Design, synthesis, and evaluation of oxyanion-hole selective inhibitor substituents for the S1 subsite of factor Xa

Article abstract of DOI:10.1016/j.bmcl.2004.07.054

Placement of an anionic group within the oxyanion hole region of the Factor Xa catalytic site substantially enhances activity, with small flexible groups favored over bulkier ones.

Full text of DOI:10.1016/j.bmcl.2004.07.054

Bioorganic & Medicinal Chemistry Letters 14 (2004) 5165–5170
Design, synthesis, and evaluation of oxyanion-hole
selective inhibitor substituents for the S1 subsite of factor Xa
Sochanchingwung Rumthao,a,b Oukseub Lee,a Qi Sheng,a WenTao Fu,a
b
a,
*
Debbie C. Mulhearn,a DavidCrich, Andrew D. Mesecara andMichael E. Johnson
aCenter for Pharmaceutical Biotechnology, University of Illinois at Chicago,
900 S. Ashland Avenue, m/c 870, Chicago, IL 60607-7173, USA
bDepartment of Chemistry, University of Illinois at Chicago, 845 West Taylor Street, Chicago, IL 60607-7061, USA
Received23 February 2004; revised26 July 2004; accepted27 July 2004
Available online 14 August 2004
Abstract—We have designed, synthesized, and evaluated the factor Xa inhibitory activities of p-amidinophenyl-sulfones, amines,
and alcohols intended to take advantage of the polarity and hydrogen-bonding potential of the oxyanion hole region of the S1 spe-
cificity pocket. We demonstrate that placement of an anionic group within the oxyanion hole region of the catalytic site substantially
enhances activity, with small flexible groups favoredover bulkier ones. Ab initio p Ka calculations suggest that the hydroxyl substit-
uent frequently used for benzamidine moieties may be ionized to form an anionic group, consistent with the general trend. One
nonamidine based substituent also shows promising activity.
Ó 2004 Elsevier Ltd. All rights reserved.
Factor Xa, the penultimate enzyme in the coagulation
cascade, has received increasing attention in the devel-
opment of new anticoagulant agents.1–6 The enzymes
of the blood coagulation cascade, including thrombin,
factor Xa, andfactor VIIa are known to have specificity
pockets (S1 subsites) very similar to those of other serine
proteases such as trypsin andplasmin. The Asp189 res-
idue at the bottom of the pocket binds ionically with
arginyl or benzamidinium ionic groups. To enhance
affinity and specificity, it is desirable to generate addi-
tional hydrogen bonding or ionic interactions with other
functional groups within the S1 specificity pocket. In
prior theoretical analysis, we have proposedthat hydro-
gen bonding with the catalytic Ser 195 hydroxyl in the
oxyanion hole region can provide an additional stabili-
zation mechanism for a properly positionedH-bonddo-
nor.7 More recently, we have shown that addition of an
anionic phosphinic acidmoiety to benzamidine as a tet-
rahedral analog in the vicinity of the oxyanion hole en-
hances inhibitory affinity by factors of 5–10 against
several serine proteases.8 Other workers have also
shown empirically that addition of hydrogen bonding
groups (OH or NH),9–14 amine,14 carboxyl,15,16 andsul-
fonyl17 functionalities at positions within or adjacent to
the S1 subsite also enhance activity. However, to date
there has been no systematic evaluation of the optimum
ionic/H-bonding functionality, or of the best positioning
of such a group within the S1 subsite. Accordingly, we
have undertaken such a survey, using the well-character-
ized benzamidine group as an anchor to the Asp 189 side
chain. This moiety has been demonstrated to bind in the
S1 specificity pocket with very little variation in posi-
tioning for a variety of benzamidine-based factor Xa
inhibitors.9,12,18–22 We have also exploredthe effects of
a couple of benzamidine replacements that have been
incorporatedinto factor Xa inhibitors, including iso-
quinoline23,24 andphenyl-dimethylamine
25,26
groups.
Whenever satisfactory literature syntheses were located
for proposedsubstrates they were followed( 1,27,28
8,29,30 17,30,31 18,30,32 and 1928,33), with the other mem-
bers of the series obtainedby the routes set out below.
In general, in the following syntheses, the Pinner reac-
tion was the first methodassayedfor conversion of nit-
riles to amidines. When this failed, transformation of the
nitrile group to the thioamide with diethyldithiophos-
phate–H2O followedby S-methylation then aminolysis
Keywords: p-Hydroxybenzamidine; Serine protease inhibitor; Factor
Xa; Oxyanion hole; S1 specificity pocket.
*
Corresponding author. Tel.: +1 312 996 9114; fax: +1 312 413
9303; e-mail: mjohnson@uic.edu
8
was the methodof choice. In the event that neither of
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.07.054

5166
S. Rumthao et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5165–5170
these methods was satisfactory, resort was made to the
conversion of nitrile groups to N-acetoxy amidines with
hydroxylamine and acetic anhydride followed by
hydrogenation.34
Amidine 11 was obtainedfrom the known azide 934,35 by
a straightforwardprocedure ( Scheme 3).
The homologous aminoamidine 16 was obtainedfrom
1236 by the sequence shown in Scheme 4. Thus, Curtius
rearrangement of 12 afforded N-Boc nitrile 13. This was
convertedto the N-acetoxyamidine 14, hydrogenation
of which afforded 15. Finally, removal of the Boc group
with TFA yielded 16 (Scheme 4).
4-Cyanobenzyl methyl sulfone (2) was preparedfrom 4-
cyanobenzaldehyde,27 which was treatedwith diethyl
dithiophosphate–H2O8 to affordthe thioamiadte
Alkylation with methyl iodide, then exposure to
NH4OAc finally afforded the amidine 4 (Scheme 1).
3.
Palladium mediated phosphorylation37 of 6-bromo-1-
amino-isoquinoline24 20 afforded phosphonate 21,
which on removal of the benzoyl andisopropyl groups
under acidic conditions gave aminoisoquinoline 22
(Scheme 5).
The homologous sulfone 7 was preparedfrom bromide
5 by a sequence beginning with sodium methyl sulfide
followedby H O2 oxidation.27,28 The resulting nitrile 6
2
was treated with diethyl dithiophosphate–H2O to afford
the thioamidate, which following methylation and sub-
sequent aminolysis with NH4OAc, gave amidine 7
(Scheme 2).
The N,N-dimethylamino phosphinic acid 28 was ob-
tainedby metallation of commercially available 24, fol-
SO2Me
SO2Me
SO2Me
NH2
MeI, Acetone,∆
HSP(S)(OEt)2-H2O
80%
NH4OAc, CH3CN, rt
86%
CN
S
NH2
HN
3
4
2
Scheme 1.
SO2Me
SO2Me
Br
HSP(S)(OEt)2-H2O
98%
NaSMe, EtOH, 90 °C
89%
H2O2, AcOH, 0 °C-rt
MeI, Acetone, ∆
NH4OAc, CH3CN, rt
85%
91%
CN
CN
HN
NH2
5
6
7
Scheme 2.
N3
NH2
NH2
NH2
HCl, MeOH, 0 °C
H
2 / 10% Pd -C
NH3-MeOH, 75 °C
76%
MeOH-CHCl3
90%
CN
CN
HN
10
11
9
Scheme 3.
NH2
NH2
NHBoc
NHBoc
COOH
NHBoc
NH2OH, Py,
H2O, EtOH,
Na2CO3, 84%
TFA, DCM,
0 °C-rt
H2, Pd-C,
54%
DPPA, TEA,
t-BuOH, ∆, 92%
Ac2O, AcOH,
85%
CN
CN
HN
HN
NHOAc
HN
NH2
12
14
15
16
13
Scheme 4.

S. Rumthao et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5165–5170
5167
O
O
P
O
P
Br
O P H ,
Pd(PPh3)4
O
HO
Conc. HCl, ∆
N
N
N
95%
Et3N, Tol, 110 °C,
87%
HN
O
HN
21
O
NH2
22
20 Ph
Ph
Scheme 5.
N
N
N
n-BuLi, THF, -78 °C
O
Conc. HCl, ∆
P
N
Cl
Br
P
O
N
P
O
OH
28
24
25
26
Scheme 6.
38
lowedby quenching with the phosphoryl amidate
25,
The sulfone 1 shouldbe essentially isosteric with the pre-
viously described AMPA system,8 but lacks the negative
charge. Modeling calculations suggest that the sulfone
shouldbe positionedin the oxyanion hole region similar
to the phosphinic acidof AMPA. It exhibits about a 5-
foldhigher Ki than benzamidine 27, in contrast to the 5-
folddecrease in Ki (enhancement of activity) for AMPA
over benzamidine,8 confirming that charge is an impor-
tant determinant of binding affinity in this region. Add-
ing an ethylene spacer between the sulfone andthe phenyl
ring (7) increases activity by about a factor of 10, but a
single methylene spacer (4) results in the loss of activity,
indicating that moving the sulfone out of the oxyanion
hole with a flexible spacer of sufficient length allows
the formation of a more favorable contact interaction
between the sulfone andthe protein, but that a short
spacer probably induces bumping collisions that cannot
be compensatedby conformational rearrangement.
and final amide hydrolysis in concd HCl at reflux. This
protocol was foundto be more convenient than an alter-
native literature synthesis for this compound( Scheme
6).
Compounds were evaluated for their potency as FXa
inhibitors using the chromogenic substrate, MeO-CO-
D-CHG-Gly-Arg-pNA. Activities were determined as
the initial rate of cleavage of the peptide p-nitroanilide
by the enzyme. All substrate concentrations usedwere
equal to the Km values under the present assay condi-
tions; Ki values are listedin Table 1.
Table 1. In vitro inhibitory activities of benzamidine-based analogs
andrelatedcompounds against factor Xa
HN
R
H2N
Placing an amine in the oxyanion hole (8) also produces
about a twofolddecrease in Ki over benzamidine (27).
Aniline exhibits a pKa of about 4.6, andthe strong elec-
tron-withdrawing character of the amidine group para
to the amine shouldsubstantially further reduce the
pKa of 8, thus producing a hydrogen-bonding group
that will have no cationic character. However, adding
one (11) or two (16) methylene spacers between the phe-
nyl andthe amine produces subsequent ꢀ2-foldand ꢀ4-
foldreductions in activity, respectively. The methylene
spacers will separate the amine from the electron-with-
drawing effects of the benzamidine, with the pKa of
the resulting alkyl amine probably in the 8–10 range,
producing a protonated amine cationic group in the
vicinity of the oxyanion hole that is consequently disad-
vantageous to binding.
Compoundno. R-group or inhibitor structure FXa Ki (lM)
27
1
R = H
349
1800
>5000
193
197
429
750
30
R = SO2CH3
R = CH2SO2CH3
R = (CH2)2SO2CH3
R = NH2
4
7
8
11
16
17
18
19
R = CH2NH2
R = (CH2)2NH2
R = OH
R = CH2OH
R = (CH2)2OH
55
607
N
23
22
226
NH2
NH2
O
P
OH
>5000
N
The most significant enhancement in activity results
from placing a hydroxyl group in the oxyanion hole
(17). It is unclear whether this is due simply to advanta-
geous hydrogen bonding, or if it is due to deprotonation
of the hydroxyl to form the p-benzamidinophenate
OH
P O
28
4100
N

5168
S. Rumthao et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5165–5170
anion. The pKa of p-nitrophenol is about 7.2, andthe
amidine group should exhibit similar or stronger elec-
tron-withdrawing characteristics, suggesting that the
hydroxyl of 17 may exist as an anion within the oxyan-
ion hole. Accordingly, we have used ab initio meth-
41
ods39,40 andGaussian 03 to calculate the pKa for the
hydroxyl of 17, andfinda value of 7.8, supporting this
suggestion. Adding a single methylene to form 18 re-
duces the activity by about a factor of two, while an
ethylene spacer reduces it by a further factor of 10, to
approximately half that of benzamidine itself. A single
methylene spacer will still permit hydroxyl hydrogen
bonding within the S1 catalytic region, while the ethyl
spacer pushes the hydroxyl up out of the S1 pocket,
probably explaining their relative activities. Compared
to benzamidine, the small p-hydroxyl group imparts
about two times more activity than the bulkier p-phos-
phinic acid group that we have previously described.8
Molecular modeling comparisons (not shown), indicate
that p-hydroxybenzamidine is less likely to exhibit minor
bumping collisions than either the phosphinic acidor its
isosteric sulfone analog 1.
Less polar replacements for the benzamidine have also
been exploredby a number of workers. To obtain a
more quantitative analysis of the effectiveness of some
such replacements, we have also evaluatedtwo substitu-
tions––the replacement of benzamidine with 1-amino-
isoquinoline, and N,N-dimethylaniline. Although 1-ami-
noisoquinoline is much less basic than benzamidine, sur-
prisingly 23 is nearly a factor of two more active than
benzamidine (27). We speculate that the added aromatic
bulk fills the S1 pocket somewhat more than benzami-
dine, with the enhanced van der Waals interactions
compensating for any loss in ionic bonding. Disappoint-
ingly, addition of the methyl phosphinic acid at a posi-
tion structurally equivalent to that in AMPA to form
22 eliminates activity in FXa, probably due to bumping
collisions. (Interestingly, 22 has about 50% higher inhib-
itory activity for trypsin than 27, suggesting that slight
structural differences between the enzymes can drasti-
cally affect activity (data not shown). Replacement of
the amidine with dimethylamine and addition of the
methyl phosphinic acidgroup to form the AMPA ana-
log 28 produces barely detectable activity, indicating
that replacement of the cationic amidine with the formal
charge of the dimethyl amine does not retain critical
interaction with the anionic Asp189 side chain
carboxylate.
Figure 1. (a) 2Fo–Fc electron density map of trypsin–AMSO surround-
ing the S1 site. The electron density is contoured at the 1.2 r level. (b)
Superposition of the structure of the trypsin–AMSO complex with the
structure of trypsin–AMPA complex.8 Residues of trypsin–AMSO are
coloredredwhile those of trypsin–AMPA are coloredgreen. The
AMPA–trypsin andAMSO–trypsin structures have been deposited
with the protein data bank as 1TX7Æpdb and 1TX8Æpdb, respectively.
AMSO (Fig. 1a). The final structure has 169 water mole-
cules and1 Ca 2+. The amidino group of the inhibitor
forms a salt bridge with Asp 189 located at the bottom
of S1 pocket as usual. One of the oxygens of the sulfonyl
group forms direct hydrogen bonds with the side chain
˚
One of the more puzzling aspects of the results in Table
1 is the very low activity of sulfone 1. Referencedto
benzamidine, it is nearly 25-fold less active than its
oxygen of Ser 195 (2.45A) andthe oxygen of a single
˚
water (2.79A). To compare the structure differences of
the trypsin–AMSO complex with that of trypsin–
AMPA complex, the two structures were superimposed
in Figure 1b. The side chain of Gln 192 is flipped down
in the trypsin–AMSO structure comparedto the tryp-
sin–AMPA structure, anda water molecular is in a dif-
ferent position, eliminating hydrogen bonds for one of
the two sulfonyl oxygens. Since both oxygens of phos-
phinic acidin the trypsin– AMPA structure form hydro-
gen bonds with neighboring atoms,8 the reduced
hydrogen bonding for AMSO is probably a contributing
factor for the lower inhibitory activity of AMSO, com-
8
structurally similar phosphinic acidanalog. To clarify
this difference, we have determined the crystal structure
of 1 (AMSO) in complex with trypsin, following
protocols similar to that previously usedfor structure
determination of trypsin complexed with p-amidinophen-
ylmethylphosphinic acid( AMPA). The X-ray crystallo-
graphic data collection, processing and refinement
statistics for the structure of bovine trypsin complexed
with AMSO followedour publishedmethods. 8 The elec-
tron density is well ordered and unambiguous for

S. Rumthao et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5165–5170
5169
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paredto AMPA. The reduced hydrogen bonding is
probably a consequence of its lower polarity, consistent
with the hypothesis that polar interactions within or
adjacent to the oxyanion hole region will enhance bind-
ing affinity andinhibitory activity. The near identity of
the binding geometries for AMSO and AMPA thus sug-
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almost certainly the result of its lower polarity andlack
of anionic character.
In summary, we have developed a systematic character-
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in the development of various FXa inhibitors. Compar-
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placement of an anionic group within the oxyanion hole
substantially enhances inhibitory activity. Surprisingly,
we findthat the small hydroxyl group is a more effective
substituent for placement in the oxyanion hole region
than the somewhat bulkier andhighly acidic phosphinic
acidgroup. Our Gaussian calculations suggest that the
electron-withdrawing character of the benzamidine
may shift the hydroxyl pKa to a value low enough for
at least partial ionization, effectively providing a
hydroxylic anion at the oxyanion hole. This is consistent
with literature observations on potency enhancements
resulting from 4-OH addition to benzamidine.6,9,14 The
bulkier 1-amino-isoquinoline also provides an effective
replacement for benzamidine, although further derivati-
zation can be problematic due to steric restrictions.
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Acknowledgements
We thank Randy Powley and Steve Hirschfeld of Hewl-
ett Packardfor assistance with the Gaussian
calculations.
21. Mueller, M. M.; Sperl, S.; Sturzebecher, J.; Bode, W.;
Moroder, L. Biol. Chem. 2002, 383, 1185.
Supplementary data
22. Matter, H.; Defossa, E.; Heinelt, U.; Blohm, P. M.;
Schneider, D.; Muller, A.; Herok, S.; Schreuder, H.;
Liesum, A.; Brachvogel, V.; Lonze, P.; Walser, A.;
Al-Obeidi, F.; Wildgoose, P. J. Med. Chem. 2002, 45,
2749.
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.bmcl.
2004.07.054.
23. Zhang, P.; Zuckett, J. F.; Woolfrey, J.; Tran, K.; Huang,
B.; Wong, P.; Sinha, U.; Park, G.; Reed, A.; Malinowski,
J.; Hollenbach, S.; Scarborough, R. M.; Zhu, B. Y.
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