Design, synthesis, and biological activity of piperidine diamine derivatives as factor Xa inhibitor

Article abstract of DOI:10.1016/j.bmcl.2007.11.037

Previously, we identified cyclohexane diamine derivative 1 as orally bioavailable factor Xa inhibitor. We have investigated two racemic cis-piperidine diamine derivatives 2 and 3 based on 1. Compounds 2a-e showed higher fXa inhibitory activity, anticoagulant activity, and aqueous solubility than 3a-e having same substituent. Compounds 2a, 2c, 2e, and 2g-m having sp2 nitrogen, especially amide and urea derivatives, showed potent anticoagulant activity. Compounds 2h and 2k showed high oral activities in rats.

Full text of DOI:10.1016/j.bmcl.2007.11.037

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 782–787
Design, synthesis, and biological activity of piperidine
diamine derivatives as factor Xa inhibitor
Akiyoshi Mochizuki,a,* Yumi Nakamoto,a Hiroyuki Naito,b
Kouichi Uotob and Toshiharu Ohtac
aMedicinal Chemistry Research Laboratory I, Daiichi Sankyo Co., Ltd, 1-2-58, Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
bMedicinal Chemistry Research Laboratory II, Daiichi Sankyo Co., Ltd, 1-16-13, Kita-Kasai, Edogawa-ku, Tokyo 134-8630, Japan
cBiological Research Laboratories I, Daiichi Sankyo Co., Ltd, 1-2-58, Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
Received 4 July 2007; revised 22 October 2007; accepted 12 November 2007
Available online 17 November 2007
Abstract—Previously, we identified cyclohexane diamine derivative 1 as orally bioavailable factor Xa inhibitor. We have investi-
gated two racemic cis-piperidine diamine derivatives 2 and 3 based on 1. Compounds 2a–e showed higher fXa inhibitory activity,
anticoagulant activity, and aqueous solubility than 3a–e having same substituent. Compounds 2a, 2c, 2e, and 2g–m having sp2 nitro-
gen, especially amide and urea derivatives, showed potent anticoagulant activity. Compounds 2h and 2k showed high oral activities
in rats.
ꢀ 2007 Elsevier Ltd. All rights reserved.
Thrombosis-related diseases, such as myocardial infarc-
tion, deep vein thrombosis, and unstable angina, are
major causes of mortality in the industrialized world.
Current therapies, using heparin and warfarin, are based
on indirect inhibition of thrombin, the terminal enzyme
in the coagulation cascade. These therapies are limited
by side effect, mode of administration, careful monitor-
ing of drug level, slow onset of action, and excessive
bleeding.1 Therefore, safer and more effective orally
administered anticoagulant agents to combat these dis-
eases are required in clinical treatment.
not sufficiently absorbed in oral administration.5 This
insufficient absorption is due to its strongly basic ami-
dine groups,6 so it is only used as an injectable formula-
tion in clinical studies. Therefore, non-amidine type fXa
inhibitors were continuously studied by us7,8 and other
groups.9 Recently cyclicdiamine derivatives were re-
ported by our group8 and Qiao et al.9a,b We reported
that the (À)-(1R,2S)-cyclohexanediamine derivative 1
showed selective fXa inhibition activity (Fig. 1).8 Its
in vivo activity and anticoagulant activity were inade-
quate, we thought that its weak in vivo activity was
due to its low solubility in neutral water and the replace-
ment of lipophilic cyclohexane into piperidine would
increase the solubility. Then we designed two types of
cis-piperidine diamine derivatives 2 and 3, and their var-
ious N-substituted derivatives were synthesized as cis
racemic compounds.
Factor Xa (fXa) is a serine protease that is situated in
convergence of the intrinsic and the extrinsic coagula-
tion pathways. This enzyme plays an important role in
the conversion of prothrombin to thrombin, which is a
highly amplified process.2 It is thought that the inhibi-
tion of fXa may be more effective and involve a lower
risk of abnormal bleeding.3
Piperidine diamine derivatives were prepared from com-
mon intermediate 9. The synthesis of intermediates is
shown in Scheme 1. Commercially available tetrahydro-
pyridine (4) was protected with Boc group. Compound 5
was oxidized with OsO4 to give diol 6. Diol 6 was trans-
formed to diamine 9 via mesylate 7 and azide 8. To
introduce 5-chloroindole as S1 ligand and 5-methyl-
4,5,6,7-tetrahydrothiazolo[5,4-c]pyridine as S4 ligand
into diamine 9, nitrophenol esters 11 and 13 were pre-
pared from each corresponding carboxylic acid.7b,10
DX-9065a is a potent fXa inhibitor and exerts anti-
thrombotic effects in animal models.4 But DX-9065a is
Keywords: Factor Xa inhibitor; Anticoagulant; cis-Piperidine diamine
derivative.
*
Corresponding author. E-mail: mochizuki.akiyoshi.yk@daiichisankyo.
co.jp
0960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.11.037

A. Mochizuki et al. / Bioorg. Med. Chem. Lett. 18 (2008) 782–787
783
R
cis racemate
N
N
Cl
Cl
O
S
N
Cl
N
O
N
N
N
S
O
N
N
2
N
N
N
N
cis racemate
1
R
O
O
S
N
N
N
N
3
O
Figure 1. Structure of cyclohexane derivative 1 and cis-piperidine diamine derivatives 2 and 3.
to give 2n and 2o, and the hydrolysis of 2o gave acid
derivative 2p.
Boc
N
Boc
N
H
N
a
b
c
HO
In Table 1, in vitro anti-fXa, anticoagulant activity,
aqueous solubility of compounds 1, 2a–e, and 3a–e are
summarized. Except 2a and 3a, these compounds
showed higher solubility in the aqueous neutral solution
(JP2: Japanese Pharmacopoeia Second fluid, pH 6.8)
than 1. In general compounds 2a–e showed higher
anti-fXa and anticoagulant activities than 3a–e for sim-
ilar piperidyl substituents. Furthermore, compounds
2b–e showed higher aqueous solubility. Particularly, 2c
showed exceedingly potent anticoagulant activity more
than six times the activity of 1. Additional analogs of
compounds that represent structure 2 were also evalu-
ated (Table 2).
OH
4
5
6
Boc
N
Boc
N
Boc
N
d
e
MsO
N3
H2N
OMs
N3
NH2
9
7
8
Cl
O
O
Cl
O
f
f
N
O2N
N
HO
H
H
10
11
Compounds 2g–p showed higher aqueous solubility
than 1. Carbamate (2a, 2e), amide (2c, 2g–j), and urea
(2k–m) derivatives showed much higher anti-fXa activ-
ity than 1, especially amide (2c, 2g–j), urea (2k–m) deriv-
atives showed high anti-fXa activity with clotting
activity (<1.1 lM). Unsubstituted piperidyl analog
(2b), sulfonyl amine (2d), and alkyl analogs (2n–p) were
comparatively weaker. A possible explanation for the
high affinity could be attributable to the sp2 hybridiza-
tion of the piperidyl nitrogen.
O
S
O
N
S
N
N
O2N
O
N
LiO
12
13
Scheme 1. Synthesis of intermediates 9, 11, and 13. Reagents and
conditions: (a) Boc2O, 10% Na2CO3 aqueous rt 92%; (b) OsO4, NMO,
MeCN-H2O rt 70%; (c) MsCl, Et3N, CH2Cl2 rt 94%; (d) NaN3, DMF
rt 74%; (e) H2, Pd(C), MeOH rt 89%; (f) 1—SOCl2, cat. DMF,
CH2Cl2, reflux, 2—nitrophenol, Et3N, CH2Cl2 50–93%.
T1/2 in human liver microsome and intrinsic clearance
(CLint) were also evaluated for active compounds
(Table 3).15,16 Carbamate derivatives 2a and 2e were
very unstable. Many other compounds showed mod-
erate stability at the same level as compound 1,
but, 2g and 2h were clearly shown to be much supe-
rior to 1.
Diamine 9 was acylated by using nitrophenol ester 11 or
13 under basic condition to get mono acylated piperi-
dine diamines 14 and 17 as main product, respectively.11
Piperidine diamine derivatives 2 and 3 were prepared as
shown in Scheme 2. Compounds 14 and 17 were con-
densed with 10 or 12 to obtain N-Boc piperidine deriva-
tives 2a and 3a. N-Boc piperidine derivatives 2a and 3a
were treated with hydrochloric acid to give NH piperi-
dine derivatives 2b and 3b, respectively. N-acyl, N-sulfo-
nyl, and N-carboxyl piperidines (2c–f, 2h–k, 3c–e) were
obtained by treating 2b or 3b with acid chlorides. The
hydrolysis of 2f gave alcohol derivative 2g. Urea deriva-
tives 2k–m were obtained by treating with dimethyl ami-
no chloroformate or corresponding isocyanates. Also 2b
was N-alkylated with alkyl bromide in basic condition
The oral activities of 2g, 2h, 2k, and 2l were examined
after the administration to rats at a dose of 10 mg/kg
(Table 4). Compounds 2h and 2k displayed potent
anti-fXa activities in plasma, on the other hand, 2g
and 2l containing hydrogen donating groups showed
low anti-fXa activities in plasma.
Compound 2h showed oral activity in rat, and the nota-
ble improvements of anti-fXa activity, anticoagulant

784
A. Mochizuki et al. / Bioorg. Med. Chem. Lett. 18 (2008) 782–787
Boc
N
Boc
N
Boc
N
Cl
Cl
a
O
O
H
N
H
N
9
+
+
H2N
N
N
H
H
HN
HN
NH2
N
N
H
H
O
O
Cl
Cl
14 (46%)
15 (17%)
16 (12%)
Boc
Boc
N
Boc
N
N
O
O
O
N
b
S
N
N
S
N
N
N
H2N
9
+
+
H
N
H
NH2
HN
HN
N
N
S
N
S
O
17 (38%)
18 (14%)
19 (3%)
a: R = Boc
b: R = H
R
N
d
e
cis racemate
S
f
l: R = CONHEt,
m: R = CONHtBu
Cl
O
c
N
g
14
n: R = CH2CH2OMe
o: R = CH2CO2Me
H
h
N
HN
N
N
H
p: R = CH 2CO2H
g: R = COCH2OH
O
2
i
c: R = COMe
f: R = COCH 2OAc
h: R = COCH2OMe
i: R = COCH2CH3
j; R = COCH(CH3)2
k: R = CONMe2
d: R = SO Me
2
e: R = CO Et
2
a: R = Boc
b: R = H
cis racemate
S
R
N
Cl
d
e
O
j
17
N
c: R = COMe
d: R = SO2Me
e: R = CO2Et
H
HN
N
N
N
H
3
O
Scheme 2. Synthesis of cis-piperidine diamine derivatives 2 and 3. Reagents and conditions: (a) 11, NMM, DMF rt; (b) 13, NMM, DMF rt; (c) 12,
WSCI, HOBt, NMM, DMF rt 66%; (d) HCl, EtOH rt 98–99%; (e) R-Cl, Et3N, CH2Cl2; (f) RNCO, TEA, CH2Cl2 65–82%; (g) R-Br, K2CO3, DMF
38–67%; (h) LiOH, THF, H2O rt 89%; (i) NaOH, THF, H2O rt 65%; (j) 10, WSCD, HOBt, NMM, DMF rt 87%.
Table 1. In vitro fXa inhibitory activity (IC50), anticoagulant activity (PTCT2), and aqueous solubility of 1, 2a–e, and 3a–e
X
Cl
Y
O
S
N
H
N
HN
N
N
H
O
Compound
X
Y
In vitro
Solubility JP2c (lg/ml)
PTCT2b (lM)
a
IC50 (nM)
1
CH2
CH2
CH2
CH2
CH2
41
16
4.4
2.9
3.5
6.2
1.3
>20
0.67
5.8
2.5
8.0
1.4
11
17
16
(À)-1
2a
3a
2b
3b
2c
N-CO2t-Bu
CH2
NH
10
3.0
<1
996
830
421
290
103
70
N-CO2t-Bu
CH2
NH
130
62
CH2
NCOCH3
CH2
530
8.6
48
CH2
NCOCH3
CH2
3c
2d
3d
2e
NSO2CH3
CH2
28
45
NSO2CH3
CH2
NCO2Et
NCO2Et
CH2
4.5
110
43
37
3e
Except for (À)-1, these are racemic compounds.
a The method of measuring anti-fXa activity was described in Ref. 12.
b Anti-coagulant activity was evaluated with the human plasma clotting time doubling concentration (PTCT2). The measurement of PTCT2 was
conducted according to Ref. 8.
c Aqueous neutral solution, JP2 (pH 6.8), was prepared as described in Ref. 13. The method of measuring solubility was described in Ref. 14.

A. Mochizuki et al. / Bioorg. Med. Chem. Lett. 18 (2008) 782–787
785
Table 2. In vitro fXa inhibitory activity (IC50) and anticoagulant
activity (PTCT2) of 2g–p
Table 4. Ex vivo anti-fXa activity on oral administration to rats
(10 mg/kg)
Compound
Anti-fXa activity (%)
1 h 2 h
R
N
0.5 h
4 h
Cl
O
2 g
2h
2k
2l
0.76 1.5 À2.0 1.3 À2.5 1.8 À7.3 2.0
S
N
68.0 2.6
80.2 1.3
16.7 4.9
50.0 2.6
78.6 0.9
11.4 2.8
38.0 4.2
73.7 0.3
5.3 2.6
16.0 2.2
68.1 0.7
4.3 2.7
H
N
HN
N
N
H
O
The methods for measuring the ex vivo anti-fXa activities were
described in Ref. 17. Values are expressed as means SE from three
rats.
Compound
R
IC50 fXaa PTCT2b Solubility
(nM)
(lM)
JP2c (lg/ml)
2g
2h
2i
COCH2OH
9.3
5.8
9.0
5.6
8.4
7.8
4.8
0.9
410
510
160
66
Table 5. Monkey pharmacokinetic profiles (1.0 mg/kg po)
COCH2OCH3
COCH2CH3
COCH(CH3)2
CONMe2
0.78
0.78
1.1
Compound
AUC0–24 h (ng h/ml)
Cmax (ng/ml)
Tmax (h)
2j
(À)-1a
56.6
83.6
23.3
36.4
2.0
1.2
2k
2l
0.79
0.80
0.78
3.8
56
2hb
CONHEt
CONH t-Bu
220
39
Hydrogen chloride salts were used. The method of measuring con-
centration was described in Ref. 18.
2m
2n
2o
2p
CH2CH2OCH3 47
130
28
a The data of compound (À)-1 were calculated by normalizing the data
CH2CO2CH3
CH2CO2H
56
26
2.1
1.8
at a dose of 3.0 mg/kg as single dose.
b Compound 2h was co-administered with other compounds.
723
a The method of measuring anti-fXa activity was described in Ref. 12.
b Anti-coagulant activity was evaluated with the human plasma clot-
ting time doubling concentration (PTCT2). The measurement of
PTCT2 was conducted according to Ref. 8.
c Aqueous neutral solution, JP2 (pH 6.8), was prepared as described in
Ref. 13. The method of measuring solubility was described in Ref. 14.
value than (À)-1, the exposure level was insufficient. We
thought that further improvement of metabolic stability
would be needed to raise the inhibitor exposure in oral
administration.
Table 3. T1/2 in human liver microsome and calculated intrinsic
clearance (CLint)
In summary, two types of racemic cis-piperidine diamine
derivatives 2 and 3 were shown to possess potent anti-
fXa activity and good solubility. Also, compounds 2a–e
showed higher anti-fXa and clotting activity, solubility
when compared to compounds 1 and 3. In general,
carbamate, amide, and urea derivatives showed higher
anti-fXa activities than 1. Moreover, amide and urea
derivatives showed potent anticoagulant activities at less
than 1 lM, even though these compounds were racemic.
In addition, methoxyacetyl derivative 2h and dimethylu-
rea derivative 2k showed oral activity in rats (ex vivo),
and 2h showed higher metabolic stability than 1. On
the basis of these results, the cis-piperidine derivative
was considered a promising lead compound for explora-
tion as a fXa inhibitor. Further efforts will be reported
in separate publications.
X
Cl
Y
O
S
N
H
N
HN
N
N
H
O
a
T1/2 (min) Clint,h
a
Compound
X
Y
(ml/min/kg)
1
CH2
CH2
CH2
CH2
CH2
CH2
CH2
7.70
6.42
4.44
6.54
4.53
81.0
97.2
140.4
95.4
137.7
41.4
45.9
102.6
98.1
92.7
86.4
99.9
(À)-1
2a
2c
2e
2g
2h
2i
NCO2t-Bu
NCOCH3
NCO2Et
NCOCH2OH
NCOCH2OCH3 CH2 13.6
CH2 15.1
NCOCH2CH3
NCOCH(CH3)2 CH2
CH2
6.08
6.36
6.73
7.22
6.24
Acknowledgments
2j
2k
2l
NCONMe2
NCONHEt
NCONH t-Bu
CH2
CH2
CH2
We thank anti-coagulants group in Biological Research
Laboratories I for performing the biological assays. We
thank also Dr. Kanno for helpful discussions and review
of this manuscript.
2m
Except for (À)-1, these are racemic compounds.
In vitro metabolic stability was measured in human liver microsome
according to Ref. 15.
Substrate concentration, 1 lM; human liver microsomes, 1 mg of
protein/ml.
a T1/2 and Clint,h were estimated from the remaining ratio (X%) of the
substrate as described in Ref. 16.
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solution in aqueous DMSO solution (1:1 (v/v)) were
prepared to make calibration curve. The measurement
sample solution, 5 lmol solution, and 100 lmol solution
were assayed using HPLC methodologies (Analytical
Column: X Terra? MSC18 3.5 lm, 3.0· 30 mm, Waters;
Mobile Phase: 10 mM ammonium acetate buffer (pH 4.5)/
0.05% acetic acid in acetonitrile = 95:5–10:90 v/v; Wave-
length: PDA 220–420 nm). The solubilities were analyzed
using Millenium software program (Waters).
10. Haginoya, N.; Komoriya, S.; Osanai, K.; Yoshino, T.;
Nagata, T.; Nagamochi, M.; Muto, R.; Yamaguchi, M.;
Kanno, H.; Nagahara, T. Heterocycles 2004, 63, 1555.
11. The structures of acylated compounds (14 and 17) were
determined by comparison with NOE measurements
between major and minor products.
15. Obach, R. S. Drug Metab. Dispos. 1999, 27, 1350.
16. T1/2 and CLint were calculated using the remaining ratio
(X%) of the substrate after 5 min. kel (/min): disappearance
rate constant. Cm, concentration of microsome. We
assumed that human liver microsome weight is 45 mg/g
of liver and human liver weight is 20 g/kg of body weight.
12. Anti-fXa activity in vitro was measured by using a
chromogenic substrate S-2222 (Chromogenix, Inc.) and
human fXa (Enzyme Research Laboratories). Aqueous
DMSO (5%, 10 lL) or inhibitors in aqueous DMSO (10 lL)
and 0.0625 U/mL human fXa (10 lL) were mixed with
0.1 M Tris–0.3 M NaCl–0.2% BSA buffer (pH 7.4; 40 lL).
The reaction was started by the addition of 0.75 M S-2222
(40 lL). The absorbance (O.D.) at 405 nm was monitored
every 10 s with a microplate spectrophotometer SPEC-
TRAmax 340 (Molecular Devices, Sunnyvale, CA, USA) at
room temperature and the reaction velocity (mO.D./min)
was obtained. Anti-fXa activity (inhibition %) was calcu-
lated as follows: Anti-fXa activity = (1 À (reaction velocity
of sample) Ä (reaction velocity of control)) · 100. The IC50
value was obtained by plotting the inhibitor concentration
against the anti-fXa activity.
13. The Japanese Pharmacopoeia Second fluid was prepared
as follows. 0.2 mol/L sodium hydroxide solution (118 mL)
was added to dihydrogenphosphonate potassium (6.80 g).
To the mixture, water was added to make colorless clear
liquid (1000 mL). The pH of this liquid was confirmed to
be 6.70–6.90. Before use, the liquid was filtered by
membrane filter.
14. The solubilities were determined by HPLC analysis. Ten
millimolar of compound solution in DMSO (50 ll) was
freeze-dried. To the residue the Japanese Pharmacopoeia
Second fluid (250 ll, pH 6.8) was added, and the mixture
was stirred by pipette operation. The mixture was stored
under shade for 12 h. After filtration of the mixture, the
filtrate was diluted by 20 times by adding aqueous DMSO
solution (1:1 (v/v)) to obtain measurement sample solu-
tion. Five micromolar of compound solution in aqueous
DMSO solution (1:1 (v/v)) and 100 lmol compound
X/100 = exp(Àkel 5). kel (/min) = Àln(X/100)/5. T1/
*
2 = ln2/kel = 5 ln2/ln(100/X). CLint = kel/Cm = Àln [(X/
*
100)/5]/1.0 45 20.
*
*
17. Anti-fXa activity in rat ex vivo. Male Wistar rats were
fasted overnight. Synthetic compounds were dissolved in
0.5% methylcellulose solution and administered orally to
rats with a stomach tube. For control rats, 0.5% methyl-
cellulose solution was administered orally. The rats were
anesthetized with thiopental sodium (100 mg/kg, i.v.)
15 min after the oral administration. Blood samples
(450 lL) were collected from the jugular vein into syringes
containing 50 lL of 3.13% trisodium citrate dihydrate 0.5,
1, 2, and 4 h after dosing. Plasma was prepared by
centrifugation. Anti-fXa activity in plasma was measured
as follows. Plasma sample (5 lL) was mixed with 0.1 M
Tris–0.3 M NaCl–0.2% BSA buffer (pH 7.4; 40 lL), H2O
(5 lL), and 0.1 U/mL human fXa (10 lL). The reaction
was started by the addition of 0.75 M S-2222 (40 lL). The
reaction velocity and anti-fXa activity (inhibition %) were
obtained as mentioned above.
18. Synthetic compound was administered orally to monkey
(1 mg/kg) in aqueous solution. Blood samples were
collected using Labospeed tubes (Toyo-kizai Inc.) at 0.5,
1, 2, 4, 8, and 24 h after oral dosing. Respective samples at
each time points were collected in n = 3. Serum concen-
trations for synthetic compound were determined by LC–
MS/MS using Sciex API 365 (Sciex Inc.) coupled with
Alliance 2690 HPLC system (Waters Inc.). Compound
was separated on a Symmetry C18 column (Waters Inc.).
The quantitation limit was 7 ng/mL. Respective pharma-
cokinetic parameters were measured using Top Fit ver. 2.0
(Gustav Fischer Inc.).