Potent direct inhibitors of factor Xa based on the tetrahydroisoquinoline scaffold

Article abstract of DOI:10.1016/j.ejmech.2012.06.032

Direct inhibition of coagulation factor Xa (FXa) carries significant promise for developing effective and safe anticoagulants. Although a large number of FXa inhibitors have been studied, each can be classified as either possessing a highly flexible or a rigid core scaffold. We reasoned that an intermediate level of flexibility will provide high selectivity for FXa considering that its active site is less constrained in comparison to thrombin and more constrained as compared to trypsin. We studied several core scaffolds including 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid for direct FXa inhibition. Using a genetic algorithm-based docking and scoring approach, a promising candidate 23 was identified, synthesized, and found to inhibit FXa with a K_i of 28 μM. Optimization of derivative 23 resulted in the design of a potent dicarboxamide 47, which displayed a K_i of 135 nM. Dicarboxamide 47 displayed at least 1852-fold selectivity for FXa inhibition over other coagulation enzymes and doubled PT and aPTT of human plasma at 17.1 μM and 20.2 μM, respectively, which are comparable to those of clinically relevant agents. Dicarboxamide 47 is expected to serve as an excellent lead for further anticoagulant discovery.

Full text of DOI:10.1016/j.ejmech.2012.06.032

European Journal of Medicinal Chemistry 54 (2012) 771e783
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European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Potent direct inhibitors of factor Xa based on the tetrahydroisoquinoline scaffold
*
Rami A. Al-Horani, Akul Y. Mehta, Umesh R. Desai
Department of Medicinal Chemistry and Institute for Structural Biology and Drug Discovery, Virginia Commonwealth University, Richmond, VA 23298, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Direct inhibition of coagulation factor Xa (FXa) carries significant promise for developing effective and
safe anticoagulants. Although a large number of FXa inhibitors have been studied, each can be classified
as either possessing a highly flexible or a rigid core scaffold. We reasoned that an intermediate level of
flexibility will provide high selectivity for FXa considering that its active site is less constrained in
comparison to thrombin and more constrained as compared to trypsin. We studied several core scaffolds
including 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid for direct FXa inhibition. Using a genetic
algorithm-based docking and scoring approach, a promising candidate 23 was identified, synthesized,
and found to inhibit FXa with a Ki of 28 mM. Optimization of derivative 23 resulted in the design of
a potent dicarboxamide 47, which displayed a Ki of 135 nM. Dicarboxamide 47 displayed at least 1852-
fold selectivity for FXa inhibition over other coagulation enzymes and doubled PT and aPTT of human
plasma at 17.1 mM and 20.2 mM, respectively, which are comparable to those of clinically relevant agents.
Received 14 March 2012
Received in revised form
12 June 2012
Accepted 15 June 2012
Available online 23 June 2012
Keywords:
Anticoagulant
Direct inhibition
Structureeactivity relationships
Coagulation enzymes
Tetrahydroisoquinoline synthesis
Dicarboxamide 47 is expected to serve as an excellent lead for further anticoagulant discovery.
Ó 2012 Elsevier Masson SAS. All rights reserved.
1. Introduction
occurrence earlier in the coagulation pathway suggests that a FXa
inhibitor may be more effective in blocking progression of a coag-
Anticoagulants represent the basis for treatment and prevention
of thromboembolic disorders [1]. A priori, the inhibition of any
coagulation enzyme can be expected to reduce or prevent clotting,
yet most strategies have targeted two serine proteases, factor IIa
(FIIa, or thrombin) and factor Xa (FXa), which belong to the common
pathway of the coagulation cascade [2]. Of these, thrombin plays
a major role in a number of physiologically relevant responses that
rely on its catalytic activity [3,4]. Thus, inhibition of thrombin not
only reduces cleavage of fibrinogen to fibrin, a key aspect of clotting,
but also other processes such as platelet activation, platelet aggre-
gation and angiogenesis. On the other hand, FXa has a rather limited
role in comparison, which is to generate thrombin.
ulation signal than a thrombin inhibitor [7,8]. Recent results with
indirect parenteral and direct oral FXa inhibitors have shown
reduced bleeding complications [9]. Also, thrombin inhibitors have
been associated with rebound hypercoagulability [10,11], while
peptidomimetic inhibitors have been shown to inhibit both free as
well as clot-bound FXa [12,13].
FXa can be inhibited through two major pathways including an
indirect, antithrombin-dependent pathway [14,15] or a direct FXa
targeting pathway [16e18]. Designing indirect FXa inhibitors that
activate antithrombin is challenging and has been achieved
primarily with heparin-based molecules [14,19], although efforts
are afoot to design effective non-saccharide-based antithrombin
activators [15,20,21]. In contrast, designing direct inhibitors of FXa
has been much more productive and has led to several clinically
relevant peptidomimetics, e.g., rivaroxaban [22], apixaban [23],
DPC423 [24], and others, that target FXa’s active site.
Several lines of evidence suggest that FXa may represent a better
target for anticoagulation. FXa’s contribution to amplification of the
coagulation signal is higher than that of thrombin [5,6]. Its
A large number of scaffolds have been studied for direct inhi-
bition of FXa including the aminopiperidines [25], piperazines [26],
diaminocycloalkanes [27], lactams [28,29], oxazolidinones [30],
Abbreviations: ABH3CA, 2-azabicyclo[2.2.1]heptane-3-carboxylic acid; APTT,
activated partial thromboplastin time; FIIa, factor IIa (thrombin); FXa, factor Xa; HS,
hill slope; P2CA, piperidine-2-carboxylic acid; P3CA, piperidine-3-carboxylic acid;
PA, phenylalanine; PT, prothrombin time; THIQ, 1,2,3,4-tetrahydroisoquinoline;
THIQ3CA, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid; THP2CA, 1,2,3,6-
tetrahydropyridine-2-carboxylic acid.
* Corresponding author. Department of Medicinal Chemistry, Virginia
Commonwealth University, 800 East Leigh Street, Suite 212, PO Box 980133, Rich-
mond, VA 23219, United States. Tel.: þ1 804 828 7328; fax: þ1 804 827 3664.
E-mail address: urdesai@vcu.edu (U.R. Desai).
amino acids (e.g., glycine, proline, and
b-aminoproionate) [31,32],
anthranilamides [33], isoxazoles [34], pyrazoles [24,35], indazoles
[36], indoles [37,38], and dihydropyrazolopyridinones [23,39].
The overall philosophy in the design of these scaffolds is to have
a three-component system, which includes a core scaffold and two
hydrophobic arms that provide a non-linear geometry considered
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.06.032

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R.A. Al-Horani et al. / European Journal of Medicinal Chemistry 54 (2012) 771e783
important for FXa recognition. In addition, a key design principle
has also been flexibility of the core scaffold. The core scaffolds
studied to date can be classified into either a highly flexible class or
a fairly rigid class.
It is known that trypsin’s active site is much open than FXa’s,
which in turn possesses an active site that is more open than that of
thrombin. This allows trypsin to work upon practically any arginine
containing sequence, while thrombin prefers a fairly rigid proline
containing sequence. Thus, we hypothesized that an intermediate
level of flexibility in the core scaffold will engineer high selectivity
for FXa inhibition.
In this work, we studied several core scaffolds with variable
flexibility to design potent direct FXa inhibitors. Of the
scaffolds studied, the 1,2,3,4-tetrahydroisoquinoline-3-carboxylic
acid (THIQ3CA) scaffold possessing an intermediate level of flexi-
bility was found to be the best. Molecular modeling-based identi-
fication of an initial hit 23 was transformed into the discovery of
a potent direct FXa inhibitor 47 with a Ki of 135 nM. This molecule
selectively targeted FXa in comparison to the enzymes of the
coagulation and digestive systems, and doubled clotting times at
17e20 mM, which are comparable to those of the inhibitors being
studied in clinical trials [40]. The work with a library of 39 molecules
has revealed interesting structureeactivity relationships (SAR) that
identify optimal structural units for engineering good FXa inhibition
properties. The lead THIQ3CA dicarboxamide 47 is expected to serve
as an excellent starting point for further advanced design.
2. Results and discussion
2.1. Designing the tetrahydroisoquinoline-3-carboxylic acid scaffold
as a potential factor Xa inhibitor scaffold
Fig. 1. Virtual screening of a library of potential THIQ3CA-based inhibitors docked and
scored onto the active site of FXa (PDB: 2P16) using GOLD resulted in the design of 23
(A: GOLD score of 69.3, Carbons in pink) that appeared to mimick the binding of well
known potent FXa inhibitors. The virtual library of THIQ3CAebased dicarboxamides
contained structural modifications in arms AN and AC. Apixaban (GOLD score of 93.7,
Carbons in blue) and rivaroxaban (GOLD score of 87.2, structure not shown) were also
docked and scored as positive controls. Systematic structural modifications led to the
design of the most potent THIQ3CA dicarboxamide 47 (B: GOLD score of 77.2, Carbons
in yellow). See text for details.
The glycine and
b-aminopropionate-based inhibitors are
examples of a highly flexible core scaffold, while aromatic scaffolds,
e.g., aminobenzoic acids, pyrazoles, and indazoles, are examples of
fairly rigid scaffolds. An intermediate level of flexibility in the core
scaffold has not been studied to date for direct FXa inhibition. A
large number of scaffolds possess intermediate level of flexibility
including tetrahydroisoquinoline (THIQ), tetrahydroquinoline, tet-
rahydroquinazoline, dihydrocoumarin, and dihydroindole. In each
of these, the five- or six-membered rings are flexible, yet full flex-
ibility is restricted due to ring fusion. Of these, we focused on the
THIQ scaffold, which is a widely explored privileged structure
[15,41e44]. To enable a non-linear structure for FXa recognition, we
introduced a carboxylic acid group at the 3-position of the THIQ
scaffold to arrive at THIQ3CA scaffold.
The designed THIQ3CA scaffold was first investigated in silico to
assess its potential in binding in factor Xa active site as well as to
identify a potential inhibitor for synthesis. A library of THIQ3CA
structures was prepared by introducing substituents at positions 2
and 3. A large group of aromatic and non-aromatic carboxylic acid
and amine armsatpositions 2 and 3 were studied to identifypotential
‘hits’ (see Supplementary Information). The virtual libraryofresulting
THIQ3CA dicarboxamides was then computationally docked into
active site of human FXa and scored using genetic algorithm-based
screening technique developed in our laboratory earlier [15]. As
positive controls, two well-established direct FXa inhibitors, apix-
aban and rivaroxaban, were also studied in an identical manner.
The GOLD-based docking and scoring strategy identified several
the S1 subsite and the 4-(piperidin-2-one)-N1-methylaniline
moiety oriented into the S4 subsite. Comparison of the binding
geometry with rivaroxaban suggested similar corresponding
features (not shown). THIQ3CA dicarboxamide 23 was synthesized
in three high yielding steps and evaluated for its direct human FXa
inhibition potential (see below). An IC50 of 55.9
mM, corresponding
to a Ki of 28 M, was measured validating the potential of the
m
THIQ3CA core scaffold in direct human FXa inhibition investigation.
2.2. Synthesis of THIQ3CA-based potential FXa inhibitors
Based on the structure of 23, a library of THIQ3CA dicarbox-
amides was designed. The synthesis of the targeted THIQ3CA
analogs was achieved using a facile three-step strategy of amida-
tion, deprotection, and amidation that relied on the availability of
the two arms, AN and AC, at positions 2 and 3, respectively [45].
Each AN structure designed as a part of the library was commer-
cially available and was introduced using standard amidation
reaction (see Scheme 1). In contrast, arm AC at position 3 of
THIQ3CA required pre-assembly. This arm was synthesized by
aromatic nucleophilic substitution in which 4-bromoaniline
derivatives 4ae4c were reacted with either valerolactam 5a or
morpholin-3-one 5b to quantitatively give the corresponding 4-
substituted aniline derivatives 8ae8g [46]. Likewise, substituted
anilines 8he8j containing the piperazine side chain were also
hits, of which THIQ3CA dicarboxamide 23, containing
a 5-
chlorothiophen-2-yl moiety (also present in rivaroxaban), was
identified as a promising candidate. Fig. 1A shows a comparison of
the predicted bound form of 23 with apixaban. Both molecules
adopted
a rather similar binding mode in which the 5-
chlorothiophenyl-carbonyl group was found to be oriented toward

R.A. Al-Horani et al. / European Journal of Medicinal Chemistry 54 (2012) 771e783
773
Scheme 1. a) Appropriate amine (8ae8g), HOBt$H2O, DMAP, EDCI, CH2Cl2, rt/overnight, 73e89%, b) For Cbz-protected intermediates: 10% Pd(OH)2, (1:1) CH3OH: t-butanol, H2, rt/
overnight, 65e72%. For Boc-protected intermediates: (1:1) TFA:CH2Cl2, rt/4 h, 75%, c) Appropriate organic acid, EDCI, DMAP, CH2Cl2, rt/overnight, 70e95%.
included in the study (see Supplementary Information Scheme S1
for synthetic and characterization details).
To understand the importance of the aromatic ring of the
THIQ3CA scaffold, and yet mimic its flexibility, we targeted 1)
To introduce either arm AN or AC through an amidation reaction,
the THIQ3CA core was first appropriately protected, which involved
either esterification of the 3-carboxylic acid or t-butox-
ycarbonylation (Boc group) or carbonylbenzyloxylation (Cbz group)
of the ring nitrogen. For example, THIQ3CAs 9e11 were amidated
using amines 8ae8g to yield Boc- or Cbz-protected THIQ3CA mono-
carboxamides 12e21 in 73e89% yield (Scheme 1). The Boc or Cbz
protecting group of 12e21 was then removed using either mild acid
[47] or catalytic hydrogenation [48], respectively, to afford the
corresponding free form in 65e75% yield. The free form of 12e21
was then amidated at the 2 position with twenty one carboxylic
acids in the presence of EDCI to yield the targeted THIQ3CA dicar-
boxamides 22e49 in 70e95% yield (Scheme 1). This strategy was
also exploited for the synthesis of THIQ3CA dicarboxamides 50e55,
59, and 62 that contain additional variations on arms AN and AC (see
Supplementary Information Scheme S2 and S3).
a partially unsaturated, cyclic ring system, i.e., (S)-1,2,3,6-
tetrahydropyridine-2-carboxylic acid (THP2CA) and 2) a bridged
system, i.e., (1R,3S,4R)-2-azabicyclo[2.2.1]heptane-3-carboxylic
acid (ABH3CA) (Scheme 3). The synthesis of either scaffold star-
ted with the Boc-protected carboxylic acid 82 or 85, which was
converted in three steps to dicarboxamide 84 or 87, respectively, in
excellent yield.
Overall, we synthesized twenty eight THIQ3CA, one PA, five
P3CA, three P2CA, one THP2CA and one ABH3CA dicarboxamides
using a simple three-step protocol. The molecules were purified
using standard flash chromatography system and were character-
ized using 1H and 13C NMR spectroscopy and ESI-MS (see
Supplementary Information).
2.3. Inhibition potential of the library of FXa inhibitors
To assess the effect of flexibility for FXa inhibition, we targeted
three core scaffolds that afford greater flexibility than the THIQ3CA
scaffold as well as can help assess the importance of configurational
geometry. These scaffolds were: 1) a ring opened variant of
THIQ3CA core, the phenylalanine (PA) derivative 62; 2) mono-cyclic
dicarboxamides 69e73 belonging to the piperidine-3-carboxylic
acid (P3CA) class; and 3) mono-cyclic dicarboxamides 78, 79 and
81 belonging to the piperidine-2-carboxylic acid (P2CA) class
(Scheme 2). These molecules were synthesized in a manner similar
to the THIQ3CA scaffold using the amidation, deprotection, and
amidation strategy.
Direct inhibition of FXa was measured by a chromogenic
substrate hydrolysis assay in 20 mM TrisHCl buffer, pH 7.4, at 37 ꢀC,
as reported earlier [49]. In this assay, hydrolysis of the substrate by
FXa results in a linear increase in absorbance at 405 nm, the slope of
which corresponds to residual enzyme activity. The change in
residual enzyme activity as a function of the concentration of the
potential inhibitor is plotted on a logarithmic scale and fitted by the
logistic doseeresponse relationship 2 to derive the potency (IC50),
efficacy (
D
Y ¼ YM ꢁ Y0) and Hill Slope (HS) of inhibition [18,49].
Fig. 2 shows representative inhibition profiles for 20, 47, 49, and 51,
which are THIQ3CA derivatives, and 62 and 81, which are PA and

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R.A. Al-Horani et al. / European Journal of Medicinal Chemistry 54 (2012) 771e783
Scheme 2. a) Benzyl chloroformate, Et3N, THF, rt/5 h, 70e73%, b) 8b or 8c, HOBt$H2O, DMAP, EDCI, CH2Cl2, rt/overnight, 73e89%, c) For Cbz-protected intermediates: 10% Pd(OH)2, (1:1)
CH3OH: t-butanol, H2, rt/overnight, 65e72%. For Boc-protected intermediates: (1:1) TFA:CH2Cl2, rt/4 h, 75%, d) Appropriate organic acid, EDCI, DMAP, CH2Cl2, rt/overnight, 70e95%.
P2CA derivatives, respectively. Similar profiles were measured for
other THIQ3CA, P2CA, P3CA, THP2CA, and ABH3CA derivatives,
except for the variation in potency of inhibition. The HS of inhibi-
tion was found to be in the range of 0.7e1.2, which suggests
absence of the complications arising from non-specific binding or
aggregation. The IC50 values of the FXa inhibitors studied in this
2.4. Structural optimization of arm AC of the THIQ3CA scaffold
Our early molecular modeling studies suggested that arm AC at
the 3-position of the THIQ3CA core structure bound in the S4
subsite of the FXa active site. An intermediate 13, synthesized early
in the library and containing N-(N-methylanilin-4-yl)piperidin-2-
work were found to exhibit a wide range of activity (high
mM to
one as the AC arm, had shown a reasonable IC50 of 16.4
mM. This
nM), which provide valuable structureeactivity relationships.
formed the starting point for studying the S4 subsite substitutions
Scheme 3. a) 8b, HOBt$H2O, DMAP, EDCI, CH2Cl2, rt/overnight, b) (1:1) TFA:CH2Cl2, rt/4 h, c) 4-Chlorophenyl acetic acid, EDCI, DMAP, CH2Cl2, rt/overnight, 70e95%.

R.A. Al-Horani et al. / European Journal of Medicinal Chemistry 54 (2012) 771e783
775
Table 1
Optimization of the AC arm (position 3) of THIQ3CA scaffold for FXa inhibition
.
Inhibitor
R1
R2
IC50 (mM)
Fig. 2. Direct inhibition of FXa by designed THIQ3CA and related dicarboxamides. The
inhibition of human FXa was determined spectrophotometrically through the chro-
mogenic substrate hydrolysis assay at pH 7.4 and 37 ꢀC. Solid lines represent sigmoidal
fits to the data to obtain IC50, YM, Y0, and HS, as described in the Experimental methods
section. Experiments were performed in duplicate or triplicate (SE < 20%). See text for
details.
12
eH
9.9 ꢂ 0.2
16.4 ꢂ 0.4
20 ꢂ 1
(Table 1). To optimize the structure of the AC arm, two levels of
structural modifications were introduced. These included 1) varia-
tion in the carboxamide N-substituent and 2) variation in the para-
substituent of the aniline moiety. Replacing the N-methyl group of
13 with ethyl (inhibitor 14) and isopropyl (inhibitor 15) groups
resulted in no significant change, however, N-demethylation as in
12 reduced the IC50 by 1.7-fold suggesting the possibility of the
formation of a hydrogen bond. This was however not substantiated
by molecular modeling (not shown) and we speculated that the
alkyl group on the nitrogen may be inducing a conformational
preference that is not favorable for binding in the S4 subsite.
To optimize the para-substituent of the aniline moiety, we relied
on the extensive literature that abounds in such optimizations
[2,16,50]. The moieties preferred at this position include the
piperidone, morpholinone, piperidine, piperazine and other rings.
Yet, introducing piperidine and piperazine moieties at the para
position of the aniline was catastrophic. The piperidineecontaining
derivative 53 and the piperazineecontaining derivatives 54 and 55
13
14
15
18
19
eCH3
eCH2CH3
eCH(CH3)2
eCH3
19 ꢂ 2
12.8 ꢂ 0.2
19 ꢂ 6
eCH2CH3
displayed an un-quantifiable IC50 of >500 mM (Table 1). Molecular
modeling suggested that the para substituent helps orient the
adjacent aromatic ring for better interaction with Tyr99, Phe174,
and Trp215 of the S4 subsite (not shown), in a manner similar to
that observed with rivaroxaban and apixaban [23,30]. Thus, we
studied the morpholin-3-one moiety (derivative 20, Table 1), which
20
50
eH
eH
6.0 ꢂ 0.8
>500
eCl
exhibited a much improved IC50 of 6.0 mM. The results indicated
a significant contribution of the carbonyl group of morpholin-3-one
for the THIQ3CA inhibitors.
53
54
eH
eH
>500
>500
2.5. Structural optimization of arm AN of the THIQ3CA scaffold
Molecular modeling suggested that the AN arm at position 2 of
the THIQ3CA core fits into S1 subsite of the FXa active site. The
initial hit 13 (IC50 ¼ 16.4
mM) containing the Cbz group as the AN
arm was used as the starting point for studying the S1 subsite
substitutions. Considering that the AN arm of 13 is structurally less
defined than its AC arm, we decided to explore a wider range of
moieties to replace the Cbz group. Thus, optimal chain length of AN,
the nature of its aromatic ring, and the position and number of
substituents on the aromatic ring were studied.
55
eH
>500
Replacing the AN arm of 13 with thiophenyl carbonyl (22),
5-chlorothiophenyl carbonyl (23), p-chlorophenyl carbonyl (24),
p-methylphenyl carbonyl (25) or p-methoxyphenyl carbonyl (26)
arm resulted in 1.2e12.7-fold reduction in potency (Table 2).
Extending the length of the linker by one carbon atom resulted in
significant improvement in IC50. Dicarboxamide 27 containing
a 4-chlorophenyl acetyl side chain exhibited an IC50 of 1.3 M,
M) designed
in the THIQ3CA series. Yet, most subsequent modifications failed to
enhance potency further. For example, one or more electron
m
which was the first high affinity inhibitor (Ki ¼ 0.65
m

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R.A. Al-Horani et al. / European Journal of Medicinal Chemistry 54 (2012) 771e783
Table 2
donating (eCH3, eOCH3) as well as one or more electron with-
drawing (eCl, eCF3, eNO2) substituents on the phenylacetyl side
chain weakened the potency by 1e262-fold (Table 2).
Optimization of the AN arm (position 2) of THIQ3CA scaffold containing the piper-
idone moiety in the AC arm for FXa inhibition
Considering the observations made with the AC structural
variants, we studied the morpholin-3-one containing THIQ3CA
derivatives also for optimization of the AN arm. Table 3 shows the
results obtained in the morpholin-3-one series. Replacing the Cbz
group of parent 18 (IC50 ¼ 12.8
mM) with thiophenyl carbonyl (40),
p-methoxyphenyl propanoyl (43), or 2,4-dimethoxyphenyl prop-
anoyl (48) resulted in considerable loss of activity. Yet, as in the
piperidone series described above, introducing a 4-chlorophenyl
acetyl side chain for the Cbz group resulted in an IC50 of 1.1 mM.
.
Analysis of the above results indicated that the best inhibitors
carried a 7-atom arm AN (at position 2), e.g., 13, 26, 27, 28, and 41.
Within this category, a 7-atom arm containing a small, lipophilic
electron-withdrawing group (eCl) is optimal. This group appears to
be the p-chlorophenylacetyl moiety, as exemplified by a consider-
able increase in affinity observed with 27, 34 and 41, and a loss in
affinity for derivative 49 (Tables 2 and 3). Thus, FXa appears to
preferentially recognize the chlorophenyl moiety in the THIQ3CA
series of inhibitors.
Inhibitor
R3
IC50 (mM)
16
221 ꢂ 19
22
23
24
42 ꢂ 2
56 ꢂ 2
2.6. Rational design of analog 47
The majority of inhibitors described above possessed (S)-
chirality at position 3. To assess whether an (R)-isomer would be
197 ꢂ 39
Table 3
25
26
209 ꢂ 31
19 ꢂ 2
Optimization of the AN arm (position 2) of THIQ3CA scaffold containing the mor-
pholinone moiety in the AC arm for FXa inhibition
27
28
1.3 ꢂ 0.1
14 ꢂ 3
.
Inhibitor
R1
R3
IC50 (mM)
29
30
31
32
34 ꢂ 1
85 ꢂ 19
335 ꢂ 50
246 ꢂ 58
40
eCH3
36 ꢂ 3
41
42
eCH3
eH
1.1 ꢂ 0.1
5.4 ꢂ 0.9
190 ꢂ 16
43
eCH3
33
34
18 ꢂ 2
45
48
eCH3
eCH3
>500
>500
1.3 ꢂ 0.2
49
eH
2.9 ꢂ 0.4
38
103 ꢂ 8

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777
better, we studied THIQ3CA derivative 35, which is a stereoisomer
of 27 (IC50 ¼ 1.3 M). This stereochemical inversion resulted in
P2CA, P3CA, THP2CA, ABH3CA, and THIQ3CA inhibitors. Overall, the
potency of PA, P2CA, and P3CA dicarboxamide inhibitors was found
m
a 1.5-fold increase in the activity (Table 4). This led to a hypothesis
that introducing optimal features derived on arms AN and AC into
a single THIQ3CA derivative may lead to the most potent FXa
inhibitor designed so far. Thus, analog 47 was designed, which
contained a morpholin-3-one moiety, a p-chlorophenylacetyl unit,
(R)-chirality at the 3-position, and NH-containing carboxamide at
the 3-position. THIQ3CA derivative 47 displayed an IC50 of 270 nM
(Ki ¼ 135 nM) and represents the highest potency observed in the
THIQ3CA series.
to be weak (23e371
THIQ3CA inhibitors were higher (0.27e56
m
M), while that for THP2CA, ABH3CA and
M). Comparison of the
m
unsaturated or bridged six-membered scaffold with the THIQ3CA
scaffold shows that the latter is the most optimal structure. For
example, THP2CA derivative 84 and ABH3CA derivative 87 exhibit
IC50s of 9.1 and 10
mM, which are 33e37-fold greater than the
0.27 M IC50 of the corresponding THIQ3CA derivative 47 (Table 5).
m
Within these broad results, we noticed interesting
structureeactivity dependence across the different core scaffolds
studied here. Nearly all 5-chloro-thiophenyl containing derivatives,
e.g., 23 and 40, display weak inhibition potency in comparison to
most p-chlorophenyl containing derivatives, e.g., 27 and 41
(Tables 2 and 3). This was against our expectation based on the well
established high affinity of rivaroxaban, which contains the 5-
chloro-thiophenyl side chain [22,30]. Another interesting obser-
vation was that morpholin-3-one ring consistently induces higher
potency than the piperid-2-one ring. For example, morpholin-3-
one-containing THIQ3CA derivatives 18, 20, and 40 have better
activity than the corresponding piperid-2-one-containing deriva-
tives 13, 12, and 23, respectively (see Tables 1e3).
The design of 47 reflects a truly additive phenomenon. Inhibitor
47 exhibits w60-fold decrease in IC50 relative to the starting inhib-
itor 13 (IC50 ¼ 16.4
mM), which can be rationalized from the 1.5-fold
(morpholin-3-one) ꢃ 2-fold (unsubstituted carboxamide) ꢃ 1.5-fold
(stereochemical inversion) ꢃ 13-fold (4-chlorophenylacetyl moiety)
increases found independently earlier. This is an interesting obser-
vation and appears to have considerable consistency across the
series of molecules studied here.
2.7. THIQ3CA core is the most optimal scaffold
The above results suggest that the THIQ3CA core scaffold was the
most optimal for FXa inhibition following optimization of its two
arms AN and AC. Yet, the THP2CA and ABH3CA scaffolds are inter-
esting. These are scaffolds are new, readily synthesizable and are
relatively unexplored. Screening of a wider chemical space would
help better understand their true potential for FXa inhibition.
Based on the number of flexible carbons constituting the core
scaffold, we reasoned that the open-chain PA core will be more
flexible than the six-membered cyclic P2CA and P3CA cores, which
in turn will exhibit greater flexibility than the unsaturated or
bridged THP2CA and ABH3CA cores. The THIQ3CA core was likely to
be similar to the unsaturated or bridged cores in terms of confor-
mational flexibility. Table 5 displays the potency of selected PA,
2.8. Dicarboxamide 47 selectively inhibits factor Xa in comparison
to other proteases of the coagulation and digestive systems
Table 4
Simultaneous optimization of both AN AC arms of THIQ3CA scaffold for optimal FXa
inhibition
A critical goal of the molecular modeling-based rational design
of THIQ3CA inhibitors was selectivity for targeting factor Xa. This
enzyme is
a trypsin-like serine protease with considerable
homology with enzymes of the coagulation and digestive systems
including FIIa, FVIIa, FIXa, FXIa, FXIIa, trypsin and chymotrypsin.
Chromogenic substrate hydrolysis assays for each of these enzymes
were performed in a manner similar to that for FXa. The assays were
conducted at 37 ꢀC using literature reported buffer systems that are
as close to physiological conditions as possible (see Experimental
methods for details). Initial screening was performed at a high,
fixed concentration of the inhibitor (100e1000 mM) and fractional
.
residual enzyme activity was measured from the initial rate of
hydrolysis in the presence of the inhibitor to that in its absence.
The THIQ3CA inhibitors showed high selectivity for FXa inhibi-
tion. No coagulation enzyme was found to be inhibited by THIQ3CA
Inhibitor R1
X
Y
R3
Chirality (*) IC50 (mM)
35
36
eCH3
eCH2e eC(O)e
eCH2e eC(O)e
(R)-
(R)-
0.9 ꢂ 0.1
311 ꢂ 56
inhibitors at concentrations less than 100
digestive enzymes, moderate inhibition of chymotrypsin
M) was noted with 4-chlorophenyl side chain con-
mM. With regard to
(IC50 ¼ 5e100
m
eCH3
eH
taining inhibitors. The most potent FXa inhibitor 47 demonstrated
a selectivity of at least 1852-fold over FIIa, FVIIa, FIXa, FXIa, and FXIIa
(Table 6). Against trypsin and chymotrypsin, the selectivity of 47
was found to be at least 370- and 279-fold, respectively. Few
rationally designed inhibitors have been able to achieve such high
selectivity for factor Xa against other homologous enzymes [51,52].
37
eCH2e eC(O)e
(S)-
13.3 ꢂ 1.4
46
47
52
eCH2CH3 eOe eC(O)e
(S)-
(R)-
(S)-
1.6 ꢂ 0.2
0.27 ꢂ 0.03
151 ꢂ 36
2.9. Prolongation of plasma clotting times by THIQ3CA, THP2CA and
ABH3CA inhibitors
eH
eOe eC(O)e
eCH2e eSO2e
Clotting assays, prothrombin and activated partial thrombo-
plastin time (PT and aPTT, respectively), are routinely used to assess
anticoagulation potential of new enzyme inhibitors in an in vitro
setting [18,53,54]. Whereas PT measures the effect of an inhibitor on
the extrinsic pathway of coagulation, aPTT measures the effect on
eCH3

778
R.A. Al-Horani et al. / European Journal of Medicinal Chemistry 54 (2012) 771e783
Table 5
Comparison of the FXa inhibition potential of different scaffolds
.
Inhibitor
A
IC50
(
mM)
Inhibitor
B
IC50
(
m
M)
Inhibitor
C
IC50 (mM)
27
1.3 ꢂ 0.1
23
56 ꢂ 2
47
0.27 ꢂ 0.03
59
62
72
7.8 ꢂ 0.4
54 ꢂ 5
78
79
69
358 ꢂ 16
99 ꢂ 7
81
84
87
23 ꢂ 2
9 ꢂ 2
371 ꢂ 59
69 ꢂ 7
10 ꢂ 3
the intrinsic pathway. The prolongation of the human plasma clot-
ting time as a function of the concentration of the inhibitors followed
a pattern typical of other well-studied anticoagulants (data not
shown), except for the range of active concentrations. Table 7 lists
the concentrations of selected potent FXa inhibitors required to
double the PT and aPTT. Overall, for the inhibitors studied, doubling
structure of two arms AN and AC of a bifunctional cyclic ring system.
We discovered interesting structural preference for both arms and
a fine additive relationship that culminated in the design of
THIQ3CA analog 47, which displays direct inhibition IC50 of 270 nM
(KI ¼ 135 nM). Synthetically, the FXa inhibitors were readily
assembled using three high yielding steps, which required room
temperature conditions and avoided harsh reactions. Our work
suggests that the morpholin-3-one ring engineers better inhibitor
potential than any other ring system; that the nitrogen of the car-
boxamide group at position-3 should be optimally unsubstituted;
that a (R)-stereochemistry at position-3 is more preferred than its
(S)-enantiomer; and that a 7-atom AN arm containing a para-chloro
substituted aromatic ring is critical for potent, direct FXa inhibition.
It is also worth mentioning that the AN and AC arms should be
adjacent to each other (1,2-substitution) rather than one carbon
away (1,3-disubstitution). These conclusions may help design more
potent molecules.
the PT required 17e2347
mM concentration, while doubling of aPTT
required 20e810 M suggesting that most inhibitors essentially
m
affect both pathways equally (Fig. 3). This is to be expected because
of their selectivity for targeting FXa, which is an enzyme of the
common pathway. The inhibitors studied included THIQ3CA deriv-
atives 20, 27, 34, 42, 47, and 49, and THP2CA/ABH3CA derivatives 84
and 87. Inhibitor 47 doubled the PTclotting time at 17.1
mM, which is
comparable to razaxaban (3.8 M) and DPC423 (4.9 M), two direct
m
m
FXa inhibitors being pursued in clinical trials [51].
3. Significance
Our work investigates the dependence of direct FXa inhibition
on the flexibility of core scaffold and attempts to optimize the
Table 7
Effect of designed FXa dicarboxamide inhibitors on
human plasma clotting time.
Table 6
Inhibitor
PT (
m
M)
aPTT (mM)
NDa
80
260
NDa
20.2
614
810
NDa
Selectivity of inhibition by THIQ3CA dicarboxamide 47.
20
27
34
42
47
49
84
87
1387
71
219
2160
17.1
645
952
Protease
IC50
(
m
M)
Selectivity index
Human FIIa
Human FVIIa
Human FIXa
>500
>1852
>3703
>3703
>1852
>1852
>370
>1000
>1000
>500
>500
>100
Human FXIa
Human FXIIa
Bovine trypsin
Bovine chymotrypsin
2347
75.3 ꢂ 13.4
279
a
Not determined.

R.A. Al-Horani et al. / European Journal of Medicinal Chemistry 54 (2012) 771e783
779
million (ppm), are either relative to the internal standard (tetra-
methyl silane, TMS) or to the residual peak of the solvent. The NMR
data are reported as chemical shift (ppm), multiplicity of signal
(s ¼ singlet, d ¼ doublet, t ¼ triplet, q ¼ quartet, dd ¼ doublet of
doublet, m ¼ multiplet), coupling constants (Hz), and integration.
Compounds with dicarboxamide functionalities exhibit amide
rotamerism resulting in complexity of NMR signals. ESI-MS of
compounds were recorded using Waters Acquity TQD MS spec-
trometer in positive ion mode. Samples were dissolved in methanol
and infused at a rate of 20 mL/min. Mass scans were obtained in the
range of 200e700 amu with a scan time of 1 s and a scan rate of
500 amu/s. The capillary voltage was varied between 3 and 4 kV,
while the cone voltage ranged from 38 to103 V. Ionization condi-
tions were optimized for each compound to maximize the ioniza-
tion of the parent ion. Generally, the extractor voltage was set to
3 V, the Rf lens voltage was 0.1 V, the source block temperature was
set to 150 ꢀC, and the desolvation temperature was about 250 ꢀC.
The purity of each final compound was greater than 95% as deter-
mined by uPLC-MS.
4.3. Proteins
Human plasma proteinases including FIIa, FXa, FXIa, FIXa, FVIIa,
and recombinant tissue factor was obtained from Haematologic
Technologies (Essex Junction, VT). FXIIa was purchased from
Enzyme Research Laboratories (South Bend, IN). Bovine a-chymo-
trypsin and bovine trypsin were obtained from SigmaeAldrich (St.
Louis, MO). The substrates Spectrozyme TH, Spectrozyme FXa,
Spectrozyme FXIIa, Spectrozyme FIXa, Spectrozyme VIIa, and
Spectrozyme CTY were obtained from American Diagnostica
(Greenwich, CT). Factor XIa substrate, S-2366, trypsin substrate, S-
2222, were obtained from Diapharma (West Chester, OH). Factor Xa
and FVIIa were prepared in 20 mM TrisHCl buffer, pH 7.4, con-
taining 100 mM NaCl, 2.5 mM CaCl2, 0.1% PEG8000, and 0.02%
Tween80. FIXa was prepared in 20 mM TrisHCl buffer, pH 7.4,
containing 100 mM NaCl, 2.5 mM CaCl2, 0.1% PEG8000, 0.02%
Tween80, and 33% v/v Ethyleneglycol. Other enzymes were
prepared in 50 mM TrisHCl buffer, pH 7.4, containing 150 mM NaCl,
0.1% PEG8000, and 0.02% Tween80.
Fig. 3. Prolongation of clotting time as
a function of concentration of designed
THIQ3CA and related dicarboxamides in either prothrombin time assay (PT) (A) or
activated partial thromboplastin time assay (aPTT) (B). Solid lines are trend lines from
which the concentration necessary to double clotting time was deduced. Clotting
assays were performed in duplicate (SE ꢄ 10%) as described in Experimental methods.
4. Experimental methods
4.4. Modeling factor Xa and the virtual library of (S)-THIQ3CA
dicarboxamides
4.1. Chemicals, reagents, and analytical chemistry
Anhydrous CH2Cl2, THF, CH3CN, DMF, toluene, and acetone were
purchased from SigmaeAldrich (Milwaukee, WI) or Fisher (Pitts-
burgh, PA) and used as such. Other solvents used were of reagent
gradient and used as purchased. Analytical TLC was performed
using UNIPLATEÔ silica gel GHLF 250 um pre-coated plates
(ANALTECH, Newark, DE). Column chromatography was performed
using silica gel (200e400 mesh, 60 Å) from SigmaeAldrich.
Chemical reactions sensitive to air or moisture were carried out
under nitrogen atmosphere in oven-dried glassware. Reagent
solutions, unless otherwise noted, were handled under a nitrogen
atmosphere using syringe techniques. Flash chromatography was
performed using Teledyne ISCO (Lincoln, NE) Combiflash RF system
Sybyl 8.1 (Tripos Associates, St. Louis, MO) was used for
modeling of FXa-inhibitor complexes. The structure of human FXa
(ID: 2P16) [23] was acquired from the Protein Data Bank (www.
rcsb.org). To prepare the protein structure for modeling experi-
ments, hydrogen atoms were added, while inorganic ions and
water molecules were removed. Individual atoms were assigned
GasteigereHückel charges. Energy minimization was then per-
formed using a Tripos force field so as to reach a terminating
gradient of 0.5 kcal/mol Å2 or a maximum of 100,000 iterations.
(S)-THIQ3CA was modified in silico at positions 2 and 3 with
appropriate substituents to “synthesize” a virtual chemical library of
nearly 150 dicarboxamide derivatives. The different combinations at
2- and 3-positions included substituted derivatives of aromatic
carboxylic acids and aromatic amines, respectively. These acids and
amines were further substituted at varying positions with halogen,
t-butyl, cyclopropyl, methoxy, amino-methyl, substituted phenyl,
substituted imidazole, pyridine, pyridine-2-one, N-oxide pyridine,
N-methyl piperazine, or cyclic amides such as piperidone and
morpholinone (See Supplementary Information). The resulting
molecules were assigned GasteigereHückel charges and were then
energetically minimized using a Tripos force field until a terminating
gradient of 0.5 kcal/mol Å2 or a maximum of 100,000 iterations.
and disposable normal silica cartridges of 30e50
m particle size,
230e400 mesh size and 60 Å pore size. The flow rate of the mobile
phase was in the range of 18e35 ml/min and mobile phase
gradients of ethyl acetate/hexanes and CH2Cl2/CH3OH were used
to elute compounds.
4.2. Chemical characterization of compounds
1H and 13C NMR were recorded on Bruker-400 MHz spectrom-
eter in either CDCl3, CD3OD, or acetone-d6. Signals, in part per

780
R.A. Al-Horani et al. / European Journal of Medicinal Chemistry 54 (2012) 771e783
4.5. Docking and scoring
at 25 ꢀC and pH 7.4. For selectivity analysis, a single concentration
point assays was utilized in which 0.5e1 mM FXa inhibitor was
tested in duplicate. The fractional residual enzyme activity was
measured and if found to be less than 50%, the inhibition profile was
measured over a range of inhibitor concentrations to determine the
IC50 of the enzymeeinhibitor complex. The KM of the substrate for
its enzyme was used to identify the concentration of the substrate
to be used for inhibition studies. The concentrations of enzymes and
Docking of the (S)-THIQ3CA dicarboxamides onto the active site
of FXa was performed with GOLD 5.1 (Cambridge Crystallographic
Data Center, UK). The docking experiment was performed as
reported earlier [15]. The binding site in human FXa was defined to
include all atoms within 6 Å of the co-crystallized ligand. The
docking protocol was validated by docking ligand for which the
adopted docking pose was found to have RMSD <1 Å relative to that
reported in the crystal structure [23]. Docking was driven by the
GOLD scoring function, while for ranking the docked solutions;
a linear, modified form of the same scoring function Eq. (1) was
used, as reported earlier [15]. In this equation, HBEXT and VDWEXT
are the “external” (nonbonded interactions taking place between
the ligand and the target protein) hydrogen bonding and van der
Waals terms, respectively.
substrates in microplate cells were: 6 nM and 50
0.765 nM and 345 M for FXIa; 5 nM and 125 M for FXIIa; 89 nM
and 850 M for FIXa; 8 nM and 1000 M for FVIIa (along with 40 nM
recombinant tissue factor); 72.5 ng/ml and 80 M for bovine
trypsin; and 500 ng/ml and 240 M for bovine chymotrypsin.
mM for FIIa;
m
m
m
m
m
m
4.8. Prothrombin time (PT) and activated partial thromboplastin
time (aPTT)
GOLDScore ¼ HBEXT þ 1:375 ꢃ VDWEXT
(1)
Clotting time was measured in a standard one-stage recalcifi-
cation assay with a BBL Fibrosystem fibrometer (BectoneDickinson,
Sparles, MD), as described previously [18]. For PT assays, throm-
boplastin was reconstituted according to the manufacturer’s
4.6. FXa inhibition studies
directions and warmed to 37 ꢀC. A 10
to give the desired concentration, was brought up to 100
citrated human plasma, incubated for 30 s at 37 ꢀC followed by
addition of 200 L of prewarmed thromboplastin. For the aPTT
assay, 10 L of inhibitor was mixed with 90 L of citrated human
plasma and 100 L of prewarmed aPTT reagent (0.2% ellagic acid).
After incubation for 4 min at 37 ꢀC, clotting was initiated by adding
100 L of prewarmed 25 mM CaCl2 and time to clot noted. The data
were fit to a quadratic trendline, which was used to determine the
concentration of the inhibitor necessary to double the clotting time.
Clotting time in the absence of an anticoagulant was determined in
similar fashion using 10 mL of deionized water and/or appropriate
organic vehicle and was found to be 21.0 s for PT and 46.4 s for aPTT.
m
L sample of the FXa inhibitor,
Direct inhibition of FXa was measured by a chromogenic
substrate hydrolysis assay, as reported earlier [49] using a micro-
plate reader (FlexStation III, Molecular Devices) at wavelength of
405 nm and incubating temperature of 37 ꢀC. Generally, each well of
m
L with
m
m
m
the 96-well microplate had 185
potential FXa inhibitor (or solvent reference) was added, to which
L FXa (stock conc. 43.5 nM) was further added. After 10 min
incubation at 37 ꢀC, 5
L FXa substrate (stock conc. 5 mM) was
mL pH 7.4 buffer to which 5 mL
m
5
m
m
m
rapidly added and the residual FXa activity was measured from the
initial rate of increase in absorbance at 405 nm. The concentration
of the organic solvent in which the inhibitors were dissolved was
maintained constant and was less than 2.5% (v/v). Stocks of FXa
inhibitors were prepared at 20 mM concentration and then serially
diluted to give twelve different aliquots spanning a range of
4.9. General procedure for nucleophilic amidation of halogenated
aniline derivatives 8ae8g
0.015e500
mM in the plate wells. Each inhibitor was studied in
duplicate or triplicate at each concentration. Relative residual FXa
activity at each concentration of the inhibitor was calculated from
the ratio of FXa activity in the presence and absence of the inhibitor.
Logistic Eq. (2) was used to fit the dose-dependence of residual
An oven-dried, two-neck round bottom flask fitted with
a condenser was charged with CuI (0.05 mmol) and K2CO3
(2.0 mmol) under nitrogen atmosphere. N, N0-dimethylethylene-
diamine (0.10 mmol), 4-haloaniline derivative (4ae4c) (1.0 mmol),
piperidin-2-one (5a) or morpholin-3-one (5b) (1.5 mmol), and
anhydrous toluene (1 mL) were added. The reaction mixture was
stirred and refluxed overnight. The resulting mixture was allowed
to reach RT and filtered through Celite, which was further washed
with methanol (10 mL). The organic filtrate was concentrated in
vacuo and the residue was purified by flash chromatography using
gradient mobile phase of EtOAc/hexanes to afford the corre-
sponding para-amidated aniline derivatives (8ae8g) as solid
products in 65e90% yield. The full spectral characterization of these
products is provided as Supplementary Information.
proteinases activity to obtain the potency (IC50) and efficacy (DY) of
inhibition. In this equation, Y is the ratio of residual factor Xa activity
in the presence of inhibitor to that in its absence (fractional residual
activity), YM and Y0 are the maximum and minimum possible values
of the fractional residual proteinase activity, IC50 is the concentra-
tion of the inhibitor that results in 50% inhibition of enzyme activity,
and HS is the Hill slope (which was between 0.7 and 1.2 in all
measurements). Nonlinear curve fitting resulted in YM, Y0, IC50 and
HS values. The reported IC50 is an average of the two or three
measurements with standard error (SE) of <20%. The efficacy of
inhibition
D
Y was found to be >80% for each studied FXa inhibitor.
YM ꢁ Y0
Y ¼ Y0 þ
(2)
4.9.1. 1-(4-Aminophenyl)piperidin-2-one (8a)
Log ½Iꢅ ꢁLog IC ðHSÞ
ð
50Þ
0
1 þ 10
1H NMR (CDCl3, 400 MHz): 7.00 (d, J ¼ 8.76 Hz, 2 H), 6.56 (d,
J ¼ 8.76 Hz, 2 H), 3.56 (t, J ¼ 5.44 Hz, 2 H), 2.52 (t, J ¼ 5.24 Hz, 2 H),
1.92e1.89 (m, 4 H). 13C NMR (CDCl3, 100 MHz): 170.11, 146.25,
132.89, 127.19, 113.35, 52.17, 32.88, 23.04, 21.20. MS (ESI) calculated
for C11H14N2O, [M þ H]þ, m/z 191.25, found for [M þ H]þ, m/z 191.98.
4.7. Inhibition of proteases of the coagulation and digestive systems
The potential of the FXa inhibitors against coagulation enzymes
including FIIa, FVIIa, FIXa, FXIa, and FXIIa, and digestive enzymes
including trypsin and chymotrypsin was performed using chro-
mogenic substrate hydrolysis assays reported in the literature [55].
These assays were performed using substrates appropriate for the
enzyme being studied under conditions closest to the physiological
condition (37 ꢀC and pH 7.4), except for FIIa, which was performed
4.10. General procedure for catalytic hydrogenation of nitro group
for synthesis of 8i and 8j
4-Nitrophenylpiperazine (7a or 7b) and 10% Pd/C was mixed in
methanol (10 mL) containing concentrated HCl (2 mL). Hydrogen

R.A. Al-Horani et al. / European Journal of Medicinal Chemistry 54 (2012) 771e783
781
gas was then pumped into the mixture at RT. After stirring the
solution for 5 h, the catalyst was filtered on Celite and the organic
filtrate concentrated in vacuo to afford the corresponding aniline
derivative (8h and 8j) in w100% yield. The full spectral character-
ization of these products is provided as Supplementary
Information.
solution after 5 h. The reaction mixture was then partitioned
between acidified water (15 mL) and EtOAc (20 mL). The aqueous
layer was further washed with EtOAc (2 ꢃ 20 mL). The organic
extracts were combined, dried over anhydrous Na2SO3, filtered, and
concentrated in vacuo. The N-Cbz protected product (65 or 66) was
isolated as white solids by flash chromatography using gradient of
EtOAc/hexanes as eluant in 70e73% yield.
4.10.1. 1-(4-(4-Aminophenyl)piperazin-1-yl)ethanone (8i)
1H NMR (CDCl3, 400 MHz): 6.81 (d, J ¼ 8.76 Hz, 2 H), 6.66 (d,
J ¼ 8.76 Hz, 2 H), 3.75 (t, J ¼ 5.08 Hz, 2 H), 3.60 (t, J ¼ 4.92 Hz, 2 H),
3.01 (t, J ¼ 5.20 Hz, 2 H), 2.98 (t, J ¼ 5.12 Hz, 2 H), 2.13 (s, 3 H). 13C
NMR (CDCl3, 100 MHz): 168.98, 144.04, 140.90, 119.32, 116.17, 51.55,
51.14, 46.51, 41.61, 21.33. MS (ESI) calculated for C12H17N3O,
[M þ H]þ, m/z 220.29, found for [Mþ]þ, m/z 220.15.
4.13.1. (S)-1-(Benzyloxycarbonyl)piperidine-3-carboxylic acid (65)
1H NMR (acetone-d6, 400 MHz): 7.43e7.30 (m, 5 H), 5.15 (s, 2 H),
4.35e4.04 (m, 1 H), 3.93 (d, J ¼ 12.08 Hz, 1 H), 3.18e3.02 (m, 1 H),
3.01e2.92 (m, 1 H), 2.53e2.46 (m, 1 H), 2.09e2.062 (m, 1 H),
1.75e1.66 (m, 2 H), 1.53e1.45 (m, 1 H). 13C NMR (acetone-d6,
100 MHz): 174.88, 155.77, 138.25, 129.30, 128.70, 128.60, 67.49,
46.62, 44.90, 41.64, 27.87, 24.98. MS (ESI) calculated for C14H17NO4,
[M þ H]þ, m/z 264.30, found for [M þ H]þ, m/z 264.37.
4.10.2. (4-(4-Aminophenyl)piperazin-1-yl)(thiophen-2-yl)
methanone (8j)
1H NMR (acetone-d6, 400 MHz): 7.57 (d, J ¼ 4.96 Hz,1 H), 7.34 (d,
J ¼ 3.56 Hz, 1 H), 7.03 (d, J ¼ 4.32 Hz, 1 H), 6.86 (d, J ¼ 8.64 Hz, 2 H),
6.54 (d, J ¼ 8.60 Hz, 2 H), 3.77 (t, J ¼ 4.96 Hz, 4 H), 3.08 (t, J ¼ 5.16 Hz,
4 H). 13C NMR (acetone-d6,100 MHz): 163.60,148.27, 138.75,129.72,
127.70, 122.94, 121.33, 120.06, 118.43, 116.14, 52.39, 51.20. MS (ESI)
calculated for C15H17N3OS, [M þ H]þ, m/z 288.39, found for
[M þ H]þ, m/z 288.25.
4.13.2. (R)-1-(Benzyloxycarbonyl)piperidine-3-carboxylic acid (66)
1H NMR (acetone-d6, 400 MHz): 7.43e7.30 (m, 5 H), 5.15 (s, 2 H),
4.35e4.04 (m, 1 H), 3.93 (d, J ¼ 12.08 Hz, 1 H), 3.18e3.02 (m, 1 H),
3.01e2.92 (m, 1 H), 2.53e2.46 (m, 1 H), 2.09e2.062 (m, 1 H),
1.75e1.66 (m, 2 H), 1.53e1.45 (m, 1 H). 13C NMR (acetone-d6,
100 MHz): 174.88, 155.77, 138.25, 129.30, 128.70, 128.60, 67.49,
46.62, 44.90, 41.64, 27.87, 24.98. MS (ESI) calculated for C14H17NO4,
[M þ H]þ, m/z 264.30, found for [M þ Na]þ, m/z 286.28.
4.11. General procedure for deprotection of t-butyloxycarbonyl
(Boc) group of 16, 17, 21, 61, 76, 77, 80, 83, or 86
4.14. General procedure for amidation of 3-carboxylic acid to yield
12e21, 50, 53e55, 59, 61, 67, 68, 76, 77, 80, 83, or 86
To a solution of 1,2,3,4-tetrahydroisoquinoline carboxamides
(16, 17, 21), phenylalanine carboxamide (61), piperidine-2-
To a stirred solution of the free carboxylic acid of (9e11, 58, 60,
65, 66, 74, 75, 82, or 85) (1.0 mmol) in anhydrous CH2Cl2 (5 mL) was
added hydrated N-hydroxybenzotrizole (HOBT.H2O, 1.1 mmol),
DMAP (1.1 mmol), and 1-ethyl-3-(3-dimethylaminopropyl)carbo-
diimide (EDCI, 1.1 mmol) at RT under nitrogen atmosphere.
Appropriate amine (8ae8j or 4-chloroaniline) (1.1 mmol) in anhy-
drous CH2Cl2 (5 mL) was then added dropwise. After stirring
overnight, the reaction mixture was partitioned between 2.0 N HCl
solution (20 mL) and CH2Cl2 (30 mL). The organic layer was washed
further with 2 N HCl (2 ꢃ 10 mL) and saturated NaCl solution
(20 mL), dried using anhydrous Na2SO4, and concentrated to give
a crude, which purified by flash chromatography using gradient of
CH2Cl2/CH3OH as eluant to give the desired carboxamide product in
73e89% yield. The full spectral characterization of these products is
provided as Supplementary Information.
carboxamides
carboxamides
(76,
(83),
77,
or
80), 1,2,3,6-tetrahydropyridine-2-
2-azabicyclo[2.2.1] heptane-3-
carboxamides (86) (1.0 mmol) in CH2Cl2 (5 mL), trifluoroacetic
acid (TFA, 5 mL) was added drop-wise at 0 ꢀC, and the mixture was
warmed to RT. After stirring for 4 h, the reaction mixture was
diluted with CH2Cl2 (25 mL) and neutralized by drop-wise addition
of saturated aqueous NaHCO3 (20 mL). The organic layer was
separated and the aqueous phase was extracted with EtOAc
(2 ꢃ 25 mL). The organic extracts were combined, washed with
saturated NaCl solution (25 mL), and dried over anhydrous Na2SO4.
Removal of the solvent under reduced pressure afforded the
desired unprotected carboxamides in quantitative yields and
sufficient purity (as indicated by TLC) to be directly used in the next
reactions without any further treatment.
4.12. General procedure for deprotection of carbobenzyloxy (Cbz)
group of 12e15, 18e20, 50, 67 or 68
4.14.1. (S)-Benzyl 3-(4-(2-oxopiperidin-1-yl)phenylcarbamoyl)-3,4-
dihydroisoquinoline-2(1H)-carboxylate (12)
1H NMR (CDCl3, 400 MHz): 7.50e7.27 (m, 6 H), 7.25e7.08 (m, 5
H), 7.06 (d, J ¼ 8.44, 2 H), 5.23e5.12 (m, 2 H), 4.96e4.54 (m, 3 H),
3.55 (t, J ¼ 5.00 Hz, 2 H), 3.40e3.30 (m, 1 H), 3.20e3.09 (m, 1 H),
2.52 (t, J ¼ 5.00 Hz, 2 H), 2.00e1.87 (m, 4 H). 13C NMR (CDCl3,
100 MHz): 170.23, 169.22, 156.94, 139.31, 136.09, 135.47, 134.33,
133.32, 128.64, 128.32, 128.07, 127.76, 127.23, 126.90, 126.58, 125.99,
120.87, 68.04, 64.35, 51.75, 45.08, 32.82, 30.31, 23.51, 21.41. MS (ESI)
calculated for C29H29N3O4, [M þ H]þ, m/z 484.57, found for
[M þ Na]þ, m/z 506.3.
1,2,3,4-tetrahydroisoquinoline carboxamides (12e15, 18, 19, 50)
or piperidine-3-carboxamides (67 or 68) and 10% Pd(OH)2 on
activated charcoal were mixed in CH3OH: tert-butanol (1:1)
mixture (10 mL). Hydrogen gas was then pumped into the mixture
at RT. After stirring the solution overnight, the catalyst was filtered
on Celite and the organic filtrate concentrated in vacuo to afford the
corresponding desired unprotected carboxamides in quantitative
yields and sufficient purity (as indicated by TLC) to be directly used
in the next reactions without any further treatment.
4.14.2. (S)-Benzyl 3-(4-chlorophenylcarbamoyl)-3,4-dihydroisoquinoline-
2(1H)-carboxylate (50)
4.13. General procedure for amine protection by carbobenzyloxy
(Cbz) group in the synthesis of 65 or 66
1H NMR (CDCl3, 400 MHz): 7.50e7.35 (m, 3 H), 7.34e7.26 (m, 2
H), 7.25e7.18 (m, 4 H), 7.17e7.00 (m, 4 H), 5.39e5.10 (m, 2 H),
4.96e4.49 (m, 3 H), 3.35e3.21 (m, 1 H), 3.20e3.3.03 (m, 1 H). 13C
NMR (CDCl3, 100 MHz): 169.12, 157.30, 136.24, 135.95, 133.44,
132.70, 129.08, 128.81, 128.70, 128.57, 128.43, 128.11, 127.77, 126.78,
125.94, 121.13, 68.19, 57.04, 45.11, 28.30. MS (ESI) calculated for
Piperidine-3-carboxylic acid (63 or 64) (1 mmol) was dissolved
in dry THF (10 mL) and stirred at RT. Tri-ethylamine (Et3N) (2 mmol)
was then added followed by benzyl chloroformate (1.5 mmol). The
reaction mixture became initially cloudy and turned to clear

782
R.A. Al-Horani et al. / European Journal of Medicinal Chemistry 54 (2012) 771e783
C24H21ClN2O3, [M þ H]þ, m/z 421.90, found for [M þ Na]þ, m/z
J ¼ 7.16 Hz, J ¼ 13.32, 1 H), 2.71e2.66 (dd, J ¼ 7.00 Hz, J ¼ 13.36 Hz,
1 H), 2.59 (t, J ¼ 6.40, 2 H), 2.05e1.95 (m, 4 H). 13C NMR (CDCl3,
100 MHz): 171.30, 170.07, 169.63, 143.01, 140.21, 136.00, 133.15,
130.62, 129.24, 128.95, 128.44, 127.95, 127.25, 126.90, 51.42, 51.19,
42.76, 38.91, 37.60, 32.93, 23.53, 21.40. MS (ESI) calculated for
C29H30ClN3O3, [M þ H]þ, m/z 505.03, found for [M þ Na]þ, m/z
526.33.
443.26.
4.14.3. (S)-tert-Butyl 6-(4-(3-oxomorpholino)phenylcarbamoyl)-
5,6-dihydropyridine-1(2H)-carboxylate (83)
1H NMR (CDCl3, 400 MHz): 7.50 (d, J ¼ 8.80 Hz, 2 H), 7.22 (d,
J ¼ 8.64 Hz, 2 H), 5.85e5.80 (m, 1 H), 4.65e4.60 (m, 1 H), 5.00e4.93
(m, 1 H), 4.28 (s, 2 H), 4.20e4.07 (m, 1 H), 3.94 (t, J ¼ 4.95 Hz, 2 H),
3.65 (t, J ¼ 4.40 Hz, 2 H), 3.60e3.63 (m, 1 H), 2.60e3.72 (m, 1 H),
2.34e2.28 (m, 1 H). 13C NMR (CDCl3, 100 MHz): 169.54, 166.82,
156.90, 137.99, 126.12, 123.36, 122.20, 120.36, 81.41, 68.54, 64.11,
61.4, 49.79, 41.50, 28.39, 24.10. MS (ESI) calculated for C22H29N3O5,
[M þ H]þ, m/z 402.46, found for [M þ Na]þ, m/z 424.36.
4.15.4. (1R, 3S, 4R)-2-(2-(4-Chlorophenyl)acetyl)-N-(4-(3-
oxomorpholino)phenyl)-2-azabicyclo [2.2.1]heptane-3-carboxamide (87)
1H NMR (CDCl3, 400 MHz): 7.52 (d, J ¼ 8.64 Hz, 2 H), 7.32 (d,
J ¼ 8.32 Hz, 2 H), 7.23 (d, J ¼ 8.36 Hz, 2 H), 7.22 (d, J ¼ 8.20 Hz, 2H),
4.32 (s, 2 H), 4.26 (s, 2 H), 4.01 (t, J ¼ 4.96 Hz, 2 H), 3.76e3.65 (m,
4 H), 3.11e3.95 (m, 1 H), 2.04e1.99 (m, 1 H), 1.89e1.74 (m, 3 H),
1.63e1.45 (m, 3 H). 13C NMR (CD3OD, 100 MHz): 171.24, 170.63,
169.48,138.89,138.48,135.06,133.86,132.02,129.63,127.56,122.03,
69.07, 67.43, 65.09, 60.21, 51.20, 43.46, 41.58, 36.43, 32.14, 28.55. MS
(ESI) calculated for C25H26ClN3O4, [M þ H]þ, m/z 468.95, found for
[M þ Na]þ, m/z 490.27.
4.15. General procedure for amidation at the ring nitrogen of the
core structure to yield 7a, 7b, 22e49, 51, 58, 62, 69e73, 78, 79, 81,
84, or 87
To a stirred solution of organic acid (1.0 mmol) in anhydrous
CH2Cl2 (5 mL) was added 1-Ethyl-3-(3-dimethylaminopropyl) car-
bodiimide (EDCI, 1.1 mmol) and DMAP (1.1 mmol) at RT under
nitrogen atmosphere. The unprotected form (free amine) of (6,
12e21, 50, 57, 61, 67, 68, 76, 77, 80, 83, or 86) (1.1 mmol) in anhy-
drous CH2Cl2 (5 mL) was then added drop-wise. After stirring
overnight, the reaction mixture was partitioned between 2.0 N HCl
solution (20 mL) and CH2Cl2 (30 mL). The organic layer was washed
further with 2 N HCl (2 ꢃ 10 mL) and saturated NaCl solution
(20 mL), dried using anhydrous Na2SO4, and concentrated to give
a crude, which purified by flash chromatography using gradient of
CH2Cl2/CH3OH as eluting phase to give the desired dicarboxamide
product in 70e95% yield. The full spectral characterization of these
products is provided as Supplementary Information.
Acknowledgments
This work was supported by grants RC1 HL099420, R01
HL090586 and P01 HL107152 from the National Institutes of Health.
Appendix A. Supplementary material
Supplementary material associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/j.ejmech.
2012.06.032.
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