Synthesis and antiproteasomal activity of novel O-benzyl salicylamide-based inhibitors built from leucine and phenylalanine

Article abstract of DOI:10.1016/j.ejmech.2017.04.027

Inhibition of protein degradation is one of strategies for suppression of uncontrolled proliferation of cancer cells. Proteolytic degradation in cells is mainly ensured by proteasome and its inhibition by bortezomib showed benefit in clinical use for the treatment of multiple myeloma. We report here the library of antiproteasomal O-benzyl salicylamides built from leucine and phenylalanine. Prepared compounds displayed antiproliferative activity on K562, CEM and U266 cancer cell lines, ranging from high micromolar to submicromolar GI₅₀ values. The most potent compounds (series 4 and 6) were further assayed for their inhibition of chymotrypsin-like protease activity of the 26S proteasome in U266 cells. The majority of compounds inhibited the proteasome in mid-nanomolar concentrations (IC₅₀ ranging from 57 to 197 nM) and it correlated with cellular potency. In a cell based assay involving green fluorescence protein (GFP) fused to a short degron that is rapidly degraded by a proteasome the compounds induced accumulation of GFP, visualised and quantified by live-cell imaging. Levels of polyubiquitinated proteins in U266 cells treated by compound 4m were also analyzed by immunoblotting, revealing a typical high molecular mass smear of ubiquitin conjugates. Finally, apoptotic cell death in treated U266 cells was detected biochemically by measuring the activity of caspases 3 and 7 in lysates and by immunoblotting of caspase 7, its substrate poly(ADP-ribose)polymerase, and Mcl-1, which all together showed changes typical for apoptosis. All these observations were in agreement with expected cellular mechanism of action and confirmed proteasome targeting by prepared O-benzyl salicylamides.

Full text of DOI:10.1016/j.ejmech.2017.04.027

European Journal of Medicinal Chemistry 135 (2017) 142e158
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Synthesis and antiproteasomal activity of novel O-benzyl
salicylamide-based inhibitors built from leucine and phenylalanine
a
b
b
b
ꢁ a
a, *
ꢀ
ꢀ
ꢀ
Radek Jorda , Jan Dusek , Eva Rezníckova , Karel Pauk , Pratibha P. Magar ,
b,
**
ꢀ
ꢀ
Ales Imramovský
, Vladimír Krystof
a
ꢁ
Laboratory of Growth Regulators, Centre of the Region Hana for Biotechnological and Agricultural Research, Palacký University & Institute of Experimental
ꢀ
ꢂ
Botany ASCR, Slechtitelu 27, 78371 Olomouc, Czech Republic
b
ꢁ
Institute of Organic Chemistry and Technology, Faculty of Chemical Technology, University of Pardubice, Studentska 573, 532 10 Pardubice, Czech Republic
a r t i c l e i n f o
a b s t r a c t
Article history:
Inhibition of protein degradation is one of strategies for suppression of uncontrolled proliferation of
cancer cells. Proteolytic degradation in cells is mainly ensured by proteasome and its inhibition by
bortezomib showed benefit in clinical use for the treatment of multiple myeloma. We report here the
library of antiproteasomal O-benzyl salicylamides built from leucine and phenylalanine. Prepared
compounds displayed antiproliferative activity on K562, CEM and U266 cancer cell lines, ranging from
high micromolar to submicromolar GI50 values. The most potent compounds (series 4 and 6) were
further assayed for their inhibition of chymotrypsin-like protease activity of the 26S proteasome in
U266 cells. The majority of compounds inhibited the proteasome in mid-nanomolar concentrations (IC50
ranging from 57 to 197 nM) and it correlated with cellular potency. In a cell based assay involving green
fluorescence protein (GFP) fused to a short degron that is rapidly degraded by a proteasome the com-
pounds induced accumulation of GFP, visualised and quantified by live-cell imaging. Levels of poly-
ubiquitinated proteins in U266 cells treated by compound 4m were also analyzed by immunoblotting,
revealing a typical high molecular mass smear of ubiquitin conjugates. Finally, apoptotic cell death in
treated U266 cells was detected biochemically by measuring the activity of caspases 3 and 7 in lysates
and by immunoblotting of caspase 7, its substrate poly(ADP-ribose)polymerase, and Mcl-1, which all
together showed changes typical for apoptosis. All these observations were in agreement with expected
cellular mechanism of action and confirmed proteasome targeting by prepared O-benzyl salicylamides.
© 2017 Elsevier Masson SAS. All rights reserved.
Received 23 January 2017
Received in revised form
7 April 2017
Accepted 9 April 2017
Available online 15 April 2017
Keywords:
Proteasome
Inhibitor
Apoptosis
Ubiquitin
Salicylamide
1. Introduction
treatment.
The proteasome is a multi-subunit protein complex composed
Cancer is associated with deregulation or dysfunction of many
cellular processes that have been validated successfully as targets of
modern therapeutics [1,2]. One of these processes relates to protein
stability and degradation. In cells, more than 80% of proteins are
degraded by a large protein complex known as a proteasome [3].
Inhibition of its proteolytic function has been shown to affect
numerous different processes and pathways, such as cell cycle
progression, cell migration, cell adhesion, viability and apoptosis
of one 20S cylindrical catalytic core and two 19S regulatory sub-
units [5]. The catalytic part of the proteasome contains several
type subunits. Three of them, namely 1, 2, and 5 are responsible
b
-
b
b
b
for the caspase-like, trypsin-like and chymotrypsin-like proteolytic
activities of proteasomes, signifying the preference for client pep-
tide. While the chymotrypsin-like activity is responsible for the
cleavage of peptide bonds after hydrophobic amino acid residues,
the trypsin-like activity causes cleavage after basic residues and the
caspase-like activity after acidic residues of amino acids [13].
Proteasomal inhibitors (Fig. 1) can be divided into distinct
chemical groups; most of them target the active site by mimicking a
substrate and interacting with catalytic Thr1 of the proteasome.
While peptide aldehydes (e.g. fellutamide B, MG132) are rapidly
oxidised in cells and not so suitable for therapeutic use, peptide
boronates (e.g. bortezomib, ixazomib, delanzomib), forming
[4], and therefore it offers
a promising strategy for cancer
* Corresponding author (biochemistry).
** Corresponding author (chemistry).
E-mail addresses: Ales.Imramovsky@upce.cz (A. Imramovský), vladimir.krystof@
ꢀ
upol.cz (V. Krystof).
http://dx.doi.org/10.1016/j.ejmech.2017.04.027
0223-5234/© 2017 Elsevier Masson SAS. All rights reserved.

R. Jorda et al. / European Journal of Medicinal Chemistry 135 (2017) 142e158
143
pseudo covalent adducts with Thr1, display improved bioavail-
2. Results and discussion
ability and excellent potency towards the proteasome. Next, pep-
tide epoxyketones (e.g. carfilzomib, oprozomib) are characterised
by nearly no off-target effects, and the group of peptide vinyl
sulphonates by better specificity for trypsin-like active sites. In
addition, there are several groups of natural proteasome inhibitors
2.1. Chemistry
The synthesis of the starting compound was based on the
repetition of a versatile two-step synthetic methodology starting
from O-benzyl-5-chloro-salicylic acid [18] (Scheme 1). First was the
condensation of acid 1 with chosen amino acid methyl ester to
produce methyl ester 2. In comparison with the original approach
[15] liberation of free base was not carried out in situ but externally.
This procedure minimises racemisation during the reaction. The
second step consists of basic hydrolysis of the ester 2, to obtain acid
1 with one amino acid. This reaction cycle can be repeated; in this
way esters 2 as well as acids 1 with various numbers of amino acid
units were synthesised and characterised (Scheme 1).
Esters 2 and acids 1 are starting materials for further modifi-
cation. Series of the esters 2 (containing one or two amino acids
(described previously) or three amino acids 2a-d) were synthesised
and characterised as original organic compounds. Hydrolysis of
prepared esters 2aed led to appropriate acids 1 (containing one 1a,
b or two 1ceh eventually three 3i, j amino acids). Both esters and
acids are versatile intermediates for further modification and syn-
thesis of targeted compounds. As our initial thoughts were also to
prepare compounds with an aldehyde functional moiety (Fig. 1) the
first approach to obtain these compounds was partial reduction of
esters 2 using DIBAL [19]. The non-selectivity of this process
encouraged the use of an alternative methodology. The preparation
of targeted aldehydes began from methyl esters 2, which upon
treatment with lithium bis(trimethylsilyl)amide (LiHMDS) and N,O-
dimethylhydroxylamine hydrochloride were converted to the cor-
responding N-methyl-O-methyl amides 3ael in good to excellent
yield [20]. It was also found that alternative synthesis using i-Pro-
MgCl was not suitable for our compounds due to slow conversion as
well as low selectivity. Problematic separation of desired products
was another disadvantage of this technique [21]. N-methyl-O-
methyl amides are generally known compounds and they serve as
versatile intermediates [22,23].
e.g. b-lactones (marizomib) [5e8] and other compounds carrying a
non-peptide scaffold [9,10].
Bortezomib (Velcade®, PS-341, Fig. 1) is a dipeptide boronic acid
that reversibly binds mainly to the chymotrypsin-like active site of
the b5 subunit of the proteasome. It exhibits nanomolar potency;
therefore, bortezomib has been approved since 2003 for the
treatment of multiple myeloma and mantle cell lymphoma [7,11].
Later, proteasome inhibition by bortezomib has been validated as
suitable treatment for other malignancies, but like other drugs,
bortezomib has several limitations and produces side-effects in
patients [12]. Thus, there is a demand for the development of new
generations of proteasome inhibitors.
Inspired by the success of bortezomib (Velcade®), a second
generation of promising proteasome inhibitors are currently being
evaluated in clinical trials, namely marizomib (salinosporamide A),
oprozomib (ONX 0912) or delanzomib (CEP-18770) and recently
carfilzomib (PR-171, Kyprolis®) and ixazomib (MLN9708, Ninlaro®)
have been approved for the treatment of patients with relapsed or
refractory multiple myeloma [13].
Our previous study showed that the 2-hydroxy-N-(arylalkyl)
benzamides induce apoptosis in cancer cell lines [14]. Their further
modification yielded O-benzyl salicylamide dipeptides with
aliphatic endings, which also displayed moderate activity [15]. Our
intention, to prepare compounds based on substituted salicylic acid
linked to a short peptide chain by an amidic bond, has remained.
We designed, synthesised and fully characterised a new series of
compounds, consisting a short dipeptide/tripeptide moiety bonded
to O-benzyl salicylic acid on the one site of molecule and carrying
various functional groups, including aldehyde, that has been found
to be highly promising in several recent studies [16,17].
Weinreb amides 3ael were transformed using LiAlH4 to obtain
Fig. 1. Example of known inhibitors of proteasome with different reactive groups.

144
R. Jorda et al. / European Journal of Medicinal Chemistry 135 (2017) 142e158
Scheme 1. General reaction scheme for synthesis of targeted compounds. Integrated L- or D-leucine (R1 ¼ i-pro) and L- or D-phenylalanine (R1 ¼ Ph) in various combinations.
Conditions: a ¼ EDCI$HCl (0.95 eq), HOBt (1 eq), RT, CH2Cl2, 2 h; b ¼ LiOH (3 eq), H2O:1,4-dioxane (vol. 1:1), RT, 2 h; c ¼ LiHMDS (1 M in THF, 7 eq), dry THF, CH3ONHCH3$HCl (2.5
eq), inert atm., 1 h ꢀ20 ꢁC, then 3 h RT; d ¼ LiAlH4 (3 eq), dry THF, 3 h ꢀ20 ꢁC, then 1 h RT; e ¼ 10% Pd/H2, EtOAc, RT, overnight.
O-benzyl protected aldehydes 4aep in moderate to good yields. In
cases where we wanted to synthesise aldehydes without benzyl
group 6, deprotection of 3 was first performed, and deprotected
Weinreb amides 5aeh were transformed to the corresponding al-
dehydes 6aec using LiAlH4 solution. Under these reaction condi-
tions two examples of alcohols 7 were also isolated as products of
the reduction process (Scheme 1).
the supplementary information to this paper.
2.2. Antiproliferative activity of novel salicylamides
In our previous studies [14,15] we described two major groups
of substituted salicylamides with dipeptide chains that showed
moderate antiproliferative activity against the panel of cancer cell
lines. The first study on chloro-substituted salicylamides (alter-
nately 2-hydroxy-N-(arylalkyl)benzamides) documented increased
cytotoxicity of derivatives bearing halogenated mono-substitution
at the terminal end of the molecule. In the following study of O-
benzyl substituted salicylamides we investigated the presence of
further terminal functional groups, namely esters, carboxylic acids
or allyl, isobutyl, isopropyl or n-propyl groups. All substitutions
mentioned seemed to display mid-micromolar antiproliferative
activities.
An alternative approach to synthesis of 3 was carried out by
reaction of the 5-chloro-O-benzyl salicylic acid 1 and L-leucine or L-
phenylalanine with preformed Weinreb amides of selected amino
acids. This approach seems to be more universal than the procedure
described in previous paragraphs. However, yields of the prepared
compounds and their purity have been in many cases lower than
those from the reaction sequence using the sequential building of
the target molecules. The final products were structurally identical
to compounds obtained by the above-mentioned process. The re-
sults of biological testing were also identical. Because of the above
disadvantages this procedure has not been applied to the synthesis
of other derivatives. The general scheme, experimental procedures
and spectral characterization of original intermediates are listed in
To follow our recent studies we synthesised and fully charac-
terised a new series of O-benzyl salicylamides having dipeptide or
tripeptide chain containing optically pure leucines or phenylala-
nines (or a combination) and terminated with various functional

R. Jorda et al. / European Journal of Medicinal Chemistry 135 (2017) 142e158
145
groups, namely esters (series 2), Weinreb amides (series 3) and
aldehydes (series 4). The library was also complemented by a
number of unsubstituted derivatives (series 5e7). All novel salicy-
lamides were tested for their antiproliferative properties against
various cancer cell lines derived from hematopoietic malignancies
(K562, CEM, U266) and the resulting data are presented in Table 1.
Our results show that esters (series 2) and Weinreb amides
(series 3 and 5) have retained GI50 values in mid-micromolar range.
While we did not observe significant changes in GI50 values with
increasing length of peptide chain for all O-benzyl derivatives, the
antiproliferative activity of unsubstituted Weinreb amides 5g, h
having three integrated amino acids was higher (GI50 ranging from
The U266 cell line was shown to be the most sensitive to the tested
compounds and GI50 value determined for the most active deriv-
ative 4m was 0.15 mM.
Results suggested that the length of peptide chain of aldehyde-
based salicylamides is important for cellular activity, but we also
studied how the type of integrated amino acids affects anti-
proliferative properties of compounds. Analysis of data for active
aldehydes (4h-o) on U266 cells showed that salicylamides bearing
phenylalanine positioned next to the aldehyde group showed
higher GI50 than the corresponding compounds having L-leucine at
this position (compare GI50 (U266) for 4n, o with 4k-m or 4j with 4g-
i). The same trend was observed for the pair of unsubstituted sal-
icylamides 6b and 6c.
16.4 to 32.9
mM) than for di- or mono-peptide based amides 5aef
(GI50 ranging from 48.2 to 100.0
mM). Reasonable cellular activities
were observed for diamides bearing terminal aldehyde group (se-
ries 4 and 6). All aldehydes having tripeptide chain displayed GI50
values reaching mid-nanomolar values in comparison to dipeptide
derivatives. This trend was similar for unsubstituted derivatives 6.
2.3. In vitro antiproteasomal and apoptotic activities of novel
salicylamides
The structural analogy of prepared salicylamides with known
Table 1
Antiproliferative and antiproteasomal activities of prepared salicylamides.
Series
Cmpd
n
Integrated amino acid/s
GI50
(
m
M)a
Proteasomal inhibition
IC50 (m
M)a
K562
CEM
U266
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
6
6
6
7
7
a
b
c
d
e
f
a
b
c
d
e
f
g
h
i
j
k
l
a
b
c
d
e
f
g
h
i
3
3
3
3
3
3
1
1
2
2
2
2
3
3
3
3
3
3
1
1
2
2
2
2
3
3
3
3
3
3
3
3
3
1
1
2
2
2
2
3
3
2
3
3
3
3
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
-Leu-
-Leu-
-Leu-
-Leu-
-Leu-
-Leu-
-Leu
-Phe
-Leu-
-Leu-
L
L
L
L
L
L
-Leu-
-Leu-
-Leu-
-Phe-
-Phe-
-Phe-
L
-Leu
-Leu
-Phe
-Leu
-Leu
-Phe
87.5
>100
70.3
58.5
>100
72.0
42.7
50.9
33.2
22.5
25.8
29.6
55.6
34.0
44.2
16.4
28.9
34.7
23.6
33.3
21.0
16.3
17.3
27.1
0.61
0.76
0.98
1.05
1.04
0.73
0.57
1.45
1.28
>100
99.1
98.1
93.6
90.9
79.5
30.9
22.2
57.9
2.10
6.90
22.4
27.3
0.007
48.4
51.0
31.4
48.5
68.2
48.0
45.2
65.2
22.2
19.5
32.2
28.6
30.6
11.5
15.1
19.0
17.6
35.7
24.3
17.7
16.3
11.0
15.1
15.5
0.49
0.41
0.58
0.79
0.43
0.40
0.28
0.44
0.40
58.6
55.5
63.0
48.2
64.1
54.2
17.2
16.3
27.7
0.78
2.30
8.45
10.8
0.001
84.9
82.0
72.2
83.7
88.5
82.1
54.1
92.3
35.2
81.9
67.4
72.1
55.2
23.7
47.2
48.6
22.6
56.3
55.8
63.8
37.0
14.8
17.7
21.7
0.29
0.20
0.38
0.54
0.17
0.21
0.15
0.40
0.46
>100
72.4
69.0
79.2
71.6
68.7
32.8
23.1
64.9
0.67
2.23
11.8
18.9
0.001
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
>1
D
L
L
D
L
L
-Leu
-Phe
L
-Phe-
-Phe-
D
-Leu
-Phe
L
-Leu-
-Leu-
-Leu-
-Leu-
-Leu-
-Leu-
-Leu
L
-Leu-
-Leu-
-Leu-
L
-Leu
-Leu
-Phe
-Leu
L
L
L
L
L
D
L
-Phe-
-Phe-
-Phe-
L
D
-Leu
-Phe
L
-Phe
>1
>1
>1
>1
-Leu-DL-Leu
-Leu- -Phe
-Phe-DL-Leu
-Phe- -Phe
-Leu-DL-Leu
L
L
>1
-Leu-
-Leu-
-Leu-
-Leu-
-Leu-
-Leu-
-Leu-
-Leu-
-Leu-
-Leu
L
0.099
0.057
0.182
0.197
0.067
0.075
0.090
0.110
0.168
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
>1
L
L
L
L
L
L
L
L
-Leu-
-Leu-
-Leu-DL-Phe
-Phe-
-Phe-
-Phe-DL-Leu
-Phe- -Phe
L
-Leu
D
-Leu
j
k
l
L
-Leu
-Leu
D
m
n
o
a
b
c
d
e
f
g
h
a
b
c
a
b
L
-Phe-DL-Phe
-Phe
-Leu-
-Leu-
-Phe-
-Phe-DL-Phe
-Leu-
-Leu-
-Leu-
-Leu-
-Leu-
-Leu-
-Leu-
L
-Leu
-Phe
L
L
-Leu
L
L
L
L
L
L
L
-Phe-
-Phe-
-Phe
-Phe-
-Phe-
-Phe-
-Phe-
L
-Leu
-Phe
L
L
L
L
L
-Leu
-Phe
-Leu
-Phe
0.237
0.740
n.t.
n.t.
0.004
Bortezomib
a
Tested at least in duplicate; n.t. - not tested.

146
R. Jorda et al. / European Journal of Medicinal Chemistry 135 (2017) 142e158
proteasomal inhibitors (see Fig. 1), together with strong anticancer
cytotoxicity in vitro, especially of derivatives with terminal alde-
hyde function (series 4 and 6), led us to the hypothesis that their
mechanism of action may be connected with inhibition of protea-
some. All active compounds were therefore subjected to different
assays in order to address this hypothesis. Bortezomib and MG132
Finally, we studied the induction of cell death by 4m in
U266 cells. Bortezomib has been shown to promote apoptotic cell
death in several cancer cell lines [29,30]. Activation of apoptosis
was quantified biochemically by measuring the activity of caspases
3 and 7 in lysates of treated cells; results showed us that compound
4m can activate apoptotic machinery in a time-dependent manner
(Fig. 4). In addition, we analyzed the expression pattern of some
apoptosis-relevant proteins. We found that caspase 7 and its sub-
strate poly(ADP-ribose)polymerase (PARP-1) were cleaved, and
Mcl-1 was downregulated in cells treated for 16e24 h (Fig. 3).
These observations were in agreement with results from previous
assays and in all aspects followed the trends observed with pro-
teasomal inhibitor bortezomib.
are known to bind with very high affinity to the
proteasome with chymotrypsin-like activity. We therefore studied
inhibition of the 5 site of the 20S proteasome subunit in U266 cells
b5 site of 20S
b
treated with most potent compounds (compounds 4 and 6) using a
luminescent assay that measured the chymotrypsin-like protease
activity (Proteasome-Glo™ assay). Results in Table 1 show that the
majority of compounds displayed IC50 in mid-nanomolar range
(IC50 ranging from 57 to 197 nM), that is over 10 times higher than
that of bortezomib. Compounds bearing phenylalanine near the
terminal aldehyde group (both substituted and unsubstituted)
2.4. Conclusion
showed higher IC50 values than compounds having
L
-leucine at this
A collection of 46 novel O-benzyl salicylamides built from
leucine and phenylalanine was generated. The most potent com-
pounds having terminal aldehyde group displayed nanomolar
inhibitory activities against proteasome. We found a strong corre-
lation between proteasome inhibition and antiproliferative effect in
all three cell lines assayed. The most potent compounds demon-
strated cellular effects corresponding to proteasome inhibition,
including dose-dependent accumulation of polyubiquitinylated
proteins and induction of apoptosis in multiple myeloma cells.
position. Importantly, proteasome inhibition correlated strongly
with antiproliferative activity of all active compounds (4g-o, 6b-c);
linear regression coefficients R2 of log transformed IC50 values
equal to 0.716, 0.796 and 0.920 for K562, CEM and U266,
respectively.
In addition, the effect of compounds 4 and 6 in U266 cells was
studied also on the other two subunits of proteasome, namely
b1-
caspase-like and 2-trypsin-like. Cell-based assays using fluoro-
b
genic substrates revealed that the compounds display preference
towards the chymotrypsin-like subunit (supplementary Table S1).
Supplementary video related to this article can be found at
http://dx.doi.org/10.1016/j.ejmech.2017.04.027.
3. Experimental part
3.1. Chemistry
In order to confirm proteasome targeting by an independent
method, we set up a cell-based assay involving green fluorescence
protein (GFP) fused to a short degron [24] that is rapidly degraded
by a proteasome; similar assays were reported recently [25e27].
Accumulation of GFP in cells treated with proteasome inhibitors
(corresponding to proteasomal inhibition) was visualised and
quantified by live-cell imaging. All results were compared with
untreated cells and cells treated with bortezomib (Fig. 2A, B). As
shown in Fig. 2A and Fig. S1, the GFP signal in cells emerged
approximately 9 h after the compound addition and all tested
compounds (4g, j, k, m-o; 6b, c) inhibited proteasomal activity in
cells in a dose-dependent manner (see also Supplementary Video
1). One of the most potent compounds, 4m, inhibited the protea-
some activity already at 200 nM concentration. The maximum
concentration used (6.25 mM) was toxic for cells after 16 h treat-
ment; this was probably the reason why decreased GFP signal was
observed (Fig. 2A).
We simultaneously monitored proliferation of treated cells over
72 h (Fig. 2A and Fig. S2). Our results showed that over the period
when the inhibition of proteasome was evident, the proliferation
All reagents and solvents were purchased from commercial
sources (TCI Europe, Sigma-Aldrich, Acros Organics, Fluorochem,
Merck, Lach-Ner). Commercial grade reagents were used without
further purification. Reactions were monitored by thin layer chro-
matography plates coated with 0.2 mm silica gel 60 F254 (Merck).
TLC plates were visualised by the UV irradiation (254 nm). All
melting points were determined on a Melting Point B-540 appa-
ratus (Büchi, Switzerland) and are uncorrected. Specific optical
rotation was determined on Perkin-Elmer Inc. Model 341 Polar-
imeter with Na lamp (589 nm) in ethyl acetate or water. The con-
centration of compound is displayed as g/100 mL. The IR spectra
were recorded on a Nicolet 6700 FT-IR spectrometer (Thermo
Fisher Scientific, Wal-tham, MA, USA) over the range of
400e4000 cmꢀ1 using the ATR technique. The NMR spectra were
measured in D2O or CD2Cl2 or CDCl3 or DMSO solutions at ambient
temperature on a Bruker Avance™ III 400 spectrometer at fre-
quencies 1H (400 MHz) and 13C (100.26 MHz) or Bruker Ascend™
500 spectrometer at frequencies 1H (500.13 MHz), 13C{1H}
(125.76 MHz). The chemical shifts, d, are given in ppm, related to
was not altered. Compound 4m (up to 1
m
M concentration) did not
the residual solvent peaks D2O - 4.79, CD2Cl2 - 5.32, CDCl3 - 7.27,
DMSO - 2.5, eventually to tetramethylsilane (TMS) as an internal
standard. The coupling constants (J) are reported in [Hz]. Elemental
analyses (C, H, N) were performed on an automatic microanalyser
Flash 2000 Organic elemental analyzer. Mass spectrometry with
high resolution was determined by “dried droplet” method using
MALDI mass spectrometer LTQ Orbitrap XL (Thermo Fisher Scien-
tific) equipped with nitrogen UV laser (337 nm, 60 Hz). Spectra
were measured in positive ion mode and in regular mass extent
with resolution 100,000 at m/z ¼ 400. 2,5-dihydrobenzoic acid
(DBH) was used as the matrix.
interfere with proliferation during the first 24 h, while over the
following 48 h cells ceased to proliferate and died. 200 nM con-
centration of 4m did not influence the viability of treated cells over
the full 72 h.
Next, we monitored the levels of polyubiquitinated proteins in
treated cells by immunoblotting, using antibody specific towards
free ubiquitin and ubiquitin conjugates, forming a typical high
molecular mass smear. As shown in Fig. 3, compound 4m induced
accumulation of polyubiquitinated proteins in treated U266 cells
and the observed effect displayed a time-dependent tendency,
similarly to bortezomib. Besides stabilisation of unspecified pro-
teins, we also observed a dose-dependent increase of p27KIP, a
short-lived cell cycle regulating protein, known to be degraded by
proteasome [28]. In contrast, levels of proteins with slow turnover,
3.2. General scheme and procedures for benzyl-protected methyl
esters 2a-f
such as
b
-actin and
a
-tubulin, remained unchanged (Fig. 3).
Chosen
5-Chloro-O-benzyl-salicylic
acid-L-leucine-L-

R. Jorda et al. / European Journal of Medicinal Chemistry 135 (2017) 142e158
147
Fig. 2. Inhibition of ubiquitineproteasome system by salicylamide 4m (A) and bortezomib as a control (B) in U2OS cells. U2OS cells, stably expressing green fluorescence protein
(GFP) fused to a short degron that is rapidly degraded by the proteasome, were treated with different doses of 4m or bortezomib for 24 h. Inhibition of the proteasome caused dose-
dependent accumulation of GFP in treated cells that was acquired with IncuCyte ZOOM® live-cell imaging microscope and expressed as a number of green objects per 1 mm2 (left
panels). Cell proliferation was monitored by analyzing the occupied area (% of confluence) of images over time up to 72 h (right panels).
phenylalanine acid 1 (3.015 g, 6.17 mmol) was dissolved in
dichloromethane (100 mL) at ambient temperature. EDCI$HCl
(1.183 g, 6.17 mmol) and HOBt (0.833 g, 6.17 mmol) were added. The
H), 6.81 (2H, t, J ¼ 9.2 Hz, Ar-H), 5.20e5.12 (2H, ABq, J ¼ 10.4 Hz, O-
CH2-Ph), 4.60e4.51 (1H, m, NH-CH-CHH-CH-(CH3)2), 4.49e4.35
(2H, m, NH-CH-CHH-CH-(CH3)2, NH-CH-CHH-CH-(CH3)2), 3.72 (3H,
s, O-CH3), 1.78e1.70 (1H, m, NH-CH-CHH-CH-(CH3)2), 1.67e1.44
(6H, m, NH-CH-CHH-CH-(CH3)2, NH-CH-CHH-CH-(CH3)2, NH-CH-
CHH-CH-(CH3)2), 1.44e1.31 (1H, m, NH-CH-CHH-CH-(CH3)2),
1.21e1.11 (1H, m, NH-CH-CHH-CH-(CH3)2, 0.95 (9H, m, 3xCH3), 0.85
(3H, d, J ¼ 6.4 Hz, CH3-CH-CH3), 0.79 (3H, d, J ¼ 6.4 Hz, CH3-CH-
CH3), 0.72 (3H, d, J ¼ 6.4 Hz, CH3-CH-CH3). 13C NMR (100.62 MHz,
L-phenylalanine amino acid methylester (0.896 g, 6.17 mmol) was
added in the solution in dichloromethane (20 mL). The reaction
mixture was stirred at RT for 3 h and then concentrated under
reduced pressure. The crude product was dissolved in ethyl acetate
(70 mL) and extracted with H2O (2 ꢂ 30 mL), sat. NaHCO3
(2 ꢂ 30 mL), 5% citric acid (2 ꢂ 30 mL), brine (1 ꢂ 30 mL) and H2O
(1 ꢂ 30 mL). The organic phase was dried over anhydrous Na2SO4,
filtered and concentrated. The residue was purified by column
chromatography (silica gel 120 g, n-hexane/ethyl acetate (2:1)) to
afford the product 2f in yield 70% as white solid. Other derivatives
2a-f were isolated as white solids in yields 72e94%.
CDCl3-d1): d 173.2, 172.1, 171.7, 164.7, 134.9, 133.1, 132.3, 129.4, 129.3,
128.8, 128.6, 127.3, 122.2, 114.1, 72.1, 52.7, 52.4, 51.9, 50.9, 41.5, 40.3,
39.9, 24.93, 24.87, 23.1, 23.0, 22.05, 22.03. CHN Analysis: Calc. for
C33H46ClN3O6 (616.19): C, 64.32; H, 7.52; N, 6.82. Found: C,
64.28 0.02; H, 7.60 0.02; N, 6.78 0.02. HRMS: m/z calc. for
C
33H46ClN3O6: 616.31479 [MþH]þ; found: 616.31586 [MþH]þ.
3.2.1. Characterization of (2S)-methyl 2-((2S)-2-((2S)-2-(2-
(benzyloxy)-5-chlorobenzamido)-4-methylpentanamido)-4-
methylpentanamido)-4-methylpentanoate 2a
3.2.2. Characterization of (2R)-methyl 2-((2S)-2-((2S)-2-(2-
(benzyloxy)-5-chlorobenzamido)-4-methylpentanamido)-4-
methylpentanamido)-4-methylpentanoate 2b
White solid; yield 89%; mp 139e143 ꢁC; ½aD20ꢃ ¼ ꢀ37.3ꢁ (c 1.00,
EtOAc); Rf (hex/EtOAce1/1) ¼ 0.53. IR (ATR):3293, 2957, 2361, 1744,
White solid; yield 72%; mp 160.4e163.1 ꢁC; ½aD20ꢃ ¼ ꢀ16.3ꢁ (c
1.00, EtOAc); Rf (hex/EtOAce1/1) ¼ 0.51. IR (ATR): 3262, 3077, 2956,
2930, 2870, 1731, 1621, 1541, 1303, 1247, 1216, 1125, 1023, 750, 732,
1638, 1533, 1470, 1272, 1244, 1159, 1009, 809, 697 cmꢀ1 1H NMR
.
(400 MHz, CDCl3-d1):
d
8.16 (1H, d, J ¼ 2.8 Hz, NH-CH-CHH-CH-
(CH3)2), 8.20 (1H, d, J ¼ 6.8 Hz, NH-CH-CHH-CH-(CH3)2), 7.41e7.47
715, 700, 660 cmꢀ1
.
1H NMR (400 MHz, CDCl3-d1):
d
8.22 (1H, d,
(6H, m, NHeCH-CHH-CH-(CH3)2, Ar-H), 7.04 (1H, d, J ¼ 8.8 Hz, Ar-
J ¼ 7.2 Hz, NH-CH-CHH- CH-(CH3)2), 8.20 (1H, d, J ¼ 2.8 Hz, NH-CH-

148
R. Jorda et al. / European Journal of Medicinal Chemistry 135 (2017) 142e158
Fig. 3. Immunoblotting analysis of ubiquitinylated proteins and proteins involved in apoptosis in multiple myeloma U266 cells treated by compound 4m in a dose (A) and a time (B)
dependent manner. Bortezomib was used as a positive control (C).
b-actin or
a-tubulin levels are included as a control for equal loading.
(1H, m, NH-CH-CHH-CH-(CH3)2), 1.01 (3H, d, J ¼ 6.4 Hz, CH3-CH-
CH3), 0.99 (3H, d, J ¼ 6.4 Hz, CH3-CH-CH3), 0.96 (3H, d, J ¼ 6.4 Hz,
CH3-CH-CH3), 0.91 (3H, d, J ¼ 6.4 Hz, CH3-CH-CH3), 0.83 (3H, d,
J ¼ 6.4 Hz, CH3-CH-CH3), 0.77 (3H, d, J ¼ 6.4 Hz, CH3-CH-CH3). 13C
NMR (100.62 MHz, CDCl3-d1):
d 173.5, 172.1, 171.9, 165.2, 155.8,
134.8, 133.3, 132.5, 129.6, 129.4, 128.7, 127.3, 122.1, 114.2, 72.2, 53.6,
52.3, 51.7, 51.0, 41.9, 40.3, 40.0, 25.1, 25.0, 24.9, 23.3, 23.1, 23.0, 21.9,
21.75, 21.68. CHN Analysis: Calc. for C33H46ClN3O6 (616.19): C,
64.32; H, 7.52; N, 6.82. Found: C, 64.36 0.01; H 7.53 0.01; N,
6.82 0.01. HRMS: m/z calc. for C33H46ClN3O6: 638.2973 [MþNa]þ;
found: 638.29778 [MþNa]þ.
3.2.3. Characterization of (2S)-methyl 2-((2S)-2-((2S)-2-(2-
(benzyloxy)-5-chlorobenzamido)-4-methylpentanamido)-4-
methylpentanamido)-3-phenylpropanoate 2c
White solid; yield 74%; mp 161e163 ꢁC; ½aD20ꢃ ¼ ꢀ12.9ꢁ (c 1.00,
EtOAc); Rf (hex/EtOAce1/1) ¼ 0.51. IR (ATR): 3297, 2956,1742, 1634,
1621, 1541, 1232, 1123, 996, 754, 736, 697 cmꢀ1. 1H NMR (400 MHz,
CDCl3-d1):
d
8.17 (1H, d, J ¼ 2.8 Hz, NH-CH-CHH-CH-(CH3)2), 8.13
(1H, d, J ¼ 6.8 Hz, NH-CH-CHH-CH-(CH3)2), 7.47e7.39 (6H, m, NH-
CH-CHH-Ph, Ar-H), 7.30e7.19 (3H, m, Ar-H), 7.13e7.08 (2H, m, Ar-H),
7.04 (1H, d, J ¼ 8.8 Hz, Ar-H), 6.68 (1H, d, J ¼ 7.6 Hz, Ar-H), 6.63 (1H,
d, J ¼ 8 Hz, Ar-H), 5.19e5.12 (2H, ABq, J ¼ 10.4 Hz, O-CH2-Ph),
4.85e4.80 (1H, m, NH-CH-CH2-Ph), 4.50e4.43 (1H, m, NH-CH-
CHH-CH-(CH3)2), 4.38e4.30 (1H, m, NH-CH-CHH-CH-(CH3)2), 3.70
(3H, s, O-CH3), 3.14 (1H, dd, J ¼ 6 Hz, J ¼ 14 Hz, NH-CH-CHH-Ph),
3.08 (1H, dd, J ¼ 6 Hz, J ¼ 14 Hz, NH-CH-CHH-Ph), 1.69e1.60 (1H, m,
NH-CH-CHH-CH-(CH3)2), 1.59e1.32 (4H, m, NH-CH-CHH-CH-
(CH3)2, NH-CH-CHH-CH-(CH3)2), 1.24e1.13 (1H, m, NH-CH-CHH-
CH-(CH3)2, 0.86 (3H, d, J ¼ 6.4 Hz, CH3-CH-CH3), 0.83e0.77 (6H, m,
2xCH3), 0.72 (3H, d, J ¼ 6.4 Hz, CH3-CH-CH3). 13C NMR (100.62 MHz,
Fig. 4. Relative caspase-3/7 activity in U266 multiple myeloma cells treated with
compound 4m in a time dependent manner. Bortezomib was used as a positive control.
CHH-CH-(CH3)2), 7.54e7.47 (6H, m, NH-CH-CHH-CH-(CH3)2, Ar-H),
7.12 (1H, d, J ¼ 8.8 Hz, Ar-H), 6.92 (1H, t, J ¼ 8 Hz, Ar-H), 6.49 (1H, t,
J ¼ 8.4 Hz, Ar-H), 5.23e5.21 (2H, ABq, J ¼ 8.8 Hz, O-CH2-Ph),
4.65e4.57 (1H, m, NH-CH-CHH-CH-(CH3)2), 4.56e4.48 (1H, m, NH-
CH-CHH-CH-(CH3)2), 4.45e4.38 (1H, m, NH-CH-CHH-CH-(CH3)2),
3.73 (3H, s, O-CH3), 1.91e1.82 (1H, m, NH-CH-CHH-CH-(CH3)2),
1.81e1.68 (3H, m, NH-CH-CHH-CH-(CH3)2, NH-CH-CHH-CH-
(CH3)2), 1.66e1.50 (3H, m, NH-CH-CHH-CH-(CH3)2, NH-CH-CHH-
CH-(CH3)2), 1.47e1.35 (1H, m, NH-CH-CHH-CH-(CH3)2), 1.26e1.16
CDCl3-d1):
d 172.1, 171.8, 171.6, 164.7, 155.6, 136.0, 134.9, 133.1, 132.3,
129.44,129.41,129.3,128.7, 128.6,127.3, 122.3,114.2, 72.1, 53.4, 52.5,

R. Jorda et al. / European Journal of Medicinal Chemistry 135 (2017) 142e158
149
52.4, 52.0, 40.7, 39.7, 38.0, 24.87, 24.84, 23.1, 22.0, 21.9. CHN Anal-
ysis: Calc. for C36H44ClN3O6 (650.20): C, 66.50; H, 6.82; N, 6.46.
Found: C, 66.3 0.02; H, 6.83 0.02; N, 6.35 0.02. HRMS: m/z
calc. for C36H44ClN3O6: 650.29914 [MþH]þ; found: 650.30049
[MþH]þ.
CH-CHH-Ph), 7.54 (1H, d, J ¼ 2.8 Hz, NH-CH-CHH-Ph), 7.52e7.42
(5H, m, Ar-H), 7.35e7.24 (3H, m, Ar-H), 7.18e7.06 (8H, m, Ar-H), 6.88
(1H, d, J ¼ 7.6 Hz, Ar-H), 6.61 (1H, d, J ¼ 8 Hz, Ar-H), 5.20e5.14 (2H,
ABq, J ¼ 10 Hz, O-CH2-Ph), 4.91e4.83 (1H, m, NH-CH-CH2-Ph),
4.72e4.64 (1H, m, NH-CH-CH2-Ph), 4.46e4.39 (1H, m, NH-CH-
CHH-CH-(CH3)2), 3.74 (3H, s, O-CH3), 3.20e3.02 (4H, m, NH-CH-
CH2-Ph, NH-CH-CH2-Ph), 1.52e1.43 (1H, m, NH-CH-CHH-CH-
(CH3)2, 1.41e1.30 (1H, m, NH-CH-CHH-CH-(CH3)2, 1.17e1.08 (1H, m,
NH-CH-CHH-CH-(CH3)2), 0.80 (3H, d, J ¼ 6.4 Hz, CH3-CH-CH3), 0.70
(3H, d, J ¼ 6.4 Hz, CH3-CH-CH3). 13C NMR (100.62 MHz, CDCl3-d1):
3.2.4. Characterization of (2S)-methyl 2-((2S)-2-((2S)-2-(2-
(benzyloxy)-5-chlorobenzamido)-4-methylpentanamido)-3-
phenylpropanamido)-4-methylpentanoate 2d
White solid; yield 94%; mp 127e130 ꢁC; ½aD20ꢃ ¼ ꢀ54.0ꢁ (c 1.00,
EtOAc); Rf (hex/EtOAce1/1) ¼ 0.42. IR (ATR): 3292, 2956, 1744,
1645, 1525, 1272, 1226, 745, 701 cmꢀ1. 1H NMR (400 MHz, CDCl3-
d
172.0, 171.6, 170.6, 164.6, 155.7, 136.8, 136.7, 136.1, 134.9, 134.8,
133.1, 132.5, 132.4, 129.5, 129.4, 129.3, 128.7, 128.6, 127.2, 126.8,
122.1, 114.1, 72.1, 54.2, 53.5, 52.5, 52.4, 39.3, 38.0, 37.5, 24.8, 23.0,
21.8. CHN Analysis: Calc. for C39H42ClN3O6 (684.22): C, 68.46; H,
6.19; N. 6.14. Found: C, 68.42 0.02; H, 6.22 0.02; N, 6.15 0.02.
HRMS: m/z calc. for C39H42ClN3O6: 684.28349 [MþH]þ; found:
684.28561 [MþH]þ.
d1):
d
8.06 (1H, d, J ¼ 6.8 Hz, NH-CH-CHH-CH-(CH3)2), 8.04 (1H, d,
J ¼ 2.8 Hz, NH-CH-CHH- CH-(CH3)2), 7.48 (1H, d, J ¼ 2.8 Hz, NH-CH-
CHH-Ph), 7.46e7.37 (5H, m, Ar-H), 7.13e6.97 (6H, m, Ar-H),
6.75e6.65 (2H, m, Ar-H), 5.17e5.09 (2H, ABq, J ¼ 10.4 Hz, O-CH2-
Ph), 4.68e4.62 (1H, m, NH-CH-CH2-Ph), 4.58e4.51 (1H, m, NH-CH-
CHH-CH-(CH3)2), 4.38e4.31 (1H, m, NH-CH-CHH-CH-(CH3)2), 3.69
(3H, s, O-CH3), 3.7e3.03 (2H, m, NH-CH-CH2-Ph), 1.67e1.40 (4H, m,
NH-CH-CH2-CH-(CH3)2, NH-CH-CH2-CH-(CH3)2), 1.31e1.21 (1H, m,
NH-CH-CHH-CH-(CH3)2), 1.11e1.01 (1H, m, NH-CH-CHH-CH-
(CH3)2), 0.91 (6H, d, J ¼ 6 Hz, 2xCH3), 0.72 (3H, d, J ¼ 6.4 Hz, CH3-CH-
CH3), 0.65 (3H, d, J ¼ 6.4 Hz, CH3-CH-CH3). 13C NMR (100.62 MHz,
3.3. General scheme and procedure for benzyl-protected Weinreb
amides 3a-l
According to [20], the L-phenylalanine methylester derivative
CDCl3-d1):
d
173.0, 171.8, 170.8, 164.8, 155.8, 136.7, 134.7, 133.2,
(1.034 g, 2.44 mmol) was dissolved in a secured three-neck round-
bottom flask in the suspension of O,N-dimethylhydroxylamine
hydrochloride (CH3ONH(CH3)$HCl, 0.595 g, 6.1 mmol) in dry tet-
rahydrofurane (45 mL) under nitrogen atmosphere. The reaction
mixture was cooled to ꢀ20 ꢁC and LiHMDS (1 M in dry THF,12.2 mL,
12.2 mmol) was added dropwise via syringe pump over 15 min. The
mixture was stirred at ꢀ20 ꢁC for 1 h, then slightly heated to ꢀ10 ꢁC
and stirred for 2 h. The reaction mixture was quenched with
saturated solution of NH4Cl in H2O (30 mL) and extracted with ethyl
acetate (3 ꢂ 30 mL). The combined organic phase was washed with
H2O (2 ꢂ 20 mL) and dried over Na2SO4. The crude product was
purified by column chromatography (silica gel 110 g, n-hexane/
132.4, 129.5, 129.4, 129.3, 128.6, 128.5, 127.2, 126.9, 121.9, 114.1, 72.2,
54.0, 52.9, 52.4, 51.1, 41.2, 39.5, 37.2, 24.85, 24.81, 23.1, 23.0, 21.9,
21.7. CHN Analysis: Calc. for C36H44ClN3O6 (650.20): C, 66.50; H,
6.82; N, 6.46. Found: C, 66.34 0.02; H, 7.01 0.02; N, 6.35 0.02.
HRMS: m/z calc. for C36H44ClN3O6: 650.29914 [MþH]þ; found:
650.30134 [MþH]þ.
3.2.5. Characterization of (2R)-methyl 2-((2S)-2-((2S)-2-(2-
(benzyloxy)-5-chlorobenzamido)-4-methylpentanamido)-3-
phenylpropanamido)-4-methylpentanoate 2e
White solid; yield 78%; mp 171.4e174.2 ꢁC; ½aD20ꢃ ¼ ꢀ28.7ꢁ (c
1.00, EtOAc); Rf (hex/EtOAce1/1) ¼ 0.50. IR (ATR): 4, 3064, 2955,
2932, 2870, 1744, 1635, 1536, 1482, 1243, 1216, 1125, 1013, 802, 746,
ethyl acetate (1:1)) to afford -phenylalanine Weinreb amide 3b in a
L
69% yield as colourless oil. Other derivatives were isolated in
following yields: 3a in yield 70% as colourless oils, 3c-f in yields
51e82% as colourless oils and 3g-l in yields 72e91% as colourless
oils or white solids. All derivatives were prepared under similar
conditions except for those with three amino acids (3g-l). The
equivalent of used LiHMDS (1 M in dry THF) was increased from 5
equivalents for compounds with one or two amino acids to 7
equivalents.
720, 660 cmꢀ1 1H NMR (400 MHz, CDCl3-d1):
. d 8.07 (1H, d,
J ¼ 2.4 Hz, NH-CH-CHH-CH-(CH3)2), 8.05 (1H, d, J ¼ 5.2 Hz, NH-CH-
CHH-CH-(CH3)2), 7.48 (1H, d, J ¼ 2.8 Hz, NH-CH-CHH-Ph), 7.46e7.38
(5H, m, Ar-H), 7.15e7.01 (6H, m, Ar-H), 6.79 (1H, d, J ¼ 8 Hz, Ar-H),
6.50 (1H, d, J ¼ 8 Hz, Ar-H), 5.13e5.12 (2H, ABq, J ¼ 8.2 Hz, O-CH2-
Ph), 4.76e4.67 (1H, m, NH-CH-CH2-Ph), 4.60e4.52 (1H, m, NH-CH-
CHH-CH-(CH3)2), 4.28e4.21 (1H, m, NH-CH-CHH-CH-(CH3)2), 3.69
(3H, s, O-CH3), 3.16 (1H, dd, J ¼ 6.4 Hz, J ¼ 14.4 Hz, NH-CH-CHH-Ph),
3.08 (1H, dd, J ¼ 7.6 Hz, J ¼ 14.4 Hz, NH-CH-CHH-Ph),1.68e1.56 (3H,
m, NH-CH-CH2-CH-(CH3)2, NH-CH-CHH-CH-(CH3)2), 1.39e1.30 (1H,
m, NH-CH-CHH-CH-(CH3)2), 1.23e1.14 (1H, m, NH-CH-CHH-CH-
(CH3)2), 1.03e0.96 (1H, m, NH-CH-CHH-CH-(CH3)2), 0.95e0.87 (6H,
m, 2xCH3), 0.69 (3H, d, J ¼ 6.4 Hz, CH3-CH-CH3), 0.63 (3H, d,
3.3.1. Characterization of (2S)-2-benzyloxy-5-chloro-N-(1-
(methoxy(methyl)amino)-4-methyl-1-oxopentan-2-yl)benzamide
3a
Colourless oil; yield 70%; ½a2D0ꢃ ¼ ꢀ6.5ꢁ (c 1.00, EtOAc); Rf (hex/
EtOAc - 1/1) ¼ 0.47. IR (ATR): 3396, 2957, 1651, 1524, 1481, 1467,
1386, 1271, 1226, 1121, 988, 810, 751, 706 cmꢀ1. 1H NMR (400 MHz,
J ¼ 6.4 Hz, CH3-CH-CH3). 13C NMR (100.62 MHz, CDCl3-d1):
d 173.3,
171.7, 170.9, 165.1, 155.8, 136.8, 134.8, 133.3, 132.6, 129.6, 129.4,
129.2, 128.7, 128.6, 127.3, 126.8, 121.8, 114.0, 72.2, 53.6, 52.3, 51.1,
41.0, 39.7, 37.3, 24.8, 23.1, 22.9, 21.9, 21.6. CHN Analysis: Calc. for
CDCl3-d1):
d
8.32 (1H, d, J ¼ 8 Hz, NH-CH-CHH-CH-(CH3)2), 8.23 (1H,
d, J ¼ 2.8 Hz, Ar-H), 7.60e7.52 (2H, m, Ar-H), 7.51e7.40 (4H, m, Ar-
H), 7.05 (1H, d, J ¼ 8.8 Hz, Ar-H), 5.30e5.10 (3H, m, O-CH2-Ph, NH-
CH-CHH-CH-(CH3)2), 3.91 (3H, s, O-CH3), 3.25 (3H, s, N-CH3),
1.55e1.40 (2H, m, NH-CH-CHH-CH-(CH3)2, NH-CH-CH2-CH-(CH3)2),
1.37e1.26 (1H, m, NH-CH-CHH-CH-(CH3)2, 0.92 (3H, d, J ¼ 6.4 Hz,
CH3-CH-CH3), 0.79 (3H, d, J ¼ 6.4 Hz, CH3-CH-CH3). 13C NMR
C
36H44ClN3O6 (650.20): C, 66.5; H, 6.82; N, 6.46. Found: C,
66.67 0.02; H 6.90 0.01; N, 6.45 0.02. HRMS: m/z calc. for
C
36H44ClN3O6: 672.28108 [MþNa]þ; found: 672.28256 [MþNa]þ.
3.2.6. Characterization of (2S)-methyl 2-((2S)-2-((2S)-2-(2-
(benzyloxy)-5-chlorobenzamido)-4-methylpentanamido)-3-
phenylpropanamido)-3-phenylpropanoate 2f
(100.79 MHz, CDCl3-d1):
d 173.3, 164.0, 155.7, 135.2, 132.6, 132.3,
129.1,129.0,128.5,127.0,123.0,114.3, 71.9, 61.7, 48.5, 41.2, 32.3, 25.0,
23.4, 21.7. CHN Analysis: Calc. for C22H27ClN2O4 (418.91): C, 63.08;
White solid; yield 70%; mp 157e160 ꢁC; ½aD20ꢃ ¼ ꢀ30.0ꢁ (c 1.00,
EtOAc); Rf (hex/EtOAce1/1) ¼ 0.42. IR (ATR): 3283, 2957, 1638,
H, 6.50; N, 6.69. Found: C, 62.95
0.02; H 6.44
0.02; N,
1529, 1216, 743, 702 cmꢀ1. 1H NMR (400 MHz, CDCl3-d1):
d
8.19 (1H,
6.61 0.02. HRMS: m/z calc. for C22H27ClN2O4: 441.15516 [MþNa]þ;
d, J ¼ 2.8 Hz, NH-CH-CHH-CH-(CH3)2), 8.08 (1H, d, J ¼ 6.8 Hz, NH-
found: 441.15505 [MþNa]þ.

150
R. Jorda et al. / European Journal of Medicinal Chemistry 135 (2017) 142e158
3.3.2. Characterization of (2S)-2-benzyloxy-5-chloro-N-(1-
(methoxy(methyl)amino)-1-oxo-3-phenylpropan-2-yl)benzamide
3b
65.81 0.02; H 6.40 0.01; N, 7.42 0.02. HRMS: m/z calc. for
C
31H36ClN3O5: 588.22357 [MþNa]þ; found: 588.22384 [MþNa]þ.
Colourless oil; yield 69%; ½a2D0ꢃ ¼ ꢀ10.1ꢁ (c 1.00, EtOAc); Rf (hex/
EtOAce1/1) ¼ 0.40. IR (ATR): 3389, 3030, 2939, 2362, 2340, 1645,
3.3.5. Characterization of 2-benzyloxy-5-chloro-N-((2S)-1-(((2S)-
1-(methoxy(methyl)amino)-4-methyl-1-oxopentan-2-yl)amino)-1-
oxo-3-phenylpropan-2-yl)benzamide 3e
1519, 1480, 1270, 1228, 1121, 989, 810, 749, 699 cmꢀ1 1H NMR
.
(400 MHz, CDCl3-d1):
d
8.53(1H, d, J ¼ 7.2 Hz, NH-CH-CHH-Ph), 8.19
Colourless oil; yield 74%; ½a2D0ꢃ ¼ ꢀ15.0ꢁ (c 1.00, EtOAc); Rf (hex/
EtOAce1/1) ¼ 0.27. IR (ATR): 3306, 2958, 2360, 2342, 1690, 1514,
(1H, d, J ¼ 2.8 Hz, Ar-H), 7.56e7.46 (5H, m, Ar-H), 7.46e7.41 (1H, m,
Ar-H), 7.38 (1H, dd, J ¼ 2.8 Hz, J ¼ 8.8 Hz, Ar-H), 7.30e7.20 (2H, m,
Ar-H), 7.08 (1H, d, J ¼ 8.8 Hz, Ar-H), 7.03 (1H, d, J ¼ 6.4 Hz, Ar-H),
6.98 (1H, d, J ¼ 8.8 Hz, Ar-H), 5.47e5.37 (1H, m, NH-CH-CHH-Ph),
5.32e5.15 (2H, m, O-CH2-Ph), 3.83 (3H, s, O-CH3), 3.25 (3H, s, N-
CH3), 3.10 (1H, dd, J ¼ 5.6 Hz, J ¼ 13.6 Hz, NH-CH-CHH-Ph), 2.81 (1H,
dd, J ¼ 8 Hz, J ¼ 13.6 Hz, NH-CH-CHH-Ph). 13C NMR (100.79 MHz,
CDCl3-d1): 163.8, 155.6, 136.8, 135.5, 133.4, 132.6, 132.2, 129.4, 129.3,
129.2, 129.0, 128.5, 128.1, 127.0, 123.0, 114.5, 71.7, 61.7, 51.5, 38.0,
32.3. CHN Analysis: Calc. for C25H25ClN2O4 (452.93): C, 66.29; H,
5.56; N, 6.18. Found: C, 66.21 0.02; H 5.61 0.01; N, 5.97 0.02.
HRMS: m/z calc. for C25H25ClN2O4: 475.13951 [MþNa]þ; found:
475.14011 [MþNa]þ.
1469, 1271, 1225, 988, 742, 698, 669 cmꢀ1 1H NMR (400 MHz,
.
CDCl3-d1):
d
8.34 (1H, d, J ¼ 7.2 Hz, NH-CH-CHH-CH-(CH3)2), 8.15
(1H, d, J ¼ 2.8 Hz, NH-CH-CHH-Ph), 7.45e7.30 (6H, m, Ar-H),
7.24e7.13 (3H, m, Ar-H), 7.06e7.00 (2H, m, Ar-H), 6.91 (1H, d,
J ¼ 8.8 Hz, Ar-H), 6.65 (1H, d, J ¼ 8.4 Hz, Ar-H), 5.17e5.12 (2H, ABq,
J ¼ 16.2 Hz, O-CH2-Ph), 5.07e4.96 (1H, m, NH-CH-CHH-Ph),
4.90e4.82 (1H, m, NH-CH-CHH-CH-(CH3)2), 3.77 (3H, s, O-CH3),
3.20 (3H, s, N-CH3), 3.11 (1H, dd, J ¼ 8 Hz, J ¼ 14 Hz, NH-CH-CHH-
Ph), 2.88 (1H, dd, J ¼ 8 Hz, J ¼ 14 Hz, NH-CH-CHH-Ph), 1.63e1.52
(1H, m, NH-CH-CHH-CH-(CH3)2), 1.51e1.43 (2H, m, 2xNH-CH-CHH-
CH-(CH3)2, NH-CH-CHH-CH-(CH3)2), 0.91 (3H, d, J ¼ 6.4 Hz, CH3-
CH-CH3), 0.87 (3H, d,
(100.79 MHz, CDCl3-d1):
J
d
¼
6.4 Hz, CH3-CH-CH3). 13C NMR
170.6, 163.9, 155.3, 136.8, 135.2, 132.6,
3.3.3. Characterization of 2-benzyloxy-5-chloro-N-((2S)-1-(((2S)-
1-(methoxy(methyl)amino)-4-methyl-1-oxopentan-2-yl)amino)-4-
methyl-1-oxopentan-2-yl)benzamide 3c
132.1, 129.2, 129.0, 128.8, 128.4, 127.6, 126.9, 126.7, 122.6, 114.3, 71.5,
61.6, 55.1, 47.7, 41.8, 37.5, 32.1, 24.8, 23.2, 21.8. CHN Analysis: Calc.
for C31H36ClN3O5 (566.09): C, 65.77; H, 6.41; N, 7.42. Found: C,
65.75 0.01; H 6.51 0.01; N, 7.36 0.02. HRMS: m/z calc. for
Colourless oil; yield 51%; ½a2D0ꢃ ¼ ꢀ20.4ꢁ (c 1.00, EtOAc); Rf (hex/
EtOAce1/1) ¼ 0.33. IR (ATR): 3306, 2958, 2360, 2342, 1643, 1526,
1480, 1272, 1228, 991, 698, 669 cmꢀ1. 1H NMR (400 MHz, CDCl3-d1):
C
31H36ClN3O5: 588.22357 [MþNa]þ; found: 588.22380 [MþNa]þ.
d
8.23 (1H, d, J ¼ 2.8 Hz, NH-CH-CHH-CH-(CH3)2), 8.17 (1H, d,
3.3.6. Characterization of 2-benzyloxy-5-chloro-N-((2S)-1-(((2S)-
1-(methoxy(methyl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-1-
oxo-3-phenylpropan-2-yl)benzamide 3f
J ¼ 7.6 Hz, NH-CH-CHH-CH-(CH3)2), 7.55e7.42 (6H, m, Ar-H), 7.06
(1H, d, J ¼ 8.8 Hz, Ar-H), 6.75 (1H, d, J ¼ 8.4 Hz, Ar-H), 5.23e5.20
(2H, ABq, J ¼ 12.2 Hz, O-CH2-Ph), 5.08e5.00 (1H, m, NH-CH-CHH-
CH-(CH3)2), 4.67e4.57 (1H, m, NH-CH-CHH-CH-(CH3)2), 3.83 (3H, s,
O-CH3), 3.24 (3H, s, N-CH3), 1.72e1.62 (1H, m, NH-CH-CHH-CH-
(CH3)2), 1.62e1.52 (3H, m, 2 x NH-CH-CHH-CH-(CH3)2, NH-CH-
CHH-CH-(CH3)2), 1.52e1.42 (1H, m, NH-CH-CHH-CH-(CH3)2),
1.35e1.24(1H, m, NH-CH-CHH-CH-(CH3)2), 0.96 (3H, d, J ¼ 6.4 Hz,
CH3-CH-CH3), 0.95 (3H, d, J ¼ 6.4 Hz, CH3-CH-CH3), 0.87 (3H, d,
J ¼ 6.4 Hz, CH3-CH-CH3), 0.78 (3H, d, J ¼ 6.4 Hz, CH3-CH-CH3). 13C
Colourless oil; yield 54%; ½a2D0ꢃ ¼ ꢀ26.2ꢁ (c 1.00, EtOAc); Rf (hex/
EtOAce1/1) ¼ 0.36. IR (ATR): 3397, 2360, 2342, 1646, 1521, 1508,
989, 742, 699 cmꢀ1 1H NMR (400 MHz, CDCl3-d1):
. d 8.32 (1H, d,
J ¼ 7.6 Hz, NH-CH-CHH-Ph), 8.22 (1H, d, J ¼ 2.8 Hz, NH-CH-CHH-
Ph), 7.50e7.35 (6H, m, Ar-H), 7.27e7.10 (8H, m, Ar-H), 7.06e6.95
(4H, m, Ar-H), 5.30e5.21 (1H, m, NH-CH-CHH-Ph), 5.18e5.14 (2H,
ABq, J ¼ 16.2 Hz, O-CH2-Ph), 4.92e4.84 (1H, m, NH-CH-CHH-Ph),
3.71 (3H, s, O-CH3), 3.23 (3H, s, N-CH3), 3.15e3.06 (2H, m, NH-CH-
CHH-Ph, NH-CH-CHH-Ph), 3.00e2.92 (1H, m, NH-CH-CHH-Ph),
2.84 (1H, dd, J ¼ 8 Hz, J ¼ 14 Hz, NH-CH-CHH-Ph). 13C NMR
NMR (100.79 MHz, CDCl3-d1):
132.4, 129.2, 128.5,127.1, 122.8,114.1, 72.0, 61.8, 52.4, 47.9, 41.9, 40.5,
32.4, 24.9, 24.8, 23.5, 23.2, 21.9, 21.8. CHN Analysis: Calc. for
d 172.0, 164.2, 155.6, 135.1, 132.8,
(100.79 MHz, CDCl3-d1): d 170.6, 164.2, 155.5, 137.0, 136.4, 135.3,
C
28H38ClN3O5 (532.07): C, 63.21; H, 7.20; N, 7.90. Found: C,
132.8, 132.4, 129.6, 129.2, 129.0, 128.6, 128.4, 127.9, 127.1, 126.9,
126.87, 122.6, 114.5, 71.7 (2xC), 61.8, 55.0, 50.7, 38.4, 37.2, 32.3. CHN
Analysis: Calc. for C34H34ClN3O5 (600.10): C, 68.05; H, 5.71; N, 7.00.
Found: C, 67.92 0.02; H 5.78 0.01; N, 6.90 0.01. HRMS: m/z
calc. for C34H34ClN3O5: 622.20792 [MþNa]þ; found: 622.20888
[MþNa]þ.
63.24 0.01; H 7.38 0.02; N, 7.89 0.02. HRMS: m/z calc. for
C
28H38ClN3O5: 554.23922 [MþNa]þ; found: 554.24014 [MþNa]þ.
3.3.4. Characterization of 2-benzyloxy-5-chloro-N-((2S)-1-(((2S)-
1-(methoxy(methyl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-4-
methyl-1-oxopentan-2-yl)benzamide 3d
Colourless oil; yield 82%; ½a2D0ꢃ ¼ ꢀ30.6ꢁ (c 1.00, EtOAc); Rf (hex/
EtOAce1/1) ¼ 0.38. IR (ATR): 3296, 2926, 1643, 1544, 1481, 1247,
3.3.7. Characterization of N-((5S,8S,11S)-5,8-diisobutyl-3,13-
dimethyl-4,7,10-trioxo-2-oxa-3,6,9-triazatetradecan-11-yl)-2-
benzyloxy-5-chlorobenzamide 3g
1226, 989, 700 cmꢀ1 1H NMR (400 MHz, CDCl3):
. d 8.21 (1H, d,
J ¼ 2.8 Hz, NH-CH-CHH-CH-(CH3)2), 8.03 (1H, d, J ¼ 7.2 Hz, NH-CH-
CHH-Ph), 7.45e7.37 (6H,m, Ar-H), 7.15e7.08 (5H, m, Ar-H), 7.02 (1H,
d, J ¼ 8.8 Hz, Ar-H), 6.94 (1H, d, J ¼ 8 Hz, Ar-H), 5.23e5.17 (1H, m,
NH-CH-CHH-Ph), 5.17e5.14 (2H, ABq, J ¼ 10.4 Hz, O-CH2-Ph),
4.56e4.47 (1H, m, NH-CH-CHH-CH-(CH3)2), 3.69 (3H, s, O-CH3),
3.18 (3H, s, N-CH3), 3.12e3.05 (1H, m, NH-CH-CHH-Ph), 2.95e2.85
(1H, m, NH-CH-CHH-Ph), 1.50e1.41 (1H, m, NH-CH-CHH-CH-
(CH3)2), 1.40e1.32 (1H, m, NH-CH-CHH-CH-(CH3)2), 1.21e1.12 (1H,
m, NH-CH-CHH-CH-(CH3)2), 0.78 (3H, d, J ¼ 6.4 Hz, CH3-CH-CH3),
0.70 (3H, d, J ¼ 6.4 Hz, CH3-CH-CH3). 13C NMR (100.62 MHz, CDCl3):
Colourless oil; yield 75%; ½a2D0ꢃ ¼ ꢀ39.7ꢁ (c 1.00, EtOAc); Rf (hex/
EtOAce1/1) ¼ 0.33. IR (ATR): 3293, 2957, 2360, 2342, 1640, 1527,
1468, 1272, 1234, 1122, 990, 809, 749, 696 cmꢀ1. 1H NMR (400 MHz,
CDCl3-d1):
d
8.16 (1H, d, J ¼ 2.8 Hz, NH-CH-CHH-CH-(CH3)2), 8.12
(1H, d, J ¼ 7.2 Hz, NH-CH-CHH-CH-(CH3)2), 7.48e7.38 (6H, m, NH-
CH-CHH-CH-(CH3)2, Ar-H), 7.02 (1H, d, J ¼ 8.8 Hz, Ar-H), 6.69 (1H, d,
J ¼ 8 Hz, Ar-H), 6.61 (1H, d, J ¼ 8 Hz, Ar-H), 5.16e5.13 (2H, ABq,
J ¼ 10.4 Hz, OeCH2-Ph), 5.30e4.96 (1H, m, NH-CH-CHH-CH-
(CH3)2), 4.53e4.46 (1H, m, NH-CH-CHH-CH-(CH3)2), 4.43e4.35 (1H,
m, NH-CH-CHH-CH-(CH3)2), 3.78 (3H, s, O-CH3), 3.18 (3H, s, N-CH3),
1.70e1.45 (7H, m, 3xNH-CH-CH2-CH-(CH3)2, NH-CH-CH2-CH-
(CH3)2), 1.45e1.34 (1H, m, NH-CH-CH2-CH-(CH3)2), 1.27e1.15 (1H,
m, NH-CH-CH2-CH-(CH3)2), 0.93 (3H, d, J ¼ 6.8 Hz, CH3-CH-CH3),
0.91 (3H, d, J ¼ 6.8 Hz, CH3-CH-CH3), 0.87 (3H, d, J ¼ 6.4 Hz, CH3-CH-
d
171.6, 164.2, 155.6, 136.5, 135.0, 132.8, 132.5, 129.6, 129.5, 129.3,
128.5, 128.4, 127.1, 126.9, 122.6, 114.1, 72.0 (2xC), 61.8, 52.2, 50.8,
40.0, 38.3, 32.3, 24.8, 23.1, 21.8. CHN Analysis: Calc. for
C
31H36ClN3O5 (566.09): C, 65.77; H, 6.41; N, 7.42. Found: C,

R. Jorda et al. / European Journal of Medicinal Chemistry 135 (2017) 142e158
151
CH3), 0.83 (3H, d, J ¼ 6.4 Hz, CH3-CH-CH3), 0.80 (3H, d, J ¼ 6.4 Hz,
3.3.10. Characterization of N-((5S,8S,11S)-8-benzyl-5-isobutyl-3,13-
CH3-CH-CH3), 0.73 (3H, d, J ¼ 6.4 Hz, CH3-CH-CH3). 13C NMR
dimethyl-4,7,10-trioxo-2-oxa-3,6,9-triazatetradecan-11-yl)-2-
benzyloxy-5-chlorobenzamide 3j
(100.62 MHz, CDCl3-d1):
d 173.0, 172.0, 171.8, 164.5, 155.6, 134.9,
133.0, 132.3, 129.4, 129.3, 128.6, 127.2, 122.5, 114.2, 72.1, 61.7, 52.4,
51.9, 47.8, 41.9, 40.9, 39.3, 32.3, 24.9, 23.5, 23.2, 23.0, 22.0, 21.8. CHN
Analysis: Calc. for C34H49ClN4O6 (645.23): C, 63.29; H, 7.65; N, 8.68.
Found: C, 63.02 0.02; H 7.84 0.02; N, 8.58 0.02. HRMS: m/z
calc. for C34H49ClN4O6: 667.32328 [MþNa]þ; found: 667.32208
[MþNa]þ.
White solid; yield 74%; mp 63.3e65.6 ꢁC; ½aD20ꢃ ¼ ꢀ43.9ꢁ (c 1.00,
EtOAc); Rf (hex/EtOAce1/1) ¼ 0.35. IR (ATR): 3297, 3067, 2957,
2871, 2362, 1621, 1529, 1500, 1482, 1272, 1234, 1122, 989, 743,
699 cmꢀ1. 1H NMR (500 MHz, CDCl3-d1):
d
8.29 (1H, d, J ¼ 3 Hz, NH-
CH-CHH-CH-(CH3)2), 8.20 (1H, d, J ¼ 6.5 Hz, NH-CH-CHH-CH-
(CH3)2), 7.67e7.55 (6H, m, NH-CH-CHH-Ph, Ar-H), 7.32e7.27 (2H, m,
Ar-H), 7.34e7.17 (4H, m, Ar-H), 7.06 (1H, d, J ¼ 8 Hz, Ar-H), 6.84 (1H,
d, J ¼ 8.5 Hz, Ar-H), 5.31e5.29 (2H, ABq, J ¼ 7.6 Hz, O-CH2-Ph),
5.21e5.15 (1H, m, NH-CH-CHH-Ph), 4.88e4.80 (1H, m, NH-CH-
CHH-CH-(CH3)2), 4.64e4.57 (1H, m, NH-CH-CHH-CH-(CH3)2), 3.96
(3H, s, O-CH3), 3.37 (3H, s, N-CH3), 3.32e3.29 (2H, m, NH-CH-CH2-
Ph), 1.77e1.58 (4H, m, 2xNH-CH-CH2-CH-(CH3)2, 1.52e1.42 (1H, m,
NH-CH-CH2-CH-(CH3)2), 1.29e1.22 (1H, m, NH-CH-CH2-CH-(CH3)2),
1.11 (3H, d, J ¼ 6.5 Hz, CH3-CH-CH3), 1.08 (3H, d, J ¼ 6.5 Hz, CH3-CH-
CH3), 0.92 (3H, d, J ¼ 6.5 Hz, CH3-CH-CH3), 0.84 (3H, d, J ¼ 6.5 Hz,
3.3.8. Characterization of N-((5R,8S,11S)-5,8-diisobutyl-3,13-
dimethyl-4,7,10-trioxo-2-oxa-3,6,9-triazatetradecan-11-yl)-2-
benzyloxy-5-chlorobenzamide 3h
Colourless oil; yield 83%; ½a2D0ꢃ ¼ ꢀ18.0ꢁ (c 1.00, EtOAc); Rf (hex/
EtOAce1/1) ¼ 0.29. IR (ATR): 3379, 3297, 3067, 2956, 2935, 2870,
1620, 1521, 1467, 1271, 128, 988, 809, 750, 696, 534 cmꢀ1. 1H NMR
(400 MHz, CDCl3-d1):
d
8.24 (1H, d, J ¼ 2.8 Hz, NH-CH-CHH-CH-
(CH3)2), 8.20 (1H, d, J ¼ 6 Hz, NH-CH-CHH-CH-(CH3)2), 7.55e7.43
(6H, m, NH-CH-CHH-CH-(CH3)2, Ar-H), 7.09 (1H, d, J ¼ 8.8 Hz, Ar-H),
6.89 (1H, d, J ¼ 8.4 Hz, Ar-H), 6.78 (1H, d, J ¼ 8 Hz, Ar-H), 5.22e5.20
(2H, ABq, J ¼ 10.4 Hz, O-CH2-Ph), 5.05e4.93 (1H, m, NH-CH-CHH-
CH-(CH3)2), 4.58e4.50 (1H, m, NH-CH-CHH-CH-(CH3)2), 4.50e4.43
(1H, m, NH-CH-CHH-CH-(CH3)2), 3.86 (3H, s, O-CH3), 3.23 (3H, s, N-
CH3), 1.85e1.72 (2H, m, 2xNH-CH-CHH-CH-(CH3)2), 1.70e1.63 (1H,
m, NH-CH-CHH-CH-(CH3)2), 1.62e1.51 (4H, m, 3xNH-CH-CHH-CH-
(CH3)2, NH-CH-CH2-CH-(CH3)2), 1.47e1.37 (1H, m, NH-CH-CH2-CH-
(CH3)2), 1.27e1.18 (1H, m, NH-CH-CH2-CH-(CH3)2), 1.01 (3H, d,
J ¼ 6.4 Hz, CH3-CH-CH3), 1.00 (3H, d, J ¼ 6.4 Hz, CH3-CH-CH3), 0.95
(3H, d, J ¼ 6.4 Hz, CH3-CH-CH3), 0.91 (3H, d, J ¼ 6.4 Hz, CH3-CH-
CH3), 0.84 (3H, d, J ¼ 6.4 Hz, CH3-CH-CH3), 0.78 (3H, d, J ¼ 6.4 Hz,
CH3-CH-CH3). 13C NMR (125.77 MHz, CDCl3-d1):
d 172.6,171.7,170.5,
164.4, 155.5, 136.5, 134.5, 132.9, 132.3, 129.3, 129.2, 128.4, 128.3,
127.0, 126.6, 121.8, 113.8, 71.9, 61.6, 54.0, 52.1, 47.7, 41.5, 39.1, 37.3,
32.1, 24.7, 24.6, 23.3, 22.8, 21.6. CHN Analysis: Calc. for
C37H47ClN4O6 (679.25): C, 65.42; H, 6.97; N, 8.25. Found: C,
65.60 0.02; H 7.00 0.01; N, 8.21 0.02. HRMS: m/z calc. for
C
37H47ClN4O6: 701.30763 [MþNa]þ; found: 701.30958 [MþNa]þ.
3.3.11. Characterization of N-((5R,8S,11S)-8-benzyl-5-isobutyl-3,13-
dimethyl-4,7,10-trioxo-2-oxa-3,6,9-triazatetradecan-11-yl)-2-
benzyloxy-5-chlorobenzamide 3k
Colourless oil; yield 91%; ½a2D0ꢃ ¼ ꢀ27.0ꢁ (c 1.00, EtOAc); Rf (hex/
EtOAce1/1) ¼ 0.35. IR (ATR): 3377, 3293, 3064, 2955, 2933, 2869,
1630, 1519, 1481, 1467, 1385, 1270, 1226, 987, 808, 744, 697,
CH3-CH-CH3). 13C NMR (100.62 MHz, CDCl3-d1):
d 173.3,172.3,172.1,
165.0,155.7,134.9,133.1, 132.5,129.4,129.3,128.7, 127.2, 122.4,114.1,
72.2, 61.7, 53.2, 51.8, 48.3, 41.2, 40.9, 40.0, 32.4, 25.1, 25.0, 24.9, 23.5,
23.3, 23.1, 21.8, 21.7. CHN Analysis: Calc. for C34H49ClN4O6 (645.23):
C, 63.29; H, 7.65; N, 8.68. Found: C, 63.08 0.02; H 7.74 0.02; N,
534 cmꢀ1. 1H NMR (400 MHz, CDCl3-d1):
d
8.23 (1H, d, J ¼ 2.8 Hz,
NH-CH-CHH-CH-(CH3)2), 8.09 (1H, d, J ¼ 5.6 Hz, NH-CH-CHH-CH-
(CH3)2), 7.53e7.43 (6H, m, NH-CH-CHH-Ph, Ar-H), 7.24e7.11 (5H, m,
Ar-H), 7.09 (1H, d, J ¼ 8.8 Hz, Ar-H), 6.92 (1H, d, J ¼ 8 Hz, Ar-H), 6.77
(1H, d, J ¼ 8 Hz, Ar-H), 5.18e5.17 (2H, ABq, J ¼ 8.8 Hz, O-CH2-Ph),
5.02e4.92 (1H, m, NH-CH-CHH-Ph), 4.84e4.75 (1H, m, NH-CH-
CHH-CH-(CH3)2), 4.44e4.36 (1H, m, NH-CH-CHH-CH-(CH3)2), 3.87
(3H, s, O-CH3), 3.35e3.20 (4H, m, N-CH3, NH-CH-CHH-Ph), 3.01 (1H,
dd, J ¼ 8.4 Hz, J ¼ 14 Hz, NH-CH-CHH-Ph), 1.68e1.45 (3H, m, NH-
CH-CH2-CH-(CH3)2, NH-CH-CHH-CH-(CH3)2), 1.44e1.35 (1H, m,
NH-CH-CHH-CH-(CH3)2), 1.34e1.25 (1H, m, NH-CH-CH2-CH-
(CH3)2), 1.11e1.01 (1H, m, NH-CH-CH2-CH-(CH3)2), 0.98 (3H, d,
J ¼ 6.4 Hz, CH3-CH-CH3), 0.97 (3H, d, J ¼ 6.4 Hz, CH3-CH-CH3), 0.77
(3H, d, J ¼ 6.4 Hz, CH3-CH-CH3), 0.70 (3H, d, J ¼ 6.4 Hz, CH3-CH-
8.49
0.02. HRMS: m/z calc. for C34H49ClN4O6: 667.32328
[MþNa]þ; found: 667.32406 [MþNa]þ.
3.3.9. Characterization of N-((5S,8S,11S)-5-benzyl-8-isobutyl-3,13-
dimethyl-4,7,10-trioxo-2-oxa-3,6,9-triazatetradecan-11-yl)-2-
benzyloxy-5-chlorobenzamide 3i
Colourless oil; yield 72%; ½a2D0ꢃ ¼ ꢀ29.7ꢁ (c 1.00, EtOAc); Rf (hex/
EtOAce1/1) ¼ 0.36. IR (ATR): 3735, 3648, 3628, 3587, 3566, 3300,
2956, 2871, 2360, 2342, 1642, 1525, 1481, 1456, 1272, 1227, 990, 810,
750, 699, 669 cmꢀ1 1H NMR (400 MHz, CDCl3-d1):
. d 8.18 (1H, d,
J ¼ 2.4 Hz, NH-CH-CHH-CH-(CH3)2), 8.15 (1H, d, J ¼ 6 Hz, NH-CH-
CHH-CH-(CH3)2), 7.48e7.36 (6H, m, NH-CH-CHH-Ph, Ar-H),
7.27e7.23 (2H, m, Ar-H), 7.22e7.17 (1H, m, Ar-H), 7.16e7.12 (2H,m,
Ar-H), 7.02 (1H, d, J ¼ 7.2 Hz, Ar-H), 6.95e6.90 (1H, m, Ar-H),
6.85e6.80 (1H,m, Ar-H), 5.25e5.19 (1H, m, NH-CH-CHH-Ph),
5.16e5.13 (2H, ABq, J ¼ 7.6 Hz, O-CH2-Ph), 4.56e4.49 (1H, m, NH-
CH-CHH-CH-(CH3)2), 4.43e4.36 (1H, m, NH-CH-CHH-CH-(CH3)2),
3.63 (3H, s, O-CH3), 3.15 (3H, s, N-CH3), 3.10e3.04 (1H, m, NH-CH-
CHH-Ph), 2.97e2.91 (1H, m, NH-CH- CHH-Ph), 1.62e1.43 (4H, m, 2 x
NH-CH-CH2-CH-(CH3)2, 1.43e1.35 (1H, m, NH-CH-CH2-CH-(CH3)2),
1.27e1.18 (1H, m, NH-CH-CH2-CH-(CH3)2), 0.85 (3H, d, J ¼ 5.2 Hz,
CH3-CH-CH3), 0.80 (6H, d, J ¼ 4.8 Hz, CH3-CH-CH3), 0.73 (3H, d,
CH3). 13C NMR (100.79 MHz, CDCl3-d1):
d 173.1, 171.9, 171.1, 164.9,
155.7, 137.0, 134.8, 133.1, 132.7, 129.4, 129.32, 129.27, 128.64, 128.59,
127.2, 126.8, 122.2, 114.0, 72.1, 61.7, 54.1, 53.0, 48.4, 41.2, 39.7, 38.0,
32.4, 24.83, 24.78, 23.5, 23.0, 21.8, 21.7. CHN Analysis: Calc. for
C37H47ClN4O6 (679.25): C, 65.42; H, 6.97; N, 8.25. Found: C,
65.63 0.02; H 7.01 0.01; N, 8.17 0.02. HRMS: m/z calc. for
C
37H47ClN4O6: 701.30763 [MþNa]þ; found: 701.30889 [MþNa]þ.
3.3.12. Characterization of N-((5S,8S,11S)-5,8-dibenzyl-3,13-
dimethyl-4,7,10-trioxo-2-oxa-3,6,9-triazatetradecan-11-yl)-2-
benzyloxy-5-chlorobenzamide 3l
White solid; yield 74%; mp 72.8e76.5 ꢁC; ½aD20ꢃ ¼ ꢀ35.1ꢁ (c 1.00,
EtOAc); Rf (hex/EtOAce1/1) ¼ 0.38. IR (ATR): 3307, 3064, 3031,
J ¼ 5.2 Hz, CH3-CH-CH3). 13C NMR (100.61 MHz, CDCl3-d1):
d 172.0,
171.5, 164.6, 155.6, 136.4, 134.9, 133.0, 132.4, 132.1, 129.6, 129.4,
129.3,128.5, 127.2, 127.0, 122.4, 114.2, 72.1, 61.7, 52.3, 52.0, 50.4, 41.1,
39.7, 38.4, 32.3, 24.8, 23.2, 23.1, 22.0, 21.9. CHN Analysis: Calc. for
2955, 2360, 2342, 1645, 1522, 1272, 1230, 989, 810, 744, 699 cmꢀ1
.
1H NMR (500 MHz, CDCl3-d1):
d
8.36 (1H, d, J ¼ 2.5 Hz, NH-CH-
CHH-CH-(CH3)2), 8.19 (1H, d, J ¼ 7 Hz, NH-CH-CHH-Ph), 7.67e7.55
(6H, m, NH-CH-CHH-Ph, Ar-H), 7.47e7.37 (3H, m, Ar-H), 7.32e7.25
(4H, m, Ar-H), 7.24e7.20 (4H, m, Ar-H), 7.07 (1H, d, J ¼ 7.5 Hz, Ar-H),
6.84 (1H, d, J ¼ 8 Hz, Ar-H), 5.43e5.35 (1H, m, NH-CH-CHH-Ph),
C
37H47ClN4O6 (679.25): C, 65.42; H, 6.97; N, 8.25. Found: C,
65.38 0.02; H 6.92 0.01; N, 8.23 0.02. HRMS: m/z calc. for
C
37H47ClN4O6: 701.30763 [MþNa]þ; found: 701.30860 [MþNa]þ.

152
R. Jorda et al. / European Journal of Medicinal Chemistry 135 (2017) 142e158
5.31e5.29 (2H, ABq, J ¼ 7.6 Hz, O-CH2-Ph), 4.83e4.77 (1H, m, NH-
CH-CHH-Ph), 4.65e4.57 (1H, m, NH-CH-CHH-CH-(CH3)2), 3.83 (3H,
s, O-CH3), 3.34 (3H, s, N-CH3), 3.28e3.22 (2H, m, NH-CH-CH2-Ph),
3.16e3.05 (2H, m, NH-CH-CH2-Ph), 1.65e1.57 (1H, m, NH-CH-CHH-
CH-(CH3)2, 1.54e1.43 (1H, m, NH-CH-CH2-CH-(CH3)2), 1.32e1.25
(1H, m, NH-CH-CHH-CH-(CH3)2, 0.94 (3H, d, J ¼ 6.5 Hz, CH3-CH-
CH3), 0.86 (3H, d, J ¼ 6.5 Hz, CH3-CH-CH3). 13C NMR (125.78 MHz,
CHH-CH-(CH3)2), 1.46e1.37 (1H, m, NH-CH-CHH-CH-(CH3)2),
1.17e1.07 (1H, m, NH-CH-CHH-CH-(CH3)2), 0.82 (3H, d, J ¼ 6.4 Hz,
CH3-CH-CH3), 0.74 (3H, d, J ¼ 6.4 Hz, CH3-CH-CH3). 13C NMR
(100.79 MHz, CDCl3-d1):
d 199.8, 164.1, 155.6, 134.8, 132.8, 132.2,
129.2, 129.1, 128.5, 127.1, 122.2, 114.0, 72.0, 57.7, 37.3, 24.7, 23.1, 21.7.
CHN Analysis: Calc. for C20H22ClNO3 (359.85): C, 66.75; H, 6.16; N,
3.89. Found: C, 66.52 0.02; H 6.21 0.01; N, 3.81 0.01. HRMS: m/
z calc. for C20H22ClNO3: 360.13610 [MþH]þ; found: 360.13638
[MþNa]þ.
CDCl3-d1):
d 171.9, 171.2, 170.2, 164.4, 155.5, 136.5, 136.3, 134.6,
132.9, 132.4, 129.4, 129.24, 129.20, 129.16, 128.4, 128.3, 127.0, 126.9,
126.6, 122.0, 113.9, 71.9, 61.6, 54.1, 52.0, 50.3, 39.0, 38.2, 37.6, 32.0,
24.6, 22.9, 21.6. CHN Analysis: Calc. for C40H45ClN4O6 (713.26): C,
67.21; H, 6.36; N, 7.86. Found: C, 67.21 0.01; H 6.31 0.01; N,
3.4.2. Characterization of ((2S)-2-benzyloxy-5-chloro-N-(1-oxo-3-
phenylpropan-2-yl)benzamide 4b
7.86
0.02. HRMS: m/z calc. for
C
40H45ClN4O6: 735.29198
Colourless oil; yield 78%; ½a2D0ꢃ ¼ ꢀ1.5ꢁ (c 1.00, EtOAc); Rf (hex/
EtOAce3/1) ¼ 0.15. IR (ATR): 3376, 3063, 2924, 1730, 1645, 1520,
[MþNa]þ; found: 735.29319 [MþNa]þ.
1478, 1270, 1224, 1124, 992, 808, 741 cmꢀ1 1H NMR (400 MHz,
.
3.4. General scheme and procedure for benzyl-protected aldehydes
4a-o
CDCl3-d1):
d
9.53 (1H, s, CHO), 8.34 (1H, d, J ¼ 6 Hz, NH-CH-CHH-
Ph), 8.11 (1H, d, J ¼ 2.8 Hz, Ar-H), 7.35e7.25 (4H, m, Ar-H), 7.20e7.10
(5H, m, Ar-H), 6.98e6.93 (2H, m, Ar-H), 6.87 (1H, d, J ¼ 8.8 Hz, Ar-H),
5.04e5.01 (2H, ABq, J ¼ 10.6 Hz, O-CH2-Ph), 4.72 (1H, q, J ¼ 6.4 Hz,
NH-CH-CHH-Ph), 3.07 (1H, dd, J ¼ 6.4 Hz, J ¼ 14.4 Hz, NH-CH-CHH-
Ph), 2.92 (1H, dd, J ¼ 6.4 Hz, J ¼ 14.4 Hz, NH-CH-CHH-Ph). 13C NMR
Reduction of Weinreb amides with one amino acid to aldehyde
was done according to similar reaction described in Ref. [19]. So-
lution of L-phenylalanine Weinreb amide (0.6 g, 1.42 mmol) in
dichlormethane (30 mL) was cooled to ꢀ78 ꢁC and DIBAL (0.89M in
THF, 3.19 mL, 2.84 mmol) was added dropwise over 15 min. The
reaction was stirred for 1 h at this temperature and then quenched
with saturated solution of NH4Cl in H2O (20 mL) and slowly heated
to ambient temperature. The crude product was extracted with
dichlormethane (3 ꢂ 20 mL). The combined organic phase was
washed with H2O (2 ꢂ 20 mL) and dried over Na2SO4. The crude
product was purified by column chromatography (silica gel 40 g, n-
hexane/ethyl acetate (3:1)) to afford aldehyde 4b in a 78% yield as
(100.79 MHz, CDCl3-d1): d 198.8, 164.0, 155.5, 135.9, 135.0, 132.8,
132.1, 129.2, 129.0, 128.8, 128.7, 127.8, 127.1, 127.0, 122.2, 114.4, 71.7,
60.5, 34.8. CHN Analysis: Calc. for C23H20ClNO3 (393.86): C, 70.14;
H, 5.12; N, 3.56. Found: C, 69.89 0.03; H 5.17 0.02; N, 3.52 0.03.
HRMS: m/z calc. for C23H20ClNO3: 394.12045 [MþH]þ; 416.10239
[MþNa]þ; found: 394.12090 [MþH]þ; 416.10286 [MþNa]þ.
3.4.3. Characterization of 2-benzyloxy-5-chloro-N-(2S)-4-methyl-
1-((4-methyl-1-oxopentan-2-yl)amino)-1-oxopentan-2-yl)
benzamide 4c
colourless oil for L-phenylalanine. L-leucine derivative 4a was ob-
tained in a 46% yield as a colourless oil.
Reduction of Weinreb amide with two amino acids to aldehyde
was done according to similar reaction described in Ref. [31]:
Product is a mixture of two diastereomers, A and B. Colourless
oil; yield 88%; ½aD20
ꢃ
¼ ꢀ9.3ꢁ (c 0.667, EtOAc); Rf (hex/EtOAce1/
L-
1) ¼ 0.51. IR (ATR): 3380, 3312, 2955, 2930, 2870, 1638, 1505, 1480,
Phenylalanine- -phenylalanine Weinreb amide (0.256 g,
L
1466, 1295, 1227, 1121, 995, 809, 744, 696 cmꢀ1. 1H NMR (400 MHz,
0.427 mmol) was dissolved in a secured three-neck round-bottom
flask in dry tetrahydrofurane (30 mL) under nitrogen atmosphere
and cooled to ꢀ20 ꢁC. Via syringe the suspension of LiAlH4
(0.0225 g, 0.593 mmol) in dry tetrahydrofurane (10 mL) was added
dropwise and stirred for 1 h. The reaction was diluted with ethyl
acetate (30 mL) and washed with 0.1 M HCl (3 ꢂ 15 mL). The water
phase was washed with ethyl acetate (3 ꢂ 15 mL). Organic phases
were combined and washed with H2O (3 ꢂ 15 mL) and dried over
Na2SO4. The crude product was purified by column chromatog-
raphy (silica gel 30 g, n-hexane/ethyl acetate (1:1)) to afford alde-
hydes 4c-f in yield 55e97% as colourless oil or white solid.
CDCl3-d1):
d
9.60e9.54 (1H, m, (CHO)AþB), 8.28e8.15 (2H, m, (2 x
NH-CH-CHH-CH-(CH3)2)AþB), 7.50e7.40 (6H, m, (Ar-H)AþB),
7.15e7.06 (1H, m, (Ar-H)AþB), 7.04e6.92 (1H, m, (Ar-H)AþB),
5.30e5.13 (2H, m, (O-CH2-Ph)AþB), 4.69e4.59 (1H, m, (NH-CH-
CHH-CH-(CH ) )
3 2 AþB), 4.52e4.41 (1H, m, (NH-CH-CHH-CH-
(CH ) )
3 2 AþB), 1.83e1.67 (2H, m, (2 x NH-CH-CHH-CH-(CH3)2)AþB),
1.66e1.56 (1H, m, (NH-CH-CHH-CH-(CH3)2)AþB), 1.56e1.41 (2H, m,
(2 x NH-CH-CHH-CH-(CH3)2)AþB), 1.32e1.21 (1H, m, (NH-CH-CHH-
CH-(CH ) )
3 2 AþB), 1.06e0.91 (6H, m, (CH3-CH-CH3)AþB), 0.90e0.83
(3H, m, (CH3-CH-CH3)AþB), 0.83e0.75 (3H, m, (CH3-CH-CH3)AþB).
13C NMR (100.79 MHz, CDCl3-d1):
d
199.8, 172.47, 172.35, 164.68,
Procedure for Weinreb amides with three amino acids follows
the same condition as above mentioned for derivatives with two
amino acids, however the procedure was slightly altered in several
aspects. Reaction temperature was increased from ꢀ20 ꢁC to ꢀ8 ꢁC
and reaction time extended to 5 h. The equivalent of added LiAlH4
was increased from 1.5 to 3. Also LiAlH4 was not added via syringe
as a suspension but as a solid under the inflow of nitrogen in three
portions over the period of 2 h. In other aspect as work up the
procedure stays unchanged. The aldehydes 4g-o were obtained in
yields 20e75% as colourless oils or white solids.
164.58, 155.68, 155.64, 134.86, 133.07, 133.04, 132.34, 132.28, 131.25,
130.50, 129.66, 129.38, 129.33, 128.57, 128.52, 127.28, 122.40, 114.25,
114.20, 72.18, 72.12, 57.51, 57.47, 52.20, 42.29, 39.88, 39.77, 37.85,
24.96, 24.90, 24.88, 24.85, 23.24, 23.19, 22.98, 22.04, 21.96. HRMS:
m/z calc. for
C
26H33ClN2O4: 473.22016 [MþH]þ; 495.20211
[MþNa]þ; found: 473.22068 [MþH]þ; 495.20256 [MþNa]þ.
3.4.4. Characterization of 2-benzyloxy-5-chloro-N-((2S)-4-methyl-
1-oxo-1-(((2S)-1-oxo-3-phenylpropan-2-yl)amino)pentan-2-yl)
benzamide 4d
White solid; yield 97%; mp 161e165 ꢁC; ½aD20ꢃ ¼ ꢀ51.8ꢁ (c 0.4,
EtOAc); Rf (hex/EtOAc - 1/1) ¼ 0.36. IR (ATR): 3356, 3068, 2965,
2828, 1732, 1688, 1656, 1644, 1530, 1514, 1278, 1242, 1219, 987, 757,
3.4.1. Characterization of (2S)-2-benzyloxy-5-chloro-N-(4-methyl-
1-oxopentan-2-yl)benzamide 4a
Colorless oil; yield 46%; Rf (hex/EtOAce3/1) ¼ 0.31. IR (ATR):
741, 715, 700 cmꢀ1 1H NMR (400 MHz, CD2Cl2-d2):
. d 9.64 (1H, s,
3371, 2960, 1725, 1640, 1527, 1481, 1273, 1220, 990, 824, 744,
CHO), 8.19 (1H, d, J ¼ 2.8 Hz, NH-CH-CHH-CH-(CH3)2), 8.08 (1H, d,
J ¼ 7.2 Hz, NH-CH-CHH-Ph), 7.56 (1H, dd, J ¼ 2.8 Hz, J ¼ 8.8 Hz, Ar-
H), 7.53e7.46 (5H, m, Ar-H), 7.25e7.13 (6H, m, Ar-H), 6.97 (1H, d,
J ¼ 4.8 Hz, Ar-H), 5.22e5.21 (2H, ABq, J ¼ 1.6 Hz, O-CH2-Ph),
4.60e4.50 (2H, m, NH-CH-CH2-Ph, NH-CH-CHH-CH-(CH3)2), 3.23
719 cmꢀ1 1H NMR (400 MHz, CDCl3-d1):
. d 9.51 (1H, s, CHO),
8.30e8.15 (2H, m, NH-CH-CHH-Ph, Ar-H), 7.48e7.37 (6H, m, Ar-H),
7.04 (1H, d, J ¼ 8.8 Hz, Ar-H), 5.17e5.15 (2H, ABq, J ¼ 8.4 Hz, O-CH2-
Ph), 4.58e4.50 (1H, m, NH-CH-CHH-Ph), 1.56e1.47 (1H, m, NH-CH-

R. Jorda et al. / European Journal of Medicinal Chemistry 135 (2017) 142e158
153
(1H, dd, J ¼ 5.6 Hz, J ¼ 14 Hz, NH-CH-CHH-Ph), 3.03 (1H, dd,
3.4.7. Characterization of 2-benzyloxy-5-chloro-N-((2S)-4-methyl-
1-(((2S)-4-methyl-1-((4-methyl-1-oxopentan-2-yl)amino)-1-
oxopentan-2-yl)amino)-1-oxopentan-2-yl)benzamide 4g
J ¼ 5.6 Hz, J ¼ 14 Hz, NH-CH-CHH-Ph), 1.56e1.49 (1H, m, NH-CH-
CH2-CH-(CH3)2), 1.25e1.15 (1H, m, NH-CH-CHH-CH-(CH3)2),
1.11e1.01 (1H, m, NH-CH-CHH-CH-(CH3)2), 0.85 (3H, d, J ¼ 6.4 Hz,
CH3-CH-CH3), 0.79 (3H, d, J ¼ 6.4 Hz, CH3-CH-CH3). 13C NMR
Product is a mixture of two diastereomers, A and B. White solid;
yield 65%; mp 62.6e66.2 ꢁC; ½aD20ꢃ ¼ ꢀ11.9ꢁ (c 1.00, EtOAc); Rf (hex/
EtOAce1/1) ¼ 0.53. IR (ATR): 3289, 3069, 2957, 2871, 1733, 1635,
(100.62 MHz, CD2Cl2-d2):
d 199.2, 172.1, 164.3, 155.8, 136.3, 135.1,
133.0, 132.1, 129.4, 129.3, 129.2, 128.6, 128.6, 126.9, 126.8, 122.4,
114.3, 72.1, 59.9, 52.0, 38.9, 34.8, 24.7, 22.8, 21.6. CHN Analysis: Calc.
for C29H31ClN2O4 (507.02): C, 68.70; H, 6.16; N, 5.53. Found: C,
68.53 0.02; H 6.41 0.01; N, 5.48 0.02. HRMS: m/z calc. for
1537, 1483, 1468, 1272, 1245, 1123, 1007, 809, 752, 697 cmꢀ1 1H
.
NMR (400 MHz, CDCl3-d1):
d
9.53e9.48 (1H, m, (CHO)AþB),
8.25e8.05 (2H, m, 2 x (NH-CH-CHH-CH-(CH3)2)AþB), 7.55e7.35 (6H,
m, (NH-CH-CHH-CH-(CH3)2)AþB, (Ar-H)AþB), 7.1e7.05 (1H, m, (Ar-
H)AþB), 7.05e6.90 (1H, m, (Ar-H)AþB), 6.60 (1H, m, (Ar-H)AþB),
5.24e5.06 (2H, m, (O-CH2-Ph)AþB), 4.50e4.20 (3H, m, 3 x (NH-CH-
C
29H31ClN2O4: 507.20451 [MþH]þ; 529.18646 [MþNa]þ; found:
507.20525 [MþH]þ; 529.18719 [MþNa]þ.
CH2-CH-(CH3)2)AþB), 1.82e1.10 (9H,
(CH3)2)AþB), 1.00e0.80 (12H, m, 4 x (CH3)AþB), 0.73e0.72 (3H, m,
(CH3)AþB), 0.71e0.70 (3H, m, (CH3)AþB). 13C NMR (100.62 MHz,
CDCl3-d1): 200.35, 200.03, 172.47, 172.37, 172.23, 165.28, 165.00,
m 3 x (NH-CH-CH2-CH-
3.4.5. Characterization of 2-benzyloxy-5-chloro-N-((2S)-1-((4-
methyl-1-oxopentan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)
benzamide 4e
d
155.79, 155.75, 134.77, 133.43, 133.37, 132.4, 132.26, 132.07, 129.60,
129.55, 129.01, 128.88, 127.38, 127.35, 121.98, 121.91, 114.2, 72.26,
72.21, 57.52, 57.40, 53.64, 53.15, 52.03, 51.8, 40.13, 39.97, 39.91,
37.91, 37.44, 29.88, 25.17, 24.96, 24.89, 24.80, 23.31, 23.25, 23.16,
23.02, 22.99, 21.94, 21.88, 21.83, 21.74, 21.71. CHN Analysis: Calc. for
Product is a mixture of two diastereomers, A and B. Colourless
oil; yield 84%; ½aD20
ꢃ
¼ ꢀ5.2ꢁ (c 0.667, EtOAc); Rf (hex/EtOAce1/
1) ¼ 0.51. IR (ATR): 3297, 2957, 2360, 2342, 1734, 1636, 1539, 1271,
1003, 810, 747, 698 cmꢀ1 1H NMR (400 MHz, CDCl3-d1):
.
C32H44ClN3O5 (586.16): C, 65.57; H, 7.57; N, 7.17. Found: C,
d
9.45e9.35 (1H, m, (CHO)AþB), 8.44e8.40 (1H, m, (NH-CH-CHH-
65.54 0.01; H 7.79 0.01; N, 7.24 0.01. HRMS: m/z calc. for
CH-(CH ) )
3 2 AþB), 8.13 (1H, m, (NH-CH-CHH-Ph)AþB), 7.48e7.33 (6H,
C
32H44ClN3O5: 608.28617 [MþNa]þ; found: 608.25668 [MþNa]þ.
m, (Ar-H)AþB), 7.23e7.14 (3H, m, (Ar-H)AþB), 7.08e7.00 (2H, m, (Ar-
H)AþB), 6.98e6.92 (1H, m, (Ar-H)AþB), 6.82e6.67 (1H, m, (Ar-H)AþB),
5.24e5.07 (2H, m, (O-CH2-Ph)AþB), 4.93e4.84 (1H, m, (NH-CH-
3.4.8. Characterization of 2-benzyloxy-5-chloro-N-((2S)-4-methyl-
1-(((2S)-4-methyl-1-(((2S)-4-methyl-1-oxopentan-2-yl)amino)-1-
oxopentan-2-yl)amino)-1-oxopentan-2-yl)benzamide 4h
CHH-CH-(CH ) )
3 2 AþB), 4.38e4.24 (1H, m, (NH-CH-CHH- Ph)AþB),
3.07e2.95 (1H, m, (NH-CH-CHH-Ph)AþB), 2.90e2.82 (1H, m, (NH-
CH-CHH-Ph)AþB), 1.63e1.50 (1H, m, (NH-CH-CHH-CH-(CH3)2)AþB),
1.50e1.40 (1H, m, (NH-CH-CHH-CH-(CH3)2)AþB), 1.40e1.29 (1H, m,
(NH-CH-CHH-CH-(CH3)2)AþB), 0.90e0.80 (6H, m, (CH3-CH-
White solid; yield 75%; mp 60.6e65.2 ꢁC; ½aD20ꢃ ¼ ꢀ22.9ꢁ (c 1.00,
EtOAc); Rf (hex/EtOAce1/1) ¼ 0.53. IR (ATR): 3289, 3069, 2957,
2871, 1733, 1635, 1537, 1483, 1468, 1272, 1245, 1123, 1007, 809, 752,
697 cmꢀ1. 1H NMR (400 MHz, CDCl3-d1):
d 9.54 (1H, s, CHO), 8.24
CH3)AþB). 13C NMR (100.79 MHz, CDCl3-d1):
d 199.90,199.82,136.80,
(1H, d, J ¼ 5.6 Hz, NH-CH-CHH-CH-(CH3)2), 8.19 (1H, d, J ¼ 2.8 Hz,
NH-CH-CHH-CH-(CH3)2), 7.58e7.40 (6H, m, NH-CH-CHH-CH-
(CH3)2, Ar-H), 7.13 (1H, d, J ¼ 8.8 Hz, Ar-H), 7.10e7.04 (1H, m, Ar-H),
6.55 (1H, d, J ¼ 7.6 Hz, Ar-H), 5.24e5.21 (2H, ABq, J ¼ 9.2 Hz, O-CH2-
Ph), 4.55e4.38 (3H, m, 3 x NH-CH-CHH-CH-(CH3)2), 1.90e1.80 (1H,
m, NH-CH-CHH-CH-(CH3)2), 1.80e1.70 (2H, m, NH-CH-CH2-CH-
(CH3)2), 1.70e1.50 (4H, m, 3 x NH-CH-CHH-CH-(CH3)2, NH-CH-CH2-
CH-(CH3)2), 1.48e1.35 (1H, m, NH-CH-CH2-CH-(CH3)2), 1.26e1.16
(1H, m, NH-CH-CH2-CH-(CH3)2), 1.08e0.95 (9H, m, 3 x CH3), 0.92
(3H, d, J ¼ 6.4 Hz, CH3), 0.84 (3H, d, J ¼ 6.4 Hz, CH3), 0.78 (3H, d,
136.78, 135.17, 135.14, 133.02, 132.20, 132.18, 129.34, 129.30, 129.27,
129.18, 129.16, 128.76, 128.12, 128.05, 127.11, 127.08, 122.47, 122.44,
114.57, 114.55, 71.90, 71.85, 57.52, 57.41, 55.30, 55.18, 37.71, 37.69,
37.63, 37.58, 24.78, 24.65, 23.19, 23.15, 22.02, 21.97, 14.39. CHN
Analysis: Calc. for C29H31ClN2O4 (507.02): C, 68.70; H, 6.16; N, 5.53.
Found: C, 68.60 0.02; H 6.39 0.02; N, 5.31 0.02. HRMS: m/z
calc. for C29H31ClN2O4: 507.20451 [MþH]þ; 529.18646 [MþNa]þ;
found: 507.20490 [MþH]þ; 529.18675 [MþNa]þ.
J ¼ 6.4 Hz, CH3). 13C NMR (100.62 MHz, CDCl3-d1):
d 200.3, 172.4,
3.4.6. Characterization of 2-benzyloxy-5-chloro-N-((2S)-1-oxo-1-
(((2S)-1-oxo-3-phenylpropan-2-yl)amino)-3-phenylpropan-2-yl)
benzamide 4f
165.0, 155.7, 134.8, 133.4, 132.3, 129.6, 129.5, 129.4, 128.8, 128.7,
127.4, 122.0, 114.2, 72.2, 57.4, 53.2, 52.0, 40.1, 39.9, 37.7, 37.4, 29.9,
25.2, 25.1, 24.9, 24.8, 23.3, 23.2, 23.0, 21.9. CHN Analysis: Calc. for
White solid; yield 55%; mp 152e154 ꢁC; ½aD20ꢃ ¼ ꢀ54.8ꢁ (c 0.25,
EtOAc); Rf (hex/EtOAc - 2/3) ¼ 0.58. IR (ATR): 3290, 1738, 1633,
C32H44ClN3O5 (586.16): C, 65.57; H, 7.57; N, 7.17. Found: C,
65.52 0.01; H 7.69 0.02; N, 7.14 0.02. HRMS: m/z calc. for
1535, 1243, 1004, 756, 698 cmꢀ1. 1H NMR (500 MHz, CDCl3):
d
9.49
C
32H44ClN3O5: 608.28617 [MþNa]þ; found: 608.25662 [MþNa]þ.
(1H, s, CHO), 8.28 (1H, d, J ¼ 5.6 Hz, NH-CH-CHH-Ph), 8.09 (1H, d,
J ¼ 2.4 Hz, NH-CH-CHH-Ph), 7.45e7.36 (4H,m, Ar-H), 7.34e7.28 (2H,
m, Ar-H), 7.22e7.15 (3H, m, Ar-H), 7.13e7.07 (3H, m, Ar-H),
7.06e7.00 (2H, m, Ar-H), 6.99e6.92 (3H, m, Ar-H), 6.84 (1H, d,
J ¼ 5.2 Hz, Ar-H), 5.10e5.08 (2H, ABq, J ¼ 8.2 Hz, O-CH2-Ph), 4.81
(1H, q, J ¼ 5.4 Hz, NH-CH-CHH-Ph), 4.52 (1H, q, J ¼ 5.4 Hz, NH-CH-
CHH-Ph), 3.09 (1H, dd, J ¼ 6 Hz, J ¼ 11.2 Hz, NH-CH-CHH-Ph), 3.01
(1H, dd, J ¼ 6 Hz, J ¼ 11.6 Hz, NH-CH-CHH-Ph), 2.96 (1H, dd, J ¼ 6 Hz,
J ¼ 11.6 Hz, NH-CH-CHH-Ph), 2.77 (1H, dd, J ¼ 6 Hz, J ¼ 11.2 Hz, NH-
3.4.9. Characterization of 2-benzyloxy-5-chloro-N-((2S)-4-methyl-
1-(((2S)-4-methyl-1-(((2R)-4-methyl-1-oxopentan-2-yl)amino)-1-
oxopentan-2-yl)amino)-1-oxopentan-2-yl)benzamide 4i
Colourless oil; yield 52%; ½a2D0ꢃ ¼ ꢀ13.5ꢁ (c 1.00, EtOAc); Rf (hex/
EtOAce2/1) ¼ 0.51. IR (ATR): 3280, 3067, 3036, 2955, 2930, 2870,
1734, 1625, 1526, 1499, 1481, 1467, 1385, 1270, 1226, 1122, 1001, 808,
745, 696 cmꢀ1. 1H NMR (400 MHz, CDCl3-d1):
d 9.54 (1H, s, CHO),
8.25 (1H, d, J ¼ 5.2 Hz, NH-CH-CHH-CH-(CH3)2), 8.15 (1H, d,
J ¼ 2.8 Hz, NH-CH-CHH-CH-(CH3)2), 7.58e7.40 (5H, m, NH-CH-
CHH-CH-(CH3)2, Ar-H), 7.13 (1H, d, J ¼ 8.8 Hz, Ar-H), 7.07 (1H, d,
J ¼ 7.6 Hz, Ar-H), 6.55 (1H, d, J ¼ 8.4 Hz, Ar-H), 5.23e5.21 (2H, ABq,
J ¼ 9.2 Hz, O-CH2-Ph), 4.57e4.45 (2H, m, 2 x NH-CH-CHH-CH-
(CH3)2), 4.44e4.36 (1H, m, NH-CH-CHH-CH-(CH3)2), 1.92e1.69 (3H,
m, 3 x NH-CH-CHH-CH-(CH3)2), 1.65e1.50 (4H, m, 3 x NH-CH-CHH-
CH-(CH3)2 NH-CH-CH2-CH-(CH3)2), 1.43e1.35 (1H, m, NH-CH-CH2-
CH-CHH-Ph). 13C NMR (125.78 MHz, CDCl3):
d 198.9, 171.2, 164.2,
155.4, 136.6, 135.6, 134.9, 132.9, 132.2, 129.2, 129.12, 129.09, 129.09,
129.07, 128.63, 128.60, 127.9, 126.94, 129.91, 122.0, 114.2, 71.7, 59.8,
54.7, 36.8, 34.9. CHNAnalysis: Calc. for C32H29ClN2O4 (541.04): C,
71.04; H, 5.40; N, 5.18. Found: C, 71.02 0.02; H 5.42 0.01; N,
5.18 0.01. HRMS: m/z calc. for C32H29ClN2O4: 563.17081 [MþNa]þ;
found: 563.17087 [MþNa]þ.

154
R. Jorda et al. / European Journal of Medicinal Chemistry 135 (2017) 142e158
CH-(CH3)2), 1.25e1.15 (1H, m, NH-CH-CH2-CH-(CH3)2), 1.27e1.18
(1H, m, NH-CH-CH2-CH-(CH3)2), 1.03 (3H, d, J ¼ 6.4 Hz, CH3-CH-
CH3), 1.00 (3H, d, J ¼ 6.4 Hz, CH3-CH-CH3), 0.97 (3H, d, J ¼ 6.4 Hz,
CH3-CH-CH3), 0.92 (3H, d, J ¼ 6.4 Hz, CH3-CH-CH3), 0.83 (3H, d,
J ¼ 6.4 Hz, CH3-CH-CH3), 0.77 (3H, d, J ¼ 6.4 Hz, CH3-CH-CH3). 13C
3.4.12. Characterization of 2-benzyloxy-5-chloro-N-((2S)-4-
methyl-1-(((2S)-1-(((2R)-4-methyl-1-oxopentan-2-yl)amino)-1-
oxo-3-phenylpropan-2-yl)amino)-1-oxopentan-2-yl)benzamide 4l
White solid; yield 46%; mp 147.5e151 ꢁC; ½aD20ꢃ ¼ ꢀ28.9ꢁ (c 1.00,
EtOAc); Rf (hex/EtOAce1/1) ¼ 0.46. IR (ATR): 3279, 3065, 3033,
2956, 2929, 2869, 1733, 1635, 1531, 1498, 1482, 1466, 1384, 1270,
NMR (100.62 MHz, CDCl3-d1):
d 200.0, 172.5, 172.2, 165.3, 155.8,
134.8, 133.4, 132.3, 129.6, 129.4, 128.7, 127.4, 121.9, 114.2, 72.3, 57.5,
53.6, 51.8, 40.2, 40.0, 37.4, 25.2, 25.0, 24.9, 23.3, 23.2, 23.0, 21.9,
21.74, 21.70. CHN Analysis: Calc. for C32H44ClN3O5 (586.16): C,
65.57; H, 7.57; N, 7.17. Found: C, 65.46 0.02; H 7.69 0.02; N,
.
1243, 1006, 808, 746, 696 cmꢀ1 1H NMR (400 MHz, CDCl3-d1):
d
9.52 (1H, s, CHO), 8.16 (1H, d, J ¼ 4.8 Hz, NH-CH-CHH-CH-(CH3)2),
8.02 (1H, d, J ¼ 2.8 Hz, NH-CH-CHH-CH-(CH3)2), 7.58e7.438 (6H, m,
NH-CH-CHH-Ph, Ar-H), 7.27e7.22 (1H, m, Ar-H), 7.20e7.11 (4H, m,
Ar-H), 7.10e7.05 (1H, m, Ar-H), 7.04e7.00 (1H, m, Ar-H), 6.56 (1H, d,
J ¼ 8.4 Hz, Ar-H), 5.22e5.19 (2H, ABq, J ¼ 12.6 Hz, O-CH2-Ph),
4.81e4.72 (1H, m, NH-CH-CHH-Ph), 4.49e4.42 (1H, m, NH-CH-
CHH-CH-(CH3)2), 4.32e4.25 (1H, m, NH-CH-CHH-CH-(CH3)2),
3.27e3.10 (2H, m, NH-CH-CH2-Ph), 1.76e1.65 (2H, m, 2 x NH-CH-
CHH-CH-(CH3)2), 1.65e1.53 (1H, m, NH-CH-CHH-CH-(CH3)2),
1.48e1.39 (1H, m, NH-CH-CHH-CH-(CH3)2), 1.30e1.20 (1H, m, NH-
CH-CH2-CH-(CH3)2), 1.13e1.03 (1H, m, NH-CH-CH2-CH-(CH3)2),
1.03e0.97 (6H, m, 2 x CH3), 0.75 (3H, d, J ¼ 6.4 Hz, CH3-CH-CH3),
0.69 (3H, d, J ¼ 6.4 Hz, CH3-CH-CH3). 13C NMR (100.79 MHz, CDCl3-
7.14
0.02. HRMS: m/z calc. for C32H44ClN3O5: 608.28617
[MþNa]þ; 586.30423 [MþH]þ; found: 608.287235 [MþNa]þ;
586.30546 [MþH]þ.
3.4.10. Characterization of 2-benzyloxy-5-chloro-N-((S)-4-methyl-
1-(((S)-4-methyl-1-oxo-1-((1-oxo-3-phenylpropan-2-yl)amino)
pentan-2-yl)amino)-1-oxopentan-2-yl)benzamide 4j
Product is a mixture of two diastereomers, A and B. White solid;
yield 69%; mp 141.3e151.7 ꢁC; ½aD20
ꢃ
¼ ꢀ13.9ꢁ (c 1.00, EtOAc) Rf
(hex/EtOAce1/1) ¼ 0.35. IR (ATR):3292, 2957, 2360, 2341, 1652,
d1): d 199.8, 171.7, 171.4, 165.2, 155.8, 136.5, 134.7, 133.4, 132.5, 129.7,
1646, 1539, 1522, 1272, 1232, 748, 699 cmꢀ1
.
1H NMR (400 MHz,
129.4, 129.2, 128.8, 128.7, 127.4, 127.0, 121.5, 114.0, 72.3, 57.6, 53.7,
53.7, 39.7, 37.4, 37.1, 24.9, 24.7, 23.3, 23.0, 21.8, 21.6. CHN Analysis:
Calc. for C35H42ClN3O5 (620.17): C, 67.78; H, 6.83; N, 6.78. Found: C,
67.78 0.02; H 6.87 0.01; N, 6.71 0.02. HRMS: m/z calc. for
CDCl3-d1): 9.60e9.55 (1H, m, (CHO)AþB), 8.70e8.08 (2H, m, 2 x
d
(NH)AþB), 7.50e7.39 (6H, m, (NH, Ar-H)AþB), 7.30e7.12 (5H, m, (Ar-
H)AþB), 7.08e6.92 (2H, m, (Ar-H)AþB), 6.70e6.58 (1H, m, (Ar-H)AþB),
5.20e5.19 (2H, m, (O-CH2-Ph)AþB), 4.67e4.58 (1H, m, (NH-CH)AþB),
4.45e4.32 (2H, m, 2 x (NH-CH)AþB), 3.24e3.03 (2H, m, (NH-CH-
CH2-Ph)AþB),1.75e1.61 (1H, m, (NH-CHH-CH-(CH3)2)AþB),1.55e1.30
C
35H42ClN3O5: 620.28858 [MþH]þ; found: 620.28986 [MþH]þ.
3.4.13. Characterization of 2-benzyloxy-5-chloro-N-((2S)-4-
methyl-1-(((2S)-1-((4-methyl-1-oxopentan-2-yl)amino)-1-oxo-3-
phenylpropan-2-yl)amino)-1-oxopentan-2-yl)benzamide 4m
Product is a mixture of two diastereomers, A and B. Colourless
(4H, m, (NH-CHH-CH-(CH3)2)AþB
1.23e1.11 (1H, m, (NH-CHH-CH-(CH3)2)AþB), 1.00e0.65 (12H, m, 4 x
(CH3)AþB). 13C NMR (100.79 MHz, CDCl3-d1):
199.21, 199.16,
,
(NH-CHH-CH-(CH3)2)AþB),
d
172.36, 172.26, 172.20, 165.03, 164.92, 155.74, 155.70, 136.19, 136.10,
134.82, 133.29, 132.31, 132.26, 129.5, 129.47, 129.43, 129.35, 128.87,
128.67, 128.62, 127.31, 127.17, 122.10, 122.07, 114.20, 72.19, 60.02,
59.75, 53.12, 52.81, 52.12, 51.81, 40.44, 39.86, 39.66, 35.14, 35.08,
25.15, 25.00, 24.99, 24.87, 23.11, 23.03, 21.89, 21.83. CHN Analysis:
Calc. for C35H42ClN3O5 (620.18): C, 67.78; H, 6.83; N, 6.78. Found: C,
67.57 0.01; H 6.91 0.01; N, 6.69 0.01. HRMS: m/z calc. for
oil; yield 30%; ½aD20
ꢃ
¼ ꢀ25.2ꢁ (c 1.00, EtOAc); Rf (hex/EtOAce1/
1) ¼ 0.50. IR (ATR): 3276, 3066, 2957, 2871, 1734, 1635, 1598, 1541,
1271, 1246, 1124, 1008, 811, 750, 697 cmꢀ1
CD2Cl2-d2): 9.51e9.49 (1H, m, (CHO)AþB), 8.25e8.17 (1H, m, (NH-
CH-CHH-CH-(CH3)2)AþB), 8.05e7.95 (1H, m, (NH-CH-CHH-CH-
(CH ) )
.
1H NMR (400 MHz,
d
3 2 AþB), 7.65e7.44 (6H, m, (NH-CH-CHH-Ph)AþB), (Ar-H)AþB),
7.25e6.98 (7H, m, (Ar-H)AþB), 6.61e6.47 (1H, m, (Ar-H)AþB),
5.31e5.17 (2H, m, (O-CH2-Ph)AþB), 4.75e4.62 (1H, m, (NH-CH-CH2-
C
35H42ClN3O5: 642.27052 [MþNa]þ; found: 642.27215 [MþNa]þ.
Ph)AþB), 4.43e4.20 (2H, m,
2 x NH-CH-CH2-CH-(CH3)2)AþB),
3.4.11. Characterization of 2-benzyloxy-5-chloro-N-((2S)-4-methyl-
1-(((2S)-1-(((2S)-4-methyl-1-oxopentan-2-yl)amino)-1-oxo-3-
phenylpropan-2-yl)amino)-1-oxopentan-2-yl)benzamide 4k
Colourless oil; yield 20%; ½a2D0ꢃ ¼ ꢀ50.7ꢁ (c 1.00, EtOAc); Rf (hex/
EtOAce1/1) ¼ 0.50. IR (ATR): 3276, 3066, 2957, 2871, 1734, 1635,
3.28e3.09 (2H, m, (NH-CH-CH2-Ph)AþB), 1.76e1.11 (6H, m, 2 x (NH-
CH-CH2-CH-(CH3)AþB), 1.05e0.66 (12H, m, 4 x (CH3)AþB). 13C NMR
(100.79 MHz, CD2Cl2-d2):
d 200.84, 200.21, 171.58, 171.51, 171.40,
171.36, 165.16, 165.11, 156.02, 136.73, 136.50, 134.95, 133.44, 133.41,
132.06, 132.01, 129.36, 129.33, 129.18, 129.15, 128.85, 128.84, 121.63,
114.32, 72.28, 72.26, 57.61, 57.46, 53.69, 39.74, 37.28, 37.23, 37.10,
36.89, 24.85, 24.80, 24.60, 24.47, 23.19, 23.07, 22.75, 22.71, 22.26,
21.56, 21.48, 21.36, 21.31. CHN Analysis: Calc. for C35H42ClN3O5
1598, 1541, 1271, 1246, 1124, 1008, 811, 750, 697 cmꢀ1 1H NMR
.
(400 MHz, CD2Cl2-d2):
d
9.50 (1H, s, CHO), 8.22 (1H, d, J ¼ 4.8 Hz,
NH-CH-CHH-CH-(CH3)2), 7.99 (1H, d, J ¼ 2.8 Hz, NH-CH-CHH-CH-
(CH3)2), 7.65e7.48 (6H, m, NH-CH-CHH-Ph, Ar-H), 7.25e6.95 (7H,
m, Ar-H), 6.49 (1H, d, J ¼ 8 Hz, Ar-H), 5.28e5.25 (2H, ABq,
J ¼ 10.4 Hz, O-CH2-Ph), 4.73e4.63 (1H, m, NH-CH-CHH-Ph),
4.40e4.32 (1H, m, NH-CH-CHH-CH-(CH3)2), 4.30e4.20 (1H, m, NH-
CH-CHH-CH-(CH3)2), 3.27e3.10 (2H, m, NH-CH-CH2-Ph), 1.70e1.52
(3H, m, NH-CH-CH2-CH-(CH3)2, NH-CH-CH2-CH-(CH3)2), 1.50e1.40
(1H, m, NH-CH-C-HH-CH-(CH3)2), 1.35e1.25 (1H, m, NH-CH-C-HH-
CH-(CH3)2), 1.20e1.11 (1H, m, NH-CH-CH2-CH-(CH3)2), 1.03 (3H, d,
J ¼ 6 Hz, CH3-CH-CH3), 1.00 (3H, d, J ¼ 6 Hz, CH3-CH-CH3), 0.79 (3H,
d, J ¼ 6.4 Hz, CH3-CH-CH3), 0.74 (3H, d, J ¼ 6.4 Hz, CH3-CH-CH3). 13C
(620.17): C, 67.78; H, 6.83; N, 6.78. Found: C, 67.69
0.02; H
6.99
0.02; N, 6.89
0.01. HRMS: m/z calc. for C35H42ClN3O5:
642.27052 [MþNa]þ; found: 642.27105 [MþNa]þ.
3.4.14. Characterization of 2-benzyloxy-5-chloro-N-((2S)-4-
methyl-1-oxo-1-(((2S)-1-oxo-1-(((2S)-1-oxo-3-phenylpropan-2-yl)
amino)-3-phenylpropan-2-yl)amino)pentan-2-yl)benzamide 4n
Colourless oil; yield 21%; ½aD20
ꢃ
¼ ꢀ45.6ꢁ (c 0.125, EtOAc); Rf
(hex/EtOAce1/1) ¼ 0.33. IR (ATR): 3259, 3063, 2926, 2360, 2341,
1726, 1636, 1534, 1498, 1489, 1454, 1276, 1257, 803, 739, 728,
NMR (100.79 MHz, CD2Cl2-d2):
d
200.8, 171.5, 171.4, 165.1, 156.0,
701 cmꢀ1. 1H NMR (400 MHz, CD2Cl2-d2):
d 9.59 (1H, s, CHO), 8.14
136.5, 134.9, 133.5, 132.0, 129.6, 129.4, 129.1, 128.8, 128.6, 127.0,
125.6, 121.5, 114.3, 72.3, 57.5, 53.8, 39.6, 37.3, 36.8, 30.3, 24.8, 24.5,
23.2, 22.7, 21.4, 21.3. CHN Analysis: Calc. for C35H42ClN3O5 (620.17):
C, 67.78; H, 6.83; N, 6.78. Found: C, 67.59 0.01; H 6.89 0.02; N,
(1H, d, J ¼ 5.6 Hz, NH-CH-CHH-Ph), 8.11 (1H, d, J ¼ 2.8 Hz, NH-CH-
CHH-CH-(CH3)2), 7.59 (1H, dd, J ¼ 2.8 Hz, J ¼ 8.8 Hz, Ar-H),
7.57e7.48 (7H, m, NH-CH-CHH-Ph, Ar-H), 7.40e7.25 (5H, m, Ar-H),
7.24e7.15 (3H, m, Ar-H), 7.07e6.97 (2H, m, Ar-H), 6.64 (1H, d,
J ¼ 6.2 Hz, Ar-H), 5.24e5.22 (2H, ABq, J ¼ 10.4 Hz, O-CH2-Ph),
4.71e4.58 (2H, m, 2 x NH-CH-CHH-Ph), 4.35e4.25 (1H, m, NH-CH-
6.69
0.02. HRMS: m/z calc. for C35H42ClN3O5: 642.27052
[MþNa]þ; found: 642.27185 [MþNa]þ.

R. Jorda et al. / European Journal of Medicinal Chemistry 135 (2017) 142e158
155
CHH-CH-(CH3)2), 3.30e3.20 (1H, m, NH-CH-CHH-Ph), 3.20e3.10
(1H, m, NH-CH-CHH-Ph), 3.10e3.00 (2H, m, NH-CH-CH2-Ph),
1.48e1.35 (1H, m, NH-CH-CHH-CH-(CH3)2), 1.35e1.25 (1H, m, NH-
CH-CHH-CH-(CH3)2), 1.180e1.05 (1H, m, NH-CH-CH2-CH-(CH3)2),
0.80 (3H, d, J ¼ 6.4 Hz, CH3-CH-CH3), 0.74 (3H, d, J ¼ 6.4 Hz, CH3-CH-
CHH-CH-(CH3)2, NH-CH-CH2-CH-(CH3)2), 1.67e1.56 (1H, m, NH-
CH-CHH-CH-(CH3)2, 1.10e0.98 (6H, m, CH3-CH-CH3). 13C NMR
(100.79 MHz, CDCl3-d1):
d 173.6, 169.2, 160.1, 134.0, 126.1, 123.3,
119.7, 114.8, 61.9, 48.2, 40.9, 32.4, 25.1, 23.5, 21.6. CHN Analysis:
Calc. for C15H21ClN2O4 (328.79): C, 54.79; H, 6.44; N, 8.52. Found: C,
54.82 0.02; H 6.52 0.01; N, 8.57 0.02. HRMS: m/z calc. for
CH3). 13C NMR (100.79 MHz, CD2Cl2-d2):
d 199.2, 171.3, 171.0, 164.9,
156.0, 136.5, 135.0, 133.0, 132.1, 129.4, 129.3, 129.2, 128.7, 128.6,
127.0, 126.9, 125.6, 121.8, 121.7, 114.3, 72.2, 59.9, 53.9, 53.1, 39.4, 37.1,
34.8, 30.3, 24.8, 22.8, 21.4. CHN Analysis: Calc. for C38H40ClN4O5
C
15H21ClN2O4: 351.10821 [MþNa]þ; found: 351.10863 [MþNa]þ.
3.5.2. Characterization of (2S)-5-chloro-2-hydroxy-N-(1-
(methoxy(methyl)amino)-1-oxo-3-phenylpropan-2-yl)benzamide
5b
(654.19): C, 69.77; H, 6.16; N, 6.42. Found: C, 69.82
0.02; H
6.30
0.02; N, 6.37
0.02. HRMS: m/z calc. for C38H40ClN3O5:
676.25487 [MþNa]þ; found: 676.25641 [MþNa]þ.
White solid; yield 54%; mp 106e109 ꢁC; ½aD20ꢃ ¼ ꢀ31.8ꢁ (c 1.00,
EtOAc); Rf (hex/EtOAce1/1) ¼ 0.50. IR (ATR): 3448, 3294, 3065,
2942, 1630, 1588, 1558, 1541, 1486, 1474, 1365, 1296, 1232, 984,
3.4.15. Characterization of 2-benzyloxy-5-chloro-N-((2S)-4-
methyl-1-oxo-1-(((2S)-1-oxo-1-((1-oxo-3-phenylpropan-2-yl)
amino)-3-phenylpropan-2-yl)amino)pentan-2-yl)benzamide 4o
Product is a mixture of two diastereomers, A and B. Colourless
oil; yield 24%; ½a2D0ꢃ ¼ ꢀ22.5ꢁ (c 0.125, EtOAc); Rf (hex/EtOAce1/
1) ¼ 0.33. IR (ATR): 3259, 3063, 2926, 2360, 2341, 1726, 1636, 1534,
821 cmꢀ1. 1H NMR (400 MHz, DMSO-d6):
d 12.10 (1H, brs, Ar-OH),
9.10 (1H, d, J ¼ 8 Hz, NH-CH-CHH-Ph), 8.03e7.98 (1H, m, Ar-H), 7.43
(1H, dd, J ¼ 2.4 Hz, J ¼ 8.8 Hz, Ar-H), 7.34e7.163 (5H, m, Ar-H), 6.93
(1H, d, J ¼ 8.8 Hz, Ar-H), 5.22e5.10 (1H, m, NH-CH-CHH-Ph), 3.77
(3H, s, O-CH3), 3.14 (3H, s, N-CH3), 3.10e2.94 (2H, m, NH-CH-CH2-
1498, 1489, 1454, 1276, 1257, 803, 739, 728, 701 cmꢀ1
.
1H NMR
Ph. 13C NMR (100.79 MHz, DMSO-d6):
d 167.0, 160.2, 158.3, 138.0,
(400 MHz, CD2Cl2-d2):
d
9.61e9.55 (1H, s, (CHO)AþB), 8.16e8.06 (2H,
134.1, 134.0, 129.7, 129.0, 128.8, 128.2, 127.3, 123.2, 119.8, 117.8, 52.0,
37.0. CHN Analysis: Calc. for C18H19ClN2O4 (362.80): C, 59.59; H,
5.28; N, 7.72. Found: C, 59.20 0.02; H 5.20 0.02; N, 7.40 0.02.
HRMS: m/z calc. for C18H19ClN2O4: 385.09256 [MþNa]þ; found:
385.09324 [MþNa]þ.
m, 2 x (NH-CH)AþB), 7.62e7.57 (1H, m, (NH-CH)AþB), 7.56e7.48 (6H,
m, (Ar-H)AþB), 7.40e7.05 (11H, m, (Ar-H)AþB), 6.75e6.62 (1H, m,
(Ar-H)AþB), 5.29e5.160 (2H, m, (O-CH2-Ph)AþB), 4.74e4.65 (1H, m,
(NH-CH)AþB), 4.64e4.56 (1H, m, (NH-CH)AþB), 4.37e4.23 (1H, m,
(NH-CH)AþB), 3.30e3.00 (4H, m,
1.49e1.05 (3H, m, NH-CH-CH2-CH-(CH3)2)AþB), 0.84e0.65 (6H, m, 2
x (CH3)AþB). 13C NMR (100.79 MHz, CD2Cl2-d2):
199.2, 171.3, 171.0,
164.9, 156.0, 136.5, 135.0, 133.0, 132.1, 129.4, 129.3, 129.2, 128.7,
128.6, 127.0, 126.9, 125.6, 121.8, 121.7, 114.3, 72.2, 59.9, 53.9, 53.1,
39.4, 37.1, 34.8, 30.3, 24.8, 22.8, 21.4. CHN Analysis: Calc. for
2 x (NH-CH-CH2-Ph)AþB),
3.5.3. Characterization of 5-chloro-2-hydroxy-N-((2S)-1-(((2S)-1-
(methoxy(methyl)amino)-4-methyl-1-oxopentan-2-yl)amino)-4-
methyl-1-oxopentan-2-yl)benzamide 5c
d
White solid; yield 78%; mp 244.5e248.1 ꢁC; ½aD20ꢃ ¼ ꢀ4ꢁ (c 0.5,
EtOAc); Rf (hex/EtOAce1/1) ¼ 0.56. IR (ATR): 3335, 3245, 3076,
2958, 2360, 1636, 1590, 1549, 1474, 1370, 1233, 903, 826, 758, 719,
C
38H40ClN3O5 (654.19): C, 69.77; H, 6.16; N, 6.42. Found: C,
69.80 0.02; H 6.20 0.01; N, 6.31 0.02. HRMS: m/z calc. for
699 cmꢀ1. 1H NMR (400 MHz, CDCl3-d1):
d 12.33 (1H, brs, Ar-OH),
C
38H40ClN4O5: 676.25487 [MþNa]þ; found: 676.25649 [MþNa]þ.
7.89 (1H, d, J ¼ 6 Hz, NH-CH-CHH-CH-(CH3)2), 7.57 (1H, d, J ¼ 2.4 Hz,
NH-CH-CHH-CH-(CH3)2), 7.32 (1H, dd, J ¼ 2 Hz, J ¼ 8.4 Hz, Ar-H),
6.94 (1H, d, J ¼ 7.6 Hz, Ar-H), 6.86 (1H, d, J ¼ 8.8 Hz, Ar-H), 5.71e5.60
(1H, m, NH-CH-CHH-CH-(CH3)2), 4.85e5.75 (1H, m, NH-CH-CHH-
CH-(CH3)2), 3.87 (3H, s, O-CH3), 3.30 (3H, s, N-CH3), 1.82e1.70 (3H,
m, NH-CH-CHH-CH-(CH3)2, NH-CH-CHH-CH-(CH3)2, NH-CH-CH2-
CH-(CH3)2), 1.70e1.58 (3H, m, NH-CH-CHH-CH-(CH3)2, NH-CH-
CHH-CH-(CH3)2, NH-CH-CH2-CH-(CH3)2), 1.07e0.90 (12H, m, 4 x
3.5. General scheme and procedure for deprotected Weinreb amides
5a-h
Modified method of [32] was used: Benzyl-protected L-phenyl-
alanine Weinreb amide (0.6 g, 1.32 mmol) was dissolved in ethyl
acetate (100 mL). After addition of 10% Pd/C (0.1 g) the hydrogen
atmosphere was introduced to the sealed reaction mixture and the
reaction mixture was stirred at ambient temperature for 5 h. Pd/C
was separated by filtration. Volume of the solution was reduced
under reduced pressure. The residue p urified via column chro-
matography (silica gel 110 g, n-hexane/ethyl acetate (2:1)) to afford
CH3). 13C NMR (100.79 MHz, CDCl3-d1):
d 173.0, 172.7, 168.8, 160.1,
134.1, 126.0, 123.5, 119.9, 115.1, 61.8, 52.0, 48.3, 41.9, 41.8, 32.5, 25.0,
24.9, 23.5, 23.2, 21.9, 21.6. CHN Analysis: Calc. for C21H32ClN3O5
(441.95): C, 57.07; H, 7.30; N, 9.51. Found: C, 57.35
0.03; H
7.50
0.02; N, 9.42
0.03. HRMS: m/z calc. for C21H32ClN3O5:
debenzylated
L-phenylalanine Weinreb amide 5b in a 54% yield as a
464.19227 [MþNa]þ; found: 464.19309 [MþNa]þ.
white solid. Other derivatives were isolated in following yields: 5a
in yield 54% as white solid, 5c-f in yields 32e78% as white solids
and 5g,h in yields 77e93% as white solids. All derivatives were
prepared under similar conditions except for those with two and
three amino acids (5c-h). Instead of purification via column chro-
matography they were purified via crystallization.
3.5.4. Characterization of 5-chloro-2-hydroxy-N-((2S)-1-(((2S)-1-
(methoxy(methyl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-4-
methyl-1-oxopentan-2-yl)benzamide 5d
White solid; yield 78%; mp 222e226 ꢁC; ½aD20ꢃ ¼ ꢀ15.3ꢁ (c 1.00,
EtOAc); Rf (hex/EtOAce1/1) ¼ 0.33. IR (ATR):3337, 3251, 3071, 2947,
2360, 2341, 2360, 2341, 1638, 1590, 1552, 1479, 1368, 1261, 1231,
3.5.1. Characterization of (2S)-5-chloro-2-hydroxy-N-(1-
(methoxy(methyl)amino)-4-methyl-1-oxopentan-2-yl)benzamide
5a
903, 829, 716 cmꢀ1. 1H NMR (400 MHz, CDCl3-d1):
d 12.33 (1H, brs,
Ar-OH), 7.60 (1H, d, J ¼ 8 Hz, NH-CH-CHH-CH-(CH3)2), 7.55 (1H, d,
J ¼ 2 Hz, NH-CH-CHH-Ph), 7.37e7.32 (1H, m, Ar-H), 7.32e7.17 (5H,
m, Ar-H), 7.08 (1H, d, J ¼ 8.4 Hz, Ar-H), 6.91 (1H, d, J ¼ 8.8 Hz, Ar-H),
5.41e5.33 (1H, m, NH-CH-CHH-CH-(CH3)2), 4.80e4.70 (1H, m, NH-
CH-CHH-Ph), 3.77 (3H, s, O-CH3), 3.29 (3H, s, N-CH3), 3.22 (1H, dd,
J ¼ 5.6 Hz, J ¼ 13.6 Hz, NH-CH-CHH-Ph), 3.05 (1H, dd, J ¼ 5.6 Hz,
J ¼ 13.6 Hz, NH-CH-CHH-Ph), 1.79e1.66 (2H, m, NH-CH-CHH-CH-
(CH3)2, NH-CH-CH2-CH-(CH3)2), 1.65e1.55 (1H, m, NH-CH-CHH-
CH-(CH3)2), 0.98e0.89 (6H, m, CH3-CH-CH3). 13C NMR (100.79 MHz,
Colourless solid; yield 45%; mp 108.6e112.7 ꢁC; ½aD20ꢃ ¼ þ28.6ꢁ
(c 1.00, EtOAc); Rf (hex/EtOAce1/1) ¼ 0.45. IR (ATR):3279, 3073,
2959, 2871, 2360, 2342, 1639, 1592, 1551, 1477, 1366, 1296, 1232,
820, 719 cmꢀ1. 1H NMR (400 MHz, CDCl3-d1):
d 12.26 (1H, brs, Ar-
OH), 8.31 (1H, d, J ¼ 8 Hz, NH-CH-CHH-CH-(CH3)2), 7.55 (1H, d,
J ¼ 2.4 Hz, Ar-H), 7.26 (1H, dd, J ¼ 2.4 Hz, J ¼ 8.8 Hz, Ar-H), 6.74 (1H,
d, J ¼ 8.8 Hz, Ar-H), 5.35e5.26 (1H, m, NH-CH-CHH-CH-(CH3)2),
4.00 (3H, s, O-CH3), 3.37 (3H, s, N-CH3), 1.91e1.79 (2H, m, NH-CH-
CDCl3-d1): d 172.1,170.1,168.9,160.3,136.8,134.2,129.7,128.6,127.2,

156
R. Jorda et al. / European Journal of Medicinal Chemistry 135 (2017) 142e158
125.9, 123.1, 120.0, 114.8, 61.8, 52.0, 50.9, 45.1, 43.2, 32.3, 24.9, 23.2,
21.9. CHN Analysis: Calc. for C24H30ClN3O5 (475.97): C, 60.56; H,
6.35; N, 8.83. Found: C, 60.79 0.01; H 6.39 0.02; N, 8.83 0.03.
HRMS: m/z calc. for C24H30ClN3O5: 498.17662 [MþNa]þ; found:
498.17737 [MþNa]þ.
(CH3)2, NH-CH-CHH-CH-(CH3)2), 0.97 (9H, d, J ¼ 6.4 Hz, 3 x CH3),
0.92 (3H, d, J ¼ 6.4 Hz, CH3). 13C NMR (100.79 MHz, CDCl3-d1):
d
172.7, 171.7, 170.9, 169.0, 160.3, 136.9, 134.2, 129.5, 128.3, 127.0,
126.7, 123.3, 119.9, 115.6, 61.9, 54.1, 52.0, 47.3, 42.4, 41.2, 39.3, 32.5,
25.03, 25.01, 23.4, 23.0, 22.4, 22.2. CHN Analysis: Calc. for
C30H41ClN4O6 (589.12): C, 61.16; H, 7.01; N, 9.51. Found: C,
3.5.5. Characterization of 5-chloro-2-hydroxy-N-((2S)-1-(((2S)-1-
(methoxy(methyl)amino)-4-methyl-1-oxopentan-2-yl)amino)-1-
oxo-3-phenylpropan-2-yl)benzamide 5e
60.96 0.02; H 6.82 0.02; N, 9.80 0.02. HRMS: m/z calc. for
C
30H41ClN4O6: 611.26068 [MþNa]þ; found: 611.26186 [MþNa]þ.
White solid; yield 32%; mp 221.7e224.4 ꢁC; ½aD20ꢃ ¼ ꢀ42.3ꢁ (c
1.00, EtOAc); Rf (hex/EtOAce1/1) ¼ 0.43. IR (ATR):3345, 3253, 3069,
2970, 2359, 2341, 1616, 1596, 1552, 1477, 1224, 827, 820, 743, 717,
3.5.8. Characterization of N-((5S,8S,11S)-5,8-dibenzyl-3,13-
dimethyl-4,7,10-trioxo-2-oxa-3,6,9-triazatetradecan-11-yl)-5-
chloro-2-hydroxybenzamide 5h
698 cmꢀ1. 1H NMR (400 MHz, CDCl3-d1):
d
12.20 (1H, brs, Ar-OH),
White solid; yield 93%; mp 130e133.7 ꢁC; ½aD20ꢃ ¼ ꢀ20.0ꢁ (c 1.00,
EtOAc); Rf (hex/EtOAce1/1) ¼ 0.37. IR (ATR):3291, 3066, 2957, 2361,
7.96 (1H, d, J ¼ 7.6 Hz, NH-CH-CHH-CH-(CH3)2), 7.51 (1H, d,
J ¼ 2.4 Hz, NH-CH-CHH-Ph), 7.33e7.21 (6H, m, Ar-H), 7.19 (1H, d,
J ¼ 8.8 Hz, Ar-H), 6.84 (1H, d, J ¼ 8.8 Hz, Ar-H), 5.25e5.15 (1H, m,
NH-CH-CHH-Ph), 5.06e4.99 (1H, m, NH-CH-CHH-CH-(CH3)2), 3.81
(3H, s, O-CH3), 3.31 (3H, s, N-CH3), 3.20 (2H, d, J ¼ 6.4 Hz, NH-CH-
CH2-Ph), 1.75e1.65 (1H, m, NH-CH-CH2-CH-(CH3)2), 1.65e1.55 (2H,
m, NH-CH- CH2-CH-(CH3)2, 1.00 (3H, d, J ¼ 6.4 Hz, CH3-CH-CH3),
0.92 (3H, d, J ¼ 6.4 Hz, CH3-CH-CH3). 13C NMR (100.79 MHz, CDCl3-
2343, 1619, 1591, 1535, 1493, 1478, 1290, 1222, 824, 743, 699 cmꢀ1
.
1H NMR (400 MHz, CDCl3-d1):
d 12.50 (1H, brs, Ar-OH), 8.18e8.02
(2H, m, NH-CH-CHH-CH-(CH3)2, NH-CH-CHH-Ph), 7.62 (1H, d,
J ¼ 2.4 Hz, NH-CH-CHH-Ph), 7.33 (1H, dd, J ¼ 2.5 Hz, J ¼ 8.8 Hz, Ar-
H), 7.26e7.05 (6H, m, Ar-H), 7.04e6.82 (6H, m, Ar-H), 5.35e5.22
(2H, m, 2 x NH-CH-CHH-Ph), 4.83e4.73 (1H, m, NH-CH-CHH-CH-
(CH3)2), 3.55 (3H, s, O-CH3), 3.14 (3H, s, N-CH3), 3.10e2.85 (4H, m, 2
x NH-CH-CH2-Ph), 1.78e1.65 (2H, m, NH-CH-CHH-CH-(CH3)2, NH-
CH-CH2-CH-(CH3)2), 1.61e1.50 (1H, m, NH-CH-CHH-CH-(CH3)2),
0.96e0.85 (6H, m, 2 x CH3). 13C NMR (100.79 MHz, CDCl3-d1):
d1): d 173.1, 171.3, 168.6, 160.1, 136.5, 134.2, 129.5, 128.7, 127.3, 126.0,
123.5, 119.9, 115.1, 61.9, 54.6, 48.2, 42.0, 38.7, 32.5, 25.0, 23.4, 21.8.
CHN Analysis: Calc. for C24H30ClN3O5 (475.97): C, 60.56; H, 6.35; N,
8.83. Found: C, 60.79 0.01; H 6.43 0.01; N, 8.79 0.01. HRMS: m/
z calc. for C24H30ClN3O5: 498.17662 [MþNa]þ; found: 498.17758
[MþNa]þ.
d
171.9, 171.2, 170.9, 169.2, 160.5, 136.3, 136.1, 134.2, 129.6, 129.5,
128.5, 128.3, 127.1, 127.0, 126.6, 123.2, 120.0, 115.7, 61.8, 54.1, 51.9,
50.2, 41.1, 39.8, 39.2, 32.5, 25.1, 23.3, 22.1. CHN Analysis: Calc. for
C33H39ClN4O6 (623.14): C, 63.61; H, 6.31; N, 8.99. Found: C,
3.5.6. Characterization of 5-chloro-2-hydroxy-N-((2S)-1-((1-
(methoxy(methyl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-1-
oxo-3-phenylpropan-2-yl)benzamide 5f
63.81 0.01; H 6.49 0.01; N, 8.75 0.02. HRMS: m/z calc. for
C
33H39ClN4O6: 645.24503 [MþNa]þ; found: 645.24582 [MþNa]þ.
Product is a mixture of two diastereomers, A and B. White solid;
3.6. General scheme and procedure for deprotected aldehydes 6a-c
and alcohols 7a,b
yield 42%; mp 197.8e200.2 ꢁC; ½aD20
ꢃ
¼ ꢀ51.0ꢁ (c 0.4, EtOAc); Rf
(hex/EtOAce1/1) ¼ 0.22. IR (ATR): 3305, 3067, 2939, 2352, 2341,
1657, 1633, 1592, 1547, 1493, 1227, 823, 748, 720, 699, 669 cmꢀ1. 1H
Reduction of Weinreb amide to aldehyde was done according to
NMR (400 MHz, CDCl3-d1):
d
12.19e12.16 (1H, m, (Ar-OH)AþB),
similar reaction described in Ref. [31]. L-leucine-L-phenylalanine
7.62e7.52 (1H, m, (NH-CH-CHH-Ph)AþB), 7.48e7.38 (1H, m, (NH-
CH-CHH-Ph)AþB), 7.40e7.18 (9H, m, (Ar-H)AþB), 7.18e7.09 (2H, m,
(Ar-H)AþB), 6.95e6.82 (2H, m, (Ar-H)AþB), 5.37e5.25 (1H, m, (NH-
CH-CHH-Ph)AþB), 4.98e4.33 (1H, m, (NH-CH-CHH-Ph)AþB),
3.75e3.60 (3H, m, (N-CH3)AþB), 3.32e3.25 (3H, m, (O-CH3)AþB),
3.24e2.90 (4H, m, 2 x (NH-CH-CH2-Ph)AþB). 13C NMR (100.79 MHz,
Weinreb amide 5d (0.166 g, 0.349 mmol) was dissolved in a secured
three-neck round-bottom flask in dry tetrahydrofurane (40 mL)
under nitrogen atmosphere and cooled to ꢀ5 ꢁC. LiAlH4 (0.039 g,
1.03 mmol) was added in two portions (0.026 g at the beginning
and 0.013 g after 45 min) under the inflow of nitrogen. The reaction
was stirred for the total of 90 min, then quenched with saturated
solution of NH4Cl in H2O (20 mL) and 10% HCl (3 mL) and extracted
with ethyl acetate (3 ꢂ 40 mL). The combined organic phase was
washed with H2O (2 ꢂ 20 mL) and dried over Na2SO4. The product
6a was isolated by reducing the volume of solvent and drying on
rotary vacuum evaporator in 83% yield as colourless oil.
Derivatives with three amino acids undergo the reaction under
same conditions except for the reaction period, which was
extended to 1 h. From the reaction mixture aldehydes 6b,c in yields
between 30 and 48% were isolated as white solid or colourless oil.
Also corresponding alcohols 7a,b in yields between 40 and 44%
were isolated.
CDCl3-d1):
d 170.9, 170.5, 168.6, 160.2, 136.4, 136.0, 134.4, 129.6,
129.5, 128.8, 128.6, 127.4, 127.3, 125.8, 123.5, 120.1, 115.0, 61.8, 54.6,
50.9, 38.5, 38.4, 32.4. CHN Analysis: Calc. for C27H28ClN3O5
(509.98): C, 63.59; H, 5.53; N, 8.24. Found: C, 63.58
0.01; H
5.55
0.01; N, 8.24
0.01. HRMS: m/z calc. for C27H28ClN3O5:
532.16097 [MþNa]þ; found: 532.16205 [MþNa]þ.
3.5.7. Characterization of N-((5S,8S,11S)-8-benzyl-5-isobutyl-3,13-
dimethyl-4,7,10-trioxo-2-oxa-3,6,9-triazatetradecan-11-yl)-5-
chloro-2-hydroxybenzamide 5g
White solid; yield 77%; mp 130.4e134.6 ꢁC; ½aD20ꢃ ¼ ꢀ31.5ꢁ (c
1.00, EtOAc); Rf (hex/EtOAce1/1) ¼ 0.41. IR (ATR): 3298, 3067, 2957,
2870, 2361, 2341, 1637, 1592, 1538, 1474, 1367, 1290, 1220, 824,
3.6.1. Characterization of 5-Chloro-2-hydroxy-N-((2S)-4-methyl-1-
oxo-1-(((2S)-1-oxo-3-phenylpropan-2-yl)amino)pentan-2-yl)
benzamide 6a
699 cmꢀ1. 1H NMR (400 MHz, CDCl3-d1):
d 12.45 (1H, brs, Ar-OH),
8.08 (1H, d, J ¼ 8 Hz, NH-CH-CHH-CH-(CH3)2), 7.95 (1H, d, J ¼ 8 Hz,
NH-CH-CHH-CH-(CH3)2), 7.69 (1H, d, J ¼ 2 Hz, NH-CH-CHH-Ph),
7.39 (1H, dd, J ¼ 2.5 Hz, J ¼ 8.8 Hz, Ar-H), 7.07e6.97 (5H, m, Ar-H),
6.97e6.91 (1H, m, Ar-H), 6.88 (1H, d, J ¼ 8.4 Hz, Ar-H), 5.30e5.20
(1H, m, NH-CH-CHH-CH-(CH3)2), 5.20e5.10 (1H, m, NH-CH-CHH-
CH-(CH3)2), 4.84e4.72 (1H, m, NH-CH-CHH-Ph), 3.81 (3H, s, O-CH3),
3.28 (3H, s, N-CH3), 2.99 (2H, d, J ¼ 7.2 Hz, NH-CH-CH2-Ph),
1.82e1.58 (4H, m, NH-CH-CHH-CH-(CH3)2, NH-CH-CHH-CH-(CH3)2,
2 x NH-CH-CH2-CH-(CH3)2), 1.58e1.48 (2H, m, NH-CH-CHH-CH-
Colourless oil; yield 83%; ½a2D0ꢃ ¼ ꢀ25.8ꢁ (c 1.00, EtOAc); Rf (hex/
EtOAce1/1) ¼ 0.38. IR (ATR): 3302, 3066, 2958, 2928, 1736, 1640,
1593, 1536, 1485, 1366, 1290, 1255, 1228, 1114, 823, 750, 721,
701 cmꢀ1. 1H NMR (400 MHz, CDCl3-d1):
d 12.11 (1H, s, Ar-OH), 9.72
(1H, s, CHO), 7.62 (1H, d, J ¼ 8 Hz, NH-CH-CHH-CH-(CH3)2), 7.52
(1H, d, J ¼ 2.8 Hz, NH-CH-CHH-Ph), 7.33e7.18 (6H, m, Ar-H), 6.90
(1H, d, J ¼ 7.2 Hz, Ar-H), 6.70 (1H, d, J ¼ 6.8 Hz, Ar-H), 4.84 (1H, q,
J ¼ 6.4 Hz, NH-CH-CHH-Ph), 4.74e4.63 (1H, m, NH-CH-CHH-CH-

R. Jorda et al. / European Journal of Medicinal Chemistry 135 (2017) 142e158
157
(CH3)2), 3.33e3.15 (2H, m, NH-CH-CH2-Ph), 1.85e1.68 (3H, m, NH-
CH-CH2-CH-(CH3), NH-CH-CH2-CH-(CH3), 1.05e0.80 (6H, m, CH3-
d 198.2, 172.7, 169.1,
160.1, 135.1, 134.5, 129.5, 129.0, 127.5, 125.9, 123.6, 120.1, 114.8, 60.1,
42.3, 39.3, 31.4, 30.2, 29.7, 24.6, 23.3, 22.9, 22.4. CHN Analysis: Calc.
for C28H38ClN3O5 (532.07): C, 63.21; H, 7.20; N, 7.90. Found: C,
63.32 0.02; H 7.29 0.02; N, 7.82 0.02. HRMS: m/z calc. for
CH-CH3). 13C NMR (100.79 MHz, CD2Cl2-d2):
C
28H38ClN3O5: 532.25728 [MþH]þ; found: 532.25776 [MþH]þ.
52.0, 40.9, 35.2, 25.0, 23.1, 22.1. CHN Analysis: Calc. for
C
22H25ClN2O4 (416.90): C, 63.38; H, 6.04; N, 6.72. Found: C,
3.6.5. Characterization of N-((2S)-1-(((2S)-1-(((2S)-1-hydroxy-3-
phenylpropan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-4-
methyl-1-oxopentan-2-yl)-5-chloro-2-hydroxybenzamide 7b
Colourless oil; yield 40%; Rf (hex/EtOAce1/1) ¼ 0.20. IR (ATR):
3304, 3066, 2958, 2361, 2339, 1726, 1640, 1597, 1546, 1536, 1494,
1288, 1257, 1228, 1113, 1034, 903, 823, 744, 700, 669 cmꢀ1. 1H NMR
63.68 0.02; H 7.15 0.02; N, 5.82 0.02. HRMS: m/z calc. for
C
22H25ClN2O4: 439.13951 [MþNa]þ; found: 439.14021 [MþNa]þ.
3.6.2. Characterization of N-((2S)-4-methyl-1-(((2S)-1-(((2S)-4-
methyl-1-oxopentan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)
amino)-1-oxopentan-2-yl-5-chloro-2-hydroxybenzamide 6b
White solid; yield 48%; mp 145e148.2 ꢁC; ½aD20ꢃ ¼ ꢀ44.3ꢁ (c 1.00,
EtOAc) Rf (hex/EtOAce1/1) ¼ 0.47. IR (ATR): 3307, 3066, 2958, 2871,
2360, 2342, 1734, 1637, 1596, 1533, 1496, 1489, 1368, 1287, 1227,
(400 MHz, CD2Cl2-d2):
d
12.10 (1H, s, Ar-OH), 7.80 (1H, d, J ¼ 6 Hz,
NH), 7.66 (1H, d, J ¼ 2.4 Hz, NH), 7.47e7.35 (2H, m, NH, Ar-H),
7.35e7.15 (10H, m, Ar-H), 7.00 (1H, d, J ¼ 8.8 Hz, Ar-H), 6.68 (1H, d,
J ¼ 7.2 Hz, Ar-H), 4.90e4.70 (2H, m, 2 x NH-CH), 4.20e4.10 (1H, m,
NH-CH), 3.50e3.40 (2H, m, CH2-OH), 3.20e3.05 (2H, m, NH-CH-
CH2-Ph), 3.85e3.70 (2H, m, NH-CH-CH2-Ph), 2.31 (1H, brs, CH2-OH),
1.80e1.60 (2H, m, NH-CH-CHH-CH-(CH3)2), 1.40e1.30(1H, m, NH-
1114, 824, 746, 700, 669 cmꢀ1
d
.
1H NMR (400 MHz, CD2Cl2-d2):
12.01 (1H, s, Ar-OH), 9.44 (1H, s, CHO), 7.61 (1H, d, J ¼ 8 Hz, NH-
CH-CHH-Ph), 7.44 (1H, d, J ¼ 2.8 Hz, NH-CH-CHH-CH-(CH3)2), 7.42
(1H, d, J ¼ 2.8 Hz, NH-CH-CHH-CH-(CH3)2), 7.35e7.19 (5H, m, Ar-H),
7.12e7.03 (1H, m, Ar-H), 6.99 (1H, d, J ¼ 8.8 Hz, Ar-H), 6.75e6.65
(1H, m, Ar-H), 4.88 (1H, q, J ¼ 7.2 Hz, NH-CH-CHH-Ph), 4.80e4.72
(1H, m, NH-CH-CHH-CH-(CH3)2), 4.47e4.39 (1H, m, NH-CH-CHH-
CH-(CH3)2), 3.25e3.12 (2H, m, NH-CH-CH2-Ph), 1.80e1.60 (5H, m, 2
x NH-CH-CH2-CH-(CH3) 2, NH-CH-CH2-CH-(CH3)2), 1.45e1.30 (1H,
m, NH-CH-CH2-CH-(CH3)2), 1.00e0.87 (12H, m, 4 x CH3). 13C NMR
CH-CHH-CH-(CH3)2), 1.00e0.85 (6H, m,
(100.79 MHz, CDCl3-d1): 173.5, 171.0, 168.8, 159.6, 137.5, 136.3,
2 x
CH3). 13C NMR
d
135.9, 134.4, 129.5, 129.4, 129.3, 129.0, 128.9, 128.8, 126.8, 126.2,
123.9, 120.1, 63.0, 59.9, 55.4, 53.1, 52.2, 41.3, 38.8, 36.9, 35.3, 29.9,
25.0, 23.3, 22.1. CHN Analysis: Calc. for C31H36ClN3O5 (566.09): C,
65.77; H, 6.41; N, 7.42. Found: C, 64.49 0.02; H 6.45 0.02; N,
6.62
0.02. HRMS: m/z calc. for C31H36ClN3O5: 588.22357
(100.79 MHz, CD2Cl2-d2):
d
199.5, 171.9, 171.1, 168.8, 159.9, 136.3,
[MþNa]þ; found: 588.22440 [MþNa]þ.
134.4, 129.4, 128.8, 127.2, 126.3, 123.6, 120.0, 115.3, 57.5, 54.6, 52.1,
41.1, 38.3, 37.7, 25.0, 24.7, 22.9, 22.8, 21.9, 21.7. CHN Analysis: Calc.
for C28H36ClN3O5 (530.06): C, 63.45; H, 6.85; N, 7.93. Found: C,
63.31 0.02; H 6.86 0.01; N, 7.89 0.01. HRMS: m/z calc. for
3.7. Antiproliferative assays
Cancer cell lines of different histological origin were cultivated
according to the manufacturer's instructions. The cells were
assayed with compounds using three-fold dilutions in triplicate.
Treatment lasted for 72 h, followed by addition of Calcein AM so-
lution, and measurement of the fluorescence of live cells at 485/
538 nm (ex/em) with a Fluoroskan Ascent microplate reader
(Labsystems). The GI50 value, the drug concentration lethal to 50%
of the tumour cells, was calculated from the obtained dose
response curves.
C
28H36ClN3O5: 552.22357 [MþNa]þ; found: 552.22415 [MþNa]þ.
3.6.3. Characterization of N-((2S)-4-methyl-1-oxo-1-(((2S)-1-oxo-
1-(((2S)-1-oxo-3-phenylpropan-2-yl)amino)-3-phenylpropan-2-yl)
amino)pentan-2-yl)-5-chloro-2-hydroxybenzamide 6c
Colourless oil; yield 30%; Rf (hex/EtOAce1/1) ¼ 0.38. IR (ATR):
3289, 3065, 2960, 2925, 1633, 1593, 1537, 1494, 1258, 1081, 1017,
797, 708, 697, 649 cmꢀ1. 1H NMR (400 MHz, CD2Cl2-d2):
d 12.08 (1H,
s, Ar-OH), 9.52 (1H, s, CHO), 7.65e7.58 (2H, m, 2 x NH), 7.43e7.10
(12H, m, NH, Ar-H), 6.99e6.93 (1H, m, Ar-H), 6.85e6.70 (1H, m, Ar-
H), 4.95e4.83 (1H, m, NH-CH), 4.82e4.70 (1H, m, NH-CH),
4.70e4.60 (1H, m, NH-CH), 3.2e3.00 (4H, m, 2 x NH-CH-CH2-Ph),
1.79e1.70 (3H, m, NH-CH-CH2-CH-(CH3)2), 1.00e0.88 (6H, m, 2 x
3.8. Proteasome-Glo
™ chymotrypsin-like cell based assay
Multiple myeloma U266 cells were plated at the appropriate
density (20,000 cells/well) in 96-well LumiNunc™ plates and
treated with different concentrations of tested compounds for
1 h at 37 ꢁC in duplicate. The activity of the 26S proteasome was
CH3). 13C NMR (100.79 MHz, CD2Cl2-d2):
d 198.5, 172.3, 171.0, 168.9,
159.9, 136.2, 135.8, 134.3, 129.45, 129.35, 128.9, 128.8, 127.3, 126.4,
123.5, 119.9, 60.0, 54.7, 52.1, 41.0, 38.6, 35.1, 25.0, 22.9, 21.8. CHN
Analysis: Calc. for C31H34ClN3O5 (564.07): C, 66.01; H, 6.08; N, 7.45.
Found: C, 65.97 0.02; H 6.17 0.01; N, 7.29 0.01. HRMS: m/z calc.
measured immediately by hydrolysis of the b5 (chymotrypsin-like)
substrate Suc-LLVY-aminoluciferin in the presence of luciferase
using the Proteasome-Glo™ assay reagents according to the man-
ufacturer's instructions (Promega). Luminescence was measured
using a TECAN Infinite® M200 Multi-mode Microplate Reader.
Proteasome activity was normalised against activity of untreated
cells and the IC50 values were calculated from the obtained dose
response curves.
for
C
31H34ClN3O5: 586.20792 [MþNa]þ; found: 586.20897
[MþNa]þ.
3.6.4. Characterization of N-((2S)-1-(((2S)-1-(((2S)-1-hydroxy-4-
methylpentan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-4-
methyl-1-oxopentan-2-yl)-5-chloro-2-hydroxybenzamide 7a
Colourless oil; yield 44%; Rf (hex/EtOAce1/1) ¼ 0.18. IR (ATR):
3307, 3066, 2958, 2871, 2360, 2341, 1734, 1636, 1595, 1540, 1533,
1496, 1473, 1457, 1367, 1288, 1230, 1114, 1068, 1031, 903, 823, 745,
3.9. Time lapse microscopy
For time lapse microscopy, cells were plated at the appropriate
density (10,000 cells/well) in 96- plates and treated with different
concentrations of tested compounds for 72 h. Time lapse images
and videos were captured by IncuCyte ZOOM® (Essen BioScience).
An acquisition of images (1 image per hour) in a green channel and
further an integrated software analysis was used for quantification
of cells expressing GFP. Cell proliferation was monitored by ana-
lysing the occupied area (% of confluence) of cell images over time
with confluence image mask (Gold) using IncuCyte™ cell
700, 669 cmꢀ1. 1H NMR (500 MHz, CDCl3-d1):
d 12.85 (1H, brs, Ar-
OH), 9.38 (1H, brs, NH), 7.44 (1H, brs, NH), 7.42 (1H, brs, NH),
7.50e7.23 (6H, m, Ar-H), 7.23e7.00 (2H, m, Ar-H), 5.40e5.20 (1H, m,
NH-CH), 5.20e5.00 (1H, m, NH-CH), 4.00e3.80 (1H, m, NH-CH),
3.39 (2H, brs, CH2-OH), 3.24 (1H, brs, CH2-OH),1.90e1.30 (8H, m, 2 x
CH2-CH-(CH3)2, CH2-Ph), 1.10e0.60 (12H, m, 4 x CH3). 13C NMR
(125.78 MHz, CDCl3-d1):
d 172.5, 172.2, 171.1, 168.2, 166.1, 135.6,
134.1, 133.5, 129.4, 128.8, 127.5, 126.9, 124.4, 64.4, 61.6, 51.1, 47.5,

158
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The manuscript was written through contributions of all au-
thors. All authors have given approval to the final version of the
manuscript.
Acknowledgements
The authors wish to acknowledge the institutional support of
the Faculty of Chemical Technology, University of Pardubice, the
Ministry of Education, Youth and Sports of the Czech Republic
(National Program of Sustainability IeGrant LO1204), and Palacky
University in Olomouc (IGA_PrF_2017_014).
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Appendix A. Supplementary data
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Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2017.04.027.
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