Discovery of a novel series of pyridine and pyrimidine carboxamides as potent and selective covalent inhibitors of Btk

Article abstract of DOI:10.1016/j.bmcl.2018.09.033

Btk is an attractive target for the treatment of a range of Bcell malignancies as well as several autoimmune diseases such as murine lupus and rheumatoid arthritis. Several covalent irreversible inhibitors of Btk are currently in development including ibrutinib which was approved for treatment of B-cell malignancies. Herein, we describe our efforts using X-ray guided structure based design (SBD) to identify a novel chemical series of covalent Btk inhibitors. The resulting pyridine carboxamides were potent and selective inhibitors of Btk having excellent enzymatic and cellular inhibitory activity.

Full text of DOI:10.1016/j.bmcl.2018.09.033

Accepted Manuscript
Discovery of a novel series of pyridine and pyrimidine carboxamides as potent
and selective covalent inhibitors of Btk
Richard Caldwell, Lesley Liu-Bujalski, Hui Qiu, Igor Mochalkin, Reinaldo
Jones, Constantin Neagu, Andreas Goutopoulos, Roland Grenningloh, Theresa
Johnson, Brian Sherer, Anna Gardberg, Ariele Viacava Follis, Federica
Morandi, Jared Head
PII:
S0960-894X(18)30775-3
https://doi.org/10.1016/j.bmcl.2018.09.033
BMCL 26054
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
4 June 2018
22 September 2018
26 September 2018
Please cite this article as: Caldwell, R., Liu-Bujalski, L., Qiu, H., Mochalkin, I., Jones, R., Neagu, C., Goutopoulos,
A., Grenningloh, R., Johnson, T., Sherer, B., Gardberg, A., Follis, A.V., Morandi, F., Head, J., Discovery of a novel
series of pyridine and pyrimidine carboxamides as potent and selective covalent inhibitors of Btk, Bioorganic &
Medicinal Chemistry Letters (2018), doi: https://doi.org/10.1016/j.bmcl.2018.09.033
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Discovery of a novel series of pyridine and pyrimidine carboxamides as potent and selective covalent
inhibitors of Btk.
Richard Caldwell*a, Lesley Liu-Bujalskia, Hui Qiua,*, Igor Mochalkina, Reinaldo Jonesa, Constantin
Neagua, Andreas Goutopoulosa, Roland Grenningloha,Theresa Johnsona, Brian Sherera, Anna Gardbergb,
Ariele Viacava Follisa, Federica Morandic, Jared Heada
aEMD Serono Research & Development Institute, Inc.,45A Middlesex Turnpike, Billerica,01821, MA, USA
bConstellation Pharmaceuticals, 215 First Street, Suite 200, Cambridge, MA, 02142, USA
cF. Hoffmann-La Roche AG, Konzern-Hauptsitz, Grenzacherstrasse 124, CH-4070 Basel, Switzerland
*Corresponding Author: richard.caldwell@emdserono.com
Keywords: Covalent, Irreversible, Btk inhibitor, Fragment
Bruton’s Tyrosine Kinase (Btk) is a member of the Tec family of tyrosine kinases and is expressed
in B cells, macrophages, neutrophils, and monocytes, but not T cells1. Btk activity is critical for B-cell
receptor (BCR) signaling in B-cells and FCRγ signaling in myeloid cells2, 3. Several Btk inhibitors are
currently in clinical development for the treatment of a range of B-cell malignancies4, the most
advanced of which, is the recently approved ibrutinib (Imbruvica).5 Ibrutinib was shown clinically to
mitigate multiple B-cell mediated forms of cancer including chronic lymphoid leukemia (CLL)6, mantle-
cell lymphoma (MCL)7, and Waldenström’s macroglobulinemia (WM)8. In addition, inhibition of Btk
signaling in preclinical models of autoimmune diseases such as murine lupus9 and rheumatoid arthritis10
demonstrate Btk’s utility as a target for the treatment of these diseases.
Ibrutinib and related compounds (Figure 1) target Btk specifically by formation of a covalent
bond between the Cys481 residue of Btk and a chemically reactive warhead moiety, such as the
acrylamide of ibrutinib and propargylamide of tirabrutinib 2. Ibrutinib also binds covalently to several
other kinases that possess a similarly accessible cysteine residue including Egfr, BrbB2, Itk, Jak3, Blk, Tec,
and Bmx.11, 12 Additionally, ibrutinib possess a promiscuous hinge binding moiety that enables the
molecule to bind potently to other kinases that lack the cysteine residue (Lck, Lyn, Fyn, Src, Abl,13, 14 and
Flt315) further contributing to the promiscuous kinase activity profile of the therapeutic. In addition to these
undesirable off target effects, reports have also linked atrial fibrillation via inhibition of PI3k-Akt16-18
cardiac signaling, as well other undesirable adverse effects such as drug-drug interaction with
verapamil19 to treatment with ibrutinib. Taken together, these data underscore the need to find new
classes of covalent Btk inhibitors with improved selectivity and the potential to provide safer and more
attractive treatment options for patents.
O
O
O
O
NH2
NH2
N
HN
N
N
N
N
O
N
N
N
N
N
N
NH
H
F
O
N
N
O
O
1
3
2
Ibrutinib
Spebrutinib
Tirabrutinib
N
O
NH
O
N
NH2
O
NH
N
N
N
N
N
O
NH2
N
4
5
Acalabrutinib
Evobrutinib

Fig. 1. Structures of covalent irreversible Btk inhibitors that have been approved (compound 1) and are currently in clinical development
(compounds 2-5).
Previously we have described the discovery of the highly selective clinical candidate M2951
(evobrutinib)20 that is currently being developed for chronic treatment of autoimmune diseases such as
rheumatoid arthritis. Herein, we describe our early efforts to identify a backup chemical series of
covalent inhibitors to our front runner evobrutinib. These efforts resulted in the identification of a series
of pyrimidine and pyridine carboxamide inhibitors of Btk. These new compounds displayed excellent
biochemical and cellular potency, as well as superior kinase selectivity, and improved drug-like properties
relative to ibrutinib.
To identify starting points for a potential backup series a fragment-based screening approach
was used to identify small molecule fragments that were competitively displaced by the front runner,
evobrutinib from the binding pocket of Btk. One goal of this work was to discover a suitable
replacement for the amino pyrimidine hinge binding moiety present in the front runner, evobrutinib.
One of the most attractive hits from the fragment screening was fragment A which inhibited full-length
human Btk (Carna Biosciences; Natick, MA) by 71% at 100 µM.
HN
N
H2N
O
Fragment A
Fig. 2: Fragment A inhibited wild type Btk 71% at 100 µM, LE=0.35.
The crystal structure of fragment A bound to Btk (Figure 3) revealed that the carboxamide
moiety was bound to the hinge of the ATP binding pocket of full length Btk. Analysis of the structure
suggested that this binding mode would provide a possible vector to the Cys481 residue. Modeling
indicated that potential attachment points for a war head would be the 2 or 3 positions of the
cyclohexyl linker and that a trans configuration of substituents would be preferred. Alternatively, 5
membered rings, such as 3-amino pyrrolidine might also provide an acceptable vector for access to the
Cys481 residue. Based on this hypothesis, we prepared a set of compounds to explore several linkers to
append an acrylamide warhead with the aim of achieving covalency.
Fig. 3: Crystal structure of fragment A bound to Btk. (PDB ID:6DI0)
Initial analogs of fragment A in which an acrylamide war head was appended to either the 2, 3,
or 4 positions of the six membered cyclohexyl linker, in both the cis and trans configuration, lacked

significant potency (data not shown). However, reducing the ring size of the linker moiety and
appending the warhead to a five membered 3-amino-pyrrolidine linker led to significant increases in
potency (Table 1 compound 7). Interestingly, the receptor displayed slight stereochemical preference for
the (S) enantiomer of the pyrrolidine linker in pyrimidine compounds 7 and 8. Adding aliphatic moieties
in the R2 position of the pyridine ring completely abolished activity, as in compound 9. By contrast,
adding an aniline moiety in the R2 position on the pyrimidine ring, as in compound 10, gave single digit
nanomolar potency. The combination of the warhead and the R2 position aniline moieties provided a
breakthrough in reaching low nanomolar potencies.
Table 1.
Exploration of linkers to achieve covalency with CYS481 of Btk.
R1
X
N
R2
NH2
O
Compound
R1
R2
H
X
C
Btk enzyme potency (nM)
>6000
6
HN
Fragment A
O
HN
7
N
N
C
N
180
500
>6000
8
NH2
NH2
N
N
N
N
O
HN
8
O
HN
9
Fig. 4:
N
H
Overlay of
fragment A
and
compound 7
bound to
ATP binding
O
F
HN
10
N
H
pocket of Btk. The structure shows that the acrylamide warhead of compound 7 attained covalency with CYS481 residue. (PDB ID:6DI1)
The x-ray crystal structure of compound 7 bound to full length Btk clearly demonstrated
covalency of the acrylamide warhead with Cys481 (Figure 4.) The structure also demonstrated a binding
mode in which the R2 position amine in compound 7 is oriented towards the solvent exposed pocket of
Btk. Based on these results we began to focus on further improving the potency and properties of these
analogs. Since the (S) 3-amino pyrrolidine linker provided the desired access to the selectivity enhancing
Cys481 residue we then began to focus on optimization of the aniline R2 moieties (Figure 5.)
Cys481
Cys481
O
HN
N
HN
N
O
N
N
O
O
N
H2N
Hinge Binding
Region
Solvent Exposed
Pocket
H
Solvent Exposed
Pocket
O
NH2
Hinge Binding
Region

Fig. 5: Hit optimization strategy for pyridine and pyrimidine carboxamides based on crystal structure.
An extensive exploration of R2 position aniline moieties revealed that a wide range of
substituents was tolerated for Btk inhibitory activity (Table 2.) Compounds bearing highly lipophilic R2
moieties such as phenolic ether in 11 provided reasonably potent Btk inhibition in the enzyme assay. To
improve solubility of these analogs more polar R2 moieties such as the morpholine carboxamides in 12
were prepared. This resulted in a 10-fold increase in enzymatic potency but did not improve
permeability. The same stereochemical preference for the (S) enantiomer that was seen in the early
fragment derived compounds was also observed for the more elaborated analogs (entries 12 and 13).
The pyrimidine analog 14 was threefold more potent than matched pair pyridine analog 12. Omission of
the acrylamide warhead in 15 resulted in >100-fold loss in activity. Substitution on the morpholine ring
in compound 16 was tolerated but did not lead to significant changes in enzymatic potency. Removing
the carboxamide of 16 and attaching a substituted morpholine ring directly to the aromatic ring
provided compound 17 and central pyridine ring fluorinated analog 18, both of which had excellent
enzyme and cellular potency, acceptable efflux ratios, and acceptable permeability. Compounds with
polar moieties, such as in entries 19-24, had excellent enzyme and cellular potency, regardless of which
R1 linker was used. However, all these compounds had very high efflux ratios and poor permeability.
Other ring sizes and heterocycles at the R2 solvent exposed pocket position were also explored. Generally, 6-
membered aromatic rings were preferred and usually led to good inhibitory activity for Btk. However,
we wanted to explore alternatives to the aniline type R2 moieties21 22 in order to mitigate any potential
liabilities with these structures. Heterocyclic R2 position analogs such as compound 24 gave good
potency against Btk but suffered from poor permeability. Based on these data, compound 17 was
selected for further profiling (Table 3).
Table 2
Selected SAR of pyrimidine carboxamide series.
R1
X
N
R2
O
NH2
Btk Enzyme
IC50 (nM)
PBMC
IC50 (nM)
Caco-2 a>b
(10-6cm/s)/
Compound
R1
R2
X
Efflux Ratio
O
O
HN
11
C
11
-
0.63
89
N
H
N
O
O
HN
N
N
12
13
C
C
1.5
-
-
0.2
0.4
52
O
O
N
H
N
O
O
N
16.2
228
N
H
N

O
O
O
O
HN
N
N
N
14
15
16
N
C
C
0.5
-
0.13
38
O
O
O
N
H
N
H2N
1600
-
-
-
N
O
N
H
HN
HN
HN
HN
N
1.1
44
4
18
N
H
N
O
O
N
N
17
18
19
20
C
0.6
1.6
1.9
1.3
6.1
14.0
7.0
41
9
5.4
9.2
13
cis
N
N
H
O
O
CF
6
cis
N
N
H
N
N
N
O
C
0.3
N
N
H
O
C
0.07
266
N
O
N
H
HN
21
22
C
C
0.09
4.9
0.02
0.06
822
979
N
N
H
O
O
HN
N
16
-
O
N
H
N

O
O
N
N
N
23
24
C
C
0.3
0.1
4.3
1.4
0.05
0.04
376
205
N
H
N
N
N
N
H
Comparison of compound 17 with ibrutinib (Table 3) demonstrated similar potency ranges for
the enzymatic, cellular, and whole blood assays for Btk. The intrinsic clearance of compound 17 was
dramatically improved in all species tested as compared to that of ibrutinib. In addition to these
encouraging results, compound 17 showed no significant CYP induction. The hERG inhibition for
compound 17 was better than that of ibrutinib. Compound 17 was relatively selective as compared to
ibrutinib in a 270-member kinase panel only inhibiting 9/270 kinase at >50% at 1 uM. Unfortunately,
compound 17 did not show significant improvement in oral bioavailability relative to ibrutinib in a low
dose mouse pK study. Efforts to improve the low oral bioavailability served as the basis for the future
lead optimization strategy for this series. This work will be described in a forthcoming publication.
Table 3
Comparison of pyridine analog 17 to ibrutinib.
O
O
HN
NH2
Compound
N
O
N
N
N
N
N
cis
N
N
H
NH2
N
O
O
17
Ibrutinib
Btk/ hPBMC/ hWB IC50 [nM]
Caco-2 a to b (efflux ratio)
0.6/6.1/52
9.1 (5.4)
0.2/4.6/14
7.2 (1.0)
PPB (h/m/r) fu %
3/5.6/3.1
2.5/1.7/2
Clint (h/m/r) [µl/min/mg]
hERG [µM]
<10/18/<10
920/>1000/330
1.1
20% inhibition (10uM)
2C9 (8.4), 2D6 (8.5)
No issue
CYP inh [µM]
2B6 (11.1), 2C19 (19), 2C8 (7.9), 2C9 (8.5), 3A4 (11)
CYP induction mRNA [µM]
3A4 (0.3)
Mouse pK (0.5 mg/kg) AUC (ng/ml*h), CL; Vss; t1/2;
F%)
14/1.57/1.61/1.2/5
26/1.68/1.07/0.5/15
Kinase Selectivity (1uM)
9/270
35/270

BocHN
BocHN
N
H
Cl
O
O
Cl
O
N
N
O
X
28
N
ii
i
N
N
N
O
X
+
N
X
cis
Cl
cis
cis
NH2
N
N
H
NH2
H
NH2
O
NH2
25
27
26
29
X=C,N
O
+H3N
Cl-
O
N
HO
O
N
O
iii
, iv
31
N
N
N
O
X
N
cis
cis
N
N
X
H
N
NH2
H
O
NH2
30
17
Scheme 1. Reagents. i. LiHMDS, THF, -78°C to rt 24 h; ii. DIPEA, DMF, 125°C, 24h; iii. HCl, MeOH, rt, 3h, quantitative; iv. T3P, DIEA, DCM, rt, 15
min.
The pyridine carboxamide analogs were prepared as described in Scheme 1 above.23 For
example, starting from commercially available 2,6-dichloro-pyridine-3-carboxamide, selective
displacement at the 2 position was accomplished using 1 M NaHMDS in THF at -78°C for 30 minutes followed
by stirring at room temperature overnight in THF under inert atmosphere. Displacement of the second
chlorine was accomplished at 125°C with primary and secondary amines in the presence of Hunig’s base in DMA in
good to moderate yields. Deprotection of the amine functionality of the pyrrolidine ring under acid
conditions, followed by acylation with acrylic acid and propyl phosphonic anhydride 50% wt. in ethyl
acetate provided the desired pyrimidine and pyridine carboxamides.
In conclusion we have discovered a novel series of covalent and selective Btk inhibitors. These
compounds were based on the elaboration of a fragment that bound to the ATP active site of Btk. The
crystal structure of fragment A guided the design of compounds that were able to achieve covalent
attachment of an acrylamide warhead with Cys481 residue of Btk. Further elaboration of these
compounds led to compound 17 with excellent biochemical, cellular, and whole blood potency.
Unfortunately, compound 17 did not attain sufficient oral bioavailability in a low dose mouse pK study to
be considered a lead compound. Further optimization of these analogs to improve the pK profile has
been completed and these results will be described in a forthcoming publication.
References
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2. Hendriks RW, Yuvaraj S, Kil LP. Targeting Bruton's tyrosine kinase in B cell malignancies. Nat Rev Cancer. 2014;14(4): 219-
232.
3. Katz FE, Lovering RC, Bradley LA, et al. Expression of the X-linked agammaglobulinemia gene, btk in B-cell acute
lymphoblastic leukemia. Leukemia. 1994;8(4): 574-577.
4. Zhang Z, Zhang D, Liu Y, et al. Targeting Bruton's tyrosine kinase for the treatment of B cell associated malignancies and
autoimmune diseases: Preclinical and clinical developments of small molecule inhibitors. Arch Pharm (Weinheim). 2018;351(7):
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Patients with Relapsed Chronic Lymphocytic Leukemia. Am Health Drug Benefits. 2015;8(Spec Feature): 66-69.

6. Byrd JC, Furman RR, Coutre SE, et al. Targeting BTK with ibrutinib in relapsed chronic lymphocytic leukemia. N Engl J Med.
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7. Wang ML, Rule S, Martin P, et al. Targeting BTK with ibrutinib in relapsed or refractory mantle-cell lymphoma. N Engl J
Med. 2013;369(6): 507-516.
8. Treon SP, Tripsas CK, Meid K, et al. Ibrutinib in previously treated Waldenstrom's macroglobulinemia. N Engl J Med.
2015;372(15): 1430-1440.
9. Katewa A, Wang Y, Hackney JA, et al. Btk-specific inhibition blocks pathogenic plasma cell signatures and myeloid cell-
associated damage in IFN-driven lupus nephritis. JCI Insight. 2017;2(7): e90111.
10. Di Paolo JA, Huang T, Balazs M, et al. Specific Btk inhibition suppresses B cell- and myeloid cell-mediated arthritis. Nat
Chem Biol. 2011;7(1): 41-50.
11. Dasmahapatra G, Patel H, Dent P, Fisher RI, Friedberg J, Grant S. The Bruton tyrosine kinase (BTK) inhibitor PCI-32765
synergistically increases proteasome inhibitor activity in diffuse large-B cell lymphoma (DLBCL) and mantle cell lymphoma
(MCL) cells sensitive or resistant to bortezomib. Br J Haematol. 2013;161(1): 43-56.
12. Liu Q, Sabnis Y, Zhao Z, et al. Developing irreversible inhibitors of the protein kinase cysteinome. Chem Biol. 2013;20(2):
146-159.
13. Cheng G, Ye ZS, Baltimore D. Binding of Bruton's tyrosine kinase to Fyn, Lyn, or Hck through a Src homology 3 domain-
mediated interaction. Proc Natl Acad Sci U S A. 1994;91(17): 8152-8155.
14. Backesjo CM, Vargas L, Superti-Furga G, Smith CI. Phosphorylation of Bruton's tyrosine kinase by c-Abl. Biochem Biophys
Res Commun. 2002;299(3): 510-515.
15. Pillinger G, Abdul-Aziz A, Zaitseva L, et al. Targeting BTK for the treatment of FLT3-ITD mutated acute myeloid leukemia.
Sci Rep. 2015;5: 12949.
16. McMullen JR, Boey EJ, Ooi JY, Seymour JF, Keating MJ, Tam CS. Ibrutinib increases the risk of atrial fibrillation,
potentially through inhibition of cardiac PI3K-Akt signaling. Blood. 2014;124(25): 3829-3830.
17. Boriani G, Corradini P, Cuneo A, et al. Practical management of ibrutinib in the real life: Focus on atrial fibrillation and
bleeding. Hematol Oncol. 2018.
18. Lee HJ, Chihara D, Wang M, Mouhayar E, Kim P. Ibrutinib-related atrial fibrillation in patients with mantle cell lymphoma.
Leuk Lymphoma. 2016;57(12): 2914-2916.
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ibrutinib and verapamil: a case report. J Clin Pharm Ther. 2016;41(1): 104-105.
20. Gaudio E. The two novel BTK-inhibitors M2951 and M7583 show in vivo anti-tumor activity in pre-clinical models of B cell
lymphoma. Proceedings of the American Association for Cancer Research Annual Meeting 77. Washington, DC; 2017.
21. Kalgutkar AS, Gardner I, Obach RS, et al. A comprehensive listing of bioactivation pathways of organic functional groups.
Curr Drug Metab. 2005;6(3): 161-225.
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Discov Devel. 2005;8(1): 44-50.
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2015:372pp.
Supplemental Material
Discovery of a novel series of pyridine and pyrimidine carboxamides as potent and selective covalent
inhibitors of BTK.
Richard Caldwella, Lesley Liu-Bujalskia, Hui Qiua,*, Igor Mochalkina, Reinaldo Jonesa, Constantin
Neagua, Andreas Goutopoulosa, Roland Grenningloha,Theresa Johnsona, Brian Sherera, Anna Gardbergb,
Ariele Viacava Follisa, Federica Morandic, Jared Heada
Synthesis of Compound 17

BocHN
BocHN
N
H
Cl
O
O
Cl
O
N
N
O
X
28
N
ii
i
N
N
N
O
X
+
N
X
cis
Cl
cis
cis
NH2
N
N
H
NH2
H
NH2
O
NH2
25
27
26
29
X=C,N
O
+H3N
Cl-
O
N
HO
O
N
O
iii
, iv
31
N
N
N
O
X
N
cis
cis
N
N
X
H
N
NH2
H
O
NH2
30
17
Scheme 1 Reagents. i. LiHMDS, THF, -78°C to rt 24 h; ii. DIPEA, DMF, 125°C, 24h; iii. HCl, MeOH, rt, 3h,
quantitative; iv. T3P, DIEA, DCM, rt, 15 min.
O
Cl
N
N
O
cis
N
H
NH2
6($!497D939F-8798-472D-B0D4-C98792846637!$)-Chloro-2-[4-(2,6-dimethyl-morpholin-4-yl)-
phenylamino]-nicotinamide (compound 27; step i)
($!BD9DC063-B6A3-432E-8372-7A7A108F6BF6!$)4-(2,6-dimethyl-morpholin-4-yl)-phenylamine (834.1
mg; 4.0 mmol; 1.02 eq.) and 2($!820E6720-7BA2-4944-AB9B-AA3B2B483DA3!$),6-Dichloro-
nicotinamide (750.2 mg; 3.9 mmol; 1.0 eq.) were dissolved in 60 ml T($!3A682932-586B-4188-8BEA-
CA34885EAD5A!$)HF and the solution was cooled to -78°C under nitrogen. S($!53B69357-926E-45D3-9B83-
D45DD2099196!$)odium bis(trimethylsilyl)amide (3.0 ml; 11.8 mmol; 3.0 eq.) was then added dropwise over
15 minutes. The mixture was stirred at -78°C for 30 minutes. The reaction mixture was removed from the
dry ice bath and stirred overnight at rt. The resulting dark solids were treated with 5 ml water and 18 ml
of 6N HCl until the pH of the solution was 2-3 as judged by pH paper. The resulting suspension was then
extracted three times with 50 ml ethyl acetate. The organics were combined and dried over MgSO4. The

drying agent was removed via filtration and the solvent was removed under reduced pressure to give [4-
(3-carbamoyl-6-chloro-pyridin-2-ylamino)-phenyl]-carbamic acid tert-butyl ester (236.1 mg, 16%) as a
light yellow solid. MS: m/z = 361 [M+H]+.
BocHN
O
N
N
N
O
cis
N
H
NH2
6($!7D46C035-B1E9-4111-9B7E-814E84518155!$)-((S)-3-Amino-pyrrolidin-1-yl)-2-[4-(2,6-dimethyl-
morpholin-4-yl)-phenylamino]-nicotinamide hydrochloride (compound 29; step ii)
To a 10 ml microwave reaction vessel was combined 6($!497D939F-8798-472D-B0D4-C98792846637!$)-
chloro-2-[4-(2,6-dimethyl-morpholin-4-yl)-phenylamino]-nicotinamide (236.1 mg; 0.6 mmol; 1.0 eq.),
(($!FBE74B91-1680-411F-92A1-FA8CBDED7201!$)S)-3-(boc-amino)pyrrolidine (146.3 mg; 0.79 mmol; 1.2
eq.),
n($!6C4485BE-577F-47EC-9EBD-CAC9E9EC7520!$),n-dimethyl-acetamide
(2.6
ml.),
and
D($!47453CBD-12CD-4719-AC71-6F15D512517F!$)IPEA (0.3 ml; 2.0 mmol; 3.0 eq.). The vessel was
sealed, and the mixture was stirred at 125°C using conventional heating for 24h. The reaction was then
cooled to room temperature and diluted with 15 ml ethyl acetate. The reaction was then washed with a
solution of saturated sodium hydrogen carbonate in water (15 ml) followed by two additional washes
with water (15-20 ml each). The organic layer was then collected and dried over anhydrous MgSO4. The
solvent was then removed under reduced pressure and crude material was purified with 11g of KPNH
silica using a gradient of 5-95% ethyl acetate to hexane to give ((S)-1-{5-carbamoyl-6-[4-(2,6-dimethyl-
morpholin-4-yl)-phenylamino]-pyridin-2-yl}-pyrrolidin-3-yl)-carbamic acid tert-butyl ester (236.0 mg;
70%) as a tan solid. MS: m/z = 511 [M+H]+.
+H3N
Cl-
O
N
N
N
O
cis
N
H
NH2
6($!39C48834-F645-4CCE-83EC-47F9F5FE22D2!$)-((S)-3-Amino-pyrrolidin-1-yl)-2-[4-(2,6-dimethyl-
morpholin-4-yl)-phenylamino]-nicotinamide hydrochloride (compound 30; step iii)

To a dry 10 ml round bottom flask containing ((S)-1-{5-carbamoyl-6-[4-(2,6-dimethyl-morpholin-4-yl)-
phenylamino]-pyridin-2-yl}-pyrrolidin-3-yl)-carbamic acid tert-butyl ester (0.4 mmol; 1.00eq.; 236 mg) in
4.0 ml m($!9E88E0D7-40AE-49AF-BE81-B5D0F0DC8CD9!$)ethanol was added
4
N
HCl in
dioxane($!B4C799FA-B385-4047-BD88-4902F61CC3BB!$) (1.7 ml; 6.5 mmol; 10.0 eq.). The reaction was
stirred at room temperature for 3h before it was concentrated and carried to the next step without
further purification. The procedure gave 6-((S)-3-amino-pyrrolidin-1-yl)-2-[4-(2,6-dimethyl-morpholin-4-
yl)-phenylamino]-nicotinamide hydrochloride (209.1 mg) crude product as a white solid. MS: m/z = 411
[M+H]+.
O
N
O
N
N
N
O
cis
N
H
NH2
6-((S)-3-Acryloylamino-pyrrolidin-1-yl)-2-[4-(2,6-dimethyl-morpholin-4-yl)-phenylamino]-nicotinamide
(compound 17; step iv)
To a dry 10 ml round bottom flask containing a stirbar and 1($!5EDFC288-2B3F-40C2-8220-
8B44DDE45830!$),2-dichloroethane (8.0 ml; 50.5 mmol; 77.7 eq.) was added 6-((S)-3-amino-pyrrolidin-
1-yl)-2-[4-(2,6-dimethyl-morpholin-4-yl)-phenylamino]-nicotinamide hydrochloride (0.5 mmol; 1.00 eq.;
209.1 mg), acrylic acid (0.5 mmol; 1.0 eq.; 33.7 mg; 3 µL), and ethyl-diisopropyl-amine (2.3 mmol; 5.0
eq.;
302.2
mg;
40
µL).
($!C406BEB5-5DB8-40F5-A284-6D867FE98754!$)2,4,6-Tripropyl-
[1,3,5,2,4,6]trioxatriphosphinane 2,4,6-trioxide (0.5 mmol; 1.0 eq.; 148.8 mg) was then added to the
stirring solution. The resulting solution was stirred at rt for 15 min before it was concentrated to
dryness. The residue was then dissolved in 5 ml of DMSO and purified using preperative reverse phase
chromatography on a X-Bridge Prep C18 5 micron 3-x150 mm column (Part No. 186003284) using a
gradient of 10-80% CH3CN/H2O (0.1% ammonium hydroxide modifier) to give 6-((S)-3-acryloylamino-
pyrrolidin-1-yl)-2-[4-(2,6-dimethyl-morpholin-4-yl)-phenylamino]-nicotinamide (46.0 mg; 21%) as a
white solid. HPLC: 93.7% , RT= 3.69 min. MS: m/z = 465 [M+H]+.1H NMR (400 MHz, DMSO-d6) δ 11.49 (s,
1H), 8.40 (d, J = 7.0 Hz, 1H), 7.89 (d, J = 8.8 Hz, 1H), 7.63 – 7.50 (m, 2H), 6.96 – 6.77 (m, 2H), 6.34 – 5.94
(m, 2H), 5.84 (d, J = 8.6 Hz, 1H), 5.61 (dd, J = 9.9, 2.5 Hz, 1H), 4.48 (h, J = 5.4 Hz, 1H), 3.80 – 3.44 (m, 6H),
2.20 (dt, J = 11.8, 8.3 Hz, 3H), 2.04 – 1.81 (m, 1H), 1.15 (d, J = 6.2 Hz, 6H).

Analytical Data:
1H-NMR spectra were recorded on a Bruker Avance III 400 MHz. Chemical shifts are expressed in parts
per million (ppm, δ units). Coupling constants are in units of Hertz (Hz). Splitting patterns describe
apparent multiplicities and are designated as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet),
or br (broad).
Mass spectra were obtained on Agilent 1200 Series LC and Agilent G6120B Series mass spectrometers
from Agilent technologies, using either Atmospheric Chemical Ionization (APCI) or Electrospray
Ionization (ESI). Column: XBridge C8, 3.5 µm, 4.6 x 50 mm; Solvent A: water + 0.1 % TFA; Solvent B: CAN ;
Flow: 2 ml/min; Gradient: 0 min: 5 % B, 8 min: 100 % B, 8.1 min: 100 % B, 8.5 min: 5% B, 10 min 5% B.
HPLC data were obtained using Agilent 1100 series HPLC from Agilent technologies using XBridge
column (C18, 3.5 µm, 4.6 x 50 mm). Solvent A: water + 0.1 % TFA; Solvent B: ACN; Flow: 2 ml/min;
Gradient: 0 min: 5 % B, 8 min: 100 % B, 8.1 min: 100 % B, 8.5 min: 5% B, 10 min 5% B.
Assay Methods:
Assay A: BTK IC50 Enzyme Assay
The following describes a microfluidic, off-chip mobility shift kinase assay used to measure inherent
potency of compounds against BTK enzyme.
2.5X stocks of full-length human BTK (08-080) from CarnaBio USA, Inc., Natick, MA, 1.6X ATP and
appropriate kinKDR peptide substrate (FITC-AHA-EEPLYWSFPAKKK-NH2) were prepared in kinase
reaction buffer consisting of 25 mM MgCl2, 0.015% Brij-35 (30%), 100 mM Hepes, pH 7.5, and 10 mM
DTT.
5 µL of enzyme buffer and 7.5 µL of ATP/kinKDR peptide substrate mix were added to Matrix (#115304)
384-well, sterile, polypropylene plates (Thermo Fisher Scientific, Hudson, NH) with 125 nL of serially
diluted compounds prepared in 100% DMSO, and incubated for 90 min. at 27C. Following the incubation
period, reactions were stopped by adding 60 µL stop buffer consisting of 100 mM Hepes, pH 7.5, 0.015%
Brij-35 (30%), 0.277% Coating Reagent #3 (Caliper Life Sciences, Mountain View, CA), 5% DMSO.
Stopped reactions were monitored at -2 PSI, -3000 V/-700 V in a LabChip 3000 plate reader from Caliper
Life Sciences, a PerkinElmer Company (Hopkinton, MA), and the activity was measured by off-chip
mobility shift assay measuring the charge/mass difference between substrate and product resulting
from peptide phosphorilation. IC50 and efficacy were determined by plotting log [Inhibitor] vs. %
Activity in GeneData Screener (Basel, Switzerland). Compounds described herein were assayed using this
protocol and the data from the same is recorded in Tables 1, 2, and 3 within the column labeled: “ BTK
Enzyme Assay IC50.”

Assay B: Time Dependent PMBC IC50 Assay
Btk is critical for mediating the signalling of B cell antigen receptor (BCR) after anti
IgM stimulation. Based on this principle, a functional cell-based assay was established to
determine the potency of compounds at inhibiting anti-IgM-induced expression of CD69, adownstream
BCR signaling event, in freshly isolated human peripheral blood mononuclear cells
(PBMCs). In the assay, a 90 pi PBMC suspension containing 2.5x 10 cells was pre-treated with
10 pi of test compound at various concentrations for an hour, and then incubated overnight
(approximateiy 16-18 hours) with 5 pi 420 pg/mi affiniPure F(ab )z fragment goat anti-human
IgM Fc fragment per well (Dianova, Cat.No. : 109-006-129). After the incubation, the cells were
washed and immunostained with an APC-labeled mouse anti-human CD69 (BD Biosciences;
clone: FN50), a PerCP-Cy5. 5 labelled mouse anti-human CD19 (BD Biosciences; clone:
SJ25C1) and a FITC-labelled mouse anti-human CD3 (BD Biosciences; clone: HIT3a), and fixed
for flow cytometric analysis of CD69 expression on CD19 positive cells (B cells). The
percentage of CD69 expressing CD19 positive cells was plotted against the concentrations of test
compounds to obtain a concentration response curve, and calculate an ICso value as a measure of
the potency of test compounds in the assay.
BTK crystallization methods
BTK catalytic domain (residues 328-369), 10 mg/ml in 20 mM tris/HCl, 50 mM NaCl, 3 mM DTT pH 8.0
was incubated overnight with 2mM -standard- replace MSCID (2% v/v final DMSO). Crystals were grown
in 20% w/v PEG 3350; 0.1 M bis tris propane, pH 7.5; 0.2 M sodium acetate using either hanging or sitting drop
vapor diffusion methods. Mature crystals were transferred to cryoprotectant drops containing 25% PEG 3350 and
back-soaked with a final concentration of 1 mM compound. Crystals were flash frozen and diffraction data was
collected at a temperature of 100 °K at the Swiss Light Source, Paul Scherrer Institut, using the PXII beamline,
equipped with a Pilatus6M detector. Data was integrated and scaled using the XDS or Global Phasing Autoproc
programs. Refinement was performed using the programs Buster or Refmac in reciprocal space and coot in direct
space.




Novel series of covalent and selective pyridine and pyrimidine Btk inhibitors was
discovered
Compounds were based on the elaboration of a fragment that bound to the ATP active
site of Btk
Optimization led to compound 17 with excellent biochemical, cellular, and whole blood
potency
Compound 17 did not attain sufficient oral bioavailability in a low dose mouse pK study
to be considered a lead compound
Graphical Abstract

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Discovery of a novel series of pyridine and pyrimidine
carboxamides as potent and selective covalent
Leave this area blank for abstract info.
inhibitors of Btk.
Richard Caldwella, Lesley Liu-Bujalskia, Hui Qiua,*, Igor Mochalkina, Reinaldo Jonesa, Constantin Neagua, Andreas
Goutopoulosa, Roland Grenningloha,Theresa Johnsona, Brian Sherera, Anna Gardbergb, Ariele Viacava Follisa, Federica
Morandic, Jared Heada
O
HN
HN
N
O
N
O
N
N
O
N
H
H2N
O
NH2
71% inhibition at 100 mM 14 Btk IC50= 0.5 nM
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