Note: Descriptions are shown in the official language in which they were submitted.
~ 7~ `7
Th~s Applicalion is a Divisional of Canadian Patent
Application Serial Number 4B4,086, Filed June 14, 1985
The present invention relat~s to new 3-amino-3,4,6-
trideoxyglycals, to processes for pr~paration thereof and
to anthracyclines obtained ~ith the aid of these ~lycals.
The anthracyclines constitute a very important
group of antibiotic and antitumor drugs. There belong to
this group, for example, doxorubicin, a hiqhly effective
cytostatic antibiotic, or daunorubicin, an antimitotic
antibiotic particularly indicated against leukemias,
Hodgkin's disease, reticulosarcoma or lymphosarcomas.
M~ny investigations have been performed on these
molecules to determine the relationship bet~een their
structure and their biological activity. From these
investigations, it emerges that modifications to the sugars
fre~uensly lead to analogs ~hich are pharmacologically
more useful than the starting substance. These modifica-
tions bring about, in e~fect, a decrease in the therapeu-
tically effective dose, a more pronounced antitumor acti-
vity and decreased side reactions, especiaLly cardiotoxi-
city (cf. in particular the s~udies of F. Arcamone "Doxo-
rubicin", Academic Press, Ne~ York, 1981 on daunosamine,the sugar of daunorubicin and doxorubic1n or adriamycin~.
Among the modifications proposed, there appears~
inter alia, deoxygenation of C-4' of the sugar ~cf. Farmi-
tal;a Euro~ Pat. 0,048,967), and according to the authors
of this patent the produtt 3 sho~n in Fig. I belo~ pos-
sesses very useful antitumor activity.
O OH n
CH2~2
CH3 ~ ~
_ 0~1'
I -t J2,
I H2,HCl
~i~7~ 7
_ 3 -
1 ~ R1 ~- OH; R2 - H (daunorubicin)
2 = R~ = R2 ~ OH (doxorubicin)
3 = R1 ~ R2 = H (E~JroP~ Pat. 0,0~8,967~.
Inversion of the configuration of the sugar at
the 3' and/or 4' carbon atoms - see Fig. II beLo~ -:
CH2 R2
-CH 3 .OH ~ 1 '
1--o~1
R~l ~
~HCl
4 ,R = OH; R~ = H; R2 = NH2
R = R2 = H; R1 = NH2
aLso contributes to the preDaration of der;vatives uhich
are pharmacologically very useful. The glycoside 4 has
a strong antitumor activity and the deoxygenated product
S has formed the subject of a f~ir~y recent patent CCarlo
Erba, Jpn. Kokai Tokkyû Koho Jp 57-142-981(82-~42-981),
Farmi~aLia~ ~eLg ~E 891,837].
The value of the tuo anino-deoxy sugars ~hich con-
stitute the sugar part of the anthracrclin~s 3 and 5 i s
hence not in doubt. Unfortunately, the manner in ~hich
these su3ars were prepared in ~he ~bovementioned pa~ents
considerably li~its their potentialit~. Thus, for ~xample,
the processea used start:
- either from the ~nthracycline itself ~and deoxy-
genation at C-4', for example, in product 1 can ehen find
~pplicution exclusively for preparing the ~roduct 3);
- or from the sugars, uhich are modif1ed. It is
elear that this second alternative is much more useful,
~nd far ~ore general. Ho~ever, the process proposed is
~;~7S~ 7
_ 4 -
economical~y a~together non-viable, since it is very com~
plicated, comprising many stages and involving, in parti
cular, the formation of halo slJgars, ~hich products are
difficult to synthesize and very fre~uently unstable.
S The chosen aim of the present invention ~as conse-
quently to provide for a synthesis of sugars which would
be simple, straightfor~ard and economical, so as to be
ab~e to synthesize many glycosides.
The present invention has as its subject a ne~
1û family of sugars, and in particular 3-amino-3,4,6-trideoxy-
Qlycals of gener~l for~ulae ~4 Dr 15 belo~:
--O ~;~ O
R~
l4 lS
in uhich R or R , ~hich may be identical or different,
denote a hydrogen atom or a substituted or unsubstituted
alkyl or acyl group,
or else R ~nd R tooether denote the group
~0
-
to for~ uith the nitro~en atom a morpholino group.
Recent publ;c3tions have, in fact, shovn that the
repl~cemene of thC 2mino (NH2) group in position 3 of
daunorubicin by a ~orpholino ( - ~ ) grouP results
ln greater po~er (in v;vo) for thc ~ntitu~or oc~ivity (cf.
;n particular ~he studies of C.~. ~osher et al., J. Med.
Chem. 25, 18, 1982).
According to one especially ~dvantageous e~bodi-
~ent of the subjece of the invention, R and/or R denote
a cOCF3 group.
The presen~ invention also has as its subject a
proces~ ~or preparing 3-amino-3,4,6-trideoxyglycals
~.~7~8~1~
-- 5
according to the present invention, in which Drocess there
are prepared, during a first stage, ~S-meth~l)dithio-
carbonates at position 4, of general formu~ae 12 and 13
belo~:
CH3S-~ \ CH3S-~!~;~
~RFI i2 13
~R and R' having the same significance as above), by
treating the gLycals 10 and 11 belo~:
H9/~ \ HO~/~
~ \J'
~RR'
11
(R and R' having the sa~e significance as above) ~ith a
mixture of sodium hydride and carbon disulfide and then
~ith methyl iodide, and in uhich process, during a second
stage, the (S-methyl)diehiocarbonate ~roups are reduc-
tively eliminatcd by the action of tributyltin hydride in
the presence of a~obis(isobutyronitri~e).
The glycals 10 and 11 are knovn and their prepa
ration is described by J. ~oivin, ~. Pais and C. Monneret,
Carbohydr. Res., 79, 193 (1980).
The present invention has for a further subject
another variant of Che process for preparing 3-amino-3,4,6-
trideoxyglycaLs according to the present invention, in~hich variant, during a first stage, there are prepared
the esters of general formulae 18 and 19:
~7~
<G;~ ,~_ OCORl ~COR
RR'
1~ 19
(R and R' h~ving the same significance as above and R1 de-
noting 2 hydrogen atom or a substituted or unsubstituted alkyl
or aryl grouP), by the ~ction of an acid anhydride or an acid
halide, in the presence of a base, on amino sugars of general
for~ulae 16 and 17 belo~:
} QH ~ H
NRR' ~6 17
(R ~nd R~ having the 5~ne significance as above)~ and in
~hich variant, during a second stage, the desired glycals
14 and 15 are prepared by heating the esters 18 and 19
respectively.
According to an advantageous procedure for this
embodiment, ~he heating of the esters 18 and 19 takes
place in the presence of an adsorbent, such as silica SiO2,
for example. The amino sugars 16 ~nd 17 are kno~n, and
their preparation is described by H.Ha Baer and H.R.
Hanna, Can. J~ Chem., 58, 1751, (1980).
The present invention furthermore has as its sub-
ject the anthr~cyclines obtained ~ith the aid of glycals
according to ~he present invention, and the use of ~hese
anthr~cyclines~ alone or ~ixed uith other ~ctive princi-
pals, ~s drugs.
In addition to the above measures, the invention
also includes further ~easures uhich ~ill emerge from the
descriPtion which follo~s.
The present invention rela~es to ne~ 3-amino-3,4,6-
~7~ 7
trideoxyglycals~ the processes for preparation thereof,the anthracyclines obtained ~ith the aid of these glycals
by glycosidation of the anthracyclinones, the drugs
cont3ining these anthracyclines and also the appropriate
reans for preparing and using these Droducts~
The invention ~ill be better unslerstood ~ith the
help of the additional description which follo~s, which
relates to examples of implementing the proc~sses accord-
ing to the present invention, and aLso to a report of
pharmacological experiments.
It should, ho~ever, be cLearly ~nderstood that
these examples of implementation and this report are
given exclusively by way of i~Lustration of the subiect
of the invention, Df ~hich they in no ~ay constitute a
limitation.
EXAMPLE 1
Preparation of 2~3,6-trideoxy-3-tr;fluoroacetamido-04_
tmethylthio)thiocarbonyl-L-arabino-1-hexenitol 1Z (R = H~
R" - COCF3~
To a solution of 10 (R = H, R' = COCF3) (1.5 9;
~.6 mmol) in anhydrous tetrahydrofuran (200 ml) there is
added 80X strength sodium hydride (400 mg, 12 mmol), and
the mixture is left ~ith stirring under an atmosphere of
argon at 0C for 2 h. Carbon disulfide (0.75 ml,
12 mmol) is then added, follo~ed by methyl iodide (0.75 ml,
12.1 mmol). After one hour's stirring at 0C, the reac-
tion ~edium is diluted uith water and then extracted ~ith
ether. This gives 1.7 9 of crude product uhich is fil-
tered on silica. Elution uith a 2:1 hexane/dichLoro-
methane mixture gives 0.8 9 (yld. 40X) of pure crystal-
li~ed 12 tR ~ H, R' s COCF3): m.p. 145C after resrys-
tallization in hexane; (~ )20 - 170 (c 1, chLoro-
form); IR ~ max Nu~ol 3260 (NH), 1690 and 1655 cm~~
(NCOCF3); N~R (270 HHz, CDCl3): 6.55 (lH, NH, m);
6.4û (1H, ddo J = 5.5, J' = 1.8, H - 1); 5.89 (1H, t, J =
J' = 9, H - 4); 4.88 (1H, ~, J = J' - 9, J" ~ 2, J"' = 1.8,
H - 3)~ 4.68 (1H, dd, J ~ 5.5, J' = 1.8, H - 2); 4.18 (qd,
J = ~.5, J' = ~ H 5); 2.57 (3H, s, scH3); 1.32 (3H, d~
J = 6.5, CH~-6).
* Denotes t rade mark
-- 8
_XAMPLE 2
Preparation of 2~3~4r6-te~radeoxy-3-trifluoroac~tamido-L-
-
thr~o-1-hexenitol 14 ~R = H, R~ - COCF3)-
To a solu~ion of 12 (R = H, R' = COCF3) (0.7 g;
2 mmol) in anhydrous tolu~ne t100 ml) there ~r~ added,
under argon~ azobistisobutyronitriLe) (50 mg) and then
tributyltin hydride (1 ml~. The reaction ~edium is heated
under reflux overnight and then evaporat~d to dryness,
and the r~sidue is resuspended in dichloromethane and
adsorbed on silica (1 9). Filtration on 3.5 9 of silica
enables the e~cess tributyltin hydride to be eLuted in
the first fractions (he~ane ~luant). In th~ following
fractions (2:1 hexane/dichloromethane eluant) there are
isolaSed 440 mg of 14 (R = H, R' = COCF3) (yield 95%).
_._
A sample is recrystallized for anal~sis in h~ane: m.p.
101 - 1û2C; ~ ~~ 29 (c 1, chloroform), IR max
Nu joLo 3330 (NH); 1690 and 1640 cm~1 (NHCOCF3); NMR
~270 MHz, CDCl3). 6.~2 t~H~ d, ~ = 6, H-1); 6.15 (com-
pLex, NH);~4.67 (1H, m, J = 10, J' Y 9, J~ = S, J"' ~ 2,
H-3); 4.55 (1H, dd, J = 6, J' = 2, H-2); 4.08 (~d, J =
6.5, J' = 10, J" = 1, H-5); 2.32 (m, J = 14, J~ = ~, J" =
1, H-be); 1.48 (lH, m, J = 14, J' = J" = 10, H-4a); 1.30
(3H, d, J = 6.5, CH3-6).
EXAMPLE 3
,
Prepar3tion of ?,3~6-trideoxy-3-trifLùoroac~tanido-O~-
(methylthioSthiorarbonyl-L-ribo-1-hex~nitoL 13 (R = H, -
~ R' = COCF3).
_
The product 13 (R = H, R' = tDCF3) is obtained from
11 (R = H, R' = COCF3) in the same manner as the product 12
(R = H, R' = COCF3) from 10 (R = H, R' ~ COCF3). M.p.
92C (CH2Cl2); ~ ~D ~ 73 (c 1, chloroform);
IR cf 12, N~R (CDCl3, 270 MHz); 6.58 (1H, d, J = 5.5,
J' ~ 1, H-1); 6.56 ~1H, m, J = 10, NH); 5.86 (lH, dd, J =
8, J~ ~ 5~ H-4); 4.92 (1H, ~, J = 10~ J' ~ J" ~ SO H-3);
4.?3 (1H, ~d, J = 5.5, J' = S, H-2); 4.25 ~1H, qd, J ~
6.5, J' = 8, U-5); 2.57 (3H, 5, ScH3); 1.35 (3H, d, J ~
S.5, CH3-6)-
EXAMPLE 4
Preparation of 2 3,4L6-t~eradeo~y-3-trifluoro~rJetamido~
~L27~8Z'7
9 _
erythro-1-hexenitol 15 (R = H R' = COCF3)
The product is obtained from 13 (R = H, R' =
CQCF3) like 14 (R = ~1, R' = COCF3~ from 12 (R = H,
R' = COCF3~. M~p. 110 - 112C (hexane); ~ 2DO -
76 (c 1, chloroform); IR Ymax Nu jol: cf 14; NMR
t270 MH2, CDCl3): 6.49 SlH, d, J = 5.5, H-1); 6.46 (1H,
complex, NH); 4.75 (1H, m, J = J' = 5, J" = 1, H-2); 4.33
(1H~ m~ H-3); 3.~5 ~1H, m. J ~ 605, J' = 10, J" - 205,
H~5); 1.95 (2H, m, J = 14, J' ~ 2.5, J" = 2.5, H-4e); 1.78
10 (2H, m, H-43); 1.33 (3H, d, CH3-6) .
EXAMPLE 5
pr-eDaration of 01-acetyl-2,3,/.,6-tetradeoxy-3-trif luoro-
acetamido-lf-threo-hexose 18 (R = H~ 2' = COCF3, R1 = CH3).
A soLution of 2,3,b,6-tetradeo~y-3-tri~luoro-
acetamido-L-threo-hexose (16) (R = H~ R' = COCF3) (2.2 9,
0 01 mol) in 80 ml of pyridine and 30 m~ of acetic anhyd-
ride is stirrcd overnight at room temper3turc. Conven-
tion~l extraction ~ith methylene c~loride Leads to 1.8 9
~70%) of acetates, 18 ( L and L anomers).
M.p. mixt~re 130 - 155C.
M.S.: m/z = 269 (M, absent); 209 (54%); 194 (20X); 180
(43X); 140 (100X).
IR: (CH2Clz): 3440, 3060, 2995, 1750, 1730, 1560,
1450~ 1430~ 1380, 1370~ 1330, 1320,
1280, 1230, 1175, 1150, 1115, 1060,
1040, 1020 cm~1.
NMR tCDCl3)S~-L ~nomer: o.62 ppm (NH); 6.22 (L1/2 =
6 Hz, H-1); 2.10 ~singlet,
tûCH3); 1.2Z (doublet, J =
6 Hz, CH3).
~3-L ~ nome r
M.p. 162 - 163C
NMR (CDCl3~: 6.27 (NH); 5.67 (doublet of doublet, J ~
10 Hz; J z 2.5 Hz, H-1); 2.08 (singlet,
CCH3); 1.27 (doublct, J ~ 6 Hz, CH3).
EXAMF'LE 6
threo-1-hexenitol (14) ~R = H~ R' ~ COCF3~-
A solution of acet~te 18 ~600 mg; 2.23 mmol) in
~7~
o 10 -
100 ~l of toluene in the presence of 10 9 of silica gelis brought to boi ling in a f lask equipped ~ith a Dean-
Stark apparatus. A~ter one hour, the silica gel is fil-
tered and ~Jashed ~i~h e~hyl acetate~ The organic solu-
tion is ~ashed ~ith bicarbonate so~ution ~nd then ~ith~ater. After the solution is dried and the solvents
are eva~orated, the crude product obtained is purified
by rapid pass3ge on a silica gel column. There are thus
obtained 210 mg (45%) of glycal 14 (R ~ ~, R' = COCF3),
the parameters of uhich are those already siven in Ex-
ample 2.
SYNTHESES OF ANTHRACYCLINES
The coupling of the glycals 1~ (R = H, R' = COCF3)
or 15 (R = H, R' = COCF3~ ~ith snthracyelinones Leads
to compound anthracyclines which are exceptional in res
pect of their antitumor activity. This synthesis gene-
ra~ly comPrises two stages:
a) glycosida~ion
b) deprotection of functional groups, since if R and
R' of the glycals 1$ and 15 both denote hydrogen atoms,
it is necessary~ for the reaction to take place, to pro-
tect at least one hydrogen atom.
By uay of example, daunomycinone 20 reacts ~ith
the qlycal 14 (R = H, R' = COCF3) in various anhydrous
solvents,(for e%ample CH2cl2~ ben~ene, etc.) in the
_ presence of acid (for example para-toluenesulfonic acid~
at room te~perature.
Besides the comPound 21, already described and
prepared, the new compounds 22a and 23a are obtained
~ l4>
MeO O OH OH MeûO OH O
~J
HR
21 ~ COCF3
-- 2~ P -- 2-
~Z7~
MeO O OH ~)H ~MeO O OH ~ ~
~ cJoc~
HR HR
~ R = cvCr 23 ~ R - CO~F3
22 b ~ - ~ 3
EXAUPLE 7
Glycosidation reaction of daunomycinone 20 ~ith the
glycal 14 (R = H, R' = COCF3)
t~lycosidation stage)
To a solution of daunomycinone 20 (71 mg; 0.178
mmol) and glycal 14 (R = H, R' = COCF3) (149 mg; 0.71
mmol) in 15 mL of CH2C~2 distilled o~er P20s, there
is added a catalytic amount of anhydrous p-toluenesulfonic
acid (2.3 mg; 0.013 mmol).
1û After 4 hours' stirrin3 at room temoerature, the
reaction mixture is poured into sodium bicarbonate solu-
tion and then extracted in the conventional manner ~ith
methylene chloride. Purification on silica plates t2luant:
50:50 - hexane/ethyl acetate) enables the three glycosides
formed to be separated:
- 07-t2',3',4',6'-tetradeoxy-3'-trifluoroacet-
amido-~ -L-threo-hexo~yranosyl)daunomycinone 21a t40X; Rf =
0.26, 50:50 - hexane/ethyl acetate).
- 09-t2',3',4',6'-tetradeoxy-3'-trifluoroacet-
amido-~ -L-threo-hexopyranosyl)daunomycinone 22a (35X; Rf =
0.28, 50:50 - hex~ne/ethyl acetate).
_ 07 0~-bis(2',3l,4',6'-tetr~deoxy-3'-trifluoro-
acetamido- ~-L threo-hexopyranosyl)daunomycinone 23a (23X;
Rf - 0~34, 50:50 - hexane/ethyl ~cetate).
Product 21a: yield 4ûX
.p. 140 - 145C (dec)
D = 458 (C = 0.23; CHCl3)
~R (~Br): 35QO, 3330, 2~40, 1705 tbroad),
7~
- 12 -
1610, 1570, 1400, 1350, 1280,
1190, 1120, 1030, 980 cm 1.
N~R (CDCl3): 1.3 (doublet, 3H); 2A4 (sing-
Let 3H), ~.4 (singlot, 1H),
5.22 ~multiplet~ L1/z = 7 Hz,
1H)~ 5-56 (multiplet~ L1l2 =
b.5 Hz~ 1H); 6~1û (broad~ 1H);
13.1 (singlet~ 1H); 13.85
(singlet~ 1H).
Rf = 0.7 ~CH2Cl2/MeOH, 96:b)
Rf = 0.2b thexane/ethyl acetate, 50:50~.
Product 22a: yield 35X
M.p. 164 - 169C (dec3
~ D = + 290 (C = 0.18; CHCl3)
IR (Ker): 3320 (broad)~ 2980~ 29~0, 1720,
1710, 1610, 1580, 1550, 1~50
1415, 1380, 1350~ 1290, 1250,
1210, 11~0, 1070, 1040~ 1010
990, 920 cm~1
NMR (CDCl3): 2.37 (singl~ 3H); 4.03
(singlet~ 3H)~ 4.98 (multi-
plet~ L1/2 3 8 H;~ 1H);
5.42 (multiplet~ L1t2 =
8 HZ~ 1H); 13.27 (singlet~
1H); 13.79 (singlet~ lH).
Rf = 0.66 ~CH2Cl2/MeOH~ 96:4)
_ Rf = 0.28 (hexane/ethyl acetate~ 50:53).
Product 23a: yield 23~
M.p. 227 - 228C tdec)
~XD = ~0-5 (C ~ 0.27, CHCl3)
IR (KBr): 3440~ 3320~ 2980~ 2920~ t725, 1710,
1605, 1580~ 9550~ 1410, ~350~ 1295~
1270, t 230, 1200, 1150, 1100, 1080,
1060, 1030~ lOOOo 980~ 915 cm 1.
WMR ScDcl3) 0.53 ppm td~ J ~ 6 HZ~ 3H);
1.32(d~ J = 6 H2~ 3H~; 2.31
(s~ 3H); 4.03 (S~ 3H); 4.97
(~ultiplet~ L1/2 ~ 8 Hz, 2H);
5.47 (~ultiPlet L1/2 = 8 Hz~
2''q~
- 13 -
2H); 6.ZO (broad, NH, 2H);
13.23 (singlet, 1H~; 13079
(singlet, 1H).
Rf = 0.58 (CH2Cl2/MeOH, 96:4).
Rf = 0.34 (hexane/ethyl acetate, 50:50).
EXAMPLE 8
Preparation of the hydrochloride of 07,09-bis(3'-amino-
2',3',4' 6' tetradeoxy~ -L threo-hexopvranosyl)daunomy-
cinone. 23b
(Deprotection st~ge)
~ eprotection, i.e~ the elimination of the protec-
tive (COCF3) groups of the compounds 21a and 23a is Der-
formed in aqueous basic ~edium.
To a solution of glycoside 23a (20 ~9; 0.024 mmoL)
in tetrahydrofur3n (8 ~ 0.1 N caustic soda solution
t8 ml~ is added at 0C and under an atmosphere of nitro-
gen. ~fter 14 hours at 0C, the pH is ~djusted to 4 -
4.5 ~ith 0.1 N hydrochloric acid solution. The aqueous
phase is ~ashed several times ~ith methylene chloride~
made alkaline with sodium bicarbonate solution and extrac-
ted ~ith methylene chloride in the usual manner.
The 07,04-bis(3~ amino-2~3~4~,6'-tetradeoxy-
-L-threo-hexopyranosyl)daunomycinone 23b thus obtained
and dissolved in anhydrous CH2Cl2 (2 ml) is treated ~ith
an equivalen~ amount of HCl in 0.05 N solu~ion in methanol.
The hydrochloride formed precipitates in ether ~m.p. 173 -
_ 174C (dec)~.
EX~MPLE 9
Prep~ration o~ the hydrochloride of 09-(3' amino-2',3'-
4'!6~-tetradeoxy-~ L-threo-hexopyranosyl)daunomycinone. _b
The hydrochloride ~m.p. 175 - 177C tdec)] is
obtainecl by ehe same method as that described in ExamPle 8.
NMR (CDCl3) 2.3 pPm (sin~let, 3H~; 4.0 (singlet, 3H);
4.90 (multiplet lH); 5.32 (multiplet, 1H).
EXAMPLE 10
Preparation of 07-(3'-~mino-2' 3',4',~'-te~r~deoxy-~ -
L-threo-~exopyranosyl)daunomycinone (or 4'-deoxydauno-
ycin~ _b.
This compound, obtained by the same method as
31 ~7~
-- 1 4 -
that described in Example 8, is purified by chromatography
on silica plates (eluant CHCl3/MeOH/NH40H, 180:20:1)~
NMR (CDCl3): 2.37 (singlet, 3H); 4.0 tsinglet, 3H);
5.25 (multiplet, L1t2 = 7 Hz, 1H);
5.50 (mul~iplet L1/2 = 8 Hz, 1H).
EX~MPLE 11
Preparation of the hydrochloride of 07-(3'-morpholino-
_
2',3',~',6'-tetradeoxy- ~-L-threo-hexoPyranosyl3daunomy-
cinone (or 3'-deamino-4'-deoxy-3'-morpholinodaunomyc-n) (?4
0
OMe O OH O
~ 24
A 4'-deoxydaunomycin 21b t38 mg; 0.074 mmol) dis-
solved in 2 ml of DMF is treated uith dit2-iodoethyl)
ether t~11 ng; 1.26 ~mol) and triethylamine t22 mg; 0.22
mmol) at room temperature for 36 hours. After extraction
and purlfication on silica plates (eluant CHCl3/MeOH/
_ NH~OH), the derivative 24 (60X) is obtained. The latter
disso~ved in CH2C~2 (2 ml) is treated ~ith an e~u;va-
lent 3mount of HC~ in 0.05 ~ solution in methanol. The
hydrochloride ~ormed precipit~tes in ether.
M.p. 145 - 150C (dec).
N~R (CDCl3~ 24: 1.27 (doublet, 3H, CH3); 2.40
(singlet, 3H, CH3); 2.44 t~ulti-
pletJ CH2 x 2); 3.~1 t~ultiplet~
CH2 x 2); 4-78 (~ultipl~t, 1H);
5.25 (multiplet, 1H); 5.55 (multi-
plet, 1H); 13.82 (singlet, 1H).
XA~PLE 12
CouDling reaction of the ~lycal i5.
1~7~8Z7
- 15 -
The aglycone 25 prepared by Jean-Claude Florent
and Claude Monneret ~French Pat. ~4/03634) reacts uith
the glycal 15 (R = H, R' = COCF3) in various anhydrous
solvents in the presence of p-toluenesulfonic acid at room
temper~ture.
Three ne~ compounds are oDtained, the anthra-
cycline~ 26, 27 and 28.
__ _ _
~ -; ~
O OH OH
'
O OH
~O~CCC~3
O OH
26
~H ~aH
Q OH O O OH O
_ ~ ~ 2
COCF3
To a solution of 25 (70 mg; 0.176 nmol) and gly-
cal 15 ~R ~ H, R' - COCF3) (70 mg; 0.33 mmol) in anhyd-
rous benzene (15 ml), p-toluenesulfonic acid (73 mg;
0.41 mmol) is added. After 2 hours' stirrinp ~t room
temperature, the reaction medium is ~oured into sod;um bi-
carbonate solution and thcn extract~d ~ith dichloromethane
in the conventional manner. After the solution is ~ashed
and dr~ed ~nd the organic solvent evaporated under reduced
2~7
- 16 -
pressure, 1SO mg sf crude product are isoL2ted.
Chromatography on siLit:a (eluan~: pure toluene)
enables the three glycosides formed to be separated:
- 013~2',3',41,6'-~etradeo~y-3-trifluoro3cetamido-
S ~/~-L-erythro-hexopyranosyl)-6,11-dihydro-5,12-dihydroxy-
2-hydroxymethylnaphthacene-6~11-dione (25X, Rf 3.43,
toluene/acetone - 90:10) 26.
- (1S,3S)-01~2',3',~',6'~tetradeoxy-3-trifluoro-
acetamido-~ -L-erythro-hexopyranosyl)-1,2~3,4,6,11-hex3-
hydro-1,3,5,1Z-tetrah~droxy-3-hydroxymethylnaphth3cene-6,11-
dione (45%, Rf 0.15, toluenetacetone - 90:10) 27.
- (1 S ,3 S) -o1 (2 ' ,3 ' ,b ' ,6 ' -tetradeoxy-3-trifluoro-
acetamido-¦3-L-erythro-hexopyranosyl)-1,2,3,4,6,11-hexa-
hydro-1,3,5,12-tetrahydroxy-3-hydroxyMethyLnaphthacene-6~11-
dione t25X, R~ 0.08, toluene/acetone - 90:10) 28.
I - Anthracycline 26: û13-(2~3~t4~6~-te~radeoxy-
3-trifLuoroacetam;do~ L-~rythro-hexopyrano~yl)-6!11
dihydro-5,12-dihydroxy~2-hydroxymethyLnaphthacene-6,11-
dione, crystallized ~ixture, m.p. 155 ~ 157c (t~lu~ne):
~ ~D ~ 17 (c 0.03, THF);
IR (CHCl3): 3380, 2920, 1720, 1590, 1540, 1510, 1460
(broad), 1410, 1340, 1265, 117û, 1115, 104û,
980 cm~l;
NMR (CDCl3~:15.~5 (OH), 8.55 - 7.77 (muLtiplet, 7H, aro-
ma~ic 7H, and NH); 5.17 tsingLet, broadened,
H-1' e~) and 5.ûO tdoublet, H-1' ax)~ 4.95
~ (doubLet) and 4.73 (doubLet) (A8 syst.,
CH2-13); 4.00 multiPlet~ 2H, H-5' and
H-3'~; 2.00 - 1.44 (muLtiplet, CH2-2' and
4'); 1.28 - 1.15 (muLti~let, 6H, CH3-6').
~ass spectrum ~DCI/NH3): m/2 547 (M+18, ?X); 530(M~1, 10%);
321 ~agLycone +1, 15X); 245 (~ugar ~18, 15X);
227 (susar, 25%); 210 (su~ar -18, 100X).
Rf = 0.64 (CH~CL2/MeOH, 98:2)
R~ = 0.25 (h~xane/ethyL acetate, 66:33).
II - AnthracycLine 27
M.P. 1S2 - 133C (hexane); ~ ~D + 139 (c 0.02 THF)
IR (CHCL3~: 3400~ 2920, 1720, 1625, 1590, 1530, 1405,
1370, 11709 990 and 970 cm-1.
- 17
NMR (CDCl3): 13.53 and 13.26 (7 singlets, 2~, phenolic
OH); 8.33 (mult;plet, 2H, Ar); 7.78 (multi~
plet, 2H, Ar); 5~45 (multiplet 1H, L1/2 =
7 Hz, H-7); 5.38 (doublet, 1H, J - 12,
J' = 3, H-1'); i"40 - 4.15 (multiplet~ 2H,
H-3' and OH); 4 00 (muLtiplet, 1H, H-SI);
3~81 (doublet, 1H, H-13); 3.45 (doublet,
1H, H-13); 3.32 tspLit doublet, 1H, H-10);
2.78 (sp~it doublet, 1H, H-10); 2.12 -
1~35 (multiplet, 6H, 3 CH2); 1.23 (doub-
let, 3H, J = 6.5 Hz, CH3~6').
Mass spectrum (DCI/NH3): ~/2 583 (M+18, ~ 1X~; 565 (M+~
trace); b54 (7.5X); 374 (aglycone ~ 18,
trace); 356 ~aglycone, 5X); 3b1 (aglycone
- 15, lX); 338 ~aglycone - 18, trace)~
245 (sugar + 18, 90X); 227 (sugar, 25X);
210 (sugar - 18, 100X).
R~ ~ D.27 (CH2Cl2/MeQH, 98:2)
Rf = 0.10 (hexane/ethyl aceta~e, 66.33~.
20 III - ~nthr~cycline 28
M.p. 162 - 164C (acetone); C~ ~2DO+ 140 (c 0.02 THF)
IR (CHCL3): cf ~0
NMR (CDCl3): 13.53 ~nd 13.26 (2 singlets, 2H, phenolic
OH); 8.34 tmultiplet, 2H~ Ar); 7.83 tmulti-
plet, 2H, Ar); 5.33 (doublet, 1H, J = 3 H~,
H-1'); 5.07 (multiplet 1H, Ltl2 = 7 Hz,
_ H-7); 4.44 - 4.22 (multiplet, 2H, H-3' and
OH); 4.06 (~ultiplet, H-5'); 3.90 (doubletO
1H, J = 10 Hz, H-13); 3.45 (doublet, lH,
J ~ 10 H2, H-13); 3.2U (split douDlet~ 1H,
J = 18~ J' s 1 5 H~, H-10); 2.64 (doublet,
1H, J - 18, H-10); 2.12 - 1.35 (~ultiplet,
6H, CH2-2',4' and 2); 1.23 (doublet, 1H,
J = 6.5 Hz, CH3-6').
Mass spectrum (DCI/NH3)o ~/~ 583 (M + 18, 2%); 566 (M +
1, 1X); 454 (SX); 374 (~glycone ~ 18~ 16%);
35S (aglycone~ 35X); 339 (aglyeone 18,
7X); 245 (sugar ~ 18, 30~); 227 (sugar,
15X); 210 ~sugar - 18, 100X).
~ 7~?8~ ~
-- 1 8 --
Rf = 0.20 (CH2Cl2/MeOH, ~8:2)
Rf ~ 0.05 (hexane/et.hyl acetater 66:33).
:1~7 C~ ~ Z,
_ 19 _
PHARMACOLOGICAL REPORT:
I. - Effect on L 1210 leuk~mia cells
A. - Procedure:
The test is performed 3ccording to the method of
Hamburger and Salmon, modified in the following manner:
The medium used is replaced by McCoy's medium 5 A.
The number of cells placed in the dishes is reduced to
5 x 102/dish, on account of the ra~id growth of L 1210
Leukemia cells.
The cells are incubatecl for 1 hour at 37C in
the presence of different concentrations of the substance
studied. The cells ~re then ~ashed t~ice ~ith McCoyls
~edium 5 A and placed on agarose plates according to the
method of Hamburger and Salmon.
Furthermore, the tese ;s carried out in parallel
uith continuous incubation ~i~h various concentrations of
the substance studied by introducing the substance in a
layer of agarose before setting up the ceLl cultures.
The dishes are placed in the incuba~or at 37C
in an atmosphere of 5% C02 and 20X C02, and relative
humi~ity of 95%, for 5 to 7 days. After this period, the
colonies of diameler greater than 60 ~m are counted by
means of zn inverted microscope.
~. - Results:
The results su~marized in Table I beLo~ are ex-
pressed as percentage of colonies formed from treated
_ L 1210 cells re~ative to untreated contro~s. The coeffi-
cient of variation, uhen the experi~ents Dre repeated, is
less than 15X.
~L~t7~ t~
-~ 20 -
T~lble I
REsllLlrs IC50 ( yg/ml )
Substance Continuous
exposure
Product 22 b ~ - H
in the form of ~ ~y ~ J~
hydrochloride ~ ~ I 0~8
OCH3
NH ~Cl~
22b -
i .
Product 23 b OH O r -~
in the form of ~ ~
~h drochloride ~ ~ ~ J ~ 0.31
_ ~
Product 24 ~ OH O
in the form of ~ ~ I
hydroch~oride ~ ~ 1.6 x 10
~ OC~3 O O~ O
~ '
C~ 24
. . _ ~ ~_
Adriamycin 2 x 10 2
. . . __ . . ....... _ . .
The IC50 values are calculated from the dose-response curves
after one hour s continuous incu~ation.
'~;1
~7~8~
-- 21
II, - Proliferation test (incorDoration of thymidine)
A. ~ Procedure:
L 1210 leukemia cells in exponential gro~th phase
(S x 103/ml in RPMI 16hO) are incubated on a microtitra-
tion piate containing 96 ~elLs for 72 hours (37C, 5X
COz, 95X relative hunidity) with different concentra-
tions of each of the substances tested. The controls com-
prise exposed cells in fresh culture medium. For each
concentration, ~ ~ells are prepared. After 65 hours, 50 ul
of (14C)-thymidine (1.5 ~Ci/ml) are added so as to Label
~he cell DNAa After 7 hours' jncubation, the cells are
harvested (Multimask cell harvester, Dynatec), nashed ~ith
5X strength trichloroacetic acid, ~ater and methanol, and
dried at 50C (2 hours~. The scintillation index is
determined using 5 mL of a scintillation li~uid (Rotis-
zint 11).
The results are expressed in the form of ratio of
scintillation index after incubation uith the substances
studied relative to controls.
The coefficient of variation for repeated experi-
ments is less than 15X.
P. - _esults:
The IC50 values are caLculated fro~ the dose-
response curv~s.
TABLE I I
R E S l) L T S ~.,Z'7~8Z7
source IC50 ~3/m~)
__ _ ~ 1~ ~ _ . _
in the form of ~ I ~ _ ¦
hydrochloricl~ OCH3 ~ 0.26
N~3 Cl
_ __ .
Produ~t 23 b
in the form of ¦ ¦ ¦ ~ ~ ~
hydr_cbLoride ~ ~ 0.3
NIS ~C~-
_ _2~b
Product 24
in the form of ~
hydr cbl_ride ~CH3 ~ ~H ~ 3 x 10 3
24 ~ ~
~r _ _ ~ _ _ . . ~
Adr;amycin 2.2 x 10 2
__ . _ _
~ . .
~ A~ 7 ~ 7
- 23 -
The results summari~ed in Tables 1 and II are
very promising, especially as regards the product 24.
The daily dosage in man is of the order of 1 to
2 mg/kg.
As emerges from the above, the invention is in
no ~ay Limited to those modes of implementation of embodi-
ment and application ~hich have just been described more
explic;tly; on the contrary, it embraces all the Yariants
~hich ~ay occur to the specialist in the subieGt~ ~ithout
departing from either the scope or the Gompass of the
present invention.
