Total syntheses of cathepsin D inhibitory izenamides A, B, and C and structural confirmation of izenamide B

Article abstract of DOI:10.3390/molecules24193424

The first total syntheses of izenamides A, B, and C, which are depsipeptides inhibitor of cathepsin D, were accomplished. In addition, the stereochemistry of izenamide B was confirmed by our syntheses. The key features of our synthetic route involve the avoidance of critical 2,5-diketopiperazine (DKP) formation and the minimization of epimerization during the coupling of amino acids for the target peptides.

Full text of DOI:10.3390/molecules24193424

molecules
Article
Total Syntheses of Cathepsin D Inhibitory
Izenamides A, B, and C and Structural Confirmation
of Izenamide B
Changjin Lim
College of Pharmacy, CHA University, 120 Haeryong-ro, Pocheon 11160, Gyeonggi-do, Korea; koryoi@cha.ac.kr;
Tel.: +82-31-881-7193
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editor: Vincenzo Piccialli
Received: 29 August 2019; Accepted: 19 September 2019; Published: 20 September 2019
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: The first total syntheses of izenamides A, B, and C, which are depsipeptides inhibitor of
cathepsin D, were accomplished. In addition, the stereochemistry of izenamide B was confirmed by our
syntheses. The key features of our synthetic route involve the avoidance of critical 2,5-diketopiperazine
(DKP) formation and the minimization of epimerization during the coupling of amino acids for the
target peptides.
Keywords: izenamides; cathepsin D; depsipeptide; total synthesis; structural confirmation
1. Introduction
Cathepsin D is an aspartic protease and has attracted considerable attention from both biologists
and chemists due to its interesting biological functions [
protein catabolism and required in certain epithelial cells for tissue remodeling and renewal, regulates
lysosomal proteolysis and endogenous fibrinolysis [ ]. Cathepsin D also maintains hormone
and antigen processing [ ]. In particular, cathepsin D is involved in various diseases, including
1,2]. This aspartic protease, which is involved in
3–6
7–9
breast cancer metastasis [10
–
12], atherosclerosis [
8,13], Alzheimer’s disease [14–16] and neuronal
ceroid lipofuscinosis (NCL) [
6,
17–19]. Therefore, therapeutic modulators of the proteolytic activity of
cathepsin D have been extensively investigated for the development of new drugs although there have
been few reports regarding inhibitors of cathepsin D [20–24].
Recently, three new depsipeptides, namely, izenamides A (
the marine cyanobacterium 1605-5 by Suenaga and coworkers (Figure 1) [25]. Interestingly, izenamides
A ( ) and B ( ) were shown to inhibit cathepsin D in vitro. These two depsipeptides consist of seven
monomers, including four amino acids, two hydroxy acids, and a -amino- -hydroxy acid called
statine. Izenamide C ( ), which lacks a statine moiety and possesses a glycine unit instead of the
alanine seen in izenamides A ( ) and B ( ), has no inhibitory activity against cathepsin D. The absolute
1), B (2), and C (3), were isolated from
1
2
γ
β
3
1
2
configuration of the statine moiety in izenamide B (
by Suenaga and coworkers [25].
2) was indirectly determined by numerous efforts
In our pursuit of developing novel cathepsin D inhibitors, we recently became interested in
establishing an efficient synthetic route towards izenamides A, B, and C. We were also interested in
confirming the structure of izenamide B to ensure the stereochemistry of the statine moiety before we
started medicinal chemistry studies. We herein report the first total syntheses of izenamides A, B, and
C as well as the structural confirmation of izenamide B.
Molecules 2019, 24, 3424; doi:10.3390/molecules24193424
www.mdpi.com/journal/molecules

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Figure 1. Structures of Izenamides.
2. Results and Discussion
2.1. Synthetic Strategy for Izenamides A (1), B (2), and C (3)
Our retrosynthetic analysis is outlined in Scheme 1. We envisioned that the target izenamides
could be accessed via a versatile convergent approach. Izenamides A ( ) and B ( ) would be synthesized
by assembling three fragments, tetrapeptide , statine , and esters or . Considering that izenamide
C ( ) has a very similar structure except for the statine moiety, it could also be convergently synthesized
by the amide coupling of tetrapeptide and ester . Tetrapeptides and were anticipated to be
derived from commercially available amino acids and NMe-d-Phe, which could be obtained from d-Phe
). Protected statine could be prepared from Boc-l-leucinal (10) through the known procedure [26 27].
Esters and can be conveniently obtained by the esterification of protected hydroxy acids 11 and 12
(or 13).
1
2
4
6
7
8
3
5
7
4
5
(9
6
,
7
8
Scheme 1. Retrosynthetic analysis for the synthesis of izenamides 1, 2 and 3.
2.2. Syntheses of Fragments
The synthesis of izenamides commenced with synthesizing tetrapeptides
4 and 5. Initially,
NMe-d-Phe 16 was prepared from commercially available d-Phe (9) as shown in Scheme 2. Amine

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protection of d-Phe
9
with Boc anhydride and N-methylation of resulting carbamate 14 afforded acid
15, which was esterified to produce desired monomer 16 in 84% yield over three steps.
Scheme 2. Preparation of NMe-d-Phe monomer 16.
With monomer 16 in hand, we turned our attention to the synthesis of tetrapeptides
4 and 5
(Scheme 3). To avoid facile DKP formation upon sequential elongation from the C-terminal NMe-Phe
and Pro residue, we synthesized tetrapeptides and according to the optimized sequence of
4
5
fragment couplings. Amidation of l-alanine methyl ester hydrochloride (17) and glycine methyl ester
hydrochloride (18) with Boc-Ile-OH followed by ester hydrolysis with LiOH afforded dipeptides 21
and 22 in high yields. Deprotection of 16 and coupling of resulting free amine 23 with acids 21 and
22 produced tripeptides 24 and 25 in 77 and 87% yields over two steps, respectively. Unfortunately,
epimerization was observed during the amidation to form 24. After thorough optimization of the
coupling conditions, amidation in the presence of DEPBT in CH2Cl2 at 0 ◦C provided the best results in
terms of high chemical yield and minimal epimerization (2.8:1). Tripeptides 24 and 25 were subjected
to hydrolysis and then amidation with l-proline methyl ester hydrochloride to afford tetrapeptides
and in 84 and 81% yield over two steps, respectively. In general, the N-Me peptide backbone near the
C-terminal is prone to -epimerization upon carboxy activation due to steric hindrance and electronic
4
5
α
effects [28]. However, we could obtain the desired tetrapeptides without epimerization under the
optimized conditions (DEPBT, CH2Cl2, 0 ◦C).
Scheme 3. Synthesis of tetrapeptides 4 and 5.

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Optically pure statine
6
was prepared by the known procedure [26
,
27] (Scheme 4). An elegant
highly diastereoselective allyl addition to N-Boc-l-leucinal in the presence of SnCl4 [26] provided
desired threo isomer 27. The reaction of homoallylic alcohol 27 with 2,2-dimethoxypropane in the
presence of catalytic PPTS followed by RuO4-mediated oxidative degradation [27] afforded monomer
acid 6.
Scheme 4. Synthesis of statine 6.
The synthesis of esters 7 and 8, which are the C-terminal fragments of izenamides, began with
the preparation of silyl-protected valic acid 11 (Scheme 5). Global TBS-protection of commercially
available l-valic acid (29) and hydrolysis of resulting silyl ester 30 produced C-terminal acid 11 in
94% yield over 2 steps. d-Allo-isoleucic acid 32 [29], which was prepared through the diazotization
of commercially available d-allo-Ile (31), and d-valic acid (33) were converted to corresponding allyl
esters 12 (64% for 2 steps) and 13 (91%), respectively. The EDC-mediated esterification of acid 11 with
alcohol 12 afforded ester 7. Unfortunately, a satisfactory yield for this esterification was not achieved
due the production of undesired esters by inevitable silyl transfer between hydroxyl groups.
Scheme 5. Synthesis of Ester 7.

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Therefore, we decided to change the TBS protecting group to a bulkier TBDPS group, which was
anticipated to minimize silyl transfer (Scheme 6). To our delight, the silyl transfer was minimized in
the coupling reactions, and fragments 36 and 37 were obtained in 92% and 94% yields, respectively.
Finally, allyl deprotection of esters 36 and 37 with N-Me-aniline in the presence of Pd(0) produced
acids 38 in 88% and 39 in 92% yield, respectively.
Scheme 6. Synthesis of fragments 38 and 39 with minimized silyl transfer.
2.3. Completion of the Syntheses
We finally assembled the prepared fragments of izenamide C as shown in Scheme 7.
The EDC-mediated amide coupling of amine 40, which was prepared from tetrapeptide 5, with
acid 38 followed by silyl-deprotection produced izenamide C (3) in 81% over 3 steps.
Scheme 7. Completion of the synthesis of izenamide C (3).
The completed synthesis of izenamides A (
1) and B (
2) is illustrated in Scheme 8. Boc-deprotection
of tetrapeptide with TFA and amide coupling with acid
4
6 afforded pentapeptide 42 in 77% yield over
two steps. Global deprotection under acidic conditions followed by amidation of the resulting amine
with acids 39 or 38 produced the corresponding heptapeptides. Finally, desilylation of heptapeptides
afforded izenamides A (
synthesized depsipeptides
Moreover, the absolute configuration of the statine moiety in
13C NMR spectra of some compounds are in the Supplementary Materials.
1
) and B (
2
3
) in 66% and 61% over 3 steps, respectively. Spectral data of the
were all identical with the reported data of natural and 3 [25]
was identical to that of
. The 1H and
1,
2, and
1
1
,
2
.
2

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Scheme 8. Completion of the syntheses of izenamides A (1) and B (2).
3. Materials and Methods
3.1. General Information
Unless noted otherwise, all starting materials and reagents were obtained from commercial
suppliers (Sigma-Aldrich, St. Louis, MO, USA; TCI, Tokyo, Japan; Combi-Blocks, San Diego, CA,
USA) and were used without further purification. Tetrahydrofuran and Et2O were distilled from
sodium benzophenone ketyl. Dichloromethane, chloroform and acetonitrile were freshly distilled
from calcium hydride. All solvents used for routine isolation of products and chromatography were
reagent grade and glass distilled. Reaction flasks were dried at 100 ◦C. Air and moisture sensitive
reactions were performed under argon atmosphere. Flash column chromatography was performed
using silica gel 60 (230–400 mesh, Merck, Kenilworth, NJ, USA) with the indicated solvents. Thin-layer
chromatography was performed using 0.25 mm silica gel plates (Merck, Kenilworth, NJ, USA). Optical
rotations were measured with JASCO P-2000 digital polarimeter (Tokyo, Japan) at ambient temperature
using cylindrical cell of 10 mm or 100 mm pathlength. Infrared spectra were recorded on a JASCO
FT-IR-4200 spectrometer (Tokyo, Japan). High resolution mass spectra were obtained with JEOL
1
JMS-700 (Tokyo, Japan) and Agilent Q TOF 6530 (Santa Clara, CA, USA) instruments. H and 13
C
NMR spectra were recorded using BRUKER AVANCE-800 (Billerica, MA, USA). Chemical shifts are
expressed in parts per million (ppm, ) downfield from tetramethylsilane and are referenced to the
deuterated solvent (CHCl3, 1H 7.24, 13 77.0; MeOH-d4, 1H 3.30, 13 49.00). 1H-NMR data
δ
δ
C
δ
δ
C δ
were reported in the order of chemical shift, multiplicity (s, singlet; d, doublet; t, triplet; q, quartet; m,
multiplet and/or multiple resonances; br, broad signal), coupling constant in hertz (Hz) and number
of protons.

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3.2. Experimental Part
Boc-NMe-d-Phe-OMe (16). To a solution of d-Phe
9
(1.7 g, 10.3 mmol) and Boc2O (3.5 mL, 15.4 mmol)
in a mixture THF and H2O (1:1, 50 mL) was added NaOH (0.6 g, 15.4 mmol) at room temperature.
After stirring overnight, the reaction mixture was quenched with 1N HCl and extracted with EtOAc.
The combined organic layer was washed with brine, dried over MgSO4, and concentrated in vacuo.
To a solution of above crude Boc-d-Phe-OH 14 (10.3 mmol) in dry THF (20 mL) was added NaH (60%
dispersion in mineral oil, 2.1 g, 51.5 mmol) at room temperature. After stirring for 1 h, iodomethane
(3.2 mL, 51.5 mmol) was added to the reaction mixture. The reaction mixture was stirred for 12 h,
quenched with 1N HCl, and extracted with EtOAc. The combined organic layer was washed with
brine, dried over MgSO4, and concentrated in vacuo. The residue was used in the next step without
further purification. To a solution of crude acid 15 (10.3 mmol) in dry DMF (20 mL) were added
iodomethane (1.3 mL, 20.6 mmol) and K2CO3 (2.8 g, 20.6 mmol) at room temperature. After stirring
overnight, the reaction mixture was quenched with 1N HCl and extracted with Et2O. The combined
organic layer was washed with brine, dried over MgSO4, and concentrated in vacuo. The residue was
purified by flash column chromatography (EtOAc/Hexane = 1:20) to give 2.5 g (84% for 3 steps) of
20
ester 16 as a colorless oil. [
α
]
= +109.84 (c 1.00, CHCl3); 1H-NMR (800 MHz, CDCl3, 3:2 mixture of
7.28–7.20 (m, 2H), 7.19–7.07 (m, 3H), 4.50 (dd, J = 10.4, 3.8 Hz, 1H),
D
two rotamers). Major rotamer
δ
3.70 (s, 3H), 3.23 (dd, J = 14.2, 4.4 Hz, 1H), 3.01–2.94 (m, 1H), 2.68 (s, 3H), 1.29 (s, 9H), minor rotamer
7.28–7.20 (m, 2H), 7.19–7.07 (m, 3H), 4.89 (dd, J = 10.6, 5.2 Hz, 1H), 3.68 (s, 3H), 3.27 (dd, J = 14.4,
5.1 Hz, 1H), 3.01–2.94 (m, 1H), 2.66 (s, 3H), 1.33 (s, 9H); 13C-NMR (200 MHz, CDCl3, 3:2 mixture of two
rotamers). Major rotamer 171.4, 154.8, 137.5, 128.9, 128.4, 126.5, 80.1, 61.5, 52.0, 35.4, 32.4, 28.0, minor
rotamer
δ
δ
δ
171.7, 155.6, 137.2, 128.8, 128.2, 126.3, 79.8, 59.4, 52.0, 34.9, 31.8, 28.1; IR (thin film, neat) νmax
2977, 1746, 1698, 1393, 1332, 1227, 1145, 751 cm−1; LR-MS (ESI+) m/z 316 (M + Na+); HR-MS (ESI+)
calcd for C16H23NNaO4 (M + Na+) 316.1519; found 316.1523.
Boc-L-Ile-L-Ala-OH (21). To a solution of l-alanine methyl ester hydrochloride 17 (1.7 g, 12.2 mmol),
Boc-l-Ile-OH (2.3 g, 9.9 mmol), DIPEA (5.2 mL, 29.8 mmol), and HOAt (1.4 g, 10.4 mmol) in CH2Cl2
(40 mL) was added EDC HCl (3.8 g, 19.9 mmol) at room temperature. After stirring for 4 h, the reaction
·
mixture was quenched with 1N HCl, extracted with CH2Cl2. The combined organic layer was washed
with aqueous NaHCO3, dried over MgSO4, and concentrated in vacuo. The residue was purified by
flash column chromatography (EtOAc/Hexane = 1:2 to 1:1) to give 2.8 g (88%) of dipeptide 19 as white
solid. [α] δ 6.40 (s, 1H), 5.03 (d, J = 6.7 Hz,
20 = +16.54 (c 1.00, CHCl3); 1H NMR (800 MHz, CDCl3)
D
1H), 4.56 (dq, J = 7.2, 7.1 Hz, 1H), 3.94 (t, J = 6.2 Hz, 1H), 3.72 (s, 3H), 1.88–1.81 (m, 1H), 1.53–1.44 (m,
1H), 1.42 (s, 9H), 1.39 (d, J = 7.2 Hz, 3H), 1.17–1.09 (m, 1H), 0.92 (d, J = 6.8 Hz, 3H), 0.89 (t, J = 7.4 Hz,
3H); 13C NMR (200 MHz, CDCl3)
δ 173.1, 171.1, 155.7, 79.9, 59.1, 52.4, 48.0, 37.3, 28.3, 24.7, 18.3, 15.4,
11.4; IR (thin film, neat) νmax 3313, 2977, 1751, 1682, 1651, 1528, 1367, 1252, 870 cm−1; LR-MS (ESI+)
m/z 317 (M + H+); HR-MS (ESI+) calcd for C15H29N2O5 (M + H+) 317.2071; found 317.2072. To a
solution of Boc-l
-Ile-l-Ala-OMe 19 (2.0 g, 6.3 mmol) in a mixture of THF, MeOH and H2O (1:1:1, 10 mL)
was added LiOH
·
H2O (0.6 g, 14.3 mmol) at room temperature. After stirring for 2 h, the reaction
mixture was quenched with 1N HCl and extracted with EtOAc. The combined organic layer was dried
over MgSO4 and concentrated in vacuo to afford 1.9 g (99%) of Boc-l-Ile-l-Ala-OH 21 as a white solid.
The free acid 21 was used in the next step without further purification.
Boc-L-Ile-Gly-OH (22). To a solution of glycine methyl ester hydrochloride 18 (1.5 g, 11.9 mmol),
Boc-l-Ile-OH (2.3 g, 9.9 mmol), DIPEA (5.2 mL, 29.8 mmol), and HOAt (1.4 g, 10.4 mmol) in CH2Cl2
(40 mL) was added EDC HCl (3.8 g, 19.9 mmol) at room temperature. After stirring for 5 h, the reaction
·
mixture was quenched with 1N HCl and extracted with CH2Cl2. The combined organic layer was
washed with aqueous NaHCO3, dried over MgSO4, and concentrated in vacuo. The residue was
purified by flash column chromatography (EtOAc/Hexane = 1:2 to 1:1) to give 2.7 g (91%) of dipeptide
20
20 as a white solid. [
α
]
= δ 6.81 (s, 1H), 5.17 (d,
5.85 (c 1.00, CHCl3); 1H-NMR (800 MHz, CDCl3)
−
D
J = 7.5 Hz, 1H), 4.08–3.91 (m, 3H), 3.70 (s, 3H), 1.84 (s, 1H), 1.51–1.45 (m, 1H), 1.39 (s, 9H), 1.13–1.06
(m, 1H), 0.91 (d, J = 6.9 Hz, 3H), 0.86 (t, J = 7.4 Hz, 3H); 13C-NMR (200 MHz, CDCl3)
δ
172.1, 170.1,

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155.8, 79.9, 59.0, 52.2, 41.0, 37.2, 28.2, 24.6, 15.4, 11.3; IR (thin film, neat) νmax 3324, 2968, 1759, 1687,
1661, 1527, 1367, 1173, 862 cm−1; LR-MS (ESI+) m/z 303 (M + H+); HR-MS (ESI+) calcd for C14H27N2O5
(M + H+) 303.1914; found 303.1923. To a solution of Boc-l
-
Ile-Gly-OMe 20 (2.0 g, 6.6 mmol) in a
mixture of THF, MeOH, and H2O (1:1:1, 33 mL) was added LiOH H2O (0.6 g, 14.3 mmol) at room
·
temperature. After stirring for 2 h, the reaction mixture was quenched with 1N HCl and extracted
with EtOAc. The combined organic layer was dried over MgSO4 and concentrated in vacuo to afford
1.9 g (99%) of Boc-l-Ile-Gly-OH 22 as white solid. The free acid 22 was used in the next step without
further purification.
Boc-l-Ile-l-Ala-NMe-d-Phe-OMe (24). To a solution of Boc-NMe-d-Phe-OMe 16 (440 mg, 1.5 mmol)
in CH2Cl2 (6.0 mL) was added TFA (1.5 mL) dropwise at room temperature. After stirring for 1 h, the
reaction mixture was concentrated in vacuo. The residue was used in the next step without further
purification. To a solution of above amine salt 23 (1.5 mmol), Boc-l-Ile-l-Ala-OH 21 (544 mg, 1.8 mmol),
and DIPEA (0.8 mL, 4.6 mmol) in CH2Cl2 (7.5 mL) was added DEPBT (898 mg, 3.0 mmol) at 0 ◦C. After
stirring for overnight at 0 ◦C, the reaction mixture was quenched with 1N HCl, extracted with CH2Cl2.
The combined organic layer was washed with aqueous NaHCO3, dried over MgSO4, and concentrated
in vacuo. The residue was purified by flash column chromatography (EtOAc/Hexane = 1:1) to give
407 mg (57% for 2 steps) of tripeptide 24 as white solid and 145 mg (20% for 2 steps) of its epimer as
20
white solid. [
α
]
D
= +61.58 (c 1.00, CHCl3); 1H-NMR (800 MHz, CDCl3, a mixture of rotamers) Major
rotamer
δ
7.24 (t, J = 7.4 Hz, 2H), 7.18 (t, J = 7.4 Hz, 1H), 7.13 (d, J = 7.3 Hz, 2H), 6.81 (d, J = 7.1 Hz, 1H),
5.29 (d, J = 7.1 Hz, 1H), 4.98 (d, J = 8.3 Hz, 1H), 4.69 (dq, J = 6.9, 6.8 Hz, 1H ), 3.95 (dd, J = 7.3, 6.1 Hz,
1H), 3.73 (s, 3H), 3.39 (dd, J = 14.8, 5.0 Hz, 1H), 3.01 (dd, J = 14.7, 11.8 Hz, 1H), 2.83 (s, 3H), 1.87–1.79 (m,
1H), 1.74–1.64 (m, 1H), 1.40 (s, 9H), 1.12–1.02 (m, 1H), 0.88 (d, J = 6.8 Hz, 3H), 0.87 (t, J = 7.4 Hz, 3H),
0.84 (d, J = 6.8 Hz, 3H).; 13C-NMR (200 MHz, CDCl3, a mixture of rotamers) Major rotamer
δ 172.9,
170.7, 170.5, 155.6, 136.4, 128.7, 128.6, 126.9, 79.8, 59.2, 58.3, 52.4, 45.5, 37.6, 34.7, 32.5, 28.3, 24.6, 17.9,
15.6, 11.5; IR (thin film, neat) νmax 3319, 2970, 1743, 1710, 1637, 1498, 1366, 1173, 1017, 871 cm−1; LR-MS
(ESI+) m/z 478 (M + H+); HR-MS (ESI+) calcd for C25H40N3O6 (M + H+) 478.2912; found 478.2915.
Boc-l-Ile-Gly-NMe-d-Phe-OMe (25). To a solution of Boc-NMe-d-Phe-OMe 16 (426 mg, 1.5 mmol)
in CH2Cl2 (6.0 mL) was added TFA (1.5 mL) dropwise at room temperature. After stirring for 1 h, the
reaction mixture was concentrated in vacuo. The residue was used in the next step without further
purification. To a solution of above amine salt 23 (1.5 mmol), Boc-l-Ile-Gly-OH 22 (502 mg, 1.7 mmol)
and DIPEA (0.8 mL, 4.4 mmol) in CH2Cl2 (7.3 mL) was added DEPBT (869 mg, 2.9 mmol) at 0 ◦C.
After stirring overnight at 0 ◦C, the reaction mixture was quenched with 1N HCl and extracted with
CH2Cl2. The combined organic layer was washed with aqueous NaHCO3, dried over MgSO4, and
concentrated in vacuo. The residue was purified by flash column chromatography (EtOAc/Hexane =
20
1:1) to give 586 mg (87% for 2 steps) of tripeptide 25 as white solid. [
α
]
= +38.96 (c 1.00, CHCl3);
D
1H-NMR (800 MHz, CDCl3, a mixture of rotamers) Major rotamer
δ
7.21 (t, J = 7.5 Hz, 2H), 7.15 (t,
J = 7.4 Hz, 1H), 7.09 (d, J = 7.3 Hz, 2H), 6.84 (s, 1H), 5.19 (dd, J = 10.7, 5.3 Hz, 1H), 5.10 (d, J = 8.4 Hz,
1H), 4.04–4.01 (m, 1H), 3.95 (dd, J = 17.9, 3.7 Hz, 1H), 3.81 (dd, J = 17.7, 3.1 Hz, 1H), 3.67 (s, 3H), 3.32
(dd, J = 14.6, 5.4 Hz, 1H), 2.98 (dd, J = 14.6, 10.9 Hz, 1H), 2.74 (s, 3H), 1.81 (s, 1H), 1.44–1.36 (m, 1H), 1.37
(s, 9H), 1.07–1.02 (m, 1H), 0.85 (d, J = 6.9 Hz, 3H), 0.83 (t, J = 7.4 Hz, 3H); 13C-NMR (200 MHz, CDCl3,
a mixture of rotamers) Major rotamer δ 171.3, 170.6, 168.4, 155.5, 136.4, 128.5, 128.5, 126.8, 79.5, 58.9,
58.7, 52.3, 41.2, 37.5, 34.4, 31.7, 28.1, 24.5, 15.4, 11.4; IR (thin film, neat) νmax 3328, 2967, 1743, 1712, 1643,
1497, 1366, 1172, 1016, 867 cm−1; LR-MS (ESI+) m/z 464 (M + H+); HR-MS (ESI+) calcd for C24H38N3O6
(M + H+) 464.2755; found 464.2751.
Boc-l
-
Ile-l-Ala-NMe-d
-
Phe-l
-Pro-OMe (
4
). To a solution of Boc-l
-
Ile-l
-
Ala-NMe-d
-Phe-OMe 24
(491 mg, 1.0 mmol) in a mixture of THF, MeOH, and H2O (1:1:1, 10 mL) was added LiOH
·
H2O (86 mg,
2.1 mmol) at room temperature. After stirring for 2 h, the reaction mixture was quenched with 1N
HCl and extracted with EtOAc. The combined organic layer was dried over MgSO4 and concentrated
in vacuo. The free acid 26 was used in the next step without further purification. To a solution of above
acid 26 (1.0 mmol), l-proline methyl ester hydrochloride (203 mg, 1.3 mmol), and DIPEA (0.6 mL,

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3.4 mmol) in CH2Cl2 (10 mL) was added DEPBT (615 mg, 2.1 mmol) at 0 ◦C. After stirring for overnight
at 0 ◦C, the reaction mixture was quenched with 1N HCl and extracted with CH2Cl2. The combined
organic layer was washed with aqueous NaHCO3, dried over MgSO4, and concentrated in vacuo.
The residue was purified by flash column chromatography (EtOAc/Hexane = 1:1 to 2:1) to give 496 mg
20
(84% for 2 steps) of tetrapeptide
4
as white solid. [
α
]
= +45.90 (c 1.00, CHCl3); 1H-NMR (800 MHz,
D
CDCl3, a mixture of rotamers) Major rotamer
δ
7.23–7.13 (m, 5H), 6.64 (d, J = 7.2 Hz, 1H), 5.66 (dd,
J = 9.5, 6.4 Hz, 1H), 4.98 (d, J = 8.2 Hz, 1H), 4.74 (dq, J = 7.1, 6.9 Hz, 1H), 4.42 (t, J = 7.5 Hz, 1H), 3.90 (t,
J = 6.4 Hz, 1H), 3.70 (s, 3H), 3.47–3.42 (m, 1H), 3.22–3.15 (m, 1H), 2.98 (s, 3H), 2.94 (dd, J = 14.4, 9.6 Hz,
1H), 2.22–2.17 (m, 1H), 1.97–1.76 (m, 6H), 1.39 (s, 9H), 1.08–1.01 (m, 1H), 0.84 (t, J = 7.4 Hz, 3H), 0.83 (d,
J = 6.8 Hz, 3H), 0.82 (d, J = 6.9 Hz, 3H); 13C-NMR (200 MHz, CDCl3, a mixture of rotamers) Major
rotamer
δ 172.3, 172.2, 170.5, 168.1, 155.5, 136.7, 129.5, 128.2, 126.6, 79.7, 59.2, 59.0, 55.5, 52.2, 46.9, 45.1,
37.6, 34.6, 30.2, 28.8, 28.2, 25.3, 24.6, 17.7, 15.4, 11.5; IR (thin film, neat) νmax 3316, 2971, 1748, 1709,
1643, 1497, 1365, 1245, 1174, 1045, 870 cm−1; LR-MS (ESI+) m/z 575 (M + H+); HR-MS (ESI+) calcd for
C30H47N4O7 (M + H+) 575.3439; found 575.3442.
Boc-l
-
Ile-Gly-NMe-d
-
Phe-l
-
Pro-OMe (
5). To a solution of Boc-l
-Ile-Gly-NMe-d-Phe-OMe 25
(511 mg, 1.0 mmol) in a mixture of THF, MeOH, and H2O (1:1:1, 11 mL) was added LiOH
·
H2O (92 mg,
2.2 mmol) at room temperature. After stirring for 2 h, the reaction mixture was quenched with 1N
HCl and extracted with EtOAc. The combined organic layer was dried over MgSO4 and concentrated
in vacuo. The free acid 27 was used in the next step without further purification. To a solution of
above acid 27 (1.1 mmol), l-proline methyl ester hydrochloride (216 mg, 1.4 mmol) and DIPEA (0.6 mL,
3.4 mmol) in CH2Cl2 (11 mL) was added DEPBT (660 mg, 2.2 mmol) at 0 ◦C. After stirring overnight at
0 ◦C, the reaction mixture was quenched with 1N HCl, extracted with CH2Cl2. The combined organic
layer was washed with aqueous NaHCO3, dried over MgSO4, and concentrated in vacuo. The residue
was purified by flash column chromatography (EtOAc/Hexane = 1:1 to 2:1) to give 501 mg (81% for
= +69.20 (c 1.00, CHCl3); 1H-NMR (800 MHz, CDCl3,
20
2 steps) of tetrapeptide
5
as a white solid. [
α
]
D
a mixture of rotamers) Major rotamer
δ
7.23 (t, J = 7.4 Hz, 2H), 7.20 (d, J = 6.9 Hz, 2H), 7.17 (t, J = 7.2 Hz,
1H), 6.77 (s, 1H), 5.56 (t, J = 7.6 Hz, 1H), 5.00 (d, J = 8.0 Hz, 1H), 4.40 (dd, J = 8.5, 5.7 Hz, 1H), 4.09–4.01
(m, 1H), 3.92–3.85 (m, 1H), 3.70 (s, 3H), 3.38–3.34 (m, 1H), 3.33–3.29 (m, 1H), 3.25 (dd, J = 13.7, 8.0 Hz,
1H), 2.96 (s, J = 6.5 Hz, 3H), 2.81 (dd, J = 13.7, 7.1 Hz, 1H), 2.16–2.11 (m, 1H), 1.95–1.74 (m, 6H), 1.42 (s,
9H), 1.12–1.02 (m, 1H), 0.90 (d, J = 6.8 Hz, 3H), 0.88 (t, J = 7.4 Hz, 3H).; 13C-NMR (200 MHz, CDCl3,
a mixture of rotamers) Major rotamer δ 172.4, 171.5, 167.9, 167.9, 155.7, 136.9, 129.4, 128.4, 126.7, 79.9,
59.2, 58.9, 56.2, 52.2, 46.8, 41.1, 37.5, 34.9, 29.7, 28.8, 28.3, 25.0, 24.5, 15.6, 11.6; IR (thin film, neat) νmax
3328, 2968, 1748, 1710, 1640, 1497, 1365, 1246, 1174, 1044, 869 cm−1; LR-MS (ESI+) m/z 561 (M + H+);
HR-MS (ESI+) calcd for C29H45N4O7 (M + H+) 561.3283; found 561.3290.
Allyl (2R,3S)-2-hydroxy-3-methylpentanoate (12). To a solution of d-allo-Ile 31 (1.4 g, 10.7 mmol) in
dilute sulfuric acid (0.8N in H2O, 26.7 mL, 21.3 mmol) was added NaNO2 (2.2 g, 32.0 mmol) slowly at
0◦C. After stirring for 6 h at the same temperature, the reaction mixture was diluted with Et2O and
extracted with Et2O. The combined organic layer was dried over MgSO4 and concentrated in vacuo.
The residue was used in the next step without further purification. To a solution of crude acid 32
(10.7 mmol) and TBAI (0.79 g, 2.13 mmol) in dry DMF (500 mL) were added allyl bromide (1.8 mL,
21.3 mmol) and K2CO3 (3.0 g, 21.3 mmol) at room temperature. After stirring for overnight, the reaction
mixture was quenched with 1N HCl and extracted with Et2O. The combined organic layer was washed
with brine, dried over MgSO4, and concentrated in vacuo. The residue was purified by flash column
chromatography (EtOAc/Hexane = 1:10) to give 1.2 g (65% for 2 steps) of ester 12 as a colorless oil.
20
[α
]
= +45.94 (c 0.50, CHCl3); 1H-NMR (800 MHz, CDCl3)
δ 5.89 (ddt, J = 17.1, 10.5, 5.9 Hz, 1H), 5.31
D
(ddd, J = 17.2, 2.9, 1.5 Hz, 1H), 5.24 (dd, J = 10.4, 2.3, 1.2 Hz, 1H), 4.67 (ddt, J = 13.0, 5.9, 1.2 Hz, 1H),
4.63 (ddt, J = 13.1, 5.9, 1.3 Hz, 1H), 4.17 (d, J = 3.0 Hz, 1H), 2.61 (s, 1H), 1.83–1.75 (m, 1H), 1.50 (ddq,
J = 13.8, 7.4, 7.2 Hz, 1H), 1.28 (ddq, J = 13.7, 7.5, 7.4 Hz, 1H), 0.91 (t, J = 7.5 Hz, 3H), 0.78 (d, J = 7.0 Hz,
3H); 13C-NMR (200 MHz, CDCl3)
δ 175.0, 131.5, 119.1, 72.9, 66.0, 38.5, 25.9, 13.1, 11.8; IR (thin film,

Molecules 2019, 24, 3424
10 of 15
neat) νmax 3524, 2966, 1742, 1461, 1384, 1200, 1138, 934 cm−1; LR-MS (ESI+) m/z 173 (M + H+); HR-MS
(ESI+) calcd for C29H45N4O7 (M + H+) 173.1172; found 173.1163.
Allyl (R)-2-hydroxy-3-methylbutanoate (13). To a solution of d-valic acid 33 (1.2 g, 10.2 mmol) and
TBAI (0.75 g, 2.0 mmol) in dry DMF (25 mL) were added allyl bromide (1.8 mL, 20.3 mmol) and K2CO3
(2.8 g, 20.3 mmol) at room temperature. After stirring overnight, the reaction mixture was quenched
with 1N HCl and extracted with Et2O. The combined organic layer was washed with brine, dried
over MgSO4 and concentrated in vacuo. The residue was purified by flash column chromatography
20
(EtOAc/Hexane = 1:20) to give 1.5 g (93%) of ester 13 as a colorless oil. [
α
]
D
= +24.77 (c 1.00, CHCl3);
1H-NMR (800 MHz, CDCl3)
δ
5.84 (ddt, J = 17.1, 10.6, 5.9 Hz, 1H), 5.26 (dd, J = 17.2, 1.3 Hz, 1H), 5.18
(dd, J = 10.4, 1.1 Hz, 1H), 4.61 (dd, J = 13.1, 5.9 Hz, 1H), 4.57 (dd, J = 13.2, 5.9 Hz, 1H), 3.98 (d, J = 3.9 Hz,
1H), 2.87 (s, 1H), 2.01 (dtd, J = 13.9, 6.9, 3.7 Hz, 1H), 0.94 (d, J = 7.3 Hz, 3H), 0.79 (d, J = 7.2 Hz, 3H);
13C-NMR (200 MHz, CDCl3)
δ 174.4, 131.4, 118.9, 74.9, 65.8, 32.0, 18.6, 15.8; IR (thin film, neat) νmax
3516, 2968, 1743, 1468, 1372, 1197, 1130, 933 cm−1; LR-MS (ESI+) m/z 181 (M + Na+); HR-MS (ESI+)
calcd for C8H14NaO3 (M + Na+) 181.0835; found 181.0834.
(S)-2-((tert-Butyldiphenylsilyl)oxy)-3-methylbutanoic acid (35). To a solution of l-valic acid (1.2 g,
10.2 mmol) and DMAP (0.12 g, 1.0 mmol) in dry DMF (5.0 mL) were added imidazole (3.5 g, 50.8 mmol)
and TBDPSCl (7.9 mL, 30.5 mmol) at room temperature. After stirring overnight, the reaction mixture
was quenched with 1N HCl and extracted with Et2O. The combined organic layer was washed with
brine, dried over MgSO4, and concentrated in vacuo. The residue was used in the next step without
further purification. To a solution of crude silyl ether 34 (10.2 mmol) in a mixture of MeOH and H2O
(3:1, 40 mL) was added K2CO3 (2.1 g, 15.2 mmol) at room temperature. After stirring for 1 h, the
reaction mixture was quenched with 1N HCl and extracted with Et2O. The combined organic layer was
washed with brine, dried over MgSO4 and concentrated in vacuo. The residue was purified by flash
column chromatography (EtOAc/Hexane = 1:10) to give 1.1 g (92% for 2 steps) of acid 35 as a colorless
20
1
oil. [
J = 6.9 Hz, 2H), 7.42 (dt, J = 13.4, 7.4 Hz, 2H), 7.39–7.33 (m, 4H), 4.12 (d, J = 3.8 Hz, 1H), 1.95–1.85 (m,
1H), 1.11 (s, 9H), 0.88 (d, J = 7.0 Hz, 3H), 0.86 (d, J = 6.9 Hz, 3H); 13C-NMR (200 MHz, CDCl3)
174.4,
α
]
=
−
12.98 (c 1.00, CHCl3); H-NMR (800 MHz, CDCl3) δ 7.64 (d, J = 6.9 Hz, 2H), 7.60 (d,
D
δ
135.9, 135.8, 132.7, 132.2, 130.2, 130.1, 127.8, 127.8, 77.4, 33.2, 27.0, 19.4, 17.6, 17.3; IR (thin film, neat)
νmax 2962, 1720, 1471, 1428, 1183, 1113, 1071, 939 cm−1; LR-MS (ESI+) m/z 355 (M - H+); HR-MS (ESI+)
calcd for C21H27OSi (M - H+) 355.1735; found 355.1742.
Allyl (2R,3S)-2-(((S)-2-((tert-Butyldiphenylsilyl)oxy)-3-methylbutanoyl)oxy)-3-methylpentanoate (36).
To a solution of acid 35 (591 mg, 1.7 mmol), alcohol 12 (238 mg, 1.4 mmol), and DMAP (338 g, 2.8 mmol)
in CH2Cl2 (14 mL) was added EDC HCl (530 g, 2.8 mmol) at room temperature. After stirring for 2 h,
·
the reaction mixture was quenched with 1N HCl and extracted with CH2Cl2. The combined organic
layer was washed with aqueous NaHCO3, dried over MgSO4, and concentrated in vacuo. The residue
was purified by flash column chromatography (EtOAc/Hexane = 1:20) to give 649 mg (92%) of ester
20
36 as a colorless oil. [
α
]
D
= δ 7.67–7.64 (m, 4H),
23.21 (c 1.00, CHCl3); 1H NMR (800 MHz, CDCl3)
−
7.42–7.38 (m, 2H), 7.36–7.33 (m, 4H), 5.84 (ddt, J = 17.2, 10.4, 5.8 Hz, 1H), 5.29 (ddd, J = 17.2, 2.9, 1.4 Hz,
1H), 5.21 (ddd, J = 10.4, 2.3, 1.1 Hz, 1H), 4.82 (d, J = 3.5 Hz, 1H), 4.59 (ddt, J = 13.1, 5.7, 1.3 Hz, 1H),
4.52 (ddt, J = 13.1, 5.9, 1.3 Hz, 1H), 4.25 (d, J = 4.3 Hz, 1H), 2.06–1.96 (m, 1H), 1.90–1.85 (m, 1H), 1.36
(ddq, J = 13.8, 7.4, 7.3 Hz, 1H), 1.16 (ddq, J = 13.7, 7.6, 7.5 Hz, 1H), 1.14 (d, J = 7.5 Hz, 3H), 1.10 (s, 9H),
0.92 (d, J = 7.0 Hz, 3H), 0.89 (d, J = 6.9 Hz, 3H), 0.86 (t, J = 7.5 Hz, 3H); 13C-NMR (200 MHz, CDCl3)
δ
172.0, 169.2, 136.0, 135.9, 133.5, 133.3, 131.7, 129.7, 129.6, 127.5, 127.4, 118.7, 77.6, 75.1, 65.5, 36.7, 33.3,
26.9, 25.7, 19.6, 18.1, 17.1, 14.4, 11.7; IR (thin film, neat) νmax 2965, 1756, 1463, 1428, 1192, 1113, 1008,
+
935, 822 cm−1; LR-MS (ESI+) m/z 528 (M + NH4+); HR-MS (ESI+) calcd for C30H46NO5Si (M + NH4
)
528.3140; found 528.3149.
Allyl (R)-2-(((S)-2-((tert-Butyldiphenylsilyl)oxy)-3-methylbutanoyl)oxy)-3-methylbutanoate (37). To a
solution of acid 35 (741 g, 2.1 mmol), alcohol 13 (274 mg, 1.7 mmol) and DMAP (423 mg, 3.5 mmol)
in CH2Cl2 was added EDC
reaction mixture was quenched with 1N HCl, extracted with CH2Cl2. The combined organic layer
·
HCl (664 mg, 3.5 mmol) at room temperature. After stirring for 2 h, the

Molecules 2019, 24, 3424
11 of 15
was washed with aqueous NaHCO3, dried over MgSO4 and concentrated in vacuo. The residue was
purified by flash column chromatography (EtOAc/Hexane = 1:20) to give 809 mg (94%) of ester 37 as a
20
colorless oil. [
α
]
= +2.49 (c 1.00, CHCl3); 1H-NMR (800 MHz, CDCl3)
δ 7.64 (td, J = 8.4, 1.2 Hz, 4H),
D
7.40 (tdt, J = 7.7, 7.6, 1.3 Hz, 2H), 7.34 (dd, J = 7.2, 7.0 Hz, 4H), 5.83 (ddt, J = 17.2, 10.4, 5.8 Hz, 1H), 5.28
(dq, J = 17.2, 1.4 Hz, 1H), 5.20 (ddt, J = 10.4, 1.2, 1.0 Hz, 1H), 4.58 (dt, J = 13.1, 5.8, 1.3 Hz, 1H), 4.58 (d,
J = 4.4 Hz, 1H), 4.51 (ddt, J = 13.2, 5.9, 1.2 Hz, 1H), 4.22 (d, J = 4.4 Hz, 1H), 2.12–2.06 (m, 1H), 2.05–1.98
(m, 1H), 1.09 (s, 9H), 0.92 (d, J = 6.9 Hz, 3H), 0.92 (d, J = 6.9 Hz, 3H), 0.90 (d, J = 6.9 Hz, 3H), 0.90 (d,
J = 7.0 Hz, 3H); 13C-NMR (200 MHz, CDCl3)
δ 172.0, 168.9, 136.0, 135.9, 133.4, 133.3, 131.6, 129.7, 129.6,
127.5, 127.4, 118.8, 77.6, 77.0, 65.5, 33.3, 30.1, 26.9, 19.6, 18.5, 18.3, 17.4, 17.2; IR (thin film, neat) νmax
2965, 1754, 1471, 1428, 1185, 1113, 990, 934, 822 cm−1; LR-MS (ESI+) m/z 519 (M + Na+); HR-MS (ESI+)
calcd for C29H40NaO5Si (M + Na+) 519.2537; found 519.2543.
(2R,3S)-2-(((S)-2-((tert-Butyldiphenylsilyl)oxy)-3-methylbutanoyl)oxy)-3-methylpentanoic acid (38). To a
solution of ester 36 (345 mg, 0.7 mmol) and N-methylaniline (0.15 mL, 1.4 mmol) in dry THF (6.8 mL)
was added Pd(PPh3)4 (78 mg, 0.1 mmol) at room temperature. After stirring for 2 h, the reaction
mixture was quenched with 1N HCl and extracted with EtOAc. The combined organic layer was
washed with brine, dried over MgSO4, and concentrated in vacuo. The residue was purified by flash
column chromatography through a short pad of silica gel (CH2Cl2/MeOH = 16:1) to afford 280 mg
20
(88%) of the acid 38 as a yellow oil. [
α
]
= δ
15.06 (c 1.00, CHCl3); 1H-NMR (800 MHz, CDCl3)
−
D
7.64–7.62 (m, 4H), 7.42–7.36 (m, 2H), 7.35–7.29 (m, 4H), 4.84 (d, J = 3.3 Hz, 1H), 4.25 (d, J = 4.1 Hz, 1H),
2.03–1.97 (m, 1H), 1.89 (dtd, J = 7.5, 7.0, 3.3 Hz, 1H), 1.36 (ddq, J = 13.9, 7.3, 7.1 Hz, 1H), 1.18 (ddq,
J = 13.8, 7.6, 7.4 Hz, 1H), 1.09 (s, 9H), 0.92 (d, J = 7.0 Hz, 3H), 0.91 (d, J = 6.9 Hz, 3H), 0.90 (d, J = 6.9 Hz,
3H), 0.86 (t, J = 7.4 Hz, 3H); 13C-NMR (200 MHz, CDCl3)
δ 174.8, 172.0, 136.0, 135.9, 133.5, 133.3, 129.7,
129.7, 127.5, 127.5, 77.4, 74.4, 36.6, 33.3, 26.9, 25.8, 19.6, 18.4, 17.1, 14.3, 11.7; IR (thin film, neat) νmax
2965, 1760, 1730, 1463, 1428, 1182, 1113, 1000, 822 cm−1; LR-MS (ESI+) m/z 488 (M + NH4+); HR-MS
(ESI+) calcd for C27H42NO5Si (M + NH4+) 488.2827; found 488.2829.
(R)-2-(((S)-2-((tert-Butyldiphenylsilyl)oxy)-3-methylbutanoyl)oxy)-3-methylbutanoic acid (39). To a
solution of ester 37 (362 mg, 0.7 mmol) and N-methylaniline (0.2 mL, 1.5 mmol) in dry THF (7.3 mL)
was added Pd(PPh3)4 (84 mg, 0.1 mmol) at room temperature. After stirring for 2 h, the reaction
mixture was quenched with 1N HCl and extracted with EtOAc. The combined organic layer was
washed with brine, dried over MgSO4, and concentrated in vacuo. The residue was purified by flash
column chromatography through a short pad of silica gel (CH2Cl2/MeOH = 16:1) to afford 306 mg
20
(92%) of the acid 39 as a yellow oil. [
α
]
D
= δ 7.63
11.79 (c 1.00, CHCl3); 1H-NMR (800 MHz, CDCl3)
−
(td, J = 8.0, 1.1 Hz, 4H), 7.41–7.36 (m, 2H), 7.32 (dt, J = 9.0, 7.6 Hz, 4H), 4.61 (d, J = 4.3 Hz, 1H), 4.24
(d, J = 4.2 Hz, 1H), 2.14–2.09 (m, 1H), 2.06–1.98 (m, 1H), 1.08 (s, 9H), 0.93 (d, J = 7.2 Hz, 3H), 0.92 (d,
J = 7.0 Hz, 6H), 0.92 (d, J = 6.9 Hz, 3H); 13C-NMR (200 MHz, CDCl3)
δ 173.6, 172.0, 136.0, 135.9, 133.4,
133.3, 129.7, 129.7, 127.5, 127.4, 77.4, 76.3, 33.3, 30.0, 26.9, 19.6, 18.6, 18.4, 17.2, 17.1; IR (thin film, neat)
νmax 2965, 1760, 1726, 1470, 1428, 1181, 1113, 999, 822 cm−1; LR-MS (ESI+) m/z 474 (M + NH4+); HR-MS
(ESI+) calcd for C26H40NO5Si (M + NH4+) 474.2670; found 474.2674.
Izenamide C (3). To a solution of Boc-l-Ile-Gly-NMe-d-Phe-l-Pro-OMe 5 (34 mg, 0.1 mmol) in
CH2Cl2 (0.8 mL) was added TFA (0.2 mL) dropwise at room temperature. After stirring for 1 h, the
reaction mixture was concentrated in vacuo. To a solution of above amine salt 40 (0.1 mmol), acid 38
(37 mg, 0.1 mmol), DIPEA (0.1 mL, 0.2 mmol), and HOAt (11 mg, 0.1 mmol) in CH2Cl2 (1.0 mL) was
added EDC HCl (24 mg, 0.1 mmol) at room temperature. After stirring overnight, the reaction mixture
·
was quenched with 1N HCl and extracted with CH2Cl2. The combined organic layer was washed with
aqueous NaHCO3, dried over MgSO4, and concentrated in vacuo. The residue was used in the next
step without further purification. To a solution of crude hexapeptide (0.1 mmol) in THF (1.0 mL) was
added TBAF (1M in THF, 0.2 mL, 0.2 mmol) at room temperature. After stirring for 4 h, the reaction
mixture was quenched with H2O and extracted with EtOAc. The combined organic layer was washed
with brine, dried over MgSO4, and concentrated in vacuo. The residue was purified by flash column
chromatography (Acetone/Hexane = 1:3) to give 33 mg (83% for 3 steps) of izenamide C (3) as white

Molecules 2019, 24, 3424
12 of 15
20
solid. [
α
]
= +21.50 (c 0.10, MeOH); 1H-NMR (800 MHz, CD3OD, a mixture of rotamers) Major
D
rotamer
δ
7.25–7.19 (m, 4H), 7.18–7.14 (m, 1H), 5.57 (t, J = 7.6 Hz, 1H), 5.06 (d, J = 4.5 Hz, 1H), 4.37
(dd, J = 8.3, 5.6 Hz, 1H), 4.33 (d, J = 7.0 Hz, 1H), 4.15 (d, J = 17.0 Hz, 1H), 4.11 (d, J = 4.4 Hz, 1H), 3.90
(d, J = 17.0 Hz, 1H), 3.70 (s, 3H), 3.44 (dt, J = 10.2, 6.9 Hz, 1H), 3.39 (dt, J = 10.2, 5.6 Hz, 1H), 3.19 (dd,
J = 13.7, 7.8 Hz, 1H), 3.02 (s, 3H), 2.84 (dd, J = 13.7, 7.4 Hz, 1H), 2.21–2.17 (m, 1H), 2.11 (qqd, J = 6.9, 6.8,
4.5 Hz, 1H), 1.97–1.89 (m, 3H), 1.88–1.81 (m, 2H), 1.50–1.43 (m, 1H), 1.44 (ddq, J = 13.8, 7.4, 7.3 Hz,
1H), 1.28 (ddq, J = 13.7, 7.6, 7.5 Hz, 1H), 1.18–1.11 (m, 1H), 1.01 (d, J = 6.9 Hz, 3H), 0.95 (t, J = 6.7 Hz,
3H), 0.95 (d, J = 6.5 Hz, 3H), 0.93 (d, J = 6.9 Hz, 3H), 0.91 (d, J = 6.8 Hz, 3H), 0.89 (t, J = 7.4 Hz, 3H);
13C-NMR (200 MHz, CD3OD, a mixture of rotamers) Major rotamer
δ 175.2, 174.0, 173.4, 172.2, 170.2,
170.1, 138.6, 130.5, 129.4, 127.6, 78.0, 76.5, 60.6, 58.9, 57.8, 52.7, 48.3, 41.9, 38.5, 37.9, 35.7, 33.3, 30.5, 29.9,
27.0, 26.0, 25.7, 19.4, 16.9, 16.1, 14.7, 11.8, 11.4; IR (thin film, neat) νmax 3317, 2960, 2877, 1744, 1637,
1519, 1447, 1199, 1176, 1137, 1030, 753 cm−1; LR-MS (ESI+) m/z 675 (M + H+); HR-MS (ESI+) calcd for
C35H55N4O9 (M + H+) 675.3964; found 675.3949.
Pentapeptide (42). To a solution of Boc-l-Ile-l-Ala-NMe-d-Phe-l-Pro-OMe 4 (140 mg, 0.2 mmol)
in CH2Cl2 (1.6 mL) was added TFA (0.4 mL) dropwise at room temperature. After stirring for 1 h,
the reaction mixture was concentrated in vacuo. The residue was used in the next step without
further purification. To a solution of above amine salt 41 (0.2 mmol), acid
DIPEA (0.1 mL, 0.7 mmol), and HOAt (43 mg, 0.3 mmol) in CH2Cl2 (2.4 mL) was added EDC
6
(100 mg, 0.3 mmol),
HCl
·
(93 mg, 0.5 mmol) at room temperature. After stirring overnight, the reaction mixture was quenched
with 1N HCl and extracted with CH2Cl2. The combined organic layer was washed with aqueous
NaHCO3, dried over MgSO4, and concentrated in vacuo. The residue was purified by flash column
chromatography (Acetone/Hexane = 1:3) to give 145 mg (77% for 2 steps) of pentapeptide 42 as white
20
1
solid. [
rotamer
α
]
= +67.88 (c 0.50, CHCl3); H-NMR (800 MHz, CDCl3, a mixture of rotamers) Major
7.23–7.18 (m, 4H), 7.16 (t, J = 7.2 Hz, 1H), 6.64 (s, 1H), 6.51 (d, J = 6.8 Hz, 1H), 5.66 (dd, J = 9.5,
D
δ
6.5 Hz, 1H), 4.72 (dq, J = 7.2, 7.0 Hz, 1H), 4.44 (dd, J = 8.1, 6.7 Hz, 1H), 4.27–4.24 (m, 1H), 4.22–4.19 (m,
1H), 3.71 (s, 3H), 3.65–3.60 (m, 1H), 3.48–3.42 (m, 1H), 3.21 (dt, J = 10.6, 7.4 Hz, 1H), 3.19 (dd, J = 14.5,
6.4 Hz, 1H), 2.98 (s, 3H), 2.95 (dd, J = 14.4, 9.6 Hz, 1H), 2.58–2.51 (m, 1H), 2.48–2.40 (m, 1H), 2.23–2.18
(m, 1H), 1.93–1.87 (m, 1H), 1.87–1.75 (m, 4H), 1.61 (s, 3H), 1.52–1.46 (m, 5H), 1.46 (s, 9H), 1.46–1.42 (m,
1H), 1.12–1.02 (m, 1H), 0.90 (d, J = 6.2 Hz, 3H), 0.88 (d, J = 6.8 Hz, 3H), 0.85 (t, J = 7.4 Hz, 3H), 0.83
(d, J = 6.8 Hz, 3H), 0.82 (d, J = 6.9 Hz, 3H); 13C-NMR (200 MHz, CDCl3)
δ 172.6, 172.4, 172.2, 169.8,
168.1, 156.3, 136.6, 129.5, 128.3, 126.7, 93.8, 79.3, 70.4, 59.3, 57.7, 55.6, 52.3, 52.2, 46.9, 45.2, 41.6, 40.1, 37.5,
34.7, 30.9, 30.3, 28.8, 28.4, 25.3, 24.9, 24.8, 24.8, 23.1, 22.1, 17.6, 15.3, 11.4; IR (thin film, neat) νmax 2961,
2875, 1751, 1700, 1632, 1532, 1455, 1366, 1257, 1175, 1047, 859 cm−1; LR-MS (ESI+) m/z 789 (M + NH4+);
HR-MS (ESI+) calcd for C41H69N6O9 (M + NH4+) 789.5121; found 789.5134.
Izenamide A (1). To a solution of pentapeptide 42 (41 mg, 0.1 mmol) in CH2Cl2 (0.8 mL) was
added TFA (0.2 mL) dropwise at room temperature. After stirring for 1 h, the reaction mixture was
concentrated in vacuo. To a solution of above amine salt 43 (0.1 mmol), acid 39 (32 mg, 0.1 mmol),
DIPEA (0.1 mL, 0.2 mmol), and HOAt (10 mg, 0.1 mmol) in CH2Cl2 (1.0 mL) was added EDC HCl
·
(21 mg, 0.1 mmol) at room temperature. After stirring for 6 h, the reaction mixture was quenched with
1N HCl and extracted with CH2Cl2. The combined organic layer was washed with aqueous NaHCO3,
dried over MgSO4 and concentrated in vacuo. The residue was used in the next step without further
purification. To a solution of crude heptapeptide (0.1 mmol) in THF (1.0 mL) was added TBAF (1M in
THF, 0.2 mL, 0.2 mmol) at room temperature. After stirring for 6 h, the reaction mixture was quenched
with H2O and extracted with EtOAc. The combined organic layer was washed with brine, dried
over MgSO4 and concentrated in vacuo. The residue was purified by flash column chromatography
(Acetone/Hexane = 1:3 to 1:1) to give 29 mg (66% for 3 steps) of izenamide A (
1
) as white solid.
20
[
α
]
= δ
17.15 (c 0.20, MeOH); 1H-NMR (800 MHz, CD3OD, a mixture of rotamers) Major rotamer
−
D
7.26–7.19 (m, 4H), 7.18–7.13 (m, 1H), 5.69 (dd, J = 10.0, 5.9 Hz, 1H), 4.71 (d, J = 5.9 Hz, 1H), 4.68 (q,
J = 7.1 Hz, 1H), 4.41 (dd, J = 7.7, 7.4 Hz, 1H), 4.19 (d, J = 7.6 Hz, 1H), 4.11 (d, J = 4.4 Hz, 1H), 4.00–3.95
(m, 2H), 3.71 (s, 3H), 3.47–3.45 (m, 1H), 3.42–3.37 (m, 1H), 3.11 (dd, J = 14.3, 5.9 Hz, 1H), 3.06 (s, 3H),

Molecules 2019, 24, 3424
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2.93 (dd, J = 14.3, 10.0 Hz, 1H), 2.41–2.34 (m, 2H), 2.29–2.23 (m, 1H), 2.17–2.12 (m, 1H), 2.11–2.07 (m,
1H), 1.97–1.90 (m, 1H), 1.87–1.82 (m, 2H), 1.80–1.73 (m, 1H), 1.60–1.52 (m, 2H), 1.48 (ddd, J = 13.4, 7.6,
3.4 Hz, 1H), 1.31 (ddd, J = 9.3, 7.5, 4.3 Hz, 1H), 1.13 (ddd, J = 13.6, 9.5, 7.3 Hz, 1H), 1.01 (d, J = 6.8 Hz,
3H), 1.00 (d, J = 6.8 Hz, 6H), 0.92 (d, J = 6.8 Hz, 3H), 0.92 (d, J = 6.5 Hz, 3H), 0.89 (d, J = 6.3 Hz, 3H),
0.87 (t, J = 7.4 Hz, 3H), 0.86 (d, J = 6.7 Hz, 3H), 0.82 (d, J = 7.0 Hz, 3H); 13C-NMR (200 MHz, CD3OD,
a mixture of rotamers) Major rotamer
δ 175.8, 174.6, 173.9, 173.8, 173.0, 171.8, 170.4, 138.3, 130.8, 129.3,
127.6, 80.6, 76.4, 71.3, 60.9, 58.7, 57.2, 52.6, 52.2, 48.5, 46.6, 41.4, 41.4, 38.3, 35.6, 33.3, 31.7, 31.1, 30.0, 26.2,
26.0, 25.7, 23.8, 22.2, 19.3, 19.2, 18.1, 17.2, 16.6, 15.8, 11.4; IR (thin film, neat) νmax 3318, 2960, 2878, 1742,
1637, 1544, 1450, 1366, 1265, 1198, 1038, 753 cm−1; LR-MS (ESI+) m/z 854 (M + Na+); HR-MS (ESI+)
calcd for C43H69N5NaO11 (M + Na+) 854.4886; found 854.4888.
Izenamide B (2). To a solution of pentapeptide 42 (46 mg, 0.1 mmol) in CH2Cl2 (0.8 mL) was
added TFA (0.2 mL) dropwise at room temperature. After stirring for 1 h, the reaction mixture was
concentrated in vacuo. To a solution of above amine salt 43 (0.1 mmol), acid 38 (36 mg, 0.1 mmol),
DIPEA (0.1 mL, 0.2 mmol), and HOAt (11 mg, 0.1 mmol) in CH2Cl2 (1.0 mL) was added EDC HCl
·
(23 mg, 0.1 mmol) at room temperature. After stirring for 6 h, the reaction mixture was quenched
with 1N HCl and extracted with CH2Cl2. The combined organic layer was washed with aqueous
NaHCO3, dried over MgSO4, and concentrated in vacuo. The residue was used in the next step
without further purification. To a solution of crude heptapeptide (0.1 mmol) in THF (1.0 mL) was
added TBAF (1M in THF, 0.2 mL, 0.2 mmol) at room temperature. After stirring for 6 h, the reaction
mixture was quenched with H2O and extracted with EtOAc. The combined organic layer was washed
with brine, dried over MgSO4, and concentrated in vacuo. The residue was purified by flash column
chromatography (Acetone/Hexane = 1:3 to 1:1) to give 31 mg (61% from S5) of izenamide B (
2
) as white
20
solid. [
α
]
D
= δ 7.25–7.19 (m, 4H), 7.18–7.13 (m,
11.62 (c 1.30, MeOH); 1H-NMR (800 MHz, CD3OD)
−
1H), 5.69 (dd, J = 10.0, 5.9 Hz, 1H), 4.93 (d, J = 4.4 Hz, 1H), 4.67 (q, J = 7.0 Hz, 1H), 4.41 (dd, J = 7.8,
7.3 Hz, 1H), 4.19 (d, J = 7.5 Hz, 1H), 4.13 (d, J = 4.3 Hz, 1H), 4.00–3.96 (m, 2H), 3.71 (s, 3H), 3.48–3.44
(m, 1H), 3.42–3.38 (m, 1H), 3.11 (dd, J = 14.3, 5.9 Hz, 1H), 3.06 (s, 3H), 2.93 (dd, J = 14.3, 10.0 Hz, 1H),
2.41–2.33 (m, 2H), 2.28–2.23 (m, 1H), 2.11–2.07 (m, 1H), 1.96–1.89 (m, 2H), 1.88–1.81 (m, 2H), 1.79–1.74
(m, 1H), 1.59–1.53 (m, 2H), 1.51–1.43 (m, 2H), 1.33–1.27 (m, 2H), 1.18–1.10 (m, 1H), 1.01 (d, J = 6.9 Hz,
3H), 0.97 (d, J = 6.9 Hz, 3H), 0.95 (t, J = 7.5 Hz, 3H), 0.92 (d, J = 6.4 Hz, 3H), 0.92 (d, J = 6.8 Hz, 3H), 0.89
(d, J = 6.2 Hz, 3H), 0.87 (t, J = 7.4 Hz, 3H), 0.86 (d, J = 6.8 Hz, 3H), 0.82 (d, J = 7.1 Hz, 3H); 13C-NMR
(200 MHz, CD3OD, a mixture of rotamers) Major rotamer
δ 175.7, 174.6, 173.9, 173.8, 173.0, 171.9, 170.3,
138.3, 130.8 129.3 127.6, 78.3, 76.4, 71.4, 60.9, 58.7, 57.2, 52.6, 52.3, 48.5, 46.6, 41.5, 41.4, 38.4, 38.3, 35.6,
33.3, 31.0, 30.0, 27.2, 26.2, 26.1, 25.7, 23.8, 22.2, 19.3, 17.1, 16.6, 15.8, 14.7, 11.9, 11.5; IR (thin film, neat)
νmax 3308, 2960, 2877, 1743, 1636, 1533, 1448, 1361, 1265, 1195, 1038, 753 cm−1; LR-MS (ESI+) m/z 846
(M + H+); HR-MS (ESI+) calcd for C44H72N5O11 (M + H+) 846.5223; found 846.5229.
4. Conclusions
In conclusion, we have accomplished the first total syntheses of izenamides A (1), B (2), and C (3)
and confirmed the stereochemistry of izenamide B, which was originally suggested by Suenaga and
coworkers. The key features of the syntheses include the DKP formation-free amide coupling sequence
and amidation with minimal epimerization. Our versatile and convergent synthesis of izenamides
is expected to be widely utilized for the efficient synthesis of izenamides and their analogs for the
development of novel cathepsin D inhibitors. Further studies on the structure-activity relationship of
izenamides are currently underway and successful results will be reported in due course.
Supplementary Materials: The following are available online. The 1H and 13C NMR spectra of 16
4, 5, 12, 13, 35, 36, 37, 38, 39, 3, 42, 1 and 2.
, 19, 20, 24, 25,
Funding: This research was funded by National Research Foundation of Korea, grant number 2019M3A9A8067070.
Acknowledgments: We thank Young-Ger Suh of Seoul National University for his helpful discussions in
management of this work and preparation of manuscript.

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Conflicts of Interest: The authors declare no conflict of interest.
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©
2019 by the author. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).