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Expedient Access to Type II Kinase Inhibitor Chemotypes by Microwave-Assisted Suzuki Coupling

Expedient Access to Type II Kinase Inhibitor Chemotypes by Microwave-Assisted Suzuki Coupling Communication Expedient Access to Type II Kinase Inhibitor Chemotypes by Microwave-Assisted Suzuki Coupling Lorenza Destro , Ross Van Melsen, Alex Gobbi, Andrea Terzi, Matteo Genitoni and Alfonso Zambon * Department of Chemical and Geological Sciences, University of Modena and Reggio Emilia, Via G. Campi 103, 41125 Modena, Italy; lorenza.destro@unimore.it (L.D.); rvanmels@unimore.it (R.V.M.); 256915@studenti.unimore.it (A.G.); 229348@studenti.unimore.it (A.T.); 227048@studenti.unimore.it (M.G.) * Correspondence: alfonso.zambon@unimore.it Abstract: Functionalized pyrazole-urea scaffolds are a common type II chemotype for the inhibition of protein kinases (PKs), binding simultaneously into the ATP-binding pocket with an ATP bioisostere and into a vicinal allosteric pocket with a pyrazole group. Standard approaches to the scaffold require multi-step synthesis of the ATP bioisostere followed by phosgene or triphosgene-mediated coupling with the substituted pyrazole group. Here we report an expedient approach to the chemotype, characterized by an optimized MW-assisted Suzuki coupling on easily accessed bromo-phenyl pyrazole ureas. The new protocol allowed quick access a large library of target analogues covering a broad chemical space of putative protein kinases inhibitors (PKIs). Keywords: protein kinase inhibitors; pyrazole-ureas; microwave synthesis 1. Introduction Citation: Destro, L.; Van Melsen, R.; Protein phosphorylation in eukaryotes plays a key role in cell signaling, gene expres- Gobbi, A.; Terzi, A.; Genitoni, M.; sion, and differentiation. Protein phosphorylation is also involved in the global control of Zambon, A. Expedient Access to DNA replication during the cell cycle, as well as in the mechanisms that cope with stress- Type II Kinase Inhibitor Chemotypes induced replication blocks [1]. The role of kinases is to phosphorylate serine, threonine, or by Microwave-Assisted Suzuki tyrosine residues of specific protein substrates via the transfer of the -phosphate group of Coupling. Appl. Biosci. 2022, 1, 64–72. adenosine triphosphate (ATP) or, in specific cases, GTP [2]. Protein kinases are, therefore, https://doi.org/10.3390/ key enzymes in the function of cellular signaling pathways and are crucial in the regulation applbiosci1010004 of key functions such as cell proliferation, differentiation, and apoptosis [3]. Academic Editor: Robert Henry Aberration of PK-mediated cellular pathways is a most common factor in the onset and progression of cancer [4,5], and starting from the early 2000s PKs have emerged as Received: 29 April 2022 prominent targets for the development of cancer therapies, with 43 protein kinase inhibitors Accepted: 28 May 2022 (PKI) approved by FDA for the treatment of solid and liquid tumors [6]. To date, an Published: 31 May 2022 estimated 20–33% of the global drug discovery efforts are directed at the development Publisher’s Note: MDPI stays neutral of protein kinase inhibitors [7–11]. Depending on their binding mode within the ATP with regard to jurisdictional claims in binding site and in proximal or distal allosteric pockets, PKIs are classified as Type I, Type published maps and institutional affil- I 1/2, and Type II-VI, with Type I and Type II being first binding modes identified and iations. the most common ones [12]. Type II inhibitors, in particular, bind simultaneously to the ATP-binding pocket and to an adjacent allosteric pocket when the kinase in an inactive, DFG-out conformation, in contrast with Type I inhibitors that bind to the active, DFG-in Copyright: © 2022 by the authors. conformation of the kinase and only into the ATP-binding pocket. Of the 27 co-crystal Licensee MDPI, Basel, Switzerland. structures of FDA-approved PKIs available in 2019, eight were Type II [6]. As inactive This article is an open access article protein kinase conformations exhibit greater structural variation than the conserved active distributed under the terms and conformation to which Type I PKIs bind, Type II PKIs are considered potentially more conditions of the Creative Commons selective than Type I ones [13,14]. Most Type II PKIs share a common chemotype enticing a Attribution (CC BY) license (https:// hydrophobic element that forms Van der Waals interactions with the allosteric pocket and creativecommons.org/licenses/by/ a bridge system (e.g., an amine or a urea) able to form hydrogen bonds with the conserved 4.0/). Appl. Biosci. 2022, 1, 64–72. https://doi.org/10.3390/applbiosci1010004 https://www.mdpi.com/journal/applbiosci Appl. Biosci. 2022, 1, FOR PEER REVIEW 2 Appl. Biosci. 2022, 1, FOR PEER REVIEW 2 Appl. Biosci. 2022, 1 65 a bridge system (e.g., an amine or a urea) able to form hydrogen bonds with the conserved a bridge system (e.g., an amine or a urea) able to form hydrogen bonds with the conserved salt bridge adjacent to the PK active site and an ATP bioisostere that occupies the ATP salt bridge adjacent to the PK active site and an ATP bioisostere that occupies the ATP salt bridge adjacent to the PK active site and an ATP bioisostere that occupies the ATP binding pocket [15–18]. binding pocket [15–18]. binding pocket [15–18]. The introduction of a pyrazole-urea moiety as an allosteric binder in Type II PKIs has The introduction of a pyrazole-urea moiety as an allosteric binder in Type II PKIs has The introduction of a pyrazole-urea moiety as an allosteric binder in Type II PKIs been shown to confer activity against a range of protein targets and to improve the phar- been shown to confer activity against a range of protein targets and to improve the phar- has been shown to confer activity against a range of protein targets and to improve the macokinetic properties of the scaffold, allowing for the progression to the clinic of several macokinetic properties of the scaffold, allowing for the progression to the clinic of several pharmacokinetic properties of the scaffold, allowing for the progression to the clinic of compounds of this type. The general structure of pyrazole-urea PKIs entices a central py- compounds of this type. The general structure of pyrazole-urea PKIs entices a central py- several compounds of this type. The general structure of pyrazole-urea PKIs entices a razole-phenyl urea core substituted on the pyrazole by aliphatic (X) and aromatic and razole-phenyl urea core substituted on the pyrazole by aliphatic (X) and aromatic and central pyrazole-phenyl urea core substituted on the pyrazole by aliphatic (X) and aromatic aliphatic (Y) groups and bearing on the phenyl ring a range of substituents usually com- aliphatic (Y) groups and bearing on the phenyl ring a range of substituents usually com- and aliphatic (Y) groups and bearing on the phenyl ring a range of substituents usually prising a variously substituted aromatic group (Z), as depicted in Figure 1 [19–21]. prising a variously substituted aromatic group (Z), as depicted in Figure 1 [19–21]. comprising a variously substituted aromatic group (Z), as depicted in Figure 1 [19–21]. Figure 1. General structure of Type II pyrazole-urea PKI. Figure 1. General structure of Type II pyrazole-urea PKI. Figure 1. General structure of Type II pyrazole-urea PKI. In general, access to this chemotype requires the multi-step synthesis of the aniline In general, access to this chemotype requires the multi-step synthesis of the aniline In general, access to this chemotype requires the multi-step synthesis of the aniline ATP isostere followed by phosgene or triphosgene-mediated coupling to urea with the ATP isostere followed by phosgene or triphosgene-mediated coupling to urea with the ATP isostere followed by phosgene or triphosgene-mediated coupling to urea with the pyrazole group. This approach requires lengthy synthetic and purification procedures, pyrazole pyrazolegr goup. roupThis . This appr app oach roacrhequir requ es ire lengthy s lengthy syn synthetic thetic and puri and purification ficatpr ion procedu ocedures, the res, the optimization of reaction conditions for each analogue, and the use of hazardous rea- the optimization of reaction conditions for each analogue, and the use of hazardous rea- optimization of reaction conditions for each analogue, and the use of hazardous reagents gents for the coupling step, which often occurs with sub-optimal yields [22–27]. We posit for gent the s for coupling the couplin step,g st which ep, wh often ich occurs often occurs with sub-optimal with sub-opti yields mal yiel [22ds –27 [22– ]. W 27 e ]. posit We posi thatt that direct reaction of the pyrazole group with commercial Bromophenyl Isocyanates fol- dir tha ect t di reaction rect reac of tion theof the pyrazole pyraz gro oup le group with commer with com cial mercial Brom Bromophenyl ophenyl I Isocyanates socyan followed ates fol- by lowed by a Suzuki coup a Suzuki coupling (Scheme ling (Scheme 1) wou 1) ld woul provide d provid an expedient e an expedien access t access to this chemo- to this chemotype. lowed by a Suzuki coupling (Scheme 1) would provide an expedient access to this chemo- type. Diversity points can be easily introduced at each step, allowing for the rapid synthe- Diversity points can be easily introduced at each step, allowing for the rapid synthesis of a type. Diversity points can be easily introduced at each step, allowing for the rapid synthe- library sis of a of lib analogues rary of an[ alo 28,g 29 ue ].s [28,29]. sis of a library of analogues [28,29]. Scheme 1. Proposed route to pyrazole-ureas analogues. Scheme 1. Proposed route to pyrazole-ureas analogues. Scheme 1. Proposed route to pyrazole-ureas analogues. Suzuki-Miyaura coupling allows the formation of new C-C bonds between an aryl Suzuki-Miyaura coupling allows the formation of new C-C bonds between an aryl Suzuki-Miyaura coupling allows the formation of new C-C bonds between an aryl halide halide and and an anaryl arylbor boron on species speciesand andis isparticularl particulary lysuitable suitable for for our our purpo purpose se due due t tooits its halide and an aryl boron species and is particularly suitable for our purpose due to its rrobustness and obustness and tolerance tolerance of of functional functional groups groups. . Al Although though this reacti this reaction on has has alr alrea eady dy been been robustness and tolerance of functional groups. Although this reaction has already been extensively investigated, reactions on substrates containing an aromatic urea group are extensively investigated, reactions on substrates containing an aromatic urea group are extensively investigated, reactions on substrates containing an aromatic urea group are scantly scantly rep reported, orted, and no e and no examples xamples in in w which hich one of t one of the two he tw aro arom omaticat rings ic rin isga s i pyrazole s a pyrazole are scantly reported, and no examples in which one of the two aromatic rings is a pyrazole rare reported eported in the in the liter literaturatur e [30 e [3 ,310,31] The ] The affinity affinof ity of p palladium alladium for for th the ur e ure ea gr aoup grou suggests p suggests a are reported in the literature [30,31] The affinity of palladium for the urea group suggests possible coordination of the catalyst by our substrate, and thus possible deactivation of the a possible coordination of the catalyst by our substrate, and thus possible deactivation of a possible coordination of the catalyst by our substrate, and thus possible deactivation of former. Here we report the optimization of the problematic Suzuki-Muyara reaction on the former. Here we report the optimization of the problematic Suzuki-Muyara reaction the former. Here we report the optimization of the problematic Suzuki-Muyara reaction pyrazole ureas, and the consequent synthesis of a library of analogues easily accessed by on pyrazole ureas, and the consequent synthesis of a library of analogues easily accessed on pyrazole ureas, and the consequent synthesis of a library of analogues easily accessed the variation of substituents on the pyrazole and phenylboronic reagents. by the variation of substituents on the pyrazole and phenylboronic reagents. by the variation of substituents on the pyrazole and phenylboronic reagents. 2. Materials and Methods 2. Materials and Methods 2. Materials and Methods General Methods: Commercial building blocks, reagents, and solvents for reactions General Methods: Commercial building blocks, reagents, and solvents for reactions General Methods: Commercial building blocks, reagents, and solvents for reactions were reagent grade and used as purchased or purified according to methods in the literature. were reagent grade and used as purchased or purified according to methods in the litera- were reagent grade and used as purchased or purified according to methods in the litera- Reactions were monitored by Pre-coated TLC sheets ALUGRAM Sil G/UV (0.20 mm ® 254 ture. Reactions were monitored by Pre-coated TLC sheets ALUGRAM Sil G/UV254 (0.20 ture. Reactions were monitored by Pre-coated TLC sheets ALUGRAM Sil G/UV254 (0.20 thickness). Dry solvents were prepared by overnight standing on freshly activated 4 Å mm thickness). Dry solvents were prepared by overnight standing on freshly activated 4 mm thickness). Dry solvents were prepared by overnight standing on freshly activated 4 molecular sieves under argon atmosphere or purchased. Flash chromatography was Appl. Biosci. 2022, 1 66 1 13 conducted using a Merk 60, 230–400 mesh silica gel. H- and C-NMR spectra were recorded at 298K on Bruker FT-NMR Advance 400 (400.13 MHz) e Bruker FT-NMR Advance III HD 600 (600.13 MHz). Chemical shift values are given in ppm relative to TMS and were determined by taking as reference the isotopic impurity signals of CDCl (7.26 ppm for 1 13 1 13 H and 77.16 for C) and DMSO-d (2.50 ppm for H and 39.52 ppm for C). Data are presented as follows: chemical shift () in ppm, multiplicity, coupling constants (J) given in hertz. LCMS data were acquired using a 6130A quadrupole ion trap analyzer Ion Trap LC-MS(n) by Agilent Technologies. Docking analysis was carried out using the Glide Docking Module of Maestro (Schroedinger) in the standard precision (SP) mode. Detailed synthetic procedures and full characterization of all the synthesized com- pounds are reported in Supplementary Information. 3. Results As discussed above, Suzuki coupling on aromatic ureas is rarely reported, and to our knowledge never on a substituted pyrazole scaffold [30,31]. We thus set out to optimize the coupling step using as model reaction the coupling of easily obtained 1-(4-bromophenyl)-3- (3-(tert-butyl)-1-phenyl-1H-pyrazol-5-yl)urea 1a with boronic 3,4-dimethoxyphenylboronic 0 0 acid 2a as coupling partner to 1-(3-(tert-butyl)-1-phenyl-1H-pyrazol-5-yl)-3-(3 ,4 -dimethoxy- [1,1 -biphenyl]-4-yl)urea 3a (Table 1). We first tried the protocol reported by Al-Masoudi et al. [30] for structurally close bis-phenyl ureas, carrying out the reaction under reflux with readily available reagents (Entry 1) but did not observe any product formation af- ter 18h. We then tried varying base, catalyst, and solvent system replacing K CO with 2 3 Na CO (Entry 2), Pd(PPh ) with Pd(dppf)Cl (Entry 3) and finally the solvent with a 2 3 3 4 2 1:1=H O:DMF mixture (Entry 4). Again, under all the conditions explored we did not observe any conversion to product, suggesting a considerable deactivation of the substrate towards Suzuki coupling under thermal conditions. We then set out to explore a range of conditions starting from those reported by Brunner et al. [31] in which coupling of phenyl bromo ureas is carried out under microwave (MW) irradiation. The use of MW is known to improve the reaction rates and thus could overcome the deactivation of the system observed under thermal conditions [32]. As the starting conditions, we used MeCN as solvent, Pd(PPh ) as catalyst, Na CO as base and 3 4 2 3 run the reaction for 1 h at 100 C under microwave condition as reported (Entry 5) [32]. Unfortunately, this reaction did not lead to the desired results, most likely due to the poor solubility of urea 1a in the solvent medium. We then changed the solvent system by replacing the MeCN with a mixture of a 2M aqueous solution of K CO and 1,4-dioxane in 2 3 a ratio of 1:2, in which our urea proved more soluble (Entry 6). Encouragingly, we observed the formation of some product in the reaction mixture, albeit with incomplete conversion and 30% isolated yield. After several attempts, we were then able to further optimize the reaction conditions by changing both the base by switching K CO with Na CO , a slightly 2 3 2 3 stronger base, and the catalyst by replacing Pd(PPh ) with Pd(dppf)Cl (Entry 7–8), a 3 4 2 catalyst that was proven stable and effective under microwave conditions [33]. We finally identified the best conditions with the use of Pd(dppf)Cl as catalyst and Na CO as base 2 2 3 with complete spectroscopic conversion and 50% yield after chromatography (Entry 9). After identifying the optimal solvent, base, and catalyst, we decided to investigate if the reaction could be performed at lower temperatures (70 C instead of 100 C, Entry 10) and with shorter reaction times (45 min instead of 1.25 h, Entry 11). Lowering the temperature significantly affected the conversion, with only 15% of urea 1a converted to 3a, but we found that reaction times could be shortened to 30 min (Entry 11), with ~99% conversion of urea 1a to 3a, in line with that observed for Entry 9. Appl. Biosci. 2022, 1, FOR PEER REVIEW 4 Appl. Biosci. 2022, 1 67 Table 1. Optimization of the reaction conditions for the Suzuki coupling of 1a and 2a. Table 1. Optimization of the reaction conditions for the Suzuki coupling of 1a and 2a. Entry Conditions Time (h) T (°C) Solvent Catalyst Base Yield Entry Conditions Time (h) T ( C) Solvent Catalyst Base Yield 1 T1 18 101 1,4-dioxane Pd(PPh3)4 K2CO3 N.R. 1 T 18 101 1,4-dioxane Pd(PPh ) K CO N.R. 1 3 4 2 3 2 T2 18 101 1,4-dioxane Pd(PPh3)4 Na2CO3 N.R. 2 T 18 101 1,4-dioxane Pd(PPh ) Na CO N.R. 2 3 4 2 3 3 T3 18 101 1,4-dioxane Pd(dppf)Cl2 K2CO3 N.R. 3 T 4 T4 18 18 101 100 1,4-dioxane 1:1 DMF/H2O Pd(dppf)Cl Pd(PPh3)4 K2K CO CO 3 N.R. N.R. 3 2 2 3 5 MW 1 100 MeCN Pd(PPh3)4 Na2CO3 N.R. 4 T 18 100 1:1 DMF/H O Pd(PPh ) K CO N.R. 4 2 3 4 2 3 6 MW 1.25 100 1:2 H2O/1,4-dioxane Pd(PPh3)4 Na2CO3 30% 5 MW 1 100 MeCN Pd(PPh ) Na CO N.R. 3 4 2 3 7 MW 1.25 100 1:2 H2O/1,4-dioxane Pd(PPh3)4 K2CO3 N.R. 6 MW 1.25 100 1:2 H O/1,4-dioxane Pd(PPh ) Na CO 30% 2 3 4 2 3 8 MW 1.25 100 1:2 H2O/1,4-dioxane Pd(dppf)Cl2 K2CO3 N.R. 9 MW5 1.25 100 1:2 H2O/1,4-dioxane Pd(dppf)Cl2 Na2CO3 50% 7 MW 1.25 100 1:2 H O/1,4-dioxane Pd(PPh ) K CO N.R. 2 3 4 2 3 10 MW6 1.25 70 1:2 H2O/1,4-dioxane Pd(dppf)Cl2 Na2CO3 15% 8 MW 1.25 100 1:2 H O/1,4-dioxane Pd(dppf)Cl K CO N.R. 2 2 2 3 11 MW7 0.5 100 1:2 H2O/1,4-dioxane Pd(dppf)Cl2 Na2CO3 ~99% 9 MW 1.25 100 1:2 H O/1,4-dioxane Pd(dppf)Cl Na CO 50% 5 2 2 2 3 a b 1 Isolated yield (column purification) spectroscopic yield ( H-NMR) N.R. = Not reacted; only Start- 10 MW ing materials reco 1.25 vered. 70 1:2 H O/1,4-dioxane Pd(dppf)Cl Na CO 15% 6 2 2 2 3 11 MW 0.5 100 1:2 H O/1,4-dioxane Pd(dppf)Cl Na CO ~99% 7 2 2 2 3 We then set out to perform the synthesis of a small library of analogues of 1a taking a b 1 Isolated yield (column purification) spectroscopic yield ( H-NMR) N.R. = Not reacted; only Starting advantage of the newly optimized Suzuki procedure. Although reaction time could be materials recovered. lowered at 30 min, we deemed it preferable to keep it at 1.25 h to account for the potential We then set out to perform the synthesis of a small library of analogues of 1a taking lower reactivity of the alternative boronic agents. advantage of the newly optimized Suzuki procedure. Although reaction time could be The overall synthetic route to this library of compounds is depicted in Scheme 2 and lowered at 30 min, we deemed it preferable to keep it at 1.25 h to account for the potential involves the condensation of an oxo nitrile 4a-b with a hydrazine 5a-c to give pyrazole 6a- lower reactivity of the alternative boronic agents. f, followed by the formation of Br-substituted ureas 1a-h by reaction with isocyanates 7a- The overall synthetic route to this library of compounds is depicted in Scheme 2 and b. These steps are already well reported in the scientific literature and gave high yields involves the condensation of an oxo nitrile 4a-b with a hydrazine 5a-c to give pyrazole 6a-f, for all the substrates explored [34]. followed by the formation of Br-substituted ureas 1a-h by reaction with isocyanates 7a-b. These steps are already well reported in the scientific literature and gave high yields for all the substrates explored [34]. Ureas 1a-l were finally reacted following the optimized Suzuki protocol with selected boronic acids 2a-e to afford para-substituted (3a-f) and meta-substituted (3g-l) final com- pounds in good 35–60% yields as reported in Table 2 (para-substituted compounds) and Table 3 (meta-substituted compounds). For a preliminary evaluation of the potential of our compounds as PKIs, we carried out docking of 3a-l in the co-crystal structure (pdb code 1KV2) of p38, a validated target for autoimmune diseases, with pyrazole-urea BIRB-796 [21]. All compounds faithfully mirrored the binding mode of BIRB-796 and registered high docking scores, suggesting a high affinity of the library for p38 (Figure 2). The best affinities were calculated for compounds 3g, 3h, 3l and 3a, with docking scores ranging from 11.7 and 8.9 kcal/mol. Appl. Biosci. 2022, 1, FOR PEER REVIEW 5 Appl. Appl. B Appl. iosci. Biosci. B 2022 iosci. 2022 , 1 2022 , FO , 1, FO , R 1, FO PEER R PEER R RE PEER RE VIEW RE VIEW VIEW 5 5 5 Appl. Biosci. 2022, 1, FOR PEER REVIEW 5 Appl. Appl. Biosci. Biosci. 2022 2022 , 1, FO , 1, FO R PEER R PEER RE RE VIEW VIEW 5 5 Appl. Biosci. 2022, 1 68 Appl. Biosci. 2022, 1, FOR PEER REVIEW 5 Scheme 2. General synthetic route to pyrazole urea 3a-l. Reagents and conditions: (a) Toluene, 116 Scheme Scheme Scheme 2. 2. General synthetic r 2. General synthetic r General synthetic r oute to pyrazole urea 3 oute to pyrazole urea 3 oute to pyrazole urea 3 a-l. Re a-l. Re a-l agents and . Re agents and agents and con con ditions: ( con ditions: ( ditions: ( a) Toluene, a) Toluene, a) Toluene, 116 116 116 Scheme 2. General synthetic route to pyrazole urea 3a-l. Reagents and conditions: (a) Toluene, 116 Scheme Scheme 2. 2. General synthetic r General synthetic r oute to pyrazole urea 3 oute to pyrazole urea 3 a-la-l . Re . Re agents and agents and con con ditions: ( ditions: ( a) Toluene, a) Toluene, 116 116 °C,72 h; (b) anhydrous DCM, room temperature, 24 h; (c) 1:2 H2O/1,4-dioxane, 100 °C, 1.25 h. °C,72 h; ( °C,72 h; ( b) an b) an hydrous DCM, room hydrous DCM, room temperature, 24 h; ( temperature, 24 h; ( c) 1:2 H c) 1:2 H 2O/1,4- 2O/1,4- diox diox ane, 100 °C ane, 100 °C , 1., 1 25. h. 25 h. °C,72 h; (b) anhydrous DCM, room temperature, 24 h; (c) 1:2 H2O/1,4-dioxane, 100 °C, 1.25 h. °C,72 h; ( °C,72 h; ( b) an b) an hydrous DCM, room hydrous DCM, room temperature, 24 h; ( temperature, 24 h; ( c) 1:2 H c) 1:2 H 2O/1,4- 2O/1,4- diox diox ane, 100 °C ane, 100 °C , 1.2 , 1 5 h. .25 h. °C,72 h; (b) anhydrous DCM, room temperature, 24 h; (c) 1:2 H2O/1,4-dioxane, 100 °C, 1.25 h. Ureas 1a-l were finally reacted following the optimized Suzuki protocol with selected Ureas Ureas Ureas 1a-l 1a were -l 1a were -l were fina fina llfy rea ill na y rea llc y rea ted f cted f c o ted f llo ol wi lo ol wi ng lowi ng the opti ng the opti the opti mimi zed Suzuki mi zed Suzuki zed Suzuki protoc protoc protoc ol w ol w iol w th selec ith selec ith selec ted te d te d Ureas 1a-l were finally reacted following the optimized Suzuki protocol with selected Ureas Ureas 1a1a -l were -l were fina fina lly rea lly rea cted f cted f oll o o lwi lowi ng ng the opti the opti mi mi zed Suzuki zed Suzuki protoc protoc ol w ol w ith selec ith selec ted te d boronic acids 2a-e to afford para-substituted (3a-f) and meta-substituted (3g-l) final com- boronic a boronic a boronic a cids cids 2a cids - 2a e t -2a e o t a -o e ffo t ao ffo rd affo par rd par rd a par -sub a-sub ast -sub it st uit tst ed ( uit ted ( u3a ted ( - 3a f) and -3a f) and -f) meta-sub and meta-sub meta-sub stituted stituted stituted (3g-l (3g-l ) f (3g-l i) f na i) f na l com- il na com- l com- boronic acids 2a-e to afford para-substituted (3a-f) and meta-substituted (3g-l) final com- boronic a boronic a cids cids 2a2a -e t -e o t a offo affo rdrd par par a-sub a-sub stit st u it tu ed ( ted ( 3a3a -f)- and f) and meta-sub meta-sub stituted stituted (3g-l (3g-l ) fi ) f na ina l com- l com- Scheme 2. General synthetic route to pyrazole urea 3a-l. Reagents and conditions: (a) Toluene, 116 pounds in good 35–60% yields as reported in Table 2 (para-substituted compounds) and Scheme 2. General synthetic route to pyrazole urea 3a-l. Reagents and conditions: (a) Toluene, pounds in good 35–60% yields as reported in Table 2 (para-substituted compounds) and pounds i pounds i n good 35– n good 35– 60% yiel 60% yiel ds as ds as reported reported in Tain bl T e 2 a b (p lear 2a- (p sub ara- stsub ituts etd com ituted com pounds) pou an nds) d and pounds i pounds i n good 35– n good 35– 60% yiel 60% yiel ds as ds as reported reported in T in a T bla eb 2 le (p 2 ar (p a- ar sub a-sub stitsu titteu d com ted com pou pnds) ounds) an an d d pounds in good 35–60% yields as reported in Table 2 (para-substituted compounds) and °C,72 h; (b) anhydrous DCM, room temperature, 24 h; (c) 1:2 H2O/1,4-dioxane, 100 °C, 1.25 h. Table 3 (meta-substituted compounds). 116 C, 72 h; (b) anhydrous DCM, room temperature, 24 h; (c) 1:2 H O/1,4-dioxane, 100 C, 1.25 h. TaTa ble 3 ble 3 (m (eta- meta- sub sub stituted stituted compounds). compounds). Table 3 (meta-substituted compounds). 2 Tabl Ta e 3 ble 3 (m ( eta- meta- sub sub stituted stituted compounds). compounds). Table 3 (meta-substituted compounds). Ureas 1a-l were finally reacted following the optimized Suzuki protocol with selected Table 2. Structures and yields of the final step of compounds 3a-f. Table 2. Structures and yields of the final step of compounds 3a-f. Table 2. Table 2. Table 2. Struct Struct Struct ures and yiel ures and yiel ures and yiel ds of th ds of th ds of th e fie f naie f l s nat il s ep o na tl s ep o ft co ep o f co mpo f co mpo umpo nd und s u 3a-f s nd 3a-f s . 3a-f . . Table 2. Structures and yields of the final step of compounds 3a-f. Table 2. Structures and yields of the final step of compounds 3a-f. Table 2. Structures and yields of the final step of compounds 3a-f. boronic acids 2a-e to afford para-substituted (3a-f) and meta-substituted (3g-l) final com- pounds in good 35–60% yields as reported in Table 2 (para-substituted compounds) and Table 3 (meta-substituted compounds). Table 2. Structures and yields of the final step of compounds 3a-f. Compound R1 R2 R3 Yield Compound R R R Yield 1 2 3 Compound R1 R2 R3 Yield ComCompoundpou Rnd R 1 R 1 R 2 R 2 R 3 Yield 3 Yield ComCompoupndound R R 1 R 1 R 2 R 2 R 3 Yield 3 Yield Compound R1 R2 R3 Yield 3a 35% 3a 3a 3a 3a 35% 35% 35% 35% 3a 35% 3a 35% 3a 35% Compound R 1 R 2 R3 Yield 3a 35% 3b 43% 3b 43% 3b 3b 43% 43% 3b 3b 43% 43% 3b 3b 43% 43% Appl. Biosci. 2022, 1, FOR PEER REVIEW 6 Appl. Appl. Biosci. Biosci. 2022 2022 , 1, FO , 1, FO R PEER R PEER RE RE VIEW VIEW 6 6 3b 43% 3c 3c 3c 3c 36% 36% 36% 36% 3d3d 3d 52% 52% 52% 3e 3e 3e -Me -Me -Me 48% 48% 48% 3f 3f -Me -Me 58% 58% 3f -Me 58% Table 3. Table 3. Struct Struct ures and yiel ures and yiel ds of th ds of th e fie f nail s natl s ep o tep o f co f co mpo mpo und und s 3g-l s 3g-l . . Table 3. Structures and yields of the final step of compounds 3g-l. Compound R1 R2 R3 Yield ComCompoupoundnd R R 1 R 1 R 2 R 2 R 3 Yield 3 Yield 3g3g 3g 36% 36% 36% 3h3h 48% 48% 3h 48% 3i 36% 3i 3i 36% 36% Appl. Biosci. 2022, 1, FOR PEER REVIEW 5 Scheme 2. General synthetic route to pyrazole urea 3a-l. Reagents and conditions: (a) Toluene, 116 °C,72 h; (b) anhydrous DCM, room temperature, 24 h; (c) 1:2 H2O/1,4-dioxane, 100 °C, 1.25 h. Ureas 1a-l were finally reacted following the optimized Suzuki protocol with selected boronic acids 2a-e to afford para-substituted (3a-f) and meta-substituted (3g-l) final com- Appl. Biosci. 2022, 1 69 pounds in good 35–60% yields as reported in Table 2 (para-substituted compounds) and Table 3 (meta-substituted compounds). Table 2. Structures and yields of the final step of compounds 3a-f. Table 2. Cont. Appl. B Appl. Appl. iosci. B B 2022 ii osci. osci. , 2022 1 2022 , FO , , R 11, FO , FO PEER R R PEER RE PEER VIEW RE REVIEW VIEW 6 6 6 Appl. Biosci. 2022, 1, FOR PEER REVIEW 6 Appl. Biosci. 2022, 1, FOR PEER REVIEW 6 Appl. Biosci. 2022, 1, FOR PEER REVIEW 6 Appl. Biosci. 2022, 1, FOR PEER REVIEW 6 Appl. Biosci. 2022, 1, FOR PEER REVIEW 6 Appl. Biosci. 2022, 1, FOR PEER REVIEW 6 Appl. Appl. B Biiosci. osci. 2022 2022, , 1 1, FO , FOR R PEER PEER RE REVIEW VIEW 6 6 Appl. Appl. Appl. Biosci. B Biiosci. 2022 osci. 2022 , 2022 1, FO , , 1 1, FO R , FO PEER R R PEER PEER REVIEW RE REVIEW VIEW 6 6 6 3c 3c 36% 36% 3c 36% 3c 3c 36% 36% 3c 3c 36%36% 3c 36% 3c 36% 3c 3c 36% 36% CompoundCompound R R 1 R R 2 R R 3 Yield Yield 3c 3c 3c 1 2 3 36% 36% 36% 3a 35% 3d 3d 3d 52% 52% 52% 3d 52% 3d3d 52% 52% 3d 3d 52%52% 3d 52% 3d 52% 3d 3d 52% 52% 3d 3d 3d 52% 52% 52% 3e -Me 48% 3e 3e -Me -Me 48% 48% 3e 3e 3e -Me -Me -Me 48% 48% 48% 3e 3e -Me -Me 48%48% 3b 43% 3e -Me 48% 3e 3e 3e -Me -Me -Me 48% 48% 48% 3e 3e -Me -Me 48% 48% 3e -Me 48% 3f -Me 58% 3f 3f -Me -Me 58% 58% 3f 3f -Me -Me 58% 58% 3f 3f 3f -Me -Me -Me 58% 58%58% 3f -Me 58% 3f 3f 3f -Me -Me -Me 58% 58% 58% 3f -Me 58% 3f 3f -Me-Me 58%58% Table 3. Structures and yields of the final step of compounds 3g-l. Table 3. Table 3. Struct Struct ures and yiel ures and yiel ds of th ds of th e fina e f l sitna ep o l stfep o compo f cou mpo ndsu 3g-l nds. 3g-l. Table 3. Table 3. Struct Struct ures and yiel ures and yiel ds of th ds of th e fie f nail s natl s ep o tep o f co f co mpo mpo und und s 3g-l s 3g-l . . Table 3. Table 3. Struct Struct ures and yiel ures and yiel ds of th ds of th e fina e f l s ina tep o l stfep o compo f compo undsu 3g-l nds. 3g-l. Table 3. Structures and yields of the final step of compounds 3g-l. Table 3. Structures and yields of the final step of compounds 3g-l. Table 3. Table 3. Table 3. Struct Struct Struct ures and yiel ures and yiel ures and yiel ds of th ds of th ds of th e fina e f e f l s iina na tep o l s l sttep o ep o f compo ff co compo mpo undu u snd nd 3g-l ss 3g-l . 3g-l. . Table 3. Structures and yields of the final step of compounds 3g-l. Table 3. Table 3. Struct Struct ures and yiel ures and yiel ds of th ds of th e fina e f l s ina tep o l step o f compo f compo unds u nd 3g-l s . 3g-l. ComComCompoundppouou R ndnd R R 1 R 11 R R 2 R 22 R R 3 Yield 33 Yield Yield Compound R1 R2 R3 Yield Compound R1 R2 R3 Yield ComCompoundpou R nd R 1 R 1 R 2 R 2 R 3 Yield 3 Yield CompoundCompound R R 1 R R 2 R R 3 Yield Yield 1 2 3 Compound R1 R2 R3 Yield ComComppououndnd R R 11 R R 22 R R 33 Yield Yield ComComCompouppndouou R ndnd R R 1 R 11 R R 2 R 22 R R 3 Yield 33 Yield Yield 3g 3g 36% 36% 3g 36% 3g3g 36% 36% 3g 3g 36%36% 3g 36% 3g 36% 3g 3g 3g 36% 36% 36% 3g 3g 36% 36% 3g 36% 3h 48% 3h 3h 48% 48% 3h 48% 3h 48% 3h 3h 48%48% 3h 48% 3h 3h 3h 48% 48% 48% 3h 48% 3h 3h 3h 48% 48% 48% 3i 3i 3i 36% 36% 36% 3i 36% 3i 36% 3i 36% 3i 36% 3i 36% 3i 36% 3i 3i 36% 36% 3i 3i 3i 36% 36% 36% Appl. Biosci. 2022, 1, FOR PEER REVIEW 6 Appl. Appl. Biosci. Biosci. 20222022 , 1, FO , 1R , FO PEER R PEER REVIEW REVIEW 6 6 3c 36% 3c 3c 36% 36% Appl. Biosci. 2022, 1, FOR PEER REVIEW 6 3d 52% 3d 3d 52% 52% 3e -Me 48% 3e 3e -Me -Me 48% 48% 3c 36% 3f -Me 58% 3f 3f -Me -Me 58% 58% 3d 52% Table 3. Structures and yields of the final step of compounds 3g-l. Table 3. Table 3. Struct Struct ures and yiel ures and yiel ds of th ds of th e fina e f l s ina tep o l step o f compo f compo undu s nd 3g-l s 3g-l . . 3e -Me 48% 3f -Me 58% Appl. Biosci. 2022, 1 70 Compound R1 R2 R3 Yield ComCompoupndou R nd R 1 R 1 R 2 R 2 R 3 Yield 3 Yield Table Table 3. 3. Cont. Structures and yields of the final step of compounds 3g-l. 3g 36% 3g 3g 36% 36% 3h 48% Appl. Biosci. 2022, 1, FOR PEER REVIEW 7 3h 3h 48% 48% Compound R1 R2 R3 Yield Compound R R R Yield 1 2 3 3g 36% 3i 36% 3i 3i 3i 36% 36% 36% 3l -Me 41% Appl. Ap Biosci. pl. Biosci. 2022 202 , 1, 2 FO , 1, R FO PE R ER PE RE ER VIEW REVIEW 7 7 For a preliminary evaluation of the potential of our compounds as PKIs, we carried 3h 48% out docking of 3a-l in the co-crystal structure (pdb code 1KV2) of p38, a validated target 3l -Me 41% 3l 3l -Me-Me 41% 41% for autoimmune diseases, with pyrazole-urea BIRB-796 [21]. All compounds faithfully mirrored the binding mode of BIRB-796 and registered high docking scores, suggesting a high affinity of the library for p38 (Figure 2). The best affinities were calculated for com- pounds 3g, 3h, 3l and 3a, with docking scores ranging from −11.7 and −8.9 kcal/mol. For For a pr a el pr imi elimi nary nary evalu evati alu on atiof on th of e th po e ten potial tential of o of ur ocom ur com pounds pounds as PKI as PKI s, w se , w ca e rried carried 3i 36% out out dockin dockin g of g 3a of -l3a in -l th ine th co e -cryst co-cryst al struct al struct ure ure (pdb (pdb code code 1KV2) 1KV2) of p38 of p38 , a v , alid a valid ated ated target target for for auto au imm toimm une une dise das ise es, as e with s, with pyr pyr azoaz le-o ure le-ure a BI a RB BI-RB 796 -796 [21]. [21]. Al l Al com l com pounds pounds faith fai fully thfully mirrored the binding mode of BIRB-796 and registered high docking scores, suggesting a mirrored the binding mode of BIRB-796 and registered high docking scores, suggesting a high high affiaffi nity n ity of th of e th libr e li ar br yar for y for p38p (38 Fi g (u Fi re gu 2) re . Th 2). e Th be e sbe t affi st affi nities nities were we cre alcculated alculated for for com com - - pounds pounds 3g, 3g 3h, , 3h 3l , and 3l and 3a, 3a with , with dock dock ing sco ing sco res res ranra gin ng f gin rom g from −11.7 and −11.7 and −8.9 −8 kc .9 al kc /mo al/l. mo l. Figure 2. Docking pose of compound 3g (gray) into the binding pocket of p38 (pdb code 1KV2) Figure 2. Docking pose of compound 3g (gray) into the binding pocket of p38 (pdb code 1KV2) superposed to BIRB-796 (pink). superposed to BIRB-796 (pink). 4. Conclusions 4. Conclusions In conclusion, we report a novel, efficient synthetic route to a privileged scaffold for In conclusion, we report a novel, efficient synthetic route to a privileged scaffold for protein kinase inhibitors based on a novel, robust microwave-assisted protocol for the Su- protein kinase inhibitors based on a novel, robust microwave-assisted protocol for the Figure Figure 2. Dock 2. Dock ing ing pose pose of co of m co pou mpou nd 3g nd (g 3g ray (g) ray into ) into the the bind bind ing ing pocket pocket of p38 of p38 (pdb (pdb code co 1KV2) de 1KV2) zuki reaction of pyrazole ureas, which allowed quick to access a structurally varied library Suzuki reaction of pyrazole ureas, which allowed quick to access a structurally varied superp superp osed os to ed BI to RB BI -RB 796- 796 (pink (pink ). ). of putative kinase inhibitors. Although this route is limited to the synthesis of biphenyl library of putative kinase inhibitors. Although this route is limited to the synthesis of pyrazole ureas, it compares favorably with previous literature in terms of yields and over- biphenyl pyrazole ureas, it compares favorably with previous literature in terms of yields 4. Co 4. n Co clus nclus ions ions all ease of synthesis. and overall ease of synthesis. In con In con clus cl ion us, ion we , we repo repo rt a rt nov a nov el, ef el, fief cie fint cie synthet nt synthet ic route ic route to a to priv a priv ilegil ed eg sc ed affol scaffol d fo d r for Supplementary Materials: The following supporting information can be downloaded at: propro tein tein kinas kinas e inh e inh ibitors ibitors basb ed as on ed a on nov a nov el, robust el, robust micm rowave icrowave -assi -assi sted sted propro tocol tocol for fo thr e th Su- e Su- www.mdpi.com/xxx/s1. zukzu i re kaction i reaction of pyra of pyra zole zo ule rea us rea , which s, which allowed allowed quick quick to acces to acces s a stru s a stru cturall cturall y var y v iear d ili ebr d li ar br yar y of putative kinase inhibitors. Although this route is limited to the synthesis of biphenyl of putative kinase inhibitors. Although this route is limited to the synthesis of biphenyl Author Contributions: Conceptualization, L.D. and A.Z.; methodology, L.D., A.Z. and R.V.M.; val- pyra pyra zole zol ur eeas, ureas, it com it com pare pare s favorab s favorab ly w ly ith wprev ith prev ious ious litera litture erature in ter inms terms of yield of yield s an s d an ov d er- over- idation, L.D. and A.Z.; investigation, L.D., R.V.M., M.G., A.T. A.G. and A.Z.; resources, L.D. and all e all ase e of ase s of ynt sy hesis nthesis . . A.Z.; data curation, L.D. and A.Z.; writing—original draft, L.D. and A.Z.; writing—review & editing Preparation, L.D., R.V.M., M.G., A.T. and A.Z.; supervision, A.Z. All authors have read and agreed SupSu plement pplement ary ary Materials: Materials: The The follo fol wlo ing wing suppor suppor tingti ng inform inform ation ation can can be be downlo downlo adeade d at d : at: to the published version of the manuscript. www.mdpi. www.mdpi. com co /xxx/s1 m/xxx/s1 . . Funding: This research received no external funding. Author Author Contri Contri bution bution s: Con s: Con ceptu ceali ptu zatio alizatio n, Ln .D , L . and .D. and A.Z A .; .m Z.; ethodo methodo logy lo , gy L.D , L ., .A D..Z , A . and .Z. and R.V R .M .V .;. M va.; l- val- Institutional Review Board Statement: Not applicable. idation, L.D. and A.Z.; investigation, L.D., R.V.M., M.G., A.T. A.G. and A.Z.; resources, L.D. and idation, L.D. and A.Z.; investigation, L.D., R.V.M., M.G., A.T. A.G. and A.Z.; resources, L.D. and Informed Consent Statement: Not applicable. A.ZA .; .dat Z.; a dat cur a at cur ioat n,io Ln .D , L . an .Dd . an Ad .Z A .; .w Zriti .; w ng riti — ng original —original draft d,raft L.D , L . and .D. and A.Z A .; .w Zr .;it w ing rit— ing revi —r ew evi& ew editin & editin g g Preparation Preparation , L.D , L ., R .D..V , R .M .V ., M .M.., M G., A .G..T , A . an .Td . an Ad .Z A .; .s Z up .; s ervi upervi sion si , on A.Z. , A Al .Z. l Al auth l auth ors ors have have read read and aagr nd eed agr eed Conflicts of interest: The authors declare no conflict of interest. to the publishe to the publishe d vers d vers ion of ion of the th me anu manu script. script. Funding: Funding: This This rese rese arch rec arch rec eived eived no extern no extern al fu al fu nding nding . . InstInst itutional itutional Review Boar Review Boar d Statement: d Statement: No t ap Not ap plicab plile cab . le. Info Info rmed rmed Cons Co ent Statement nsent Statement : No : t No appli t appli cablcab e. le. Conflic Conflic ts of ts in of terest: interest: The The auth auth ors ors declare declare no co no nfli coct nfli of interest. ct of interest. Appl. Biosci. 2022, 1 71 Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/applbiosci1010004/s1. Author Contributions: Conceptualization, L.D. and A.Z.; methodology, L.D., A.Z. and R.V.M.; validation, L.D. and A.Z.; investigation, L.D., R.V.M., M.G., A.T., A.G. and A.Z.; resources, L.D. and A.Z.; data curation, L.D. and A.Z.; writing—original draft, L.D. and A.Z.; writing—review & editing Preparation, L.D., R.V.M., M.G., A.T. and A.Z.; supervision, A.Z. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Conflicts of Interest: The authors declare no conflict of interest. References 1. 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Expedient Access to Type II Kinase Inhibitor Chemotypes by Microwave-Assisted Suzuki Coupling

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10.3390/applbiosci1010004
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Communication Expedient Access to Type II Kinase Inhibitor Chemotypes by Microwave-Assisted Suzuki Coupling Lorenza Destro , Ross Van Melsen, Alex Gobbi, Andrea Terzi, Matteo Genitoni and Alfonso Zambon * Department of Chemical and Geological Sciences, University of Modena and Reggio Emilia, Via G. Campi 103, 41125 Modena, Italy; lorenza.destro@unimore.it (L.D.); rvanmels@unimore.it (R.V.M.); 256915@studenti.unimore.it (A.G.); 229348@studenti.unimore.it (A.T.); 227048@studenti.unimore.it (M.G.) * Correspondence: alfonso.zambon@unimore.it Abstract: Functionalized pyrazole-urea scaffolds are a common type II chemotype for the inhibition of protein kinases (PKs), binding simultaneously into the ATP-binding pocket with an ATP bioisostere and into a vicinal allosteric pocket with a pyrazole group. Standard approaches to the scaffold require multi-step synthesis of the ATP bioisostere followed by phosgene or triphosgene-mediated coupling with the substituted pyrazole group. Here we report an expedient approach to the chemotype, characterized by an optimized MW-assisted Suzuki coupling on easily accessed bromo-phenyl pyrazole ureas. The new protocol allowed quick access a large library of target analogues covering a broad chemical space of putative protein kinases inhibitors (PKIs). Keywords: protein kinase inhibitors; pyrazole-ureas; microwave synthesis 1. Introduction Citation: Destro, L.; Van Melsen, R.; Protein phosphorylation in eukaryotes plays a key role in cell signaling, gene expres- Gobbi, A.; Terzi, A.; Genitoni, M.; sion, and differentiation. Protein phosphorylation is also involved in the global control of Zambon, A. Expedient Access to DNA replication during the cell cycle, as well as in the mechanisms that cope with stress- Type II Kinase Inhibitor Chemotypes induced replication blocks [1]. The role of kinases is to phosphorylate serine, threonine, or by Microwave-Assisted Suzuki tyrosine residues of specific protein substrates via the transfer of the -phosphate group of Coupling. Appl. Biosci. 2022, 1, 64–72. adenosine triphosphate (ATP) or, in specific cases, GTP [2]. Protein kinases are, therefore, https://doi.org/10.3390/ key enzymes in the function of cellular signaling pathways and are crucial in the regulation applbiosci1010004 of key functions such as cell proliferation, differentiation, and apoptosis [3]. Academic Editor: Robert Henry Aberration of PK-mediated cellular pathways is a most common factor in the onset and progression of cancer [4,5], and starting from the early 2000s PKs have emerged as Received: 29 April 2022 prominent targets for the development of cancer therapies, with 43 protein kinase inhibitors Accepted: 28 May 2022 (PKI) approved by FDA for the treatment of solid and liquid tumors [6]. To date, an Published: 31 May 2022 estimated 20–33% of the global drug discovery efforts are directed at the development Publisher’s Note: MDPI stays neutral of protein kinase inhibitors [7–11]. Depending on their binding mode within the ATP with regard to jurisdictional claims in binding site and in proximal or distal allosteric pockets, PKIs are classified as Type I, Type published maps and institutional affil- I 1/2, and Type II-VI, with Type I and Type II being first binding modes identified and iations. the most common ones [12]. Type II inhibitors, in particular, bind simultaneously to the ATP-binding pocket and to an adjacent allosteric pocket when the kinase in an inactive, DFG-out conformation, in contrast with Type I inhibitors that bind to the active, DFG-in Copyright: © 2022 by the authors. conformation of the kinase and only into the ATP-binding pocket. Of the 27 co-crystal Licensee MDPI, Basel, Switzerland. structures of FDA-approved PKIs available in 2019, eight were Type II [6]. As inactive This article is an open access article protein kinase conformations exhibit greater structural variation than the conserved active distributed under the terms and conformation to which Type I PKIs bind, Type II PKIs are considered potentially more conditions of the Creative Commons selective than Type I ones [13,14]. Most Type II PKIs share a common chemotype enticing a Attribution (CC BY) license (https:// hydrophobic element that forms Van der Waals interactions with the allosteric pocket and creativecommons.org/licenses/by/ a bridge system (e.g., an amine or a urea) able to form hydrogen bonds with the conserved 4.0/). Appl. Biosci. 2022, 1, 64–72. https://doi.org/10.3390/applbiosci1010004 https://www.mdpi.com/journal/applbiosci Appl. Biosci. 2022, 1, FOR PEER REVIEW 2 Appl. Biosci. 2022, 1, FOR PEER REVIEW 2 Appl. Biosci. 2022, 1 65 a bridge system (e.g., an amine or a urea) able to form hydrogen bonds with the conserved a bridge system (e.g., an amine or a urea) able to form hydrogen bonds with the conserved salt bridge adjacent to the PK active site and an ATP bioisostere that occupies the ATP salt bridge adjacent to the PK active site and an ATP bioisostere that occupies the ATP salt bridge adjacent to the PK active site and an ATP bioisostere that occupies the ATP binding pocket [15–18]. binding pocket [15–18]. binding pocket [15–18]. The introduction of a pyrazole-urea moiety as an allosteric binder in Type II PKIs has The introduction of a pyrazole-urea moiety as an allosteric binder in Type II PKIs has The introduction of a pyrazole-urea moiety as an allosteric binder in Type II PKIs been shown to confer activity against a range of protein targets and to improve the phar- been shown to confer activity against a range of protein targets and to improve the phar- has been shown to confer activity against a range of protein targets and to improve the macokinetic properties of the scaffold, allowing for the progression to the clinic of several macokinetic properties of the scaffold, allowing for the progression to the clinic of several pharmacokinetic properties of the scaffold, allowing for the progression to the clinic of compounds of this type. The general structure of pyrazole-urea PKIs entices a central py- compounds of this type. The general structure of pyrazole-urea PKIs entices a central py- several compounds of this type. The general structure of pyrazole-urea PKIs entices a razole-phenyl urea core substituted on the pyrazole by aliphatic (X) and aromatic and razole-phenyl urea core substituted on the pyrazole by aliphatic (X) and aromatic and central pyrazole-phenyl urea core substituted on the pyrazole by aliphatic (X) and aromatic aliphatic (Y) groups and bearing on the phenyl ring a range of substituents usually com- aliphatic (Y) groups and bearing on the phenyl ring a range of substituents usually com- and aliphatic (Y) groups and bearing on the phenyl ring a range of substituents usually prising a variously substituted aromatic group (Z), as depicted in Figure 1 [19–21]. prising a variously substituted aromatic group (Z), as depicted in Figure 1 [19–21]. comprising a variously substituted aromatic group (Z), as depicted in Figure 1 [19–21]. Figure 1. General structure of Type II pyrazole-urea PKI. Figure 1. General structure of Type II pyrazole-urea PKI. Figure 1. General structure of Type II pyrazole-urea PKI. In general, access to this chemotype requires the multi-step synthesis of the aniline In general, access to this chemotype requires the multi-step synthesis of the aniline In general, access to this chemotype requires the multi-step synthesis of the aniline ATP isostere followed by phosgene or triphosgene-mediated coupling to urea with the ATP isostere followed by phosgene or triphosgene-mediated coupling to urea with the ATP isostere followed by phosgene or triphosgene-mediated coupling to urea with the pyrazole group. This approach requires lengthy synthetic and purification procedures, pyrazole pyrazolegr goup. roupThis . This appr app oach roacrhequir requ es ire lengthy s lengthy syn synthetic thetic and puri and purification ficatpr ion procedu ocedures, the res, the optimization of reaction conditions for each analogue, and the use of hazardous rea- the optimization of reaction conditions for each analogue, and the use of hazardous rea- optimization of reaction conditions for each analogue, and the use of hazardous reagents gents for the coupling step, which often occurs with sub-optimal yields [22–27]. We posit for gent the s for coupling the couplin step,g st which ep, wh often ich occurs often occurs with sub-optimal with sub-opti yields mal yiel [22ds –27 [22– ]. W 27 e ]. posit We posi thatt that direct reaction of the pyrazole group with commercial Bromophenyl Isocyanates fol- dir tha ect t di reaction rect reac of tion theof the pyrazole pyraz gro oup le group with commer with com cial mercial Brom Bromophenyl ophenyl I Isocyanates socyan followed ates fol- by lowed by a Suzuki coup a Suzuki coupling (Scheme ling (Scheme 1) wou 1) ld woul provide d provid an expedient e an expedien access t access to this chemo- to this chemotype. lowed by a Suzuki coupling (Scheme 1) would provide an expedient access to this chemo- type. Diversity points can be easily introduced at each step, allowing for the rapid synthe- Diversity points can be easily introduced at each step, allowing for the rapid synthesis of a type. Diversity points can be easily introduced at each step, allowing for the rapid synthe- library sis of a of lib analogues rary of an[ alo 28,g 29 ue ].s [28,29]. sis of a library of analogues [28,29]. Scheme 1. Proposed route to pyrazole-ureas analogues. Scheme 1. Proposed route to pyrazole-ureas analogues. Scheme 1. Proposed route to pyrazole-ureas analogues. Suzuki-Miyaura coupling allows the formation of new C-C bonds between an aryl Suzuki-Miyaura coupling allows the formation of new C-C bonds between an aryl Suzuki-Miyaura coupling allows the formation of new C-C bonds between an aryl halide halide and and an anaryl arylbor boron on species speciesand andis isparticularl particulary lysuitable suitable for for our our purpo purpose se due due t tooits its halide and an aryl boron species and is particularly suitable for our purpose due to its rrobustness and obustness and tolerance tolerance of of functional functional groups groups. . Al Although though this reacti this reaction on has has alr alrea eady dy been been robustness and tolerance of functional groups. Although this reaction has already been extensively investigated, reactions on substrates containing an aromatic urea group are extensively investigated, reactions on substrates containing an aromatic urea group are extensively investigated, reactions on substrates containing an aromatic urea group are scantly scantly rep reported, orted, and no e and no examples xamples in in w which hich one of t one of the two he tw aro arom omaticat rings ic rin isga s i pyrazole s a pyrazole are scantly reported, and no examples in which one of the two aromatic rings is a pyrazole rare reported eported in the in the liter literaturatur e [30 e [3 ,310,31] The ] The affinity affinof ity of p palladium alladium for for th the ur e ure ea gr aoup grou suggests p suggests a are reported in the literature [30,31] The affinity of palladium for the urea group suggests possible coordination of the catalyst by our substrate, and thus possible deactivation of the a possible coordination of the catalyst by our substrate, and thus possible deactivation of a possible coordination of the catalyst by our substrate, and thus possible deactivation of former. Here we report the optimization of the problematic Suzuki-Muyara reaction on the former. Here we report the optimization of the problematic Suzuki-Muyara reaction the former. Here we report the optimization of the problematic Suzuki-Muyara reaction pyrazole ureas, and the consequent synthesis of a library of analogues easily accessed by on pyrazole ureas, and the consequent synthesis of a library of analogues easily accessed on pyrazole ureas, and the consequent synthesis of a library of analogues easily accessed the variation of substituents on the pyrazole and phenylboronic reagents. by the variation of substituents on the pyrazole and phenylboronic reagents. by the variation of substituents on the pyrazole and phenylboronic reagents. 2. Materials and Methods 2. Materials and Methods 2. Materials and Methods General Methods: Commercial building blocks, reagents, and solvents for reactions General Methods: Commercial building blocks, reagents, and solvents for reactions General Methods: Commercial building blocks, reagents, and solvents for reactions were reagent grade and used as purchased or purified according to methods in the literature. were reagent grade and used as purchased or purified according to methods in the litera- were reagent grade and used as purchased or purified according to methods in the litera- Reactions were monitored by Pre-coated TLC sheets ALUGRAM Sil G/UV (0.20 mm ® 254 ture. Reactions were monitored by Pre-coated TLC sheets ALUGRAM Sil G/UV254 (0.20 ture. Reactions were monitored by Pre-coated TLC sheets ALUGRAM Sil G/UV254 (0.20 thickness). Dry solvents were prepared by overnight standing on freshly activated 4 Å mm thickness). Dry solvents were prepared by overnight standing on freshly activated 4 mm thickness). Dry solvents were prepared by overnight standing on freshly activated 4 molecular sieves under argon atmosphere or purchased. Flash chromatography was Appl. Biosci. 2022, 1 66 1 13 conducted using a Merk 60, 230–400 mesh silica gel. H- and C-NMR spectra were recorded at 298K on Bruker FT-NMR Advance 400 (400.13 MHz) e Bruker FT-NMR Advance III HD 600 (600.13 MHz). Chemical shift values are given in ppm relative to TMS and were determined by taking as reference the isotopic impurity signals of CDCl (7.26 ppm for 1 13 1 13 H and 77.16 for C) and DMSO-d (2.50 ppm for H and 39.52 ppm for C). Data are presented as follows: chemical shift () in ppm, multiplicity, coupling constants (J) given in hertz. LCMS data were acquired using a 6130A quadrupole ion trap analyzer Ion Trap LC-MS(n) by Agilent Technologies. Docking analysis was carried out using the Glide Docking Module of Maestro (Schroedinger) in the standard precision (SP) mode. Detailed synthetic procedures and full characterization of all the synthesized com- pounds are reported in Supplementary Information. 3. Results As discussed above, Suzuki coupling on aromatic ureas is rarely reported, and to our knowledge never on a substituted pyrazole scaffold [30,31]. We thus set out to optimize the coupling step using as model reaction the coupling of easily obtained 1-(4-bromophenyl)-3- (3-(tert-butyl)-1-phenyl-1H-pyrazol-5-yl)urea 1a with boronic 3,4-dimethoxyphenylboronic 0 0 acid 2a as coupling partner to 1-(3-(tert-butyl)-1-phenyl-1H-pyrazol-5-yl)-3-(3 ,4 -dimethoxy- [1,1 -biphenyl]-4-yl)urea 3a (Table 1). We first tried the protocol reported by Al-Masoudi et al. [30] for structurally close bis-phenyl ureas, carrying out the reaction under reflux with readily available reagents (Entry 1) but did not observe any product formation af- ter 18h. We then tried varying base, catalyst, and solvent system replacing K CO with 2 3 Na CO (Entry 2), Pd(PPh ) with Pd(dppf)Cl (Entry 3) and finally the solvent with a 2 3 3 4 2 1:1=H O:DMF mixture (Entry 4). Again, under all the conditions explored we did not observe any conversion to product, suggesting a considerable deactivation of the substrate towards Suzuki coupling under thermal conditions. We then set out to explore a range of conditions starting from those reported by Brunner et al. [31] in which coupling of phenyl bromo ureas is carried out under microwave (MW) irradiation. The use of MW is known to improve the reaction rates and thus could overcome the deactivation of the system observed under thermal conditions [32]. As the starting conditions, we used MeCN as solvent, Pd(PPh ) as catalyst, Na CO as base and 3 4 2 3 run the reaction for 1 h at 100 C under microwave condition as reported (Entry 5) [32]. Unfortunately, this reaction did not lead to the desired results, most likely due to the poor solubility of urea 1a in the solvent medium. We then changed the solvent system by replacing the MeCN with a mixture of a 2M aqueous solution of K CO and 1,4-dioxane in 2 3 a ratio of 1:2, in which our urea proved more soluble (Entry 6). Encouragingly, we observed the formation of some product in the reaction mixture, albeit with incomplete conversion and 30% isolated yield. After several attempts, we were then able to further optimize the reaction conditions by changing both the base by switching K CO with Na CO , a slightly 2 3 2 3 stronger base, and the catalyst by replacing Pd(PPh ) with Pd(dppf)Cl (Entry 7–8), a 3 4 2 catalyst that was proven stable and effective under microwave conditions [33]. We finally identified the best conditions with the use of Pd(dppf)Cl as catalyst and Na CO as base 2 2 3 with complete spectroscopic conversion and 50% yield after chromatography (Entry 9). After identifying the optimal solvent, base, and catalyst, we decided to investigate if the reaction could be performed at lower temperatures (70 C instead of 100 C, Entry 10) and with shorter reaction times (45 min instead of 1.25 h, Entry 11). Lowering the temperature significantly affected the conversion, with only 15% of urea 1a converted to 3a, but we found that reaction times could be shortened to 30 min (Entry 11), with ~99% conversion of urea 1a to 3a, in line with that observed for Entry 9. Appl. Biosci. 2022, 1, FOR PEER REVIEW 4 Appl. Biosci. 2022, 1 67 Table 1. Optimization of the reaction conditions for the Suzuki coupling of 1a and 2a. Table 1. Optimization of the reaction conditions for the Suzuki coupling of 1a and 2a. Entry Conditions Time (h) T (°C) Solvent Catalyst Base Yield Entry Conditions Time (h) T ( C) Solvent Catalyst Base Yield 1 T1 18 101 1,4-dioxane Pd(PPh3)4 K2CO3 N.R. 1 T 18 101 1,4-dioxane Pd(PPh ) K CO N.R. 1 3 4 2 3 2 T2 18 101 1,4-dioxane Pd(PPh3)4 Na2CO3 N.R. 2 T 18 101 1,4-dioxane Pd(PPh ) Na CO N.R. 2 3 4 2 3 3 T3 18 101 1,4-dioxane Pd(dppf)Cl2 K2CO3 N.R. 3 T 4 T4 18 18 101 100 1,4-dioxane 1:1 DMF/H2O Pd(dppf)Cl Pd(PPh3)4 K2K CO CO 3 N.R. N.R. 3 2 2 3 5 MW 1 100 MeCN Pd(PPh3)4 Na2CO3 N.R. 4 T 18 100 1:1 DMF/H O Pd(PPh ) K CO N.R. 4 2 3 4 2 3 6 MW 1.25 100 1:2 H2O/1,4-dioxane Pd(PPh3)4 Na2CO3 30% 5 MW 1 100 MeCN Pd(PPh ) Na CO N.R. 3 4 2 3 7 MW 1.25 100 1:2 H2O/1,4-dioxane Pd(PPh3)4 K2CO3 N.R. 6 MW 1.25 100 1:2 H O/1,4-dioxane Pd(PPh ) Na CO 30% 2 3 4 2 3 8 MW 1.25 100 1:2 H2O/1,4-dioxane Pd(dppf)Cl2 K2CO3 N.R. 9 MW5 1.25 100 1:2 H2O/1,4-dioxane Pd(dppf)Cl2 Na2CO3 50% 7 MW 1.25 100 1:2 H O/1,4-dioxane Pd(PPh ) K CO N.R. 2 3 4 2 3 10 MW6 1.25 70 1:2 H2O/1,4-dioxane Pd(dppf)Cl2 Na2CO3 15% 8 MW 1.25 100 1:2 H O/1,4-dioxane Pd(dppf)Cl K CO N.R. 2 2 2 3 11 MW7 0.5 100 1:2 H2O/1,4-dioxane Pd(dppf)Cl2 Na2CO3 ~99% 9 MW 1.25 100 1:2 H O/1,4-dioxane Pd(dppf)Cl Na CO 50% 5 2 2 2 3 a b 1 Isolated yield (column purification) spectroscopic yield ( H-NMR) N.R. = Not reacted; only Start- 10 MW ing materials reco 1.25 vered. 70 1:2 H O/1,4-dioxane Pd(dppf)Cl Na CO 15% 6 2 2 2 3 11 MW 0.5 100 1:2 H O/1,4-dioxane Pd(dppf)Cl Na CO ~99% 7 2 2 2 3 We then set out to perform the synthesis of a small library of analogues of 1a taking a b 1 Isolated yield (column purification) spectroscopic yield ( H-NMR) N.R. = Not reacted; only Starting advantage of the newly optimized Suzuki procedure. Although reaction time could be materials recovered. lowered at 30 min, we deemed it preferable to keep it at 1.25 h to account for the potential We then set out to perform the synthesis of a small library of analogues of 1a taking lower reactivity of the alternative boronic agents. advantage of the newly optimized Suzuki procedure. Although reaction time could be The overall synthetic route to this library of compounds is depicted in Scheme 2 and lowered at 30 min, we deemed it preferable to keep it at 1.25 h to account for the potential involves the condensation of an oxo nitrile 4a-b with a hydrazine 5a-c to give pyrazole 6a- lower reactivity of the alternative boronic agents. f, followed by the formation of Br-substituted ureas 1a-h by reaction with isocyanates 7a- The overall synthetic route to this library of compounds is depicted in Scheme 2 and b. These steps are already well reported in the scientific literature and gave high yields involves the condensation of an oxo nitrile 4a-b with a hydrazine 5a-c to give pyrazole 6a-f, for all the substrates explored [34]. followed by the formation of Br-substituted ureas 1a-h by reaction with isocyanates 7a-b. These steps are already well reported in the scientific literature and gave high yields for all the substrates explored [34]. Ureas 1a-l were finally reacted following the optimized Suzuki protocol with selected boronic acids 2a-e to afford para-substituted (3a-f) and meta-substituted (3g-l) final com- pounds in good 35–60% yields as reported in Table 2 (para-substituted compounds) and Table 3 (meta-substituted compounds). For a preliminary evaluation of the potential of our compounds as PKIs, we carried out docking of 3a-l in the co-crystal structure (pdb code 1KV2) of p38, a validated target for autoimmune diseases, with pyrazole-urea BIRB-796 [21]. All compounds faithfully mirrored the binding mode of BIRB-796 and registered high docking scores, suggesting a high affinity of the library for p38 (Figure 2). The best affinities were calculated for compounds 3g, 3h, 3l and 3a, with docking scores ranging from 11.7 and 8.9 kcal/mol. Appl. Biosci. 2022, 1, FOR PEER REVIEW 5 Appl. Appl. B Appl. iosci. Biosci. B 2022 iosci. 2022 , 1 2022 , FO , 1, FO , R 1, FO PEER R PEER R RE PEER RE VIEW RE VIEW VIEW 5 5 5 Appl. Biosci. 2022, 1, FOR PEER REVIEW 5 Appl. Appl. Biosci. Biosci. 2022 2022 , 1, FO , 1, FO R PEER R PEER RE RE VIEW VIEW 5 5 Appl. Biosci. 2022, 1 68 Appl. Biosci. 2022, 1, FOR PEER REVIEW 5 Scheme 2. General synthetic route to pyrazole urea 3a-l. Reagents and conditions: (a) Toluene, 116 Scheme Scheme Scheme 2. 2. General synthetic r 2. General synthetic r General synthetic r oute to pyrazole urea 3 oute to pyrazole urea 3 oute to pyrazole urea 3 a-l. Re a-l. Re a-l agents and . Re agents and agents and con con ditions: ( con ditions: ( ditions: ( a) Toluene, a) Toluene, a) Toluene, 116 116 116 Scheme 2. General synthetic route to pyrazole urea 3a-l. Reagents and conditions: (a) Toluene, 116 Scheme Scheme 2. 2. General synthetic r General synthetic r oute to pyrazole urea 3 oute to pyrazole urea 3 a-la-l . Re . Re agents and agents and con con ditions: ( ditions: ( a) Toluene, a) Toluene, 116 116 °C,72 h; (b) anhydrous DCM, room temperature, 24 h; (c) 1:2 H2O/1,4-dioxane, 100 °C, 1.25 h. °C,72 h; ( °C,72 h; ( b) an b) an hydrous DCM, room hydrous DCM, room temperature, 24 h; ( temperature, 24 h; ( c) 1:2 H c) 1:2 H 2O/1,4- 2O/1,4- diox diox ane, 100 °C ane, 100 °C , 1., 1 25. h. 25 h. °C,72 h; (b) anhydrous DCM, room temperature, 24 h; (c) 1:2 H2O/1,4-dioxane, 100 °C, 1.25 h. °C,72 h; ( °C,72 h; ( b) an b) an hydrous DCM, room hydrous DCM, room temperature, 24 h; ( temperature, 24 h; ( c) 1:2 H c) 1:2 H 2O/1,4- 2O/1,4- diox diox ane, 100 °C ane, 100 °C , 1.2 , 1 5 h. .25 h. °C,72 h; (b) anhydrous DCM, room temperature, 24 h; (c) 1:2 H2O/1,4-dioxane, 100 °C, 1.25 h. Ureas 1a-l were finally reacted following the optimized Suzuki protocol with selected Ureas Ureas Ureas 1a-l 1a were -l 1a were -l were fina fina llfy rea ill na y rea llc y rea ted f cted f c o ted f llo ol wi lo ol wi ng lowi ng the opti ng the opti the opti mimi zed Suzuki mi zed Suzuki zed Suzuki protoc protoc protoc ol w ol w iol w th selec ith selec ith selec ted te d te d Ureas 1a-l were finally reacted following the optimized Suzuki protocol with selected Ureas Ureas 1a1a -l were -l were fina fina lly rea lly rea cted f cted f oll o o lwi lowi ng ng the opti the opti mi mi zed Suzuki zed Suzuki protoc protoc ol w ol w ith selec ith selec ted te d boronic acids 2a-e to afford para-substituted (3a-f) and meta-substituted (3g-l) final com- boronic a boronic a boronic a cids cids 2a cids - 2a e t -2a e o t a -o e ffo t ao ffo rd affo par rd par rd a par -sub a-sub ast -sub it st uit tst ed ( uit ted ( u3a ted ( - 3a f) and -3a f) and -f) meta-sub and meta-sub meta-sub stituted stituted stituted (3g-l (3g-l ) f (3g-l i) f na i) f na l com- il na com- l com- boronic acids 2a-e to afford para-substituted (3a-f) and meta-substituted (3g-l) final com- boronic a boronic a cids cids 2a2a -e t -e o t a offo affo rdrd par par a-sub a-sub stit st u it tu ed ( ted ( 3a3a -f)- and f) and meta-sub meta-sub stituted stituted (3g-l (3g-l ) fi ) f na ina l com- l com- Scheme 2. General synthetic route to pyrazole urea 3a-l. Reagents and conditions: (a) Toluene, 116 pounds in good 35–60% yields as reported in Table 2 (para-substituted compounds) and Scheme 2. General synthetic route to pyrazole urea 3a-l. Reagents and conditions: (a) Toluene, pounds in good 35–60% yields as reported in Table 2 (para-substituted compounds) and pounds i pounds i n good 35– n good 35– 60% yiel 60% yiel ds as ds as reported reported in Tain bl T e 2 a b (p lear 2a- (p sub ara- stsub ituts etd com ituted com pounds) pou an nds) d and pounds i pounds i n good 35– n good 35– 60% yiel 60% yiel ds as ds as reported reported in T in a T bla eb 2 le (p 2 ar (p a- ar sub a-sub stitsu titteu d com ted com pou pnds) ounds) an an d d pounds in good 35–60% yields as reported in Table 2 (para-substituted compounds) and °C,72 h; (b) anhydrous DCM, room temperature, 24 h; (c) 1:2 H2O/1,4-dioxane, 100 °C, 1.25 h. Table 3 (meta-substituted compounds). 116 C, 72 h; (b) anhydrous DCM, room temperature, 24 h; (c) 1:2 H O/1,4-dioxane, 100 C, 1.25 h. TaTa ble 3 ble 3 (m (eta- meta- sub sub stituted stituted compounds). compounds). Table 3 (meta-substituted compounds). 2 Tabl Ta e 3 ble 3 (m ( eta- meta- sub sub stituted stituted compounds). compounds). Table 3 (meta-substituted compounds). Ureas 1a-l were finally reacted following the optimized Suzuki protocol with selected Table 2. Structures and yields of the final step of compounds 3a-f. Table 2. Structures and yields of the final step of compounds 3a-f. Table 2. Table 2. Table 2. Struct Struct Struct ures and yiel ures and yiel ures and yiel ds of th ds of th ds of th e fie f naie f l s nat il s ep o na tl s ep o ft co ep o f co mpo f co mpo umpo nd und s u 3a-f s nd 3a-f s . 3a-f . . Table 2. Structures and yields of the final step of compounds 3a-f. Table 2. Structures and yields of the final step of compounds 3a-f. Table 2. Structures and yields of the final step of compounds 3a-f. boronic acids 2a-e to afford para-substituted (3a-f) and meta-substituted (3g-l) final com- pounds in good 35–60% yields as reported in Table 2 (para-substituted compounds) and Table 3 (meta-substituted compounds). Table 2. Structures and yields of the final step of compounds 3a-f. Compound R1 R2 R3 Yield Compound R R R Yield 1 2 3 Compound R1 R2 R3 Yield ComCompoundpou Rnd R 1 R 1 R 2 R 2 R 3 Yield 3 Yield ComCompoupndound R R 1 R 1 R 2 R 2 R 3 Yield 3 Yield Compound R1 R2 R3 Yield 3a 35% 3a 3a 3a 3a 35% 35% 35% 35% 3a 35% 3a 35% 3a 35% Compound R 1 R 2 R3 Yield 3a 35% 3b 43% 3b 43% 3b 3b 43% 43% 3b 3b 43% 43% 3b 3b 43% 43% Appl. Biosci. 2022, 1, FOR PEER REVIEW 6 Appl. Appl. Biosci. Biosci. 2022 2022 , 1, FO , 1, FO R PEER R PEER RE RE VIEW VIEW 6 6 3b 43% 3c 3c 3c 3c 36% 36% 36% 36% 3d3d 3d 52% 52% 52% 3e 3e 3e -Me -Me -Me 48% 48% 48% 3f 3f -Me -Me 58% 58% 3f -Me 58% Table 3. Table 3. Struct Struct ures and yiel ures and yiel ds of th ds of th e fie f nail s natl s ep o tep o f co f co mpo mpo und und s 3g-l s 3g-l . . Table 3. Structures and yields of the final step of compounds 3g-l. Compound R1 R2 R3 Yield ComCompoupoundnd R R 1 R 1 R 2 R 2 R 3 Yield 3 Yield 3g3g 3g 36% 36% 36% 3h3h 48% 48% 3h 48% 3i 36% 3i 3i 36% 36% Appl. Biosci. 2022, 1, FOR PEER REVIEW 5 Scheme 2. General synthetic route to pyrazole urea 3a-l. Reagents and conditions: (a) Toluene, 116 °C,72 h; (b) anhydrous DCM, room temperature, 24 h; (c) 1:2 H2O/1,4-dioxane, 100 °C, 1.25 h. Ureas 1a-l were finally reacted following the optimized Suzuki protocol with selected boronic acids 2a-e to afford para-substituted (3a-f) and meta-substituted (3g-l) final com- Appl. Biosci. 2022, 1 69 pounds in good 35–60% yields as reported in Table 2 (para-substituted compounds) and Table 3 (meta-substituted compounds). Table 2. Structures and yields of the final step of compounds 3a-f. Table 2. Cont. Appl. B Appl. Appl. iosci. B B 2022 ii osci. osci. , 2022 1 2022 , FO , , R 11, FO , FO PEER R R PEER RE PEER VIEW RE REVIEW VIEW 6 6 6 Appl. Biosci. 2022, 1, FOR PEER REVIEW 6 Appl. Biosci. 2022, 1, FOR PEER REVIEW 6 Appl. Biosci. 2022, 1, FOR PEER REVIEW 6 Appl. Biosci. 2022, 1, FOR PEER REVIEW 6 Appl. Biosci. 2022, 1, FOR PEER REVIEW 6 Appl. Biosci. 2022, 1, FOR PEER REVIEW 6 Appl. Appl. B Biiosci. osci. 2022 2022, , 1 1, FO , FOR R PEER PEER RE REVIEW VIEW 6 6 Appl. Appl. Appl. Biosci. B Biiosci. 2022 osci. 2022 , 2022 1, FO , , 1 1, FO R , FO PEER R R PEER PEER REVIEW RE REVIEW VIEW 6 6 6 3c 3c 36% 36% 3c 36% 3c 3c 36% 36% 3c 3c 36%36% 3c 36% 3c 36% 3c 3c 36% 36% CompoundCompound R R 1 R R 2 R R 3 Yield Yield 3c 3c 3c 1 2 3 36% 36% 36% 3a 35% 3d 3d 3d 52% 52% 52% 3d 52% 3d3d 52% 52% 3d 3d 52%52% 3d 52% 3d 52% 3d 3d 52% 52% 3d 3d 3d 52% 52% 52% 3e -Me 48% 3e 3e -Me -Me 48% 48% 3e 3e 3e -Me -Me -Me 48% 48% 48% 3e 3e -Me -Me 48%48% 3b 43% 3e -Me 48% 3e 3e 3e -Me -Me -Me 48% 48% 48% 3e 3e -Me -Me 48% 48% 3e -Me 48% 3f -Me 58% 3f 3f -Me -Me 58% 58% 3f 3f -Me -Me 58% 58% 3f 3f 3f -Me -Me -Me 58% 58%58% 3f -Me 58% 3f 3f 3f -Me -Me -Me 58% 58% 58% 3f -Me 58% 3f 3f -Me-Me 58%58% Table 3. Structures and yields of the final step of compounds 3g-l. Table 3. Table 3. Struct Struct ures and yiel ures and yiel ds of th ds of th e fina e f l sitna ep o l stfep o compo f cou mpo ndsu 3g-l nds. 3g-l. Table 3. Table 3. Struct Struct ures and yiel ures and yiel ds of th ds of th e fie f nail s natl s ep o tep o f co f co mpo mpo und und s 3g-l s 3g-l . . Table 3. Table 3. Struct Struct ures and yiel ures and yiel ds of th ds of th e fina e f l s ina tep o l stfep o compo f compo undsu 3g-l nds. 3g-l. Table 3. Structures and yields of the final step of compounds 3g-l. Table 3. Structures and yields of the final step of compounds 3g-l. Table 3. Table 3. Table 3. Struct Struct Struct ures and yiel ures and yiel ures and yiel ds of th ds of th ds of th e fina e f e f l s iina na tep o l s l sttep o ep o f compo ff co compo mpo undu u snd nd 3g-l ss 3g-l . 3g-l. . Table 3. Structures and yields of the final step of compounds 3g-l. Table 3. Table 3. Struct Struct ures and yiel ures and yiel ds of th ds of th e fina e f l s ina tep o l step o f compo f compo unds u nd 3g-l s . 3g-l. ComComCompoundppouou R ndnd R R 1 R 11 R R 2 R 22 R R 3 Yield 33 Yield Yield Compound R1 R2 R3 Yield Compound R1 R2 R3 Yield ComCompoundpou R nd R 1 R 1 R 2 R 2 R 3 Yield 3 Yield CompoundCompound R R 1 R R 2 R R 3 Yield Yield 1 2 3 Compound R1 R2 R3 Yield ComComppououndnd R R 11 R R 22 R R 33 Yield Yield ComComCompouppndouou R ndnd R R 1 R 11 R R 2 R 22 R R 3 Yield 33 Yield Yield 3g 3g 36% 36% 3g 36% 3g3g 36% 36% 3g 3g 36%36% 3g 36% 3g 36% 3g 3g 3g 36% 36% 36% 3g 3g 36% 36% 3g 36% 3h 48% 3h 3h 48% 48% 3h 48% 3h 48% 3h 3h 48%48% 3h 48% 3h 3h 3h 48% 48% 48% 3h 48% 3h 3h 3h 48% 48% 48% 3i 3i 3i 36% 36% 36% 3i 36% 3i 36% 3i 36% 3i 36% 3i 36% 3i 36% 3i 3i 36% 36% 3i 3i 3i 36% 36% 36% Appl. Biosci. 2022, 1, FOR PEER REVIEW 6 Appl. Appl. Biosci. Biosci. 20222022 , 1, FO , 1R , FO PEER R PEER REVIEW REVIEW 6 6 3c 36% 3c 3c 36% 36% Appl. Biosci. 2022, 1, FOR PEER REVIEW 6 3d 52% 3d 3d 52% 52% 3e -Me 48% 3e 3e -Me -Me 48% 48% 3c 36% 3f -Me 58% 3f 3f -Me -Me 58% 58% 3d 52% Table 3. Structures and yields of the final step of compounds 3g-l. Table 3. Table 3. Struct Struct ures and yiel ures and yiel ds of th ds of th e fina e f l s ina tep o l step o f compo f compo undu s nd 3g-l s 3g-l . . 3e -Me 48% 3f -Me 58% Appl. Biosci. 2022, 1 70 Compound R1 R2 R3 Yield ComCompoupndou R nd R 1 R 1 R 2 R 2 R 3 Yield 3 Yield Table Table 3. 3. Cont. Structures and yields of the final step of compounds 3g-l. 3g 36% 3g 3g 36% 36% 3h 48% Appl. Biosci. 2022, 1, FOR PEER REVIEW 7 3h 3h 48% 48% Compound R1 R2 R3 Yield Compound R R R Yield 1 2 3 3g 36% 3i 36% 3i 3i 3i 36% 36% 36% 3l -Me 41% Appl. Ap Biosci. pl. Biosci. 2022 202 , 1, 2 FO , 1, R FO PE R ER PE RE ER VIEW REVIEW 7 7 For a preliminary evaluation of the potential of our compounds as PKIs, we carried 3h 48% out docking of 3a-l in the co-crystal structure (pdb code 1KV2) of p38, a validated target 3l -Me 41% 3l 3l -Me-Me 41% 41% for autoimmune diseases, with pyrazole-urea BIRB-796 [21]. All compounds faithfully mirrored the binding mode of BIRB-796 and registered high docking scores, suggesting a high affinity of the library for p38 (Figure 2). The best affinities were calculated for com- pounds 3g, 3h, 3l and 3a, with docking scores ranging from −11.7 and −8.9 kcal/mol. For For a pr a el pr imi elimi nary nary evalu evati alu on atiof on th of e th po e ten potial tential of o of ur ocom ur com pounds pounds as PKI as PKI s, w se , w ca e rried carried 3i 36% out out dockin dockin g of g 3a of -l3a in -l th ine th co e -cryst co-cryst al struct al struct ure ure (pdb (pdb code code 1KV2) 1KV2) of p38 of p38 , a v , alid a valid ated ated target target for for auto au imm toimm une une dise das ise es, as e with s, with pyr pyr azoaz le-o ure le-ure a BI a RB BI-RB 796 -796 [21]. [21]. Al l Al com l com pounds pounds faith fai fully thfully mirrored the binding mode of BIRB-796 and registered high docking scores, suggesting a mirrored the binding mode of BIRB-796 and registered high docking scores, suggesting a high high affiaffi nity n ity of th of e th libr e li ar br yar for y for p38p (38 Fi g (u Fi re gu 2) re . Th 2). e Th be e sbe t affi st affi nities nities were we cre alcculated alculated for for com com - - pounds pounds 3g, 3g 3h, , 3h 3l , and 3l and 3a, 3a with , with dock dock ing sco ing sco res res ranra gin ng f gin rom g from −11.7 and −11.7 and −8.9 −8 kc .9 al kc /mo al/l. mo l. Figure 2. Docking pose of compound 3g (gray) into the binding pocket of p38 (pdb code 1KV2) Figure 2. Docking pose of compound 3g (gray) into the binding pocket of p38 (pdb code 1KV2) superposed to BIRB-796 (pink). superposed to BIRB-796 (pink). 4. Conclusions 4. Conclusions In conclusion, we report a novel, efficient synthetic route to a privileged scaffold for In conclusion, we report a novel, efficient synthetic route to a privileged scaffold for protein kinase inhibitors based on a novel, robust microwave-assisted protocol for the Su- protein kinase inhibitors based on a novel, robust microwave-assisted protocol for the Figure Figure 2. Dock 2. Dock ing ing pose pose of co of m co pou mpou nd 3g nd (g 3g ray (g) ray into ) into the the bind bind ing ing pocket pocket of p38 of p38 (pdb (pdb code co 1KV2) de 1KV2) zuki reaction of pyrazole ureas, which allowed quick to access a structurally varied library Suzuki reaction of pyrazole ureas, which allowed quick to access a structurally varied superp superp osed os to ed BI to RB BI -RB 796- 796 (pink (pink ). ). of putative kinase inhibitors. Although this route is limited to the synthesis of biphenyl library of putative kinase inhibitors. Although this route is limited to the synthesis of pyrazole ureas, it compares favorably with previous literature in terms of yields and over- biphenyl pyrazole ureas, it compares favorably with previous literature in terms of yields 4. Co 4. n Co clus nclus ions ions all ease of synthesis. and overall ease of synthesis. In con In con clus cl ion us, ion we , we repo repo rt a rt nov a nov el, ef el, fief cie fint cie synthet nt synthet ic route ic route to a to priv a priv ilegil ed eg sc ed affol scaffol d fo d r for Supplementary Materials: The following supporting information can be downloaded at: propro tein tein kinas kinas e inh e inh ibitors ibitors basb ed as on ed a on nov a nov el, robust el, robust micm rowave icrowave -assi -assi sted sted propro tocol tocol for fo thr e th Su- e Su- www.mdpi.com/xxx/s1. zukzu i re kaction i reaction of pyra of pyra zole zo ule rea us rea , which s, which allowed allowed quick quick to acces to acces s a stru s a stru cturall cturall y var y v iear d ili ebr d li ar br yar y of putative kinase inhibitors. Although this route is limited to the synthesis of biphenyl of putative kinase inhibitors. Although this route is limited to the synthesis of biphenyl Author Contributions: Conceptualization, L.D. and A.Z.; methodology, L.D., A.Z. and R.V.M.; val- pyra pyra zole zol ur eeas, ureas, it com it com pare pare s favorab s favorab ly w ly ith wprev ith prev ious ious litera litture erature in ter inms terms of yield of yield s an s d an ov d er- over- idation, L.D. and A.Z.; investigation, L.D., R.V.M., M.G., A.T. A.G. and A.Z.; resources, L.D. and all e all ase e of ase s of ynt sy hesis nthesis . . A.Z.; data curation, L.D. and A.Z.; writing—original draft, L.D. and A.Z.; writing—review & editing Preparation, L.D., R.V.M., M.G., A.T. and A.Z.; supervision, A.Z. All authors have read and agreed SupSu plement pplement ary ary Materials: Materials: The The follo fol wlo ing wing suppor suppor tingti ng inform inform ation ation can can be be downlo downlo adeade d at d : at: to the published version of the manuscript. www.mdpi. www.mdpi. com co /xxx/s1 m/xxx/s1 . . Funding: This research received no external funding. Author Author Contri Contri bution bution s: Con s: Con ceptu ceali ptu zatio alizatio n, Ln .D , L . and .D. and A.Z A .; .m Z.; ethodo methodo logy lo , gy L.D , L ., .A D..Z , A . and .Z. and R.V R .M .V .;. M va.; l- val- Institutional Review Board Statement: Not applicable. idation, L.D. and A.Z.; investigation, L.D., R.V.M., M.G., A.T. A.G. and A.Z.; resources, L.D. and idation, L.D. and A.Z.; investigation, L.D., R.V.M., M.G., A.T. A.G. and A.Z.; resources, L.D. and Informed Consent Statement: Not applicable. A.ZA .; .dat Z.; a dat cur a at cur ioat n,io Ln .D , L . an .Dd . an Ad .Z A .; .w Zriti .; w ng riti — ng original —original draft d,raft L.D , L . and .D. and A.Z A .; .w Zr .;it w ing rit— ing revi —r ew evi& ew editin & editin g g Preparation Preparation , L.D , L ., R .D..V , R .M .V ., M .M.., M G., A .G..T , A . an .Td . an Ad .Z A .; .s Z up .; s ervi upervi sion si , on A.Z. , A Al .Z. l Al auth l auth ors ors have have read read and aagr nd eed agr eed Conflicts of interest: The authors declare no conflict of interest. to the publishe to the publishe d vers d vers ion of ion of the th me anu manu script. script. Funding: Funding: This This rese rese arch rec arch rec eived eived no extern no extern al fu al fu nding nding . . InstInst itutional itutional Review Boar Review Boar d Statement: d Statement: No t ap Not ap plicab plile cab . le. Info Info rmed rmed Cons Co ent Statement nsent Statement : No : t No appli t appli cablcab e. le. Conflic Conflic ts of ts in of terest: interest: The The auth auth ors ors declare declare no co no nfli coct nfli of interest. ct of interest. Appl. Biosci. 2022, 1 71 Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/applbiosci1010004/s1. Author Contributions: Conceptualization, L.D. and A.Z.; methodology, L.D., A.Z. and R.V.M.; validation, L.D. and A.Z.; investigation, L.D., R.V.M., M.G., A.T., A.G. and A.Z.; resources, L.D. and A.Z.; data curation, L.D. and A.Z.; writing—original draft, L.D. and A.Z.; writing—review & editing Preparation, L.D., R.V.M., M.G., A.T. and A.Z.; supervision, A.Z. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Conflicts of Interest: The authors declare no conflict of interest. References 1. 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Journal

Applied Biosciences – Multidisciplinary Digital Publishing Institute

Published: May 31, 2022

Keywords: protein kinase inhibitors; pyrazole-ureas; microwave synthesis

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