HDAC

Article Cite This: J. Med. Chem. 2017, 60, 8336-8357

pubs.acs.org/jmc

Design and Synthesis of Ligand Efficient Dual Inhibitors of Janus Kinase (JAK) and Histone Deacetylase (HDAC) Based on Ruxolitinib and Vorinostat Lianbin Yao,† Nurulhuda Mustafa,‡,∞ Eng Chong Tan,§,∥,∞ Anders Poulsen,⊥,# Prachi Singh,∇ Minh-Dao Duong-Thi,∇ Jeannie X. T. Lee,‡ Pondy Murugappan Ramanujulu,†,○ Wee Joo Chng,‡,◆,¶ Jeffrey J. Y. Yen,§ Sten Ohlson,*,∇ and Brian W. Dymock*,† †

Department of Pharmacy, National University of Singapore, 18 Science Drive 4, 117543, Singapore Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, 1E Kent Ridge Road, NUHS Tower Block Level 10, 117549, Singapore § Institute of Biomedical Sciences, Academia Sinica, Taipei 115, Taiwan ∥ Taiwan International Graduate Program in Molecular Medicine, National Yang-Ming University and Academia Sinica, Taipei, 115, Taiwan ⊥ Experimental Therapeutics Centre, 31 Biopolis Way, 03-01 Nanos, 138669, Singapore # Department of Chemistry, National University of Singapore, 3 Science Drive 3, 117543, Singapore ∇ School of Biological Sciences, Nanyang Technological University (NTU), 637551, Singapore ○ Centre for Life Sciences Level 5, Life Sciences Institute, National University of Singapore, 28 Medical Drive, 117456, Singapore ◆ Cancer Science Institute, National University of Singapore, 117599, Singapore ¶ National University Cancer Institute of Singapore, National University Health System, 119074, Singapore ‡

S Supporting Information *

ABSTRACT: Concomitant inhibition of multiple oncogenic pathways is a desirable goal in cancer therapy. To achieve such an outcome with a single molecule would simplify treatment regimes. Herein the core features of ruxolitinib (1), a marketed JAK1/2 inhibitor, have been merged with the HDAC inhibitor vorinostat (2), leading to new molecules that are bispecific targeted JAK/ HDAC inhibitors. A preferred pyrazole substituted pyrrolopyrimidine, 24, inhibits JAK1 and HDACs 1, 2, 3, 6, and 10 with IC50 values of less than 20 nM, is 100 15.2 ± 14.4 ±

0.08 4.8 0.87 0.07 0.3 2.9

0.3 7.9

HCT-116 2.0 1.85 ± >100 46.2 ± 8.99 ± 2.32 ± 11.8 ± 41.1 ± 23.0 ± >100 19.4 ± 25.3 ±

0.52 11.3 1.23 1.03 1.4 16.8 3.3 8.2 6.6

PC3 8.6 0.85 ± >100 >100 18.8 ± 2.41 ± 12.9 ± 54.3 ± >50 >100 23.9 ± 17.0 ±

0.37

4.6 0.10 6.4 5.6

8.5 3.9

Values are the mean ± SD of at least three determinations. Compound 1 is >10 μM in all assays. bHDAC6 selective inhibitor.31 cPan-HDAC inhibitor.15 a

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DOI: 10.1021/acs.jmedchem.7b00678 J. Med. Chem. 2017, 60, 8336−8357

Journal of Medicinal Chemistry

Article

Table 7. Cell Proliferation Inhibition Assay Data of Selected Compounds in Three Leukemia Cell Lines

Table 9. Cell Proliferation Assay Data of Compounds in Two Normal Cell Lines

IC50 (μM)a b

compd

HL-60

pacritinibe tubastatinf 1h 2i 3j 24 38 41

1.78 ± 0.62 >4g >4g 2.28 ± 1.28 c.4g,k 7.36 ± 1.78 >10g >10g

HEL92.1.7 1.17 >4g >4g 0.49 0.94 1.33 >4g >4g

c

± 0.03

± 0.10 ± 0.22 ± 0.14

compd Jurkat 1.09 >4g >4g 0.59 1.19 0.47 c.4 >4g

d

d

2 pacritinibe 3f 24

± 0.08

TAMH IC50 (μM)a,b

AC10 IC50 (μM)a,c

± ± ± ±

79.2 ± 43 2.02 ± 0.15 2.07 ± 0.10 >100 (56% viable)

7.86 3.68 9.57 4.63

2.2 0.88 1.00 0.91

Average ± SD of four determinations. bMouse hepatocyte. Cardiomyocyte. dPan-HDAC inhibitor.15 eJAK2/FLT3 inhibitor.32 f JAK2/HDAC dual inhibitor.13 a

± 0.17 ± 0.22 ± 0.15

c

both the JAK-STAT and HDAC pathways in two different cell lines (Figure 7). Cell lines from multiple myeloma (KMS-12BM) and acute myeloid leukemia (MOLM-14) were used to demonstrate target engagement of 24 (Figure 7). Compounds were compared using acetylated histone 3 (Ac-H3) as a marker for HDAC 1, 2, or 3 inhibition35 and using acetylated tubulin (Ac-Tub) as a marker for HDAC6 inhibition.36 Compound 2 was used as an HDAC1−3 positive control and tubastatin as a selective HDAC6 control.23 Effects on HDAC1−3 were observed at concentrations just above the antiproliferative IC50 in both cell lines (Figure 7A,B). Inhibition of HDAC6 appeared to be more sensitive in these cell lines with increases in Ac-Tub seen at concentrations lower than that required for Ac-H3 induction. JAK pathway inhibition (contributions from both JAK1 and JAK237) were clearly apparent with p-STAT3 levels being reduced at submicromolar concentrations (Figure 7C,D). Taken together, these data support the engagement of JAK1/2 and HDACs 1−3, 6 in these two cell lines. It is important to note that the effects of the compound are dependent on the cell line and the pathways that are active in that cell line (see Figure S2 for effects of 24 and reference compounds on HEL92.1.7 cells and HeLa cells). Poly(ADP-ribose) polymerase (PARP) has been implicated in cell death pathways via apoptosis where it is cleaved by caspases to give a N-terminal DNA binding domain. Increase in cleaved PARP following treatment of cells by a therpaeutic agent indicates cell death via apoptosis.38,39 Treatment of KMS12BM and MOLM-14 cells with 24 led to dose related increases in cleaved PARP at concentrtinos from 1 to 6 μM (Figure 7E,F). ADME Properties. Compound 24 was selected for further profiling (Table 10). With a low molecular weight and hydrogen bond donors (three) and acceptors (eight), 24 has potential to be an oral drug. Calculated log P was 1.45 with polar surface area of 108.7 Å2. Unfortunately, the PAMPA assay was unable to detect any compound, a result that could be due

a

Antiproliferative inhibitory activities are the average of at least three determinations. b Acute myeloid leumekia. c Erythroleukemia (JAK2V617F). dAcute T-cell leukemia. eJAK2/FLT3 inhibitor.32 f HDAC6 selective inhibitor.31 gTop concentration tested. hJAK1/ JAK2 inhibitor.5 iPan-HDAC inhibitor.15 jJAK2/HDAC inhibitor.13 k 47% inhibition at 4 μM.

selected for further study in a wider range of hematological cell lines (Table 8). Three multiple myeloma cell lines (KMS-12-BM, OPM-2, XG-6), three AML cell lines (KG-1, MOLM-14, MV4-11), and two natural killer T-cell lymphoma cell lines (NKYS, KHYG) were tested against 24 as well as four reference compounds against selected cell lines: the JAK1/2 inhibitor 1, pan-HDAC inhibitor 2, JAK2/HDAC inhibitor 3, and JAK2-FLT3 inhibitor pacritinib. Potency ranged from 0.15 to 2.16 μM for 24, which compares very favorably with the reference compounds. Compound 24 was either more potent or similarly potent (within 2- to 3-fold) to each reference compound for all cell lines with the exception of MOLM-14 where 24 (IC50 = 0.74 μM) is nearly 10-fold less active than the very potent pacritinib (IC50 = 0.079 μM). Normal Cells. Having shown that 24 is a broad antiproliferative agent in a range of cancer cells, it was then determined if these effects also applied to two normal cell lines, transforming growth factor α mouse hepatocytes (TAMH)33 and the cardiomyocyte AC1034 (Table 9). Compound 24 stopped the growth of TAMH cells at a concentration higher than its average anticancer potency (IC50 = 4.63 μM), whereas for AC10 cells 24 was considerably less potent (56% at 100 μM). These data suggest that dual inhibitors such as 24 may have a therapeutic window. Intracellular Target Engagement. Having shown that compound 24 had a desirable antiproliferative profile, it was next determined whether or not the compound was inhibiting

Table 8. Cell Proliferation Inhibition Assay Data of 24 and Reference Standards in a Range of Hematological Cell Lines IC50 (μM)a compd e

1 2j pacritinibk 3m 24

KMS-12-BMb f

>10 0.94 0.75 2.11 2.02

OPM-2b

XG-6b

KG-1c

g

± ± ± ±

0.24 0.24 0.91 1.07

>10 2.91 1.21 2.05 2.16

MOLM-14c >10

± ± ± ±

0.87 0.65 0.89 0.57

2.01 ± 0.99

1.48l 1.63 ± 0.53 0.15 ± 0.03

h

0.079l 1.14 ± 0.39 0.74 ± 0.21

MV4-11c >10

NKYSd

KHYGd

1.60l 1.08 ± 0.22 0.45 ± 0.16

1.24l 1.09 ± 0.45 0.57 ± 0.22

i

0.46 ± 0.08

a

Antiproliferative inhibitory activities are the average of at least three determinations. bMultiple myeloma. cAcute myeloid leukemia. dNatural killer T-cell lymphoma. eJAK1/2 inhibitor.5 f59% viability at 10 μM. g74% viability at 10 μM. Blank cells = not tested. h73% viability at 10 μM. i52% viability at 10 μM. jPan-HDAC inhibitor.15 kJAK2/FLT3 inhibitor.32 lSingle determination of an 8-dose response in triplicate. mJAK2/HDAC dual inhibitor.13 Blank cells = not tested. 8346



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Figure 7. Compound 24 effectively blocks dual signaling pathways in MM and AML cell lines. (A) KMS-12-BM and (B) MOLM-14 were treated with 24 at the respective concentrations for 16 h. After lysis, acetylated tubulin (Ac-tubulin) and acetylated histone 3 (Ac-H3) were detected by immunoblotting. Subsequently, as a loading control the same membranes were reprobed with tubulin and H3, respectively. Compound 2 was used as a postive control for the inhibtion of HDACs. (C) KMS-12-BM and (D) MOLM-14 were pretreated with 24 for 3 h and then were treated with 10 ng/mL of IL-6 for 15 min. Compound 1 was used as a positive control for the inhibition of the JAK2 pathway at 3 μM (sub-IC50 concentration). After lysis, p-STAT3 was detected by immunoblotting. The same membranes were reprobed with STAT3 to detect total protein levels. (E) KMS-12BM and (F) MOLM-14 cells were treated with 24 at the respective concentrations for 24 h and then lysed. Tubastatin and 1 were utilized as negative and positive controls, respectively. Cleaved PARP was detected by immunoblotting.

microsomes (RLMs) was excellent with half-lives in either male or female RLM of over 200 min (see Supporting Information). Pharmacokinetics. Given the encouraging aqueous solubility and microsomal stability of 24, pharmacokinetics (PK) in rats was determined for 24. The compound was dosed at 5 mg/kg orally (po) and 1 mg/kg intravenously (iv). Concentrations of 24 were quantifiable up to 2 h postdose but were below the limit of quantitation between 4 and 24 h postdose (Figure 8). The compound was rapidly cleared (CL = 95 mL min−1 kg−1), with a moderate volume of distribution at steady state (Vss= 1.5 L/kg) and a short terminal half-life of ∼0.4 h. The high systemic clearance was unexpected given the slow turnover in rat liver micosomes. This suggests that 24 is cleared by other routes, such as biliary or renal clearance. Oral bioavailability was measurable but poor at 1.4% possibly indicative of low permeability. In an oral/iv pharmacokinetic study in rats, increased bioavailability was observed for 45; however clearance was high indicating further optimization of these templates is required prior to oral efficacy studies (see Supporting Information). In conclusion, by use of the core scaffold of the marketed 1 as a base template, HDAC activity was added by linking a hydroxamate to the pyrazole nitrogen. Optimization of the linker between the pyrazole and hydroxamate led to the

Table 10. Physicochemical Properties and in Vitro ADME Data for 24 property

value

molecular weight no. of HBDa no. of HBAb cLogPc TPSA (Å2)d PAMPA Pe (×10−6 cm/s)e aq solubility (μg/mL, pH 7.4, 3 h)f aq solubility (μg/mL, pH 7.4, 24 h)f RLM (t1/2, min)g

328.4 3 8 1.45 108.7 73 >200

a

Hydrogen bond donors. bHydrogen bond acceptors. cCalculated as miLogP using Molinspiration property engine version 2014.11 (http://www.molinspiration.com). dTopological polar surface area (http://www.molinspiration.com). eParallel artificial membrane permeability assay; LOD = 1 nM. fThermodynamic aqueous solubility. g Rat liver microsomes.

to low solubility. Hence solubilty in pH 7.4 PBS buffer was quantified using a UV assay at 3 and 24 h. This indicated a moderate solubilty of 66 μg/mL which increased to over 73 μg/mL after stirring for 24 h. Metabolic stability in rat liver 8347



Article

Figure 8. Pharmacokinetics of 24 in Wistar rats. (A) Semilogarithmic plot of mean plasma concentration versus time following a single iv dose of 1 mg/kg and oral dose of 5 mg/kg. Error bars are ± SEM. (B) Intravenous PK parameters. (C) Oral PK parameters. 60 (0.040−0.063 mm) purchased from SiliCycle or Merck. Melting points were determined using an OMEGA (MPA100) automated melting point system. 1H NMR and 13C NMR spectra were recorded on a Bruker AMX400 (400 MHz) NMR spectrometer at ambient atmosphere. The deuterated solvent used was CDCl3 unless otherwise stated. Chemical shifts are reported in parts per million (ppm), and residual undeuterated solvent peaks were used as internal reference: proton (7.26 ppm for CDCl3, 2.50 ppm for DMSO-d6), carbon (77.0 ppm for CDCl3, 39.52 ppm for DMSO-d6). 1H NMR coupling constants (J) are reported in hertz (Hz), and multiplicities are presented as follows: s (singlet), d (doublet), t (triplet), m (multiplet), and br (broad). Low resolution mass spectra were obtained on an Agilent 6130B quadrupole LC/MS in ESI mode with an Agilent 1260 Infinity LC system using a ThermoScientific Hypersil 150 mm × 2.1 mm, 5 μm column. High resolution electrospray ionization (ESI) mass spectra were obtained on a Shimadzu MS-IT-TOF spectrometer. Shimadzu formula predictor software was used to process the data. MS and HRMS were reported in units of mass to charge ratio (m/z). Mass samples were dissolved in CH3CN/DMSO mixture (HPLC grade, 90% CH3CN) unless otherwise stated. The purity of new compounds that underwent biological testing was determined by HPLC using an Agilent 1200 SL instrument. All tested compounds had purity of >95%. The analysis was performed with a flow rate of 0.5 mL/min, an Agilent C-18 column of dimensions 250 mm × 4.6 mm, a particle size of 5 μm, and a loop volume of 20 μL. The detector was set at 254 nm. The mobile phase consisted of phase A (Milli-Q H2O, 18.0 MΩ, TFA 0.05%) and phase B (95% CH3CN, 5% phase A). The gradient elution was performed as reported: 0 min, %B = 5; 0−10 min, %B = 95; 10− 12 min, %B = 95; 12−13 min, %B = 5; 13−15 min, %B = 5. Methyl 4-(4-(7-((2-(Trimethylsilyl)ethoxy)methyl)-7Hpyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)butanoate (6). A mixture of compound 4 (see Supporting Information) (100 mg, 0.317 mmol), potassium carbonate (132 mg, 0.951 mmol, 3.0 equiv), and methyl 4-bromobutanoate (115 mg, 0.634 mmol, 2.0 equiv) in DMF (15 mL) was stirred at room temperature overnight. The reaction mixture was suspended in ethyl acetate (30 mL) and washed with H2O (3 × 30 mL). Drying of the organic phase over Na2SO4 followed by removal of the solvent in vacuo afforded the crude product. The crude was purified by column chromatography (DCM/MeOH, 40:1) to yield 6 (120 mg, 0.289 mmol, 91%) as a colorless oil. LRMS (ESI) m/ z 416.4 [M + H]+. 1H NMR (400 MHz, CDCl3) δ 8.81 (s, 1H), 8.23 (s, 1H), 8.19 (s, 1H), 7.36 (d, J = 3.7 Hz, 1H), 6.77 (d, J = 3.7 Hz, 1H), 5.64 (s, 2H), 4.28 (t, J = 6.7 Hz, 2H), 3.66 (s, 3H), 3.53 (t, J = 8 Hz, 2H), 2.36 (dd, J = 10.7, 3.8 Hz, 2H), 2.27−2.24 (m, 2H), 0.90 (t, J = 8 Hz, 2H), −0.08 (s, 9H). 13C NMR (100 MHz, CDCl3) δ 173.7,

identification of 24 (7-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)1H-pyrazol-1-yl)-N-hydroxyheptanamide, YLB243B), a highly lipophilic efficient compound selectively inhibiting JAK1 and HDACs 1, 2, 3, 6, and 10 with IC50 values of less than 20 nM. JAK1/JAK2 selectivity was about 5-fold with >10-fold selectivity against TYK2 and JAK3. Proposed binding modes in the hinge of JAK1/2 and the substrate site of HDACs1/6 support the observed activity profile. Compound 24 was shown to inhibit JAK2 and HDACs 1−3 in a range of cancer cell types supporting its broad antiproliferative mechanism of action. Low dose pharmacokinetics in rats indicated oral exposures detectable for several hours postdose. Given the low molecular weight and high ligand and lipophilic efficiency, 24 represents a promising lead candidate suitable for optimization. Addition of a methyl group led to 45 (7-(4-(7H-pyrrolo[2,3-d]pyrimidin-4yl)-1H-pyrazol-1-yl)-N-hydroxyoctanamide, YLB343B), with a more selective enzyme profile than 24 but with increased cLogP and increased bioavailability. The highly efficient template shared by 24 and 45 represents an opportunity for dual pathway control with a single molecule. Further optimization could lead to selective suppression of a number of oncogenic targets in a single clinical candidate for the treatment of a range of proliferative disorders.

■

EXPERIMENTAL SECTION

General Experimental. Unless stated otherwise, all nonaqueous reactions were performed in oven-dried round-bottom flasks under an inert nitrogen atmosphere. Commercially available AR grade solvents or anhydrous solvents packed in resealable bottles were used as received. All reaction temperatures stated in the procedures are external bath temperatures unless otherwise stated. Commercial reagents were purchased from Sigma-Aldrich, Alfa Aesar, TCI Chemicals, Combi-Blocks, or Strem Chemicals and used as received without further purification unless otherwise stated. Compound 1 and tubastatin were purchased from Selleckchem (www.selleckchem.com). Compound 3 was prepared previously in our laboratory.13 Compound 2 was prepared in-house. Yields refer to chromatographically and spectroscopically homogeneous materials unless otherwise stated. Reaction progress was monitored by analytical thin layer chromatography (TLC) with 0.25 mm Merck precoated silica gel plates (60F254) using UV light (254 nm) as visualizing agent and ceric ammonium molybdate or potassium permanganate solutions as developing stains. Flash chromatography was performed on silica gel 8348



Article

J = 0.7 Hz, 2H), 8.08 (d, J = 8.4 Hz, 2H), 7.73 (d, J = 4.1 Hz, 1H), 7.29 (d, J = 8.1 Hz, 2H), 6.83 (d, J = 4.1 Hz, 1H), 4.17 (t, J = 7.1 Hz, 2H), 3.64 (s, 3H), 2.36 (s, 3H), 2.26 (t, J = 7.5 Hz, 2H), 1.95−1.80 (m, 2H), 1.65−1.55 (m, 2H), 1.29−1.24 (m, 12H). 13C NMR (100 MHz, CDCl3) δ 174.9, 153.9, 153.0, 152.5, 146.5, 139.7, 135.5, 130.7, 130.5, 128.9, 126.9, 121.3, 116.4, 104.5, 53.4, 52.1, 34.7, 30.8, 29.9, 29.9, 29.8, 29.7, 29.7, 27.2, 25.5, 22.3. Methyl 4-(4-(7H-Pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1yl)butanoate (12). To a RBF was added 6 (120 mg, 0.289 mmol), acetonitrile (20 mL) and H2O (2 mL), and the mixture was degassed three times backfilling with nitrogen each time before being treated with LiBF4 (271 mg, 2.89 mmol, 10.0 equiv) at room temperature. The resulting reaction mixture was heated to gentle reflux for 24 h. After cooling to room temperature, the reaction mixture was concentrated under reduced pressure. The residue was purified by column chromatography (DCM/MeOH, 60:1) to yield 12 (41 mg, 0.144 mmol, 50%) as a colorless oil. LRMS (ESI) m/z 286.3 [M + H]+. 1H NMR (400 MHz, CDCl3) δ 11.44 (s, 1H, NH), 8.83 (s, 1H), 8.26 (d, J = 4.3 Hz, 2H), 7.41 (d, J = 3.6 Hz, 1H), 6.78 (d, J = 3.4 Hz, 1H), 4.30 (t, J = 6.7 Hz, 2H), 3.67 (s, 3H, CH3), 2.38 (dd, J = 10.8, 3.8 Hz, 2H), 2.27 (t, J = 6.9 Hz, 2H). 13C NMR (100 MHz, CDCl3) δ 173.7, 153.0, 151.6, 151.5, 140.2, 131.0, 126.5, 122.1, 114.7, 101.1, 52.4, 46.9, 31.3, 23.4. N-((Tetrahydro-2H-pyran-2-yl)oxy)-5-(4-(7-((2(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-4yl)-1H-pyrazol-1-yl)pentanamide (13). To a solution of 7 (141 mg, 0.328 mmol) in THF (20 mL) and H2O (5 mL) was added LiOH (79 mg, 3.282 mmol, 10.0 equiv). The mixture was stirred overnight at room temperature. When the reaction was deemed complete, as determined by TLC, the mixture was neutralized with 1 M aqueous HCl and concentrated under reduced pressure. To the residue was added DMSO (20 mL), triethylamine (332 mg, 3.282 mmol, 10.0 equiv), HOBT (67 mg, 0.429 mmol, 1.5 equiv), O-(tetrahydro-2Hpyran-2-yl)hydroxylamine (77 mg, 0.656 mmol, 2.0 equiv), and EDC (126 mg, 0.656 mmol, 2.0 equiv), and the mixture was stirred at room temperature overnight. The crude was purified by column chromatography (DCM/MeOH, 40:1) to yield 13 (110 mg, 0.214 mmol, 65%) as a colorless oil. LRMS (ESI) m/z 515.2 [M + H]+. 1H NMR (400 MHz, CDCl3) δ 9.01 (s, 1H), 8.81 (s, 1H), 8.22 (s, 2H), 7.37 (d, J = 3.7 Hz, 1H), 6.79 (d, J = 3.7 Hz, 1H), 5.65 (s, 2H), 4.94 (s, 1H), 4.22 (t, J = 6.8 Hz, 2H), 3.92 (s, 1H), 3.59 (s, 1H), 3.54 (t, J = 8 Hz, 2H), 2.13 (s, 2H), 2.05−1.90 (m, 2H), 1.77−1.54 (m, 8H), 0.90 (t, J = 8.4 Hz, 2H), −0.07 (s, 9H). 13C NMR (100 MHz, CDCl3) δ170.5, 152.8, 152.4, 151.8, 140.0, 130.8, 128.9, 122.1, 114.7, 103.1, 101.6, 73.4, 67.2, 63.2, 52.8, 33.1, 30.1, 28.7, 25.6, 22.9, 19.2, 18.4, −0.8. N-((Tetrahydro-2H-pyran-2-yl)oxy)-8-(4-(7-((2(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-4yl)-1H-pyrazol-1-yl)octanamide (14). To a solution of 10 (220 mg, 0.466 mmol) in THF (30 mL) and H2O (5 mL) was added LiOH (112 mg, 4.66 mmol, 10.0 equiv). The mixture was stirred overnight at room temperature. When the reaction was deemed complete as determined by TLC, the mixture was neutralized with 1 M aqueous HCl and concentrated under reduced pressure. To the residue was added DMSO (20 mL), triethylamine (472 mg, 4.66 mmol, 10.0 equiv), HOBT (95 mg, 0.699 mmol, 1.5 equiv), O-(tetrahydro-2Hpyran-2-yl)hydroxylamine (82 mg, 0.699 mmol, 1.5 equiv), and EDC (179 mg, 0.933 mmol, 2.0 equiv), and the mixture was stirred at room temperature overnight. The crude was purified by column chromatography (DCM/MeOH, 40:1) to yield 14 (169 mg, 0.302 mmol, 65%) as a pale-yellow oil. LRMS (ESI) m/z 557.7 [M + H]+. 1 H NMR (400 MHz, CDCl3) δ 9.09 (s, 1H), 8.80 (s, 1H), 8.21 (d, J = 2.4 Hz, 2H), 7.36 (d, J = 3.7 Hz, 1H), 6.79 (d, J = 3.7 Hz, 1H), 5.64 (s, 2H), 4.93 (s, 1H), 4.19 (t, J = 7.0 Hz, 2H), 3.95−3.85 (m, 1H), 3.60− 3.50 (m, 1H), 3.52 (t, J = 8.0 Hz, 2H), 2.15−1.95 (m, 2H), 1.95−1.85 (m, 2H), 1.80−1.65 (m, 2H), 1.62−1.54 (m, 6H), 1.40−1.25 (m, 6H), 0.90 (t, J = 8.0 Hz, 2H), −0.08 (s, 9H). 13C NMR (100 MHz, CDCl3) δ 171.2, 152.8, 152.5, 151.9, 139.8, 130.6, 128.9, 122.0, 114.7, 103.1, 101.6, 73.4, 67.2, 63.1, 53.3, 33.7, 30.6, 29.3, 29.1, 28.7, 26.9, 25.6, 19.3, 18.4, −0.8.

152.8, 152.5, 151.8, 140.1, 130.8, 128.8, 122.2, 114.7, 101.5, 73.4, 67.2, 52.4, 52.1, 31.2, 26.1, 18.4, −0.8. Methyl 5-(4-(7-((2-(Trimethylsilyl)ethoxy)methyl)-7Hpyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)pentanoate (7). A mixture of 4 (110 mg, 0.501 mmol), potassium carbonate (208 mg, 1.503 mmol, 3.0 equiv), and methyl 5-bromopentanoate (195 mg, 1.002 mmol, 2.0 equiv) in DMF (15 mL) was stirred at room temperature overnight. The crude was purified by column chromatography (DCM/MeOH, 40:1) to yield 7 (202 mg, 0.470 mmol, 94%) as a colorless oil. LRMS (ESI) m/z 430.2 [M + H]+. 1H NMR (400 MHz, CDCl3) δ 8.83 (s, 1H), 8.23 (d, J = 5.3 Hz, 2H), 7.38 (d, J = 3.7 Hz, 1H), 6.80 (d, J = 3.7 Hz, 1H), 5.67 (s, 2H), 4.24 (t, J = 7.0 Hz, 2H), 3.66 (s, 3H), 3.55 (t, J = 8.0 Hz, 2H), 2.37 (t, J = 7.3 Hz, 2H), 2.00−1.96 (m, 2H), 1.71−1.65 (m, 4H), 0.92 (t, J = 8.4 Hz, 2H), −0.06 (s, 9H). 13C NMR (100 MHz, CDCl3) δ 174.2, 152.9, 152.5, 151.9, 140.0, 130.6, 128.9, 122.2, 114.8, 101.7, 73.5, 67.2, 52.9, 52.3, 34.1, 30.3, 22.6, 18.4, −0.8. Ethyl 6-(4-(7-Tosyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)hexanoate (8). A mixture of 5 (100 mg, 0.295 mmol), potassium carbonate (82 mg, 0.589 mmol, 2.0 equiv), and ethyl 6bromohexanoate (132 mg, 0.589 mmol, 2.0 equiv) in DMF (15 mL) was stirred at room temperature overnight. The crude was purified by column chromatography (DCM/MeOH, 40:1) to yield 8 (115 mg, 0.239 mmol, 81%) as a pale-yellow oil. LRMS (ESI) m/z 482.2 [M + H]+. 1H NMR (400 MHz, CDCl3) δ 8.89 (s, 1H), 8.13 (d, J = 1.6 Hz, 2H), 8.09 (d, J = 8.4 Hz, 2H), 7.74 (d, J = 4.1 Hz, 1H), 7.30 (d, J = 8 Hz, 2H), 6.84 (d, J = 4.1 Hz, 1H), 4.19 (t, J = 7.0 Hz, 2H), 4.09 (q, J = 7.1 Hz, 2H), 2.38 (s, 3H), 2.28 (t, J = 7.4 Hz, 2H), 2.00−1.85 (m, 2H), 1.75−1.65 (m, 2H), 1.40−1.30 (m, 2H), 1.21 (t, J = 7.1 Hz, 3H). 13 C NMR (100 MHz, CDCl3) δ 174.1, 153.9, 153.0, 152.5, 146.5, 139.8, 135.6, 130.8, 130.5, 128.9, 127.0, 121.4, 116.5, 104.5, 61.0, 53.1, 34.7, 30.5, 26.7, 25.0, 22.3, 14.9. Methyl 7-(4-(7-Tosyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1Hpyrazol-1-yl)heptanoate (9). A mixture of 5 (68 mg, 0.2 mmol), potassium carbonate (83 mg, 0.6 mmol, 3.0 equiv), and methyl 7bromoheptanoate (134 mg, 0.4 mmol, 2.0 equiv) in DMF (10 mL) was stirred at room temperature overnight. The crude was purified by column chromatography (DCM/MeOH, 45:1) to yield 9 (94 mg, 0.195 mmol, 97%) as a pale-yellow oil. LRMS (ESI) m/z 482.2 [M + H]+. 1H NMR (400 MHz, CDCl3) δ 8.89 (s, 1H), 8.13 (s, 2H), 8.09 (d, J = 8.4 Hz, 2H), 7.74 (d, J = 4.1 Hz, 1H), 7.30 (d, J = 8 Hz, 2H), 6.84 (d, J = 4.1 Hz, 1H), 4.18 (t, J = 7.2 Hz, 2H), 3.64 (s, 3H), 2.38 (s, 3H), 2.28 (t, J = 7.2 Hz, 2H), 2.00−1.85 (m, 2H), 1.65−1.55 (m, 2H), 1.40−1.30 (m, 4H). 13C NMR (100 MHz, CDCl3) δ 174.7, 153.9, 153.0, 152.5, 146.5, 139.8, 135.6, 130.8, 130.5, 128.9, 127.0, 121.4, 116.5, 104.5, 53.3, 52.1, 34.5, 30.6, 29.2, 26.9, 25.3, 22.3. Methyl 8-(4-(7-((2-(Trimethylsilyl)ethoxy)methyl)-7Hpyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)octanoate (10). A mixture of 4 (180 mg, 0.571 mmol), potassium carbonate (158 mg, 1.141 mmol, 2.0 equiv), and methyl 8-bromooctanoate (271 mg, 1.141 mmol, 2.0 equiv) in DMF (20 mL) was stirred at room temperature overnight. The crude was purified by column chromatography (DCM/MeOH, 35:1) to yield 10 (220 mg, 0.466 mmol, 82%) as a pale-yellow oil. LRMS (ESI) m/z 472.5 [M + H]+. 1 H NMR (400 MHz, CDCl3) δ 8.83 (s, 1H), 8.23 (s, 1H), 8.19 (s, 1H), 7.37 (d, J = 3.7 Hz, 1H), 6.80 (d, J = 3.7 Hz, 1H), 5.66 (s, 2H), 4.20 (t, J = 7.1 Hz, 2H), 3.65 (s, 3H), 3.54 (t, J = 8 Hz, 2H), 2.30− 2.27 (m, 2H), 1.95−1.85 (m, 2H), 1.65−1.50 (m, 2H), 1.40−1.25 (m, 6H), 0.91 (t, J = 8 Hz, 2H), −0.06 (s, 9H). 13C NMR (100 MHz, CDCl3) δ 174.8, 152.9, 152.6, 152.0, 139.8, 130.5, 128.8, 122.1, 114.7, 101.6, 73.5, 67.2, 53.4, 52.1, 34.7, 30.8, 29.6, 29.4, 27.1, 25.5, 18.4, −0.8. Methyl 11-(4-(7-Tosyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1Hpyrazol-1-yl)undecanoate (11). A mixture of 5 (57 mg, 0.168 mmol), potassium carbonate (47 mg, 0.336 mmol, 2.0 equiv), and methyl 11-bromoundecanoate (94 mg, 0.336 mmol, 2.0 equiv) in DMF (15 mL) was stirred at room temperature overnight. The crude was purified by column chromatography (DCM/MeOH, 50:1) to yield 11 (77 mg, 0.143 mmol, 85%) as a yellow oil. LRMS (ESI) m/z 538.6 [M + H]+. 1H NMR (400 MHz, CDCl3) δ 8.88 (s, 1H), 8.12 (d, 8349



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4-(4-(7H-Pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-N((tetrahydro-2H-pyran-2-yl)oxy)butanamide (15). To a solution of 12 (41 mg, 0.144 mmol) in THF (20 mL) and H2O (5 mL) was added LiOH (35 mg, 1.44 mmol, 10.0 equiv).The mixture was stirred overnight at room temperature. When the reaction was deemed complete as determined by TLC, the mixture was neutralized with 1 M aqueous HCl and concentrated under reduced pressure. To the residue were added DMSO (10 mL), triethylamine (146 mg, 1.44 mmol, 10.0 equiv), HOBT (29 mg, 0.216 mmol, 1.5 equiv), EDC (55 mg, 0.288 mmol, 2.0 equiv), and O-(tetrahydro-2H-pyran-2-yl)hydroxylamine (26 mg, 0.216 mmol, 2.0 equiv), and the mixture was stirred at room temperature overnight. The crude was purified by column chromatography (DCM/MeOH, 30:1) to yield 15 (39 mg, 0.106 mmol, 74%) as a colorless oil. LRMS (ESI) m/z 371.4 [M + H]+. 1H NMR (400 MHz, CDCl3) δ 11.13 (s, 1H), 10.23 (s, 1H), 8.72 (s, 1H), 8.17 (d, J = 12.3 Hz, 2H), 7.34 (d, J = 2.7 Hz, 1H), 6.68 (s, 1H), 5.05 (s, 1H), 4.30 (s, 2H), 4.05−3.85 (m, 1H), 3.63−3.58 (m, 1H), 2.30−2.20 (m, 2H), 2.25−2.10 (m, 2H), 1.90−1.75 (m, 2H), 1.75−1.50 (m, 4H). 13C NMR (100 MHz, CDCl3) δ 170.2, 152.8, 151.7, 140.1, 131.2, 128.4, 126.3, 122.2, 114.7, 102.9, 101.1, 63.1, 51.7, 30.4, 28.7, 26.9, 25.7, 19.2. 5-(4-(7H-Pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-N((tetrahydro-2H-pyran-2-yl)oxy)pentanamide (16). To a solution of 13 (110 mg, 0.214 mmol) in THF (20 mL) was added TBAF (560 mg, 2.14 mmol, 10.0 equiv). The resulting reaction mixture was heated to gentle reflux for 48 h. After cooling to room temperature, the reaction mixture was concentrated under reduced pressure. The residue was purified by column chromatography (DCM/MeOH, 20:1) to yield 16 (60 mg, 0.156 mmol, 73%) as a colorless oil. LRMS (ESI) m/z 385.2 [M + H]+. 1H NMR (400 MHz, CDCl3) δ 10.51 (s, 1H), 9.01 (s, 1H), 8.80 (s, 1H), 8.23 (d, J = 10.5 Hz, 2H), 7.37 (d, J = 3.6 Hz, 1H), 6.77 (d, J = 3.6 Hz, 1H), 4.96 (s, 1H), 4.23 (t, J = 6.4 Hz, 2H), 4.05−3.80 (m, 1H), 3.65−3.60 (m, 1H), 2.14 (s, 2H), 1.99 (s, 2H), 1.76−1.56 (m, 8H). 6-(4-(7H-Pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-N((tetrahydro-2H-pyran-2-yl)oxy)hexanamide (17). To a solution of 8 (115 mg, 0.239 mmol) in THF (20 mL) and H2O (5 mL) was added LiOH (57 mg, 2.39 mmol, 10.0 equiv). The mixture was stirred overnight at room temperature. When the reaction was deemed complete as determined by TLC, the mixture was neutralized with 1 M aqueous HCl and concentrated under reduced pressure. To the residue were added DMSO (10 mL), triethylamine (242 mg, 2.39 mmol, 10.0 equiv), HOBT (48 mg, 0.359 mmol, 1.5 equiv), O(tetrahydro-2H-pyran-2-yl)hydroxylamine (42 mg, 0.359 mmol, 1.5 equiv), and EDC (92 mg, 0.478 mmol, 2.0 equiv), and the mixture was stirred at room temperature overnight. The crude was purified by column chromatography (DCM/MeOH, 35:1) to yield 17 (20 mg, 0.050 mmol, 21%) as a colorless oil. LRMS (ESI) m/z 399.2 [M + H]+. 1H NMR (400 MHz, CDCl3) δ 10.65 (s, 1H), 9.16 (s, 1H), 8.81 (s, 1H), 8.23 (d, J = 9.3 Hz, 2H), 7.37 (d, J = 2.3 Hz, 1H), 6.78 (d, J = 3.3 Hz, 1H), 4.95 (s, 1H), 4.20 (t, J = 6.3 Hz, 2H), 4.05−3.80 (m, 1H), 3.60−3.55 (m, 1H), 2.00−2.10 (m, 2H), 1.93−1.56 (m, 10H), 1.25−1.40 (m, 2H). 13C NMR (100 MHz, CDCl3) δ 171.0, 152.9, 152.0, 151.8, 140.0, 130.8, 126.2, 122.0, 114.7, 103.2, 101.3, 63.2, 53.0, 33.6, 30.4, 28.7, 26.7, 25.7, 25.3, 19.3. 7-(4-(7H-Pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-N((tetrahydro-2H-pyran-2-yl)oxy)heptanamide (18). To a solution of 9 (94 mg, 0.195 mmol) in THF (10 mL) and H2O (2 mL) was added LiOH (47 mg, 1.952 mmol, 10.0 equiv). The mixture was stirred overnight at room temperature. When the reaction was deemed complete as determined by TLC, the mixture was neutralized with 1 M aqueous HCl and concentrated under reduced pressure. To the residue were added DMSO (10 mL), triethylamine (198 mg, 1.952 mmol, 10.0 equiv), HOBT (40 mg, 0.293 mmol, 1.5 equiv), O(tetrahydro-2H-pyran-2-yl)hydroxylamine (34 mg, 0.293 mmol, 1.5 equiv), and EDC (75 mg, 0.390 mmol, 2.0 equiv), and the mixture was stirred at room temperature overnight. The crude was purified by column chromatography (DCM/MeOH, 35:1) to yield 18 (26 mg, 0.063 mmol, 32%) as a pale-yellow solid. LRMS (ESI) m/z 413.2 [M + H]+. 1H NMR (400 MHz, CDCl3) δ 9.89 (s, 1H), 9.34 (s, 1H), 8.83

(s, 1H), 8.32 (s, 1H), 8.26 (s, 1H), 7.39−7.38 (m, 1H), 6.82 (d, J = 2.3 Hz, 1H), 5.01 (s, 1H), 4.24 (t, J = 6.7 Hz, 2H), 4.05−3.80 (m, 1H), 3.60−3.55 (m, 1H), 2.15−2.00 (m, 2H), 1.93−1.57 (m, 10H), 1.38− 1.32 (m, 4H). 13C NMR (100 MHz, CDCl3) δ 171.2, 153.0, 152.0, 151.8, 139.9, 130.9, 126.3, 122.1, 114.7, 103.1, 101.3, 63.1, 52.8, 33.4, 30.6, 28.8, 28.5, 26.3, 25.7, 19.3. 8-(4-(7H-Pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-N((tetrahydro-2H-pyran-2-yl)oxy)octanamide (19). To a solution of 14 (169 mg, 0.302 mmol) in THF (20 mL) was added TBAF (790 mg, 3.02 mmol, 10.0 equiv). The resulting reaction mixture was heated to gentle reflux for 48 h. After cooling to room temperature, the reaction mixture was concentrated under reduced pressure. The residue was purified by column chromatography (DCM/MeOH, 20:1) to yield 19 (82 mg, 0.192 mmol, 64%) as a colorless oil. LRMS (ESI) m/z 427.5 [M + H]+. 1H NMR (400 MHz, CDCl3) δ 10.91 (s, 1H), 9.27 (s, 1H), 8.82 (s, 1H), 8.25 (d, J = 1.8 Hz, 2H), 7.39 (d, J = 3.5 Hz, 1H), 6.79 (d, J = 3.5 Hz, 1H), 4.98 (s, 1H), 4.19 (t, J = 7.0 Hz, 2H), 3.96 (t, J = 9.0 Hz, 1H), 3.70−3.60 (m, 1H), 2.10−2.00 (m, 2H), 1.91−1.78 (m, 4H), 1.75−1.50 (m, 6H), 1.45−1.20 (m, 6H). 13C NMR (100 MHz, CDCl3) δ 171.3, 153.0, 152.1, 151.9, 139.9, 130.7, 126.1, 122.1, 114.6, 103.3, 101.3, 63.2, 53.3, 33.8, 30.6, 29.4, 29.1, 28.8, 27.0, 25.7, 24.8, 19.3. 11-(4-(7H-Pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-N((tetrahydro-2H-pyran-2-yl)oxy)undecanamide (20). To a solution of 11 (76 mg, 0.141 mmol) in THF (20 mL) and H2O (5 mL) was added LiOH (34 mg, 1.41 mmol, 10.0 equiv). The mixture was stirred at 50 °C overnight. When the reaction was deemed complete as determined by TLC, the mixture was neutralized with 1 M aqueous HCl and concentrated under reduced pressure. To the residue were added DMSO (15 mL), triethylamine (143 mg, 1.41 mmol, 10.0 equiv), HOBT (29 mg, 0.212 mmol, 1.5 equiv), O-(tetrahydro-2Hpyran-2-yl)hydroxylamine (33 mg, 0.282 mmol, 2.0 equiv), and EDC (54 mg, 0.282 mmol, 2.0 equiv), and the mixture was stirred at room temperature overnight. The crude was purified by column chromatography (DCM/MeOH, 30:1) to yield 20 (44 mg, 0.093 mmol, 66%) as a white solid. LRMS (ESI) m/z 469.6 [M + H]+. 1H NMR (400 MHz, CDCl3) δ 11.08 (s, 1H), 9.17 (s, 1H), 8.85 (s, 1H), 8.25 (d, J = 10.3 Hz, 2H), 7.39 (d, J = 3.4 Hz, 1H), 6.80 (s, 1H), 4.98 (s, 1H), 4.20 (t, J = 7.1 Hz, 2H), 3.95 (t, J = 9.4 Hz, 1H), 3.65−3.55 (m, 1H), 2.15−1.95 (m, 2H), 1.96−1.88 (m, 2H), 1.84−1.76 (m, 2H), 1.61−1.57 (m, 6H), 1.29−1.21 (m, 12H). 13C NMR (100 MHz, CDCl3) δ 171.4, 153.0, 152.1, 151.8, 139.9, 130.6, 126.2, 122.1, 114.7, 103.3, 101.3, 63.3, 53.4, 34.1, 30.8, 30.0, 29.8, 29.8, 29.6, 28.7, 27.1, 26.0, 25.7, 19.3. 4-(4-(7H-Pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-Nhydroxybutanamide (21). To a solution of intermediate 15 (39 mg, 0.106 mmol) in 1,4-dioxane (10 mL) was added HCl (75 μL, 0.3 mmol, 4 M in dioxane, 3.0 equiv), and the reaction mixture was stirred at room temperature overnight. The solids were collected by filtration to afford compound 21 (25 mg, 0.873 mmol, 82%) as a pink solid. LRMS (ESI) m/z 287.3 [M + H]+. 1H NMR (400 MHz, CD3OD) δ 8.88 (s, 1H), 8.83 (s, 1H), 8.45 (s, 1H), 7.86 (d, J = 3.6 Hz, 1H), 7.32 (d, J = 3.6 Hz, 1H), 4.40 (t, J = 6.8 Hz, 2H), 2.47−2.10 (m, 4H). 13C NMR (100 MHz, CD3OD) δ176.1, 153.4, 145.5, 144.8, 140.9, 134.6, 132.5, 114.0, 113.9, 104.6, 53.0, 31.5, 26.5. HPLC purity >99%. 5-(4-(7H-Pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-Nhydroxypentanamide (22). To a solution of 16 (60 mg, 0.156 mmol) in 1,4-dioxane (10 mL) was added HCl (117 μL, 0.468 mmol, 4 M in dioxane, 10.0 equiv), and the reaction mixture was stirred at room temperature overnight. The solids were collected by filtration to afford compound 22 (30 mg, 0.100 mmol, 64%) as a pale-yellow solid. LRMS (ESI) m/z 301.1 [M + H]+. 1H NMR (400 MHz, DMSO-d6) δ 12.41 (s, 1H), 10.37 (s, 1H), 8.74 (d, J = 4.1 Hz, 2H), 8.34 (s, 1H), 7.68 (s, 1H), 7.07 (d, J = 2.0 Hz, 1H), 4.24 (t, J = 6.8 Hz, 2H), 2.00 (t, J = 7.2 Hz, 2H), 1.90−1.75 (m, 2H), 1.55−1.43 (m, 2H). 13C NMR (100 MHz, DMSO-d6) δ 168.8, 151.9, 149.2, 148.5, 138.9, 131.6, 127.8, 118.5, 112.5, 100.7, 51.3, 31.7, 29.3, 22.2. HPLC purity >95%. 6-(4-(7H-Pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-Nhydroxyhexanamide (23). To a solution of intermediates 17 (16 mg, 0.040 mmol) in 1,4-dioxane (5 mL) was added HCl (30 μL, 0.120 8350



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mmol, 4 M in dioxane, 3.0 equiv), and the reaction mixture was stirred at room temperature overnight. The solids were collected by filtration to afford compound 23 (7 mg, 0.022 mmol, 55%) as a pale-yellow solid. LRMS (ESI) m/z 315.1 [M + H]+. 1H NMR (400 MHz, DMSO-d6) δ 13.47 (s, 1H), 10.39 (s, 1H), 9.21 (s, 1H), 8.94 (s, 1H), 8.77 (s, 1H), 8.00 (dd, J = 3.6, 2.4 Hz, 1H), 7.42 (dd, J = 3.6, 1.6 Hz, 1H), 4.28 (t, J = 6.9 Hz, 2H), 1.95 (t, J = 7.4 Hz, 2H), 1.87 (dd, J = 14.9, 7.8 Hz, 2H), 1.60−1.48 (m, 2H), 1.30−1.20 (m, 2H). 13C NMR (100 MHz, DMSO-d6) δ 169.1, 151.7, 144.4, 144.1, 139.9, 133.5, 131.1, 112.9, 111.8, 103.2, 51.9, 32.09, 29.2, 25.5, 24.6. HPLC purity >99%. 7-(4-(7H-Pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-Nhydroxyheptanamide (24). To a solution of 18 (26 mg, 0.063 mmol) in 1,4-dioxane (10 mL) was added HCl (47 μL, 0.189 mmol, 4 M in dioxane, 3.0 equiv), and the reaction mixture was stirred at room temperature overnight. The solids were collected by filtration to afford compound 24 (15 mg, 0.046 mmol, 72%) as a pale-yellow solid. LRMS (ESI) m/z 329.2 [M + H]+. HRMS (ESI-MS) m/z [M + H]+ calculated for C16H21N6O2+, 329.1726; found, 329.1722. Mp 180.5− 182.0 °C. 1H NMR (400 MHz, DMSO-d6) δ 12.73 (s, 1H), 10.32 (s, 1H), 8.83 (s, 2H), 8.42 (s, 1H), 7.78 (d, J = 2.3 Hz, 1H), 7.18 (d, J = 2.2 Hz, 1H), 4.24 (t, J = 7.0 Hz, 3H), 1.93 (t, J = 7.0 Hz, 2H), 1.88− 1.78 (m, 2H), 1.54−1.39 (m, 2H), 1.34−1.17 (m, 4H). 13C NMR (100 MHz, DMSO-d6) δ 169.3, 151.8, 147.6, 147.0, 139.1, 132.1, 128.9, 116.5, 112.3, 101.5, 51.7, 32.1, 29.4, 28.0, 25.6, 24.9. HPLC purity >95%. 8-(4-(7H-Pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-Nhydroxyoctanamide (25). To a solution of 19 (82 mg, 0.192 mmol) in 1,4-dioxane (10 mL) was added HCl (144 μL, 0.576 mmol, 4 M in dioxane. 3.0 equiv), and the reaction mixture was stirred at room temperature overnight. The solids were collected by filtration to afford 25 (49 mg, 0.144 mmol, 75%) as a pale-yellow solid. LRMS (ESI) m/z 343.4 [M + H]+. 1H NMR (400 MHz, DMSO-d6) δ 13.19 (s, 1H), 10.33 (s, 1H), 9.07 (s, 1H), 8.91 (s, 1H), 8.63 (s, 1H), 7.94 (s, 1H), 7.34 (s, 1H), 4.27 (t, J = 7.0 Hz, 2H), 1.92 (t, J = 7.3 Hz, 2H), 1.95− 1.81 (m, 2H), 1.50−1.42 (m, 2H), 1.28−1.21 (m, 6H). 13C NMR (100 MHz, DMSO-d6) δ 169.0, 151.6, 145.0, 139.8, 133.2, 130.6, 111.8, 102.7, 51.9, 32.2, 29.4, 28.4, 28.1, 25.7, 25.0. HPLC purity >99%. 11-(4-(7H-Pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-Nhydroxyundecanamide (26). To a solution of 20 (21 mg, 0.045 mmol) in 1,4-dioxane (10 mL) was added HCl (34 μL, 0.135 mmol, 4 M in dioxane, 3.0 equiv), and the reaction mixture was stirred at room temperature overnight. The solids were collected by filtration to afford compound 26 (16 mg, 0.042 mmol, 93%) as a yellow solid. LRMS (ESI) m/z 385.2 [M + H]+. Mp 151.0−153.0 °C. 1H NMR (400 MHz, DMSO-d6) δ 13.25 (s, 1H), 10.31 (b, 1H), 9.11 (s, 1H), 8.91 (s, 1H), 8.68 (s, 1H), 7.95 (s, 1H), 7.35 (s, 1H), 4.27 (t, J = 6.8 Hz, 2H), 1.95− 1.80 (m, 4H), 1.50−1.40 (m, 2H), 1.40−1.05 (m, 12H). 13C NMR (100 MHz, DMSO-d6) δ 169.1, 151.6, 144.9, 144.4, 139.8, 133.3, 130.7, 113.4, 111.7, 102.8, 51.9, 32.2, 29.4, 28.8, 28.6, 28.5, 28.4, 25.8, 25.1, 24.4. HPLC purity >99%. tert-Butyl (4-(3-(4-(7-Tosyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)1H-pyrazol-1-yl)propyl)phenyl)carbamate (27). A mixture of 5 (34 mg, 0.1 mmol), potassium carbonate (28 mg, 0.2 mmol, 2.0 equiv), and tert-butyl (4-(3-iodopropyl)phenyl)carbamate (44 mg, 0.12 mmol, 1.2 equiv) in DMF (10 mL) was stirred at room temperature overnight. The crude was purified by column chromatography (DCM/MeOH, 50:1) to yield 27 (43 mg, 0.075 mmol, 75%) as a white solid. LRMS (ESI) m/z 573.2 [M + H]+. 1H NMR (400 MHz, CDCl3) δ 8.90 (s, 1H), 8.16 (s, 1H), 8.09 (m, 3H), 7.75 (d, J = 4.1 Hz, 1H), 7.29 (m, 4H), 7.08 (d, J = 8.5 Hz, 2H), 6.83 (d, J = 4.1 Hz, 1H), 6.49 (s, 1H), 4.17 (t, J = 7.0 Hz, 2H), 2.58 (t, J = 7.5 Hz, 2H), 2.38 (s, 3H), 2.35−2.15 (m, 2H), 1.51 (s, 9H). 13C NMR (100 MHz, CDCl3) δ 171.8, 153.9, 153.5, 153.0, 152.5, 146.5, 139.9, 137.3, 135.8, 135.6, 131.0, 130.5, 129.6, 129.0, 127.0, 119.6, 116.5, 104.5, 81.1, 52.5, 32.5, 32.2, 29.0, 22.3. 4-(1-(2-(1,3-Dioxolan-2-yl)ethyl)-1H-pyrazol-4-yl)-7-tosyl7H-pyrrolo[2,3-d]pyrimidine (28). A mixture of 5 (200 mg, 0.589 mmol), potassium carbonate (163 mg, 1.178 mmol, 2.0 equiv), and 2-

(2-bromoethyl)-1,3-dioxolane (213 mg, 1.178 mmol, 2.0 equiv) in DMF (10 mL) was stirred at room temperature overnight. The crude was purified by column chromatography (DCM/MeOH, 40:1) to yield 28 (230 mg, 0.524 mmol, 89%) as pale-yellow oil. LRMS (ESI) m/z 440.1 [M + H]+. 3-(4-(7-Tosyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1yl)propanal (29). To a mixture of 28 (190 mg, 0.432 mmol) in acetone (20 mL) was added HCl (2.16 mL, 1 M in H2O, 2.16 mmol, 5.0 equiv), and the reaction mixture was stirred at 55 °C overnight. The mixture was extracted with DCM (3 × 20 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to yield 29 (128 mg, 0.324 mmol, 75%) as pale-yellow oil which was used directly in the next step. LRMS (ESI) m/z 396.1 [M + H]+. Methyl 8-Oxo-8-((4-(3-(4-(7-tosyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)propyl)phenyl)amino)octanoate (30). To a mixture of 27 (32 mg, 0.056 mmol) in ethyl acetate (20 mL) was added H2SO4 (10 μL, 0.168 mmol, 3.0 equiv), and the reaction mixture was stirred at room temperature overnight. The mixture was then neutralized with 5 N aqueous NaHCO3 and extracted with DCM (3 × 20 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to yield 4-(3-(4-(7-tosyl-7Hpyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)propyl)aniline (26 mg, 0.055 mmol, 98%) as a pale-yellow solid which was used directly in the next step. LRMS (ESI) m/z 473.2 [M + H]+. A mixture of 4-(3-(4-(7tosyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)propyl)aniline (121 mg, 0.256 mmol), 8-methoxy-8-oxooctanoic acid (58 mg, 0.307 mmol, 1.2 equiv), triethylamine (52 mg, 0.512 mmol, 2.0 equiv), and HATU (146 mg, 0.384 mmol, 1.5 equiv) in DMF (15 mL) was stirred at 50 °C overnight. The crude was purified by column chromatography (DCM/MeOH, 40:1) to yield 30 (135 mg, 0.21 mmol, 82%) as a brown oil. LRMS (ESI) m/z 643.3 [M + H]+. 1H NMR (400 MHz, CDCl3) δ 8.87 (s, 1H), 8.14 (s, 1H), 8.13−8.05 (m, 3H), 7.72 (d, J = 4.1 Hz, 1H), 7.70 (s, 1H), 7.43 (d, J = 8.4 Hz, 2H), 7.28 (d, J = 8 Hz, 2H), 7.06 (d, J = 8.4 Hz, 2H), 6.81 (d, J = 4.1 Hz, 1H), 4.15 (t, J = 7.0 Hz, 2H), 3.63 (s, 3H), 2.56 (t, J = 7.4 Hz, 2H), 2.35 (s, 3H), 2.34− 2.24 (m, 4H), 2.25−2.15 (m, 2H), 1.72−1.64 (m, 2H), 1.64−1.55 (m, 2H), 1.40−1.28 (m, 4H). 13C NMR (100 MHz, CDCl3) δ 174.8, 172.1, 153.8, 152.9, 152.4, 146.5, 139.9, 137.0, 136.8, 135.4, 130.9, 130.5, 129.4, 128.8, 126.9, 121.2, 120.7, 116.4, 104.4, 52.4, 52.1, 38.0, 34.5, 32.5, 32.1, 29.4, 29.3, 26.0, 25.3, 22.3. Methyl 8-((4-(4-(4-(7H-Pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)butanamido)phenyl)amino)-8-oxooctanoate (31). To a solution of 12 (83 mg, 0.289 mmol) in THF (20 mL) and H2O (5 mL) was added LiOH (69 mg, 2.89 mmol, 10.0 equiv). The mixture was stirred at room temperature overnight. When the reaction was deemed complete as determined by TLC, the mixture was neutralized with 1 M aqueous HCl and concentrated under reduced pressure. To the residue were added DMF (15 mL), N,N-diisopropylethylamine (374 mg, 2.89 mmol, 10.0 equiv), methyl 8-((4-aminophenyl)amino)8-oxooctanoate (89 mg, 0.318 mmol, 1.1 equiv), and HATU (165 mg, 0.434 mmol, 1.5 equiv), and the mixture was stirred at 60 °C overnight. The crude was purified by column chromatography (DCM/ MeOH, 15:1) to yield 31 (87 mg, 0.164 mmol, 57%) as a pink solid. LRMS (ESI) m/z 532.6 [M + H]+. 1H NMR (400 MHz, DMSO-d6) δ 12.06 (s, 1H), 9.83 (s, 1H), 9.75 (s, 1H), 8.67 (d, J = 6.3 Hz, 2H), 8.29 (s, 1H), 7.56 (dd, J = 3.5, 2.4 Hz, 1H), 7.47 (s, 4H), 6.97 (dd, J = 3.6, 1.7 Hz, 1H), 4.28 (t, J = 6.7 Hz, 2H), 3.57 (s, 3H), 2.31−2.23 (m, 6H), 2.20−2.12 (m, 2H), 1.58−1.52 (m, 4H), 1.28 (m, 4H). 13C NMR (100 MHz, DMSO-d6) δ 173.3, 170.8, 169.9, 152.0, 150.9, 150.1, 138.7, 134.6, 134.4, 131.0, 126.5, 120.7, 119.4, 119.3, 112.7, 99.8, 51.1, 51.1, 36.2, 33.2, 33.0, 28.3, 28.2, 25.6, 24.9, 24.3. Methyl 8-Oxo-8-((4-((3-(4-(7-tosyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)propyl)amino)phenyl)amino)octanoate (32). A mixture of 29 (100 mg, 0.253 mmol, 1.5 equiv), methyl 8-((4-aminophenyl)amino)-8-oxooctanoate (47 mg, 0.169 mmol) in MeOH (15 mL) was stirred at room temperature for 3 h. Then sodium cyanoborohydride (53 mg, 0.843 mmol, 5.0 equiv) was added, and the reaction mixture was stirred at room temperature overnight. The mixture was suspended in DCM (20 mL) and washed with H2O (20 mL). Drying of the organic phase over Na2SO4 followed 8351



Article

N1-(4-(3-(4-(7H-Pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1yl)propyl)phenyl)-N8-hydroxyoctanediamide (36). To a solution of 33 (28 mg, 0.049 mmol) in 1,4-dioxane (10 mL) was added HCl (61 μL, 0.244 mmol, 4 M in dioxane, 3.0 equiv), and the reaction mixture was stirred at room temperature overnight. The solids were collected by filtration to afford 36 (9 mg, 0.018 mmol, 38%) as a pale yellow solid. LRMS (ESI) m/z 490.2 [M + H]+. 1H NMR (400 MHz, DMSO-d6) δ 12.23 (s, 1H), 10.32 (s, 1H), 9.78 (s, 1H), 8.71 (d, J = 3.2 Hz, 2H), 8.33 (s, 1H), 7.63−7.59 (m, 1H), 7.50 (d, J = 8.4 Hz, 2H), 7.13 (d, J = 8.5 Hz, 2H), 7.02 (d, J = 2.0 Hz, 1H), 4.23 (t, J = 6.9 Hz, 2H), 2.58−2.48 (m, 2H), 2.26 (t, J = 7.4 Hz, 2H), 2.23−2.13 (m, 2H), 2.00−1.90 (m, 2H), 1.60−1.50 (m, 2H), 1.50−1.45 (m, 2H), 1.33−1.23 (m, 4H). 13C NMR (100 MHz, DMSO-d6) δ 171.0, 169.1, 152.0, 150.1, 149.3, 138.8, 137.3, 135.5, 131.3, 128.4, 127.1, 119.2, 112.6, 100.3, 51.1, 36.3, 36.1, 32.2, 31.4, 31.4, 28.4, 25.0, 25.0. HPLC purity >95%. N1-(4-(4-(4-(7H-Pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1yl)butanamido)phenyl)-N8-hydroxyoctanediamide (37). To a solution of 34 (19 mg, 0.031 mmol) in 1,4-dioxane (5 mL), THF (5 mL) was added HCl (23 μL, 0.93 mmol, 4 M in dioxane, 3.0 equiv), and the reaction mixture was stirred at room temperature overnight. The solids were collected by filtration to afford 37 (10 mg, 0.019 mmol, 61%) as a pink solid. LRMS (ESI) m/z 533.3 [M + H]+. 1H NMR (400 MHz, DMSO-d6) δ 12.95 (s, 1H), 10.18 (s, 1H), 9.88 (s, 1H), 9.78 (s, 1H), 8.97 (s, 1H), 8.88 (s, 1H), 8.55 (s, 1H), 7.87 (s, 1H), 7.70 (s, 1H), 7.47 (s, 4H), 7.27 (s, 1H), 4.35 (t, J = 6.8 Hz, 2H), 2.32−2.23 (m, 6H), 2.25−2.15 (m, 2H), 1.58−1.52 (m, 4H), 1.33− 1.23 (m, 4H). HPLC purity >95%. N1-(4-((3-(4-(7H-Pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1yl)propyl)amino)phenyl)-N8-hydroxyoctanediamide (38). To a solution of 35 (40 mg, 0.068 mmol) in 1,4-dioxane (5 mL) and THF (5 mL) was added HCl (51 μL, 0.204 mmol, 4 M in dioxane, 3.0 equiv), and the reaction mixture was stirred at room temperature overnight. The solids were collected by filtration to afford 38 (8 mg, 0.016 mmol, 23%) as a pink solid. LRMS (ESI) m/z 505.3 [M + H]+. 1 H NMR (400 MHz, DMSO-d6) δ 12.19 (s, 1H), 10.32 (s, 1H), 9.47 (s, 1H), 8.70 (s, 2H), 8.32 (s, 1H), 7.60 (s, 1H), 7.29 (d, J = 8.7 Hz, 2H), 6.99 (d, J = 2.5 Hz, 1H), 6.53 (d, J = 8.5 Hz, 2H), 4.33 (t, J = 6.7 Hz, 2H), 3.02 (t, J = 6.7 Hz, 2H), 2.20 (t, J = 7.3 Hz, 2H), 2.16−2.08 (m, 2H), 1.97−1.89 (m, 2H), 1.54−1.48 (m, 4H), 1.31−1.21 (m, 4H). 13 C NMR (100 MHz, DMSO-d6) δ 170.3, 169.1, 152.0, 150.2, 149.5, 138.8, 131.3, 129.3, 127.0, 120.9, 119.9, 112.7, 112.5, 100.1, 49.5, 40.7, 36.2, 32.2, 29.2, 28.4, 28.4, 25.2, 25.0. HPLC purity >97%. (E)-Methyl 3-(4-((3-(4-(7-Tosyl-7H-pyrrolo[2,3-d]pyrimidin-4yl)-1H-pyrazol-1 -yl)propyl)amino)phenyl)acrylate (39). A mixture of 29 (100 mg, 0.253 mmol, 1.5 equiv) and (E)-methyl 3-(4aminophenyl)acrylate (30 mg, 0.169 mmol) in MeOH (15 mL) was stirred at room temperature for 3 h. Then sodium cyanoborohydride (53 mg, 0.846 mmol, 5.0 equiv) was added, and the reaction mixture was stirred at room temperature overnight. The mixture was suspended in DCM (20 mL) and washed with H2O (20 mL). Drying of the organic phase over Na2SO4 followed by removal of the solvent in vacuo afforded the crude product. The crude was purified by column chromatography (DCM/MeOH, 50:1) to yield 39 (67 mg, 0.120 mmol, 71%) as a pale yellow oil. LRMS (ESI) m/z 557.2 [M + H]+. 1H NMR (400 MHz, CDCl3) δ 8.90 (s, 1H), 8.16 (d, J = 8.3 Hz, 2H), 8.13−8.07 (m, 2H), 7.74 (d, J = 4.1 Hz, 1H), 7.58 (d, J = 15.9 Hz, 1H), 7.35−7.29 (m, 4H), 6.78 (d, J = 4.1 Hz, 1H), 6.54 (d, J = 8.7 Hz, 2H), 6.20 (d, J = 15.9 Hz, 1H), 4.33 (t, J = 6.5 Hz, 2H), 3.77 (s, 3H), 3.22 (t, J = 6.4 Hz, 2H), 2.39 (s, 3H), 2.29−2.17 (m, 2H). 13C NMR (100 MHz, CDCl3) δ 168.9, 153.9, 152.7, 152.5, 150.4, 146.6, 145.8, 140.1, 135.5, 131.1, 130.7, 130.6, 129.0, 127.1, 124.4, 121.7, 116.5, 113.4, 113.2, 104.3, 52.1, 50.7, 41.0, 30.2, 22.4. (E)-3-(4-((3-(4-(7H-Pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol1-yl)propyl)amino)phenyl)-N-((tetrahydro-2H-pyran-2-yl)oxy)acrylamide (40). To a solution of 39 (211 mg, 0.379 mmol) in THF (30 mL) and H2O (10 mL) was added LiOH (91 mg, 3.79 mmol, 10.0 equiv). The mixture was stirred at 50 °C for 24 h, neutralized with 1 M aqueous HCl, and concentrated under reduced pressure. To the residue was added DMF (15 mL), triethylamine (384 mg, 3.79 mmol,

by removal of the solvent in vacuo afforded the crude product. The crude was purified by column chromatography (DCM/MeOH, 15:1) to yield 32 (85 mg, 0.129 mmol, 76%) as a colorless oil. LRMS (ESI) m/z 658.3 [M + H]+. 1H NMR (400 MHz, CDCl3) δ 8.89 (s, 1H), 8.16 (s, 1H), 8.14 (s, 1H), 8.12−8.06 (m, 2H), 7.74 (d, J = 4.2 Hz, 1H), 7.31 (d, J = 8.2 Hz, 3H), 7.25 (d, J = 8.8 Hz, 1H), 7.05 (s, 1H), 6.78 (d, J = 4.1 Hz, 1H), 6.54 (d, J = 8.8 Hz, 1H), 4.32 (t, J = 6.5 Hz, 2H), 3.66 (s, 3H), 3.14 (t, J = 6.3 Hz, 2H), 2.39 (s, 3H), 2.32−2.28 (m, 4H), 2.25−2.15 (m, 2H), 1.77−1.67 (m, 2H), 1.68−1.58 (m, 2H), 1.45−1.30 (m, 4H). 13C NMR (100 MHz, CDCl3) δ 174.9, 171.7, 153.9, 152.8, 152.5, 146.5, 140.1, 135.5, 131.2, 130.5, 129.4, 129.0, 127.1, 122.9, 121.6, 116.5, 114.0, 104.4, 52.2, 50.8, 41.7, 38.1, 34.6, 30.3, 29.5, 29.4, 26.1, 25.4, 22.4. N1-(4-(3-(4-(7H-Pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1yl)propyl)phenyl)-N8-((tetrahydro-2H-pyran-2-yl)oxy)octanediamide (33). To a solution of 30 (120 mg, 0.187 mmol) in THF (20 mL) and H2O (10 mL) was added LiOH (45 mg, 1.87 mmol, 10.0 equiv). The mixture was stirred at 50 °C for 36 h, neutralized with 1 M aqueous HCl, and concentrated under reduced pressure. To the residue were added DMSO (20 mL), triethylamine (189 mg, 1.87 mmol, 10.0 equiv), HOBT (38 mg, 0.28 mmol, 1.5 equiv), O-(tetrahydro-2H-pyran-2-yl)hydroxylamine (44 mg, 0.373 mmol, 2.0 equiv), and EDC (72 mg, 0.373 mmol, 2.0 equiv), and the mixture was stirred at room temperature overnight. The crude was purified by column chromatography (DCM/MeOH, 15:1) to yield 33 (28 mg, 0.049 mmol, 26%) as a pale yellow solid. LRMS (ESI) m/z 574.3 [M + H]+. 1H NMR (400 MHz, CDCl3) δ 9.64 (s, 1H), 8.82 (s, 1H), 8.74 (s, 1H), 8.25 (s, 1H), 8.14 (s, 1H), 7.43 (d, J = 8.3 Hz, 2H), 7.36 (d, J = 3.6 Hz, 2H), 7.12 (d, J = 8.3 Hz, 2H), 6.78 (s, 1H), 5.05− 4.91 (m, 1H), 4.21 (t, J = 6.8 Hz, 2H), 4.03−3.89 (m, 1H), 3.67−3.59 (m, 1H), 2.69−2.59 (m, 2H), 2.33−2.26 (m, 4H), 2.17−2.07 (m, 2H), 1.84−1.74 (m, 2H), 1.65−1.56 (m, 8H), 1.42−1.30 (m, 4H). N1-(4-(4-(4-(7H-Pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1yl)butanamido)phenyl)-N8-((tetrahydro-2H-pyran-2-yl)oxy)octanediamide (34). To a solution of 31 (72 mg, 0.135 mmol) in THF (20 mL) and H2O (5 mL) was added LiOH (32 mg, 1.35 mmol, 10.0 equiv). The mixture was stirred at room temperature overnight. When the reaction was deemed complete as determined by TLC, the mixture was neutralized with 1 M aqueous HCl and concentrated under reduced pressure. To the residue were added DMF (15 mL), triethylamine (137 mg, 1.35 mmol, 10.0 equiv), O-(tetrahydro-2Hpyran-2-yl)hydroxylamine (32 mg, 0.271 mmol, 2.0 equiv), and HATU (78 mg, 0.203 mmol, 1.5 equiv), and the mixture was stirred at room temperature overnight. The crude was purified by column chromatography (DCM/MeOH, 30:1) to yield 34 (19 mg, 0.031 mmol, 23%) as a white solid which was used directly in the next step. N1-(4-((3-(4-(7H-Pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1yl)propyl)amino)phenyl)-N8-((tetrahydro-2H-pyran-2-yl)oxy)octanediamide (35). To a solution of 32 (64 mg, 0.097 mmol) in THF (20 mL) and H2O (5 mL) was added LiOH (23 mg, 0.97 mmol, 10.0 equiv). The mixture was stirred at 50 °C for 48 h. When the reaction was deemed complete as determined by TLC, the mixture was neutralized with 1 M aqueous HCl and concentrated under reduced pressure. To the residue were added DMF (15 mL), triethylamine (98 mg, 0.973 mmol, 10.0 equiv), O-(tetrahydro-2Hpyran-2-yl)hydroxylamine (23 mg, 0.194 mmol, 2.0 equiv), and HATU (56 mg, 0.146 mmol, 1.5 equiv), and the mixture was stirred at room temperature overnight. The crude was purified by column chromatography (DCM/MeOH, 30:1) to yield 35 (28 mg, 0.047 mmol, 48%) as a white solid. LRMS (ESI) m/z 589.3 [M + H]+. 1H NMR (400 MHz, CDCl3) δ 8.66 (s, 1H), 8.21 (d, J = 5.8 Hz, 2H), 7.31 (d, J = 3.6 Hz, 1H), 7.22 (dd, J = 8.9, 2.3 Hz, 2H), 6.68 (d, J = 3.6 Hz, 1H), 6.51 (d, J = 8.6 Hz, 2H), 4.88 (s, 1H), 4.30 (t, J = 6.6 Hz, 2H), 4.00−3.85 (m, 1H), 3.60−3.50 (m, 1H), 3.08 (t, J = 6.5 Hz, 2H), 2.23 (t, J = 7.4 Hz, 2H), 2.30−2.10 (m, 2H), 2.15−1.95 (m, 2H), 1.73 (t, J = 9.7 Hz, 3H), 1.61−1.51 (m, 7H), 1.35−1.25 (m, 4H). 13C NMR (100 MHz, CDCl3) δ 172.7, 171.8, 152.3, 151.5, 151.3, 145.6, 140.1, 131.3, 129.1, 126.7, 122.8, 121.8, 114.7, 113.7, 102.9, 101.0, 63.0, 41.4, 37.4, 33.4, 30.2, 28.8, 28.7, 28.5, 25.9, 25.6, 19.1. 8352



Article

7-(4-(7H-Pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-Nhydroxyoctanamide (45). To a solution of 44 (101 mg, 0.237 mmol) in 1,4-dioxane (10 mL) was added HCl (132 μL, 0.711 mmol, 4 M in dioxane), and the reaction mixture was stirred at room temperature overnight. The reaction mixture was concentrated to afford compound 45 (75 mg, 0.218 mmol, 92%) as a pale-yellow solid. LRMS (ESI) m/z 343.2 [M + H]+. HRMS (ESI-MS) m/z [M + H]+ calculated for C17H23N6O2+, 343.1882; found, 343.1879. 1H NMR (400 MHz, DMSO-d6) δ13.27 (s, 1H), 10.28 (s, 1H), 9.13 (s, 1H), 8.91 (s, 1H), 8.69 (s, 1H), 7.96−7.94 (m, 1H), 7.40−7.37 (m, 1H), 4.59−4.49 (m, 1H), 1.90 (t, J = 7.3 Hz, 2H), 1.83−1.70 (m, 2H), 1.50 (d, J = 6.7 Hz, 3H), 1.48−1.38 (m, 2H), 1.34−1.14 (m, 4H). 13C NMR (100 MHz, DMSO-d6) δ169.0, 151.6, 144.7, 144.4, 139.7, 131.9, 130.7, 111.7, 102.9, 58.4, 35.8, 32.1, 28.1, 25.1, 24.9, 20.9. HPLC purity >99%. Molecular Modeling Experimental. The human JAK1 X-ray structure (4IVB)40 JAK2 X-ray structure (3FUP),22 human HDAC1 structure (4BKX),41 human HDAC2 structure (4LXZ),42 and zebrafish HDAC6 structures (5EEI (second catalytic domain) and 5G0G (first catalytic domain))43 were downloaded from the Protein Data Bank (http://www.rcsb.org) and prepared using the Protein Preparation Wizard in Maestro version 10.6 using standard settings.44 This included the addition of hydrogen atoms, bond assignments, removal of water molecules further than 5 Å from the ligand, protonation state assignment, optimization of the hydrogen bond network, and restrained minimization using the OPLS3 force field.45 The HDAC proteins were superimposed using structural alignment, and the HDAC2 structure ligand 2 was duplicated into HDAC1. The HDAC6 protein sequence (identifier Q9UBN7) was downloaded from Uniprot (http://www.uniprot.org). A human HDAC6 homology model of each of the catalytic domains was built using the zebrafish structures as template. The homology between human and zebrafish HDAC6 catalytic domains is 75% with 60% sequence identity and 0% gaps, and alignments are shown in Supporting Information. The energy based algorithm of Prime version 4.444 with default settings was used for homology building. Inhibitors and metal ions were included in the models. The cocrystallized or soaked ligands were used as templates for manual docking. For HDAC modeling, the hydroxamate and linker part of 2 were retained and the remaining part of ligands 24 and 45 were built upon this template. In the JAK1 and JAK2 structures the pyrrolopyrimidine hinge binder was used as template. The JAK1, JAK2, HDAC1, and HDAC6 inhibitor−protein complexes were finally minimized using Macromodel 11.2.44 In the HDAC complexes, distance constraints between both the hydroxamate oxygen atoms and zinc (2.3 Å) as well as the three residues that hydrogen-bond with the hydroxamate (2.8 Å) were included. All residues more than 9 Å from the ligand were constrained before the complex was subjected to 500 steps of Polak−Ribiere conjugate gradient46 minimization using the OPLS3 force field and GB/SA continuum solvation model.47 Enzyme Assays. Enzyme inhibition assays for HDAC and kinase enzymes (kinase “Hotspot” profiling) were carried out by Reaction Biology Corporation (RBC) using an ATP concentration of 10 μM, and kinase panel profiling was carried out by DiscoveRx, as previously described.13 Affinity-LC/MS (WAC) was carried out as follows: JAK2 (20 mM Tris-HCl, pH 8.0, 10% glycerol, and 0.4 M urea) protein was purchased from Prospec Bio, Israel. All reagents used were analytical grade. Compounds were dissolved in dimethyl sulfoxide (DMSO; Merck, NJ, USA) at a concentration of either 10 or 20 mM based on solubility. Samples for affinity-LC/MS (WAC) were prepared by further dilution of compounds to a working concentration between 10 and 50 μM in Milli-Q water. For chromatography, 20 mM ammonium acetate (AmAc; pH 6.8) (Merck, NJ, USA) was used as mobile phase. For protein immobilization, periodic acid and sodium cyanoborohydride (NaBH3CN) were purchased from Sigma-Aldrich (MO, USA). Preparation of Protein Affinity Columns. Spherical porous diol silica particles (average diameter of 5 μm, 300 Å pore size, and a surface area of 100 m2/g) from Kromasil, EKA Chemicals, Bohus, Sweden, were used as supporting matrix. JAK2 was immobilized on the

10.0 equiv), O-(tetrahydro-2H-pyran-2-yl)hydroxylamine (89 mg, 0.758 mmol, 2.0 equiv), and HATU (217 mg, 0.569 mmol, 1.5 equiv), and the mixture was stirred at room temperature overnight. The crude was purified by column chromatography (DCM/MeOH, 30:1) to yield 40 (105 mg, 0.215 mmol, 57%) as a pale yellow solid. LRMS (ESI) m/z 488.2 [M + H]+. 1H NMR (400 MHz, CDCl3) δ 11.11 (s, 1H), 8.73 (s, 1H), 8.24 (s, 1H), 8.18 (s, 1H), 7.54 (d, J = 15.7 Hz, 1H), 7.37−7.27 (m, 1H), 7.25−7.15 (m, 2H), 6.65 (d, J = 3.5 Hz, 1H), 6.51−6.41 (m, 2H), 6.16 (br, 1H), 4.99 (s, 1H), 4.29 (t, J = 6.5 Hz, 2H), 4.04−3.92 (m, 1H), 3.63−3.57 (m, 1H), 3.18−3.08 (m, 2H), 2.80−2.45 (br m, 2H), 2.25−2.09 (m, 2H), 1.86−1.72 (m, 2H), 1.60−1.45 (m, 2H). 13C NMR (100 MHz, CDCl3) δ 152.7, 152.5, 151.5, 150.3, 140.1, 131.2, 130.4, 126.7, 126.5, 124.2, 122.0, 114.7, 113.0, 112.7, 103.5, 101.0, 63.3, 50.5, 40.6, 30.0, 28.7, 25.6, 19.3. (E)-3-(4-((3-(4-(7H-Pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol1-yl)propyl)amino)phenyl)-N-hydroxyacrylamide (41). To a solution of 40 (105 mg, 0.215 mmol) in 1,4-dioxane (10 mL), THF (10 mL) was added HCl (161 μL, 0.645 mmol, 4 M in dioxane, 3.0 equiv), and the reaction mixture was stirred at room temperature overnight. The solids were collected by filtration to afford 41 (78 mg, 0.193 mmol, 90%) as a yellow solid. LRMS (ESI) m/z 404.2 [M + H]+. 1H NMR (400 MHz, DMSO-d6) δ 13.47 (s, 1H), 9.28 (s, 1H), 8.95 (s, 1H), 8.80 (s, 1H), 8.01 (dd, J = 3.6, 2.4 Hz, 1H), 7.42 (dd, J = 3.5, 1.5 Hz, 1H), 7.38−7.30 (m, 3H), 6.82 (s, 2H), 6.24 (d, J = 15.4 Hz, 1H), 4.44 (t, J = 6.7 Hz, 2H), 3.18−3.12 (m, 2H), 2.24−2.16 (m, 2H). 13C NMR (100 MHz, DMSO-d6) δ 163.5, 151.6, 143.8, 143.4, 140.3, 138.5, 134.1, 131.4, 128.9, 114.5, 112.3, 111.6, 103.3, 49.8, 48.6, 28.3. HPLC purity >95%. Methyl 7-(4-(7-Tosyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1Hpyrazol-1-yl)octanoate (43). A mixture of 5 (265 mg, 0.78 mmol), potassium carbonate (216 mg, 1.56 mmol), and intermediate ester 42 (443 mg, 1.56 mmol) in DMF (20 mL) was stirred at room temperature overnight. The reaction mixture was suspended in ethyl acetate (30 mL) and washed with H2O (3 × 30 mL). Drying of the organic phase over Na2SO4 followed by removal of the solvent in vacuo afforded the crude product. The crude was purified by column chromatography (ethyl acetate/hexane, 1:2) to yield 43 (114 mg, 0.363 mmol, 46%) as a colorless oil. LRMS (ESI) m/z 496.2 [M + H]+. 1H NMR (400 MHz, CDCl3) δ 8.90 (s, 1H), 8.21 (s, 1H), 8.14 (s, 1H), 8.09 (d, J = 8.4 Hz, 2H), 7.76 (d, J = 4.1 Hz, 1H), 7.30 (d, J = 8.1 Hz, 2H), 6.86 (d, J = 4.1 Hz, 1H), 4.39−4.35 (m, 1H), 3.63 (s, 3H), 2.38 (s, 3H), 2.25 (t, J = 7.5 Hz, 2H), 2.01−1.86 (m, 1H), 1.86− 1.69 (m, 1H), 1.60−1.50 (m, 5H), 1.35−1.20 (m, 4H). 13C NMR (100 MHz, CDCl3) δ174.0, 153.0, 152.2, 151.8, 145.8, 138.8, 134.9, 129.8, 128.6, 128.3, 126.4, 115.7, 103.9, 59.1, 51.4, 36.7, 33.8, 28.6, 25.7, 24.6, 21.7, 21.2. 7-(4-(7H-Pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-N((tetrahydro-2H-pyran-2-yl)oxy)octanamide (44). To a solution of 43 (180 mg, 0.363 mmol) in THF (20 mL) and H2O (5 mL) was added LiOH (87 mg, 3.63 mmol, 10 equiv). The mixture was stirred at room temperature overnight. When the reaction was deemed complete as determined by TLC, the mixture was neutralized with 1 M aqueous HCl and concentrated under reduced pressure. To the residue were added DMSO (15 mL), triethylamine (367 mg, 3.63 mmol), HOBT (74 mg, 0.545 mmol), O-(tetrahydro-2H-pyran-2-yl)hydroxylamine (85 mg, 0.726 mmol), and EDC (139 mg, 0.726 mmol), and the mixture was stirred at room temperature overnight. The reaction mixture was suspended in ethyl acetate (30 mL) and washed with H2O (3 × 30 mL). Drying of the organic phase over Na2SO4 followed by removal of the solvent in vacuo afforded the crude product. The crude was purified by column chromatography (DCM/MeOH, 30:1) to yield 44 (101 mg, 0.237 mmol, 65%) as a pale yellow oil. LRMS (ESI) m/z 427.2 [M + H]+. 1H NMR (400 MHz, CDCl3) δ11.06 (s, 1H), 8.84 (s, 1H), 8.37 (d, J = 18.0 Hz, 1H), 8.26 (s, 1H), 7.41 (d, J = 3.1 Hz, 1H), 6.82 (d, J = 1.9 Hz, 1H), 5.02−4.96 (m, 1H), 4.43−4.38 (m, 1H), 4.01−3.83 (m, 1H), 3.65−3.50 (m, 1H), 2.05−1.95 (m, 2H), 1.87−1.72 (m, 4H), 1.70−1.48 (m, 8H), 1.40−1.03 (m, 8H). 13C NMR (100 MHz, CDCl3) δ170.6, 152.3, 151.0, 150.7, 139.0, 128.6, 125.8, 120.5, 113.9, 102.3, 100.9, 62.5, 58.7, 36.8, 36.7, 32.6, 29.7, 28.0, 25.0, 21.3, 18.6. 8353



Article

tprotein (tJAK2) = tR of the analyte on the affinity column and tRef (taldehyde or tethanolamine) = tR on the reference column.27

silica by the Schiff base method. Before immobilization, the storage buffer of JAK2 (20 mM Tris-HCl, pH 8.0, 10% glycerol, and 0.4 M urea) was exchanged with 100 mM sodium phosphate, 300 mM sodium chloride, pH 7.5 (PBS), by centrifugation using Amicon Ultra0.5 centrifugal filter (Merck Millipore, Darmstadt, Germany) with molecular weight cutoff of 3000 Da. The centrifugation was performed three times (14 000g for 30 min each) at 4 °C with PBS (500 μL). The concentration of JAK2 was measured after buffer exchange based on absorbance at 280 nm measured on a NanoDrop 2000 (Thermo Fisher Scientific, Wilmington, DE, USA). The diol silica was first oxidized with periodic acid, 100 mg/mL for 2 h at room temperature (22 °C) to produce aldehyde silica. After oxidation, the aldehyde silica slurry was allowed to react with JAK2 (0.28 mg/mL, 214 μL final slurry) and NaBH3CN (final concentration of 10 mg/mL) for 116 h on a rotator at 4 °C. The reaction was stopped by centrifugation (2000g for 2 min) and the supernatant removed. The resulting slurry was then washed with PBS (5 × 200 μL). The supernatant and waste were pooled together, and absorbance was measured at 280 nm on a NanoDrop 2000 to indirectly calculate the immobilization yield. Two reference columns with the same dimensions were produced by a similar procedure except 200 μL PBS (to produce aldehyde reference column) or 200 μL 1 M ethanolamine (to produce reference ethanolamine column) was used during immobilization instead of the JAK2 solution. The resulting silica with immobilized JAK2 was packed into a stainless steel capillary column (35 mm × 0.5 mm) by an air-driven liquid pump (Haskel MS-110, Burbank, CA, USA) at 340 bar in 1 h using PBS as packing mobile phase. Columns were stored in 0.01% sodium azide in PBS (pH 7.4) at 4 °C when not in use. Zonal Screening of Compounds on Single Quad LC/MS. Compounds were tested on a LC/MS system fitted with a combination of HPLC (Agilent 1260 infinity) and a single quadrupole mass spectrometry (MS) detector (Agilent 6130B; Agilent Technologies, Germany). Ultraviolet (UV) detection was done at two different wavelengths of 214 nm for DMSO and 254 nm for other compounds (reference wavelength as 360 ± 4 nm). For MS detection, compound ionization was achieved by atmospheric pressure ionization electrospray (API-ES) in positive mode. MS parameters were set with dry gas temperature of 300 °C, nitrogen (N2) gas flow at 8 L/min and a nebulizer pressure of 25 psig. Capillary voltage of 3000 V and fragmentor of 110 V were used in positive mode. Compounds were evaluated in selected ion monitoring (SIM) mode with m/z set at [M + H]+ ions for each analyte based on their identification during study on the reference column. Samples were analyzed at a flow rate of 20 μL/min on JAK2 and 15 μL/min on the reference column in 20 mM AmAc (pH 6.8) mobile phase with an injection volume of 0.4 μL. Chromatograms were acquired and analyzed with the Agilent OpenLab version A.02.01 (1.3.3) chromatography data system. Compounds were analyzed at a final concentration of 10 μM (with 0.05% DMSO) except for 41 which was tested at 50 μM (0.25% DMSO). Reference sample S13 (see Supporting Information) was injected intermittently as a positive control to monitor the performance of the affinity column. Additionally, DMSO was monitored in each run as a void marker. Individual compounds were tested in triplicate (n = 3). Compounds were analyzed on the reference columns for 60−90 and 90 min (except for 41 which was run for 300 min) on the affinity column. Data Acquisition. Compound retention times (tR) were obtained based on the peak apex of the extracted ion chromatograms (EIC). Calculation of apparent dissociation constant (KD) of the analytes was based on their affinity toward the binding sites available on the protein surface as mentioned in eq 1. KD =

Btot tnetF

tnet = t JAK2 − t Ref

(2)

tJAK2 or tRef was calculated by subtraction of the tR of the void marker (DMSO) from the retention of the analyte on the affinity and reference columns, respectively. This is mathematically expressed as t JAK2 = tcompound − t DMSO

(3)

t Ref = tcompound − t DMSO

(4)

The coupling yield for the JAK2 affinity-LC/MS column was estimated at 10.3 mg/g (1.95 nmol) corresponding to a protein density of 0.33 mM. The percent loading of protein onto the column, expressed as Btot, was estimated at 50% (0.97 nmol).48 On the basis of the immobilization achieved on the JAK2 column and the analysis time, the effective affinity range that can be quantified is approximately within the range of 0.6−125 μM. Cellular Assays. Cell Proliferation Inhibition Assays. Human breast cancer cell lines MCF-7 and MDA-MB231, prostate cancer cell line PC-3, and colon cell line HCT-116 were purchased from ATCC (Rockville, MD) and assays carried out as previously described.13 Acute myeloid leukemia (AML) HL-60, erythroleukemia HEL92.1.7, acute T-cell leukemia jurkat, multiple myeloma cell lines KMS12BM, OPM2, XG-6, KG-1, AML cell lines KG1, and MOLM14 and NKTcell lymphoma cell lines NKYS and KHYG were used as for assays previously described.13 Immunoblotting. Western blot analyses of KMS-12-BM and MOLM-14 cell lines were performed as previously described.13 Determination of Toxicity in Transforming Growth Factor α Mouse Hepatocyte (TAMH) and AC10 Human Cardiomyocyte Cells. TAMH cell assay was carried out as previously described.13 AC10 cells were seeded at a cell density of 5000 cells/well (75 000 cells/ mL) in a 96-well plate (NUNC) a day before drug treatment. Cells were then treated across a wide concentration range starting from a top concentration of 100 μM (final DMSO %: 0.5%, 0.5 μL of drug in 99.5 μL of media in every well). Stock concentrations used were 100 μM, 10 μM, and 1 μM), and final dilute concentrations were 100, 50, 25, 12.5, 6.25, 3.125, 1.5, 0.78, and 0.315 μM. Drug-treated cells were then incubated at 37 °C for 24 h. After 24 h, cell viability was determined with CellTiter-Glo cell viability assay (Promega Corporation) as per manufacturer’s instructions. The cell−reagent mixture was then transferred to a solid white flat-bottom 96-well plate (Greiner) for luminescence reading. Luminescence was then recorded with an integration time of 0.25 s with a Tecan Infinite M200 microplate reader. On the basis of the analyses from the abovementioned concentration range, cell viability assay was repeated with a more focused drug concentration range as required to obtained consistent IC50 data. Determination of in Vitro Metabolic Stability in Male (MRLM) and Female Rat Liver Microsomes (FRLM). Liver microsomal incubations were conducted in triplicate as previously described.13 Aqueous Solubility. A 20 mM stock solution of 24 was prepared in DMSO. Then 10 μL of the stock solution was added to 990 μL of buffer. The mixture was sonicated and vortexed. From the resulting turbid solution, 300 μL was transferred to three wells of a MultiScreen HTS- PCF filter plate. The plate was covered and incubated with gentle shaking (250 rpm, 3 or 24 h) at room temperature (22.5 ± 2.5 °C). After the period of incubation, the filter plate was placed on a vacuum manifold and the contents were filtered into a 96-well UV plate. After filtration, 80 μL of filtrate was transferred from each well to another well in a 96-well UV plate. Acetonitrile (20 μL) was added to each well, and the sample solutions were further diluted by adding another 200 μL of universal buffer. Absorbances of the solutions were read at 310 nm. A calibration curve was determined with concentrations of 200, 100, 50, 25, 12.5, and 6.25 μM and the solubility of the samples determined in triplicate. Pharmacokinetics in BALB/c Mice. This work was carried out by TheraIndx Lifesciences Pvt Ltd., India. This study was performed

(1)

Equation 1 is valid when Ca ≪ KD, where Ca (μM) indicates the concentration of the analyte, Btot is the amount of available sites on the affinity column (nmol), F is the flow rate (μL/min), and tnet is the net retention time (min). Estimation of tnet was done as per eq 2, where 8354



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following all ethical practices as laid down in the guidelines for animal care (Registration No. 1852/PO/Rc/S/16/CPCSEA). The study was approved by the Institutional Animals Ethics Committee (IAEC) of the test facility (Protocol No. IAEC/01/2016/024). The pharmacokinetics (PK) of 24 and 45 were characterized in BALB/c mice following single intravenous (iv) and oral (po) doses of 2 and 10 mg/ kg, respectively. Animals were housed at a constant temperature (range 22 ± 2 °C) and humidity (range 45−60%). Animals were provided with 12 h light and 12 h dark artificial photoperiod. For po dosing suspensions, the vehicle used was 0.25% CMC containing 0.25% Tween 80 in in water. For iv dosing solutions (2 mg/kg), the vehicle used was DMSO/PEG400/ethanol (absolute)/sterile water at ratio of 10:40:20:30 (v/v). A clear solution of concentration 0.4 mg/ mL was obtained. The formulations were prepared on the day of the study. Groups of animals (n = 12/route) were administered single iv bolus doses of 2.0 mg/kg (5 mL/kg dose volume) or oral dose of 10 mg/kg by gavage, at a dose volume of 10 mL/kg. Postdose, blood samples were collected from mice at 2 min (iv) and 5 min (oral), 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, and 24 h. At the indicated time points, and prior to sampling, mice were anesthetized with isoflurane before sampling. From each mouse, two blood samples were drawn; the first was nonterminal (∼0.1 mL blood) done by saphenous vein puncture and the second terminal, by retro-orbital plexus puncture using heparinized mice capillary tubes. Blood was collected in 2 mL Eppendorf tubes containing 0.01 mL of 10% K 2 EDTA as anticoagulant, mixed gently, and placed in ice before centrifugation. Blood was centrifuged at 10 000 rpm for 5 min, and plasma was harvested and stored at −80°C until analysis. Levels of each compound in plasma were quantified by LSMSMS using a Shimadzu SIL-HTc with an Atlantis dC18 (4.6 mm × 50 mm, 3 μm) column and a API-5500 (ABS Sciex) mass spectrometer. Mobile phase used was acetonitrile and 0.2% formic acid in Milli-Q water.

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Article

AUTHOR INFORMATION

Corresponding Authors

*S.O.: e-mail, [email protected]. *B.W.D.: e-mail, [email protected]. ORCID

Anders Poulsen: 0000-0002-2790-9340 Brian W. Dymock: 0000-0002-2374-5756 Author Contributions ∞

N.M. and E.C.T. contributed equally.

Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS The authors gratefully acknowledge Sheela David Packiaraj and Associate Professor Ho Han Kiat for providing TAMH and AC10 data and Dr Yang Shilli and Professor Paul Ho for RLM data. This research was funded by a generous grant from the National Medical Research Council of Singapore (NMRC CBRG Grant NMRC/CBRG12Nov114 (B.W.D./S.O.)). W.J.C. is supported by a NMRC Clinician Scientist Investigator Award and partly supported by a Singapore Cancer Syndicate Grant, the National Research Foundation Singapore, and the Singapore Ministry of Education under the Research Centers of Excellence initiative. J.J.Y.Y. is supported by Research Grant NSC 102-2320-B-001-007-MY3 from the Ministry of Science and Technology, Taiwan.

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ABBREVIATIONS USED DML, designed multiple ligand; JAK, Janus kinase; HDAC, histone deacetylase

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.7b00678. Additional synthesis of compounds, NMR and HPLC spectra for final compounds, additional immunoblotting data, selected IC50 curves, RLM and oral PK data for 24, docking of 45 to HDAC1/6 and JAK1/2, and PK of 45 (PDF) Molecular formula strings and some data (CSV) Coordinates information for structure representation (PDB) Coordinates information for structure representation (PDB) Coordinates information for structure representation (PDB) Coordinates information for structure representation (PDB) Coordinates information for structure representation (PDB) Coordinates information for structure representation (PDB) Coordinates information for structure representation (PDB) Coordinates information for structure representation (PDB) Coordinates information for structure representation (PDB) Coordinates information for structure representation (PDB)

REFERENCES

(1) Stewart, B. W.; Wild, C. P. World Cancer Report 2014. World Health Organization, International Agency for Research on Cancer, 2014. (2) Al-Lazikani, B.; Banerji, U.; Workman, P. Combinatorial drug therapy for cancer in the post-genomic era. Nat. Biotechnol. 2012, 30, 679−692. (3) Anighoro, A.; Bajorath, J.; Rastelli, G. Polypharmacology: challenges and opportunities in drug discovery. J. Med. Chem. 2014, 57, 7874−7887. (4) Kiu, H.; Nicholson, S. E. Biology and significance of the JAK/ STAT signalling pathways. Growth Factors 2012, 30, 88−106. (5) Harrison, C.; Kiladjian, J. J.; Al-Ali, H. K.; Gisslinger, H.; Waltzman, R.; Stalbovskaya, V.; McQuitty, M.; Hunter, D. S.; Levy, R.; Knoops, L.; Cervantes, F.; Vannucchi, A. M.; Barbui, T.; Barosi, G. JAK inhibition with ruxolitinib versus best available therapy for myelofibrosis. N. Engl. J. Med. 2012, 366, 787−798. (6) Dowty, M. E.; Jesson, M. I.; Ghosh, S.; Lee, J.; Meyer, D. M.; Krishnaswami, S.; Kishore, N. Preclinical to clinical translation of tofacitinib, a Janus kinase inhibitor, in rheumatoid arthritis. J. Pharmacol. Exp. Ther. 2014, 348, 165−173. (7) Kim, M. K.; Shin, H.; Park, K.-s.; Kim, H.; Park, J.; Kim, K.; Nam, J.; Choo, H.; Chong, Y. Benzimidazole derivatives as potent JAK1selective inhibitors. J. Med. Chem. 2015, 58, 7596−7602. (8) Dymock, B. W.; Yang, E. G.; Chu-Farseeva, Y.; Yao, L. Selective JAK inhibitors. Future Med. Chem. 2014, 6, 1439−1471. (9) Gao, S. M.; Chen, C. Q.; Wang, L. Y.; Hong, L. L.; Wu, J. B.; Dong, P. H.; Yu, F. J. Histone deacetylases inhibitor sodium butyrate inhibits JAK2/STAT signaling through upregulation of SOCS1 and SOCS3 mediated by HDAC8 inhibition in myeloproliferative neoplasms. Exp. Hematol. 2013, 41, 261−270. (10) Quintás-Cardama, A.; Kantarjian, H.; Estrov, Z.; Borthakur, G.; Cortes, J.; Verstovsek, S. Therapy with the histone deacetylase

8355



Article

inhibitor pracinostat for patients with myelofibrosis. Leuk. Res. 2012, 36, 1124−1127. (11) Wang, H.; Dymock, B. W. New patented histone deacetylase inhibitors. Expert Opin. Ther. Pat. 2009, 19, 1727−1757. (12) Marks, P. A.; Breslow, R. Dimethyl sulfoxide to vorinostat: development of this histone deacetylase inhibitor as an anticancer drug. Nat. Biotechnol. 2007, 25, 84−90. (13) Yang, E. G.; Mustafa, N.; Tan, E. C.; Poulsen, A.; Ramanujulu, P. M.; Chng, W. J.; Yen, J. J.; Dymock, B. W. Design and synthesis of janus kinase 2 (JAK2) and histone deacetlyase (HDAC) bispecific inhibitors based on pacritinib and evidence of dual pathway inhibition in hematological cell lines. J. Med. Chem. 2016, 59, 8233−8262. (14) Zwergel, C.; Stazi, G.; Valente, S.; Mai, A. Histone deacetylase inhibitors: updated studies in various epigenetic-related diseases. J. Clin. Epigenet. 2016, 2, 1−15. (15) Guha, M. HDAC inhibitors still need a home run, despite recent approval. Nat. Rev. Drug Discovery 2015, 14, 225−226. (16) Evrot, E.; Ebel, N.; Romanet, V.; Roelli, C.; Andraos, R.; Qian, Z.; Dolemeyer, A.; Dammassa, E.; Sterker, D.; Cozens, R.; Hofmann, F.; Murakami, M.; Baffert, F.; Radimerski, T. JAK1/2 and Pandeacetylase inhibitor combination therapy yields improved efficacy in preclinical mouse models of JAK2V617F-driven disease. Clin. Cancer Res. 2013, 19, 6230−6241. (17) de Lera, A. R.; Ganesan, A. Epigenetic polypharmacology: from combination therapy to multitargeted drugs. Clin. Epigenet. 2016, 8, 1− 21. (18) Bieliauskas, A. V.; Pflum, M. K. Isoform-selective histone deacetylase inhibitors. Chem. Soc. Rev. 2008, 37, 1402−1413. (19) 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. Mutation of Jak3 in a patient with SCID: essential role of Jak3 in lymphoid development. Science 1995, 270, 797−800. (20) Alicea-Velazquez, N. L.; Boggon, T. J. The use of structural biology in Janus kinase targeted drug discovery. Curr. Drug Targets 2011, 12, 546−555. (21) Lai, C. J.; Bao, R.; Tao, X.; Wang, J.; Atoyan, R.; Qu, H.; Wang, D. G.; Yin, L.; Samson, M.; Forrester, J.; Zifcak, B.; Xu, G. X.; DellaRocca, S.; Zhai, H. X.; Cai, X.; Munger, W. E.; Keegan, M.; Pepicelli, C. V.; Qian, C. CUDC-101, a multitargeted inhibitor of histone deacetylase, epidermal growth factor receptor, and human epidermal growth factor receptor 2, exerts potent anticancer activity. Cancer Res. 2010, 70, 3647−3656. (22) Williams, N. K.; Bamert, R. S.; Patel, O.; Wang, C.; Walden, P. M.; Wilks, A. F.; Fantino, E.; Rossjohn, J.; Lucet, I. S. Dissecting specificity in the janus kinases: the structures of JAK-specific inhibitors complexed to the JAK1 and JAK2 protein tyrosine kinase domains. J. Mol. Biol. 2009, 387, 219−232. (23) Butler, K. V.; Kalin, J.; Brochier, C.; Vistoli, G.; Langley, B.; Kozikowski, A. P. Rational design and simple chemistry yield a superior, neuroprotective HDAC6 inhibitor, tubastatin A. J. Am. Chem. Soc. 2010, 132, 10842−10846. (24) Hopkins, A. L.; Groom, C. R.; Alex, A. Ligand efficiency: a useful metric for lead selection. Drug Discovery Today 2004, 9, 430− 431. (25) Tarcsay, A. k.; Nyíri, K.; Keserű , G. r. M. Impact of lipophilic efficiency on compound quality. J. Med. Chem. 2012, 55, 1252−1260. (26) Cai, X.; Zhai, H. X.; Wang, J.; Forrester, J.; Qu, H.; Yin, L.; Lai, C. J.; Bao, R.; Qian, C. Discovery of 7-(4-(3-ethynylphenylamino)-7methoxyquinazolin-6-yloxy)-N-hydroxyheptanamide (CUDC-101) as a potent multi-acting HDAC, EGFR, and HER2 inhibitor for the treatment of cancer. J. Med. Chem. 2010, 53, 2000−2009. (27) Duong-Thi, M.-D.; Meiby, E.; Bergström, M.; Fex, T.; Isaksson, R.; Ohlson, S. Weak affinity chromatography as a new approach for fragment screening in drug discovery. Anal. Biochem. 2011, 414, 138− 146. (28) Ohlson, S.; Duong-Thi, M.-D. Emerging Technologies for Fragment Screening; Wiley-VCH Verlag: Weinheim, Germany, 2016; Vol. 67, pp 173−195.

(29) Fabian, M. A.; Biggs, W. H.; Treiber, D. K.; Atteridge, C. E.; Azimioara, M. D.; Benedetti, M. G.; Carter, T. A.; Ciceri, P.; Edeen, P. T.; Floyd, M. A small molecule−kinase interaction map for clinical kinase inhibitors. Nat. Biotechnol. 2005, 23, 329−336. (30) Karaman, M. W.; Herrgard, S.; Treiber, D. K.; Gallant, P.; Atteridge, C. E.; Campbell, B. T.; Chan, K. W.; Ciceri, P.; Davis, M. I.; Edeen, P. T.; Faraoni, R.; Floyd, M.; Hunt, J. P.; Lockhart, D. J.; Milanov, Z. V.; Morrison, M. J.; Pallares, G.; Patel, H. K.; Pritchard, S.; Wodicka, L. M.; Zarrinkar, P. P. A quantitative analysis of kinase inhibitor selectivity. Nat. Biotechnol. 2008, 26, 127−132. (31) Vishwakarma, S.; Iyer, L. R.; Muley, M.; Singh, P. K.; Shastry, A.; Saxena, A.; Kulathingal, J.; Vijaykanth, G.; Raghul, J.; Rajesh, N.; Rathinasamy, S.; Kachhadia, V.; Kilambi, N.; Rajgopal, S.; Balasubramanian, G.; Narayanan, S. Tubastatin, a selective histone deacetylase 6 inhibitor shows anti-inflammatory and anti-rheumatic effects. Int. Immunopharmacol. 2013, 16, 72−78. (32) William, A. D.; Lee, A. C.; Blanchard, S.; Poulsen, A.; Teo, E. L.; Nagaraj, H.; Tan, E.; Chen, D.; Williams, M.; Sun, E. T.; Goh, K. C.; Ong, W. C.; Goh, S. K.; Hart, S.; Jayaraman, R.; Pasha, M. K.; Ethirajulu, K.; Wood, J. M.; Dymock, B. W. Discovery of the macrocycle 11-(2-pyrrolidin-1-yl-ethoxy)-14,19-dioxa-5,7,26-triazatetracyclo[19.3.1.1(2,6). 1(8,12)]heptacosa1(25),2(26),3,5,8,10,12(27),16,21,23-decaene (SB1518), a potent Janus kinase 2/fms-like tyrosine kinase-3 (JAK2/FLT3) inhibitor for the treatment of myelofibrosis and lymphoma. J. Med. Chem. 2011, 54, 4638−4658. (33) Coe, K. J.; Jia, Y.; Ho, H. K.; Rademacher, P.; Bammler, T. K.; Beyer, R. P.; Farin, F. M.; Woodke, L.; Plymate, S. R.; Fausto, N.; Nelson, S. D. Comparison of the cytotoxicity of the nitroaromatic drug flutamide to its cyano analogue in the hepatocyte cell line TAMH: evidence for complex I inhibition and mitochondrial dysfunction using toxicogenomic screening. Chem. Res. Toxicol. 2007, 20, 1277−1290. (34) Davidson, M. M.; Nesti, C.; Palenzuela, L.; Walker, W. F.; Hernandez, E.; Protas, L.; Hirano, M.; Isaac, N. D. Novel cell lines derived from adult human ventricular cardiomyocytes. J. Mol. Cell. Cardiol. 2005, 39, 133−147. (35) West, A. C.; Johnstone, R. W. New and emerging HDAC inhibitors for cancer treatment. J. Clin. Invest. 2014, 124, 30−39. (36) Hubbert, C.; Guardiola, A.; Shao, R.; Kawaguchi, Y.; Ito, A.; Nixon, A.; Yoshida, M.; Wang, X.-F.; Yao, T.-P. HDAC6 is a microtubule-associated deacetylase. Nature 2002, 417, 455−458. (37) Clark, J. D.; Flanagan, M. E.; Telliez, J. B. Discovery and development of janus kinase (JAK) inhibitors for inflammatory diseases. J. Med. Chem. 2014, 57, 5023−5038. (38) D’Amours, D.; Sallmann, F. R.; Dixit, V. M.; Poirier, G. G. Gainof-function of poly(ADP-ribose) polymerase-1 upon cleavage by apoptotic proteases: implications for apoptosis. J. Cell Sci. 2001, 114, 3771−3778. (39) Danial, N. N.; Korsmeyer, S. J. Cell death: critical control points. Cell 2004, 116, 205−219. (40) Zak, M.; Hurley, C. A.; Ward, S. I.; Bergeron, P.; Barrett, K.; Balazs, M.; Blair, W. S.; Bull, R.; Chakravarty, P.; Chang, C.; Crackett, P.; Deshmukh, G.; DeVoss, J.; Dragovich, P. S.; Eigenbrot, C.; Ellwood, C.; Gaines, S.; Ghilardi, N.; Gibbons, P.; Gradl, S.; Gribling, P.; Hamman, C.; Harstad, E.; Hewitt, P.; Johnson, A.; Johnson, T.; Kenny, J. R.; Koehler, M. F.; Bir Kohli, P.; Labadie, S.; Lee, W. P.; Liao, J.; Liimatta, M.; Mendonca, R.; Narukulla, R.; Pulk, R.; Reeve, A.; Savage, S.; Shia, S.; Steffek, M.; Ubhayakar, S.; van Abbema, A.; Aliagas, I.; Avitabile-Woo, B.; Xiao, Y.; Yang, J.; Kulagowski, J. J. Identification of C-2 hydroxyethyl imidazopyrrolopyridines as potent JAK1 inhibitors with favorable physicochemical properties and high selectivity over JAK2. J. Med. Chem. 2013, 56, 4764−4785. (41) Millard, C. J.; Watson, P. J.; Celardo, I.; Gordiyenko, Y.; Cowley, S. M.; Robinson, C. V.; Fairall, L.; Schwabe, J. W. Class I HDACs share a common mechanism of regulation by inositol phosphates. Mol. Cell 2013, 51, 57−67. (42) Lauffer, B. E.; Mintzer, R.; Fong, R.; Mukund, S.; Tam, C.; Zilberleyb, I.; Flicke, B.; Ritscher, A.; Fedorowicz, G.; Vallero, R.; Ortwine, D. F.; Gunzner, J.; Modrusan, Z.; Neumann, L.; Koth, C. M.; 8356



Article

Lupardus, P. J.; Kaminker, J. S.; Heise, C. E.; Steiner, P. Histone deacetylase (HDAC) inhibitor kinetic rate constants correlate with cellular histone acetylation but not transcription and cell viability. J. Biol. Chem. 2013, 288, 26926−26943. (43) Hai, Y.; Christianson, D. W. Histone deacetylase 6 structure and molecular basis of catalysis and inhibition. Nat. Chem. Biol. 2016, 12, 741−747. (44) Schrödinger, L L.C., 2014. New York, NY, U.S.http://www. schrodinger.com (accessed April 10, 2016). (45) Harder, E.; Damm, W.; Maple, J.; Wu, C.; Reboul, M.; Xiang, J. Y.; Wang, L.; Lupyan, D.; Dahlgren, M. K.; Knight, J. L.; Kaus, J. W.; Cerutti, D. S.; Krilov, G.; Jorgensen, W. L.; Abel, R.; Friesner, R. A. OPLS3: a force field providing broad coverage of drug-like small molecules and proteins. J. Chem. Theory Comput. 2016, 12, 281−296. (46) Polak, E.; Ribiere, G. Note sur la convergence de méthodes de directions conjuguées. Rev. Fr. Info Res. Op. 1969, 3, 35−43. (47) Hasel, W.; Hendrickson, T. F.; Still, W. C. A rapid approximation to the solvent accessible surface areas of atoms. Tetrahedron Comput. Methodol. 1988, 1, 103−116. (48) Bergström, M.; Liu, S.; Kiick, K. L.; Ohlson, S. Cholera toxin inhibitors studied with high−performance liquid affinity chromatography: a robust method to evaluate receptor−ligand interactions. Chem. Biol. Drug Des. 2009, 73, 132−141.

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