Design, synthesis and biological activity of a targeted library of potential tryptase inhibitors

Article abstract of DOI:10.1039/b403629h

We have designed, synthesized, and tested two small collections of potential tryptase inhibitors. The first library consists of diversely N-substituted 3-aminopiperidin-2-ones 6, and the second (compounds 7) was prepared by dimerising compounds 6 through the 3-amino function using diverse carbon chains. We have established efficient routes for obtaining 6 both in solution and on solid supports. We have also compared the dimerisation on-resin and in solution. Four of the compounds showed a high degree of tryptase inhibition at 1 μM, but none surpassed the tryptase inhibition activity of BABIM.

Full text of DOI:10.1039/b403629h

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Design, synthesis and biological activity of a targeted library of
potential tryptase inhibitors
Mónica García,a,b Xavier del Río,a,b Sandra Silvestre,b Mario Rubiralta,a,b Estrella Lozoya,c
Victor Segarra,c Dolors Fernández,c Montserrat Miralpeix,c Mònica Aparicic and
Anna Diez*a,b
a Laboratori de Química Orgànica, Facultat de Farmàcia, Universitat de Barcelona,
Av. Joan XXIII s/n, 08028 Barcelona, Spain
b Institut de Recerca Biomèdica de Barcelona, Parc Científic de Barcelona, c/ Josep Samitier,
1-5, 08028 Barcelona, Spain. E-mail: adiez@pcb.ub.es; Fax: ϩ34-934037104;
Tel: ϩ34-934037105
c Drug Discovery Division, Research Centre, Almirall Prodesfarma, Cardener, 68-74,
08024 Barcelona, Spain
Received 10th March 2004, Accepted 15th April 2004
First published as an Advance Article on the web 12th May 2004
We have designed, synthesized, and tested two small collections of potential tryptase inhibitors. The first library
consists of diversely N-substituted 3-aminopiperidin-2-ones 6, and the second (compounds 7) was prepared by
dimerising compounds 6 through the 3-amino function using diverse carbon chains. We have established efficient
routes for obtaining 6 both in solution and on solid supports. We have also compared the dimerisation on-resin
and in solution. Four of the compounds showed a high degree of tryptase inhibition at 1 µM, but none surpassed
the tryptase inhibition activity of BABIM.
Introduction
Tryptases are the major serine proteases of human mastocytes,
and are known to be involved in numerous inflammatory
processes. Tryptases are tetrameric proteins, whose monomers
define a flat rectangle and orient their active sites towards the
central pore.1 Owing to their structure, the substrate specificity
of tryptases is governed by the quaternary architecture rather
than by the structure of the active sites. This peculiar geometry,
which impedes access of proteins to the active centers, also
seems to be responsible for the resistance of tryptases to protein
inhibitors. So far, the only site-directed proteinaceous inhibitor
known for human tryptase is LDTI (leech-derived tryptase
inhibitor, a 46 residue peptide).2
Over the last decade, low molecular weight synthetic tryptase
inhibitors have been largely studied to control inflammatory
diseases such as allergies and asthma.3 The first generation
of inhibitors (Fig. 1) consisted of bis-benzamidines such as
BABIM (1),4 peptidilphosphonates (2),5 benzisothiazolones
(3),6 or APC 366 (4).7 All of these bind in different ways to the
Fig. 2
active sites of tryptase. The second generation of tryptase
inhibitors was based on the assumption first proposed by Axys
structure of these compounds consists of a central linker that
Pharm Corp., that larger compounds could interact with active
sites of two adjacent monomers which are spatially close
(Fig. 2).8
binds two identical or very similar moieties. These moieties
contain a terminal base function, attached to an aromatic
ring (“R-base”), which is also bound to the linker through a
scaffold.
APC 2059, which belongs to this generation of tryptase
inhibitors (5b, Fig. 2), is in phase II clinical trials.9 The general
In connection with our previous work on the application of
3-aminolactams as pseudopeptides,10 we planned to use this
heterocycle as a surrogate of the piperazine moiety in
compounds 5. Thus, the monomeric and dimeric series 6 and 7
were designed as potential tryptase inhibitors (Fig. 3).
Compounds 6 consist of a 3-aminovalerolactam scaffold
bearing a terminal basic moiety, which not only has all the
requisites for interaction as a monomer, but could also be used
to produce the dimerised species 7.
We selected from among already reported compounds the
linkers and the “R-base” moieties which had given the best
biological activity,11 and attached them to the piperidone
scaffold. Theoretically, the combination of these two factors
Fig. 1
T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 4
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 1 6 3 3 – 1 6 4 2
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Fig. 5 Flexible superposition of 5a with compound 7f (n = 7).
of these items would provide a small library of 32 dimers of
type 7, which should provide data on the relevance of the four
parameters considered (the base, the aromatic ring, the
carbonyl group, and the length of the linker).
We planned to carry out the synthesis of compounds 6 both
in solution (SS) and on solid phase (SPS). Compounds 7 were
prepared in solution, but we also attempted an on-resin
dimerisation of compounds of type 6 to produce a resin-bound
library of dimers 7.
Results and discussion
Synthesis of compounds 6a–g in solution
Compounds 6a–c were prepared in three steps from com-
pound 8 (Scheme 1). Compound 811 was coupled to the chosen
“R–NH2” moieties using PyBOP and NMM as the activating
agents. In this manner we obtained compounds 9a–c in good
yields, which were Boc-deprotected by treatment with TFA.
Hydrogenation of compounds 10a–c in the presence of 10%
Pd–C led to the target compounds 6a–c, which were fully
characterized.
Fig. 3
should give us a small but efficient library of compounds. For
the terminal base, we planned to test amines and guanidines.
Concerning the R moiety, we decided to use a phenyl ring, as
well as a non-aromatic valerolactam and an acyclic carbon
chain. Another variable to consider was the need for the polar
carbonyl group in R. Thus, we selected the eight radicals shown
in Fig. 3 to prepare monomers type 6.
To evaluate the feasibility of this scaffold replacement, we
first performed a molecular overlay of compound 5a and some
of the designed compounds. For this purpose, and because 5a is
a very flexible molecule, we docked it to the tryptase active site
(PDB accession code: 1A0L) in order to obtain a possible
bioactive conformation (Fig. 4).
Scheme 1
Compounds 6d,e were prepared from Boc-cyclo-ornithine12
in three steps. Thus, selective alkylation of Boc-cyclo-ornithine
yielded the corresponding amines 11a,b. Treatment of com-
pounds 11a,b with TFA, and hydrogenation of the resulting
amines 12a,b, yielded the target lactams 6d,e in good yields
(Scheme 2).
The guanidino derivatives 6f and 6g were prepared from the
nitro precursors 9c and 11a (Scheme 3). Reduction of the nitro
group followed by reaction of the resulting anilines 13 and 14
with N,NЈ-bis(benzyloxycarbonyl)guanidinium triflate in the
presence of NMM led to the fully protected guanidines 15
(71%) and 16 (75%), which presented NMR signals at δ ∼163
(13C), and at δ ∼10–11 (1H, two signals), characteristic of the
guanidino group. Removal of the Cbz protecting groups and
TFA cleavage of the Boc yielded the target lactams 6f and 6g
(86 and 97%, respectively).
Fig. 4 Docking (InsightII package) of compound 5a in the tryptase
active site (PDB accession code: 1A0L). The two guanidino groups of
5a are accommodated very well in two of the tryptase subunits.
The conformation of 5a which provided better results in the
docking study was used as a template for comparison to
compounds 7. As an example, Fig. 5 shows the superposition of
this conformation of 5a with compound 7f (n = 7), which shows
a good overlay of the different functional groups.
In view of these results, we chose to use diacids ranging from
6 to 9 carbons long,8 to create amide bonds on the exocyclic
C3-amino group of the piperidone scaffold. The combination
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 1 6 3 3 – 1 6 4 2
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N1–C2 bond. For this purpose, enantiomerically pure (S)-5-
hydroxynorvaline (17)15 was anchored to a previously function-
alised benzhydrylamine resin 1816 by means of a carbamate
function to give compound 19. The extent of this coupling was
70%, calculated from amino acid analysis using Ala as the
internal reference. Subsequent mesylation and nucleophillic
substitution with 4-aminobenzylamine yielded compound
20. The complete formation of the product was verified by a
Kaiser test, and the identity of compound 20 was confirmed by
1
cleavage from the resin and H-NMR characterization of the
resulting benzylamino amino acid.
Compound 20 was then treated with HOBt/DIPCDI to
promote the intramolecular amino acid coupling, and
subsequent TFA cleavage released the desired 1-substituted
3-aminolactam 6d in 68% crude yield, based on the loading
of resin 18. The purity of the crude product was 75% as
determined by HPLC analysis.
Scheme 2
To anchor compounds 6 through the terminal function we
used resins 18 and 22,17 which would release the suitable amino
and guanidino functions (Scheme 5). The benzyl alcohol 2318
was anchored to the solid support 18 through a carbamate
function to give compound 24 (Scheme 5). To quantify the
loading of the resin-bound amino alcohol, we acetylated
and cleaved (TFA) a sample of compound 24. The resulting
trifluoroacetate was analyzed by quantitative mass spectro-
metry and by HPLC.19 Surprisingly, the conditions used in the
conversion of 18 to 19 did not give a satisfactory yield when
applied to the anchoring of compound 23. After optimization,
the best results were obtained when carbonate activation
was extended to 24 h and its reaction with compound 23 was
performed without additional base for 24 h.
Once the solid support was satisfactorily loaded (>80%),
compound 24 was mesylated and the on-resin nucleophilic
substitution with Alloc-cyclo-ornithine20 was performed. Com-
pound 2821 was obtained in 70–80% yield using the lithium
amidate of p-methoxyacetanilide22 as the base. Lower yields
were obtained when the bromide was used instead of the
mesylate. Compound 6e was obtained in 57% yield from 24,
after removal of the Alloc protecting group and final cleavage
with TFA/CH2Cl2. HPLC analysis indicated that this material
was 75% pure.
Scheme 3
SPS synthesis of compounds 6e, 6d and 6h
Regarding the SPS synthesis,13,14 we planned to anchor the
monomers of type 6 both through the exocyclic C3-amino
group and through the terminal base function (amino or
guanidino). The first series would allow easy diversification
on the lactam nitrogen atom, whereas the second would do so
on the C3-amino substituent.
To establish the first SPS synthetic route, we aimed at
obtaining compound 6d (Scheme 4). Our approach involved
attachment of (S)-5-hydroxynorvaline to a solid support via its
N-terminus and building the lactam ring by formation of the
In parallel, benzyl alcohol 2318 was anchored on resin 22,17
using 2-chloro-1-methylpyridinium iodide and DIEA (Scheme
5).23 Subsequent mesylation of the alcohol, reaction with
Alloc-cyclo-ornithine, Alloc deprotection, and cleavage using
triisopropylsilane (TIS), led to the expected compound 6h in
85% crude yield from 25. Compound 6h was 95% pure
according to HPLC analysis, and was identified from its
spectral data.
Synthesis of a small library of dimers 7
Once we had established the SPS methods to obtain the
“monomer” moieties 6, we tried the on-resin conversion of
compound 30 to the resin-bound dimer type 7 (32, Scheme 6).
Despite the fact that working on-resin theoretically imitates
infinite dilution conditions,24 the existence of two precedents
for this sort of cross-linking,25 encouraged us to test the assay.
Compound 30 was treated with 0.5 equivalents of azelaic acid
in the presence of DIPCDI and HOBt as the activating agents.
The resulting resin 32 showed a very encouraging negative
ninhydrin test. Cleavage using 95% TFA in CH2Cl2 for 20 min
yielded compound 7e, which was identified from its spectral
data. However, a dramatic loss of weight was observed, and 7e
was obtained only in 15% yield. Longer reaction times for the
cleavage did not improve the result. In order to find a possible
explanation for this low yield, we evaluated how much lactam
resin 32 actually contained. For this, we performed an amino
acid analysis of resin 32 using pure lactam 6e (previously
obtained in solution) as the reference.
Scheme 4
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 1 6 3 3 – 1 6 4 2
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Scheme 5
by treatment of compound 10c with adipic acid, in the presence
of PyBOP and NMM, which led to the corresponding dimer in
75% yield. Then compounds 10a–c,12a,b, and Boc-deprotected
15 and 16 were treated in parallel under the same conditions,
with the four chosen diacids (7 × 4 = 28 reactions). Final
hydrogenation to deprotect the terminal functions, using the
catalysts already established in the monomeric series, yielded
the target library (Scheme 7). There was one exception: cyclo-
arginine-derived compound 10b yielded the acyclic arginine
compounds 7a in the four couplings. The results are sum-
marised in Table 1. Compounds 7 showed the characteristic
NMR signals observed in their corresponding monomers 6,
and additional aliphatic signals corresponding to the central
linkers.28 Structural assignment of the dimers was confirmed by
ES mass spectrometry.
Scheme 6
Resin 32 showed only traces of the lactam, which indicated
that the cleavage had occurred in the previous step. Modifi-
cation of the coupling conditions, by using HBTU or HATU
and DIPEA,26 or by adding a previously prepared mixture of
azelaic acid and the activating agents, did not improve the
results either. We also tested the on-resin dimerisation using a
Wang resin. For this, we anchored compound 23 to the CDI-
activated Wang resin17 and followed the synthetic sequence
described in Scheme 527 to obtain the analogue of compound
30, attached to the Wang resin (90% total yield). Unfortunately,
dimerisation on this resin also produced the target dimer in
poor yields.
In view of these results we decided to test the sensitivity of
our compounds to the coupling reagents. Compound 30 was
therefore coupled to Fmoc-Ala-OH in HATU/DIEA and
HOBt/DIPCDI conditions. The yield of this reaction was
calculated by the method of dibenzofulvene–piperidine adduct
quantification, to be 70%, which confirmed that compound
30 was stable in the coupling conditions and that the on-resin
dimerisation was not a suitable option for preparative purposes.
In contrast, the library of compounds 7 was satisfactorily
prepared in solution. The experimental conditions were tested
Scheme 7
Tryptase inhibition activity tests
A total of 54 compounds (24 dimers of type 7 (a, and c–g), 6
“intermediate” dimers shown in Scheme 7, and all the lactams
we have prepared) were evaluated as tryptase inhibitors using
a colorimetric technique4 (see Experimental). In the first test,
only 4 compounds 7e (n = 5, 6, 7) and 7f (n = 7) showed a
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 1 6 3 3 – 1 6 4 2
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Table 1 Synthesis of the library of dimers. The chemical yields given correspond to the 2 steps
Substrate
7a (n = 4) 44%
7a (n= 4) 40%
7a (n = 5) 40%
7a (n = 5) 20%
7a (n = 6) 38%
7a (n = 6) 33%
7a (n = 7) 34%
7a (n = 7) 22%
7c (n = 4) 75%
7c (n = 5) 15%
7c (n = 6) 37%
7c (n = 7) 26%
7d (n = 4) 66%
7e (n = 4) 57%
7d (n = 5) 70%
7e (n = 5) 65%
7d (n = 6) 63%
7e (n = 6) 55%
7d (n = 7) 66%
7e (n = 7) 37%
7f (n = 4) <5%
7g (n = 4) <5%
7f (n = 5) <5%
7g (n = 5) <5%
7f (n = 6) <5%
7g (n = 6) <5%
7f (n = 7) 34%
7g (n = 7) 37%
Table 2 IC50 values (µM) of the four active compounds
(δ) relative to Me4Si. Mass spectra were determined on
a Hewlett Packard 5988A mass spectrometer by electronic
impact (EIMS), or on a Micromass Vplatform 2 system by
electrospay (ESMS). TLC was performed on SiO2 (silica gel
60 F254, Macherey-Nagel) and developed with the eluent
described for column chromatography. The spots were located
with ninhydrin, potassium hexachloroplatinate, anisaldehyde,
or KMnO4. Organic solutions were dried over anhydrous
Na2SO4 (Panreac, ref. 141716.0416), prior to evaporation of the
solvent. Unless otherwise indicated, flash chromatography was
done over SiO2 (60 A C.C 35–70 µm, SDS). HPLC analyses
were performed on a Waters system using Symmetry C18 5 µm
reversed phase column (4.6 × 150 mm). Purification of reagents
and solvents was performed according to standard methods.
Microanalyses were performed on a Carlo Erba 1106 analyzer
at the Serveis Científico-Tècnics (Universitat de Barcelona).
Amino acid analyses were performed using a Beckman system
Gold 6300 at the Serveis Científico-Tècnics (UB).
Compound
IC50/µM
7e (n = 5)
7e (n = 6)
7e (n = 7)
0.32
7f (n = 7)
1.094
0.31
0.36
percentage of inhibition at 1 µM of 50% or higher. Their IC50
was then calculated (Table 2).
From these biological results, we can conclude that, as
expected, the dimers are more active than the monomers, that
the most efficient base function is the benzyl amino group, and
that the preferred length of the linker is n = 5, 6 or 7. However,
since the aminolactams are less potent than the reference
compound BABIM (see Experimental), new linkers and amino
groups need to be explored.
Conclusion
We have designed and builts a targeted library of diversely
substituted 3-aminolactam-derived monomeric (6) and dimeric
(7) compounds, and have tested their activity as tryptase
inhibitors. We have established the synthesis of the monomers
of type 6, both in solution and on solid phase. The two SPS
methods, anchorage through the C3-amino group and anchor-
age through the terminal base function, resulted in equally
efficient syntheses, and furnished the target monomeric com-
pounds 6 in very pure form. However, the on-resin synthesis
of dimers of type 7 gave very poor yields (15%), and parallel
synthesis in solution was used to obtain the library of dimers.
Some of the compounds assayed showed moderate tryptase
inhibition in the submicromolar range. Therefore, the amino-
lactam may be a bioisoster replacement of the piperazino
group, although both the base group and linker remain to be
optimised.
General method for HPLC analysis
The purity of cleaved compounds was determined by C-18
reverse phase HPLC, using 90% solvent A (0.05% TFA in H2O)
and 10% solvent B (0.05% TFA in CH3CN) for 10 min, then a
gradient to 70% solvent A and 30% solvent B over 20 min with
a flow rate of 1 mL minϪ1 monitored at 214 and 280 nm.
Boc-cyclo-Orn-Gly-Arg(NO2)-OMe (9a). To a solution of
compound 812 (1 g, 3.68 mmol) in dry DMF (10 mL) cooled at
0 ЊC, NMM (1.40 mL, 12.88 mmol) and PyBOP (1.91 g, 3.68
mmol) were added. Commercial Arg(NO2)-OMe (994 mg, 3.64
mmol) was then added to the solution, and the mixture was
stirred overnight, at rt. The DMF was removed and the residue
was partitioned in AcOEt and saturated aqueous NaHCO3.
The organic phase was washed with H2O, dried and evaporated,
to yield an oil which was flash chromatographed (AcOEt :
MeOH, 99 : 1) to obtain compound 9a (71%) as a foam. IR
(CHCl3): νmax (cmϪ1) 3320, 1634, 1682, 1745; 1H NMR: δ 1.42 (s,
9H, C(CH3)3), 1.68 (br s, 2H, H-β), 1.75–1.87 (m, 1H, H-4),
1.94–1.99 (m, 4H, H-5, H-γ), 2.27 (br s, 1H, H-4Ј), 3.29 (dd,
J = 13.2 and 6.4 Hz, 2H, H-δ), 3.38 (dd, J = 8.4 and 4.4 Hz, 1H,
H-6), 3.50–3.57 (m, 1H, H-6Ј) 3.74 (s, 3H, CO2CH3), 3.83 (d,
J = 16 Hz, 1H, N(CH2)CO), 4.12 (dd, J = 14.4 and 7.2 Hz, 1H,
H-3), 4.30 (d, J = 16 Hz, 1H, N(CH2Ј)CO), 4.53 (d, J = 3.6 Hz,
Experimental
General
Optical rotations were measured with a Perkin-Elmer 241
polarimeter, at 23 ЊC. IR spectra were recorded on a Nicolet
1
FT-IR spectrophotometer. Unless otherwise indicated, H and
13C NMR spectra were recorded in CDCl3, on a Mercury 400
instrument. Chemical shifts are expressed in parts per million
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 1 6 3 3 – 1 6 4 2
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1H, H-α), 5.62 (br s, 1H, NHBoc), 7.30 (d, J = 5.6 Hz, 1H,
CONH ), 8.45 (br s, 3H, HNC( = NH )NH ); 13C NMR: δ 21.2,
24.9, 28.1, 28.3, 28.6, 40.6, 49.7, 51.4, 51.8, 52.5, 79.8, 155.9,
159.2, 168.7, 171.0, 172.1. m/z (ESϩ): 488 (MHϩ), 388 (100%).
C19H33N7O8 requires: C, 46.81%; H, 6.82%; N, 20.11%; found:
C, 46.35%; H, 6.76%; N, 19.97%.
(CD3OD): δ 1.76–1.87 (m, 1H, H-4), 1.90–2.01 (m, 3H, H-β,
H-γ), 2.05 (br s, 2H, H-5), 2.22 (dt, J = 13.6 and 6.8 Hz, 1H,
H-βЈ), 2.30 (ddd, J = 12, 6.4 and 4.8 Hz, 1H, H-4Ј), 3.42–3.51
(m, 2H, H-δ), 3.69–3.77 (m, 1H, H-6), 3.95 (dd, J = 11.6 and
6 Hz, 1H, H-3), 4.15 (d, J = 13.2 Hz, 1H, N(CH2)CO), 4.17 (d,
Ј
J = 13.2 Hz, 1H, N(CH2 )CO), 4.22 (ddd, J = 10, 4.4 and 2.4 Hz,
1H, H-6Ј), 4.68 (dt, J = 11.6 and 7.6 Hz, 1H, H-α); 13C NMR
(CD3OD): δ 21.3, 21.4, 26.2, 26.5, 45.7, 49.9, 50.6, 51.3, 52.7,
160.0, 168.1, 170.3, 176.5; m/z (ESϩ): 356.4 (MHϩ), 388
(100%). C13H21N7O5ؒCF3COOH requires: C, 38.38%; H, 4.72%;
N, 20.89%. Found: C, 38.75%; H, 4.86%; N, 20.79%
Boc-cyclo-Orn-Gly-cyclo-Arg(NO2) (9b). Operating as above,
from compound 812 (1 g, 3.68 mmol) in dry DMF (10 mL),
NMM (1.40 mL, 12.88 mmol), PyBOP (1910 mg, 3.68 mmol),
and cyclo-Arg(NO2) (1160 mg, 3.68 mmol), an oil was obtained
which was flash chromatographed (AcOEt : MeOH, 99 : 1) to
yield piperidone 9b (85%) as a foam. IR (CHCl3): νmax (cmϪ1
)
p-Nitrobenzylamide 10c. Operating as above from compound
9c (200 mg, 0.49 mmol), and TFA : CH2Cl2 (1 : 1, 2 mL),
compound 10c was obtained quantitatively as a foam. IR
3387, 1651, 1666, 1681, 1698; 1H NMR: δ 1.40 (s, 9H, C(CH3)3),
1.80–2.04 (m, 6H, H-4, H-5, H-β, H-γ), 2.26–2.35 (m, 2H, H-4Ј,
H-βЈ), 3.31–3.37 (m, 1H, H-6), 3.50 (td, J = 10.8 and 3.6 Hz, 1H
H-6Ј), 3.65–3.77 (m, 2H, H-δ, N(CH2)CO), 3.85 (dt, J = 9,6 and
5,6 Hz, 1H, H-3), 4.42 (2dt, J = 14 and 5.6 Hz, 1H, H-δЈ), 4.59
1
(CHCl3): νmax (cmϪ1) 3409, 1775, 1679, 1519, 1347; H NMR:
δ 1.86–2.09 (m, 3H, H-5, H-4), 2.27–2.34 (m, 1H, H-4Ј), 3.39–
3.57 (m, 2H, H-6), 3.95 (ddd, J = 11.7 Hz, J = 6.3 and 2.7 Hz,
1H, H-3), 4.07 (d, J = 16.5 Hz, 1H, N(CH2)CO), 4.18 (d, J =
16.2 Hz, 1H, N(CH2Ј)CO), 4.52 (dd, J = 15.9 Hz, 2H, NHCH2-
C6H4(NO2)), 7.53 (d, J = 9 Hz, 2H, C6H4(NO2)-o), 8.17 (d, J = 9
Hz, 2H, C6H4(NO2)-m); 13C NMR: δ 21.5, 26.5, 43.5, 50.4, 51.1,
51.3, 124.5, 129.2, 147.6, 148.4, 168.1, 170.6. m/z (EI-MS): 307
(MHϩ, 3%), 289 (10), 261 (20), 99 (30), 70 (100), 56 (51).
C12H18N4O4ؒCF3COOH requires: C, 45.72%; H, 4.56%; N,
13.33%. Found: C, 45.62%; H, 4.41%; N, 13.21%.
Ј
(d, J = 15.6 Hz, 1H, N(CH2 )CO), 4.75 (dt, J = 11.6 and 8 Hz,
1H, H-α), 5.52 (d, J = 6.4 Hz, 1H, NHBoc), 7.45 (t, J = 7.2 Hz,
1H, NH-cyclo-Arg), 9.5 (br s, 1H, NC(᎐NH )NH), 10.4 (br s,
᎐
1H, NC(᎐NH)NH ); 13C NMR: δ 20.0, 21.1, 24.6, 27.9, 43.6,
᎐
49.1, 51.1, 51.4, 52.7, 79.8, 155.9, 158.5, 168.6, 170.1, 175.7,
176.1. m/z (ESϩ): 456 (MHϩ), 388 (100%). C18H29N7O7
requires: C, 47.47%; H, 6.42%; N, 21.53%. Found: C, 47.13%;
H, 6.05%; N, 21.13%.
p-Nitrobenzylamide 9c. Operating as above, from compound
812 (1 g, 3.68 mmol) in dry DMF (10 mL), NMM (1.40 mL,
3.68 mmol), PyBOP (1.91 g, 3.68 mmol), and p-nitrobenzyl-
amine (696 mg, 3.68 mmol), an oil was obtained which was
flash chromatographed (AcOEt : MeOH, 99 : 1) to obtain pip-
eridone 9c (85%) as a foam. IR (CHCl3): νmax (cmϪ1) 1651, 1666,
1681, 1698, 1521, 1344; 1H NMR: δ 1.30 (s, 9H, C(CH3)3), 1.80–
1.91 (m, 1H, H-5), 1.96 (dt, J = 14 and 3.6 Hz, 1H, H-5Ј), 2.04–
2.07 (m, 1H, H-4), 2.14–2.22 (m, 1H, H-4Ј), 3.30 (d, J = 11.6
Hz, 1H, H-6), 3.46 (d, J = 16.4 Hz, 1H, N(CH2)CO), 3.53 (td,
J = 11.2 and 4 Hz, 1H, H-6Ј), 3.73 (dt, J = 10.4 and 6.8 Hz, 1H,
H-3), 4.50 (dd, J = 15.6 and 6 Hz, 1H, NHCH2C6H4(NO2)),
4.57 (dq, J = 15.6 and 6 Hz, 1H, NHCH2ЈC6H4(NO2)), 4.85 (d,
J = 16 Hz, 1H, N(CH2Ј)CO), 5.61 (d, J = 6.4 Hz, 1H, NHBoc),
7.46 (d, J = 8.4 Hz, 2H, C6H4(NO2)-o), 7.84 (br s, 1H, NHCH2-
C6H4(NO2)), 8.16 (d, J = 8.8 Hz, 2H, C6H4(NO2)-m); 13C NMR:
δ 21.3, 28.2, 28.3, 42.5, 49.7, 51.9, 52.4, 80.3, 123.7, 127.9,
146.2, 146.9, 156.1, 168.5, 168.8. m/z (ESϩ): 407 (MHϩ), 351
(100%). C19H26N4O6 requires: C, 56.15%; H, 6.45%; N, 13.79%.
Found: C, 55.67%; H, 6.34%; N, 13.74%.
cyclo-Orn-Gly-Arg-OMe (6a). To a solution of 10a (120 mg,
0.25 mmol) in MeOH (10 mL), Pd black (5%) and concentrated
HCl (37%, 0.1 mL) were added. The mixture was hydrogenated
at rt until disappearance of the substrate (tlc monitoring). The
mixture was filtered through Celite® and the filtrate was
evaporated. The residue was washed with Et2O to yield com-
pound 6a as a foam (59%). IR (CHCl3): νmax (cmϪ1) 3389, 1681,
1650, 1633; 1H NMR (CD3OD): δ 1.68 (br s, 2H, H-β), 1.80 (br
s, 1H, H-4), 1.95 (br s, 2H, H-γ), 2.07 (br s, 2H, H-5), 2.32 (br s,
1H, H-4Ј), 3.22 (br s, 2H, H-δ), 3.29 (br s, 2H, H-6), 3.72 (s, 3H,
CO2CH3), 3.98 (br s, 1H, H-3), 4.07 (br s, 1H, N(CH2)CO), 4.22
(br s, 1H, N(CH2Ј)CO), 4.44 (br s, 1H, H-α); 13C NMR
(CD3OD): δ 20.9, 25.5, 25.8, 28.5, 41.2, 49.6, 50.2, 50.7, 52.4,
52.5, 157.4, 167.1, 169.5, 172.4. m/z (ESϩ): 343 (MHϩ), 165
(100%). C14H26N6O4ؒ2HCl requires: C, 44.15%; H, 7.67%; N,
22.07%. Found: C, 44.00%; H, 7.83%; N, 21.59%.
cyclo-Orn-Gly-cyclo-Arg (6b). Operating as above, from
compound 10b (70 mg, 0.15 mmol) in MeOH (10 mL),
Pd-black (5%) and concentrated HCl (37%, 0.1 mL) compound
6b was obtained quantitatively as a foam. IR (CHCl3): νmax
(cmϪ1) 3584, 1731, 1693, 1650; 1H NMR (CD3OD): δ 2.00 (br s,
1H, H-4), 2.07 (br s, 6H, H-β, H-γ, H-5), 2.21 (br s, 1H, H-4),
2.32 (br s, 1H, H-4Ј), 3.44–3.49 (m, 2H, H-δ), 3.81 (br s, 2H,
H-6), 3.97 (br s, 1H, H-3), 4.16 (s, 2H, N(CH2)CO), 4.62 (br s,
1H, H-α); 13C NMR (CD3OD): δ 20.4, 20.6, 25.5, 25.7, 49.0,
49.7, 50.4, 51.0, 51.8, 157.9, 166.9, 169.3, 173.1; m/z (ESϩ):
311.4 (MHϩ), 133 (100%). C13H22N6O3ؒ2HCl requires: C,
44.89%; H, 6.95%; N, 24.16%. Found: C, 44.45%; H, 6.63%; N,
24.01%
cyclo-Orn-Gly-Arg(NO2)-OMe (10a). Compound 9a (200
mg, 0.41 mmol) was dissolved in CH2Cl2 : TFA (1 : 1, 2 mL) and
the mixture was stirred at rt until disappearance of the
substrate (tlc monitoring). The solvent was evaporated and
the residue was washed with Et2O to yield quantitatively com-
pound 10a as a foam. IR (CHCl3): νmax (cmϪ1) 3395, 1740, 1679;
1H NMR (CD3OD): δ 1.68–1.80 (m, 3H, H-β, H-γ), 1.87–1.97
(m, 2H, H-βЈ, H-4), 2.02–2.06 (m, 2H, H-5), 2.30 (dd, J = 8.8
and 2.8 Hz, 1H, H-4Ј), 3.29 (t, J = 6.4 Hz, 2H, H-δ), 3.41 (dt,
J = 8.4 and 3.6 Hz, 1H, H-6), 3.50 (td, J = 10.4 and 5.6 Hz, 1H,
H-6Ј), 3.72 (s, 3H, CO2CH3), 3.96 (dd, J = 11.2 and 5.6 Hz, 1H,
H-3), 3.97 (d, J = 17.6 Hz, 1H, N(CH2)CO), 4.23 (d, J = 16 Hz,
1H, N(CH2Ј)CO), 4.48 (d, J = 4.8 Hz, 1H, H-α); 13C NMR
(CD3OD): δ 21.5, 26.0, 26.5, 29.5, 41.6, 50.4, 50.9, 51.3, 52.8,
53.3, 160.8, 168.2, 170.4, 173.4. m/z (EI-MS): 386 (MHϪ), 69
(100), 51 (66). C14H25N7O6ؒCF3COOH requires: C, 36.97%; H,
4.96%; N, 20.12%. Found: C, 37.01%; H, 5.05%; N, 19.87%.
p-Aminobenzylamide 6c. Operating as above, from compound
10c (75 mg, 0.17 mmol), MeOH (10 mL) and 10% Pd/C (5%),
compound 6c was obtained quantitatively as a foam. IR
(NaCl): νmax (cmϪ1) 3318, 3069, 1668; 1H NMR (CD3OD):
δ 1.85–2.04 (m, 3H, H-5, H-4), 2.22–2.30 (m, 1H, H-4Ј), 3.35–
3.51 (m, 2H, H-6), 3.92 (dd, J = 10 and 6.4 Hz, 1H, H-3), 3.98
(d, J = 16.4 Hz, 1H, N(CH2)CO), 4.15 (d, J = 16.4 Hz, 1H,
N(CH2Ј)CO), 4.46 (dd, J = 14.8 and 14.4 Hz, 2H, NHCH2Ј-
C6H4(NH2)), 7.34 (d, J = 8.7 Hz, 2H, C6H4(NH2)-o), 7.45 (d,
J = 8.7 Hz, 2H, C6H4(NH2)-m); 13C NMR (CD3OD): δ 21.4,
26.5, 43.4, 50.3, 51.0, 51.3, 124.0, 130.0, 131.2, 140.8, 168.1,
170.4. m/z (EI-MS): 276 (Mϩ, 9%), 121 (85), 106 (100), 70 (91).
cyclo-Orn-Gly-cyclo-Arg(NO2) (10b). Operating as above
from compound 9b (200 mg, 0.44 mmol), and TFA : CH2Cl2
(1 : 1, 2 mL), compound 10b was obtained quantitatively as a
foam. IR (CHCl3): νmax (cmϪ1) 3366, 1697, 1652, 1609; 1H NMR
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 1 6 3 3 – 1 6 4 2
1638

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C14H20N4O2ؒCF3COOH requires: C, 49.23%; H, 5.42%; N,
14.35%. Found: C, 49.13%; H, 5.15%; N, 14.11%.
(d, J = 8.4 Hz, 2H, C6H4(CN)-o), 7.70 (d, J = 8.4 Hz, 2H
C6H4(CN)-m); 13C NMR (CD3OD): δ 21.5, 26.4, 49.2, 51.1,
51.4, 112.2, 119.5, 128.4, 129.6, 143.5, 167.7. m/z (ESϩ): 230
(MHϩ, 100%). C13H15N3OؒCF3COOH requires: C, 52.48%; H,
4.70%; N, 12.24%. Found: C, 52.56 %; H, 4.75 %; N, 11.95 %.
p-Nitrobenzylpiperidin-2-one 11a. To a solution of Boc-cyclo-
Orn12 (1 g, 4.67 mmol) in dry THF (30 mL) cooled at 0 ЊC,
LiHMDS (1 M in THF, 5.6 mL, 5.6 mmol) was added under Ar
atmosphere. The solution was stirred for 1 h. p-Nitrobenzyl-
bromide (2.02 g, 9.34 mmol) was then added to the solution,
and the mixture was stirred overnight at rt. The THF was
evaporated, the residue was dissolved in CH2Cl2 and washed
with 2 M aqueous NaHSO4. The organic phase was dried and
evaporated, to yield an oil which was flash chromatographed
(hexane : AcOEt, 7 : 3 to 0 : 1) to obtain compound 11a (86%),
as a foam. IR (CHCl3): νmax (cmϪ1) 1712, 1648 cmϪ1; 1H NMR:
δ 1.45 (s, 9H, C(CH3)3), 1.68 (ddd, J = 12.6, 8.4, and 3.6 Hz, 1H,
H-4), 1,92 (dddd, J = 13.8, 8.7, 3.9 and 2.4 Hz, 2H, H-5), 2.48
(ddd, J = 12.6, 10.2, and 4.2 Hz, 1H, H-4Ј), 3.29 (td, J = 6.3 and
1.2 Hz, 2H, H-6), 4.09 (dt, J = 11.7 and 5.7 Hz, 1H, H-3), 4.68
(s, 2H, CH2C6H4(NO2)), 5.09 (d, J = 3.9 Hz, 1H, NHBoc), 7.42
(d, J = 9 Hz, 2H, C6H4(NO2)-o), 8.18 (d, J = 9 Hz, 2H,
C6H4(NO2)-m); 13C NMR: δ 20.9, 27.8, 28.3, 47.4, 50.3, 52.0,
79.7, 123.8, 128.5, 144.2, 148.3, 155.8, 170.0. m/z (EI-MS): 350
(MHϩ), 293 (15), 204 (37), 57 (100). C17H23N3O5 requires: C,
58.44%; H, 6.64%; N, 12.03%. Found: C, 58.71%; H, 6.73%; N,
11.71%.
p-Aminobenzylpiperidin-2-one d6. Method A (SS). Operating
as for the preparation of compound 6c, from nitrobenzyl-
piperidone 12a (100 mg, 0.27 mmol) and 10% Pd–C (5%) in
MeOH (10 mL), compound 6d was quantitatively obtained as a
foam, which was purified by precipitation from Et2O. Method B
(SPS). A solution of (S)-5-hydroxynorvaline (17, 10 equiv-
alents) and DIEA (20 equivalents) in DMF/NMP (1/1) was
added to resin 18, which had previously been suspended in
DMF. The reaction was shaken for 7 h under Ar atmosphere.
The resin was then drained and rinsed sequentially with DMF
(×4), CH2Cl2 (×4), MeOH (×4), Et2O (×4), and dried in vacuo
to obtain compound 19. Resin 19 was then suspended in
CH2Cl2, purged with Ar and cooled to 0 ЊC. MsCl (20 equiv-
alents) and pyridine (20 equivalents) were added and the
reaction was shaken at 0 ЊC for 1 h, and at rt for an additional
24 h. The mesylated resin was drained and washed with the
above solvent sequence. For the alkylation reaction the resin
was suspended in NMP, purged with Ar and heated to 75 ЊC.
4-Aminobenzylamine (20 equivalents) was added and the reac-
tion was gently stirred at 75 ЊC for 2 days. The resin was then
drained and washed as described before to obtain compound
20. Resin 20 was suspended in DMF, purged with Ar and
cooled to 0 ЊC. DIPCDI (10 equivalents) was added and the
mixture was shaken at 0 ЊC for 1 h before the addition of HOBt
(10 equivalents). The coupling was carried out for 24 h. After
draining and washing, resin 21 was subjected to cleavage with
a mixture of TFA/CH2Cl2 (1/1) for 5 h. Compound 6d was
isolated by precipitation from Et2O after evaporation of the
filtrates. Purity of the cleaved compound determined by HPLC
(see general procedure) was 75%. Piperidone 6d IR (NaCl): νmax
p-Cyanobenzyl lactam 11b. Operating as above, from Boc-
cyclo-Orn12 (1 g, 4.67 mmol), LiHDMS (1 M in THF, 5.6 mL,
5.6 mmol), and p-cyanobenzylbromide (1830 mg, 9.34 mmol) in
THF (30 mL), an oil was obtained which was flash chromato-
graphed (hexane : AcOEt, 7 : 3 to 0 : 1) to yield compound 11b
(84%) as a foam. IR (NaCl): νmax (cmϪ1) 1712, 1648; 1H NMR:
δ 1.46 (s, 9H, C(CH3)3), 1.66 (ddd, J = 12, 8.1, and 4.2 Hz, 1H,
H-4), 1.90 (dddd, J = 14.4, 8.4, 4.5 and 2.1 Hz 2H, H-5), 2.48
(ddd, J = 12.6, 9.9, and 4.5 Hz, 1H, H-4Ј), 3.29 (td, J = 6.3 and
2.1 Hz, 2H, H-6), 4.08 (dt, J = 11.7 and 5.4 Hz, 1H, H-3), 4.59
(d, J = 16.5 Hz, 1H, CH2C6H4(CN)), 4.66 (d, J = 15.3, 1H,
CH2ЈC6H4(CN)), 5.50 (br s, 1H, NHBoc), 7.36 (d, J = 8.1 Hz,
2H, C6H4(CN)-o), 7.62 (d, J = 8.1 Hz, 2H, C6H4(CN)-m); 13C
NMR: δ 20.9, 27.8, 28.3, 47.3, 50.5, 52.0, 79.7, 111.4, 118.5,
128.4, 132.4, 142.2, 155.8, 169.9. m/z (EI-MS): 330 (MHϩ, 2%),
184 (42), 116 (57), 57 (100). C18H23N3O3 requires: C, 65.63%; H,
7.04%; N, 12.76%. Found: C, 65.14 %; H, 7.06%; N, 12.31%.
1
(cmϪ1) 3441, 2924, 1677; H NMR (CD3OD): δ 1.79–1.91 (m,
1H, H-4), 1.95–2.06 (m, 2H, H-5), 2.29 (ddd, J = 11.7, 6.3, and
4.2 Hz, 1H, H-4Ј), 3.34 (dd, J = 6.3 and 4.2 Hz, 2H, H-6), 3.98
(dd, J = 12 and 6.3 Hz, 1H, H-3), 4.59 (d, J = 15 Hz, 1H,
CH2C6H4(NH2)), 4.68 (d, J = 15 Hz, 1H, CH2ЈC6H4(NH2)), 7.35
(d, J = 8.4 Hz, 2H, C6H4(NH2)-o), 7.45 (d, J = 8.4 Hz, 2H,
C6H4(NH2)-m); 13C-NMR (CD3OD): δ 21.5, 26.5, 49.8, 50.9,
51.4, 124.7, 130.6, 131.5, 138.7, 167.5. m/z (EI-MS): 219 (Mϩ,
1%), 106 (100), 69 (67), 51 (53). C12H17N3OؒCF3CO2H requires:
C, 50.45%; H, 5.44%; N, 12.61%. Found: C, 50.36%; H, 5.40%;
N, 12.35%. The enantiomerically pure sample obtained by SPS
showed [α]D Ϫ16 (c = 1, CH3OH).
p-Nitrobenzylpiperidin-2-one 12a. Operating as for the
preparation of compound 10a, from piperidone 11a (200 mg,
0.57 mmol), CH2Cl2 : TFA (1 : 1, 2 mL), an oil was obtained
which was washed with Et2O to yield quantitatively compound
12a as a foam. IR (CHCl3): νmax (cmϪ1) 2924, 1652, 1519, 1343;
1H NMR (CD3OD): δ 1.96 (2dd, dd, J = 12 and 5.1 Hz, 1H,
H-4), 2.04–2.18 (m, 2H, H-5), 2.41 (dddd, J = 12, 5.2, 4.2, and
2.4 Hz, 1H, H-4Ј), 3.48 (dd, J = 7.5 and 4.5 Hz, 2H, H-6), 4.11
(dd, J = 11.7 and 6 Hz, 1H, H-3), 4.78 (d, J = 15.6 Hz, 1H,
CH2C6H4(NO2)), 4.87 (d, J = 15,6 Hz, 1H, CH2ЈC6H4(NO2)),
7.63 (d, J = 8.7 Hz, 2H, C6H4(NO2)-o), 8.31 (d, J = 8.7 Hz, 2H,
C6H4(NO2)-m); 13C NMR: δ 21.5, 26.5, 49.8, 50.9, 51.4, 124.7,
129.8, 145.4, 148.7, 167.5. m/z (EI-MS): 250 (MHϩ, 3%), 204
(22), 69 (85), 56 (100). C12H15N3O3ؒCF3COOH requires: C,
46.29%; H, 4.44%; N, 11.57%. Found: C, 46.12%; H, 4.33%; N,
11.23%.
p-Aminomethylbenzylpiperidin-2-one 6e. Method
A (SS).
Operating as for the preparation of compound 6a, from cyano-
benzylpiperidone 12b (145 mg, 0.44 mmol), 10% Pd–C (5%),
concentrated HCl (37%, 0.1 mL), and MeOH (5 mL), com-
pound 6e was quantitatively obtained as an foam. Method B
(SPS). A solution of p-(hydroxymethyl)benzylamine 23 (10
equivalents) in dry DMF was added to resin 18, which had been
previously suspended in DMF and purged with Ar. The
reaction mixture was shaken for 24 h, and the resin was then
drained and washed sequentially with DMF (×4), CH2Cl2 (×4),
MeOH (×4), Et2O (×4), to obtain compound 24. Resin 24 was
suspended in CH2Cl2, purged with Ar and cooled to 0 ЊC. MsCl
(20 equivalents) and pyridine (20 equivalents) were added, and
the reaction was gently stirred at 0 ЊC for 1 h, and at rt for
additional 24 h. The resulting support-bound mesylated
compound 26 was drained and washed sequentially with the
above solvent sequence. In a separate flask, n-BuLi (2.5 M in
hexanes, 15 equivalents) was added dropwise to a solution of
N-(4-methoxyphenyl)acetamide (15 equivalents) in dry THF,
purged with Ar and cooled to Ϫ78 ЊC. The suspension was
stirred for 45 min, and became slightly yellow. DMF was added
p-Cyanobenzylpiperidin-2-one 12b. Operating as for the prep-
aration of compound 10a, from piperidone 11b (200 mg, 0.62
mmol), CH2Cl2 : TFA (1 : 1, 2 mL), compound 12b was
obtained quantitatively as a foam. IR (NaCl): νmax (cmϪ1) 3440,
2229, 1696; 1H NMR (CD3OD): δ 1.81–1.94 (m, 1H, H-4),
2.96–2.08 (m, 2H, H-5), 2.30 (ddd, J = 11.4, 6.3, and 4.2 Hz,
1H, H-4Ј), 3.36 (td, J = 4.5, and 1.5 Hz, 2H, H-6), 4.01 (dd,
J = 11.4 and 5.7 Hz, 1H, H-3), 4.64 (d, J = 15.3 Hz, 1H,
CH2C6H4(CN)), 4.72 (d, J = 15.6 Hz, 1H, CH2ЈC6H4(CN)), 7.46
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 1 6 3 3 – 1 6 4 2
1639

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to dissolve the precipitate, and the mixture was stirred at
Ϫ78 ЊC for another 15 min. This solution was transferred via
cannula into a solution of Alloc-cyclo-Orn20 (10 equivalents) in
dry DMF, under Ar atmosphere, cooled to 0 ЊC. The reaction
was stirred at 0 ЊC for 2 h, warmed to rt and then transferred via
cannula to the syringe containing resin 26, pre-swollen in DMF.
The mixture was gently shaken for 2 days and the solid-
supported compound 28 was then drained and washed with the
usual solvent sequence (see above). The Alloc group was
cleaved by treating compound 28 with PhSiH3 (24 equivalents)
and Pd(PPh3)4 (0.1 equivalent), in dry CH2Cl2 under Ar
(2 × 20 min), and washing with CH2Cl2 and dioxane:H2O (9 :
1). Quantitative deprotection was confirmed by the Kaiser test.
Final cleavage from the solid support was effected in TFA :
CH2Cl2 (1 : 1) for 5 h to yield compound 6e as the tri-
fluoroacetate salt. This salt was purified by precipitation with
Et2O. Piperidone 6e: 1H NMR (CD3OD): δ 1.92–2.01 (br s, 3H,
H-5, H-4), 2.34 (br s, 1H, H-4Ј), 3.34 (br s, 2H, H-6), 4.01 (br s,
1H, H-3), 4.12 (s, 2H, C6H4(CH2NH2)), 4.59 (d, J = 14.8 Hz,
1H, CH2C6H4(CH2NH2)), 4.69 (d, J = 14.8, 1H, CH2ЈC6H4-
(CH2NH2)), 7.38 (d, J = 7.6 Hz, 2H, C6H4(CH2NH2)-o), 7.47
(d, J = 7.2 Hz, 2H, C6H4(CH2NH2)-p); 13C NMR (CD3OD):
δ 20.4, 25.4, 42.9, 48.5, 49.8, 50.4, 128.6, 129.4, 132.7, 137.7,
166.4; m/z (ESϩ): 234 (MHϩ), 217 (100%). C13H19N3Oؒ2HCl
requires: C, 57.66%; H, 7.85%; N, 15.52%. Found: C, 57.17%;
H, 7.80 %; N, 15.35%.
aqueous Na2CO3 and brine, dried and evaporated, to yield an
oil which was flash chromatographed (CH2Cl2, CH2Cl2 : MeOH
98 : 2) to obtain compound 15 (71%). IR (NaCl) νmax (cmϪ1
)
1723, 1720, 1679, 1649, 1636; 1H NMR: δ 1.36 (s, 9H, C(CH3)3),
1.80–1.97 (m, 3H, H-5, H-4), 2.20 (br s, 1H, H-4Ј), 3.27 (br s,
1H, H-6), 3.44 (td, J = 11.4 and 4.2 Hz, 1H, H-6Ј), 3.51 (d,
J = 12 Hz, 1H, NHCH2CO), 3.77 (dt, J = 10.2 and 6.6 Hz, 1H,
H-3), 4,28 (dd, J = 15 and 5.4 Hz, 1H, NHCH2C6H4), 4.49 (dd,
J = 15 and 6.6 Hz, 1H, NHCH2ЈC6H4), 4.63 (d, J = 15.6 Hz,
1H, N(CH2Ј)CO), 5.13 (s, 2H, Cbz), 5.23 (s, 2H, Cbz), 5.49 (d,
J = 6 Hz, 1H, NHBoc), 7.24 (d, J = 7.8 Hz, 2H, C6H4-o), 7.31
(m, 10H, (Cbz)2), 7.49 (d, J = 8.1 Hz, 2H, C6H4-m), 10.2 (s, 1H,
NHGuan), 11.8 (s, 1H, NHGuan); 13C NMR: δ 21.1, 28.0, 28.2,
42.6, 49.3, 51.8, 67.3, 68.4, 79.9, 122.6, 127.8, 127.9, 128.1,
128.3, 128.4, 128.6, 128.8, 13.3, 135.1, 135.6, 136.3, 153.5,
153.7, 155.9, 163.6, 168, 169.9. m/z (ESϩ): 687 (MHϩ, 100%).
C36H42N6O8 requires: C, 62.96%; H, 6.16%; N, 12.24%. found:
C, 62.50%; H, 6.05%; N, 11.87%.
p-Guanidinobenzylpiperidin-2-one 16. Operating as above,
from compound 15 (470 mg, 1.47 mmol), N,NЈ-bis(benzyloxy-
carbonyl)guanidinium triflate (609 mg, 1.32 mmol), and NMM
(0.16 mL, 1.47 mmol), in dry CH2Cl2 (8 mL), an oil was
obtained which was flash chromatographed (CH2Cl2, CH2Cl2 :
MeOH 98 : 2) to obtain piperidone 16 (75%) as a foam. IR
(NaCl) νmax (cmϪ1) 1717, 1647; 1H NMR: δ 1.45 (s, 9H,
C(CH3)3), 1.59 (td, J = 12 and 3.6 Hz, 1H, H-4), 1.82–1.85 (m,
2H, H-5), 2.47 (dt, J = 12 and 4.8 Hz, 1H, H-4Ј), 3.13–3.25
(m, 2H, H-6), 4.06 (dt, J = 11.7 and 5.1 Hz, 1H, H-3), 4.48 (d,
J = 14.4 Hz, 1H, CH2C6H4), 4.56 (d, J = 14.4 Hz, 1H,
CH2ЈC6H4), 5.14 (s, 2H, Cbz), 5.23 (s, 2H, Cbz), 5.50 (br s, 1H,
NHBoc), 7.20 (d, J = 8.4 Hz, 2H, C6H4-o), 7.32 (m, 10H,
(Cbz)2), 7.52 (d, J = 8.4, Hz, 2H, C6H4-m), 10.25 (s, 1H,
NHGuan), 11.9 (s, 1H, NHGuan); 13C NMR: δ 20.7, 27.8, 28.3,
46.5, 50.0, 51.9, 67.4, 68.5, 79.5, 122.7, 128.0, 128.3, 128.4,
128.7, 128.8, 133.7, 134.3, 135.5, 136.3, 153.5, 153.8, 155.9,
163.7, 169.6. m/z (ESϩ): 630 (MHϩ, 100%). C34H39N5O7
requires: C, 64.85%; H, 6.24%; N, 11.12% found: C, 64.37 %; H,
6.32%; N, 10.85%.
p-Aminobenzylamide 13. Operating as for the preparation of
compound 6a, from piperidone 9c (250 mg, 0.61 mmol), 10%
Pd/C (5%), and MeOH (15 mL), compound 13 was obtained
(97%) as a foam. IR (NaCl): νmax (cmϪ1) 3423, 1697, 1681, 1650;
1H NMR: δ 1.36 (s, 9H, C(CH3)3), 1.85–1.93 (m, 3H, H-5, H-4),
2.19–2.22 (m, 1H, H-4Ј), 3.28 (d, J = 11.2 Hz, 1H, H-6), 3.45
(dd, J = 13.6 and 7.2 Hz, 1H, H-6Ј), 3.62 (d, J = 15.2 Hz, 1H,
N(CH2Ј)CO), 3.88 (d, J = 6.4 Hz, 1H, H-3), 4.24 (dd, J = 14.4
and 6 Hz, 1H, NHCH2C6H4(NH2)), 4.33 (dd, J = 14.8 and 6 Hz,
1H, NHCH2ЈC6H4(NH2)), 4.48 (d, J = 15.6 Hz, 1H, N(CH2Ј)-
CO), 4.74 (br s, 2H, C6H4(NH2)), 5.74 (d, J = 4.4 Hz, 1H, NH-
Boc), 6.73 (d, J = 8.4 Hz, 2H, C6H4(NH2)-o), 7.03 (d, J = 8.4 Hz,
2H, C6H4(NH2)-m), 7.45 (br s, 1H, NHCH2C6H4(NH2)); 13C
NMR: δ 21.0, 27.9, 28.2, 29.5, 42.7, 49.1, 51.6, 79.7, 114.9,
128.0, 128.6, 145.4, 155.8, 167.8, 169.9. m/z (EI-MS): 376 (Mϩ,
2%), 161 (36), 121 (100), 106 (77). C19H28N4O4 requires: C,
60.62%; H, 7.50%; N, 14.88% found: C, 60.81%; H, 7.89%; N,
14.97%.
p-Guanidinobenzylamide 6f. Operating as for the preparation
of compound 6a, from piperidone 15 (100 mg, 0.15 mmol) and
10% Pd–C (5%) the corresponding Cbz-deprotected guanidine
was obtained (86%) as a foam. 1H NMR: δ 1.35 (s, 9H,
C(CH3)3), 1.87 (br s, 3H, H-5, H-4), 2.18 (br s, 1H, H-4Ј), 3.29
(br s, 1H, H-6), 3.44 (br s, 1H, H-6Ј), 3,67–3.74 (m, 1H,
N(CH2)CO), 3.92 (br s, 1H, H-3), 4,28 (br s, 3H, N(CH2Ј)CO,
NHCH2C6H4), 6.78 (br s, 2H, C6H4-o), 7.11 (br s, 2H, C6H4-m),
7.26 (s, 6H, NH2, NH ); 13C NMR: δ 21.3, 28.6, 29.9, 42.8, 49.5,
51.6, 79.9, 124.3, 128.8, 156.3, 168,8, 170.8. The Boc group was
then cleaved following the protocol described for the prepar-
ation of compound 11a, obtaining an oil which was washed
p-Aminobenzylpiperidin-2-one 14. Operating as for the
preparation of compound 6a, from piperidone 11a (225 mg,
0.64 mmol), 10% Pd/C (5%), and MeOH (15 mL), compound
14 was obtained (97%) as a foam. IR (NaCl) νmax (cmϪ1) 3354,
1711, 1647; 1H NMR: 1.45 (s, 9H, C(CH3)3), 1.55 (ddd, J = 12,
8.7, and 4 Hz, 1H, H-4), 1.76–1.85 (m, 2H, H-5), 2.45 (dt,
J = 17.4 and 5.1 Hz, 1H, H-4Ј), 3.15 (dd, J = 12.6 and 7.2 Hz,
1H, H-6), 3.21 (ddd, J = 12.6, 5.4, and 2.1 Hz, 1H, H-6Ј), 3.70
(br s, 2H, NH2), 4.05 (dt, J = 12.3 and 5.4 Hz, 1H, H-3), 4.40 (d,
J = 14.4 Hz, 1H, CH2C6H4(NH2)), 4.49 (d, J = 14.1 Hz, 1H,
CH2ЈC6H4(NH2)), 5.58 (br s, 1H, NHBoc), 6.62 (d, J = 8.4 Hz,
1
with Et2O to yield quantitatively piperidone 6f as a foam. H
NMR (CD3OD): δ 1.84 (ddd, J = 16.4, 11.6, and 5.2 Hz, 1H,
H-4), 1.95–1.99 (m, 2H, H-5), 2.18 (ddd, J = 12, 6.8, and 4 Hz,
1H, H-4Ј), 3.20 (td, J = 8.4 and 3.6 Hz, 1H, H-6), 3.42 (td,
J = 10.8 and 6.4 Hz, 1H, H-6Ј), 3.86 (dd, J = 11.6 and 6 Hz, 1H,
H-3), 3.98 (d, J = 16.8 Hz, 1H, N(CH2)CO), 4.08 (d, J = 16.4
Hz, 1H, N(CH2Ј)CO), 4.35 (s, 2H, NHCH2C6H4), 7.15 (d,
2H, C6H4(NH2)-o), 7.03 (d, J = 8.7 Hz, 2H, C6H4(NH2)-m); 13
C
NMR: δ 20.6, 27.7, 28.3, 46.0, 50.0, 51.9, 79.4, 115.0, 126.4,
129.3, 145.7, 155.8, 169.3. m/z (EI-MS): 319 (Mϩ, 1%), 174 (20),
106 (20), 59 (28). C17H25N3O3 requires: C, 63.93%; H, 7.89%; N,
13.16% found: C, 63.53%; H, 7.93%; N, 12.85%.
J = 8.4 Hz, 2H, C6H4-o), 7.31 (d, J = 8.4 Hz, 2H, C6H4-m); 13
C
NMR (CD3OD): δ 20.2, 25.3, 42.3, 49.1, 49.9, 50.1, 125.5,
128.8, 133.9, 138.3, 157.0, 167.0, 169.2. m/z (ESϩ): 319 (MHϩ),
137 (100%). C15H22N6O2ؒCF3COOH requires: C, 47.22%; H,
5.36%; N, 19.44% found: C, 47.02%; H, 5.15%; N, 19.30%
p-Guanidinobenzylamide 15. To a solution of compound
13 (380 mg, 1.01 mmol) in dry CH2Cl2 (4 mL) under Ar
atmosphere, N,NЈ-bis(benzyloxycarbonyl)guanidinium triflate
(417 mg, 0.91 mmol) and NMM (0.11 mL, 1.01 mmol) were
added. The solution was stirred overnight at rt. The solution
was evaporated and the residue was partitioned in AcOEt and
0.1 M aqueous HCl. The organic solution was washed with 10%
p-Guanidinobenzylpiperidin-2-one 6g. Operating as for the
preparation of compound 6a, from piperidone 16 (100 mg, 0.16
mmol) and 10% Pd–C (5%) the corresponding Cbz-deprotected
guanidine was obtained (97%) as a foam. 1H NMR: 1.44 (s, 9H,
C(CH3)3), 1.62 (dt, J = 8.8 and 6 Hz, 1H, H-4), 1.85 (br s, 2H,
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 1 6 3 3 – 1 6 4 2
1640

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H-5), 2.38 (br s, 1H, H-4Ј), 3.24 (br s, 2H, H-6), 4.06 (m, 1H,
H-3), 4.43 (d, J = 14.4 Hz, 1H, CH2C6H4) 4.53 (d, J = 14.4 Hz,
1H, CH2ЈC6H4), 6.89 (d, J = 7.6 Hz, 2H, C6H4-o), 7.13 (d,
J = 7.6 Hz, 2H, C6H4-m), 7.26 (s, 4H, HNC(NH )NH2); 13C
NMR: δ 21.1, 28.0, 28.6, 47.2, 50.5, 51.9, 79.8, 124.3, 129.3,
132.7, 154.7, 156.3, 170.1. The Boc group was then cleaved
following the protocol described for the preparation of
compound 11a, and piperidone 6g was obtained quantitatively:
1H NMR (CD3OD): δ 1.78 (2dd, J = 12 and 4 Hz, 1H, H-4),
1.87 (dt, J = 8.4 and 2.8 Hz, 1H, H-5), 1.95 (dt, J = 8 and 4 Hz,
1H, H-5Ј), 2.27 (ddd, J = 11.6, 6, and 3.6 Hz, 1H, H-4Ј), 3.28
(ddd, J = 7.6, 4, and 1.2 Hz, 2H, H-6), 3.91 (dd, J = 11.6 and 6
Hz, 1H, H-3), 4.48 (d, J = 15.2 Hz, 1H, CH2C6H4), 4.63 (d,
J = 14.8 Hz, 1H, CH2ЈC6H4), 7.18 (d, J = 8.4 Hz, 2H, C6H4-o),
7.32 (d, J = 8.4 Hz, 2H, C6H4-m); 13C NMR (CD3OD): 20.3,
25.3, 47.1, 49.6, 50.3, 125.6, 129.4, 134.4, 136.0, 156.9, 166.4,
166. m/z (ESϩ): 262.4 (MHϩ, 100%). C13H19N5OؒCF3COOH
requires: C, 48.00%; H, 5.37%; N, 18.66% found: C, 47.56%; H,
5.11%; N, 18.33%
pH 8.2, 100 mM NaCl and 0.05% Tween20) containing 100 µg
mlϪ1 heparin, freshly prepared, to stabilize the tryptase. The
Tosyl-GPL-pNA was diluted to 2 mM with TNT buffer. One
unit of activity was defined as the amount of enzyme which
gave a change of 1.0 absorbance units minϪ1 at 405 nm.
The effects of the compounds were evaluated as follows:
25 µl of compound, diluted with TNT buffer containing 10%
DMSO, were pre-incubated for 1 hour at room temperature
with 50 µl of TNT buffer containing 25 µl of 2.5 µg mlϪ1 human
lung tryptase enzyme. The reaction was started by addition of
25 µl of 2 mM Tosyl-GPL-pNA substrate, and absorbance at
405 nm was monitored every 2 min for 10 min using a kinetic
microtiter plate reader (CERES UV 900, Bio Tek Instruments,
Inc.) at room temperature. Reaction rates were linear during
this period (linear regression correlations >99%).
Inhibitory activities were first tested in duplicate at a single
dose, 1 µM. The compounds that showed an inhibition effect of
about 50% or higher were then evaluated using eight different
concentrations in duplicate. IC50 values were obtained by non-
linear regression using Prism 3.03 software. In all experiments
the inhibitory effect of BABIM on tryptase activity was used as
a positive control (IC50 values in the range of 10–25 nM).
Stock solutions (10Ϫ2 M) of the tested compounds were
prepared in 100% DMSO. Further dilutions were performed in
TNT containing 10% DMSO. The same amount of DMSO
(10%; 2.5% is the final concentration in the assay) was also
included in the control conditions of the assay: basal tryptase
activity (without enzyme and compounds) and maximum
tryptase activity (with enzyme and without compounds).
p-Guanidinomethylbenzylpiperidin-2-one 6h. Resin 2217
(1 equivalent) was swollen in DMF, and a solution of 2-chloro-
1-methylpyridinium iodide23 (Mukaiyama’s reagent, 10 equiv-
alents) and DIEA (5 equivalents) in dry DMF was added. After
gently shaking for 2 h at rt, the resin was rinsed with DMF (×8).
The S-alkylation process was repeated twice. A solution of
p-(hydroxymethyl)benzylamine 23 (15 equivalents), 2-chloro-
1-methylpyridinium iodide23 (5 equivalents), and DIEA
(10 equivalents) in DMF was added, the resin was shaken at rt
for 18 h, and washed with DMF (×5), DCM (×5), MeOH (×5),
THF (×5), Et2O (×5), THF (×5), and Et2O (×5). Subsequent
mesylation, alkylation, deprotection and cleavage were
performed as previously described for compound 6e from resin
24. Final cleavage from the solid support was effected in
TFA : CH2Cl2 (1 : 1) for 5 h to yield compound 6h as the
trifluoroacetate salt, which was purified by precipitation in
Et2O. Compound 6h (85% from resin 22): 1H NMR (CD3OD):
1.70 (m, 1H), 1.80 (m, 2H), 2.2 (m, 1H), 3.30 (m, 2H), 3.50 (t,
J = 13.4 Hz, 1H), 4.10 (m, 2H), 4.25 (m, 2H), 4.5 (d, J = 4.8 Hz,
2H), 5.15 (dd, J = 1.2 and 1 Hz, 1H), 5.28 (dd, J = 1.2 and 1 Hz,
1H), 5.9 (m, 1H), 6.80 (d, J = 7.6 Hz, 2H), 7.35 (d, J = 9.20 Hz,
2H); 13C NMR (CD3OD) 22.1, 28.75, 50, 52, 55, 58, 65.3, 113,
128.5, 130.1, 132.3, 158.1, 162.1, 174.9; ESMS calcd. for
C18H23N5O3 m/z 357. Found: m/z 357.4.
Acknowledgements
Support for this research was provided by the CIRIT
(Generalitat de Catalunya) through grants 1999SGR-00077,
2001SGR-00082, and a CDOC contract to M.G., as well as by
the MCYT (Ministerio de Ciencia y Tecnología, Spain) through
grants PB97-0976, 2FD97-0293, and BQU2001-3228. We also
thank the University of Barcelona for a fellowship to X. d. R.
References
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General method for the preparation of compounds 7. Coupling
of compounds 10a, 10b, 10c, 12a, 12b, deBoc-15, and deBoc-16
with the diacids was performed using the protocol reported for
the preparation of compound 9a. The target dimers 7 were
obtained by cleavage of the protecting groups following
the protocols reported for deprotection of the monomers.
Compounds 7 were obtained as a mixture of isomers owing to
the epimerisation of the starting Boc-cyclo-Orn.12 Thus, each
sample of 7 contained a meso form and a pair of enantiomers,
as shown by analytical HPLC (2 peaks). The samples were
washed with Et2O, but the isomers were not separated. Com-
pounds 7 were identified by ESMS, and by comparison of their
spectral data with those of their monomeric precursors. Thus,
the NMR spectra showed splitting of the signals corresponding
to the respective monomers, and aliphatic signals characteristic
of the additional carbon chain of the linkers. The samples were
used as such for the bioassays.
Tryptase activity assay and determination of the inhibiting
activity of compounds on tryptase. The hydrolytic activity of
human lung tryptase (Promega) was measured using the
chromogenic
substrate
Tosyl-Gly-Pro-Lys-p-nitroanilide
(Tosyl-GPL-pNA, Sigma).4 Assays were performed in 96 well
microtiter plates, in a final volume of 0.1 ml. Tryptase enzyme
was diluted to 2.5 µg mlϪ1 with TNT buffer (50 mM Tris-HCl
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 1 6 3 3 – 1 6 4 2
1641

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11 J. E. Semple, D. C. Rowley, T. K. Brunck, T. Ha-Uong, N. K.
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see: (a) C. Flamant-Robin, Q. Wang and N. A. Sasaki, Tetrahedron
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Mimetics and Peptidomimetics, JAY Press Inc, 1997, vol. 1, p. 193.
13 The only precedent of a lactamisation on solid phase was reported
as a side-reaction in the context of an intramolecular amino
acid coupling: Y. Lee and R. B. Silverman, Synthesis, 1999,
1495–1499.
14 M. García, A. Serra, M. Rubiralta, A. Diez, V. Segarra, E. Lozoya,
H. Ryder and J. M. Palacios, Tetrahedron: Asymmetry, 2000, 11,
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15 The resin was functionalised as an active carbonate according to:
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19 Alloc-cyclo-Orn was synthesized in solution by cyclodehydration of
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21, 2443–2446 and subsequent protection of the α-amino with an
allyloxycarbonyl group following the protocol by E. J. Corey and
J. W. Suggs, J. Org. Chem., 1973, 38, 3223–3224. The sample was not
optically pure.
20 Compound 28 was characterised by the NMR and HPLC data of
the product cleaved from an aliquot of the resin. The purity
determined by HPLC was higher than 95%.
21 (a) C. G. Boojamra, K. M. Burow, L. A. Thompson and
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and J.-A. Fehrentz, EDK, Paris, France, 2001; Proceedings of the
26th European Peptide Symposium, Montpellier, 2000.
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26 The loading of the resin was 0.9 mmol gϪ1
.
18 For the procedure and preliminary results see: M. García,
M. Rubiralta, A. Diez, H. Ryder, E. Lozoya and V. Segarra, in
Innovation and Perspectives in Solid Phase Synthesis & Combinatorial
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Organic Chemical Diversity, Collected Papers, International
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27 The manipulation of the resulting dimer was delicate, mainly
because of its size and polarity.
28 We expected to obtain three stereoisomers for compounds 7
prepared from racemic lactams: the racemic RR and SS forms and
the meso epimer RS. However, we could not detect the
diastereomeric mixture by standard methods such as NMR and
analytical HPLC. Since the compounds turned out not to be
sufficiently active, we did not pursue this issue.
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 1 6 3 3 – 1 6 4 2
1642