Asymmetric synthesis and DNA intercalation of (-)-6-[[(aminoalkyl)oxy]methyl]-4-demethoxy-6,7-dideoxydaunomycinones

DOI: 10.1021/jo960606z

Source and publish data:

Journal of Organic Chemistry p. 6958 - 6970 (1996)

Update date:2022-08-03

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Authors:

Dienes, Zoltan

Vogel, Pierre

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Article abstract of DOI:10.1021/jo960606z

The BF₃·Et₂O-promoted Diels-Alder addition of 1-acetylvinyl RADO(Et)-ate (RADO(Et)-ate = 3-ethyl-2-oxo-6,8-dioxa-3-azabicyclo[3.2.1]octane-7-exo-carboxylate) to 1-(dimethoxymethyl)-2,3,5,6-tetramethylidene-7-oxabicyclo[2.2.1]heptan e led to one major monoadduct that added to 1,2-didehydrobenzene and was converted into (-)-4-demethoxy-7-deoxydaunomycinone and (2R)-12-acetoxy-2-acetyl-5-(bromomethyl)-1,2,3,4-tetrahydronaphthacen- 2-yl RADO(Et)-ate. The latter compound was used to construct (8R)-8-acetyl-6,8-dihydroxy-11-[[(3'-[(aminopropyl)oxy]-, -4'-[(aminobutyl)oxy], and -5'-[(aminopentyl)oxy]methyl]-7,8,9,10-tetrahydronaphthacene-5,12-dion e hydrochloride (-)-8, (-)-9, (-)-16, respectively, as well as (8R)-8-acetyl-6,8-dihydroxy-11-[[[2'-[(3''-aminopropyl)amino]ethyl]oxy ]-((-)-11) and -[[3'-[(3''-aminopropyl)amino]propyl]oxy]methyl]-7,8,9,10-tetrahydrona phthacene-5,12-dione hydrochloride ((-)-12). (8R)-8-Acetyl-6,8-dihydroxy-11-[[(α-L-daunosaminyl)oxy]methyl]-7,8,9, 10-tetrahydronaphthacene-5,12-dione hydrochloride ((-)-13), a mimic of idarubicin, was also prepared. Absorbance and fluorescence titration experiments showed (-)-8, (-)-9, and (-)-16 to intercalate calf thymus DNA whereas (-)-11, (-)-12, and (-)-13 did not. The best intercalator was (-)-9 (K(b) = (1.1 ± 0.1) x 10⁵ M^-1) with the [(4'-aminobutyl)oxy]-methyl chain. Inhibition of topoisomerase II-induced DNA strand religation was observed for (-)-8 at a concentration of 50 μM.

Full text of DOI:10.1021/jo960606z

6958

J . Org. Chem. 1996, 61, 6958-6970

Asym m etr ic Syn th esis a n d DNA In ter ca la tion of

(-)-6-[[(Am in oa lk yl)oxy]m eth yl]-4-d em eth oxy-6,7-

d id eoxyd a u n om ycin on es^1,†

Zolta´n Dienes²and Pierre Vogel*

Section de Chimie de l′Universite´, BCH, 1015 Lausanne-Dorigny, Switzerland

Received April 1, 1996^X

The BF₃·Et₂O-promoted Diels-Alder addition of 1-acetylvinyl RADO(Et)-ate (RADO(Et)-ate )

3-ethyl-2-oxo-6,8-dioxa-3-azabicyclo[3.2.1]octane-7-exo-carboxylate) to 1-(dimethoxymethyl)-2,3,5,6-

tetramethylidene-7-oxabicyclo[2.2.1]heptane led to one major monoadduct that added to 1,2-

didehydrobenzene and was converted into (-)-4-demethoxy-7-deoxydaunomycinone and (2R)-12-

acetoxy-2-acetyl-5-(bromomethyl)-1,2,3,4-tetrahydronaphthacen-2-yl RADO(Et)-ate. The latter

compound was used to construct (8R)-8-acetyl-6,8-dihydroxy-11-[[(3′-[(aminopropyl)oxy]-, -4′-

[(aminobutyl)oxy], and -5′-[(aminopentyl)oxy]methyl]-7,8,9,10-tetrahydronaphthacene-5,12-dione

hydrochloride (-)-8, (-)-9, (-)-10, respectively, as well as (8R)-8-acetyl-6,8-dihydroxy-11- [[[2′-[(3′′-

aminopropyl)amino]ethyl]oxy]- ((-)-11) and -[[3′-[(3′′-aminopropyl)amino]propyl]oxy]methyl]-7,8,9,

10-tetrahydronaphthacene-5,12-dione hydrochloride ((-)-12). (8R)-8-Acetyl-6,8-dihydroxy-11-[[(R-

L-daunosaminyl)oxy]methyl]-7,8,9,10-tetrahydronaphthacene-5,12-dione hydrochloride ((-)-13), a

mimic of idarubicin, was also prepared. Absorbance and fluorescence titration experiments showed

(-)-8, (-)-9, and (-)-10 to intercalate calf thymus DNA whereas (-)-11, (-)-12, and (-)-13 did

not. The best intercalator was (-)-9 (K_b) (1.1 ( 0.1) × 10⁵M^-1) with the [(4′-aminobutyl)oxy]-

methyl chain. Inhibition of topoisomerase II-induced DNA strand religation was observed for (-)-8

at a concentration of 50 µM.

The clinical utility of the anthracycline antitumor

antibiotics such as adriamycin (1) and daunomycin (2)

is well demonstrated.³Their effectiveness, however, is

restricted due to acute bone marrow toxicity, cardiotox-

icity, and drug resistance development.⁴More than 2000

analogues have been synthesized and tested during the

last 30 years. Among them, idarubicin (3), 4-demethoxy-

adriamycin (4),^5,6and carminomycine (5)⁷have compa-

rable or better in vivo activity than 1 and 2 at lower dose.

Simple 1,4-bis[(aminoalkyl)amino]-9,10-anthraquino-

nes⁸such as mitoxantrone (6)⁹and iosoxantrone (7)¹⁰also

^†Dedicated to Prof. Horst Prinzbach on the occasion of his 65th

birthday, with thanks and admiration.

have useful anticancer properties. These observations

together with the fact that polyamines are able to inhibit

cancer cell growth¹¹led us to conceive a new series of

anthracycline analogues (-)-8 - (-)-12 bearing ami-

noalkyl chains through a benzyl ether link at C(6). We

have also prepared the R-^L-daunosaminyl derivative (-)-

13 and have evaluated the ability of these new analogues

to bind calf thymus DNA through interacation¹²and, for

some of them, their ability to inhibit topoisomerisase

^XAbstract published in Advance ACS Abstracts, September 15, 1996.

(1) For preliminary communications see: (a) Dienes, Z.; Antonsson,

T.; Vogel, P. Tetrahedron Lett. 1993, 34, 1013. (b) Dienes, Z.; Vogel, P.

Bioorg. Med. Chem. Lett. 1995, 5, 547.

(2) Part of the Ph.D. Thesis of Z. Dienes, University of Lausanne.

Present address: Department of Chemistry, Ecole Polytechnique

Fe´de´rale de Lausanne, CH 1015 - Lausanne-Ecublens, Switzerland.

(3) See, e.g.: (a) Lown, J . W. Chem. Soc. Rev. 1993, 165. Priebe, W.

Antracycline Antibiotics: New Analogues, Method of Delivery, and

Mechanisms of Action; ACS Symposium Series 574; American Chemical

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19, 670.

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Beck, W. T. Biochem. Pharmacol. 1987, 36, 2879. Keizer, H. G.; Pinedo,

H. M.; Schuurhuis, G. J .; J oenje, H. Pharmacol. Ther. 1990, 47, 219.

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Pratesi, G.; Arcamone, F.; Bernardi, L.; Franchi, G.; Giardino, P.;

Patelli, B.; Penco, S. Cancer Treat. Rep. 1978, 62, 375. Matsumoto, T.;

Ohsaki, M.; Suzuki, M.; Kimura, Y.; Terashima, S. Chem. Pharm. Bull.

1986, 34, 4613. Adams, N.; Blake, C.; Broadhurst, M. J .; Bushnell, D.

J .; Hassall, C. H.; Hartmann, H. R.; Keech, E.; Stratton, A. R.; Thomas,

G. J . J . Med. Chem. 1990, 33, 2375. See also: Florent, J .-C.; Gaudel,

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36, 1364. Monneret, C.; Florent, J .-C. Synlett 1994, 304.

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er. Rep. Part I 1974, 58, 255.

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R. E.; Durr, F. E.; Citarella, R. V. J . Med. Chem. 1979, 22, 1024.

(9) See, e.g.: Showalter, H. D. H.; J ohnson, J . L.; Hoftiezer, J . M.;

Tumer, W. R.; Werbel, L. M.; Leopold, W. R.; Shillis, J . L.; J ackson, R.

C.; Elslager, E. F. J . Med. Chem. 1987, 30, 121.

(10) Leopold, W. R.; Nelson, M. J .; Plowman, J .; J ackson, R. C.

Cancer Res. 1985, 45, 5532. Showalter, H. D. H.; J ohnson, J . L.;

Weiberl, L. M.; Leopold, W. R.; J ackson, R. C.; Elslager, E. F. J . Med.

Chem. 1984, 27, 255. Showalter, H. D. H.; J ohnson, J . L.; Hoftiezer, J .

M. J . Heterocycl. Chem. 1986, 23, 1491. Zhang, L-h.; Meier, W. E.;

Watson, E. J .; Gibson, E. P. Tetrahedron Lett. 1994, 35, 3675.

(11) Tabor, C. W.; Tabor H. Ann. Rev. Biochem. 1984, 53, 749. Porter

C. W.; Cavanaugh, P. F.; Stolowich, N.; Ganis, B.; Kelly, E.; Bergeron,

R. J . Cancer Res. 1985, 45, 2050. Bergeron, R. J .; McManis, J . S.; Liu,

C. Z.; Feng, Y.; Weimar, W. R.; Luchetta, G. R.; Wu, Q.; Ortiz-Ocasio,

J .; Vinson, J . R. T.; Kramer, D.; Porter, C. J . Med. Chem. 1994, 37,

3464. Bergeron, R. J .; McManis, J . S.; Weimar, W. R.; Schreier, K. M.;

Gao, F.; Wu, Q.; Ortiz-Ocasio, J .; Luchetta, G. R.; Porter, C.; Vinson,

J . R. T. J . Med. Chem. 1995, 38, 2278.

Synthesis of Substituted Daunomycinones

J . Org. Chem., Vol. 61, No. 20, 1996 6959

I-induced relaxation of circular plasmids and topoiso-

merase II-induced DNA strand religation. The newly

developed synthetic approach in this work has led us to

propose a new synthesis of (-)-(R)-4-demethoxy-7-deoxy-

daunomycinone,¹a known precursor¹³of idarubicin (3).

While (-)-11 - (-)-13 do not intercalate calf thymus

DNA, (-)-8 - (-)-10 do bind with DNA.

Since preliminary experiments with optically pure

Lewis acids such as (-)-BINAP-TiCl₂and (+)-Eu(tfc)₃

failed to induce useful asymmetry in the Diels-Alder

additions of 15 to 1-acetylvinyl p-nitrobenzoate, we

explored the possibility to apply optically pure 1-acetylvi-

nyl esters as dienophiles to generate optically active

monoadducts.

In 1979, our group¹⁴demonstrated that the readily

available tetraene 14 can be converted through two

successive Diels-Alder additions with two different

dienophiles into a variety of linearly condensed polycyclic

systems including racemic anthracyclinone derivatives.

The method has been applied to the synthesis of (()-4-

demethoxydaunomycinone,¹⁵(()-daunomycinone,¹⁶and

(()-11-deoxydaunomycinone.¹⁷1-(Dimethoxymethyl)-

2,3,5,6-tetramethylidene-7-oxabicyclo[2.2.1]heptane (15),

a tetraene obtained readily from furfural,¹⁸was envisaged

as a precursor of anthracyclinone analogues bearing a

carbon substituent at C(6), as required for (-)-8-(-)-13.

Desym m etr iza tion th r ou gh Diels-Ald er Ad d i-

tion s. The cycloadditions of tetraene 15 to 1-acetylvinyl

esters of type 16 are expected to generate eight diaster-

eomeric monoadducts of type 17-20. There are 16 modes

of addition corresponding to the “para” (f 17 + 18) and

“meta” (f 19 + 20) orientations, to the “Alder” or “anti-

Alder” rule and whether the exo or endo face of the

bicyclic exocyclic diene is involved. Under thermal

conditions the Diels-Alder addition of methyl vinyl

ketone to 15 is not stereoselective and gives a mixture

of all possible monoadducts.¹⁸However, when methyl

vinyl ketone was coordinated to a strong Lewis acid some

selectivity could be observed, the best results being

obtained for EtAlCl₂. It was found also that the regiose-

lectivity (product ratio) depends on the solvent.¹⁸A single

monoadduct (17a ) was obtained for the cycloaddition of

15 to 1-acetylvinyl p-nitrobenzoate¹⁹precomplexed with

BF₃·Et₂O.²⁰For all these Diels-Alder additions the

formation of the bis-adduct of 15 were at least 100 times

slower than the additions of the first equivalent of the

dienophile.²¹

Condensation of diacetyl with the optically pure acyl

chlorides 21b-f in toluene (pyr., 0 °C) gave the corre-

sponding dienophiles 16b-f. Compound 16e added

slowly and with low stereoselectivity to tetraene 15 on

heating. Mixture of eight monoadducts were obtained

in all cases. When mixed with Me₃Al, Et₂AlCl, EtAlCl₂,

TiCl₄, Ti(O-i-Pr)Cl₃, BF₃·Me₂O, BBr₃, BBr(OMe)₂, or Et₂-

BBr, polymerization of 15 and 16 occurred between -78

and -50 °C. For the reaction of 15 with 16c²²coordi-

nated to Ti(O-i-Pr)₂Cl₂, a 19:10:5:3 mixture of four

diastereomeric monoadducts (64% yield) was obtained.

Lewis acids such as (t-Bu)Me₂SiOSO₂CF₃(-30 °C),

B(OMe)₃(25 °C), or Eu(tfc)₃(25 °C) did not promote the

cycloadditions of 15 to 16e. However, in the presence of

a large excess of BF₃·Et₂O, 15 added to 16b-f giving the

corresponding monoadducts 17b-f and 17′b-f (see Table

1) with less than 10% (by 360 MHz ¹H NMR) of the other

stereomeric adducts, thus confirming the high regio- and

stereoselectivity of the Lewis-acid-promoted Diels-Alder

additions. The best diastereoselectivity was observed

with 16e²²(product ratio 17e/17′e 87:13) in CH₂Cl₂.

Changing the solvent from pure CH₂Cl₂to CH₂Cl₂/MeNO₂

6:1 (-78 °C) led to a higher yield of 17e + 17′e but with

lower diastereoselectivity (83:17). In CH₂Cl₂/hexane 5:1

(-55 °C) and CH₂Cl₂/CF₂Cl₂2:1 (-78 °C) the yields and

diastereoselectivity were not improved (51% (81:19) and

20% (82:18), respectively). Surprisingly, in pure propi-

onitrile the yield climbed to 90% but with lower and

reversed diastereoselectivity (43:57). The structure and

absolute configuration of the major adduct 17e have been

established as shown in Table 1.

(12) Leng, F.; Savkur, R.; Fokt, I.; Przewloka, T.; Priebe, W.; Chaires,

J . B. J . Am. Chem. Soc. 1996, 20, 4731 and references cited therein.

Roche, C. J .; Thompson, J . A.; Crothers, D. M. Biochemistry 1994, 33,

926.

(13) Tamamoto, K.; Sugimori, M.; Terashima, S. Tetrahedron 1984,

40, 4617.

(14) Carrupt, P.-A.; Vogel, P. Tetrahedron Lett. 1979, 20, 4533.

(15) Bessie`re, Y.; Vogel, P. Helv. Chim. Acta 1980, 63, 232.

(16) Tamariz, J .; Vogel, P. Angew. Chem., Int. Ed. Engl. 1984, 23,

74.

(17) Tornare, J .-M.; Vogel, P. Helv. Chim. Acta 1985, 68, 1069.

(18) Me´tral, J .-L.; Lauterwein, J .; Vogel, P. Helv. Chim. Acta 1986,

69, 1287.

The 87:13 mixture of 17e and 17e′ (CHCl₃, drying with

3 Å molecular sieves) reacted with didehydrobenzene

(generated through nitrosation of anthranilic acid with

(19) Tamariz, J .; Vogel, P. Helv. Chim. Acta 1981, 64, 188. Aguilar,

R.; Reyes, A.; Tamariz, J .; Birbaum, J .-L. Tetrahedron Lett. 1987, 28,

865.

(20) Antonsson, T.; Vogel, P. Tetrahedron Lett. 1990, 31, 89. In this

paper this compound was wrongly assigned to 18a .

(21) Vogel, P. In Advances in Theoretically Interesting Molecules;

Thummel, R. P., Ed.; J AI Press, Inc.: Greenwich, 1989; Vol. 1, p 201.

(22) Reymond, J .-L.; Vogel, P. Tetrahedron: Asymmetry 1990, 1, 729.

6960 J . Org. Chem., Vol. 61, No. 20, 1996

Dienes and Vogel

F igu r e 1. Superimposed CD spectra (CHCl₃) of (-)-23 and

(-)-24.

Ta ble 1. Yield s a n d Dia ster eoselectivity (P r od u ct Ra tio

17/17′) for th e BF ₃‚Et₂O (15 Equ iv)-p r om oted

amount of 29. The direct transformation of (-)-25 into

30 was accomplished using Bu₄NMnO₄in pyridine as

oxidant,²⁸followed by esterification with CH₂N₂. Unfor-

tunately, the yield never surpassed 45%. The reduction

of aldehyde (-)-25 with NaBH₃CN in AcOH/MeOH/

CHCl₃(20 °C) was selective (no reduction of the methyl

ketone moiety) and afforded the benzyl alcohol (-)-31

(93%), which was converted into bromide (-)-32 (97%)

on treatment with AcBr in CHCl₃.

Cycloa d d ition s of 15 to 16 (1.2 Equ iv) in CH₂Cl₂(7 d a ys)

dienophile

T (°C)

yield of 17 + 17′ (%) 53

[17]/[17′]

16b

-78

16c

-55

16d

-78

16d

-50

16e

-78

16f

-78

56:44 36:64 85:15 81:19 87:13 84:16

isopentyl nitrite in 1,2-dimethoxyethane (DME)/CHCl₃).

After treatment with 2,3-dichloro-5,6-dicyanobenzoquino-

ne (DDQ in DME, 55 °C) the naphthacenyl methyl ketone

(-)-22 ((2R,5S,12R)-2-acetyl-5-(dimethoxymethyl)-1,2,

3,4,5,12-hexahydro-5,12-epoxynaphthacen-2-yl RADO-

(Et)-ate) was obtained (60%, purified by medium-pressure

liquid chromatography). Saponification of (-)-22 with 1

M NaOH/EtOH gave (-)-23, the esterification of which

with p-methoxycinnamic anhydride (DMAP, CH₂Cl₂, 25

°C, 50 h) afforded (-)-24. The CD spectrum of (-)-24

showed a typical exciton type of Cotton effect²³(see

Figure 1) consistent with a negative chirality, which

implies the (R) configuration at C(2). The relative

configuration of center C(2) was confirmed by NOE

measurements in the ¹H NMR spectrum observed be-

tween the aromatic protons at C(6) and C(11) and the

protons of the RADO(Et) moiety of (-)-22.

The absolute configuration of C(2) in (-)-22 was

further confirmed by converting it into the known (-)-

7-deoxyidarubicinone ((-)-27).²⁴The treatment of crude

(-)-22 with Me₃SiOSO₂CF₃in CH₂Cl₂(0 °C), followed by

acetylation (Ac₂O/pyridine, 25 °C) and two recrystalliza-

tion from EtOAc furnished the diastereomerically pure

naphthacenyl methyl ketone (-)-25 (55%). Baeyer-

Villiger oxidation with SeO₂and H₂O₂led to the formate

(-)-26 (94%), which upon oxidation (4 N J ones-reagent,

acetone, 0-20 °C) and saponification (1 M NaOH/THF,

20 °C) afforded (-)-27 in 80% yield.

Syn t h esis of 6-[[(Am in oa lk yl)oxy]m et h yl]-4-d e-

m eth oxy-6,7-d id eoxyd a u n om ycin on es. Oxidation of

the dimethyl acetal of naphthacenecarbaldehyde (-)-25

with PDC (pyridium dichromate²⁵) led to a mediocre

yield (33%) of the corresponding methyl ester 28. The

reaction was accompanied with the formation of the

naphthacene-5,12-quinone 29. Oxidation of (-)-25 with

RuCl₃/NaIO₄or with KMnO₄/18-crown-6 ether/ben-

zene²⁷generated a complex mixture containing a small

(23) Harada, N.; Nakanishi, K. Circular Dichroic Spectroscopy-

Exciton Coupling in Organic Chemistry; University Science Books: Hill

Valley, CA, 1983. Wiesler, W. T.; Nakanishi, K. J . Am. Chem. Soc.

1989, 111, 9205.

(24) Tamoto, K.; Sugimori, M.; Terashima, S. Tetrahedron 1984, 40,

4617. Suzuki, M.; Matsumoto, T.; Abe, R.; Kimura, Y.; Terashima, S.

Chem. Lett. 1985, 57.

Alcoholysis of bromide (-)-32 with HOCH₂CH₂CH₂-

NHBoc in anhydrous THF and in the presence of AgOSO₂-

(25) O’Connor, B.; J ust, G. Tetrahedron Lett. 1987, 28, 3235.

Synthesis of Substituted Daunomycinones

J . Org. Chem., Vol. 61, No. 20, 1996 6961

CF₃gave (-)-33 (71%). Saponification of (-)-33 with

applied to the synthesis of analogues (-)-9, (-)-10, and

(-)-11. The reactions of (-)-32 with HO(CH₂)₄NHCOCF₃,

HO(CH₂)₅NHCOCF₃, and HO(CH₂)₂N(COCF₃)(CH₂)₃-

NHCOCF₃(AgOTf, THF) gave (-)-50 (79%), (-)-51 (63%),

and (-)-52 (75%), respectively. Oxidation (J ones) pro-

vided (-)-53 (82%), (-)-54 (77%), and (-)-55 (76%),

respectively. These compounds were hydrolyzed as above

into (-)-9 (26%), (-)-10 (30%), and (-)-11 (60%), respec-

tively. Amines (-)-9 and (-)-10 were characterized as

their p-nitrobenzamide (-)-56 (81%) and (-)-57 (81%),

respectively.

aqueous 1 M NaOH in THF (0 °C) cleaved selectively the

RADO(Et) ester group with formation of (-)-34 (84%).

At this stage we failed in our attempts to deprotect the

amino moiety.³⁰When (-)-34 was treated with gaseous

HCl in CH₂Cl₂(0 °C)³¹the benzyl chloride (-)-35 was

obtained in 90% yield. Aqueous 3 M HCl in AcOEt (20

°C, 12 h) provided the corresponding alcohol (-)-36 (74%).

These experiments showed that the Boc protective group

does not suit our purposes; we thus prepared the Alloc³²

-protected amine (-)-37 (80%) as above by treatment of

bromide (-)-32 with HO(CH₂)₃NHCOOCH₂CHdCH₂and

AgOTf. Selective saponification of (-)-37 with NaOH/

H₂O/THF provided (-)-38 (84%). Palladium-catalyzed

(Pd(Ph₃P)₄) reduction (Bu₃SnH) of the Alloc group,³³

followed by treatment with aqueous HCl (pH g 3.0 to

avoid ether cleavage) furnished (-)-39 (66%). In a

similar way, the analogues (-)-40 (60%), (-)-41 (53%),

and (-)-42 (70%) were prepared.

Because the direct oxidation of the anthracene units

of (-)-39 and (-)-42 led only to decomposition, we turned

back to the protected amines (-)-37 and (-)-40. Oxida-

tion of (-)-37 with aqueous 4 N J ones reagent (CrO₃/H₂-

SO₄) in acetone (0 °C, 30 min, 20 °C, 1.5 h) led to the

corresponding anthraquinone (-)-43 (91%). Saponifica-

tion of (-)-43 with LiOH/H₂O/THF gave (-)-44, which

was contaminated with about 10% of an unknown

compound. Treatment of this crude product with Pd-

(Ph₃P)₂(OAc)₂and Bu₃SnH in wet CH₂Cl₂(20 °C, 20 min)

afforded the unprotected amine. After acidification with

0.1 M aqueous HCl (to pH ) 2.3-3.0) and purification

by flash chromatography on silica gel, the pure hydro-

chloride (-)-8 was obtained in 45% yield. The structure

of this compound was given by its spectral data (see

Experimental Part) and was further characterized as its

p-nitrobenzamide (-)-45. Under similar conditions we

prepared also the diamine double hydrochloride (-)-12.

The reaction of bromide (-)-32 with N,N-bis(allyloxy)-

carbamate of 3-[(3′-aminopropyl)amino]propanol³⁴and

AgOTf (THF, molecular sieves) gave (-)-46 (84%). Oxi-

dation (J ones) led to anthraquinone (-)-47 (94%), the

saponification of which with aqueous 1 M LiOH in THF

(0 °C), followed by palladium-catalyzed reduction pro-

vided (-)-12 (16%) after acidification with aqueous HCl.

Another route toward the anthracycline analogue (-)-8

was opened that relies on the nucleophilic displacement

of bromide (-)-32 with 3-(trifluoroacetamido)propanol

(AgOTf, THF, 20 °C). The benzyl ether (-)-48 (87%) so-

obtained was oxidized (J ones) into (-)-49 (85%). Treat-

ment of (-)-49 with LiOH (THF, H₂O, 0 °C) cleaved the

RADO(Et) and acetate esters. Without isolating the

corresponding alcohol, the mixture was then treated with

aqueous 0.02 M NaOH (25 °C) and finally with aqueous

HCl to furnish (-)-8 (62%). The same method was

Glycosidation of the benzyl alcohol (-)-31 with the

appropriate protected ^L-daunosamine derivative (-)-58³⁵

in the presence of Me₃SiOTf in THF (-78 to -25 °C)

provided a 3:1 mixture of R- and â-pyranosides 60 in 25%

yield only. Yields were lower under Terashima’s condi-

tions³⁶(CH₂Cl₂/ether 2:1, -40 °C), probably because of

solubility problems. Adding CH₃CN led to a loss of

selectivity with formation of 1:1 mixture of R- and

â-anomers, probably because of the nitrilium ion effect.³⁷

Finally, we found that the slow addition of (-)-31 in

CHCl₃at -25 °C to a solution of (-)-58 pretreated with

Me₃SiOTf in THF at 0 °C (formation of intermediate 59)

led to a 2:1 mixture of the R- and â-anomers with 75%

yield. The R/â selectivity could be raised to 7:1 on

carrying out the glycosidation at 0 °C. Unfortunately,

the yield dropped to 15% under these conditions. The

desired glycoside (-)-60 was separated from its â-anomer

by high-pressure liquid chromatography and was isolated

in 50% yield. Oxidation of (-)-60 (J ones) gave (-)-61

(71%). Ester cleavage with LiOH/H₂O/THF and hydroly-

sis of the trifluoroacetamido moiety with aqueous 0.02

M NaOH, followed by acidification with aqueous 0.1 M

HCl, provided (-)-13 (66%; 23% overall yield based on

(-)-31).

(26) Carlsen, H. J .; Katsuki, T.; Martin, V. S.; Sharpless, K. B. J .

Org. Chem. 1981, 46, 3936.

(27) Lifshitz, E.; Goldfarb, D.; Vega, S.; Luz, Z.; Zimmermann, M.

J . Am. Chem. Soc. 1987, 109, 7280.

(28) Sala, T.; Sargent, M. V. J . Chem. Soc., Chem. Commun. 1978,

253.

(29) Wakup, R. D.; Cunnigham, R. T. Tetrahedron Lett. 1987, 28,

4019.

DNA In ter ca la tion . The molecular recognition of

DNA by small molecules is an important macromolecular

(30) Gutte, B.; Merrifield, R. B. J . Am. Chem. Soc. 1969, 91, 501.

(31) Houghten, R. A.; Beckman, A.; Ostresh, J . M. Int. J . Peptide

Protein Res. 1986, 27, 653.

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J . Org. Chem. 1987, 52, 4984.

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D. W. J . Org. Chem. 1977, 42, 3653. Schallenberg, E.; Calvin, M. J .

Am. Chem. Soc. 1955, 77, 2779.

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R.; Terashima, S. Bull. Chem. Soc. J pn. 1986, 59, 423.

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6962 J . Org. Chem., Vol. 61, No. 20, 1996

Dienes and Vogel

requirement for intercalation of these 2-napthacenyl

methyl ketones. Among the monoamine derivatives,

(-)-9 with a butyl chain seems to be the best adapted to

calf thymus DNA structure under the studied conditions.

It probably realizes a suitable arrangement for interac-

tion between its ammonium unit and the phosphate

backbone of DNA on one hand and simultaneous inter-

calation of its naphthacenequinone moiety on the other

hand. The fact that the analogues (-)-11 and (-)-12 with

diamino chains do not intercalate DNA suggests that

these systems have geometries that do not allow their

naphthacenequinone moieties to intercalate because their

bis-ammonium systems are held by the phosphate anions

away from the suitable base sequence. The inability of

the R-daunosaminyl analogue (-)-13 to intercalate shows

the severe steric demand of DNA for the 6-substituted

2-naphthacenyl methyl ketone system to enter between

the suitable base pairs.

F igu r e 2. Equilibrium binding isotherms of (-)-8 (9), (-)-9

(b) and (-)-10 (2) (r: concentration ratio of intercalator/DNA

in base pairs; free intercalator concentration).

receptor-drug interaction in the field of chemotherapy.

Intercalation is one of the ways that a foreign molecule

can interact with DNA.^12,38It involves compounds pos-

sessing a planar aromatic chromophore stacked between

the adjacent base pairs of DNA and stabilized by van der

Waals forces. Hydrogen bonding or ionic interaction

involving substituents attached to the chromophore

frequently impart further stabilization and even DNA

sequence selectivity to the binding process.^39,40The

relationship between structural alterations of DNA caused

by intercalators and biological activity such as frameshift

mutagenicity, B-DNA-transition, and antitumor activity

is now clearly established. Changing the structure of

RNA and DNA causes inhibition of some very important

enzymes such as DNA, RNA polymerases, and DNA

topoisomerases.⁴¹

Effect on Top oisom er a ses. Intercalators induce

supercoils in circular plasmids. Topoisomerase I enzyme

catalyzes the relaxation of supercoils, a phenomenon that

depends on the concentration and affinity of the inter-

calator and that can be monitored by electrophoresis

(unwinding assay).⁴⁸pGEM plasmid was taken in en-

tirely supercoiled form and was incubated (37 °C, 30 min,

in the dark) with yeast topoisomerase I and different

concentrations (50, 100, 200, 500 µM) of compounds (-)-

8, (-)-13, (-)-39, and (-)-42.⁴⁹After protein separation,

samples were extracted (phenol/CHCl₃) and subjected to

agarose gel electrophoresis using daunomycin (2) as

standard. The topoisomerase I-induced relaxation was

inhibited only with (-)-8 at 200 µM. Under the same

conditions, daunomycin inhibited the DNA unwinding at

20 µM. DNA intercalators also inhibit topoisomerase II,

an important enzyme that catalyzes many topological

interconversions of DNA loops during replication and

transcription.⁵⁰Anthracyclines are known to inhibit both

DNA relaxation and DNA strand religation by topo-

isomerase II.⁵¹Inhibition of these two activities was

tested for (-)-8, (-)-11, (-)-12, and (-)-13⁴⁹using human

topoisomerase II enzyme and pGEM DNA. Samples of

different concentrations (50-500 µM) were incubated at

37 °C in the dark. After protein digestion (proteinase

K) and extraction (phenol/CHCl₃), aliquots were subjected

to agarose gel electrophoresis. In order to assay the

topoisomerase II-induced relaxation inhibition, agarose

without ethidium bromide was used, whereas agarose

with ethidium bromide was employed in the topo-

isomerase II-induced DNA cleavage assays (distinction

between linear DNA band and relaxed DNA bands).⁵²

Topoisomerase II-induced relaxation was inhibited at

150, 50, 150, 400, and 5 µM for (-)-8, (-)-11, (-)-12, (-)-

13, and 2, respectively. Inhibition of the topoisomerase

II-induced DNA strand religation was observed only for

(-)-8 at a concentration of 50 µM (2 µM for 2 under the

same conditions).

Binding affinities of naphthacenequinones (-)-8-(-)-

13 and of naphthacene derivatives (-)-39 and (-)-42 to

calf thymus DNA Type XV were evaluated by absorbance

titration⁴²and by fluorescence titration.⁴³The data were

analyzed by Scatchard-plots.⁴⁴The dashed lines shown

in Figure 2 for (-)-8, (-)-9, and (-)-10 were calculated

according to the neighbor exclusion model⁴⁵and were

obtained with n ) 7.0 ( 0.1, 4.6 ( 0.4, and 2.0 ( 0.2,

respectively, as exclusion parameters in base pairs. In

the cases of (-)-8 and (-)-9 (data were not accurate

enough for (-)-10) we calculated K_b) (6.1 ( 0.1) × 10⁴

M^-1and (1.1 ( 0.1) × 10⁵M^-1, respectively, as intrinsic

binding constants. These results suggests that (-)-8 and

(-)-9 are better intercalators than daunomycinone (K_b

) (1-3) × 10⁴M^-1

)

but weaker intercalators than

adriamycin (1, K_b) (2-4) × 10⁶M^-1) and daunomycin

(2, K_b) (4-6) × 10⁶M^-1or 1.2 × 10⁶M^-1under our

conditions⁴⁷).

Compounds (-)-39 and (-)-42 did not intercalate calf

thymus DNA. This shows that the quinonic moiety is a

(38) Pullman, B., J ortner, J ., Eds. Molecular Basis of Specificity in

Nucleic Acid-Drug Interactions; Kluwer Academic Publishers: Dor-

drecht, The Netherlands, 1990.

(39) Lerman, L. S. J . Mol. Biol. 1961, 3, 18.

(47) Chaires, J . B. Biopolymers 1985, 24, 403. Capranico, G.; Zunino,

F.; Kohn, K. W.; Pommier, Y. Biochemistry 1990, 29, 562.

(48) Corbett, A. H.; Hong, D.; Osheroff, N. J . Biol. Chem. 1993, 268,

14394.

(49) Other amines were not assayed because of lack of material.

(50) Liu, L. F. Ann. Rev. Biochem. 1989, 58, 351. Gasser, S. M.;

Larouche, T., Falquet, J .; Boy de la Tour, E.; Laemmli, U. K. J . Mol.

Biol. 1986, 188, 613.

(51) Pommier, Y.; Minford, J . K.; Schwartz, R. E.; Zwelling, L. A.;

Kohn, K. W. Biochemistry 1985, 24, 6410. See also: D’Inalci, M. Curr.

Opin. Oncol. 1993, 5, 1023.

(52) Muller, M. T.; Spitzner, J . R.; DiDonato, J . A.; Mehta, V. B.;

Tsuitsui, K. Biochemistry 1988, 27, 8369.

(40) Lavery, R.; Pullman, B. Int. J . Quantum Chem. 1981, 20, 259.

Kumar, C. V.; Asuncion, E. H. J . Am. Chem. Soc. 1993, 115, 8547.

Neidle, S.; Abraham, Z. CRC Critical Rev. Biochem. 1984, 17, 73.

(41) Hurley, L. H. J . Med. Chem. 1989, 9, 2027.

(42) Chaires, J . B.; Dattagupta, N.; Crothers, D. M. Biochemistry

1982, 21, 3933.

(43) Strothkamp, K. G.; Strothkamp, R. E. J . Chem. Educ. 1994,

71, 77.

(44) Scatchard, G. Ann. N.Y. Acad. Sci. 1949, 51, 660.

(45) McGhee, J . D.; Von Hippel, P. H. J . Mol. Biol. 1974, 86, 469.

(46) Roche, C. J .; Berkowitz, D.; Sulikowski, G. A.; Danishefsky, S.

J .; Crothers, D. M. Biochemistry 1994, 33, 936.

Synthesis of Substituted Daunomycinones

J . Org. Chem., Vol. 61, No. 20, 1996 6963

aqueous 0.02 M NaOH (0.2 mL, changed to red) at 25 °C for

20 min. It was then neutralized (aqueous 0.1 M HCl) and

washed with Et₂O (10 mL, five times). The aqueous layer was

evaporated, and the yellow residue was taken up with EtOH

(1 mL) to allow filtration of the insoluble NaCl. After filtration

The nonquinonic analogues (-)-39 and (-)-42 were also

assayed as above. They did not show any inhibiting

activity (up to 500 µM concentration) toward topo-

isomerase II.

Our results demonstrate that intercalation is a neces-

sary condition for our anthracycline analogues to act as

inhibitors of topoisomerase II-mediated DNA strand

religation. The weak inhibiting activities observed for

the nonintercalating systems (-)-11, (-)-12, and (-)-13

toward topoisomerase II-induced DNA relaxation might

be assigned to the association of the ammonium ions of

these compounds with the phosphate DNA backbone, a

nonspecific effect.

(Celite), Et₂O was added to precipitate 6 mg (62%), yellow

powder: [R]²⁵) -53 (c ) 0.25, MeOH); H NMR (400 MHz,

CD₃OD) δ 8.23, 7.89 (2m), 4.98 (2d, J ) 10), 3.91 (t, J ) 5),

3.30-2.90 (m), 2.41 (s), 2.15-1.85 (m).

(8R)-8-Acet yl-6,8-d ih yd r oxy-11-[(4′-a m in ob u t yloxy)-

m eth yl]-7,8,9,10-tetr a h yd r on a p h th a cen e-5,12-d ion e Hy-

d r och lor id e ((-)-9). (-)-53 (10 mg, 13 µmol) was dissolved

in degassed THF/H₂O 3/1 (2 mL). After the solution was cooled

to 0 °C, LiOH·H₂O (4 mg, 95 µmol) was added and the red

mixture was stirred in the dark for 20 min. After addition of

saturated aqueous solution of NH₄Cl (5 mL), the product was

extracted (CHCl₃, 15 mL, three times). The organic extracts

were dried (MgSO₄), and solvent evaporation yielded a yellow

solid that was immediately dissolved in THF (0.2 mL) and H₂O

(2 mL). The THF was evaporated and the suspension was

treated with aqueous 0.02 M NaOH (0.1 mL, changed to red)

at 25 °C for 30 min. It was then neutralized (aqueous 0.1 M

HCl) and washed with Et₂O (10 mL, five times). The aqueous

layer was evaporated, the yellow residue was taken up with

EtOH (99.8%, 1 mL), and the insoluble NaCl was filtered off

(cotton-wool). The filtrate was concentrated, and Et₂O was

added to precipitate about 2 mg (30%) of (-)-9 as a yellow

powder. The quantity was measured more precisely (1.92 mg)

by ¹H NMR using tris-HCl as internal standard. The product

can be purified in low yield by FC in the dark (CH₂Cl₂/MeOH

4/1, R_f) 0.4, UV, alizarin). Other eluants such as i-PrOH/

MeCN or EtOH/MeCN with 1% of CF₃COOH led to decompo-

sition. Separation by HPLC on reversed phase (MN, CC 125/

4, Nucleosil 100-5, C18 AB) was also attempted. With MeCN/

H₂O 1/1 as eluant, no separation was achieved, while with a

more polar mixture, no fractions of the desired product could

be detected (probably due to low sensibility of Kontron IOTA

2 refractometer). Product degradation can be observed on

storing in the solid state under argon at -18 °C: [R]²⁵_D) -55

(c ) 0.2, EtOH 95%); ¹H NMR (400 MHz, CD₃OD) δ 8.29, 7.91

(2m), 5.07 (d, ²J ) 10), 3.79 (m), 3.30-2.90 (m), 2.42 (s), 2.10-

1.90, 1.81 (2m).

Con clu sion

Optically pure (-)-6-[[(aminoalkyl)oxy]methyl]-4-

demethoxy-6,7-dideoxydaunomycinone derivatives have

been obtained through asymmetric Diels-Alder additions

of 1-(dimethoxymethyl)-2,3,5,6-tetramethylidene-7-oxa-

bicyclo[2.2.1]heptane. Those bearing (3′-aminopropyl)-

oxy ((-)-8), (4′-aminobutyl)oxy ((-)-9), and (5′-aminopen-

tyl)oxy ((-)-10) chains are calf thymus DNA intercala-

tors, whereas analogues with two amine units such as

[N-(3′′-aminopropyl)-2′-aminoethyl]oxy ((-)-11) or [N-(3′′-

aminopropyl)-3′-aminopropyl]oxy ((-)-12) chain attached

at the 6-methyl moiety do not intercalate DNA. This was

also the case for the 6-[(R-daunosaminyloxy)methyl]-4-

demethoxy-6,7-dideoxydaunomycinone ((-)-13). Only

compounds that were calf thymus DNA intercalators

were inhibitors of human topoisomerase II-induced DNA

strand religation. The best results were obtained with

the (4′-aminobutyl)oxy derivative (-)-9 that showed an

intrinsic binding constant with calf thymus DNA K_b)

(1.1 ( 0.1) × 10⁵M^-1(n ) 4.6 ( 0.4); this is about 10

times smaller than the binding constant measured for

daunomycin.

(8R)-8-Acetyl-6,8-d ih yd r oxy-11-[[(5′-a m in op en tyl)oxy]-

m eth yl]-7,8,9,10-tetr a h yd r on a p h th a cen e-5,12-d ion e Hy-

d r och lor id e ((-)-10). The same procedure as for the prepa-

ration of (-)-9 was followed, starting with (-)-54 (12 mg, 16

µmol). FC (-18 °C, in the dark, silica gel, CH₂Cl₂/MeOH 4/1,

Exp er im en ta l Section

Gen er a l Rem a r k s. See ref 53. None of the procedures

were optimized. Flash column chromatography (FC) was

performed on Merck silica gel (230-400 mesh). Thin layer

chromatography (TLC) was carried out on silica gel (Merck

aluminum foils). ¹H NMR signal assignments were confirmed

by double irradiation experiments and, when required, by 2-D-

NOESY and COSY spectra. J values are given in Hz.

(8R)-8-Acetyl-6,8-d ih yd r oxy-11-[(3′-a m in op r op yloxy)-

m eth yl]-7,8,9,10-tetr a h yd r on a p h th a cen e-5,12-d ion e Hy-

d r och lor id e ((-)-8). Meth od A. A mixture of (-)-44 (12 mg,

24 µmol), Pd(Ph₃P)₂(OAc)₂(catalytic amount), and Bu₃SnH (13

µL, 2 equiv) was stirred in wet CH₂Cl₂(2 mL) in the dark at

20 °C for 20 min. After solvent evaporation, the residue was

dissolved in H₂O (3 mL) and acidified to pH 2.5-3.0 with

aqueous 0.1 M HCl. It was then washed with Et₂O (10 mL,

seven times) and evaporated to give a yellow solid that can be

purified by FC (silica gel, 5 g, CH₂Cl₂/MeOH 4/1, R_f0.19

(alizarine)) to yield 5 mg of (-)-8 (45%). The product is very

sensitive to light.

Meth od B. (-)-49 (16 mg, 21 µmol) was dissolved in

degasssed THF (1 mL). After the solution was cooled to 0 °C

aqueous 1 M LiOH (0.1 mL) was added and the mixture was

stirred in the dark for 20 min. After addition of saturated

aqueous solution of NH₄Cl (5 mL) the product was extracted

(CHCl₃, 15 mL, three times). The organic extracts were dried

(MgSO₄). Solvent evaporation yielded a yellow solid that was

immediately dissolved in THF (0.2 mL) and H₂O (2 mL). The

THF was evaporated, and the suspension was treated with

R_f) 0.33, UV, alizarin) gave 2 mg (26%) of a yellow gum:

[R]²⁵) -49 (c ) 0.2, EtOH 95%); H NMR (400 MHz, CD₃-

OD) δ 8.22, 7.86 (2m), 5.02, 4.96 (2d, ²J ) 10), 3.71, 3.16 (2m),

3.09, 2.95 (2d, J ) 18.5), 2.39 (s), 2.08-1.90, 1.70, 1.50 (3m).

(8R)-8-Acetyl-6,8-d ih yd r oxy-11-[[2′-[(3′′-a m in op r op yl)-

a m in o]eth oxy]m eth yl]-7,8,9,10-tetr a h yd r on a p h th a cen e-

5,12-d ion e Hyd r och lor id e ((-)-11). The same procedure as

for the preparation of (-)-9 was followed, starting with (-)-

55 (8 mg, 9 µmol): yield 3 mg (60%); [R]²⁵) -6 (c ) 0.1,

EtOH 95%); ¹H NMR (400 MHz, CD₃OD) δ 8.29, 7.92 (2m),

4.92 (d, ²J ) 10), 4.08 (br s), 3.70, 3.20-2.90 (2m), 2.41 (s),

2.12-1.95, 1.65 (2m).

(8R)-8-Acetyl-6,8-d ih yd r oxy-11-[[[3′-[(3′′-a m in op r op yl)-

a m in o)p r op yl]oxy]m e t h yl]-7,8,9,10-t e t r a h yd r on a p h -

th a cen e-5,12-d ion e Hyd r och lor id e ((-)-12). (-)-46 (19 mg,

22 µmol) was dissolved in degassed THF (2 mL). After the

solution was cooled to 0 °C, aqueous 1 M LiOH (0.1 mL) was

added and the mixture was stirred in the dark for 10 min.

After addition of a saturated aqueous solution of NH₄Cl (5 mL)

the product was extracted (CHCl₃, 15 mL, three times) and

the combined organic extracts were dried (MgSO₄). Solvent

evaporation yielded a yellow solid with about 10% impurity

that could not be separated by chromatography or crystalliza-

tion. The product obtained above, Pd(Ph₃P)₂(OAc)₂(catalytic

amount) and Bu₃SnH (20 µL, 74 µmol), was stirred in wet CH₂-

Cl₂(2 mL) in the dark at 20 °C for 40 min. After solvent

evaporation the residue was dissolved in H₂O (3 mL) and

acidified to pH 2.5-3.0 with aqueous 0.1 M HCl. It was then

washed with Et₂O (10 mL, seven times) and evaporated to give

(53) Gasparini, F.; Vogel, P. J . Org. Chem. 1990, 55, 2451. Sevin,

A.-F.; Vogel, P. Ibid. 1994, 59, 5920.

6964 J . Org. Chem., Vol. 61, No. 20, 1996

Dienes and Vogel

2 mg (16%) of a yellow solid: mp 130-135 °C (Et₂O/EtOH);

[R]²⁵_D) -2 (c ) 0.1, EtOH 95%); ¹H NMR (400 MHz, CD₃OD)

δ 8.29, 7.92 (2m), 4.92 (d), 3.91 (t, ²J ) 2.5), 3.35-2.90 (m),

2.41 (s), 2.12-1.95 (m).

5.18, 4.90 (2s), 3.55 (dd, ²J ) 12.5, ³J ) 2.5), 3.22 (d, ³J )

12.5), 2.95, 2.39 (2s).

3′-Oxobu t-1′-en -2′-yl (1S,5R,7S)-6,8-Dioxa -2-oxo-3-p r o-

pyl-3-azabicyclo[3.2.1]octan e-7-exo-car boxylate (3-oxobu t-

1-en -2-yl RADO(P r )-a te, (-)-16f). RADO(Pr)-OH⁴¹(215 mg,

1 mmol) was boiled under reflux in SOCl₂(15 mL) for 1 h.

The excess of reagent was distilled off to yield a yellowish oil

that was used without further purification. A mixture of

diacetyl (0.1 mL, 1.12 mmol), Et₃N (0.3 mL, 2 mmol), and the

acid chloride obtained above was stirred for 24 h at 20 °C in

anhydrous toluene (3 mL). It was then poured into EtOAc

(20 mL) and washed with aqueous 0.1 M HCl (15 mL, three

times), with H₂O (15 mL, three times), and with brine (15 mL,

three times). After drying (MgSO₄) and solvent evaporation,

FC (silica gel, 10 g, EtOAc/light petroleum 1/1 (KMnO₄)) gave

(8R)-8-Acetyl-6,8-d ih yd r oxy-11-[[(2′,3′,6′-tr id eoxy-4′-h y-

d r oxy-3′-a m in o-r-^L-lyxo-h e xop yr a n osyl)oxy]m e t h yl]-

7,8,9,10-tetr a h yd r on a p h th a cen e-5,12-d ion e Hyd r och lo-

r id e ((-)-13). (-)-61 (11 mg, 11 µmol) was dissolved in

degassed THF (0.1 mL). After the solution was cooled to 0

°C, aqueous 1 M LiOH (0.1 mL) was added and the mixture

was stirred in the dark for 20 min. After addition of a

saturated aqueous solution of NH₄Cl (5 mL) the product was

extracted (CHCl₃, 15 mL, three times), and the combined

organic extracts were dried (MgSO₄). Solvent evaporation

yielded a yellow solid that was immediately dissolved in THF

(2 mL) and H₂O (2 mL). THF was then evaporated, and the

suspension was treated with aqueous 0.02 M NaOH (0.2 mL,

changed to red) at 25 °C for 20 min. It was then neutralized

(aqueous 0.1 M HCl) and washed with Et₂O (10 mL, five

times). The aqueous layer was evaporated, and the yellow

residue was taken up with EtOH (1 mL) to allow filtration of

the insoluble NaCl. After filtration (Celite), Et₂O was added

119 mg (42%) of a colorless oil: [R]²⁵) -50 (c ) 1.0, CHCl₃).

(1′R,4′R,8′S)-4′-Acet yl-8′-(d im et h oxym et h yl)-9′,10′-d i-

m eth ylid en e-11′-oxa tr icyclo[6.2.1.0²′^,7′]u n d ec-2′(7′)-en -4′-

yl (1R,5S,7R)-3-eth yl-2-oxo-6,8-d ioxa -3-a za bicyclo[3.2.1]-

octa n e-7-exo-ca r boxyla te ((-)-17e). (-)-16e⁴¹(500 mg, 1.86

mmol) was dissolved in anhydrous CH₂Cl₂(9 mL) under Ar.

The solution was cooled to -78 °C, and BF₃‚Et₂O (3.5 mL, 28

mmol, 15 equiv) was added under vigorous stirring. After 30

min, a solution of 15⁴⁰(490 mg, 2.23 mmol, 1.2 equiv) in

anhydrous CH₂Cl₂(9 mL) was introduced slowly. The mixture

was stirred for 1 week at -78 °C, and then it was poured at

once into an ice-cold mixture of EtOAc and saturated aqueous

solution of NaHCO₃(100 mL, 1/1). The organic layer was

washed with aqueous NaHCO₃(50 mL), with H₂O (50 mL,

twice), and finally with brine (50 mL, three times). After

drying (MgSO₄) and evaporation of the solvent, the residue

was purified on Florisil (50 g, 60-100 mesh, EtOAc/light

petroleum 1/1) to give 50 mg of 15 (R_f0.9) and 825 mg of a

mixture of isomeric adducts (R_f0.6). Two successive chro-

matographies (Lobar, CH₂Cl₂/EtOAc) gave an analytical sample

of (-)-17e (69%) as an amorphous solid: mp 77-80 °C (hexane/

Et₂O); [R]²⁵_D) -1 (c ) 1.0, CHCl₃); ¹H NMR (250 MHz, CDCl₃)

δ 5.80 (d, ³J ) 2), 5.37, 5.30, 5.28, 5.01 (4s), 4.95, 4.82 (2s),

to precipitate 4 mg (66%) of a yellow powder: mp 172-174

°C; [R]²⁵) -22 (c ) 0.1, MeOH); H NMR (400 MHz, CD₃-

OD) δ 8.23, 7.90 (2m), 5.48 (d, ³J ) 11), 5.30 (m), 5.22 (d, ³J )

1), 5.00-4.80 (m), 4.26, 3.20-1.95 (2m), 2.42 (s), 2.20-1.95

(m), 1.28 (d, J ) 6).

3′-Oxobu t-1′-en -2′-yl (2R)-2-Meth oxy-2-p h en yla ceta te

((+)-16b). A mixture of (+)-2-methoxy-2-phenylacetic acid

(100 mg, 0.6 mmol), DMF (0.047 mL, 0.6 mmol), and oxalyl

chloride (0.25 mL, 0.3 mmol) was stirred for 1 h in hexane at

20 °C. It was then decanted from the white precipitate and

evaporated to give 90 mg of a colorless oil that was dissolved

without further purification in anhydrous toluene (5 mL) at 0

°C, and diacetyl (0.053 mL, 0.6 mmol) with Et₃N (0.18 mL,

1.2 mmol) were added. The mixture was stirred at 20 °C for

24 h. It was then poured into EtOAc (10 mL) and washed

with aqueous 0.1 M HCl (10 mL, three times), with H₂O (10

mL, three times), and with brine (10 mL, three times). After

drying (MgSO₄), evaporation, FC (silica gel, 10 g, light

4.60, 4.54 (2d, J ) 0.5), 3.59, 3.57 (2s), 3.47 (dd, J ) 2, J )

12), 3.40, 3.38 (2q, J ) 7.2), 3.20 (d, J ) 12), 3.01 (dt, J )

18.5, ³J ) 2), 2.60-2.38 (m), 2.20-2.05 (m), 2.08 (s), 1.90-

petroleum/EtOAc 5/1 (KMnO₄)) gave 49 mg (35%) of a colorless

1.70 (m), 1.09 (t, J ) 7.2).

oil: [R]²⁵) +4 (c ) 1.0, CHCl₃); H NMR (250 MHz, CDCl₃)

(2 ′R ,5 ′S ,1 2 ′R )-2 ′-Ac e t y l -5 ′-(d i m e t h o x y m e t h y l )-

1′,2′,3′,4′,5′,12′-h exa h yd r o-5′, 12′-ep oxyn a p h t h a cen -2′-yl

(1R,5S,7R)-3-E t h yl-2-oxo-6,8-d ioxa -3-a za b icyclo[3.2.1]-

octa n e-7-exo-ca r boxyla te ((-)-22). A mixture of crude

adduct (-)-17e and isomers (650 mg, 1.33 mmol), molecular

sieves (3 Å, 10 pieces), isopentyl nitrite (250 µL, 1.86 mmol,

1.4 equiv), and anthranilic acid (430 mg in MeOCH₂CH₂OMe

(DME, 2 mL), 3.13 mmol, 2.4 equiv) was stirred for 45 min in

CHCl₃(6 mL) at 0 °C. A deep red precipitate was formed.

The mixture was heated rapidly under vigourous stirring to

55 °C and allowed to react until the end of the gas evolution

(about 25 min). It was then diluted with CHCl₃(15 mL), and

3,6-dichloro-2,3-dicyanobenzoquinone (DDQ, 328 mg in DME

(2 mL), 1.45 mmol, 1.1 equiv) was added. After 35 min at 55

°C, it was cooled, diluted (CHCl₃, 50 mL), and washed with

5% aqueous NaHSO₃(30 mL, three times), with a saturated

aqueous solution of NaHCO₃(30 mL, three times), and finally

with brine (30 mL, three times). Drying and solvent evapora-

tion gave an oil that was subjected to chromathography (silica

δ 7.56-7.30 (m), 5.97, 5.58 (2d, J ) 3), 4.99, 3.52, 2.22 (3s).

3′-Oxobu t-1′-en -2′-yl (1S)-3-Oxo-4,7,7-tr im eth yl-2-oxa -

bicyclo[2.2.1]h ep ta n e-1-ca r boxyla te ((-)-16c). A mixture

of diacetyl (0.1 mL, 1.12 mmol), Et₃N (0.3 mL, 2 mmol), and

camphanic acid chloride (217 mg, 1 mmol) was stirred at 20

°C in anhydrous toluene (3 mL) for 24 h. It was then poured

into EtOAc (20 mL) and washed with aqueous 0.1 M HCl (15

mL, three times), with H₂O (15 mL, three times), and finally

with brine (15 mL, three times). After drying (MgSO₄) and

solvent evaporation, FC (silica gel, 10 g, EtOAc/light petroleum

1/1 (KMnO₄)) gave 157 mg (60%) of colorless crystals: mp 61-

64 °C (Et₂O); [R]²⁵_D) -2 (c ) 1.0, CHCl₃); ¹H NMR (250 MHz,

CDCl₃) δ 6.02, 5.75 (2d, J ) 2.8), 2.48 (ddd, J ) 13.5, J )

11, 4.5), 2.09 (ddd, ²J ) 13.5, ³J ) 9.5, 4.5), 1.96 (ddd, ²J )

13.5, ³J ) 11, 4.5), 1.68 (ddd, ²J ) 13.5, ³J ) 9.5, 4.5), 2.4,

1.12, 1.10, 1.08 (3s).

3′-Oxobu t-1′-en -2′-yl (1S,5R,7S)-3-Meth yl-2-oxo-6,8-d i-

oxa -3-a za bicyclo[3.2.1]octa n e-7-exo-ca r boxyla te (3-Ox-

obu t-1-en -2-yl RADO(Me)-a te, (-)-16d ). RADO(Me)-OH⁴¹

(187 mg, 1 mmol) was boiled under reflux in SOCl₂(15 mL)

for 1 h. The excess reagent was distilled off, and the residue

was dissolved in EtOAc and precipitated with Et₂O to yield a

pale yellow solid that was used without further purification.

A mixture of diacetyl (0.1 mL, 1.12 mmol), Et₃N (0.3 mL, 2

mmol), and the acid chloride obtained above was stirred for

36 h at 20 °C in anhydrous toluene (3 mL). It was then poured

into EtOAc (20 mL) and washed with aqueous 0.1 M HCl (15

mL, three times), with H₂O (15 mL, three times), and finally

with brine (15 mL, three times). After drying (MgSO₄) and

solvent evaporation, FC (silica gel, 10 g, EtOAc/light petroleum

3/1 (KMnO₄)) gave 153 mg (60%) of colorless crystals: mp 113-

gel, 50 g, EtOAc/light petroleum 1/1, then 3/1, R_f0.6).

mixture of starting material and different isomers of (-)-22

was obtained and then separated on a Lobar column (EtOAc/

light petroleum 1/1) to yield 180 mg of 17e (72% conversion),

370 mg of pure (-)-22, and 80 mg of isomers of (-)-22.

Recrystallization of the second fraction ((-)-22) gave colorless

crystals: mp 98-102 °C (Et₂O); [R]²⁵_D) -31 (c ) 1.0, CHCl₃);

¹H NMR (250 MHz, CDCl₃) δ 7.72, 7.52 (2s), 7.72 (m), 7.38,

7.36 (2d, J ) 3.2), 5.53, 5.05 (2s), 4.82 (d, J ) 2), 4.00, 3.42,

3.75, 3.65 (4s), 3.20-2.90 (m), 2.77 (dd, J ) 12, J ) 2), 2.62

(dm, J ) 15), 2.50 (d, J ) 12), 2.40 (dm, J ) 15), 2.20-2.00

(m), 2.05 (s), 1.90-1.70 (m), 0.95 (t, J ) 7).

(2R,5S,12R)-5-(Dim eth oxym eth yl)-1,2,3,4,5,12-h exa h y-

d r o-2-h yd r oxy-5,12-ep oxyn a p h th a cen -2-yl Meth yl Ke-

ton e ((-)-23). (-)-22 (28 mg, 0.05 mmol) was dissolved in

114 °C (Et₂O); [R]²⁵) -77 (c ) 1.0, CHCl₃); ¹H NMR (250

MHz, CDCl₃) δ 6.00, 5.76 (2d, ²J ) 3.0), 5.97 (d, ³J ) 2.5),

Synthesis of Substituted Daunomycinones

J . Org. Chem., Vol. 61, No. 20, 1996 6965

EtOH (3 mL, at 25 °C). Aqueous 0.1 M NaOH (0.5 mL) was

added, and the mixture was stirred for 10 min. It was then

neutralized with aqueous 0.2 M HCl, and EtOH was evapo-

rated. The residue was dissolved in EtOAc (20 mL) and

washed with brine (10 mL, three times). Drying (MgSO₄) and

) 2.5), 3.08 (d, ²J ) 12), 2.58 (s), 2.60-2.50 (m), 2.27 (s), 2.20-

2.05 (m), 1.03 (t, J ) 7).

(8R)-8-Acetyl-7,8,9,10-tetr ah ydr o-6,8,11-tr ih ydr oxyn aph -

th a cen e-5,12-d ion e ((-)-27). (-)-25 (16 mg, 28 µmol) was

dissolved in acetone (4 mL, 0 °C). J ones reagent (4 N, 0.12

mL, 0.17 mmol) was added dropwise, and the reaction mixture

was stirred in the dark for 30 min at 0 °C and for 3 h at 20 °C.

The excess of the oxidant was destroyed with i-PrOH (1 mL,

changed to green). After solvent evaporation, the residue was

taken up with EtOAc (20 mL) and washed with 10% aqueous

NaHCO₃(10 mL) and finally with brine (10 mL, four times).

After drying (MgSO₄) and solvent evaporation, the yellow

residue was immediately treated with 0.2 mL of aqueous 1 M

NaOH in THF/H₂O (2 mL, 1/1) at 0 °C. After being stirred

for 4 h at 20 °C, the solution was neutralized with 0.1 M

aqueous HCl and extracted with CHCl₃(10 mL, three times).

The combined extracts were washed with brine (15 mL, three

times). Drying (MgSO₄), solvent evaporation, and FC (silica

gel, 1 g, CH₂Cl₂/EtOAc 6/1, R_f0.45 (red spot)) gave 8 mg (80%)

of red crystals, mp 216 °C (benzene) (lit.²⁴mp 218-219 °C

(benzene)). Spectral data were in agreement with those

reported for this compound.²⁴

solvent evaporation yielded 17 mg (89%) of colorless crystals:

mp 68-70 °C (Et₂O); [R]²⁵) -19 (c ) 1.0, CHCl₃); H NMR

(250 MHz, CDCl₃) δ 7.70 (m), 7.66, 7.51 (2s), 7.41 (m), 5.54,

5.08, 3.73, 3.66 (4s), 2.90 (ddd, J ) 18, J ) 4, 3), 2.62 (dm,

²J ) 18), 2.21 (s), 2.15-2.00 (m), 1.98-1.79 (m), 1.62-1.52

(m).

(2R,5S,12R)-2-Acetyl-5-(d im eth oxym eth yl)-2-h yd r oxy-

1,2,3,4,5,12-h exa h yd r o-5,12-ep oxyn a p h th a cen -2-yl 3-(4′-

Meth oxyp h en yl)p r op -2-en oa te ((-)-24). (-)-23 (9 mg, 24

µmol) was dissolved in anhydrous Et₂O (1 mL) at 0 °C.

Paramethoxycinnamic acid anhydride (25 mg, 48 µmol) and

4-(dimethylamino)pyridine (2 mg) were added. After being

stirred at 40 °C for 50 h, the mixture was poured into ice-

water and extracted with EtOAc (10 mL, three times). The

combined organic extracts were washed with aqueous 0.1 M

HCl (10 mL, twice), a saturated aqueous solution of NaHCO₃

(10 mL, twice), and finally with brine (10 mL, twice). Drying

(MgSO₄), solvent evaporation, FC (silica gel, 1 g, EtOAc/light

petroleum 1/1, R_f0.5 (KMnO₄)) afforded 4 mg (31%) of a pale

yellow solid: mp 99-102 °C (Et₂O); ¹H NMR (250 MHz, CDCl₃)

δ 7.68, 7.50 (2s), 7.62 (m), 7.22 (m), 6.86 (d, ³J ) 15), 6.62 (m),

5.58 (s), 5.05 (s), 5.04 (d, ³J ) 15), 3.82, 3.72, 3.68 (3s), 2.93

(dm, ²J ) 18), 2.62 (m), 2.42 (d, ²J ) 18), 2.18 (m), 2.15 (s),

2.10-1.97 (m).

(2′R )-12′-Ac e t o x y -2′-a c e t y l-5′-(m e t h o x y c a r b o n y l)-

1′,2′,3′,4′-tetr a h yd r on a p h th a cen -2′-yl (1R,5S,7R)-3-Eth yl-

2-oxo-6,8-d ioxa -3-a za b icyclo[3.2.1]oct a n e -7-exo-ca r b -

oxyla te (28). (-)-25 (20 mg, 36 mmol) was dissolved in DMF

(2 mL) at 20 °C. Activated molecular sieves (3 Å), MeOH (10

mL), and PDC (80 mg, 6 equiv) was added, and the mixture

was stirred for 24 h. It was then diluted with CH₂Cl₂and

filtered (Celite), and the solvent was evaporated. The residue

was directly subjected to FC (silica gel, 10 g, CH₂Cl₂/EtOAc

3/1) to give 7 mg (33%) of 28 (R_f0.5) and 4 mg of 29 (R_f0.85).

(2′R)-12′-Acetoxy-2′-a cetyl-5′-for m yl-1′,2′,3′,4′-tetr a h y-

d r on a p h th a cen -2′-yl (1R,5S,7R)-3-Eth yl-2-oxo-6,8-d ioxa -

3-azabicyclo[3.2.1]octan e-7-exo-car boxylate ((-)-25). Crude

adduct 22 (563 mg, 1 mmol) was dissolved in anhydrous CH₂-

Cl₂(70 mL) at 0 °C. TMSOTf (1.0 mL, 6 equiv) was added at

once, and the red mixture was stirred for 45 min. It was then

rapidly poured into a mixture of EtOAc and saturated aqueous

solution of NaHCO₃(100 mL, 4:1, 0 °C). The organic layer

was washed with a saturated aqueous solution of NaHCO₃(30

mL), with H₂O (30 mL, twice), and with brine (30 mL, three

times). After drying (MgSO₄), the solvent was evaporated. The

brown solid was immediately treated with pyridine (5 mL) and

Ac₂O (5 mL) under Ar at 0 °C. After 3 h of stirring at 20 °C,

the solution was poured into ice-water and extracted with

EtOAc (30 mL, three times). The combined organic extracts

were washed with aqueous 1 M H₂SO₄(30 mL, three times),

with saturated aqueous solution of NaHCO₃(30 mL, three

times), and finally with brine (30 mL, three times), dried

(MgSO₄), and evaporated. The residue was purified by two

successive crystallizations (EtOAc) to yield 250 mg of pure (de

> 95% by ¹H NMR: ¹³C-¹H satellites) (-)-25. The filtrate

was purified by FC (silica gel, 15 g, CH₂Cl₂/EtOAc 3/1, R_f0.38

(yellow spot)) to give 120 mg of a yellow solid from which 60

mg of pure (-)-25 can be obtained by two recrystallizations

from EtOAc. Yield: 310 mg (55%) of optically pure (-)-25 and

60 mg (11%) of a 1/1 mixture of diastereoisomers. Compound

(-)-25 was a yellow powder: mp 216 °C (EtOAc); [R]²⁵_D) -22

(c ) 1.0, CHCl₃); ¹H NMR (250 MHz, CDCl₃) δ 11.1, 9.50, 8.32

(3s), 8.05, 7.50 (2m), 5.70 (d, ²J ) 2), 4.81, 4.72 (2s), 3.60-

Data for 28: white solid; H NMR (250 MHz, CDCl₃) δ 8.34,

8.28 (2s), 7.98, 7.48 (2m), 5.80 (d, ³J ) 2), 4.85, 4.70, 4.12 (3s),

3.41 (dd, ²J ) 12, ³J ) 2), 3.40-3.10 (m), 3.12 (d, ²J ) 12),

2.60 (s), 2.60-2.50 (m), 2.26 (s), 2.25-2.20 (m), 1.08 (t, J )

7).

(2′R)-2′-Acet yl-5′,12′-d ioxo-1′,2′,3′,4′-t et r a h yd r on a p h -

th a cen -2′-yl (1R,5S,7R)-3-Eth yl-2-oxo-6,8-d ioxa -3-a za bi-

cyclo[3.2.1]octa n e-7-exo-ca r boxyla te (29): yellow solid; ¹H

NMR (250 MHz, CDCl₃) δ 8.65, 8.64 (2s), 8.07, 7.68 (2m), 5.88

(d, ³J ) 2), 4.91, 4.72 (2s), 3.48 (dd, ²J ) 12, ³J ) 2), 3.40-

3.30 (m), 3.19 (d, ²J ) 12), 3.15-2.88 (m), 2.62-2.50 (m), 2.24

(s), 2.10-2.00 (m), 1.10 (t, J ) 7).

(2′R)-12′-Acetoxy-2′-a cetyl-5′-(m eth oxyca r bon yl)-6′,11′-

d ioxo-1′,2′,3′,4′-tetr a h yd r on a p h th a cen -2′-yl (1R,5S,7R)-3-

Eth yl-2-oxo-6,8-dioxa-3-azabicyclo[3.2.1]octan e-7-exo-car -

boxyla te (30). (-)-25 (10 mg, 18 µmol) in pyridine (1 mL)

was treated with 19 mg of n-Bu₄MnO₄(3 equiv) at 20 °C. After

1 h of stirring, it was diluted with CHCl₃(10 mL) and washed

with aqueous 1 M H₂SO₄(10 mL, three times), with 5%

aqueous NaHSO₃(10 mL, three times), and finally with brine

(10 mL, three times). After drying (MgSO₄) and solvent

evaporation, the residue was dissolved in THF (3 mL). CH₂N₂

(1 mL, 0.6 M in Et₂O) was added, and 15 min later, the excess

of the reagent was destroyed with AcOH (0.5 mL). After

solvent evaporation, FC (silica gel, 5 g, CH₂Cl₂/EtOAc, R_f0.55)

gave 5 mg (45%) of a yellow solid: ¹H NMR (250 MHz, CDCl₃)

δ 8.21, 7.80 (2m), 5.82 (d, J ) 2), 5.77, 4.85, 4.70, 4.09 (4s),

3.30 (m), 3.42 (dd, J ) 12, J ) 2.5), 3.31, 3.28 (2q, J ) 7),

3.50-3.30 (m), 3.16 (d, J ) 12), 2.56 (s), 2.60-2.50 (m), 2.26

3.12 (d, J ) 12), 2.58 (s), 2.60-2.50 (m), 2.26 (s), 2.30-2.10

(m), 1.05 (t, J ) 7).

(s), 2.20-2.00 (m), 1.10 (t, J ) 7).

(2′R)-12′-Acetoxy-2′-acetyl-5′-(h ydr oxym eth yl)-1′,2′,3′,4′-

tetr a h yd r on a p h th a cen -2′-yl (1R,5S,7R)-3-Eth yl-2-oxo-6,8-

d ioxa -3-a za bicyclo[3.2.1]octa n e-7-exo-ca r boxyla te ((-)-

31). Na(CN)BH₃(20 mg, 0.27 mmol) in MeOH (1 mL) was

added to (-)-25 (150 mg, 0.268 mmol) in CHCl₃(10 mL) at 20

°C, and the mixture was acidified to pH ) 6 with AcOH (20%

in MeOH). The reaction was followed by TLC (R_f0.5, EtOAc);

more AcOH must be added if the reaction stops. After ca. 4 h,

the mixture was diluted with CH₂Cl₂(100 mL) and washed

with brine (20 mL, four times). The combined organic extracts

were dried (MgSO₄), and the solvent was evaporated. The

residue was recrystallized from EtOAc (10 mL) to give 140 mg

(2′R)-12′-Acetoxy-2′-a cetyl-5′-(for m yloxy)-1′,2′,3′,4′-tet-

r a h yd r on a p h t h a cen -2′-yl (1R,5S,7R)-3-E t h yl-2-oxo-6,8-

d ioxa -3-a za bicyclo[3.2.1]octa n e-7-exo-ca r boxyla te ((-)-

26). A mixture of (-)-25 (30 mg, 54 µmol), SeO₂(1 mg, 10

µmol), and 30% aqueous H₂O₂(10 µL, 100 µmol) was stirred

vigorously for 24 h at 20 °C in CH₂Cl₂(3 mL). It was then

diluted with EtOAc (20 mL) and washed with 5% aqueous

NaHSO₃(10 mL, twice), with 10% aqueous Na₂CO₃(10 mL,

twice), and finally with brine (10 mL, twice). After drying

(MgSO₄) and solvent evaporation, the residue was recrystal-

lized from Et₂O, giving 29 mg (94%) of colorless crystals: mp

240-241 °C (Et₂O); [R]²⁵) -13 (c ) 1.0, CHCl₃); H NMR

(93%) of colorless needles: mp 194-196 °C (EtOAc); [R]²⁵

)

(250 MHz, CDCl₃) δ 8.60, 8.38, 8.30 (3s), 8.00, 7.51 (2m), 5.78

(d, ²J ) 2), 4.72, 4.68 (2s), 3.60-2.90 (m), 3.35 (dd, ²J ) 12, ³J

-45 (c ) 1.0, CHCl₃); ¹H NMR (250 MHz, CDCl₃) δ 8.89, 8.33

3 2

(2s), 8.03, 7.48 (2m), 5.70 (d, J ) 2), 5.38, 5.24 (2d, J ) 12),

6966 J . Org. Chem., Vol. 61, No. 20, 1996

Dienes and Vogel

4.60 (s), 3.65-3.05 (m), 3.02 (d, J ) 12), 2.50-2.40 (m), 2.58,

vigorous stirring. After 5 min, the white precipitate was

filtered off (rinsing with EtOAc), and the filtrate was washed

with brine (20 mL). The solution was dried (MgSO₄), and the

solvent was evaporated. FC (silica gel 10 g, washing off the

excess of alcohol with EtOAc/light petroleum 1/1, then 3/1)

gave a main fraction (R_f0.58), 36 mg (80%) as colorless

2.28 (2s), 2.12-1.98 (m), 0.98 (t, J ) 7).

(2′R)-12′-Acetoxy-2′-a cetyl-5′-(br om om eth yl)-1′,2′,3′,4′-

tetr a h yd r on a p h th a cen -2′-yl (1R,5S,7R)-3-Eth yl-2-oxo-6,8-

d ioxa -3-a za bicyclo[3.2.1]octa n e-7-exo-ca r boxyla te ((-)-

32). Freshly distilled AcBr (0.5 mL) was added to a solution

of (-)-31 (56 mg, 0.1 mmol) in CHCl₃(10 mL) at 20 °C. After

being stirred for 15 min, the mixture was poured into brine

(30 mL). The aqueous layer was washed with CHCl₃(10 mL,

four times). The combined organic extracts were dried (Mg-

SO₄), and the solvent was evaporated to give a yellow solid

that was recrystallized from EtOAc, yielding 60 mg (97%)

crystals: mp 175 °C (Et₂O); [R]²⁵) -23 (c ) 1.0, CHCl₃); ¹H

NMR (250 MHz, CDCl₃) δ 8.70, 8.29 (2s), 7.98, 7.48 (2m), 5.80

(d, J ) 2), 5.82 (m), 5.30-5.02 (m), 5.11, 4.70, 4.62 (3s), 4.45

(dm, ³J ) 5.5), 3.73 (t, ³J ) 6.0), 3.40 (dd, ²J ) 12, ³J ) 2),

3.45-3.20 (m), 3.10 (d, J ) 12), 2.59 (s), 2.55-2.50 (m), 2.27

(s), 2.22-2.15 (m), 1.85 (m), 1.04 (t, J ) 7).

(TLC, EtOAc/light petroleum 1/1, R_f0.63) of yellow needles:

(2R)-2-Acet yl-5-[[[3′-[[(a llyloxy)ca r b on yl]a m in o]p r o-

p yl]oxy]m eth yl]-1,2,3,4-tetr a h yd r on a p h th a cen -12-yl Ac-

eta te ((-)-38). (-)-37 (28 mg, 0.040 mmol) was dissolved in

THF (4 mL) and cooled to 0 °C. Aqueous 1 M NaOH (0.5 mL)

was added, and the mixture was stirred at 20 °C for 3 h. It

was then neutralized with aqueous 1 M HCl, and the THF

was evaporated. After dilution with H₂O, the mixture was

mp 173-174 °C; [R]²⁵) -65 (c ) 1.0, CHCl₃); H NMR (250

MHz, CDCl₃) δ 8.64, 8.33 (2s), 8.05, 7.51 (2m), 5.78 (d, ³J )

2), 5.15, 5.08 (2d, J ) 12), 4.67, 4.64 (2s), 3.47 (dd, J ) 12,

³J ) 2), 3.40-3.10 (m), 3.10 (d, J ) 12), 2.65-2.55 (m), 2.58,

2.27 (2s), 2.12-1.98 (m), 1.04 (t, J ) 7).

(2′R)-12′-Acetoxy-2′-a cetyl-5′-[[[3′′-[[(ter t-bu tyloxy)ca r -

b on yl]a m in o]p r op yl]oxy]m et h yl]-1′,2′,3′,4′-t et r a h yd r o-

n a p h t h a cen -2′-yl (1R,5S,7R)-3-E t h yl-2-oxo-6,8-d ioxa -3-

a za bicyclo[3.2.1]octa n e-7-exo-ca r boxyla te ((-)-33). To a

mixture of (-)-32 (40 mg, 64 µmol), the N-(tert-butyloxy)-

carbamate of 3-aminopropanol (56 mg, 0.32 mmol), activated

molecular sieves (4 Å), and CaH₂(20 mg) in anhydrous THF

(1 mL) at 20 °C was added a solution of AgOTf (16 mg, 64

µmol) in THF (0.5 mL) under vigorous stirring. After 5 min,

the white precipitate was filtered off (rinsing with EtOAc), and

the filtrate was washed with brine (20 mL). After drying

(MgSO₄) and solvent evaporation, FC (silica gel 10 g, washing

off the excess of alcohol with light petroleum/EtOAc 3/1, then

1/1, R_f0.11) gave 32 mg (71%) of colorless crystals: mp 182-

extracted with CHCl₃

(10 mL, three times), and the combined

extracts were washed with brine (10 mL, three times). Drying

(MgSO₄), solvent evaporation, and FC (silica gel, 5 g, light

petroleum/EtOAc 3/1, R_f0.81) gave 17 mg (84%) of yellowish

crystals: mp 79-80 °C (CH₂Cl₂/hexane); [R]²⁵_D) -17 (c ) 1.0,

CHCl₃); ¹H NMR (250 MHz, CDCl₃) δ 8.70, 8.29 (2s), 8.00, 7.46

(2m), 5.82 (m), 5.30-5.02 (m), 5.10 (s), 4.45 (dm, ³J ) 5.5),

3.74 (t,

1.84 (3m).

³J ) 5.5), 3.45-2.85 (m), 2.57, 2.35 (2s), 2.20, 2.05,

(2R)-2-Acet yl-2-h yd r oxy-5-[[(3′-a m in op r op yl)oxy]m e-

th yl]-1,2,3,4-tetr a h yd r on a p h th a cen -12-yl Aceta te Hyd r o-

ch lor id e ((-)-39). A mixture of (-)-38 (10 mg, 19 µmol),

Pd(Ph₃P)₄(catalytic amount), and Bu₃SnH (4 µL, 2 equiv) was

stirred in the dark at 20 °C in wet CH₂Cl₂(2 mL) for 20 min.

After solvent evaporation, the residue was dissolved in H₂O/

MeOH 9/1 (3 mL) and acidified to pH 2.5-3.0 with aqueous

0.1 M HCl. The MeOH was evaporated and the suspension

washed with Et₂O (10 mL, seven times) and evaporated to give

a pale yellow solid that was purified by FC (silica gel 5 g, CH₂-

Cl₂/MeOH 4/1, R_f0.19 (alizarin)) to yield 6 mg (66%) as a

yellowish solid: mp 152-154 °C (EtOH/Et₂O); [R]²⁵_D) -18 (c

183 °C (Et₂O); [R]²⁵) -38 (c ) 1.0, CHCl₃); ¹H NMR (250

MHz, CDCl₃) δ 8.72, 8.29 (2s), 8.01, 7.49 (2m), 5.78 (d, ³J )

2), 5.11, 4.68, 4.66 (3s), 3.74 (t, J ) 5.7), 3.49 (dd, J ) 12, J

) 2), 3.45-3.20 (m), 3.10 (d, ²J ) 12), 2.55-2.50 (m), 2.58,

2.26 (2s), 2.22-2.15 (m), 1.84 (m), 1.35 (s), 1.05 (t, J ) 7).

(2R)-2-Acetyl-2-h yd r oxy-5-[[[3′-[[(ter t-bu tyloxy)ca r bo-

n yl]a m in o]p r op yl]oxy]m et h yl]-1,2,3,4-t et r a h yd r on a p h -

th a cen -12-yl Aceta te ((-)-34). (-)-33 (32 mg, 45 µmol) was

dissolved in THF (4 mL) and cooled to 0 °C. Aqueous 1 M

NaOH (0.5 mL) was added, and the mixture was stirred at 20

°C for 3 h. It was then neutralized with aqueous 1 M HCl.

After evaporation of the THF, the mixture was extracted with

CHCl₃(10 mL, three times) and washed with brine (10 mL,

three times). Drying (MgSO₄), solvent evaporation, and FC

(silica gel, flash, EtOAc/light petroleum 1/1, R_f0.75) gave 20

) 1.0, EtOH 95%); H NMR (250 MHz, CD₃OD) δ 8.79, 8.39

(2s), 8.08, 7.50 (2m), 5.20 (s), 3.82 (t, ³J ) 5.5), 3.45-2.85 (m),

3.03 (t, J ) 5.5), 2.61, 2.40 (2s), 2.20-1.90 (m).

(2′R)-12′-Acetoxy-2′-a cetyl-5′-[[[3′′-[N-[(a llyloxy)ca r bo-

n y l]-N -(h y d r o x y p r o p y l)a m in o ]p r o p y l]o x y ]m e t h y l]-

1′,2′,3′,4′-tetr a h yd r on a p h th a cen -2′-yl (1R,5S,7R)-3-Eth yl-

2-o x o -6,8-d io x a -3-a za b ic y c lo [3.2.1]o c t a n e -7-exo-c a r -

boxyla te ((-)-40). To a mixture of (-)-32 (30 mg, 53 µmol),

the N-allyloxycarbamate of 1-dipropanolamine (53 mg, 0.32

mmol), and activated molecular sieves (4 Å) in anhydrous THF

(1 mL) at 20 °C was added a solution of AgOTf (14 mg, 0.055

mmol) in THF (0.5 mL) under vigorous stirring. After 5 min,

the white precipitate was filtered off (rinsing with EtOAc), and

mg (84%) of a yellowish solid: mp 89-91 °C (Et₂O); [R]²⁵

)

-18 (c ) 1.0, CHCl₃); ¹H NMR (250 MHz, CDCl₃) δ 8.72, 8.28

(2s), 8.04, 7.47 (2m), 5.12 (s), 3.75 (t, ³J ) 5.7), 3.45-2.85 (m),

2.57, 2.36 (2s), 2.20 (m), 2.05 (m), 1.83 (m), 1.45 (s).

(2R)-2-Acetyl-2-h yd r oxy-5-(h yd r oxym eth yl)-1,2,3,4-tet-

r a h yd r on a p h th a cen -12-yl Aceta te ((-)-36). Meth od A.

(-)-34 (6 mg, 11 mmol) was dissolved in a mixture of EtOAc

(1 mL) and aqueous 3 M HCl (1 mL) and stirred at 20 °C for

12 h. The organic phase was diluted, separated, and washed

with brine (10 mL, 5 times). Drying and solvent evaporation

gave 3 mg (74%) of a yellowish solid.

Meth od B. (-)-31 (15 mg, 0.027 mmol) was dissolved in

THF (5 mL) and cooled to 0 °C. Aqueous 1 M NaOH (0.5 mL)

was added, and the mixture was stirred at 20 °C for 2 h. It

was then neutralized with aqueous 1 M HCl. After evapora-

tion of the THF, the mixture was extracted with CHCl₃(10

mL, three times) and washed with brine (10 mL, three times).

After drying (MgSO₄) and solvent evaporation, FC (silica gel,

5 g, EtOAc/light petroleum 3/1, R_f0.53) yielded 8 mg (79%) of

a yellowish solid: mp 84-87 °C (Et₂O); [R]²⁵_D) -32 (c ) 0.25,

CHCl₃).

(2′R)-12′-Acetoxy-2′-acetyl-5′-[[[3′′-[[(allyloxy)car bon yl]-

a m in o]p r op yl]oxy]m e t h yl]-1′,2′,3′,4′-t e t r a h yd r on a p h -

th a cen -2′-yl (1R,5S,7R)-3-Eth yl-2-oxo-6,8-d ioxa -3-a za bi-

cyclo[3.2.1]octa n e-7-exo-ca r boxyla te ((-)-37). To a mix-

ture of (-)-32 (40 mg, 64 µmol), the N-(allyloxy)carbamate of

3-aminopropanol (48 mg, 0.32 mmol), and activated molecular

sieves (4 Å) in anhydrous THF (1 mL) at 20 °C was added a

solution of AgOTf (16 mg, 64 µmol) in THF (0.5 mL) under

the filtrate was washed with brine (20 mL). Drying (MgSO₄

solvent evaporation, and FC (silica gel 10 g, washing off the

excess of alcohol with EtOAc/light petroleum 3/1, then EtOAc,

R_f0.19) gave a main fraction, which provided 26 mg (about

60%) of a 1:1 mixture of protected dipropanolamine/(-)-40:

[R]²⁵) -25 (as mixture, c ) 1.0, CHCl₃); H NMR of (-)-40

(250 MHz, CDCl₃) δ 8.70, 8.28 (2s), 7.98, 7.48 (2m), 5.80 (d, ³J

) 2), 5.82 (m), 5.30-5.02 (m), 5.10, 4.72, 4.70 (3s), 4.56 (dm),

3.70-3.20 (m), 3.11 (d, J ) 12), 2.59 (s), 2.55-2.50 (m), 2.25

(s), 2.22-2.15 (m), 1.90-1.70 (m), 1.05 (t, J ) 7).

(2R)-2-Acetyl-2-h yd r oxy-5-[[[3′-[N-[(a llyloxy)ca r bon yl]-

N-(h yd r oxyp r op yl)a m in o]p r op yl]oxy]m eth yl]-1,2,3,4-tet-

r a h yd r on a p h th a cen -12-yl Aceta te ((-)-41). (-)-40 with

some alcohol impurity (26 mg, about 32 µmol) was dissolved

in THF (4 mL) and cooled to 0 °C. Aqueous 1 M NaOH (0.5

mL) was added, and the mixture was stirred at 20 °C for 3 h.

It was then neutralized with aqueous 1 M HCl, and after

evaporation of the THF, it was extracted with CHCl₃(10 mL,

three times) and washed with brine (10 mL, three times). After

drying (MgSO₄) and solvent evaporation, FC (silica gel, 10 g,

EtOAc, R_f0.50) yielded 9 mg (32% for two steps) of a yellowish

solid: mp 47-50 °C (Et₂O/hexane); [R]²⁵) -14 (c ) 1.0,

CHCl₃); ¹H NMR (250 MHz, CDCl₃) δ 8.70, 8.28 (2s), 7.98, 7.48,

Synthesis of Substituted Daunomycinones

J . Org. Chem., Vol. 61, No. 20, 1996 6967

5.85, 5.30-5.02 (4m), 5.11 (s), 4.56 (dm, J ) 5.5), 3.68, 3.42

(2′R)-12′-Acetoxy-2′-a cetyl-5′-[[[3′′-[N-[(a llyloxy)ca r bo-

n yl]-N-[3′′′-[[(a llyloxy)ca r b on yl]a m in o]p r op yl]a m in o]-

p r op yl]oxy]m et h yl]-1′,2′,3′,4′-t et r a h yd r on a p h t h a cen -2′-

yl (1R,5S,7R)-3-Eth yl-2-oxo-6,8-d ioxa -3-a za bicyclo[3.2.1]-

octa n e-7-exo-ca r boxyla te ((-)-46). A solution of AgOTf (16

mg, 64 µmol) in 0.5 mL of THF was added to a stirred mixture

of (-)-32 (40 mg, 64 µmol), the N,N-bis(allyloxy)carbamate of

3-[(3′-aminopropyl)amino]propanol (100 mg, 0.33 mmol), and

activated molecular sieves (4 Å) in anhydrous THF (1 mL) at

20 °C. After 5 min, the white precipitate was filtered off

(rinsing with EtOAc), and the filtrate was washed with brine

(20 mL). Drying (MgSO₄) and solvent evaporation gave a

residue that was purified by FC (silica gel 10 g, washing off

the excess of alcohol with acetone/light petroleum 1/1, then

3/1, (R_f0.32)) to give 40 mg of a mixture of (-)-46 and the

starting alcohol (yield: about 84%). An analytical sample was

obtained by recrystallization from EtOAc/hexane as an amor-

(2t, J ) 5.5), 3.45-2.85 (m), 2.58, 2.36 (2s), 2.20, 2.05, 1.90,

1.52 (4m).

(2R)-2-Acet yl-2-h yd r oxy-5-[[[3′-[(3′′-h yd r oxyp r op yl)-

am in o]pr opyl]oxy]m eth yl]-1,2,3,4-tetr ah ydr on aph th acen -

12-yl Aceta te Hyd r och lor id e((-)-42). A mixture of (-)-41

(10 mg, 19 µmol), Pd(Ph₃P)₄(catalytic amount), and Bu₃SnH

(4 µL, 2 equiv) was stirred in the dark at 20 °C in wet CH₂Cl₂

(3 mL) for 20 min. After solvent evaporation, the residue was

dissolved in H₂O/MeOH 9/1 (3 mL) and acidified to pH 2.5-

3.0 with aqueous 0.1 M HCl. The MeOH was evaporated, and

the suspension was washed with Et₂O (10 mL, seven times)

and evaporated to give a pale yellow solid that was purified

by FC (silica gel, 5 g, CH₂Cl₂/MeOH 4/1, R_f0.55 (alizarin)) to

yield 6 mg (70%) of a yellowish solid: mp 140-144 °C (EtOH/

Et₂O); [R]²⁵) -21 (c ) 0.1, EtOH 95%); ¹H NMR (400 MHz,

CD₃OD) δ 8.77, 8.39 (2s), 8.10, 7.51 (2m), 5.18 (s), 3.85 (t, J

) 5.5), 3.48, 3.45-2.85, 3.10, 2.95 (4m), 2.62, 2.40 (2s), 2.20-

1.65 (m).

phous solid: mp ca. 27 °C; [R]²⁵) -33 (c ) 1.0, CH₃CN); ¹H

NMR (250 MHz, CDCl₃) δ 8.71, 8.29 (2s), 7.98, 7.48, 5.90 (3m),

5.78 (d, ³J ) 2), 5.30-5.10 (m), 5.11, 5.08 (2d, ²J ) 7), 4.68,

(2′R)-12′-Acetoxy-2′-a cetyl-6′,11′-d ioxo-5′-[[[3′′-[[(a llyl-

oxy)ca r b on yl]a m in o]p r op yl]oxy]m et h yl]-1′,2′,3′,4′-t et -

r a h yd r on a p h t h a cen -2′-yl (1R,5S,7R)-3-E t h yl-2-oxo-6,8-

d ioxa -3-a za bicyclo[3.2.1]octa n e-7-exo-ca r boxyla te ((-)-

43). (-)-37 (40 mg, 57 µmol) was dissolved in acetone (10 mL)

and cooled to 0 °C. J ones reagent (4 N, 0.25 mL, 0.34 mmol)

was added dropwise, and the reaction mixture was stirred in

the dark at 0 °C for 30 min and at 20 °C for 1 h. It was cooled

again to 0 °C, and 40 µL of J ones reagent was added. After

the mixture was stirred at 20 °C for 1 h, the excess of the

oxidant was destroyed with i-PrOH (1 mL, changed to green).

Solvent evaporation afforded a residue that was taken up with

EtOAc (20 mL) and washed with 10% aqueous NaHCO₃(10

mL) and finally with brine (10 mL, four times). After drying,

4.67 (2s), 4.55, 4.52 (2d, J ) 5.5), 3.68 (m), 3.38 (dd, J ) 12,

³J ) 2), 3.45-3.00 (m), 3.10 (d, J ) 12), 2.58 (s), 2.55-2.50

(m), 2.26 (s), 2.22-2.15, 1.90-1.60 (2m), 1.04 (t, J ) 7).

(2′R)-12′-Acet oxy-2′-a cet yl-6′,11′-d ioxo-5′-[[[3′′-[N-[(a l-

lyloxy)ca r b on yl]-N-[3′′′-[(a llyloxyca r b on yl]a m in o]p r o-

p yl]a m in o]p r op yl]oxy]m eth yl]-1′,2′,3′,4′-tetr a h yd r on a p h -

th a cen -2′-yl (1R,5S,7R)-3-Eth yl-2-oxo-6,8-d ioxa -3-a za bi-

cyclo[3.2.1]octa n e-7-exo-ca r boxyla te ((-)-47). (-)-46 (40

mg, 47 µmol) was dissolved in acetone (10 mL) and then cooled

to 0 °C. J ones reagent (4 N, 0.21 mL, 0.28 mmol) was added

dropwise, and the reaction mixture was stirred in the dark at

0 °C for 30 min and at 20 °C for 1 h. It was cooled again to 0

°C, and 40 µL of J ones reagent was added. After the mixture

was stirred at 20 °C for 1 h, the excess of the oxidant was

destroyed with i-PrOH (1 mL, changed to green). After solvent

evaporation, the residue was dissolved in EtOAc (20 mL) and

washed with 10% aqueous NaHCO₃(10 mL) and finally with

brine (10 mL, four times). After drying, solvent evaporation

gave 39 mg (94%) of an amorphous yellow solid: mp ∼27 °C

solvent evaporation gave 36 mg (91%) of a yellowish gum:

[R]²⁵) -13 (c ) 1.0, CHCl₃); H NMR (250 MHz, CDCl₃) δ

8.13, 7.73, 5.90 (3m), 5.82 (d, ³J ) 2), 5.20 (m), 4.98 (br s),

4.75, 4.70 (2s), 4.51 (d, ³J ) 5.5), 3.75 (t, ³J ) 5.5), 3.42 (dd, ²J

) 12, J ) 2), 3.40-3.00 (m), 3.15 (d, J ) 12), 2.55, 2.26 (2s),

2.15-2.00, 1.85 (2m), 1.10 (t, J ) 7).

(Et₂O); [R]²⁵) -14 (c ) 1.0, CHCl₃); ¹H NMR (250 MHz,

(8R)-8-Acetyl-6,8-d ih yd r oxy-11-[[[3′-[[(a llyloxy)ca r bo-

n yl]a m in o]p r op yl]oxy]m eth yl]-7,8,9,10-tetr a h yd r on a p h -

th a cen e-5,12-d ion e ((-)-44). Meth od A. A mixture of (-)-

37 (20 mg, 32 µmol) and HO(CH₂)₃NHAlloc (27 mg, 6 equiv)

in THF at -30 °C was treated with NaH (85% in oil, 3 mg, 3

equiv). The temperature was allowed to rise to 20 °C. After

being stirred for 6 h, the red solution was neutralized with

aqueous 1 M HCl and evaporated. The residue was dissolved

in EtOAc (2 mL) and treated with aqueous 1 M HCl (2 mL).

After being stirred overnight, the organic phase was separated,

washed with brine (10 mL, three times), and dried (MgSO₄),

and the solvent was evaporated. The residue was purified by

FC (silica gel, 5 g, light petroleum/EtOAc 1/1, R_f0.53 (UV,

green with vanillin)) to yield 6 mg (37%) of (-)-44 with some

impurity. Meth od B. (-)-43 (36 mg, 49 µmol) was dissolved

in degassed THF (2 mL). After the solution was cooled to 0

°C, aqueous 1 M LiOH (0.1 mL) was added, and the mixture

was stirred in the dark for 10 min. After addition of a

saturated aqueous solution of NH₄Cl (5 mL), the product was

extracted with CHCl₃(15 mL, three times) and the combined

extracts were dried (MgSO₄). Solvent evaporation yielded 24

mg of a yellow solid with about 10% impurity, which could

CDCl₃) δ 8.13, 7.72, 5.90 (3m), 5.83 (d, ³J ) 2), 5.40-5.10 (m),

5.05, 4.88 (2d, ²J ) 10), 4.76, 4.68 (2s), 4.60 (dt, ²J ) 5.5, ³J )

1.2), 4.51 (d, J ) 5.5), 3.67 (t, J ) 5.5), 3.42 (dd, J ) 12, J

) 2), 3.40-3.00 (m), 3.15 (d, ²J ) 12), 2.52, 2.24 (2s), 2.15-

2.00 (m), 1.90, 1.70 (2m), 1.10 (t, J ) 7).

(2′R)-12′-Acetoxy-2′-acetyl-5′-[[[3′′-(tr iflu or oacetam ido)-

p r op yl]oxy]m et h yl]-1′,2′,3′,4′-t et r a h yd r on a p h t h a cen -2′-

yl (1R,5S,7R)-3-Eth yl-2-oxo-6,8-d ioxa -3-a za bicyclo[3.2.1]-

octa n e-7-exo-ca r boxyla te ((-)-48). A solution of AgOTf (16

mg, 64 µmol) in THF (0.5 mL) was added to a stirred mixture

of (-)-32 (40 mg, 64 µmol), 3-(trifluoroacetamido)propanol (55

mg, 0.32 mmol), and activated molecular sieves (4 Å) in

anhydrous THF (1 mL) at 20 °C. After 5 min, the white

precipitate was filtered off (rinsing with EtOAc), and the

filtrate was washed with brine (20 mL). After drying (MgSO₄

)

and solvent evaporation, FC (silica gel 10 g, washing off the

excess of alcohol with light petroleum/EtOAc 2/1, then 1/1)

gave 44 mg (87%) of colorless crystals: mp 181 °C (Et₂O/

hexane 5/1); [R]²⁵_D) -12 (c ) 0.5, CHCl₃); ¹H NMR (250 MHz,

CDCl₃) δ 8.68, 8.30 (2s), 7.98, 7.46 (2m), 5.77 (d, J ) 2), 5.20,

5.06 (2d, J ) 12), 4.65, 4.40 (2s), 3.76 (t, J ) 6.0), 3.50-3.10

(m), 3.08 (d,

2.05 (m), 1.89 (m), 1.01 (t, J ) 7).

²J ) 12), 2.58 (s), 2.55-2.40 (m), 2.26 (s), 2.18-

not be separated by chromatography or crystallization: [R]²⁵

) -7 (c ) 0.5, CHCl₃); ¹H NMR (250 MHz, CDCl₃) δ 13.81 (s),

8.24, 7.80, 5.83 (3m), 5.49 (br. t), 5.28-5.10 (m), 5.10, 4.83

(2d, ²J ) 10), 4.48 (dm, ³J ) 5.5), 3.92 (s), 3.75, 3.40-3.11 (2m),

(2′R)-12′-Acetoxy-2′-a cetyl-6′,11′-d ioxo-5′-[[[3′′-(tr iflu o-

r oa cet a m id o)p r op yl]oxy]m et h yl]-1′,2′,3′,4′-t et r a h yd r o-

n a p h t h a cen -2′-yl (1R,5S,7R)-3-E t h yl-2-oxo-6,8-d ioxa -3-

a za bicyclo[3.2.1]octa n e-7-exo-ca r boxyla te ((-)-49). (-)-

48 (22 mg, 31 µmol) was dissolved in acetone (5 mL) and then

cooled to 0 °C. J ones reagent (4 N, 0.12 mL, 0.17 mmol) was

added dropwise, and the reaction mixture was stirred in the

dark at 0 °C for 30 min and at 20 °C for 1 h. It was cooled

again to 0 °C, and J ones reagent (20 µL) was added. After

being stirred at 20 °C for 1 h, the excess of the oxidant was

destroyed with i-PrOH (1 mL, changed to green). Solvent

evaporation afforded a residue that was taken up with EtOAc

(10 mL) and washed with brine (10 mL, four times). Drying

3.11, 2.98 (2d, J ) 18), 2.40 (s), 2.12-1.90, 1.84 (2m).

(8R)-8-Acetyl-6,8-dih ydr oxy-11-[[[3′-(p-n itr oben zam ido)-

p r op yl]oxy]m et h yl]-7,8,9,10-t et r a h yd r on a p h t h a cen e-5,

12-d ion e ((-)-45). (-)-8 (5 mg, 13 µmol) was dissolved in

anhydrous CH₂Cl₂(1 mL) at 0 °C. Et₃N (2 µL, 13 µmol) and

p-nitrobenzoyl chloride (3 mg, 16 µmol) were added. After

being stirred for 30 min, the mixture was diluted with CH₂-

Cl₂and washed with aqueous 0.1 M HCl (10 mL, three times)

and brine (10 mL, three times). Drying (MgSO₄) and solvent

evaporation gave 5 mg (67%) of yellow prisms: mp 86-88 °C

(hexane/CHCl₃1/1); [R]²⁵) -36 (c ) 0.25, CHCl₃).

6968 J . Org. Chem., Vol. 61, No. 20, 1996

Dienes and Vogel

and solvent evaporation gave 18 mg (85%) of an amorphous

0 °C for 30 min and at 20 °C for 2 h. The excess of the oxidant

was destroyed with i-PrOH (1 mL). Solvent evaporation

afforded a residue that was taken up with EtOAc (10 mL) and

washed with brine (10 mL, four times). After drying and

solvent evaporation the product still contained nonoxidized

pale yellow solid: mp 35-40 °C (CH₂Cl₂/hexane); [R]²⁵_D) -14

(c ) 1.0, CHCl₃); H NMR (250 MHz, CDCl₃) δ 8.13 (m), 8.00

(br s), 7.76 (m), 5.82 (d, J ) 2), 4.99, 4.82 (2 br. d, J ) 10),

4.68, 4.62 (2s), 3.80 (t, ³J ) 5.5), 3.42 (dd, ²J ) 12, ³J ) 2),

3.60-3.10 (m), 3.15 (d, ²J ) 12), 2.55, 2.26 (2s), 2.15-1.90 (m),

starting (-)-50 (about 15% by H NMR), so it was redissolved

1.10 (t, J ) 7).

in acetone (5 mL) and treated in the same manner with 110

µL of 4 N J ones reagent. The usual workup gave 30 mg (82%)

of an amorphous pale yellow solid: mp 74-77 °C (CH₂Cl₂/Et₂O/

(2′R)-12′-Acetoxy-2′-acetyl-5′-[[[4′′-(tr iflu or oacetam ido)-

b u t yl]oxy]m et h yl]-1′,2′,3′,4′-t et r a h yd r on a p h t h a cen -2′-

yl (1R,5S,7R)-3-Eth yl-2-oxo-6,8-d ioxa -3-a za bicyclo[3.2.1]-

octa n e-7-exo-ca r boxyla te ((-)-50). A solution of AgOTf (16

mg, 64 µmol) in THF (0.5 mL) was added to a stirred mixture

of (-)-32 (40 mg, 64 µmol), 4-(trifluoroacetamido)butanol (80

mg, 0.43 mmol), and activated molecular sieves (4 Å) in

anhydrous THF (1.5 mL) at 20 °C. After 5 min, the white

precipitate was filtered off (rinsing with EtOAc), and the

filtrate was washed with brine (20 mL, three times). The

combined aqueous fractions were reextracted with EtOAc (as

many times as needed, checked by TLC). After drying (MgSO₄)

and solvent evaporation, FC (silica gel 10 g, washing off the

excess of alcohol with light petroleum/EtOAc 2/1, then 1/1, then

1/3) afforded the main fraction (R_f0.54, UV), which gave 37

hexane); [R]²⁵) -10 (c ) 1.0, CHCl₃).

(2′R)-12′-Acetoxy-2′-a cetyl-6′,11′-d ioxo-5′-[[[5′′-(tr iflu o-

r oa cet a m id o)p en t yl]oxy]m et h yl]-1′,2′,3′,4′-t et r a h yd r o-

n a p h t h a cen -2′-yl (1R,5S,7R)-3-et h yl-2-oxo-6,8-d ioxa -3-

a za bicyclo[3.2.1]octa n e-7-exo-ca r boxyla te ((-)-54) was

prepared by the same procedure as for the preparation of (-)-

53, starting with (-)-51 (20 mg, 27 µmol): yield 16 mg (77%)

of an amorphous, pale yellow solid; mp 73-75 °C (CH₂Cl₂/Et₂O/

hexane); [R]²⁵) -11 (c ) 1.0, CHCl₃).

(2′R)-12′-Acet oxy-2′-a cet yl-6′,11′-d ioxo-5′-[[[2′′-[N-(t r i-

flu or oa cetyl)-N-[3′′′-(tr iflu or oa ceta m id o)p r op yl]a m in o]-

et h yl]oxy]m et h yl]-1′,2′,3′,4′-t et r a h yd r on a p h t h a cen -2′-

yl (1R,5S,7R)-3-eth yl-2-oxo-6,8-d ioxa -3-a za bicyclo[3.2.1]-

octa n e-7-exo-ca r boxyla te ((-)-55) was prepared by the

same procedure as for the preparation of (-)-53, starting with

(-)-52 (15 mg, 17 µmol): yield 12 mg (76%) of an amorphous

yellow solid; mp ca. 29 °C (Et₂O); [R]²⁵_D) -10 (c ) 1.0, CHCl₃).

(8R)-8-Acetyl-6,8-dih ydr oxy-11-[[[4′-(p-n itr oben zam ido)-

bu tyl]oxy]m eth yl]-7,8, 9,10-tetr a h yd r on a p h th a cen e-5,12-

d ion e ((-)-56) was prepared by the same procedure as for

the preparation of (-)-45, starting with (-)-9 (2 mg, 4 µmol):

yield 2 mg (81%), recrystallization from CH₂Cl₂/hexane; mp

mg (79%) of colorless crystals: mp 158-160 °C (EtOAc/

hexane); [R]²⁵) -15 (c ) 1.0, CHCl₃); H NMR (250 MHz,

CDCl₃) δ 8.68, 8.30 (2s), 7.98, 7.48, 7.25 (3m), 5.78 (d, J ) 2),

5.19, 5.05 (2d, ²J ) 12), 4.68, 4.55 (2s), 3.68 (m), 3.50-3.10

(m), 3.10 (d, J ) 12), 2.58 (s), 2.55-2.40 (m), 2.26 (s), 2.18-

2.05, 1.66 (2m), 1.03 (t, J ) 7).

(2′R)-12′-Acet oxy-2′-a cet yl-5′-[[[5′′-(t r iflu or oa cet a m i-

d o)p en tyl]oxy]m eth yl]-1′,2′,3′,4′-tetr a h yd r on a p h th a cen -

2′-yl (1R,5S,7R)-3-Eth yl-2-oxo-6,8-dioxa-3-azabicyclo[3.2.1]-

octa n e-7-exo-ca r boxyla te ((-)-51). A solution of AgOTf (16

mg, 64 µmol) in THF (0.5 mL) was added to a stirred mixture

of (-)-32 (40 mg, 64 µmol), 5-(trifluoroacetamido)pentanol (90

mg, 0.45 mmol), and activated molecular sieves (4 Å) in

anhydrous THF (1.5 mL) at 20 °C. After 5 min, the white

precipitate was filtered off (rinsing with EtOAc), and the

filtrate was washed with brine (20 mL, three times). The

combined aqueous fractions were reextracted with EtOAc (as

many times as needed, checked by TLC). After drying (MgSO₄)

and solvent evaporation, FC (silica gel 20 g, washing off the

excess of alcohol with light petroleum/EtOAc 2/1, the 1/2, then

1/3) afforded the main fraction (R_f0.52, UV), which gave 30

89-92 °C; [R]²⁵) -23 (c ) 0.2, CHCl₃).

(8R)-8-Acetyl-6,8-dih ydr oxy-11-[[[5′-(p-n itr oben zam ido)-

p en tyl]oxy]m eth yl]-7,8, 9,10-tetr a h yd r on a p h th a cen e-5,

12-d ion e ((-)-57) was prepared by the same procedure as for

the preparation of (-)-45, starting with (-)-10 (2 mg, 4 µmol):

yield 2 mg (81%) of a yellow gum; [R]²⁵_D) -40 (c ) 0.2, CHCl₃).

(2′R)-12′-Acet oxy-2′-a cet yl-5′-[(2′′,3′′,6′′-t r id eoxy-4′′-O-

(p -n it r ob en zoyl)-3′′-(t r iflu or oa cet a m id o)-r-^L-lyxo-h ex-

opyr an osyl]oxy]m eth yl]-1′,2′,3′,4′-tetr ah ydr on aph th acen -

2′-yl (1R,5S,7R)-3-Eth yl-2-oxo-6,8-dioxa-3-azabicyclo[3.2.1]-

octa n e-7-exo-ca r boxyla te ((-)-60). Fifty mg (93 µmol) of

(-)-58 (2,3,6-trideoxy-4-O-(p-nitrobenzoyl)-3-(trifluoroaceta-

mido)-R-^L-lyxo-hexopyranosyl p-nitrobenzoate) was dissolved

under Ar in anhydrous THF (3 mL). After the mixture was

cooled to -78 °C, Me₃SiOSO₂CF₃(42 µL, 0.2 mmol) was added

and the temperature was allowed to rise to -25 °C. After the

mixture was stirred at -25 °C for 30 min, (-)-31 (42 mg in

CHCl₃(1 mL), 74 µmol) was introduced slowly and the mixture

was stirred at -25 °C for 3 h. It was then poured into a

saturated aqueous solution of NaHCO₃(20 mL, cooled to 0 °C)

and extracted with EtOAc (15 mL, three times). The combined

organic extracts were washed with brine (15 mL, three times)

and dried (MgSO₄). After solvent evaporation, FC (silica gel,

10 g, EtOAc/light petroleum 1/1, R_f0.66) gave two anomers

(52 mg, 75%, R/â ) 2/1) that could only be separated by HPLC

(Nucleosil 100-3, EtOAc/light petroleum 3/2) to give 34 mg

(50%) of (-)-60 and 16 mg of its â-anomer.

mg (63%) of colorless crystals: mp 151 °C (EtOAc/hexane);

[R]²⁵) -18 (c ) 1.0, CDCl₃); H NMR (250 MHz, CDCl₃) δ

8.72, 8.28 (2s), 7.98, 7.46 (2m), 5.70 (d, ³J ) 2), 5.10 (br s),

4.67 (s), 3.62 (dt, J ) 2.2, J ) 6.0), 3.40-3.00 (m), 2.58 (s),

2.55-2.40 (m), 2.27 (s), 2.25-2.10, 1.63, 1.35 (3m), 1.02 (t, J

) 7).

(2′R)-12′-Acetoxy-2′-a cetyl-5′-[[[2′′-[N-(tr iflu or oa cetyl)-

N -[3′′′-(t r iflu or oa ce t a m id o)p r op yl]a m in o]e t h yl]oxy]-

m eth yl]-1′,2′,3′,4′-tetr a h yd r on a p h th a cen -2′-yl (1R,5S,7R)-

3-Eth yl-2-oxo-6,8-d ioxa -3-a za bicyclo[3.2.1]octa n e-7-exo-

ca r boxyla te ((-)-52). A solution of AgOTf (16 mg, 0.064

mmol) in THF (0.5 mL) was added to a stirred mixture of (-)-

32 (20 mg, 32 µmol), the N,N-bis(trifluoroacetamide) of 2-[(3′-

aminopropyl)amino]propanol (100 mg, 0.33 mmol), and acti-

vated molecular sieves (4 Å) in anhydrous THF (1 mL) at 20

°C. After being stirred for 5 min, the white precipitate was

filtered off (rinsing with EtOAc) and the filtrate was washed

with brine (20 mL). After drying (MgSO₄) and solvent

evaporation, FC (silica gel 10 g, washing off the excess of

alcohol with EtOAc/light petroleum 1/1, then 3/1, (R_f0.32))

afforded 20 mg (75%) of an amorphous solid: mp ca. 30 °C

(from CH₂Cl₂/hexane); [R]²⁵_D) -12 (c ) 1.0, CHCl₃); ¹H NMR

(250 MHz, CDCl₃) δ 8.64, 8.30 (2s), 7.98, 7.48 (2m), 5.81 (d, ³J

) 2), 5.15, 5.13 (2d, ²J ) 7), 4.65, 4.53 (2s), 3.88, 3.79, 3.61

(3t, ³J ) 5), 3.45-2.95 (m), 2.58 (s), 2.55-2.50 (m), 2.26 (s),

Data for (-)-60: colorless crystals; mp 260 °C dec (Et₂O);

[R]²⁵) -45 (c ) 1.0, CHCl₃); H NMR (400 MHz, CDCl₃) δ

8.79, 8.32 (2s), 8.40-8.20, 8.02, 7.47 (3m), 6.50 (d, ³J ) 7),

5.80 (d, ³J ) 2), 5.47 (br s), 5.30 (s), 5.07 (d, ³J ) 1), 4.85-4.65

(m), 4.66, 4.58 (2s), 4.37 (q, J ) 6), 3.40-3.10 (m), 2.59 (s),

2.55-2.45 (m), 2.27 (s), 2.15-1.80 (m), 1.26 (d, J ) 6), 1.01

(t, J ) 7).

(2′R)-12′-Acet oxy-2′-a cet yl-6′,11′-d ioxo-5′-[[[2′′,3′′,6′′-

tr id eoxy-4′′-O-(p-n itr oben zoyl)-3′′-(tr iflu or oa ceta m id o)-

r-^L-lyxo-h exop yr a n osyl]oxy]m et h yl]-1′,2′,3′,4′-t et r a h y-

d r on a p h th a cen -2′-yl (1R,5S,7R)-3-Eth yl-2-oxo-6,8-d ioxa -

3-a za bicyclo[3.2.1]octa n e-7-exo-ca r boxyla te ((-)-61). (-)-

60 (15 mg, 16 µmol) was dissolved in acetone (4 mL) and then

cooled to 0 °C. J ones reagent (4 N, 0.07 mL, 0.1 mmol) was

added dropwise, and the reaction mixture was stirred in the

dark at 0 °C for 20 min and at 20 °C for 30 min. The excess

of the oxidant was destroyed with i-PrOH (0.5 mL). After

solvent evaporation, the residue was taken up with EtOAc (20

2.22-2.15, 1.60 (2m), 1.04 (t, J ) 7).

(2′R)-12′-Acetoxy-2′-a cetyl-6′,11′-d ioxo-5′-[[[4′′-(tr iflu o-

r oacetam ido)bu tyl]oxy]m eth yl]-1′,2′,3′,4′-tetr ah ydr on aph -

th a cen -2′-yl (1R,5S,7R)-3-Eth yl-2-oxo-6,8-d ioxa -3-a za bi-

cyclo[3.2.1]octa n e-7-exo-ca r boxyla te ((-)-53). (-)-50 (35

mg, 48 µmol) was dissolved in acetone (5 mL) and then cooled

to 0 °C. J ones reagent (4 N, 0.22 mL, 0.31 mmol) was added

dropwise, and the reaction mixture was stirred in the dark at

Synthesis of Substituted Daunomycinones

J . Org. Chem., Vol. 61, No. 20, 1996 6969

F igu r e 3. Normalized UV spectra of (-)-8 with increasing

amounts of CT-DNA. At 406 nm wavelength ꢀ_f) 3740 ( 180

and ꢀ_b) 2740 ( 240.

F igu r e 5. Effect of DNA intercalators on topoisomerase II

strand passing activity: (A) 1 kd ladder; (B) pGEM supercoiled

DNA without enzyme; (C) with enzyme; (D) daunomycin in 2

µM, (E) (-)-8 in 50 µM, (F) in 150 µM, and (G) in 500 µM

concentration.

aliquots of potential intercalator solution (about 100 µM in

Tris-HCl buffer) were added to a solution of CT-DNA (about

200 µM in Tris-HCl buffer) under continous stirring. The

base-line absorbance was recorded and subtracted from all

subsequent readings. All the spectrum was transferred into

Microcal Origin software. For the calculation of free and

bound intercalator concentration, only absorbance at the λ_max

wavelength, the exact concentration of the intercalator, and

the DNA in base pairs are necessary. Three readings were

recorded at each addition and averaged. ꢀ_bwas determined

by extrapolation to high DNA concentration. The titration was

repeated by addition of aliquots of DNA solution to intercalator

solution (see Figure 3 for an example).

F igu r e 4. Normalized fluorescence spectra of (-)-8 with

increasing amounts of CT-DNA. Excitation wavelength λ_ex

)

(B) F lu or escen ce Titr a tion . Titration experiments were

408 nm.

performed on

a Perkin-Elmer LS-50B spectrofluorometer

maintained at 25 °C. A slit width of 4 nm was used with λ_ex

) λ_maxof the UV spectra. Excitation and emission spectra of

potential intercalator alone were recorded and used to deter-

mine the wavelength of maximum intensity in fluorescence

(λ_em, I_max). Every 15 min aliquots of intercalator solution

(about 10 µM in Tris-HCl buffer) were added to a solution of

CT-DNA (about 20 µM in Tris-HCl buffer) under continous

stirring. The base-line fluorescence was recorded and sub-

stracted from all subsequent readings. For the calculation of

free and bound intercalator concentration, fluorescence inten-

sity at the I_maxwavelength, the exact concentration of the

intercalator and the DNA in base pairs are only necessary.

Three readings were recorded at each addition and averaged.

k_bwas determined by extrapolation to high DNA concentra-

tion. The titration was repeated by addition of aliquots of DNA

solution to intercalator solution. Data obtained from absor-

bance and fluorescence titrations were used to calculate the

free intercalator concentration (see Figure 4 for an example).

mL) and washed with 10% aqueous NaHCO₃(10 mL) and then

with brine (10 mL, four times). After drying, solvent evapora-

tion gave 11 mg (71%) of a yellow powder: mp 130-134 °C

(Et₂O); [R]²⁵) -73 (c ) 1.0, CHCl₃); ¹H NMR (250 MHz,

CDCl₃) δ 8.40-8.20, 8.15, 7.75 (3m), 6.54 (d, J ) 7), 5.85 (d,

³J ) 2), 5.50 (br s), 5.35, 5.18 (2d, J ) 12), 5.28 (d, J ) 1),

4.72, 4.65 (2s), 4.60-4.35, 3.42-3.14 (2m), 2.55 (s), 2.55-2.40

(m), 2.25 (s), 2.25-1.90 (m), 1.27 (d, J ) 6), 1.05 (t, J ) 7).

Exa m p le of In ter ca la tion Assa y by Absor ba n ce Titr a -

tion . The electronic absorption spectrum of (-)-8 in the

presence of increasing amounts of CT-DNA shows strong

decrease of the peak intensity (Figure 3). This hypochromicity

attains 60% with only 2 mm red shift of the absorbance

maximum.

Exa m p le of In ter ca la tion Assa y by F lu or escen ce Ti-

tr a tion . An interesting property of the fluorescence emission

spectrum was the increase of intensity (P ) 2.38) with

increasing amount of DNA without any change in the shape

of the spectrum (Figure 4).

In ter ca la tion Assa ys. Calf thymus DNA type XV was

purchased from Sigma. Purity was checked by measuring the

ratio of the absorbance at 260-280 nm (1.9). Extinction

coefficient at 260 nm: 12824 M^-1in base pairs. All samples

were dissolved in 10 mM Tris-HCl buffer, 1 mM Na₂EDTA,

100 mM NaCl pH 7.4. Yeast topoisomerase I and pGEM

plasmid (Promega) were a generous gift of Dr. Y. Vassetzky

and D. Bragulia (ISREC, Lausanne), while human topo-

isomerase II and other reagents for inhibition assays were

received in a Topoisomerase II drug screening kit (TopoGEN,

Inc., Columbus, OH).

Top oisom er a se In h ibition . (A) Un w in d in g Assa y. Re-

actions were carried out in 20 µL assay buffer (10 mM Tris-

HCl, pH 7.9, 50 mM NaCl, 50 mM KCl, 5 mM MgCl₂, 0.1 mM

EDTA, and 2.5 wt % glycerol) that contained 270 ng of pGEM

plasmid, increasing quantities of the potential inhibitor (50,

100, 200, 500 µM final concentration), and 10 units of topo-

isomerase I. Following a 30 min incubation at 37 °C (in the

dark), samples were extracted with phenol/chloroform and

subjected to electrophoresis (1% agarose, TRIS-borate, with-

out ethidium bromide, 10 V/cm).

(A) Absor ba n ce Titr a tion . Absorbance titration experi-

ments were performed on a Hewlett-Packard 8450A diode

array spectrometer maintained at 25 °C. Every 15 min,

(B) Top oisom er a se II Assa y. Reactions were carried out

in 20 µL cleavage buffer (10× to detect enzyme-mediated

6970 J . Org. Chem., Vol. 61, No. 20, 1996

Dienes and Vogel

clevage, 1× contained 30 mM Tris-HCl, pH 7.6, 3 mM ATP,

60 mM NaCl, 8 mM MgCl₂, and 15 mM mercaptoethanol) or

in assay buffer (10× to detect relaxation, 1× contained 50 mM

Tris-HCl, pH 8.0, 0.5 mM ATP, 120 mM KCl, 10 mM MgCl₂,

and 0.5 mM dithiothreitol) that contained about 300 ng of

pGEM plasmid, increasing quantity of potential inhibitor (2-

500 µM final concentration), and four units of topoisomerase

II. Following 40 min incubation at 37 °C (in the dark), the

reaction was stopped by addition of 2 µL of SDS (10%), and

proteins were digested on adding proteinase K (50 µg/mL) in

30 min at 37 °C. Samples were then extracted with phenol/

chloroform and subjected to electrophoresis (1% agarose,

TRIS-borate, with or without ethidium bromide (0.5 µg/mL),

10 V/cm). After running, agarose gels were examined under

a UV lamp (when no ethidium bromide was added before the

electrophoresis, it was added afterwards for visualization) and

photographed (see Figure 5 for an example).

Ack n ow led gm en t . We thank the Swiss National

Science Foundation, the Fonds Herbette (Lausanne),

and Hoffmann-La-Roche (Basel) for financial support.

We are also most grateful to Dr. Ce´cile Pasquier, Pierre-

Yves Musard (Universite´ de Neuchaˆtel, Switzerland),

Dr. Yegor Vassetzky (Institut Suisse de Recherche

Expe´rimentales sur le Cancer (ISREC), Epalinges,

Switzerland), and Miss Isabelle Rochat for their advice

and technical help.

Su p p or tin g In for m a tion Ava ila ble: Spectral data and

elemental analysis for various compounds (30 pages). This

material is contained in libraries on microfiche, immediately

follows this article in the microfilm version of the journal, and

can be ordered from the ACS; see any current masthead page

for ordering information.

J O960606Z

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