Discovery and SAR studies of a novel class of cytotoxic 1,4-disubstituted piperidines via Ugi reaction

Article abstract of DOI:10.1016/j.ejmech.2014.06.026

Herein we report a novel class of 1,4-disubstituted piperidines as potential anticancer agents. One-step and efficient synthesis of a structurally diverse library of piperidine-based analogs with five points of diversity has been developed using the Ugi four-component reaction. A structure-activity relationship (SAR) study showed that the presence of a benzyl or a Boc group at the N-1 position together with two or three aromatic groups at the C-4 position of the piperidine ring are important for optimal cytostatic properties. Compounds 20, 22, 27 and 29 were found to be the most potent with a 50% inhibitory concentration (IC₅₀) ranging between 3 and 9.5 μM in the cancer cell lines evaluated. The NCI60 screen confirmed this 50% cytostatic concentration range for compound 20, irrespective of the nature of the tumor cell lines evaluated. The NCI COMPARE algorithm did not show any significant correlation between the growth inhibition profile of compound 20 with the NCI database compound profiles suggesting a potential novel mechanism of action.

Full text of DOI:10.1016/j.ejmech.2014.06.026

Accepted Manuscript
Discovery and SAR studies of a novel class of cytotoxic 1,4-disubstituted piperidines
via Ugi reaction
Sonia de Castro, Dr. María-José Camarasa, Jan Balzarini, Sonsoles Velázquez, Dr.
PII:
S0223-5234(14)00545-5
10.1016/j.ejmech.2014.06.026
EJMECH 7069
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 25 April 2014
Revised Date: 4 June 2014
Accepted Date: 12 June 2014
Please cite this article as: S. de Castro, M.-J. Camarasa, J. Balzarini, S. Velázquez, Discovery and SAR
studies of a novel class of cytotoxic 1,4-disubstituted piperidines via Ugi reaction, European Journal of
Medicinal Chemistry (2014), doi: 10.1016/j.ejmech.2014.06.026.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Discovery and SAR studies of a novel class of
cytotoxic 1,4-disubstituted piperidines via Ugi
reaction
a
a
b
a
Sonia de Castro, * María-José Camarasa, Jan Balzarini, and Sonsoles Velázquez *
a
Instituto de Química Médica (C.S.I.C.), Juan de la Cierva 3, E-28006 Madrid, Spain
Rega Institute for Medical Research, KU Leuven, Minderbroedersstraat 10, B-3000 Leuven,
Belgium
b
Abbreviations- Alloc, allyloxycarbonyl; Boc, tert-butoxycarbonyl; Caki-1, human kidney carcinoma
cell lines; Cbz, benzyloxycarbonyl; CEM, human T-lymphocyte cell lines; CNS, central nervous system;
Fmoc, fluorenylmethoxycarbonyl; HeLa, human cervix carcinoma cell lines; Huh-7, human liver
hepatoma cell lines; MCRs, multicomponent condensation reactions; NCI60, National Cancer Institute
Cancer-60 screen; NCI, National Cancer Institute; SAR, structure–activity relationship.
*
Corresponding author: Dr. Sonsoles Velázquez
Instituto de Química Médica (CSIC), C/ Juan de la Cierva 3
E-28006 Madrid (Spain) Phone number: (+34) 912587458
e-mail: iqmsv29@iqm.csic.es
Dr. Sonia de Castro.
Instituto de Química Médica (CSIC), C/ Juan de la Cierva 3,
E-28006 Madrid, Spain. Phone: (+34)-915622900
e-mail: sonia@iqm.csic.es
1

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Abstract— Herein we report a novel class of 1,4-disubstituted piperidines as potential anticancer
agents. One-step and efficient synthesis of a structurally diverse library of piperidine-based analogues
with five points of diversity has been developed using the Ugi four-component reaction. A structure-
activity relationship (SAR) study showed that the presence of a benzyl or a Boc group at the N-1
position together with two or three aromatic groups at the C-4 position of the piperidine ring are
important for optimal cytostatic properties. Compounds 20, 22, 27 and 29 were found to be the most
potent with a 50% inhibitory concentration (IC ) ranging between 3-9.5 µM in the cancer cell lines
5
0
evaluated. The NCI60 screen confirmed this 50% cytostatic concentration range for compound 20,
irrespective of the nature of the tumor cell lines evaluated. The NCI COMPARE algorithm did not show
any significant correlation between the growth inhibition profile of compound 20 with the NCI database
compound profiles suggesting a potential novel mechanism of action.
Keywords- Disubstituted piperidines, Ugi reaction, Anticancer, Structure-activity relationships
2

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1. Introduction
Convergent strategies to synthesize diverse libraries of drug-like compounds for biological
screening is an important goal in contemporary medicinal chemistry. In this context,
multicomponent condensation reactions (MCRs) constitute a powerful tool for the convergent
synthesis of small molecule libraries, because products are formed in a single step by
simultaneous reactions of several condensation reagents and the molecular diversity required for
such combinatorial libraries can be easily achieved by simply varying each component [1-3].
Undoubtedly one of the most studied and widely used MCRs is the Ugi four-component reaction
which combines a carboxylic acid, an amine, an isocyanide and a carbonyl compound to afford
dipeptide-like structures containing a high number of substitution diversities [4,5]. Aldehydes are
widely used as carbonyl components but there are relatively fewer examples of the use of
ketones as inputs [6-8].
The piperidine nucleus is an attractive drug template because of its useful biological
applications as anti-histaminic, anti-inflammatory, fungicidal, bactericidal, anticancer, analgesic,
CNS stimulant and depressant activities [9-12]. Thus, piperidine moiety-containing molecules
play an important role in the field of medicinal chemistry.
In an effort to discover novel lead compounds as potential anticancer drugs, several
components of our diverse in-house library were tested against proliferation of different tumor
cell lines. Following this approach, we identified the disubstituted piperidine-based compounds 1
and 2 (Figure 1) as potential hits with moderate cytostatic activity. These compounds can be
easily synthesized by a one-step Ugi four-component reaction from an amine, an isocyanide, N-
substituted piperidones and chiral amino acids as ketone and carboxylic acid components,
respectively.
3

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These results and the versatility of the Ugi reaction prompted us to explore the biological
potential of this readily available heterocyclic system. In order to study the influence of the
substituents on the cytotoxic and anti-proliferative activity of these compounds we herein
described the synthesis and evaluation of a diverse library of novel 1,4-disubstituted piperidine
analogues of hit compounds 1 and 2 by using the Ugi four-component reaction. In this library
five points of diversity were introduced from readily commercially available starting materials.
The goal of this study was to obtain more potent cytostatic analogs with a piperidine scaffold and
to study the initial structure–activity relationship (SAR) for cytostatic activity. A total of 48
analogs were synthesized and their anti-proliferative activities against murine leukemia (L1210),
human T-lymphocyte (CEM) and human cervix carcinoma (HeLa) tumor cell cultures were
determined. The most active analogs were further tested for their ability to inhibit the
proliferation of other tumor cell types including human kidney Caki-1 and human liver Huh-7
tumor cells. Also, one of the most promising compounds has been the subject of the broad NCI
Cancer-60 screen. Among the most effective compounds were 20, 22, 27 and 29 showing a 5–10
fold improvement over the initial hit structures.
2. Results and Discussion.
2.1. Chemistry. The general synthetic route used to access the piperidine- or pyrrolidine-based
analogues (E) is outlined in Scheme 1. Target compounds were efficiently prepared in a single
step by using the Ugi four-component reaction. Each of the compounds 1-38 (Table 1) were
synthesized in moderate to good yields by reaction of N-substituted 4-piperidone or 3-
pirrolidinones (A), alkyl isocyanides (B), aromatic and aliphatic primary amines (C) and a
variety of polar, hydrophobic or aromatic natural (L) amino acids as carboxylic acids (D) in
methanol at room temperature over 72 h, followed by chromatographic purification.
4

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Commercially available reagents used for the synthesis of the 1,4-disubstituted piperidine library
are also detailed in the Scheme.
In order to introduce more variability at the R substituent, hit compound 1 was used as
4
starting material to synthesize derivatives 39-42 bearing polar groups such as either carboxylic
acid or amido groups (Scheme 2). Thus, the methyl ester group of compound 1 was further
transformed into the free acid 39 in excellent yield (90%) by treatment with lithium hydroxide in
dry THF at room temperature overnight. Alternatively, reaction of the methyl ester 1 with
methanol saturated with ammonia, N-methyl or N,N-dimethylamines in a sealed tube at 50 °C for
1
2-24 h afforded the amides 40-42 in 57%, 60% and 45% yields, respectively (Scheme 2).
To determine whether the N-1 (R ) substituent and/or the R substituent of the amino group of
1
5
the amino acid at the C-4 position were necessary for the cytostatic activity, deprotected
piperidine analogues 43-48 were prepared (Scheme 3). The allyloxycarbonyl (Alloc)
deprotection at the N-1 position of compound 3 was performed by using Pd(PPh ) in the
3
4
presence of PhSiH as a neutral allyl group scavenger to give compound 43 in 83% yield.
3
Deprotection of the tert-butoxycarbonyl (Boc) group of compounds 1, 29 and 26 using a solution
of 4N HCl in 1,4-dioxane at 0° C for 3 h gave the deprotected analogues 44, 45 and 46 in good
yields. Finally, removal of the benzyloxycarbonyl (Cbz) group of compound 27 was carried out
by catalytic hydrogenation (H , Pd/C) in ethanol at 30° C for 30 min to yield a mixture of
2
deprotected derivatives 47 and 48 in 58% and 87% yields, respectively.
2.2. Biological studies
All the target piperidine-based derivatives 1-48 were initially tested for their anti-proliferative
activity against murine leukemia (L1210), human T-lymphocyte (CEM) and human cervix
5

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carcinoma (HeLa) cancer cell cultures (Tables 2-4). The hit compounds 1 and 2 were found to
have moderate 50% inhibitory concentration (IC ) values ranging between 18 and 49 µM.
5
0
In vitro anti-proliferative activity of piperidine analogues modified at the R , R or R groups
1
2
3
(compounds 3-16 and 43) are shown in Table 2. The benzyl substituent at the N-1 position is an
important structural component since elimination (compound 43) or replacement by an alloc
group (compound 3) is detrimental to preserve cytostatic activity. However, substitution by a
hydrophobic Boc group (compound 4) maintained the anti-proliferative activity. Evaluation of
the pyrrolidine-based derivatives 5a,b demonstrated that the piperidine nucleus could be replaced
by a five-membered ring without loss of activity. No difference of cytostatic activity could be
found between the two diastereoisomers. When investigating the R substituent at the C-4
2
position of the piperidine, the benzyl group of compound 1 was replaced by different alkyl
groups such as n-propyl, t-butyl and cyclohexyl groups (compounds 6-8) by using the
corresponding commercially available isocyanides. These compounds showed similar anti-
proliferative activity compared with hit compound 1. In contrast, compound 9 bearing a
carboxymethylester group at this position showed poor if any cytostatic activity.
Regarding modifications at the R
carboxymethyl methyl group (compound 2) resulted in a 2-3-fold decrease in cytostatic potency
Table 2). However, replacement of the benzyl group at the R substituent by a variety of
3
benzyl group of hit compound 1, its substitution by a polar
(
3
aromatic or alkyl groups (compounds 10-12, 14 and 15) or its elimination (compound 13) by
using the appropriate commercially available primary amines, afforded compounds exhibiting
similar cytostatic activity than hit compound 1 with IC50 values in the range of 11-31 µM.
Interestingly, the cyclopropyl derivative 16 proved to be 2-fold more potent than hit compound
1
. Thus, while modifications at the R or R benzyl groups, in general, did not result in a
2 3
6

ACCEPTED MANUSCRIPT
significant improvement on the cytostatic activity, resulting in a quite flat SAR, a benzyl or a
hydrophobic Boc group at the N-1 position proved essential for the anti-proliferative activity.
Modifications at the R and R substituent of the amino acid moiety had a pronounced
4
5
influence on anti-proliferative activity (Table 3). While elimination of the R carboxymethyl
4
methyl group of compound 1 (glycine derivative 17) maintained the potency, elimination of the
R substituent (deprotected derivatives 44-47) or simultaneous deprotection at the R and R
1
5
5
substituents (compound 48) generally compromised the cytostatic activity. The conformationally
restricted proline analogue 18 showed a marked decrease in cytostatic activity. Similarly,
4
replacement of the R carboxymethyl methyl group of 1 by carboxylic acid and amide methyl
groups (compounds 39-42) also weakened the activity. In contrast, introduction of an aromatic
moiety at the R substituent (i.e., the phenylalanine 20, tyrosine 21 or tryptophane 22 analogues)
4
markedly enhanced the cytostatic activity against all cancer cell lines showing a 5- to 10-fold
improvement compared with the parental hit compounds 1 and 2. Interestingly, the hydrophobic
isoleucine derivative 19 also showed pronounced activity. Similarly, substitution of the Boc
5
protecting group of the amino group of the amino acid (R ) by aromatic groups such as Cbz or
Fmoc (compounds 23 and 24) also increased the cytostatic activity (Table 3).
The preliminary SAR studies for hit compound 1 are summarized in Figure 2.
Finally, in order to determine the minimum number of phenyl rings and their best location in
the piperidine framework required for cytostatic activity, compounds 25-38 were prepared and
evaluated (Table 4). These compounds combine the optimal structural requirements observed in
the preliminary SAR studies mentioned above including one or two phenyl groups at the R4
5 1 5
and/or R substituent of the amino acid moiety. The presence of a phenyl group at each R to R
position (5 phenyls in total) (compound 25) or a total of 4 phenyl groups (20, 23) or three phenyl
7

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groups (27-31) result in potent cytostatic activity, especially against the L1210 and CEM cell
cultures. The anti-proliferative activity against HeLa cells is often somewhat slightly
compromised for unclear reasons.
Strikingly, compound 34, bearing two phenyl groups at positions 1 and 5 in the molecule is
devoid of any significant cytostatic activity, being in sharp contrast with compounds 33 and 32.
Clearly, subtile differences in the nature of the other substituent groups eventually seem to
determine the anti-proliferative activity against each of the tumor cell lines.
Since compounds 20, 22, 23, 27, 24 and 29 were endowed with the most pronounced anti-
proliferative activity against L1210, CEM and HeLa cell cultures, it was decided to expand the
set of investigated cell lines to be tested in the presence of 20, 27, 22 and 29. In fact, these
compounds also suppressed kidney (Caki-1) and human liver (Huh-7) tumor cell growth equally
well as they did against L1210, CEM and HeLa. Compound 20 has been also submitted for the
-4
-8
NCI60 cancer screen. Five different concentrations ranging between 10 and 10 M were
evaluated for their cytostatic/cytotoxic activity against 9 groups of cancer cell lines including
leukemic, non-small cell lung, colon, central nervous system, melanoma, ovarian, renal, prostate
and breast cancer (see Supplementary material). The compound invariably showed cytostatic
-5
-6
activity at concentrations between 10 and 10 M, irrespective of the nature of the tumor cell
line evaluated (Figure 3). For a few tumor cell lines (i.e. colon cancer HCC-2998 and HT29, and
-5
-4
the melanoma SK-MEL-5 cells) concentrations of 10 and/or 10 of compound 20 were also
markedly cytotoxic.
The pattern of activity (fingerprint) of compound 20 has been also analyzed using the
accessible online tool NCI COMPARE analysis [13] to investigate whether there is resemblance
over the 175 known anticancer drugs from the NCI standard agent database compounds with
8

ACCEPTED MANUSCRIPT
various modes of action [14]. The results of the standard COMPARE analysis for compound 20
showed a poor Pearson’s coefficient correlation (PCC ≤ 0.5) with various known anticancer
agents. This suggests that piperidine analogue 20 may act by a potential novel mechanism of
action or act by more than one mechanism as no standard NCI anticancer compounds were found
with a similar activity pattern.
3. Conclusions
We herein describe the first steps in the investigation of a novel class of piperidine-based
cytotoxic compounds. Forty-eight novel analogues with different substituents on the N-1 and C-4
positions of the piperidine ring were efficiently synthesized using the Ugi four-component
reaction and evaluated against a wide variety of tumor cell lines. Among them, four compounds
2
0, 22, 27 and 29 showed improved anti-profilerative activity as compared with the hit
compounds 1 and 2 and should be considered as lead compounds for further investigation as
potential anticancer drugs. The NCI60 screen revealed a consistent 50% inhibitory activity of
-5
-6
compound 20 at concentrations ranging between 10 and 10 M, irrespective of the tumor cell
line evaluated. The mechanism of action of compound 20 is currently unknown and further
studies are ongoing.
4. Experimental Section
4.1. Chemical Procedures. Microanalyses were obtained with a Heraeus CHN-O-RAPID
instrument and the analytical results were within 0.4% of the theoretical values. Electrospray
mass spectra were measured on a quadropole mass spectrometer equipped with an electrospray
source (Hewlett Packard, LC/MS HP 1100). Analytical thin-layer chromatography (TLC) was
performed on silica gel 60 F254 (Merck). Compounds were purified by flash column
chromatography with silica gel 60 (230-400 mesh) (Merck) or by MPLC using SNAP 12 g KP-
9

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C18-HS cartridges in an Isolera One system (Biotage). The purity of the compounds was
analyzed using an analytical Agilent Technologies (model 1120 Compact LC) ACE 5 C18-300
column (150 mm x 4.6 mm). Gradient conditions used: mobile phase CH CN/H O (0.05% TFA);
3
2
flow rate, 1 mL/min; detection, UV (254 and 217 nm). All retention times are quoted in minutes.
HPLC-MS was performed by HPLC (Waters) coupled to a single quadrupole ESI-MS
(Micromass ZQ 2000). Spectra were recorded with a Varian Inova-400 or a Varian system-500
1
13
spectrometers operating at 400 or at 500 MHz for H NMR and at 100 or at 125 MHz for
C
NMR with Me Si as internal standard. The purity of novel compounds was determined to be
4
>
95% by elemental analysis. Chemicals and reagents were obtained from commercial sources
and were used without further purification.
.2. General Procedure for the Ugi reaction
4
To a solution of the ketone (1.32 mmol) in methanol (2 mL) were added successively 2 equiv
of the corresponding amine, 2 equiv of the aminoacid and 2 equiv of the isocyanide. The
resulting mixture was stirred at room temperature for 4 days. Then, a 1.2 M solution of HCl in
MeOH was added and the mixture was stirred at room temperature for 30 min. The solvent was
removed under reduced pressure. The residue was redissolved in ethyl acetate and was
successively washed with saturated NaHCO (3 x 10 mL) and brine (3 x 10 mL). The organic
3
phase was dried (MgSO ), filtered and evaporated to dryness. The final residue was purified by
4
flash column chromatography (hexane:ethyl acetate, 4:1 to 0:1) to give the target products 1-38.
4.2.1.
Methyl
(S)-4-(benzyl(N-benzyl-4-(benzylcarbamoyl)piperidin-4-yl)amino)-3-((tert-
butoxycarbonyl)amino)-4-oxobutanoate (1). Following the general procedure, a solution of N-
benzyl-4-piperidone (1.32 mmol, 245 µL), benzylamine (2.64 mmol, 288 µL), Boc-Asp(OMe)-
OH (2.64 mmol, 653 mg) and benzyl isocyanide (2.64 mmol, 322 µL) in MeOH (2 mL) was
10

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1
reacted. The final residue was purified to give 1 (747 mg, 88%) as a white foam. H NMR [400
MHz, CDCl ] δ: 7.30 (m, 15H), 6.75 (s, 1H), 5.34 (m, 1H), 5.11 (m, 1H), 4.80 (m, 3H), 4.44 (m,
3
1
3
2
H), 3.65 (s, 3H), 3.50 (m, 1H), 2.60 (m, 8H), 2.03 (m, 2H), 1.66 (m, 1H9, 1.43 (s, 9H).
C
NMR [100 MHz, CDCl ] δ: 173.2, 173.1, 171.6, 155.1, 138.7, 138.3, 129.1, 128.5, 127.4, 127.1,
3
+
9
4.4, 80.5, 64.6, 62.9, 52.1, 50.2, 49.8, 48.7, 43.4, 37.4, 32.8, 32.4, 31.7. MS (ESI ) m/z 643.6
+
.
[
M+1] . HPLC 8.09 min (99%) (H O/CH CN from 10/90 to 0/100 in 10 min, then isocratic for 2
2 3
min; flow rate of 1 mL/min). Anal. for C37
.2.2. Methyl (S)-4-((N-benzyl-4-(benzylcarbamoyl)piperidin-4-yl)(2-methoxy-2-
oxoethyl)amino)-3-((tert-butoxycarbonyl)amino)-4-oxobutanoate (2). Following the general
46 4 6
H N O (C, H, N).
4
(
procedure, a solution of N-benzyl-4-piperidone (1.32 mmol, 245 µL), H-Gly-OMe·HCl (2.64
mmol, 331 mg), Boc-Asp(OMe)-OH (2.64 mmol, 653 mg), and benzyl isocyanide (2.64 mmol,
3
22 µL) in MeOH (2 mL) was stirred for 4 days. The final residue was purified to give 2 (798
1
mg, 88%) as a white foam. H NMR [400 MHz, CDCl ] δ: 7.28 (m, 10H), 5.29 (m, 1H), 4.67-
3
4
.29 (m, 7H), 3.74 (s, 3H), 3.58 (s, 3H), 3.50 (m, 3H), 2.70 (m, 4H), 2.04 (m, 4H), 1.38 (s, 9H).
1
3
3
C NMR [100 MHz, CDCl ] δ: 173.0, 171.1, 161.0, 155.2, 1392, 129.3, 128.7, 128.4, 128.3,
1
28.2, 127.9, 127.7, 127.0, 80.4, 63.8, 62.5, 53.0, 51.8, 48.0, 45.4, 43.2, 42.1, 37.2, 28.4. MS
+
.
(
ES+) m/z 625.4 (M+1) . HPLC 7.64 min (95%) (H O/CH CN from 10/90 to 0/100 in 10 min,
2 3
then isocratic for 2 min; flow rate of 1 mL/min). Anal. for C H N O (C, H, N).
3
3
44
4
8
4.2.3. Allyl (S)-4-(N-benzyl-2-((tert-butoxycarbonyl)amino)-4-methoxy-4-oxobutanamido)-4-
(benzylcarbamoyl)piperidine-1-carboxylate (3). Following the general procedure, a solution of
N-Alloc-4-piperidone (1.32 mmol, 241 mg), benzylamine (2.64 mmol, 288 µL), Boc-Asp(OMe)-
OH (2.64 mmol, 653 mg) and benzyl isocyanide (2.64 mmol, 322 µL) in MeOH (2 mL) was
1
stirred for 4 days. The final residue was purified to give 3 (628 mg, 75%) as a white foam. H
11

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NMR [500 MHz, (CD
3
2
) SO] δ: 7.91 (m, 1H), 7.47-7.19 (m, 10H), 5.75 (m, 1H), 5.09 (m, 2H),
4
2
.75 (m, 3H), 4.37 (s, 2H), 4.25 (s, 2H), 3.69 (m, 1H), 3.57 (s, 4H), 3.34 (bs, 1H), 2.85 (m, 1H),
1
3
.50 (m, 2H), 2.19 (m, 1H), 1.66 (m, 1H), 1.42 (m, 1H), 1.25 (s, 6H), 1.16 (m, 1H). C NMR
[
125 MHz, (CD ) SO ] δ: 172.2, 171.3, 170.9, 154.9, 154.1, 139.9, 138.3, 133.4, 128.5, 128.1,
3 2
1
3
0
27.1, 127.0, 126.9, 126.5, 116.6, 78.4, 64.9, 63.5, 51.5, 49.5, 47.71, 42.4, 40.8, 40.2, 39.7, 36.3,
+
.
1.6, 27.9. MS (ES+) m/z 659.48 (M+23) . HPLC 9.30 min (99%) (H O/CH CN from 10/90 to
2
3
44 4 8
/100 in 10 min, then isocratic for 2 min; flow rate of 1 mL/min). Anal. for C34H N O (C, H,
N).
4.2.4.
tert-Butyl
(S)-4-(N-benzyl-2-((tert-butoxycarbonyl)amino)-4-methoxy-4-
oxobutanamido)-4-(benzylcarbamoyl)piperidine-1-carboxylate (4). Following the general
procedure, a solution of N-Boc-4-piperidone (1.32 mmol, 263 mg), benzylamine (2.64 mmol,
2
88 µL), Boc-Asp(OMe)-OH (2.64 mmol, 653 mg) and benzyl isocyanide (2.64 mmol, 322 µL)
in MeOH (2 mL) was reacted. The final residue was purified to give 4 (731 mg, 85%) as a white
1
foam. H NMR [500 MHz, (CD ) SO] δ: 7.92 (m, 1H), 7.46 (d, J = 7.5 Hz, 1H9, 7.33 (t, J = 7.5
3
2
Hz, 2H), 7.31 (t, J = 7.5, 3H), 7.24 (m, 4H), 4.75 (m, 3H), 4.23 (m, 2H), 3.64 (m, 1H), 3.55 (s,
H), 3.51 (m, 1H), 3.33 (s, 2H), 2.84 (dd, J = 16.5, 5.5 Hz, 1H), 2.53 (d, J = 8.0 Hz, 1H), 2.45 (d,
J = 12.3 Hz, 1H), 2.17 (d, J = 12.3 Hz, 1H), 1.61 (m, 1H), 1.36 (m, 1H), 1.26 (s, 9H), 1.23 (s,
3
1
3
9
1
6
H). C NMR [125 MHz, (CD ) SO] δ: 172.3, 171.2, 170.9, 154.9, 153.9, 139.9, 138.3, 128.4,
3 2
28.1, 127.1, 126.9, 126.5, 78.4, 63.6, 51.5, 49.4, 47.7, 42.3, 36.2, 31.7, 27.9. MS (ES+) m/z
+
.
75.3 (M+23) . HPLC 8.85 min (98%) (H O/CH CN from 10/90 to 0/100 in 10 min, then
2
3
isocratic for 2 min; flow rate of 1 mL/min). Anal. for C H N O (C, H, N).
3
5
48
4
8
4.2.5. Methyl (3S)-4-(benzyl(N-benzyl-3-(benzylcarbamoyl)pyrrolidin-3-yl)amino)-3-((tert-
butoxycarbonyl)amino)-4-oxobutanoate (5a and 5b). Following the general procedure, a solution
12

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of N-benzyl-3-pyrrolidinone (1.32 mmol, 217 µL), benzylamine (2.64 mmol, 288 µL), Boc-
Asp(OMe)-OH (2.64 mmol, 653 mg) and benzyl isocyanide (2.64 mmol, 322 µL) in MeOH (2
mL) was stirred for 4 days and the final residue was purified.
1
The fastest moving fractions afforded 183 mg (22%) of 5a as an amorphous white solid. H
NMR [500 MHz, (CD ) SO] δ: 8.03 (bs, 1H), 7.34-7.15 (m, 15H), 7.00 (m, 3H), 5.32 (m, 1H),
3
2
5
3
.26 (m, 2H), 5.00 (d, J = 16.0 Hz, 1H), 4.60 (m, 7H), 4.40 (m, 4H), 3.60 (s, 3H), 3.56 (s, 3H),
13
.40 (m, 2H), 2.92-2.56 (m, 9H), 2.34 (m, 6H), 1.39 (s, 9H), 1.28 (s, 9H). C NMR [126 MHz,
SO] δ: 173.7, 173.1, 171.5, 154.8, 138.7, 138.6, 138.5, 138.1, 128.9, 128.8, 128.5, 127.8,
27.4, 126.0, 80.3, 70.7, 63.7, 63.5, 59.4, 53.5, 52.0, 49.1, 43.8, 37.7, 34.5, 28.3. MS (ES+) m/z
3 2
(CD )
1
6
+
.
29.48 (M+1) . HPLC 8.36 min (98%) (H O/CH CN from 10/90 to 0/100 in 10 min, then
2
3
isocratic for 2 min; flow rate of 1 mL/min). Anal. for C H N O (C, H, N).
3
6
44
4
6
From the slowest moving fractions 174 mg (21%) of 5b as an amorphous white solid was
1
isolated. H NMR [500 MHz, (CD ) SO] δ: 7.75 (bs, 1H), 7.34-7.15 (m, 15H), 7.00 (m, 3H),
3
2
5
9
1
1
.32 (m, 1H), 4.60 (m, 7H), 4.40 (m, 4H), 3.60 (s, 3H), 3.56 (s, 3H), 3.40 (m, 2H), 2.78-2.56 (m,
1
3
3 2
H), 2.34 (m, 6H), 1.28 (s, 9H). C NMR [125 MHz, (CD ) SO] δ: 173.7, 173.5, 173.1, 171.5,
54.8, 138.7, 138.6, 138.5, 138.4, 138.1, 128.9, 128.8, 128.5, 127.8, 127.7, 127.4, 127.3, 126.0,
25.9, 80.3, 70.9, 63.7, 63.5, 59.4, 53.5, 52.0, 50.8, 49.4, 43.8, 37.7, 34.6, 34.5, 28.3. MS (ES+)
+
.
m/z 629.48 (M+1) . HPLC 8.17 min (97%) (H O/CH CN from 10/90 to 0/100 in 10 min, then
2
3
isocratic for 2 min; flow rate of 1 mL/min). Anal. for C H N O (C, H, N).
3
6
44
4
6
4.2.6.
Methyl
(S)-4-(benzyl(N-benzyl-4-(butylcarbamoyl)piperidin-4-yl)amino)-3-((tert-
butoxycarbonyl)amino)-4-oxobutanoate (6). Following the general procedure, a solution of N-
benzyl-4-piperidone (1.32 mmol, 245 µL), benzylamine (2.64 mmol, 288 µL, Boc-Asp(OMe)-
OH (2.64 mmol, 653 mg), and propyl isocyanide (2.64 mmol, 322 µL) in MeOH (2 mL) was
13

ACCEPTED MANUSCRIPT
1
reacted. The final residue was purified to give 6 (913 mg, 88%) as a white foam. H NMR [500
MHz, (CD ) SO] δ: 7.47 (d, J = 7.5 Hz, 2H), 7.37 (t, J = 7.5 Hz, 2H), 7.28 (m, 2H), 7.24 (m,
3
2
2
2
H), 7.19 (m, 1H), 7.13 (m, 2H), 5.75 (s, 1H), 4.71 (m, 3H), 3.55 (s, 3H), 3.34 (s, 1H), 3.28 (s,
H), 2.98 (q, J = 6.5 Hz, 2H), 2.82 (dd, J = 16.5, 6.0 Hz, 1H), 2.45 (m, 2H), 2.41 (m, 1H), 2.32
(d, J = 12.0 Hz, 1H), 2.20 (t, J = 11.5 Hz, 1H), 2.13 (d, J = 11.5 Hz, 1H), 1.75 (td, J = 12.5, 3.5
Hz, 1H), 1.54 (m, 1H), 1.34 (quint, J = 6.5 Hz, 3H), 1.28 (s, 7H), 1.23 (q, J = 7.5 Hz, 2H), 0.86
1
3
(
t, J = 7.5 Hz, 3H). C NMR [125 MHz, (CD
3
)
2
SO] δ: 171.9, 170.9, 154.9, 138.9, 138.4, 128.7,
1
3
28.4, 128.3, 126.9, 126.7, 126.7, 78.363.7, 61.7, 56.0, 54.9, 51.4, 49.7, 49.4, 49.2, 47.4, 38.4,
+
.
6.3, 31.9, 31.8, 31.1, 27.9, 19.6, 18.5, 13.7. MS (ES+) m/z 609.3 (M+1) . HPLC 7.86 min
(
95%) (H O/CH CN from 10/90 to 0/100 in 10 min, then isocratic for 2 min; flow rate of 1
2 3
mL/min). Anal. for C H N O (C, H, N).
3
4
48
4
6
4.2.7. Methyl (S)-4-(benzyl(N-benzyl-4-((tert-butylcarbamoyl)piperidin-4-yl)amino)-3-((tert-
butoxycarbonyl)amino)-4-oxobutanoate (7). Following the general procedure, a solution of N-
benzyl-4-piperidone (1.32 mmol, 245 µL), benzylamine (2.64 mmol, 299 µL), Boc-Asp(OMe)-
OH (2.64 mmol, 653 mg), and tert-butyl isocyanide (2.64 mmol, 322 µL) in MeOH (2 mL) was
1
stirred for 4 days. The final residue was purified to give 7 (587 mg, 73%) as a white foam. H
NMR [500 MHz, (CD ) SO] δ: 7.51 (d, J = 7.5 Hz, 2H), 7.36 (t, J = 7.5 Hz, 2H), 7.27 (t, J = 7.5
3
2
Hz, 2H), 7.24 (d, J = 7.0 Hz, 2H), 7.18 (t, J = 7.0 Hz, 2H), 6.17 (bs, 1H), 5.73 (s, 1H), 4.79 (d, J
18.0 Hz, 1H), 4.69 (q, J = 7.0 Hz, 1H), 4.62 (d, J = 18.0 Hz, 1H), 3.09 (s, 3H), 2.84 (dd, J =
6.5, 7.0 Hz, 1H), 2.48 (m, 4H), 2.18 (m, 2H), 2.05 (m, 2H), 1.80 (m, 1H), 1.56 (m, 1H), 1.41 (s,
=
1
1
1
5
1
3
H), 1.30 (s, 9H), 1.16 (s, 9H). C NMR [125 MHz, (CD ) SO] δ: 171.5, 171.2, 170.9, 155.0,
3
2
39.1, 138.5, 128.7, 128.6, 128.5, 128.1, 128.0, 128.0, 127.1, 126.8, 126.7, 78.5, 64.3, 61.8, 54.9,
1.5, 49.89, 49.9, 49.7, 49.2, 49.1, 46.9, 36.0, 32.1, 31.6, 28.1, 28.0. MS (ES+) m/z 609.3
14

ACCEPTED MANUSCRIPT
+
.
(
M+1) . HPLC 8.29 min (95%) (H
2 3
O/CH CN from 10/90 to 0/100 in 10 min, then isocratic for 2
min; flow rate of 1 mL/min). Anal. for C H N O (C, H, N).
3
4
48
4
6
4.2.8. Methyl (S)-4-(benzyl(N-benzyl-4-(cyclohexylcarbamoyl)piperidin-4-yl)amino)-3-((tert-
butoxycarbonyl)amino)-4-oxobutanoate (8). Following the general procedure, a solution of N-
benzyl-4-piperidone (1.32 mmol, 245 µL), benzylamine (2.64 mmol, 288 µL), Boc-Asp(OMe)-
OH (2.64 mmol, 653 mg) and cyclohexyl isocyanide (2.64 mmol, 328 µL) in MeOH (2 mL) was
1
reacted. The final residue was purified to give 8 (486 mg, 58%) as a white foam. H NMR [500
3 2
MHz, (CD ) SO] δ: 7.47 (d, J = 7.0 Hz, 1H), 7.37 (t, J = 7.0 Hz, 2H), 7.31-7.14 (m, 6H), 6.77 (d,
J = 7.0 Hz, 1H), 4.69 (m, 3H), 4.33 (m, 1H), 3.56 (s, 3H), 3.46-3.25 (m, 6H), 2.84 (dd, J = 16.5,
5
1
3
1
3
.5 Hz, 1H), 2.45 (m, 6H), 2.15 (t, J = 11.3 Hz, 1H), 2.08 (d, J = 11.3 Hz, 1H), 1.79 (m, 1H),
.63 (m, 1H), 1.56 (m, 1H), 1.49 (m, 1H), 1.43 (m, 1H), 1.31 (s, 3H), 1.21 (m, 3H), 1.09 (m,
1
3
H). C NMR [125 MHz, (CD ) SO] δ: 171.5, 170.9, 155.0, 138.8, 138.4, 128.7, 128.5, 128.0,
3
2
27.1, 126.8, 126.7, 78.3, 63.7, 61.7, 60.7, 57.9, 51.5, 49.8, 49.3, 49.0, 47.5, 47.4, 36.1, 35.8,
+
.
2.3, 32.1, 31.6, 29.2, 28.0, 25.3, 24.8. MS (ES+) m/z 635.6 (M+1) . HPLC 8.39 min (99%)
O/CH CN from 10/90 to 0/100 in 10 min, then isocratic for 2 min; flow rate of 1 mL/min).
Anal. for C36
.2.9. Methyl
(H
2
3
H
50
N
4
O
6
(C, H, N).
4
(S)-4-(benzyl(N-benzyl-4-((2-methoxy-2-oxoethyl)carbamoyl)piperidin-4-
yl)amino)-3-((tert-butoxycarbonyl)amino)-4-oxobutanoate (9). Following the general procedure,
a solution of N-benzyl-4-piperidone (1.32 mmol, 245 µL), benzylamine (2.64 mmol, 288 µL),
Boc-Asp(OMe)-OH (2.64 mmol, 653 mg) and methyl isocyanoacetate (2.64 mmol, 240 µL) in
MeOH (2 mL) was reacted. The final residue was purified to give 9 (507 mg, 62%) as a white
1
foam. H NMR [500 MHz, (CD
3
)
2
SO] δ: 7.71 (m, 1H), 7.46 (d, J = 7.3 Hz, 2H), 7.36 (t, J = 7.3
Hz, 2H), 7.28 (d, J = 7.3 Hz, 1H), 7.23 (m, 3H), 7.17 (t, J = 7.3 Hz, 1H), 7.11 (d, J = 7.3 Hz, 2H),
15

ACCEPTED MANUSCRIPT
4
.71 (m, 3H), 3.77 (dd, J = 17.0, 5.5 Hz, 1H), 3.68 (s, 3H), 3.53 (s, 3H), 2.79 (dd, J = 16.5, 6.5
Hz, 1H), 3.43 (m, 3H), 2.31 (m, 3H), 2.19 (m, 1H), 1.71 (m, 1H), 1.54 8m, 1H), 1.35 (m, 1H),
1
3
1
1
4
.24 (s, 9H), 1.13 (m, 1H). C NMR [125 MHz, (CD ) SO] δ: 172.7, 171.3, 170.9, 170.6, 154.9,
3 2
38.7, 138.5, 128.6, 128.6, 128.5, 128.1, 127.0, 126.7, 78.4, 63.5, 61.6, 51.6, 51.4, 49.4, 49.0,
+
.
7.4, 40.8, 36.3, 31.6, 27.9.MS (ES+) m/z 625.45 (M+1) . HPLC 7.13 min (99%) (H O/CH CN
2
3
from 10/90 to 0/100 in 10 min, then isocratic for 2 min; flow rate of 1 mL/min). Anal. for
C
33
H
44
N
4
O
8
(C, H, N).
4.2.10. Methyl (S)-4-((N-benzyl-4-(benzylcarbamoyl)piperidin-4-yl)(4-fluorobenzyl)amino)-3-
((tert-butoxycarbonyl)amino-4-oxobutanoate (10). Following the general procedure, a solution of
N-benzyl-4-piperidone (1.32 mmol, 245 µL), 4-fluorobenzylamine (2.64 mmol, 302 µL), Boc-
Asp(OMe)-OH (2.64 mmol, 653 mg) and benzyl isocyanide (2.64 mmol, 322 µL) in MeOH (2
1
mL) was reacted. The final residue was purified to give 10 (715 mg, 82%) as a white foam. H
NMR [500 MHz, (CD ) SO] δ: 7.81 (t, J = 5.0 Hz, 1H), 7.53 (m, 2H), 7.30 (t, J = 7.5 Hz, 3H),
3
2
7
2
9
1
4
.24 (m, 5H), 7.21 (m, 2H), 7.15 (m, 2H), 4.72 (m, 3H), 4.24 (m, 2H), 3.54 (s, 2H), 3.29 (s, 3H),
.80 (dd, J = 16.5, 5.7 Hz, 1H), 2.44 (m, 3H), 2.21 (m, 2H), 1.77 (m, 1H), 1.55 (m, 1H), 1.26 (s,
1
3
H), 1.17 (m, 1H). C NMR [125 MHz, (CD
3 2
) SO] δ: 172.3, 171.5, 170.9, 162.2, 160.3, 154.9,
40.0, 138.4, 134.9, 128.6, 128.1, 127.0, 126.7, 126.4, 115.2, 115.1, 78.4, 63.7, 61.7, 51.5, 49.6,
+
.
9.4, 49.1, 46.9, 42.3, 36.4, 31.9, 27.9. MS (ES+) m/z 661.3 (M+1) . HPLC 8.09 min (99%)
H O/CH CN from 10/90 to 0/100 in 10 min, then isocratic for 2 min; flow rate of 1 mL/min).
(
2
3
Anal. for C H FN O (C, H, N).
3
7
45
4
6
4.2.11.
Methyl
(S)-4-((N-benzyl-4-(benzylcarbamoyl)piperidin-4-yl-)(3,4,5-
trimethoxybenzyl)amino)-3-((tert-butoxycarbonyl)amino)-4-oxobutanoate (11). Following the
general procedure, a solution of N-benzyl-4-piperidone (1.32 mmol, 245 µL), 3,4,5-
16

ACCEPTED MANUSCRIPT
trimethoxybenzylamine (2.64 mmol, 451 µL), Boc-Asp(OMe)-OH (2.64 mmol, 653 mg) and
benzyl isocyanide (2.64 mmol, 322 µL) in MeOH (2 mL) was stirred for 4 days. The final
1
residue was purified to give 11 (270 mg, 28%) as a white foam. H NMR [500 MHz, (CD ) SO]
3
2
δ: 7.75 (m, 1H), 7.32-7.13 (m, 12H), 6.78 (m, 3H), 4.70 (m, 3H), 4.23 (m, 2H), 3.77 (s, 6H), 3.64
s, 3H), 3.53 (s, 3H), 3.44 (t, J = 6.0 Hz, 1H), 3.37 (m, 1H), 3.28 (m, 2H), 2.81 (m, 1H), 2.17 (t, J
10.0 Hz, 1H), 2.09 (d, J = 10.0 Hz, 1H), 1.91 (m, 1H), 1.57 (m, 2H), 1.42 (m, 1H), 1.26 (s,
(
=
1
3
9
1
4
3 2
H). C NMR [125 MHz, (CD ) SO] δ: 172.4, 171.5, 171.0, 155.1, 153.0, 140.0, 138.4, 136.3,
34.3, 128.7, 128.1, 126.9, 126.8, 126.5, 104.1, 78.3, 63.8, 61.4, 60.7, 59.9, 57.9, 55.8, 51.5,
9.8, 49.5, 48.7, 47.7, 42.2, 35.9, 35.8, 31.7, 31.4, 29.2, 27.9. MS (ES+) m/z 733.3
+
.
(
M+1) . HPLC 7.97 min (99%) (H O/CH CN from 10/90 to 0/100 in 10 min, then isocratic for 2
2 3
min; flow rate of 1 mL/min). Anal. for C H N O (C, H, N).
4
0
52
4
9
4.2.12. Methyl (S)-4-((N-benzyl-4-(benzylcarbamoyl)piperidin-4-yl)(2-phenethyl)amino)-3-
((tert-butoxycarbonyl)amino)-4-oxobutanoate (12). Following the general procedure, a solution
of N-benzyl-4-piperidone (1.32 mmol, 245 µL), 4-(2-aminoethyl)aniline (2.64 mmol, 345 µL),
Boc-Asp(OMe)-OH (2.64 mmol, 653 mg) and benzyl isocyanide (2.64 mmol, 322 µL) in MeOH
(2 mL) was stirred for 4 days. The final residues was purified to give 12 (407 mg, 46%) as a
1
white foam. H NMR [500 MHz, (CD ) SO] δ: 7.81 (m, 1H), 7.31-7.24 (m, 4H), 7.20 (m, 2H),
3
2
7
1
1
.08 (t, J = 8.0 Hz, 2H), 6.91 (d, J = 8.0 Hz, 1H), 6.65 (d, J = 8.0 Hz, 1H), 6.56 (t, J = 7.3 Hz,
H), 5.73 (bs, 1H), 4.77 (m, 1H), 4.21 (m, 2H), 3.70 (m, 1H), 3.57 (m, 5H), 3.46 (t, J = 6.5 Hz,
H), 3.39 (m, 1H), 3.32 (m, 1H), 2.76 (m, 2H), 2.59 (m, 2H), 2.41 (m, 1H), 2.11 (m, 1H), 1.75
1
3
(
m, 1H), 1.57 (quint, J = 6.0 Hz, 1H), 1.43 (m, 1H), 1.37, 1.32 (2s, 9H). C NMR [125 MHz,
CD SO] δ: 172.9, 172.7, 171.9, 170.9, 170.7, 155.4, 155.1, 148.4, 139.9, 137.6, 128.9, 128.2,
28.0, 127.1, 126.9, 126.4, 116.2, 112.4, 78.4, 78.1, 62.5, 61.5, 60.7, 57.9, 51.6, 51.4, 50.5, 49.9,
(
3 2
)
1
17

ACCEPTED MANUSCRIPT
4
9.0, 48.9, 43.6, 43.2, 42.2, 36.3, 35.8, 35.7, 31.9, 31.5, 29.2, 28.1, 28.1. MS (ES+) m/z 672.5
+
.
(
M+1) . HPLC 8.44 min (99%) (H O/CH CN from 10/90 to 0/100 in 10 min, then isocratic for 2
2 3
min; flow rate of 1 mL/min). Anal. for C H N O (C, H, N).
3
8
49
5
6
4.2.13.
Methyl
(S)-4-((N-benzyl-4-(benzylcarbamoyl)piperidin-4-yl)amino)-3-((tert-
butoxycarbonyl)amino)-4-oxobutanoate (13). Following the general procedure, a solution of N-
benzyl-4-piperidone (1.32 mmol, 245 µL), benzyl carbamate (2.64 mmol, 399 mg), Boc-
Asp(OMe)-OH (2.64 mmol, 653 mg) and benzyl isocyanide (2.64 mmol, 322 µL) in MeOH (2
1
mL) was reacted. The final residue was purified to give 13 (182 mg, 25%) as a white foam. H
3 2
NMR [500 MHz, (CD ) SO] δ: 7.24 (m, 1H), 7.10 (m, 2H), 5.48 (m, 1H), 4.47 (m, 2H), 4.35 (dd,
J = 14.8, 5.6 Hz, 1H), 3.50 (s, 2H), 3.46 (s, 2H), 3.43 (s, 2H), 3.00 (dd, J = 17.2, 5.2 Hz, 1H),
13
2
.81 (dd, J = 17.2, 4.4 Hz, 1H), 2.74 (m, 2H), 2.26 (m, 2H), 2.14 (m, 3H), 1.40 (s, 9H). C NMR
[
125 MHz, (CD ) SO] δ: 172.4, 171.7, 169.5, 155.7, 138.5, 138.4, 129.0, 128.5, 128.3, 127.8,
3
2
1
5
27.2, 127.1, 82.4, 80.7, 62.9, 52.3, 50.6, 48.7, 48.6, 43.4, 36.5, 32.3, 31.9, 28.4. MS (ES+) m/z
+
.
54.4 (M+1) . HPLC 7.34 min (95%) (H O/CH CN from 10/90 to 0/100 in 10 min, then isocratic
2
3
for 2 min; flow rate of 1 mL/min). Anal. for C30
40 4 6
H N O (C, H, N).
4.2.14. Methyl (S)-4-((N-benzyl-4-(benzylcarbamoyl)piperidin-4-yl)(methyl)amino)-3-((tert-
butoxycarbonyl)amino)-4-oxobutanoate (14). Following the general procedure, a solution of N-
benzyl-4-piperidone (1.32 mmol, 245 µL), Boc-Asp(OMe)-OH (2.64 mmol, 653 mg), benzyl
isocyanide (2.64 mmol, 322 µL) and methylamine 2 M in MeOH (2.64 mmol, 1.5 mL) was
1
stirred for 4 days. The final residue was purified to give 14 (501 mg, 67%) as a white foam. H
NMR [400 MHz CDCl ] δ: 7.29-7.17 (m, 10H), 6.88 (t, J = 5.8 Hz, 1H), 5.20 (d, J = 9.2 Hz, 1H),
3
4
1
.88 (m, 1H), 4.37 (m, 2H), 3.51 (s, 3H), 3.45 (s, 2H), 3.11 (s, 3H), 2.73 (dd, J = 16.2, 8.2 Hz,
13
H), 2.66 (m, 1H), 2.55 (m, 1H), 2.35 (m, 3H), 2.24 (m, 3H), 1.40 (s, 9H). C NMR [100 MHz,
18

ACCEPTED MANUSCRIPT
CDCl
3
] δ: 173.3, 173.1, 171.6, 155.1, 138.9, 138.4, 129.2, 128.5, 128.3, 127.5, 127.2, 127.1,
8
0.4, 64.5, 62.9, 52.0, 50.0, 49.9, 48.8, 43.4, 37.3, 32.8, 32.6, 31.8, 28.4. MS (ES+) m/z 567.47
+
.
(
M+1) . HPLC 7.14 min (98%) (H O/CH CN from 10/90 to 0/100 in 10 min, then isocratic for 2
2 3
min; flow rate of 1 mL/min). Anal. for C H N O (C, H, N).
3
1
42
4
6
4.2.15. Methyl (3S)-4-((N-benzyl-4-(benzylcarbamoyl)piperidin-4-yl)(sec-butyl)amino)-3-
((tert-butoxycarbonyl)amino)-4-oxobutanoate (15). Following the general procedure, a solution
of N-benzyl-4-piperidone (1.32 mmol, 245 µL), sec-butylamine (2.64 mmol, 267 µL), Boc-
Asp(OMe)-OH (2.64 mmol, 653 mg) and benzyl isocyanide (2.64 mmol, 322 µL) in MeOH (2
1
mL) was reacted. The final residue was purified to give 15 (603 mg, 75%) as a white foam. H
NMR [300 MHz CDCl ] δ: 7.30 (m, 10H), 5.30 (s, 1H), 5.13 (m, 1H), 4.90 (m, 1H), 4.40 (m,
3
2
0
1
4
H), 3.50 (m, 6H), 2.55 (m, 6H), 2.24 (m, 1H), 2.03-1.56 (m, 6H), 1.42 (s, 9H), 1.27 (m, 1H),
1
3
.94 (t, J = 6.0 Hz, 2H), 1.02 (m, 1H). C NMR [75 MHz, CDCl ] δ: 170.88, 154.69, 138.74,
3
29.30, 128.62, 128.36, 127.91, 127.87, 127.33, 127.21, 80.22, 66.31, 62.82, 52.07, 50.80, 50.38,
3.57, 33.68, 32.85, 31.03, 30.02, 28.36, 28.33, 21.16, 19.62, 12.57, 12.38. MS (ES+) m/z 609.7
+
.
(
M+1) . HPLC 5.31 min (96%) (H
min; flow rate of 1 mL/min). Anal. for C34
.2.16. Methyl (S)-4-((N-benzyl-4-(benzylcarbamoyl)piperidin-4-yl)(cyclopropyl)amino)-3-
(tert-butoxycarbonyl)amino)-4-oxobutanoate (16). Following the general procedure, a solution
2
O/CH
3
CN from 10/90 to 0/100 in 10 min, then isocratic for 2
48 4 6
H N O
(C, H, N).
4
(
of N-benzyl-4-piperidone (1.32 mmol, 245 µL), cyclopropylamine (2.64 mmol, 183 µL), Boc-
Asp(OMe)-OH (2.64 mmol, 653 mg) and benzyl isocyanide (2.64 mmol, 322 µL) in MeOH (2
mL) was stirred for 4 days. The final residues was purified to give 16 (494 mg, 71%) as a white
1
foam. H NMR [500 MHz (CD
3
)
2
SO] δ: 7.75 (m, 1H), 7.28 (q, J = 7.5Hz, 2H), 7.25 (m, 3H),
7
.17 (m, 4H), 5.31 (dd, J = 14.0, 8.0 Hz, 1H), 4.20 (d, J = 4.5 Hz, 2H), 3.55 (s, 3H), 3.40 (dd, J =
19

ACCEPTED MANUSCRIPT
1
1
3.5, 4.5 Hz, 2H), 3.31 (s, 1H), 2.77 (dd, J = 16.0, 5.5 Hz, 1H), 2.74 (m, 1H), 2.52 (d, J = 8.5 Hz,
13
H), 2.43 (m, 1H), 3.29 (m, 4H), 2.12 (m, 2H), 1.37 (s, 9H), 0.88 (m, 3H). C NMR [125 MHz,
(
CD ) SO] δ: 175.5, 173.4, 170.9, 155.1, 139.5, 138.6, 128.6, 128.1, 126.8, 126.5, 78.2, 65.2,
3 2
6
2.0, 51.5, 50.1, 49.6, 49.5, 42.3, 35.7, 31.9, 31.8, 28.2, 28.17, 10.36, 10.0. MS (ES+) m/z 593.4
+
.
(
M+1) . HPLC 7.57 min (98%) (H O/CH CN from 10/90 to 0/100 in 10 min, then isocratic for 2
2 3
min; flow rate of 1 mL/min). Anal. for C H N O (C, H, N).
3
3
44
4
6
4
.2.17.
oxoethyl)carbamate (17). Following the general procedure, a solution of N-benzyl-4-piperidone
1.32 mmol, 245 µL), benzylamine (2.64 mmol, 288 µL), Boc-Gly-OH (2.64 mmol, 462 mg) and
tert-Butyl
(2-(benzyl(N-benzyl-4-(benzylcarbamoyl)piperidin-4-yl)amino)-2-
(
benzyl isocyanide (2.64 mmol, 322 µL) in MeOH (2 mL) was reacted. The final residue was
1
purified to give 17 (475 mg, 63%) as a white foam. H NMR [400 MHz CDCl ] δ: 7.26 (m,
3
1
4
1
1
4
1
5H), 6.72 (t, J = 4.6 Hz, 1H), 5.34 (m, 1H), 4.50 (s, 2H), 4.41 (d, J = 5.6 Hz, 2H), 3.90 (d, J =
.4 Hz, 1H), 3.40 (s, 1H), 2.66 (m, 2H), 2.53 (m, 2H), 2.30 (t, J = 11.4 Hz, 2H), 1.92 (dd, J =
13
3.2, 3.8 Hz, 2H), 1.41 (s, 9H). C NMR [100 MHz, CDCl ] δ: 171.9, 171.3, 155.8, 138.4,
3
37.9, 137.4, 129.2, 129.2, 128.7, 128.3, 127.9, 127.7, 127.5, 127.2, 125.8, 79.8, 65.3, 62.8, 50.3,
+
.
7.4, 43.9, 32.6, 28.4. MS (ES+) m/z 571.5 (M+1) . HPLC 7.78 min (99%) (H
0/90 to 0/100 in 10 min, then isocratic for 2 min; flow rate of 1 mL/min). Anal. for C H N O
4
2 3
O/CH CN from
3
4
42
4
(C, H, N).
4.2.18.
tert-Butyl
(S)-2-(benzyl(N-benzyl-4-(benzylcarbamoyl)piperidin-4-
yl)carbamoyl)pyrrolidine-1-carboxylate (18). Following the general procedure, a solution of N-
benzyl-4-piperidone (1.32 mmol, 245 µL), benzylamine (2.64 mmol, 288 µL), Boc-Pro-OH
(2.64 mmol, 568 mg) and benzyl isocyanide (2.64 mmol, 322 µL) in MeOH (2 mL) was stirred
1
for 4 days. The final residue was purified to give 18 (654 mg, 81%) as a white foam. H NMR
20

ACCEPTED MANUSCRIPT
[400 MHz CDCl
3
] δ: 7.40-7.21 (m, 5H), 5.01 (m, 1H), 4.59 (m, 1H), 4.51 (dd, J = 15,2, 4.8 Hz,
1
H), 4.29 (m, 3H), 3.58-3.32 (m, 5H), 2.81-2.58 (m, 5H), 2.30 (m, 1H), 2.11 (m, 2H), 2.04-1.82
13
(
m, 7H), 1.76-1.63 (s, 3H), 1.38 (s, 9H). C NMR [100 MHz, CDCl ] δ: 176.9, 176.6, 173.2,
3
1
1
5
2
0
71.5, 15487, 153.8, 139.3, 138.9, 138.7, 138.5, 129.3, 128.9, 128.9, 128.7, 128.5, 128.3, 128.2,
28.0, 127.5, 127.3, 127.2, 127.1, 126.9, 126.2, 125.9, 80.4, 79.7, 65.3, 65.1, 62.9, 62.8, 58.1,
1.4, 50.8, 50.5, 49.8, 48.5, 48.3, 47.5, 47.4, 43.9, 43.4, 33.3, 33.1, 32.7, 32.3, 30.8, 28.8, 28.5,
+
.
4.6, 23.2. MS (ES+) m/z 611.5 (M+1) . HPLC 8.33 min (99%) (H
2
O/CH
3
CN from 10/90 to
(C, H,
/100 in 10 min, then isocratic for 2 min; flow rate of 1 mL/min). Anal. for C37
46 4 4
H N O
N).
4.2.19. tert-butyl ((2S,3S)-1-(benzyl(N-benzyl-4-(benzylcarbamoyl)piperidin-4-yl)amino)-3-
methyl-1-oxopentan-2-yl)carbamate (19). Following the general procedure, a solution of N-
benzyl-4-piperidone (1.32 mmol, 245 µL), benzylamine (2.64 mmol, 288 µL), Boc-Ile-OH (2.64
mmol, 610 mg) and benzyl isocyanide (2.64 mmol, 322 µL) in MeOH (2 mL) was stirred for 4
1
days to give 19 (643 mg, 78%) as a white foam. H NMR [500 MHz (CD ) SO] δ: 7.84 (t, J =
3
2
5
.5 Hz, 1H), 7.36 (q, J = 7.5, 3H), 7.24 (m, 6H), 7.21 (m, 2H), 7.16 (m, 3H), 4.83 (q, J = 18.5
Hz, 3H), 4.36 (dd, J = 15.0, 6.3 Hz, 1H), 4.14 (dd, J = 15.0, 5.7 Hz, 1H), 3.32 (s, 2H), 3.30 (s,
H), 2.52 (m, 1H), 2.36 (m, 5H), 2.22 (m, 1H), 1.82 (m, 2H), 1.59 (m, 1H), 1.38 (m, 1H), 1.32
2
1
3
(
s, 9H), 1.15 (m, 2H), 0.90 (m, 1H), 0.72 (t, J = 7.0 Hz, 3H), 0.70 (t, J = 7.0 Hz, 3H). C NMR
[
125 MHz, (CD ) SO] δ: 173.2, 172.2, 155.4, 139.9, 139.3, 138.4, 128.7, 128.4, 128.1, 128.0,
3
2
1
2
1
27.1, 126.9, 126.7, 126.6, 126.4, 78.1, 63.6, 61.7, 55.9, 49.7, 49.3, 47.4, 42.5, 36.7, 32.3, 31.6,
+
.
8.0, 23.5, 15.5, 10.8. MS (ES+) m/z 627.5 (M+1) . HPLC 8.83 min (98%) (H O/CH CN from
2
3
50 4 4
0/90 to 0/100 in 10 min, then isocratic for 2 min; flow rate of 1 mL/min). Anal. for C38H N O
(C, H, N).
21

ACCEPTED MANUSCRIPT
4.2.20. tert-Butyl (S)-(1-(benzyl(N-benzyl-4-(benzylcarbamoyl)piperidin-4-yl)amino)-1-oxo-3-
phenylpropan-2-yl)carbamate (20). Following the general procedure, a solution of N-benzyl-4-
piperidone (1.32 mmol, 245 µL), benzylamine (2.64 mmol, 288 µL), Boc-Phe-OH (2.64 mmol,
6
99 mg) and benzyl isocyanide (2.64 mmol, 322 µL) in MeOH (2 mL) was reacted. The final
1
residue was purified to give 20 (663 mg, 76%) as a white foam. H NMR [300 MHz (CD ) SO]
3
2
δ: 7.33 (m, 14H), 7.04 (m, 6H), 4.98 (m, 1H), 4.56 (m, 6H), 3.42 (m, 2H), 2.37 (m, 6H), 2.44 (m,
+
.
1
8
H), 2.20 (m, 2H), 1.81 (m, 2H), 1.63 (m, 2H), 1.37 (s, 9H). MS (ES+) m/z 661.7 (M+1) . HPLC
.88 min (99%) (H O/CH CN from 10/90 to 0/100 in 10 min, then isocratic for 2 min; flow rate
(C, H, N).
.2.21. tert-Butyl (S)-(1-(benzyl(N-benzyl-4-(benzylcarbamoyl)piperidin-4-yl)amino)-3-(4-
2
3
48 4 4
of 1 mL/min). Anal. for C41H N O
4
hydroxyphenyl)-1-oxopropan-2-yl)carbamate (21). Following the general procedure, a solution
of N-benzyl-4-piperidone (1.32 mmol, 245 µL), benzylamine (2.64 mmol, 288 µL), Boc-Tyr-OH
(2.64 mmol, 742 mg) and benzyl isocyanide (2.64 mmol, 322 µL) in MeOH (2 mL) was stirred
1
for 4 days. The final residue was purifed to give 21 (668 mg, 83%) as a white foam. H NMR
[
500 MHz (CD
3
)
2
SO] δ: 9.11 (s, 1H), 7.89 (t, J = 5.3 Hz, 1H), 7.33-7.16 (m, 12H), 7.13 (d, J =
7
.0 Hz, 1H), 7.06 (d, J = 8.3 Hz, 1H), 6.80 (d, J = 8.3 Hz, 2H), 6.55 (d, J = 8.3 Hz, 2H), 4.90 (d, J
=
19.0 Hz, 1H), 4.76 (s, 1H), 4.74 (d, J = 19.0 Hz, 1H), 4.26 (m, 3H), 3.32 (s, 2H), 3.28 (s, 2H),
3
.82 (dd, J = 14.0, 4.0 Hz, 1H), 2.56 (dd, J = 13.7, 9.3 Hz, 1H), 2.40 (m, 3H), 2.26 (m, 1H), 1.74
1
3
(
m, 1H), 1.50 (m, 1H), 1.25 (s, 9H), 0.93 (s, 2H). C NMR [125 MHz, (CD ) SO] δ: 173.46,
3 2
1
1
72.3, 155.8 155.4, 139.9, 139.0, 138.4, 130.1, 128.7, 128.5, 128.1, 127.9, 127.1, 126.9, 126.8,
26.5, 126.4, 114.8, 78.1, 63.4, 61.7, 54.4, 49.6, 49.4, 47.6, 42.4, 36.7, 32.2, 31.8, 28.1, 27.4. MS
+
.
(
ES+) m/z 677.5 (M+1) . HPLC 7.63 min (99%) (H
2
O/CH
3
CN from 10/90 to 0/100 in 10 min,
(C, H, N).
then isocratic for 2 min; flow rate of 1 mL/min). Anal. for C41
48 4 5
H N O
22

ACCEPTED MANUSCRIPT
4.2.22. tert-Butyl (S)-(1-(benzyl(N-benzyl-4-(benzylcarbamoyl)piperidin-4-yl)amino)-3-(1H-
indol-3-yl)-1-oxopropan-2-yl)carbamate (22). Following the general procedure, a solution of N-
benzyl-4-piperidone (1.32 mmol, 245 µL), benzylamine (2.64 mmol, 288 µL), Boc-Trp-OH
(2.64 mmol, 803 mg) and benzyl isocyanide (2.64 mmol, 322 µL) in MeOH (2 mL) was stirred
1
for 4 days. The final residue was purified to give 22 (760 mg, 83%) as a white foam. H NMR
[500 MHz (CD ) SO] δ: 10.81 (s, 1H), 7.86 (s, 1H), 7.20 (m, 15H), 7.01 (t, J = 7.5 Hz, 1H), 6.83
3 2
(
t, J = 7.5 Hz, 1H), 4.92 (d, J = 18.5 Hz, 1H), 4.82 (d, 1H, J = 18.5 Hz), 4.79 (d, J = 18.5 Hz,
H), 4.39 (m, 1H), 4.23 (m, 1H), 3.37 (s, 1H), 3.27 (s, 2H), 3.13 (dd, J = 14.5, 4.5 Hz, 1H), 2.87
dd, J = 9.0, 8.7 Hz, 1H), 2.47 (m, 3H), 2.27 (m, 1H), 1.97 (s, 1H), 1.69 (m, 1H), 1.52 (m, 1H),
1
(
1
3
1
1
1
4
.25 (s, 9H), 0.71 (s, 2H). C NMR [125 MHz, (CD ) SO] δ: 173.6, 172.4, 161.1, 155.4, 153.6,
3 2
40.0, 138.8, 138.4, 135.9, 128.8, 128.5, 128.4, 128.2, 128.1, 127.3, 127.2, 127.1, 126.9, 126.8,
26.5, 126.4, 124.1, 120.8, 118.2, 111.3, 110.0, 78.1, 63.4, 61.8, 59.8, 53.2, 49.7, 49.4, 47.5,
+
.
2.4, 40.7, 32.2, 31.8, 28.1, 27.8, 27.1. MS (ES+) m/z 700.7 (M+1) . HPLC 8.41 min (99%)
(
H O/CH CN from 10/90 to 0/100 in 10 min, then isocratic for 2 min; flow rate of 1 mL/min).
2
3
Anal. for C43
.2.23.
((benzyloxy)carbonyl)amino)-4-oxobutanoate (23). Following the general procedure, a solution
H
49
N
5
O
3
(C, H, N).
4
Methyl (S)-4-(benzyl(N-benzyl-4-(benzylcarbamoyl)piperidin-4-yl)amino)-3-
(
of N-benzyl-4-piperidone (1.32 mmol, 245 µL), benzylamine (2.64 mmol, 288 µL), Cbz-
Asp(OMe)-OH (2.64 mmol, 742 mg) and benzyl isocyanide (2.64 mmol, 322 µL) in MeOH (2
1
mL) was reacted. The final residue was purified to give 23 (760 mg, 88%) as a white foam. H
NMR [500 MHz (CD ) SO] δ: 7.34 (m, 1H), 7.79 (d, J = 8.0 Hz, 1H), 7.51 (d, J = 7.5 Hz, 2H),
3
2
7
1
.40-7.18 (m, 16H), 7.12 (d, J = 7.0 Hz, 2H), 5.75 (s, 1H), 4.99 (d, J = 12.5 Hz, 1H), 4.90 (d, J =
2.5 Hz, 1H), 4.77 (m, 3H), 4.24 (d, J = 6.0 Hz, 2H), 3.54 (s, 3H), 3.34 (s, 2H), 3.29 (d, J = 13.5
23

ACCEPTED MANUSCRIPT
Hz, 1H), 3.25 (d, J = 13.5 Hz, 1H), 2.89 (dd, J = 16.5, 5.0 Hz, 1H), 2.55 (dd, J = 16.5, 8.3 Hz,
1
3
2
1
1
3
1
H), 2.46 (m, 1H), 2.22 (m, 2H), 1.80 (m, 1H), 1.54 (m, 1H). C NMR [125 MHz, (CD ) SO] δ:
3 2
72.2, 171.3, 170.7, 155.7, 139.9, 138.6, 138.4, 136.7, 128.7, 128.5, 128.3, 128.1, 128.0, 127.8,
27.7, 127.1, 127.0, 126.7, 126.4, 65.7, 63.7, 61.6, 51.6, 49.9, 49.7, 48.9, 47.5, 42.3, 36.0, 32.1,
+
.
1.8. MS (ES+) m/z 677.6 (M+1) . HPLC 8.11 min (99%) (H O/CH CN from 10/90 to 0/100 in
2
3
0 min, then isocratic for 2 min; flow rate of 1 mL/min). Anal. calcd for C H N O (C, H, N).
4
0
44
4
6
4.2.24. Methyl (S)-3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-(benzyl(N-benzyl-4-
(benzylcarbamoyl)piperidin-4-yl)amino)-4-oxobutanoate (24). Following the general procedure,
a solution of N-benzyl-4-piperidone (1.32 mmol, 245 µL), benzylamine (2.64 mmol, 288 µL),
Fmoc-Asp(OMe)-OH (2.64 mmol, 975 mg), and benzyl isocyanide (2.64 mmol, 322 µL) in
MeOH (2 mL) was reacted. The final residue was purified to give 24 (858 mg, 88%) as a white
1
foam. H NMR [500 MHz, (CD ) SO] δ: 7.87 (d, J = 7.0 Hz, 2H), 7.81 (t, J = 5.3 Hz, 1H), 7.70
3
2
(d, J = 7.5 Hz, 2H), 7.49 (d, J = 7.5 Hz, 2H), 7.39 (t, J = 7.5 Hz, 2H), 7.30 (m, 6H), 7.23 (t, J =
7
3
.2 Hz, 4H), 7.17 (m, 2H), 7.11 (d, J = 7.0 Hz, 2H), 4.76 (m, 3H), 4.24 (m, 3H), 4.14 (m,2H),
.54 (s, 1H), 3.27 (d, J = 13.0 Hz, 1H); 3.24 (d, J = 13.0 Hz, 1H), 3.16 (d, J = 4.5 Hz, 1H), 2.89
(dd, J = 16.5, 4.5 Hz, 1H), 2.57 (dd, J = 16.5, 8.0 Hz, 1H), 2.48 (m, 4H), 2.21 (m, 2H), 1.81 (m,
1
3
1
1
1
3
1
H), 1.54 (m, 1H). C NMR [126 MHz, (CD ) SO] δ: 172.2, 171.4, 170.8, 155.7, 143.7, 140.7,
3 2
39.9, 138.6, 138.4, 128.6, 128.5, 128.1, 128.0, 127.6, 127.1, 127.0, 127.0, 126.7, 126.7, 126.4,
25.3, 125.3, 120.1, 65.8, 63.7, 61.6, 51.6, 49.8, 49.7, 49.0, 48.6, 47.5, 46.6, 42.3, 36.1, 32.1,
+
.
1.8. MS (ES+) m/z 765.3 (M+1) . HPLC 9.13 min (95%) (H O/CH CN from 10/90 to 0/100 in
2
3
0 min, then isocratic for 2 min; flow rate of 1 mL/min). Anal. for C H N O (C, H, N).
4
7
48
4
6
4.2.25. Benzyl (S)-(1-(benzyl(N-benzyl-4-(benzylcarbamoyl)piperidin-4-yl)amino)-1-oxo-3-
phenylpropan-2-yl)carbamate (25). Following the general procedure, a solution of N-benzyl-4-
24

ACCEPTED MANUSCRIPT
piperidone (1.32 mmol, 245 µL), benzylamine (2.64 mmol, 288 µL), Cbz-Phe-OH (2.64 mmol,
7
90 mg) and benzyl isocyanide (2.64 mmol, 322 µL) in MeOH (2 mL) was stirred for 4 days.
1
The final residue was purified to give 25 (460 mg, 60%) as a white foam. H NMR [500 MHz
(
CD ) SO] δ: 7.91 (t, J = 4.0 Hz, 1H), 7.83 (d, J = 8.1 Hz, 1H), 7.36-7.05 (m, 20H), 4.91 (dd, J =
3 2
1
4
2.3 Hz, 2H), 4.90 (dd, J = 16.2 Hz, 2H), 4.41 (td, J = 12.3, 4.2 Hz, 1H), 4.29 (t, J = 4.0 Hz, 1H),
.28 (m, 1H), 3.31 (m, 2H), 3.01 (m, 1H), 2.73 (dd, J = 14.5 Hz, 1H), 2.44-2.45 (m, 4H), 2.27
1
3
(
m, 1H), 1.81 (m, 1H), 1.55 (m, 1H). C NMR [126 MHz, (CD
3
)
2
SO] δ: 173.1, 172.3, 156.0,
39.9, 138.8, 138.4, 137.8, 136.9, 129.2, 128.7, 128.6, 128.3, 128.3, 127.7, 127.1, 126.8, 126.5,
26.5, 126.4, 65.3, 63.5, 61.7, 54.71, 5.68, 5.28, 5.62, 4.38, 4.2, 32.3, 31.9. MS (ES+) m/z
1
1
6
+
.
95.6(M+1) . HPLC 8.89 min (98%) (H O/CH CN from 10/90 to 0/100 in 10 min, then isocratic
2
3
for 2 min; flow rate of 1 mL/min). Anal. for C H N O (C, H, N).
4
4
46
4
4
4.2.26. tert-Butyl ((2S)-1-((N-benzyl-4-(benzylcarbamoyl)piperidin-4-yl)(sec-butyl)amino)-1-
oxo-3-phenylpropan-2-yl)carbamate (26). Following the general procedure, a solution of N-
benzyl-4-piperidone (1.32 mmol, 245 µL), sec-butylamine (2.64 mmol, 199 µL), Boc-Phe-OH
(2.64 mmol, 699 mg), and benzyl isocyanide (2.64 mmol, 322 µL) in MeOH (2 mL) was stirred
1
for 4 days. The final residue was purified to give 26 (732 mg, 89 %) as a white foam. H NMR
[
500 MHz (CD ) SO] δ: 0.77 (t, J = 7.5 Hz, 1H), 0.88 (t, J = 7.5 Hz, 1H), 1.04 (s, 2H), 1.25 (s,
3 2
6
1
H), 1.32 (d, 1H), 1.37 (d, 1H), 1.45 (m, 1H), 1.76-2.06 (m, 5H), 2.23-2.46 (m, 6H), 2.66 (m,
1
3
H), 2.91 (m, 1H), 3.41 (m, 4H), 4.24 (m, 3H), 4.63 (m, 1H), 7.25 (m, 15H), 7.80 (bs, 1H). C
NMR [126 MHz, (CD ) SO] δ: 154.7, 153.0, 139.9, 139.9, 138.2, 138.2, 137.7, 137.7, 129.6,
3
2
1
7
29.3, 128.8, 128.8, 128.1, 128.1, 128.0, 127.2, 127.1, 127.1, 126.8, 126.8, 126.5, 126.5, 126.2,
7.9, 77.9, 65.3, 65.2, 61.9, 55.0, 49.7, 49.6, 42.3, 28.1, 27.5, 12.2. MS (ES+) m/z 627.7
25

ACCEPTED MANUSCRIPT
+
.
(
M+1) . HPLC 8.77 min (99%) (H
2 3
O/CH CN from 10/90 to 0/100 in 10 min, then isocratic for 2
min; flow rate of 1 mL/min). Anal. for C H N O (C, H, N).
3
8
50
4
4
4.2.27. Methyl (3S)-4-((N-benzyl-4-(benzylcarbamoyl)piperidin-4-yl)(sec-butyl)amino)-3-
(((benzyloxy)carbonyl)amino)-4-oxobutanoate (27). Following the general procedure, a solution
of N-benzyl-4-piperidone (1.32 mmol, 245 µL), sec-butylamine (2.64 mmol, 199 µL), Cbz-
Asp(OMe)-OH (2.64 mmol, 742 mg) and benzyl isocyanide (2.64 mmol, 322 µL) in MeOH (2
mL) was stirred for 4 days. The final residue was purified to give 27 (570 mg, 66%) as a white
1
foam. H NMR [500 MHz (CD
3
)
2
SO] δ: 7.31 (m, 15 H), 5.02 (m, 3H), 4.87 (m, 2H), 4.24 (m,
3
2
H), 3.56 (s, 4H), 3.40 (s, 3H), 3.34 (s, 1H), 3.18 (d, J = 5.0 Hz, 1H), 2.71 (m, 2H), 2.45 (m, 1H),
1
3
.34 (m, 2H), 2.24 (m, 3H), 1.42 (d, J = 7.0 Hz, 2H), 1.36 (m, 2H), 0.87 (m, 4H). C NMR [125
MHz, (CD ) SO] δ: 172.5, 170.6, 155.5, 139.9, 138.4, 136.9, 128.7, 128.3, 128.1, 128.0, 127.8,
3
2
1
2
27.7, 127.0, 127.0, 126.8, 126.4, 126.4, 65.4, 62.0, 59.7, 51.6, 49.9, 49.8, 48.6, 42.4, 32.3, 29.9,
+
.
9.2, 20.7, 20.0, 19.2, 14.1, 12.48, 12.3. MS (ES+) m/z 657.3 (M+1) . HPLC 8.05 min (98%)
(
H O/CH CN from 10/90 to 0/100 in 10 min, then isocratic for 2 min; flow rate of 1 mL/min).
2 3
Anal. for C37
.2.28. tert-Butyl (S)-4-(N-benzyl-2-((tert-butoxycarbonyl)amino)-3-phenylpropanamido)-4-
benzylcarbamoyl)piperidine-1-carboxylate (28). Following the general procedure, a solution of
N-Boc-4-piperidone (1.32 mmol, 263 mg), benzylamine (2.64 mmol, 288 µL), Boc-Phe-OH
46 4 6
H N O (C, H, N).
4
(
(2.64 mmol, 699 mg) and benzyl isocyanide (2.64 mmol, 322 µL) in MeOH (2 mL) was reacted.
1
The final residue was purified to give 28 (770 mg, 87%) as a white foam. H NMR [500 MHz
(
CD ) SO] δ: 7.96 (M, 1H), 7.34-7.09 (m, 15H), 7.09 (d, J = 7.5 Hz, 2H), 4.85 (s, 1H), 4.40 (m,
3 2
1
3
H), 4.31 (dd, J = 15.5, 5.8 Hz, 1H), 4.25 (dd, J = 15.5, 5.8 Hz, 1H), 3.67 (m, 1H), 3.52 (m, 1H),
.00 (dd, J = 14.0, 3.5 Hz, 1H), 2.72 (dd, J = 13.5, 10.5 Hz, 1H), 2.45 (m, 1H), 2.32 (m, 1H),
26

ACCEPTED MANUSCRIPT
1
3
1
.65 (m, 1H), 1.36 (m, 1H), 1.25, 1.23 (2s, 18H), 0.87 (s, 2H). C NMR [126 MHz, (CD
3 2
) SO]
δ: 172.9, 172.3, 155.4, 153.9, 139.8, 138.5, 138.0, 129.2, 128.5, 128.1, 128.0, 127.1, 126.6,
1
6
26.5, 126.2, 78.5, 78.1, 63.3, 54.1, 47.7, 42.3, 37.1, 32.1, 31.6, 28.0, 27.9, 27.3. MS (ES+) m/z
+
.
93.5 (M+23) . HPLC 10.56 min (96%) (H O/CH CN from 10/90 to 0/100 in 10 min, then
2
3
isocratic for 2 min; flow rate of 1 mL/min). Anal. for C H N O (C, H, N).
3
9
50
4
6
4
.2.29.
benzylcarbamoyl)piperidine-1-carboxylate (29). Following the general procedure, a solution of
N-Alloc-4-piperidone (1.32 mmol, 241 mg), benzylamine (2.64 mmol, 288 µL), Boc-Phe-OH
Allyl
(S)-4-(N-benzyl-2-((tert-butoxycarbonyl)amino)-3-phenylpropanamido)-4-
(
(2.64 mmol, 699 mg) and benzyl isocyanide (2.64 mmol, 322 µL) in MeOH (2 mL) was reacted.
1
The final residue was purified to give 29 (673 mg, 78%) as a white foam. H NMR [500 MHz
(CD ) SO] δ: 8.12 (bs, 1H), 7.96 (bs, 1H), 7.32-7.08 (m, 15H), 6.58 (s, 1H), 5.79 (m, 1H), 5.10
3 2
(
m, 1H), 4.86 (m, 2H), 4.40 (m, 3H), 4.32 (dd, J = 15.5, 5.5 Hz, 1H), 4.25 (dd, J = 15.5, 5.5 Hz,
H), 3.74 (d, J = 12.3 Hz, 1H), 3.59 (d, J = 12.3 Hz, 1H), 3.23 (m, 1H), 2.99 (m, 2H), 2.75 (dd, J
13.5, 10.0 Hz, 1H), 2.34 (d, J = 11.5 Hz, 1H), 1.71 (m, 1H), 1.36 (m, 2H), 1.23 (s, 9H), 0.88 (s,
1
=
1
3
2
1
4
H). C NMR [126 MHz, (CD
3 2
) SO] δ: 173.1, 172.2, 155.4, 154.1, 139.8, 138.5, 138.0, 133.4,
29.2, 128.5, 128.1, 128.0, 127.1, 126.7, 126.5, 126.2, 116.6, 78.1, 77.9, 64.9, 63.3, 54.9, 54.1,
+
.
7.7, 42.4, 40.7, 40.3, 37.2, 32.0, 31.5, 28.0, 27.3. MS (ES+) m/z 669.6 (M+1) . HPLC 10.11
min (99%) (H O/CH CN from 10/90 to 0/100 in 10 min, then isocratic for 2 min; flow rate of 1
2
3
mL/min). Anal. for C H N O (C, H, N).
3
8
46
4
6
4
.2.30.
methylpentanamido)-4-(benzylcarbamoyl)piperidine-1-carboxylate (30). Following the general
procedure, a solution of N-Boc-4-piperidone (1.32 mmol, 263 mg), benzylamine (2.64 mmol,
88 µL), Cbz-Ile-OH (2.64 mmol, 700 mg) and benzyl isocyanide (2.64 mmol, 322 µL) in
tert-Butyl
4-((2S,3R)-N-benzyl-2-(((benzyloxy)carbonyl)amino)-3-
(
2
27

ACCEPTED MANUSCRIPT
MeOH (2 mL) was stirred for 4 days. The final residue was purified to give 30 (638 mg, 72%) as
1
a white foam. H NMR [500 MHz (CD ) SO] δ: 7.96 (t, J = 5.8 Hz, 1H), 7.40-7.24 (m, 15H),
3
2
7
1
.20 (m, 1H), 5.04 (d, J = 13.0 Hz, 1H), 4.92 (d, J = 13.0 Hz, 1H), 4.85 (m, 1H), 4.35 (dd, J =
5.0, 6.5 Hz, 1H), 4.25 (t, J = 7.5 Hz, 1H), 4.12 (dd, J = 15.0, 5.0 Hz, 1H), 3.69 (bs, 1H), 3.54
(bs, 1H), 3.18- 2.85 (m, 3H), 2.48 (d, J = 12.5 Hz, 1H), 2.40 (d, J = 12.5 Hz, 1H), 1.90 (m, 1H),
1
3
1
.69 (m, 1H), 1.39 (m, 1H), 1.26 (s, 9H), 1.38 (m, 1H), 0.73 (t, J = 6.5 Hz, 3H), 0.71 (s, 1H). C
SO] δ: 172.6, 172.2, 156.3, 153.9, 139.8, 138.8, 137.0, 128.4, 128.3,
28.1, 127.7, 127.5, 127.1, 126.7, 126.5, 78.6, 65.5, 63.5, 56.7, 47.6, 42.4, 36.3, 27.9, 23.3, 15.6,
3 2
NMR [125 MHz, (CD )
1
1
+
.
2 3
0.9. MS (ES+) m/z 693.5 (M+23) . HPLC 10.57 min (97%) (H O/CH CN from 10/90 to 0/100
in 10 min, then isocratic for 2 min; flow rate of 1 mL/min). Anal. for C H N O (C, H, N).
3
9
50
4
6
4.2.31. Benzyl (S)-(1-((N-benzyl-4-(tert-butylcarbamoyl)piperidin-4-yl)(cyclopropyl)amino)-1-
oxo-3-phenylpropan-2-yl)carbamate (31). Following the general procedure, a solution of N-
benzyl-4-piperidone (1.32 mmol, 245 µL), cyclopropylamine (2.64 mmol, 183 µL), Cbz-Phe-OH
(2.64 mmol, 790 mg) and tert-butylisocyanide (2.64 mmol, 298 µL) in MeOH (2 mL) was stirred
1
for 4 days. The final residue was purified to give 31 (717 mg, 89%) as a white foam. H NMR
[500 MHz (CD
3 2
) SO] δ: 7.70 (bs, 1H), 7.34-7.22 (m, 13H), 7.16 (m, 2H), 5.17 (m, 1H), 5.05 (d,
J = 12.5 Hz, 1H), 4.96 (m, 1H), 3.37 (m, 3H), 3.03 (m, 1H), 2.87 (m, 1H), 2.74 (m, 1H), 2.56 (m,
1
3
2
H), 2.16 (m, 2H), 2.01 (m, 2H), 1.17 (m, 9H), 0.95 (m, 2H), 0.84 (m 2H). C NMR [125 MHz,
(
CD ) SO] δ: 177.3, 173.5, 156.2, 138.2, 137.0, 129.4, 128.7, 128.3, 128.2, 128.0, 127.7, 127.4,
3
2
1
26.8, 126.4, 65.8, 65.2, 61.9, 49.7, 49.2, 49.1, 36.69, 31.9, 28.0. MS (ES+) m/z 611.4
+
.
(
M+1) . HPLC 8.36 min (99%) (H O/CH CN from 10/90 to 0/100 in 10 min, then isocratic for 2
2 3
46 4 4
min; flow rate of 1 mL/min). Anal. for C37H N O (C, H, N).
28