Design, synthesis and biological evaluation of new peptide-based ureas and thioureas as potential antagonists of the thrombin receptor PAR1

Article abstract of DOI:10.1016/j.ejmech.2012.10.015

By applying a diversity oriented synthesis strategy for the search of new antagonists of the thrombin receptor PAR1, a series of peptide-based ureas and thioureas, including analogues of the PAR1 reference antagonist RWJ-58259, has been designed and synthesized. The general synthetic scheme involves reduction of basic amino acid-derived amino nitriles by hydrogen transfer from hydrazine monohydrate in the presence of Raney Ni, followed by reaction with diverse isocyanates and isothiocyanates, and protecting group removal. All new compounds have been evaluated as inhibitors of human platelet aggregation induced by the PAR1 agonist SFLLRN. Some protected peptide-based ureas displayed significant antagonist activity.

Full text of DOI:10.1016/j.ejmech.2012.10.015

European Journal of Medicinal Chemistry 58 (2012) 98e111
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis and biological evaluation of new peptide-based ureas and
thioureas as potential antagonists of the thrombin receptor PAR1
Pilar Ventosa-Andrés a, Ángel M. Valdivielso a, Ioannis Pappos b, M. Teresa García-López a,
,
Nikos E. Tsopanoglou b, Rosario Herranz a
*
a Instituto de Química Médica (CSIC), Juan de la Cierva 3, 28006 Madrid, Spain
b Department of Pharmacology, Medical School, University of Patras, 26500 Rio-Patras, Greece
a r t i c l e i n f o
a b s t r a c t
Article history:
By applying a diversity oriented synthesis strategy for the search of new antagonists of the thrombin
receptor PAR1, a series of peptide-based ureas and thioureas, including analogues of the PAR1 reference
antagonist RWJ-58259, has been designed and synthesized. The general synthetic scheme involves
reduction of basic amino acid-derived amino nitriles by hydrogen transfer from hydrazine monohydrate
in the presence of Raney Ni, followed by reaction with diverse isocyanates and isothiocyanates, and
protecting group removal. All new compounds have been evaluated as inhibitors of human platelet
aggregation induced by the PAR1 agonist SFLLRN. Some protected peptide-based ureas displayed
significant antagonist activity.
Received 6 September 2012
Received in revised form
2 October 2012
Accepted 9 October 2012
Available online 17 October 2012
Keywords:
PAR1 antagonists
Peptide-derived ureas
Peptide-derived thioureas
Ó 2012 Elsevier Masson SAS. All rights reserved.
a
-Amino nitriles
Platelet antiaggregant activity
1. Introduction
now, PAR1 antagonists have been searched almost exclusively in
relation to the cardiovascular system [3,18,19]. However, numerous
In addition to the key role of thrombin in the blood coagulation
cascade [1], this serine protease regulates multiple effects on an
increasing variety of cells, such as: platelets [2,3], endothelial and
smooth muscle cells [2,4], neurons and astrocytes in the nervous
system [2,4e7], immune and inflammatory cells [8,9], osteoblasts
[10], and tumor cells [11e14]. These cellular effects are mainly
mediated by the activation of the protease-activated receptor PAR1
[15]. This is a G-protein coupled receptor (GPCR) that is activated by
the thrombin-catalysed cleavage of the N-terminal extracellular
domain at the Arg41/Ser42 peptide bond, which unveils the recog-
nition sequence SFLLRN that acts as a tethered activation ligand.
The conformational changes induced by activation favours the
coupling of PAR1 with heterotrimeric G-proteins, which conse-
quently activate complex cellular signaling cascades [16,17].
studies have shown that PAR1 is overexpressed in invasive and
metastatic tumors and that its expression levels directly correlate
with the degree of invasiveness of the cancer [20e28]. Based on
these facts, this receptor is starting to be also considered a prom-
ising target for cancer therapy [15], particularly in the search of
angiogenesis inhibitors [29].
The first potent PAR1 antagonists were SFLLRN-based peptido-
mimetic ureas, represented by the optimized antagonist RWJ-
58259 (Fig. 1) [19]. This antagonist showed protection against
thrombus formation in nonhuman primates, providing the first
in vivo proof for supporting the potential clinical utility of PAR1
antagonists, although, its low oral bioavailability stopped its clinical
development [19]. Later, several laboratories have reported a few
series of antagonists obtained from HTS of diverse libraries of non-
peptide small molecules, followed by optimization [19,30]. The
most advanced of these antagonists is SCH-530348 (named vor-
apaxar, Fig. 1), derived from the natural product himbacine, which
currently is undergoing Phase III clinical trials in patients with
acute coronary syndrome and in patients with atherosclerosis [31e
33]. Up to now, there is no structural information on the binding
sites of these PAR1 antagonists to be used for structure-based
design of new antagonists. However, recent mutagenesis studies
Since PAR1 is mainly expressed in platelets, where its activation
induces aggregation, it has been proposed that PAR1 antagonists
could be good antithrombotic agents without the hemorrhagic
drawbacks of thrombin inhibitors. Based on this suggestion, up to
* Corresponding author. Tel.: þ34 912587537; fax: þ34 915644853.
E-mail address: rosario@iqm.csic.es (R. Herranz).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.10.015

P. Ventosa-Andrés et al. / European Journal of Medicinal Chemistry 58 (2012) 98e111
99
SFLLRN
F
EtO
F
N
NH
O
F
O
O
N
H
N
N
Cl
(S)
H
H
H
H
N
(S)
N
H
N
H
N
H
Ph
O
Indz
NH2
O
Cl
O
(RWJ-58259)
(SCH-530348)
O
X
O
H
H
N
N
R2
C
N
(S)
(S)
Ph
Ph
*
*
N
H
N
H
N
H
N
H
(CH2)m
(CH2)m
(CH2)n
(CH2)n
R1
N
N
R1
H
H
1
: m =1
A
2: m =2
X = O, S
: n = 3, R1 = Boc
b: n = 4, R1 = Boc
c: n = 3, R1 = C(NH2)N-Pbf
a
R1 = H, C(NH)NH2
R2 = Ar, (CH2)-Ar, (CH2)2-Ar, Indz
* Configuration (R) and (S)
Me
O
Me
O
S
Pbf =
O
H
Me
Me
Me
Fig. 1. Selected PAR1 antagonists and proposed new structures.
on thrombin and/or PAR1 [34e42], X-ray of thrombin crystallized
with diverse N-terminal fragments of PAR1 [43,44], and NMR
studies on the (Ala26eHse103) N-terminal sequence of PAR1 [45]
have shown that the first thrombin/PAR1 interaction is produced
between the exosite I of thrombin and the hirudin-like sequence of
PAR1 (K51YEPF55), and that this first interaction is essential and
determinant for high affinity. It seems that the hydrophobic resi-
dues F34, I82, L65 and Y76, and the basic residues R67 and R73, of
the exosite I of thrombin are important for high affinity. Taking into
account this knowledge, we started a project directed to the search
of new PAR1 antagonists based on these hot spots of the exosite I of
thrombin for PAR1. As these hot spots are discontinuous and are not
localized in a defined secondary structure, we decided to use
a diversity oriented synthesis (DOS) strategy for the search of
peptidomimetics. For this purpose, we planned the synthesis of
diverse small directed libraries of different scaffolds able to display,
at least, one or two aromatic groups and one or two basic groups at
variable distances and orientations. Among these structures, our
first objective was the synthesis of ureas and thioureas of general
formula A, which could be considered as analogues of the reference
antagonist RWJ-58259. To this aim, we devised a synthetic scheme
amino nitriles 1 and 2, followed by reaction of the resulting primary
amines with isocyanates or isothiocyanates and subsequent
removal of protecting groups. The starting basic amino acid-derived
a-amino nitriles 1 and 2 were obtained by a modified Strecker
reaction as (1:1) epimeric mixtures at the cyano-supporting ster-
eocenter, except for the arginine derivatives 1c, which were ob-
tained in an (R)/(S) ratio of (1:3) [47]. Only the epimeric mixtures
(RS)-1a,b could be chromatographically resolved into the respec-
tive (R)- and (S)-epimers. Therefore, (RS)-1c and (RS)-2a,b were
used as such epimeric mixtures through the synthetic pathway.
The epimeric mixture (RS)-1a was used as a model for opti-
mizing the cyano reduction. Initially, this was attempted by 10%
Pd(C) or Raney Ni catalysed hydrogenation. However, as shown in
Scheme 1 and Table 1, the HPLC MS analysis of the crude reaction
showed that the expected primary amines (RS)-3a were obtained
along with variable percentages of 5a and 6a, resulting from retro-
Strecker reaction and reduction of the intermediate imine after
removal of HCN, respectively. The difficulties in the purification of
(RS)-3a from the crude reaction mixtures moved us to try the
reduction by hydrogen transfer, using hydrazine monohydrate as
hydrogen source and Raney Ni as catalyst [48]. After the study of
reaction conditions (Table 1), the formation of the side product 6a
could be completely avoided, but not that of the retro-Strecker
reaction 5a. This side reaction was minimized to a 5% by carrying
out the reduction under refluxing MeOH for 5 min, using 20
equivalents of hydrazine monohydrate and 100 mg/mmol of Raney
Ni (entry 7). These optimized conditions were applied to the
using basic amino acid derived a-amino nitriles 1 and 2 (Fig. 1) as
key diversity generation intermediates [46]. Herein, we report the
synthesis of ureas and thioureas A and their evaluation as human
PAR1 antagonists in a platelet aggregation assay.
2. Results and discussion
reduction of the other starting a-amino nitriles. It is interesting to
2.1. Synthesis
point out the importance of the Raney Ni activation, as well as the
efficiency of the stirring, to make the reduction as faster as possible,
in order to minimize the presence of the amino nitrile within the
reaction medium, for minimizing the retro-Strecker reaction.
The proposed general synthetic scheme for the preparation of
ureas and thioureas A involves cyano-reduction of the protected a-

100
P. Ventosa-Andrés et al. / European Journal of Medicinal Chemistry 58 (2012) 98e111
O
O
O
H
N
H
N
(S)
(S)
+
H2N
+
(S)
Ph
Ph
Ph
*
N
H
N
H
N
H
H2N
O
(CH2)m
(CH2)m
H
N
a), b) or c)
N
(CH2)n
(CH2)n
(CH2)n
(S)
Ph
Ph
*
Ph
N
H
N
H
Boc
N
H
Boc
N
H
Boc
(CH2)m
(CH2)n
(
)-3
5
6
RS : m =1
: m =1
(
)-4
7
RS : m =2
: m =2
N
H
Boc
(RS)-1: m =1
(RS)-2: m =2
d)
a
: n = 3
O
O
O
b
: n = 4
H
N
H
N
H
(S)
(S)
Ph
N
N
Ph
+
Ph
*
N
H
N
Ph
N
H
H
H
O
(CH2)m
(CH2)n
(CH2)n
Ph
N
H
Boc
N
H
Boc
(
)-8
RS : m =1
)-9
RS : m =2
10
(
e)
O
O
H
(S)
Ph
N
N
Ph
*
N
H
N
H
H
(CH2)m
(CH2)n
Ph
NH2·2HCl
(
)-11
RS
: m =2
Scheme 1. Reagents and conditions: (a) H2, Pd(C), MeOH, rt. (b) H2, Raney Ni, MeOH, rt. (c) NH2NH2$H2O, Raney Ni, MeOH or EtOH, T and time as indicated in Table 1. (d) PhNCO,
CH2Cl2, 0 ꢀC. (e) 3 M HCl, EtOAc, rt.
Besides, in the phenylacetaldehyde derivatives 3 (m ¼ 1), the
reduction of the (R)-epimer was slower than that of the corre-
sponding (S)-epimer and, consequently, its percentage of degra-
dation via retro-Strecker was significantly higher [see for example
different yields for reduction of (R)- and (S) epimers of 1a and 1b in
Table 2]. In the case of the Arg derivative 3c, the difference in
reactivity between epimers made that the minor (R)-epimer were
lost in the reduction.
strategy was studied in the phenylacetaldehyde derivatives (R)-
and (S)-3a,b and (RS)-3c. At this point, it is important to note that
when the Fmoc-protection was carried out under the standard
basic conditions, using Na2CO3 as HCl acceptor in dioxane/water
[49], almost complete epimerization of one of the two chiral
centres was observed. This epimerization was completely avoided
Due to the difficulties found in the separation of the primary
amines 3 and 4 from the side product of the retro-Strecker reaction
5, the crude reduction mixtures were initially used without puri-
fication for the synthesis of the desired ureas 8 and 9. However, the
difficulties in the purification of these ureas remained, and several
column chromatographies were required for their separation from
the ureas 10, byproducts derived from the amino amides 5, with
considerable loss in the yield. Only the epimeric mixture of
ornithine-derived ureas (RS)-9a could be resolved during this
chromatographic purification. In view of these results, to avoid the
purification difficulties, we decided to isolate the reduction inter-
mediate amines as the corresponding Fmoc-derivatives. This
Table 2
Optimization of the synthesis of ureas 8, 9 and 13e17.
Amino nitrile
Diaminea
Nꢀ
(RS)-3a (1:3)e
(R)-3a
Ureab
Nꢀ
(RS)-8a (1:3)e
(R)-8a
%
A (%)c
B (%)d
(RS)-1a (1:1)e
(R)-1a
74
55
92
73
60
87
64
55
92
60
87
60
87
60
87
74
74
36
e
52
17
67
44
44
57
25
25
51
40
52
42
61
40
62
e
(S)-1a
(S)-3a
(S)-8a
e
(RS)-1b (1:1)e
(R)-1b
(RS)-3b (1:1.3)e
(R)-3b
(RS)-8b (1:1.3)e
(R)-8b
21
e
(S)-1b
(S)-3b
(S)-3c
(R)-3a
(S)-3a
(R)-3b
(S)-3b
(R)-3b
(S)-3b
(S)-8b
(S)-8c
(R)-13a
(S)-13a
(R)-14b
(S)-14b
(R)-15b
(S)-15b
e
(RS)-1c (1:3)e
(R)-1a
e
e
(S)-1a
(R)-1b
(S)-1b
(R)-1b
(S)-1b
(R)-1b
e
e
e
Table 1
e
Optimization of the reduction conditions for (RS)-1a.
e
T (ꢀC) t (min) Yield (%)a
(RS)-3a 5a 6a
(R)-3b
(R)-16b
e
Entry Catalyst
H2 source
(S)-1b
(S)-3b
(S)-16b
e
(RS)-2a (1:1)e
(RS)-2b (1:1)e
(RS)-4a (1:1)e
(RS)-4b (1:1)e
(RS)-9a (1:1)e
(RS)-9b (1:1)e
27
29
1
2
3
4
5
6
7
10% Pd(C)
Raney Ni (100 mg/mmol) H2
H2
rt
rt
24 h
24 h
30
30
30
80
54
37
67
73
92
95
8
6
63
33
27
8
12
40
e
e
a
Yields determined by HPLC-MS analyses.
Isolated yields.
Overall yield of reduction, followed by reaction with the corresponding isocy-
Raney Ni (100 mg/mmol) NH2NH2$H2O 85
Raney Ni (100 mg/mmol) NH2NH2$H2O rt
Raney Ni (200 mg/mmol) NH2NH2$H2O rt
Raney Ni (100 mg/mmol) NH2NH2$H2O rt
Raney Ni (100 mg/mmol) NH2NH2$H2O 65
b
c
e
e
anate (Method A).
90
5
e
d
Overall yield of reduction, followed by sequential Fmoc-protection, isolation,
5
e
Fmoc-deprotection, and reaction with the corresponding isocyanate (Method B).
a
e
Determined by HPLC-MS analysis.
(R:S)-epimer ratio.

P. Ventosa-Andrés et al. / European Journal of Medicinal Chemistry 58 (2012) 98e111
101
by using propylene oxide as HCl acceptor in CH2Cl2, to obtain the
Fmoc-protected amines (R)- and (S)-12a,b and (S)-12c in 30e72%
overall yield from the amino nitriles (R)- and (S)-3a,b and (RS)-3c
(Scheme 2).
The optimized method B of synthesis of urea derivatives 17a was
similarly applied to the synthesis of the thiourea analogues (R)- and
(S)-27a, by replacing the phenylisocyanate by phenyl iso-
thiocyanate (Scheme 4).
The Fmoc-removal in 12aec, followed by reaction with different
aromatic isocyanates (Scheme 2) led to the desired ureas 8 and 13e
16 in significantly higher overall yields than by the direct synthesis
of the urea after the cyano-reduction (compare yields of methods A
and B in Table 2).
2.2. Biological evaluation
Since PAR1 is mainly expressed in platelets, to evaluate the PAR1
antagonist activity, all new compounds were screened as inhibitors
Removal of the N-Boc protection of the basic side chain in the
Orn and Lys derived ureas 8a,b, 9a,b and 13a,be16b by treatment
with 3 M HCl solution in EtOAc, yielded the corresponding depro-
tected compounds 11a,b and 17a,be21b (Schemes 1 and 2). In view
of the poor biological results of these deprotected ornithine and
lysine derivatives (see below) and the low overall yield of the
arginine-derived urea 8c, this urea was not deprotected.
The lysine-derived Fmoc protected diamine 12b was also used
for the synthesis of the PAR1 reference antagonist RWJ-58259
analogue (S)-25b (Scheme 3). This urea was synthesized by
applying our recently reported procedure for the preparation of
RWJ-58259 [50]. This procedure involves the in situ formation of
the indazole-derived isocyanate 23, by reaction of the required 6-
amino-indazole 22 with triphosgene in the presence of propylene
oxide as HCl acceptor, followed by reaction with the deprotected
diamines (R)- and (S)-3b. Interestingly, as commented for the
reduction of the starting amino nitriles, the application of this
methodology gave considerably higher yield for (S)-24b [85%
overall from (S)-12b] than for its epimer (R)-24b [20% overall yield
from (R)-12b]. Due to this low yield, this epimer was reserved for
the biological evaluation and was not deprotected.
of human platelet aggregation induced by a 30
the PAR1 agonist SFLLRN. The antagonist RWJ-58259 was used as
a reference. At a 10 M concentration this antagonist inhibited 98%
the platelet aggregation. All compounds were tested at an initial
concentration of 0.1 mg/mL (z150 M). Unfortunately, none of the
mM concentration of
m
m
deprotected ureas or thioureas inhibited the platelet aggregation.
However, as shown in Fig. 2, some of the protected ureas, and
particularly the Fmoc intermediates 12, showed significant inhibi-
tion. Thus, the Fmoc derivatives (S)-12a and (S)-12c inhibited a 51%
and a 36%, respectively, the platelet aggregation. Among the pro-
tected ureas and thioureas, the most potent were the urea (R)-8b
and the thiourea (S)-26a, which showed 38 and 33% of inhibition,
respectively. Although these results do not allow to establish
defined structureeactivity relationships, it seems that in the Fmoc
derivatives 12 and thioureas 26a, the (S)-epimers showed higher
inhibition % than the respective (R). However, in the case of the urea
derivatives there is not a clear stereochemistryeactivity relation-
ship, as the results varied depending on the urea substitution. At
this position, the phenyl group was the best (compare ureas 8b
with 14be16b and 24b). Respecting the influence of the basic
amino acid moiety, from the comparison of analogue (S)-epimers,
: n = 3, R1 = Boc
a
O
b: n = 4, R1 = Boc
H
N
N
(S)
Ph
c: n = 3, R1 = C(NH2)N-Pbf
*
N
H
Me
O
Me
(CH2)n
Ph
N
H
R1
O
S
Pbf =
O
H
Me
Me
(
)-
( )-1
S
R
and
Me
a)
O
O
O
H
N
H
N
(S)
H2N
(S)
Fmoc
+
(S)
Ph
Ph
Ph
*
*
N
H
N
H
H2N
b)
N
H
c)
N
H
(CH2)n
(CH2)n
(CH2)n
Ph
Ph
R1
N
H
R1
N
R1
N
H
H
5
(R)- and (S)-3
(R)- and (S)-12
O
H
O
O
O
H
N
R2
(S)
(S)
N
d)
R2
e)
Ph
Ph
*
*
N
N
H
N
H
N
H
N
H
N
H
H
(CH2)n
(CH2)n
Ph
Ph
N
R1
NH2·2HCl
H
: R2 = Ph
: R2 = Bn
R
and S
and S
and S
and S
: R2 = Ph
(
R
)-
(
)-8a,b
(
)-
)-
)-
)-
(
)-17a,b
)-18a
and S
)-8c
: R2 = Ph
R
: R2 = Bn
(
S
(
(
: R2 = 2,4-di(Cl)-Ph
(
R
)-
(
)-13a
(
(
)-19b
and S
and S
R
: R2 = 2,4-di(Cl)-Ph
R
: R2 = 4-MeO-Ph-(CH2)2
(
R
)-
(
)-14b
(
(
)-20b
(R)- and (S)-15b: R2 = 4-MeO-Ph-(CH2)2
(R)- and (S)-16b: R2 = 4-F-Ph-(CH2)2
(R)- and (S)-21b: R2 = 4-F-Ph-(CH2)2
Scheme 2. Reagents and conditions: (a) NH2NH2$H2O, Raney Ni, MeOH, 65 ꢀC. (b) Fmoc-Cl, propylene oxide, CH3CN, 0 ꢀC. (c) Et2NH, CH2Cl2, rt. (d) R2NCO, CH2Cl2, 0 ꢀC. (e) 3 M HCl,
EtOAc, rt.

102
P. Ventosa-Andrés et al. / European Journal of Medicinal Chemistry 58 (2012) 98e111
(R)- and (S)-12b
b)
O
H
(S)
N
Ph
*
N
H
H2N
Ph
(CH2)4
N
H
Boc
N
N
N
(R)- and (S)-3b
O
O
N
N
Cl
Cl
H
N
a)
N
N
N
Cl
(S)
NH2
NCO
Ph
*
N
c)
N
H
N
H
N
H
22
23
(CH2)4
Ph
Cl
Cl
N
H
R1
Cl
: R1 = Boc (20%)
(
)-24b
R
(S)-24b: R1 = Boc (85%)
d)
: R1 = H (100%)
(S)-25b
Scheme 3. Reagents and conditions: (a) (CCl3O)2CO, propylene oxide, THF, 0 ꢀC. (b) Et2NH, CH2Cl2, rt. (c) THF, 0 ꢀC (d) 3 M HCl, EtOAc, rt.
in the Fmoc-derivatives 12, the order of activity was
Orn(a) > Arg(c) > Lys(b), while in the urea-derivatives 8, the order
was Arg(c) ꢁ Lys(b) > Orn(a).
4. Experimental
4.1. General
In the structural comparison of the inactive indazole-derived
urea (S)-25b with the family of peptidomimetic urea PAR1 antag-
onists, to which the reference antagonist RWJ-58259 belongs [51],
the main difference is the replacement of the di(F)-Phe residue of
RWJ-58259 by the 1-amino-3-phenyl-propan-2-yl moiety in (S)-
25b (see Fig. 1). This replacement involves the loss of the carbonyl
group of the peptide bond, which could be important for the
interaction with the PAR1 receptor, participating in H-bond
formation or for the appropriate orientation of the pharmacophoric
groups.
All reagents were of commercial quality. Solvents were dried
and purified by standard methods. Analytical TLC was performed
on aluminium sheets coated with a 0.2 mm layer of silica gel 60
F
254. Silica gel 60 (230e400 mesh) was used for flash chromatog-
raphy. Analytical RP-HPLC was performed on Sunfire C18
m) column, with a flow rate of 1 mL/min, using
a
(4.6 ꢂ 150 mm, 3.5
m
a tunable UV detector set at 214 and 254 nm and a (10e100%,
30 min) gradient of CH3CN (solvent A) and 0.05% TFA in H2O
(solvent B) as mobile phase. HPLC-MS was performed on a Sunfire
C18 (4.6 ꢂ 50 mm, 3.5
m
m) column at 30 ꢀC, with a flow rate of 1 mL/
min (10e100%, 5 min) Gradient of CH3CN with 0.08% of formic acid
(solvent A) in 0.1% of formic acid in H2O (solvent B) was used as
mobile phase. Electrospray in positive mode was used for ioniza-
tion. Melting points were taken on a Mettler Toledo M170 appa-
ratus and are uncorrected. Elemental analyses were obtained on
a CH-O-RAOID apparatus. Optical rotations were determined in
a Perkin Elmer 141 polarimeter. NMR spectra were recorded using
Varian Inova 300, Varian Inova or Mercury 400, and Varian Unity
500 spectrometers. The NMR spectra assignment was based on
COSY, HSQC, and HMBC spectra.
3. Conclusion
A
series of peptide-based ureas and thioureas, including
analogues of the PAR1 reference antagonist RWJ-58259, has been
designed and synthesized as potential PAR1 antagonists. A DOS
strategy has been applied for the synthesis of these urea deriva-
tives, consisting of reduction of basic amino acid-derived amino
nitriles, followed by reaction with diverse isocyanates or iso-
thiocyanates. To evaluate the PAR1 antagonist activity, all new
synthesized compounds have been screened as inhibitors of human
platelet aggregation induced by the PAR1 agonist SFLLRN. Although
none of the designed peptide-based ureas inhibited aggregation,
some of their protected derivatives showed moderate anti-
aggregant activity. These results could be a good clue for the search
of new potent PAR1 antagonists and to explore the thrombin/PAR1
interaction.
4.2. General procedure for the synthesis of basic amino acid-derived
ureas. Method A. Synthesis of ureas (RS)-8a,b and -9a,b, and (R)-
and (S)-9a
Raney Ni (30 mg) and hydrazine monohydrate (0.186 mL,
6 mmol) were added to a solution of the corresponding amino
O
H
O
S
H
N
Et2NH
PhNCS
(S)
(S)
N
Fmoc
Ph
Ph
Ph
*
*
N
H
N
H
N
H
Ph
N
H
N
H
(CH2)3
(CH2)3
Ph
N
Boc
N
H
R1
H
(R)- and (S)-12a
R
and
: R1 = Boc (68-72%)
(
)-
( )-26a
S
HCl
(R)- and (S)-27a: R1 = H (100%)
Scheme 4. Synthesis of thiourea derivatives.

P. Ventosa-Andrés et al. / European Journal of Medicinal Chemistry 58 (2012) 98e111
103
Fig. 2. Inhibition (%) of human platelet aggregation induced by a 30 mM concentration of SFLLRN.
nitrile (RS)-1a,b and (RS)-2a,b (0.3 mmol) in MeOH (20 mL) and the
resulting reaction mixture was refluxed for 5 min. Then, the
mixture was filtered through celite, this was washed with MeOH
(3 ꢂ 75 mL), and the filtrates were evaporated to dryness. The
residue was dissolved in dry CH2Cl2 (10 mL) and the solution was
cooled at 0 ꢀC. The corresponding isocyanate (0.3 mmol) was added
to the solution that was stirred at this temperature for 2 h. Then, the
solvent was evaporated and the residue was dissolved in EtOAc
(50 mL). This solution was successively washed with H2O (20 mL)
and brine (20 mL), dried over Na2SO4, and evaporated to dryness.
The residue was purified by flash chromatography, using 20e100%
EtOAc gradient in hexane as eluant to afford the ureas (RS)-8a,b and
(RS)-9a,b. Only both epimers of the ornithine-derived ureas (R)-9a
and (S)-9a could be separated in this purification. The two epimers
of (RS)-8a,b were obtained as optical pure compounds from each
epimer of the starting amino nitrile (R)-1a,b and (S)-1a,b, by
applying method B described below. The characterization data of
(R)-8a,b and (S)-8a,b are also described below.
C2), 58.3 (CH, Ca), 79.8 (C, Boc), 119.8e129.2 (15CH, Ph), 138.5, 139.6
and 141.9 (3C, Ph), 156.2 (HNCONH), 157.1 (CO, Boc), 175.1 (CONH);
ES-MS m/z [M þ 1]þ calcd. for C34H45N5O4, 688.35; found 688.17
(100%); Anal. calcd. for C34H45N5O4: C, 69.48; H, 7.72; N, 11.92.
Found: C, 69.67; H, 7.86; N, 11.78.
4.2.2. Nd-Boc-Na-[(2S)-4-phenyl-1-(3-phenylureido)butan-2-yl]-
ornithine benzyl amide [(S)-9a]
Foam (25 mg, 14%); HPLC tR 18.89; [
(400 MHz, CDCl3)
and
4H, 1-H (Bu),
a
]
D
20 þ5 (c 1, MeOH); 1H NMR
d
1.42 (s, 9H, Boc), 1.46e1.80 [m, 6H, 3-H (Bu), g-
b
-H (Orn)], 2.51e2.63 [m, 3H, 2-H and 4-H (Bu)], 2.92e3.31 [m,
- and
a
d
-H (Orn)], 4. 26 [dd, 1H, J ¼ 6 and 15 Hz, CH2
(Bn)], 4. 31 [dd, 1H, J ¼ 6 and 15 Hz, CH2 (Bn)], 4.73 (bs, 1H, NH-Boc),
5.84, and 7.70 (2 bs, 2H, HNCONH), 6.92e7.52 (m, 16 H, Ph and NH-
Bn); 13C NMR (100 MHz, CDCl3)
d 28.6 ( CH2, Cg), 28.7 (CH3, Boc), 32.2
(CH2, Cb), 32.4 (CH2, C4), 35.6 (CH2, C3), 40.3 (CH2, Cd), 42.5 (CH2, C1),
43.4 [CH2, (Bn)], 57.4 (CH, C2), 59.9 (CH, Ca), 80.1 (C, Boc),119.6e129.2
(15CH, Ph), 138.6, 139.6 and 141.9 (3C, Ph), 156.7 (HNCONH), 157.2
(CO, Boc), 174.9 (CONH); ES-MS m/z [M þ 1]þ calcd. for C34H45N5O4,
688.35; found 688.27 (100%); Anal. calcd. for C34H45N5O4: C, 69.48;
H, 7.72; N, 11.92. Found: C, 69.59; H, 7.82; N, 11.85.
4.2.1. Nd-Boc-Na-[(2R)-4-phenyl-1-(3-phenylureido)butan-2-yl]-
ornithine benzyl amide [(R)-9a]
Foam (23 mg, 13%); HPLC tR 18.54; [
NMR (400 MHz, CDCl3)
(Bu), - and
2.95e3.43 [m, 4H, 1-H (Bu),
a
]
20 þ7.5 (c 0.9, MeOH); 1H
D
d
1.40 (s, 9H, Boc), 1.29e1.81 [m, 6H, 3-H
4.2.3. Nε-Boc-Na-[4-phenyl-1-(3-phenylureido)butan-2-yl]-lysine
benzyl amide [(RS)-9b]
g
b-H (Orn)], 2.49e2.78 [m, 3H, 2-H and 4-H (Bu)],
a
- and -H (Orn)], 4.32 [m, 2H, CH2
d
Foam (52 mg, 29%); HPLC tR 18.70 [(R)-9b] and 19.06 min [(S)-
(Bn)], 4.92 (bs, 1H, NH-Boc), 5.86, and 7.67 (2 bs, 2H, HNCONH),
9b]; 1H NMR (400 MHz, CDCl3)
d
1.41 and 1.43 (2s, 9H, Boc), 0.94e
-H (Lys)],1.50e1.85 [m, 6H, - and -H (Lys), 3-H (Bu)],
2.45e2.74 [m, 3H, 2-H and 4-H (Bu)], 2.90e3.17 [m, 2H, ε-H (Lys)],
3.17e3.27 [m, 1.5H, 1-H (Bu) and -H (Lys)], 3.38 [m, 0.5H,1-H (Bu)],
6.95e7.56 (m, 16 H, Ph and NH-Bn); 13C NMR (100 MHz, CDCl3)
1.33 [m, 2H,
d
g
b
d
28.6 (CH3, Boc and CH2, Cg), 31.6 (CH2, Cb), 32.3 (CH2, C4), 35.7
(CH2, C3), 39.5 (CH2, Cd), 43.1 (CH2, C1), 43.5 [CH2, (Bn)], 57.1 (CH,
a

104
P. Ventosa-Andrés et al. / European Journal of Medicinal Chemistry 58 (2012) 98e111
4.36 [m, 2H, CH2 (Bn)], 4.66 and 4.84 (2bs, 1H, NH-Boc), 5.80, 7.84
and 8.10 (3 bs, 2H, HNCONH), 6.86e7.54 (m, 16 H, Ph and NH-Bn);
4.3.3. Nε-Boc-Na-[(2R)-3-phenyl-1-(Fmoc)amino-propan-2-yl]-
lysine benzyl amide [(R)-12b]
20
13C NMR (100 MHz, CDCl3)
d
22.9 (CH2, Cg), 26.4 and 28.5 (CH3, Boc),
Foam (132 mg, 64%); HPLC tR 20.68 min; [
MeOH); 1H NMR (400 MHz, CDCl3)
-H (Lys)], 1.44 (s, 9H, Boc), 2.48e2.83 [m, 3H, 2-H (Pr) and ε-H
(Lys)], 2.88e3.02 [m, 2H, 3-H (Pr)], 3.06e3.19 [m, 3H, 1-H (Pr) and
a
]
þ8.2 (c 0.8,
D
29.8 (CH2, Cd), 32.1 (CH2, C4), 33.0 (CH2, Cb), 34.8 (CH2, C3), 40.0
(CH2, Cε), 42.0 and 43.0 (CH2, C1), 43.3 [CH2, (Bn)], 56.9 and 58.8
(CH, C2), 60.1 (CH, Ca), 80.1 (C, Boc), 119.1e128.9 (15CH, Ph), 139.1
and 141.3 (3C, Ph), 156.2 and 156.7 (2CO, Boc and HNCONH), 174.9
(CONH); ES-MS m/z [M þ 1]þ calcd. for C35H47N5O4, 602.36; found
602.27 (100%); Anal. calcd. for C35H47N5O4: C, 69.86; H, 7.87; N,
11.64. Found: C, 69.97; H, 7.84; N, 11.45.
d 0.91e164 [m, 6H, b-H, g-H and
d
a
-H (Lys)], 4.17 [t, 1H, J ¼ 7.5 Hz, CH (Fmoc)], 4.28e4.47 [m, 4H, CH2
(Fmoc) and CH2 (Bn)], 4.60 (bs, 1H, NH-Boc), 5.18 (1bs, 1H, NH-
Fmoc), 7.09 (1bs, 1H, NH-Bn), 7.11e7.75 (m, 19H, aromatics); 13C
NMR (100 MHz, CDCl3) d 22.4 (CH2, Cg), 28.3 (3CH3, Boc), 29.6 (CH2,
Cd), 33.1 (CH2, Cb), 38.9 (CH2, C3), 39.9 (CH2, Cε), 42.9 (CH2, Bn), 44.6
(CH2, C1), 47.1 (CH, Fmoc), 59.2 (CH, C2), 61.1 (CH, Ca), 66.4 (CH2,
Fmoc), 78.9 (C, Boc), 119.8e143.8 (18CH and 6C, aromatics), 155.9
(CO, Boc), 156.7 (NHCOO, Fmoc), 174.4 (CONH); ES-MS m/z [M þ 1]þ
calcd for C42H50N4O5, 691.38; found 691.59 (100%); Anal. calcd. for
4.3. General procedure for the synthesis of the N-Fmoc-protected
amines (R)-12a,b and (S)-12aec
Raney Ni (30 mg) and hydrazine monohydrate (0.186 mL,
6 mmol) were added to a solution of the corresponding amino
nitrile (R)- and (S)-1aec (0.3 mmol) in MeOH (20 mL) and the
resulting reaction mixture was refluxed for 5 min. Then, the
mixture was filtered through celite, this was washed with MeOH
(3 ꢂ 75 mL), and the filtrates were evaporated to dryness. The
respective crude reaction mixture of amines (R)-3a,b and (S)-3aec
impurified with 5aec was dissolved in CH3CN (10 mL) and cooled at
C42H50N4O5: C, 73.02; H, 7.29; N, 8.11. Found: C, 72.89; H, 7.32;
N, 8.02.
4.3.4. Nε-Boc-Na-[(2S)-3-phenyl-1-(Fmoc)amino-propan-2-yl]-
lysine benzyl amide [(S)-12b]
20
Foam (145 mg, 70%); HPLC tR 21.21 min; [
MeOH); 1H NMR (400 MHz, CDCl3)
-H (Lys)], 1.42 (s, 9H, Boc), 2.42e2.68 [m, 2H, 3-H (Pr)], 2.72 [m, 1H,
2-H (Pr)], 2.88e3.44 [m, 4H,1-H (Pr) and ε-H (Lys)], 3.22 [m,1H, -H
a
]
þ12.3 (c 0.9,
D
d
1.16e1.86 [m, 6H, b-H, g-H and
0
ꢀC. Propylene oxide (56
mL, 8 mmol) and Fmoc-Cl (78 mg,
d
0.3 mmol) were added. The solution was stirred for 2 h. Then, the
solvent was evaporated and the residue was dissolved in EtOAc
(50 mL). This solution was successively washed with H2O (20 mL)
and brine (20 mL), dried over Na2SO4, and evaporated to dryness.
The residue was purified by flash chromatography, using 10e40%
EtOAc gradient in hexane as eluant to give the corresponding
Fmoc-protected amine (R)-12a,b and (S)-12aec.
a
(Lys)], 3.83 [dd, 1H, J ¼ 7 and 15 Hz, CH2 (Bn)], 4.19 [t, 1H, J ¼ 7.5 Hz,
CH (Fmoc)], 4.35 [dd, 1H, J ¼ 7, 15 Hz, CH2 (Bn)], 4.41 [d, 2H,
J ¼ 7.5 Hz, CH2 (Fmoc)], 4.57 (bs, 1H, NH-Boc), 5.19 (1bs, 1H, NH-
Fmoc), 7.04 (1bs, 1H, NH-Bn), 7.07e7.77 (m, 19H, aromatics); 13C
NMR (100 MHz, CDCl3) d 22.5(CH2, Cg), 28.4 (3CH3, Boc), 29.8 (CH2,
Cd), 33.5 (CH2, Cb), 39.8 (CH2, C3), 41.5 (CH2, Cε), 42.7 (CH2, Bn), 43.5
(CH2, C1), 47.2 (CH, Fmoc), 59.3 (CH, C2), 60.2 (CH, Ca), 66.7 (CH2,
Fmoc), 79.1 (C, Boc), 119.9e143.7 (18CH and 6C, aromatics), 156.2
(CO, Boc),157.0 (CO, Fmoc),174.4 (CONH); ES-MS m/z [M þ 1]þ calcd
for C42H50N4O5, 691.38; found 691.59 (100%) [M þ 1]þ; Anal. calcd.
for C42H50N4O5: C, 73.02; H, 7.29; N, 8.11. Found: C, 73.14; H, 7.38;
N, 7.96.
4.3.1. Nd-Boc-Na-[(2R)-3-phenyl-1-(Fmoc)amino-propan-2-yl]-
ornithine benzyl amide [(R)-12a]
Foam (112 mg, 55%); HPLC tR 20.56 min; [
a
]
20 þ7.1 (c 1, MeOH);
D
b
1H NMR (400 MHz, CDCl3)
d
1.10e1.98 [m, 4H,
-H and
g
-H (Orn)],
1.36 (s, 9H, Boc), 2.76e3.14 [m, 5H, 2-H, 3-H (Pr) and
d-H (Orn)],
3.24 [m, 1H, -H (Orn)], 3.26e3.55 [m, 1H, 1-H (Pr)], 4.19 [t, 1H,
a
J ¼ 7 Hz, CH (Fmoc)], 4.37 [d, 2H, J ¼ 7 Hz, CH2 (Fmoc)], 4.42 [dd, 2H,
J ¼ 5 and 15 Hz, CH2 (Bn)], 4.71 (bs, 1H, NH-Boc), 5.75 (1bs, 1H, NH-
Fmoc), 7.14e7.76 (m, 19H, aromatics and NH-Bn); 13C NMR
4.3.5. Nd-Pbf-Na-[(2S)-3-phenyl-1-(Fmoc)amino-propan-2-yl]-
arginine benzyl amide [(S)-12c]
Foam (222 mg, 85%); HPLC [Sunfire C18, 3.9 ꢂ 50 mm, 3.5
mm,
(100 MHz, CDCl3)
d
26.0(CH2, Cg), 28.3 (3CH3, Boc), 30.7 (CH2, Cb),
(10e100%, 5 min) gradient of solvent A in solvent B] tR 4.65 min;
20
39.1 (CH2, C3), 39.5 (CH2, Cd), 43.1 (CH2, Bn), 44.6 (CH2, C1), 47.1 (CH,
Fmoc), 59.6 (CH, C2), 60.2 (CH, Ca), 66.5 (CH2, Fmoc), 79.2 (C, Boc),
119.9e143.9 (18CH and 6C, aromatics), 156.2 (CO, Boc), 156.8 (CO,
Fmoc), 174.2 (CONH); ES-MS m/z [M þ 1]þ calcd for C41H48N4O5,
677.36; found 677.59 (100%); Anal. calcd. for C41H48N4O5: C, 72.76;
H, 7.15; N, 8.28. Found: C, 72.93; H, 7.34; N, 8.05.
[a
]
þ6.4 (c 1, MeOH); 1H NMR (500 MHz, CDCl3)
d (ppm): 1.12e
D
1.80 [m, 4H, b-H and g-H (Arg)], 1.45 [s, 6H, 2 CH3 (Pbf)], 2.09 [s,
3H, CH3 (Pbf)], 2.51 [s, 3H, CH3 (Pbf)], 2.59 [s, 3H, CH3 (Pbf)], 2.66e
2.84 [m, 2H, 1-H (Pr)], 2.73 [m, 1H, 2-H (Pr)], 2.92 [s, 2H, CH2 (Pbf)],
3.02e3.55 [m, 4H, 1-H (Pr) and d-H(Arg)], 3.38 [m, 1H, a-H (Arg)],
3.85 [dd, 1H, i ¼ 7.5 and 15 Hz, CH2 (Bn)], 4.17 [t, 1H, J ¼ 7 Hz, CH2
(Fmoc)], 4.29 [dd,1H, J ¼ 7.5 and 15 Hz, CH2 (Bn)], 4.25e4.53 [m, 2H,
CH2 (Fmoc)], 5.60 (bs, 1H, NH-Fmoc), 6.28 (bs, 3H, guanidine NH),
4.3.2. Nd-Boc-Na-[(2S)-3-phenyl-1-(Fmoc)amino-propan-2-yl]-
ornithine benzyl amide [(S)-12a]
6.97e7.78 (m, 15H, aromatics); 13C NMR (125 MHz, CDCl3)
d (ppm):
Foam (162 mg, 80%); HPLC tR 21.26 min; [
1H NMR (400 MHz, CDCl3)
and
2.97e3.16 [m, 2H,
a
]
20 þ15.4 (c 1, MeOH);
12.9, 18.3, 19.7, 28.9 (5CH3, Pbf), 25.7 (CH2, Cg), 30.1 (CH2, Cb), 31.3
(CH2, C3), 40.3 (CH2, Cd), 43.1 (CH2, C1), 43.6 (CH2, Pbf), 47.6 (CH,
Fmoc), 59.2 (CH, C2), 59.5 (CH, Ca), 67.1 (CH2, Fmoc), 86.77, 117.93,
125.0, 132.6, 133.3 (5C, Pbf), 126.9e139.2 (15CH and 5C, aromatics
and Pbf), 156.7 (CO, Fmoc), 157.7e159.2 (C]NH, guanidine), 174.8
(CONH); ES-MS m/z [M þ 1]þ calcd for C50H58N6O6S: 871.77; found
871.77 (100%); Anal. calcd. for C50H58N6O6S: C, 68.94; H, 6.71; N,
9.65. Found: C, 69.07; H, 6.82; N, 9.46.
D
d
1.41 (s, 9H, Boc), 1.46e1.59 [m, 4H, b-H
g-H (Orn)], 2.41e2.71 [m, 2H, 3-H (Pr)], 2.77 [m, 1H, 2-H (Pr)],
d-H (Orn)], 3.16e3.43 [m, 3H, -H and 1-H (Orn)],
a
3.83 ]dd, 2H, J ¼ 7, 15 Hz, CH2(Bn)], 4.20 [t, 1H, J ¼ 6.5 Hz, CH
(Fmoc)], 4.35 [dd, 2H, J ¼ 5, 15 Hz, CH2 (Bn)], 4.42 [d, 2H, J ¼ 6.5 Hz,
CH2 (Fmoc)], 4.75 (bs, 1H, NH-Boc), 5.32 (1bs, 1H, NH-Fmoc), 6.91
(1bs, 1H, NH-Bn), 7.04e7.77 (m, 19H, aromatics); 13C NMR
(100 MHz, CDCl3)
d 26.3(CH2, Cg), 28.4 (3CH3, Boc), 30.9 (CH2, Cb),
39.6 (CH2, C3), 40.1 (CH2, Cd), 42.7 (CH2, Bn), 43.1 (CH2, C1), 47.2 (CH,
Fmoc), 58.7 (CH, C2), 59.1 (CH, Ca), 66.7 (CH2, Fmoc), 79.3 (C, Boc),
119.9e143.8 (18CH and 6C, aromatics), 156.3 (CO, Boc), 157.1 (CO,
Fmoc), 174.2 (CONH); ES-MS m/z [M þ 1]þ calcd for C41H48N4O5,
677.36; found 677.59 (100%); Anal. calcd. for C41H48N4O5: C, 72.76;
H, 7.15; N, 8.28. Found: C, 72.89; H, 7.32; N, 8.12.
4.4. General procedure for the synthesis of basic amino acid-derived
ureas. Method B. Synthesis of ureas (R)-8a,b, (S)-8aec, (R)- and
(S)-13a, and (R)- and (S)-14e16b
Diethyl amine (0.32 mL, 3 mmol) was added to a solution of
the corresponding Fmoc-protected amine 12aec (0.3 mmol) and

P. Ventosa-Andrés et al. / European Journal of Medicinal Chemistry 58 (2012) 98e111
105
the reaction mixture was stirred at rt for 2 h. Then, the solvent
4.4.4. Nε-Boc-Na-[(2S)-3-phenyl-1-(3-phenylureido)propan-2-yl]-
was evaporated to dryness and the residue was dissolved in
EtOAc (50 mL). The solution was successively washed with H2O
(20 mL) and brine (20 mL), dried over Na2SO4, and evaporated to
dryness. The residue was dissolved in dry CH2Cl2 (10 mL), cooled
at 0 ꢀC. The corresponding isocyanate (0.3 mmol) was added to
this solution and the mixture was stirred for 15 mine2 h. Then,
the solvent was evaporated and the residue was dissolved in
EtOAc (50 mL). This solution was successively washed with H2O
(20 mL) and brine (20 mL), dried over Na2SO4, and evaporated to
dryness. The residue was purified by flash chromatography, using
20e100% gradient of EtOAc in hexane as eluant to give the cor-
responding urea (R)-8a,b, (S)-8aec, (R)- and (S)-13a, and (R)- and
(S)-14e16b.
lysine benzyl amide [(S)-8b]
20
Foam (143 mg, 81%); HPLC tR 18.49 min; [
a
]
þ4.0 (c 0.9,
D
MeOH); 1H NMR (400 MHz, CDCl3)
d 1.00e1.90 [m, 6H, b-H, g-H and
d
-H (Lys)], 1.46 (s, 9H, Boc), 2.51e2.68 [m, 2H, 3-H (Pr)], 2.73 [m, 1H,
2-H (Pr)], 2.90e3.18 [m, 2H, ε-H (Lys)], 3.18e3.50 [m, 3H, 1-H (Pr)
and
a
-H (Lys)], 3.90 [dd, 1H, J ¼ 7 and 15 Hz, CH2 (Bn)], 4.35 [dd, 1H,
J ¼ 4 and 15 Hz, CH2 (Bn)], 4.88 (bs, 1H, NH-Boc), 6.82e7.20 (m, 16H,
aromatics and NH-Bn), 5.98 and 8.09 (2bs, 2H, HNCONH); 13C NMR
(100 MHz, CDCl3)
d 21.1 (CH2, Cg), 26.3 (3CH3, Boc), 27.6 (CH2, Cd),
27.9 (CH2, Cb), 30.9 (CH2, C3), 38.1 (CH2, Cε), 39.8 (CH2, C1), 40.7 (CH2,
Bn), 58.0 (CH, C2), 58.4 (CH, Ca), 77.4 (C, Boc), 117.0e137.3 (15CH and
3 C, aromatics), 154.7 (CO, Boc), 154.8 (HNCONH), 172.1 (CONH); ES-
MS m/z [M þ 1]þ calcd for C34H45N5O4, 588.35; found, 588.55
(100%); Anal. calcd. for C34H45N5O4: C, 69.48; H, 7.72; N, 11.92.
Found: C, 69.59; H, 7.84; N, 12.85.
4.4.1. Nd-Boc-Na-[(2R)-3-phenyl-1-(3-phenylureido)propan-2-yl]-
ornithine benzyl amide [(R)-8a]
20
Foam (53 mg, 31%); HPLC tR 17.45 min; [
1H NMR (400 MHz, CDCl3)
1.10e2.41 [m, 4H,
1.40 (s, 9H, Boc), 2.58e2.76 [m, 2H, 3-H (Pr)], 2.81 [m, 1H, 2-H (Pr)],
2.84e3.02 [m, 5H, 1-H (Pr), -H and
a
]
D
þ7.2 (c 1, MeOH);
4.4.5. Nε-Pbf-Na-[(2S)-3-phenyl-1-(3-phenylureido)propan-2-yl]-
d
b-H and
g
-H (Orn)],
arginine benzyl amide [(S)-8c]
Foam (68 mg, 29%); HPLC tR 19.95 min; [
a
]
b
20 þ2.5 (c 1, MeOH); 1H
D
a
d
-H (Orn)], 4.29 [dd, 1H, J ¼ 6
NMR (500 MHz, CDCl3) 0.61e1.63 [m, 4H,
d
- and
g
-H (Arg)], 1.38,
and 15 Hz, CH2 (Bn)], 4.41[ dd, 1H, J ¼ 6 and 15 Hz, CH2 (Bn)], 4.62
2.01, 2.39 and 2.45 [4s,15H, CH3 (Pbf)], 2.74 [m,1H, 2-H (Pr)], 2.85 [s,
2H, CH2 (Pbf)], 2.92e3.17 [m, 4H, 3-H (Pr) and -H (Arg)], 3.20e3.42
[m, 2H, 1-H (Pr)], 3.99 [bs, 1H,
(bs, 1H, NH-Boc), 6.89e7.42 (m, 16H, aromatics and NH-Bn), 5.77
d
and 7.57 (2bs, 2H, NHCONH); 13C NMR (100 MHz, CDCl3)
d
26.0
a
-H (Arg)], 4.17 [d, 1H, J ¼ 6 and
(CH2, Cg), 28.4 (3CH3, Boc), 30.7 (CH2, Cb), 39.2 (CH2, Cd), 39.4 (CH2,
C3), 43.2 (CH2, Bn), 43.9 (CH2, C1), 60.2 (CH, Ca), 60.4 (CH, C2), 79.4
(C, Boc), 119.5e139.2 (15CH and 3 C, aromatics), 156.2 (HNCONH),
156.4 (CO, Boc), 175.0 (CONH); ES-MS m/z [M þ 1]þ calcd for
14.5 Hz, CH2 (Bn)], 4.30 [dd, 1H, J ¼ 4.5 and 14.5 Hz, CH2 (Bn)], 6.20
[bs, 3H, guanidino], 6.85 (t, 1H, J ¼ 6 Hz, NH-Bn), 7.00e7.34 (m, 15H,
aromatics), 7.78 and 8.15 (2bs, 2H, NHCONH); 13C NMR (125 MHz,
CDCl3) d 12.9,18.3,19.7, 28.9 (CH3, Pbf), 25.7 (CH2, Cg), 30.1 (CH2, Cb),
C
C
33H43N5O4, 574.33; found 574.27 (100%); Anal. calcd. for
33H43N5O4: C, 69.08; H, 7.55; N, 12.21. Found: C, 69.13; H, 7.62; N,
31.3 (CH2, C3), 40.3 (CH2, Cd), 43.6 (CH2, C1), 43.6 (CH2, Pbf), 59.2 (CH,
C2), 59.5 (CH, Ca), 86.77, 117.93, 125.0, 132.6, 133.3 (5C, Pbf), 126.9e
139.2 [15CH and 4C, aromatics and Pbf], 156.4 (HNCONH), 159.2
(guanidino); ES-MS m/z [M þ 1]þ calcd. for C42H53N7O5S, 767.38;
found, 768.81 (100%). Anal. calcd. for C42H53N7O5S: C, 65.69; H, 6.96;
N, 12.77. Found: C, 65.82; H, 7.04; N, 12.68.
12.08.
4.4.2. Nd-Boc-Na-[(2S)-3-phenyl-1-(3-phenylureido)propan-2-yl]-
ornithine benzyl amide [(S)-8a]
Foam (144 mg, 84%); HPLC tR 18.15 min; [
a
]
20 þ6.7 (c 1.1, MeOH);
D
1H NMR (400 MHz, CDCl3)
d
1.10e1.74 [m, 4H,
b
-H and
g
-H (Orn)],
4.4.6. Nd-Boc-Na-[(2R)-1-(3-benzylureido)-3-phenylpropan-2-yl]-
1.42 (s, 9H, Boc), 2.40e2.75 [m, 2H, 3-H (Pr)], 2.81e3.14 [m, 2H, 1-H
(Pr)], 2.93 [m, 1H, 2-H (Pr)], 3.14e3.48 [m, 3H, -H and -H (Orn)],
3.70 [dd,1H, J ¼ 7 and 15 Hz, CH2 (Bn)], 4.20 (dd,1H, J ¼ 5 and 15 Hz,
CH2 (Bn)], 4.94 (bs, 1H, NH-Boc), 6.86e7.47 (m, 15H, aromatics),
6.04 and 8.62 (2bs, 2H, NHCONH), 7.75 (bs, 1H, NH-Bn); 13C NMR
ornithine benzyl amide [(R)-13a]
a
d
Foam (80 mg, 45%); HPLC tR 18.47 min; [
a]
20 þ6.5 (c 1.1, MeOH);
D
1H NMR (500 MHz, CDCl3)
d
1.19e1.69 [m, 4H,
b
-H and
g
-H (Orn)],
1.39 (s, 9H, Boc), 2.61e2.65 [m, 2H, 3-H (Pr)], 2.80 [m, 1H, 2-H (Pr)],
2.87e3.20 [m, 4H, 1-H (Pr), and -H (Orn)], 3.21 [m, 1H, -H (Orn)],
d
a
(100 MHz, CDCl3)
d
26.2 (CH2, Cg), 28.7 (3CH3, Boc), 30.8 (CH2, Cb),
4.27 [m, 2H, CH2 (Bn-ureido)], 4.32 [dd, 1H, J ¼ 6 and 15 Hz, CH2
(Bn)], 4.40 [ dd,1H, J ¼ 6 and 15 Hz, CH2 (Bn)], 4.58 (bs, 1H, NH-Boc),
4.73 and 5.06 (2bs, 2H, NHCONH), 7.07e7.36 (m,16H, aromatics and
40.2 (CH2, Cd), 40.6 (CH2, C3), 42.4 (CH2, Bn), 42.9 (CH2, C1), 59.5 (CH,
C2), 59.6 (CH, Ca), 79.9 (C, Boc), 119.6e139.6 (15CH and 3C,
aromatics), 156.8 (HNCONH), 156.9 (CO, Boc), 174.7 (CONH); ES-MS
m/z [M þ 1]þ calcd for C33H43N5O4, 574.33; found 574,52 (100%),
699.43 (10%) [M þ Na]þ; Anal. calcd. for C33H43N5O4: C, 69.08; H,
7.55; N, 12.21. Found: C, 69.17; H, 7.60; N, 12.13.
NH-Bn); 13C NMR (100 MHz, CDCl3)
d 26.1 (CH2, Cg), 28.4 (3CH3, Boc),
30.6 (CH2, Cb), 39.4 (CH2, C3), 39.5 (CH2, Cd), 43.2 (CH2, Bn), 43.9 [CH2,
(Bn-ureido)], 44.4 (CH2, C1), 59.4 (CH, C2), 59.9 (CH, Ca), 79.4 (C, Boc),
127.2e138.7 (15CH and 3 C, aromatics), 156.2 (CO, Boc), 158.2
(HNCONH),174.6 (CONH); ES-MS m/z [M þ 1]þ calcd for C34H45N5O4,
588.35; found 588.48 (100%); Anal. calcd. for C34H45N5O4: C, 69.48;
H, 7.72; N, 11.92. Found: C, 69.61; H, 7.62; N,12.08.
4.4.3. Nε-Boc-Na-[(2R)-3-phenyl-1-(3-phenylureido)propan-2-yl]-
lysine benzyl amide [(R)-8b]
20
Foam (121 mg, 69%); HPLC tR 18.29 min; [
MeOH); 1H NMR (400 MHz, CDCl3)
-H (Lys)], 1.37 (s, 9H, Boc), 2.53e2.87 [m, 3H, 3-H and 2-H (Pr)],
2.89e3.00 [m, 2H, 1-H (Pr)], 3.01e3.42 [m, 3H, -H and ε-H (Lys)],
a
]
D
þ6.5 (c 0.6,
d
0.73e1.65 [m, 6H,
b
-H,
g
-H and
4.4.7. Nd-Boc-Na-[(2S)-1-(3-benzylureido)-3-phenylpropan-2-yl]-
d
ornithine benzyl amide [(S)-13a]
20
a
Foam (112 mg, 64%); HPLC tR 18.86 min; [
MeOH); 1H NMR (400 MHz, CDCl3)
4H, -H and -H (Orn)], 2.45e2.81 [m, 2H, 3-H (Pr)], 2.74 [m, 1H, 2-
H (Pr)], 2.86e3.34 [m, 4H, 1-H (Pr) and -H (Orn)], 3.42 [m, 2H, -H
a]
þ7.0 (c 0.9,
D
4.29 [dd,1H, J ¼ 6 and 15 Hz, CH2 (Bn)], 4.39 [dd,1H, J ¼ 6 and 15 Hz,
CH2 (Bn)], 4.59 (s, 1H, NH-Boc), 5.51 (bs, 1H, HNCONH), 6.87e7.34
(m, 16H, aromatics and HNCONH), 7.56 (bs, 1H, NH-Bn); 13C NMR
d 1.31 (s, 9H, Boc), 1.41e1.85 [m,
b
g
d
a
(100 MHz, CDCl3)
d
22.6 (CH2, Cg), 28.4 (3CH3, Boc), 29.6 (CH2, Cd),
(Orn)], 3.78 [dd, 1H, J ¼ 7 and 15 Hz, CH2 (Bn)], 4.30 (dd, 1H, J ¼ 4
and 15 Hz, CH2 (Bn)], 4.37 [m, 2H, CH2 (Bn-ureido)], 4.94 (bs, 1H,
NH-Boc), 6.86e7.47 (m, 15H, aromatics), 5.81 (1bs, 2H, NHCONH),
33.3 (CH2, Cb), 39.0 (CH2, C3), 39.9 (CH2, Cε), 43.1 (CH2, Bn), 43.8
(CH2, C1), 59.1 (CH, C2), 61.0 (CH, Ca), 79.3 (C, Boc), 122.8e138.4
(15CH and 3C, aromatics), 156.0 (HNCONH), 156.2 (CO, Boc), 174.0
(CONH); ES-MS m/z [M þ 1]þ calcd for C34H45N5O4, 588.35; found
588.62 (100%); Anal. calcd. for C34H45N5O4: C, 69.48; H, 7.72; N,
11.92. Found: C, 69.62; H, 7.89; N, 12.75.
6.96 (bs, 1H, NH-Bn); 13C NMR (100 MHz, CDCl3)
d 25.8 (CH2, Cg),
28.3 (3CH3, Boc), 30.5 (CH2, Cb), 39.8 (CH2, Cd), 40.3 (CH2, C3), 41.7
(CH2, Bn), 42.6 [CH2, (Bn-ureido)], 44.3 (CH2, C1), 58.6 (CH, C2), 59.2
(CH, Ca), 79.7 (C, Boc), 126.3e139.7 (15CH and 3C, aromatics), 156.7

106
P. Ventosa-Andrés et al. / European Journal of Medicinal Chemistry 58 (2012) 98e111
(CO, Boc), 159.2 (HNCONH), 174.4 (CONH); ES-MS m/z [M þ 1]þ
(Pr) and ε-H (Lys)], 3.29e3.50 [m, 1H,
a
-H (Lys)], 3.40 [dd, 2H, J ¼ 7
calcd for C34H45N5O4, 588.35; found 588.62 (100%); Anal. calcd. for
and 13 Hz, 1-H (ethyl)], 3.80 (s, 3H, OMe), 3.91 [dd, 1H, J ¼ 7 and
15 Hz, CH2 (Bn)], 4.35 [dd,1H, J ¼ 7 and 15 Hz, CH2 (Bn)], 4.85 (bs,1H,
NH-Boc), 5.15 and 5.29 (2bs, 2H, HNCONH), 6.75e7.44 (m, 15H,
C
34H45N5O4: C, 69.48; H, 7.72; N, 11.92. Found: C, 69.63; H, 7.84; N,
11.87.
aromatics and NH-Bn); 13C NMR (75 MHz, CDCl3)
d 23.6 (CH2, Cg),
4.4.8. Nε-Boc-Na-[(2R)-1-(3-(2,4-dichlorophenyl)ureido)-3-
28.8 (3CH3, Boc), 30.1 (CH2, Cd), 30.5 (CH2, Cb), 33.5 (CH2, C3), 35.5
[CH2, C2 (ethyl)], 40.5 (CH2, Cε), 40.8 [CH2, C1 (ethyl)], 42.2 (CH2, Bn),
43.1 (CH2, C1), 55.6 (CH3, OMe), 60.6 (CH, C2), 60.8 (CH, Ca), 79.9 (C,
Boc), 114.3e139.5 (14CH and 4C, aromatics), 157.1 (CO, Boc), 159.5
(HNCONH),174.9 (CONH); ES-MS m/z [M þ 1]þ calcd for C37H51N5O5,
646.39; found, 646.52 (100%); Anal. calcd. for C37H51N5O5: C, 68.81;
H, 7.96; N, 10.84. Found: C, 68.93; H, 8.01; N, 10.69.
phenylpropan-2-yl]-lysine benzyl amide [(R)-14b]
Foam (125 mg, 63%); HPLC tR 20.05 min; [
a
]
20 þ7.5 (c 1, MeOH);
D
1H NMR (400 MHz, CDCl3)
d
0.73e1.65 [m, 6H, b-H, g-H and d-H
(Lys)],1.42 (s, 9H, Boc), 2.63 [dd,1H, J ¼ 6 and 13.5 Hz, 3-H (Pr)], 2.72
[dd, 1H, J ¼ 6 and 13.5 Hz, 3-H (Pr)], 2.83e3.14 [m, 5H, 1-H (Pr), 2-H
(Pr), and ε-H (Lys)], 3.32 [m, 1H,
a
-H (Lys)], 4.30 [dd, 1H, J ¼ 6 and
15 Hz, CH2 (Bn)], 4.40 [dd, 1H, J ¼ 6 and 15 Hz, CH2 (Bn)], 4.55 (s, 1H,
NH-Boc), 5.96 (bs, 2H, HNCONH), 7.04e7.31 (m,13H, aromatics), 7.33
4.4.12. Nε-Boc-Na-[(2R)-1-((3-(4-fluorophenethyl)ureido)-3-
phenyl)propan-2-yl]-lysine benzyl amide [(R)-16b]
(bs, 1H, NH-Bn); 13C NMR (100 MHz, CDCl3)
d 22.5 (CH2, Cg), 28.4
(3CH3, Boc), 29.6 (CH2, Cd), 33.6 (CH2, Cb), 39.2 (CH2, C3), 39.8 (CH2,
Cε), 43.2 (CH2, Bn), 43.9 (CH2, C1), 58.9 (CH, C2), 60.8 (CH, Ca), 79.4 (C,
Boc), 121.5e138.2 (13CH and 5C, aromatics), 154.9 (HNCONH), 156.3
Foam (118 mg, 63%); HPLC tR 18.47 min; [
a
]
20 þ7.6 (c 1, MeOH);
D
1H NMR (300 MHz, CDCl3)
d
0.81e2.11 [m, 6H, b-H, g-H and d-H
(Lys)], 1.46 (s, 9H, Boc), 2.56e2.89 [m, 3H, 2-H and 3-H (Pr)], 2.77 [t,
-H and ε-H
(CO, Boc), 175.0 (CONH); ES-MS m/z [M
þ
1]þ calcd for
2H, J ¼ 7 Hz, 2-H (ethyl)], 2.93e3.28 [m, 5H, 1-H (Pr),
a
C
34H43Cl2N5O4, 656.27; found, 656.66 (100%), 658.62 (70%)
(Lys)], 3.32 [dd, 2H, J ¼ 7 and 13 Hz, 1-H (ethyl)], 4.41 [d, 2H,
J ¼ 7 Hz, CH2 (Bn)], 4.58 (bs, 1H, NH-Boc), 4.80 (bs, 2H, HNCONH),
6.89e7.48 (m, 14H, aromatics), 7.52 (bs, 1H, NH-Bn); 13C NMR
[M þ 3]þ, 660.65 (10%) [M þ 5]þ; Anal. calcd. for C34H43Cl2N5O4: C,
62.19; H, 6.60; N, 10.67. Found: C, 62.32; H, 6.75; N, 10.48.
(75 MHz, CDCl3)
d 22.3 (CH2, Cg), 28.0 (3CH3, Boc), 29.3 (CH2, Cd),
4.4.9. Nε-Boc-Na-[(2S)-1-(3-(2,4-dichlorophenyl)ureido)-3-
30.5 (CH2, Cb), 32.9 [CH2, C2 (ethyl)], 35.2 (CH2, C3), 38.9 (CH2, Cε),
41.2 [CH2, C1 (ethyl)], 42.7 (CH2, Bn), 43.8 (CH2, C1), 59.1 (CH, C2),
60.7 (CH, Ca), 79.8 (C, Boc), 114.7e159.5 (14CH and 4C, aromatics),
155.7 (CO, Boc), 157.9 (HNCONH), 174.4 (CONH); ES-MS m/z
[M þ 1]þ calcd for C36H48FN5O4, 634.37; found, 634.68 (100%);
Anal. calcd. for C36H48FN5O4: C, 68.22; H, 7.63; N, 11.05. Found: C,
68.38; H, 7.75; N, 10.89.
phenylpropan-2-yl]-lysine benzyl amide [(S)-14b]
20
Foam (147 mg, 74%); HPLC tR 20.03 min; [
MeOH); 1H NMR (300 MHz, CDCl3)
-H (Lys)], 1.45 (s, 9H, Boc), 2.66e2.91 [m, 3H, 2-H and 3-H (Pr)],
3.00e3.54 [m, 5H, 1-H (Pr),
a
]
þ3.0 (c 0.8,
D
d
0.86e2.19 [m, 6H, b-H, g-H and
d
a
-H and ε-H (Lys)], 4.00 [dd, 1H, J ¼ 6.5
and 15 Hz, CH2 (Bn)], 4.37 [dd, 1H, J ¼ 6.5 and 15 Hz, CH2 (Bn)], 4.86
(s, 1H, NH-Boc), 6.33 (bs, 2H, HNCONH), 6.90e7.58 (m, 13H,
aromatics), 7.70 (bs, 1H, NH-Bn); 13C NMR (100 MHz, CDCl3)
d
20.8
4.4.13. Nε-Boc-Na-[(2S)-1-((3-(4-fluorophenethyl)ureido)-3-
(CH2, Cg), 28.5 (3CH3, Boc), 29.7 (CH2, Cb and Cd), 36.6 (CH2, C3), 39.7
(CH2, Cε), 40.0 (CH2, Bn), 42.9 (CH2, C1), 60.2 (CH, C2), 60.4 (CH, Ca),
79.9 (C, Boc), 121.4e138.5 (13CH and 5C, aromatics), 154.7 (CO,
Boc),157.2 (HNCONH), 170.2 (CONH); ES-MS m/z [M þ 1]þ calcd for
phenyl)propan-2-yl]-lysine benzyl amide [(S)-16b]
20
Foam (168 mg, 89%); HPLC tR 18.57 min; [
MeOH); 1H NMR (300 MHz, CDCl3)
and -H (Lys)], 1.45 (s, 9H, Boc), 2.58e2.73 [m, 3H, 2-H and 3-H
(Pr)], 2.73 [t, 2H, J ¼ 7 Hz, 2-H (ethyl)], 2.94e3.28 [m, 4H, 1-H
(Pr) and ε-H (Lys)], 3.28e3.37 [m, 1H, -H (Lys)], 3.40 [dd, 2H,
a
]
þ3.5 (c 1.1,
D
d
0.77e2.23 [m, 6H, b-H, g-H
d
C
34H43Cl2N5O4, 656.27; found, 656.46 (100%), 658.48 (80%)
[M þ 3]þ, 660.49 (15%) [M þ 5]þ; Anal. calcd. for C34H43Cl2N5O4: C,
a
62.19; H, 6.60; N, 10.67. Found: C, 62.29; H, 6.71; N, 10.55.
J ¼ 7 and 13 Hz, 1-H (ethyl)], 3.89 [dd, J ¼ 7 and 15 Hz, CH2 (Bn)],
4.34 [dd, J ¼ 7 and 15 Hz, CH2 (Bn)], 4.88 (bs, 1H, NH-Boc), 5.26 and
5.41 (2bs, 2H, HNCONH), 6.88e7.60 (m, 15H, aromatics and NH-Bn);
4.4.10. Nε-Boc-Na-[(2R)-1-(3-(4-methoxyphenethyl)ureido)-3-
phenylpropan-2-yl]-lysine benzyl amide [(R)-15b]
13C NMR (75 MHz, CDCl3)
d 23.6 (CH2, Cg), 28.8 (3CH3, Boc), 30.1
20
Foam (127 mg, 66%); HPLC tR 18.29 min; [
MeOH); 1H NMR (400 MHz, CDCl3)
and
2.66e2.82 [m, 3H, 2-H and 3-H (Pr)], 2.91e3.18 [m, 5H,1-H (Pr),
a]
þ8.5 (c 1.1,
(CH2, Cd), 30.5 (CH2, Cb), 33.6 [CH2, C2 (ethyl)], 36.2 (CH2, C3), 40.5
(CH2, Cε), 40.9 [CH2, C1 (ethyl)], 42.0 (CH2, Bn), 43.1 (CH2, C1), 60.6
(CH, C2), 60.8 (CH, Ca), 79.8 (C, Boc), 115.5e139.6 (14CH and 4C,
aromatics), 157.1 (CO, Boc), 160.3 (HNCONH), 175.0 (CONH); ES-MS
m/z [M þ 1]þ calcd for C36H48FN5O4, 634.37; found, 634.43 (100%);
Anal. calcd. for C36H48FN5O4: C, 68.22; H, 7.63; N, 11.05. Found: C,
68.36; H, 7.78; N, 10.95.
D
d
0.78e1.71 [m, 6H,
b
-H,
g
-H
d-H (Lys)], 1.42 (s, 9H, Boc), 2.66 [t, 2H, J ¼ 7 Hz, 2-H (ethyl)],
a
-H
and ε-H (Lys)], 3.28 [dd, 2H, J ¼ 7 and 13 Hz,1-H (ethyl)], 3.76 (s, 3H,
OMe), 4.32 [dd, 1H, J ¼ 6 and 14.5 Hz, CH2 (Bn)], 4.41 [dd, 1H, J ¼ 6
and 14.5 Hz, CH2 (Bn)], 4.55 (bs, 1H, NH-Boc), 4.70 (bs, 2H,
HNCONH), 7.04e7.33 (m, 14H, aromatics), 7.57 (bs, 1H, NH-Bn); 13
C
NMR (100 MHz, CDCl3)
d
22.6 (CH2, Cg), 28.4 (3CH3, Boc), 29.7 (CH2,
4.5. General procedure for the N-Boc-deprotection of protected
ureas (RS)-9b, (R)- and (S)-8a,b, (R)-, (S)-9a and -13a, (R)- and
(S)-14e16b. Synthesis of urea hydrochlorides (R)- and (S)-11a,
(RS)-11b, (R)- and (S)-17a,b, (R)- and (S)-18a, (R)- and (S)-19e21b
Cd), 30.0 (CH2, Cb), 35.4 (CH2, C3), 35.5 [CH2, C2 (ethyl)], 38.9 [CH2, Cε
and C1 (ethyl)], 43.1 (CH2, Bn), 43.9 (CH2, C1), 55.2 (CH3, OMe), 59.5
(CH, C2), 60.9 (CH, Ca), 79.1 (C, Boc), 113.9e138.5 (14CH and 4C,
aromatics), 156.0 (CO, Boc), 158.3 (HNCONH), 174.6 (CONH); ES-MS
m/z [M þ 1]þ calcd for C37H51N5O5, 646.39; found, 646.72 (100%);
Anal. calcd. for C37H51N5O5: C, 68.81; H, 7.96; N, 10.84. Found: C,
68.96; H, 8.07; N, 10.48.
The corresponding protected urea (0.10 mmol) was dissolved in
a 3 M solution of HCl in EtOAc (2 mL) and stirred at rt for 2 h. Then,
the solvent was evaporated under reduced pressure, the residue
was dissolved in H2O (3 mL) and the solution was lyophilized.
4.4.11. Nε-Boc-Na-[(2S)-1-(3-(4-methoxyphenethyl)ureido)-3-
phenylpropan-2-yl]-lysine benzyl amide [(S)-15b]
4.5.1. Na-[(2R)-4-Phenyl-1-(3-phenylureido)butan-2-yl]-ornithine
Foam (143 mg, 87%); HPLC tR 18.43 min; [
a
]
20 þ5.9 (c 1, MeOH);
benzyl amide dihydrochloride [(R)-11a]
D
20
1H NMR (300 MHz, CDCl3)
d
1.09e2.23 [m, 6H,
b-H,
g
-H and
d-H
Amorphous solid (56 mg, 100%); [
NMR (400 MHz, DMSO-d6)
(Orn)], 2.64e2.94 [m, 4H, 4-H (Bu) and
a
]
þ6.9 (c 0.9, MeOH); 1H
D
(Lys)], 1.43 (s, 9H, Boc), 2.60e2.69 [m, 2H, 3-H (Pr)], 2.65e2.72 [m,
2H, 2-H (Pr)], 2.76 [t, 2H, J ¼ 7 Hz, 2-H (ethyl)], 2.99e3.17 [m, 4H,1-H
d 1.51e2.06 [m, 6H, 3-H (Bu), g- and b-H
d-H (Orn)], 3.08 [m, 1H, 2-H

P. Ventosa-Andrés et al. / European Journal of Medicinal Chemistry 58 (2012) 98e111
107
þ
(Bu)], 3.22e3.45 [m, 2H, 1-H (Bu)], 4.09 [m, 1H,
a
-H (Orn)], 4.25 [dd,
aromatics), 6.94 and 7.10 (2bs, 2H, NHCOþNH), 8.12 [bs, 3H,
d
ꢃ NH3
1H, J ¼ 5 and 15 Hz, CH2 (Bn)], 4.40 [dd, 1H, J ¼ 4 and 15 Hz, CH2
(Orn)], 9.41 and 9.50 [2bs, 2H,
a
ꢃ NH2 (Orn)], 9.64 (bs, 1H, NH-
(Bn)], 6.74 and 6.91 (2bs, 2H, HNCONH), 6.97e7.47 (m, 15H, Phþ),
Bn); 13C NMR (100 MHz, DMSO-d6)
d 22.7 (CH2, Cg), 27.3 (CH2, Cb),
þ
7.95 [bs, 3H,
d
ꢃ NH3 (Orn)], 9.11 and 9.43 [2bs, 2H,
a
ꢃ NH2
22.7
34.9 (CH2, C3), 37.7 (CH2, Cd), 38.8 (CH2, Bn), 42.7 (CH2, C1), 57.7 (CH,
(Orn)], 9.33 (bs, 1H, NH-Bn); 13C NMR (100 MHz, DMSO-d6)
d
C2), 59.0 (CH, Ca), 117.8e140.2 (15CH and 3 C, aromatics), 156.2
(CH2, Cg), 27.2 (CH2, Cb), 28.1 (CH2, C4), 29.4 (CH2, C3), 31.0 (CH2, Cd),
38.1 [CH2, (Bn)], 42.6 (CH2, C1), 56.6 (CH, C2), 57.8 (CH, Ca), 118.7e
140.7 (15CH, and 3C, Ph), 156.5 (HNCONH), 166.9 (CONH); ES-MS
m/z [M þ 1]þ calcd. for C29H37N5O2, 488.29; found, 488.50 (100%).
(HNCONH), 167.2 (CONH); ES-MS m/z [M
28H35N5O2, 474.28; found, 574.52 (100%).
þ
1]þ calcd for
C
4.5.6. Na-[(2R)-3-Phenyl-1-(3-phenylureido)propan-2-yl]-lysine
benzyl amide dihydrochloride [(R)-17b]
4.5.2. Na-[(2S)-4-Phenyl-1-(3-phenylureido)butan-2-yl]-ornithine
Amorphous solid (56 mg, 100%); HPLC tR 12.78 min; [
(c 1.6, MeOH); 1H NMR (400 MHz, DMSO-d6)
(Lys)],1.50e1.70 [m, 2H, -H (Lys)],1.72e2.08 [m, 2H,
[m, 2H, ε-H (Lys)], 2.77e2.92 [m, 1H, 3-H (Pr)], 3.08e3.34 [m, 3H, 1-
H and 3-H (Pr)], 3.24 [m, 1H, 2-H (Pr)], 4.20 [m, 1H, -H (Lys)], 4.28
a]
20 þ16.5
D
benzyl amide dihydrochloride [(S)-11a]
d 1.25e1.48 [m, 2H, g-H
20
Amorphous solid (55 mg, 100%); [
NMR (400 MHz, DMSO-d6)
(Orn)], 2.55e2.96 [m, 4H, 4-H (Bu) and
(Bu)], 3.35e3.71 [m, 2H, 1-H (Bu)], 4.25 [m, 1H,
a
]
þ11.3 (c 1, MeOH); 1H
d
b-H (Lys)], 2.72
D
d
1.61e2.09 [m, 6H, 3-H (Bu), g- and b-H
d
-H (Orn)], 3.05 [m, 1H, 2-H
a
a
-H (Orn)], 4.33 [d,
[dd, 1H, J ¼ 6 and 15 Hz, CH2 (Bn)], 4.45 [dd, 1H, J ¼ 5 and 15 Hz, CH2
2H, J ¼ 5, CH2 (Bn)], 6.92 and 7.32 (2bs, 2H, HNCONH), 7.12e7.64 (m,
(Bn)], 6.89 (bs, 2þH, HNCONH), 7.09e7.49 (m, 15H, aromatics), 8.01
þ
þ
15H,þPh), 8.07 [bs, 3H,
d
ꢃ NH3 (Orn)], 9.11 and 9.43 [2bs, 2H,
a
ꢃ
[bs, 3H,
d
ꢃ NH3 (Lys)], 9.01 and 9.77 [2bs, 2H,
a
ꢃ NH2 (Lys)],
NH2 (Orn)], 9.19 (bs, 1H, NH-Bn); 13C NMR (100 MHz, DMSO-d6)
9.47 (bs, 1H, NH-Bn); 13C NMR (100 MHz, DMSO-d6)
d 22.0 (CH2, Cg),
d
22.4 (CH2, Cg), 27.1 (2CH2, C4 and Cb), 30.5 (CH2, C3), 30.9 (CH2, Cd),
27.1 (CH2, Cd), 30.2 (CH2, Cb), 34.3 (CH2, C3), 38.9 (CH2, Cε), 38.9 (CH2,
Bn), 43.3 (CH2, C1), 57.9 (CH, C2), 60.3 (CH, Ca), 118.9e140.7 (15CH
and 3C, aromatics), 157.3 (HNCONH), 167.8 (CONH); ES-MS m/z
[M þ 1]þ calcd for C29H37N5O2, 488.29; found, 488.50 (100%).
37.8 [CH2, (Bn)], 42.4 (CH2, C1), 57.2 (CH, C2), 57.5 (CH, Ca), 117.7e
139.9 (15CH, and 3C, Ph), 156.0 (HNCONH), 166.5 (CONH); ES-MS
m/z [M þ 1]þ calcd. for C29H37N5O2, 488.29; found, 488.50 (100%).
4.5.3. Na-[4-Phenyl-1-(3-phenylureido)butan-2-yl]-lysine benzyl
amide dihydrochloride [(RS)-11b]
4.5.7. Na-[(2S)-3-Phenyl-1-(3-phenylureido)propan-2-yl]-lysine
benzyl amide dihydrochloride [(S)-17b]
Amorphous solid (57 mg, 29%); HPLC tR 13.26 [(R)-9b] and
Amorphous solid (57 mg, 100%); HPLC tR 13.26 min; [
(c 1.5, MeOH); 1H NMR (400 MHz, DMSO-d6)
H (Lys)], 1.51e1.69 [m, 2H, -H (Lys)], 1.74e2.01 [m, 2H,
a
]
20 þ19.3
D
13.45 min [(S)-9b]; 1H NMR (400 MHz, DMSO-d6)
d
1.23e1.40 [m,
-H (Lys)], 1.70e1,96 [m, 4H, -H
(Lys) and 3-H (Bu)], 2.58e2.78 [m, 4H, 4-H (Bu) and ε-H (Lys)],
2.91e3.06 [m, 1H, 2-H (Bu)], and -H (Lys)], 3.38e3.65 [m, 2H, 1-H
(Bu)], 4.03 and 4.16 [2m, 1H,
d 1.28e1.49 [m, 2H, g-
2H,
g-H (Lys)], 1.43e1.62 [m, 2H,
b
d
d
b
-H (Lys)],
2.69 [m, 2H, ε-H (Lys)], 2.78e2.94 [m, 1H, 3-H (Pr)], 2.99e3.18 [m,
2H, 1-H and 3-H (Pr)], 3.23 [m, 1H, 2-H (Pr)], 3.51e3.60 [m, 1H, 1-H
a
a
-H (Lys)], 4.26 [dd, 0.5H, J ¼ 7 and
(Pr)], 4.23e4.48 [m, 1H,
6.80e7.48 [m, 18H, aromatics and
a
-H (Lys)], 4.33 [d, 2H, J ¼ 5 Hz, CH2 (Bn)],
þ
15 Hz, CH2 (Bn)], 4.30 [d, 1H, J ¼ 5 Hz, CH2 (Bn)], 4.35 [dd, 0.5H, J ¼ 7
d
ꢃ NH3 (Lys)], 6.89 and 7.15
þ
and 15 Hz, CH2 (Bn)], 6.77 (bs, 2H, HNCONH), 7.10e7.43 (m, 15 H,
(2bs, 2H, NHCONH), 9.09 and 9.51 [2bs, 2H,
a
ꢃ NH2 (Lys)], 9.51
Ph), 7.92þ[bs, 3H,
d
ꢃ NH3 (Lys)], 8.93, 8.77, 9.33 and 9.46 [4bs, 2H,
(bs, 1H, NH-Bn); 13C NMR (100 MHz, DMSO-d6)
d 22.1 (CH2, Cg), 27.0
þ
a
ꢃ NH2 (Lys)], 9.32 (bs, 1H, NH-Bn); 13C NMR (100 MHz, DMSO-
(CH2, Cd), 30.4 (CH2, Cb), 35.7 (CH2, C3), 38.5 (CH2, Cε), 38.9 (CH2, Bn),
43.3 (CH2, C1), 58.9 (CH, C2), 59.8 (CH, Ca), 118.5e140.9 (15CH and
3C, aromatics),157.0 (HNCONH),168.0 (CONH); ES-MS m/z [M þ 1]þ
calcd for C29H37N5O2, 488.29; found, 488.50 (100%).
d6) d 22.9 (CH2, Cg), 29.8 (CH2, Cd), 32.1 (CH2, C4), 33.0 (CH2, Cb), 34.8
(CH2, C3), 40.0 (CH2, Cε), 42.0 and 43.0 (CH2, C1), 43.3 [CH2, (Bn)],
56.9 and 58.8 (CH, C2), 60.1 (CH, Ca), 119.1e141.3 (15CH, and 3C, Ph),
156.2 and 156.7 (HNCONH), 174.9 (CONH); ES-MS m/z [M þ 1]þ
calcd. for C30H39N5O2, 502.31; found, 502.50 (100%).
4.5.8. Na-[(2R)-1-(3-Benzylureido)-3-phenylpropan-2-yl]-
ornithine benzyl amide dihydrochloride [(R)-18a]
4.5.4. Na-[(2R)-3-Phenyl-1-(3-phenylureido)propan-2-yl]-
Amorphous solid (56 mg,100%); HPLC tR 12.87 min; [
1, MeOH); 1H NMR (500 MHz, DMSO-d6)
(Orn)], 1.90e2.10 [m, 2H,
-H (Orn)], 3.23 [m, 1H, 2-H (Pr)], 3.09e3.22 [m, 2H, 1-H (Pr)], 4.30
[m, 2H, CH2 (Bn-ureido)], 4.32e4.38 [m, 1H, -H (Orn)], 4.35 [dd,
a]
20 þ5.2 (c
D
ornithine benzyl amide dihydrochloride [(R)-17a]
d 1.56e1.82 [m, 2H, g-H
Amorphous solid (54 mg, 100%); HPLC tR 12.87 min; [
1.3, MeOH); 1H NMR (400 MHz, DMSO-d6)
(Orn)], 1.80e2.07 [m, 2H,
H (Pr) and
-H(Orn)], 4.25 [dd, 1H, J ¼ 6 and 15 Hz, CH2 (Bn)], 4.33
[m, 2H,
a
]
20 þ5.5 (c
b-H (Orn)], 2.74e3.02 [m, 4H, 3-H (Pr) and
D
d
1.56e1.79 [m, 2H,
g
-H
d
b-H (Orn)], 2.69e3.31 [m, 7H, 1-H, 2-H, 3-
a
d
1H, J ¼ 6.5 and 15 Hz, CH2 (Bn)], 4.60 [dd, 1H, J ¼ 5 and 15 Hz, CH2
a
-H (Orn)], 4.50 [dd, 1H, J ¼ 6 and 15 Hz, CH2 (Bn)], 6.89 (bs,
(Bn)], 6.72 and 7.00 (2bs, 2H, þNHCONH), 7.21e7.65 (m, 15H,
2H, NHþCONH), 6.79e7.48 (m, 15H, aromatics), 8.08 [bs, 3H,
aromatics), 8.08 [bs, 3H,
a
d
ꢃ NH3 (Orn)], 9.56 and 9.81 [2bs, 2H,
ꢃ NH2 (Orn)], 9.56 (bs, 1H, NH-Bn); 13C NMR (100 MHz, DMSO-
þ
þ
d
ꢃ NH3 (Orn)], 9.15 and 9.78 [2bs, 2H,
a
ꢃ NH2 (Orn)], 9.65 (bs,
1H, NH-Bn); 13C NMR (100 MHz, DMSO-d6)
d
22.1 (CH2, Cg), 27.1
d6) d 22.7 (CH2, Cg), 27.2 (CH2, Cb), 28.9 (CH2, C3), 33.5 (CH2, Cd), 38.1
(CH2, Cb), 28.2 (CH2, C3), 33.5 (CH2, Cd), 38.0 (CH2, Bn), 42.6 (CH2,
C1), 56.7 (CH, C2), 59.5 (CH, Ca), 118.1e148.0 (15CH and 3C,
aromatics), 156.2 (HNCONH), 167.0 (CONH); ES-MS m/z [M þ 1]þ
calcd for C28H35N5O2, 474.28; found, 574.27 (100%).
(CH2, Bn), 42.6 [CH2, (Bn-ureido)], 43.1 (CH2, C1), 56.7 (CH, C2), 60.4
(CH, Ca), 126.9e140.2 (15CH and 3C, aromatics), 159.7 (HNCONH),
166.9 (CONH); ES-MS m/z [M þ 1]þ calcd for C29H37N5O2, 488.29;
found, 488.52 (100%).
4.5.5. Na-[(2S)-3-Phenyl-1-(3-phenylureido)propan-2-yl]-
4.5.9. Na-[(2S)-1-(3-Benzylureido)-3-phenylpropan-2-yl]-ornithine
ornithine benzyl amide dihydrochloride [(S)-17a]
benzyl amide dihydrochloride [(S)-18a]
Amorphous solid (55 mg, 100%); HPLC tR 12.89 min; [
(c 0.9, MeOH); 1H NMR (400 MHz, DMSO-d6)
H (Orn)], 1.82e2.06 [m, 2H,
and -H (Orn)], 2.99e3.18 [m, 1H, 3-H (Pr)], 3.11 [m, 1H, 1-H (Pr)],
3.23 [m, 1H, 2-H (Pr)], 3.53 [m, 1H, 1-H (Pr)], 4.27e4.44 [m, 1H, -H
(Orn)], 4.35 [d, 2H, J ¼ 6.5 Hz, CH2 (Bn)], 6.86e7.45 (m, 15H,
a]
20 þ 26.6
Amorphous solid (55 mg, 100%); HPLC tR 12.95 min; [
1.5, MeOH); 1H NMR (500 MHz, DMSO-d6)
(Orn)],1.80e1.99 [m, 2H,
and -H (Orn)], 3.18 [m, 1H, 2-H (Pr)], 3.38e3.46 [m, 1H, 1-H (Pr)],
4.20 [dd,1H, J ¼ 6 and 15 Hz, CH2 (Bn-ureido)], 4.28 [d, 2H, J ¼ 6 CH2
(Bn)], 4.32 [m,1H,
-H (Orn)], 4.41 [dd, 1H, J ¼ 6 and 15 Hz, CH2 (Bn-
a
]
20 þ2.1 (c
D
D
d
1.67e1.78 [m, 2H,
g-
d 1.58e1.74 [m, 2H, g-H
b-H (Orn)], 2.74e2.96 [m, 3H, 3-H (Pr)
b-H (Orn)], 2.74e3.10 [m, 5H, 1-H, 3-H (Pr)
d
d
a
a

108
P. Ventosa-Andrés et al. / European Journal of Medicinal Chemistry 58 (2012) 98e111
ureido)], 4.60 [ dd, 1H, J ¼ 5 and 15 Hz, CH2 (Bn)], 6.94 and 7.11 (2bs,
2H, NHþCONH), 7.18e7.30 (m, 15H, aromaticsþ), 8.09 [bs, 3H,
2.71 [m, 2H, 2-H (ethyl)], 2.96 [m,1H, 3-H (Pr)], 3.03 [m, 3H,1-H and
3-H (Pr)], 3.15 [d, 2H, J ¼ 7 Hz,1-H (ethyl)], 3.20 [m,1H, 2-H (Pr)], 3.69
d
ꢃ NH3 (Orn)], 9.19 and 9.86 [2bs, 2H,
a
ꢃ NH2 (Orn)], 9.54 (bs,
(s, 3H, OMe), 4.21 [m,1H, a-H (Lys)], 4.36 [m, 2H, CH2 (Bn)], 6.57 and
1H, NH-Bn); 13C NMR (100 MHz, DMSO-d6)
d
23.3 (CH2, Cg), 27.9
6.76 (2bs, 2H, HNCONH), 7.14e7.3 (m, 14H, aromatics), 8.03 [bs, 3H,
(CH2, Cb), 28.8 (CH2, C3), 35.4 (CH2, Cd), 38.6 (CH2, Bn), 43.4 [CH2,
(Bn-ureido)], 43.7 (CH2, C1), 58.5 (CH, C2), 60.6 (CH, Ca), 127.4e140.9
(15CH and 3C, aromatics), 160.5 (HNCONH), 167.9 (CONH); ES-MS
m/z [M þ 1]þ calcd for C29H37N5O2, 488.29; found, 488.38 (100%).
d
ꢃ NH3þ (Lys)], 9.00 and 9.92 [2bs, 2H,
a
ꢃ NH2þ (Lys)], 9.45 (bs,1H,
NH-Bn); 13C NMR (125 MHz, DMSO-d6)
d 21.3 (CH2, Cg), 26.3 (CH2, Cd),
29.6 (CH2, Cb), 34.7 [CH2, C2 (ethyl)], 34.9 (CH2, C3), 38.1 [CH2, C1
(ethyl)], 38.7 (CH2, Cε), 40.1 (CH2, Bn), 42.6 (CH2, C1), 54.9 (CH3, OMe),
58.4 (CH, C2), 60.0 (CH, Ca), 113.8e157.6 (14CH and 4C, aromatics),
159.8 (HNCONH), 167.4 (CONH); ES-MS m/z [M þ 1]þ calcd for
4.5.10. Na-[(2R)-1-(3-(2,4-Dichlorophenyl)ureido)-3-
phenylpropan-2-yl]-lysine benzyl amide dihydrochloride [(R)-19b]
C32H43N5O3, 546.34; found, 546.62 (100%).
Amorphous solid (62 mg, 100%); HPLC tR 14.85 min; [
0.4, MeOH); 1H NMR (500 MHz, DMSO-d6)
(Lys)], 1.53e1.71 [m, 2H, -H (Lys)], 1.82e2.05 [m, 2H,
2.80 [m, 2H, ε-H (Lys)], 2.91 [m, 1H, 3-H (Pr)], 3.23 [m, 1H, 2-H (Pr)],
3.27e3.40 [m, 3H, 1-H and 3-H (Pr)], 3.26 [m, 1H, -H (Lys)], 3.26
a
]
20 þ9.8 (c
D
d
1.37e1.49 [m, 2H,
g
-H
4.5.14. Na-[(2R)-1-((3-(4-Fluorophenethyl)ureido)-3-phenyl)
d
b
-H (Lys)],
propan-2-yl]-lysine benzyl amide dihydrochloride [(R)-21b]
Amorphous solid (60 mg, 100%); HPLC tR 13.65 min; [
(c 0.8, MeOH); 1H NMR (500 MHz, DMSO-d6)
H (Lys)], 1.50e1.67 [m, 2H, -H (Lys)], 1.75e1.98 [m, 2H,
a
]
20 þ15.2
D
a
d 1.30e1.41 [m, 2H, g-
[dd, 1H, J ¼ 6 and 15 Hz, CH2 (Bn)], 4.52 [dd, 1H, J ¼ 5 and 15 Hz, CH2
d
b
-H (Lys)],
(Bn)], 7.18e7.44 (m, 13H, aromatics), 7.59 (bs, 2H, HNCONH), 7.94
2.68 [t, 2H, J ¼ 7 Hz, 2-H (ethyl)], 2.71 [m, 2H, ε-H (Lys)], 2.78 [m,1H,
þ
þ
[bs, 3H,
d
ꢃ NH3 (Lys)], 8.99 and 9.67 [2bs, 2H,
a
ꢃ NH2 (Lys)],
3-H (Pr)], 3.04 [m, 1H, 2-H (Pr)], 3.08e3.26 [m, 5H, 1-H, 3-H (Pr),
9.43 (bs, 1H, NH-Bn); 13C NMR (125 MHz, DMSO-d6)
d
21.3 (CH2, Cg),
and 1-H (ethyl)], 4.21 [m, 1H,
a
-H (Lys)], 4.28 [dd, 1H, J ¼ 6.5 and
26.5 (CH2, Cd), 29.4 (CH2, Cb), 32.0 (CH2, C3), 37.5 (CH2, Cε), 38.3 (CH2,
Bn), 42.6 (CH2, C1), 57.2 (CH, C2), 58.8 (CH, Ca), 116.9e140.2 (13CH
and 5C, aromatics), 155.9 (HNCONH), 167.1 (CONH); ES-MS m/z
[M þ 1]þ calcd for C29H35Cl2N5O2, 556.22; found, 556.63 (100%),
558.52 (64%) [M þ 3]þ, 560.42 (10%) [M þ 5]þ.
15 Hz, CH2 (Bn)], 4.46 [dd, 1H, J ¼ 5 and 15 Hz, CH2 (Bn)], 6.43 and
6.59 (2bs, 2H, HNCONH), 7.02e7.36 (m, 14H, aromatiþcs), 7.95 [bs,
þ
3H,
d
ꢃ NH3 (Lys)], 9.42 and 9.84 [2bs, 2H,
a
ꢃ NH2 (Lys)], 9.48
(bs, 1H, NH-Bn); 13C NMR (75 MHz, DMSO-d6)
d 21.6 (CH2, Cg), 26.8
(CH2, Cd), 30.0 (CH2, Cb), 33.9 (CH2, C3), 35.3 [CH2, C2 (ethyl)], 38.2
[CH2, C1 (ethyl)], 38.7 (CH2, Cε), 41.7 (CH2, Bn), 42.9 (CH2, C1), 57.6
(CH, C2), 61.2 (CH, Ca), 115.2e160.1 (14CH and 4C, aromatics), 162.2
4.5.11. Na-[(2S)-1-(3-(2,4-Dichlorophenyl)ureido)-3-
phenylpropan-2-yl]-lysine benzyl amide dihydrochloride [(S)-19b]
(HNCONH), 167.5 (CONH); ES-MS m/z [M
C31H40FN5O2, 534.32; found, 534.65 (100%).
þ
1]þ calcd for
Amorphous solid (63 mg, 100%); HPLC tR 14.09 min; [
(c 1.1, MeOH); 1H NMR (400 MHz, DMSO-d6)
(Lys)], 1.46e1.681 [m, 2H, -H (Lys)], 1.68e1.98 [m, 2H,
2.61e2.95 [m, 2H, 3-H (Pr)], 2.71 [m, 2H, ε-H (Lys)], 2.96e3.19 [m,
2H, 1-H (Pr)], 3.23 [m, 1H, 2-H (Pr)], 4.3 [m, 1H, -H (Lys)], 4.36 [d,
a
]
20 þ12.3
D
d
1.17e1.42 [m, 2H, g-H
d
b
-H (Lys)],
4.5.15. Na-[(2S)-1-((3-(4-Fluorophenethyl)ureido)-3-phenyl)
propan-2-yl]-lysine benzyl amide dihydrochloride [(S)-21b]
a
Amorphous solid (59 mg,100%); HPLC tR 13.69 min; [
1.3, MeOH); 1H NMR (500 MHz, DMSO-d6)
1.36 [m, 2H,
1.59 [m, 2H, -H (Lys)],1.84 [m, 2H,
a
]
20 þ21.5 (c
D
2H, J ¼ 5 Hz, CH2 (Bn)], 7.11e7.39 (m, 15H, aromatics and HNCONH),
d
g-H (Lys)],
þ
þ
7.92 [bs, 3H,
d
ꢃ NH3 (Lys)], 9.16 [bs, 2H,
a
ꢃ NH2 (Lys)], 9.41 (bs,
d
b
-H (Lys)], 2.68 [t, 2H, J ¼ 7 Hz, 2-H
1H, NH-Bn); 13C NMR (100 MHz, DMSO-d6)
d
21.9 (CH2, Cg), 26.9
(ethyl)], 2.72 [m, 2H, ε-H (Lys)], 2.78 [m,1H, 3-H (Pr)], 2.97 [m,1H,1-H
(CH2, Cd), 29.5 (CH2, Cb), 35.0 (CH2, C3), 38.3 (CH2, Bn), 38.8 (CH2, Cε),
43.1 (CH2, C1), 58.5 (CH, C2), 59.0 (CH, Ca), 116.5e141.5 (13CH and
5C, aromatics),156.2 (HNCONH),167.9 (CONH); ES-MS m/z [M þ 1]þ
calcd for C29H35Cl2N5O2, 556.22; found, 556.63 (100%), 558.52
(64%) [M þ 3]þ, 560.42 (10%) [M þ 5]þ.
(Pr)], 3.04 [m, 1H, 3-H (Pr)], 3.15 [m, 1H, 2-H (Pr)], 3.18 [m, 1H, 1-H
(Pr)], 3.22 [t, 2H, J ¼ 7 Hz, 1-H (ethyl)], 4.23 [m, 1H,
a-H (Lys)], 4.36
[d, 2H, J¼ 6Hz, CH2 (Bn)], 6.63and6.82(2bs, 2H,HNCONH), 7.10e7.34
(m,14H, aromatics), 8.07 [bs, 3H,
d
ꢃ NH3þ (Lys)], 9.02 and 9.91 [2bs,
2H,
a
ꢃ NH2þ (Lys)], 9.48 (bs, 1H, NH-Bn); 13C NMR (75 MHz, DMSO-
d6) d 20.1 (CH2, Cg), 26.7 (CH2, Cd), 31.8.0 (CH2, Cb), 35.2 (CH2, C3), 35.5
4.5.12. Na-[(2R)-1-(3-(4-Methoxyphenethyl)ureido)-3-
phenylpropan-2-yl]-lysine benzyl amide dihydrochloride [(R)-20b]
[CH2, C2 (ethyl)], 38.7 [CH2, C1 (ethyl)], 39.1 (CH2, Cε), 40.6 (CH2, Bn),
43.0 (CH2, C1), 58.9 (CH, C2), 60.4 (CH, Ca),115.4e160.3 (14CH and 4C,
aromatics), 162.2 (HNCONH), 167.9 (CONH); ES-MS m/z [M þ 1]þ
calcd for C31H40FN5O2, 534.32; found, 534.65 (100%).
Amorphous solid (61 mg, 100%); HPLC tR 13.46 min; [
(c 0.8, MeOH); 1H NMR (500 MHz, DMSO-d6)
H (Lys)], 1.39e1.64 [m, 2H, -H (Lys)], 1.74e1.95 [m, 2H,
a
]
20 þ15.6
D
d
1.27e1.39 [m, 2H, g-
d
b-H (Lys)],
2.68e2.79 [m, 1H, 3-H (Pr)], 2.63 [t, 2H, J ¼ 7 Hz, 2-H (ethyl)], 2.71
4.6. Synthesis of the indazole-containing protected ureas (R)- and
(S)-24b
[m, 2H, ε-H (Lys)], 3.03e3.25 [m, 6H, 1-H, 2-H, 3-H (Pr) and 1-H
(ethyl)], 3.69 (s, 3H, OMe), 4.09 [m, 1H, a-H (Lys)], 4.28 [dd, 1H,
J ¼ 6 and 15 Hz, CH2 (Bn)], 4.47 [dd, 1H, J ¼ 5 and 15 Hz, CH2 (Bn)],
Diethyl amine (0.22 mL, 2.1 mmol) was added to a solution of
the corresponding Fmoc-protected amine (R)- and (S)-12b
(0.21 mmol) and the reaction mixture was stirred at rt for 2 h. Then,
the solvent was evaporated to dryness and the residue was dis-
solved in EtOAc (50 mL). The solution was successively washed with
H2O (20 mL) and brine (20 mL), dried over Na2SO4, and evaporated
to dryness to give the deprotected amines (R)- and (S)-3b, which
6.37 and 6.50 (2bs, 2H, HNCONH), 6.69e7.38 (m, 14H, aromatics),
7.80 [bs, 3H,
d
ꢃ NH3þ (Lys)], 9.25 [bs, 2H,
a
ꢃ NH2þ (Lys)], 9.45 (bs,
1H, NH-Bn); 13C NMR (125 MHz, DMSO-d6)
d 21.6 (CH2, Cg), 27.2
(CH2, Cd), 30.5 (CH2, Cb), 34.3 (CH2, C3), 35.4 [CH2, C2 (ethyl)], 38.0
[CH2, Cε and C1 (ethyl)], 38.0 (CH2, Cε), 38.8 (CH2, Bn), 41.9 (CH2, C1),
55.4 (CH, C2), 55.4 (CH3, OMe), 56.8 (CH, Ca), 114.0e158.0 (14CH and
4C, aromatics), 161.5 (HNCONH), 167.9 (CONH); ES-MS m/z [M þ 1]þ
calcd for C32H43N5O3, 546.34; found, 546.62 (100%).
were reserved. Apart, propylene oxide (118
mL, 1.68 mmol) was
added to a 0 ꢀC cooled solution of 1-(2,6-dichlorobenzyl)-3-(pyr-
rolidin-1-ylmethyl)-1H-indazole-6-amine [50] (22) (79 mg,
0.21 mmol) in dry THF (5 mL). Then, a solution of bis(tri-
chloromethyl)carbonate (20 mg, 0.07 mmol) in dry THF (1 mL) was
added dropwise and stirring was maintained at 0 ꢀC for 15 min.
Afterwards, the mixture was added dropwise to a 0 ꢀC cooled
solution of (R)- and (S)-3b (98 mg, 0.21 mmol) in dry THF (5 mL)
4.5.13. Na-[(2S)-1-(3-(4-Methoxyphenethyl)ureido)-3-
phenylpropan-2-yl]-lysine benzyl amide dihydrochloride [(S)-20b]
Amorphous solid (62 mg,100%); HPLC tR 13.52 min; [
0.7, MeOH); 1H NMR (500 MHz, DMSO-d6)
1.35 [m, 2H,
1.594[m,2H, -H(Lys)],1.83[m,2H,
a
]
20 þ19.7 (c
D
d
g-H (Lys)],
d
b
-H(Lys)], 2.62 [m, 2H,ε-H(Lys)],

P. Ventosa-Andrés et al. / European Journal of Medicinal Chemistry 58 (2012) 98e111
109
and stirred for 1 h. Then, the reaction mixture was diluted with
EtOAc (50 mL), washed successively with H2O (20 mL) and brine
(20 mL), dried over Na2SO4, and evaporated to dryness. The residue
was purified by reversed phase chromatography, using 10e100%
CH3CN gradient in 0.05% TFA solution in H2O as mobile phase.
Then, the solvent was evaporated under reduced pressure, the
residue was dissolved in H2O (3 mL) and the solution was lyophi-
lized to give the urea (S)-25b (131 mg, 100%). HPLC tR 13.11 min;
HPLC-MS tR 2.73 min; [
DMSO-d6)
1.74 [m, 2H,
a]
20 þ1.6 (c 1.2, MeOH); 1H NMR (500 MHz,
D
d
(ppm): 1.32 [m, 2H, g-H (Lys)], 1.53 [m, 2H, d-H (Lys)],
b-H (Lys)], 1.77e1.95 [m, 4H, 2CH2 (pyrrolidine)], 2.66
4.6.1. Nε-Boc-Na-[(2R)-1-(3-(1-(2,6-dichlorobenzyl)-3-(pyrrolidin-
1-ylmethyl)-1H-indazol-6-yl)ureido)-3-phenylpropan-2-yl]-lysine
benzyl amide [(R)-24b]
[m, 2H, ε-H (Lys)], 2.84 [dd, 1H, J ¼ 10 and 13 Hz, 3-H (Pr)], 3.04 [m,
4H, 2CH2 (pyrrolidine)], 3.08e3.16 [m, 1H, 3-H (Pr)], 3.12 [m, 1H, 2-
H (Pr)], 3.19e3.59 [m, 2H, 1-H (Pr)], 4.14 [m, 1H, a-H (Lys)], 4.32 [t,
Foam (36 mg, 20%); HPLC tR 16.33 min; HPLC-MS tR 3.26 min;
2H, J ¼ 7 Hz, CH2 (Bn)], 4.54 (s, 2H, CH2epyrrolidine), 5.57 (s, 2H,
[
a
]
20 þ5.6 (c 1, MeOH); 1H NMR (400 MHz, CDCl3)
d
1.02e1.46 [m,
CH2-diClPh), 7.02e7.95 (m, 16H, aromatics), 8.01 and 9.66 (2s, 2H,
D
þ
4H,
g- and
d-H (Lys)], 1.31 (s, 9H, Boc), 1.69, 1.85, 3.02, 3.45 (4m, 8H,
HNCONH), 9.08 (s, 1H, NH-Bn), 9.43 and 10.58 [2bs, 2H,
a
ꢃ NH2
pyrrolidine), 1.75e1.94 [m, 2H,
(Lys)], 2.74e3.07 [m, 2H, 3-H (Pr)], 3.27 [m, 1H, 2-H (Pr)], 3.21e3.43
[m, 2H, 1-H (Pr)], 3.93 [bs, 1H,
-H (Lys)], 4.29 [d, J ¼ 7 Hz, 2H, CH2
b
-H (Lys)], 2.74e2.92 [m, 2H, ε-H
(Lys)]. 13C NMR (125 MHz, DMSO-d6)
d
(ppm): 21.4 (CH2, Cg), 22.6
(2CH2, pyrrolidine), 26.4 (CH2, Cd), 29.7 (CH2, Cb), 35.1 (CH2, C3),
38.2 (CH2, Cε), 38.3 (CH2, C1), 42.6 (CH2, Bn), 47.4 (CH2, diClPh), 47.7
(CH2, CH2epyrrolidine), 52.7 (2CH2, pyrrolidine), 58.2 (CH, Ca), 58.8
(CH, C2), 98.4 [C7 (indazole)], 114.6 [C5 (indazole)], 117.8 [C4 (inda-
zole)], 120.6 [C3a (indazole)], 127.1e131.4 (CH, aromatics), 135.5 (C,
Bn), 136.1 (C, diClPh), 136.2 (C, Ph), 138.2 [C5 (indazole)], 138.4 (2C,
diClPh), 139.7 [C6 (indazole)], 141.3 [C7a (indazole)], 156.1
a
(Bn)], 4.36 (s, 2H, CH2epyrrolidine), 4.64 (s, 1H, NH-Boc), 5.52 (s,
2H, CH2-diClPh), 6.94 [d, J ¼ 7 Hz,1H, 5-H (indazole)], 7.36e7.00 (m,
13H, Ph), 7.41 [d, J ¼ 7 Hz, 1H, 4-H (indazole)], 7.87 [s, 1H, 7-H
(indazole)], 8.12 (s, 1H, NH-Bn), 8.72 and 11.8 (2bs, 2H, HNCONH);
13C NMR (100 MHz, CDCl3) 21.7 (CH2, Cg), 23.4 (CH2, pyrrolidine),
28.3 (CH3, Boc), 29.1 (CH2, Cd), 29.5 (CH2, Cb), 34.9 (CH2, C3), 40.8
(CH2, C1), 43.8 (CH2, Bn), 47.7 (CH2-diClPh), 48.1, 52.3 (CH2, pyrro-
lidine and CH2epyrrolidine), 59.3 (CH, Ca), 60.2 (CH, C2), 79.5 (C,
Boc), 98.8 [C7 (indazole)], 116.3[C5 (indazole)], 119.2 [C4 (indazole)],
127.6e129.2 (CH, Ph), 130.1 (C, Ph), 131.2 (C, diClPh), 133.7 [C5
(indazole)], 136.8 (C, Bn), 137.3 (2C, diClPh), 138.8 [C6 (indazole)],
141.6 [C7a (indazole)], 157.4 2 (HNCONH), 162.2 (CO, Boc), 167.2
(CONH); ES-MS m/z [M þ 1]þ calcd. for C47H58Cl2N8O4, 869.40;
found, 435.29 (100%) [(M þ 2)/2]þ, 869.49 (6%) [M þ 1]þ. Anal.
calcd. for C47H58Cl2N8O4: C, 64.89; H, 6.72; N, 12.88. Found: C,
64.95; H, 6.82; N, 12.95.
(HNCONH), 167.3 (CONH); ES-MS m/z [M
þ
1]þ calcd for
C
42H50Cl2N8O2, 769.34; found, 385.28 (60%) [(M þ 2)/2]þ, 769.37
(6%) [M þ 1]þ.
4.7. General procedure for the synthesis of thioureas (R)- and (S)-
26a
Diethyl amine (0.32 mL, 3 mmol) was added to a solution of the
corresponding Fmoc-protected amine (R)- and (S)-12a (0.3 mmol)
and the reaction mixture was stirred at rt for 2 h. Then, the solvent
was evaporated to dryness and the residue was dissolved in EtOAc
(50 mL). The solution was successively washed with H2O (20 mL)
and brine (20 mL), dried over Na2SO4, and evaporated to dryness to
give the deprotected amines (R)- and (S)-3a. These amines were
dissolved in dry CH2Cl2 (10 mL) and the solution was cooled at 0 ꢀC.
Phenyl isothiocyanate (40.5 mg, 0.3 mmol) was added and the
mixture was stirred for 2 h. Then, the solvent was evaporated and
the residue was dissolved in EtOAc (50 mL). This solution was
successively washed with H2O (20 mL) and brine (20 mL), dried
over Na2SO4, and evaporated to dryness. The residue was purified
by flash chromatography, using 20e100% EtOAc gradient in hexane
as eluant to give the corresponding thiourea (R)- and (S)-26a.
4.6.2. Nε-Boc-Na-[(2S)-1-(3-(1-(2,6-dichlorobenzyl)-3-(pyrrolidin-
1-ylmethyl)-1H-indazol-6-yl)ureido)-3-phenylpropan-2-yl]-lysine
benzyl amide [(S)-24b]
Foam (155 mg, 85%); HPLC tR 16.51 min; HPLC-MS tR 3.26 min;
[
a]
20 þ8.4 (c 1, MeOH); 1H NMR (500 MHz, CDCl3)
d 1.08e1.42 [m,
D
4H, g- and d-H (Lys)], 1.29 (s, 9H, Boc), 1.70, 1.87, 2.98, 3.45 (4m, 8H,
pyrrolidine), 1.76e1.83 [m, 2H,
(Lys)], 2.74e3.07 [m, 2H, 3-H (Pr)], 3.23 [m, 1H, 2-H (Pr)], 3.18e3.31
[m, 1H, 1-H (Pr)], 3.55 [m, 1H, 1-H (Pr)], 4.25 [bs, 1H, -H (Lys)], 4.30
b-H (Lys)], 2.72e2.86 [m, 2H, ε-H
a
[dd, J ¼ 5 and 15 Hz, 2H, CH2 (Bn)], 4.36 (s, 2H, CH2epyrrolidine),
4.70 (s, 1H, NH-Boc), 5.50 (s, 2H, CH2-diClPh), 6.99 [d, J ¼ 7 Hz,
1H, 5-H (indazole)], 7.09e7.34 (m,13H, Ph), 7.43 [d, J ¼ 7 Hz,1H, 4-H
(indazole)], 7.80 [s, 1H, 7-H (indazole)], 9.18 (s, 1H, NH-Bn), 8.92 and
4.7.1. Nd-Boc-Na-[(2R)-3-phenyl-1-(3-phenylthioureido)propan-2-
yl]-ornithine benzyl amide [(R)-26a]
11.78 (2bs, 2H, HNCONH); 13C NMR (125 MHz, CDCl3)
d
21.9 (CH2,
Foam (121 mg, 68%); HPLC tR 18.42 min; [
1H NMR (500 MHz, CDCl3)
and -H (Orn)], 2.71e2.91 [m, 3H, 3-H (Pr) and
a
]
D
20 þ3.0 (c 1, MeOH);
Cg), 23.6 (CH2, pyrrolidine), 28.5 (CH3, Boc), 29.3 (CH2, Cd), 30.7
(CH2, Cb), 30.7 (CH2, Cb), 36.6 (CH2, C3), 39.7 (CH2, Cε), 40.3 (CH2, C1),
44.2 (CH2, Bn), 48.0 (CH2-diClPh), 48.6 (CH2epyrrolidine), 52.8
(CH2, pyrrolidine), 60.6 (CH, Ca), 61.5 (CH, C2), 79.2 (C, Boc), 98.8 [C7
(indazole)], 116.4 [C5 (indazole)], 119.3 [C4 (indazole)], 119.8 [C3a
(indazole)], 167.6 (CONH), 127.8e129.5 (CH, Ph), 130.5 (C, Ph), 131.6
(C, diClPh), 133.9 [C5 (indazole)], 134.3 (C, Bn), 137.0 (2C, diClPh),
138.8 [C6 (indazole)], 141.7 [C7a (indazole)], 158.5 (C, HNCONH),
162.1 (CO, Boc), 167.6 (CONH); ES-MS m/z [M þ 1]þ calcd. for
d 1.38 (m, 9H, Boc), 1.10e1.65 [m, 4H, b-H
g
d-H (Orn)], 2.97 [dd,
1H, J ¼ 7.5 and 13 Hz, 3-H (Pr)], 3.09e3.30 [m, 2H,
a
-H (Orn) and 2-
H (Pr)], 3.63 [d, 2H, J ¼ 5 Hz, 1-H (Pr)], 4.21 [dd, J ¼ 6.5 and 15 Hz,
1H, CH2 (Bn)], 4.43 [dd, 1H, J ¼ 5 and 15 Hz, CH2 (Bn)], 4.53 (s, 1H,
NH-Boc), 6.79 and 7.85 (2s, 2H, HnCSNH), 7.04e7.63 (m, 16H,
aromatics and NH-Bn); 13C NMR (125 MHz, CDCl3)
d 25.9 (CH2, Cg),
28.3 (CH3, Boc), 30.5 (CH2, Cb), 39.4 (CH2, Cd), 43.2 (CH2, Bn), 58.1
(CH, Ca), 59.7 (CH, C2), 79.3 (C, Boc), 125.0e138.3 (15CH and 3 C,
aromatics), 156.3 (CO, Boc), 174.9 (CONH), 181.2 (CS); ES-MS m/z
[M þ 1]þ calcd. for C33H44N5O3S, 590.31; found, 590.51 (100%).
Anal. calcd. for C33H43N5O3S: C, 67.20; H, 7.35; N, 11.87. Found: C,
67.24; H, 7.55; N, 11.93.
C
47H58Cl2N8O4, 869.40; found, 435.29 (100%) [(M þ 2)/2]þ, 869.49
(5%) [M þ 1]þ; Anal. calcd. for C47H58Cl2N8O4: C, 64.89; H, 6.72; N,
12.88. Found: C, 65.02; H, 6.85; N, 12.92.
4.6.3. N-Boc deprotection of (S)-24b. Synthesis of Na-[(2S)-1-(3-(1-
(2,6-dichlorobenzyl)-3-(pyrrolidin-1-ylmethyl)-1H-indazol-6-yl)
ureido)-3-phenylpropan-2-yl]-lysine benzyl amide trihydrochloride
[(S)-25b]
The protected urea (S)-24b (130 mg, 0.15 mmol) was dissolved
in a 3 M solution of HCl in EtOAc (2 mL) and stirred at rt for 2 h.
4.7.2. Nd-Boc-Na-[(2S)-3-phenyl-1-(3-phenylthioureido)propan-2-
yl]-ornithine benzyl amide [(S)-26a]
Foam (127 mg, 72%); HPLC tR 18.67 min; [
1H NMR (500 MHz, CDCl3)
and
a
]
20 þ7.5 (c 1.1, MeOH);
D
d 1.38 (s, 9H, Boc), 1.23e1.90 [m, 4H, b-H
g
-H (Orn)], 2.87e3.17 [m, 6H, 1- and 3-H (Pr), and d-H (Orn)],

110
P. Ventosa-Andrés et al. / European Journal of Medicinal Chemistry 58 (2012) 98e111
3.40 [m, 1H, 2-H (Pr)], 3.62 [m, 1H,
a
-H (Orn)], 4.10 [dd, J ¼ 7 and
aggregation value at the end of 5 min was recorded. Aggregation
15 Hz, 1H, CH2 (Bn)], 4.38 [dd, J ¼ 5 and 15 Hz, 1H, CH2 (Bn)], 4.71 (s,
response to SFLLRN plus compound was recorded and compared to
control (SFLLRN/vehicle) to determine the % of inhibition. Each
compound was tested twice and the results are the mean of the two
assays.
1H, NH-Boc), 6.69 and 8.05 (2s, 2H, HNCSNH), 7.02e7.54 (m, 16H,
aromatics and NH-Bn); 13C NMR (125 MHz, CDCl3)
d 26.2 (CH2, Cg),
28.4 (CH3, Boc), 29.3 (CH2, Cb), 39.5 (CH2, Cd), 40.5 (CH2, C3), 43.1
(CH2, Bn), 58.3 (CH, Ca), 59.3 (CH, C2), 79.5 (C, Boc), 124.5e138.4
(15CH and 3C, aromatics), 156.4 (CO, Boc), 173.4 (CONH), 181.3
(CS); ES-MS m/z [M þ 1]þ calcd. for C33H43N5O3S, 590.31; found,
590.58 (100%). Anal. calcd. for C33H43N5O3S: C, 67.20; H, 7.35; N,
11.87. Found: C, 67.15; H, 7.45; N, 11.86.
Acknowledgements
This work was supported by the Spanish Ministerio de Ciencia e
Innovación grant SAF2009-09323. P.V.-A. held a FPI fellowship from
the Ministerio de Ciencia e Innovación.
4.8. N-Boc-deprotection of (R)- and (S)-26a. Synthesis of the
thiourea hydrochlorides (R)- and (S)-27a
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It was carried out by applying the above described methodology
for N-Boc removal in the urea analogues.
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4.8.1. Na-[(2R)-3-Phenyl-1-(3-phenylthioureido)propan-2-yl]-
ornithine benzyl amide dihydrochloride [(R)-27a]
Amorphous solid (56 mg, 100%); HPLC tR 13.29 min; [
(c 1, MeOH); 1H NMR (500 MHz, DMSO-d6)
(Orn)],1.78e2.06 [m, 2H,
-H (Orn)], 3.46e3.66 [m, 2H, 1-H (Pr)], 3.80 [m, 1H, 2-H (Pr)], 4.25
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1.50e1.76 [m, 2H, g-H
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d
[dd, 1H, J ¼ 6 and 15 Hz, CH2(Bn)], 4.37 [m, 1H,
a
1H, J ¼ 5 and 15 Hz, CH2(Bn)], 7.10 (s, 1H, HNCSNH), 6.93e7.55 (m,
þ
16H, aromatics and HNCSNH), 8.05 [bs, 3H,
d
ꢃ NH3 (Orn)], 8.75
þ
and 10.22 [2bs, 2H,
a
ꢃ NH2 (Orn)], 9.41 (s, 1H, NH-Bn); 13C NMR
(125 MHz, DMSO-d6)
d
22.8 (CH2, Cg), 27.1 (CH2, Cb), 28.2 (CH2, C3),
34.1 (CH2,Cd), 38.1 (CH2, Bn), 42.7 (CH2, C1), 57.0 (CH, C2), 58.3 (CH,
Ca),117.7e139.9 (15CH and 3C, aromatics), 166.9 (CONH), 181.0 (CS);
ES-MS m/z [M þ 1]þ calcd. for C28H35N5OS, 490.26; found
246.13(100%) [(M þ 2)/2]þ, 490.48 (15%) [M þ 1]þ.
4.8.2. Na-[(2S)-3-Phenyl-1-(3-phenylthioureido)propan-2-yl]-
ornithine benzyl amide dihydrochloride [(S)-27a]
Amorphous solid (54 mg, 100%); HPLC tR 13.40 min; [
0.7, MeOH); 1H NMR (500 MHz, DMSO-d6)
and -H (Orn)], 2.73e3.22 [m, 5H, 2- and 3-H (Pr), and
3.52e3.60 [m, 2H, 1-H (Pr)], 4.29 [m, 1H, -H (Orn)], 4.40 [d, 2H,
a
]
20 þ2.9 (c
D
d
1.51e2.06 [m, 4H, b-
g
d-H (Orn)],
a
J ¼ 6 Hz, CH2 (Bn)], 7.10 (s, 1H, HNCSNH), 6.69e7.71 (m, 15H,
þ
þ
aromatics and HNCSNH), 7.94 [bs, 3H,
d
a
ꢃ NH3 (Orn)], 9.42 (s, 1H,
NH-Bn), 9.19 and 9.54 [2bs, 2H,
(125 MHz, DMSO-d6)
ꢃ NH2 (Orn)]; 13C NMR
d
22.7 (CH2, Cg), 27.1 (CH2, Cb), 31.6 (CH2, C3),
38.3 (CH2,Cd), 38.9 (CH2, Bn), 42.8 (CH2, C1), 57.0 (CH, C2), 58.8 (CH,
Ca), 123.4e138.7 (15CH and 3C, aromatics), 166.9 (CONH), 181.7
(CS); ES-MS m/z [M þ 1]þ calcd. for C28H35N5OS, 490.26; found
246.13 (100%) [(M þ 2)/2]þ, 490.48 (15%) [M þ 1]þ.
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