Design, synthesis, and structure-activity relationships of unsubstituted piperazinone-based transition state factor Xa inhibitors

Article abstract of DOI:10.1016/S0960-894X(02)01037-5

A series of novel transition state factor Xa inhibitors containing a variety of lactam ring systems as central templates was synthesized in an expedient manner and allowed for a great deal of structural variability. Among them, the piperazinone-based inhibitors were found to be not only active against factor Xa but also selective over thrombin. Optimization of the P4 moiety yielded several potent compounds with IC₅₀ below 1 nM against factor Xa.

Full text of DOI:10.1016/S0960-894X(02)01037-5

Bioorganic & Medicinal Chemistry Letters 13 (2003) 723–728
Design, Synthesis, and Structure–Activity Relationships of
Unsubstituted Piperazinone-Based Transition State Factor
Xa Inhibitors
Wenrong Huang,a,* Mary Ann Naughton,a Hua Yang,a Ting Su,a Suiko Dam,b
Paul W. Wong,b Ann Arfsten,b Susan Edwards,b Uma Sinha,b Stanley Hollenbach,c
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
cDepartment of In Vivo Science, Millennium Pharmaceuticals Inc., 256 East Grand Ave., South San Francisco, CA 94080, USA
Received12 February 2002; accepted1 November 2002
Abstract—A series of novel transition state factor Xa inhibitors containing a variety of lactam ring systems as central templates was
synthesized in an expedient manner and allowed for a great deal of structural variability. Among them, the piperazinone-based
inhibitors were foundto be not only active against factor Xa but also selective over thrombin. Optimization of the P4 moiety yiel-
ded several potent compounds with IC50 below 1 nM against factor Xa.
# 2003 Elsevier Science Ltd. All rights reserved.
Factor Xa is a trypsin-like serine protease positionedat
the convergence point of both intrinsic andextrinsic
coagulation pathways. It plays a pivotal role in the
bloodcoagulation cascade andin maintaining homeo-
stasis.1 Factor Xa forms a prothrombinase complex
with factor Va, Ca2+ andphospholipids. It is the active
enzyme in this complex that catalyzes the conversion of
inactive prothrombin to active thrombin. Thrombin is
the final serine protease in the coagulation pathway that
ultimately leads to blood clotting. Factor Xa has
recently emergedas an attractive pharmacological tar-
get for the development of improved antithrombotic
agents.2ꢀ9 Highly potent andspecific factor Xa inhibi-
tors have been shown to be efficacious in several animal
thrombosis models.10 They can effectively block both
venous andarterial thrombosis formation andmay have
a much wider therapeutic window than direct thrombin
inhibitors, heparin andLMWH, andwarfarin.
andselective (against thrombin) inhibitors for factor
Xa. For example, compound 1 has a Ki value of 13 pM
andan IC
value of 10,000 nM against factor Xa and
50
thrombin, respectively. However, concerns with its pep-
tidic features and highly basic character of the two gua-
nidinium groups led us to design compounds with better
physiochemical properties. In this letter, the design,
synthesis, andSAR of a series of novel lactam based
transition state factor Xa inhibitors will be presented.
Although the X-ray structure of 1 with factor Xa has
not been determined, we believe there are many inter-
actions that leadto the high inhibitory activity. It is
likely that the ketone carbonyl group forms a reversible
covalent bondwith the hydroxyl group of Ser-195 of
factor Xa. The thiazole group may also form a hydro-
gen bondwith the imidazole ring of His-57. The P1 Arg
side chain would insert into the S1 specific pocket and
form ionic interactions with the carboxylic acidside
chain of Asp-189. Both the P3-(d)-Arg side chain and the
P4 sulfonamide could occupy the S4 aryl-binding pocket
of factor Xa through hydrophobic interactions and the
guanidinium group of (d)-Arg might also form ionic
interactions with the S40cation hole of factor Xa. This
hypothesis ledus to believe that the highly positively
chargedP3-( d)-Arg couldbe replacedby hydrophobic or
Previously, we reporteda series of tripeptide transition
state factor Xa inhibitors, R-SO2-(d)-Arg-Gly-Arg-
Thiazole.2,11 This class of compounds is highly potent
*Corresponding author. Fax: +1-650-244-9287; e-mail: bingyan.zhu
@mpi.com
0960-894X/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0960-894X(02)01037-5

724
W. Huang et al. / Bioorg. Med. Chem. Lett. 13 (2003) 723–728
polar amino acids such as (d)-Phe, (d)-Cha, and
(d)-Trp. These compounds retained the potency against
factor Xa, but they are significantly less selective over
thrombin.2,12 For example, compound 2 has IC50 values
of 2 and870 nM for factor Xa andthrombin, respec-
tively. Nevertheless, these results indicated that the S4
pocket of factor Xa couldaccommodate a variety of
motifs, thus, the P3-P4 part of a factor Xa inhibitor
couldbe reaidly manipulatedwithout sacrificing the
Scheme 2. (a) HN(OCH3)(CH3), BOP, DIEA, CH2Cl2; (b) thiazole,
n-BuLi, THF, ꢀ78 ꢁC; (c) THF; (d) TFA, CH2Cl2.
potency. Inspiredby the pioneering work in the elastase
13ꢀ15
andthrombin inhibitors areas,
we introduced the
structural constraints into the molecules. Utilizing dif-
ferent cyclization approaches, we designed several sub-
stitutedlactam ring systems as the templates to mimic
the P2–P3–P4 region of our tripeptide transition state
inhibitors (Fig. 1). By cyclizing the P3-(d)-Arg side
chain and P2–P3 back bone amide nitrogen, compounds
with the a-amino lactam, such as compound 3a, were
obtained. Similarly, cyclization of the sulfonamide
nitrogen with the P2–P3 back bone amide nitrogen
produced the piperazinone based transition state inhi-
bitors (3b). The subsequent article16 exploredthis
approach further andidentifieda series of substituted
piperazinone transition state factor Xa inhibitors.
Scheme 3. (a) BnBr, DIEA, DMF; (b) piperidine, DMF; (c) BnSO2Cl,
DIEA, CH2Cl2, ꢀ78 ꢁC; (d) H2, 10% Pd/C, EtOH.
Scheme 4. (a) (i) NaH, THF, ꢀ78 ꢁC; (ii) ICH2CO2Et; (b) H2 (40 psi),
Pd/C, MeOH; (c) BnSO2Cl, Et3N, DMF, 0 ꢁC; (d) 0.2 N LiOH, H2O,
dioxane.
Scheme 5.17 (a) EtONa, EtOH, 75 ꢁC; (b) (i) aq NaOH; (ii) concd
HCl; (iii) CH3CN, reflux; (c) H2 (40 psi), PtO2, MeOH, H2O; (d)
Boc2O, Et3N, CH2Cl2; (e) (i) LiN(TMS)2, THF, ꢀ78 ꢁC; (ii) ICH2-
CO2Et; (f) LiOH, EtOH.
Figure 1. Design of novel factor Xa inhibitors featuring lactam ring
systems as P2–P3 mimetic.
Scheme 6. (a) (i) LiN(TMS)2, THF, ꢀ78 ꢁC; (ii) ICH2CO2Et; (b) H2,
Pd/C, MeOH; (c) BnSO2Cl, DIEA, CH2Cl2, ꢀ78 ꢁC (4b and 4g), or
BnBr, Et3N, THF (4f); (d) LiOH, EtOH.
Two general strategies were usedto prepare the inhibi-
tors reportedin this communication. Our initial
approach utilizedsynthetic methods to construct the
P2–P3–P4 segment containing a variety of lactams
before they were connectedto the P1 segment. For
Scheme 7. (a) ICH2CO2Et, DIEA, DMF, 60 ꢁC; (b) TFA, CH2Cl2; (c)
BnSO2Cl, DIEA, CH2Cl2, ꢀ78 ꢁC; (d) LiOH, EtOH.
Scheme 1. (a)13 (i) H2 (45 psi), 10% Pd/C, MeOH/H2O/AcOH (10/6/
1), OHCCO2H; (ii) MeOH, 60 ꢁC; (b) BnBr, DIEA, DMF; (c) TFA,
CH2Cl2; (d) BnSO2Cl, DIEA, CH2Cl2, ꢀ78 ꢁC; (e) H2, 10% Pd/C,
EtOH; (f) 5, BOP, DIEA, DMF; (g) HF.
Scheme 8. (a) 1.6 N aq HCl, reflux, 4 h; (b) Boc2O, Na2CO3, dioxane/
H2O; (c) 5, BOP, DIEA, DMF; (d) TFA, CH2Cl2; (e) R0SO2Cl, DIEA,
DMF, ꢀ78 to 0 ꢁC or R0COCl, DIEA, CH2Cl2, 0 ꢁC; (f) HF.

W. Huang et al. / Bioorg. Med. Chem. Lett. 13 (2003) 723–728
725
example, to make compound 3a (Scheme 1), the key
intermediate (2-oxo-3-phenylmethanesulfonyl-piperidin-
1-yl)-acetic acid( 4a) was formedthrough a five-step
sequence from a-Boc-o-Cbz-l-Orn. It was then assem-
bledwith NH 2-Arg(Tos)-thiazole (5) through BOP
coupling, followedby HF deprotection to remove the
tosyl group giving the desired inhibitor 3a. Compound 5
was obtainedfrom the TFA treatment of Boc-NH-
Arg(Tos)-thiazole (8), which was obtainedthrough
a-position of the lactam, showedmodest potency for
factor Xa but excellent selectivity between factor Xa
andthrombin. To our edlight, when the neutral P4
moiety was movedinto the b-position of the lactam, the
resulting compounds, 3b and 3g, displayed the desired
potency against factor Xa as well as the selectivity over
thrombin. Although compound 3g is more selective
than 3b, compound 3b was chosen as the leadfor fur-
ther structure modifications and SAR studies due to the
relative synthetic accessibility of the piperazinone tem-
plate. Compound 3h demonstrated the importance of
the ring carbonyl group towards activity. The carbonyl
group presumably acts in the following two ways: to
eliminate the positive charge of the nitrogen andto
provide hydrogen bonding with factor Xa.
reaction of 2-lithiothiazole andthe Weinreb amide
(Scheme 2). Compounds 3b–3h were preparedin a
7
similar fashion from the corresponding acids 4b–4h,
which were synthesizedaccording to Schemes 3–7.
The secondsynthetic approach was developedfor mak-
ing a large number of piperazinone compounds con-
taining a variety of P4 moieties. As shown in Scheme 8,
the readily available diacid 9 was cyclized under acidic
conditions.18 After neutralization of the reaction solu-
tion, the Boc group was installedto give the acid 10.
BOP coupling of 10 with 5 ledto the key intermediate
11. Compound 11 was subjectedto TFA treatment,
followedby N-acylation or N-sulfonylation andsub-
sequent HF deprotection to give the desired inhibitors
12–46. This process allowedus to produce many analo-
gues rapidly by using commercially available acid
chlorides and sulfonyl chlorides.
The SAR of the piperazinone-basedinhibitors with
modified P4 will be the focus of the remainder of this
report. First, the effect of different P4 linkers on inhibi-
tory activities was examined( Table 2). Unsubstituted
amine 12 is 500 times less potent than 3b indicating that
the P4 moiety provides crucial binding for the inhibitory
activity. We compared carboxamide, sulfonamide and
methylene moiety as linkers, anddiscoveredthat the
sulfonamide linkage generally led to more potent factor
Xa inhibitors. For example, sulfonamide 14 is nearly
100 and40 times more potent than compounds 3f and
13, respectively. In the sulfonamide series, extending the
length of the P4 moiety only slightly increases the
potency for factor Xa (3b vs 14). By contrast, this has a
more positive influence on the inhibitory activity for the
carboxamide series. Compound 17 is about 15 times
more active than 13, and 18 is 7 times more active than
15 against factor Xa.
Table 1 summarizes the in vitro IC50 data11,19 of the
compounds containing different lactams. The a-amino-
lactam compounds containing a nonbasic P4 moiety
(BnSO2), 3a, 3c, and 3d, are potent against factor Xa
but have little selectivity over thrombin. Interestingly,
compound 3e, which has a basic group installedat the
We noticedthat a chloro substituent on the phenyl ring
improvedthe potency in the sulfonamide series ( 14 ver-
sus 16). This promptedfurther investigation into the
effect of substituents on the in vitro activities (Table 3).
When a halogen substituent is placedat the 4-position
of the phenyl ring (19, 16, and 20), a modest increase in
both potency andselectivity is observedrelative to the
unsubstitutedphenylsulfonameid 14. The 4-bromo
compound 20 displayed a significant 10- and 20-fold
increase in activity for factor Xa andselectivity against
Table 1. Inhibitory activities for compounds with different central
templates
CompdStructure
IC
50 (nM)
Thrombin
Factor Xa
29
3a
138
3c
3d
3e
3b
3g
3h
3
38
8
Table 2. Effect of linkers on the inhibitory activities for piper-
azinone-basedinhibitors
3
65
12,000
390
CompdAr
X
IC
(nM)
50
Factor Xa
Thrombin
4
5
12
3f
—
H
22,000
771
320
8
220,000
14,000
7000
417
5000
1000
2000
11,000
390
Phenyl
Phenyl
Phenyl
4-Cl-phenyl
4-Cl-phenyl
Benzyl
CH2
CO
SO2
CO
SO2
CO
CO
SO2
13
14
15
16
17
18
3b
2000
118
2
21
17
24,000
>500,000
4-Cl-benzyl
Benzyl
4

726
W. Huang et al. / Bioorg. Med. Chem. Lett. 13 (2003) 723–728
Table 3. Effect of substituents on the P4 phenyl sulfonamide on
inhibitory activities for piperazinone-basedinhibitors
Moderate loss of activity was observed for compounds
30, 31, and 32 containing 4-CN, 4-SO2Me anda sub-
stitutedphenoxyl phenylsulfonamied, respectively. A
4-CO2H (33) substituent at the phenyl ring dramatically
reduces the inhibitory activity towards both enzymes,
indicating that an acidic group is not well tolerated in
the S4 binding pocket.
CompdX
IC
50 (nM)
Thrombin
Factor Xa
We also investigatedthe structure–activity relationships
of piperazinone-basedfactor Xa inhibitors with aro-
matic bicyclic and other sulfonamide derivatives at P4
position. As shown in Table 4, compounds containing a
wide variety of sulfonamide at P4 are highly potent
factor Xa inhibitors. This indicates that the hydro-
phobic S4 pocket of factor Xa can accommodate large
aromatic ring systems. SAR of the regioisomers of
naphthlene sulfonamide compounds indicates that the
1-naphthlene sulfonamide is slightly preferred over
2-naphthlene for potency. However, the 2-naphthlene
sulfonamide leads to more selective factor Xa inhibitor.
For example, compound 36 is 4 times more active than
34 for factor Xa, but about 10 times less selective
between factor Xa andthrombin. Insertion of an ethyl
unit between the 1-naphthlene andthe sulfonyl group of
36 yielded 38 with comparable potency andselectivity.
Again, a chloro substituent on the naphthlene ring
enhancedthe activity for factor Xa ( 35 vs 34 and 37 vs
36). Similar to 36, 5-isoquinoline compound 39,
8-quinoline 40 and4-benzo [2,1,3]oxadiazole 41 dis-
playedhigh potency for factor Xa but less desirable
selectivity over thrombin. The benzothiophene sulfona-
mide 42 is the most active factor Xa inhibitor in this
investigation. It is also highly selective over thrombin.
High potency andselectivity was also displayedby the
biaryl compound 43. Compound 44 can be visualizedas
a close analogue of compound 34 that removes part of
the naphthyl ring. Both compounds exhibited compar-
able in vitro activities. Compounds 45 and 46, which
contain substitutedheterocyclic sulfonamides at P4, are
only moderate factor Xa inhibitors.
14
19
16
20
21
22
23
24
25
26
27
28
29
30
31
32
33
H
4-F
4-Cl
4-Br
8
2
2
0.7
5
1
417
751
1000
809
134
1000
573
3-Cl
3,4-diCl
2,4,5-triCl
4-OMe
4-CF3
4-NO2
4-NH2
1
0.89
10
6
792
6000
3000
5000
7000
5000
3000
4000
4000
17,000
6
4-NHOH
4-NHCOCH3
4-CN
4-SO2Me
4-O(3-Cl-2-CN)Ph
4-CO2H
10
116
32
85
38
510
thrombin, respectively. A 3-chloro substituent at the
phenyl ring (21) has little effect on the potency but sig-
nificantly reduces the selectivity. Multiple chloro sub-
stituents (22 and 23) do not further enhance the
inhibitory activities.
Non-halogen substituents also have a noticeable influ-
ence on the in vitro activities. The 4-OMe group (24)
increases the inhibitory activity for factor Xa by 10 fold
when comparedto 14. Compounds 25–28 containing
4-CF3, 4-NO2, 4-NH2, or 4-NHOH phenyl sulfonamide,
displayed comparable inhibitory activities for factor Xa
with much improvedselectivity over thrombin. Inter-
estingly, acylating the amino group of 27 afforded 29,
which is 20 times less active andselective than 27.
Basedon the in vitro IC
data, selected compounds
50
Table 4. Inhibitory activities for piperazinone-basedinhibitors with
other P4 moieties
were profiledin a number of in vitro andin vivo studies.
Table 5 summarizes the results of detailed in vitro factor
Xa binding kinetic studies, enzyme selectivity data, in
vitro anticoagulant activity studies, and in vivo studies
of antithrombotic efficacy andpharmacokinetic proper-
ties. Ki studies20 unveiledthat these piperazinone based
compounds are considerably less potent than the tri-
peptide compound 1. They have comparable Kon’s, but
the piperazinone basedcompounds have much faster off
rates (Koff) than 1. Besides the thrombin selectivity that
we have already discussed in the above context, these
compounds display high selectivity over activated pro-
tein C (aPC), relatively high selectivity over tissue plas-
minogen activator (tPA) andplasmin (except for
compound 40). Their selectivity towards plasma kallik-
rein is less desirable. All of these transition state factor
Xa inhibitors suffer from low selectivity towards trypsin,
which makes them unsuitable for oral administration.
CompdAr
IC
(nM)
50
Factor Thrombin
Xa
34
35
36
37
38
39
40
41
42
43
44
45
46
2-Naphthyl
5-Chloro-naphthalen-2-yl
1-Naphthyl
5-Chloro-naphthalen-1-yl
2-Naphthalen-1-yl-ethyl
5-Isoquinolinyl
8
0.79
2
0.62
3
1
2
2
6000
2000
118
185
375
267
569
719
751
4000
7000
4000
669
8-Quinolinyl
Benzo[2,1,3]oxadiazol-4-yl
5-Chloro-3-methyl-benzo[b]thiophen-2-yl 0.51
5-Pyridin-2-yl-thiophen-2-yl
trans-1-Styryl
2-Acetylamino-4-methyl-thiazol-5-yl
3,5-Dimethyl-isoxazol-4-yl
4
13
58
69
The anticoagulant activity of these compounds were
testedboth in vitro in the plasma basedthrombin

W. Huang et al. / Bioorg. Med. Chem. Lett. 13 (2003) 723–728
Table 5. In vitro andin vivo profiles for selectedcompounds
727
Compd
1
3b
16
34
35
40
42
In vitro factor Xa binding assays
IC50 (nM)
Ki (nM)
Kon (mM-1 S-1)
Koff (S-1)
0.5
0.013
6.5
3.1 ꢂ 105
4
1.4
2.3
0.013
2
8
2.7
6.3
0.132
0.8
1.3
NA
NA
2
4.4
2.9
0.107
0.5
0.17
NA
NA
2.3
3.4
0.19
Enzyme selectivity data IC50 (nM)
Thrombin
Trypsin
tPA
aPC
Plasmin
Kallikrein
10,000
0.8
329
6000
309
390
33
4010
>180,000
12,100
677
1000
10.8
1880
>180,000
10,700
406
6000
39.1
1850
>180,000
6960
390
2000
20.5
740
170,000
1880
34
569
2
150
751
20
9960
169,000
1300
9.4
>180,000
10
240
27
In vitro functional assays
2X TG (mM)
2X PT (mM)
2X APTT (mM)
Plasma protein binding (%)
0.18
6.8
0.5
43
NA
11.7
2.8
0.52
11.2
4.2
NA
14.5
13.5
95
NA
12.5
12.5
>99
2.5
10.4
2
5.46
14.5
15.5
>99
NA
82
NA
In vivo anticoagulant efficacy in rabbits andpharmacokinetics in rats
% inhibition
Plasma concentration (mM)
IV t1/2 in rats (min)
100
0.07
24
35
0.4
10
90
0.5
22
0
0.8
15
0
0.2
51
0
0.4
29
0
1.6
5
generation (TG),21 prothrombin time (PT),22 andacti-
vatedpartial thromboplastin time (APTT) 22 assays, and
in vivo in a rabbit venous thrombosis model.22 The tri-
peptide 1, being extremely potent in binding assays and
highly hydrophilic, displayed high TG and APTT
activity but poor PT activity. It showedcomplete
inhibition of thrombosis formation at a very low plasma
concentration (70 nM) in vivo. The piperazinone com-
pound 16 with a Ki of 2 nM against factor Xa and82%
plasma protein binding, displayed 90% inhibition of
thrombosis formation at 500 nM plasma concentration.
It is noteworthy that although compound 16 is sig-
nificantly less efficacious in vivo than compound 1, they
have similar activity in the PT assay. The APTT assay did
show the same degree of difference in activity between
these two compounds. Compounds 34, 35, and 40, which
have similar factor Xa binding affinity to 16, did not dis-
play significant inhibition of thrombosis formation in
vivo. Even compound 42 with a Ki of 0.17 nM does not
have the desired in vivo efficacy. These compounds are
highly plasma protein bound, which hampered the
effectiveness of translating high factor Xa binding affi-
nity into significant in vivo antithrombotic efficacy in
the rabbit model. However, the PT and APTT data for
compounds 3a, 16, 34, 35, 40, and 42 are comparable
anddo not correlate to their in vivo efficacy. Thus, PT
andAPTT assays are not sensitive enough to differ-
entiate andpredict the in vivo antithrombotic potentials
of these piperazinone basedtransition state factor Xa
inhibitors. A similar phenomenon was observedfor
fondaparinux, a indirect factor Xa inhibitor.23,24 For
example, at fully efficacious doses that produce a
plasma concentration of 300 nM, no PT or APTT is
extended by fondaparinux. It showed a 2X APTT
value of 29 mM andonly exteneddPT by 7% at
58 mM. TG assay has been demonstrated to be more
reliable in predicting the in vivo antithrombotic activ-
ity of factor Xa inhibitors. Fondaparinux displayed a
2X TG of 300 nM. Compound 16 is 5 and10 times
more active than compounds 40 and 42 in the TG
assay, respectively.
The pharmacokinetic properties of this class of novel fac-
tor Xa inhibitors were evaluatedin Sprague–Dawley rats.
When administered by an IV bolus dose at 1 mg/kg, these
compounds exhibited relatively short half life, ranging
from 5 min for compound 42 to 51min for compound 35.
In summary, during our effort to modify compound 1 to
reduce its peptidic character and high basicity, we have
discovered and developed a series of novel transition
state factor Xa inhibitors containing a-lactam ring sys-
tem as P2–P3 mimetic. Among the lactams being stud-
ied, the piperazinone-based compounds drew the most
attention with the following advantages: (1) they con-
tain only one chiral center andone positively charged
guanidinium group, (2) they can be rapidly synthesized
with readily available starting materials, and (3) they
displayed the desired in vitro profile in terms of inhibi-
tory potency for factor Xa andselectivity against
thrombin. Modifications of the P4 moiety led to highly
potent inhibitors with IC50 below 1 nM for factor Xa.
Some of the compounds achieved high specificity for
factor Xa versus thrombin. Compound 16 also exhib-
itedsignificant inhibition of thrombosis formation in a
rabbit venous thrombosis model.
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