Note: Descriptions are shown in the official language in which they were submitted.
- ETOPOSI DE ANALOGUES 2 0 4 ~ 2 1 1
This invention relates to compounds
that are analogs of etoposide having antitumor
activity. This invention also relates to a
method for treatinq tumors by administering a
S safe and effective amount of the etoposide
analog compounds.
BACKGROUND OF THE INVENTION
Podophyllotoxin is a naturally
occurring compound extracted from the mandrake
plant. Recently two therapeutically useful
semi-synthetic glycosides of podopyllotoxin,
etoposide (also known as VP-16), shown below,
and teniposide (also known as VM-26), have been
developed.
R
~0
~0~0
< ~0
0
R-CH, ~Etoposide)
R- ~ ~Teniposide)
ThesQ compounds exhibit therapeutic activity in
several human neoplasms, includinq small cell
carcinomas of the lung, testicular tumors,
Hodgkin~s disease, Papillomavirus, and diffuse
histiocytic lymphoma.
It is believed that these drugs block
the catalytic activity of DNA topoisomerase II
by stabilizing an enzyme-DNA complex in which
the DNA is cleaved and covalently linked to the
enzyme. See Chen, G. L., Yang, L., Rowe T. C.,
Halligan, B.D., Tewey, K., and Liu, L., _. Biol.
20~211
Che~., 259, 13560 (1984); R~ss, W., Rowe, T.,
Glisson, B., Yalowich, J., and Liu, L., cancer
Res., 44, 5857 (1984); Rowe, T., Kuppfer, G.,
and Ross, W., Biochem. Pharmacol., 34, 2483
(1985), which are all herein specifically
incorporated by reference. By way of
background, topoisomerases are enzymes which
control the topological state of DNA. Type II
topoisomerases catalyze DNA strand passage
through transient double strand breaks in the
DNA. The resulting change in the linking number
of DNA allows these enzymes to mediate DNA
interconversions, such as supercoiling and
relaxation of supercoiling, catenation and
decatenation, knotting, and unknotting. See
Wang, J. C., Annu. Rev. Biochem., 54, 665 (1985)
and Maxwell, A., and Gellert, M., Adv. Protein
Chem., 38, 69 (1986), which are herein
specifically incorporated by reference.
Type II DNA topoisomerase enzy~es have
been shown to be involved in a number of vital
cellular processes, including DNA replication
and transcription, and chromosomal segregation.
These enzymes, therefore, are a critical target
for the action of a wide variety of anticancer
drugs, including etoposide and teniposid~. The
key step leading to cell death may be the
capability of these drugs to block the catalytic
activity of DNA topoisomerase II, as noted
above.
Structure-activity studies have
demonstrated a direct correlation between
cytotoxicity, DNA breakage, and murine-derived
topoisomerase II inhibition activities among the
podophyllotoxin analogues. See Minocha, A., and
Long, B., Biochem Res. Comm., 122, 165 (1984),
which is herein specifically incorporated by
2044211
reference. The isolation and purification of
human type II topoisomerase from lymphocytic
leuk~mia cells has provided the means to use
this enzyme as a target to investigate the
structure-activity relationships among etoposide
and related congeners.
It has been shown that the substitution
of etoposide's glycosidic moiety by an 4-alkoxy
group, as in 4'-demethyl-epipodophyllotoxin
ethyl ether, preserves the inhibitory activity
of DNA topoisomerase II intact at higher
concentrations. See Thurston, L.S., Irie, H.,
Tani, S., Han, F. S., Liu, Z. C., Cheng, Y.C.,
and Lee, X. H., J. ~P~L_ÇbSm~, 29, 1547 (1986),
which is herein specifically incorporated by
reference. However, it has also been shown that
a series of 4-acyl congeners are less active,
even though some of them possessed potent
cytotoxicity. See Thurston, L. S., Imakura, Y.,
Haruna, M., Li, D. H., Liu, Z. C., Liu , S . Y .,
2~ Cheng, Y. C., and Lee, K. H., J. Med~ Chem., 31,
(1988), which is herein specifically
incorporated by reference.
Summary of the Inyçntio~
~he present invention provides novel
compounds which exhibit antitumor activity. The
compounds are analogs of etoposide. More
specifically, pre~erred compounds of the present
invention are etoposide analogs wherein the
glycosidic moiety is replaced by various
substituents, such as a
2"-hydroxyethylamino chain, a 2n-
methoxyethylamino chain, a 4 " -fluoroanilinyl
chain, a chlorine atom, or a bromine atom.
2~4211
present invention. The compounds of the present
invention have been shown to inhibit type II
human topoisomerase and also to cause cellular
protein-linked DNA breakage and, therefore, may
be useful in the treatment of tumors~ The
compounds may also be useful in the treatment of
papilloma virus.
In accordance with the present
invention, there are provided compounds of the
formula:
<~
H ~ ~ ~3
wherein
R, is B-OCH,CH2NH, B-NHCH(CH,)CH,OH,
B-NHCH,CH(CH,)OH, B-Cl, B-Br, B-OH, ~-OH, B-NH~,
~-NH" B-NHCH,CH~OH, ~-NHCH,CHIOH, B-NHCH2CHjCH"
B-NHCH,CH,OCH" B-NHCH2CH=CH" B-NHCH,CH(OH)CH" B-
NHCH,CH,CH,OH, B-OCH,CH20H,
~o o~ r r
~0.114~ ~ IN~r ~~ ,B-IIII~ J~llll~ )
C 11 O~
' IIII~CO~C~ J ~ 1111~ )
2 ~
e l~ ~
11 0 t ,l3~ ~oc~a
Rl is H, or Br;
R, is H, or Br;
R, is H, or 8r;
~ is H, or Br; and
~ is H, or -CH,.
The present invention i~ also ~or a
process for treatinq tumors in humans and lower
animals by administering a safe and effective
amount of a compound as described above.
A preferred group of compounds within
the present invention exhibit inhibitory
activity on human type II DNA topoisomerase to
an equal or greater extent than etoposide and
are of the formula:
~1
<0~'~
~0
CH~0 0C~
Olî
wherein
R, is -NHCH,CH,OH, -NHCH,CH,OCH"
NHCH,CH(OH)CH" NH~H(CH,)CH,OH, Cl,
OCH,CH,NH,
Il o o 11
~i~ ) ""~ ) Illl~o~ ~ J ~ ~el~
o~
~l~r ~ ~o ~ ~\o
2~42~1
Particularly preferred compounds include
4'-Demethyl-4B-amino-4-desoxypodophyllotoxin;
4'-Demethyl-4B-[2"-hydroxyethylamino]-4-desoxy-
podophyllotoxin; 4'-Demethyl-4B-
[2~-hydroxypropylamino]-4-desoxy-
podophyllotoxin; 4'-Demethyl-4B-[l"-methyl-2"-
hydroxyethylamino]-4-desoxypodophyllotoxin; 4B-
Chloro-4-desoxy-podophyllotoxin; 4'-demethyl-
4B-Chloro-4-desoxypodophyllotoxin; 4'-Demethyl-
4B-[3~'-hydroxyanilinyl]-4-desoxypodophyllotoxin;
4'-Demethyl-4B-[2"-hydroxyanilinyl]-
4-desoxypodophyllotoxin; 4'-Demethyl-4B-
[4"-hydroxyanilinyl]-4-desoxypodophyllotoxin;
4'-Demethyl-4~-[2"-fluoroanilinyl]-4-
desoxypodophyllotoxin; 4'-Demethyl-4~-[3"-
fluoroanilinyl]-4-desoxypodophyllotoxin; 4'-
Demethyl-4~-~4"-fluoroanilinyl]-4-
desoxypodophyllotoxin; 4'-Demethyl-4~-[3",5"-
difluoroanilinyl]-4-desoxypodophyllotoxin; 4'-
Demethyl-4~-[4"-chloroanilinyl]-4-
desoxypodophyllotoxin; 4'-Demethyl-4~-[4"-
bromoanilinyl]-4-desoxypodophyllotoxin; 4'-
Demethyl-4~-anilinyl-4-desoxypodophyllotoxin;
4'-Demethyl-4~-[4"-cyanoanilinyl]-4-
desoxypodophyllotoxin; 4'-Demethyl-4~-~3"-
cyanoanilinyl]-4-desoxypodophyllotoxin; 4'-
Demethyl-4~-t4"-ethoxycarbonylanilinyl]-4-
desoxypodophyllotoxin; 4'-Demethyl-4~-t4"-
morpholinoanilinyl]-4-desoxypodophyllotoxin; 4'-
Demethyl-4~-[3",4"-methylenedioxyanilinyl]-4-
desoxypodophyllotoxin; 4'-Demethyl-4~-~3",4"-
dimethoxyanilinyl]-4-desoxypodophyllotoxin; 4'-
Demethyl-4~-t3"-pyridylamino]-4-
20~4211
desoxypodophyllotoxin; and 4'-Demethyl-4~-[3"-
quinolinylamino]-4-desoxypodophyllotoxin.
Additional objects and advantages of
the invention will be set forth in part in the
description which follows, and in part will be
obvious from the description, or may be learned
from the practice of the invention.
Description of The Prefç~ed Embodiments
Reference will now be made in detail to
the presently preferred embodiments of the
invention, which tog~ther with the following
examples, serve to explain the principles of the
invention.
As noted above, the present invention
relates to compounds of the formula:
<~
N,~
wherein
R, is B-OCH,CH~H, B-NHCH(CH,)CH,OH,
B-NHCH,CH(CH,)OH, B-Cl, B-Br~ B-OH, ~-OH, B-NH"
~-NH" ~-NHCH,CH,OH, B-NHCH,CH,CH3, B-NHCH,CH,OCH"
B-NHCH,CH-CH" B-NHCH,CH(OH)CH" B-NHCH,CH,CH,OH, or
-OCH,CH,OH,
2 ~ 2 ~ 1
p ~
R, is H, or Br;
R, is H, or Br;
R, is H, or Br;
Rs is H, or Br; and
R~ is H, or -CH,.
As noted above, a preferred group of
compounds within the present invention exhibit
inhibitory activity on human type II DNA
topoisomerase to an equal or greater extent as
etoposide and are of the formula:
<0~
C H ~ 0~0 C H
01
whereln
R, is NHCH,CH,OH, NHCH,CH,OCH"
NHCH~CH(OH)CH" NHCH(CH,)CH,OH, Cl,
OCH,CH,NH"
1~0 011 f
IIN~3 j #N~ ~~011 ~ 1111~ ) #II~C 1~
`l
~"~r ~ #1~3t~ O or
2~4~
Another preferred group of compounds
under the present invention will possess a 4'-
demethyl-4~-substituted anilinyl 4-
desoxypodophyllotoxin wherein the compound
possesses the following formula.
'~i ~
~O~
c~,o 1, oc~
wherein Ar is an aryla~ine as in the formula
below,
- N~
wherein R, i8 H, OH, F, Cl, Br, CO,CH" CO,C2H"
CN, NO" NH" N(CH,)" OCH" CH,OH, CH" CF"
CH,CH,OH, COCH" CH2NH,;
R, is H, OH, F, Cl, Br, CO,CH" CO,C,H" CN, NO"
NH" N~CH,)" OCH" CH,OH, CH" CF" CH,CH,OH, COCH"
CH,NH" CHOHCH" SCH" CH" CO,CH,;
R, is H, ON, F, Cl, Br, I, CO,CH" CO,C,H" CN, NO"
NH2, N(CH,~" OCH" CH,OH, CH" CF" CH,CH,OH, COCH"
CH,NH" ~ . , ~ ,
N~CH,CH,OH)" CH,;
20~211
R, is H, F, Cl, OH, OCH" CO,CH" CO2C2H" CH3,
CF" NO2, NH" C l;
R, is H, F, Cl, CH" CF" OH, OCH" NO2;
R, and R, are OCH,O or OCH,CH,O.
The preferred compou~ds of the present
invention can also be more generally described
as a compound of the formula:
<0~
CH~OJ~OCH~
0~
wherein R is a flat aromatic group which may
contain a heteroatom or alternatively may
contain electron donating substituents at the 3"
or 4" position of the aromatic ring.
Specifically the substituents may be oxygen
containing groups, or the aromatic group may be
pyridine.
The compounds of the present invention
are analogs of etoposide wherein the glycosidic
moiety has been replaced. Compounds exhibiting
potent inhibition of human DNA topoisomerase II
result from replacing the glycosidic moiety with
a 2~-hydroxyethylamino chain, a 2"-
methoxyethylamino chain, or a substituted
arylamine at the C-4B position. Inhibitory
activity can also be increased by substituting
the glycosidic moiety by chlorine, bromine, or
an amino group at the C-4B position. It is
believed that the stereochemistry of the 4B-
substituents plays an important role in
204~211
11
determininq inhibitory potency. In general, B-
isomers exhibit greater activity than the
corresponding ~-isomers. Another factor
affecting the potency is the length of the
S substituent group at the C-4 position and the
substitution on that group. This distance
factor is different for halide and hydroxy
substituted arylamines. For the hydroxy
substituted arylamine, the meta position showed
the most potency, while in the halogens, para
substitution showed the most potency. A second
potency factor regarding the halogen substituted
aryl amines is the size of the halogen.
Fluorine, the smallest has the most potency,
lS while Iodine, the largest has the least. In
addition, substitution of bromine at either one
or more of the R" R" R" and ~ positions will
result in compounds having significant
inhibitory activity. ~ can be varied by
substituting hydrogen with a methyl group.
These modifications will produce changes in
inhibitory activity which can be readily
determined by assays known in the prior art
through the exercise of routine skill in light
of the teachings contained herein.
The compounds of the present invention
were tested for their degree of inhibitory
activity on human type II DNA topoisomerase,
their effect on the formation of protein-linked
DNA breakage, and their cytotoxicity. The
inhibitory activity for compounds of the present
invention correlated with the ability of the
compounds to cause DNA strand breakage.
However, the ~n vitro cytotoxicity of the
compounds tested did not appear to correlate
with the enzyme inhibitory activity and DNA
strand break activity. The results of the tests
2~211
12
on some of the compounds of the present
invention are shown in Tables I and III. For a
description of the assays used with respect to
the compounds listed in Tables I and III see
Thurston, L.S., Irie, H., Tani, S., Han, F. s.,
Liu , Z . C ., Cheng, Y.c., and Lee, K. H.,
Antitumor Agents 78. Inhibition of Human DNA
Topoisomerase II by Podophyllotoxin and ~-
Peltatin Analogues, 3. Med. Chem. 29, 1547
(1986), and the references cited therein.
Tab}es I, and III illustrate the
inhibitory activity, DNA strand breakage
ability, as well as the cytotoxicity of
etoposide and some of the compounds of the
present invention. As shown in Tables I and
III, the inhibitory activity of 4'-Demethyl-4B-
amino-4-desoxypodophyllotoxin (Example 3), 4'-
Demethyl-4B-[2"-hydroxyethylamino]-4-desoxy-
podophyllotixin (Example 5), 4'-Demethyl-4B-
t2"-hydroxypropylamino]-4-desoxypodophyllotoxin
(Example 10), and 4'-Demethyl-4B-tln-methyl-2~-
hydroxyethylamino]-4-desoxypodophyllotoxin
(Example 11), 4'-Demethyl-4B-t2n-aminoethoxy]-
4-desoxypodophyllotoxin (Example 13); 4'-
Demethyl-4B-t3n-hydroXyanilinyl~-4-
desoxypodophyllotoxin ~Example 33), 4'-Demethyl-
4B-t2~-hydroxyanilinyl~-4-desoxypodophyllotoxin
(Example 34), 4'-Demethyl-4~-[4"-
hydroxyanilinyl~-4-desoxypodophyllotoxin
(Example 35), 4'-Demethyl-4~-t3"-
fluoroanilinyl]-4-desoxypodophyllotoxin (Example
26); 4'-Demethyl-4~-t4"-fluoroanilinyl]-4-
desoxypodophyllotoxin (Example 28); 4'-Demethyl-
4~-t4"-chloroanilinyl]-4-desoxypodophyllotoxin
(Example 38); 4'-Demethyl-4~-anilinyl-4-
desoxypodophyllotoxin (Example 19); 4'-Demethyl-
4~-t4 N -cyanoanilinyl]-4-desoxypodophyllotoxin
2~21 ~
13
(Example 20~; 4'-Demethyl-4~-~3~-cyanoanilinyl]-
4-desoxypodophyllotoxin (Example 21); 4~-
Deme.hyl-4~-[4"-ethoxycarbonylanilinyl]-4-
desoxypodophyllotoxin (Example 22); 4'-Demethyl-
4~-[4~'-morpholinoanilinyl]-4-
desoxypodophyllotoxin (Example 23); 4~-Demethyl-
4B-~3~4~-methylenedioxyanilinyl]-4-
desoxypodophyllotoxin (Example 24); 4'-Demethyl-
4~-t3 n, 4 " -dimethoxyanilinyl~-4-
desoxypodophyllotoxin (Example 25); 4~-Demethyl-
4,~-t3"-pyridylamino]-4-desoxypodophyllotoxin
(Example 30); 4'-Demethyl-4~-t2"-pyridylamino]-
4-desoxypodophyllotoxin (Example 31); and 4'-
Demethyl-4~-[3"-quinolinylamino]-4-
desoxypodophyllotoxin (Example 32) equals or
exceeds that of etoposide. In addition, as
shown in Tables I and III, the DNA strand
breakage abilities of 4'-Demethyl-4B-t2"-
hydroxyethylamino]-4-desoxypodophyllotoxin
(Example 5), 4'-Demethyl-4B-t2"-
methoxyethylamino]-4-desoxypodophyllotoxin
(Example 8), 4'-Demethyl-4B-t2"-
hydroxypropylamino]-4-desoxy-podophyllotoxin
(Example 10), and 4'-Demethyl-4B-t1"-methyl-2"-
hydroxyethylamino]-4-desoxypodophyllotoxin
(Example 11), 4'-Demethyl-4B-~2"-aminoethoxy]-
4-desoxypodophyllotoxin (Example 13);4~-
Demethyl-4B-~3-hydroxyanilinyl]-4-
dexoypodophyllotoxin (Exa~ple 33), 4'-Demethyl-
4B-t2-hydroxyanilinyl]-4-desoxypodophyllotoXin
(Example 34), 4'-Demethyl-4B-t4-
hydroxyanilinyl]-4-desoxypodophyllotoxin
(Example 35), 4'-Demethyl-4~-t3"-
fluoroanilinyl]-4-desoxypodophyllotoxin (Example
26); 4'-Demethyl-4~-[2"-fluoroanilinyl]-4-
desoxypodophyllotoxin (Example 27); 4'-Demethyl-
4~-~4"-fluoroanilinyl]-4-desoxypodophyllotoxin
20~42t~
14
S (Example 28j; 4'-Demethyl-4~-[3",5"-
difluoroanilinyl]-4-desoxypodophyllotoxin
(Example 29); 4'-Demethyl-4~-anilinyl-4-
desoxypod~phyllotoxin (Example 19); 4'-Demethyl-
4B-[4"-cyanoanilinyl~-4-desoxypodophyllotoxin
(Example 20); 4'-De~ethyl-4~-t3"-cyanoanilinyl~-
4-desoxypodophyllotoxin (Example 21); 4'-
Dem~thyl-4~-~4"-ethoxycarbonylanilinyl~-4-
desoxypodophyllotoxin ~Example 22); 4~-Demethyl-
4~-~4"-morpholinoanilinyl]-4-
desoxypodophyllOtOXin (Example 23); 4'-Demethyl-
4~-~3",4~-methylenedioxyanilinyl]-4-
desoxypodophyllotoxin (Example 24); 4'-Demethyl-
4~-~3",4"-dimethoxyanilinyl]-4-
desoxypodophyllotoxin (Example 25); 4'-Demethyl-
20 4~ _ [ 3 n -pyridylamino]-4-desoxypodophyllotoxin
~Example 30); 4'-Demethyl-4~-t3"-
quinolinylamino]-4-desoxypodophyllotoxin
(Example 32); and 4'-Demethyl-4~-[4~-
bromoanilinyl]-4-desoxypodophyllotoxin (Example
- 25 40) greatly exceeds that of etoposide.
Table II compares the relative DNA
topoisomerase II inhibitory activity of several
compounds of the present invention with
etoposide. As shown in Table II, the compounds
test-d exhibited inhibitory activity exceoding
that of etopo-ide by two to eight times.
Preparation of compounds within the
5COp- of the present invention appear in the
following examples.
~A~ilE_L
Preparation of 4'-Demethylepipodophyllotoxin.
5 g. (12.1 mmol) of podophyllotoxin
were dissolved in 75 ml of anhydrous CH,Cl,. Dry
hydrogen bromide gas was then bubbled through
the solution to saturation. The reaction
2 ~ 2 1 1
mixture was then capped and allowed to stand at
room temperature for 48 hours. Removal of the
solvent yielded a residue which was then treated
with 25 ml of water, 50 ml of acetone and 5 g.
of BaC0" and refluxed for one hour. The
reaction ~ixture was extracted with chloroform
and chromatographed on a silica gel column. The
product was obtained by elution with chloroform-
methanol (30:1) and recrystallized from
CH,Cl,/ethylether to give 2.5 g. (52t) of 4'-
Demethylepipodophyllotoxin. Spectral data
agreed with that described by Kuhn, M., Keller-
Julsen, C., and von Wartburg, A., ~çlv. Chi~-
aç~, 52, 944 (1969), which is herein
specifically incorporated by reference.
(See Scheme 1)
- EX~PL~ 2
Preparation of 4'-Demethylpodophyllotoxin.
4'-Demethylpodophyllotoxin was obtained
using the silica gel column of Example 1 by
further elution with chloroform-methanol (30:1).
The product obtained by elution was crystallized
from acetone in 5% (0.5 g) yield. Spectral data
agreed with that dQscribod by Kuhn, M., and von
25 Wartburg, A., H~ly~_Ch1m~ Acta, 52, 948 (1969)
(hereinafter Kuhn and von Wartburg), which is
herein specifically incorporated by reference.
~AMpL~ 3
Preparation of 4'-DQmothyl-4B-amino-4-desoxy-
podophyllotoxin. (Scheme II)
A. Preparation of 4'-0-Carbobenzoxyepipodo-
phyllotoxin.
A solution of 2 g. of 4'-Demethy-
lepipodophyllotoxin (5 mmol) in 200 ml of
anhydrous dichloromethane was cooled in an ice
2 ~ 2 ~ 1
16
bath, and treated with 2.5 ml of triethylamine
~18 mmol), and 2.5 ml of carbobenzoxychloride
(17.5 mmol). The reaction mixture was stirred
at room temperature for 2 hours after which time
100 ml of water was added. The organic layer
was dried using MgS0" concentrated, and purified
using silica gel column chromatography. The
product was obtained upon elution with
chloroform and recrystallized from
chloroformtethanol to give 2.4 g. (89%).
Spectral data agreed with that described by Kuhn
and von Wartburg.
B. Preparation of 4'-0-Carbobenzoxy-4-
epia~idopodophyllotoxin.
A solution of 3 g. (5.6 mmol) of
4'-0-Carbobenzoxy-4-epipodophyllotoxin (the
product of Example 3) in 100 ml of anhydrous
methylene chloride was cooled in an ice bath and
treated successively with 1.5 ml (10.8 mmol) of
triethylamine, and 1.2 ml (15.5 mmol) of
methanesulfonylchloride. The ice bath was then
removed and the reaction mixture was stirred at
room temperature for one hour. This mixture was
then evaporated in vacuo to dryness, and 40 ml
of anhydrous DMF was added along with 3 g~ ~46
mmol) of sodium azide. The reaction mixture was
stirred overnight at room temperature and then
partitioned between water (100 ml) and
ethylacetate. The organic layer was washed with
water, dried using MgS0" and concentrated to
yield a crude residue, which was checked by TLC
and NMR analyses to be a mixture of 4~- and 4B-
azido isomers (ca.1:3). Crystallization from
chloroform/ethanol provided the pure B-isomer
4'-0-Carbobenzoxy-4-epiazidopodophyllotoxin (2.3
g, 73%) having the following properties:
17 2~
mp.202-204 C; MS, m/z 559 (~), 424, and 382; IR
(KBr) 29SO, 2900, 2100 (azide), 1770 (carbonate
C=O), 1745 (lactone C=O), 1600, and 1475
(aromatic C=C) cm l;'H NMR (CDCl,) ~ 7.40 (m, 5H,
cbz aromatic), 6.82 (s, lH, 5-H), 6.58 (s, lH,
8-H), 6.27 (s, 2H, 2'.6'-H), 6.03 (ABq, J=2.4
Hz, 2H, O-CH,-O), 5.25 (s, 2H, OCH2Ph), 4.77 (d,
J=4 Hz, lH, 4-H), 4.65 (d, J-5 Hz, lH, l-H),
4.31 (d, J-9 Hz, 2H, 11, ll'-H), 3.66 (8, 6H,
3',5'-OCH,), 3.2(dd, J=5,14 Hz, lH, 2-H), and
2.90 (m,lH, 3-H); Anal. (C"H"O,N, 1/2 H,O), C.H.
C. Preparation of 4'-Demethyl-4B-amino-4-
desoxy-podophyllotoxin.
500 mg of 10% palladium on carbon was
added to a solution of the crude 4'-demethyl-4-
azidopodophyllotoxin (2.3g, 4.1 mmol), obtained
according to steps A and B, and 200 ml of
ethylacetate. This mixture was shaken under 40
psi of hydrogen for four hours. The reaction
mixture was then filtered over celite and the
filtrate evaporated in vacuo. The residue was
chromatographed on a silica gel column and
eluted first with a chloroform/ethylacetate
(2:1) solvent system to remove the non-polar
products. Further elution wlth a
chloroform/methanol (19:1) mobile phase ylelded
0.85g, (52%) of the desired product. The
product wa~ then crystallized from methylene
chloride/ethylether and had the following
properties: mp 132-135 C; MS m/z 399 (~); IR
(KBr) 3360 (OH), 3290 (primary amine), 2900
(aliphatic C-H), 1745 (lactone), 1590 (aromatic
C-H) cm l;'H NMR (CDCl,) ~ 6.81 (s, lH,5-H), 6.49
(s, lH, 8-H~, 6.30 (s, 2H, 2~,6~-H), 5.96 (ABq,
3s J-l Hz, 2H, OCH,O), 5.3 (s, lH, OH, D,O
exchangeable), 4.55 (d, J=5.2 Hz, lH, l-H), 4.28
2~21~
(d, J--9.5 Hz, 2H, 11,11'-H), 4.17 (d, J=4.1 Hz,
lH, 4-H) 3.77 (s, 6H, 3',5'-OCH,), 3.28 (dd,
J=5.2,14 Hz, lH, 2-H), and 2.85 (m, lH, 3-H),
Anal. (Cl,HI,O~NH2Q), C.H.
ExA~LE-~
Preparation of 4'-Demethyl-4~-amino-4-desoxy-
podophyl lotoxin .
4'-Demethyl-4~-amino-4-desoxy-
podophyllotoxin was obtained from the column
used in Example 3 by further elution with a
chloroform/methanol (19:1) mobile phase. The
pure product (0.34g, 20%) was crystallized from
methylene chloride/ethylether and had the
following properties: mp 133-135 C; MS m/z 399
(~); IR (KBr) 3360 (OH), 3295 (NH,), 2900
(aliphatic C-H), 1743 (lactone), 1590 (aromatic
C-H) cm l;'H NMR (CDCl,) ~ 7.14 (s, lH, 5-H),
6.54 (s, lH, 8-H), 6.20 (s, 2H, 2',6'-H), 6.00
(ABq, J=l Hz, 2H, OCH,O), 4.63 (d, J-5.1 Hz, lH,
l-H), 4.61 (d, J=9.0 Hz, lH, ll~-H), 4.07 (dd,
J=9.0,10.4 Hz, lH, llB-H), 3.83 (d, J=10.3 Hz,
lH, 4-H), 3.81 (s, 6H, 3',5'-OCH,), 2.85 (dd,
J=5.1,14.1 Hz, lH, 2-H), and 2.57(m, lH, 3-H);
Anal. (C"~,O,N H,O), C.H.
~
Preparation o~ 4-alkylamino-4-desoxy-
podophyllotoxins. (Scheme III)
The 4-alkylamino-4-desoxy-
podophyllotoxins specified in Examples 5-12 were
prepared according to the following procedure.
A solution of podophyllotoxin (5 g, 12.1 mmol)
in 50 ml of anhydrous methylenechloride was kept
at room temperature and dry hydrogen bromide gas
was bubbled through the solution until
saturation was achieved. The flask was then
20~2~
19
capped and allowed to stand for 48 hours after
which time dry nitrogen was bubbled through the
solution to drive off excess HBr. Then 2 g. of
anhydrous BaCO, and 2 ml of the appropriate amine
were added. Vigorous evolution of gas was
observed. The mixtur~ was allowed to stand for
5 hours at room temperaturs after which the
reaction mixture was filtered, washed with
water, dried, and purified via column
chromatography. Yields ranged from 5-10%. The
products obtained in these examples had the
characteristics listed below.
EXAMP~ 5
4'-Demethyl-4B-[2"-hydroxyethylamino]-4-desoxy-
podophyllotoxin.
Amorphous powder from CH,Cl,-ether : mp
120 C; MS m/z 443 (M-~; IR (KBr) 3420 (NH,OH),
2900 (aliphatic C-H~, 1755 (lactone), 1600, and
1475 (aromatic C=C) cm 1;'H NMR (CDCl,) ~ 6.82
20 (s, lH, 5-H), 6.49 (s, lH, 8-H), 6.29 (s, 2H,
2',6'-H), 5.97 (ABq, J=1.0,4.4 Hz, 2H, OCH,O),
4.57 (d, J35.0 Hz, lH, 1-H), 4.35 (m, 2H,
11,11'-H), 3.93 (d, J=4.0 Hz, lH, 4-H), 3.79 (s,
6H, 3',5'-OCH,), 3.76 (m, 2H, 2"-H), 3.3 (dd,
25 J=5.0,13.5 Hz, lH, 2-H), 3.09 (m. lH, 3-H), and
2.75 (m, 2H, 1"-H); Anal. (C7,H"O,N l/2 H70), C.H.
EXAMPLE 6
4'-Demethyl-4~-[2"-hydroxyethylamino]-4-desoxy-
podophyllotoxin.
Crystals rom CH,Cl7-ether : mp 230-
234 C; MS m/z 443 (M-); IR (KBr) 3425 (NH,OH),
2900 (aliphatic C H), 1753 (lactone), 1600, and
1475 (aromatic C=C) cm l;'H NMR (CD,OD) ~ 6.83
~s, lH, 5-H), 6.47 (s, lH, 8-H), 6.38 (s, 2H,
35 2',6'-H), 5.g2 (ABq, J-1.0,14.3 Hz, 2H, OCH70),
2~4~
4.40 (s, lH, ll-H), 4.25 (s, lH, ll'-H), 3.75
(s, 6H, 3',5'- OCH,), 3.65 (m, 4H, 1 ",2"-H),
3.56 (m, lH, l-H), 3.49 (dd, J=6.1,11.3 Hz, lH,
4-H), 2.87 (ddd, J-5.1, S.3, 13.5, lH, 3-H), and
2.67 (dd, J=6.1, 8.2, lH, 2-H); Anal.
(C2,H"O~N-l/2 H2O), C.H.
EX~MPLE 7
4'-Demethyl-4B-propylamino-4-
desoxypodophyl lotoxin.
Crystals from CH,Cl,-ether: mp 153-
156C; MS m/z 441 (M+); IR (KBr) 3470 (OH), 3320
(NH), 1750 (lactone), 1600, and 1475 (aromatic
C=C) cm~'; 'H NMR (CDCl,) ~ 6.77 (s, lH, 5-H),
6.47 (s, lH, 8-H), 6.28 (s, 2H, 2',6'-H), 5.95
(ABq, J=1.2,5.0 Hz, 2H, OCH,O), 4.30 (d, J=5.0
Hz, lH, l-H), 4.30 (d, J=4.0 Hz, lH, 4-H),
4.28(m, 2H, 11,11'-H), 3.78(8, 6H, 3',5'-OCH3),
3.30 (dd, J=5.0, 13.9 Hz, lH, 2-H~, 2.83 (m, 2H,
1"-H), 2.52 (m, lH, 3-H), 1.55~m, 2H, 2"-H), and
0.95 (t, J=7.6 Hz, 3H, 3"-H); Anal. (C"H2,O,N
1/2H,O), C.H.
EX~MpLE 8
4'-Demethyl-4B-[2"-methoxyethylamino]-4-desoxy-
podophyllotoxin.
Crystals from CH,Cl,-ether : mp 202-
204 C; MS m/z 457 (~); IR (KBr) 3440 (OH, NH),
1750 (lactone), 1600, and 1475 (aromatic C=C)
cm l;'H NMR (CDCl,) ~ 6.80 (5, lH, 5-H), 6.44 (s,
lH, 8-H), 6.25 (s, 2H, 2',6'-H), 5.92 (ABq,
J=1.0,5.0 Hz, 2H, OCH,O), 4.50 (d, J=5.4 Hz, lH,
1-H), 4.28 (m, 2H, ll,11'-H), 3.88 (d, J=4.0 Hz,
lH, 4-H), 3.75 (s, 6H, 3',5'-OCH,), 3.52 (m, 2H,
2"-H), 3.37 (s, 3H, 3"-H), 3.38 (dd, J=14.4,5.4
Hz, lH, 2-H), 3.05 (m, lh, 3-H), and 2.75 (m,
2H, l"~H); Anal. (C"H"O,N 1/4 H,O), C.H.
2~2~ ~
21
EX~MPL~:_9
4'-Demethyl-4B-allylamino-4-desoxypodophyllo-
toxin.
Amorphous powder from CH2Cl,-ether: mp
225-228 C; MS m/z 439 (M-); IR (KBr) 3340 (OH,
NH), 2885 (aliphatic C-H~, 174~ (lactone), 1600,
and 1475 (aromatic C=C) cm l;'H NMR (CDCl,) ~
6.77 (s, lh, 5-H), 6.49 (s, lH, 8-H), 6.28 (s,
2H, 2',6'-H), 5.95 (ABq, J=1.0, 4.5 Hz, 2H,
OCH20), 5.90 (m, lH, 2"-H), 5.4 (m, lH, N-H, D20
exchangeable), 5.22 (dd, J=4.0, 17.5 Hz, 2H, 3"-
H), 4.53 (d, J=5.5 Hz, lH, l-H), 4.30 (m, 2H,
11,11'-H), 3.88 (d, J=3.6 Hz, lH, 4-H), 3.75 (s,
6H, 3',5'-OCH,), 3.30 (dd, J=5.4, 14.4 Hz, lH, 2-
15 H), 3.30 (m, lH, 1"-H), and 2.80 (m, lH, 3-H);
Anal. (C"H2,0,N 2.2 H20), C.H.
EXAM~eLE 1~
4'-Demethyl 4B-[2"-hydroxypropylamino]-4-desoxy-
podophyllotoxin.
Crystals from CH,Cl2-ether: mp 145-
150 C; MS m/z 457 (M-); IR (KBr) 3330 (OH, NH),
2890 Saliphatic C-H), 1750 (lactone), 1600, and
1475 (aromatic C=C) cm 1;'H NMR (CDCl,) ô 6.83
(c, lh, 5-H), 6.47 (~, lH, 8-H), 6.27 (s, 2H,
25 2',6'-H), 5.95 (ABq, J~1.0, 6.3 Hz, 2H, OCH~O),
5.40 (m, lh, N-H, D~O exchangeable ), 4.54 (d,
J34.6 Hz, lHI, 1-H), 4.30 (m, 2H, 11,11'-H), 3.85
(~, lH, 2"-11), 3.85 (d, J--3.8 Hz, lH, 4-H), 3.75
(s, 6H, 3',5'-OCH,), 3.25 (dd, J=4.6, 13.8 Hz,
30 lH, 2-H), 2.85 (dd, J=6.8, 12.5 Hz, lH, l"-H),
2.82 (m, lh, 3-H), 2.63 (dd, J=3.8, 12.5 Hz, lH,
1'l-H), and 1.20 (d, J=6.3 Hz, 3H, 3"-H); Anal.
(C2,H"O,N î/2 H~O), C.H.
2~211
EXAMPLE 11
4'-Demethyl-4~-[1"-methyl-2"-hydroxyethylamino]-
4-desoxypodophyllotoxin.
Amorphous powder from CH2Cll-ether : mp
220-225O C; MS m/z 457 (~);'H NMR (CDCl,) ~ 6.89
(s, lH, 5-H), 6.47 (s, lH, 8-H), 6.27 (s, 2H,
2',6'-H3, 5.95 (ABq, J=1.0, 4.5 Hz, 2H, OCH,O),
5.40 (m, lH, N-H, D,O exchangeable), 4.52 (d,
J=4.8 Hz, lH, l-H), 4.30 ~d, J-9.0 Hz, 2H,
11,11'-H), 4.00 (d, J=4.0 Hz, lH, 4-H), 3.74 (s,
6H, 3',5'-OCH,), 3.50 (m, 2H, 2"-H), 3.22 (dd,
J=4.8, 13.5 Hz, lH, 2-H), 2.85 (m, lH, 3-H),
2.82 (m, lH, 1"-H)m and 1.05 (d, J=6.3 Hz, 3H,
1'-CH,); Anal. (C,~H"O,N 1/2 H,O), C.H.
E~AM~LE 12
4'-Demethyl-4B-[3"-hydroxypropylamino]-4-desoxy-
podophyllotoxin.
Crystals from CH,Cl,-ether : mp 193-
196 C; MS m/z 457 (~); IR (KBr) 3460 (OH) 3320
(NH), 2900 (aliphatic C-H), 1740 (lactone),
1600, and 1475 (aromatic C=C) cm l;'H NMX (CDCl,)
~ 6.75 (s, lH, 5-H), 6.46 (8, lH, 8-H), 6.24 (s,
2H, 2',6'-H), 5.94 (ABq, J-1.0,4.4 Hz, 2H,
OCHaO), 4.52 (d, J=5.3 Hz, lH, l-H), 4.33 ~dd,
J-7.9,8.0 Hz, lH, ll-H), 4.23 ~dd, J~8.0,10.7
Hz, ll'-H), 3.78 ~d, J~4.0 Hz, lH, 4-H), 3.73
(o, 6H, 3',5'-OCH,), 3.72 (t, 2H, 3"-H), 3.21
~dd, J=5.3,14.0 Hz, lH, 2-H), 3.11 (dd,
J=5.9,11.4 Hz, lH, l"-H), 2.64 (ddd,
J-14.0,7.0,11.0 Hz, lH, ll'-H) and 1.75 (m, 2H,
2"-H); Anal. (C,~H"O,N 3/4 H,O), C.H.
EXAMPLE 13
Preparation of 4'-Demethyl-4B(2"-aminoethoxy)-
4-desoxypodophyllotoxin. ~Scheme I~')
2~21~
~3
4'-Demethyl-4B(2l'-aminoethoxy)-4-
desoxypodophyllotoxi~ was prepared according to
the following procedure.
A. 4'-D~methyl-4B-(2"-bromoethoxy)-4-desoxy-
podophyllotoxin.
Podophyllotoxin (500 mg) was suspendsd
in anhydrous dichloromet~ane (15 ml~. Dry
hydrogen bromide gas was bubbled through the
mixture until saturation was achieved. The
flask was then capped and allowed to stand at
room temperature for 48 h. After bubbling
nitrogen gas through the solution to drive off
excess hydrogen bromide gas, barium carbonate
(1.50 g) and 2-bromoethanol (1 ml) were added
and stirred at room temperature for 10 h. The
mixture was diluted with dichloromethane,
filtered and evaporated to dryness. The syrupy
residue was purified by silica gel column
chromatography eluting with chloroform-acetone
~30 : 1 v/v). For further purification the
product was chromatographed on silica gel column
using toluene-ethylacetate (5 : 1 v/v) as an
eluant. Yield (218 mg). mp 194-195C. 'H-NMR
(CDC1,): 6 6.79 (lH, S, 5-H), 6.56 (lH, S, 8-
H), 6.25 (2H, S, 2', 6'-H), 6.00-5.97 (each d,
J= 1.2 Hz, OCHzO), 5.40 (H, S, 4-OH), 4.88 (lH,
dd, J= 7.60, 8.34 Hz, 11-H), 4.61 (lH, d, J= 5.2
Hz, l-H), 4.50 (2H, ~), 4.04 (lH, m), 3.80 (lH,
m), 3.77 (6H, S, 2 x OCH,), 3.47 (2H, m), 3.42
(lH, dd, J- 5.39, 14.08 Hz, 2-H), 2.90 (lH, m,
3-H)
Anal. Calcd. for C"H2,BrO, : C, S4.45; H, 4.57.
Found: C, 54.35; H, 4.60.
~. 4'-Demethyl-4B-(2"-azidoethoxy)-4-desoxy-
podophyllctoxin.
2~2~
24
The mixture of 4' demethyl-4~-(2-
bromoethyl~ epipodophyllotoxin (157 mg) and
sodium azide (150 mg) in N,N-dimethylformamide
(6 ml) was stirred for 10 h at room temperature.
Pouring the reaction mixture into water and
stirring gave a white precipitate, which was
collected by filtration and dried in the air.
Recryst llization from chloroform-ether gave
pure product (120 mg). mp 215-217C IR (CHCl,)
cm': 3538 (OH), 2205 (N,), 1770 (lactone), 1615
(aromatic C=C) Anal. Calcd. for C~,H~,N,O,: C,
58.84; H, 4.93; N, 8.95.
Found: C, 58.78; H, 4.98; N, 9.28.
C. 4'-Demethyl-4B(2"-aminoethoxy)-4-desoxy-
podoplyllotoxin.
A mixture of 4'-Demethyl-4B-(2"-
azidoethyl)epipodophyllotoxin (108 mg) and 10 %
palladium in carbon (55 mg) in ethylacetate (20
ml~ was stirred under a hydrogen atmosphere for
5 h. After the removal of the catalyst by
filtration, the filtrate was evaporated to
dryness under reduced pressure. The crude
product was purified by silica column
chromatography eluting with chloroform-methanol
(5 : 1 v/v) to obtain pure material ~82 mg)
having the Eollowing characteristics.
mp 143-145C
Anal. Cald. for C,~H"NO, H,O: C, 58.72; H, 6.00;
N, 2.97.
Found: C, 58.74; H, 5.98; N, 2.97.
EXAMPLE 14
Preparation of 4'-Demethyl-4B-(2"-hydroxy-
ethoxy)-4-desoxypodophyllotoxin. (Scheme V)
Podophyllotoxin (200 mg) was suspended
in anhydrous dichloromethane (15 ml). Dry
2~2~1
hydrogen bromide gas was bubbled through the
mixture until saturation was achieved. The
flask was then capped and allowed to stand at
room temperature for 48 h. After bubbling
nitrogen gas through the solution to drive off
excess hydrogen bromide gas, anhydrous barium
carbonate (500 mg) and ethyleneglycol (500 mg)
were added and stirred at room temperature for
10 h. The mixture was diluted with
dichloromethane, filtered, washed with water and
the organic layer was dried over anhydrous MgSO,.
The removal of solvent gave a syrup, which was
purified by silica gel column chromatography
eluting with chloroform-acetone (30 : 1 v/v) to
obtain pure product (80 mg). The resulting
product had the following properties:
'H-NMR (CDC1,): ~ 6.82 (lH, S, 5-H), 6.51 (lH, S,
8-H), 6.23 (2H, S, 2', 6'-H), 5.95 (2H,
S,OCH~O), 5.52 (lH, S, OH), 4.70 4.15 (m), 3.72
(s, 6H, 3',5'-OCH,).
.
EXAMPLE 15
Preparation of 4'-demethyl-4~-Chloro-4-
desoxypodophyllotoxin. (Scheme VI)
Methylsulfide (0.3 ml) and N-chloro-
succinimide (60 mg, 0.45 mmole) were added at
0C to a solution of 4'-demethylpodophyllotoxin
(100 mg, 0.25 mmole) in methylene chloride ~15
ml). The mixture was otirred for 5 h at 0C
under a nitrogen atmosphere. After the removal
of the volatile reagents by evaporation in
vacuo, the residue was purified by silica gel
chromatography eluting with methylene chloride
and acetone to obtain the pure product (82 mg,
78.5%)~ The product had the following
properties:
IR(CHCl,) cm':3540(0H), 1770(1actone)
2~42~1
26
'H-NMR(CDCl3) S:6.57 (s, lH, 8-H), 6.53 (s, lH,
5-H), ~.30 (s, 2H, 2'6'-H), 6.02, 5.98 (each d,
2H, J=1.3 Hz, OCH~O), 5.42 (s, lH, OH), 4.62 (d,
lH, J=5.2 Hz, l-H), 4.53 (m, 2H, ll-CH7), 4.45
(d, lH, J-3.3 Hz, 4-H), 3.80 (s, 6H, 3', 5'-
OCH,), 3.41 (dd, lH, J-5.3, 14 Hz, 2-H), 2.86 (m,
lH, 3-H)
EXAMPLES 16. 17, AND 18
Preparation of 4, 5, 8, 2 '-tetrabromo-4 B-
desoxypodophyllotoxin, 5, 2', 6'-tribromo-4B-
desoxypodophyllotoxi~, and 5, 8, 2',6'-
tetrabromo-4B-desoxypodophyllotoxin.(Scheme VII)
The compounds of Examples 16,17, and 18 were
prepared according to the following procedure.
To a solution of podophyllotoxin (200 mg, 0.54
mMol) in CHC1, (7 ml) was added bromine (0.5 ml,
9.70 mMol). After stirring for 2 h. at room
temperature, the mixture was poured into ice-
water, and extracted with CHC1" followed by
washing with 5% sodium hydrosulfite to remove
excess bromine. The organic layer was dried
over anhydrous magnesium sufate, filtered, and
concentrated ~ vacuo to yield a crude product.
Thi8 was purified by use of preparative TLC
(CHCl,-acetone 15:1) to obtain the compounds of
Example 16, 17, and 18.
4, 5, 8, 2'-tetrabromo-4B-
desoxypodophyllotoxin: 16 mg; NMR (CDC1,)~ 6.21
(d, J-1.0 Hz, 2H, OCH,O), 5.85 (s, lH, 6'-H),
5.74 (d, J=3.2 Hz, lH, 4-H), 5.45 (d, J=6.4 Hz,
lH, 1-H), 4.49 (~, 2H, 11,11'-H), 3.95 (s, 3H,
3'-OCH,), 3.92 (s, 3H, 4'-OCH,), 3.68 (s, 3H, 5'-
OCH,), 3.44 (dd, J=6.4, 14.4 Hz, lH, 2-H) and
20~4211
3 . 35 (m, lH, 3-H); IR (CHC1,) no OH barld, 1785
( lactone) cm ' .
EXAMp~ Z
5, 2', 6'-tribromo-4B-
desoxypodophyllotoxin: 60 mg: NMR (CDC1,)6 6.33
(s, lH, 8-H), 6.04 (d, J=l.OHz, 2H, OCH,O), 5.72
(d, J=9.5 Hz, lH, 1-H), 5.25 lbr.s, lH, 4-H),
4.40 (m, 2H, 11, ll'-H), 4.95 (B, 3H, 3'-OCH3),
3.94 (s, 3H, 5'-OCH,), 3.81 (s, 3H, 4'-OCH,),
3.70 (m, lH, 3-H), and 3.55 (dd, J=9.5, 15.0 Hz,
lH, 2-H: IR (CHC1,) 3600 (OH), 1776 (lactone)
C~' .
~X~pL~ 18~
5, 8, 2', 6'-tetrabromo-4B-
lS desoxydophyllotoxin: 16 mg; NMR (CDCl,)~ 6.14
(d, J=2.2, 2H, OCH2O), 5.70 (d, J=7.6, lH, 1-H),
5.21 (d, J=2.8 Hz, lH, 4-H), 4.40 (m, 2H,
11/11'-H 3.93 (s, 6H, 3'and 5'-OCH,), 3.78 (8,
3H, 4'-OCH,), 3.65 (m, lH, 3-H) and 3.49 (dd,
. 20 J=9.5, 15 Hz, lH, 2-H); IR (CHC1,) 3600 (OH),
1776 (lactone) cm'.
16, R,~R,=R,~R,-Br, R,=H
17, R,-OH, R,-R,-R,~Br, R,=H
18, R,-OH, Rl=R,-R,=R,=Br
Ex~eh~ 19 - 41
Preparation of 4'demethyl-4~-
(arylaminoS-4-desoxypodophyllotoxins (19-41).
(Scheme VIII)
The 4'-demethyl-4~-(arylamino)-4-
deoxypodopohyllotoxins specified in examples 19
- 41 were prepared according to the following
procedure:
~42~1
28
A solution of 4'-demethyl~
epipodophyllotoxin (10 g, 24 mmol) in 250 ml of
dry dichloromethane was kept at 0C, and dry
hydrogen bromidP gas was bubbled through the
solution. After 30 min., nitrogen was bubbled
through the solution to drive off excess
hydrogen bromide. The solution was then
evaporated in vacuum to dryness by means of
azeotropic distillation with benzen~.
The desired product (11.5 g) was
obtained and then used for the next step
reaction without any further purification.
Spectral data agreed with that
described by M. Xuhn and A. Von Wartburg. Helv.
Chim. Acta, 52, 944 (1969).
A solution containing 4'-demethyl-4~-
bromo-4-deoxypodophyllotoxin (300 mg, 0.65
mmol), barium carbonate (153 mg, 0.78 mmol) and
the appropriate arylamines (0.78 mmol) in 7 ml
of dry 1, 2-dichloroethane was stirred overnight
at room temperature. The reaction mixture was
filtered, diluted with ethyl acetate, washed
with water, dried and puri~ied via column
chromatography. The products (19-41) obtained
in the examples had the characteristics listed
below.
~XAMPLE 19
4'-Demethyl-4~-anilinyl-4-desoxypodophyllotoxin.
Crystals from methanol; mp 172-173,
[~]"D-120 (C-1.0, CHCI,); IR (RBr) 3500 (OH),
3360 (NH), 2900 (aliphatic C-H), 1755 (lactone),
1595, 1500 and 1475 (aromatic c=c) cm': 'H NMR
CDCI, ~ 7.22 (t, J=7.5 Hz, 2H, 3", 5"-H), 6.80
(m, 2H, 4" -H and 5-H), 6.50 (m, 3H, 2"-H, 6"-H
and 8-H), 6.33 (s, 2H, 2', 6'-H), 5.97 (ABa,
J=1.3, 3.6 Hz, OCH,O), 5.42 (s, lH, exchangeable,
2~421~
29
4'-O~), 4.68 (br, lH, 4-H), 4.60 (d, J=4.9 ~z,
1-H), 4.38 (t, J=8.4 Hz, lH, 11-H), 4.01 (t,
J=8.4 Hz, 1~, 11-H), 3.85 (br, lH, exchangeable,
NH), 3.79 (s, 6H, 3', 5'-OCH,), 3.16 (dd, J=5.0,
14.0 Hz, lH, 2-H), 3.00 (m, lH, 3-H~.
Anal. (C2,H"NO7), C.H.N.
EXAMPLE_~0
4'-Demethyl-4~-[4"-cyanoanilinyl]-4-
desoxypodophyllotoxin.
Crystals from ethanol; mp 187-189,
[~]2~D-1~50 (C=1.0, CHCI,); IR (KBr) 3500 (OH),
3360 (NH), 2890 (aliphatic C-H), 2210 (lactone),
1600~ 1510 and 1475 (aromatic C=C) cm': 'H NMR
CDCI, ~ 7.50 (d, J-8.7 Hz, 2H, 3", 5"-H), 6.74
15 (s, lH, 5-H), 6.57 (d, J=8.7 Hz, 2H, 2", 6"-H),
6.55 (s, lH, 8-H), 6.32 (s, 2H, 2', 6'-H), 5.99
(AB~, J=1.2, 8.3 Hz, 2H, OCH20), 5.44 (8, lH,
exchangeable, 4'-OH), 4.78 (~, lH, exchangeable,
NH), 4.63 (d, J=4.2 Hz, lH, 4-H), 4.36 (m, 2H,
20 ll-H), 3.85 (m, lH, 1-H), 3.79 (s, 6H, 3', 5'-
OCH,), 3.09 (dd, lH, 2-H), 3.05 (m, lH, 3-H).
Anal. (C2,H24NO7), C.H.N.
EXA~LE_2L
4'-Demethyl-4~-[3"-cyanoanilinyl]-4-
desoxypodophyllotoXin.
Cry~tals from methanol; mp 191-192,
t~]2'D-117 (C~0.33, CHCI,); IR (KBr) 3450 (OH),
3360 (NH), 2900 (aliphatic C-H), 2225 (CN), 1750
(lactone), 1595, 1500 and 1450 (aromatic C=C) cm~
30 ': 'H NMR CDCI, ~ 7.31 (t, J27.6 Hz, 5"-H), 7.07
(d, J=7.6 Hz, 4"-H), 6.80 (d, 2H, 2"-H and 6"-
H), 6.74 (s, lH, 5-H), 6.55 (s, lH, 8-H), 6.33
(s, 2H, 2', 6'-H), 6.00 (d, J-7.0 Hz, 2H, OCH20),
5.48 (s, lH exchangeable, 4'-OH), 4.69 (d, Ja3.8
Hz, lH, 4-H), 4.62 (d, J=4.5 Hz, lH, 2-H), 4.41
2~21~
(t, J=8.s Hz, lH, 11-H), 3.92 (t, J=8.5 Hz, lH,
11-H), 3.~1 (s, 6H, 3', 5'-OCH,), 3.14-3.00 tm,
2H, 2-H and 3-H).
Anal. (C,~H2~N2O~)-1/2 H,O, C.H.N.
EXAMPLE 22
4'-Demethyl-4~-[4"-ethoxycarbonylanilinyl]-4-
desoxypodophyllotoxin.
Crystals from ethanol; mp 270-27i,
[~2'D-145 (C=0.33, CHCI,); IR (KBr) 3500 (OH),
10 3370 (NH), 2940 (aliphatic C-H), 1762 (lactone~,
1695 (ester), 1610, 1520 and 1480 (aromatic C=C~
cm-': 'H NMR CDCI, ~ 7.92 (d, J=8.8 Hz, 2H, 3",
5"-H), 6.77 (s, lH, 5-H), 6.55 (d, J=8.8 Hz, 2H,
2", 6'~-H), 6.54 (s, lH, 8-H), 6.33 (s, 2H, 2',
lS 6'-H), 5.99 (AB~, J=l.1, 8.2 Hz, 2H, OCH,O), 5.44
(s, lH, exchangeable, 4' OH), 4.78 (d, J=3.3 Hz,
lH, 4-H), 4.62 (d, J=4.5 Hz, lH, 1-H), 4.40 ~m,
2H, 4-H and 11-H), 4.37 (g, J=7.1 Hz, 2H,
CO2CH2CH,), 4.32 (d, J-7.1, lH, exchangeable, NH),
20 3.92 (t, J=7.5 Hz, lH, ll-H), 3.80 (s, 6H, 3',
5'-OCH,), 3.10 (dd, lH, 2-H), 3.08 (m, lH, 3-H),
1.38 (t, J=7.1 Hz, 3H, CO,CH~L).
Anal. (C~oH2~0~), C.H.N.
EXAMPLE 23
4~-Demethyl-4~-~4"-morpholinoanilinyl]-4-
desoxypodophyllotoxin.
Crystals from ethanol; mp 235-237,
t~]"D-129 (C=l, CHCI,); IR (K~r) 3500 (OH), 3300
(NH), 2880 (aliphatlc C-H), 1755 (lactone),
30 16~0, 1510 and 1475 (aromatic C=C) cm-': 'H NMR
CDCI, ~ 6.86 (d, J=9.5 Hz, 2H, 3", 5"-H), 6.76
(s, lH, 5-H), 6.52 (br, 3H, 8-H and 2", 6"-H),
6.35 (s, 2H, 2', 6'-H), 5.96 (d, J=66.7 Hz, 2H,
OCH,O), 5.44 (s, lH, exchangeable, 4~-OH), 4.61
(m, 2H, 4-H and 1-H), 4.37 (t, J=7.0 Hz, lH, 11-
H), 4.08 (t, J=7.0 Hz, lH, ll-H), 3.82 (br, 4H,
2 1 ~
31
~ , 3.80 ~s, 6H, 3', 5'-3CH,), 3.22-2.90 (m,
2H, 2-H, 3-H and 4H, ~ ~;
Anal. (C,,H,IN,O,), C.H.N.
EXAMPLE 24
4'-Demethyl-4~-[3", 4"-
(methylenedioxy)anilinyl~-4-
desoxypodophyllotoxin.
Crystals from methanol; mp 247-2497,
[~]2~D-126 (C=1, CHCI,); IR (KBr) 3500 (OH), 3340
10 (NH), 2900 (aliphatic C-H), 1752 (lactone),
1605, 1496 and 1475 (aromatic C=C) cm~': 'H NMR
CDCI" ~ 6.76 (s, lH, 5-H), 6.68 (d, J=8.1 Hz,
lH, 5n-H), 6.52 (s, lH, 8-H), 6.33 (s, 2H, 2',
6'-H), 6.17 (d, J31.2 Hz, lH, 2"-H), 5.96 (q,
15 J=1.2, 8.1 Hz, 3H, 6"H and OCH,O), 5.90 (s, 2H,
7"-H), 5.43 (s, lH exchangeable, 4'-OH3, 4.59
(d, J=4.9 Hz, lH, 4-H), 4.56 (d, J=3.9 Hz, lH,
l-H), 4 . 37 (t, lH, 11-H), 4.05 (t, lH, 11-H),
3.79 (s, 6H, 3', 5'-OCH,), 3.15 (dd, lH, 2-H),
20 2.95 (m, lH, 3-H).
Anal. (C2,H"NO~), C.H.N.
EXAMP~E 25
4'-Demethyl-4~-[3", 4"-dimethoxyanilinyl]~4-
desoxypodophyllotoxin.
Cry~tals from methanol; mp 233-~34
tdec); t~]2'D-118D (C=1, CHCI,); IR (KBr) 3500
(OH), 3360 (NH), 2920 (aliphatic C-H), 1770
(lactone), 1605, and 1505 (aromatic C=C) cm': 'H
NMR CDCI" ~ 6.78 (s, lH, 5-H), 6.75 (d, J=8.5
30 Hz, lH, 5"-H), 6.53 (s, lH, 8-H), 6.34 (s, 2H,
2', 6'-H), 6.17 (s, lH, 2"-H), 6.05 (d, J=8.5
Hz, lH, 6"-H), 5.96 (d, J=2.4 Hz, 2H, OCH2O),
5.43 (s, exchangeable, 4'-OH), 4.60 (d, 2H, 4-H
and l-H), 4.38 (t, J=8.3 Hz, lH, 11-H), 4.05
2~4~2~1
32
(t, J=8.3 Hz, 1, ll-H), 3.83 (s, 3H, 4"-OCH,),
3.81 (s, 3H, 3"-OCH3), 3.80 (s, 6H, 3', 5'-OCH,),
3.18 (dd, J=5.0, 14.0 Hz, lH, 2-H), 2.96 (m, lH,
3-H).
Anal. (Cl,H"NOq), C.H.N.
EXA~PLE 26
4'-Demethyl-4~-[3"-fluoroanilinyl]-4-
desoxypodophyllotoxin.
Crystals from methanol; mp 201-203C
10 (dec.);[~]2'D-132 tc = 1, CHCl,); IR (KBr) 3500
(OH), 3360 (NH), 2900 (aliphatic C-H), 1750
(lactone), 1605, 1500 1475 (aromatic C=C) cm-';
'HNMR (CDCl,) ~ 7.15 (t, J = 7.4 Hz, lH, 5"-H),
6.76 (s, lH, 5-H), 6.53 (s, lH, 8-H), 6.49 (dd,
H = 1.2, 7.4 Hz, lH, 4"-H), 6.40 (s, 2H, 2', 6'-
H), 6.32 (d, J = 1.2 Hz, lH, 2"-H), 6.24 (dd, J
= 1.2, 7.4 Hz, lH, 6l'-H), 5.97 (ABq, J = 1.2,
7.9 Hz, 2H OCH~O), 5.44 (s, lH, exchangeable, 4'-
OH), 4.67 (s, lH, exchangeable, NH), 4.63 (d, J
= 4.0 Hz, lH, 4-H), 4.59 (d, J = 5.0 Hz, lH, 1-
H), 4.39 (t, J = 8.5 Hz, lH, 11-H) 3.98 (t, J =
8.5 Hz, lH, 11-H), 3.79 (8, 6H, 3',5'-OCH,), 3.11
(dd, J = 5.0, 14.0 Hz, lH, 2-H), 3.00 (m, lH, 3-
H). Anal. calcd for Cl7H"FNO7:C,65.71;H, 4.90; N,
25 2.84. Found: C, 66.81; H, 4.94; N, 2.79.
~Z~
4'-Demethyl-4~-t2"-fluoroanilinyl]-4-
desoxypodophyllotoxin.
Crystals from methanol; mp 197-
198C;[~'D-128 (c = 0.25, CHCl,); IR (KBr) 3500
(OH), 4480 (NH), 2890 (aliphatic C-H), 1755
(lactone), 1610, 1505 and (aromatic C=C) cm~'; 'H
NMR (CDCl,) ~ 7.04 (m, 2H, 3",6"-H), 6.76
(s, lH, 5H), 6.72 (m, lH 5"-H), 6.60 (t, J = 7.2
35 Hz, lH, 4"-H), 6.54 (s, lH, 8-H), 6.34 (s, 2H,
2~2~1 1
33
2',6'-H), 5.97 (d, J = 7.3 Hz, 2H, OCH,O), C.46
(s, lH, exchangeable, 4'-OH), 4.69 (d, J = 4.2
Hz, lH, 4-H), 4.62 (d, J = 4.9 Hz, lH, 1-H),
4.38 tt, J = 8.2 Hz, lH, 11-H) 4.10 (t, lH,
exchan~eable, NH), 3.82 (t, J = 8.2 Hz, lH, 11-
H) 3.79 (s, 6H, 3',5'-OCH,), 3.15 (dd, J = 5.0,
14.0 Hz, lH, 2-H), 3.00 (m, lH, 3-H). Anal.
calcd for C"H,4FNO,:C,65.71;H, 4.90; N, 2.84.
Found: C, 66.80; H, 4.95; N, 2.84.
EXAMPLE 28
4'-Demethyl-4~-~4"-fluoroanilinyl]-4-
desoxypodophyllotoxin.
Crystals from ethanol; mp 176-
177C;[]~'D-100 (c - 0.8, CHCl,); IR (KBr) 3540
15 (OH), 3420 (NH), 2900 (aliphatic C-H), 1740
(lactone), 1610, 1500 1480 (aromatic C=C) cm~';
'HNMR (CDCl,) ~ 6.94 (t, J = 6.7, 2-H, 3",5",-
H), 6.75 (s, lH, 5-H), 6.53 (s, lH, 8H), 6.49
(q, J = 2.2, 6.2 Hz, 2H, 2",6"-H), 6.33 (s, 2H,
2',6'-H), 5.96 (ABq, J = 1.2, 7.5 Hz, 2H OCH,O),
5.43 (s, lH, exchangeable, 4'-OH), 4.60 (d, 2H,
4-H and l-H), 4.37 (t, J s 7.5 Hz, lH, ll-H)
3.99 (t, J = 7.5 Hz, lH, ll-H), 3.79 (æ, 6H,
3',5'-OCH,), 3.73 (br, lH, exchangeable, NH),
25 3.13 (t, J ~ 5.0, 14.0 Hz, lH, 2-H), 3.00 (m,
lH, 3-H). Anal. C~7H,~FNO7), C.H.N.
EXAMPLE 29
4'-Demethyl-4~-t3", 5"-difluoroanilinyl]-4-
desoxypodophyllotoxin.
Crystals from ethanol; mp 180-
183C;~]"D-132 (c = 0.33, CHCl,); IR (KBr) 3500
(OH), 3370 (NH), 2890 (aliphatic C-H), 1750
(lactone), 1620, 1590, 1500 and 1475 (aromatic
C=C) cm~~; 'HNMR (CDCl,) ~ 6.75 (s, lH, 5-H), 6.54
(s, lH, 8-H), 6.32 (s, 2H, 2',6'-H), 6.23 (m,
2~
34
lH, 4l'-H), 6.07 (m, 2H, 2",6"-H), 5.98 (ABq, J =
1.3, 9.o Hz, 2H OCH2O), 5.45 (s, lH,
exchangeable, 4'-OH), 4.61 (m, 2H, 4-H and 1-
H), 4.39 (t, J = 8.5 Hz, lH, 11 H) 4.10 (d, J -
6.1 Hz, lH, lH, exchangeable, NH), 3.85 (t, J =
8.5 Hz, lH, ll-H), 3.81 (s, 6H, 3',5'-OCH,), 3.08
(dd, J = 4.8, 14.1 Hz, lH, 2-H), 3.02 (m, lH, 3-
H). Anal. C2,H2,NF2OI). C.H.N.
EXAM~LE 30
4'-Demethyl-4~-[3"-pyridylamino]-4-
desoxypodophyllotoxin.
Crystals from ethanol; mp 179-181
(dec); [~]2~D-99o (C=0.33, CHCI,); IR (KBr~ 3500
(OH), 3350 (NH), 2900 (aliphatic C-H), 1765
(lactone), 1575, 1500 and 1470 (aromatic C=C~N)
cm': 'H NMR CDCI, ~ 8.08 (d, J=5.5 Hz, lH, 6"-
H), 8.02 (br, lH, 2"-H), 7.16 (m, lH, 5"-H),
6.85 (dd, lH, 4"-H), 6.75 (g, lH, 5-H), 6.55
(s, lH, 8-H), 6.32 (s, 2H, 2', 6'H), 5.98 (AB~,
J=1.3, 7.3 Hz, 2H, OCH2O), 4.65 (d, J=4.9 Hz, lH,
4-H), 4.60 (m, lH, 1-H), 4.20 (t, J=8.2 Hz, lH,
ll-H), 3.90 (m, 2H, 11-H and NH), 3.80 (s, 6H,
3', 5'-OCH,), 3.18 (dd, J=5.0, 14.1 Hz, lH, 2-
H), 3.03 (m, lH, 3-H).
Anal. (C,~H~,N,O,) 1/2 H~O, C.H.N.
EXAMPLE 31
4'-Demethyl-4~-~2"-pyridylamino]-4-
desoxypodophyllotoxin.
Crystals from ethanol; mp 215-218
(dec); [a~ -82 (C=0.33, CHCI,); IR (KBr) 3500
(OH), 3360 (NH), 2950 (aliphatic C-H), 1760
(lactone), 1690, 1645, 1600 and 1460 (aromatic
C=C=N) cm~': 'H NMR CDCI, ~ 8.11 (d, lH, 4"-H),
7.45 (m, lH, 4"-H), 6.81 (s, lH, 5-H), 6.67 (m,
lH, 5"-H), 6.55 (s, lH, 8-H), 6.45 (d, lH, 3"-
20~42~l1
H), 6.34 (s, 2H, 2', 6'-H3, 5.97 (AB~, J-1.3, 6.7
Hz, 2H, OCH,O), 5.43 (br, lH, exchangeable, 4'-
OH), 5.35 (m, lH, exchangeable, NH), 4.60 ~d,
J=4.2 Hz, lH, 4-H), 4.24 (m, 2H, l-H and llNH)
3.85 (m, lH, llH), 3.78 (s, 6H, 3', S'-OCH,),
3.05 (m, 2H, 2-H and 3-H).
Anal. (C26H,~N,O~), 1/2 H,O, C.H.N.
4'-Demethyl-4~-[3"-quinolinylamino]-4-
desoxypodophyllotoxin.
Crystals from ethanol-ether; mp 243-
246 (dec); [~]"n~l79 (C=0.5, CHCI,); IR (RBr)
3460 (OH~, 3380 (NH), 2900 (aliphatic C-H), 1775
(lactone), 1605, 1510 and 1480 (aromatic C=C=N)
cm-': 'H NMR CDCI, ~ 8.46 (d, J=2.9 Hz, 2"-H),
7.97 (m, lH, 4"-H), 7.65 (m, lH, 7"-H), 7.48 (m,
2H, 5", 6"-H), 6.g9 (d, J=2.9 Hz, 8'-H), 6.76
(s, lH, 5-H), 6.57 (5, lH, 8-H), 6.35 (s, 2H,
2', 6'-H), 5.99 (AB~, J=1.1, 8.0 Hz, 2H, OCH,O),
5.48 (s, lHj exchangeable, 4'-OH), 4.78 (d,
J=4.8 Hz, lH, l-H), 4.45 (t, lH, ll-H), 4.23 (d,
lH, exchangeable, NH), 3.99 (t, lH, ll-H), 3.81
(s, 6H, 3', 5'-OCH,), 3.15 (m, 2H, 2-H and 3-H).
Anal. (C,oH2,N,O7) 1/2~,0, C.H.N.
~a~LE_~l
4'-Demethyl-4B-[3 " -hydroxyanilinyl]-4-
desoxypodophyllotoxin.
Amorphous powder from ether: mp 163-
166C; IR (KBr) 3480 (OH), 3380 (NH), 2900
(aliphatic CH), 1750 (lactone), 1590, 1475
(aromatic C=C) cm-';'H NMR (CDCl,) ~
7.05(t,J=8Hz,lH,5"-H), 6.78 (s,lH,5-H),
6.52(s,1H,8-H), 6.33(s,2H,2',6'-H), 6.24
(dd,J=2.2, 8Hz,lH,4"-H), 6.15(dd,J=1.7,
2 ~ 1
36
8H~,lH,6"-H), 6.07(t,J=2.2Hz,1H,2"-H),
5.97(d,J=4.4Hz,2H,
OCH,O), 5 . 43(s, exchangeabl e), 4 . 82 (s,
excha~geable), 4.65 (d,J=3.9Hz,lH,4-H),
4.58(d,J=4.8Hz,lH,l-H), 4.37(t,J=8.7Hz,lH,11-
H), 4.0(t,J=8.7Hz,lH,ll-H), 3.79(s,6H,3'5'-OCH,),
3.1(dd, J=4.8, 14.1 Hz, lH, 2-H), 2.98(m,1H,3-
H); MS, m/z=491 (m+). Anal. Calcd for
C"H2,NO,.H20: C,63.65jH,5.30. Found: C,63.35;
H,5.44.
EXAMPLE_34
4'-Demethyl-4~-[2 " -hydroxyanilinyl]-
4-desoxypodophyllotoxin.
Amorphous crystals from ether: mp
175C; IR (KBr) 3360 (OH, NH), 2900 (aliphatic C-
H), 1750 (lactone, 1600, 1475 (aromatic C=C) cm-
';'H NMR (CDCI,) ~ 6.88(t,J=7.4H2,lH,4"-H),
6.78(s,1H,5-H), 6.65(m,2H,3",6"-H), 6.5(m,2H,8-
H,5"-H), 6.35(s,2H,2'6'-H), 5.96(AB~
J=1.2Hz,3.5Hz,2H,OCH,O), 5.44(s.exchangeable),
5.10(s, exchangeable), 4.67(d,J=4Hz,lH,4-H),
4.61(d,J=4.8Hz,lH,l-H), 4.38(t,J=8.5Hz,lH,ll-
H), 3.98(t,J=8.5Hz,lH,ll-H), 3.79(s,6H,3',5'-
OCH,), 3.24 (dd,J=4.8,14Hz,lH,2-H), 3.02(m,1H,3-
H), MS, m/z-491(m+). Anal. Calcd for C,7H"NO,:
C,65.99; H,5.09. Found: C, 65.85; H,5.18.
EXAMPLE ~
4'-Demethyl-4B-~4 " -hydroxyanilinyl]-4-
desoxypodophyllotoxin.
Amorphous powder from ether mp 162-165C;IR
(RBr) 3525 (OH), 3345 (NH), 3010 (aromatic CH),
2900 (aliphatic CH), 1745 (lactone), 1600, 1475
(aromatic C=C) cm~';'H NMR (DMSO d~, D,O exchange)
~ 6.69~s,1H,5-H), 6.55(s,4H,2",3",5",6"-H),
6.48(s,1H,8-H), 6.23(s,2H,2'6'-H),
37 2~2~
5.94~d,J=9.7Hz,2H,O-cH2-O), 4.68(d,J=4.3Hz, lH, 4
H), 4.46(d,J=5.4HZ,lH,l-H), 4.29(t,J=7.6,1H,ll-
H), 3.76(t,J=7.6Hz,lH,ll-H), 3.61(s,6H,3',5'-
OCH,), 3.28(dd,J=5.4,15.8Hz,lH,2~H), 2.95(m,1H,3-
H).
ExAMpLE ~
4'~Demethyl-4B-[2"-chloroanilinyl]-4-
desoxypodophyllotoxin.
Crystals from ethyl acetate/ether, mp
253-255C;[~]"D-90 (c = 1.0, CHCl,); IR (KBr)
3500 (OH), 3450 (NH), 2895 (aliphatic CH), 1751
(lactone), 1590, 1500 1472 (aromatic C=C) cm~';
'H NMR (CDCl,) 7.31 (dd, J = 1.4, 7.9 Hz, lH, 3"-
H), 7.18 (t, J = 8.8, Hz, lH, 5"-H), 6.76 (s,
lH, 5-H), 6.73 (t, J = 9.0 Hz, 4l'-H) 6.58 (d, J
= 8.2 Hz, lH, 6"-H), 6.54 (s, lH, 8-H), 6.35 (s,
2H, 2',6'-H), 5.98 (ABq, J = 1.2, 4.2 Hz, 2H
OCOCH,O), 5.44 (s, lH, exchangeahle, 4'-OH), 4.73
(t, J = 4.9 Hz, lH, 4-H) 4.64 (d, J - 4.9 Hz,
lH, l-H), 4.49 (d, J = 6.0 Hz, lH, exchangeable,
NH), 4.36 (t, J = 8.3 Hz, lH, ll-H), 3.91 (t, J
= 8.3, Hz, lH, ll-H), 3.80 (s, 6H, 3,'5'-OCH,~,
3.17 (dd, J = 4.8, 14.0 Hz, lH, 2-H), 3.04 ~m,
lH, 3-H). Anal. C"H"CINO, C.H.N.
EXA~ Z
4'--Demethyl-4~-[3"-chloroanilinyl]-4-
desoxypodophyllotoxin.
Crystals from ethyl acetate/ether, mp
174-176C;[~]"D-112 (C = 1.0, CHCl,); IR (KBr)
30 3500 (OH), 3360 (NH), 2920 (aliphatic CH), 1752
(lactone), 1580, and 1452 (aromatic C=C) cm~'; 'H
NMR (CDCl,) 7.12 (t, J = 8.1, Hz, lH, 5"-H), 6.76
(s, lH, 5-H), 6.74 (dd, J = 1.0, 8.1 Hz, lH 4"-
H), 6.53 (br, 2H, 8-H and 2"-H), 6.42 (dd, J =
35 1.6, 6.5 Hz, lH, 6"-H), 6.33 (s, 2H, 2',6'-H),
2~2~
38
5.97 (ABq, J 5 1.0~ 8.7 Hz, 2H OCH,O), 5.43 (s,
lH, exchangeable, 4'-OH), 4.66 (br, lH, 4-H),
4.59 (d, J - 4.8 Hz, lH, 1-H), 4.39 (t, J = 7.7
Hz, lH, ll-H), 3.99 (t, J = 7.7 Hz, lH, 11-H)
3.96 (br, lH, exchangeable, NH~, 3.79 ts, 6H,
3,'5'-OCH,), 3.11 (dd, J - 5.8, 14.0 Hz, H, 2-
H), 3.01 (m, lH, 3-H). Anal. C"H2,CINO,) C.H.N.
EXAMPLE 38
4'-Demethyl-4B-~4"-chloroanilinyl]-4-
desoxypodophyllotoxin.
Crystals from ethyl acetate/ether, mp
25~-255Cj[~]"D-125 (c = 0.75 CHCl,); IR (KBr)
3500 (OH), 3360 (NH), 2920 (aliphatic CH), 1758
(lactone), 1605, 1590 and 1475 (aromatic C=C) cm-
15 ; H NMR (CDCl,) 8 7.17 (d, J - 8.7, Hz, 2H,
3",5"-H), 6.74 (5, lH, 5-H), 6.53 (s, lH, 8-H),
6.48 (d, J = 8.7 Hz, 2H, 2",6"-H), 6.32 (s,
2'6'-H), 5.96 (ABq, J = 1.0, 6.8 Hz, 2H OCH,O),
5.43 (s, lH, exchangeable, 4'-OH), 4.63 (d, J =
4.2 Hz, lH, 4-H), 4.59 (d, J = 4.9 Hz, lH, 1-
H), 4.38 (t, J 2 8.0 Hz, lH, ll-H) 3.96 (t,J =
8.0 Hz, lH, ll-H), 3.79 (s, 6H 3,'5'-OCH,), 3.12
(dd, J = 4.9, 14.1 Hz, H, 2-H), 2.99 (m, lH, 3-
H). Anal. C"Hl,CINOT) C.H.N.
~ EL~
4'-Demethyl-4B-t3"-bromoanilinyl~-4-
desoxypodophyllotoxin.
Crystals from methanol/ether; mp 177-
179C;t~]"D-105 (c = 1, CHCl,); IR (RBr) 3450
(OH), 3340 (NH), 2900 (aliphatic CH), 1740
(lactone), 1590, 1500 and 1475 (aromatic C=C) cm~
'; 'H NMR (CDCl,) 8 7.07 (t, J = 8.0, Hz, lH, 5"-
H), 6.90 (dd, J = 0.9, 7.9 Hz, lH, 4"-H), 6.75
(s, lH, 5-H), 6.70 (br, lH, 2"-H), 6.53 (s, lH,
8-H), 6.47 (dd, J = 1.7, 8.3 Hz, lH 6"-H), 6.33
2~2~:~
39
(s, 2H, 2',6'-H), 5.97 (dd, J = 1.2, 9.3 Hæ, 2H,
OCH,O), 5.43 (s, lH, exchangeable, 4'-OH), 4.65
(d, J = 4.2 Hz, lH, 4-H), 4.6Q (d, J = 4.8 Hz,
lH, 1-H), 4.39 (t, J = 7.3 Hz, lH, 11-H) 3.96
(t, J = 7.3 Hz, lH, 11-H), 3.90 (d, J = 6.2, Hz,
lH, exchangeable, NH), 3.R0 (s, 6H, 3,'5'-OCH,),
3.10 (dd, J = 4.9, 14.0 Hz, lH, 2-H), 3.02 (m,
lH, 3-H). Anal. C"H"BrNO7) C.H.N.
EXAMPLE 4Q
4'-Demethyl~4~-[4"-bromoanilinyl]-4-
desoxypodophyllotoxin.
Crystals from ethyl acetate/ethanol; mp
227-230C,[~]"D-110 (c = 0.5, CHCl,); IR (KBr)
3500 (OH), 3330 (NH), 2900 (aliphatic CH), 1755
(lactone), 1605, 1590 and 1475 (aromatic C-C) cm~
'; 'H NMR (CDCl,) ~ 7.30 (d, J = 8.9, Hz, 2H,
3",5"-H), 6.75 (s, lH, 5-H), 6.53 (s, lH, 8-H),
6.44 (d, J = 8.9 Hz, 2H, 2",6"-H), 6.32 (s, 2H,
2',6'-H), 5.98 (A8q, J = 1.3, 8.3 Hz, 2H, OCH~O),
5-42 (5, lH, exchangeable, 4'-OH), 4.62 (m, 2H,
4-H and 1-H), 4.36 (t, J = 8.5 Hz, lH, 11-H),
3.95 (t, 8.5 Hz, 11-H), 3.86 (d, J = 708, lH,
exchan~eable, NH), 3.73 (s, 6H, 3,'5'-OCH,), 3.11
(dd-, J s 4.8, 14.1 Hz, lH, 2-H), 3.00 (m, lH, 3-
H). Anal. C~H,4BrNO,) C.H.N.
EX~MP~E 41
4'-De~ethyl-4B-t4"-iodoanilinyl]-4-
desoxypodophyllotoxin.
Cry~tals from ethanol; mp 198-
200C(dec.);~]"D-111 (c = 0.5, CHCl,); IR (KBr)
3540 (OH), 3420 (NH), 2900 (aliphatic CH), 1770
(lactone), 1610, 1585 and 1480 (aromatic C=C) cm~
'; 'H NMR (CDCl,) ~ 7.46 (d, J = 8.8 Hz, 2H,
2",6"-H), 6.32 (s, 2H, 2',6' H), 5.59 (ABq, J =
1.1, 8.9 Hz, 2H, OCHiO), 5.44 (s, lH,
20~2~
exchangeabl~, 4'-OH), 4.62 (~, lH, 4-H), 4.58
td, J = 4.9 Hz, lH, 1-H), 4.34 (t, J = 8.5 Hz,
lH, l1-H), 3.94 (m, 2H, 11-H and NH), 3.78 (s,
6H, 3~,5~-OCH,), 3.09 (dd, J = 4.9, 14.1 Hz, lH,
2-H), 2.99 (m, lH, 3-H). Anal. C"H"BrNO,) C.H.N.
EXAMP~ 42 - 44
(Scheme IX)
Podophyllotoxin (500mg, 1.2 mmol) was
dissolved in dry dichloromethane (lOml) and kept
at 0C. Hydrogen bromide gas was introduced
into the solution for 45 min., after which time
the solvent was evaporated in vacuo, anhydrous
tetrahydrofuran (15 ml), anhydrous barium
carbonate (474 mg, 2.4 mmol) and the appropriate
hydroxyaniline ~142mg, 1.3 mmol) was added. The
mixture stood at room temperature overnight, and
then was filtered and concentrated. The crude
product was purified using column chromatography
tsilica gel 45 g with dichloromethane-acetone-
ethyl acetate 100:5:5 as an eluant). Theproducts (42-44) obtained in the examples had
the characteristic~ listed below.
~Z
4B-~2"-hydroxyanilinyl]-4-
de60xypodophyllotoxin,
Amorphous crystals from ether: mp 145-
148C; IR (KBr) 3480 (OH), 3410 (NH), 2900
(aliphatic CH), 1760 (lactone), 1580, 1475
(aromatic C=C) cm'; H NMR (CDCl,) ~ 6.90 (t,J =
6.6 Hz, lH, 4"-H), 6.78 (s, lH, 5-H), 6.65 (~,
2H, 3",6"-H), 6.53 (m, 2H,8-H, 5"-H), 6.34 (s,
2H 2',6'H), 5.96 (ABq, J = 1.0, 3.5 Hz,2H,
OCH,O), 5.02 (s, lH, exchangeable, 2"-OH), 4.68
(m, lH, 4-H), 4.62 (d, J = 4.9 Hz, lH, l-H),
2~2~ ~
41
4.38 (t, J = 8.6 Hz, lH, ll-H), 4.33 (m, lH,
exchangeable, NH), 4.00 (t, J = 8.6 Hz, lH,
llH), 3.82 (s, 3H, 4'-OCH,), 3.76 (s, 6H, 3',5'-
OCH,), 3.25 ~dd, J = 5.1, 14.0 Hz, lH, 2-H), 3.05
(m, lH, 3-H). MS, m/z = 505 (m+). Anal.
(C"H"NOl 3/2 H2O) C.H.
EXAMPLE 43
4B-[3"-hydroxyanilinyl]~-
Desoxypodophyllotoxin.
Amorphous powder from ether, mp 148-
150C; IR(RBr) 3370 (OH,NH), 2900 (aliphatic
CH), 1760 (lactone), 158S, 1475 (aromatic C=C)
cm~'; 'H NMR (CDCl,) ~ 7.05 (t, J = 8.0 Hz, lH,
5"-H), 6.77 (8, lH, 5H), 6.52 (s, lH, 8-H), 6.32
(s, 2H, 2' 6'-H), 6.25 (dd, J = 2.2, 8.0 Hz, lH,
4"-H), 6.14 (dd, J = 2.2, 8.0 Hz, lH, 6"-H),
6.05 (t = 2.2 Hz, lH, 2"-H), 5.96 (ABq, J = 1.3,
3.8 Hz, 2H OCH,O), 4.64 (d, J = 3.9 lH, 4-H),
4.49 (d, J = 5.0, Hz, lH, l-H), 4.4 (t, J = 8.7
Hz, lH, 11-H), 4.03 (t, J = 8.7 Hz, lH, 11-H),
3.81 (s, 3H, 4'0CH,), 3.76 (s, 6H, 3',5'-OCH,),
3.18 (s, 6H, 3',5'-OCH,), 3.18 (dd, J = 5.0 Hz,
14.0 Hz, lH,2-H), 3.02 (m, lH, 3-H); MS, m/z =
505 (m+). Anal. (C,tH"N~, H,O) C.H.
~XAX~J~ 44
4B-[4"-hydroxyanilinyl]-4-
de~oxypodophyllotoxin.
Crystals from chloroform; mp 145-150C;
IR(KBr) 3310 (OH,NH), 3010 (aromatic CH), 2900
(aliphatic CH), 1730 (latone), 1575, 1475
(aromatic C=H) cm-'; 'H NMR (CDCl,) D,O exchange)
6.75 (d, J = 8.3 Hz, 3H, 5-H, 3",5"-H), 6.53 (s,
lH, 8H), 6.45 (d, J - 8.3 Hz, 2H, 2",6"-H), 6.23
(s, 2H 2'6'-H), 5.95 (ABq, J = 1.0, 4.0 Hz, 2H
OCH,O), 4.60 (d, J = 4.2 Hz, lH, 4-H), 4.57 (d, J
= 4.6 Hz, lH, 1-H), 4.38 (t, J = 6.0 Hz, lH, 11-
42 2 a~42
H), 4.05 (t, J = 6.0 Hz, lH, llH) 3.83 (s, 3H,
4-OCH,3, 3.75 (s, 6H, 3',5'-OCH,), 3.18 (dd, J =
4.6 Hz, 14.0 Hz, lH,2-H), 3.0 (m, lH, 3-H).
Anal. (C,~H,7NO, 1/2 H~O) C.H.
Isolation of Human DNA Topç~somerase II.
Human DNA topoiso~erase II was isolated
from peripheral blast cells of a patient with
acute leukemia. The isolation procedure is
described in Thurston, L., Imakura, Y., Haruna,
M., Li, Z. C., Liu, S. Y., and Lee, K. H., J.
Med. Chem., 31, COMPLETE (1988) and is a partial
combination of the procedure described in Goto,
T., Laiapia, P. and Wang, J., J. Biol Chem~,
259, 10422 (1984) and Halligan, B., Edwards, X.,
and Liu, L., 2l_~inL-_She~., 260, 2475 (1985)
which are herein specifically incorporated by
reference.
Preparations of Druas.
Drugs were dissolved in Me,SO at a
concentration of 20 mM as the stock solution and
diluted before use with water to the desired
concentration of each druq.
DNA TQpoiso~e~ase II Assay.
The P4 unknotting reaction was a
modification Or the procedure described by
Hseih, T., ~ iol~ m~, 258, 8413 ~1985),
which i8 herein specifically incorporated by
reference.
The reaction mixture (20 ~L), which
contained 50 mM HEPES, pH 7.0, 50 mM KCI, 100 mM
NaCl, 0.1 mM EDTA, 10 mM MgCl" 1.0 mM ATP, 50
~g/mL bovine serum albumin, 0.4 ~g P4 knotted
DNA, and enzyme, was incubated with or without
drugs.
2 ~
43
The reaction mixture was incubated at
37 C for 30 min and terminated by adding 5.0 ~1
of a stop solution 52~ sodium dodecyl sulfate,
20% glycerol, 0. 05~ bromophenol blue). These
samples were loaded onto a 1% agarose gel and
electrophoresed at 55 V overnight with an
electrophoresis buffer that contained 90 mM
Tris-boric acid, pH 8.3, and 2.5 mM EDTA. At
completion, the gel was stained in 0.5 ~g/mL of
ethidium bromide. Then a photograph was taken
of the DNA bands visualized with fluorescence
induced by a long-wavelength W lamp. The data
reported in Table 1 reflect a lOo ~M drug
concentration.
K-SpS Precipi~ation Assay_or P~otein-DNA
CompLç~çs.
The intracellular formation of covalent
topoisomerase II-DNA complexes was quantitated
using the potassium SDS precipitation assay, a
procedure adapted from the method described in
Rowe, T.C., Chen, G. L., ~siang, Y. H., and Liu,
L., Cancer RÇs., 46, 2021 (1986) (hereinafter
Rowe et al.), which is herein specifically
incorporated by reference. KB ATCC cells were
prelabeled with 0.05 mCi/ml 14C-thymidine
~specific activity 50.5 mCi/mmol) for 18 hr. A
~inal concentration of 5xlO' cells/sample were
treated with 10 ~M of the drugs at 37 C for 1
hr and proceeded according to the procedure
described by Rowe et al. to detect the protein-
linked DNA levels.
It will be apparent to those skilled in
the art that various modifications and
variations can be made in the processes and
products of the present invention. Thus, it is
intended that the present invention cover the
20~42~1
44
modifications and variations of this invention
provided that they come within the scope of the
appended claims and their equivalents.
TABLE I
BIOLOGICAL EVALUATION OF 4'-DEMETHYL-4-ALKYLAMINO-
PODOPHYLLOTOXIN ANALOGUES
R
< C~
O
o
H3CO~OCH3
OH DNAC~LLULAR
TOPO-PROTEIN-
CYTO- ISOMERASE II DNA
TOXICITY- AC~IVITYCOMPLEX
COMPOUND R ED,~KB(~g/ml) % INHIBITION~ FORMATIO~%
.
Etoposlde O ~ 2a +++ 100.0
Example:
1 B-OH 0.34 ++ 42.2
2 ~-OH 0.045 + 3.3
3 B-NH2 1.0 ++++36.4
4 ~-NH, 0.42 ~ 8.0
~-NHC~,CH,OH 1.6 ++++121.4
6 ~-NHCH,CH,OH 710.0 - 0.0
7 B-NHCH,CH,CH, <0.4 ++ 69.7
8 B-NHCH,CH,OCH, >4.0 +++110.8
9 ~-NHCH,CH~CH, 3.4 +++ 84.1
B-NHCH,CH(OH)CH, 3.6 ++++ 167.2
11 8-NHCH(CH,)CH,OH 2.3 ++++ 161.7
12 B-NHCH,CH,CH,OH 4.0 ++ 89.2
13 B-OCH,CH,NH, 0.1 ++++300.0
14 B-OCH,CH,OH 0.7 ++ 50.0
' ED,o is the concentration of drug which affords 50%
reduction in cell number after 3 days incubation.
+, ++, +++, ++++ and - denote for 25%, 50%, 75~, >75%, and
0% inhibition.
' Relative activities of cellular protein-DNA complex formation
in KB ATCC tissue culture cells measured at 10 ~M drug
concentration as compared the complex formed by 10 ~M of
etoposide.
20~211
46
TABLE II
COMPOUNDS WITH POTENT TOPOISOMERASE II
INHIBITORY ACTIVITY
DNA
TOPOISOMERASE DNA TOPOISOMERASE
II
INHIBITORY ACTIVITY'
COMPOUNDS (RELATIVE POTENCY)
.. .. _
Etoposide 1.0
2.0 - 6.0
16 8.0
17 2.0
18 2.0
'~e o~
0.
OH
15 R~Cl 16 R,~R,=R,-R,=Br,R,-H
17 R,-OH, R,~R,-R,-Br,R,-H
18 R,~OH, R,-R,-R4=R,~Br
Several different concentrations of tested compounds were
employed for the determination of their potency. The
relative potency with respect to etoposide as expressed in
Table I was the relative concentration of compounds tested to
achieve the same degree inhibition by etoposide in the range
of 25 to 400 ~M.
2~21~
q7
Table ILE~
~iological Evaluation of 4-Demethyl-4B(arylamino)-4-desoxy Podopyllotoxin
It~
<0~O
O
CH~O ~ OC~
OH
Compound R, DNA Cellular Cytotoxicity
Topoisomerase II Protein-DNA IDso ~
Activity Complex (uM)
% Inhi~ition Formation
Etoposide +++ 100 0.2
19 ~ ~ +++ 243 0.71
20 ~ +++~ 211 0.64
21 ~ ~ +++ 137 0.6g
22 ~ ~ eo~c~ +++ 207 <0.10
23 ~ +++ 140 0.66
24 .~? ++++ 164 <1 0
oc~
~ ++ 180 <1.0
~6 ~ +++ 158 0.23
2n~4~
48
Compound R, DNA Cellular Cytotoxicity
Topoisomerase II Protein-DNA ID50 K~
Activity Complex (uM)
% Inhibition Formation
27 ~ ~ ++ 121 0.25
28 ~f ++++ 213 0.24
29 ~ ~ ++ 115 1.08
f
~ +++ 148 0.24
31 ~ ~ ++ 97 0.71
>~
32 ~ ++ 123 <1.0
o~l
33 ~ ++++ 290 0.45
110
34 ~ ~ ++++ 151 4.54
35~ ~ o~ +++ 211 2.26
tl
36 ~ + 32 2.34
2~4211
49
Compound R, DNA Cellular cytotoxicity
Topoisomerase II Protein-DNA ID,
Activity Complex (uM)'
% Inhibition~ FormationC
37 ~ ~ ++ 51 2.29
38 ~ ~ cl +++ 99 0.22
~1
,--
39 ~ ~ ~ + 62 2.36
~ ' +~ 179 0.22
41 ~ + 6~ 0.34
' ID,o is the concentration of drug which affords 50% reduction
in cell number ater 3 days incubation.
~ Activities of cellular protein-DNA complex formation in KB
ATCC tissue culture cells relative to Etoposide.
' +,++,+++,++++ denote 0-24%,25-49%,50-74%, and 275% inhibition
respectively.
204~211
Tabl~ IV
Biological Evaiuation 014~ (arylamino)-4-desoxypodophyl~otoxins
<~0
O
H3CO~OCH3
OcH3
Compound R DNA Cellubr Cytoloxicily
Topoisolnerase l l prolein DNA 1~5O KB (~lM)
Activity Compl~x
Formatbn
~ Inhibitbn
Etoposlde ~ 100 0.2
OH ,
42 NH~ ~ 6 4.11
OH
43 NH~ ~ 37 0.31
44 ~ 21 0.31
2~421~
51
Scherre I
OH ~ ~
<~ ~ H~O X~0
H3CO~ocH3 H3COJ~OCH3 H3CO~OCH~
OCH3 OH OH
~2) fl . r~ OH
Scherne 11
OH SO,CH,
CBz - Cl ~ CH3S02C1 ~
<~.""( <O~V ~
O _ O
H3COJ~OCH~ H3CO~OCH3
OCBZ Oc82
N;~N3 < ~O 2, < ~O
H3CO~oCH3 H3CO~OcH3
OC82 OH
~3) R ~ B-NH2
~) R ~ NH2
20~211
52
Scheme 111
NHR
RNH2 < ~
O
HaCO OCH3
(S 12)
Scheme IV
OCH2CH2Elr OCH2,CH2N3
HOCH2CH2Br ~1~ NaN3
Il <0~ "~ ~ ~
H3COJ~oCH3 H3COJ~oCH3
OH OH
~V~ ~)
OcH2cH2NH2
H2 <~
H3CO~OCH3
OH
(13)
2~4~2~
SCheme V
OCH2CH2C~I
HOCH2cH20H < ~
o .."(
o
H3CO~ oc~3
OH
Schen~ Vl
a
o~o<O~o
a - o
- ' ~
H3CO~OCH3
~ 15 )
Schem~ Vll
R2 R
Br2 < ~
RJ ' O ~ 16 ) RlnR2~R3~Rs~B~, R~.H
R~ ~, Rs
( 17 ) R~.OH, R2.R~.Rs.Br, R3-H
H3CO~OCH3 ~1B ) R~.OH, R2.~3.R~.Rs..Br
OCH3
20~23 ~
54
Schernl~ Vlil
I~HAr
H8r NH2A~ O ~
11 <~ 0
~ O
H3CO J~ OCH3
( 1~1 41 )
Schen~ IX
Br NHAr
HBr<~,~ NH2Ar < ~O
. ""~ O "'~(
H3CO~ocH3 H3COJ~H3
OCH3 OCH3
(42-~4)
