Discovery of thienopyrimidine-based inhibitors of the human farnesyl pyrophosphate synthase - Parallel synthesis of analogs via a trimethylsilyl ylidene intermediate

Article abstract of DOI:10.1016/j.bmc.2013.02.006

Thienopyrimidine-based bisphosphonates were identified as a new class of nitrogen-containing bisphosphonate (N-BP) inhibitors of the human farnesyl pyrophosphate synthase (hFPPS). Analogs were prepared via cyclization of 2-(1-(trimethylsilyl)ethylidene)malononitrile to 2-amino-4-(trimethylsilyl) thiophene-3-carbonitrile in the presence of elemental sulfur. Direct ipso-iododesilylation of this intermediate led to selective iodination at C _β of the sulfur atom in high efficiency. The synthetic protocols developed were used in the parallel synthesis of structurally diverse thieno[2,3-d]pyrimidin-4-amine-based bisphosphonate inhibitors of hFPPS.

Full text of DOI:10.1016/j.bmc.2013.02.006

Bioorganic & Medicinal Chemistry xxx (2013) xxx–xxx
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery of thienopyrimidine-based inhibitors of the human
farnesyl pyrophosphate synthase—Parallel synthesis of analogs
via a trimethylsilyl ylidene intermediate
Chun-Yuen Leung a, Adrienne M. Langille a, John Mancuso a,c, Youla S. Tsantrizos a,b,
⇑
a Department of Chemistry, McGill University, 801 Sherbrooke Street West, Montreal, QC, Canada H3A 0B8
b Department of Biochemistry, McGill University, 3649 Promenade Sir William Osler, Montreal, QC, Canada H3G 0B1
c Tranzyme Pharma, 3001 12th Avenue North, Sherbrooke, QC, Canada J1H 5N4
a r t i c l e i n f o
a b s t r a c t
Article history:
Thienopyrimidine-based bisphosphonates were identified as
a
new class of nitrogen-containing
Received 4 January 2013
Revised 1 February 2013
Accepted 11 February 2013
Available online xxxx
bisphosphonate (N-BP) inhibitors of the human farnesyl pyrophosphate synthase (hFPPS). Analogs were
prepared via cyclization of 2-(1-(trimethylsilyl)ethylidene)malononitrile to 2-amino-4-(trimethylsilyl)
thiophene-3-carbonitrile in the presence of elemental sulfur. Direct ipso-iododesilylation of this interme-
diate led to selective iodination at Cb of the sulfur atom in high efficiency. The synthetic protocols devel-
oped were used in the parallel synthesis of structurally diverse thieno[2,3-d]pyrimidin-4-amine-based
bisphosphonate inhibitors of hFPPS.
Keywords:
Thienopyrimidine bisphosphonates
Modified Gewald synthesis
2-(1-(Trimethylsilyl)ethylidene)
malononitrile
Ó 2013 Elsevier Ltd. All rights reserved.
Farnesyl pyrophosphate synthase
1. Introduction
potential inhibitors of the human farnesyl pyrophosphate synthase
(hFPPS) and/or geranylgeranyl pyrophosphate synthase (hGGPPS);
The thienopyrimidine core is an integral part of numerous
biologically active compounds, as evidenced by the plethora of
literature and industrial patents disclosing the use of this heterocy-
cle in agrochemicals1–3 and human therapeutics (e.g., antifungal4
and antiviral5 agents). Thienopyrimidines are often used as
bioisosteres of purine nucleobases (e.g., adenine),5 and have been
employed extensively in the design of various kinase inhibitors,
including Aurora kinase,6 tyrosine kinases,7,8 cyclin-dependent
kinase,9 c-Jun N-terminal kinases,10–12 PERK,13 phosphoinositide
these prenyl synthase enzymes are functionally closely related.
The development of synthetic methodologies amenable to
parallel synthesis of structurally diverse libraries of thienopyrimi-
O
PO(OH)2
NH2
N
HN
N
R5
S
PO(OH)2
3-kinase
a (PI3Ka
),14,15 and EGFR/ErbB-2.16 Many of these inhibi-
R5
S
tors are currently under pre-clinical or clinical investigations for
the treatment of cancer, inflammatory, autoimmune and neurode-
generative diseases. Thienopyrimidine-based compounds have
N
R6
R6
PO(OH)2
PO(OH)2
N
N
3
N
H
1
2
also been reported as inhibitors of
c
-secretase17 and antagonists
of the adenosine A2A receptor,18–20 and implicated in the potential
treatment or prevention of Alzheimer’s disease.
In our own investigations, we focus on the identification of struc-
turally diverse molecules that selectively inhibit mammalian prenyl
synthase enzymes. Inspired by the privileged biopharmaceutical
properties of the thienopyrimidine scaffold (1; Fig. 1), we decided
to explore thienopyrimidine-based bisphosphonates (2; Fig. 1), as
(HO)2OP
PO(OH)2
4
Figure 1. Structures of thieno[2,3-d]pyrimid-4-amines (general structure 1), thie-
nopyrimidine-based bisphosphonate inhibitors of hFPPS/hGGPPS (general structure
2), hFPPS inhibitor 3 and hGGPPS inhibitor 4.
⇑
Corresponding author. Tel.: +1 514 398 3638; fax: +1 514 398 3797.
E-mail address: youla.tsantrizos@mcgill.ca (Y.S. Tsantrizos).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.02.006
Please cite this article in press as: Leung, C.-Y.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.02.006

2
C.-Y. Leung et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
R4
CN
NC
CN
NC
R4
CN
O
S8, Base
R4
NH2
7a, R4 = (CH2)2CH3, R5 = H
7b
R5
S
5a
, R4 = (CH2)2CH3
5c, R4 = p-NO2-C6H4
6
, R4 = CH3, R5 = CH2CH3
7c, R4 = p-NO2-C6H4, R5 = H
O
N
HO
S
NC
CN
NH
7d, R4 = R5 = H
HCO2H,
conc. H2SO4
uW 100 oC
S
S
8
OH
9
1) Br2, AcONa
reflux
2) Zn, AcOH
H2O
R4
Br
O
Cl
I
Br
1) i-PrMgBr, 0 o
C
POCl3
N
N
NH
uW, 100 o
C
N
S
S
S
2) I2
N
N
12
11a
11b
, R4 = Cl
10
NH4OH
90 o
C
, R4 = NH2
Scheme 1. Gewald synthesis of 2-aminothiophene-3-carbonitriles (7) and halo-substituted thienopyrimidines.
dine bisphosphonates (2) were required to support our medicinal
chemistry efforts. We anticipated that the final compounds could
be made from highly substituted thieno[2,3-d]pyrimidin-4-amine
intermediates (1), after coupling of the exocyclic amine (at C-4)
with the bisphosphonate moiety; the latter step could be easily
achieved using previously developed procedures for the prepara-
tion of 2-aminopyridine bisphosphonates (e.g., hFPPS inhibitor 3;
Fig. 1).21,22
Traditionally, thieno[2,3-d]pyrimid-4-amines (1) have been
synthesized via a multistep process from 2-aminothiophenes (7;
Scheme 1). However, preparation of highly substituted thienopyr-
imidine libraries is usually hampered by limitations in selectively
ing bicyclic heterocycle, is unknown. Recent reports on the prepa-
ration of 3-bromothiophenes usually describe the generation of the
a-lithium-b-bromothiophene from the 2-bromothiophene using
the (so-called) base-catalyzed halogen dance (BCHD) reaction.
28–32
This approach has also been used in the synthesis of
4-chloro-5-iodothieno[2,3-d]pyrimidine (12) from the LDA-medi-
ated BCHD rearrangement of the 6-bromo-4-chlorothieno
[2,3-d]pyrimidine first to the 5-bromo analog 11a, followed by
treatment with a Grignard reagent and quenching of the anion
with iodine (Scheme 1).1,3 Several alternative approaches have
been reported, most commonly involving 2,3-dibromination of 9,
followed by reductive dehalogenation at the more reactive C po-
a
functionalizing the C and/or Cb positions of the thiophene core
sition of the thiophene with Zn in acetic acid to give intermediate
10 (Scheme 1).10,11 Two additional synthetic steps are required to
convert 10–11b, which can then be used to prepare C-5 mono-
substituted thienopyrimidines. Herein, we report an alternative
a
(7). Similarly, direct and selective substitution at those positions
in the corresponding thieno[2,3-d]pyrimidine is very challenging
(i.e., the C-5 and C-6 of 1).
The 2-aminothiophene core (7) can be easily prepared by the
classical Gewald method (Scheme 1).23 One-step and two-step
procedures, under either thermal or microwave conditions, have
been reported with various optimizations for the condensation of
the Knoevenagel ylidene 6 with elemental sulfur to give the thio-
phene 7. The drawbacks to this elegant protocol can be regioselec-
tivity in the cyclization step, when non-symmetrical ketones (5)
are used with an alkyl or a benzylic substituent, and the chemical
stability of ylidene 6. For example, the one-pot condensation of
pentane-2-one (5a) with malononitrile, sulfur and catalytic
amounts of imidazole, was recently reported to produce a mixture
of thiophenes 7a and 7b in 1:3.4 ratio and in modest yield (Scheme
1).24 Furthermore, acetophenones bearing electron withdrawing
substituents on the phenyl ring (e.g., 5c) often produce very low
yields of the desired 2-aminothiophene (7c) under standard Ge-
wald reaction conditions.25 The low yields have been attributed,
in part, to decomposition and dimerization of ylidene 6, under high
temperatures and basic conditions.25
protocol that allows selective disubstitution at either C or Cb of
a
the thiophene core and is amenable to modular parallel synthesis
of structurally diverse libraries of thieno[2,3-d]pyrimidin-4-
amines (1). We also provide preliminary biological data which
indicates that the thienopyrimidine-based bisphosphonates de-
scribed herein are inhibitors of the human FPPS and new leads
for drug discovery. Up-regulation of hFPPS has been linked to
numerous human disorders,33,34 including cancer35 and neurode-
generative diseases, thus inhibitors of this enzyme have the poten-
tial of becoming valuable human therapeutics.
2. Results
2.1. Chemistry
As part of our continued interest in developing efficient, robust
methodologies that are amenable to parallel synthesis of structur-
ally diverse libraries of biologically active compounds,21,22 we
modified the classical Knoevenagel/Gewald approach by incorpo-
rating the trimethylsilyl ylidine 14 as the precursor to the 2-ami-
no-thiophene-3-carbonitrile scaffold (15; Scheme 2). The ylidine
2-(1-(trimethylsilyl)ethylidene)malononitrile (14) was prepared
in nearly quantitative yield via condensation of acetyltrimethylsi-
lane (13) with malononitrile under acidic conditions (Scheme 2).
Ylidine 14 was found to be stable at ꢀ20 °C for several months
Alternatively, the unsubstituted 2-amino-thiophene-3-carboni-
trile core (7d) has also been prepared from 2,5-dihydroxy-1,4-
dithiane (8) and subsequently elaborated to substituted thie-
no[2,3-d]pyrimidines.26,27 Chlorination or bromination at C of
a
the thiophene sulfur can be easily achieved with NCS or NBS,
respectively. However, direct and regiospecific halogenation at
the b-carbon, or the equivalent carbon on any thiophene-contain-
Please cite this article in press as: Leung, C.-Y.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.02.006

C.-Y. Leung et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
3
Table 1
NC
CN
O
a
Mini-library of thieno[2,3-d]pyrimidin-4-amines (1) synthesized
TMS
TMS
R5
NH2
N
14
13
R6
b
S
N
R2
TMS
CN
R
S
15
TMS
Br
CN
1a, R2 = a, R5 = R6 = H
1b
1i, R2 = R6 = H, R5 = e
1j
1k, R2 = H, R5 = g, R6 = a
1l, R2 = H, R5 = h, R6 = a
, R=NH2
16, R = NCHNMe2
f
e
c
a
f
, R2 = R6 = H, R5 =
, R2 = R6 = H, R5
=
N
N(CH3)2
N(CH3)2
N(CH3)2
1c, R2 = R5 = H, R6 = Br
1d, R2 = R5 = H, R6 = c
S
17
NH2
N
R5
S
1e
, R2 = R5 = H, R6
e
1m
j
a
=
=
, R2 = H, R5
1n, R2 = H, R5 = k, R6 = a
1o a
=
=
, R6
g
1f, R2 = R5 = H, R6 = f
1g
i
b
, R2 = R5 = H, R6
=
, R2 = H, R5
=
, R6
TMS
R5
CN
N
h, c
N
1h, R2 = R6 = H, R5 = d
20a
, R5 = TMS
S
a
k
R2, R5 and/or R6 = H or a fragment from to
d
18
, R5
a k
-
=
20b
, R5 = Iodine
CH3
NO2
CF3
i, j
N
R4
R5
CN
N
N
S
NH2
N
R5
a
b
c
d
e
f
g
19a
a k
-
, R4 = Iodine, R5
=
g
R6
19b, R4 = a-k, R5 = a-k
S
N
c
O
1
S
N
N
k, l
PO(OH)2
g
h
i
j
k
HN
R5
S
PO(OH)2
N
R6
N
an ipso-iododesilylation reaction with silver trifluoroacetate and
elemental iodine; however, this reaction resulted in mostly unre-
acted starting material. Alternatively, excellent yields (>98%) of
20b were obtained when 20a was treated with ICl in DCM at
ꢀ10 °C and the reaction was quenched at low temperatures (below
0 °C) with H2O.38,39 We noted that if the reaction mixture was
allowed to warm-up above 0 °C, the isolated yield was significantly
lower and mixtures of di-halogenated side products were observed
by LC–MS. The iodo product 20b was found to be a useful building
block for various cross-coupling reactions in the synthesis of C-5
mono-substituted thieno[2,3-d]pyrimidin-4-amines (e.g., 1b, 1h,
1i, 1j, Table 1). It is noteworthy that fragment 20b was obtained
from 13 in four steps, with an average of 50–60% overall isolated
yield, using our protocol (Scheme 2). This methodology is signifi-
cantly more efficient in preparing analogs of 1 that are selectively
mono-substituted at C-5 as compared to those reported previously
(e.g., examples shown in Scheme 1).
2
Scheme 2. Modular synthesis of highly substituted thieno[2,3-d]pyrimidin-4-
amines; the stuctures of substituent a–k are shown in Table 1. Reagents and
conditions: (a) CH2(CN)2, NH4OAc, AcOH, C6H6, Dean–Stark trap, 95 °C, 24 h (90%);
(b) S8, Et2NH, pyridine, rt, 18 h (85%); (c) HCONH2, 130 °C, 48 h (75–85%); (d) ICl,
DCM, ꢀ10 °C (>98%); (e) (CH3O)2CHN(CH3)2, DMF, rt, 4 h (90%); (f) NBS, DMF, rt,
13 h in the dark (80%); (g) various cross-coupling reactions under standard Suzuki,
Buchwald–Hartwig, Sonogashira and Stille conditions, isolated yields varied from
50% to 95%, reactions were not individually optimized (for the conversion of 20b or
18 to 1, R6 = H or R5 = H, respectively); (h) TBAF, THF, 0 °C to rt, 3 h (>95%); (i)
CF3CO2Ag, THF, ꢀ78 °C, 15 min; (j) I2, THF, ꢀ78 °C, 3 h (>98%); (k) CHOEt3,
diethylphosphite, 130 °C, 24 h (50–75%); (l) TMSBr, MeOH, rt, 72 h (>85%).
and at RT for several weeks without any evidence of decomposi-
tion. Condensation of 14 with elemental sulfur in pyridine
provided the 2-amino-4-(trimethylsilyl)-thiophene-3-carbonitrile
(15) as a dark orange/brown solid in >85% yield.
In order to explore direct cross-coupling reactions selectively at
the Cb of the sulfur in the thiophene core (i.e., where intermediate
1 is mono-substituted at C-5), we initially focused on preparing
the iodothiophene core, as a precursor to the 5-iodothienopyrimi-
dine 20b (Scheme 2). Efforts to iodinate intermediate 15 at Cb,
using silver trifluoroacetate and elemental iodine,36,37 lead to
decomposition of the starting material at temperatures ranging
from 0 to ꢀ78 °C. Previous examples of ipso-iododesilylation of
thiophenes under such conditions were reported to give poor
yields when the TMS was adjacent to substituents with sp charac-
ter.37 Cyclization of 15 with formamide produced the 5-(trimethyl-
silyl)thieno[2,3-d]pyrimidin-4-amine (20a) in 75–80% yield
(Scheme 2; step c). Mindful of the facile decomposition of
thiophenes upon exposure to light, this reaction was usually
carried out in the dark. Subsequently, we reattempted to perform
In the course of these studies, we noted that thiophene 15 was
somewhat chemically unstable. Interestingly, protection of the
2-amino moiety with N,N-dimethylformamide-dimethylacetal,
provided intermediate 16, which is chemically stable and easy to
prepare in high yield and purity (Scheme 2). Bromination of the
protected thiophene 16 was also carried-out in >80% yield, provid-
ing intermediate 17 (Scheme 2). Compound 17 is a suitable build-
ing block for a variety of cross-coupling reactions. We explored
selective cross-coupling with various boronic acids or boronate
esters to obtain intermediates of general structure 18, which upon
ipso-iododesilylation at C-4 (i.e., leading to 19a), followed by
Suzuki, Buchwald–Hartwig, Sonogashira, Stille or other cross-cou-
pling reactions, gave the disubstituted thiophenes 19b.
Finally, cyclization of 19b provided a library of novel and
structurally diverse C-5/C-6 di-substituted thieno[2,3-d]pyrimi-
Please cite this article in press as: Leung, C.-Y.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.02.006

4
C.-Y. Leung et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
Figure 2. Inhibition data for human FPPS and GGPPS of select thienopyrimide-based bisphosphonates 2a–2f (R2, R5 and R6 substituents are selected from those shown in
Table 1). (a) Superposition of analog 2e with the bioactive conformation of inhibitor 3 (PDB: 4DEM). The carbon skeleton of 2e and 3 are highlighted in light and dark green
colour, respectively; nitrogen, oxygen, sulphur and phosphorus atoms are indicated in blue, red, yellow and orange colour, respectively. (b) Structures of key thienopyrimide-
based bisphosphonate compounds. (c) Inhibition data for hFPPS and hGGPPS at 10
lM of inhibitor; the hFPPS and hGGPPS inhibitors 3 and 4, respectively (Fig. 1) were used as
+ve/ꢀve controls (% inhibition at 10 M concentration of inhibitor; average values of three determinations; standard deviation 10%).
l
din-4-amines (1k–o, Table 1) in good overall yield. For example,
preparation of analogs 1l from 15 was achieved in seven steps
(including cross coupling under typical Suzuki and Sonogashira
conditions at C-5 and C-4, respectively) in an overall isolated yield
of 50%. Similarly, analog 1m (involving Buchwald–Hartwig amina-
tion in the conversion of 19a–19b) was prepared from 15 in 30%
isolated overall yield. It should be noted that these reactions were
not individually optimized during preparation of our library. Ana-
log 1a (Table 1) was prepared by a different synthetic scheme,
involving direct condensation of 2-aminothiophene-3-carbonitrile
with benzonitrile under basic condition and at high temperature,
as previously reported.19
To gain some insight as to the potential for developing favour-
able structure–activity-relationship (SAR) for thienopyrimidine-
based inhibitors of hFPPS and/or hGGPPS, a small cluster of analogs
from this library were converted to the corresponding bisphospho-
nates 2 (Scheme 2). Treatment of fragments 1 with triethyl ortho-
formate and diethylphosphite, followed by hydrolysis of the
tetraethyl bisphosphonate esters with TMSBr/MeOH resulted in
the formation of bisphosphonic acids 2 (Scheme 2); details of this
two-step protocol were previously described in the synthesis of the
hFPPS inhibitor 3.21
a single concentration of 10
l
M (as previously reported),21 along
M in hGGPPS)
M in
with inhibitors 3 (IC50 = 28 nM in hFPPS; IC50 >100
l
and 4 (IC50 = 410 nM in our own hGGPPS assay;21 IC50 >10
l
hFPPS40) as the positive controls. The inhibition data observed
are summarized in Figure 2c (% inhibition of hFPPS and hGGPPS
at 10 lM of compound; average values of three determinations;
standard deviation of approximately 10%). Significant inhibition
of hFPPS was observed with the parent molecule 2a, as well as
the C-2 and C-6 phenyl analogs 2b and 2d, respectively (75–80%
inhibition at 10
lM). However, analog 2c exhibited <20% inhibi-
tion at 10 M (Fig. 2b and c). These preliminary data suggest that
l
C-2 and C-6 derivatives of the thienopyrimidine core may be ben-
eficial in developing inhibitors of hFPPS, whereas favourable sub-
stitution at C-5 may be limited. In order to gain further insight
as to the volume of space within the hFPPS binding pocket occu-
pied by the substituents at C-6, the tolyl and naphthyl moieties
(2e and 2f, respectively) were also investigated. These modifica-
tions resulted in modest improvement in inhibiting hFPPS
(Fig. 2c). The full dose response for hFPPS inhibition was subse-
quently determined for analogs 2e and 2f and their IC50 values
were calculated to be 390 and 250 nM, respectively. Interestingly,
this modest SAR optimization (i.e., compounds 2e and 2f) also
improved the selectivity in inhibiting hFPPS as compared to
hGGPPS (Fig. 2c).
2.2. Biological results and discussion
Superposition of the hFPPS-bound conformation of inhibitor 3
(IC50 = 28 nM; PDB: 4DEM),21 with the thienopyrimidine analog
2e strongly suggests that these compounds cannot adopt the same
Initially, the bisphosphonic acid analogs 2a–f (Fig. 2b) were
evaluated in our routine hFPPS and hGGPPS inhibition assays at
Please cite this article in press as: Leung, C.-Y.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.02.006

C.-Y. Leung et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
5
binding mode in the active site of hFPPS (Fig. 2a). Therefore, the
thienopyrimidine-based bisphosphonates 2e and 2f represent
novel leads for drug discovery into hFPPS inhibitors. To fully ex-
plore the SAR potential of these compounds, synthesis of a larger
and structurally diverse library of bisphosphonate analogs is cur-
rently in progress. Concurrently, we are pursuing a number of
structural investigations in order to determine the mechanism by
which these compounds inhibit hFPPS.
Method (homogeneity analysis using a Waters Atlantis T3 C18
5
lm column):
Solvent A: H2O, 0.1% formic acid.
Solvent B: CH3CN, 0.1% formic acid.
Mobile phase: linear gradient from 95% A and 5% B to 5% A and
95% B in 13 min, then 2 min at 100% B.
Flow rate: 1 mL/min.
3. Conclusion
4.2. Synthesis of key fragments
Currently, nitrogen-containing bisphosphonates (N-BPs) are the
only clinically validated drugs that target the human FPPS, thus
blocking prenylation.33,34 Although these drugs are mainly used
for the treatment of bone-related diseases, recent clinical data pro-
vides evidence that N-BPs are also disease modifying agents that
improve the survival of cancer patients (particularly of patients
with multiple myeloma35) via mechanisms unrelated to their ske-
letal effects. In an effort to identify compounds with improved bio-
pharmaceutical properties (as compared to those of the current
drugs), we designed a new class of bisphosphonates based on the
thienopyrimidine scaffold 1. Thienopyrimidines are usually pre-
pared via the Gewald method. However, this approach is not easily
amenable to parallel synthesis of highly substituted and structu-
rally diverse analogs. In addition, structural diversity is typically
dictated by the starting ketones 5 (Scheme 1); thus the synthesis
is linear and does not allow preparation of permutation libraries.
Furthermore, selective mono-substitution at the Cb of the thio-
phene sulfur is challenging due to the inherent higher reactivity
4.2.1. 2-(1-(Trimethylsilyl)ethyl)malononitrile (14)
Acetyltrimethylsilane (1.460 g, 12.56 mmol), malononitrile
(1.140 g, 12.56 mmol) and ammonium acetate (262.1 mg,
2.386 mmol) were dissolved in acetic acid (0.58 mL, 10.05 mmol)
and benzene (30 mL) in a 100 mL round bottom flask attached to
a Dean–Stark trap and filled with benzene. The reaction mixture
was stirred and heated to 95 °C for 24 h. The resulting orange solu-
tion was cooled and diluted with ethyl acetate (20 mL). The organic
layer was washed with saturated sodium bicarbonate solution
(15 mL), water (45 mL), brine (15 mL) and dried over MgSO4. The
product was purified by column chromatography (25% ethyl acet-
ate/hexanes) to give the desired product as clear pale yellow oil in
90% yield (1.973 g).
1H NMR (400 MHz, CDCl3) d 2.34 (s, 3H), 0.36 (s, 9H).
13C NMR (126 MHz, CDCl3) d 188.3, 113.2, 111.5, 94.4, 24.3,
ꢀ2.2.
HRMS (ESIꢀ) Calcd for C8H11N2Si m/z [MꢀH]ꢀ: 163.06970,
found m/z 163.06875 and HRMS (ESIꢀ) Calcd 327.14667 for
of the C carbon. We modified the classical Gewald methodology
a
by employing 2-(1-(trimethylsilyl)-ethylidene)-malononitrile (14)
as a novel synthon in the preparation of thieno[2,3-d]pyrimid-4-
amine libraries (1; Scheme 2). Preparation of 5-iodothieno[2,3-
d]pyrimidin-4-amine (20b) was achieved in high yields and signif-
icantly fewer steps as compared to previous reports. This metho-
C
16H23N4Si2, found m/z 327.14695 [2MꢀH]ꢀ.
4.2.2. 2-Amino-4-(trimethylsilyl)thiophene-3-carbonitrile (15)
2-(1-(Trimethylsilyl)ethyl)malononitrile (1.21 g, 7.365 mmol)
and sulfur (248.02 mg, 7.734 mmol) were dissolved in pyridine
(25 mL) at room temperature. To this, diethylamine (0.762 mL,
7.365 mmol) was added dropwise. The reaction mixture stirred
at room temperature for 18 h. Evaporation of pyridine afforded
the crude thiophene, which was dissolved in ethyl acetate
(20 mL) and washed with water (45 mL), brine (15 mL), and dried
over MgSO4. Purification by column chromatography (25% ethyl
acetate/hexanes, Rf = 0.58) afforded the desired product as an or-
ange oil in 85% yield (803.5 mg).
dology was then used to prepare
a
small cluster of
thienopyrimidine-based bisphosphonate compounds (2) and probe
the ability to such N-BPs to inhibit the hFPPS. In depth SAR studies
and structural investigations are currently in progress in order to
evaluate the potential of these compounds for further optimization
into potent and selective inhibitors of hFPPS.
4. Experimental
4.1. General procedures for characterization of compounds
1H NMR (300 MHz, CDCl3) d 6.38 (s, 1H), 4.74 (br s, 2H), 0.30 (s,
All intermediate compounds were purified by normal phase
flash column chromatography on silica gel using a CombiFlash in-
strument and the solvent gradient indicated. Key thienopyrimidine
building blocks, bisphosphonate esters and all final bisphosphonic
acid inhibitors were analyzed by reverse-phase HPLC and fully
characterized by 1H, 13C and 31P NMR, and MS (HR-MS for key frag-
ments and final inhibitors). Chemical shifts (d) are reported in ppm
relative to the internal deuterated solvent (1H, 13C) or external
H3PO4 (d 0.00 31P), unless indicated otherwise. High-resolution
MS spectra were recorded using electrospray ionization (ESI+/ꢀ)
and Fourier transform ion cyclotron resonance mass analyzer
(FTMS). Melting points (when relevant) were determined using a
conventional capillary melting point analyzer. The homogeneity
of the bisphosphonate tetra esters and the final bisphosphonic acid
inhibitors was confirmed by HPLC to >90% (using the conditions in-
dicated below); IC50 values were determined only for samples with
>95% homogeneity. HPLC analysis was performed using a Waters
ALLIANCEÒ instrument (e2695 with 2489 UV detector and 3100
mass spectrometer).
9H).
13C NMR (126 MHz, CDCl3) d 164.3, 141.1, 116.8, 116.7, 92.3,
ꢀ1.3.
HRMS (ESI+) Calcd 197.05632 for C8H13N2SSi, found m/z
197.05615 [M+H]+.
4.2.3. 5-(Trimethylsilyl)thieno[2,3-d]pyrimidin-4-amine (20a)
Fragment 20a was obtained in two different ways: (a) 2-Amino-
4-(trimethylsilyl)thiophene-3-carbonitrile
2.04 mmol, 1 equiv) was added to formamide (8.1 mL, 200 mmol,
100 equiv) in a pressure vessel. The reaction mixture was sealed
and stirred at 145 °C in the dark for 16 h. (b) 3-Cyano-4-(trimethyl-
(15a,
400 mg,
silyl)thiophen-2-yl)-N,N-dimethylformimidamide
(79.3 mg,
0.315 mmol) was reacted with formamide (anhydrous, 2.5 mL,
63.08 mmol) in a dry 15 mL pressure vessel. The vessel was flushed
with argon and the mixture stirred at 130 °C for 45 h. The dark red
solution was diluted with ethyl acetate, washed with water
(25 mL), brine (10 mL), and dried over Na2SO4. The crude mixture
Please cite this article in press as: Leung, C.-Y.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.02.006

6
C.-Y. Leung et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
was purified by flash column chromatography (5–30% EtOAc/hex-
anes, dry loading) to afford the desired product as a pink solid in
80% yield (59.0 mg).
1H NMR (500 MHz, CDCl3) d 7.63 (s, 1H), 3.09 (s, 6H), 0.44 (s,
9H).
13C NMR (126 MHz, CDCl3) d 168.4, 154.6, 138.9, 116.7, 106.2,
101.9, 40.8, 35.3, 0.2.
1H NMR (400 MHz, CDCl3) d 8.47 (s, 1H), 7.43 (s, 1H), 5.36 (br s,
2H), 0.46 (s, 9H).
HRMS (ESI+) Calcd 330.00903 for C11H17N3BrSSi, found m/z
330.00926 [M+H]+.
13C NMR (126 MHz, CDCl3) d 170.9, 158.8, 153.6, 133.2, 131.3,
119.8, 0.4.
HRMS (ESI+) Calcd 224.06722 for C8H13N2SSi, found m/z
4.2.7. 5-Bromo-3-cyanothiophen-2-yl-N,N-
dimethylformimidamide
224.06705 [M+H]+.
A 1 M solution of TBAF (5.13 mL) in THF was added dropwise to
a
solution of 5-bromo-3-cyano-4-(trimethylsilyl)thiophen-2-yl-
4.2.4. 5-Iodothieno[2,3-d]pyrimidin-4-amine (20b)
N,N-dimethylformimidamide (17, 1.61 g, 4.89 mmol) in THF
(100 mL) cooled to 0 °C. The reaction mixture was warmed to room
temperature and stirred in the dark for 3 h. The volume of THF was
reduced in vacuo and EtOAc was added, washed with water
(3 ꢁ 50 mL), brine (25 mL), and dried over Na2SO4. The desired
crude product was obtained as a red oil in 95% yield (1.20 g) and
used as such for the synthesis of analogs 1, where R5 = H without
any further purification.
A solution of 5-(trimethylsilyl)thieno[2,3-d]pyrimidin-4-amine
(220.4 mg, 0.987 mmol, 1 equiv) in dichloromethane (2 mL) was
stirred at ꢀ10 °C ice slurry for 5 min. 1 M Iodine monochloride in
dichloromethane (2.96 mL, 2.96 mmol, 3 equiv) was added to the
reaction mixture drop-wise and the reaction mixture was stirred
at ꢀ10 °C for 30 min. Ice cooled water (30 mL) was directly added
to the reaction mixture to quench the reaction. Dichloromethane
(2 ꢁ 20 mL) at room temperature was added to the mixture and
the entire mixture was filtered through a Whatman #5 2.5
lm fil-
1H NMR (400 MHz, CDCl3) d 7.64 (s, 1H), 6.86 (s, 1H), 3.11 (s,
ter paper. The yellowish solid was washed with dichloromethane
(2 ꢁ 10 mL) and recrystallized with methanol to give the desired
product 5-iodothieno[2,3-d]pyrimidin-4-amine as a yellow colored
solid (271.9 mg, >98% yield).
6H).
13C NMR (300 MHz, CDCl3) d 167.0, 154.2, 128.3, 115.1, 99.4,
96.5, 40.8, 35.2.
MS (ESI+) m/z: 258.06 [M+H]+.
1H NMR (300 MHz, CD3CN): d 8.33 (1H, s), 7.79 (1H, s), 7.44 (br
4.2.8. N0-(3-Cyano-4-iodo-5-phenylthiophen-2-yl)-N,N-
dimethylformimidamide (19a, R5 = a)
s, 2H).
13C NMR (126 MHz, acetone-d6) d 159.5, 154.7, 128.6, 116.4,
106.1, 70.5.
Silver trifluoroacetate (93.2 mg, 0.42 mmol) was added to a so-
lution of N0-(3-cyano-5-phenyl-4-(trimethylsilyl)thiophen-2-yl)-
N,N-dimethylformimidamide (69.1 mg, 0.21 mmol) in THF
(20 mL) cooled to ꢀ78 °C and stirred under argon for 15 min. Io-
dine (214.2 mg, 0.84 mmol) dissolved in THF (10 mL) was added
dropwise to the cold mixture and stirred in the dark at ꢀ78 °C
for 4 h. Ethyl acetate was added and the mixture was filtered
through Celite. The filtrate was washed with 2 M sodium thiosul-
fate, brine, and dried over Na2SO4. The crude mixture was purified
by flash column chromatography (5–30% EtOAc/hexanes, solid
loading) to afford the desired product as an orange solid in >98%
yield (80.0 mg).
HRMS (ESI+) Calcd 277.92434 for C6H4IN3S, found m/z
277.92353 [M+H]+.
Melting point: 196 °C (dec).
4.2.5. 3-Cyano-4-(trimethylsilyl)thiophen-2-yl)-N,N-
dimethylformimidamide (16)
To a solution of 2-amino-4-(trimethylsilyl)thiophene-3-carbo-
nitrile (15, 372.40 mg, 1.90 mmol) in DMF (20 mL) was added
DMF–DMA (2.5 mL, 18.97 mmol). After stirring at room tempera-
ture for 4 h, the reaction mixture was diluted with ethyl acetate,
washed with water (60 mL), brine (20 mL), and dried over MgSO4.
Solvent was removed in vacuo to afford the desired product as a
brown-yellow solid in 90% yield (440.1 mg).
1H NMR (400 MHz, CDCl3) d 7.77 (s, 1H), 7.58–7.51 (m, 2H), 7.40
(m, 3H), 3.13 (s, 6H).
13C NMR (75 MHz, CDCl3) d 167.2, 154.7, 134.1, 130.7, 129.5,
128.7, 128.6, 116.8, 105.7, 78.1, 40.9, 35.3.
1H NMR (500 MHz, CDCl3) d 7.72 (s, 1H), 6.63 (s, 1H), 3.10 (s,
3H), 3.09 (s, 3H), 0.32 (s, 9H).
HRMS (ESI+) Calcd 381.98694 for
C14H13N3IS, found m/z
13C NMR (126 MHz, CDCl3) d 168.9, 154.8, 141.6, 120.7, 117.5,
101.2, 40.7, 35.2, ꢀ1.3.
381.98667 [M+H]+.
29Si NMR (99 MHz, CDCl3) d ꢀ6.69.
HRMS (ESI+) Calcd 252.09852 for C11H18N3SSi, found m/z
252.09781 [M+H]+.
4.3. Synthesis of thieno[2,3-d]pyrimidin-4-amines—general
protocols
4.3.1. Suzuki coupling reactions using fragment 20b
4.2.6. 5-Bromo-3-cyano-4-(trimethylsilyl)thiophen-2-yl-N,N-
dimethylformimidamide (17)
Fragment 20b is not very soluble in non-polar solvents; conse-
quently all reactions were carried out in MeOH. To an argon
flushed microwave reactor vial, fragment 20b, the boronic acid or
boronate ester (1.4 equiv) and Pd(PPh3)4 (0.1 equiv) and KF
(2.5 equiv) were added and flushed again with argon. Argon
flushed methanol was added to the reaction mixture and the reac-
tion was stirred at 120 °C for 20 min (120 W). The crude mixture
was filtered through celite and concentrated to dryness under va-
cuum. The crude product was purified by normal phase flash col-
umn chromatography on silica gel (silica gel was pre-washed
N-Bromosuccinimide (121.2 mg, 0.68 mmol) was added to a so-
lution of 3-cyano-4-(trimethylsilyl)thiophen-2-yl-N,N-dimethyl-
formimidamide (163.0 mg, 0.648 mmol) in DMF (7 mL). The
yellow solution was stirred in the absence of light at room tem-
perature for 13 h. The mixture was diluted with ethyl acetate,
washed with water (25 mL), brine (10 mL), and dried over MgSO4.
Solvent was removed in vacuo to afford the desired product as a
brown-orange solid in 80% yield (177.1 mg).
Please cite this article in press as: Leung, C.-Y.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.02.006

C.-Y. Leung et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
7
with hexanes) using a CombiFlash instrument and a solvent gradi-
ent from 100% hexanes to 100% EtOAc.
from 2% EtOAc in hexanes to 100% EtOAc (unless otherwise indi-
cated) to afford the desired product.
4.3.2. General protocol for the Suzuki coupling reactions using
fragment 17
4.3.6. General protocol for the cyclization of C-4 and/or C-5
substituted fragments 18 or 19b to thieno[2,3-d]pyrimidin-4-
amines 1
In order to probe the versatility and stability of fragment 17, Suzu-
ki coupling reactions were carried out using two different reaction
conditions: (a) The boronic acid or boronate ester (1.5 equiv),
Pd(PPh3)4 (0.1 equiv) and fragment 17 were dissolved in toluene/
ethanol (3:1) (approximate concentration with respect to 17 of
0.1 M). The mixture was degassed and flushed with Argon. Aqueous
2 M Na2CO3 (2.5 equiv) was added and the mixture was again de-
gassed and flushed with Argon. The reaction mixture was stirred at
85 °C overnight. The crude was filtered through a plug of celite, rinsed
with 10 mL of solvent and concentrated under vacuum. The residue
was purified on silica gel using a CombiFlash instrument to give the
desired products (the common solvent gradient was from 2% EtOAc
in hexanes to 100% EtOAc, unless otherwise indicated). (b) The proto-
col described for the Suzuki reactions using fragment 20b was also
used successfully; presumably the TMS group survives these condi-
tions due to high solvation of the fluoride ion in methanol.
The mono-substituted or di-substituted fragments 18 or 19b,
respectively, (0.04 mmol) and dry formamide (excess, >200 equiv)
were added to a dry 15 mL pressure vessel. The vessel was flushed
with argon and the mixture stirred at 130 °C for 48 h. The dark red
solution was diluted with EtOAc, washed with water (25 mL), brine
(10 mL), and dried over Na2SO4. The crude mixture was purified by
flash column chromatography (5–100% EtOAc/hexanes, solid load-
ing) to afford the desired product.
4.4. Spectral data of key synthetic intermediates 18
The R5 groups indicated are taken from the fragments shown in
Table 1.
4.4.1. N0-(3-Cyano-5-phenyl-4-(trimethylsilyl)thiophen-2-yl)-
N,N-dimethylformimidamide (18, R5 = a)
Isolated as a beige solid in 90% yield (179 mg).
4.3.3. General protocol for Stille cross-coupling reactions using
fragment 19a
1H NMR (400 MHz, CDCl3) d 7.71 (s, 1H), 7.36–7.33 (m, 5H), 3.12
(s, 3H), 3.08 (s, 3H), 0.12 (s, 9H).
Palladium acetate (1 equiv) and XPhos (2 equiv) in DME (2 mL)
were heated under argon to 85 °C in a 2-dram vial for 10 min. To
this mixture cesium carbonate (2 equiv), and fragment 19a
(1 equiv 0.3 mmol) were added and stannane (1.77 equiv). The vial
was flushed with argon and the reaction mixture was stirred at
80 °C for 15 h. The reaction mixture was diluted with EtOAc
(10 mL), washed with water (3 ꢁ 10 mL) and brine (10 mL), dried
over Na2SO4, and concentrated under vacuum. The crude residue
was purified by column chromatography on silica gel using a Com-
biFlash instrument and a solvent gradient from% EtOAc in hexanes
to 100% EtOAc, unless otherwise indicated.
13C NMR (126 MHz, CDCl3) d 167.8, 154.6, 139.1, 136.3, 135.9,
130.6, 128.5, 128.1, 118.0, 102.7, 40.7, 35.2, 0.4.
HRMS (ESI+) Calcd 328.12982 for C17H22N3SSi, found m/z
328.12920 [M+H]+.
4.4.2. N0-(3-Cyano-5-(p-tolyl)-4-(trimethylsilyl)thiophen-2-yl)-
N,N-dimethylformimidamide (18, R5 = c)
Isolated as a pale yellow solid in 75% yield (115 mg).
1H NMR (400 MHz, CDCl3) d 7.79 (s, 1H), 7.21 (d, J = 8 Hz, 2H),
7.15 (d, J = 8 Hz, 2H), 3.32 (s, 3H), 3.20 (s, 3H), 2.38 (s, 3H),
0.14 (s, 9H).
4.3.4. General protocol for Sonogashira cross-coupling reactions
using fragment 19a
13C NMR (126 MHz, CDCl3) d 167.6, 154.5, 139.3, 138.3, 135.9,
132.8, 130.4, 128.7, 117.9, 102.6, 40.6, 35.0, 21.4, 0.5.
HRMS (ESI+) Calcd 342.14547 for C18H24N3SSi, found m/z
342.14458 [M+H]+.
Triethylamine (0.5 mL) was added to a vial of the iodide 19a
(0.081 mmol), acetylene (0.018 mL, 0.16 mmol), copper(II) bro-
mide (2.7 mg, 0.012 mmol), and Pd(PPh3)4 (9.4 mg, 0.008 mmol).
The vial was purged with argon, capped, and heated to 90 °C for
13 h. Extracted with EtOAc (3 ꢁ 10 mL) and washed with saturated
sodium bicarbonate (5 mL), water (3 ꢁ 5 mL), brine (5 mL), and
dried over Na2SO4. The crude mixture was purified by flash column
chromatography on silica gel (solid loading) using a solvent gradi-
ent from 2% EtOAc in hexanes to 100% EtOAc (unless otherwise in-
dicated) to afford the desired product.
4.4.3. N0-(3-Cyano-5-(4-(trifluoromethyl)phenyl)-4-
(trimethylsilyl)thiophen-2-yl)-N,N-dimethylformimidamide
(18, R5 = e)
Isolated as a pale brown solid in 71% yield (49.6 mg).
1H NMR (300 MHz, CDCl3) d 7.72 (s, 1H), 7.61 (d, J = 8 Hz, 2H),
7.46 (d, J = 8 Hz, 2H), 3.12 (d, J = 12 Hz, 6H), 0.14 (s, 9H).
13C NMR (75 MHz, CDCl3) d 168.3, 154.7, 139.7, 137.4, 136.8,
130.5 (q, JCF = 33 Hz), 125.0 (q, JCF = 4 Hz), 124.1 (q,
JCF = 272 Hz), 117.7, 103.1, 40.8, 35.2, 0.5.
4.3.5. General protocol for Buchwald–Hartwig amination
reactions using fragment 19a
The amine (5 equiv) was added to a degassed solution of frag-
ment 19a (usually on a 0.03 mmol scale), Pd2(dba)3 (5 mol %),
XantPhos (11 mol %), and cesium carbonate (1.7 equiv) in toluene
(1 mL). The vial was purged with argon and the reaction mixture
stirred at 100 °C for 18 h. A second portion of Pd2(dba)3 (5 mol %)
and XantPhos (11 mol %) were added and the reaction mixture
was stirred at 100 °C for an additional 18 h. The reaction mixture
was diluted with EtOAc (10 mL), washed with water (3 ꢁ 10 mL)
and brine (10 mL), dried over Na2SO4, and concentrated under va-
cuum. The crude residue was purified by column chromatography
on silica gel using a CombiFlash instrument and a solvent gradient
HRMS (ESI+) Calcd 396.11775 for C18H21F3N3SSi, found m/z
396.11519 [M+H]+.
4.4.4. N0-(3-Cyano-5-(naphthalen-2-yl)-4-
(trimethylsilyl)thiophen-2-yl)-N,N-dimethylformimidamide
(18, R5 = f)
Isolated as an orange solid in 63% yield (72.1 mg).
Please cite this article in press as: Leung, C.-Y.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.02.006

8
C.-Y. Leung et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
1H NMR (400 MHz, CDCl3): d 7.87–7.81 (m, 5H), 7.74 (s, 1H),
13C NMR (126 MHz, CDCl3) d 164.2, 154.6, 134.0, 133.3, 131.9,
128.8, 128.7, 128.5, 128.2, 127.5, 122.9, 116.6, 115.5, 100.9,
94.7, 83.3, 40.9, 35.3.
7.53–7.51 (m, 2H), 7.46 (m, 1H), 3.15 (s, 3H), 3.10 (s, 3H) 0.13
(9H, s).
13C NMR (75 MHz, CDCl3): d 167.9, 154.6, 139.1, 136.6, 133.3,
133.0, 132.8, 129.6, 128.4, 128.1, 127.9, 127.7, 126.7, 126.6,
40.7, 35.2, 0.6.
HRMS (ESI+) Calcd 356.12159 for
C22H18N3S, found m/z
356.12098 [M+H]+.
HRMS (ESI+) Calcd 378.14547 for C21H24N3SSi, found m/z
378.14412 [M+H]+.
4.5.3. N0-(3-Cyano-4-(cyclopropylethynyl)-5-phenylthiophen-2-
yl)-N,N-dimethylformimidamide (19b, R4 = h, R5 = a)
Isolated beige solid in 90% yield (15 mg, based on the recovery
of some starting material).
4.4.5. N0-(4-Cyano-3-(trimethylsilyl)-[2,30-bithiophen]-5-yl)-
N,N-dimethylformimidamide(18, R5 = i)
Isolated as light brown solid in 75% yield (53.1 mg).
1H NMR (500 MHz, CDCl3) d 7.78–7.75 (m, 3H), 7.38–7.35 (m,
2H), 7.30–7.26 (m, 1H), 3.10 (s, 6H), 1.52–1.46 (m, 1H), 0.91–
0.87 (m, 2H), 0.87–0.83 (m, 2H).
1H NMR (400 MHz, CDCl3): d 7.70 (s, 1H), 7.31–7.29 (m, 1H),
7.23 (m, 1H), 3.12 (3H, s), 3.09 (3H, s), 0.15 (s, 9H).
13C NMR (75 MHz, CDCl3): d 167.6, 154.7, 154.6, 137.3, 135.7,
132.9, 130.0, 125.5, 125.3, 117.8, 102.5, 40.7, 35.2, 0.2.
HRMS (ESI+) Calcd 334.08624 for C15H20N3S2Si, found m/z
334.08530 [M+H]+.
13C NMR (126 MHz, CDCl3) d 163.8, 154.5, 133.30, 132.9, 128.6,
127.9, 127.1, 117.2, 115.6, 101.4, 99.6, 69.4, 40.9, 35.2, 9.1, 0.7.
HRMS (ESI+) Calcd 320.12159 for
C19H18N3S, found m/z
320.12139 [M+H]+.
4.5.4. N0-(3-Cyano-4-morpholino-5-phenylthiophen-2-yl)-N,N-
dimethylformimidamide (19b, R4 = j, R5 = a)
4.4.6. N0-(3-Cyano-4-(trimethylsilyl)-5-vinylthiophen-2-yl)-N,N-
dimethylformimidamide (18, R5 = h)
Isolated as a beige solid in 90% yield (9.5 mg).
Isolated as beige solid in 75% yield (63 mg) and 95% purity (as
determined by LC–MS). The impurity was determined to be the de-
halogenated thiophene, however, this compound was eliminated
from the library for the subsequent steps.
1H NMR (400 MHz, CDCl3) d 7.74 (s, 1H), 7.50 (d, J = 7.3 Hz, 2H),
7.35 (t, J = 7.5 Hz, 2H), 7.29 (t, J = 7.3 Hz, 1H), 3.75–3.71 (m, 4H),
3.12 (s, 3H), 3.10 (s, 3H), 3.10–3.07 (m, 4H).
13C NMR (126 MHz, CDCl3) d 164.4, 153.9, 143.8, 133.6, 129.3,
128.4, 127.6, 118.4, 116.3, 96.6, 67.5, 51.7, 40.9, 35.2.
HRMS (ESI+) Calcd 341.14306 for C18H21ON4S, found m/z
341.14218 [M+H]+.
1H NMR (400 MHz, CDCl3) d 7.72 (s, 1H), 6.86 (dd, J = 16.9,
10.8 Hz, 1H), 5.36 (d, J = 17.0 Hz, 1H), 5.10 (d, J = 10.8 Hz, 1H),
3.12 (s, 3H), 3.10 (s, 3H), 0.41 (s, 9H).
13C NMR (75 MHz, CDCl3) d 166.3, 154.8, 138.1, 137.5, 130.7,
117.5, 113.6, 103.0, 40.7, 35.1, 0.8.
4.5.5. N0-(4-(Benzyl(methyl)amino)-3-cyano-5-phenylthiophen-
2-yl)-N,N-dimethylformimidamide (19b, R4 = k, R5 = a)
HRMS (ESI+) Calcd 278.11417 for C13H20N3SSi, found m/z
278.11400 [M+H]+.
The crude residue was purified by column chromatography on a
CombiFlash instrument using a solvent gradient from 3% EtOAc/
hexanes to 100% EtOAc with 0.1% triethylamine. The desired pro-
duct was obtained in 45% yield (19 mg), 70% purity (as determined
by 1H NMR). The impurity was determined to be the dehaloge-
nated thiophene and was eliminated in the following step.
4.5. Spectral data of key synthetic intermediates 19
The R4 and/or R5 groups indicated are taken from the fragments
shown in Table 1.
4.5.1. N0-(3-Cyano-5-phenyl-4-(pyrazin-2-yl)thiophen-2-yl)-
N,N-dimethylformimidamide (19b, R4 = b, R5 = a)
Isolated as beige solid in 41% yield (8.0 mg) and 95% purity (as
determined by LC–MS).
1H NMR (400 MHz, CDCl3) d 7.76 (s, 1H), 7.50–7.44 (m, 1H),
7.38–7.27 (m, 5H), 7.25–7.21 (m, 4H), 4.16 (s, 2H), 3.14–3.07
(m, 8H), 2.74 (s, 3H).
13C NMR (75 MHz, CDCl3) d 164.2, 153.9, 144.6, 138.4, 133.7,
129.1, 128.9, 128.3, 128.2, 127.6, 127.3, 127.1, 125.3, 121.9,
120.1, 116.6, 96.9, 60.1, 41.1, 35.2.
1H NMR (400 MHz, CDCl3): d 8.67 (dd, J = 2.6, 1.6 Hz, 1H), 8.46
(d, J = 2.6 Hz, 1H), 8.36 (d, J = 1.5 Hz, 1H), 7.85 (s, 1H), 7.28–
7.25 (m, 3H), 7.17–7.14 (m, 2H), 3.15 (s, 3H), 3.15 (s, 3H).
13C NMR (75 MHz, CDCl3): d 166.9, 154.6, 149.3, 145.8, 144.5,
143.1, 132.7, 132.2, 131.1, 129.3, 129.0, 128.4, 115.9, 99.2,
40.9, 35.3.
HRMS (ESI+) Calcd 375.16379 for
C22H23N4S, found m/z
375.16296 [M+H]+.
4.6. Spectral data of key thieno[2,3-d]pyrimidin-4-amines (1)
HRMS (ESI+) Calcd 334.11209 for
C18H16N5S, found m/z
334.11144 [M+H]+.
The yields indicated are for the isolated pure fragments after the
cyclization step of 19b–1 from the library synthesis. For the key
building blocks 5-(trimethylsilyl)thieno[2,3-d]pyrimidin-4-amine
(20a) and 5-iodothieno[2,3-d]pyrimidin-4-amine (20b), refer to
Sections 4.2.3 and 4.2.4, respectively.
4.5.2. N0-(3-Cyano-5-phenyl-4-(phenylethynyl)thiophen-2-yl)-
N,N-dimethylformimidamide (19b, R4 = g, R5 = a)
Isolated as a red-orange solid in 95% yield (28.0 mg).
4.6.1. 2-Phenylthieno[2,3-d]pyrimidin-4-amine (1a)
1H NMR (400 MHz, CDCl3) d 7.86–7.75 (d, J = 7.4 Hz, 3H), 7.83 (s,
1H), 7.57–7.51 (m, 2H), 7.42 (t, J = 7.5 Hz, 2H), 7.37–7.30 (m,
4H), 3.15 (s, 3H), 3.15 (s, 3H).
Synthesis of derivative 20c was achieved via direct condensa-
tion of 2-aminothiophene-3-carbonitrile with benzonitrile, as
previously reported.12b To an argon flushed pressure vessel, 2-
Please cite this article in press as: Leung, C.-Y.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.02.006

C.-Y. Leung et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
9
aminothiophene-3-carbonitrile (150 mg, 1.21 mmol, 1 equiv),
benzonitrile (174.4 mg, 1.69 mmol, 1.4 equiv) and potassium
tert-butoxide (135.3 mg, 1.21 mmol, 1 equiv) were added and
the mixture was flushed again with argon. Anhydrous dioxane
(1 mL) was added and the reaction mixture was stirred at
150 °C for 14 h. The crude mixture was concentrated to dryness
under vacuum and was purified by flash column chromatogra-
phy (0–50% EtOAc/hexanes, solid loading) to afford the final pro-
duct in 27% yield (37.7 mg).
1H NMR (500 MHz, DMSO-d6): d 8.28 (s, 1H), 8.18 (s, 1H), 8.15
(s, 1H), 8.06–8.01 (m, 2H), 7.96–7.94 (m, 1H), 7.84–7.83 (m,
1H), 7.62–7.53 (m, 4H). 13C NMR (126 MHz, DMSO-d6) d
165.9, 158.3, 154.3, 133.0, 132.6, 130.7, 129.0, 128.1, 127.7,
127.0, 126.7, 124.4, 123.4, 117.1, 116.3, 104.6.
HRMS (ESI+) Calcd 278.07464 for
C16H11N3S, found m/z
278.07371 [M+H]+.
4.6.7. 6-(Thiophen-3-yl)thieno[2,3-d]pyrimidin-4-amine (1g)
1H NMR (500 MHz, CDCl3): d 8.45–8.42 (m, 2H), 7.48–7.45 (m,
3H), 7.26 (d, J = 6.0 Hz, 1H), 7.14 (d, J = 6.0 Hz, 1H), 5.55 (s, 2H).
13C NMR (126 MHz, CDCl3): d 168.9, 160.3, 157.8, 137.9, 130.1,
128.4, 128.2, 123.3, 117.4, 114.3.
Isolated as a brown solid in 28% yield (9.6 mg).
1H NMR (400 MHz, DMSO-d6): d 8.24 (s, 1H), 7.78 (m, 2H), 7.73
(dd, J = 5.0, 2.9 Hz, 1H), 7.50 (br s, 2H), 7.41 (dd, J = 5.0, 1.4 Hz, 1H).
13C NMR (126 MHz, DMSO-d6) d 165.3, 158.1, 154.0, 134.6,
133.1, 128.3, 125.4, 121.9, 116.6, 115.3.
MS (ESI+) m/z: 228.1 [M+H]+.
HRMS (ESI+) Calcd 234.01542 for
C10H7N3S2, found m/z
4.6.2. 5-Phenylthieno[2,3-d]pyrimidin-4-amine (1b)
234.01433 [M+H]+.
Isolated as pale yellow solid in 22% yield (27 mg).
1H NMR (500 MHz, CD3OD): d 8.29 (s, 1H), 7.51–7.46 (m, 5H),
7.31 (s, 1H).
4.6.8. 5-(4-Nitrophenyl)thieno[2,3-d]pyrimidin-4-amine (1h)
Isolated as orange solid in 40% yield (14.2 mg); all NMR data
was consistent with previous reports.24
13C NMR (126 MHz, CD3OD) d 168.3, 160.0, 154.6, 137.2, 136.8,
130.2, 130.1, 129.8, 122.1, 114.9.
MS (ESI+) m/z: 228.4 [M+H]+.
1H NMR (500 MHz, DMSO-d6): d 8.37 (s, 1H), 8.34 (d, J = 8.2 Hz,
2H), 7.73 (d, J = 8.2 Hz, 2H), 7.69 (s, 1H), 7.06 (s, 2H).
13C NMR (75 MHz, CDCl3) d 167.7, 158.3, 154.0, 147.1, 142.0,
133.2, 130.1, 123.9, 122.9, 112.2.
4.6.3. 6-Bromothieno[2,3-d]pyrimidin-4-amine (1c)
Isolated as a pale yellow solid in 35% yield (321 mg).
MS (ESI+) m/z: 273.1 [M+H]+.
1H NMR (300 MHz, DMSO-d6): d 8.22 (1H, s), 7.72 (1H, s), 7.56
(2H, s).
4.6.9. 5-(4-(Trifluoromethyl)phenyl)thieno[2,3-d]pyrimidin-4-
amine (1i)
13C NMR (300 MHz, DMSO-d6): d 167.5, 157.7, 154.8, 123.3,
116.7, 109.7.
Isolated as a pale yellow solid in 57% yield (28.2 mg).
MS (ESI+) m/z: 229.9 and 232.0 [M+H]+.
1H NMR (400 MHz, DMSO-d6): d 8.36 (s, 1H), 7.87 (d, J = 8.0 Hz),
7.70 (d, J = 8.0 Hz), 7.62 (s, 1H).
4.6.4. 6-(p-Tolyl)thieno[2,3-d]pyrimidin-4-amine (1d)
13C NMR (126 MHz, DMSO-d6) d 167.5, 158.3, 153.9, 139.6,
133.6, 129.1, 128.5 (q, J = 32.1 Hz), 125.7 (q, J = 3.8 Hz), 124.3
(q, J = 272.1 Hz), 122.2, 112.4.
Isolated as a beige solid in 65% yield (49.6 mg).
1H NMR (400 MHz, DMSO-d6): d 8.28 (s, 1H), 8.18 (s, 1H), 8.15
(s, 1H), 8.06–8.01 (m, 2H), 7.96–7.94 (m, 1H), 7.84–7.83 (m,
1H), 7.62–7.53 (m, 4H).
HRMS (ESI+) Calcd 296.04638 for
C13H8F3N3S, found m/z
294.04552 [M+H]+.
13C NMR (75 MHz, DMSO-d6) d 165.5, 162.9, 158.2, 154.0, 138.2,
130.5, 129.9, 125.5, 117.0, 114.9, 20.8.
4.6.10. 5-(Naphthalen-2-yl)thieno[2,3-d]pyrimidin-4-amine (1j)
HRMS (ESI+) Calcd for C13H12N3S m/z [M+H]+: 242.07464, found
m/z 242.07383.
Isolated as a pale yellow solid in 54% yield (27.0 mg).
1H NMR (500 MHz, DMSO-d6): d 8.37 (s, 1H), 8.08–8.00 (m, 4H),
7.62–7.59 (m, 4H).
4.6.5. 6-(4-(Trifluoromethyl)phenyl)thieno[2,3-d]pyrimidin-4-
amine (1e)
13C NMR (75 MHz, DMSO-d6) d 167.3, 158.4, 153.8, 134.9, 133.0,
132.8, 132.4, 128.5, 128.0, 127.8, 127.7, 126.8, 126.7, 126.6,
121.1, 113.0.
Isolated as a pale yellow solid in 56% yield (18.6 mg).
HRMS (ESI+) Calcd 278.07464 for
C16H11N3S, found m/z
1H NMR (500 MHz, DMSO-d6): d 8.29 (s, 1H), 8.14 (s, 1H), 7.86
(s, 4H), 7.66 (s, 2H). 13C NMR (126 MHz, DMSO-d6) d 166.3,
158.5, 154.6, 137.1, 135.7, 128.31 (q, J = 32.0 Hz), 126.3 (q,
J = 3.7 Hz), 124.09 (q, J = 272.0 Hz), 117.8, 116.9.
278.07371 [M+H]+.
4.6.11. 6-Phenyl-5-(phenylethynyl)thieno[2,3-d]pyrimidin-4-
amine (1k)
HRMS (ESI+) Calcd 296.04638 for
C13H9F3N3S, found m/z
296.04552 [M+H]+.
Isolated as a brown solid in 85% yield (12 mg).
1H NMR (500 MHz, CDCl3) d 8.45 (s, 1H), 7.96–7.94 (m, 1H),
7.94–7.93 (m, 1H), 7.51–7.47 (m, 4H), 7.45–7.37 (m, 4H),
ꢂ6.30 (br s, 2H).
4.6.6. 6-(Naphthalen-2-yl)thieno[2,3-d]pyrimidin-4-amine (1f)
Isolated as a brown solid in 35% yield (15.4 mg).
Please cite this article in press as: Leung, C.-Y.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.02.006

10
C.-Y. Leung et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
13C NMR (126 MHz, CDCl3) d 165.3, 158.4, 154.6, 143.9, 133.0,
4.7. Synthesis of bisphosphonic acids 2
131.4, 129.4, 129.3, 129.0, 128.9, 128.6, 122.0, 116.5, 108.8,
94.8, 85.2.
4.7.1. Standard 2-step procedure for the conversion of
thienopyrimidin-4-amines 1 to inhibitors 2
The R2, R5 and R6 groups indicated are taken from the fragments
shown in Table 1.
HRMS (ESI+) Calcd 328.09029 for
C20H14N3S, found m/z
328.09015 [M+H]+.
Step (a):
A solution of the thienopyrimidin-4-amine 1
(0.2 mmol) in anhydrous toluene (10 mL) was flushed with argon.
Triethyl orthoformate (1.5 equiv) and diethylphosphite (7 equiv)
was added to the reaction mixture via syringe and the reaction
mixture was flushed again with argon, sealed, and stirred for
130 °C in the dark for 48 h. The crude mixture was concentrated
to dryness under vacuum and purified by flash column chromato-
graphy (20–100% EtOAc/hexanes to 0–20% MeOH/EtOAc, solid
loading) to afford the desired tetraethyl bisphosphonate ester.
Step (b): A solution of the tetraethyl bisphosphonate ester
(1 equiv) in CH2Cl2 was cooled to 0 °C and trimethylsilyl bromide
(15 equiv) was added via syringe. The reaction mixture was stirred
at room temperature for 3–5 days; completion of conversion was
monitored by 31P NMR. The mixture was then concentrated under
vacuum, diluted with HPLC grade MeOH (ꢂ5 mL), and the solvent
was evaporated to dryness; this step was repeated four times. The
organic solvents were evaporated under vacuum, the residue was
suspended in 0.5 mL MeOH, excess water (ꢂ5 mL Milli-Q grade)
was added to induce full precipitation of the final bisphosphonic
acid. The amorphous powder was collected by filtration, washed
with de-ionized water (2ꢁ), with HPLC-grade CH3CN (2ꢁ), with
distilled Et2O or toluene (2ꢁ) and dried under vacuum to give
the final compound as a white solid.
4.6.12. 5-(Cyclopropylethynyl)-6-phenylthieno[2,3-
d]pyrimidin-4-amine (1l)
Isolated as a beige solid in 85% yield (11 mg).
1H NMR (500 MHz, CDCl3): d 8.39 (s, 1H), 7.88–7.81 (m, 2H),
7.49–7.35 (m, 3H), 1.52 (m, 1H), 0.98–0.92 (m, 2H), 0.84 (m,
2H).
13C NMR (126 MHz, CDCl3): d 164.9, 158.4, 154.4, 143.0, 133.1,
129.1, 128.8, 128.4, 116.7, 109.3, 99.5, 71.6, 8.8, 0.55.
HRMS (ESI+) Calcd 292.09029 for
C17H14N3S, found m/z
292.09030 [M+H]+.
4.6.13. 5-Morpholino-6-phenylthieno[2,3-d]pyrimidin-4-amine
(1m)
The crude mixture was purified by flash column chromatogra-
phy (5–100% EtOAc/hexanes with 0.1% Et3N, solid loading) to af-
ford the desired product as a beige solid in 50% yield (20.5 mg).
1H NMR (400 MHz, CDCl3) d 8.42 (s, 1H), 7.49–7.41 (m, 5H), 3.84
(d, J = 10.6 Hz, 2H), 3.60 (td, J = 11.5, 2.3 Hz, 2H), 3.03 (td,
J = 11.6, 2.8 Hz, 2H), 2.95 (d, J = 11.8 Hz, 2H), 1.60 (s, 1H).
13C NMR (75 MHz, CDCl3) d 164.5, 158.8, 154.3, 138.3, 133.4,
131.2, 131.0, 129.2, 128.6, 113.9, 67.8, 53.2.
4.7.2. (Thieno[2,3-d]pyrimidin-4-ylamino)methylene
bisphosphonic acid (2a)
The tetraethyl ((5-(trimethylsilyl)thieno[2,3-d]pyrimidin-4-
yl)amino)methylene bisphosphonate was prepared from inter-
mediate 20a; isolated in 60% yield (36.7 mg).
HRMS (ESI+) Calcd 313.11176 for C16H17ON4S, found m/z
313.11125 [M+H]+.
4.6.14. N5-Benzyl-N5-methyl-6-phenylthieno[2,3-d]pyrimidine-
4,5-diamine (1n)
The crude mixture was purified by flash column chromatogra-
phy (5–100% EtOAc/hexanes with 0.1% Et3N, solid loading) to af-
ford the desired product as a pale orange solid in 53% yield
(9.0 mg).
1H NMR (400 MHz, CD3OD): d 8.50 (s, 1H), 7.45 (s, 1H), 5.98 (td,
J = 22.0, 9.9 Hz, 1H), 5.78 (d, J = 10.3 Hz, 1H), 4.28–4.12 (m, 8H),
1.26 (td, J = 7.1, 1.7 Hz, 12H), 0.52 (s, 9H).
13C NMR (126 MHz, CD3OD): d 170.1, 156.2, 152.7, 133.0, 131.3,
120.3, 63.4 (d, JCP = 122.5 Hz), 44.2 (t, JCP = 583 Hz), 16.3, 0.09.
31P NMR (81 MHz, CD3OD): d 14.81.
MS (ESI) m/z 532.15 [M+Na]+.
1H NMR (500 MHz, CDCl3) d 8.41 (s, 1H), 7.47–7.36 (m, 5H),
7.29–7.21 (m, 3H), 7.12–7.06 (m, 2H), 3.99 (d, J = 13.3 Hz, 1H),
3.66 (d, J = 13.3 Hz, 1H), 2.77 (s, 3H).
A
solution of the bisphosphonate tetraester (47.4 mg,
13C NMR (126 MHz, CDCl3) d 164.4, 158.6, 154.3, 139.3, 138.1,
133.6, 130.8, 130.6, 128.9, 128.9, 128.7, 128.46, 127.7, 114.2,
60.7, 43.4.
0.093 mmol) in THF (2 mL) was then cooled to 0 °C and 0.1 mL
(23.6 mg of a 1 M solution, 0.1 mmol) of TBAF in THF was added.
After stirring for 20 min at 0 °C, the mixture was warmed to ambi-
ent temperature and stirred for and additional 4 h in the dark. THF
was removed under vacuum and the crude mixture was dissolved
in 10 mL of EtOAc, washed with water (1 ꢁ 5 mL) and brine
(1 ꢁ 5 mL), and dried over anhydrous sodium sulfate. The desily-
lated product was obtained quantitatively as a pale yellow solid
(41.2 mg).
HRMS (ESI+) Calcd 347.13249 for
C20H19N4S, found m/z
347.13188 [M+H]+.
4.6.15. 6-Phenyl-5-(pyrazin-2-yl)thieno[2,3-d]pyrimidin-4-
amine (1o)
Isolated as a beige solid in 95% yield (7.0 mg).
1H NMR (500 MHz, CD3OD): d 8.47 (s, 1H), 7.72 (d, J = 6.0 Hz,
1H), 7.57 (d, J = 6.0 Hz 1H), 6.00 (t, J = 23.7 Hz, 1H), 4.20 (m,
8H), 1.26 (m, 12H).
1H NMR (500 MHz, CDCl3) d 8.68 (dd, J = 2.6, 1.5 Hz, 1H), 8.50 (d,
J = 2.6 Hz, 1H), 8.48 (s, 1H), 8.29 (d, J = 1.4 Hz, 1H), 7.36–7.30
(m, 3H), 7.22–7.19 (m, 2H).
13C NMR (126 MHz, CD3OD): d 167.6, 157.6, 154.1, 125.1, 119.8,
118.5, 65.16, 45.6 (t, JCP = 150.1 Hz), 16.6.
13C NMR (126 MHz, CDCl3) d 167.3, 159.1, 154.1, 150.8, 149.0,
143.1, 143.0, 141.9, 132.9, 130.2, ꢂ129.4 (3 ꢁ C), 126.1, 115.9.
HRMS: Calcd 306.08079 for C16H12N5S, found m/z 306.08165
[M+H]+.
31P NMR (81 MHz, CD3OD): d 18.52.
HRMS (ESI+) Calcd 460.08370 for C15H25N3NaO6P2S, found
461.08320 [M+Na]+.
Please cite this article in press as: Leung, C.-Y.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.02.006

C.-Y. Leung et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
11
The final inhibitor, thieno[2,3-d]pyrimidin-4-ylamino methy-
lene bisphosphonic acid (2a) was isolated as a white solid in 70%
yield (20.8 mg).
1H NMR (500 MHz, D2O): d 8.17 (s, 1H), 7.68 (s, 1H), 7.54 (d,
J = 7.0 Hz, 2H), 7.43 (t, J = 7.6 Hz, 2H), 7.35 (d, J = 7.4 Hz, 1H),
7.12 (s, 1H).
13C NMR (126 MHz, D2O): d 165.0, 153.2, 135.3, 129.4, 129.1,
128.3, 120.7, 113.9, 50.6 (t, J = 123.6).
1H NMR (400 MHz, D2O, 10% ND4OD, 10% DMSO internal stan-
dard): d 8.284 (s, 1H), 7.549 (d, J = 5.9 Hz, 1H), 7.427 (d,
J = 6.1 Hz, 1H), 4.594 (t, J = 19.1 Hz, 1H).
31P NMR (81 MHz, D2O): d 13.4.
HRMS (ESIꢀ) Calcd 399.9928 for C13H12N3P2O6S, found m/z
13C NMR (126 MHz, D2O, 20% ND4OD, 10% DMSO internal stan-
dard): d 164.3, 157.3, 154.2, 123.4, 119.8, 117.9, 51.7 (t,
JCP = 125.6 Hz).
399.9918 [MꢀH]ꢀ.
31P NMR (81 MHz, D2O, 20% ND4OD): d 13.308.
HRMS (ESIꢀ) Calcd 323.96873 for C7H8SN3O6P2, found
323.96125 [MꢀH]ꢀ.
4.7.5. (6-Phenylthieno[2,3-d]pyrimidin-4-ylamino)methylene
bisphosphonic acid (2d)
The tetraethyl ((6-phenylthieno[2,3-d]pyrimidin-4-yl)amino)-
methylene bisphosphonate was isolated in 65% yield (38.0 mg).
4.7.3. (((2-Phenylthieno[2,3-d]pyrimidin-4-
yl)amino)methylene)diphosphonic acid (2b)
The tetraethyl (((2-phenylthieno[2,3-d]pyrimidin-4-yl)amino)-
methylene)bis(phosphonate) was isolated as an orange oil 61%
(52.0 mg).
1H NMR (500 MHz, CD3OD) d 8.45 (s, 1H), 8.04 (s, 1H), 7.75 (d,
J = 7.3 Hz, 2H), 7.48 (t, J = 7.6 Hz, 2H), 7.41 (d, J = 7.3 Hz, 1H),
6.00 (t, J = 23.6 Hz, 1H), 4.22 (dd, J = 7.7, 3.2 Hz, 8H), 1.45–1.09
(m, 12H).
13C NMR (126 MHz, CD3OD) d 167.0, 157.2, 154.1, 142.7, 134.6,
130.4, 130.1, 127.2, 119.8, 115.2, 65.2, 45.6, 16.7.
31P NMR (81 MHz, CD3OD) d 17.004.
1H NMR (500 MHz, MeOD-d4): d 8.43–8.41 (m, 2H), 7.71 (d,
J = 6.0 Hz, 1H), 7.51 (d, J = 6.0 Hz, 1H), 7.49–7.47 (m, 4H), 6.21
(t, J = 23.7 Hz, 1H), 4.22–4.19 (m, 8H), 1.25–1.22 (m, 12H).
13C NMR (126 MHz, MeOD-d4): d 169.5, 160.5, 157.3, 139.0,
131.5, 129.5, 129.1, 124.8, 119.7, 116.8, 65.1 (m), 45.5 (t,
J = 150.1 Hz), 16.7.
MS (ESI) m/z 536.15 [M+Na]+.
The final inhibitor (6-phenylthieno[2,3-d]pyrimidin-4-ylami-
no)methylene bisphosphonic acid (2d) was isolated as a white so-
lid in 63% yield (18.8 mg).
31P NMR (81 MHz, MeOD-d4): d 17.6.
MS (ESI+) m/z: 514.7 [M+H]+.
1H NMR (400 MHz, D2O, 10% ND4OD): d 8.18 (s, 1H), 7.80 (s,
1H), 7.71 (d, J = 7.4 Hz, 2H), 7.42 (t, J = 7.6 Hz, 2H), 7.33 (t,
J = 7.4 Hz, 1H).
The final product (((2-phenylthieno[2,3-d]pyrimidin-4-yl)ami-
no)methylene)diphosphonic acid (2b) was isolated as a pale yellow
solid in 66% yield (22.0 mg).
13C NMR (126 MHz, D2O, 10% ND4OD): d 163.1, 156.2, 153.6,
139.2, 133.2, 129.3, 128.6, 125.9, 118.64, 114.8.
31P NMR (81 MHz, D2O, ND4OD) d 13.29.
1H NMR (300 MHz, D2O): d 8.15–8.12 (m, 2H), 7.44–7.40 (m,
4H), 7.28 (d, J = 6.0 Hz, 1H), 4.86 (t, J = 19.0 Hz, 1H).
13C NMR (126 MHz, D2O): d 166.3, 161.5, 157.8, 138.4, 131.4,
129.6, 129.1, 128.8, 123.6, 119.9, 116.6, 105.9, 50.6 (t, J = 123.6).
31P NMR (81 MHz, D2O): d 14.2.
HRMS (ESIꢀ) Calcd 399.99166 for
C13H12SN3O6P2, found
399.99268 [MꢀH]ꢀ.
4.7.6. (((6-(p-Tolyl)thieno[2,3-d]pyrimidin-4-
yl)amino)methylene)diphosphonic acid (2e)
The tetraethyl (((6-(p-tolyl)thieno[2,3-d]pyrimidin-4-yl)ami-
no)methylene)bis(phosphonate) was isolated as a light brown
powder 94% (27.3 mg).
HRMS (ESIꢀ) Calcd 399.9928 for C13H12N3P2O6S, found m/z
399.9922 [MꢀH]ꢀ.
4.7.4. (((5-Phenylthieno[2,3-d]pyrimidin-4-
yl)amino)methylene)diphosphonic acid (2c)
The tetraethyl (((5-phenylthieno[2,3-d]pyrimidin-4-yl)amino)-
methylene)bis(phosphonate) was isolated as a pale yellow solid
24% yield (61 mg).
1H NMR (500 MHz, MeOD-d4): d 8.43 (s, 1H), 7.97 (s, 1H),
7.60 (d, J = 8.1 Hz, 2H), 7.27 (d, J = 7.8 Hz, 1H), 6.00 (t,
J = 23.6 Hz, 1H), 4.24–4.21 (m, 8H), 2.37 (s, 3H), 1.31–1.24
(m, 12H).
13C NMR (126 MHz, MeOD-d4): d 166.8, 157.0, 153.9, 142.9,
140.4, 131.8, 130.9, 127.1, 119.8, 114.4, 65.10 (m), 45.6 (t,
J = 150 Hz), 21.3, 16.7 (m).
1H NMR (500 MHz, MeOD-d4): d 8.52 (s, 1H), 7.60–7.55 (m, 3H),
7.52–7.50 (m, 2H), 7.45 (s, 1H), 5.80 (t, J = 22.4 Hz, 1H), 4.11–
4.03 (m, 8H), 1.22–1.19 (m, 12H).
31P NMR (81 MHz, MeOD-d4): d 17.4.
13C NMR (75 MHz, MeOD-d4): d 166.9, 152.8, 135.4, 134.4,
129.2, 128.9, 128.8, 122.0, 63.7 (m), 43.6 (t, J = 150.1 Hz), 15.2.
31P NMR (81 MHz, MeOD-d4): d 16.1.
MS (ESI+) m/z: 528.2 [M+H]+.
The final inhibitor (((6-(p-tolyl)thieno[2,3-d]pyrimidin-4-yl)a-
mino)methylene)diphosphonic acid (2e) was isolated as a white
solid in 94% yield (27.3 mg).
MS (ESI+) m/z: 515.3 [M+H]+.
The final inhibitor (((5-phenylthieno[2,3-d]pyrimidin-4-yl)ami-
no)methylene)diphosphonic acid (2c) was isolated as a white solid
in 54% yield (5.1 mg)
1H NMR (500 MHz, D2O): d 8.22 (s, 1H), 7.80 (s, 1H), 7.66 (d,
J = 8.2 Hz, 2H), 7.30 (d, J = 8.0 Hz, 2H), 2.33 (s, 3H).
Please cite this article in press as: Leung, C.-Y.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.02.006

12
C.-Y. Leung et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
13C NMR (126 MHz, D2O): d 164.3, 157.6, 155.0, 140.8, 140.7,
131.9, 127.5, 120.2, 115.7, 21.8.
6. McClellan, W. J.; Dai, Y.; Abad-Zapatero, C.; Albert, D. H.; Bouska, J. J.; Glaser, K.
B.; Magoc, T. J.; Marcotte, P. A.; Osterling, D. J.; Stewart, K. D.; Davidsen, S. K.;
Michaelides, M. R. Bioorg. Med. Chem. Lett. 2011, 21, 5620.
7. Dai, Y.; Guo, Y.; Frey, R. R.; Ji, Z.; Curtin, M. L.; Ahmed, A. A.; Albert, D. H.;
Arnold, L.; Arries, S. S.; Barlozzari, T.; Bauch, J. L.; Bouska, J. J.; Bousquet, P. F.;
Cunha, G. A.; Glaser, K. B.; Guo, J.; Li, J.; Marcotte, P. A.; Marsh, K. C.; Moskey, M.
D.; Pease, L. J.; Stewart, K. D.; Stoll, V. S.; Tapang, P.; Wishart, N.; Davidsen, S. K.;
Michaelides, M. R. J. Med. Chem. 2005, 48, 6066.
31P NMR (81 MHz, D2O): d 13.72.
HRMS (ESIꢀ) Calcd 414.00840 for C14H14N3P2O6S, found m/z
414.00863 [MꢀH]ꢀ.
8. Luke, R. W.; Ballard, P.; Buttar, D.; Campbell, L.; Curwen, J.; Emery, S. C.; Griffen,
A. M.; Hassall, L.; Hayter, B. R.; Jones, C. D.; McCoull, W.; Mellor, M.; Swain, M.
L.; Tucker, J. A. Bioorg. Med. Chem. Lett. 2009, 19, 6670.
9. Horiuchi, T.; Chiba, J.; Uoto, K.; Soga, T. Bioorg. Med. Chem. Lett. 2009, 19, 305.
10. Sham, H. L.; Konradi, A. W.; Hom, R. K.; Probst, G. D.; Bowers, S.; Truong, A.;
Neitz, R. J.; Sealy, J.; Toth, G. WO 2010/091310.
4.7.7. (((6-(Naphthalen-2-yl)thieno[2,3-d]pyrimidin-4-
yl)amino)methylene)diphosphonic acid (2f)
The tetraethyl (((6-(naphthalen-2-yl)thieno[2,3-d]pyrimidin-4-
yl)amino)methylene)bis(phosphonate) was isolated as a white
powder 71% (30.8 mg)
11. Bowers, S.; Truong, A. P.; Neitz, R. J.; Hom, R. K.; Sealy, J. M.; Probst, G. D., et al
Bioorg. Med. Chem. Lett. 2011, 21, 5521.
12. Angell, R. M.; Atkinson, F. L.; Brown, M. J.; Chuang, T. T.; Christopher, J. A.;
Cichy-Knight, M.; Dunn, A. K.; Hightower, K. E.; Malkakorpi, S.; Musgrave, J. R.;
Neu, M.; Rowland, P.; Shea, R. L.; Smith, J. L.; Somers, D. O.; Thomas, S. A.;
Thompson, G.; Wang, R. Bioorg. Med. Chem. Lett. 2007, 17, 1296.
13. Axten, J. M.; Grant, S. W.; Heerding, D. A.; Medina, J. R.; Romeril, S. P.; Tang, J.
WO 2011/119663.
1H NMR (500 MHz, MeOD-d4): d 8.45 (s, 1H), 8.14 (s, 2H), 7.94–
7.83 (m, 4H), 7.53–7.47 (m, 2H), 6.02 (t, J = 23.6 Hz, 1H), 4.24–
4.21 (m, 8H), 1.31–1.24 (m, 12H).
13C NMR (126 MHz, MeOD-d4): d 167.1, 157.1, 154.2, 142.6,
135.0, 134.8, 131.9, 130.0, 129.3, 128.8, 128.0, 127.9, 126.3,
124.6, 119.9, 115.6, 65.1 (m), 45.7 (t, J = 150 Hz), 16.7 (m).
31P NMR (81 MHz, MeOD-d4): d 17.4.
14. Edgar, K. A.; Wallin, J. J.; Berry, M.; Lee, L. B.; Prior, W. W.; Sampath, D.;
Friedman, L. S.; Belvin, M. Cancer Res. 2010, 70, 1164.
15. Heffron, T. P.; Wei, B. Q.; Olivero, A.; Staben, S. T.; Tsui, V.; Do, S.; Dotson, J.;
Folkes, A. J.; Goldsmith, P.; Goldsmith, R.; Gunzner, J.; Lesnick, J.; Lewis, C.;
Mathieu, S.; Nonomiya, J.; Shuttleworth, S.; Sutherlin, D. P.; Wan, N. C.; Wang,
S.; Wiesmann, C.; Zhu, B.-Y. J. Med. Chem. 2011, 54, 7815.
16. Rheault, T. R.; Ceferro, T. R.; Dickerson, S. H.; Donaldson, K. H.; Gaul, M. D.;
Goetz, A. S.; Mullin, R. J.; McDonald, O. B.; Petrov, K. G.; Rusnak, D. W.;
Shewchuk, L. M.; Spehar, G. M.; Truesdale, A. T.; Vanderwall, D. E.; Wood, E. R.;
Uehling, D. E. Bioorg. Med. Chem. Lett. 2009, 19, 817.
17. Fischer, C.; Shah, S.; Hughes, B. L.; Nikov, G. N.; Crispino, J. L.; Middleton, R. E.;
Szewczak, A. A.; Munoz, B.; Shearman, M. S. Bioorg. Med. Chem. Lett. 2011, 21,
773.
18. Barbay, J. K.; Leonard, K.; Chakravarty, D.; Shook, B. C.; Wang, A. WO 2010/
045009.
MS (ESI+) m/z: 564.3 [M+H]+.
The final inhibitor (((6-(naphthalen-2-yl)thieno[2,3-d]pyrimi-
din-4-yl)amino)methylene)diphosphonic acid (2f) was isolated as
a white solid in 86% yield (29.5 mg).
1H NMR (500 MHz, D2O): d 8.19 (s, 1H), 8.13 (s, 1H), 7.91–7.83
(m, 5H), 7.50–7.44 (m, 2H).
13C NMR (126 MHz, D2O): d 164.8, 157.8, 155.1, 140.7, 134.7,
134.3, 132.2, 130.4, 129.6, 129.3, 128.6, 128.3, 126.2, 125.3,
120.3, 116.8.
19. Chakravarty, D.; Shook, B. C. WO 2010/045017.
20. Barbay, J. K.; Chakravarty, D.; Shook, B. C.; Wang, A. WO 2010/045006.
21. Lin, Y.-S.; Park, J.; De Schutter, J. W.; Huang, X. F.; Berghuis, A. M.; Sebag, M.;
Tsantrizos, Y. S. J. Med. Chem. 2012, 55, 3201.
22. De Schutter, J. W.; Zaretsky, S.; Welbourn, S.; Pause, A.; Tsantrizos, Y. S. Biorg.
Med. Chem. Lett. 2010, 20, 5781.
23. Gewald, K.; Schinke, E.; Boettcher, H. Chem. Ber. 1966, 99, 94.
24. Huang, X.-G.; Liu, J.; Ren, J.; Wang, T.; Chen, W.; Zeng, B.-B. Tetrahedron 2011,
67, 6202.
31P NMR (81 MHz, D2O): d 13.74.
HRMS (ESIꢀ) Calcd 450.0084 for C17H14N3P2O6S, found m/z
450.0085 [MꢀH]ꢀ.
25. Barnes, D. M.; Haight, A. R.; Hameury, T.; McLaughlin, M. A.; Mei, J.; Tedrow, J.
S.; Toma, J. D. R. Terahedron 2006, 62, 11311.
26. Hesse, S.; Perspicace, E.; Kirsch, G. Tetrahedron Lett. 2007, 48, 5261.
27. Tranberg, C. E.; Zickgraf, A.; Giunta, B. N.; Luetjens, H.; Figler, H.; Murphree, L.
J.; Falke, R.; Fleischer, H.; Linden, J.; Scammells, P. J.; Olsson, R. A. J. Med. Chem.
2002, 45, 382.
28. Miller, R. E.; Rantanen, T.; Ogilvie, K. A.; Groth, U.; Snieckus, V. Org. Lett. 2010,
12, 2198.
Acknowledgments
Financial support for this work was provided by NSERC and
FQRNT (research Grants to Y.S.T). A.L. is grateful to NSERC for grad-
uate scholarship.
29. Getmanenko, Y.; Tongwa, P.; Timofeeva, T. V.; Marder, S. R. Org. Lett. 2010, 12,
2136.
30. Schmürch, M.; Spina, M.; Khan, A. F.; Mihoviloviv, M. D.; Stanetty, P. Chem. Soc.
Rev. 2007, 36, 1046.
31. Schlosser, M. Angew. Chem., Int. Ed. 2005, 44, 376.
Supplementary data
32. Lukevics, E.; Arsenyan, P.; Belyakov, S.; Popelis, J.; Pudova, O. Tetrahedron Lett.
2001, 42, 2039.
33. Rondeau, J.-M.; Bitsch, F.; Bourgier, E.; Geiser, M.; Hemmig, R.; Kroemer, M.;
Lehmann, S.; Ramage, P.; Rieffel, S.; Strauss, A.; Green, J. R.; Jahnke, W.
ChemMedChem 2006, 1, 267.
34. Kavanagh, K. L.; Guo, K.; Dunford, J. E.; Wu, X.; Knapp, S.; Ebetino, F. H.; Rogers,
M. J.; Russell, R. G. G.; Oppermann, U. Proc. Natl. Acad. Sci. U.S.A. 2006, 103,
7829.
35. Morgan, G. J.; Davies, F. E.; Gregory, W. M.; Cocks, K.; Bell, S. E.; Szubert, A. J.;
Navarro-Coy, N.; Drayson, M. T.; Owen, R. G.; Feyler, S.; Ashcroft, A. J.; Ross, F.;
Byrne, J.; Roddie, H.; Rudin, C.; Cook, G.; Jackson, G. H.; Child, J. A. Lancet 2010,
376, 1989.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2013.02.006.
References and notes
1. Marugan, J. J.; Zheng, W.; Southall, N.; Huang, W.; McCoy, J. G.; Titus, S.;
Patnaik, S. WO 2012/044993.
2. Brewster, W. K.; Demeter, D. A.; Erickson, W. R.; Klittich, C. J. R.; Lowe, C. T.;
Rieder, B. J.; Nugent, J. S.; Yerkes, C. N.; Zhu, Y. WO 2007/046809.
3. Brewster, W. K.; Klittich, C. J. R.; Balko, T.W.; Breax, N. T.; Erickson, W. R.;
Hunter, J. E.; Lowe, C. T.; Ricks, M. J.; Siddall, T. L.; Yerkes, C. N.; Zhu, Y. U.S.
Patent Application 2006/0089370.
36. Feng, X. J.; Wu, P. L.; Tam, H. L.; Li, K. F.; Wong, M. S.; Cheah, K. W. Chem. Eur. J.
2009, 15, 11681.
4. Tani, N.; Rahnasto-Rilla, M.; Wittekindt, C.; Salminen, K. A.; Ritvanen, A.;
Ollakka, R.; Koskiranta, J.; Raunio, H.; Juvonen, R. O. Eur. J. Med. Chem. 2012, 47,
270.
5. Babu, Y.; Chand, P.; Wu, M.; Kotian, P. L.; Kumar, V. S.; Lin, T.-H.; El-Kattan, Y.;
Ghosh, A. K. WO 2006/050161.
37. Ye, X.-S.; Wong, H. N. C. J. Org. Chem. 1997, 62, 1940.
38. Bair, J. S.; Palchaudhuri, R.; Hergenrother, P. J. J. Am. Chem. Soc. 2010, 132, 5469.
39. Wilhelms, W. Synthesis 2006, 10, 1578.
40. Wiemer, A. J.; Tong, H.; Swanson, K. M.; Hohl, R. J. Biochem. Biophys. Res.
Commun. 2007, 353, 921.
Please cite this article in press as: Leung, C.-Y.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.02.006