Approaching the active conformation of 1,3-diaminopyrimidine based covalent inhibitors of Bruton's tyrosine kinase for treatment of Rheumatoid arthritis

Article abstract of DOI:10.1016/j.bmcl.2016.03.011

By applying conformational restrictions, we were able to discover highly potent 1,3-diaminopyrimidine based covalent inhibitors of BTK, such as 8a (IC₅₀ = 3.76 nM), and providing useful information of its active conformation. We are developing these novel small molecule covalent inhibitors of BTK toward oral agents for Rheumatoid arthritis.

Full text of DOI:10.1016/j.bmcl.2016.03.011

Accepted Manuscript
Approaching the Active Conformation of 1,3-Diaminopyrimidine Based Cova-
lent Inhibitors of Bruton’s Tyrosine Kinase for Treatment of Rheumatoid
Arthritis
Zhenhua Huang, Qian Zhang, Lianzhong Yan, Guizhen Zhong, Linqi Zhang,
Xiaojuan Tan, Yanli Wang
PII:
S0960-894X(16)30231-1
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.03.011
BMCL 23654
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
31 December 2015
22 February 2016
4 March 2016
Please cite this article as: Huang, Z., Zhang, Q., Yan, L., Zhong, G., Zhang, L., Tan, X., Wang, Y., Approaching
the Active Conformation of 1,3-Diaminopyrimidine Based Covalent Inhibitors of Bruton’s Tyrosine Kinase for
Treatment of Rheumatoid Arthritis, Bioorganic & Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/
10.1016/j.bmcl.2016.03.011
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Bioorganic & Medicinal Chemistry Letters
Approaching the Active Conformation of 1,3-Diaminopyrimidine Based
Covalent Inhibitors of Bruton’s Tyrosine Kinase for Treatment of Rheumatoid
Arthritis
Zhenhua Huang, Qian Zhang, Lianzhong Yan, Guizhen Zhong, Linqi Zhang, Xiaojuan Tan and Yanli
Wang
KBP Biosciences, C201, No. 750, Shunhua Road, Jinan, Shandong Province, 250101, China
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
By applying conformational restrictions, we were able to discover highly potent 1,3-
diaminopyrimidine based covalent inhibitors of BTK, such as 8a (IC50 = 3.76 nM), and
providing useful information of its active conformation. We are developing these novel small
molecule covalent inhibitors of BTK toward oral agents for Rheumatoid arthritis.
Accepted
Available online
2015 Elsevier Ltd. All rights reserved.
Keywords:
Bruton’s tyrosine kinase (BTK)
Rheumatoid arthritis (RA)
Collagen-induced arthritis (CIA)
1,3-Diamino-pyrimidine
Bruton’s tyrosine kinase (BTK) is a cytoplasmic, non-receptor
tyrosine kinase and belongs the Tec kinase family.1 BTK is
expressed in B cells, mast cells, monocytes/macrophages,
neutrophils, dendritic cells, erythroid cells, platelets,
hematopoietic stem cells, and multipotent progenitors.2 It has
been reported that BTK plays a critical role in B cell antigen
receptor (BCR) activation, and that loss of function mutations in
the BTK gene prevent the development of B cells, causing a
depletion of B cells and circulating immunoglobulins.3
erythematosus (SLE).4 Several BTK inhibitors are currently
under clinical development.1 The most advanced BTK inhibitor,
Ibrutinib (1, Figure 1) has been approved for the treatment of
mantle cell lymphoma in 2013, and for chornic lymphocytic
leukemia in 2014, and for Waldenström's macroglobulinemia, a
form of non-Hodgkins lymphoma in January 2015.5 Also in
clinical development is Spebrutinib (2, Figure 1), and it is the
first BTK inhibitor to be tested in patients with RA.6 Both
Ibrutinib and Spebrutinib are irreversible covalent inhibitors of
BTK, and they represent the new trend of effective and safe
covalent drugs in development.7
O
O
O
In search for novel small molecule BTK covalent inhibitor
with improved selectivity and pharmacokinetic properties for the
treatment of RA, we formulated our working hypothesis as
follows: restriction of the free rotation of C-N single bonds
between aromatic moieties of Spebrutinib would result in
conformationally defined molecules that might have better
alignment of its reactive group inside the BTK active site. Since
the nitrogen atom at the 4-position of the pyrimidine is closer to
the reactive site, we decided to explore this position first rather
than the nitrogen atom at 2 position. Taken into consideration
that this structural space has been heavily patented in recent
years, we carried out an extensive search for novel chemotypies
with better defined conformational space. In this letter, we report
HN
N
N
N
N
NH2
N
N
H
F
N
N
NH
1
2
O
O
Figure 1. BTK inhibitor Ibrutinib (1) and Spebrutinib (2)
Because of these findings, BTK has been targeted for the
treatment of autoimmune diseases, such as Rheumatoid arthritis
(RA), and several research groups have recently reported small
molecule BTK inhibitors which are efficacious in rodent models
of collagen-induced arthritis (CIA) and systemic lupus
———
 Corresponding author. Tel.: +86-531-8889-0505; e-mail: kbp@kbpbioscience.com

Scheme 1. Reaction keys: a) Acryloyl chloride, K2CO3, THF; b) HCl, DCM;
c) NaOAc, EtOH; d) HOAc, n-BuOH, 110˚C; e) H2O2, HOAc; f) m-CPBA,
DCM.
the discovery of novel covalent BTK inhibitors based on
conformationally restricted 1,3-diaminopyrimidine.
Comparative conformational analysis revealed that to adopt
the active conformation close to that of Ibrutinib, the benzene
ring of benzene-1,3-diamine in Spebrutinib is most likely rotated
away from the fluorine atom on the pyrimidine ring. It is
conceivable that this particular conformation is stabilized by the
intramolecular H-bond between the NH and electronegative
fluorine atom (Figure 1, dotted line).8 Based on this analysis, we
decided, first, to replace the fluorine atom with sterically more
demanding substituents, and second, to cyclize from this position
onto the amino group (Figure 2). A quick SAR study showed that
bulkier sulfur atom is a good replacement for fluorine.
Table
methanethioethers
1
shows the activities of some representative
(7), methanesulfoxides (8), and
methanesulfones (9). It is clear that the sulfones have reduced
activities as compared to corresponding thioethers and sulfoxides,
whereas the thioethers and the corresponding sulfoxides are
similar in enzyme inhibitory activities. Further evaluation in B-
Cell proliferation assay, we found that the thioethers (7) have
bigger right shifts as compared to its sulfoxide analogs (8),
probably due to the differences in unspecific reactivity. The
sulfoxides showing in the table are racemates. Using chiral
HPLC or chiral synthesis described earlier, we were able to
resolve some of these racemic mixtures and obtain enantiomers.
However, we found no noticeable differences between
enantiomers with regard to BTK inhibition and B cell
proliferation inhibition (data not shown).
X
Figure 2. Conformationally restricted BTK inhibitor designs
HN
O
N
N
HN
HN
Y
The synthesis of 1,3-diaminopyrimidine-4-sulfur substituted
analogs (7, 8, and 9) is outlined in Scheme 1. The starting mono-
Boc-protected 1,3-diaminobenzene was reacted with acryloyl
chlodride under the conditions of potassium carbonate in THF.
Product 4 was deprotected under acidic conditions to yield mono-
acryloylated 1,3-diaminobenzene 5. Aniline 5 was then reacted
with 1,3-dichloro-4-(methylthio)pyrimidine in ethanol and
anhydrous sodium acetate, and resulting pyrimidine 2-chloride 6
was treated with various para-substituted anilines in n-butanol
with catalytic amounts of glacier acetic acid under mild heating
to yield sulfide product 7a-d. Oxidation of the sulfide 7a-d to the
corresponding sulfoxides (8a-d) was accomplished by treating
the sulfides with hydrogen peroxide in acetic acid with mild
heating, and enantiomerically enriched sulfoxide 8a-d were
obtained by asymmetric oxidation with either (D)-(-)-DET or (L)-
(+)-DET ligand in the presence of titanium tetraisopropoxide and
oxidizing agent TBHP. Selected sulfoxides were further oxidized
to the corresponding sulfones with mCPBA.9
BTK
B Cell
Compd
Inhibition
Proliferation
-X-
-Y
IC50 (nM)a
IC50 (nM)a
2b
7a
8a
9a
7b
8b
F
S
4.61
6.60
3.76
31.2
1.08
0.903
3.28
30.6
5.85
N/Ac
64.0
3.55
O
O
SO
SO2
S
O
N
SO
SO
SO
1.48
5.65
14.9
19.4
8c
O
O
N
8d
H
N
aValues reported are the mean of a minimum of two replicates with a
standard deviation <25% of the mean, and the same for data in other tables.
bReported values, see ref. 10; cNot available, and same in other tables.
Table 1 BTK inhibition of sulfur containing analog 7, 8 and 9 in Scheme 1
Although there was no noticeable improvement in BTK
inhibition from that of the original lead Spebrutinib, we were
quite encouraged to observe that, by and large, structural
modification at that particular site can be tolerated. We then
applied cyclization strategy outlined earlier to further restrict the
accessible conformation of the lead compound.
The synthesis of the cyclized derivatives is outlined in
Scheme 2. The starting 5-bromopyrimidine-2,4-dione was
substituted with alkylamines under the conditions of potassium
carbonate in THF. Product 11a-b was treated with phosphoryl
trichloride to yield 2,4-dichloropyrimidine 12a-b, which was
then reacted with mono protected1,3-benzendiamine in ethanol
and with catalytic amounts of glacier acetic acid and heating to
yield sulfide product 13a-b. The resulting 2-chloropyrimidine
was then substituted with various aromatic amines under

microwave heating. Cyclization was accomplished by heating the
product (14a-g) with oxylylchloride. Final product 16a-g were
obtained after removal of Boc protecting group under acidic
conditions, followed by acylation with acryloyl chloride to install
the reactive Michael acceptor for covalent inhibition.9
toward plasma proteins.10 So, for plasma stability, samples with
initial concentration of 1M was incubated in SD rat, beagle dog
and human plasma samples for up to 4 h at 37 ℃. At the desired
time points, an aliquot from the incubation was quenched by
adding 10 volumes of 100% methanol supplemented with
internal standard. Following the last collection, the samples were
centrifuged at 12,000 rpm for 5 min and the supernatants were
transferred to a new plate containing an equal volume of water
for analysis by liquid chromatography coupled to triple
quadrupole mass spectrometry (LC-MS/MS).11 The results of
representative compounds were summarized in Table 3.
R
N
N
O
X
N
N
N
H
O
N
H
BTK
B Cell
Compd
Inhibition
IC50 (nM)
Proliferation
IC50 (nM)
R-, -X
O
O
NH
CH3
-
13.0
4.11
13.7
6.84
16a
N
16b
16c
16d
16e
O
CH3-
O
Scheme 2. Reaction keys: a) R-NH2, K2CO3, THF; b) POCl3; c) n-BuOH, ;
d) X-NH2, microwave, 110˚C; e) (COCl)3; f) HCl; g) Acryloyl Chloride,
DIEA, THF.
31.0
30.6
222
70.6
818.4
N/A
N
O
The synthesis of the cyclized derivatives is outlined in
Scheme 2. The starting 5-bromopyrimidine-2,4-dione was
substituted with alkylamines under the conditions of potassium
carbonate in THF. Product 11a-b was treated with phosphoryl
trichloride to yield 2,4-dichloropyrimidine 12a-b, which was
then reacted with mono protected1,3-benzendiamine in ethanol
and with catalytic amounts of glacier acetic acid and heating to
yield sulfide product 13a-b. The resulting 2-chloropyrimidine
was then substituted with various aromatic amines under
microwave heating. Cyclization was accomplished by heating the
product (14a-g) with oxylylchloride. Final product 16a-g were
obtained after removal of Boc protecting group under acidic
conditions, followed by acylation with acryloyl chloride to install
the reactive Michael acceptor for covalent inhibition.9
N
N
O
N
O
O
O
265
318
N/A
N/A
16f
N
Although some of these cyclic imidazolinone derivatives, such
as 16b, are rather potent BTK inhibitors (Table 2), they do not
show improvement in terms of intrinsic activities when compared
to the acyclic sulfur containing analogs (7 and 8).
16g
O
N
Table 2 BTK inhibition of imidazolinone derivative 16.
These results are consistent with our proposed active
conformation depicted in Figures 1 and 2. One could further
argue that, although acyclic, our sulfoxide derivatives are also
stabilized by the same intramolecular hydrogen bond in adopting
the conformation necessary for covalent inhibition. We also
believe that by fine tuning of the juxtaposition of reactive
acrylamide and the binding domain of the molecule with various
conformation restriction will eventually lead to more potent
covalent inhibitors of BTK.
Beagle
dog
Compd
Human
SD rat
86.7%
42.2%
40.8%
94.3%
90.7%
27.5%
80.5%
8a
16.2%
93.4%
16b
16c
Optimization of irreversible kinase inhibitors is somewhat
different from that of reversible inhibitors, because not all
ADME parameters are of equal importance.7 Since, by definition,
covalent inhibitors are reactive in nature, it is imperative that we
examine the non-specific reactivity of these new inhibitors
% remaining after 4 h incubation in plasma at 37˚C with initial
concentration of 1M.
Table 3. In vitro plasma stability of representative compounds.

References
1, Burger, J. A. Current Hematologic Malignancy Reports 2014,
Compound 8a showed remarkable stability across different
species. However, somewhat unexpected, cyclic imidazolinone
analog 16b has much reduced stability across the species, and
16c has good stability in dog and SD rat plasma, by not in human
plasma. These results indicated that for cyclic analogs (16), non-
specific reactivity is moderate to high, and this could be due to
the slightly increased reactivity of the acrylamide or the more
easily accessible of its reactive group from conformational
restriction.
9, 44.
2, Smith, C. R.; Dougan, D. R.; Komandla, M.; Kanouni, T.;
Knight, B.; Lawson, J. D.; Sabat, M.; Taylor, E. R.; Vu, P.;
and Wyrick, C. Journal of Medicinal Chemistry 2015, 58,
5437.
We further studied the pharmacokinetic properties of these
compounds, and results were summarized in Table 4. Not too
surprisingly, these compounds showed moderate plasma
clearance and very low oral bioavailability. Clearly these
parameters need optimizing, however, we were delighted to
observe that these compounds have short half-lives in rats. Since
no excessive circulating levels are required to maintain efficacy
with irreversible inhibitors, rapid clearance should lead to a lower
propensity for off-target related adverse effects, provided that
covalent inhibitors have fast on-rate (kon) and slow off-rate
(koff).12
3, Hendriks, R. W.; Yuvaraj, S.; and Kil, L. P. Nature Reviews
Cancer 2014, 4, 219.
4, Lou, Y.; Han, X.; Kuglstatter, A.; Kondru, R. K.; Sweeney, Z.
K.; Soth, M.; McIntosh, I.; Litman, R.; Suh, J.; Kocer, B.;
Davis, D.; Park, J.; Frauchiger, S.; Dewdney, N.; Zecic, H.;
Taygerly, J. P.; Sarma, K.; Hong, J.; Hill, R. J.; Gabriel, T.;
Goldstein, D. M.; and Owens, T. D. Journal of Medicinal
Chemistry 2015, 58, 512.
5, Pan, Z.; Scheerens, H.; Li, S. J.; Schultz, B. E.; Sprengeler, P.
A.; Burrill, L. C.; Mendonca, R. V.; Sweeney, M. D.; Scott, J.
C.; Grothaus, P. G.; Jeffery, D. A.; Spoerke, J. M.; Honigberg,
L. A.; Young, P. R.; Dalrymple, S. A.; and Palmer, J. T.
ChemMedChem 2007, 2, 58.
Pharmacokinetic Parameter
CL
(L/h/kg)
6.5±1.2
t1/2
(hr)
Foral
(%)
Cmax
(ng/mL)
AUC0-t
(ng*hr/mL)
73±16
Compd
8a
1.3±0.5
23±4.0
55±7.0
1.8±0.1
2.4±2.3 9.0±1.1
1.4±0.9 20±1.1
107±112
41±24
100±91
27±17
16a
16b
6, Evans, E. K.; Tester, R.; Aslanian, S.; Karp, R.; Sheets, M.;
Labenski, M. T.; Witowski, S. R.; Lounsbury, H.; Chaturvedi,
P.; Mazdiyasni, H.; Zhu, Z,; Nacht, M,; Freed, M. I.; Petter,
R, C.; Dubrovskiy, A.; Singh, J.; and Westlin, W. F. Journal
of Pharmacology and Experimental Therapeutics 2013, 346,
219.
15.2±4.6
Data are presented as Mean±SD. Clp, plasma clearance after iv dosing;
t1/2, half life after po dosing; Foral, oral bioavailability; Cmax, peak plasma
concentration after indicated oral dosing; AUC0-t, area-under-curve after
indicated oral dosing. Pharmacokinetic parameters were obtained following
an IV (2 mg/kg) in 2%DMSO/15%Kolliphor HS15/5%Glucose /Water
(pH=4.0) or P.O. (2 mg/kg) dose (amorphous free base) in 2%DMSO/15%
Kolliphor HS15/5%Glucose /Water (pH=4.0).
7, Barf, T.; and Kaptein, A. Journal of Medicinal Chemistry
2015, 55, 6243.
Table 4. Pharmacokinetic Parameters of representative compounds in SD
rats.
8, Razgulin, A. V.; Mecozzi, S. Journal of Medicinal Chemistry
2006, 49, 7902.
In summary, we have discovered novel covalent BTK
inhibitors based on conformationally restricted 2-Amino-7,9-
dihydro-8H-purin-8-one, which led toward better understanding
of the reactive conformation of the covalent inhibitors. Some of
our inhibitors are highly potent and have shown good stability in
plasma, indicating little unspecific covalent labeling of plasma
proteins. We are working to expand the SAR's to new cyclic
systems, and new structure motifs for better overall profiles. We
are advancing the BTK program by carrying out in vivo efficacy
studies in rat CIA models, and eventually to preclinical candidate
nomination.
9, All new compounds were characterized by 1H NMR and LC-
MS prior to submission for biological evaluation.
10, Karp, R.; Evans, E.; Aslanian, S.; Chaturvedi, P.; Mazdiyasni,
H.; Nacht, M.; Petter, R.; Sheets, M.; Singh, J.; Tester R.; and
Westlin, W. 2010 IRA, Poster#A113.
11, Wang, J.; Li-Chan, X. X.; Atherton, J.; Deng, L.; Espina, R.;
Yu, L.; Horwatt, P,; Ross, S.; Lockhead, S,; Ahmad, S.;
Chandrasekaran, A.; Oganesian, A.; Scatina, J.; Mutlib, A.;
and Talaat. R. Drug Metabolism and Disposition 2010, 38,
1083.
Acknowledgments
12, Konsoula, R.; Jung, M. International Journal of
Pharmaceutics 2008, 361, 19.
We thank Dr. Gui-Bai Liang for his valuable advices and
suggestions to the project; KBP DMPK group for
pharmacokinetic studies; Chem Partner, Inc. for service of in
vitro enzymatic assays; and CrownBio, Inc. for service of in vitro
cell based assays.
13, Smith, A. J.; Zhang, X.; Leach, A. G.; Houk, K. N. Journal of
Medicinal Chemistry 2009, 52, 225.

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Approaching the Active Conformation of
1,3-Diaminopyrimidine Based Covalent
Inhibitors of Bruton’s Tyrosine Kinase for
Treatment of Rheumatoid Arthritis
Leave this area blank for abstract info.
Zhenhua Huang, Qian Zhang, Lianzhong Yan, Guizhen Zhong, Linqi Zhang, Xiaojuan Tan and Yanli Wang
X
8a, X = MeSO-, IC50 = 3.76 nM
HN
O
N
N
R
16b, X = -N(Me)CO-, IC50 = 4.11 nM
HN
HN
O