Synthesis and biological evaluation of pyrido[3′,2′:4,5]furo[3,2-d]pyrimidine derivatives as novel PI3 kinase p110α inhibitors

Article abstract of DOI:10.1016/j.bmcl.2007.02.032

4-Morpholin-4-ylpyrido[3′,2′:4,5]thieno[3,2-d]pyrimidine 2a was discovered in our chemical library as a novel p110α inhibitor with an IC₅₀ of 1.4 μM. By structural modification of 2a, the 2-aryl-4-morpholinopyrido[3′,2′:4,5]furo[3,2-d]pyrimidin

Full text of DOI:10.1016/j.bmcl.2007.02.032

Bioorganic & Medicinal Chemistry Letters 17 (2007) 2438–2442
Synthesis and biological evaluation of pyrido[30,20:4,5]furo
[3,2-d]pyrimidine derivatives as novel PI3 kinase p110a inhibitors
Masahiko Hayakawa,a,* Hiroyuki Kaizawa,a Hiroyuki Moritomo,a Tomonobu Koizumi,a
Takahide Ohishi,a Mayumi Yamano,a Minoru Okada,a Mitsuaki Ohta,a
Shin-ichi Tsukamoto,a Florence I. Raynaud,b Paul Workman,b
Michael D. Waterfieldc and Peter Parkerd
aDrug Discovery Research, Astellas Pharma Inc., 5-2-3 Tokodai, Tsukuba, Ibaraki 300-2698, Japan
bCancer Research UK Centre for Cancer Therapeutics, The Institute of Cancer Research, Sutton, Surrey SN2 5NG, United Kingdom
cLudwig Institute for Cancer Research, 91 Riding House Street, London W1W 7BS, United Kingdom
dCancer Research UK London Research Institute, 44 Lincoln’s Inn Fields, London WC2A 3PX, United Kingdom
Received 30 November 2006; revised 7 February 2007; accepted 13 February 2007
Available online 15 February 2007
Abstract—4-Morpholin-4-ylpyrido[30,20:4,5]thieno[3,2-d]pyrimidine 2a was discovered in our chemical library as a novel p110a
inhibitor with an IC50 of 1.4 lM. By structural modification of 2a, the 2-aryl-4-morpholinopyrido[30,20:4,5]furo[3,2-d]pyrimidine
derivative 10e was discovered as a p110a inhibitor with approximately 400-fold greater potency than 2a. Evaluation of isoform selec-
tivity showed that 10e is a potent inhibitor of p110b. Furthermore, 10e showed anti-proliferative activity in various cell lines, includ-
ing multi-drug resistant MCF7/ADR-res cells, and was effective against HeLa human cervical tumor xenografts in nude mice.
Ó 2007 Elsevier Ltd. All rights reserved.
O
O
Phosphoinositide 3-kinase (PI3K) is an enzyme that cat-
alyzes phosphorylation of the 3-hydroxyl position of
phosphatidylinositides (PIs) and plays a crucial role in
mitogenic signal transduction.1–4 PI3K is negatively reg-
ulated by the lipid-phosphatase PTEN, which is one of
the most commonly mutated proteins in human can-
cers,5–7 and therefore PI3K inhibitors are considered
to be potential anti-cancer agents (Fig. 1).
O
O
MeO
O
N
O
O
O
H
O
O
Wortmannin
LY294002
Among the various subtypes of PI3K identified to
date,8–11 class Ia PI3Ks (p110a, p110b, and p110d) are
known to play critical roles in cell growth and survival.12
The PIK3CA gene that encodes p110a is amplified and
overexpressed in ovarian and other cancers,13,14 and is
mutated in a variety of cancers.15–18 Therefore, inhibi-
tors of class Ia PI3Ks, and particularly PI3K p110a,
are likely to be useful in cancer treatment.
O
O
N
S
N
N
N
N
S
N
OH
N
1
2a
Several non-selective PI3K inhibitors have been identi-
fied, including wortmannin and LY294002,19–21 but
Figure 1. Structures of PI3K inhibitors.
few isoform-specific PI3K inhibitors are available.22,23
We have reported that the thieno[3,2-d]pyrimidine deriv-
ative 1 is a highly potent and selective p110a inhibitor,
but it is not effective in vivo because of a poor
Keywords: PI3 kinase; p110a; Inhibitor; Anti-cancer agent.
Corresponding author. Tel.: +81 29 865 7124; fax: +81 029 847
8313; e-mail: masahiko.hayakawa@jp.astellas.com
*
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.02.032

M. Hayakawa et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2438–2442
2439
pharmacokinetic profile due to a short half-life of less
than 10 min.24,25 In screening our chemical library, we
identified 2a as another lead compound with structural
similarity to 1. Through structural modification of 2a,
the novel pyrido[30,20:4,5]furo[3,2-d]pyrimidine 10e was
identified as a potent p110a inhibitor that is also effec-
tive in HeLa xenografts in mice. Although biological
evaluation of 10e has been recently reported after our
publication of the patent,26–28 the generation of 10e
and SAR of its derivatives has not yet been described.
Herein, we report the synthesis, SAR, and biological
data for a series of pyrido[30,20:4,5]furo[3,2-d]pyrimidine
derivatives as novel PI3K p110a inhibitors.
described.24 Acylation of 5 with the appropriate aryl
acid chloride, ester hydrolysis, chlorination with SOCl2,
and subsequent treatment with NH4OH afforded the
series of amides 8. Cyclization of 8 under previously re-
ported conditions using MeOH as a solvent24 provided 9
in low yield due to cleavage of the amide linkage of 8,
while reaction in 2-PrOH instead of MeOH gave cyc-
lized 9 in better yield. Chlorination of 9 with phospho-
rus oxychloride and substitution with morpholine gave
the desired compounds 10a–b and 10h. The amino deriv-
ative 10g was synthesized from the corresponding nitro
derivative via a reduction with iron. Demethylation of
9a with HBr, acetylation, chlorination, and treatment
with morpholine afforded compounds 10d–f. Alkylation
of the phenol derivative 10e gave 10i–k. The acetic acid
derivative 10l was prepared from the corresponding es-
ter derivative by hydrolysis under basic conditions.
The lead compound 2a was synthesized according to the
literature29 and its furan analogue, 2b, was prepared as
shown in Scheme 1. Treatment of 2-chloronicotinonitri-
le 3 with 4 in the presence of DBU afforded a bicyclic
acid ester 5a, which was treated with formamide to give
a pyrimidone 6. Chlorination of 6 with phosphorus oxy-
chloride followed by substitution with morpholine gave
the desired product 2b. The synthesis of compounds 10,
each of which has an aryl group on the pyrimidine ring,
is illustrated in Scheme 2. Synthetic methods for the
derivatives were generally analogous to those previously
Inhibition of p110a was determined by a scintillation
proximity assay (SPA), as previously reported,24,25
which gave similar results to those obtained by a con-
ventional TLC method.21 The lead compound 2a had
an IC50 of 1.4 lM for p110a in the SPA assay. In this
assay, LY294002 inhibited p110a with an IC50 of
0.63 lM, showing approximately 2-fold greater potency
O
O
N
CO2Et
NH2
O
N
Cl
a
b
O
c
O
NH
N
N
N
N
N
CN
N
5a
6
2b
3
Scheme 1. Reagents and conditions: (a) HOCH2CO2Et 4, DBU, EtOH, reflux; (b) formamide, reflux; (c) i) POCl3, reflux; ii) morpholine, toluene,
reflux.
CO2Et
NH2
CO2Et
NH
CONH2
NH
Y
Y
Y
a
b
N
N
N
O
O
R
R
5:Y = O or S
8
7
O
O
N
N
Y
d or d, e
c
NH
N
Y
N
N
R
N
R
O
9
10a, b, g, h
O
O
O
N
N
N
f, d
N
g or h
O
O
NH
N
N
N
N
N
N
OMe
OH
OR'
9a
10d-f
10i-l
Scheme 2. Reagents and conditions: (a) ArCOCl, Et3N or pyridine, THF or CHCl3; (b) i) 1 N NaOH, EtOH; ii) SOCl2, reflux; iii) NH4OH, THF; (c)
2 N NaOH, MeOH or 2-PrOH, reflux; (d) i) POCl3, D; ii) morpholine, toluene or neat, reflux; (e) Fe, NH4Cl, aqueous EtOH, reflux; (f) i) HBr,
AcOH, reflux; ii) Ac2O, AcONa, reflux; (g) R0-Cl, K2CO3, DMF; (h) i) BrCH2CO2Et, K2CO3, DMF; ii) 1 N NaOH, MeOH, THF.

2440
M. Hayakawa et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2438–2442
Table 2. Inhibition of p110a by pyridofuropyrimidine derivatives
than 2a. Since the morpholino group is important for
p110a inhibitory activity in derivatives of 1,24 the mor-
pholino group in 2a was retained and other groups in
2a were modified with the goal of obtaining p110a inhib-
itors with greater potency.
O
N
O
N
N
N
R
First, modifications on the tricyclic ring of 2a were
investigated (Table 1). Replacement of sulfur with oxy-
gen at Y in 2a resulted in a 3-fold increase in p110a
inhibitory activity (2b: IC50 = 0.56 lM), whereas the
pyrazole derivative 2c (Y = NH) was inactive against
p110a. Introduction of a phenyl group at R of the thio-
phene derivative 2a retained similar p110a inhibitory
activity (10a: IC50 = 1.7 lM), whereas the furan deriva-
tive 10b with a phenyl ring at C2 was about 10-fold more
potent than 2a (10b: IC50 = 0.16 lM). The benzothieno-
pyrimidine derivative 2d (X = C) showed comparable
potency to 2a; however, introduction of a benzene ring
at C2 (10c) eliminated the p110a inhibitory activity.
d
Compound
R
IC50 (lM)
p110a
A375
10ba
10da
10eb
10fb
10gc
10hb
10ic
10jc
10kc
10lb
H
2-OH
0.16
0.40
NT
>30
3-OH
4-OH
0.0036
0.026
0.14
0.33
>30
3-NH2
3-OMe
3.87
15.78
9.67
12.32
11.76
>30
0.10
0.17
3-(Piperidinoethoxy)
3-(Morpholinoethoxy)
3-(Thiomorpholinoethoxy)
3-OCH2CO2H
0.016
0.079
0.024
NT, not tested.
a Free base.
b HCl salt.
The 3-hydroxy group on the benzene ring of 1 was pre-
viously found to be important for p110a inhibitor activ-
ity.24 Considering the structural similarity between 1
and 10b, introduction of a hydroxy group on the ben-
zene ring of 10b was investigated (Table 2). The 2-hy-
droxy derivative 10d showed about a 3-fold decrease
in potency compared with 10b. As expected, the 3-hy-
droxy derivative 10e and the 4-hydroxy derivative 10f
were approximately 45- and 6-fold more potent p110a
inhibitors compared with the unsubstituted 10b (10e,
10f: IC50 = 3.6, 26 nM, respectively). Since the hydroxy
group at the 3-position on the benzene ring of 10e was
especially effective in producing p110a inhibitory activ-
ity, other hydrophilic substituents at C3 on the benzene
ring were introduced, based on the possibility of a
hydrophilic binding pocket in the binding site. The
3-amino derivative 10g showed almost the same p110a
activity compared with 10b. Introduction of a 3-meth-
oxy group at C3 on the benzene ring of 10b retained
the same p110a inhibitory activity, but most compounds
with ether substituents at this position showed increased
c 2HCl salt.
d IC50 values represent means of at least two separate determinations
with typical variations of less than 20% for the p110a enzyme and
A375 cell proliferation assays.
activity. Among these compounds, the morpholinoeth-
oxy derivative 10j and the carboxylic acid derivative
10l were potent p110a inhibitors, with IC50 values of
16 and 24 nM, respectively. Preliminary studies indi-
cated that the inhibitors are ATP competitive but fur-
ther work is required to investigate the inhibition
kinetics in more detail.
The inhibitory activities of these derivatives were evalu-
ated against serum-induced proliferation of A375 hu-
man melanoma cells. The 3-hydroxy derivative 10e
showed a potent anti-proliferative effect in vitro with
an IC50 of 0.33 lM, whereas compounds 10f, 10j, 10k,
and 10l, which had relatively potent p110a inhibitory
activities in the enzyme assay, did not show cellular
activity; it can be speculated that this may be due to
poor permeability through the cell membrane.
Table 1. Inhibition of p110a by tricyclic derivatives
O
N
Further evaluation of compound 10e was performed,
since this was the most potent p110a inhibitor in the ser-
ies and was effective as an anti-proliferative agent
in vitro. To examine selectivity for p110a, the inhibitory
activity of 10e was evaluated against other PI3K iso-
forms. As shown in Table 3, 10e was also a potent inhib-
itor of p110b (class Ia), which is also a potential target
for cancer treatment.12 Although 10e showed excellent
selectivity for p110a over p110c (class Ib), the selectivity
for p110a over PI3K C2b (class II) was not very high. It
is of interest that the thienopyrimidine derivative 1,
which is structurally similar to 10e, is a more isoform-
selective p110a inhibitor than 10e. Concerning selectiv-
ity against protein kinases, 10e was inactive at 100 lM
against protein kinases such as KDR, PKA, PKCa,
and cyclin E/CDK2. Recent publications reported that
10e also inhibited other kinases such as DNA-PK or
mTOR.27,28 Although we have not yet determined the
Y
N
X
N
R
c
Compound
X
Y
R
IC50 (lM)
p110a
LY294002a
2aa
2ba
2ca
0.63
1.4
N
N
N
N
N
C
S
H
O
H
0.56
>30
1.7
NH
S
H
10aa
10ba
2db
Ph
Ph
H
O
S
0.16
2.4
10ca
C
S
Ph
>30
a Free base.
b HCl salt.
c IC50 values represent means of at least two separate determinations
with typical variations of less than 20%.

M. Hayakawa et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2438–2442
Table 3. Selectivity of 10e against PI3Ks and protein kinases
2441
c
Compound
IC50 (lM)
p110a
p110b
p110c
PI3K C2b
KDR
PKA
PKCa
Cyclin E/CDK2
LY294002a
10eb
1b
0.63
0.0036
0.0025
0.34
0.0030
0.016
1.6
2.1
0.010
0.22
NT
>100
91
NT
>100
3.4
NT
>100
466
NT
>100
28
0.25
0.66
NT, not tested.
a Free base.
b HCl salt.
c IC50 values represent means of at least two separate determinations with typical variations of less than 20%.
Table 4. Inhibition of human tumor cell proliferation in vitro by 10e
b
Compound
IC50 (lM)
A375 (Melanoma)
0.33
HeLa (Cervix)
0.54
A549 (Lung)
0.54
MCF7 (Breast)
0.18
MCF7 ADR-res (Breast)
0.13
10ea
a HCl salt.
b IC50 values represent means of at least two separate determinations with typical variations of less than 20%.
inhibitory activity of 10e and its derivatives against
mTOR and DNA-PK, the inhibitory potencies of the
compounds in A375 melanoma cell proliferation assay
were well correlated with those in a PKB phosphoryla-
tion assay (data not shown). This observation suggested
that the tumor suppressive actions of 10e and its deriv-
atives may depend mainly on PI3K inhibition. The
involvement of mTOR and DNA-PK inhibition should
be further investigated.
el when dosed intraperitoneally at 100 mg/kg daily for 2
weeks, and did not cause any associated weight loss
(Fig. 2).
In summary, structural modification of 4-morpholino-
pyrido[30,20:4,5]thieno[3,2-d]pyrimidine 2a led to the dis-
covery of 10e as a p110a inhibitor of approximately 400-
fold greater potency than 2a. Compound 10e is also a
potent inhibitor of p110b and showed selectivity over
several tested protein kinases. Furthermore, 10e exhib-
ited anti-tumor activity against the MCF7 ADR-res tu-
mor cell line, which shows multi-drug resistance to anti-
cancer drugs, and was effective in a HeLa human cervi-
cal cancer xenograft model without causing weight loss.
Evaluation of the effects of 10e in several human tumor
cell lines showed that 10e exerted anti-proliferative
activity at submicromolar concentrations (Table 4).
Notably, 10e was active in MCF7 ADR-res human
breast cancer cells, which display a high level of
resistance to doxorubicin and several other anti-cancer
agents due to overexpression of P-glycoprotein.30,31 Fi-
nally, the activity of 10e was evaluated in vivo using a
HeLa human cervical tumor xenograft model in mice.
The half-life of 10e, as measured by HPLC/MS/MS,
was 4.1 h, when administered intraperitoneally. 10e sig-
nificantly suppressed tumor growth by 45% in this mod-
Acknowledgments
We thank Drs. K. Matsuda and N. Taniguchi for their
advice and members of the Division of Analytical Re-
search for performing instrumental analyses. We also
thank Sonia Alex and Angela Hayes for help with the
HPLC/MS/MS. This work was funded in part by Cancer
Research UK [CUK] Programme Grant C308/A2187
and Paul Workman is a Cancer Research UK Life
Fellow.
500
Vehicle
10e, 100 mg/kg i.p.
400
300
200
100
0
References and notes
1. Leevers, S. J.; Vanhaesebroeck, B.; Waterfield, M. D.
Curr. Opin. Cell Biol. 1999, 11, 219.
2. Lawlor, M. A.; Alessi, D. R. J. Cell Sci. 2001, 114, 2903.
3. Vanhaesebroeck, B.; Leevers, S. J.; Ahmadi, K.; Timms,
J.; Katso, R.; Driscoll, P. C.; Woscholski, R.; Parker, P. J.;
Waterfield, M. D. Annu. Rev. Biochem. 2001, 70, 535.
4. Cantley, L. C. Science 2002, 296, 1655.
0
7
14
Time (days)
5. Hopkins, K. Science 1998, 282, 1027.
6. Maehama, T.; Dixon, J. E. Trends Cell Biol. 1999, 9, 125.
7. Simpson, L.; Parsons, R. Exp. Cell Res. 2001, 264, 29.
8. Domin, J.; Waterfield, M. D. FEBS Lett. 1997, 410, 91.
9. Vanhaesebroeck, B.; Waterfield, M. D. Exp. Cell Res.
1999, 253, 239.
Figure 2. Effect of compound 10e on the growth of HeLa human
cervical tumor xenografts. Compound 10e (100 mg/kg) suspended in
20% hydroxypropyl-b-cyclodextrin/saline was administered intraperi-
toneally daily for 2 weeks to nude mice carrying a subcutaneous HeLa
xenograft. Error bars show SE.

2442
M. Hayakawa et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2438–2442
10. Vanhaesebroeck, B.; Leevers, S. J.; Panayotou, G.;
Waterfield, M. D. Trends Biochem. Sci. 1997, 22, 267.
11. Fruman, D. A.; Meyers, R. E.; Cantley, L. C. Annu. Rev.
Biochem. 1998, 67, 481.
21. Vlahos, C. J.; Matter, W. F.; Hui, K. Y.; Brown, R. F.
J. Biol. Chem. 1994, 269, 5241.
22. Ward, S.; Sotsios, Y.; Dowden, J.; Bruce, I.; Finan, P.
Chem. Biol. 2003, 10, 207.
12. Vivanco, I.; Sawyers, C. L. Nat. Rev. Cancer 2002, 2, 489.
13. Shayesteh, L.; Lu, Y.; Kuo, W.-L.; Baldocchi, R.; God-
frey, T.; Collins, C.; Pinkel, D.; Powell, B.; Mills, G. B.;
Gray, J. W. Nat. Genet. 1999, 21, 99.
14. Ma, Y.-Y.; Wei, S.-J.; Lin, Y.-C.; Lung, J.-C.; Chang, T.-
C.; Whang-Peng, J.; Liu, J. M.; Yang, D.-M.; Yang, W.
K.; Schen, C.-Y. Oncogene 2000, 19, 2739.
15. Samuels, Y.; Wang, Z.; Bardelli, A.; Silliman, N.; Ptak, J.;
Szabo, S.; Yan, H.; Gazdar, A.; Powell, S. M.; Riggins, G.
J.; Willson, J. K.; Markowitz, S.; Kinzler, K. W.;
Vogelstein, B.; Velculescu, V. E. Science 2004, 304, 554.
16. Campbell, I. G.; Russell, S. E.; Choong, D. Y.; Mont-
gomery, K. G.; Ciavarella, M. L.; Hooi, C. S.; Cristiano,
B. E.; Pearson, R. B.; Phillips, W. A. Cancer Res. 2004, 64,
7678.
23. Ward, S. G.; Finan, P. Curr. Opin. Pharmacol. 2003, 3,
426.
24. Hayakawa, M.; Kaizawa, H.; Moritomo, H.; Koizumi, T.;
Ohishi, T.; Okada, M.; Ohta, M.; Tsukamoto, S.; Parker,
P.; Workman, P.; Waterfield, M. Bioorg. Med. Chem.
2006, 14, 6847.
25. Hayakawa, M.; Kaizawa, H.; Kawaguchi, K.; Ishikawa,
N.; Koizumi, T.; Ohishi, T.; Yamano, M.; Okada, M.;
Ohta, M.; Tsukamoto, S.; Raynaud, F. I.; Waterfield, M.
D.; Parker, P.; Workman, P. Bioorg. Med. Chem. 2007, 15,
403.
26. Hayakawa, M.; Kaizawa, H.; Moritomo, H; Kawaguchi,
K; Koizumi, T.; Yamano, M.; Matsuda, K.; Okada, M.;
Ohta, M. U.S. Patent 6,608,053 B2, 2003.
27. Knight, Z. A.; Gonzalez, B.; Feldman, M. E.; Zunder, E.
R.; Goldenberg, D. D.; Williams, O.; Loewith, R.; Stokoe,
D.; Balla, A.; Toth, B.; Balla, T.; Weiss, W. A.; Williams,
R. L.; Shokat, K. M. Cell 2006, 125, 733.
28. Fan, Q.-W.; Knight, Z. A.; Goldenberg, D. D.; Yu, W.;
Mostov, K. E.; Stokoe, D.; Shokat, K. M.; Weiss, W. A.
Cancer Cell 2006, 9, 341.
17. Kang, S.; Bader, A. G.; Vogt, P. K. Proc. Natl. Acad. Sci.
U.S.A. 2005, 102, 802.
18. Parsons, D. W.; Wang, T.-L.; Samuels, Y.; Bardelli, A.;
Cummins, J. M.; DeLong, L.; Silliman, N.; Ptak, J.;
Szabo, S.; Willson, J. K.; Markowitz, S.; Kinzler, K. W.;
Vogelstein, B.; Lengauer, C.; Velculescu, V. E. Nature
2005, 436, 792.
19. Schultz, R. M.; Merriman, R. L.; Andis, S. L.; Bonjouk-
lian, R.; Grindey, G. B.; Rutherford, P. G.; Gallegos, A.;
Massey, K.; Powis, G. Anticancer Res. 1995, 15, 1135.
20. Wymann, M. P.; Bulgarelli-Leva, G.; Zvelebil, M. J.;
Pirola, L.; Vanhaesebroeck, B.; Waterfield, M. D.; Pan-
ayotou, G. Mol. Cell Biol. 1996, 16, 1722.
29. Schneller, S. W.; Clough, F. W. J. Heterocycl. Chem. 1974,
11, 975.
30. Fairchild, C. R.; Ivy, S. P.; Kao-Shan, C.-S.; Whang-Peng,
J.; Rosen, N.; Israel, M. A.; Melera, P. W.; Cowan, K. H.;
Goldsmith, M. E. Cancer Res. 1987, 47, 5141.
31. Scudiero, D. A.; Monks, A.; Sausville, E. A. J. Natl.
Cancer Inst. 1998, 90, 862.