Design, synthesis, and evaluation of orally active inhibitors of plasminogen activator inhibitor-1 (PAI-1) production

Article abstract of DOI:10.1016/j.bmcl.2008.10.067

A novel series of butadiene-imide 1 (T-686) derivatives having an inhibitory activity against PAI-1 production was synthesized and evaluated their biological activities and DMPK profiles, in which 15k (T-2639) was selected as the best compound based on its strong antithrombotic activity and good bioavailability.

Full text of DOI:10.1016/j.bmcl.2008.10.067

Bioorganic & Medicinal Chemistry Letters 18 (2008) 6419–6422
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis, and evaluation of orally active inhibitors of plasminogen
activator inhibitor-1 (PAI-1) production
a
a
b
b
Hiroshi Miyazaki a, Tsuyoshi Ogiku a, , Hiroshi Sai , Hiroshi Ohmizu , Jun Murakami , Akio Ohtani
*
a Medicinal Chemistry Laboratory, Mitsubishi Tanabe Pharma Co., Ltd, 3-16-89, Kashima, Yodogawa, Osaka 532-8505, Japan
b Pharmacology Laboratory, Mitsubishi Tanabe Pharma Co., Ltd, 2-2-50, Kawagishi, Toda, Saitama 335-8505, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of butadiene-imide 1 (T-686) derivatives having an inhibitory activity against PAI-1 pro-
duction was synthesized and evaluated their biological activities and DMPK profiles, in which 15k
(T-2639) was selected as the best compound based on its strong antithrombotic activity and good
bioavailability.
Received 16 September 2008
Revised 14 October 2008
Accepted 16 October 2008
Available online 19 October 2008
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Plasminogen activator inhibitor-1 (PAI-1)
Inhibitor of PAI-1 production
T-2639
Plasminogen activator inhibitor-1 (PAI-1), a specific inhibitor of
both tissue-type plasminogen activator and urokinase-type plas-
minogen activator, plays an important role in the regulation of
the fibrinolytic system.1 Elevated levels of PAI-1 in plasma have
been observed in patients with deep vein thrombosis2 and unsta-
ble angina.3 Furthermore, a number of animal studies have shown
that PAI-1 is the factor which disturbs fibrinolytic activity in the
thrombotic and prethrombotic states.4 Thus, inhibition of PAI-1
activity or reduction of PAI-1 production may shift the balance be-
tween thrombogenesis and thrombolysis towards thrombolysis. In
fact, it has been reported that an antibody against PAI-1 can en-
hance clot lysis and decrease thrombus growth in animal models
of venous thrombosis5 and arterial thrombosis.6 Recently a number
of small molecules have been reported to inhibit PAI-17 and show
in vivo efficacy in animal thrombosis models.8
tural features of the butadiene template. In this paper, we report
the design, synthesis and biological activities of these butadiene
analogues.
We have reported the syntheses of the butadiene derivatives
(1–4, Fig. 1).12 By using a similar method to that of 2, butadiene
analogues (8, 9, 11, and 12) possessing methyl group on 1- or 4-po-
sition of the butadienes were synthesized (Scheme 1). The E- and
Z-dimethyl benzylidenesuccinates (6 and 7) were synthesized by
the first Stobbe condensation13 of acetophenone with dimethyl
succinate followed by esterification. These isomers were separated
We previously described that a new butadiene-imide derivative
1 (T-686) inhibited the production of PAI-1 in cultured vascular
endotherial cells9 and could prevent the shutdown of fibrinolysis
in an experimental hypofibrinolytic model.10 Furthermore,
1
showed antithrombotic activity in both rat venous thrombosis
and arterio-venous shunt models without affecting bleeding
time.11 These results suggest the possibility that inhibitors of
PAI-1 production would provide safe antithrombotic agents.
Compound 1 has, however, some defects such as poor water sol-
ubility (9 lg/ml), low oral bioavailability (<3% in dog), and chromo-
somal aberration. In order to improve these defects, we started to
identify alternative novel inhibitors that incorporated key struc-
* Corresponding author.
E-mail address: ogiku.tsuyoshi@me.mt-pharma.co.jp (T. Ogiku).
Figure 1. T-686 and its derivatives.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.10.067

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H. Miyazaki et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6419–6422
Scheme 1. Reagents and conditions: (a) dimethyl succinate, t-BuOK, t-BuOH, rt, 1 h; (b) SOCl2, MeOH, rt, 16 h; (c) 3,4,5-trimethoxybenzaldehyde, t-BuOK, t-BuOH, rt, 1 h; (d)
SOCl2, CHCl3, reflux, 30 min; (e) 28% NH4OH, 0 °C–rt, 30 min; (f) 30,40,50-trimethoxyacetophenone, t-BuOK, t-BuOH, rt, 1 h.
by chromatography and used for the next step. The second Stobbe
condensation of succinate (6 and 7) with 3,4,5-trimethoxybenzal-
dehyde provided 2E-isomeric carboxylic acids as major products,
which were respectively treated with SOCl2 followed by aqueous
NH3 to afford the corresponding amides (8 and 9). Also, the buta-
diene analogues (11 and 12) were synthesized via the Stobbe con-
densation of 1012 with 30,40,50-trimethoxyacetophenone, where the
isomers were separated by chromatography after the amidation.
The syntheses of 15a–l are summarized in Scheme 2. The key
intermediate (2Z,3E)-carboxylic acid 14 was once isolated by chro-
matography as its methoxymethyl (MOM) ester 13 due to the dif-
ficulty in the separation of the initially produced carboxylic acid
isomers. Treatment of 13 with HCl afforded 14,14 which was trans-
formed into the desired amides and hydrazones 15a–k by using
the conventional method. The free form of 15k was oxidized with
meta-chloroperbenzoic acid (mCPBA) to give the corresponding N-
oxide 15l.
Compounds 21a and 21b were synthesized via the alternative
method shown in Scheme 3. Esterification of the crude product
of the Stobbe condensation of 30,50-dimethoxyacetophenone with
dimethyl succinate provided the E-diester 17 predominantly.
Photo-irradiation to the solution of E-isomer 17 afforded a mixture
Scheme 2. Reagents and conditions: (a) 30,50-dimethoxyacetophenone, t-BuOK, t-BuOH/THF, 50 °C, 4 h; (b) MOMCl, i-Pr2NEt, CH2Cl2, rt, 0.5 h; (c) concd HCl, THF, rt, 1 h;
(d) CDI, CH2Cl2, rt, 2 h, then 3- or 4-picolylamine, rt, 8 h; (e) (COCl)2, DMF (cat.), CH2Cl2, rt, 1 h, then HNR1R2, Et3N, THF, rt, 0.5 h; (f) 4 N-HCl/EtOAc, rt; (g) mCPBA, CH2Cl2,
ꢀ50 °C–rt, 1 h.

H. Miyazaki et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6419–6422
6421
Scheme 3. Reagents and conditions: (a) dimethyl succinate, t-BuOK, t-BuOH, 50 °C, 4 h; (b) MOMCl, i-Pr2NEt, CH2Cl2, rt, 0.5 h; (c) h
m
, MeCN, rt, 8 h; (d) LDA, THF, ꢀ78 °C,
0.5 h, then 3- or 4-pyridinecarboxaldehyde, THF, ꢀ100 °C, 20 min; (e) MsCl, Et3N, CH2Cl2, 0 °C, 0.5 h, then DBU, CH2Cl2, rt, 16 h; (f) concd HCl, THF, rt, 1 h; (g) CDI, THF, rt, 16 h,
then 28% NH4OH, 0 °C, 2 h; (h) 4 N-HCl/EtOAc, rt.
of Z- and E-isomers, which were separated by chromatography to
give Z-isomer 18 in 36% yield. The adducts of the aldol reaction
of 18 with 3- and 4-pyridinecarboxaldehydes were mesylated
and treated with 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) to af-
ford (2Z,3E)-isomers of pyridylbutadiene derivatives as major
products, which were purified by chromatography to give 20a,b
respectively. Compounds 20a,b were transformed into 21a,b by
using the method described above.
phenyl ring. We next introduced the methyl group onto the buta-
diene scaffolds. While 8, 9, and 11 were not effective, it was
revealed that 1-Me-(2Z,3E)-butadiene derivative 12 possessed
good potency and, as expected, excellent stability in dog blood
(96% remain). Furthermore, it was found that 12 exhibited im-
proved properties in comparison with 1 (Table 3. 1 vs 12), that
is, enhanced solubility in water (12, 106 lg/ml; 1, 9 lg/ml), im-
proved bioavailability (12, 9%; 1, 3% in dog), and negative result
We started our research with the optimization of the butadiene
scaffold. The relative inhibitory activity against lipopolysaccha-
ride(LPS)-induced PAI-1 production in cultured vascular endothe-
in the chromosomal abnormality test.
Encouraged by these fundamental data on the templates, we
next intensified our efforts in optimizing the substituents of 12.
In the segment A (Fig. 2), we anticipated that removal of one of
the three methoxy groups should improve metabolic stability be-
cause the methoxy group had been generally known to be the typ-
ical metabolic target leading to demethylated phenol metabolite.15
In the segment B, we tried replacing the phenyl ring with the pyr-
idine ring to enhance solubility. In the segment C, we planned to
introduce other amide moieties that, if possible, could form a salt
to improve solubility.
lial cells (compound concentration: 10 lM) and the stability in
dog blood are summarized in Table 1. During the course of the ini-
tial screening of the compounds related to imide 1, it was proved
that the amide-ester type compound 2 also showed good PAI-1
production inhibitory activity. Although the ester moiety seemed
to be unstable to hydrolysis (0% remain in dog blood), this result
encouraged us to synthesize the amide-ester type compounds be-
cause the good crystalline properties of imide led to low water sol-
ubility, which seemed to be one of the reasons for the low
bioavailability of 1. Next, our search for more attractive templates
focused on the stereoisomeric structures of 2. Although the corre-
sponding (2Z,3E)- and (2E,3Z)-butadiene derivatives 3 and 4
exhibited decreased activity, it was found that 3 possessed largely
increased stability in dog blood compared with 2 and 4 (3, 75%; 2
and 4, 0% remain). It could be elucidated that the ester hydrolysis
of 3 was sterically blocked by the (Z)-positioned 3,4,5-trimethoxy-
The relative inhibitory activity against LPS-induced PAI-1 pro-
duction in cultured vascular endothelial cells (compound concen-
Table 2
Inhibitory activities on LPS-induced PAI-1 production in cultured vascular endothelial
cells.
Compound
Relative potencya
1 (T-686)
12
14
1.0
1.5
0.4
1.3
2.1
1.8
2.2
0.1
0.1
0.4
0.6
0.3
0.8
1.5
0.2
1.5
0.8
Table 1
Inhibitory activities of PAI-1 production and stability in dog blood.
15a
15b
15c
15d
15e
15f
15g
15h
15i
15j
15k
15l
21a
21b
Compound
Relative potencya
Stability in dog blood % of remainb
1 (T-686)
2
3
4
8
9
11
12
1.0
0.9
0.7
0.7
0.2
0.3
0.3
1.0
79
nd
75
nd
nt
nt
nt
96
nd, not detected.
nt, not tested.
a
Relative inhibitory activities on LPS-induced PAI-1 production in cultured
a
vascular endothelial cells (compound concentration: 10
lM) are shown using T-686
Relative inhibitory activities on LPS-induced PAI-1 production in cultured
as a reference compound.
vascular endothelial cells (compound concentration: 1
as a reference compound.
lM) are shown using T-686
b
% of remains are shown in dog blood after 1 h at 37 °C.

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H. Miyazaki et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6419–6422
Table 3
Solubility, stability in dog blood, bioavailability, geno-toxicity, antithrombotic activity and toxicity in rats of selected compounds.
Compound
Water solubility
Stability in dog blood
% remaina
Bioavailability
(%) (dog)
Chromosomal
aberration test
Antithrombotic activity % decrease
in thrombus weightb
Toxicity in male rats
at 300 mg/kg po
(
lg/ml)
1 (T-686)
12
15a
15b
15c
15d
15k
21a
9
106
1600
>2000
>3000
>2000
>4000
>4000
79
96
87
76
87
83
89
88
3
9
+
nt
nt
53
nt
58
nt
50
nt
nt
nt
ꢀ
ꢀ
+
ꢀ
nt
ꢀ
+
21
28
52
17
49
78
Decrease in locomotor activity
nt
Decrease in locomotor activity
nt
No toxicity
nt
nt, not tested.
a
% of remains are shown in dog blood after 1 h at 37 °C.
Antithrombotic effects on the rat venous thrombosis model (3 mg/kg/day ꢁ 3 days) are shown. See Ref. 16.
b
Figure 2. Optimization of the substituents of 12.
4. (a) Krishnamurti, C.; Barr, C. F.; Hassett, M. A.; Young, G. D.; Alving, B. M. Blood
1987, 69, 798; (b) Reilly, C. F.; Fujita, T.; Mayer, E. F.; Siefried, M. E. Arterioscler.
Thromb. 1991, 11, 1276; (c) Carmeliet, P.; Stassen, J. M.; Schoonjans, L.; Ream,
B.; Van den Oord, J. J.; De Mol, M.; Mulligan, R. C.; Collen, D. J. Clin. Invest. 1993,
92, 2756.
5. Levi, M.; Biemond, B. J.; Van Zonnenveld, A. J.; Ten Cate, J. W.; Pannekoek, H.
Circulation 1992, 85, 305.
6. Biemond, B. J.; Levi, M.; Coronel, R.; Janse, M. J.; Ten Cate, J. W.; Pannekoek, H.
Circulation 1995, 1, 1175.
7. (a) Folkes, A.; Roe, M. B.; Sohal, S.; Golec, J.; Faint, R.; Brooks, T.; Charlton, P.
Bioorg. Med. Chem. Lett. 2001, 11, 2589; (b) Folkes, A.; Brown, S. D.; Canne, L. E.;
Chan, J.; Engelhardt, E.; Epshteyn, S.; Faint, R.; Golec, J.; Hanel, A.; Kearney, E.;
Leahy, J. W.; Mac, M.; Matthews, D.; Prisbylla, M. P.; Sanderson, J.; Simon, R. J.;
Tesfai, Z.; Vicker, N.; Wang, S.; Webb, R. R.; Charlton, P. Bioorg. Med. Chem. Lett.
2002, 12, 1063; (c) Wang, S.; Golec, J.; Miller, W.; Milutinovic, S.; Folkes, A.;
Williams, S.; Brooks, T.; Hardman, K.; Charlton, P.; Wren, S.; Spencer, J. Bioorg.
Med. Chem. Lett. 2002, 12, 2367; (d) De Nanteuil, G.; Lila-Ambroise, C.; Rupin,
A.; Vallez, M. O.; Verbeuren, T. J. Bioorg. Med. Chem. Lett. 2003, 13, 1705; (e) Ye,
B.; Bauer, S.; Buckman, B. O.; Ghannam, A.; Griedel, B. D.; Khim, S. K.; Lee, W.;
Sacchi, K. L.; Shaw, K. J.; Liang, A.; Wu, Q.; Zhao, Z. Bioorg. Med. Chem. Lett. 2003,
13, 3361; (f) Ye, B.; Chou, Y. L.; Karanjawala, R.; Lee, W.; Lu, S. F.; Shaw, K. J.;
Jones, S.; Lentz, D.; Liang, A.; Tseng, J. L.; Wu, Q.; Zhao, Z. Bioorg. Med. Chem. Lett.
2004, 14, 761; (g) Gopalsamy, A.; Kincaid, S. L.; Ellingboe, J. W.; Groeling, T. M.;
Antrilli, T. M.; Krishnamurthy, G.; Aulabaugh, A.; Friedrichs, G. S.; Crandall, D. L.
Bioorg. Med. Chem. Lett. 2004, 14, 3477; (h) Hu, B.; Jetter, J. W.; Wrobel, J. E.;
Antrilli, T. M.; Bauer, J. S.; Di, L.; Polakowski, S.; Jain, U.; Crandall, D. L. Bioorg.
Med. Chem. Lett. 2005, 15, 3514.
tration: 1 lM) is summarized in Table 2. The synthetic intermedi-
ate carboxylic acid 14 showed poor potency. Among the amide
derivatives, while 3- and 4-picolyl amides (15a,b) and 3- and
4-pyridyl amides (15c,d) were revealed to possess equal to en-
hanced potency compared with 12, other amides and hydrazone
(15e–h) exhibited no strong activity. Interestingly, hydrazone
derivative 15k exhibited good potency although the corresponding
amide derivatives (15i,j) and N-oxide 15l showed decreased po-
tency. Replacement of the phenyl ring in the segment B with 4-pyr-
idyl ring (21a) led to equal activity, and with 3-pyridyl ring (21b)
to decreased activity compared with 12.
In order to select the best compound, the physicochemical and
pharmacokinetic properties of these potent compounds were eval-
uated and listed in Table 3. The solubility of these compounds was
largely enhanced and stability of most compounds in dog blood
was also improved compared with 1. As a result, the bioavailability
of the compounds was quite improved, and 15a–c, 15k and 21a
satisfied our criterion (>20%). Next, chromosomal aberration was
tested for these five compounds and the negative result was shown
in 15a,c and 15k. The antithrombotic activity of these three com-
pounds was examined in a rat model of venous thrombosis. All
three compounds inhibited the thrombus weight by around 50%
in the rat venous thrombosis model. In the toxicity study in male
rats at 300 mg/kg po, the two compounds 15a,c showed decrease
in locomotor activity as a side effect.
8. Elokdah, H.; Abou-Gharbia, M.; Hennan, J. K.; McFarlane, G.; Mugford, C. P.;
Krishnamurthy, G.; Crandall, D. L. J. Med. Chem. 2004, 47, 3491.
9. Ohtani, A.; Takagi, T.; Hirano, A.; Murakami, J.; Sasaki, Y. J. Biochem. 1996, 120,
1203.
10. Ohtani, A.; Murakami, J. Fibrinolysis Proteol. 1997, 11, 45.
11. Ohtani, A.; Murakami, J.; Hirano-Wakimoto, A. Eur. J. Pharmacol. 1997, 330,
151.
Based on these results, we selected 15k (T-2639) as the best
compound for further investigation.
12. Sai, H.; Ogiku, T.; Ohmizu, H.; Ohtani, A. Chem. Pharm. Bull. 2006, 54, 1686.
13. Johnson, W. S.; Daube, G. H. Org. React. 1951, 6, 1.
14. Crystal data of 14; C22H22O6, M = 382.40, monoclinic, space group P21/c,
a = 12.967 (2), b = 12.0214(2), c = 13.360 (1) Å, b = 101.12 (1)°, V = 2043.4
(5) Å3, Dc = 1.243 mg mꢀ3, Z = 4, R = 0.046, Rw = 0.165, CCDC Reference No.
705024.
15. We saw during another project that the 4-methoxy group was predominantly
demethylated to phenol metabolite in the 3,4,5-trimethoxyphenyl group
(unpublished result).
16. Compounds (3 mg/kg per day) were orally administered for three consecutive
days. Two hours after the last administration, thrombi were induced and their
dry weights were measured. See Ref. 11.
References and notes
1. Schneiderman, J.; Loskutoff, D. J. Trends Cardiovasc. Med. 1991, 1, 99.
2. (a) Nilsson, I. M.; Ljungner, H.; Tengborn, L. Br. J. Med. 1985, 290, 1453; (b)
Wiman, B.; Ljungberg, S.; Chmielewska, J.; Urden, G.; Blomback, M.; Johnsson,
H. J. Lab. Clin. Med. 1985, 105, 265; (c) Juhan-Vague, I.; Valadier, J.; Alessi, M. C.;
Aillaud, M. F.; Ansaldi, J.; Philip-Joet, C.; Holvet, J.; Serradimigni, A.; Collen, D.
Thromb. Haemost. 1987, 57, 67.
3. Wieczorek, I.; Ludlam, C. A.; Fox, K. A. A. Am. J. Cardiol. 1994, 74, 424.