome P450 (rhCYP) isoforms (1A2, 2C9, 2C19,
2D6, and 3A4),18 suggesting low potential for drugꢀdrug interactions. Compound 12f showed a high
metabolic stability in human liver microsomes (Clint = 2.4 µl/min/mg prot.), a good indicator for
obtaining an appropriate pharmacokinetic profile in humans. It had also a good permeability in Cacoꢀ2
cells (Papp = 407 nm/s) and an efflux ratio of 0.7, indicating that it was not a Pꢀgp substrate.
Additionally, 12f did not show in vitro cytotoxic potential in the MTT and Neutral Red uptake assays
when tested up to 100 µM in HepG2 cells,19 and lacked genotoxic potential in the SOS/umu and Ames
bacterial mutation assays.20 As in the previous series, this good in vitro ADMET profile was
complemented by a low (IC50 > 10 ꢀM) blockage of the human EtherꢀaꢀgoꢀgoꢀRelated Gene (hERG)
potassium channel.21
Compound 12f (Figure 3) complied Lipinski’s rules, showing a low lipophilicity which together with
its good affinity provided one of the best lipophilic ligand efficiencies (LLE)22 ever described for a σ1R
ligand (LLE: 6.1) and at the same time, its total polar surface area (tPSA) was maintained in the desired
range for a good blood−brain barrier (BBB) penetration (59.2). In accordance with this reduced polarity,
its thermodynamic solubility was high in phosphate buffer both at pH 7.4 (1.5 mg/mL) and pH 2 (2.4
mg/mL).
In view of its good in vitro profile, compound 12f was advanced to in vivo testing in three pain
models in mice in comparison to 1 (Table 5). It showed a similar activity to 1, both i.p. and p.o., in the
formalin model, where the intraplantar (i.pl.) injection of formalin elicits an early phase (phase I, results
not shown) and a delayed phase (phase II) of pain characterized by paw licking, biting and other
behaviours.23 Compound 12f was also active, after oral administration, in the mouse model of
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mechanical hypersensitivity induced by i.pl. capsaicin24 and in the partial sciatic nerve ligation (PSNL)
model in mice,25 where it exhibited a substantial antiallodynic activity. The range of activity in the three
models was similar to that shown by 1, a result that reinforces the link of antagonistic behavior for the
σ1R and analgesia.
Conclusions
A change of the acylamino groups in the 4 position of the pyrazolo[3,4ꢀd]pyrimidine scaffold, by
cyclic substituents, led to highly active σ1R ligands with improved chemical stability. The SAR study
showed that phenyl or pyrazolyl groups were the best in terms of affinity for the σ1R. Introduction of
nitrogen atoms in the six membered rings was detrimental and only a few five membered heterocycles
provided a good affinity. The 4ꢀ(1ꢀmethylpyrazolꢀ5ꢀyl) derivative, 12f, was the most selective derivative
both over the σ2R and a wider panel of receptors. The compound showed as well a low lipophilicity,
which together with its good affinity, provided one of the best lipophilic ligand efficiencies (LLE) ever
described for a σ1R ligand. These properties resulted in a good physicochemical profile, including a
good thermodynamic solubility, a good ADMET profile and potent antinociceptive properties in several
pain models in mice, indicative of its antagonistic nature.
Experimental
Unless otherwise noted, all materials were obtained from commercial suppliers and used without
further purification. Microwave assisted reactions were conducted with an Explorer Synthesizer from
CEM. Flash chromatography was performed with a forced flow of the indicated solvent system on SDS
silica gel Chromagel 60 ACCꢀ(230ꢀ400 mesh) or on a CombiFlash Companion system with Redisep Rf
disposable columns. 1H spectra were recorded on an Agilent UNITY 300 MHz (spectrometer fitted with
a 5 mm H/F/X ATB probe) or an Agilent Mercury 400 MHz (spectrometer fitted with a 5 mm ID/PFG
probe) with 2 H lock in deuterated solvents. Chemical shifts (δ) are in parts per million. Commercially
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available reagents and solvents (HPLC grade) were used without further purification for all the
analytical tests. Analytical UHPLC−MS were performed on a Waters Acquity HꢀClassꢀMS ZQ system
using reverse phase C18 columns. Several analytical methods (1 to 6) were used depending on the
particular compound, as detailed in the ESI. Mass spectra were obtained over the range m/z 100−800 at
a sampling rate of 0.3 scans per second using Waters ZQ. Data were integrated and reported using
Waters MassLynx software. All compounds display purity higher than 95 % as determined by these
methods. Accurate mass measurements were carried out using an Agilent 6540 UHD AccurateꢀMass
QTOF system and obtained by electrospray ionization (ESI) in positive mode.
Calculation of molecular descriptors. clogP was calculated using ChemDraw Ultra 10.0.3. LLE was
calculated with the equation: LLE = pIC50– cLogP.
Determination of solubility. Solubility was measured as thermodynamic solubility from solid
compound in phosphate buffer after 24 h of stirring at pH = 7.4 using HPLC.
Determination of chemical stability. Chemical stability was determined from 10 mM DMSO
solution of test compounds in phosphate buffer at pH = 7.4 or 0.01 M HCl at pH = 2 by LC/MS.
4ꢀChloroꢀ1ꢀ[2ꢀ(piperidinꢀ1ꢀyl)ethyl]ꢀ1Hꢀpyrazolo[3,4ꢀd]pyrimidine (5). To a stirred solution of 4ꢀ
chloroꢀ1Hꢀpyrazolo[3,4ꢀd]pyrimidine (0.2 g, 1.29 mmol) in anhydrous THF (10 mL), 2ꢀ(piperidinꢀ1ꢀ
yl)ethanol (0.258 mL, 1.94 mmol) and triphenylphosphine (0.51 g, 1.94 mmol) were sequentially added.
The reaction mixture was cooled to 0 ºC and diisopropylazodicarboxylate (0.38 mL, 1.94 mmol) was
added dropwise. The mixture was stirred for 30 min. at 0 ºC and kept overnight at 4 ºC. The solvent was
removed at reduced pressure and the residue was dissolved in dichloromethane and washed with 1 M
HCl. The aqueous phase was separated, basified and extracted with dichloromethane. The organic phase
was separated, dried and the solvent was removed under reduced pressure to give a residue, that was
purified by flash chromatography eluting with (EtOAc/Petroleum ether, 8:2) to yield 5 (146 mg, 42 %)
1
as an oil. H NMR (400 MHz, CDCl3) δ 8.76 (1H, s, 6ꢀH), 8.15 (1H, s, 3ꢀH), 4.67 (2H, t, J=6.7 Hz, 1ꢀ
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Et), 2.93 (2H, t, J=6.6 Hz, 2ꢀEt), 2.65 – 2.42 (4H, m, 2'ꢀH, 6'ꢀH), 1.59 – 1.50 (4H, m, 3'ꢀH, 5'ꢀH), 1.47 –
1.34 (2H, m, 4'ꢀH). LC/MS (Method 7) Rt 2.70 min, purity 100%. MS (ESI): m/z [M+H]+ 266.2
1ꢀ[2ꢀ(Piperidinꢀ1ꢀyl)ethyl]ꢀ1Hꢀpyrazolo[3,4ꢀd]pyrimidinꢀ4ꢀamine (8). NaH (60 % in mineral oil,
651 mg, 16.3 mol) was added under argon atmosphere to a solution of 4ꢀaminoꢀ1Hꢀpyrazolo[3,4ꢀ
d]pyrimidine (6, 2.0 g, 14.8 mmol) in anhydrous DMF (25 mL) at 0 ºC. The mixture was left to reach
room temperature for 1 h, and then, after cooling to 0 ºC again, a solution of 1ꢀ(2ꢀchloroethyl)piperidine
(7, 2.19 g, 14.8 mmol) in anhydrous DMF (10 mL) was added dropwise. The mixture was stirred at
room temperature for 18 h and then quenched with H2O. The mixture was concentrated in vacuum and
partitioned between EtOAc and H2O. The combined organic layers were dried over anhydrous Na2SO4,
1
filtered and concentrated to dryness to yield 8 as a solid (1.63 g, 45% yield). H NMR (400 MHz,
CDCl3) δ 8.38 (1H, s, 3ꢀH), 7.94 (1H, s, 6ꢀH), 5.55 (2H, br s, NH2), 4.62 (2H, t, J=7.0 Hz, 1Et), 3.05 –
2.98 (2H, m, 2Et), 2.66 – 2.51 (4H, m, 2'ꢀH, 6'ꢀH), 1.66 – 1.61 (4H, m, 3'ꢀH, 5'ꢀH), 1.47 – 1.42 (2H, m,
4'ꢀH) LC/MS (Method 7) Rt 1.79 min, purity 100%. MS (ESI): m/z [M+H]+ 247.2.
Method
A:
4ꢀ(4,4ꢀDifluoropiperidinꢀ1ꢀyl)ꢀ1ꢀ[2ꢀ(piperidinꢀ1ꢀyl)ethyl]ꢀ1Hꢀpyrazolo[3,4ꢀ
d]pyrimidine (9b). A suspension of 4,4ꢀdifluoropiperidine hydrochloride (44 mg, 0.28 mmol) and
K2CO3 (78 mg, 0.56 mmol) in acetonitrile (3 mL) was stirred for 15 min. Then, compound 5 (50 mg,
0.19 mmol) in acetonitrile (2 mL) was added and the mixture stirred at room temperature overnight. The
suspension thus obtained was filtered and the solvent was removed under reduced pressure to give 9b as
1
an oil (65 mg, quantitative). H NMR (400 MHz, CDCl3) δ 8.38 (1H, s, 6ꢀH), 7.94 (1H, s, 3ꢀH), 4.54
(2H, t, J=7.2 Hz, 1Et), 4.14 – 4.06 (4H, m, 2''ꢀH, 6''ꢀH), 2.85 (2H, t, J=7.2 Hz, 2Et), 2.48 (4H, t, J=5.4
Hz, 2'ꢀH, 6'ꢀH), 2.20 – 2.05 (4H, m, 3'ꢀH, 5'ꢀH), 1.59 – 1.48 (4H, m, 3''ꢀH, 5''ꢀH), 1.45 – 1.36 (2H, m, 4''ꢀ
H). 13C NMR(CDCl3, 101 MHz): δ (ppm) 156.73 (Cꢀ4), 155.06 (Cꢀ6), 154.45 (Cꢀ7a), 131.96 (Cꢀ3),
121.65 (t, J=242.2 Hz, Cꢀ4'), 100.62 (Cꢀ3a), 57.77 (2Et), 54.63 (Cꢀ2'', Cꢀ6')', 44.87 (1Et), 42.61 (t, J=4.7
Hz, Cꢀ2', Cꢀ6'), 34.11 (t, J=23.5 Hz, Cꢀ3', Cꢀ5'), 26.09 (Cꢀ3'', Cꢀ5''), 24.38 (Cꢀ4''). LC/MS MS (Method
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1) Rt 1.69 min, purity 98 %. HRMS (ESI): m/z calculated for C17H25N6F2 [M+H]+ 351.2103, found
351.2108.
Method B: 1ꢀ{1ꢀ[2ꢀ(Piperidinꢀ1ꢀyl)ethyl]ꢀ1Hꢀpyrazolo[3,4ꢀd]pyrimidinꢀ4ꢀyl}pyrrolidinꢀ2ꢀone
(9f). A mixture of compound 5 (50 mg,0.19 mmol), pyrrolidinꢀ2ꢀone (0.07 mL), palladium diacetate
(4.22 mg, 1.7 mmol), Xantphos (16 mg, 2.7 mmol) and Cs2CO3 (67 mg, 0.20 mmol) in dry toluene (3
mL) was stirred at 120 ºC for 20 min under microwave irradiation (150 W) and argon atmosphere. The
mixture was filtered on decalite and the solvent was removed under reduced pressure to give a crude
product that was purified by flash chromatography to give 9f (12 mg, 20 % yield). 1H NMR (400 MHz,
CDCl3) δ 8.68 (1H, s, 6'ꢀH), 8.63 (1H, s, 3'ꢀH), 4.63 (2H, t, J=7.1 Hz, 1Et), 4.20 (2H, t, J=7.1 Hz, 5ꢀH),
2.93 (2H, t, J=7.1 Hz, 2Et), 2.75 (2H, t, J=8.1 Hz, 3ꢀH), 2.60 – 2.46 (4H, m, 6''ꢀH, 2''ꢀH), 2.35 – 2.15
(2H, m, 4ꢀH), 1.66 – 1.48 (4H, m, 5''ꢀH, 3''ꢀH), 1.48 – 1.35 (2H, m, 4''ꢀH).13C NMR (101 MHz, CDCl3):
δ (ppm) 175.17 (Cꢀ2), 154.53 (Cꢀ4'), 154.30 (Cꢀ6'), 151.52 (Cꢀ7a'), 136.71 (Cꢀ3'), 104.80 (Cꢀ3a'), 57.55
(2Et), 54.51 (Cꢀ6'', Cꢀ2''), 48.12 (Cꢀ5), 44.59 (1Et), 33.57 (Cꢀ3), 25.81 (Cꢀ5'', Cꢀ3''), 24.19 (Cꢀ4''), 18.46
(Cꢀ4). LC/MS (Method 1) Rt 1.45 min, purity 96 %. HRMS (ESI): m/z calculated for C16H23N6O
[M+H]+ 315.1955, found 315.1933.
Method C: 4ꢀ(1HꢀImidazolꢀ1ꢀyl)ꢀ1ꢀ[2ꢀ(piperidinꢀ1ꢀyl)ethyl]ꢀ1Hꢀpyrazolo[3,4ꢀd]pyrimidine (9g).
Potassium tertꢀbutoxide (51 mg, 0.45 mmol) was added to a solution of 1Hꢀimidazole (31 mg, 0.45
mmol) in acetonitrile (5 mL) at room temperature and the mixture was stirred for 15 min. Then,
compound 5 (100 mg, 0.37 mmol) was added and the mixture was stirred overnight at room
temperature. Water (0.5 mL) was added and the solvent was removed at reduced pressure. The resulting
solid was extracted with dichloromethane and the organic phase was washed with brine and removed
1
under reduced pressure to give 9g as a pale yellow solid (110 mg, quantitative). H NMR (400 MHz,
CDCl3) δ 8.83 (1H, s, 6ꢀH), 8.65 (1H, dd, J=1.3, 0.9 Hz, 2'ꢀH), 8.25 (1H, s, 3ꢀH), 7.93 (1H, t, J=1.5 Hz,
4'ꢀH), 7.31 (1H, dd, J=1.6, 0.9 Hz, 5'ꢀH), 4.67 (2H, t, J=6.9 Hz, 1Et), 2.89 (2H, t, J=6.9 Hz, 2Et), 2.47
(4H, t, J=5.3 Hz, 6''ꢀH, 2''ꢀH), 1.54 – 1.44 (4H, m, 5''ꢀH, 3''ꢀH), 1.45 – 1.33 (2H, m, 4''ꢀH). 13C NMR
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(101 MHz, CDCl3) δ (ppm) 155.32 (Cꢀ4), 154.91 (Cꢀ6), 149.16 (Cꢀ7a), 136.53 (Cꢀ3), 131.90 (Cꢀ2'),
130.93 (Cꢀ4’), 117.08 (Cꢀ5'), 103.26 (Cꢀ3a), 57.70 (2Et), 54.62 (Cꢀ6'', Cꢀ2''), 45.38 (1Et), 26.07 (Cꢀ5'',
Cꢀ3''), 24.34 Cꢀ(4''). LC/MS (Method 1) Rt 1.10 min, purity 97 %. HRMS (ESI): m/z calculated for
C15H20N7 [M+H]+ 298.1775, found 298.1778.
Method
D:
4ꢀ(2,5ꢀDimethylꢀ1Hꢀpyrrolꢀ1ꢀyl)ꢀ1ꢀ[2ꢀ(piperidinꢀ1ꢀyl)ethyl]ꢀ1Hꢀpyrazolo[3,4ꢀ
d]pyrimidine (9i). A mixture of compound 8 (50 mg, 0.20 mmol) in hexaneꢀ2,5ꢀdione (1.5 mL) was
stirred at 180 ºC for 20 min under microwave irradiation (150 W). The mixture was acidified with 1M
HCl and extracted with dichloromethane. The aqueous phase was basified with 20% NaOH and
extracted with dichloromethane. The organic phase was removed under reduced pressure and the residue
1
was purified by flash chromatography to give 9i as a brown solid (21 mg, 32%). H NMR (400 MHz,
CDCl3) δ 8.83 (1H, s, 6ꢀH), 8.29 (1H, br s, 4'ꢀH), 8.18 (1H, s, 3ꢀH), 6.46 – 6.33 (1H, m, 3'ꢀH), 4.62 (2H,
t, J=6.8 Hz, 1Et), 2.96 (2H, t, J=6.9 Hz, 2Et), 2.57 (3H, s, Me), 2.54 (4H, t, J=5.3 Hz, 2''ꢀH, 6''ꢀH), 2.29
(3H, s, Me), 1.62 – 1.49 (4H, m, 3''ꢀH, 5''ꢀH), 1.49 – 1.34 (2H, m, 4''ꢀH). 13C NMR (101 MHz, CDCl3) δ
(ppm) 158.78 (Cꢀ4), 154.96 (Cꢀ6), 153.25 (Cꢀ7a), 133.56 (Cꢀ3), 132.05 (Cꢀ5'), 127.27 (Cꢀ2'), 117.34 (Cꢀ
4'), 110.87 (Cꢀ3'), 107.36 (Cꢀ3a), 57.86 (2Et), 54.63 (Cꢀ2'', Cꢀ6''), 44.30 (1Et), 25.86 (Cꢀ3'', Cꢀ5''), 24.30
(Cꢀ4''), 14.19 (Me), 13.07 (Me). LC/MS (Method A) Rt 1.33 min, purity 97 %. HRMS (ESI): m/z
calculatedd for C18H25N6 [M+H]+ 325.2135, found 325.2141.
Method E: 4ꢀPhenylꢀ1ꢀ[2ꢀ(piperidinꢀ1ꢀyl)ethyl]ꢀ1Hꢀpyrazolo[3,4ꢀd]pyrimidine (10a). A mixture
of compound 5 (50 mg,0.19 mmol), phenylboronic acid (73 mg, 0.60 mmol) and anhydrous K2CO3 (82
mg, 0.59 mmol) in toluene (3 mL) was introduced in a microwave vial. It was degassed by argon for 30
min followed by the addition of Pd(PPh3)4 (4.0 mg, 0.003 mmol), after which the mixture was degassed
for 10 additional min. The mixture was stirred at 150 ºC for 30 min under microwave irradiation (150
W). After cooling to rt, the mixture was filtered on decalite and the solvent was removed at reduced
pressure. The crude was purified by flash chromatography to provide 10a as a white solid (19.1 mg,
33%). 1H NMR (300 MHz, CD3OD) δ 9.00 (1H, s, 6ꢀH), 8.53 (1H, s, 3ꢀH), 8.32 – 8.16 (2H, m, Ph), 7.63
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(3H, dq, J=5.3, 2.0 Hz, Ph), 4.70 (2H, t, J=6.8 Hz, 1Et), 2.94 (2H, t, J=6.8 Hz, 2Et), 2.54 (4H, t, J=5.2
Hz, 6'ꢀH, 2'ꢀH), 1.64 – 1.49 (4H, m, 5'ꢀH, 3'ꢀH), 1.49 – 1.37 (2H, m, 4'ꢀH). LC/MS (Method 1) Rt 1.60
min, purity 99 %. HRMS (ESI): m/z calculated for C18H22N5 [M+H]+ 308.1870, found 308.1876.
Method
E:
4ꢀ(1ꢀMethylꢀ1Hꢀpyrazolꢀ5ꢀyl)ꢀ1ꢀ[2ꢀ(piperidinꢀ1ꢀyl)ethyl]ꢀ1Hꢀpyrazolo[3,4ꢀ
d]pyrimidine (12f). A mixture of compound 5 (0.28 g, 1.05 mmol), 1ꢀmethylꢀ5ꢀ(4,4,5,5ꢀtetramethylꢀ
1,3,2ꢀdioxaborolanꢀ2ꢀyl)ꢀ1Hꢀpyrazole (0.33 g, 1.58 mmol), Na2CO3 (0.28 g, 2.63 mmol) and
PdCl2(dppf)·CH2Cl2 (0.09 g, 0.10 mmol) in CH3CN/H2O (1:1, 3mL), was placed in a vial, capped and
heated at 110 ºC for 10 min under microwave irradiation (5ꢀ250 W). After cooling to room temperature,
the mixture was poured into water (5 mL) and extracted with CH2Cl2 (4x5 mL). The combined organic
layers were dried over anhydrous Na2SO4, filtered and concentrated. The crude residue was purified by
medium pressure flash chromatography (Biotage SP1 25+M SiO2 column, 0ꢀ10% MeOH/CH2Cl2) to
1
furnish compound 12f as a pale yellow solid (215 mg, 66% yield). H NMR (400 MHz, CDCl3) δ 9.03
(1H, s, 6ꢀH), 8.24 (1H, s, 3ꢀH), 7.64 (1H, d, J=2.1 Hz, 3'ꢀH), 7.01 (1H, d, J=2.1 Hz, 4'ꢀH), 4.66 (2H, t,
J=7.0 Hz, 1Et), 4.38 (3H, s, NMe), 2.90 (2H, t, J=7.0 Hz, 2Et), 2.49 (4H, t, J=5.2 Hz, 2''ꢀH, 6''ꢀH), 1.57
– 1.46 (4H, m, 3''ꢀH, 5''ꢀH), 1.45 – 1.36 (2H, m, 4''ꢀH).
13C NMR (101 MHz, CDCl3) δ (ppm) 154.62 (Cꢀ6), 153.56 (Cꢀ7a), 151.39 (Cꢀ4), 138.82 (Cꢀ3'),
137.50 (Cꢀ5'), 132.51 (Cꢀ3), 111.34 (Cꢀ3a), 110.29 (Cꢀ4'), 57.78 (2Et), 54.64 (Cꢀ2'', Cꢀ6''), 45.00 (1Et),
40.37 (NMe), 26.06 (Cꢀ3'', Cꢀ5''), 24.34 (Cꢀ4''). The assignment of signals is according to the numbering
of Figure 3. LC/MS (Method 6) Rt 18.15 min, purity 99 %. HRMS (ESI): m/z calculated for C16H22N7
[M+H]+ 312.1931, found 312.1938.
Method F: 2ꢀ{1ꢀ[2ꢀ(Piperidinꢀ1ꢀyl)ethyl]ꢀ1Hꢀpyrazolo[3,4ꢀd]pyrimidinꢀ4ꢀyl}oxazole (12l). A
mixture of compound 5 (50 mg, 0.188 mmol), 2ꢀ(triꢀnꢀbutylstannyl)oxazole (0.059 mL, 0.282 mmol) in
toluene (3 mL) was introduced in a microwave vial. It was degassed by argon for 30 min followed by
the addition of Pd(PPh3)4 (22 mg, 19 mmol), after which the mixture was degassed for 10 additional
min. The mixture was stirred at 100 ºC for 30 min under microwave irradiation (150 W). The solvent
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was removed and the crude thus obtained was purified by flash chromatography to provide 12l as a
creamꢀcolored solid (37 mg, 66 %). 1H NMR (300 MHz, CDCl3) δ 9.12 (1H, s, 6'ꢀH), 8.71 (1H, s, 3'ꢀH),
8.09 – 7.88 (1H, m, 5ꢀH), 7.60 – 7.39 (1H, m, 4ꢀH), 5.06 – 4.68 (2H, m, 1Et), 3.10 (2H, s, 2Et), 2.76 –
2.35 (4H, m, 2''ꢀH, 6''ꢀH), 1.86 – 1.52 (4H, m, 3''ꢀH, 5''ꢀH), 1.52 – 1.32 (2H, m, 4''ꢀH). LC/MS (Method
1) Rt 1.46 min, purity 98 %. HRMS (ESI): m/z calculated for C15H19N6O [M+H]+ 299.1615, found
299.1620.
In vitro and in vivo tests: In vitro and in vivo (formalin, capsaicin and PSNL) tests were performed
using the same methods described in the previous article.6
Acknowledgment.
We thank Adriana Port, Raquel Enrech, Xavier Monroy, Pilar Pérez, Enrique Hernández, Eva Ayet,
Ariadna Balada, Raquel FernándezꢀReinoso, Sandra Yeste, Enrique Portillo, Beatriz de la Puente and
Daniel Zamanillo, for their expert contribution to analytical, in vitro and in vivo studies, Joan Andreu
Morató, Monica Carro, and Edmundo Ortega for their excellent technical assistance and Carlos Pérez
and Eduardo Villarroel for their contribution to compound management. We thank also the support from
Centro de Desarrollo Tecnológico e Industrial (CDTI, PROJECT IDIꢀ20110577).
Abbreviations.
ADMET
BBB
Absorption, Distribution, Metabolism, Excretion and Toxicity
BloodꢀBrain Barrier
Clint
Intrinsic clearance
cLogP
CNS
Calculated logarithm of the octanol/water partition coefficient
Central Nervous System
rhCYP
Recombinant human cytochrome P450
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3D
Threeꢀdimensional
hERG
human EtherꢀaꢀgoꢀgoꢀRelated Gene
High Resolution Mass Spectrometry
Human Liver Microsomes
HRMS
HLM
LLE
Lipophilic Ligand Efficiency
Apparent permeability coefficient
Partial Sciatic Nerve Ligation
StructureꢀActivity Relationships
Retention time
Papp
PSNL
SAR
Rt
σR, σ1R, σ2R Sigma, Sigmaꢀ1 and Sigmaꢀ2 Receptor, respectively
tPSA Total Polar Surface Area
References and Notes.
(1) R. Quirion, W. D. Bowen, Y. Itzhak, J. L. Junien, J. M. Musacchio, R. B. Rothman, T. P. Su, S.
W. Tam, D. P. A. Taylor, Trends Pharmacol. Sci. 1992, 13, 85.
(2) D. Zamanillo, E. PortilloꢀSalido, J.M. Vela, L. Romero, in Therapeutic Targets. Modulation,
Inhibition and Activation. Botana, J. M., Loza, M., Eds.; John Wiley & Sons, Inc.: Hoboken, NJ, 2012;
pp 225ꢀ278.
(3) T. Hayashi, T. P. Su. Cell 2007, 131, 596.
(4) D. Zamanillo, L. Romero, M. Merlos, J. M. Vela, Eur. J. Pharmacol. 2013, 716, 78.
(5) C. Almansa, J. M. Vela, Future Med. Chem. 2014, 6, 1179.
(6) J. L. Díaz, J. Corbera, R. Cuberes, M. Contijoch, R. Enrech, S. Yeste, A. Montero, A. Dordal,
X. Monroy, C. Almansa, Med. Chem. Commun. Previous paper in this issue.
(7) M. J. Waring, Expert Opin. Drug Discov. 2010, 5, 235.
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(8) S. Brune, S. Pricl, B. Wünsch, J. Med. Chem. 2013, 56, 9809.
(9) H. R. Schmidt, S. Zheng, E. Gurpinar, A. Koehl, A. Manglik, A. C. Kruse, Nature, 2016, 532,
527.
(10) R. A. Glennon, S. A. Ablordeppey, A. M. Ismaiel, M. B. ElꢀAshmawy, J. B. Fischer, K. B.
Howie, J. Med. Chem. 1994, 37, 1214.
(11) C. Laggner, C. Schieferer, B. Fiechtner, G. Poles, R. D. Hoffmann, H. Glossmann, T. Langer,
E. F. Moebius, J. Med. Chem. 2005, 48, 4754.
(12) M. Abadias, M. Escriche, A. Vaqué, M. Sust, G. Encina, Br. J. Clin. Pharmacol. 2013, 75, 103.
(13) (a) C. Almansa, J. L Díaz, J. Corbera, PCT Int. Appl. (2014), WO2014/076170. (b) D.
Martínez, J. L Díaz, J. Corbera, C. Almansa, A. Montero, A. Dordal, I. Álvarez, Poster communication
SpanishꢀItalian Medicinal Chemistry Congress (SIMCC), Barcelona, 2015.
(14) D. L. DeHavenꢀHudkins, L. C. Fleissner, F. Y. FordꢀRice, Eur. J. Pharmacol. 1992, 227, 371ꢀ
378.
(15) G. Ronsisvalle, A. Marrazzo, O. Prezzavento, A. Cagnotto, T. Mennini, C. Parenti, G. M. Scoto,
Pur. Appl. Chem. 2001, 73, 1499.
(16) C. Laggner, C. Schieferer, B. Fiechtner, G. Poles, R. D. Hoffmann, H. Glossmann, T. Langer,
E. F. Moebius, J. Med. Chem. 2005, 48, 4754.
(17) Compound 12f failed to show any significant activity at 10 µM in the standard Ricerca panel of
65 receptors, ion channels and enzymes.
(18) D. M. Stresser, in Optimization in Drug Discovery. In vitro methods; Yan, Z.; Caldwell, G. W.;
Eds. Humana Press: Totowa, New Jersey, 2004; pp 215ꢀ230.
(19) K. P. Putman, Toxicology in Vitro 2000, 16, 599.
(20) (a) D. M. Maron, B. N. Ames, Mutation Research, 1983, 113, 173; (b) G. Reifferscheid, J. Heil,
Mutation Research 1996, 369, 129.
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(21) G. J. Diaz, K. Daniell, S. T. Leitza, R. L. Martin, Z. Su, J. S. McDermott, JB. F. Cox, G. A.
Gintant, J. Pharmacol. Toxicol. Methods 2004, 50, 187.
(22) P. D. Leeson, B. Springthorpe, Nat. Rev. Drug Discovery 2007, 6, 881.
(23) J. H. Rosland, A Tjolsen, B. Maehle, K. Hole, Pain 1990, 42, 235.
(24) J. M. Entrena, E. J. Cobos, F. R. Nieto, C. M. Cendán, G. Gris, E. Del Pozo, D. Zamanillo, J.
M. Baeyens, Pain 2009, 143, 252.
(25) A. B. Malmberg, A. I. Basbaum, Pain 1998, 76, 215.
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Table 1. Cyclic nitrogenated derivatives in position 4.
a
Ki σ1
% 1 ꢀM
σ2 (gp)
comp
1
b
(h, nM)
17
ꢀꢀꢀ
ꢀꢀꢀ
67
67
58
71
ꢀꢀꢀ
51
200
2
Piperidinꢀ1ꢀyl
9a
9b
9c
9d
9e
9f
4,4ꢀDifluoropiperidinꢀ1ꢀyl
3,3ꢀDimethylpyrrolidinꢀ1ꢀyl
3ꢀPhenylpyrrolidinꢀ1ꢀyl
3ꢀMethylaminopyrrolidinꢀ1ꢀyl
2ꢀOxopyrrolidinꢀ1ꢀyl
108
165
294
25770
6194
Imidazolꢀ1ꢀyl
Benzimidazolꢀ1ꢀyl
13803
112
ꢀꢀꢀ
66
ꢀꢀꢀ
9g
9h
9i
2,5ꢀDimethylpyrrolꢀ1ꢀyl
740
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aBinding affinity (Ki) to human σ1R in transfected HEKꢀ293 membranes
using [3H](+)ꢀpentazocine as radioligand. Each value is the mean of two
b
determinations. Binding affinity (% inhibition at 1 ꢀM) to σ2R in guinea pig
brain membranes using [3H]ꢀdiꢀoꢀtolylguanidine as radioligand. Each value is
the mean of two determinations.
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Table 2. Substituted phenyl moieties in 4ꢀposition.
a
Ki σ1
% 1 ꢀM
σ2 (gp)
comp
10a
R
cLogP
b
(h, nM)
H
100
2.8
3.1
3.4
2.9
3.0
2.9
2.3
3.4
3.6
3.9
3.6
3.2
69
83
82
81
75
58
47
92
74
95
89
83
2ꢀMethyl
3ꢀMethyl
74
82
10b
10c
10d
10e
10f
10g
10h
10i
2ꢀEthoxy
300
95
4ꢀFluoro
4ꢀMethoxy
4ꢀCyano
531
558
76
2ꢀChloro
4ꢀChloro
43
2ꢀChloroꢀ5ꢀmethyl
2,5ꢀDimethyl
2,4ꢀDifluoro
31
10j
10k
10l
63
93
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4ꢀFluoroꢀ2ꢀmethoxy
39
48
87
86
73
87
86
2.6
3.2
3.8
3.9
3.8
10m
10n
10o
10p
10q
4ꢀFluoroꢀ2ꢀmethyl
4ꢀChloroꢀ2ꢀfluoro
112
57
4ꢀFluoroꢀ3ꢀ
trifluoromethyl
2ꢀTrifluoromethyl
125
a,bSee footnotes of Table 1.
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Table 3. 6ꢀMembered heterocycles in 4ꢀposition.
a
Ki σ1
comp
11a
R
(h, nM)
Pyridinꢀ3ꢀyl
926
2ꢀMethylpyridinꢀ3ꢀyl
2ꢀMethoxypyridinꢀ5ꢀyl
4ꢀMethylpyridinꢀ3ꢀyl
5ꢀMethylpyridinꢀ3ꢀyl
2ꢀMethoxypyridinꢀ3ꢀyl
Isoquinolinꢀ4ꢀyl
732
38293
707
11b
11c
11d
11e
11f
11g
11h
11i
447
112
666
Pyridinꢀ2ꢀyl
634
6ꢀMethylpyridinꢀ2ꢀyl
Pyridinꢀ4ꢀyl
709
1717
11j
aSee footnote of Table 1.
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Table 4. 5ꢀMembered heterocycles in 4ꢀposition.
a
b
Ki σ1
% 1 ꢀM σ2
comp
12a
R
cLogP
(h, nM)
(gp)
475
40
ꢀꢀꢀ
1.5
802
ꢀ0.2
12b
12c
9460
ꢀꢀꢀ
1.1
1.9
2915
ꢀꢀꢀ
76
12d
72
41
1.0
1.3
12e
12f
>1000 nMc
137
72
2.3
12g
N
31
78
ꢀꢀꢀ
2.2
1.0
12h
12i
715
11843
ꢀꢀꢀ
1.0
12j
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Cl
35
75
3.5
12k
S
23832
ꢀꢀꢀ
ꢀꢀꢀ
0.7
1.4
12l
1834
12m
a,bSee footnotes of Table 1. cKi value
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Table 5. In vivo activity of 12f in comparison to 1.
Formalina
40 mg/kg i.p.
Phase II
Formalina
80 mg/kg p.o.
Phase II
Capsaicinb
PSNLc
80 mg/kg p.o. 80 mg/kg p.o.
50 ± 13
66 ± 9
48 ± 7
43 ± 2
62 ± 17
46 ± 21
1
67 ± 8
54 ± 15
12f
aPercentage of antinociception in the formalin test evaluated as the lickingꢀbiting
b
time in drugꢀtreated animals vs vehicle. Percentage reduction of mechanical
hypersensitivity in the capsaicin test evaluated as the latency time to the paw
withdrawal response to upward pressure by von Frey filament stimulation in drugꢀ
c
treated animals vs vehicle. Percentage reduction of mechanical hypersensitivity
induced by partial sciatic nerve ligation evaluated as the pressure threshold (grams)
required to elicit the paw withdrawal response following von Frey filament
stimulation in drugꢀtreated animals after compound administration vs response 8
days after surgery (pain condition, vehicle treatment) and before nerve ligation
(baseline normal conditions).
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Figure 1. Reference compound 1 and lead compound 2.
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Figure 2. 3D superposition of compound 2 with ligands 9a (A), 9b (B) and 12f (C) correlated with the Laggner
model
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Figure 3. Physicochemical properties of compound 12f
.
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Scheme 1
Reagents and conditions: a) Diisopropylazodicarboxylate, PPh3, THF, 4 ºC, 18 h; b) Method A: K2CO3,
ACN, rt; c) Method B: Amine, Pd(OAc)2, Xantphos, Cs2CO3, Tol, MW, 150 ºC, 30 min; d) Method C:
Amine, ButOK, ACN, rt; e) NaH, DMF, rt, 18 h; f) Method D: Hexaneꢀ2,5ꢀdione, MW; g) Method E:
ArylB(OH)2 (or ester), Pd(PPh3)4, K2CO3, Tol, MW, 150 ºC, 30 min; h) Method F: ArylSnBu3,
Pd(PPh3)4, K2CO3, Tol, MW, 150 ºC, 30 min.
29