Synthesis and biological evaluation of novel FK228 analogues as potential isoform selective HDAC inhibitors

Article abstract of DOI:10.1016/j.ejmech.2016.05.031

Novel C4- and C7-modified FK228 analogues were efficiently synthesized in a highly convergent and unified manner. This synthesis features the amide condensation of glycine-d-cysteine-containing segments with d-valine-containing segments for the direct assembly of the corresponding seco-acids, which are key precursors of macrolactones. The HDAC inhibition assay and cell-growth inhibition analysis of the synthesized analogues revealed novel aspects of their structure-activity relationship. This study demonstrated that simple modification at the C4 and C7 side chains in FK228 is effective for improving both HDAC inhibitory activity and isoform selectivity; moreover, potent and highly isoform-selective class I HDAC1 inhibitors were identified.

Full text of DOI:10.1016/j.ejmech.2016.05.031

Accepted Manuscript
Synthesis and biological evaluation of novel FK228 analogues as potential isoform
selective HDAC inhibitors
Koichi Narita, Keisuke Matsuhara, Jun Itoh, Yui Akiyama, Singo Dan, Takao Yamori,
Akihiro Ito, Minoru Yoshida, Tadashi Katoh
PII:
S0223-5234(16)30427-5
10.1016/j.ejmech.2016.05.031
EJMECH 8623
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 24 February 2016
Revised Date: 14 May 2016
Accepted Date: 16 May 2016
Please cite this article as: K. Narita, K. Matsuhara, J. Itoh, Y. Akiyama, S. Dan, T. Yamori, A. Ito,
M. Yoshida, T. Katoh, Synthesis and biological evaluation of novel FK228 analogues as potential
isoform selective HDAC inhibitors, European Journal of Medicinal Chemistry (2016), doi: 10.1016/
j.ejmech.2016.05.031.
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ACCEPTED MANUSCRIPT
Graphical abstract:

ACCEPTED MANUSCRIPT
Synthesis and biological evaluation of novel FK228 analogues as potential isoform
selective HDAC inhibitors
Koichi Narita a, Keisuke Matsuhara a, Jun Itoh a, Yui Akiyama a, Singo Dan b, Takao Yamori b,c,
Akihiro Ito d, Minoru Yoshida d, Tadashi Katoh a,*
a Laboratory of Synthetic and Medicinal Chemistry, Faculty of Pharmaceutical Sciences, Tohoku Medical
and Pharmaceutical University, 4-4-1 Komatsushima, Aoba-ku, Sendai 981-8558, Japan
b Division of Molecular Pharmacology, Cancer Chemotherapy Center, Japanese Foundation for Cancer
Research, 3-10-6 Ariake, Koto-ku, Tokyo 135-8550, Japan
c Pharmaceuticals and Medical Devices Agency (PMDA), 3-3-2 Kasumigaseki, Chiyoda-ku, Tokyo
100-0013, Japan
d Chemical Genetics Laboratory, RIKEN, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan
Abstract: Novel C4- and C7-modified FK228 analogues were efficiently synthesized in a
highly convergent and unified manner. This synthesis features the amide condensation of
glycine-D-cysteine-containing segments with D-valine-containing segments for the direct
assembly of the corresponding seco-acids, which are key precursors of macrolactones. The
HDAC inhibition assay and cell-growth inhibition analysis of the synthesized analogues
revealed novel aspects of their structure-activity relationship. This study demonstrated that
simple modification at the C4 and C7 side chains in FK228 is effective for improving both
HDAC inhibitory activity and isoform selectivity; moreover, potent and highly
isoform-selective class I HDAC1 inhibitors were identified.
Key words:
FK228 analogues
Histone deacetylase inhibitors
Structure-activity relationship
Bicyclic depsipeptide
Total synthesis
1

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1. Introduction
The reversible acetylation and deacetylation of histones plays an important role for
regulating gene expression by altering chromatin architecture [1]. Histone acetyltransferases
(HATs) catalyse the transfer of acetyl groups from acetyl-CoA to the ε-amino groups on histone
lysine residues, which relaxes the chromatin structure and epigenetically promotes gene
transcription. Conversely, histone deacetylases (HDACs) catalyse the removal of acetyl groups
from the N-acetyl lysine residues on histones, which contracts the chromatin structure and
epigenetically suppresses gene transcription. The inhibition of HDAC enzymatic activity has
been demonstrated to affect transcriptional events that are involved in growth arrest,
differentiation, proliferation, cell cycle regulation, protein turnover and/or apoptosis in
transformed tumour cell cultures. Consequently, HDAC-inhibiting compounds are considered to
be potential drugs for targeted cancer therapy [2].
There are 18 human HDAC isoforms, which are grouped into four major classes: class I
(HDACs 1, 2, 3 and 8), class II (class IIa: HDACs 4, 5, 7 and 9; class IIb: HDACs 6 and 10),
and class IV (HDAC 11) are Zn2+-dependent metallohydrolases, whereas class III HDACs
(SirTs 1–7) are NAD+-dependent sirtuins [3]. Inhibiting class I HDACs is considered to be an
effective mechanism for anticancer agents, whereas inhibiting class II HDACs may cause
undesirable side effects such as serious cardiac hypertrophy [4]; therefore, in cancer
chemotherapy, the potent and selective inhibition of class I HDACs is highly desirable.
In 2009, the U.S. Food and Drug Administration (FDA) approved the bicyclic depsipeptide
HDAC inhibitor FK228 (romidepsin, 1, Fig. 1) for treating cutaneous T-cell lymphoma and
other peripheral T-cell lymphomas [5]; however, FK228 is known to be associated with an
unresolved cardiotoxicity issue [5b,6], which highlights the requirement for further identifying
and developing new HDAC inhibitors with high efficacy and low toxicity. FK228 was first
discovered by researchers from Fujisawa Pharmaceutical Co. Ltd. (now Astellas Pharma Inc.) in
1994, who isolated it from a culture broth of Chromobacterium violaceum (No. 968) [7].
Yoshida et al. proposed a molecular mechanism by which FK228 inhibits HDAC. [8] In that
mechanism, FK228, which itself serves as a stable prodrug, is activated by reductive cleavage of
the disulfide bond after incorporation into the cells, and the released sulfhydryl group in the
butenyl side chain interacts with the zinc cation located at the binding pocket of the HDACs,
resulting in a potent inhibitory effect. The structural features of FK228 are a 16-membered
bicyclic depsipeptide comprising dehydrobutyrine (Dhb),
L-valine, D-cysteine, D-valine,
(3S,4E)-3-hydroxy-7-mercapto-4-heptenoic acid (Hmh) and a characteristic disulfide bond
linkage. Since the discovery of FK228, several structurally similar bicyclic depsipeptide HDAC
inhibitors have been isolated from bacterial fermentations, including spiruchostatins A–D (2–5)
[9], FR901375 (6) [10], burkholdacs A (7) and B (8) [11] and thailandepsins A–F (7–12) [12]
(thailandepsins A and C are identical to burkholdacs B and A, respectively). The potentially
2

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Fig. 1. Structures of FK228 (romidepsin; 1), spiruchostatins A–D (2–5), FR901375 (6), burkholdacs A
(thailandepsin C; 7) and B (thailandepsin A; 8), and thailandepsins E (9), B (10), D (11) and F (12). i-Pr =
isopropyl, s-Bu = sec-butyl, i-Bu = isobutyl.
therapeutic biological properties and unique structural features of 1–12 have made them
attractive targets for total synthesis, and several such syntheses of these natural products have
been published in the literature [13–19].
During our research on the synthesis and biological evaluation of bicyclic depsipeptide
HDAC inhibitors such as 1–5, 7 and 8 [13a,14c,15b,16,18a], we became interested in
synthesizing FK228 analogues and evaluating their isoform selectivity in order to identify the
potent and class I-selective HDAC inhibitors. There are several studies in the literature on
synthesizing FK228 analogues [20]. Note that the synthesis and evaluation of FK228 analogues,
which was reported by Ganesan et al., revealed novel aspects of their structure–activity
relationship (SAR) [20c]. From this SAR study, three primary critical features that were related
3

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to the HDAC inhibitory activity of 1 were identified; i) the 16-membered macrocyclic scaffold
is indispensable, ii) the unsaturated Dhb moiety can be replaced with other amino acids and iii)
the C4 isopropyl group in the L-valine moiety is not always necessary. Although these results
are very useful for designing novel FK228 analogues, no information on the effects of the
stereochemistry in the amino acid components was reported.
Based on the results presented by Ganesan et al., we designed eight novel C4- and
C7-modified FK228 analogues, which were denoted as FK-A1 to FK-A8 (13a–h, Fig. 2). Note
that the
L-valine and Dhb moieties of 1 were replaced by simple amino acids such as glycine,
D
-/L-alanine,
D
-/ -phenylalanine and -/ -leucine. We envisaged that 13a–h could be readily
L
D L
synthesized via the total synthesis we had previously developed for bicyclic depsipeptide
natural products.
In this study, we describe the synthesis of FK228 analogues 13a–h using a synthetic strategy
that was developed previously in our laboratory. Then, to investigate the effect of the C7
stereochemistry and amino acid substituent groups, these synthesized analogues were subjected
to an HDAC inhibition assay and antiproliferative analysis.
Fig. 2. Structures of the novel FK228 analogues FK-A1 to FK-A8 (13a–h). Bn = benzyl.
2. Results and discussion
2.1. Chemistry
2.1.1. General synthetic strategy for the novel FK228 analogues FK-A1 to FK-A8 (13a–h)
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The selection of the method that was used for constructing the 16-membered macrocyclic
ring is crucial to successfully achieve the total synthesis of this class of natural products. In our
previously reported total syntheses of FK228 (1) [13a], spiruchostatins A–D (2–5) [14c,15b,16]
and burkholdacs A (7) and B (8) [18a], we employed two different reliable methods such as
Shiina’s macrocyclization [21] and Mitsunobu macrocyclization [22]. For synthesizing 2–5, 7
and 8, we employed Shiina’s macrocyclization, whereas for synthesizing 1, we employed
Mitsunobu macrocyclization. Note that Shiina’s macrocyclization was superior to the
Mitsunobu macrocyclization in terms of chemical yield, reaction cleanness and ease of
purification; however, in the case of 1, all attempts to conduct Shiina’s macrocyclization proved
unsuccessful, most possibly because of the sterically hindered isopropyl group in the
unit. Remarkably, using Shiina’s macrolactonization, Ganesan et al. successfully synthesized
FK228 analogues in which the -valine moiety was replaced by glycine [20c]; therefore, we
L-valine
L
decided to perform the key macrolactonization using Shiina’s method in our synthesis.
Our general synthetic plan is outlined in Scheme 1. We envisioned that the target molecules
13a–h could be synthesized by Shiina’s macrolactonization of the corresponding seco-acids
14a–h, followed by internal disulfide bond formation. The key element of this scheme is the
highly convergent assembly of 14a–h by the direct condensation of the
glycine-
D
-cysteine-containing segments 15a–h and the
D-valine-containing segment 16.
Segments 15a–h are formed by the condensation of the
D-cysteine derivative 17, amino acid 18
and glycine methyl ester hydrochloride (19). The remaining segment 16 is obtained using our
5

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Scheme 1. Retrosynthetic analysis for 13a–h. Tr = triphenylmethyl, PMB = 4-methoxylbenzyl, Boc =
tert-butoxycarbonyl.
previously described total synthesis of 1 [13a] and 5 [16].
2.1.2. Synthesis of FK-A1 (13a)
First, the synthesis of segment 15a was performed from L-threonine (18a), as shown in
Scheme 2. Protection of the amino group in 18a with a 9-fluorenylmethoxycarbonyl (Fmoc)
group, which was followed by condensation of the resulting carbamate 20 with glycine methyl
ester hydrochloride (19), provided the desired dipeptide 21 in 88% yield over the two steps.
After deprotection of the N-Fmoc group in 21, the free amine 22 was subjected to condensation
with the D-cysteine derivative 17, which resulted in the tripeptide 23 in 53% yield over the two
steps. At this stage, the threonine part of 23 is converted to an unsaturated Dhb moiety via a
two-step sequence that involves mesylation of the hydroxy group (94% yield) followed by
elimination of the resulting mesylate 24 (89% yield), thus affording the desired product 25.
Deprotection of the N-Boc group in 25 is efficiently achieved using BF3·OEt2, leading to the
desired segment 15a in 88% yield.
Scheme 2. Synthesis of segment 15a. a) FmocCl, 10% aq. Na2CO3, dioxane, rt; b) 19, PyBOP, i-Pr2NEt,
MeCN, rt, 88% (2 steps); c) Et2NH, MeCN, rt; d) 17, PyBOP, i-Pr2NEt, MeCN, rt, 53% (2 steps); e)
MsCl, Et3N, CH2Cl2, 0 ºC, 94%; f) DABCO, THF, rt, 89%; g) BF3·Et2O, CH2Cl2, rt, 88%. FmocCl =
9-fluorenylmethoxycarbonyl chloride, PyBOP
=
(benzotriazol-1-yloxy)tripyrrolidinophosphonium
hexafluorophosphate, MsCl = methanesulfonyl chloride, DABCO=1,4-diazabicyclo-[2.2.2]octane.
6

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Subsequently, the synthesis of FK-A1 (13a) was accomplished by assembling the two
segments 15a and 16, as shown in Scheme 3. Treating 15a and 16 with HATU (1.3 equiv) and
HOAt (1.3 equiv) in the presence of i-Pr2NEt (2.6 equiv) in CH2Cl2/MeCN at −20 ºC for 5 h
afforded the desired condensation product 26a in 80% yield without appreciable epimerization
at the C21 stereogenic centre [13a]. Then, the condensation product 26a was converted to the
required seco-acid 14a with an overall yield of 87% via the successive removal of both the
PMB and methyl-protecting groups. The critical macrolactonization of 14a is successfully
performed using Shiina’s method [MNBA (1.3 equiv), DMAP (3.0 equiv), CH2Cl2 (1.0 mM), rt,
12 h] [21], and the desired cyclization product 28a is obtained in 64% yield. Finally, disulfide
bond formation accompanied by oxidative S-Tr deprotection of 28a is efficiently achieved by
brief exposure to I2 in a dilute CH2Cl2/MeOH solution (0.5 mM) at room temperature, which
results in the production of the desired FK-A1 (13a) in 97% yield.
Scheme 3. Synthesis of FK-A1 (13a). a) HATU, HOAt, i-Pr2NEt, CH2Cl2/MeCN, −20 ºC, 80%; b) DDQ,
CH2Cl2/H2O, rt, 90%; c) LiOH, THF/ H2O, 0 ºC, 97%; d) MNBA, DMAP, CH2Cl2, rt, 64%; e) I2, MeOH/
CH2Cl2,
hexafluorophosphate,
rt,
97%.
HATU
HOAt
=
O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
1-hydroxy-7-azabenzotriazole, DDQ
=
=
2,3-dichloro-5,6-dicyano-1,4-benzoquinone, MNBA = 2-methyl-6-nitrobenzoic anhydride, DMAP =
4-dimethylaminopyridine.
7

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2.1.3. Synthesis of FK-A2 to FK-A8 (13b–h)
As shown in Scheme 4, using glycine (18b: R1 = R2 = H),
-/ D L
R2 = H), -phenylalanine (18e,f: R1 = α-/β-Bn, R2 = H) and
D-/
L
-alanine (18c,d: R1 = α-/β-Me,
-/
-leucine (18g,h: R1 =
D
L
α-/β-i-Bu, R2 = H) as starting materials, the remaining FK228 analogues, i.e. FK-A2 to FK-A8
(13b–h), were efficiently synthesized in a manner similar to that described for synthesizing
FK-A1 (13a).
Scheme 4. Synthesis of FK-A2 to FK-A8 (13b–h). a) FmocCl, 10% aq. Na2CO3, dioxane, rt; b) 18,
PyBOP, i-Pr2NEt, MeCN, rt; c) Et2NH, MeCN, rt; d) 17, PyBOP, i-Pr2NEt, MeCN, rt; e) BF3·Et2O,
CH2Cl2, rt; f) 16, HATU, HOAt, i-Pr2NEt, CH2Cl2/MeCN, −20 ºC; g) DDQ, CH2Cl2/H2O, rt; h) LiOH,
THF/ H2O, 0 ºC; i) MNBA, DMAP, CH2Cl2, rt, 64%; j) I2, MeOH/ CH2Cl2, rt.
8

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2.2. Biological evaluation
2.2.1. HDAC inhibition assay
To determine their potency and isoform selectivity, the synthesized FK228 analogues 13a–h
were tested for their HDAC inhibitory activity against HDAC1 (class I) and HDAC6 (class IIb).
In this assay, the clinically approved FK228 (1) was used as a positive control, and the results
are summarized in Table 1. FK-A1 (13a), which lacks the C4 isopropyl group that is present in
1, exhibited high HDAC1 inhibitory activity (IC50 = 4.7 nM), although its potency is
approximately half of that of 1 (IC50 = 2.4 nM). This observation is in good agreement with a
previous SAR study that was conducted by Ganesan et al. [20c]. On the other hand, FK-A2
(13b), which lacks both the C4 isopropyl and the C7 ethylidene groups present in 1, exhibits
much lower inhibition potency (IC50 = 92.4 nM) compared to 1. These results indicate that the
C4 substituent is not responsible for HDAC inhibitory activity, whereas the C7 substituent is
indispensable.
Table 1. HDAC inhibitory activity of FK228 analogues FK-A1–FK-A8 (13a–h).
a
IC50
(
n
M)
Compound
HDAC1 (class I)b HDAC6 (class IIb)b
SId
c
2.4 ± 0.16
4.7 ± 0.88
92.4 ± 30.0
2.1 ± 1.0
184 ± 69.1
0.54 ± 0.10
40.8 ± 4.9
0.96 ± 0.14
11.4 ± 4.1
45
182
14
480
6
102
9
109 ± 35.8
859 ± 117
131 ± 178
1008 ± 461
1022 ± 38.6
54.9 ± 14.5
355 ± 82
FK228 (1)
FK-A1 (13a
FK-A2 (13b
FK-A3 (13c
FK-A4 (13d
FK-A5 (13e
FK-A6 (13f
FK-A7 (13g
FK-A8 (13h
)
)
)
)
)
)
)
)
8452 ± 1066
2255 ± 577
8804
198
a The concentration that induces 50% inhibition against HDACs.
b The enzyme assay was performed in the presence of 0.1 mM dithiothreitol (DTT).
c Positive control used in this study was synthesized in our laboratory [13a].
d Selectivity index (HDAC6 IC50/HDAC1 IC50) as the selectivity toward class I HDAC1 over class II HDAC6.
With respect to the effect of the R1 substituent (including the C7 stereochemistry), FK-A3
(13c), FK-A5 (13e) and FK-A7 (13g), which were synthesized from the corresponding D-amino
acids, exhibit excellent potency against HDAC1 in the low nanomolar range (IC50 = 0.54–2.1
nM). Importantly, the potencies of 13c, 13e and 13g were estimated to be equal to or two-fold
higher than that of 1. FK-A5 shows the highest potency (IC50 = 0.54 nM) among the analogues
that were tested in this assay; however, FK-A4 (13d), FK-A6 (13f) and FK-A8 (13h), which
were synthesized from the corresponding
activity (IC50 = 11.4–184 nM). These results suggest that the C7 stereochemistry plays an
important role in conferring potency and that the -amino acid side chains are preferable as the
R1 substituents. To the best of our knowledge, the SAR of the C7 stereochemistry in FK228 has
L-amino acids, showed considerably lower inhibitory
D
9

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not been previously reported. Thus, these findings provide new insight for designing novel
synthetic depsipeptide HDAC inhibitors.
As we expected, 13a–h exhibit much lower HDAC6 inhibitory activity, with IC50 values in
the range of 55–8452 nM. For convenience, isoform selectivity (class I HDAC1/class IIb
HDAC6) is expressed as a selective index (SI) value (HDAC6 IC50/HDAC1 IC50). The isoform
selectivity of 13a (SI = 182) is four-fold higher than that of 1 (SI = 45), suggesting that removal
of the C4 substituent is effective for increasing isoform selectivity. However, FK-A2 (13b)
exhibits lower isoform selectivity (SI = 14), suggesting that removal of the C7 substituent
reduces isoform selectivity. Among the remaining analogues 13c–h, compounds 13c, 13e and
13g, which possess
D-amino acid side chains at the C7 position, exhibit higher isoform
selectivity (SI = 102–8804) compared to compounds 13d, 13f and 13h, which possess
L-amino
acid side chains (SI = 6–198). Note that FK-A7 (13g, SI = 8,804) exhibits approximately
200-fold higher isoform selectivity compared to 1 (SI = 45), which, to the best our knowledge,
represents the highest level of class I/II selectivity among the naturally occurring and synthetic
bicyclic depsipeptides that have been reported to date.
These results reveal that a simple transformation of the C7 substituent (an ethylidene group)
of 1 into a D-amino acid side chain is effective for improving both the HDAC inhibitory activity
and the isoform selectivity. This structural alternation may confer a preferable conformational
change onto the macrocyclic depsipeptide scaffold, thereby enhancing the discrimination and
interactions between the macrocycle and the HDAC1 enzyme ‘rim’ located outside the active
site binding pocket [23].
2.2.2. Cell-growth inhibition assay
The growth inhibitory activity of the synthesized analogues 13a–h was simultaneously
evaluated with FK228 (1) as a reference compound, using a panel of 39 human cancer cell lines
at the Japanese Foundation for Cancer Research [24]. The organs from which the cell lines were
derived were as follows (number of cells in parentheses): breast (5), central nervous system (6),
colon (5), lung (7), melanoma (1), ovary (5), kidney (2), stomach (6) and prostate (2).
Dose-response curves were measured at 5 different concentrations (from 10−11 to 10−7 M, from
10−10 to 10−6 M or from 10−8 to 10−4 M) for each compound, and the concentration that caused
50% cell growth inhibition (GI50) was compared with that of the control.
The GI50 values of the tested analogues 13a–h together with 1 are shown in Table 2. The
potency order of these analogues was estimated using the MG-MID value (mean value of GI50
over all of the cell lines tested) to be 1 (6.2 nM) > 13e (9.5 nM) > 13c (43 nM) > 13g (56 nM) >
13a (115 nM) >> 13f (1071 nM) > 13h (1259 nM) >> 13b (14,000 nM) > 13d (20,000 nM).
10

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Table 2. Growth inhibition of FK228 analogues FK-A1 to FK-A8 (13a–h) against a panel of 39 human
cancer cell lines.
GI50a [ nM ]
Origin of cancer Cell line
1
13a
13b
13c
13d
13e
13f
13g
13h
6.9
8.5
13
170
200
630
190
53
13,000
18,000
39,000
33,000
9200
17,000
12,000
40,000
3300
30
36
160
37
21
19
17,000
20,000
21,000
17,000
16,000
17,000
17,000
51,000
15,000
14,000
13,000
13,000
15,000
12,000
37
36
84
62
8.7
22
5.0
21
3.3
8.7
8.6
3.2
2.8
3.0
690
1300
1900
1900
1200
1500
220
70
69
270
52
1400
1500
1700
1600
1200
330
HBC-4
BSY-1
HBC-5
MCF-7
MDA-MB-231
U-251
SF-268
SF-295
Breast
4.2
5.5
3.9
4.9
4.0
3.6
7.2
9.6
3.1
3.4
3.3
39
120
110
280
10b
11
Central nervous
system (brain)
31
71
820
1800 1100c
1500
1500
1000
1800
1300
1100
690
10b
10b
10b
10b
14
860
1300
540
1100
740
57
43
30
27
33
33
SF-539
10b
10b
32
950
SNB-75
SNB-78
HCC2998
KM-12
610b
13,000
12,000
2700
Colon
Lung
45
42
20
13
570
HT-29
450c 20,000c
100,000c 6700c
100,000c 100c
26,000c 590
17,000c
340
1200
1600
200
1100
720
1400
680
HCT-15
HCT-116
NCI-H23
NCI-H226
NCI-H522
NCI-H460
A549
DMS273
DMS114
LOX-IMVI
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
SK-OV-3
RXF-631 L
ACHN
St-4
MKN1
MKN7
MKN28
MKN45
MKN74
DU-145
PC-3
3.1
4.6
8.9
1.8b
3.0
2.6
5.8
3.6
2.5
4.6
26
130
410
2500
36,000
61,000
680
15,000
10,000
47,000
5200
10b
31
110
11,000
27,000
21,000
1700b
43,000
18,000
47,000
15,000
4500
16,000
22,000
15,000
31,000
16,000
100,000c
100,000c
62,000
14,000
43,000
24,000
100,000c
18,000
15,000
13,000
2.6
4.4
250
1100
1200
200
1100
880
22
39
59
10b
39
23
74
36
23
40
87
31
42
23
68
200
230
21
280
270
240
44
4.8
10b
10b
15
15
89
17
11
22
0.72b
6.2
56
52
230
43
25
64
270
49
54
3.4
20
4.1
2.2
1500
620
2900
160b
1100
1900
1400
1400
1200
1800
11,000
2000
290
180b
1500
1600
1200
1500
1500
3200
9400
2100
1100
1800
1800
2200
1500
830
Melanoma
Ovary
13,000
46,000
3100
17
27
6.8
4.6
2.4
7.9
24
34
2.6
35
78
64
20
100
14
31
2.8
5.5
3.3
6.6
20
22
3.2
4.9
17
14
3.0
6.0
18
18,000
12,000
91,000
43,000
81,000
12,000
47,000
95,000
100,000c
16,000
5400
63
19
230
460
1100
73
380
1400
1900
100
39
110
270
410
36
260
440
500
35
Kidney
Stomach
1900
2100
1800
500
590
1100
5.9
3.6
5.7
21
70
37
69
Prostate
170
11,000
1400
MG-MIDd
6.2
115
14,000
43
20,000
9.5
1071
56
1259
a Concentration that induces 50% inhibition of cell growth compared to the control.
b The most sensitive cell.
c The least sensitive cell.
d Mean value of GI50 over all cell lines tested.
These results correspond reasonably with the abilities of the analogues to inhibit HDAC1, as
discussed above. Analogues 13c, 13e and 13g, which possess -amino acid side chains, exhibit
considerably more potency (GI50 = 9.5–56 nM) compared to analogues 13d, 13f and 13h, which
possess -amino acid side chains (GI50 = 1071–20,000 nM). Interestingly, analogue 13g, which
D
L
exhibits the highest HDAC isoform selectivity (SI = 8804), exhibits the lowest sensitivity
against SF-295 (brain cancer) among the 39 cell lines. This result is significantly different from
the analogues 13a–f and 13h, all of which exhibit their lowest sensitivity against HCT-15 (colon
11

ACCEPTED MANUSCRIPT
cancer). The change in the cell-growth inhibition pattern for 13g might be caused by its HDAC
isoform selectivity. Moreover, analogue 13e, which exhibits the highest MG-MID value (GI50 =
9.5 nM) among the tested analogues, exhibits two-fold higher sensitivity to NCI-H522 (lung
cancer, GI50 = 0.72 nM) compared to that of 1 (GI50 = 1.8 nM). One possible explanation for
this observation is that analogue 13e, which possesses an aromatic ring in the C7 side chain, has
an additional molecular target (e.g. a different type of protein kinase) [25]; therefore, further
investigation is necessary. Considering both HDAC inhibitory and antiproliferative activities,
13c, 13e and 13g are promising candidates for developing novel anticancer agents with high
efficacy and low toxicity.
3. Conclusion
We synthesized eight new FK228 analogues, i.e. FK-A1 to FK-A8 (13a–f), in a highly
convergent and unified manner. The present preliminary biological evaluation revealed novel
aspects of the SAR for this class of depsipeptide HDAC inhibitor. In particular, replacement of
the C4 isopropyl and C7 ethylidene substituents of FK228 by a hydrogen atom and a D-amino
acid side chain, respectively (13c, 13e and 13g), was quite effective for improving HDAC1
inhibitory activity and isoform selectivity. Furthermore, incorporating an aromatic ring into the
C7 side chain (13e) was demonstrated to result in an improvement of the specificity to cancer
cell lines. These findings are useful for designing and developing new anticancer agents that
work by the isoform-selective inhibition of HDACs. Further investigation of the activity of the
synthesized analogues 13c, 13e and 13g using an animal model is currently underway in our
laboratories and will be reported in due course.
4. Experimental
4.1. Chemistry
All reactions involving air- and moisture-sensitive reagents were carried out using oven
dried glassware and standard syringe-septum cap techniques. Routine monitorings of reaction
were carried out using glass-supported Merck silica gel 60 F254 TLC plates. Flash column
chromatography was performed on Kanto Chemical Silica Gel 60N (spherical, neutral 40–50
nm) with the solvents indicated.
All solvents and reagents were used as supplied with following exceptions. Tetrahydrofuran
(THF) was freshly distilled from Na metal/benzophenone under argon. CH2Cl2, MeCN, Et2NH,
and i-Pr2NEt were distilled from calcium hydride under argon.
Measurements of optical rotations were performed with a JASCO DIP-370 automatic digital
1
polarimeter. H and 13C NMR spectra were measured with a JEOL AL-400 (400 MHz)
spectrometer. Chemical shifts were expressed in ppm using Me4Si (δ = 0) as an internal standard.
12

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The following abbreviations are used: singlet (s), doublet (d), triplet (t), quartet (q), multiplet
(m) and broad (br). Infrared (IR) spectral measurements were carried out with a JASCO
FT/IR-4100 spectrometer. Low- and High-resolution mass (HRMS) spectra were measured on a
JEOL JMS-700 high resolution mass spectrometer.
4.1.1. Fmoc-
A solution of 9-fluorenylmethyl chloroformate (FmocCl) (652 mg, 2.5 mmol) in dioxane (10
mL) was added dropwise to a stirred solution of -threonine ( -Thr) (18a) (300 mg, 2.5 mmol)
L-Thr–Gly-OMe (21)
L
L
in 10% aqueous Na2CO3 (10 mL) at room temperature. After 2 h, the reaction mixture was
diluted with H2O (130 mL), and the resulting mixture was washed with Et2O (2 × 40 mL).
Concentrated H2SO4 was slowly added to the aqueous layer until pH was 2–3, and the resulting
solution was extracted with EtOAc (3 × 100 mL). The combined extracts were washed with
H2O (2 × 50 mL) and brine (2 × 50 mL), then dried over Na2SO4. Concentration of the solvent
in vacuo afforded Fmoc-L-Thr (20) (859 mg) as a colorless amorphous solid, which was used
for the next reaction without further purification.
i-Pr2NEt (1.71 mL, 10 mmol) was added dropwise to a stirred solution of the crude 20 (859
mg) and glycine methyl ester (Gly-OMe) hydrochloride (19) (348 mg, 2.8 mmol) in MeCN (25
mL) containing (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate
(PyBOP) (1.97 g, 3.8 mmol) at room temperature under argon. After 1 h, concentration of the
solvent in vacuo afforded a residue, which was purified by column chromatography
27
(hexane/EtOAc 1:1) to give 21 (912 mg, 88%, 2 steps) as a colorless amorphous solid. [α]D
1
−29.1 (c 1.0, CHCl3); H NMR (400 MHz, CDCl3): δ 1.18 (3H, d, J = 6.3 Hz), 3.62 (1H, s),
3.70 (3H, s), 4.02 (2H, d, J = 5.4 Hz), 4.21 (2H, q, J = 6.8 Hz), 4.37–4.40 (3H, m), 5.99 (1H, d,
J = 7.8 Hz), 7.15 (1H, br t, J = 4.9 Hz), 7.29 (2H, t, J = 7.3 Hz), 7.38 (2H, t, J = 7.6 Hz), 7.58
13
(2H, d, J = 7.3 Hz), 7.75 (2H, d, J = 7.3 Hz); C NMR (100 MHz, CDCl3): δ 18.1, 41.1, 47.1,
52.4, 58.8, 67.0, 67.2, 120.0 (2C), 125.0 (2C), 127.0 (2C), 127.71, 127.72, 141.3 (2C), 143.59,
143.64, 156.8, 170.2, 171.3; IR (neat): 3331, 3017, 2975, 2952, 1724, 1714, 1667, 1530, 1449,
1215, 1106, 758, 740 cm−1; HRMS (EI): m/z calcd for C22H24N2O6 (M+) 412.1634, found
412.1628.
4.1.2. Boc-D-Cys–L-Thr–Gly-OMe (23)
Et2NH (2.3 mL, 22 mmol) was added to a stirred solution of 21 (912 mg, 2.2 mmol) in
MeCN (30 mL) at room temperature. After 3 h, the reaction mixture was concentrated in vacuo
to afford NH2-L-Thr–Gly-OMe (22) (421 mg) as a colorless oil, which was used for the next
reaction without further purification.
i-Pr2NEt (0.98 mL, 5.8 mmol) was added dropwise to a stirred solution of the crude amine
22 (421 mg, 2.2 mmol) and N-Boc-
D
-Cys(Tr)-OH (17) (1.23 g, 2.7 mmol) in MeCN (22 mL)
13

ACCEPTED MANUSCRIPT
containing PyBOP (1.50 g, 2.9 mmol) at room temperature under argon. After 1 h, concentration
of the solvent in vacuo afforded a residue, which was purified by column chromatography
28
(hexane/EtOAc 1:1) to give 23 (745 mg, 53%, 2 steps) as a colorless amorphous solid. [α]D
1
−27.5 (c 0.10, CHCl3). H NMR (400 MHz, CDCl3): δ 1.12 (3H, d, J = 6.3 Hz), 1.40 (9H, s),
2.59 (1H, dd, J = 12.9, 4.9 Hz), 2.78 (1H, dd, J = 12.4, 7.8 Hz), 3.30 (1H, br s), 3.60 (1H, br d, J
= 4.9 Hz), 3.65–3.74 (4H, m), 4.04 (1H, dd, J = 17.6, 5.4 Hz), 4.23 (1H, d, J = 7.8 Hz), 4.46
(1H, br s), 5.00 (1H, d, J = 6.3 Hz), 6.82 (1H, d, J = 7.8 Hz), 7.21–7.31 (9H, m), 7.39–7.44 (7H,
m); 13C NMR (100 MHz, CDCl3): δ 18.5, 28.2 (3C), 33.5, 41.0, 52.3, 54.3, 57.8, 66.6, 67.3,
80.7, 127.0 (3C), 128.1 (6C), 129.5 (6C), 144.2 (3C), 155.5, 170.2, 171.4 (2C); IR (neat): 3387,
3303, 3275, 2979, 2921, 2852, 1685, 1676, 1654, 1642, 1541, 1533, 1508, 1219, 1163, 1101,
877, 700 cm−1; HRMS (FAB): m/z calcd for C34H42N3O7S (M++H) 636.2738, found 636.2759.
4.1.3. Boc-D-Cys(Tr)-L-Thr(OMs)-Gly-OMe (24)
Methanesulfonyl chloride (MsCl) (0.245 L, 3.2 mmol) was added slowly to a stirred
solution of 23 (745 mg, 1.2 mmol) in THF (23 mL) containing Et3N (0.98 mL, 7.0 mmol) at
0 °C under argon, and stirring was continued for 1 h at room temperature. The reaction was
quenched with 1 M HCl (5 mL) at 0 °C. The resulting mixture was extracted with CHCl3 (3 ×
30 mL), and the combined extracts were washed with brine (2 × 20 mL), then dried over MgSO4.
Concentration of the solvent in vacuo afforded a residue, which was purified by column
chromatography (hexane/EtOAc 2:1) to give 24 (785 mg, 94%) as a colorless amorphous solid.
[α]D27 −21.0 (c 1.0, CHCl3); 1H NMR (400 MHz, CDCl3): δ 1.35–1.44 (12H), 2.60 (1H, dd, J =
13.3, 5.3 Hz), 2.84 (1H, dd, J = 13.5, 7.7 Hz), 3.02 (3H, s), 3.21–3.34 (1H, m), 3.50 (1H, dd, J =
17.4, 5.3 Hz), 3.68 (3H, s), 4.06 (1H, dd, J = 17.4, 6.3 Hz), 4.60 (1H, d, J = 8.7 Hz), 4.87 (1H, d,
J = 4.4 Hz), 5.44 (1H, qd, J = 6.5, 1.4 Hz), 6.38 (1H, d, J = 8.7 Hz), 7.24–7.34 (9H, m), 7.40–
7.42 (6H, m), 7.69 (1H, br s); 13C NMR (100 MHz, CDCl3): δ 18.5, 28.2 (3C), 31.5, 33.0, 37.7,
41.0, 52.2, 54.8, 56.5, 67.6, 81.2, 127.2 (3C), 128.2 (6C), 129.4 (6C), 144.0 (3C), 155.8, 168.9,
169.4, 171.2; IR (neat): 3330, 3057, 2979, 2935, 1752, 1675, 1517, 1491, 1364, 1211, 1175, 975,
912, 742, 700 cm−1; HRMS (FAB): m/z calcd for C35H42N3O9S2 (M+–H) 712.2357, found
712.2363.
4.1.4. Boc-D-Cys(Tr)–Dhb–Gly-OMe (25)
1,4-Diazabicyclo[2.2.2]octane (DABCO) (478 mg, 4.3 mmol) was added to a stirred solution
of 24 (761 mg, 1.1 mmol) in THF (21 mL) at room temperature under argon. After 24 h,
concentration of the solvent in vacuo afforded a residue, which was purified by column
14

ACCEPTED MANUSCRIPT
chromatography (hexane/EtOAc 2:1) to give 25 (585 mg, 89%) as a colorless amorphous solid.
[α]D29 −3.6 (c 1.0, CHCl3); 1H NMR (400 MHz, CDCl3): δ 1.40 (9H, s), 1.69 (3H, d, J = 7.3 Hz),
2.64 (1H, dd, J = 12.7, 5.9 Hz), 2.80 (1H, dd, J = 12.7, 6.8 Hz), 3.64–3.73 (4H, m), 3.87 (2H, d,
J = 5.4 Hz), 4.90 (1H, d, J = 5.4 Hz), 6.86 (1H, q, J = 6.8 Hz), 7.12–7.22 (2H, m), 7.22–7.32
13
(9H, m), 7.41–7.43 (6H, m); C NMR (100 MHz, CDCl3): δ 13.5, 28.2 (3C), 33.2, 41.2, 52.1,
54.3, 67.4, 81.0, 127.1 (3C), 128.0, 128.2 (6C), 129.4 (6C), 134.0, 144.1 (3C), 155.9, 164,2,
169.4, 170.1; IR (neat): 3390, 3345, 3235, 3059, 3019, 2974, 2932, 1739, 1713, 1671, 1659,
1650, 1538, 1518, 1495, 1440, 1367, 1218, 1167, 1063, 910, 738, 700 cm−1; HRMS (FAB): m/z
calcd for C34H40N3O6S (M++H) 618.2632, found 618.2628.
4.1.5. H2N-D-Cys(Tr)–Dhb–Gly-OMe (15a)
A solution of BF3·Et2O (0.41 mL, 3.2 mmol) in CH2Cl2 (3.2 mL) was added dropwise to a
stirred solution of 25 (571 mg, 0.93 mmol) in CH2Cl2 (46 mL) at 0 °C, and stirring was
continued for 5 h at room temperature. The reaction was quenched with saturated aqueous
NaHCO3 (15 mL) at 0 °C. The resulting mixture was extracted with CHCl3 (3 × 40 mL), and the
combined extracts were washed with brine (2 × 20 mL), then dried over MgSO4. Concentration
of the solvent in vacuo afforded a residue, which was purified by column chromatography
(CHCl3/MeOH 20 :1) to give 15a (422 mg, 88%) as a colorless amorphous solid. [α]D27 +2.0 (c
1.0, CHCl3); 1H NMR (400 MHz, CDCl3): δ 1.62 (2H, br s), 1.67 (3H, d, J = 7.3 Hz), 2.65 (1H,
dd, J = 12.2, 4.4 Hz), 2.77 (1H, dd, J = 12.2, 4.4 Hz), 3.25 (1H, dd, J = 6.1, 4.4 Hz), 3.67 (3H,
s), 3.79 (1H, dd, J = 18.0, 5.4 Hz), 3.92 (1H, dd, J = 18.0, 5.9 Hz), 6.63 (1H, q, J = 7.3 Hz),
13
6.97 (1H, br t, J = 4.9 Hz), 7.19–7.30 (9H, m), 7.40–7.42 (6H, m), 8.53 (1H, br s); C NMR
(100 MHz, CDCl3): δ 13.5, 37.2, 41.1, 52.1, 53.9, 66.9, 126.8 (3C), 127.9 (6C), 128.8, 129.4
(6C), 130.7, 144.3 (3C), 164.6, 170.2, 172.1; IR (neat): 3304, 3057, 3015, 2951, 1749, 1669,
1642, 1525, 1488, 1443, 1212, 1033, 847, 745, 700 cm−1; HRMS (FAB): m/z calcd for
C29H32N3O4S (M++H) 518.2108, found 518.2127.
4.1.6. (5E,7S,11R,14S,17Z)-Methyl 17-ethylidene-11-isopropyl-7-(4-methoxybenzyloxy)-
9,12,15,18-tetraoxo-1,1,1-triphenyl-14-tritylthiomethyl-2-thia-10,13,16,19-tetraazahenicos-5-en
-21-oate (26a)
i-Pr2NEt (0.16 mL, 0.96 mmol) was added dropwise to a stirred solution of 15a (190 mg,
0.37 mmol) and 16 (234 mg, 0.37 mmol) in CH2Cl2/MeCN 2:1 (8.0 mL) containing
O-(7-azabenzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium hexafluorophosphate (HATU) (182
mg, 0.48 mmol) and 1-hydroxy-7-azabenzotriazole (HOAt) (65 mg, 0.48 mmol) at −20 °C
under argon. After 5 h, the reaction mixture was diluted with CHCl3 (80 mL). The organic layer
was washed successively with 10% aqueous HCl (2 × 20 mL), saturated aqueous NaHCO3 (2 ×
15

ACCEPTED MANUSCRIPT
20 mL) and brine (2 × 20 mL), then dried over MgSO4. Concentration of the solvent in vacuo
afforded a residue, which was purified by column chromatography (hexane/EtOAc 2:1) to give
26a (334 mg, 80%) as a colorless amorphous solid. [α]D29 0 (c 1.0, CHCl3); 1H NMR (400 MHz,
CDCl3): δ 0.71 (3H, d, J = 7.0 Hz), 0.99 (3H, d, J = 6.8 Hz), 1.69 (3H, d, J = 6.8 Hz), 1.91–2.27
(5H, m), 2.37–2.45 (2H, m), 2.54 (1H, dd, J = 12.9, 5.6 Hz), 2.93 (1H, dd, J = 12.7, 6.3 Hz),
3.64 (3H, s), 3.79 (3H, s), 3.85–4.03 (5H, m), 4.21 (1H, d, J = 11.2 Hz), 4.46 (1H, d, J = 10.7
Hz), 5.27 (1H, dd, J = 15.4, 8.0 Hz), 5.51 (1H, dt, J = 15.1, 6.8 Hz), 6.64 (1H, d, J = 6.8 Hz),
6.81–6.87 (3H, m), 7.07 (1H, t, J = 5.6 Hz), 7.17–7.29 (21H, m), 7.36–7.41 (12H, m), 7.68 (1H,
13
s); C NMR (100 MHz, CDCl3): δ 13.5, 17.7, 19.4, 29.4, 31.25, 31.30, 32.9, 41.3, 42.3, 52.0,
53.0, 55.3, 59.7, 66.6, 67.2, 70.0, 76.4, 113.9 (2C), 126.6 (3C), 127.0 (3C), 127.9 (6C), 128.1
(6C), 128.3, 129.4 (6C), 129.5 (7C), 129.6, 129.8 (2C), 133.6, 133.7, 144.1 (3C), 144.8 (3C),
159.4, 164.2, 168.7, 170.3, 171.9, 172.3; IR (neat): 3271, 3057, 3016, 2959, 2930, 1752, 1634,
1536, 1512, 1443, 1247, 1216, 1078, 1034, 768, 744, 700 cm−1; HRMS (FAB): m/z calcd for
C68H73N4O8S2 (M++H) 1137.4864, found 1137.4889.
4.1.7. (5E,7S,11R,14S,17Z)-Methyl 17-ethylidene-7-hydroxy-11-isopropyl-9,12,15,18-tetraoxo-
1,1,1-triphenyl-14-tritylthiomethyl-2-thia-10,13,16,19-tetraazahenicos-5-en-21-oate (27a)
2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) (112 mg, 0.49 mmol) was added in small
portions to a stirred solution of 26a (373 mg, 0.33 mmol) in CH2Cl2/H2O 9:1 (16 mL) at room
temperature. After 3 h, the mixture was diluted with CHCl3 (60 mL), and the organic layer was
washed with saturated aqueous NaHCO3 (2 × 20 mL) and brine (2 × 20 mL), then dried over
MgSO4. Concentration of the solvent in vacuo afforded a residue, which was purified by
column chromatography (hexane/EtOAc 1:5) to give 27a (299 mg, 90%) as a colorless
29
1
amorphous solid. [α]D +0.19 (c 1.0, CHCl3); H NMR (400 MHz, CDCl3): δ 0.93 (3H, d, J =
6.8 Hz), 0.98 (3H, d, J = 6.3 Hz), 1.71 (3H, d, J = 7.3 Hz), 1.97–2.31 (6H, m), 2.36 (1H, dd, J =
13.2, 2.0 Hz), 2.64 (1H, dd, J = 12.7, 4.9 Hz), 2.75 (1H, dd, J = 12.7, 7.8 Hz), 3.61 (3H, s), 3.79
(1H, br s), 3.84 (1H, dd, J = 18.0, 4.9 Hz), 4.02 (1H, dd, J = 18.0, 5.4 Hz), 4.11 (1H, t, J = 5.6
Hz), 4.20 (1H, td, J = 7.8, 5.4 Hz), 4.29–4.37 (1H, m), 5.35 (1H, dd, J = 15.1, 5.9 Hz), 5.49 (1H,
dt, J = 15.1, 6.3 Hz), 6.32 (1H, d, J = 5.9 Hz), 6.67 (1H, q, J = 7.1 Hz), 6.90 (1H, t, J = 5.4 Hz),
7.13–7.29 (19H, m), 7.35–7.41 (12H, m), 7.78 (1H, br s); 13C NMR (100 MHz, CDCl3): δ 13.2,
17.8, 19.5, 29.1, 31.3 (2C), 33.1, 41.3, 43.9, 52.2, 52.5, 60.2, 66.6, 67.0, 69.6, 126.6 (3C), 126.9
(3C), 127.8 (6C), 128.0 (6C), 129.0, 129.4 (6C), 129.5 (6C), 129.9, 132.5, 132.6, 144.3 (3C),
144.8 (3C), 164.7, 169.2, 170.7, 171.4, 173.1; IR (neat): 3284, 3057, 3017, 2962, 2928, 1747,
1634, 1531, 1489, 1443, 1215, 1033, 745, 700 cm−1; HRMS (FAB): m/z calcd for C60H65N4O7S2
(M++H) 1017.4289, found 1017.4280.
4.1.8. (5E,7S,11R,14S,17Z)-17-Ethylidene-7-hydroxy-11-isopropyl-9,12,15,18-tetraoxo-1,1,1-
16

ACCEPTED MANUSCRIPT
triphenyl-14-tritylthiomethyl-2-thia-10,13,16,19-tetraazahenicos-5-en-21-oic acid (14a)
LiOH·H2O (24.7 mg, 0.59 mmol) was added dropwise to a stirred solution of 27a (299 mg,
0.94 mmol) in THF/H2O 4:1 (15 mL) at 0 ºC. After 4 h, 10% aqueous HCl was added to the
mixture at 0 °C until pH was 6. The resulting mixture was extracted with EtOAc (3 × 30 mL),
and the combined extracts were washed with saturated aqueous NaHCO3 (2 × 15 mL) and brine
(2 × 15 mL), then dried over Na2SO4. Concentration of the solvent in vacuo afforded a residue,
which was purified by column chromatography (CHCl3/MeOH 6:1) to give 14a (289 mg, 97%)
as a colorless amorphous solid. [α]D29 +5.5 (c 1.0, CHCl3); 1H NMR (400 MHz, CDCl3/CD3OD
10 : 1): δ 0.83 (3H, d, J = 5.9 Hz), 0.85 (3H, d, J = 5.4 Hz), 1.65 (3H, d, J = 6.8 Hz), 1.96–2.09
(3H, m), 2.12–2.22 (2H, m), 2.33 (2H, d, J = 6.3 Hz), 2.49–2.71 (2H, m), 3.57–3.82 (2H, m),
4.00–4.19 (2H, m), 4.33 (1H, q, J = 5.9 Hz), 5.37 (1H, dd, J = 15.1, 5.9 Hz), 5.50 (1H, dt, J =
15.1, 6.3 Hz), 6.67 (1H, q, J = 5.9Hz), 7.18–7.28 (19H, m), 7.33–7.40 (15H, m); 13C NMR (100
MHz, CDCl3/CD3OD 10 : 1): δ 13.3, 17.7, 19.0, 30.0, 31.2, 31.3, 32.3, 43.1, 43.3, 53.1, 59.0,
66.4, 66.9, 68.9, 126.5 (3C), 126.8 (3C), 127.7 (6C), 127.9 (6C), 128.5, 129.3 (6C), 129.4 (6C),
131.9, 132.1, 132.6, 144.1 (3C), 144.7 (3C), 164.7, 169.8, 172.9, 176.0, 183.3; IR (neat): 3290,
3056, 3016, 2963, 2927, 1628, 1528, 1489, 1443, 1317, 1217, 1033, 744, 700 cm−1; HRMS
(FAB): m/z calcd for C59H63N4O7S2 (M++H) 1003.4133, found 1003.4124.
4.1.9. (9S,12R,16S,Z)-6-Ethylidene-12-isopropyl-16-[(E)-4-tritylthiobut-1-enyl]-9-
tritylthiomethyl-1-oxa-4,7,10,13-tetraazacyclohexadecane-2,5,8,11,14-pentaone (28a)
A solution of 14a (154 mg, 0.15 mmol) in CH2Cl2 (15 mL) was added very slowly to a
stirred solution of 2-methyl-6-nitrobenzoic anhydride (MNBA) (68.7 mg, 0.20 mmol) in CH2Cl2
(150 mL, 1.0 mM concentration) containing 4-dimethylaminopyridine (DMAP) (56.3 mg, 0.46
mmol) at toom temperature over 10 h. After 2 h, the mixture was diluted with CH2Cl2 (150 mL),
and the organic layer was washed with saturated aqueous NaHCO3 (2 × 50 mL), water (2 × 20
mL) and brine (2 × 50 mL), then dried over MgSO4. Concentration of the solvent in vacuo
afforded a residue, which was purified by column chromatography (EtOAc) to give 28a (97.3
28
1
mg, 64%) as a colorless amorphous solid. [α]D −24.0 (c 1.0, CHCl3); H NMR (400 MHz,
CDCl3/CD3OD 10:1): δ 0.86 (3H, d, J = 6.8 Hz), 0.94 (3H, d, J = 6.8 Hz), 1.64 (3H, d, J = 6.8
Hz), 1.94–2.26 (5H, m), 2.44 (1H, dd, J = 14.6, 2.0 Hz), 2.58 (1H, dd, J = 14.1, 10.2 Hz), 2.80
(1H, dd, J = 13.7, 4.9 Hz), 2.86 (1H, dd, J = 13.7, 8.8 Hz), 3.27–3.38 (1H, m), 3.71 (1H, dd, J =
16.6, 3.9 Hz), 4.24–4.41 (2H, m), 5.37 (1H, dd, J = 15.6, 6.3 Hz), 5.54–5.62 (2H, m), 6.85 (1H,
q, J = 6.8 Hz), 7.15–7.32 (20H, m), 7.38–7.42 (13H, m), 8.16 (1H, d, J = 6.3 Hz); 13C NMR
(100 MHz, CDCl3/CD3OD 10 : 1): δ 13.2, 17.2, 19.3, 30.9, 31.1, 31.2, 31.8, 41.3, 41.6, 53.9,
58.2, 66.5, 67.0, 72.2, 126.5 (3C), 126.6, (3C), 127.7 (6C), 127.8 (6C), 128.0, 129.27 (6C),
129.34 (7C), 132.6, 135.3, 144.2 (3C), 144.6 (3C), 164.2, 168.9, 169.0, 170.5, 173.0; IR (neat):
3316, 3057, 3018, 2964, 2929, 1749, 1667, 1659, 1650, 1644, 1538, 1488, 1444, 1217, 1193,
17

ACCEPTED MANUSCRIPT
1033, 768, 700 cm−1; HRMS (FAB): m/z calcd for C59H61N4O6S2 (M++H) 985.4027, found
985.4037.
4.1.10. FK-A1 (13a)
A solution of 28a (87.7 mg, 89 ꢀmol) in CH2Cl2/MeOH 9:1 (22 mL) was added dropwise to
a vigorously stirred solution of I2 (226 mg, 0.89 mmol) in CH2Cl2/MeOH 9:1 (178 mL, 0.5 mM
concentration) over 10 min at room temperature. After 10 min, the reaction was quenched with
0.2 M ascorbic acid/citric acid buffer (10 mL, adjusted to pH 4.0) at room temperature. The
resulting mixture was diluted with CH2Cl2 (100 mL) and water (50 mL). The organic layer was
washed with brine (2 × 50 mL) and dried over MgSO4. Concentration of the solvent in vacuo
afforded a residue, which was purified by column chromatography (CHCl3/MeOH, 15:1) to give
13a (42.8 mg, 97%) as a colorless amorphous solid. [α]D28 +54.9 (c 1.0, CHCl3); 1H NMR (400
MHz, CDCl3/CD3OD 10:1): δ 1.11 (3H, d, J = 6.3 Hz), 1.12 (3H, d, J = 6.3 Hz), 1.76 (3H, d, J
= 6.8 Hz), 2.24 (1H, dq, J = 12.7, 6.8 Hz), 2.41–2.53 (1H, m), 2.66–2.78 (2H, m), 2.81 (1H, dd,
J = 13.2, 6.3 Hz), 2.93 (1H, ddd, J = 13.2, 8.8, 3.4 Hz), 3.13–3.25 (3H, m), 3.97 (1H, dd, J = 4.9,
3.4 Hz), 4.10 (1H, dd, J = 18.0, 3.9 Hz), 4.26 (1H, dd, J = 17.6, 4.9 Hz), 4.82 (1H, q, J = 5.4
Hz), 5.72–5.91 (3H, m), 6.71 (1H, q, J = 7.3 Hz), 7.03 (1H, t, J = 4.1 Hz), 7.67 (1H, d, J = 6.3
13
Hz), 8.01 (1H, d, J = 2.9 Hz), 8.55 (1H, s); C NMR (100 MHz, CDCl3/CD3OD 10:1): δ 13.6,
18.8, 18.9, 29.2, 30.4, 35.5, 37.3, 38.0, 42.8, 56.3, 62.5, 69.7, 128.0, 129.0, 129.5, 132.6, 164.2,
166.9, 168.7, 171.6, 172.1; IR (neat): 3434, 3356, 3011, 2965, 2929, 1748, 1650, 1519, 1406,
1252, 979, 753 cm−1; HRMS (FAB): m/z calcd for C21H31N4O6S2 (M++H) 499.1680, found
499.1695.
4.1.11. Fmoc-Gly–Gly-OMe (30b)
Compound 30b was synthesized from Gly (18b) (300 mg, 4.0 mmol) in a manner similar to
that described for the synthesis of 21. Purification by column chromatography (CHCl3/MeOH
1
30:1) gave 30b (1.12 g, 78%, 2 steps) as colorless amorphous solid. H NMR (400 MHz,
CDCl3): δ 3.76 (3H, s), 3.93 (2H, d, J = 4.9 Hz), 4.06 (2H, d, J = 4.9 Hz), 4.23 (1H, t, J = 6.8
Hz), 4.45 (2H, d, J = 6.8 Hz), 5.47 (1H, br s), 6.48 (1H, br s), 7.31 (2H, td, J = 7.3, 1.0 Hz), 7.40
(2H, t, J = 7.3 Hz), 7.59 (2H, d, J = 7.3 Hz), 7.77 (2H, d, J = 7.3 Hz); 13C NMR (100 MHz,
CDCl3): δ 41.1, 44.3, 47.1, 52.4, 67.2, 120.0 (2C), 125.0 (2C), 127.0 (2C), 127.7 (2C), 141.3
(2C), 143.7 (2C), 156.6, 169.3, 170.1; IR (KBr): 3387, 3294, 3076, 2948, 1742, 1715, 1653,
1555, 1536, 1285, 1243, 1234, 1166, 756, 733 cm−1; HRMS (EI): m/z calcd for C20H20N2O5
(M+) 368.1372, found 368.1364.
4.1.12. Boc-D-Cys(Tr)–Gly–Gly-OMe (32b)
Compound 32b was synthesized from 30b (500 mg, 1.4 mmol) in a manner similar to that
18

ACCEPTED MANUSCRIPT
described for the synthesis of 23. Purification by column chromatography (CHCl3/MeOH 20:1)
26
gave 32b (588 mg, 73%, 2 steps) as a colorless amorphous solid. [α]D −14.4 (c 1.0, CHCl3).
1H NMR (400 MHz, CDCl3): δ 1.40 (9H, s), 2.63 (1H, dd, J = 12.9, 5.4 Hz), 2.76 (1H, dd, J =
12.7, 7.3 Hz), 3.65–3.73 (4H, m), 3.89 (2H, d, J = 5.9 Hz), 3.95 (2H, t, J = 5.4 Hz), 4.79 (1H, br
d, J = 4.4 Hz), 6.51 (1H, br t, J = 5.9 Hz), 7.00 (1H, br s), 7.22–7.32 (9H, m), 7.40–7.42 (6H,
m); 13C NMR (100 MHz, CDCl3): δ 28.2 (3C), 33.6, 40.8, 42.9, 52.2, 54.0, 67.3, 80.7, 127.0
(3C), 128.1 (6C), 129.5 (6C), 144.2 (3C), 155.6, 169.0, 169.9, 170.9; IR (neat): 3317, 3008,
2978, 1749, 1667, 1521, 1490, 1443, 1367, 1247, 1214, 1166, 1033, 852, 752, 700 cm−1; HRMS
(FAB): m/z calcd for C32H38N3O6S (M++H) 592.2476, found 592.2475.
4.1.13. H2N-D-Cys(Tr)–Gly–Gly-OMe (15b)
Compound 15b was synthesized from 32b (1.46 g, 2.5 mmol) in a manner similar to that
described for the synthesis of 15a. Purification by column chromatography (CHCl3/MeOH
28
1
20:1) gave 15b (873 mg, 72%) as a colorless amorphous solid. [α]D −13.8 (c 1.1, CHCl3); H
NMR (400 MHz, CDCl3): δ 1.48 (2H, br s), 2.69 (2H, d, J = 5.9 Hz), 3.16 (1H, t, J = 5.9 Hz),
3.70 (3H, s), 3.80–3.99 (4H, m), 6.82 (1H, t, J = 5.9 Hz), 7.20–7.31 (9H, m), 7.41–7.43 (6H, m),
7.69 (1H, t, J = 5.9 Hz); 13C NMR (100 MHz, CDCl3): δ 37.3, 40.8, 42.8, 52.2, 53.8, 66.9, 126.8
(3C), 128.0 (6C), 129.5 (6C), 144.4 (3C), 169.2, 169.9, 173.8; IR (neat): 3353, 3317, 3057,
2951, 1748, 1660, 1520, 1489, 1443, 1372, 1212, 1183, 1033, 984, 886, 848, 745, 700 cm−1;
HRMS (FAB): m/z calcd for C27H30N3O4S (M++H) 492.1952, found 492.1942.
4.1.14. (7S,11R,14S,E)-Methyl 11-isopropyl-7-(4-methoxybenzyloxy)-9,12,15,18-tetraoxo-1,1,1-
triphenyl-14-tritylthiomethyl-2-thia-10,13,16,19-tetraazahenicos-5-en-21-oate (26b)
Compound 26b was synthesized from 15b (77.1 mg, 0.16 mmol) in a manner similar to that
described for the synthesis of 26a. Purification by column chromatography (CHCl3/MeOH
27
1
30:1) gave 26b (152 mg, 87%) as a colorless amorphous solid. [α]D +10.4 (c 1.0, CHCl3); H
NMR (400 MHz, CDCl3): δ 0.73 (3H, d, J = 6.8 Hz), 0.82 (3H, d, J = 6.8 Hz), 1.95 (1H, dq, J =
13.2, 6.8 Hz), 2.06–2.16 (2H, m), 2.21–2.25 (2H, m), 2.44 (2H, d, J = 5.9 Hz), 2.58 (1H, dd, J =
13.2, 5.9 Hz), 2.78 (1H, dd, J = 13.2, 6.8 Hz), 3.65 (3H, s), 3.75 (3H, s), 3.81–3.89 (3H, m),
3.96 (2H, dt, J = 18.0, 6.3 Hz), 4.08 (1H, q, J = 6.3 Hz), 4.14 (1H, t, J = 6.8 Hz), 4.21 (1H, d, J
= 10.7 Hz), 4.41 (1H, d, J = 10.7 Hz), 5.29 (1H, dd, J = 15.1, 8.3 Hz), 5.54 (1H, dt, J = 15.1, 6.8
Hz), 6.81 (2H, d, J = 8.3 Hz), 6.97 (1H, d, J = 7.3 Hz), 7.02 (1H, t, J = 5.6 Hz), 7.15–7.30 (22H,
m), 7.35–7.42 (12H, m); 13C NMR (100 MHz, CDCl3): δ 17.8, 19.3, 29.9, 31.2, 31.3, 33.3, 40.8,
42.3, 42.8, 52.1, 52.6, 55.2, 59.0, 66.5, 67.0, 70.0, 76.5, 113.7 (2C), 126.5 (3C), 126.8 (3C),
127.8 (6C), 128.0 (6C), 129.4 (6C), 129.5 (6C), 129.6, 129.8 (2C), 129.9, 133.2, 144.2 (3C),
144.7 (3C), 159.3, 169.1, 169.9, 170.2, 171.5, 171.6; IR (neat): 3287, 3082, 3058, 3016, 2960,
2931, 1744, 1691, 1634, 1513, 1490, 1443, 1247, 1216, 1181, 1079, 1034, 975, 822, 752, 700
19

ACCEPTED MANUSCRIPT
cm−1; HRMS (FAB): m/z calcd for C66H71N4O8S2 (M++H) 1111.4708, found 1111.4731.
4.1.15. (7S,11R,14S,E)-Methyl 7-hydroxy-11-isopropyl-9,12,15,18-tetraoxo-1,1,1-triphenyl-14-
tritylthiomethyl-2-thia-10,13,16,19-tetraazahenicos-5-en-21-oate (27b)
Compound 27b was synthesized from 26b (108 mg, 97 ꢀmol) in a manner similar to that
described for the synthesis of 27a. Purification by column chromatography (CHCl3/MeOH
30:1) gave 27b (95.6 mg, 99%) as a colorless amorphous solid. [α]D26 −17.0 (c 1.0, CHCl3); 1H
NMR (400 MHz, CDCl3): δ 0.89 (3H, d, J = 6.8 Hz), 0.93 (3H, d, J = 6.8 Hz), 2.03–2.08 (2H,
m), 2.12–2.22 (3H, m), 2.28 (1H, dd, J = 13.7, 9.8 Hz), 2.39 (1H, dd, J = 13.7, 2.9 Hz), 2.61–
2.69 (2H, m), 3.60 (3H, s), 3.73 (1H, dd, J = 17.1, 5.4 Hz), 3.79 (1H, dd, J = 18.0, 5.4 Hz), 3.89
(1H, br s), 3.96 (1H, dd, J = 18.0, 5.9 Hz), 4.03 (1H, dd, J = 16.6, 6.3 Hz), 4.18 (1H, t, J = 6.3
Hz), 4.23 (1H, q, J = 7.1 Hz), 4.33–4.43 (1H, m), 5.38 (1H, dd, J = 15.1, 5.9 Hz), 5.53 (1H, dt, J
13
= 15.1, 6.3 Hz), 6.54 (1H, d, J = 6.8 Hz), 7.14–7.28 (20H, m), 7.31–7.41 (13H, m); C NMR
(100 MHz, CDCl3): δ 17.7, 19.4, 29.4, 31.2 (2C), 33.4, 40.8, 43.0, 43.7, 52.2, 52.3, 59.5, 66.5,
66.8, 69.4, 126.5 (3C), 126.8 (3C), 127.8 (6C), 127.9 (6C), 129.4 (6C), 129.5 (6C), 129.7, 132.6,
144.2 (3C), 144.7 (3C), 169.5, 170.46, 170.50, 171.3, 172.8; IR (neat): 3289, 3082, 3058, 3016,
2962, 2929, 1744, 1635, 1531, 1489, 1443, 1216, 1183, 1082, 1033, 973, 851, 752, 700 cm−1;
HRMS (FAB): m/z calcd for C58H63N4O7S2 (M++H) 991.4133, found 991.4120.
4.1.16. (7S,11R,14S,E)-7-Hydroxy-11-isopropyl-9,12,15,18-tetraoxo-1,1,1-triphenyl-14-
tritylthiomethyl-2-thia-10,13,16,19-tetraazahenicos-5-en-21-oic acid (14b)
Compound 14b was synthesized from 27b (106 mg, 0.11 mmol) in a manner similar to that
described for the synthesis of 14a. Purification by column chromatography (CHCl3/MeOH 5:1)
gave 14b (79.5 mg, 76%) as a colorless amorphous solid. [α]D29 −12.5 (c 1.0, CHCl3). 1H NMR
(400 MHz, CD3OD/CDCl3 5:1): δ 0.93 (3H, d, J = 5.8 Hz), 0.94 (3H, d, J = 6.3 Hz), 1.98–2.07
(2H, m), 2.09–2.21 (3H, m), 2.35 (1H, dd, J = 13.5, 4.3 Hz), 2.46 (1H, dd, J = 13.5, 9.2 Hz),
2.62 (1H, dd, J = 12.6, 8.7 Hz), 2.70 (1H, dd, J = 12.6, 5.8 Hz), 3.74 (1H, d, J = 16.9 Hz), 3.76
(2H, s), 3.96 (1H, d, J = 16.9 Hz), 4.12–4.15 (2H, m), 4.36–4.40 (1H, m), 5.42 (1H, dd, J = 15.0,
6.3 Hz), 5.54 (1H, dt, J = 15.0, 6.8 Hz), 7.13–7.24 (18H, m), 7.24–7.39 (12H, m); 13C NMR
(100 MHz, CD3OD/CDCl3 5:1, 40 °C): δ 18.4, 19.7, 30.9, 32.2, 32.4, 34.0, 42.1, 43.4, 44.1,
54.0, 60.6, 67.6, 67.9, 70.2, 127.5 (3C), 127.7, (3C), 128.6 (6C), 128.8 (6C), 130.4 (6C), 130.46,
130.50 (6C), 134.1, 145.5 (3C), 146.0 (3C), 171.3, 172.2, 173.3, 173.5, 174.5; IR (neat): 3299,
3057, 3017, 2964, 2928, 1725, 1635, 1536, 1488, 1444, 1218, 1033, 971, 752, 700 cm−1; HRMS
(FAB): m/z calcd for C57H61N4O7S2 (M++H) 977.3976, found 977.3962.
4.1.17. (9S,12R,16S)-12-Isopropyl-16-[(E)-4-tritylthiobut-1-enyl]-9-tritylthiomethyl-1-oxa-
4,7,10,13-tetraazacyclohexadecane-2,5,8,11,14-pentaone (28b)
20

ACCEPTED MANUSCRIPT
Compound 28b was synthesized from 14b (72.1 mg, 74 ꢀmol) in a manner similar to that
described for the synthesis of 28a. Purification by column chromatography (CHCl3/MeOH
30:1) gave 28b (59.9 mg, 85%) as a colorless amorphous solid. [α]D29 −24.5 (c 1.0, CHCl3); 1H
NMR (400 MHz, CDCl3): δ 0.79 (3H, d, J = 6.8 Hz), 0.91 (3H, d, J = 6.3 Hz), 1.85–2.03 (2H,
m), 2.08–2.24 (3H, m), 2.42 (1H, dd, J = 13.7, 9.8 Hz), 2.47–2.54 (1H, m), 2.75 (1H, dd, J =
13.2, 3.9 Hz), 2.86 (1H, dd, J = 13.2, 9.8 Hz), 3.32–3.42 (1H, m), 3.42–3.55 (2H, m), 4.30 (1H,
dd, J = 17.1, 8.3 Hz), 4.46 (1H, dd, J = 16.6, 9.3 Hz), 4.72 (1H, dd, J = 9.3, 3.9 Hz), 5.34 (1H,
dd, J = 15.6, 6.3 Hz), 5.49–5.64 (2H, m), 6.30 (1H, d, J = 8.8 Hz), 7.12–7.28 (20H, m), 7.33–
7.39 (12H, m), 8.08 (1H, d, J = 5.9 Hz); 13C NMR (100 MHz, CDCl3): δ 16.7, 19.6, 30.9, 31.4,
31.6, 32.4, 40.9, 42.7, 43.6, 54.8, 56.6, 66.7, 67.0, 72.4, 126.6 (3C), 126.7, (3C), 127.6, 127.8
(6C), 127.9 (6C), 129.4 (6C), 129.5 (6C), 132.5, 144.5 (3C), 144.7 (3C), 169.8, 169.9, 170.2,
170.4, 172.7; IR (neat): 3300, 3057, 3018, 2964, 2931, 1737, 1661, 1539, 1489, 1444, 1214,
1033, 974, 744, 700 cm−1; HRMS (FAB): m/z calcd for C57H59N4O6S2 (M++H) 959.3871, found
959.3891.
4.1.18. FK-A2 (13b)
Compound 13b was synthesized from 28b (59.9 mg, 62 ꢀmol) in a manner similar to that
described for the synthesis of 13a. Purification by column chromatography (CHCl3/MeOH
10:1) gave 13b (22.0 mg, 75%) as a colorless amorphous solid. [α]D24 −39.7 (c 0.70, CHCl3); 1H
NMR (400 MHz, CDCl3): δ 1.09 (3H, d, J = 3.4 Hz), 1.10 (3H, d, J = 2.9 Hz), 2.30 (1H, dq, J =
12.1, 6.3 Hz), 2.45–2.60 (1H, m), 2.63–2.80 (2H, m), 2.88–2.99 (2H, m), 3.08 (1H, ddd, J =
13.0, 9.7, 2.9 Hz), 3.18 (1H, dd, J = 15.0, 4.3 Hz), 3.27 (1H, dd, J = 15.5, 10.1 Hz), 3.51 (1H,
dd, J = 16.4, 3.4 Hz), 4.04 (1H, t, J = 3.9 Hz), 4.10–4.21 (2H, m), 4.44 (1H, dd, J = 16.4, 8.7
Hz), 4.76–4.84 (1H, m), 5.68–5.82 (2H, m), 5.82–5.95 (1H, m), 6.78 (1H, d, J = 2.9 Hz), 6.95
(1H, t, J = 4.3 Hz), 7.49 (1H, d, J = 6.8 Hz), 7.68 (1H, dd, J = 8.2, 2.9 Hz); 13C NMR (100 MHz,
CDCl3): δ 18.6, 19.4, 29.3, 31.1, 37.1, 37.5, 39.2, 42.3, 43.5, 55.9, 62.3, 69.7, 129.1, 129.5,
167.0, 168.9, 170.3, 171.2, 171.3; IR (neat): 3298, 3011, 2965, 2932, 1742, 1658, 1529, 1258,
1191, 1031, 979, 754 cm−1; HRMS (EI): m/z calcd for C19H28N4O6S2 (M+) 472.1450, found
472.1463.
4.1.19. Fmoc-
D-Ala–Gly-OMe (30c)
Compound 30c was synthesized from
D
-alanine (D-Ala) (18c) (500 mg, 5.6 mmol) in a
manner similar to that described for the synthesis of 21. Purification by column chromatography
29
(hexane/EtOAc 2:3) gave 30c (1.98 g, 92%, 2 steps) as a colorless amorphous solid. [α]D
1
+13.5 (c 1.1, CHCl3); H NMR (400 MHz, CDCl3): δ 1.40 (3H, d, J = 6.8 Hz), 3.70 (3H, s),
4.01 (2H, d, J = 4.8 Hz), 4.19 (1H, t, J = 6.8 Hz), 4.29–4.40 (1H, m), 4.39 (2H, d, J = 6.8 Hz),
5.61 (1H, d, J = 7.7 Hz), 6.82 (1H, br s), 7.29 (2H, t, J = 7.2 Hz), 7.38 (2H, t, J = 7.2 Hz), 7.57
21

ACCEPTED MANUSCRIPT
13
(2H, dd, J = 7.2, 3.6 Hz), 7.74 (2H, d, J = 7.7 Hz); C NMR (100 MHz, CDCl3): δ 18.5, 41.2,
47.1, 50.4, 52.4, 67.1, 120.0 (2C), 125.0 (2C), 127.1 (2C), 127.7 (2C), 141.3 (2C), 143.7 (2C),
156.0, 170.1, 172.5; IR (KBr): 3323, 3293, 3067, 2982, 2951, 2940, 2890, 1731, 1720, 1693,
1648, 1541, 1449, 1329, 1301, 1266, 1254, 1105, 1089, 1051, 1023, 758, 741, 670, 645 cm−1;
HRMS (EI): m/z calcd for C21H22N2O5 (M+) 382.1529, found 382.1526.
4.1.20. Boc-D-Cys(Tr)–D-Ala–Gly-OMe (32c)
Compound 32c was synthesized from 30c (200 mg, 0.52 mmol) in a manner similar to that
described for the synthesis of 23. Purification by column chromatography (hexane/EtOAc 1:2)
gave 32c (260 mg, 82%, 2 steps) as a colorless amorphous solid. [α]D24 +5.3 (c 1.0, CHCl3); 1H
NMR (400 MHz, CDCl3): δ 1.36 (3H, d, J = 6.8 Hz), 1.41 (9H, s), 2.61 (1H, dd, J = 12.9, 5.9
Hz), 2.70 (1H, dd, J = 12.7, 6.8 Hz), 3.69 (3H, s), 3.70–3.84 (2H, m), 3.96 (1H, dd, J = 17.6, 5.9
Hz), 4.47 (1H, dq, J = 7.4, 7.2 Hz), 4.81 (1H, br s), 6.35 (1H, br s), 6.96 (1H, br s), 7.21–7.32
13
(9H, m), 7.41–7.43 (6H, m); C NMR (100 MHz, CDCl3): δ 17.9, 28.1 (3C), 33.7, 40.8, 48.6,
52.0, 53.4, 67.0, 80.2, 126.8 (3C), 127.9 (6C), 129.3 (6C), 144.1 (3C), 155.3, 169.8, 170.2,
172.0; IR (neat): 3285, 3058, 2978, 2928, 1756, 1680, 1645, 1523, 1491, 1445, 1367, 1209,
1167, 1049, 1020, 851, 751, 700 cm−1; HRMS (FAB): m/z calcd for C33H40N3O6S (M++H)
606,2632, found 606.2641.
4.1.21. H2N-D-Cys(Tr)–D-Ala–Gly-OMe (15c)
Compound 15c was synthesized from 32c (70.0 mg, 0.12 mmol) in a manner similar to that
described for the synthesis of 15a. Purification by column chromatography (CHCl3/MeOH
20:1) gave 15c (52.0 mg, 89%) as a colorless amorphous solid. [α]D25 +26.9 (c 1.0, CHCl3); 1H
NMR (400 MHz, CDCl3): δ 1.35 (3H, d, J = 6.8 Hz), 1.42 (2H, br s), 2.58 (1H, dd, J = 12.6, 8.2
Hz), 2.70 (1H, dd, J = 12.6, 4.3 Hz), 3.00 (1H, dd, J = 8.2, 4.3 Hz), 3.71 (3H, s), 3.89 (1H, dd, J
= 17.9, 5.3 Hz), 3.98 (1H, dd, J = 17.9, 5.3 Hz), 4.42 (1H, quint, J = 7.2 Hz), 6.87 (1H, t, J = 5.3
Hz), 7.20–7.31 (9H, m), 7.39–7.45 (7H, m); 13C NMR (100 MHz, CDCl3): δ 17.4, 37.1, 41.0,
48.3, 52.2, 53.9, 67.0, 126.8 (3C), 128.0 (6C), 129.5 (6C), 144.5 (3C), 170.0, 172.2, 173.4; IR
(neat): 3311, 3057, 3016, 2952, 1751, 1652, 1514, 1444, 1369, 1211, 1182, 1032, 982, 886, 848,
746, 700 cm−1; HRMS (FAB): m/z calcd for C28H32N3O4S (M++H) 506.2108, found 506.2106.
4.1.22. (7S,11R,14S,17R,E)-Methyl 11-isopropyl-7-(4-methoxybenzyloxy)-17-methyl-
9,12,15,18-tetraoxo-1,1,1-triphenyl-14-tritylthiomethyl-2-thia-10,13,16,19-tetraazahenicos-
5-en-21-oate (26c)
Compound 26c was synthesized from 15c (101 mg, 0.20 mmol) in a manner similar to that
described for the synthesis of 26a. Purification by column chromatography (CHCl3/MeOH
27
1
30:1) gave 26c (193 mg, 86%) as a colorless amorphous solid. [α]D −0.39 (c 1.0, CHCl3); H
22

ACCEPTED MANUSCRIPT
NMR (400 MHz, CDCl3): δ 0.72 (3H, d, J = 6.8 Hz), 0.79 (3H, d, J = 6.8 Hz), 1.33 (3H, d, J =
6.8 Hz), 1.96 (1H, dq, J = 13.4, 6.7 Hz), 2.05–2.15 (2H, m), 2.21–2.25 (2H, m), 2.38–2.52 (3H,
m), 2.70 (1H, dd, J = 13.2, 7.3 Hz), 3.65 (3H, s), 3.76 (3H, s), 3.89 (2H, d, J = 5.4 Hz), 4.00 (1H,
td, J = 4.4, 2.9 Hz), 4.08 (1H, q, J = 6.3 Hz), 4.19 (1H, d, J = 10.7 Hz), 4.30 (1H, t, J = 7.1 Hz),
4.34 (1H, d, J = 10.7 Hz), 4.72 (1H, dq, J = 7.2, 7.1 Hz), 5.28 (1H, dd, J = 16.4, 7.8 Hz), 5.51
(1H, dt, J = 15.1, 6.8 Hz), 6.81 (2H, d, J = 8.3 Hz), 6.91 (1H, br s), 7.14–7.28 (22H, m), 7.35–
7.42 (12H, m), 7.49 (1H, br s); 13C NMR (100 MHz, CDCl3): δ 17.9, 18.3, 19.2, 30.6, 31.2, 31.3,
34.0, 41.0, 42.5, 48.7, 52.0, 52.1, 55.2, 58.8, 66.5, 67.0, 69.9, 76.4, 113.7 (2C), 126.6 (3C),
126.8 (3C), 127.8 (6C), 128.0 (6C), 129.4 (6C), 129.5 (6C), 129.7, 129.8 (2C), 130.0, 133.1,
144.2 (3C), 144.8 (3C), 159.2, 169.4, 170.0, 171.3 (2C), 172.3; IR (neat): 3277, 3082, 3059,
3029, 3018, 2960, 2931, 1752, 1692, 1631, 1537, 1489, 1444, 1246, 1215, 1181, 1080, 1034,
976, 822, 752, 700 cm−1; HRMS (FAB): m/z calcd for C67H73N4O8S2 (M++H) 1125.4864, found
1125.4873.
4.1.23. (7S,11R,14S,17R,E)-Methyl 7-hydroxy-11-isopropyl-17-methyl-9,12,15,18-tetraoxo-
1,1,1-triphenyl-14-tritylthiomethyl-2-thia-10,13,16,19-tetraazahenicos-5-en-21-oate (27c).
Compound 27c was synthesized from 26c (193 mg, 0.17 mmol) in a manner similar to that
described for the synthesis of 27a. Purification by column chromatography (CHCl3/MeOH
27
1
40:1) gave 27c (140 mg, 81%) as a colorless amorphous solid. [α]D −1.6 (c 1.0, CHCl3); H
NMR (400 MHz, CDCl3): δ 0.89 (3H, d, J = 7.3 Hz), 0.96 (3H, d, J = 6.8 Hz), 1.40 (3H, d, J =
7.3 Hz), 2.05–2.10 (2H, m), 2.20–2.28 (4H, m), 2.36 (1H, dd, J = 13.7, 2.9 Hz), 2.53–2.63 (2H,
m), 2.92 (1H, br s), 3.67 (3H, s), 3.81 (1H, dd, J = 18.0, 5.4 Hz), 3.96 (1H, dd, J = 18.0, 5.9 Hz),
4.10–4.20 (2H, m), 4.29–4.33 (1H, m), 4.42 (1H, quint, J = 7.3 Hz), 5.36 (1H, dd, J = 15.6, 6.3
Hz), 5.39 (1H, dt, J = 15.6, 6.3 Hz), 6.20 (1H, d, J = 6.8 Hz), 6.81 (1H, d, J = 7.8 Hz), 7.01 (1H,
t, J = 4.9 Hz), 7.05 (1H, d, J = 7.3 Hz), 7.17–7.30 (18H, m), 7.36–7.42 (12H, m); 13C NMR (100
MHz, CDCl3): δ 17.9, 18.1, 19.2, 30.4, 31.30, 31.32, 33.9, 41.0, 43.5, 49.0, 52.0, 52.2, 58.8,
66.6, 66.8, 69.6, 126.6 (3C), 126.9 (3C), 127.8 (6C), 128.0 (6C), 129.4 (6C), 129.5 (6C), 130.0,
132.5, 144.2 (3C), 144.8 (3C), 169.7, 170.3, 171.3, 172.2, 172.3; IR (neat): 3279, 3083, 3060,
3030, 3017, 2963, 2928, 1746, 1691, 1632, 1537, 1488, 1444, 1217, 1183, 1081, 1033, 971, 853,
751, 699 cm−1; HRMS (FAB): m/z calcd for C59H65N4O7S2 (M++H) 1005.4289, found
1005.4321.
4.1.24. (7S,11R,14S,17R,E)-7-Hydroxy-11-isopropyl-17-methyl-9,12,15,18-tetraoxo-1,1,1-
triphenyl-14-tritylthiomethyl-2-thia-10,13,16,19-tetraazahenicos-5-en-21-oic acid (14c)
Compound 14c was synthesized from 27c (106 mg, 0.11 mmol) in a manner similar to that
described for the synthesis of 14a. Purification by column chromatography (CHCl3/MeOH 5:1)
gave 14c (134 mg, 97%) as a colorless amorphous solid. [α]D29 −15.4 (c 1.0, CHCl3); 1H NMR
23

ACCEPTED MANUSCRIPT
(400 MHz, CD3OD/CDCl3 3:1): δ 0.91 (3H, d, J = 6.8 Hz), 0.93 (3H, d, J = 7.3 Hz), 1.34 (3H, d,
J = 6.8 Hz), 2.01–2.06 (2H, m), 2.10–2.21 (3H, m), 2.36 (1H, dd, J = 13.7, 4.4 Hz), 2.46 (1H,
dd, J = 13.7, 8.8 Hz), 2.57 (1H, dd, J = 11.7, 8.8 Hz), 2.64 (1H, dd, J = 12.2, 5.9 Hz), 3.69 (1H,
d, J = 17.6 Hz), 3.79 (1H, d, J = 18.0 Hz), 4.16 (1H, d, J = 5.9 Hz), 4.18–4.26 (1H, m), 4.30–
4.43 (2H, m), 5.43 (1H, dd, J = 15.6, 5.9 Hz), 5.53 (1H, dt, J = 15.1, 6.3 Hz), 7.14–7.24 (18H,
m), 7.33–7.39 (12H, m); 13C NMR (100 MHz, CD3OD/CDCl3 3:1): δ 18.0, 18.3, 19.7, 30.9,
32.2, 32.3, 34.0, 42.5, 44.1, 50.2, 53.7, 60.4, 67.5, 67.8, 70.3, 127.5 (3C), 127.7, (3C), 128.6
(6C), 128.8 (6C), 130.36 (6C), 130.44 (6C), 130.5, 133.9, 145.4 (3C), 145.9 (3C), 171.4, 173.4,
173.9, 174.1, 174.2; IR (neat): 3291, 3057, 3017, 2965, 2929, 1727, 1636, 1527, 1490, 1444,
1405, 1217, 1034, 971, 754, 700 cm−1; HRMS (FAB): m/z calcd for C58H62N4O7S2Na (M++Na)
1013.3952, found 1013.3991.
4.1.25. (6R,9S,12R,16S)-12-Isopropyl-6-methyl-16-[(E)-4-tritylthiobut-1-enyl]-9-
tritylthiomethyl-1-oxa-4,7,10,13-tetraazacyclohexadecane-2,5,8,11,14-pentaone (28c)
Compound 28c was synthesized from 14c (134 mg, 0.14 mmol) in a manner similar to that
described for the synthesis of 28a. Purification by column chromatography (CHCl3/MeOH
27
1
40:1) gave 28c (108 mg, 85%) as a colorless amorphous solid. [α]D −2.2 (c 1.0, CHCl3); H
NMR (400 MHz, CDCl3): δ 0.85 (3H, d, J = 6.8 Hz), 0.92 (3H, d, J = 6.8 Hz), 1.44 (3H, d, J =
7.3 Hz), 1.92–2.17 (5H, m), 2.38 (1H, dd, J = 14.1, 9.8 Hz), 2.43–2.52 (1H, m), 2.64 (1H, dd, J
= 12.7, 5.4 Hz), 2.81 (1H, dd, J = 12.2, 8.8 Hz), 3.56 (1H, q, J = 6.8 Hz), 3.90–4.02 (2H, m),
4.16 (1H, quint, J = 7.3 Hz), 4.25 (1H, t, J = 7.1 Hz), 5.31 (1H, dd, J = 15.6, 6.3 Hz), 5.49–5.61
(2H, m), 6.33 (1H, d, J = 8.3 Hz), 7.16–7.30 (20H, m), 7.36–7.39 (12H, m), 7.81 (1H, br s); 13C
NMR (100 MHz, CDCl3, 40 °C): δ 16.7, 18.0, 19.6, 30.9, 31.1, 31.4, 31.8, 41.7, 42.4, 50.2, 56.0,
58.4, 66.7, 67.2, 72.1, 126.6 (3C), 126.9 (3C), 127.9 (6C), 128.0, 128.1 (6C), 129.4 (6C), 129.6
(6C), 132.5, 144.4 (3C), 144.9 (3C), 168.9, 169.8, 170.2, 172.2, 173.0; IR (neat): 3307, 3057,
3016, 2964, 2932, 1745, 1660, 1651, 1538, 1490, 1444, 1265, 1215, 1033, 972, 750, 700 cm−1.
HRMS (FAB): m/z calcd for C58H60N4O6S2Na (M++Na) 995.3846, found 995.3847.
4.1.26. FK-A3 (13c)
Compound 13c was synthesized from 28c (108 mg, 0.11 mmol) in a manner similar to that
described for the synthesis of 13a. Purification by column chromatography (CHCl3/MeOH
20:1) gave 13c (42.4 mg, 79%) as a colorless amorphous solid. [α]D25 +19.8 (c 1.0, CHCl3); 1H
NMR (400 MHz, CDCl3): δ 1.09 (6H, d, J = 5.9 Hz), 1.54 (3H, d, J = 6.8 Hz), 2.23 (1H, dq, J =
12.7, 6.8 Hz), 2.49–2.59 (1H, m), 2.66–2.77 (2H, m), 2.94–3.03 (3H, m), 3.11 (1H, ddd, J =
13.2, 9.3, 3.4 Hz), 3.33 (1H, dd, J = 16.1, 11.2 Hz), 3.77 (1H, q, J = 6.8 Hz), 3.88 (1H, dd, J =
17.6, 2.0 Hz), 4.06 (1H, dd, J = 5.9, 4.4 Hz), 4.41 (1H, dd, J = 17.6, 6.3 Hz), 4.76 (1H, ddd, J =
11.2, 7.3, 3.9 Hz), 5.69–5.80 (2H, m), 5.80–5.91 (1H, m), 6.64 (1H, d, J = 3.9 Hz), 7.11 (1H, d,
24

ACCEPTED MANUSCRIPT
J = 3.9 Hz), 7.59 (1H, d, J = 7.3 Hz), 7.73 (1H, d, J = 6.3 Hz); 13C NMR (100 MHz, CDCl3): δ
15.1, 19.3, 19.5, 29.2, 31.1, 34.2, 37.5, 38.1, 43.1, 51.2, 55.1, 62.0, 69.4, 129.7, 130.3, 168.0,
170.03, 170.05, 170.8, 172.4; IR (neat): 3342, 3012, 2965, 2934, 1742, 1659, 1525, 1260, 1186,
1024, 979, 754 cm−1; HRMS (EI): m/z calcd for C20H30N4O6S2 (M+) 486.1607, found 486.1616.
4.1.27. Fmoc-
L-Ala–Gly-OMe (30d)
Compound 30d was synthesized from
L
-alanine (L-Ala) (18d) (500 mg, 5.6 mmol) in a
manner similar to that described for the synthesis of 21. Purification by column chromatography
29
(hexane/EtOAc, 2:3) gave 30d (1.99 g, 93%, 2 steps) as a colorless amorphous solid. [α]D
1
−13.2 (c 1.0, CHCl3); H NMR (400 MHz, CDCl3): δ 1.41 (3H, d, J = 6.3 Hz), 3.72 (3H, s),
4.02 (2H, d, J = 4.9 Hz), 4.20 (1H, t, J = 6.8 Hz), 4.27–4.38 (1H, m), 4.40 (2H, d, J = 6.3 Hz),
5.51 (1H, d, J = 6.3 Hz), 6.71 (1H, br s), 7.30 (2H, t, J = 7.3 Hz), 7.39 (2H, t, J = 7.3 Hz), 7.58
13
(2H, dd, J = 6.8, 2.4 Hz), 7.75 (2H, d, J = 7.3 Hz); C NMR (100 MHz, CDCl3): δ 18.5, 41.2,
47.1, 50.4, 52.4, 67.1, 120.0 (2C), 125.0 (2C), 127.1 (2C), 127.7 (2C), 141.3 (2C), 143.7 (2C),
156.0, 170.0, 172.5; IR (KBr): 3322, 3293, 3067, 2983, 2951, 2941, 2890, 1731, 1720, 1693,
1648, 1541, 1449, 1329, 1301, 1266, 1254, 1105, 1089, 1051, 1023, 758, 741, 671, 645 cm−1;
HRMS (EI): m/z calcd for C21H22N2O5 (M+) 382.1529, found 382.1520.
4.1.28. Boc-D-Cys(Tr)–L-Ala–Gly-OMe (32d)
Compound 32d was synthesized from 30d (1.65 g, 4.3 mmol) in a manner similar to that
described for the synthesis of 23. Purification by column chromatography (hexane/EtOAc 1:2)
gave 32d (2.24 g, 86%, 2 steps) as a colorless amorphous solid. [α]D25 −31.4 (c 1.0, CHCl3); 1H
NMR (400 MHz, CDCl3): δ 1.36 (3H, d, J = 6.8 Hz), 1.40 (9H, s), 2.55 (1H, dd, J = 12.9, 5.4
Hz), 2.75 (1H, dd, J = 12.0, 6.8 Hz), 3.66–3.73 (4H, m), 3.82 (1H, dd, J = 18.0, 5.4 Hz), 4.00
(1H, dd, J = 18.0, 5.4 Hz), 4.48 (1H, dq, J = 7.2, 7.1 Hz), 4.90 (1H, br s), 6.39 (1H, br s), 7.04
(1H, br s), 7.04–7.31 (9H, m), 7.39–7.41 (6H, m); 13C NMR (100 MHz, CDCl3): δ 17.9, 28.0
(3C), 33.7, 40.8, 48.5, 51.9, 53.6, 66.8, 80.0, 126.7 (3C), 127.8 (6C), 129.3 (6C), 144.1 (3C),
155.2, 169.9, 170.2, 172.2; IR (neat): 3299, 3058, 3006, 2978, 2932, 1752, 1652, 1518, 1491,
1445, 1367, 1213, 1166, 1049, 1021, 854, 751, 700 cm−1; HRMS (FAB): m/z calcd for
C33H40N3O6S (M++H) 606,2632, found; 606.2625.
4.1.29. H2N-D-Cys(Tr)–L-Ala–Gly-OMe (15d)
Compound 15d was synthesized from 32d (1.83 g, 3.0 mmol) in a manner similar to that
described for the synthesis of 15a. Purification by column chromatography (CHCl3/MeOH
25
1
15:1) gave 15d (1.43 g, 94%) as a colorless amorphous solid. [α]D −64.5 (c 1.0, CHCl3); H
NMR (400 MHz, CDCl3): δ 1.34 (3H, d, J = 6.8 Hz), 1.47 (2H, br s), 2.64 (2H, d, J = 4.9 Hz),
3.05 (1H, t, J = 5.6 Hz), 3.64 (3H, s), 3.78 (1H, dd, J = 17.6, 5.4 Hz), 3.90 (1H, dd, J = 17.6, 5.9
25

ACCEPTED MANUSCRIPT
Hz), 4.49 (1H, dq, J = 7.2, 7.1 Hz), 7.18–7.28 (10H, m), 7.39–7.41 (6H, m), 7.58 (1H, d, J = 7.8
13
Hz); C NMR (100 MHz, CDCl3): δ 17.6, 37.3, 40.7, 48.2, 51.9, 53.8, 66.7, 126.7 (3C), 127.8
(6C), 129.3 (6C), 144.3 (3C), 169.8, 172.3, 173.0; IR (neat): 3308, 3057, 3016, 2951, 1750,
1654, 1509, 1444, 1369, 1211, 1183, 1032, 981, 886, 848, 745, 700 cm−1; HRMS (FAB): m/z
calcd for C28H32N3O4S (M++H) 506.2108, found 506.2109.
4.1.30. (7S,11R,14S,17S,E)-Methyl 11-isopropyl-7-(4-methoxybenzyloxy)-17-methyl-9,12,15,18-
tetraoxo-1,1,1-triphenyl-14-tritylthiomethyl-2-thia-10,13,16,19-tetraazahenicos-5-en-21-oate
(26d)
Compound 26d was synthesized from 15d (119 mg, 0.24 mmol) in a manner similar to that
described for the synthesis of 26a. Purification by column chromatography (CHCl3/MeOH
27
1
40:1) gave 26d (240 mg, 91%) as a colorless amorphous solid. [α]D −4.0 (c 1.0, CHCl3); H
NMR (400 MHz, CDCl3): δ 0.69 (3H, d, J = 6.8 Hz), 0.79 (3H, d, J = 6.8 Hz), 1.34 (3H, d, J =
7.3 Hz), 2.27 (1H, dq, J = 13.2, 6.6 Hz), 2.05–2.14 (2H, m), 2.21–2.25 (2H, m), 2.47 (2H, d, J =
5.4 Hz), 2.56 (1H, dd, J = 12.7, 5.4 Hz), 2.78 (1H, dd, J = 13.4, 7.1 Hz), 3.64 (3H, s), 3.77 (3H,
s), 3.82 (1H, dd, J = 18.0, 5.4 Hz), 3.89–3.99 (2H, m), 4.06 (1H, q, J = 6.7 Hz), 4.16 (1H, t, J =
6.6 Hz), 4.23 (1H, d, J = 10.7 Hz), 4.41 (1H, d, J = 10.7 Hz), 4.57 (1H, dq, J = 7.4, 7.2 Hz),
5.29 (1H, dd, J = 15.4, 8.0 Hz), 5.55 (1H, dt, J = 15.1, 6.8 Hz), 6.78 (1H, d, J = 8.3 Hz), 6.82
(2H, d, J = 8.8 Hz), 7.01 (1H, d, J = 7.3 Hz), 7.16–7.28 (22H, m), 7.33–7.42 (12H, m); 13C
NMR (100 MHz, CDCl3): δ 17.7, 17.8, 19.3, 30.3, 31.2, 31.3, 33.7, 40.9, 42.4, 48.9, 52.1, 52.5,
55.2, 58.9, 66.5, 67.0, 69.9, 76.4, 113.8 (2C), 126.6 (3C), 126.8 (3C), 127.8 (6C), 128.0 (6C),
129.45 (6C), 129.48 (6C), 129.7, 129.8 (2C), 129.9, 133.1, 144.3 (3C), 144.8 (3C), 159.3, 169.5,
170.1, 171.2, 171.6, 172.4; IR (neat): 3291, 3058, 3016, 2961, 2931, 1752, 1635, 1513, 1490,
1444, 1390, 1368, 1247, 1215, 1181, 1079, 1034, 975, 847, 823, 751, 700 cm−1; HRMS (FAB):
m/z calcd for C67H73N4O8S2 (M++H) 1125.4864, found 1125.4873.
4.1.31. (7S,11R,14S,17S,E)-Methyl 7-hydroxy-11-isopropyl-17-methyl-9,12,15,18-tetraoxo-
1,1,1-triphenyl-14-tritylthiomethyl-2-thia-10,13,16,19-tetraazahenicos-5-en-21-oate (27d)
Compound 27d was synthesized from 26d (240 mg, 0.21 mmol) in a manner similar to that
described for the synthesis of 27a. Purification by column chromatography (CHCl3/MeOH
27
1
50:1) gave 27d (213 mg, 99%) as a colorless amorphous solid. [α]D −37.8 (c 1.0, CHCl3); H
NMR (400 MHz, CDCl3): δ 0.95 (3H, d, J = 6.8 Hz), 1.00 (3H, d, J = 6.8 Hz), 1.42 (3H, d, J =
7.3 Hz), 2.03–2.08 (2H, m), 2.18–2.37 (4H, m), 2.42 (1H, dd, J = 13.2, 2.0 Hz), 2.64 (2H, d, J =
6.8 Hz), 3.54 (3H, s), 3.61 (1H, dd, J = 18.0, 4.4 Hz), 4.00 (1H, br s), 4.08–4.10 (2H, m), 4.33–
4.45 (3H, m), 5.38 (1H, dd, J = 15.6, 5.9 Hz), 5.59 (1H, dt, J = 15.6, 6.3 Hz), 6.01 (1H, d, J =
5.8 Hz), 6.96 (1H, t, J = 5.6 Hz), 7.02 (1H, d, J = 6.8 Hz), 7.16–7.30 (19H, m), 7.33–7.42 (12H,
m); 13C NMR (100 MHz, CDCl3): δ 16.1, 17.4, 19.5, 29.0, 31.1, 31.3, 33.6, 40.7, 43.7, 48.8,
26

ACCEPTED MANUSCRIPT
51.9, 52.1, 59.9, 66.5, 66.7, 69.3, 126.5 (3C), 126.6 (3C), 127.7 (6C), 127.8 (6C), 129.38 (6C),
129.42 (6C), 129.7, 132.7, 144.3 (3C), 144.7 (3C), 170.3, 170.7, 170.8, 172.9, 173.0; IR (neat):
3291, 3082, 3058, 3017, 2964, 2930, 1745, 1633, 1531, 1490, 1444, 1391, 1370, 1216, 1183,
1081, 1033, 972, 850, 751, 700 cm−1; HRMS (FAB): m/z calcd for C59H65N4O7S2 (M++H)
1005.4289, found 1005.4293.
4.1.32. (7S,11R,14S,17S,E)-7-Hydroxy-11-isopropyl-17-methyl-9,12,15,18-tetraoxo-1,1,1-
triphenyl-14- tritylthiomethyl-2-thia-10,13,16,19-tetraazahenicos-5-en-21-oic acid (14d)
Compound 14d was synthesized from 27d (213 mg, 0.21 mmol) in a manner similar to that
described for the synthesis of 14a. Purification by column chromatography (CHCl3/MeOH 5:1)
gave 14d (209 mg, 99%) as a colorless amorphous solid. [α]D30 −16.9 (c 1.0, CHCl3); 1H NMR
(400 MHz, CD3OD/CDCl3 3:1): δ 0.94 (3H, d, J = 5.4 Hz), 0.95 (3H, d, J = 6.3 Hz), 1.37 (3H, d,
J = 6.8 Hz), 2.01–2.06 (2H, m), 2.09–2.21 (3H, m), 2.36 (1H, dd, J = 13.7, 4.4 Hz), 2.48 (1H,
dd, J = 13.7, 9.6 Hz), 2.57 (1H, dd, J = 12.2, 8.8 Hz), 2.64 (1H, dd, J = 12.7, 5.9 Hz), 3.79 (2H,
s), 4.08 (1H, d, J = 5.4 Hz), 4.13 (1H, dd, J = 8.3, 6.3 Hz), 4.29 (1H, q, J = 6.8 Hz), 4.39–4.43
(1H, m), 5.43 (1H, dd, J = 15.1, 6.3 Hz), 5.55 (1H, dt, J = 15.1, 6.3 Hz), 7.15–7.26 (18H, m),
13
7.35–7.39 (12H, m); C NMR (100 MHz, CD3OD/CDCl3 3:1, 45 °C): δ 17.6, 18.2, 19.6, 30.5,
32.0, 32.2, 33.8, 41.7, 43.9, 50.0, 53.7, 60.4, 67.4, 67.7, 69.8, 127.3 (3C), 127.5, (3C), 128.4
(6C), 128.6 (6C), 130.2 (6C), 130.26, 130.27 (6C), 133.7, 145.3 (3C), 145.7 (3C), 171.3, 172.5,
173.1, 174.2, 174.3; IR (neat): 3297, 3057, 3018, 2964, 2928, 1725, 1638, 1531, 1490, 1444,
1217, 1033, 970, 750, 700 cm−1; HRMS (FAB): m/z calcd for C58H63N4O7S2 (M++H) 991.4133,
found 991.4153.
4.1.33. (6S,9S,12R,16S)-12-Isopropyl-6-methyl-16-[(E)-4-tritylthiobut-1-enyl]-9-
tritylthiomethyl-1-oxa-4,7,10,13-tetraazacyclohexadecane-2,5,8,11,14-pentaone (28d)
Compound 28d was synthesized from 14d (104 mg, 0.11 mmol) im a manner similar to that
described for the synthesis of 28a. Purification by column chromatography (CHCl3/MeOH
40:1) gave 28d (87.6 mg, 86%) as a colorless amorphous solid. [α]D27 −16.3 (c 1.0, CHCl3); 1H
NMR (400 MHz, CDCl3): δ 0.85 (3H, d, J = 6.3 Hz), 0.95 (3H, d, J = 6.3 Hz), 1.20 (3H, d, J =
7.2 Hz), 1.88–2.18 (5H, m), 2.42–2.57 (2H, m), 2.75 (1H, dd, J = 12.6, 3.4 Hz), 2.91 (1H, dd, J
= 12.6, 9.7 Hz), 3.49 (1H, dd, J = 16.4, 3.4 Hz), 3.56–3.64 (1H, m), 4.29 (1H, dd, J = 16.4, 8.2
Hz), 4.34–4.51 (2H, m), 5.37 (1H, dd, J = 15.5, 6.3 Hz), 5.47–5.62 (2H, m), 6.17 (1H, br d, J =
5.3 Hz), 7.13–7.27 (20H, m), 7.33–7.38 (12H, m), 7.67 (1H, br s); 13C NMR (100 MHz, CDCl3,
40 °C): δ 17.2, 17.6, 19.8, 31.1, 31.4, 32.1, 32.2, 42.1, 42.5, 48.5, 54.6, 57.7, 66.7, 66.9, 72.0,
126.6 (3C), 126.8 (3C), 127.8 (6C), 127.9 (6C), 128.0, 129.5 (6C), 129.6 (6C), 132.8, 144.6
(3C), 144.8 (3C), 169.0, 169.2, 169.8, 172.3, 172.8; IR (neat): 3289, 3058, 3016, 2965, 2933,
1734, 1654, 1540, 1489, 1444, 1216, 1195, 1033, 980, 751, 700 cm−1; HRMS (FAB): m/z calcd
27

ACCEPTED MANUSCRIPT
for C58H60N4O6S2Na (M++Na) 995.3846, found 995.3822.
4.1.34. FK-A4 (13d)
Compound 13d was synthesized from 28d (87.0 mg, 93 ꢀmol) in a manner similar to that
described for the synthesis of 13a. Purification by column chromatography (CHCl3/MeOH
20:1) gave 13d (40.0 mg, 89%) as a colorless amorphous solid. [α]D27 −30.4 (c 1.0, CHCl3); 1H
NMR (400 MHz, CDCl3): δ 1.09 (3H, d, J = 4.4 Hz), 1.10 (3H, d, J = 3.9 Hz), 1.42 (3H, d, J =
7.3 Hz), 2.30 (1H, dq, J = 12.2, 6.8 Hz), 2.46–2.57 (1H, m), 2.62–2.76 (2H, m), 2.89–2.96 (2H,
m), 3.08 (1H, ddd, J = 13.2, 8.8, 2.9 Hz), 3.14 (1H, dd, J = 15.1, 3.9 Hz), 3.27 (1H, dd, J = 15.1,
10.2 Hz), 4.06 (1H, t, J = 4.4 Hz), 4.11 (1H, dd, J = 17.6, 5.4 Hz), 4.20 (1H, dd, J = 17.6, 5.4
Hz), 4.54 (1H, q, J = 7.3 Hz), 4.80 (1H, ddd, J = 10.2, 6.8, 3.9 Hz), 5.70–5.81 (2H, m), 5.83–
5.94 (1H, m), 6.48 (1H, d, J = 2.9 Hz), 6.90 (1H, t, J = 4.9 Hz), 7.43 (1H, d, J = 6.8 Hz), 7.48
(1H, d, J = 8.3 Hz); 13C NMR (100 MHz, CDCl3): δ 17.2, 18.7, 19.4, 29.3, 31.3, 37.5, 37.6, 39.5,
42.3, 49.5, 56.0, 62.2, 69.5, 128.9, 129.7, 167.3, 169.6, 171.0, 171.1 171.9; IR (neat): 3297,
3009, 2966, 2933, 1742, 1655, 1524, 1253, 1190, 1136, 1024, 979, 754 cm−1; HRMS (EI): m/z
calcd for C20H30N4O6S2 (M+) 486.1607, found 486.1601.
4.1.35.Fmoc-
D-Phe–Gly-OMe (30e)
Compound 30e was synthesized from
D
-phenylalanine (D-Phe) (18e) (500 mg, 3.0 mmol) in
a manner similar to that described for the synthesis of 21. Purification by column
chromatography (CHCl3/MeOH 30:1) gave 30e (971 mg, 70%, 2 steps) as a colorless
25
1
amorphous solid. [α]D +11.1 (c 1.0, CHCl3); H NMR (400 MHz, CDCl3): δ 3.07–3.11 (2H,
m), 3.68 (3H, s), 3.89–4.02 (2H, m), 4.15 (1H, t, J = 6.8 Hz), 4.26–4.30 (1H, m), 4.37–4.41 (1H,
m), 4.52 (1H, d, J = 5.9 Hz), 5.55 (1H, br d, J = 6.8 Hz), 6.57 (1H, br s), 7.19–7.29 (7H, m),
7.38 (2H, t, J = 7.3 Hz), 7.50 (2H, t, J = 7.3 Hz), 7.74 (2H, d, J = 7.3 Hz); 13C NMR (100 MHz,
CDCl3): δ 38.4, 41.1, 46.3, 52.2, 55.9, 67.0, 119.9 (2C), 125.0, 126.9 (2C), 127.0 (2C), 127.6
(2C), 128.6 (2C), 129.2 (2C), 136.3, 141.2 (2C), 143.6 (2C), 156.0, 169.8, 171.3; IR (neat):
3301, 3064, 3029, 2952, 1748, 1660, 1538, 1449, 1259, 1213, 1036, 758, 740, 700 cm−1; HRMS
(FAB): m/z calcd for C27H27N2O5 (M++H) 459.1920, found 459.1924.
4.1.36. Boc-D-Cys(Tr)–D-Phe–Gly-OMe (32e)
Compound 32e was synthesized from 30e (775 mg, 1.7 mmol) in a manner similar to that
described for the synthesis of 23. Purification by column chromatography (hexane/EtOAc 1:1)
gave 32e (1.07 g, 92%, 2 steps) as a colorless amorphous solid. [α]D24 +15.4 (c 1.0, CHCl3); 1H
NMR (400 MHz, CDCl3): δ 1.34 (9H, s), 2.52-2.62 (2H, m), 3.08 (1H, dd, J = 14.2, 6.3Hz),
3.15 (1H, dd, J = 13.9, 6.8 Hz), 3.60 (1H, dd, J = 11.7, 5.9 Hz), 3.65 (3H, s), 3.71 (1H, dd, J =
17.8, 5.9 Hz), 3.93 (1H, dd, J = 18.1, 5.9 Hz), 4.64 (1H, d, J = 5.4 Hz), 4.73 (1H, dd, J = 14.9,
28