A prodrug approach towards the development of tricyclic-based FBPase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2010.03.017

For the purpose of reducing the strong CYP3A4 inhibitory potency of diamide prodrug 4, cyclic prodrugs of tricyclic-based FBPase inhibitors were synthesized. Extensive SAR studies led to the discovery of pyridine-containing cyclic prodrug 20, which strong

Full text of DOI:10.1016/j.bmcl.2010.03.017

Bioorganic & Medicinal Chemistry Letters 20 (2010) 2938–2941
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
A prodrug approach towards the development of tricyclic-based
FBPase inhibitors
Tomoharu Tsukada a, Kazuhiko Tamaki a, Jun Tanaka b, Toshiyuki Takagi c, Taishi Yoshida b, Akira Okuno b,
Takeshi Shiiki d, Mizuki Takahashi e, Takahide Nishi a,
*
a Medicinal Chemistry Research Laboratories I, Daiichi Sankyo Co., Ltd, 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
b Biological Research Laboratories II, Daiichi Sankyo Co., Ltd, 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
c Clinical Development Department I, Daiichi Sankyo Co., Ltd, 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
d Drug Metabolism & Pharmacokinetics Research Laboratories, Daiichi Sankyo Co., Ltd, 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
e 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:
For the purpose of reducing the strong CYP3A4 inhibitory potency of diamide prodrug 4, cyclic prodrugs
of tricyclic-based FBPase inhibitors were synthesized. Extensive SAR studies led to the discovery of pyr-
idine-containing cyclic prodrug 20, which strongly inhibited glucose production in monkey hepatocytes
and also showed weak CYP3A4 inhibitory potency.
Received 25 December 2009
Revised 3 March 2010
Accepted 4 March 2010
Available online 7 March 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
FBPase
Prodrug
CYP3A4
Prodrug is a useful approach to overcoming a drug’s defect
including physiochemical, pharmacokinetic and pharmacodynamic
properties.1 Many kinds of prodrugs were designed to improve
insufficient chemical stability, poor solubility or insufficient oral
absorption. With the aim of improving insufficient oral absorption
of compounds containing polar groups such as alcohols, carboxylic
acids and phosphonic acids, a prodrug approach is frequently
adopted to enhance membrane permeability. In particular, a phos-
phonate moiety possessing properties such as high ionization at
physiologic pH and accompanying low membrane permeability
requires a prodrug approach to obtain good oral bioavailability.2
We have developed tricyclic-based phosphonates and corre-
sponding prodrugs as fructose-1,6-bisphosphatase (FBPase)
inhibitors.3 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. There
are several small-molecule inhibitors of FBPase,4–9 in which the
prodrug of MB05032 (1) (CS-917, 2) lowered blood glucose levels
in animal models and was in clinical development (Fig. 1).10 In
previous papers, we reported that the prodrug of phosphonate 3
(4) strongly inhibited glucose production in hepatocytes and
lowered blood glucose levels in fasted cynomolgus monkeys
(Figure 2).3c However, subsequent tests revealed that diamide
prodrug 4 strongly inhibited cytochrome P450 3A4 (CYP3A4).
In this Letter, we describe our efforts to solve the problem of the
strong CYP3A4 inhibition of diamide prodrug 4. CYP3A4 is respon-
sible for metabolizing over 50% of drugs and the inhibition of
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 MB05032 and CS-917.
O
P
NH
EtO
N
H
(HO)2P
O
O
O
N
N
O
S
S
EtO
O
4
3
* 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.2010.03.017

T. Tsukada et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2938–2941
2939
CYP3A4 is the major mechanism of drug–drug interactions, which
can cause serious side effects in clinical practice. We expected that
the conversion of the prodrug moiety of 4 to other prodrug moie-
ties was beneficial in order to reduce the strong CYP3A4 inhibitory
potency of 4. Among many classes of phosphonate prodrugs, we
are interested in cyclic 1-aryl-1,3-propanyl ester (HepDirect) pro-
drugs with respect to their application to pradefovir, which is in
clinical trial for the therapy of hepatitis B infection.11 This type of
prodrug has an interesting property of being oxidatively cleaved
by CYP in the liver, where our target enzyme FBPase is mainly
expressed. We focused our attention on introducing various cyclic
1-aryl-1,3-propanyl ester moieties to the parent phosphonate 3 in
order to reduce the strong CYP3A4 inhibitory potency of diamide
prodrug 4.
Initially, various 1-aryl-1,3-propanediols were synthesized
according to Scheme 1. The condensation of ethyl acetate to the
starting material aldehydes 5a–g resulted in b-hydroxyesters 6a–
g, which were transformed into racemic diols 7a–g by reduction
with LiAlH4. Pyridinediols containing a Me group (9a,b) were pre-
pared from corresponding bromo-pyridinediols 7f,g via ketaliza-
tion, methylation and subsequent deketalization. In addition, (S)-
1-(3-chlorophenyl)propane-1,3-diol 10 (prodrug moiety of prade-
fovir) was prepared following the literature method.11a Next, pro-
drugs 16–20 were prepared by the construction of tricyclic
scaffold and subsequent introductions of prodrug moieties
(Scheme 2). Acylation of 3,4-dimethyl phenol 11 followed by a
Fries rearrangement and the introduction of a diethyl phosphonate
unit resulted in diethyl phosphonate 12, which was transformed
into tricyclic thiazole 13 via bromination and cyclization with thio-
formamide (in situ generation). Cleaving phosphonate ethyl groups
of 13 followed by dichlorination and subsequent condensation
with corresponding chiral diol 10 or racemic diols 7a–e, 9a,b affor-
ded cyclic prodrugs 14 (mixtures of two stereoisomers) or 15a–g
(mixtures of four stereoisomers), respectively. Enantiomers 16
and 17 were obtained by silica gel separation of diastereomeric
mixture 14. In addition, the facile separation of four stereoisomers
15a–g with silica gel chromatography led to racemic cis isomers
18a–g and racemic trans isomers 19a–g. Moreover, the chiral HPLC
separation of racemic cis isomer 18f afforded (2R,4S)-enantiomer
20, the structure of which was determined by single-crystal X-
ray analysis (Fig. 3).
Scheme 2. Synthesis of prodrugs 16–20. Reagents and conditions: (a)
ClCH2CH2COCl, pyridine, CH2Cl2, 72%; (b) AlCl3, 180 °C, 78%; (c) (EtO)2P(O)CH2OTs,
K2CO3, DMF, 80 °C, 78%; (d) CuBr2, EtOH, 60 °C, 98%; (e) P2S5, HCONH2, THF, reflux,
58%; (f) TMSBr, CH2Cl2; (g) (COCl)2, DMF, CH2Cl2; (h) diol, DIPEA, CH2Cl2, 0 °C, 19–
61% over three steps; (i) silica gel separation; (j) chiral HPLC separation (CHIRALPAK
AD-H).
and racemic trans isomers, were evaluated. Only the cis isomer of
4-chloro substituted analogue (18a) modestly inhibited glucose
production in hepatocytes (IC50 = 66 lM), whereas other prodrugs
including 3-methoxy analogues showed no inhibitory activities. In
addition, these prodrugs showed strong CYP3A4 inhibitory poten-
cies and poor aqueous solubilities. It is known that the potency of
CYP3A4 inhibition correlates with compound lipophilicity.12 We
thought that decreasing lipophilicity of our prodrugs was benefi-
cial in order to reduce the potency of CYP3A4 inhibition and also
to improve aqueous solubility, which prompted us to introduce a
pyridine ring to the prodrug instead of a benzene ring.
Prodrugs containing a benzene ring showed strong CYP3A4
inhibitory potencies and poor aqueous solubilities (Table 1). Ini-
tially, the prodrug moiety of pradefovir was introduced to our tri-
cyclic phosphonate. The (2R,4S)-enantiomer 16 inhibited glucose
production in hepatocytes (IC50 = 15
CYP3A4 inhibitory potency (IC50 = 0.42
diamide prodrug 4 (IC50 = 0.58 M). Next, other substituted ben-
lM), but also showed a strong
l
M) almost equal to that of
l
zene-containing prodrugs, which were prepared as racemic cis
a
b
O
OH OH
OH
O
Ar
H
Ar
OEt
Ar
5a-g
Ar =
6a-g
7a-g
a: 4-Cl-Ph, b: 3-OMe-Ph, c: 2-pyridyl, d: 3-pyridyl,
e: 4-pyridyl, f: 2-Br-3-pyridyl, g: 4-Br-3-pyridyl
c
d, e
OH OH
O
O
OH OH
Br
Br
Me
N
N
N
7f,g
8a,b
9a,b
Scheme 1. Synthesis of diols 7, 9. Reagents and conditions: (a) iPr2NH, nBuLi, EtOAc, THF, À78 °C to rt; (b) LiAlH4, THF, 0 °C, 17–94% over two steps; (c) Me2C(OMe)2, TsOH,
acetone, reflux, 62–85%; (d) nBuLi, MeI, THF, À78 °C to rt; (e) HClaq, MeOH, 39–79%, over two steps.

2940
T. Tsukada et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2938–2941
(Table 2). Although both racemic cis and racemic trans isomers of
pyridine-containing prodrugs were evaluated, racemic trans iso-
mers 19c–g did not inhibit glucose production in hepatocytes (data
not shown). The efforts to incorporate unsubstituted pyridine rings
(18c, 18d, 18e) led to a decrease of CYP3A4 inhibitory potencies
and an improvement in aqueous solubility relative to benzene-
containing prodrugs. Among these unsubstituted pyridine ana-
logues, 3-pyridyl analogue 18d most potently inhibited glucose
production in hepatocytes (IC50 = 8.8 lM). Introducing the methyl
group into ortho positions of the pyridine nitrogen showed oppo-
site effects on the inhibition of glucose production in hepatocytes,
including little effect with the 2-Me group (18f, IC50 = 11
detrimental effect with the 4-Me group (18g, IC50 = >100
These methyl-substituted pyridine analogues 18f and 18g showed
weak CYP3A4 inhibitory potencies (IC50 = 5.8, 5.2 M, respectively)
compared with the corresponding unsubstituted pyridine analogue
18d (IC50 = 2.9 M), presumably due to the steric effects on pyri-
lM) and a
l
M).
l
l
dine nitrogen, which is supposed to bind to the CYP heme iron.
Moreover, (2R,4S)-enantiomer of 18f (20) more strongly inhibited
glucose production in hepatocytes (IC50 = 4.0
lM) and showed
weak CYP3A4 inhibitory potency (IC50 = 15 M) relative to 18f.
l
Compared with diamide prodrug 4, pyridine-containing cyclic pro-
drug 20 more potently inhibited glucose production in hepato-
cytes, and showed an over 25 times weaker CYP3A4 inhibitory
potency. These results revealed that prodrug 20 would have a re-
duced risk of problematic drug–drug interactions.
Figure 3. ORTEP view of prodrug 20 from single-crystal X-ray analysis.
Pyridine-containing prodrug 20 appears to have more favorable
physicochemical properties than other types of prodrugs (Table 3).
In a previous paper, we revealed the importance of the physico-
Prodrugs containing the pyridine ring demonstrated improved
results compared with prodrugs containing the benzene ring
Table 1
SAR of prodrugs containing a benzene ring13
O
P
O
4
O
N
2
O
R
S
4
2
3
Compound
R
P2, C4 stereochemistry
Hepatocytes
CYP3A4
Solubility (lg/ml)
a
IC50
(lM)
IC50 (lM)
JP1 (pH 1.2)
JP2 (pH 6.8)
4
16
17
18a
19a
18b
19b
—
3-Cl
3-Cl
4-Cl
4-Cl
3-OMe
3-OMe
—
6.8
15
0.58
0.42
2.7
0.65
0.73
0.27
0.3
27
19
<0.5
4.4
<0.5
17
66
cis-(2R,4S)
trans-(2S,4S)
Racemic cis
Racemic trans
Racemic cis
Racemic trans
<0.5
<0.5
<0.5
<0.5
0.8
>100
66
>100
>100
>100
<0.5
<0.5
a
Inhibition of glucose production in primary monkey hepatocytes.
Table 2
SAR of prodrugs containing a pyridine ring13
O
P
O
4
O
2
N
Ar
O
S
Compound
Ar
P2, C4 stereochemistry
Hepatocytes
CYP3A4
Solubility (lg/ml)
a
IC50
(l
M)
IC50
(lM)
JP1 (pH 1.2)
JP2 (pH 6.8)
18c
18d
18e
18f
18g
20
2-Py
3-Py
4-Py
2-Me-3-Py
4-Me-3-Py
2-Me-3-Py
Racemic cis
Racemic cis
Racemic cis
Racemic cis
Racemic cis
cis-(2R,4S)
22
8.8
41
11
>100
8.4
2.9
2.2
5.8
5.2
15
86
86
88
87
>100
88
73
84
51
83
81
84
4.0
a
Inhibition of glucose production in primary monkey hepatocytes.

T. Tsukada et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2938–2941
2941
Table 3
Physicochemical properties of prodrugsa
Compound
MW
HB-Db
HB-Ac
c Log P
PSAd
Hepatocytes
CYP3A4
Solubility (
JP1 (pH 1.2)
l
g/ml)f
e
f
IC50
(
lM)
IC50
(l
M)
JP2 (pH 6.8)
4
16
20
509.56
461.90
442.47
2
0
0
9
5
6
2.93
4.39
2.76
153.9
95.7
108.59
6.8
15
4.0
0.58
0.42
15
27
19
88
66
<0.5
84
a
b
c
d
e
f
Calculated using ACD/labs version 9.0 (Advanced Chemistry Development, Inc.).
Hydrogen bond donors.
Hydrogen bond acceptors.
Polar surface area (Å2).
Inhibition of glucose production in primary monkey hepatocytes (experimental values).
Experimental values.
4. (a) Wright, S. W.; Hageman, D. L.; McClure, L. D.; Carlo, A. A.; Treadway, J. L.;
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Nestor, N. B.; McPherson, R. K.; Pandit, J.; Pustilnik, L. R.; Schulte, G. K.; Soeller,
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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.
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chemical parameter of prodrugs and also suggested that the low PSA
values related to high membrane permeability of the prodrugs was
probably critical to the strong inhibitory effect on glucose produc-
tion in hepatocytes.3c With reference to physicochemical parame-
ters of cyclic prodrugs, it is apparent that benzene-containing
prodrug 16 has a high c Log P value (4.39), which perhaps resulted
in the strong CYP3A4 inhibitory potency and poor aqueous solubility
of 16. Compared with benzene-containing prodrug 16, pyridine-
containing prodrug 20 has a low c Log P value (2.76), which probably
led to the weak CYP3A4 inhibitory potency and improved aqueous
solubility of 20. Furthermore, the low PSA value of pyridine-contain-
ing prodrug 20 (108.59) relative to that of diamide prodrug 4 (153.9)
presumably contributed to the strong inhibitory effect on glucose
production in hepatocytes.
In summary, we developed cyclic prodrugs of tricyclic-based
FBPase inhibitors in order to reduce the strong CYP3A4 inhibitory
potency of diamide prodrug 4. In contrast to prodrugs containing
a benzene ring, prodrugs containing a pyridine ring showed weak
CYP3A4 inhibitory potency and high aqueous solubility, which
was presumably linked to low c Log P values of the prodrugs. Pyr-
idine-containing cyclic prodrug 20 more potently inhibited glucose
production in hepatocytes and also showed weaker CYP3A4 inhib-
itory potency than diamide prodrug 4. These results not only re-
vealed that prodrug 20 would have a reduced risk of problematic
drug–drug interactions, but also demonstrated the importance of
optimizing a prodrug to improve a drug’s defect.
11. (a) Erion, M. D.; Reddy, K. R.; Boyer, S. H.; Matelich, M. C.; Gomez-Galeno,
J.; Lemus, R. H.; Ugarkar, B. G.; Colby, T. J.; Schanzer, J.; van Poelje, P. D. J.
Am. Chem. Soc. 2004, 126, 5154; (b) Reddy, K. R.; Matelich, M. C.; Ugarkar,
B. G.; Gomez-Galeno, J. E.; DaRe, J.; Ollis, K.; Sun, Z.; Craigo, W.; Colby, T.
J.; Fujitaki, J. M.; Boyer, S. H.; van Poelje, P. D.; Erion, M. D. J. Med. Chem.
2008, 51, 666.
References and notes
12. Gleeson, M. P. J. Med. Chem. 2008, 51, 817.
1. Ettmayer, P.; Amidon, G. L.; Clement, B.; Testa, B. J. Med. Chem. 2004, 47, 2393.
2. Hecker, S. J.; Erion, M. D. J. Med. Chem. 2008, 51, 2328.
13. (a) For details of assays to measure the inhibitory effects on glucose production
in monkey hepatocytes, see Ref. 3c; (b) The potency of test compounds to
inhibit expressed CYP3A4 (Supersomes, BD Gentest, MA, USA) was measured
using 7-benzyloxy-4-trifluoromethylcoumarin as substrate; (c) The solubility
of test compounds was assayed using the first (JP1, pH 1.2) and second (JP2, pH
6.8) fluids of Japanese Pharmacopoeia, which simulate gastric and intestinal pH
conditions, respectively.
3. (a) Tsukada, T.; Takahashi, M.; Takemoto, T.; Kanno, O.; Yamane, T.; Kawamura,
S.; Nishi, T. Bioorg. Med. Chem. Lett. 2009, 19, 5909; (b) Tsukada, T.; Takahashi,
M.; Takemoto, T.; Kanno, O.; Yamane, T.; Kawamura, S.; Nishi, T. Bioorg. Med.
Chem. Lett. 2010, 20, 1004; (c) Tsukada, T.; Kanno, O.; Yamane, T.; Tanaka, J.;
Yoshida, T.; Okuno, A.; Shiiki, T.; Takahashi, M.; Nishi, T., in preparation.