The synthetic and biological studies of discorhabdins and related compounds

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PAPER

The synthetic and biological studies of discorhabdins and related compounds†

Yasufumi Wada,^aYu Harayama,^aDaigo Kamimura,^aMasako Yoshida,^aTomoyuki Shibata,^c

Kousaku Fujiwara,^cKoji Morimoto,^bHiromichi Fujioka*^aand Yasuyuki Kita*^a,b

Received 11th January 2011, Accepted 11th April 2011

DOI: 10.1039/c1ob05058c

Various analogues of the marine alkaloids, discorhabdins, have been synthesized. The strategy contains

spirocyclization with phenyliodine(III) bis(triﬂuoroacetate) (PIFA), oxidative fragmentation of the

b-amino alcohols with the hypervalent iodine reagent C₆F₅I(OCOCF₃)₂, the detosylation and

dehydrogenation reaction of the pyrroloiminoquinone unit in the presence of a catalytic amount of

NaN₃and the bridged ether synthesis with HBr–AcOH as the key reactions. All the synthesized

compounds were evaluated by in vitro MTT assay for cytotoxic activity against the human colon cancer

cell line HCT-116. Furthermore, the discorhabdin A oxa analogues were also evaluated against four

kinds of tumor model cells, a human colon cancer cell line (WiDr), a human prostate cancer cell line

(DU-145) and murine leukemia cell lines (P388 and L1210). For the identiﬁcation of the target,

discorhabdin A and the discorhabdin A oxa analogue were evaluated by an HCC panel assay. In the

test, discorhabdins could have a novel mode of action with the tumor cells.

Introduction

Discorhabdin alkaloids were isolated from marine sponges, such

as New Zealand sponges of the genus Latrunculia, the Okinawan

sponge Prianos melanos, the Fijian sponge Zyzzya cf. Marsailis,

etc.^1–17All of them have a unique structure of the azacarbocyclic

spirocyclohexanone and pyrroloiminoquinone system. Among the

various isolated discorhabdins (A–X), discorhabdin A (1a),^2,3

B

(1b),^2,3D (1d),⁴H,⁶¹I,⁴⁵J,⁴⁶L,⁴⁵M,⁴⁶N,⁴⁶Q,¹³R¹⁴and X¹⁷have

a sulfur-containing fused ring system. Discorhabdin S (2), T and

U have a methyl sulﬁde moiety, while the others have no sulfur

atom. The discorhabdins F,⁶Q, S,¹⁶T¹⁶and prianosin B (1c)⁵

have the 16,17-dehydropyrroloiminoquinone moiety. Because of

their prominent potent antitumor activity, the discorhabdins have

attracted considerable attention (Fig. 1).

Many studies have reported the synthesis of the discorhabdins.

Several partial and total syntheses of discorhabdin C (3) have

appeared in the past decade by Knolker, Heathcock, Yamamura

and our group, etc.^18–35We have accomplished the ﬁrst total

syntheses of discorhabdin A (1a) in 2003 and prianosin B (1c)

in 2009.^36–41The discorhabdins have a strong cytotoxic activity

in vitro. Currently, many discorhabdin alkaloids have been isolated

and synthesized. However, only a few biological studies including

structure activity relationships or the mode of action of the

Fig. 1 The structure of the discorhabdins.

^aGraduate School of Pharmaceutical Sciences, Osaka University, 1-6

Yamada-oka, Suita, Osaka, 565-0871, Japan

^bCollege of Pharmaceutical Sciences, Ritsumeikan University, 1-1-1 Nojihi-

gashi, Kusatsu, Shiga, 525-8577, Japan

^cShinagawa R&D Center, Daiichi Sankyo Co. Ltd., 1-2-58 Hiromachi,

Shinagawa, Tokyo, 140-0005, Japan

† Electronic supplementary information (ESI) available: Experimental

details. See DOI: 10.1039/c1ob05058c

Fig. 2 Four types of discorhabdin analogues.

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discorhabdins have been reported.^42–44As part of our research

program on new potential antitumor drugs, we focused our

efforts on the preparation of the discorhabdin analogues which

were categorized in four groups, i.e., the phenylethylamine

type (4), spirodienone type (5), sulfur cross-linked type (6)

and oxa type (7) (Fig. 2).⁴⁵To clarify the best location for the

chemical modiﬁcations, we began to study the structure activity

relationships (SAR) of the discorhabdin analogues.

Scheme 2 Syntheses of tyramine derivatives.

Results and discussion

Chemistry

The retrosynthetic analysis to prepare the analogues is shown

in Scheme 1. The coupling reaction between the tyramine or

tyrosine derivative 8 and quinone or iminoquinone derivative

9 would give rise to the corresponding phenylethylamine type

(4). The spirodienone type (5) could be synthesized from the

phenylethylamine type (4) via the spirocyclization reaction. The

S or O bridge formation reaction would produce the sulfur cross-

linked type (6) and oxa type (7) from the spirodienone type (5)

(Scheme 1).

Scheme 3 Syntheses of tyrosine derivatives.

reduction of 15a–d with diisobutylaluminium hydride (DIBAL).

Disilylation of the phenolic and aliphatic hydroxyl groups of 16a–

d produced compounds 17a–d. The syntheses of phenols 18a–d

were accomplished by selective desilylation of the phenolic silyl

ether of 17a–d with tetra-n-butylammonium ﬂuoride (TBAF).

The syntheses of the quinones are shown in Scheme 4. Quinone

20 was synthesized from 5-hydroxyquinoline 19 according to

Barret’s method.⁴⁷Quinone 23 was prepared from 4-hydroxyindole

by the tosylation of 21 followed by oxidation with phenylio-

dine(III)diacetate (PIDA).

Scheme 1 Retrosynthetic analysis.

The syntheses of tyramine or the tyrosine derivatives 8 are

shown in Schemes 2 and 3. The starting materials were the

commercially available tyramine (10), (L)-tyrosine methyl ester

hydrochloride (11) and 3-iodo-(L)-tyrosine (12). The preparations

of dibromotyramine (13) and diiodotyramine (14) were achieved

from tyramine (10) in a single step. Treatment of tyramine with

I₂and 30% H₂O₂in H₂O for 3 h at 55 ^◦C produced the diiodo

compound 14 (yield 99%).

The preparations of the non- or monohalogenated tyrosine

derivatives 18a–d are shown in Scheme 3. N-Tritylation of the

(L)-tyrosine methyl ester hydrochloride 11 with TrCl and Et₃N

in DMF produced compound 15a. N-Tritylation of 11 followed

by ortho-bromination afforded the bromo compound 15b. ortho-

Chlorination of 11 followed by N-tritylation gave the chloro

compound 15c. The iodo compound 15d was synthesized by es-

teriﬁcation of the carboxyl group of 3-iodo-L-tyrosine 12 followed

by N-tritylation. The aminoalcohols 16a–d were obtained by the

Scheme 4 Syntheses of quinones 20 and 23.

Iminoquinone 32 was prepared by our previously developed

PIFA-induced pyrroloiminoquinone formation (Scheme 5).⁴⁸The

starting material was commercially available vanillin 24. The

nitro compound 25 was prepared from 24 by acetylation of the

hydroxyl group followed by selective nitration with fuming HNO₃.

Deacetylation of compound 25 and the subsequent methylation

of the resulting phenolic hydroxyl group gave the dimethoxy

compound 26. The nitro-aldol reaction of 26 with CH₃NO₂under

basic conditions afforded the dinitro compound 27. The synthesis

of indole 28 was accomplished by the reductive cyclization of

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Scheme 5 Synthesis of iminoquinone 32.

27 with HCO₂NH₄, Pd/C and HCO₂H.⁴⁹Compound 29 was

synthesized from the indole 28 by the reaction with oxalyl chloride

and subsequent quenching by EtOH. The produced ketoester 29

was reduced to the ethylalcohol 30 by lithium aluminium hydride

(LAH). Azide 31 was synthesized by a 2-step procedure, i.e.,

Mitsunobu azidation followed by N-tosylation. The iminoquinone

32 was prepared by the PIFA-induced pyrroloiminoquinone

formation. In this reaction, PIFA was activated by TMSOTf.

Coupling reactions of tyramine or the tyrosine derivatives with

the quinones or iminoquinone to produce the phenylethylamine-

type compounds are shown in Schemes 6 and 7. Syntheses of the

phenylethylamine-type-1 reactions gently proceeded to produce

the desired products in about 60% yields. The reaction conditions

for the coupling reactions of tyramine or the tyrosine derivatives

were different. In the case of the tyramine derivatives, i.e., the N-H

compound (for example 10), the coupling reaction only proceeded

in MeOH. The reactions of the tyramine ammonium salts 13 or 14

needed the addition of Et₃N. In the cases of the tyrosine derivatives

i.e., the N-3;Tr compounds, deprotection was carried out using

HCl–MeOH before the coupling reaction. Iminoquinone 32 was

so unstable that it decomposed within 1 day in air. The coupling

reaction with the iminoquinone 32 selectively proceeded at the

methoxyl position. However, in the case of the quinones 20 or 23,

two regioisomers were obtained in the ratio of ca. 2 : 1 (the major

isomers were 36a, 37a, 39a and 40a).³⁵

Scheme 6 Syntheses of phenylethylamine type-1.

Scheme 7 Syntheses of phenylethylamine type-2.

were obtained. For the phenols (38a–d), the reactions proceeded

smoothly, but the diastereoselectivities were lower (dr = 1.5–1.0).

The reactions of the silyl ether (38¢b) proceeded slowly, but the

diastereoselectivity was better (dr = ~4.8) (Scheme 9).^36,37

The sulfur cross-linked-type compounds were synthesized from

the spiro-type compounds. The desilylation reaction of compound

49b by BF₃·Et₂O in CH₂Cl₂afforded the aminoalcohol 50b. The

reaction using TBAF did not give good results because of its

basicity. Compound 49b or 50b was unstable under the basic

conditions. The N,O-acetal compound 51 was synthesized by the

oxidative fragmentation reaction of 50b with C₆F₅I(OCOCF₃)₂

in CH₃CN–MeOH.^38,39The resulting N,O-acetal was a mixture

The PIFA-induced spirocyclization reaction of the

phenylethylamine-type compounds gave the spiro-type

compounds (Schemes 8 and 9). The spirocyclization reaction

of the tyramine derivatives 33–37 proceeded with PIFA in

CF₃CH₂OH to give the spirodienones 41–43 and 47–48.^18,23,50The

reaction times of compounds 33–35, having iminoquinone as the

partial structure, were longer than those of compounds 36–37

containing the quinone (Scheme 8).

On the other hand, the spirocyclization of the tyrosine types

needed MK10 as an additive and two spirodienones 49 and 49¢

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Scheme 8 Syntheses of spirodienone type-1.

Scheme 10 Synthesis of sulfur cross-linked type.

Scheme 9 Synthesis of spirodienone type-2.

of a- and b-methoxyl isomers due to the route via the iminium

intermediate. The sulfur cross-linked reaction was carried out by

the addition of p-MeOBnSH at the 8-position, Michael addition

of the thioether to the dienone followed by the removal of the p-

MeOBn group with aq. MeNH₂to give the sulfur cross-linked type

compounds 52a,b and 53.³⁷Two constitutional isomers (52a,b)

were produced by the addition of the thiol at the 8 position (dr =

1 : 1.5). Detosylation of 52a was performed with NaOMe–MeOH

in THF to give (+)-discorhabdin A (1a).

(-)-Discorhabdin A (1¢a) was also synthesized from the D-

tyrosine methyl ester hydrochloride (Scheme 10).

The syntheses of oxa-type compounds are shown in Schemes 11

and 12. Aminoalcohols 50a–d were synthesized by desilylation of

compounds 49a–d using BF₃·Et₂O. The ether bridged formation

reactions were carried out with 30% HBr–AcOH, and the oxa-

type compounds 54a–d were obtained. The N-H and N-Ms oxa-

type compounds 55 and 56 were synthesized from a non-halogen

compound 54a. The 16,17-dehydro oxa-type compound 57 was

synthesized from compound 54b (Scheme 11).

Scheme 11 Synthesis of oxa type-1.

58 and its constitutional isomer 58¢ were synthesized from the D-

tyrosine methyl ester hydrochloride (Scheme 12).

The absolute conﬁguration of the oxa-type compound 54d was

determined as follows. Although four stereoisomers would be

possible for 54d, the presence of the NOE effect between the

Compounds 54¢b–d, which are the constitutional isomers of

54b–d, were also synthesized from compounds 50¢b–d. Compound

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Scheme 13 Synthesis of compounds 59 and 60.

activity relationships of the discorhabdins using the derivatives of

the natural discorhabdin C (3) (Scheme 14 (1)).⁶They reported

that the conversion of the dienone to a dienol caused a dramatic

alteration in the cytotoxicity and conﬁrmed the importance of the

dienone/enone system in the observed biological properties of the

discorhabdins. The azepine derivatives X, Y, Z were not effective.

Copp et al. also reported the structure activity relationships using

the derivatives of the natural discorhabdins B (1b) and C (3)

(Scheme 14 (2)).⁵²

Scheme 12 Synthesis of oxa type-2.

hydrogen atoms at the C-5 and C-a positions suggested A or B

for 54d. We next determined the relative conﬁguration between

the C-4 and C-5 hydrogen atoms to be trans, because the coupling

constant J_4,5of 54d was 13.0 Hz. It is reported that J_4,5of the trans

conﬁguration was 13.5 Hz and J_4,5of the cis conﬁguration was

4.3 Hz in epinardin B.⁵¹Therefore, we determined that the absolute

conﬁguration of the oxa-type compound 54d was A (Fig. 3).

Fig. 3 Determination of absolute conﬁguration of oxa type compound

54d.

Compounds 59 and 60 were synthesized from the aminoalcohol

50b. Protection of the aliphatic hydroxyl group of 50b by BzCl and

Et₃N gave the benzoate 59 in 57% yield. The Mitsunobu azidation

of 50b gave the azide compound 60 in 45% yield (Scheme 13).

Biological activity

Although many synthetic studies of the discorhabdin alkaloids

have been reported, the biological studies including the structure

activity relationships or the mode of action in the discorhabdins

did not signiﬁcantly proceed.^42–44Blunt et al. reported the structure

Scheme 14 Structure activity relationships of discorhabdin derivatives

against P388.

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Table 1 SAR of spirodienone type against HCT-116

It is suggested that the sulfur cross-linked structure is important.

However, the discorhabdin alkaloids were isolated in such small

amounts that it was difﬁcult to undergo more systematic and

detailed biological studies. As for the mode of action of the

discorhabdins, Ireland et al. reported that the inhibitive activity

of DNA topoisomerase II by discorhabdin A was weak, whereas

the makaluvamines, in the same class of pyrroloiminoquinone

alkaloids as the discorhabdins, showed a cytotoxicity toward the

tumor cell line by inhibition of topoisomerase II.⁸The inability

of discorhabdin A to inhibit topoisomerase II is indicative of the

fact that this potent cytotoxicity most probably acts by a different

mechanism than the makaluvamines.^53–57

in vitro MTT assay

The in vitro cytotoxic potency of the phenylethylamine-type

compounds, type A to type C, against HCT-116 was ﬁrst evaluated.

The results are expressed as IC₅₀values. We examined the inﬂuence

of the conversion of the pyrroloiminoquinone moiety to the

quinone moiety and of the R substitution. The type B compounds

(36a, 39a, 40a) showed a stronger cytotoxicity than the type C ones

(36b, 39b, 40b). From the comparison of R = H, CO₂Me, CH₂OH,

it can be seen that the strength of the biological activity was in

the order of H (36a, b) > CO₂Me (39a, b) > CH₂OH (40a, b).

However, overall, the phenylethylamine-type compounds showed

a lower activity than discorhabdin A (1a). One of the reasons may

be that it is the difference in the mode of action with discorhabdin

A (1a) (Fig. 4).

substituted R¹was next investigated; see the results of 44, 45 and

41. No notable effect of the N-substituent was observed, but it was

the preferred electron-withdrawing group. The effect of the R²and

R³substituent was next investigated. The presence of the halogen

atom was necessary for the biological activity. Comparing the

nonhalogen compound (41) to the dibromo compound (42) or the

diiodo compound (43), compound 42 or 43 was about ten times

stronger than 41. Among the halogens, the natural-type bromo

substituent, compound 42, showed the strongest IC₅₀value. We

think that the side chain at the 8-position, i.e., the R⁴substituent,

does not have a signiﬁcant effect on the biological activity; see the

results of 49b, 50b, 59 and 60 (Table 1).

The stereochemistry of how the discorhabdins inﬂuenced the

activity among the sulfur cross-linked-type compounds was

investigated. (+)-Discorhabdin A (1a) had the same activity as

(-)-discorhabdin A (1¢a). From this result, the stereochemistry at

the C-6 and C-8 positions did not affect the activity very much. In

these compounds, the position of the carbonyl oxygen of the enone

is similar. The bromo substituent may not have a steric effect, but

an electronic effect on the biological activity (Scheme 15).^58–60

From the structural comparison of makaluvamine F (62),^8,61

discorhabdins C (3),¹P (46)¹²and discorhabdin A (1a),^2,3the

difference in the activity might come from the ﬂexibility of the

compounds. The order of strength of the activity was discorhabdin

A, discorhabdins C and P then makaluvamine F. We estimated that

one of the active sites is the spiro enone moiety. Discorhabdins C

and P have ﬂexible spiro-centers. On the other hand, discorhabdin

Fig. 4 SAR of phenylethylamine type compounds against HCT-116.

We next examined the spirodienone-type compounds. The activ-

ity and stability of the spirodienone-type compounds were better

than the phenylethylamine-type compounds. We ﬁrst examined the

inﬂuence of the conversion of the pyrroloiminoquinone moiety to

a variety of quinone moieties; see the results of 44, 47a,b, 48a,b

and 61. If the quinone compounds have a strong cytotoxicity, it

is ideal, because the quinone compounds are easily synthesized

and stable. From the results, however, the pyrroloiminoquinone

moiety had a signiﬁcant biological activity. The effect of the N-

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Table 2 SAR of oxa-type against HCT-116

Scheme 15 SAR of sulfur cross-linked type against HCT-116.

A has a rigid spiro-center (Scheme 16). C. M. Ireland et al.

also reported that the activity of makaluvamine C, D, E, F

and discorhabdin A dramatically demonstrated an increasing

cytotoxicity concurrent with the structural elaboration.^7,8,25

The clogP values were calculated by ChemBioDraw Ultra 12.0.

Scheme 16 Phenylethylamine vs. spiro vs. sulfur cross-linked type against

HCT-116.

results. Their IC₅₀values against HCT-116 (0.04 mM, 0.05 mM:

see Table 2) are almost the same as that of discorhabdin A (0.03

mM). To our surprise, their IC₅₀values against L1210 (0.01 mM,

0.02 mM) are higher than that of discorhabdin A (1a) (0.06 mM)

(Table 3).

These results indicated signiﬁcant structural information about

the biological activity. The spiro and pyrroloiminoquinone struc-

tures play an important role in the biological activity. It may be

that these units are active sites. Next, the halogen substitution

is signiﬁcant, especially the bromo substitution on R¹or R²,

and the electron-withdrawing group on R³. The sulfur or oxa-

bridged structure is necessary for ﬁxing the spiro-enone structure.

However, it does not signiﬁcantly affect the stereochemistries at

the 6- and 8-positions (Fig. 5).

We then examined the oxa-type compounds (Table 2). Almost

all the oxa-type compounds had strong activities. (1) From a

comparison of R¹(H (55), Ms (56) and Ts (54a)), it was shown

that the electron-withdrawing groups as the N-substituent R¹are

better. (2) The presence of the halogen atom in the R²or R³

substituent was preferable in the oxa-type compounds (54a,b,c,d

and 54¢b,c,d). The activity of the compounds was not caused by

the variation of the halogen, but the bromo substituent showed the

highest activity. The stereochemistry at the C-6 and C-8 positions

did not signiﬁcantly affect the activity. It is interesting that the

oxa-aromatic (57¢) had a very weak activity. We think that the

decrease in the value of ClogP affects the shift of the drug

into a tumor cell during the absorption process or weakens the

hydrophobic interaction of the pyrroloiminoquinone unit and the

target protein.^62–66

With the oxa-type compounds in hand, we examined their

cytotoxicity in vitro against four kinds of tumor model cells, WiDr

(human colon adenocarcinoma), DU-145 (human prostate tumor

cell), P388 and L1210 (murine leukemia). As a reference, the IC₅₀

values of discorhabdin A in vitro for each cell, WiDr = 0.03 mM,

DU-145 = 0.09 mM, P388 = 0.03 mM and L1210 = 0.04 mM, are

shown. All the oxa-type compounds exhibited good IC₅₀values.

Especially, compound 54¢b and its enantiomer 58¢ gave the best

Fig. 5 Information on the structure for biological activity.

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Table 3 Biological activity of oxa-type against tumor cells

IC₅₀values (mM)

Entry

Compound

R¹

R²

R³

WiDr

DU-145

P388

L1210

1

2

3

4

5

6

7

55

H

Br

H

Br

H

Br

H

Br

1.23

0.22

0.06

0.08

0.30

0.18

0.03

0.71

0.18

0.38

0.15

0.33

0.17

0.09

0.38

0.13

0.09

0.14

0.29

0.03

0.80

0.14

0.12

0.01

0.12

0.02

0.04

54a

Ts

54b

54¢b

58

58¢

discorhabdin A (1a)

In vivo assay

Acutetoxicity test. On the basis of our favorable proﬁle invitro,

compounds 54a and discorhabdin P (46) were tested in the mouse

in vivo assay.‡ We ﬁrst evaluated the single-dose toxicity test. For

the test, the model animal is BALB/c-nu/nu. The single-dose

toxicity of compound 54a was tested using two kinds of doses (a;

58 mg kg^-1¥ 2, b; 58 mg kg^-1). The results are as follows: a; It was

observed that the model mouse became pale, hypothermic and

stertorous after a dose. It was also observed that the ability of the

model mouse to exercise had decreased and it was conﬁrmed dead.

b; It was observed that the dose made the model mouse pale and

hypothermic and it had a decreased ability to exercise. Next, the

single-dose toxicity of compound 46 was tested using four kinds of

doses (a; 100 mg kg^-1, b; 80 mg kg^-1, c; 60 mg kg^-1, d; 40 mg kg^-1).

The results are as follows: a, b, c; The dose made the model mouse

immediately go into rigor contraction. After 20 min, an abnormal

behavior, for example the mouse was jumping up and down and a

general stiffness, was observed. Paramyotonia was observed and

the mouse was conﬁrmed dead after 1 h. d; In this case, it was

milder than for the cases with highest doses. The mortality rate

was 50%. Suffering from diarrhea was not observed by the time of

death.

Fig. 6 Drug efﬁcacy test.

was BALB/c-nu/nu (n = 2), i.v. The dose of compound 54a was

10, 20 and 40 mg kg^-1. The blood collecting times were 5, 30, 60,

120 and 240 min. In the test, the detection limit was 0.3 mg mL^-1.

The result was that only 0.36 mg mL^-1was detected at 5 min for the

dose of 40 mg kg^-1. The dose of compound 46 was only 10 mg kg^-1.

The blood collecting times were 15, 30, 60, 120 and 240 min. In

the test, the detection limit was 0.1 mg mL^-1. The result was that

there was no detection at every point.

Drug efﬁcacy test. Next, we assessed the drug efﬁcacy. The

assessed model was the human colorectal tumor HCT-116

xenograft subcutaneously implanted in BALB/c-nu/nu mice.

The compound administration was started when the tumor was

established on day 7. Compound 54a was injected by i.v. 40 mg kg^-1

(5 times/week), 20 mg kg^-1(5 times/week) and 10 mg kg^-1

(5 times/week). We assessed the drug efﬁcacy for compound 46.

Compound 46 was injected by i.v. 20 mg kg^-1, 10 mg kg^-1, 5 mg kg^-1

(3 times/week ¥ 2w), 2 mg kg^-1(3 times/week ¥ 2w) and 1 mg kg^-1

(3 times/week ¥ 2w). The changing of the tumor volumes are

shown in Fig. 6. Regrettably, from these results, no effect by

compound 54a was observed. In the case of compound 46, no

shrinking of the tumor cell by the various administration methods

was observed. A temporary decrease in body weight for the doses

of 20 mg kg^-1and 10 mg kg^-1was observed. This may be the

onset of the cell cytotoxicity against healthy cells (see supporting

information†).

From the in vivo testing, we reasoned that the oxa-type

compound 54a and discorhabdin P (46) were so unstable inside

a living body that they were immediately decomposed

We think that this result was attributed to the iminoquinone

structure, because the iminoquinone moiety was unstable. On

the other hand, discorhabdin D (64) has an activity in vivo,⁴

because the iminoquinone structure is stabilized by the cross-

linking between the nitrogen atom of the iminoquinone and the

a-carbon of the spiroenone.

Mode of action of discorhabdins

We estimated the HCC panel assay, the inhibition of protein ki-

nase, histone deacetylase (HDAC), farnesyltransferase, telomerase

and proteasome assays for discorhabdin A (1a) and the oxa-type

compound (54b). We tried to determine the mode of action of the

discorhabdins by comparison to the existing anticancer drugs.

Pharmacokinetic test. We also examined the pharmacokinetic

test. The examination conditions are as follows. The model animal

(A) Inhibition of protein kinase assay. In the assay, we examined

the selective inhibitive activity of the protein kinase. The activity

of the multiprotein phosphoenzyme could be detected at the same

time, which is connected to the intercellular signaling.⁶⁷We also

examined the inhibitive activity of the tyrosine kinase of the

EGF receptor and VEGF receptor (Flt-1) by the easy-to-use

‡ All the animal care and experiments were conducted under the standard

operational protocol of the former Sankyo Institutional Animal Care and

Use Committee.

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Table 4 Inhibition of protein kinase assay^a,b

assay result

Conc. (mM)

eEF2K

PKC

PTK

PKA

EGFR

Flt-1

Judgement positive; <1 mM

1a

100

10

1

100

10

1

+

—

+

—

2+

+

—

2+

+

M1Y1F2 negative

54b

2+

—

+

—

M1Y1F2 negative

—

^aControls: Herbimycin A (PTK), H89 (PKA), AG1478 (EGFR), SU4984 (Flt-1). Abbreviations: A (PKA), C (PKC), M (eEF2K), Y (PTK), E (EGFR),

F (Flt-1). ^b>50% inh. At 0.01mM; 5+, 0.1mM; 4+, 1mM; 3+, 10mM; 2+, 100mM; 1+, 2+: >80% inhibition, +: 50~80% inhibition, -: <50% (not inhibition).

Table 6 Inhibition of farnesyltransferase assay^a

in vitro autophosphorylation reaction. The method determines

the phosphorylated inhibition and the inhibitive pattern. The test

Conc. (mM)

Radioactivity (dpm)

% Control

compounds were drawn up to 100 mM. The test compounds having

the inhibitive activity and selectivity were examined again by using

a ten times diluted solution at three more levels. The case of more

than a 50% inhibition activity at less than a 1 mM concentration

was determined as positive. The result was that these compounds

(1a, 54b) were negative (Table 4).

1a

0

10

0

1805

100

52.4

100

78.5

1a

945.5

3528.5

2770

54b

10

^aStrong activity; IC₅₀value < 100 nM. Relatively strong activity; IC₅₀

value100 nM–10 mM. Weak activity; IC₅₀value >10 mM. Positive control;

IC₅₀value 0.73 nM. Sample result; 1a: IC₅₀value > 10 mM, 54b: IC₅₀

value > 10 mM.

(B) Inhibition of histone deacetylase (HDAC) assay. It is

believed that the reason for the pathogenic mechanism of the

cancer is the mutations of the oncogenes or antioncogenes. The

control of the cell growth, adhesion and apoptosis are out of

order because of the accumulation of changes in base sequence.

On the other hand, impaired expression of genes without changing

in base sequence was revealed; epigenetic alteration signiﬁcantly

contributes to the pathogenic mechanism of the cancer. Among

them, the histone deacetylation, which controls the activity of the

chromatin, plays an important role.⁶⁸The HDAC controlling the

level of histone acetylation is the focus of attention as a molecular

target of an antitumor drug. The histone deacetylation occurs at

the e-amino group of the lysine residue which is in the core histone

amino-terminal domain and affects the transcriptive activity by

controlling the chromatin structure. The HDAC is the hydrolase

of this acetylated lysine and is very involved in the impaired

expression of genes during the carcinogenesis process. Therefore,

the activity of the inhibition of the HDAC by compounds 1a and

54b was examined. The ﬁrst test compounds were drawn up to

100 mM. The test compounds having an inhibitive activity were

examined again using more diluted solutions at every level and

the IC₅₀value determined. The results showed no activity (IC₅₀

value > 100 mM) (Table 5).

(C) Inhibition of farnesyltransferase assay. The Ras is the low-

molecular-weight G protein and is connected to cell growth and

migration. This protein is activated by farnesylation, which is cat-

alyzed by the farnesyltransferase (FPTase). Therefore, the FPTase

inhibitor is expected to be an anticancer drug by controlling the

overgrowth, metastasis and invasion of the cancer cells.⁶⁹In the

assay, the sample compound was added at 10 mM. In the case of

IC₅₀value < 1 mM, it was positive. However, the results showed a

weak activity (Table 6).

(D) The inhibition of telomerase assay. The telomerase

(telomea synthetase) is activated in the cancer cells at the amount

of 80–90% and supports the unlimited proliferation of cancer cells

by means of resolving the problem of the genetic replication at the

end of the liner DNA.⁷⁰The cancer cells caused by the activity

of the telomerase due to the aging and apoptosis with shortened

telomeres. The inhibition activity of the telomerase by compounds

1a and 54b was examined. However, the results were negative

(Table 7).

(E) The inhibition of proteasome assay. The proteasome is

a selective degrading enzyme of the intracellular ubiquitinated

protein and plays a main role in the cell cycle and the degradation

of proteins which controls apoptosis.⁷¹There is clinical proof that

Table 5 Inhibition of HDAC assay^a

R

VELCADEꢀ (the inhibitor of the proteasome) has an anticancer

efﬁcacy against multiple myeloma. A great deal of attention has

been focused on the inhibitor of the proteasome as a molecular

target of antitumor drugs.⁷²In this assay, the enzyme inhibition

with the human origin 20S proteasome was examined. However,

the results were negative (Table 8). From these results, the mode of

action of 54a or 1a is not the inhibition of protein kinase, HDAC,

farnesyltransferase, telomerase and proteasome.

Conc. (mM)

Fluorescence intensity

% Control

1a

0

100

0

58.15

54.67

58.15

50.13

100

94.02

100

54b

100

86.22

^aTrichostatin A: IC₅₀value is 0.029 mM (as control). Very strong activity

(+++); IC₅₀value < 0.1 mM. Strong activity (++); IC₅₀value 0.1–

1 mM. Relatively strong activity (+); IC₅₀value 1–10 mM. No activity; IC₅₀

value > 10 mM. Autogenic ﬂuorescent compound cannot be measured.

(F) HCC panel assay. One of the accounts of the Human

Cancer Cell Line (HCC) panel assay is that a drug having a similar

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Table 7 Inhibition of telomerase assay^a

Conc. mM

Telomea product (A)

Internal standard (B)

A/B

% Control

0

4252, 6048

4104, 3253

3562, 3574

729

5109, 5450

5850, 5742

5241, 5865

5285

0.971

0.634

0.645

0.138

100

1a

10

1

65.3

66.4

14.2

54b

MST-204

^aIC₅₀value < 10 mM; positive, IC₅₀value ≥ 10 mM; negative 1a and 54b do not inhibit PCR. MST-204 was used as control.

Table 8 inhibition of proteasome assay^a

Table 9 HCC panel assay for discorhabdin A (1a) and oxa analogue (54b)

[Chymotrypsin-like activity]

GI₅₀

TGI

LC₅₀

Conc. (mM)

% Control

IC₅₀(mM)

Discorhabdin A (1a)

Oxa analogue (54b)

MG-MID

Delta

Range

MG-MID

Delta

Range

-5.39

0.43

1.25

-6.39

1.12

-4.94

0.55

1.31

-5.94

0.96

-4.32

0.87

1.19

-5.33

0.99

1a

10

106.1

91.3

0

> 10

0.039

0.011

54b

clasto-Lactacystin b-lactone 10

MG132

[Caspase-like activity]

10

0

2.01

1.77

2.32

Conc. (mM)

10

% Control

115.5

98.1

IC₅₀(mM)

> 10

1a

However, the r value is < 0.5. As a result, the oxa analogue 54b is

estimated to have a novel mode of action (Fig. 7).

54b

[trypsin-like activity]

Conc. (mM)

10

% Control

95.5

124.9

IC₅₀(mM)

> 10

1a

54b

^aVery strong activity (+++); IC₅₀value < 0.1 mM. Strong activity (++);

IC₅₀value 0.1–1 mM. Relatively strong activity (+); IC₅₀value 1–10 mM. No

activity; IC₅₀value > 10 mM.

mode of action statistically indicates a similar correlated ﬁnger-

print pattern.⁷³The mean cytostatic and cytotoxic parameters of

the oxa analogue 54b and discorhabdin A (1a) were measured

in a 39 human cancer cell line panel. The cytostatic parameters

include GI₅₀and TGI, which are the concentrations of the drug

required for a 50% growth inhibition and total growth inhibition,

respectively. The cytotoxic parameter is the LC₅₀, which is the

concentration required for a 50% cell kill. Also, the Table 9 range

values, which represent the log of the maximum concentration

difference between the least-sensitive and the most-sensitive cell

line, are provided. This range parameter has been used to gain

insights into the selectivity of the antitumor agents, because it

provides a measure of histological speciﬁcity. The COMPARE

analysis provided insights into what might be the molecular target

of the active analogues exhibiting a high histologic selectivity.

The COMPARE analysis correlates the mean graph data with the

known molecular target levels in cell lines and thereby generates

hypotheses concerning the agent’s mechanism of action. First,

the COMPARE correlations indicated that the cytostatic and

cytotoxic parameters of the oxa analogue (54b) and discorhabdin

A (1) represent three mechanistically distinct classes. The GI₅₀,

TGI, and LC₅₀parameters of the two compounds correlate

(correlation coefﬁcient r = 0.815) with the same molecular target.

The COMPARE correlations using the oxa analogue as a seed

against the drug database (83 drugs: see supporting information)

revealed no compound correlations. The third high ranking drugs

which have similar r values of the oxa analogue, are vincristine

(r = 0.433), actinomycin-D (r = 0.430) and vinblastine (r = 0.392).

Fig. 7 The GI₅₀, TGI and LC₅₀values of oxa analogue 54b.

Conclusions

We synthesized various discorhabdin analogues, i.e., phenylethy-

lamine (4), spirodienone (5), sulfur cross-linked (6) and the oxa-

type (7). We designed and synthesized various stable oxa-type

compounds with the oxygen cross-linked spiro-fused ring system.

All compounds were evaluated by in vitro MTT assay for the

structure activity relationships. The discorhabdin oxa analogues

exhibited a strong activity against tumor model cells and some

of them were the same as discorhabdin A (1a). However, the

oxa analogue 54a and discorhabdin P (46) were not active in

vivo. For target identiﬁcation of the discorhabdins, we evaluated

discorhabdin A (1a) and the oxa analogue (54b) using six types of

assays, i.e., the inhibition of protein kinase, histone deacetylase,

farnesyltransferase, telomerase, proteasome and the HCC panel

assay. From these assays, it was revealed that the discorhabdin oxa

analogue (54b) could have a novel mode of action in tumor cells.

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hexane/AcOEt (3/1) as the eluent to give 15c (123 mg, 69%) as a

colorless oil: ¹H NMR (300 MHz, CDCl₃) d: 2.60 (1H, br s), 2.85

(2H, d, J = 6.6 Hz), 3.05 (3H, s), 3.47–3.55 (1H, m), 5.64 (1H, s),

6.91 (1H, d, J = 8.2 Hz), 6.99 (1H, d, J = 8.2 Hz), 7.09–7.23 (10H,

m), 7.40 (6H, d, J = 7.2 Hz); ¹³C NMR (75 MHz, CDCl₃) d: 41.0,

51.4, 58.0, 70.9, 115.9, 126.3, 127.7, 128.7, 129.7, 130.1, 130.5,

145.7, 150.2, 174.8; IR (KBr): 3533, 3342, 1730, 1595, 1500 cm^-1;

HRMS (FAB) calcd for C₂₉H₂₇ClNO₃[M+H]⁺: 472.1679, found

472.1686.

Experimental section

General experimental procedures

Column chromatography was carried out on MERCK Kieselgel

60 (70–230 mesh ASTM), Fuji Silysia Chemical silica gel BW-300,

Kanto Chemical silica gel 60 N (spherical, neutral, 40–50 mm)

and Fuji Silysia Chromatography silica gel NH (100–200 mesh)

using n-hexane/AcOEt or CH₂Cl₂/MeOH as an eluent. TLC

analysis was carried out using aluminium sheet precoated with

silica gel 60 F₂₅₄(Merck, Darmstadt, Germany). UV-Vis spectra

were obtained on a SIMAZU UV-2200. Optical rotations were

measured on a JASCO P-1020 polarimeter. HRMS were obtained

Methyl-3-(3-iodo-4-hydroxyphenyl)-2-(tritylamino)propionate

(15d). SOCl₂(0.52 mL, 7.16 mmol) was added dropwise to a

solution of 3-iodo-L-tyrosine (12) (2.00 g, 6.51 mmol) in MeOH

(13.0 mL). The suspension was heated at a reﬂux for 3 h. The

mixture was evaporated in vacuo to gi_◦ve precursor 15d (2.30 g,

on a JEOL JMS-D300 or JEOL JMS-600. H and ¹³C NMR

1

spectra were recorded on a JEOL JNM-LA500, JNM-ECS-400,

JNM-AL300 and JNM-EX270. Samples were dissolved in CDCl₃,

CD₃OD, (CD₃)₂CO or DMSO-d₆. IR spectra were taken as KBr

pellets using a SIMAZU FTIR-8400 spectrophotometer.

1

99%) as a colorless solid; mp 201–202 C; H NMR (300 MHz,

CD₃OD) d: 3.03 (1H, dd, J = 14.4, 7.5 Hz), 3.14 (1H, dd, J =

14.4, 6.0 Hz), 3.81 (3H, s), 4.24 (1H, dd, J = 7.5, 6.0 Hz), 6.82

(1H, d, J = 8.4 Hz), 7.07 (1H, dd, J = 8.4, 1.8 Hz), 7.22 (1H, d,

J = 1.8 Hz); ¹³C NMR (75 MHz, DMSO-d₆) d: 34.3, 52.6, 53.2,

84.8, 115.0, 126.8, 130.5, 139.5, 156.0, 169.4; IR (KBr): 3200,

1741, 1600, 1570, 1502 cm^-1; HRMS (FAB) calcd for C₁₀H₁₃INO₃

[M+H]⁺: 321.9940, found 321.9941 (as free base).

Total synthesis of prianosin

B (1c). NaN₃(1.1 mg,

0.0175 mmol) was added to a solution of compound 51b (99.7 mg,

0.175 mmol) in DMF (0.3 mL) at rt under N₂. The mixture was

allowed to warm to 70 ^◦C and stirred for 1 h. The reaction mixture

was quenched by H₂O and extracted by AcOEt. Organic phase

was washed by H₂O (¥3) and brine (¥1). Organic phase was dried

over Na₂SO₄and evaporated in vacuo. The residue was puriﬁed

by SiO₂column chromatography (CH₂Cl₂/MeOH = 20/1) to give

prianosin B (1c) (35.1 mg, 48%) as red solid; mp 253–255 ^◦C;

Et₃N (1.78 mL, 13.02 mmol) was added to a solution of

precursor 15d (2.3 g, 6.51 mmol) in DMF (32 mL) at rt under N₂.

After being stirred for 10 min, TrCl (1.8 g, 6.51 mmol) was added

to the resulting solution. The mixture was stirred at rt for 3 h. The

reaction was quenched with H₂O and extracted with AcOEt. The

organic layer was washed with H₂O and brine, dried over Na₂SO₄,

and evaporated in vacuo. The residue was puriﬁed by SiO₂column

chromatography using hexane/AcOEt (3/1) as the eluent to give

15d (3.62 g, quant.) as a colorless solid; mp 95–98 ^◦C; [a]_D^26.7+15.2

[a]²_D^3.0+362 (c 0.405, CHCl₃); H NMR (500 MHz, CDCl₃) d:

1

2.87–2.94 (3H, m), 2.98 (1H, dd, J = 16.5, 4.0 Hz), 4.80 (1H,

dd, J = 12.0, 6.5 Hz), 5.49–5.58 (1H, m), 6.30 (1H, br s), 7.54

(1H, d, J = 5.5 Hz), 7.78 (1H, s), 8.03 (1H, s), 8.49 (1H, d, J =

5.5 Hz); ¹³C NMR (125 MHz, CDCl₃) d: 40.0, 45.6, 50.8, 56.5,

61.7, 113.7, 118.2, 119.6, 120.2, 125.3, 129.0 (2C), 143.0, 143.6,

146.1, 155.7, 167.6, 188.3; IR (KBr): 3057, 2924, 2853, 1682, 1645,

1595, 1472, 1303 cm^-1; HRMS (FAB) calcd for C₁₈H₁₃BrN₃O₂S

[M+H]⁺: 413.9912, found 413.9920.

1

(c 2.95, CHCl₃); H NMR (300 MHz, CDCl₃) d: 2.55 (1H, br

s), 2.84 (2H, dd, J = 6.5, 2.6 Hz), 3.06 (3H, s), 3.50–3.55 (1H,

m), 5.32 (1H, s), 6.92 (1H, d, J = 8.1 Hz), 7.07 (1H, dd, J = 8.1,

1.6 Hz), 7.17–7.24 (9H, m), 7.39 (6H, d, J = 7.2 Hz), 7.55 (1H, d,

J = 1.6 Hz); ¹³C NMR (75 MHz, CDCl₃) d: 40.6, 51.4, 57.9, 70.9,

85.2, 114.6, 126.4, 127.8, 128.7, 131.5, 131.6, 139.3, 145.7, 153.6,

174.7; IR (KBr): 3340, 3057, 3030, 2949, 2925, 2848, 1726, 1597,

1574, 1489, 1467, 1417 cm^-1; HRMS (FAB) calcd for C₂₉H₂₇INO₃

[M+H]⁺: 564.1036, found 564.1043.

Methyl-3-(3chloro-4-hydroxyphenyl)-2-(tritylamino)propionate

(15c). SO₂Cl₂(0.38 mL, 4.74 mmol) was added to a solution of

11 (1.0 g, 4.32 mmol) in AcOH (7.7 mL) and Et₂O (0.86 mL) at

0

^◦C under N₂. The resulting solution was warmed to rt under

stirring. Then the solution was ﬁltered through a Celite pad, and

washed with Et₂O. The ﬁltrate was concentrated in vacuo to give

precursor 15c (910 mg, 79%) as a colorless solid; mp 191–192 ^◦C;

¹H NMR (500 MHz, CD₃OD) d: 3.06 (1H, dd, J = 14.4, 7.5 Hz),

3.16 (1H, dd, J = 14.4, 6.0 Hz), 3.81 (3H, s), 4.26 (1H, dd, J = 7.5,

6.0 Hz), 6.90 (1H, d, J = 8.4 Hz), 7.01 (1H, dd, J = 8.4, 2.1 Hz),

7.22 (1H, d, J = 2.1 Hz); ¹³C NMR (100 MHz, DMSO-d₆) d:

34.5, 52.6, 53.2, 116.7, 119.5, 126.0, 129.0, 130.8, 152.4, 169.4; IR

(KBr): 3100, 1743, 1613, 1580, 1510 cm^-1; HRMS (FAB) calcd for

C₁₀H₁₃ClNO₃[M+H]⁺: 230.0584, found 230.0565 (as free base).

Et₃N (103 mL, 0.750 mmol) was added to a solution of precursor

15c (100.0 mg, 0.375 mmol) in DMF (1.8 mL) at rt under N₂.

After being stirred for 10 min, trityl chloride (TrCl) (104.5 mg,

0.375 mmol) was added to the resulting solution. The mixture

was stirred at rt for 3 h. The reaction was quenched with H₂O

and extracted with AcOEt. The organic layer was washed with

H₂O and brine, dried over Na₂SO₄, and evaporated in vacuo.

The residue was puriﬁed by SiO₂column chromatography using

General procedure for the syntheses of 17a,c,d from 15a,c,d

Diisobutylaluminium hydride (DIBAL) (0.94 M solution in hex-

ane, 3.23 equiv) was added dropwise to a solution of 15 (1.0 equiv)

in dry CH₂Cl₂(0.053 M solution) at -78 ^◦C under N₂. The resulting

solution was warmed to rt and stirred for 5 h. The mixture was

cooled to 0 ^◦C and quenched with H₂O. The precipitate was ﬁltered

through a Celite pad and the ﬁltrate was evaporated in vacuo.

The residue was dissolved in CH₂Cl₂and the mixture was washed

with sat. aq. NaHCO₃and brine, then dried over Na₂SO₄and

evaporated in vacuo. The residue was puriﬁed by SiO₂column

chromatography to give 16.

16a (5.6 g, 95%) was obtained from 15a (6.3 g, 14.4 mmol),

DIBAL (50.5 mL, 47.5 mmol) and dry CH₂Cl₂(280 mL). Eluent:

1

hexane/AcOEt (2/1). 16a: Colorless oil: H NMR (CDCl₃): d =

2.17 (dd, 1H, J = 13.2, 4.6 Hz), 2.42 (dd, 1H, J = 13.2, 9.5 Hz),

2.72–2.75 (m, 1H), 2.92 (dd, 1H, J = 10.8, 3.8 Hz), 3.11 (dd, 1H,

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J = 10.8, 2.2 Hz), 4.92 (br s, 1H), 6.62 (d, 2H, J = 8.4 Hz), 6.76 (d,

2H, J = 8.4 Hz), 7.17–7.31 (m, 9H), 7.53 (d, 6H, J = 7.3 Hz).

16c (3.9 g, 51%) was obtained from 15c (8.15 g, 17.3 mmol),

DIBAL (64.0 mL, 60.5 mmol) and dry CH₂Cl₂(86 mL). Eluent:

CH₂Cl₂(12.7 mL). Eluent: hexane/AcOEt (20/1). 17d: Colorless

oil: ¹H NMR (300 MHz, CDCl₃) d: -0.35 (3H, s), -0.33 (3H, s),

0.00 (6H, s), 0.61 (9H, s), 0.80 (9H, s), 2.26–2.34 (3H, m), 2.44–

2.45 (1H, m), 2.66 (1H, dd, J = 9.6, 3.3 Hz), 6.42 (1H, d, J =

8.3 Hz), 6.61 (1H, dd, J = 8.3, 2.1 Hz), 6.90–7.04 (9H, m), 7.21

(1H, d, J = 2.1 Hz), 7.33 (6H, d, J = 6.9 Hz); ¹³C NMR (75 MHz,

CDCl₃) d: -5.4, -4.0, 18.1, 18.3, 25.8, 25.9, 34.6, 37.4, 55.2, 62.3,

71.1, 90.1, 117.8, 126.3, 127.8, 128.7, 130.3, 134.2, 140.5, 147.2,

153.2; IR (KBr): 3327, 1712, 1682, 1595, 1487, 1471, 1446 cm^-1;

HRMS (FAB) calcd for C₄₀H₅₄INNaO₂Si₂[M+Na]⁺: 786.2662,

found 786.2635.

1

hexane/AcOEt (2/1). 16c: Colorless oil: H NMR (300 MHz,

CDCl₃) d: 2.14 (1H, dd, J = 13.2, 9.6 Hz), 2.41 (1H, dd, J = 13.2,

9.6 Hz), 2.64–2.80 (1H, m), 2.97 (1H, dd, J = 10.8, 3.9 Hz), 3.10

(1H, dd, J = 10.8, 2.5 Hz), 6.71 (1H, dd, J = 8.1, 1.5 Hz), 6.80 (1H,

d, J = 8.1 Hz), 6.83 (1H, d, J = 1.5 Hz), 7.16–7.30 (9H, m), 7.55

(6H, d, J = 7.5 Hz); ¹³C NMR (75 MHz, CDCl₃) d: 37.8, 55.2,

67.9, 71.3, 115.9, 119.5, 126.5, 127.9, 128.6, 129.3, 129.7, 132.1,

146.4, 149.7; IR (KBr): 3247, 1700, 1590, 1575, 1500 cm^-1; HRMS

(FAB) calcd for C₂₈H₂₇ClNO₂[M+H]⁺: 444.1730, found 444.1721.

16d (680 mg, 90%) was obtained from 15d (792 g, 1.40 mmol),

DIBAL (4.46 mL, 4.20 mmol) and dry CH₂Cl₂(7.0 mL). Eluent:

General procedure for the syntheses of 38a,c,d from 17a,c,d

TBAF (1.0 M solution in THF, 1.0 equiv) was added to a solution

of 17 (1.0 equiv) in dry THF (0.063 M solution) at 0 ^◦C under

N₂. The mixture was stirred at the same temperature for 0.5 h.

The reaction was quenched with sat. aq. NH₄Cl and extracted

with AcOEt. The organic layer was washed with brine, dried over

Na₂SO₄and evaporated in vacuo. The residue was puriﬁed by SiO₂

column chromatography to give 18.

1

hexane-AcOEt (2/1). 16d: Colorless solid: H NMR (300 MHz,

CDCl₃) d: 2.12 (1H, dd, J = 13.2, 4.5 Hz), 2.40 (1H, dd, J =

13.2, 9.5 Hz), 2.72–2.73 (1H, m), 2.98 (1H, dd, J = 10.9, 3.9 Hz),

3.10 (1H, dd, J = 10.9, 2.0 Hz), 6.73 (1H, d, J = 8.3 Hz), 6.78

(1H, dd, J = 8.3, 1.2 Hz), 7.22–7.34 (10H, m), 7.54 (6H, d, J =

7.5 Hz); ¹³C NMR (100 MHz, CDCl₃) d: 37.5, 55.2, 67.9, 71.3,

85.3, 114.6, 126.6, 127.9, 128.6, 131.0, 132.9, 138.9, 146.4, 153.3;

IR (KBr): 3500, 3315, 3057, 3030, 2937, 2889, 1703, 1597, 1574,

1487, 1446 cm^-1; HRMS (FAB) calcd for C₂₈H₂₇INO₂[M+H]⁺:

536.1087, found 536.1082.

tert-Butyldimethylsilyl chloride (TBSCl) (3.0 equiv) was added

to a solution of 16 (1.0 equiv) and 1,8-diazabicyclo[5.4.0]undec-

7-ene (DBU) (5.0 equiv) in dry CH₂Cl₂(0.13 M solution) at 0 ^◦C

under N₂. The mixture was stirred at the same temperature for

2.5 h. The reaction was quenched with sat. aq. NaHCO₃and

extracted with CH₂Cl₂. The organic layer was washed with brine,

dried over Na₂SO₄and evaporated in vacuo. The residue was

puriﬁed by SiO₂column chromatography to give 17.

18a (818 mg, 95%) was obtained from 17a (1.05 g, 1.64 mmol),

TBAF (1.64 mL, 1.64 mmol) and dry THF (26 mL). Eluent:

1

hexane/AcOEt (10/1). 18a: Colorless oil: H NMR (500 MHz,

CDCl₃) d: -0.25 (3H, s), -0.22 (3H, s), 0.74 (9H, s), 2.12 (1H, br

s), 2.37 (1H, dd, J = 9.6, 4.6 Hz), 2.44–2.52 (3H, m), 2.83 (1H, dd,

J = 9.6, 3.1 Hz), 6.65 (2H, d, J = 8.4 Hz), 6.77 (2H, d, J = 8.4 Hz),

7.06–7.20 (9H, m), 7.46 (6H, d, J = 7.5 Hz).

18c (2.75 g, 99%) was obtained from 17c (3.35 g, 4.98 mmol),

TBAF (4.98 mL, 4.98 mmol) and dry THF (79 mL). Eluent:

1

hexane/AcOEt (10/1). 18c: Colorless oil; H NMR (300 MHz,

CDCl₃) d: -0.02 (3H, s), -0.01 (3H, s), 0.98 (9H, s), 2.23 (1H, br

s), 2.60–2.68 (3H, m), 2.80–2.82 (1H, m), 3.00 (1H, dd, J = 9.3,

3.0 Hz), 6.91–6.97 (2H, m), 7.08 (1H, s), 7.26–7.40 (9H, m), 7.66

(6H, d, J = 7.5 Hz); ¹³C NMR (75 MHz, CDCl₃) d: -5.4, 18.1,

25.9, 37.7, 55.1, 62.3, 71.1, 115.6, 119.3, 126.3, 127.8, 128.7, 129.6,

129.9, 132.9, 147.1, 149.4; IR (KBr): 3541, 3327, 3057, 3030, 2927,

2854, 1595, 1498, 1470, 1448 cm^-1.

17a (7.3 g, 84%) was obtained from 16a (5.6 g, 13.6 mmol),

TBSCl (6.2 g, 40.8 mmol), DBU (10.2 mL, 68.0 mmol) and dry

CH₂Cl₂(90 mL). Eluent: hexane/AcOEt (20/1). 17a: Colorless

1

solid: H NMR (300 MHz, CDCl₃): d = -0.30 (s, 3H), -0.27 (s,

3H), 0.10 (s, 6H), 0.69 (s, 9H), 0.81 (s, 9H), 2.10 (br s, 1H), 2.36

(dd, 1H, J = 9.0, 3.0 Hz), 2.45–2.50 (m, 1H), 2.51 (m, 1H), 2.80

(dd, 1H, J = 9.6, 3.0 Hz), 6.52 (d, 2H, J = 8.1 Hz), 6.7 (d, 2H, J =

8.1 Hz), 7.00–7.14 (m, 9H), 7.43 (d, 6H, J = 8.4 Hz); ¹³C NMR

(CDCl₃): d = -5.6 (2C), -4.6, 18.0 (2C), 25.5, 25.7, 37.9, 55.3,

62.3, 70.9, 119.5, 126.0, 127.6, 128.6, 130.3, 132.2, 147.1, 153.5;

IR (KBr): 2953, 2928, 1606, 1508, 1488, 1471 cm^-1;HRMS (FAB)

calcd for C₄₀H₅₆NO₂Si₂(M+H⁺) 638.3850. found 638.3834.

17c (4.9 g, 83%) was obtained from 16c (3.9 g, 8.78 mmol),

TBSCl (3.9 g, 26.3 mmol), DBU (3.9 mL, 26.3 mmol) and dry

CH₂Cl₂(44 mL). Eluent: hexane/AcOEt (20/1). 17c: Colorless

oil: ¹H NMR (300 MHz, CDCl₃) d: -0.31 (3H, s), -0.29 (3H, s),

0.00 (6H, s), 0.67 (9H, s), 0.83 (9H, s), 1.99 (1H, br s), 2.31–2.39

(3H, m), 2.48–2.50 (1H, m), 2.73 (1H, d, J = 9.3 Hz), 6.52 (1H,

d, J = 8.5 Hz), 6.57 (1H, d, J = 8.5 Hz), 6.81 (1H, s), 6.95–7.09

(9H, m), 7.38 (6H, d, J = 7.5 Hz); ¹³C NMR (75 MHz, CDCl₃)

d: -5.5, -5.4, -4.3, 18.1, 18.3, 25.7, 25.9, 37.8, 55.2, 62.3, 71.1,

120.2, 124.9, 126.3, 127.8, 128.6, 128.7, 131.2, 133.6, 147.2, 149.5;

IR (KBr): 3327, 1596, 1494, 1471, 1462, 1448 cm^-1; HRMS (FAB)

calcd for C₄₀H₅₅ClNO₂Si₂[M+H]⁺: 672.3460, found 672.3453.

17d (883 mg, 91%) was obtained from 16d (680 mg, 1.27 mmol),

TBSCl (564 mg, 3.81 mmol), DBU (0.564 mL, 3.81 mmol) and dry

18d (2.82 g, 99%) was obtained from 17d (3.35 g, 4.39 mmol),

TBAF (4.39 mL, 4.39 mmol) and dry THF (70 mL). Eluent:

hexane-AcOEt (10/1). 18d: Colorless oil: H NMR (300 MHz,

1

CDCl₃) d: -0.02 (3H, s), -0.01 (3H, s), 0.98 (9H, s), 2.47–2.56

(3H, m), 2.80 (1H, m), 3.00 (1H, dd, J = 9.3, 3.0 Hz), 6.91–6.97

(2H, m), 7.08 (1H, s), 7.27–7.41 (9H, m), 7.66 (6H, d, J = 7.5 Hz);

¹³C NMR (75 MHz, CDCl₃) d: -5.4, 18.1, 25.9, 37.4, 55.1, 62.2,

71.1, 85.3, 114.4, 126.3, 127.8, 128.7, 131.3, 133.8, 139.1, 147.1,

152.9; IR (KBr): 3541, 3327, 3057, 3030, 2927, 2854, 1595, 1498,

1470, 1448 cm^-1; HRMS (FAB) calcd for C₃₄H₄₁INO₂Si [M+Na]⁺:

650.1951, found 650.1964.

A solution of 18 (1.2 equiv) in 0.1 M HCl/MeOH (1.44 equiv)

was stirred at rt for 0.5 h under N₂. This solution was added

dropwise to a solution of 32 (1.0 equiv) in MeOH (0.345 M

solution). The mixture was stirred at rt for 16 h and evaporated

in vacuo. The residue was puriﬁed by SiO₂column chromatography

to give 38.

38a (228 mg, 83%) was obtained from 18a (446 mg, 0.851 mmol),

0.1 M HCl/MeOH (9.3 mL), 32 (260 mg, 0.709 mmol) and MeOH

(1.6 mL). Eluent: CH₂Cl₂/MeOH/Et₃N (100/5/0.1). 38a: Red

1

solid: H NMR (300 MHz, CDCl₃) d: 0.00 (3H, s), 0.01 (3H, s),

4970 | Org. Biomol. Chem., 2011, 9, 4959–4976

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0.92 (9H, s), 2.44 (3H, s), 2.61 (2H, d, J = 6.9 Hz), 2.80 (2H, t, J =

7.2 Hz), 3.30–3.32 (1H, m), 3.47 (2H, d, J = 3.0 Hz), 4.14 (2H, t, J =

7.2 Hz), 5.63 (1H, s), 5.95 (1H, br s), 6.74 (2H, d, J = 8.4 Hz), 6.84

(2H, d, J = 8.4 Hz), 7.34 (2H, d, J = 8.4 Hz), 7.54 (1H, s), 8.06 (2H,

d, J = 8.4 Hz); ¹³C NMR (75 MHz, CD₃OD) d: -5.5, -5.4, 17.9,

18.2, 21.7, 25.8, 34.2, 48.3, 54.9, 61.4, 94.6, 116.0, 118.0, 122.8,

125.9, 126.3, 128.3, 128.8, 129.7, 130.1, 134.4, 145.2, 145.8, 155.7,

156.1, 169.4; IR (KBr): 3375, 1666, 1614, 1579, 1531, 1514, 1494,

1462, 1380 cm^-1; HRMS (FAB) calcd for C₃₂H₄₀N₃O₅SSi [M+H]⁺:

606.2458, found 606.2465.

17.5, 18.3, 21.7, 25.7, 37.5, 40.8, 49.5, 66.3, 105.5, 118.5, 121.9,

125.8, 126.4, 127.5, 128.6, 129.7, 134.6, 141.9, 145.7, 152.9, 157.3,

168.7, 186.1; IR (KBr): 3394, 1660, 1620, 1595, 1574, 1524, 1460,

1435, 1379 cm^-1; HRMS (FAB) calcd for C₃₂H₃₈N₃O₅SSi [M+H]⁺:

604.2301, found 604.2323.

49c (24.7 mg, 24%) and 49¢c (18.2 mg, 17%) were obtained from

38c (105.6 mg, 0.165 mmol), PIFA (85.1 mg, 0.198 mmol), and

CF₃CH₂OH (5.5 ml). Eluent: hexane/AcOEt/Et₃N (100/50/0.1).

49c: Red solid: mp > 300 ^◦C; [a]²_D^6.3+225 (c 1.84, MeOH); ¹H NMR

(300 MHz, CDCl₃) d: 0.01 (3H, s), 0.02 (3H, s), 0.84 (9H, s), 1.46–

1.65 (2H, m), 2.33 (3H, s), 2.52 (2H, t, J = 7.3 Hz), 3.38–3.45 (2H,

m), 3.64–3.76 (2H, m), 3.97–4.04 (1H, m), 6.19 (1H, d, J = 9.9 Hz),

6.88 (1H, dd, J = 9.9, 2.7 Hz), 6.93 (1H, d, J = 2.7 Hz), 7.22 (2H,

d, J = 8.4 Hz), 7.36 (1H, s), 7.72 (1H, d, J = 8.4 Hz), 7.94 (1H,

d, J = 8.4 Hz); ¹³C NMR (75 MHz, CDCl₃) d: -5.4, 17.5, 18.3,

21.7, 25.9, 36.6, 41.8, 42.9, 48.1, 49.5, 53.4, 66.1, 118.5, 121.8,

125.4, 126.1, 126.3, 128.6, 129.7, 130.2, 134.5, 145.8, 152.5, 153.2,

168.4, 179.3; IR (KBr): 3387, 3153, 2928, 1798, 1660, 1597, 1574,

1528, 1460, 1379 cm^-1; HRMS (FAB) calcd for C₃₂H₃₇ClN₃O₅SSi

[M+H]⁺: 638.1912, found 638.1909.

38c (128.1 mg, 58%) was obtained from 18c (231.2 mg,

0.414 mmol), 0.1 M HCl/MeOH (4.97 mL), 32 (122.9 mg,

0.345 mmol) and MeOH (1.0 mL). Eluent: CH₂Cl₂/MeOH/Et₃N

1

(100/5/0.1). 38c: Red solid: H NMR (270 MHz, CD₃OD) d:

0.00 (6H, s), 0.91 (9H, s), 2.42 (3H, s), 2.61 (2H, d, J = 6.6 Hz),

2.77 (2H, t, J = 8.0 Hz), 3.33–3.36 (1H, m), 3.64–3.67 (2H, m),

4.11–4.12 (2H, m), 5.57 (1H, s), 6.75 (1H, d, J = 9.0 Hz), 6.82

(1H, d, J = 9.0 Hz), 7.03 (1H, s), 7.32 (2H, d, J = 8.9 Hz), 7.51

(1H, s), 8.03 (2H, d, J = 8.9 Hz); ¹³C NMR (75 MHz, CDCl₃) d:

-5.6, -5.5, 17.9, 18.2, 21.7, 25.8, 34.0, 48.1, 54.7, 61.3, 74.0, 86.1,

94.4, 107.4, 117.0, 117.9, 120.8, 122.7, 126.0, 126.3, 128.5, 128.8,

129.7, 130.0, 134.2, 145.9, 151.4, 156.0, 162.6, 166.8; IR (KBr):

3375, 3018, 2928, 2856, 1666, 1614, 1580, 1537, 1495, 1462, 1445,

1379 cm^-1; HRMS (FAB) calcd for C₃₂H₃₉ClN₃O₅SSi [M+H]⁺:

640.2068, found 640.2095.

38d (71.7 mg, 49%) was obtained from 18d (171.3 mg,

0.264 mmol), 0.1 M HCl/MeOH (3.17 mL), 32 (78.4 mg,

0.200 mmol) and MeOH (0.5 mL). Eluent: CH₂Cl₂/MeOH/Et₃N

(100/5/0.1). 38d: Red solid: ¹H NMR (500 MHz, CDCl₃) d: 0.01

(3H, s), 0.13 (3H, s), 0.85 (9H, s), 2.32 (3H, s), 2.63 (2H, d, J =

6.3 Hz), 2.77 (2H, t, J = 8.1 Hz), 3.37–3.40 (1H, m), 3.48–3.54

(2H, m), 4.03 (2H, t, J = 8.1 Hz), 5.48 (1H, s), 6.79 (1H, d,

J = 7.8 Hz), 6.89 (1H, d, J = 7.8 Hz), 7.28 (1H, s), 7.30 (2H,

d, J = 9.0 Hz), 7.34 (1H, s), 8.00 (2H, d, J = 9.0 Hz); ¹³C NMR

(75 MHz, CDCl₃) d: -5.5, -5.4, 18.0, 18.2, 18.8, 21.8, 23.9, 25.9,

45.4, 45.6, 53.7, 56.4, 62.0, 97.1, 107.6, 117.2, 118.0, 119.3, 126.5,

128.7, 128.9, 129.8, 130.6, 131.0, 135.4, 137.0, 139.2, 158.1, 164.5,

175.7, 186.2; IR (KBr): 3368, 2953, 2928, 2856, 1709, 1666, 1616,

1578, 1558, 1534, 1491, 1464, 1443, 1379 cm^-1; HRMS (FAB) calcd

for C₃₂H₃₉IN₃O₅SSi [M+H]⁺: 732.1424, found 732.1416.

1

49¢c: Red solid: H NMR (300 MHz, CDCl₃) d: 0.00 (3H, s),

0.02 (3H, s), 0.84 (9H, s), 1.45–1.62 (2H, m), 2.33 (3H, s), 2.52 (2H,

t, J = 7.5 Hz), 3.40–3.46 (2H, m), 3.64–3.73 (2H, m), 3.94–3.41

(1H, m), 5.27 (1H, br s), 6.24 (1H, d, J = 9.6 Hz), 6.82 (1H, d, J =

9.6 Hz), 7.06 (1H, s), 7.23 (2H, d, J = 7.8 Hz), 7.36 (1H, s), 7.93

(2H, d, J = 7.8 Hz); ¹³C NMR (125 MHz, CDCl₃) d: -5.4, -5.4,

17.5, 18.3, 21.7, 25.9, 36.9, 43.0, 49.4 53.4, 66.1, 77.2, 125.4, 126.4,

128.6, 129.6, 129.7, 133.0, 134.6, 142.1, 143.5, 145.8, 148.2, 157.2,

179.2; IR (KBr): 3393, 3153, 2955, 2930, 2856, 1661, 1595, 1574,

1529, 1462, 1379 cm^-1; HRMS (FAB) calcd for C₃₂H₃₇ClN₃O₅SSi

[M+H]⁺: 638.1912, found 638.1926.

49d (21.8 mg, 25%) and 49¢d (10.8 mg, 12%) were obtained

from 38d (88.4 mg, 0.121 mmol), PIFA (63.3 mg, 0.145 mmol), and

CF₃CH₂OH (4.0 ml). Eluent: hexane/AcOEt/Et₃N (100/50/0.1).

49d: Red solid: [a]²_D^7.4-164 (c 4.50, MeOH); ¹H NMR (500 MHz,

CDCl₃) d: -0.23 (3H, s), 0.21 (3H, s), 0.84 (9H, s), 1.36–1.68

(2H, m), 2.33 (3H, s), 2.53 (2H, t, J = 7.1 Hz), 3.38–3.49 (2H,

m), 3.63–3.82 (2H, m), 3.94–4.04 (1H, m), 5.99 (1H, br s), 6.19

(1H, d, J = 9.7 Hz), 6.90 (1H, dd, J = 9.7, 2.6 Hz), 7.23 (2H,

d, J = 7.4 Hz), 7.36 (1H, s), 7.49 (1H, d, J = 2.6 Hz), 7.94

(2H, d, J = 7.4 Hz); ¹³C NMR (67.8 MHz, CDCl₃) d: -5.3,

-5.2, 17.6, 18.4, 21.6, 21.8, 24.7, 25.9, 29.7, 36.7, 45.1, 49.6, 66.1,

104.0, 118.5, 121.7, 123.2, 124.1, 125.5, 126.0, 126.3, 128.5, 129.5,

129.6, 129.7, 134.4, 139.0, 143.3, 145.6, 152.7, 157.2, 160.1, 168.1,

179.5; IR (KBr): 3395, 2928, 2856, 1659, 1574, 1529, 1461 cm^-1;

HRMS (FAB) calcd for C₃₂H₃₇IN₃O₅SSi [M+H]⁺: 730.1268, found

730.1266.

General procedure for the syntheses of 49a, c–d, 49¢c–d from

38a,c,d

PIFA (1.2 equiv) and montmorillonite K10 (1/4 mg of 38 (mg))

was added to a solution of 38 (1.0 equiv) in CF₃CH₂OH (0.03 M

solution) at rt under N₂. The mixture was stirred at rt for 0.5 h

and evaporated in vacuo. The residue was puriﬁed by SiO₂column

chromatography to give 49.

1

49¢d: Red solid: H NMR (270 MHz, CDCl₃) d: 0.03 (3H, s),

0.06 (3H, s), 0.88 (9H, s), 1.60–1.65 (2H, m), 2.38 (3H, s), 2.55 (2H,

t, J = 7.5 Hz), 3.43–3.46 (2H, m), 3.66–3.77 (2H, m), 3.97–4.04

(1H, m), 6.05 (1H, br s), 6.26 (1H, d, J = 9.6 Hz), 6.87 (1H, dd,

J = 9.6, 2.5 Hz), 7.25 (2H, d, J = 8.4 Hz), 7.38 (1H, s), 7.69 (1H,

d, J = 2.5 Hz), 7.96 (2H, d, J = 8.4 Hz); ¹³C NMR (67.8 MHz,

CDCl₃) d: -5.2 (2C), 17.7, 18.4, 21.8, 24.7, 26.0, 29.8, 36.7, 45.1,

49.4, 49.6, 66.1, 104.1, 118.5, 121.8, 123.3, 125.5, 126.0, 126.3,

128.6, 129.2, 129.6, 129.8, 134.4, 143.4, 145.7, 152.7, 157.1, 160.0,

168.1, 179.5; IR (KBr): 3391, 3153, 2928, 2856, 1794, 1655, 1575,

1528, 1462, 1382 cm^-1; HRMS (FAB) calcd for C₃₂H₃₇IN₃O₅SSi

[M+H]⁺: 730.1268, found 730.1284.

49a (66.0 mg, 57%) was obtained from 38a (115 mg,

0.190 mmol), PIFA (98 mg, 0.228 mmol), and CF₃CH₂OH(6.3 ml).

1

Eluent: hexane/AcOEt/Et₃N (100/50/0.1). 49a: Red solid: H

NMR (300 MHz, CDCl₃) d: -0.02 (3H, s), 0.02 (3H, s), 0.84 (9H,

s), 1.41 (1H, d, J = 13.2 Hz), 1.61 (1H, d, J = 13.2 Hz), 2.33 (3H, s),

2.52 (2H, t, J = 7.2 Hz), 3.43–3.45 (2H, m), 3.63–3.75 (3H, m), 3.99

(1H, t, J = 7.2 Hz), 5.99 (1H, br s), 6.12 (1H, dd, J = 9.9, 3.0 Hz),

6.13 (1H, dd, J = 9.9, 3.0 Hz), 6.80 (1H, dd, J = 9.9, 3.0 Hz), 6.89

(1H, dd, J = 9.9, 3.0 Hz), 7.23 (2H, d, J = 8.4 Hz), 7.35 (1H, s), 7.95

(2H, d, J = 8.4 Hz); ¹³C NMR (75 MHz, CDCl₃) d: -5.4, -5.3,

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General procedure for the syntheses of 50a, c–d, 50¢c–d from 49a,

c–d, 49¢c–d

HRMS (FAB) calcd for C₂₆H₂₃IN₃O₅S [M+H]⁺: 616.0403, found

616.0403.

50¢d (6.3 mg, 68%) was obtained from 49¢d (10.6 mg,

0.015 mmol), BF₃·Et₂O (16.7 mL, 0.145 mmol), and dry CH₂Cl₂

(0.7 mL). Eluent: CH₂Cl₂/MeOH/Et₃N (100/5/0.1). 50¢d: Red

BF₃·Et₂O (9.4 equiv) was added to a solution of 49 (1.0 equiv) in

dry CH₂Cl₂(0.055 M solution) at 0 ^◦C under N₂. The mixture was

allowed to warm to rt for 7 h. The reaction was quenched with

NaHCO₃powder, ﬁltered and evaporated in vacuo. The residue

was puriﬁed by SiO₂column chromatography to give 50.

1

solid: H NMR (300 MHz, CDCl₃) d: 1.93–1.98 (1H, m), 2.15–

2.22 (1H, m), 2.36 (3H, s), 2.53–2.62 (2H, m), 3.51–3.55 (3H, m),

3.79–3.84 (2H, m), 6.30 (1H, d, J = 9.8 Hz), 6.90 (1H, d, J =

9.8 Hz), 7.23 (1H, s), 7.28 (2H, d, J = 7.5 Hz), 7.71 (1H, s), 7.95

(2H, d, J = 7.5 Hz); ¹³C NMR (125 MHz, (CD₃)₂CO) d: 18.0,

21.6, 37.6, 46.2, 46.7, 50.3, 50.4, 65.3, 104.2, 119.6, 123.5, 127.4,

128.3, 129.4, 130.2, 130.7, 131.3, 147.0, 153.9, 158.9, 162.1, 169.3;

IR (KBr): 3370, 2924, 1655, 1574, 1528, 1458, 1377 cm^-1; HRMS

(FAB) calcd for C₂₆H₂₃IN₃O₅S [M+H]⁺: 616.0403, found 616.0413.

50a (35.3 mg, 87%) was obtained from 49a (50.1 mg,

0.0829 mmol), BF₃·Et₂O (0.10 mL, 0.78 mmol), and dry CH₂Cl₂

(1.5 mL). Eluent: CH₂Cl₂/MeOH/Et₃N (100/5/0.1). 50a: Red

solid: ¹H NMR (270 MHz, (CD₃)₂CO) d: 1.53 (1H, d, J = 12.0 Hz),

1.81 (1H, t, J = 12.0 Hz), 2.64 (3H, s), 2.83 (2H, m), 3.57–3.82 (4H,

m), 4.32 (1H, m), 6.06 (2H, dd, J = 10.2, 2.2 Hz), 6.96 (1H, dd,

J = 10.2, 2.2 Hz), 7.47 (2H, d, J = 8.4 Hz), 7.63 (1H, s), 8.06 (2H,

d, J = 8.4 Hz); ¹³C NMR (75 MHz, (CD₃)₂CO) d: 18.0, 21.5, 38.4,

41.6, 50.4, 65.5, 111.9, 119.6, 122.5, 126.8, 127.3, 127.8, 129.4,

130.7, 135.6, 140.0, 147.0, 153.8, 158.0, 169.4, 185.6; IR (KBr):

3305, 1659, 1614, 1595, 1573, 1525, 1487, 1461, 1375 cm^-1; HRMS

(FAB) calcd for C₂₆H₂₄N₃O₅S [M+H]⁺: 490.1437, found 490.1429.

50c (9.0 mg, 65%) was obtained from 49c (16.7 mg,

0.0262 mmol), BF₃·Et₂O (35 mL, 0.276 mmol), and dry CH₂Cl₂

(1.4 mL). Eluent: CH₂Cl₂/MeOH/Et₃N (100/5/0.1). 50c: Red

solid: ¹H NMR (300 MHz, CDCl₃) d: 1.55 (1H, d, J = 12.0 Hz),

1.79 (1H, d, J = 12.0 Hz), 2.36 (3H, s), 2.57–2.62 (2H, m), 3.54–

3.56 (2H, m), 3.78–3.83 (2H, m), 4.00–4.03 (1H, m), 6.03 (1H, br

s), 6.22 (1H, d, J = 9.9 Hz), 6.92 (1H, dd, J = 9.9, 2.4 Hz), 6.98

(1H, d, J = 2.4 Hz), 7.27 (2H, d, J = 8.4 Hz), 7.40 (1H, s), 7.95

(2H, d, J = 8.4 Hz); ¹³C NMR (67.8 MHz, CDCl₃) d: 17.8, 21.7,

28.9, 29.6, 38.6, 44.2, 49.4, 59.6, 68.1, 77.2, 80.1, 118.6, 122.0,

123.3, 125.4, 126.0, 128.5, 129.8, 134.5, 145.9, 152.5, 157.4, 168.3,

191.2; IR (KBr): 3389, 2930, 1713, 1661, 1595, 1573, 1529, 1485,

1462, 1377 cm^-1; HRMS (FAB) calcd for C₂₆H₂₃ClN₃O₅S [M+H]⁺:

524.1047, found 524.1066.

General procedure for the synthesis of 54a, c–d, 54¢c–d from 50a,

c–d, 50¢c–d

30% HBr–AcOH (0.75 mL/1.0 mmol of 50) was added to a

solution of 50 (1 equiv) in dry CH₂Cl₂(0.02 M solution) at 0 ^◦C

under N₂. The mixture was stirred warming to rt for 36 h. The

reaction was quenched with sat. aq. NaHCO₃and extracted with

CH₂Cl₂. The organic layer was washed with brine, dried over

Na₂SO₄and evaporated in vacuo. The residue was puriﬁed by

SiO₂column chromatography to give 54.

54a (23.5 mg, 66%) was obtained from 50a (35.3 mg,

0.0721 mmol), 30% HBr-AcOH (67 mL), and dry CH₂Cl₂(3.6 mL).

Eluent: hexane/AcOEt (1/1). 54a: Red solid: mp > 300 ^◦C; [a]_D^26.5

+956 (c 0.541, MeOH); ¹H NMR (300 MHz, CDCl₃) d: 1.69 (1H,

d, J = 12.9 Hz), 1.92 (1H, d, J = 12.9 Hz), 2.34 (3H, s), 2.53–2.58

(3H, m), 2.84 (1H, dd, J = 16.8, 12.9 Hz), 3.55 (1H, s), 3.73–3.79

(2H, m), 4.15 (1H, dd, J = 16.8, 6.0 Hz), 4.28 (1H, dd, J = 9.6,

6.0 Hz), 5.85 (1H, s), 5.89 (1H, d, J = 10.2 Hz), 7.03 (1H, d,

J = 10.2 Hz), 7.25 (2H, d, J = 8.4 Hz), 7.40 (1H, s), 7.95 (2H,

d, J = 8.4 Hz); ¹³C NMR (75 MHz, CDCl₃) d: 17.8, 21.7, 27.9,

36.5, 37.5, 44.0, 44.5, 49.5, 53.4, 68.0, 75.0, 118.6, 122.1, 124.8,

125.5, 126.7, 128.6, 129.7, 134.6, 145.8, 148.3, 152.7, 157.6, 168.5,

174.9, 197.5; IR (KBr): 3392, 2941, 2842, 1660, 1595, 1569, 1523,

1488, 1458 cm^-1; HRMS (FAB) calcd for C₂₆H₂₄N₃O₅S [M+H]⁺:

490.1437, found 490.1439.

54c (3.5 mg, 71%) was obtained from 50c (4.9 mg,

0.00935 mmol), 30% HBr-AcOH (7 mL), and dry CH₂Cl₂

(0.47 mL). Eluent: hexane/AcOEt (1/1). 54c: Red solid: ¹H NMR

(300 MHz, CDCl₃) d: 1.91 (1H, d, J = 12.9 Hz), 2.34 (1H, dd,

J = 12.9, 2.1 Hz), 2.44 (3H, s), 2.85–2.99 (2H, m), 3.67–4.15 (7H,

m), 5.48 (1H, br s), 6.36 (1H, d, J = 10.6 Hz), 7.06 (1H, d, J =

10.6 Hz), 7.45 (2H, d, J = 8.4 Hz), 7.91 (1H, s), 8.09 (2H, d, J =

8.4 Hz); ¹³C NMR (75 MHz, CDCl₃) d: 17.8, 21.7, 28.9, 29.6,

38.6, 44.2, 49.4, 59.6, 68.1, 77.2, 80.1, 118.6, 122.0, 123.3, 125.4,

126.0, 129.8, 134.5, 145.9, 152.5, 157.4, 168.3, 191.2; IR (KBr):

3395, 2928, 2853, 1682, 1658, 1574, 1526, 1487, 1462, 1379 cm^-1;

HRMS (FAB) calcd for C₂₆H₂₃ClN₃O₅S [M+H]⁺: 524.1047, found

524.1057.

54¢c (2.4 mg, 57%) was obtained from 50¢c (4.2 mg,

0.00801 mmol), 30% HBr-AcOH (6 mL), and dry CH₂Cl₂

(0.40 mL). Eluent: hexane/AcOEt (1/1). 54¢c: Red solid: ¹H NMR

(300 MHz, CDCl₃) d: 1.87 (1H, d, J = 13.5 Hz), 2.38 (1H, dd, J =

13.5, 2.1 Hz), 2.44 (3H, s), 2.78 (1H, dd, J = 16.8, 4.8 Hz), 2.93–2.99

(2H, m), 3.36 (1H, dd, J = 16.8, 13.2 Hz), 3.65–4.02 (5H, m), 4.32

(1H, dd, J = 13.2, 5.3 Hz), 7.22 (1H, s), 7.45 (2H, d, J = 8.7 Hz), 7.91

50¢c (5.5 mg, 65%) was obtained from 49¢c (11.9 mg,

0.0160 mmol), BF₃·Et₂O (21.4 mL, 0.169 mmol), and dry CH₂Cl₂

(0.86 mL). Eluent: CH₂Cl₂/MeOH/Et₃N (100/5/0.1). 50¢c: Red

1

solid: H NMR (300 MHz, CDCl₃) d: 1.56–1.57 (1H, m), 1.80–

1.84 (1H, m), 2.37 (3H, s), 2.57–2.62 (2H, m), 3.54–3.59 (2H, m),

3.79–3.85 (2H, m), 3.98–4.01 (1H, m), 4.72 (1H, br s), 6.29 (1H, d,

J = 9.9 Hz), 6.86 (1H, d, J = 9.9 Hz), 7.10 (1H, s), 7.27 (2H, d, J =

7.8 Hz), 7.41 (1H, s), 7.95 (1H, d, J = 7.8 Hz); ¹³C NMR (75 MHz,

(CD₃)₂CO) d: 18.0, 21.5, 37.6, 39.8, 42.4, 44.0, 47.9, 50.2, 65.2,

119.6, 125.8, 127.4, 129.4, 130.7, 131.7, 135.6, 147.0, 150.0, 158.7,

169.3, 178.9; IR (KBr): 3385, 3130, 1660, 1650, 1595, 1575, 1525,

1485, 1460, 1375 cm^-1; HRMS (FAB) calcd for C₂₆H₂₃ClN₃O₅S

[M+H]⁺: 524.1047, found 524.1060.

50d (14.9 mg, 81%) was obtained from 49d (22.1 mg,

0.030 mmol), BF₃·Et₂O (39.3 mL, 0.303 mmol), and dry CH₂Cl₂

(1.5 mL). Eluent: CH₂Cl₂/MeOH/Et₃N (100/5/0.1). 50d: Red

solid: ¹H NMR (300 MHz, CDCl₃) d: 1.65 (1H, d, J = 12.0 Hz),

1.78 (1H, d, J = 12.0 Hz), 2.36 (3H, s), 2.58 (2H, t, J = 7.2 Hz),

3.54–3.57 (2H, m), 3.79–3.85 (2H, m), 4.00–4.05 (1H, m), 6.23

(1H, d, J = 9.7 Hz), 6.94 (1H, dd, J = 9.7, 2.6 Hz), 7.27 (1H, d,

J = 8.4 Hz), 7.41 (1H, s), 7.53 (1H, d, J = 2.6 Hz), 7.96 (2H, d,

J = 8.4 Hz); ¹³C NMR (125 MHz, (CD₃)₂CO) d: 18.0, 21.6, 37.1,

45.9, 50.0, 50.4, 65.4, 65.5, 119.6, 122.6, 124.3, 126.2, 127.4, 129.4,

130.2, 130.7, 135.6, 147.0, 153.8, 154.2, 166.4, 169.2, 179.7; IR

(KBr): 3387, 2925, 2853, 1794, 1655, 1572, 1528, 1460, 1379 cm^-1;

4972 | Org. Biomol. Chem., 2011, 9, 4959–4976

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(1H, s), 8.09 (2H, d, J = 8.7 Hz); ¹³C NMR (75 MHz, CDCl₃) d:

17.8, 21.7, 27.5, 29.7, 30.9, 37.7, 38.2, 44.3, 49.7, 68.2, 74.5, 118.6,

122.0, 125.9, 128.6, 129.8, 134.5, 140.8, 145.9, 152.4, 153.7, 174.9,

190.2; IR (KBr): 3400, 2928, 2853, 1682, 1659, 1595, 1574, 1526,

1489, 1462, 1377 cm^-1; HRMS (FAB) calcd for C₂₆H₂₃ClN₃O₅S

[M+H]⁺: 524.1047, found 524.1071.

Organic phase was washed by H₂O (¥3) and brine (¥1). Organic

phase was dried over Na₂SO₄. and evaporated in vacuo. Residue

was puriﬁed by SiO₂column chromatography (CH₂Cl₂/MeOH =

20/1) to give 57¢ (5.07 mg, 50%) as a red solid; mp >300 ^◦C; ¹H

NMR (300 MHz, CDCl₃) d: 2.10 (1H, d, J = 12.8 Hz), 2.25 (1H, d,

J = 12.8 Hz), 2.97–3.00 (1H, m), 3.15–3.23 (1H, m), 3.86–4.05 (2H,

m), 4.45–4.59 (1H, m), 5.23–5.30 (1H, m), 6.32 (1H, br s), 7.26–

7.49 (2H, m), 7.87–7.94 (2H, m), 8.41 (1H, d, J = 5.9 Hz); ¹³C NMR

(125 MHz, CDCl₃) d: 27.4, 29.1, 39.7, 40.0, 52.4, 80.8, 110.6, 112.3,

118.0, 123.6, 125.0, 129.2, 142.9, 143.3, 146.2, 158.6, 165.3, 190.5,

191.0; IR (KBr): 3057, 2930, 2856, 1682, 1645, 1599, 1535, 1504,

1485, 1461 cm^-1; HRMS (FAB) calcd for C₁₉H₁₅BrN₃O₃[M+H]⁺:

412.0297, found 412.0293.

54d (2.8 mg, 76%) was obtained from 50d (3.7 mg,

0.00601 mmol), 30% HBr-AcOH (9 mL), and dry CH₂Cl₂

(0.30 mL). Eluent: hexane/AcOEt (1/1). 54d: Red solid: [a]_D^21.0

+96.8 (c 0.322, CHCl₃); ¹H NMR (500 MHz, CDCl₃) d: 1.78 (1H,

d, J = 13.0 Hz), 2.00 (1H, d, J = 13.0 Hz), 2.43 (3H, s), 2.61–2.70

(2H, m), 2.92 (1H, dd, J = 16.8, 13.0 Hz), 3.61 (1H, s), 3.73 (1H,

d, J = 12.5 Hz), 3.80–3.84 (1H, m), 3.88 (1H, d, J = 12.5 Hz), 4.23

(1H, dt, J = 17.5, 6.0 Hz), 4.36 (1H, dd, J = 13.0, 5.5 Hz), 5.89 (1H,

br s), 5.97 (1H, d, J = 10.0 Hz), 7.11 (1H, d, J = 10.0 Hz), 7.33 (2H,

d, J = 8.0 Hz), 7.48 (1H, s), 8.03 (2H, d, J = 8.0 Hz); ¹³C NMR

(125 MHz, CDCl₃) d: 17.9, 21.7, 28.1, 36.6, 37.6, 44.5, 49.6, 68.1,

75.1, 109.4, 118.6, 122.2, 124.9, 125.5, 125.7, 128.7, 129.8, 134.7,

143.5, 145.8, 152.8, 157.6, 168.6, 198.5; IR (KBr): 2928, 2853,

2359, 2341, 2253, 1659, 1570, 1523, 1489, 1458; HRMS (FAB)

calcd for C₂₆H₂₃IN₃O₅S [M+H]⁺: 616.0403, found 616.04011.

54¢d (1.4 mg, 67%) was obtained from 50¢d (2.1 mg,

0.0341 mmol), 30% HBr-AcOH (3 mL), and dry CH₂Cl₂(0.17 mL).

Eluent: hexane/AcOEt (1/1). 54¢d: Red solid: mp > 300 ^◦C; [a]_D^22.0

-76.0 (c 0.626, CHCl₃); ¹H NMR (500 MHz, CDCl₃) d: 1.79 (1H,

d, J = 12.8 Hz), 2.04 (1H, d, J = 12.8 Hz), 2.41 (3H, s), 2.67 (2H,

t, J = 7.5 Hz), 2.88 (1H, dd, J = 16.5, 6.0 Hz), 3.01 (1H, dd, J =

16.8, 13.0 Hz), 3.61 (1H, s), 3.72 (1H, d, J = 12.4 Hz), 3.82–3.99

(2H, m), 4.25 (1H, dt, J = 18.0, 6.5 Hz), 4.31 (1H, dd, J = 12.5,

5.0 Hz), 5.85 (1H, br s), 7.32 (2H, d, J = 8.3 Hz), 7.47 (1H, s), 7.74

(1H, s), 8.01 (2H, d, J = 8.3 Hz); ¹³C NMR (125 MHz, CDCl₃) d:

17.8, 21.7, 27.1, 29.7, 36.3, 44.4, 49.8, 63.4, 68.2, 74.8, 98.4, 102.1,

118.5, 124.4, 125.9, 128.7, 129.8, 129.8, 145.8, 152.3, 154.1, 154.3,

165.5, 168.4, 185.0, 191.4; IR (KBr): 3018, 1710, 1659, 1526, 1487,

1460; HRMS (FAB) calcd for C₂₆H₂₃IN₃O₅S [M+H]⁺: 616.0403,

found 616.0408.

Discorhabdin oxa analogue (55). 5 M NaOMe in MeOH (9.60

ml, 0.0480 mmol) was added to a solution of 54a in dry THF

(1.6 ml) at 0 ^◦C under N₂. The mixture was stirred at 0 ^◦C for 0.5 h

and evaporated in vacuo. The residue was puriﬁed by SiO₂column

chromatography (CH₂Cl₂/MeOH = 5/1) to give 55 (10.3 mg, 64%)

as a green solid: ¹H NMR (300 MHz, CD₃OD) d: 2.09 (1H, d, J =

12.3 Hz), 2.44 (1H, d, J = 12.3 Hz), 2.63–2.66 (3H, m), 3.04–3.14

(1H, m), 3.60–3.66 (4H, m), 3.88 (1H, d, J = 10.8 Hz), 4.23 (1H,

d, J = 8.4 Hz), 5.94 (1H, d, J = 9.6 Hz), 6.70 (1H, s), 7.08 (1H,

d, J = 9.6 Hz); ¹³C NMR (75 MHz, CDCl₃) d: 19.4, 28.9, 37.7,

38.8, 46.6, 63.7, 67.7, 76.2, 103.2, 120.3, 124.1, 124.3, 124.8, 125.9,

128.5, 155.5, 156.7, 169.4, 200.5; IR (KBr): 3350, 2931, 2852, 1651,

1602, 1556, 1537, 1523, 1488, 1434 cm^-1; HRMS (FAB) calcd for

C₁₉H₁₈N₃O₃[M+H]⁺: 336.1348, found 336.1370.

Discorhabdin oxa analogue (56). NaH (0.902 mg,

0.0228 mmol) was added to a solution of 55 (5.10 mg,

0.0152 mmol) in_◦dry THF (0.75 ml) at 0 ^◦C under N₂. The mixture

was stirred at 0 C for 10 min and MsCl (1.40 ml, 0.0182 mmol)

was added to the mixture. The mixture was stirred at 0 ^◦C for

30 min and evaporated in vacuo. The residue was puriﬁed by

SiO₂column chromatography (CH₂Cl₂/MeOH = 20/1) to give

1

56 (3.40 mg, 54%) as red solid; H NMR (300 MHz, CDCl₃) d:

1.78 (1H, d, J = 12.6 Hz), 1.99 (1H, d, J = 12.6 Hz), 2.61–2.66

(3H, m), 2.89 (1H, m), 3.57 (3H, s), 3.58–3.58 (2H, m), 3.71–3.89

(2H, m), 4.20 (1H,m), 4.35 (1H, dd, J = 12.9, 5.1 Hz), 5.89–5.92

(1H, br s), 5.94 (1H, d, J = 10.2 Hz), 7.08 (1H, d, J = 10.2 Hz),

7.26 (1H, s); ¹³C NMR (75 MHz, CDCl₃) d: 17.7, 28.0, 31.8, 36.7,

37.6, 42.3, 44.6, 49.7, 68.1, 74.2, 109.8, 118.4, 122.1, 124.9, 125.5,

145.9, 152.8, 157.5, 159.4, 165.4, 198.5; IR (KBr): 3400, 2933,

2849, 1651, 1614, 1568, 1523, 1494, 1462 cm^-1; HRMS (FAB)

calcd for C₂₀H₂₀N₃O₅S [M+H]⁺: 414.1124, found 414.1138.

Discorhabdin oxa-aromatic analogue (57). NaN₃(0.248 mg,

0.00382 mmol) was added to a solution of 54b (20.8 mg,

0.0366 mmol) in DMF (0.063 mL) at rt under N₂. The mixture

◦

was allowed to warm to 70 C and stirred for 1 h. The reaction

mixture was quenched by H₂O and extracted by AcOEt. Organic

phase was washed by H₂O (¥3) and brine (¥1), dried over Na₂SO₄

and evaporated in vacuo. The residue was puriﬁed by SiO₂column

chromatography (CH₂Cl₂/MeOH = 20/1) to give 57 (8.15 mg,

1

54%) as a red solid; H NMR (300 MHz, CDCl₃) d: 2.10 (1H,

d, J = 11.7 Hz), 2.29 (1H, d, J = 11.7 Hz), 2.79–2.82 (2H, m),

4.01–4.06 (1H, m), 4.14 (1H, d, J = 10.4 Hz), 4.57 (1H, d, J =

10.4 Hz), 5.97 (1H, s), 6.56 (1H, d, J = 9.7 Hz), 6.57 (1H, br s),

7.04 (1H, d, J = 9.7 Hz), 7.34 (1H, d, J = 5.8 Hz), 7.90 (1H, s),

8.31 (1H, d, J = 5.8 Hz); ¹³C NMR (125 MHz, DMSO-d₆) d: 26.0,

30.0, 35.5, 42.7, 57.7, 72.8, 80.6, 101.9, 114.9, 117.2, 117.3, 117.7,

119.3, 124.2, 132.5, 170.7, 178.1, 190.6, 191.8; IR (KBr): 2924,

1651, 1601, 1531, 1548, 1454 cm^-1.

Compound 59. BzCl (5.00 ml, 0.0450 mmol) was added to

a solution of 50b (17.0 mg, 0.0300 mmol) and Et₃N (6.30 ml,

0.0450 mmol) in dry CH₂Cl₂(1.0 ml) at 0 ^◦C under N₂. The

resulting solution was warmed to rt for 3.0 h. The reaction

was quenched with H₂O and extracted with AcOEt. The organic

layer was washed with brine, dried over Na₂SO₄and evaporated

in vacuo. The residue was puriﬁed by NH column chromatography

(n-hexane/AcOEt = 2/1) to give 59 (11.5 mg, 57%) as red solid; ¹H

NMR (400 MHz, CDCl₃) d: 2.00–2.07 (2H, m), 2.42 (3H, s), 2.66

(2H, t, J = 8.0 Hz), 3.63–3.66 (2H, m), 4.01 (2H, t, J = 8.0 Hz),

4.15–4.17 (1H, m), 6.12 (1H, d, J = 10.4 Hz), 7.14 (1H, dd, J =

10.4, 2.8 Hz), 7.32 (2H, d, J = 8.4 Hz), 7.38 (1H, s), 7.47–7.56 (3H,

m), 7.61 (1H, d, J = 7.2 Hz), 8.03 (2H, d, J = 8.4 Hz), 8.08 (2H, d,

Discorhabdin oxa-aromatic analogue (57¢). NaN₃(0.205 mg,

0.00315 mmol) was added to a solution of 54¢b (14.0 mg,

0.0246 mmol) in DMF (0.042 mL) at rt under N₂atmosphere. The

◦

mixture was allowed to warm to 70 C. and stirred for 1 h. The

reaction mixture was quenched by H₂O and extracted by AcOEt.

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J = 8.4 Hz); ¹³C NMR (100 MHz, DMSO-d₆) d: 15.4, 22.3, 25.9,

29.7, 52.6, 72.5, 76.2, 93.4, 98.7, 102.0, 115.5, 117.6, 123.7, 129.3,

145.4, 145.8, 147.2, 147.8, 147.9, 148.3, 155.7, 157.9, 158.3, 163.7,

165.5, 169.3, 180.0, 191.7, 198.3; IR (KBr): 2963, 2924, 1719, 1655,

1595, 1481, 1458 cm^-1; HRMS (FAB) calcd for C₃₃H₂₆BrN₃O₆S

[M]⁺: 671.0726, found 671.0690.

¹³C NMR (100 MHz, CD₃OD) d: 19.7, 21.7, 42.5, 47.2, 54.9, 94.6,

115.0, 119.4, 125.5, 128.3, 128.7, 130.0, 130.7, 131.0, 135.2, 135.8,

146.1, 148.1, 150.1, 156.6, 170.8; IR (KBr): 3262, 3053, 2101, 1681,

1614, 1566, 1556, 1531, 1525, 1494, 1469, 1446 cm^-1.

PIFA (55.0 mg, 0.129 mmol) was added to a solution of 35

(1.0 equiv) in CF₃CH₂OH (4.0 ml) at rt under N₂. The mixture was

stirred at rt for 3.0 h and evaporated in vacuo. The residue was pu-

riﬁed by SiO₂column chromatography (CH₂Cl₂/MeOH/Et₃N =

Compound 60. PPh₃(19.4 mg, 0.0740 mmol), DEAD (33.6 ml,

0.0740 mmol) and DPPA (16.0 ml, 0.0740 mmol) were added to

a solution of 50b (28.0 mg, 0.0490 mmol) in toluene (1.0 ml) at

0 ^◦C under N₂. The resulting solution was warmed to rt for 7.0 h

and evaporated in vacuo. The residue was puriﬁed by NH column

chromatography (CH₂Cl₂/MeOH = 20/1) to give 60 (13.1 mg,

45%) as red solid; ¹H NMR (400 MHz, CDCl₃) d: 1.88 (1H, d, J =

12.8 Hz), 1.94 (1H, d, J = 12.8 Hz), 2.43 (3H, s), 2.66 (2H, t, J =

7.2 Hz), 3.67–3.85 (3H, m), 4.11–4.23 (2H, m), 5.07 (1H, d, J =

12.4 Hz), 5.94 (1H, br s), 6.01 (1H, d, J = 10.4 Hz), 7.14 (1H, d,

J = 10.4 Hz), 7.33 (2H, d, J = 8.4 Hz), 7.49 (1H, s), 8.01 (2H, d, J =

8.4 Hz); ¹³C NMR (DMSO-d₆) d: 17.3, 21.2, 43.6, 49.2, 55.1, 67.5,

80.2, 100.1, 100.7, 111.0, 121.9, 126.2, 126.8, 128.0, 130.0, 143.9,

146.0, 152.0, 155.5, 157.2, 158.9, 168.1, 189.3, 195.0; IR (KBr):

1659, 1575, 1525, 1493, 1462, 1369 cm^-1.

1

100/1/0.1) to give 43 (20.6 mg, 27%) as a red solid; H NMR

(500 MHz, CDCl₃) d: 1.92–1.95 (2H, m), 2.43 (3H, s), 2.64 (2H, t,

J = 7.5 Hz), 3.48–3.54 (2H, m), 3.99 (2H, t, J = 7.5 Hz), 5.81 (1H,

br s), 7.33 (2H, d, J = 8.0 Hz), 7.48 (1H, s), 7.71 (2H, s), 8.01 (2H, d,

J = 8.0 Hz); ¹³C NMR (75 MHz, CDCl₃) d: 21.8, 29.7, 32.8, 37.5,

48.5, 49.8, 96.4, 102.2, 111.3, 118.6, 121.7, 125.6, 126.4, 126.5,

127.2, 128.6, 129.8, 134.6, 142.1, 145.9, 162.6, 168.4, 174.1; IR

(KBr): 3391, 2926, 2853, 1659, 1574, 1528, 1495, 1460, 1435 cm^-1;

HRMS (FAB) calcd for C₂₅H₂₀I₂N₃O₄S [M+H]⁺: 711.9264, found

711.9258.

Compounds 36a, 36b. Quinoline-5,8-dione (20) (25.1 mg,

0.158 mmol) was added to a solution of tyramine (10) (26.0 mg,

0.189 mmol) in MeOH (2.0 ml) at rt under N₂. The mixture was

stirred at rt for 16 h and evaporated in vacuo. The residue was

puriﬁed by SiO₂column chromatography (CH₂Cl₂/MeOH = 20/1)

to give 36a and 36b (less polar; (13.3 mg, 24%), polar (27.3 mg,

49%).

Discorhabdin P (46). K₂CO₃(16.3 mg, 0.118 mmol) and MeI

(3.70 ml, 0.0593 mmol) was added to a solution of discorhabdin C

(3) (5.50 mg, 0.0118 mmol) in dry acetone (0.50 ml). The mixture

was stirred at 40 ^◦C for 12 h and evaporated in vacuo. The residue

was puriﬁed by SiO₂column chromatography (CH₂Cl₂/MeOH =

10/1) to give discorhabdin P (3.60 mg, 64%) as a red solid; mp >

300 ^◦C; ¹H NMR (300 MHz, CD₃OD) d: 1.87–1.89 (2H, m), 2.51

(2H, t, J = 7.5 Hz), 3.43–3.47 (2H, m), 3.73 (2H, t, J = 7.5 Hz), 3.82

(3H, s), 6.79 (1H, s), 7.56 (2H, s); ¹³C NMR (125 MHz, CDCl₃)

d: 18.0, 35.4, 37.6, 43.1, 45.9, 71.1, 97.1, 99.2, 106.3, 128.2, 133.1,

141.2, 149.9, 155.3, 168.0, 186.0, 193.2; UV/Vis (MeOH) lmax =

488 (log e 0.18), 341 (1.60), 246 (3.29), 211 (3.41) nm; IR (KBr):

3387, 2926, 2503, 1649, 1566, 1523, 1493 cm^-1; HRMS (FAB) calcd

for C₁₉H₁₈Br₂N₃O₂[M+H]⁺: 477.9766, found 477.9581.

1

36b (less polar): brown solid: H NMR (300 MHz, CDCl₃) d:

2.92 (2H, t, J = 7.5 Hz), 3.46 (2H, t, J = 7.5 Hz), 5.83 (1H, s),

6.80 (2H, d, J = 8.4 Hz), 7.07 (2H, d, J = 8.4 Hz), 7.72 (1H, dd,

J = 7.8, 4.5 Hz), 8.44 (1H, dd, J = 7.8, 1.8 Hz), 8.87 (1H, dd,

J = 4.5, 1.8 Hz); ¹³C NMR (75 MHz, CDCl₃) d: 32.2, 43.5, 98.5,

114.9, 128.2, 128.6, 129.4, 130.0, 133.1, 146.3, 148.6, 152.3, 155.5,

179.4, 180.1; IR (KBr): 3297, 2982, 2947, 1769, 1759, 1703, 1605,

1581, 1564, 1514, 1454 cm^-1; HRMS (FAB) calcd for C₁₇H₁₅N₂O₃

[M+H]⁺: 295.1083, found 295.1079.

36a (polar): brown solid: ¹H NMR (300 MHz, CDCl₃) d: 2.90

(2H, t, J = 6.9 Hz), 3.44 (2H, t, J = 6.9 Hz), 5.94 (1H, s), 6.11

(1H, br s), 6.80 (2H, d, J = 8.4, 2.1 Hz), 7.05 (2H, d, J = 8.4 Hz),

7.58 (1H, dd, J = 7.8, 4.8 Hz), 8.34 (1H, dd, J = 7.8, 1.8 Hz), 8.99

(1H, dd, J = 4.8, 1.8 Hz); ¹³C NMR (125 MHz, CDCl₃) d: 33.5,

43.9, 101.7, 115.8, 126.4, 127.4, 128.6, 129.7, 134.3, 147.6, 149.2,

155.0, 155.7, 181.3, 181.4; IR (KBr): 3556, 2986, 2086, 1757, 1606,

1568 cm^-1; HRMS (FAB) calcd for C₁₇H₁₅N₂O₃[M+H]⁺: 295.1083,

found 295.1089.

Compound 14. I₂(8.20 g, 32.3 mmol) and 30% H₂O₂(7.20 ml)

were added to a solution of tyramine (10) (4.00 g, 29.2 mmol) in

H₂O (140 ml). The resulting solution was warmed to 55 ^◦C for

3.0 h. The reaction mixture was quenched by sat. aq. Na₂S₂O₃and

sat aq. NaHCO₃. The solid was ﬁltered and washed with H₂O dried

in vacuo to give 14 (10.8 g, 99%) as a brown solid; mp 208–212 ^◦C;

¹H NMR (300 MHz, DMSO-d₆) d: 2.68 (2H, t, J = 7.8 Hz), 2.98

(2H, t, J = 7.8 Hz), 3.32 (1H, br s), 7.59 (2H, s), 8.01 (2H, br s);

¹³C NMR (100 MHz, DMSO-d₆) d: 30.9, 40.0, 87.7, 132.7, 139.3,

154.8; IR (KBr): 3349, 3127, 2997, 1589, 1471, 1454, 1300 cm^-1;

HRMS (FAB) calcd for C₈H₁₀I₃NNaO [M+Na]⁺: 539.7794, found

539.7800.

Compounds 47a, 47b. PIFA (74.3 mg, 0.173 mmol) was added

to a solution of 36a, 36b (42.4 mg, 0.144 mmol) in CF₃CH₂OH

(7.2 ml) at rt under N₂. The mixture was stirred at rt for 1.0 h

and evaporated in vacuo. The residue was puriﬁed by SiO₂column

chromatography (CH₂Cl₂/MeOH = 15/1) to give 47a, 47b (less

polar: 9.80 mg, 23%. polar: 20.1 mg, 48%).

Compound 43. Et₃N (64.3 ml, 0.464 mmol) was added to a

solution of 14 (240 mg, 0.464 mmol) in MeOH (5.5 ml) at rt for

10 min under N₂. This solution was added dropwise to a solution

of 32 (138 mg, 0.387 mmol). The mixture was stirred at rt for 16 h

and evaporated in vacuo. The residue was puriﬁed by SiO₂column

chromatography (CH₂Cl₂/MeOH/Et₃N = 100/5/0.1) to give 35

47b (less polar): brown solid; mp 246–249 ^◦C; ¹H NMR

(300 MHz, CDCl₃) d: 1.99 (2H, t, J = 5.7 Hz), 3.65 (2H, t, J =

5.7 Hz), 6.42 (2H, d, J = 9.9 Hz), 6.57 (1H, br s), 6.96 (2H, d, J =

9.9 Hz), 7.64 (1H, dd, J = 7.8, 4.5 Hz), 8.35 (1H, dd, J = 7.8, 1.8 Hz),

8.91 (1H, dd, J = 4.5, 1.8 Hz); ¹³C NMR (125 MHz, DMSO-d₆)

d: 32.8, 37.0, 48.6, 107.0, 126.8, 128.6, 130.6, 133.6, 146.1, 146.7,

152.4, 154.7, 176.9, 179.0, 185.1; IR (KBr): 3242, 2928, 1693,

1659, 1595, 1556, 1514, 1437, 1404 cm^-1; HRMS (FAB) calcd for

C₁₇H₁₃N₂O₃[M+H]⁺: 293.0926, found 293.0932.

1

(76.6 mg, 23%) as a red solid; H NMR (270 MHz, CD₃OD) d:

2.42 (3H, s), 2.88 (2H, t, J = 7.0 Hz), 3.04 (2H, d, J = 7.0 Hz), 3.09

(2H, d, J = 7.0 Hz), 3.62 (2H, t, J = 7.0 Hz), 5.48 (1H, s), 7.41 (2H,

d, J = 8.6 Hz), 7.48 (2H, s), 7.72 (1H, s), 8.05 (2H, d, J = 8.6 Hz);

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47a (polar): brown solid: ¹H NMR (300 MHz, CDCl₃) d: 1.98

(2H, t, J = 5.7 Hz), 3.62 (2H, t, J = 5.7 Hz), 6.39 (2H, d, J = 9.9 Hz),

7.00 (2H, d, J = 9.9 Hz), 7.59 (1H, dd, J = 7.8, 4.8 Hz), 8.35 (1H,

dd, J = 7.8, 1.8 Hz), 8.93 (1H, dd, J = 4.8, 1.8 Hz); ¹³C NMR

(75 MHz, CDCl₃) d: 33.3, 37.3, 39.7, 126.3, 126.6, 127.9, 130.1,

133.9, 137.3, 148.7, 153.2, 155.0, 177.2, 180.2, 186.2; IR (KBr):

3265, 2927, 2860, 2359, 2341, 2248, 2067, 1655, 1618, 1593, 1566,

1517, 1434 cm^-1; HRMS (FAB) calcd for C₁₇H₁₂N₂NaO₃[M+Na]⁺:

315.0746, found 315.0735.

J = 8.0 Hz), 7.56 (1H, dd, J = 7.2, 4.4 Hz), 8.28 (1H, d, J = 7.2 Hz),

8.76 (1H, d, J = 3.6 Hz); ¹³C NMR (100 MHz, CD₃OD) d: 36.8,

57.8, 63.4, 101.4, 116.2, 116.3, 128.0, 129.7, 131.4, 135.8, 150.1,

150.3, 155.3, 157.2, 182.0, 182.6; IR (KBr): 3336, 2924, 2853,

1732, 1685, 1600, 1568, 1514, 1462 cm^-1; HRMS (FAB) calcd for

C₁₈H₁₆N₂NaO₄[M+Na]⁺: 347.1008, found 347.1008.

Compound 48b. 1-(Toluene-4-sulfonyl)-1H-indole-4,7-dione

(23) (228 mg, 0.755 mmol) was added to a solution of tyramine

(10) (114 mg, 0.831 mmol) in MeOH (8.3 ml) at rt under N₂.

The mixture was stirred at rt for 18 h and evaporated in vacuo.

The residue was puriﬁed by SiO₂column chromatography (n-

hexane/AcOEt = 3/1) to give 37a (polar: 116 mg, 32%), 37b (less

polar: 60.7 mg, 17%).

PIFA (65.8 mg, 0.153 mmol) was added to a solution of 37b

(60.7 mg, 0.139 mmol) in CF₃CH₂OH (7.0 ml) at rt under N₂.

The mixture was stirred at rt for 1.0 h and evaporated in vacuo.

The residue was puriﬁed by SiO₂column chromatography (n-

hexane/AcOEt = 3/1) to give 48b (28.3 mg, 47%) as brown solid.

¹H NMR (400 MHz, CDCl₃) d: 1.82 (2H, t, J = 6.0 Hz), 2.42

(3H, s), 3.46–3.50 (2H, m), 6.07 (1H, br s), 6.28 (2H, dd, J = 8.4,

1.6 Hz), 6.62 (1H, d, J = 4.0 Hz), 6.76 (2H, d, J = 8.4, 1.6 Hz), 7.27

(2H, d, J = 8.4 Hz), 7.63 (1H, d, J = 3.2 Hz), 7.95 (2H, dd, J = 8.4,

1.6 Hz); ¹³C NMR (125 MHz, CDCl₃) d: 21.8, 34.0, 37.6, 39.4,

105.8, 106.7, 126.0, 127.8, 127.9, 129.3, 129.6, 132.7, 133.8, 143.8,

145.9, 153.2, 171.4, 177.8, 185.9; IR (KBr): 3372, 2359, 2341,

1658, 1620, 1589, 1541, 1510, 1462 cm^-1; HRMS (FAB) calcd for

C₂₃H₁₉N₂O₅S [M+H]⁺435.1015, found 435.1021.

Compounds 39a, 39b. Et₃N (0.200 ml, 1.47 mmol) was added

to a solution of 11 (340 mg, 1.47 mmol) in MeOH (7.5 ml) at

rt for 10 min under N₂. This solution was added dropwise to

a solution of quinoline-5,8-dione (20) (213 mg, 1.33 mmol) in

MeOH (7.5 ml). The mixture was stirred at rt for 16 h and

evaporated in vacuo. The residue was puriﬁed by SiO₂column

chromatography (CH₂Cl₂/MeOH = 10/1) to give 39a and 39b

(less polar; (140 mg, 27%), polar (223 mg, 43%)).

1

39b (less polar): brown solid; H NMR (300 MHz, CDCl₃) d:

3.10 (1H, dd, J = 13.8, 6.6 Hz), 3.21 (1H, dd, J = 13.8, 5.4 Hz),

3.77 (3H, s), 4.29 (1H, dd, J = 13.8, 5.7 Hz), 5.74 (1H, s), 6.46 (1H,

d, J = 7.8 Hz), 6.82 (2H, d, J = 8.4 Hz), 7.00 (2H, d, J = 8.4 Hz),

7.17 (1H, br s), 7.67 (1H, dd, J = 7.5, 4.5 Hz), 8.43 (1H, dd, J = 7.8,

1.8 Hz), 8.90 (1H, dd, J = 4.5, 1.5 Hz); ¹³C NMR (75 MHz, CDCl₃)

d: 36.6, 52.9, 56.4, 101.5, 116.0, 126.0, 128.6, 130.3, 130.4, 134.6,

146.3, 147.0, 153.0, 155.9, 170.5, 179.3, 181.9; IR (KBr): 3306,

3015, 2926, 2853, 1742, 1693, 1607, 1566, 1514, 1443 cm^-1; HRMS

(FAB) calcd for C₁₉H₁₇N₂O₅[M+H]⁺: 353.1137, found 353.1146.

39a (polar): brown solid; ¹H NMR (500 MHz, CDCl₃) d: 3.07

(1H, dd, J = 14.0, 7.0 Hz), 3.17 (1H, d, J = 14 Hz), 3.76 (3H,

s), 4.28 (1H, dd, J = 14.0, 7.0 Hz), 5.82 (1H, s), 6.35 (1H, d, J =

8.0 Hz), 6.79 (2H, d, J = 8.0 Hz), 6.95 (2H, d, J = 8.0 Hz), 7.57

(1H, dd, J = 7.5, 4.5 Hz), 8.32 (1H, dd, J = 7.5, 1.5 Hz), 8.95 (1H,

dd, J = 4.5, 1.5 Hz); ¹³C NMR (125 MHz, CDCl₃) d: 37.5, 53.6,

57.0, 103.5, 116.8, 126.7, 127.3, 128.0, 130.9, 135.2, 147.1, 149.4,

155.6, 156.6, 171.4, 181.4, 182.3; IR (KBr): 3252, 2953, 1741, 1682,

1607, 1572, 1514, 1443 cm^-1; HRMS (FAB) calcd for C₁₉H₁₇N₂O₅

[M+H]⁺: 353.1137, found 353.1138.

MTT assay. The cytotoxicity was tested against HCT-116

human colon cancer cells, WiDr human colon tumor cells, DU-

145 human prostage tumor cells, P388 and L1210 mouse leukemia

cells using the microculture tetrazolium (MTT) assay. Growing

cells for HCT-116, WiDr and DU-145 were incubated for 24 h

after seeding, and for 72 h in the presence of the test compounds

in DMSO. The growing cells for P388 and L1210 were incubated

in the presence of the test compounds in DMSO for 96 h.

HCC panel assay cell lines. Human breast cancer: HBC-

4, BSY-1, HBC-5, MCF-7, MDA-MB-231. Central nervous

system cancer: U251, SF-268, SF-295, SF-539, SNB-75, SNB-

78. Colon cancer: HCC2998, KM-12, HT-29, HCT-15, HCT-116.

Lung cancer: NCI-H23, NCI-H226, NCI-H522, NCI-H460,

A549, DMS273, DMS114. Melanoma: LOX-IMVI. Ovarian

cancer: OVCAR-3, OVCAR-4, OVCAR-5, OVCAR-8, SK-OV-3.

Renal cancer: RXF-631L, ACHN. Stomach cancer: St-4, MKN1,

MKN7, MKN28, MKN45, MKN74. Prostate cancer: DU-145,

PC-3.

Compounds 40a, 40b. Et₃N (0.120 ml, 0.893 mmol) was added

to a solution of tyrosinol (182 mg, 0.893 mmol) in MeOH (4.5 ml)

at rt for 10 min under N₂. This solution was added dropwise

to a solution of quinoline-5,8-dione (20) (129 mg, 0.812 mmol)

in MeOH (4.5 ml). The mixture was stirred at rt for 3.0 h and

evaporated in vacuo. The residue was puriﬁed by SiO₂column

chromatography (CH₂Cl₂/MeOH = 15/1) to give 40a and 40b

(less polar; (63.7 mg, 22%), polar (116 mg, 40%))

40b (less polar): brown solid; ¹H NMR (500 MHz, CD₃OD) d:

2.80 (1H, dd, J = 14.0, 8.0 Hz), 2.91 (1H, dd, J = 14.0, 6.0 Hz),

3.63–3.75 (3H, m), 5.76 (1H, s), 6.67 (2H, d, J = 8.5 Hz), 7.08 (2H,

d, J = 8.5 Hz), 7.77 (1H, dd, J = 8.0, 5.0 Hz), 8.39 (1H, dd, J =

7.5, 1.5 Hz), 8.81 (1H, dd, J = 5.0, 1.5 Hz); ¹³C NMR (125 MHz,

CD₃OD) d: 36.8, 57.9, 63.5, 100.4, 102.0, 116.3, 126.6, 129.8,

130.0, 131.4, 135.5, 150.6, 153.5, 175.2, 179.6, 183.1; IR (KBr):

3287, 2922, 2853, 1730, 1693, 1605, 1566, 1556, 1514, 1462 cm^-1;

HRMS (FAB) calcd for C₁₈H₁₇N₂O₄[M+H]⁺: 325.1188, found

325.1173.

Acknowledgements

This work was ﬁnancially supported by a Grant-in-Aid for Scien-

tiﬁc Research (A) and (B) and Scientiﬁc Research for Exploratory

Research from the Japan Society for the Promotion of Science

Y. W. We thank the Japan Society for the Promotion of Science

(JSPS) for the Research Fellowship for Young Scientists. We thank

Dr Takao Yamori of the Screening Committee of Anticancer

Drugs supported by a Grant-in-Aid for Scientiﬁc Reserch on

Priority Area “Cancer” from The Ministry of Education, Culture,

Sports, Science and Technology, Japan, for the HCC panel assay.

40a (polar): brown solid; ¹H NMR (400 MHz, CD₃OD) d: 2.70

(1H, dd, J = 13.6, 6.0 Hz), 2.81 (1H, dd, J = 13.6, 6.0 Hz), 3.53–

3.65 (3H, m), 5.74 (1H, s), 6.57 (2H, d, J = 8.0 Hz), 6.97 (2H, d,

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40 Y. Harayama and Y. Kita, Curr. Org. Chem., 2005, 9, 1567.

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4976 | Org. Biomol. Chem., 2011, 9, 4959–4976

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Article Doi

The synthetic and biological studies of discorhabdins and related compounds

DOI: 10.1039/c1ob05058c

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