Discovery and optimization of thieno[2,3-d]pyrimidines as B-Raf inhibitors

Article abstract of DOI:10.1016/j.bmcl.2011.03.006

The serine/threonine specific protein kinase B-Raf is part of the MAPK pathway and is an interesting oncology target. We have identified thieno[2,3-d]pyrimidines as a core scaffold of small molecule B-Raf inhibitors. The SAR of analogs in this series will be described.

Full text of DOI:10.1016/j.bmcl.2011.03.006

Bioorganic & Medicinal Chemistry Letters 22 (2012) 747–752
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery and optimization of thieno[2,3-d]pyrimidines as B-Raf inhibitors
⇑
⇑
Garrick K. Packard , Patrick Papa, Jennifer R. Riggs , Paul Erdman, Lida Tehrani, Dale Robinson, Roy Harris,
Graziella Shevlin, Sophie Perrin-Ninkovic, Robert Hilgraf, Margaret A. McCarrick, Tam Tran, Yuedi Fleming,
April Bai, Samantha Richardson, Jason Katz, Yang Tang, Jim Leisten, Mehran Moghaddam, Brian Cathers,
Dan Zhu, Steven Sakata
Celgene Corporation, San Diego, 4550 Towne Centre Ct, San Diego, CA 92121, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
The serine/threonine specific protein kinase B-Raf is part of the MAPK pathway and is an interesting
oncology target. We have identified thieno[2,3-d]pyrimidines as a core scaffold of small molecule
B-Raf inhibitors. The SAR of analogs in this series will be described.
Received 17 January 2011
Revised 28 February 2011
Accepted 2 March 2011
Available online 6 March 2011
Ó 2011 Published by Elsevier Ltd.
Keywords:
B-Raf inhibitor
Thieno[2,3-d]pyrimidine
PDE4 inhibitor
Raf proteins are components of the mitogen activated protein
(MAP) kinase pathway, downstream of the membrane-bound small
G-protein Ras. Upon activation by extracellular signals, Ras stimu-
lates the activation of Raf protein kinases, which trigger a series
of phosphorylation events to regulate cell proliferation, differentia-
tion, senescence, and apoptosis.1 Among the three Raf isoforms in
humans (A-Raf, B-Raf, and C-Raf), B-Raf is the most critical isoform
Our screening lead 5 was identified from a series of PDE4
inhibitors and is nearly equipotent against both B-Raf and PDE4.
PDE4 inhibitors are known to induce emesis in animal species en-
dowed with a vomiting reflex,6 therefore we wished to identify
potent B-Raf inhibitors with little or no PDE4 inhibitory activity.
It was quickly determined that the C-5 amino and the C-6 carbox-
amide are both required for B-Raf inhibitory activity, therefore we
to mediate Ras activity and is important for tumor progression.2
A
significant fraction of melanoma, colorectal, and thyroid cancers
have activating B-Raf mutations, particularly at valine 600.3 A neg-
atively charged amino acid substitution mimics the active form of
the B-Raf enzyme thereby making it constitutively active.4 The
V600E B-Raf mutation is by far the most prevalent in human can-
cers.3 In this publication, we present our efforts to discover V600E
B-Raf inhibitors from a thieno[2,3-d]pyrimidine series.5
A general synthetic approach to the thieno[2,3-d]pyrimidine
series is shown in Scheme 1. Condensation of an appropriately
substituted benzaldehyde with 2-methyl isothiourea and ethyl
cyanoacetate afforded intermediate 2. Conversion of the 4-hydroxy
substituent to a chloro followed by reaction and cyclization with
2-mercaptoacetamide gave intermediate 3. The thiomethyl substi-
tuent at C-2 of the pyrimidine was oxidized with m-CPBA and then
displaced using either an amine and diisopropylethylamine or an
alcohol and sodium hydride to give the desired products 4.
R1
R1
a
b, c, d
CN
OH
N
2
O
H
S
N
1
R1
R1
NH2
NH2
4
e, f
5
O
O
N
N
3
R2
X2
N
S
NH2
S
NH2
S
N
4
X = NH, O
Scheme 1. Reagents and conditions: (a) 2-methyl-isothiouronium sulfate, ethyl
cyanoacetate, K2CO3, EtOH, 80 °C; (b) POCl3, 1,4-dioxane, reflux; (c) 2-mercapto-
acetamide, Na2CO3, EtOH, 60 °C; (d) sodium ethoxide, EtOH, 75 °C; (e) m-CPBA,
CHCl3; (f) R2NH2, DIPEA, DMF, 90 °C, or R2OH, NaH, THF.
⇑
Corresponding authors. Tel.: +1 858 795 4854; fax: +1 858 795 4719 (J.R. Riggs).
E-mail addresses: gkpackardca@gmail.com (G.K. Packard), jriggs@celgene.com
(J.R. Riggs).
0960-894X/$ - see front matter Ó 2011 Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2011.03.006

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G. K. Packard et al. / Bioorg. Med. Chem. Lett. 22 (2012) 747–752
Table 1
C-2 SAR of the thienopyrimidine scaffold
Cl
Cl
NH2
O
N
S
NH2
R
N
Hepat. stab.d
8.8 2.9
a
b
c
#
R
B-Raf IC50
(
lM)
PDE4 IC50
(lM)
pMEK IC50 (lM)
5
0.316 0.059
0.261 0.045
4.74 1.01
N
H
6
7
0.507 0.103
0.407
11.8e
>60e
NDf
H
0.077e
17.6e
53 10
H2N
0.023e
ND
ND
N
H
8
0.718 0.273
HO
3.72e
22.4e
7.91e
ND
N
H
9
0.275 0.018
0.110 0.017
H2N
N
10
9.75 2.88
100 27
H
HO
HO
11
12
0.067 0.024
0.112e
8.42e
4.84e
3.82e
9.64e
35
34
2
5
N
H
N
H
13
14
HO
HO
0.050 0.008
0.219e
ND
6.88e
14.5e
17
57
2
4
O
11.9e
a
b
c
d
e
f
In vitro enzyme activity (V600E B-Raf 33P enzyme assay).5
In vitro enzyme activity (U937 cell-derived PDE4 enzyme assay).5
(Biomarker for B-Raf inhibition) In vitro cellular activity (A375 pMEK1/2 meso scale assay).5
In vitro rat hepatocyte assay (% remaining after 1 h).7
Compound was tested once in this assay.
No data.
examined the SAR of substituents at C-2 and C-4 (Tables 1 and 2).
A small amine substituent is required at C-2 for potent PDE4 inhi-
bition (Table 1). Replacing the C-2 amine substituent with H (ana-
log 6) greatly reduced PDE4 inhibitory activity. Primary amine 7
and methyl amine 8 are both more potent inhibitors of PDE4 than
B-Raf. An increase in B-Raf enzyme potency and a decrease in
PDE4 enzyme potency were observed by extending the C-2 sub-
stituent from NHMe 8 to ethanolamine 9. Further probing the
amine linkers, we found variations of the ethanolamine 10–14
were well tolerated and offered improved rat hepatocyte stability
compared to the cyclopropyl amine 5. In general, oxygen-linked
compounds were less stable in the rat hepatocyte assay than
the corresponding amine-linked analogs (see analog 13 vs analog
11). Carbon-linked analogs were generally less potent than both
the corresponding amine and oxygen-linked analogs (see analog
14 vs analogs 13 and 11). Table 2 details some of our initial ef-
forts to optimize the C-4 aryl substituent. For SAR purposes, we
chose to hold the cyclopropyl amine at C-2 constant while vary-
ing C-4 substituent. The 3,4-difluoro analog 15 is almost 10 times
less potent than the 3,4-dichloro analog 5. The 3-phenol analog
16 gave a significant boost in B-Raf potency. Close analogs of
the 3-phenol such as 4-phenol 17, 3-methoxy 18, 3-pyridyl 19,
and 3-amino 20, resulted in a dramatic drop in B-Raf potency.
Combining the 3-chloro with the 3-hydroxy substituent resulted
in analog 21 which afforded a ꢀ10-fold boost in B-Raf and pMEK
biomarker potency. This analog represents the most potent ana-
log to date but suffers from poor hepatocyte stability correlating
to rapid in vivo clearance.8,9
Our general strategy was guided by SBDD of compound 5
docked into the published crystal structure of B-Raf ( Fig. 1).10
The C-6 carboxamide of 5 acts as hydrogen bond donor and accep-
tor to CYS 531 in the hinge region of the kinase while the C-2 sub-
stituent occupies a solvent exposed region. The C-4 substituent of
the pyrimidine occupies an interior pocket formed by residues
Val470, Ala480, the backbone of Val481, Lys482, Leu513, Ile526,
and the ‘gatekeeper’ residue Thr528. This pocket is fairly large ow-
ing to the small gatekeeper residue, Thr528, and it easily accom-
modates a 3,4-dichlorophenyl substituent at the C-4 position as
in compound 5. When a 3-phenol is at C-4, the OH group is mod-
eled on the other side of the phenyl ring where it can form a key

G. K. Packard et al. / Bioorg. Med. Chem. Lett. 22 (2012) 747–752
749
Table 2
C-4 SAR of the thienopyrimidine scaffold
R
N
NH2
O
N
S
NH2
N
H
#
R
B-Raf IC50
(
l
M)
PDE4 IC50
(
l
M)
pMEK IC50
4.74 1.01
(l
M)
Hepat. stab.
Cl
F
Cl
5
0.316 0.059
0.261 0.045
8.8 2.9
F
15
16
17
1.21a
ND
ND
ND
OH
0.014 0.001
0.982a
0.850a
0.212a
13.9a
7.5 0.4
OH
0.954a
ND
OMe
NH2
18
19
20
0.724 0.367
1.12a
0.342a
2.30a
4.34a
11.7a
ND
ND
ND
ND
N
1.54a
ND
Cl
OH
21
0.0020 0.0002
0.469a
0.025 0.005
7.8 3.5
a
Compound was tested once in this assay.
hydrogen bond to the backbone carbonyl of Asp593. The 3-Cl,
5-OH analog 21 combines both the lipophilic interactions and the
phenol hydrogen bond; we postulate that this is the reason for
its increased potency.
Compound 21 possesses significant pMEK biomarker potency
making it useful as a proof-of-concept molecule. An in vivo
biomarker study was performed in SCID mice bearing A375 mela-
noma tumors measuring the phosphorylation of both MEK and
ERK.11 After ip dosing (50 mg/kg) compound 21 demonstrated sig-
nificant knockdown of both pMEK and pERK, 60% and 80%, respec-
tively, at 30-min post dose.
From metabolite identification data, the C-4 phenol of com-
pounds 16 and 21 was identified as the major site of metabo-
lism (glucuronidation). Some of our efforts to stabilize or
replace the phenol are presented in Table 3. Substituting ortho
to the phenol was attempted to either sterically or electroni-
cally block glucuronidation. These analogs (22–24) maintain
good B-Raf enzyme potency, but stability was not improved.
Efforts to replace the phenol, such as benzyl alcohol 25, cyana-
mide 26, and benzimidazole analog 27, were unsuccessful.
Figure 1. Compound 5 docked into the published crystal structure of B-Raf.

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G. K. Packard et al. / Bioorg. Med. Chem. Lett. 22 (2012) 747–752
Table 3
C-4 phenol modifications
R
N
NH2
O
N
S
NH2
N
H
#
R
B-Raf IC50
(
l
M)
PDE4 IC50
1.30a
(l
M)
pMEK IC50
0.271a
(
lM)
Hepat. stab.
8.4 0.2
Cl
OH
OH
22
0.005 0.001
23
0.004a
1.31a
0.202 0.011
11
1
1
OH
F
24
25
0.004 0.001
1.33a
3.77a
ND
0.111 0.077
17
OH
ND
ND
H
N
CN
26
1.60a
ND
ND
ND
N
NH
27
28
15.4a
ND
ND
ND
22
OMe
N
0.017 0.006
1.06a
0.685 0.167
4
a
Compound was tested once in this assay.
Interestingly, the 5-methoxypyridin-3-yl analog 28 proved to be
a potent and moderately stable B-Raf inhibitor.
34 and 35, continued to suffer from rapid clearance.9 The urea-con-
taining C-4 analog 37 had slower clearance and good potency in
both the enzyme and cellular assay. Unfortunately this analog
exhibited poor exposure upon oral administration in rats.12 Our
most promising compound to date was obtained with the C-4
methoxypyridine analog 39 which has excellent hepatocyte stabil-
ity and is cleared much more slowly than the C-4 phenols. In addi-
tion, further analysis of this compound showed it to be moderately
orally bioavailable in rats (17%).12
In summary, a series of thieno[2,3-d]pyrimidines were identi-
fied as B-Raf inhibitors. Our research showed that small amine sub-
stituents at C-2 are essential for potent PDE4 inhibitory activity.
Either removal of the C-2 substituent or use of larger substituents
favored B-Raf activity. C-4 meta-phenol derivatives proved to be
very potent B-Raf inhibitors and were selective against PDE4. Phe-
nol derivative 21 proved to be a useful proof-of-concept compound
showing significant knockdown of the biomarkers pMEK and pERK
in vivo. Further research led to C-4 methoxypyridine and C-4 aryl
urea analogs which proved to be potent and metabolically stable
B-Raf inhibitors. Ultimately a combination of potent and stable
C-2 and C-4 substituents led to analog 39 which proved to be a
moderately bioavailable B-Raf inhibitor. The thieno[2,3-d]pyrimi-
dine template described above is a novel addition to the list of
Further efforts to replace the phenol led us to explore a series of
ureas at C-4 (Table 4). Replacing the phenol with an acetamide 29
and ethyl urea 30 resulted in a loss in potency. The addition of an
aryl ring to the urea moiety 31 afforded a gain in enzyme potency,
but the gain did not translate into acceptable cellular potency.
Substituting the aryl urea with either a para-methyl or para-tri-
fluoromethyl group, 32 and 33, further improved both the enzyme
and cellular potency of this series. The aryl urea analogs were
much more potent in the B-Raf time-dependent assay, possibly
indicating a slower on-rate binding event. This observation is con-
sistent with movement of the activation loop of the B-Raf enzyme
to the DFG-out conformation as described for ‘second-generation
type II’ B-Raf inhibitors.3
Through our exploration of the C-4 position of the thienopyrim-
idine template we identified several different very potent substit-
uents: phenols, methoxypyridine, and aryl ureas. Our exploration
of the C-2 position identified ethanolamine derivatives with good
potency and moderate stability. The ethanolamine substituents
also generally improved the solubility of the template. The SAR
of combinations of some of our best C-2 and C-4 substituents are
presented in Table 5. Compounds bearing the aryl phenol at C-4,

G. K. Packard et al. / Bioorg. Med. Chem. Lett. 22 (2012) 747–752
751
Table 4
C-4 urea SAR
R
N
NH2
O
N
S
NH2
N
H
a
#
R
B-Raf IC50
2.45b
(
lM)
B-Raf T-dep IC50
3.13b
(l
M)
pMEK IC50
ND
(lM)
H
N
29
O
H
N
H
N
30
31
32
4.73b
>30b
ND
O
H
N
H
N
0.032 0.019
0.028 0.001
0.343 0.032
0.038b
0.004b
0.003b
10.1b
O
H
N
H
N
2.03 0.080
O
H
N
H
N
33
0.546 0.184
O
F3C
a
B-Raf time-dependent assay (10Â compound and 10Â enzyme were allowed to pre-incubate for 1 h before initialization of the kinase reaction. Preincubation time for the
normal assay is 15 min).
b
Compound was tested once in this assay.
Table 5
C-2 and C-4 substituents pairing
#
B-Raf IC50
(lM)
pMEK IC50
(lM)
Hepat. stab.
Rat Cla
Rat F (%)
HO
Cl
NH2
34
0.003b
0.103b
23
2
76 14
ND
O
N
N
HO
S
NH2
N
H
HO
N
N
F
NH2
35
0.005 0.001
0.332 0.127
66
1
119
9
ND
O
HO
S
NH2
N
H
H
H
N
N
O
F3C
NH2
36
0.014c
1.54 0.44
111 17
34
4
ND
O
N
HO
S
NH2
N
H
N
(continued on next page)

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G. K. Packard et al. / Bioorg. Med. Chem. Lett. 22 (2012) 747–752
Table 5 (continued)
#
B-Raf IC50
(
lM)
pMEK IC50
(lM)
Hepat. stab.
101 11
Rat Cla
Rat F (%)
H
N
H
N
O
F3C
0.001c
0.327b
12
1
0.3 0.1
NH2
37
O
N
S
NH2
H
N
MeO
N
NH2
38
0.040b
2.51b
ND
ND
ND
O
N
HO
S
NH2
N
H
N
MeO
N
NH2
39
0.058 0.006
1.01 0.08
90
5
32
7
17
5
O
N
HO
S
NH2
N
H
N
a
Rat CLiv (mL/min/kg).9
Compound was tested once in this assay.
Compound was tested once using the B-Raf time-dependent assay.
b
c
9. The total body clearance (CL) was assessed in male CD-IGS rats following a
bolus intravenous (iv) injection via jugular vein catheter at 2 mg/kg in a vehicle
containing 15% DMA/85% PEG 400. The clearance was calculated based on the
known B-Raf inhibitors and may aid in the design of future useful
templates.
following equation: CL = dose/AUC(0–infinity)
, where dose referred to the
intravenous dose of the drug and AUC(0–infinity) was the area under the
plasma drug concentration curve from time zero to time infinity following the
iv dosing.
Acknowledgment
The authors gratefully acknowledge Kirsten Blumeyer for
assistance with data extraction for this Letter.
10. Initial molecular modeling coordinates of B-Raf came from the RCSB published
crystal structure complex 1UWH containing wild-type B-Raf and sorafenib:
Wan, P. T.; Garnett, M. J.; Roe, S. M.; Lee, S.; Niculescu-Duvaz, D.; Good, V. M.;
Jones, C. M. Marshall, C. J.; Springer, C. J.; Barford, D.; Marais, R. Cell 2004, 116,
855. Protein chain A was used for modeling. All molecular modeling was
performed using MOE software: Molecular Operating Environment (MOE),
version 2001.01; Chemical Computing Group Inc., 1010 Sherbooke St. West,
Suite #910, Montreal, QC, Canada H3A 2R7, 2005. Compounds were docked
into the protein coordinates from 1UWH so that the carboxamide functionality
formed hydrogen bonds to the hinge residue CYS 531. The inhibitor and all B-
Raf residues within 6 Å of the inhibitor were allowed to minimize using the
MMFF94Â force field and a distance-dependent dielectric constant, while the
rest of the protein was held fixed.
References and notes
1. Inamdar, G. S.; Madhunapantula, S. V.; Robertson, G. P. Biochem. Pharmacol.
2010, 80, 624.
2. Gollob, J. A.; Wilhelm, S.; Carter, C.; Kelley, S. L. Semin. Oncol. 2006, 382.
3. Li, H.; Chen, Y.; Rao, S.; Chen, X.; Liu, H.; Qin, J.; Tang, W.; Wang, Y.; Zhou, X.; Lu,
T. Curr. Med. Chem. 2010, 17, 1618.
4. Mercer, K. E.; Pritchard, C. A. Biochim. Biophys. Acta 2003, 1653, 25.
5. For experimental details of compound synthesis and in vitro assays please see:
McKenna, J.; Papa, P. W.; Sakata, S. T.; Erdman, P. E.; Packard, G. K. WO 2007/
084560.
11. For a representative procedure see: Pratilas, C. A.; Taylor, B. S.; Ye, Q.; Viale, A.;
Sander, C.; Solit, D. B.; Rosen, N. Proc. Natl. Acad. Sci. 2009, 11, 4519.
6. Robichaud, A.; Tattersall, F. D.; Choudhury, I.; Rodger, I. W. Neuropharmacology
1999, 38, 289.
12. Oral protocol: Test compounds, prepared as
a
suspension in 0.5%
carboxymethyl cellulose + 0.25% Tween80 in water, were dosed via oral
gavage at 10 mg/kg (dose volume of 5 mL/kg) to non-fed male CD-IGS rats
(n = 3 or 4). Blood samples were collected at 0.5, 1, 2, 3, 6, 9, and 26 h post dose
via a jugular vein cannula and subsequently processed into plasma for analysis
by LC–MS/MS. IV protocol: Test compounds, prepared as a solution in 15% DMA
and 85% PEG 400, were dosed via jugular vein cannula as an iv bolus at 2 mg/kg
(dose–volume of 2 mL/kg) to non-fasted male CD-IGS rats (n = 3). Blood
samples were collected at 0.0833, 0.333, 0.75, 1.5, 3, 5, 7, 9, and 26 h post dose
via a jugular vein cannula and subsequently processed into plasma for analysis
by LC–MS/MS. Plasma concentration–time profiles for both oral and
intravenous studies were constructed and the area under the curve (AUC)
was calculated based on the ‘linear trapezoidal–linear interpolation’ method
(using WINNONLIN software). The absolute bioavailability of a drug is defined by
the AUC of a dose delivered by one method (e.g., oral) divided by the AUC of an
iv bolus, corrected by different dose levels.
7. Test compounds and control (7-ethoxycoumarin) were incubated in triplicate
in cryopreserved male sprague–dawley rat hepatocytes (In Vitro
Technologies). Thawed cryopreserved hepatocytes were re-suspended in
Krebs-Henseleit Buffer (KHB), pH 7.4 at a density of 1 Â 106 cells/mL (final
cell density = 233,000 cells/well) and
a final compound concentration of
3 lM. Percentage of parent disappearance was determined after 60 min of
incubation at 37 °C by removing two aliquots (one at time zero and one at
60 min) and quenching the reaction with three volumes of 100% cold
acetonitrile. Samples were mixed on a multi-tube vortexer for one minute
and a volume of the mixture was filtered through a Captiva (Varian) 0.45
lm
pore size polypropylene filter plate. Sample extracts were analyzed by LC–
MS/MS to determine parent compound levels, reported as
(mean SD).
% remaining
8. Rat CLiv = 116 mL/min/kg.