Design, Synthesis, and Development of Pyrazolo[1,5-a]pyrimidine Derivatives as a Novel Series of Selective PI3Kδ Inhibitors: Part II—Benzimidazole Derivatives

Abstract

Phosphoinositide 3-kinase (PI3K) is the family of lipid kinases participating in vital cellular processes such as cell proliferation, growth, migration, or cytokines production. Due to the high expression of these proteins in many human cells and their involvement in metabolism regulation, normal embryogenesis, or maintaining glucose homeostasis, the inhibition of PI3K (especially the first class which contains four subunits: α, β, γ, δ) is considered to be a promising therapeutic strategy for the treatment of inflammatory and autoimmune diseases such as systemic lupus erythematosus (SLE) or multiple sclerosis. In this work, we synthesized a library of benzimidazole derivatives of pyrazolo[1,5-a]pyrimidine representing a collection of new, potent, active, and selective inhibitors of PI3Kδ, displaying IC₅₀ values ranging from 1.892 to 0.018 μM. Among all compounds obtained, CPL302415 (6) showed the highest activity (IC₅₀ value of 18 nM for PI3Kδ), good selectivity (for PI3Kδ relative to other PI3K isoforms: PI3Kα/δ = 79; PI3Kβ/δ = 1415; PI3Kγ/δ = 939), and promising physicochemical properties. As a lead compound synthesized on a relatively large scale, this structure is considered a potential future candidate for clinical trials in SLE treatment.

1. Introduction

Phosphoinositide 3-kinase delta (PI3Kδ), the lipid kinase, is a member of the family of PI3K enzymes divided into three classes: I (PI3Kα, PI3Kβ, PI3Kγ, PI3Kδ), II, and III. Due to their involvement in catalyzing the phosphorylation of phosphatidylinositol-4,5-diphosphate (PIP2) to phosphatidylinositol-3,4,5-triphosphate (PIP3), PI3Ks start a cascade of downstream activities to induce various types of biological processes such as cell growth, survival, proliferation, or differentiation [1,2,3,4,5,6]. All class I PI3K isoforms occur as a heterodimer of one regulatory subunit (p85) with the corresponding catalytic subunit (p110α, p110β, and p110δ). The p110 subunits of PI3K isoforms have been thoroughly characterized [7]. The ATP-binding site of p110δ in PI3Kδ comprises a few functionalities such as a hinge pocket, an affinity pocket, and a hydrophobic region, which lies below a non-conserved rim of the active site. Interaction with the large and flat hydrophobic face of a conserved tyrosine residue (Tyr-876) was reported for most PI3K inhibitors. Moreover, many of them also have additional hydrophobic interactions with the affinity pocket for the enzyme, where they can form hydrogen bonds with Lys-833 or other hydrophilic residues caused by the presence of adequate heteroatom [8,9]. Most selective PI3Kδ inhibitors exhibit interaction between crucial amino acids (Trp-760 and Met-752) while entering the active pocket [9,10,11]. Interaction with the tryptophan shelf (Trp-760) impacts the PI3Kδ selectivity. Steric blockage in the tryptophan region leads to selectivity for PI3Kδ because of the disfavored binding to other PI3K isoforms. It has been proven that these structural determinants are crucial in the activity and selectivity of PI3Kδ and thus are used for designing PI3Kδ inhibitors [5,8,10,11].

In this study, we designed, developed, and described a family of PI3Kδ inhibitor structures, based on the pyrazolo[1,5-a]pyrimidine core with different modifications, which can occupy the affinity pocket of the enzyme.

The basis for inflammatory and autoimmune diseases, such as systemic lupus erythematosus (SLE) or rheumatoid arthritis (RA), is dysregulation, including overactivity of the immune system [8]. These alterations are typically progressive and cause much burdensomeness for the patients. The overproduction of autoantibodies manifests in SLE due to uncontrolled cellular action in which T lymphocytes and B lymphocytes play a crucial role [4,12,13,14,15,16,17]. The activity of PI3Kδ in T cells of SLE diagnosed patients is risen by approximately 70% [15]. Due to the engagement of the p110δ subunit of the PI3Kδ in human Th17 cells for the production of IL-17, this PI3K subfamily can be viewed as a promising molecular target for future therapies, including SLE [18,19,20,21]. Moreover, a well-recognized mechanism of the PI3Kδ interaction at the molecular level can be efficiently utilized for the rational design, synthesis, and development of new anti-inflammatory drugs [18,19,20,22].

Selective PI3Kδ inhibitors can be obtained by appropriate modifications of a heterocycle that occupies the affinity pocket of the enzyme [5,10,23]. Despite the formation of hydrogen bonds between the indazole group of well-known inhibitor GDC-0941 (Figure 1) [24] and two amino acids Asp-787 and Tyr-813, the PI3Kγ/δ selectivity was poor [10]. It was reported that changing the indazole group of GDC-0941 for 2-methylbenzimidazole group helped to obtain a more selective inhibitor of PI3Kδ (PI3Kγ/δ = 29) which demonstrated good potency in cellular assays [5,10]. Moreover, it was shown that optimization of interactions with Trp-760 helped to improve the selectivity of PI3Kδ inhibitors as candidates for further development with good pharmacokinetic properties [10,11,25,26]. The new compounds with different substituents at the benzimidazole ring’s C(2) position were obtained and described [10]. It was reported that the inclusion of large, bulky groups at the benzimidazole’s C(2) position could reduce the inhibition of PI3Kδ [10]. These structure–activity relationships highlight the crucial role of the amine and benzimidazole subunit in determining PI3Kδ activity and selectivity for an obtained series of compounds.

Many bicyclic cores-based compounds were reported as effective and active PI3K inhibitors. Most of them, including thienopyrimidines [23,27] or pyridopyrimidines [10,23,24], were described as pan-PI3K inhibitors. Due to the problems with time-dependent CYP inhibition [5,23], selectivity, bioavailability, or solubility [23,28], other bicyclic cores such as isoxazolopyrimidines [23,29], imidazopyrimidines [23,30], or pyrazolopyrimidines [23,31] have also been designed, synthesized, and reported. A large number of PI3K inhibitors showed the potential of applying morpholine moiety as a H-bond acceptor in the hinge-binding motif [23,24]. In our docking studies in the previous paper [25], the crucial role of binding between the morpholine system and Val-828 was observed. Since 2012, many morpholine-based inhibitors of the PI3K kinase have been published [23]. Moreover, in 2012, a group of 2-(difluoromethyl)-1H-benzimidazole derivatives enriched a library of known PI3K inhibitors [23,32,33,34]. These structures are based on the 1,3,5-triazine monocyclic core and a morpholine ring in the hinge region. Evaluation of mono-, bi-, or higher-cyclic cores with a different arrangement of the substituents allowed for more active and selective compounds [10,23,28].

In our work, we focused on the pyrazolo[1,5-a]pyrimidine core with various amine substituents in position C(2) and different benzimidazole groups in the C(5) position at the core region. It was reported that pyrazolo[1,5-a]pyrimidines are promising medical pharmacophores in structures as potential drugs in the treatment of cancer, as well as inflammatory or viral diseases [35,36]. Our previous study [25] described the development of pyrazolo[1,5-a] pyrimidine derivatives with different substituents (heteroaromatic systems) at position 5 of the mentioned core. It was reported that 5-indole-pyrazolo [1,5-a] pyrimidines as inhibitors of PI3Kδ were the most selective structures of the obtained series. On the other hand, we identified a 2-difluoromethylbenzimidazole derivative 1 as the most active compound (Figure 1). It was identified as a moderate PI3Kδ inhibitor (IC₅₀ = 475 nM) with poor selectivity toward the alpha isoform. We reported that modifications of a mentioned core with many different substituents could contribute to inhibitors’ activity and selectivity enhancement. In this work, we synthesized and described more than thirty new, active, and potent selective PI3Kδ inhibitors in the extended structure–active relationship (SAR) study, keeping the scaffold of 1 as a starting point.

2. Results and Discussion

2.1. Chemistry

2.1.1. Synthesis of Compounds 5–9 and 11–12

Our research shows that modifications of benzimidazole groups and amine subunits play a crucial role in the activity and selectivity of PI3Kδ inhibitors. During the SAR exploration and docking calculations, we found two amine subunits at the C(2) position of the pyrazolo[1,5-a]pyrimidine core, N-tert-butylpiperazin-1-ylmethyl and 2-(4-piperidin-1-ylmethyl)-2-propanol, have the most promising potency in PI3Kδ inhibition. Due to the observed high activity of the mentioned families of compounds against PI3Kδ and unexplored chemical space, new benzimidazole derivatives were synthesized according to Scheme 1.

Starting from ethyl 5-chloro-7-(morpholin-4-yl)pyrazolo[1,5-a]pyrimidine-2-carboxylate, structures 5–9 and 11–12 were obtained after four-step synthesis. In the first step, alcohol 2 was synthesized by the ester group reduction with sodium borohydride and an almost quantitative yield (99%, Scheme 1). Next, alcohol 2 was oxidized into the corresponding aldehyde 3 using Dess–Martin periodinane (46% yield). Subsequently, the amine subunits derivatives 4 and 10 were obtained in reductive amination reactions by engaging appropriate amine in the presence of sodium triacetoxyborohydride (good, 84%, and 63% yields, respectively). In the last step, the received substituted 5-chloro-pyrazolo[1,5-a]pyrimidines 4 and 10 were transferred into the final structures by utilizing the Buchwald–Hartwig reaction conditions with corresponding benzimidazoles. This palladium-catalyzed reaction, conducted under microwave irradiation, gave compounds 5–9 and 11–12 (un-optimized yields in the range of 34–93%).

2.1.2. Synthesis of Compounds 16–38 and 40–54

Benzimidazole derivatives were prepared in a multistep synthesis that branched into two pathways depending on the group selected in the core C(2) position (Scheme 2).

Structures 16–38 and 40–57 were synthesized according to the general pathway depicted in Scheme 2. Compounds 16–38 were obtained after four-step synthesis from 5-chloro-7-(morpholin-4-yl)pyrazolo[1,5-a]pyrimidine-2-carboxylate (commercially available) which was coupled with 2-(difluoromethyl)-1H-benzimidazole in the presence of tetraethylammonium chloride and potassium carbonate to provide ethyl 5-[2-(difluoromethyl)-1H-1,3-benzimidazol-1-yl]-7-(morpholin-4-yl)pyrazolo[1,5-a]pyrimidine-2-carboxylate (13) as a crucial intermediate (89% yield). Then, the resulting product 13 was reduced to alcohol 14 under treatment with a lithium aluminum hydride solution (89% yield). We observed that the double bond within the imidazole ring of benzimidazole substituent was reduced concomitantly with the ester group. Interestingly, the oxidation of alcohol 14 to aldehyde 15 with Dess–Martin periodinane (or with activated manganese(IV) oxide) was accompanied by full restoration of aromaticity within the benzimidazole heterocycle. Final structures 16–38 were obtained by reductive amination reactions (Scheme 2) with different amines such as N-tert-butylpiperazine, morpholine, or 4-methylpiperidin-4-ol (see tables below for details) with un-optimized yields (38–93%). Based on our previous SAR studies, we observed that the carbonyl group in position C(2) of pyrazolo[1,5-a]pyrimidine derivatives may enhance the activity of the obtained structures. While keeping this in mind, further research focused on the replacement of the (-CH₂) group with a (-CO) group leading to structures 40–57 in two steps from intermediate 13. Unlike for compounds 16–38 synthesis, the ester group of 13 was hydrolyzed with the lithium hydroxide to 5-[2-(difluoromethyl)-1H-benzimidazol-1-yl]-7-(morpholin-4-yl)pyrazolo[1,5-a]pyrimidine-2-carboxylic acid (39, 98% yield). Subsequently, carboxyl derivatives were converted into a series of final amides 40–57 (see tables below to trace the selection of substituents) using 2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU) and triethylamine as a base (un-optimized 33–81% yields).

2.2. Docking Study

Crystal structure analysis, combined with data from biochemical and cellular assays, has been used to understand the molecular basis of observed inhibitors’ activities and selectivities. Utilizing the Auto-Dock Vina program for docking studies [35], we wished to investigate the binding mode of our compounds with the PI3Kδ isoform. Based on the available crystallographic structures of PI3K (for example PDB ID: 2WXL) and reference papers regarding in silico calculations [5,8,10,23,24], we gained valuable information about protein–ligand interactions in the active site and chose to focus on the pyrazolo[1,5-a]pyrimidine core.

Over the course of our computer-assisted studies, we found that the morpholine ring at position 7 of pyrazolo[1,5-a]pyrimidine core is required for interaction with PI3K at the catalytic site. More specifically, the most crucial interaction is the critical hydrogen bond between the oxygen atom of the morpholine group and Val-828 in the hinge region of the enzyme. In our previous work [25], we observed an existing hydrogen bond between the C(5)-indole pyrazolo[1,5-a]pyrimidine derivatives and the Asp-787. However, benzimidazole derivatives, presented in this work, lack that interaction when targeted toward this region. Instead, we observed the possibility of hydrogen bond formation between the nitrogen atom at the third position of the benzimidazole ring system and Lys-779.

In addition, several regions have been identified in the active site of the enzyme that have a profound impact on the activity and selectivity toward PI3Kδ. Due to critical structural determinants, depending on the substituent type (R¹, R², Scheme 1 and Scheme 2, respectively), we observed different interactions of our structures with the tryptophan shelf (Trp-760) and selected amino acids within the active pocket. For example, 2-hydroxypropyl residue of compound 11 keeps close proximity to Trp-760 (the tryptophan shelf interaction, Figure 2A) by locating the hydroxyl group conformationally away from the amino acid. On the other hand, the piperazine fragment of compound 17 (Figure 2B) takes the most distant position from the tryptophan shelf, supported by the polar amide group bond with aspartic acid (Asp-897). Moreover, among some structures showing no Trp-760 interaction, due to different types of the amine substituents, a shift towards other amino acids, e.g., Ser-831, was observed.

Compounds containing a donor fragment, such as hydroxyl, amine, or the amide group near the piperazine or piperidine ring (such as 11, 35, or 36), are found to have higher IC₅₀ values and therefore lower potential for activity due to the poor interaction between the aliphatic fragment and the tryptophan shelf (Trp-760). A similar situation is observed for structure 30 (Figure 2C), in which the aliphatic component targets the Trp-760 indole ring, but the hydroxyl group is too far to form a hydrogen bond with the polar amino acids located at the opposite side of the enzyme pocket.

A shift beyond the tryptophan region of the piperidine ring was also observed for compound 49, with the carboxyl group introduced in place of the methylene group. Such arrangement is additionally supported by the formation of a hydrogen bond between the hydroxyl group of the 4-hydroxy-4-methylpiperidinyl subunit and aspartic acid (Asp-832, Figure 2D).

In connection with the described dependencies, our research suggests that due to the shift of the amine ring relative to the Trp-760 and the formation of a hydrogen bonding with the aforementioned Asp-832 and Asp-897, compounds with a carboxyl group in the C(2) position of the pyrazolo[1,5-a]pyrimidine (linking the amine group) are more preferred in terms of kinase activity and selectivity than compounds with a methylene group at the same position.

Compound 6 (Figure 2E), containing the tert-butyl piperazine ring, gave different outcomes in our docking studies. Interaction between that aliphatic fragment and Trp-760 translates into the properties of this compound, such as potency, activity, and selectivity towards PI3Kδ. Moreover, in this structure, we observed the characteristic bond between the oxygen atom located at the morpholine ring (playing the role of an H-bond acceptor in the hinge-binding motif) and Val-828. Interaction of the benzimidazole residue nitrogen atom and Lys-779 has also been recognized.

A mix of conformational interactions was assigned to compound 40 (Figure 2F), which binds to Val-828 and Lys-779, including compound 6. However, the replacement of the methylene bridge with the carbonyl function was associated with the loss of Trp-760 interaction and the simultaneous loss of biological activity.

As a result of all relationships described, compound 6 turned out to be the most active and promising structure of the entire library obtained.

2.3. Biological Evaluation

In Vitro PI3 Kinase Inhibition Assays

All compounds were tested in a biochemical assay that measured the inhibition of phosphatidylinositol (4,5)-bisphosphate (PIP2) production by PI3K isoforms using the ADP-Glo kinase assay (Promega). In addition, the effects of synthesized compounds on B cell proliferation were measured.

All newly synthesized compounds proved to be active PI3Kδ inhibitors (IC₅₀ = 1.892–0.018 μM) and, additionally, eleven of obtained structures turned out to be highly active, reaching the value of IC₅₀ below 100 nM. For this reason, PI3Kδ final inhibitors, which are very potent with drug-like physical properties, are considered drug candidates in SLE and other autoimmune and inflammatory diseases.

Two positions of the pyrazolo[1,5-a]pyrimidine core: C(2) (R¹) and C(5) (R²) were optimized and described in this paper. Regarding the first C(2) optimization, many benzimidazole derivatives (for two chosen amine subunits: piperazines and piperidines) were designed and synthesized (Table 1). It was observed that within the nano- and micro-molar IC₅₀ value range, the potency of obtained inhibitors is different despite the substituent size at the C(2) position, for example in pairs 5 and 8 or 6 and 9. On the other hand, a selected pair of examples (compounds 11 and 12) indicates that the selectivity against PI3Kδ activity is relatively insensitive to the steric bulkiness of the substituent placed at the C(5) core’s position. Of the whole series of PI3Kδ inhibitors obtained, compounds 6 and 11 turned out to be the most potent, with IC₅₀ values of 18 and 52 nM, respectively. Moreover, structure 6 shows the best selectivity towards other PI3K isoforms among all the compounds tested. For the above reasons, 2-(difluoromethyl)-1H-benzimidazole was selected as the most optimal and promising R¹ substituent in pyrazolo[1,5-a]pyrimidine ring. The next step of our studies was the expansion of the compound library with the determination of R² groups keeping the constant 2-(difluoromethyl)-1H-benzimidazole R¹ substituent at C(5) position.

The R² substituent has been optimized using different substituents which aim to simultaneously increase selectivity and activity. Optimization focused on modifying the amine subunit; thus, piperazines, piperidines, five-member rings, bulky amine groups, and other available amines were used (Table 2). Among all modifications, we found the piperazine and piperidine derivatives as the most promising. Compounds containing an amine group with a five-membered ring showed the IC₅₀ values in the 1892–1896 nM range; however, their PI3Kγ/δ selectivities remained lower than for the other amine groups with a six-member ring in their structure (Table 2). This observation suggests that replacing the five- with a six-membered ring is more favorable for PI3Kδ inhibition. Moreover, heterocycles based on the six-membered ring as R², with a nitrogen atom in the 1- or 1- and 4-position(s), are generally more active and selective. As an excellent example, this thesis can serve compound 27, with the nitrogen atom shifted outside the six-membered ring. In this individual case, a significant potency drop against PI3Kδ below 1000 nM threshold was noted (Table 2). Modifications of substituted amine heterocycles are significant because they directly affect the PI3Kδ enzyme’s affinity pocket interactions. Our preliminary research suggested that the presence of the 2-(4-piperidyl)-2-propanol hydroxylic group could play a crucial role in gaining the inhibition of PI3Kδ. We believed, based on in silico studies, that the bond formed between the hydroxyl group and amine group of Ser-831 within the selectivity pocket of the enzyme should increase both the activity and selectivity. Therefore, a set of compounds containing proton donor groups was synthesized. Unfortunately, this rationale failed, as it can be clearly seen while searching the IC₅₀ values of compounds: 29–31 and 34–36 (Table 2). The observed loss of potency could be explained by the conformational freedom provided by the methylene linkage, allowing the escape from the tryptophan shelf position and simultaneous polar interactions with side amino acids within the active pocket. To confront this possibility, the methylene bridge was converted into carbonyl function, leading to conformationally restrained cyclic amide derivatives which lack possible conformational changes in the C(2) position of pyrazolo[1,5-a]pyrimidine.

For that reason, a library of compounds with multiple amine substituents was designed and synthesized (Table 3). Within this set, the lowest IC₅₀ values were observed for examples 40, 42, 43, and 55, (measured at 84, 74, 63, and 82 nM, respectively). These structures also had good PI3Kγ/δ selectivity.

The consequence of the methylene bridge to carbonyl group interchange can be tracked separately in groups of compounds divided into those with or without hydrogen bond donor (HBD) capabilities within the R² substituent.

The compounds lacking the HBD functionally tend to have better potency when the methylene bridge at the C(2) core position is present within a stable substituent environment. Although some exceptions have been found, including the pairs 33 and 52 or 37 and 56, for all other pairs, such as 6 and 40 or 38 and 57, the PI3Kδ activity drops when the methylene bridge is replaced with its carbonyl structural equivalent (Scheme 3). The measured potency is positively correlated with the increasing spherical lipophilic volume present at position 4 in the six-membered heterocyclic ring of the amino substituent. As an example, compounds 37, 38 (Table 2), and 6 (Table 1), bearing methyl, iso-propyl, and tert-butyl motif, can be named, for which the IC₅₀ value at concentrations of 351, 38, and 18 nM was measured, respectively.

The activity dependence on the relationship between the methylene bridge and carbonyl interchange is not so clear for the compounds with structural capabilities of HBD interaction within the R² substituent. Since it is challenging to isolate the bulkiness of the substituent alone and the accompanying HBD interplay with the surrounding polar environment, both those elements might affect the IC₅₀ value. As seen in Scheme 4, there is only a slight prevalence of increased activity toward the carbonyl (amide) functionality. Therefore, both structural motifs (the methylene bridge and the carbonyl function) should be considered equally essential modifications for SAR exploration.

A detailed analysis of the entire library of synthesized compounds led to the selection of five the most promising structures: 6, 11, 16, 17, and 18 (Table 4). All of them turned out to be the derivatives of 2-(difluoromethyl) -1H-benzimidazole at the C(5) position of pyrazolo[1,5-a] pyrimidine core bearing amines of the six-membered ring as the R² can substitute a methylene bridge (CH₂) as a linkage (Table 4). Their IC₅₀ values against PI3Kδ were found in the nanomolar range (18–52 nM), good selectivity in relation to other PI3K isoforms, and preserved CD19 cellular activity (for details see Table 4).

Besides the best enzymatic and cellular activity, compound 6 was chosen for further development based on acceptable solubility, microsomal stability, permeability, and the plasma protein binding range (for details see Table 5). Attempts to scale up the synthesis turned out to be chemically and economically viable. As a result, the lead compound 6 (CPL302415) was obtained in the amount exceeding one kilogram and purity suitable for future toxicological studies.

Due to the prediction of metabolism, the calculation of in vitro clearance, or the identification of the correlation of metabolites with hepatic stability in the ADMET studies [37,38], many important parameters were determined for our lead compound CPL302415 (Table 5). The metabolic stability was evaluated by measuring the intrinsic clearance and t_1/2 in mice and human liver microsomes (MLM and HLM), which were reported as very promising for CPL302415 (details in Table 5). Moreover, this structure has good solubility, permeability, and optimistic plasma protein binding parameters ((PPBs) in the range of 79–83% depending on the species; Table 5). Additionally, CPL302415 (6) was checked for bioavailability in mice (F > 55%) and dogs (F > 90%), depending on the formulation form. All these parameters make CPL302415 ready for toxicological studies and, hopefully, for future clinical trials.

3. Materials and Methods

3.1. Chemistry

3.1.1. General Information

Chemicals (at least 95% purity) were purchased from ABCR (Dallas, TX, USA), Acros (Geel, Belgium), Alfa Aesar (Haverhill, MA, USA), Combi-Blocks (San Diego, CA, USA), Fluorochem (Hadfield, UK), (Buchs, Switzerland), Merck (Darmstadt, Germany), and Sigma Aldrich (Saint Louis, MI, USA), and were used without additional purification. Solvents were purified according to standard procedures if required. Air- or moisture-sensitive reactions were carried out under an argon atmosphere. All reaction progresses were routinely checked by thin-layer chromatography (TLC). TLC was performed using silica-gel-coated plates (Kieselgel F254) and visualized using UV light. Flash chromatography was performed using Merck silica gel 60 (230–400 mesh ASTM). ¹H NMR spectra were acquired using a Varian Inova 300 MHz NMR spectrometer, a JOEL JNMR-ECZS 400 MHz spectrometer, a JOEL JNMR-ECZR 600 MHz spectrometer, and a Bruker DRX 500 NMR spectrometer with ¹H being observed at 300 MHz, 400 MHz, 600 MHz, and 500 MHz, respectively. ¹³C NMR spectra were recorded similarly at 75 MHz, 101 MHz, 151 MHz, and 126 MHz frequencies for ¹³C, respectively. Due to the poor solubility of some final compounds, usual characterization was omitted using ¹³C NMR. Chemical shifts for ¹H and ¹³C NMR spectra were reported in δ (ppm) using tetramethylsilane as an internal standard or according to the residual undeuterated solvent signal (2.50 ppm for DMSO-d₆ and 7.26 ppm for CDCl₃). The abbreviations for spin interaction coupled ¹H signals are as follows: s (singlet), d (doublet), t (triplet), m (multiplet), dd (doublet of doublets), dt (doublet of triplet), and q (quartet). Coupling constants (J) are expressed in Hertz. The ¹³C NMR spectrum was recorded with the use of the JEOL Royal HFX probehead that allows measurements to be taken with the simultaneous decoupling of both ¹H and ¹⁹F nuclei [39]. Mass spectra (atmospheric pressure ionization electrospray (API-ES) and electrospray ionization (ESI-MS)) were obtained using the Agilent 6130 LC/MSD spectrometer or Agilent 1290 UHPLC coupled with the Agilent QTOF 6545 mass spectrometer. All spectra of final compounds are in Supplementary Materials.

3.1.2. Synthesis

Procedure for 5-chloro-7-(morpholin-4-yl)pyrazolo[1,5-a]pyrimidin-2-yl]methanol (2)

Calcium chloride (10.4 g, 93.6 mmol) and sodium borohydride (7.56 g, 190 mmol) were added to the suspension of ethyl 5-chloro-7-(morpholin-4-yl)pyrazolo[1,5-a]pyrimidine-2-carboxylate (10.0 g, 31.2 mmol) in EtOH (150 mL). The mixture was stirred and heated to reflux for 3 h. Then, the reaction was cooled to RT and quenched with NH₄Cl_aq (150 mL) and 1 M HCl (150 mL). The aqueous phase was extracted three times with AcOEt. The combined extracts were washed with water and dried over Na₂SO₄, filtered, and concentrated to give 2 as a white solid (8.36 g, 31.1 mmol) with a 99% yield. ¹H NMR (600 MHz, DMSO-d₆) δ 6.43 (s, 1H, Ar-H), 6.35 (s, 1H, Ar-H), 5.32 (t, J = 5.9 Hz, 1H, -OH), 4.59 (dd, J = 5.9, 0.3 Hz, 2H, CH₂), 3.82–3.80 (m, 4H, morph.), 3.78–3.77 (m, 4H, morph.).

Procedure for 5-chloro-7-(morpholin-4-yl)pyrazolo[1,5-a]pyrimidine-2-carbaldehyde (3)

To a solution of compound 2 (3.00 g, 10.9 mmol) in DMF (30.0 mL) in argon atmosphere, Dess–Martin periodinane (97%, 5.74 g, 13.1 mmol) was added. The resulting mixture was stirred at room temperature for 2 h. The solvent was evaporated. The residue was washed with AcOEt and filtered. The filtrate was concentrated and the crude product was purified by flash chromatography (0–100% AcOEt gradient in heptane) to give 3 (1.34 g, 5.02 mmol) with a 46% yield. ¹H NMR (500 MHz, DMSO-d₆) δ 10.09 (s, 1H, -CHO), 6.97 (s, 1H, Ar-H), 6.63 (s, 1H, Ar-H), 3.90–3.85 (m, 4H, morph.), 3.86–3.78 (m, 4H, morph.).

General Procedure for the Reductive Amination Reaction

Amine derivative (1.2 eq) was added to the solution of the corresponding aldehyde (1.0 eq) in dry DCM (10 mL/1 g corresponding aldehyde) and then stirred at room temperature. After 1 h, sodium triacetoxyborohydride (1.5 eq) was added and the mixture was stirred at room temperature for a further 15 h. Water was added to the reaction mixture and phases were separated. The aqueous phase was extracted three times with DCM. Combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by flash chromatography.

• 4-{2-[(4-tert-butylpiperazin-1-yl)methyl]-5-chloropyrazolo[1,5-a]pyrimidin-7-yl}morpholine (4)

Compound 4 was prepared from aldehyde 3 (1.70 g, 2.15 mmol), 1-tert-butylpiperazine (0.36 g, 2.58 mmol), and DCM (17.0 mL) with sodium triacetoxyborohydride (0.68 g, 3.22 mmol), according to the general procedure for the reductive amination reaction. The crude product was purified by flash chromatography (0–10% MeOH gradient in AcOEt) to give 4 (1.42 g, 3.61 mmol) as a white solid with a 84% yield. ¹H NMR (600 MHz, DMSO-d₆) δ 6.38 (s, 1H, Ar-H), 6.36 (s, 1H, Ar-H), 3.83–3.80 (m, 4H, morph.), 3.80–3.78 (m, 4H, morph.), 3.59 (s, 2H, -CH₂), 2.50–2.46 (m, 4H, piperaz.), 2.45–2.37 (m, 4H, piperaz.), 0.98 (s, 9H, t-Bu.).

General Procedure for the Buchwald–Hartwig Reaction

To a pressure, microwave vessel 5-chloro-pyrazolo[1,5-a]pyrimidine (1.0 eq), amine (1.5 eq), tris(dibenzylideneacetone)dipalladium (0.05 eq), 9,9-dimethyl-4,5-bis(diphenylphosphino)xanthene (0.1 eq), cesium carbonate (2.0 eq), and solvent (10 mL/1 g pyrazolo[1,5-a]pyrimidine) were simultaneously added. The reaction vessel was then sealed and heated to 150 °C for 6 h in a microwave (power 200 W). Then, the reaction mixture was filtered through Celite^® and concentrated, and the crude product was purified using flash chromatography.

• 1-{2-[(4-tert-butylpiperazin-1-yl)methyl]-7-(morpholin-4-yl)pyrazolo[1,5-a]pyrimidin-5-yl}-2-methyl-1H-benzimidazole (5)

Compound 5 was synthesized from 4 (0.22 g, 0.56 mmol) and 2-methyl-benzimidazole (0.11 g, 0.83 mmol) as an amine, tris(dibenzylideneacetone)dipalladium (26.3 mg, 0.027 mmol), 9,9-dimethyl-4,5-bis(diphenylphosphino)xanthene (33.9 mg, 0.055 mmol), cesium carbonate (0.37 g, 1.11 mmol), and o-xylene (2.20 mL), according to the general procedure for the Buchwald–Hartwig reaction. The crude product was purified by flash chromatography (0–100% AcOEt gradient in heptane) to give the title compound 5 as a white solid (0.19 g, 0.38 mmol) with a 69% yield. ¹H NMR (300 MHz, CDCl₃) δ 7.79–7.70 (m, 1H, Ar-H), 7.50–7.43 (m, 1H, Ar-H), 7.35–7.21 (m, 2H, Ar-H), 6.60 (s, 1H, Ar-H), 6.17 (s, 1H,Ar-H), 4.02–3.95 (m, 4H, morph.), 3.90–3.83 (m, 4H, morph.), 3.82 (s, 2H, CH₂), 2.76 (s, 3H, CH₃), 2.71–2.59 (m, 8H, piperaz.), 1.08 (s, 9H, t-Bu.). ¹³C{¹H}NMR (75 MHz, CDCl₃) δ 155.1, 151.4, 151.2, 151.0, 150.2, 148.5, 142.7, 134.5, 122.9, 119.4, 110.3, 96.5, 87.6, 66.1, 56.3, 53.6, 48.4, 45.5, 25.8, 15.6. HRMS (ESI/MS): m/z calculated for C₂₇H₃₆N₈O [M + H]⁺ 489.3084 found 489.3088.

• 1-{2-[(4-tert-butylpiperazin-1-yl)methyl]-7-(morpholin-4-yl)pyrazolo[1,5-a]pyrimidin-5-yl}-2-(difluoromethyl)-1H-benzimidazole (6)

Compound 6 was synthesized from 4 (0.27 g, 0.68 mmol), 2-(difluoromethyl)benzimidazole (0.17 g, 1.01 mmol), tris(dibenzylideneacetone)dipalladium (31.8 mg, 0.033 mmol), 9,9-dimethyl-4,5-bis(diphenylphosphino)xanthene (39.0 mg, 0.067 mmol), cesium carbonate (0.44 g, 1.35 mmol), and o-xylene (2.70 mL), according to the general procedure for the Buchwald–Hartwig reaction. The crude product was purified by flash chromatography (0–100% AcOEt gradient in heptane; amino-functionalized gel column) and crystallization (AcOEt) to give 6 (0.33 g, 0.63 mmol) as a white solid with a 93% yield. ¹H NMR (600 MHz, CDCl₃) δ 7.92–7.90 (m, 1H, Ar-H), 7.65–7.64 (m, 1H, Ar-H), 7.43–7.38 (m, 2H, Ar-H), 7.25 (t, J = 53.5 Hz, 1H, CHF₂), 6.59 (s, 1H, Ar-H), 6.29 (s, 1H, Ar-H), 3.99–3.97 (m, 4H, morph.), 3.90–3.89 (m, 4H, morph.), 3.80 (s, 2H, CH₂), 2.64 (s, 8H), 1.07 (s, 9H, t-Bu.). ¹³C{¹H, ¹⁹F}NMR (151 MHz, CDCl₃) δ 155.5, 151.3, 150.0, 147.5, 144.7, 141.8, 134.6, 125.7, 124.1, 121.5, 111.7, 109.2 (CF₂), 96.7, 87.2, 66.2, 56.3, 53.9, 53.6, 48.5, 45.6, 25.9 (t-Bu.). HRMS (ESI/MS): m/z calculated for C₂₇H₃₄F₂N₈O [M + H]⁺ 525.2896 found 525.2904.

• 1-{2-[(4-tert-butylpiperazin-1-yl)methyl]-7-(morpholin-4-yl)pyrazolo[1,5-a]pyrimidin-5-yl}-2-ethyl-1H-benzimidazole (7)

Compound 7 was synthesized from 4 (0.17 g, 0.42 mmol), 2-ethyl-benzimidazole (93.0 mg, 0.64 mmol), tris(dibenzylideneacetone)dipalladium (20.0 mg, 0.021 mmol), 9,9-dimethyl-4,5-bis(diphenylphosphino)xanthene (25.8 mg, 0.042 mmol), cesium carbonate (0.28 g, 0.85 mmol), and o-xylene (1.7 mL), according to the general procedure for the Buchwald–Hartwig reaction. The crude product was purified by flash chromatography (0–100% AcOEt gradient in heptane; amino-functionalized gel column) to give the title compound 7 as a white solid (85.0 mg, 0.17 mmol) with a 40% yield. ¹H NMR (300 MHz, CDCl₃) δ 7.77 (dd, J = 6.9, 1.6 Hz, 1H, Ar-H), 7.42 (dd, J = 6.8, 1.5 Hz, 1H, Ar-H), 7.26 (qd, J = 7.3, 3.7 Hz, 2H, Ar-H), 6.57 (s, 1H, Ar-H), 6.16 (s, 1H, Ar-H), 4.00–3.94 (m, 4H, morph.), 3.87–3.79 (m, 6H), 3.10 (q, J = 7.5 Hz, 2H, CH₂), 2.77 (s, 8H), 1.40 (t, J = 7.5 Hz, 3H, CH₃), 1.17 (s, 9H, t-Bu.). ¹³C{¹H}NMR (75 MHz, CDCl₃) δ 156.2, 154.9, 151.2, 150.3, 148.6, 142.7, 134.6, 122.9, 119.5, 110.2, 99.8, 96.4, 87.9, 66.1, 56.0, 52.5, 48.4, 45.6, 25.4, 22.1, 11.9. HRMS (ESI/MS): m/z calculated for C₂₈H₃₈N₈O [M + H]⁺ 503.3241 found 503.3242.

• 1-{2-[(4-tert-butylpiperazin-1-yl)methyl]-7-(morpholin-4-yl)pyrazolo[1,5-a]pyrimidin-5-yl}-2-cyclopropyl-1H-benzimidazole (8)

Compound 8 was synthesized from 4 (100 mg, 0.25 mmol), 2-cyclopropyl-benzimidazole (59.2 mg, 0.37 mmol), tris(dibenzylideneacetone)dipalladium (11.8 mg, 0.012 mmol), 9,9-dimethyl-4,5-bis(diphenylphosphino)xanthene (15.2 mg, 0.025 mmol), cesium carbonate (168 mg, 0.51 mmol), and o-xylene (1.0 mL), according to the general procedure for the Buchwald–Hartwig reaction. The crude product was purified by flash chromatography (0–20% MeOH gradient in AcOEt) to give the title compound 8 as a white solid (93.0 mg, 0.18 mmol) with a 72% yield. ¹H NMR (400 MHz, CDCl₃) δ 7.70–7.68 (m, 1H, Ar-H), 7.53–7.51 (m, 1H, Ar-H), 7.29–7.20 (m, 2H, Ar-H), 6.61 (s, 1H, Ar-H), 6.28 (s, 1H, Ar-H), 3.99–3.97 (m, 4H, morph.), 3.87–3.84 (m, 4H, morph.), 3.82 (s, 2H, CH₂), 2.66 (s, 8H), 2.39–2.32 (m, 1H, CH), 1.39–1.35 (m, 2H, CH₂), 1.12–1.06 (m, 11H). ¹³C{¹H}NMR (101 MHz, CDCl₃) δ 156.4, 155.1, 151.2, 150.4, 148.8, 142.7, 134.9, 123.0, 122.8, 119.2, 110.6, 96.6, 88.4, 66.2, 56.3, 53.7, 48.5, 45.7, 25.8 (t-Bu.), 9.8, 8.9. HRMS (ESI/MS): m/z calculated for C₂₉H₃₈N₈O [M + H]⁺ 515.3241 found 515.3239.

• 1-{2-[(4-tert-butylpiperazin-1-yl)methyl]-7-(morpholin-4-yl)pyrazolo[1,5-a]pyrimidin-5-yl}-2-(trifluoromethyl)-1H-benzimidazole (9)

Compound 9 was synthesized from 4 (0.20 g, 0.51 mmol), 2-(trifluoromethyl)-benzimidazole (0.14 g, 0.76 mmol), tris(dibenzylideneacetone)dipalladium (24.1 mg, 0.025 mmol), 9,9-dimethyl-4,5-bis(diphenylphosphino)xanthene (29.5 mg, 0.051 mmol), cesium carbonate (0.34 g, 1.02 mmol), and toluene (2.0 mL), according to the general procedure for the Buchwald–Hartwig reaction. The crude product was purified by flash chromatography (0–100% AcOEt gradient in heptane; amino-functionalized gel column) to give the title compound 9 as a white solid (12.0 mg, 0.02 mmol) with a 4% yield. ¹H NMR (600 MHz, CDCl₃) δ 7.94–7.93 (m, 1H, Ar-H), 7.55–7.53 (m, 1H, Ar-H), 7.46–7.42 (m, 2H, Ar-H), 6.63 (s, 1H, Ar-H), 6.16 (s, 1H, Ar-H), 3.98–3.97 (m, 4H, morph.), 3.90–3.89 (m, 4H, morph.), 3.82 (s, 2H, CH₂), 2.67 (d, J = 2.1 Hz, 8H), 1.13–1.06 (m, 9H, t-Bu.). ¹³C{¹H, ¹⁹F}NMR (151 MHz, CDCl₃) δ 151.2, 150.2, 147.0, 141.0, 139.9, 135.4, 126.3, 124.5, 121.7, 119.7, 118.0, 111.9, 97.2, 88.1, 66.2, 56.3, 53.8, 48.6, 45.7, 45.0, 29.7, 25.9, 25.8. HRMS (ESI/MS): m/z calculated for C₂₇H₃₃F₃N₈O [M + H]⁺ 543.2802 found 543.2806.

• 2-(1-{[5-chloro-7-(morpholin-4-yl)pyrazolo[1,5-a]pyrimidin-2-yl]methyl}piperidin-4-yl)propan-2-ol (10)

Compound 10 was prepared from 3 (3.4 g, 12.5 mmol), 2-(4-piperidyl)-2-propanol (2.24 g, 15.0 mmol) as an amine, DCM (34.0 mL), and sodium triacetoxyborohydride (4.09 g, 18.7 mmol), according to the general procedure for the reductive amination reaction. The crude product was purified by flash chromatography (0–10% MeOH gradient in AcOEt) to give 10 (3.1 g, 7.87 mmol) with a 63% yield. ¹H NMR (300 MHz, CDCl₃) δ 6.48 (s, 1H, Ar-H), 6.03 (s, 1H, Ar-H), 3.94 (dd, J = 5.9, 3.6 Hz, 4H), 3.78 (dd, J = 5.9, 3.6 Hz, 4H), 3.72 (s, 2H, CH₂), 3.11–3.02 (m, 2H, CH₂), 2.09–1.98 (m, 2H,CH₂), 1.78–1.66 (m, 4H), 1.50–1.21 (m, 4H), 1.16 (s, 6H, 2xCH₃).

• 2-[1-({5-[2-(difluoromethyl)-1H-benzimidazol-1-yl]-7-(morpholin-4-yl)pyrazolo[1,5-a]pyrimidin-2-yl}methyl)piperidin-4-yl]propan-2-ol (11)

Compound 11 was synthesized from 10 (0.50 g, 1.24 mmol), 2-(difluoromethyl)-benzimidazole (0.31 g, 1.87 mmol), tris(dibenzylideneacetone)dipalladium (58.7 mg, 0.63 mmol), 9,9-dimethyl-4,5-bis(diphenylphosphino)xanthene (75.8 mg, 0.12 mmol), cesium carbonate (0.82 g, 2.49 mmol), and toluene (5.0 mL), according to the general procedure for the Buchwald–Hartwig reaction. The crude product was purified by flash chromatography (0–15% MeOH gradient in AcOEt) and crystallization (AcOEt) to give 11 (0.43 g, 0.81 mmol) as a white solid with a 66% yield. ¹H NMR (300 MHz, DMSO-d₆) δ 7.85 (dd, J = 20.1, 7.3 Hz, 2H, Ar-H), 7.55 (s, J = 54.0, 1H, Ar-H), 7.51–7.40 (m, 2H), 6.66 (s, 1H, Ar-H), 6.53 (s, 1H, Ar-H), 3.94 (s, 4H, morph.), 3.84 (s, 4H, morph.), 3.65 (s, 2H, CH₂), 2.97 (d, J = 10.5 Hz, 2H, CH₂), 1.93 (d, J = 10.8 Hz, 1H), 1.65 (d, J = 11.8 Hz, 2H), 1.36–1.10 (m, 3H), 1.02 (s, 6H, 2xCH₃). ¹³C{¹H}NMR (75 MHz, DMSO-d₆) δ 155.4, 150.8, 149.6, 146.9, 144.6 (t, J = 47.5 Hz), 141.1, 134.0, 125.4, 123.8, 120.6, 112.3, 108.5 (t, J = 177.7 Hz), 95.2, 87.6, 70.1, 65.5, 56.1, 53.8, 48.1, 46.8, 26.8, 26.5. HRMS (ESI/MS): m/z calculated for C₂₇H₃₃F₂N₇O₂ [M + H]⁺ 526.2736 found 526.2741.

• 2-(1-{[5-(2-methyl-1H-1,3-benzimidazol-1-yl)-7-(morpholin-4-yl)pyrazolo[1,5-a]pyrimidin-2-yl]methyl}piperidin-4-yl)propan-2-ol (12)

Compound 12 was synthesized from 10 (0.15 g, 0.38 mmol), 2-methyl-benzimidazole (75.5 mg, 0.57 mmol), tris(dibenzylideneacetone)dipalladium (18.0 mg, 0.019 mmol), 9,9-dimethyl-4,5-bis(diphenylphosphino)xanthene (23.2 mg, 0.038 mmol), cesium carbonate (0.25 g, 0.56 mmol), and o-xylene (1.5 mL), according to the general procedure for the Buchwald–Hartwig reaction. The crude product was purified by flash chromatography (0–20% MeOH gradient in AcOEt) to give the title compound 12 as a white solid (97.0 mg, 0.20 mmol) with a 52% yield. ¹H NMR (300 MHz, CDCl₃) δ 7.79–7.72 (m, 1H, Ar-H), 7.52–7.45 (m, 1H, Ar-H), 7.34–7.20 (m, 2H), 6.61 (s, 1H, Ar-H), 6.18 (s, 1H, Ar-H), 4.04–3.95 (m, 4H, morph.), 3.91–3.82 (m, 4H, morph.), 3.79 (s, 2H, CH₂), 3.18–3.09 (m, 2H), 2.77 (s, 3H, CH₃), 2.16–2.03 (m, 2H, CH₂), 1.82–1.71 (m, 2H, CH₂), 1.55–1.37 (m, 2H), 1.37–1.23 (m, 1H), 1.19 (s, 6H, 2xCH₃). ¹³C{¹H}NMR (75 MHz, CDCl₃) δ 155.2, 151.5, 151.2, 150.2, 148.5, 142.6, 134.4, 123.0, 119.3, 110.3, 96.5, 87.6, 72.3, 66.1, 56.4, 53.9, 48.4, 47.0, 29.6, 26.7, 15.5. HRMS (ESI/MS): m/z calculated for C₂₇H₃₅N₇O₂ [M + H]⁺ 490.2925 found 490.2956.

Procedure for 5-[2-(difluoromethyl)-1H-1,3-benzimidazol-1-yl]-7-(morpholin-4-yl)pyrazolo[1,5-a]pyrimidine-2-carboxylate (13)

Compound 13 was synthesized from ethyl 5-chloro-7-(morpholin-4-yl)pyrazolo[1,5-a]pyrimidine-2-carboxylate (100 mg, 0.31 mmol), 2-(difluoromethyl)-1H-benzimidazole (79.5 mg, 0.47 mmol), tris(dibenzylideneacetone)dipalladium (14.4 mg, 0.015 mmol), 9,9-dimethyl-4,5-bis(diphenylphosphino)xanthene (19.2 mg, 0.03 mmol), cesium carbonate (0.21 g, 0.63 mmol), and toluene (2.0 mL), according to the general procedure for the Buchwald–Hartwig reaction. The crude product was purified by flash chromatography (0–50% AcOEt gradient in heptane; amino-functionalized gel column) to give the title compound 13 as a light yellow solid (65.0 mg, 0.31 mmol) with a 47% yield.

Alternatively, 13 can be synthesized as follows. Ethyl 5-chloro-7-(morpholin-4-yl)pyrazolo[1,5-a]pyrimidine-2-carboxylate (100 g, 312 mmol), 2-(difluoromethyl)-1H-benzimidazole (79.5 g, 473 mmol), tetra ethyl ammonium chloride (78.0 g, 471 mmol), potassium carbonate (87.0 g, 623 mmol), and DMF (1000 mL) were added to a reactor (2000 mL volume). The reaction was heated at 160 °C for 3 h. Then, the reaction was cooled to room temperature, filtered through Celite^®, and washed with AcOEt (1.5 l). Water (3.0 mL) was added to the filtrate and phases were separated. The organic layer was concentrated. The solid was dissolved in 20% MeOH in DCM and the crude product was then purified by filtration through silica gel (0.72 kg) (20% MeOH in DCM) and macerated in TBME to give 13 (123 g, 312 mmol) as a light yellow solid with an 89% yield. ¹H NMR (300 MHz, CDCl₃) δ 7.95–7.89 (m, 1H, Ar-H), 7.71–7.65 (m, 1H, Ar-H), 7.46–7.39 (m, 2H, Ar-H), 7.30 (t, J = 54.0 Hz, 1H, CHF₂), 7.11 (s, 1H,Ar-H), 6.47 (s, 1H), Ar-H, 4.48 (q, J = 7.1 Hz, 2H, CH₂₂), 4.03–3.99 (m, 4H, morph.), 3.98–3.93 (m, 4H, morph.), 1.45 (dd, J = 8.1, 6.2 Hz, 3H, CH₃).

Procedure for {5-[2-(difluoromethyl)-2,3-dihydro-1H-1,3-benzodiazol-1-yl]-7-(morpholin-4-yl)pyrazolo[1,5-a]pyrimidin-2-yl}methanol (14)

Lithium aluminum hydride solution (1M in THF, 8.00 mL, 7.20 g, 8.00 mmol) was added to the suspension of 13 (2.40 g, 5.30 mmol) in dry THF (28.0 mL) at 0 °C. The suspension was stirred at 0 °C for 3 h. The reaction was quenched with 1.0 M HCl (14.0 mL). Then, water (70 mL) and AcOEt (90 mL) were added and the mixture was then allowed to warm to room temperature and stirred for 0.5 h. The organic layer was separated, dried over Na₂SO₄, and filtered. The solvent was removed under reduced pressure. The solid was macerated with DCM to give the title compound 14 (1.90 g, 4.72 mmol) as a light yellow solid with an 89% yield. ¹H NMR (300 MHz, DMSO-d₆) δ 7.70 (d, J = 7.7 Hz, 1H, Ar-H), 6.74 (td, J = 7.6, 1.2 Hz, 1H), 6.62 (ddd, J = 8.7, 6.9, 2.4 Hz, 3H), 6.12 (s, J = 54.0 Hz, 1H, Ar-H), 6.10 (s, 1H, Ar-H), 5.13 (t, J = 5.9 Hz, 1H, OH), 4.47 (d, J = 5.9 Hz, 2H, CH₂), 3.74 (t, J = 4.5 Hz, 4H, morph.), 3.65–3.54 (m, 4H, morph.).

Procedure for 5-[2-(difluoromethyl)-1H-1,3-benzimidazol-1-yl]-7-(morpholin-4-yl)pyrazolo[1,5-a]pyrimidine-2-carbaldehyde (15)

Dess–Martin reagent (2.90 g, 6.63 mmol) was added to the solution of 14 (1.30 g, 3.23 mmol) in dry DMF (33 mL). The whole mixture was stirred at room temperature for 1 h. The solid was filtered off and then washed with ethyl acetate (25 mL). The obtained solution was concentrated under reduced pressure. The crude product was purified by flash chromatography (0–70% ethyl acetate gradient in heptane) to give 15 (1.02 g, 2.56 mmol) as a white solid with a 78% yield.

Alternatively, 15 can be synthesized as follows. Activated toluene:butyl acetate 1:1 (700 mL) manganese(IV) oxide (58.4 g, 667 mmol) was added to the solution of 14 (27.2 g, 67.1 mmol). The mixture was stirred at reflux (set temp: 120 °C) for 1.5 h. The reaction was then filtered through Celite^®_. Celite^® was washed with DCM (200 mL). Organic phases were combined, and concentrated to give 15 (18.2 g, 45.7 mmol) as a creamy solid with a 68% yield. ¹H NMR (300 MHz, CDCl₃) δ 10.21 (s, 1H, CHO), 7.97–7.90 (m, 1H, Ar-H), 7.73–7.67 (m, 1H, Ar-H), 7.48–7.42 (m, 2H, Ar-H), 7.29 (t, J = 54.0 Hz, 1H, CHF₂), 7.11 (s, 1H, Ar-H), 6.53 (s, 1H, Ar-H), 4.03 (dd, J = 6.1, 2.7 Hz, 4H, morph.), 3.96 (dd, J = 6.3, 2.9 Hz, 4H, morph.).

General Procedure for the Amidation Reaction

Corresponding amine (1.05 eq), 2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU) (1.1 eq), and triethylamine (1.5 eq) were added to the solution of substituted 5-chloro-pyrazolo[1,5-a]pyrimidine derivative (1.0 eq) in solvent (10 mL/1 g pyrazolo[1,5-a]pyrimidine derivative). The mixture was stirred at room temperature for 2 h. Water was added to the reaction mixture and phases were separated. The aqueous phase was extracted three times with the solvent. Combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by flash chromatography.

• 2-(difluoromethyl)-1-{2-[(4-methanesulfonylpiperazin-1-yl)methyl]-7-(morpholin-4-yl)pyrazolo[1,5-a]pyrimidin-5-yl}-1H-1,3-benzimidazole (16)

Compound 16 was prepared from aldehyde 15 (0.48 g, 1.18 mmol), 1-methanesulfonylpiperazine (0.24 g, 1.42 mmol) as an amine, DCM (4.80 mL), and sodium triacetoxyborohydride (0.38 g, 1.87 mmol), according to the general procedure for the reductive amination reaction. The crude product was purified by flash chromatography (0–100% AcOEt gradient in heptane) and crystallization (AcOEt) to give the title compound 16 (0.35 g, 0.63 mmol) as a white solid with a 54% yield. ¹H NMR (600 MHz, CDCl₃) δ 7.93–7.91 (m, 1H, Ar-H), 7.66–7.65 (m, 1H, Ar-H), 7.45–7.40 (m, 2H, Ar-H), 7.29 (t, J = 52.6 Hz, 1H, CHF₂), 6.58 (s, 1H, Ar-H), 6.33 (s, 1H, Ar-H), 4.00–3.98 (m, 4H, morph.), 3.91–3.89 (m, 4H, morph.), 3.83 (s, 2H, CH₂), 3.29 (t, J = 4.6 Hz, 4H), 2.78 (s, 3H, CH₃), 2.70 (t, J = 4.8 Hz, 4H). ¹³C{¹H, ¹⁹F}NMR (151 MHz, CDCl₃) δ 155.1, 151.4, 150.2, 147.7, 144.6, 141.9, 134.6, 125.7, 124.2, 121.6, 111.7, 109.3 (CF₂), 96.3, 87.5, 66.2, 56.2, 52.4, 48.6, 45.9, 34.3. HRMS (ESI/MS): m/z calculated for C₂₄H₂₈F₂N₈O₃S [M + H]⁺ 547.2045 found 547.2048.

• 2-[4-({5-[2-(difluoromethyl)-1H-1,3-benzimidazol-1-yl]-7-(morpholin-4-yl)pyrazolo[1,5-a]pyrimidin-2-yl}methyl)piperazin-1-yl]-2-methylpropanamide (17)

Compound 17 was prepared from aldehyde 15 (0.50 g, 1.23 mmol), 2-methyl-2-(piperazin-1-yl)propanamide dihydrochloride (0.38 g, 1.48 mmol) as an amine, DCM (5.0 mL), and sodium triacetoxyborohydride (0.40 g, 1.85 mmol), according to the general procedure for the reductive amination reaction. The crude product was purified by flash chromatography (0–15% MeOH gradient in AcOEt) to give 17 (0.49 g, 0.88 mmol) as a light yellow solid with a 72% yield. ¹H NMR (400 MHz, CDCl₃) δ 7.93–7.90 (m, 1H, Ar-H), 7.67–7.63 (m, 1H, Ar-H), 7.45–7.39 (m, 2H, Ar-H), 7.23 (t, J = 53.6 Hz, 1H, CHF₂), 7.13 (d, J = 5.2 Hz, 1H), 6.59 (s, 1H, Ar-H), 6.32 (s, 1H, Ar-H), 5.46 (d, J = 5.1 Hz, 1H), 4.00–3.96 (m, 4H, morph.), 3.93–3.89 (m, 4H, morph.), 3.79 (s, 2H), 2.60 (s, 8H), 1.22 (s, 6H, 2xCH₃). ¹³C{¹H, ¹⁹F}NMR (101 MHz, CDCl₃) δ 180.1, 155.5, 151.3, 150.1, 147.6, 144.7, 141.8, 134.5, 125.7, 124.2, 121.6, 111.7, 109.3 (CF₂), 96.5, 87.3, 66.2, 63.5, 56.4, 53.9, 48.5, 46.6, 20.6. HRMS (ESI/MS): m/z calculated for C₂₇H₃₃F₂N₉O₂ [M + H]⁺ 554.2798 found 554.2800.

• 2-{2-[(4-cyclopropanecarbonylpiperazin-1-yl)methyl]-7-(morpholin-4-yl)pyrazolo[1,5-a]pyrimidin-5-yl}-2-(difluoromethyl)-1H-1,3-benzimidazole (18)

Compound 18 was prepared from aldehyde 15 (0.50 g, 1.23 mmol), 1-(cyclopropylcarbonyl)piperazine (0.21 mL, 0.23 g, 1.48 mmol) as an amine, DCM (5.0 mL), and sodium triacetoxyborohydride (0.40 g, 1.85 mmol), according to the general procedure for the reductive amination reaction. The crude product was purified by flash chromatography (0–15% MeOH gradient in AcOEt) and crystallization (AcOEt) to give 18 (0.45 g, 0.84 mmol) as a white solid with a 68% yield. ¹H NMR (400 MHz, CDCl₃) δ 7.93–7.90 (m, 1H, Ar-H), 7.67–7.64 (m, 1H, Ar-H), 7.46–7.39 (m, 2H, Ar-H), 7.23 (t, J = 53.6 Hz, 1H, CHF₂), 6.61 (s, 1H, Ar-H), 6.33 (s, 1H, Ar-H), 4.00–3.96 (m, 4H, morph.), 3.93–3.89 (m, 4H, morph.), 3.82 (s, 2H, CH₂), 3.73–3.69 (m, 4H), 2.60 (d, J = 24.3 Hz, 4H), 1.76–1.70 (m, 1H, CH), 1.00–0.96 (m, 2H, CH₂), 0.77–0.73 (m, 2H, CH₂). ¹³C{¹H, ¹⁹F}NMR (101 MHz, CDCl₃) δ 171.9, 155.3, 151.4, 150.1, 147.7, 144.6, 141.9, 134.6, 125.7, 124.2, 121.6, 111.7, 109.3 (CF₂), 96.4, 87.4, 66.2, 56.4, 48.5, 10.9, 7.4. HRMS (ESI/MS): m/z calculated for C₂₇H₃₀F₂N₈O₂ [M + H]⁺ 537.2532 found 537.2541.

• [1-({5-[2-(difluoromethyl)-1H-1,3-benzimidazol-1-yl]-7-(morpholin-4-yl)pyrazolo[1,5-a]pyrimidin-2-yl}methyl)pyrrolidin-2-yl]methanol (19)

Compound 19 was prepared from aldehyde 15 (0.50 g, 1.23 mmol), 2-pyrrolidinylmethanol (0.14 mL, 0.15 g, 1.48 mmol) as an amine, DCM (5.0 mL), and sodium triacetoxyborohydride (0.40 g, 1.85 mmol), according to the general procedure for the reductive amination reaction. The crude product was purified by flash chromatography (0–20% MeOH gradient in AcOEt) to give 19 (0.33 g, 0.68 mmol) as a white solid with a 55% yield. ¹H NMR (400 MHz, CDCl₃) δ 7.93–7.90 (m, 1H, Ar-H), 7.67–7.65 (m, 1H, Ar-H), 7.45–7.38 (m, 2H, Ar-H), 7.29 (t, J = 53.6 Hz, 1H, CHF₂), 6.56 (s, 1H, Ar-H), 6.33 (s, 1H, Ar-H), 4.14 (d, J = 14.3 Hz, 1H, OH), 4.00–3.98 (m, 4H), 3.90–3.82 (m, 6H), 3.70 (dd, J = 11.0, 3.5 Hz, 1H), 3.47 (dd, J = 11.0, 3.2 Hz, 1H), 3.20–3.16 (m, 1H), 2.90–2.85 (m, 1H), 2.73–2.72 (m, 1H), 2.59–2.54 (m, 1H), 1.97–1.92 (m, 1H), 1.82–1.75 (m, 2H, CH₂). ¹³C{¹H, ¹⁹F}NMR (101 MHz, CDCl₃) δ 156.5, 151.4, 150.1, 147.6, 144.7, 141.8, 134.5, 125.7, 124.2, 121.6, 111.7, 109.3 (CF₂), 96.2, 87.5, 66.2, 64.3, 62.3, 54.8, 51.7, 48.6, 27.7, 25.3, 23.5. HRMS (ESI/MS): m/z calculated for C₂₄H₂₇F₂N₇O₂ [M + H]⁺ 484.2267 found 484.2271.

• 1-({5-[2-(difluoromethyl)-1H-1,3-benzimidazol-1-yl]-7-(morpholin-4-yl)pyrazolo[1,5-a]pyrimidin-2-yl}methyl)pyrrolidin-3-amine (20)

Compound tert-butyl N-[1-({5-[2-(difluoromethyl)-1H-1,3-benzodiazol-1-yl]-7-(morpholin-4-yl)pyrazolo[1,5-a]pyrimidin-2-yl}methyl)pyrrolidin-3-yl]carbamate (Boc-20) was prepared from aldehyde 15 (0.50 g, 1.23 mmol), 3-(Boc-amino)pyrrolidine (0.27 g, 1.48 mmol) as an amine, DCM (5.0 mL), and sodium triacetoxyborohydride (0.40 g, 1.85 mmol), according to the general procedure for the reductive amination reaction. The crude product was purified by flash chromatography (0–10% MeOH gradient in AcOEt) to give Boc-20 (0.50 g, 0.88 mmol) as a white solid with a 71% yield. ¹H NMR (400 MHz, CDCl₃) δ 7.93–7.91 (m, 1H, Ar-H), 7.67–7.65 (m, 1H, Ar-H), 7.45–7.39 (m, 2H, Ar-H), 7.23 (t, J = 52.5 Hz, 1H, CHF₂), 6.58 (s, 1H, Ar-H), 6.32 (s, 1H, Ar-H), 4.91–4.90 (m, 1H), 4.20 (d, J = 3.4 Hz, 1H), 4.00–3.96 (m, 4H), 3.93–3.82 (m, 6H), 2.97 (d, J = 3.0 Hz, 1H), 2.72 (s, 2H, CH₂), 2.47 (d, J = 8.0 Hz, 1H), 2.32–2.27 (m, 1H), 1.67–1.63 (m, 1H), 1.43 (s, 9H, t-Bu.). ¹³C{¹H, ¹⁹F}NMR (101 MHz, CDCl₃) δ 156.0, 155.4, 151.4, 150.1, 147.6, 144.7, 141.8, 134.6, 125.7, 124.2, 121.5, 111.7, 109.3 (CF₂), 96.2, 87.4, 66.2, 61.0, 53.4, 52.8, 48.5, 32.7, 28.4 (t-Bu). HRMS (ESI/MS): m/z calculated for C₂₈H₃₄F₂N₈O₃ [M + H]⁺ 569.2794 found 569.2803.

The solution of Boc-20 (0.40 g, 0.69 mmol) in trifluoroacetic acid (2.08 mL, 3.09 g, 26.9 mmol) was heated at 50 °C for 3 h. The reaction was then cooled to room temperature, stopped with 15% NaOH (15 mL). The aqueous mixture was extracted with DCM (3 × 15 mL). The combined organic extracts were washed with water and dried over Na₂SO₄, filtered, and concentrated to give 20 as a white solid (0.30 g, 0.64 mmol) with a 93% yield. ¹H NMR (400 MHz, DMSO-d₆) δ 8.04 (s, 2H, NH₂), 7.89–7.87 (m, 1H, Ar-H), 7.81 (dd, J = 6.9, 1.4 Hz, 1H, Ar-H), 7.59 (t, J = 52.6 Hz, 1H, CHF₂), 7.49–7.41 (m, 2H, Ar-H), 6.69 (s, 1H, Ar-H), 6.66 (s, 1H, Ar-H), 3.94–3.89 (m, 6H), 3.84–3.82 (m, 5H), 3.73 (d, J = 5.0 Hz, 1H), 2.80 (s, 2H, CH₂), 2.13–2.23 (1H), 1.68–1.80 (1H), 1.22 (s, 1H). ¹³C{¹H, ¹⁹F}NMR (101 MHz, DMSO-d₆) δ 158.0, 150.9, 149.7, 147.2, 144.7, 141.2, 134.1, 125.5, 124.0, 120.7, 117.3, 112.4, 108.6, 95.5, 88.0, 65.6, 51.9, 48.3, 29.2. HRMS (ESI/MS): m/z calculated for C₂₃H₂₆F₂N₈O [M + H]⁺ 469.2270 found 469.2273.

• (3S)-1-({5-[2-(difluoromethyl)-1H-1,3-benzimidazol-1-yl]-7-(morpholin-4-yl)pyrazolo[1,5-a]pyrimidin-2-yl}methyl)pyrrolidin-3-ol (21)

Compound 21 was prepared from aldehyde 15 (0.50 g, 1.23 mmol), (S)-3-pyrrolidinol (0.16 mL, 0.17 g, 1.85 mmol) as an amine, DCM (5.0 mL), and sodium triacetoxyborohydride (0.40 g, 1.85 mmol), according to the general procedure for the reductive amination reaction. The crude product was purified by flash chromatography (0–100% AcOEt gradient in heptane, amino-functionalized gel column) to give 21 (0.22 g, 0.47 mmol) as a light yellow solid with a 38% yield. ¹H NMR (400 MHz, CDCl₃) δ 7.94–7.90 (m, 1H, Ar-H), 7.67–7.64 (m, 1H, Ar-H), 7.45–7.39 (m, 2H, Ar-H), 7.23 (t, J = 52.5 Hz, 1H, CHF₂), 6.60 (s, 1H, Ar-H), 6.32 (s, 1H, Ar-H), 4.41–4.37 (m, 1H, OH), 4.00–3.96 (m, 4H, morph.), 3.93–3.89 (m, 6H), 3.07–3.01 (m, 1H), 2.84 (dd, J = 10.1, 1.6 Hz, 1H, CH), 2.74 (dd, J = 10.1, 5.2 Hz, 1H), 2.53 (td, J = 8.9, 6.2 Hz, 1H), 2.46–2.31 (m, 1H), 2.27–2.19 (m, 1H), 1.84–1.77 (m, 1H). ¹³C{¹H, ¹⁹F}NMR (101 MHz, CDCl₃) δ 156.0, 151.4, 150.1, 147.6, 144.7, 141.8, 134.6, 125.7, 124.2, 121.6, 111.7, 109.3 (CF₂), 96.3, 87.4, 71.5, 66.2, 62.8, 53.3, 52.4, 48.5, 35.1. HRMS (ESI/MS): m/z calculated for C₂₃H₂₅F₂N₇O₂ [M + H]⁺ 470.2110 found 470.2134.

• (3R)-1-({5-[2-(difluoromethyl)-1H-1,3-benzimidazol-1-yl]-7-(morpholin-4-yl)pyrazolo[1,5-a]pyrimidin-2-yl}methyl)pyrrolidin-3-ol (22)

Compound 22 was prepared from aldehyde 15 (0.50 mg, 1.23 mmol), (R)-3-pyrrolidinol (0.16 mL, 0.17 g, 1.85 mmol) as an amine, DCM (5.0 mL), and sodium triacetoxyborohydride (0.40 g, 1.85 mmol), according to the general procedure for the reductive amination reaction. The crude product was purified by flash chromatography (0–100% AcOEt gradient in heptane, amine gel column) to give 22 (0.34 g, 0.73 mmol) as a white solid with a 60% yield. ¹H NMR (400 MHz, CDCl₃) δ 7.94–7.90 (m, 1H, Ar-H), 7.68–7.63 (m, 1H, Ar-H), 7.45–7.39 (m, 2H, Ar-H), 7.23 (t, J = 52.5 Hz, 1H, CHF₂), 6.60 (s, 1H, Ar-H), 6.31 (s, 1H, Ar-H), 4.40–4.36 (m, 1H, OH), 4.00–3.97 (m, 4H, morph.), 3.92–3.88 (m, 6H), 3.04–2.99 (m, 1H), 2.82 (dd, J = 10.0, 2.1 Hz, 1H), 2.73 (dd, J = 10.1, 5.1 Hz, 1H), 2.51 (td, J = 8.9, 6.2 Hz, 1H), 2.27–2.18 (m, 2H), 1.83–1.75 (m, 1H). ¹³C{¹H, ¹⁹F}NMR (101 MHz, CDCl₃) δ 156.2, 151.4, 150.1, 147.6, 144.7, 141.8, 134.6, 125.7, 124.2, 121.5, 111.7, 109.3 (CF₂), 96.3, 87.4, 71.5, 66.2, 62.9, 53.4, 52.4, 48.5, 35.1. HRMS (ESI/MS): m/z calculated for C₂₃H₂₅F₂N₇O₂ [M + H]⁺ 470.2110 found 470.2115.

• 2-(difluoromethyl)-1-[7-(morpholin-4-yl)-2-(morpholin-4-ylmethyl)pyrazolo[1,5-a]pyrimidin-5-yl]-1H-1,3-benzimidazole (23)

Compound 23 was prepared from aldehyde 15 (0.50 g, 1.23 mmol), morpholine (0.13 mL, 0.13 g, 1.48 mmol) as an amine, DCM (5.00 mL), and sodium triacetoxyborohydride (0.40 g, 1.85 mmol), according to the general procedure for the reductive amination reaction. The crude product was purified by flash chromatography (0–15% AcOEt gradient in heptane, amino-functionalized gel column) and crystallization) (AcOEt) to give the title compound 23 (0.28 g, 0.60 mmol) as a white solid with a 50% yield. ¹H NMR (300 MHz, CDCl₃) δ 7.89–7.81 (m, 1H, Ar-H), 7.63–7.54 (m, 1H, Ar-H), 7.38–7.30 (m, 2H, Ar-H), 7.23 (d, J = 54.0 Hz, 1H, CHF₂), 6.55 (s, 1H, Ar-H), 6.25 (s, 1H, Ar-H), 3.92 (dd, J = 6.0, 2.9 Hz, 4H, morph.), 3.83 (dd, J = 6.1, 3.0 Hz, 4H, morph.), 3.72 (s, 2H, CH₂), 3.71–3.66 (m, 4H, morph.), 2.58–2.49 (m, 4H, morph.). ¹³C{¹H}NMR (75 MHz, CDCl₃) δ 155.6, 151.5, 150.3, 147.8, 144.84 (t, J = 26.2 Hz), 142.1, 134.8, 125.0, 124.3, 121.7, 111.9, 109.5 (t, J = 238.5 Hz) (CF₂), 96.7, 87.5, 77.6, 77.2, 76.7, 67.1, 66.4, 57.1, 53.8, 48.7, 31.0. HRMS (ESI/MS): m/z calculated for C₂₃H₂₅F₂N₇O₂ [M + H]⁺ 470.2110 found 470.2113.

• 2-(difluoromethyl)-1-(2-{[(3S)-3-methylmorpholin-4-yl]methyl}-7-(morpholin-4-yl)pyrazolo[1,5-a]pyrimidin-5-yl)-1H-1,3-benzimidazole (24)

Compound 24 was prepared from aldehyde 15 (0.50 g, 1.23 mmol), (R)-3-methylmorpholine (0.15 g, 1.48 mmol) as an amine, DCM (5.0 mL), and sodium triacetoxyborohydride (0.40 g, 1.85 mmol), according to the general procedure for the reductive amination reaction. The crude product was purified by flash chromatography (0–100% AcOEt gradient in heptane) and crystallization (AcOEt) to give 24 (0.32 g, 0.67 mmol) as a white solid with a 54% yield. ¹H NMR (400 MHz, CDCl₃) δ 7.93–7.90 (m, 1H, Ar-H), 7.67–7.64 (m, 1H, Ar-H), 7.45–7.39 (m, 2H, Ar-H), 7.23 (t, J = 52.5 Hz, 1H, CHF₂), 6.57 (s, 1H, Ar-H), 6.32 (s, 1H, Ar-H), 4.07 (d, J = 14.4 Hz, 1H, CH), 4.00–3.97 (m, 4H, morph.), 3.93–3.89 (m, 4H, morph.), 3.84 (d, J = 14.4 Hz, 1H), 3.79 (dt, J = 11.2, 2.6 Hz, 1H), 3.73–3.63 (m, 2H), 3.30 (dd, J = 11.2, 9.3 Hz, 1H), 2.78 (dt, J = 11.8, 2.5 Hz, 1H), 2.60–2.51 (m, 2H), 1.15 (d, J = 6.3 Hz, 3H, CH₃). ¹³C{¹H, ¹⁹F}NMR (101 MHz, CDCl₃) δ 155.1, 151.2, 150.0, 147.6, 144.7, 141.9, 134.5, 125.7, 124.2, 121.6, 111.7, 109.3 (CF₂), 96.8, 87.3, 73.0, 67.4, 66.2, 54.5, 51.7, 51.6, 49.3, 48.5, 14.4. HRMS (ESI/MS): m/z calculated for C₂₄H₂₇F₂N₇O₂ [M + H]⁺ 484.2267 found 484.2269.

• 2-(difluoromethyl)-1-(2-{[(3R)-3-methylmorpholin-4-yl]methyl}-7-(morpholin-4-yl)pyrazolo[1,5-a]pyrimidin-5-yl)-1H-1,3-benzimidazole (25)

Compound 25 was prepared from aldehyde 15 (0.50 g, 1.23 mmol), (S)-3-methylmorpholine (0.16 g, 1.51 mmol) as an amine, DCM (5.0 mL), and sodium triacetoxyborohydride (0.40 g, 1.85 mmol), according to the general procedure for the reductive amination reaction. The crude product was purified by flash chromatography (0–100% AcOEt gradient in heptane) and crystallization (AcOEt) to give 25 (0.33 g, 0.68 mmol) as a white solid with a 55% yield. ¹H NMR (400 MHz, CDCl₃) δ 7.93–7.90 (m, 1H, Ar-H), 7.68–7.64 (m, 1H, Ar-H), 7.45–7.39 (m, 2H, Ar-H), 7.23 (t, J = 52.5 Hz, 1H, CHF₂), 6.58 (s, 1H, Ar-H), 6.32 (s, 1H, Ar-H), 4.07 (d, J = 14.4 Hz, 1H, CH), 4.00–3.97 (m, 4H, morph.), 3.93–3.89 (m, 4H, morph.), 3.85 (d, J = 14.4 Hz, 1H), 3.81–3.77 (m, 1H), 3.73–3.63 (m, 2H), 3.30 (dd, J = 11.1, 9.3 Hz, 1H), 2.78 (dt, J = 11.8, 2.5 Hz, 1H), 2.61–2.51 (m, 2H, CH₂), 1.15 (d, J = 6.3 Hz, 3H, CH₃). ¹³C{¹H, ¹⁹F}NMR (101 MHz, CDCl₃) δ 155.1, 151.3, 150.0, 147.6, 144.7, 141.9, 134.6, 125.7, 124.2, 121.6, 111.7, 109.3 (CF₂), 96.8, 87.3, 73.0, 67.4, 66.2, 54.5, 51.7, 51.6, 48.5, 14.4. HRMS (ESI/MS): m/z calculated for C₂₄H₂₇F₂N₇O₂ [M + H]⁺ 484.2267 found 484.2268.

• 1-({5-[2-(difluoromethyl)-1H-1,3-benzodiazol-1-yl]-7-(morpholin-4-yl)pyrazolo[1,5-a]pyrimidin-2-yl}methyl)piperidine-3-carboxylate (26)

Compound 26 was prepared from aldehyde 15 (0.50 g, 1.23 mmol), ethyl piperidine-3-carboxylate (0.23 mL, 0.24 g, 1.48 mmol) as an amine, DCM (5.0 mL), and sodium triacetoxyborohydride (0.40 g, 1.85 mmol), according to the general procedure for the reductive amination reaction. The crude product was purified by flash chromatography (0–100% AcOEt gradient in heptane) to give 25 (0.56 g, 1.04 mmol) as a white solid with a 84% yield. ¹H NMR (400 MHz, CDCl₃) δ 7.93–7.91 (m, 1H, Ar-H), 7.67–7.64 (m, 1H, Ar-H), 7.45–7.40 (m, 2H, Ar-H), 7.24 (t, J = 53.6 Hz, 1H, CHF₂), 6.59 (s, 1H, Ar-H), 6.30 (s, 1H, Ar-H), 4.15–4.10 (m, 2H, CH2), 4.00–3.98 (m, 4H, morph.), 3.91–3.89 (m, 4H, morph.), 3.80 (s, 2H), 3.09 (dd, J = 10.9, 2.9 Hz, 1H), 2.88–2.86 (m, 1H), 2.65–2.58 (m, 1H), 2.38–2.33 (m, 1H), 2.20 (td, J = 10.9, 2.9 Hz, 1H), 1.98–1.93 (m, 1H), 1.80–1.73 (m, 1H), 1.69–1.58 (m, 1H), 1.26–1.23 (m, 3H, CH₃). ¹³C{¹H, ¹⁹F}NMR (101 MHz, CDCl₃) δ 174.1, 156.0, 151.3, 150.0, 147.5, 144.7, 141.9, 134.6, 125.7, 124.1, 121.5, 111.7, 109.2 (CF₂), 96.4, 87.2, 66.2, 60.3, 56.8, 55.4, 53.8, 48.5, 41.9, 26.8, 24.6, 14.2. HRMS (ESI/MS): m/z calculated for C₂₈H₃₄F₂N₈O₂ [M + H]⁺ 540.2529 found 540.2536.

• N-({5-[2-(difluoromethyl)-1H-1,3-benzimidazol-1-yl]-7-(morpholin-4-yl)pyrazolo[1,5-a]pyrimidin-2-yl}methyl)oxan-4-amine (27)

Compound 27 was prepared from aldehyde 15 (0.50 g, 1.23 mmol), 4-aminotetrahydropyran (0.16 mL, 0.16 g, 1.51 mmol) as an amine, DCM (5.0 mL), and sodium triacetoxyborohydride (0.40 g, 1.85 mmol), according to the general procedure for the reductive amination reaction. The crude product was purified by flash chromatography (0–10% MeOH gradient in AcOEt) to give 27 (0.45 g, 0.95 mmol) as a white solid with a 77% yield. ¹H NMR (600 MHz, DMSO-d₆) δ 7.88 (d, J = 7.6 Hz, 1H, Ar-H), 7.82–7.80 (m, 1H, Ar-H), 7.58 (t, J = 52.6 Hz, 1H, CHF₂), 7.47–7.42 (m, 2H, Ar-H), 6.64 (s, 1H, Ar-H), 6.60 (s, 1H, Ar-H), 3.93 (d, J = 7.7 Hz, 7H), 3.84–3.81 (m, 6H), 3.26 (td, J = 11.4, 2.1 Hz, 2H, CH₂), 2.71–2.66 (m, 1HCH), 1.80 (dd, J = 12.5, 1.7 Hz, 2H, CH₂), 1.32–1.26 (m, 2HCH₂). ¹³C{¹H, ¹⁹F}NMR (151 MHz, DMSO-d₆) δ 157.9, 150.9, 149.6, 146.9, 144.7, 141.2, 134.1, 125.5, 123.9, 120.7, 112.4, 108.6, 94.5, 87.6, 65.7, 65.6, 52.4, 48.1, 43.6, 33.0. HRMS (ESI/MS): m/z calculated for C₂₄H₂₇F₂N₇O₂ [M + H]⁺ 484.2267 found 484.2266.

• 2-(difluoromethyl)-1-{2-[(4,4-difluoropiperidin-1-yl)methyl]-7-(morpholin-4-yl)pyrazolo[1,5-a]pyrimidin-5-yl}-1H-1,3-benzimidazole (28)

Compound 28 was prepared from aldehyde 15 (0.50 g, 1.23 mmol), 4,4-difluoropiperidine (0.24 g, 1.48 mmol) as an amine, DCM (5.0 mL), and sodium triacetoxyborohydride (0.40 g, 1.85 mmol), according to the general procedure for the reductive amination reaction. The crude product was purified by flash chromatography (0–100% AcOEt gradient in heptane) and crystallization (AcOEt) to give 28 (0.41 g, 0.81 mmol) as a white solid with a 66% yield. ¹H NMR (400 MHz, CDCl₃) δ 7.93–7.91 (m, 1H, Ar-H), 7.67–7.65 (m, 1H, Ar-H), 7.46–7.39 (m, 2H, Ar-H), 7.23 (t, J = 54.0 Hz, 1H,CHF₂), 6.59 (s, 1H, Ar-H), 6.33 (s, 1H, Ar-H), 4.00–3.98 (m, 4H, morph.), 3.91–3.89 (m, 4H, morph.), 3.84 (s, 2H, CH₂), 2.70 (t, J = 5.6 Hz, 4H), 2.09–1.99 (m, 4H). ¹³C{¹H, ¹⁹F}NMR (101 MHz, CDCl₃) δ 155.9, 151.5, 150.3, 147.8, 144.8, 142.0, 134.7, 125.9, 124.4, 121.8, 111.9, 109.5 (CF₂), 96.5, 96.4, 87.6, 66.4, 55.9, 50.2, 48.7, 34.2. HRMS (ESI/MS): m/z calculated for C₂₄H₂₅F₄N₇O [M + H]⁺ 504.2194 found 504.2131.

• 1-({5-[2-(difluoromethyl)-1H-1,3-benzimidazol-1-yl]-7-(morpholin-4-yl)pyrazolo[1,5-a]pyrimidin-2-yl}methyl)piperidine-4-carboxamide (29)

Compound 29 was prepared from aldehyde 15 (0.50 g, 1.23 mmol), 4-piperidinecarboxamide (0.19 g, 1.48 mmol) as an amine, DCM (5.0 mL) and sodium triacetoxyborohydride (0.40 g, 1.85 mmol), according to the general procedure for the reductive amination reaction. The crude product was purified by crystallization (AcOEt/DCM, 90:10, v/v) to give 29 (0.40 g, 0.78 mmol) as a white solid with a 64% yield. ¹H NMR (300 MHz, CDCl₃) δ 7.95–7.88 (m, 1H, Ar-H), 7.70–7.62 (m, 1H, Ar-H), 7.46–7.37 (m, 2H,Ar-H), 7.30 (t, J = 54.0 Hz, 1H, CHF₂), 6.59 (s, 1H, Ar-H), 6.31 (s, 1H, Ar-H), 5.48 (s, 2H, NH₂), 3.98 (m, 4H, morph.), 3.90 (m, 4H, morph.), 3.78 (s, 2H, CH₂), 3.06 (d, J = 11.7 Hz, 2H, CH₂), 2.18 (t, J = 11.3 Hz, 3H), 1.96–1.73 (m, 4H). ¹³C{¹H}NMR (75 MHz, CDCl₃) δ 177.3, 156.1, 151.5, 150.3, 147.7, 144.8 (t, J = 26.2 Hz), 142.0, 134.8, 125.9, 124.3, 121.7, 111.9, 109.5 (t, J = 237.0 Hz, CF₂), 96.6, 87.5, 66.4, 56.8, 53.3, 48.7, 31.7, 29.1, 22.8. HRMS (ESI/MS): m/z calculated for C₂₅H₂₈F₂N₈O₂ [M + H]⁺ 511.2376 found 511.2377.

• 1-({5-[2-(difluoromethyl)-1H-1,3-benzimidazol-1-yl]-7-(morpholin-4-yl)pyrazolo[1,5-a]pyrimidin-2-yl}methyl)-4-methylpiperidin-4-ol (30)

Compound 30 was prepared from aldehyde 15 (0.50 g, 1.23 mmol), 4-methylpiperidin-4-ol (0.18 g, 1.48 mmol) as an amine, DCM (5.0 mL), and sodium triacetoxyborohydride (0.40 g, 1.85 mmol), according to the general procedure for the reductive amination reaction. The crude product was purified by flash chromatography (50–100% AcOEt gradient in heptane) and crystallization (AcOEt) to give 30 (0.33 g, 0.66 mmol) as a white solid with a 54% yield. ¹H NMR (400 MHz, CDCl₃) δ 7.93–7.91 (m, 1H, Ar-H), 7.67–7.65 (m, 1H, Ar-H), 7.45–7.40 (m, 2H, Ar-H), 7.23 (t, J = 54.0 Hz, 1H, CHF₂), 6.60 (s, 1H, Ar-H), 6.30 (s, 1H, Ar-H), 4.00–3.96 (m, 4H, morph.), 3.93–3.89 (m, 4H, morph.), 3.81 (s, 2H, CH₂), 2.74–2.69 (m, 2H, CH₂), 2.57–2.51 (m, 2H, CH₂), 1.77–1.70 (m, 2H, CH₂), 1.63 (d, J = 13.2 Hz, 2H, CH₂), 1.26 (s, 3H, CH₃). ¹³C{¹H, ¹⁹F} NMR (101 MHz, CDCl₃) δ 156.1, 151.3, 150.1, 147.5, 144.7, 141.9, 134.6, 125.7, 124.1, 121.6, 111.7, 109.3 (CF₂), 96.5, 87.2, 67.7, 66.2, 56.6, 49.8, 48.5, 38.8. HRMS (ESI/MS): m/z calculated for C₂₅H₂₉F₂N₇O₂ [M + H]⁺ 498.2423 found 498.2422.

• [1-({5-[2-(difluoromethyl)-1H-1,3-benzimidazol-1-yl]-7-(morpholin-4-yl)pyrazolo[1,5-a]pyrimidin-2-yl}methyl)piperidin-4-yl]methanol (31)

Compound 31 was prepared from aldehyde 15 (0.50 g, 1.23 mmol), 4-piperidinemethanol (0.17 g, 1.48 mmol) as an amine, DCM (5.0 mL), and sodium triacetoxyborohydride (0.40 g, 1.85 mmol), according to the general procedure for the reductive amination reaction. The crude product was purified by flash chromatography (0–10% MeOH gradient in AcOEt) and crystallization (i-PrOH) to give 31 (0.34 g, 0.68 mmol) as a white solid with a 56% yield. ¹H NMR (600 MHz, CDCl₃) δ 7.93–7.91 (m, 1H, Ar-H), 7.67–7.64 (m, 1H, Ar-H), 7.44–7.39 (m, 2H, Ar-H), 7.26 (t, J = 26.8 Hz, 1H, CHF₂), 6.60 (s, 1H, Ar-H), 6.30 (s, 1H, OH), 3.99–3.98 (m, 4H, morph.), 3.91–3.89 (m, 4H, morph.), 3.78 (s, 2H, CH2), 3.51 (d, J = 6.5 Hz, 2H, CH₂), 3.04 (d, J = 11.5 Hz, 2H CH₂ 2.16–2.11 (m, 2H, CH₂), 1.76–1.74 (m, 2H, CH₂), 1.70–1.59 (m, 1H), 1.56–1.48 (m, 1H), 1.37–1.31 (m, 2H). ¹³C{¹H, ¹⁹F} NMR (151 MHz, CDCl₃) δ 156.2, 151.3, 150.1, 147.5, 144.7, 141.8, 134.6, 125.7, 124.1, 121.5, 111.7, 109.3 (CF₂), 96.5, 87.2, 67.9, 66.2, 56.9, 53.6, 48.5, 38.4, 28.8. HRMS (ESI/MS): m/z calculated for C₂₅H₂₉F₂N₇O₂ [M + H]⁺ 498.2423 found 498.2426.

• 1-({5-[2-(difluoromethyl)-1H-1,3-benzimidazol-1-yl]-7-(morpholin-4-yl)pyrazolo[1,5-a]pyrimidin-2-yl}methyl)piperidine-4-carbonitrile (32)

Compound 32 was prepared from aldehyde 15 (0.50 g, 1.23 mmol), 4-cyanopiperidine (0.17 mL, 0.16 g, 1.48 mmol) as an amine, DCM (5.0 mL), and sodium triacetoxyborohydride (0.40 g, 1.85 mmol), according to the general procedure for the reductive amination reaction. The crude product was purified by flash chromatography (0–100% AcOEt gradient in heptane) and crystallization (AcOEt) to give 32 (0.50 g, 1.01 mmol) as a white solid with a 82% yield. ¹H NMR (600 MHz, CDCl₃) δ 7.93–7.91 (m, 1H, Ar-H), 7.67–7.65 (m, 1H, Ar-H), 7.45–7.41 (m, 2H, Ar-H), 7.30 (t, J = 53.4 Hz, 1H, CHF₂), 6.58 (s, 1H, Ar-H), 6.32 (s, 1H, Ar-H), 4.00–3.98 (m, 4H, morph.), 3.91–3.89 (m, 4H, morph.), 3.79 (s, 2H, CH₂), 2.79 (s, 2H, CH₂), 2.69 (s, 1H, CH), 2.51 (d, J = 1.3 Hz, 2H,CH₂), 2.00–1.89 (m, 4H). ¹³C{¹H, ¹⁹F} NMR (151 MHz, CDCl₃) δ 155.6, 151.4, 150.2, 147.7, 144.7, 141.9, 134.6, 125.7, 124.2, 121.7, 121.6, 111.7, 109.3 (CF₂), 96.4, 87.4, 66.2, 56.7, 51.3, 48.6, 28.8, 26.0. HRMS (ESI/MS): m/z calculated for C₂₅H₂₆F₂N₈O [M + H]⁺ 493.2270 found 493.2281.

• 2-(difluoromethyl)-1-[7-(morpholin-4-yl)-2-{[4-(morpholin-4-yl)piperidin-1-yl]methyl}pyrazolo[1,5-a]pyrimidin-5-yl]-1H-1,3-benzimidazole (33)

Compound 33 was prepared from aldehyde 15 (0.50 g, 1.23 mmol), 4-morpholinopiperidine (0.26 g, 1.48 mmol) as an amine, DCM (5.0 mL), and sodium triacetoxyborohydride (0.40 g, 1.85 mmol), according to the general procedure for the reductive amination reaction. The crude product was purified by flash chromatography (0–20% MeOH gradient in AcOEt) and crystallization (i-PrOH) to give 33 (0.37 g, 0.66 mmol) as a white solid with a 54% yield. ¹H NMR (600 MHz, CDCl₃) δ 7.92–7.91 (m, 1H, Ar-H), 7.66–7.65 (m, 1H, Ar-H), 7.44–7.39 (m, 2H, Ar-H), 7.26 (t, J = 53.4 Hz, 1H, CHF₂), 6.59 (s, 1H, Ar-H), 6.30 (s, 1H, Ar-H), 3.99–3.98 (m, 4H, morph.), 3.91–3.89 (m, 4H, morph.), 3.77 (s, 2H, CH₂), 3.72 (t, J = 4.6 Hz, 4H, morph.), 3.07 (d, J = 11.9 Hz, 2H, CH₂), 2.55 (t, J = 4.6 Hz, 4H, morph.), 2.21 (t, J = 11.4, 3.8 Hz, 1H, CH), 2.13 (td, J = 11.8, 2.1 Hz, 2H, CH₂), 1.84–1.82 (m, 2H, CH₂), 1.64–1.57 (m, 2H, CH₂). ¹³C{¹H, ¹⁹F} NMR (151 MHz, CDCl₃) δ 156.2, 151.3, 150.1, 147.5, 144.7, 141.9, 134.6, 125.7, 124.2, 121.6, 111.7, 109.3 (CF₂), 96.5, 87.3, 67.3, 66.2, 62.1, 56.5, 53.2, 49.8, 48.5, 28.2. HRMS (ESI/MS): m/z calculated for C₂₈H₃₄F₂N₈O₂ [M + H]⁺ 553.2845 found 553.2846.

• 1-({5-[2-(difluoromethyl)-1H-1,3-benzimidazol-1-yl]-7-(morpholin-4-yl)pyrazolo[1,5-a]pyrimidin-2-yl}methyl)piperidin-4-ol (34)

Compound 34 was prepared from aldehyde 15 (0.50 g, 1.23 mmol), 4-hydroxypiperidine (0.15 g, 1.48 mmol) as an amine, DCM (5.0 mL), and sodium triacetoxyborohydride (0.40 g, 1.85 mmol), according to the general procedure for the reductive amination reaction. The crude product was purified by flash chromatography (0–20% MeOH gradient in AcOEt) and crystallization (i-PrOH) to give 34 (0.32 g, 0.66 mmol) as a white solid with a 54% yield. ¹H NMR (300 MHz, CDCl₃) δ 7.96–7.87 (m, 1H, Ar-H), 7.70–7.60 (m, 1H, Ar-H), 7.46–7.36 (m, 2H, Ar-H), 7.29 (t, J = 54.0 Hz, 1H, CHF₂), 6.60 (s, 1H, Ar-H), 6.30 (s, 1H, Ar-H), 4.02–3.94 (m, 4H, morph.), 3.94–3.86 (m, 4H morph. 3.79 (s, 2H, CH₂), 3.77–3.66 (m, 1H), 2.95–2.81 (m, 2H, CH₂), 2.40–2.25 (m, 2H, CH₂), 1.99–1.85 (m, 2H, CH₂), 1.74–1.55 (m, 3H). ¹³C{¹H} NMR (75 MHz, CDCl₃) δ 156.3, 151.5, 150.3, 147.7, 144.8 (t, J = 27.0 Hz), 142.0, 134.8, 125.8, 124.3, 121.7, 109.4 (t, J = 240.0 Hz, CF₂), 96.6, 87.4, 67.9, 66.3, 56.6, 51.2, 48.7, 34.6. HRMS (ESI/MS): m/z calculated for C₂₄H₂₇F₂N₇O₂ [M + H]⁺ 484.2267 found 484.2272.

• N-[1-({5-[2-(difluoromethyl)-1H-1,3-benzimidazol-1-yl]-7-(morpholin-4-yl)pyrazolo[1,5-a]pyrimidin-2-yl}methyl)piperidin-4-yl]acetamide (35)

Compound 35 was prepared from aldehyde 15 (0.50 g, 1.23 mmol), 4-acetylamino-piperidine (0.22 g, 1.48 mmol) as an amine, DCM (5.0 mL), and sodium triacetoxyborohydride (0.40 g, 1.85 mmol), according to the general procedure for the reductive amination reaction. The crude product was purified by flash chromatography (0–15% MeOH gradient in AcOEt) and crystallization (AcOEt) to give 35 (0.51 g, 0.97 mmol) as a white solid with a 79% yield. ¹H NMR (600 MHz, CDCl₃) δ 7.92–7.91 (m, 1H, Ar-H), 7.66–7.64 (m, 1H, Ar-H), 7.44–7.39 (m, 2H, Ar-H), 7.25 (t, J = 53.4 Hz, 1H, CHF₂), 6.58 (s, 1H,Ar-H), 6.31 (s, 1H, Ar-H), 5.39 (d, J = 8.0 Hz, 1H, NH), 3.99–3.97 (m, 4H, morph.), 3.90–3.89 (m, 4H, morph.), 3.84–3.78 (m, 1H, CH),3.76 (s, 2H, CH₂), 2.95 (d, J = 11.8 Hz, 2H, CH₂), 2.29–2.25 (m, 2H, CH₂), 1.96 (s, 3H, CH₃), 1.96–1.93 (m, 2H, CH₂), 1.52–1.45 (m, 2H, CH₂). ¹³C{¹H, ¹⁹F} NMR (151 MHz, CDCl₃) δ 169.3, 156.0, 151.3, 150.1, 147.6, 144.7, 141.9, 134.6, 125.7, 124.2, 121.5, 111.7, 109.3 (CF₂), 96.4, 87.3, 66.2, 56.5, 52.5, 48.5, 46.5, 32.4, 23.5. HRMS (ESI/MS): m/z calculated for C₂₆H₃₀F₂N₈O₂ [M + H]⁺ 525.2532 found 525.2890.

• 2-[4-({5-[2-(difluoromethyl)-1H-1,3-benzimidazol-1-yl]-7-(morpholin-4-yl)pyrazolo[1,5-a]pyrimidin-2-yl}methyl)piperazin-1-yl]-N-methylacetamide (36)

Compound 36 was prepared from aldehyde 15 (0.50 g, 1.23 mmol), N-methyl-2-(1-piperazinyl)acetamide (0.24 g, 1.48 mmol) as an amine, DCM (5.0 mL), and sodium triacetoxyborohydride (0.40 g, 1.85 mmol), according to the general procedure for the reductive amination reaction. The crude product was purified by flash chromatography (0–20% MeOH gradient in AcOEt) and crystallization (AcOEt) to give 36 (0.44 g, 0.82 mmol) as a white solid with a 66% yield. ¹H NMR (600 MHz, CDCl₃) δ 7.92–7.90 (m, 1H, Ar-H), 7.66–7.64 (m, 1H, Ar-H), 7.44–7.39 (m, 2H,Ar-H), 7.25 (t, J = 53.4 Hz, 1H, CHF₂), 7.10–7.08 (m, 1H, NH), 6.59–6.58 (m, 1H, Ar-H), 6.32 (s, 1H, Ar-H), 3.99–3.98 (m, 4H, morph.), 3.91–3.89 (m, 4H, morph.), 3.80 (s, 2H, CH₂), 3.02 (s, 2H, CH₂), 2.83 (d, J = 5.1 Hz, 3H, CH₃), 2.61 (d, J = 17.8 Hz, 8H). ¹³C{¹H, ¹⁹F} NMR (151 MHz, CDCl₃) δ 170.8, 155.6, 151.3, 150.1, 147.6, 144.6, 141.9, 134.6, 125.7, 124.2, 121.6, 111.7, 109.3 (CF₂), 96.5, 87.3, 66.2, 61.5, 56.4, 53.6, 53.2, 48.5, 25.7. HRMS (ESI/MS): m/z calculated for C₂₆H₃₁F₂N₉O₂ [M + H]⁺ 540.2641 found 540.2644.

• 2-(difluoromethyl)-1-{2-[(4-methylpiperazin-1-yl)methyl]-7-(morpholin-4-yl)pyrazolo[1,5-a]pyrimidin-5-yl}-1H-1,3-benzimidazole (37)

Compound 37 was prepared from aldehyde 15 (0.20 g, 0.49 mmol), 1-methylpiperazine (0.066 mL, 59.7 mg, 0.59 mmol) as an amine, DCM (2.0 mL), and sodium triacetoxyborohydride (0.17 g, 0.80 mmol), according to the general procedure for the reductive amination reaction. The crude product was purified by flash chromatography (0–30% AcOEt gradient in heptane, amino-functionalized gel column) to give 37 (0.19 g, 0.40 mmol) as a white solid with a 81% yield. ¹H NMR (600 MHz, CDCl₃) δ 7.92–7.91 (m, 1H, Ar-H), 7.66–7.64 (m, 1H, Ar-H), 7.44–7.39 (m, 2H,Ar-H), 7.29 (t, J = 54.0 Hz, 1H, CHF₂), 6.60 (s, 1H, Ar-H), 6.30 (s, 1H, Ar-H), 3.99–3.98 (m, 4H, morph.), 3.91–3.89 (m, 4H, morph.), 3.79 (s, 2H, CH₂), 2.56 (d, J = 85.2 Hz, 8H, piperaz.), 2.30 (s, 3H, CH₃). ¹³C{¹H, ¹⁹F} NMR (151 MHz, CDCl₃) δ 155.7, 151.2, 150.0, 147.4, 144.6, 141.7, 134.5, 125.6, 124.0, 121.4, 111.6, 109.1 (CF₂), 96.4, 87.1, 66.1, 56.3, 55.0, 53.0, 48.4, 45.9. HRMS (ESI/MS): m/z calculated for C₂₄H₂₈F₂N₈O [M + H]⁺ 483.2426 found 483.2429.

• 2-(difluoromethyl)-1-[7-(morpholin-4-yl)-2-{[4-(propan-2-yl)piperazin-1-yl]methyl}pyrazolo[1,5-a]pyrimidin-5-yl]-1H-1,3-benzimidazole (38)

Compound 38 was prepared from aldehyde 15 (1.50 g, 3.69 mmol), 1-isopropylpiperazine (0.65 mL, 0.58 g, 4.43 mmol) as an amine, DCM (15.0 mL), and sodium triacetoxyborohydride (1.25 g, 5.90 mmol), according to the general procedure for the reductive amination reaction. The crude product was purified by flash chromatography (0–30% AcOEt gradient in heptane, amino-functionalized gel column) and crystallization (AcOEt) to give 38 (1.30 g, 3.45 mmol) as a white solid with a 93% yield. ¹H NMR (400 MHz, DMSO-d₆) δ 7.89–7.87 (m, 1H, Ar-H), 7.82–7.80 (m, 1H, Ar-H), 7.60 (t, J = 52.6 Hz, 1H, CHF₂), 7.46–7.40 (m, 2H, Ar-H), 6.65 (s, 1H, Ar-H), 6.52 (s, 1H, Ar-H), 3.94–3.92 (m, 4H, morph.), 3.84–3.82 (m, 4H, morph.), 3.66 (s, 2H, CH₂), 2.58 (t, J = 6.5 Hz, 1H, CH), 2.44 (s, 8H, piperaz.), 0.94 (d, J = 6.5 Hz, 6H, 2xCH₃). ¹³C{¹H, ¹⁹F} NMR (101 MHz, DMSO-d₆) δ 155.0, 150.9, 149.6, 147.0, 144.7, 141.2, 134.0, 125.4, 123.9, 120.7, 112.4, 108.5, 108.5, 95.4, 87.8, 65.6, 55.9, 53.5, 53.1, 48.2, 47.9, 18.2. HRMS (ESI/MS): m/z calculated for C₂₆H₃₂F₂N₈O [M + H]⁺ 511.2739 found 511.2743.

Procedure for 5-[2-(difluoromethyl)-1H-1,3-benzimidazol-1-yl]-7-(morpholin-4-yl)pyrazolo[1,5-a]pyrimidine-2-carboxylic acid (39)

The solution of lithium hydroxide monohydrate (28.4 g, 678 mmol) was added to the suspension of 13 (60.0 g, 136 mmol) in MeOH (1000 mL) in water (200 mL). The mixture was stirred at room temperature for 18 h. The solvent was removed under reduced pressure and the reaction was quenched with water (600 mL) and 2.5 M HCl (60 mL). The solid was collected by filtration and dried to give 39 (52.1 g, 126 mmol) as a white solid with a 93% yield. ¹H NMR (300 MHz, DMSO-d₆) δ 7.93–7.81 (m, 2H, Ar-H), 7.56 (t, J = 54.0 Hz, 1H, CHF₂), 7.47 (ddd, J = 10.4, 5.2, 3.6 Hz, 2H, Ar-H), 7.03 (s, 1H), 6.86 (s, 1H), 4.02–3.96 (m, 4H, morph.), 3.90–3.77 (m, 6H), 3.57 (s, 1H).

• 1-[2-(4-tert-butylpiperazine-1-carbonyl)-7-(morpholin-4-yl)pyrazolo[1,5-a]pyrimidin-5-yl]-2-(difluoromethyl)-1H-1,3-benzimidazole (40)

Compound 40 was prepared from 39 (0.20 g, 0.48 mmol), N-t-butylpiperazine (0.10 g, 0.72 mmol), HATU (0.21 g, 0.57 mmol), TEA (0.13 mL, 96.5 mg, 0.75 mmol), and DMF (2.0 mL), according to the general procedure for the amidation reaction. The crude product was purified by flash chromatography (0–100% AcOEt gradient in heptane, amino-functionalized gel column) to give 40 (0.13 g, 0.23 mmol) as a white solid with a 51% yield. ¹H NMR (400 MHz, DMSO-d₆) δ 7.88 (dd, J = 7.1, 1.5 Hz, 1H, Ar-H), 7.84–7.82 (m, 1H, Ar-H), 7.61 (t, J = 52.5 Hz, 1H, CHF₂), 7.48–7.41 (m, 2H, Ar-H), 6.83 (s, 1H, Ar-H), 6.80 (s, 1H, Ar-H), 3.93–3.91 (m, 4H), 3.86–3.83 (m, 4H), 3.72–3.64 (m, 4H), 2.59–2.51 (m, 4H), 1.02 (s, 9H, t-Bu). ¹³C{¹H, ¹⁹F} NMR (101 MHz, DMSO-d₆) δ 161.5, 151.2, 150.4, 149.2, 147.8, 144.7, 141.2, 134.0, 125.6, 124.1, 120.7, 112.4, 108.5, 108.5, 96.7, 89.3, 65.6, 59.7, 48.5, 46.1, 25.6, 20.7, 14.1. HRMS (ESI/MS): m/z calculated for C₂₇H₃₂F₂N₈O₂ [M + H]⁺ 539.2689 found 539.2736.

• 2-(4-{5-[2-(difluoromethyl)-1H-1,3-benzimidazol-1-yl]-7-(morpholin-4-yl)pyrazolo[1,5-a]pyrimidine-2-carbonyl}piperazin-1-yl)propan-2-ol (41)

Compound 41 was prepared from 39 (1.50 g, 3.62 mmol), 2-(4-piperidyl)-2-propanol (0.57 g, 3.83 mmol), HATU (1.51 mg, 3.98 mmol), TEA (0.76 mL, 0.55, 5.44 mmol), and DCM (15.0 mL), according to the general procedure for the amidation reaction. The crude product was purified by flash chromatography (0–10% MeOH gradient in AcOEt) to give 41 (0.66 g, 1.22 mmol) as a light yellow solid with a 34% yield. ¹H NMR (300 MHz, CDCl₃) δ 7.94–7.89 (m, 1H, Ar-H), 7.69–7.65 (m, 1H, Ar-H), 7.46–7.39 (m, 2H, Ar-H), 7.29 (t, J = 54.0 Hz, 1H, CHF₂), 6.86 (s, 1H, Ar-H), 6.42 (s, 1H, Ar-H), 4.91 (d, J = 13.0 Hz, 1H), 4.49 (d, J = 13.5 Hz, 1H), 4.00–3.95 (m, 4H, morph.), 3.94–3.86 (m, 4H, morph.), 3.08 (t, J = 11.8 Hz, 1H), 2.76 (td, J = 12.8, 2.6 Hz, 1H), 1.97–1.78 (m, 2H, CH₂), 1.68–1.56 (m, 1H, CH), 1.49–1.34 (m, 3H), 1.22 (d, J = 6.0 Hz, 6H, 2xCH₃). ¹³C{¹H} NMR (75 MHz, CDCl₃) δ 162.7, 151.6, 149.7, 148.5, 144.7(t, J = 27.0 Hz), 142.0, 134.7, 126.0, 124.5, 121.7, 111.9 (t, J = 238.5 Hz, CF₂), 97.8, 88.6, 72.2, 66.3, 48.9, 47.8, 43.2, 27.6 (d, J = 16.8 Hz), 26.9 (d, J = 5.1 Hz). HRMS (ESI/MS): m/z calculated for C₂₇H₃₁F₂N₇O₃ [M + H]⁺ 540.2529 found 540.2533.

• 2-(4-{5-[2-(difluoromethyl)-1H-1,3-benzimidazol-1-yl]-7-(morpholin-4-yl)pyrazolo[1,5-a]pyrimidine-2-carbonyl}piperazin-1-yl)-2-methylpropanamide (42)

Compound 42 was prepared from 39 (0.20 g, 0.48 mmol), 2-methyl-2-(piperazin-1-yl)propanamide dihydrochloride (0.13 g, 0.49 mmol), HATU (0.20 g, 0.52 mmol), TEA (0.23 mL, 0.16 g, 1.66 mmol), and DCM (2.0 mL), according to the general procedure for the amidation reaction. The crude product was purified by flash chromatography (0–20% MeOH gradient in AcOEt) to give 42 (0.18 g, 0.31 mmol) as a white solid with a 67% yield. ¹H NMR (600 MHz, DMSO-d₆) δ 7.89 (d, J = 7.6 Hz, 1H, Ar-H), 7.82 (d, J = 7.9 Hz, 1H, Ar-H), 7.58 (t, J = 52.6 Hz, 1H, CHF₂), 7.49–7.42 (m, 2H), 7.25 (d, J = 2.9 Hz, 1H), 6.99 (d, J = 2.8 Hz, 1H, Ar-H), 6.84 (s, 1H, Ar-H), 6.80 (s, 1H), 3.93–3.91 (m, 4H, morph.), 3.85–3.84 (m, 4H, morph.), 3.79 (s, 2H, CH₂), 3.73 (s, 2H, CH₂), 2.52–2.50 (m, 2H, CH₂), 2.46 (t, J = 4.4 Hz, 2H, CH₂), 1.09 (s, 6H, 2xCH₃). ¹³C{¹H, ¹⁹F}NMR (151 MHz, DMSO-d6) δ 177.7, 161.6, 151.1, 150.3, 149.2, 147.7, 144.6, 141.2, 134.0, 125.5, 124.0, 120.6, 112.3, 108.5, 96.6, 89.2, 79.1, 65.5, 62.6, 48.4, 47.1, 46.4, 42.2, 20.5. HRMS (ESI/MS): m/z calculated for C₂₇H₃₁F₂N₉O₃ [M + H]⁺ 568.2590 found 568.2586.

• 1-[2-(4-cyclopropanecarbonylpiperazine-1-carbonyl)-7-(morpholin-4-yl)pyrazolo[1,5-a]pyrimidin-5-yl]-2-(difluoromethyl)-1H-1,3-benzimidazole (43)

Compound 43 was prepared from 39 (0.30 g, 0.71 mmol), 1-(cyclopropylcarbonyl)piperazine (0.11 mL, 0.11 g, 0.74 mmol), HATU (0.30 g, 0.78 mmol), TEA (0.15 mL, 100 mg, 1.06 mmol), and DCM (3.0 mL), according to the general procedure for the amidation reaction. The crude product was purified by flash chromatography (0–10% MeOH gradient in AcOEt) to give 43 (0.24 g, 0.44 mmol) as a white solid with a 62% yield. ¹H NMR (600 MHz, DMSO-d₆) δ 7.89–7.88 (m, 1H, Ar-H), 7.83 (d, J = 7.8 Hz, 1H, Ar-H), 7.59 (t, J = 52.5 Hz, 1H, CHF₂), 7.49–7.43 (m, 2H, Ar-H), 6.89 (s, 1H, Ar-H), 6.82 (s, 1H, Ar-H), 3.92 (d, J = 4.5 Hz, 4H), 3.85 (s, 4H), 3.78 (s, 4H), 3.67–3.56 (m, 3H), 1.98 (s, 1H, CH), 1.16 (d, J = 7.1 Hz, 1H), 0.77–0.73 (m, 4H). ¹³C{¹H, ¹⁹F} NMR (151 MHz, DMSO-d₆) δ 171.3, 151.2, 150.1, 149.3, 147.8, 144.7, 141.2, 134.0, 125.6, 124.1, 120.7, 112.4, 108.5, 97.0, 89.4, 65.6, 48.5, 45.8, 10.4, 7.1. HRMS (ESI/MS): m/z calculated for C₂₇H₂₈F₂N₈O₃ [M + H]⁺ 551.2332 found 551.2333.

• (1-{5-[2-(difluoromethyl)-1H-1,3-benzodiazol-1-yl]-7-(morpholin-4-yl)pyrazolo[1,5-a]pyrimidine-2-carbonyl}pyrrolidin-2-yl)methanol (44)

Compound 44 was prepared from 39 (0.50 g, 1.18 mmol), pyrrolidin-2-ylmethanol (0.13 mL, 0.13 g, 1.31 mmol), HATU (0.49 g, 1.30 mmol), TEA (0.25 mL, 0.18 g, 1.79 mmol), and DCM (5.0 mL), according to the general procedure for the amidation reaction. The crude product was purified by flash chromatography (0–10% MeOH gradient in AcOEt) to give 44 (0.16 g, 0.33 mmol) as a white solid with a 28% yield. ¹H NMR (600 MHz, DMSO-d₆) δ 7.89 (d, J = 7.8 Hz, 1H, Ar-H), 7.85–7.83 (m, 1H,Ar-H), 7.60 (t, J = 52.3 Hz, 1H, CHF₂), 7.49–7.42 (m, 2H, Ar-H), 6.92–6.90 (m, 1HAr-H) 6.81 (s, 1H, Ar-H), 4.85–4.81 (m, 1H, OH), 4.24–4.17 (m, 1H), 3.96–3.90 (m, 4H, morph.), 3.87–3.83 (m, 4H, morph.), 3.68–3.59 (m, 1H), 3.54–3.45 (m, 1H), 3.37–3.35 (m, 2H, CH₂), 2.09–1.82 (m, 4H). ¹³C{¹H, ¹⁹F} NMR (151 MHz, DMSO-d₆) δ 161.0, 160.7, 151.2, 149.0, 144.7, 141.2, 134.0, 125.6, 124.1, 120.7, 112.4, 108.5, 97.3, 89.2, 65.7, 60.8, 59.7, 59.1, 49.1, 48.5, 26.3, 24.2. HRMS (ESI/MS): m/z calculated for C₂₄H₂₅F₂N₇O₃ [M + H]⁺ 498.2059 found 498.2066.

• 1-{5-[2-(difluoromethyl)-1H-1,3-benzodiazol-1-yl]-7-(morpholin-4-yl)pyrazolo[1,5-a]pyrimidine-2-carbonyl}pyrrolidin-3-amine (45)

Compound N-(1-{5-[2-(difluoromethyl)-1H-1,3-benzodiazol-1-yl]-7-(morpholin-4-yl)pyrazolo[1,5-a]pyrimidine-2-carbonyl}pyrrolidin-3-yl)carbamate (Boc-45) was prepared from 39 (0.50 g, 1.18 mmol), 3-(Boc-amino)pyrrolidine (0.24 g, 1.30 mmol), HATU (0.49 g, 1.30 mmol), TEA (0.25 mL, 0.18 g, 1.79 mmol), and DCM (5.0 mL), according to the general procedure for the amidation reaction. The crude product was purified by flash chromatography (0–10% MeOH gradient in AcOEt) to give Boc-45 (0.37 g, 0.64 mmol) as a light yellow solid with a 55% yield. ¹H NMR (300 MHz, DMSO-d₆) δ 7.93–7.82 (m, 2H, Ar-H), 7.56 (t, J = 54.0 Hz, 1H, CHF₂), 7.52–7.41 (m, 2H, Ar-H), 6.92 (d, J = 0.6 Hz, 1H, Ar-H), 6.82 (s, 1H, Ar-H), 4.87–4.79 (m, 1H), 4.71–4.16 (m, 1H), 3.99–3.77 (m, 11H), 3.71–3.58 (m, 1H), 3.58–3.32 (m, 2H), 2.14–1.79 (m, 9H, t-Bu).

The solution of Boc-45 (0.35 g, 0.60 mmol) in trifluoroacetic acid (2.09 mL, 3.11 g, 27.0 mmol, 45.0 eq) was heated at 50 °C for 3 h. The reaction was then cooled to room temperature, and stopped with 15% NaOH (15.0 mL). The aqueous mixture was extracted with DCM (3 × 15 mL). The combined organic extracts were washed with water and dried over Na₂SO₄, filtered, and concentrated. The crude product was purified by flash chromatography (50–100% AcOEt gradient in heptane, amino-functionalized gel column) to give 45 (0.14 g, 0.29 mmol) as a white solid with a 48% yield. ¹H NMR (600 MHz, DMSO-d₆) δ 7.89 (d, J = 7.6 Hz, 1H, Ar-H), 7.84–7.83 (m, 1H, Ar-H), 7.60 (t, J = 52.5 Hz, 1H, CHF₂), 7.47–7.42 (m, 2H,Ar-H), 6.91 (d, J = 3.5 Hz, 1H, Ar-H), 6.80 (s, 1H, Ar-H), 4.00–3.93 (m, 5H), 3.90–3.83 (m, 5H), 3.71–3.66 (m, 1H), 3.62 (dd, J = 12.2, 5.8 Hz, 1H), 3.55–3.48 (m, 2H), 3.24–3.15 (m, 1H, CH), 2.03–1.94 (m, 1H, CH), 1.73–1.61 (m, 1H, CH). ¹³C{¹H, ¹⁹F} NMR (151 MHz, DMSO-d₆) δ 160.5, 151.1, 148.9, 147.6, 144.6, 141.1, 133.9, 125.4, 123.9, 120.6, 112.3, 108.4, 97.1, 89.1, 65.5, 56.7, 54.8, 51.3, 48.9, 46.7, 45.0, 34.7, 32.0. HRMS (ESI/MS): m/z calculated for C₂₃H₂₄F₂N₈O₂ [M + H]⁺ 483.2063 found 483.2063.

• 2-(difluoromethyl)-1-[7-(morpholin-4-yl)-2-(morpholine-4-carbonyl)pyrazolo[1,5-a]pyrimidin-5-yl]-1H-1,3-benzimidazole (46)

Compound 46 was prepared from 39 (1.50 g, 3.62 mmol), morpholine (0.34 mL, 0.34 g, 3.90 mmol), HATU (1.52 mg, 3.99 mmol), TEA (0.76 mL, 0.55 g, 5.44 mmol), and DCM (15.0 mL), according to the general procedure for the amidation reaction. The crude product was purified by flash chromatography (50–100% AcOEt gradient in heptane) to give 46 (937.0 mg, 1.94 mmol) as a light yellow solid with a 53% yield. ¹H NMR (600 MHz, CDCl₃) δ 7.93–7.91 (m, 1H, Ar-H), 7.68–7.66 (m, 1H, Ar-H), 7.46–7.41 (m, 2H, Ar-H), 7.28 (t, J = 54.0 Hz, 1H, CHF₂), 6.91 (s, 1H, Ar-H), 6.45 (s, 1H, Ar-H), 3.98–3.96 (m, 4H, morph.), 3.92–3.86 (m, 8H, morph.), 3.84–3.82 (m, 2H, morph.), 3.73–3.72 (m, 2H, morph.). ¹³C{¹H, ¹⁹F} NMR (151 MHz, CDCl₃) δ 162.9, 151.7, 150.9, 149.9, 148.7, 144.7, 142.1, 134.7, 126.2, 124.6, 121.8, 112.0, 109.7 (CF₂), 98.4, 89.0, 67.2, 66.4, 48.9, 43.1. HRMS (ESI/MS): m/z calculated for C₂₃H₂₃F₂N₇O₃ [M + H]⁺ 484.1903 found 484.1912.

• 2-(difluoromethyl)-1-[2-(4,4-difluoropiperidine-1-carbonyl)-7-(morpholin-4-yl)pyrazolo[1,5-a]pyrimidin-5-yl]-1H-1,3-benzimidazole (47)

Compound 47 was prepared from 39 (1.50 g, 3.62 mmol), 4,4-difluoropiperidine (0.63 g, 3.99 mmol), HATU (1.52 mg, 3.99 mmol), TEA (1.31 mL, 0.95 g, 9.35 mmol), and DCM (15.0 mL), according to the general procedure for the amidation reaction. The crude product was purified by flash chromatography (0–50% AcOEt gradient in heptane, amino-functionalized gel column) to give 47 (934.0 mg, 1.80 mmol) as a light yellow solid with a 50% yield. ¹H NMR (300 MHz, CDCl₃) δ 7.96–7.88 (m, 1H, Ar-H), 7.71–7.66 (m, 1H, Ar-H), 7.48–7.41 (m, 2H, Ar-H), 7.29 (t, J = 54.0 Hz, 1H, CHF₂), 6.93 (s, 1H, Ar-H), 6.47 (s, 1H, Ar-H), 4.03–3.95 (m, 8H), 3.93–3.87 (m, 4H, morph.), 2.22–1.98 (m, 4H). ¹³C{¹H} NMR (75 MHz, CDCl₃) δ 163.3, 152.1, 150.3, 149.1, 145.1 (t, J = 27.0 Hz), 142.5, 135.2, 126.5, 125.0, 122.0 (t, J = 240.7 Hz), 112.4, 110.1 (t, J = 238.5 Hz), 98.9, 89.6, 66.7, 49.3, 44.4, 40.1, 35.6 (t, J = 24.7 Hz), 34.5 (t, J = 22.5 Hz). HRMS (ESI/MS): m/z calculated for C₂₄H₂₃F₄N₇O₂ [M + H]⁺ 518.1922 found 518.1924.

• 1-{5-[2-(difluoromethyl)-1H-1,3-benzimidazol-1-yl]-7-(morpholin-4-yl)pyrazolo[1,5-a]pyrimidine-2-carbonyl}piperidine-4-carboxamide (48)

Compound 48 was prepared from 39 (1.50 g, 3.62 mmol), 4-piperidinecarboxamide (0.51 g, 3.90 mmol), HATU (1.52 mg, 3.99 mmol), TEA (0.76 mL, 0.55 g, 5.44 mmol), and DCM (15.0 mL), according to the general procedure for the amidation reaction. The crude product was purified by flash chromatography (0–20% MeOH gradient in AcOEt) to give 48 (0.73 g, 1.39 mmol) as a light yellow solid with a 39% yield. ¹H NMR (600 MHz, DMSO-d₆) δ 7.89–7.88 (m, 1H, Ar-H), 7.84–7.82 (m, 1H, Ar-H), 7.59 (t, J = 52.5 Hz, 1H, CHF₂), 7.49–7.42 (m, 2H), 7.31 (s, 1H, Ar-H), 6.83 (s, 1H, Ar-H), 6.82 (s, 1H), 6.80 (s, 1H), 4.48 (d, J = 13.0 Hz, 2H, CH₂), 4.25 (d, J = 13.6 Hz, 2H, CH₂), 3.93–3.91 (m, 4H, morph.), 3.84 (t, J = 4.7 Hz, 4H, morph.), 3.18 (s, 2H), 2.90 (d, J = 2.5 Hz, 2H), 2.43 (s, 1H). ¹³C{¹H, ¹⁹F} NMR (151 MHz, DMSO-d₆) δ 175.8, 161.8, 151.2, 150.6, 149.2, 147.7, 144.7, 141.2, 134.0, 125.5, 124.0, 120.7, 112.4, 108.5, 96.4, 89.3, 65.6, 48.4, 46.2, 41.4, 29.0, 28.2. HRMS (ESI/MS): m/z calculated for C₂₅H₂₆F₂N₈O₃ [M + H]⁺ 525.2168 found 525.2171.

• 1-{5-[2-(difluoromethyl)-1H-1,3-benzimidazol-1-yl]-7-(morpholin-4-yl)pyrazolo[1,5-a]pyrimidine-2-carbonyl}-4-methylpiperidin-4-ol (49)

Compound 49 was prepared from 39 (1.50 g, 3.62 mmol), 4-methylpiperidin-4-ol (0.47 g, 3.89 mmol), HATU (1.52 mg, 3.99 mmol), TEA (0.76 mL, 0.55 g, 5.44 mmol), and DCM (15.0 mL), according to the general procedure for the amidation reaction. The crude product was purified by flash chromatography (0–10% MeOH gradient in AcOEt) to give 49 (0.90 g, 1.76 mmol) as a light yellow solid with a 49% yield. ¹H NMR (600 MHz, CDCl₃) δ 7.93–7.91 (m, 1H, Ar-H), 7.67 (dd, J = 7.4, 1.8 Hz, 1H,Ar-H), 7.46–7.41 (m, 2H, Ar-H), 7.29 (t, J = 54.0 Hz, 1H, CHF₂), 6.87 (s, 1H, Ar-H), 6.42 (s, 1H, Ar-H), 4.45–4.41 (m, 1H), 4.12–4.09 (m, 1H), 3.98–3.96 (m, 4H, morph.) 3.91–3.90 (m, 4H, morph.), 3.65–3.61 (m, 1H), 3.42–3.38 (m, 1H), 1.78–1.71 (m, 2H, CH₂), 1.68–1.62 (m, 3H, CH₃), 1.36 (s, 1H), 1.34 (s, 3H). ¹³C{¹H, ¹⁹F} NMR (151 MHz, CDCl₃) δ 162.8, 151.7, 149.8, 148.5, 144.8, 142.1, 134.8, 126.1, 124.5, 121.8, 112.0, 109.7 (CF₂), 97.9, 88.8, 68.4, 66.4, 48.9, 43.8, 39.5, 39.1, 38.6, 30.6. HRMS (ESI/MS): m/z calculated for C₂₅H₂₇F₂N₇O₃ [M + H]⁺ 512.2216 found 512.2222.

• (1-{5-[2-(difluoromethyl)-1H-1,3-benzimidazol-1-yl]-7-(morpholin-4-yl)pyrazolo[1,5-a]pyrimidine-2-carbonyl}piperidin-4-yl)methanol (50)

Compound 50 was prepared from 39 (1.50 g, 3.62 mmol), 4-piperidinemethanol (0.46 g, 3.95 mmol), HATU (1.52 mg, 3.99 mmol), TEA (0.76 mL, 0.55 g, 5.44 mmol), and DCM (15.0 mL), according to the general procedure for the amidation reaction. The crude product was purified by flash chromatography (0–10% MeOH gradient in AcOEt) to give 50 (0.76 g, 1.54 mmol) as a light yellow solid with a 43% yield. ¹H NMR (600 MHz, DMSO-d₆) δ 7.90–7.87 (m, 1H, Ar-H), 7.83 (dd, J = 7.0, 1.3 Hz, 1H, Ar-H), 7.61 (t, J = 52.5 Hz, 1H, CHF₂), 7.49–7.41 (m, 2H, Ar-H), 6.81 (s, 1H, Ar-H), 6.80 (s, 1H), 4.53 (t, J = 5.3 Hz, 2H, CH₂), 4.24 (d, J = 13.3 Hz, 1H), 3.92–3.91 (m, 4H, morph.), 3.85–3.83 (m, 5H), 3.30–3.28 (m, 2H), 3.15–3.08 (m, 1H), 2.85–2.78 (m, 1H), 1.78 (d, J = 13.1 Hz, 1H), 1.70 (d, J = 10.4 Hz, 2H), 1.12–1.09 (m, 1H). ¹³C{¹H, ¹⁹F} NMR (151 MHz, DMSO-d₆) δ 170.6, 161.9, 151.4, 149.5, 148.0, 144.9, 141.4, 134.2, 125.8, 124.3, 120.9, 112.7, 108.7, 96.6, 89.4, 65.8, 60.0, 49.0, 46.9, 42.0, 38.6, 29.5, 28.7. HRMS (ESI/MS): m/z calculated for C₂₅H₂₇F₂N₇O₃ [M + H]⁺ 512.2216 found 512.2218.

• 1-{5-[2-(difluoromethyl)-1H-1,3-benzimidazol-1-yl]-7-(morpholin-4-yl)pyrazolo[1,5-a]pyrimidine-2-carbonyl}piperidine-4-carbonitrile (51)

Compound 51 was prepared from 39 (1.50 g, 3.62 mmol), 4-cyanopiperidine (0.44 mL, 0.43 g, 3.96 mmol), HATU (1.52 mg, 3.99 mmol), TEA (0.76 mL, 0.55 g, 5.44 mmol), and DCM (15.0 mL), according to the general procedure for the amidation reaction. The crude product was purified by flash chromatography (50–100% AcOEt gradient in heptane) to give 51 (0.86 g, 1.69 mmol) as a light yellow solid with a 47% yield. ¹H NMR (400 MHz, CDCl₃) δ 7.93–7.91 (m, 1H, Ar-H), 7.69–7.66 (m, 1H, Ar-H), 7.47–7.40 (m, 2H, Ar-H), 7.28 (t, J = 54.0 Hz, 1H, CHF₂), 6.90 (s, 1H, Ar-H), 6.46 (s, 1H, Ar-H), 4.08–3.97 (m, 6H), 3.91–3.83 (m, 6H), 3.02–2.98 (m, 1H, CH), 2.08–1.92 (m, 4H). ¹³C{¹H, ¹⁹F} NMR (101 MHz, CDCl₃) δ 162.6, 151.5, 150.6, 149.7, 148.5, 144.5, 141.8, 134.5, 126.0, 124.4, 121.6, 120.6, 111.8, 109.5 (CF₂), 98.1, 88.9, 66.1, 48.7, 45.1, 40.4, 29.3, 28.3, 26.4. HRMS (ESI/MS): m/z calculated for C₂₅H₂₄F₂N₈O₂ [M + H]⁺ 507.2063 found 507.2068.

• 2-(difluoromethyl)-1-[7-(morpholin-4-yl)-2-[4-(morpholin-4-yl)piperidine-1-carbonyl]pyrazolo[1,5-a]pyrimidin-5-yl]-1H-1,3-benzimidazole (52)

Compound 52 was prepared from 39 (1.50 g, 3.62 mmol), 4-morpholinopiperidine (0.68 g, 3.90 mmol), HATU (1.52 mg, 3.99 mmol), TEA (0.76 mL, 0.55 g, 5.44 mmol), and DCM (15.0 mL), according to the general procedure for the amidation reaction. The crude product was purified by flash chromatography (0–20% MeOH gradient in AcOEt) to give 52 (0.70 g, 1.23 mmol) as a light yellow solid with a 34% yield. ¹H NMR (400 MHz, DMSO-d₆) δ 7.90–7.88 (m, 1H, Ar-H), 7.83 (dd, J = 7.2, 1.3 Hz, 1H, Ar-H), 7.61 (t, J = 52.5 Hz, 1H, CHF₂), 7.49–7.44 (m, 2H), 6.85 (s, 1H, Ar-H), 6.82 (s, 1H, Ar-H), 4.21–4.65 (m, 2H, CH₂), 3.92 (d, J = 3.3 Hz, 5H), 3.86–3.84 (m, 5H), 3.66–3.61 (m, 4H, morph.), 3.15–3.07 (m, 4H, morph.), 2.86 (d, J = 1.2 Hz, 1H, CH), 1.78–2.06 (m, 2H, CH₂), 1.37–1.57 (m, 2H, CH₂). ¹³C{¹H, ¹⁹F} NMR (101 MHz, DMSO-d₆) δ 161.6, 151.2, 149.2, 147.8, 144.7, 141.2, 134.0, 125.5, 124.1, 120.7, 112.4, 108.5, 108.5, 89.3, 65.6, 48.7, 48.4, 45.7, 26.8, 8.6. HRMS (ESI/MS): m/z calculated for C₂₈H₃₂F₂N₈O₃ [M + H]⁺ 567.2638 found 567.2643.

• 1-{5-[2-(difluoromethyl)-1H-1,3-benzimidazol-1-yl]-7-(morpholin-4-yl)pyrazolo[1,5-a]pyrimidine-2-carbonyl}piperidin-4-ol (53)

Compound 53 was prepared from 39 (1.50 g, 3.62 mmol), 4-hydroxypiperidine (0.41 g, 4.06 mmol), HATU (1.52 mg, 3.99 mmol), TEA (0.76 mL, 0.55 g, 5.44 mmol), and DCM (15.0 mL), according to the general procedure for the amidation reaction. The crude product was purified by flash chromatography (0–15% MeOH gradient in AcOEt) to give 53 (0.86 g, 1.72 mmol) as a light yellow solid with a 48% yield. ¹H NMR (600 MHz, DMSO-d₆) δ 7.90–7.88 (m, 1H, Ar-H), 7.84–7.82 (m, 1H, Ar-H), 7.59 (t, J=52.5 Hz, 1H, CHF₂), 7.49–7.43 (m, 2H, Ar-H), 6.83 (s, 1H, Ar-H), 6.81 (s, 1H, Ar-H), 4.83 (d, J = 4.0 Hz, 1H, OH), 4.08–4.03 (m, 2H, CH₂), 4.02–3.97 (m, 2H, CH₂), 3.93–3.92 (m, 4H, morph.), 3.85–3.84 (m, 4H, morph.), 3.81–3.77 (m, 1H, CH), 3.44–3.37 (m, 2H, CH₂), 3.32–3.28 (m, 2H, CH₂). ¹³C{¹H, ¹⁹F} NMR (151 MHz, DMSO-d₆) δ 162.0, 151.5, 150.9, 149.5, 148.0, 145.0, 141.5, 134.3, 125.8, 124.3, 121.0, 112.7, 108.8, 96.7, 89.5, 65.9, 65.7, 48.7, 44.3, 35.0, 34.2. HRMS (ESI/MS): m/z calculated for C₂₄H₂₅F₂N₇O₃ [M + H]⁺ 498.2059 found 498.2060.

• N-(1-{5-[2-(difluoromethyl)-1H-1,3-benzimidazol-1-yl]-7-(morpholin-4-yl)pyrazolo[1,5-a]pyrimidine-2-carbonyl}piperidin-4-yl)acetamide (54)

Compound 54 was prepared from 39 (1.50 g, 3.62 mmol), 4-acetylamino-piperidine (0.57 g, 3.89 mmol), HATU (1.52 mg, 3.99 mmol), TEA (0.76 mL, 0.55 g, 5.44 mmol), and DCM (15.0 mL), according to the general procedure for amidation reaction. The crude product was purified by flash chromatography (0–20% MeOH gradient in AcOEt) to give 54 (0.80 g, 1.48 mmol) as a light yellow solid with a 41% yield. ¹H NMR (600 MHz, CDCl₃) δ 7.93–7.91 (m, 1H, Ar-H), 7.68–7.66 (m, 1H, Ar-H), 7.46–7.42 (m, 2H, Ar-H), 7.29 (t, J = 54.0 Hz, 1H, CHF₂), 6.87 (s, 1H, Ar-H), 6.44 (s, 1H, Ar-H), 5.55 (d, J = 7.8 Hz, 1H, NH), 4.74 (d, J = 13.7 Hz, 2H, CH₂), 4.43 (d, J = 15.1 Hz, 2H CH₂ 4.13–4.07 (m, 1H, CH), 3.98–3.96 (m, 4H, morph.), 3.91–3.89 (m, 4H, morph.), 3.31–3.27 (m, 1H), 3.03–2.99 (m, 1H), 2.10–2.05 (m, 1H), 2.00 (s, 3H, CH₃), 1.55–1.40 (m, 1H). ¹³C{¹H, ¹⁹F} NMR (151 MHz, CDCl₃) δ 169.3, 162.6, 151.4, 150.9, 149.5, 148.3, 144.4, 141.7, 134.4, 125.8, 124.2, 121.5, 111.7, 109.4 (CF₂), 97.8, 88.6, 66.1, 48.6, 46.7, 45.9, 41.4, 32.9, 31.7, 23.3. HRMS (ESI/MS): m/z calculated for C₂₆H₂₈F₂N₈O₃ [M + H]⁺ 539.2352 found 539.2325.

• 2-(4-{5-[2-(difluoromethyl)-1H-1,3-benzimidazol-1-yl]-7-(morpholin-4-yl)pyrazolo[1,5-a]pyrimidine-2-carbonyl}piperazin-1-yl)-N-methylacetamide (55)

Compound 55 was prepared from 39 (1.50 g, 3.62 mmol), N-methyl-2-(1-piperazinyl)acetamide (0.65 g, 3.97 mmol), HATU (1.52 mg, 3.99 mmol), TEA (0.76 mL, 0.55 g, 5.44 mmol), and DCM (15.0 mL), according to the general procedure for the amidation reaction. The crude product was purified by flash chromatography (0–15% MeOH gradient in AcOEt) to give 55 (0.66 g, 1.18 mmol) as a light yellow solid with a 33% yield. ¹H NMR (600 MHz, DMSO-d₆) δ 7.89–7.88 (m, 1H, Ar-H), 7.83–7.82 (m, 1H, Ar-H), 7.79 (s, 1H, Ar-H), 7.59 (t, J = 52.5 Hz, 1H, CHF₂), 7.49–7.42 (m, 2H, Ar-H), 6.84 (s, 1H, Ar-H), 6.81 (s, 1H, Ar-H), 3.92–3.91 (m, 4H, morph.), 3.85–3.83 (m, 4H), 3.80–3.73 (m, 4H), 2.98 (s, 2H), 2.62 (d, J = 4.7 Hz, 3H, CH₃), 2.54 (s, 2H, CH₂), 1.09 (s, 2H, CH₂). ¹³C{¹H, ¹⁹F} NMR (151 MHz, DMSO-d₆) δ 161.6, 151.0, 150.1, 149.1, 147.6, 144.5, 141.1, 133.9, 125.4, 123.9, 120.6, 112.3, 108.4, 96.6, 89.2, 71.9, 65.4, 53.0, 52.4, 48.6, 48.3, 45.7, 26.6, 25.2. HRMS (ESI/MS): m/z calculated for C₂₆H₂₉F₂N₉O₃ [M + H]⁺ 554.2434 found 554.2434.

• 2-(difluoromethyl)-1-[2-(4-methylpiperazine-1-carbonyl)-7-(morpholin-4-yl)pyrazolo[1,5-a]pyrimidin-5-yl]-1H-1,3-benzimidazole (56)

Compound 56 was prepared from 39 (0.20 g, 0.48 mmol), 1-methylpiperazine (0.05 mL, 50.2 mg, 0.49 mmol), HATU (0.20 g, 0.52 mmol), TEA (0.09 mL, 72.2 mg, 0.71 mmol), and DCM (2.0 mL), according to the general procedure for the amidation reaction. The crude product was purified by flash chromatography (0–35% MeOH gradient in AcOEt) to give 56 (0.13 g, 0.27 mmol) as a white solid with a 57% yield. ¹H NMR (600 MHz, DMSO-d₆) δ 7.89 (m, 1H, Ar-H), 7.83–7.81 (m, 1H, Ar-H), 7.58 (t, J = 52.4 Hz, 1H, CHF₂), 7.49–7.42 (m, 2H, Ar-H), 6.83 (s, 1H, Ar-H), 6.81 (s, 1H, Ar-H), 3.92 (t, J = 4.7 Hz, 4H, morph.), 3.84 (t, J = 4.7 Hz, 4H, morhp.), 3.76–3.69 (m, 4H, morph.), 2.43–2.37 (m, 4H, morph.), 2.23 (s, 3H, CH₃). ¹³C{¹H, ¹⁹F} NMR (151 MHz, DMSO-d₆) δ 161.7, 151.1, 150.3, 149.2, 147.7, 144.6, 141.2, 134.0, 125.5, 124.0, 120.6, 112.3, 108.5, 96.6, 89.2, 65.5, 54.8, 48.4, 46.5, 45.5, 41.7, 30.8. HRMS (ESI/MS): m/z calculated for C₂₄H₂₆F₂N₈O₂ [M + H]⁺ 497.2219 found 497.2229.

• 2-(difluoromethyl)-1-[7-(morpholin-4-yl)-2-[4-(propan-2-yl)piperazine-1-carbonyl]pyrazolo[1,5-a]pyrimidin-5-yl]-1H-1,3-benzimidazole (57)

Compound 57 was prepared from 39 (0.20 g, 0.48 mmol), 1-isopropylpiperazine (0.07 mL, 65.7 mg, 0.49 mmol), HATU (0.20 g, 0.52 mmol), TEA (0.09 mL, 72.2 mg, 0.71 mmol), and DCM (2.0 mL), according to the general procedure for the amidation reaction. The crude product was purified by flash chromatography (0–30% MeOH gradient in AcOEt) to give 57 (0.20 g, 0.38 mmol) as a white solid with a 81% yield. ¹H NMR (600 MHz, DMSO-d₆) δ 7.89–7.88 (m, 1H, Ar-H), 7.83–7.82 (m, 1H, Ar-H), 7.58 (t, J = 52.5 Hz, 1H, CHF₂), 7.49–7.42 (m, 2H, Ar-H), 6.83 (s, 1H, Ar-H), 6.80 (s, 1H, Ar-H), 3.93–3.91 (m, 4H, morph., morph.), 3.85–3.84 (m, 4H, morph.), 3.73–3.71 (m, 2H, CH₂), 3.67–3.66 (m, 2H, CH₂), 2.72–2.68 (m, 1H, CH), 2.53–2.50 (m, 2H, CH₂), 2.47 (d, J = 4.8 Hz, 2H, CH₂), 0.98 (d, J = 6.6 Hz, 6H, 2xCH₃). ¹³C NMR (151 MHz, DMSO-d₆) δ 161.6, 151.1, 150.4, 149.2, 147.7, 144.6, 141.2, 134.0, 125.5, 124.0, 120.6, 112.3, 108.4, 96.6, 89.2, 65.5, 53.6, 48.5, 48.4, 47.9, 47.0, 42.2, 18.0. HRMS (ESI/MS): m/z calculated for C₂₆H₃₀F₂N₈O₂ [M + H]⁺ 525.2532 found 525.2544.

3.2. Docking Study

The docking procedure was performed using the PI3Kδ protein from the Protein Data Bank (PDB: 2WXL) with the Auto-Dock Vina program [40]. All figures with examples of 3D modeling of a possible binding mode of selected compounds were prepared based on the calculated pK_a from the Instant JChem 21.13.0 program [39]. More specifically, all structures depicted in the respective figures have not had protons added, but the appropriate state of protonation has been maintained.

3.3. Biology

3.3.1. In Vitro PI3K Inhibition Assays

The potency and selectivity of compounds were assessed by measuring the ability of PI3K kinases to convert ATP to ADP during an enzymatic reaction in the presence of decreasing doses of tested compounds. The experiments were carried out using the ADP-Glo kinase assay kit (Promega), according to the manufacturer’s protocol. PI3Kα, PI3Kβ, PI3Kδ, and PI3Kγ have been purchased from Merck Millipore and phosphoinositol-4,5-bisphosphate (PIP2) lipid vesicles with phosphoserine from ThermoFisher Scientific were used as a substrate in the enzymatic reaction. The composition of the reaction mixture and reaction conditions for individual kinases are listed in the table below (Table 6).

After the reaction, the ADP-Glo reagent and the kinase detection reagent were added sequentially with 40 min of incubation (25 °C, 600 rpm) after adding each reagent. Finally, the luminescence intensity was measured and the IC₅₀ was calculated using GraphPad Prism 7 software. The presented results are the mean value of IC₅₀ from at least two independent experiments.

3.3.2. Influence of Selected Compounds on B Cells Proliferation

CD19 cells were isolated from PBMCs using magnetic beads (Stem Cell (Vancouver, Canada)) and then labeled with 2 µM of CFSE (Invitrogen (Waltham, MA, USA)).

Then, 1 × 105 cells were seeded on 96-well plates, activated by 2 µg/mL of αIgM (Jackson ImmunoResearch (West Grove, PA, USA)) and 1 µg/mL of ODN2006 (InvivoGen (San Diego, CA, USA)), and incubated with increasing concentrations of drugs (0.1, 0.3, 1.0, 3.3, 10, 33, 100, 333, 1000, 3333, and 10000 nM). After 4 days, cells were stained with LIVE/DEAD™ kit (Invitrogen (Waltham, MA, USA)). Samples were acquired using Attune NxT flow cytometer (Invitrogen) and analyzed using FlowJo software. Each biological assay was performed with cells isolated from a different donor. The presented results constitute the average percentage values of proliferating cells from 3 independent experiments.

3.4. Metabolic Stability and Solubility

3.4.1. Metabolic Stability Assay

The metabolic phase I stability in mouse (CD-1™) and human microsomes (Thermo-Fisher Scientific (Waltham, MA, USA)) was assessed on 96-well non-binding plates (Greiner (Kremsmuster, Austria)) at a 1 μM concentration for verapamil (positive control) and donepezil (negative control) and tested compounds. Unless otherwise stated, all chemicals and materials were ordered from Merck Life Science (Sheboygan, WI, USA). Each biological replicate was prepared in triplicates [40,41]. Briefly, compounds were incubated in 100 mM potassium phosphate buffer with microsomes (0.5 mg/mL) and NADPH (1–1.2 mM) on a plate shaker (500 rpm) in the dark at 37 °C. On a 4× solution of NADPH, the cofactor for metabolic enzymes was prepared directly prior to the experiment by reducing NADP with G6P dehydrogenase (13.2 mM MgCl₂, 13.2 mM G6P, 5.2 mM NADP, 3.2 U/mL G6P dehydrogenase, 20 min at 30 °C, 500 rpm). The negative control contained buffer instead of NADPH solution. Samples were collected at 0, 10, 20, and 40 min or 0 and 40 min for the negative and double negative controls. The reaction was stopped by protein precipitation in 2 volumes of ice-cold MeOH with 200 nM of imipramine (as an internal standard for LC-MS analysis). Then, the extract was mixed (1 min, 1000 rpm), filtered through a 0.22 μm filter on a 96-well plate vacuum manifold, and subjected to LC-MS analysis.

3.4.2. Lymphocyte B Proliferation Assessment

Human PBMCs were isolated from buffy coats of healthy donors obtained from the Regional Blood Donation and Blood Medicine Center in Warsaw. Lymphocytes B: CD19+ cells were isolated from PBMCs on magnetic beads (Stem Cell), labeled with 2 µM CFSE (Invitrogen), and seeded on a 96-well plate (1 × 10⁵ cells/well). B cells were activated by 2 µg/mL of αIgM (Jackson ImmunoResearch) and 1 µg/mL of ODN2006 (InvivoGen). Cells were incubated with increasing concentrations of compounds (range 0.03–10,000 nM). After 4 days, B cells were stained with LIVE/DEAD™ kit (Invitrogen), acquired using the Attune NxT flow cytometer (Invitrogen), and analyzed using FlowJo10 software. For data normalization, the percentage value of proliferating cells in each sample was divided by the average percentage value of positive control in a single experiment. IC₅₀ values were calculated using a three-parameter dose–response inhibition function in GraphPad Prism.

3.4.3. Kinetic Stability Assay

The kinetic solubility was determined using the shake-flask protocol [42,43]. The appropriate compounds (500 µM) were incubated in an aqueous buffer (0.1 M phosphate-buffered saline pH 7.4) at 25 °C with stirring at 500 rpm. The samples were taken at the start time and after 24 h of incubation, filtered through 0.22 µm filters, and diluted with 2 volumes of acetonitrile. Sample concentrations were determined by UHPLC-UV/Vis. A calibration curve was prepared in order to quantify the contents of the compound in the test solution.

4. Conclusions

A new family of substituted pyrazolo[1,5-a]pyrimidines was prepared in multi-step synthesis utilizing the Buchwald–Hartwig reaction or reductive amination as the crucial synthetic steps. The SAR studies were performed firstly at the C(5) position of the pyrazolo[1,5-a]pyrimidine and then the final optimization was turned up using a careful sterical amino group at the C(2) position adjustment. The biological activities were measured for each new compound against four PI3K isoforms: α, β, γ, and δ, providing comprehensive information on the selectivity of the obtained structures. Eleven compounds with an IC₅₀ value below the 100 nM threshold within the new compounds’ library were synthesized in this work. Five of them, with an IC₅₀ value below or equal to 52 nM, were assumed as hits. Molecular modeling studies provide a rational explanation for the interaction of active structures within the PI3Kδ ATP binding site. CPL302415 (1-{2-[(4-tert-butylpiperazin-1-yl)methyl]-7-(morpholin-4-yl)pyrazolo[1,5-a]pyrimidin-5-yl}-2-(difluoromethyl)-1H-benzimidazole, compound 6) proved to be the most potent structure with excellent activity (IC₅₀ = 18 nM), good selectivity (PI3Kα/PI3Kδ = 79; PI3Kb/δ = 1415; PI3Kγ/PI3Kδ = 939), and other promising parameters (Table 5). Therefore, CPL302415 was selected as a lead compound for toxicological studies and as a candidate for further development in phase I clinical trials in SLE treatment. More detailed biological and physicochemical studies and their outcomes are the subjects of a separate paper under preparation.

Author Contributions

Synthesis, M.S., M.Z., S.M., N.O. and M.D.; biological evaluation M.B., P.T., P.G., D.Z.-B., A.S., U.K. and B.Z.; analytical evaluation K.M., D.S., W.M., L.G.-B. and A.L.; investigation, M.S., M.Z., P.G., D.Z.-B. and B.Z.; writing—original draft preparation, M.S., M.Z. and S.M.; writing—review and editing, M.M., P.G., D.Z.-B. and Z.O.; visualization, W.P.; supervision, M.W. and Z.O.; project administration, M.Z., P.G., A.S., B.Z., K.D., J.P., M.W. and Z.O.; funding acquisition, M.W. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data is contained within the article and supplementary material.

Acknowledgments

We would like to thank Aleksandra Świderska (Celon Pharma S.A.) for NMR analyses and practical suggestions. This work was supported by The National Centre for Research and Development (POIR.01.01.01-00-1341/15) in Poland.

Conflicts of Interest

The authors declare the following financial interest/personal relationships which may be considered as potential competing interests. All contributors to this work at the time of their direct involvement in the project were the full-time employees of Celon Pharma S.A. A patent application WO 2016/157091 A1, based on the present observations, has been filed. M. Wieczorek is the CEO of Celon Pharma S.A. Some of the authors are the shareholders of Celon Pharma S.A. This work was financially supported by The National Centre for Research and Development (POIR.01.01.01-00-1341/15).

Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures, Schemes and Tables

Figure 1. Structure of the PI3Kδ active inhibitor and GDC-0941.

Scheme 1. Synthesis of benzimidazole derivatives. Reagents and conditions: (i) CaCl2, NaBH4, EtOH, reflux, 3 h, 99%; (ii) Dess–Martin periodinane, DMF, RT, 2 h, 46%; (iii) 1-tert-butylipiperazine, sodium triacetoxyborohydride, DCM, RT, 18 h, 84%; (iv) benzimidazole derivative, tris(dibenzylideneacetone)dipalladium(0), 9,9-dimethyl-4,5-bis(diphenylphosphino)xanthene, Cs2CO3, toluene, 150 °C, 6 h, 200 W, MW, 4–93%; (v) 2-(4-piperidyl)-2-propanol, sodium triacetoxyborohydride, DCM, RT, 63%; (vi) benzimidazole derivative, tris(dibenzylideneacetone)dipalladium(0), 9,9-Dimethyl-4,5-bis(diphenylphosphino)xanthene, Cs2CO3, toluene, 150 °C, 6 h, 200 W, MW, 52–66%.

Scheme 2. Synthesis of 5-(2-difluoromethylobenzimidazo-1-yl)pyrazolo[1,5-a]pyrimidine derivatives. Reagents and conditions: (i) 2-(difluoromethyl)-1H-benzimidazole, TEACl, K2CO3, DMA, 160 °C, 3 h, 89%; (ii) LiAlH4, THF, 0 °C, 3 h, 89%, (iii) Dess–Martin periodinane, DMF, RT, 1 h, 78% or MnO2, toluene:buthyl acetate, reflux 1.5 h, 68%; (iv) amine, sodium triacetoxyborohydride, DCM, 18 h, 38–93%; (v) LiOH, MeOH, H2O, 98%; (vi) amine, HATU, TEA, RT, 2 h, 33–81%.

Figure 2. The most important interactions in the PI3K binding site for selected structures. An example of 3D modeling possible interaction found for selected compounds (PDB ID:2WXL): (A)—compound 11; (B)—compound 17; (C)—compound 30; (D)—compound 49; (E)—compound 6; (F)—compound 40. No protons were added, but the appropriate state of protonation was maintained.

Scheme 3. The correlation of the potencies given for the compounds lacking HBD interaction within the heterocyclic system.

Scheme 4. The correlation of the potencies given for the compounds lacking HBD interaction within the heterocyclic system.

Table 1

Activity and selectivity of benzimidazole derivatives (5–12).

[Image omitted. Please see PDF.]
Compound	R1	R2	IC50 PI3Kδ[nM]	IC50 PI3Kα[nM]	IC50 PI3Kβ[nM]	IC50 PI3Kγ[nM]	Fold Selectivity
							α/ δ	β/ δ	γ / δ
5	[Image omitted. Please see PDF.]	[Image omitted. Please see PDF.]	236	10,642	298,400	175,187	45	1264	742
6	[Image omitted. Please see PDF.]	[Image omitted. Please see PDF.]	18	1428	25,475	16,904	79	1415	939
7	[Image omitted. Please see PDF.]	[Image omitted. Please see PDF.]	385	28,677	265,699	258,277	74	690	671
8	[Image omitted. Please see PDF.]	[Image omitted. Please see PDF.]	1072	82,502	259,143	271,097	77	242	253
9	[Image omitted. Please see PDF.]	[Image omitted. Please see PDF.]	907			135,500			149
11	[Image omitted. Please see PDF.]	[Image omitted. Please see PDF.]	52	1729		6347	33		122
12	[Image omitted. Please see PDF.]	[Image omitted. Please see PDF.]	878	30,511	194,624	119,254	35	222	136

IC₅₀ values were determined as the mean based on two independent experiments.

Table 2

PI3Kδ and PI3Kg activity of pyrazolo[1,5-a]pyrimidine derivatives with (-CH₂) groups.

[Image omitted. Please see PDF.]
Compound	R2	IC50 PI3Kδ[nM]	IC50 PI3Kγ[nM]	FoldSelectivity γ/δ
16	[Image omitted. Please see PDF.]	43	111	2.6
17	[Image omitted. Please see PDF.]	31	2197	71
18	[Image omitted. Please see PDF.]	24	156	6.5
19	[Image omitted. Please see PDF.]	1070	4474	4.2
20	[Image omitted. Please see PDF.]	1892	22,134	12
21	[Image omitted. Please see PDF.]	956	10,944	11
22	[Image omitted. Please see PDF.]	979	8420	8.6
23	[Image omitted. Please see PDF.]	135	1380	10
24	[Image omitted. Please see PDF.]	254	588	2.3
25	[Image omitted. Please see PDF.]	647	1972	3.0
26	[Image omitted. Please see PDF.]	203	4283	21
27	[Image omitted. Please see PDF.]	1805	20,526	11
28	[Image omitted. Please see PDF.]	443	1709	3.9
29	[Image omitted. Please see PDF.]	240	1855	7.7
30	[Image omitted. Please see PDF.]	615	7553	12
31	[Image omitted. Please see PDF.]	593	8318	14
32	[Image omitted. Please see PDF.]	192	3155	16
33	[Image omitted. Please see PDF.]	276	4800	17
34	[Image omitted. Please see PDF.]	561	4317	7.7
35	[Image omitted. Please see PDF.]	445	1503	3.4
36	[Image omitted. Please see PDF.]	63	1247	20
37	[Image omitted. Please see PDF.]	351	17,658	50
38	[Image omitted. Please see PDF.]	38	13,330	351

IC₅₀ values were determined as the mean based on two independent experiments.

Table 3

PI3Kδ and PI3Kg activity of pyrazolo[1,5-a]pyrimidine derivatives with (-CO) groups.

[Image omitted. Please see PDF.]
Compound	R2	IC50 PI3Kδ[nM]	IC50 PI3Kγ[nM]	FoldSelectivity γ/δ
40	[Image omitted. Please see PDF.]	84	48,777	581
41	[Image omitted. Please see PDF.]	101	2483	25
42	[Image omitted. Please see PDF.]	74	3593	48
43	[Image omitted. Please see PDF.]	63	3831	61
44	[Image omitted. Please see PDF.]	459	2024	4.4
45	[Image omitted. Please see PDF.]	1704	23,290	14
46	[Image omitted. Please see PDF.]	226	8536	38
47	[Image omitted. Please see PDF.]	801	35,750	45
48	[Image omitted. Please see PDF.]	314	2793	9
49	[Image omitted. Please see PDF.]	213	1818	8.5
50	[Image omitted. Please see PDF.]	90	1782	20
51	[Image omitted. Please see PDF.]	344	1629	4.7
52	[Image omitted. Please see PDF.]	92	5946	65
53	[Image omitted. Please see PDF.]	195	2081	11
54	[Image omitted. Please see PDF.]	141	1689	12
55	[Image omitted. Please see PDF.]	82	2170	26
56	[Image omitted. Please see PDF.]	768	11,397	94
57	[Image omitted. Please see PDF.]	271	25,527	15

IC₅₀ values were determined as the mean based on two independent experiments.

Table 4

Activity and selectivity of the most promising compounds.

[Image omitted. Please see PDF.]
Compound	R2	IC50 PI3Kδ[nM]	IC50 PI3Kα[nM]	IC50 PI3Kβ[nM]	IC50 PI3Kγ[nM]	α/ δ	β/ δ	γ / δ	IC50 CD19[nM]
6	[Image omitted. Please see PDF.]	18	1428	25,475	16,904	79	1415	939	41
11	[Image omitted. Please see PDF.]	52	1729		6347	33		122
16	[Image omitted. Please see PDF.]	43	44	13,577	111	1.0	316	2.6	114
17	[Image omitted. Please see PDF.]	31	624	44,753	2197	20	1444	71	52
18	[Image omitted. Please see PDF.]	24	73	47,360	156	3.0	1973	6.5	58

IC₅₀ values were determined as the mean based on two independent experiments.

Table 5

Selected parameters measured for CPL302415 (6).

	Kinetic SolubilitypH 7.4 [mM]	Metabolic Stability				PAMPA $[{10}^{-6}\mathbf{cm}\times {\mathbf{s}}^{-1}]$	Plasma Protein Binding [%]
		MLM t½[min]	MLM CL $[\mathbf{mL}\times {\mathbf{mn}}^{-1}\times {\mathbf{mg}}^{-1}]$	HLM t½[min]	HLM CL $[\mathbf{mL}\times {\mathbf{min}}^{-1}\times {\mathbf{mg}}^{-1}]$		Human	Monkey	Mice	Rat
6	>500	378	3.7	145	9.6	13.3	79	81	83	82

Table 6

Reaction conditions and compositions of mixtures for individual kinases.

KINASE	Kinase Concentration [ng per Reaction]	Reaction Temperature and Time	Substrate PIP2 [Final Concentration μM]	Reaction Buffer
PI3Kα(Carna Biosciences)	7.5 ng	25 °C, 1 h	30 μM	50 mM of HEPES pH 7.550 mM of NaCl3 mM of MgCl20.025 mg/mL of BSA
PI3Kδ (Merck Millipore)	10 ng	25 °C, 1 h	30 μM	50 mM of HEPES pH 7.550 mM of NaCl3 mM of MgCl20.025 mg/mL of BSA
PI3Kβ(Merck Millipore)	15 ng	30 °C, 1 h	50 μM	50 mM of HEPES pH 7.550 mM of NaCl3 mM of MgCl20.025 mg/mL of BSA
PI3Kα(Merck Millipore)	30 ng	30 °C, 1 h	50 μM	40 nM of Tris pH 7.520 mM of MgCl20.1 mg/mL of BSA1 mM of DTT

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ph15080927/s1, 1H NMR, 13C NMR and HRMS supplementary figures of the final compounds 5–9, 11–12, 16–38, and 40–54.

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