Synthesis of daunosamyl anthraquinone and anthracyclinone analogs

Four groups of aglycones (cyclopenta-annulated anthraquinones, mono-and bis-hydroxyalkyl-anthraquinones, steffinycinone) were glycosylated with daunosamine chloride. In all cases, the major products were the respective α -glycosides ( α , β -ratio ca . 9:1). In some cases, the β - glycosides rearranged to furanosides under the glycosylation conditions.


Introduction
New synthetic anthracyclines continue to be of great interest to overcome resistance and to reduce toxicity of these important anti-cancer drugs. 1 In connection with our work on the total synthesis of anthracyclinones (for reviews, see refs 2-13) and the aglycones of the important anthracycline antitumor antibiotics (for reviews, see refs 14-36), we started an investigation on the glycosylation of structurally diverse synthetic anthraquinone derivatives prepared by our group.(For a selection of other syntheses of anthracycline derivatives see refs 37-47).The general aim of this research was to get insight into the DNA binding capability as a function of distinct structural parameters and to correlate these binding data with cytotoxic activity.This investigation will be published elsewhere.In the present study we, describe the glycosylation step, the ratio of the α-to β-glycosides formed, and a new rearrangement of the N-trifluoroacetyl daunosamine β-glycosides to the respective furanoside glycosides.Trifluoroacetyl-protected daunosamine chloride 2 was used as the sugar component in all the reactions (Scheme 1).The activated protected sugar 2 was prepared from daunosamine hydrochloride, a gift from Prof. Arcamone, and was also prepared on a larger scale using the procedure of Horton and  Weckerle. 48Silver triflate, introduced by Hanessian into glycosylation chemistry, 49 served as the catalyst, as described by Arcamone et al. in the early stages of synthetic anthracycline analogs.[52]
The first aglycone, the cyclopenta-anthraquinone, rac-1, was prepared stereoselectively by reaction of leuco-quinizarine with α,β-unsaturated aldehydes such as methacrolein (see the Experimental Section).The trans-configuration of the methyl and hydroxyl group was deduced from the coupling constants of J 1,2 = 3. 4 Hz in the 1 H NMR spectrum.From the glycosylation of rac-1, only a single α-glycoside, 3, was isolated in pure form and the assignment shown in Scheme 1 is tentative.The largest group of non-natural daunosamyl glycosides was prepared from achiral hydroxymethyl anthraquinones 4-10 and the chiral racemic 1-hydroxybutylanthraquinone, rac-27.4][55][56] The glycosylation reactions were studied in great detail in repeated reactions and by careful analysis of the reaction products.In the reaction of the enantiomerically pure halo-sugar 2 with the achiral hydroxymethyl anthraquinones 4-10, only the α-glycosides and the respective β-glycosides were expected.In fact, in many cases these two products (11-17: α-glycosides) and (18-22: β-glycosides) could be isolated, with the α-glycosides being mostly the less polar products (Scheme 2).However, careful analysis of some of the polar reaction products revealed a surprising phenomenon.The compounds 23-26 turned out to be furanosides as shown in Scheme 2. In two cases (aglycone 7 and 9), no β-glycoside could be isolated from the reaction mixture.In contrast, all three possible products were isolated from the reaction of aglycones 8 and 10.In the case of the chiral 1-hydroxybutyl-quinizarine rac-27, the glycoslyation led to the diastereoisomeric α-glycosides 28 and 29.The minor β-glycoside were not isolated in this case, and the stereochemical assignment is arbitrary.The configuration of the furanosides 23-26 could in part be solved by comparison with the stereoisomeric methyl 2,3,6-trideoxy-3-(trifluoroacetamido)-β-and -α-D-ribo-hexo-furanosides, A and B, and the β-and α-L-lyxo-furanosides C and D (Scheme 3), prepared by El Khadem and Matsuura via the corresponding dithioacetals of epidaunosamin and daunosamine, respectively. 57s can be seen from Table 1, in which the relevant 1 H NMR data of the furanoses A-D was obtained from the literature 57 are compared with those of our furanoses 23-26, the coupling constant, J = 4.0 Hz, for the β-glycoside A compares well with that of our furanose glycosides (J = 4.0-4.2).In addition, the corresponding signals for the anomeric protons of the α-glycosides B and D appear as multiplets with two different coupling constants (see Table 1).Thus, the βconfiguration of our furanosides can be deduced unambiguously.On the assumption that the other stereocenters of the original daunosamine sugar did not change, the β-L lyxo configurations for 23-26 and 34 are proposed as shown in Schemes 2 and 4. The data shown in Table 1 also show the consistency of the relevant 1 H NMR data of the synthetic furanoses, demonstrating their stereochemical identity.
The mechanism of the rearrangement of the hexoses into the furanoses during the silverpromoted glycosylation is not yet clear.However, since the major α-pyranosides are stable under the reaction conditions, a rearrangement of the β-anomers during the glycosylation reaction, or the presence of a furanoside structure of the daunosamyl halide, seems most probable.Two examples of bis-hydroxymethylated achiral anthraquinones 30 and 32 were reacted with an excess of the sugar halide 2. The bis-glycosides are very attractive candidates to probe the possibility of stronger intercalation into double-stranded DNA.Since in principle each glycoside can appear in three different forms, a complex mixture was expected.However, to our delight, in the case of the 2,3-bis-hydroxymethyl-quinizarine 30, 55 which has both hydroxymethyl groups on the same side of the molecule, one major bis-α-glycoside 31 was isolated in highly pure form.However, in the case of bis-hydroxymethyl-1,5-dihydroxy-anthraquinone 32, 54 where the two hydroxymethyl groups are on opposite sides and on different aryl rings of the molecular scaffold, careful analysis of the glycosylation products revealed the formation of two major products 33 and 34.In the analogue 33, both sugar moieties were attached in α-glycosidic form, whereas in the analogue 34, one α-glycoside and one rearranged β-L-lyxo-furanoside were present in the same molecule.Finally, the naturally occurring anthracyclinone steffimycinone, 35, 58 was converted into the daunosamyl glycoside 36 by reaction with the halo-sugar 2 (Scheme 5).As expected, the homochiral aglycone was converted into one major α-glycoside, as seen from the 1 H NMR spectrum.Since the natural anthracycline steffimycin does not have an amino sugar moiety, for reasons of comparison, it was considered advantageous to have the homologous series of daunosamyl glycosides.In addition, the hybrid glycoside 34 offered the chance to probe the influence of the different substitution pattern of the hydroaromatic ring A, quite different from the standard daunorubicine, on DNA binding, and also to compare the amino-sugar glycoside 36 with the natural steffimycins 58 which have no amino-sugar moiety.In summary, the four groups of selected aglycones were converted into daunosamyl glycosides.In all cases, the major products were the α-glycosides (α:β-ratio ca 3-9:1).In five cases, the β-glycosides rearranged to β-L-lyxo-furanosides under the glycosylation conditions employed.

Experimental Section
General Procedures.Melting points were determined with an Electrothermal apparatus and are uncorrected.The IR spectra (KBr) were measured with a Perkin-Elmer 297, and the NMR spectra (CDCl 3 ) with a Bruker WH 270 (270 MHz) or Bruker Avance-500 NMR spectrometer, with TMS as internal standard (mc = centered multiplet).EI mass spectra were obtained on a MAT 8200 mass spectrometer at 70 eV.UV spectra were measured with a Perkin-Elmer Lambda 45 instrument and the UV/VIS data of representative compounds are listed in Table 2.
General procedure for glycoside synthesis. 51A solution (or suspension) of the respective aglycone in dry CH 2 Cl 2 (distilled over P 2 O 5 ) (10-30 mL) was treated first with a threefold molar excess of daunosamyl chloride (2), prepared according to the procedure of Horton and Weckerle. 48Then a solution of silver trifluorosulfonate (threefold excess) in dry diethyl ether (2-5 mL) was added dropwise.The mixture was stirred under exclusion of light and monitored by TLC (for reaction times see the individual compounds).The reaction was quenched by addition of excess aq.NaHCO 3 solution (ca. 5 mL) and the organic phase was dried (Na 2 SO 4 ) and evaporated at reduced pressure.To cleave the trifluoroacetate ester, the residue was dissolved in dry methanol (20 mL) and heated under reflux for 30 min.The solvent was removed under reduced pressure, the residue redissolved in CH 2 Cl 2 (10 mL), washed twice with water (10 mL), dried (Na 2 SO 4 ) and separated by preparative TLC (1 mm silica gel plates, Schleicher and Schüll).CH 2 Cl 2 was normally used as the eluent; for more polar compounds 15-25 % of diethyl ether-, and for very polar compounds an additional 1-2 % of methanol, was added.The plates were developed 15-20 times to separate the very similar isomeric glycosides.The major products usually consisted of the diastereomeric α-L-glycosides, and the minor components were either the β-L-glycosides or rearranged β-furanoside.Some of the glycosides could be crystallized from diethyl ether while others were precipitated with petroleum to afford a noncrystalline powder.These powders showed a melting range of 120-130 °C.All samples were dried for 6-7 h at 60 °C under high vacuum (1 mm Hg).In some cases, traces of solvent (CH 2 Cl 2 , diethyl ether) could not be removed completely without decomposition of the glycosides.(24), 213 (10), 180 (10), 169 (18), 155 (24), 140 (40), 113 (20)

Table 1 .
1H-NMR data for assignment of the configuration of the furanoses