Structure based design of 4-(3-aminomethylphenyl)piperidinyl-1-amides: Novel, potent, selective, and orally bioavailable inhibitors of βII tryptase

Article abstract of DOI:10.1016/j.bmc.2005.02.014

Tryptase is a serine protease found almost exclusively in mast cells. It has trypsin-like specificity, favoring cleavage of substrates with an arginine (or lysine) at the P1 position, and has optimal catalytic activity at neutral pH. Current evidence suggests tryptase β is the most important form released during mast cell activation in allergic diseases. It is shown to have numerous pro-inflammatory cellular activities in vitro, and in animal models tryptase provokes broncho-constriction and induces a cellular inflammatory infiltrate characteristic of human asthma. Screening of in-house inhibitors of factor Xa (a closely related serine protease) identified β-amidoester benzamidines as potent inhibitors of recombinant human βII tryptase. X-ray structure driven template modification and exchange of the benzamidine to optimize potency and pharmacokinetic properties gave selective, potent and orally bioavailable 4-(3-aminomethyl phenyl)piperidinyl-1-amides.

Full text of DOI:10.1016/j.bmc.2005.02.014

Bioorganic & Medicinal Chemistry 13 (2005) 2859–2872
Structure based design of 4-(3-aminomethylphenyl)piperidinyl-1-
amides: novel, potent, selective, and orally bioavailable inhibitors
of bII tryptase
Julian Levell,* Peter Astles, Paul Eastwood, Jennifer Cairns, Olivier Houille,
Suzanne Aldous, Gregory Merriman, Brian Whiteley, James Pribish, Mark Czekaj,
Guyan Liang, Sebastien Maignan, Jean-Pierre Guilloteau, Alain Dupuy, Jane Davidson,
Trevor Harrison, Andrew Morley, Simon Watson, Garry Fenton, Clive McCarthy,
Joseph Romano, Rose Mathew, Darren Engers, Michael Gardyan, Keith Sides,
Jennifer Kwong, Joseph Tsay, Sam Rebello, Liduo Shen, Jie Wang, Yongyi Luo,
Odessa Giardino, Heng-Keang Lim, Keith Smith and Henry Pauls
Drug Innovation and Approval, Aventis Pharmaceuticals, 1041 Route 202-206, Mail Stop N-103A, Bridgewater, NJ 08807-6800, USA
Received 17 January 2005; revised 8 February 2005; accepted 9 February 2005
Abstract—Tryptase is a serine protease found almost exclusively in mast cells. It has trypsin-like specificity, favoring cleavage of
substrates with an arginine (or lysine) at the P1 position, and has optimal catalytic activity at neutral pH. Current evidence suggests
tryptase b is the most important form released during mast cell activation in allergic diseases. It is shown to have numerous pro-
inflammatory cellular activities in vitro, and in animal models tryptase provokes broncho-constriction and induces a cellular inflam-
matory infiltrate characteristic of human asthma. Screening of in-house inhibitors of factor Xa (a closely related serine protease)
identified b-amidoester benzamidines as potent inhibitors of recombinant human bII tryptase. X-ray structure driven template mod-
ification and exchange of the benzamidine to optimize potency and pharmacokinetic properties gave selective, potent and orally
bioavailable 4-(3-aminomethyl phenyl)piperidinyl-1-amides.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
genous inhibitors of b-tryptase—possibly due to its
tetrameric structure.2 Following allergen cross-linking
of IgE on the IgE receptor, tryptase is exocytosed from
mast cell granules and released extracellularly, where it
has been shown to have a variety of pro-inflammatory
functions.1 Levels of tryptase are highly up-regulated
in allergic disease states [e.g., 100- to 200-fold increase
in human broncho alveolar lavage fluid (BALF) after
antigen challenge (asthmatics vs control)].1 It is shown
to have numerous pro-inflammatory cellular activities
in vitro, and in animal models tryptase provokes bron-
cho-constriction and induces a cellular inflammatory
infiltrate characteristic of human asthma.1 Clinical evi-
dence (APC-366) suggests that inhibition of the enzy-
matic activity of tryptase can attenuate the asthmatic
response.3 In chronic asthma and other long term respi-
ratory disorders, all the known activities of tryptase
could potentially drive the profound changes observed
in the airways such as epithelial desquamation, fibrosis,
Tryptase1 is a serine protease found almost exclusively
in mast cells. It has trypsin-like specificity, favoring
cleavage of substrates with an arginine (or lysine) at
the P1 position, and has optimal catalytic activity at
neutral pH. It is stored primarily as a tetramer, stabi-
lized by heparin. There are eight human tryptase homo-
logues cloned and identified, to date—a, b (I), b (II), b
(III), c (hTMT), d (mMCP-7-like) (two forms), and e
(epithelial cells). Current evidence suggests tryptase b
is the most important form released during mast cell
activation in allergic disease (bI and bII have only one
amino acid difference—N103 in bI is K103 in bII). Un-
like most other proteases there are no known endo-
Keywords: Tryptase; Inhibitors; Asthma; X-ray.
*
Corresponding author. Tel.: +1 908 231 4045; fax: +1 908 231
3576; e-mail: julian.levell@aventis.com
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.02.014

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J. Levell et al. / Bioorg. Med. Chem. 13 (2005) 2859–2872
NH
NH2
NH2
O
O
O
O
HN
HN
O
O
N
N
2
1
rH βΙΙ-tryptase Ki = 497nM
rH βΙΙ-tryptase Ki = 12nM
factor Xa Ki = 190nM
trypsin Ki > 2900 nM
Figure 2. Initial hits from factor Xa library.
Figure 1. Tryptase (TPSB2), factor Xa (F10), and trypsin (PRSS1)
sequence comparison.4 Highlighted residues represents trypsin homo-
logy, red bII-tryptase residues highlight the S1 pocket, magenta bII-
tryptase residues—sequence variation versus aII tryptase. #—bII-
tryptase S1 pocket sequence variation versus aII-tryptase (G vs D);
*—K to N variation versus bI tryptase; ꢀ—Asp-189 and Gly-219.
corresponds to serine-190 in tryptase) makes the S1
pocket smaller and more polar for tryptase, and limits
the ability to parallel our factor Xa optimization ra-
tional with neutral P1 groups.7 The only alternative P1
group that showed significant activity was the 40-fold
less active meta-benzylamine analog 2 (tryptase Ki =
497 nM). In order to form a salt bridge to aspartate
189 at the base of the S1 pocket, the basic moiety needs
to be protonated at physiological pH. The pKa of benz-
amidine is ꢀ11.6, therefore the equilibrium of proton-
ated versus unprotonated form at pH 7.4 is about
10,000:1. Benzylamine has a pKa of ꢀ8.9, so should also
favor the protonated form at pH 7.4, albeit in a lesser
ratio (ꢀ50:1 at equilibrium).
and thickening of underlying tissues.1 These changes are
not addressed by currently available therapeutics (e.g.,
steroids).
Our previous work on factor Xa, another serine prote-
ase with high homology to both trypsin and tryptase
(Fig. 1),4 offered us a more rational approach to screen
for leads versus high throughput screening. The primary
goal of the discovery program was an orally efficacious
drug, so it was decided to focus on monomeric and non-
peptidic molecules so as to avoid some of the obvious
liabilities of these templates, which have been generally
targeted for inhaled delivery.3,5
A rat PK study for compound 2 showed a short half life
and low bioavailability. A dual cannulated rat study
showed that the compound was not found in the hepatic
portal vein (HPV) or jugular vein post p.o. dosing. A
number of explanations are possible, that is, the ester
was poorly absorbed, metabolized in the GI epithelium
or gut lumen (ester to acid transformation), not stable
the GI tract (e.g., low stomach pH) or not stable in
blood (hydrolytic enzymes). Plasma hydrolysis to the
corresponding acid 3 (inactive vs b-tryptase) shows a
conventional plasma conc./time profile. Benzamidine
and benzylamine were retained in further small explo-
ratory libraries, which varied the hydrophobic moiety,
the ester functionality (amides etc.), and the a substitu-
ent (Fig. 3) in order to address the potency and also
establish SAR for eADME and PK parameters. How-
ever, simplification of the template to give it improved
metabolic stability (e.g., removal of the ester) resulted
in a huge drop in activity versus tryptase. Compound
6 was the only analog to retain submicromolar Ki, pre-
sumably due to a hydrogen bond with the catalytic triad,
but this compound was not followed up because of low
metabolic stability.
2. Results and discussion
Approximately 2500 proprietary factor Xa inhibitors
were screened in a medium throughput chromogenic
tryptase assay against recombinant human (rH) b-II
tryptase. A number of hits were identified and filtered
for reasonable early absorption–distribution–metabo-
lism–excretion (eADME) properties (e.g., solubility, S9
stability, and Caco-2 transport) and selectivity profiles
(e.g., trypsin, factor Xa, chymase, thrombin). The b-
amidoester 1 (tryptase Ki = 12 nM), was identified as a
lead for optimization. The synthesis of this template
and subsequent analogs was carried out according to
the previously described protocols (Fig. 2).6
During our previous work on the factor Xa program6
we investigated replacing the benzamidine moiety in
order to improve the pharmacokinetic (PK) characteris-
tics. Therefore, a small number of basic and nonbasic
benzamidine replacements were also investigated for
tryptase (a number of the potential replacements were
also screened amongst the original 2500 compound
set). A key amino acid difference between the factor
Xa and tryptase S1 pockets (alanine-190 in factor Xa
Rational design based on crystallography of the b-
amidoesters in factor Xa,6 suggested that conformation-
ally restricted analogs in the form of achiral piperidine
derivatives could act as surrogates of this template
(Scheme 1). This strategy had been previously employed
for factor Xa inhibitors to good effect.7d,e Loss of po-
tency from removal of the ester side chain could be com-

J. Levell et al. / Bioorg. Med. Chem. 13 (2005) 2859–2872
2861
A convergent synthesis of the aforementioned phenyl-
ethynyl piperidine is highlighted in Scheme 2. Thus,
Suzuki coupling of 3-cyanophenyl boronic acid (11)
(from 3-bromobenzonitrile, 10) with the triflate of N-
Boc-piperidin-4-one (13) gave N-Boc-4-(3-cyanophen-
yl)-1,2,3,6-tetrahydropyridine (14). Conversion of the
nitrile to benzamidine under Pinner conditions also
facilitated removal of the Boc protecting group. Subse-
quent catalytic hydrogenation gave 3-piperidin-4-
ylbenzamidine (15), which was selectively coupled at
the piperidine nitrogen with the requisite carboxylic acid
using TBTU as the coupling agent. These products
could also be accessed by initial palladium coupling of
the Cbz protected piperidine enol triflate with 3-cyano-
phenyl boronic acid, followed by tandem deprotection
and catalytic hydrogenation of the endocyclic double
bond. Subsequent amide coupling with the appropriate
carboxylic acid, and conversion of the nitrile to benz-
amidine under Pinner conditions gave the desired mate-
rials. Two distinct routes were required because upon
scale-up, certain carboxylic acids would couple to both
the piperidine and the benzamidine groups. However,
not all R groups would tolerate Pinner conditions at
the end of the synthetic scheme.
HN
NH2
H2N
H2N
HN
O
O
HO
Me2N
N
N
O
O
HN
O
N
O
HN
O
N
Ph
Ph
Ph
3
5
4
rH βII Tryptase
rH βII Tryptase
rH βII Tryptase
Ki > 30000 nM
Ki > 30000 nM
Ki = 4893 nM
H2N
H2N
HO
HN
O
N
O
N
HN
Ph
Ph
7
6
rH βII Tryptase
rH βII Tryptase
Ki = 2233 nM
Ki = 467 nM
Compound 9a (tryptase Ki = 53 nM; factor Xa
Ki = 380 nM) showed ꢀ10-fold selectivity versus factor
Xa and at least 40-fold versus trypsin. Re-analysis of
the potential for replacing the benzamidine with alterna-
tive basic P1 groups and neutral P1 groups (e.g., target-
ing tyrosine 228) was once again unfruitful. However,
replacing the benzamidine with benzylamine on the
piperidine template resulted in only a 3-fold loss of
activity (16a, tryptase Ki = 180 nM) rather than the 40-
fold loss observed on the b-amidoester template (2 vs
1). Further profiling also revealed that 16a (Fig. 5)
was apparently inactive against a select panel of serine
proteases, including both factor Xa and trypsin (Table
2).
Figure 3. Selected phenylethynyl-pyridine amide substituted b-amido-
ester analogs.
Me
NH
NH
MeO2C
O
O
H2N
H2N
R
N
N
R
8
9
Scheme 1. b-Amidoester to piperidine template modification.
pensated for by the boost in potency from locking the
molecule into its binding conformation.
The benzylamine template was accessed in a similar
manner to the benzamidine, except that the coupling
partners used in the Suzuki coupling were N,N-di-Boc-
3-bromobenzylamine (18) and the pinacolato boronic
ester derivative of the Cbz protected piperidine triflate
(22) (Scheme 3).8 Subsequent tandem reduction–depro-
tection to piperidine 24 and coupling, followed by
The hydrophobic moiety which gave the best selectivity
for tryptase on the b-amidoester template was the meta-
substituted phenylethynyl-pyridine (1), so the corre-
sponding benzamidine piperidine (9) was targeted.
NH
(i)
NC
Br
NC
B(OH)2
NH
NH2
NH2
CN
10
11
(iv)
(iii)
(v)
O
OTf
(vi)
.2HCl
(ii)
N
N
Boc
14
N
H
15
O
N
Boc
12
N
Boc
13
N
9a
Scheme 2. Synthesis of benzamidinepiperidine template and subsequent coupling. Reagents and conditions: (i) a. n-BuLi, B(OCHMe2)3, THF, b. aq
HCl; (ii) LDA, PhNTf2, THF; (iii) (Ph3P)4Pd, Na2CO3, MeCN/H2O; (iv) a. MeOH–HCl, b. MeOH–NH3; (v) 5% Pd/C, H2, MeOH; (vi) Et3N,
TBTU, 3-phenylethynyl-pyridinecarboxylic acid, DMF.

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J. Levell et al. / Bioorg. Med. Chem. 13 (2005) 2859–2872
Boc
N
Boc
Me
NH2
NH
Boc
(ii)
(i)
Br
NH2
NH
Me
N
Boc
B
O
O
Br
Br
18
19
17
N
N
N
N
N
O
R
O
R
O
R
O
R
O
N
Cbz
20
OTf
O
O
(ii)
(iii)
B
N
N
N
Me
N
Cbz
21
NH2
NH2
NH2
NH2
22
Me
Cbz
N
N
Boc
N
N
Boc
N
N
O
O
R
Boc
R
O
R
O
R
Boc
(iv)
(v)
18 + 22
or
19 + 21
H2N
NH2
NH2
N
Cbz
23
N
H
24
N
NH2
N
O
N
Scheme 3. Synthetic route to N,N-di-Boc protected 4-(3-aminomethy-
phenyl)piperidine template. Reagents and conditions: (i) NaH,
(Boc)2NH, THF; (ii) bis-pinacolato-diboron, PdCl2dppfÆDCM com-
plex, dppf, KOAc, dioxane; (iii) LDA, PhNTf2, THF; (iv)
PdCl2dppfÆDCM complex, K2CO3, DMF; (v) H2, Pd/C, EtOH.
R
R
N
N
O
O
R
O
R
Figure 4. Inactive benzamidine, benzylamine, and piperidine analogs.
removal of the two Boc protecting groups gave the de-
sired targets (16). Alternative Suzuki coupling protocols
were also examined, e.g., 18 was converted to the pina-
colato boronic ester (19) and coupled to the Cbz-pro-
tected piperidine triflate (21) to give 23.
idine template (Fig. 5). The fully reduced phen-
ethylpyridine (16b) gave a 12-fold boost in activity (tryp-
tase Ki = 15 nM) versus the acetylene. Subsequent N-
oxide formation on the phenethylpyridine (16c) reversed
this (tryptase Ki = 233 nM). Replacement of the pyr-
idine with a phenyl resulted in a 2-fold boost on the
acetylene (16d, tryptase Ki = 106 nM), but 4-fold loss
on the saturated ethyl analog (16e, tryptase Ki =
53 nM). Partial reduction of 16d to the meta-E-alkene
gave 16f (tryptase Ki = 367). An attempt to replace the
acetylene with a heterocyclic benzoxazole ring (16g)
(Fig. 6) resulted in an 8-fold loss of activity (tryptase
Ki = 400 nM).
Many close analogs and simple derivatives of the benzyl-
amine and piperidine moieties caused loss of activity
(Fig. 4—all compounds were Ki > 10 lM). Analysis of
potential piperidine replacements (e.g., 8-aza-nortropane
and piperazine) also resulted in 10- to 20-fold loss of
activity. Ester substitution on the 3 position of the piper-
idine (cf. closer to the b-amidoester parent) resulted in a
10- to 1000-fold loss in activity, depending on the diaste-
reomer (the trans-piperidine mixture (3R,4S and 3S,4R)
was the most active versus the cis-piperidine mixture
(3R,4R and 3S,4S); the ester imparted metabolic instabil-
ity, so the pure diastereomers were not pursued). Access
to aspartate-189 is restricted by serine-190, and the angle
of approach of the benzylamine is key to the ability of the
molecule to form a good salt bridge to this residue. Pre-
sumably substitution ortho to the benzylamine causes a
steric effect on the conformation of the benzylamine.
Docking experiments also suggest that if the benzyl-
amine were allowed to take up the preferred conforma-
tion, then there would be a steric clash of even small
substituents ortho to the benzylamine (intramolecular
as well as with the protein). Therefore, the template
was fixed at 4-(3-aminomethylphenyl)piperidine, and
the optimization for potency versus tryptase was ad-
dressed by varying the hydrophobic group.
Small diverse libraries of sulfonamide and urea deriva-
tives of the piperidine benzylamine were prepared using
solid phase synthesis techniques to investigate the poten-
tial for replacement of the piperidine amide. Unfortu-
nately, almost all of the urea and sulfonamides were
d. R =
e. R =
N
N
a. R =
b. R =
NH2
*
*
*
*
Ph
Ph
Ph
Ph
N
O
N
O
R
f. R =
c. R =
16
Ph
*
Ph
*
A number of close analogs of the phenylethynyl-pyr-
idine amide were synthesized on the benzylamine piper-
Figure 5. Compound 16a and initial analogs.

J. Levell et al. / Bioorg. Med. Chem. 13 (2005) 2859–2872
2863
inactive in the tryptase assay. The sulfonamides intro-
duce an sp3 sulfur atom bridging the piperidine and
the (usually aromatic) hydrophobic binding group. In
factor Xa this amide would bind to glycine-219 and
modeling into tryptase suggested that this would be an
unfavorable hinge angle in order to point the benzyl-
amine down into the S1 pocket. The urea introduces
an extra chain atom between the piperidine and the
hydrophobic group, which may not be easily tolerated.
An alkyl bridge would probably give the correct angle,
but would lack the hydrogen bonding capability for
the crucial H-bond to glycine-219.
vein (HPV) post p.o. dosing, even though it was below
the limit of quantitation in the jugular vein—that is,
the compound was well absorbed, but efficiently cleared
by first pass metabolism in the liver. The acid was the
main metabolite identified, which was predictably inac-
tive against tryptase.
Analysis of a carefully chosen subset of the benzyla-
mines via a combi-PK cassette dosing regimen, showed
that there was potential to improve the bioavailability
to a good level with 16g, even if the potency against
the target was somewhat weaker for this compound.
Rat PK showed ꢀ90% bioavailability, although the
i.v. half life was still rather short. This result suggested
that the benzylamine was not necessarily a metabolic
soft spot on the molecule. Furthermore, the hypothesis
was supported by literature data on DuPont–Merckꢁs
benzylamine factor Xa inhibitor DPC4239 (they have re-
cently followed up with discussion of DPC602, another
orally bioavailable factor Xa inhibitor with a benzyl-
amine moiety).10
The initial rat PK study on 16b showed low bioavailabi-
lity and short i.v. half life (Fig. 6). However, further ana-
lysis in a dual cannulated rat study showed that the
compound was 88% absorbed into the hepatic portal
NH2
NH2
We were fortunate to be able to obtain crystals of our
protein–inhibitor complex with compound 16b by co-
N
N
N
˚
crystallization to give diffraction resolution at 2.2 A
O
O
O
(Fig. 7—in stereo). Only one molecule of 16b was ob-
served per monomer of the tetramer, and the electron
density for the inhibitor is unambiguous. The benzyl-
amine sits in the S1 specificity pocket and the amine
group forms a salt bridge with aspartic acid-189
(ASP189) and three hydrogen bonds with the glycine-
219 (GLY219) carbonyl oxygen atom, the serine-190
(SER190) carbonyl oxygen atom and a water molecule.
The piperidine is in the chair conformation and the car-
bonyl oxygen atom of the amide forms a hydrogen bond
N
16b
16g
rH βII tryptase Ki = 15nM
rH βII tryptase Ki = 400nM
solubility > 40 µM
Caco-2 = 2.8% (medium)
S9 (rat) = 35% remaining
S9 (human) = 25% remaining
i.v. half-life = 0.32h
solubility = 31 µM
Caco-2 = 0.96% (low)
S9 (rat) = 65% remaining
S9 (human) = 62% remaining
i.v. half-life = 1.13h
Bioavailability = 3.93%
Bioavailability = 90%
Figure 6. Initial eADME and PK.
Figure 7. X-ray structure of 16b in rH bII tryptase (stereo view).

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J. Levell et al. / Bioorg. Med. Chem. 13 (2005) 2859–2872
with the GLY219 nitrogen. The pyridine nitrogen does
not interact with the protein but is seen to form a hydro-
gen bond with a water molecule which bridges to an-
other inhibitor molecule bound in the active site of the
adjacent protein monomer. The distance between two
Boc
N
N
F
F
Boc
(i)
(ii)
F
O
O
16
O
R
N
F
˚
inhibitor pyridine nitrogen atoms is ꢀ5.5 A. The phenyl-
O
R
ethyl plane is nearly 90ꢁ from the pyridine and makes
Van der Waals contacts with proline-600A, aspartic
acid-601A, and glutamic acid-217C at the interface of
two monomers.
25
26
(i) 24, DCM; (ii) TFA, DCM
Scheme 4. Target synthesis via TFP-resin loaded carboxylic acids.
This crystal structure helped to explain much of the pre-
viously observed SAR:
the p-nitrophenyl carbonate Wang resin) and removal
of the Teoc protecting group with TBAF to give 31.
The resin bound template underwent standard amide
coupling to carboxylic acids, and subsequent removal
from the solid phase with TFA gave the target com-
pounds (Table 1).
• Size and nature of the S1 pocket was confirmed,
which rationalized the intolerance of groups other
than benzylamine/benzamidine.
• Loss of activity upon oxidation of 16b to the N-oxide
(16c); and the drop in activity of the phenethylphenyl
versus phenethylpyridine amide was presumably due
to the loss of an intermolecular water mediated
hydrogen bond between two different inhibitor mole-
cules bound into the active sites of adjacent protein
monomers in the tetramer.
• Drop in activity when changing from piperidine to
piperazine or aza-nortropane—the basic piperazine
nitrogen was at an unfavorable location and changed
the angle of access into the S1 pocket. The aza-nor-
tropane bridging atoms could not be easily accommo-
dated (steric clash with glutamine-92).
A representative sample of the compounds synthesized
via these two methodologies is shown in Table 1. As
was predicted from the crystal structure, a wide variety
of hydrophobic groups are tolerated in this shallow sur-
face pocket. This template allows a wide range of acti-
vities to be accessed. The salt bridge of the benzylamine
and the hydrogen bonding network of the amide gives
micromolar affinity, but optimization of the hydrophobe
is required to achieve activity at the nanomolar level.
This gave us the handle of diversity to allow parallel
optimization for potency, toxicity, and pharma-
cokinetics.
It was also clear from this X-ray structure that the sur-
face hydrophobic pocket of the enzyme was quite large
and should be tolerant of a wide variety of hydrophobic
groups. A number of diversity oriented amide libraries
were synthesized to explore this hydrophobic pocket.
3. Conclusions
Knowledge from work on factor Xa provided a basis for
identifying a series of moderately active (albeit unselec-
tive) tryptase inhibitors. Template and P1 binding group
modification of the hit b-amidoester benzamidine gave
achiral benzylamine piperidines with good potency and
considerable selectivity versus many closely related ser-
ine proteases. Poor PK characteristics could be ad-
dressed whilst retaining a reasonable level of activity
against the target. Initial in-house X-ray co-crystal
structures have opened a number of viable ways forward
for optimization of this series of novel potent, selective,
and orally bioavailable inhibitors of bII tryptase. Subse-
quent synthesis of hydrophobe diversity explosion li-
braries has expanded the scope of this novel class of
Library synthesis: libraries were synthesized by two
routes. The first involved treating TFP resin loaded
carboxylic acids11 with 24 in dichloromethane (DCM),
followed by deprotection with TFA (Scheme 4).
The second route involved tethering the template to the
solid phase (Scheme 5): N-Teoc-protected 4-piperidi-
none was converted to the triflate (28), and coupled,
with the same Suzuki type methodology as previously
described, to give the N-Teoc-protected 4-(3-cyanophe-
nyl)-1,2,3,6-tetrahydropyridine (29). Tandem reduction
of the nitrile and the endocyclic double bond was fol-
lowed by loading this material onto Wang resin (via
O
CN
NH2
OTf
N
H
O
HO OH
(vii)
(v)
(iv)
(i)
(iii)
16
(vi)
(viii)
(ii)
N
N
Teoc
H .HCl
N
Teoc
29
N
Teoc
30
N
H
27
28
31
Scheme 5. Target synthesis via resin bound template (i) 2-trimethylsilylethyl-p-nitrophenylcarbonate, Et3N, DMAP, MeCN; (ii) Li(TMS)2, PhNTf2,
THF; (iii) m-cyanophenyl boronic acid, Na2CO3(aq), LiCl, Pd(PPh3)4, MeCN; (iv) H2, Pd/C, concd HCl/EtOH; (v) p-nitrophenylcarbonate Wang
resin, DIEA, DMAP, DMF; (vi) TBAF, THF; (vii) RCO2H, DIC, HOBt, DMF; (viii) TFA, DCM.

J. Levell et al. / Bioorg. Med. Chem. 13 (2005) 2859–2872
2865
Table 1. rH b-II tryptase Ki values for selected active, diversity
oriented, amide hydrophobes
Table 2. Selectivity data (IC50/lM)
Factor Xa Thrombin Trypsin Elastase Chymase
*
*
16h
>30 lM
Me
1
9a
0.35
0.7
>100
>100
>100
>100
>100
>100
>100
5
20
ND
>100
>100
>100
>100
>100
>100
>100
Cl
Cl
>100
>100
>100
>100
>100
>100
16i
48 nM
16a
16b
16d
16e
16i
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
*
*
16j
170 nM
500 nM
N
16k
O
OMe
4.1. Tryptase inhibition activity (in vitro)
Cl
Tryptase inhibition activity is confirmed using recombi-
nant human bII tryptase expressed in yeast cells. The
assay procedure employs a 96-well microplate (Costar
3590) using L-pyroglutamyl-L-prolyl-L-arginine-para-
nitroanilide (S2366: Quadratech) as substrate (essen-
tially as described by McEuen et al.12). Assays were per-
formed at room temperature using 0.5 mM substrate
(2 · Km) and the microplate was read on a microplate
reader (Beckman Biomek Plate reader) at 405 nm wave-
length. Materials and Methods for Tryptase primary
screen (Chromogenic assay): assay buffer—50 mM Tris
(pH 8.2), 100 mM NaCl, 0.05% Tween 20, 50 lg/mL
heparin; substrate-S2366 (stock solutions of 2.5 mM);
purified recombinant bII tryptase enzyme stocks of
310 lg/mL. Protocol (single point determination): Add
60 lL of diluted substrate (final concentration of
500 lM in assay buffer) to each well, add compound
in duplicates, final concentration of 20 lM, volume
20 lL, add enzyme at a final concentration of 50 ng/
mL in a volume of 20 lL. Total volume for each well
is 100 lL. Agitate briefly to mix and incubate at room
temp in the dark for 30 min. Read absorbances at
405 nM. Each plate has the following controls—totals:
60 lL of substrate, 20 lL of buffer (with 0.2% final con-
centration of DMSO), 20 lL of enzyme. Nonspecific:
60 lL of substrate, 40 lL of buffer (with 0.2% DMSO).
Totals: 60 lL of substrate, 20 lL of buffer (no DMSO),
20 lL of enzyme. Nonspecific: 60 lL of substrate, 40 lL
of buffer (no DMSO). Protocol (IC50 determination): The
protocol is essentially the same as above except that the
compound is added in duplicates at the following final
concentrations: 0.01, 0.03, 0.1, 0.3, 1, 3, 10 lM (all dilu-
tions carried out manually). For every assay, whether
single point or IC50 determination, a standard com-
pound is used to derive IC50 for comparison. For a com-
petitive inhibitor, the IC50 value was converted into Ki
according to the equation: Ki = IC50/(1 + [S]/Km).
16l
700 nM
550 nM
70 nM
*
*
N
H
16m
16n
16o
*
*
*
O
S
O
N
150 nM
N
O
16p
1000 nM
Cl
*
*
16q
16r
16s
16t
933 nM
270 nM
70 nM
*
S
*
750 nM
O
*
O
16u
16v
130 nM
330 nM
*
*
S
16w
16x
300 nM
290 nM
S
S
N
*
*
N
16y
733 nM
N
4.2. In vitro permeability and metabolism assays
In vivo absorption through the intestine was modeled
using the caco-2 cell line.13 In vivo metabolism was mod-
eled using the S9 homogenates from human and rat
liver,14 compounds were incubated for 15 min at 37 ꢁC.
inhibitors, yielding opportunities for further structure
based optimization.
4. Experimental
4.3. General experimental discussion
Atomic coordinates for the X-ray structure of rH bII
tryptase with compound 16b will be deposited at the
Protein Data Bank upon publication.
TLC analyses were performed with Merck DC-F254 sil-
ica gel plates, with visualization by UV light, iodine,
alkaline permanganate, and/or molybdophosphoric

2866
J. Levell et al. / Bioorg. Med. Chem. 13 (2005) 2859–2872
acid. Flash chromatography was performed with Merck
silica gel 60 (0.040–0.063 lm). NMR spectra were re-
corded on Gemini-300 spectrometers in CDCl3, unless
tetrakistriphenyl phosphine (0.81 g, 0.7 mmol) at 90 ꢁC
for 1 h. The reaction was cooled, filtered warm, and
the filtrate concentrated to an oil. The oil was extracted
with methylene chloride and the solvent removed under
vacuum. The residue was chromatographed (15% ethyl
acetate/hexane) to give 14 as a white solid (2.7 g,
1
otherwise stated. H signals are reported in ppm from
tetramethylsilane (s, d, t, m, and br for singlet, doublet,
triplet, multiplet, and broad, respectively). Mass spectral
data were collected at 70 eV on a Finnigan MAT 4600,
Mat TSQ-700 or VG Analytical Ltd ZAB2-SE mass
spectrometer and computerized for HRMS. Combus-
tion analyses were performed using a Perkin–Elmer
Model 2400 elemental analyzer and fell within 0.4%
of calculated values. The organic extracts were dried
over magnesium sulfate or sodium sulfate prior to evap-
oration in vacuum on a rotary evaporator. Anhydrous
solvents were purchased from Aldrich Chemical Co. or
Acros organics and used as obtained. Any noncommer-
cially available known starting materials were synthe-
sized as referenced. TFP resin is commercially
available from Polymer Labs. All polymer loaded tetra-
fluorophenoxy (PL-TFP) resin loaded carboxylic acids
were prepared by the method of Salvino et al.11 Any
final products not specifically described herein were syn-
thesized via the TFP resin protocol or the solid phase
template protocol, and were isolated as library members
characterized by LC/MS (overall purity >90%, no single
impurity >5%). Solubility measurements: a 10 mM stock
solution of the compound (in DMSO) was diluted 200-
fold with LC/MS mobile phase to give a 50 lM standard
solution; a separate portion of the 10 mM stock solution
was diluted 200-fold with phosphate buffered saline, and
this sample was mixed vigorously and centrifuged. Com-
parison of the area under the curve of the parent com-
pound from LC/MS analysis of the two solutions gives
a relative estimate of aqueous solubility (up to 50 lM).
1
9.5 mmol). H NMR d 7.63 (s, 1H), 7.55 (m, 2H), 7.44
(t, 1H), 6.10 (br, 1H), 4.09 (m, 2H), 3.63 (t, 2H), 2.50
(br, 2H). MS(EI): 285 (M++1).
4.3.3. 3-(1,2,3,6-Tetrahydropyridin-4-yl)benzamidine di-
hydrochloride (15). Compound 14 (3.4 g, 12.0 mmol) was
dissolved in anhydrous methanol (300 mL), cooled in an
ice bath and saturated with a stream of HCl gas. The
reaction vessel was sealed and the solution was stirred
overnight at ambient temperature. The solvent was re-
moved and the residue was redissolved in methanol
(300 mL). The resulting solution was saturated with
ammonia gas, the vessel was sealed and stirred overnight
at room temperature. The solvent was removed in vacuo
and the residue was dissolved in dilute aqueous HCl and
purified by reverse phase HPLC (acetonitrile/H2O,
2:98). Fractions containing the product were lyophilized
1
to yield 15 as a white solid (1.7 g, 6.2 mmol). H NMR
(DMSO): d 9.48 (s, 2H), 9.38 (s, 2H), 9.15 (s, 2H), 7.90
(s, 1H), 7.80 (d, 1H), 7.73 (d, 1H), 7.58 (t, 1H), 6.39 (s,
1H), 3.76 (br, 2H), 3.3 (br, 2H), 1.9 (m, 4H). MS(EI):
201 (M+).
4.3.4. 3-Piperidin-4-ylbenzamidine dihydrochloride. Com-
pound 15 (1.0 g, 5.0 mmol) was dissolved in methanol
(50 mL) treated with 5% Pd on carbon and stirred over-
night under an atmosphere of hydrogen gas. The reac-
tion mixture was filtered; the filtrate was concentrated
to give 3-piperidin-4-ylbenzamidine dihydrochloride as
a white foam (1.30 g, 4.7 mmol). 1H NMR (DMSO-
d6): d 9.40 (s, 2H), 9.2 (br, 4H), 7.67 (br, 2H), 7.56 (br,
2H), 3.4 (br, 2H), 3.0 (m, 3H), 2.7 (br, 4H). MS(Ion
spray): 204 (M++1).
CAUTION!—many intermediates prepared herein con-
tain the 4-phenyl-1,2,5,6-tetrahydropiperidine substruc-
ture, which is closely related to the known Parkinsonꢁs
inducing compound MPTP.15 Although none of these
particular unsaturated piperidine intermediates have
known toxicity, extra care should be taken (especially
upon scale-up).
4.3.5. 3-(Piperidin-4-yl)-benzonitrile. A solution of benz-
yl 4-oxo-1-piperidinecarboxylate (20) (11.4 g, 49 mmol)
in THF (100 mL) was cannulated dropwise into a cooled
(ꢁ78 ꢁC), vigorously stirring (overhead stirrer) solution
of LDA (36.7 mL of 1.5 M solution in cyclohexanes,
55 mmol) in THF (100 mL), under N2. The reaction
mixture was left at ꢁ78 ꢁC for 30 min before adding a
solution of phenyl trifluorosulfonimide (19.1 g,
53.5 mmol) in THF (50 mL). Then the reaction mixture
was warmed to room temperature and stirred for 3 h
and concentrated directly in vacuo (water bath at
30 ꢁC). The reaction mixture was flash columned on alu-
mina with ramped dilution of neat hexanes to 4%
EtOAc/hexanes. Product fractions were combined and
concentrated to give benzyl 1,2,3,6-tetrahydro-4-(tri-
fluoromethylsulfonyloxy)-pyridine-1-carboxylate (21)
4.3.1. 3-Cyanophenyl boronic acid (11). 3-Bromobenzo-
nitrile (10) (3.6 g, 20 mmol) in anhydrous THF
(100 mL) was treated with triisopropyl borate (13 mL,
56 mmol), followed by 1.6 M butyllithium in hexane
(14 mL, 22 mmol) over 15 min at ꢁ78 ꢁC. The reaction
was stirred at ꢁ78 ꢁC for 45 min then warmed to room
temperature over 60 min. The reaction mixture was
poured into 2 N HCl (40 mL) and the solid precipitate
collected. The filter cake was washed with water and
1
methylene chloride to yield 11 (2.3 g, 15.6 mmol). H
NMR d 8.4 (br, 2H), 8.13 (s, 1H), 8.07 (d, 1H), 7.85
(d, 1H), 7.55 (t, 1H). MS(EI): 147 (M+).
1
4.3.2. 4-(3-Cyanophenyl)-3,6-dihydro-2H-pyridin-1-car-
boxylic acid tert-butyl ester (14). Compound 11 (1.8 g,
12.2 mmol) and 4-(trifluoromethanesulfonyloxy)-3,6-
dihydro-2H-pyridine-1-carboxylic acid tert-butyl ester
(13) (5.0 g, 15.2 mmol) were dissolved in acetonitrile
(60 mL) and 0.4 M aqueous sodium carbonate (60 mL).
The solution was degassed and treated with palladium
(11.34 g) as a white solid (after cooling overnight). H
NMR d 7.30–7.38 (5H, m), 5.75 (H, br s), 5.15 (2H, s),
4.13 (2H, m), 3.70 (2H, t), 2.46 (2H, br s). A portion
of this material (3.65 g, 10 mmol) was dissolved in anhy-
drous 1,2-dimethoxyethane (30 mL) and treated with 11
(1.47 g, 10 mmol), lithium chloride (1.27 g, 30 mmol),
2.0 M aqueous sodium carbonate (10 mL) and palla-

J. Levell et al. / Bioorg. Med. Chem. 13 (2005) 2859–2872
2867
dium tetrakistriphenylphosphine (0.73 g, 0.6 mmol).
The reaction mixture was heated at reflux for 2.5 h,
cooled and concentrated under vacuum. The residue
was partitioned between dichloromethane (2 · 100 mL)
and 2.0 M aqueous sodium carbonate (100 mL) contain-
ing concd ammonium hydroxide (6 mL). The combined
organic extracts were dried (magnesium sulfate), con-
centrated under vacuum and the resultant oil was chro-
matographed on silica gel (ethyl acetate/pentane 2:5) to
yield a yellow oil (2.31 g). This material was dissolved in
ethanol (70 mL), treated with 10% palladium on carbon
(1.0 g) and stirred at ambient temperature under an
atmosphere of hydrogen for 5 h. The reaction mixture
was filtered through a short pad of hyflo and concen-
trated under vacuum to give the title compound as a col-
43.74 mmol, 3 equiv), 1,10-bis-(diphenylphosphino)fer-
rocene (0.242 g, 0.437 mmol, 0.03 equiv), [1,10-bis-(diph-
enylphosphino)ferroceno]dichloropalladium(II)–dichlo-
romethane complex (0.32 g, 0.437 mmol, 0.03 equiv)
and bis-pinacolato diborane (4.07 g, 16.04 mmol,
1.1 equiv) and the reaction mixture heated at 80 ꢁC
overnight under N2. Added water (200 mL), extracted
into EtOAc, dried (MgSO4), and presorbed crude mate-
rial onto silica and flash columned with ramped neat
hexanes to 50% EtOAc/hexanes (5% steps)—product
off at 20–25%. Combined pure product fractions and
concentrated in vacuo to give a white solid (3.58 g,
72%). MS ESI: 385 (+MeCN), 344 (parent), 300
(base).1H NMR d 7.24–7.38 (m, 5H); 6.45 (d, H); 5.13
(s, 2H); 4.02 (q, 2H); 3.51 (t, 2H); 2.24 (br s, 2H); 1.25
(s, 12H).
1
orless oil (1.26 g). H NMR (500 MHz): d 9.78 (br m,
1H), 7.50 (m, 4H), 4.49 (br m, 1H), 3.65 (br m, 1H),
3.00 (m, 2H), 2.82 (m, 1H), 2.21–1.75 (m, 4H). MS(EI):
187 (M++H).
4.3.9. 4-{3-[N,N-Bis-(tert-butoxycarbonyl)aminomethyl]-
phenyl}-piperidine (24). Compound 21 (0.84 g, 2.3 mmol)
was dissolved in anhydrous DMF (20 mL) and the solu-
tion was treated with 19 (1.0 g, 2.3 mmol), potassium
carbonate (0.96 g, 6.7 mmol) and [1,10-bis-(diph-
enylphosphino)ferroceno]dichloropalladium(II)–dichlo-
romethane complex (0.1 g, 0.14 mmol). This mixture
was heated at 80 ꢁC under an atmosphere of nitrogen
for 18 h, then cooled and then concentrated under vac-
uum. The residue was partitioned between ethyl acetate
(two lots of 100 mL) and water (100 mL) containing
concentrated ammonium hydroxide (6 mL). The com-
bined organic extracts were dried over magnesium
sulfate and then concentrated under vacuum. The
resultant oil was subjected to chromatography on silica
gel eluting with a mixture of ethyl acetate and pentane
(1:4, v/v) to yield compound 23 as a yellow oil (0.9 g).
1H NMR (300 MHz): d 7.40–7.14 (m, 9H), 6.00 (d,
H), 5.18 (s, 2H), 4.77 (s, 2H), 4.18–4.07 (m, 2H), 3.70
(t, 2H), 2.52 (br s, 2H), 1.43 (s, 18H). MS(EI): 545
(M++Na). This material was dissolved in ethanol
(20 mL) and the solution was treated with 10% palla-
dium on carbon (20 mg) then stirred at ambient temper-
ature under an atmosphere of hydrogen for 5 h. The
reaction mixture was filtered through a short pad of
hyflo and the filtrate was concentrated under vacuum
4.3.6.
3-Bromo-N,N-bis-(tert-butoxycarbonyl)benzyl-
amine (18). A solution of 3-bromobenzylbromide (17)
(7.5 g, 30 mmol) and di-tert-butyliminodicarboxylate
(6.5 g, 30 mmol) in anhydrous tetrahydrofuran (80 mL)
was treated portionwise with sodium hydride (1.2 g,
60% dispersion in mineral oil). After stirring at ambient
temperature for 7 h the reaction mixture was partitioned
between saturated aqueous ammonium chloride solu-
tion (90 mL) and ethyl acetate (two lots of 250 mL).
The combined organic layers were washed with brine
(75 mL), then dried over magnesium sulfate and concen-
trated under vacuum. The residue was subjected to chro-
matography on silica gel eluting with a mixture of
pentane and dichloromethane (2:1, v/v) to give 18 as a
1
pale yellow oil (9.52 g). H NMR d 7.43 (s, 1H), 7.36
(dt, 1H), 7.23–7.18 (m, 1H), 7.17 (dd, 1H), 4.72 (s,
2H), 1.45 (s, 18H). MS(EI): 386/388 (M++H).
4.3.7. B-{3-[N,N-Bis-(tert-butoxycarbonyl)aminomethyl]-
phenyl}-pinacolato-boron (19). Compound 18 (2.0 g,
5.2 mmol) was dissolved in anhydrous dimethylsulfoxide
(30 mL) and the solution was treated with potassium
acetate (1.52 g, 15.5 mmol), bis(pinacolato)diboron
(1.45 g, 5.7 mmol), and [1,10-bis-(diphenylphosph-
ino)ferroceno]dichloropalladium(II)–dichloromethane
complex (0.13 g, 0.16 mmol). This mixture was stirred at
80 ꢁC under an atmosphere of nitrogen for 5 h, then
cooled and then partitioned between water (100 mL)
and diethyl ether (four lots of 75 mL). The combined
organic layers were washed twice with brine (75 mL), then
dried over magnesium sulfate and then concentrated
under vacuum. The residue was subjected to chromatogra-
phy on silica gel eluting with a mixture of pentane and
dichloromethane (2:1, v/v) to give 19 as a colorless oil
1
to give 24 as colorless oil (0.54 g). H NMR (500 MHz):
d 7.10 (m, 4H), 4.80 (s, 2H), 4.45 (br m, 1H), 3.20 (br
m, 1H), 2.98 (br m, 1H), 2.75 (br m, 1H), 1.90 (m,
1H), 1.75–1.60 (m, 3H), 1.42 (s, 18H). MS(EI): 391
(M++H). Compound 24 could also be synthesized by
utilizing this same coupling methodology, but using 18
and 22 as reactants instead of 19 and 21 (all reactants
and reagents were used in the same molar equivalents).
The yield of compound 23 and 24 in this case were
80% and 85%, respectively, and gave identical analytical
data to those above.
1
(1.08 g). H NMR (500 MHz): d 7.78 (s, 1H), 7.70 (m,
1H), 7.39 (m, 1H), 7.30 (m, 1H), 4.79 (s, 2H), 1.27 (s,
4.3.10. 5-Phenylethyl-pyridine-3-carboxylic acid. A solu-
tion of 5-phenylethynyl-pyridine-3-carboxylic acid (2 g,
8.9 mmol) in tetrahydrofuran (50 mL) was treated with
10% palladium on carbon (200 mg) and stirred at ambi-
ent temperature under an atmosphere of hydrogen for
5 h. The reaction mixture was filtered through a short
pad of hyflo and the filtrate was concentrated under vac-
uum to give 5-phenylethyl-pyridine-3-carboxylic acid as
18H), 1.35 (s, 12H). MS(EI): 434 (M++H).
4.3.8. 4-(4,4,5,5-Tetramethyl-[1,3,2]dioxaborolan-2-yl)-
3,6-dihydro-2H-pyridine-1-carboxylic acid benzyl ester
(22). Compound 21 (5.32 g, 14.58 mmol) was dissolved
in dioxane (90 mL) and added under nitrogen to a
degassed mixture of potassium acetate (4.29 g,

2868
J. Levell et al. / Bioorg. Med. Chem. 13 (2005) 2859–2872
a white solid (2 g). 1H NMR (DMSO-d6, 500 MHz):
8.90 (m, 1H), 8.60 (m, 1H), 8.12 (m, 1H), 7.21 (m,
5H), 3.38 (br s, 1H), 2.95 (m, 4H).
4.3.13. 4-(3-Aminomethyl-phenyl)-piperidine-1-carboxylic
acid 2-trimethylsilanyl-ethyl ester hydrochloride (30).
Compound 29 (10.40 g, 32 mmol) was dissolved in etha-
nol (250 mL), concentrated hydrochloric acid (3 mL,
35 mmol) and 10% palladium on carbon (50% wet,
5.0 g) was added. The mixture was hydrogenated at
50 psi for 4 h then filtered through Celite and concen-
trated. The oily solid obtained was triturated with
ether/pentane and 30 was obtained as a white solid
4.3.11. 4-Oxo-piperidine-1-carboxylic acid (trimethylsil-
yl) ethyl ester. A solution of 4-piperidone monohydrate
hydrochloride (27) (13.55 g, 88 mmol) and 2-trimethyl-
silylethyl-p-nitrophenylcarbonate (25.00 g, 88 mmol) in
acetonitrile (300 mL) was treated with triethylamine
(50 mL, 359 mmol) and dimethylaminopyridine (10.78
g, 88 mmol) and heated to reflux for 2 h. The solution
was cooled and concentrated to an oil. The residue
was dissolved in dichloromethane (300 mL) and washed
twice with 1 N hydrochloric acid and twice with 1 N
sodium hydroxide until all of the yellow color was
removed from the organics. The organics were then
washed with brine, dried over magnesium sulfate, and
concentrated under vacuum to give 4-oxo-piperidine-1-
carboxylic acid (trimethylsilyl) ethyl ester as colorless
oil (19.35 g). 1H NMR d 4.24 (2H, t), 3.78 (4H, t),
2.45 (4H, t), 1.05 (2H, t), 0.05 (9H, s). MS(EI): 284
(M+CH3CN).
1
(7.10 g). H NMR (DMSO-d6): d 8.38 (br s, 2H), 7.20–
7.40 (m, 4H), 4.10 (t, 4H), 3.98 (s, 2H), 2.63–3.00 (m,
3H), 1.75 (m, 2H), 1.50 (m, 2H), 0.94 (t, 2H), 0.02 (s,
9H). LC–MS(ES): 335 (M++H), 93% TIC.
4.3.14. (3-Piperidin-4-yl-benzyl) carbamate Wang resin
(31). p-Nitrophenyl carbonate Wang resin (11.00 g,
15 mmol) and anhydrous DMF (100 mL) were placed
in a peptide synthesis vessel and the resin was allowed
to swell for 15 min. This was then treated with 30
(7.50 g, 21 mmol) in DMF (50 mL), DMAP (0.72,
6 mmol) and diisopropylethylamine. The peptide vessel
was shaken at room temperature for 24 h, then washed
thoroughly with DMF (·5), methanol (·2), DMF (·2),
methanol (·2), dichloromethane (·3), methanol (·2),
dichloromethane (·2), methanol (·3), and dried. The re-
sin was then treated with THF to swell the resin and
then allowed to drain. Then anhydrous tetrahydrofuran
(100 mL) and tetrabutylammonium fluoride (75 mL,
1 M in tetrahydrofuran) were added and the resin sha-
ken for 18 h. The resin was drained and washed with tet-
rahydrofuran (·5), methanol (·3), dichloromethane
(·3), methanol (·3), dichloromethane (·3), methanol
(·3), and dried to give 31 (12.30 g).
4.3.12.
4-(3-Cyanophenyl)-3,6-dihydro-2H-pyridine-1-
carboxylic acid 2-trimethylsilanyl-ethyl ester (29). A solu-
tion of lithium hexamethyldisilazide (60 mmol) in anhy-
drous tetrahydrofuran (150 mL), at ꢁ78 ꢁC, was treated
dropwise with a solution of 4-oxo-piperidine-1-carbox-
ylic acid (trimethylsilyl) ethyl ester (13.30 g, 55 mmol)
in anhydrous tetrahydrofuran (50 mL). The addition
was over 20 min maintaining the internal temperature
at ꢁ65 to ꢁ70 ꢁC. This solution was stirred at ꢁ78 ꢁC
for 45 min and then treated with a solution of N-phenyl-
trifluoromethanesulfonimide (19.65 g, 55 mmol) in
anhydrous tetrahydrofuran (75 mL). The solution was
warmed to 0 ꢁC, then stirred at 0 ꢁC for 3 h and then
concentrated under vacuum. The residue was dissolved
in dichloromethane and washed with water, dried over
magnesium sulfate, and concentrated to give 2-(trimeth-
ylsilyl) ethyl 1,2,3,6-tetrahydro-4-(trifluoromethylsulfon-
4.4. General procedure for solution phase amide coupling
of carboxylic acids to piperidine benzylamine and piper-
idine benzamidine templates, and subsequent deprotection
2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyluronium
tetrafluoroborate (TBTU) (120 mg, 0.38 mmol) was
added to a room temperature solution of carboxylic acid
(0.36 mmol) and diisopropylethylamine (140 mg,
1.08 mmol) dissolved/suspended in DMF (2 mL), under
nitrogen. The reaction mixture was stirred for 10 min
at room temperature before adding a solution of 24
(140 mg, 0.36 mmol) and diisopropylethylamine
(140 mg, 1.08 mmol) dissolved in DMF (2 mL). The
reaction mixture was stirred at room temperature for
16 h, concentrated to dryness in vacuo, and subjected
to dry flash column chromatography on silica with
50:50, dichloromethane–ethyl acetate. The pure frac-
tions were combined and concentrated to give the 1-
acyl-4-(3-(N,N-di-tert-butoxycarbonylaminomethyl)phen-
yl)piperidine, which was generally isolated as a colorless
oil. This intermediate was dissolved in dichloromethane
(20 mL), cooled at 0 ꢁC, and treated with trifluoroacetic
acid (2 mL). The reaction mixture was stirred at room
temperature under nitrogen for 2 h, and concentrated
to dryness in vacuo. The residue was dissolved in 20%
acetonitrile/water (containing 0.1% trifluoroacetic acid)
(9 mL) and purified by preparative reverse-phase HPLC
(C-18, 10 lm reverse-phase column), eluting with 10–
100% acetonitrile/water (containing 0.1% trifluoroacetic
yloxy)-pyridine-1-carboxylate (28) as
a yellow oil
(22.1 g). The material was used crude, as column chro-
matography on silica gel or alumina caused the material
to deteriorate. A portion of 28 (20.65 g, 55 mmol) was
dissolved in acetonitrile (300 mL) and the solution was
treated with 11 (8.90 g, 60 mmol), 2 M sodium carbon-
ate (82.5 mL, 165 mmol) and lithium chloride (6.98 g,
165 mmol). The nonhomogenous mixture was stirred
vigorously and flushed with nitrogen for 5 min, then
added tetrakistriphenylphosphine palladium(0) (3.10 g,
3 mmol). The mixture was heated to reflux (90 ꢁC oil
bath) for 90 min, then cooled and filtered. The red fil-
trate was concentrated and the residue was partitioned
between dichloromethane (three lots 100 mL) and 2 M
sodium carbonate (200 mL). The combined organic ex-
tracts were dried over magnesium sulfate and then con-
centrated under vacuum. The resultant oil was subjected
to chromatography silica gel eluting with a mixture of
ethyl acetate, heptane, and dichloromethane (1:5:1,
1
v/v) to give 29 as a yellow oil (10.46 g). H NMR d
7.40–7.65 (m, 4H), 6.10 (m, 1H), 4.23 (t, 2H), 4.15
(d, 2H), 3.70 (t, 2H), 2.45 (m, 2H), 1.12 (t, 2H), 0.05
(s, 9H).

J. Levell et al. / Bioorg. Med. Chem. 13 (2005) 2859–2872
2869
acid). The product fractions were combined and the ace-
tonitrile removed in vacuo. The aqueous residue was
frozen and lyophilized to give the products as amor-
phous white solids.
and saturated aqueous sodium bicarbonate (45 mL).
The organic layer was dried (magnesium sulfate), con-
centrated under vacuum and the residue chromato-
graphed on silica gel (dichloromethane/methanol 39:1)
1
to give the title compound as a yellow oil (0.53 g). H
This procedure was also used for couplings with the
benzamidine template.
NMR (500 MHz): d 8.80 (s, 1H), 8.63 (s, 1H), 8.00 (s,
1H), 7.50 (m, 9H), 4.90 (br m, 1H), 3.85 (br m, 1H),
3.28 (m, 1H), 2.90 (m, 2H), 1.91 (m, 1H), 1.75 (m,
3H). MS(EI): 392 (M++H).
TBTU was used interchangeably with O-(7-aza-
benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexaflu-
orophosphate (HATU).
4.6.2. 3-[1-(5-Phenylethynyl-pyridine-3-carbonyl)-piper-
idin-4-yl]-benzamidine hydrochloride (9a). A solution of
3-[1-(5-phenylethynyl-pyridine-3-carbonyl)-piperidin-4-
yl]-benzonitrile (0.51 g, 1.3 mmol) in methanol (20 mL)
was cooled to 0 ꢁC and saturated with hydrogen chlo-
ride gas. The reaction vessel was sealed and allowed to
stand at 0 ꢁC for 18 h. The reaction mixture was concen-
trated to dryness under vacuum, redissolved in methanol
(20 mL), and cooled to 0 ꢁC. The solution was saturated
with ammonia gas and the vessel sealed before being al-
lowed to stand at 0 ꢁC for 48 h. The reaction mixture
was concentrated to dryness under vacuum and the res-
idue chromatographed on silica gel (dichloromethane/
methanol 9:1) to give the title compound as a light yel-
low amorphous solid (0.42 g). 1H NMR (DMSO-d6,
500 MHz): d 8.85 (s, 1H), 8.67 (s, 1H), 8.08 (s, 1H),
7.75 (s, 1H), 7.67 (m, 2H), 7.62 (m, 2H), 7.56 (m, 1H),
7.48 (m, 2H), 7.47 (m, 1H), 4.67 (br m, 1H), 3.65 (br
m, 1H), 2.94 (m, 3H), 1.96 (m, 1H), 1.68 (m, 3H).
MS(EI): 409 (M++H).
Deprotection could also be effected by dissolving the
intermediate (0.25 mmol) in ethyl acetate (20 mL), cool-
ing the solution to 0 ꢁC, and saturating with HCl(g)
.
After stirring for 4 h at room temperature the solvent
was removed and the residue purified by preparative re-
verse-phase HPLC.
4.5. General procedure for coupling of TFP-resin bound
carboxylic acids to piperidine benzylamine template, and
subsequent deprotection
TFP resin (125 mg of 1.25 mmol/g resin, with 100%
loading of acid, 156 mmol)11 was swollen in dichloro-
methane (2.5 mL) for 15 min then treated with a solu-
tion of 24 (40 mg, 100 mmol) in dichloromethane
(2.5 mL). The mixture was sealed in the reaction vessel
then left shaking for 8 h. The resin was filtered, and
washed with dichloromethane (2 mL) [TLC (5%
MeOH/EtOAc) showed single product spot (no baseline
amine)]. The filtrate was treated with trifluoroacetic acid
(0.5 mL). After shaking/stirring for 2 h TLC showed no
residual intermediate and the reaction mixture was evap-
orated to give the product directly as the trifluoroacetic
acid salt.
4.6.3. [4-(3-Aminomethyl-phenyl)-piperidin-1-yl]-(5-phen-
ylethynyl-pyridin-3-yl)-methanone di-hydrochloride (16a).
Solution phase HATU coupling of 5-phenylethynyl-pyr-
idine-3-carboxylic acid—intermediate isolated as a yel-
low oil (0.25 g). 1H NMR (DMSO-d6, 500 MHz): d
8.82 (s, 1H), 8.62 (s, 1H), 8.02 (s, 1H), 7.61 (m, 2H),
7.45 (m, 3H), 7.30 (m, 1H), 7.20 (m, 1H), 7.15 (m,
1H), 7.04 (m, 1H), 4.68 (s, 2H), 4.62 (br m, 1H), 3.60
(br m, 1H), 3.23 (br m, 1H), 2.85 (m, 2H), 1.83 (br m,
1H), 1.65 (m, 3H), 1.39 (s, 18H). MS(EI): 596
(M++H). HCl deprotection gave the product as a white
4.6. General procedure for coupling Wang resin bound
piperidine benzylamine template to carboxylic acids, and
subsequent cleavage from the resin
Compound 31 (60 mg, 0.075 mmol) suspended in DMF
(3 mL) and carboxylic acid (0.38 mmol), diisopropylcar-
bodiimide (48 mg, 0.38 mmol), and 1-hydroxybenzo-
triazole (50 mg, 0.38 mmol) were added. The mixture
was shaken at room temperature overnight and washed
with DMF (·5), methanol (·5), dichloromethane then
methanol (repeat five times). The resin was treated with
trifluoroacetic acid and dichloromethane (1:1 v/v, 4 mL)
for 45 min and filtered. The filtrate was concentrated to
give the product directly as the trifluoroacetic acid salt.
1
solid. H NMR (DMSO-d6, 500 MHz): d 8.83 (s, 1H),
8.63 (s, 1H), 8.08 (s, 1H), 7.61 (m, 2H), 7.45 (m, 3H),
7.44 (s, 1H), 7.37 (m, 3H), 4.64 (br m, 1H), 4.00 (m,
2H), 3.62 (br m, 1H), 3.25 (br m, 1H), 2.90 (br m,
2H), 1.88 (br m, 1H), 1.70 (m, 3H). MS(EI): 396
(M++H).
4.6.4. [4-(3-Aminomethyl-phenyl)-piperidin-1-yl]-(5-phen-
ethyl-pyridin-3-yl)-methanone di-hydrochloride (16b).
Solution phase HATU coupling of 5-phenylethyl-pyr-
idine-3-carboxylic acid—intermediate isolated as a white
4.6.1. 3-[1-(5-Phenylethynyl-pyridine-3-carbonyl)-piper-
idin-4-yl]-benzonitrile. A solution of 5-phenylethynyl-
pyridine-3-carboxylic acid (0.75 g, 3.3 mmol) in anhy-
drous DMF (9 mL) was treated with HATU (1.27 g,
3.3 mmol) and diisopropylethylamine (1.1 mL, 6.1
mmol). The reaction mixture was stirred 15 min at ambi-
ent temperature before being treated with a solution of
3-(piperidin-4-yl)-benzonitrile (0.6 g, 3 mmol) in DMF
(6 mL). The reaction mixture was stirred at ambient
temperature for 2 h, concentrated under vacuum and
the residue partitioned between ethyl acetate (200 mL)
1
amorphous solid. H NMR (DMSO-d6, 500 MHz): d
8.50 (s, 1H), 8.41 (s, 1H), 7.61 (m, 1H), 7.30–7.05 (m,
9H), 4.68 (s, 2H), 4.62 (br m, 1H), 3.48 (br m, 1H),
3.35 (s, 4H), 3.18 (br m, 1H), 2.85 (m, 2H), 1.82 (br
m, 1H), 1.65 (br m, 1H), 1.58 (br m, 2H), 1.39 (s,
18H). MS(EI): 600 (M++H). HCl deprotection gave
the product as a white solid. 1H NMR (DMSO-d6,
500 MHz): d 8.90 (m, 2H), 8.42 (s, 1H), 7.58 (m, 1H),
7.38–7.20 (m, 8H), 4.62 (br m, 1H), 4.00 (m, 2H), 3.45
(br m, 1H), 3.20 (br m, 1H), 3.17 (m, 2H), 3.00 (m,

2870
J. Levell et al. / Bioorg. Med. Chem. 13 (2005) 2859–2872
2H), 2.90 (br m, 2H), 1.88 (br m, 1H), 1.70 (br m, 3H).
MS(EI): 400 (M++H).
partitioned between ethyl acetate (three lots of 50 mL)
and water (20 mL). The combined organic layers were
washed with brine (50 mL), then dried over magnesium
sulfate and then concentrated under vacuum. The resi-
due was subjected to chromatography on silica gel elut-
ing with a mixture of cyclohexane and ethyl acetate (3:2,
v/v) to give N,N-bis-(tert-butoxycarbonyl)-3-[1-(3-phen-
ylethynyl-benzoyl)-piperidin-4-yl]-benzylamine as a yel-
4.6.5. [4-(3-Aminomethyl-phenyl)-piperidin-1-yl]-(1-oxy-
5-phenethyl-pyridin-3-yl)-methanone hydrochloride (16c).
A solution of N,N-bis-(tert-butoxycarbonyl)-3-[1-(5-
phenylethyl-pyridine-3-carbonyl)-piperidin-4-yl]-benzyl-
amine (100 mg, 0.17 mmol) in dichloromethane (10 mL)
was treated with meta-chloroperbenzoic acid (80%,
80 mg, 0.37 mmol). After stirring for 18 h at ambient
temperature the reaction mixture was diluted with
dichloromethane (40 mL) and then washed three times
with saturated aqueous sodium bicarbonate solution
(20 mL). The organic phase was dried over magnesium
sulfate and then concentrated under vacuum. The resi-
due was subjected to chromatography on silica gel elut-
ing with a mixture of dichloromethane and methanol
(98:2, v/v) to give N,N-bis-(tert-butoxycarbonyl)-3-[1-
(1-oxy-5-phenylethyl-pyridine-3-carbonyl)-piperidin-4-
yl]-benzylamine as a colorless oil (70 mg). This material
was dissolved in ethyl acetate (10 mL) and the solution
was cooled to 0 ꢁC, then saturated with hydrogen chlo-
ride gas, then stirred at ambient temperature for 4 h and
then concentrated to dryness under vacuum. The residue
was treated with ethyl acetate (10 mL) and the solvent
removed under vacuum. This process was repeated twice
to leave 16c as a white solid (45 mg). 1H NMR (DMSO-
d6, 500 MHz): d 8.39 (s, 1H), 8.30 (s, 1H), 7.45 (s, 1H),
7.40 (s, 1H), 7.38–7.18 (m, 8H), 4.59 (br m, 1H), 4.00 (m,
2H), 3.50 (br m, 1H), 3.20 (br m, 1H), 2.98 (s, 4H), 2.85
(br m, 2H), 1.84 (br m, 1H), 1.68 (br m, 3H). MS(EI):
416 (M++H).
1
low oil (0.23 g). H NMR (500 MHz): d 7.58 (m, 4H),
7.40 (m, 5H), 7.25 (m, 1H), 7.10 (m, 3H), 4.90 (br m,
1H), 4.79 (s, 2H), 3.80 (br m, 1H), 3.15 (br m, 1H),
2.84 (br m, 1H), 2.78 (br m, 1H), 1.95 (m, 1H), 1.80
(m, 3H), 1.42 (s, 18H). MS(EI): 617 (M++Na).
4.6.9. [4-(3-Aminomethyl-phenyl)-piperidin-1-yl]-(3-phen-
ylethynyl-phenyl)-methanone hydrochloride (16d). A solu-
tion of N,N-bis-(tert-butoxycarbonyl)-3-[1-(3-phen-
ylethynyl-benzoyl)-piperidin-4-yl]-benzylamine (100 mg,
0.17 mmol) in methanol (10 mL), cooled to 0 ꢁC, was
saturated with hydrogen chloride gas. The mixture was
stirred at ambient temperature for 4 h then concentrated
to dryness under vacuum. The residue was triturated
with a mixture of dichloromethane and diethyl ether
1
to 16d as a white amorphous solid (46 mg). H NMR
(DMSO-d6, 500 MHz): d 7.61–7.30 (m, 13H), 4.62 (br
m, 1H), 4.00 (s, 2H), 3.62 (br m, 1H), 3.25 (br m, 1H),
2.85 (br m, 2H), 1.88 (br m, 1H), 1.70 (br m, 3H).
MS(EI): 395 (M++H).
4.6.10.
phenethyl-phenyl)-methanone hydrochloride (16e).
[4-(3-Aminomethyl-phenyl)-piperidin-1-yl]-(3-
A
solution of N,N-bis-(tert-butoxycarbonyl)-3-[1-(3-phen-
ylethynyl-benzoyl)-piperidin-4-yl]-benzylamine (110 mg,
0.18 mmol) in ethanol (10 mL) was treated with 10%
palladium on carbon (20 mg) and then stirred at ambi-
ent temperature under an atmosphere of hydrogen for
8 h. The reaction mixture was filtered through a short
pad of hyflo and then concentrated under vacuum
to give a white amorphous solid (0.54 g). HCl deprotec-
tion gave the product as a white amorphous solid
4.6.6. 4-(3-Aminomethyl-phenyl)-piperidin-1-yl]-quinolin-
3-yl-methanone di-hydrochloride (16j). Solution phase
HATU coupling of quinoline-3-carboxylic acid, fol-
lowed by HCl deprotection gave the product as a white
1
solid. H NMR (DMSO-d6, 500 MHz): d 9.10 (s, 1H),
8.78 (s, 1H), 8.20 (m, 2H), 7.98 (m, 1H), 7.80 (m, 1H),
7.50 (s, 1H), 7.35 (m, 3H), 4.72 (br m, 1H), 4.00 (m,
2H), 3.80 (br m, 1H), 3.30 (br m, 1H), 2.90 (br m,
2H), 1.90 (br m, 1H), 1.75 (br m, 3H). MS(EI): 346
(M++H).
1
(25 mg). H NMR (DMSO-d6, 500 MHz): d 7.40–7.15
(m, 13H), 4.62 (br m, 1H), 4.00 (s, 2H), 3.60 (br m,
1H), 3.10 (br m, 1H), 2.80 (br m, 2H), 1.85 (br m,
1H), 1.60 (br m, 3H). MS(EI): 399 (M++H).
4.6.7. N,N-Bis-(tert-butoxycarbonyl)-3-[1-(3-ethynyl-ben-
zoyl)-piperidin-4-yl]-benzylamine. Solution phase HATU
coupling of 3-ethynyl-benzoic acid,16 isolated as a white
4.6.11. [4-(3-Aminomethyl-phenyl)-piperidin-1-yl]-(3-ben-
zoxazol-2-yl-phenyl)-methanone hydrochloride (16g).
Solution phase HATU coupling of 3-(benzooxazo-2-
yl)-benzoic acid,17 followed by HCl deprotection gave
the product as a white solid. 1H NMR (DMSO-d6,
500 MHz): d 8.28 (m, 1H), 8.19 (s, 1H), 7.81 (m, 2H),
7.50 (m, 2H), 7.43 (m, 3H), 7.30 (m, 3H), 4.66 (br m,
1H), 4.00 (m, 2H), 3.70 (br m, 1H), 3.25 (br m, 1H),
2.92 (br m, 1H), 2.82 (br m, 1H), 1.90 (br m, 1H), 1.70
(br m, 3H). MS(EI): 412 (M++H).
1
amorphous solid. H NMR (500 MHz): d 7.76 (m, 2H),
7.40 (m, 2H), 7.25 (m, 2H), 7.10 (m, 4H), 4.95 (br m,
1H), 4.79 (s, 2H), 3.80 (br m, 1H), 3.10 (br m, 1H),
2.84 (br m, 1H), 2.78 (br m, 1H), 1.90 (m, 1H), 1.75
(m, 3H), 1.42 (s, 18H). MS(EI): 541 (M++Na).
4.6.8. N,N-Bis-(tert-butoxycarbonyl)-3-[1-(3-phenylethy-
nyl-benzoyl)-piperidin-4-yl]-benzylamine. A mixture of
N,N-bis-(tert-butoxycarbonyl)-3-[1-(3-ethynyl-benzoyl)-
piperidin-4-yl]-benzylamine (0.24 g, 0.46 mmol), iodo-
benzene (95 mg, 0.46 mmol), dichlorobis(triphenylphos-
phine)palladium(II) (35 mg, 0.05 mmol), copper(I)
iodide (26 mg, 0.14 mmol), triethylamine (0.57 mL,
4.1 mmol) and anhydrous DMF (8 mL) was stirred at
ambient temperature under nitrogen for 18 h. The sol-
vent was removed under vacuum and the residue was
4.6.12. [4-(3-Aminomethyl-phenyl)-piperidin-1-yl]-[3-((E)-
styryl)-phenyl]-methanone hydrochloride (16f). Solution
phase HATU coupling of 3-(2-E-phenylethenyl)-benzoic
acid,18 followed by HCl deprotection gave the product
as a white solid. 1H NMR (DMSO-d6, 500 MHz): d
7.71 (m, 1H), 7.63 (m, 3H), 7.50 (m, 2H), 7.40–7.25
(m, 9H), 4.63 (br m, 1H), 4.00 (s, 2H), 3.71 (br m,

J. Levell et al. / Bioorg. Med. Chem. 13 (2005) 2859–2872
2871
1H), 3.10 (br m, 1H), 2.84 (br m, 2H), 1.88 (br m, 1H),
1.70 (br m, 3H). MS(EI): 397 (M++H).
(m, 2H), 1.86–1.65 (m, 2H). MS(Ion spray): 351
(M++1).
4.6.13.
3-[4-(3-Aminomethyl-phenyl)-piperidine-1-car-
References and notes
bonyl]-1-benzopyran-2-one trifluoroacetate (16t). Solu-
tion phase HATU coupling of coumarin-3-carboxylic
acid, followed by TFA deprotection gave the product
as a yellow oil. LC–MS: tR = 3.15 min (>86% by
ELSD). MS(ES+): 363 (MH+).
1. (a) Cairns, J. A. Pulm. Pharmacol. Ther. 2005, 18(1), 55–
66, and references cited therein; (b) Norris, A. Expert
Opin. Invest. Drugs 2004, 13(7), 739–741; (c) Martin, T. J.
In Highlights in Bioorganic Chemistry; Schmuck, C.,
Wennemers, H., Eds.; Wiley–VCH: Weinheim, Germany,
2004; pp 227–239.
2. Pereira, P. J. B.; Bergner, A.; Macedo-Ribeiro, S.; Huber,
R.; Matschiner, G.; Fritz, H.; Sommerhoff, C. P.; Bode,
W. Nature 1998, 392(6673), 306–311.
4.6.14. [4-(3-Aminomethyl-phenyl)-piperidin-1-yl]-naph-
thalen-2-yl-methanone trifluoroacetate (16r). Solution
phase HATU coupling of 2-naphthoic acid, followed
by TFA deprotection gave the product as a yellow oil.
LC–MS: tR = 3.66 min (100% by ELSD). MS(ES+):
345 (MH+).
3. (a) Rice, K. D.; Tanaka, R. D.; Katz, B. A.; Numerof, R.
P.; Moore, W. R. Curr. Pharm. Des. 1998, 4(5), 381–396;
(b) Newhouse, B. J. IDrugs 2002, 5(7), 682–688.
4. Yousef, G. M.; Elliott, M. B.; Kopolovic, A. D.; Serry, E.;
Diamandis, E. P. Biochim. Biophys. Acta 2004, 1698(1),
77–86.
5. Burgess, L. E.; Newhouse, B. J.; Ibrahim, P.; Rizzi, J.;
Kashem, M. A.; Hartman, A.; Brandhuber, B. J.; Wright,
C. D.; Thomson, D. S.; Vigers, G. P. A.; Koch, K. Proc.
Nat. Acad. Sci. U.S.A. 1999, 96(15), 8348–8352.
4.6.15. [4-(3-Aminomethyl-phenyl)-piperidin-1-yl]-phenyl-
methanone trifluoroacetate (16q). Solution phase HATU
coupling of benzoic acid, followed by TFA deprotection
gave the product as a yellow oil. LC–MS: tR = 3.39 min
(>95% by ELSD). MS(ES+): 295 (MH+).
6. (a) Klein, S. I.; Czekaj, M.; Gardner, C. J.; Guertin, K. R.;
Cheney, D. L.; Spada, A. P.; Bolton, S. A.; Brown, K.;
Colussi, D.; Heran, C. L.; Morgan, S. R.; Leadley, R. J.;
Dunwiddie, C. T.; Perrone, M. H.; Chu, V. J. Med. Chem.
1998, 41(4), 437–450; (b) Guertin, K. R.; Gardner, C. J.;
Klein, S. I.; Zulli, A. L.; Czekaj, M.; Gong, Y.; Spada, A.
P.; Cheney, D. L.; Maignan, S.; Guilloteau, J.-P.; Brown,
K. D.; Colussi, D. J.; Chu, V.; Heran, C. L.; Morgan, S.
R.; Bentley, R. G.; Dunwiddie, C. T.; Leadley, R. J.;
Pauls, H. W. Bioorg. Med. Chem. Lett. 2002, 12(12), 1671–
1674; (c) Maignan, S.; Guilloteau, J.-P.; Pouzieux, S.;
Choi-Sledeski, Y.-M.; Becker, M. R.; Klein, S. I.; Ewing,
W. R.; Pauls, H. W.; Spada, A. P.; Mikol, V. J. Med.
Chem. 2000, 43(17), 3226–3232.
7. (a) Choi-Sledeski, Y.-M.; Kearney, R.; Poli, G.; Pauls, H.;
Gardner, C.; Gong, Y.; Becker, M.; Davis, R.; Spada, A.;
Liang, G.; Chu, V.; Brown, K.; Collussi, D.; Leadley, R.,
Jr.; Rebello, S.; Moxey, P.; Morgan, S.; Bentley, R.;
Kasiewski, C.; Maignan, S.; Guilloteau, J.-P.; Mikol, V. J.
Med. Chem. 2003, 46(5), 681–684; (b) Maignan, S.;
Guilloteau, J.-P.; Choi-Sledeski, Y.-M.; Becker, M. R.;
Ewing, W. R.; Pauls, H. W.; Spada, A. P.; Mikol, V. J.
Med. Chem. 2003, 46(5), 685–690; (c) Choi-Sledeski, Y.
M.; Becker, M. R.; Green, D. M.; Davis, R.; Ewing, W.
R.; Mason, H. J.; Ly, C.; Spada, A.; Liang, G.; Cheney,
D.; Barton, J.; Chu, V.; Brown, K.; Colussi, D.; Bentley,
R.; Leadley, R.; Dunwiddie, C.; Pauls, H. W. Bioorg.
Med. Chem. Lett. 1999, 9, 2539; (d) Gong, Y.; Pauls, H.
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Liang, G.; Maignan, S.; Guilloteau, J.-P.; Chu, V.;
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P. C.; Eastwood, P. R. 1-Aroyl Piperidinyl Benzamidines
as Factor Xa Inhibitors. PCT Int. Appl. 0181310, 11/01/
01.
4.6.16. [4-(3-Aminomethylphenyl)piperidin-1-yl]-(3,4-di-
chlorophenyl)methanone trifluoroacetate (16i). TFP resin
coupling and TFA deprotection gave the product as
an amorphous white solid. 1H NMR (DMSO-d6): d
8.15 (br s, 3H), 7.72–7.69 (m, 2H), 7.40 (dd, 1H),
7.36–7.25 (m, 4H), 4.66–4.51 (m, 1H), 4.05–3.96 (m,
2H), 3.69–3.48 (m, 1H), 3.30–3.11 (m, 1H), 2.91–2.73
(m, 2H), 1.90–1.54 (m, 4H). MS(Ion spray): 363 and
365 (M++1).
4.6.17. 1-[4-(3-Aminomethyl-phenyl)-piperidin-1-yl]-1-(5-
chloro-1H-indol-2-yl)-methanone trifluoroacetate (16l).
Solution phase TBTU coupling of 5-chloro-1H-indole-
2-carboxylic acid, followed by TFA deprotection gave
the product as an amorphous white solid. 1H NMR
(DMSO-d6): d 11.78 (s, H); 8.14 (br s, 3H, NH3+),
7.63 (s, H), 7.43–7.22 (m, 5H), 7.16 (dd, H), 6.76 (s,
H), 4.55 (br d, 2H), 4.00 (q, 2H), 3.14 (br s, 2H),
2.94–2.80 (m, H), 1.90–1.79 (m, 2H), 1.73–1.54 (m,
2H). MS(Ion spray): 368 and 370 (M++1).
4.6.18. 6-{1-[4-(3-Aminomethyl-phenyl)-piperidin-1-yl]-
methanoyl}-2,3-dihydro-thiazolo[3,2-a]pyrimidin-5-one
(16o). Solution phase TBTU coupling of using 5-
oxo-2,3-dihydro-5H-thiazolo[3,2-a]pyrimidine-6-carbox-
ylic acid, followed by TFA deprotection gave the prod-
uct as an amorphous white solid. 1H NMR [CD3OD]: d
7.97 (s, H), 7.44–7.29 (m, 4H), 4.76 (br d, H), 4.59–4.51
(m, 2H), 4.07 (s, 2H), 3.83 (br d, H), 3.69–3.57 (m, 2H),
3.41–3.21 (m, H), 3.02–2.85 (m, 2H), 2.05–1.67 (m, 4H).
MS(Ion spray): 371 (M++1).
4.6.19.
1-[4-(3-Aminomethyl-phenyl)-piperidin-1-yl]-1-
8. Eastwood, P. R. Tetrahedron Lett. 2000, 41(19), 3705–
3708.
benzo[b]thiophen-2-yl-methanone-trifluoroacetate (16s).
Solution phase TBTU coupling of benzo(b)thiophene-
2-carboxylic acid, followed by TFA deprotection gave
the product as an amorphous white solid. 1H NMR
(CD3OD): d 7.98–7.81 (m, 2H), 7.62 (s, 1H), 7.45–7.32
(m, 5H), 7.31–7.22 (m, 1H), 4.80–4.40 (m, 2H), 4.10 (s,
2H), 3.28–3.05 (m, 1H), 3.02–2.90 (m, 2H), 2.02–1.88
9. Pinto, D. J. P.; Orwat, M. J.; Wang, S.; Fevig, J. M.;
Quan, M. L.; Amparo, E.; Cacciola, J.; Rossi, K. A.;
Alexander, R. S.; Smallwood, A. M.; Luettgen, J. M.;
Liang, L.; Aungst, B. J.; Wright, M. R.; Knabb, R. M.;
Wong, P. C.; Wexler, R. R.; Lam, P. Y. S. J. Med. Chem.
2001, 44(4), 566–578.

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