Design, synthesis, and evaluation of a novel series of macrocyclic inhibitors of norovirus 3CL protease

Article abstract of DOI:10.1016/j.ejmech.2016.12.033

Norovirus infections have a major impact on public health worldwide, yet there is a current dearth of norovirus-specific therapeutics and prophylactics. This report describes the discovery of a novel class of macrocyclic inhibitors of norovirus 3C-like pr

Full text of DOI:10.1016/j.ejmech.2016.12.033

Accepted Manuscript
Design, synthesis, and evaluation of a novel series of macrocyclic inhibitors of
norovirus 3CL protease
Vishnu C. Damalanka, Yunjeong Kim, Anushka C. Galasiti Kankanamalage, Gerald
H. Lushington, Nurjahan Mehzabeen, Kevin P. Battaile, Scott Lovell, Kyeong-Ok
Chang, William C. Groutas
PII:
S0223-5234(16)31039-X
10.1016/j.ejmech.2016.12.033
EJMECH 9124
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 26 October 2016
Revised Date: 1 December 2016
Accepted Date: 16 December 2016
Please cite this article as: V.C. Damalanka, Y. Kim, A.C. Galasiti Kankanamalage, G.H. Lushington,
N. Mehzabeen, K.P. Battaile, S. Lovell, K.-O. Chang, W.C. Groutas, Design, synthesis, and evaluation
of a novel series of macrocyclic inhibitors of norovirus 3CL protease, European Journal of Medicinal
Chemistry (2017), doi: 10.1016/j.ejmech.2016.12.033.
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ACCEPTED MANUSCRIPT
O
O
H
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O
N
H
O
HN
O
Cl
IC50 = 1.5 µM
EC50 = 1.8 µM
ꢀ
ꢀꢀ

ACCEPTED MANUSCRIPT
Design, Synthesis, and Evaluation of a Novel Series of
Macrocyclic Inhibitors of Norovirus 3CL Protease.
a
b
a
Vishnu C. Damalanka, Yunjeong Kim, Anushka C. Galasiti Kankanamalage, Gerald
c
,
d
e
d
H. Lushington
Nurjahan Mehzabeen, Kevin P. Battaile, Scott Lovell, Kyeong-Ok
b,
a,
Chang, ** William C. Groutas, *
a
Department of Chemistry, Wichita State University, Wichita, Kansas 67260, USA
b
Department of Diagnostic Medicine & Pathobiology, College of Veterinary Medicine,
Kansas State University, Manhattan, Kansas 66506, USA
c
LiS Consulting, Lawrence, KS 66046, USA
d
Protein Structure Laboratory, The University of Kansas, Lawrence, Kansas 66047,
USA
e
IMCA-CAT, Hauptman-Woodward Medical Research Institute, APS Argonne National
Laboratory, Argonne, IL 60439, USA
authors to whom correspondence should be addressed.
*
Department of Chemistry, Wichita State University, Wichita, KS 67260
Tel. (316) 978 7374; Fax: (316) 978 3431
e-mail: bill.groutas@wichita.edu
**
Department of Diagnostic Medicine & Pathobiology, Manhattan, KS 66506
Tel. (785) 532 3849; Fax: (785) 532 4039
e-mail: kchang@vet.ksu.edu
1

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ABSTRACT
Norovirus infections have a major impact on public health worldwide, yet there is a
current dearth of norovirus-specific therapeutics and prophylactics. This report
describes the discovery of a novel class of macrocyclic inhibitors of norovirus 3C-like
protease, a cysteine protease that is essential for virus replication. SAR, structural, and
biochemical studies were carried out to ascertain the effect of structure on
pharmacological activity and permeability. Insights gained from these studies have laid
a solid foundation for capitalizing on the therapeutic potential of the series of inhibitors
described herein.
2

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1. Introduction
Noroviruses belong to the family Caliciviridae and are classified into at least six
genogroups (GI-GVI) [1-3]. Human noroviruses are the primary cause of non-bacterial
acute gastroenteritis worldwide [4-5], and are associated with high morbidity and a
heavy economic burden [6-8]. In the U.S. alone noroviruses account for >20 million
cases annually and impact most severely the young and elderly, as well as
immunocompromised individuals [9-12]. Combating norovirus infections presents a
formidable challenge because of their robustness, high infectivity, ease of transmission
via
multiple
routes
[13-14],
the
current
dearth
of
norovirus-specific
therapeutics/prophylactics and vaccines [15-21], as well as a sub-optimal understanding
of norovirus biology and pathophysiology [22-26].
Noroviruses are small, single-stranded, positive sense RNA viruses whose genome
(7-8 kb) is covalently linked to a viral protein (VPg, virion protein, genome-linked) at the
5’ end and polyadenylated at the 3’ end [1,24]. The genome consists of three open
reading frames (ORFs) that encode a 200 kDa polyprotein (ORF1), a major capsid
protein VP1 (ORF2) and a small basic protein VP2 (ORF3) [1,24]. The polyprotein is
processed by a virus-encoded protease to generate six mature non-structural proteins,
pro
including the viral protease (3C-like protease, 3CLpro or NS6 ) and the RNA
pol
dependent RNA polymerase (NS7 ). Co- and post-translational processing of the
polyprotein by 3CLpro is essential for virus replication, consequently, 3CLpro is an
attractive target for the discovery of anti-norovirus small molecule therapeutics and
prophylactics [15-20].
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Norovirus 3CLpro, including Norwalk virus (NV) 3CLpro, is a cysteine protease with
a Cys-His-Glu catalytic triad, an extended binding site, and a chymotrypsin-like fold [27-
3
1]. The protease displays a primary substrate specificity requirement for a P glutamine
1
or glutamic acid residue, or equivalent [32]. We have recently described an array of
transition state inhibitors and transition state mimics of NV 3CLpro [15]. In continuing
our studies in this area, we describe herein the structure-guided design of a novel series
of macrocyclic inhibitors of the protease, as well as pertinent structural, biochemical,
and cell-based studies.
2. Results and Discussion
2.1. Inhibitor design rationale.
Cocrystal structures of NV 3CLpro with peptidyl inhibitors reveal a network of
backbone hydrogen bonds involving Ala158, Ala160, Gln110 and His30, as well as two
critical hydrogen bonds between His157 and Thr134 and the carbonyl oxygen of the P1
Gln side chain [27,29,33]. The backbone hydrogen bonds mimic an antiparallel β-sheet
and serve to correctly orient and position the inhibitor to the active site [34-35]. It was
envisaged that the construction of a macrocyclic structure (Figure 1, structure (I))
capable of (a) maintaining the aforementioned favorable binding interactions and, (b)
having a flexible diversity site that is well-suited to exploiting H-bonding and
hydrophobic interactions with the S -S subsites and modulating physicochemical
3
4
properties, could potentially result in the identification of a molecule that displays high
potency and drug-like characteristics [36-37]. Additional advantages frequently accrued
by macrocyclization include higher pharmacological activity and selectivity, enhanced
permeability, and improved stability [38-40]. The interaction of inhibitor (I) with the
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2 1
protease was further probed using different warheads, ring sizes, and P residues, a P
alkoxyamide side chain replacement for the P Gln side chain that could potentially
1
engage in intramolecular H-bonding, thereby attenuating cellular permeability [41-46]
and, finally, structural variants focused on R which projects toward the S -S subsites
1
3
4
and affords opportunities for favorable binding interactions.
2.2. Chemistry.
The synthesis of inhibitors 11-21 by coupling compounds 3a-k (Scheme 1) with
compound 6a (Scheme 2B) to yield acyclic compounds 7a-k (Scheme 3). The reaction
sequence outlined in Scheme 1 is flexible and permits ready manipulation of the ring
size using appropriate alkenyl Grignard reagents. Furthermore, the nature of the P2
2
residue (R ) could be readily manipulated by reacting intermediates 1a-f (Scheme 1)
with an appropriate amino acid ester isocyanate 4a-d (Scheme 2A). Ring closure using
a metathesis reaction furnished compounds 8a-k which, upon sequential catalytic
hydrogenation, reduction with lithium borohydride, and Dess-Martin periodinane
oxidation yielded aldehydes 11-21. With the exception of compounds 14 and 36, these
were obtained as diastereomeric mixtures. Aldehydes 23-24 having an unsaturated
linker were synthesized in an analogous manner (Scheme 4). Aldehyde bisulfite
adducts 25-29 and α-ketoamide 31 were synthesized as shown in Scheme 5. Finally,
coupling of intermediate 3c with 6b (Scheme 2C) followed by further elaboration of the
product yielded aldehyde 36 (Scheme 6).
2.3. Biochemical Studies.
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The inhibitory activity of the synthesized compounds against NV 3CLpro and
their anti-norovirus activity in a cell-based replicon system, were evaluated as described
in the experimental section [33,47]. The determined IC in enzyme assay, EC against
50
50
NV in the replicon harboring cells (HG23 cells) or murine norovirus (MNV) in RAW264.7
cells, and CC values in HG23 cells are listed in Tables 1 and they are the average of
5
0
at least two determinations.
The low cellular permeability and susceptibility to proteolytic degradation of
peptide-based inhibitors provided the impetus behind the design of macrocyclic inhibitor
(I). It is evident from the results shown in Table 1 that, with the exception of compounds
18, 26, 19, and 27 (Table 1) that were inactive, the rest of the compounds inhibited NV
3CLpro and displayed antiviral activity in cell based assays in the replicon harboring
cells (NV) as well as RAW264.7 cells (MNV) with IC50 and EC50 values in the low
micromolar range. The antiviral activities against NV and MNV in cell based assays
demonstrate that the compounds have good permeability in different cell types
(hepatoma cells [HG23] and macrophage-like cells [RAW264.7]). The aldehyde and
aldehyde bisulfite adducts had comparable potency, however, replacement of the
warhead with an α-ketoamide (Table 1, compounds 13 and 25 versus α-ketoamide 31)
diminished activity. These observations are congruent with the results of previous
studies with peptidyl and macrocyclic inhibitors of NV 3CLpro [48-49]. Replacement of
the P
This is contrast to the significant boost in potency observed in the dipeptidyl series of
acyclic inhibitors having a P Cha [33]. As expected, a P Ala (R ) residue resulted in
2 2
Leu (R ) residue with cyclohexylalanine (Cha) had a minor effect on potency.
2
2
2
greatly diminished potency, a reflection of the strong preference of NV 3CLpro for a Cha
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or Leu residue at the
P
2
position [32]. Surprisingly, substitution of
a
spirocyclohexylglycine (1-aminocyclohexaneglycine) at P (R ) resulted in a dramatic
2
2
loss of activity (Table 1, compounds 18, 26, 19, and 27). In those instances, where the
diastereomers were separable, these displayed comparable potencies (Table 1,
compounds 14A-B and 36A-B).
Delineation of the structural determinants impacting pharmacological activity, as
well as demonstration of the mechanism of action, was made possible by determining a
high resolution cocrystal structure of inhibitor 13 with NV 3CLpro. Examination of the
active site revealed the presence of prominent difference electron density with inhibitor
13 covalently bound to Cys 139 and the entire inhibitor could be traced (Figure 2). The
interactions between NV 3CLpro and 13 are shown in Figure 3 and electrostatic surface
representations of the NV 3CLpro with the inhibitor nestled in the active site are shown
in Figure 4. A network of H-bonds involving the backbone of inhibitor 13 and residues
Gln110, Ala158, and Ala160 that serve to position the inhibitor correctly at the active
site are clearly evident. Surprisingly, the His157 and Thr134 residues that are present
in the vicinity of the Gln side chain and ordinarily engage in H-bonding interactions with
the Gln side chain oxygen [33] are absent and seemingly displaced by Pro136, which
forms a H-bond with the oxygen of the tetrahedral adduct (Figure 3). Thus, the loss of
the pair of H-bonds between the Gln side chain oxygen and the His157 and Thr134
residues may account for the observed potency (vide infra). The inhibitor is covalently
bonded to the active site Cys139 residue providing unequivocal demonstration of the
mechanism of action of (I). The m-chlorophenyl group of inhibitor 13 is positioned within
a hydrophobic cleft, as shown in Figure 5. Extending the position of the phenyl ring
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(Table 1, compounds 11-12, 20 and 28) resulted in a two to four-fold decrease in
potency. Furthermore, one of the macrocycles having an unsaturated linker (compound
4, Table 1) was more potent than the corresponding macrocycle with a saturated linker
Table 1/compounds 14A-B) while the second one (Table 1/23) was ~2-fold more potent
2
(
than compound 12 (Table 1). In order to enhance binding and permeability, the P Gln
1
side chain was modified by introducing an additional H-bonding site. The structural
modification did not have a major effect on potency (Table 1, compounds 36A-B).
The m-chlorophenyl moiety occupies a predominantly hydrophobic pocket
(Figure 5), consequently, this site was probed by synthesizing macrocycles having an n-
pentyl chain. The resulting compounds were found to be ~ 2-fold less active (Table 1,
compounds 21/29 and compounds 13/25). Inhibitor 21 is bound to the active site
similarly to inhibitor 13 (Figures 6 and 7) and engages in similar H-bonding interactions
(Figure 8), however, the Pro136 H-bond is missing. The latter, along with the higher
entropic penalty associated with the alkyl chain, partially accounts for the lower affinity.
The n-pentyl chain is clearly shown to engage in hydrophobic interactions (Figure 9).
Modification of this portion of the inhibitor to facilitate the formation of new hydrogen
bonds with the side chains of Thr161 or Thr168 could potentially enhance affinity
(Figures 5 and 9).
3. Conclusions
Noroviruses are a leading cause of acute gastroenteritis in all age groups
worldwide and have a significant impact on public health. There are currently no
norovirus-specific therapeutics or prophylactics. In this report we describe the design,
8

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synthesis, and anti-norovirus activity of a novel class of macrocyclic inhibitors of NV
3CLpro. These studies provide new insights into the interaction of macrocyclic inhibitors
with NV 3CLpro and demonstrate the nuanced interplay of structure, pharmacological
activity, and cellular permeability. They also lay the ground work for conducting further
studies related to the development of anti-norovirus therapeutics.
4. Experimental section
4.1. General
Reagents and dry solvents were purchased from various chemical suppliers
(Aldrich, Acros Organics, ChemImpex, TCI America, Oakwood chemicals, Bachem, and
Fisher) and were used as obtained. Silica gel (230-450 mesh) used for flash
chromatography was purchased from Sorbent Technologies (Atlanta, GA). Thin layer
1
chromatography was performed using Analtech silica gel plates. The H spectra were
recorded in CDCl or DMSO-d on a Varian XL-400 NMR spectrometer and are reported
3
6
relative to TMS (δH= 0.00 ppm). High resolution mass spectra (HRMS) were performed
at the University of Kansas Mass Spectrometry lab using an LCT Premier mass
spectrometer (Waters, Milford, MA) equipped with a time of flight mass analyzer and an
electrospray ion source or a G6230B TOF MS (Agilent Technologies, Santa Clara, CA).
Visualization was accomplished using UV light and/ or iodine.
4.1.1. Synthesis of compounds 1a – f. General procedure.
To a 250 mL round bottom flask kept under nitrogen atmosphere was added a
.5 M solution (35 mmol) of the appropriate Grignard reagent (3-butenyl magnesium
0
bromide or 4-pentenyl magnesium bromide) in THF and the solution was cooled to 0 – 5
9

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ºC. A solution of the appropriate aldehyde (35 mmol) in dry THF (20 mL) was added
dropwise to the cooled Grignard solution over ~ 1 h. The reaction mixture was allowed
to warm up to room temperature and stirred for 5 h under nitrogen. The disappearance
of the aldehyde was monitored by TLC. The reaction mixture was cooled to 0 – 5ºC and
acidified to pH ~3.0 using 5% aqueous hydrochloric acid. The organic solvent was
evaporated off and the residue was extracted with ethyl acetate (2 x 150 mL). The
combined organic extracts were washed with brine (50 mL) dried over anhydrous
2 4
Na SO , filtered, and concentrated. The crude product was purified by column
chromatography to yield alcohols 1a-1f as oils.
4.1.1.1. 1-phenylpent-4-en-1-ol 1a
1
Oil (yield 80%); H NMR (400 MHz, CDCl -d) δ ppm 1.68 - 1.99 (m, 2 H), 1.99 - 2.33 (m,
3
2
H), 4.58 - 4.74 (m, 1 H), 4.90 - 5.11 (m, 2 H), 5.73 - 5.94 (m, 1 H), 7.20 - 7.50 (m, 5
+
H). HRMS (ESI) calcd for C H O: [M+H] : 163.1123 Found: 163.1126.
11
15
4.1.1.2. 1-phenylhex-5-en-2-ol 1b
1
Oil (yield 80%); H NMR (400 MHz, CDCl -d) 1.53 - 1.69 (m, 2 H), 1,91 -2.02 (m,
3
2
H), 2.70 - 2.93 (m, 2 H), 3.95 - 4.08 (m, 1 H), 4.88 - 5.09 (m, 3 H), 5.67 - 5.91 (m, 1
+
H), 7.23 - 7.34 (m, 5 H). HRMS (ESI) calcd for C H O: [M+H] : 177.1279 Found:
12
17
177.1285.
4.1.1.3. 1-(3-chlorophenyl) pent-4-en-1-ol 1c
1
Oil, yield (80%), H NMR (400 MHz, CDCl ) δ ppm 1.71 - 1.96 (m, 2 H), 2.04 -
3
2.24 (m, 2 H), 4.69 (s, 1 H), 4.97 - 5.13 (m, 2 H), 5.77 - 5.93 (m, 1 H), 7.17 - 7.50 (m, 4
+
H). HRMS (ESI) calcd for C11
H
14ClO: [M+H] : 197.0733 Found: 197.0735.
4.1.1.4. 1-(3-chlorophenyl) hex-5-en-1-ol 1d
1
0

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1
Oil (yield 75%); H NMR (400 MHz, CDCl
3
-d) δ ppm 1.26 - 1.62 (m, 1 H), 1.59 -
1.85 (m, 1 H), 1.83 - 1.89 (m, 2 H), 2.02 - 2.13 (m, 2 H), 4.61 - 4.71 (m, 1 H), 4.95 - 5.08
(m, 3 H), 5.69 - 5.86 (m, 1 H), 7.16 - 7.30 (m, 3 H), 7.35 (s, 1 H). HRMS (ESI) calcd for
+
C
12
H
16ClO: [M+H] : 211.0890 Found: 211.0894.
4.11.5. 1-phenyloct-7-en-4-ol 1e
1
Oil (yield 55%); H NMR (400 MHz, CDCl -d) δ ppm 1.41 - 1.59 (m, 4 H), 1.61 -
3
1.86 (m, 2 H), 2.04 – 2.26 (m, 2 H), 2.57 - 2.71 (t, 2 H), 3.59 – 3.70 (m, 1 H), 4.91 - 5.10
(m, 3 H), 5.76 - 5.89 (m, 1 H), 7.14 - 7.20 (d, 2 H), 7.23 – 7.30 (t, 3 H). HRMS (ESI)
+
calcd for C14
H
21O: [M+H] : 205.1592 Found: 205.1598.
4.1.1.6. Dec-1-en-5-ol 1f
1
Oil (yield 93%); H NMR (400 MHz, CDCl -d) δ ppm 0.82 – 0.91(t, 2 H), 1.21 -
3
1
.33 (m, 4 H), 1.35 – 1.44 (m, 2 H), 1.47 - 1.61 (m, 2 H), 2.05 – 2.25 (m, 2 H),3.55 –
.63 (m, 1 H), 4.90 - 5.07 (m, 3 H), 5.76 - 5.89 (m, 1 H). HRMS (ESI) calcd for C H O:
3
10
21
+
[
M+H] : 157.1592 Found: 157.1954.
4.1.2. Synthesis of carbamates 2a – k. General procedure.
A solution of compound 1 (35 mmol) in dry acetonitrile (60 mL) was treated with
trimethylamine (7.1 g; 70 mmol) followed by an appropriate amino acid methyl ester
isocyanate 4 (35 mmol). The resulting reaction mixture was refluxed for 3 h and then
allowed to cool to room temperature. The disappearance of the alcohol was monitored
by TLC. The solvent was evaporated and the residue was taken up in ethyl acetate (250
mL) and the organic layer was washed with 5% aqueous HCl (2 x 50 mL) and saturated
NaCl (50 mL). The organic layer was dried over anhydrous sulfate, filtered, and
concentrated to yield an oily product. Purification by flash chromatography yielded
esters 2a-k as colorless oils.
4.1.2.1. Methyl (((1-phenylpent-4-en-1-yl) oxy) carbonyl)-L-leucinate 2a
1
1

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1
Oil (yield 80%); H NMR (400 MHz, CDCl
3
-d) δ ppm 0.68 - 1.05 (d, 6 H), 1.40 -
1.73 (m, 1 H), 1.73 - 1.95 (m, 2 H), 1.95 - 2.21 (m, 4 H), 3.64 - 3.86 (s,3 H), 4.45 - 4.53
(m, 1 H), 4.86 - 5.20 (m, 2 H), 5.54 - 5.69 (m, 1 H), 5.69 - 5.92 (m, 1 H), 7.17 - 7.35 (m,
+
5
3
4
H), 7.40 – 7.50 (d, 1 H). HRMS (ESI) calcd for C19
H
28NO
4
: [M+H] : 334.2018 Found:
34.2021.
.1.2.2. Methyl (((1-phenylhex-5-en-2-yl) oxy) carbonyl)-L-leucinate 2b
1
Oil (yield 80%); H NMR (400 MHz, CDCl ) δ ppm 0.81 - 0.97 (d, 6 H), 1.53 -
3
1.69 (m, 5 H), 1,91 -2.02 (m, 2 H), 2.70 - 2.93 (m, 2 H), 3.71 – 3.78 (s, 3 H), 4.18 - 4.28
(m, 1 H), 4.88 - 5.09 (m, 3 H), 5.67 - 5.91 (m, 1 H), 6.20 – 6 60 (br, 1 H), 7.13 - 7.43 (m,
+
5
3
4
H), 7.71 – 7.80 (d, 1 H). HRMS (ESI) calcd for C H NO : [M+H] : 348.2175 Found:
20
30
4
48.2180.
.1.2.3. Methyl (((1-(3-chlorophenyl) pent-4-en-1-yl) oxy) carbonyl)-L-leucinate 2c
1
Oil (yield 80%); H NMR (400 MHz, CDCl -d) δ ppm 0.84 - 1.06 (d, 6 H), 1.41 -
3
1.72 (m, 1 H), 1.73 - 2.24 (m, 5 H), 2.42 - 2.57 (m, 2 H),3.62 - 3.77 (d, 3 H), 4.23 - 4.42
(m, 1 H), 4.60 - 4.74 (m, 1 H), 4.92 - 5.24 (m, 1 H), 5.54 - 5.68 (m, 1 H), 5.72 - 5.98 (m,
+
1
3
4
H), 7.15 - 7.32 (m, 3 H), 7.41 (s, 1 H). HRMS (ESI) calcd for C19
4
H27ClNO : [M+H] :
68.1629 Found: 368.1633.
.1.2.4. Methyl (2S)-2-((((1-(3-chlorophenyl) pent-4-en-1-yl) oxy) carbonyl) amino)-3-
cyclohexylpropanoate 2d
1
Oil (yield 75%); H NMR (400 MHz, CDCl -d) δ ppm 0.73 - 0.93 (m, 4 H), 1.03 -
3
1.31 (m, 2H), 1.31 - 1.55 (m, 2 H),1.55 - 1.75 (m, 2 H), 1.74 - 1.89 (m, 4 H), 2.00 - 2.21
(m, 2 H), 3.52 - 3.61 (s, 3H), 4.44 - 4.50 (m, 1 H), 4.95 - 5.18 (m, 3 H), 5.67 - 5.89 (m, 2
H), 7.14 - 7.35 (m, 4 H), 7.44 - 7.49 (s, 1 H). HRMS (ESI) calcd for C H ClNO : [M+H]
22
31
4
+
:
408.1942 Found: 408.1947.
4.1.2.5. Methyl (((1-(3-chlorophenyl) hex-5-en-1-yl) oxy) carbonyl)-L-leucinate 2e
1
Oil (yield 80%); H NMR (400 MHz, CDCl
3
-d) δ ppm 0.87 - 0.92 (d, 6 H), 1.30 -
1.62 (m, 3 H), 1.61 - 1.84 (m, 2 H), 1.97 - 2.16 (m, 4 H), 3.61 - 3.70 (s, 3 H), 4.60 - 4.70
(m, 1 H), 4.89 - 5.06 (m, 2 H), 5.66 - 5.87 (m, 2 H), 7.13 - 7.38 (m, 4 H), 7.40 – 7.45 (s,
+
1
H). HRMS (ESI) calcd for C20
H
29ClNO
4
: [M+H] : 382.1785 Found: 382.1789.
4.1.2.6. Methyl (((1-(3-chlorophenyl) pent-4-en-1-yl) oxy) carbonyl)-L-alaninate 2f
1
2

ACCEPTED MANUSCRIPT
1
Oil (yield 80%); H NMR (400 MHz, CDCl
3
-d) δ ppm 1.42 (d, J=7.03 Hz, 3 H),
1.75 - 1.92 (m, 2 H), 1.90 - 2.14 (m, 2 H), 3.74 (br d, J=16.01 Hz, 3 H), 4.27 - 4.39 (m, 1
H), 4.94 - 5.09 (m, 2 H), 5.24 - 5.44 (d, 1 H), 5.55 - 5.68 (m, 1 H), 5.72 - 5.87 (m, 1 H),
+
7
.26 (dd, J=4.10, 0.98 Hz, 4 H). HRMS (ESI) calcd for C16
H
21ClNO
4
: [M+H] : 326.1159
Found: 326.1162.
4.1.2.7. Methyl (((1-(3-chlorophenyl) hex-5-en-1-yl) oxy) carbonyl)-L-alaninate 2g
1
Oil (yield 80%); H NMR (400 MHz, CDCl -d) δ ppm 1.27 - 1.56 (m, 5 H), 1.66 -
3
2.14 (m, 4 H), 3.71 (br d, J=16.01 Hz, 3 H), 4.24 - 4.39 (m, 1 H), 4.89 - 5.06 (m, 2 H),
5.29 - 5.49 (m, 1 H), 5.54 - 5.83 (m, 2 H), 7.10 - 7.36 (m, 4 H). 8.10 – 8.15 (br s, 1 H).
+
HRMS (ESI) calcd for C H ClNO : [M+H] : 340.1316 Found: 340.1322.
17
23
4
4.1.2.8. Methyl 1-((((1-(3-chlorophenyl) pent-4-en-1-yl) oxy) carbonyl) amino)
cyclohexane-1-carboxylate 2h
1
Oil (yield 70%); H NMR (400 MHz, CDCl -d) δ ppm 1.18 - 1.66 (m, 6H), 1.67 -
3
2.21 (m, 6 H), 2.23 - 2.40 (m, 2 H), 3.59 (s, 3 H), 4.53 - 4.73 (m, 1 H), 4.93 - 5.13 (m, 2
H), 5.48 - 5.64 (m, 1 H), 5.71 - 5.93 (m, 1 H), 7.24 (m, 4 H). HRMS (ESI) calcd for
+
C
20
H
27ClNO
4
: [M+H] : 380.1629 Found: 380.1635.
4.1.2.9. Methyl 1-((((1-(3-chlorophenyl) hex-5-en-1-yl) oxy) carbonyl) amino)
cyclohexane-1-carboxylate 2i
1
Oil, yield (65%); H NMR (400 MHz, CDCl
3
-d) δ ppm 1.30 - 2.16 (m, 16 H), 3.57
-
3.65 (s, 3 H), 4.62 - 4.71 (m, 1 H), 4.88 - 5.08 (m, 2 H), 5.48 - 5.63 (m, 1 H), 5.67 -
5.87 (m, 1 H), 7.11 - 7.30 (m, 3 H), 7.32 - 7.39 (s, 1 H). HRMS (ESI) calcd for
+
C H ClNO : [M+H] : 394.1785 Found: 394.1790.
21
29
4
4.1.2.10. Methyl (((1-phenyloct-7-en-4-yl) oxy) carbonyl)-L-leucinate 2j
1
Oil (yield 88%); H NMR (400 MHz, CDCl
3
-d) δ ppm 0.83 – 0.96 (d, 6 H), 1.37 –
1.92 (m, 7 H), 1.95 - 2.07 (m, 3 H), 2.49 – 2.65 (t, 2 H), 3.63 – 3.73 (s, 3 H), 4.28 – 4.48
(m, 1 H), 4.71 – 4.81 (m, 1 H), 4.88 - 5.08 (m, 2 H), 5.48 - 5.63 (m, 1 H), 5.67 - 5.87 (m,
1
H), 7.08 - 7.18 (m, 2 H), 7.18 - 7.28 (m, 3 H). HRMS (ESI) calcd for C H NO : [M+H]
22 34 4
+
:
376.2488 Found: 376.2452.
4.1.2.11. Methyl ((dec-1-en-5-yloxy) carbonyl)-L-leucinate 2k
1
Oil (yield 91%); H NMR (400 MHz, CDCl -d) δ ppm 0.83 – 0.90 (t, 2 H),0.90 –
3
0.98 (d, 6 H), 1.20 – 1.35 (m, 6 H), 1.45 - 1.70 (m, 4 H), 1.98 – 2.15 (m, 4 H), 3.69 –
1
3

ACCEPTED MANUSCRIPT
3.76 (s, 3 H), 4.30 – 4.43 (m, 1 H), 4.70 – 4.80 (m, 1 H), 4.90 - 5.05 (m, 2 H), 5.70 - 5.80
+
(
m, 1 H), 7.80 - 7.89 (d, 1 H). HRMS (ESI) calcd for C H NO : [M+H] : 328.2488
18
34
4
Found: 328.2454.
4.1.3. Synthesis of acids (3a-g and 3j-k). General procedure A.
A solution of ester 2 (10 mmol) in tetrahydrofuran (15 mL) was treated with 1M
aqueous LiOH (20 mL). The reaction mixture was stirred for 3 h at room temperature
while monitoring the disappearance of the ester by TLC. Most of the solvent was
evaporated off and the solution was acidified to pH ~3 using 5% hydrochloric acid (10
mL). The aqueous layer was extracted with ethyl acetate (2 x 100 mL) and the
combined organic layer was washed with brine (50 mL). The organic layer was dried
over anhydrous sodium sulfate, filtered, and concentrated to yield compounds 3a-g and
3j-k as colorless oils.
4.1.3.1. (((1-phenylpent-4-en-1-yl) oxy) carbonyl)-L-leucine 3a
1
Oil (yield 92%); H NMR (400 MHz, CDCl
3
-d) δ ppm 0.68 - 1.05 (d, 6 H), 1.40 -
1.73 (m, 1 H), 1.73 - 1.95 (m, 2 H), 1.95 - 2.21 (m, 4 H), 4.45 - 4.53 (m, 1 H), 4.86 - 5.20
(m, 2 H), 5.54 - 5.69 (m, 1 H), 5.69 - 5.92 (m, 1 H), 7.17 - 7.35 (m, 5 H), 7.40 – 7.50 (d,
+
1
H). HRMS (ESI) calcd for C18
H
26NO
4
: [M+H] : 320.1862 Found: 320.1865.
4.1.3.2. (((1-phenylhex-5-en-2-yl) oxy) carbonyl)-L-leucine 3b
Oil (yield 91%); 1H NMR (400 MHz, CDCl -d) δ ppm 0.81 - 0.97 (d, 6 H), 1.53 -
3
1.69 (m, 5 H), 1,91 -2.02 (m, 2 H), 2.70 - 2.93 (m, 2 H), 4.18 - 4.28 (m, 1 H), 4.88 - 5.09
(m, 3 H), 5.67 - 5.91 (m, 1 H), 6.20 – 6 60 (br., 1 H), 7.13 - 7.43 (m, 5 H), 7.71 – 7.80 (d,
+
1
H). HRMS (ESI) calcd for C H NO : [M+H] : 334.2018 Found: 334.2022.
19
28
4
4.1.3.3. (((1-(3-chlorophenyl) pent-4-en-1-yl) oxy) carbonyl)-L-leucine 3c
1
Oil (yield 92%); H NMR (400 MHz, CDCl
3
-d) δ ppm 0.84 - 1.06 (d, 6 H), 1.41 -
1.72 (m, 1 H), 1.73 - 2.24 (m, 5 H), 2.42 - 2.57 (m, 2 H), 4.23 - 4.42 (m, 1 H), 4.60 - 4.74
1
4

ACCEPTED MANUSCRIPT
(m, 1 H), 4.92 - 5.24 (m, 1 H), 5.54 - 5.68 (m, 1 H), 5.72 - 5.98 (m, 1 H), 7.15 - 7.32 (m,
+
3
3
H), 7.41 (s, 1 H). HRMS (ESI) calcd for C H ClNO : [M+H] : 354.1472 Found:
18
25
4
54.1475.
.1.3.4. (2S)-2-((((1-(3-chlorophenyl) pent-4-en-1-yl) oxy) carbonyl) amino)-3-
4
cyclohexylpropanoic acid 3d
1
Oil (yield 90%); H NMR (400 MHz, CDCl -d) δ ppm 0.77 - 1.30 (m, 7 H), 1.58 -
3
2.21 (m, 8 H), 4.25 - 4.44 (m, 2 H), 4.65 - 4.73 (m, 1 H), 4.95 - 5.22 (m, 2 H), 5.53 - 5.71
(m, 1 H), 5.71 - 5.88 (m, 1 H), 7.14 - 7.41 (m, 4 H), 7.44 - 7.49 (s, 1 H). HRMS (ESI)
+
calcd for C H ClNO : [M+H] : 394.1785 Found: 394.1791.
21
29
4
4.1.3.5. (((1-(3-chlorophenyl) hex-5-en-1-yl) oxy) carbonyl)-L-leucine 3e
1
Oil (yield 95%); H NMR (400 MHz, CDCl
3
-d) δ ppm 0.85 - 1.00 (d, 6 H), 1.41 -
1.78 (m, 5 H), 1.95 - 2.17 (m, 4 H), 4.29 - 4.48 (m, 1 H), 4.63 - 4.73 (m, 1 H), 4.84 - 5.01
(m, 1 H), 5.18 - 5.30 (m, 2 H), 5.72 - 5.85 (m, 2 H), 7.14 - 7.40 (m, 4 H). HRMS (ESI)
+
calcd for C H ClNO : [M+H] : 368.1629 Found: 368.1637.
19
27
4
4.1.3.6. (((1-(3-chlorophenyl) pent-4-en-1-yl) oxy) carbonyl)-L-alanine 3f
1
Oil (yield 92%); H NMR (400 MHz, CDCl -d) δ ppm 1.34 - 1.47 (d, 3 H), 1.75 -
3
1.91 (m, 2 H), 1.91 - 2.11 (m, 2 H), 4.28 - 4.46 (m, 1 H), 4.97 - 5.13 (m, 2 H), 5.35 - 5.46
(m, 1 H), 5.57 - 5.66 (m, 1 H), 5.73 - 5.87 (m, 1 H), 7.28 (br d, J=5.47 Hz, 3 H), 7.31 -
+
7
.42 (s, 1 H). HRMS (ESI) calcd for C H ClNO : [M+H] : 312.1003 Found: 312.1010.
15
19
4
4.1.3.7. (((1-(3-chlorophenyl) hex-5-en-1-yl) oxy) carbonyl)-L-alanine 3g
1
Oil (yield 92%); H NMR (400 MHz, CDCl
3
-d) δ ppm 1.27 - 1.56 (m, 5 H), 1.66 -
2.14 (m, 4 H), 4.24 - 4.39 (m, 1 H), 4.89 - 5.06 (m, 2 H), 5.29 - 5.49 (m, 1 H), 5.54 - 5.83
(m, 2 H), 7.10 - 7.36 (m, 4 H). 8.10 – 8.15 (br s, 1 H). HRMS (ESI) calcd for
+
C H ClNO : [M+H] : 326.1159 Found: 326.1168.
16
21
4
4.1.3.8. (((1-phenyloct-7-en-4-yl) oxy) carbonyl)-L-leucine 3j
1
5

ACCEPTED MANUSCRIPT
1
Oil (yield 94%); H NMR (400 MHz, CDCl
.92 (m, 7 H), 1.95 - 2.07 (m, 3 H), 2.49 – 2.65 (t, 2 H), 4.28 – 4.48 (m, 1 H), 4.71 – 4.81
m, 1 H), 4.88 - 5.08 (m, 2 H), 5.48 - 5.63 (m, 1 H), 5.67 - 5.87 (m, 1 H), 7.08 - 7.18 (m,
3
-d) δ ppm 0.83 – 0.96 (d, 6 H), 1.37 –
1
(
2
H), 7.18 - 7.28 (m, 3 H), 8.10 – 8.16 (d, 1 H). HRMS (ESI) calcd for C21
4
H32NO : [M+H]
+
:
362.2331 Found 362.2333.
4.1.3.9. ((Dec-1-en-5-yloxy) carbonyl)-L-leucine 3k
1
Oil (yield 91%); H NMR (400 MHz, CDCl
3
-d) δ ppm 0.83 – 0.90 (t, 2 H),0.90 –
0
4
7
.98 (d, 6 H), 1.20 – 1.35 (m, 6 H), 1.45 - 1.70 (m, 4 H), 1.98 – 2.15 (m, 4 H), 4.30 –
.43 (m, 1 H), 4.70 – 4.80 (m, 1 H), 4.90 - 5.05 (m, 2 H), 5.70 - 5.80 (m, 1 H), 7.80 -
+
.89 (d, 1 H). HRMS (ESI) calcd for C17
H
32NO
4
: [M+H] : 314.2331 Found 314.2339.
4.1.3. Synthesis of acids 3h-i. General procedure B.
A solution of ester 2a or 2i (10 mmol) in tetrahydrofuran (10 mL) and methanol
(10 mL) was treated with 2M aqueous LiOH (20 mL) and the reaction mixture was
heated to 60ºC for 12 h with stirring. The disappearance of the ester was monitored by
TLC. The reaction mixture was cooled to room temperature, the solvent was removed
under reduced pressure, and the solution was acidified to pH ~3 using 5% hydrochloric
acid (~20 mL). The aqueous layer was extracted with ethyl acetate (2 x 100 mL) and the
combined extracts were washed with brine (50 mL). The organic layer was dried over
2 4
anhydrous Na SO , filtered, and concentrated to yield compounds 3h-i as oils.
4.1.3.10. 1-((((1-(3-chlorophenyl) pent-4-en-1-yl) oxy) carbonyl) amino) cyclohexane-1-
carboxylic acid 3h
1
Oil (yield 90%); H NMR (400 MHz, CDCl
3
) δ ppm 1.18 - 1.66 (m, 6H), 1.67 -
2.21 (m, 6 H), 2.23 - 2.40 (m, 2 H), 4.69 (dd, J=7.62, 5.27 Hz, 1 H), 4.94 - 5.09 (m, 2 H),
5.57 - 5.62 (m, 1 H), 5.72 - 5.90 (m, 1 H), 7.14 - 7.17 (m, 1 H), 7.15 - 7.31 (m, 4 H).
+
HRMS (ESI) calcd for C H ClNO : [M+H] : 366.1472 Found 366.1478.
19
25
4
1
6

ACCEPTED MANUSCRIPT
4.1.3.11. 1-((((1-(3-chlorophenyl) hex-5-en-1-yl) oxy) carbonyl) amino) cyclohexane-1-
carboxylic acid 3i
1
Oil (yield 95%); H NMR (400 MHz, CDCl ) δ ppm 1.30 - 2.16 (m, 16 H), 4.62 -
3
4.71 (m, 1 H), 4.88 - 5.08 (m, 2 H), 5.48 - 5.63 (m, 1 H), 5.67 - 5.87 (m, 1 H), 7.11 - 7.30
+
(
m, 3 H), 7.32 - 7.39 (s, 1 H). HRMS (ESI) calcd for C H ClNO : [M+H] : 380.1629
20
27
4
Found 380.1637.
4.1.4. Synthesis of amino acid methyl ester isocyanates 4a-d. General procedure.
The appropriate amino acid methyl ester hydrochloride (55 mmol) was placed in
a dry 500-mL RB flask and then dried overnight on the vacuum pump. The flask was
flushed with nitrogen and dry dioxane (150 mL) was added followed by trichloromethyl
chloroformate (16.26 g, 82.5 mmol). After refluxing for 12 h, the solvent was removed
on the rotary evaporator and the residue was vacuum distilled to yield pure isocyanates
4a-d as colorless oils.
4.1.5. Synthesis of compounds 5a–b. General procedure.
To a solution of Boc-L-glutamic acid α-methyl ester (80 mmol) in dry DMF (200
mL) were added EDCI (19.93 g; 104 mmol), HOBt (15.92 g; 104 mmol) and the reaction
mixture was stirred for 30 min at room temperature. Following the sequential addition of
allylamine/O-allylhydroxylamine hydrochloride (120 mmol) and DIEA (20.68 g; 160
mmol), the reaction mixture was stirred for 16 h at room temperature. Completion of the
reaction was monitored by TLC. The solvent was removed and the residue was
partitioned between ethyl acetate (400 mL) and 5% aqueous HCl (100 mL). The layers
were separated and the organic layer was further washed with saturated aqueous
3
NaHCO (2 x 100 mL), followed by saturated NaCl (100 mL). The organic layer was
1
7

ACCEPTED MANUSCRIPT
dried over anhydrous sodium sulfate, filtered and concentrated to yield a yellow-colored
oily product. Purification by flash chromatography yielded a white solid.
5
2
4
.1.5.1. Methyl N -allyl-N -(tert-butoxycarbonyl)-L-glutaminate 5a
o
1
White solid (yield 85%); m.p. 69-70 C; H NMR (400 MHz, CDCl
3
-d) δ ppm 1.42 -
1.50 (s, 9 H), 1.90 – 2.00 (m, 1 H), 2.16 – 2.28 (m. 1 H), 3.74 - 3.80 (s, 3 H), 3.91 – 4.00
(t, 2 H), 4.34 – 4.41 (m, 1 H), 5.09 - 5.22 (m, 2 H), 5.30 – 5.40 (br. s, 1 H), 5.78 – 5.94
+
(
m, 1 H), 6.10 - 6.20 (br. s, 1 H). HRMS (ESI) calcd for C H N O : [M+H] : 301.1763
14
25
2
5
Found 301.1771.
5
2
4
.1.5.2. Methyl N -(allyloxy)-N -(tert-butoxycarbonyl)-L-glutaminate 5b
1
3
Sticky oil (yield 54%); H NMR (400 MHz, CDCl -d) δ ppm 1.41 - 1.50 (s, 9 H), 1.78 –
1.96 (br. s, 1 H),2.12 – 2.28 (m. 4 H), 3.72 - 3.80 (s, 3 H), 4.24 – 4.37 (m, 2 H), 4.44 –
4.51 (m, 1 H), 5.29 - 5.50 (m, 2 H), 5.92 – 6.07 (m, 1 H), 9.29 - 9.42 (br. s, 1 H). HRMS
+
(
ESI) calcd for C H N O : [M+H] : 317.1713 Found 317.1718.
14
25
2
6
4.1.6. Synthesis of compounds 6a–b. General procedure.
To a solution of compound 5 (43 mmol) in dry DCM (30 mL) was added a
solution of 4 M HCl in dioxane (130 mL) with stirring. The reaction mixture was stirred
for 3 h at room temperature. The disappearance of the starting material was monitored
by TLC. The solvent was evaporated under reduced pressure and compound 6 was
used in the next step without further purification.
4.1.7. Synthesis of acyclic compounds 7a–k and 32.
General procedure. To a solution of compound 3 (15 mmol) in dry DMF (40 mL)
was added EDCI (3.74 g, 19.5 mmol, 1.30 eq), HOBt (2.97 g, 19.5 mmol, 1.30 eq) and
the mixture was stirred for 30 min at room temperature. In a separate flask, a solution of
1
8

ACCEPTED MANUSCRIPT
o
compound 6a (3.55 g, 15 mmol) in DMF (20 mL) cooled to 0-5 C was treated with
diisopropyl ethylamine (DIEA) (7.75 g, 60 mmol, 4 eq), stirred for 30 min, and then
added to the reaction mixture containing acid 3. The reaction mixture was stirred for 16
h while monitoring the reaction by TLC. The solvent was removed and the residue was
partitioned between ethyl acetate (300 mL) and 10% citric acid (50 mL). The layers
were separated and the organic layer was further washed with saturated aqueous
3
NaHCO (2 x 50 mL), followed by brine (50 mL). The organic layer was dried over
anhydrous sodium sulfate, filtered and concentrated to yield a yellow-colored oily
product. Purification by flash chromatography yielded esters 7a-k.
5
2
4
.1.7.1. Methyl N -allyl-N -((((1-phenylpent-4-en-1-yl) oxy) carbonyl)-L-leucyl)-L-
glutaminate 7a
1
Oil (yield 62%); H NMR (400 MHz, CDCl
.73 (m, 3 H), 1.73 - 1.95 (m, 2 H), 1.95 - 2.21 (m, 6 H), 3.64 - 3.76 (s,3 H), 4.45 - 4.53
m, 4 H), 4.86 - 5.20 (m, 4 H), 5.54 - 5.69 (m, 2 H), 5.69 - 5.92 (m, 2 H), 7.17 - 7.35 (m,
H), 7.40 – 7.50 (d, 1 H), 7.90 – 7. 80 (d, 1 H). HRMS (ESI) calcd for C H N O :
3
-d) δ ppm 0.68 - 1.05 (d, 6 H), 1.40 -
1
(
5
27
40
3
6
+
[
M+H] : 502.2917 Found 502.2921.
5
2
4
.1.7.2. Methyl N -allyl-N -((((1-phenylhex-5-en-2-yl) oxy) carbonyl)-L-leucyl)-L-
glutaminate 7b
1
Oil (yield 60%); H NMR (400 MHz, CDCl -d) δ ppm 0.81 - 0.97 (d, 6 H), 1.53 -
3
1.69 (m, 5 H), 1,91 -2.02 (m, 6 H), 2.70 - 2.93 (m, 2 H), 3.73 (s, 3 H), 3.80 - 3.94 (t, 2
H), 4.40 - 4.62 (m, 2 H), 4.88 - 5.04 (m, 2 H), 5.06 - 5.26 (m, 2 H), 5.68 - 5.87 (m, 3 H),
6.20 - 6.41 (br. d, 1 H), 6.91 - 7.10 (d, 1 H), 7.14 - 7.36 (m, 5 H), 7.85 – 7.92 (d, 1 H).
+
HRMS (ESI) calcd for C28
H N
42 3
O
6
: [M+H] : 516.3074 Found 516.3080.
5
2
4
.1.7.3. Methyl N -allyl-N -((((1-(3-chlorophenyl) pent-4-en-1-yl) oxy) carbonyl)-L-
leucyl)-L-glutaminate 7c
1
9

ACCEPTED MANUSCRIPT
1
Oil (yield 65%); H NMR (400 MHz, CDCl
.13 (m, 4 H), 2.13 - 2.38 (m, 6 H), 3.60 - 3.71 (s, 3 H), 4.07 - 4.24 (m, 1 H), 4.41 - 4.61
m, 2 H), 4.91 - 5.25 (m, 1 H), 5.25 - 5.42 (m, 2 H), 5.50 - 5.68 (m, 2 H), 5.70 - 5.92 (m,
3
-d) δ ppm 0.74 - 1.02 (d, 6 H), 1.43 -
2
(
2
8
5
H), 6.07 - 6.26 (m, 2 H), 6.30 - 6.39 (m, 1 H), 6.96 - 7.34 (m, 4 H), 7.7 - 7.8 (d, 1 H),
+
.1 – 8.2 (d, 1 H). HRMS (ESI) calcd for C H ClN O : [M+H] : 536.2527 Found
27
39
3
6
36.2534.
.1.7.4. Methyl N -allyl-N -((2S)-2-((((1-(3-chlorophenyl) pent-4-en-1-yl) oxy) carbonyl)
5
2
4
amino)-3-cyclohexylpropanoyl) glutaminate 7d
1
Oil (yield70%); H NMR (400 MHz, CDCl -d) δ ppm 0.75 - 1.59 (m, 4 H), 1.55 -
3
1.73 (m, 4 H), 1.74 - 1.89 (m, 4 H), 1.89 - 2.14 (m, 5 H), 2.14 - 2.31 (m, 4 H), 3.65 - 3.76
(d, 3 H), 3.76 - 4.02 (m, 1 H), 4.07 - 4.29 (m, 1 H), 4.40 - 4.61 (m, 2 H), 4.93 - 5.38 (m, 2
H), 5.43 - 5.64 (m, 2 H), 5.69 - 5.90 (m, 3 H), 6.10 - 6.20 (d, 1 H), 6.35 - 6.44 (d, 1 H),
+
6
.86 - 7.05 (d, 1 H), 7.12 - 7.37 (m, 4 H). HRMS (ESI) calcd for C H ClN O : [M+H] :
30
43
3
6
576.2840 Found 576.2848.
5
2
4
.1.7.5. Methyl N -allyl-N -((((1-(3-chlorophenyl) hex-5-en-1-yl) oxy) carbonyl)-L-leucyl)-
L-glutaminate 7e
1
Oil (yield 75%); H NMR (400 MHz, CDCl -d) δ ppm 0.82 - 1.03 (d, 6 H), 1.20 -
3
2
4
-
.32 (m, 13 H), 3.73 (br d, J=12.89 Hz, 3 H), 4.03 - 4.20 (m, 2 H), 4.38 - 4.59 (m, 2 H),
.89 - 5.03 (m, 2 H), 5.08 - 5.32 (m, 2 H), 5.48 - 5.67 (m, 1 H), 5.68 - 5.94 (m, 1 H), 5.98
6.12 (m, 1 H), 6.21 - 6.34 (br s, 1 H), 6.86 - 7.03 (br s, 2 H), 7.13 - 7.37 (m, 4 H).
+
HRMS (ESI) calcd for C28H41ClN
3
O
6
: [M+H] : 550.2684 Found 550.2691.
5
2
4
.1.7.6. Methyl N -allyl-N -((((1-(3-chlorophenyl) pent-4-en-1-yl) oxy) carbonyl)-L-
alanyl)-L-glutaminate 7f
1
Oil (yield 80%); H NMR (400 MHz, CDCl -d) δ ppm 1.39 (d, J=7.03 Hz, 3 H),
3
1
.75 - 1.91 (m, 4 H), 1.91 - 2.11 (m, 4 H), 3.66 - 3.71 (s, 3 H), 4.17 - 4.27 (d, 2 H), 4.41 -
.61 (m, 2 H), 4.92 - 5.25 (m, 4 H), 5.47 - 5.65 (m, 1 H), 5.71 - 5.88 (m, 2 H), 6.15- 6.20
4
(br s, 1 H), 6.40 - 6.45 (br s, 1 H), 7.12 - 7.32 (m, 4 H), 7.50 – 7.60 (d, 1 H). HRMS (ESI)
+
calcd for C H ClN O : [M+H] : 494.2058 Found 494.2067.
24
33
3
6
2
0

ACCEPTED MANUSCRIPT
5
2
4
.1.7.7. Methyl N -allyl-N -((((1-(3-chlorophenyl) hex-5-en-1-yl) oxy) carbonyl)-L-alanyl)-
L-glutaminate 7g
1
Oil (yield 80%); H NMR (400 MHz, CDCl -d) δ ppm 1.30- 1.39 (m, 2 H), 1.39 –
3
1
4
-
.42 (d, J=7.03 Hz, 3 H), 1.75 - 1.91 (m, 4 H), 1.91 - 2.11 (m, 4 H), 3.66 - 3.71 (s, 3 H),
.17 - 4.27 (d, 2 H), 4.41 - 4.61 (m, 2 H), 4.92 - 5.25 (m, 4 H), 5.47 - 5.65 (m, 1 H), 5.71
5.88 (m, 2 H), 6.15- 6.20 (br s, 1 H), 6.40 - 6.45 (br s, 1 H), 7.12 - 7.32 (m, 4 H), 7.50 –
+
7
.60 (d, 1 H). HRMS (ESI) calcd for C25
H
35ClN
3
O
6
: [M+H] : 508.2214 Found 508.2218.
5
2
4
.1.7.8. Methyl N -allyl-N -(1-((((1-(3-chlorophenyl) pent-4-en-1-yl) oxy) carbonyl)
amino) cyclohexane-1-carbonyl)-L-glutaminate 7h
1
Oil (yield 55%); H NMR (400 MHz, CDCl ) δ ppm 1.20 - 1.48 (m, 5 H), 1.49 -
3
1.74 (m, 5 H), 1.75 - 1.90 (m, 4 H), 1.90 - 2.35 (m, 4 H), 3.71 (s, 3 H), 3.76 - 3.91 (m, 2
H), 4.41 - 4.61 (m, 1 H), 4.95 - 5.26 (m, 4 H), 5.72 - 5.91 (m, 3 H), 6.29 - 6.45 (br. d, 1
H), 6.57 - 6.71 (br. s, 1 H), 6.95 - 7.06 (d, 1 H), 7.15 - 7.35 (m, 4 H). HRMS (ESI) calcd
+
for C H ClN O : [M+H] : 548.2527 Found 548.2531.
28
39
3
6
5
2
4
.1.7.9. Methyl N -allyl-N -(1-((((1-(3-chlorophenyl) hex-5-en-1-yl) oxy) carbonyl) amino)
cyclohexane-1-carbonyl)-L-glutaminate 7i
1
Oil (yield 57%); H NMR (400 MHz, CDCl -d) δ ppm 1.16 - 1.53 (m, 7 H), 1.54 -
3
1.74 (m, 7 H), 1.75 - 2.34 (m, 6 H), 3.71 (s, 3 H), 3.73 - 3.90 (m, 2 H), 4.39 - 4.63 (m, 1
H), 4.90 - 5.27 (m, 4 H), 5.41 - 5.61 (m, 1 H), 5.65 - 5.91 (m, 2 H), 6.29 - 6.44 (br. s, 1
H), 6.60 - 6.66 (d, 1 H), 6.89 - 7.03 (d, 1 H), 7.13 - 7.18 (m, 3 H), 7.21 - 7.31 (s, 1 H).
+
HRMS (ESI) calcd for C H ClN O : [M+H] : 562.2684 Found 562.2685.
29
41
3
6
5
2
4
.1.7.10. Methyl N -allyl-N -((((1-phenyloct-7-en-4-yl) oxy) carbonyl)-L-leucyl)-L-
glutaminate 7j
o
1
White solid (yield 62%); m.p 99 – 100 C. H NMR (400 MHz, CDCl -d) δ ppm
3
0
.90 – 0.99 (d, 6 H), 1.46 - 1.74 (m, 7 H), 1.75 – 1.82 (t, 2 H),1.90 – 2.12 (m, 4 H), 2.14
2.35 (m, 2 H), 2.52 – 2.60 (t, 2 H), 3.70 – 3.75 (s, 3 H), 3.80 - 3.90 (d, 2 H), 4.10 -4.20
–
(m, 1 H), 4.48 - 4.59 (m, 1 H), 4.71 – 4.84 (m, 1 H), 4.90 - 5.23 (m, 4 H), 5.70 - 5.90 (m,
2
H), 6.20 - 6.30 (br. s, 1 H), 6.30 – 6.40 (br. s, 1 H), 6.93 - 6.70 (d, 1 H), 7.11 - 7.20 (d,
2
1

ACCEPTED MANUSCRIPT
+
2
H), 7.22 - 7.30 (m, 3 H). HRMS (ESI) calcd for C30
46 3 6
H N O : [M+H] : 544.3387 Found
544.3390.
5
2
4
.1.7.11. Methyl N -allyl-N -(((dec-1-en-5-yloxy) carbonyl)-L-leucyl)-L-glutaminate 7k
o
1
White solid (yield 70%); m.p 103 – 104 C. H NMR (400 MHz, CDCl -d) δ ppm
3
0.83 – 0.91 (t, 2 H),0.91 – 0.99 (d, 6 H), 1.20 – 1.35 (m, 6 H), 1.45 - 1.70 (m, 4 H), 1.98
– 2.12 (m, 4 H), 2.14 – 2.35 (m, 2 H), 2.52 – 2.60 (t, 2 H), 3.72 – 3.76 (s, 3 H), 3.80 -
3.90 (d, 2 H), 4.10 - 4.20 (m, 1 H), 4.50 - 4.60 (m, 1 H), 4.66 – 4.78 (m, 1 H), 4.91 - 5.24
(m, 4 H), 5.71 - 5.90 (m, 2 H), 6.35 – 6.50 (br. s, 2 H), 6.91 – 7.00 (d, 1 H). HRMS (ESI)
+
calcd for C H N O : [M+H] : 496.3387 Found 496.3388.
26
46
3
6
5
2
4
.1.7.12. Methyl N -(allyloxy)-N -((((1-(3-chlorophenyl) pent-4-en-1-yl)oxy)carbonyl)-L-
leucyl)-L-glutaminate 32
1
Oil (yield 52%); H NMR (400 MHz, CDCl -d) δ ppm 0.83 – 1.00 (dd, 6 H), 1.23 –
3
1.29 (t, 2 H), 1.43 – 2.15 (m, 5 H), 2.14 – 2.35 (m, 4 H), 3.69 – 3.80 (d, 3 H), 4.03 – 4.
22 (m, 2 H), 4.32 – 4.48 (m, 2 H), 4.93 – 5.06 (m, 2 H), 5.23 – 5.44 (m, 2 H), 5.50 - 5.61
(m, 1 H), 5.73 – 5.84 (m, 1 H), 5.87 – 6.05 (m, 1 H), 6.76 – 6. 89 (br. s, 1 H), 7.13 – 7.20
(s, 1 H), 7.23 – 7.33 (m, 3 H), 9.22 – 9.29 (d, 1 H), 9.43 – 9.51 (d, 1 H). HRMS (ESI)
+
calcd for C H ClN O : [M+H] : 552.2477 Found 552.2479.
27
39
3
7
4.1.8. Synthesis of compounds 8a–k and 33. General procedure for ring closing
metathesis.
A solution of acyclic diene 7 (1.21 mmol) in dry DCM (1.5 L) was degassed for
0 min using nitrogen. Chloro dicyclohexyl borane (1 M in hexane) (1.21 mL; 1.21
3
nd
mmol) and Grubb’s 2 generation catalyst (104 mg; 10 mol%) were added and
degassing was continued for 10 min. The reaction mixture was heated to 45 ºC and
nd
stirred for 30 min under a nitrogen atmosphere. An additional portion of Grubb’s 2
generation catalyst (52 mg; 5 mol%) was added and the solution stirred for 16 h at 45
ºC under a nitrogen atmosphere. The reaction was quenched by adding activated
2
2

ACCEPTED MANUSCRIPT
charcoal (600 mg) and stirring the reaction for 18 h at room temperature. The reaction
mixture was filtered through a Celite bed and the solvent was removed on the rotary
evaporator. The crude residue was purified by flash chromatography to give macrocyclic
esters 8a-k as off-white solids.
4.1.8.1.
Methyl
(4S,7S,
E)-4-isobutyl-2,5,10-trioxo-17-phenyl-1-oxa-3,6,11-
triazacycloheptadec-13-ene-7-carboxylate 8a
o
1
Off-white solid (yield 65%); m.p 79 – 80 C. H NMR (400 MHz, CDCl -d) δ ppm
3
0
.68 - 1.05 (d, 6 H), 1.40 - 1.73 (m, 3 H), 1.73 - 1.95 (m, 2 H), 1.95 - 2.21 (m, 6 H), 3.64
3.76 (s, 3 H), 3.80 – 3.89 (m, 2 H), 4.45 - 4.53 (m, 2 H), 5.51 - 5.74 (m, 2 H), 5.76 -
.90 (m, 1 H), 7.17 - 7.35 (m, 5 H), 7.40 – 7.50 (d, 1 H), 7.90 – 7. 80 (d, 1 H), 8.25 –
-
5
8
+
.35 (d, 1 H). HRMS (ESI) calcd for C H N O : [M+H] : 474.2604 Found: 474.2608.
25
36
3
6
4.1.8.2.
Methyl
(4S,7S,
E)-17-benzyl-4-isobutyl-2,5,10-trioxo-1-oxa-3,6,11-
triazacycloheptadec-13-ene-7-carboxylate 8b
o
1
3
Off-white solid (yield 68%); m.p 70 - 71 C. H NMR (400 MHz, CDCl -d) δ ppm
0
–
5
7
4
.81 - 0.97 (d, 6 H), 1.53 - 1.69 (m, 4 H), 1,91 -2.02 (m, 5 H), 2.70 - 2.93 (m, 2 H), 3.73
3.80 (s, 3 H), 3.80 - 3.94 (m, 2 H), 4.40 - 4.62 (m, 2 H), 4.88 - 5.04 (m, 1 H), 5.06 -
.26 (m, 2 H), 5.68 - 5.87 (m, 2 H), 6.20 - 6.41 (br. d, 1 H), 6.91 - 7.10 (d, 1 H), 7.14 -
+
.36 (m, 5 H), 7.85 – 7.92 (d, 1 H). HRMS (ESI) calcd for C H N O : [M+H] :
26
38
3
6
88.2761 Found: 488.2767.
.1.8.3. Methyl (4S,7S, E)-17-(3-chlorophenyl)-4-isobutyl-2,5,10-trioxo-1-oxa-3,6,11-
4
triazacycloheptadec-13-ene-7-carboxylate 8c
o
1
Off-white solid (yield 70%); m.p 84 - 85 C. H NMR (400 MHz, CDCl
3
-d) δ ppm
0
.74 - 0.99 (d, 6 H), 1.42 - 1.83 (m, 3 H), 2.11 - 2.43 (m, 6 H),2.47 - 2.64 (m, 2 H), 3.28 -
.40 (m, 2 H), 3.69 - 3.81 (s, 3 H), 3.81 - 3.96 (t, 2 H), 4.62 - 4.77 (m, 1 H), 5.24 - 5.35
3
(m, 1 H), 5.54 - 5.75 (m, 1 H), 5.91 - 6.03 (m, 1 H), 6.14 - 6.24 (d, 1 H), 6.96 - 7.34 (m, 4
H), 8.02 – 8.11 (d, 1 H), 8.60 – 8.65 (d, 1 H). HRMS (ESI) calcd for C H ClN O :
25
35
3
6
+
[
M+H] : 508.2214 Found: 508.2219.
2
3

ACCEPTED MANUSCRIPT
4.1.8.4. Methyl (4S,7S, E)-17-(3-chlorophenyl)-4-(cyclohexylmethyl)-2,5,10-trioxo-1-oxa-
3,6,11-triazacycloheptadec-13-ene-7-carboxylate 8d
o
o
1
Off-white solid (yield 69%); m.p (A: 74 - 75 C), (B: 177- 178 C). H NMR (400
MHz, CDCl -d) δ ppm 0.66 - 0.93 (m, 11 H), 0.95 - 2.32 (m, 10 H), 3.61 - 3.74 (s, 3 H),
.74 - 3.86 (d, 2 H), 4.00 - 4.20 (m, 1 H), 4.29 - 4.51 (m, 1 H), 4.84 - 5.01 (m, 1 H), 5.42
3
3
-
5.83 (m, 2 H), 5.94 - 6.11 (m, 1 H), 6.22 - 6.31 (d, 1 H), 6.75 - 6.92 (d, 1 H), 7.04 - 7.27
+
(
m, 4 H). HRMS (ESI) calcd for C28
H
38ClN
3
NaO
6
: [M+Na] : 570.2347 Found: 570.2346.
4.1.8.5. Methyl (4S,7S, E)-18-(3-chlorophenyl)-4-isobutyl-2,5,10-trioxo-1-oxa-3,6,11-
triazacyclooctadec-13-ene-7-carboxylate 8e
o
1
Off-white solid (yield 70%); m.p 63 - 64 C. H NMR (400 MHz, CDCl
3
-d) δ ppm 0.82 -
1.03 (d, 6 H), 1.20 - 2.32 (m, 7 H), 2.35 - 2.49 (m, 6 H), 3.76 (s, 3 H), 4.01 - 4.25 (m, 2
H), 4.49 - 4.58 (m, 2 H), 5.48 - 5.86 (m, 3 H),6.32 – 6.38 (d, 1 H), 6.89 – 6.93 (d, 1 H),
3 6
7.13 - 7.44 (m, 4 H), 8.05 – 8.11 (br s, 1 H). HRMS (ESI) calcd for C26H37ClN O : [M+H]
+
:
522.2371 Found: 522.2378.
4.1.8.6. Methyl (4S,7S, E)-17-(3-chlorophenyl)-4-methyl-2,5,10-trioxo-1-oxa-3,6,11-
triazacycloheptadec-13-ene-7-carboxylate 8f
o
1
Off-white solid (yield 70%); m.p 105 - 106 C. H NMR (400 MHz, CDCl -d) δ ppm 1.32 -
3
1.50 (d, 3 H), 1.55 - 2.44 (m, 4 H), 2.44 - 2.75 (m, 4 H), 3.66 – 3.70 (s, 3 H), 4.05 - 4.10
(d, 2 H), 4.50 - 4.61 (m, 1 H), 4.66 - 4.82 (m, 1 H), 5.46 - 5.73 (m, 2 H), 5.89 - 6.01 (m, 1
H), 6.20 – 6.25 (d, 1 H),6.91 – 6.95 (br s, 1 H), 7.07 - 7.42 (m, 4 H), 8.50 – 8.54 (br s, 1
+
H). HRMS (ESI) calcd for C H ClN O : [M+H] : 466.1745 Found: 466.1751.
22
29
3
6
4.1.8.7. Methyl (4S,7S, E)-18-(3-chlorophenyl)-4-methyl-2,5,10-trioxo-1-oxa-3,6,11-
triazacyclooctadec-13-ene-7-carboxylate 8g
o
1
Off-white solid (yield 70%); m.p 74 – 75 C. H NMR (400 MHz, CDCl -d) δ ppm
3
1
3
6
.20 – 1.30 (m, 2 H), 1.37 (br d, J=7.03 Hz, 3 H), 1.56 - 2.49 (m, 8 H), 3.76 (s, 3 H),
.81 - 3.90 (m, 2 H), 4.54 - 4.86 (m, 2 H), 5.48 - 5.80 (m, 3 H), 6.14 - 6.32 (br d, 1 H),
.90 - 7.02 (d, 1 H), 7.06 - 7.38 (m, 4 H), 7.70 – 7.75 (br s, 1 H). HRMS (ESI) calcd for
+
C
23
H
31ClN
3
O
6
: [M+H] : 480.1901 Found: 480.1907.
2
4

ACCEPTED MANUSCRIPT
4.1.8.8. Methyl (20S, E)-10-(3-chlorophenyl)-8,17,22-trioxo-9-oxa-7,16,21-triazaspiro
[
5.16] docos-13-ene-20-carboxylate 8h
o
1
Off-white solid (yield 70%); m.p 105 – 106 C. H NMR (400 MHz, CDCl -d) δ
3
ppm 1.20 - 1.26 (m, 4 H), 1.35 - 1.76 (m, 4 H), 1.77 - 1.90 (m, 5 H), 2.26 – 2.49 (m, 6
H), 3.69 - 3.72 (s, 3 H), 3.381 - 3.88 (m, 2 H), 4.35 - 4.49 (m, 1 H), 5.46 - 5.70 (m, 2 H),
5.91 - 6.04 (m, 1 H), 6.08 - 6.15 (d, 1 H), 7.14 - 7.38 (m, 4 H), 7.87 - 7.96 (d, 1 H).
+
HRMS (ESI) calcd for C26H35ClN
3
O
6
: [M+H] : 520.2214 Found: 520.2222.
4.1.8.9. Methyl (21S, E)-10-(3-chlorophenyl)-8,18,23-trioxo-9-oxa-7,17,22-triazaspiro
[
5.17] tricos-14-ene-21-carboxylate 8i
o
1
3
Off-white solid (yield 72%); m.p 87 – 88 C. H NMR (400 MHz, CDCl -d) δ ppm
1.20 - 1.26 (m, 4 H), 1.35 - 1.55 (m, 6 H), 1.55 - 1.74 (m, 5 H), 2.05 - 2.36 (m, 4 H), 2.37
-
2.55 (m, 2 H), 3.76 (d, J=2.34 Hz, 3 H), 3.80 - 3.87 (m, 2 H), 4.53 - 4.74 (m, 1 H), 5.59
-
5.75 (m, 2 H), 6.38 - 6.51 (d, 1 H), 6.51 - 6.61 (d, 1 H), 6.83 - 6.91 (d, 1 H), 7.13 - 7.39
+
(
m, 4 H). HRMS (ESI) calcd for C H ClN O : [M+H] : 534.2371 Found: 534.2381.
27
37
3
6
4.1.8.10. Methyl (4S, 7S, Z)-4-isobutyl-2,5,10-trioxo-17-(3-phenylpropyl)-1-oxa-3,6,11-
triazacycloheptadec-13-ene-7-carboxylate 8j
o
1
Off-white solid (yield 68%); m.p 72 – 73 C. H NMR (400 MHz, CDCl -d) δ ppm
3
0.86 – 1.02 (d, 6 H), 1.46 - 1.86 (m, 7 H), 2.04 – 2.36 (m, 8 H), 2.52 – 2.60 (t, 2 H), 3.70
– 3.77 (s, 3 H), 3.82 - 3.93 (d, 2 H), 4.13 -4.23 (m, 1 H), 4.63 – 4.80 (m, 1 H), 5.00 -
5.08 (t, 1 H), 5.33 - 5.63 (m, 2 H), 5.73 – 5.83 (d, 1H), 6.00 - 6.06 (d, 1 H), 6.93 - 6.70
(
d, 1 H), 7.10 - 7.20 (d, 2 H), 7.22 - 7.32 (m, 3 H). HRMS (ESI) calcd for C H N O :
28 42 3 6
+
[
M+H] : 516.3074 Found: 516.3079.
4.1.8.11.
Methyl
(4S,
7S,
Z)-4-isobutyl-2,5,10-trioxo-17-pentyl-1-oxa-3,6,11-
triazacycloheptadec-13-ene-7-carboxylate 8k
o
1
3
Off-white solid (yield 54%); m.p 64 – 65 C. H NMR (400 MHz, CDCl -d) δ ppm
0.85 – 0.92 (t, 2 H),0.93 – 1.04 (d, 6 H), 1.22 – 1.37 (m, 6 H), 1.43 - 1.92 (m, 6 H), 2.12
– 2.40 (m, 4 H), 2.52 – 2.64 (m, 2 H), 3.70 – 3.78 (s, 3 H), 3.80 - 3.90 (d, 2 H), 4.15 -
4.25 (m, 1 H), 4.50 - 4.60 (m, 1 H), 4.62 – 4.78 (m, 2 H), 4.91 - 5.24 (m, 1 H), 5.42 -
2
5

ACCEPTED MANUSCRIPT
5.50 (m, 2 H), 5.84 – 5.89 (d, 1 H), 6.03 – 6.09 (d, 1 H), 6.31 – 6.37 (br. s, 1 H). HRMS
+
(
ESI) calcd for C H N O : [M+H] : 468.3074 Found: 468.3081.
24
42
3
6
4.1.8.12.
Methyl
(6S,9S,E)-13-(3-chlorophenyl)-9-isobutyl-3,8,11-trioxo-1,12-dioxa-
2,7,10-triazacyclooctadec-16-ene-6-carboxylate 33
o
1
Off-white solid (yield 52%); m.p 160 – 161 C. H NMR (400 MHz, CDCl
3
-d) δ
ppm 0.84 – 0.99 (d, 6 H), 1.23 – 1.29 (t, 2 H), 1.49 – 2.57 (t, 8 H), 3.72 – 3.79 (s, 3 H),
3
5
7
.82 – 3.90 (d, 1 H), 4.03 – 4. 17 (d, 2 H), 4.24 – 4.43 (m, 1 H), 4.59 – 4.74 (m, 1 H),
.18 – 5.24 (d, 1 H), 5.52 - 5.67 (m, 2 H), 5.68 – 5.96 (m, 1 H), 6. 24 – 6. 32 (d, 1 H),
.13 – 7.21 (s, 1 H), 7.25 – 7.36 (m, 3 H), 9.24 – 9.30 (d, 1 H). HRMS (ESI) calcd for
+
C
25
H
35ClN
3
O
7
: [M+H] : 524.2164 Found: 524.2171.
4.1.9. Synthesis of compounds 9a-k and 34. General procedure for the hydrogenation
reaction.
To a solution of the appropriate olefin 8 (1.0 mmol) in anhydrous ethanol (10 mL)
was added palladium on carbon (10% Pd-C, 2.0 eq) and the mixture was stirred under a
hydrogen atmosphere (a balloon was used) at room temperature and atmospheric
pressure for 18 h. The mixture was filtered through Celite and the filtrate was
concentrated in vacuo to yield a crude product which was purified by flash
chromatography to yield compounds 9a-k as white solids.
4.1.9.1.
Methyl
(4S,7S)-4-isobutyl-2,5,10-trioxo-17-phenyl-1-oxa-3,6,11-
triazacycloheptadecane-7-carboxylate 9a
o
1
White solid (yield 92%); m.p 85 – 86 C. H NMR (400 MHz, CDCl -d) δ ppm 0.68
3
-
1.05 (d, 6 H), 1.40 - 1.73 (m, 5 H), 1.73 - 1.95 (m, 4 H), 1.95 - 2.21 (m, 6 H), 3.20 –
.29 (t, 2 H), 3.64 - 3.76 (s, 3 H), 4.45 - 4.53 (m, 2 H), 5.76 - 5.90 (m, 1 H), 7.17 - 7.35
3
(m, 5 H), 7.40 – 7.50 (d, 1 H), 8.25 – 8.35 (d, 1 H), 8. 72 – 8.80 (d, 1 H). HRMS (ESI)
+
calcd for C H N O : [M+H] : 476.2761 Found: 476.2768.
25
38
3
6
2
6

ACCEPTED MANUSCRIPT
4.1.9.2.
Methyl
(4S,7S)-17-benzyl-4-isobutyl-2,5,10-trioxo-1-oxa-3,6,11-
triazacycloheptadecane-7-carboxylate 9b
o
1
White solid (yield 95%); m.p 190 – 191 C. H NMR (400 MHz, CDCl ) δ ppm 0.81
3
-
0.97 (d, 6 H), 1.53 - 1.69 (m, 6 H), 1,91 -2.02 (m, 5H), 2.20 – 2.31 (4 H), 2.70 - 2.93
m, 2 H), 3.21 – 3.31 (t, 2 H), 3.73 – 3.80 (s, 3 H), 4.40 - 4.62 (m, 2 H), 4.88 - 5.04 (m, 1
(
H), 6.20 - 6.41 (br. d, 1 H), 6.91 - 7.10 (d, 1 H), 7.14 - 7.36 (m, 5 H), 7.85 – 7.92 (d, 1
+
H). HRMS (ESI) calcd for C26
H
40
N
3
O
6
: [M+H] : 490.2917 Found: 490.2921.
4.1.9.3.
Methyl
(4S,7S)-17-(3-chlorophenyl)-4-isobutyl-2,5,10-trioxo-1-oxa-3,6,11-
triazacycloheptadecane-7-carboxylate 9c
o
1
3
White solid (yield 95%); m.p 149 – 150 C. H NMR (400 MHz, CDCl -d) δ ppm
0
.74 - 0.99 (d, 6 H), 1.42 - 1.83 (m, 3 H), 2.11 - 2.43 (m, 6 H),2.47 - 2.64 (m, 2 H), 3.28 -
.40 (m, 2 H), 3.69 - 3.81 (s, 3 H), 3.81 - 3.96 (t, 2 H), 4.50 – 5.60 (m, 1 H), 4.62 - 4.77
3
(m, 1 H), 5.91 - 6.03 (m, 1 H), 6.14 - 6.24 (d, 1 H), 6.96 - 7.34 (m, 4 H), 8.02 – 8.11 (d, 1
+
H), 8.60 – 8.65 (d, 1 H). HRMS (ESI) calcd for C H ClN O : [M+H] : 510.2371 Found:
25
37
3
6
510.2378.
4.1.9.4. Methyl (4S,7S)-17-(3-chlorophenyl)-4-(cyclohexylmethyl)-2,5,10-trioxo-1-oxa-
3,6,11-triazacycloheptadecane-7-carboxylate 9d
o
o
1
White solid (yield 95%); m.p (A: 71 – 72 C, B: 161 – 162 C). H NMR (400 MHz,
CDCl
3
-d) δ 0.66 - 0.93 (m, 12 H), 0.95 - 2.32 (m, 10 H), 3.18 – 3.21 (t, 2 H), 3.63 - 3.81
(s, 3 H), 3.96 - 4.10 (t, 2 H), 4.00 - 4.20 (m, 1 H), 4.29 - 4.51 (m, 1 H), 4.84 - 5.01 (m, 1
H), 5.94 - 6.11 (m, 1 H), 6.22 - 6.31 (d, 1 H), 6.75 - 6.92 (d, 1 H),, 7. 12 - 7.27 (m, 4 H),
3 6
8.25 - 8.40 (br. s, 1 H), 8.82 - 8.90 (d, 1 H). HRMS (ESI) calcd for C28H41ClN O : [M+H]
+
:
550.2684 Found: 550.2689.
4.1.9.5. Methyl (4S,7S)-18-(3-chlorophenyl)-4-isobutyl-2,5,10-trioxo-1-oxa-3,6,11-
triazacyclooctadecane-7-carboxylate 9e
o
1
White solid (yield 94%); m.p 122 – 123 C. H NMR (400 MHz, CDCl -d) δ ppm
3
0
.74 - 0.99 (d, 6 H), 1.42 - 1.83 (m, 5 H), 2.11 - 2.43 (m, 6 H),2.47 - 2.64 (m, 2 H), 3.28 -
.40 (m, 2 H), 3.69 - 3.81 (s, 3 H), 3.81 - 3.96 (t, 2 H), 4.50 – 5.60 (m, 1 H), 4.62 - 4.77
3
2
7

ACCEPTED MANUSCRIPT
(m, 1 H), 5.91 - 6.03 (m, 1 H), 6.14 - 6.24 (d, 1 H), 6.96 - 7.34 (m, 4 H), 8.02 – 8.11 (d, 1
+
H), 8.60 – 8.65 (d, 1 H). HRMS (ESI) calcd for C H ClN O : [M+H] : 525.2527 Found:
26
39
3
6
524.2533.
4.1.9.6.
Methyl
(4S,7S)-17-(3-chlorophenyl)-4-methyl-2,5,10-trioxo-1-oxa-3,6,11-
triazacycloheptadecane-7-carboxylate 9f
o
1
White solid (yield 93%); m.p 131 – 132 C. H NMR (400 MHz, CDCl -d) δ ppm
3
0
2
-
.82 - 0.99 (m, 4 H), 1.40 (d, J=6.64 Hz, 3 H), 1.52 - 1.84 (m, 2 H), 1.82 - 2.06 (m, 2 H),
.09 - 2.50 (m, 4 H), 3.12 - 3.31 (m, 2 H), 3.70 - 3.81 (s, 3 H), 4.65 - 4.77 (m, 1 H), 5.18
5.26 (m, 1 H), 5.84 - 5.93 (m, 1 H), 6.17 - 6.33 (br s, 1 H), 6.52 - 6.63 (d, 1 H), 7.10 -
+
7
.30 (m, 4 H), 7.30 - 7.44 (m, 1 H). HRMS (ESI) calcd for C22
H
31ClN
3
O
6
: [M+H] :
468.1901 Found: 468.1911.
4.1.9.7.
Methyl
(4S,7S)-18-(3-chlorophenyl)-4-methyl-2,5,10-trioxo-1-oxa-3,6,11-
triazacyclooctadecane-7-carboxylate 9g
o
1
White solid (yield 93%); m.p 100 - 101 C. H NMR (400 MHz, CDCl -d) δ ppm
3
0
.75 - 0.96 (m, 4 H), 1.37 - 1.58 (d, m, 5 H), 1.65 - 1.81 (m, 2 H), 1.81 - 2.10 (m, 2 H),
.10 - 2.54 (m, 4 H), 3.21 - 3.34 (t, 2 H), 3.75 (s, 3 H), 4.17 - 4.28 (m, 1 H), 4.56 - 4.70
2
(m, 1 H), 5.25 - 5.40 (br s, 1 H), 5.47 - 5.82 (m, 1 H), 6.14 - 6.29 (br s, 1 H), 7.01 - 7.51
+
(
m, 4 H). 7.90 – 8.00 (br s, 1 H). HRMS (ESI) calcd for C H ClN O : [M+H] : 482.2058
23
33
3
6
Found: 482.2069.
4.1.9.8. Methyl (20S)-10-(3-chlorophenyl)-8,17,22-trioxo-9-oxa-7,16,21-triazaspiro [5.16]
docosane-20-carboxylate 9h
o
1
3
White solid (yield 95%); m.p 105 - 106 C. H NMR (400 MHz, CDCl -d) δ ppm
0
3
7
.84 - 0.97 (m, 2 H), 1.09 - 1.40 (m, 7 H), 1.40 - 2.65 (m, 13 H), 3.10 - 3.28 (m, 2 H),
.67 - 3.70 (s, 3 H), 4.31 - 4.44 (m, 1 H), 5.81 - 5.90 (m, 1 H), 6.47 - 6.62 (t, 1 H), 7.11 -
.39 (m, 4 H), 8.26 – 8. 30 (br. S, 1 H), 8.64 - 8.79 (br. s, 1 H). HRMS (ESI) calcd for
+
C H ClN O : [M+H] : 522.2371 Found: 522.2389.
26
37
3
6
4.1.9.9. Methyl (21S)-10-(3-chlorophenyl)-8,18,23-trioxo-9-oxa-7,17,22-triazaspiro [5.17]
tricosane-21-carboxylate 9i
2
8