Design and synthesis of factor Xa inhibitors and their prodrugs

Article abstract of DOI:10.1016/S0960-894X(02)00921-6

In addition to our previously reported fluoro acrylamides Xa inhibitors 2 and 3, a series of potent and novel cyclic diimide amidine compounds has been identified. In efforts to improve their oral bioavailability, replacement of the amidine group with methyl amidrazone gives compounds of moderate potency (14, IC₅₀=0.028 μM). In the amidoxime prodrug approach, the amidoxime compounds show good oral bioavailability in rats and dogs. High plasma level of prodrug 26 and significant concentration of active drug 26a were obtained upon oral administration of prodrug 26 in rats.

Full text of DOI:10.1016/S0960-894X(02)00921-6

Bioorganic & Medicinal Chemistry Letters 13 (2003) 297–300
Design and Synthesis of Factor Xa Inhibitors and Their Prodrugsy
Yonghong Song,a,* Lane Clizbe,a Chhaya Bhakta,a Willy Teng,a Paul Wong,b
Brian Huang,b Katherine Tran,b Uma Sinha,b Gary Park,b Andrea Reed,b
Robert M. Scarborougha andBing-Yan Zhu a
aDepartment of Medicinal Chemistry, Millennium Pharmaceuticals, Inc., 256 East Grand Ave., South San Francisco, CA 94080, USA
bDepartment of Biology, Millennium Pharmaceuticals, Inc., 256 East Grand Ave., South San Francisco, CA 94080, USA
Received6 August 2002; accepted16 September 2002
Abstract—In addition to our previously reported fluoro acrylamides Xa inhibitors 2 and 3, a series of potent andnovel cyclic diimide
amidine compounds has been identified. In efforts to improve their oral bioavailability, replacement of the amidine group with
methyl amidrazone gives compounds of moderate potency (14, IC50=0.028 mM). In the amidoxime prodrug approach, the ami-
doxime compounds show good oral bioavailability in rats and dogs. High plasma level of prodrug 26 andsignificant concentration
of active drug 26a were obtained upon oral administration of prodrug 26 in rats.
# 2002 Elsevier Science Ltd. All rights reserved.
Factor Xa is a serine proteinase which cleaves pro-
thrombin to thrombin, leading to blood clot formation
within the bloodcoagulation cascaed. It is the sole
enzyme responsible for activation of thrombin in the
cascade. Because of its important role in blood coagul-
ation, factor Xa has emergedas an attractive target for
development of new antithrombotic agents.2 Our
research objective is to discover and develop orally
active factor Xa inhibitors for treatment of thrombotic
disorders.
In our search for Xa inhibitors basedon substituted
acrylamides, we serendipitously discovered the cyclic
diimide compound 1 as a potent Xa inhibitor with IC50
of 0.002 mM, as shown in Figure 1. Previously, we have
also reported identification of the fluoro acrylamides 2
and 3 as potent, selective andefficacious Xa inhibi-
tors.3,4 However, these amidine compounds generally
suffer from low bioavailability. In this communication,
we will discuss modifications of the amidine group
with the goal of improving the bioavailability of these
amidine-containing compounds. Previous literature has
shown that amidoximes can be converted to corre-
sponding amidines in vivo (Fig. 1), thus serving as the
prodrugs of amidines.5 In this study, the amidoxime
Figure 1. Cyclic diimide 1, fluoro acrylamides 2 and 3, andtheir ami-
doxime prodrugs.
prodrug approach has also been pursued to overcome
the low bioavailability of our amidine compounds.
In an attemptedsynthesis of a carboxylate substituted
acrylamide as factor Xa inhibitor (Scheme 1), a surpris-
ing ring closure provided the cyclic diimide compound
1. This novel compoundshowedpotent anti-Xa activity
(IC50 of 0.002 mM) andmoedrate selectivity against
thrombin andtrypsin. To determine the importance of
the ring structure on potency, the open chain diamide 4
was synthesized. As shown in Table 1, compound 4 is
50-foldless active than 1, indicating that cyclic struc-
ture is optimal for potency. Reduction of the double
yFor a preliminary account of this work, see ref 1.
*Corresponding author. Fax: +1-650-244-9287; e-mail: yonghong.
song@mpi.com
bondgave the saturatedanalogue 5 as a mixture of
stereoisomers with decreased activity, indicating that
0960-894X/03/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00921-6

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Y. Song et al. / Bioorg. Med. Chem. Lett. 13 (2003) 297–300
more active than 8. Unfortunately, compound 9 does
not show improvedbioavailability in rats. The ami-
doxime 10 was also synthesizedas prodrug of 1. How-
ever, its bioavailability is also low (1.1%).
We have previously reporteda series of fluoro sub-
stitutedacrylamides as potent andefficacious Xa inhibi-
tors.3,4 However, these amidine-containing compounds
also have low bioavailability (<3%). Replacement of
the amidine group with amidrazone and alkylami-
drazone was performed in this study. As shown in Table
3, the best compoundis again the methyl amidrazone 14
with IC50 of 0.028 mM. The unsubstitutedamidrazones
11–13 are 2–4-foldless active than 14. Compounds with
larger substituting groups (15 and 16) anddi-substi-
tution (17) all give reduced potency. The aminoindazole
18, which couldbe viewedas a cyclizedamidrazone, is
about 100-foldless active than open chain analogues
(11–13).
Scheme 1. Synthesis of cyclic diimide compounds and 4. Reagents and
conditions: (a) diethyl succinate, KOtBu, HOtBu, reflux; (b) biphenyl-
amine, BOP, triethylamine, DMF; (c) MeOH/HCl (g); (d) NH4OAc
(or NH2OH, or NH2R), MeOH; (e) NH3, MeOH, R=H.
the geometry provided by the double bond also con-
tributes to the potency. To explore P4 modification, the
sulfonylphenyl was replacedwith t-butyl (6) andpyrro-
lidylcarbonyl (7). However, the replacement reduced the
potency by 14- and90-fol,d respectively. Although
compound 1 shows potent activity, its bioavailability is
low (1.4%) in rats.
Modification of the amidine group by alkylation (19–
22) resulted in dramatic decrease in activity, as shown
in Table 4. The smaller cyano group (22) is the most
tolerant.
Previous literature has shown that replacement of ami-
dine group with amidrazone gave rise to potent and
orally bioavailable thrombin inhibitors.6 In our case,
the amidrazone compound
8 shows an IC50 of
0.078 mM, a 39-fold decrease from the amidine 1 (Table
2). The methyl amidrazone compound 9 is about 2-fold
Previous literature has shown that in many cases, ami-
doxime compounds can be used as prodrugs of the
corresponding amidine compounds, for the amidoxime
compounds can be converted to parent amidines in vivo
by liver microsomal enzymes. In certain cases, these
amidoxime prodrugs have been shown to possess good
Table 1. SAR of cyclic diimides
Structure
CompdFXa IIa (IC 50, mM) Trypsin F (%)
Table 3. Amidrazone analogues of amidine 2
1
0.002
0.67
1.6
0.18
2.5
1.4
4
5
6
7
0.099
0.12
R
X
CompdFXa (IC
50, mM)
0.001
0.11
0.041
0.057
0.028
0.75
H
NH2
NH2
NH2
NHCH3
NHPh
NHCH2CF3
N(CH3)2
H
F
2
11
12
13
14
15
16
17
Cl
Br
H
H
H
H
0.029
0.18
0.076
0.50
1.9
1.7
0.42
0.49
10.7
18
5.91
Table 4. Modification of fluoro acrylamide amidine 2
Table 2. Modification of cyclic diimide amidine 1
R
CompdFXa (IC
50, mM)
NHCH3
19
20
0.89
1.61
R
CompdFXa (IC
50, mM)
0.078
0.048
0.71
F (%)
NH2
NHCH3
OH
8
9
10
21
22
0.89
1.1
1.1
NHCN
0.253

Y. Song et al. / Bioorg. Med. Chem. Lett. 13 (2003) 297–300
299
Table 5. Amidoxime prodrugs
active drug is relatively low, indicating that the in vivo
conversion of the prodrug to active drug was not opti-
mal (about 20% conversion).
In summary, in addition to our previously reported
fluoro acrylamides Xa inhibitors, a series of potent and
novel cyclic diimide amidine compounds have been
identified. In efforts to improve their oral bioavail-
ability, replacement of the amidine group with methyl
amidrazone gives compounds of moderate potency (14,
IC50=0.028 mM). In the amidoxime prodrug approach,
the amidoxime compounds show good oral bioavail-
ability in rats and dogs. High plasma level of prodrug 26
andsignificant concentration of active drug 26a were
obtained upon oral administration of prodrug 26 in
rats. Whether improvedoral bioavailability of prodrug
26 will translate into in vivo oral activity is to be
demonstrated. However, the in vivo conversion of the
prodrug to active drug is not complete and could afford
significant inter-subject variability.
R
X
CompdFXa (IC
Amidoxime
50, mM)
Amidine
F (%, rat)9
CH3
CH3
CH3
CH3
H
F
Cl
Br
23
24
25
26
0.44
1.59
0.75
1.18
0.001 (2)
2.5
6
16
52
0.0002 (24a)
0.0005 (25a)
0.0005 (26a)
H
27
3.79
0.001 (3)
25 (dog)
oral bioavailability.5 In this study, we have applied the
amidoxime prodrug strategy to our amidine-containing
Xa inhibitors. As shown in Table 5, the amidoxime
prodrugs (23–27) are generally inactive, 400–8000-fold
less potent than the corresponding active drugs (2, 3,
24a–26a). Although analogue 23 does not have good
bioavailability, the bioavailability of additional pro-
drugs 24–26 in rats increases with halogen substitutions
within the P4 biphenylsulfonamide moiety, from fluoro
(24, 6%), to chloro (25, 16%), to bromo (26, 52%).7 A
similar effect of halogen substitution on bioavailability
has also been observedin our previous study in which
the P1 amidine was replaced by aminoisoquinoline.8
Interestingly, the amidoxime prodrug 27 of the pyr-
azyl acrylamide 3 also shows improvedbioavailabilty
in dogs (F, 25%) as compared to the parent amidine
3 (F, <5% in dog). These data demonstrate again
that modification of the highly basic amidine group
couldimprove the oral absorption of parent amidine
compounds.
In an attemptedsynthesis of a carboxylate substituted
acrylamide as shown in Scheme 1, Stobbe condensation
of 3-cyano acetophenone with diethyl succinate gave the
mono acidas a mixture of E/Z isomers with a ratio of
10:7. After separation by chromatography, the Z isomer
was coupledwith the biphenylamine by using BOP to
give the amide. However, during the transformation of
the nitrile to corresponding amidine under Pinner con-
ditions, the amide underwent cyclization in methanol
saturatedwith hydrogen chloride, to yieldthe cyclic
diimide as methyl imidate. Treatment of the imidate
with ammonium acetate, hydroxylamine, and hydrazine
gave corresponding amidine, amidoxime and ami-
drazone analogues, respectively. It was also found that
the cyclic diimide ring could be opened by nucleophiles
such as ammonia at the less conjugatedcarbonyl site,
providing the open chain diamide 4.
Upon oral administration of prodrug 26 in rats, high
plasma concentration of the prodrug (Cmax=630 ng/
mL) was obtained; at the same time, significant level of
active amidine compound 26a (Cmax=168 ng/mL) was
also achievedas shown in Figure 2. As comparedto
the concentration of the prodrug, the level of the
Synthesis of the fluoro acrylamide compounds is shown
in Scheme 2. In case of R1=CH3, Horner–Emmons
reaction of the cyanoacetophenone with triethyl
2-fluoro-2-phosphonoacetate gave the acrylates in favor
of the desired isomer (with the cyanophenyl cis to the
carboxylate). In case of R1=1-pyrazole, 3-cyano-
benzoyl chloride was treated with pyrazole to give the
acylpyrazole. Interestingly the acylpyrazole was found
to undergo Horner–Emmons reaction with triethyl
Scheme 2. Synthesis of fluoro acrylamide compounds. Reagents and
conditions: (a) triethyl 2-fluoro-2-phosphonoacetate, KN(Me3Si)2,
THF, ꢀ78 ꢁC; (b) biphenylamine, X=H, F, Cl, Br, AlMe3, CH2Cl2;
(c) MeOH/HCl (g); (d) NH2OH (or NH2R), MeOH; (e) pyrazole,
triethylamine, CH2Cl2.
Figure 2. Plasma concentrations of prodrug 26 andactive drug 26a
versus time upon po administration of prodrug 26 to four conscious
rats at a dose of 6 mg/kg, as determined by LC/MS/MS.

300
Y. Song et al. / Bioorg. Med. Chem. Lett. 13 (2003) 297–300
2-fluoro-2-phosphonoacetate, giving the desired acrylate
as a minor isomer. Similar Wittig reaction of acyl-
imidazole with phosphine ylide was observed previously
in literature, indicating that these acyl azoles behave
as ketones rather than acylating agents.10 Weinreb
amidation of these acrylates with the biphenylamine
generatedthe acrylamides. Conversion of the nitrile to
corresponding amidine, amidoxime and amidrazone
was similarly performed under Pinner conditions.
National Meeting of the American Chemical Society, San
Diego, CA, April 1–5, 2001; American Chemical Society:
Washington, DC, 2001; MEDI 063.
4. Song, Y.; Clizbe, L.; Bhakta, C.; Teng, W.; Li, W.; Wu, Y.;
Jia, Z. J.; Zhang, P.; Wang, L.; Doughan, B.; Su, T.; Kanter, J.;
Woolfrey, J.; Wong, P.; Huang, B.; Tran, K.; Sinha, U.; Park,
G.; Reed, A.; Malinowski, J.; Hollenbach, S.; Scarborough,
R. M.; Zhu, B.-Y. Bioorg. Med. Chem. Lett. 2002, 12, 1511.
5. Weller, T.; Alig, L.; Beresini, M.; Blackburn, B.; Bunting,
S.; Hadvary, P.; Muller, M. H.; Knopp, D.; Levet-Trafit, B.;
Lipari, M. T.; Modi, N. B.; Muller, M.; Refino, C. J.; Schmitt,
M.; Schonholzer, P.; Weiss, S.; Steiner, B. J. Med. Chem.
1996, 39, 3139.
6. Oh, Y. S.; Yun, M.; Hwang, S. Y.; Hong, S.; Shin, Y.; Lee,
K.; Yoon, K. H.; Yoo, Y. J.; Kim, D. S.; Lee, S. H.; Lee,
Y. H.; Park, H. D.; Lee, C. H.; Lee, S. K.; Kim, S. Bioorg.
Med. Chem. Lett. 1998, 8, 631.
7. One of the referees askedwhether we made any halogenated
analogues of compound 10 for the cyclic diimide series, to see
whether the same trendholds for that series. Unfortunately, we
do not have such data at this point of time. However, we see the
same trendin our aminoisoquinoline series. 8
8. Song, Y.; Clizbe, L.; Bhakta, C.; Teng, W.; Li, W.; Wong,
P.; Huang, B.; Sinha, U.; Park, G.; Reed, A.; Scarborough,
R. M.; Zhu, B.-Y. Bioorg. Med. Chem. Lett. 2002, 12, 2043.
9. The prodrugs were administered orally whereas the active
forms were administered intravenously. Bioavailability was
calculatedbasedon the AUC for the active form of the drug.
10. Strehlke, P.; Bohlmann, R.; Schneider, M.; Nishino, Y.;
Muhn-Seipoldy, H.-P. US Patent 5,344,834, 1994.
References and Notes
1. Song, Y.; Clizbe, L.; Bhakta, C.; Teng, W.; Wong, P.;
Huang, B.; Tran, K.; Sinha, U.; Park, G.; Reed, A.; Scar-
borough, R. M.; Zhu, B.-Y. Books of Abstracts, 223th
National Meeting of the American Chemical Society,
Orlando, FL, April 7–11, 2002; American Chemical Society:
Washington, DC, 2002; MEDI 26.
2. (a) Zhu, B.-Y.; Scarborough, R. M. Annu. Rep. Med.
Chem. 2000, 35, 83. (b) Zhu, B.-Y.; Scarborough, R. M. Curr.
Opin. Cardiovasc., Pulm. Renal Invest. Drugs 1999, 1, 63. (c)
Sanderson, P. E. J. Annu. Rep. Med. Chem. 2001, 36, 79.
3. Song, Y.; Clizbe, L.; Bhakta, C.; Teng, W.; Li, W.; Wu, Y.;
Jia, Z. J.; Zhang, P.; Wang, L.; Doughan, B.; Su, T.; Kanter,
J.; Woolfrey, J.; Wong, P.; Huang, B.; Tran, K.; Sinha, U.;
Park, G.; Reed, A.; Malinowski, J.; Hollenbach, S.; Scar-
borough, R. M.; Zhu, B.-Y. Books of Abstracts, Part 2, 221st