Structure-based design of novel guanidine/benzamidine mimics: Potent and orally bioavailable factor Xa inhibitors as novel anticoagulants

Article abstract of DOI:10.1021/jm020578e

As part of an ongoing effort to prepare orally active factor Xa inhibitors using structure-based drug design techniques and molecular recognition principles, a systematic study has been performed on the pharmacokinetic profile resulting from replacing the

Full text of DOI:10.1021/jm020578e

J . Med. Chem. 2003, 46, 4405-4418
4405
Str u ctu r e-Ba sed Design of Novel Gu a n id in e/Ben za m id in e Mim ics: P oten t a n d
Or a lly Bioa va ila ble F a ctor Xa In h ibitor s a s Novel An ticoa gu la n ts
Patrick Y. S. Lam,* Charles G. Clark, Renhua Li, Donald J . P. Pinto, Michael J . Orwat, Robert A. Galemmo,
J ohn M. Fevig, Christopher A. Teleha, Richard S. Alexander, Angela M. Smallwood, Karen A. Rossi,
Matthew R. Wright, Stephen A. Bai, Kan He, J oseph M. Luettgen, Pancras C. Wong, Robert M. Knabb, and
Ruth R. Wexler
Bristol-Myers Squibb Company, P.O. Box 5400, Princeton, New J ersey 08542-5400
Received December 20, 2002
As part of an ongoing effort to prepare orally active factor Xa inhibitors using structure-based
drug design techniques and molecular recognition principles, a systematic study has been
performed on the pharmacokinetic profile resulting from replacing the benzamidine in the P1
position with less basic benzamidine mimics or neutral residues. It is demonstrated that
lowering the pKa of the P1 ligand resulted in compounds (3-benzylamine, 15a ; 1-aminoiso-
quinoline, 24a ; 3-aminobenzisoxazole, 23a ; 3-phenylcarboxamide, 22b; and 4-methoxyphenyl,
22a ) with improved pharmacokinetic features mainly as a result of decreased clearance,
increased volume of distribution, and enhanced oral absorption. This work resulted in a series
of potent and orally bioavailable factor Xa inhibitors that ultimately led to the discovery of
SQ311, 24a . SQ311, which utilizes a 1-aminoisoquinoline as the P1 ligand, inhibits factor Xa
with a Ki of 0.33 nM and demonstrates both good in vivo antithrombotic efficacy and oral
bioavailability.
In tr od u ction
calcium, and phospholipids, a prothrombinase complex
is formed, which converts prothrombin to thrombin.
Thrombin, in turn, converts fibrinogen to fibrin and
activates platelets, eventually leading to the formation
of thrombus or blood clots. Inhibition of FXa has been
demonstrated to result in antithrombotic efficacy in both
animal models9 and clinical settings.10 Thus, FXa has
emerged as a promising target for the discovery of oral
anticoagulants.11,12
Arginine plays an important role in many inhibitor/
ligand and macromolecule interactions. Consequently,
guanidine- and benzamidine-containing compounds have
been extensively utilized in the rational design of
biologically active compounds. Some representative
examples that are illustrated in Figure 1 include factor
Xa (FXa) inhibitors (1),2 glycoprotein IIb/IIIa integrin
antagonists (2),3 nitric oxide synthase inhibitors (3),4
urokinase inhibitors (4),5 and oligoarginines as antago-
nists in HIV TAT/TAR interactions (5).6 However,
because of the high basicity of the guanidine (pKa ) 13)
and benzamidine (pKa ) 11.6), which renders these
compounds to be very polar, it has been a challenge to
identify compounds with good pharmacokinetic profiles
from these series. The high basicity of guanidine/
benzamidine groups has translated into a high desol-
vation cost during passive absorption through the
epithelial layer of the gut wall, which results in poor
oral absorption. Overall, the short duration of action and
poor oral absorption have precluded the development
of these compounds as oral agents. As such, many
pharmaceutical companies have invested significant
efforts to identify less basic and neutral mimics to try
to improve the pharmacokinetic properties of these
compounds.
Our ability to design less basic and neutral P1 ligands
was facilitated by the availability of a large number of
X-ray structures of trypsin-like enzyme/inhibitor com-
plexes.11 The strategy employed was to drive the potency
of our inhibitor to the picomolar range and then replace
the benzamidine moiety with a less basic or neutral P1
ligand.
More specifically we recently reported the design and
synthesis of SN429, a picomolar pyrazole-based FXa
inhibitor (Figure 2) containing a benzamidine in the P1
position.2,14 However SN429 is only 4% orally bioavail-
able and has a short half-life (iv t1/2 of 0.82 h in dogs,
typical of benzamidine-containing compounds). A com-
prehensive effort was initiated to search for potent and
orally bioavailable benzamidine mimics. The high po-
tency of the pyrazole (FXa Ki ) 13 pM) afforded us the
opportunity of being able to sacrifice 1-2 orders of
magnitude in affinity during the process of benzamidine
replacement while maintaining subnanomolar FXa
inhibition. This work culminated in a subnanomolar
FXa inhibitor with a 1-aminoisoquinoline as the P1
moiety that demonstrated improved pharmacokinetic
properties. During the course of our investigations,
several other research groups have also reported sub-
nanomolar benzamidine mimics with improved phar-
macokinetic features.11b,c,13
Herein we report on our systematic and comprehen-
sive search for novel benzamidine mimics with reduced
basicity that arose from our efforts to develop orally
active FXa inhibitors suitable for clinical development.
FXa was chosen as the target because it is the crucial
enzyme at the convergent point of the intrinsic and
extrinsic coagulation pathways.7,8 Together with FVa,
* Author to whom correspondence should be addressed [telephone
(609) 818-5720; fax (609) 818-3550; e-mail patrick.lam@bms.com].
10.1021/jm020578e CCC: $25.00 © 2003 American Chemical Society
Published on Web 09/17/2003

4406 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21
Lam et al.
F igu r e 1. Literature examples of guanidine/benzamidine-containing compounds.
F igu r e 2. Factor Xa inhibitor design.
Design
The interaction of the benzamidine group of the
mimics, it is preferable to maintain both components
in order to minimize the loss in binding affinity.
Figure 3 presents a representative series of designed
benzamidine mimics and nonbenzamidines, which were
synthesized and evaluated, in the order of decreasing
pKa. To maintain the Coulombic interaction of the mimic
with Asp189, one would prefer the mimic to have a pKa
above the physiological pH of 7.4 so that at least 50%
of the protonated mimic is available for binding. How-
ever, because of the phenomenon of pKa perturbation
as a result of ion-pairing interaction,16 a base with a
pKa 1-2 units lower than 7.4 may also be sufficiently
inhibitors with the carboxylate of Asp189 located at the
bottom of the S1 of the FXa pocket is a reinforced
interaction consisting of two components (Figure 3).15
The first is the Coulombic interaction of positive and
negative charges (ion pairing), and the second is the
bidentate hydrogen-bonding interaction. In general, a
1-2 orders of magnitude loss in Ki will result if one of
the two components is lost. When both components are
lost, there is usually a decrease in Ki of 3-4 orders of
magnitude. Thus, in the design of potent benzamidine

Novel Guanidine/ Benzamidine Mimics
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21 4407
F igu r e 3. Design of guanidine/benzamidine mimics.
Sch em e 1a
a
Reagents: (a) (i) NaNO2, HCl, 0 °C; (ii) SnCl2; (b) EtOH, 11, reflux; (c) (i) 3 N NaOH, dioxane, (ii) (ClCO)2, DMF or BOP, TEA,
CH2Cl2, then 13; (d) AlMe3, 13, CH2Cl2.
protonated when it is involved in an ion-pairing interac-
tion with Asp189.
the S1 pocket is positioned close to the 4-position of the
P1 phenyl group of the FXa inhibitors. Therefore,
4-substituted phenyl P1 moieties were designed to
introduce selectivity against trypsin.
Benzamidine mimics were also explored with the goal
of increasing selectivity against other trypsin-like pro-
teases. Trypsin was used as a prototypical example for
other trypsin-like proteases. One unique structural
feature of FXa (and also thrombin) as compared with
other trypsin-like enzymes is that FXa (and also throm-
bin) contains Ala190 at the bottom of the S1 pocket. In
contrast, several trypsin-like enzymes, such as trypsin,
FIXa, and FVIIa, contain Ser190.17 Hence, our strategy
was to take advantage of the larger S1 specificity pocket
in FXa (one oxygen-atom size difference) and introduce
selectivity against trypsin-like proteases by preparing
FXa inhibitors with a larger P1 moiety. The Ala190 of
Syn th esis
The synthesis of pyrazoles 14a -m was performed
according to the procedures shown in Scheme 1 begin-
ning either from the commercially available anilines
9a -g or from hydrazines 10h -m . Anilines 9a -g18 were
converted to their hydrazine analogues via diazotization
and stannous chloride reduction to give compounds
10a -g. The hydrazines 10a -m were next condensed
with ethyl 2-(methoxyimino)-4-oxopentanoate 112 to
give pyrazole esters 12a -m in a regioselective manner.

4408 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21
Lam et al.
Sch em e 2a
a
Reagents: (a) TFA, reflux; (b) (i) TFA, reflux, (ii) Pd(C), H2, EtOH; (c) (i) chloramine-T, then TFA, (ii) H2SO4; (d) (i) acetone oxime,
t-BuOK, DMF, (ii) 6 N HCl aq, EtOH, 80 °C; (e) (i) H2NNH2‚H2O, n-BuOH, 105 °C, (ii) TFA, reflux; (f) H2SO4; (g) (i) HCl, MeOH, (ii)
H2NOH; (h) (i) NaH, THF, (ii) n-BuLi, -78 °C, then (i-PrO)3B, (iii) HCl aq (i) (i) m-CPBA, acetone, (ii) PtO2, H2, EtOH, AcOH, (iii) TFA,
reflux; (j) (i) m-CPBA, acetone, (ii) TsCl, pyridine, (iii) ethanolamine, 50 °C, (iv) TFA, reflux; (k) (i) NaH, H2NC(dNH)NH2‚HCl, DMAC,
reflux, (ii) TFA, reflux; (l) (i) HC(dNH)NH2‚AcOH, DMAC, reflux, (ii) TFA, reflux.
Ta ble 1. Basic P1
The pyrazole ester was coupled with biarylaniline 13
via the Weinreb procedure19 to afford pyrazole amides
14a -m . Alternatively, ester hydrolysis yielded the
pyrazole carboxylic acid, which was activated with the
BOP reagent and coupled with biarylaniline 13 to
furnish pyrazole amides 14a -m . On the other hand, the
pyrazolecarboxylic acid was also converted to the acyl
chloride prior to coupling with biarylaniline 13.
Scheme 2 describes the conversion of pyrazoles 14a-m
to the inhibitors found in Tables 1-4. The synthesis of
basic inhibitor 15a has been reported by our research
group earlier.2 Inhibitors 15b-d of Table 1 were ob-
tained from the corresponding aryl nitro compounds by
sulfonamide deprotection in hot trifluoroacetic acid
followed by hydrogenation over palladium on carbon.
Butylamine 15e was synthesized as described in Scheme
3. More specifically, protection of amine 16, followed by
unmasking of the aldehyde gave 18. The aldehyde was
reductively aminated to give hydrazine 20, which fol-
lowed by condensation and deprotection resulted in
pyrazole 15e. Piperidinyl inhibitor 15f resulted from the
N-oxidation of pyridine 14m , followed by reduction over
Pt2O and deprotection.
The neutral inhibitors 22a ,c-e,g of Table 2 were
prepared in a straightforward manner by removal of the
sulfonamide tert-butyl group using trifluoroacetic acid
heated to reflux. Amide 22d was prepared via removal
of the sulfonamide tert-butyl group and concomitant
nitrile hydrolysis using concentrated sulfuric acid.
3-Methylsulfinimidoylphenyl 22f was synthesized from
methylthiophenyl pyrazole 14d by sulfimidation with
chloramine-T, followed by acidic hydrolysis.
The bidentate inhibitors 23a ,b,d in Table 3 were
prepared from the regioisomeric cyanofluorophenylpyra-
zoles 14f and 14g. Aminobenzisoxazole 23a was syn-
thesized using acetone oxime and base to replace aryl
fluoride followed by acidic ring closure and accompany-
ing sulfonamide deprotection. Aminoindazoles 23b and

Novel Guanidine/ Benzamidine Mimics
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21 4409
Ta ble 4. Basic Bidentate Hydrogen-Bonding P1
Ta ble 2. Neutral P1
sulfonamide deprotection. Phenylboronic acid 23c was
obtained from the bromide 14i via lithiation, followed
by quenching with borate and aqueous hydrolysis.
The basic inhibitors 24a and 24e were synthesized
according to Scheme 4. 7-Aminoisoquinoline 25 was
converted to the hydrazine 26 via diazotization and
stannous chloride reduction followed by condensation
with ethyl 2-(methoxyimino)-4-oxopentanoate, 11, to
give isoquinolinepyrazole ester 27. Amino and diami-
noquinazolines 24b,c were synthesized from 14f via
displacement and condensation with either formamidine
or guanidine, respectively. Benzamidoxime 24d was
prepared from 14c via Pinner reaction with hydrox-
ylamine. Aminopyridine 24f was the result of amination
of pyridine 14m .
Ta ble 3. Bidentate Hydrogen-Bonding P1
Resu lts a n d Discu ssion
Table 1 summarizes a list of basic amine P1 moieties
that were examined as benzamidine mimics. Benzyl-
amine 15a 2 was the most potent FXa inhibitor in this
series (FXa Ki of 2.7 nM compared to 0.013 nM for
SN429, 1), having a loss of only 200-fold binding affinity
as compared with compound 1. Among the anilines, the
3-anilino derivative 15b was the most potent but was
20-fold less potent than the corresponding m-benzyl-
amine 15a . All of the remaining amines that were
evaluated were determined to be significantly less
potent. Surprisingly, n-butylamine 15e was found to be
ineffective with a FXa Ki of 12 µM. We speculate that
this is a result of the steric congestion of the 1,2-
regiochemistry on the pyrazole ring, leading to a distor-
tion of the binding conformation of the flexible P1 alkyl
side chain of 15e. Finally, piperidine 15f was also
determined to be a very weak inhibitor, probably
because the piperidine nitrogen is not close enough to
interact with Asp189.
23d were prepared from heating hydrazine hydrate with
14f or 14g, respectively, in n-butanol followed by

4410 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21
Lam et al.
Sch em e 3a
a
Reagents: (a) TFAA, pyr, CH2Cl2; (b) HCO2H, H2O, CHCl3; (c) NH2NHBoc, PhH, reflux; (d) (i) BH3‚THF, (ii) HCl; (e) EtOH, 11,
reflux; (f) (i) TFA, reflux, (ii) K2CO3, MeOH aq, (iii) HCl.
Sch em e 4a
a
Reagents: (a) (i) NaNO2, HCl, 0 °C, (ii) SnCl2; (b) AcOH, 11, reflux; (c) AlMe3, 13, CH2Cl2; (d) (i) m-CPBA, acetone, (ii) TsCl, pyridine,
(iii) ethanolamine, (iv) TFA, reflux.
A series of pyrazole inhibitors with neutral non-
benzamidine P1 ligands were explored (Table 2). It was
found that 22a with a p-methoxyphenyl P1 moiety has
a FXa Ki of 11 nM, a loss of only 850-fold as compared
with benzamidine 1 (SN429). This was the most potent
neutral P1 ligand in our pyrazole series of FXa inhibi-
tors.20 Compound 22a resulted from a P1 screen of
phenols and anisoles, which was initiated on the basis
of the analysis of our X-ray structure of FXa/r-tick
anticoagulant protein.21 The details of the design will
be published elsewhere.20 Other compounds containing
neutral non-benzamidine P1 ligands, which were found
to be relatively potent inhibitors, were the 3-carboxa-
midophenyl derivative 22b and the 3-chlorophenyl
derivative 22c (FXa affinities of 19 and 37 nM, respec-
tively). Compounds that utilize the 3-cyanophenyl P1
22e, 3-sulfiniminephenyl P1 22f, and 3-sulfonami-
dophenyl P1 22g ligands were weaker FXa binders than
22d , which has an unsubstituted phenyl group in the
P1 position.
A series of benzamidine mimics capable of forming
bidentate hydrogen bonding were designed (Table 3) to
be able to achieve two hydrogen-bonding interactions
with Asp189 similar to the benzamidine and hence were
anticipated to be potent FXa inhibitors. Indeed, 3-ami-
nobenzisoxazole, 23a , was found to be a potent FXa
inhibitor with a Ki of 1.4 nM, a loss of only 110-fold
compared with benzamidine 1. 3-Aminoindazole, 23b,
was determined to be a weaker FXa binder, with a Ki
of 29 nM. Pyrazole 23c, with phenylboronic acid in the
P1 position, was a surprisingly weak inhibitor (Ki of 61
nM) even though it contains the bidentate hydrogen-
bond donor feature. This may be because phenylboronic
acid prefers to exist in the form of a negatively charged
borate and as a result cannot interact favorably with
Asp189. Not surprisingly, aminoindazole 23d , with a
Ki of 2800 nM, was found to be a much poorer binder
than the corresponding 23b, which contains the correct
regiochemistry.
Combining the bidentate hydrogen-bonding feature
with the basic amine feature, a series of interesting
benzamidine mimics were designed (Table 4). It was
anticipated this combination ought to provide the best
FXa affinity. Indeed, 1-aminoisoquinoline 24a (SQ311)
was found to be a subnanomolar inhibitor (FXa Ki of
0.33 nM), showing only a 25-fold loss in affinity relative
to the parent benzamidine 1 (Ki ) 0.013 nM). Thus,
1-aminoisoquinoline is the best benzamidine mimic in
this series in terms of FXa affinity. In addition, trypsin
selectivity was improved (7900- versus 1200-fold for 1).
We believe this is due to the negative steric interaction
incurred for the fused bicyclic P1 as a result of the
replacement of Ala190 in FXa with Ser190 in trypsin.
The addition of nitrogens as in 1-aminoquinazoline 24c
or 1,3-diaminoquinazoline 24b yielded weaker inhibitors
with FXa Ki of 29 and 16 nM, respectively. The
importance of the amino group of 1-aminoisoquinoline
P1 is evidenced by the weaker binding affinity of
isoquinoline 24e (FXa Ki of 370 nM). Benzamidoxime
24d was found to be 1000-fold less potent than benza-
midine 1. The significantly reduced FXa affinity may
be a result of the intramolecular hydrogen bonding of
the amidoxime functional group, preventing a favorable
interaction in a bidentate fashion with Asp189. 4-Ami-
nopyridine 24f, a benzamidine mimic used frequently
in the design of thrombin inhibitors,13b was ineffective
in our case.
To confirm our design strategy, the X-ray structure
of the complex of 24a (SQ311) bound in the active site
of thrombin was solved (Figure 4).22 In the S1 pocket,
1-aminoisoquinoline was determined to interact with
Asp189 in a bidentate fashion (2.5 and 2.9 Å) as
designed. This represents the first published 1-ami-
noisoquinoline (or the analogous aminopyridine) P1
interaction with Asp189 in the serine protease literature
where the interaction is truly bidentate, precisely
mimicking the guanidine/carboxylate bidentate interac-
tion in nature. The pKa of 24a was measured at 6.7.

Novel Guanidine/ Benzamidine Mimics
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21 4411
Ta ble 5. Relationship of pKa and Pharmacokinetic Parameters
in Dogs
F igu r e 4. Crystal structure of 24a (SQ311) in thrombin.
site is similar to that of the X-ray structure of the24a /
thrombin complex. The distance between the 4-carbon
of 1-aminoisoquinoline and the methyl carbon of Ala190
is 3.9 Å. This is approximately the van der Waals
contact distance and as anticipated introduces selectiv-
ity against trypsin, because trypsin has a slightly
smaller S1 pocket because of Ser190. Thus, for the
conversion of benzamidine (SN429) to 1-aminoisoquino-
line (24a ), a loss of 25-fold in FXa Ki and a 22-fold loss
in thrombin Ki were oberved (Table 4). In contrast, the
identical conversion gave a bigger loss of 163-fold in
trypsin Ki, indicating better selectivity of 24a against
trypsin.
P h a r m a cok in etics
A representative set of less basic and neutral benz-
amidine replacements were evaluated in dog pharma-
cokinetic studies and compared to benzamidine 1. These
compounds are listed in order of decreasing pKa in Table
5. The compounds were dosed both intravenously and
orally. All of the non-benzamidines have lower clear-
ances, ranging from 0.42 (15a ) to 0.09 (22a ) L/h/kg
relative to benzamidine 1 (0.67 L/h/kg). Also shown in
Table 5, lowering the pKa also resulted in a concomitant
increase in lipophilicity as measured by logP.23 The
volume of distribution at steady state (Vdss) of benz-
amidine 1 is very small (0.29 L/kg). As might be
expected on the basis of the higher logP values, the
non-benzamidines have larger volumes of distribution
at steady state (Vdss), ranging from 0.52 L/kg (22a ) to
1.6 L/kg (15a ). The combined effect of smaller clearance
and larger Vdss of non-benzamidines results in longer
iv half-lives (2.8-4.5 h), compared with 1 (0.82 h). As
can be seen in Figure 6a, where the pharmacokinetics
following intravenous dosing are normalized to a dose
of 1 mg/kg, the compounds separate into three clusters.
The neutral non-benzamidines have the best pharma-
cokinetic profile, followed by the moderately basic
benzamidine mimics. The highly basic benzamidine 1
has the poorest exposure in plasma and shortest half-
life.
F igu r e 5. (a, top) Model of 24a (SQ311) in FXa. (b, bottom)
Model of aminoisoquinoline SQ311 binding at the FXa active
site.
With a pKa of 6.7, only 13% of the 1-aminoisoquinoline
is protonated at the physiological pH of 7.4. The high
potency and the bidentate interaction of the 1-aminoiso-
quinoline P1 group, as seen from the X-ray complex,
implies that the 1-aminoisoquinoline is probably fully
protonated in its interaction with Asp189, due to ion-
pairing perturbation as described under Design.16 In
the crystal structure, the 1-aminoisoquinoline ring and
the pyrazole ring are found to form a dihedral angle of
73°. The amide carbonyl accepts a hydrogen bond (3.4
Å) from the backbone N-H of Gly216. The biaryl P4
moiety is situated in the S4 hydrophobic aryl-binding
pocket similar to 1, as detailed in our earlier published
work.2
Because the S1 pockets of FXa and thrombin are
similar, a model of 24a /FXa complex was obtained
(Figure 5a).15 The interaction of 24a with the FXa active

4412 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21
Lam et al.
An tith r om botic Effica cy
Among the various benzamidine mimics and non-
benzamidines in this study, the aminoisoquinoline, 24a
(SQ311), was chosen for further evaluation on the basis
of its sub-nanomolar FXa Ki (0.33 nM), excellent selec-
tivity profile, good human protein binding (89%), and
improved pharmacokinetic profile relative to benzami-
dine 1. In a rabbit arterial-venous (AV) shunt anti-
thrombosis model,9 24a inhibits thrombus formation
with an ID50 of 1.2 µmol/kg/h and with an IC50 (concen-
tration required to inhibit thrombus formation by 50%)
of 690 nM. Thus, 24a is an effective antithrombotic
agent in the rabbit AV shunt thrombosis model.
Su m m a r y
In summary, we have performed a systematic study
of the effect of lowering the pKa of the benzamidine P1
ligand by employing benzamidine mimics and non-
benzamidines as it relates both to potency and to the
pharmacokinetic profile of the compounds examined. We
have demonstrated that lowering the pKa improves the
pharmacokinetic features mainly as a result of three
parameters: the decrease in clearance and the increases
in Vdss and oral absorption as a result of the associated
decreasing polarity and desolvation cost. As a result of
this work a series of potent and orally bioavailable
benzamidine replacements have been identified. Fur-
ther optimization to potential clinical candidates is in
progress and will be reported elsewhere.2,20 Among these
benzamidine mimics and non-benzamidines, 1-ami-
noisoquinoline SQ311, 24a , was shown to be a highly
potent and selective orally bioavailable inhibitor of FXa.
Exp er im en ta l Section
Syn th esis. All reactions were run under an atmosphere of
dry nitrogen unless otherwise noted. Solvents and reagents
were obtained from commercial vendors in the appropriate
grade and used without further purification unless otherwise
indicated. NMR spectra were obtained on VXR or Unity 300
MHz instruments (Varian Instruments, Palo Alto, CA) with
chemical shift in parts per million downfield from TMS as an
internal reference standard. 1H assignment abbreviations are
as follows: singlet (s), doublet (d), triplet (t), quartet (q), pentet
(p), broad singlet (bs), doublet of doublets (dd), doublet of
triplets (dt), and multiplet (m). Elemental analyses were
performed by Quantitative Technologies, Inc., Whitehouse, NJ .
Mass spectra were measured with an HP 5988A mass spec-
trometer with particle beam interface using NH3 for chemical
ionization or a Finnigan MAT 8230 mass spectrometer with
NH3-DCI or VG TRIO 2000 for ESI. High-resolution mass
spectra were measured on a VG 70-VSE instrument with NH3
ionization. Flash chromatography was done using EM Science
silica gel 60 (230-400 mesh). Preparative thin-layer chroma-
tography was done on EM Science 60 plates F254 (2 mm; 20 ×
20 cm). HPLC purification was performed on a J asco 900 series
instrument using a C18 reverse phase column with acetoni-
trile/water (containing 0.05% TFA) as a mobile phase. All
compounds were found to be >97% pure by HPLC analysis
unless otherwise noted. Melting points were determined on a
Thomas-Hoover melting point apparatus and are uncorrected.
Compounds 11,2 13,14b and 22a ,d 20 were obtained as described
previously.
F igu r e 6. Pharmacokinetics in dogs: (a) intravenous dosing
normalized to 1 mg/kg; (b) oral dosing normalized to 1 mg/kg.
In terms of oral absorption, benzamidine 1 is only 4%
bioavailable, typical of benzamidine-containing com-
pounds. On the other hand, all of the non-benzamidines
have higher oral bioavailabilities ranging from 13% (15a
and 24a ) to 48% (22a ) due to a lower desolvation cost,
leading to higher lipid permeability, as seen from the
higher logP of the nonbenzamidines. The t1/2 (po) values
of the non-benzamidine compounds range from 3.2 h
(23a ) to 9.3 h (15a ), substantially longer than that of
benzamidine 1 (1.3 h). The normalized pharmacokinet-
ics for dogs dosed orally are shown in Figure 6b. To a
first approximation, the pharmacokinetics improve as
the pKa is decreased.24 Thus, the pharmacokinetic
features improve as one lowers the pKa from benza-
midines to less basic benzamidine mimics to the neutral
nonbenzamidines. This improvement is mainly a result
of three parameters: the decrease in clearance and the
increases in Vdss and oral absorption as a result of the
respective decreasing polarity and desolvation cost upon
lowering pKa.
Rep r esen ta tive P r oced u r e for P yr a zole In ter m ed ia tes
14a -m . Eth yl 1-(3-Ch lor op h en yl)-3-m eth yl-1H-p yr a zole-
ca r boxyla te (12h ). Ethyl 2-(methoxyimino)-4-oxopentanoate,
11 (217 mg, 1.16 mmol), and 3-chlorophenylhydrazine hydro-
chloride, 10h (416 mg, 2.32 mmol), in absolute ethanol (15 mL)
were heated at reflux for 5 h. The solvent was evaporated
under reduced pressure, and the residue was dissolved in

Novel Guanidine/ Benzamidine Mimics
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21 4413
dichloromethane and applied to a silica gel column. Elution
with a gradient of hexane to 4:1 hexane/ethyl acetate gave 305
mg (99%) of ethyl 1-(3-chlorophenyl)-3-methyl-1H-pyrazole-5-
carboxylate, 12h : 1H NMR (CDCl3) δ 7.45-7.26 (m, 4H), 6.82
(s, 1H), 4.26 (dd, 2H, J ) 7 Hz), 2.35 (s, 3H), 1.26 (t, 3H, J )
7 Hz); MS, 265.1 (M + H)+.
(8.05 g, 50 mmol), in methylene chloride (10 mL) cooled to 0
°C was added pyridine (4.48 mL, 55 mmol); trifluoroacetic
anhydride (7.77 mL, 55 mmol) was then added dropwise via
an addition funnel. After the addition, the reaction was slowly
warmed to room temperature and stirred for an additional 3
h. The reaction mixture was then partitioned between meth-
ylene chloride (80 mL) and water (40 mL), washed with 1 N
HCl (30 mL) and brine (30 mL), dried over sodium sulfate,
filtered, and concentrated to give 12.3 g (96%) of N-(4,4-
diethoxybutyl)-2,2,2-trifluoroacetamide, 17. This compound
(12.3 g, 47.8 mmol) was dissolved in chloroform (20 mL), and
85% formic acid (5 mL) was added. The reaction mixture was
stirred at room temperature for 14 h, after which the reaction
mixture was partitioned between chloroform (80 mL) and
water (40 mL), washed once with brine (40 mL), dried over
sodium sulfate, filtered, and concentrated to give 7.0 g (80%)
of 2,2,2-trifluoro-N-(4-oxobutyl)acetamide, 18. This compound
(5.0 g, 27.2 mmol) was dissolved in benzene (50 mL), and tert-
butyl carbazate (3.6 g, 27.2 mmol) was added. The reaction
was brought to reflux and stirred for 14 h. The reaction was
cooled to room temperature, the benzene was evaporated under
reduced pressure to ∼20 mL, and the resulting white solid that
precipitated was isolated by filtration. The solids were washed
once with diethyl ether (20 mL) and air-dried to give 5.5 g
(68%) of tert-butyl 2-{4-[(trifluoroacetyl)amino]butylidene}-
hydrazinecarboxylate, 19. This compound (5.0 g, 16.8 mmol)
was added to 18 mL of a borane THF complex (1 M). The
reaction mixture was stirred at room temperature for 14 h,
and the solvent was removed under reduced pressure. The
residue was purified via flash chromatography on silica gel
using 1:1 ethyl acetate/hexane to give 3.2 g (95%) of tert-butyl
2-{4-[(trifluoroacetyl)amino]butyl}hydrazinecarboxylate. This
compound (3.2 g, 16 mmol) was dissolved in methanol (20 mL),
cooled to 0 °C, and HCl gas was bubbled through the solution
for 10 min. The solution was slowly warmed to room temper-
ature and stirred for another 10 min. The solvent was
evaporated to give 2.5 g of 2,2,2-trifluoro-N-(4-hydrazinobutyl)-
acetamide hydrochloride, 20, as a brown oil (63%): LRMS
(AP+), 200.1 (M + H)+; 1H NMR (CDCl3) δ 7.06 (bs, 1H), 4.45
(bs, 2H), 3.39 (q, 2H), 3.54 (t, 2H), 1.82-1.76 (m, 2H), 1.64-
1.58 (t, 2H). This compound was converted to 21 according to
the procedure for the conversion of 14h to 22c. Compound 21
(250 mg, 0.431 mmol) was dissolved in trifluoroacetic acid (4
mL) and brought to reflux for 1.5 h. The solution was cooled
to ambient temperature and the solvent evaporated under
reduced pressure. The residue was taken up in ethyl acetate
and saturated sodium bicarbonate, the phases were separated,
and the organic phase was washed once with water, dried over
sodium carbonate, filtered, and evaporated to give 200 mg
(89%) of the corresponding deprotected sulfonamide. This
compound (200 mg, 0.382 mmol) was dissolved in methanol
(3 mL), and potassium carbonate (211 mg, 1.52 mmol) and
water (3 mL) were added and stirred for 14 h. The methanol
was evaporated, and ethyl acetate and water were added; the
phases were separated, and the organic phase was washed
with water and brine, dried over sodium sulfate, filtered, and
evaporated. The residue was purified by silica gel flash
chromatography to give 80 mg (49%) of 1-(4-aminobutyl)-N-
[2′-(aminosulfonyl)-1,1′-biphenyl-4-yl]-3-methyl-1H-pyrazole-
5-carboxamide, which was converted to its hydrochloride salt
by treating a methanol solution with 1 M HCl in ether followed
by evaporation to give the title compound: 1H NMR (CDCl3)
δ 8.12 (d, 1H, J ) 8.1 Hz), 7.68 (d, 2H, J ) 8.4 Hz), 7.59 (td,
H, J ) 7.2, 1.1 Hz), 7.52-7.43 (m, 3H), 7.34-7.30 (m, 2H),
6.57 (s, 1H), 4.49 (t, 2H, J ) 7.2 Hz), 2.78 (bs, 4H), 2.65 (t,
2H, J ) 7.2 Hz), 2.29 (s, 3H), 1.85 (p, 2H, J ) 7.5 Hz), 1.44 (p,
2H, J ) 7.5 Hz); LRMS (ESI+), 428.3 (M + H)+(100%). HRMS
calcd 428.1756; found 428.173774 for C21H26N5O3S (M + H)+.
N-{2′-[(ter t-Bu tyla m in o)su lfon yl]-1,1′-bip h en yl-4-yl}-1-
[3-ch lor op h en yl]-3-m et h yl-1H -p yr a zole-5-ca r b oxa m id e
(14h ). To a solution of 4′-amino-N-(tert-butyl)-1,1′-biphenyl-
2-sulfonamide, 13 (115 mg, 0.378 mmol), in dichloromethane
(5 mL) at ambient temperature was added dropwise trimethy-
laluminum (0.38 mL, 2.0 M in hexane, 0.756 mmol). This
mixture was stirred for 15 min at ambient temperature, and
then 12h (100 mg, 0.378 mmol) in dichloromethane (5 mL)
was added. The reaction was heated to 40 °C and stirred for
18 h. The reaction was cooled and carefully added to 20 mL of
a 10% methanol/chloroform solution. The solvent volume was
reduced to ∼2 mL, and the residue was purified by preparative
TLC (eluent 50% ethyl acetate/hexane) to give 170.5 mg (70%)
of the title compound as a colorless solid: 1H NMR (CD3OD)
δ 8.10 (d, 1H, J ) 8.7 Hz), 7.64-7.31 (m, 11H), 6.84 (s, 1H),
2.37 (s, 3H), 1.01 (s, 9H). Anal. Calcd (C27H26N4O3SCl‚H2O)
C, H, N.
1-[3-(Am in om eth yl)p h en yl]-N-[2′-(a m in osu lfon yl)-1,1′-
bip h en yl-4-yl]-3-m eth yl-1H-p yr a zole-5-ca r boxa m id e, Tr i-
flu or oa cetic Acid Sa lt (15a ). 15a was prepared according
to the method of Pinto et al.2
1-(3-Am in oph en yl)-N-[2′-(am in osu lfon yl)-1,1′-biph en yl-
4-yl]-3-m eth yl-1H-p yr a zole-5-ca r boxa m id e (15b). 14k was
synthesized from 10k according to the procedure described for
the conversion of 10h to 14h . A solution of 14k in trifluoro-
acetic acid (5 mL) was heated to reflux for 1 h. The reaction
was cooled to ambient temperature, and the solvent was
concentrated under reduced pressure. The residue was taken
up in ethyl acetate and washed with saturated aqueous sodium
bicarbonate, water, and brine, dried over sodium sulfate, and
filtered, and the solvent was evaporated under reduced pres-
sure. N-[2′-(Aminosulfonyl)-1,1′-biphenyl-4-yl]-3-methyl-1-(3-
nitrophenyl)-1H-pyrazole-5-carboxamide was judged to be of
sufficient purity and used without purification. To this com-
pound (64.3 mg, 0.135 mmol) in absolute ethanol (5 mL) under
N2 was added 5% Pd on carbon (60 mg), and the flask was
evacuated and charged with H2 (1 atm) via a double-walled
latex balloon. After 3 h, the reaction was judged to be complete
by TLC, the flask was purged with N2, and the contents were
filtered through a pad of Celite. The pad was washed with
methanol and the solvent concentrated under reduced pres-
sure. The residue was purified by preparative TLC (eluent 10%
methanol/chloroform) to give 50 mg (83%) of the title compound
as a colorless solid: 1H NMR (CD3OD) δ 8.0 (d, 1H, J ) 7.8
Hz), 7.61-7.58 (m, 3H), 7.50 (t, 1H, J ) 7.8 Hz), 7.38 (d, 2H,
J ) 6.9 Hz), 7.31 (d, 1H, J ) 7.5 Hz), 7.13 (t, 1H, J ) 8.1 Hz),
6.78 (s, 1H), 6.72-6.69 (m, 3H), 2.33 (s, 3H). HRMS calcd
448.1443; found 448.144 for C23H22N5O3S (M + H)+.
1-(2-Am in oph en yl)-N-[2′-(am in osu lfon yl)-1,1′-biph en yl-
4-yl]-3-m eth yl-1H-p yr a zole-5-ca r boxa m id e (15c). 15c was
synthesized from 10j according to the procedure described for
the conversion of 10k to 15b: 1H NMR (CD3OD) δ 8.13 (d, 1H,
J ) 7.5 Hz), 8.04 (d, 1H, J ) 8.1 Hz), 7.58-7.28 (m, 6H), 7.17
(d, 2H, J ) 8.4 Hz), 6.93 (s, 1H), 6.75 (d, 2H, J ) 9.0 Hz), 2.47
(s, 3H); LRMS (ESI+), 448.12 (M + H)+(60%), 470.16 (M +
Na)+(100%).
1-(4-Am in oph en yl)-N-[2′-(am in osu lfon yl)-1,1′-biph en yl-
4-yl]-3-m eth yl-1H-p yr a zole-5-ca r boxa m id e, Tr iflu or oa ce-
tic Acid Sa lt (15d ). 15d was synthesized from 10l according
to the procedure described for the conversion of 10k to 15b:
1
mp 234.9 °C; H NMR (CD3OD) δ 8.10 (dd, 1H, J ) 7.8, 1.2
Hz), 7.63-4.49 (m, 4H), 7.40-7.31 (m, 3H), 7.15 (d, 2H, J )
8.7 Hz), 6.75-7.70 (m, 3H), 2.34 (s, 3H); LRMS (ESI+), 448.4
(M + H)+(100%). HRMS calcd 448.1443; found 448.1444 for
N-[2′-(Am in osu lfon yl)-1,1′-bip h en yl-4-yl]-3-m eth yl-1-(3-
p ip er d in yl)-1H-p yr a zole-5-ca r boxa m id e (15f). 14m was
synthesized from 10m according to the procedure described
for the conversion of 10h to 14h . To a solution of 14m (0.5 g,
1.02 mmol) in acetone (20 mL) was added m-chloroperbenzoic
C
23H22N5O3S (M + H)+.
1-(4-Am in obu tyl)-N-[2′-(a m in osu lfon yl)-1,1′-bip h en yl-
4-yl]-3-m eth yl-1H-p yr a zole-5-ca r boxa m id e, Hyd r och lo-
r id e (15e). To a solution of 4,4-diethoxy-1-butanamine, 16

4414 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21
Lam et al.
acid (0.28 g of 70% grade, 1.12 mmol), and the reaction was
brought to reflux for 1.5 h. The reaction was cooled to ambient
temperature and solvent removed under reduced pressure. The
residue was taken up in ethyl acetate and saturated sodium
bicarbonate, and the phases were separated. The organic phase
was washed with saturated sodium bicarbonate and water,
dried over sodium sulfate, and filtered, and the solvent was
removed under reduced pressure to give 470 mg (91%) of the
N-oxide of sufficient purity to be used without purification.
This compound (50 mg, 0.099 mmol) was subjected to hydro-
genation in absolute ethanol/acetic acid 5:1 (6 mL) with PtO2
(10 mg, 0.044 mmol) under 20 lb/in.2 of H2 for 14 h. The
reaction was filtered through a pad of Celite, the pad washed
with methanol, and the filtrate evaporated. The residue was
taken up in ethyl acetate and saturated sodium bicarbonate,
and the phases were separated. The organic phase was washed
with water, dried over sodium sulfate, and filtered, and the
solvent was removed under reduced pressure. The residue was
subjected to silica gel flash column chromatography eluting
with a gradient of 10-20% methanol in chloroform to give 30
mg (61%) of the piperdinyl compound. A solution of this
compound (30 mg, 0.061 mmol) in trifluoroacetic acid (2 mL)
was heated to reflux for 1 h. The reaction was allowed to cool
to ambient temperature, and the solvent was concentrated
under reduced pressure. The residue was taken up in ethyl
acetate and washed with saturated aqueous sodium bicarbon-
ate and water, dried over sodium sulfate, and filtered, and the
solvent was evaporated under reduced pressure to give 17 mg
(64%) of the title compound: 1H NMR (CD3OD) δ 8.02 (dd,
1H, J ) 7.8, 1.2 Hz), 7.62 (d, 2H, J ) 8.4 Hz), 7.53 (td, 1H, J
) 7.5, 1.5 Hz), 7.44 (td, 1H, J ) 7.8, 1.5 Hz), 7.34 (d, 2H, J )
8.7 Hz), 7.26 (dd, 1H, J ) 7.5, 1.5 Hz), 6.73 (s, 1H), 5.40 (m,
1H), 3.45 (d, 2H, J ) 5.7 Hz), 3.14-3.08 (m, 2H), 2.23 (s, 3H),
2.14 (dd, 2H, J ) 37.2, 5.4 Hz), 1.87 (m, 1H), 1.74 (m, 1H);
LRMS (ESI+), 440.1 (M + H)+(100%). HRMS calcd 440.1756;
found 440.176811 for C22H26N5O3S (M + H)+.
N-[2′-(Am in osu lfon yl)-1,1′-b ip h en yl-4-yl]-3-m et h yl-1-
p h en yl-1H-p yr a zole-5-ca r boxa m id e (22d ). 22d was pre-
pared according to the method of Pruitt.20
N-[2′-(Am in osu lfon yl)-1,1′-biph en yl-4-yl]-1-[3-cyan oph e-
n yl]-3-m eth yl-1H-p yr a zole-5-ca r boxa m id e (22e). 22e was
synthesized from 9c according to the procedure for the
conversion of 10h to 22c: 1H NMR (CDCl3) δ 8.17 (dd, 2H, J
) 8.1,1.5 Hz), 7.86 (m, 3H), 7.66 (d, 2H, J ) 8.8 Hz), 7.61 (m,
4H), 7.50 (d, 2H, J ) 8.4 Hz), 7.35 (dd, 1H, J ) 7.3, 1.5 Hz),
6.72 (s, 1H), 4.24 (s, 2H), 1.56 (s, 3H); LRMS (ESI+) 458.18
(M + H)+, 480.13 (M + Na)+. HRMS calcd 458.1287; found
458.1274 for C24H20O3N5S1 (M + H)+. Anal. Calcd (C24H19O3N5S‚
0.1H2O) C, H, N.
N-[2′-(Am in osu lfon yl)-1,1′-bip h en yl-4-yl]-3-m eth yl-1-[3-
(m eth ylsu lfin im id oyl)p h en yl]-1H-p yr a zole-5-ca r boxa m -
id e, Tr iflu or oa cetic Acid Sa lt (22f). 14d was synthesized
from 9d according to the procedure described for the conver-
sion of 10h to 14h . To a solution of 14d (300 mg, 0.56 mmol)
in 10 mL of methanol was added chloramine-T hydrate (153
mg, 0.67 mmol), and the resulting solution was stirred at 60
°C for 1 h. The reaction was allowed to cool and then was
diluted with ethyl acetate, washed with 1 N sodium hydroxide
and brine, dried over magnesium sulfate, and concentrated to
afford 250 mg (63%) of an approximate 1:1 mixture of two
products. This mixture was dissolved in 5 mL of trifluoroacetic
acid and was stirred at 80 °C for 15 min. After cooling to
ambient temperature, the reaction was concentrated in vacuo,
and the residue was purified by HPLC and lyophilized to afford
45 mg (20%) of the desired N-[2′-(aminosulfonyl)-1,1′-biphenyl-
4-yl]-3-m et h yl-1-(3-{m et h yl[(4-m et h ylph en yl)su lfon yl]-
sulfinimidoyl}phenyl)-1H-pyrazole-5-carboxamide: 1H NMR
(CDCl3) δ 8.65 (s, 1H), 8.10 (d, 1H, J ) 7.0 Hz), 7.80 (s, 1H),
7.68-7.60 (m, 4H), 7.59-7.51 (m, 2H), 7.50-7.44 (m, 2H), 7.37
(d, 2H, J ) 8.1 Hz), 7.28 (m, 1H), 7.16 (d, 2H, J ) 8.1 Hz),
6.78 (s, 1H), 4.64 (s, 2H), 2.82 (s, 3H), 2.32 (s, 3H), 2.29 (s,
3H); LRMS (ES+), 648 (M + H)+. To this compound (30 mg,
0.046 mmol) was added 0.10 mL of concentrated H2SO4, and
this reaction mixture was stirred at ambient temperature until
it was homogeneous. Ether was added, and a solid precipitated
out of solution. The solvent was decanted, and the solid was
further triturated with ether and dried in vacuo. The solid was
then purified by HPLC and lyophilized to afford 20 mg (71%)
of the title compound: 1H NMR (CDCl3) δ 10.65 (s, 1H), 8.15
(s, 1H), 7.99 (dd, 1H, J ) 8.1, 1.5 Hz), 7.93-7.89 (m, 1H), 7.79-
7.70 (m, 2H), 7.66 (d, 2H, J ) 8.8 Hz), 7.61-7.53 (m, 2H), 7.33
(d, 2H, J ) 8.8 Hz), 7.28-7.25 (m, 1H), 7.01 (s, 1H), 6.69 (s,
1H), 3.34 (s, 3H), 2.31 (s, 3H); LRMS (ES)+, 494.0 (M + H)+.
HRMS (ES+) calcd 494.1321; found 494.1323 for C24H24N5O3S2
(M + H)+.
N-[2′-(Am in osu lfon yl)-1,1′-bip h en yl-4-yl]-3-m eth yl-1-(3-
m eth oxyp h en yl)-1H-p yr a zole-5-ca r boxa m id e (22a ). 22a
was prepared according to the method of Pruitt.20
1-[3-(Am in oca r b on yl)p h en yl]-N-[2′-(a m in osu lfon yl)-
1,1′-b ip h en yl-4-yl]-3-m et h yl-1H -p yr a zole-5-ca r b oxa m -
id e (22b). 14c was synthesized from 9c according to the
procedure described for the conversion of 10h to 14h . To 14c
(185 mg, 0.361 mmol) was added concentrated H2SO4 (5 mL)
at ambient temperature and the nitrile dissolved slowly. After
24 h, ice and water were added and the precipitated solids
were isolated by filtration. The solids were taken up in ethyl
acetate and water. The phases were separated, and the
aqueous fraction was extracted three times with ethyl acetate.
The combined organic fraction was washed with water,
saturated sodium bicarbonate, and brine, dried over magne-
sium sulfate, and filtered, and the solvent was removed under
reduced pressure. The residue was purified by flash column
chromatography (gradient elution 1-10% methanol in meth-
ylene chloride) and afforded 88 mg (52%) of the title com-
pound: 1H NMR (DMSO-d6) δ 10.63 (s, 1H), 8.12 (s, 1H), 8.04
(m, 2H), 7.90 (m, 1H), 7.69 (d, 2H, J ) 8.4 Hz), 7.62-7.52 (m,
5H), 7.36 (d, 2H, J ) 8.4 Hz), 7.32 (m, 1H), 7.24 (s, 2H), 6.93
(s, 1H), 2.50 (s, 3H); LRMS (ESI+), 476.3 (M + H)+. HRMS
calcd 476.1392; found 476.1392 for C24H22O3N5S (M + H)+.
Anal. Calcd (C24H21O4N5S) C, H, N.
N-[2′-(Am in osu lfon yl)-1,1′-biph en yl-4-yl]-1-[3-ch lor oph e-
n yl]-3-m eth yl-1H-p yr a zole-5-ca r boxa m id e (22c). A solu-
tion of 14h (28.0 mg, 0.0536 mmol) in trifluoroacetic acid (3
mL) was heated at reflux for 1 h. The reaction was evaporated,
taken up in ethyl acetate, and washed with 1 N sodium
hydroxide solution. The organic solution was dried over sodium
sulfate, filtered, and evaporated. The residue was purified by
preparative TLC (eluent 10% methanol/chloroform) to give 17.5
mg (70%) of the title compound as a colorless solid: 1H NMR
(CD3OD) δ 8.09 (d, 1H, J ) 8.1 Hz), 7.64-7.51 (m, 6H), 7.48-
7.41 (m, 6H), 7.30 (d, 1H, J ) 8.1 Hz), 2.37 (s, 3H); LRMS
(ES+) 467.2 (M + H)+ (100%). HRMS calcd 467.0945; found
467.0915 for C23H19N4O3SCl (M + H)+.
1-[3-(Am in osu lfon yl)ph en yl]-N-[2′-(am in osu lfon yl)-1,1′-
biph en yl-4-yl]-3-m eth yl-1H-pyr azole-5-car boxam ide (22g).
22g was synthesized from 9e according to the procedure
described for the conversion of 10h to 22c: 1H NMR (CD3OD)
δ 8.08 (dd, 1H, J ) 7.8, 0.9 Hz), 7.65-7.30 (m, 15H), 6.88 (s,
1H), 2.37 (s, 3H); LRMS (ES+), 512.07 (M + H)+(20%), 534.07
(M + Na)+(100%).
1-(3-Am in o-1,2-ben zisoxazol-5-yl)-N-[2′-(am in osu lfon yl)-
1,1′-b ip h en yl-4-yl]-3-m et h yl-1H -p yr a zole-5-ca r b oxa m -
id e (23a ). 14f was synthesized from 9f according to the
procedure described for the conversion of 10h to 14h . To a
solution of acetone oxime (86 mg, 1.18 mmol) in DMF (6 mL)
was added sodium tert-butoxide (1 M in THF, 1.18 mL). The
mixture was stirred at room temperature for 30 min followed
by the addition of a solution of 14f (0.21 g, 0.39 mmol) in DMF
(4 mL). The reaction was stirred at room temperature for 5 h.
The reaction mixture was then partitioned between ethyl
acetate and aqueous HCl (1.4 M), washed once with aqueous
HCl (1.4 M), four times with water, and once with brine, dried
over sodium sulfate, filtered, and concentrated. Flash chro-
matography (30% ethyl acetate/hexane) gave N-{2′-[(tert-
butylamino)sulfonyl]-1,1′-biphenyl-4-yl}-1-(3-cyano-4-isopro-
p ylid e n e a m in ooxyp h e n yl)-3 -m e t h yl-1 H -p yr a zole -5 -
carboxamide (0.19 g, 81% yield) in sufficient purity to be used
without purification. This compound (0.19 g, 0.32 mmol) was

Novel Guanidine/ Benzamidine Mimics
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21 4415
dissolved in ethanol (4 mL), and to the solution was added 6
N HCl (4 mL). The reaction mixture was stirred at 80 °C for
3 h and cooled to room temperature. The white precipitate that
formed was filtered and recrystallized from methanol to give
0.14 g (80%) of the title compound: 1H NMR (CD3OD) δ 8.17
(d, 1H, J ) 1.8 Hz), 8.07 (dd, 1H, J ) 7.9, 1.2 Hz), 7.94 (dd,
1H, J ) 9.0, 1.2 Hz), 7.64 (d, 2H, J ) 8.7 Hz), 7.61-7.50 (m,
3H), 7.38 (d, 2H, J ) 8.4 Hz), 7.30 (dd, 1H, J ) 7.5, 1.0 Hz),
6.97 (s, 1H), 2.38 (s, 3H); LRMS (ESI+), 489.1 (M + H)+(100%).
Anal. Calcd (C24H20N6O4S1‚H2O) C, H.
carboxamide, 24e (3.50 g, 6.49 mmol), was dissolved in 60 mL
of acetone to which was added m-chloroperbenzoic acid (70%)
(1.86 g, 7.55 mmol), and the reaction was allowed to stir at
ambient temperature. Upon completion, as determined by
TLC, the solvent was removed under reduced pressure and
the residue taken up in 100 mL each of ethyl acetate and
saturated sodium bicarbonate. The phases were separated, and
the organic layer was dried over sodium sulfate, filtered, and
evaporated to give the N-oxide as a light pink solid in
quantitative yield and of sufficient purity for the next step:
MS (ES+), 556.20 (M + H)+ (15%), 578.21 (M + Na)+ (100%).
1-(3-Am in o-1H-in d a zol-5-yl)-N-[2′-(a m in osu lfon yl)-1,1′-
biph en yl-4-yl]-3-m eth yl-1H-pyr azole-5-car boxam ide (23b).
14f was synthesized from 9f according to the procedure
described for the conversion of 10h to 14h . To a solution of
14f (95 mg, 0.179 mmol) in n-butanol (5 mL) was added
hydrazine monohydrate (0.3 mL) and the solution brought to
reflux for 4 h. The reaction was cooled to ambient temperature
and partitioned between ethyl acetate and water. The organic
phase was washed once with water, dried over sodium sulfate,
and filtered, and the solvent was evaporated under reduced
pressure. The residue was subjected to silica gel flash column
chromatography eluting with 10% methanol in chloroform to
give 50 mg (52%) of 1-(3-amino-1H-indazol-5-yl)-N-{2′-[(tert-
butylamino)sulfonyl]-1,1′-biphenyl-4-yl}-3-methyl-1H-pyrazole-
5-carboxamide. This compound (45 mg, 0.083 mmol) was
treated with trifluoroacetic acid (3 mL) at reflux for 1 h. The
solvent was evaporated and the residue taken up in ethyl
acetate and saturated sodium bicarbonate. The organic phase
was washed twice with water, dried over sodium sulfate,
filtered, and evaporated to yield 31 mg (77%) of the title
compound: 1H NMR (CD3OD) δ 8.07 (dd, 1H, J ) 7.5, 1.2 Hz),
7.76 (s, 1H), 7.59-7.54 (m, 3H), 7.50 (dd, 1H, J ) 7.5, 1.5 Hz),
7.35 (d, 4H, J ) 7.8 Hz), 7.29 (dd, 1H, J ) 7.5, 1.2 Hz), 6.81
(s, 1H), 2.36 (s, 3H); LRMS (ESI+), 488.1 (M + H)+(100%).
HRMS calcd 448.1505; found 448.150485 for C24H22N7O3S (M
+ H)+. Anal. Calcd (C24H21N7O3S1‚H2O‚MeOH) C, H.
The N-oxide was dissolved in 110 mL of anhydrous pyridine,
tosyl chloride (1.64 g, 8.63 mmol) was added in three equal
portions, and the reaction was allowed to stir at ambient
temperature. The pyridine was removed under reduced pres-
sure, and to the residue was added 45 mL of ethanolamine;
the reaction was stirred at ambient temperature. Upon
completion, as determined by TLC, the reaction was poured
onto cracked ice, and the solids were isolated by filtration and
dried under vacuum to yield 2.33 g (65% yield) of a mixture of
the 1-aminoisoquinoline (major) and 4-aminoisoquinoline (mi-
nor) products as a tan solid: MS (ES+) 555.22 (M + H)+
(100%). HRMS calcd 555.2178; found 555.21858 for C30H30N6O3S
(M + H)+.
To 20 mL of trifluoroactic acid was added the mixture of
the aminoisoquinoline compounds and the reaction brought
to reflux for 4 h. The solvent was removed under reduced
pressure and the residue made basic with cold aqueous sodium
carbonate, extracted with ethyl acetate (3 × 40 mL), dried over
sodium sulfate, and evaporated. The tan solid was purified by
silica gel flash column chromatography eluting with 15%
MeOH/CHCl3 to give 1.60 g (76% yield) of the title compound
as a light tan solid.
The product was then treated with 1 equiv of methane-
sulfonic acid in THF. Evaporation of the solvent gave 24a : mp
1
195 °C; H NMR (CD3OD) δ 8.27 (s, 1H), 8.07 (d, 1H, J ) 7.5
3-[5-({[Am in o s u lfo n y l)-1,1′-b ip h e n y l-4-y l]a m in o }-
ca r bon yl)-3-m eth yl-1H-p yr a zol-1-yl]p h en ylbor on ic a cid
(23c). 14i was synthesized from 10i according to the procedure
described for the conversion of 10h to 14h . To a solution of
14i (100 mg, 0.196 mmol) in THF (15 mL) was added sodium
hydride (47 mg of 60% dispersion in oil, 1.2 mmol) and the
reaction stirred at ambient temperature for 20 min. The
reaction was cooled to -78 °C, and n-butyllithium (0.25 mL
of 1.6M in hexanes, 0.40 mmol) was added dropwise followed
by stirring at this temperature for 30 min and warming to 0
°C for 10 min, followed by cooling again to -78 °C. Triisopropyl
borate (0.11 mL, 0.49 mmol) was next added and the cooling
bath removed, allowing the reaction to warm to ambient
temperature for 14 h. The reaction was cooled to 0 °C, 10%
HCl (5 mL) was added, and the reaction was allowed to warm
to ambient temperature and stirred for 1.5 h. The reaction was
quenched into an excess of saturated sodium bicarbonate, ethyl
acetate was added, and the phases were separated. The organic
phase was washed once with water and brine, dried over
sodium sulfate, filtered, and evaporated under reduced pres-
sure. The residue was subjected to preparative TLC to give
35 mg (37%) of the title compound: 1H NMR (CDCl3) δ 8.13
(d, 1H, J ) 8.1 Hz), 7.75 (s, 2H), 7.59-7.39 (m, 11H), 6.75 (s,
1H), 2.40 (s, 3H); LRMS (ESI+), 477.1 (M + H)+(40%), 499.0
(M + Na)+(100%). HRMS calcd 533.1666; found 533.167468
for glycerol ester C26H26BN4O6S (M + H)+.
1-(3-Am in o-1H-in d a zol-6-yl)-N-[2′-(a m in osu lfon yl)-1,1′-
biph en yl-4-yl]-3-m eth yl-1H-pyr azole-5-car boxam ide (23d).
14g was synthesized from 9g according to the procedure
described for the conversion of 10h to 14h . 23d was synthe-
sized from 14g according to the procedure for the conversion
of 14f to 23b: 1H NMR (CD3OD) δ 8.12 (d, 1H, J ) 7.8 Hz),
7.75-7.31 (m, 8H), 7.11 (d, 1H, J ) 7.8 Hz), 6.83 (s, 1H), 2.41
(s, 3H); LRMS (ESI+), 510.1 (M + Na)+(100%).
1-(1-Am in o-7-isoqu in olyl)-N-[2′-(a m in osu lfon yl)-1,1′-bi-
ph en yl-4-yl]-3-m eth yl-1H-p yr a zole-5-ca r boxa m id e, Meth -
a n esu lfon ic Acid Sa lt (24a ). N-{2′-[(tert-Butylmino)sulfonyl]-
1,1′-biphenyl-4-yl}-1-(7-isoquinolyl)-3-methyl-1H-pyrazole-5-
Hz), 7.80-7.68 (m, 6H), 7.62-7.49 (m, 5H), 7.36 (d, 2H, J )
8.4 Hz), 7.29 (d, 1H, J ) 7.5 Hz), 7.02 (d, 1H, J ) 6.0 Hz),
6.91 (s, 1H), 2.38 (s, 3H); LRMS (ESI+), 499.14 (M + H)+
(100%). HRMS calcd 499.1552; found 499.1535 for C26H22N6O3S
(M + H)+. Anal. Calcd (C26H22N6O3S‚MsOH‚3H2O) C, H, N.
N-[2′-(Am in osu lfon yl)-1,1′-biph en yl-4-yl]-1-(2,4-diam in o-
6-qu in azolin yl)-3-m eth yl-1H-pyr azole-5-car boxam ide, Tr i-
flu or oa cetic Acid Sa lt (24b). 14f was synthesized from 9f
according to the described procedure for the conversion of 10h
to 14h . To a solution of 14f (60 mg, 0.112 mmol) in dimethy-
lacetamide (5 mL) was added guanidine hydrochloride (40 mg,
0.419) and sodium hydride (25 mg of a 60% dispersion in oil,
1.7 mmol), and the reaction was brought to reflux for 14 h.
The reaction was cooled to ambient temperature, methanol
was added (2 mL), and the reaction was partitioned between
ethyl acetate and saturated sodium bicarbonate. The organic
phase was dried over sodium sulfate, filtered, and evaporated
under reduced pressure. The residue was purified by prepara-
tive TLC (eluent 10% methanol/chloroform) to give 15 mg
(24%) of the 2,4-diamino-6-quinazolinyl compound. This com-
pound (15 mg, 0.026 mmol) in trifluoroacetic acid (2 mL) was
heated to reflux for 1 h. The reaction was allowed to cool to
ambient temperature, and the solvent was concentrated under
reduced pressure. The residue was taken up in ethyl acetate
and washed with saturated aqueous sodium bicarbonate and
water, dried over sodium sulfate, and filtered, and the solvent
was evaporated under reduced pressure to give 6 mg (44%) of
N-[2′-(aminosulfonyl)-1,1′-biphenyl-4-yl]-1-(2,4-diamino-6-
quinazolinyl)-3-methyl-1H-pyrazole-5-carboxamide. The resi-
due was purified by reverse phase HPLC and lyophilized to
give the title compound: 1H NMR (CD3OD) δ 8.31 (d, 1H, J )
2.4 Hz), 8.10 (d, 1H, J ) 7.8 Hz), 7.87 (d, 1H, J ) 8.7 Hz),
7.66-7.32 (m, 13H), 6.99 (s, 1H), 2.40 (s, 3H); LRMS (ESI+),
515.2 (M + H)+ (100%). HRMS calcd 515.1614; found 515.1604
for C25H22N8O3S (M + H)+.
1-(4-Am in o-6-qu in a zolin yl)-N-[2′-(a m in osu lfon yl)-1,1′-
bip h en yl-4-yl]-3-m eth yl-1H-p yr a zole-5-ca r boxa m id e, Tr i-
flu or oa cetic Acid Sa lt (24c). 14f was synthesized from 9f

4416 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21
Lam et al.
according to the procedure described for the conversion of 10h
to 14h . To a solution of 14f (53 mg, 0.10 mmol) in dimethy-
lacetamide (5 mL) was added formamidine acetate (104 mg,
1.00 mmol), and the reaction was heated to 120 °C for 14 h.
The reaction was cooled to ambient temperature, ethyl acetate
and saturated aqueous sodium bicarbonate were added, and
the phases were separated. The organic phase was dried over
sodium sulfate and filtered and the solvent evaporated under
reduced pressure to give 30 mg (54%) of the 4-amino-6-
quinazolinyl compound. This compound (30 mg, 0.054 mmol)
in trifluoroacetic acid (3 mL) was heated to reflux for 1 h. The
reaction was allowed to cool to ambient temperature, and the
solvent was concentrated under reduced pressure. The residue
was purified by reverse phase HPLC and lyophilized to afford
18 mg (54%) of the title compound: 1H NMR (CD3OD) δ 10.63
(s, 1H), 8.80 (s, 1H), 8.62 (d, 1H, J ) 2.1 Hz), 8.25 (s, 2H),
8.04 (d, 2H, J ) 6.6 Hz), 7.84 (d, 1H, J ) 9 Hz), 7.71 (d, 2H,
J ) 8.7 Hz), 7.60-7.53 (m, 2H), 7.36 (d, 2H, J ) 8.7 Hz), (d,
2H, J ) 8.7 Hz), 7.30 (dd, 1H, J ) 7.2, 1.5 Hz), 7.19 (s, 2H),
7.09 (s, 1H), 2.39 (s, 3H); LRMS (ESI+), 500.2 (M + H)+
(100%). HRMS calcd 500.1505 (M + H)+; found 500.1485 for
dropwise trimethylaluminum (10.9 mL, 21.6 mmol, 2 M in
hexane). The solution was stirred for 30 min at ambient
temperature, and then ethyl 1-(isoquinolyn-7′-yl)-3-methyl-5-
pyrazole carboxylate (2.02 g, 7.19 mmol), 27, in 70 mL of
anhydrous dichloromethane was added dropwise; the reaction
warmed to 40 °C and allowed to stir for 14 h. The reaction
was cooled to 0 °C and carefully quenched with 50 mL of 1 N
hydrochloric acid, diluted with 50 mL of water, and made basic
with solid sodium carbonate. The phases were separated, and
the aqueous fraction was extracted with dichloromethane (3
× 30 mL), dried over sodium sulfate, and evaporated to give
N-{2′-[(tert-butylamino)sulfonyl]-1,1′-biphenyl-4-yl}-1-(7-iso-
quinolyl)-3-methyl-1H-pyrazole-5-carboxamide (3.50 g, 90%
yield), 24e, as a brown solid of sufficient purity for the next
step. The material was further purified by silica gel flash
chromatography eluting with 5% methanol in chloroform: MS
(ES+), 540.22 (M + H)+ (100%). 24e (26.3 mg, 0.0487 mmol)
in trifluoroacetic acid (3 mL) was heated to reflux for 1 h. The
reaction was allowed to cool to ambient temperature and the
solvent concentrated under reduced pressure. The residue was
taken up in ethyl acetate and washed with saturated aqueous
sodium bicarbonate and water, dried over sodium sulfate, and
filtered, and the solvent was evaporated under reduced pres-
sure. The residue was purified by preparative TLC (eluent 10%
methanol/chloroform) to give 14.4 mg (61%) of the title
compound: 1H NMR (CD3OD) δ 9.27 (s, 1H), 8.46 (d, 1H, J )
6.0 Hz), 8.18 (s, 1H), 8.09-8.01 (m, 2H), 7.90-7.86 (m, 2H),
7.63-7.46 (m, 4H), 7.37 (d, 2H, J ) 8.4 Hz), (dd, 1H, J ) 7.8,
1.5 Hz), 6.92 (s, 1H), 2.40 (s, 3H); LRMS (ESI+), 484.12 (M +
H)+(100%). HRMS calcd 484.1443; found 484.143007 for (M
+ H)+.
1-(2-Am in o-5-p yr id yl)-N-[2′-(a m in osu lfon yl)-1,1′-bip h e-
n yl-4-yl]-3-m eth yl-1H-p yr a zole-5-ca r boxa m id e (24f). 14m
was synthesized from 10m according to the procedure de-
scribed for the conversion of 10h to 14h . 14m was oxidized by
m-CPBA according to the procedure for 15f. The N-oxide (110
mg, 0.218 mmol) was dissolved in anhydrous pyridine (4 mL),
tosyl chloride (54 mg, 0.283 mmol) was added, and the reaction
was allowed to stir at ambient temperature. The pyridine was
removed under reduced pressure, and to the residue was added
ethanolamine (4 mL); the reaction was stirred at 50 °C for 24
h. The reaction was purified by silica gel flash chromatography
eluting with 7% methanol in chloroform to give 75 mg (67%)
of the 2-aminopyridin-5-yl compound. A solution of this
compound (40 mg, 0.077 mmol) in trifluoroacetic acid (3 mL)
was heated to reflux for 1 h. The reaction was allowed to cool
to ambient temperature, and the solvent was concentrated
under reduced pressure. The residue was taken up in ethyl
acetate, washed with saturated aqueous sodium bicarbonate
and water, dried over sodium sulfate, and filtered, and the
solvent was evaporated under reduced pressure to give
29 mg (84%) of the title compound: 1H NMR (CDCl3) δ 8.00
(d, 1H, J ) 7.2 Hz), 7.83 (d, 1H, J ) 2.4 Hz), 7.63 (d, 2H, J )
8.4 Hz), 7.65-7.52 (m, 2H), 7.39 (dd, 1H, J ) 9.0, 3.0 Hz), 7.30
(d, 2H, J ) 8.7 Hz), 7.20 (s, 2H), 6.82 (s, 1H), 6.44 (d, 1H, J )
8.7 Hz), 6.18 (s, 2H), 2.26 (s, 3H); LRMS (ESI+), 449.1 (M +
H)+(100%). HRMS calcd 449.1395; found 449.139317 for
C22H21N6O3S (M + H)+.
C
25H22N7O3S (M + H)+.
N -[2′-(Am in osu lfon yl)-1,1′-b ip h e n yl-4-yl]-1-{3-[(h y-
d r oxya m in o)(im in o)m eth yl]p h en yl}-3-m eth yl-1H-p yr a -
zole-5-ca r boxa m id e, Tr iflu or oa cetic Acid Sa lt (24d ). 14c
was synthesized from 9c according to the procedure described
for the conversion of 10h to 14h . To a solution of 14c (184
mg, 0.403 mmol) in absolute ethanol (2 mL) was added
hydroxylamine hydrochloride (100 mg, 0.145 mmol) and
sodium carbonate (72 mg, 0.68 mmol) and the reaction heated
to reflux for 14 h. The reaction was filtered and the filtrate
purified by reverse phase HPLC to give 54 mg (27%) of the
desired compound after removal of the solvent: 1H NMR
(DMSO-d6) δ 10.65 (s, 1H), 8.04 (dd, 1H, J ) 7.7, 1.5 Hz), 7.85
(s, 1H), 7.70 (d, 2H, J ) 8.4 Hz), 7.62 (m, 6H), 7.37 (d, 2H, J
) 8.4 Hz), 7.32 (dd, 2H, J ) 7.3, 1.5 Hz), 7.27 (s, 2H), 6.98 (s,
1H), 2.34 (s, 3H); LRMS (ESI+), 491.2 (M + H)+(100%), 513.2
(M + Na)+(50%); HPLC purity >97%. HRMS calcd 491.1501;
found 491.1490 for C24H22N6O4S (M + H)+. Anal. Calcd
(C24H22N6O4S‚TFA‚0.7H2O) C, H, N.
N -[2′-(Am in o s u lfo n y l)-1,1′-b ip h e n y l-4-y l]-1-(7-is o -
q u in olyl)-3-m et h yl-1H -p yr a zole-5-ca r b oxa m id e (24e).
7-Aminoisoquinoline25 (6.26 g, 43.4 mmol), 25, was added to
40 mL of concentrated hydrochloric acid at 0 °C. Sodium nitrite
(3.0 g, 43.4 mmol) was dissolved in 15 mL of water, cooled to
0 °C, and added dropwise to the solution of 25 in HCl. The
reaction was stirred for 30 min at 0 °C. Stannous chloride
dihydrate (29.3 g, 130.2 mmol, 3 equiv) was dissolved in 25
mL of concentrated hydrochloric acid, and the solution was
cooled to 0 °C and added dropwise to the solution containing
25 and allowed to stand at 0 °C for 1 h. The resulting
precipitate was isolated by filtration and washed with 100 mL
of ice-cold brine followed by 100 mL of a 2:1 petroleum ether/
ethyl ether solution. The brown solid was dried under dynamic
vacuum to give 26. The isoquinoline salt 26 (9.0 g, 26 mmol)
was suspended in 100 mL of glacial acetic acid, and ethyl
2-(methoxyimino)-4-oxopentanoate, 11 (4.0 g, 21.3 mmol), was
added dropwise. The reaction was heated to reflux for 14 h,
after which the acetic acid was evaporated; to the residue was
added 100 mL of water, and the suspension was cooled to 0
°C and neutralized by the addition of solid sodium bicarbonate.
The solution was extracted with ethyl acetate (6 × 50 mL),
dried over sodium sulfate, and evaporated to give the title
compound as a brownish solid (5.15 g, 86% yield), which was
>85% of the desired pyrazole regioisomer. This material was
purified by silica gel flash chromatography eluting with 5%
methanol in chloroform to give 27: 1H NMR (CDCl3) δ 9.29
(s, 1H,), 8.58 (s, 1H, J ) 5.9 Hz), 8.05 (d, 1H, J ) 2.0 Hz), 7.89
(d, 1H, J ) 8.8 Hz), 7.75 (dd, 1H, J ) 8.8, 2.2 Hz), 7.70 (d, 1H,
J ) 5.9 Hz), 6.89 (s, 1H), 4.24 (q, 2H, J ) 7.1 Hz), 2.40 (s, 3H),
1.24 (t, 3H, J ) 7.1 Hz); MS (ES+), 282.1 (M + H)+ (100%).
P h a r m a cok in etic Stu d y. Compounds were administered
intravenously over 60 min to two dogs or orally to two dogs
(0.5 mg/kg iv, 0.2 mg/kg po) in an N-in-1 format. The formula-
tion used for both intravenous and oral dosing was 70% water,
10% propylene glycol, 10% ethanol, and 10% N,N-dimethylac-
etamide. Blood samples were collected into sodium citrate
tubes predose and at 0.5, 1, 1.1, 1.25, 1.5, 2, 3, 4, 5, 6, 8, and
12 h for the intravenous dosingg or predose and at 1, 2, 3, 4,
5, 6, 8, and 12 h for oral dosing. Plasma concentrations for
the compounds were determined by LC-MS/MS using multiple
reaction monitoring in positive electrospray ionization mode
on a Micromass Quattro Ultima mass spectrometer coupled
with a Shimadzu HPLC system and an HTC PAL autosampler.
Each compound was separated on a Supelco Discovery C18
column. The quantitation limit was 1 nM for all compounds.
To a solution of 4′-amino-N-(tert-butyl)-1,1′-biphenyl-2-sul-
fonamide, 13 (2.19 g, 7.19 mmol), in 100 mL of anhydrous
dichloromethane under an atmosphere of nitrogen was added

Novel Guanidine/ Benzamidine Mimics
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21 4417
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R. R. Design and Synthesis of Isoxazoline Derivatives as Factor
Xa Inhibitors. 1. J . Med. Chem. 1999, 42, 2752-2759. (c) Quan,
M. J .; Ellis, C. D.; Liauw, A. Y.; Alexander, R.; Knabb, R. M.;
Lam, G. N.; Wong, P. C.; Wexler, R. R. Design and Synthesis of
Isoxazoline Derivatives as Factor Xa Inhibitors. 2. J . Med. Chem.
1999, 42, 2760-2773. (d) Pruitt, J . R.; Pinto, D. J .; Quan, M. L.;
Estrella, M. J .; Bostrom, L. L.; Knabb, R. M.; Wong, P. C.;
Wexler, R. R. Isoxazolines and Isoxazoles as Factor Xa Inhibi-
tors. Bioorg. Med. Chem. Lett. 2000, 10, 685-689. (e) Fevig, J .
M.; Pinto, D. J .; Han, Q.; Quan, M. L.; Pruitt, J . R.; J acobson, I.
C.; Galemmo, R. A., J r.; Wang, S.; Orwat, M. J .; Bostrom, L. L.;
Lam, P. Y. S.; Knabb, R. M.; Wong, P. C.; Wexler, R. R. Synthesis
and SAR of Benzamidine Factor Xa Inhibitors Containing a
Vicinal-Substituted Heterocyclic Core. Bioorg. Med. Chem. Lett.
2001, 11, 641-645. (f) Quan, M. L.; Wexler, R. R. The Design
and Synthesis of Noncovalent Factor Xa Inhibitors. Curr. Top.
Med. Chem. 2001, 1, 131-149.
(15) Energy-minimized molecular models were performed using the
Insight II program developed by Molecular Simulations Inc.
molecular modeling program (version 97.2). For similar models
refer to: Padmanabhan, K.; Padmanabhan, K. P.; Tulinsky, A.;
Park, C. H.; Bode, W.; Blankenship, D. T.; Cardin, A. D.; Kisiel,
Structure of human Des (1-45) factor Xa at 2.2 Å resolution W.
J . Mol. Biol. 1993, 232, 947-966.
(16) A good example is the crucial ion-pairing interaction of Asp29/
Arg8′ or Asp29′/Arg8 at the dimeric interface of HIV protease.
The pKa of Asp29 was titrated to be 2.1 instead 3.7, the pKa of
normal aspartic acid. This pKa suppression of aspartic acid is
attributed to the ion-pairing effect, which suppresses the pKa of
the acid partner and elevates the pKa of the base partner. See:
Yamazaki, T.; Nicholson, L. K.; Torchia, D. A.; Wingfield, P.;
Stahl, S. J .; Kaufman, J . D.; Eyermann, C. J .; Hodge, C. N.; Lam,
P. Y. S.; Ru, Y.; J adhav, P. K.; Chang, C.-H.; Weber, P. C. NMR
(9) (a) Wong, P. C.; Quan, M. L.; Crain, E. J .; Watson, C. A.; Wexler,
R. R.; Knabb, R. M. Nonpeptide Factor Xa Inhibitors: I. Studies
with SF303 and SK549, a New Class of Potent Antithrombotics.
J . Pharmacol. Exp. Ther. 2000, 292, 351-357. (b) Wong, P. C.;
Crain, E. J .; Knabb, R. M.; Meade, R. P.; Quan, M. L.; Watson,
C. A.; Wexler, R. R.; Wright, M. R.; Slee, A. M. Nonpeptide Factor
Xa Inhibitors: II. Antithrombotic Evaluation in a Rabbit Model
of Electrically Induced Carotid Artery Thrombosis. J . Pharmacol.
Exp. Ther. 2000, 295, 212-218.
(10) Murayama, N.; Tanaka, M.; Kunitada, S.; Yamada, H.; Inoue,
T.; Terada, Y.; Fujita, M.; Ikeda, Y. Tolerability, Pharmacoki-
netics, and Pharmacodynamics of DX-9065a, a New Synthetic
Potent Anticoagulant and Specific Factor Xa Inhibitor, in
Healthy Male Volunteers. Clin. Pharmacol. Ther. 2000, 66, 258-
264.
(11) For reviews and references see: (a) Vacca J . P. Thrombosis and
Coagulation. Annu. Rep. Med. Chem. 1998, 33, 81-90. (b) Fevig,
J . M.; Wexler, R. R. Anticoagulants: Thrombin and Factor Xa
inhibitors. Annu. Rep. Med. Chem. 1999, 34, 81-100. (c) Zhu,
B.-Y.; Scarborough, R. M. Factor Xa Inhibitors: Recent Advances
in Anticoagulant Agents. Annu. Rep. Med. Chem. 2000, 35, 83-
102. (d) Sanderson, P. E. J . Anticoagulants: Inhibitors of
Thrombin and Factos Xa. Annu. Rep. Med. Chem. 2001, 36, 79-
98.
(12) Currently there are no oral antithrombotic agents on the market
based solely on FXa inhibition. More than 20 companies
worldwide are performing research with the aim of producing
orally bioavailable FXa inhibitors as clinical agents.
(13) For review specifically on nonbenzamidines see: (a) Menear, K.
Progress Towards the Discovery of Orally Active thrombin
Inhibitors. Curr. Med. Chem. 1998, 5, 457-468. (b) Rewinkel,
J . B. M.; Adang, A. E. P. Strategies and Progress Towards the
Ideal Orally Active Thrombin Inhibitor. Curr. Pharm. Design
1999, 5, 1043-1075. (c) Peterlin-Masic, L.; Kikelj, D. Arginine
Mimetics. Tetrahedron 2001, 57, 7073-7105. It is our belief that
for benzamidine mimics to be useful as anticoagulants in clinics,
they should have subnanomolar FXa Ki. For examples of papers
containing nonbenzamidne thrombin and FXa inhibitors that

4418 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 21
Lam et al.
and X-ray Evidence That the HIV Protease Catalytic Aspartyl
Groups Are Protonated in the Complex Formed by the Protease
and a Non-Peptide Cyclic Urea-Based Inhibitor. J . Am. Chem.
Soc. 1994, 116, 10791-10792.
PEG 8000 buffered with 100 mM sodium phosphate in the
reservoir. Crystal growth was initiated by macroseeding with
small thrombin-hirudin crystals. Crystals of a usable size grew
in 1 week. Data were collected on an R Axis II image plate
mounted on a Rigaku RU200 rotating anode. Data were pro-
cessed with the HKL suite (Otwinowski, Z.; Minor, W. Processing
of X-ray Diffraction Data Collected in Oscillation Mode. Methods
Enzymol. 1997, 276 (Macromolecular Crystallography, Part A),
307-326.) Crystals diffracted to 2.1 Å with an R-merge of 7.4%.
Unambiguous density for the inhibitor was observed in the active
site. The inhibitor was built using the program Quanta (Molec-
ular Structure Inc.). Data were refined using the program CNX
(Molecular Structure Inc.) with a crystallographic R factor of
21.0% with an R-free of 25.8%.
(17) Mann, Kenneth G.; Lorand, Laszlo. Blood coagulation. Methods
Enzymol. 1993, 222 (Proteolytic Enzymes in Coagulation, Fi-
brinolysis, and Complement Activation, Part A), 121-123.
(18) Compound 9e was obtained from 3-nitro-benzenesulfonyl chlo-
ride via reaction with tert-butylamine/TEA/CH2Cl2 then reduc-
tion Pd(C)/H2/EtOH.
(19) Basha, A.; Lipton, M.; Weinreb, S. M. Tetrahedron Lett. 1977,
48, 4171-4174.
(20) A comprehensive list of neutral non-benzamidines with p-
methoxyphenyl being the best neutral non-benzamidine can be
found in: Pruitt, J . R.; Galemmo, R. A., J r.; Lam, P. Y. S.; Pinto,
D. J .; Dominguez, C.; Alexander, R. S.; Rossi, K. A.; Wells, B.
L.; Drummond, S.; Bostrom, L. L.; Wong, P. C.; Wright, M. R.;
Bai, S.; Knabb, R. M.; Wexler, R. R. Discovery of 2-(2-Amino-
methyl-phenyl)-5-trifluoromethyl-2H-pyrazole-3-carboxylic acid
(3-fluoro-2′-sulfamoyl-biphenyl-4-yl)-amide (DPC602), a Potent,
Selective and Orally Bioavailable Factor Xa Inhibitor. J . Med.
Chem. 2003, in press.
(23) HPLC logP was performed by J . Gerry Everlof: Partition
coefficients were determined using reversed-phase high-perfor-
mance liquid chromatography (RP-HPLC). A series of standards
with known literature Log P values were eluted through a C18
column using an isocratic methanol/water mobile phase. There
is a linear correlation between the Log K′ and Log P. In reversed-
phase liquid chromatography the compounds’ distribution be-
tween the mobile and stationary phase is governed by hydro-
phobic forces; these parameters can be regarded as a measure
of the compounds’ lipophilicity.
(24) The normal correlation of increasing oral absorption with
increasing Caco2 permeability cannot be performed here due to
the relatively low water solubility of the less polar inhibitors,
preventing permeability measurements.
(25) 7-Aminoisoquinoline 25 was obtained from 3,4-dihydroisoquino-
line according to the method of McCoubrey (Isoquinolines. Part
III. The Nitration of 3,4-Dihydro- and 1,2,3,4-Tetrahydroiso-
quinolines J . Chem. Soc. 1951 2851-2853). 3,4-Dihydroiso-
quinoline was obtained from commercially available 1,2,3,4-
tetrahydroisoquinoline according to the method of Yamazaki
(Oxidation of secondary amines with nickel sulfate-potassium
persulfate [NiSO4-K2S2O8]. Chem. Lett. 1992, 5, 823-826).
(21) Wei, A.; Alexander, R. S.; Duke, J .; Ross, H.; Rosenfeld, S. A.;
Chang, C.-H. Unexpected Binding Mode of Tick Anticoagulant
Peptide Complexed to Bovine Factor Xa. J . Mol. Biol. 1998, 283,
147-154.
(22) Human R-thrombin was purchased from Enzyme Research Labs
and used without further purification. Acetylhirudin (54-65)
sulfated was purchased from Bachem (catalog no. H-7415). The
thrombin-hirudin complex was prepared and concentrated to
10 mg/mL. This complex was diluted in half with 41 mM SQ311
in 0.375 M NaCl, 0.05M sodium phosphate, pH 7.3, 0.02% NaN3,
and 4% DMSO. Crystallization conditions were similar to those
described by Skrzypczak-J ankun et al. (Skrzypczak-J ankun, E.;
Carperos, V. E.; Ravichandran, K. G.; Tulinsky, A.; Westbrook,
M.; Maraganore, J . M. Structure of the hirugen and hirulog 1
complexes of R-thrombin. J . Mol. Biol. 1991, 221, 1379). Crystals
were grown using hanging drop vapor diffusion from 27 to 30%
J M020578E