Structure-based drug design of tricyclic 8H-indeno[1,2-d][1,3]thiazoles as potent FBPase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2009.12.056

With the goal of improving metabolic stability and further enhancing FBPase inhibitory activity, a series of tricyclic 8H-indeno[1,2-d][1,3]thiazoles was designed and synthesized with the aid of structure-based drug design. Extensive SAR studies led to th

Full text of DOI:10.1016/j.bmcl.2009.12.056

Bioorganic & Medicinal Chemistry Letters 20 (2010) 1004–1007
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Structure-based drug design of tricyclic 8H-indeno[1,2-d][1,3]thiazoles 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 goal of improving metabolic stability and further enhancing FBPase inhibitory activity, a series
of tricyclic 8H-indeno[1,2-d][1,3]thiazoles was designed and synthesized with the aid of structure-based
drug design. Extensive SAR studies led to the discovery of 19a with an IC50 value of 1 nM against human
FBPase. X-ray crystallographic studies revealed that high affinity of 19a was due to the hydrophobic
interaction arising from better shape complementarity and to the hydrogen bonding network involving
the side chain on the tricyclic scaffold.
Received 22 October 2009
Revised 8 December 2009
Accepted 11 December 2009
Available online 21 December 2009
Keywords:
FBPase
Ó 2010 Elsevier Ltd. All rights reserved.
Diabetes
Fructose-1,6-bisphosphatase (FBPase) is one of the enzymes in-
volved in the rate-limiting steps of hepatic gluconeogenesis, and
catalyzes the hydrolysis of fructose-1,6-bisphosphate to fructose-
6-phosphate.1 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 Type 2 diabetes mellitus. Several
classes of small-molecule inhibitors of FBPase have been re-
ported.2–6 Among them, AMP mimetic MB05032 (1) exhibited high
inhibitory activity. A prodrug of MB05032 (CS-917, 2) lowered
blood glucose levels in animal models and was entered into clinical
development (Fig. 1).7
In the previous Letter, we described the design and synthesis of
tricyclic thiazoles as FBPase inhibitors, and a series of SAR studies
led to the identification of phosphate 3 exhibiting potent FBPase
inhibitory activity (IC50 = 13 nM) (Fig. 2).8 In addition, we reported
the finding of non-hydrolyzable difluoromethylenephosphonate 4
which also showed high inhibitory activity (IC50 = 47 nM).8 How-
ever, subsequent attempts to evaluate the inhibitory effect on cel-
In order to address the problem of the metabolically unstable
amino group on tricyclic scaffolds, we examined the conversion
of the amino group. Our previous SAR studies led to the identifica-
tion of compound 6 which possessed the non-hydrolyzable oxy-
methylphosphonate moiety and showed moderate FBPase
inhibitory activity (IC50 = 124 nM).8 Therefore, we selected com-
pound 6 for the modification of the amino group. An X-ray crystal
NH2
S
NH2
S
O
P
O
P
NH
N
N
HO
HO
EtO
O
O
N
H
O
EtO
O
1 (MB05032)
2 (CS-917)
Figure 1. Structures of known FBPase inhibitors.
lular glucose production of
4 and corresponding prodrug
compound 5 were not successful. These results were rationalized
by considering the metabolism of the amino group on the tricyclic
scaffold. In fact, the amino groups of these compounds were read-
ily metabolized by N-acetyltransferases. In order to improve meta-
bolic stability and to enhance FBPase inhibitory activity, we turned
our attention to further modification of our tricyclic-based inhibi-
tors with the aid of structure-based drug design.
NH2
O
(HO)2㪧
N
NH2
N
S
㪦
X
Y2P
O
S
6
3
4
5
X = O, Y = OH
X = CF2, Y = OH
X = CF2, Y = (S)-NHCH(Me)CO2Et
* Corresponding author. Tel.: +81 3 3492 3131; fax: +81 3 5436 8563.
E-mail address: nishi.takahide.xw@daiichisankyo.co.jp (T. Nishi).
Figure 2. Structures of tricyclic-based FBPase inhibitors.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.12.056

T. Tsukada et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1004–1007
1005
structure of human liver FBPase in complex with 6 suggested the
possibility of removing the amino group (Fig. 3).9 The complex
structure suggested that the amino group of 6 interacted with a
none 7 was converted to diethyl phosphonate 8. Bromination of 8
followed by cyclization with thioacetamide, thioformamide (in situ
generation), and thiourea gave 2-methythiazole (9a), thiazole (9b),
and 2-aminothiazole (9c) intermediates, respectively. The hydroly-
sis of intermediates 9a,b afforded the desired compounds 10a,b. In
addition, 2-halogen thiazole analogues 11a,b were prepared from
9c by a Sandmeyer reaction followed by hydrolysis.
carbonyl oxygen of Val17 and a side chain oxygen (Oc) of Thr31
through hydrogen-bonding interaction. On the other hand, the
amino group was located near the hydrophobic surface area
formed by hydrophobic residues of Val17, Leu34, and Met177. In
addition, the carbonyl oxygen of Val17 and O
c
of Thr31 forms
The inhibitory activities of the compounds with the modifica-
tion of the amino group against human liver FBPase are summa-
rized in Table 1.10 Replacement of the amino group of 6 with
hydrophobic substituent such as a methyl group (10a) led to a sub-
stantial loss of activity (IC50 = 995 nM). Similarly, replacement with
halogen groups (11a, 11b) resulted in a considerable loss of activity
(IC50 = 908, 772 nM, respectively). These results were presumably
due to the loss of hydrogen-bonding interaction between the ami-
no group and FBPase. In contrast, to our surprise, 10-fold increase
in activity (IC50 = 12 nM) was achieved by removing the amino
group of 6 (10b), and the activity of 10b was almost equal to that
of MB05032.
hydrogen bonds to backbone amide nitrogen of Gly21 and a water
molecule, respectively, suggesting that the interaction to the ami-
no group of compound 6 might not be crucial in retaining the pro-
tein structure to the inhibited conformation. Based on these
observations, we hypothesized that hydrophobic interaction in-
stead of the interaction through the amino group might be benefi-
cial to obtain high affinity. This led us to focus on replacing the
amino group of compound 6 with other hydrophobic substituents.
Tricyclic thiazoles without amino group were synthesized
according to Scheme 1. Commercially available 7-hydroxy-1-inda-
In order to obtain insights into the binding mode of desamino
compound 10b, an X-ray crystal structure of human liver FBPase
in complex with 10b was determined and was compared to that
of corresponding amino compound 6 ( Fig. 4). The phosphonate
group of 10b occupies the same position as that of 6, and interacts
Table 1
FBPase inhibitory activity of compounds 10–11
R1
O
(HO)2㪧
N
㪦
S
R1
IC50 (nM)
a
Compound
MB05032
6
10a
11a
11b
10
124
995
908
772
12
NH2
Me
Cl
Br
H
10b
a
Inhibition of human liver FBPase.
Figure 3. X-ray crystal structure of human liver FBPase in complex with 6.
b, c (for R1 = Me),
b, d (for R1 = H) or
OH
(EtO)2P
O
O
O
O
b, e (for R1 = NH2)
a
8
7
R1
R1
N
(EtO)2㪧
O
O
(HO)2㪧
N
f
㪦
㪦
S
S
S
9a-c
10a,b
R1 = Me, H
g, f (for R1 = Cl) or
h, f (for R1 = Br)
R1
(EtO)2㪧
O
O
(HO)2㪧
NH2
N
N
㪦
㪦
S
9c
11a,b
R
1 = Cl, Br
Scheme 1. Synthesis of compounds 10–11. Reagents and conditions: (a) (EtO)2-
P(O)CH2OTs, K2CO3, DMF, 80 °C, 70%; (b) CuBr2, EtOH, 60 °C, 98%; (c) thioacetamide,
DMF, 50 °C, 41%; (d) P2S5, HCONH2, THF, reflux, 66%; (e) thiourea, NaOAc, EtOH,
reflux, 46%; (f) TMSBr, CH2Cl2, 90–98%; (g) tBuONO, CuCl2, MeCN, 33%; (h) tBuONO,
CuBr2, MeCN, 44%.
Figure 4. X-ray crystal structure of human liver FBPase in complex with 10b.
(Overlay of 6. Compound 6 and residues Leu34, Val17 and Met177 in complex with
6 are colored in blue.)

1006
T. Tsukada et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1004–1007
with the backbone amides and the side chains of FBPase in a sim-
ilar way to 6. In contrast, when they were superimposed, a small
but distinct shift of the tricyclic scaffold was observed. The tricyclic
scaffold of 10b shifts to the hydrophobic surface area formed by
hydrophobic residues of Val17, Leu34, and Met177. In addition,
side chains of these residues appear to be moved slightly to im-
prove the shape complementarity to 10b. The shift of tricyclic scaf-
fold of 10b and induced fit of hydrophobic side chains led to the
formation of the hydrophobic interaction between the hydropho-
bic surface area and the thiazole ring of the tricyclic scaffold. This
hydrophobic interaction reinforced by better shape complemen-
tarity would compensate for the loss of the hydrogen-bonding
interactions between the amino group and FBPase. In other words,
the amino group of 6 contributes not only to hydrogen-bonding
interaction but also to steric reputation to some extent.
Moreover, the X-ray co-crystal structure of 10b provided impor-
tant clues for further development. The complex structure sug-
gested that there was a cavity in the direction of the 7-position
of tricyclic scaffold (p-position of oxymethylphosphonate moiety)
and also showed that there were some water molecules in this
space. We expected higher affinity might be obtained by introduc-
ing the side chains which occupied the cavity, especially the side
chains with the potential to interact to the water molecules. Based
on these considerations, we focused our attention on introducing
side chains mainly to the 7-position of tricyclic scaffold to further
enhance its FBPase inhibitory activity.
Tricyclic thiazoles with side chains were synthesized according
to Scheme 2. The compounds with alkyl or halogen side chains (
15a–f) were prepared from corresponding phenols 12a–g. Acyla-
tion of 12a–g followed by a Fries rearrangement and the introduc-
tion of a diethyl phosphonate unit resulted in 13a–g, which were
transformed into intermediates 14a–g and desired compounds
15a–f by the subsequent steps already described in Scheme 1.
The compounds with aromatic side chain (16a–e) were obtained
from intermediate 14g by Suzuki couplings with corresponding
boronic acids/esters followed by hydrolysis. The compounds with
amide side chain (19a,b) were obtained from intermediate 13g.
CO insertion of 13g followed by a transesterification reaction re-
sulted in allyl ester 17, which was transformed into carboxylic acid
18 via bromination followed by cyclization and deesterification.
Amidation of 18 with corresponding amines and subsequent
hydrolysis afforded desired compounds 19a,b.
The inhibitory activities of the compounds containing the side
chains are summarized in Table 2. The attempts to occupy the cav-
ity with hydrophobic substituents such as alkyl groups and halo-
gen groups were slightly effective to ineffective in increasing
inhibitory activity. The insertion of Me (15a), Et (15b), F (15c),
and Cl (15d) at 7-position showed similar inhibitory activities
(IC50 = 8, 11, 15, 8 nM, respectively) to lead compound 10b. With
disubstitution, 6,7-disubstitution such as 6,7-di Me (15e) and
6,7-di F (15f) were tolerated (IC50 = 10, 9 nM, respectively). The ef-
forts to incorporate aromatic ring at the 7-position led to a range of
results, including detrimental effects with Ph (16a) and 2-Py (16b)
(IC50 = 28, 40 nM, respectively), minor effects with 3-Py (16c) and
4-Py (16d) (IC50 = 9, 9 nM, respectively), and fourfold boost in po-
tency with 3,5-pyrimidine (16e) (IC50 = 3 nM). These results sug-
gested that the incorporation of a hydrogen bond acceptor into
its proper location was beneficial to increase inhibitory activity,
whereas only occupying the cavity was relatively ineffective. On
the basis of these findings, we introduced other hydrogen bond
acceptor such as a carbonyl group. The efforts to introduce amide
groups (19a, 19b) provided a major boost in inhibitory activity
(IC50 = 1, 2 nM, respectively), and 19a was over 10-fold more po-
tent than lead compound 10b.
OH
(EtO)2P
O
O
O
(EtO)2㪧
O
N
a, b, c
d, e
㪦
S
R2
R2
R2
R3
R3
R3
12a-g
13a-g
14a-g
(HO)2㪧
O
N
f
㪦
S
R2
R3
15a-f
O
(EtO)2㪧
O
(HO)2㪧
N
S
N
g, f
㪦
㪦
S
R3
Br
O
Table 2
16a-e
14g
FBPase inhibitory activity of compounds 15–19
O
(HO)2㪧
(EtO)2P
O
(EtO)2P
O
O
N
O
O
㪦
h, i
d, e, j
S
6
R2
7
R3
Br
COOAllyl
17
13g
Compound
R2
R3
IC50 (nM)
a
10b
15a
15b
15c
15d
15e
15f
16a
16b
16c
16d
16e
19a
19b
H
H
H
H
H
Me
F
H
H
H
H
H
H
H
H
Me
Et
F
Cl
12
8
11
15
8
10
9
28
40
9
O
(HO)2㪧
O
(EtO)2㪧
N
S
N
k, f
㪦
㪦
S
Me
F
Ph
2-Py
3-Py
4-Py
3,5-Pyrimidine
CONH2
CONHMe
COOH
18
CONHR4
19a,b
Scheme 2. Synthesis of compounds 15–19. Reagents and conditions: (a)
ClCH2CH2COCl, pyridine, CH2Cl2, 56–98%; (b) AlCl3, 180 °C, 32–90%; (c) (EtO)2P(O)-
CH2OTs, K2CO3, DMF, 80 °C, 57–98%; (d) CuBr2, EtOH, 60 °C, 90–98%; (e) P2S5,
HCONH2, THF, reflux, 45–71%; (f) TMSBr, CH2Cl2, 66–98%; (g) boronic acid/ester,
Pd(PPh3)4, Na2CO3, EtOH–H2O, reflux, 17–62%; (h) Pd(OAc)2, dppp, CO, Et3N,
TMSCH2CH2OH, DMF, 70 °C, 52%; (i) TBAF, allyl bromide, Et3N, 93%; (j) Pd(PPh3)4,
pyrrolidine, MeCN, 79%; (k) amine, WSC, HOBt, DMF, 47–73%.
9
3
1
2
a
Inhibition of human liver FBPase.

T. Tsukada et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1004–1007
1007
activity compared to 10b. This high affinity would be obtained by
forming the hydrogen bonding network involving the side chain.
The X-ray co-crystal structures of lead compounds (6, 10b) pro-
vided us with the structural information which was beneficial to
further developments, and these results demonstrated the useful-
ness of structure-based drug design. Further efforts on identifying
various phosphonate prodrugs of the tricyclic 8H-indeno[1,2-
d][1,3]thiazoles to investigate in vivo activity are underway.
Acknowledgments
We thank Professor Noriyoshi Sakabe of the Structural Biology
Sakabe Project and Professor Soichi Wakatsuki of the Institute of
Materials Structure Science for the use of the facilities at the Pho-
ton Factory.
References and notes
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Figure 5. X-ray crystal structure of human liver FBPase in complex with 19a.
In order to understand the determinants of high affinity of 19a
with amide side chain, an X-ray crystal structure of human liver
FBPase in complex with 19a was determined (Fig. 5). The position
of phosphonate group and tricyclic scaffold of 19a is similar to
those of 10b with no side chain, which suggests the formation of
the hydrophobic interaction as in the case of 10b. In addition, the
amide side chain of 19a makes hydrogen bonds with the side chain
of Asp178 and two water molecules, as expected. One water mol-
ecule interacts with the backbone carbonyl oxygen of Val160, the
backbone nitrogen (NH) of Asp178 and the side chain of Asp178,
and the other water molecule interacts with the backbone nitrogen
(NH) of Cys179 and the side chain of Glu20. As a result, the side
chain of 19a forms the hydrogen bonding network involving
Val160, Asp178, Cys179, Glu20, and two water molecules. This
hydrogen bonding network would contribute to increase affinity.
In summary, we further developed a series of tricyclic 8H-inde-
no[1,2-d][1,3]thiazoles as potent FBPase inhibitors with the aid of
structure-based drug design. In order to enhance the metabolic
stability, lead compound 6 was modified to desamino compound
10b which showed 10-fold increase in inhibitory activity relative
to 6. The X-ray co-crystal structure of 10b suggested that hydro-
phobic interaction would compensate for the loss of the hydro-
gen-bonding interactions through the amino group. Furthermore,
introducing side chain with hydrogen bonding capability to 10b
led to the discovery of 19a which exhibited over 10-fold increased
8. Tsukada, T.; Takahashi, M.; Takemoto, T.; Kanno, O.; Yamane, T.; Kawamura, S.;
Nishi, T. Bioorg. Med. Chem. Lett. 2009, 19, 5909.
9. The X-ray crystallographic study was accomplished according to the procedure
described in Ref. 8. The FBPase-6, 10b, 19a cocrystals were diffracted to 2.45,
2.8, 2.25 Å in resolution, respectively. The coordinates and statistics are
available from the PDB using accession codes 3kbz, 3kc0, and 3kc1,
respectively.
10. Inhibition assays using recombinant human liver FBPase were performed
according to the methods described in Ref. 7c.