Synthesis, SAR, and X-ray structure of tricyclic compounds as potent FBPase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2009.08.081

With the aim of discovering a novel class of fructose-1,6-bisphosphatase (FBPase) inhibitors, a series of compounds based on tricyclic scaffolds was synthesized. Extensive SAR studies led to the finding of 8l with an IC₅₀ value of 0.013 μM against human FBPase. An X-ray crystallographic study revealed that 8l bound at AMP binding sites of human liver FBPase with hydrogen bonding interactions similar to AMP.

Full text of DOI:10.1016/j.bmcl.2009.08.081

Bioorganic & Medicinal Chemistry Letters 19 (2009) 5909–5912
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis, SAR, and X-ray structure of tricyclic compounds as potent
FBPase inhibitors
Tomoharu Tsukada a, Mizuki Takahashi b, Toshiyasu Takemoto a, Osamu Kanno a, Takahiro Yamane a,
Sayako Kawamura b, Takahide Nishi a,
*
a Medicinal Chemistry Research Laboratories I, Daiichi Sankyo Co., Ltd, 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
b Exploratory Research Laboratories I, Daiichi Sankyo Co., Ltd, 1-16-13 Kitakasai, Edogawa-ku, Tokyo 134-8630, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
With the aim of discovering a novel class of fructose-1,6-bisphosphatase (FBPase) inhibitors, a series of
Received 16 June 2009
Revised 17 August 2009
Accepted 18 August 2009
Available online 27 August 2009
compounds based on tricyclic scaffolds was synthesized. Extensive SAR studies led to the finding of 8l
with an IC50 value of 0.013 lM against human FBPase. An X-ray crystallographic study revealed that 8l
bound at AMP binding sites of human liver FBPase with hydrogen bonding interactions similar to AMP.
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Fructose-1,6-bisphosphatase (FBPase)
Gluconeogenesis
AMP
Type 2 diabetes mellitus (T2DM), which accounts for more than
90% of all diabetes, is characterized by high levels of glucose in the
plasma. The global incidence of this disease is predicted to rise to
more than 366 million by the year 2030.1 T2DM usually leads to
complications such as retinopathy, nephropathy, and neuropathy.
Clinical studies have suggested that fasting hyperglycemia in
T2DM is associated with excessive glucose production through glu-
coneogenesis.2 Thus, the inhibition of gluconeogenesis is a poten-
tially useful approach to reducing the increased blood glucose
levels in patients with T2DM.
Fructose-1,6-bisphosphatase (FBPase), which is predominantly
expressed in the liver and the kidney and catalyzes the hydrolysis
of fructose-1,6-bisphosphate (F1,6BP) to fructose-6-phosphate
(F6P), is one of the rate-limiting enzymes of gluconeogenesis.3
FBPase is a homotetramer and exists in two distinct conforma-
tional states, an active R-state and an inactive T-state. F1,6BP and
F6P in combination with metal cations stabilize the active R-state,
while adenosine 50-monophosphate (AMP, 1) and substrate ana-
logue fructose-2,6-bisphosphate (F2,6BP) synergistically stabilize
the inactive T-state.4 AMP and/or F2,6BP are natural inhibitors of
FBPase which bind to the allosteric site and the substrate-binding
site, respectively, and control blood glucose levels by regulating
the activity of FBPase. FBPase inhibitors would lower blood glucose
levels by reducing hepatic glucose output and are expected to be a
novel class of drugs for the treatment of T2DM.
Several classes of small-molecule inhibitors of FBPase have been
reported. These inhibitors can be structurally classified into two
groups; non-phosphorus-based inhibitors and phosphorus-based
inhibitors. In the former group, several chemotypes including ani-
linoquinazoline,5 indole dicarboxylic acid,6 piperazinedione,7 and
benzoxazole benzenesulfonamide8 were reported, although they
showed only modest inhibitory potency against FBPase. Also in this
group, bissulfonylurea9 was described as a potent FBPase inhibitor
with novel interaction. In the latter group, in contrast, AMP mi-
metic MB05032 (2) exhibited high inhibitory activity. A prodrug
of MB05032 (CS-917, 3) lowered blood glucose levels in animal
models and was entered into clinical development (Fig. 1).10
With the intention of developing a novel class of FBPase inhib-
itors, we turned our attention to phosphorus-based AMP mimetics
in recognition of their in vitro and in vivo potencies. From the bind-
ing mode of AMP and FBPase, the hydrogen bonding interactions of
the NH2 and PO3H2 groups of AMP seemed to play an important
role in realizing high affinity.4a Based on this structural informa-
tion, we focused our attention on placing the two groups at the
appropriate position to obtain such high affinity. AMP mimetic
MB05032 has a rotatable biheteroaryl moiety; we expected that
increasing the structural rigidity of the compound would be useful
to fix the NH2 and PO3H2 groups in the appropriate binding orien-
tation. This led us to design rigid tricyclic scaffolds, and the three
kinds of the scaffolds were designed by varying the central ring
size from a five- to a seven-membered ring (Fig. 2). In order to facil-
itate changing the positions of the PO3H2 group, a benzene ring in-
stead of a furan ring was introduced to the tricyclic scaffolds.
* Corresponding author. Tel.: +81 3 3492 3131; fax: +81 3 5436 8563.
E-mail address: nishi.takahide.xw@daiichisankyo.co.jp (T. Nishi).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.08.081

5910
T. Tsukada et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5909–5912
tosylate.11 Bromination of 5–7 followed by cyclization with thio-
urea and subsequent hydrolysis afforded the final compounds
8a–u.
The inhibitory activities against human FBPase of the tricyclic
derivatives are summarized in Table 1. Initially, to optimize the po-
sition of the PO3H2 group, a variety of benzene phosphonic acid
derivatives were prepared (8a–g). Most of these compounds
showed low activity, but only 8g, possessing a relatively flexible
scaffold with a seven-membered central ring, showed moderate
NH2
N
N
N
O
P
HO
HO
O
N
O
HO
OH
1 (AMP)
activity (IC50 = 0.169 lM). This result suggested that some degree
NH2
S
NH2
S
of structural flexibility was beneficial in improving inhibitory
activity and prompted us to introduce a spacer group of 1–2 atoms
in length connecting the PO3H2 group with the tricyclic scaffolds.
Due to synthetic feasibility, linkers such as –O– and –CH2O– were
selected for further investigation (8h–u). Introducing the spacer
groups showed a tendency to increase activity, and several com-
pounds bearing –O– linker (i.e., phosphate compounds) exhibited
enhanced activity (8j, 8l, and 8n, IC50 = 0.070, 0.013, and
O
P
O
P
NH
N
N
HO
HO
EtO
O
O
N
H
O
EtO
O
2
(MB05032)
3 (CS-917)
Figure 1. Structures of AMP and known FBPase inhibitors.
0.022 lM, respectively). In particular, 8l exhibited the highest po-
tency, almost equal to that of MB05032.
In order to obtain insights into the binding mode of this novel
series of tricyclic inhibitors, an X-ray crystal structure of human li-
ver FBPase in complex with 8l was determined (Fig. 3).12 As ex-
pected, 8l binds at four AMP binding sites of FBPase tetramer
and the 8l-bound FBPase exists in the inactive T-state. The phos-
phate group of 8l occupies a similar position to that of the phos-
phate group of AMP. Similarly, the amino group of 8l is placed in
the same position as the 6NH2 of AMP. The phosphate group inter-
acts with the backbone nitrogens (NH) of Gly26, Thr27, Gly28,
Glu29, Leu30, Thr31 and the side chains of Thr27, Lys112, Tyr113
and Arg140, and some of these interactions are formed via water
molecules. A water molecule placed between phosphate and the
aromatic N atom also contributes to the hydrogen-bonding net-
work among the phosphate group and FBPase. The amino group
of 8l makes hydrogen bonds with the carbonyl oxygen of Val17
O
NH2
(HO)2
P
N
S
(
)
n
n = 1, 2, 3
Figure 2. General structure of new FBPase inhibitors based on tricyclic scaffolds.
A series of tricyclic derivatives was efficiently synthesized
according to Scheme 1. The bicyclic phenols 4a–g were converted
to diethyl phosphonate 5a–g via triflation and Pd-catalyzed phos-
phonylation. Diethyl phosphate 6a–g were obtained by direct
phosphorylation of 4a–g, and diethyl phosphonate 7a–g were ac-
quired by condensation of 4a–g with diethoxyphosphorylmethyl
Table 1
FBPase inhibitory activity of compounds 8a–u
O
P
O
P
(EtO)2
O
HO
O
(EtO)2
O
O
c
a, b
O
NH2
(HO)2P
X
4
N
(
)
(
)
(
)
n
n
n
5
6
S
5a-g
4a-g
6a-g
(
)
n
d
a
O
(EtO)2
Compound
Position
n
X
IC50 (lM)
P
MB05032
8a
8b
8c
8d
8e
8f
8g
8h
8i
8j
8k
8l
8m
8n
8o
8p
8q
8r
8s
8t
8u
—
6
5
4
6
5
4
5
6
5
4
6
5
4
5
6
5
4
6
5
4
5
—
1
1
1
2
2
2
3
1
1
1
2
2
2
3
1
1
1
2
2
2
3
—
0.010
91.9
18.7
28.4
354
9.32
354
0.169
146
O
O
Nothing
Nothing
Nothing
Nothing
Nothing
Nothing
Nothing
O
O
O
O
O
(
)
n
7a-g
O
NH2
(HO)2P
X
4
N
e, f, g
307
5
0.070
112
0.013
335
0.022
112
S
5a-g, 6a-g, 7a-g
6
(
)
n
O
O
8a-u
position = 4, 5, 6
n = 1, 2, 3
X = nothing, O, CH2O
CH2O
CH2O
CH2O
CH2O
CH2O
CH2O
CH2O
112
0.124
16.4
19.1
21.5
41.7
Scheme 1. Synthesis of compounds 8a–u. Reagents and conditions: (a) 2,6-lutidine,
4-dimethylaminopyridine, Tf2O, CH2Cl2, 46–97%; (b) Pd(PPh3)4, (EtO)2P(O)H, Et3N,
DMF, 60 °C, 91–98%; (c) (EtO)2P(O)H, Et3N, CCl4, 87–95%; (d) (EtO)2P(O)CH2OTs,
K2CO3, DMF, 80 °C, 28–72%; (e) PhNMe3Br3, CH2Cl2; (f) thiourea, NaOAc, EtOH,
reflux, 14–57% over two steps; (g) TMSBr, CH2Cl2, 26–91%.
a
Inhibition of human liver FBPase.

T. Tsukada et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5909–5912
5911
O
O
O
O
O
a, b, c
HO
d, e
HO
4e
9
10
O
O
O
f, g
h
(EtO)2P
O
(EtO)2P
O
11
12
O
F
F
i, h
(EtO)2P
O
11
13
NH2
S
N
j, k, l
X
(HO)2P
O
12, 13
14a,b
X = CH2, CF2
Scheme 2. Synthesis of compounds 14a,b. Reagents and conditions: (a) 2,6-
lutidine, 4-dimethylaminopyridine, Tf2O, CH2Cl2, 98%; (b) ethylene glycol, TsOH,
toluene, reflux, 76%; (c) PdCl2(dppf), vinylmagnesium bromide, THF; (d) K2OsO4,
NMO, NaHCO3, KIO4, tBuOH, THF, H2O; (e) NaBH4, MeOH, 57% over three steps; (f)
PPh3, CBr4, CH2Cl2, 0 °C, 84%; (g) (EtO)3P, 150 °C, 51%; (h) PPTS, acetone, H2O, reflux,
85–88%; (i) NaHMDS, N-fluorobenzenesulfonimide, THF, ꢀ78 °C, 80%; (j)
PhNMe3Br3, CH2Cl2; (k) thiourea, NaOAc, EtOH, reflux, 44–64% over two steps; (l)
TMSBr, CH2Cl2, 81–93%.
through dihydroxylation, oxidative cleavage, and subsequent
reduction. Diethyl phosphonate 11 was obtained by bromination
of 10 and subsequent Arbuzov reaction. Deketalization of 11 pro-
vided ketone 12, and difluorination of 11 followed by deketaliza-
tion led to ketone 13. Bromination of 12, 13 followed by
cyclization with thiourea and subsequent hydrolysis afforded the
final compounds 14a,b.
Figure 3. X-ray crystal structure of human liver FBPase in complex with 8l. (a)
Overlay of 8l and AMP. (b) Details of the interaction of 8l with FBPase. The residues
that interact with 8l are shown as a stick model.
(Val17CO) and Thr31Oc. These hydrogen bonds may contribute to
causing the quaternary conformational change of FBPase towards
the inactive T-state.4b,c This complex structure suggests that the
tricyclic scaffold of 8l is beneficial in retaining the proper distance
and directions between the phosphate group and the amino group.
In addition, this planar tricyclic scaffold can fit well to the pocket of
the AMP-binding site, resulting in relatively high affinity and
therefore high inhibitory activity of 8l.
The inhibitory activities against human FBPase of these analogs
are summarized in Table 2. Methylene analog 14a displayed re-
duced activity (IC50 = 0.840
methylene analog 14b showed similar potency (IC50 = 0.047
8l. Interestingly, the second pKa of benzylic -difluorophosphon-
lM) relative to 8l. In contrast, difluoro-
lM) to
a,a
ic acid (5.71) is more like that of phenylphosphate (6.22) than that
Although 8l exhibited high inhibitory activity, 8l was found to
be metabolically unstable due to the hydrolysis of the phosphate
group (data not shown), which prompted us to modify the phos-
phate moiety of 8l. Comparison of the activities between three
compounds bearing no or different spacer groups (8e, 8l, and 8s,
Table 2
FBPase inhibitory activity of compounds 14a,b
NH2
N
IC50 = 9.32, 0.013, and 19.1 lM, respectively) and the X-ray struc-
ture of 8l bound to FBPase suggested that a spacer group of 1 atom
in length was favorable for potent inhibitory activity. Therefore, we
decided to evaluate phosphate mimics such as methylenephosph-
onate and difluoromethylenephosphonate.
The syntheses of these phosphate mimics are illustrated in
Scheme 2. Methylene analog 14a and difluoromethylene analog
14b were prepared from 7-hydroxy-1-tetralone 4e. Triflation of
4e followed by ketalization and Pd-catalyzed coupling with vinyl
bromide gave olefin 9. Olefin 9 was transformed into alcohol 10
S
X
(HO)2P
O
Compound
X
IC50 (l
M)a
8l
14a
14b
O
CH2
CF2
0.013
0.840
0.047
a
Inhibition of human liver FBPase.

5912
T. Tsukada et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5909–5912
of benzylic phosphonic acid (7.72),13 which suggests that the high
charge density on the PO3H2 group is favorable to the enzyme-
inhibitor interaction. Contrary to the phenyl phosphate structure
in 8l, the difluoromethylenephosphonate structure in 14b is chem-
ically stable to hydrolysis. This finding provided us with the possi-
bility for further development of this tricyclic scaffold as an FBPase
inhibitor.
In summary, we designed and synthesized a series of tricyclic
compounds as FBPase inhibitors. Compound 8l exhibited high
inhibitory activity, and an X-ray crystallographic study revealed
that 8l binds in the AMP binding site with hydrogen bonding inter-
actions similar to those of AMP. In order to enhance metabolic sta-
bility, the structure of 8l was modified to difluoromethylene analog
14b which showed inhibitory activity similar to 8l. Further studies
on enhancing the inhibitory activity and developing prodrug com-
pounds are being intensively conducted at this time.
B. J. Biol. Chem. 2005, 280, 19737; (d) Pilkis, S. J.; El-Maghrabi, M. R.; Pilkis, J.;
Claus, T. H. J. Biol. Chem. 1981, 256, 3619.
5. (a) Wright, S. W.; Hageman, D. L.; McClure, L. D.; Carlo, A. A.; Treadway, J. L.;
Mathiowetz, A. M.; Withka, J. M.; Bauer, P. H. Bioorg. Med. Chem. Lett. 2001, 11,
17; (b) Wright, S. W.; Carlo, A. A.; Carty, M. D.; Danley, D. E.; Hageman, D. L.;
Karam, G. A.; Levy, C. B.; Mansour, M. N.; Mathiowetz, A. M.; McClure, L. D.;
Nestor, N. B.; McPherson, R. K.; Pandit, J.; Pustilnik, L. R.; Schulte, G. K.; Soeller,
W. C.; Treadway, J. L.; Wang, I.-K.; Bauer, P. H. J. Med. Chem. 2002, 45, 3865.
6. Wright, S. W.; Carlo, A. A.; Danley, D. E.; Hageman, D. L.; Karam, G. A.; Mansour,
M. N.; McClure, L. D.; Pandit, J.; Schulte, G. K.; Treadway, J. L.; Wang, I.-K.;
Bauer, P. H. Bioorg. Med. Chem. Lett. 2003, 13, 2055.
7. Choe, J.-Y.; Nelson, S. W.; Arienti, K. L.; Axe, F. U.; Collins, T. L.; Jones, T. K.;
Kimmich, R. D. A.; Newman, M. J.; Norvell, K.; Ripka, W. C.; Romano, S. J.; Short,
K. M.; Slee, D. H.; Fromm, H. J.; Honzatko, R. B. J. Biol. Chem. 2003, 278, 51176.
8. (a) von Geldern, T. W.; Lai, C.; Gum, R. J.; Daly, M.; Sun, C.; Fry, E. H.; Abad-
Zapatero, C. Bioorg. Med. Chem. Lett. 2006, 16, 1811; (b) Lai, C.; Gum, R. J.; Daly,
M.; Fry, E. H.; Hutchins, C.; Abad-Zapatero, C.; von Geldern, T. W. Bioorg. Med.
Chem. Lett. 2006, 16, 1807.
9. Hebeisen, P.; Kuhn, B.; Kohler, P.; Gubler, M.; Huber, W.; Kitas, E.; Schott, B.;
Benz, J.; Joseph, C.; Ruf, A. Bioorg. Med. Chem. Lett. 2008, 18, 4708.
10. (a) Erion, M. D.; van Poelje, P. D.; Dang, Q.; Kasibhatla, S. R.; Potter, S. C.; Reddy,
M. R.; Reddy, K. R.; Jiang, T.; Lipscomb, W. N. Proc. Natl. Acad. Sci. 2005, 102,
7970; (b) Erion, M. D.; Dang, Q.; Reddy, M. R.; Kasibhatla, S. R.; Huang, J.;
Lipscomb, W. N.; van Poelje, P. D. J. Am. Chem. Soc. 2007, 129, 15480; (c) Dang,
Q.; Rao Kasibhatla, S.; Reddy, K. R.; Jiang, T.; Reddy, M. R.; Potter, S. C.; Fujitaki,
J. M.; van Poelje, P. D.; Huang, J.; Lipscomb, W. N.; Erion, M. D. J. Am. Chem. Soc.
2007, 129, 15491; (d) van Poelje, P. D.; Dang, Q.; Erion, M. D. Drug Discovery
Today Ther. Strateg. 2007, 4, 103.
Acknowledgements
We thank Professor Noriyoshi Sakabe at the Photon Factory for
help with the beam line station (BL-6B). We are also grateful to Dr.
Kazuhiko Tamaki for useful comments and suggestions.
11. For preparation of diethoxyphosphorylmethyl tosylate, see: Farrington, G. K.;
Kumar, A.; Wedler, F. C. J. Med. Chem. 1985, 28, 1668.
12. The X-ray crystallographic study was accomplished according to the
procedure described in Ref. 4a. Crystals of FBPase-8l complex were
References and notes
grown using the hanging drop vapor diffusion method at 22 °C from
protein solution (6–10 mg/mL FBPase, 20 mM Tris/HCl (pH 8.5), 1 mM DTT,
0.1 mM EDTA, 1 mM 8l) combined with an equal volume of reservoir
solution (8–10% PEG3350, 0.15 M NaCl, and 0.1 M Tris/HCl (pH 8.5)). The
FBPase-8l cocrystal was diffracted to 2.6 Å in resolution and the structure
was solved by the molecular replacement method with a tetramer model
of the published human FBPase structure (PDB 1fta). The coordinate and
statistics of FBPase-8l complex are available from the PDB using accession
code 3a29.
a
1. Wild, S.; Roglic, G.; Green, A.; Sicree, R.; King, H. Diabetes Care 2004, 27, 1047.
2. (a) Magnusson, I.; Rothman, D.; Katz, L.; Shulman, R.; Shulman, G. J. Clin. Invest.
1992, 90, 1323; (b) Consoli, A.; Nurjhan, N.; Capani, F.; Gerich, J. Diabetes 1989,
38, 550; (c) Wajngot, A.; Chandramouli, V.; Schumann, W. C.; Ekberg, K.; Jones,
P. K.; Efendic, S.; Landau, B. R. Metabolism 2001, 50, 47.
3. Benkovic, S. J.; deMaine, M. M. Adv. Enzymol. Relat. Areas Mol. Biol. 1982, 53, 45.
4. (a) Gidh-Jain, M.; Zhang, Y.; van Poelje, P. D.; Liang, J.-Y.; Huang, S.; Kim, J.;
Elliott, J. T.; Erion, M. D.; Pilkis, S. J.; El-Maghrabi, M. R.; Lipscomb, W. N. J. Biol.
Chem. 1994, 269, 27732; (b) Zhang, Y.; Liang, J.; Huang, S.; Lipscomb, W. N. J.
Mol. Biol. 1994, 244, 609; (c) Iancu, C. V.; Mukund, S.; Fromm, H. J.; Honzatko, R.
a
13. Smyth, M. S.; Ford, H.; Burke, T. R. Tetrahedron Lett. 1992, 33, 4137.