Six acyl esters of cephalotaxine have been synthesized by ordinary and standard procedures, and all have demonstrated chemotherapeutic activity against leukemia in animals.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
This invention relates to a group of synthetic esters of cephalotaxine and 
the use of these esters as chemotherapeutic agents for the remission of 
leukemia in animals. 
2. Description of the Prior Art 
Among the alkaloids which have been isolated from Cephalotaxus harringtonia 
plant material are cephalotaxine and a number of its esters [Powell et 
al., Tetrahedron Lett. 4081 (1969); Powell et al., Tetrahedron Lett. 815 
(1970); Mikolajczak et al., Tetrahedron 28: 1995 (1972); U.S. Pat. No. 
3,793,454; and U.S. Pat. No. 3,870,727]. Though cephalotaxine itself is 
inactive, some of its esters which are derived from relatively complex 
dicarboxylic acid moieties have been found to exhibit significant activity 
against experimental leukemia systems [Powell et al., J. Pharm. Sci. 
61(8): 1227-1230 (August 1972)]. Two of the esters, harringtonine and 
homoharringtonine, have been approved for preclinical evaluation at the 
National Cancer Institute. However, plant material from which to extract 
the active esters is in critically short supply. 
Cephalotaxine has been synthesized [Weinreb et al., J. Am. Chem. Soc. 97: 
2503 (1975); Semmelhack et al., J. Am. Chem. Soc. 97: 2507 (1975); and 
Weinreb and Semmelhack, Acc. Chem. Res. 8: 158 (1975)] thereby stimulating 
efforts to convert it to some of its active, naturally occurring esters by 
reaction with appropriate acid compounds. However, very unfavorable steric 
(and perhaps electronic) intereactions at the reaction sites of both the 
cephalotaxine and the acyl moiety preclude direct esterification 
[Mikolajczak et al., J. Pharm. Sci. 63: 1280 (1974)]. By means of 
complicated and indirect routes, cephalotaxine has been converted to the 
active esters, deoxyharringtonine [U.S. Pat. No. 3,959,312; Mikolajczak et 
al., Tetrahedron Lett. 283 (1974); and Li et al., Hua Hsueh Hsueh Pao 33: 
75 (1975)]; and harringtonine [Anonymous, K'o Hsueh T'ung Pao 20: 437 
(1975); Chem. Abstr. 84: 105859Z (1976)]. 
SUMMARY OF THE INVENTION 
We have now surprisingly found a group of synthetic acyl esters of 
cephalotaxine which show activity against leukemia in animals, and which 
can be prepared from cephalotaxine by ordinary and standard procedures 
because they are not subject to the severe steric requirements of the 
prior art compounds. These alkaloid compounds are characterized by the 
following structural formula: 
##STR1## 
where R is selected from the group consisting of 
##STR2## 
It is therefore an object of this invention to obtain from cephalotaxine 
synthetic esters which have activity against leukemia. 
Another object of the invention is to prepare these active cephalotaxine 
esters by ordinary and simple procedures. 
It is also an object of the invention to administer the novel alkaloid 
compounds to animals in order to cause remission of leukemia therein. 
Other objects and advantages of the invention will become readily apparent 
from the ensuing description.

DETAILED DESCRIPTION OF THE INVENTION 
Cephalotaxine is characterized by the following structural formula: 
##STR3## 
where R equals H. We have found that by substituting selected acyl 
groupings for the hydroxyl hydrogen, the chemotherapeutically inactive 
cephalotaxine can be converted to antileukemic alkaloids. It would appear 
from preliminary investigations that the activity of cephalotaxine esters 
is attributable primarily to the (-)-cephalotaxine enantiomer. This is the 
enantiomer isolated from natural sources. Cephalotaxine produced 
synthetically, such as by one of the procedures taught in the references 
discussed above, occurs as a racemic mixture. While it is preferred to use 
the (-)-isomer, either isolated from plants or separated from a synthetic 
(+)-mixture, it is understood that the (+)-mixture itself would be a 
suitable starting material. 
The acylating agents are preferably acid chlorides or anhydrides which are 
commercially available, or else readily obtainable by conventional 
modification of available precursor compounds, such as the appropriate 
acid, ester, or half ester. Such modifications are illustrated in the 
examples below. 
Acylation and recovery procedures for obtaining the instant cephalotaxyl 
esters do not in themselves constitute novelty within the instant 
invention. However, these have been illustrated in detail in the 
accompanying examples for each of the disclosed species. It is understood 
that certain obvious alterations and variations of these procedures, 
involving the reagents, proportions, solvent systems, and other reaction 
conditions and parameters may be made without altering the nature of the 
final products. For instance, we have found that methyl cephalotaxyl 
fumarate 
##STR4## 
may be prepared with either methyl hydrogen fumarate or maleic anhydride 
as the acylating agent, due to isomerization of the double bond in the 
anhydride. 
In the examples which follow, anhydrous reagents, solvents, and solutions 
of reactants were prepared by drying for at least 4 hours over type 3A or 
4A molecular sieve. Extracts of aqueous systems were routinely dried with 
MgSO.sub.4. All purification steps were monitored by thin layer 
chromatography, and in most instances by infrared analysis (IR). All the 
isolated cephalotaxine esters were amorphous solids, and each gave an IR, 
nuclear magnetic resonance, and mass spectrum (MS) consistent with its 
structure (see Table I for MS analysis). High-resolution mass spectral 
analyses were performed with a Nuclide 12-90G spectrometer. 
EXAMPLE 1 
Preparation of ethyl cephalotaxyl oxalate 
##STR5## 
(-)-Cephalotaxine (5.0 g.) and 1.34 g. of pyridine in 20 ml. of anhydrous 
CH.sub.2 Cl.sub.2 were cooled in an ice bath. Ethyl oxalyl chloride (2.38 
g.) in 10 ml. of CH.sub.2 Cl.sub.2 was added dropwise over 1 hour. 
Stirring at 0.degree. C. was continued 3 more hours and then at room 
temperature overnight. The mixture was poured into 100 ml. of pH 7.0 
phosphate buffer solution, and the solution was extracted with CH.sub.2 
Cl.sub.2. Evaporation of the CH.sub.2 Cl.sub.2 afforded pure ethyl 
cephalotaxyl oxalate. The yield was 71% based on the (-)-cephalotaxine 
used. 
EXAMPLE 2 
Preparation of methyl cephalotaxyl fumarate 
##STR6## 
Fumaric acid (40.0 g.) in 150 ml. of anhydrous benzene and 75 ml. of 
dioxane was treated with 18 ml. (25% excess over the amount needed to 
esterify one carboxyl group) of MeOH and 2 ml. of concentrated H.sub.2 
SO.sub.4. The mixture was refluxed under a Dean-Stark trap until no more 
water collected in the trap, about 4 hours. The solvent was concentrated 
to 25 ml., water was added, and the mixture was extracted with diethyl 
ether. After evaporation of ether, the residue was separated in 3.0-g. 
batches on a 2.5.times.35 cm. column of silica gel with 300 ml. of ethyl 
acetate:benzene (10:90) followed by 400 ml. of ethyl acetate:benzene 
(20:80). Yield of methyl hydrogen fumarate was 15%. 
Methyl hydrogen fumarate (3.22 g.) was treated overnight with stirring at 
room temperature with 10 ml. of oxalyl chloride. Excess oxalyl chloride 
was evaporated in vacuo, and the residue (the acid chloride) was dissolved 
in 10 ml. of CH.sub.2 Cl.sub.2. A solution of 3.1 g. of (-)-cephalotaxine 
and 3 ml. of pyridine in 10 ml. of CH.sub.2 Cl.sub.2 was added to the acid 
chloride slowly over 30 minutes., and the resulting solution stirred 
overnight. The mixture was poured into 75 ml. of 5% Na.sub.2 CO.sub.3 
solution and extracted with CH.sub.2 Cl.sub.2. After evaporation of 
CH.sub.2 Cl.sub.2, the residue was dissolved in diethyl ether:petroleum 
ether (75:25) and run through a 1.times.10 cm. neutral alumina (Woelm, 
grade III) column. The yield of methyl cephalotaxyl fumarate was 68% based 
on the (-)-cephalotaxine used. 
EXAMPLE 3 
Preparation of methyl cephalotaxyl itaconate 
##STR7## 
Itaconic acid (248 g.) was treated with 246 ml. of anhydrous methanol and 
4.0 ml. acetyl chloride at reflux for 20 minutes. Excess MeOH was 
evaporated in vacuo and the residue was crystallized by adding 200 ml. of 
benzene, followed by 300 ml. of petroleum ether (b.p. 
30.degree.-60.degree. C.) and chilling the solution to 0.degree. C. The 
crystals were recrystallized from benzene:petroleum ether (3:2) and gave a 
m.p. of 66.degree.-69.degree. C. The yield of methyl hydrogen itaconate 
was 42% (115 g.). 
Methyl hydrogen itaconate (3.17 g.) was dissolved in anhydrous ether and 
dried over type 4A molecular sieve. The ether solution was removed from 
the sieve and all solvent removed in vacuo. The residue was treated neat 
with 10 ml. of oxalyl chloride overnight at room temperature with stirring 
(magnetic). Excess oxalyl chloride was removed in vacuo and the acid 
chloride was dissolved in anhydrous CH.sub.2 Cl.sub.2 and cooled in an ice 
bath. To this cold solution of the acid chloride was added dropwise a 
solution of 3.15 g. of (-)-cephalotaxine and 4 ml. of pyridine in 10 ml. 
of CH.sub.2 Cl.sub.2. The mixture was allowed to warm to room temperature 
and was stirred overnight. 
This mixture was poured into 50 ml. of 5% Na.sub.2 CO.sub.3 solution, and 
the solution was extracted with CH.sub.2 Cl.sub.2. The crude product was 
dissolved in diethyl ether:CH.sub.2 Cl.sub.2 (2:1) and was run through a 
neutral alumina (Woelm, grade III) column of 1.times.10 cm. The yield of 
pure methyl cephalotaxyl itaconate was 83% based on the (-)-cephalotaxine 
used. 
EXAMPLE 4 
Preparation of cephalotaxyl trans,trans-sorbate 
##STR8## 
trans,trans-Sorbic acid (2.24 g.) was treated with oxalyl chloride for 2 
hours. Excess oxalyl chloride was evaporated in vacuo. The residue was 
dissolved in 10 ml. of CH.sub.2 Cl.sub.2 and cooled in an ice bath. A 
solution of 3.1 g. of (-)-cephalotaxine and 3 ml. of pyridine in CH.sub.2 
Cl.sub.2 (10 ml.) was added slowly (30 minutes) to the cold acid chloride 
solution and the mixture stirred at room temperature overnight. The 
mixture was poured into 75 ml. of 5% Na.sub.2 CO.sub.3 solution and 
extracted with CH.sub.2 Cl.sub.2. The crude product remaining after 
removal of CH.sub.2 Cl.sub.2 was purified by chromatography on a 
1.times.10 cm. neutral alumina (Woelm, grade III) column with diethyl 
ether. This was followed by chromatography on a silica gel column, 
2.5.times.35 cm., with CH.sub.2 Cl.sub.2. The yield of cephalotaxyl 
trans,trans-sorbate was 36% based on (-)-cephalotaxine used. 
EXAMPLE 5 
Preparation of cephalotaxyl L-mandelate, trichloroethylcarbonate ester 
##STR9## 
L-Mandelic acid (10 g.), 6.91 g. of benzyl alcohol, and 75 mg. of 
p-toluenesulfonic acid in 70 ml. of benzene was refluxed under a 
Dean-Stark trap for a total of 28 hours on 4 consecutive days. The crude 
benzyl ester was obtained by successively concentrating the liquor and 
cooling it to about 10.degree. C. a number of times. The fractions melting 
between 97.degree. C. and 105.degree. C. were combined, dissolved in 
CHCl.sub.3 and washed twice with 15-ml. portions of 5% aqueous NaHCO.sub.3 
solution. After evaporation of the CHCl.sub.3, the product was 
recrystallized from benzene to give benzyl L-mandelate, m.p. 
104.degree.-106.degree. C., yield 42%. 
6.5 Grams of benzyl L-mandelate was dissolved in CH.sub.2 Cl.sub.2 (25 ml.) 
and was treated dropwise with 5.90 g. of trichloroethoxycarbonyl chloride 
in 15 ml. of CH.sub.2 Cl.sub.2. The mixture was stirred overnight at room 
temperature and then was poured into 75 ml. of 5% Na.sub.2 CO.sub.3 
solution and extracted with CH.sub.2 Cl.sub.2. The crude product 
##STR10## 
was purified by chromatography of 2-g. batches on a 2.5.times.35 cm. 
silica gel column with 200 ml. of benzene:petroleum ether, 25:75, 200 ml. 
of 35:65 and enough of 45:55 to completely elute the desired ester. The 
ester was then crystallized from diethyl ether at 0.degree. C. to give a 
68% yield of the trichloroethylcarbonate ester of benzyl L-mandelate, m.p. 
96.degree.-97.degree. C. Hydrogenolysis of this ester (to remove the 
benzyl ester grouping) was done by hydrogenating 2-g. batches dissolved in 
10 ml. of tetrahydrofuran with 200 mg. of 10% Pd/C until one molar 
equivalent of H.sub.2 was consumed. This gave a 97% yield of 
##STR11## 
4.1 Grams of above acid was treated at reflux for 1 hour with 15 ml. of 
oxalyl chloride and the excess reagent removed in vacuo. The residue was 
dissolved in CH.sub.2 Cl.sub.2 (20 ml.) and cooled in an ice bath. A 
solution of 2.52 g. of (-)-cephalotaxine and 2 ml. of pyridine in 20 ml. 
of CH.sub.2 Cl.sub.2 was added slowly. The mixture was allowed to warm to 
room temperature and was then stirred overnight at room temperature. The 
mixture was poured into 75 ml. of 5% Na.sub.2 CO.sub.3 solution which was 
then extracted with CH.sub.2 Cl.sub.2. After evaporation of the CH.sub.2 
Cl.sub.2, the crude cephalotaxyl L-mandelate, trichloroethylcarbonate 
ester was run through a 1.times.10 cm. neutral alumina (Woelm, grade III) 
column in 2-g. batches with diethyl ether. The yield was 91% based on 
(-)-cephalotaxine used. 
EXAMPLE 6 
Preparation of cephalotaxyl trichloroethylcarbonate 
##STR12## 
(-)-Cephalotaxine (2.40 g.) and 2 ml. of pyridine in 20 ml. of CH.sub.2 
Cl.sub.2 (anhydrous) was cooled in an ice bath. Then 1.80 g. of 
trichloroethoxycarbonyl chloride in 5 ml. of CH.sub.2 Cl.sub.2 was added 
dropwise with stirring at 0.degree. C. Stirring was continued at 0.degree. 
C. for 3 more hours and then at room temperature overnight. The mixture 
was poured into 100 ml. of pH 7.0 phosphate buffer solution and the 
solution was extracted with CH.sub.2 Cl.sub.2. Evaporation of the CH.sub.2 
Cl.sub.2 yielded pure cephalotaxyl trichloroethylcarbonate. The yield was 
96% based on cephalotaxine used. 
Table I 
______________________________________ 
Isolated 
yield, High resol. MS 
Example %.sup.a Formula M.sup.+ calc. 
M.sup.+ obsd. 
______________________________________ 
1 71 C.sub.22 H.sub.25 NO.sub.7 
415.163 
415.162 
2 68 C.sub.23 H.sub.25 NO.sub.7 
427.163 
427.162 
3 83 C.sub.24 N.sub.27 NO.sub.7 
441.179 
441.179 
4 36 C.sub.24 H.sub.27 NO.sub.5 
409.189 
409.188 
5 91 C.sub.29 H.sub.28 NO.sub.8 Cl.sub.3 
.sup.b .sup.b 
6 96 C.sub.21 H.sub.22 NO.sub.6 Cl.sub.3 
491.048 
491.047 
______________________________________ 
.sup.a Based on cephalotaxine; not optimized. 
.sup.b M.sup.+ -191 (--C.sub.3 H.sub.2 O.sub.3 Cl.sub.3 group); calc. 
432.179, obsd. 432.181. 
Chemotherapeutic acitivity of each of the compounds prepared in Example 1-6 
was determined in mice which were implanted with lymphocytic leukemia 
cells of the strain P388, according to the National Cancer Institute 
Protocols [Geran et al., Cancer Chemother. Rep., Part 3, 3:9 (1972)]. 
Starting 24 hours after the tumor implantation, previously determined 
dosages of each compound were injected intraperitoneally once a day for 9 
days. The results are shown in Table II. Survival time of treated leukemic 
mice is compared to that of untreated leukemic mice (T/C.times.100). A T/C 
value of 100% indicates no activity. A T/C value greater than 100% means 
that the treated mice are surviving longer than the control mice. A 
compound giving a T/C value greater than or equal to 125% is indicative of 
activity as defined by the NCI Protocols, supra. 
Table II 
__________________________________________________________________________ 
Biological Test Data for Activity of Cephalotaxine 
Esters Against P388 Lymphocytic Leukemia in Mice 
Animal weight 
Vehi- 
Dose difference 
Example 
R Group cle.sup.a 
mg./kg./inj..sup.b 
T-C T/C.sup.c, 
__________________________________________________________________________ 
% 
1A D 20 0.1 135 
##STR13## D 20 -0.2 211 
C D 13 -0.8 154 
D T 20 -0.1 129 
2A 
##STR14## B 80 -0.7 145 
B B 40 -0.9 134 
C B 20 -1.0 125 
D B 10 -1.2 136 
E D 4.4 -1.5 147 
F D 1.9 0.5 134 
3A D 365 -3.0 198.sup.d 
B 
##STR15## D 240 -1.4 169 
C D 160 -0.1 183 
D D 160 -1.1 167 
E D 80 -0.1 173 
F D 40 -1.3 135 
4A D 80 0.9 150 
B 
##STR16## D 40 -1.8 125 
C D 20 0.9 130 
5A A 320 -2.3 136 
B A 160 1.2 154 
C 
##STR17## A 80 -1.0 138 
6A D 320 -1.0 172 
B 
##STR18## D 160 -0.9 162 
C D 160 -0.3 155 
D D 80 -0.9 183 
E D 80 -1.3 160 
F D 40 -0.4 140 
G D 40 0.9 183 
H D 20 -2.7 128 
I D 20 -1.5 160 
J D 20 -1.0 195 
K D 13 -0.5 138 
L D 8.8 -0.3 170 
__________________________________________________________________________ 
.sup.a A = saline, B = water + alcohol + acetone, C = water + acetone, D 
water + alcohol, T = saline + Tween 80. 
.sup.b One intraperitoneal injection per day for 9 days; DBA/2 mice. 
.sup.c T/C = meansurvival time of test animals/mean survival time of 
control animals; 125% or above considered active. Unaccountable variation 
in T/C values among duplicate tests were sometimes observed; these may 
possibly be due to solubility properties of the esters in the vehicles 
used. 
.sup.d One 30-day cure was reported. 
Dose levels other than those indicated in Table II were tested but gave 
either toxic or inactive responses. The terms "effective amount" and 
"effective dose" as referring to the treatment of animals is defined 
herein to mean those quantities of cephalotaxine ester which will cause 
remission of leukemia in the animal to which it is administered, without 
imparting a toxic response. The effective amount will vary with the 
particular esters, the injection vehicle, the strain of leukemia, and 
other related factors. Generally for the instant esters, an effective dose 
will be in the range of about 1.5-380 mg./kg. of body weight/day. The 
preferred dose range for a given ester is defined by the lowest and 
highest dose shown in Table II for that ester. 
The activities of these novel cephalotaxyl esters are not predictable, and 
in some cases are totally unexpected from structure-activity correlations 
based upon the naturally occurring active esters and certain other 
synthetic esters. For instance, all of the naturally occurring active 
esters, such as harringtonine, homoharringtonine, and deoxyharringtonine, 
contain a dicarboxylic acyl group and a tertiary hydroxy group alpha to 
the carboxyl esterified with the cephalotaxine molecule. The hydroxyl 
group is notably absent in all of our compounds, and the 
trans,trans-sorbate of Example 4 and the trichloroethylcarbonate of 
Example 6 do not exhibit the dicarboxylic acid moiety. Eighteen other acyl 
esters, synthesized at approximately the same time as the subject acyl 
esters, were inactive. For example, the cephalotaxyl ester of mandelic 
acid, without the trichloroethylcarbonate moiety appearing in the compound 
of Example 5, had no activity. In addition, toxicity observed during 
administration of the mandelic acid ester at doses above 80 
mg./kg./injection is not present with the trichloroethylcarbonate mandelic 
acid ester at doses up to 320 mg./kg./injection. On the other hand, the 
trichloroethylcarbonate of cephalotaxyl .alpha.-hydroxy-.alpha.-methyl 
butyrate is inactive. Likewise, the ethyl carbonate, benzyl carbonate, and 
chloro acetate esters of cephalotaxine are inactive. The same 
unpredictability occurs with compounds related to the itaconate ester of 
Example 3 by virtue of having .alpha.,.beta.-unsaturation or 
.alpha.,.beta.-unsaturation coupled with a second carboxyl group. Thus, 
the methyl 2-methyl-2-butendioate, methyl muconate, acrylate, 
methacrylate, cinnamate, and p-nitrocinnamate esters of cephalotaxine are 
all inactive. 
It is understood that the foregoing detailed description is given merely by 
way of illustration and that modification and variations may be made 
therein without departing from the spirit and scope of the invention.