RA-XXV and RA-XXVI, Bicyclic Hexapeptides from Rubia cordifolia L.: Structure, Synthesis, and Conformation

Article abstract of DOI:10.1002/asia.201801466

Two RA-series bicyclic hexapeptides, RA-XXV (4) and RA-XXVI (5), which have no N-methyl group at Tyr-5, were isolated from the roots of Rubia cordifolia L. Their amino acid compositions and sequences were determined by interpretation of MS, and 1D and 2D NMR data and their relative structures were elucidated by XRD analysis of 4 and RA-XXVI acetate (6). The absolute stereochemistry of 4 was established by the total synthesis of 4, and that of 5, by the chemical correlation with 4. Peptides 4 and 5 exhibited cytotoxicity toward human promyelocytic leukemia HL-60 (IC₅₀=0.062 and 0.066 μm, respectively) and human colonic carcinoma HCT-116 (IC₅₀=0.028 and 0.051 μm, respectively) cell lines. Analysis of the conformational structures of 4 and 6 in the crystalline state and those of 4 and 5 in solution revealed that the N-methyl group at Tyr-5 functions to make this series of peptides preferentially adopt the active conformation.

Full text of DOI:10.1002/asia.201801466

CHEMISTRY
AN ASIAN JOURNAL
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Accepted Article
Title: RA-XXV and RA-XXVI, Bicyclic Hexapeptides from Rubia
cordifolia L. Structure, Synthesis, and Conformation
Authors: Yukio Hitotsuyanagi, Masahito Hirai, Masumi Odagiri, Miho
Komine, Tomoyo Hasuda, Haruhiko Fukaya, and Koichi
Takeya
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10.1002/asia.201801466
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RA-XXV and RA-XXVI, Bicyclic Hexapeptides from Rubia
cordifolia L. Structure, Synthesis, and Conformation
Yukio Hitotsuyanagi,*[a] Masahito Hirai, Masumi Odagiri, Miho Komine, Tomoyo Hasuda, Haruhiko
Fukaya, and Koichi Takeya
Abstract: Two new RA-series bicyclic hexapeptides, RA-XXV (4)
and RA-XXVI (5), which have no N-methyl group at Tyr-5, were
isolated from the roots of Rubia cordifolia L. Their amino acid
compositions and sequences were determined by interpretation of
MS and 1D and 2D NMR data and their relative structures were
elucidated by X-ray crystallography of 4 and RA-XXVI acetate (6).
The absolute stereochemistry of 4 was established by the total
synthesis of 4, and that of 5, by the chemical correlation with 4.
Peptides 4 and 5 exhibited cytotoxicity toward human promyelocytic
leukemia HL-60 (IC50=0.062 and 0.066 μM, respectively) and human
colonic carcinoma HCT-116 (IC50=0.028 and 0.051 μM, respectively)
cell lines. Analysis of the conformational structures of 4 and 6 in the
crystalline state and those of 4 and 5 in solution revealed that the N-
methyl group at Tyr-5 functions to make this series of peptides
preferentially adopt the active conformation.
established their absolute structures. Their conformational
structures in crystals were studied by X-ray diffraction analysis
and those in solution were elucidated by ROESY experiments
and computational methods. We also evaluated their cytotoxic
activities on HL-60 and HCT-116 cells.
Results and Discussion
Isolation: A methanol extract obtained from the dried roots of R.
cordifolia (50 kg) was partitioned between chloroform and water.
The chloroform-soluble portion was subjected to a series of
column chromatographic separations using silica gel, alumina,
and then aminopropyl-bonded silica gel, employing a series of
CHCl3/MeOH mixtures as the eluent. After removal of the
solvent, the residue of the CHCl3 eluate and the residue of the
CHCl3/MeOH (9:1) eluate of the aminopropyl-bonded silica gel
column chromatography were each crystallized from MeOH to
give crystals of crude RAs and the mother liquors. By reversed-
phase HPLC separation, the mother liquor from the CHCl3 eluate
gave RA-XXV (4, 16.9 mg, 3.4 × 10−5%) and that from the
CHCl3/MeOH (9:1) eluate afforded RA-XXVI (5, 15.1 mg, 3.0 ×
Introduction
RA-VII (1) is a bicyclic hexapeptide isolated from rubiaceous
plants Rubia cordifolia L. and R. akane Nakai (Rubiaceae).[1,2]
Structurally related peptides, bouvardin (NSC 259968, 2) and
deoxybouvardin (RA-V, 3), were isolated from Bouvardia
ternifolia (Cav.) Schltdl., a plant of the same family (Fig. 1).[3,4]
These peptides have a structurally unique cycloisodityrosine unit
in which the two phenyl rings of tyrosyl-tyrosine are connected
by an ether linkage to form a 14-membered cyclophane
macrocycle. These peptides exhibit potent antitumor activity;
peptide 1 underwent phase I clinical trials as an anticancer drug
in Japan from the late 80s to the early 90s.[5] The antitumor
activity of these peptides is believed to be due to the inhibition of
protein synthesis through interaction with eukaryotic
ribosomes.[6] Peptide 1 was also shown to cause conformational
changes in F-actin, which stabilize actin filaments and induce G2
arrest,[7] and peptide 3 was found to inhibit angiogenesis by
down-regulating ERK1/2 phosphorylation in HUVEC and HMEC-
1 endothelial cells.[8]
In the present study, we examined minor constituents in the
methanol extracts of the roots of R. cordifolia L. We isolated two
new bicyclic hexapeptides, RA-XXV (4) and RA-XXVI (5), and
[a]
Prof. Dr. Y. Hitotsuyanagi, M. Hirai, M. Odagiri, M. Komine, Dr. T.
Hasuda, H. Fukaya, and Prof. Dr. K. Takeya
School of Pharmacy
Figure 1. Structures of RA-VII (1), bouvardin (2), deoxybouvardin (3), RA-XXV
(4), RA-XXVI (5), and RA-XXVI acetate (6).
Tokyo University of Pharmacy and Life Sciences
1432-1 Horinouchi, Hachioji, Tokyo 192-0392 (Japan)
E-mail: yukioh@toyaku.ac.jp
Supporting information for this article is given via a link at the end of
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Table 1 NMR data of RA-XXV (4) and RA-XXVI (5)[a]
RA-XXV (4)
RA-XXVI (5)
conformer I
conformer II
conformer I
conformer II
Residue
D-Ala-1
δH [ppm]
δC [ppm]
δH [ppm]
δC [ppm]
δH [ppm]
δC [ppm]
δH [ppm]
4.34[b]
δC [ppm]
α
4.57 (quintet, 7.2)
1.22 (d, 7.2, 3H)
47.2
16.4
172.8
4.33 (quintet, 6.9)
1.27 (d, 6.9, 3H)
47.7
4.57 (quintet, 7.1)
1.22 (d, 7.1, 3H)
47.3
47.6
β
16.03
172.2
16.47
172.8
1.26 (d, 7.0, 3H)
16.03
172.3
C=O
NH
α
5.79 (brs)
6.52 (d, 6.9)
4.46[b]
5.82 (brd, 6.0)
6.57[b]
Ala-2
Tyr-3
4.48 (quintet, 6.7)
1.46 (d, 6.9, 3H)
46.1
45.6
4.49 (quintet, 6.6)
1.47 (d, 6.9, 3H)
46.2
4.43 (brm)
1.25 (d, 6.9, 3H)
45.6
β
15.99
173.1
1.25 (d, 6.9, 3H)
15.99
173.0
16.03
173.2
15.96
173.0
C=O
NH
α
7.38 (d, 5.7)
3.59 (brm)
3.48[b]
n.o.
7.45[b]
n.o.
69.3
32.6
3.58 (dd, 8.7, 7.1)
3.40[b]
3.40[b]
69.1
32.8
3.59 (brm)
3.48[b]
69.2
32.6
3.58 (dd, 9.2, 6.6)
3.40[b]
3.40[b]
69.1
32.8
βa
βb
γ
3.36 (dd, 13.4, 4.3)
3.36 (dd, 13.9, 4.4)
130.4
130.2[c]
114.1[c]
158.5
170.0
39.6
130.9
130.3[c]
113.9[c]
158.3
170.2
39.4
130.2
130.2[c]
114.1[c]
158.5
170.0
39.6
130.85
130.3[c]
113.9[c]
158.3
170.2
39.5
δ
7.11 (d, 8.6, 2H)
6.85 (d, 8.6, 2H)
7.04 (d, 8.6, 2H)
6.813 (d, 8.6, 2H)
7.11 (d, 8.6, 2H)
6.85 (d, 8.6, 2H)
7.04 (d, 8.5, 2H)
6.81 (d, 8.5, 2H)
ε
ζ
C=O
NMe
OMe
α
2.77 (s, 3H)
2.72 (s, 3H)
3.78 (s, 3H)
3.92[b]
2.77 (s, 3H)
2.72 (s, 3H)
3.78 (s, 3H)
3.92 (brm)
3.80 (s, 3H)
55.3
55.2
3.80 (s, 3H)
55.3
55.2
Ala-4
Tyr-5
3.69 (quintet, 7.1)
1.72 (d, 7.4, 3H)
52.6
52.5
3.71 (quintet, 7.1)
1.70 (d, 7.4, 3H)
52.4
52.4
β
16.5
1.61 (brd, 7.1, 3H)
17.6
16.50
173.0
1.61 (brs, 3H)
17.6
C=O
NH
α
173.1
172.8
173.1
6.88 (brd, 6.2)
5.17 (m)
7.13 (d, 8.0)
4.81 (m)
3.48[b]
3.00[b]
6.88 (brd, 6.4)
5.17 (m)
7.15 (brd, 6.2)
4.80 (m)
3.50[b]
3.02[b]
51.7
36.9
52.5
40.1
51.5
37.0
52.6
39.9
β (pro-S) 3.24 (t, 12.5)
3.22 (t, 12.8)
3.32 (dd, 12.8, 5.4)
β (pro-R) 3.34 (dd, 12.5, 5.1)
γ
134.8
131.6
131.5
127.4
123.7
157.6
171.5
135.2
132.5
130.7
124.5
125.8
158.2
170.6
135.3
131.7
131.6
127.2
123.5
157.3
171.5
135.6
132.7
130.76
124.4
125.7
157.8
170.4
δa
δb
εa
εb
ζ
7.43 (dd, 8.3, 2.1)
7.50 (dd, 8.6, 2.1)
7.28 (dd, 8.3, 2.4)
7.05 (dd, 8.6, 2.4)
7.26[b]
7.43 (dd, 8.2, 2.1)
7.48 (dd, 8.6, 2.1)
7.26 (dd, 8.2, 2.4)
6.98 (dd, 8.6, 2.4)
7.26[b]
7.46 (dd, 8.3, 2.1)
6.90 (dd, 8.3, 2.4)
7.18 (dd, 8.3, 2.4)
7.44[b]
6.85[b]
7.13 (dd, 8.4, 2.4)
C=O
NH
α
8.01 (brd, 7.0)
n.o.
7.95 (brd, 8.0)
n.o.
Tyr-6
3.42 (dd, 10.8, 4.0)
67.4
32.7
4.36 (dd, 11.9, 3.7)
2.80[b]
3.38[b]
56.9
33.2
3.42 (dd, 10.4, 4.3)
3.00 (dd, 14.9, 4.3)
3.13 (dd, 14.9, 10.4)
67.1
33.0
4.36[b]
2.79[b]
3.34[b]
57.0
33.3
β (pro-S) 3.02 (dd, 14.7, 4.0)
β (pro-R) 3.19 (dd, 14.7, 10.8)
γ
129.7
121.7
119.1
111.8
152.0
146.6
169.5
39.9
127.9
121.2
113.5
112.2
153.1
146.4
170.4
28.9
129.0
122.2
119.0
115.4
149.8
143.0
169.4
39.5
127.4
121.9
113.1
115.7
151.1
142.8
170.4
28.9
δa
6.63 (dd, 8.2, 1.9)
4.87 (s)
6.62[b]
6.57 (dd, 8.1, 1.9)
5.02 (s)
6.56[b]
δb
4.30 (d, 1.9)
6.809 (d, 8.5)
4.30 (d, 1.7)
6.83 (d, 8.3)
εa
6.75 (d, 8.2)
6.78 (d, 8.1)
εb
ζ
C=O
NMe
OMe
OH
2.78 (s, 3H)
3.93 (s, 3H)
2.76 (s, 3H)
3.94 (s, 3H)
2.69 (s, 3H)
5.86[d]
2.76 (s, 3H)
5.86[d]
56.11
56.14
[a] Recorded at 600 MHz for 1H NMR and 150 MHz for 13C NMR in CDCl3. Conformers I and II are the most populated and the second-most populated conformers in CDCl3, respectively.
J -values given in Hz in parentheses. n.o. = not observed. [b] Multiplicity not determined due to overlapping signals. [c] Two carbons. [d] Very broad signal.
10−5%) along with known RA-series peptides.
exhibited signals for three secondary methyl groups (δH/δC
1.22/16.4, 1.46/15.99, 1.72/16.5 ppm), two N-methyl groups
(δH/δC 2.77/39.6, 2.78/39.9 ppm), two O-methyl groups (δH/δC
3.80/55.3, 3.93/56.11 ppm), three methylenes (δH/δC 3.02 and
3.19/32.7, 3.24 and 3.34/36.9, 3.36 and 3.48/32.6 ppm), six N-
substituted methines (δH/δC 3.42/67.4, 3.59/69.3, 3.69/52.6,
4.48/46.1, 4.57/47.2, 5.17/51.7 ppm), eleven aromatic methines
(δH/δC 4.87/119.1, 6.63/121.7, 6.75/111.8, 6.85/114.1 (×2),
7.05/123.7, 7.11/130.2 (×2), 7.28/127.4, 7.43/131.6, 7.50/131.5),
seven aromatic quaternary carbons (δC 129.7, 130.4, 134.8,
146.6, 152.0 157.6, 158.5 ppm), and six carbonyl groups (δC
169.5, 170.0, 171.5, 172.8, 173.1 (×2) ppm) (Table 1).
25
Structures of RA-XXV (4) and RA-XXVI (5): RA-XXV (4), [α]D
=−155 (c=0.063 in MeOH), was obtained as colorless plates. Its
molecular formula was determined to be C40H48N6O9 from the
[M+Na+] peak at m/z 779.3398 (calcd for C40H48N6O9+Na+,
1
779.3380) in the high-resolution ESI-MS (HR-ESIMS). The H
NMR spectrum of 4 in CDCl3 indicated the presence of four
spectroscopically distinguishable conformers in the ratio of
69:25:3:3 at 300 K. The planar structure of 4 was determined by
analyzing the NMR signals from the most populated conformer
(conformer I).[9] The 1H NMR, 13C NMR, and HSQC spectra of 4
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Figure 2. 1H-1H COSY and selected HMBC correlations for RA-XXV (4).
Inspection of the 1H-1H COSY and HMBC spectra and its
molecular formula with 20 degrees of unsaturation suggested
that peptide 4 had a bicyclic structure composed of three
alanines, one tyrosine, and two N,O-dimethyltyrosines of which
one had a 1,2,4-trisubstituted phenyl ring. Analysis of the HMBC
correlations of the NH, NMe, and/or Hα proton of the amino acid
residue (i) to the i−1 carbonyl carbon atom revealed that peptide
4 had the sequence shown in Figure 2. The presence of a
cyclophane structure at Tyr-5 and Tyr-6 was suggested by the
fact that the signals of the four aromatic methine groups of Tyr-5
had different chemical shifts in the NMR spectra. The Hδb of Tyr-
6 displayed a marked upfield signal at δH 4.87 ppm due to
shielding by the Tyr-5 phenyl ring, which is characteristic of a
14-membered cycloisodityrosine structure of RA-series peptides
having a diphenyl ether linkage between the phenolic oxygen of
Tyr-5 and the Cεb of Tyr-6.[10,11] From these observations, the
planar structure of 4 was determined as shown in Figure 2.
Although the planar structure of 4 corresponded to the des-N-
methyl derivative of 1, we could not elucidate its relative
configuration by comparison of its NMR spectra with that of 1,[11]
because their spectra were dissimilar. The relative
stereochemistry of 4 was determined by the X-ray diffraction
analysis of 4 to be as shown in Figure 3,[12] revealing that
peptide 4 has the same relative configuration as 1–3 at all the
amino acid chiral centers.
Figure 3. ORTEP representation of RA-XXV (4) and RA-XXVI acetate (6).
hydroxyl group in 5. Treatment of 5 with acetic anhydride in
pyridine gave crystalline acetate 6, which was subjected to X-ray
diffraction analysis to assign the relative configuration of 6
unambiguously (Fig. 3).[12] Thus, the structure and the relative
configuration of RA-XXVI (5) were determined to be as shown in
Figure 1.
Absolute Configuration of RA-XXV (4) and RA-XXVI (5): The
absolute structure of RA-XXV (4) was established by the total
synthesis of this peptide with D-alanine at residue 1, L-alanines
at residues 2 and 4, and modified L-tyrosines at residues 3, 5,
and 6. We synthesized peptide 4 on the basis our previous
synthesis of allo-RA-V,[13,14] a related compound but with a
structurally different cycloisodityrosine unit in which a diphenyl
ether bond was formed between the carbon atom at the ε
position of Tyr-5 and the phenolic oxygen at the ζ position of
Tyr-6. This synthesis was characterized by the diaryl ether
formation of the core cycloisodityrosine unit by copper(II)
acetate-mediated
intramolecular
phenol/arylboronic
acid
coupling[15,16] and the macrocyclization of the linear hexapeptide
between the Tyr-6 and D-Ala-1 residues to construct the 18-
membered cyclopeptide ring.
3-Iodo-L-tyrosine (7) was N-protected with a Cbz group (8)
and subsequently treated with paraformaldehyde in the
presence of p-toluenesulfonic acid in toluene to give crystalline
oxazolidinone 9 (Scheme 1). Reductive cleavage of the
oxazolidinone ring of 9 with triethylsilane in a trifluoroacetic acid-
chloroform mixture[17] and subsequent (trimethylsilyl)diazome-
thane treatment gave the N,O-dimethyliodotyrosine derivative 10.
Compound 10 was converted into boronic acid 11 through the
substitution of a pinacolboryl group for the iodine atom and
subsequent hydrolysis of the boronic acid pinacol ester. Amine
12, obtained by removal of the Cbz group from 11, was coupled
with Boc-L-tyrosine to give dipeptide 13, which was then treated
with copper(II) acetate and 4-(dimethylamino)pyridine in the
RA-XXVI (5), [α]D25=−170 (c=0.048 in MeOH), was obtained as a
white amorphous solid. Its molecular formula, C39H46N6O9, which
was deduced from the [M+H+] peak at m/z 743.3405 (calcd for
C39H46N6O9+H+, 743.3399) in the HR-ESIMS, contained one
methylene unit less than that of RA-XXV (4). Its 1H NMR spectra
measured in CDCl3 showed that it was a mixture of four
1
conformers in the 68:28:3:1 ratio. The H and 13C NMR spectra
of 5 were very similar to those of 4; the only differences were
that the signal for the O-methyl group at Tyr-6 was missing and
a phenolic hydroxyl signal newly appeared at δH 5.86 as a very
broad signal in its 1H NMR spectrum. These observations
indicated that the methoxy group at Tyr-6 in 4 was replaced by a
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Scheme 1. Synthesis of cycloisodityrosine 14. Reagents and conditions: a)
Cbz-OSu, Et3N, 1,4-dioxane/H2O,
0 °C, 1 h, then RT, 17 h, 98%; b)
paraformaldehyde, p-TsOH·H2O, PhMe, RT, 3 d, 89%; c) (i) Et3SiH, TFA,
CHCl3, RT, 3 d, (ii) (trimethylsilyl)diazomethane, MeCN/MeOH/Et2O, RT, 4 d,
76%; d) (i) bis(pinacolato)diboron, PdCl2(dppf)·CH2Cl2, KOAc, DMSO, 80 °C,
20 h, (ii) NaIO4, NH4OAc, acetone/H2O, RT, 20 h, 94%; e) H2, Pd/C, EtOH, RT,
20 min, 99%; f) Boc-L-Tyr-OH, EDC·HCl, HOAt, DMF, RT, 7 d, 85%; g)
Cu(OAc)2, DMAP, 4Å MS, CH2Cl2, RT, 2 d, 37%. For a list of abbreviations,
see Ref. 23.
Scheme 2. Synthesis of RA-XXV (4). Reagents and conditions: a) (i) LiOOH,
MeOH/H2O, 0 °C, 1 h, then RT, 23 h, (ii) BnOH, diethyl azodicarboxylate, PPh3,
THF/toluene, 0 °C, 1 h, then RT, 10 h, 75%; b) (i) TFA, RT, 2 h, (ii) 16,
EDC·HCl, HOOBt, DMF, RT, 10 d, 70%; c) TFA, RT, 2.5 h, 77%; d) (i) H2,
Pd/C, EtOH, RT, 2.5 h, (ii) EDC·HCl, HOOBt, DMF, 0 °C, 2 h, then RT, 3 d,
42%; e) MeI, K2CO3, acetone/MeOH, 40 °C,
abbreviations, see Ref. 23.
6 h, 94%. For a list of
presence of 4Å molecular sieves to afford cycloisodityrosine 14
in 37% yield. The structure of 14 was confirmed by X-ray
crystallography.[12] As (S,S)-cycloisodityrosine is known to
readily convert into its thermodynamically more stable epimer at
the C-terminal residue under basic conditions, we converted at
this stage the methyl ester group of 14 into a benzyl ester group
that can be removed under neutral conditions at the final
macrocyclization stage. This conversion was effected by treating
14 with lithium hydroperoxide and subsequent O-benzylation of
the acid under Mitsunobu conditions (Scheme 2). The Boc group
in 15 was removed and the resulting amine was coupled with
tetrapeptide 16 to provide hexapeptide 17. Deprotection of the
N-terminus gave amine 18. Removal of the benzyl protecting
group of 18 by catalytic hydrogenolysis and subsequent
cyclization with EDC·HCl (8 equiv) and HOOBt (8 equiv) in DMF
under dilution conditions (0.0013 M) gave peptide 4 in 42% yield.
Thus obtained 4, [α]D25=−152 (c=0.050 in MeOH), was shown to
The absolute configuration of RA-XXVI (5) was established by
chemical correlation of with 4. Treatment of with
iodomethane and K2CO3 afforded
product, [α]D25=−157
(c=0.020 in MeOH), that was shown to be identical to natural 4
on the basis of their spectroscopic data and optical rotations.
Thus, peptide 5 was proved to have the same absolute
configuration as 4 at all six amino acid chiral centers.
5
5
a
Structures of 4 and 6 in Crystals: The crystal structures of 4
and 6 exhibited disorder in the Tyr-3 residue (Fig. 3). The
structure of 4 comprised two structures, 4A and 4B, with
occupancies of 0.509(7) and 0.491(7), respectively, and that of 6
comprised two structures, 6A and 6B, with occupancies of
0.750(6) and 0.250(6), respectively. In each pair, the positions of
the p-methoxyphenyl ring in Tyr-3 were somewhat different.
Comparison of the crystal structures of 4 and 6 revealed that 4
and 6 have quite different structures in their peptide backbones
in the crystalline state. The backbone dihedrals of the crystal
structures of peptides 1, 4, and 6 are summarized in Table 2. In
the crystal structure of 4, all six peptide bonds are in trans
conformation. This amide conformation pattern has not been
observed in natural RA-series peptides with an N-methyl group
at Tyr-5.[18] Figure 4 shows an overlay of the crystal structures of
1, 4A, and 6A. Due to the trans amide bond between Tyr-5 and
Tyr-6, peptide 4 has a different structure from peptide 1.[11,20] It
adopts a type II β-turn at Tyr-6 and D-Ala-1, which is stabilized
1
be identical to natural 4 by comparison of their H and 13C NMR
spectra, IR spectra, mass spectra, and optical rotations.
Accordingly, the absolute structure of RA-XXV (4) was
determined to be as shown in Figure 1. Although the compound
having structure 4 has already been reported as a synthetic des-
N-methyl analogue of RA-VII,[18] the reported NMR data for this
analogue did not match the data for our natural compound 4.
The reported analogue may be the diastereoisomer of 4.[19]
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Table 2
Backbone dihedrals (degree) in RA-XXV (4), RA-XXVI acetate (6), and RA-VII (1)
residue
D-Ala-1
4a
4b
6c
6d
1e
φ
85.5(5)
−10.6(6)
178.2(4)
−135.9(4)
65.2(5)
99.5
−38.9
−169.3
−78.3
109.8
−167.0
50.8
106.1(4)
−156.7(3)
−178.1(3)
−98.4(4)
121.3(3)
139.64(17)
−164.43(15)
−177.30(16)
−90.1(2)
ψ
ω
φ
Ala-2
Tyr-3
Ala-4
Tyr-5
Tyr-6
ψ
ω
φ
114.02(17)
−176.18(15)
57.3(2)
−176.2(4)
53.5(5)
179.1(5) −176.4(11)
57.4(10)
38.3(10)
46(3)
49(2)
ψ
ω
φ
−121.9(4)
−170.7(4)
−62.6(5)
−29.3(5)
170.0(4)
−86.6(4)
126.8(4)
−174.7(4)
−43.2(5)
131.3(4)
178.2(4)
36.7
35.7(2)
−175.7
80.6
−169.1(5) −173.9(10)
−167.3(3)
165.6(3)
−173.67(16)
−161.11(16)
158.73(15)
−178.87(15)
−121.84(17)
110.53(17)
−13.4(2)
ψ
ω
φ
−24.4
−172.3
−80.9
107.8
−162.6
48.9
170.9(3)
−113.7(3)
118.5(3)
ψ
ω
φ
−25.9(5)
−68.8(4)
−79.9(2)
Figure 5. Key ROESY correlations observed in the CDCl3 solution of 4. The
structure shown for conformer I represents the structure of the most stable
conformer found in the Monte Carlo conformational search for 4, and that
shown for conformer II was built from the crystal structure of 6 by replacing the
acetyl group by a methyl group.
ψ
ω
33.0
−163.3(3)
170.6(3)
156.78(15)
178.62(16)
−179.3
a In crystal structure 4A. b In the most stable structure found by Monte Carlo conformational
search and optimized by DFT calculations. c In crystal structure 6A. d In crystal structure 6B.
e In crystal structure, reference 13.
tyrosine aromatic rings of both peptides occupy almost the same
space.
Structures of 4 and 5 in Solution: The solution structures of
peptides 4 and 5 were examined by analysis of the data from
ROESY experiments (Fig. 5). In the ROESY spectrum of 4,
cross-peaks from the most populated conformer (conformer I)
with population 69% were observed between Ala-2 Hα and Tyr-3
NMe and between Tyr-5 Hα and Tyr-6 NMe, indicating that this
conformer had trans amide bonds between Ala-2 and Tyr-3 and
between Tyr-5 and Tyr-6 as was observed in the crystal
structure of 4 (Fig. 3). However, the crystal structure appeared
not to reflect the solution structure of 4 in CDCl3. There were
some inconsistencies between the interproton distances of the
crystal structure and the ROESY data. Although strong cross-
peaks between D-Ala-1 H3β and Ala-4 Hα and between Tyr-3
NMe and Ala-4 NH were observed in the ROESY spectrum, the
calculated distances from Ala-4 Hα to the closest proton of D-Ala-
1 H3β and from Ala-4 NH to that of Tyr-3 NMe in the crystals
were 8.20 Å and 5.25 Å, respectively, and were not sufficiently
short to generate intense cross-peaks in the ROESY spectrum.
The most stable conformation obtained from a Monte Carlo
conformational search of the structure of 4 using MacroModel
software and the MMFFs force field[22] was then subjected to
geometry optimization at the B3LYP level of density functional
theory (DFT) using the 6-31G(d) basis set. The thus-obtained
structure is illustrated in Figure 5 with observed ROESY
correlations designated by arrows. In this structure, the
distances between those protons were estimated to be 2.32 Å
and 2.80 Å, respectively. Other ROESY correlations observed
were between D-Ala-1 NH and Tyr-6 NMe, Tyr-3 Hα and Tyr-3
NMe,Tyr-5 Hβ (pro-S) and Tyr-5 NH, and Tyr-5 Hδb and Tyr-6
NMe, and the distances between those protons were all less
than 2.9 Å in the calculated most stable conformation. Thus, the
observed ROE interactions for conformer I seemed to be
Figure 4. Overlay of crystal structures of RA-VII (1, red), RA-XXV (4A, green),
and RA-XXVI acetate (6A, blue).
by an intramolecular hydrogen bond between Ala-2 NH and Tyr-
5 C=O (N–O distance, 2.71 Å; N–H–O angle, 152°), and a type
II′ β-turn at Tyr-3 and Ala-4. This crystal structure of 4 is also
stabilized by a water molecule that forms hydrogen bonds with
D-Ala-1 NH (N–O distance, 2.89 Å; N–H–O angle, 171°) and Tyr-
3 C=O (O–O distance, 2.79 Å; O–H–O angle, 171°).
The structure of 6 has a cis amide between Tyr-5 and Tyr-6,
and the other five peptide bonds are in all trans conformation.
This amide conformation pattern is the same as that of the
crystal structure of 1, which was identified as the active
conformation of peptide 1.[16b,18,21] The backbone structure of 6
bears a good resemblance to that of 1, namely, the three
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consistent with the conformation shown in Figure 5. This most
stable conformation obtained from a Monte Carlo conformational
search was stabilized by the intramolecular hydrogen bonds
between Ala-2 NH and Tyr-5 C=O (N–O distance, 2.96 Å; N–H–
O angle, 154°), between Ala-4 NH and D-Ala-1 C=O (3.18 Å;
155°), and between Tyr-5 NH and Ala-2 C=O (2.89 Å; 156°), and
was calculated to be 4.8 kcal/mol more stable than the DFT-
optimized crystal structure of 4.
population 83%. Peptides
4
and
5
adopted
a
similar
conformation to the active conformation of 1 in CDCl3 solution,
which could be responsible for the activity, but their populations
were only 25% and 28%, respectively.
Conclusions
The ROESY cross-peaks derived from the second-most
populated conformer (conformer II) with population 25% were
observed between Ala-2 Hα and Tyr-3 NMe and between Tyr-5
Hα and Tyr-6 Hα, which indicated the presence of trans and cis
amide bonds between Ala-2 and Tyr-3 and between Tyr-5 and
Tyr-6, respectively. A weak ROESY cross-peak between D-Ala-1
Hα and Ala-4 H3β was observed. This transannular cross-peak
was characteristic of the major conformer of RA-VII (1) and the
distance between D-Ala-1 Hα and the nearest methyl proton of
Ala-4 was estimated to be 2.92 Å in the crystal structure of 6.
Additional cross-peaks were observed between D-Ala-1 NH and
Tyr-6 Hα, Ala-2 H3β and Tyr-3 H2δ, Tyr-3 Hα and Tyr-3 NMe, Tyr-
3 Hα and Ala-4 NH, and Tyr-3 NMe and Ala-4 NH. The
calculated distances between those protons in the crystal
structure of 6 were all less than 3.0 Å. These observations
indicated that the solution structure of conformer II resembled
the crystal structure of 6. These observations for RA-XXV (4)
agreed with the results of a ROESY study of RA-XXVI (5). Thus,
the conformational nature of 4 and 5 was found to be essentially
identical as expected from the high similarity of the structures of
4 and 5.
We have isolated two new RA-series peptides RA-XXV (4) and
RA-XXVI (5) from the roots of Rubia cordifolia L. Their relative
structures were determined on the basis of spectroscopic data
and X-ray crystallography of RA-XXV (4) and RA-XXVI acetate
(6), and their absolute stereochemistry was elucidated by the
total synthesis of 4 and the chemical correlation between 4 and
5, revealing that peptides 4 and 5 correspond to the des-N-
methyl congeners of RA-VII (1) and deoxybouvardin (3) at Tyr-5,
respectively. This is the first isolation of RA-series peptides
lacking an N-methyl group at Tyr-5 residue from a natural source.
Investigation of their conformational features showed that in
solution, peptides 4 and 5 preferentially adopt a backbone
conformation not seen in known natural peptides of this series,
and the population of the active conformer is small. Thus, the N-
methyl group at Tyr-5 is necessary for this series of peptides to
take the active conformation preferentially. These findings will be
useful to design the active synthetic analogues of peptide 1.[18]
Experimental Section
General Experimental Procedures Melting points were determined on
a Yanaco MP-3 apparatus and recorded uncorrected. Optical rotations
were measured on a JASCO P-1030 digital polarimeter. NMR spectra
Cytotoxic activity: RA-XXV (4), RA-XXVI (5), and RA-XXVI
acetate (6) were evaluated for their cytotoxic activity on human
promyelocytic leukemia HL-60 and human colonic carcinoma
HCT-116 cells with peptides 1 and 3 for comparison, and their
IC50 values are summarized in Table 3. The cytotoxic activities of
RA-XXV (4) and RA-XXVI (5) on those cell lines were significant
but weaker than those of the corresponding N-methyl congeners.
RA-XXV (4) was 7–19 times less cytotoxic than RA-VII (1), and
RA-XXVI (5) was 7–12 times less cytotoxic than deoxybouvardin
(3). RA-XXVI acetate (6) expressed almost the same activity as
5. In addition to their structural differences, the weaker cytotoxic
activity of 4 and 5 relative to that of 1 and 3 would partly be
accounted for by their weaker tendency to adopt the active
conformation. In CDCl3 solution, RA-VII (1) adopted the active
conformation, demonstrated by the crystal structure of 1,[13] with
were recorded on
a Bruker AV-600, a DRX-500, or a DPX-400
spectrometer at 300 K unless otherwise indicated. The 1H chemical shifts
in CDCl3, C5D5N, or CD3OD were each referenced to the residual CHCl3
(δ=7.26 ppm), C5D4HN (δ=7.21 ppm), or CD2HOD (δ=3.31 ppm)
resonance, and the 13C chemical shifts to the solvent resonance
(δ=77.03, 135.5, or 49.0 ppm). IR spectra were recorded on a JASCO
FT/IR-620 spectrometer and UV spectra, on
a JASCO V-530
spectrophotometer. Mass spectra were obtained with a Micromass LCT
spectrometer. Single-crystal X-ray analysis was carried out on a Bruker
AXS APEX II ULTRA CCD area detector diffractometer with a rotating
anode source (MoKα radiation, λ=0.71073 Å). Preparative HPLC was
carried out on a Shimadzu LC-6AD pump unit equipped with an SPD-10A
UV detector (λ=254 nm) and a pre-packed ODS column (5 μm, 20 × 250
mm) eluted with a solvent mixture at the flow rate of 10 mL min−1
.
Plant Material The roots of Rubia cordifolia L. were commercially
obtained in Tokyo in March 2004. The material was identified by
Professor Koichi Takeya, and a voucher specimen (Tko-0403-01) was
deposited at the Herbarium of Tokyo University of Pharmacy and Life
Sciences.
Table 3
Cytotoxic activity[a]
Compound
HL-60
HCT-116
1
3
4
5
6
0.0032
0.0053
0.062
0.066
0.051
0.0043
0.0070
0.028
0.051
0.066
Extraction and Isolation The dried roots (50 kg) of R. cordifolia were
extracted with MeOH (3 × 175 L). After removal of MeOH under reduced
pressure, the residue (3.6 kg) was partitioned between chloroform and
water. The chloroform-soluble portion (993 g) was placed on a column of
silica gel (Merck, 70–230 mesh, 3.6 kg) and eluted with CHCl3 (9 L),
EtOAc (18 L), and CHCl3/MeOH (9:1, 27 L) sequentially to give three
fractions. After removal of the solvent, the residue of the CHCl3/MeOH
[a] IC50 (μM).
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(9:1) fraction (152 g) was subjected to alumina (Merck, 3 kg) column
chromatography (CC), eluting sequentially with CHCl3 (2 L) and
CHCl3/MeOH (9:1, 12 L). After evaporation, the CHCl3/MeOH (9:1)
fraction (41.9 g) was subjected to aminopropyl-bonded silica gel
(Chromatorex, 200–350 mesh, 300 g) CC, eluting sequentially with
CHCl3 (6 L) and CHCl3/MeOH (9:1, 1 L). The residue obtained after
removal of the solvent of the CHCl3 eluate was crystallized from MeOH to
give crystals of crude RAs (8.5 g) and the mother liquor (ML). After
removal of the solvent, ML (10.2 g) was subjected to ODS HPLC using
MeOH/H2O (60:40, then 100:0) as eluent to give five fractions: M1 (0.42
g), M2 (2.44 g, mostly deoxybouvardin), M3 (0.33 g), M4 (0.11 g, mostly
RA-VII), and M5 (1.12 g). Fraction M3 was separated by repeated ODS
HPLC using MeCN/H2O (30:70) as eluent to give RA-XXV (4, 16.9 mg).
The residue obtained after removal of the solvent of the CHCl3/MeOH
(9:1) eluate from the aminopropyl-bonded silica gel CC was crystallized
from MeOH to give crystals of crude RAs (2.4 g) and ML. After removal
of the solvent, ML (6.2 g) was subjected to ODS HPLC (MeOH/H2O
60:40) to give four fractions: F1 (0.52 g), F2 (1.07 g), F3 (0.31 g), and F4
(0.17 g). Fraction F2 was subjected to ODS HPLC using MeCN/H2O
(28:72) and then MeOH as eluent to give nine fractions: F21 (45 mg),
F22 (424 mg, RA-I), F23 (40 mg), F24 (209 mg), F25 (124 mg), F26 (37
mg), F27 (61 mg), F28 (28 mg), and F29 (MeOH eluate, 39 mg).
Fractions F24 and F25 were each separated by ODS HPLC using
MeOH/H2O (55:45) as eluent to afford RA-XXVI (5, total 15.1 mg).
7.15 (dd, J=8.3, 2.4 Hz, 1H; Tyr-5 Hεb), 7.04 (app d, J=8.6 Hz, 2H; Tyr-3
H2δ), 6.93 (d, J=8.2 Hz, 1H; Tyr-6 Hεa), 6.90 (dd, J=8.4, 2.4 Hz, 1H; Tyr-5
Hεa), 6.81 (app d, J=8.6 Hz, 2H; Tyr-3 H2ε), 6.66 (dd, J=8.2, 1.9 Hz, 1H;
Tyr-6 Hδa), 6.55 (d, J=7.0 Hz, 1H; D-Ala-1 NH), 4.79 (m, 1H; Tyr-5 Hα),
4.43 (d, J=1.9 Hz, 1H; Tyr-6 Hδb), 4.40 (m, 1H; Ala-2 Hα), 4.34 (Tyr-6 Hα,
overlapped), 4.33 (quintet, J=7.0 Hz, 1H; D-Ala-1 Hα), 3.87 (brm, 1H; Ala-
4 Hα), 3.78 (s, 3H; Tyr-3 OMe), 3.58 (dd, J=8.9, 6.8 Hz, 1H; Tyr-3 Hα),
3.50 (Tyr-5 pro-S Hβ, overlapped), 3.45 (Tyr-6 pro-R Hβ, overlapped),
3.40 (Tyr-3 H2β, overlapped), 3.00 (Tyr-5 pro-R Hβ, overlapped), 2.80
(Tyr-6 pro-S Hβ, overlapped), 2.79 (s, 3H; Tyr-6 NMe), 2.71 (s, 3H; Tyr-3
NMe), 2.40 (s, 3H; Tyr-6 Ac), 1.66 (brd, J=6.9 Hz, 3H; Ala-4 H3β), 1.27 (d,
J=7.0 Hz, 3H; D-Ala-1 H3β), 1.25 ppm (d, J=7.0 Hz, 3H; Ala-2 H3β); 13C
NMR (150 MHz, CDCl3) conformer I: δ=173.2 (2C; Ala-2 C=O and Ala-4
C=O), 172.8 (D-Ala-1 C=O), 171.5 (Tyr-5 C=O), 169.97 (Tyr-3 C=O),
169.4 (Tyr-6 C=O), 169.1 (Tyr-6 COCH3), 158.5 (Tyr-3 Cζ), 157.5 (Tyr-5
Cζ), 153.9 (Tyr-6 Cεb), 137.5 (Tyr-6 Cζ), 135.9 (Tyr-6 Cγ), 135.2 (Tyr-5 Cγ),
131.7 (Tyr-5 Cδb), 131.4 (Tyr-5 Cδa), 130.4 (Tyr-3 Cγ), 130.2 (2C; Tyr-3
Cδ), 127.1 (Tyr-5 Cεa), 123.5 (Tyr-5 Cεb), 122.7 (Tyr-6 Cεa), 121.8 (Tyr-6
Cδa), 120.5 (Tyr-6 Cδb), 114.1 (2C; Tyr-3 Cε), 69.3 (Tyr-3 Cα), 67.4 (Tyr-6
Cα), 55.3 (Tyr-3 OMe), 52.7 (Ala-4 Cα), 51.6 (Tyr-5 Cα), 47.2 (D-Ala-1 Cα),
46.1 (Ala-2 Cα), 40.0 (Tyr-6 NMe), 39.6 (Tyr-3 NMe), 36.8 (Tyr-5 Cβ),
33.2 (Tyr-6 Cβ), 32.6 (Tyr-3 Cβ), 20.82 (Tyr-6 COCH3), 16.5 (Ala-4 Cβ),
16.3 (D-Ala-1 Cβ), 15.94 ppm (Ala-2 Cβ); conformer II: δ=172.9 (Ala-2
C=O), 172.8 (Ala-4 C=O), 172.3 (D-Ala-1 C=O), 170.2 (2C; Tyr-5 C=O
and Tyr-6 C=O), 170.03 (Tyr-3 C=O), 169.0 (Tyr-6 COCH3), 158.3 (Tyr-3
Cζ), 158.1 (Tyr-5 Cζ), 155.2 (Tyr-6 Cεb), 137.0 (Tyr-6 Cζ), 135.5 (Tyr-5 Cγ),
134.1 (Tyr-6 Cγ), 132.6 (Tyr-5 Cδa), 130.9 (Tyr-3 Cγ), 130.8 (Tyr-5 Cδb),
130.3 (2C; Tyr-3 Cδ), 125.5 (Tyr-5 Cεb), 124.4 (Tyr-5 Cεa), 122.9 (Tyr-6
Cεa), 121.6 (Tyr-6 Cδa), 114.6 (Tyr-6 Cδb), 113.9 (2C; Tyr-3 Cε), 69.1 (Tyr-
3 Cα), 56.8 (Tyr-6 Cα), 55.2 (Tyr-3 OMe), 52.9 (Ala-4 Cα), 52.5 (Tyr-5 Cα),
47.6 (D-Ala-1 Cα), 45.7 (Ala-2 Cα), 40.1 (Tyr-5 Cβ), 39.4 (Tyr-3 NMe),
33.5 (Tyr-6 Cβ), 32.8 (Tyr-3 Cβ), 29.0 (Tyr-6 NMe), 20.79 (Tyr-6 COCH3),
17.4 (Ala-4 Cβ), 15.89 (Ala-2 Cβ), 15.6 ppm (D-Ala-1 Cβ); IR (film):
νmax=3296, 2985, 2934, 1766, 1661, 1631, 1512, 1246, 1200, 1113, 755
cm−1; HR-ESIMS: m/z calcd for C41H48N6O10+H+: 785.3510 [M+H+];
found: 785.3528.
Characteristics of New Peptides
RA-XXV (4): Colorless plates; m.p. 293–295 °C (MeOH/H2O);
[α]D25=−155 (c=0.063 in MeOH); IR (film): νmax=3291, 3000, 2936, 2837,
1634, 1514, 1445, 1249, 1214, 1128, 1031, 754 cm−1; UV (MeOH): λmax
(ε)=224sh (25100), 277 (3200), 282sh (2700 mol−1dm3cm−1) nm; HR-
ESIMS: m/z calcd for C40H48N6O9+Na+: 779.3380 [M+Na+]; found:
779.3398.
RA-XXVI (5): White amorphous solid; [α]D25=−170 (c=0.048 in MeOH); IR
(film): νmax=3290, 2934, 1637, 1514, 1446, 1247 cm−1; UV (MeOH): λmax
(ε)=225sh (26900), 277 (3700), 283sh (3300 mol−1dm3cm−1) nm; HR-
ESIMS: m/z calcd for C39H46N6O9+H+: 743.3399 [M+H+]; found: 743.3405.
Cbz-3-iodo-L-tyrosine (8): Triethylamine (3.0 mL, 22.0 mmol) was
added to a cooled solution (0 °C) of 3-iodo-L-tyrosine (7) (4.51 g, 14.7
mmol) in 1,4-dioxane/H2O (1:1, 54 mL) and the solution was stirred at
this temperature for 5 min. N-(Benzyloxycarbonyloxy)succinimide (4.03 g,
16.2 mmol) was added to the solution and the mixture was stirred at 0 °C
for 1 h and then at room temperature for 17 h. The mixture was acidified
(pH ~2) by adding aqueous KHSO4 (5%) at 0 °C and then extracted with
EtOAc (3 × 90 mL). The combined organic extracts were washed with
brine (30 mL), dried over MgSO4, and filtered, and the solvent was
removed in vacuo. The residue was subjected to CC (silica gel,
hexane/EtOAc/AcOH 15:15:1) to give 8 (6.36 g, 98%) as a white
crystalline solid: m.p. 123–125 °C; [α]D25=+14 (c=0.56 in MeOH); 1H NMR
(400 MHz, CD3OD): δ=7.57 (d, J=1.9 Hz, 1H), 7.35–7.24 (m, 5H), 7.04
(dd, J=8.2, 1.9 Hz, 1H), 6.73 (d, J=8.2 Hz, 1H), 5.08 (d, J=12.5 Hz, 1H),
5.01 (d, J=12.5 Hz, 1H), 4.35 (dd, J=9.1, 4.9 Hz, 1H), 3.07 (dd, J=14.0,
4.9 Hz, 1H), 2.81 ppm (dd, J=14.0, 9.1 Hz, 1H); 13C NMR (100 MHz,
CD3OD): δ=175.0 (s), 158.4 (s), 156.8 (s), 141.0 (d), 138.2 (s), 131.40 (s),
131.38 (d), 129.5 (d, 2C), 128.9 (d), 128.5 (d, 2C), 115.6 (d), 84.4 (s),
67.5 (t), 56.8 (d), 37.2 ppm (t); IR (film): νmax=3323, 3032, 2926, 1694,
1503, 1414, 1344, 1255, 1217, 1060, 752 cm−1; HR-ESIMS: m/z calcd for
C17H16INO5+Na+: 463.9971 [M+Na+]; found: 463.9959; elemental
analysis calcd (%) for C17H16INO5: C 46.28, H 3.66, N 3.17; found: C
46.30, H 3.83, N 3.24.
Synthetic Study
RA-XXVI acetate (6): RA-XXVI (5) (4.6 mg, 0.0062 mmol) was treated
with acetic anhydride (0.3 mL) in pyridine (0.6 mL) and the mixture was
stirred at room temperature for 20 h. MeOH (1 mL) was added to the
solution and the volatiles were removed in vacuo. The residue was
crystallized from a MeOH/H2O mixture to give acetate 6 (4.3 mg, 88%) as
colorless plates: m.p. 277–281 °C; [α]D25=−129 (c=0.085 in 1,4-dioxane);
1H NMR (600 MHz, CDCl3) conformer I: δ=8.01 (brd, J=7.6 Hz, 1H; Tyr-5
NH), 7.47 (dd, J=8.4, 2.1 Hz, 1H; Tyr-5 Hδb), 7.42 (dd, J=8.2, 2.1 Hz, 1H;
Tyr-5 Hδa), 7.30 (d, J= 5.6 Hz, 1H; Ala-2 NH), 7.27 (dd, J=8.2, 2.4 Hz, 1H;
Tyr-5 Hεa), 7.11 (app d, J=8.6 Hz, 2H; Tyr-3 H2δ), 7.03 (dd, J=8.4, 2.4 Hz,
1H; Tyr-5 Hεb), 6.90 (Ala-4 NH, overlapped), 6.89 (d, J=8.0 Hz, 1H; Tyr-6
Hεa), 6.85 (app d, J=8.6 Hz, 2H; Tyr-3 H2ε), 6.68 (dd, J=8.0, 1.9 Hz, 1H;
Tyr-6 Hδa), 5.79 (brs, 1H; D-Ala-1 NH), 5.15 (m, 1H; Tyr-5 Hα), 5.07 (s,
1H; Tyr-6 Hδb), 4.57 (m, 1H; D-Ala-1 Hα), 4.47 (quintet, J=6.5 Hz; 1H, Ala-
2 Hα), 3.81 (s, 3H; Tyr-3 OMe), 3.67 (quintet, J=7.2 Hz, 1H; Ala-4 Hα),
3.59 (brm, 1H; Tyr-3 Hα), 3.48 (Tyr-3 Hβa, overlapped), 3.41 (dd, J=11.1,
4.1 Hz, 1H; Tyr-6 Hα), 3.37 (dd, J=13.8, 4.3 Hz, 1H; Tyr-3 Hβb), 3.33 (dd,
J=13.0, 5.3 Hz, 1H; Tyr-5 pro-R Hβ), 3.22 (m, 2H; Tyr-5 pro-S Hβ and Tyr-
6 pro-R Hβ), 3.07 (dd, J=14.6, 4.1 Hz, 1H; Tyr-6 pro-S Hβ), 2.77 (s, 3H;
Tyr-3 NMe), 2.73 (s, 3H; Tyr-6 NMe), 2.41 (s, 3H; Tyr-6 Ac), 1.72 (d,
J=7.4 Hz, 3H; Ala-4 H3β), 1.46 (d, J=6.9 Hz, 3H; Ala-2 H3β), 1.22 ppm (d,
J=7.1 Hz, 3H; D-Ala-1 H3β); conformer II: δ=7.45 (dd, J=8.4, 2.1 Hz, 1H;
Tyr-5 Hδb), 7.26 (Tyr-5 Hδa, overlapped), 7.17 (d, J=6.7 Hz, 1H; Ala-4 NH),
Oxazolidinone 9: p-Toluenesulfonic acid monohydrate (157 mg, 0.825
mmol) was added to a suspension of Cbz-3-iodo-L-tyrosine (8) (1.82 g,
4.12 mmol) and paraformaldehyde (2.12 g, 70.6 mmol) in toluene (40
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mL) and the mixture was stirred at room temperature for 3 days. The
mixture was diluted with EtOAc (240 mL) and the solution was washed
sequentially with saturated aqueous NaHCO3 (3 × 40 mL), water (40 mL),
and brine (40 mL), dried over MgSO4, and filtered. The solvent was
removed in vacuo and the residue was crystallized from EtOAc to give 9
(1.66 g, 89%) as colorless fine needles: m.p. 140–141 °C; [α]D26=+158
(c=0.62 in CHCl3); 1H NMR (500 MHz, C5D5N, 360 K): δ=11.79 (brs, 1H),
7.86 (d, J=1.9 Hz, 1H), 7.50 (app d, J=7.5 Hz, 2H), 7.42 (app t, J=7.5 Hz,
2H), 7.35 (app t, J=7.5 Hz, 1H), 7.13 (dd, J=8.1, 1.9 Hz, 1H), 6.99 (d,
J=8.1 Hz, 1H), 5.42 (d, J=4.0 Hz, 1H), 5.37 (d, J=13.0 Hz, 1H), 5.35 (d,
J=13.0 Hz, 1H), 4.78 (d, J=4.0 Hz, 1H), 4.72 (m, 1H), 3.38 (dd, J=14.1,
5.7 Hz, 1H), 3.18 ppm (dd, J=14.1, 3.3 Hz, 1H); 13C NMR (125 MHz,
C5D5N, 360 K): δ=172.2 (s), 157.8 (s), 153.2 (s), 141.1 (d), 136.9 (s),
131.4 (d), 129.2 (d, 2C), 128.8 (d), 128.71 (d, 2C), 128.68 (s), 116.1 (d),
86.1 (s), 78.6 (t), 68.1 (t), 57.0 (d), 35.2 ppm (t); IR (film): νmax=3362,
3031, 2919, 1800, 1712, 1693, 1418, 1359, 1129, 1051, 751 cm−1; HR-
ESIMS: m/z calcd for C18H16INO5+Na+: 475.9971 [M+Na+]; found:
475.9976; elemental analysis calcd (%) for C18H16INO5: C 47.70, H 3.56,
N 3.09; found: C 47.61, H 3.69, N 3.16.
stirring at room temperature for 2 h, a solution of sodium (meta)periodate
(658 mg, 3.08 mmol) in water (70 mL) was added and the mixture was
stirred at room temperature for 18 h. The mixture was concentrated in
vacuo to remove acetone and the residue was extracted with CHCl3 (3 ×
50 mL). The combined organic extracts were washed with brine (50 mL),
dried over MgSO4, and filtered, and the solvent was removed in vacuo.
The residue was subjected to CC (silica gel, hexane/EtOAc 3:2) to afford
11 (1.16 g, 94%) as a colorless amorphous solid: [α]D25=−55 (c=0.51 in
CHCl3); 1H NMR (400 MHz, CDCl3, mixture of two rotamers) major
rotamer: δ=7.68 (d, J=1.8 Hz, 1H), 7.34–7.18 (m, 6H), 6.80 (d, J=8.5 Hz,
1H), 6.30 (s, 2H), 5.11 (d, J=12.6 Hz, 1H), 5.07 (d, J=12.6 Hz, 1H), 5.00
(dd, J=10.9, 5.3 Hz, 1H), 3.87 (s, 3H), 3.74 (s, 3H), 3.31 (dd, J=14.6, 5.3
Hz, 1H), 3.01 (dd, J=14.6, 10.9 Hz, 1H), 2.82 ppm (s, 3H); minor rotamer:
δ=7.68 (d, J=1.8 Hz, 1H), 7.34–7.18 (m, 5H), 7.15 (dd, J=8.5, 1.8 Hz, 1H),
6.75 (d, J=8.5 Hz, 1H), 6.27 (s, 2H), 5.05 (d, J=12.6 Hz, 1H), 5.02 (d,
J=12.6 Hz, 1H), 4.80 (dd, J=10.8, 4.8 Hz, 1H), 3.87 (s, 3H), 3.67 (s, 3H),
3.26 (dd, J=14.4, 4.8 Hz, 1H), 2.96 (dd, J=14.4, 10.8 Hz, 1H), 2.84 ppm
(s, 3H); 13C NMR (100 MHz, CDCl3, mixture of two rotamers) major
rotamer: δ=171.5 (s), 163.3 (s), 156.6 (s), 137.2 (d), 136.7 (s), 133.0 (d),
129.5 (s), 128.4 (d, 2C), 127.8 (d), 127.5 (d, 2C), 118.4 (s), 110.1 (d),
67.2 (t), 60.2 (d), 55.5 (q), 52.3 (q), 33.9 (t), 31.6 ppm (q); minor rotamer:
δ=171.3 (s), 163.3 (s), 155.9 (s), 137.2 (d), 136.4 (s), 133.1 (d), 129.6 (s),
128.4 (d, 2C), 127.9 (d, 2C), 127.8 (d), 118.6 (s), 110.08 (d), 67.4 (t),
60.7 (d), 55.5 (q), 52.3 (q), 34.3 (t), 32.1 ppm (q); IR (film): νmax=3408,
3030, 2952, 2842, 1742, 1698, 1607, 1493, 1421, 1331, 1236, 1042,
Cbz-3-iodo-N,O-dimethyl-L-Tyr-OMe (10): Triethylsilane (1.6 mL, 10
mmol) and TFA (21 mL) were added to a solution of 9 (1.55 g, 3.42
mmol) in CHCl3 (21 mL), and the mixture was stirred at room temperature
for 3 days. Volatiles were removed in vacuo and the residue was
dissolved
in
MeCN/MeOH
(9:1,
14
mL).
To
this,
;
1008, 755, 698 cm−1 HR-ESIMS: m/z calcd for C20H24BNO7+H+:
(trimethylsilyl)diazomethane (2.0 M in diethyl ether, 5.1 mL, 10.2 mmol)
was added. The mixture was stirred at room temperature for 2 days and
concentrated in vacuo. The residue was dissolved in MeCN/MeOH (9:1,
14 mL) again and treated with (trimethylsilyl)diazomethane (2.0 M in
diethyl ether, 1.7 mL, 3.4 mmol). After stirring for 2 days at room
temperature, the solution was concentrated in vacuo. The residue was
subjected to CC (silica gel, hexane/EtOAc 7:2) to afford 10 (1.26 g, 76%)
as a colorless amorphous solid: [α]D25=−47 (c=0.63 in CHCl3); 1H NMR
(400 MHz, CDCl3, mixture of two rotamers) major rotamer: δ=7.61 (s, 1H),
7.37–7.20 (m, 5H), 7.13 (d, J=8.3 Hz, 1H), 6.70 (d, J=8.3 Hz, 1H), 5.13 (d,
J=12.6 Hz, 1H), 5.09 (d, J=12.6 Hz, 1H), 4.92 (dd, J=10.4, 5.5 Hz, 1H),
3.84 (s, 3H), 3.74 (s, 3H), 3.25 (dd, J=14.7, 5.5 Hz, 1H), 2.94 (dd, J=14.7,
10.4 Hz, 1H), 2.81 ppm (s, 3H); minor rotamer: δ=7.59 (s, 1H), 7.37–7.20
(m, 5H), 7.02 (d, J=8.3 Hz, 1H), 6.65 (d, J=8.3 Hz, 1H), 5.07 (d, J=12.5
Hz, 1H), 5.04 (d, J=12.5 Hz, 1H), 4.74 (dd, J=10.5, 4.8 Hz, 1H), 3.84 (s,
3H), 3.67 (s, 3H), 3.20 (dd, J=14.9, 4.8 Hz, 1H), 2.89 (dd, J=14.9, 10.5
Hz, 1H), 2.84 ppm (s, 3H); 13C NMR (100 MHz, CDCl3, mixture of two
rotamers) major rotamer: δ=171.2 (s), 157.0 (s), 156.6 (s), 139.8 (d),
136.6 (s), 131.3 (s), 129.9 (d), 128.5 (d, 2C), 127.9 (d), 127.6 (d, 2C),
110.9 (d), 85.7 (d), 67.3 (t), 60.3 (d), 56.3 (q), 52.3 (q), 33.5 (t), 31.8 ppm
(q); minor rotamer: δ=171.0 (s), 157.0 (s), 155.8 (s), 139.7 (d), 136.3 (s),
131.3 (s), 130.0 (d), 128.5 (d, 2C), 128.1 (d), 128.0 (d, 2C), 110.8 (d),
86.0 (d), 67.5 (t), 60.6 (d), 56.3 (q), 52.3 (q), 33.9 (t), 32.1 ppm (q); IR
(film): νmax=3030, 3006, 2951, 2839, 1742, 1702, 1492, 1454, 1401, 1317,
1281, 1254, 1218, 1181, 1137, 1049, 1016, 699 cm−1; HR-ESIMS: m/z
calcd for C20H22INO5+Na+: 506.0440 [M+Na+]; found: 506.0436.
402.1724 [M+H+]; found: 402.1707.
Amine 12: Palladium (10%) on charcoal catalyst (48.9 mg) was added to
a solution of 11 (489 mg, 1.22 mmol) in EtOH (13 mL). Then, the reaction
mixture was stirred at room temperature under an atmosphere of
hydrogen for 20 min. The catalyst was filtered off, the filtrate was
concentrated to dryness, and the residue was crystallized from EtOH to
give 12 (291 mg) as
a white crystalline solid. Chromatographic
separation (silica gel, CHCl3/MeOH 30:1) of ML gave additional 12 (30
mg, total 321 mg, 99%): m.p. 268–273 °C (decomp.); [α]D25=+25 (c=0.72
in CHCl3); 1H NMR (400 MHz, CDCl3): δ=7.65 (d, J=2.3 Hz, 1H), 7.24 (dd,
J=8.5, 2.3 Hz, 1H), 6.84 (d, J=8.5 Hz, 1H), 3.89 (s, 3H), 3.69 (s, 3H),
3.44 (t, J=6.7 Hz, 1H), 2.94 (dd, J=13.7, 6.4 Hz, 1H), 2.91 (dd, J=13.7,
6.9 Hz, 1H), 2.36 ppm (s, 3H); 13C NMR (100 MHz, CDCl3): δ=174.8 (s),
163.5 (s), 137.5 (d), 133.5 (d), 129.7 (s), 110.0 (d), 64.7 (d), 55.6 (q),
51.7 (q), 38.5 (t), 34.7 ppm (q); IR (film): νmax=3515, 3321, 2950, 1734,
1605, 1582, 1492, 1418, 1335, 1236, 1177, 1046, 818, 756 cm−1; HR-
ESIMS: m/z calcd for C12H18BNO5+H+: 268.1356 [M+H+]; found:
268.1368.
Dipeptide 13: EDC·HCl (290 mg, 1.51 mmol) was added to a solution of
12 (202 mg, 0.756 mmol), Boc-L-Tyr-OH (425 mg, 1.51 mmol), and HOAt
(206 mg, 1.51 mmol) in DMF (3.7 mL) and the mixture was stirred at
room temperature for 7 days. The solvent was removed in vacuo. The
residue was treated with CHCl3 (10 mL) and aqueous citric acid (10%, 20
mL) and extracted with CHCl3 (3 × 20 mL). The combined organic
extracts were washed with brine (20 mL), dried over MgSO4, and filtered,
and the solvent was removed in vacuo. The residue was subjected to
HPLC (MeCN/H2O 30:70) to give 13 (341 mg, 85%) as a colorless
amorphous solid: [α]D25=−48 (c=0.32 in CHCl3); 1H NMR (500 MHz,
C5D5N, major rotamer): δ=11.29 (s, 1H), 9.17 (s, 2H), 8.22 (d, J=2.1 Hz,
1H), 8.10 (d, J=9.1 Hz, 1H), 7.37 (app d, J=8.4 Hz, 2H), 7.29 (dd, J=8.4,
2.1 Hz, 1H), 7.12 (app d, J=8.4 Hz, 2H), 6.84 (d, J=8.4 Hz, 1H), 5.22 (m,
1H), 5.20 (m, 1H), 3.64 (s, 3H), 3.60 (s, 3H), 3.48 (dd, J=14.2, 5.7 Hz,
1H), 3.39 (dd, J=13.7, 6.6 Hz, 1H), 3.28 (dd, J=14.2, 9.5 Hz, 1H), 3.18
(dd, J=13.7, 7.4 Hz, 1H), 3.04 (s, 3H), 1.45 ppm (s, 9H); 13C NMR (125
MHz, C5D5N, major rotamer): δ=172.9 (s), 171.3 (s), 163.4 (s), 157.6 (s),
156.2 (s), 137.8 (d), 132.8 (d), 131.1 (d, 2C), 130.2 (s), 128.4 (s), 122.0
Boronic acid 11: PdCl2(dppf)·CH2Cl2 (378 mg, 0.463 mmol) was added
to a solution of 10 (1.49 g, 3.08 mmol), bis(pinacolato)diboron (1.02 g,
4.02 mmol), and potassium acetate (909 mg, 9.26 mmol) in dimethyl
sulfoxide (21 mL) and the mixture was stirred at 80 °C for 20 h under an
atmosphere of argon. Water (250 mL) was added to the solution, and the
whole was extracted with EtOAc (4 × 36 mL). The combined organic
extracts were washed with brine (36 mL), dried over MgSO4, and filtered,
and the solvent was removed in vacuo. The residue was subjected to CC
(silica gel). After evaporation, the residue of the fraction eluted with
hexane/EtOAc (3:1) was dissolved in acetone (140 mL) and to this, a
solution of ammonium acetate (476 mg, 6.18 mmol) and sodium
(meta)periodate (658 mg, 3.08 mmol) in water (70 mL) was added. After
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10.1002/asia.201801466
Chemistry - An Asian Journal
FULL PAPER
(s), 116.1 (d, 2C), 110.6 (d), 78.5 (s), 61.1 (d), 55.3 (q), 53.0 (d), 51.8 (q),
38.3 (t), 34.2 (q), 34.2 (t), 28.4 ppm (q, 3C); IR (film): νmax=3346, 3013,
2978, 1740, 1695, 1635, 1613, 1517, 1494, 1420, 1367, 1238, 1170,
1046, 1020, 822, 757 cm−1; HR-ESIMS: m/z calcd for C26H35BN2O9+Na+:
553.2333 [M+Na+]; found: 553.2333.
583.2411; elemental analysis calcd (%) for C32H36N2O7: C 68.55, H 6.47,
N 5.00; found: C 68.61, H 6.47, N 4.98.
Hexapeptide 17: A solution of 15 (12.2 mg, 0.0218 mmol) in TFA (1.0
mL) was stirred at room temperature for 2 h. TFA was removed in vacuo.
The residue was treated with CHCl3 (5 mL) and saturated aqueous
NaHCO3 (10 mL) and the whole was extracted with CHCl3 (3 × 10 mL).
The combined CHCl3 extracts were washed with brine (10 mL), dried
over MgSO4, and filtered, and the solvent was removed in vacuo. The
residue, 16 (17.1 mg, 0.0327 mmol), and HOOBt (5.3 mg, 0.032 mmol)
were dissolved in DMF (0.5 mL) and to this, EDC·HCl (6.3 mg, 0.033
mmol) was added at 0 °C. The mixture was stirred at room temperature
for 10 days. Saturated aqueous NaHCO3 (10 mL) was added to the
solution and the whole was extracted with CHCl3 (3 × 10 mL). The
combined organic extracts were washed sequentially with aqueous citric
acid (10%, 2 × 10 mL) and brine (10 mL), dried over Na2SO4, and filtered,
and the solvent was removed in vacuo. The residue was separated by
CC (silica gel, hexane/CHCl3/MeOH 20:20:1) and then by HPLC
(MeCN/H2O 55:45) to give 17 (14.6 mg, 70%) as a colorless amorphous
solid: [α]D26=−99 (c=0.13 in CHCl3); 1H NMR (500 MHz, CDCl3, mixture of
rotamers): δ=8.16 (d, J=8.3 Hz), 7.54–7.25 (m), 7.23 (dd, J=8.3, 1.9 Hz),
7.17 (d, J=8.2 Hz), 7.10 (d, J=8.4 Hz), 7.00 (d, J=8.4 Hz), 6.91 (d, J=8.3
Hz), 6.90 (d, J=8.3 Hz), 6.84 (d, J=8.5 Hz), 6.79 (d, J=8.3 Hz), 6.74 (d,
J=8.1 Hz), 6.61–6.54 (m), 5.85 (brm), 5.57 (brm), 5.29 (brm), 5.13 (s),
5.02 (s), 4.84 (dd, J=11.3, 2.4 Hz), 4.84–4.76 (m), 4.74–4.66 (m), 4.60 (d,
J=10.1 Hz), 4.54 (septet, J=7.8 Hz), 4.50 (d, J=10.8 Hz), 4.44–4.28 (m),
4.34 (s), 4.23 (brm), 3.93 (s), 3.78 (s), 3.74 (s), 3.41–3.18 (m), 3.08 (dd,
J=11.8, 3.7 Hz), 3.02–2.87 (m), 2.89 (s), 2.86 (s), 2.59 (s), 2.57 (s), 1.44
(s), 1.43 (s), 1.40 (d, J=6.3 Hz), 1.36 (d, J=6.4 Hz), 1.30 (d, J=7.1 Hz),
0.47 ppm (d, J=6.6 Hz); 13C NMR (125 MHz, CDCl3, mixture of rotamers,
selected data): δ=174.5 (s), 174.1 (s), 173.5 (s), 173.4 (s), 173.1 (s),
172.2 (s), 171.1 (s), 170.8 (s), 170.6 (s), 169.0 (s), 168.8 (s), 158.6 (s),
158.5 (s), 158.10 (s), 158.05 (s), 156.0 (s), 155.9 (s), 153.11 (s), 153.06
(s), 146.4 (s), 146.3 (s), 135.4 (s), 135.3 (s), 135.1 (s), 132.6 (d), 132.3
(d), 130.8 (d), 130.7 (d), 130.3 (d), 130.1 (d), 129.8 (s), 129.7 (s), 128.7
(d), 128.64 (d), 128.61 (d), 128.58 (d), 128.5 (d), 127.9 (s), 127.7 (s),
126.0 (d), 124.5 (d), 120.6 (d), 114.4 (d), 114.1 (d), 113.6 (d), 113.5 (d),
112.3 (d), 80.4 (s), 79.9 (s), 68.0 (t), 67.9 (t), 65.3 (d), 63.4 (d), 57.7 (d),
57.5 (d), 56.2 (q), 55.34 (q), 55.25 (q), 52.2 (d), 51.7 (d), 50.4 (d), 50.1
(d), 49.9 (d), 49.3 (d), 45.8 (d), 44.9 (d), 39.7 (t), 36.8 (q), 32.9 (t), 32.7 (t),
32.41 (t), 32.36 (t), 29.6 (q), 28.39 (q), 28.35 (q), 28.2 (q), 18.0 (q), 17.6
(q), 17.1 (q), 16.5 (q), 16.0 ppm (q); IR (film): νmax=3301, 2978, 2933,
1742, 1635, 1515, 1249, 1177, 1029, 754 cm−1; HR-ESIMS: m/z calcd for
C52H64N6O12+Na+: 987.4480 [M+Na+]; found: 987.4496.
Cycloisodityrosine 14: A mixture of 13 (55.9 mg, 0.105 mmol), DMAP
(64.1 mg, 0.525 mmol), 4Å powdered molecular sieves (300 mg) in
CH2Cl2 (10 mL) was stirred at room temperature for 30 min. Copper(II)
acetate (19.1 mg, 0.105 mmol) was added and the mixture was stirred at
room temperature for 2 days. Insoluble materials were removed by
filtration and aqueous KHSO4 (5%, 10 mL) was added to the filtrate. The
whole was extracted with CHCl3 (3 × 10 mL). The combined extracts
were washed with brine (10 mL), dried over MgSO4, and filtered, and the
solvent was removed in vacuo. The residue was separated by CC (silica
gel, hexane/EtOAc 1:5) and then by HPLC (MeCN/H2O 48:52) to give 14
(18.7 mg, 37%) as colorless prisms: m.p. 184–185 °C; [α]D26=−103
(c=0.29 in CHCl3); 1H NMR (500 MHz, CDCl3, major rotamer): δ=7.44 (dd,
J=8.4, 2.2 Hz, 1H), 7.23 (dd, J=8.4, 2.2 Hz, 1H), 7.16 (dd, J=8.4, 2.4 Hz,
1H), 6.92 (dd, J=8.4, 2.4 Hz, 1H), 6.82 (d, J=8.2 Hz, 1H), 6.61 (app d,
J=8.2 Hz, 1H), 5.12 (d, J=9.6 Hz, 1H), 4.45 (dd, J=12.1, 3.3 Hz, 1H), 4.41
(m, 1H), 4.33 (d, J=2.1 Hz, 1H), 3.94 (s, 3H), 3.70 (s, 3H), 3.26 (t, J=11.3
Hz, 1H), 3.22 (dd, J=17.9, 3.3 Hz, 1H), 3.06 (dd, J=11.7, 4.0 Hz, 1H),
2.99 (dd, J=17.9, 12.1 Hz, 1H), 2.61 (s, 3H), 1.44 ppm (s, 9H); 13C NMR
(100 MHz, CDCl3, major rotamer): δ=173.8 (s), 171.4 (s), 158.2 (s), 154.6
(s), 153.1 (s), 146.3 (s), 134.9 (s), 132.4 (d), 130.5 (d), 127.9 (s), 125.9
(d), 124.7 (d), 120.6 (d), 113.6 (d), 112.3 (d), 79.8 (s), 57.4 (d), 56.2 (q),
53.1 (d), 52.7 (q), 40.8 (t), 32.6 (t), 28.4 (q, 3C), 28.2 ppm (q); IR (film):
νmax=3310, 2977, 2933, 2838, 1747, 1708, 1643, 1518, 1499, 1265, 1205,
1169, 1129, 754 cm−1
;
HR-ESIMS: m/z calcd for C26H32N2O7+H+:
485.2289 [M+H+]; found: 485.2288; elemental analysis calcd (%) for
C26H32N2O7: C 64.45, H 6.66, N 5.78; found: C 64.38, H 6.64, N 5.83.
Benzyl ester 15: A LiOOH solution [LiOH·H2O (3.5 mg, 0.083 mmol)
dissolved in aqueous H2O2 (35%, 0.1 mL)] was slowly added to a cooled
(0 °C) solution of 14 (10.2 mg, 0.0211 mmol) in MeOH (0.4 mL). The
solution was stirred at 0 °C for 1 h and then at room temperature for 23 h.
Aqueous NaHSO3 (5%, 0.5 mL) and aqueous citric acid (10%, 1 mL)
were added to the solution at 0 °C. After stirring for 10 min, the mixture
was extracted with CHCl3 (3 × 10 mL). The combined organic extracts
were washed with brine (5 mL), dried over MgSO4, and filtered, and the
solvent was removed in vacuo. The residue, triphenylphosphine (22.0 mg,
0.0839 mmol), and benzyl alcohol (9.0 µL, 0.087 mmol) were dissolved in
THF (0.4 mL) and to this, diethyl azodicarboxylate (2.2 M in toluene, 38
µL, 0.084 mmol) was slowly added at 0 °C. The mixture was stirred at
0 °C for 1 h and then at room temperature for 10 h. The solvent was
removed in vacuo and the residue was subjected to HPLC (MeCN/H2O
60:40) to give 15 (8.8 mg, 75%) as a white crystalline solid: m.p. 205–
206 °C; [α]D25=−82 (c=0.41 in CHCl3); 1H NMR (500 MHz, CDCl3, major
rotamer): δ=7.45 (dd, J=8.4, 2.2 Hz, 1H), 7.39–7.29 (m, 5H), 7.24 (dd,
J=8.4, 2.2 Hz, 1H), 7.16 (dd, J=8.4, 2.4 Hz, 1H), 6.92 (dd, J=8.4, 2.4 Hz,
1H), 6.79 (d, J=8.4 Hz, 1H), 6.58 (dd, J=8.4, 1.9 Hz, 1H), 5.16 (d, J=9.6
Hz, 1H), 5.15 (d, J=12.0 Hz, 1H), 5.05 (d, J=12.0 Hz, 1H), 4.50–4.43 (m,
2H), 4.32 (d, J=1.9 Hz, 1H), 3.93 (s, 3H), 3.26 (t, J=11.3 Hz, 1H), 3.20
(dd, J=18.0, 2.9 Hz, 1H), 3.07 (dd, J=11.9, 4.0 Hz, 1H), 2.96 (dd, J=18.0,
12.1 Hz, 1H), 2.61 (s, 3H), 1.46 ppm (s, 9H); 13C NMR (125 MHz, CDCl3,
major rotamer): δ=174.0 (s), 170.9 (s), 158.2 (s), 154.6 (s), 153.1 (s),
146.3 (s), 135.2 (s), 134.9 (s), 132.5 (d), 130.5 (d), 128.7 (d, 2C), 128.6
(d), 128.5 (d, 2C), 127.8 (s), 126.0 (d), 124.7 (d), 120.6 (d), 113.6 (d),
112.3 (d), 79.8 (s), 67.7 (t), 57.6 (d), 56.2 (q), 53.0 (d), 40.8 (t), 32.5 (t),
28.5 (q, 3C), 28.3 ppm (q); IR (film): νmax=3436, 3315, 2977, 2932, 1746,
Amine 18: A solution of 17 (12.8 mg, 0.0133 mmol) in TFA (1.0 mL) was
stirred at room temperature for 2.5 h. TFA was removed in vacuo. The
residue was treated with CHCl3 (5 mL) and saturated aqueous NaHCO3
(5 mL) and the whole was extracted with CHCl3 (3 × 10 mL). The
combined CHCl3 extracts were washed with brine (10 mL), dried over
MgSO4, and filtered, and the solvent was removed in vacuo. The residue
was separated by CC (silica gel, CHCl3/MeOH 20:1) to give amine 18
(8.8 mg, 77%) as a colorless amorphous solid: [α]D26=−100 (c=0.09 in
1
CHCl3); H NMR (400 MHz, CDCl3, major rotamer): δ=8.52 (d, J=8.9 Hz,
1H), 7.60 (d, J=4.8 Hz, 1H), 7.46 (dd, J=8.3, 2.3 Hz, 1H), 7.43–7.31 (m,
5H), 7.28 (d, J=9.1 Hz, 1H), 7.18 (dd, J=8.3, 2.3 Hz, 1H), 7.17 (dd, J=8.3,
2.3 Hz, 1H), 7.01 (app d, J=8.7 Hz, 2H), 6.89 (dd, J=8.3, 2.3 Hz, 1H),
6.79 (app d, J=8.7 Hz, 3H), 6.58 (dd, J=8.3, 2.1 Hz, 1H), 5.14 (s, 2H),
4.83 (dd, J=11.2, 3.1 Hz, 1H), 4.70 (m, 1H), 4.60 (dq, J=8.9, 7.3 Hz, and
overlapping signal, 3H), 4.51 (dd, J=12.2, 3.3 Hz, 1H), 4.34 (d, J=2.1 Hz,
1H), 4.30 (qd, J=6.8, 4.8 Hz, 1H), 3.93 (s, 3H), 3.75 (s, 3H), 3.66 (q,
J=7.0 Hz, 1H), 3.38 (dd, J=14.8, 3.0 Hz, 1H), 3.20 (m, 1H), 3.03–2.86 (m,
4H), 2.90 (s, 3H), 2.55 (s, 3H), 1.43 (d, J=7.3 Hz, 3H), 1.34 (d, J=7.0 Hz,
3H), 0.50 ppm (d, J=6.8 Hz, 3H); 13C NMR (100 MHz, CDCl3, major
1707, 1644, 1518, 1498, 1280, 1265, 1218, 1200, 1172, 1129, 754 cm−1
HR-ESIMS: m/z calcd for C32H36N2O7+Na+: 583.2420 [M+Na+]; found:
;
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rotamer): δ=177.5 (s), 173.4 (s), 172.9 (s), 172.3 (s), 171.1 (s), 168.9 (s),
158.6 (s), 158.0 (s), 153.0 (s), 146.3 (s), 135.5 (s), 135.3 (s), 132.4 (d),
130.7 (d), 130.3 (d, 2C), 130.1 (s), 128.6 (d, 4C), 128.4 (d), 127.8 (s),
126.1 (d), 124.4 (d), 120.7 (d), 114.3 (d, 2C), 113.5 (d), 112.2 (d), 68.0 (t),
63.5 (d), 57.4 (d), 56.2 (q), 55.3 (q), 52.0 (d), 50.5 (d), 50.4 (d), 44.6 (d),
39.7 (t), 32.8 (t), 32.4 (t), 29.5 (q), 28.2 (q), 21.1 (q), 18.2 (q), 15.8 ppm
(q); IR (film): νmax=3295, 2960, 2931, 1740, 1644, 1633, 1515, 1262,
1202, 1128, 1092, 1030, 803, 756 cm−1; HR-ESIMS: m/z calcd for
C47H56N6O10+Na+: 887.3956 [M+Na+]; found: 887.3956.
(DMSO) (1, 0.3, 0.1, 0.03, 0.01, 0.003, 0.001, 0.0003, 0.0001 μg mL−1)
were prepared and 10 μL of the test solution or DMSO (control) was
added to each well. The plate was kept in an incubator for 48 h. After
termination of the cell culture by adding 0.5% MTT in PBS (20 μL) to
each well, the plate was kept in the incubator for 4 h. After addition of
100 μL of 10% SDS-0.01 N aqueous HCl to each well, the plate was read
on a microplate reader (MPR A4i, Tosoh Co., Japan) at 550/700 nm. A
dose-response curve was plotted for each compound and concentrations
exhibiting 50% inhibition of cell growth (IC50) were recorded.
The assay using human colon carcinoma HCT-116 cells was
performed in the same manner except that McCoy’s 5A medium
(Invitrogen Co., USA) supplemented with 10% heat-inactivated fetal
bovine serum (FBS) and kanamycin (100 mg L−1) was used for preculture
and that the cell suspension used was 3 × 105 cells mL−1, 100 μL, i.e., 3
RA-XXV (4): Palladium (10%) on charcoal catalyst (17.6 mg) was added
to a solution of 18 (8.8 mg, 0.010 mmol) in EtOH (2 mL). Then, the
reaction mixture was stirred at room temperature under an atmosphere of
hydrogen for 2.5 h. The catalyst was filtered off, the filtrate was
concentrated to dryness, and the residue was dissolved in DMF (7.7 mL).
To this solution were added HOOBt (13.1 mg, 0.0803 mmol) and
EDC·HCl (15.3 mg, 0.0798 mmol) at 0 °C. After stirring at 0 °C for 2 h
and then at room temperature for 3 days, the solvent was removed under
reduced pressure. The residue was treated with CHCl3 (5 mL) and
aqueous citric acid (10%, 10 mL) and the whole was extracted with
CHCl3 (3 × 10 mL). The combined organic extracts were washed
sequentially with saturated aqueous NaHCO3 (10 mL) and brine (10 mL),
dried over MgSO4, and filtered, and the solvent was removed in vacuo.
The residue was separated by CC (silica gel, hexane/EtOAc 3:2, then
CHCl3/MeOH 5:1), and then by HPLC (MeOH/H2O 58:42) to give a
compound [3.2 mg, 42%, [α]D25=−152 (c=0.050 in MeOH)] that was
shown to be identical to natural 4 by comparison of their 1H (600 MHz)
and 13C NMR (150 MHz) spectra, IR spectra, mass spectra, and optical
rotations.
× 104 cells well−1
.
Acknowledgements
This work was supported by JSPS KAKENHI Grant Number
JP16K08308.
Conflict of interest
The authors declare no conflict of interest.
Keywords: Rubia cordifolia • RA-XXV • RA-XXVI • synthesis •
O-Methylation of RA-XXVI (5): Iodomethane (4.5 µL, 0.072 mmol) and
K2CO3 (2.0 mg, 0.014 mmol) were added to a suspension of RA-XXVI (5)
(2.6 mg, 0.0035 mmol) in acetone/MeOH (3:1, 0.2 mL) and the mixture
was stirred at 40 °C for 6 h. The mixture was passed through a CBA
cartridge column with MeOH and the eluate was separated by HPLC
(MeCN/H2O 40:60) to give a compound [2.5 mg, 94%, [α]D25=−157
(c=0.020 in MeOH)] that was shown to be identical to natural 4 by
comparison of their 1H (600 MHz) and 13C NMR (150 MHz) spectra, IR
spectra, mass spectra, and optical rotations.
conformation
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Monte Carlo conformational search and DFT calculations
The software used for the Monte Carlo (MC) conformational search was
MacroModel ver. 8.1 (Schrödinger, Portland, OR). The MC search was
configured using the “automatic setup” routine in MacroModel with all
amide configuration in the macrocycle fixed as determined by the
ROESY experiments. Calculation consisted of 50000 MC steps with 500
iterations per step using the MMFFs force field and the PR conjugate
gradient (PRCG) with no solvation. DFT calculations were performed
using Gaussian 09W (revision B.01) software[24] with B3LYP functional
and 6-31G(d) basis set.
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Assays for Cytotoxic Activity
Assays for cytotoxic activity were performed by using the 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay
method. Human promyelocytic leukemia HL-60 cells were precultured in
RPMI 1640 medium (Nissui Co. Ltd., Japan) supplemented with 10%
heat-inactivated fetal bovine serum (FBS) and kanamycin (100 mg L−1) in
a humidified atmosphere of 93% air and 7% CO2 at 37 °C. The cell
suspension (3 × 104 cells mL−1, 100 μL) was added to each well (3 × 103
cells well−1) of a 96-microwell plate (flat-bottomed, polystyrene-treated)
and incubated for 24 h. Test compound solutions in dimethyl sulfoxide
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[23] Abbreviations:
DMAP=4-(dimethylamino)pyridine,
(dimethylaminopropyl)carbodiimide hydrochloride, HOAt=1-hydroxy-7-
azabenzotriazole, HOOBt=3-hydroxy-1,2,3-benzotriazin-4(3H)-one,
Cbz-OSu=N-(benzyloxycarbonyloxy)succinimide,
EDC·HCl=1-ethyl-3-
PdCl2(dppf)·CH2Cl2=[1,1´-
bis(diphenylphosphino)ferrocene]dichloropalladium(II) dichloromethane
complex, TFA=trifluoroacetic acid.
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Two new RA-series bicyclic
Yukio Hitotsuyanagi,* Masahito Hirai,
Masumi Odagiri, Miho Komine, Tomoyo
Hasuda, Haruhiko Fukaya, and Koichi
Takeya
hexapeptides, RA-XXV (4) and RA-
XXVI (5), which have no N-methyl
group at Tyr-5, were isolated from the
roots of Rubia cordifolia L. and their
absolute structures were established
by synthetic methods. Investigation of
their conformational property and
cytotoxic acitivity revealed that the N-
methyl group at Tyr-5 is necessary for
this series of peptides to take the
active conformation preferentially.
Page No. – Page No.
RA-XXV and RA-XXVI, Bicyclic
Hexapeptides from Rubia cordifolia L.
Structure, Synthesis, and
Conformation
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