O'-Methyl derivatives of arabinosylcytosine

Vol. 46, No. 4, 1972

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

O’dWl!HXL DEBIVATIVES OF ARABINOSYUXl’CSINE E. Darzynkiewicz,

J. T. Ku&eierek and D. shugar

Department of Organic Chemistry, University

of Wsrsaw, Werszawa 22;

Institute of Biochemistry snd Biophysics, Acadof Sciences, Warszawa 12; and Department of Biophysics, University of Warsaw, Wars5aaa 22, Poland.

Received January 31, 1972 %mnsgy,r Dimethylsulphat e treatment of 1-p -Dsrablnofursnosylcytosine (ara-C) in aqueous alkaline medium, followed by colum chrcmatography, has led to isolation of all possible O’lmethylated derivatives of ararCo The three mono-methylated derivatives were obtained in crystalline form and identified

on the basis of NMRspectra and other criteria.

The 3*,5’-&L-O-

-methyl derivative was distinguished from the 2’,5’and 2*,3*on the basis of its susceptibility to acidrcatalyzed desmination. The potential antimetabolic properties

of these new ari+C snalogues are discussed.

Several procedures have now been described for the methylation of the 21 and 3’0 cis hgdroryls

In rlbonucleosides,

treatment in aqueous or methanolic tiethoxyethane. a method, based on the relative

resistance

to alkylation

aqueous medium of the ring N of l-substituted 3

feasible to obtain all auy cytosine nucleoside. alkaline readily

possible 0’0alkyl This involves

in alkaline

cytosines,

which makes it

(methyl or ethyl) dialkylsulphate

medium; the corresponding O'-alkyl aocessible by stiple

based on diazaeethene l-6 We have developed

uracil

derivatives

treatment in aqueous

nucleosides me then

deamfnation. 78

The present cosxzunication describes the application forego&g

to the preparation

10~0~arabinofPrePosylc therapeutio

properties

The utiUty

of

of

of all

of the

the possible O’-metbyl derivatives

ytosine(A, ara-C) . The autlmetabolic of ara=C are too well known to requ5re

of

and camnent.

9,lO

preparing B1%c walogues in which the sugar hgdroxyls are 1734

Copyrigbt@1972,by AcademicPress,Inc.

Vol. 46, No. 4, 1972

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

has been elsewhere discussed,

alkylated

11

and is further

emphasized by the

recently

reported superior antileukemic ma immunosuppressive activities 12 some 5'-acyl esters of era-C.

of

OR, 1 R,=Rz=R3=H 2 R,=CH,, R,=R,=H 3 R,=CH,, R,=R,=H 4 R&H,, R,=R,=H 5 R,=R,=CH,,R,=H f( R,=R,=CH,,R,=H l R,=R,=CH,,R,=H & R,=R,zR,=CH, Chromatoma~

was carried out with solvent system A

(watezcsaturat ea @utsnol) B (chloroform-methanol, silica

on Whatmsnpaper No. 1; and with solvent system

4:1, v/v)

by thin-layer

chromatography on HF254

gel.

Methvlation (a,

3 R,=H,R,=CH3 10 R,=R,=CH,

Drocedure:

1.12 gm (4 mM) of the HCl salt of ara-C

prepesed according to Ksnai -et -0) al "

To the vigorously

&tired

was dissolved in 200 ml 1 N KOH.

solution was added, dropwise, over a period of

3 hours, 6 ml dimetbyl sulphate, The reaction mixture was left hours at room temperature, following

several

which chromatography with solvent A

revealed four spots with Rf values (and relative uv lamp) of 0.15 (- 4%)) 0.30 (- 4%),

intensities

under a dark

0.50 (- 2%)) ma 0.60 (traces),

1735

Vol. 46, No. 4, 1972

tentatively

BIOCHEMICAL

identified

-methyl-ara-C

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

as unreacted ara-C, mono-O*-methyl-era-C,

ana tri-O~-methyl-ara-C.

The reactim

&i-O@-

mixture was neutralized

with 10 N H2S04 end concentrated under vacuum to about 20 ml. To this was added 150 ml methanol and the resulting

inorganic

washed with methanol, The combined filtrates

salt filtered

off and

were brought to dryness and

the residue dissolved in 500 ml water and further

desalted, The solution

was then deposited on a 30 x 2-2 cm column of Dowex 50 Xx2 200/400 (Ii+) and the columu washed with water until

the effluent

was neutral.

were then eluted with 1 N KOH, and the 300 ml fraction bing material

containing W-absor

in turn passed through a 25 x 4.5 cm column of Dowex 50 HxS

20/50 (NH+4), which was eluted with water until W absorption.

The nucleosides

the eluate exhibited

no

The eluate was brought to dryness, dissolved in 10 ml water

(26,000 ODunits at 270 run) and deposited on a 55 x 6 cm column of Dowex lx2 200/400 (OH-), previously

equilibrated

described by Dekker. 4,14 Elution

with 33% aqueous methanol, as

with aqueous methanol gave the pattern

shown in Fig. 1. Isolation

aa Identification

Chrmtography

of Methylaticn

and W spectroscopy:

A of all the numbered peaks from the elution possible to group them tentatively EIono-O'-methyl-era-C!: Di-O*-methyl-ara-C; Tri-O'-methyl-ara-Cr

ically

profile

in Fig. lmade it

VI (Rf 0.27),

(Rf O&3), III

(Rf 0.46).

VIII

(Rf 0.29).

IV

(Rf 0.50).

I
Uniaent ifie at

VII

Dnreacted ara-C:

Rf 0.15.

All the peaks I-VIII,

Chromatography with solvent

as follows:

V (Rf 0.39). II

Products

including

(Rf Oe12)

the unidentified

VII,

homogeneousin solventa A and B; while their

values showed that the cytosine rings in all on N3, In the case of VII

were chromatographspectra at various pH

of them were not methylated

there was somesuspicion that the exocyclic

group may have been monomethylated (cf.

ref.7*8);

1736

this is being further

amino

270

T

.A30%

I

OD.9.4

CH,OH-+-----

50% CH,OH

.‘i

300

85% CH,OH-lM+

320

in each peak (total

material

deposited on column, 26.0~0

ODno -w*

sulphate in alkaU.ne medium, as described in text. F'ra&i.ons of 100 ml collected at 10 min intervals. Remannumerals beside each peak correspond to those used in text. Arabic numerals present the number of OD270units

pig. 1. Elution pattern, on a 55 x 6 cm0 column of Dowex lx2 (OH-), 200/400 mesh, of products of methylatim of ara-C with dimethyl-

CH,OH-40%

Fraction

340

ara C

P 000

360

Number

1 I

NaOH

Vol. 46, No. 4, 1972

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

studied and is omitted from additional Crystallization

consideration

below.

of mono-O'-methyl derivatives

of sra-Cr

The

eluates corresponding to peaks V, VI and VIII

were each brought to dryness,

The residue from peak V was then crystallized

from anhydrous ethanol as the

HCl salt to yield

37 mg of colorless needles, m.p. lg6-lgg".

from peak VI was crystallized

directly

from anhydrous ethanol, giving

in the form of needles, m.p. 19C-193°. crystallized

The residue 14 mg

The residue from peak VIII,

yielded 85 mg needles, m.p, 260-263'.

as for VI,

Elementary analyses on al

three samples agreed with the presence

in each of one methyl group0 Deamination in acetate buffer: that ara-C readily

undergoes hydrolytic

conditions where cytidine that a significant

contribution

derivatives

resistant,

the interpretation

is due to iatrsmolecular

from this finding

vsxious O'-methyl

desmination in acidic medium, under

is relatively

participation

for preUminary identification

by

of the

of ara-C.

The rate of deamination was followed spectrally buffer

being

the data of Notari IL-* et al 15~6 and

the 2.9OK of ara-C. Wehave verified have profited

It was shownby Notari --• et al 15*16

in 1 I acetate

pH 3.5 clnd 80°C for 2-3 hours, and noting the decrease in optical

density at 290 nm and the corresponding increase at 260 nmb Susceptibility to deemination was observed, apart fran ara=C itself, VI end VIII,

only for peeks III,

with rates increasing in the order given. CombSnaticm

of these results with the chrcmatographic data suggest

that V is 2'-O=methyl-ara-C 2',5'-di-O-methyl-ara-C these contain a 2*--ethyl By contrast,

III

(9);

(3;

II

and IV are the 2',3'-

I is 2',3.,5'-tri-O-methyl-aC substituent

and are resists&

is 3',5'-di-O-methyl-aC

5'0 (4) and/or 3'0O-methyl-ara-C Identification possible, by application

(3;

(2) and/or (a

(all

of

to deamination),

whereas VI and VIII

are

(2).

of peak VI,

5'~Cknethy1-ara-C (98

It proved

of the procedure of Ksnai et al. 13 to prepare 4 17

1738

Vol. 46, No. 4, 1972

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

by unambiguous synthesis frcm 5.-O-methylcytidine.

78 This product melted at

190-192° and showedno depression on mixing with crystalline products were also spectrally Identification

and chromatographically

of peak VIII,

as 5c4-methyl-ara-C

identified

above, on the basis of desmination rates,

We now show that VIII

is,

in fact,

(3:

With VI

to V and VIII,

identified

ively.

identical.

3'-&methyl-era-C

(s>, we revert

VI. Both

tentatively

as 2 anal,

2 on the basis of its

respectconversion

-D-arabinofursnosyl)-3-methyluracil (ILJ).~ P in 2.5 ml 1 H-9 COOHwas heated in a sealed

to the known l-(FO-methyl-

30 mg of VIII

smpoule on a water bath for 5 hours, following

which chromatography with

solvent B demonstrated disappearance of the product (Rf - 0.5) and appearance of a new spot (Rf - 0.7)) uridine

the eluate of which gave a typical

spectrum19 showing that desmination had occurred to the srabinosyl

nucleoside (n).

The solvent was removed and the residue brought to dryness

several times from water to remove C COOH.It was then dissolved in water 3 and methylated with BP ethereal solution of diascmethane, the reaction being followed chramatographically.

After

about 90%methylatlon,

products began to appear, end the reaction was interrupted, phase was chromatographed on a preparative

traces of side The aqueous

scale with solvent B, the main

product eluted with anbydrous ethanol, brought to small volume snd filtered to remove traces of gel. It pound; but its

did not prove possible to crystallize

spectral properties

Codington --et alo I.8 for 2. ion at 25' with a Q2

this corn-

corresponded to those reported by

Furthermore, in O.lN RaOHit underwent degradat-

of 22.5 mins. as compared to 22 mins. reported by

the foregoing authors, Identification from identification

of peak V, 2'4-nlethylA3,ra-C

of peak VIII

as 3'-O-methyl-ara-C,

(a:

This follows

the resistance

2 to desmination (see above), and from RMRdata presented in the next paragraphs The 100 MIis NM3spectra of the 2*-, 3'0 and 5’-O-momxnethyl

1739

of

Vol. 46, No. 4, 1972

derivatives

BIOCHEMICAL

(2, 2 anad,

presence of s

methyl

3.45 ppm (relative

shielding

reported

in D20 solution, singlet

2'0,

similar

by Co&in&on

exhibited respectively,

--et al,l*

to detailed

of Oc-metbylation

for l-(20O-methyl-

di-O*-methyl

of any correlation affinities

3*-&methyl

and tri-O*-metbyl

Of same theoretical

analogues,

Such a correlation

are

to the influence

interest

of al.1 three

of the corresponding is the apparent

between the acidities a&We

was

conformation.

as well as to the preparation

for a strongly

methylationr

to free

and d(H-3’)

is being extended to identification

snalogues,

derivatives.

relative

-Darabinofuranosyl)-3P snalogue (in IMSO), These

IWR analyses in relation

on nucleoside

The foregoing

at 3.37, 3.50 and

between 6(H-2')

as well as the di-O.-methyl

being subjected

in each case the

3'- snd 5'- protons,

relation

and the corresponding

derivatives,

RESEARCH COMMUNICATIONS

DSS); as well as the expected enhanced

of the corresponu

-methyluracil

OI-ethyl

located,

to internal

ara-C. A qualitatively

AND BIOPHYSICAL

of the &r&M! hydroxyls, resin, 4,14 and susoeptibilities

appeers to exist

absence based on to

in the case of ribose

nucleosides.l~ The potential autimetabolites

value of O'-alkylated

ia testified

to by the observation

who found that 5*-+methyl-aracC

is at least

enzymatic

or oytitie,

deamination

analoguea of ar*C

than sr*C

N-fold

as

of Dr. r$ i!?wlerkowski, more resistant

using a cytidine

to

deaminase

from mouse kidney.

This explains, in part, the improved therapeutic activity noted by Gish --et al. 12 for 5~-&acyl esters of sr&C, Enzymatic and biological

tests

on all

the foregoing

are now in progress.

Ac~owledgments: We wre indebted to I& J. GLziewicz for assistsnce in the identification of 5*-O-methylcar%C, and to Mr P. Remin for the R?dRspectra. Cne of us (E. D.) wishes to thank l?rofo Stefaais Drabarek for her interest end encouregement. This study was supported by the Polish Academy of Sciences (Projeot 0!3,3.1.) and also profited from the support of the World Health Organization, The Wellcome Trust, and the Agricultural Research Service, U.& Dept. of Agriculture. 1740

Vol. 46, No. 4. 1972

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

REFERENCES 1. Broom, A.D. end Robins, R.K,, J.Am.Chem.Soco, a, 1145 (1965) 2. Khwaja, T.A. and Robins, R.K., J.Am.Chem.Soc., 2, 3640 (1966) 3. Martin, D.&G., Reese, C.B. end Stephenson, G.F., Biochemistry, 1, 1406

( 1968) 4. Gin, J.B. and Dekker, C.A., Biochemistry, 1, 1413 (1968) 5. Robins, l&J. and Naik, S.R., Biochim.Biophys.Acta, *, 341 (1971) 6. Robins, M.J. and Naik, S.R., Biochemistry, g, 3591 (1971) 7. Ku&mierek, J.T. and Shugar, D., Acta Biochim.Polon., & 4l3 (1971) 8. Kusmierek, J.T., Gizieticz, J. and Shugar, D., in preparation 9. Cohen, SOS., Prog.Nucl,Acid Res., 2, 1 (1966) 10. Roy-Bunnan, P., Analogues of Nucleic Acid Components, Springer-Verlag, New York, Heidelberg, Berlin (1970), pp. 66-69 11. Shugar, Do, in "'Virus-Cell

Interactions

and Viral

Antimetabolitesw

(Symp. No.3, 7th Annual Meeting, FEBS, Varna, Sept. 1971), D. Shuger (ea.), Academic Press, London and New York (1972) 12. Gish, D.T., Kelly, 2,

13. Qnai,

R.C., Camiener, G.W. and Wechter,

W.J., J.Med.Chem.,

1159 (1971)

To, Koj-,

T., Maruyama, 0. and Ichino,

I.,

Chem.Pharm,Bull.

(Tokyo), 2, 2569 (1970) 14. Dekker, C.A., J.Am.Chem.Soc., a, 15. Noteri,

4027 (1965) R.E., Chin, M.L. and Cardoni, A., Tetrahedron Letters,

No 40,

34% ( 1969) 16. Notari, R.E., Chin, M.L. and Cardoni, A., JoPhazm.Sci., a, 28 (1970) 17. Giziewicz, J., Kusmierek, J.T. and Shugar, D., In preparation 18. Codington, JeF., Cnshley, R.J. and Fox, J.J., J.Org.Chemo, 3, 466 (1968) 19. Fox, J.J. and Shugsr, I)., Bioohim.Biophys.Acta 2, 369 (1952)

1741