Structure determination of indolocarbazole alkaloids by NMR spectroscopy

TevahedronVol. 47.No.22,~p.3565-3574. 1991

0040s4020,91 $3.00+.00

Printedin GreatBritain

PergamonLess plc

STRUCTURE DETERWINATION OF INDOLOCARBAZOLEALKALOIDS BY NYR SPECTROSCOPY Shigetoshi

Nicrobiology

Tsubotani*,

Research

Laboratories,

Takeda 2-17-85

Seiichi

Chemical

Jusohonrachi,

Tanida

and

Research

Setsuo

Harada

& Development

Industries

Division,

Ltd.,

Yodogawa-ku,

Osaka

532,

Japan

(Received in USA 3 December 1990)

The structures of two indolocarbazole TAN-1030 A and TAN-999, properties, analysis.

Abstract: activating spectral

TAN-1030

A and

StreptoHyces

SP.

d5,

K-252

are

the

first

compounds having

having

the

same

so

also

Fig.

1.

produced Structures

have

by of

been

A,

such

reported

Strepto#yces

TAN-1030

sp.

TAN-999

by

respectiveas

C-71799. and

SF-

staurosporine.

on.

reported series

propertiesl.

detail

deals

in

the

struc-

with

determination

TAN-1030 (Fig.11

A and using

kOCH3 N

of

NMR

TH CH3

'OH

TAN-999 tech-

TAN-1030

A

niques.

tS

C-71425,

TAN-999

in this

paper

ture

was

and

chrosophore

produced

alkaloids

dassonvillei

racrophage-acti-

vating This

and

A and

indolocarbazole

Nocardiopsis

a,b,c

UCN-016

TAN-1030

new

and

, which

111urosporine2*3

are

C-71793

Compounds

23704,

TAN-999

alkaloids with macrophagewere determined based on NMR

TAN-999

R=OCH3

Staurosporine

R=H

r c TAN-1030

290-295°C C27H22N404

A

was

(dec). on

the

obtained The basis

as

molecular of

colorless formula

secondary

crystals of

ion 3565

TAN-1030 sass

with

a

A was

spectrum

melting

point

determined

[m/z

467;

to

of be

(M+H>'I,

3566

S. TSUROTANIet al.

l

Table

‘H

1.

NfdR spectral

data

TAN-99gb

Position 1 i 4 6 7

Fi

:*

,

3&H 4’-OCH; IO-OCH3 3’=NOH a: C:

e:

7.27 7.46 7.35 9.40 6.28e 4.96 7.75 6.96

(d,J=8.0) (t> (t> (d,J=7.8) (s) (ABq,J=l7.0) (d,J=8.5) (dd,J=2.0,8.5)

7.46 6.53 2.38 2.75 3.35 3.87

(d,J=2.0) (d,J=5.6) (II> (dd,J=4.0,14.7) (II> (d,J=3.5)

2.33 1.57 3.44 3.95

(s) (s) (s) (s)

Coupling constants in Hz d: These signals DfdSO-d6. This signal shifted according 2.

data

TAN-999*

TAN-1030Ab

106.89 124.88 119.65 126.50 123.55 114.88 118.58 173.81 45.92 131.48 114.24 118.92 120.94 107.94 157.49

4” :;: 4c ! 7a 7b 7c 8 9 10 a:

In

elemental

CDC13.

b:

d d d d s s s s t s s s d d s ln

analysis H,5.07;N,11.70)

Presence

of

sorption

NH and

maxima

in

of

108.88 125.21 119.52 125.61 122.85 114.98 119.15 171.77 45.30 132.25 114.02 123.79 120.72 120.13 124.64

StaurosporineC

;.;;

((ij~=8.2)

7:32d ;.;;

(t> ;ijJ=7.8)

7.56 (d,J=8.1) 7.45 (t> 7.27d (t> 9.30 (d,J=7.9) 8.53 (s) 4.96 (s like) 7.96d(d,J=7.3) z: 7:98 6.68 2.49

::; (d,J=8.4) (s like) (II)

3.24 4.04

4.73

(5)

2.47

(s)

3.43

(s)

10.45

(s)

TAN-999

cm> (d,J=3.2) (s) (s) (s)

2.29 1.44 3.32

and

TAN-1030

Position

b:

A (75

100.72 141.04 130.63 127.09 136.67 80.12 30.29 50.36 84.26 91.06 29.89 33.38 57.24 55.78

,

d* 3’ 4’ 5’ 6’ 3’-NCH3 4’-0CH3 IO-0CH3

In

CDC13.

Mz).

TAN-99ga

11 lla 12a 12b 13a

d d d d s s s s t s s s d d d

MHz)*.

TAN-1030Ab d s s s s d t d d s q q q q

115.58 139.82 128.03 124.60 136.01 82.17 29.71 145.12 83.57 98.16 28.59

d s s s s d t s d s q

58.29

q

DRSO-d6. (Calcd

C.67.23;

(300

are given in parentheses. overlapped. to the concentration.

13C NMR spectral

Position

TAN-1030A

4196 (s like) 7.98 (d,J=8.0) 7.32d(t> 7.44 (t> 8.01 (d, J=9.1> 7.04 (d,J=5.2) 3.01 (dd,J=5.2,14.0) 3.63 (d,J=l4.0)

In

Table

and

TAN-999

TAN-1030AC

10

11 1’ 2’

of

for and

mono-hydrate:

OH (3430cme1) methanol

C,66.93;H,4.99;N,ll.56, The

13C NMR spectrum. and were

amide observed

(1680c11-~) at

Found:

1R spectrum

233nm

showed

functions. (E

29,400).

the

UV ab244

3567

Indolocarbazolealkaloids

263

(sh,28,000), (sh,13,400). 1030

333

A has

(17,700).

a chrorophore TAN-1030

porine2. lich

(sh.31.300).

reagents

A

and

275

352

(12,100)

sisilar

to

showed

color

and

the

positive

negative

(sh,42,000), 369

289

(13,400)

showing

indolocarbazole

color

roiety

reactions

reactions

with

(71,000).

with

that in

and

TAN-

stauros-

Barton

ninhydrin

319

and

Ehr-

Dragendorff

reagents.

cate

ill and

13C

the

presence

nineteen

are

one

quaternary

(H-6.8.58

H-3,7.32

and

H-11,8.01)

signsent

of

Fig.

only and

other

four

moieties

1=60msec

spectra

which

were

spectroscopy of TAN-i030

A.

and

off

by

via

long

range

methines, contains

two sany

revealed:

la,

H-2,7.50;

H-9,7.32;

H-10,7.44 The

3.63).

by

cut

the

(H-1,7.70;

H-2',3.01, achieved

indiof

'Ii-ltl correlation

(H-8,7.98;

was

A

were

[la,-CH=CH-CH=CH-

data

(eleven

two

from

structures

(H-1',7.04

The

TAN-1030

Therefore

signals

2.

carbons

carbon,

group.

IIla,-CH=CH-CH=CH-

carbon

1 and sp2

quaternary

partial

H-7,4.96);

IVa,-yHCH2-

a correlation 2. INEPT

one

nethoxy

one

Tables

nineteen

heteroatoas.

proton-bonded

with

heteroatoms,

and

in

group,

carbons),

H-4,9.31); and

suaaarized

aaide

and

(COSY>

-CONHCH2-

are

one

methyl

carbons

spectroscopy

connect

data of

quaternary

sethylenes,

and

NHR

'H-13C

quaternary couplings

as-

COSY.

To

carbons

or

7

tcoLoc)

3568

S. TSUROTANI er al.

.-.

-

-

__ -_

Yn-

--

I

_

I -_

_

z

Indolocarbazole

was

For

used.

pling

is

cussed

a

(20,

when

40

and

ticulary giving

the

coupling

most

These

with data

carbons

a

stituted

the

aethylene

benzene

139.82).

and

-CONHCHy

benzene with

A qethoxv

carbon

structures

two

a

amide

were

quaternary

of TAN-1030

coupled

with

long

a

and (H-4’,4.73)

-yHCHz-

IIIa IVa

Ib

IIb

IIIb

OH IVb

oy3 ‘; N ,’ _,;, \oH’;:...

-

‘H ,13C

d..--L N‘,E

long range

which

1,2-disub-

(C-7c,123.79

-CH=CH-CH=CH-

la.

quaternary IIa,

formed

a qethine

as in

Two

A.

IIa

I

range

C-7a,132.25)

with

carbons

pardelay

(H-6,8.58)

(lb).

also

I

structures and

correlated

best

(Fig.3).

proton

IIIa

the

C-H

partial

f-lactam

(Ilb).

(S58.29)

-CH=CH-CH=CH-

Ia

an

to

is delay

intensity as

the

the

refo-

experi-

the

best

cou-

a

signals

2Oasec

(C-4c,119.15

C-13a.136.01)

(Illb)

4. Partial

and la

the

COLOC experiment

carbons

structure

1,2-disubstisuted

gave

expanded

range via

We varied

Therefore,

the

(H-7,4.98)

the

carbon

We chose

COLOC experiment quaternary

of

that

long transfer)

1/4JC6.

signals.

Hz in

C-H performed

polarization

to

spectrum

the

was

enhancerent

signals.

12.5

of

by

equal

carbon to

the

(C-4a,122.85 a

the set

carbon

Two

extended

indicated

Fig.

find

equal of

(Fig.4).

coupled

is

quaternary

set

result

follows

to

quaternary

was

The

I

choice

optimization

enhanced

experiwent

delay

8Owsec)

the

This nuclei

this

the

for

isent,

point.

In

(Fig.2).

observed

COLOC exper

(’ in t ensive

INEPT*

sent

lla,

a

difficult

3569

alkaloids

coupling

C-

3570

S. TSUROTANIet

and

the

methine

(13.43).

carbon

This

observed

indicates

between

exchangeable carbon

was

also

and

a

data

combined carbon of

these

data of

1Vb

the

indicate

connected

correlated

extended

iVa

rethine

(C-l',

to

these

a

the

with

the

six-membered

82.17) carbons

and

IVa

group.

oxine

and

the

Furthermore

group

(H-6', (H-l')

methyl

(IVb).

are

bound

to

heteroatoms

(J=12.5Hz)

ten

of

the

of

IVa. were

quaternary

of

carbon

a

2.471,

group a

(H-l')

moiety

quaternary

The

that

the

was

oxide

qethoxynethine

the

addition,

methine ring

the

coupling

deuteriun

aethine

and In

(C-5').

protons

moiety.

of

Methyl

nethoxymethine

carbon

a

oxime

oxime

a

and

range

indicates

the

with

methoxy

and

(H-4') This

through

the

Long

an

methine IVa.

rethoxynethine

IVa,

was

revealed

coupled

quaternary

that

of

the

(C-3',145.12)

the

96.16)

the

that

the

(C-lZb,124.60)

shifts in

of

showed

with

are

(C-5',

(H-4')

via

which

with

moiety.

carbon

(H-1',7.04)

moiety

qethine

These

correlated

carbon

coupled

methoxymethine

(610.451,

nethine

lethoxymethine quaternary

a

quaternary

proton

moiety

the

a

83.57)

(C-4',

al.

IVa.

All

Downfield

(C-5',96.16) (Xl,

X2

and

x3>. From carbons

the were

Fig.

5.

COLOC

experinent

connected. COLOC spectrum

Two of

of

TAN-1030

remaining A

three

thirteen

quaternary

quaternary

carbons

(J=4.2Hz).

-------(I

I

1

5.n

6.ll

?.fl

8.0

9. D

~

3571

Indolocarbazolealkaloids

(6114.98,

114.02

went

the

when

COLOC

and

delay

experiwent

expected,

in

8,7.98),

Downfield (C-lla,139.82)

set

COLOC

observed

equal range

two

arosatic Therefore

shifts

of

show

two

set

quaternary

IIb

Illb

and

Ilb

4.2Hz

Ilc

bound

the

(C-4b,114.98

and

and

IIIb

(H-

TIC

and

to

(C-13a,136.01) to

next As

extended

in

are

in

experi-

(Fig.5).

(H-4,9.31)

were

carbons

carbons

INEPT

Thus

at

carbons

in

these

refocussed

(Fiw.2).

was

protons

quaternary

that

the

BOwsec

coupling

spectrum

with

at

in

and

heteroatows

(X4

X5). To

connect

Overhauser

the

effect

aethylene

8.7.98) the

were

long

respectively.

111~

the

C-H

coupled

IIIC.

and

I was

the this

C-6b,114.02)

128.03)

of

111~

the

tively.

when

the

Thus

oxime at

the

showed

X2

proton

is

difference

(6

was

in

111~

that to

10.45)

(H-6,8.58)

spectra

X4

and of

Illc

IIlc, and

X3

the

TAN-1030

the

IVb),

Ilc

is

equal NOE

to

were X5.

en-

respec-

connected

in

Furthermore,

enhancement

(H-6',2.47)

(H-

In addition

(H-1'.7.04)

(H-1,7.70),

TIC

When

proton

enhanced.

and

nuclear

(Fig.6).

aromatic

methine

and

methyl A.

the

IVb

irradiated,

and

followed

were

and

(H-11,8.01)

lb,

was

3.43)

Ilc,

irradiated,

(H-6',2.47)

equal

(6

nethoxy

F13. 6. Now

(Ib,

experiwents

proton

methyl

protons

data

Ib

amide

the

aromatic

order.

of

the

of

These

this

served

and

structures

differential

(H-7,4.96)

irradiation

hanced

partial (NOE)

of

was IVb.

obThis

3572

S. TSUROTANI er al.

indicates

the

structure

V (Fig.4).

constitute are

marked

mined

to

as

*

as

in

lows:

was

368

similar

ltl and of

the

benzene benzene (63.95)

between staurosporine.

TAN-999

was

moiety was and

data

carbon

other

the

pale

five

lead

of

to

partial

(C-12a,128.03)

quaternary

structure

UV

are of

Yellow

+42”

(KBr)

similar

moiety

these

must

carbons

TAN-1030

which

A was

deter-

TAN-999 as

(c

0.50,

3430~~~1

298

(80,300),

spectrum

in

summarized to

of

(H-8,7.75,

the

Tables

TAN-999 1 and

has

2.

(amide),

UV

(sh,16,200) a chroaophore

replaced and

by

H-11,7.46) molecular

confirmed

the

methoxv

(J=4.2Hz).

of

the

The

except

in

I

cm-’ 352

difference

position

Fig. 7. COLOC spectrum of TAN-999

fol-

molecular

1880

(19,500),

that

was

staurosporine

as

staurosporine.

H-9,6.98 The

and

staurosporine,

staurosporine

properties

dinethylforaanide), 341

in

that

with

(NH)

indicates

moiety

observed.

To determine

crystals

rmax

indolocarbazole

13C NMR data

TAN-999

stituted

The

the

Therefore,

(~31,200).

(11,000). to

signal

IR

245nm

with

Calm

(dec),

of

quaternary

Fig.1. of

C2gH2gN404,

(MeOH)

tuted

in

obtained

mp 221°C

trum

V.

shown

All

relaining

ring

determination

formula

and

The

aromatic

TAN-999

lmax

stereochemistry.

an

be

Structure

oxime

methoxy

a

IH NMR spec-

the

l,&-disub-

1,2,4-trisubstiand

a

formula

methoxy (OCH2)

substitution substituent,

of ‘H-lH

3573

Indolocnrbazolealkaloids

lIi-13C

COSY,

experiments TAN-1030

were A.

Fig.7),

performed

In

the

the

with

a NOESY

of

NOE

protons in

the

NOESY

aore,

an

NOE

was

proton

(H-11) data

The

NOESY

that

of

detersined

and

methyl

spectrum

to

that also

(66.53) be

as

and shown

of Hz,

l

in

(63.95)

the

G$D

the

of

.

CIH CH3

aromatic

'xa3c *-..-wucs

(H-6'.2.33).

qethoxy

showed

the

H-l

(67.27).

group

correlation Thus,

OCH3

V-

and

Further-

the

--._ \ /+$ N W._._.-cti;

.-__V0

the

(H-9

(Fig.9).

protons

case

between

protons

between

-_-. *. ** ,

was

experiments,

found

spectrum

indicate

H-l'

was

Fig. 8. COLOC and NOESY data of TAN-999.

Fig.8).

aromatic

observed

case

(5~4.2

protons

(H-8.

applied

peak and

the

COLOC

(C-10.157.49)

gethoxy

was

H-11)

in

and

spectrum

differential

A cross

nethoxy

as

COLOC

proton

experiment

TAN-999.

INEPT

carbon

the

aromatic

Instead

These

refocussed

a quaternary

correlated and

COSY,

is

bound of

the

H-7

to

LOng range coupling

the

IO-position.

(64.96)

structure

and

of

TAN-999

in Fig.1.

Fig. 9. NOESY spectrum of TAN-999.

0

0

L

0

0

_

1.0

-

5.0

-

6.0

_

7.0

0

r

H-8

6.0

and was

3574

S. TSUB~TANI et al.

Experimental For pared.

the

NMR experiments,

All

NMR experiments

programs ments

on

data

256

The was

of

12.5Hz

and

the

NOE

the

NOEDIFF

200;

total in

differential program,

at

points

130

Hz.

of

experiments

case

were

the

For the

run

sample

standard

the

INEPT

16hr

2Omin The

performed

using

experiments were

2.

Oaura,S.;lwai,Y.;Hirano,A.;Nakagawa,A.;Awaya,J.;Tsuchiya,H.;Takahashi,

follows:

the

case

of

in

NOESY program

.;Takizawa,M.;Takahashi,T.;Tsubotani,S.;Harada,S.J.Antibiotics

1989,42,1619-1630. Y.;Masuna,R.J.Anti~iotics 3.

1977,30,275-282.

Furusaki,A.;Hashiba,N.;Matsumoto,T.;Hirano,A.:twai,Y.;Omura,S.J.Che~. Sot.

Chew. CoBNun. 1978,800-801.

4.

Sezaki,M.;Sasaki,T.;Nakagawa,T.;Takeda.U.;Iwata,M.;Watanabe,T.;Koyaaa,

5.

Yasuzawa,T.;lida.T.;Yoshida,M.;Hirauama,N.;Takahashi,M.;Shirahata,K.;

M.;Kai.F.;Shomura,T

Sano,H.

J.4ntibiotic.s

.;Kojima,M.J.Anti~iotics

1986,

1985,38,1437-1439.

39,1072-1078.

6.

Takahashi.l.;Kobayashi,E.;Asano,K.;Yoshida.M.;Nakano,H.j.Antibiotics

7.

Kess1er.H

1987.

40.1782-1784. .;Griesinger,C.;Zarbock,J

.;Loosli,H.R.J.Magn.Reson.1984,57,

331-336. 8.

Morris,G.A

. ;Freeman,R.

J.As.Ches.Soc.

1979,

101,760-762.

using

as

References Tanida,S

were run

NOESY experiments the

X 4K

were

respectively.

1.

experi128

XHCORR program

COLOC experiments

COLOC experiments

prenicro-

homonuclear

with

J=4.2Hz.

was

Bruker

heteronuclear

refocussed

tine, of

for

were

The

measuring the

the

24OC.

and

The

program.

conditions

12min

scans, 17hr

to The

COLOC program.

number

spectroneter

INEPTRD

of

using

COSY experiments

equal the

solution

performed

X 1K data

lH-13C

set

using

were

AC-300

were

points.

and lJCH performed the

a Bruker

matrixes

O.lM

~PPPOX.

J= and and