Triazolopyridines as Selective JAK1 Inhibitors: From Hit Identification

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Triazolopyridines as selective JAK1 inhibitors: from hit identification to GLPG0634 Christel Jeanne Menet, Stephen Robert Fletcher, Guy Van Lommen, Raphael geney, Javier Blanc, Koen Smits, Nolwenn Jouannigot, Ellen Van der Aar, Philippe ClementLacroix, Liên Lepescheux, René Galien, Béatrice Vayssiere, Luc Nelles, Thierry Christophe, Reginald Brys, Luc Van Rompey, Fabrice Ciesielski, Murielle Uhring, and Pierre Deprez J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 04 Nov 2014 Downloaded from http://pubs.acs.org on November 5, 2014

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Journal of Medicinal Chemistry

Triazolopyridines as selective JAK1 inhibitors: From hit identification to GLPG0634 Christel J. Menet*1, Stephen R, Fletcher2, Guy Van Lommen1, Raphael Geney3, Javier Blanc1, Koen Smits1, Nolwenn Jouannigot1, Pierre Deprez3, Ellen M. van der Aar1, Philippe ClementLacroix3, Liên Lepescheux3, René Galien3, Béatrice Vayssiere3, Luc Nelles1, Thierry Christophe1, Reginald Brys1, Muriel Uhring4, Fabrice Ciesielski4, Luc Van Rompaey1

1

Galapagos NV, Generaal de Wittelaan L11A3, 2800 Mechelen, Belgium

2

BioFocus, Chesterford Research Park, Saffron Walden, Essex, CB10 1XL, United Kingdom

3

Galapagos SASU, 102 Avenue Gaston Roussel, 93230 Romainville, France

4

NovAliX, Bld Sébastien Brant, BP 30170, F-67405 Illkirch CEDEX, France

*Corresponding author: email: [email protected], tel : +32 15 342 938

1 ACS Paragon Plus Environment


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Abstract: Janus kinases (JAK1, JAK2, JAK3, TYK2) are involved in the signaling of multiple cytokines important in cellular function. Blockade of the JAK-STAT pathway with a small molecule has been shown to provide therapeutic immunomodulation. Having identified JAK1 as a possible new target for arthritis at Galapagos, the compound library was screened against JAK1 resulting in the identification of a triazolopyridine-based series of inhibitors represented by 3. Optimization within this chemical series led to identification of GLPG0634 (65, filgotinib), a selective JAK1 inhibitor currently in Phase 2B development for RA and Phase 2A development for Crohn’s Disease (CD).


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Janus kinases (JAKs) are cytoplasmic non-receptor tyrosine kinases. Four human JAK family members are known: JAK1, JAK2, JAK3 and TYK2 (tyrosine kinase 2). It is well documented that JAK kinases play a central role in cytokine receptor signaling through phosphorylation and activation of signal transducer and activator of transcription (STAT) proteins.1 JAKs are constitutively bound to the cytoplasmic tail of the cytokine receptors. Upon binding of the cytokine, JAK family members auto- and/or trans-phosphorylate, leading to phosphorylation of the receptors at specific tyrosine (Tyr) residues. The resulting activated STATs migrate to the nucleus and modulate transcription.

JAK1, JAK2, and TYK2 kinases are ubiquitously

expressed, whereas JAK3 is limited to the lymphoid lineage. Many cytokines relying on JAKs for intracellular signal transduction have key patho-physiological roles in auto-immune and inflammatory diseases. Such cytokines include IL-2 (interleukine), IL-6, IL-12, IL-23, IFN-α, IFN-γ (interferon) and GM-CSF (Granulocyte macrophage colony-stimulating factor).1 Therapeutic benefit from JAK kinase inhibition has been established in rheumatoid arthritis (RA) with tofacitinib, 1 (XeljanzTM) and in the treatment of myelofibrosis with ruxolitinib, 2 (JakafiTM). Tofacitinib generally reported as a JAK1/JAK3 inhibitor is found in house (figure 1) to have a JAK1/2/3selectivity profile. Ruxolitinib, 2 (JakafiTM) generally known as a JAK1/JAK2 inhibitor has been shown in different literature references to have a JAK1/JAK2/TYK2 profile (figure 1).2,3 In 1998, Schreiber et al. showed using JAK1-/- cells that although responsive to many cytokines JAK1 is necessary for cells to respond effectively to multiple immune-relevant cytokines; these include all class II cytokine receptors, receptors that utilize the γc subunit for signaling and the family of cytokine receptors that depend on the gp130 subunit signaling.4 Moreover, in 2001 Yoshimura et al. conducted in vivo CIA (CollagenInduced

Arthritis)

experiments

by

periarticular

injection


of

adenovirus

carrying


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CIS3/SOCS3/SSI3 to inhibit JAK tyrosine kinase activity.5 This study showed drastic reduction of the severity of the CIA disease, leading them to conclude that a JAK1 inhibitor may be a useful therapeutic reagent for RA (rheumatoid arthritis). The work carried out by these two groups validated the Galapagos approach to the identification of JAK1 as a possible new target in arthritis using viral siRNA libraries in functional screening. Additional recent findings suggest that JAK1 dominates IL-2 induced JAK1/JAK3/STAT5 signaling and IL-6 induced JAK1/JAK2/TYK2/STAT1 signaling. This implies that JAK1 inhibition alone can provide in vivo efficacy towards immune-inflammatory diseases.6,7,8,4,9,10,11 Since JAK2 inhibition leads to unwanted side effects such as anemia, thrombocytopenia and neutropenia, selective JAK1 inhibition could provide an increased therapeutic window, allowing higher dosing and efficacy, while avoiding dose-limited pharmacology observed with pan JAK inhibitors (i.e. compounds inhibiting multiple JAKs).12,13,14,15 Herein we describe the finding and characterization of a triazolopyridine series as JAK inhibitor along with our initial efforts at achieving the desired JAK1 selectivity profile within this chemotype. Figure 1: tofacitinib, 1; ruxolitinib, 2: *Biochemical IC50 determination: recombinant JAK1 (Invitrogen), JAK2 (Carna Biosciences), The ATP concentration was equivalent to Km value.

CN

Compound

N N N

N

CN O

N N

N H

N

JAK2, IC50 (nM)*

1

0.8

0.6

2

1.2

0.2

N H

N 1

JAK1, IC50 (nM)*

2


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To identify chemical starting points for JAK1 inhibitors, the BioFocus kinase-focused library collection was screened against the kinase domain of JAK1 in an in vitro biochemical assay at 10 µM initially. All compounds having a percentage inhibition above 75% were further evaluated in dose response tests with measurement of IC50 values. Triazolo[1,5-a]pyridine 3 exhibited encouraging JAK1 inhibitory activity (70 nM), and owing to its modular nature, emerged as a good platform to initiate chemistry (low molecular weight: 308, appropriate xLogP316: 2.35). Furthermore, 3 appeared to be slightly more potent on JAK1 than on the other JAKs in the biochemical assays (Figure 2). A similar selectivity profile has since been reported by Siu et al. on the same triazolo-pyridine scaffold.17 The catalytically active JH1 (JAK Homology) kinase domain carries out the phosphorylation events responsible for the physiological function of the JAK kinases, and this selective series of molecules appeared to be ATP competitive inhibitors binding within this region. However, compound 3 was not optimal, showing a moderate potency on JAK1 kinase and low metabolic stability (6% of compound remaining after 60 min in human microsomes). Figure 2. Compound 3, hit identified during our HTS; *LiPE (lipophilic efficiency) = pIC50-xlogP3

Hit: compound 3 N

hJAK1 IC50 = 70 ± 14 nM

N N

NH O

hJAK2 IC50 = 138 ± 22 nM hJAK3 IC50 = 528 ± 82 nM hTYK2 IC50 = 519 ± 55 nM

O

LiPE* = 4.65

Exploiting the subtle sequence differences in the active sites of the JAK family kinases via a combination of structure-activity relationships (SAR) and structure-based design, we report a series of modifications to the exocyclic amine and the 5-position of compound 3. To the best of 5 ACS Paragon Plus Environment


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our knowledge, at that time no JAK1 selective inhibitor was yet described and no help from known SAR could be used. Nevertheless sequential improvements in potency together with achieving a balance of drug-like features, led to the discovery of compound 65 (GLPG0634, filgotinib), a potent, selective, and orally active JAK1 inhibitor with an excellent PK profile in two preclinical species (rat and dog). In addition, compound 65 showed good in vivo efficacy in a rat CIA model. Chemistry: Analogs of 3 were synthesized according to the procedure depicted in Scheme 1, starting from the commercially available 2-amino-6-bromo-pyridine 4. The central scaffold was constructed by reaction of ethoxycarbonyl isothiocyanate with 4 to form the thiourea 5 which was then reacted with hydroxylamine to yield, after heating, the desired intermediate 6.18 Scheme 1. Synthesis of the triazolo-pyridine scaffold 8 S

i Br

N 4

NH 2

Br

N 5

N H

OEt N H

O

ii

7 6

1 N

N N 2 5 3 Br 6

NH2

i) ethoxycarbonyl isothiocyanate, DCM, RT; ii) hydroxylamine hydrochloride, DIPEA, EtOH/MeOH, reflux

Next, para-methoxyphenyl boronic acid was first coupled via a Suzuki reaction to the bromoamino-triazolopyridine scaffold to provide 7. Derivatives of 7 with various groups in the 2position were easily accessible using different functionalization of the free amine. Acylation was carried out with the acyl chlorides and the initial double acylation adducts easily hydrolyzed with an ammonia solution in methanol to provide the mono-amides (Scheme 2). Palladium coupling was carried out by first transforming the free amine to iodine by a Sandmeyer reaction, followed by Buchwald or Suzuki reactions (Scheme 2). To vary the 5-position, the triazolo-pyridine scaffold 6 was acylated with cyclopropanecarboxyl chloride to give 9, followed by a Suzuki reaction (Scheme 2).


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Scheme 2. Synthesis of the triazolo-pyridine derivatives

N N Br

N

i

N NH2

N

iia,b,c NH 2

N N

NH A R

N N

W

n

7

6

W=O n = 0, 1 A= C, CH2

O

O vi

25, 28, 30, 32, 33, 36

iii

N N N

NH

N O

N N

iv

Br

NH R

9 N vii

27, 35

O

N

N N N

I

N N

O

NH

v

N N

8

O

N

Ar 34 3, 37- 47, 55 - 65

O

i) para-methoxyphenyl boronic acid, Pd(dppf)Cl2, K2CO3, 1,4-dioxane/water, 100°C; ii) a. acid chloride, Et3N, CH3CN; b. NH3 (7N) in MeOH; b. 4-nitrophenyl chloroformate, DIPEA, methylamine, DCM; c. aldehyde, Ti(OiPr)4, Na(CN)BH3, EtOH;19 iii) HI (57% aqueous solution); NaNO2, DMSO; iv) Pd(OAc)2, Cs2CO3, BINAP, aniline, 1,4-dioxane or toluene; v) Pd(dppf)Cl2, K2CO3, boronic acid, 1,4-dioxane, vi) Cyclopropanecarbonyl chloride, Et3N, CH3CN; vii) para-methoxyphenyl boronic acid, Pd(dppf)Cl2, K2CO3, 1,4-dioxane/water, 100°C

Examples such as 49 with additional substitution on the scaffold were synthesized similarly, starting from the amino-pyridine with the desired substitution, followed by sequential coupling, acylation and Suzuki reaction (Scheme 3). Scheme 3. Synthesis of the compound 49



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NH2

H N

i

N

O

N

O Cl 10

H N S

Cl

O

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N

ii

O 11

Cl

N N

O

NH 2

12

N

N iii

N N

O

iv

NH O

NH

N N

O

Cl

O

49

13 O

i) ethoxycarbonyl isothiocyanate, DCM, RT; ii) hydroxylamine hydrochloride, DIPEA, EtOH/MeOH, reflux; iii) acid chloride, Et3N, CH3CN; iv) para-ethoxyphenyl boronic acid, Pd(dppf)Cl2, K2CO3, 1,4-dioxane/water, 100°C

The isomeric scaffold with substitution at the 8-position (compounds 50 and 52) was obtained starting from the isomeric pyridine with or without substitution. Compound 54 was made by the same sequence, starting from amino-pyrazine. For the synthesis of the triazolo-pyrimidine scaffold, compound 51, it was necessary to start the synthesis via a Suzuki coupling on 6-amino-2-chloropyrimidine, as the cyclisation (step ii) would not proceed in the presence of the chlorine (Scheme 4). Scheme 4. Synthesis of compound 51, triazolo-pyrimidine scaffold NH2

NH2 N

N

i

N

H N N

S

N

15

16

O O

N

iii N

N N

NH2

O

ii

N

Cl 14

H N

N

iv N

NH

N N

O

17

51 O

O


O

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i) para-ethoxyphenyl boronic acid, Pd(dppf)Cl2, K2CO3, 1,4-dioxane/water, 100 °C, ii) ethoxycarbonyl isothiocyanate, DCM, RT; iii) hydroxylamine hydrochloride, DIPEA, EtOH/MeOH, reflux; iv) acid chloride, Et3N, CH3CN

An alternative approach to the synthesis of compound 53 was required as the methods above failed to give the desired product. Instead, acetophenone 18 was reacted with dimethylformamide dimethyl acetal 19 to give compound 20, which was condensed with di-aminotriazole 21 to yield 22. Acylation of 22 gave the required product 53 (Scheme 5). Scheme 5. Synthesis of compound 53 O

O

O i +

O

N

H N N

+

N

18

19 N

ii

20

N N

21 N

N NH2

iii

N N N

22

NH 2

N

H2 N

H N O

53

i) 150°C, MW (Wmax:300; Pmax:150PSI); ii) EtOH, 100 °C; iii) acid chloride, Et3N, CH3CN

Results and Discussion The goal of this work was to develop a potent and selective JAK1 inhibitor possessing appropriate ADME/DMPK properties to enable clinical development via oral dosing for the treatment of immune-inflammatory disorders. Moreover it was reasoned that, in order to be a potential drug candidate, any new compounds should reach levels of potency in the low nanomolar range like the existing drug 1 or 2 with more importantly a greater JAK1 selectivity. Indeed, the accepted dose by the FDA of 1 is low (5 mg), but some observation of reduced hemoglobin levels at higher doses (15 mg BID) during clinical trials was attributed to possible effects on hematopoiesis through inhibition of JAK2 and consequently its lack of selectivity.



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This led us to hypothesise that an increased degree of selectivity for JAK1 over JAK2 may be sufficient to provide a suitable therapeutic window. Analogues of 3 were primarily screened in JAK1 and JAK2 biochemical assays. Selectivity against JAK3 and TYK2 biochemical assay was subsequently evaluated for selected compounds. Biochemical JAK2 inhibition was assessed early in the screening cascade, due to the essential role of JAK2 in hematopoiesis, including EPO receptor signaling and red blood cell homeostasis as mentioned above.13,14 Assessment against a selectivity panel of 14 kinases was also employed for compounds of interest. The series appeared to be selective towards the JAK kinases, as none of an initial target list of 14 kinases were inhibited by the compound 3 at an IC50 < 2 µM (~>30 fold of selectivity) (Table 1). Table 1. Selectivity towards 14 kinase panel (biochemical assay)

Kinase

Cpd 3 IC50 (nM)

Kinase

Cpd 3 IC50 (nM)

FGFR3

>5,741

PYK2

3,184

GSK3β

7,114

RIPK2

50,978

IKKβ

24,689

ROCK1

>10,000

LCK

4,440

SYK

2,272

MAPKAPK5

>10,000

TAK1

>10,000

p38α

>10,000

TBK1

>4,000

PKA

39,986

CDK2

>10000

From Hit to potent JAK inhibitors (SAR) 10 ACS Paragon Plus Environment

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For initial SAR development towards enhancing JAK1 selectivity, both the cyclopropyl and para-methoxy phenyl positions were explored via carefully designed parallel synthesis efforts.

Table 2. Substitution on exocyclic NH. N/A: compound not active against JAK2; NT: not tested against JAK2. Compounds were tested at least twice unless indicated otherwise (n=1); * (JAK2 IC50)/(JAK1 IC50). Biochemical IC50 determination: recombinant JAK1 (Invitrogen), JAK2 (Carna Biosciences), the ATP concentration was equivalent to Km value. N R

N N

O

Compound

3

R

JAK1 IC50 (nM) ± s.e.m (JAK2 selectivity index)*

O

70 ± 14 (2)

Compound

R

JAK1 IC50 (nM) ± s.e.m (JAK2 selectivity index)*

O

30

N H

>10,000

N H

>10,000 (N/A) 7

NH2

3,055 ± 716 (0.6)

31

24

N

>10,000 (N/A)

32

>10,000 (NT)

33

>10,000 (N/A)

34

N

N H O

O

25

>10,000 (N/A)

N H

N H

413 (0.7) (n=1)

N H

O

26

N

>10,000 (N/A)

N H N

O

27

N H

O S

5,740 (N/A) (n=1)

35

>10,000 (N/A)

36

O

N H

O

28

N H

O

O

29

N H

2.8 ± 0.27 (0.9)

>4,000 (N/A)


N H

1,979 ± 691 (0.5)


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We first started investigating the 2-position of the triazolo-pyridine through replacement of the cyclopropylamide of the original hit. SAR rationalization efforts were supported by modeling using a proprietary docking model based on the JAK1 co-crystal structure incorporating 1.20 The validity of this docking model was later confirmed by the crystal structure of JAK2 with compound 65, Figure 4. Synthesized compounds without a free hydrogen atom on the amine at the 2-position (24, 26 and 31) essentially lost all activity on JAK1. Modeling studies indicate that an H-bond is formed with the main chain carbonyl of Leu959 of the hinge region, as shown on Figure 3A. Figure 3. A. Model of compound 3 docked in the JAK1 JH1 kinase domain; B. Model of compound 3 docked in the JAK1 JH1 kinase domain. B. Molecular surface and side chains of residues within 5 ångströms of compound 3.

A B

From our early investigations on the JAK1 binding mode (Figure 3A/B), it was initially expected that the core could be substituted with various solubilizing groups at the 2-position which would drive potency and improve the ADME profile, as this portion of the molecule would be directed toward the solvent region. However, replacement of the cyclopropyl amide moiety displayed in


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Table 2 showed the importance of this position on the activity and selectivity of the compounds. Surprisingly, close amide analogs, such as isopropylamide 28 or acetamide 29 displayed much reduced potency against JAK1. Only the crystal structure obtained later (Figure 4), gave a possible explanation for this SAR cliff. It was hypothesized that the superior ability of the cyclopropane ring to donate an H-bond could explain the SAR observations.

Indeed, the

65/JAK2 crystal structure shows a putative H-bond between the CH carbon atom of the cyclopropylamide and the carbonyl oxygen of Leu932 and of Pro933 in JAK2 (Pro960 and Leu959 in JAK1): bond length 3.3-3.4 Å. This type of interaction has been previously described in the literature.21 Cyclopropanes and other hydrocarbons containing three-membered rings have a certain sp2 character and gain substantial H-bond acidity. Furthermore compound 25 (methylcyclopropyl) and compound 28 (isopropylamide) support this hypothesis by having little or no activity and being unable to form this non-classical hydrogen bond.

Urea 33 bearing an

additional H-donor is more potent than the ethylamide analog 36, but it should be noted that 33 loses selective against JAK2. Directly linking alkyl groups to the exo-nitrogen (e.g. 32) is not tolerated. Thus, potency on JAK1 is lost. This is postulated to be due to the difference in pKa of the NH, preventing a strong H-bond with the carbonyl of the hinge and/or maybe due to a higher flexibility of these substituents. In addition, the carbonyl in the amide functionality is directly involved in H-bond interactions with neighboring water molecules as observed in the X-ray structure of the JAK2/65 crystal (Figure 4). The loss of such interactions in the alkyl analogue could be detrimental for potent binding.



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Larger replacement of the cyclopropyl like benzylamides or replacement of the carbonyl by sulfonamides resulted in poorly active or inactive compounds towards JAK1, possibly due to sub-optimal interaction with the hinge. The only suitable replacement of the cyclopropylamide group was the aniline 35, increasing hugely the IC50 to 2.8 nM, however the selectivity profile shifted from JAK1 to JAK2 (further details on this JAK2 selective series have been described by others).17 Some other groups reported that bulkier groups in the hinge region were better tolerated in JAK2 than in JAK1 due to a larger available space in that area for JAK2. In summary the cyclopropyl moiety appears to be a preferred substituent for JAK1 selectivity over JAK2.25

Table 3. Investigation on 5-position; NT = not tested; Compounds were tested at least twice unless indicated; * (JAK2 IC50)/(JAK1 IC50), (JAK3 IC50)/(JAK1 IC50), (TYK2 IC50)/(JAK1 IC50). Biochemical IC50 determination: recombinant JAK1, TYK2 (Invitrogen), JAK2, and JAK3 (Carna Biosciences)

N N N

NH O

Ar

Selectivity Index* Compound

Ar

3

JAK1 IC50 (nM)

JAK2

JAK3

TYK2

70 ± 14

2.0

7.5

7.4

184 ± 30

2.3

7.1

4.1

371 ± 68

1.5

2.0

2.8

O

37

O

38


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Cl

39

40

142 ± 29

1.1

6.7

2.4

156 (n=1)

3.1

7.4

28.2

2,554 ± 532

0.7

1.5

>1.6

547 ± 136

1.5

4.4

6.2

975 ± 469

2.1

>4.1

>4.1

1,351

1.1

2.1

>3.0

572 (n=1)

0.9

3.8

2.8

103 ± 39

1.0

3.0

18

>10,000

NT

NT

NT

Cl

41

N

O N

42

O

43

N

44

N

N N H

O

45

O

46

47

O

We next turned our attention to the SAR around the phenyl group at the 5-position of the triazolo[1,5-a]pyridine core (Table 3).

Replacement of phenyl by more polar heterocycles

(compounds 42 to 44) resulted in a substantial (8 to 20 fold) reduction in potency. This is consistent with modeling studies from which it was evident that the phenyl ring sits in a hydrophobic sub-pocket of JAK1, forming hydrophobic contacts with Leu881 (Leu855 in 15 ACS Paragon Plus Environment


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JAK2), Val938 (Val863 in JAK2), Ser963 (Ser936 in JAK2), Leu1010 (Leu983 in JAK2) and Gly1020 (Gly993 in JAK2) (Figure 3B) . Relative to the unsubstituted phenyl derivative 37, substituents in the ortho-position caused a two-fold decrease in potency for small substituents. This drop in potency was even higher for larger groups (for example compound 47). This may be explained by a steric clash observed with the triazolopyridine scaffold, forcing an unfavorable off-planar conformation of the phenyl ring. In comparison, adding a chloro-substituent at the meta-position of the phenyl ring (compound 39) has no effect on the potency of JAK1 inhibition, but seems to be more favorable towards JAK2 inhibition. Consequently, it was observed that the most potent and most selective compounds were those with the 5-phenyl substituted at the para-position. Modeling studies confirmed that substituents in this position point toward the glycine-rich loop where it is possible to make stabilizing interactions or have a positive impact on the water network. Indeed, this loop has been reported in numerous kinase crystal structures to be involved in a network of interactions with inhibitors, and displays remarkable conformational flexibility allowing accommodation of a vast array of ligands.

Table 4. Changes on scaffold; N/A= not Active; * (JAK2 IC50)/(JAK1 IC50). Biochemical IC50 determination: recombinant JAK1 (Invitrogen), JAK2 (Carna Biosciences)

N

N

NH O

O

Compound

structure

Compound

JAK1 IC50 (nM) (JAK2 selectivity


structure

JAK1 IC50 (nM) (JAK2 selectivity

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index)*

index)*

N

O

NH

N N

O

27 ± 9 (2)

48

52 F

O

N/A

N F

NH

N N

O

F

N NH

N N

O

O

3,012 ± 492 (>1)

49

53

N N N

O

N/A NH

N O

N

N

O

N N

NH O

67 ± 17 (2.2)

50

3,098 (1)

54

N N N

NH

O

O

N N

N N

NH O

418 ± 125 (1.5)

51 O

Substitution at the 6-position of the scaffold appears not to be tolerated for JAK1 inhibition, as shown for compound 49 (Table 4). The reason lies in the skewed torsion of the phenyl ring at the 5-position provoked by the adjacent methoxy group. An isomeric scaffold was made by repositioning the ring junction nitrogen. Analogs of this isomeric scaffold, 8-substituted 2amino-[1,2,4]triazolo[1,5-a]pyridine, had similar or slightly reduced potency (48 vs 50), and similar ADME properties (data not presented). On this scaffold, additional substitution also led to loss of potency (compound 52). Finally, modification of the scaffold by addition of an extra nitrogen atom showed no inhibition of JAK1. It appears that the electronics of the pyrazolopyridine scaffold are optimal for binding in the JAK ATP pocket.



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Page 18 of 70

Encouraged by these preliminary results of the 5-phenyl substituent, we then focused the SAR exploration on the para-position of this substitution.

Table 5. SAR of the para-position; *Not measured due to precipitation; * ASOL = kinetic solubility measure with 2% DMSO; PPB = Plasma Protein Binding. Biochemical IC50 determination: recombinant JAK1, TYK2 (Invitrogen), JAK2 and JAK3 (Carna Biosciences); ** LiPE (lipophilic efficiency) = pIC50-xlogP3 N NH

N N

O

R

Index selectivity Compound

R

HN O S O

55

56

N N O

N H

57

OH

PPB % (rat)

xlogP3

ASOL* pH=7.4

LiPE **

90

1.89

>80

5.74

66

99

3.6

4

5.62

-

-

-

3.61

28

4.47

11.4

26

9

99

3.5

9.4

5.35

147 (n=1)

7.5

-

-

-

3.63

-

3.20

92 ± 12

1.8

9.5

6.1

71

1.84

>38

5.19

JAK1 IC50 (nM)

JAK2

JAK3

TYK2

23±8

1.3

37

3.0

0.6±0.2

5.3

21

8.3 (n=1)

1.7

1.4 (n=1)

OH

HN

58

CN

N

59

O CN

60

N O


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An illustrative selection of the synthesized substitutions at the para-position is presented in Table 5. Substitution seems to increase the potency when large enough to contact the glycine rich loop. Sulfonamides in the para-position (e.g., 55) gave high potencies. Unfortunately, permeability was found to be compromised for this subseries with high efflux observed (Caco2 assay: permeability apparent (A2B) 3.7 cm.10-6sec-1; efflux ratio: 16.5). The best potency and selectivity for JAK1 was obtained with groups consisting of a long chain terminated by an aromatic group such as compound 56 or 58 or 59. JAK1 has a histidine in the p-loop that can have a favorable interaction with aromatic moieties. In the number of substitutions that were explored, high selectivity was achieved for compound 58 with 11 fold selectivity for JAK1 over JAK2 inhibition (the phenyl can directly interact with the histidine and the nitrile with the glycine rich loop) and low selectivity was obtained if the aromatic was substituted by an alkoxy like compound 57 (less favorable in the interaction with the histidine). This lipophilic motif, unfortunately, was not compatible with good ADME properties, giving low solubility, low LiPE ( 90 min; no efflux with high permeability and good solubility and PPB of 70% in rat. Good aqueous solubility and PPB with improved potency. Encouraged by the good ADME properties, we sought alternative of the morpholine moiety. 60 was used as a reference compound for probing the structure-activity relationships of a diverse subset of amines to assess if identification of a compound with high potency and improved



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physical properties could be attained. In this small library, all compounds conserve the JAK1selectivity, but only few examples show attractive potency on JAK1.

Table 6. SAR of the para-phenyl substitution. *RLM: Rat Liver Microsome (%remaining after 60 min): *** Caco2: permeability (A2B) 0.3 cm.10-6sec-1, efflux ratio, 69. NT = not tested (racemic mixture). Biochemical IC50 determination: recombinant JAK1, TYK2 (Invitrogen), JAK2, and JAK3 (Carna Biosciences); ** LiPE (lipophilic efficiency) = pIC50-xlogP3; + in-house data N N N

NH O

index JAK1-selectivity Compound

Ar

N

60

JAK1 IC50 (nM)

JAK3

TYK2

ASOL pH_7.4

RLM %*

PPB % (rat)

LiPE**

JAK2

92 ± 12

1.8

9.5

6.1

>38

38

71

5.19

>4,000

-

-

-

-

-

-

3.38

16 ± 4

2.6

9.5

6.2

>80

80

83

5.19

27 ± 4

1.1

8.7

3.2

27

26

70

4.20

41 ± 14

2.1

8

9.6

>85

85

NT

6.07

O

61

N N

NH F

62 O

F

F

63

N F F

NH

64*** S O O


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N

65

1

S O

O

cf Figure 1

10 ± 0.8

2.8

81

11.6

>85

65

47.5

6.64

0.8

0.8

0.4

42

-

27+

33+

7.77

The nature of the para-substitution on the 5-phenyl substituent, in particular lipophilic groups, was found to have a pronounced effect on potency, as shown above.

To extend the

understanding of this effect, compound 60 bearing a morpholino-benzyl group was used as a starting point for the optimization of the LiPE and ADME properties of the series (Table 6). Extended amines with strong basicity such as piperazine 61 appeared to be completely inactive against JAK1. The less basic piperidine analogue, 4-difluoropiperidine 63 and the non-basic trifluoro-amide gave rise to good potency but loss of selectivity and again decreased LiPE. After extensive studies, the cyclic sulfone moiety was found to offer a good balance of selectivity and with a LiPE slightly lower than compound 1, but still higher than 6 (Table 6). Thus compounds 65 and 64 reached a JAK1 potency below 50 nM with an improved selectivity of ~2/3-fold over JAK2 in contrast to the potent but unselective 1. Compound 64 was not profiled further due to the very low permeability observed in Caco2 and less attractive selectivity toward the other JAK-members (the two enantiomers were not separated). Compound 65 showed the best in vitro stability in rat; however compounds 60, 63 and 65 were all three advanced into rat PK studies. Table 7. Pharmacokinetics in rats; formulation was in methyl cellulose (MC) 0.5%

Compound

IV

PO

IV

PO

PO

1 mg/kg

5 mg/kg

1 mg/kg

5 mg/kg

5 mg/kg

60

65


63


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C0 or Cmax (ng/mL)

863

1,320

1,407

310

547

Tmax (h)

_

0.33

_

2.2

0.25

AUC(0-z) (ng.h/mL)

470

1,437

739

1,681

690

Cl (L/h/kg)

2.12

_

1.35

_

_

Vss (L/kg)

1.46

_

1.76

_

_

T1/2 (h)

0.74

0.92

1.6

3.9

0.92

F (%)

62

45

N/A

The PK properties of the 3 selected compounds are summarized in Table 7. Both 60 and 65 showed good oral bioavailability with compound 65 displaying a longer half-life with a lower total plasma clearance than compound 60 as expected from in vitro data (Table 6). Therefore compound 65 was profiled further in a dog PK demonstrating good oral bioavailability of 67% (Table 8). Table 8: Pharmacokinetics in dog; formulation was in methyl cellulose (MC) 0.5% IV

PO

1 mg/kg

5 mg/kg

Compound

65

C0 or Cmax (ng/mL)

1,143

1,807

Tmax (h)

_

1.5

AUC(0-z) (ng.h/mL)

4,098

13,908

Cl (L/h/kg)

0.25

_

Vss (L/kg)

1.7

_

T1/2 (h)

7.5

5.2

F (%)

67


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The biochemical potency of the described compounds was measured at the Km of ATP whereupon compound 65 showed only a selectivity of 2.8 fold against JAK2, lower than for recently reported JAK1 selective inhibitors.22,23 Discrepancies between enzyme and cellular data for JAK kinases has been recognized for some time, but only recently have potential explanations supported by experimentation been published to address these inconsistencies.24 A first explanation stipulates that in the cell, the concentration of ATP is accepted to be in the 1 to 5 mM range. Consequently, neither a Ki nor an IC50 measurement at ATP Km in an enzymatic assay represents a good evaluation of cellular activity for ATP competitive inhibitors.3 The second explanation is due to the role of the different JAK within the cells. In recent years the importance of the JAKs in the heterodimeric pair has been the source of intensive research.6,11 Thus different groups have demonstrated the hierarchical role of the JAKs in the cytokine pathway, in particularly the dominant role of JAK1 in the IL2, IL22 or IL6 pathways. These later observations show that different selectivity profiles of inhibitors is obtained when looking at different cytokine pathways. Finally, shifts in biochemical potency and cellular potency between JAK2 and JAK1 have since been reported on other compound series by a number of research groups.23,24,25 Consequently, to assess the profile of compound 65 in potentially more relevant cellular settings, its potency and selectivity was determined in cellular and whole blood assays. Cellular assays were developed for cytokines employing different combinations of JAK hetero- and homodimers for signaling. Cell lines were pre-incubated with 65 and treated with cytokines that employ different JAK heterodimeric or JAK2 homodimeric complexes for signaling (Table 9). Compound 65 inhibited JAK1/JAK3/γc and JAK1/TYK2 signaling pathways most potently (IC50 values from 150 to 760



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Page 24 of 70

nM), whereas JAK1/JAK2 signaling mediated by type II and gp130 receptor complexes or JAK2 signaling were inhibited with low micromolar potencies. Compound 65 does not inhibit JAK2 homodimer–mediated signaling induced by EPO or PRL (IC50 > 10 µM). Due to the selectivity of compound 65 against JAK3 and TYK2 in the biochemical assay and precedent literature that showed JAK1 dominance,6,11 it can be concluded that inhibition of JAK/STAT signaling involving JAK1, JAK3 and TYK2 is certainly due to JAK1 inhibition. Hence, compound 65 preferentially inhibits JAK/STAT signaling involving JAK1 over JAK2 kinase in a cellular context. This observation was recently confirmed elsewhere with a selectivity of >14 reported for compound 65 in cell assays using IL-6/pSTAT1 for JAK1 pathway and EPO/pSTAT5 for JAK2.3 Table 9: Cell assays employing different JAK heterodimeric or homodimeric complex.8,26 Copyright 2013. The American Association of Immunologists, Inc. JAK involved

Cell type

Trigger

Read-out

IC50, nM (pIC50 ± s.e.m)

JAK1-JAK3

THP-1

IL-4

pSTAT6

154, 203

JAK1-JAK3

NK-92

IL-2

pSTAT5

148, 757, 367

JAK1-TYK2

U2OS

IFN-αB2

pSTAT1

494, 436

JAK1-JAK2

HeLa

OSM

STAT1 reporter

1,045

JAK1-JAK2

U2OS

INFγ

pSTAT1

3,364

JAK2

TF-1

IL-3

pSTAT5

3,524

JAK2

BaF3

IL-3

Proliferation

4,546

EPO

pSTAT5

>10,000

PRL

pSTAT5

>10,000

UT7JAK2 EPO JAK2

22Rv1


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Human blood was pre-incubated with the different compounds, and IL-6 or GM-CSF was added to stimulate respectively JAK1 or JAK2 signaling pathways (Table 10). STAT1 and STAT5 phosphorylation levels respectively were measured in CD4+ lymphocytes and CD33+ monocytes. Table 10. Human whole blood potency on JAK1 and JAK2 signaling pathway. Compound 65 displayed a high selectivity for JAK1 over JAK2 in a cellular environment

IL6-induced pSTAT1

GM-CSF-induced pSTAT5

IC50 (nM)

IC50 (nM)

65

629 (n =7)

17,453 (n =7)

60

5,590 (n=3)

>30,000 (n=3)

62

4,467 (n=3)

>30,000 (n=3)

64

6,152 (n=3)

>30,000 (n=3)

63

2,749 (n=3)

>10,000 (n=2), >30,000 (n=1)

1

74 (n =16)

740 (n=7)

2

1,053 (n=5)

4,982 (n=2)

Compounds

Once again, in this setting compound 65 showed a higher selectivity (> 27 fold) for JAK1 over JAK2 (Table 10). We also confirmed that compound 65 was the most potent compound on JAK1 signaling within the series of compounds with improved ADME properties (60, 62, 63 and 64 IC50 > 2,000). In the same assay compounds 1 and 2 do not reach the same level of selectivity as compound 65, with 65 being 3 fold more selective than 1. This key finding provided optimism for the first time that JAK1 selectivity could be achieved in vivo within this series. Remarkably, compound 2 only showed a weak potency in this assay, being 2 fold less potent than compound 65, which was not predicted from its biochemical data. This apparent discrepancy was also shown elsewhere.3



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Extensive ADME profiling of 658: The in vitro ADME and safety pharmacology properties of compound 65 were assessed. Metabolic stability in vitro is favorable with 65%, 45% and 87% of compound left respectively in rat, dog and human liver microsomes after 60 min incubation and a half-life of more than 200 min in hepatocytes for all species. Permeability in a Caco-2 assay was found to be 3.5 10-6 cm/s (Papp (A2B)) with an efflux ratio of 16 indicating that 65 is a substrate of P-gp (permeability increased in presence of verapamil, P-gp inhibitor). However, despite the high efflux, good solubility of GLPG0634 allows high absorption in animal species (Table 7 and Table 8). Thermodynamic solubilities of the compound in aqueous buffer solutions of pH 3 and pH 7.4 are 243 µg/mL and 40 µg/mL respectively. Plasma protein bindings are 47.5% in rat, 28.9% in dog and 31.8% in human. An initial assessment of potential cardiovascular toxicity was performed using a hERG patch clamp assay, in which compound 65 displayed a safe profile (IC50 >100 µM). Inhibition of the major cytochrome P450 (CYP) isosymes (1A2, 2C9, 2C19, 2D6, and 3A4) is notably low with an IC50 >70 µM for each CYP450 tested. Kinase profiling: Following the identification of 65 as a lead molecule, in vitro selectivity was studied extensively. The compound was tested at 1 µM against a panel of 170 kinases. Of the 170 kinases tested, only weak inhibition was observed for hFLT3, hFLT4 and hCSF1R, with IC50’s of 338 nM, 274 nM and 489 nM, respectively. No off-target liabilities were identified upon screening a diverse panel of 99 GPCRs, ion channels, transporters and non-kinase enzymes (data not shown). Crystallography To explore the binding mode of our selected inhibitor to the enzyme, 65 was co-crystallized with JAK2 JH1 domain crystals, as JAK1 co-crystal was not readily available at that time and since


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our modeling predicted a similar binding mode. As shown in Figure 4, 65 binds to the ATP binding pocket of JAK2 with the amino triazolopyridine core interacting with the hinge region of the kinase. The triazolopyridine ring is positioned against the hinge, and forms via the exocyclic NH and the triazolo nitrogen N3 two hydrogen bonds with the main chain atoms of Leu932. The phenyl ring sits within a hydrophobic environment. The terminal thiomorpholine dioxide group packs against the glycine rich loop, forming polar interactions with main chain atoms of this flexible loop (Gly861, Ser862), the side chain of Val863, the catalytic Lys882 and Asp994 of the DFG segment. This same binding should occur in all JAK ATP pockets and may explain the low selectivity observed in biochemical assay. In conclusion, the crystal structure does not explain the high selectivity observed in the whole blood assay. Figure 4: Crystal structure of JAK2 with 65; A) Crystal structure of JAK2 with compound 65; B) Detailed view of the X-ray crystal structure of compound 65 bound to the ATP-binding site of JAK2. Presumed hydrogen bonds involving compound 65 are represented as yellow lines with interatomic distances indicated in angstroms (PDB: 4P7E)

B

A



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Page 28 of 70

In vivo efficacy assessment:8 The rat collagen-induced arthritis (CIA) model was used to assess the potential therapeutic utility of 65 for the treatment of autoimmune arthritis, as it was shown previously that selective JAK1 inhibitor can be efficacious.23 We measured disease progression following daily oral administration of 65 at doses of 0.1, 0.3, 1, 3, 10 and 30 mg/kg in 3 separate studies. Disease severity was assessed periodically, scoring clinical signs of disease and comparing to a vehicle and an etanercept-treated group as positive control (Enbrel® 10 mg/kg, 3 times/week). Data obtained from the 3 studies were normalized to the vehicle data for each experiment, allowing a meta-analysis of the 3 studies (Figure 5). A dose-dependent effect was observed in all readouts. A statistically significant reduction of the clinical score was measured, surprisingly even at doses as low as 0.1 mg/kg and 0.3 mg/kg.

Figure 5. CIA rat model.8,27 Difference between the clinical score at day 0 and day 15 (Meta-analysis of combined data obtained for the three studies after 15 d of treatment. E, etanercept; V, vehicle. 65 doses are indicated on the xaxis. The numbers of animals used to obtain a meta-analysis score for V, E, 0.1, 0.3, 1, 3, 10, and 30 mg/kg were 29, 10, 20, 20, 29, 20, 10, and 29, respectively. *p , 0.05, **p , 0.01, ***p , 0.001 versus vehicle, Student t test). Copyright 2013. The American Association of Immunologists, Inc.

Only at 10 mg/kg the concentration of 65 in blood covered whole blood IC50 for almost 8 hrs (Figure 6). At doses up to 3 mg/kg, the in vivo exposure of the compound barely reaches the IC50 28 ACS Paragon Plus Environment

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in the whole blood assay (IL-6/pSTAT1). Note that the potency of in the rat biochemical assay is in the same range as human (29 nM ± 8.9)). In particular, showing efficacy for 65 in the rat CIA model at doses as low as 0.1 and 0.3 mg/kg, the latter confirmed in two separate experiments, was surprising. It is difficult to understand the strong efficacy of the compound at these low doses. As discussed by Van Rompaey et al. we cannot provide a definitive explanation for the exceptional efficacy of compound 65.8 One can surmise that the very low protein binding of 65 allows for a rapid and extensive penetration into tissues, including the inflamed joints. FLT3 and FLT4 are the closest off-target kinases and their inhibition could contribute to the antiinflammatory effects, but the >25 fold selectivity for JAK1 inhibition does not support this hypothesis.8 The compound is also known to form an active metabolite (compound 66, Scheme 6).28 This metabolite is formed by simple hydrolysis of the electron deficient aniline in position 2 of the triazolo-pyridine and was found to be less potent than compound 65, while retaining a good JAK1 selectivity. The PK profile of 66 in humans show a high and sustained exposure, well exceeding the exposure of parent 65, and this metabolite is understood to contribute to the overall clinical efficacy.28 However, at doses below 3 mg/kg in rats, plasma levels would still remain well below its IC50 for JAK1 inhibition. Thus, none of the current hypotheses are truly satisfactory, and future work will need to identify more likely explanations. Figure 6: Steady state PK (day 9) of 65 in rat CIA. WBA IC50 pSAT1/IL6 and GM_CSF/pSTAT5 are indicated



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N NH2

N N

Page 30 of 70

hJAK1 IC50 = 307 nM IL-6/pSTAT1 = 11.9 µM GM-CSF/pSTAT5 = >100 µM

N S

O

O

Scheme 6: structure of compound 66, metabolite of compound 65.29

Conclusion: A potent and novel series of JAK1 inhibitors was discovered from an initial screening hit to lead SAR study. Importantly, this has led to the identification of GLPG0634, the first reported selective JAK1 inhibitor in clinical trials. GLPG0634 exhibits: good potency in enzyme and cellular systems; selectivity against JAK2 in whole blood assays; good in vitro ADME properties; oral bioavailability in animal species; and dose-dependent efficacy in CIA with oral dosing. Results from two Phase 2A studies in RA have shown an encouraging efficacy and safety 30 ACS Paragon Plus Environment

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profile, and compound 65 is being currently tested in Phase 2B clinical trials in rheumatoid arthritis and Phase 2A in inflammatory bowel disease. 26

Acknowledgement: We acknowledge the expert technical or editorial assistance of Cécile Belleville, Marie Christine Cecotti, Christelle David, Francois Gendrot, Didier Merciris, Alain Monjardet, Isabelle Orlans, Isabelle Parent, Laetitia Perret, Emanuelle Wakselmann (affiliated with Galapagos SASU) and of Katja Conrath, Annick Hagers, Annelies Iwens, Daisy Liekens, Maarten Van Balen, Kris Van Beeck (affiliated with Galapagos NV). We thank GSK for their financial support and especially Mike Skynner and Phil Jeffrey for the scientific discussions. AbbVie has provided funding to Galapagos for development of compound 65.

Experimental section

Biochemical assays All detail were reported in reference 8

Chemistry procedures All reagents were of commercial grade and were used as received without further purification, unless otherwise stated. Commercially available anhydrous solvents were used for reactions conducted under inert atmosphere. Reagent grade solvents were used in all other cases, unless



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Page 32 of 70

otherwise specified. Column chromatography was performed on silica gel 60 (35-70 µm). Thin layer chromatography was carried out using pre-coated silica gel F-254 plates (thickness 0.25 mm).

1

H NMR spectra were recorded on a Bruker DPX 400 NMR spectrometer (400 MHz).

Chemical shifts (δ) for 1H NMR spectra are reported in parts per million (ppm) relative to tetramethylsilane (δ 0.00) or the appropriate residual solvent peak, i.e. CHCl3 (δ 7.27), as internal reference. Multiplicities are given as singlet (s), doublet (d), triplet (t), quartet (q), multiplet (m) and broad (br). Coupling constants (J) are given in Hz. Electrospray MS spectra were obtained on a Micromass platform LC/MS spectrometer. Columns Used for LCMS analysis: Hichrom, Kromasil Eternity, 2.5µm C18, 150 x 4.6mm, Waters Xbridge 5µm C18 (2), 250 x 4.6mm (ref 86003117), Waters Xterra MS 5µm C18, 100 x 4.6mm (Plus guard cartridge) (ref 186000486), Gemini-NX 3 µm C18 100 x 3.0 mm (ref 00D-4453-Y0), Phenomenex Luna 5µm C18 (2), 100 x 4.6mm. (Plus guard cartridge) (ref 00D-4252-E0), Kinetix fused core 2.7µm C18 100 x 4.6 mm (ref 00D-4462-E0), Supelco, Ascentis® Express C18 (ref 53829-U), or Hichrom Halo C18, 2.7µm C18, 150 x 4.6mm (ref 92814-702). LC-MS were recorded on a Waters Micromass ZQ coupled to a HPLC Waters 2795, equipped with a UV detector Waters 2996. LC were also run on a HPLC Agilent 1100 coupled to a UV detector Agilent G1315A. Preparative HPLC: Waters XBridge Prep C18 5µm ODB 19mm ID x 100mm L (Part No.186002978). All the methods are using MeCN/H2O gradients. H2O contains either 0.1% TFA or 0.1% NH3.

General procedure for mono-acylation To a solution of the triazolo[1,5-a]pyridin-2-ylamine intermediate (1 eq.) in dry CH3CN at 5 ºC is added Et3N (2.5 eq.) followed by the acyl chloride (2.5 eq.). The reaction mixture is then allowed to warm to rt and stirred until all starting material was consumed. If required, further 32 ACS Paragon Plus Environment

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Et3N (1 eq) and acyl chloride (1 eq) are added to ensure complete reaction. Following solvent evaporation in vacuum, the resultant residue is treated with 7 N methanolic ammonia solution and stirred at rt (for 1-16 h) to hydrolyse any bis-acylated product. Product isolation is made by removal of volatiles in vacuum followed by trituration with Et2O. The solids are collected by filtration, washed with H2O, acetone and Et2O, then dried in vacuum to give the required desired compound. General procedure for Suzuki coupling: The appropriate boronic acid or boronic pinacol ester (2 eq.) is added to a solution of the halide intermediate in 1,4-dioxane/water (5:1). K2CO3 (2 eq.) and PdCl2dppf (0.05 eq.) are added to the solution. The resulting mixture is then heated in a microwave at 140 °C for 30 min (this reaction can also be carried out by traditional heating in an oil bath at 90 °C for 16h under N2). Water is added and the solution is extracted with EtOAc. The organic layers are dried over anhydrous MgSO4 and evaporated in vacuum. The compound is obtained after purification by flash chromatography or preparative HPLC. HPLC: Waters XBridge Prep C18 5 µm ODB 19mm ID x 100mm L (Part No.186002978). All the methods are using MeCN/H2O gradients. H2O contains either 0.1% TFA or 0.1% NH3.

Preparation of 1-(6-Bromo-pyridin-2-yl)-3-carboethoxy-thiourea (5)

To a solution of 2-amino-6-bromopyridine (4) (253.8 g, 1.467 mol) in DCM (2.5 L) cooled to 5 ºC was added ethoxycarbonyl isothiocyanate (173.0 mL, 1.467 mol) dropwise over 15 min. The reaction mixture was then allowed to warm to room temp. (20 ºC) and stirred for 16 h.



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Evaporation in vacuum gave a solid which was collected by filtration, thoroughly washed with petrol (3 × 600 mL) and air-dried to afford (5) (100% of conversion observed by UPLC). The thiourea was used as such for the next step without any purification (purity, 99 % by UPLC). 1H (400 MHz, CDCl3) δ 12.03 (1H, br s, NH), 8.81 (1H, d, J 7.8 Hz, H-3), 8.15 (1H, br s, NH), 7.60 (1H, t, J 8.0 Hz, ArH), 7.32 (1H, dd, J 7.7 and 0.6 Hz, ArH), 4.31 (2H, q, J 7.1 Hz, CH2), 1.35 (3H, t, J 7.1 Hz, CH3).

Preparation of 5-Bromo-[1,2,4]triazolo[1,5-a]pyridin-2-ylamine (6) To a suspension of hydroxylamine hydrochloride (101.8 g, 1.465 mol) in EtOH/MeOH (1:1, 900 mL) was added N,N-diisopropylethylamine (145.3 mL, 0.879 mol) and the mixture was stirred at rt (20 ºC) for 1 h. 1-(6-Bromo-pyridin-2-yl)-3-carboethoxy-thiourea (5) (89.0 g, 0.293 mol) was then added and the mixture slowly heated to reflux (Note: bleach scrubber is required to quench H2S evolved). After 3 h at reflux, the mixture was allowed to cool and filtered to collect the precipitated solid. Further product were collected by evaporation under vacuum of the filtrate, addition of H2O (250 mL) and filtration. The combined solids were washed successively with H2O (250 mL), EtOH/MeOH (1:1, 250 mL) and Et2O (250 mL) then dried under vacuum to afford the triazolopyridine derivative (6) as a solid (100 % conversion observed by UPLC. The compound was used as such for the next step without any purification (99 % purity by UPLC). 1

H (400 MHz, DMSO-d6) δ 7.43-7.34 (2H, m, ArH), 7.24 (1H, dd, J 6.8 and 1.8 Hz, ArH), 6.30

(2H, br, NH2); LCMS m/z 213/215 (1:1, M+H+, 100%).

Preparation of cyclopropanecarboxylic acid (5-bromo-[1,2,4]triazolo[1,5-a]pyridin-2-yl)-amide 9.


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Starting from 5-bromo-[1,2,4]triazolo[1,5-a]pyridin-2-ylamine, 6 (0.15 mg, 0.7 mmol), cyclopropanecarbonylacid chloride (0.159 mL, 1.76 mmol) and triethylamine (0.25 mL, 1.76 mmol), compound 9 was prepared in 79 % yield (99 % purity by UPLC), using the general procedure for mono-acylation. 1H (400 MHz, d6-DMSO) δ 11.18 (1H, br s, NH), 7.71 (1H, dd, J 8.5 and 1.3 Hz, ArH), 7.56 (1H, t, J 7.6 Hz, ArH); 7.48 (1H, dd, J 7.6 and 1.3 Hz, ArH), 2.05 (1H, br, CH), 0.84-0.83 (4H, m, 2xCH2); LCMS m/z 281.0/283.0 (1:1, M+H+). Preparation

of

cyclopropanecarboxylic

acid

[5-(4-methoxy-phenyl)-[1,2,4]triazolo[1,5-

a]pyridin-2-yl]-amide, 3 Starting from 4-methoxyphenyl boronic acid (0.132 mg, 0.85 mmol) and cyclopropanecarboxylic acid (5-bromo-[1,2,4]triazolo[1,5-a]pyridin-2-yl)-amide 9 (0.120 mg, 0.42 mmol), compound 3 was prepared using the general procedure for the Suzuki coupling reaction. Purification by silica chromatography (EtOAc/petrol ether; 50:50) afforded the desired compound in 15 % yield (99 % purity by UPLC). LCMS m/z 309.8 (M+H+). 1H (400 MHz, CDCl3) 9.12 (1H, br s, NH), 7.97 (2H, d, J 8.5 Hz, ArH), 7.60 (21H, m, ArH), 7.08 (3H, m, ArH), 7.09 (2H, d, J 8.8 Hz, ArH), 3.90 (3H, s, OCH3), 1.60 (1H, under water peak, CH), 1.00-0.81 (4H, m, 2xCH2).

Preparation of 5-(4-methoxy-phenyl)-[1,2,4]triazolo[1,5-a]pyridin-2-ylamine 7 Starting from 4-methoxyphenyl boronic acid (0.282 mmol, 42.80 mg) and 5-bromo[1,2,4]triazolo[1,5-a]pyridin-2-ylamine 6 (0.141 mmol, 30 mg), compound 7 was prepared using the general procedure for the Suzuki coupling reaction. Recrystallisation with MeOH/EtOAc afforded the desired compound in 82 % yield (28 mg, 99 % purity by UPLC). LCMS m/z 241.1 (M+H+).



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Preparation of 2-Iodo-5-(4-methoxy-phenyl)-[1,2,4] triazolo[1,5-a]pyridine 8 A solution of 57% aqueous HI (8.4 mL) in DMSO (20 mL) was added dropwise to a solution of 5-(4-methoxy-phenyl)-[1,2,4]triazolo[1,5-a]pyridin-2-ylamine 7 (5.4 g, 23 mmol) in a mixture of NaNO2 in DMSO (6.3 g in 30 mL of DMSO) at 35 ºC with stirring. The mixture was stirred at 35 ºC for 10 min (monitored by LCMS) and then it was transferred to a solution containing K2CO3 (10 g) in 80 mL of water. The reaction mixture was taken up in EtOAc and the extract was washed with water, dried (MgSO4), filtered and concentrated under vacuum. The crude product was obtained in 72 % yield and was used without further purification (99 % purity by UPLC). LCMS m/z 351.8 (M+H+).

Preparation

of


acid


a]pyridin-2-yl]-methyl-amide 26 and 5-(4-Methoxy-phenyl)-[1,2,4]triazolo[1,5-a]pyridin-2-yl]dimethyl-amine 24 To compound 3 (25 mg, 0.080 mmol) in dry THF (0.5 mL) at 0 °C was added NaH (60 % dispersion, 0.20 mmol, 6 mg) followed by MeI (6 µL, 0.097 mmol) and the mixture was stirred at rt for 2 h. The solution was quenched with water, extracted with ethyl acetate, dried (MgSO4), filtered and concentrated under vacuum. The crude material contained both title compounds 26 and 24 that were purified by preparative HPLC (26:7 mg, 28 % yield; 24: 4 mg, 13 % yield) Compound 24: (99 % purity by UPLC). LCMS m/z 269.1 (M+H+); 1H (400 MHz, CDCl3) 8.04 (2H, d, J 8.9 Hz, ArH), 7.37-7.40 (2H, m, ArH), 7.05 (2H, d, J 9.0 Hz, ArH), 6.87 (1H, m, ArH), 3.9 (3H, s, OCH3), 3.16 (6H, s, 2xCH3).


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Compound 26 (99 % purity by LCMS): LCMS m/z 323.1 (M+H+); 1H (400 MHz, CDCl3) 7.97 (2H, d, J 8.8 Hz ArH), 7.62 (2H, m, ArH), 7.12 (1H, dd, J 2.8, 5.8 Hz ArH), 7.06 (2H, d, J 8.8 Hz, ArH), 3.92 (3H, s, OCH3), 3.53 (3H, s, CH3), 2.65-2.57 (1H, m, CH), 1.17-1.12 (2H, m, CH2), 0.85-0.81 (2H, m, CH2).

Preparation

of

1-methyl-cyclopropanecarboxylic

acid

[5-(4-methoxy-phenyl)-

[1,2,4]triazolo[1,5-a]pyridin-2-yl]-amide 25 Step i: preparation of 1-methyl-cyclopropanecarbonyl chloride O

O

Cl

OH

1-Methyl-cyclopropanecarboxylic acid (84 mg, 0.83 mmol) was dissolved in DCM with a catalytic amount of DMF (1 drop) under nitrogen. The mixture was cooled to 5 °C and SOCl2 (84 µL, 1.16 mmol) was added dropwise. The excess of solvent and reagent was evaporated under vacuum and the obtained acid chloride was used without further purification. Step

ii:

Preparation

of

1-methyl-cyclopropanecarboxylic

acid

[5-(4-methoxy-phenyl)-

[1,2,4]triazolo[1,5-a]pyridin-2-yl]-amide 25 Using the previously made acid chloride and the amine 7, compound 25 was obtained in 48 % yield using the general procedure for mono-acylation (99% purity by UPLC). 1H (400 MHz, CDCl3) 9.19 (1H, br s, NH), 7.99 (2H, d, J 8.8 Hz, ArH), 7.66 (1H, dd, J 8.8, 7.6 Hz, ArH), 7.60 (1H, d, J 8.4 Hz, ArH), 7.18 (1H, dd, J 7.2, 1.2 Hz, ArH), 7.09 (2H, d, J 8.8 Hz, ArH), 3.88 (3H, s, OCH3), 1.50 (3H, s, CH3), 1.41 (2H, dd, J 7.2, 4.4 Hz, CH2), 0.75 (2H, dd, J 6.8, 4.4 Hz, CH2); LCMS m/z 323.0 (M+H+). 37 ACS Paragon Plus Environment


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Page 38 of 70

Preparation of cyclopropanesulfonic acid [5-(4-methoxy-phenyl)-[1,2,4]triazolo[1,5-a]pyridin2-yl]-amide 27 A mixture of compound 8 (100 mg, 0.28 mmol), cyclopropanesulfonamide (42 mg, 0.34 mmol), Pd(OAc)2 (28 mg, 0.12 mmol), Xantphos (72 mg, 0.12 mmol) and Cs2CO3 (446 mg, 1.36 mmol) in dry 1,4-dioxane (1.8 mL) was degassed under nitrogen and then stirred at 120 °C until completion. The resulting mixture was diluted with EtOAc and washed with water. The organic layer was dried (MgSO4), filtered and concentrated in vacuum. Purification by preparative HPLC gave the desired product (3 mg, 3 % yield) (86 % purity by UPLC). 1H (400 MHz, CDCl3) δ 7.96 (2H, d, J 8.8 Hz, ArH), 7.72 (1H, dd, J 8.8, 1.2 Hz, ArH), 7.57 (1H, dd, J 8.8, 7.2 Hz, ArH), 7.08-7.04 (3H, m, ArH), 3.90 (3H, s, OCH3), 3.08-3.02 (1H, m, CH), 1.47-1.43 (2H, m, CH2), 1.08-1.03 (2H, m, CH2); LCMS m/z 344.9 (M+H+). Preparation of N-[5-(4-methoxy-phenyl)-[1,2,4]triazolo[1,5-a]pyridin-2-yl]-isobutyramide 28 Starting from compound 7 (20 mg, 0.083 mmol) and isobutyryl chloride (0.022 mL, 0.208 mmol), compound 28 was obtained in 85 % yield using the general procedure for monoacylation (22 mg, 99 % purity by HPLC). 1H (400 MHz, d6-DMSO) δ 10.71 (1H, s, NH), 8.03 (2H, d, J 8.8 Hz, ArH), 7.70 (1H, dd, 8.8, 7.2 Hz, ArH), 7.64 (1H, dd, J 8.8, 1.6 Hz, ArH), 7.27 (1H, dd, J 7.2, 1.2 Hz, ArH), 7.13 (2H, d, J 8.8 Hz, ArH), 3.86 (3H, s, OCH3), 2.77 (1H, br, CH), 1.11-1.08 (6H, m, 2xCH3); LCMS m/z 311.0 (M+H+).

Preparation of N-[5-(4-methoxy-phenyl)-[1,2,4]triazolo[1,5-a]pyridin-2-yl]-acetamide 29 Step i: Preparation of N-(5-bromo-[1,2,4]triazolo[1,5-a]pyridin-2-yl)-acetamide 38 ACS Paragon Plus Environment

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O N N N

N NH2

N N

Br

N H

Br

Starting from compound 6 (50 mg, 0.235 mmol) and acetyl chloride (0.042 mL, 0.587 mmol), the title compound was obtained in 73% yield using the general procedure for mono-acylation (44 mg). LCMS m/z 255.1/257.1 (1:1; M+H+). The compound was used in the next step without further purification. Step ii: Starting from N-(5-bromo-[1,2,4]triazolo[1,5-a]pyridin-2-yl)-acetamide (44 mg, 0.172 mmol) and 4-methoxyphenyl boronic acid (52.4 mg, 0.345 mmol), compound 29 was obtained in 25 % yield using the general procedure for Suzuki coupling (13 mg) (99 % purity by UPLC). 1H (400 MHz, d6-DMSO) δ 10.69 (1H, br s, NH), 8.01 (2H, d, J 8.8 Hz, ArH), 7.69 (1H, dd, J 8.8, 6.8 Hz, ArH), 7.64 (1H, dd, J 8.8, 1.2 Hz, ArH), 7.26 (1H, dd, J 7.2, 1.2 Hz, ArH), 7.12 (2H, d, J 8.8 Hz, ArH), 3.86 (3H, s, OCH3), 2.14 (3H, br s, CH3); LCMS m/z 283.1 (M+H+).

Preparation of N-[5-(4-methoxy-phenyl)-[1,2,4]triazolo[1,5-a]pyridin-2-yl]-benzamide 30 Starting from compound 7 (20 mg, 0.083 mmol) and benzoyl chloride (0.024 mL, 0.208 mmol), compound 30 was prepared in 21 % yield using the general procedure for mono-acylation (7 mg) (99 % purity established by UPLC). 1H (400 MHz, CDCl3) δ 10.9 (1H, br s, NH), 8.01-7.99 (4H, m, ArH), 7.67-7.50 (5H, m, ArH), 7.15 (1H, d, J 6.4 Hz, ArH), 7.07 (2H, d, J 8.8 Hz, ArH), 3.88 (3H, s, OCH3); LCMS m/z 345.1 (M+H+). Preparation of 5-(4-methoxy-phenyl)-2-piperidin-1-yl-[1,2,4]triazolo[1,5-a]pyridine 31



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Page 40 of 70

A mixture of 2-chloro-5-(4-methoxy-phenyl)-[1,2,4]triazolo[1,5-a]pyridine (30 mg, 0.116 mmol), piperidine (14 µL, 0.114 mmol), DIPEA (0.22 mmol) in t-BuOH (0.5 mL) was heated at 120 °C for 14 days. The resulting mixture was diluted with EtOAc and washed with water (3 times). The organics were dried (MgSO4), filtered and concentrated to give the desired compound in 31 % yield (99 % purity by UPLC). 1H (400 MHz, CD3OD) δ 8.00 (2H, d, J 9.2 Hz, ArH), 7.85 (1H, dd, J 8.8, 7.6 Hz, ArH), 7.52 (1H, dd, J 8.8, 1.2 Hz, ArH), 7.37 (1H, dd, J 7.6, 1.6 Hz, ArH), 7.12 (2H, d, J 8.8 Hz, ArH), 3.91 (3H, s, OCH3), 3.59 (1H, br, 2xCH2), 1.73 (6H, br, 3xCH2); LCMS m/z 309.0 (M+H+). Preparation of cyclopropylmethyl-[5-(4-methoxy-phenyl)-[1,2,4]triazolo[1,5-a]pyridin-2-yl]amine 32 A mixture of cyclopropanecarboxaldehyde (15 µL, 0.166 mmol), compound 7 (20 mg, 0.083 mmol) and Ti(OiPr)4 (64 µL, 0.194 mmol) was stirred at rt. After 1 h, the mixture was diluted with EtOH (0.1 mL), Na(CN)BH3 (6 mg, 0.083 mmol) was added and the solution was stirred for 20 h. Water (2 mL) was added with stirring and the resulting precipitate was filtered and washed with EtOH. The filtrate was concentrated, dissolved in EtOAc, filtered to remove the remaining inorganic solids and concentrated under vacuum. Purification by preparative HPLC afforded the desired compound in 28 % yield (99 % purity by UPLC). 1H (400 MHz, d6-DMSO) δ 7.98 (2H, d, J 9.2 Hz, ArH), 7.55 (1H, dd, J 8.8, 7.6 Hz, ArH), 7.37 (1H, dd, J 8.8, 1.2 Hz, ArH), 7.11-7.06 (3H, m, ArH), 3.85 (3H, s, OCH3), 3.12 (1H, d, J 6.8 Hz, CH2), 1.11-1.04 (1H, m, CH), 0.440.39 (2H, m, CH2), 0.24-0.21 (2H, m, CH2); LCMS m/z 295.1 (M+H+). Preparation of 1-[5-(4-methoxy-phenyl)-[1,2,4]triazolo[1,5-a]pyridin-2-yl]-3-methyl-urea 33


Page 41 of 70

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To a mixture of compound 7 (30 mg, 0.125 mmol) in DCM (0.3 mL) was added DIPEA (50 µL, 0.289 mmol) followed by 4-nitrophenyl chloroformate (31 mg, 0.150 mmol) and the mixture was stirred for 20. Then methylamine (27 mg, 0.875 mmol) and DIPEA (0.15 mL, 0.875 mmol) were added. After completion, the resulting mixture was diluted with EtOAc and washed with aq. sat. NaHCO3 (3x). The organic was dried (MgSO4), filtered and concentrated under vacuum. Purification by preparative HPLC afforded the desired compound in 16 % yield (99 % purity by UPLC). 1H (400 MHz, d6-DMSO) δ 9.88 (1H, s, NH), 8.14 (1H, app. d, J 5.2 Hz, NH), 7.96 (2H, d, J 8.8 Hz, ArH), 7.71 (1H, dd, J 8.8, 7.2 Hz, ArH), 7.62 (1H, dd, J 8.8, 1.6 Hz, ArH), 7.24 (1H, dd, J 7.2, 1.2 Hz, ArH), 7.13 (2H, d, J 8.8 Hz, ArH), 3.86 (3H, s, OCH3), 2.78 (3H, d, J 4.4 Hz, CH3); LCMS m/z 298.0 (M+H+). Preparation of 5-(4-methoxy-phenyl)-2-pyridin-3-yl-[1,2,4]triazolo[1,5-a]pyridine 34 A mixture of the 2-pyridine boronic acid (29 mg, 0.23 mmol), 2-chloro-5-(4-methoxy-phenyl)[1,2,4]triazolo[1,5-a]pyridine (30 mg, 0.23 mmol), K2CO3 (80 mg, 0.58 mmol) and PdCl2dppf (13 mg, 0.012 mmol) in DME/water/EtOH (7:3:2 (v:v:v), 0.5 mL) was heated for 30 min at 140 °C in a microwave reactor. The resulting mixture was diluted with EtOAc and washed with water (3 times). The organic was dried (MgSO4), filtered and evaporated under vacuum. Purification by preparative HPLC afforded the desired compound in 43 % yield (99 % purity by UPLC). 1H (400 MHz, CDCl3) δ 9.61 (1H, s, ArH), 8.98 (1H, d, J 7.6 Hz, ArH), 8.79 (1H, d, J 4.8 Hz, ArH), 7.71 (2H, d, J 8.4 Hz, ArH), 7.80-7.54 (2H, m, ArH), 7.67 (1H, dd, J 8.4, 7.6 Hz, ArH), 7.18 (1H, dd, J 7.2, 1.2 Hz, ArH), 7.11 (2H, d, J 8.4 Hz, ArH), 3.94 (3H, s, OCH3); LCMS m/z 303.0 (M+H+). Preparation of [5-(4-methoxy-phenyl)-[1,2,4]triazolo[1,5-a]pyridin-2-yl]-phenyl-amine 35



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Page 42 of 70

A mixture of 2-chloro-5-(4-methoxy-phenyl)-[1,2,4]triazolo[1,5-a]pyridine (30 mg, 0.086 mmol), N-(4-amino-phenyl)-acetamide (14.2 mg, 0.095 mmol), Pd(OAc)2 (2 mg, 0.0086 mmol), BINAP (5.4 mg, 0.0086 mmol) and Cs2CO3 (140 mg, 0.43 mmol) in toluene (0.5 mL) was degassed under nitrogen and then stirred at 100 °C for 15 h. The resulting mixture was diluted with EtOAc and washed with water (3 times). The organic layer was dried (MgSO4), filtered and concentrated under vacuum. Purification by preparative HPLC to give the desired product in 31 % yield (10 mg, 99 % purity by UPLC). 1H (400 MHz, DMSO-d6) δ 9.76 (1H, s, NH); 9.51 (1H, s, NH); 8.04 (2H, d, J 8.8 Hz , ArH); 7.60 (3H , m, J 9.0, 8.8, 7.4 Hz, ArH,); 7.51 (1H, dd, J 8.8, 1.2 Hz, ArH); 7.44 (2H, d, J 9.2 Hz, ArH); 7.14 (2H, d, J 8.9 Hz, ArH); 7.13 (1H, dd, J 7.32, 1.3 Hz, ArH); 3.87 (3H, s, OCH3); 2.01 (3H, s, CH3).. Preparation of N-[5-(4-methoxy-phenyl)-[1,2,4]triazolo[1,5-a]pyridin-2-yl]-propionamide 36 Starting from compound 7 (20 mg, 0.083 mmol) and propionyl chloride (0.029 mL, 0.332 mmol), compound 33 was prepared in 44 % yield using the general procedure for monoacylation (12 mg) (99 % purity by UPLC). 1H (400 MHz, DMSO-d6) δ 10.68 (1H, br s, NH), 8.01 (2H, d, J 9.1 Hz, ArH), 7.69 (1H, dd, J 7.3, 8.8 Hz, ArH), 7.62 (1H, dd, J 1.3, 8.8 Hz, ArH), 7.26 (1H, dd, J 1.5, 7.1 Hz, ArH), 7.12 (2H, d, J 8.8 Hz, ArH), 3.86 (3H, s, OCH3), 3.5 (peak of CH2 under peak of water), 1.06 (3H, t, J 7.5 Hz, CH3); LCMS m/z 297.1 (M+H+). Preparation of cyclopropanecarboxylic acid (5-phenyl-[1,2,4]triazolo[1,5-a]pyridin-2-yl)-amide 37 Starting from compound 9 (120 mg, 0.427 mmol)) and phenylboronic acid (104 mg, 0.854 mmol), compound 37 was prepared using the general procedure for the Suzuki coupling reaction. Purification by silica chromatography (EtOAc/petrol ether; 1:1) followed by trituration with 42 ACS Paragon Plus Environment

Page 43 of 70

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petrol ether afforded the desired compound (33.7 mg, 28 % yield, 99 % purity by UPLC). 1H (400 MHz, CDCL3 ) δ 9.30 (1H, brs, NH), 7.98 (2H, m, ArH), 7.54 (5H, m, ArH), 7.01 (1H, m, ArH), 1.90 (1H, under water peak), 1.20 (2H, m, CH2), 0.93 (2H, m, CH2); LCMS m/z 279.1 (M+H+). Preparation

of


acid


a]pyridin-2-yl]-amide 38 Starting from compound 9 (40 mg, 0.142 mmol) and 2-methoxyphenylboronic acid (64.87 mg, 0.285 mmol), compound 35 was prepared using the general procedure for the Suzuki coupling reaction. Purification by preparative HPLC afforded the desired compound (43 mg, 99 % yield) (99 % pure by UPLC). 1H (400 MHz, d6-DMSO ) δ 11.10 (1H, br s, NH), 7.70 (2H, d, J 3.6 Hz, ArH), 7.53 (1H, t, J 7.6 Hz, ArH), 7.44 (1H, d, J 7.2 Hz, ArH), 7.21 (1H, d, J 8.4 Hz, ArH), 7.11-7.08 (2H, m, ArH), 3.72 (3H, s, OCH3), 1.99 (1H, br, CH), 0.79 (4H, app. d, J 5.6 Hz, 2xCH2); LCMS m/z 309.1 (M+H+). Preparation of cyclopropanecarboxylic acid [5-(3-chloro-phenyl)-[1,2,4]triazolo[1,5-a]pyridin2-yl]-amide 39 Starting from compound 9 (100 mg, 0.35 mmol) and 3-chlorophenylboronic acid (82 mg, 0.53 mmol), compound 36 was prepared using the general procedure for the Suzuki coupling reaction. Purification by silica chromatography (EtOAc/petrol ether; 70:30) afforded the desired compound (84.6 mg, 77 % yield) (99 % purity by UPLC). 1H (400 MHz, d6-DMSO ) δ 11.04 (1H, br s, NH), 8.13-8.12 (1H, m, ArH), 7.97-7.95 (1H, m, ArH), 7.74-7.72 (2H, m, ArH), 7.627.60 (2H, m, ArH), 7.36 (1H, dd, J 4.8, 4.0 Hz, ArH), 2.05 (1H, br, CH), 0.83-0.81 (4H, m, 2xCH2); LCMS m/z 313.1 (M+H+). 43 ACS Paragon Plus Environment


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Page 44 of 70

Preparation of cyclopropanecarboxylic acid [5-(4-chloro-phenyl)-[1,2,4]triazolo[1,5-a]pyridin2-yl]-amide 40 Starting from compound 9 (30 mg, 0.10 mmol) and 4-chlorophenylboronic acid (24 mg, 0.15 mmol), compound 37 was prepared using the general procedure for the Suzuki coupling reaction. Purification by preparative HPLC afforded the desired compound (23 mg, 73 % yield) (purity 99 % by UPLC). LCMS m/z 313.1 (M+H+).1H (400 MHz, CDCL3 ) δ 9.0 (1H, br s, NH), 7.95 (2H, d, J 8.5 Hz, ArH), 7.63 (2H, m, ArH), 7.53 (2H, d, J 8.8 Hz, ArH), 7.11 (1H, d, J 8.6 Hz, ArH), 2.22 (1H, under water peak, CH), 1.21 (2H, m, CH2), 0.96 (2H, m, CH2); LCMS m/z 309.1 (M+H+) Preparation of cyclopropanecarboxylic acid (5-quinolin-5-yl-[1,2,4]triazolo[1,5-a]pyridin-2-yl)amide 41 Starting from compound 9 (30 mg, 0.10 mmol) and quinoline-5-boronic acid (26 mg, 0.15 mmol), 41 was prepared using the general procedure for the Suzuki coupling reaction. Purification by preparative HPLC afforded the desired compound (34 mg, 99 % yield, 99 % puirity by UPLC). LCMS m/z 329.4 (M+H+). 1H (400 MHz, d6-DMSO ) δ 11.00 (1H, b, NH), 9.03 (1H, b, ArH), 8.27 (1H, d, J 8.3 Hz, ArH), 7.99 (1H, m, ArH), 7.89 (4H, m, ArH), 7.57 (1H, m, ArH), 7.32 (1H, d, J 6.40 Hz, ArH), 1.91 (1H, b, CH), 0.75 (4H, m, 2xCH2). Preparation of cyclopropanecarboxylic acid [5-(6-methoxy-pyridin-3-yl)-[1,2,4]triazolo[1,5a]pyridin-2-yl]-amide 42 Starting from compound 9 (40.0 mg, 0.142 mmol) and 6-methoxypyridin-3-ylboronic acid (43.3 mg, 0.285 mmol), compound 42 was prepared using the general procedure for the Suzuki coupling reaction. Purification by preparative HPLC afforded the desired compound (43.9 mg, 44 ACS Paragon Plus Environment

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46 % yield, 99 % purity by UPLC). 1H (400 MHz, d6-DMSO ) δ 11.11 (1H, s, NH), 8.87 (1H, s, ArH), 8.39 (1H, d, J 8.8 Hz, ArH), 7.75-7.67 (2H, m, ArH), 7.37 (1H, d, J 6.8 Hz, ArH), 7.02 (1H, d, J 8.8 Hz, ArH), 3.95 (3H, s, OCH3), 2.02 (1H, br, CH), 0.82 (4H, app. d, 2xCH2); LCMS m/z 310.1 (M+H+). Preparation of cyclopropanecarboxylic acid [5-(2-methoxy-pyrimidin-5-yl)-[1,2,4]triazolo[1,5a]pyridin-2-yl]-amide 43 Starting from compound 9 (30.0 mg, 0.10 mmol) and 2-methoxypyrimidin-5-ylboronic acid (23.0, 15 mmol) compound 43 was prepared using the general procedure for the Suzuki coupling reaction. Purification by preparative HPLC afforded the desired compound 43 (11.6 mg, 37% yield, 99% purity by UPLC). 1H (400 MHz, d6-DMSO ) δ 8.20 (2H, s, ArH), 7.91 (1H, br s, NH), 7.71 (1H, dd, J 9.2, 1.6 Hz, ArH), 7.63 (1H, dd, J 8.8, 7.2 Hz, ArH), 7.12 (1H, dd, J 7.2, 1.2 Hz, ArH), 4.14 (3H, s, OCH3), 1.67 (1H, br, CH), 1.24-1.20 (2H, m, CH2), 1.00-0.96 (2H, m, CH2); LCMS m/z 311.1 (M+H+). Preparation of cyclopropanecarboxylic acid [5-(1H-pyrazol-4-yl)-[1,2,4]triazolo[1,5-a]pyridin2-yl]-amide 44 Starting from compound 9 (30.0 mg, 0.10 mmol) and 4-pyrazoleboronic acid pinacol ester (30.0 mg, 0.15 mmol), compound 44 was prepared using the general procedure for the Suzuki coupling reaction. Purification by preparative HPLC afforded the desired compound (14.2 mg, 53 % yield, 99 % purity by UPLC). LCMS m/z 269.1 (M+H+). 1H (400 MHz, d6-DMSO ) δ 11.17 (1H, b, NH), 8.7 (2H, brs, ArH), 7.67 (1H, m, ArH), 7.61 (1H, d, J 7.0 Hz, ArH), 7.52 (1H, d, J 8.6 Hz, ArH), 2.08 (1H, b, CH), 0.87 (4H, m, 2xCH2).



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 46 of 70

Preparation of cyclopropanecarboxylic acid [5-(3-methoxymethyl-phenyl)-[1,2,4]triazolo[1,5a]pyridin-2-yl]-amide 45 Starting from compound 9 (30.0 mg, 0.10 mmol) and 3-(methoxymethyl)phenylboronic acid (24.8 mg, 0.15 mmol), compound 45 was prepared using the general procedure for the Suzuki coupling reaction. Purification by preparative HPLC afforded the desired compound (19.8 mg, 62 % yield, 99 % purity by UPLC). 1H (400 MHz, CDCl3) δ 7.96 (1H, d, J 7.6 Hz, ArH), 7.90 (1H, s, ArH), 7.75 (1H, app. t, J 8.8 Hz, ArH), 7.67 (1H, d, J 8.8 Hz, ArH), 7.54 (1H, t, J 7.6 Hz, ArH), 7.49 (1H, d, 7.6 Hz, ArH), 7.27 (1H, d, J 8.0 Hz, ArH), 4.55 (2H, s, OCH2), 3.43 (3H, s, OCH3), 2.01 (1H, br, CH), 1.18-1.15 (2H, m, CH2), 0.96-0.91 (2H, m, CH2); LCMS m/z 323.1 (M+H+). Preparation

of


acid

[5-(3-benzyloxy-phenyl)-[1,2,4]triazolo[1,5-

a]pyridin-2-yl]-amide 46 Starting from compound 9 (30.0 mg, 0.10 mmol) and 3-benzyloxyphenylboronic acid (34.0 mg, 0.15 mmol), compound 46 was prepared using the general procedure for the Suzuki coupling reaction. Purification by preparative HPLC afforded the desired compound (14.9 mg, 39 % yield) (purity 94 % by UPLC). LCMS m/z 385.2 (M+H+); 1H (400 MHz, CDCl3) δ 9.59 (1H, b, NH), 7.70-7.28 (10H, m, ArH), 7.21 (2H, m, ArH), 5.17 (2H, s, CH2), 2.06 (1H, b, CH), 1.20 (2H, m, CH2), 0.93 (2H, m, CH2). Preparation

of


acid

[5-(2-tert-butoxymethyl-phenyl)-

[1,2,4]triazolo[1,5-a]pyridin-2-yl]-amide 47 Starting from compound 9 (30.0 mg, 0.10 mmol) and 2-(tert-butoxymethyl)phenylboronic acid (31.2 mg, 0.15 mmol), compound 47 was prepared using the general procedure for the Suzuki 46 ACS Paragon Plus Environment

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coupling reaction. Purification by silica chromatography (EtOAc/petrol ether; 1:1) afforded the desired compound (24.3 mg, 66 % yield, 99 % purity by UPLC). LCMS m/z 365.2 (M+H+). Preparation

of

N-(5-(4-ethoxyphenyl)-[1,2,4]triazolo[1,5-a]pyridin-2-yl)cyclopropane

carboxamide compound 48 Starting from compound 9 (30.0 mg, 0.10 mmol) and 4-ethoxyphenylboronic acid (25.0 mg, 0.15 mmol), compound 48 was prepared using the general procedure for the Suzuki coupling reaction. Purification by silica chromatography (EtOAc/petrol ether; 7:3) afforded the desired compound (24.5 mg, 76 % yield). (99 % purity by UPLC); LCMS m/z 323.1 (M+H+).1H (400 MHz, d6DMSO ) δ 10.99 (1H, b, NH), 8.01 (2H, d, J 8.8 Hz, ArH), 7.68 (1H, dd, J 8.9, 7.4 Hz, ArH), 7.63 (1H, dd, J 8.9, 1.2 Hz, ArH), 7.25 (1H, dd, J 1.2, 7.6 Hz, ArH), 7.10 (2H, d, J 9.1 Hz, ArH), 4.14 (2H, q, J 7.0 Hz, CH2), 1.98 (1H, b, CH), 1.38 (3H, t, J 7.0 Hz, CH3), 0.82 (4H, m, 2xCH2). Preparation of 1-(6-methoxy-pyridin-2-yl)-3-carboethoxy-thiourea 11 To a solution of 6-chloro-5-methoxy-pyridin-2-ylamine (100 mg, 0.63 mmol) in 1,4-dioxane (3 mL) at 0 ºC was added ethoxycarbonyl isothiocyanate (73 µL, 0.63 mmol) dropwise and the reaction mixture was allowed to warm to rt and stirred for 16 h. Evaporation in vacuum gives a solid which was thoroughly washed with petrol and dried to afford the desired compound in 89% yield (99 % pure by UPLC). LCMS m/z 290.0 (M+H+). It was used in the next step without further purification. Preparation of 5-chloro-6-methoxy-[1,2,4]triazolo[1,5-a]pyridin-2-ylamine 12 To a suspension of hydroxylamine hydrochloride (194 mg, 2.80 mmol) in EtOH/MeOH (1:1, 3 mL) is added N,N-diisopropylethylamine (0.28 mL, 1.68 mmol) and the mixture was stirred at rt



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Page 48 of 70

for 1 h. 1-(6-Methoxy-pyridin-2-yl)-3-carboethoxy-thiourea 11 (162 mg, 0.56 mmol) was then added and the mixture was heated at reflux for 3 h. The mixture was allowed to cool to rt and filtered to collect the precipitated solid. Further product was collected by evaporation under vacuum of the filtrate, addition of H2O (2 mL) and filtration. The combined solids were washed successively with H2O (2 mL), EtOH/MeOH (1:1 (v:v), 2 mL) and Et2O (2 mL) then dried under vacuum to afford the triazolopyridine derivative 12 as a white solid in 62 % yield (69 mg, 99 % pure by UPLC). LCMS m/z 199.0 (M+H+). It was used in the next step without further purification. Preparation of cyclopropanecarboxylic acid (5-chloro-6-methoxy-[1,2,4]triazolo[1,5-a]pyridin2-yl)-amide 13 Starting from 5-chloro-6-methoxy-[1,2,4]triazolo[1,5-a]pyridin-2-ylamine 12

(69 mg, 0.347

mmol) and cyclopropanecarbonyl chloride (80 µL, 0.868 mmol), compound 13 was prepared in 65 % yield using the general procedure for mono-acylation (60 mg, 99 % purity by UPLC). LCMS m/z 267.0 (M+H+). Preparation

of


acid

[5-(4-ethoxy-phenyl)-6-methoxy-

[1,2,4]triazolo[1,5-a]pyridin-2-yl]-amide 49 Starting from cyclopropanecarboxylic acid (5-chloro-6-methoxy-[1,2,4]triazolo[1,5-a]pyridin-2yl)-amide 13 (30.0 mg, 0.11 mmol) and 4-ethoxyphenylboronic acid (28.0 mg, 0.17 mmol), compound 49 was prepared using the general procedure for the Suzuki coupling reaction. Purification by preparative HPLC afforded the desired compound in 60 % yield (23.0 mg, 99 % purity by UPLC). 1H (400 MHz, CDCl3) δ 8.52 (1H, br s, NH), 7.70 (2H, d, J 8.0 Hz, ArH), 7.59 (1H, br d, ArH), 7.50 (1H, br d, ArH), 7.05 (2H, d, J 8.4 Hz, ArH), 4.14 (2H, q, J 7.1 Hz, OCH2), 48 ACS Paragon Plus Environment

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3.82 (3H, s, CH3), 1.90 (1H, br, CH), 1.48 (3H, t, J 6.8 Hz, CH3), 1.17 (2H, br s, CH2), 0.91 (2H, br s, CH2); LCMS m/z 354.0 (M+H+). Preparation of cyclopropanecarboxylic acid [8-(4-ethoxy-phenyl)-[1,2,4]triazolo[1,5-a]pyridin2-yl]-amide 50 Step i: Preparation of cyclopropanecarboxylic acid (8-bromo-[1,2,4]triazolo[1,5-a]pyridin-2yl)-amide Starting

from

8-bromo-[1,2,4]triazolo[1,5-a]pyridin-2-ylamine

(1g,

4.69

mmol)

and

cyclopropanecarbonyl chloride (1.07 mL, 11.73 mmol), compound was prepared in 57 % yield using the general procedure for mono-acylation (0.76 g). LCMS m/z 283.0 (M+H+). It was used in the next step without further purification. Step ii: Starting from cyclopropanecarboxylic acid (8-bromo-[1,2,4]triazolo[1,5-a]pyridin-2-yl)amide (30.0 mg, 0.10 mmol) and 4-ethoxyphenylboronic acid (26.0 mg, 0.15 mmol), compound 49 was prepared using the general procedure for the Suzuki coupling reaction. Purification by preparative HPLC afforded the desired compound in 46 % yield (15.0 mg, 99 % purity by UPLC). 1H (400 MHz, CDCl3) δ 9.26 (1H, br s, NH), 8.56 (1H, s, ArH), 7.88 (2H, d, J 8.6 Hz, ArH), 7.61 (1H, d, J 7.3 Hz, ArH), 7.10-7.02 (3H, m, ArH), 4.11 (2H, q, J 6.8 Hz, OCH2), 1.90 (1H, br, CH), 1.47 (3H, t, J 6.8 Hz, CH3), 1.14 (2H, br, CH2), 0.85 (2H, br, CH2); LCMS m/z 323.1 (M+H+). Preparation

of


acid

[5-(4-ethoxy-phenyl)-[1,2,4]triazolo[1,5-

c]pyrimidin-2-yl]-amide 51 Step i: Preparation of 2-(4-ethoxy-phenyl)-pyrimidin-4-ylamine



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Page 50 of 70

Starting from 2-chloro-pyrimidin-4-ylamine (50.0 mg, 0.38 mmlol) and 4-ethoxyphenylboronic acid (64.0 mg, 0.38 mmol), title compound was prepared using the general procedure for the Suzuki coupling reaction. Purification by silica chromatography (EtOAc/petrol ether; 20:80 to 40:60) afforded the compound in 50 % yield (52 mg, 90 % purity). LCMS m/z 216.1 (M+H+). Step ii: Preparation of [2-(4-ethoxy-phenyl)-pyrimidin-4-yl]-3-carboethoxy-thiourea To a solution of 2-(4-ethoxy-phenyl)-pyrimidin-4-ylamine (39 mg, 0.18 mmol) in 1,4-dioxane (0.9 mL) at 0 ºC was added ethoxycarbonyl isothiocyanate (30 µL, 0.27 mmol) dropwise and the reaction mixture was then allowed to warm to rt and stirred for 16 h. Evaporation in vacuum gives the crude compound that was used as such in the next step (50 % purity by UPLC). LCMS m/z 347.1 (M+H+). Step iii: Preparation of 5-(4-ethoxy-phenyl)-[1,2,4]triazolo[1,5-c]pyrimidin-2-ylamine To a suspension of hydroxylamine hydrochloride (60 mg, 0.87 mmol) in EtOH/MeOH (1:1 (v:v), 1 mL) was added N,N-diisopropylethylamine (85 µL, 0.62 mmol) and the mixture was stirred at rt for 1 h. [2-(4-Ethoxy-phenyl)-pyrimidin-4-yl]-3-carboethoxy-thiourea (crude, 0.18 mmol) was then added and the mixture heated to reflux for 1 h. The mixture was allowed to cool and filtered to collect the precipitated solid. Further product was collected by evaporation in vacuum of the filtrate, addition of H2O (2 mL) and filtration. The combined solids were washed successively with H2O (2 mL), EtOH/MeOH (1:1 (v:v), 2 mL) and Et2O (2 mL) then dried in vacuum to afford the triazolopyridine derivative as a white solid in 28 % yield. LCMS m/z 256.1 (M+H+). Step iv: Starting from 5-(4-ethoxy-phenyl)-[1,2,4]triazolo[1,5-c]pyrimidin-2-ylamine (13.0 mg, 0.051 mmol) and cyclopropanecarbonyl chloride (11.6 µL, 0.127 mmol), compound 51 was prepared using the general procedure for mono-acylation. Purification by preparative HPLC 50 ACS Paragon Plus Environment

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afforded the desired compound in 18 % yield (3 mg, 99 % purity by UPLC). 1H (400 MHz, CDCl3) δ 8.61 (2H, d, J 9.2 Hz, ArH), 8.48 (1H, br s, NH), 7.25 (1H, d, J 6.4 Hz, ArH), 7.36 (1H, d, J 6.4 Hz, ArH), 6.99 (2H, d, J 9.2 Hz, ArH), 4.07 (2H, q, J 6.8 Hz, OCH2), 1.40 (3H, t, J 6.8 Hz, CH3), 1.18-1.16 (3H, m, CH & CH2), 0.93-0.91 (2H, m, CH2); LCMS m/z 324.1 (M+H+). Preparation

of


acid

[8-(4-methoxy-phenyl)-6-trifluoromethyl-

[1,2,4]triazolo[1,5-a]pyridin-2-yl]-amide 52 Step

i:Preparation

of


acid

(8-chloro-6-trifluoromethyl-

[1,2,4]triazolo[1,5-a]pyridin-2-yl)-amide Starting from 8-chloro-6-trifluoromethyl-[1,2,4]triazolo[1,5-a]pyridin-2-ylamine (100 mg, 0.42 mmol) and cyclopropanecarbonyl chloride (97 µL, 1.06 mmol), the title compound was prepared in quantitative yield using the general procedure for mono-acylation (83 % purity). It was used in the next step without further purification. Step

ii:

Starting

from


acid

(8-chloro-6-trifluoromethyl-

[1,2,4]triazolo[1,5-a]pyridin-2-yl)-amide (140.0 mg, 0.46 mmol) and 4-methoxyphenylboronic acid (84.0 mg, 0.55 mmol), 52 was prepared using the general procedure for the Suzuki coupling reaction. Purification by preparative HPLC afforded the desired compound in 4 % yield (7 mg, 99 % purity by UPLC). 1H (400 MHz, d6-DMSO) δ 11.36 (1H, br s, NH), 9.46 (1H, s, ArH), 8.18 (2H, d, J 8.8 Hz, ArH), 8.03 (1H, s, ArH), 7.10 (2H, d, J 8.8 Hz, ArH), 3.85 (3H, s, OCH3), 2.02 (1H, br, CH), 0.85 (4H, app. d, CH2); LCMS m/z 376.9 (M+H+). Preparation of N-(7-phenyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-yl)cyclopropanecarboxamide 53



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Page 52 of 70

Step i :acetophenone (200 mg, 1.66 mmol) and N,N-dimethylformamide diethyl acetal (198 mg, 1.66 mmol) was heated in a sealed tube in microwave (300 W) 6 min. After cooling to rt, the product was isolated by recrystallization in diethyl ether (185 mg, 64 % yield). The compound was used in the next step without further purification. Step ii: Dimethyl-((E)-3-phenyl-propenyl)-amine (100 mg, 0.57 mmol) and 1H-[1,2,4]Triazole3,5-diamine (57 mg, 0.57 mmol) were dissolved in ethanol (2 mL) and the reaction was subjected to reflux until completion. The solvent was removed under vacuum to yield the expected product (120 mg, 100 % yield). It was used in the next step without further purification. Step iii: Starting from 7-Phenyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-ylamine (50 mg, 0.24 mmol) and cyclopropanecarbonyl chloride (65 µL, 0.72 mmol), the title compound was prepared in 33% yield using the general procedure for mono-acylation (22 mg, 99 % purity). 1H (400 MHz, d6DMSO) δ 11.20 (1H, b, NH), 8.77 (1H, d, J 4.8 Hz, ArH), 8.19 (2H, m, ArH), 7.60 (3H, m, ArH), 7.49 (1H, d, J 4.8 Hz, Ar), 2.01 (1H, b, CH), 0.81 (4H, m, 2 xCH2) Preparation

of

Cyclopropanecarboxylic

acid

[5-(4-ethoxy-phenyl)-[1,2,4]triazolo[1,5-

a]pyrazin-2-yl]-amide 54 Step i: Preparation of (6-chloro-pyrazin-2-yl)-3-carboethoxy-thiourea To a solution of 6-chloro-pyrazin-2-ylamine (1.0 g, 8.23 mmol) in 1,4-dioxane (10 mL) at 0 ºC was added ethoxycarbonyl isothiocyanate (0.74 mL, 6.56 mmol) dropwise and the reaction mixture was allowed to warm to rt and then heated at reflux for 2 h. Evaporation in vacuum gives a solid which was thoroughly washed with petrol and dried to afford the desired compound in 51% yield. Used in the next step without further purification.


Page 53 of 70

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Step ii:Preparation of 5-chloro-[1,2,4]triazolo[1,5-a]pyrazin-2-ylamine To a suspension of hydroxylamine hydrochloride (1.5 g, 21.6 mmol) in EtOH/MeOH (1:1 (v:v), 20 mL) is added N,N-diisopropylethylamine (1.8 mL, 10.9 mmol) and the mixture was stirred at rt for 1 h. (6-Chloro-pyrazin-2-yl)-3-carboethoxy-thiourea (0.88 g, 3.38 mmol) was then added and the mixture was heated to reflux for 16 h. The mixture was allowed to cool and filtered to collect the precipitated solid. Further product was collected by evaporation in vacuum of the filtrate, addition of H2O (2 mL) and filtration. The combined solids were washed successively with H2O (2 mL), EtOH/MeOH (1:1, 2 mL) and Et2O (2 mL) then dried in vacuum to afford the title compound as a white solid in 64 % yield (99 % pure by UPLC). It was used in the next step without further purification Step iii: Preparation of cyclopropanecarboxylic acid (5-chloro-[1,2,4]triazolo[1,5-a]pyrazin-2yl)-amide Starting from 5-chloro-[1,2,4]triazolo[1,5-a]pyrazin-2-ylamine (0.37 g, 2.19 mmol) and cyclopropanecarbonyl chloride (395 µL, 4.38 mmol), the title compound was prepared using the general procedure for mono-acylation. Purification by silica chromatography (EtOAc/petrol ether; 1:4) afforded the compound in 15 % yield (80 mg). It was used in the next step without further purification. Step iv: Starting from cyclopropanecarboxylic acid (5-chloro-[1,2,4]triazolo[1,5-a]pyrazin-2yl)-amide (40 mg, 0.169 mmol) and 4-ethoxyphenylboronic acid (56 mg, 0.338 mmol), compound 53 was prepared using the general procedure for the Suzuki coupling reaction. Purification by preparative HPLC afforded the desired compound in 14 % yield (7 mg, 99 % purity by UPLC). LCMS m/z 324.1 (M+H+).1H (400 MHz, d6-DMSO) δ 11.35 (1H, b, NH), 9.13 53 ACS Paragon Plus Environment


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Page 54 of 70

(1H, s, ArH), 8.41 (1H, s, ArH), 8.11 (2H, d, J 8.9 Hz, ArH), 7.14 (2H, d, J 8.8, ArH), 4.15 (2H, q, J 7.0 Hz, CH2), 2.05 (1H, b, CH), 1.38 (3H, t, J 7.0 Hz, CH3), 0.85 (4H, m, 2xCH2) Preparation

of


acid

[5-(4-cyclopropanesulfonylamino-phenyl)-

[1,2,4]triazolo[1,5-a]pyridin-2-yl]-amide 55 Step i: Preparation of {4-[2-(cyclopropanecarbonyl-amino)-[1,2,4]triazolo[1,5-a]pyridin-5-yl]phenyl}-carbamic acid tert-butyl ester Starting from compound 9 and 4-(N-Boc-amino)phenylboronic acid pinacol ester, the title compound was prepared using the general procedure for the Suzuki coupling reaction. Purification by silica chromatography (EtOAc/petrol ether; 70:30) afforded the compound in 53 % yield. LCMS m/z 338.0 (M+H+). Step ii:Preparation of cyclopropanecarboxylic acid [5-(4-amino-phenyl)-[1,2,4]triazolo[1,5a]pyridin-2-yl]-amide To a solution of the {4-[2-(cyclopropanecarbonyl-amino)-[1,2,4]triazolo[1,5-a]pyridin-5-yl]phenyl}-carbamic acid tert-butyl ester (200 mg, 0.51 mmol) in MeOH (2 mL) was added conc. HCl (0.5 mL) and the mixture was stirred at 50 ºC for 4 h. After concentration in vacuum, the title compound was obtained in quantitative yield. LCMS m/z 293.9 (M+H). Step iii: To cyclopropanecarboxylic acid [5-(4-amino-phenyl)-[1,2,4]triazolo[1,5-a]pyridin-2yl]-amide (25 mg, 0.085 mmol) in DCM (1 mL) at 0 ºC was added triethylamine (42 µL, 0.306 mmol) followed by cyclopropylsulfonyl chloride (32 µL, 0.153 mmol). The resulting mixture was stirred at rt for 72 h and then concentrated in vacuum. Purification by preparative HPLC afforded 52 in 12 % yield (99 % purity by UPLC). 1H (400 MHz, d6-DMSO) δ 11.08 (1H, br s,


Page 55 of 70

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NH), 10.13 (1H, br s, NH), 8.03 (2H, d, J 8.8 Hz, ArH), 7.71-7.67 (3H, m, ArH), 7.39 (2H, d, J 8.8 Hz, ArH), 7.29 (1H, dd, J 7.2, 1.6 Hz, ArH), 2.88 (1H, br, CH), 2.01 (1H, br, CH), 1.02-1.00 (4H, m, 2xCH2), 0.83-0.82 (4H, m, CH2); LCMS m/z 398.0 (M+H+). Preparation

of

4-(2-(cyclopropanecarboxamido)-[1,2,4]triazolo[1,5-a]pyridin-5-yl)-N-(2-(1-

phenyl-1H-pyrazol-4-yl)ethyl)benzamide 56 Step i: preparation of 4-[2-(Cyclopropanecarbonyl-amino)-[1,2,4]triazolo[1,5-a]pyridin-5-yl]benzoic acid Starting from compound 9 (400 mg, 1.49 mmol) and, 4-(ethoxycarobonyl)phenylboronic acid (283 mg, 1.70 mmol) the title compound was prepared using the general procedure for the Suzuki coupling reaction. Purification by silica chromatography (EtOAc/petrol ether; 50:50) afforded the compound in 93 % yield (764 mg, 95 % purity by UPLC). The obtained compound was dissolved in THF/H20/MeOH (2 mL/1 mL/ 1 mL), NaOH solution (2N), 3.5 mL was added to the solution. After 1h30 of stirring at room temperature, the reaction was completed. Solvents were removed. 12 mL of HCl solution (1N) was added to precipitate the expected product. Filtration gave the pure title compound (100 % yield, 90 % purity by UPLC) Step ii: preparation of 4-[2-(Cyclopropanecarbonyl-amino)-[1,2,4]triazolo[1,5-a]pyridin-5-yl]benzoyl chloride 4-[2-(Cyclopropanecarbonyl-amino)-[1,2,4]triazolo[1,5-a]pyridin-5-yl]-benzoic acid (150 mg, 0.46 mmol) was dissolved in DCM (1.5 mL) at room temperature and a drop of DMF was added under nitrogen atmosphere. Oxalyl chloride (50 µL, 0.56 mmol) was added dropwise to the solution. The reaction mixture was stirred for 2h at room temperature. The solvent was removed under vacuum and the residue was used directly in the next step without further isolation. 55 ACS Paragon Plus Environment


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Step iii: 4-[2-(Cyclopropanecarbonyl-amino)-[1,2,4]triazolo[1,5-a]pyridin-5-yl]-benzoyl chloride (30 mg, 0.09 mmol) was dissolved in DCM (1 mL). Et3N (20 µL, 0.14 mmol) was added followed by 2-(1-Phenyl-1H-pyrazol-4-yl)-ethylamine (26 mg, 0.14 mmol). The reaction was stirred at room temperature for 2 hrs. Water was added, the organic layer was separated, dried over MgSO4. The crude was purified by preparative HPLC (15 mg, 33 %). LCMS m/z 514.1 (M+23); 1

H (400 MHz, d6-DMSO) δ 11.05 (1H, b, NH), 8.79 (1H, bt, J 5.5 Hz, NH), 8.39 (1H, s, ArH),

8.12 (2H, d, J 8.5 Hz, ArH), 8.01 (2H, d, J 8.5 Hz, ArH), 7.80 (2H, m, ArH) 7.74 (1H, d, J 2.0 Hz, ArH), 7.73 (1H, s, ArH), 7.65 (1H, s, ArH), 7.48 (2H, m, ArH), 7.36 (1H, dd, J 3.3, 5.3 Hz, ArH), 7.27 (1H, m, ArH), 3.54 (2H, q apparent, CH2), 2.81 (2H, t, J 7.3 Hz, CH2), 1.99 (1H, b, CH), 0.82 (4H, d, J 6.04 Hz, 2x CH2) Preparation of 57: Cyclopropanecarboxylic acid {5-[4-(4-hydroxy-3-hydroxymethyl-benzyl)-p henyl]-[1,2,4]triazolo[1,5-a]pyridin-2-yl}-amide Step i: 2-Hydroxymethyl-4-[4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-benzyl]-phenol To 2-Hydroxymethyl-phenol (167 mg, 1.35 mmol) in dry DCM/MeOH (5/1 mL) at RT was added NaH (60 % dispersion, 2.02 mmol, 29 mg) followed by 2-(4-Bromomethyl-phenyl)4,4,5,5-tetramethyl-[1,3,2]dioxaborolane (200 mg, 0.67 mmol) and the mixture was stirred at rt for 16 h. The solution was quenched with water, extracted with ethyl acetate, dried (MgSO4), filtered and concentrated under vacuum. The crude material was used in the next step without further purification Step 2:


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Starting from compound made in step 1 (0.673 mmol) and compound 9 (172 mg, 0.61 mmol) the title compound was prepared using the general procedure for the Suzuki coupling reaction. Purification by preparative HPLC afforded the compound in 57% yield (144mg, 100% purity by UPLC). . 1H (400 MHz, d6-DMSO) δ 7.9 (2H, d, J 8.6 Hz, ArH), 7.67 (1H, dd, J 8.9, 7.3, ArH), 7.57 (1H, m, ArH), 7.33 (2H, d, J 8.5 Hz, ArH), 7.20 (1H, m, ArH), 7.13 (1H, m, ArH), 6.95 (1H, dd, J 8.3, 2.3, AeH), 6.69 (1H, d, J 8.2, ArH), 4.6 (2H, s, CH2), 4.5 (1H, b, OH), 3.94 (2H, s, CH2), 1.9 (1H, b, CH), 0.99 (2H, m, CH2), 0.88 (2H, m, CH2). Preparation

of

N-(5-(4-((2-(cyanomethyl)phenylamino)methyl)phenyl)-[1,2,4]triazolo[1,5-

a]pyridin-2-yl)cyclopropanecarboxamide 58 Step i: To a mixture of (2-amino-phenyl)-acetonitrile (178 mg, 1.347 mmol) and N,Ndiisopropylethylamine (0.235 mL, 1.347 mmol) in DCM (5 mL) and MeOH (1 mL) was added 2-(4-bromomethyl-phenyl)-4,4,5,5-tetramethyl-[1,3,2]dioxaborolane (200 mg, 0.673 mmol) slowly and the reaction was stirred at rt for 18 h. The resulting mixture was concentrated in vacuum, rediluted with EtOAc, washed with water, dried (MgSO4), filtered and concentrated in vacuum to give quantitatively the desired compound that was used as such in the next step. Step ii: Starting from compound 9 (172 mg, 0.612 mmol) and compound from step i (234 mg, 0.673 mmol), compound 58 was prepared using the general procedure for the Suzuki coupling reaction. Purification by silica chromatography followed by trituration with Et2O afforded the desired compound in 29 % yield (75 mg, 99 % purity by UPLC). LCMS m/z 423.1 (M+H+).1H (400 MHz, d6-DMSO) δ 10.98 (1H, b, NH) 7.96 (2H, d, J 8.0 Hz, ArH) 7.68 (2H, m, ArH) 7.56 (2H, d, J 8.15 Hz, ArH) 7.27 (1H, m, ArH) 7.19 (1H, m, ArH) 7.07 (1H, m, ArH) 6.60 (1H, m,



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ArH) 6.52 (1H, d, J 8.3 Hz, ArH) 6.11 (1H, m, NH) 4.48 (2H, d, J 5.8 Hz CH2) 3.93 (2H, s, CH2) 2.02 (1H, b, CH) 0.80 (4H, m, 2xCH2) Preparation

of

N-(5-(4-((3-((4-cyanophenoxy)methyl)azetidin-1-yl)methyl)phenyl)-

[1,2,4]triazolo[1,5-a]pyridin-2-yl)cyclopropanecarboxamide 59 Step i: Preparation of 4-{1-[4-(4,4,5,5-Tetramethyl-[1,3,2 ]dioxaborolan-2-yl)-benzyl]-azetidin-3ylmethoxy}-benzonitrile

4-(Azetidin-3-ylmethoxy)-benzonitrile(50 mg, 0.27 mmol)) was dissolved in DCM (6 mL);. diisopropylethylamine (235 µL, 1.34 mmol) was added to the solution followed by 2-(4Bromomethyl-phenyl)-4,4,5,5-tetramethyl-[1,3,2]dioxaborolane (39 mgn 0.13 mmol). The reaction mixture was stirred for 16 h at room temperature. Solvent was removed under vacuum. after addition of water, the organics were extracted with EtOAc. Solvent was evaporated under vacuum and the crude was used without further purification. LCMS m/z 405.0 (M+H+). Step 2: Preparation of N-(5-(4-((3-((4-cyanophenoxy)methyl)azetidin-1-yl)methyl)phenyl)[1,2,4]triazolo[1,5-a]pyridin-2-yl)cyclopropanecarboxamide Starting

from

4-{1-[4-(4,4,5,5-Tetramethyl-[1,3,2

]dioxaborolan-2-yl)-benzyl]-azetidin-3-

ylmethoxy}-benzonitrile

Starting from compound 9 (33.9mg, 0.12 mmol) and 4-(4-morpholinomethyl)-phenylboronic acid pinacol ester hydrochloride (53.7 mg, 0.13 mmol), compound 59 was prepared using the general procedure for the Suzuki coupling reaction. Purification by preparative HPLC afforded the desired compound in 10 % yield (6 mg, 99 % purity by UPLC). LCMS m/z 479 (M+H+). 1H (400 MHz, d6-DMSO) δ 11.04 (1H, bs, NH), 8.22 (1Hn s, ArH), 7.94 (2H, d, J 8.6 Hz, ArH), 7.76 (2H, m, ArH) 7.69 (2H, m, ArH), 7.44 (2H, d, J, 8.3, ArH), 7.27 (1H, dd, J 1.8, 6.5 Hz, 58 ACS Paragon Plus Environment

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ArH), 7.13 (2H, m, ArH), 4.24 (2H, d, J 7.1 Hz, CH2), 3.67 (2H, s, CH2), ~3.4 (2H, under DMSO peak) 3.07 (2H, m, CH2), 2.8 (1H, m, CH), 2.0 (1H, m, CH), 0.8 (4H, m, 2xCH2). Preparation

of

N-(5-(4-(morpholinomethyl)phenyl)-[1,2,4]triazolo[1,5-a]pyridin-2-

yl)cyclopropanecarboxamide 60 Starting from compound 9 (30 mg, 0.10 mmol) and 4-(4-morpholinomethyl)-phenylboronic acid pinacol ester hydrochloride (51 mg, 0.15 mmol), compound 60 was prepared using the general procedure for the Suzuki coupling reaction. Purification by preparative HPLC afforded the desired compound in 100 % yield (38 mg, 99 % purity by UPLC). 1H (400 MHz, CDCl3) δ 10.22 (1H, br s, NH), 8.06 (2H, d, J 8.4 Hz ArH), 7.78 (2H, m, ArH), 7.62 (2H, d, J 8.3 Hz, ArH) 7.22 (1H, d, J 6.8 Hz, ArH), 4.28 (2H, s, CH2), 3.99 (4H, s, 2xCH2), 3.50 (2H, br s, CH2), 2.97 (2H, br s, CH2), 1.97 (1H, br, CH), 1.18-1.16 (2H, m, CH2), 0.96-0.94 (2H, m, CH2); LCMS m/z 378.2 (M+H+). Preparation of N-(5-(4-((4-methylpiperazin-1-yl)methyl)phenyl)-[1,2,4]triazolo[1,5-a]pyridin-2yl)cyclopropanecarboxamide 61 Step i: preparation of Cyclopropanecarboxylic acid [5-(4-formyl-phenyl)-[1,2,4]triazolo[1,5a]pyridin-2-yl]-amide Starting from compound 9 (1 g, 3.56 mmol) and 4-formylphenylboronic acid (0.639 g, 4.63 mmol), the title compound was obtained in 74 % yield using the general procedure for the Suzuki coupling reaction used in the next step without further purification (85 % pure by UPLC). Step ii: Cyclopropanecarboxylic acid [5-(4-formyl-phenyl)-[1,2,4]triazolo[1,5-a]pyridin-2-yl]amide (30 mg, 0.098 mmol) was stirred with N-methylpiperazine (9.82 mg, 0.098 mmol) in



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Page 60 of 70

titanium (IV) isopoxide (40 µL). After 1 hour of stirring at room temperature, ethanol (0.1 mL) was added followed by Na(CN)BH3 (6 mg, 0.098 mmol). After completion of the reaction, 2 mL of water was added and the suspension was filtered. The white solid was washed with ethanol and ethyl acetate. The solvent was removed under vacuum to afford the expected product (15 mg, 40 % yield) (99% purity by LCMS). LCMS m/z 391.1 (M+H+). 1H (400 MHz, d6-DMSO) δ 11.07 (1H, broad s, NH), 8.02 (2H, d, J 8.3 Hz, ArH), 7.70 (2H, m, ArH), 7.51 (2H, d, J 8.2 Hz, ArH), 7.28 (1H, dd, J 2.0, 6.1 Hz, ArH), 3.73 (4H, broad, 2xCH2), 3.03 (4H, broad, 2xCH2), 2.79 (3H, s, CH3), 1.99 (1H, b, CH), 0.81 (4H, m, 2xCH2) Preparation of N-(5-(4-((2,2,2-trifluoroacetamido)methyl)phenyl)-[1,2,4]triazolo[1,5-a]pyridin2-yl)cyclopropanecarboxamide 62 Step i: Cyclopropanecarboxylic acid [5-(4-aminomethyl-phenyl)-[1,2,4]triazolo[1,5-a]pyridin-2yl]-amide Starting from compound 9 (0.10 mmol, 30 mg)) and 4-(aminomethyl)phenylboronic acid hydrochloride (28 mg, 0.15 mmol), the title compound was obtained in 6 % yield using the general procedure for the Suzuki coupling reaction used in the next step without further purification (2 mg) Step

ii:

N-(5-(4-((2,2,2-trifluoroacetamido)methyl)phenyl)-[1,2,4]triazolo[1,5-a]pyridin-2-

yl)cyclopropanecarboxamide The benzylamine derivative (30 mg, 0.097 mmol) obtained above and Et3N (0.489 mmol, 68 µL) are dissolved in DCM under N2 and cooled at 0 °C. Trifluoroacteic anhydride (0.146 mmol, 30 mg) dissolved in DCM is added dropwise to this solution. The reaction is stirred at room temperature for 16 h. After this time, the reaction is complete. The compound is extracted with 60 ACS Paragon Plus Environment

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EtOAc and water, washed with brine and dried over MgSO4. Organic layers are filtered and evaporated. The final compound is isolated by preparative HPLC, 33 % yield (99 % purity by UPLC); LCMS m/z 404.1 (M+H+). 1H (400 MHz, d6-DMSO) δ 11.00 (1H, br, NH), 10.13 (1H, brt, NH), 7.98 (2H, d, J 8.4 Hz, ArH), 7.70 (2H, m, ArH), 7.45 (2H, d, J 8.0 Hz, ArH), 7.28 (1H, m, ArH), 4.49 (2H, d, J 6.4 Hz, CH2), 2.0 (1H, b, CH), 0.80 (4H, m, 2x CH2)

Preparation of cyclopropanecarboxylic acid {5-[4-(4,4-difluoro-piperidin-1-ylmethyl)-phenyl][1,2,4]triazolo[1,5-a]pyridin-2-yl}-amide 63 Step i: Preparation of (2-fluoro-phenyl)-[4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)benzyl]-amine To a mixture of 2-fluoro-phenylamine (129 µL, 1.347 mmol) and N,N-diisopropylethylamine (235 µL, 1.347 mmol) in DCM (5 mL) and MeOH (1 mL) was added 2-(4-bromomethylphenyl)-4,4,5,5-tetramethyl-[1,3,2]dioxaborolane (200 mg, 0.673 mmol) slowly and the reaction was stirred at rt for 18 h. The resulting mixture was concentrated under vacuum, rediluted with EtOAc, washed with water, dried (MgSO4), filtered and concentrated under vacuum to give the desired compound in quantitative yield. Step ii: Preparation of 4,4-difluoro-1-[4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-benzyl]piperidine To a mixture of 4,4-difluoro-piperidine (212 mg, 1.347 mmol) and N,N-diisopropylethylamine (235 µL, 1.347 mmol) in DCM (5 mL) and MeOH (1 mL) was added 2-(4-bromomethylphenyl)-4,4,5,5-tetramethyl-[1,3,2]dioxaborolane (200 mg, 0.673 mmol) slowly and the reaction



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Page 62 of 70

was stirred at rt for 18 h. The resulting mixture was concentrated under vacuum, rediluted with EtOAc, washed with water, dried (MgSO4), filtered and concentrated under vacuum to give the desired compound that was used as such in the next step. Step

iii:

Starting

from

compound

9

and

4,4-difluoro-1-[4-(4,4,5,5-tetramethyl-

[1,3,2]dioxaborolan-2-yl)-benzyl]-piperidine, compound 63 was prepared using the general procedure for the Suzuki coupling reaction. Purification by preparative HPLC afforded the compound in 35 % yield (99 % purity by UPLC). 1H (400 MHz, d6-DMSO) δ 11.00 (1H, br s, NH), 7.98 (2H, d, J 8.4 Hz, ArH), 7.73-7.66 (2H, m, ArH), 7.50 (2H, d, J 8.4 Hz, ArH), 7.29 (1H, dd, J 6.8, 1.6 Hz, ArH), 3.65 (2H, s, CH2), 2.56-2.52 (4H, m, 2xCH2), 2.04-1.94 (5H, m, 2xCH2 and CH), 0.82-0.81 (4H, m, 2xCH2); LCMS m/z 412.1 (M+H+). Preparation of cyclopropanecarboxylic acid (5-{4-[(1,1-dioxo-tetrahydrothiophen-3-ylamino)methyl]-phenyl}-[1,2,4]triazolo[1,5-a]pyridine-2-yl)-amide 64 Step i: 2-(4-Bromomethyl-phenyl)-4,4,5,5-tetramethyl-[1,3,2]dioxaborolane (0.674 mmol, 200 mg) and DIPEA (1.347 mmol, 234 µL)) were dissolved in DCM/MeOH (4:1 v:v) under N2 and 1,1-dioxotetrahydrothiophen-3-ylamine (1.347 mmol, 200 mg)) was added dropwise. The resulting solution was stirred at room temperature for 16h. After this time, the reaction was complete. The solvent was evaporated. The compound was extracted with EtOAc and water, washed with brine and dried over MgSO4. Organic layers were filtered and evaporated. The final compound was isolated by flash chromatography. (231 mg, 97 % yield). Used in the next step without further purification.


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Step ii: Starting from compound 9 (172 mg, 0.612 mmol) and compound described above (225 mg, 0.673 mmol), the title compound was obtained in 51 % yield using the general procedure for the Suzuki coupling reaction (99% purity by LCMS). LCMS m/z 416 (M+H+); 1H (400 MHz, d6DMSO) δ 10.99 (1H, b, NH), 7.97 (2H, d, J 8.6 Hz, ArH), 7.69 (2H, m, 2 x ArH), 7.53 (2H, d, J 8.6 Hz, ArH), 7.28 (1H, dd, J 1.8, 6.8 Hz ArH), 3.90 (1H, b, NH), 3.83 (2H, m, CH2), 3.48 (1H, m, CH), 3.34 (1H, m, CH), 3.26 (1H, m, CH), 3.05 (1H, m, CH), 2.94 (1H, m, CH), 2.28 (1H, m, CH), 2.03 (2H, m, 2x CH), 0.81 (4H, m, 2 x CH2) Preparation of cyclopropanecarboxylic acid {5-[4-(1,1-dioxo-1lambda*6*-thiomorpholin-4ylmethyl)-phenyl]-[1,2,4]triazolo[1,5-a]pyridin-2-yl}-amide 65 Step1:

Preparation

of

4-[4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-benzyl]-

thiomorpholine 1,1-dioxide To

a

mixture

of

thiomorpholine

1,1-dioxide

(455

mg,

3.367

mmol)

and

N,N-

diisopropylethylamine (0.58 mL, 3.367 mmol) in DCM (15 mL) and MeOH (3 mL) was added 2-(4-bromomethyl-phenyl)-4,4,5,5-tetramethyl-[1,3,2]dioxaborolane (500 mg, 1.683 mmol) slowly and the reaction was stirred at rt for 18 h. The resulting mixture was concentrated in vacuo, rediluted with EtOAc, washed with water, dried (MgSO4), filtered and concentrated in vacuo to give the desired compound that was used as such in the next step. Step ii: Starting from compound 9 and 4-[4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)benzyl]-thiomorpholine 1,1-dioxide, compound 65 was prepared using the general procedure for the Suzuki coupling reaction. Purification by silica chromatography followed by trituration with Et2O afforded the desired compound in 59 % yield (99 % purity by UPLC). 1H (400 MHz, d6DMSO) δ 11.15 (1H, br s, NH), 8.11 (2H, m, ArH), 7.78 (2H, m, ArH), 7.76 (1H, dd, J 8.8, 6.5 63 ACS Paragon Plus Environment


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Hz, ArH), 7.73 (1H, dd, J 8.8, 2.1 Hz, ArH), 7.36 (1H, dd, J 6.5, 2.1 Hz, ArH), 4.44 (2H, s, CH2), 3.53 (8H, m, 4xCH2), 2.03 2.03 (1H, br, CH), 0.82-0.81 (4H, m, 2xCH2); LCMS m/z 426.0 (M+H+).

13

C (400 MHz, d6-DMSO) δ 171.5, 157.5, 149.9, 138.8, 132.7, 132.1, 131.1,

130.6, 129.3, 114.1, 113.7, 57.7, 49.7, 47.8, 13.8, 7.7. LCMS m/z 426 (M+H+). CHN analysis C%: 48.64, H%: 5.81, N%: 13.41, Cl%: 6.88, S%: 6.37 (theoretical: C%:48.88, H%: 8.86, N%: 13.57, Cl%: 6.87, S%: 6.21) References: (1)

O’Shea, J. J.; Plenge, R. JAK and STAT Signaling Molecules in Immunoregulation and Immune-Mediated Disease. Immunity 2012, 36, 542–550.

(2)

West, K. CP-690550, a JAK3 Inhibitor as an Immunosuppressant for the Treatment of Rheumatoid Arthritis, Transplant Rejection, Psoriasis and Other Immune-Mediated Disorders. Curr. Opin. Investig. Drugs Lond. Engl. 2000 2009, 10, 491–504.

(3)

Clark, J. D.; Flanagan, M. E.; Telliez, J.-B. Discovery and Development of Janus Kinase (JAK) Inhibitors for Inflammatory Diseases. J. Med. Chem. 2014.

(4)

Rodig, S. J.; Meraz, M. A.; White, J. M.; Lampe, P. A.; Riley, J. K.; Arthur, C. D.; King, K. L.; Sheehan, K. C.; Yin, L.; Pennica, D.; Johnson, E. M., Jr; Schreiber, R. D. Disruption of the Jak1 Gene Demonstrates Obligatory and Nonredundant Roles of the Jaks in Cytokine-Induced Biologic Responses. Cell 1998, 93, 373–383.

(5)

Shouda, T.; Yoshida, T.; Hanada, T.; Wakioka, T.; Oishi, M.; Miyoshi, K.; Komiya, S.; Kosai, K.; Hanakawa, Y.; Hashimoto, K.; Nagata, K.; Yoshimura, A. Induction of the Cytokine Signal Regulator SOCS3/CIS3 as a Therapeutic Strategy for Treating Inflammatory Arthritis. J. Clin. Invest. 2001, 108, 1781–1788.


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(6)

Haan, C.; Rolvering, C.; Raulf, F.; Kapp, M.; Drückes, P.; Thoma, G.; Behrmann, I.; Zerwes, H.-G. Jak1 Has a Dominant Role over Jak3 in Signal Transduction through ΓcContaining Cytokine Receptors. Chem. Biol. 2011, 18, 314–323.

(7)

Ghoreschi, K.; Jesson, M. I.; Li, X.; Lee, J. L.; Ghosh, S.; Alsup, J. W.; Warner, J. D.; Tanaka, M.; Steward-Tharp, S. M.; Gadina, M.; Thomas, C. J.; Minnerly, J. C.; Storer, C. E.; LaBranche, T. P.; Radi, Z. A.; Dowty, M. E.; Head, R. D.; Meyer, D. M.; Kishore, N.; O’Shea, J. J. Modulation of Innate and Adaptive Immune Responses by Tofacitinib (CP690,550). J. Immunol. Baltim. Md 1950 2011, 186, 4234–4243.

(8)

Van Rompaey, L.; Galien, R.; van der Aar, E. M.; Clement-Lacroix, P.; Nelles, L.; Smets, B.; Lepescheux, L.; Christophe, T.; Conrath, K.; Vandeghinste, N.; Vayssiere, B.; De Vos, S.; Fletcher, S.; Brys, R.; van ’t Klooster, G.; Feyen, J. H. M.; Menet, C. Preclinical Characterization of GLPG0634, a Selective Inhibitor of JAK1, for the Treatment of Inflammatory Diseases. J. Immunol. Baltim. Md 1950 2013, 191, 3568–3577.

(9)

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Triazolopyridines as Selective JAK1 Inhibitors: From Hit Identification

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