Structure-based design and synthesis of 1H-pyrazolo[3,4-d]pyrimidin-4-amino derivatives as Janus kinase 3 inhibitors

Accepted Manuscript Structure-Based Design and Synthesis of 1H-pyrazolo[3,4-d]pyrimidin-4-amino Derivatives as Janus Kinase 3 Inhibitors Yuan Yin, Cheng-Juan Chen, Ru-Nan Yu, Zhi-Jian Wang, Tian-Tai Zhang, DaYong Zhang PII: DOI: Reference:

S0968-0896(18)30147-0 https://doi.org/10.1016/j.bmc.2018.04.005 BMC 14288

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Bioorganic & Medicinal Chemistry

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23 January 2018 30 March 2018 2 April 2018

Please cite this article as: Yin, Y., Chen, C-J., Yu, R-N., Wang, Z-J., Zhang, T-T., Zhang, D-Y., Structure-Based Design and Synthesis of 1H-pyrazolo[3,4-d]pyrimidin-4-amino Derivatives as Janus Kinase 3 Inhibitors, Bioorganic & Medicinal Chemistry (2018), doi: https://doi.org/10.1016/j.bmc.2018.04.005

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Structure-Based Design and Synthesis of 1Hpyrazolo[3,4-d]pyrimidin-4-amino Derivatives as Janus Kinase 3 Inhibitors

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Yuan Yina, Cheng-Juan Chenb, Ru-Nan Yua, Zhi-Jian Wanga, Tian-Tai Zhang b *, Da-Yong Zhang a * a Institute of Pharmaceutical Science, China Pharmaceutical University, Nanjing, P. R. China; b Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China.

Bioorganic & Medicinal Chemistry

Click and type the title of your paper, only capitalize first word Yuan Yina , Cheng-Juan Chenb , Ru-Nan Yua , Zhi-Jian Wanga , Tian-Tai Zhang b,  and Da-Yong Zhang a,  a b

Institute of Pharmaceutical Science, China Pharmaceutical University, Nanjing, P. R. China Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China

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ABSTRACT

Article history: Received Received in revised form Accepted Available online

Janus kinases (JAKs) regulate various inflammatory and immune responses and are targets for the treatment of inflammatory and immune diseases. Here we report the discovery and optimization of 1H-pyrazolo[3,4-d]pyrimidin-4-amino as covalent JAK3 inhibitors that exploit a unique cysteine (Cys909) residue in JAK3. Our optimization study gave compound 12a, which exhibited potent JAK3 inhibitory activity (IC50 of 6.2 nM) as well as excellent JAK kinase selectivity (>60 fold). In cellular assay, 12a exhibited potent immunomodulating effect on IL-2stimulated T cell proliferation (IC50 of 9.4 μM). Further, Compound 12a showed efficacy in delayed hypersensitivity assay. The data supports the further investigation of these compounds as novel JAKs inhibitors.

Keywords: Janus kinase covalent JAK3 inhibitors 1H-pyrazolo[3,4-d]pyrimidin-4-amino derivatives

1. Introduction The Janus kinases (JAKs) and their downstream effectors, signal transducer and activator of transcription proteins (STATs), form a critical cell signaling circuit, which control survival, proliferation, and differentiation of a variety of cells. 1 There are four JAK family members in mammals, JAK1, JAK2, JAK3, and tyrosine kinase 2 (TYK2), each of which can bind to distinct cytokines and/or growth factor receptors.2, 3 A notable feature of JAK signaling is the requirement for a dimer of JAK within the cytokine receptor complex, and cytokine receptors signaling through JAK1/JAK3, JAK1/JAK2, JAK1/TYK2, JAK2/TYK2, and JAK2/JAK2 have been described.4 JAK/STAT signaling is of fundamental importance in innate immunity, inflammation, and hematopoiesis, and dysregulation is frequently observed in immune disease and cancer. 5 The JAKs have been the subject of extensive drug discovery efforts. However, only several small molecule ATP-competitive inhibitors have entered clinical evaluation for autoimmune diseases as well as various cancers and myeloproliferative disorders, and resulting in just two FDA approvals to date (tofacitinib and ruxolitinib) (Figure 1).6 The high degree of structural conservation of the JAK ATP binding pockets has

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posed a considerable challenge to medicinal chemists seeking to develop highly selective inhibitors as pharmacological probes and as clinical drugs.7 Compared with other members of the JAK family, JAK3 has been pursued as a target for the treatment of autoimmune and inflammatory diseases. This is based on two facts. First, JAK3 is the only JAK family member with a restricted expression pattern and function, being localized in hematopoietic system. 3 Second, JAK3 was restricted to the gamma chain (γc) subfamily of cytokines. This protein associates exclusively with the γc chain, and in both humans and mice, JAK3 deficiency phenocopies γc deficiency, resulting in X-linked severe combined immunodeficiency (X-SCID) due to complete abrogation of IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21 signaling.8, 9 In addition, JAK3 has also been explored as a potential anticancer target; for example, JAK3 mutations have been found in T-cell acute lymphoblastic leukemia (T-ALL) and T-cell prolymphocytic leukemia (T-PLL).9, Herein, we report the design and synthesis of 1Hpyrazolo[3,4-d]pyrimidin-4-amino derivatives as potent JAK3 inhibitors that exploit an acrylamide electrophile to form a covalent bond to Cys909. A series of modifications at the 1- and

 Corresponding author. Tel.: +86-25-83271315(D. Zhang), +86-10-63035779 (T. Zhang); e-mail: [email protected] (D. Zhang), [email protected] (T. Zhang)

Figure 1. Representative JAK inhibitors.

Figure 2. Design of the target compounds.

3-positions of 1H-pyrazolo[3,4-d]pyrimidin-4-amino core utilizing structure-based drug design (SBDD) led to the discovery of 1-(3-(4-amino-3-(1-methyl-1H-pyrazol-4-yl)-1H-pyrazolo[3,4d]pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-one (12a) as a potent JAK3 inhibitor with potent immunomodulating effect on IL-2-stimulated T cell proliferation. The design hypothesis was later confirmed by docking calculations. Furthermore, Compound 12a showed efficacy in delayed hypersensitivity assay. 2. Design of 1H-pyrazolo[3,4-d]pyrimidin-4-amino derivatives The molecular structure of the JAK inhibitors is divided into four parts marked as A, B, C and D, Figure 2.11 The part A binding to the hinge region is generallycritical for kinase inhibitory activity, and modification of the scaffold moiety around hinge region may increase the affinity to JAK3. The part B and C could be the key to discover new lead structures. However, when the part A become bicyclic fused heteroaryl scaffold, resulting in the missing of the part C (e.g., pyrrolopyrimidine of tofacitinib). The tail D emerged mainly as amide and cyano group. By means of modification in part D was

important for interaction with the hydrophobic cavity and increase of JAK3 inhibitory activity. Compound 5 has excellent biological activities against JAK3, both in vitro and in vivo. According to the strategy of cyclization, we synthesized compound 7 by a simple method, however, which showed a weaker activities against JAK3 than tofacitinib. Based on the structure comparision between approved kinase inhibitor ibrutinib and compound 7, copmpound 8 was designed with the strategy of scaffold hopping. Furthermore, a series of 1Hpyrazolo[3,4-d]pyrimidin-4-amino derivatives was synthesised as JAK3 inhibitors. 3. Results and discussion 3.1. Chemistry A series of 4-amino-1H-pyrazolopyrimidin derivatives and their intermediates were synthesised according to the pathways described in Schemes 1-5. Synthesis of the 4-Amino-1H-pyrazolopyrimidin derivatives possessing piperidin-3-ylmethyl group, piperidin-4-ylmethyl group, 3-pyrrolidinyl group, 3-piperidinyl group, or 4-piperidinyl

Scheme 1. Reagents and conditions: (a) formamide, 210°C, 2 h; (b) DMF, NBS, 80 °C, 8 h; (c) 1,4-dioxane/H2O, Pd(PPh3)4, K2CO3, 1-methyl-4-(4,4,5,5tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole, 100 °C, 12 h; (d) THF, triphenylphosphine, DIAD, tert-butyl 3-(hydroxymethyl) piperidine-1-carboxylate/ tert-butyl 4-(hydroxymethyl) piperidine-1-carboxylate, 0 °C to rt, 12 h; (e) 4 N HCl in EtOAc, rt, 1 h; (f) THF, triphenylphosphine, DIAD, tert-butyl 3hydroxypyrrolidine-1-carboxylate, 0 °C to r.t, 12 h; (g) THF, triphenylphosphine, DIAD, tert-butyl 3-hydroxypiperidine-1-carboxylate/ tert-butyl 4hydroxypiperidine-1-carboxylate, 0 °C to rt, 12 h.

Scheme 2. Reagents and conditions: (a) MeCN, 1-bromo-2-methoxyethane, K2CO3, KI, 82 °C, overnight; (b) THF, 2-isopropoxy-4,4,5,5tetramethyl[1,3,2]dioxaborolane, i-PrMgCl, 0 °C to r.t, 2.5 h; (c) 1,4-dioxane/H2O, Pd(PPh3)4, K2CO3, 100 °C, 12 h; (d) THF, triphenylphosphine, DIAD, tertbutyl 3-hydroxypiperidine-1-carboxylate, 0 °C to r.t, 12 h; (e) 4 N HCl in EtOAc, rt, 1 h.

Scheme 3. Reagents and conditions. Method A: (a) acryloyl chloride, DIEA, DCM, -5 °C, 30 min. Method B: RCOOH, HATU, DIEA, DMF, 0 °C-r.t, 16 h. Method C: BrCN, KOAc, MeOH, 0 °C-r.t, 16 h.

Scheme 4. Reagents and conditions: acryloyl chloride, DIEA, DCM, -5 °C, 30 min.

Scheme 5. Reagents and conditions. Method A: (a) acryloyl chloride, DIEA, DCM, -5 °C, 30 min. Method B: RCOOH, HATU, DIEA, DMF, 0 °C-r.t, 16 h. Method C: BrCN, KOAc, MeOH, 0 °C-r.t, 16 h.

group at the 1-position is described in Scheme 1 and 2. Commercially available 1H-pyrazolo[3,4-d]pyrimidin-4-amine (S1), which was readily prepared from 3-amino-4-cyanopyrazole, was reacted with N,N-dimethylformamide (DMF) to afford bromide S2. As shown in Scheme 2, 4-iodo-1H-pyrazole was treated with 1-bromo-2-methoxyethane to give the pyrazoles S9. Halogen-magnesium exchange of S9 followed by addition of borate afforded the pyrazole pinacol borate S10 in good yield. Then Suzuki couplings of S2 to a variety of pyrazole pinacol borate provide intermediate S3 in moderate to good yield (75−80%). The key intermediates (S4-S8, S12) were synthesized from S3 or S11 via a Mitsunobu parallel medicinal chemistry or singleton chemical synthesis. Subsequent Boc deprotection using 4 N HCl in EA cleanly provided the pyrazolopyrimidin HCl salt in nearly quantitative yield. As indicated in Schemes 3-5, our key steps in the synthesis of 3-(pyrazol-4-yl)-1H-pyrazolo[3,4d]pyrimidin-4-amine derivatives, involved three kind of reactions. Method A shows synthesis of the derivatives having acrylamide in N1-position of the pyrrolidin or the piperidine. Intermediates S4-S8 were reacted with acryloyl chloride to give compounds 9a-9b, 10a-10b, 11a-11b and 12a-12b under alkaline condition. Method B shows synthesis of the derivatives having cyanoacetamide or N,N-dimethylacetamide in N1-position of the pyrrolidin or the piperidine. Amide couplings using standard

HATU conditions provided compounds in good yield. Compounds 9g-9h, 10g-10h, 12g-12h and 13g-13h were synthesized as shown in Method C. Treatment of S4-S8 with cyanogen bromide (BrCN) gave the targets 9g-9h, 10g-10h, 12g12h and 13g-13h. 3.1. In vitro enzymatic inhibitory activities The newly synthesized 1H-pyrazolo[3,4-d]pyrimidin-4-amine derivatives (9a-13h) were tested in enzymatic assays measuring JAK kinase activity. The preliminary in vitro screening indicated that some derivatives showed good JAK3 inhibitory activity and selectivity compared with the positive controls. The IC 50 values in JAK enzyme assays are summarized in Tables 1 and 2. Among the 1-(piperidin-3-ylmethyl)-1H-pyrazolo[3,4d]pyrimidin-4-amine series, most of the target compounds exhibited stronger JAK3 inhibitory activity than JAK1-2 and TYK2 with selectivity varied 1.4 to 206.5-fold. While substituent variation at R1 position appeared tolerant with less than 1.9-fold differences on potency (9a, 9b), diversification at R2 position could significantly affect the inhibitory activity and JAK enzyme selectivity. Acrylamide (9a) was replaced with a cyanoacetamide (9c), N,N-dimethylacetamide (9e) or cyano (9g); as a result, a significant decrease in JAK3 inhibitory activity (4 to 31 fold) and

Table 1. SARs of 1-(piperidin-3-ylmethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine derivatives.

IC50 a (nM) Compd

R1

R2

7 8

-

-

9a

CH3

19.2

9b

CH2CH2OCH3

36.3

9c

CH3

56.1

9d

CH2CH2OCH3

87.4

9e

CH3

591.2

9f

CH2CH2OCH3

9g

JAK1 [JAK1/JAK3] NDb NDb 1946.9 [101.4] 1674.7 [46.1] 146.8 [2.6] 496.8 [8.6] 2291.5 [3.9]

JAK2 [JAK2/JAK3] NDb NDb 808.3 [42.1] 1420.6 [39.1] 78.4 [1.4] 696.8 [12.1] 1947.0 [3.3]

TYK2 [TYK2/JAK3] NDb NDb 3964.8 [206.5] 3296.4 [90.8] 2352.1 [41.9]

787.5

NDb

NDb

NDb

CH3

189.3

NDb

NDb

9h

CH2CH2OCH3

143.2

NDb

10a

CH3

197.2

NDb

10b

CH2CH2OCH3

324.3

NDb

10c

CH3

278.4

NDb

867.3 [4.6] 1397.6 [9.8] 469.3 [2.4] 497.4 [1.5] NDb

10d

CH2CH2OCH3

315.3

NDb

NDb

NDb

10e

CH3

>1000

NDb

NDb

NDb

10f

CH2CH2OCH3

>1000

NDb

NDb

NDb

>1000

b

b

NDb

b

NDb

CH3

10g 10h

CH2CH2OCH3

Tofacitinib C a

Values are means of three experiments.

b

ND = not determined.

c

Tofacitinib, positive control.

JAK3 267.4 154.2

>1000 2.1

ND

b

ND 1.6 [0.8]

ND

ND 4.3 [3.6]

>3000 NDb

NDb NDb NDb NDb

35.5

Table 2. SARs of 1-(piperidin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine derivatives.

IC50 a (nM) Compd

R1

R2

11a

CH3

9.9

11b

CH2CH2OCH3

15.6

12a

CH3

6.2

12b

CH2CH2OCH3

21.3

12c

CH3

32.3

12d

CH2CH2OCH3

57.7

12e

CH3

582.6

12f

CH2CH2OCH3

12g

JAK1 [JAK1/JAK3] 694.0 [70.1] 1067.0 [68.4] 618.1 [99.7] 1354.7 [63.6] 342.4 [10.6] 496.8 [8.6] 1372.5 [2.4]

JAK2 [JAK2/JAK3] 499.0 [50.4] 508.6 [32.6] 396.2 [63.9] 590.0 [27.7] 590.0 [6.7] 696.8 [12.1] 1932.5 [3.3]

TYK2 [TYK2/JAK3] 1419.7 [143.4] 1722.2 [110.4] 1175.5 [189.6] 2117.2 [99.4] 2427.4 [75.1] 3219.4 [55.8]

756.3

NDb

NDb

NDb

CH3

115.4

NDb

NDb

12h

CH2CH2OCH3

144.2

NDb

13c

CH3

394.8

NDb

13d

CH2CH2OCH3

373.7

NDb

168.4 [1.5] 197.4 [1.4] 281.7 [0.7] 367.2 [1.0]

13e

CH3

>1000

NDb

>1000

NDb

13f

CH2CH2OCH3

>1000

CH3

94.3

13h

CH2CH2OCH3

124.4

NDb 103.5 [1.1] 146.8 [1.2] 4.3 [3.6]

NDb

13g

NDb 165.4 [1.8] 112.3 [0.9] 1.6 [0.8]

Tofacitinib C a

Values are means of three experiments.

b

ND = not determined.

c

Tofacitinib, positive control.

JAK3

2.1

JAK3 selectivity was observed. The replacement of acrylamide (9a, 9b) with cyanoacetamide (9c, 9d) led to a dramatic loss of potency on JAK3 and loss of selectivity, which is consistent with the reported SAR of compound 4.3 The piperidine-3-yl (9a) showed good JAK3 inhibitory activity with an IC50 of 19.2 nM. The scaffold hopping of the piperidine-3-yl with piperidine-4-yl (10a) also resulted in a 10-fold decrease in potency and loss of selectivity. Among this series, compound 9a (IC50 = 19.2 nM) displayed an almost 4-fold reduction in JAK3 potency compared to tofacitinib, along with 101.4-fold JAK1/ 3, 42.1-fold JAK2/ 3 and 206.5-fold TYK2/ JAK3 selectivity. As for 1-(piperidin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-4amine series, similar SAR was concluded at the preliminary stage (Table 2). The piperidine-3-yl (12a) with pyrrolidine-3-yl (11a) resulted in a similar enzyme potency. Compound 12a was well potent on JAK3 (IC50 = 6.2 nM) and showed an unprecedented selectivity within the JAK. The selectivity for JAK3 seems to be general as indicated by compounds 9a-9b and 11a-11b, which were also most active on JAK3. To examine the selectivity of compounds 9a-9b, 11a-11b and 12a-12b on JAK3 and BTK, these compounds were screened

NDb

NDb NDb NDb

NDb NDb 35.5

against BTK. As shown in Table 3, compound 12a exhibited well selectivity versus BTK (IC50 = 180.3 nM, 29.1-fold). These data suggest that compound 12a may be a selective target JAK3 kinase inhibitor. The docking study of the related 1H-pyrazolo[3,4d]pyrimidin-4-amine 12a with the JAK3 kinase domain furnished a rationale for the selectivity of compounds 9a-9b, 11a-11b and 12a-12b within the JAK kinase family (Figure 3, see Table 2 for activity of 12a in enzymatic JAK assays). The pyrazolopyrimidin ring binds to the hinge part of the kinase through two hydrogen bonds with adjacent backbone amines (Glu903, Leu905). The 1methylpyrazole group optimally fits into a small pocket delimited by the side chains of Val836, Lys855, Val844, Met902 and Gly969. Most important for the observed selectivity is a covalently bind between the acrylamide warhead and Cys909. The unique residue Cys909 in human JAK3 have been successfully targeted by covalent JAK kinase inhibitors. 9 In this pyrazolopyrimidin series, 12a was found to display well potent on JAK3 and showed an unprecedented JAK3 selectivity than positive control tofacitinib. Based on the above SAR, we proposed the modification of R1 group could improve the potency

and selectivity of 1H-pyrazolo[3,4-d]pyrimidin-4-amine derivatives. Subsequent structure optimization is in progress.

12a further showed the excellent antiproliferative activity of the T cell lines with IC50 value of 9.4 μM, which was nearly 7.8-fold reduction active than tofacitinib. 3.3. Delayed-type hypersensitivity Having identified leading compound 12a showing excellent JAK3 inhibitory activity, we conducted a delayed hypersensitivity test. To our delight, compound 12a also exhibited potent inhibitory activity in delayed hypersensitivity assay (Figure 4). Moreover, compound 12a showed a lack of influence on hematological parameters (Figure 5). 4. Conclusion

Figure 3. Compound 12a bound to the active site of JAK3. Compound 12a is shown with yellow carbon atoms while the amino acid is colored in white. Green dotted lines represent intermolecular hydrogen bonds.

Table 3. BTK enzymatic and cellular assay of 1Hpyrazolo[3,4-d]pyrimidin-4-amine derivatives. Compd

BTK IC50 a (nM)

T cell proliferation IC50b (μM)

9a

481.2

97.3

9b

420.4

126.1

11a

203.1

16.2

11b

126.4

72.4

12a

180.3

9.4

12b

159.0

77.3

We described the design, synthesis and biological screening of two novel series of 1-(piperidin-3-ylmethyl)-1H-pyrazolo[3,4d]pyrimidin-4-amine and 1-(piperidin-3-yl)-1H-pyrazolo[3,4d]pyrimidin-4-amine derivatives as JAK3 inhibitors. Primary SAR studies resulted in the discovery of a novel class of 1(piperidin-1-yl)prop-2-en-1-one based JAK3 inhibitors with well potency (IC50 = 6.2 nM) and higher selectivity (>60 fold) to other JAK kinase than tofacitinib. Further T-cell proliferation assay showed compound 12a could inhibit the T-cell proliferation effectively. Our further effort will be devoted on exploiting the comprehensive SARs, design of more highly efficient and selective agents with satisfactory druggability and safety profiles. 5. Experimental 5.1. General synthesis 1

Tofacitinib

1.2

a

Values are means of three experiments. b Inhibitory effect on IL-2-stimulated T cell proliferation using spleen cells.

3.2. Rat T cell proliferation Due to its high kinase selectivity, we selected compounds 9a9b, 11a-11b and 12a-12b for further evaluation. In the cellular assay of IL-2-stimulated T cell proliferation, less potent inhibition was exerted by 9a (IC50 =97.3 μM) and 11a (IC50 =16.2 μM) compared to 12a despite retaining good JAK3 inhibitory activity. Compounds 9b, 11b and 12b tested were demonstrated low potency for the T cell lines. The most potent

H-NMR and 13C-NMR spectra were recorded with Bruker ARX-300 spectrometer with TMS as the internal standard in deuterium chloroform (CDCl3), deuterium methanol (MeOD) or deuterium dimethyl sulfoxide (DMSO-d6). HR-ESI-MS was run on a Bruker micro-TOF-Q mass spectrometer. Most chemicals were purchased from Sigma–Aldrich and Fluka. All commercially available reagents were used without further purification. The solvents used were all AR grade and were redistilled under positive pressure of dry nitrogen atmosphere in the presence of proper desiccant when necessary. Silica gel (200300 mesh, Qingdao city, China) was used for column chromatography. The progress of the reactions was monitored by analytical thin-layer chromatography (TLC) on HSGF254 precoated silica gel plates. 5 . 1 . 1 . 1H-pyrazolo[3,4-d]pyrimidin-4-amine (S1) A solution of 3-amino-4-cyanopyrazole (9.8 g, 0.09 mol) and formamide (100 mL, 2.5 mol) was stirred at 210 °C for 2 h. After cooling to room temperature, water(200 mL) was added and the obtained solid was filtered. The crude product was suspended in hot water (100 mL) and conc HCl (5 mL), then charcoal (10 g) was added and the mixture was boiled for 15 min. After charcoal

Figure 4. Delayed-type hypersensitivity assay of compound 12a. Compound 12a was orally administered at 100 mg/kg ( ), twice a day.

Figure 5. Analysis of hematologic parameters in the mouse DTH experiment.

filtration, sodium hydroxide (2 N) was added (pH= 8) and the precipitated solid was filtered, giving S1 as a white solid (10 g, 82%); 1H NMR (300 MHz, DMSO-d6) δ 13.35 (s, 1H), 8.13 (s, 1H), 8.07 (s, 1H), 7.62 (s, 2H). 5.1.2. 3-Bromo-1H-pyrazolo[3,4-d]pyrimidin-4-amine (S2) The mixture of S1 (10 g, 74 mmol) and N-bromosuccinimide (15.8 g, 89 mmol) in DMF (100 mL) was stirred at 80 °C for 8 h. The resulting mixture was allowed to cool to room temperature and then diluted with 300 mL of water. The precipitate was filtered and washed with 2 × 60 mL of saturated aqueous sodium sulfite, 2 × 100 mL of water, respectively, and dried under vacuum to yield S2 as a light-yellow solid (10.2 g, 64.4%), which was used directly without further purification. 1H NMR (300 MHz, DMSO-d6) δ 13.78 (s, 1H), 8.19 (s, 1H), 6.89 (s, 2H). 5.1.3. 3-(1-methyl-1H-pyrazol-4-yl)-1H-pyrazolo[3,4d]pyrimidin-4-amine (S3) To a mixture of S2 (10 g, 47.0 mmol) and 1,4-dioxane/H2O (150 mL, v/v, 5/1) was added 1-methyl-4-(4,4,5,5-tetramethyl1,3,2-dioxaborolan-2-yl)-1H-pyrazole (14.7 g, 70.4 mmol), K2CO3 (13.0 g, 93.9 mmol), and Pd(PPh 3)4 (2.7 g, 2.3 mmol). The reaction mixture was placed into an oil bath preheated to 100 °C, with stirring at this temperature for 12 h under argon. TLC showed the completion of the reactin. The reaction mixture was filtered through a Celite bed, and the filtrate was concentrated and purified by silica gel column chromatography (eluting with 0-10% MeOH in DCM) to afford S3 as a white solid (8.0 g, 79%). MS (ESI) m/z: 216.1 [M + H]+.

General procedure for the preparation of intermediates S4, S5, S6, S7, and S8. 5.1.4. 3-(1-methyl-1H-pyrazol-4-yl)-1-(piperidin-3-ylmethyl)1H-pyrazolo[3,4-d] pyrimidin-4-amine (S4) To a mixture of triphenylphosphine (18.29 g, 69.7 mmol), tertbutyl 3-(hydroxymethyl) piperidine-1-carboxylate (10.1 g, 46.7 mmol) and S3 (5.0 g, 23.2 mmol) in THF (300 mL) was added diisopropyl azodicarboxylate (14.1 mL, 69.7 mmol) at 0 °C. The reaction mixture was stirred at this temperature for 0.5 h under argon. After that, the reaction mixture was allowed to warm to room temperature with stirring for 12 h. The resulting mixture was then concentrated to afford the crude product, which was purified by silica gel column chromatography (eluting with 010% MeOH in DCM) to provide the desired Boc-protected monoamine as a white solid (7.3 g, 76%). Then to the solid in EtOAc (10 mL) was added 4 N HCl in EtOAc (30 mL). The reaction mixture was stirred at room temperature for 1 h. After complete conversion of the starting material, excess EtOAc was removed under vacuum. The residue was diluted in EtOAc and water. The water layer was basified with 2 N NaHCO3 solution and extracted with EtOAc (2 × 100 mL). The organic layers were then washed with water followed by brine. The organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated to provide S4 as a white solid (4.9 g, 89%) without further purification. MS (ESI) m/z: 313.2 [M + H]+. 5.1.5. 3-(1-methyl-1H-pyrazol-4-yl)-1-(piperidin-4-ylmethyl)1H-pyrazolo[3,4-d]pyrimidin-4-amine (S5) Synthesis was according to intermediate S4. Yield 68%. MS (ESI) m/z: 313.2 [M + H]+.

5.1.6. 3-(1-methyl-1H-pyrazol-4-yl)-1-(pyrrolidin-3-yl)-1Hpyrazolo[3,4-d]pyrim- idin-4-amine (S6) Synthesis was according to intermediate S4. Yield 73%. MS (ESI) m/z: 285.2 [M + H]+. 5.1.7. 3-(1-methyl-1H-pyrazol-4-yl)-1-(piperidin-3-yl)-1Hpyrazolo[3,4-d]pyram-iddddin-4-amine (S7) Synthesis was according to intermediate S4. Yield 71%. MS (ESI) m/z: 299.2 [M + H]+. 5.1.8. 3-(1-methyl-1H-pyrazol-4-yl)-1-(piperidin-4-yl)-1Hpyrazolo[3,4-d]pyramidin-4-amine (S8) Synthesis was according to intermediate S4. Yield 70%. MS (ESI) m/z: 299.2 [M + H]+. 5.1.9. 4-iodo-1-(2-methoxyethyl)-1H-pyrazole (S9) To a solutin of 4-iodo-1H-pyrazole (10 g, 51.6 mmol), K2CO3 (10.7 g, 77.3 mmol) and KI (1.7 g, 10.3 mmol) in MeCN (130 mL) was added 1-bromo-2-methoxyethane (8.6 g, 61.9 mmol) at room temperature. The reaction mixture was stirred overnight at 82 °C. TLC showed the completion of the reactin. The resulting mixture was removed under vacuum. Water (100 mL) was added to the residue, and the resultant mixture was extracted with EtOAc (2 × 100 mL). The organic layers were then washed with water followed by brine. The organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated to provide S9 as a yellow oil (12.7 g, 98%) without further purification. 1H NMR (300 MHz, DMSO-d6) δ 7.87 (s, 1H), 7.52 (s, 1H), 4.26 (t, J = 6 Hz, 2H), 3.65 (t, J = 6 Hz, 2H), 3.21 (s, 3H). 5.1.10. 1-(2-methoxyethyl)-4-(4,4,5,5-tetramethyl-1,3,2dioxaborolan-2-yl)-1H-pyrazole (S10) To a solution of i-PrMgCl (2 M in THF, 10 mL, 20 mmol) at 0 °C was added slowly a solution of S9 (2.51 g, 10 mmol) in THF (100 mL). The resulting solution was stirred at this temperature under Ar for 1 h. 2-isopropoxy-4,4,5,5tetramethyl[1,3,2]dioxaborolane (2.8 g, 15 mmol) was then added slowly to maintain the temperature below 0 °C. After the addition, the reaction was stirred at room temperature for 1.5 h and then quenched slowly by the addition of aqueous ammonium chloride solution (80 mL). To the mixture was added H 2O (80 mL), and extraction was done twice with EtOAc (100 mL). The organic layer was washed with brine, dried over anhydrous sodium sulfate, and the solvent was concentrated to provide S10 as a pale yellow oil. MS (ESI) m/z: 253.2 [M + H] +. 5.1.11. 3-(1-(2-methoxyethyl)-1H-pyrazol-4-yl)-1Hpyrazolo[3,4-d]pyrimidin-4-amine (S11) S11 was prepared in a manner similar to that described for S3 in 77% yield as a white solid. MS (ESI) m/z: 260.2 [M + H] +. 5.1.12. 3-(1-(2-methoxyethyl)-1H-pyrazol-4-yl)-1-(pyrrolidin3-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (S12) S12 was prepared in a manner similar to that described for S4 in 83% yield as a white solid. MS (ESI) m/z: 343.2 [M + H] +. 5.2. Chemistry Method A: General procedure for the synthesis of targets 8, 9a-9b, 10a-10b, 11a-11b and 12a-12b. 5.2.1. 1-(3-((4-amino-3-(1-methyl-1H-pyrazol-4-yl)-1Hpyrazolo[3,4-d]pyrimidin-1-yl)methyl)piperidin-1-yl)prop-2en-1-one (9a) To a solution of S4 (25 mg, 0.08 mmol) in DCM (5 mL) were added DIEA(11.8 mg, 0.05 mmol) and acryloyl chloride (8.2 mg, 0.08 mmol) at -5 °C. The resulting mixture was stirred for 30 min. Then it was quenched by MeOH (2 mL), concentrated, and purified by silica gel column chromatography (0−10% MeOH in

DCM) to afford the title compound 9a (24.6 mg, 84%) as a white solid. 1H NMR (300 MHz, CDCl3) δ 8.28 (s, 1H), 7.78 (s, 1H), 7.75 (s, 1H), 6.57- 6.36 (m, 1H), 6.20- 6.13 (m, 1H), 5.63- 5.52 (m, 1H), 4.42- 4.34 (m, 1H), 4.28 (d, J = 6 Hz, 2H), 3.97 (s, 3H), 3.84- 3.80 (m, 1H), 3.09- 2.95 (m, 1H), 2.84- 2.63 (m, 1H), 2.26 (m, 1H), 1.80- 1.76 (m, 2H), 1.48- 1.39 (m, 1H), 1.36- 1.28 (m, 1H); 13C NMR (75 MHz, CDCl3) δ 165.58, 158.24, 155.76, 154.48, 138.38, 136.80, 129.85, 129.57, 127.86, 114.63, 98.72, 52.55, 49.71, 49.48, 46.59, 45.75, 33.84, 25.23; HR-ESIMS: m/z 367.19957 [M+H]+ (Calcd for C18H22N8O, 366.1917). 5.2.2. 1-(3-((4-amino-3-(1-(2-methoxyethyl)-1H-pyrazol-4-yl)1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)piperidin-1yl)prop-2-en-1-one (9b) The procedure outlined in method A was followed to afford the desired compound with 80% yield. 1H NMR (300 MHz, CDCl3) δ 8.24 (s, 1H), 7.83 (s, 1H), 7.78 (s, 1H), 6.55- 6.35 (m, 1H), 6.17- 6.10 (m, 1H), 5.60- 5.49 (m, 1H), 4.39 (m, 1H), 4.33 (m, 2H), 4.26 (d, J = 6 Hz, 2H), 3.82- 3.77 (m, 1H), 3.73 (t, J = 6 Hz, 2H), 3.30 (s, 3H), 3.06- 2.93 (m, 1H), 2.83- 2.62 (m, 1H), 2.24 (m, 1H), 1.76- 1.72 (m, 2H), 1.46- 1.33 (m, 1H), 1.29- 1.19 (m, 1H); 13C NMR (75 MHz, CDCl3) δ 165.62, 158.29, 155.79, 154.31, 138.46, 136.81, 129.86, 129.76, 127.81, 114.31, 98.56, 70.89, 58.97, 52.45, 49.62, 49.36, 46.48, 45.74, 33.75, 25.18; HR-ESIMS: m/z 411.22474 [M+H]+ (Calcd for C20H26N8O2, 410.2179). 5.2.3. 1-(4-((4-amino-3-(1-methyl-1H-pyrazol-4-yl)-1Hpyrazolo[3,4-d]pyrimidin-1-yl)methyl)piperidin-1-yl)prop-2en-1-one (10a) The procedure outlined in method A was followed to afford the desired compound with 82% yield. 1H NMR (300 MHz, CDCl3) δ 8.23 (s, 1H), 7.74 (s, 1H), 7.70 (s, 1H), 6.47 (dd, J = 10.5 Hz, 16.8 Hz, 1H), 6.14 (dd, J = 1.5 Hz, 16.8 Hz, 1H), 5.56 (dd, J = 1.5 Hz, 10.5 Hz, 1H), 4.58- 4.53 (m, 1H), 4.22 (d, J = 9 Hz, 2H), 3.92 (s, 3H), 2.97- 2.89 (m, 1H), 2.58- 2.50 (m, 1H), 2.24 (m, 1H), 1.62- 1.57 (m, 2H), 1.30- 1.20 (m, 2H); 13C NMR (75 MHz, CDCl3) δ 165.36, 158.33, 155.78, 154.32, 138.28, 136.53, 129.51, 127.77, 127.45, 114.50, 98.55, 51.73, 45.42, 41.74, 39.24, 36.73, 30.32, 29.24; MS (ESI) m/z: 367.2 [M + H]+. 5.2.4. 1-(4-((4-amino-3-(1-(2-methoxyethyl)-1H-pyrazol-4-yl)1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)piperidin-1yl)prop-2-en-1-one (10b) The procedure outlined in method A was followed to afford the desired compound with 80% yield. 1H NMR (300 MHz, CDCl3) δ 8.23 (s, 1H), 7.81 (s, 1H), 7.77 (s, 1H), 6.48 (dd, J = 9 Hz, 16.5 Hz, 1H), 6.15 (dd, J = 1.5 Hz, 16.5 Hz, 1H), 5.57 (dd, J = 1.5 Hz, 9 Hz, 1H), 4.59- 4.55 (m, 1H), 4.32 (t, J = 6 Hz, 2H), 4.22 (d, J = 9 Hz, 2H), 3.93- 3.89 (m, 1H), 3.71 (t, J = 6 Hz, 2H), 3.29 (s, 3H), 2.98- 2.90 (m, 1H), 2.59- 2.51 (m, 1H), 2.25 (m, 1H), 1.63- 1.57 (m, 2H), 1.30- 1.21 (m, 1H); 13C NMR (75 MHz, CDCl3) δ 165.38, 158.32, 155.74, 154.26, 138.43, 136.69, 129.74, 127.80, 127.45, 114.28, 98.59, 70.84, 58.93, 52.42, 51.74, 45.56, 41.76, 36.75, 30.34, 29.26; MS (ESI) m/z: 411.2 [M + H]+. 5.2.5. 1-(3-(4-amino-3-(1-methyl-1H-pyrazol-4-yl)-1Hpyrazolo[3,4-d]pyrimidin-1-yl)pyrrolidin-1-yl)prop-2-en-1one (11a) The procedure outlined in method A was followed to afford the desired compound with 79% yield. 1H NMR (300 MHz, DMSO-d6) δ 8.25 (s, 1H), 8.09 (s, 1H), 7.74 (s, 1H), 6.71- 6.54 (m, 1H), 6.21- 6.14 (m, 1H), 5.73- 5.64 (m, 1H), 5.55- 5.43 (m, 1H), 4.15- 4.09 (m, 1H), 3.93 (s, 3H), 3.85- 3.72 (m, 2H), 3.633.54 (m, 1H), 3.42 (s, 1H), 2.49- 2.34 (m, 1H); 13C NMR (75 MHz, DMSO-d6) δ 163.42, 158.29, 155.74, 153.90, 137.74,

136.85, 130.34, 129.55, 127.05, 113.65, 97.87, 55.16, 53.69, 50.32, 45.12, 38.79; HR-ESIMS: m/z 339.16792 [M+H]+ (Calcd for C16H18N8O, 338.1604). 5.2.6. 1-(3-(4-amino-3-(1-(2-methoxyethyl)-1H-pyrazol-4-yl)1H-pyrazolo[3,4-d]pyrimidin-1-yl)pyrrolidin-1-yl)prop-2-en1-one (11b) The procedure outlined in method A was followed to afford the desired compound with 79% yield. 1H NMR (300 MHz, DMSO-d6) δ 8.13 (s, 1H), 7.97 (s, 1H), 7.62 (s, 1H), 6.59- 6.42 (m, 1H), 6.09- 6.01 (m, 1H), 5.61- 5.52 (m, 1H), 5.43- 5.31 (m, 1H), 4.37 (t, J = 6 Hz, 2H), 4.03- 3.97 (m, 1H), 3.75 (t, J = 6 Hz, 2H), 3.72- 3.60 (m, 1H), 3.51- 3.42 (m, 1H), 3.30 (m, 1H), 3.22 (s, 3H), 2.37- 2.30 (m, 1H) , 2.29- 2.22 (m, 1H); 13C NMR (75 MHz, DMSO-d6) δ 163.49, 158.36, 155.81, 153.97, 137.81, 136.92, 130.41, 129.62, 127.12, 113.72, 97.94, 71.10, 58.53, 55.23, 53.16, 50.39, 50.14, 38.86; MS (ESI) m/z: 383.2 [M + H]+. 5.2.7. 1-(3-(4-amino-3-(1-methyl-1H-pyrazol-4-yl)-1Hpyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-one (12a) The procedure outlined in method A was followed to afford the desired compound with 76% yield. 1H NMR (300 MHz, MeOD) δ 8.29 (s, 1H), 8.05 (s, 1H), 7.87 (s, 1H), 6.91- 6.67 (m, 1H), 6.28- 6.17 (m, 1H), 5.86- 5.68 (m, 1H), 4.35- 4.22 (m, 1H), 4.06 (s, 3H), 3.93- 3.90 (m, 1H), 3.81- 3.70 (m, 1H), 3.55- 3.48 (m, 1H), 3.33- 3.25 (m, 1H), 2.40- 2.36 (m, 1H), 2.27- 2.23 (m, 1H), 2.14- 2.11 (m, 1H), 1.78- 1.76 (m, 1H); 13C NMR (75 MHz, CDCl3) δ 165.58, 158.24, 155.76, 154.48, 138.38, 136.80, 129.85, 129.53, 127.86, 114.63, 98.72, 52.55, 49.71, 46.59, 45.75, 33.84, 25.23; HR-ESIMS: m/z 353.18396 [M+H]+ (Calcd for C17H20N8O, 352.1760). 5.2.8. 1-(3-(4-amino-3-(1-(2-methoxyethyl)-1H-pyrazol-4-yl)1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1one (12b) The procedure outlined in method A was followed to afford the desired compound with 78% yield. 1H NMR (300 MHz, MeOD) δ 8.29 (s, 1H), 8.10 (s, 1H), 7.89 (s, 1H), 6.87 (m, 1H), 6.29- 6.23 (m, 1H), 5.89- 5.72 (m, 1H), 4.47 (t, J = 6 Hz, 2H), 4.23 (m, 1H), 3.86 (t, J = 6 Hz, 2H), 3.81- 3.75 (m, 2H), 3.41 (m, 1H), 3.37 (s, 3H), 3.30- 3.25 (m, 1H), 2.41 (m, 1H), 2.25 (m, 1H), 2.10 (m, 1H), 1.76 (m, 1H); 13C NMR (75 MHz, CDCl3) δ 165.80, 158.24, 155.69, 153.78, 138.55, 136.85, 130.27, 129.88, 127.61, 114.34, 98.40, 70.90, 58.99, 52.48, 49.98, 47.90, 47.77, 33.21, 25.30; HR-ESIMS: m/z 397.20798 [M+H]+ (Calcd for C19H24N8O2, 396.2022). 5.2.9. N-(3-((4-amino-3-(1-(2-methoxyethyl)-1H-pyrazol-4-yl)1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)phenyl)acrylamide (8) The procedure outlined in method A was followed to afford the desired compound with 80% yield. 1H NMR (300 MHz, MeOD) δ 8.32 (s, 1H), 8.10 (s, 1H), 7.90 (s, 1H), 7.50 (s, 1H), 7.30 (s, 1H), 7.13- 7.10 (m, 2H), 6.69- 6.67 (m, 3H), 5.52 (m, 2H), 4.47 (t, J = 6 Hz, 2H), 3.86 (t, J = 6 Hz, 2H), 3.38 (s, 3H); MS (ESI) m/z: 419.2 [M + H]+. Method B: General procedure for the synthesis of targets 9c-9f, 10c-10f, 12c-12f and 13c-13f. 5.2.10. 3-(3-((4-amino-3-(1-methyl-1H-pyrazol-4-yl)-1Hpyrazolo[3,4-d]pyrimidin-1-yl)methyl)piperidin-1-yl)-3oxopropanenitrile (9c) To a stirred solution of S4 (25 mg, 0.08 mmol) in DMF (4 mL) was added DIEA (10.4 mg, 0.08 mmol), HATU (76.13 mg, 0.2 mmol), and 2-cyanoacetic acid (8.2 mg, 0.1 mmol) at 0 °C. The reaction mixture was stirred at room temperature for 16 h. It

was quenched with water and extracted with ethyl acetate (3 × 30 mL). The organic layer was washed with water (2 × 10 mL) followed by brine (1 × 10 mL), dried over anhydrous sodium sulfate, and was evaporated to dryness. The crude mass was purified by silica gel column chromatography (0−10% MeOH in DCM) to afford compound 9c (22.5 mg, 74%) as a white solid. 1 H NMR (300 MHz, CDCl3) δ 8.32 (s, 1H), 7.80 (s, 1H), 7.77 (s, 1H), 4.32 (d, J = 9 Hz, 2H), 4.00 (s, 3H), 3.67- 3.61 (m, 1H), 3.47 (m, 2H), 3.28- 3.22 (m, 1H), 3.19- 3.10 (m, 1H), 2.73- 2.65 (m, 1H), 2.36- 2.27 (m, 1H), 1.86- 1.74 (m, 2H), 1.57- 1.51 (m, 2H); 13C NMR (75 MHz, CDCl3) δ 160.57, 158.23, 156.00, 154.66, 138.43, 135.61, 129.76, 119.26, 114.42, 98.81, 53.54, 49.68, 47.24, 46.27, 43.33, 36.81, 29.80, 25.10; MS (ESI) m/z: 380.2 [M + H]+. 5.2.11. 3-(3-((4-amino-3-(1-(2-methoxyethyl)-1H-pyrazol-4yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)piperidin-1-yl)3-oxopropanenitrile (9d) The procedure outlined in method B was followed to afford the desired compound with 78% yield. 1H NMR (300 MHz, CDCl3) δ 8.33 (s, 1H), 7.88 (s, 1H), 7.82 (s, 1H), 4.38 (t, J = 6 Hz, 2H), 4.32 (t, J = 9 Hz, 2H), 3.78 (t, J = 6 Hz, 2H), 3.59- 3.53 (m, 1H), 3.47 (m, 2H), 3.36 (s, 3H), 3.21- 3.14 (m, 2H), 2.752.67 (m, 1H), 2.36- 2.31 (m, 1H), 1.91- 1.81 (m, 2H), 1.58- 1.50 (m, 2H); 13C NMR (75 MHz, CDCl3) δ 160.66, 158.24, 156.06, 154.52, 138.45, 135.50, 129.96, 119.98, 114.18, 98.90, 70.96, 59.10, 52.64, 49.74, 49.00, 47.27, 43.36, 36.87, 29.75, 25.08; MS (ESI) m/z: 424.2 [M + H]+. 5.2.12. 3-(4-((4-amino-3-(1-methyl-1H-pyrazol-4-yl)-1Hpyrazolo[3,4-d]pyrimidin-1-yl)methyl)piperidin-1-yl)-3oxopropanenitrile (10c) To a stirred solution of S5 (25 mg, 0.08 mmol) in DMF (4 mL) was added DIEA (10.4 mg, 0.08 mmol), HATU (76.13 mg, 0.2 mmol), and 2-cyanoacetic acid (8.2 mg, 0.1 mmol) at 0 °C. The reaction mixture was stirred at room temperature for 16 h. It was quenched with water and extracted with ethyl acetate (3 × 30 mL). The organic layer was washed with water (2 × 10 mL) followed by brine (1 × 10 mL), dried over anhydrous sodium sulfate, and was evaporated to dryness. The crude mass was purified by silica gel column chromatography (0−10% MeOH in DCM) to afford compound 10c (21.6 mg, 71%) as a white solid. 1 H NMR (300 MHz, CDCl3) δ 8.30 (s, 1H), 7.78 (s, 1H), 7.75 (s, 1H), 4.52 (m, 1H), 4.29 (t, J = 9 Hz, 2H), 3.98 (s, 3H), 3.68- 3.64 (m, 1H), 3.47 (s, 2H), 3.13- 3.04 (m, 1H), 2.65- 2.56 (m, 1H), 2.34- 2.26 (m, 1H), 1.71- 1.67 (m, 2H), 1.44- 1.30 (m, 2H); 13C NMR (75 MHz, CDCl3) δ 159.96, 158.24, 156.01, 154.53, 138.39, 136.74, 129.64, 114.57, 114.08, 98.73, 51.61, 46.33, 42.43, 39.40, 36.51, 29.95, 29.11, 25.10; MS (ESI) m/z: 380.2 [M + H]+. 5.2.13. 3-(4-((4-amino-3-(1-(2-methoxyethyl)-1H-pyrazol-4yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)piperidin-1-yl)3-oxopropanenitrile (10d) The procedure outlined in method B was followed to afford the desired compound with 72% yield. 1H NMR (300 MHz, CDCl3) δ 8.31 (s, 1H), 7.86 (s, 1H), 7.82 (s, 1H), 4.53 (m, 1H), 4.37 (t, J = 6 Hz, 2H), 4.30 (d, J = 6 Hz, 2H), 3.77 (t, J = 6 Hz, 2H), 3.70- 3.65 (m, 1H), 3.47 (s, 2H), 3.34 (s, 3H), 3.15- 3.06 (m, 1H), 2.66- 2.58 (m, 1H), 2.35- 2.28 (m, 1H), 1.73- 1.68 (m, 2H), 1.44- 1.31 (m, 2H); 13C NMR (75 MHz, CDCl3) δ 159.95, 158.12, 155.78, 154.51, 138.54, 136.89, 129.85, 114.50, 113.99, 98.65, 70.92, 59.08, 52.62, 51.64, 46.36, 42.45, 36.54, 30.00, 29.15, 25.09; MS (ESI) m/z: 424.2 [M + H] +. 5.2.14. 3-(3-(4-amino-3-(1-methyl-1H-pyrazol-4-yl)-1Hpyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)-3oxopropanenitrile (12c)

To a stirred solution of S7 (25 mg, 0.08 mmol) in DMF (4 mL) was added DIEA (10.4 mg, 0.08 mmol), HATU (76.13 mg, 0.2 mmol), and 2-cyanoacetic acid (8.2 mg, 0.1 mmol) at 0 °C. The reaction mixture was stirred at room temperature for 16 h. It was quenched with water and extracted with ethyl acetate (3 × 30 mL). The organic layer was washed with water (2 × 10 mL) followed by brine (1 × 10 mL), dried over anhydrous sodium sulfate, and was evaporated to dryness. The crude mass was purified by silica gel column chromatography (0−10% MeOH in DCM) to afford compound 12c (20.5 mg, 67%) as a white solid. 1 H NMR (300 MHz, DMSO-d6) δ 8.22 (s, 1H), 8.06 (s, 1H), 7.66 (s, 1H), 4.76- 4.41 (m, 1H), 4.10 (m, 2H), 4.01- 3.85 (m, 1H), 3.83 (s, 3H), 3.70- 3.57 (m, 1H), 3.16- 3.02 (m, 1H), 2.93- 2.82 (m, 1H), 2.15- 2.06 (m, 2H),1.86- 1.67 (m, 2H); 13C NMR (75 MHz, DMSO-d6) δ 161.81, 157.45, 156.70, 153.62, 138.56, 136.64, 130.17, 116.21, 110.07, 97.27, 52.83, 52.27, 48.92, 45.71, 29.24, 25.04, 24.16; MS (ESI) m/z: 366.2 [M + H] +. 5.2.15. 3-(3-(4-amino-3-(1-(2-methoxyethyl)-1H-pyrazol-4-yl)1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)-3oxopropanenitrile (12d) The procedure outlined in method B was followed to afford the desired compound with 72% yield. 1H NMR (300 MHz, CDCl3) δ 8.27 (s, 1H), 7.82 (s, 1H), 7.76 (s, 1H), 4.61- 4.58 (m, 1H), 4.31 (t, J = 6 Hz, 2H), 4.09- 4.06 (m, 1H), 3.96- 3.93 (m, 2H), 3.72 (t, J = 6 Hz, 2H), 3.53- 3.47 (m, 1H), 3.30 (s, 3H), 3.15-3.08 (m, 1H), 2.87- 2.82 (m, 1H), 2.11- 2.03 (m, 2H), 1.851.75 (m, 2H); 13C NMR (75 MHz, DMSO-d6) δ 161.63, 158.31, 155.78, 153.66, 137.86, 136.93, 130.20, 116.04, 113.34, 97.78, 70.39, 57.99, 51.26, 50.27, 46.17, 43.00, 28.47, 25.27, 24.58; MS (ESI) m/z: 410.2 [M + H]+. 5.2.16. 3-(4-(4-amino-3-(1-methyl-1H-pyrazol-4-yl)-1Hpyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)-3oxopropanenitrile (13c) To a stirred solution of S8 (25 mg, 0.08 mmol) in DMF (4 mL) was added DIEA (10.4 mg, 0.08 mmol), HATU (76.13 mg, 0.2 mmol), and 2-cyanoacetic acid (8.2 mg, 0.1 mmol) at 0 °C. The reaction mixture was stirred at room temperature for 16 h. It was quenched with water and extracted with ethyl acetate (3 × 30 mL). The organic layer was washed with water (2 × 10 mL) followed by brine (1 × 10 mL), dried over anhydrous sodium sulfate, and was evaporated to dryness. The crude mass was purified by silica gel column chromatography (0−10% MeOH in DCM) to afford compound 13c (21.7 mg, 71%) as a white solid. 1 H NMR (300 MHz, MeOD) δ 8.20 (s, 1H), 7.96 (s, 1H), 7.77 (s, 1H), 4.99- 4.92 (m, 1H), 4.62- 4.58 (m, 1H), 3.96 (s, 3H), 3.90 (m, 1H), 3.40- 3.35 (m, 1H), 3.31- 3.27 (m, 2H), 3.00- 2.90 (m, 1H), 2.32- 2.22 (m, 1H), 2.20- 2.09 (m, 1H), 2.05- 1.99 (m, 2H); 13 C NMR (75 MHz, DMSO-d6) δ 161.33, 158.24, 155.47, 153.31, 137.60, 136.31, 130.19, 116.22, 113.72, 97.71, 59.56, 52.86, 44.49, 40.75, 31.09, 30.59, 24.80; MS (ESI) m/z: 366.2 [M + H]+. 5.2.17. 3-(4-(4-amino-3-(1-(2-methoxyethyl)-1H-pyrazol-4-yl)1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)-3oxopropanenitrile (13d) The procedure outlined in method B was followed to afford the desired compound with 70% yield. 1H NMR (300 MHz, DMSO-d6) δ 8.24 (s, 1H), 8.12 (s, 1H), 7.79 (s, 1H), 4.89- 4.80 (m, 1H), 4.61- 4.57 (m, 1H), 4.37 (t, J = 6 Hz, 2H), 3.89 (m, 1H), 3.75 (t, J = 6 Hz, 2H), 3.36- 3.32 (m, 1H), 3.28 (s, 3H), 3.223.17 (m, 2H), 2.86- 2.82 (m, 1H), 2.21- 2.09 (m, 2H), 2.06- 1.92 (m, 2H); 13C NMR (75 MHz, DMSO-d6) δ 161.17, 157.40, 156.70, 154.15, 138.86, 136.67, 130.47, 116.06, 114.08, 97.34, 70.34, 59.42, 57.79, 51.54, 44.97, 42.15, 35.99, 30.94, 24.75; MS (ESI) m/z: 410.2 [M + H]+.

5.2.18. 1-(3-((4-amino-3-(1-methyl-1H-pyrazol-4-yl)-1Hpyrazolo[3,4-d]pyrimidin-1-yl)methyl)piperidin-1-yl)-2(dimethylamino)ethanone (9e) To a stirred solution of S4 (25 mg, 0.08 mmol) in DMF (4 mL) was added DIEA (10.4 mg, 0.08 mmol), HATU (76.13 mg, 0.2 mmol), and 2-(dimethylamino)acetic acid (10.4 mg, 0.1 mmol) at 0 °C. The reaction mixture was stirred at room temperature for 16 h. It was quenched with water and extracted with ethyl acetate (3 × 30 mL). The organic layer was washed with water (2 × 10 mL) followed by brine (1 × 10 mL), dried over anhydrous sodium sulfate, and was evaporated to dryness. The crude mass was purified by silica gel column chromatography (0−10% MeOH in DCM) to afford compound 9e (17.0 mg, 53%) as a white solid. 1H NMR (300 MHz, CDCl3) δ 8.19 (s, 1H), 7.70 (s, 1H), 7.68 (s, 1H), 4.32 (m, 1H), 4.16 (m, 2H), 3.88 (s, 3H), 3.73- 3.69 (m, 1H), 3.19-3.12 (m, 1H), 3.062.90 (m, 2H), 2.84- 2.76 (m, 1H), 2.65 (m, 1H), 2.26 (s, 3H), 2.01 (s, 3H), 1.73- 1.63 (m, 2H), 1.35- 1.21 (m, 2H); 13C NMR (75 MHz, CDCl3) δ 167.66, 158.21, 155.65, 154.15, 138.08, 136.52, 129.46, 114.31, 98.43, 61.59, 59.90, 49.46, 44.92, 42.41, 39.13, 37.19, 36.27, 29.47, 28.38; MS (ESI) m/z: 398.3 [M + H]+. 5.2.19. 1-(3-((4-amino-3-(1-(2-methoxyethyl)-1H-pyrazol-4yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)piperidin-1-yl)2-(dimethylamino)ethanone (9f) The procedure outlined in method B was followed to afford the desired compound with 52% yield. 1H NMR (300 MHz, CDCl3) δ 8.24 (s, 1H), 7.85 (s, 1H), 7.76 (s, 1H), 4.33 (t, J = 6 Hz, 2H), 4.22 (d, J = 9 Hz, 2H), 4.13- 4.09 (m, 1H), 3.71 (t, J = 6 Hz, 2H), 3.50- 3.47 (m, 1H), 3.28 (s, 3H), 3.24- 3.20 (m, 1H), 3.00- 2.87 (m, 1H), 2.84- 2.71 (m, 2H), 2.59- 2.51 (m, 1H), 2.45 (s, 3H), 2.19 (s, 3H), 1.76- 1.67 (m, 2H), 1.43- 1.31 (m, 2H); 13C NMR (75 MHz, CDCl3) δ 167.03, 158.24, 155.63, 154.31, 138.42, 136.85, 129.82, 114.13, 98.55, 70.83, 61.26, 58.92, 52.40, 49.52, 45.04, 42.98, 39.37, 37.13, 36.36, 29.60, 28.33; MS (ESI) m/z: 442.3 [M + H]+. 5.2.20. 1-(4-((4-amino-3-(1-methyl-1H-pyrazol-4-yl)-1Hpyrazolo[3,4-d]pyrimidin-1-yl)methyl)piperidin-1-yl)-2(dimethylamino)ethanone (10e) To a stirred solution of S5 (25 mg, 0.08 mmol) in DMF (4 mL) was added DIEA (10.4 mg, 0.08 mmol), HATU (76.13 mg, 0.2 mmol), and 2-(dimethylamino)acetic acid (10.4 mg, 0.1 mmol) at 0 °C. The reaction mixture was stirred at room temperature for 16 h. It was quenched with water and extracted with ethyl acetate (3 × 30 mL). The organic layer was washed with water (2 × 10 mL) followed by brine (1 × 10 mL), dried over anhydrous sodium sulfate, and was evaporated to dryness. The crude mass was purified by silica gel column chromatography (0−10% MeOH in DCM) to afford compound 10e (17.0 mg, 53%) as a white solid. 1H NMR (300 MHz, CDCl3) δ 8.18 (s, 1H), 7.70 (s, 1H), 7.68 (s, 1H), 4.45- 4.41 (m, 1H), 4.17 (d, J = 9 Hz, 2H), 3.95 (m, 1H), 3.89 (s, 3H), 3.11- 2.98 (m, 2H), 2.87- 2.79 (m, 1H), 2.46- 2.38 (m, 1H), 2.23 (m, 1H), 2.18 (s, 6H), 1.55- 1.51 (m, 2H), 1.25- 1.16 (m, 2H); 13C NMR (75 MHz, CDCl3) δ 167.79, 158.27, 155.58, 154.09, 138.18, 136.30, 129.45, 114.41, 98.44, 61.87, 51.71, 45.32, 45.06, 41.47, 39.15, 36.55, 30.18, 29.34; MS (ESI) m/z: 398.2 [M + H] +. 5.2.21. 1-(4-((4-amino-3-(1-(2-methoxyethyl)-1H-pyrazol-4yl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)piperidin-1-yl)2-(dimethylamino)ethanone (10f) The procedure outlined in method B was followed to afford the desired compound with 52% yield. 1H NMR (300 MHz, DMSO-d6) δ 8.17 (s, 1H), 8.06 (s, 1H), 7.73 (s, 1H), 4.45 (m, 1H), 4.29 (t, J = 6 Hz, 2H), 4.15 (d, J = 9 Hz, 2H), 3.87 (m, 1H),

3.68 (t, J = 6 Hz, 2H), 3.21 (s, 3H), 3.05- 3.01 (m, 3H), 2.622.54 (m, 1H), 2.21 (s, 6H), 2.07 (m, 1H), 1.52- 1.48 (m, 2H), 1.34-1.16 (m, 2H); 13C NMR (75 MHz, CDCl3) δ 167.66, 158.15, 155.46, 153.97, 138.06, 136.18, 129.33, 114.29, 98.32, 70.24, 61.75, 58.89, 50.60, 45.20, 44.94, 41.35, 39.03, 36.43, 30.06, 29.22; MS (ESI) m/z: 442.3 [M + H]+. 5.2.22. 1-(3-(4-amino-3-(1-methyl-1H-pyrazol-4-yl)-1Hpyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)-2(dimethylamino)ethanone (12e) To a stirred solution of S7 (25 mg, 0.08 mmol) in DMF (4 mL) was added DIEA (10.4 mg, 0.08 mmol), HATU (76.13 mg, 0.2 mmol), and 2-(dimethylamino)acetic acid (10.4 mg, 0.1 mmol) at 0 °C. The reaction mixture was stirred at room temperature for 16 h. It was quenched with water and extracted with ethyl acetate (3 × 30 mL). The organic layer was washed with water (2 × 10 mL) followed by brine (1 × 10 mL), dried over anhydrous sodium sulfate, and was evaporated to dryness. The crude mass was purified by silica gel column chromatography (0−10% MeOH in DCM) to afford compound 12e (16.4 mg, 51%) as a white solid. 1H NMR (300 MHz, CDCl3) δ 8.14 (s, 1H), 8.02 (s, 1H), 7.66 (s, 1H), 4.37- 4.32 (m, 1H), 4.11- 4.07 (m, 1H), 3.85 (s, 3H), 3.56- 3.48 (m, 1H), 3.15 (s, 2H), 3.01- 2.93 (m, 1H), 2.70- 2.62 (m, 1H), 2.21 (s, 3H), 2.13 (s, 3H), 2.06 (m, 2H), 1.82- 1.78 (m, 1H), 1.58- 1.48 (m, 1H); 13C NMR (75 MHz, CDCl3) δ 168.21, 158.19, 155.27, 153.31, 138.15, 136.45, 129.46, 114.30, 98.51, 61.83, 53.00, 52.19, 49.53, 45.34, 41.79, 39.06, 30.02, 29.64; MS (ESI) m/z: 384.3 [M + H] +. 5.2.23. 1-(3-(4-amino-3-(1-(2-methoxyethyl)-1H-pyrazol-4-yl)1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)-2(dimethylamino)ethanone (12f) The procedure outlined in method B was followed to afford the desired compound with 54% yield. 1H NMR (300 MHz, CDCl3) δ 8.21 (s, 1H), 7.83 (s, 1H), 7.76 (s, 1H), 4.45- 4.41 (m, 1H), 4.32 (t, J = 6 Hz, 2H), 4.15- 4.11 (m, 1H), 3.69 (t, J = 6 Hz, 2H), 3.60- 3.51 (m, 1H), 3.28 (s, 3H), 3.16 (s, 2H), 3.09- 3.05 (m, 1H), 2.75- 2.69 (m, 1H), 2.34 (s, 3H), 2.23 (s, 3H), 2.13 (m, 2H), 1.87 (m, 2H); 13C NMR (75 MHz, CDCl3) δ 168.40, 158.24, 155.46, 153.57, 138.37, 136.66, 129.74, 114.10, 98.86, 70.77, 61.81, 58.83, 53.11, 52.30, 49.55, 45.48, 41.91, 30.03, 29.75; MS (ESI) m/z: 428.3 [M + H]+. 5.2.24. 1-(4-(4-amino-3-(1-methyl-1H-pyrazol-4-yl)-1Hpyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)-2(dimethylamino)ethanone (13e) To a stirred solution of S8 (25 mg, 0.08 mmol) in DMF (4 mL) was added DIEA (10.4 mg, 0.08 mmol), HATU (76.13 mg, 0.2 mmol), and 2-(dimethylamino)acetic acid (10.4 mg, 0.1 mmol) at 0 °C. The reaction mixture was stirred at room temperature for 16 h. It was quenched with water and extracted with ethyl acetate (3 × 30 mL). The organic layer was washed with water (2 × 10 mL) followed by brine (1 × 10 mL), dried over anhydrous sodium sulfate, and was evaporated to dryness. The crude mass was purified by silica gel column chromatography (0−10% MeOH in DCM) to afford compound 13e (18.3 mg, 57%) as a white solid. 1H NMR (300 MHz, CDCl3) δ 8.21 (s, 1H), 7.70 (s, 1H), 7.68 (s, 1H), 4.90- 4.83 (m, 1H), 4.66- 4.62 (m, 1H), 4.21- 4.16 (m, 1H), 3.90 (s, 3H), 3.13- 3.05 (m, 3H), 2.76- 2.68 (m, 1H), 2.22 (s, 6H), 2.19- 2.06 (m, 2H),1.97- 1.95 (m, 2H); 13C NMR (75 MHz, CDCl3) δ 168.19, 158.27, 155.42, 153.35, 138.25, 136.35, 129.53, 114.54, 98.86, 62.13, 53.73, 45.47, 44.51, 41.03, 39.17, 31.68, 31.09; MS (ESI) m/z: 384.2 [M + H]+. 5.2.25. 1-(4-(4-amino-3-(1-(2-methoxyethyl)-1H-pyrazol-4-yl)1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)-2(dimethylamino)ethanone (13f)

The procedure outlined in method B was followed to afford the desired compound with 53% yield. 1H NMR (300 MHz, CDCl3) δ 8.14 (s, 1H), 7.74 (s, 1H), 7.67 (s, 1H), 4.85- 4.77 (m, 1H), 4.59- 4.55 (m, 1H), 4.23 (t, J = 6 Hz, 2H), 4.13- 4.08 (m, 1H), 3.63 (t, J = 6 Hz, 2H), 3.20 (s, 3H), 3.13- 3.04 (m, 3H), 2.72- 2.62 (m, 1H), 2.22 (s, 6H), 2.12- 2.00 (m, 2H), 1.93- 1.90 (m, 2H); 13C NMR (75 MHz, CDCl3) δ 167.70, 158.12, 155.16, 153.12, 138.14, 136.29, 129.52, 114.06, 98.62, 70.56, 61.57, 58.58, 53.46, 52.04, 45.26, 44.25, 40.84, 31.40, 30.80; MS (ESI) m/z: 428.3 [M + H]+. Method C: General procedure for the synthesis of targets 9g-9h, 10g-10h, 12g-12h and 13g-13h. 5.2.26. 3-((4-amino-3-(1-methyl-1H-pyrazol-4-yl)-1Hpyrazolo[3,4-d]pyrimidin-1-yl)methyl)piperidine-1carbonitrile (9g) To a stirred solution of S4 (25 mg, 0.08 mmol) in MeOH (5 mL) was added KOAc (11.8 mg, 0.12 mmol) and BrCN (9.3 mg, 0.09 mmol) at 0 °C. The reaction mixture was stirred at room temperature for 16 h. It was quenched with water, and was evaporated to dryness. The crude mass was purified by silica gel column chromatography (0−10% MeOH in DCM) to afford compound 9g (25.1 mg, 93%) as a white solid. 1H NMR (300 MHz, DMSO-d6) δ 8.22 (s, 1H), 8.07 (s, 1H), 7.74 (s, 1H), 4.23 (d, J = 9 Hz, 2H), 3.91 (s, 3H), 3.26- 3.18 (m, 2H), 3.03- 2.88 (m, 2H), 2.23 (m, 1H), 1.69- 1.59 (m, 2H), 1.49- 1.45 (m, 1H), 1.201.13 (m, 1H); 13C NMR (75 MHz, DMSO-d6) δ158.27, 155.75, 154.26, 137.62, 136.64, 130.23, 118.07, 113.62, 97.34, 51.93, 49.08, 48.27, 35.28, 26.12, 22.91, 21.00; MS (ESI) m/z: 338.2 [M + H]+. 5.2.27. 3-((4-amino-3-(1-(2-methoxyethyl)-1H-pyrazol-4-yl)1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)piperidine-1carbonitrile (9h) The procedure outlined in method C was followed to afford the desired compound with 91% yield. 1H NMR (300 MHz, DMSO-d6) δ 8.01 (s, 1H), 7.86 (s, 1H), 7.53 (s, 1H), 4.50 (t, J = 6 Hz, 2H), 4.02 (d, J = 9 Hz, 2H), 3.60 (t, J = 6 Hz, 2H), 3.12 (s, 3H), 3.05- 3.98 (m, 2H), 2.82- 2.67 (m, 2H), 2.02 (m, 1H), 1.491.38 (m, 2H), 1.31- 1.22 (m, 1H), 1.03- 0.87 (m, 1H); 13C NMR (75 MHz, DMSO-d6) δ 158.76, 156.24, 154.75, 138.11, 137.13, 130.72, 118.56, 114.11, 97.83, 73.34, 57.92, 51.25, 49.57, 48.76, 39.20, 35.77, 26.61, 23.40; MS (ESI) m/z: 382.3 [M + H]+. 5.2.28. 4-((4-amino-3-(1-methyl-1H-pyrazol-4-yl)-1Hpyrazolo[3,4-d]pyrimidin-1-yl)methyl)piperidine-1carbonitrile (10g) To a stirred solution of S5 (25 mg, 0.08 mmol) in MeOH (5 mL) was added KOAc (11.8 mg, 0.12 mmol) and BrCN (9.3 mg, 0.09 mmol) at 0 °C. The reaction mixture was stirred at room temperature for 16 h. It was quenched with water, and was evaporated to dryness. The crude mass was purified by silica gel column chromatography (0−10% MeOH in DCM) to afford compound 10g (24.6 mg, 91%) as a white solid. 1H NMR (300 MHz, DMSO-d6) δ 8.30 (s, 1H), 8.19 (s, 1H), 7.86 (s, 1H), 4.57 (m, 1H), 4.27 (d, J = 9 Hz, 2H), 3.99 (s, 3H), 3.34 (m, 1H), 3.183.14 (m, 1H), 2.74- 2.66 (m, 1H), 2.34- 2.20 (m, 1H), 1.65- 1.47 (m, 2H), 1.43- 1.34 (m, 2H); 13C NMR (75 MHz, DMSO-d6) δ 158.37, 155.86, 154.32, 137.86, 136.40, 130.12, 118.26, 113.59, 97.43, 51.26, 49.85, 48.50, 34.91, 28.21; MS (ESI) m/z: 338.2 [M + H]+. 5.2.29. 4-((4-amino-3-(1-(2-methoxyethyl)-1H-pyrazol-4-yl)1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)piperidine-1carbonitrile (10h) The procedure outlined in method C was followed to afford the desired compound with 94% yield. 1H NMR (300 MHz,

DMSO-d6) δ 8.24 (s, 1H), 8.12 (s, 1H), 7.79 (s, 1H), 4.36 (m, 2H), 4.23 (m, 2H), 3.74 (m, 2H), 3.35 (m, 2H), 3.27 (s, 3H), 2.99 (m, 2H), 2.08- 1.92 (m, 1H), 1.52 (m, 2H), 1.33 (m, 2H); 13C NMR (75 MHz, DMSO-d6) δ 158.37, 155.86, 154.32, 137.86, 136.40, 130.12, 118.26, 113.59, 97.43, 70.43, 58.00, 51.26, 50.97, 48.50, 34.91, 28.21; MS (ESI) m/z: 382.2 [M + H]+. 5.2.30. 3-(4-amino-3-(1-methyl-1H-pyrazol-4-yl)-1Hpyrazolo[3,4-d]pyrimidin-1-yl)piperidine-1-carbonitrile (12g) To a stirred solution of S7 (25 mg, 0.08 mmol) in MeOH (5 mL) was added KOAc (11.8 mg, 0.12 mmol) and BrCN (9.3 mg, 0.09 mmol) at 0 °C. The reaction mixture was stirred at room temperature for 16 h. It was quenched with water, and was evaporated to dryness. The crude mass was purified by silica gel column chromatography (0−10% MeOH in DCM) to afford compound 12g (24.7 mg, 91%) as a white solid. 1H NMR (300 MHz, DMSO-d6) δ 8.23 (s, 1H), 8.07 (s, 1H), 7.73 (s, 1H), 4.81 (m, 1H), 3.91 (s, 3H), 3.62- 3.58 (m, 1H), 3.51- 3.48 (m, 1H), 3.41- 3.38 (m, 1H), 3.18- 3.12 (m, 1H), 2.49 (m, 2H), 2.14- 2.06 (m, 2H), 1.85- 1.81 (m, 2H); 13C NMR (75 MHz, DMSO-d6) δ 158.31, 155.74, 153.80, 137.72, 136.78, 130.36, 117.63, 113.56, 97.71, 51.79, 51.15, 48.71, 28.44, 23.40; MS (ESI) m/z: 324.2 [M + H]+. 5.2.31. 3-(4-amino-3-(1-(2-methoxyethyl)-1H-pyrazol-4-yl)1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidine-1-carbonitrile (12h) The procedure outlined in method C was followed to afford the desired compound with 94% yield. 1H NMR (300 MHz, DMSO-d6) δ 8.25 (s, 1H), 8.10 (s, 1H), 7.77 (s, 1H), 4.87- 4.78 (m, 1H), 4.35 (t, J = 6 Hz, 2H), 3.73 (t, J = 6 Hz, 2H), 3.63- 3.57 (m, 1H), 3.53- 3.45 (m, 1H), 3.41- 3.37 (m, 1H), 3.26 (s, 3H), 3.19- 3.10 (m, 1H), 2.17- 2.07 (m, 2H), 1.91- 1.76 (m, 2H); 13C NMR (75 MHz, DMSO-d6) δ 157.93, 155.23, 153.57, 137.77, 136.79, 130.05, 117.48, 113.29, 97.71, 70.33, 57.92, 51.72, 51.20, 48.66, 28.31, 23.27; MS (ESI) m/z: 368.2 [M + H]+. 5.2.32. 3-(4-amino-3-(1-methyl-1H-pyrazol-4-yl)-1Hpyrazolo[3,4-d]pyrimidin-1-yl)piperidine-1-carbonitrile (13g) To a stirred solution of S8 (25 mg, 0.08 mmol) in MeOH (5 mL) was added KOAc (11.8 mg, 0.12 mmol) and BrCN (9.3 mg, 0.09 mmol) at 0 °C. The reaction mixture was stirred at room temperature for 16 h. It was quenched with water, and was evaporated to dryness. The crude mass was purified by silica gel column chromatography (0−10% MeOH in DCM) to afford compound 13g (24.9 mg, 92%) as a white solid. 1H NMR (300 MHz, MeOD) δ 8.29 (s, 1H), 8.06 (s, 1H), 7.88 (s, 1H), 5.55 (m, 1H), 4.06 (s, 3H), 3.71- 3.66 (m, 2H), 3.44- 3.37 (m, 2H), 2.502.39 (m, 2H), 2.12- 2.05 (m, 2H); 13C NMR (75 MHz, DMSOd6) δ158.20, 155.69, 153.44, 137.47, 136.35, 130.25, 117.24, 113.38, 97.62, 54.70, 50.63, 45.13, 42.266, 30.28, 27.99; MS (ESI) m/z: 324.2 [M + H]+. 5.2.33. 3-(4-amino-3-(1-(2-methoxyethyl)-1H-pyrazol-4-yl)1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidine-1-carbonitrile (13h) The procedure outlined in method C was followed to afford the desired compound with 92% yield. 1H NMR (300 MHz, MeOD) δ 8.24 (s, 1H), 8.12 (s, 1H), 7.80 (s, 1H), 5.47 (m, 1H), 4.37 (t, J = 6 Hz, 2H), 3.75 (t, J = 6 Hz, 2H), 3.58- 3.54 (m, 2H), 3.40- 3.33 (m, 2H), 3.28 (s, 3H), 2.26- 2.17 (m, 2H), 1.98- 1.87 (m, 2H); 13C NMR (75 MHz, DMSO-d6) δ 158.32, 156.60, 153.26, 137.88, 136.30, 130.16, 117.15, 113.71, 97.80, 70.47, 58.05, 52.73, 51.30, 43.81, 42.36, 30.52, 28.03; MS (ESI) m/z: 366.2 [M + H]+. 5.3. Computational analysis

5.3.1. Docking calculation We used the X-ray cocrystal structure of JAK3 in the Protein Data Bank (PDB ID: 3PJC). The protein structure for the docking study was prepared with Protein Preparation Workflow. All crystallographic waters were removed and chain A was kept. Hybridization states, charges, and angles were assigned in the protein structure with missing bond orders, and hydrogen atoms were added. The energy of the protein structure was minimized in 100 steps of the smart minimize method. Docking grids were generated and defined based on the centroid of compound 12a in the ATP binding site incorporating hydrogen-bond constraints to the hinge and hydrophobic regions. Ligands were prepared using Discovery Studio 3.0 (DS 3.0). energy-minimized conformation of each ligands were used to docking calculation input molecules. LigandFit and CDOCKER docking programs implemented in DS 3.0 were used in this study. Other docking parameters were kept to the default values. The top-scoring pose was employed for discussions. 5.4. Bioactivity 5.4.1. JAK1-3 and TYK2 Enzyme Assay Human JAK1-3 and TYK2 kinase were obtained from Invitrogen and assays were performed using HTRF (Homogenous Time-Resolved Fluorescence) detection technology. Briefly, the enzyme reaction was run in reaction buffer, which consisted of 50 mM HEPES (pH7.0), 5 mM MgCl2, 1 mM DTT, 0.1% NaN3 and 0.1% orthovanadate. The assay was done by a 384-well plate (10 μL) assay format. The end concentration of enzyme, TK-substrate-biotin, ATP was 1 ng/μl, 100 nM, 4 μM for JAK1, 0.004 ng/μl, 100 nM, 4 μM for JAK2, 0.012 ng/μl, 100 nM, 1.43 μM for JAK3, 0.012 ng/μl, 100 nM, 1.43 μM for TYK2, respectively. Compounds were screened at serial diluted concentration in the presence of 2% DMSO with a 5 min pre-incubation of kinase and compounds. All reactions were started by the addition of ATP and TK-substrate-biotin, incubated at 30 °C for 30 min and quenched with the stop buffer containing 25 nM Strep-XL665 and TK Ab-Cryptate. The plates were incubated for 1 h. Time-resolved fluorescence was monitored with an Synergy H1 (Biotek) by excitation at 330 nm and emission donor at 620 nm or emission acceptor at 665 nm, respectively. The files recorded by the Synergy H1 were read with Excel and contained the acceptor and donor counts for each sample. And IC50 values were determined using the Graphpad Prism 5.0 Software. 5.4.2. BTK Enzyme Assay The BTK enzyme assay of compounds 9a-9b, 11a-11b and 12a-12b were performed by HTRF assay. Briefly, The assay buffer was composed of 50 mM HEPES (pH7.0), 5 mM MgCl2, 1 mM DTT, 0.1% NaN3 and 0.1% orthovanadate. The assay was done by a 384-well plate assay format. Then 4 μl, 0.5 ng/μl BTK, 2 μl, 22.4 μM ATP and various concentrations of compounds were added to each well. Incubated at 30 °C for 30 min and quenched with the stop buffer containing 25 nM Strep-XL665 and TK Ab-Cryptate. The plates were incubated for 1 h before being read on synergy H1 (Biotek) using standard HTRF settings. And IC50 values were determined using the GraphPad Prism 5.0 software. 5.4.3. Rat T cell proliferation Spleen cells were suspended in RPMI1640 medium, supplemented with 10% fetal calf serum. 100 U/mL penicillin, 100 μg/mL streptomycin, and 50 μM 2-mercaptoethanol at a density of 1.5 ×106 cells/mL. Splenocytes were cultured with Concanavalin A for 24 h at 37 °C in 5% CO2 to induce IL-2 receptor expression and then incubated with IL-2 and test

compounds at designated concentrations in 96-well tissue culture plates. After incubation for 72h, cell viability was monitored using the CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS) (Promega, Madison, WI) according to the manufacturer’s recommended protocols. Colorimetric assay of each well was read using microplate reader synergy H1 (Biotek). Values were transformed to percent inhibition relative to vehicle control, and IC50 curves were fitted according to nonlinear regression analysis of the data using PRISM GraphPad 5.0. 5.4.4. Delayed-type hypersensitivity Animals were sensitized on the abdomen and subsequently challenged on the pinna with dinitrofluorobenzene (n= 5-8 per group for treated mice and n = 2 for naives)). Compounds (100 mg/kg, by mouth, twice per day) were administered just prior to and during the challenge phase. Two-tailed Student t tests were used to compare individual treatment groups.

Acknowledgments The authors are grateful to financial supports from Natural Science Foundation of China (81573445, 81172934). References and notes 1.

2. 3. 4.

5.

(a) Ihle, J. N.; Kerr, I. M. Trends Genet. 1995, 11, 69. (b) Ihle, J. N. Semin. Immunol. 1995, 7, 247. (c) Yu, H.; Pardoll, D.; Jove, R. Nat. Rev. Cancer. 2009, 9, 798. Shuai, K.; Liu, B. Nat. Rev. Immunol. 2003, 3, 900. Ghoreschi, K.; Laurence, A.; O’Shea, J. J. Immunol. Rev. 2009, 228, 273. Thorarensen, A.; Dowty, M. E.; Banker, M. E.; Juba, B.; Jussif, J.; Lin, T.; Vincent, F.; Czerwinski, R. M.; Unwalla, R.; Trujillo, J. I.; Liang, S.; Leung, P.; Yang, X.; Soucy, S.; Hegen, M.; Coe, J.; Langille, J.; Vajdos, F.; Chrencik, J.; Telliez, J. B. J. Med. Chem. 2017, 60, 1971. (a) Johnston, J. A.; Bacon, C. M.; Riedy, M. C.; O’Shea, J. J. J. Leukocyte. Biol. 1996, 60 (4), 441. (b) Baker, S. J.; Rane, S. G.; Reddy, E. P. Oncogene. 2007, 26, 6724.(c) Aringer, M.; Cheng, A.; Nelson, J. W.; Chen, M.; Sudarshan, C.; Zhou, Y. J.; O’Shea, J. J. Life Sci. 1999, 64, 2173. (d) O'Shea, J. J.; Schwartz, D. M.; Villarino, A. V.; Gadina, M.; McInnes, I. B.; Laurence, A. Annu.

Rev. Med. 2015, 66, 311. (e) Schindler, C. W. J. Clin. Invest. 2002, 109, 1133. 6. (a) Clark, J. D.; Flanagan, M. E.; Telliez, J. B. J. Med. Chem. 2014, 57, 5023. (b) Flanagan, M. E.; Blumenkopf, T. A.; Brissette, W. H.; Brown, M. F.; Casavant, J. M.; Shang-Poa, C.; Doty, J. L.; Elliott, E. A.; Fisher, M. B.; Hines, M.; Kent, C.; Kudlacz, E. M.; Lillie, B. M.; Magnuson, K. S.; McCurdy, S. P.; Munchhof, M. J.; Perry, B. D.; Sawyer, P. S.; Strelevitz, T. J.; Subramanyam, C.; Sun, J.; Whipple, D. A.; Changelian, P. S. J. Med. Chem. 2010, 53, 8468. (c) Quintas-Cardama, A.; Vaddi, K.; Liu, P.; Manshouri, T.; Li, J.; Scherle, P. A.; Caulder, E.; Wen, X.; Li, Y.; Waeltz, P.; Rupar, M.; Burn, T.; Lo, Y.; Kelley, J.; Covington, M.; Shepard, S.; Rodgers, J. D.; Haley, P.; Kantarjian, H.; Fridman, J. S.; Verstovsek, S. Blood. 2010, 115, 3109. 7. (a) Menet, C. J.; Rompaey, L. V.; Geney, R. Prog. Med. Chem. 2013, 52, 153. (b) Norman, P. Expert. Opin. Invest. Drugs. 2014, 23, 1067. 8. (a) Russell, S. M.; Tayebi, N.; Nakajima, H.; Riedy, M. C.; Roberts, J. L.; Aman, M. J.; Migone, T. S.; Noguchi, M.; Markert, M. L.; Buckley, R. H.; O’Shea, J. J.; Leonard, W. J. Science. 1995, 270, 797. (b) O’Shea, J. J.; Pesu, M.; Borie, D. C.; Changelian, P. S. Nat. Rev. Drug. Discov. 2004, 3, 555. (c) Kulagowski J J, Blair W, Bull R J, et al. J. Med. Chem. 2012, 55(12): 5901-5921. 9. Tan, L.; Akahane, K.; McNally, R.; Reyskens, K. M.; Ficarro, S. B.; Liu, S.; Herter-Sprie, G.; Koyama, S.; Pattison, M. J.; Labella, K.; Johannessen, L.; Arthur, E. A.; Eck, M. J.; Gray, N. S. J. Med. Chem. 2015, 58, 6589. 10. Bains, T.; Heinrich, M. C.; Loriaux, M. M.; Beadling, C.; Nelson, D.; Warrick, A.; Neff, T. L.; Tyner, J. W.; Dunlap, J.; Corless, C. L.; Fan, G. Leukemia. 2012, 26, 2144. 11. Wang Y, Huang W, Xin M, et al. Bioorg. Med. Chem. 2017, 25(1): 75-83

Supplementary Material Supplementary material that may be helpful in the review process should be prepared and provided as a separate electronic file. That file can then be transformed into PDF format and submitted along with the manuscript and graphic files to the appropriate editorial office.

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Highlights A series of 1H-pyrazolo[3,4-d]pyrimidin-4amino derivatives has been synthesized.

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An acrylamide group increased the selectivity of the JAK kinase family.

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Some compounds displayed exhibited potent JAK3 inhibitory activity as well as excellent JAK kinase selectivity.