A 5′-(trifluoromethyl)anthracycline glycoside: Synthesis of antitumor-active 7-O-(2,6-dideoxy-6,6,6-trifluoro-α-L-lyxo-hexopyranosyl)adriamycinone

Article abstract of DOI:10.1021/jm960177x

7-O-(2,6-Dideoxy-6,6,6-trifluoro-α-L-lyxo-hexopyranosyl)adriamycinone (3), whose substituent at C-5′ is a lipophilic trifluoromethyl group, has been prepared by coupling of 3,4-di-O-acetyl-2,6-dideoxy-6,6,6-trifluoro-α-L-lyxo-hexopyranosyl bromide (20) wi

Full text of DOI:10.1021/jm960177x

1582
J . Med. Chem. 1996, 39, 1582-1588
A 5′-(Tr iflu or om eth yl)a n th r a cyclin e Glycosid e: Syn th esis of An titu m or -Active
7-O-(2,6-Did eoxy-6,6,6-tr iflu or o-r-L-lyxo-h exop yr a n osyl)a d r ia m ycin on e
Yasushi Takagi,† Ken Nakai,† Tsutomu Tsuchiya,*,† and Tomio Takeuchi‡
Institute of Bioorganic Chemistry, 1614 Ida, Nakahara-ku, Kawasaki, 211, J apan, and Institute of Microbial Chemistry,
3-14-23 Kamiosaki, Shinagawa-ku, Tokyo 141, J apan
Received March 4, 1996X
7-O-(2,6-Dideoxy-6,6,6-trifluoro-R-L-lyxo-hexopyranosyl)adriamycinone (3), whose substituent
at C-5′ is a lipophilic trifluoromethyl group, has been prepared by coupling of 3,4-di-O-acetyl-
2,6-dideoxy-6,6,6-trifluoro-R-L-lyxo-hexopyranosyl bromide (20) with 14-O-(tert-butyldimeth-
ylsilyl)adriamycinone under the Koenigs-Knorr conditions. The key step in this synthesis
was the C-trifluoromethylation of 5-O-acetyl-2,3-di-O-benzyl-4-deoxy-aldehydo-L-erythro-pentose
(10), derived from D-lyxose in 10 steps, with (trifluoromethyl)trimethylsilane in the presence
of tetrabutylammonium fluoride, whereupon 1,1,1-trifluoro-L-arabino-hexitol (11) was obtained
along with its 2-epimer. The synthetic product 3 showed remarkable antitumor activity in
vivo in a low dose range compared to the analogs including doxorubicin. The fact may be
ascribed to the presence of a trifluoromethyl group at C-5′, suggesting the importance of the
group in view of biological activity.
In tr od u ction
sidic bond by decreasing the electron density of the
glycosidic oxygen as experienced for F-2′, and a trifluo-
romethyl group was chosen as the candidate. Another
reason for this selection is in its high lipophilicity29
compared to a methyl group; the CF3 at C-5′ was
expected to enhance the cellular uptake of the drugs
bearing the group and facilitate effective transportation
into organs or tumor cells when administered in vivo.
We report here on the preparation and antitumor
activity of 7-O-(2,6-dideoxy-6,6,6-trifluoro-R-L-lyxo-hex-
opyranosyl)adriamycinone (3).
Recently several promising anticancer drugs such as
calicheamicins,1 camptothecin analogs,2 dynemicins,3
esperamicins,4 etoposide,5 and taxol6 have been devel-
oped; however, doxorubicin (DOX), an anthracycline
glycosidic antibiotic, is still one of the most important
anticancer drugs in chemotherapy. Its clinical use,
however, is limited due to drug-cumulative cardiotox-
icity, myelosuppression, and other undesirable side
effects as well as occurrence of natural and acquired
resistance to this drug in tumor cells. To overcome
these drawbacks, a number of chemical and biosyn-
thetical modifications have been undertaken on both the
aglycon and sugar portions for over two decades.7-22
Among the studies, Horton and co-workers synthesized,
in 1984, 3′-deamino-3′-hydroxydoxorubicin23 (1) which
had fairy good antitumor activity with less toxicity,
albeit it has no amino group. This indicates that the
C-3′ amino group is not essential in its activity. On the
2′-position, most of the DOX analogs including 1 have
no substituent, and this will be the reason why these
antibiotics are unstable in acidic conditions, giving
inactive aglycons. This problem, however, was solved
by introducing a strongly electron-withdrawing fluorine
at C-2′, as illustrated by 7-O-(2,6-dideoxy-2-fluoro-R-L-
talopyranosyl)adriamycinone24,25 (2), which was stable
against acid26 and exhibited stronger antitumor activity
with less toxicity in comparison to those for DOX.
Additionally, 2 was found to be absorbed27 more rapidly
and accumulated more readily into tumor cells than
DOX. 4′-O-Glycosylated derivatives of 2 were also
prepared.28 Although the precise relationships between
the chemical stability and antitumor activity in vivo in
DOX analogs is not clear, the above result of 2 encour-
aged us to search for another type of stable compounds.
We considered that introduction of an electron-with-
drawing group at C-5′ may also strengthen the glyco-
Resu lts a n d Discu ssion
The synthesis was accomplished by coupling a 2,6-
dideoxy-6,6,6-trifluoro sugar with a protected adriamy-
cinone derivative (Scheme 1). During the past 6 years,
several 6-deoxy-6,6,6-trifluoro sugars have been syn-
thesized from simple sugars30,31 or nonsugar building
blocks.32-36 In this study, we adopted D-lyxose as a
starting material for preparation of 2,6-dideoxy-6,6,6-
trifluoro-L-lyxo-hexopyranose. Methyl 4-O-(p-tolylsul-
fonyl)-R-D-lyxopyranoside37,38 (4), prepared from D-lyxose
in four steps, was treated with LiAlH4 to give the
4-deoxy derivative 5.39 After protection of the hydroxyl
groups with benzyl ethers (BnBr, NaH in DMF), the
resulting compound 6 was hydrolyzed (0.4 M HCl in
aqueous 80% AcOH, 80 °C) to give the free sugar 7.
Treatment of 7 with 1,3-propanedithiol in the presence
* Author to whom correspondence should be sent.
† Institute of Bioorganic Chemistry.
‡ Institute of Microbial Chemistry.
X Abstract published in Advance ACS Abstracts, April 1, 1996.
0022-2623/96/1839-1582$12.00/0 © 1996 American Chemical Society

A 5′-(Trifluoromethyl)anthracycline Glycoside
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 8 1583
Sch em e 1a
a
(a) LiAlH4, THF, reflux (65%); (b) BnBr, NaH, DMF (81%); (c) 1:4 aqueous 2 M HCl/AcOH, 80 °C (82%); (d) HS(CH2)3SH, BF3‚Et2O,
Cl(CH2)2Cl, 60 °C (64%); (e) Ac2O, Py (97%); (f) Hg(ClO4)2‚3H2O, CaCO3, aqueous THF (99%); (g) (i) TMSCF3, Bu4NF‚3H2O, THF; (ii)
aqueous 80% AcOH, 50 °C (37% of 11, 37% of 12); (h) MeONa, MeOH (99%); (i) (i) (CF3SO2)2O, Py, CH2Cl2, 0 °C; (ii) NaNO2, DMF, 95 °C
(43%); (j) MeONa, MeOH (quantitative); (k) (i) TMSCl, DMAP, Py; (ii) CrO3, Py, CH2Cl2, 0 °C; (iii) 1:10 aqueous 1.1 M HCl/1,4-dioxane
(98%); (l) The same with (k) (92%); (m) Ac2O, Py (88%); (n) H2, Pd black, 10:1 aqueous 90% 1,4-dioxane/AcOH (98%); (o) Ac2O, Py
(quantitative); (p) 30% HBr in AcOH (90%); (q) 14-O-(tert-butyldimethylsilyl)adriamycinone, HgBr2, HgO(yellow), molecular sieves 3A,
Cl(CH2)2Cl (26% of 21, 25% of 22); (r) (i) MeONa, MeOH; (ii) aqueous 80% AcOH, 80 °C (69%).
of BF3‚Et2O in Cl(CH2)2Cl gave the dithioacetal 8, which
was then acetylated to give the 5-acetate 9. After
removal of the dithioacetal group with Hg(ClO4)2-
CaCO3 in aqueous THF, the resulting aldehyde 10 was
trifluoromethylated according to the procedure reported
by Prakash et al.:40 treatment of 10 with CF3Si(CH3)3
in the presence of a catalytic amount of Bu4NF in THF,
followed by deprotection of the resulting trimethylsilyl
group (aqueous 80% AcOH, 50 °C), gave, after chroma-
tography, the desired 1,1,1-trifluoro-L-arabino-hexitol
derivative (11) together with its 2-epimer (the 6,6,6-
trifluoro-D-ribo-hexitol derivative 12) in a ratio of 1:1
in 74% overall yield. Compound 12 was able to be
converted into 11 by treatment of the triflate of 12 with
8.2% H-5 signal increase). Compound 12 was similarly
converted to the D-ribo-hexopyranose derivative 16
through 14, and the structure was confirmed by the
NMR study (see the Experimental Section). Acetylation
of 15 gave the 1-acetate 17 and successive catalytic
hydrogenolysis (to give 18) and reacetylation gave the
triacetate 19. Several attempts to couple 19 or other
1-esters or 1-halides with 14-O-(tert-butyldimethylsilyl)-
adriamycinone23 in the presence of an appropriate
catalyst, however, did not proceed well, as demonstrated
by lack of coupling of 19 with the aglycon in the presence
of trimethylsilyl triflate. This may be the reason why
no couplings of 5-CF3 sugars to anthracyclinones have
ever been reported. Further attempts to couple using
either the protected phenylthioglycoside (in the presence
of N-bromosuccinimide43 or N-iodosuccinimide-CF3-
SO3H44) or the glycosyl fluoride (in the presence of Cp2-
ZrCl2-AgClO445) failed to give the condensation product.
However, the most classical coupling using the glycosyl
bromide 20 (prepared from 19 with 30% HBr-AcOH)
and 14-O-(tert-butyldimethylsilyl)adriamycinone under
the Koenigs-Knorr conditions [HgO(yellow), HgBr2,
molecular sieves 3A in Cl(CH2)2Cl] gave, after chroma-
tography, the desired R-L-glycoside 21 (J 1′,2′ax 3.5 Hz)
along with the â-L-glycoside 22 (J 1′,2′ax 10 Hz) in 26%
and 25% yield, respectively. Deacetylation (MeONa-
MeOH) followed by desilylation (aqueous 80% AcOH,
41
NaNO2 in 43% yield. After deacetylation (MeONa-
MeOH) of 11, the primary hydroxyl group of 13 was
selectively oxidized according to the procedure of Mahr-
wald et al.42 To do that, compound 13, after trimeth-
ylsilylation (TMSCl, DMAP in pyridine), was oxidized
(Collins oxidation), and the resulting aldehyde was
desilylated (0.1 M HCl in aqueous 1,4-dioxane) to give
an anomeric mixture of 3,4-di-O-benzyl-2,6-dideoxy-
6,6,6-trifluoro-L-lyxo-hexopyranose (15; 98% overall yield
1
based on 13). The structure (and the C4(L) conforma-
tion) was confirmed by a large coupling constant J 2ax 3
,
1
(12 Hz) in its H NMR spectrum as well as the existence
of NOE between H-3 and H-5 (saturation of H-3 showed

1584 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 8
Takagi et al.
Ta ble 1. Growth Inhibitory Effect of 3 and DOX against Various Cell Lines in Vitroa
IC50 (µg/mL)
compound
K562
HMV-1
KB
MKN-1
PC-14
T24
P388
P388/ADR
3
0.049
0.045
0.14
0.063
0.045
0.033
0.21
0.076
0.21
0.19
0.060
0.061
<0.0098
0.017
0.17
0.51
DOX‚HCl
a
IC50 values (50% inhibition concentration) were determined by MTT assay on day-3 culture.
Ta ble 2. Antitumor Activities (T/C, %; 60 Day Survivor Numbers/Treated Numbers of Mice) of 3 and DOX against L1210a
dose (mg/kg/day)
compound
5
2.5
1.25
0.6
0.3
0.15
0.08
0.04
3
T/C
survivor
T/C
135c
0/4
194c
0/4
203
0/4
330
0/4
>406
1/4
208
0/4
>606
3/4
132
0/4
>612
3/4
140
0/4
148
0/4
114
0/4
DOX‚HClb
177c
0/4
273c
0/4
survivor
a
Leukemia L1210 cells (105) were inoculated into CDF1 mice intraperitoneally. Drugs were administered daily, starting 24 h after
inoculation, from day 1 to 9, intraperitoneally. See ref 46. c Toxic.
b
and 19F were measured, respectively, downfield from internal
Me4Si, Me4Si, and CFCl3. Mass spectra were recorded with a
J EOL SX-102 spectrometer. TLC was performed on Kieselgel
60 F254 (Merck) and column chromatography, on Kieselgel 60
(230-400 mesh, Merck).
80 °C) gave the trifluoromethyl analog 3 as a red
powder. The compound was fairly stable against acid;
in monitoring by TLC, DOX was completely hydrolyzed
within 3.5 h in 0.5 M HCl in 1:1 DMSO/H2O at 60 °C,
whereas 3 was only partially hydrolyzed even after 7 h
in the same conditions.
Meth yl 4-Deoxy-â-L-er yth r o-p en top yr a n osid e (5). To a
cold (0 °C) suspension of LiAlH4 (1.03 g, 27 mmol) in dry
tetrahydrofuran (THF, 4 mL, freshly distilled from sodium
benzophenone ketyl) was added slowly a solution of methyl
4-O-(p-tolylsulfonyl)-R-D-lyxopyranoside37 (4, 2.14 g, 6.7 mmol)
in THF (11 mL), and the mixture was refluxed for 1.5 h. After
cooling to 0 °C, aqueous 10% NH4Cl (11 mL) and EtOAc (25
mL) were added, the mixture was stirred for 1 h at room
temperature and filtered through a pad of Celite, and the
filtrate was concentrated to give a residue that was chromato-
graphed (10:1 CHCl3/MeOH) to give 5 as a syrup (0.64 g, 65%),
together with methyl R-D-lyxopyranoside (0.13 g, 12%). Com-
pound 5 had [R]24 +101° (c 0.2, H2O) [lit.37 [R]21 +39.2° (c
Antitumor activity of 3 was examined by growth
inhibition assay against human and murine tumor cell
lines. As shown in Table 1, 3 exhibited comparative or
slightly-weaker activity than DOX against six human
cell lines [K562 (human leukemia), HMV-1 (human
melanoma), KB (human nasopharyngeal carcinoma),
MKN-1 (human gastric adenocarcinoma), PC-14 (human
lung carcinoma), and T24 (human bladder carcinoma)]
although 3-fold stronger activity than DOX against
P388/ADR (DOX-resistant P388 murine leukemia).
Compound 3, however, displayed significant antitumor
activity against L1210 murine leukemia in vivo in
comparison to DOX (Table 2). It is noteworthy that, in
a low dose range (0.6-0.15 mg/kg/day), seven mice out
of twelve survived 60 days, whereas no survivors were
observed in DOX. 3′-Deamino-3′-hydroxydoxorubicin 1,
whose substituent at C-5′ is a methyl, was reported23
to show the highest T/C value (462%) at 25 mg/kg (P388
murine leukemia by single ip injection on the first day;
no long-term survivors were reported). In the case of
2, 60-day survivors were observed in a relatively high
dose range (2.5-5 mg/kg/day).24 Comparison of these
results indicates that 3 has a remarkably high potency
in vivo. The higher activity of 3 versus 1 or 2 may be
reasonably ascribed to the presence of a trifluoromethyl
group at C-5′, whose contribution to its stability as well
as high lipophilicity may enhance the cellular uptake
of this analog, facilitating the transportation into organs
as well.
D
D
0.2, H2O); this value is incorrect]; 1H NMR (CD3OD) δ 1.60
(ddt, J ) 13, 4.5, 4.5, and 3 Hz, 1H, H-4eq), 1.90 (dddd, J )
13, 10, 8.5, and 5.5 Hz, 1H, H-4ax), 3.36 (s, 3H, OMe), 3.55
(apparently t, J ) 3.5 and 3 Hz, 1H, H-2), 3.61-3.77 (m, 2H,
H-5ax,5eq), 3.90 (ddd, J ) 10, 4.5, and 3 Hz, 1H, H-3), 4.57
(d, J ) 3.5 Hz, 1H, H-1). Anal. (C6H12O4) C, H.
Meth yl 2,3-Di-O-ben zyl-4-d eoxy-â-L-er yth r o-p en top y-
r a n osid e (6). A mixture of 5 (2.75 g, 18.6 mmol) and NaH
(60% in mineral oil, 3.74 g, 94 mmol) in dry N,N-dimethylfor-
mamide (DMF, 19 mL) was stirred for 1 h at room tempera-
ture. The mixture was cooled (0° C), benzyl bromide (6.6 mL,
56 mmol) was added, and the mixture was stirred for 1 h at
room temperature. After addition of aqueous 5% AcOH (160
mL), the mixture was extracted with CHCl3. The extract was
washed with aqueous NaHCO3, dried (Na2SO4), and concen-
trated. Column chromatography (30:1 toluene/Me2CO) of the
residue gave 6 as a pale yellow syrup (4.93 g, 81%): [R]23D +35°
(c 1.9, CHCl3); TLC Rf 0.55 (12:1 toluene/Me2CO); 1H NMR
(CDCl3) δ 1.69 (br ddt, J ) 13, 4.5, 3, and 3 Hz, 1H, H-4eq),
2.14 (dddd, J ) 13, 10.5, 10, and 5 Hz, 1H, H-4ax), 3.35 (s,
3H, OMe), 3.61 (ddd, J ) 3, 2.5, and 1 Hz, 1H, H-2), 3.63-
3.79 (m, 2H, H-5a,5b), 3.83 (ddd, J ) 10.5, 4.5, and 2.5 Hz,
1H, H-3), 4.56 (s, 2H, PhCH2), 4.71 (d, J ) 3 Hz, 1H, H-1),
4.73 and 4.78 (each d of 1H, J ) 12 Hz, PhCH2), 7.20-7.45
(m, 10H, Ph). Anal. (C20H24O4) C, H.
In summary, we have been able to prepare a novel
anthracycline glycoside having a CF3 group at C-5′ with
significant antitumor activity in vivo. Short syntheses
of the sugar and further biological evaluations of 3
(together with the related analogs) are now under study.
2,3-Di-O-ben zyl-4-d eoxy-L-er yth r o-p en top yr a n ose (7).
A solution of 6 (4.81 g, 14.7 mmol) in 1:4 aqueous 2 M HCl/
AcOH (48 mL) was heated for 30 min at 80 °C and then poured
into aqueous NaHCO3 (saturated, 400 mL). Extraction of the
product with CHCl3 gave a syrup that was purified by column
chromatography (12:1 toluene/Me2CO) to give 7 as a syrup
Exp er im en ta l Section
Melting points were determined on a Kofler block and are
uncorrected. Optical rotations were determined with a Perkin-
Elmer 241 polarimeter. 1H NMR (at 250 MHz) and 19F NMR
(at 235.3 MHz) spectra were recorded with a Bruker AC 250P
spectrometer, unless otherwise stated. 1H NMR (at 500 MHz)
and 13C NMR (at 125.8 MHz) spectra were recorded with a
(3.78 g, 82%): [R]25 +24.5° (c 1.1, CHCl3, 24 h after dissolu-
D
tion); TLC Rf 0.20 (12:1 toluene/Me2CO). Anal. (C19H22O4) C,
H.
2,3-Di-O-ben zyl-4-d eoxy-L-er yth r o-p en tose Tr im eth yl-
en e Dith ioa ceta l (8). A mixture of 7 (10.3 g, 32.7 mmol),
1,3-propanedithiol (5.9 mL, 59 mmol), and BF3‚Et2O (1.2 mL,
1
Bruker AMX 500 spectrometer. Chemical shifts (δ) for H, 13C,

A 5′-(Trifluoromethyl)anthracycline Glycoside
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 8 1585
9.8 mmol) in dry Cl(CH2)2Cl (60 mL) was heated for 3.5 h at
60 °C. Chloroform (300 mL) was added, and the solution was
washed with aqueous 5% NaOH and water, dried (Na2SO4),
and concentrated. The residue was chromatographed (12:1
toluene/Me2CO) to give 8 as a colorless syrup (8.50 g, 64%):
10H, Ph); 19F NMR (CDCl3) δ -76.1 (d, J ) 7 Hz, CF3). Anal.
(C21H25F3O5) C, H, F.
P r ep a r a tion of 11 fr om 12. To a cold (0 °C) solution of
12 (42 mg, 0.1 mmol) in CH2Cl2 (0.4 mL) were added (CF3-
SO2)2O (25 µL, 0.15 mmol) and pyridine (0.05 mL), and the
solution was kept for 1.5 h at 0 °C. MeOH (0.06 mL) was
added, and after dilution with CH2Cl2, the solution was washed
successively with cold aqueous 20% KHSO4, aqueous NaHCO3,
and water, dried (Na2SO4), and concentrated to give the
5-triflate of 12 as a chromatographically homogeneous syrup
(51 mg, 93%): TLC Rf 0.5 (25:1 toluene/EtOAc); 1H NMR
(CDCl3) δ 5.35 (dq, 1H, J ) 6, 6, 6, and 3.5 Hz, H-5); 19F NMR
(CDCl3) δ -74.3 (q, 3F, J CF3SO3,F-6 ) 3.5 Hz CF3SO3), -72.1
(dq, 3F, J ) 6, 3.5, 3.5, and 3.5 Hz, F-6). A mixture of the
syrup (46 mg, 0.08 mmol) and NaNO2 (73 mg, 1.1 mmol) in
DMF (0.45 mL) was stirred for 2.5 h at 95 °C. Evaporation of
the solvent after addition of water and xylene gave a residue
that was chromatographed (25:1 toluene/EtOAc) to give 11 (16
mg, 43% based on 12), which was identical with the specimen
obtained from 10.
[R]25 -39° (c 1.1, CHCl3); TLC Rf 0.27 (12:1 toluene/Me2CO);
D
1H NMR (CDCl3) δ 1.81-1.99 (m, 3H, H-4a and SCH2CH2-
CH2S), 2.05-2.17 (m, 2H, H-4b and OH), 2.72-2.92 (m, 4H,
SCH2CH2CH2S), 3.72 (q, collapsed to t in addition of D2O, J )
5.5 Hz, 2H, H-5a,5b), 3.83 (dd, J ) 5.5 and 4.5 Hz, 1H, H-2),
3.98 (dt, J ) 5.5, 5.5 and 4.5 Hz, 1H, H-3), 4.36 (d, J ) 5.5 Hz,
1H, H-1), 4.57 and 4.65 (each d of 1H, J ) 11.5 Hz, PhCH2),
4.75 and 4.90 (each d of 1H, J ) 11 Hz, PhCH2), 7.21-7.49
(m, 10H, Ph). Anal. (C22H28O3S2) C, H, S.
5-O-Acet yl-2,3-d i-O-b en zyl-4-d eoxy-L-er yth r o-p en t ose
Tr im eth ylen e Dith ioa ceta l (9). A solution of 8 (1.03 g, 2.5
mmol) and Ac2O (0.4 mL, 4.2 mmol) in pyridine (8 mL) was
treated conventionally to give 9 as a colorless syrup (1.09 g,
97%): [R]21 -37.5° (c 0.7, CHCl3); TLC Rf 0.62 (12:1 toluene/
D
Me2CO); 1H NMR (CDCl3) δ 1.98 (s, 3H, Ac), 1.81-2.17 (m,
4H, H-4a,4b and SCH2CH2CH2S), 2.71-2.92 (m, 4H, SCH2-
CH2CH2S), 3.77 (apparently t, J ) ∼5 Hz, 1H, H-2), 3.87 (ddd,
J ) 7.5, 5, and 4 Hz, 1H, H-3), 4.13-4.23 (m, 2H, H-5a,5b),
4.36 (d, J ) 5.5 Hz, 1H, H-1), 4.53, 4.63, 4.74, and 4.90 (each
d of 1H, 2PhCH2), 7.22-7.49 (m, 10H, Ph). Anal. (C24H30O4S2)
C, H, S.
3,4-Di-O-ben zyl-1,5-d id eoxy-1,1,1-tr iflu or o-L-a r a bin o-
h exitol (13). A solution of 11 (296 mg, 0.70 mmol) in
methanolic 0.025 M MeONa (5 mL, 0.13 mmol) was kept for 1
h at room temperature. Neutralization with Dowex 50W X-2
resin (H+ form) followed by filtration and concentration gave
13 as a syrup (266 mg, 99%): [R]21 -17° (c 0.7, CHCl3); TLC
D
Rf 0.2 (10:1 toluene/Me2CO). Anal. (C20H23F3O4) C, H, F.
3,4-Di-O-ben zyl-2,6-d id eoxy-6,6,6-tr iflu or o-D-r ibo-h exi-
tol (14). Compound 12 (273 mg) was treated similarly as
described for 13 to give 14 as a syrup (246 mg, quantitative):
5-O-Acetyl-2,3-d i-O-ben zyl-4-d eoxy-a ld eh yd o-L-er yth r o-
p en tose (10). To a mixture of 9 (1.09 g, 2.45 mmol) and
CaCO3 (4.70 g) in 3:1 THF/H2O (15 mL) was added Hg(ClO4)2‚
3H2O (2.90 g, 6 mmol) in THF (9 mL), and the mixture was
stirred for 1 h at room temperature. After addition of CH2Cl2
(50 mL), the mixture was shaken with aqueous NaHCO3
(saturated, 20 mL) and filtered through a pad of Celite, and
the filtrate was washed with aqueous 10% KI and water, dried
(Na2SO4), and concentrated to give 10 as a syrup (0.87 g, 99%)
that was used without purification to the next step: [R]20D -71°
(c 1, CHCl3); 1H NMR (CDCl3) δ 1.82 (ddt, J ) 15, 7.5, 7.5,
and 3.5 Hz, 1H, H-4a), 1.96 (s, 3H, Ac), 1.97-2.11 (m, 1H,
H-4b), 3.89 (dt, J ) 9, 3.5, and 3.5 Hz, 1H, H-3), 3.95 (dd, J )
3.5 and 1.5 Hz, 1H, H-2), 4.08-4.18 (m, 2H, H-5a,5b), 4.52,
4.64, 4.64, and 4.72 (each d of 1H, J ) 11.5 Hz, 2PhCH2), 7.22-
7.49 (m, 10H, Ph), 9.71 (d, J ) 1.5 Hz, 1H, H-1); FAB-MS m/ e
357 (M + H)+.
6-O-Acetyl-3,4-d i-O-ben zyl-1,5-d id eoxy-1,1,1-tr iflu or o-
L-a r a bin o-h exitol (11) a n d 1-O-a cetyl-3,4-d i-O-ben zyl-2,6-
d id eoxy-6,6,6-tr iflu or o-D-r ibo-h exitol (12). To a cold (0 °C)
solution of 10 (0.81 g, 2.28 mmol) and CF3SiMe3 (0.5 mL, 3.4
mmol) in THF (8 mL) was added Bu4NF‚3H2O (71 mg, 0.23
mmol) in THF (2 mL), and the solution was kept for 1.5 h at
room temperature. TLC (25:1 toluene/EtOAc) of the solution
showed three spots at Rf 0.08 (12), 0.17 (11), and 0.38
(O-trimethylsilyl derivatives of 11 and 12). After concentra-
tion, the residue was extracted with CHCl3, and the solution
was washed with water, dried (Na2SO4), and concentrated. The
resulting syrup was dissolved in aqueous 80% AcOH (4 mL)
and kept for 1.5 h at 50 °C. In TLC, the spot at Rf 0.38
disappeared. Evaporation of solvents by codistillation with
toluene gave a residue that was chromatographed (25:1
toluene/EtOAc) to give 11 (0.36 g, 37%) and 12 (0.36 g, 37%)
as syrups.
[R]24 -31° (c 0.5, CHCl3). Anal. (C20H23F3O4) C, H, F.
D
3,4-Di-O-ben zyl-2,6-d id eoxy-6,6,6-tr iflu or o-L-lyxo-h ex-
op yr a n ose (15). A mixture of 13 (1.46 g, 3.8 mmol), Me3-
SiCl (1.74 mL, 13.7 mmol), and 4-dimethylaminopyridine (183
mg, 1.5 mmol) in pyridine (15 mL) was stirred for 15 h at room
temperature. The mixture showed, in TLC (10:1 toluene/Me2-
CO), a spot at Rf 0.9 (the 2,6-di-O-TMS derivative). After
concentration, the residue dissolved in EtOAc was washed with
water, dried (Na2SO4), and concentrated to give a pale yellow
syrup (2.07 g). The syrup dissolved in CH2Cl2 (3.5 mL) was
added to a mixture of dry CrO3 (1.82 g, 18.2 mmol) and
pyridine (3.0 mL, 37 mmol) in CH2Cl2 (25 mL), which was
previously stirred for 30 min at room temperature and cooled
(0 °C), and stirred for 40 min at the temperature. TLC (10:1
toluene/Me2CO) showed two spots at Rf 0.35 (15) and 0.75 (the
5-O-TMS derivative). After filtration through a pad of silica
gel with aid of EtOAc, the mixture was concentrated. The
residue dissolved in EtOAc was again treated as above to
eliminate insoluble matters. The resulting brown syrup (1.83
g) dissolved in a mixture of 1:10 aqueous 1.1 M HCl/1,4-
dioxane (18 mL) was kept for 1 h at room temperature. TLC
(10:1 toluene/Me2CO) showed a single spot (15) with disap-
pearance of the one of Rf 0.75. After neutralization (aqueous
NaHCO3), the product was extracted with CH2Cl2 to give, after
usual purification, 15 (1.42 g, 98%) as a syrup of an anomeric
mixture (R:â ∼14:1), which solidified after 1 month: mp 71.5-
72 °C; [R]24 -72° (c 0.4, CHCl3, 24 h after dissolution); 1H
D
NMR (CDCl3, only for R-L-anomer) δ 2.02 (ddt, J ) 13, 4.5,
∼1.5, and ∼1.5 Hz, 1H, H-2eq), 2.30 (ddd, J ) 13, 12, and 3.5
Hz, 1H, H-2ax), ∼2.8 (br, 1H, OH), 3.96 (ddd, J ) 12, 4.5, and
2.5 Hz, 1H, H-3), 4.11 (br s, 1H, H-4), 4.28 (br q, J ) 7 Hz,
1H, H-5), 4.60 (s, 2H, PhCH2), 4.71 and 4.91 (each d of 1H, J
) 11 Hz, PhCH2), 5.54 (br d, J ) 3.5 Hz, 1H, H-1), 7.28-7.42
(m, 10H, Ph); 19F NMR (CDCl3) δ -74.0 (d, J ) 7 Hz, ∼2.8F,
CF3 of R-L-anomer), -73.6 (d, J ) 6.5 Hz, ∼0.2F, CF3 of â-L-
anomer). Anal. (C20H21F3O4) C, H, F.
Compound 11 had [R]24D -24° (c 1, CHCl3); 1H NMR (CDCl3)
δ 2.00 (s, 3H, Ac), 1.84-2.12 (m, 2H, H-5a,5b), 3.28 (d, J )
9.5 Hz, 1H, OH, disappeared on deuteration), 3.67 (ddd, J )
7, 6, and 4 Hz, 1H, H-4), 3.83 (dd, J ) 6 and 1.5 Hz, 1H, H-3),
4.08 (ddq, J 2,F ) 7.5, J 2,OH ) 9.5, and J 2,3 ) 1.5 Hz, which
collapsed to dq on deuteration 1H, H-2), 4.12-4.26 (m, 2H,
H-6a,6b), 4.56 and 4.62 (each d of 1H, PhCH2), 4.66 and 4.72
(each d of 1H, PhCH2), 7.23-7.42 (m, 10H, Ph); 19F NMR
(CDCl3) δ -77.6 (d, J ) 7.5 Hz, CF3). Anal. (C21H25F3O5) C,
H, F.
3,4-Di-O-ben zyl-2,6-d id eoxy-6,6,6-tr iflu or o-D-r ibo-h ex-
op yr a n ose (16). Compound 14 (192 mg) was treated simi-
larly as described for 15 to give 16 as a syrup (175 mg, 92%).
An analytical sample was prepared by column chromatography
(25:1 toluene/EtOAc) followed by recrystallization (EtOAc/
Compound 12 had [R]23 -31° (c 0.9, CHCl3); 1H NMR
hexane) as needles: mp 74.5-75.5 °C; [R]24 +51° (c 0.5,
D
D
(CDCl3) δ 1.97 (s, 3H, Ac), 1.88-2.14 (m, 2H, H-2a,2b), 3.20
(br, 1H, OH), 3.85-3.94 and 4.04-4.24 (each m of 2H and 3H,
respectively, H-1a,1b,3,4,5), 4.56 and 4.67 (each d of 1H,
PhCH2), 4.62 and 4.74 (each d of 1H, PhCH2), 7.23-7.41 (m,
CHCl3, 24 h after dissolution); TLC Rf 0.2 (25:1 toluene/EtOAc;
cf. 15, Rf 0.1); 1H NMR (500 MHz, CDCl3) δ 1.54 [ddd, J )
13.5, 9.5, and 2.5 Hz, 0.2H, H-2ax(â-L-anomer)], 1.79 [ddd, J
) 14.5, 3.5, and 2.5 Hz, 0.8H, H-2ax(R-L-anomer)], 2.13 [ddd,

1586 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 8
Takagi et al.
J ) 14.5, 4, and ∼1 Hz, 0.8H, H-2eq(R)], 2.24 [ddd, J ) 13.5,
4, and 2 Hz, 0.2H, H-2eq(â)], ∼3.1 [br, 0.2H, HO-1(â)], 3.65
[dd, J ) 9.5 and 2.5 Hz, 1H, H-4(R and â)], 3.98 [m, 0.2H,
H-3(â)], 4.09 [m, 0.8H, H-3(R)], 4.39 [dq, J ) 9.5, 6.5, 6.5, and
6.5 Hz, 0.2H, H-5(â)], 4.56 [dq, J ) 9.5, 6.5, 6.5, and 6.5 Hz,
0.8H, H-5(R)], 4.69 [d, J ) 11.5 Hz, 0.2H, PhCH2(â)], 4.55, 4.62,
4.64, and 4.85 [each d of 0.8H, J ) 11.5 Hz, PhCH2(R)], 5.21
[br dd, J ) 11 and 3.5 Hz, 0.8H, H-1(R)], 5.26 [dd after
deuteration; J ) 9.5 and 2 Hz, 0.2H, H-1(â)], 5.27 [d, J ) 11
Hz, 0.8H, HO-1(R)], 7.25-7.40 (m, 10H, Ph); saturation of
H-2ax(R) showed 5.2% signal increase of H-4; 19F NMR (CDCl3)
δ -74.6 [d, J ) 6.5 Hz, 0.6F, CF3(â)], -74.2 [d, J ) 6.5 Hz,
2.4F, CF3(R)]. Anal. (C20H21F3O4) C, H, F.
1-O-Acetyl-3,4-d i-O-ben zyl-2,6-d id eoxy-6,6,6-tr iflu or o-
L-lyxo-h exop yr a n ose (17). A solution of 15 (1.34 g, 3.5
mmol) in pyridine (11 mL) was treated with Ac2O (0.5 mL,
5.3 mmol) conventionally to give 17 as a syrup (1.31 g, R-L:â-L
7:3, 88%); 1H NMR (CDCl3) δ (selected signals only) 1.96-2.13
[m, 1H, H-2eq (R- and â-L-anomer)], 2.07 [s, 2.1H, Ac(R)], 2.11
[s, 0.9H, Ac(â)], 2.31 [dt, J ) 12, 12, and 10.5 Hz, 0.3H, H-2ax-
(â)], 2.45 [ddd, J ) 13.5, 12, and 3.5 Hz, 0.7H, H-2ax(R)], 5.71
[dd, J ) 10.5 and 2.5 Hz, 0.3H, H-1(â)], 6.38 [br d, J ) 3.5 Hz,
0.7H, H-1(R)]; 19F NMR (CDCl3) δ -74.1 [d, J ) 6.5 Hz, 2.1F,
CF3(R)], -73.5 [d, J ) 6.5 Hz, 0.9F, CF3(â)]. Anal. (C22H23F3O5)
C, H, F.
1-O-Acetyl-2,6-d id eoxy-6,6,6-tr iflu or o-L-lyxo-h exop yr a -
n ose (18). A solution of 17 (1.31 g) in 10:1 aqueous 90% 1,4-
dioxane/AcOH (25 mL) was hydrogenated in the presence of
Pd black under gentle bubbling of H2 for 5 h at room
temperature. Filtration followed by evaporation of the solvents
with toluene gave 18 as a syrup (736 mg, 98%), which was
precipitated from the EtOAc solution by addition of hexane to
give a solid: 1H NMR (CD3OD) δ 1.99 [s, 2.1H, Ac(R-L)], 2.00
[s, 0.9H, Ac(â-L)], 1.66-2.12 [m, 2H, H-2ax(R),2eq(R),2ax(â),
2eq(â)], 3.72 [ddd, J ) 11.5, 5.5, and 3 Hz, 0.3H, H-3(â)], 3.82-
4.00 [m, 1.7H, H-3(R),4(R),4(â)], 4.19 (br d, J ) 7 Hz, 1H, H-5),
5.66 [dd, J ) 10 and 3 Hz, 0.3H, H-1(â)], 6.15 [br d, J ) 3.5
Hz, 0.7H, H-1(R)]; 19F NMR (CD3OD) δ -73.9 [d, J ) 7 Hz,
2.1F, CF3(R)], -73.4 [d, J ) 7 Hz, 0.9F, CF3(â)]. Anal.
(C8H11F3O5) C, H, F.
mmol) and 14-O-(tert-butyldimethylsilyl)adriamycinone23 (142
mg, 0.27 mmol) in dry Cl(CH2)2Cl (4 mL, freshly distilled from
CaH2) was stirred in the dark in the presence of yellow HgO
(498 mg, 2.3 mmol), HgBr2 (174 mg, 0.48 mmol), and molecular
sieves 3A (700 mg) for 15 h at room temperature. After
addition of CHCl3, the mixture was filtered through a pad of
Celite, which was thoroughly washed with CHCl3, and the
filtrates combined were washed successively with aqueous 30%
KI, aqueous NaHCO3, and water, dried (Na2SO4), and concen-
trated. TLC (10:1 toluene/Me2CO) of the residue showed three
spots at Rf 0.17 (an adriamycinone derivative), 0.25 (22), and
0.28 (21). Separation of the products by twice column chro-
matography (10:1 toluene/Me2CO f 30:1 CHCl3/Me2CO) gave
21 (50 mg, 26%) and 22 (49 mg, 25%) as red solids. Analytical
samples of each compound were obtained by reprecipitation
from CHCl3/hexane.
1
Compound 21: [R]22 +202° (c 0.1, CHCl3); H NMR (500
D
MHz, CDCl3) δ 0.13 and 0.15 (each s of 3H, SiMe2), 0.96 (s,
t
9H, Bu), 1.93 (br dd, J ) 12.5 and 5 Hz, 1H, H-2′eq), 1.95
and 2.15 (each s of 3H, 2Ac), 2.20 (dt, J ) 12.5, 12.5, and 3.5
Hz, 1H, H-2′ax), 2.22 (dd, J ) 15 and 4.5 Hz, 1H, H-8ax), 2.41
(ddd, J ) 15, 2.5, and 1.5 Hz, 1H, H-8eq), 3.02 (d, J ) 19 Hz,
1H, H-10ax), 3.22 (dd, J ) 19 and 1.5 Hz, 1H, H-10eq), 3.84
(s, 1H, HO-9), 4.09 (s, 3H, OMe), 4.55 (br q, J ) 6.5 Hz, 1H,
H-5′), 4.79 and 4.87 (each d of 1H, J ) 19.5 Hz, H-14a,14b),
5.06 (ddd, J ) 12.5, 5, and 3 Hz, 1H, H-3′), 5.26 (dd, J ) 4.5
and 2.5 Hz, 1H, H-7), 5.65 (br d, J ) 3 Hz, 1H, H-4′), 5.73 (br
d, J ) 3.5 Hz, 1H, H-1′), 7.40 (dd, J ) 8.5 and ∼1 Hz, 1H,
H-3), 7.79 (apparently t, J ) 8.5 and 7.5 Hz, 1H, H-2), 8.03
(dd, J ) 7.5 and ∼1 Hz, 1H, H-1), 13.20 and 13.98 (each s of
1H, HO-6,11); 19F NMR (CDCl3) δ -74.6 (d, J ) 6.5 Hz, CF3).
Anal. (C37H43F3O14Si‚0.5H2O) C, H, F.
Compound 22: [R]23 +247° (c 0.1, CHCl3); 1H NMR (C6D6)
D
t
δ 0.16 and 0.18 (each s of 3H, SiMe2), 1.06 (s, 9H, Bu), 1.52
and 1.67 (each s of 3H, 2Ac), 1.68 (m, 1H, H-2′eq), 1.91 (dd, J
) 15 and 3.5 Hz, 1H, H-8ax), 1.93 (dt, J ) 12.5, 12.5, and 10
Hz, 1H, H-2′ax), 2.25 (br d, J ) 15 Hz, 1H, H-8eq), 3.04 (dq, J
) 6, 6, 6, and ∼1 Hz, 1H, H-5′), 3.25 (d, J ) 19.5 Hz, 1H,
H-10ax), 3.32 (s, 3H, OMe), 3.33 (br d, J ) 19.5 Hz, 1H,
H-10eq), 4.60 (s, 1H, HO-9), 4.62 (dd, J ) 10 and 2.5 Hz, 1H,
H-1′), 4.69 (ddd, J ) 12.5, 5, and 3 Hz, 1H, H-3′), 4.96 and
5.11 (each d of 1H, J ) 20 Hz, H-14a,14b), 5.24 (dd, J ) 3.5
and 2.5 Hz, 1H, H-7), 5.45 (br d, J ) 3 Hz, 1H, H-4′), 6.50 (dd,
J ) 8.5 and ∼1 Hz, 1H, H-3), 7.02 (apparently t, J ) 8.5 and
8 Hz, 1H, H-2), 7.94 (dd, J ) 8 and ∼1 Hz, 1H, H-1), 13.51
and 14.68 (each s of 1H, HO-6,11); 19F NMR (C6D6) δ -73.8
(d, J ) 6 Hz, CF3). Anal. (C37H43F3O14Si‚0.5H2O) C, H, F.
7-O-(2,6-Did eoxy-6,6,6-t r iflu or o-R-L-lyxo-h exop yr a n o-
syl)a d r ia m ycin on e (3). To a suspension of 21 (40 mg, 50
µmol) in dry MeOH (3.6 mL) was added methanolic 0.25 M
MeONa (0.13 mL, 32 µmol), and the mixture was stirred for
2.5 h at room temperature. After a piece of dry ice was added,
and the mixture was concentrated. The residue was extracted
with CHCl3, and the product [TLC (1:1 toluene/Me2CO) Rf 0.55]
dissolved in aqueous 80% AcOH (1 mL) was kept for 40 min
at 80 °C. Concentration together with toluene gave a residue,
which was washed alternately with water and toluene and
1,3,4-Tr i-O-acetyl-2,6-dideoxy-6,6,6-tr iflu or o-L-lyxo-h ex-
op yr a n ose (19). A solution of 18 (0.74 g, 3.0 mmol) and Ac2O
(0.7 mL, 7.4 mmol) in pyridine (6 mL) was kept for 15 h at
room temperature. Conventional processing gave 19 as a
syrup (0.99 g, quantitative); TLC (CH2Cl2): Rf 0.35(R) and 0.2-
(â). Analytical samples were obtained by chromatographic
separation using CH2Cl2.
R-L-Anomer (solid): mp 91.5-92.5 °C; [R]19 -100° (c 0.6,
D
CHCl3); 1H NMR (CDCl3) δ 1.94 (ddt, J ) 13.5, 5, 1.5, and 1.5
Hz, 1H, H-2eq), 2.02, 2.14, and 2.15 (each s of 3H, Ac), 2.32
(ddd, J ) 13.5, 12.5, and 3.5 Hz, 1H, H-2ax), 4.36 (br q, J ) 6
Hz, 1H, H-5), 5.28 (ddd, J ) 12.5, 5, and 3 Hz, 1H, H-3), 5.66
(m, 1H, H-4), 6.44 (br d, J ) 3.5 Hz, 1H, H-1); 19F NMR (CDCl3)
δ -75.0 (d, J ) 6 Hz, CF3). Anal. (C12H15F3O7) C, H, F.
â-L-Anomer (syrup): [R]22 -26° (c 0.8, CHCl3); 1H NMR
D
(CDCl3) δ 2.03 (s, 3H, Ac), 2.16 (s, 6H, 2Ac), 1.99-2.25 (m,
2H, H-2ax,2eq), 4.05 (dq, J ) 6 and ∼1 Hz, 1H, H-5), 5.06 (ddd,
J ) 12.5, 5, and 3 Hz, 1H, H-3), 5.58 (br d, J ) 5 Hz, 1H,
H-4), 5.85 (dd, J ) 10 and 2.5 Hz, 1H, H-1); 19F NMR (CDCl3)
δ -74.4 (d, J ) 6 Hz, CF3).
dried to give 3 as a dark red solid (21 mg, 69%): [R]21 +188°
D
1
(c 0.02, pyridine); TLC Rf 0.35 (1:1 toluene/Me2CO); H NMR
(500 MHz, pyridine-d5) δ 2.40 (br dd, J ) 12 and 4.5 Hz, 1H,
H-2′eq), 2.50 (dd, J ) 14.5 and 5 Hz, 1H, H-8ax), 2.72 (dt, J )
12, 12, and 3.5 Hz, 1H, H-2′ax), 2.99 (apparently dt, J ) 14.5,
2.5, and 1.5 Hz, 1H, H-8eq), 3.44 (d, J ) 19 Hz, 1H, H-10ax),
3.55 (dd, J ) 19 and 1.5 Hz, 1H, H-10eq), 3.96 (s, 3H, OMe),
4.51 (br dt, J ) 12, ∼4, and ∼4 Hz, 1H, H-3′), 4.53 (br s, 1H,
H-4′), 5.25 (br q, J ) 7 Hz, 1H, H-5′), 5.37 and 5.42 (each d of
1H, J ) 20 Hz, H-14a,14b), 5.42 (dd, J ) 5 and 2.5 Hz, 1H,
H-7), 5.95 (br d, J ) 3.5 Hz, 1H, H-1′), 6.55 (br s, 2H, 2OH),
∼7.1 (v br, 1H, OH), 7.41 (br d, J ) 8 Hz, 1H, H-3), 7.71 (t, J
) 8 Hz, 1H, H-2), 8.06 (br d, J ) 8 Hz, 1H, H-1), 13.53 and
14.58 (each s of 1H, HO-6,11); 19F NMR (pyridine-d5) δ -72.2
(d, J ) 7 Hz, CF3); 13C NMR (pyridine-d5) δ 33.5 (C-10), 34.3
(C-2′), 37.2 (C-8), 56.6 (OCH3), 65.3 (C-3′), 65.7 (C-14), 67.3
(C-4′), 71.0 (q, J ) 30 Hz, C-5′), 71.9 (C-7), 76.3 (C-9), 103.2
(C-1′), 111.5 and 111.8 (C-5a,11a), 119.5 (C-3), 119.6 (C-1),
121.2 (C-4a), 125.6 (q, J ) 281 Hz, C-6′), 134.7, 135.6, and
3,4-Di-O-a cetyl-2,6-d id eoxy-6,6,6-tr iflu or o-R-L-lyxo-h ex-
op yr a n osyl Br om id e (20). A solution of 19 (89 mg, 0.27
mmol) in 30% HBr in AcOH (0.9 mL) was kept for 5 h at room
temperature. CHCl3 (15 mL) was added, and the solution was
washed with aqueous NaHCO3 (saturated) and water, dried
(MgSO4), and concentrated to give 20 as a pale yellow syrup
(85 mg, 90%), which was used without purification: TLC Rf
1
0.6 (CH2Cl2); H NMR (CDCl3) δ 2.02 and 2.14 (each s of 3H,
Ac), 2.30 (ddt, J ) 13.5, 5, 1, and 1 Hz, 1H, H-2eq), 2.64 (ddd,
J ) 13.5, 12, and 4 Hz, 1H, H-2ax), 4.56 (br q, J ) 6.5 Hz, 1H,
H-5), 5.47 (ddd, J ) 12, 5, and 3 Hz, 1H, H-3), 5.72 (m, 1H,
H-4), 6.72 (br d, J ) 4 Hz, 1H, H-1); 19F NMR (CDCl3) δ -74.3
(d, J ) 6.5 Hz, CF3).
14-O-(ter t-Bu tyld im eth ylsilyl)-7-O-(3,4-d i-O-a cetyl-2,6-
d id eoxy-6,6,6-tr iflu or o-r- a n d -â-L-lyxo-h exop yr a n osyl)-
a d r ia m ycin on e (21 a n d 22). A mixture of 20 (85 mg, 0.24

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1995; pp 59-77.
135.8 (C-6a,10a,12a), 136.0 (C-2), 155.6 and 157.1 (C-6,11),
161.5 (C-4), 187.1 (C-5,12), 215.2 (C-13). Anal. (C27H25F3O12
C, H, F.
)
Cell Gr ow th In h ibitor y Mea su r em en ts in Vitr o. K 562
human leukemia, HMV-1 human melanoma, KB human
nasopharyngeal carcinoma, MKN-1 human gastric adenocar-
cinoma, PC-14 human lung carcinoma, T24 human bladder
carcinoma, and P388 murine leukemia cells were cultured in
RPMI 1640 medium supplemented with 10% fetal bovine
serum, and DOX-resistant P388 cells (P388/ADR) were cul-
tured in a medium containing 2-hydroxyethyl disulfide ad-
ditionally added. Compound 3 (1 mg) and DOX‚HCl (1 mg)
were dissolved respectively in DMSO (1 mL) and water (1 mL),
and each solution was diluted with RPMI 1640 medium to a
concentration of 5-0.0098 µg/mL. The cultured cells, after
addition of each of the above solutions, were allowed to
proliferate in an incubator under the atmosphere of 5% CO2
and 95% air in 100% relative humidity at 37 °C for 72 h, and
the cell densities were measured using a MTT colorimetric
method47 to give the drug concentration inhibiting 50% cellular
growth (IC50, µg/mL).
An titu m or Activity in Vivo. L1210 murine leukemia cells
(105) were innoculated into female CDF1 mice (5 weeks old,
20 ( 1 g) intraperitoneally, and then compound 3, which was
previously suspended in 5% DMSO in physiological saline and
diluted with the same saline, or DOX‚HCl in saline was
administered intraperitoneally to each experimental group
containing four mice, daily, starting 24 h after inoculation, for
9 consecutive days. Mortality was checked daily for 60 days.
The control group with four mice was treated similarly by
replacing the drug solution with the neat saline. Antitumor
activity (T/C, %) was determined by the ratio of the median
survival time of the treated (T) and control (C) mice.
Ack n ow led gm en t. We are grateful to Pharmaceuti-
cal Research Center of Meiji Seika Co., Ltd. for carrying
out the bioassay for Table 1. We also express deep
thanks to Ms. Chisato Nosaka of IMC for antitumor
assay in vivo.
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