5-deoxy, 12-deoxy, 5,12-bisdeoxy, and 4,5,12-trisdeoxy anthracyclines: Synthesis of new analogues of daunorubicin and doxorubicin by controlled deoxygenation of the C-ring

C S I R O P U B L I S H I N G

Australian Journal

of Chemistry

Volume 53, 2000

A journal for the publication of original research

in all branches of chemistry and chemical technology

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Published by CSIRO PUBLISHING

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the Australian Academy of Science

Aust. J. Chem., 2000, 53, 25–40

5-Deoxy, 12-Deoxy, 5,12-Bisdeoxy,

and 4,5,12-Trisdeoxy Anthracyclines:

Synthesis of New Analogues of Daunorubicin and

Doxorubicin by Controlled Deoxygenation of the C-Ring*

Donald W. Cameron,^A,BGeoffrey I. Feutrill^A,Cand Peter G. Griffiths^A,D

^ASchool of Chemistry, The University of Melbourne, Parkville, Vic. 3010.

^BAuthor to whom correspondence should be addressed.

^CDeceased.

^DPresent address: Chiron Technologies Pty Ltd, Clayton, Vic. 3168.

Hydrogenation of the anthracyclines daunorubicin (1) and doxorubicin (2) gave selective deoxygenation at position

5. Hydride reduction of (1) and (2) gave complementary regiocontrol, leading to 12-deoxygenation or 5,12-bis-

deoxygenation. This chemistry allows retention of the 7-glycoside and the side-chain carbonyl groups. It has led to

new anthracycline families possessing all of the stereochemical and most of the spatial characteristics of the parent

compounds (1) and (2). These are typified by 5-deoxy (12), (15); 12-deoxy (22), (23); 5,12-bisdeoxy (34), (35); and

4,5,12-trisdeoxy systems (36). All possess high anticancer activity.

Keywords. Daunorubicin; doxorubicin; anthracycline; anticancer.

Introduction

aged by the reported formation of 5,7- and 7,12-bisdeoxy

systems (3) and (4). (For convenience of describing such

products, the quinonoid grouping in (3) and (4) is considered

as occupying a position tautomeric with its placement in (1)

and (2).) System (3) was obtained by either electrochemical

reduction of 5-iminodaunorubicin⁶or treatment of 7-deoxy-

daunorubicinone (5) with sodium dithionite.⁷In other work,

hydrogenolysis of daunorubicinone (6) (palladium on

barium sulfate) gave a product whose reported spectra also

appear to correlate with the 5,7-bisdeoxy system (3); this is

in preference to the isomeric 7,11-bisdeoxy structure origi-

nally suggested.⁸While the present investigation was in

progress, the same 5,7-deoxy chromophore was obtained as

its 13-ol (7), by reduction of (5) with sodium borohydride,

together with its 7,12-bisdeoxy analogue (8).⁹

Deoxy analogues of the important commercial antineoplas-

tic agents daunorubicin (daunomycin) (1) and doxorubicin

(adriamycin) (2)† are of interest as second-generation antitu-

mour agents.¹They modify the redox potential of the parent

compound without altering the stereochemistry, leading to an

altered and, in some cases, useful spectrum of bioactivity.

Known examples include 11-deoxy, 6-deoxy and 4-demethoxy

systems, each of which has demonstrated antitumour activity.

Gaining access to these systems, the first of which also occurs

naturally, has required multi-step total synthesis.¹

In principle, access to analogous deoxy systems might be

considered by simple reduction of parent anthracyclines.

However, significant chromophoric modification of (1) and

(2) without loss of the glycoside has been reported in only a

few cases. Thus 5-imino anthracyclines were formed by

reaction of (1) and (2) with ammonia.²The products had sig-

nificantly different reduction potentials from the parent

systems and lower cardiotoxicity in test mammals.³Another

example, leuco daunorubicin, resulted as a mixture of

diastereoisomers from mild reduction of (1) with sodium

dithionite.⁴The glycoside was also retained following acyla-

tion of (1) and subsequent catalytic hydrogenation.⁵

However, at the outset of this work there was no reported

way in which such chromophoric modification of the parent

anthracyclines (1) and (2) could be effected without prior

reductive cleavage of the 7-glycoside, an ortho-hydroxyben-

zyl system. Thus mild catalytic hydrogenation of daunoru-

bicin (1) mainly led to the 7-deoxy product (5).¹⁰Retaining

the daunosaminyloxy residue is necessary for water solubil-

ity and bioactivity.¹

Following our preliminary reports,^11,12this paper fully

describes efficient reductive synthesis of several new anthra-

cycline families, based on selective 5- and 12-deoxygenation

chemistry, without significant loss of the sugar residue.

If cleavage of the glycoside could be suppressed, direct

synthesis of deoxy anthracyclines by reductive deoxygena-

tion of anthracycline parents is chromophorically encour-

* Published in preliminary form: Tetrahedron Lett., 1988, 29, 4629; Tetrahedron Lett., 1993, 34, 4685.

† WHO names for these compounds are daunorubicin (1) and doxorubicin (2). For purposes of uniformity, common anthracycline numbering, as on

structure (1), is used throughout this paper. This differs from IUPAC numbering, which varies with functionality.

Manuscript received 3 November 1999

10.1071/CH99151 0004-9425/00/010025

26

D. W. Cameron, G. I. Feutrill and P. G. Griffiths

Analogues of Daunorubicin and Doxorubicin

27

5-Deoxy Anthracyclines

protons confirmed that 5-deoxygenation had occurred, while

reduction of the COMe group in (11) was evident from a pair

of shielded 14-methyl doublets at δ 1.30 and 1.35 (J 6.5 Hz

each), representing the two diastereoisomers of this system.

Molecular ions were shown at m/z 382 (10) and 384 (11) in

the respective e.i. mass spectra. The change of catalyst from

palladium to platinum thus afforded preferential deoxygena-

tion at position 5 over 7 for the first time, albeit accompanied

by competitive reduction at position 13.

By contrast, parallel platinum-catalysed hydrogenation of

the unprotected aglycone (6) gave 5,7-bisdeoxydaunorubici-

none (3), together with trace amounts of (4) and (7). While

retention of the 7-oxy substituent during reduction of (9)

may reflect its cyclic nature, it also seemed possible that the

difference may have derived from the steric bulk of the

phenylboronate residue. This led to experiments involving

platinum-catalysed reduction of the anthracycline glycosides

(1) and (2) themselves. It was thereby pleasingly established

that hydrogenation of the hydrochloride of daunorubicin (1)

in methanol over Adams catalyst, followed by the usual

aerial reoxidation, gave smooth conversion into a mixture of

the new 5-deoxydaunorubicin (12) and 5-deoxy-13-dihydro-

daunorubicin (13), the latter as a pair of C 13 diastereoiso-

mers. Subsequent to our original report,¹¹compound (12) has

also been derived by reduction of 5-iminodaunorubicin with

sodium dithionite.¹⁹

Glycosides (12) and (13) were separated by column chro-

matography on silica gel and each was isolated as its

hydrochloride. Their ¹H n.m.r. spectra were similar to those

of the corresponding aglycones (10) and (11) except for addi-

tional resonances due to the daunosaminyl residue. Thus the

spectra of (12) and (13) each contained an appropriate meso

proton resonance (δ 8.40, 8.39 respectively), while the 14-

methyl singlet (δ 2.30) in the spectrum of the former product

was deshielded relative to the corresponding resonance (δ

1.29) for the latter. Molecular ions were not observed in their

e.i. mass spectra, but f.a.b. mass spectrometry showed weak

M+H ions [m/z 512 for (12), 514 for (13)] and corresponding

M+Na ions. The relative yields of (12) and (13) could be

varied by changing the ratio of catalyst to substrate.

Compared to 5-deoxygenation of (1), reduction of the side-

chain carbonyl group was slow. However, by increasing the

ratio of catalyst/substrate to 1 : 1, complete conversion into

(13) was achieved in good yield (71%) and isolation did not

require chromatography.

Minimizing side-chain reduction so as to achieve

optimum yield of (12) would require protection–deprotec-

tion of the 13-keto group, as described later. However, satis-

factory formation of this product resulted from direct

reaction of unprotected (1) by lowering the catalyst ratio to

1 : 5 (20% w/w) and by addition of chloroacetic acid (4 : 1

acid/substrate). After basifying the product solution with

sodium hydrogen carbonate followed by aerial reoxidation

this led to (12) (57%) along with (13) (25%). The remainder

of the product was largely a mixture of (3), (7) and a trace of

a red, non-glycosidic component arising from a different

mode of reduction. Its ¹H n.m.r. spectrum indicated no aro-

matic proton resonances, but two broad multiplets at δ 1.78

Successful 5-deoxygenation of daunorubicin (1) and dox-

orubicin (2) was found to be effected by catalytic hydro-

genation under conditions that evolved through a series of

preliminary experiments. Initially, hydrogenation of the

hydrochloride of (1) in water over palladium on barium

sulfate, was confirmed to result in rapid hydrogenolysis of

the glycoside, with precipitation of 7-deoxydaunorubicinone

(5) (97%) as reported.¹³However, with the same catalyst in

methanol, this first product (5) was identified chromato-

graphically but it remained in solution, where it underwent

further, slower reduction to a mixture, from which 5,7-bis-

deoxydaunorubicinone (3) (88%) was obtained following

aerial reoxidation of the derived hydroquinone. A small pro-

portion (8%) of the isomeric 7,12-bisdeoxy compound (4)

was also isolated. These isomers were distinguished by their

¹H n.m.r. spectra, isomer (3) showing a single chelated

hydroxy signal at δ 13.77, whereas for (4) the signal was

considerably more deshielded (δ 15.12). For the former

isomer, its meso proton resonated as a singlet at δ 8.50. This

was deshielded relative to the corresponding proton of (4) (δ

8.02), consistent with its peri relationship to two oxygen sub-

stituents. Other signals were consistent with the assigned

structures and each isomer exhibited a molecular ion at m/z

366 in its electron-impact (e.i.) mass spectrum. The data for

(4) are consistent with relevant values for model tricyclic

compounds prepared in our earlier work.^14–16

Chromophorically, the overall deoxygenation of (5) to the

major product (3) is analogous to regioselective hydrogena-

tion of a 1,4-dihydroxy-5-methoxy-9,10-anthraquinone to

the corresponding anthrone followed by tautomerism and

reoxidation to a 9-hydroxy-5-methoxy-1,4-anthraquinone.¹⁵

With appropriately different regiochemistry, the natural

anthraquinone catenarin behaves analogously.¹⁷

In order to suppress 7-deoxygenation, experiments next

compared hydrogenation of the phenylboronate (9), in which

the 7-oxy substituent was part of a cyclic system,¹⁸with the

aglycone (6) from which it was derived. The latter has been

reported to undergo hydrogenolysis over palladium to give

(5).¹⁰The phenylboronate (9) was synthesized efficiently¹⁸

but when hydrogenated in methanol over palladium on

barium sulfate it was found to undergo only 7-deoxygenation

to (5). To exclude any possibility of boronate exchange in the

alcoholic solvent, the hydrogenation was repeated in ethyl

acetate but this led only to recovery of (9), following aerial

reoxidation of the corresponding quinol. However, hydro-

genation of (9) in ethyl acetate over Adams catalyst instead

of palladium gave a pale yellow solution, containing none of

the starting quinone. Aerial reoxidation of the filtrate gave a

red product clearly different from the orange chromophore of

(9). Acid-catalysed exchange of the phenylboronate residue

with pentane-2,4-diol and chromatography gave two red

compounds, identified as the new 5-deoxydaunorubicinone

(10) (45%) and its more polar 13-dihydro derivative (11)

(45%). Retention of the 7-hydroxy group in both products

1

was evident from their H n.m.r. spectra, which contained

deshielded multiplets due to H 7 at δ 5.19 and 5.22, respec-

tively. Singlets at δ 8.54 and 8.63 for the respective meso

28

D. W. Cameron, G. I. Feutrill and P. G. Griffiths

and 2.66 instead, integrating for four protons apiece. There

was an acetyl methyl group (δ 2.37) but no methoxy reso-

nance. The spectrum also showed two chelated hydroxy res-

onances at δ 13.15 and 13.19 and a broad hydroxy resonance

at δ 3.82, all D₂O-exchangeable. Other resonances due to the

A-ring were similar to those observed for (5). These data,

taken with an e.i. molecular ion of m/z 356, suggest the com-

pound may have the tetrahydro structure (14).

These optimum conditions were derived empirically by

varying the catalyst, reaction time and added acid. Fresh

Adams catalyst gave best results. In the absence of

chloroacetic acid, 5-deoxygenation to (12) proceeded too

slowly, prolonged hydrogenation increasing the proportion

of non-glycosidic by-products. An increased catalyst-to-sub-

strate ratio (1 : 1) gave 5-deoxygenation accompanied by

excessive side-chain reduction to (13). Adding trifluo-

roacetic or mineral acid in place of chloroacetic acid gave

rapid 7-deoxygenation, while alternative addition of acetic

acid had no significant effect.

with a mixture of diastereoisomers (2: 1). This particularly

affected signals in the δ 1.7–2.5 and 2.6–3.2 regions due to

H 8 and H 10, respectively. Its e.i. mass spectrum showed an

appropriate molecular ion at m/z 400.

Similarly 5-deoxy-13-dihydrodaunorubicinone (11) also

showed signals corresponding to a diastereoisomer ratio of

2 : 1. In particular, the C 14 methyl signal resonated as a pair

of doublets, as noted earlier. The absolute stereochemistry of

1

the respective isomers was assigned by H n.m.r. spec-

troscopy according to literature procedures for analogous

13-ols.^20–22To suppress rotational factors affecting the side

chain, the mixture (11) was converted into the corresponding

isopropylidene derivatives (20) and (21) by treatment with

2,2-dimethoxypropane and p-toluenesulfonic acid in

dioxan.^20,21These derivatives were spectroscopically differ-

entiable and chromatographically separable. For the major

ketal, chemical shift differences between the non-equivalent

methylene protons H8eq and H 8ax (δ 2.36, 1.61 respec-

tively; ∆δ 0.75), and H 10eq and H 10ax (δ 2.99, 2.53 respec-

tively; ∆δ 0.46) were consistent with values reported for

analogous anthracyclinone ketals of defined 13R configura-

tion. Corresponding ∆δ values for the minor ketal [H 8eq,

H8ax (δ 2.18, 1.96 respectively; ∆δ 0.22) and H 10eq,

H10ax (δ 3.02, 2.49 respectively; ∆δ 0.53)] likewise paral-

leled isomeric (13S)-anthracyclinone ketals.^20,21

The major ketal in the present study was thereby assigned

as the 13R-isomer (20), an outcome that was also found to be

consistent with n.O.e. experiments. In previous work involv-

ing an analogous pair of (13R)- and (13S)-anthracyclinone

ketals, irradiation of the H13 resonance led to significant

enhancement of the H 10 signal for the former isomer and of

H 8 for the latter.²²For the major isomer, now assigned as

(20), irradiation of H 13 (δ 4.20) showed largest enhance-

ments (5%) for H10eq and the adjoining methyl signal. For

the minor isomer (21), the largest enhancements (7%) were

observed for H 8eq, along with the adjoining methyl protons.

This diastereofacial bias in catalytic reduction of (1)

towards 13R stereochemistry differs from the outcome of

mammalian and microbial reduction in the metabolism of

(1). Both have been shown to give only the 13S configura-

tion.^20,21

Under these conditions in methanol, the daunosaminyl

residue of (1) both protects the 7-oxy function from

hydrogenolysis and leads to greater regioselectivity of

deoxygenation of the ^C-ring. Thus reduction of the aglycone

(6) under those same conditions gave only the 7-deoxy-

genated products (3), (4) and (7). In contrast, the products

from parallel reductions of (1) were largely glycosides (12)

and (13), and no 12-deoxygenated product analogous to (4)

1

was observed, within the limits of H n.m.r. spectroscopic

sensitivity.

The clinically important antitumour antibiotic doxoru-

bicin (2) was also hydrogenated in the same way as (1),

giving a mixture of the new 5-deoxydoxorubicin (15) (59%)

and 5-deoxy-13-dihydrodoxorubicin (16) (20%), which

were separated by chromatography and isolated as

hydrochlorides. Because of its considerable polarity, com-

pound (16) was difficult to extract from aqueous solution and

to elute from silica gel columns. By increasing the w/w ratio

ofAdams catalyst (1 : 1), (2) gave glycoside (16) exclusively.

It was thereby isolated in 76% yield as its hydrochloride,

without chromatography being required, as a mixture of C 13

diastereoisomers. Both (15) and (16) showed M+H ions by

f.a.b. mass spectrometry (m/z 528, 530 respectively) as well

as M+Na ions. Such hydrogenations were also accompanied

by minor hydrogenolysis of the sugar residue, leading

chiefly to the 5,7-bisdeoxy product (17) (15%). Its ¹H n.m.r.

spectrum resembled that of (3), the 14-methyl signal of the

latter (δ 2.38) being replaced by an AB methylene quartet (δ

4.70, 4.74, J 21 Hz).

Aqueous acidic hydrolysis of the glycosides (12), (13),

(15) and (16) gave the respective aglycones (10), (11), (18)

and (19), the first two of which were identical with the reduc-

tion products from daunorubicinone phenylboronate (9). The

latter pair of aglycones had ¹H n.m.r. spectra that were appro-

priately similar to the former pair but 5-deoxydoxorubici-

none (18) showed an AB quartet [δ 4.76 and 4.81 (J 21 Hz)]

due to the C 14 methylene group and a molecular ion at m/z

398 in its e.i. mass spectrum. The ¹H n.m.r. spectrum of its

13-dihydro derivative (19) was more complex, consistent

12-Deoxy, 5,12-Bisdeoxy, and 4,5,12-Trisdeoxy

Anthracyclines

Attention was next directed towards alternative regiocon-

trol of ^C-ring deoxygenation, so as to gain access to a new, iso-

meric series of 12-deoxy anthracyclines, represented by

12-deoxydaunorubicin (22), 12-deoxydoxorubicin (23) and

12-deoxycarminomycin (24). Compound (24), hypothetically

derivable from the parent carminomycin (25),^23,24represented

a realistic initial target since, by analogy with anthraquinone

chemistry,^15,17it was expected that 12-deoxygenation might be

preferred over 5-deoxygenation, for hydrogenation of anthra-

cyclines in which the 4-methoxy substituent was replaced by

a 4-hydroxyl. Regioselective 4-O-methylation of (24) might

then be a reasonable source of (22).

Carminomycin (25) was therefore hydrogenated in

methanol containing chloroacetic acid over Adams catalyst,

Analogues of Daunorubicin and Doxorubicin

29

analogously to (1). Oxidative workup showed poor conver-

sion into a new glycoside initially identified as 12-deoxy-

carminomycin (24), accompanied by a still more polar

ated with a pK_avalue around 5. Other potentially zwitter-

ionic anthracyclines have been reported but these have

always involved carboxy groups as the internal proton source

interacting with the amino glycosides.^25,26A synthetic amino

anthracenol has also been reported to form a zwitterionic

species in solution.²⁷

glycoside

12-deoxy-13-dihydrocarminomycin

(26).

However, the major products (46% overall) were water-

insoluble, reflecting hydrogenolysis of the glycoside and

consisting chiefly of the 7,12-bisdeoxy compounds (27) and

(28). Spectroscopic data for the latter pair were analogous to

those for their respective O-methyl counterparts (4) and (8),

except that in the ¹H n.m.r. spectrum of (27), for example, the

two phenolic hydroxy groups resonated as two singlets at δ

16.14 and 9.92. The considerable deshielding of the former

resonance is characteristic of the 5-hydroxy group flanked by

two peri oxygen substituents.¹⁴The e.i. mass spectrum of

(27) showed an appropriate molecular ion (m/z 352).

The more polar (28) was isolated as a (1 : 1) pair of

diastereoisomers with doubling of signals due to H 13 (δ

3.84, 3.66, q, q, J 6.5, 6.5 Hz) and the C 14 methyl protons (δ

1.33, 1.30, d, d, J 6.5, 6.5 Hz). As expected, the meso proton

H 12 was less deshielded than was H 5 in the 5-deoxy series.

For (27), H 12 resonated at δ 8.02 while for the diastereoiso-

mers of (28) it was observed at δ 7.93 and 7.94.

Despite being accompanied by excessive loss of the amino

sugar, all of these products reflected the desired 12-deoxy-

genation. Increasing the acidity of the hydrogenation medium,

through addition of dichloroacetic acid (4% w/v) rather than

chloroacetic acid, was empirically found to give a much better

yield of the glycoside (24) (59%). This was accompanied by a

minor proportion of the corresponding 13-dihydro product

(26), which was difficult to isolate, on account of its consider-

able polarity. However, conventional extraction of both these

products from aqueous solution, sufficiently basic to avoid

protonation of the amino group, proved particularly difficult.

Moreover, their colour behaviour differed from that of the

non-glycosidic (27) and (28). Sodium hydrogen carbonate

solutions of both glycosides were blue, and ethyl acetate

extracts were purple, spectacularly different colours from con-

ventional anthracyclines. The products were separated on

silica gel or sephadex LH20 columns, with polar mixtures of

chloroform, methanol and water as eluent, despite some loss

of material thereby occurring, due to strong binding.

Evaporated fractions gave (24) and (26) as blue solids. The

hydrochloride of (24) was isolated as a red solid after addition

of an excess of methanolic hydrogen chloride, but on redis-

solving in neutral methanol it spontaneously gave a blue solu-

tion. Analogous behaviour was observed for the more polar

compound (26), on which less work has been done. The elec-

trospray mass spectrum of (24) (acetonitrile/water) showed an

M+H ion at m/z 498 as the base peak.

Solutions of (29) in (D₄)methanol, deuterium oxide or

(D₆)dimethyl sulfoxide gave ¹H n.m.r. spectra in which every

signal except the acetyl methyl was broadened beyond recog-

nition. However, a mixture of (D)chloroform/(D₄)methanol

(2 : 1), the maximum proportion of chloroform in which (29)

remained in solution, gave a much sharpened spectrum, with

recognizable aromatic resonances in particular. The cause of

the broadening in protic and dipolar aprotic solvents is

unclear. Acidifying a (D₄)methanolic solution of (24) with

(D)trifluoroacetic acid, thereby changing the colour from

blue to red, sharpened only some signals in the spectrum. This

appears to rule out broadening by rapid electron transfer as

ascribed to both fredericamycin A²⁸and the synthetic amino

anthracenol mentioned above.²⁷Nevertheless, the dramatic

line sharpening on addition of (D)chloroform evidently

reflects suppression of radical formation in the other solvents.

In many respects compound (29) thus behaves like kinob-

scurinone, described as ‘n.m.r. silent’ in (D₆)dimethyl sulfox-

ide, (D₅)pyridine and (D)trifluoroacetic acid.²⁹

Acidic hydrolysis of (29) and (26) gave the respective red

aglycones (30) and (31), which showed none of the spectro-

scopic anomalies of the amino glycosides. They were also

obtained by conversion of carminomycinone (32) into its

phenylboronate (33) and hydrogenation of the latter over

Adams catalyst, followed by deprotection.

While hydrogenation of carminomycin (25) thus occurred

with the desired regiochemistry, the zwitterionic product

(29) proved unsatisfactory as a source of 12-deoxydaunoru-

bicin (22). Despite encouragement from simpler model

systems,^14,15selective O-methylation of (29) could not be

effected. Attention therefore turned to obtaining (22) and its

doxorubicin analogue (23) more directly, by hydride reduc-

tion of the parent anthracyclines (1) and (2). Extension of

methodology so developed has then also led to a further new

family of 5,12-bisdeoxy anthracyclines, represented by bis-

deoxydaunorubicin (34), bisdeoxydoxorubicin (35) and the

4,5,12-trisdeoxy analogue (36), this last product from analo-

gous bisdeoxygenation of the antitumour antibiotic idaru-

bicin (37). Sodium borohydride was used earlier for

converting 1,4-dihydroxy anthraquinones into 9-hydroxy-

1,4-anthraquinones.^16,29It seemed possible that, as nucle-

ophiles, such reductants might lead to different selectivity

from hydrogenolysis.

These observations, and the ¹H n.m.r. characteristics fol-

lowing, are consistent with (24) existing chiefly as the zwit-

terion (29) and it is designated as such in subsequent

discussion. An analogous zwitterionic structure is inferred

also for (26). This implies involvement of the amino sugar

with a phenolic hydroxy group of acidity unparalleled in

conventional anthracycline chemistry. The visible absorption

spectra of buffered solutions of (29) at varying pH revealed

that the chromophoric change from red to blue was associ-

Reported treatment of daunorubicin (1)³⁰and carmino-

mycin (25)³¹with sodium borohydride in basic methanol

resulted simply in reduction of the side-chain 13-keto group,

the quinone nucleus apparently being protected by ioniza-

tion. More recently,³²while the present work was in

progress, similar reduction of (1) in unbuffered methanol

was reported also to give the 7-deoxy 13-ol (38) (58%) along

with the side-chain reduced glycosidic system (39) (17%)

reported earlier.³⁰

30

D. W. Cameron, G. I. Feutrill and P. G. Griffiths

In the present investigation (1) was first treated with

doublets (δ 2.96, 3.02, both J 20 Hz). An electrospray mass

spectrum showed a strong M+H ion at m/z 514. The relative

shielding of the meso proton singlet (δ 8.04 for both isomers)

confirmed that by far the major product of reduction corre-

sponded to the desired 12-deoxygenation.

potassium or sodium borohydride in methanol containing

aqueous buffer (pH 7). With buffers of lower pH, reduction

competed with decomposition of the reagent, while in more

basic environments it was impeded by ionization of the sub-

strate. However, at pH 7 there was rapid reduction of the

chromophore at 0°, as indicated by a sharp colour change

from orange to yellow. Quenching with mineral acid to pH

1–3 caused another colour change to deep red. Workup then

gave a dark red solid, isolated as its hydrochloride (93%). ¹H

n.m.r. spectroscopy identified the major product as the new

12-deoxy-13-dihydrodaunorubicin (40) (71%) accompanied

by minor proportions of its 5-deoxy isomer (13)¹¹(14%) and

It was next sought to develop protection–deprotection

chemistry, so as to allow the side-chain 13-keto group to be

retained during reduction. Treatment of daunorubicin (1) in

ethane-1,2-diol or propane-1,3-diol with an excess of

trimethyl orthoformate and camphorsulfonic acid catalyst at

20°, gave the respective ketals (42) and (43) without signifi-

cant hydrolysis of glycoside.³³However, only the 1,3-dioxan

(43) could be deprotected to (1) without undue formation of

accompanying aglycone, by treatment with 0.1 ^Mhydrochlo-

ric acid at 20°. Indeed, despite (43) being formed efficiently

in solution, attempted isolation of (43) as its hydrochloride

always gave mixtures with deprotected (1), as differentiated

by the chemical shifts of the 14-methyl protons [δ 2.42 for (1)

and δ 1.64 for (43)]. Accordingly, the ketalization mixture

containing (43) was diluted as such, with methanol and

aqueous buffer (pH 7), and treated with potassium borohy-

1

a new glycoside (41) (8%). The H n.m.r. spectrum of (41)

showed two sharp aromatic singlets at δ 8.58 and 9.05, con-

sistent with bisdeoxygenation (discussed later). This mixture

was inseparable by chromatography but (40) was obtained

by several crystallizations as a 1 : 1 mixture of diastereoiso-

mers at C 13. Despite considerable overlapping, some reso-

nances for the individual isomers were spectroscopically

distinct. Thus H 10eq for the mixture resonated as a pair of

Analogues of Daunorubicin and Doxorubicin

31

dride at 0° under nitrogen. After acid-quenching, the interme-

diate ketal was deprotected as above and the product was iso-

lated as a mixture of hydrochlorides. Its H n.m.r. spectrum

shielded with respect to the corresponding group of (2) (δ

4.76). In situ reduction of (46) with potassium borohydride

as for (1) and deprotection in a separate step proceeded less

cleanly than for the latter series. Nevertheless, the major

product was the desired new 12-deoxydoxorubicin (23)

(52% overall) [δ 4.74, 4.78, ABq, J 17 Hz (14-CH₂); 8.05, s

(H 12)] accompanied by its 5-deoxy isomer (15) (7%) and

the new 5,12-bisdeoxydoxorubicin (35) (5%). The major

product (23), separated by fractional crystallization, showed

an M+H ion at m/z 528 in its electrospray mass spectrum.

We next sought preparative access to the new 5,12-bis-

deoxy systems (34) and (35), noted above as by-products

accompanying formation of their 12-deoxy counterparts.

Initial assessment towards (34) involved extended contact of

(1) with an excess of potassium borohydride. Surprisingly this

did not lead to a significant increase in the low yield of 5,12-

bisdeoxy-13-dihydrodaunorubicin (41). Apparently the major

yellow intermediate that led to (40) on quenching with acid is

relatively resistant to further reduction during the lifetime of

the reagent in the reaction solution. On a spectroscopic scale,

the more powerful reductant lithium borohydride in tetrahy-

drofuran was next added to (1), dissolved in tetrahydrofuran,

for solubility reasons as its free base. However, the orange

solution instantly became blue, representing ionization of (1)

by the strongly basic reagent, and spectroscopic analysis indi-

cated that only the side-chain carbonyl group was then

reduced. But, notwithstanding the instability of lithium boro-

hydride towards protic solvents in isolation, addition of a

small proportion of methanol (10%) was found to disrupt the

lithium complex, allowing reduction of the chromophore to

proceed rapidly and giving an almost colourless solution.

Quenching by addition of acetone followed by mineral acid

then gave a bright yellow solution. This gave a single, yellow,

water-soluble product, spectroscopically assigned as the bis-

deoxy 13-dihydro glycoside (41), most particularly by its two

meso proton resonances (δ 8.58, 9.05).

Preparative development of this procedure to give the new

5,12-bisdeoxy anthracycline (34), in which the 13-keto

group survived intact, involved initial protection of (1) as the

1,3-dioxan (43) as discussed above, followed by dilution of

the ketalizing mixture with methanol, then with a 10-fold

excess of tetrahydrofuran. This was followed by reduction

with aliquots of lithium borohydride until the solution

became nearly colourless. Quenching with acid and associ-

ated deprotection then gave 5,12-bisdeoxydaunorubicin (34)

as its yellow hydrochloride (69%). The chief contaminant

was 5-deoxydaunorubicin (12) (13%), which was removed

by fractional crystallization. The same major product (34)

was obtained more conveniently by in situ reduction of the

dimethyl ketal (44) with lithium borohydride. Glycoside (34)

showed an M+H ion in its f.a.b. mass spectrum at m/z 496

and an M+Na ion at m/z 518. The aromatic region of its ¹H

n.m.r. spectrum showed the two new meso protons as sharp

singlets at δ 8.58 and 9.05 and other signals consistent with

the assigned structure. A singlet acetyl methyl resonance (δ

2.40) confirmed retention of the 13-keto group.

1

was consistent with the major component being the desired

new 12-deoxydaunorubicin (22) (71%) [δ 2.40 (COMe), 8.03

(H 12)] accompanied by its 5-deoxy isomer (12) (4%) [δ 2.30,

8.40 (H 5)] and the 5,12-bisdeoxy glycoside (34) (8%) (δ

2.40, 8.58, 9.05). These compounds were chromatographi-

cally inseparable, but (22) was obtained pure by fractional

crystallization. Its electrospray mass spectrum (acetoni-

trile/water) showed a significant M+H ion at m/z 512.

Considerable streamlining of this procedure involved for-

mation of the more labile dimethyl ketal (44), by treating (1)

in a mixture of methanol, trimethyl orthoformate and an acid

catalyst. After addition of neutral buffer, this was followed

by in situ reduction and workup with aqueous mineral acid.

Under these conditions a separate deprotection step was not

required, though the 13-ketone (22) still needed to be sepa-

rated from the minor products (12) and (34) by fractional

crystallization.

While this procedure thus ensured protection of the keto

group as desired, attempted isolation of the intermediate

ketal (44) as its hydrochloride resembled (43) in always

giving mixtures with considerable proportions of depro-

1

tected (1), as shown by H n.m.r. spectroscopy. Indeed,

signals for (44) [δ 3.48, 3.50 (2×OMe) and δ 1.52 (14-Me)]

invariably represented the minor component of such mix-

tures, following isolation. However, the in situ protection of

(1) as (44) also proved compatible towards 5-deoxygenation

by catalytic hydrogenolysis. Hydrogenation of the ketaliza-

tion mixture over Adams catalyst, oxidative workup and

final deprotection with aqueous acid gave 5-deoxydaunoru-

bicin (12) (62%). Despite involving the additional protection

and deprotection steps, this overall yield was better than that

described earlier for direct hydrogenation of (1); no chro-

matography of the product was required and there was no

significant accompanying formation of the 13-o1 (13).

With these procedures in hand, attention was next directed

towards the more difficult problem of synthesizing 12-deoxy

analogues of doxorubicin (2). In the absence of side-chain

protection, deoxygenation of (2) proceeded analogously to

(1), to give the 13,14-diol (45) (95%). However, ketalization

of (2) with propane-1,3-diol was significantly slower than

for (1). Complete conversion was never achieved and with

lengthening reaction time, aglycone by-products became

dominant. Moreover, deprotection of the ketal was also slow

in 0.2 M hydrochloric acid at 20°, generating more aglycone.

Involvement of the dimethyl ketal (46) was considerably

better, particularly when 0.1 M methanolic hydrogen bromide

instead of camphorsulfonic acid was used as ketalization cat-

alyst, and a mixture of acetone and 0.25 M hydrobromic acid

at 20° for deprotection. Although the hydrochloride of (46)

was more stable towards isolation than that of analogous

(44), it was similarly not able to be obtained sufficiently pure

for characterization, because of the presence of (2).

The ¹H n.m.r. spectrum of (46) included singlets at δ 3.53

and 3.56 for the new methoxy groups and a further singlet (δ

3.98) for the 14-methylene protons, the latter appropriately

Extension to doxorubicin (2), through its dimethyl ketal

(46), gave the new 5,12-bisdeoxydoxorubicin (35) (61%),

32

D. W. Cameron, G. I. Feutrill and P. G. Griffiths

accompanied by a small amount of 5-deoxydoxorubicin

(15). Quinone (35) had distinguishing ¹H n.m.r. signals at δ

4.75, 4.79 [ABq, J 17 Hz (14-CH₂)] and at 8.58, 9.06

(H12,5). It did not show an M+H ion in its f.a.b. mass spec-

trum but an M+Na ion at m/z 534 was observed. However, a

strong M+H ion at m/z 512 was observed in its electrospray

mass spectrum.

matography on silica gel. After a brief induction period cor-

responding to formation of platinum metal from the dioxide,

the colour of the supernatant phase rapidly changed from

orange to yellow. At that point the species present in solution

was apparently the hydroquinone (52), since on contact with

air it rapidly reverted to (1). Extended hydrogenation led to

two new yellow compounds. Unlike (52), they were stable

enough to be chromatographed as such, though allowing

chromatograms then to stand in air turned them both bright

red, corresponding to formation of the 5-deoxy products (12)

and (13). The two yellow intermediates were inferred to be

the major anthrone (53) and the analogous 13-ol.

Chromatography of these compounds was assisted by

excluding air as far as possible, and the former was obtained

Reduction of the clinically important idarubicin (37)¹was

then explored. It was subjected to both of the procedures

optimized in preceding paragraphs, for 12-deoxygenation

and for 5,12-bisdeoxygenation. Formation of its dimethyl

ketal, followed by in situ reduction with potassium borohy-

dride and deprotection gave a product spectroscopically

assigned as a mixture of the isomeric deoxy glycosides (47)

and (48) (87%), together with a small amount of contami-

nating bisdeoxyidarubicin (36) (8%). The two main products

were associated with a pair of ¹H n.m.r. singlets at δ 8.11 and

8.14 (2 : 3), assigned to meso protons. The lack of significant

regioselectivity in deoxygenating (37) is to be expected,

given the absence of a 4-oxy substituent and the individual

structures (47) and (48) have not been spectroscopically dif-

ferentiated. All three products co-chromatographed and,

because there was no single major isomer, no attempt was

made at fractionating the mixture preparatively.

In situ reduction of the dimethyl ketal of idarubicin (37),

with lithium borohydride, gave the new 4,5,12-trisdeoxy

anthracycline (36) (62%), accompanied by a small amount

of recovered (37) (5%), removed by fractional crystalliza-

tion. The meso protons of (36) resonated at δ 8.63 and 8.66,

while the remaining aromatic protons formed two multiplets,

each corresponding to two hydrogens, at δ 7.64 and 8.01.

There was also an acetyl methyl singlet at δ 2.42. The f.a.b.

mass spectrum of (36) showed strong M+H and M+Na ions

at m/z 466 and 488 respectively.

1

as a yellow-brown hydrochloride. Its H n.m.r. spectrum in

(D₄)methanol showed minor signals due to (12) (15–20%),

representing adventitious oxidation, but these were distinctly

resolved from aromatic signals corresponding to the major

product. The latter included aromatic doublets at δ 7.30 and

7.89 (J 8Hz) and an apparent triplet at δ 7.47 (J 8 Hz) con-

sistent with an unaltered ^D-ring. Importantly, a two-proton

singlet at δ 4.04, assigned to a new methylene group, sup-

ported the anthrone structure (53).¹⁷This product could not

be purified further because of the ease with which it under-

went aerial oxidation, rapidly in solution or more slowly in

the solid state, to give (12).

Similar isolation of an analogous intermediate from

carminomycin (25) gave a solid, unstable hydrochloride,

whose ¹H n.m.r. data supported the anthrone structure (54).

The different orientation of its nuclear carbonyl group from

that of (53) led to less deshielding of the D-ring doublets of

1

its H n.m.r. spectrum [δ 6.86 (J 8.5 Hz), 7.03 (J 7.5 Hz)].

The remaining aromatic signal (H 2) was observed as a

doublet of doublets at δ 7.55 (J 7.5, 8.5 Hz), while the

anthrone methylene signal appeared at δ 4.26.

Hydrolysis of the 12-deoxy (22) and 5,12-bisdeoxy (34)

glycosides derived from daunorubicin (1) gave the new agly-

cones (49) and (50). Subsequent to our original communica-

tion,¹²system (50) has also been reported in the patent

These observations indicate that in the presence of plat-

inum catalyst, reduction of (1) and (25) proceeds with the

same, established selectivity as for analogous, simpler

anthraquinones,^15,17where anthrone intermediates can be

isolated more easily.¹⁷For α-methoxy quinones, deoxygena-

tion with associated change to sp³geometry occurs peri to

the methoxy group. For α-hydroxy quinones it proceeds with

the opposite regiochemistry, maximizing intramolecular H-

bonding to the anthrone carbonyl group.

1

literature,³⁴associated with a multistep synthesis. The H

n.m.r. spectrum of (49) showed a strongly deshielded, sharp

hydroxy singlet at δ 14.90 in (D)chloroform. This was absent

from the spectrum of (50). Hydrolysis of bisdeoxyidarubicin

(36) gave the yellow trisdeoxy aglycone (51), a system that

has been obtained elsewhere by multistep total synthesis.¹⁸

Formation of 5-deoxy anthracycline glycosides thereby

indicates that, under hydrogenating conditions, the hydro-

quinone (52) and its tautomers have sufficient lifetime to

undergo further reduction to anthrone (53), without signifi-

cantly competitive 1,4-elimination of daunosamine. The

latter process was postulated as occurring rapidly, to convert

(52) into the 7-deoxy product (5), through the quinone

methide (55);⁷however, subsequent work by Danishefsky’s

group has separately also concluded that, under mild condi-

tions, (52) was stable towards elimination of the sugar.⁵

Preferential 7-deoxygenation of (1) to give (5), such as does

occur by catalytic reduction over palladium, is inferred to

involve direct hydrogenolysis of the o-hydroxybenzyl

system.

Regiochemistry of Deoxygenation

The previous discussion has outlined two new modes of

anthracycline reduction. Both of them are mild enough to be

effected without loss of the glycosyl residue and both are

able alternatively to avoid, or to involve, accompanying

reduction of the 13-keto group. Whereas hydrogenation over

platinum led chiefly to 5-deoxy products, the action of

hydride reagents led to isomeric 12-deoxy glycosides or,

with more extensive reduction, to 5,12-bisdeoxy glycosides.

The following paragraphs consider these respective pro-

cesses in more detail.

The course of platinum-catalysed hydrogenation of (1)

leading to (12) was followed by analytical thin-layer chro-

Analogues of Daunorubicin and Doxorubicin

33

These outcomes are strongly contrasted by the 12-deoxy-

genation of (1), effected by potassium or sodium borohy-

dride. This latter process does not appear to involve an

anthrone or any other intermediate stable enough to allow

isolation. The yellow mixture, formed by treatment of (1)

with an excess of potassium borohydride in methanol and

aqueous buffer (pH 7) at 0° under nitrogen, slowly regained

the quinizarin chromophore on being stirred in air.

Chromatographic analysis then identified 13-dihydro-

daunorubicin (39) as the sole product.

While an early report has postulated borohydride reduction

of (1) as leading to the corresponding hydroquinone,³⁵the

yellow mixture above did not show the chromatographic char-

acteristics of either a hydroquinone or an anthrone. No stable

intermediate could be isolated from it but, on being applied

anaerobically to a t.l.c. layer of silica gel impregnated with

oxalic acid, its colour rapidly turned red and it chro-

matographed as the 12-deoxy anthracycline (40). Under these

conditions a solution of the hydroquinone (52) could not be

prevented from adventitiously oxidizing completely to (1),

while the anthrone (53) was largely stable enough to allow

chromatography as such. This chromatographic conversion

into (40) parallels the preparative procedure already described.

Borohydride reduction of (1) is thus chromophorically

reversible up to the point of quenching with acid, the latter step

evidently being the one that promotes 12-deoxygenation.

The simplest explanation for these observations is that

mild reduction of (1) by potassium borohydride leads

directly and selectively to the oxanthrone tautomer (56), or

its boron complex,^32,36as the major product. This reflects

reduction by the nucleophilic reagent kinetically favouring

attack at the more electron-deficient 12-carbonyl group, the

5-carbonyl being conjugatively affected by electron donation

from the 4-methoxy. While intermediate (56) reoxidizes in

air, on contact with acid it undergoes 1,4-elimination of

water leading to the stable 12-deoxy product (40), without

further change of oxidation level (Scheme 1).

Further reduction of (56) is more difficult than of (1) but

occurs rapidly with lithium borohydride in methanolic

tetrahydrofuran, as described for 5,12-bisdeoxygenation.

Formation of the hypothetically derived intermediate (57)

has analogy in the reduction of anthraquinone itself to the

9,10-dihydro 9,10-diol, on treatment with sodium borohy-

dride.³⁷The nearly colourless reaction solution containing

(57) then undergoes twofold elimination on quenching with

acid, aromatizing to give the bisdeoxy quinone (41)

(Scheme 1).

The borohydride methodology described here represents a

useful general basis for deoxygenating quinizarin deriva-

tives. We have applied it earlier to simpler systems¹⁶but have

not previously discussed its regiochemistry.

Conclusion

The new deoxy anthracyclines showed strong anticancer

activity, comparable with the parent compounds daunoru-

bicin (1) and doxorubicin (2). For the former series, the 5-

deoxy (12), 12-deoxy (22), 5,12-bisdeoxy (34) and

4,5,12-trisdeoxy product (36) gave ^T/C values of 196% [2.1

mg/kg], 180 [16], 155 [8] and 148 [8] against murine P388

lymphocytic leukaemia in vivo; this compared with a value

of 173% [1.6 mg/kg] for daunorubicin (1) itself. For the dox-

orubicin series, corresponding values for the 5-deoxy (13),

34

D. W. Cameron, G. I. Feutrill and P. G. Griffiths

formic acid on a V.G. Quattro quadrupole mass spectrometer in posi-

tive-ion mode. The mass of each ion is given, followed by its relative

intensity. In general, only peaks greater than 20% are quoted, apart

from the molecular ion. For all hydrochlorides the molecular ion (M) is

taken as of the free base. Analytical and preparative thin-layer chro-

matography (t.l.c., p.l.c.) were carried out on glass plates coated with a

layer of silica gel [Merck Kieselgel 60 GF₂₅₄, or Merck Kieselgel 60

GF₂₅₄containing 2% oxalic acid (oxalated silica)]. The separated com-

ponents were extracted from the silica with ethyl acetate or chloroform.

Oxalic acid was removed by washing the extracts with water and then

drying over sodium sulfate. Organic extracts generally were dried in the

same way before evaporation at reduced pressure. Column chromatog-

raphy was carried out by using Merck Kieselgel No. 9385 or Pharmacia

Sephadex LH20. All solvents were ofAR grade or were redistilled prior

to use. Petrol refers to the hydrocarbon fraction boiling in the range

40–60°. Adams catalyst refers to freshly opened platinum dioxide

obtained from the Aldrich Chemical Co. or type D platinum dioxide

from Johnson Matthey and Co.

12-deoxy (23) and 5,12-bisdeoxy compound (35) were

234% [6.4 mg/kg], 134 [4] and 177 [8], compared with a

value of 231% [5 mg/kg] for the parent doxorubicin (2).

Deoxy 13-dihydro anthracyclines were also strongly active

but at higher concentrations. Thus for the 5-deoxy 13-

dihydro compound (16) the corresponding value was 212%

[16 mg/kg].

Similarly high levels of activity also were exhibited by

selected deoxy compounds against murine L1210 lymphoid

leukaemia and B16 melanoma in vivo. For 5-deoxydoxoru-

bicin (15) the respective ^T/C values were 171% [3.2 mg/kg]

and 235 [6.4]; and for 5-deoxy-13-dihydrodoxorubicin (16)

the corresponding values were 162 [16] and 247 [24].

Disadvantageously, however, the new compounds did not

give worthwhile activity against a doxorubicin-resistant

strain of P388; while long-term administration of 5-deoxy-

doxorubicin (23) in mice showed it to be more highly car-

diotoxic than doxorubicin (2) itself. Consistent with its

unusual physical properties, the zwitterion (29) behaved dif-

ferently from the other anthracyclines. Obtaining its ^T/C value

proved impractical, because its much lower toxicity towards

mice prevented a control level from being established.

The chemistry described in this paper has efficiently

allowed controlled 5- and/or 12-deoxygenation of anthracy-

clines, without significant complication from other reductive

processes, particularly cleavage of the glycoside, that

impeded earlier work. It has led to new anthracycline fami-

lies possessing most of the skeletal features and all of the

stereochemistry of the parent compounds daunorubicin (1),

doxorubicin (2) and idarubicin (37), spatially permitting the

new systems to function as DNA-intercalators in the same

way as the parents. Reduction of the acetyl-containing sub-

strates (1) and (37), protected as 13-ketals, has given 5-

deoxy, 12-deoxy, 5,12-bisdeoxy or 4,5,12-trisdeoxy

products (12), (22), (34) and (36) respectively by one-pot

chemistry and without the need for chromatography. The

same applies to reduction of the hydroxyacetyl-containing

substrate (2), leading to the 12-deoxy or 5,12-bisdeoxy

products (23) and (35), apart from deprotection of the rela-

tively stable 13-ketals in this latter series being best per-

formed as a separate, second stage.

Associated antitumour testing in vivo was performed on B₆D₂F₁

mice.Approximately 10⁶tumour cells were injected intraperitoneally in

control and test mice. Test mice were dosed at 1, 5 and 9 days over a

range of concentrations less than the toxic dose. The antitumour activ-

ity is expressed as ^T/C% calculated from the following: T/C% = median

test animal survival time/median control animal survival time×100. The

highest ^T/C value is quoted in each case.

Hydrogenation of Daunorubicin (1) over Palladium

The hydrochloride of daunorubicin (1) (20 mg) in methanol (20 ml)

was reduced with hydrogen and palladium on barium sulfate (5%) for

5.5 h at room temperature and pressure. The mixture was filtered

through Celite and allowed to stand overnight in air. The red mixture

was concentrated under reduced pressure and preparatively chro-

matographed on oxalated silica layers with toluene/ethyl acetate (9: 1)

as eluent. The red band with R_F0.35 gave 5,7-bisdeoxydaunorubici-

none (3) as a red solid (11 mg, 88%). It formed red needles from

acetone, m.p. 231–233° (lit.¹⁰229–231°) (Found: C, 69.1; H, 5.3. Calc.

for C₂₁H₁₈O₆: C, 68.8; H, 5.0%). λ_max(logε) 275 (4.16), 280 (4.15), 307

(3.56), 478 nm (3.96). ν_max3504, 1700, 1654, 1632, 1602 cm^–1. δ 13.77,

s, OH; 8.50, br s, H5; 8.00, br d, J 8 Hz, H1; 7.59, t, J 8 Hz, H2; 7.06,

br d, J 8 Hz, H3; 4.04, s, OMe; 3.08–2.77, m, 7-CH₂, 10-CH₂; 2.38, s,

COMe; 1.98–1.86, m, 8-CH₂. m/z 366 (M, 15%), 323 (48), 305 (30),

295 (21).

The band with R_F0.1 gave 7,12-bisdeoxydaunorubicinone (4) as a

red solid (1 mg, 8%). δ 15.12, s, OH; 8.02, s, H12; 7.62, t, J 8 Hz, H2;

7.51, br d, J 8 Hz, H1; 7.07, br d, J 8 Hz, H3; 4.08, s, OMe; 3.10–2.74,

m, 7-CH₂, 10-CH₂; 2.23, s, COMe; 1.96–1.90, m, 8-CH₂. m/z 366 (M,

100%), 323 (45), 279 (37).

5-Deoxydaunorubicinone (10) and 5-Deoxy-13-

dihydrodaunorubicinone (11) via the Phenylboronate (9)

Experimental

The hydrochloride of daunorubicin (1) (25 mg) was heated in 0.2 ^M

hydrochloric acid (5 ml) at 90° for 1 h. On cooling, the orange precipi-

tate was filtered off and dried to give daunorubicinone (6) (16.5 mg,

93%), red needles from toluene, m.p. 221–225° (lit.¹⁰213–214°). This

product (16.5 mg), phenylboronic acid (6 mg) and p-toluenesulfonic

acid (1 mg) were stirred together in dichloromethane (5 ml) for 43 h at

room temperature. The mixture was washed with saturated aqueous

sodium hydrogen carbonate and water, then dried. Evaporation under

reduced pressure gave daunorubicinone phenylboronate (9) as an

orange solid (20 mg, 100%), clusters of red needles from ethyl

acetate/petrol, m.p. 257–259° (Found: C, 66.5; H, 4.8. C₂₇H₂₁BO₈

requires C, 67.0; H, 4.4%). λ_max(logε) 250 (4.39), 288 (3.87), 476

(4.04), 492 (4.05), 530 nm (3.78). ν_max1720, 1618, 1578 cm^–1. δ 13.87,

13.20, s, s, 2×OH; 8.02, dd, J 1, 7.5 Hz, H1; 7.82, dd, J 1, 7.5 Hz, H 2´,

H6´; 7.76, dd, J 7.5, 8.5 Hz, H 2; 7.44–7.30, m, H3, H3´, H4´, H 5´;

5.83, br t, J 2.5 Hz, H 7; 4.08, s, OMe; 3.34, dd, J 2, 19 Hz, H 10eq; 3.24,

d, J 19 Hz, H10ax; 2.57, s, COMe; 2.35, ddd, J 1.5, 2, 14 Hz, H8eq;

2.27, dd, J 2.5, 14 Hz, H 8ax. m/z 485 (M+H, 30%), 484 (M, 100), 483

(22), 407 (68).

General

Melting points were determined on a Kofler hot stage and are uncor-

rected. Microanalyses were carried out by Chemical and

Microanalytical Services, Geelong, or National Analytical

Laboratories, Melbourne. Electronic spectra were recorded in methanol

containing 1% formic acid (v/v) unless otherwise stated, by using a

Varian Superscan 3 spectrophotometer. Infrared spectra were recorded

as potassium bromide disks by using a Perkin–Elmer 983 G spec-

trophotometer. Proton nuclear magnetic resonance (¹H n.m.r.) spectra

were recorded at 399.65 MHz with a JEOL JNM-GX400 and a Varian

Unity plus 400 system. The solvent was (D)chloroform for water-insol-

uble compounds. Glycosides were run in (D₄)methanol/(D)chloroform

mixtures unless otherwise stated. High- and low-resolution mass

spectra of aglycones were recorded by using a V.G. Micromass 7070F

instrument or a JEOL JMS-AX505H mass spectrometer at 70 ev unless

otherwise stated. F.a.b. mass spectra of glycosides were recorded on the

latter instrument with thioglycerol as the matrix. Electrospray mass

spectra were recorded in acetonitrile/water mixture acidified with

Analogues of Daunorubicin and Doxorubicin

35

Crude phenylboronate (9) (19.5 mg) was hydrogenated over Adams

catalyst (10 mg) in ethyl acetate (5 ml) for 40 min. The mixture was then

filtered and the filtrate was evaporated to a red solid, which was dis-

solved in dichloromethane (2 ml) and stirred with pentane-2,4-diol (0.2

ml) and acetic acid (0.1 ml) at room temperature for 64 h. The red

mixture was then washed with water, dried and evaporated under

reduced pressure. The residue was preparatively chromatographed on

oxalated silica layers in toluene/ethyl acetate (7: 3). The red band with

R_F0.4 gave 5-deoxydaunorubicinone (10) (7 mg, 45%), red needles

from acetone, m.p. 289–291° (Found: C, 65.8; H, 4.9. C₂₁H₁₈O₇

requires C, 66.0; H, 4.7%). λ_max(logε) 268 (4.06), 296 (3.72), 306

(3.52), 483 nm (3.91). ν_max3454, 1712, 1654, 1633, 1603 cm^–1. δ 13.74,

s, OH; 8.54, s, H 5; 8.04, d, J 8 Hz, H1; 7.63, t, J 8 Hz, H 2; 7.09, d, J

8 Hz, H3; 5.19, d, J 4.5 Hz, H7; 4.50, br s, OH; 4.05, s, OMe; 3.65, br s,

OH; 3.08, dd, J 2, 19.5 Hz, H 10eq; 2.85, dd, J 1, 19.5 Hz, H 10ax; 2.43,

s, COMe; 2.30, td, J 1, 14.5 Hz, H 8eq; 2.08, dd, J 4.5, 14.5 Hz, H8ax.

m/z 382 (M, 58%).

The red band with R_F0.1 gave mixed diastereoisomers of 5-deoxy-

13-dihydrodaunorubicinone (11) (7 mg, 45%), red needles from

acetone, m.p. 221–224° (Found: M^+•, 384.1209. C₂₁H₂₀O₇requires M^+•,

384.1209). δ 13.91, s, OH (minor); 13.90, s, OH (major); 8.63, br s, H5;

8.07, d, J 8 Hz, H1; 7.65, t, J 8 Hz, H 2; 7.12, d, J 8 Hz, H 3; 5.22, m,

H7; 3.88, q, J 6.5 Hz, H 13 (major); 3.75, q, J 6.5 Hz, H 13 (minor);

3.10, dd, J 2, 20 Hz, H 10eq (minor); 3.09, dd, J 2, 20 Hz, H 10eq

(major); 2.56, dd, J 1, 20 Hz, H 10ax (major); 2.52, td, J 1, 15 Hz, H 8eq

(minor); 2.46, dd, J 1, 20 Hz, H 10ax (minor); 2.37, td, J 2, 15 Hz, H8eq

(major); 1.83, dd, J 5, 15 Hz, H 8ax (major); 1.76, dd, J 5, 15 Hz, H8ax

(minor); 1.35, d, J 6.5 Hz, 14-Me (minor); 1.30, d, J 6.5 Hz, 14-Me

(major). m/z 384 (M, 16%), 369 (20), 368 (68), 366 (34), 358 (20), 356

(34), 348 (100).

stirred in an ice bath. Extraction with dichloromethane (3×50 ml) and

washing of the organic phase with brine, then drying and evaporation

under reduced pressure gave a dark red solid. Separation of the products

on a column of silica gel (8×3.5 cm), with chloroform/methanol/water

as eluent [first with 100 : 20: 1, then with 80: 20: 1], gave two red

bands.

The faster moving, major band on evaporation gave 5-deoxy-

daunorubicin (12) as a dark red solid. Addition of methanolic hydrogen

chloride (1 equiv.) to a chloroform solution, followed by ether gave (12)

as its hydrochloride (26 mg, 57%), m.p. 189–191° (Found: C, 53.6; H,

6.1; N, 2.6. C₂₇H₃₀ClNO₉·3H₂O requires C, 53.9; H, 6.0; N, 2.3%). λ_max

(logε) 273sh (4.16), 295sh (3.58), 325 (3.58), 480 nm (3.93). ν_max3422,

1710, 1654, 1636, 1608 cm^–1. δ [(D₄)methanol] 8.40, s, H5; 7.94, d, J

8 Hz, H 1; 7.62, t, J 8 Hz, H 2; 7.21, d, J 8 Hz, H 3; 5.45, d, J 3.5 Hz,

H1′; 4.96, m, H7; 4.21, q, J 7 Hz, H5′; 4.02, s, OMe; 3.59, br d, J 3 Hz,

H4′; 3.53, ddd, J 3, 5, 13 Hz, H 3′; 2.92, 2.88, ABq, J 18 Hz, 10-CH₂;

2.30, s, COMe; 2.19, dd, J 3, 15 Hz, H8eq; 2.08, dd, J 5.5, 15 Hz,

H8ax; 1.99, dt, J 3.5, 13 Hz, H2′ax; 1.76, dd, J 5, 13 Hz, H2′eq; 1.23,

d, J 7 Hz, Me′. m/z (f.a.b.) 534 (M+Na, 3%), 512 (M+H, 3), 383 (20),

347 (50), 321 (40), 305 (75).

The slower moving band on evaporation gave mixed diastereoiso-

mers of 5-deoxy-13-dihydrodaunorubicin (13) as a dark red solid. As

for (12), the hydrochloride (11.5 mg) (25%) was obtained as a solvate,

m.p. 200–205° (dec.), but because of its higher polarity it retained inor-

ganic contaminant that persisted through crystallization and invariably

led to residue on combustion analysis. It was characterized as its agly-

cone (11), described earlier. λ_max(logε) 270sh (4.14), 295sh (3.86), 320

(3.82), 480 nm (3.80). ν_max3424, 1654, 1636, 1608 cm^–1. δ

[(D₄)methanol] 8.39, s, H 5; 7.96, d, J 8 Hz, H1; 7.65, t, J 8 Hz, H2;

7.23, d, J 8 Hz, H3; 5.51, br s, H1′; 4.99, br s, H7; 4.25, q, J 5.5 Hz,

H5′ (minor); 4.23, q, J 5.5 Hz, H5′ (major); 4.05, s, OMe; 3.67, m,

H13; 3.64, br d, J 3 Hz, H4′; 3.54, ddd, J 3, 5, 13 Hz, H 3′; 2.97, d, J

19 Hz, H10eq (major); 2.88, d, J 19 Hz, H10eq (minor); 2.62, d, J 19

Hz, H10ax; 2.37, br d, J 13.5 Hz, H 8eq (minor); 2.07, m, H8ax

(major), H8eq (major); 2.02, dt, J 3.5, 13 Hz, H 2′ax; 1.85, dd, J 5.5, 13

Hz, H8ax (minor); 1.78, dd, J 5, 13 Hz, H 2′eq; 1.29, m, 14-Me, Me′.

m/z (f.a.b) 536 (M+Na, 8%), 514 (M+H, 6), 367 (30), 305 (22).

Chromatographic analysis of the non-glycosidic fraction (5.3 mg,

16%), obtained above by dichloromethane extraction of the hydrogena-

tion mixture, showed it to consist chiefly of (3), (7) and a new compo-

nent. This new product was isolated by p.l.c. on oxalated silica in

toluene/ethyl acetate (4: 1). The red band, R_F0.34, gave 2-acetyl-

2,6,11-trihydroxy-1,2,3,4,7,8,9,10-octahydronaphthacene-5,12-dione

(14) (0.9 mg), which formed red needles from acetone, m.p. 290–292°

(Found: M^+•, 356.1262. C₂₀H₂₀O₆requires M^+•, 356.1260). λ_max(logε)

290 (3.84), 472 (3.78), 504 (3.87), 543 nm (3.26). ν_max3428, 1708,

1652 cm^–1. δ 13.19, 13.15, s, s, 2×OH (D₂O-exchanged); 3.82, br s, 2-

OH (D₂O-exchanged); 3.05, ddd, J 3, 5, 20 Hz, H 4eq; 2.96, ddd, J 2,

3, 20 Hz, H 1eq; 2.88–2.81, m, H 4ax; 2.83, br d, J 20 Hz, H1ax; 2.66,

br s, 7-CH₂, 10-CH₂; 2.37, s, COMe; 2.01–1.88, m, 3-CH₂; 1.78, br s,

8-CH₂, 9-CH₂. m/z 356 (M, 19%), 313 (100).

Hydrogenation of Daunorubicinone (6)

Daunorubicinone (6) (5 mg) in methanol (2 ml) containing

chloroacetic acid (20 mg) was hydrogenated over Adams catalyst (2.5

mg) for 55 min. The yellow mixture was filtered, then the filtrate was

diluted with methanol (5 ml), and saturated aqueous sodium hydrogen

carbonate solution was added dropwise to give a purple suspension.

This was stirred in air for 15 min. Water was then added and the mixture

was extracted with dichloromethane. The extract was washed with

water, dried and concentrated under reduced pressure. The residue was

fractionated by p.l.c. on oxalated silica in chloroform/methanol (19: 1)

to give major and minor red bands. The major band, R_F0.23, gave

mixed diastereoisomers of 5,7-bisdeoxy-13-dihydrodaunorubicinone

(7) as a red solid (1.4 mg). It formed red needles from acetone, m.p.

205–210° (Found: M^+•, 368.1260. C₂₁H₂₀O₆requires M^+•, 368.1260).

λ_max(logε) 275 (4.13), 308sh (3.44), 470 nm (3.83). ν_max3426, 1654,

1629, 1604 cm^–1. δ 13.88, s, OH; 8.54, d, J 1 Hz, H 5; 7.99, br d, J 8 Hz,

H1; 7.58, t, J 8 Hz, H 2; 7.05, br d, J 8 Hz, H 3; 4.03, s, OMe; 3.85, q,

J 6.5 Hz, H 13 (isomerA); 3.77, q, J 6.5 Hz, H 13 (isomer B); 3.00–2.50,

m, 7-CH₂, 10-CH₂; 2.10–1.90, m, 8-CH₂; 1.34, d, J 6.5 Hz, COMe

(isomer A); 1.31, d, J 6.5 Hz, COMe (isomer B). m/z 368 (M, 100%),

342 (20), 324 (42), 323 (92), 322 (22).

The minor band, R_F0.54, gave 5,7-bisdeoxydaunorubicinone (3)

(0.4 mg), which co-chromatographed with the product described

earlier. A trace of the 5,12-bisdeoxy isomer (4) was also observed by

analytical t.l.c. but not isolated. Carrying out the hydrogenation in ethyl

acetate chromatographically showed formation of the same three prod-

ucts, compound (3) being the major one.

Method B

A solution of the hydrochloride of daunorubicin (1) (10 mg) in

propane-1,3-diol (200 mg) containing trimethyl orthoformate (0.1 ml)

and camphorsulfonic acid (1 mg) was allowed to stand for 16 h at room

temperature. It was then diluted with water, basified with solid sodium

hydrogen carbonate and extracted with chloroform, and the extract was

washed with water, dried and concentrated to give impure ketal (43) as

an oil. This was dissolved in methanol (4 ml) containing chloroacetic

acid (40 mg) and hydrogenated as in A. After filtration, the resulting

solution was then stirred in air in an ice bath, with addition of an excess

of aqueous sodium hydrogen carbonate. The purple mixture was

extracted into chloroform and the extract was then re-extracted into

0.1 ^Mhydrochloric acid. This red acidic solution was left standing at

room temperature for 48 h, then washed with dichloromethane, basified

with solid sodium hydrogen carbonate and finally extracted into chlo-

roform. The red extract was washed with a little water, dried and con-

centrated. Filtration, addition of methanolic hydrogen chloride (1

5-Deoxydaunorubicin (12) and

5-Deoxy-13-dihydrodaunorubicin (13)

Method A

A rapidly stirred solution of the hydrochloride of daunorubicin (1)

(50 mg) in methanol (20 ml) containing chloroacetic acid (200 mg) was

hydrogenated over Adams catalyst (10 mg) for 50 min at room temper-

ature. The yellow mixture was filtered, and the filtrate was diluted with

water (50 ml) and washed with dichloromethane to remove non-glyco-

sidic materials. The aqueous fraction was then brought to pH 8.5 by

addition of saturated aqueous sodium hydrogen carbonate while being

36

D. W. Cameron, G. I. Feutrill and P. G. Griffiths

equiv.) to the filtrate and precipitation with an excess of ether gave the

hydrochloride of (12) as a red solid (5.6 mg) (62%), identical with the

product from A.

13 Hz, H3′; 3.11, dd, J 1, 20 Hz, H10eq; 2.93, dd, J 1, 20 Hz, H10ax;

2.28, dt, J 1, 15 Hz, H 8eq; 2.13, dd, J 5, 15 Hz, H8ax; 2.03, dt, J 3.5,

13 Hz, H 2′ax; 1.83, dd, J 5, 13 Hz, H2′eq; 1.28, d, J 7 Hz, Me′. m/z

(f.a.b.) 550 (M+Na, 5%), 528 (M+H, 2).

The slower moving band on evaporation and treatment with hydro-

gen chloride, as for (15), gave mixed diastereoisomers of 5-deoxy-13-

dihydrodoxorubicin (16) as its hydrochloride (9.6 mg) (20%), identical

with the product from the previous section.

5-Deoxy-13-dihydrodaunorubicin (13)

A solution of daunorubicin hydrochloride (1) (10.4 mg) in methanol

(6 ml) was hydrogenated over Adams catalyst (8.7 mg) for 70 min at

room temperature. The mixture was filtered, and the filtrate was diluted

with water (10 ml) and washed with dichloromethane. The aqueous

phase was brought to pH 8.5 by addition of saturated aqueous sodium

hydrogen carbonate, and then extracted with dichloromethane. The

extract was washed with brine, dried and evaporated, to give 5-deoxy-

13-dihydrodaunorubicin (13) as a dark red solid (6.7 mg, 71%), identi-

cal with the product from the previous section.

Non-Glycosidic Derivatives of 5-Deoxydoxorubicin

Doxorubicin hydrochloride (50 mg) was hydrogenated as in the pre-

vious section but the crude chloroform extract was partitioned rather

than chromatographed. Extraction with 0.2 ^Mhydrochloric acid gave

aqueous glycosidic and organic non-glycosidic fractions.

The latter fraction gave a red solid (13 mg). Analytical t.l.c. showed

it to consist of a major component and several minor ones. The major

component was isolated by p.l.c. on oxalated silica in toluene/acetone

(7: 3). The band with R_F0.53 was consistent with 5,7-bisdeoxydoxoru-

bicinone (17) as an incompletely characterized red solid (5 mg, 15%),

red microneedles from acetone (Found: M^+•, 382.1052. Calc. for

C₂₁H₁₈O₇: M^+•, 382.1052). ν_max3427, 1717, 1649, 1629 cm^–1. δ 13.65,

s, OH; 8.46, s, H 5; 7.95, d, J 8 Hz, H 1; 7.59, t, J 8 Hz, H 2; 7.06, d, J

8 Hz, H3; 4.74, 4.70, ABq, J 21 Hz, 14-CH₂; 4.04, s, OMe; 3.00–1.50,

m, 7-CH₂, 8-CH₂, 10-CH₂. m/z 382 (M, 54%), 323 (100).

The acidic glycoside fraction was heated on a steam bath for 1 h and

then cooled. The precipitated solid was filtered off, and then subjected

to preparative chromatography on oxalated silica in toluene/acetone

(7: 3). The red band, R_F0.53, gave 5-deoxydoxorubicinone (18) (10 mg,

28%), red needles from acetone, m.p. 188–190° (Found: M^+•,

398.1002. C₂₁H₁₈O₈requires M^+•, 398.1002). λ_max(logε) 270sh (4.13),

306sh (3.60), 482 nm (3.97). ν_max3420, 1724, 1654, 1636, 1604 cm^–1.

δ 13.80, s, OH; 8.59, s, H5; 8.04, d, J 8 Hz, H1; 7.64, t, J 8 Hz, H2;

7.10, d, J 8 Hz, H3; 5.20, br d, J 5 Hz, H7; 4.81, 4.76, ABq, J 21 Hz,

14-CH₂; 4.05, s, OMe; 3.16, dd, J 2.5, 20 Hz, H 10eq; 2.90, dd, J 1,

20 Hz, H 10ax; 2.35, td, J 1.5, 15 Hz, H8eq; 2.09, dd, J 5, 15 Hz, H8ax.

m/z 398 (M, 9%), 362 (65), 381 (100), 303 (71).

The red band with R_F0.18 gave mixed diastereoisomers of 5-deoxy-

13-dihydrodoxorubicinone (19) (5 mg, 15%), red needles from

methanol/acetone, m.p. 214–217° (Found: M^+•, 400.1158. C₂₁H₂₀O₈

requires M^+•, 400.1158). λ_max(logε) 268sh (3.99), 307sh (3.48),

482 nm (3.86). ν_max3417, 1653, 1635, 1602 cm^–1. δ 8.59, s, H5; 8.03,

br d, J 8 Hz, H1; 7.64, t, J 8 Hz, H2; 7.13, br d, J 8 Hz, H3; 5.14, br d,

J 5 Hz, H7; 4.06, s, OMe; 3.94, dd, J 4, 11.5 Hz, H_A14 (minor); 3.92,

dd, J 4, 11.5 Hz, H_A14 (major); 3.81, dd, J 7, 11.5 Hz, H_B14 (minor);

3.78, dd, J 7, 11.5 Hz, H_B14 (major); 3.63, dd, J 4, 7 Hz, H13 (major);

3.57, dd, J 4, 7 Hz, H13 (minor); 3.09, dd, J 2, 20 Hz, H10eq (major);

3.05, dd, J 2, 20 Hz, H10eq (minor); 2.73, dd, J 1, 20 Hz, H10ax

(minor); 2.72, dd, J 1, 20 Hz, H10ax (major); 2.42, br td, J 2, 15 Hz,

H8eq (minor); 2.22, br td, J 2, 15 Hz, H8eq (major); 1.98, dd, J 5,

15 Hz, H8ax (major); 1.82, dd, J 5, 15 Hz, H8ax (minor). m/z 400 (M,

5%), 382 (24), 364 (74), 333 (100).

5-Deoxy-13-dihydrodoxorubicin (16)

Doxorubicin hydrochloride (100 mg) was hydrogenated in

methanol (40 ml) over Adams catalyst (100 mg) for 90 min at room

temperature. The yellow solution was then filtered through a Celite pad

and the filtrate was cooled in an ice-bath and then stirred with saturated

aqueous sodium hydrogen carbonate (15 drops) for 10 min to give a

dark red mixture. Brine (20 ml) was added and the mixture was

extracted four times with chloroform. The combined organic layers

were washed with a little brine and then dried over anhydrous sodium

sulfate. Evaporation of the filtered extract under reduced pressure

below 30° gave

a dark red solid which was dissolved in

chloroform/methanol, filtered, and concentrated to c. 1 ml in a cen-

trifuge tube. The tube was chilled at –10° and then methanolic hydro-

gen chloride (1 equiv.) was added followed by an excess of diethyl

ether. Centrifugation gave a dark red solid which was redissolved in

methanol and precipitated again with diethyl ether to give a pair of

diastereoisomers of 5-deoxy-13-dihydrodoxorubicin (16) as the

hydrochloride (74 mg, 76%), m.p. 195–200° (dec.) (Found: C, 57.0; H,

5.7. C₂₇H₃₂ClNO₁₀requires C, 57.3; H, 5.7%). λ_max(logε) 270sh (4.07),

308sh (3.54), 480 nm (3.84). ν_max3416, 1654, 1636, 1604 cm^–1. δ 8.52,

s, H5 (minor); 8.51, s, H 5 (major); 8.02, d, J 8 Hz, H 1 (minor); 8.01,

d, J 8 Hz, H1 (major); 7.64, t, J 8 Hz, H 2; 7.03, d, J 8 Hz, H 3; 5.57, br

d, J 3 Hz, H1′; 5.09, br s, H7; 4.21, q, J 6.5 Hz, H 5′ (minor); 4.19, q,

J 6.5 Hz, H 5′ (major); 4.05, s, OMe; 3.94, dd, J 3.5, 12 Hz, H_A14; 3.78,

m, H_B14; 3.74, br d, J 3 Hz, H 4′; 3.60, m, H 13; 3.50, ddd, J 3, 5, 13

Hz, H 3′; 3.06, d, J 20 Hz, H 10eq (major); 3.04, d, J 20 Hz, H10eq

(minor); 2.72, d, J 20 Hz, H 10ax (major); 2.70, d, J 20 Hz, H 10ax

(minor); 2.48, br d, J 15 Hz, H8eq (minor); 2.21, br d, J 15 Hz, H8eq

(major); 2.07, dd, J 5, 15 Hz, H8ax (major); 2.03, dt, J 3.5, 13 Hz,

H2′ax; 1.87, br dd, J 5, 15 Hz, H 8ax (minor); 1.82, br dd, J 5, 13 Hz,

H2′eq; 1.29, br d, J 6.5 Hz, Me′. m/z (f.a.b.) 552 (M+Na, 2%), 530

(M+H, 1).

5-Deoxydoxorubicin (15) and 5-Deoxy-13-dihydrodoxorubicin (16)

A solution of the hydrochloride of doxorubicin (2) (50 mg) in

methanol (20 ml) and chloroacetic acid (200 mg) was hydrogenated by

rapidly stirring over Adams catalyst (5 mg) for 50 min at room temper-

ature. The yellow solution was filtered and the filtrate was then brought

to pH 8.5 by addition of saturated aqueous sodium hydrogen carbonate

while being stirred in an ice bath. Extraction with chloroform (4×25 ml)

and washing of the organic phase with brine, then drying and evapora-

tion under reduced pressure gave a dark red solid. Separation of the

products on a column of silica gel, with chloroform/methanol/water (ini-

tially 80 : 20 : 0.5 and then 75 :25 :0.5) as eluent, gave two red bands.

The faster moving, major band on evaporation gave a dark red solid

which was redissolved in chloroform and treated with methanolic

hydrogen chloride (1 equiv.) followed by ether to give 5-deoxydoxo-

rubicin (15) as its hydrochloride (29 mg) (59%), m.p. >200° (dec.)

(Found: C, 54.1; H, 6.4; N, 1.9. C₂₇H₃₀ClNO₁₀·3.5MeOH requires C,

54.2; H, 6.5; N, 2.1%). λ_max(logε) 272 (4.22), 310 (3.69), 480 nm

(3.99). ν_max3416, 1724, 1654, 1636, 1604 cm^–1. δ 8.53, s, H 5; 8.02, d,

J 8.5 Hz, H 1; 7.65, t, J 8.5 Hz, H 2; 7.15, d, J 8.5 Hz, H 3; 5.56, d, J 3.5

Hz, H 1′; 5.10, m, H7; 4.78, 4.74, ABq, J 20 Hz, 14-CH₂; 4.18, q, J

7 Hz, H 5′; 4.06, s, OMe; 3.75, br d, J 3 Hz, H 4′; 3.53, ddd, J 3, 5,

Ketals (20) and (21)

The hydrochloride of daunorubicin (1) (27 mg) was hydrogenated

overAdams catalyst (25 mg) in methanol as described earlier for prepa-

ration of glycoside (13). Workup led to non-glycosidic and glycosidic

fractions. The former (4.5 mg, 25%) contained largely 5,7-bisdeoxy-

13-dihydrodaunorubicinone (7) by t.l.c. The glycosidic fraction was

hydrolysed in 0.2 ^Mhydrochloric acid for 1 h at 90°. Filtration then gave

largely 5-deoxy-13-dihydrodaunorubicinone (11) as

a red solid

(10.6 mg, 57%). This mixture of diastereoisomers was boiled with 2,2-

dimethoxypropane (0.4 ml) and camphorsulfonic acid (0.5 mg) in

dioxan (2 ml) for 15 min. The mixture was then cooled, diluted with

water and extracted with dichloromethane. The extract was washed

with water, dried and evaporated to a red solid. This was subjected to

p.l.c. on silica gel in toluene/ethyl acetate (70: 30). The major band, R_F

0.58, gave the (13R)-ketal (20) as a red solid (5.5 mg). δ 13.80, s, OH;

8.59, s, H 5; 8.00, d, J 8 Hz, H1; 7.59, t, J 8 Hz, H2; 7.06, d, J 8 Hz,

H3; 5.15, d, J 4 Hz, H 7; 4.19, q, J 6.5 Hz, H 13; 4.03, s, OMe; 2.99, dd,

Analogues of Daunorubicin and Doxorubicin

37

J 1.5, 19.5 Hz, H 10eq; 2.53, d, J 19.5 Hz, H 10ax; 2.36, br d, J 14.5 Hz,

H8eq; 1.61, dd, J 5, 14.5 Hz, H 8ax; 1.54, 1.39, s, s, CMe₂; 1.31, d, J

6.5 Hz, 14-Me.

The minor band, R_F0.50, gave the (13S)-ketal (21) as a red solid

(2.6 mg). δ 13.84, s, OH; 8.60, s, H 5; 8.02, d, J 8 Hz, H 1; 7.60, t, J

8 Hz, H2; 7.07, d, J 8 Hz, H 3; 5.11, br s, H7; 4.13, q, J 6.5 Hz, H13;

4.03, s, OMe; 3.02, dd, J 1, 19 Hz, H10eq; 2.49, d, J 19 Hz, H10ax;

2.18, ddd, J 1, 2, 14 Hz, H 8eq; 1.96, dd, J 5, 14 Hz, H 8ax; 1.45, 1.42,

s, s, CMe₂; 1.33, d, J 6.5 Hz, 14-Me.

off and dried to give carminomycinone (32) as a red solid (24 mg). It

formed red needles from toluene, m.p. 225–228° (subl.) (lit.²³

233–235°). The same product was obtained alternatively by stirring

daunorubicinone (6) (33 mg) in dichloromethane (20 ml) with an excess

of powdered anhydrous aluminium chloride for 16 h at room tempera-

ture under nitrogen. The purple mixture was then stirred with saturated

aqueous oxalic acid solution for 30 min. Extraction with

dichloromethane gave carminomycinone (32) as a red solid (32 mg).

Without purification this solid (24 mg) in dichloromethane (10 ml) was

stirred with phenylboronic acid (9 mg) and p-toluenesulfonic acid (1 mg)

for 16 h at room temperature. The mixture was diluted with more

dichloromethane and washed with saturated aqueous sodium hydrogen

carbonate solution then water. Drying and evaporation gave carmino-

mycinone phenylboronate (33) (29 mg, 98%), orange needles from ethyl

acetate, m.p. 271–274° (Found: C, 66.2; H, 4.4. C₂₆H₁₉BO₈requires C,

66.4; H, 4.1%). λ_max(logε) 242 (4.25), 290 (3.63), 480sh (3.94), 490

(3.99), 506sh (3.85), 526 nm (3.80). ν_max1723, 1600 cm^–1. δ 13.35,

12.86, 12.16, s, s, s, 3×OH; 7.86, dd, J 1, 7.5 Hz, H1; 7.81, m, H 2′, H 6′;

7.68, dd, J 7.5, 8.5 Hz, H2; 7.43, m, H4′; 7.35−7.31, m, Η3, Η3′, H 5′;

5.82, dd, J 2.5, 3.0 Hz, H7; 3.37, dd, J 1.5, 19 Hz, H10eq; 3.27, d, J 19

Hz, H10ax; 2.55, s, COMe; 2.37, ddd, J 1.5, 3, 14 Hz, H8eq; 2.28, dd,

J 2.5, 14 Hz, H8ax. m/z 470 (M, 54%), 393 (25), 348 (84).

The crude phenylboronate (33) was dissolved in ethyl acetate

(15 ml) by warming. Adams catalyst (6 mg) and dichloroacetic acid

(120 mg) were added and the mixture was hydrogenated for 2 h at room

temperature. The filtrate was concentrated under reduced pressure. The

residue was dissolved in tetrahydrofuran, and then saturated aqueous

sodium hydrogen carbonate solution was added dropwise with stirring

until a purple colour was established. The mixture was stirred for 5 min

longer, then it was diluted with water and neutralized dropwise with

98% formic acid. It was extracted with ethyl acetate and the extract was

washed with water, dried and evaporated to give 12-deoxycarminomy-

cinone (30) as its phenylboronate (28 mg, 98%), which gave dark red

clusters of microneedles from ethyl acetate/petrol, m.p. 222–224°. m/z

454 (M, 16%), 332 (100). This product was treated with pentane-2,4-

diol and acetic acid in chloroform analogously to (9), to give 12-deoxy-

carminomycinone (30), dark red needles from ethyl acetate, m.p.

191–193° (Found: M^+•, 368.0896. C₂₀H₁₆O₇requires M^+•, 368.0896).

λ_max(logε) 262sh (4.24), 296sh (3.67), 310sh (3.49), 335 (3.37), 500

(3.90), 526 nm (3.87). ν_max3420, 1711, 1654, 1629, 1612, 1586 cm^–1. δ

15.95, s, OH (D₂O-exchanged); 9.79, s, OH (D₂O-exchanged); 8.03, s,

H12; 7.64, t, J 8 Hz, H 2; 7.46, d, J 8 Hz, H 1; 7.12, d, J 8 Hz, H3; 5.21,

br d, J 5 Hz, H7; 3.05, dd, J 1, 19.5 Hz, H10eq; 2.83, br d, J 19.5 Hz,

H10ax; 2.41, s, COMe; 2.31 td, J 1, 14.5 Hz, H 8eq; 2.14, dd, J 5,

14.5 Hz, H 8ax. m/z 368 (M, 13%), 332 (86). This product (30) was also

obtained by heating the glycoside (29) in 0.2 ^Mhydrochloric acid, as for

the foregoing hydrolysis of carminomycin (25).

Hydrogenation of Carminomycin (25)

Method A

The hydrochloride of carminomycin (25) (50 mg) in methanol

(20 ml) and dichloroacetic acid (200 mg) was hydrogenated while

rapidly being stirred over Adams catalyst (8 mg) for 35 min at room

temperature. The yellow filtrate was stirred in air at 0° with addition of

saturated aqueous sodium hydrogen carbonate to give a purple suspen-

sion. This was extracted with ethyl acetate and the extract was washed

with brine, dried and concentrated. The purple residue was column

chromatographed on flash silica gel in chloroform/methanol/water

(70 : 25 : 3). The major blue-purple band was recovered by evaporation,

redissolving in chloroform/methanol and precipitation with ether

several times to give 12-deoxycarminomycin (29) as a purple solid

(26.8 mg, 59%). λ_max(logε) 495 (3.65), 525 nm (3.57). λ_max(MeOH,

qual., as the purple zwitterion) 526, 550sh nm. ν_max3410, 1718, 1653,

1636 cm^–1. δ 8.08, s, H 12; 7.66, t, J 8 Hz, H 2; 7.50, d, J 8 Hz, H1; 7.13,

m, H3; 5.55, br s, J 1 Hz, H 1′; 5.11, br s, H 7; 4.23, q, J 6.5 Hz, H 5′;

3.79, br d, J 3 Hz, H 4′; 3.58, br d, J 13 Hz, H 3′; 3.06, d, J 19.5 Hz,

H10eq; 2.87, d, J 19.5 Hz, H 10ax; 2.42, s, COMe; 2.27, d, J 15 Hz,

H8eq; 2.12, dd, J 5, 15 Hz, H 8ax; 2.05, dt, J 3, 13 Hz, H 2′ax; 1.88, dd,

J 4, 13 Hz, H 2′eq; 1.31, d, J 6.5 Hz, Me′. m/z (electrospray) 499

(M+2H, 45%), 498 (M+H, 100), 369 (20).

A very polar minor purple band, difficult to elute and to obtain free

from inorganic contaminant, was presumed to be the 13-dihydro

species (26), as its zwitterion. On being heated with 0.2 ^Mhydrochloric

acid it gave an aglycone chromatographically indistinguishable from a

minor product of hydrogenation of the phenylboronate (33). This

product was inferred to be the 12-deoxy 13-dihydro compound (31).

Method B

Another hydrogenation of carminomycin hydrochloride (50 mg) but

with added chloroacetic acid (200 mg) in place of dichloroacetic acid

gave the same two glycosides together with a larger proportion of non-

glycosidic material. The latter material was isolated as the early fraction

eluted from a column of flash silica gel with chloroform/methanol/water

(70 :25 :3), as a dark red solid (15 mg, 46%). Two components were sep-

arated from this solid by p.l.c. on oxalated silica in toluene/ethyl acetate

(70 :30). The component with R_F0.40 gave 7,12-bisdeoxycarminomyci-

none (27), red needles from acetone, m.p. dec. above 230° (Found: M^+•,

352.0926. C₂₀H₁₆O₆requires M^+•, 352.0947). λ_max(logε) 276sh (3.85),

335 (3.32), 500sh (3.77), 525 nm (3.82). ν_max3490, 1690, 1650, 1625

cm^–1. δ 16.14, s, OH; 9.92, s, OH; 8.02, s, H 12; 7.61, t, J 8 Hz, H2; 7.44,

d, J 8 Hz, H1; 7.09, d, J 8 Hz, H 3; 3.03–2.79, m, 7-CH₂, 10-CH₂; 2.37,

s, COMe; 2.00–1.80, m, 8-CH₂. m/z 352 (M, 55%), 334 (52), 325 (25),

309 (100).

The component with R_F0.13 gave 7,12-bisdeoxy-13-dihydrocarmi-

nomycinone (28), red needles from acetone, m.p. 200–205° (dec.)

(Found: M^+•, 354.1082. C₂₀H₁₈O₆requires M^+•, 354.1103). λ_max(logε)

274sh (3.90), 282sh (3.89), 340 (3.31), 494 (3.94), 520 nm (3.94). ν_max

3400, 1650, 1620, 1610, 1580 cm^–1. δ 16.10 (major), 16.08 (minor), s,

s, OH, OH; 9.89, br s, OH; 7.94 (major), 7.93 (minor), s, s, H12; 7.59,

t, J 8 Hz, H2; 7.40, d, J 8 Hz, H 1; 7.07, d, J 8 Hz, H 3; 3.84, q, J 6.5 Hz,

H13 (minor); 3.66, q, J 6.5 Hz, H 13 (major); 2.96–2.46, m, 7-CH₂, 10-

CH₂; 2.16–2.00, m, 8-CH₂; 1.33, d, J 6.5 Hz, Me′ (major); 1.30, d, J

6.5 Hz, Me′ (minor). m/z 355 (M+H, 26%), 354 (M, 100), 309 (63).

12-Deoxy-13-dihydrodaunorubicin (40)

To a solution of the hydrochloride of daunorubicin (1) (50 mg) in

methanol (40 ml) was added 0.1 m aqueous phosphate buffer (pH 7, 2

ml) and nitrogen was bubbled through the mixture, cooled in an ice

bath. Addition of potassium borohydride (14.4 mg) and stirring for 30

min were followed by treatment with acetone (0.5 ml) and then drop-

wise addition of an excess of 1 ^Mhydrobromic acid until the mixture

turned red. It was then stirred at room temperature for 30 min, diluted

with water and extracted repeatedly with dichloromethane. The organic

extract was re-extracted with dilute hydrochloric acid and the combined

aqueous acidic phases were then basified with solid sodium hydrogen

carbonate and extracted with chloroform. This red extract was washed

with a small volume of aqueous sodium hydrogen carbonate, dried and

concentrated. After filtration, the concentrate was chilled in an ice bath.

Methanolic hydrogen chloride (1 equiv.) was added, followed by an

excess of ether. ¹H n.m.r. spectroscopy of the dark red precipitate

(45.5 mg) (93%) showed this crude product to consist of (40) (a 1 : 1

diastereoisomeric mixture), (13) and (41) [76: 15: 9]. It was recrystal-

lized from methanol/acetone to give 12-deoxy-13-dihydrodaunorubicin

(40) as its hydrochloride, dark red clumps of needles, m.p. 180–182°

(Found: C, 59.0; H, 5.7; N, 2.8. C₂₇H₃₂ClNO₉requires C, 59.0; H, 5.9;

12-Deoxycarminomycinone (30)

The hydrochloride of carminomycin (25) (40 mg) was heated in 0.2

M hydrochloric acid at 90° for 45 min. The precipitated solid was filtered

38

D. W. Cameron, G. I. Feutrill and P. G. Griffiths

N, 2.5%). λ_max(logε) 283sh (3.98), 297sh (3.74), 325 (3.56), 486

(3.90), 520 nm (3.81). ν_max3411, 1655, 1595 cm^–1. δ 8.04, s, H12; 7.68,

t, J 8 Hz, H 2; 7.56, d, J 8 Hz, H 1; 7.14, d, J 8 Hz, H 3; 5.58, br s, H1′;

5.15, br s, H 7; 4.21, m, H 5′; 4.08, s, OMe; 3.77, br s, H 4′; 3.68, m,

H13; 3.50, br d, J 13 Hz, H 3′; 3.02, d, J 20 Hz, H10eq (isomer A);

2.96, d, J 20 Hz, H 10eq (isomer B); 2.57, d, J 20 Hz, H 10ax (isomer

A) (B); 2.47, d, J 20 Hz, H 10ax (isomer B) (A); 2.21, br d, J 15 Hz,

H8eq; 2.04, dt, J 3.5, 13 Hz, H2′ax; 1.94, dd, J 4.5, 15 Hz, H 8ax

(isomer A) (B); 1.79, m, H 2′eq, H 8ax (isomer B) (A); 1.32, br d, J 6.5

Hz, Me′. m/z (electrospray) 514 (M + H, 48%), 386 (24), 385 (100).

phorsulfonic acid (2.5 mg) was left to stand at room temperature for

23 h. Lithium carbonate (4 mg) was added with stirring for 10 min. Dry

tetrahydrofuran (15 ml) was then added and the mixture was stirred in

an ice bath, after nitrogen was bubbled through it. Lithium borohydride

solution (70 µl of 2 ^Min tetrahydrofuran) was then added by syringe.

After 10 min another aliquot (70 µl) was added and, after then stirring

for 45 min, a pale lemon-coloured mixture was obtained. This was

treated with acetone (0.5 ml), followed dropwise by 1 ^Mhydrobromic

acid, until it gave a more strongly yellow-coloured mixture. After stir-

ring at room temperature for 45 min this was diluted with distilled water

and extracted twice with dichloromethane. The organic extract was re-

extracted with 0.1 ^Mhydrochloric acid and the combined aqueous frac-

tions were then basified with solid sodium hydrogen carbonate,

followed by final extraction into dichloromethane. The yellow organic

extract was washed with a little water, dried and concentrated. The con-

centrate was filtered and chilled to 0° prior to addition of methanolic

hydrogen chloride (1 equiv.), followed by precipitation with diethyl

ether. This gave an orange solid on centrifuging and drying (21 mg)

12-Deoxydaunorubicin (22)

Method A

A solution of the hydrochloride of daunorubicin (1) (27.7 mg) in

methanol (2 ml) containing trimethyl orthoformate (1 ml) and cam-

phorsulfonic acid (2.5 mg) was left to stand at room temperature for

23 h. The resulting solution, containing the ketal (44) (δ 3.50, 3.48, s, s,

2×OMe; 1.52, s, 14-Me), was then diluted with methanol (20 ml) and

0.1 ^Maqueous phosphate buffer pH 7 (1 ml), and a stream of nitrogen

was bubbled through the mixture in an ice bath. Potassium borohydride

(5.4 mg) was added and, after stirring for 20 min, the yellow mixture

was treated with acetone (1 ml) followed dropwise by 1 ^Mhydrobromic

acid, until it gave a red solution. This was allowed to warm to room

temperature and to stand for 60 min. Distilled water was then added and

the mixture was extracted with dichloromethane to remove aglycone.

The aqueous phase was basified with solid sodium hydrogen carbonate

and it was then extracted with dichloromethane. The red extract was

washed with a little water, dried and concentrated. The concentrate was

filtered and then chilled to 0°. Methanolic hydrogen chloride (1 equiv.)

in methanol was added followed by precipitation with ether. Filtration

1

(86%). H n.m.r. spectroscopy showed the crude product to contain a

small proportion (1: 7) of 5-deoxydaunorubicin (12), which was

removed by repeated recrystallization. Alternatively, the presence of

this contaminant was avoidable by interposing repeated rapid extrac-

tion of the first dichloromethane solution of (34) and (12) with 0.025 ^M

aqueous sodium hydroxide, until the blue colour of the extract dimin-

ished, then continuing workup as before; this gave (34) (66%). From

either procedure, the product was purified by recrystallization from

methanol/acetone to give orange platelets of 5,12-bisdeoxydaunoru-

bicin (34) as its hydrochloride, m.p. 172–175° (Found: C, 61.1; H, 6.0;

N, 2.6. C₂₇H₃₀ClNO₈requires C, 61.0; H, 5.7; N, 2.6%). λ_max(logε)

263sh (4.16), 295sh (3.95), 330 (3.50), 448 nm (3.71). ν_max3420, 1710,

1660, 1610 cm^–1. δ 9.05, s, H5; 8.58, s, H 12; 7.66, m, H 1, H2; 7.09,

m, H 3; 5.59, br d, J 3 Hz, H 1′; 5.13, br s, H 7; 4.22, q, J 6.5 Hz, H5′;

4.10, s, OMe; 3.71, br s, H4′; 3.53, br d, J 13 Hz, H 3′; 3.04, d, J

19.5 Hz, H10eq; 2.93, d, J 19.5 Hz, H10ax; 2.40, s, COMe; 2.25, br d,

J 15 Hz, H8eq; 2.14, dd, J 5, 15 Hz, H8ax; 2.05, dt, J 3, 13 Hz, H 2′ax;

1.84, dd, J 4, 13 Hz, H2′eq; 1.31, d, J 6.5 Hz, Me′. m/z (f.a.b.) 518

(M+Na, 18%), 496 (M+H, 5), 331 (30), 307 (35).

1

then gave a dark red solid (25.4 mg). H n.m.r. spectroscopy showed

this was a mixture of (22) (71%), (34) (8%) and (12) (4%). The major

product 12-deoxydaunorubicin (22) as its hydrochloride was purified

by several recrystallizations from methanol/acetone, to give red-black

cubes, m.p. 182–184° (Found: C, 59.0; H, 5.6; N, 2.3. C₂₇H₃₀ClNO₉

requires C, 59.2; H, 5.5; N, 2.6%). λ_max(logε) 263sh (4.13), 270sh

(4.07), 308sh (3.48), 492 (3.97), 520 nm (3.94). ν_max3416, 1711, 1654,

1636, 1599 cm^–1. δ 8.05, s, H 12; 7.68, t, J 8 Hz, H2; 7.65, d, J 8 Hz,

H1; 7.14, d, J 8 Hz, H 3; 5.57, d, J 4 Hz, H 1′; 5.12, m, H 7; 4.21, q, J

6.7 Hz, H 5′; 4.06, s, OMe; 3.77, br s, H 4′; 3.55, ddd, J 3, 5, 13 Hz, H 3′;

3.01, d, J 19.5 Hz, H 10eq; 2.83, d, J 19.5 Hz, H 10ax; 2.40, s, COMe;

2.24, br d, J 15 Hz, H 8eq; 2.10, dd, J 5, 15 Hz, H 8ax; 2.03, dt, J 4,

13 Hz, H2′ax; 1.82, dd, J 5, 13 Hz, H 2′eq; 1.30, d, J 6.5 Hz, Me′. m/z

(electrospray) 512 (M+H, 28%), 384 (24), 383 (100).

Method B

A mixture of the hydrochloride of daunorubicin (1) (100 mg),

propane-1,3-diol (1 g), trimethyl orthoformate (0.5 ml) and camphor-

sulfonic acid (10 mg) was stirred at room temperature for 16 h.

Methanol (6 ml) was then added to the red gel, followed by lithium car-

bonate (8 mg) and stirring was continued for 1 h. The mixture was

diluted with tetrahydrofuran (60 ml) and then degassed under nitrogen.

It was then cooled in an ice bath and lithium borohydride (780 µl of 2 ^M

in tetrahydrofuran, total) was added in three equal portions over 1.5 h.

Then acetone (2 ml) was added, followed dropwise by 1 ^Mhydrobromic

acid until it turned yellow. The resulting mixture was allowed to stand

at room temperature for 30 min, diluted with water and basified with

solid sodium hydrogen carbonate, prior to being extracted with

dichloromethane. The extract was re-extracted into 0.1 ^Mhydrochloric

acid. The resulting acidic solution was left to stand at room temperature

for 16 h, when the glycoside was recovered as its hydrochloride (65 mg,

69%), as in A. Recrystallization from methanol/acetone gave (34), iden-

tical with the product from A.

Method B

A mixture of the hydrochloride of daunorubicin (1) (104 mg),

propane-1,3-diol (2 ml), trimethyl orthoformate (0.5 ml) and camphor-

sulfonic acid (5 mg) was allowed to stand at room temperature for 16 h,

and then it was diluted with methanol (50 ml) and 0.1 ^Maqueous phos-

phate buffer (pH 7) (5 ml). A stream of nitrogen was bubbled through

the mixture in an ice bath and potassium borohydride (60 mg total) was

added in three equal portions over 1.3 h. The resulting dark yellow

mixture was then treated dropwise with 1 ^Mmethanolic hydrogen chlo-

ride until it gave a red solution. This was stirred for 10 min at room tem-

perature, diluted with water and washed with dichloromethane. The

aqueous phase was basified with solid sodium hydrogen carbonate and

extracted with dichloromethane to give a red extract. This was re-

extracted with 0.1 ^Mhydrochloric acid. This acidic phase was allowed

to stand for 16 h at room temperature, whereupon the glycosidic

hydrochloride fraction (85 mg), consisting of (22), together with minor

proportions of (34) and (12), was recovered as in A. Recrystallization

from methanol/acetone gave (22), m.p. 182–184°, identical with the

product from A.

12-Deoxy-13-dihydrodoxorubicin (45)

To a solution of the hydrochloride of doxorubicin (2) (25 mg) in

methanol (25 ml) was added 0.1 ^Maqueous phosphate buffer (pH 7,

1.25 ml) and nitrogen was bubbled through the mixture, cooled in an ice

bath. Addition of potassium borohydride (7 mg) and stirring for 25 min

gave a clear yellow solution. This was quenched with acetone (0.5 ml)

then dropwise with 1 ^Mhydrobromic acid until it gave a red solution,

which was warmed to room temperature. Water was then added and the

mixture was extracted with a little dichloromethane to remove non-gly-

cosidic by-product. The aqueous phase was basified by addition of solid

sodium hydrogen carbonate and extracted with chloroform until the

aqueous phase was colourless (typically 10 extractions). The extract

5,12-Bisdeoxydaunorubicin (34)

Method A

A solution of the hydrochloride of daunorubicin (1) (26 mg) in

methanol (1.5 ml) containing trimethyl orthoformate (0.5 ml ) and cam-

Analogues of Daunorubicin and Doxorubicin

39

was washed with a little water, dried, concentrated and filtered.

Addition of methanolic hydrogen chloride (1 equiv.) to the filtrate at 0°

followed by an excess of diethyl ether gave the product, recovered as a

dark red solid (23.2 mg) (95%). It was recrystallized from

methanol/acetone to give 12-deoxy-13-dihydrodoxorubicin (45), a 1: 1

mixture of diastereoisomers, as its hydrochloride. This was isolated as

a black powder, m.p. 195–199° (dec.) (Found: C, 57.4; H, 5.5; N, 2.7.

C₂₇H₃₂ClNO₁₀requires C, 57.3; H, 5.7; N, 2.5%). λ_max(logε) 283sh

(3.95), 296sh (3.71), 308sh (3.45), 490 nm (3.92). ν_max3422, 1654,

1636, 1598 cm^–1. δ 8.03, s, H 12; 7.68, t, J 8 Hz, H2; 7.55, d, J 8 Hz,

H1; 7.14, d, J 8 Hz, H3; 5.57, br s, H1′; 5.13, br s, H 7; 4.23, br q, J

6.5 Hz, H5′; 4.07, s, OMe; 3.99–3.92, m, 14-CH₂; 3.76, br s, H 4′;

3.68–3.60, m, H 13; 3.51, br d, J 12 Hz, H 3′; 3.08–2.96, m, H 10eq;

2.76–2.62, m, H 10ax; 2.49, br d, J 15 Hz, H8eq (isomer A); 2.22, br d,

J 15 Hz, H 8eq (isomer B); 2.10–1.78, m, H 8ax, H 2′; 1.33, d, J 6.5 Hz,

Me′. m/z (electrospray) 530 (M+H, 42%).

in tetrahydrofuran) was then added, followed by further aliquots (2×140

µl) after 1 and 2 h. After 2.5 h the pale lemon solution was treated with

acetone (0.5 ml) and then, dropwise, with 1 ^Mhydrobromic acid until

the colour turned sharply orange. The resulting solution was stirred at

room temperature (15 min). Water was added and the mixture was then

basified with solid sodium hydrogen carbonate. Extraction with

dichloromethane gave an orange solution which was washed with a

little water, dried and evaporated. The resulting solid was dissolved in

acetone (4 ml) and hydrobromic acid (0.25 ^M, 4 ml) and was left to stand

at room temperature for 66 h. The mixture was diluted with water, and

aglycones were removed by dichloromethane extraction. Further

extraction with dichloromethane, following addition of solid sodium

hydrogen carbonate, then gave the glycosidic fraction. The latter frac-

tion was washed with a little water, dried and concentrated. The residue

was re-dissolved in dichloromethane/methanol, filtered and chilled.

Methanolic hydrogen chloride solution (1 equiv.) at 0° was then added,

followed by an excess of ether. The precipitate was recovered by cen-

trifugation and drying to give 5,12-bisdeoxydoxorubicin (35) as its

hydrochloride, a dark orange solid (29 mg, 61%). Crystallization from

methanol/acetone gave dark orange platelets, m.p. 180–181° (Found: C,

59.1; H, 5.4; N, 2.4. C₂₇H₃₀ClNO₉requires C, 59.2; H, 5.5; N, 2.5%).

λ_max(logε) 293sh (3.91), 338 (3.42), 455 nm (3.79). ν_max3404, 1723,

1662, 1614 cm^–1. δ 9.06, s, H5; 8.58, s, H12; 7.64, m, H 1, H2; 7.07,

m, H3; 5.58, br d, J 3.5 Hz, H1′; 5.13, br s, H 7; 4.79, 4.75, ABq, J 17

Hz, 14-CH₂; 4.19, q, J 6.5 Hz, H5′; 4.08, s, OMe; 3.76, br d, J 3 Hz,

H4′; 3.53, m, H3′; 3.11, dd, J 4, 19.5 Hz, H10eq; 2.92, dd, J 5, 19.5 Hz,

H10ax; 2.29, br d, J 15 Hz, H8eq; 2.15, dd, J 5, 15 Hz, H 8ax; 2.04, dt,

J 3.5, 13 Hz, H2′ax; 1.84, dd, J 4.5, 13 Hz, H2′eq; 1.31, d, J 6.5 Hz,

Me′. m/z (electrospray) 512 (M+H, 84%), 383 (57). m/z (f.a.b.) 534

(M+Na, 8%).

12-Deoxydoxorubicin (23)

The hydrochloride of doxorubicin (2) (26 mg) was dissolved in

methanol by heating in a large volume of the solvent then concentrating

to 1 ml. This solution was treated with 0.1 ^Mmethanolic hydrogen

bromide (0.75 ml) and trimethyl orthoformate (0.5 ml) for 64 h at room

temperature. The mixture was then diluted with water and basified by

addition of sodium hydrogen carbonate. Extraction with chloroform,

drying and evaporation gave a red-orange solid comprising largely the

dimethyl ketal (46) (δ 3.98, s, H 14; 3.56, 3.53, s, s, 2×OCH₃). Without

purification, this was dissolved in methanol (15 ml), and then 0.1 ^M

aqueous phosphate buffer (pH 7; 0.75 ml) was added prior to a stream

of nitrogen being bubbled through the mixture in an ice bath. Potassium

borohydride (4.8 mg) was then added with stirring for 30 min to give a

yellow solution. This was quenched with acetone (0.5 ml), followed

dropwise by 1 ^Mhydrobromic acid until the colour turned sharply red.

The resulting solution was allowed to warm to room temperature over

15 min. The mixture was then diluted with water and, after adding an

excess of solid sodium hydrogen carbonate, it was then extracted with

chloroform. The extract was dried and evaporated under reduced pres-

sure to give a dark red residue, which was dissolved in 0.25 ^Mhydro-

bromic acid (2 ml) and acetone (2 ml) and allowed to stand overnight at

room temperature. It was then diluted with water and extracted with

dichloromethane to remove aglycone. The aqueous phase was then

basified with solid sodium hydrogen carbonate and extracted with chlo-

roform. The extract was washed with a little water, dried and concen-

trated under reduced pressure. The concentrate was filtered and the

filtrate was chilled in ice prior to reaction with methanolic hydrogen

chloride (1 equiv.), followed by precipitation with ether. This gave the

product (23) as a dark red solid (16 mg) (63%). ¹H n.m.r. spectroscopy

showed the crude product was a mixture of (23), (15) and (35)

(83: 15 : 12). The major product 12-deoxydoxorubicin (23) as its

hydrochloride was purified by several crystallizations from

methanol/acetone to give black microplates, m.p. 192–196° (dec.)

(Found: C, 57.3; H, 5.3; N, 2.3. C₂₇H₃₀ClNO₁₀requires C, 57.5; H, 5.4;

N, 2.5%). λ_max(logε) 275sh (4.15), 297sh (3.83), 325 (3.72), 490

(3.95), 515 nm (3.92). ν_max3419, 1724, 1658, 1635, 1598 cm^–1. δ 8.06,

s, H 12; 7.69, t, J 8 Hz, H2; 7.56, d, J 8 Hz, H 1; 7.15, d, J 8 Hz, H3;

5.57, br d, J 3.5 Hz, H 1′; 5.14, br s, H 7; 4.78, 4.74, ABq, J 17 Hz, 14-

CH₂; 4.20, q, J 6.5 Hz, H5′; 4.08, s, OMe; 3.77, br s, H 4′; 3.55, br d, J

13 Hz, H3′; 3.08, br d, J 19.5 Hz, H 10eq; 2.89, d, J 19.5 Hz, H10ax;

2.30, d, J 15 Hz, H8eq; 2.15, dd, J 5, 15 Hz, H8ax; 2.03, dt, J 3.5,

13 Hz, H 2′ax; 1.83, dd, J 5, 13 Hz, H 2′eq; 1.30, d, J 6.5 Hz, Me′. m/z

(electrospray) 529 (M+2H, 20%), 528 (M+H, 57), 512 (30), 399 (100).

4,5,12-Trisdeoxydaunorubicin (36)

To a solution of the hydrochloride of idarubicin (37) (25 mg) in

methanol (2.5 ml) was added trimethyl orthoformate (1.25 ml) and cam-

phorsulfonic acid (2.5 mg). The mixture was allowed to stand at room

temperature for 16 h. It was then cooled in ice, tetrahydrofuran (25 ml)

was added and nitrogen was bubbled through.Asolution of lithium boro-

hydride in tetrahydrofuran (72 µl of 2 ^M) was then added by syringe fol-

lowed, after 30 and then 70 min, by two further aliquots (2×72 µl). After

110 min, the pink solution was treated with acetone (0.5 ml) then drop-

wise with 1 ^Mhydrobromic acid until it gave a yellow solution, which

was stirred at room temperature for 30 min. It was then diluted with

water and extracted twice with dichloromethane. The extract was re-

extracted with 0.1 ^Mhydrochloric acid. The combined aqueous phases

were basified with solid sodium hydrogen carbonate and finally

extracted with dichloromethane, and the yellow extract was washed with

water, dried and concentrated.Addition of methanolic hydrogen chloride

(1 equiv.) at 0° and an excess of ether precipitated the hydrochloride of

4,5,12-trisdeoxydaunorubicin (36) (14.6 mg, 62%). Crystallization from

methanol/acetone gave orange platelets, m.p. 160–162° (dec.) (Found:

C, 58.9; H, 5.6; N, 2.5. C₂₆H₂₈ClNO₇·1.5 H₂O requires C, 59.0; H, 5.9;

N, 2.6%). λ_max(logε) 273 (4.11), 284 (4.13), 295 (4.12), 408 nm (3.54).

ν_max3421, 1707, 1664, 1616 cm^–1. δ 8.66, 8.63, s, s, H5, H12; 8.01, m,

H1, H4; 7.64, m, H2, H3; 5.50, br d, J 3.5 Hz, H1′; 5.05, br s, H7; 4.11,

q, J 6.5 Hz, H5′; 3.71, br s, H4′; 3.46, br td, J 2.5, 13 Hz, H 3′; 2.99, br

d, J 19.5 Hz, H10eq; 2.81, d, J 19.5 Hz, H10ax; 2.42, s, COMe; 2.16, br

d, J 15 Hz, H8eq; 2.03, dd, J 5, 13 Hz, H8ax; 1.95, dt, J 4, 13 Hz, H 2′ax;

1.75, dd, J 4.5, 13 Hz, H2′eq; 1.22, d, J 6.5 Hz, Me′. m/z (f.a.b.) 488

(M+Na, 15%), 466 (M+H, 35), 327 (31).

12-Deoxydaunorubicinone (49), 5,12-Bisdeoxydaunorubicinone

(50) and 4,5,12-Trisdeoxydaunorubicinone (51)

5,12-Bisdeoxydoxorubicin (35)

The hydrochloride of doxorubicin (2) (50 mg) was dissolved in

methanol (30 ml) by gentle reflux, and then the solution was concen-

trated under reduced pressure to a small volume (2 ml). Methanolic

hydrogen bromide (0.1 ^M, 1.5 ml) and trimethyl orthoformate (1.5 ml)

were added and the mixture was stirred at room temperature for 3 days.

It was then treated with lithium carbonate (5.5 mg) and diluted with

tetrahydrofuran (35 ml), and a stream of nitrogen was bubbled through

it at ice-bath temperature. Lithium borohydride solution (140 µl of 2 ^M

12-Deoxydaunorubicin (22), 5,12-bisdeoxydaunorubicin (34) and

4,5,12-trisdeoxydaunorubicin (36) were hydrolysed separately in 0.2 ^M

hydrochloric acid for 1 h on a steam bath. The precipitated solids were

respectively recrystallized from acetone.

12-Deoxydaunorubicinone (49) gave dark red needles, m.p.

189–191° (Found: M^+•, 382.1045. C₂₁H₁₈O₇requires M^+•, 382.1052).

λ_max(logε) 272 (3.88), 320 (3.40), 490 (3.88), 510 nm (3.88). ν_max1710,

1655, 1630, 1590 cm^–1. δ 14.90, s, OH; 8.01, s, H12; 7.65, t, J 8 Hz,

40

D. W. Cameron, G. I. Feutrill and P. G. Griffiths

⁶Berg, H., Horn, G., and Ihn, W., J. Antibiot., 1982, 35, 800;

Tresselt, D., Ihn, W., Horn, G., and Berg, H., Pharmazie, 1984, 39,

417.

H2; 7.51, d, J 8 Hz, H 1; 7.09, d, J 8 Hz, H 3; 5.22, d, J 5 Hz, H7; 4.07,

s, OMe; 3.04, dd, J 2, 19.5 Hz, H 10eq; 2,81, dd, J 1, 19.5 Hz, H 10ax;

2.41, s, COMe; 2.31, td, J 2, 14.5 Hz, H 8eq; 2.10, dd, J 5, 14.5 Hz,

H8ax. m/z 382 (M, 15%), 346 (62).

5,12-Bisdeoxydaunorubicinone (50) gave orange microneedles,

m.p. 144–146° (Found: M^+•, 366.1099. C₂₁H₁₈O₆requires M^+•,

366.1103). λ_max(logε) 260sh (4.07), 280sh (3.97), 296 (3.88), 323

(3.51), 440 nm (3.68). ν_max3434, 1709, 1665, 1615 cm^–1. δ 9.02, s, H5;

8.48, s, H12; 7.61, m, H1, H 2; 7.01, m, H 3; 5.22, br d, J 4.5 Hz, H7;

4.07, s, OMe; 3.08, dd, J 2, 19.5 Hz, H10eq; 2.85, br d, J 19.5 Hz,

H10ax; 2.42, s, COMe; 2.30, td, J 1.5, 14.5 Hz, H 8eq; 2.10, dd, J 5,

14.5 Hz, H8ax. m/z 366 (M, 44%), 330 (88), 305 (100).

4,5,12-Trisdeoxydaunorubicinone (51) gave yellow needles, m.p.

135–137° (dec.). λ_max(logε) 260sh (4.37), 270 (4.63), 296 (4.28), 380

(3.70), 412 nm (3.72). ν_max3425, 1711, 1664, 1617 cm^–1. δ 8.64, 8.61,

s, s, H 5, H 12; 8.07, m, H 1, H4; 7.71, m, H 2, H 3; 5.22, td, J 1.5, 5 Hz,

H7; 3.09, dd, J 2.5, 20 Hz, H 10eq; 2.87, dd, J 1.5, 20 Hz, H10ax; 2.42,

s, COMe; 2.31, td, J 2.5, 15 Hz, H 8eq; 2.11, dd, J 5, 15 Hz, H 8ax; 4.50,

3.70, br s, br s, 2×OH (both D₂O-exchanged). m/z 336 (M, 2.4%), 316

(26), 301 (40), 300 (79), 285 (100).

⁷Brand, D. J., and Fisher, J. F., J. Am. Chem. Soc., 1986, 108, 3088.

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1985, 63, 2697.

⁹Brand, D. J., and Fisher, J. F., J. Org. Chem., 1990, 55, 2518.

¹⁰Arcamone, F., Franceshi, G., Orezzi, P., Cassinelli, G., Barbieri, W.,

and Mondelli, R., J. Am. Chem. Soc., 1964, 86, 5334.

¹¹Cameron, D. W., Feutrill, G. I., and Griffiths, P. G., Tetrahedron Lett.,

1988, 29, 4629.

¹²Cameron, D. W., and Griffiths, P. G., Tetrahedron Lett., 1993, 34,

4685.

¹³Arcamone, F., Franceschi, G., Orezzi, P., Penco, S., and Mondelli,

R., Tetrahedron Lett., 1968, 3349.

¹⁴Cameron, D. W., Conn, C., Crossley, M. J., Feutrill, G. I., Fisher,

M. W., Griffiths, P. G., Merrett, B. K., and Pavlatos, D., Tetrahedron

Lett., 1986, 27, 2417.

¹⁵Cameron, D. W., Feutrill, G. I., Gibson, C. L., and Read, R. W.,

Tetrahedron Lett., 1985, 26, 3887.

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5593.

Isolation of Anthrone Glycosides (53) and (54)

¹⁷Cameron, D. W., Edmonds, J. S., and Raverty, W. D., Aust. J. Chem.,

1976, 29, 1535.

The hydrochloride of carminomycin (25) (5 mg) was hydrogenated

in methanol (4 ml) overAdams catalyst (1.8 mg) and dichloroacetic acid

(20 mg) for 40 min at room temperature. Analytical t.l.c. then indicated

the formation of a pale yellow, non-fluorescent product, of polarity con-

sistent with a glycoside. The mixture was filtered through Celite under

nitrogen and the filtrate was concentrated under vacuum, then diluted

with ether to give a precipitate recovered by centrifugation. The solid

was re-dissolved in methanol, then the mixture was treated with an

excess of hydrogen chloride in methanol and re-precipitated with an

excess of ether to give the anthrone (54) as its hydrochloride, a tan solid

(2.5 mg). δ (CD₃OD) 7.55, dd, J 7.5, 8.5 Hz, H 2; 7.03, br d, J 7.5 Hz,

H1; 6.86, br d, J 8.5 Hz, H3; 5.47, br d, J 4 Hz, H1′; 5.17, dd, J 3, 5 Hz,

H7; 4.27, br q, J 6.5 Hz, H 5′; 4.26, br s, anthrone CH₂; 3.65, br s, H4′;

3.60, m, H 3′; 3.10, d, J 18 Hz, H10eq; 3.05, br d, J 18 Hz, H10ax; 2.33,

s, COMe; 2.29, br d, J 14 Hz, H8eq; 2.18, dd, J 5, 14 Hz, H8ax; 2.02,

dt, J 4, 13 Hz, H2′ax; 1.88, dd, J 5, 13 Hz, H2′eq; 1.28, d, J 6.5 Hz, Me′.

The hydrochloride of daunorubicin (1) (5 mg) was hydrogenated in

methanol (2 ml) and chloroacetic acid (20 mg) over Adams catalyst

(1 mg) for 20 min at room temperature. The mixture was filtered under

nitrogen and then concentrated under reduced pressure. Dilution of the

yellow concentrate with ether gave a tan solid recovered by centrifuga-

tion. The hydrochloride was prepared in the usual way to give the

anthrone (53) as a tan solid (2 mg). Partial δ (CD₃OD) 7.89, d, J 8 Hz,

H1; 7.47, t, J 8 Hz, H 2; 7.30, d, J 8 Hz, H 3; 4.04, s, anthrone CH₂;

3.99, s, OMe; 2.29, s, COMe; 1.28, d, J 6.5 Hz, Me′.

¹⁸Thomas, G. J., Broadhurst, M. J., and Hassall, C. H., Ger. Offen.

3,030,612, Mar. 12, 1981 (Chem. Abstr., 1981, 95, 115142b);

Broadhurst, M. J., Hassall, C. H., and Thomas, G. J., J. Chem. Soc.,

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5440.

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K. D., J. Am. Chem. Soc., 1975, 97, 5955.

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²⁵Wiley, P. F., Elrod, D. W., Houser, D. J., Johnson, J. L., Pschigoda,

L. M., Krueger, W. C., and Moscowitz, A., J. Org. Chem., 1979, 44,

4030.

²⁶Yoshimoto, A., Fujii, S., Johdo, O., Kubo, K., Nishida, H., Okamoto,

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Acknowledgments

²⁸Hilton, B. D., Misra, R., and Zweier, J. L., Biochemistry, 1986, 25,

5533.

We thank Farmitalia Carlo Erba for gifts of (1), (2) and

(3), Bristol–Myers Squibb for (25), and both the Cancer

Institute, Melbourne, and Farmitalia Carlo Erba, Milan, for

biological testing. This work was carried out during the

tenure of grants from the Anti-Cancer Council of Victoria

and the Australian Research Council.

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Article Doi

5-deoxy, 12-deoxy, 5,12-bisdeoxy, and 4,5,12-trisdeoxy anthracyclines: Synthesis of new analogues of daunorubicin and doxorubicin by controlled deoxygenation of the C-ring

DOI: 10.1071/ch99151

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