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

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.


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 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 (IC 50 = 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. 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.

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. 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%).

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).

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[1,5a]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 Pharmaceuticals 2022, 15, 927 6 of 35 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 (R 1 , R 2 , Schemes 1 and 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 IC 50 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.

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 (IC 50 = 1.892-0.018 µM) and, additionally, eleven of obtained structures turned out to be highly active, reaching the value of IC 50 below 100 nM. For this reason, PI3Kδ final As a result of all relationships described, compound 6 turned out to be the most active and promising structure of the entire library obtained.   Two positions of the pyrazolo[1,5-a]pyrimidine core: C(2) (R 1 ) and C(5) (R 2 ) 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 IC 50 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 IC 50 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 R 1 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 R 2 groups keeping the constant 2-(difluoromethyl)-1H-benzimidazole R 1 substituent at C(5) position.  The R 2 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 1892-96 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 R 2 , with a nitrogen atom in the 1-or 1-and 4position(s), are generally more active and selective. As an excellent example, this thesis  The R 2 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 1892-96 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 R 2 , with a nitrogen atom in the 1-or 1-and 4position(s), are generally more active and selective. As an excellent example, this thesis  The R 2 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 1892-96 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 R 2 , with a nitrogen atom in the 1-or 1-and 4position(s), are generally more active and selective. As an excellent example, this thesis  The R 2 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 1892-96 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 R 2 , with a nitrogen atom in the 1-or 1-and 4position(s), are generally more active and selective. As an excellent example, this thesis  The R 2 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 1892-96 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 R 2 , with a nitrogen atom in the 1-or 1-and 4position(s), are generally more active and selective. As an excellent example, this thesis  The R 2 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 1892-96 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 R 2 , with a nitrogen atom in the 1-or 1-and 4position(s), are generally more active and selective. As an excellent example, this thesis  The R 2 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 1892-96 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 R 2 , with a nitrogen atom in the 1-or 1-and 4position(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  The R 2 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 1892-96 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 R 2 , with a nitrogen atom in the 1-or 1-and 4position(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  The R 2 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 1892-96 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 R 2 , with a nitrogen atom in the 1-or 1-and 4position(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  The R 2 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 1892-96 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 R 2 , with a nitrogen atom in the 1-or 1-and 4position(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 The R 2 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 IC 50 values in the 1892-1896 nM range; however, their PI3Kγ/δ selectivities remained lower than for the other amine groups with  (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 R 2 , 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 IC 50 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. 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[1,5a]pyrimidine. 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[1,5a]pyrimidine. 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[1,5a]pyrimidine. 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[1,5a]pyrimidine. 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[1,5a]pyrimidine. 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[1,5a]pyrimidine. 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[1,5a]pyrimidine. 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[1,5a]pyrimidine. 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[1,5a]pyrimidine.  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[1,5a]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. 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. 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. 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. 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. 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. 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. 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. For that reason, a library of compounds with multiple amine substituents was designed and synthesized (Table 3). Within this set, the lowest IC 50 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. IC50 values were determined as the mean based on two independent experiments.
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. 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. 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. 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. For that reason, a library of compounds with multiple amine substituents was designed and synthesized (  examples 40, 42, 43, and 55, (measured at 84, 74, 63, and 82 nM, respectively). These structures also had good PI3Kγ/δ selectivity. For that reason, a library of compounds with multiple amine substituents was designed and synthesized (  examples 40, 42, 43, and 55, (measured at 84, 74, 63, and 82 nM, respectively). These structures also had good PI3Kγ/δ selectivity. For that reason, a library of compounds with multiple amine substituents was designed and synthesized (  examples 40, 42, 43, and 55, (measured at 84, 74, 63, and 82 nM, respectively). These structures also had good PI3Kγ/δ selectivity. 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. 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 R 2 substituent.
The compounds lacking the HBD functionally tend to have better potency when the IC50 values were determined as the mean based on two independent experiments.
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 R 2 substituent.
The compounds lacking the HBD functionally tend to have better potency when the 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 R 2 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 IC 50 value at concentrations of 351, 38, and 18 nM was measured, respectively. Scheme 3. The correlation of the potencies given for the compounds lacking HBD interaction within the heterocyclic system. 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 R 2 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 IC 50 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 R 2 can substitute a methylene bridge (CH 2 ) as a linkage (Table 4). Their IC 50 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).

Scheme 4.
The correlation of the potencies given for the compounds lacking HBD interaction within the heterocyclic system. Table 4. Activity and selectivity of the most promising compounds. Therefore, both structural motifs (the methylene bridge and the carbonyl function) should be considered equally essential modifications for SAR exploration.  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 R 2 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). 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.  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 R 2 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). 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.  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 R 2 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 t1/2 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, 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 t1/2 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, 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 t1/2 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, 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.

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 silicagel-coated plates (Kieselgel F254) and visualized using UV light. Flash chromatography was performed using Merck silica gel 60 (230-400 mesh ASTM). 1 H 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 1 H being observed at 300 MHz, 400 MHz, 600 MHz, and 500 MHz, respectively. 13 C NMR spectra were recorded similarly at 75 MHz, 101 MHz, 151 MHz, and 126 MHz frequencies for 13 C, respectively. Due to the poor solubility of some final compounds, usual characterization was omitted using 13 C NMR. Chemical shifts for 1 H and 13 C 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-d 6 and 7.26 ppm for CDCl 3 ). The abbreviations for spin interaction coupled 1 H 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 13 C 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 1 H and 19 F 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.

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. 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(diphenyl phosphino)xanthene (0.1 eq), cesium carbonate (2.0 eq), and solvent (10 mL/1 g pyrazolo[1,5a]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.
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)-1Hbenzimidazole (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. 1  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 Na 2 SO 4 , 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. 1 (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.

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.

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).