Identification of Cyanamide-Based Janus Kinase 3 (JAK3) Covalent

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Identification of Cyanamide-Based Janus Kinase 3 (JAK3) Covalent Inhibitors Agustin Casimiro-Garcia, John I Trujillo, Felix F. Vajdos, Brian M Juba, Mary Ellen Banker, Ann Aulabaugh, Paul Balbo, Jonathan Bauman, Jill Chrencik, Jotham W Coe, Robert M. Czerwinski, Martin E. Dowty, John D. Knafels, Soojin Kwon, Louis Leung, Sidney Liang, Ralph P. Robinson, Jean-Baptiste Telliez, Ray Unwalla, Xin Yang, and Atli Thorarensen J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01308 • Publication Date (Web): 13 Nov 2018 Downloaded from http://pubs.acs.org on November 13, 2018

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Journal of Medicinal Chemistry

Identification of Cyanamide-Based Janus Kinase 3 (JAK3) Covalent Inhibitors Agustin Casimiro-Garcia,a,* John I. Trujillo,b Felix Vajdos,b Brian Juba,c Mary Ellen Banker,b Ann Aulabaugh,b Paul Balbo,c Jonathan Bauman,b Jill Chrencik,b Jotham W. Coe,b Robert Czerwinski,c Martin Dowty,d John D. Knafels,b Soojin Kwon,b Louis Leung,b Sidney Liang,b Ralph P. Robinson,b Jean-Baptiste Telliez,c Ray Unwalla,a Xin Yang,b Atli Thorarensena aMedicine

Design, Pfizer Inc., 1 Portland Street, Cambridge, MA 02139, United States;

bMedicine

Design, Pfizer Inc., 445 Eastern Point Rd, Groton, CT 06340, United States;

cInflammation

and Immunology Research Unit, Pfizer Inc., 1 Portland Street, Cambridge, MA

02139, United States; dMedicine Design, Pfizer Inc., 1 Burtt Road, Andover, MA 01810, United States Abstract: Ongoing interest in the discovery of selective JAK3 inhibitors led us to design novel covalent inhibitors that engage the JAK3 residue Cys909 by cyanamide, a structurally and mechanistically differentiated electrophile from other cysteine reacting groups previously incorporated in JAK3 covalent inhibitors. Through crystallography, kinetic, and computational studies, interaction of cyanamide 12 with Cys909 was optimized leading to potent and selective JAK3 inhibitors as exemplified by 32. In relevant cell-based assays, and in agreement with previous results from this group, 32 demonstrated that selective inhibition of JAK3 is sufficient to drive JAK1/JAK3-mediated cellular responses. The contribution from extrahepatic processes to the clearance of cyanamide-based covalent inhibitors was also characterized using metabolic and pharmacokinetic data for 12. This work also gave key insights into a productive approach to decrease glutathione/glutathione-S-transferase-mediated clearance, a challenge typically encountered during the discovery of covalent kinase inhibitors.

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Introduction The Janus kinases (JAKs) are a family of intracellular tyrosine kinases that have a central role in the signaling process of many cytokine receptors. Along with their downstream effectors, signal transducer and activator of transcription proteins (STATs), JAKs form a signaling pathway of fundamental importance in innate immunity, inflammation, and hematopoiesis.1-5 Dysregulation of this pathway has been implicated in the pathogenesis of immune diseases and cancers and thus JAKs are attractive targets for the treatment of a number of therapeutic indications.6-8 The JAK kinases include four family members JAK1, JAK2, JAK3, and tyrosine kinase 2 (TYK2), each selectively associated with the intracellular domain of different receptors subunits. The pairing of the JAK kinases to mediate signaling from a given cytokine receptor is an important element of their function. For example, JAK3 is always paired to JAK1 and this arrangement controls the signaling for the -common chain (c) cytokines interleukin 2 (IL-2), IL-4, IL-7, IL-9, IL-15 and IL-21.9 Another feature of the JAKs is their high degree of sequence homology, particularly in the catalytic domain, which has made challenging the identification of selective JAK inhibitors. JAK3 was the first of the JAKs to attract interest because of its essential role in immune signaling and predominant expression in the hematopoietic system.10-12 Validation of its critical role in the immune system has come from human JAK3 deficiencies. Loss-of-function mutations in c or JAK3 confer a severe combined immunodeficiency in humans (SCID).13 This strong rationale has led to the search of JAK3 inhibitors as therapeutic agents for the treatment of autoimmune and inflammatory diseases. The approval of tofacitinib (1) for the treatment of rheumatoid arthritis (RA) by the U.S. Food and Drug Administration in 2012 was an important milestone for JAK inhibition for the treatment of RA.14 This agent was originally reported as a selective JAK3 inhibitor,11 but in more recent screens was demonstrated to be a pan-JAK


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inhibitor, with predominant JAK1 inhibition.14, 15 There are now several JAK inhibitors in development for the treatment of inflammatory indications,8 but none of them is a JAK3 specific inhibitor,15 with the exception of PF-06651600 (6), as further detailed below. The identification of selective JAK3 inhibitors has continued to attract the attention of both industrial and academic groups. In addition to the potential of providing safer immunotherapeutic agents due to the restricted expression of JAK3, highly selective JAK3 inhibitors are important to dissect the complex signaling pathways involving JAK1/JAK3. These efforts have targeted the ATP binding site of the kinase for identification of selective agents. Two approaches have been pursued. The first approach has utilized reversible inhibitors that target specific regions in JAK3 with lipophilic or hydrogen bonding interactions to achieve enhanced selectivity towards JAK3.16-20 In this context, it is important to note that JAK3 has high affinity for ATP which makes it difficult to identify an inhibitor that is more than JAK3 biased. The second approach has targeted the JAK3 residue Cys909, a unique residue within the JAK family, with covalent inhibition. Several research groups have reported the identification of JAK3 covalent inhibitors which have commonly incorporated an N-aryl/heteroaryl acrylamide (Fig. 1, 2 - 4),21-23 or an -cyanoacrylamide (e.g. 5) moiety as the electrophilic, or cysteine reacting group (CRG).24, 25 Exquisite selectivity for JAK3 over other JAKs, as well as generally high kinome selectivity has been achieved with these inhibitors. Furthermore, this approach has delivered useful tools for understanding the complex biology driven by JAK3. For example, 4 was recently utilized to identify new aspects of IL-2 signaling mediated by JAK3 catalytic activity.23 Our group has focused extensive research efforts towards the discovery of JAK3 selective inhibitors based on a covalent approach to identify potential candidates for human clinical



studies. One of the important questions to address with such inhibitors is the relative JAK3 contribution to the overall efficacy observed with pan-JAK inhibitors. Our efforts recently culminated in the identification 6, a JAK3 specific inhibitor recently advanced into human clinical trials.26, 27 The potency and selectivity of 6 was confirmed using both enzyme and cellular assays. Formation of a covalent bond with Cys909 was observed in the co-crystal structure of 6 with JAK3. A high degree of kinome selectivity was observed with 6 in a panel of 305 kinases. When evaluated in a panel of 11 kinases that possess a Cys residue at the same position as that found in JAK3, 6 exhibited generally good selectivity for JAK3 with measurable activity demonstrated mainly against kinases of the Tec family.27 In addition, 6 demonstrated low propensity for covalent binding to other proteins as determined by assessing the nonspecific chemical reactivity of 6 against the proteome using human serum albumin as a surrogate protein.26 One of the key features of 6 that distinguishes it from other reported covalent JAK3 inhibitors is its favorable pharmacokinetic profile that makes it suitable for oral dosing and for studies in both preclinical and clinical arenas. This includes human blood clearance that was predicted to be approximately 5.6 mL/min/kg and human half-life of approximately 2 h, values that were obtained from a thorough understanding of clearance mechanisms and in vitro to in vivo correlations.27, 28 The favorable pharmacokinetic profile of 6 is critical because the half-life for JAK3 enzyme turnover determined in human primary CD4+ T cells was in the 3 – 4 h range,26 indicating irreversible inhibition of JAK3 would not lead to a significantly prolonged effect. As part of our ongoing efforts to discover novel JAK3 selective inhibitors based on a covalent approach, the replacement of an acrylamide with a structurally differentiated electrophile was pursued. Our interest in pursuing this work was to develop better understanding of the effect of


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different electrophiles on two key components of covalent JAK3 inhibitor design: (a) reactivity tuning to achieve optimal balance between Ki (reversible binding affinity) and kinact, and (b) pharmacokinetic properties. Our efforts targeted inhibitors based on cyanamide as the CRG for a number of reasons. For example, cyanamide derivatives have been described as covalent inhibitors of other active-site cysteine containing enzymes like cathepsins C, K and L, and Bruton’s tyrosine kinase, in which they form a reversible isothiourea ester link to the active-site cysteine residue.29-33 In addition, the covalent-reversible mechanism reported with this electrophile was anticipated to provide differentiation versus our acrylamide-based inhibitors. The ability to tune cyanamide reactivity through structural modifications to achieve the proper balance between Ki and kinact was an important consideration. As we have reported previously,27 since JAK3 has the highest affinity for ATP among the JAKs, the Ki component of target inhibition must not be the major driver for potency as this would only provide modest selectivity gains through covalent interaction with Cys909. Data reported from studies that evaluated the reactivity of diverse CRGs with either the biologically relevant nucleophile glutathione (GSH),34 or the model amine-based nucleophile N-α-acetyl-L-lysine,35 have demonstrated that cyanamide reactivity can be modulated. For example, significant reactivity differences with N-α-acetyl-Llysine, using half-life values determined from pseudo first order rate constants, were observed among cyanamides derived from azetidine (t1/2 = 2.59 h), pyrrolidine (t1/2 = 7.7 h) or piperidine (t1/2 = 11.1 h). An intriguing aspect of cyanamide-based covalent inhibitors was their pharmacokinetic properties since published reports have not provided insight into the contribution from extrahepatic processes to the clearance of this type of covalent inhibitors.29, 32 In this paper, the identification of a series of cyanamides based on an amino-indane scaffold leading to potent and selective JAK3 inhibitors is presented.



Cl

N NH

N

N

N

O

N N

O

HN N

N

H N

N H

O

N

Page 6 of 120

O O

N H

N

1

2

N H

NH

F

3

N

O

N N H

N

N

O

4

O N

O N

N

N N

NH N

O N

5

N H

N

N H

6

Figure 1. Structure of tofacitinib (1), PF-06651600 (6), a JAK3-specific inhibitor in clinical studies, and recently reported JAK3 covalent inhibitors (2 - 5).

Results and Discussion Screening sequence. The new compounds were evaluated in a panel that included JAK1, JAK2, JAK3 and TYK2 enzyme inhibition at 1 mM ATP. Our group has recently reported on the relevance of obtaining JAK1 and JAK3 enzyme inhibition at physiologically relevant ATP concentration to provide better insights into the cellular activity and selectivity of a compound.36 Assays for JAK1 and JAK3 were also performed at the corresponding Km for ATP for each kinase. These assays allowed for increased sensitivity and were used to quantify affinity of weaker inhibitors. Compounds were also assessed in a TR-FRET competition binding assay utilized to characterize inhibitor type (i.e., reversible vs irreversible). For irreversible covalent inhibitors, this assay was used to determine the second-order rate constant for enzyme


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inactivation (kinact/Ki). The new compounds were also tested in a glutathione (GSH) reactivity assay utilized to understand impact of structural modifications on inherent cysteine reactivity. A limited number of selected compounds were further evaluated in cell-based assays measuring IL15 induced STAT5 phosphorylation, or IL-10 induced STAT3 phosphoryation in peripheral blood mononuclear cells (PBMCs) performed as previously described.36 Identification of N-aryl-based cyanamides. The lead molecules for the cyanamide-based JAK3 covalent inhibitors were acrylamides 7 and 8 obtained during the evaluation of several scaffolds that eventually led to the discovery of 6.27 Acrylamide 7 was identified as a potent JAK3 inhibitor (IC50 = 33 nM) with high selectivity (>300-fold) over other JAK isoforms (Table 1). However, the potency and selectivity of this compound was attributed to high reactivity of the Naryl acrylamide scaffold. In fact, this compound showed a relatively short half-life in the GSH reactivity assay (t1/2 = 56 min). Furthermore, data obtained from whole blood stability assays in both rat and human showed 7 had a short half-life in both species (rat t1/2 = 29 min; human t1/2 = 148 min) suggesting high extrahepatic clearance primarily involving GSH conjugation mediated through glutathione-S-transferases (GSTs).28 These data were in agreement with the high in vivo clearance observed in a rat PK study with 7 (Cl = 455 mL/min/Kg). In an attempt to modulate reactivity of 7 by introducing non-conjugated substituents on the nitrogen, the benzoxazine cyclic scaffold incorporated in acrylamide 8 was prepared. Although 8 showed high potency and selectivity for JAK3 (JAK3 IC50 = 49 nM; >200-fold selectivity vs JAK1), it exhibited even higher reactivity with GSH (t1/2 = 3 min) and a much shorter half-life in blood stability assays (rat t1/2 = 4 min; human t1/2 = 5 min) than 7. The high clearance of these compounds led us to consider alternative strategies to identify novel JAK3 covalent inhibitors with differentiated



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profiles. Among them was the replacement of an acrylamide with a structurally differentiated class of electrophile, in particular cyanamide. Table 1. Data for compounds 6 - 10. IC50 (nM)

Km

Compd

Structure

6 N

Competition assay

1 mM ATP

GSH react.

t1/2 (h)

kinact/Ki (M-1 s-1)c

t1/2 (min)

JAK3a

JAK1b

JAK3

JAK1

JAK2

TYK2

0.3

1640

33

>10000

>10000

>10000

>8

3.68  105

NDd

0.5

>4163

31

>10000

>10000

>10000

>8

1.1  106

56

0.5

>10000

49

>10000

>10000

>10000

ND

ND

3

17

991

1226

6501

>10000

>10000

1.1

2.3  104

>190

NH N

O

N

N H

7 H N O

O N N H

N

8 O N O

O N N H

N

9 O N N

O N N

N H


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10

3

1942

456

>10000

>10000

>10000

1.8

4.8  104

F F O N N

O N N

a

N H

ATP concentration for JAK3 is 4 M at Km. b ATP concentration for JAK1 is 40 M at Km. c

kinact/Ki value for 6 was taken from previous report.27

d

Not determined.

The benzoxazine scaffold utilized for 8 was chosen for providing reversible binding to JAK3 (Ki) and was selected to start investigation of a CRG replacement. Following on this plan, cyanamide 9 was prepared, which to our surprise, exhibited significant loss of JAK3 potency and selectivity (IC50 = 1226 nM; ~5-fold selectivity vs JAK1, Table 1). The significant potency difference between 8 and 9, differing only by the CRG, may be derived from the intrinsic reactivity changes of the electrophile and/or sub-optimal positioning to react with Cys909 in JAK3. Indeed, data from the GSH reactivity assay indicated a large reduction in reactivity with this CRG replacement (9: t1/2 >190 min vs 8: t1/2 = 3 min). Thus, modification of this scaffold was pursued to adjust reactivity. The effects of fluorine substitution on piperidine to modulate pKa are well known, with a 3,3-difluoro substitution leading to significant reduction in the pKa value when compared to unsubstituted piperidine (pKa exp = -3.7).37 It was proposed that similar modification of 9 would lead to an adjustment of cyanamide reactivity and thus 10 was prepared. This analog provided marked improvement in both potency and selectivity (IC50 = 456 nM; >22fold selectivity vs JAK1) when compared to 9. Data from the GSH reactivity assay showed a modest increase in reactivity (10: t1/2 >120 min) when compared to 9. The interaction of 10 with JAK3 was evaluated in the competition assay (kinact/Ki = 4.8  104 M-1 s-1; t1/2 = 1.8 h) showing a profile that was not completely consistent with irreversible inhibition. The dissociation rate half-


>120


life of 10 was not in the range typically observed with irreversible inhibitors (t1/2 > 8 h), but was longer than values determined for reversible inhibitors (t1/2 ≤ 0.05 h). In an effort to better understand the interactions of this compound at a structural level, a crystal structure of 10 bound to JAK3 was solved at 2.9 Å resolution (Figure 2). In addition to the hydrogen bonding interactions between the pyrrolopyrimidine of 10 and the hinge binding region of JAK3, the structure revealed highly interesting aspects of the binding. First, it showed the formation of a covalent bond between the nitrile moiety and Cys909 to give an isothiourea adduct, similar to the interaction reported with cyanamide-based cathepsin inhibitors.30,32 Second, it showed Cys909 side chain adopted a rotamer state different from that previously observed in other JAK3 crystal structures, including the structure of 1.38 The Cys909 chi1 dihedral angle (N-C-C-S) in the structure of 10 is -65 degrees, while the angle in the structure of 1 is 75 degrees. Intrigued by this difference, a more extensive analysis of the Cys909 chi1 dihedral angle was carried out using over 160 proprietary and public JAK3 crystal structures. As shown in Figure 3, only six crystal structures were identified with dihedral angle within 30 degrees from the dihedral angle measured in the structure of 10, confirming a rather uncommon rotamer state. The most common Cys909 rotamer in JAK3 structures has a Cys909 chi1 dihedral angle in the range of 65-85 degrees. An intriguing question that emerged from this analysis was whether there was an energy difference associated with the Cys909 rotamer observed in the structure of 10. To understand the preferred rotamer states of the Cys909 residue within the JAK3 binding site, an enhanced sampling method, metadynamics, was used to sample the free energy landscape of the chi1 dihedral angle of this residue. Metadynamics is an effective sampling technique in which the potential for one or more chosen variables called collective variables (CVs) i.e. dihedral angle in this case, is modified during the simulation by periodically adding a repulsive potential of


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Gaussian shape at the location given by particular values of the variable.39,40 In this way the system is discouraged to go back to its previous steps and forces the calculation to explore the full energy landscape. Figure 4 shows the free energy plot generated from a 50 ns metadynamics simulation of the Cys909 chi1 dihedral angle. The plot shows two nearly equienergetic minimas, one near -54 degrees, which is similar to the observed rotamer state of Cys909 residue in the Xray structure of 10 (-65 degrees) and the other at 72 degrees corresponding closely to the rotamer state observed in the structure of 1. This calculation indicated that with a relative small free energy difference of ~0.4 kcal/mol between the two rotamers along with a low energy barrier of ~7.2 kcal/mol separating the two states, both rotamer states are accessible within the JAK3 binding site. Taken together, this analysis suggested either one of the Cys909 rotamer states could be targeted for covalent interaction without a significant energy penalty. However, the interaction of the nitrile in 10 with Cys909 was not considered optimal (e.g. kinetic data was not completely consistent with irreversible inhibition) and led us to pursue structural modifications that would better position the nitrile for reaction with the more common Cys909 rotamer.



Figure 2. Crystal structure of 10 (gold) bound to JAK3 (ribbons in cyan, residues in gray) at 2.9 Å resolution. Formation of covalent bond with Cys909 was observed (PDB code 6DA4).

Figure 3. Distribution of Cys909 chi1 dihedral angle in JAK3 crystal structures (n = 168, includes both proprietary and public structures).


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Figure 4: Representative free energy surface (FES) generated from a 50 ns metadynamic simulation run of the Cys909 residue chi1 dihedral as the collective variable. The small relative energy difference and the low barrier between the two rotamers suggest both states would be accessible within the JAK3 binding site.

Design of indane-based cyanamides. Analysis of the crystal structure of 10 bound to JAK3 suggested that the nitrile may be better positioned for reaction with the more common Cys909 rotamer using a one atom extended linker between the aromatic ring and the CRG. Accordingly, analogs based on an indane (11 – 15) or chromane (16 – 19) scaffold were obtained (Table 2). The 2-methoxyethyl substituent at C5 of the pyrrolopyrimidine which was present in 7 and 8 was maintained in a number of initial analogs, as it had provided a boost in potency in the N-aryl acrylamides series. The impact of substitution at the nitrogen bearing the CRG was investigated with analogs containing N-methyl substitution. Evaluation of indane-based 11 and chromanebased 16 showed both compounds possessed moderate JAK3 potency (11: IC50 = 711 nM; 16:



IC50 = 1211 nM) but no selectivity versus JAK1 (11: JAK1 IC50 = 908 nM; 16: JAK1 IC50 = 1179 nM). Furthermore, 11 and 16 were evaluated in the TR-FRET competition assay to determine the kinetics of compound binding/dissociation to JAK3 and JAK3 inactivation (kinact/Ki). Interestingly, both compounds exhibited long dissociation rate half-life values (t1/2 > 8h) supporting irreversible inhibition of JAK3. With some encouragement from these data, separation of the enantiomers of 11 by chiral supercritical fluid chromatography (SFC) was pursued affording 12 and 13. The absolute stereochemistry of these compounds was unambiguously assigned based on single crystal structures obtained for each compound (see Supporting Information). Evaluation of this pair demonstrated the JAK3 activity resided primarily with S-enantiomer 12 (IC50 = 256 nM), while the R-enantiomer 13 exhibited a significant potency drop (IC50 >5820 nM). Furthermore, these compounds exhibited significant differences in the competition binding assay (12: t1/2 > 8; 13: t1/2 =0.3 h). Following a similar procedure with the chromane scaffold, chiral separation of 15 using SFC provided 17 and 18. The absolute stereochemistry of this pair was assigned based on biological data and the separation of activity observed with 12 and 13.The S-enantiomer 17 was identified as the most potent enantiomer (17: IC50 = 781 nM vs 18: IC50 = 6110 nM), with data from the competition assay supporting irreversible inhibition for this compound (17: t1/2 > 8; 18: t1/2 = 6.5 h). The data for the S-enantiomers 12 (JAK1 IC50 = 644 nM; JAK2 IC50 = 1666 nM; TYK2 IC50 = 1839 nM) and 17 (JAK1 IC50 = 1215 nM; JAK2 IC50 = 876 nM; TYK2 IC50 = 2971 nM) against other JAK isoforms showed a modest improvement in JAK3 selectivity with 12. There was also a small GSH reactivity difference observed between these scaffolds (12: t1/2 = 69 min; 17: t1/2 =41 min). Other modifications evaluated at this stage were found to be detrimental to JAK3 activity. Incorporation of the 2-methoxyethyl substituent in both of these scaffolds led to a potency loss as


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observed with 14 (JAK3 IC50 = 1592 nM) and 19 (JAK3 IC50 = 5828 nM). Incorporation of a methyl at the nitrogen containing the nitrile was detrimental to potency leading to complete loss of measurable activity as observed with 15 (JAK3 IC50 >10000 nM). Table 2. Data for compounds 11 - 19. IC50 (nM)

Km

Compd 11

Structure N

Competition assay

1 mM ATP

GSH react.

t1/2 (h)


t1/2 (min)

JAK3a

JAK1b

JAK3

JAK1

JAK2

TYK2

6

113

711

909

1544

2890

>8

1.3  104

67

3

82

256

644

1666

1839

>8

5.7  104

69

66

96

>5820

844

1118

3144

0.3

1.1  104

81

12

493

1592

7546

>10000

>10000

6.5

1.6  104

74

N H

N N

N H

12 N N H

N N

N H

13 N N H

N N

N H

14 N N H

O N N

N H



15

Page 16 of 120

230

814

>10000

7624

8623

9487

NDc

ND

>190

7

90

1211

1179

783

2517

>8

9.5  103

36

7

37

781

1215

876

2971

>8

1.6  104

41

26

81

6110

665

470

1816

6.5

3.4  103

ND

49

369

5828

3776

>10000

>10000

>8

4.5  103

70

N N

N N

N H

16 O

N N H N

N

N H

17 O

N N H N

N

N H

18 O

N N H N

N

N H

19 O

N N H

O N N

a

N H


Not determined In order to better understand the interactions of the indane-based cyanamides, a crystal structure of 12 in complex with JAK3 was obtained at 1.66 Å resolution (Figure 5). The S-enantiomer 12 adopts a low-energy conformation, positioning the amino-indane NH to form a hydrogen bond


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with the backbone carbonyl of Arg953, with no obvious conformational or steric strain in the covalently bound conformation. As observed in the structure of 10, the crystal structure of 12 showed the formation of a covalent isothiourea adduct between the cyanamide and the thiolate of Cys909, which was similar to the interaction reported with cyanamide-based cathepsin inhibitors.30,32 This structure also revealed the nitrogen of the isothiourea is stabilized by hydrogen bonds to the side chain carboxyl of Asp912 and is within hydrogen bonding distance to the side chain guanidine group of Arg911. As described above, the amino-indane NH engages in hydrogen bonding with the backbone carbonyl of Arg953. This network of interactions suggested the potential for stabilization of the reactive conformation of the cyanamide through binding to these residues. A similar effect has been postulated in our series of aliphatic acrylamides in which the acrylamide is engaged in H-bonding to the NH of Cys909 and to a conserved water molecule, providing stabilization to the reactive conformation.27



Figure 5. Crystal structure of 12 (purple) bound to JAK3 (ribbons in cyan, residues in grey) at 1.66 Å resolution. Formation of covalent bond with lower energy Cys909 rotamer was observed (PDB code 6DUD).

The crystal structures of JAK3 bound to compounds 10 and 12, along with the data from the TRFRET competition binding assay (t1/2 = 1.8 h and > 8 h for 10 and 12, respectively) indicate a reversible covalent interaction with 10 and an irreversible covalent interaction with 12. The Cys909 chi1/chi2 (N-C-C-S/C-C-S-C) rotamer pairs for the JAK3/10 and the JAK3/12 structures are -54°/96.8° (gauche-/gauche+) and 81.7°/-128.8° (gauche+/trans), respectively. The gauche-/gauche+ configuration for 10 is predicted to be less stable than the gauche+/trans configuration for 12, as it results in a syn-pentane interaction between the backbone NH of Cys909 and the cyanamide moiety of the inhibitor. In contrast, the gauche+/trans configuration for 12 is a low energy conformer, with no steric clash between the inhibitor and protein backbone. These different rotamer values also place the Cys909 sulfhydryl in very different microenvironments, which may perturb its pKa and thereby influence the nature of the covalent interaction with the nitrile group. The pKa of Cys909 was therefore estimated using both a computational and an experimental approach. For the computational approach, the PROPKA method as implemented in MOE software (MOE2015.10001)41 was used to calculate the pKa of Cys909 for the two observed rotamers. The PROPKA method is based upon empirical shifts in pKa values associated with the different types of interactions that each ionizable group may make in the protein environment and can be computed rapidly as a simple pairwise scoring function. Briefly, the parameters used in this function consist of the pKa of the group free in aqueous solution, the desolvation component measured as a function of the group burial, the sum


Page 18 of 120

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of all potential hydrogen bonds made by the group, and a buried charge-charge interaction component. A benchmark study of PROPKA on cysteine residues for several Trx and ArsC proteins revealed a fair correlation (R2 = 0.74) with an average deviation from the experimental value of 0.88 pKa units versus experimental pKa.42 The calculated pKa values are quite different for the two rotamers (pKa = 4.5 for 12 versus pKa = 9.0 for 10). For the experimental approach, the rate of inactivation of JAK3, kinact, was determined for a potent acrylamide-containing compound at various pH values (acrylamide 125, see Experimental section). The thiol form of Cys909, which predominates when the pH is below the pKa, is a poor nucleophile and would be less reactive towards the electrophilic acrylamide moiety, whereas the thiolate anion form, which predominates when the pH is above the pKa, is a highly reactive nucleophile which should result in a measurably larger kinact. When the kinact is plotted against the pH, the midpoint of the curve yields an estimate of the thiolate pKa. The pKa measured by this method was determined to be 6.3  0.2, significantly lower than that of a typical solvent exposed Cys residue (pKa ~ 8.3). A possible mechanism for the reduced pKa value for Cys909 is a direct interaction with the helix dipole from helix 909-917. In the conformation of Cys909 observed with 10 (predicted pKa 9), the Cys909 sulfhydryl is oriented well off of the axis of helix 909-917, while in the conformation seen with 12 (predicted pKa 4.5, measured 6.2), it is oriented along the helix axis, positioned precisely at the N-terminal end where the positive helix dipole is directed. It is predicted that the positive helix dipole perturbs the pKa of the Cys909 SH group, such that at physiological pH, the thiol will have a greater propensity to ionize to the thiolate anion, thereby facilitating nucleophilic attack. Because of these steric and pKa effects, we hypothesize that the Cys909 conformation seen in 10 results in a less stable covalent interaction that leads to a reversible covalent interaction consistent with the TR-FRET results.



Pharmacokinetic evaluation of cyanamide 12. As outlined above, one of the objectives in pursuing cyanamide-based covalent JAK3 inhibitors was to develop a better understanding of the pharmacokinetic properties of this type of covalent inhibitor. Based on its profile consistent with that of an irreversible inhibitor of JAK3, including a long dissociation rate half-life value (t1/2 > 8 h) and crystallographic data showing formation of a covalent bond between the cyanamide and Cys909, cyanamide 12 was selected for profiling in blood stability assays and pharmacokinetic studies in rat. Evaluation of 12 in the blood stability assay showed longer half-life values in both rat and human (rat t1/2 = 150 min; human t1/2 >360 min) than those observed for the acrylamides 7 and 8, with the value in human reaching the upper limit of determination for the assay. These results suggested the potential for lower extrahepatic clearance derived from GST-mediated GSH conjugation for indane-based cyanamides when compared to N-aryl acrylamides. The in vitro CYP450-mediated metabolism, assessed using liver microsomes, showed relatively high clearance for this compound in both rat and human (RLM CLint, app = 100 L/min/mg; HLM CLint, app = 50 L/min/mg). The pharmacokinetic properties of 12 were evaluated in rat (Table 3). In vivo clearance was about half hepatic blood flow (Cl = 36 mL/min/kg) with a low volume of distribution (Vss = 0.35 L/kg) and short measured half-life (t1/2 = 0.27 h). In order to understand the contribution to clearance from non-CYP450 mediated pathways, the IV PK profile of 12 in the presence of the CYP450 inhibitor aminobenzotriazole (ABT) was also obtained. There was no significant difference in clearance values observed when the study was done in the presence of ABT (Cl = 34 mL/min/kg), suggesting that clearance of 12 is primarily GSH/GST-mediated. Notably, there was a significant difference in the rat in vivo clearance of 12 compared to that of acrylamide 7 (Cl = 455 mL/min/Kg). Table 3. Pharmacokinetic properties of 12 in rat.


Page 20 of 120

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Route

Dose

ABT

AUC

t1/2

F

Cl

Vss

(mg/kg)

(mg/kg)

(ng·h/mL)

(h)

(%)

(mL/min/kg)

(L/kg)

36

0.35

34

0.63

IV

1

None

466

PO

3

None

240

IV

1

100

488

0.27 16 0.75

Metabolism studies of cyanamide 12. To further expand on our understanding of the metabolic pathways of cyanamide-based covalent inhibitors, we pursued metabolism studies of 12. Following incubation of 12 in the presence and absence of either NADPH, GSH alone or in human liver microsomes incubations, the major pathways of metabolism identified were glutathione conjugates (Figure 6). Oxidative and hydrolytic metabolism of 12 was also identified but considered minor pathways. LC-MS/MS analysis of NADPH-supplemented human liver microsomal incubations containing 12 and GSH led to the detection of three major peaks including conjugates with molecular ions (MH+) at m/z 583, 759 and 454 (Figure 6). These conjugates were also observed when human liver microsomes and NADPH were omitted from the incubation mixtures, indicating intrinsic reactivity with GSH and that bioactivation was not a requirement for conjugation. The exact mass of the conjugates M-583 (m/z 583.2083, C26H30N8O6S, -0.2 ppm) is consistent with the addition of one molecule of GSH to 12, M-759 (m/z 759.2333, C31H38N10O9S2, -0.5 ppm) a molecule of GSH and cysteine-glycine GSH fragment and M-454 (m/z 454.1655, C21H23N7O3S, -0.2 ppm) the addition of the cysteine-glycine fragment of GSH. The collision-induced dissociation (CID) spectrum of M-583 and M-759 displayed diagnostic loss of the pyroglutamate



moiety (m/z 129), while M-454 displayed diagnostic loss of the glycine moiety (m/z 74), all of which represent characteristic fragment ions derived from GSH type conjugates.43 The conjugation of the thiol conjugates to the nitrile of 12 occurs through a Pinner reaction-like mechanism of nucleophilic addition. In all incubations fortified with GSH, based on UV profiles, 12 was entirely consumed over the duration of the incubation. These findings suggest that GSH conjugation to 12 was not reversible over this period under those conditions. Several minor hydrolytic and or oxidative peaks were detected in human liver microsomal incubations containing 12 with molecular ions (MH+) of m/z 294, 310, 292 and 296 (Figure 6). The hydrolysis peak M-294 with molecular ion (MH+) at m/z 294 was detected in all incubations that included human liver microsomes regardless of the presence or absence of NADPH or GSH. The exact mass of M-294 (m/z 294.1348, C16H15N5O, -0.3 ppm) and CID fragmentation patterns are consistent with the addition of H2O to nitrile moiety of 12. This reaction in the absence of cofactor in human liver microsomes suggests a role for any number of enzymatic hydrolases. Metabolite M-294 was further oxidized to peak M-310 detected with molecular ion (MH+) at m/z 310, and was detected in all incubations that included human liver microsomes and NADPH suggesting oxidation by cytochrome P450. The exact mass of M-310 (m/z 310.1299, C16H15N5O2, 0.0 ppm) and CID fragmentation patterns are consistent with the addition of oxygen to the urea moiety of M-294. The oxidative peak M-292 with molecular ion (MH+) at m/z 292, was detected in all incubations that included human liver microsomes and NADPH suggesting oxidation by cytochrome P450. The exact mass of the conjugates M-292 (m/z 292.1192, C16H13N5O, -0.3 ppm) and CID fragmentation patterns are consistent with the addition of oxygen to the cyclopentane ring of 12. The oxidative peak M-296 with molecular ion (MH+) at m/z 296, was detected in all incubations that included human liver microsomes and NADPH


Page 22 of 120

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suggesting oxidation by cytochrome P450. The exact mass of the metabolite M-296 (m/z 296.1144, C15H13N5O2, 0.7 ppm) and CID fragmentation patterns are consistent with pyrrole ring epoxidation, rearrangement and a Baeyer-Villiger reaction which results in the pyrrole ring opening, loss of carbon and addition of two oxygens to 12. O

O

S

N H

HO

N

NH

HN

N N H

N H

O

O N

N O

O O

HO

N H HN

N S

M-296 O

OH

H 2N

NH

N H

O

O

N H

HO

NH2

N H

NH2 N

S N

HO

N

N O

NH2

N

12

M-583

OH

N N H

N

N H

N NH2

HO

O

N H

N N

N H

N H

N

N H

M-292

M-294

M-759 O

O HO

N H

O

NH S

H 2N

N H

HO

H 2N N

N H

N N

N H

M-454

N

N H

M-310

Figure 6. The major in vitro metabolic pathways of cyanamide 12 identified included three conjugates (m/z 583, 759, and 454) consistent with GSH addition.43 Minor metabolic pathways included both hydrolytic and oxidative metabolism (m/z 294, 310, 292 and 296).



The results obtained with 12 supported a focus on indane-based cyanamides. The moderate JAK3 potency and limited selectivity versus other JAK isoforms with this scaffold was attributed to a relatively high Ki contribution leading to significant reversible binding across the JAKs. More importantly, the inactivation rate (kinact) was deemed suboptimal to achieve high selectivity. Further insight was gained by comparing kinact/Ki values for 6 and 12. The optimized acrylamide 6 demonstrated approximately one order of magnitude higher kinact/Ki than 12 (6, kinact/Ki = 3.68  105 M-1·s-1; 12, kinact/Ki = 5.7  104 M-1·s-1), and exhibited high JAK3 selectivity, suggesting further opportunity to improve kinact/Ki values for 12. Therefore, efforts were directed at the identification of structural modifications that could improve JAK3 potency and selectivity over other JAK isoforms through optimization of the inactivation rate. Among the modifications that were considered, substitutions of the indane at the C1, C2 and C3 positions were particularly attractive. It was hypothesized that a substituent at one of these positions would modulate favorably cyanamide reactivity, or would lead to improved positioning of the CRG. In addition, new interactions between such a substituent and protein residues through H-bonding, lipophilic contacts, or steric clash, could lead to JAK3 potency and selectivity improvements. Analogs incorporating small substituents including methyl and hydroxyl were prepared (Table 4). Addition of a cis 2-methyl on cyanamide 12 led to 20, which showed no improvement in potency or selectivity (JAK3 IC50 = 603 nM), while the trans diastereomer 21 lost potency (JAK3 IC50 = 1530 nM). Incorporation of a methyl to create a tertiary center gave 22 and led to a modest improvement in potency (JAK3 IC50 = 92 nM). However, evaluation of 22 in the competition assay showed a profile consistent with reversible inhibition (t1/2 = 0.05 h). In addition, data from the GSH reactivity assay showed a decrease in reactivity with 22 (t1/2 >120 min) when compared to 12 (t1/2 = 69 min). Intrigued by these results, 22 was separated into its


Page 24 of 120

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enantiomers 23 and 24 using chiral SFC. The absolute stereochemistries were assigned based on crystallographic data with 23 assigned as the (S)-enantiomer (see below). Evaluation of 23 and 24 confirmed JAK3 activity was mainly retained by 23, with approximately a five-fold increase in potency (23: JAK3 IC50 = 56 nM) when compared to 12 (IC50 = 256 nM). However, a potency gain with 23 was also observed across the other JAKs (JAK1 IC50 = 57 nM; JAK2 IC50 = 71 nM; TYK2 IC50 = 179 nM), thus leading to no improvement in selectivity. Profiling of 23 in the competition assay also showed a profile consistent with reversible inhibition (t1/2 ≤ 0.05 h). In keeping with the data observed with racemic 22, 23 also exhibited longer half-life than 12 in the GSH reactivity assay (t1/2 > 120 min). Evaluation of 23 in blood stability assays showed half-life values in both rat and human greater than 360 min, which suggested a major reduction in extrahepatic clearance involving GSH conjugation mediated through GSTs. This was a key result because it demonstrated for the first time in our JAK3 covalent project that steric hindrance around the electrophile provided a productive approach to decrease glutathione-S-transferase (GST)-mediated clearance. It is interesting to note that Laine and collaborators previously described their approach to improve plasma stability of cyanamide-based cathepsin inhibitors.32 Plasma instability was considered to be mediated by an esterase acting on the nitrile and they pursued reduction of the rate of reaction with the esterase by decreasing the electrophilicity and steric accessibility of the nitrile head. Supported with our better understanding of the clearance pathways of covalent inhibitors, our findings go far beyond than addressing esterase metabolism. Our findings demonstrate that steric hindrance around the electrophile can be used as a productive method to decrease one of the major clearance pathways of covalent inhibitors (GSTmediated clearance).



Page 26 of 120

The intriguing profile of 23 led us to seek a better understanding of the mode of interaction of this compound with JAK3. The initially proposed interactions with 23 and JAK3 were reexamined. Based on an initial docking model of 23, its angular methyl group was predicted to be positioned near the backbone carbonyl of Leu828 from the JAK3 G-loop (CH3 – C=O, ~4.6 Å). The proximity of these groups was expected to limit the rotation around the indanepyrrolopyrimidine bond, leading to stabilization of the reactive conformation for interaction of the nitrile with Cys909, thus improving potency with concomitant selectivity gain. Although potency was increased, the lack of selectivity improvement did not support this docking hypothesis. In order to further understand the mode of binding of 23, a crystal structure of this compound bound to JAK3 was obtained at 1.97 Å resolution (Figure 7). To our surprise, rotation of approximately 110o around the indane-pyrrolopyrimidine bond relative to our docking pose took place. The cyanamide was now positioned away from Cys909 with no possibility for a covalent interaction and now directed towards the back pocket of the ATP binding site. The NH displaying the nitrile was located within H-bond distance to the catalytic Asp967 residue (3.1 Å). The angular methyl group partly filled the lipophilic pocket formed by Leu956 on the floor of the ATP binding site identified in the crystal structure of 1, and likely contributes to the potency across all JAKs for 23. Thus, all data obtained with 23 supported its profile being that of a potent, reversible, but non-selective JAK3 inhibitor. Table 4. Data for compounds 20 - 27. IC50 (nM)

Km

Compd

Structure

JAK3a

1 mM ATP

JAK1b

JAK3

JAK1

JAK2


TYK2

Competition assay

GSH react.

t1/2 (h)

t1/2 (min)


Page 27 of 120 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

20


N

6

100

603

705

1046

2010

>8

2.8  104

NDc

13

94

1530

973

1870

4619

>8

1  104

95

1

8

92

94

97

239

0.05

ND

>120

0.7

5

56

57

71

179

120

120

203

ND

ND

ND

ND

0.2

ND

>120

N H

N N

N H

21 N N H

N N

N H

22 N N H

N N

N H

23 N N H

N N

N H

24 N N H

N N

N H



25

Page 28 of 120

1

652

163

>7318

>10000

>10000

>8

1.5  105

28

13

92

1338

1094

2455

3372

7.6

1.9  104

40

2

105

280

1266

4964

4285

>8

4.6  104

47

OH

N N H

N N

N H

26 OH

N N H

N N

N H

27 N

HO N H

N N

a

N H


Not determined.


Page 29 of 120 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


Figure 7. Crystal structure of 23 (orange) bound to JAK3 (ribbons in cyan, residues in gray) at 1.97 Å resolution. Rotation around the indane-pyrrolopyrimidine bond was observed leading to a completely different orientation and loss of covalent interaction with Cys909 (PDB code 6DB3).

In contrast to the unsuccessful attempts to improve JAK3 selectivity by incorporation of a methyl at the indane C1 or C2 positions, addition of a hydroxyl group at C3 led to more promising results (Table 4). The incorporation of a cis-3-hydroxy in 25 led to a modest improvement in JAK3 potency (IC50 = 163 nM), but more importantly, this modification led to marked selectivity gains (45-fold selectivity over JAK1 and >60-fold over JAK2 and TYK2). The stereochemistry and position of the hydroxyl group was important for both potency and selectivity. The trans-3hydroxy compound 26 was approximately eight-fold less potent (JAK3 IC50 = 1338 nM) than 25 and showed no selectivity over JAK1 (26: IC50 = 1094 nM). On the other hand, moving the hydroxyl group to C2 to give trans-2-hydroxy compound 27 (JAK3 IC50 = 280 nM) led to less than a two-fold drop in potency as compared to 25, but only five-fold selectivity over JAK1 (IC50



= 1266 nM). The three cyanamides 25 - 27 exhibited irreversible binding to JAK3 in the competition binding assay. Assessment of 25 - 27 in the GSH assay showed reactivity increases compared to 12 (25, t1/2 = 28 min; 26, t1/2 =40 min; 27, t1/2 =47 min), with 25 exhibiting the highest reactivity. The increase in chemical reactivity of 25 may contribute to the observed potency and selectivity gain achieved with this compound. These results may be explained by the conformation of the cis-3-hydroxy group. In this regard, a theoretical conformational analysis of cis-3-aminoindan-1-ol was described recently.44 This study showed that the predominant conformation of cis-3-aminoindan-1-ol has a puckered cyclopentenyl ring; where the C2 atom is located on the opposite side of the OH and NH2 groups, and is stabilized by a hydrogen bond between the NH and OH. However, the study also revealed that the cis-3-aminoindan-1-ol is a flexible system. A low energy barrier (3.5 kcal/mol) was calculated for interconversion to a second puckered conformation wherein the C2 atom is located on the same side as the OH and NH2 groups, and where no hydrogen bonding occurs. The cis-3-aminoindan-1-ol work suggested that the cis-3-hydroxy group in 25 might increase the reactivity of the cyanamide through Hbonding to the NH. Data from the GSH reactivity assay supported a higher reactivity increase going from 12 to the cis-3-hydroxy 25 versus the trans-3-hydroxy 26. Data from blood stability assays also inferred the higher reactivity of 25 (rat t1/2 = 20 min; human t1/2 = 339 min) compared to 12. The results obtained with 25 led us to consider that a hydrogen bond donor at C3 of the indane might be important to enhance potency and selectivity through modulation of cyanamide reactivity, or CRG positioning. To explore this hypothesis, a set of analogs incorporating a cis-3sulfonamide were prepared and selected examples 28 – 35 are presented in Table 5. It was encouraging to observe that the methanesulfonamide 28 exhibited a four-fold increase in JAK3


Page 30 of 120

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potency (IC50 = 38 nM) when compared to 25, and that it retained similar selectivity vs JAK1 (51-fold). Evaluation of 28 in the blood stability assay showed a reduction in half-life values in both rat and human (rat t1/2 = 31 min; human t1/2 = 208 min) when compared to 12 and 25. Further improvement in selectivity was obtained with sulfonamides possessing larger groups. Cyanamide 29, incorporating a methoxyethyl sulfonamide group, demonstrated good potency (JAK3 IC50 = 111 nM) and high selectivity vs JAK1 (78-fold). The trend was also observed with heterocyclic sulfonamides like 30, which possesses an N-methyl-5-pyrazolyl (IC50 = 38 nM; 106fold selectivity). Substituted aryl sulfonamides also demonstrated a large improvement in selectivity, with the substituent on the phenyl ring having an important effect on potency and selectivity. The most selective compounds incorporated substitution at the meta and para positions. The 3-cyano analog 31 (JAK3 IC50 = 14 nM), and 4-methoxy derivative 33 (JAK3 IC50 = 16 nM) exhibited >100-fold selectivity vs other JAK isoforms while the 3-fluorine analog 32 (JAK3 IC50 = 11 nM) demonstrated the highest selectivity observed within this series of cyanamides (246-fold selectivity vs all JAKs). Cyanamides 28 - 33 showed irreversible binding in the competition assay consistent with covalent inhibition of JAK3. These cyanamides exhibited reactivity with GSH (t1/2 = 22 - 80 min) in a similar range, or slightly higher than that observed with 12. The reasons for the large gain in selectivity with the 3-fluorophenyl sulfonamide 32 are not well understood. Chemical reactivity may not have been a factor, as 32 exhibited comparable GSH reactivity when compared to 12 (12, t1/2 = 69 min; 32, t1/2 = 80 min). It is possible that interactions of the 3-fluorophenylsulfonamide moiety with the JAK3 P-loop contribute to both potency and selectivity gains. One last tactic that was used to improve potency and selectivity in this series was the incorporation of aryl groups at C5 of the pyrrolopyrimidine. It was hypothesized that a C5 aryl or



heteroaryl substituent would provide improvements in potency and selectivity in two ways. First, pi-pi stacking between the C5 aryl/heteroaryl substituent and the indane ring may influence the conformation around the indane-pyrrolopyrimidine bond leading to improved positioning of the CRG. Second, a properly positioned substituent on the C5 aryl or heteroaryl group could pick up specific interactions with JAK3 leading to improved potency and selectivity. Additionally, such a substituent could lead to steric clashes with other JAK isoforms leading to improved selectivity. Despite the limited number of analogs prepared from this effort, the results were encouraging and suggest further improvements may be possible following this tactic. The results obtained with 34 (C5 phenyl) and 35 (C5 3-hydroxymethylphenyl) are displayed in Table 5. Both 34 (JAK3 IC50 = 49 nM) and 35 (JAK3 IC50 = 78 nM) demonstrated potency gains when compared to 12 (JAK3 IC50 = 256 nM; JAK1 IC50 = 644 nM). Selectivity vs JAK1 was also improved with 34 (JAK1 IC50 = 3345 nM, 68-fold selectivity) and even more markedly with 35 (JAK1 IC50 >9604 nM, 123-fold selectivity). Both analogs 34 and 35 exhibited irreversible binding in the competition assay consistent with covalent inhibition of JAK3. Also, both compounds showed comparable, or slightly reduced GSH reactivity (34: t1/2 = 94 min; 35: t1/2 = 69 min) when compared to 12. Evaluation of 34 and 35 in the blood stability assay showed an approximately three to seven-fold reduction in half-life values in both rat and human (34: rat t1/2 = 61 min; human t1/2 = 61 min; 35: rat t1/2 = 59 min; human t1/2 = 48 min) when compared to 12, suggesting higher affinities towards GSTs than those of C5-unsubstituted cyanamides like 12. In order to confirm the mode of binding of this set of analogs, a crystal structure of 34 bound to JAK3 was obtained at 1.66 Å resolution (Figure 8). This structure showed the C5 phenyl ring resides over the lipophilic pocket on the floor of the active site and is at a 4.3 Å distance to the indane, measured from the center of each 6-membered aromatic ring. These rings are nearly parallel to


Page 32 of 120

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each other as determined from comparing torsion angles between pyrrolopyrimidine and phenyl (49o) vs between indane and pyrrolopyrimidine (39o). As observed in the crystal structure of 12 bound to JAK3, the nitrile of 34 forms a covalent isothiourea adduct with Cys909. The imino NH of the isothiourea moiety is stabilized by hydrogen bonds to the side chain carboxyl group of Asp912, which is further stabilized by interaction with Arg911. The nitrogen attached to the indane ring engages in hydrogen bonding with the backbone carbonyl of Arg953. The five-fold increase in JAK3 potency and >20-fold improvement in selectivity vs JAK1 when 34 is compared to 12 may be explained by the interaction between the phenyl and indane rings leading to improved placement of the CRG. Strikingly, these improvements do not appear to be a consequence of increased reactivity, as 34 (t1/2 = 94 min) showed comparable half-life values in the GSH reactivity assay when compared to 12 (t1/2 = 69 min). Table 5. Data for compounds 28 - 35. IC50 (nM)

Km

Compd 28

Structure H O N S O

N

1 mM ATP

Competition assay

GSH react.

t1/2 (h)


t1/2 (min)

JAK3a

JAK1b

JAK3

JAK1

JAK2

TYK2

0.9

97

38

1918

>7448

>9443

>8

2.8  105

23

1

775

111

8652

>10000

>10000

>8

1.3  105

70

N H

N N

N H

29 H O N S O

N N H

O

N N

N H



30

Page 34 of 120

NDc

ND

38

4027

>7646

>10000

>8d

1.2  105

22

ND

ND

14

1715

2419

>9817

>8d

5.4  104

24

ND

ND

11

2703

3012

>10000

>8d

1.9  105

80

ND

ND

16

1720

2554

>10000

>8d

2.4  105

35

0.7

422

49

3345

>6578

>10000

>8

2.2  105

94

0.5

733

78

>9604

>9925

>10000

>8

7.7  103

69

H O N S O

N

N N

N H

N N H

N

31 H O N S O

N N H

N N N

N H

32 H O N S O

N N H

F

N N H

N

33 H O N S O

N N H

O N N

N H

34 N N H

N N

N H

35 N N H OH N N

a

N H


Not determined. d Data obtained using racemic compound.


Page 35 of 120 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


Figure 8. Crystal structure of 34 (gold) bound to JAK3 (ribbons in cyan, residues in gray) at 1.66 Å resolution. Covalent interaction between the nitrile and Cys909 was observed. The indane and pyrrolopyrimidine rings are nearly parallel to each other as determined from comparing torsion angles between pyrrolopyrimidine and phenyl (49o) and between indane and pyrrolopyrimidine (39o) (PDB code 6DB4).

Selected compounds were evaluated in cell-based assays using PBMCs with IL-15 stimulation, measuring STAT5 phosphorylation, or IL-10 stimulation measuring STAT3 phosphorylation (Table 6). Signaling by IL-15 is mediated through the JAK1/JAK3 heterodimer, while signaling by IL-10 is mediated through the JAK1/TYK2 heterodimer. Selected compounds were also tested in a human whole blood (HWB) IL-15-induced STAT5 phosphorylation assay. Demonstration of suitable potency in this assay has been used by our group as key criteria to advance compounds for further pre-clinical profiling.27 The IL-15 and IL-10 PBMC and IL-15 HWB assays were performed as previously described.27, 36 JAK3 and JAK1 IC50 values at 1 mM



ATP and JAK1 IC50/JAK3 IC50 ratio (J1/J3) are included to facilitate data analysis. Passive permeability was obtained using Ralph Russ Canine Kidney (RRCK) cells and results are included.45 Data for acrylamides 6 and 7 are provided for comparison. In agreement with our previous reports,36 there was a strong correlation between potency for JAK1 and JAK3, selectivity (J1/J3), and permeability with the potency determined in the PBMC IL-15 assay. It is evident from examination of the permeability data presented in Table 6, that several cyanamides exhibited low permeability values (RRCK Papp AB < 310-6 cm/sec), which in turn had a detrimental effect on cell potency. Among the cyanamides with good JAK3 selectivity (J1/J3 > 50) and good permeability (RRCK Papp AB ≥ 410-6 cm/sec), 10, 31, 32, and 35 were of particular interest. Cyanamide 10 exhibited cell potency in the PBMC IL-15 assay (IC50 = 162 nM) that was only about three-fold lower than the potency observed with acrylamide 7 (IC50 = 63 nM); also interestingly, no JAK1 activity was observed with this compound up to the top concentration of the JAK1 enzymatic assay (10 M), suggesting that the activity observed in the IL-15-stimulated PBMC assay may be primarily driven by JAK3 inhibition. Indane-based cyanamides 31, 32 and 35 exhibited potency (IC50 = 331

469 nM) in the IL-15 stimulated

PMBCs assay that was within 5-7-fold of the potency observed with acrylamide 7. In particular, among the compounds with greater than 100-fold JAK3 selectivity, 32 was identified as the most potent analog (IC50 = 331 nM) in this assay and also displayed high JAK3 selectivity (246-fold) when compared to other JAK isoforms in enzymatic assays. The high JAK3 selectivity of 32 supports its potency in the IL-15 stimulated PBMC assay as being driven primarily through JAK3 inhibition. In order to gain further understanding of the selectivity of this set of compounds at a cellular level, evaluation in PBMCs stimulated with IL-10, measuring STAT3 phosphorylation was carried out. As noted earlier, signaling by IL-10 is mediated through the


Page 36 of 120

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JAK1/TYK2 heterodimer. Cyanamides 10, 25, 28, and 31-34 exhibited no inhibition in this assay up to the top concentration tested (10 M) further supporting that their effect in PBMCs stimulated with IL-15 is primarily driven through JAK3 inhibition. A set of cyanamides was further evaluated in a human whole blood IL-15-induced STAT5 phosphorylation assay that revealed a significant liability for this series. Despite the good potency exhibited by 12, 32, 34 and 35 in the IL-15 PMBC assay (IC50 = 331- 598 nM), these cyanamides demonstrated no inhibition in the human whole blood assay up to the top tested concentration (IC50 > 10 M). The less JAK3 selective cyanamide 10 (IL-15 PMBC IC50 = 162 nM; IL-15 HWB IC50 = 7252 nM) and the non-covalent analog 23 (IL-15 PMBC IC50 = 56 nM; IL-15 HWB IC50 = 5480 nM) also demonstrated poor correlation between assays. It is important to note that 6 demonstrated potent activity in this assay (IL-15 HWB IC50 = 197 nM), indicating that JAK3 selective inhibitors can inhibit IL-15 in human whole blood, as previously reported.27 The poor translation from a cellbased PBMC assay to one conducted in human whole blood has previously been observed by our group when comparing the poorly correlated aryl aminoacrylamides versus the better correlated aliphatic aminoacrylamides.27 The reasons for the poor correlation were not fully elucidated, but it was postulated that the enhanced reactivity of aryl aminoacrylamides was a major contributor to their poor correlation. It is unlikely that reactivity may explain the poor correlation observed with the cyanamides; as a number of these analogs exhibited low reactivity in the GSH assay (10 and 23: t1/2 >120 min; 34; t1/2 = 94 min). In addition, a number of these analogs also exhibited high stability in human blood stability assay (12 and 23, t1/2 > 120 min). Thus, the reasons for the poor correlation observed with the cyanamides are unclear. Table 6. Evaluation of selected cyanamides in cell-based assays.a Cmpd

JAK3

JAK1

J1/J3

RRCKb

IL-15 PBMC

IL-10 PBMC


IL-15 HWB


IC50

IC50

(nM)

(nM)

Papp AB

IC50 (nM)

Page 38 of 120

IC50 (nM)

IC50 (nM)

( 10-6 cm/sec)

6c

33

>10000

>293

15

51

>60000d

197

7

31

>10000

>323

10

63

>10000

953

10

456

>10000

22

14

162

>10000

7252

12

256

644

3

11

598

2815

>10000

23

56

57

1

19

56

NDe

5480

25

163

>7318

>45

2

1728

>10000

ND

28

38

1918

51

10000

ND

31

14

1715

123

6

468

>10000

ND

32

11

2703

246

10

331

>10000

>10000f

33

16

1720

108

ND

947

>10000

ND

34

49

3345

69

2

458

>10000

>10000

35

78

>9604

>123

4

498

>7843

>10000

a

ATP concentration for JAK3 and JAK1 assays is 1 mM . b Passive permeability was obtained

using Ralph Russ Canine Kidney (RRCK) cells.45 c Data for 6 was reported previously.27 d Determined in total lymphocytes in human whole blood.26 e Not determined. f Data obtained using racemic compound. Evaluation of the kinome selectivity of selected cyanamides 10, 12, 25, 32 and 35 was pursued in a panel of forty kinases. The screen was run at a compound concentration of 1 M and 1 mM


Page 39 of 120 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


ATP concentration for all kinases. Data showed that in general these cyanamides demonstrate high selectivity in this panel (Figure 9), with 10, 25, 32, and 35 exhibiting >50% inhibition for only a single kinase in addition to JAK3. Kinases inhibited by these compounds include BTK (10, 75% inhibition), PRKCB2 (35, 52% inhibition), CHEK2 (25, 52% inhibition) and KDR (32, 58% inhibition). Cyanamide 12 inhibited 5/40 kinases with >50% inhibition. Cyanamides 12, 32 and 35 were selected for evaluation in a panel of kinases that contain a Cys residue at the equivalent position of Cys909 in JAK3. The IC50 values were determined in the presence of 1 mM ATP (Table 7). The data showed that high selectivity for JAK3 versus this panel of kinases was achieved. BMX was the only kinase inhibited by all compounds, and the one that exhibited the lowest margins of selectivity for 12 (IC50 = 6310 nM, 24-fold selectivity) and 35 (IC50 = 1900 nM, 24-fold selectivity). In contrast to these compounds, 32 exhibited high selectivity for JAK3 vs BMX (IC50 = 3390 nM, 308-fold selectivity). 35 inhibited three other kinases (HER4, IC50 = 3540 nM, 45-fold; TEC, IC50 = 8960 nM 115-fold; TXK, IC50 = 2990 nM 38-fold). Collectively, the data obtained for this selected set of cyanamide analogs demonstrated that high kinome selectivity can be obtained, even for kinases expected to be inhibited due to the presence of a Cys residue at the equivalent position of Cys909 in JAK3.



Figure 9. Heatmap of kinase activity profile (% inhibition at 1mM ATP) of 10, 12, 25, 32 and 35 at 1 M concentration. Red: 100% inhibition; yellow; 50% inhibition; green; 0% inhibition.

Table 7. Activity of 12, 32 and 35 against kinases containing a Cys residue at the same position as Cys909 in JAK3. Compound 12 Kinase

32

35

IC50 (nM)

BLK

>10000

>10000

>10000

BMX

6310

3390

1900

BTK

>10000

>10000

>10000

EGFR

>10000

>10000

>10000

HER2

>10000

>10000

>10000

HER4

>10000

>10000

3540

ITK

>10000

>10000

>10000

MAP2K7

>10000

>10000

>10000

TEC

>10000

>10000

8960

TXK

>10000

>10000

2990

As discussed earlier, our interest to pursue this work was to develop a better understanding of the effect of a cyanamide as the electrophile on key components of covalent JAK3 inhibitor design, including reactivity tuning to achieve an optimal balance between Ki and kinact. The results


Page 40 of 120

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obtained with the amino-indane based cyanamides showed that modulation of Ki and kinact could be achieved through structural modifications to enhance potency and selectivity. This was observed in going from 12 (JAK3 IC50 = 256 nM; 2-fold selectivity vs JAK1) to 32, with the optimized 32 exhibiting potent JAK3 inhibition (IC50 = 11 nM) and high selectivity for JAK3 vs other JAKs (246-fold). Despite the promising profile demonstrated with 32 and other analogs, the lack of potency of the indane-based cyanamides in the IL-15 human whole blood assay and lack of understanding of the factors that contribute to the poor translation between cell-based PBMC assays and the assay conducted in human whole blood led us to deprioritize this series for further progression. Chemistry The synthesis of compound 7 is described in Scheme 1. The synthesis departs with the selective bromination of 4-chloro-7H-pyrrolo[2,3-d]pyrimidine (36) with NBS at C-2 to give 37, which after reacted with nBuLi to form the dianion, was then reacted with 1-bromo-2-methoxyethane to give 38. Intermediate 38 was subsequently coupled to (3-aminophenyl)boronic acid under Suzuki conditions to provide 39,46,47 and this was followed by acylation with acryloyl chloride to give acrylamide 7. Scheme 1. Synthesis of acrylamide 7.a

Cl

b

N N a

Cl

R

N H 36: R = H 37: R = Br

R

O

H N O

c

N N 38

N

N H

N d

N H

39: R = H 7: R = COCHCH2



aReagents oC,

Page 42 of 120

and conditions: (a) NBS, DCM, rt, 14 h, 51%; (b) Br(CH2)2OMe, n-BuLi, THF, -60

1 h, 15%; (c) m-NH2PhB(OH)2, dioxane:H2O (4:1), K3PO4, PdCl2(dppf), 90 oC, 3 h, 74%; (d)

acryloyl-chloride, iPr2NEt, DMF, 0 oC, 4 h, 21%. The synthesis of compounds 8 and 9 are described in Scheme 2. 4-Chloro-5-(2-methoxyethyl)7H-pyrrolo[2,3-d]pyrimidine (38) was reacted with boronate 4048 under Suzuki cross-coupling conditions to give benzoxazinone 41. Reduction of the carbonyl group of 41 with LAH in THF afforded amine 42, which was acylated with acryloyl chloride to give 8. The synthesis of the cyanamide derivative 9 was accomplished by reacting amine 42 with CNBr/K2CO3 to provide the desired product..49-51 Scheme 2. Synthesis of acrylamide 8 and cyanamide 9.a O

O

HN O 40 O 38

B

O

O

HN O

R

O

a

O

b N

N N 41

N H

N H 42: R = H 8: R = COCHCH2 N

c d

aReagents

N

42: R = H 9: R = CN

and conditions: (a) 40, Cs2CO3, dioxane:H2O (10:1), Pd(PPh3)4, 100 oC, 30 min, 38%;

(b) LAH, THF, reflux, 18 h, 25%; (c) acryloyl chloride, THF, 0 oC, 18%; (d) BrCN, THF, K2CO3, 0 oC, 42%. The synthesis of 10 is described in Scheme 3. Thus, beginning with 2-amino-4-bromophenol (43) the hydroxyl group was selectively alkylated with CF2CHBr under basic conditions to


Page 43 of 120 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


provide the ether 44, which was treated with potassium carbonate in DMF to give the morpholino derivative 45. The bromide 45 was converted to its boronate ester 46 under standard conditions. With the boronate 46 in hand, this was then coupled to chloride 47 under Suzuki conditions to give 48. The final compound was obtained in two steps: 1) cyanation of the amine in 48 with CNBr in the presence of sodium hydride at low temperature to give 49, and 2) SEM group removal to give 10. Scheme 3. Synthesis of cyanamide 10.a Br

OH H 2N

O

O

H 2N

a

b

Br 43

F F

F F

F F

HN

Br 44

O

N

N

SEM 47

aReagents

B

O

46

F F O

O N

N O

O d

O

F F

HN

N

HN

c Br 45

F F

Cl

O

N e

N N

N 48

SEM

N

O f

N

O N

N

N 49

SEM

N H

N 10

and conditions: (a) CF2CHBr, KOH, CH3CN:H2O (7:1), 50 oC, 14 h, 98%; (b) K2CO3,

DMF, 100 oC, 14 h, 99%; (c) (BPin)2, PdCl2(dppf), KOAc, dioxane, 100 oC, 95%; (d) 47, PdCl2(dppf), K3PO4, MeTHF:H2O (4:1), 70 oC, 14 h, 27%; (e) BrCN, NaH, DMF, 0 oC, 95%; (f) TFA, DCM, 2.5 h, then NH4OH, CH3CN, 42%. The synthesis of cyanamides 12–15 are described in Scheme 4. Commencing from pyrrolopyrimidine chloride 36, Suzuki coupling with the known boronate 5052 provided



compound 51. The Boc group was removed under acidic conditions to give amine 52, which was then cyanated with cyanogen bromide and potassium carbonate to give racemic 11. The racemate 11 was purified by chiral SFC chromatography to afford the single enantiomers 12 and 13. The absolute stereochemistry of 13 was assigned as (R) based on a single X-ray structure (see Supporting Information). By inference, the stereochemistry of 12 was assigned as (S). The synthesis of compounds 14 and 15 was carried out following a similar route to that described for 11, but starting from pyrrolopyrimidine chloride 38 and boronate 50 or 58. The boronate 58 was readily prepared from 6-bromo-indanone (55) by condensation with methyl amine, followed by reduction to give amine 56. The amine was then protected as the Boc carbamate to give 57, and the bromide subsequently converted to the boronate to give 58. In order to establish the structure of the preferred tautomer (form A or B, Figure 10) of 12, an HSQC and COSY spectra (in DMSO-d6) were taken (see Supporting Information). The HSQC spectra clearly assigned all the key carbons and hydrogens. A COSY spectra showed a 1H-1H correlation between H18 and H19 supporting the tautomeric form A. Moreover, the tautomeric form A is further supported by literature precedent on similar systems.53 N

C

HN N

C

N

H18 H19 COSY

N

N N

N H

form A

N H

N form B

Figure 10. Possible tautomers of cyanamide 12. Data from COSY experiment supports tautomeric form A.


Page 44 of 120

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Scheme 4. Synthesis of cyanamides 12–15.a N

N R

Boc

N H

N H

a

36

d

b N

N N

N H

N R

N R

O

O

c

N H

N

Boc g

N H

N

N H

14: R = H 15: R = CH3

Boc

N

h

Br

Br 56

N

54: R = H 60: R = CH3

N H

Br

aReagents

N H

13

N

53: R = H 59: R = CH3

55

N

12

b N

f

N H

N

HN R

O N

O

N

N

e

38

N H

52: R = H 11: R = CN

c

+

N N

51

Boc

N H

N H

57

N R

O

B

O

50: R = H 58: R = CH3

and conditions: (a) 50, K2CO3, dioxane:H2O (8:2), Pd(PPh3)4, 110 oC, 16 h, 39%, (b)

2N HCl/Ether, 66-91%; (c) BrCN, K2CO3, THF, 40 oC, 16-26%; (d) Chiral SFC (Chiralcel OD3, CO2/MeOH + 0.05%TEA); (e) 50 or 58, K2CO3, dioxane:H2O, Pd(PPh3)4, 110 oC, 1 h, W, 50-54%; (f) MeNH2, Ti(O-iPr)4 then NaBH4, MeOH, 75%; (g) Boc2O, TEA, DCM, 0 oC, 78%; (h) (BPin)2, KOAc, PdCl2(dppf), dioxane, 80 oC, 97%. The synthesis of the chromane-based cyanamides 16–19 was carried out using a route similar to the one described above (Scheme 5), but utilizing a different boronate coupling partner. Suzuki



Page 46 of 120

coupling of pyrrolopyrimidine chloride 36 and boronate 6154 provided the Boc-protected amine 62. The Boc group was removed under acidic conditionsto give amine 63, which was then cyanated with cyanogen bromide and potassium carbonate to give 16. The racemate16 was separated by chiral SFC to provide the enantiomers 17 and 18. The absolute stereochemistry of 17 was assigned as (S) based on: (a) the JAK3 activity of this compound that differentiates it from its opposite enantiomer and resembles the potency difference observed in the indane series; and (b) a crystal structure of 17 bound to JAK3 determined at 1.99 Å resolution (data not shown). Finally, the racemic cyanamide 19 was prepared using a similar route but starting from pyrrolopyrimidine chloride 38. Scheme 5. Synthesis of cyanamides 16–19.a O

36

a

O

HN Boc O

HN R b

HN Boc

N H

O

c

O

N H 63: R = H 16: R = CN

e

17

64

N H

O O

c N

N

N H

18

N H

O

b N

N

N

H 2N

O

N H

N

O

HN Boc

N

N

O

38

N H

N

62 B

O

N

N H

N N

61

d

N

O

N

N N

N H

65

N

N H

19

Reagents and conditions: (a) 61, Cs2CO3, dioxane:H2O, Pd(PPh3)4, 110 oC, 16 h, 52%; (b) 2N HCl/Ether, 2 h, rt, 91-97%; (c) CNBr, K2CO3, THF, 40 oC, 25-27%; (d) chiral SFC (AD,


Page 47 of 120 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


MeOH/CO2 + 0.1% NH4OH); (e) 38, 61, K2CO3, dioxane:H2O, Pd(PPh3)4, 110 oC, 1 h, W, 54%. The preparation of compounds 20 and 21 are described in Scheme 6. For these compounds, the route begins with 6-bromo-2,3-dihydro-1H-inden-1-one (55), which was mono alkylated with methyl iodide in the presence of LDA to give 66. The ketone group was then converted to an amine in two steps: 1) hydroxylamine formation to give 67, and 2) reduction with zinc under acidic conditions to give 68 as a mixture of cis and trans isomers. The amino group in 68 was protected to give carbamate 69; and the bromide was converted to the boronate to give 70. Suzuki coupling of pyrrolopyrimidine chloride 36 with boronate 70 under standard conditions gave 71 (rac-trans) and 73 (rac-cis) after chromatography. Both 71 and 73 were treated with acid to remove the Boc group and provide the amines 72 and 74, respectively. The amines were then treated with cyanogen bromide in the presence of 4-methylmorpholine to afford 20 and 21. Scheme 6. Synthesis of cyanamides 20 and 21.a



55

a

O

X

b

d

Br

BocNH

R

Br

66

67: X = NOH 68: X = NH2

c

BocNH g

69: R = Br 70: R = BPin

e

R N H

h

71 N 36

f

72: R = H 20: R = CN

N N

N H

N

BocNH g

R N H

N H

h

73 N

74: R = H 21: R = CN

N N

aReagents

Page 48 of 120

N H

N

N H

and conditions: (a) 1N LDA, THF, -78 oC, then MeI, 12 % (b) NH2OH·HCl, MeOH,

reflux, 3 h, 100%; (c) Zn, 2N HCl, THF, 3 h, 87%; (d) Boc2O, TEA, DCM, 24 h, 0 oC, 18%; (e) PdCl2(dppf), (BPin)2, KOAc, dioxane, 100 oC, 73%; (f) Pd(PPh3)4, 72, Na2CO3, dioxane, 120 oC, W, 30 min, 62%; (g) TFA, DCM, 0 oC, 100%; (h) CNBr, NMM, DMF, -20 oC, 2 h, 31% The synthesis of compounds 22–24 is described in Scheme 7. The synthesis of the boronate 79 starts from indenone 55. Reaction of this ketone with methyl magnesium bromide provided cleanly the tertiary racemic alcohol 75. The alcohol 75 was converted to the azide 76, following a similar procedure to that described by Kumar et. al.55 (warning: hydrazoic acid), which was then reduced using trimethylphosphine to give amine 77. After protection of the amine as the Boc derivative to afford 78, the bromide was converted to boronate 79. Suzuki coupling of 36


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with boronate 79 provided the Boc-protected amine 80. Removal of the protecting group under acidic conditions gave amine 81, which was readily converted to cyanamide 22. The racemic Boc-protected amine 80 was separated by chiral SFC to give the enantiomeric pair 82 and 83. Both 82 and 83 were separately treated with acid to provide the free amines, which were then cyanated to give 23 and 24. The absolute stereochemistry of 23 was assigned as (S) based on the crystal structure of this compound bound to JAK3. By inference, the absolute stereochemistry of 24 was assigned as (R). Scheme 7. Synthesis of cyanamides 22–24.a

55

a

HO

R

b

d

f N

75

c

N

R

Br

Br

76: R = N3 77: R = NH2

e

78: R = Br 79: R = BPin

g h

N H

80: R = Boc 81: R = H 22: R = CN

H N

H N

R

R 80

R

Boc HN

H N

i

+ N

N N H 82: R = Boc 23: R = CN

g, h

aReagents

N H 83: R = Boc 24: R = CN N

N

g, h

and conditions: (a) 3M MeMgBr, THF, 0 oC, 2 h, 71% (b) NaN3, TFA, 0 oC to 30 oC,

3 h, 96%; (c) PMe3, THF:H2O (10:1), 30 oC, 24 h, 41%; (d) Boc2O, TEA, DCM, 24 h, 30 oC, 52%; (e) PdCl2(dppf), (BPin)2, KOAc, dioxane, 100 oC, 100%; (f) 36, 79, Pd(PPh3)4, K2CO3, dioxane:H2O, 100 oC, 14 h, 47%; (g) 4N HCl, dioxane, 25 oC, 34%; (h) CNBr, NMM, DMF, -20 oC,

2 h, 16-37%; (i) Chiral SFC (AD, CO2/EtOH + 0.1% NH4OH), 95%.



The synthesis of compounds 25 and 26 are described in Scheme 8. A key component of the synthesis of these analogs was the construction of ketone 92 with control of the absolute stereochemistry. This was accomplished using Ellman sulfinimine chemistry employing the chiral amine derivative tert-butanesulfinamide.56 Thus, 3-bromobenzaldehyde (84) was condensed with (R)-2-methylpropane-2-sulfinamide to provide imine 85. The imine 85 was then reacted with tert-butyl bromoacetate/SmI2 to give 86.57,58 Treatment of 86 under acidic conditions provided the amino acid 87, which was protected as the trifluoroacetamide derivative 88. The carboxylic acid 88 was converted to the acid chloride 89, which was subjected to Friedel-Crafts acylation to give 90 and 91 (~3:1 ratio of regioiosmers). The regiochemistry was confirmed by NMR (Supporting Information). The desired regioisomer bromide 90 was converted to boronate 92, followed by Suzuki coupling with chloride 36 to provide ketone 93. The ketone was then reduced with NaBH4 to give 94 as a mixture of cis and trans alcohols, which were separated by RP-HPLC to afford 95 (cis) and 96 (trans). The stereochemistry of the compounds was confirmed by NOE. The trifluroacetamide group in 95 was removed to give amine 97, which in turn was cyanated to give 25. Compound 96 was taken through a similar sequence as 95 to give cyanamide 26. Scheme 8. Synthesis of cyanamides 25 and 26.a


Page 50 of 120

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O S

O S

N

OH

NH

b

a

O

O

c O

Br

Br

85

Br

86

87: R = H 88: R = COCF3

d

O O F F

e

f

F

89

Br

36

O

O F

F F

N H

91

OH R

N H

R

N H

N N H

N H

N N 94

93

N H

N N

k l

aReagents

OH

j

N N

Br

N H

OH

i

h

O

O

R 90: R = Br 92: R = Bpin O

F

F F F

N H

g

F F

O

O

F F F

Cl N H

N H

O

Br

84

R

N H

95: R = COCF3 97: R = H 25: R = CN

N k l

N H

96: R = COCF3 98: R = H 26: R = CN

and conditions: (a) (R)-2-methylpropane-2-sulfinamide, p-TsOH, MgSO4, CH2Cl2, rt,

14 h, 68%; (b) 0.1M SmI2, t-BuO2CCH2Br, THF, -70 oC, 39%; (c) 4N HCl/dioxane, 14 h, rt, 100%; (d) TFAA, 2 h, rt, 67%; (e) SOCl2, 1 h, reflux, 98%; (f) AlCl3, CH2Cl2, reflux, 1 h, 90, 58%, 91, 19%; (g) PdCl2(dppf), KOAc, dioxane, 100 oC, 5 h, 100%; (h) 92, PdCl2(dppf), KF, dioxane:H2O (4:1), 90 oC, 14 h, 53%; (i) NaBH4, MeOH, 0 oC, 2 h, 55%; %; (j) RP-HPLC CH3CN:H2O + 0.05% NH4OH; (k) Na2CO3, MeOH:H2O (3:1), reflux, 14 h, 81%; (l) CNBr, NMM, CH2Cl2, -30 oC, 1.5 h, 36-47%. The synthesis of compound 27 is described in Scheme 9. The amine of the known racemic trans (1R,2R)-1-amino-6-bromo-2,3-dihydro-1H-inden-2-ol (99) was initially protected as the Boc-



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carbamate to give 100, followed by replacement of the bromide to give boronate 101. Suzuki coupling of 101 to pyrrolopyrimidine chloride 36, followed by chiral SFC provided the enantiomers 102 (Peak 2, R, R) and 104 (Peak 1, S,S). The absolute stereochemistry of 104 was determined by obtaining an X-ray crystal structure of the HCl salt of 105 (Supporting Information), after Boc deprotection of 104. This structure showed that 105 possessed 1S,2Sstereochemistry. By inference, the stereochemistry of 102 was assigned as 1R,2R. The Bocamine 102 was converted to the amine 103 and then cyanated to give enantiomerically pure 27. Scheme 9. Synthesis of cyanamide 23.a HO R

HO

HO Boc

a

H 2N

HO R

N H

c

N H

N N H

N H e

+ N

R

Br 99

aReagents

b

N

N H 102: R = Boc 103: R = H N

100: R = Br 101: R = BPin

HO

d

N N H 104: R = Boc 105: R = H

N d

N

N H

27

and conditions: (a) Boc2O, THF, 0 oC, 1 h, 38%; (b) PdCl2(dppf), KOAc, (BPin)2,

dioxane, 3 h, 100 oC, 85%; (c) 36, PdCl2(dppf), K2CO3, 2.5 h, 100 oC, 48% then SFC, 102, peak 2 carried forward; (d) 4N HCl/dioxane, 1.5 h, (e) 103, CNBr, NMM, DMF, 0 oC, 35% in two steps. The synthesis of sulfonamides 28–33 are described in Scheme 10. The synthesis of these analogs departs from the chiral ketone 93 which was converted to hydroxylamine derivative 106. Reduction with zinc under acidic conditions afforded the primary amines 107 and 108 after separation by chromatography (~4:1 cis/trans ratio as determined by NMR). The cis isomer 107 was then taken forward through a three step sequence in a parallel chemistry format: 1)


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sulfonamide formation with selected sulfonyl chlorides; 2) trifluoroacetamide removal and finally 3) cyanation to give 28–33 as single enantiomers. Scheme 10. Synthesis of sulfonamides 28–33.a X

O F F

F F

N H

F

NH2

O

b

+

N N H

c

F

N H

N

d

N 109a-f

N H

N H

108 H O N S R O

H 2N

H O N S R O

N e

N

N

aReagents

N H

107 H O N S R O

O 107

N N

93: X = O 106: X = NOH

F F

F

N H

N N

a

F F

N H

F

NH2

O

N H

N N

N H

110a-f

N

N H

28-33

and conditions: (a) NH2OH·HCl, EtOH, 1 h, reflux, 100%; (b) Zn, 4N HCl, THF, rt, 1

h, 107: 23%, 108: 5%; (c) RSO2Cl, DIPEA, DMF, 0 oC, 12 hrs, 14-75%; (d) K2CO3, EtOH:H2O (7:1), 100 oC, 8 h, 41-88%; (e) CNBr, NMM, DMF, -30 oC, 30 min, 19-38%. The synthesis of cyanamide 34 is described in Scheme 11. The preparation begins from the known (R)-6-bromo-indan-1-ol (111)59 to obtain optically active boronate 115 via four steps. It is important to note that compound 115 is the (S)-enantiomer of boronate 50 (Scheme 4). Thus, alcohol 111 was reacted with DPPA and DBU to provide azide 112, which was then reduced with tin chloride to give amine 113. Protection of the amine afforded 114 (see Supporting Information for single X-ray), which was then converted to the optically pure boronate 115.



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Suzuki coupling of pyrrolopyrimidine chloride 116, prepared as previously described,60 with boronate 115 gave 117. Finally, the Boc group was removed under acidic conditions to give amine 118, which was then cyanated to give cyanamide 34. Scheme 11. Synthesis of cyanamide 34.a

Cl R

N Boc HO

R

a

c

N H

e

N

N H

N 116 H N

Br 111

aReagents

R

Br b

112: R = N3 d 113: R = NH2

114: R = Br 115: R = Bpin

N H 117: R = Boc 118: R = H N

f g

34: R = CN

and conditions: (a) DPPA, DBU, PhMe, 4 h, 0 oC, 70%; (b) SnCl2, MeOH, 0 oC, 5 h,

78%; (c) Boc2O, Et3N, CH2Cl2, 2 h, rt, 63%; (d) (BPin)2, PdCl2(dppf), KOAc, dioxane, 3 h, 80 oC,

90%; (e) 116, PdCl2(dppf), K2CO3, dioxane:H2O (4:1), 36%; (f) TFA, CH2Cl2, 2 h, 0 oC,

78%; (g) CNBr, NMM, DMF, -30 oC, 1 h, 46%. The synthesis of compound 35 is described in Scheme 12. The starting 4-chloro-5-iodo-7-((2(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidine (119) was prepared as previously described.61 Coupling of 119 with boronic acid 120 under Suzuki conditions gave 121. The SEM protecting group was removed under acidic conditions to give 122, which was coupled to boronate 115 to give 123. Removal of the Boc-group with acid provided amine 124, which was cyanated to give 35. It is important to note that no atropoisomerism was observed for 34 and 35. The 1H NMR and HPLC of each of these compounds showed no doubling up of signals (see Supporting Information).


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Scheme 12. Synthesis of cyanamide 35.a HO

HO Cl

120

I

HO

N

B

Cl OH

a N 119

N N

N SEM

aReagents

Boc

b

N R

121: R = SEM 122: R = H

c

OH

R

N H

OH N H

d

N N 123

N N H 124: R = H 35: R = CN N

N H e

and conditions: (a) 119, PdCl2(dppf), K2CO3, dioxane:H2O (5:1), 3 h, 100%; (b) TFA,

CH2Cl2 then K2CO3, MeOH, rt, 89%; (c) 115, PdCl2(dppf), K2CO3, dioxane:H2O (5:1), 24 h, 80 oC,

43%; (d) 4N HCl/EtOAc, 2 h, rt, 11%; (e) CNBr, NMM, DMF, 0.5 h, -20 oC, 41%.

Conclusions The discovery of selective JAK3 inhibitors has attracted attention from both academic and industrial research groups for several decades due to the key role that this kinase plays in the immune system. Recently, covalent inhibitors that target the JAK3 residue Cys909, a unique residue within the JAK family, have resulted in compounds with high JAK3 selectivity, including 6, a JAK3-specific inhibitor currently ongoing human clinical trials. Further interest in the discovery of selective JAK3 inhibitors led us to design novel covalent inhibitors that engage Cys909 through a cyanamide moiety, a structurally and mechanistically differentiated electrophile from the acrylamide moiety in 6. Our results with amino-indane based cyanamides showed that modulation of two key parameters of covalent inhibitors, Ki and kinact, could be achieved through structural modifications leading to enhanced potency and selectivity. This was demonstrated in the progression from the unoptimized cyanamide 12 (JAK3 IC50 = 256 nM; 2fold selectivity vs other JAKs) to the potent and highly JAK3 selective 32 (JAK3 IC50 = 11 nM; 246-fold selectivity vs other JAKs). Cyanamide 32 also showed high kinome selectivity in a



panel of 40 kinases (1/40 kinases, >50% inhibition) and in a panel of ten kinases that contain a Cys residue at the equivalent position of Cys909 in JAK3 (>300-fold selectivity for JAK3 vs all kinases in panel). Furthermore, evaluation of 32 in IL-15-stimulated PBMC assays demonstrated that selective inhibition of JAK3 is sufficient to drive cellular activity in pathways signaled through the JAK1/JAK3 heterodimer (IL-15 PBMC IC50 = 331 nM), a result in agreement with previous reports from this group. Even with the interesting profile demonstrated by 32 and other analogs, the weak potency observed with the indane-based cyanamides in the IL-15 human whole blood assay (e.g. 32, IL-15 HWB IC50 > 10000 nM), and more importantly, the lack of understanding of the factors that contribute to the poor translation between cell-based PBMC assays and the assay conducted in human whole blood were recognized as significant liabilities leading us to deprioritize this series for further progression. Gaining further understanding of the pharmacokinetic properties of cyanamide-based JAK3 covalent inhibitors was an important component of our work. To this end, the pharmacokinetic properties of 12 were evaluated in rat. An important finding was the significant contribution to the clearance of 12 from extrahepatic processes, including GSH/GST-mediated clearance. This data suggested cyanamide-based covalent inhibitors would likely face similar challenges as do other covalent JAK3 inhibitors based on different CRGs (e.g., acrylamides) to achieve favorable pharmacokinetic properties suitable for oral dosing in humans. One of the more important results from this work was the identification of an approach to decrease GSH/GST-mediated clearance. It was demonstrated with 23 that steric hindrance around the electrophile significantly decreased GST-mediated clearance (GSH reactivity: t1/2 > 120 min; human and rat blood stability: t1/2 > 360 min). In the case of the amino-indane based cyanamide series, this modification led to loss of covalent interaction, but our group successfully


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applied this approach in the aliphatic acrylamide series leading to the identification of 6 as previously reported. Experimental Section General. All chemicals, reagents, and solvents were purchased from commercial sources when available and used without further purification. Anhydrous solvents (dioxane, dichloromethane (CH2Cl2), toluene, acetonitrile) were purchased from Aldrich (SureSeal) and used in reactions without further purification. NADPH, GSH, and magnesium chloride utilized in metabolism studies were purchased from Sigma-Aldrich (St. Louis, MO). Reactions were magnetically stirred and monitored by thin layer chromatography (TLC) using precoated Whatman 250 m silica gel plates and visualized by UV light and by GC/MS. Silica gel chromatography was performed utilizing ACS grade solvents and ISCO or Biotage purification systems and prepackaged columns. Yields refer to purified compounds. 1H NMR spectra (400 MHz field strength) and 13C NMR spectra (101 MHz field strength) were obtained on a Bruker AV III spectrometer equipped with a BBFO probe. Alternatively, 1H NMR spectra (500 MHz field strength) and 13C NMR spectra (125 MHz field strength) were obtained on a Bruker 500 Avance III HD using a 5 mm Prodigy BBO cryoprobe. Chemical shifts were referenced to the residual 1H

solvent signals (CDCl3,  7.27), (DMSO-d6,  2.50) and solvent 13C signals (CDCl3,  77.00),

(DMSO-d6,  39.51). Signals are listed as follows: chemical shift in ppm (multiplicity identified as s = singlet, br = broad, d = doublet, t = triplet, q = quartet, m = multiplet; coupling constants in Hz; integration). High resolution mass spectrometry (HRMS) was performed via atmospheric pressure chemical ionization (APCI) or electron scatter ionization (ESI) sources. Purity of final compounds was determined by HPLC or LCMS methods. All final compounds possess a purity of ≥95%, unless noted.



Synthesis of N-(3-(5-(2-methoxyethyl)-7H-pyrrolo[2,3-d]pyrimidin-4yl)phenyl)acrylamide (7). Step 1: 5-Bromo-4-chloro-7H-pyrrolo[2,3-d]pyrimidine (36). Prepared analogous to as described in WO201385802.62 To a suspension of 4-chloro-7Hpyrrolo[2,3-d]pyrimidine 36 (13.0 g, 84.65 mmol) in DCM (200 mL) was added NBS (18.08 g, 101.58 mmol). The reaction mixture was stirred at room temperature overnight. The suspension was filtered. The filter cake was suspended in saturated aq. Na2SO3 (200 mL) and stirred at room temperature for 30 minutes. The suspension was filtered again and the filter cake was washed with hot-water (200 mL) and MTBE (200 mL), dried in vacuum to afford 37 (10.0 g, 51%) as white solid. 1H NMR (300 MHz, DMSO-d6) δ 12.9 (bs, 1H), 8.6 (s, 1H), 7.9 (s, 1H); LC/MS (M + H) expected: 231.93, observed: 231.9. Step 2: 4-Chloro-5-(2-methoxyethyl)-7H-pyrrolo[2,3-d]pyrimidine (38). Prepared analogous to as described in WO2015083028.63 To a suspension of 37 (4.0 g, 17.20 mmol) in THF (200 mL) at -60 oC under N2 was added nBuLi (14.11 g, 35.27 mmol) dropwise over a period of 30 minutes. The reaction mixture was stirred at the same temperature for 30 minutes, then Br(CH2)2OMe (6.55 mL, 68.82 mmol) was added dropwise. The reaction was quenched with aq. NH4Cl (50 mL), extracted with EtOAc (200 mL×3). The organic phase was concentrated to dryness and the residue was purified by column chromatography (PE: EtOAc from 100:1 to 30:1) to afford compound 38 (400 mg, 15%) as white solid. 1H NMR (300 MHz, DMSO- d6) δ 12.3 (bs, 1H), 8.5 (s, 1H), 7.5 (s, 1H), 3.6 (t, 2H), 3.3 (s, 3H), 3.1 (t, 2H); LC/MS (M+H) expected: 212.06, observed: 212.18. Step 3: 3-(5-(2-Methoxyethyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)aniline (39). To a solution of 38 (150 mg, 0.70 mmol) and (3-aminophenyl)boronic acid (107 mg, 0.77 mmol) in dioxane/water = 4:1 (20 mL) were added K3PO4 (301 mg, 1.41 mmol) and PdCl2(dppf) (26 mg, 0.03 mmol). The


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reaction was stirred at 90 oC for 2.5 hours. After the reaction was complete, the mixture was filtered and the filtrate was concentrated to dryness. The crude product was purified by column chromatography (DCM: MeOH from 10:1 to 20:1) to afford 39 (140 mg, 74%) as a yellow oil. 1H

NMR (400 MHz, DMSO-d6)  11.92 (br. s., 1 H), 8.66 (s, 1 H), 7.33 (d, J = 1.95 Hz, 1 H),

7.11 (t, J = 7.71 Hz, 1 H), 6.57–6.79 (m, 3 H), 3.07 (t, J = 7.12 Hz, 2 H), 2.98 (s, 3 H), 2.69 (t, J = 7.22 Hz, 2 H); LC/MS (M+H) expected: 269.14, observed: 269.2 Step 4. N-(3-(5-(2-Methoxyethyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)phenyl)acrylamide (7).To a solution of 39 (130 mg, 0.48 mmol) in DMF (15 mL) was added acryloyl chloride (0.04 mL, 0.48 mmol) and DIPEA (0.43 mL, 2.42 mmol) at 0 oC. The reaction was stirred at 0 oC for 4 hours. The reaction was quenched with CH3OH (15 mL) and evaporated to dryness. The residue was dissolved in water (15 mL) and then extracted with EtOAc (3  20 mL). The combined organic layers were dried over Na2SO4 and concentrated. The crude product was purified by prep-HPLC to afford 7 (32.9 mg, 21.1%) as white solid. 1H NMR (400 MHz, DMSO- d6) δ 12.10 (s, 1H), 10.33 (s, 1H), 8.76 (s, 1H), 8.03 (s, 1H), 7.77 (d, J = 8 Hz, 1H), 7.51–7.33 (m, 2H), 7.35 (d, J = 8 Hz, 1H), 6.44–6.41 (m, 1H), 6.29–6.24 (m, 1H), 5.78–5.76 (m, 1H), 3.09–3.05 (m, 2H), 2.97 (s, 3H), 2.74–2.71 (m, 2H); LC/MS (M+H) expected: 323.14, observed: 323.1. HRMS (ESI+) m/z: calculated for C18H18N4O2 [M + H]+ 323.1503; observed 323.1500. Synthesis of 1-(6-(5-(2-methoxyethyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-2,3-dihydro4H-benzo[b][1,4]oxazin-4-yl)prop-2-en-1-one (8) and 6-(5-(2-methoxyethyl)-7Hpyrrolo[2,3-d]pyrimidin-4-yl)-2,3-dihydro-4H-benzo[b][1,4]oxazine-4-carbonitrile (9). Step 1: 6-(5-(2-Methoxyethyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-2H-benzo[b][1,4]oxazin-3(4H)-one (41). To a solution of 38 (0.5 g, 2.36 mmol) in dioxane was added 6-(4,4,5,5-tetramethyl-1,3,2dioxaborolan-2-yl)-2H-benzo[b][1,4]oxazin-3(4H)-one 4048 (0.78 g, 2.83 mmol), Cs2CO3 (2.3 g,



7.08 mmol) and water (2 mL). The mixture was purged with nitrogen for 20 min, then Pd(PPh3)4 (0.14 g, 0.118 mmol) was added and the reaction again purged with nitrogen for 10 min. The reaction was heated to 100 oC in a microwave for 30 min. The reaction mixture was poured into ice water and the aqueous mixture extracted with ethyl acetate. The organic extract was collected and washed with brine solution, dried over Na2SO4 and concentrated. The crude compound was purified by washing with CH3CN, DCM and pentane to give 41 as an off white solid (0.3 g, 38%). 1H NMR (300 MHz, DMSO-d6) δ 12. 1 (br s, 1H), 10.9 (s, 1H), 8.7 (s, 1H), 7.3–7.1 (m, 3 H), 4.7 (s, 2H), 3.2 (t, 2H), 3.1 (s, 3H), 2.7 (t, 2H); LC/MS (M+H) expected: 325.12, observed: 325.2; Step 2: 6-(5-(2-Methoxyethyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-3,4-dihydro-2Hbenzo[b][1,4]oxazine (42). To a suspension of LAH (0.05 g, 1.24 mmol) in THF (10 mL) was added 41 (0.1 g, 0.31 mmol) portion wise. After the addition was complete, the reaction was heated to reflux overnight. The reaction mixture was quenched with sat. Na2SO4 solution and filtered through Celite. The solid was washed with EtOAc. The organic layer was washed with water, brine and dried (Na2SO4). The solvent was removed and the crude material purified by column chromatography (silica, 10% MeOH/DCM) to give 42 (0.024 g, 25%) as off white solid. 1H

NMR (300 MHz, DMSO-d6) δ 11.9 (bs, 1H), 8.7 (s, 1H), 7.4 (s, 1H), 6.9 (s, 1H), 6.8–6.7 (m,

2H), 5.9 (s, 1H), 4.23–4.21 (m, 2H), 3.33–3.31 (m, 2H), 3.15 (t, 2H), 3.1 (s, 3H), 2.8 (t, 2H); LC/MS (M+H) expected: 311.14 , observed: 311.2. Step 3: 1-(6-(5-(2-Methoxyethyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-2,3-dihydro-4Hbenzo[b][1,4]oxazin-4-yl)prop-2-en-1-one (8). To a solution of 42 (0.1 g, 0.32 mmol) in THF (10 mL) was added acryloyl chloride (0.03 g, 0.32 mmol) at 0 oC and stirred for 10 min. The reaction mixture was quenched with MeOH and NaHCO3 solution. The volatiles were removed


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and the crude mixture was extracted with EtOAc. The organic layer was dried over Na2SO4 and concentrated under reduced pressure. The crude was purified by preparative TLC to provide 8 (0.04 g, 34%) as an off white solid. 1H NMR (300 MHz, DMSO-d6) δ 12.1 (bs, 1H), 8.75 (s, 1H), 8.0–7.8 (bs, 1H), 7.42–7.35 (m, 2H), 7.15–7.13 (m, 1H), 6.91–6.79 (m, 1H), 6.31–6.21 (m, 1H), 5.84–5.76 (m, 1H), 4.41 (t, 2H), 4.05 (t, 2H), 3.12 (t, 2H), 3.09 (s, 3H), 2.78 (t, 2H); LC/MS (M+H) expected: 365.15, observed: 365.30. HRMS (ESI) m/z: calculated for C20H20N4O3 [M + H]+ 365.1608; observed: m/z 365.1607. Step 4: 6-(5-(2-Methoxyethyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-2,3-dihydro-4Hbenzo[b][1,4]oxazine-4-carbonitrile (9). To a mixture of 42 (50 mg, 0.16 mmol) in DMF (3 mL) at 0 oC was added BrCN (3M in DCM, 0.16 mL). The reaction was stirred at 0 oC for 90 min and then quenched with NH4Cl (10% aq solution). The aqueous mixture was extracted (3EtOAc). The organic layer was washed with brine, dried and solvent removed to give crude product, which was purified by chromatography (silica, EtOAc/Hep) to give pure 9 (26 mg, 49%). 1H NMR (300 MHz, DMSO-d6) δ 12.1 (bs, 1H), 8.74 (s, 1H), 7.45 (s, 1H), 7.33–7.30 (m, 2H), 7.11–7.08 (m, 1H), 4.37 (t, 2H), 3.99 (t, 2H), 3.18 (t, 2H), 3.07 (s, 3H), 2.77 (t, 2H); LC/MS (M+H) expected: 336.14; observed: 336.2. Synthesis of 2,2-difluoro-6-(5-(2-methoxyethyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)2,3-dihydro-4H-benzo[b][1,4]oxazine-4-carbonitrile (10). Step 1: 5-Bromo-2-(2-bromo-1,1difluoroethoxy)aniline (44). To a solution of phenol 43 (180 mg, 0.95 mmol) in H2O/CH3CN (0.2 mL/ 1.5 mL) was added KOH (76 mg, 1.05 mmol, 1.2 eq). The reaction was stirred at room temperature for 2 min and then a solution of 2-bromo-1,1-difluoroethene (1.5 mL in CH3CN, 1.05 mmol) was added. The reaction was warmed to 50 oC and stirred overnight. After completion, the reaction was filtered through Na2SO4 and the desired product 44 isolated (300



mg, 90%). 1H NMR (400 MHz, DMSO-d6)  6.84–7.03 (m, 2 H), 6.64 (dd, J = 8.8, 2.2 Hz, 1 H), 5.49 (s, 2 H), 4.29 (t, J = 10.34 Hz, 2 H); 19F NMR (377 MHz, DMSO-d6)  ppm -72.01 (s, 2 F); LC/MS (M+H) expected: 329.89; observed: 331.8. Step 2: 6-Bromo-2,2-difluoro-3,4-dihydro-2H-benzo[b][1,4]oxazine (45). To a solution of 44 (140 mg, 0.42 mmol) in DMF (3 mL) was added K2CO3 (176 mg, 1.3 mmol). The reaction mixture was heated to 100 oC and stirred overnight. The solvent was removed and the residue dissolved in ethyl acetate. The mixture was washed with water, brine and dried (Na2SO4) to give after removal of solvent the crude product, which was purified by chromatography (silica, EtOAc/Hep) to give 45 (0.053 g, 67%). GC/MS observed: m/z 249, 251, expected 249, 251. 1H NMR (400 MHz, CDCl3-d)  6.89–7.00 (m, 2 H), 6.79–6.88 (m, 1 H), 3.97 (br. s., 1 H), 3.57 (td, J = 6.16, 3.52 Hz, 2 H); 19F NMR (377 MHz, CDCl3-d)  -74.00 (s, 2 F). Step 3: 2,2-Difluoro-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,4-dihydro-2Hbenzo[b][1,4]oxazine (46). To a round bottom flask containing 45 (390 mg, 1.56 mmol) in dioxane (7 mL) was added (BPin)2 ( 440 mg, 1.73 mmol), potassium acetate (459 mg, 4.68 mmol) and PdCl2(dppf) (60 mg, 0.078 mmol). The reaction mixture was heated to 100 oC overnight and then diluted with ethyl acetate. The mixture was washed with water, brine and dried (Na2SO4) to give the crude product, which was purified by chromatography (silica, EtOAc/Hep) to give product 46 (440 mg, 95%). 1H NMR (400 MHz, CDCl3)  7.10–7.38 (m, 1 H), 6.96 (d, J = 8.03 Hz, 1 H), 3.87 (br. s., 1 H), 3.53 (br. s., 2 H), 1.16–1.46 (m, 12 H); 19F NMR (376 MHz, CDCl3)  -73.55 (br. s., 2 F); LC/MS (M+H) expected: 298, observed: 298. Step 4: 2,2-Difluoro-6-(5-(2-methoxyethyl)-7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3d]pyrimidin-4-yl)-3,4-dihydro-2H-benzo[b][1,4]oxazine (48). To a vial containing 46 (229 mg, 0.771 mmol) and 47 (264 mg, 0.771 mmol) in MeTHF:H2O (8 mL:2 mL) was added K3PO4 (574


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mg, 2.31 mmol) and PdCl2(dppf) (37.9 mg, 0.046 mmol). The mixture was purged with nitrogen and then heated to 70 oC overnight. The mixture was cooled to room temperature and diluted with water. The aqueous mixture was separated and the organic layer washed with brine, dried (Na2SO4) and the solvent removed to give crude material, which after chromatography (silica, EtOAc/Hep) provided 48 (100 mg, 27%). 1H NMR (400 MHz, CDCl3)  8.90 (s, 1 H), 7.29 (d, J = 6.16 Hz, 1 H), 7.02–7.19 (m, 3 H), 5.68 (s, 3 H), 4.15–4.24 (m, 1 H), 3.51–3.67 (m, 4 H), 3.33 (t, J = 6.82 Hz, 2 H), 3.23 (s, 3 H), 2.72–2.89 (m, 2 H), 0.89–1.02 (m, 2 H), -0.06 (s, 9 H); 19F NMR (377 MHz, CDCl3-d)  -73.67 (s, 2 F); LC/MS (M+H) expected: 477.2, observed: 477.1. Step 5: 2,2-Difluoro-6-(5-(2-methoxyethyl)-7-((2-(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3d]pyrimidin-4-yl)-2,3-dihydro-4H-benzo[b][1,4]oxazine-4-carbonitrile (49). To a solution of 48 (49 mg, 0.10 mmol) in DMF (3 mL) at 0 oC was added NaH. After 15 min, CNBr (3M soln in DCM, 0.204 mmol) was added dropwise and the reaction stirred for 90 min. The reaction was quenched by the addition of NH4Cl (10% solution) and the subsequent aqueous mixture extracted with EtOAc (3  10 mL). The organic layers were combined, washed with brine, dried (Na2SO4) and concentrated to give 49 (49 mg, 94%). 1H NMR (400 MHz, CDCl3)  8.93 (s, 1 H), 7.63 (d, J = 1.5 Hz, 1 H), 7.49 (dd, J = 8.31, 1.5 Hz, 1 H), 7.14–7.38 (m, 2 H), 5.70 (s, 2 H), 3.89–4.23 (m, 2 H), 3.53–3.68 (m, 2 H), 3.37 (t, J = 6.85 Hz, 2 H), 3.25 (s, 3 H), 2.79 (t, J =6.60 Hz, 2 H), 1.28 (t, J = 7.09 Hz, 2 H), 0.82–1.04 (m, 3 H), -0.06 (s, 9 H); 19F NMR (377 MHz, CDCl3)  -74.62 (s, 2 F); LC/MS (M+H) expected: 502.21, observed: 501.8. Step 6: 2,2-Difluoro-6-(5-(2-methoxyethyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-2,3-dihydro-4Hbenzo[b][1,4]oxazine-4-carbonitrile (10). To a solution of 49 (49 mg, 0.098 mmol) in DCM (4 mL) at room temperature was added TFA (2 mL). The reaction was stirred for 2.5 h and then the solvent removed to give the crude product, which was then dissolved into CH3CN (3 mL) and



NH4OH (2 mL) added. The mixture was stirred for 50 minutes and the solvent removed to give the crude product. The residue was dissolved in dimethyl sulfoxide (1 mL) and purified by reversed-phase HPLC: column: Waters Sunfire C18, 19  100 mm, 5 m; mobile phase A: 0.05% TFA in water (v/v); mobile phase B: 0.05% TFA in acetonitrile (v/v); gradient: 95.0% H20/5.0% acetonitrile linear to 0% H2O/100% acetonitrile in 8.5 min, hold at 0% H20/100% acetonitrile to 10.0 min. Flow: 25 mL/min to give 10 (15 mg, 42%). 1H NMR (400 MHz, DMSO-d6)  12.46 (br. s., 1 H), 8.88 (s, 1 H), 7.38–7.63 (m, 4 H), 4.66 (t, J = 6.60 Hz, 2 H), 3.18 (t, J = 7.09 Hz, 2 H), 3.06 (s, 3 H), 2.71 (t, J = 7.09 Hz, 2 H); 19F NMR (377 MHz, DMSOd6)  -73.72 (s, 1 F) -74.57 (s, 1 F); LC/MS (M+H) expected: 372.13, observed: 372.0. HRMS (ESI) m/z: calculated for C18H15F2N5O2 [M + H]+ 372.1267; observed 372.1262. Synthesis of N-(6-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-2,3-dihydro-1H-inden-1yl)cyanamide (11), (S)-N-(6-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-2,3-dihydro-1H-inden-1yl)cyanamide (12) and (R)-N-(6-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-2,3-dihydro-1H-inden-1yl)cyanamide (13). Step 1: Tert-butyl (6-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-2,3-dihydro-1Hinden-1-yl)carbamate (51). To a solution of 36 (4 g, 26 mmol) in dioxane/water (8:2) (40 mL), was added 50 (1.69g, 26 mmol prepared as previously described)52 and K2CO3 (4.48 g, 78 mmol) at room temperature. The mixture was degassed with nitrogen for 20 min, then Pd(PPh3)4 (160 mg, 3.4 mmol) was added and nitrogen bubbling was continued for another 10 minutes. The reaction mixture was heated to 110 oC for 16 h. The reaction mixture was cooled to room temperature and diluted with water. The aqueous solution was extracted with ethyl acetate, washed with brine solution, dried over anhydrous Na2SO4 and concentrated under reduced pressure to provide crude product. The crude compound was purified by column chromatography (silica, EtOAc/Hep 10–80%) to give 51 (1.5 g, 39% yield). 1H NMR (DMSO-d6)  12.2 (s, 1 H),


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8.8 (s, 1H), 8.1–8.0 (m, 2 H), 7.8–7.3 (m, 3 H), 6.8 (s, 1 H), 5.2–5.0 (m, 1H), 3.1–2.8 (m, 2 H), 2.6–2.4 (m, 1 H), 1.8–2.0 (m, 1 H), 1.5 (s, 9 H); LC/MS (M+H) expected: 351.18, observed: 351.1. Step 2: 6-(7H-Pyrrolo[2,3-d]pyrimidin-4-yl)-2,3-dihydro-1H-inden-1-amine (52). To a flask containing 51 (1.5 g, 4.28 mmol) was added 2M HCl in diethyl ether (50 mL). After stirring at room temperature for 2h, the solvent was decanted and the solid washed with diethyl ether (3) to remove excess HCl. The solvent was evaporated under reduced pressure to give 52 as a grey solid (0.6 g as HCl salt, 66% crude yield). 1H NMR (DMSO-d6)  12.9 (bs, 1 H), 8.9 (s, 1H), 8.7 (bs, 3 H), 8.4 (s, 1 H), 8.2 (d, 1 H), 7.9 (s, 1 H), 7.6 (d, 1 H), 7.2 (s, 1 H), 4.8 (bs, 1 H), 3.3–2.8 (m, 2 H), 2.6–2.4 (m, 1 H), 2.2–2.0 (m, 1 H); LC/MS (M+H) expected: 251.12, observed: 251.1. Step 3: N-(6-(7H-Pyrrolo[2,3-d]pyrimidin-4-yl)-2,3-dihydro-1H-inden-1-yl)cyanamide (11). To a solution of 52 (0.3 g, 1.19 mmol) in THF (10 mL) was added CNBr (0.13g, 1.19 mmol) and K2CO3 (0.24 g, 1.79 mmol). The mixture was heated to 40 oC for 1 h and then poured into ice water, extracted with ethyl acetate and washed with brine. The organic layer was collected, dried (Na2SO4) and solvent removed to give the crude compound, which was purified by column and preparative TLC to give 11 (0.055 g, 16%). 1H NMR (DMSO-d6)  12.3 (bs, 1 H), 8.9 (s, 1 H), 8.2 (s, 1 H), 8.1 (d, 1 H), 7.8-7.6 (m, 1 H), 7.5–7.4 (m, 2 H), 6.85–6.90 (m, 1 H), 4.8–4.7 (m, 1 H), 3.1–3.0 (m, 1 H), 3.0–2.8 (m, 1 H), 2.6–2.5 (m, 1 H), 2.0–1.9 (m, 1 H); LC/MS (M+H) expected: 276.12, observed: 276.0. HRMS (ESI) m/z: calculated for C16H13N5 [M + H]+ 276.1244; observed 276.1243. Step 4: (S)-N-(6-(7H-Pyrrolo[2,3-d]pyrimidin-4-yl)-2,3-dihydro-1H-inden-1-yl)cyanamide (12) and (R)-N-(6-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-2,3-dihydro-1H-inden-1-yl)cyanamide(13). 9 (300 mg) was purified by chiral SFC (Chiralcel OD-3, CO2/MeOH + 0.05% TEA) to give two



peaks, 12 (peak 1, 128 mg) and 13 (peak 2, 120 mg). Data for 12: peak 1, Rt = 3.35 min; 1H NMR (DMSO-d6)  12.3 (bs, 1 H), 8.9 (s, 1 H), 8.2 (s, 1 H), 8.1 (d, 1 H), 7.8–7.6 (m, 1 H), 7.5– 7.4 (m, 2 H), 6.85–6.90 (m, 1 H), 4.8–4.7 (m, 1 H), 3.1–3.0 (m, 1 H), 3.0–2.8 (m, 1 H), 2.6–2.5 (m, 1 H), 2.0–1.9 (m, 1 H); LC/MS (M+H) expected: 276.12, observed: 276.0; Data for 13: peak 2, Rt = 3.47 min, 1H NMR (DMSO-d6)  12.3 (bs, 1 H), 8.9 (s, 1 H), 8.2 (s, 1 H), 8.1 (d, 1 H), 7.8–7.6 (m, 1 H), 7.5–7.4 (m, 2 H), 6.85–6.90 (m, 1 H), 4.8–4.7 (m, 1 H), 3.1–3.0 (m, 1 H), 3.0– 2.8 (m, 1 H), 2.6–2.5 (m, 1 H), 2.0–1.9 (m, 1 H); LC/MS (M+H) expected: 276.12, observed: 276.0. Absolute stereochemistry of 13 was determined as (R) by single crystal X-ray (see Supporting Information). By inference, the stereochemistry of 12 was assigned as (S). Synthesis of 6-(5-(2-methoxyethyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-2,3-dihydro4H-benzo[b][1,4]oxazine-4-carbonitrile (14). Step 1: Tert-butyl (6-(5-(2-methoxyethyl)-7Hpyrrolo[2,3-d]pyrimidin-4-yl)-2,3-dihydro-1H-inden-1-yl)carbamate (53). To a solution of 38 (1.0 g 2.78 mmol) in dioxane/water (4:1, 15 mL) was added 50 (588 mg, 2.78 mmol) and K2CO3 (1.15 g, 8.35 mmol). The mixture was degassed with nitrogen for 20 min and then Pd(PPh3)4 (100 mg, 0.083 mmol) was added. The reaction mixture was heated to 110 oC for 16 h. The reaction mixture was cool to room temperature and diluted with water. The aqueous mixture was extracted with ethyl acetate and the organic extracts combined, washed with brine and dried (Na2SO4). The solvent was removed to provide the crude product, which after chromatography (silica, EtOAc/Hep 10-50%) provided 53 (400 mg, 35%) as light yellow solid. 1H NMR (300 MHz, DMSO-d6)  12.1 (bs, 1 H), 8.72 (s, 1H), 7.49 (m, 5H), 5.1 (d, 1H), 3.3 (t, 2H), 3.0 (s, 4H), 2.85 (m, 2H), 2.7 (m, 2H), 1.9 (m, 1H), 1.3 (s, 9H). LC/MS expected: m/z 409.22, observed: 409.3.


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Step 2: 6-(5-(2-Methoxyethyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-2,3-dihydro-1H-inden-1-amine (54). To a flask containing 53 (400 mg, 0.98 mmol) was added 2M HCl in ether (10 mL). After 2 h, the reaction was concentrated to give 54 (274 mg, 91%). LC/MS (M+H) expected: 309.4, observed: 309.4. Step 3. N-(6-(5-(2-Methoxyethyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-2,3-dihydro-1H-inden-1yl)cyanamide: (14). To a solution of 54 (373 mg, 0.98 mmol) in DMF (3 mL) at 0 oC was added NMM (0.44 mL, 3.91 mmol) and BrCN (3M in DCM, 0.49 ml, 1.47 mmol). The reaction mixture was stirred for 2 h and then diluted with brine (25 mL) and ethyl acetate (25 mL). The layers were separated and the aqueous layer extracted with ethyl acetate (2  200 mL). The combined extracts were washed with brine, dried (Na2SO4) and the solvent removed to give the crude product, which after chromatography (silica, EtOAc/heptanes) gave the desired product 14 (140 mg, 43%). 1H NMR (300 MHz, CDCl3)  9.02 (bs, 1 H), 8.87 (s, 1 H), 7.68 (s, 1 H), 7.61 (d, 1H), 7.40 (d, 1H), 7.18 (s, 1 H), 4.87–4.82 (m, 1 H), 3.26–3.20 (m, 1H), 3.16–3.08 (m, 3H), 3.10 (s, 3 H), 2.99–2.92 (m, 2 H), 2.85–2.28 (m, 2 H), 2.65–2.59 (m, 1 H); LC/MS (M+H) observed: 334.2, expected: 334.17. Synthesis of 2,2-difluoro-6-(5-(2-methoxyethyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)2,3-dihydro-4H-benzo[b][1,4]oxazine-4-carbonitrile (15). Step 1: 6-Bromo-N-methyl-2,3dihydro-1H-inden-1-amine 6-bromo-N-methyl-2,3-dihydro-1H-inden-1-amine (59). To a stirred solution of 55 (5.0 g, 23.7mmol) in MeOH (150 mL) was added methylamine (2M in THF, 35.5 mL, 71.09 mmol) and Ti(O-iPr)4 (16.8 mL, 59.22 mmol) at rt. After stirring at room temperature for 12 h the reaction was cooled to 0 oC and NaBH4 (1.8 g, 47.38 mmol) was added in portions. The reaction was allowed to warm to rt and stirred for 10 h. The reaction mixture was quenched with water and extracted with ethyl acetate (2  25 mL). The organic extracts were collected and



washed with brine solution, dried (Na2SO4) and solvent removed to provide crude 56 as a brownish liquid (4.0 g, 75% crude yield). 1H NMR (300 MHz, CDCl3) δ 7.58-7.46 (m, 1H), 7.38-7.29 (m, 1 H), 7.18–7.11 (m, 1H), 5.31–5.21 (m, 0.5H), 4.18–4.64 (m, 0.5H), 3.11–2.84 (m, 1H), 2.82–2.71 (m, 1H), 2.58–2.35 (m, 3H), 2.00–1.81 (m, 1H); LC/MS (M+H) expected: 226.02, observed: 226.12. Step 2: Tert-butyl (6-bromo-2,3-dihydro-1H-inden-1-yl)(methyl)carbamate (57). To a solution of 56 (4 g, 17.7 mmol) in DCM (500 mL) at 0 oC was added (Boc)2O (7.72 g, 35.39 mmol), TEA (3.58g, 35.39 mmol) and DMAP (50 mg). The reaction mixture was warmed to room temperature and stirred for 16 h. The reaction mixture was diluted with DCM (1 L) and washed with water twice. The organic layer was dried over anhydrous Na2SO4 and concentrated under reduced pressure to provide crude product. The crude 57 was purified by column chromatography (silica, EtOAc/Heptanes, 0–10%) give 57 as thick brown liquid (4.5 g, 78% yield). LC/MS (M+H) expected: 326.02, observed: 326.12. Step 3: Tert-butyl methyl(6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2,3-dihydro-1H-inden1-yl)carbamate (58). To a solution of 57 (4.5 g, 13.8 mmol) in dioxane (150 mL) at room temperature was added bis(pinacolato)diboron (3.85 g, 15.18 mmol) and KOAc (4.06 g, 41.4 mmol). The reaction mixture was purged with nitrogen for 20 minutes. To the suspension was then added PdCl2(dppf) (430 mg, 0.69 mmol) in one portion. After heating to 80 oC for 16 h the reaction mixture was allowed to cool to room temperature and diluted with water (500mL). The aqueous layer was extracted with ethyl acetate, washed with brine solution, dried over anhydrous Na2SO4 and concentrated to provide 58 as a thick green liquid (5 g, 97% crude yield), which was taken onto the next step. LC/MS (M+H) expected: 374.25, observed: 374.2.


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Step 4: Tert-butyl (6-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-2,3-dihydro-1H-inden-1yl)(methyl)carbamate (59). To a solution of 38 (5 g, 13.4 mmol) in dioxane/water (8:2) (150 mL) at room temperature was added 58 (2.25 g, 14.74 mmol) and K2CO3 (5.55 g, 40.2mmol). The mixture was then degassed with nitrogen for 20 min and then Pd(PPh3)4 (774 mg, 0.67 mmol) was added and the reaction mixture heated to 110 oC for 16 h. The reaction mixture was cooled to room temperature and then diluted with water (250mL). The aqueous mixture was extracted with ethyl acetate (2  500mL). The organic extracts were washed with brine solution, dried over anhydrous Na2SO4 and concentrated under reduced pressure to provide crude 59. The crude was purified by chromatography (silica, EtOAc/PE 10–80%) to give 59 (800 mg, 16% yield). 1H NMR (400 MHz, DMSO) δ 12.21 (bs, 1H), 8.81 (s, 1H), 8.15–8.07 (m, 1H), 7.82 (bs, 1H), 7.61 (s, 1H), 7.52-7.45 (m, 1H), 6.81 (s, 1H), 5.81–5.42 (m, 2H), 3.11–2.84 (m, 2H), 2.71–2.41 (m, 3H), 2.18–1.98 (m, 2H), 1.54–1.41 (m, 9H); LC/MS (M+H) expected: 365.19, observed: 365.2. Step 5: N-Methyl-6-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-2,3-dihydro-1H-inden-1-amine (60). 2M HCl in ether (100 mL) was added to 59 (450 mg, 2.19 mmol). The reaction was stirred at room temperature for 2 h and then the solvent was decanted and the solid washed with diethyl ether (3  100mL) to remove excess HCl. The solid was dried under reduced pressure to give 60 as an off white solid (550 mg as HCl salt, 95% yield). 1H NMR (300 MHz, DMSO) δ 12.58 (br s, 1H), 9.15-9.41 (m, 2H), 8.85 (s, 1H), 8.42 (s, 1H), 8.25–8.19 (m, 1H), 7.81 (s, 1H), 7.64–7.58 (m, 1 H), 7.15 (s, 1H), 4.91–4.81 (m, 1H), 3.25–2.85 (m, 2H), 2.72–2.21 (m, 5H); LC/MS (M+H) expected: 265.14, observed: 265.04. Step 6: N-(6-(7H-Pyrrolo[2,3-d]pyrimidin-4-yl)-2,3-dihydro-1H-inden-1-yl)-N-methylcyanamide (15). To a solution of 60 (450 mg, 1.70mmol) in DMF (25mL) was added CNBr (269 mg, 2.55 mmol) and K2CO3 (470 mg, 3.4 mmol). After stirring at 40 oC for 2 h, the reaction mixture was



diluted with ethyl acetate, washed with water, dried (Na2SO4) and concentrated under reduced pressure to give a pale green liquid. The material was purified by chromatography (silica, MeOH/DCM 0-2%) to give 15 as an off-white solid (135 mg, 26% yield). 1H NMR (300 MHz, DMSO) δ 12.22 (br s, 1H), 8.82 (s, 1H), 8.18–8.11 (m, 2H), 7.71 (s, 1H), 7.58–7.51 (m, 1H), 6.95–6.88 (m, 1H), 4.84–4.78 (m, 1H), 3.18–3.09 (m, 1H), 2.98–2.90 (m, 4H), 2.61–2.43 (m, 1H), 2.18–2.04 (m, 1H); LC/MS (M+H) expected: 290.14, observed: 290.2. Synthesis of N-(6-(7H-Pyrrolo[2,3-d]pyrimidin-4-yl)chroman-4-yl)cyanamide (16). Step 1: Tert-butyl (6-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)chroman-4-yl)carbamate (62). To a solution of boronate 6154 (5 g, 13.33mmol) in dioxane/water (8:2) (100 mL) at room temperature was added 36 (2 g, 13.07 mmol) and Cs2CO3 (8.64 g,26.66 mmol). The reaction mixture was degassed with nitrogen for 20min. Pd(PPh3)4 (800 mg) was added and the reaction mixture was heated to reflux for 16 h at 110 oC. The reaction mixture was cooled to room temperature and then diluted with water (250 mL). The aqueous mixture was extracted with ethyl acetate (2  500 mL). The organic extracts were washed with brine, dried (Na2SO4) and concentrated under reduced pressure to provide crude product. The crude was purified by chromatography (silica, EtOAc/PE 10-80%) to give 62 (2.5 g, 52 % yield.). 1H NMR (DMSO-d6)  12.2 (bs, 1 H), 8.7 (s, 1 H), 8.1 (s, 1 H), 8.0 (d, 1 H), 7.65–7.60 (m, 1 H), 7.5 (d, 1 H), 6.9 (d, 1 H), 6.88–6.80 (m, 1 H), 4.9–4.8 (m, 1 H), 4.4–4.2 (m, 2 H), 2.2–1.9 (m, 2 H), 1.5 (s, 9 H); LC/MS (M+H) expected: 367.18, observed: 367.34. Step 2: 6-(7H-Pyrrolo[2,3-d]pyrimidin-4-yl)chroman-4-amine (63). A solution of 2 M HCl in ether (50 mL) was added to 62 (1 g, 2.73 mmol). After stirring at room temperature for 2 h, the solvent was decanted and solid washed with diethyl ether (3  100 mL) to remove excess HCl. The solvent was evaporated under reduced pressure to give 63 as an off-white solid (0.85 g, 97%


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yield). 1H NMR (DMSO-d6)  13.1 (bs, 1 H), 9.1 (s, 1 H), 9.0–8.9 (bs, 3 H), 8.4 (s, 1 H), 8.1 (d, 1 H), 7.9 (s, 1 H), 7.4 (s, 1 H), 7.1 (d, 1 H), 4.8–4.7 (m, 1 H), 4.5–4.3 (m, 2 H), 2.4–2.2 (m, 2 H); LC/MS (M+H) expected: 267.12, observed: 267.24 Step 3: N-(6-(7H-Pyrrolo[2,3-d]pyrimidin-4-yl)chroman-4-yl)cyanamide (16). To a solution of 63 (120 mg, 0.45 mmol) in DMF (4 mL) at 0 oC was added K2CO3 (125 mg, 0.90 mmol) followed by CNBr (52.5 mg, 0.49 mmol). The reaction mixture was stirred for 3 h and then poured into water/ethyl acetate. The layers were separated and the organic layer dried over sodium sulfate and the solvent removed to give crude 16, which was purified by RP-HPLC (column: DuraShell: 150  25 mm, 5 m; mobile phase A: 0.05% NH4OH in H2O; mobile phase B: 0.05% NH4OH in CH3CN; gradient 84:16 to 64:36 over 10 min; flow 30 mL/min) to give desired product (55 mg, 42%). 1H NMR (400 MHz, DMSO-d6)  12.24 (br. s., 1 H), 8.80 (s, 1 H), 8.21 (d, J = 1.76 Hz, 1 H), 8.09 (dd, J = 8.36, 2.20 Hz, 1 H), 7.46–7.74 (m, 2 H), 6.79–7.17 (m, 2 H), 4.58 (q, J = 4.84 Hz, 1 H), 4.09–4.41 (m, 2 H), 2.13–2.27 (m, 1 H), 1.94–2.13 (m, 1 H); LC/MS (M+H) expected: 292.11, observed: 292.0. HRMS (ESI) m/z: calculated for C16H13N5O [M + H]+ 292.1193; observed 292.1190. Step 4: (S)-N-(6-(7H-Pyrrolo[2,3-d]pyrimidin-4-yl)chroman-4-yl)cyanamide (17) and (R)-N-(6(7H-pyrrolo[2,3-d]pyrimidin-4-yl)chroman-4-yl)cyanamide (18). The racemate 16 (120 mg) was purified by SFC (AD column: 250  30 mm, 5 m; 0.1% NH4OH MeOH/CO2) to give two peaks: peak 1, 17, 40 mg, 33%, and peak 2, 18, 40 mg. 17: 1H NMR (400 MHz, DMSO-d6)  12.24 (br. s., 1 H), 8.80 (s, 1 H), 8.21 (d, J = 1.76 Hz, 1 H), 8.09 (dd, J = 8.36, 2.20 Hz, 1 H), 7.46–7.74 (m, 2 H), 6.79–7.17 (m, 2 H), 4.58 (q, J = 4.84 Hz, 1 H), 4.09–4.41 (m, 2 H), 2.13– 2.27 (m, 1 H), 1.94–2.13 (m, 1 H); LC/MS (M+H) expected: 292.11, observed: 292.1. HRMS (ESI) m/z: calculated for C16H13N5O [M + H]+ 292.1193, observed: m/z 292.1190. 18: 1H NMR



(400 MHz, DMSO-d6)  12.24 (br. s., 1 H), 8.80 (s, 1 H), 8.21 (d, J = 1.76 Hz, 1 H), 8.09 (dd, J = 8.36, 2.20 Hz, 1 H), 7.46–7.74 (m, 2 H), 6.79–7.17 (m, 2 H), 4.58 (q, J = 4.84 Hz, 1 H), 4.09– 4.41 (m, 2 H), 2.13–2.27 (m, 1 H), 1.94–2.13 (m, 1 H); LC/MS (M+H) expected: 292.11, observed: 292.1. HRMS (ESI) m/z: calculated for C16H13N5O [M + H]+ 292.1193, observed: m/z 292.1190. Absolute stereochemistry of 17 and 18 assigned based on biological activity and protein:ligand crystal structure (see Supporting Information). Synthesis of N-(6-(5-(2-methoxyethyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)chroman-4yl)cyanamide (19). Step 1: Tert-butyl (6-(5-(2-methoxyethyl)-7H-pyrrolo[2,3-d]pyrimidin-4yl)chroman-4-yl)carbamate: (64). The boronate 61 (700 mg, 1.898 mmol) and 38 (321 mg, 1.599 mmol) were combined in a microwave vial and dioxane/H2O (4:1; 10 mL) was added followed by K2CO3 (785 mg, 5.694 mmol). The mixture was purged for 20 min with argon and then Pd(PPh3)4 (64 mg, 0.056 mmol) was added. The vial was irradiated with microwave at 110 oC for 1 h. The reaction mixture was then poured into water (5 mL) and extracted with EtOAc (3  50 mL). The organic extracts were combined and washed with brine solution (10 mL), dried (Na2SO4), and the solvent removed to give crude material, which was purified by chromatography (silica, EtOAc/PE 90–100%) to give 64 (410 mg, 60%) as an off white solid. 1H NMR (300 MHz, DMSO-d6) δ 12.01 (s, 1H), 8.71 (s, 1H), 7.39–7.55 (overlapping, 4H), 6.88 (d, 1H), 4.9–4.8 (m, 1H), 4.4–4.2 (m, 2H), 3.11–3.23 (m, 2H), 3.19 (s, 3H), 2.8-2.7 (m, 2H), 2.1-1.9 (m, 2H), 1.4 (s, 9 H); LCMS: (M+H) expected: 425.21, observed: 425.3. Step 2: 6-(5-(2-Methoxyethyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)chroman-4-amine (65). To a flask containing 64 (410 mg, 0.96 mmol) in dioxane (2 mL) at 0 oC was added 2 M HCl in diethyl ether (10 mL). After stirring at room temperature for 5 h, the ether/dioxane was decanted and the residual solid was washed with ether. The solid was dissolved in water and extracted


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with 10% MeOH in DCM (3  25 mL). The organic layer was dried over sodium sulfate and the solvent removed to give crude material, which was purified by chromatography (silica, MeOH/EtOAc, 0–10%) to provide 65 (110 mg) as an off white solid. 1H NMR (300 MHz, DMSO-d6) δ 11.1 (s, 1H), 7.72 (s, 1H), 7.52–7.38 (m, 2H), 4.1–4.4 (m, 2 H), 6.84 (d, 1H), 4.41– 4.23 (m, 1H), 4.28–4.19 (m, 1H), 4.08–3.39 (m, 1H); LCMS: (M+H) expected: 325.16, observed: 325.2. Step 3: N-(6-(5-(2-Methoxyethyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)chroman-4-yl)cyanamide (19). To a flask containing 65 (105 mg, 0.32 mmol) in THF (5 mL) was added CNBr (40 mg, 0.387 mmol) and K2CO3 (66 mg, 0.483 mmol). After heating the reaction at 40 oC for 5 h, the reaction mixture was poured into water (2 mL) and extracted with EtOAc (3  25 mL). The organic extracts were dried over sodium sulfate and the solvent removed to give crude material, which was purified by preparative TLC (100 % EtOAc) to give 19 (30 mg, 26 %) as an off white solid. 1H NMR (300 MHz, DMSO-d6) δ 12.02 (s, 1H), 8.75 (s, 1H), 7.63 (s, 1H), 7.42–7.58 (overlapping, 2H), 7.42 (s, 1H), 6.98 (d, 1H), 4.43–4.55 (m, 1H), 4.42–4.31 (m, 2H), 3.19 (t, 2H), 3.05 (s, 3H), 2.78 (t, 2H), 1.98-2.23 (m, 2H). LCMS (M+H) expected: 350.16, observed: 350.1. HRMS (ESI) m/z: calculated for C19H19N5O2 [M + H]+ 350.1612; observed 350.1609. Synthesis of N-((1R,2S)-2-Methyl-6-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-2,3-dihydro1H-inden-1-yl)cyanamide (20) and N-((1R,2S)-2-methyl-6-(7H-pyrrolo[2,3-d]pyrimidin-4yl)-2,3-dihydro-1H-inden-1-yl)cyanamide (21). Step 1: 6-Bromo-2-methyl-2,3-dihydro-1Hinden-1-one (66). To a 1 N solution of LDA (120 mL, 0.24 mol) in anhydrous THF (300 mL) was added dropwise a solution of bromide 55 (50 g, 0.24 mol) in THF (300 mL) at -60 oC under N2. After addition was completed, the solution was stirred at -60 oC for 0.5 h and then MeI (33.78 g, 0.24 mol) was added. The reaction mixture was stirred at room temperature overnight.



After the reaction was completed, aqueous NH4Cl (200 mL) was added to the reaction mixture. The organic layer was separated and the aqueous layer was extracted with EtOAc (3 × 300 mL). The combined organic layers were washed with brine, dried over Na2SO4, and the solvent removed to give the crude material, which was purified by chromatography (silica, ethyl acetate/petroleum ether, 1-20%) to give 66 (6.5 g, 12.2%) as a yellow solid. Analytical data was consistent to previously reported data.64 Step 2: (Z)-6-Bromo-2-methyl-2,3-dihydro-1H-inden-1-one oxime (67). To a flask containing ketone 66 (5 g, 22 mmol) was added MeOH (80 mL) and NH2OH·HCl (6.12 g, 88 mmol). The reaction mixture was heated at reflux for 3 h, then was cooled to room temperature and diluted with ethyl acetate (80 mL). The pH was adjusted (~8-9) with addition of aqueous NaHCO3. The organic layer was separated and the aqueous phase was extracted with ethyl acetate (3 × 50 mL). The combined organic layers were dried over anhydrous Na2SO4 and concentrated in vacuo to afford crude 67 (5.60 g, 105%) as a yellow solid, which was taken onto the next step. LC/MS (M+H) expected: 240.0, observed: 240.0. Step 3: 6-Bromo-2-methyl-2,3-dihydro-1H-inden-1-amine (68). To a solution of hydroxylamine 67 (5.60 g, 23.4 mmol) in THF (200 mL) was added 2 N HCl (115 mL, 230 mmol), followed by Zn dust (14.93 g, 230 mmol). After the addition, the reaction mixture was heated at reflux for 3 h. The reaction mixture was cooled to room temperature and solvent removed. The crude material was then diluted with ethyl acetate (80 mL) and the pH ~10 with addition of NH4OH. The organic layer was separated and the aqueous phase was extracted with ethyl acetate (3 × 50 mL). The combined organic layers were dried over anhydrous Na2SO4 and the solvent removed in vacuo to afford crude amine 68 (4.60 g, 87.3%) as a yellow oil. LC/MS (M+H) expected: 226.0, observed: 226.0.


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Step 4: Tert-butyl (6-bromo-2-methyl-2,3-dihydro-1H-inden-1-yl)carbamate (69). To a solution of amine 68 (4.6 g, 20.4 mmol) and Et3N (5.7 mL, 40.8 mmol) in CH2Cl2 (50 mL) was added dropwise a solution of (Boc)2O (8.87 g, 40.8 mmol) in CH2Cl2 (40 mL) at 0℃. After addition, the mixture was stirred at room temperature overnight. The solvent was removed in vacuo and the residue was purified by chromatography (ethyl acetate/petroleum ether, 1–20%) to give 69 (1.2 g, 18%) as a yellow solid. 1H NMR (400 MHz, CDCl3)  7.28–7.52 (m, 2 H), 6.87–7.16 (m, 1 H), 5.11–5.08 (m, 0.5H), 4.53–4.85 (m, 1 H), 2.89–3.08 (m, 1 H), 2.70–2.84 (m, 0.5H), 2.38– 2.62 (m, 1 H), 2.03–2.26 (m, 1 H), 1.50 (d, J = 7.03 Hz, 9H), 1.16–1.33 (m, 2 H), 0.95 (d, J = 7.03 Hz, 2 H); LC/MS (M+H) expected: 326.08 observed: 326.1. Step 5: Tert-butyl (2-methyl-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2,3-dihydro-1Hinden-1-yl)carbamate (70). A mixture of bromide 69 (1.2 g, 3.68 mmol), bis(pinacolato)diborane (1.3 g, 5.12 mmol), KOAc (1.44 g, 14.7 mmol) and PdCl2(dppf) (300 mg, 0.37 mmol) in dioxane (30 mL) was degassed with nitrogen. The mixture was then stirred under nitrogen at 100℃ overnight. The solvent was removed in vacuo and the residue was purified by chromatography (EtOAc/Petroleum ether, 1–20%) to give 70 (1 g, 73%) as a yellow solid. 1H NMR (400Hz, DMSO-d6 + D2O): δ 7.72–7.62 (m, 2H), 7.21–7.14 (m, 1H), 5.12–5.06 (m, 1H), 4.71–4.68 (m, 1H), 3.05–2.91 (m, 1H), 2.79–2.71 (m, 1H), 2.52–2.48 (m, 1H), 1.54–1.50 (m, 9H), 1.24 (s, 12H), 1.15–1.10 (m, 3H); LC/MS (M+Na) expected: 396.2322 , observed: 396.23. Step 6: Tert-butyl ((1R,2S)-2-methyl-6-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-2,3-dihydro-1H-inden1-yl)carbamate (71) and tert-butyl ((1R,2R)-2-methyl-6-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-2,3dihydro-1H-inden-1-yl)carbamate(73).To a solution of boronate 70 (400 mg, 1.07 mmol) and 36 (166.4 mg, 1.07 mmol) in dioxane (8 mL) was added Na2CO3 (3.2 mL, 3.2 mmol). After degassing with nitrogen for 10 min, Pd(PPh3)4 (140 mg, 0.12 mmol) was added in one portion.



The reaction mixture was stirred at 120 oC under microwave irradiation for 30 min. The solvent was removed in vacuo and the residue was purified by chromatography (methanol: CH2Cl2, 1:50 to1:30) to afford a mixture of 71 and 73 (480 mg, 61%, ~1:1) as a yellow solid, which was further separated by chiral SFC (column: AD, 250  30 mm, 5 m; CO2/IPA(0.1% NH4OH), 73:27; flow: 65 mL/min) to afford 71 and 73. Data for 71, rac-trans: 1H NMR (400 MHz, DMSO-d6)  12.19 (br. s. 1 H), 8.76 (s, 1 H), 7.85–8.06 (m, 2 H), 7.61 (br. s., 1 H), 7.20–7.45 (m, 2 H), 6.77 (br. s., 1 H), 4.60 (t, J = 9.03 Hz, 1 H), 3.02 (dd, J =15.56, 7.53 Hz, 1 H), 1.43 (s, 9 H), 1.17 (d, J = 6.53 Hz, 3 H), 1.00 (d, J = 6.02 Hz, 2 H); LC/MS (M+H) expected: 365.20, observed: 365.2. Data for 73, rac-cis: 1H NMR (400 MHz, DMSO-d6)  12.29 (br. s., 1 H), 8.87 (s, 1 H), 8.09 (d, J =4.02 Hz, 2 H), 7.71 (br. s., 1 H), 7.26–7.55 (m, 2 H), 6.95 (br. s., 1 H), 5.13 (t, J = 7.78 Hz, 1 H), 2.96–3.16 (m, 1 H), 2.69–2.86 (m, 2 H), 1.50 (s, 9 H), 1.01 (d, J = 6.53 Hz, 3 H); LC/MS (M+H) expected: 365.20, observed: 365.2. Step 7: (1R,2S)-2-Methyl-6-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-2,3-dihydro-1H-inden-1-amine (72). To a solution of 71 (0.12 g, 0.33 mmol) in CH2Cl2 (10 mL) was added dropwise TFA (1.2 mL) at 0℃. After addition, the mixture was stirred at room temperature for 2 h. The solvent was removed in vacuo to give the crude product 72, which was used in the next step without further purification. LC/MS (M+H) expected: 265.1, observed: 265.1. Step 8: N-((1R,2S)-2-Methyl-6-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-2,3-dihydro-1H-inden-1yl)cyanamide (20). To a solution of amine 72 (120 mg, 0.33 mmol) and NMM (100 mg, 0.99 mmol) in anhydrous DMF (6 mL) was added CNBr (70 mg, 0.66 mmol) at -20℃. After addition, the reaction mixture was stirred at -20℃ for 2 h. The solvent was removed in vacuo and the residue was purified by preparative HPLC to afford 20 (40.3 mg, 31%) as a white solid. 1H NMR (400 MHz, DMSO) δ 12.24 (br s, 1H), 8.81 (s, 1H), 8.13-8.06 (m, 2H), 7.65–7.60 (m, 2H), 7.45–


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7.43 (m, 1H), 6.90-6.88 (m, 1H), 4.24–4.21 (m, 1H), 3.16–3.14 (m, 1H), 2.51–2.48 (m, 2H), 1.26 (d, 3H); LC/MS (M+Na) expected: 312.1, observed: 312.1. Synthesis of N-((1R,2R)-2-Methyl-6-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-2,3-dihydro1H-inden-1-yl)cyanamide (21). Step 1: Tert-butyl ((1R,2S)-2-methyl-6-(7H-pyrrolo[2,3d]pyrimidin-4-yl)-2,3-dihydro-1H-inden-1-yl)carbamate (74). To a solution of 73 (0.15 g, 0.41mmol) in CH2Cl2 (10 mL) was added dropwise TFA (1.5 mL) at 0℃. After addition, the mixture was stirred at room temperature for 2 h. The solvent was removed in vacuo to give the crude product, which was used in the next step without further purification. LC/MS (M+H) expected: 265.1, observed: 265.1. Step 2: N-((1R,2R)-2-Methyl-6-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-2,3-dihydro-1H-inden-1yl)cyanamide (21). To a solution of 74 (150 mg, 0.41 mmol) and NMM (125 mg, 1.24 mmol) in anhydrous DMF (6 mL) was added CNBr (86 mg, 0.81 mmol) at -20℃. After addition, the reaction mixture was stirred at -20℃ for 2 h. The solvent was removed in vacuo and the residue was purified by preparative HPLC to afford 21 (30 mg, 25.2%) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 12.23 (br s, 1H), 8.81 (s, 1H), 8.16–8.08(m, 2H), 7.65 (s, 1H), 7.47–7.45 (m, 1H), 7.20–7.18 (m, 1H), 6.93 (s, 1H), 4.67–4.62 (m, 1H), 3.05–3.00 (m, 1H), 2.73–2.66 (m, 2H), 1.11 (d, J = 6.4 Hz, 3H); LC/MS (M+Na) expected 312.1observed 312.1. HRMS (ESI) m/z: calculated for C17H15N5 [M + H]+ 290.1400; observed 290.1397. Synthesis of N-(1-Methyl-6-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-2,3-dihydro-1Hinden-1-yl)cyanamide (22), (R)-N-(1-methyl-6-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-2,3dihydro-1H-inden-1-yl)cyanamide (23), (S)-N-(1-methyl-6-(7H-pyrrolo[2,3-d]pyrimidin-4yl)-2,3-dihydro-1H-inden-1-yl)cyanamide (24). Step 1: 6-Bromo-1-methyl-2,3-dihydro-1Hinden-1-ol (75). To a 3 M solution of methylmagnesium bromide in THF (63 mL, 0.19 mol) was



added dropwise a solution of indanone 55 (10 g, 47.38 mmol) in THF (100 mL) at 0 oC. The solution was stirred at 25 oC for 2 h. The reaction solution was quenched with saturated aqueous NH4Cl (50 mL) and the pH adjusted to 6-7 with 1N HCl. The aqueous mixture was extracted with EtOAc (3 × 200 mL). The combined organic layers were washed with brine (2 × 200 mL), dried over Na2SO4 and solvent removed. Purification by chromatography (silica, EtOAc/petroleum ether 30:1–20:1) gave 75 (7.65 g, 71%) as a yellow solid. 1H NMR (400 MHz, DMSO-d6)  7.41 (d, J = 1.51 Hz, 1 H), 7.34 (dd, J = 8.03, 1.51 Hz, 1 H), 7.15 (d, J = 8.03 Hz, 1 H), 5.13 (s, 1 H), 2.78–2.90 (m, 1 H), 2.62–2.74 (m, 1 H), 2.03 (t, J = 7.03 Hz, 2 H), 1.37 (s, 3 H); LC/MS (M+H) expected: 227.01, observed: 227.0 Step 2: 1-Azido-6-bromo-1-methyl-2,3-dihydro-1H-indene (76). To a solution of 75 (7.65 g, 33.68 mmol) in CHCl3 (90 mL) was added NaN3 (4.38 g, 67.37 mmol) and TFA (15 mL) at 0 oC. The reaction mixture was stirred at 30 oC for 3 h. To the reaction solution was added 10% NH4OH to adjust pH to between 8-9 and then the aqueous mixture extracted with CHCl3 (2 × 100 mL). The combined organic layers were dried over Na2SO4 and the solvent removed to give 76 (8.2 g, 96% of yield) as a yellow oil, which was used for the next step directly. Step 3: 6-Bromo-1-methyl-2,3-dihydro-1H-inden-1-amine (77). To a solution of 76 (4.4 g, 17.45 mmol) in THF (200 mL) was added a 1 M solution of PMe3 in THF (19.2 mL, 19.2 mmol) and water (20 mL). The solution was stirred at 30 oC overnight. The reaction mixture was diluted with EtOAc (200 mL) and then washed with saturated NaHCO3 (3 × 100 mL) and brine (2 × 100 mL). The organic layer was collected, dried over Na2SO4, and the solvent removed to give crude material, which was purified by chromatography (silica, DCM/MeOH 100:1 to 20:1) to give 77 (1.64 g, 41% of yield) as a yellow oil. 1H NMR (400 MHz, DMSO-d6)  7.49 (d, J = 1.51 Hz, 1


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H), 7.29 (dd, J = 7.78, 1.76 Hz, 1 H), 7.12 (d, J = 8.03 Hz, 1 H), 5.76 (s, 1 H), 2.64 - 2.85 (m, 3 H), 1.85 - 2.03 (m, 3 H), 1.23 (s, 3 H); LC/MS (M+H) expected: 226.02, observed: 226.0. Step 4: Tert-butyl (6-bromo-1-methyl-2,3-dihydro-1H-inden-1-yl)carbamate (78). To a solution of 77 (2.0 g, 8.84 mmol) in DCM (80 mL) was added Et3N (1.96 g, 19.45 mmol) followed by Boc2O (3.82 g, 17.69 mmol). The solution was stirred at 30 oC overnight. The solvent was removed in vacuo and the residue was purified by chromatography (silica, EtOAc/petroleum ether, 1–50%) to give 78 (1.5 g, 52%) as a yellow oil. 1H NMR (400 MHz, CDCl3)  7.37 (s, 1 H), 7.32 (d, J = 8.0 Hz, 1 H), 7.08 (d, J = 8.03 Hz, 1 H), 4.83 (br. s., 1 H), 2.92 (dd, J = 8.78, 4.3 Hz, 1 H), 2.74–2.85 (m, 1 H), 2.62 (br. s., 1 H), 2.13 (td, J = 8.41, 4.27 Hz, 1 H), 1.53 (br. s., 3 H), 1.38 (br. s., 9 H); LC/MS (M+H) expected: 326.08, observed: 326.1. Step 5: Tert-butyl (1-methyl-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2,3-dihydro-1Hinden-1-yl)carbamate (79). To a degassed solution of bromide 78 (3.6 g, 11.0 mmol), bis(pinacolato)diborane (2.8 g, 11.0 mmol) and KOAc (3.23 g, 33.0 mmol) in dioxane (60 mL) was added PdCl2(dppf) (402 mg, 0.55 mmol). The mixture was stirred at 80 oC for 3 h. The reaction mixture was diluted with EtOAc (100 mL) and then washed with brine (3 × 50 mL). The organic layer was collected, dried over Na2SO4, and the solvent removed to give crude material, which was purified by chromatography (silica, EtOAc/petroleum ether 1–50%) to give 79 (4.2 g, 100%) as a white solid. 1H NMR (400 MHz, CDCl3) δ 7.67 (d, 2H), 7.22 (d, 1H), 4.85 (br, 1H), 2.95 (m, 1H), 2.82 (m, 1H), 2.59 (m, 1H), 2.20 (m, 1H), 1.60 (s, 3H), 1.34 (m, 21H); LC/MS (M+Na) expected: 396.23, observed: 396.3. Step 6: Tert-butyl (1-methyl-6-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-2,3-dihydro-1H-inden-1yl)carbamate (80). To a mixture of boronate 79 (1.72 g, 4.6 mmol), 36 (706 mg, 4.6 mmol) and K2CO3 (1.9 g, 13.8 mmol) in dioxane (50 mL) and water (10 mL) was added Pd(PPh3)4 (690 mg,



0.6 mmol) under N2. The mixture was degassed and then stirred at 100 oC overnight. The solution was diluted with EtOAc (100 mL), then washed with brine (3 × 100 mL), dried over Na2SO4, filtered and concentrated in vacuo. The residue was purified by column chromatography (petroleum ether /EtOAc = 10/1 to1/2) to give 80 (800 mg, 48%) as a yellow solid. 1H NMR (400 MHz, CDCl3)  10.67 (br. s., 1 H), 8.98 (s, 1 H), 7.97–8.13 (m, 2 H), 7.37–7.47 (m, 2 H), 6.88 (br. s., 1 H), 2.87–3.20 (m, 2 H), 2.69 (d, J = 6.53 Hz, 1 H), 2.25 (ddd, J = 12.92, 8.16, 5.02 Hz, 1 H), 1.68 (br. s., 3 H), 1.41 (br. s., 9 H); LC/MS (M+H) expected: 365.20, observed: 365.2. Step 7: 1-Methyl-6-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-2,3-dihydro-1H-inden-1-amine (81). To a solution of 80 (450 mg, 1.23 mmol) in dioxane (20 mL) at 0 oC was added dropwise a solution of 4 N HCl in dioxane (6.2 mL). The mixtue was stirred at 25 oC for 2 h. Additional 4 N HCl in dioxane (10 mL) was added and the mixture was stirred at 25 oC for 3 h. The reaction mixture was filtered. The wet cake was washed with EtOAc, dried in vacuo to give a green solid, which was re-crystallized from 10:1 DCM/MeOH, (30 mL) to give 81 (110 mg, 34% of yield) as a green solid. 1H NMR (400 MHz, DMSO-d6) δ 12.90 (s, 1H), 8.97 (s, 1H), 8.73 (s, 3H), 8.35 (s, 1H), 8.11 (d, 1H), 7.88 (s, 1H), 7.58 (d, 1H), 7.19 (s, 1H), 3.15 (m, 1H), 3.02 (m, 1H), 2.26 (m, 2H), 1.68 (s, 3H); LC/MS (M+H) expected: 265.15, observed: 265.14. Step 8: N-(1-Methyl-6-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-2,3-dihydro-1H-inden-1-yl)cyanamide (22). To the solution of 81 (100 mg, 0.378 mmol) in DMF (6 mL) was added NMM (115 mg, 1.13 mmol) at 25 oC, and then CNBr (80 mg, 0.756 mmol) in DCM (1 mL) at -20 oC. The reaction mixture was stirred below 0 oC for 2 h. The solvent was removed in vacuo and the residue was purified by chromatography (DCM/MeOH, 100/1 to 10/1) to give 22 (40 mg, 37%) as a white solid. 1H NMR (400 MHz, DMSO) δ 12.27 (s, 1H), 8.83 (s, 1H), 8.81–8.07 (m, 2H), 7.66 (d, 1H), 7.49–7.46 (m, 2H), 6.90 (d, 1H), 3.19–2.85 (m, 2H), 2.25–2.20 (m, 2H), 1.53 (s,


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3H); LC/MS (M+H) expected: 290.14, observed: 290.2. HRMS (ESI) m/z: calculated for C17H15N5 [M + H]+ 290.1400; observed 290.1397. Step 9: (R)-N-(1-Methyl-6-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-2,3-dihydro-1H-inden-1yl)cyanamide (23) and (S)-N-(1-methyl-6-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-2,3-dihydro-1Hinden-1-yl)cyanamide (24). 400 mg of racemic 80 was separated by chiral SFC (column: AD, 250  30 mm, 5m; mobile phase: CO2/EtOH (0.1% NH4OH), 65:35; flow: 60 mL/min) to give compound 82, peak 1 (190 mg) and 83, peak 2 (190mg). 82 and 83 were converted to cyanamides 23 and 24 following the same steps as described for 22. Data for 23 (peak 1): Chiral SFC (column: chiralpak AS-H, 150  4.6 mm, 5m; mobile phase: CO2/EtOH 5-40% (0.05% DIPEA), detection  = 220 nm): Rt = 4.55 min; 1H NMR (400 MHz, DMSO) δ 12.27 (s, 1H), 8.83 (s, 1H), 8.81–8.07 (m, 2H), 7.66 (d, 1H), 7.49–7.46 (m, 2H), 6.90 (d, 1H), 3.19–2.85 (m, 2H), 2.25–2.20 (m, 2H), 1.53 (s, 3H); LC/MS (M+H) expected: 290.14, observed: 290.2. HRMS (ESI) m/z: calculated for C17H15N5 [M + H]+ 290.1400; observed 290.1397. Data for 24 (peak 2): Chiral SFC: Rt = 4.85 min; 1H NMR (400 MHz, DMSO) δ 12.27 (s, 1H), 8.83 (s, 1H), 8.81–8.07 (m, 2H), 7.66 (d, 1H), 7.49–7.46 (m, 2H), 6.90 (d, 1H), 3.19–2.85 (m, 2H), 2.25–2.20 (m, 2H), 1.53 (s, 3H); LC/MS (M+H) expected: 290.14, observed: 290.1. HRMS (ESI) m/z: calculated for C17H15N5 [M + H]+ 290.1400; observed 290.1397. The absolute stereochemistry of 23 was assigned as (S) based on protein:ligand cocrystal. By inference, absolute stereochemistry of 24 was assigned as (R). Synthesis of N-((1S,3S)-3-Hydroxy-6-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-2,3dihydro-1H-inden-1-yl)cyanamide (25) and N-((1S,3R)-3-hydroxy-6-(7H-pyrrolo[2,3d]pyrimidin-4-yl)-2,3-dihydro-1H-inden-1-yl)cyanamide (26). Step 1: (R,Z)-N-(3Bromobenzylidene)-2-methylpropane-2-sulfinamide (85). To a solution of (R)-2-methylpropane-



2-sulfinamide (32.67 g, 0.27 mol) in DCM (500 mL) was added p-TsOH (27.8 g, 0.108 mol), MgSO4 (162 g, 1.35 mol) and then 3-bromobenzaldehyde 84 (50 g, 0.27 mol). The mixture was stirred at room temperature overnight and then filtered. The filtrate was concentrated and the residue was purified via column chromatography (petroleum ether /EtOAc, 100/1 to 20/1) to give compound 85 (53.5 g, 68% of yield) as an oil. 1H NMR (400 MHz, CDCl3)  8.53 (s, 1 H), 8.03 (s, 1 H), 7.74 (d, J = 7.53 Hz, 1 H), 7.64 (d, J = 8.03 Hz, 1 H), 7.36 (t, J = 7.78 Hz, 1 H), 1.28 (s, 9 H); LC/MS (M+H) expected: 288.01, observed: 288.0. Step 2. Tert-butyl (S)-3-(3-bromophenyl)-3-(((R)-tert-butylsulfinyl)amino)propanoate (86). To a 0.1 M solution of SmI2 in THF (800 mL,) at -70 oC under N2 was added a solution of 85 (5.75 g, 19.95 mmol) in THF (10 mL) followed by dropwise addition of a solution of tert-butyl 2bromoacetate (11.67 g, 59.84 mmol) in THF (100 mL) of at -70 oC. The reaction mixture was stirred for 3h at -70 oC and then allowed to warm to 0 oC. After 1 h, the mixture was quenched with saturated NH4Cl (200 mL) at 0 oC. The aqueous mixture was extracted with EtOAc (2 × 300 mL) and the combined organic layers were dried over anhydrous Na2SO4. The solvent was then removed and the residue was purified by chromatography (EtOAc/ petroleum ether, 1:50 to 1:5) to give compound 86 (3.2 g, 39%) as an oil. 1H NMR (400 MHz, CDCl3)  7.49 (s, 1 H), 7.42 (d, J = 8.0 Hz, 1 H), 7.16–7.31 (m, 2 H), 4.60–4.77 (m, 2 H), 2.71–2.79 (m, 2 H), 1.40 (s, 9 H), 1.24 (s, 9 H); LC/MS (M+H) expected: 404.08, observed: 403.7. Step 3. (S)-3-Amino-3-(3-bromophenyl)propanoic acid (87). To a flask containing 86 (5 g, 12.38 mmol) was added 4 N HCl in dioxane (200 mL) and the mixture was stirred overnight at room temperature. The solvent was evaporated in vacuo to afford 87 (3.2 g, > 100%) which was used in the next step without further purification. 1H NMR (400 MHz, DMSO-d6)  8.54 (br. s., 3H),


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7.71–7.92 (m, 1 H), 7.47–7.64 (m, 2 H), 7.25–7.46 (m, 1 H), 4.60 (br. s., 1 H), 2.84 - 3.13 (m, 2 H). Step 4. (S)-3-(3-Bromophenyl)-3-(2,2,2-trifluoroacetamido)propanoic acid (88). To a flask containing 87 (3.2 g, 12.38 mmol) was added TFAA (20 mL). After 2 h at room temperature, the solvent was removed in vacuo and the residue was purified by chromatography (EtOAc/ petroleum ether, 1:20 to 1:1) to give 88 (2.8 g, 67%) as a yellow solid. 1H NMR (400 MHz, CDCl3)  7.41–7.59 (m, 2 H), 7.22–7.32 (m, 1 H), 7.12–7.21 (m, 1 H), 5.35–5.46 (m, 1 H), 2.94 3.14 (m, 2 H); 19F NMR (376 MHz, CDCl3)  -75.83 (s, 3 F); LC/MS (M+H) expected: 339.98, observed: 340.0. Step 5. (S)-3-(3-Bromophenyl)-3-(2,2,2-trifluoroacetamido)propanoyl chloride (89). To a flask containing 88 (2.8 g, 8.23 mmol) was added SOCl2 (35 mL). The mixture was heated to reflux for 1 h and then concentrated in vacuo to give the crude product. The crude was azeotroped with dry DCM to give 89 (2.9 g, 98%) as a yellow solid. 1H NMR (400 MHz, CDCl3)  7.52 (d, J = 7.53 Hz, 1 H), 7.43–7.48 (m, 1 H), 7.22–7.35 (m, 3 H), 6.85 (br. s., 1 H), 5.40 (q, J = 6.53 Hz, 1 H) 3.45–3.71 (m, 2 H); 19F NMR (376 MHz, CDCl3)  -75.68 (s, 3 F). Step 6. (S)-N-(4-Bromo-3-oxo-2,3-dihydro-1H-inden-1-yl)-2,2,2-trifluoroacetamide (90) and (S)N-(6-bromo-3-oxo-2,3-dihydro-1H-inden-1-yl)-2,2,2-trifluoroacetamide (91). To a solution of 89 (2.9 g, 8.09 mmol) in CH2Cl2 (35 mL) at room temperature was added AlCl3 (2.1 g, 15.4 mmol). The mixture was heated to reflux for 1 h and then allowed to cool to room temperature. The mixture was poured into 1 N HCl (20 mL) and the aqueous mixture was extracted with ethyl acetate (80 mL). The organic layer was washed with saturated aqueous NH4Cl (50 mL) then brine and dried over sodium sulfate. The solvent was removed to give the crude product, which was purified by preparative HPLC to afford 90 (1.5 g, 58%) and 91 (0.5 g, 19%) as white solids.



Data for 90: 1H NMR (400 MHz, DMSO) δ 10.0 (d, 1H), 7.84 (s, 1H), 7.78–7.76 (m, 1H), 7.64– 7.62 (m, 1H), 5.62–5.57 (m, 1H), 3.17–3.11 (m, 1H), 2.68–2.64 (m, 1H); LC/MS (M+H) expected: 321.9691, observed: 321.9; Chiral SFC (column: AD-3, 150  4.6 mm; mobile phase: IPA + 0.05% DEA; flow = 2.5 mL/min, 5-40% CO2/IPA): Rt = 3.4 min; 20D = -46.8 (c = 2.8 mg/mL, EtOH); Data for 91: 1H NMR (400 MHz, DMSO) δ 9.99 (s, 1H), 7.76 (m, 1H), 7.64 (m, 2H), 5.58 (m, 1H), 3.17 (m, 1H), 2.67 (m, 1H); LC/MS (M+H) expected 321.9691, observed 322.0; chiral SFC (column: OJ-H, 150  4.6 mm; MeOH+0.05%DEA, flow = 2.5 mL/min, 540% CO2/MeOH): Rt = 4.6 min; 20D = -84.72 (c = 3.6 mg/mL, EtOH). Step 7. (S)-2,2,2-Trifluoro-N-(3-oxo-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2,3dihydro-1H-inden-1-yl)acetamide (92). To a solution of 90 (2.5 g, 7.76 mmol), bis(pinacolato)diboron (2.08 g, 8.15 mmol) and KOAc (2.28 g, 2.3 mmol) in dioxane (60 mL) was added PdCl2(dppf) (316 mg, 0.38 mmol) under nitrogen. The mixture was degassed with nitrogen and stirred at 100 oC for 5 h. The reaction mixture was diluted water and the aqueous mixture extracted with EtOAc (3 × 50 mL), washed with brine (3 × 30 mL), dried over Na2SO4, and concentrated in vacuo. The residue was purified by column chromatography (EtOAc/ petroleum ether) to give 92 (2.9 g, 100%) as a red solid. 1H NMR (400 MHz, CDCl3) δ 8.03 (s, 1 H), 7.98 (d, J = 7.53 Hz, 1 H), 7.79 (d, J = 7.53 Hz, 1 H), 6.69 (d, J = 7.53 Hz, 1 H), 5.68 (td, J = 7.78, 3.01 Hz, 1 H), 3.31 (dd, J = 19.07, 7.53 Hz, 1 H), 2.58 (dd, J = 19.07, 3.51 Hz, 1 H), 1.37 (bs, 12 H); LC/MS (M+H) expected: 370.0, observed: 370.0. Step 8. (S)-2,2,2-Trifluoro-N-(3-oxo-6-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-2,3-dihydro-1H-inden1-yl)acetamide (93). To a solution of 92 (2.7 g, 7.31 mmol), 36 (1.21 g, 7.85 mmol) and KF (0.85 g, 14.6 mmol) in dioxane/water (64 mL/16 mL) was added PdCl2(dppf) (268 mg, 0.37 mmol) under nitrogen. The solution was degassed with nitrogen and then heated to 90 oC


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overnight. The reaction mixture was diluted with water and the aqueous mixture was extracted with EtOAc (3 × 50 mL), washed with brine (3 × 30 mL), dried over sodium sulfate, filtered and solvent removed in vacuo. The residue was purified by column chromatography (EtOAc/ petroleum ether) to give 93 (2.0 g, 76%) as a solid. 1H NMR (400 MHz, DMSO-d6)  12.42 (br. s., 1 H), 10.12 (d, J = 7.82 Hz, 1 H), 8.92 (s, 1 H), 8.19 - 8.50 (m, 2 H), 7.63–7.98 (m, 2 H), 6.86 (d, J = 1.96 Hz, 1 H), 5.61–5.93 (m, 1 H), 3.09–3.27 (m, 1 H), 2.61–2.82 (m, 1 H); LC/MS (M+H) expected: 360.08, observed: 360.1. Step 9. 2,2,2-Trifluoro-N-((1S)-3-hydroxy-6-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-2,3-dihydro-1Hinden-1-yl)acetamide (94), 2,2,2-trifluoro-N-((1S,3R)-3-hydroxy-6-(7H-pyrrolo[2,3-d]pyrimidin4-yl)-2,3-dihydro-1H-inden-1-yl)acetamide (95) and 2,2,2-trifluoro-N-((1S,3S)-3-hydroxy-6(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-2,3-dihydro-1H-inden-1-yl)acetamide (96). To a solution of ketone 93 (2.0 g, 5.56 mmol) in MeOH (150 mL) at 0 oC was added NaBH4 (0.7 g, 16.67 mmol). After 2 h at room temperature, the reaction mixture was quenched with water (10 mL) and the mixture was extracted with EtOAc (3 × 50 mL), washed with brine (3 × 30 mL), dried over Na2SO4, filtered and the solvent removed to provide crude product 94 (1.1 g, 55%), as a mixture of cis/trans isomers. The crude was purified by preparative HPLC (column: DuraShell, 150  25 mm, 5 m; mobile phase: CH3CN/H2O+0.05% NH4OH) to give two peaks. Data for 94, cis/trans mixture: 1H NMR (400 MHz, DMSO- d6) δ 12.30 (s, 1H), 10.03–9.91 (m, 1H), 8.83 (s, 1H), 8.16–7.92 (m, 2H), 7.68–7.60 (m, 2H), 6.80 (m, 1H), 5.82–5.61 (m, 1H), 5.50–5.11 (m, 2H), 2.87–2.84 (m, 0.7H), 2.35 (m, 0.5H), 1.97–1.95 (m, 0.8H); LC/MS (M+H) expected: 363.10, observed: 363.10. Data for 96: peak 1, trans: 1H NMR (400 MHz, DMSO-d6)  12.29 (br. s., 1 H), 9.91 (d, J = 8.03 Hz, 1 H), 8.84 (s, 1 H), 8.16 (d, J = 8.03 Hz, 1 H), 8.04 (s, 1 H), 7.45–7.75 (m, 2 H), 6.82 (d, J = 3.01 Hz, 1 H), 5.62 (q, J = 7.03 Hz, 1 H), 5.48 (d, J = 6.02 Hz, 1



H), 5.22–5.38 (m, 1 H), 2.26–2.43 (m, 2 H); LC/MS (M+H) expected: 363.10, observed: 363.10. Data for 95, peak 2, cis: 1H NMR (400 MHz, DMSO-d6)  12.28 (br. s., 1 H), 10.01 (d, J = 8.03 Hz, 1 H), 8.84 (s, 1 H), 8.16 (d, J = 8.03 Hz, 1 H), 7.92 (s, 1 H), 7.48–7.78 (m, 2 H) 6.81 (d, J = 2.51 Hz, 1 H), 5.80 (d, J = 5.52 Hz, 1 H), 5.33 (d, J = 8.53 Hz, 1 H), 5.12 (d, J = 6.53 Hz, 1 H), 2.87 (d, J = 12.05 Hz, 1 H), 1.96 (d, J = 11.54 Hz, 1 H); LC/MS (M+H) expected: 363.10 , observed: 363.10. Step 10. (1R,3S)-3-Amino-5-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-2,3-dihydro-1H-inden-1-ol (97). To a solution of 95 (73.0 mg, 0.20 mmol) in MeOH (5 mL) was added a 2 M solution of NH3 in MeOH (10 mL). The mixture was stirred at 50 oC for 16 h and then concentrated in vacuo and the material carried onto the next step without further purification. 1H NMR (400 MHz, DMSOd6)  12.24 (br. s., 1 H), 8.67–8.95 (m, 1 H), 8.50 (br. s., 1 H), 7.99–8.27 (m, 2 H), 7.66 (d, J = 3.51 Hz, 1 H), 7.52 (d, J = 7.53 Hz, 1 H), 6.92 (d, J = 3.51 Hz, 1 H), 5.76 (s, 1 H) 5.52 (br. s., 1 H), 4.97 (br. s., 1 H), 2.70–2.88 (m, 1 H), 1.91–2.13 (m, 1 H), 1.51–1.68 (m, 1 H); LC/MS (M+H) expected: 267.1, observed: 266.9. Step 11. (N-((1S,3R)-3-Hydroxy-6-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-2,3-dihydro-1H-inden-1yl)cyanamide (25). To a solution of 97 (53.7 mg, 0.20 mmol) in DMF (2 mL) at -30 oC was added NMM (40.8 mg, 0.40 mmol), then BrCN (29.9 mg in 1 mL of DMF). After 1.5 h the reaction mixture was concentrated to give the crude product, which was purified by preparative HPLC (column: Durashell 150  25 mm, 5 m; mobile phase: CH3CN/H2O + 0.05% NH4OH) and lyophilized to give 25 (27.6 mg, 47%) as a white solid. 1H NMR (400 MHz, CH3OH-d4)  8.79 (s, 1 H), 8.01–8.15 (m, 2 H), 7.65 (d, J = 7.81 Hz, 1 H), 7.54 (d, J = 3.51 Hz, 1 H), 6.90 (d, J = 3.51 Hz, 1 H), 5.15 (t, J = 7.42 Hz, 1 H), 4.61 (t, J = 8.00 Hz, 1 H), 3.04 (dt, J = 12.54, 7.00 Hz, 1 H), 1.93 (dt, J = 12.54, 8.46 Hz, 1 H); 1H NMR (400 MHz, DMSO-d6)  12.28 (br. s., 1


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H), 8.66–8.98 (m, 1 H), 8.02–8.28 (m, 2 H), 7.47–7.81 (m, 3 H), 6.92 (d, J = 3.51 Hz, 1 H), 5.75 (d, J = 6.53 Hz, 1 H), 5.03 (q, J = 7.03 Hz, 1 H), 4.55 (q, J = 7.36 Hz, 1 H), 2.89 (dt, J = 12.17, 6.96 Hz, 1 H), 1.79 (dt, J = 11.92, 8.85 Hz, 1 H); LC/MS (M+H) expected: 292.1, observed: 292.0. HRMS (ESI) m/z: calculated for C16H13N5O [M + H]+ 292.1193; observed 292.1191. Step 12. (1S,3S)-3-Amino-5-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-2,3-dihydro-1H-inden-1-ol (98). To a solution of 96 (36.4 mg, 0.10 mmol) in MeOH (2.5 mL) was added a 2 M solution of NH3 in MeOH (5 mL). The reaction was heated to 50 oC for 16 h and then the solvent removed in vacuo to give a colorless oil, which was used in the next step without further purification. 1H NMR (400 MHz, DMSO-d6)  12.23 (br. s., 1 H), 8.83 (s, 1 H), 7.97–8.24 (m, 2 H), 7.66 (d, J = 3.51 Hz, 1 H), 7.51 (d, J = 7.53 Hz, 1 H), 6.93 (d, J = 3.51 Hz, 1 H), 5.66–5.92 (m, 1 H), 5.18 (s, 2 H), 3.00 (d, J = 5.02 Hz, 1 H), 2.21 (d, J = 6.53 Hz, 1 H), 1.83 - 2.07 (m, 3 H); LC/MS (M+H) expected: 267.1, observed: 266.9. Step 13. N-((1S,3S)-3-Hydroxy-6-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-2,3-dihydro-1H-inden-1yl)cyanamide (26). To a solution of 98 (26.7 mg, 0.1 mmol) in DMF (1 mL) at -30 oC was added NMM (20.3 mg, 0.20 mmol). After 10 min, BrCN (14.9 mg in 1 mL DMF) was added dropwise and the reaction stirred for 1.5 h. The solvent was removed in vacuo and the crude product was purified by preparative HPLC (column: Durashell, 150  25 mm, 5 m; mobile phase: CH3CN/H2O + 0.05 % NH4OH) to give 26 (10.48 mg, 36%) as a white solid after lyophilization. 1H

NMR (400 MHz, CH3OH-d4)  8.79 (s, 1 H), 8.04 - 8.19 (m, 2 H), 7.66 (d, J = 7.81 Hz, 1 H),

7.55 (d, J = 3.51 Hz, 1 H), 6.92 (d, J = 3.71 Hz, 1 H), 5.41 (t, J = 5.27 Hz, 1 H), 4.96 (t, J = 6.05 Hz, 1 H), 2.45 (t, J = 5.76 Hz, 2 H); 1H NMR (400 MHz, DMSO-d6)  12.28 (br. s., 1 H), 8.85 (s, 1 H), 8.07–8.28 (m, 2 H), 7.54–7.76 (m, 2 H), 7.44 (d, J = 5.38 Hz, 1 H), 6.95 (d, J = 1.96 Hz, 1 H), 5.49 (d, J = 5.87 Hz, 1 H), 5.29 (q, J = 5.38 Hz, 1 H), 4.89 (q, J = 5.87 Hz, 1 H), 2.14–2.39



(m, 2 H); LC/MS (M+H) expected: 292.1, observed: 292.0. HRMS (ESI) m/z: calculated for C16H13N5O [M + H]+ 292.1193; observed 292.1191. Synthesis of N-((1R,2R)-2-Hydroxy-6-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-2,3dihydro-1H-inden-1-yl)cyanamide (27). Step1. Rac tert-butyl((1R,2R)-6-bromo-2-hydroxy-2,3dihydro-1H-inden-1-yl)carbamate (100). To a solution of rac-trans amine 99 (2.0 g, 8.7 mmol) in THF (10 mL) at 0 oC was added TEA (1.48 mL, 10.5 mmol) and Boc2O (1.93 g, 8.77 mmol). The reaction was stirred for 1 h and then the solvent was removed to give a residue, which was triturated with heptane and filtered to give desired product 100 (1.1 g, 38%). 1H NMR (400 MHz, CDCl3)  7.32–7.42 (m, 2 H), 7.10 (d, J = 8.00 Hz, 1 H), 4.81–5.05 (m, 2 H), 4.33–4.47 (m, 1 H), 3.25 (dd, J = 16.00, 7.71 Hz, 1 H), 2.85 (dd, J = 15.91, 8.20 Hz, 1 H), 1.51 (s, 9 H); LC/MS (M+Na) expected: 350.368, observed: 350.10. Step 2. Rac tert-butyl ((1R,2R)-2-hydroxy-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2,3dihydro-1H-inden-1-yl)carbamate (101). To a solution of bromide 100 (590 mg, 1.80 mmol) in dioxane (10 mL) was added (BPin)2 (548 mg, 2.16 mmol), KOAc (535 mg, 5.39 mmol) and PdCl2(dppf) (65.9 mg, 0.09 mmol). The reaction mixture was heated to 100 oC for 3 h and then allowed to cool to room temperature. The reaction mixture was diluted with ethyl acetate and filtered through Celite. The filtrate was concentrated to afford a brown oil, which after chromatography (silica, EtOAc/Heptane) gave the desired product 101 as a white solid (570 mg, 84%). 1H NMR (400 MHz, CDCl3)  7.72 (d, J = 7.42 Hz, 1 H), 7.62 (s, 1 H), 7.23 (d, J = 7.61 Hz, 1 H), 5.15 (d, J = 3.32 Hz, 1 H), 4.88 (t, J = 5.66 Hz, 1 H), 4.35–4.47 (m, 1 H), 3.30 (dd, J = 16.20, 7.81 Hz, 1 H), 2.92 (dd, J = 16.10, 8.49 Hz, 1 H), 1.50 (s, 9 H), 1.35 (d, J = 3.30 Hz, 12 H).


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Step 3. Tert-butyl ((1R,2R)-2-hydroxy-6-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-2,3-dihydro-1Hinden-1-yl)carbamate (102) and tert-butyl ((1S,2S)-2-hydroxy-6-(7H-pyrrolo[2,3-d]pyrimidin-4yl)-2,3-dihydro-1H-inden-1-yl)carbamate (104) To a solution of boronate 101 (530 mg, 1.41 mmol) in dioxane:water (47 mL:12 mL) at room temperature was added pyrrolopyrimidine chloride 36 (238 mg, 1.55 mmol), K2CO3 (644 mg, 4.66 mmol) and PdCl2dppf (106 mg, 0.127 mmoL). After heating to 100 oC for 2.5 h, the reaction was diluted with ethyl acetate and washed with 10% NH4Cl and water. The organic extract was dried over Na2SO4 and the solvent removed to give the crude product, which after purification by chromatography (EtOAc/Heptanes) gave racemic product (250 mg, 48 %), as a mixture of racemic trans isomers. The racemate was separated by chiral SFC (column: ChiralPak AY-H, 30  250 mm; mobile phase: 65:35 CO2/MeOH; flow: 10 mL/min; detection:  = 210 nm) to give two peaks: 102: peak 2: Rt = 4.34; 1H

NMR (400 MHz, DMSO-d6)  12.21 (br. s., 1 H), 8.78 (s, 1 H), 8.00 (d, J = 7.80 Hz, 1 H),

7.92 (s, 1 H), 7.62 (d, J = 3.12 Hz, 1 H), 7.35 (d, J = 7.81 Hz, 1 H), 6.77 (d, J = 3.32 Hz, 1 H), 5.32 (d, J = 5.66 Hz, 1 H), 4.78 (t, J = 7.61 Hz, 1 H), 4.19–4.30 (m, 1 H), 3.09–3.22 (m, 2 H), 2.73 (dd, J = 15.90, 7.51 Hz, 1 H), 1.44 (s, 9 H); LC/MS (M+H) expected: 367.1770, observed: 367.2. Data for 104: peak 1: Rt = 3.60 min: 1H NMR (400 MHz, DMSO-d6)  12.21 (br. s., 1 H), 8.78 (s, 1 H), 8.00 (d, J = 8.00 Hz, 1 H), 7.92 (s, 1 H), 7.62 (d, J = 3.12 Hz, 1 H), 7.35 (d, J = 7.61 Hz, 2 H), 6.77 (d, J = 3.51 Hz, 1 H), 5.32 (d, J = 5.85 Hz, 1 H), 4.78 (t, J = 7.61 Hz, 1 H), 4.18–4.39 (m, 1 H), 3.04–3.21 (m, 2 H), 2.73 (dd, J = 15.81, 7.61 Hz, 1 H), 1.44 (s, 9 H); LC/MS (M+H) expected: 367.1770, observed: 367.2. Absolute stereochemistry of 102 and 104 was assigned based on single X-ray of 105 (see below). Step 4a. (1R,2R)-1-Amino-6-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-2,3-dihydro-1H-inden-2-ol (103). To a solution of 102 (111 mg, 0.30 mmol) in dioxane (4 mL) was added a 4 N solution of



HCl in dioxane (4 mL). The reaction mixture was stirred at room temperature for 1.5 h and then the solvent removed in vacuo to give a residue, which was triturated with ether to give a solid. The solid was filtered and dried to give the desired product as the HCl salt 103 (94 mg, 103%). 1H

NMR (400 MHz, DMSO-d6)  13.01 (br. s., 1 H), 9.00–8.87 (m, 2 H), 8.40 (s, 1 H), 8.12 (dd,

J = 7.90, 1.07 Hz, 1 H), 7.90 (br. s., 1 H), 7.57 (d, J = 7.81 Hz, 1 H), 7.24 (d, J = 1.95 Hz, 1 H), 4.51 (br. s., 2 H), 3.59–3.73 (m, 2 H), 3.31–3.51 (m, 2 H), 2.90 (dd, J = 16.49, 5.17 Hz, 1 H); LC/MS (M+H) expected: 267.1246, observed: 267.1. Step 4b. (1S,2S)-1-Amino-6-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-2,3-dihydro-1H-inden-2-ol (105). To a solution of 104 (55 mg, 0.30 mmol) in dioxane (2 mL) was added a 4 N solution of HCl in dioxane (2 mL). The reaction mixture was stirred at room temperature for 1.5 h and then the solvent removed in vacuo to give a residue, which was triturated with ether to give a solid. The solid was filtered and dried to give the desired product as the HCl salt 105 (47 mg, 103%). 1H NMR (400 MHz, DMSO-d6)  13.01 (br. s., 1 H), 9.00–8.87 (m, 2 H), 8.40 (s, 1 H), 8.12 (dd, J = 7.90, 1.1 Hz, 1 H), 7.90 (br. s., 1 H), 7.57 (d, J = 7.81 Hz, 1 H), 7.24 (d, J = 1.95 Hz, 1 H), 4.51 (br. s., 2 H), 3.59–3.73 (m, 2 H), 3.31–3.51 (m, 2 H), 2.90 (dd, J = 16.49, 5.17 Hz, 1 H); LC/MS (M+H) expected: 267.1246, observed: 267.1. The absolute stereochemistry of 105 was determined by single X-ray crystallography to be (S,S) (see Supporting Information), by inference 103 was assigned as (R,R). Step 5. N-((1R,2R)-2-Hydroxy-6-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-2,3-dihydro-1H-inden-1yl)cyanamide (27). To a solution of 103 (105 mg, 0.394 mmol) in DMF (4 mL) at 0 oC was added NMM (121 mg, 1.18 mmol) followed by CNBr (2M solution in DCM, 1.1 eq). After 20 min, the reaction was diluted with brine and ethyl acetate. The layers were separated and the aqueous layer extracted twice with ethyl acetate. The organic extracts were combined, dried over


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Na2SO4 and the solvent removed to give the crude product, which was diluted with methanol and triturated with diethyl ether to provide desired product 27 (40 mg, 35 %). 1H NMR (400 MHz, DMSO-d6)  12.23 (br. s., 1 H), 8.81 (s, 1 H), 7.95–8.14 (m, 2 H), 7.56–7.69 (m, 2 H), 7.42 (d, J = 8.39 Hz, 1 H), 6.89 (d, J = 2.15 Hz, 1 H), 5.64 (d, J = 5.46 Hz, 1 H), 4.15–4.41 (m, 2 H), 3.21 (dd, J = 16.10, 6.93 Hz, 1 H), 2.69–2.83 (m, 1 H); LC/MS (M+H) expected: 292.1198, observed: 292.0. Synthesis of N-((1R,3S)-3-Cyanamido-5-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-2,3dihydro-1H-inden-1-yl)methanesulfonamide (28), N-((1R,3S)-3-Cyanamido-5-(7Hpyrrolo[2,3-d]pyrimidin-4-yl)-2,3-dihydro-1H-inden-1-yl)-2-methoxyethane-1-sulfonamide (29), N-((1R,3S)-3-Cyanamido-5-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-2,3-dihydro-1H-inden1-yl)-1-methyl-1H-pyrazole-5-sulfonamide (30), N-((1R,3S)-3-Cyanamido-5-(7Hpyrrolo[2,3-d]pyrimidin-4-yl)-2,3-dihydro-1H-inden-1-yl)-3-fluorobenzenesulfonamide (31), N-((1R,3S)-3-Cyanamido-5-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-2,3-dihydro-1H-inden1-yl)-3-cyanobenzenesulfonamide (32), and N-((1R,3S)-3-Cyanamido-5-(7H-pyrrolo[2,3d]pyrimidin-4-yl)-2,3-dihydro-1H-inden-1-yl)-4-methoxybenzenesulfonamide (33): Step 1. (S)-2,2,2-Trifluoro-N-(3-(hydroxyimino)-6-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-2,3-dihydro-1Hinden-1-yl)acetamide (106). To a flask containing ketone 93 (13.0 g, 36 mmol) in ethanol (300 mL) was added NH2OH·HCl (6.2 g, 90 mmol). After heating the reaction mixture at reflux for 1 h, the mixture was concentrated in vacuo to give crude 106 (17 g, 100%) as a white solid, which was used in the next step without further purification. Step 2. N-((1S,3R)-3-Amino-6-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-2,3-dihydro-1H-inden-1-yl)2,2,2-trifluoroacetamide (107) and N-((1S,3S)-3-amino-6-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)2,3-dihydro-1H-inden-1-yl)-2,2,2-trifluoroacetamide (108). To a solution of hydroxylamine 106



(2.1 g, 5.54 mmol) in aqueous HCl (14.0 mL, 55.4 mmol) and THF (73 mL) was added zinc powder (3.63 g, 55.4 mmol). After stirring at 60 oC for 1.5 h, the reaction mixture was neutralized with NH4OH at 5 oC. The solution was extracted with 2:1 THF/EtOAc (6  300 mL) and water (200 mL). The organic layer was washed with brine and dried over Na2SO4. The solvent was removed to give the crude product, which was purified by preparative HPLC (CH3CN/H2O+ 0.22% formic acid) to give the trans derivative 108 (310 mg, 12%) as a brown solid formate salt and cis derivative 107 (858 mg, 34% yield) as a brown solid formate salt. Data for 107: cis (major): 1H NMR (400 MHz, DMSO-d6)  12.72 (br. s., 1 H), 10.19 (d, J = 7.82 Hz, 1 H), 8.96 (s, 1 H), 8.88 (br. s., 3 H), 8.22 (d, J = 8.31 Hz, 1 H), 8.01 (s, 1 H), 7.94 (d, J = 7.82 Hz, 1 H), 7.84 (br. s., 1 H), 6.91 (d, J = 1.96 Hz, 1 H), 5.51 (q, J = 7.83 Hz, 1 H), 4.83 (d, J = 5.38 Hz, 1 H), 3.02 (dt, J = 12.84, 7.76 Hz, 1 H), 2.17 (dt, J = 12.96, 7.70 Hz, 1 H); LC/MS (M+H) expected: 362.12, observed: 362.1. Data for 108: trans (minor): 1H NMR (400 MHz, DMSO-d6)  12.80 (br. s., 1 H), 10.08 (d, J = 7.82 Hz, 1 H), 8.98 (s, 1 H), 8.75 (br. s., 3 H), 8.22 (d, J = 7.82 Hz, 1 H), 8.07 (s, 1 H), 7.95 (d, J = 8.31 Hz, 1 H), 7.86 (br. s., 1 H), 6.91 (d, J = 1.47 Hz, 1 H), 5.79 (q, J = 7.34 Hz, 1 H), 5.03 (br. s., 1 H), 2.55 - 2.63 (m, 2 H); LC/MS (M+H) expected: 362.12, observed: 362.1. Step 3. 2,2,2-Trifluoro-N-((1S,3R)-3-(methylsulfonamido)-6-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)2,3-dihydro-1H-inden-1-yl)acetamide (109a). To a solution of amine 107 (150 mg, 0.38 mmol) in pyridine (10 mL) at 0 oC was added methylsulfonyl chloride (65.1 mg, 0.57 mmol in 0.5 mL of pyridine). After stirring at room temperature overnight, the reaction mixture was quenched by the addition of H2O (10 mL) and the mixture extracted with EtOAc (2  30 mL). The organic layer was dried over Na2SO4 and the solvent removed in vacuo to give a residue which was


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purified via column chromatography on (MeOH:DCM, 2% to 20%) to give 109a (100 mg, 60.3%) as a white solid. Step 4. N-((1R,3S)-3-Amino-5-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-2,3-dihydro-1H-inden-1yl)methanesulfonamide (110a). To a solution of 109a (100 mg, 0.23 mmol) in ethanol/water (10 mL: 1.5 mL) was added K2CO3 (94.09 mg, 0.68 mmol). After heating at reflux for 8 h, the solvent was removed in vacuo and the residue was treated with silica gel directly and purified via column chromatography (MeOH: DCM, 2% to 20%) to give 110a (60 mg, 77.06%) as a white solid. Step 5. N-((1R,3S)-3-Cyanamido-5-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-2,3-dihydro-1H-inden-1yl)methanesulfonamide (28). To a solution of 110a (60 mg, 0.175 mmol) in DMF (2 mL) was added NMM (35 mg, 0.35 mmol) at room temperature. The mixture was cooled to -30 oC and a solution of BrCN (22.3 mg, 0.21 mmol in 0.5 mL of DMF) was added dropwise. After 30 min at -30 oC, the reaction mixture was diluted with water and extracted with EtOAc (2  30 mL). The organic layer was dried over Na2SO4, concentrated and purified via column chromatography (MeOH: DCM, gradient of 2% to 10%) to afford 28 (12 mg, 18.75%) as an off-yellow solid. 1H NMR (400 MHz, DMSO) δ 12.32 (s, 1H), 8.85 (s, 1H), 8.20–8.15 (m, 2H), 7.88 (m, 1H), 7.74– 7.69 (m, 2H), 7.61 (m, 1H), 6.92 (m, 1H), 4.83 (m, 1H), 4.62 (m, 1H), 3.10 (s, 3H), 3.01 (m, 1H), 1.92–1.89 (m, 1H).; LC/MS (M+H) expected 369.11, observed 391.2. HRMS (ESI) m/z: calculated for C17H16N6O2S [M + H]+ 369.1128; observed 369.1127. Compounds 29–33 were prepared via a similar sequence as that described for 28, but done in parallel. The data for final compounds 29–33 is below: N-((1R,3S)-3-Cyanamido-5-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-2,3-dihydro-1H-inden-1-yl)2-methoxyethane-1-sulfonamide (29). 29 (25 mg, 20%) was obtained as white solid. 1H NMR



(400 MHz, DMSO) δ 12.31 (s, 1H), 8.86 (s, 1H), 8.22–8.190 (d, 1H), 8.17 (s, 1H), 7.95–7.93(m, 1H), 7.74–7.70 (m, 2H), 7.63–7.61 (d, 1H), 6.93–6.92 (d, 1H), 4.83–4.81 (m, 1H), 4.62 (s, 1H), 3.78–3.74 (m, 2H), 3.49–3.45 (m, 2H), 3.32 (s, 3H), 2.98-2.95 (m1 1H),1.94-1.91 (m, 1H). LC/MS (M+H) expected: 412.13, observed: 413.2. HRMS (ESI) m/z: calculated for C19H20N6O3S [M + H]+ 413.1390; observed 413.1388. N-((1R,3S)-3-Cyanamido-5-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-2,3-dihydro-1H-inden-1-yl)1-methyl-1H-pyrazole-5-sulfonamide (30). The residue was lyophilized to give 30 (35 mg, 34.7%) as an off-yellow solid. 1H NMR (400 MHz, DMSO) δ 12.31 (s, 1H), 9.07–9.05 (s, 1H), 8.84 (S, 1H), 8.17–8.13(m, 2H), 7.71–7.68 (m, 2H), 7.62 (m, 1H), 7.35–7.33 (m, 1H), 6.90–6.85 (m, 2H), 4.63–4.61 (m, 1H), 4.57–4.52 (m, 1H),4.08 (m, 1H), 2.52-2.49 (m, 1H), 1.73 (m, 1H); LC/MS (M+H) expected: 435.14, observed: 435.1. HRMS (ESI) m/z: calculated for C20H18N8O2S [M + H]+ 435.1346; observed 435.1346. N-((1R,3S)-3-Cyanamido-5-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-2,3-dihydro-1H-inden-1-yl)3-fluorobenzenesulfonamide (31). 1H NMR (400 MHz, DMSO-d6)  12.30 (br. s., 1 H) 8.85 (s, 1 H) 8.64 (d, J = 6.02 Hz, 1 H) 8.04–8.30 (m, 2 H) 7.53–7.88 (m, 7 H) 7.40 (d, J = 7.53 Hz, 1 H) 6.90 (d, J = 3.01 Hz, 1 H) 4.80 (d, J = 6.02 Hz, 1 H) 4.54 (br. s., 1 H) 2.39 - 2.48 (m, 1 H) 1.55– 1.81 (m, 1 H); LC/MS (M+H) expected 456.12, observed 456.1. HRMS (ESI) m/z: calculated for C23H17N7O2S [M + H]+ 456.1237; observed 456.1238. N-((1R,3S)-3-Cyanamido-5-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-2,3-dihydro-1H-inden-1-yl)3-cyanobenzenesulfonamide (32). Purified via column chromatography (CH3OH: DCM, gradient 2% to10%) to afford 32 (30 mg, 35%) as a white solid.1H NMR (400 MHz, MeOD) δ 8.81 (s, 1H), 8.38 (s, 1 H), 8.32–8.29 (m, 1 H), 8.11–8.06 (m, 3H), 7.88–7.84 (m, 1H), 7.58–7.56 (m, 1 H), 7.46–7.44 (m, 1 H), 6.91–6.90 (m, 1H), 4.96–4.91 (m, 1 H), 4.63–4.60 (m, 1 H), 2.75–


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2.72 (m, 1 H), 1.78–1.75 (m, 1 H); LC/MS (M+Na) expected: 478.11, observed: 478.0. HRMS (ESI) m/z: calculated for C22H17FN6O2S [M + H]+ 449.1190; observed 449.1188. N-((1R,3S)-3-Cyanamido-5-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-2,3-dihydro-1H-inden-1-yl)4-methoxybenzenesulfonamide (33). 1H NMR (400 MHz, DMSO-d6)  12.31 (br. s., 1 H) 8.85 (s, 1 H) 8.33 (d, J = 9.03 Hz, 1 H) 8.05–8.22 (m, 2 H) 7.87 (d, J = 8.53 Hz, 2 H) 7.59–7.74 (m, 2 H) 7.42 (d, J = 8.03 Hz, 1 H) 7.19 (d, J = 8.53 Hz, 2 H) 6.90 (d, J = 2.51 Hz, 1 H) 4.69 (d, J = 7.53 Hz, 1 H) 4.52 (d, J = 8.53 Hz, 1 H) 3.79–3.92 (m, 3 H) 2.37–2.45 (m, 1 H) 1.53–1.79 (m, 1 H); LC/MS (M+H) expected: 461.14, observed: 461.1. Synthesis of (S)-N-(6-(5-Phenyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-2,3-dihydro-1Hinden-1-yl)cyanamide (34). Step 1. (S)-1-Azido-6-bromo-2,3-dihydro-1H-indene (111). To a solution of enantiomerically pure alcohol 11059 (8.1 g, 38 mmol) in PhMe (230 mL) was added DPPA (13.6 g, 49 mmol) at 0 oC, followed by DBU (8.08 g, 53 mmol). After 4 h at room temperature, the reaction was quenched with H2O (150 mL) at 0 - 10 oC and ethyl acetate (150 mL) was added. The organic layer was separated, collected and dried over Na2SO4. The solvent was removed and the crude product was purified by column chromatography (petroleum ether) to give 112 (6.3 g, 70%) as oil. LC/MS (M+H+) expected: 238.00, observed: 238.0. Step 2. (S)-6-Bromo-2,3-dihydro-1H-inden-1-amine (113). To a solution of azide 112 (2 g, 8.4 mmol) in MeOH (60 mL) was added SnCl2 (2.8 g, 12.6 mmol) at 0 oC. After 5 h at room temperature, the reaction mixture was concentrated to dryness and aqueous 1 M NaOH (20 mL) added. The aqueous mixture was extracted with ethyl acetate (2  100 mL). Aqueous 1 M HCl (100 mL) was added to the combined organic layer and stirred for 5 min then the aqueous layer was separated and basified to pH = 9–10 with 1 M NaOH. The solution was extracted with ethyl



acetate (3  100 mL) and organic extracts were dried over Na2SO4. The solvent was removed to give 113 (1.5 g, 78 %) as oil. LC/MS (M+H+) expected: 212.01, observed: 212.0. Step 3. Tert-butyl (S)-(6-bromo-2,3-dihydro-1H-inden-1-yl)carbamate (113). To a solution of amine 113, (1.5 g, 7 mmol) in DCM (60 mL) at 0 oC was added Et3N (2.9 mL, 21 mmol) followed by Boc2O (1.98 g, 9.19 mmol). After 2 h at room temperature, the reaction mixture was quenched with H2O (20 mL) and the organic layer separated, dried over Na2SO4 and the solvent removed to give crude product. The crude product was purified by column chromatography (EtOAc: petroleum ether, 2% to 5%) to give 114 (1.4 g, 63%) as a solid. 1H NMR (400 MHz, DMSO-d6)  7.27–7.42 (m, 3 H), 7.20 (d, J = 8.20 Hz, 1 H), 4.99 (d, J = 7.81 Hz, 1 H), 2.81– 2.92 (m, 1 H), 2.72 (dt, J = 16.10, 8.15 Hz, 1 H), 2.29–2.42 (m, 1 H), 1.76–1.91 (m, 1 H), 1.45 (s, 9 H); LC/MS (M+H+) expected: 312.06, observed: 312.0; 24D = -56.5 (c = 25 mg/mL, MeOH). Step 4. Tert-butyl (S)-(6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2,3-dihydro-1H-inden-1yl)carbamate (115). To a solution of bromide 114 (1.5 g, 4.8 mmol) in dioxane (80 mL) at room temperature was added bis(pinacolato)diborane (1.71 g, 6.76 mmol) and KOAc (1.88 g, 19.2 mmol). Pd(dppf)Cl2 (0.27 g, 0.336 mmol) was added under N2. After heating the reaction mixture at 80 oC for 3 h, it was concentrated to dryness. The reaction mixture was partitioned between ethyl acetate (100 mL) and H2O (15 mL). The organic layer was collected, dried over Na2SO4 and the solvent removed in vacuo to give a residue, which was purified via chromatography (EtOAc: petroleum ether, 2% to 5%) to afford 115 (1.54 g, 90 %) as yellow oil. LC/MS (M+H+) expected: 233.14, observed: 233.1. Step 5. Tert-butyl (S)-(6-(5-phenyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-2,3-dihydro-1H-inden-1yl)carbamate (117). To a solution of boronate 115 (1.1 g, 3.06 mmol) in dioxane (80 mL) and


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H2O (20 mL) at room temperature was added chloride 116 (0.7 g, 3.06 mmol) and K2CO3 (1.2 g, 9.2 mmol). Pd(dppf)Cl2 (0.12 g, 0.15 mmol) was added under N2. After heating the reaction mixture at 90 oC for 9 h, it was concentrated to dryness and diluted with ethyl acetate (60 mL) and H2O (10 mL). The organic layer was collected, dried over Na2SO4 and the solvent removed to give a residue, which was purified by chromatography (EtOAc: petroleum ether, 10% to 50%) to afford 117 (480 mg, 36 %) as a yellow solid. LC/MS (M+H+) expected: 427.21, observed: 427.2. Step 6. (S)-6-(5-Phenyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-2,3-dihydro-1H-inden-1-amine (118). To a solution of 117 (80 mg, 0.18 mmol) in DCM (6 mL) at 0 oC was added TFA (1 mL). After 2 h, the solvent was removed in vacuo and the residue was diluted with ethyl acetate (15 mL) and H2O (20 mL). The organic layer was separated and discarded. The pH of the aqueous layer was adjusted to 9-10 with aqueous K2CO3 and then extracted with (6:1 CHCl3:CH3OH, 3  30 mL). The combined organic extracts were washed with brine (10 mL), dried over Na2SO4 and the solvent removed to give amine 118 (48 mg, 78%) as white solid. 1H NMR (400 MHz, DMSO-d6) δ 8.84 (s, 1H), 7.72 (s, 1H), 7.36–7.34 (m, 1H), 7.34–7.05 (m, 5H), 6.95–6.93 (m, 2H), 3.85– 3.81 (m, 1H), 2.83–2.76 (m, 1H), 2.68–2.60 (m, 1H), 2.26–2.21 (m, 1H), 1.46–1.44 (m, 1H); Chiral SFC (column: Chiralpak AS-H, 150  4.6 mm; mobile phase: MeOH (0.05% DEA)/CO2 55 to 40%): Rt = 4.974 min; LC/MS (M+H) expected: 327.16, observed: 326.9. Step 7. (S)-N-(6-(5-Phenyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-2,3-dihydro-1H-inden-1yl)cyanamide (34). To a solution of amine 118 (60 mg, 0.184 mmol) in DMF (8 mL) at 0 oC was added NMM (0.06 mL, 0.55 mmol). The reaction was then cooled to -25 oC and BrCN (29 mg, 0.27 mmol) was added under N2. After 1 hr at 0 oC, the reaction was quenched with H2O (8 mL) and diluted with DCM (20 mL). The organic layer was collected, dried over Na2SO4 and the



solvent removed to give the crude product, which was purified by column chromatography (EtOAc:DCM 5% to 35%) to give 34 (30 mg, 46% ) as white solid. 1H NMR (400 MHz, DMSOd6) δ 12.46 (s, 1H), 8.86 (s, 1H), 7.73 (s, 1H), 7.32 (s, 1H), 7.24–6.94 (m, 9H), 4.24–4.22 (m, 1H), 2.93–2.88 (m, 1H), 2.78–2.73 (m, 1H), 2.34–2.32 (m, 1H), 1.87–1.83 (m, 1H); Chiral SFC (column: Chiralpak AS-H, 150  4.6 mm; mobile phase: MeOH (0.05% DEA)/CO2 55 to 40%): Rt = 5.559 min.; LC/MS (M+H) expected: 352.16, observed: 352.2. Synthesis of (S)-N-(6-(5-(3-(Hydroxymethyl)phenyl)-7H-pyrrolo[2,3-d]pyrimidin-4yl)-2,3-dihydro-1H-inden-1-yl)cyanamide (35). Step 1. (3-(4-Chloro-7-((2(trimethylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-5-yl)phenyl)methanol (121). To a mixture of iodide 119 (13.3 g, 32.5 mmol), boronate 120 (5.43 g, 35.7 mmol), K2CO3 (8.97 g, 65 mmol) in dioxane:H2O (150:30 mL) at room temperature was added Pd(dppf)Cl2 (1.19 g, 1.63 mmol). After heating the reaction mixture at reflux for 3 h, the mixture was diluted with ethyl acetate (1.5 L), washed with brine (500 mL), dried over sodium sulfate and the solvent removed. The crude product was purified by chromatography (EtOAc/petroleum ether, 10 to 50%) to afford 121 (13 g, 100%) as a yellow solid. LC/MS (M+H) expected: 389.1326, observed: 389.15. Step 2. (3-(4-Chloro-7H-pyrrolo[2,3-d]pyrimidin-5-yl)phenyl)methanol (122). To a solution of 121 (13.0 g, 32.5 mmol) in DCM (66 mL) at room temperature was added dropwise TFA (33 mL). After 4 h at room temperature, the reaction solution was concentrated to afford a brown oil, The solution of crude 122 in methanol (150 mL) was adjusted to pH = 10 with addition of solid K2CO3. After 2.5 h at room temperature, the solvent was removed and the residue was partitioned between water (500 mL) and ethyl acetate (1 L). The layers were separated and the aqueous phase was extracted with ethyl acetate (2 × 500 mL). The combined organic extracts


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were dried over Na2SO4 and the solvent removed to give 122 (7.5 g, 89%) as a yellow solid, which was used for the next step without purification. LC/MS (M+H) expected: 260.0591, observed: 260.0. Step 3. Tert-butyl (S)-(6-(5-(3-(hydroxymethyl)phenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-2,3dihydro-1H-inden-1-yl)carbamate (123). To a mixture of chloride 122 (1.6 g, 5.64 mmol), chiral boronate 115 (2.02 g, 5.64 mmol), K2CO3 (1.6 g, 11.2 mmol) in dioxane:water (30:6 mL) at room temperature was added Pd(dppf)Cl2 (410 mg, 0.56 mmol). After heating the reaction mixture at 80 oC for 24 h, the mixture was cooled to room temperature and then diluted with water (50 mL). The aqueous mixture was extracted with ethyl acetate (3 × 200 mL) and the organic layers collected, dried over Na2SO4 and solvent removed. The crude product was purified by column chromatography (EtOAc/petroleum ether, 10% to 100%) to afford 123 (1.1 g, 42.8%). LC/MS (M+H) expected: 457.22, observed: 457.2. Step 4. (S)-(3-(4-(3-Amino-2,3-dihydro-1H-inden-5-yl)-7H-pyrrolo[2,3-d]pyrimidin-5yl)phenyl)methanol (124). To a solution of 123, (350 mg, 0.77 mmol) in EtOAc (5 mL) at 0 oC was added a 4 M solution of HCl in ethyl acetate (5 mL). After the addition was completed, the reaction mixture was stirred at room temperature for 2 h and then concentrated to give 360 mg of crude product, 260 mg of which was purified by preparative HPLC to afford amine 124 (30.5 mg, 11%) as white solid. 1H NMR (400 MHz, DMSO-d6) δ 8.85 (s, 1H), 7.73 (s, 1H), 7.30 (d, 1H), 7.17 (s, 1H), 7.10 (m, 3H), 6.99 (d, 1H), 6.67 (s, 1H), 4.18 (s, 2H), 3.95 (m, 1H), 2.89 (m, 1H), 2.73 (m, 1H), 2.29 (m, 1H), 1.57 (m, 1H); LC/MS (M+H) expected: 357.17, observed: 357.4. Step 5. (S)-N-(6-(5-(3-(Hydroxymethyl)phenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-2,3-dihydro1H-inden-1-yl)cyanamide (35). To a solution of amine 124 (100 mg, 0.26 mmol) as HCl salt and



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NMM (78 mg, 0.78 mmol) in DMF (5 mL) at -20 oC was added BrCN (55 mg, 0.52 mmol). After stirring the reaction mixture at -20 oC for 30 min, it was diluted with brine (20 mL). The aqueous mixture was extracted with ethyl acetate (3 × 20 mL) and the organic extracts combined, washed with brine (2 × 10 mL) and dried over Na2SO4. The solvent was removed to give a residue, which was purified by column chromatography (NH4OH/MeOH/DCM, 0/1/50 to 1/10/100) to afford 35 (41 mg, 41%) as a yellow solid. 1H NMR (400 MHz, DMSO-d6): δ 12.45 (br, 1H), 8.85 (s, 1H), 7.72 (s, 1H), 7.23 (m, 2H), 7.14 (m, 4H), 6.89 (d, 1H), 6.69 (s, 1H), 5.00 (m, 1H), 4.23 (m, 3H), 2.93 (m, 1H), 2.77 (m, 1H), 2.39 (m, 1H), 1.88 (m, 1H); LC/MS (M+H) expected: 404.1487, observed: 404.4. HRMS (ESI) m/z: calculated for C23H19N5O [M + H]+ 382.1662; observed 382.1661. The JAK enzyme data and the PBMC data were obtained as previously described.36,27 Determination of compound binding and JAK3 inactivation kinetics. The kinetics of compound binding/dissociation to JAK3 and JAK3 inactivation (kinact/Ki) was measured using a time-resolved Förster resonance energy transfer (TR-FRET) competition binding assay based on the LanthaScreen™ Eu Kinase Binding Assay (Invitrogen/Life Technologies). The assay utilizes a fluorescently labeled kinase active site probe and a JAK3 construct labeled with a Eu3+conjugated antibody. Active site occupancy by the probe elicits a TR-FRET signal between the Eu3+-labeled kinase and probe, and thus compound binding or dissociation is detected by the concomitant change in TR-FRET signal upon exchange with the probe. Experimental conditions (final assay concentrations) were: 4 nM JAK3-GST (human protein, GST-tagged, amino acids 781-1124; Invitrogen #PR7507B), and 2 nM Eu3+-labeled anti-GST antibody (Invitrogen #PV5594). Assay buffer conditions were 20 mM HEPES, pH 7.5, 10 mM MgCl2, 0.0005% Tween 20, 0.01% BSA, 1 mM DTT. Compounds were prepared at 100


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concentration in DMSO. A 1:3 dilution series was prepared for a total number of five compound concentrations in addition to a “no compound” control. Immediately prior to assay, 4 compound were prepared by dilution of the 100 DMSO solutions into buffer containing no DTT. Experiments were performed in a low volume NBS, black 384-well plate (Corning #3676). Two assays types were performed for each compound: To measure compound dissociation (20 µL total volume): 15 µL solutions containing a 1.33 final concentration of JAK3, compound and Eu+3-labeled anti-GST antibody were incubated at room temperature for 2 h, and the assay was initiated by adding 5 µL of 4 of final concentration of kinase tracer-236 (Invitrogen # PV5592) (prepared in 4% DMSO, final 150 nM). To measure compound binding (20 µL total volume): 15 µL solutions containing a 1.33 final concentration of JAK3, Eu+3-labeled anti-GST antibody, and kinase tracer-178 (Invitrogen # PV5593, 150 nM final) were incubated at room temperature for 2 h, and the assay was initiated by adding 5 µL of 4 compound. Each assay was mixed thoroughly, and then read on an EnVision plate reader (excitation wavelength, 340 nm; TR-FRET was calculated by dividing the signal from the emission peak of the probe (665 nm) by that of the europium (615 nm). Data was recorder every 60 seconds for 75 cycles. Data was analyzed by global fit of the TR-FRET data to the integrated rate equation using the DynaFit fitting program (BioKin Ltd.). For reversible inhibitors, the data were analyzed according to a simple one-step binding mechanism described by an on-rate (kon) and off-rate (koff). JAK3 inactivation by a covalent inhibitor is described by the following two-step mechanism: Step 1.

E + I = E∙I

Step 2.

E∙I → E∙I*



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Steps 1 and 2 are described by Ki (equal to koff/kon) and kinact, respectively, and the ratio (kinact/Ki) is the apparent second order rate constant for kinase inactivation. For most compounds tested, the data could not be fit to a unique solution for a two-step mechanism, and were thus analyzed according to a single step inactivation mechanism, where the observed rate constant is equal to (kinact/Ki). The t1/2 was calculated from the off-rate (ln2/koff). JAK3 Kinase kinetics. ATP, phosphoenolpyruvate (PEP), NADH, pyruvate kinase (PK), lactate dehydrogenase (LDH), and the buffer HEPES were purchased from Sigma Chemical Co. (St. Louis, MO). The JAK1 peptide substrate (KAIETDKEYYTVKD-NH2) was from AnaSpec (San Jose, CA). The enzymatic activity of JAK3 (JH1 domain was determined at 25°C using the coupled PK/LDH assay at 340 nm on a Tecan plate reader in a base assay buffer of 25 mM HEPES (pH 7.5), 10 mM MgCl2, 25 mM KCl, 0.005% Brij-35, and 1 mM DTT. The continuous assay solution contained an additional 20 units of pyruvate kinase (PK), 30 units of lactate dehydrogenase (LDH), 0.25 mM NADH, and 2 mM PEP. Determination of JAK3 Cys909 pKa by the kinetics of 125 irreversible inhibition across a range of pH values. JAK3 (2.5 M final concentration) was incubated with 5 M of 1((4aR,8aS)-4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)hexahydro-2H-pyrido[4,3-b][1,4]oxazin-6(5H)yl)prop-2-en-1-one (125, Figure 11)63 in buffer of varying pH between 5.1 and 9.7 using the multi-component system of 50 mM MES, 25 mM Tris, 25 mM CAPS and 50 mM NaCl (MTCN buffer) containing 10 mM MgCl2, 1 mM DTT, and 0.005% Brij-35. After various incubation times, 2 L aliquots were removed and diluted 1:100 into continuous assay buffer containing 1 mM ATP, and 0.2 mM JAK1 peptide substrate. JAK3 incubated with buffer served as control to determine the percent of activity remaining in each kinase-inhibitor aliquot. The percent remaining activity was plotted against time and fit to a single exponential to determine the kinact.


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The kinact at each pH was then plotted against pH, and the pKa was determined by a direct fit to equation 1, y = ymin +

(ymax ― ymin) (pK ― pH) 1 + 10 a

(1)

where ymin is the lower plateau at low pH, and ymax is the upper plateau at high pH. The data were analyzed using GraphPad Prism version 6.03 for Windows (GraphPad Software, La Jolla California). H N O

O

H N N N 125

N H

Figure 11. Structure of acrylamide 125. Determination of reactivity of compounds with glutathione. A solution of L-Glutathione reduced was prepared by dissolving L-Glutathione reduced (Sigma G-4251) in 100 mM phosphate buffer (pH = 7.4). The concentration of this solution was 5.5 mM. A 20 µM stock solution of test compound was prepared in DMSO. In a microtube kept at 37 °C in a heat-block, 450 µL of 5.5 mM L-Glutathione reduced solution or 100 mM phosphate buffer were added and warm for approximately 5 min. 50 µL of 20 µM test compound solution was added into above matrixes to start reaction. The mixture was incubated at 37 °C. The reaction was quenched by removing 10 L sample and placed into 200 L acetonitrile containing internal standard at the required time points. Sample was analyzed by LC-MS/MS. The area ratio was used for calculation.



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%inhibition: (1-(60 min instrument response of test compound in glutathione/ 60 min instrument response test compound in buffer))  100 Pharmacokinetics Studies. Plasma concentrations of cyanamide 12 were determined in male rats (n=2) after a single 1 mg/kg intravenous (IV) dose with pretreatment of 100 mg/kg oral (PO) ABT, which was conducted at comparative medicine (Pfizer) and 1 mg/kg intravenous (IV) dose without pretreatment of 100 mg/kg oral (PO) ABT, which was conducted at BioDuro (Beijing, China) according to standard protocols, respectively. Briefly, test compound and ABT were formulated in standard solubilizing vehicles (IV dose (test compound)): 10% DMSO / 50% PEG400 / 40% Water; (PO dose (ABT)): 0.5% methycellulose in water. Serial blood samples were collected at various times from 5 min to 24 h. Plasma was harvested from blood samples following centrifugation. Plasma samples (50 µL) and standards were prepared by protein precipitation with acetonitrile (1:1 v:v) containing an internal standard, propranolol or terfenadine. Samples were vortexed and centrifuged to obtain supernatant, which was analyzed using general LC-MS/MS methodology as described above (Sciex API 4000; sciex.com). Pharmacokinetic parameters were determined from individual animal data using noncompartmental analysis in Watson LIMS 7.4.1 (thermoscientific.com). Metabolism Studies. Stock solutions of cyanamide 12 were prepared in dimethyl sulfoxide/acetonitrile [25%:75%, (v/v)]. The final concentration of organic solvent in the incubation medium was 0.1% (v/v). Incubations were carried out at 37°C for 45 min in a shaking water bath. The incubation volume was 3 mL and consisted of the following: 0.1 M potassium phosphate buffer (pH 7.4) containing MgCl2 (5 mM), human liver microsomes (purchased from BD Gentest, Woburn, MA; P450 concentration 25 pmol), NADPH (1.3 mM), substrate (10 µM), and GSH (5 mM). Incubations that lacked either NADPH or GSH served as negative controls.


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Aliquots (1 mL) were removed after at 10 and 45 min and reactions were terminated by the addition of ice-cold acetonitrile (4 mL). The samples were combined and centrifuged (3,000g, 15 min), and the supernatants were dried under a steady nitrogen stream. The residue was reconstituted with mobile phase, vortexed, centrifuged (15,000g, 3 min) and analyzed for metabolite formation by liquid chromatography tandem mass spectrometry (LC-MS/MS). Qualitative assessment of the metabolism of cyanamide 12 was conducted using a Thermo Finnegan Surveyor photodiode array plus detector, Thermo Acela pump and a Thermo Acela Autosampler (Thermo Scientific, West Palm Beach, FL). The monitoring wavelength (λ) was 280 nm. Chromatography was performed on a Phenomenex Aqua C18 (2.0 mm x 150 mm, 5 μm) column. The mobile phase composed of 0.1% HCOOH (solvent A) and acetonitrile (solvent B) at a flow rate of 0.7 mL/min. The binary gradient was as follows: solvent A to solvent B ratio was held at 95:5 (v/v) for 5 min and then adjusted to 30:70 (v/v) from 5 to 40 min, 30:70 (v/v) from 40 to 43 min, and 5:95 (v/v) from 44 to 60 min. Identification of the metabolites was performed on a Thermo Orbitrap mass spectrometer operating in positive ion electrospray mode. The spray potential was 3 kV and heated capillary was at 275 °C. Xcalibur software version 2.0 was used to control the HPLC-MS system. MS2 and MS3 product ion spectra were acquired at a normalized collision energy of 20 and 40 eV, with an isolation width of 2 amu. LC-MS spectra were acquired over a mass range of 150-900 m/z. Data dependent scanning was used to trigger MS2 and MS3 analysis of molecular ions and product ions. The two most intense ion from MS and MS2 scans were selected for MS2 and MS3 scans, respectively. Searching of LC-MSn data for molecular ions representing possible metabolites of 12 was performed manually. A reference list of all possible metabolites of 12 and their expected molecular ions was used during this process. LC-MS peaks identified to be possible metabolites, based on the molecular ions, were


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compared against blank samples. In cases where the signal strength was of sufficient intensity to trigger MSn scanning, the subsequent MS2 and MS3 spectra were examined to further confirm the identity and structure of possible metabolites. In addition, LC-MS chromatograms were compared directly against the appropriate blank chromatogram to identify additional metabolite peaks. X-ray crystallography. Crystal structures of JAK3 bound to 10, 12, 23, and 34 were determined as reported previously.38 To generate the desired complexes, JAK3/CMP-6 crystals were soaked for ~24 hours in the reservoir solution supplemented with 3 mM compound, resulting in full exchange of the bound CMP-6 for the compound of interest. Data for Jak3/ 10 were collected at 100° K on a Rigaku FRE X-ray generator outfitted with a Saturn 944 CCD detector and processed with HKL2000.65 Data for JAK3/12, JAK3/23, and JAK3/34 were collected at 100° K at the Industrial Macromolecular Crystallography Association (IMCA) Collaborative Access Team Beamline 17-ID (Advanced Photon Source, Argonne National Laboratory, Argonne, IL, USA), and processed using XDS (Kabsch 2010) and AUTOPROC.66 All other data manipulations utilized the CCP4 suite of programs.67 For all data sets, a common test set of reflections corresponding to ~5% of the total were used throughout refinement. All structures were solved by rigid body refinement in REFMAC using a reference structure of JAK3 with all ligands and waters removed.68 Iterative rounds of refinement using AUTOBUSTER69 were interspersed with manual model building in COOT.70 Crystallographic data statistics and refinement results are detailed in Supporting Information S-Table 1. PDB CODES. The coordinates and structure factor amplitudes for JAK3 complexes with compounds 10, 12, 23, and 34 have been deposited with the Protein Data Bank (http://www.rcsb.org), with accession codes: 6DA4 (10), 6DUD (12), 6DB3 (23), and 6DB4


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(34). The authors will release the atomic coordinates and experimental data upon article publication. ASSOCIATED CONTENT Supporting information available: Crystallographic data collection and structure refinement statistics for structures of JAK3 bound to 10, 12, 23, or 34; single molecule X-ray crystallography for 13, 105, and 114; 1H NMR data for 90 and 91; electron density of 10, 12, 23, and 34 in complex with JAK3; determination of preferred tautomer form of 12 using 2D NMR; 1H

NMR and HPLC data for 34 and 35; Molecular formula strings and some data (CSV).

ACKNOWLEDGEMENTS We thank Tsung Lin, Jason Jussif and Nancy Wood for PBMC and human whole blood assay data, Shihua Yao for JAK3 inactivation kinetics data, and Cuiman Cai, Gaurav Arora, Arindrajit Basak, Mihir D. Parikh and Steven E. Heasley for chemistry contributions. This research used resources at the Industrial Macromolecular Crystallography Association Collaborative Access Team (IMCA-CAT) beamline 17-ID, supported by the companies of the Industrial Macromolecular Crystallography Association through a contract with Hauptman-Woodward Medical Research Institute. This research used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357. AUTHOR INFORMATION Corresponding author * Phone: (617) 665-5673. E-mail: [email protected]. Notes:



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The authors declare the following competing financial interest(s): This work was funded by Pfizer. All authors were employed by Pfizer Inc. at the time this work was done. All activities involving laboratory animals were carried out in accordance with federal, state, local, and institutional guidelines governing the use of laboratory animals in research and were reviewed and approved by Pfizer Institutional Animal Care and Use Committee. Pfizer animal care facilities that supported this work are fully accredited by AAALAC International.

ABBREVIATIONS ABT, aminobenzotriazole; ATP, adenosine triphosphate; BLK, B lymphocyte kinase; BMX, cytoplasmic tyrosine-protein kinase BMX; BTK, Bruton’s tyrosine kinase; CID, collisioninduced dissociation; COSY, homonuclear correlation spectroscopy; CRG, cysteine reacting group; Cys, cysteine; EGFR, epidermal growth factor receptor; GSH, glutathione; GST, glutathione-S-transferase; HER-2, receptor tyrosine-protein kinase erbB-2; HER-4, receptor tyrosine-protein kinase erbB-4; HSQC, heteronuclear single quantum coherence spectroscopy; ITK, interleukin-2-inducible T-cell kinase; JAK1, Janus kinase 1; JAK2, Janus kinase 2; JAK3, Janus kinase 3; PBMC, peripheral blood mononuclear cell; SCID, severe combined immunodeficiency; SFC, supercritical fluid chromatography; STAT, signal transducer and activator of transcription protein; TEC, tyrosine-protein kinase TEC; TYK2, tyrosine kinase 2; TXK, tyrosine-protein kinase TXK. References 1.

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Table of Contents Graphic H O N S O

N

N N H

12

Modulation of Ki and kinact N

N H

32 N

N H

JAK3 IC50 = 256 nM 2x selective vs other JAKs

F

N N

N H

JAK3 IC50 = 11 nM 246x selective vs other JAKs


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Identification of Cyanamide-Based Janus Kinase 3 (JAK3) Covalent

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