Design and evaluation of novel 8-oxo-pyridopyrimidine Jak1/2 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2013.08.082

A highly ligand efficient, novel 8-oxo-pyridopyrimidine containing inhibitor of Jak1 and Jak2 isoforms with a pyridone moiety as the hinge-binding motif was discovered. Structure-based design strategies were applied to significantly improve enzyme potency and the polarity of the molecule was adjusted to gain cellular activity. The crystal structures of two representative inhibitors bound to Jak1 were obtained to enable SAR exploration.

Full text of DOI:10.1016/j.bmcl.2013.08.082

Accepted Manuscript
Design and Evaluation of Novel 8-Oxo-pyridopyrimidine Jak1/2 Inhibitors
Sharada Labadie, Kathy Barrett, Wade S. Blair, Christine Chang, Gauri
Deshmukh, Charles Eigenbrot, Paul Gibbons, Adam Johnson, Jane R. Kenny,
Pawan Bir Kohli, Marya Liimatta, Patrick J. Lupardus, Steven Shia, Micah
Steffek, Savita Ubhayakar, Anne van Abbema, Mark Zak
PII:
S0960-894X(13)01022-6
DOI:
http://dx.doi.org/10.1016/j.bmcl.2013.08.082
BMCL 20820
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
13 June 2013
14 August 2013
19 August 2013
Please cite this article as: Labadie, S., Barrett, K., Blair, W.S., Chang, C., Deshmukh, G., Eigenbrot, C., Gibbons,
P., Johnson, A., Kenny, J.R., Kohli, P.B., Liimatta, M., Lupardus, P.J., Shia, S., Steffek, M., Ubhayakar, S., Abbema,
A.v., Zak, M., Design and Evaluation of Novel 8-Oxo-pyridopyrimidine Jak1/2 Inhibitors, Bioorganic & Medicinal
Chemistry Letters (2013), doi: http://dx.doi.org/10.1016/j.bmcl.2013.08.082
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Design and Evaluation of Novel 8-Oxo-pyridopyrimidine
Jak1/2 Inhibitors
Sharada Labadie,* Kathy Barrett, Wade S. Blair, Christine Chang, Gauri Deshmukh,
Charles Eigenbrot, Paul Gibbons, Adam Johnson, Jane R. Kenny, Pawan Bir Kohli,
Marya Liimatta, Patrick J. Lupardus, Steven Shia, Micah Steffek, Savita Ubhayakar,
Anne van Abbema and Mark Zak
Genentech Inc. 1 DNA Way, South San Francisco, California 94080, USA.
1

The Janus protein tyrosine kinases (Jak1, Jak2, Jak3, and Tyk2) mediate the
intracellular signaling of numerous cytokines and thereby play critical roles in a variety
of biological processes, including hematopoiesis and the regulation of immune and
inflammatory responses.1,2 Binding of a cytokine to its receptor leads to the activation of
receptor associated Jak family kinases and subsequent recruitment and
phosphorylation of STAT (Signal Transducer and Activator of Transcription) proteins.3
The phosphorylated STATs form homo- or heterodimers that translocate to the nucleus
and regulate transcription of target genes. Signaling by a large subfamily of cytokines,
which includes IL-6, always involves Jak1 activation and is implicated in the
pathogenesis of rheumatoid arthritis (RA).1 A gain-of-function mutation (V617F) in the
pseudo-kinase domain of the Jak2 isoform, which renders the kinase constitutively
active, has been found in a significant portion of patients with myeloproliferative
disorders (MPDs).4 The deregulation of the Jak2 activity leads to the pathogenesis of
MPDs.5 Recent approval of tofacitinib 16 (XeljanzTM, Figure 1) for RA and ruxolitinib 27
(Jakafi™, Figure 1) for myelofibrosis (MF) has validated Jak1/2 targets in the treatment
of these disorders. These molecules are inhibitors of both Jak1 and Jak2 isoforms and it
is suggested that compounds with the ability to inhibit both Jak1/2 isoforms may have a
broader therapeutic potential in neoplasia with disregulated Jak signaling.8 Therefore,
we were interested in the identification of novel Jak1/2 inhibitors which might
complement our previously described Jak1 and Jak2 selective compounds. 9-11
In our own efforts to discover novel potent Jak inhibitors, compounds bearing
several distinct hinge-binding motifs were investigated. For example, a series of potent
imidazopyrrolopyridine Jak1 selective inhibitors (3, Figure 2) and pyrazolopyrimidine
2

containing Jak2 selective inhibitors (4, Figure 2) were recently reported from our
laboratories.9-11 Numerous Jak1/2 inhibitors were also disclosed by several companies
and some of these entities are in advanced clinical trials for the treatment of RA and
MPDs.12-14 We were intrigued by the excellent Jak2 activity of the tricyclic pyridone 5a
(Figure 3a) described by others in the literature.15 Due to their rigid, planar structures,
these compounds sometimes suffer from undesired physicochemical properties such as
poor solubility.15 We therefore envisioned modification of the pyridone-containing
tricyclic template 5a to the bicyclic structure 5b while retaining the pyridone as the hinge
binding moiety. Additional nitrogen atoms were incorporated in the new inhibitor design
to facilitate analog synthesis and to obtain favorable direct or water mediated
interactions with Jak1/2 proteins (5c, Figure 3a, b). Herein, we describe the synthesis
of novel 8-oxo-pyridopyrimidine Jak1/2 inhibitors and their biochemical evaluation in
related assays.
To test the viability of the modified template, compound 6 (Table 1) was
synthesized and the Jak1/2 enzyme potencies were assessed. Encouragingly,
compound 6 displayed activity for both Jak1 and Jak2 isoforms with high ligand-
efficiency16 (0.51 and 0.52 for Jak1 and Jak2, respectively). The inhibitor also displayed
excellent aqueous kinetic solubility17 (225 µM). To facilitate the medicinal chemistry
efforts, a crystal structure of 6 bound to Jak1 was obtained (Figure 4). The pyridone
oxygen makes an H-bond with the backbone NH of L959 while the pyridone NH
interacts with the C=O of E957 in the Jak1 hinge region (4a). In addition, water
mediated H-bond interaction between the C=O of L959 and N-2 of the pyrimidine ring is
observed (4a). The cyclohexane moiety attached at C-4 of the core pyrimidine is
3

situated in close proximity to the Jak1 P-loop. The proximal N-H is situated within the
H-bond distance from a water molecule that makes H-bond interactions with the C=O of
G1020 and the carboxylic acid residue of D1021 (Figure 4b). The C-2 carbon of the
pyrimidine ring is 5.4 Å away from a polar Jak1 side chain (E966) whose Jak2
counterpart is also polar in nature (D939).18 These structural features suggested that
modification of the cyclohexyl moiety at C-4 and functionalization of C-2 position were
reasonable strategies to improve potency.
With the knowledge of the binding mode of the inhibitor 6, a medicinal chemistry
effort was initiated to further investigate the scope of this novel template. A systematic
study of cyclohexylamine replacements at C-4 of the pyridopyrimidine ring was carried
out to explore interactions with the P-loop region (7-31, Table 1). Among the un-
substituted alkyl and cycloalkylamines tested, the molecule bearing a cyclopentylamine
(10) moiety was approximately three-fold more potent than compound 6 for both the
Jak1 and Jak2 isoforms. 3- and 4-Aminotetrahydropyranyl moieties at C-4 reduced the
biochemical potency relative to 6 (15 and 16). Similarly, pyridopyrimidines with
benzylamine and 4-chlorophenylanilino groups at C-4 gave undesired results (17 and
18). Molecules bearing tertiary amines (19 and 20) and a cyclopentyl ether moiety (21)
exhibited loss of enzyme potencies for both Jak1 and Jak2 isoforms indicating that a
hydrogen bond donor is required for the inhibitory activity. Compound 10 displayed
excellent solubility (318 µM) and moderate permeability (MDCK Papp A:B = 2.5 x 10-6
cm/s). From these initial results, the cyclopentylamine at C-4 position appeared to be a
favored substitution and we next explored functionalization of the cyclopentyl ring.
4

Accordingly, a number of substituted cyclopentylamine analogs were assessed
as C-4 substituents in the inhibitor design. Inclusion of cyclopentylamines containing
ester and hydroxyl groups resulted in a loss of enzyme potency relative to compound 10
(22-25). However, a nitrile group appended directly to the cyclopentane ring enhanced
the biochemical potency (26-31). Among these derivatives, the cis-2-CN analog (26)
was more potent than the trans- isomer (27). These 2-substituted analogs were more
potent than the 3-substituted analogs (30 and 31). The pure cis-enantiomer 28 exhibited
low nanomolar enzyme inhibition for both Jak1 and Jak2 isoforms while the other
enantiomer 29 was ten-fold less potent for these isoforms. Encouragingly, inhibitor 28
displayed good aqueous kinetic solubility (119 µM), human liver microsomal stability (CL
= 3.3 mL/min/kg) and potency in both Jak1 (IL-6 pSAT3 EC50 = 214 nM) and Jak2
(EPO-pSTAT5 EC50 = 248 nM) driven measures of cell-based inhibition.11 In addition,
the molecule inhibited only one (MEKK2) non-Jak family kinases by >50% when tested
against 40 such enzymes at a concentration of 1 µM (>100x Jak1/2 Ki).19 Encouraged
by these results, substitution at the C-2 of the pyrimidine ring of compound 26 (cis-
racemate) was explored to further enhance enzyme and cell potencies.
Initially, a few H-bond donor/acceptor moieties were incorporated at the C-2
position of 26. The N-Me analog 32 (Table 2) exhibited a moderate boost in the enzyme
potency but a loss of cellular potency relative to 26 was observed. The loss of cell
activity was believed to be due to higher tPSA20 and an increase in the number of H-
bond donors relative to 26 and therefore, tertiary amines at the C-2 position were
explored. Piperidine and morpholine (33 and 34) moieties at C-2 deteriorated the
enzyme potencies considerably. Similarly, incorporation of a hydroxyl moiety as the C-2
5

substituent did not provide improvement in enzyme inhibition (35). The analogs 32-35
displayed large shifts between the enzyme and cellular potencies. Again, this
phenomenon is similar to the trend observed previously and is believed to be due to
increase in the polarity of the molecules.9 Therefore, a few analogs with C-linked
substituents at the C-2 position of 26 were investigated (36-38). The cis-racemate 36
with the phenyl group at C-2 exhibited Jak2 potency comparable to 26 with a slight
decrease in potency for Jak1. The 4-pyrazolyl group at C-2 (37) enhanced the
biochemical potency for both Jak1/2. This analog, however, was inactive in the cellular
assay, presumably due to poor permeability (MDCK Papp A:B = 0.2 x10-6 cm/s).
Incorporation of an ethyl group at C-2 provided compound 38 with excellent enzyme
potencies for both Jak1 and Jak2. This molecule also displayed good cell potencies
against Jak1 (IL-6 pSAT3 EC50 = 300 nM) and Jak2 (EPO-pSTAT5 EC50 = 115 nM).
The potency profile against the four Jak isoforms and in vitro DMPK properties of
compound 38 are summarized in Table 3. The inhibitor was more selective for Jak1 and
Jak2 isoforms relative to Jak3 and Tyk2. The compound had low human liver
microsomal and human hepatocyte clearance and good aqueous kinetic solubility. The
permeability of this molecule was lower than that observed for compound 10. The
compound did not inhibit Cytochrome-P450 (CYP) enzymes (1A2, 3A4, 2C9, 2C19, and
2D6; IC50 values = >10 µM) and only two out of 40 kinases (MEKK2, MEKK3) were
inhibited greater than 50% when the compound was tested in non-Jak family kinases in
a 40 membered panel tested at 1.0 µM.19
In order to understand the boost in potency obtained by incorporation of the
nitrile on the cyclopentyl ring at C-4, co-crystallizations of nitrile containing inhibitors
6

with both Jak1 and Jak2 enzymes were attempted. These activities afforded a co-crystal
of (R, S) enantiomer of 37 bound to Jak1 from the racemate (Figure 5). As expected,
the pyridone moiety makes two H-bond interactions with the hinge region similar to that
observed for compound 6. The nitrile group points towards the G1020 region making
multipolar interactions with the backbone carbonyl moieties. The backbone carbonyl
oxygen of R1007 and the backbone carbonyl carbon of G1020 make polar interactions
with the carbon atom and the nitrogen atom of the nitrile of the inhibitor 37, respectively
(Figure 5a). We surmise that these favorable polar interactions are responsible for the
observed gain in potency relative to compound 10.21 As seen in the crystal structure of
6, the N-H at C-4 makes H-bond interaction with the water molecule (Figure 5b). The
pyrazolyl hetero-atom is situated within H-bond distance (2.8 Å) of the carboxylic acid
moiety of E966 of the Jak1 protein. We hypothesize that similar interactions are also
possible with the D939 of Jak2 protein and therefore similar potencies were observed
for both Jak1 and Jak2.
The synthetic approach for compounds 5-31 is described in Scheme 1. The
synthesis commenced with protection of 3-amino-2-chloropyridine 39 with the Boc
o
group to provide 40. Treatment of 40 with n-butyl lithium at -78 C followed by
quenching with carbon dioxide afforded acid 41. Boc deprotection with TFA and
o
subsequent heating in formamide at 140 C overnight formed the pyrimidinone which,
o
upon heating in POCl3 at 100 C for 2 h, afforded dichloride 42. SNAr displacement of
the C-4 chloride in 42 with various amines in the presence of a base and subsequent
hydrolysis of the C-8 chloride by heating with 6N HCl in THF provided the pyridones.
When an acid labile group was present in the molecule, heating with NaOAc in acetic
7

acid was utilized for the hydrolysis of the 8-chloropyridylpyrimidines to the
corresponding 8-oxo-pyridopyrimidines. The O-linked compound 21 was prepared by
treating 42 with cyclopentanol in the presence of sodium hydride and subsequent
hydrolysis of the chloride as described above.
The C-2 and C-4 disubstituted 8-oxo-pyridopyrimidines were obtained as
depicted in Scheme 2. Acid 43 was prepared as described above starting from 2-
methoxy-3-aminopyridine. Treatment of acid 43 with ammonia in the presence of 1,1-
carbonyldiimidazole (CDI) provided amide 44 which was subsequently transformed to
pyrimidinedione 45. Reaction of 45 with POCl3 formed trichloropyridylpyrimidine 46,
possibly via the in-situ demethylation of the methoxy group and subsequent chlorination
by POCl3. SNAr displacement of the C-4 chloride in 46 with a mixture of cis- and trans-2-
cyanocyclopentylamine22 in the presence of a base provided 47. The cis- and trans-
isomers were separated by flash chromatography over silica gel. The displacement of
the C-2 chloride of the cis-isomer with a variety of amines in the presence of a base and
subsequent hydrolysis of the C-8 chloride in 48-51 generated compounds 32-35. Suzuki
coupling of 47 with phenyl or 4-pyrazoloboronic acid in the presence of a Pd(II) catalyst,
followed by hydrolysis provided compounds 36 and 37. Similarly, the Stille coupling of
the intermediate 47 with vinyltributylstannane in the presence of a Pd(II) catalyst and
subsequent hydrogenation followed by the hydrolysis of the C-8 chloride as described
above provided the ethyl analog 38.
In summary, we successfully designed a novel, potent and highly ligand efficient
(LE = 0.51 and 0.52 for Jak1 and Jak2, respectively) bicyclic 8-oxo-pyridopyrimidine
Jak1/Jak2 inhibitor (6). Structure-based design, based on the crystal structure of 6
8

bound to Jak1 was then applied to further boost enzyme potency which eventually led to
compound 38 with better ligand efficiency (LE = 0.56 and 0.59 for Jak1 and Jak2,
respectively). During the optimization process, the polarity of the molecules was
adjusted such that compounds not only exhibited excellent biochemical potency, but
also improved cellular activity.
Acknowledgements
We thank the small molecule analytical and purification group for their support.
References
1. Heinrich, P. C.; Behrmann, I.; Müller-Newen, G.; Schaper, F.; Graeve. L. J.
Biochem.1998, 334, 297.
2. (a) Song, L.; Schindler, C. JAK-STAT Signaling; The Handbook of Cell Signaling; 2nd
ed.; Bradshaw, R. A., Dennis, E. A., Eds.; Elsevier Inc.: San Diego, 2010; vol. 3, pp
2041. (b) Shuai, K.; Liu, B. Nat. Rev. Immunol. 2003, 3, 900; (c) Ghoreschi, K.;
Laurence, A.; O’Shea, J. J. Immunol. Rev. 2009, 228, 273.
3. Darnell, J. E., Jr. Science 1997, 277, 1630.
4. James, C.; Ugo, V.; Casadevall, N.; Constantinescu, S. N.; Vainchenker, W. Trends
Mol. Med. 2005, 11, 546.
5. Pardanani, A. Leukemia, 2008, 22, 23.
6. Flanagan, M. E.; Blumenkopf, T. A.; Brissette, W. H.; Brown, M. F.; Casavant, J. M.;
Shang-Poa, C.; Doty, J. L.; Elliott, E. A.; Fisher, M. B.; Hines, M.; Kent, C.; Kudlacz, E.
M.; Lillie, B. M.; Magnuson, K. S.; McCurdy, S. P.; Munchhof, M. J.; Perry, B. D.;
9

Sawyer, P. S.; Strelevitz, T. J.; Subramanyam, C.; Sun, J.; Whipple, D. A.; Changelian,
P. S. J. Med. Chem. 2010, 53, 8468.
7. Verstovsek, S.; Kantarjian, H.; Mesa, R. A.; Pardanani, A. D.; Cortes-Franco, J.;
Thomas, D. A.; Estrov, Z.; Fridman, J. S.; Bradley, E.;C.; Erickson-Viitanen, S.; Vaddi,
K.; Levy, R.; Tefferi, A. New Engl. J. Med. 2010, 363, 1117.
8. Liu, P. C. C.; Caulder, E.; Li, J.; Waeltz P.; Margulis, A.; Wynn, R.; Becker-Pasha, M.;
Li, Y.; Crowgey, E.; Hollis, G.; Haley, P.; Sparks, R. B.; Combs, A. P.; Rodgers, J. D.;
Burn, T. C.; Vaddi, K.; Fridman, J. S. Clin. Cancer Res. 2009, 15, 6891.
9. Labadie, S. S.; Dragovich, P. S.; Barrett, B.; Blair, W. S.; Bergeron, P.; Chang, C.;
Deshmukh, G.; Eigenbrot, C.; Ghilardi, N.; Gibbons, P.; Hurley, C.; Johnson, A.;
Kenny, J. R.; Kohli, P. B.; Kulagowski, J. J.; Liimatta, M.; Lupardus, P. J.; Mendonca,
R.; Murray, J. M.; Pulk, R.; Shia, S.; Steffek, M.; Ubhayakar, S.; Ultsch, M.; van
Abbema, A.; Ward, S.; Zak, M. Bioorg. Med. Chem. Lett. 2012, 22, 7627.
10. Hanan, E. J.; v. Abbema, A.; Barrett, K.; Blair, W. S.; Blaney, J.; Chang, C.;
Eigenbrot, C.; Flynn, S.; Gibbons, P.; Hurley, C. A.; Kenny, J. R.; Kulagowski, J.; Lee,
L.; Magnuson, S. R.; Morris, C.; Murray, J.; Pastor, R. M.; Rawson,T.; Siu, M.; Ultsch,
M.; Zhou, A.; Sampath, D.; Lyssikatos, J. P. J. Med. Chem. 2012, 55, 10090.
11. Zak, M.; Mendonca, R.; Balazs, M.; Barrett, K.; Bergeron, B.; Blair, W.; Chang, C.;
Deshmukh, G.; DeVoss, J.; Dragovich, P. S.; Eigenbrot, C.; Ghilardi, N.; Gibbons, P.;
Gradl, S.; Hamman, H.; Hanan, E. J.; Harstad, E.; Hewitt, P. R.; Hurley, C. A.; Jin, T.;
Johnson, A.; Johnson, T.; Kenny, J. R.; Koehler, M. F. T.; Kohli, P. B.; Kulagowski, J.
J.; Labadie, S.; Liao, J.; Liimatta, M.; Lin, Z.; Lupardus, P. J.; Maxey, R. J.; Murray, J.;
10

Pulk, R.; Rodriguez, M.; Savage, S.; Shia, S.; Steffek, M.; Ubhayakar, S.; Ultsch, M.;
Van Abbema, A.; Ward, S. I.; Xiao, L.; Xiao, Y. J. Med. Chem. 2012, 55, 6176.
12. (a) Wilson, L. J. Expert Opin. Ther. Patents 2010, 20, 609. (b) Dymock, B. W.; See,
C. S. Expert Opin. Ther. Patents 2013, 23, 449.
13. Ionnidis, S.; Lamb, M. L.; Wang, T.; Almeida, L.; Block, M. H.; Davies, A. M.; Peng,
B.; Su, M.; Zhang, H. J.; Hoffman, E.; Rivard, C.; Green, I.; Howard, T.; Pollard, H.;
Read, J.; Alimzhanov, M.; Bebernitz, G.; Bell, K.; Ye, M.; Huszar, D.; Zinda, M. J. Med.
Chem. 2011, 54, 262.
14. Thompson, J. E.; Cubbon, R. M.; Cummings, R. T.; Wicker, L. S.; Frankshun, R.;
Cunningham, B. R.; Cameron, P. M.; Meinke, P. T.; Liverton, N.; Weng, Y.; DeMartino,
J. A. Bioorg. Med. Chem. Lett. 2002,12,1219.
15. Siu, T.; Kozina, E. S.; Jung, J.; Rosenstein, C.; Mathur, A.; Altman, M. D.; Chan, G.;
Xu, L.; Bachman E.; Mo, J. R.; Bouthillette, M.; Rush, T.; Dinsmore, C. J.; Marshall, C.
G.; Young, J. R. Bioorg. Med. Chem. Lett. 2010, 20, 7421.
16. Kuntz, I. D.; Chen, K.; Sharp, K. A.; Kollman, P. A. Proc. Natl. Acad. Sci U.S.A.
1999, 96, 9997. (b) Hopkins, A. L.; Groom, C. R.; Alex, A. Drug Disc. Today 2004, 9,
430.
17. Kinetic solubility was measured at pH 7.4 in PBS buffer.
18. Williams, N. K.; Bamert, R. S.; Oatel, O., Wang, C.; Walden, P. M.; Wilks, A. F.;
Fantino, E.; Rossjohn, J.; Lucet, I. S. J. Mol. Biol. 2009, 387, 219.
19. Off-target kinase inhibition assessed by SelectScreen Kinase Profiling Services
(Invitrogen-Life Technologies, Madison, WI, U.S.).
20. Ertl, P.; Rohde, B.; Selzer, P. J. Med. Chem. 2000, 43, 3714.
11

21. Paulini, R.; Müller, K.; Diederich, F. Angew. Chem. Int. Ed. 2005, 44, 1788.
22. (a) Tian, Z.; Jiang, A.; Li, Z.; Song, G.; Huang, Q. J. Agric. Food Chem. 2007, 55,
143. (b) Winkler, M.; Martı´nkova´, L.; Knall, A. C.; Krahuleca, S.; Klempier, N.
Tetrahedron 2005, 61, 4249.
23. Winkler, M.; Knall, A. C.; Kulterer, M. R.; Klempier, N. J. Org. Chem. 2007, 72,
7423.
12

Figure Captions
Figure 1. Marketed Jak1/2 inhibitors.
Figure 2. Previously described Jak1 and Jak2 selective inhibitors.
Figure 3. Design of 8-oxo-pyridopyrimidine template.
Figure 4: X-ray structure of 6 in complex with Jak1 (resolution = 2.4 Å, PDB code
4K77). Figures 4a and 4b provide two visual perspectives. 4b is rotated approximately
120 degrees relative to 4a. Compound 6 is depicted in yellow. Hydrogen bonds to the
hinge region are shown as dashed red lines. Backbone hinge atoms contacting 6 are
highlighted (E957 and L959). Notable crystallographic waters are denoted as red
spheres. Hydrogen bonds to the water molecules are depicted as blue dashed lines.
The double-headed black arrow shows the distance between the C-2 position of the
ligand and the carboxylic acid moiety of the E966 in Jak1.
Figure 5: X-ray structure of 37 in complex with Jak1 (resolution = 2.73 Å, PDB code
4K6Z). Figures 5a and 5b provide two visual perspectives. 5b is rotated approximately
120 degrees relative to 5a. Compound 37 is depicted in yellow. Hydrogen bonds to the
hinge region are shown as red dashed lines. Backbone hinge atoms contacting 37 are
highlighted (E957 and L959). Dashed line in magenta shows interaction between the
C-2 pyrazole and the carboxylic acid moiety of E966. Polar interactions between the CN
and the backbone carbonyls of G1020 and R1007 are shown as cyan dashed lines.
Hydrogen bonds to the water molecules are depicted as blue dashed lines.
13

14

15

1
8
3
4
3
1
2
2
Ki
1
2
16

5b
5a
E957
Hinge
Hinge
L959
E966
G1020
R1007
D1021
R1007
E966
17

5b
5a
E957
Hinge
Hinge
L959
E966
G1020
R1007
D1021
R1007
E966
18

Scheme 1: Synthesis of compounds 6-31.
C-4
2
C-8
1
Reagents and reaction conditions: See Table 1 for R1 substituents (i) 1M NaHMDS in
THF, (Boc)2O; (ii) 2.5 M n-BuLi in hexanes, TMEDA,-78 oC, CO2; (iii) TFA, DCM; (iv)
HCONH2, 140 oC, 20 h; (v) POCl3, 100 oC; 2 h (vi) primary/secondary amines, DIPEA,
rt-60 oC, 20 h; (vii) 6N HCl, THF, reflux, 2h or NaOAc, AcOH, 80 oC, 20 h; (viii)
cyclopentyl alcohol, NaH, DMF, 0.5 h.
19

Scheme 2: Synthesis of compounds 32-38.
Reagents and reaction conditions: See Table 2 for R2 substituents (i) CDI, ammonia,
THF, 0 oC – rt; (ii) (CCl3CO)2O, 1,4-dioxane, 75 oC, 20 h; (iii) POCl3, 100 oC, 20 h; (iv) 2-
cyanocyclopentylamine(cis-, trans-mixture), DIPEA, 1,4-dioxane, 80 oC, 2 h; (v)
primary/secondary amines, DIPEA, 60 oC, 20 h; (vi) NaOAc, AcOH, 60 oC, 20 h; (vii)
RB(OH)2, PdCl2(PPh3)2, Na2CO3, 1,4-dioxane/water or tributylvinyltin, PdCl2(PPh3)2,
THF, 70 oC, 20 h and then H2(balloon)/Pd-C/EtOAc.
20

Table 1: SAR of 8-oxo-pyridopyrimidine inhibitors 6-31.
1
Compound
No.
R1
Jak1 Enzyme
Ki (nM)a,b
Jak2 Enzyme
Ki (nM)a,b
6
235
145
7
8
9
98
123
377
130
710
120
10
11
75
54
398
189
12c
13
14
313
644
151
158
550
201
15
893
766
21

16
17
18
661
720
610
574
690
380
19
20
21
3,500
>5,600
2,800
3,200
3,200
1,500
22e
23d
24d
453
1,200
408
567
1,400
575
HO
NH
25d
3,200
13
>5600
9
cis
26d,f
cis
22

27d
65
63
trans
cis-enantiomer 1
28
7
4
cis-enantiomer 2
29
68
40
N
30d,g
65
59
NH
cis
N
NH
31d,g
151
150
trans
aFor compounds with enzyme potency of >100 nM, the enzyme data are from a single
run. For those with enzyme potency of <100 nM, the data are the average of at least
two separate runs. bFor enzyme and cell based assay details see ref. 11. cexo-
racemate. dracemate. emixture of cis/trans diastereomers. fSee ref. 22 for the synthesis
of 2-cyanocyclopentyl amine. gSee ref. 23 for the synthesis of 3-cyanocyclopentyl
amine.
23

Table 2. SAR of 8-oxo-pyridopyrimidine inhibitors 32-38.
2
cis
IL6-
Jak1
Jak2
Enzyme
EPO-
Compd
No.
pSTAT3
EC50
Solubility tPSA
R2
H
Enzyme
pSTAT5
(µM)c
(Å)d
Ki (nM)a,b Ki (nM)a,b
EC50 (nM)a,b
(nM)a,b
670
26
32
13
9
230
350
104
94
4.6
1.8
1000
74
106
97
33
34
440
380
21
13
10,000
4,100
8.6
>10,000
>10,000
800
>10,000
51
19
11
106
117
94
35
36
280
18
23
>10,000
426
3.0
37
38
2
3
0.6
1.0
7,700
300
6,300
115
24
123
94
140
24

aThe data are the average of at least two separate runs. bFor enzyme and cell based assay
details see ref. 11. cKinetic solubility measured at pH 7.4 in PBS buffer. dFor tPSA see ref. 20.
25

Table 3. Enzyme potencies and in-vitro DMPK properties of 38.
MDCK
HLM
CL-hep
HH
CL-hep
CYP 450
(>10µM)
Enzyme Ki (nM)
Jak1, Jak2, Jak3, Tyk2
Papp A:B
IC50
(x10-6 cm/s) (mL/min/kg) (mL/min/kg)
3A4. 2C9,
1A2,
2C19, 2D6
3.0, 1.0, 37, 11
0.6
0.3
2.8
26

Graphical abstract
27