Synthesis and optimization of furano[3,2-d]pyrimidines as selective spleen tyrosine kinase (Syk) inhibitors

Article abstract of DOI:10.1016/j.bmcl.2016.09.077

A series of furano[3,2-d]pyrimidine Syk inhibitors were synthesized and optimized for their enzyme potency and selectivity versus other kinases. In addition, ADME properties were assessed and compounds were prepared with optimized profiles for in vivo experiments. Compound 23 was identified as having acceptable pharmacokinetic properties and demonstrated efficacy in a rat collagen induced arthritis model.

Full text of DOI:10.1016/j.bmcl.2016.09.077

Accepted Manuscript
Synthesis and optimization of furano[3,2-d]pyrimidines as selective spleen ty-
rosine kinase (Syk) inhibitors
Michael Hoemann, Noel Wilson, Maria Argiriadi, David Banach, Andrew
Burchat, David Calderwood, Bruce Clapham, Phil Cox, David B. Duignan, Don
Konopacki, Gagandeep Somal, Anil Vasudevan
PII:
S0960-894X(16)31026-5
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.09.077
BMCL 24301
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
16 August 2016
28 September 2016
29 September 2016
Please cite this article as: Hoemann, M., Wilson, N., Argiriadi, M., Banach, D., Burchat, A., Calderwood, D.,
Clapham, B., Cox, P., Duignan, D.B., Konopacki, D., Somal, G., Vasudevan, A., Synthesis and optimization of
furano[3,2-d]pyrimidines as selective spleen tyrosine kinase (Syk) inhibitors, Bioorganic & Medicinal Chemistry
Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.09.077
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Synthesis and optimization of furano[3,2-d]pyrimidines as selective
spleen tyrosine kinase (Syk) inhibitors
Michael Hoemanna,* Noel Wilsonb,* Maria Argiriadia, David Banacha, Andrew Burchata, David
Calderwooda, Bruce Claphamc, Phil Coxb, David B. Duignana, Don Konopackia, Gagandeep
Somala, Anil Vasudevanb
aAbbVie Bioresearch Center, 100 Research Dr., Worcester MA 01545
bAbbVie, 1 North Waukegan Rd., North Chicago IL 60064
cCurrent Address: Reset Therapeutics, One Corporate Drive, South San Francisco CA 94080
Abstract
A series of furano[3,2-d]pyrimidine Syk inhibitors were synthesized and optimized for their
enzyme potency and selectivity versus other kinases. In addition, ADME properties were
assessed and compounds were prepared with optimized profiles for in vivo experiments.
Compound 23 was identified as having acceptable pharmacokinetic properties and demonstrated
efficacy in a rat collagen induced arthritis model.
Spleen Tyrosine Kinase (Syk) is a non-receptor tyrosine kinase important for the amplification of
immune signaling through Fc receptors, B-cell receptors and integrin engagement.1 It is broadly
expressed, with the highest levels in hematopoietic cells including mast cells, macrophages,
neutrophils, dendritic cells, NK cells and B cells. The downstream consequences of Fc receptor
signaling such as TNF release from macrophages, oxidative burst from neutrophils, IL-17 and
prostanoid release from mast cells are all thought to contribute to the pathogenesis of rheumatoid
arthritis (RA). Therefore, Syk inhibitors could be useful as therapeutic agents for RA as well as
other immunological diseases.
Several Syk inhibitors have advanced into clinical trials. Fostamatinib is the prodrug of the
active compound tamatinib (1), which is a non-selective kinase inhibitor and therefore not
considered to be an appropriate compound to establish proof of concept for RA through
inhibition of Syk.2,3 There have been several additional Syk inhibitors reported in the clinic
including Portola’s PRT-062607 (2), Gilead’s GS-9973 (3) and Merck’s MK-8457 (4).4 This
paper will describe the design of a set of furano[3,2-d]pyrimidines that are selective inhibitors of

Syk, along with how we optimized their profile to support their progression into in vivo models
of arthritis.
Figure 1. Known Syk inhibitors and starting furano[3,2-d]pyrimdine 6
Our entry into the furano[3,2-d]pyrimidine series started from the thoroughly explored
pyrrolo[2,3-d]pyrimidine scaffold from which inhibitors for many kinases such as Jak’s 5a, Src5b,
Akt5c, BTK5d and PDK15e have been described. More recently, GSK disclosed on a series of
pyrrolo[2,3-d]pyrimidine Syk inhibitors with compound 5 being a representative example.6 It is
widely recognized that pyrrolo[2,3-d]pyrimidine kinase inhibitors suffer from poor kinase
selectivity due predominantly to the H-bond donor-acceptor-donor motif comprising the hinge-
binding element. We reasoned that replacing one of the hinge H-bond donors with a C-H would
attenuate the hinge-binding potential, and through optimization of interactions with more distal
residues afford a more selective inhibitor. To test this hypothesis, the furano[3,2-d]pyrimidine
compound 6 was synthesized and showed a ~30-fold reduction in Syk potency (Table 1). The
potency of this first analog was too weak to establish an improved selectivity profile and the

strategy focused on targeting other regions of the protein binding site to enhance binding
potency.
Table 1. Syk enzyme potency
Syk IC50 (M)a
Compound
0.017
0.561
5
6
a Syk IC50 determined using an HTRF format (see supplementary material for details)
Our optimization strategy was based on a structural model of 6 in Syk to identify potential
interactions that could improve the Syk affinity. We constructed the model from the tamatinib
crystal structure (PDB code 3FQS)7 using Glide software8. The model (Figure 2) showed two
distinct hinge interactions of the furano[3,2-d]pyrimidine core with the Ala451 NH and C=O.
The propylbenzamide was directed towards the extended hinge region while the
trifluoroethylamine was positioned under the glycine rich loop. One key structural feature in the
Syk ATP binding pocket is the relatively rare Pro455 residue with only 8 other kinases having a
proline at this position. In Syk, Pro455 creates a narrow channel at the extended hinge region
and presents an opportunity to make favorable CH/ interactions with aromatic ring systems.9
Figure 2. Model of Compound 6 in the catalytic domain of human Syk Kinase. This model was
based on crystal structure of Tamatinib bound to Syk (PDB code: 3FQS)

The general synthesis of disubstituted furano[3,2-d]pyrimidine analogs began with
commercially available 2,4-dichlorofurano[3,2-d]pyrimidine 24 as illustrated in Scheme 1.
Nucleophilic aromatic substitution (SNAr) of 24 with various amines and subsequent Buchwald
coupling or SNAr allowed rapid access to analogues 6-20, 23, and 26.10 Primary amines used in
the Buchwald reaction were commercially available or were obtained via reduction of the
corresponding commercial nitro compound using standard literature methods (Scheme 1, step
iv), or by the syntheses outlined in Schemes 3 and 4. The homochiral products 21 and 22 were
isolated using chiral HPLC of racemic intermediates 26 (Scheme 2).11
Scheme 1.

Reagents and conditions: (i) NHR1R2, TEA, 1,4-dioxane, 83-99% (ii) NH2R3, Pd2dba3, X-Phos,
K2CO3 or KOt-Bu, t-BuOH or 1,4-dioxane, 85 -110 ºC, 8-48% or (-)-camphor-10-sulfonic acid,
iPrOH, 90 ºC, 54-70% (iii) NaH, THF, 0 ºC, 30 min then SEM-Cl, 0 ºC – rt, 59-90% (iv) H2,
Pd/C, MeOH (v) 25, Pd2dba3, X-Phos, K2CO3, 1,4-dioxane, 110 ºC, 75-100% or 25, Pd2dba3, X-
Phos, KOt-Bu, 1,4-dioxane, 100 ºC, W, 65% (vi) TFA, DCM, 30 ºC, 19-60%
Scheme 2.
Reagents and conditions: (i) Chiral HPLC purification

Targets bearing an indazole in the extended hinge region required a protecting group strategy
as the unprotected indazole yielded two regioisomers as products. While Boc protection was
adequate, the best strategy was protection of commercially available indazoles, 27 and 28, with a
SEM group. Reduction of the nitro group afforded the protected aminoindazoles 29 and 30.
Palladium-catalyzed amination of 25 with the protected indazoles followed by removal of the
protecting group afforded furano[3,2-d]pyrimidines 8, 9, and 19.
Scheme 3.
Reagents and conditions: (i) Pd(OAc)2, Xantphos, Cs2CO3, TEA, 1,4-dioxane, 90 ºC, 77-91%
(ii) 1M HCl, THF, 0 ºC, 70-87%
Scheme 4.

Reagents and conditions: (i) Methylchloroformate, DIEA, DCM, 5 ºC, 81-97% (ii) H2, 10%
Pd/C, 1:1 EtOAc/MeOH, 72-100% (iii) Boc2O, NaHCO3, 5:1 DCM/water (iv) Chiral HPLC
purification, 40% over 2 steps (v) 4M HCl in 1,4-dioxane (vi) Methylchloroformate, DIEA,
DCM, 5 ºC, 100% (vii) H2, 10% Pd/C, 1.7:1 EtOAc/MeOH, 81-95%
Azepinones 33, 36 and 40 were synthesized according to Schemes 3 and 4. Amination of
benzoxazepinone 3112 using benzophenone imine as an ammonia surrogate provided amine
intermediate 33. Treatment of azepinone 3413 with methyl chloroformate provided carbamate
35. Reduction of the nitro group using Pd/C gave the racemic azepinone 36. Homochiral
intermediate 37 was obtained after protecting azepinone 34 and chiral HPLC separation. The
separated tert-butyl carbamate 37 was exchanged for the methyl carbamate to provide 39 using
standard deprotection/protection methods. Reduction of the nitro group with H2/Pd afforded
aniline 40.
The loss in Syk affinity as a result of the core switch could be regained through modification
to the extended hinge group as shown in Table 2. While maintaining the trifluoroethylamino
group at the 4-position, modification of the phenyl group to 7 or the fusion of an additional ring
as in 8, yielded about an 8-fold improvement in potency from 6. In addition, adding modest
steric bulk in the form of a methyl group to the 3-position of the indazole provided a further
modest boost in potency as shown with compound 9. In order to mitigate the presence of four
aromatic rings in compounds 8 and 9, we prepared bicyclic analogs with a saturated ring giving
10, 11, and 12. These bicycles were well tolerated and led to compounds with improved
lipophilic ligand efficiency (LLE), such as 13 (LLE = 5.10).14 In addition, as Syk enzyme
potency was enhanced, it correlated well with enhanced cellular potency (Table 2).
Table 2. SAR for initial analogs

a Syk IC50 determined using an HTRF format (see supplementary material for details)
b
Ramos B-cell IC50 determined using an Ca+2 flux format (see supplementary material for
details)
c LLE is derived from the pIC50 – cLog P (Biobyte)14
With the improved Syk affinity, we next assessed the kinome selectivity of several analogs
utilizing an internal panel of 75 kinases in a TR-FRET format (Figure 3). We evaluated the
kinome profile of the pyrrolopyrimidine 5 as well as several furanopyrimidines from Table 2.
We were encouraged to see that the furanopyrimidines displayed a much improved selectivity
profile over the pyrrolopyrimidine 5. Compound 5 has nine kinases within 10x of the enzyme
IC50 while 7 has a single kinase within 10x and 8 and 13 have none. In fact 13 has only two
kinases (Src and Fyn) at less than 30x, validating the hypothesis that removing one of the H-
bond donors from the hinge would result in an improved kinome profile.

Figure 3. Kinome profiling for furano[3,2-d]pyrimdine Syk inhibitors.
A white bar within the heatmap denotes no data for that particular kinase as the panel changed
over the course of the project (See supplemental section for data)
While 13 showed very good enzyme and cellular potency, and had acceptable properties in
terms of unbound clearance in rat microsome incubation, it suffered from poor solubility (4.96
M, Fed SSIF pH 5).15 In addition, the trifluoroethyl group contributed a significant amount of
molecular weight and calculated lipophilicity. Therefore, we set out to scan the 4-position to
find a suitable replacement (Table 3). The ethyl analog, 14, provided a significant improvement
in solubility, yet suffered from increased rat microsomal clearance. This trend continued with
extended alkyl chains 15 as well as branched alkyl chains 16, which also exhibited a loss in
potency. Incorporation of a cyclopropyl group in 18 provided the optimal balanced set of
attributes related to potency, clearance and solubility
Table 3. SAR and optimization for solubility and clearance

We obtained a crystal structure of Compound 19 bound to the catalytic domain (amino acids
363-639) of human Syk (Structure methods, data and structure refinement are described in the
Supplemental Methods; PDB Code 5T68). Compound 19 is a potent Syk inhibitor (Enzyme IC50
= 0.023 M; Ramos B-Cell EC50 = 0.033 M). The structure confirmed the donor-acceptor
hinge binding interactions of the furanopyrimidine core with distances of 2.8 Å to the acceptor
NH of Ala451 and 2.6 Å to the donor carbonyl group of Ala451. The structure also confirmed
the interaction of the indazole ring with Pro455 measured at a distance of 4.0 Å (Figure 4).
Proline-aromatic interactions are routinely observed in protein structures and also reported in
Isostar.16 An additional interaction was observed between the NH of the indazole and the
carbonyl group of glycine rich loop residue Leu377. We found that the combination of a 1)
donor-acceptor hinge interaction with 2) stabilization of the glycine rich loop via Leu377 and 3)

long range aromatic interaction with Pro455 led to an excellent selectivity profile in our internal
TR-FRET kinase panel (64 kinases with IC50’s > 1.0 M). The cyclopropyl moiety did not
appear to be making any direct interactions with the protein and most likely contributed to filling
void space in the ATP binding pocket. In silico water analysis using the Shrödinger Maestro
WaterMap software generated water with calculated energies in the apo form of Syk.
Destabilized waters (G 2 to 3 kcal/mol) were identified in the cyclopropyl binding pocket
suggesting that this area could be displaced by hydrophobic groups.17
Figure 4. X-ray crystal structure of Compound 19
As with any medicinal chemistry program, there was a need to optimize ADME properties for
this series based on data from our initial screens. In particular, for 18 and 19, it was found that
these compounds displayed significant time-dependent inhibition (TDI) of CYP3A4 in human

liver microsomes (Table 4). In general, the TDI was observed when the 4-position contained a
cyclopropylamino group. For example, 13 had a lower decrease in enzyme activity. This
observation is supported by literature reports of compounds containing cyclopropylamines
moieties that exhibit TDI of CYP3A4.18 Therefore, even though 19 exhibited low microsomal
clearance, the TDI liability led us to use other substituents at this position.
Table 4. In vitro time-dependent inhibition of CYP3A4 in human liver microsomes
Concentration
% Activity at
0 minutes
% Activity Remaining at
30 minutes
Compound
(M)
10
50
10
50
10
50
91
92
97
80
94
87
81
75
56
14
51
28
13
18
19
In addition, based on the high cLog P values and early in vivo PK data (data not shown), we
continued to look for ways to minimize the in vivo clearance of these compounds. This series
had a good in vitro / in vivo correlation between unbound rat microsomal clearance and unbound
plasma clearance from rat pharmacokinetic (PK) experiments. This enabled the use of in vitro
microsomal clearance data to select compounds to move forward into in vivo experiments. The
data suggested that the addition of an exocyclic methyl carbamate (20 to 21) could reduce mouse
and human clearance, probably due to the relative decrease in cLog P (5.09 to 3.61). For the
exocyclic carbamates, one enantiomer was more potent than the other (Compare 21 with 22) and
this was a consistent finding (~10 to 100x) irrespective of the carbamate group or the 4-
substituent. Compound 23 did not exhibit CYP3A4 TDI and had a low rate of in vitro
microsomoal clearance in both mouse and human. Due to its acceptable PK profile in both rat
and dog (Table 5), 23 was selected for further in vivo experiments. We chose to evaluate the role
of 23 in rat collagen induced arthritis (CIA) because this model shares many similarities with
human rheumatoid arthritis. In particular, the development of arthritis relies upon breach of
tolerance and generation of antibodies to collagen and self, which are modulated by Syk
dependent cellular processes. Compound 23 was dosed orally in the rat CIA model at 0.3,1, 3, 0
and 30 mg/kg b.i.d. Treatment began at first signs of clinical disease and continued for 8 days
until the end of the experiment. Administration of 23 resulted in a dose dependent decrease in
paw swelling achieving 75% inhibition at 10 mg/kg and 95% inhibition at 30 mg/kg, the same

degree of efficacy as seen with prednisolone treatment. The compound was well tolerated
throughout the study. The ED50 was 9 mg/kg corresponding to a day last AUC50 (0-12 h) of 3.9
g*hr/mL. Compound 23 did not show any liabilities in an in vitro safety panel (Ames >100
mM , hERG IC50 >20 M, and HepG2 cytotoxicity IC20 >10 M).
Table 5. Rat and Dog Pharmaokinetcs for 23
i.v.
p.o.
Dose
(mg.kg)
Species
Cl
(L/hr/kg)
Vss
(L/kg)
AUC
Cmax
T1/2 (h)
F (%)
(g*hr/mL) (ng/mL)
Rat
1
1
1.7
2.0
9.9
4.6
2.3
27
40
0.18
2.34
36
Dog
0.17
151
Figure 5. Rat CIA data for Compound 23

In summary, we report the discovery of furano[3,2-d]pyrimidines as novel and selective Syk
inhibitors. The initial lead 6 was optimized for potency as well as kinase selectivity by targeting
interactions with Pro455 in the kinase pocket. Liabilities including time–dependent CYP3A4
inhibition as well as high microsomal clearance were observed. Optimization to address these
multiple liabilities led to 23 which possessed good PK in both rat and dog and demonstrated
efficacy in a rat CIA model.
References and notes
Use of the IMCA-CAT beamline 17-ID (or 17-BM) at the Advanced Photon Source was
supported by the companies of the Industrial Macromolecular Crystallography Association
through a contract with Hauptman-Woodward Medical Research Institute.
Use of the Advanced Photon Source was supported by the U.S. Department of Energy, Office of
Science, Office of Basic Energy Sciences, under Contract No. DE-AC02-06CH11357.
Disclosures
Michael Hoemann, Noel Wilson, Maria Argiriadi, David Banach, Andrew Burchat, David
Calderwood, Phil Cox, David Duignan, Don Konopacki, Gagandeep Somal and Anil Vasudevan
are employees of Abbvie. Bruce Clapham was an employee of AbbVie at the time of the work.
The design, study conduct and financial support for this research were provided by AbbVie.
AbbVie participated in the interpretation of data, review and approval of the publication.
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Graphical abstract

Ramos B-
Cell EC50
(M)b
Syk IC50
Rat Cl,mic unbound
(L/hr/kg)
(M)a
Compound
R
LLE
6
0.561
0.061
0.346
No Data
1.73
7
0.117
96.0
2.25
8
9
0.081
0.047
0.063
0.179
0.235
0.087
0.101
0.723
15.3
No Data
21.9
2.82
2.79
3.34
3.05
10
11
10.6
12
13
0.043
0.007
0.049
0.017
52.8
<4.9
2.07
5.10

Human
Cl,mic
unbound
(L/hr/kg)
Ramos B-
Cell EC50
(M)b
Solubility
Fed SSIF
pH5 (M)
Rat Cl,mic
unbound
(L/hr/kg)
Syk IC50
Compound
R1
R2
(M)a
LLE
13
-CH2CF3
0.007
0.006
0.045
0.064
0.017
0.018
0.030
0.043
<4.9
19.1
22.4
64.5
10.2
5.5
4.96
>67
5.10
14
15
16
-CH2CH3
5.43
4.03
3.47
- CH2CH2CH3
- CH2CH2(CH3)2
18.4
30.4
>200

17
- CH2(CH3)2
0.013
0.030
4.79
18
19
20
-c-Pr
-c-Pr
-c-Pr
0.005
0.023
0.005
0.018
0.034
0.035
12.0
7.2
158
99
5.45
3.3
23.8
10.9
19.9
10.6
19.0
7.4
3.21
21
22
23
-c-Pr
-c-Pr
0.012
1.37
0.039
27
4.31
2.25
4.10
7.1
-CH2CHF2
0.020
0.026
<2.3
>67