A novel cyclization pathway in activation of neocarzinostatin chromophore by thiol under physiological conditions

TetrahedronLetters,Vol 33,No 4, pp 515518,1992 Printed1~1 GreatBrim

A NOVEL

CYCLIZATION

PATHWAY

CHROMOPHORE Hlroshl Department

tFaculty

IN ACTIVATION

OF NEOCARZINOSTATIN

BY THIOL UNDER PHYSIOLOGICAL

Suglyama,

Synthetrc

Of

00404039/92$3 00 + M) PergamonPressplc

Katsuhlro

Chemistry,

Yamashlta,

Masatoshl Nlshlt, and lsao Salto* of Engmeermg, Kyoto Umverslty, Kyoto 6U6, Japan

Faculty

of Pharmaceutxal

CONDITIONS

Sciences,

Setsunan

Unlverslty,

Osaka

573-01,

Japan

Abstract Reactron of neocarzlnostatln chromophore (1) wrth 2-mercaptoethanol buffered solutron resulted zn an efflclent formation of unprecedented cyclrratlon Zwrtterlontc rntermedrate 6 as the precursor for 5 was proposed

Neocarzmostatln and Its

earner

rcsponslble of the

(NCS)

protem

for

intermediary NMR,2b aqueous

2

the exact

the

attack

by

conducted,

has

cychzatlon

of

product

of

nature

solution still

of thlol-1 at

reaction

reaction

with

and

be

under

clarified

shown epoxlde

to form

the

We

now

2-mercaptoethanol

a dlradlcal 1 (path

opcnlng

mdacene

been

condltlons, cleavage to

In aqueous

generate

an

While the

3

by

lH

e g , m an aerated

report

an

are

usually

unprecedented

cychzatlon

buffered

species

elucldatlon

supported

experiments

of a novel

1

A) has been

would

dlradlcal

has

wish

chromophore

on the structural

m Scheme

solvent

DNA

to the formation

generate

ring

physlologlcal where

3

to

nonprotein

Based

in organic

temperature,

of

1~ belteved sugar tcyd

cychzes

1 leading

conslstlng

mechamsm

at Cl2

temperature

ambient to

thlol DNA

actlvatlon thlol

at low

chromophore

in the

the

spontaneously 2

remamed

NCS

from

adduct,

antlbtotlc

of 1 by

Actlvatlon

which

formation

buffered

antItumor

abstractlon

nucleophlllc

enyne[3]cumulene

major

l

hydrogen

chromophore-thlol

proposed,2

IS an

rn aqueous product 5

product

solution

(path

5 as a B)

Scheme 1

A solution

of NC@

incubated

at 0 ‘C

such

d(GCATOC)2

as

under

(4 mM) m 50 mM tns-HCI

(0 25 mM) and 2-mercaptoethanol aerobic were

condltlons

degraded

to

6

Under

produce 515

the

known

mcubation producte

as

condltlons already

(pH 7 0) was

DNA

substrates

reported 8

HPLC

516

analysis

of

product

dt

the

reaction

11 0 mm

as 5 on the bases product The

of II3

1H

NMR

The

data

signals

Scheme

UV

1 and

mdlcated

‘H-lH

together

as mdlcated

12

COSY

lncubntlon was

and

revealed

Isolated

by The

FTIR

Z-mercaptoethanol

the

presence

experiment

of

COSY

a pair

FABMS an

of

a couplmg

experiment

allow

the

formation

and

Its

(M+l,

756)

pathway

complete

one was

lndlcated

of

protons

lmkmg

assignment

signals

C8-C5’

of

this Hz0

(6 7 23

the

Clo-Cgh,

maJO

asslgned that

mcorporatlon

aromdtlc

C30H-C14.

of

structure

addItiona

doublet

m C3OH-Cl2,

all

and (C7-

and C5-

of the

lH

1

2

+

Scheme

HPLC,

wtth

revealed

were observed

with ‘3C-lH m Table

h

product

The NOE crosspeaks

These 13C

after This

FABMS,

between

(DMSO-d6)

Ca-ClO-Cll-Cl2) Cl3

1)

NMR,

IS a 1 1 adduct

6 99, J = 8 2 Hz)

and

mixture

(Flgurc

3

4a

0

.

0x0

(A)

5

-_A._-_

J\

a.---..+

i.-__

Flgurc 1 HPLC proflles of (A) NCS, (B) the reactlon mixture of NCS and 2-mercaptocthanol under and (C) the same reactlon as aerobic condltlons m trls-HCl buffer (pH 7 0) after 12 h Incubation, The reaction mixture was analyzed on (8) III a buffered solution contalnlng 80% lsopropanol Wnkos11 5Cls column (4 6 x 1.50 mm), elutlon was with 0 OS M ammonmm acetate (pH 4 0), 30 50 ‘%J ,~c~on~tr~lc, llncnr gradlent. 20 mm, at a flow rate of 1 5 mL/mm

517

Table 1

‘I-I (400 MHz) and 13C (100 MHz) NMR Data of 5 m DMSO-d6

Aqslgnment

13C (ppm) 115 5 136 4 139 7 112 8 882, 894’ 155 0 118 8 122 2, 1215* 139 0 82 3 84 5 50 6 16 1, 76 7* 68 7

1 2 3 4 5 6 I 8 9 10 11 12 13 14

‘H(ppm)

5 99, 6 00* (lH, s) 6 99 (d, 1 H, J = 8 2 Hz) 7 23 (d, 1 H, J = 8 2 Hz) 5 05, 5 03* 572, .571* 481. 473’ 5 56, 5 58* 4 53 (dd, 1 4 73 (dd 1

154 5, 154 3* 349, 35 1 61 0

:5

16 17 3-OH

96 ? 58 2 69 0 71 0 66 7 16 8 34 0 107 5 161 0 115 3 129 3 138 6 116 5 160 1 100 7 121 5 136 0 172 9 19 4 7”-OCH3 54 8 * Peaks due to the dlastereomenc

3 06 3 70, 7 22, 5 42 275 338 3 48 390 1 17 2 55

2 3’ 4’ 5’ 6’ 2’-NCH3

As shown m Flgure and are

m the

reactlon

product

together

with

a mmor

such

as aLId

orgamc system

solvent would

chromophore mercaptoethanol solution

hberate 1

6 98 (bs, 1 H)

252 (s, 3 H) 370, 371* (F, 3H) mixture

of

peak

at 8 8 mln was ascribed

to that of 4a as Judged by 1~

of the reactlon

mchcated

from

1 with

mixture

The ratlo of 5 YS 4a In a solvent amount

NCS to

4a

Z-merLaptoethano1

dependent 4a

80% lsopropanol

that

and, as a result,

complex

provide

that the yields

Smce 1 IS extractable

It IS possible

methanol,

IS highly

Lontammg

of 5 (5 1%)

1 from NCS complex

Isolated

mcubatlon

at Cl2

analysis

system

In lsopropanol

(m, 1 H), 2 89 (m, 1 H) 3 71, (t, 2 H, J = 7 0 Hz) 7 26* (<, 1 H) (bs, 1 H) (d, 1 H, .I = 110 Pz) (d, 1 H, J = 110 Hz) (bs, 1 H) (qd, 1 P, J = 64, 64 Hz) (d, 3 H, J = 6 4 Hz) (s, 3 H)

6 82 (bs, 1 H)

59% and 9 5%, respectively

content

H, J = 15 0, 8 3 Hz)

701 (d, 1 H, J = 92 Hz) 791 (d, 1 H, J = 92 Hz)

1, a small

Quantitative

FABMS 9

(bs. 1 H) (bs, 1 H) (bs, 1 H) (dd, 1 H, J = 8 3, 8 0 Hz) H, J = 15 0, 8 0 Hz)

bv

reported

addltlon chdnge

gave

neither

from

9 nor

the

4d

In fact reaLted

However,

to result

with

to the reactIon

ratlo

actually

product

lsopropanol was a major

NCS complex

of lsopropanol the product

procedurc2c

a5 a sole detectable

on

(45%)

NMR

of 5 and 4 a

NCS

with

2-

m aqueous

in d formation

of

518

a complex when

mixture

complexed

of multlple

with

Its apoprotem

The fact that 5

1s also

may not be mcorporated mcorporated was

mto

Incorporated

from

In

nuclcophlllc demonstrated of

NCS

for DNA

precursor

the for the

with

suggest

(Scheme

condltlons

758)

to

time

arc underway

3)

An

I2

there

5 19 only

mdlcates

are two

Further

studies

produced

When D20 These

analogous 7

would

dlgtlnct

that

out m H2l80, was used results

from

1

polar also

addltlon

molecule 180

as solvent,

deutermm

of

solvent

5 ”

has the

Present

pathways

the real active

was

that 5 1~ formed

Alternatively,

produce

arotnatlzatlon

to confmn

oxygen

the labelled

suggest

1,2,4-heptatrlen-6-yne In

of

enyne[3]cumulene

that

that

solution

anaerobic

of 5 to the extent of 80%

6

water

first

under

by FABMS (M+l,

2-mercaptoethanol

cleavage

m an aqueous

cycloaromat~zation of

results

When the reactlon was camed

into 5

at C7 posltlon

attack

These

produced

5 as revealed

zwltterlonlc

reported

products

direct results

m the

species

been

reaction

responsible

13

References and Notes (a) Ishlda, N , Mlyazakl, K , Kumagal, K , Rlklmaru, M J Antrbx~trcs, 1965, 18, 68 (b) Edo, 1) K , MtLugakl, M , Kolde, Y , Seto, H , Fuphara, K , Otake, N , Ishlda, N Tetrahedron Left 1985, 26, 331 (c) Goldberg, I H Free Radrcal Btol Med 1987, 3, 41 (d) Goldberg, I H Accountc Ckem Res 1991, 24, 191 and rcdcrences therem (A) Myers, A G Tetrahedron Lett 1987,2R, 4493 (b) Myers. A G, Proteau, P J J Am Ckem SOL 1989,111, 1146 (c) Myers, A G , P .I Proteau, P J , Handel, T M / Am Ckem Sot 1988, 110, 7212, and refercces therem Aerobic mcubatlon of 1 In methanoll~ &etlc acid was reported to provide an oxygenated 3) cycllzatlon product 4 Tanaka, T , FuJiward, K , Hirama, M Tetrahedron Lett 1990,31, 5947 4) NCS was obtamed from Pola Kasel Co R & D laboratory Concentration of NCS was 5) determined spectrophotometrlcally (~340 10,800 mM-l cm-l), see, Povlrk, L F ) Dattagupta, B C , Warf, B C, Goldberg, I H B!ockemzsrry, 1981, 20, 4007 Although NCS and 1 showed exactly the same sequence speclficlty of DNA c1eavage,7 the 6) efficiency of the cleavage for 1 was less than 10 % of that for NCS Takeshlta, M , Kappen, L S , Grollman, A P , Elsenberg, M , Goldberg, I H Bzockemrstry, 7) 1981,20, 7599 (a) Kawabata, H , Suglyama, H , Tashlro T , Takeshlta, H , Matsuura, T , Salto, I , Ito, A , 8) Kolde, Y Nuclezc Aczds Res Sym Serzes, 1988, 20, 69 (b) Kawabata, H , Takeshlta, H , FuJiwara, T , Suglyama, H , Matsuura, T, Salto, I Tetrnhedron Letters, 1989,30, 4263 (c) Saito, I, Kawabata, H , Fujlwara, T Suglyama, H , Matsuura, T J Am Chem Sot , 1989, 111, 8302 (d) Sugiyama, H , Kawabatd, H , Fupwara, T , Dannoue, Y , Matsuura, T J Am Ckem

91 4’)

10) 11) 12) 13)

Sot

1990,112,

5253

4a IH NMR (DMSO-d6) S 1 18 (d, 3 H, J = 6 5 HZ, 6”), 2 51 (s, 3 H, Z’NCH$, 2 54 (s, 3 H, S’CH3). 2 76 (rn> 2 H, 16, 2’), 2 87 (dt, 1 H, J = 12 8, 4 5 Hz, 16), 3 50 (d, 1 H, J = 3 3 Hz, 3’), 3 53 (bs, 1 H, 3 63 (m, 2 H, 17), 3 72 (6, 3 H, 7”OCH3), 4 00 (q, 1 H, J = 6 5 Hz. S), 4 37 (dd, 1 H, J = 5 5, 2 9 Hz, 14),448(m,1H,12),452(m,1H,13),443(dd,1H,J=55,29Hz,14),519(d,1H,J=28H~, lo), 5 48 (d. 1 H, J = 3 7 Hz, l’), 5 76
(Received in Japan 1 October 1991)