10-substituted ether derivatives of dihydroartemisinin, process for their preparation and their use as antiprotozoal agents

10-substituted ether derivatives of 3.alpha., 12.alpha.-Epoxy-3,4,5 5a.alpha., 6,7,8,8a.alpha.,9,10,12.beta., 12a-dodecahydro-10-hydroxy-3.beta.,6.alpha.,9.beta.-trimethylpyano-(4,3-j) (1,2) benzodioxepin, also known as Dihydroartemisinin or Dihydroquinghaosu (DHQ), pharmaceutically acceptable salts thereof, processes for their preparation and their use as chemotherapeutics against protozoal infections are provided.

This invention relates to 10-substituted ether derivatives of 3.alpha., 
12.alpha.-Epoxy-3,4,5,5a.alpha.,6,7,8,8a-dodecahydro-10-hydroxy-3.beta.,6. 
alpha.,9-62 -trimethylpyano-(4,3-j) (1,2) benzodioxepin, also known as 
Dihydroartemisinin or Dihydroquinghaosu (DHQ) and pharmaceutically 
acceptable salts thereof, processes for their preparation and their use as 
chemotherapeutics against protozoal infections. 
Artemisinin and its ethers are reviewed in Medicinal Research Reviews, Vol. 
7, No. 1, 29-52 (1987) and J. of Medicinal Chemistry, 31, 645 (1988). 
Although compounds of the prior art have been reported to possess 
antimalarial activity, they have invariably had to be administered 
parenterally for activity to be demonstrated at sufficiently low doses. 
Recrudescence was also observed at a rate of 10-30% in a month after 
administration with such parenteral forms. The use of these compounds is 
thus claimed as useful only for cerebral malaria. Surprisingly, it has now 
been determined that the novel derivatives of artemisinin described herein 
are characterized by two special qualities: 
(a) they possess potent antimalarial activity when administered orally to 
animals, becoming thus potential agents for all forms of malaria resulting 
from susceptible and resistant form of pathogenic Plasmodium strains and 
(b) they possess antiprotozoal activity in general and in particular 
antiamoebic activity against the protozoa Eimera tenella, hitherto not 
known for artemisinin or any of its known derivatives. 
Thus, the instant invention is directed to 10-substituted ether derivatives 
of dihydroquinghaosu as represented by the general formula I: 
##STR1## 
wherein, R.sub.1 is hydrogen or a lower alkyl, 
R.sub.2 is hydrogen, a lower alkyl or an alkoxy alkyl, and 
R.sub.3 is an acyl, alkoxy alkyl, alkynyl, heterocycle or heterocyclic 
alkyl, 
and pharmaceutically acceptable salts thereof. 
In the formulae presented herein, the various substituents are illustrated 
as joined to a pyrano(4,3-j)(1,2)benzodioxepin nucleus by one of two 
notations a solid line (-) indicating a substituent which is in the 
.beta.-orientation (i.e. above the plane of the molecule), and a broken 
line (- - -) indicating a substituent which is in the .alpha.-orientation 
(i.e. below the plane of the molecule). The formula have all been drawn to 
show the compounds in their absolute configuration. In as much as the 
starting materials having a pyrano(4,3-j)(1,2)benzodioxepin nucleus are 
naturally occurring or are derived from naturally occurring materials, 
they as well as the final products, have a pyrano (4,3-j) 
(1,2)benzodioxepin nucleus in the single absolute configuration depicted 
herein. The processes of the present invention, however, are intended to 
apply as well to the synthesis of pyrano)4,3-j) (1,2)benzodioxepines of 
the racemic series. 
In addition to the optical centers of a pyrano(4,3-j) (1,2)benzodioxepin 
nucleus, the substituents thereon may also contain chiral centers 
contributing the optical properties of the compounds of the present 
invention and providing a means for the resolution thereof by conventional 
methods, for example, by the use of optically active acids. A wavy line 
(.sup..about.) indicates that the substituents can either be in the 
.alpha.-orientation or .beta.-orientation. The present invention 
comprehends all optical isomers and racemic forms of the compounds of the 
present invention where such compounds have chiral centers in addition to 
those of the pyrano (4,3-j) (1,2)benzodioxepin nucleus. 
The term alkyl stands for C.sub.1 -C.sub.8 straight or branched chain 
carbon compounds such as methyl, ethyl, propyl, butyl, isopropyl, and 
t-butyl. The term alkynyl stands for straight or branched chain carbon 
compounds containing one or more triple bonds, and may in addition contain 
a double bond. Examples of alkynyl groups are 3-methyl-1-pentoynyl, 
1-butynyl, 3-methyl-1-butynyl, 2-butynyl-1-hydroxymethyl. 
The term heterocycle stands for piperazino, morpholino, piperidino, 
pyyrolidino, phthalimido, isoxazolyl, optionally substituted at one or 
more places by alkyl, alkoxy, hydroxy, halogen, or aryl groups optionally 
substituted by a carboxyl group. 
Preferred compounds of the invention are listed in Table 1, and their 
biological activity in Table 2. 
TABLE 1 
__________________________________________________________________________ 
##STR2## 
m.p. solvent for 
Yield 
No. 
R.sub.1 
R.sub.2 R.sub.3 .degree.C. 
crystallisation 
(%) 
__________________________________________________________________________ 
1. H CH.sub.3 COCH.sub.3 (.beta.) 
OIL -- 33 
2. H CH.sub.2 OCH(CH.sub.3).sub.2 
CH.sub.2 OCH(CH.sub.3).sub.2 
(.alpha.) 
OIL -- 39 
3. H CH.sub.3 C CH (.beta.) 
91-93 
Pet. ether 
35 
4. CH.sub.3 
CH.sub.3 C CH (.beta.) 
58-60 
Pet. ether 
57 
5. CH.sub.3 
C.sub.2 H.sub.5 
C CH (.beta.) 
OIL Pet. ether 
62 
6. H H 
##STR3## (.beta.) 
150-152 
Chloroform/ Diisopropyl 
32her 
7. H H 
##STR4## (.alpha. + .beta.) 
150-152 
Methylene chloride/ Pet. 
55her 
8. H H 
##STR5## (.beta.) 
106 -- 53 
9. H H 
##STR6## (.beta.) 
OIL -- 63 
__________________________________________________________________________ 
Particularly preferred compounds of the invention are: 
3.alpha.,12-60 
-Epoxy-3,4,5,5a.alpha.,6,7,8a.alpha.,9,10,12.beta.,12a-dodecahydro-10-.bet 
a.[1-acetylethoxy]-3.beta.,6.alpha.,9.beta.-trimethylpyrano(4,3-j) 
(1,2)benzodioxepin. 
3.alpha.,12.alpha.-Epoxy-3,4,5,5a.alpha.,6,7,8,8a.alpha.,9,10,12.beta.12a-d 
odecahydro-10.alpha.-[1,3-bis(isopropyoxy) 
propyl-2]oxy-3.beta.,6.alpha.9.beta.-trimethylpyrano 
(4,3-j)(1,2)benzodioxepin. 
3.alpha.,12.alpha.-Epoxy-3,4,5,5a.alpha.6,7,8,8a,9,10,12.beta.,12a-dodecahy 
dro-10.beta.-(3-butynyl-2-oxy)-3 
-butynyl-2-oxy)-3.beta.6.alpha.,9.beta.-trimethylpyrano(4,3-j) 
(1,2)benzodioxepin. 
3.alpha.,12.alpha.-Epoxy-3,4,5,5a.alpha.,6,7,8,8a.alpha.,9,10,12.beta.,12.a 
lpha.-dodecahydro-10.beta.-(2-methyl-3-butynyl-2-oxy)-3.beta.,6.alpha.,9.be 
ta.-trimethylpyrano (4,3-j)(1,2)benzodioxepin. 
3.alpha.,12.alpha.-Epoxy-3,4,5,5a.alpha.,6,7,8,8a.alpha.,9,10,12.beta.12ad 
odecahydro-10.beta.-(2-ethyl-3-butynyl-2-oxy)-3.beta.,6.alpha.,9.beta.-trim 
ethylpyrano (4,3-j)1,2)benzodioxepin. 
3.alpha.,12.alpha.-Epoxy-3,4,5,5a.alpha.,6,7,8,8a.alpha.,9,10,12.beta.,12a- 
dodecahydro-10.beta.-[2-(4-chlorophthalimido)-ethoxy]-3.beta.,6.alpha.9.bet 
a.-trimethylpyrano (4,3-j)1,2)benzodioxepin. 
5-[3.alpha.,12.alpha.-Epoxy-3,4,5,5a.alpha.,6,7,8,8a.alpha.,9,10,12.beta.,1 
2a-dodecahydro-3.beta.6.alpha.,9.beta.-trimethylpyrano 
(4,3-j)(1,2)benzodioxepin-10-oxy] methyl-3-(4-carboxyphenyl)isoxazole. 
5-[3.alpha.,12.alpha.-Epoxy-3,4,5,5a.alpha.,6,7,8,8a.alpha.,9,10,12.beta.,1 
2a-dodecahydro-3.beta.,6.alpha.9.beta.-trimethylpyrano(4,3-j)1,2)benzodioxe 
pin-10-.beta.-yl]oxymethyl-3-chloroisoxazole. 
5-[3.alpha.,12.alpha.-Epoxy-3,4,5,5a.alpha.,6,7,8,8a.alpha.,9,10,12.beta.,1 
2a-dodecahydro-3.beta.,6.alpha.,962 -trimethylpyrano 
(4,3-j)(1,2)benzodioxepin-10.beta.-yl]oxymethyl-3bromoisoxazole. 
The process for the preparation of compounds of the invention comprises 
treating compounds of the formula II with compounds of the formula III, 
wherein X and R have the same meaning as defined above, preferably in the 
presence of BF.sub.3 -etherate at a temperature of 0.degree. -10.degree. 
C. under stirring for half an our to six hours. 
##STR7## 
The reaction is preferably carried out in organic solvents such as benzene 
and chloroform. For completion of the reaction, the reaction mixture may 
be heated to the boiling point of the solvent used. The compounds of the 
invention are isolated by diluting the reaction mixture with water, 
separating the organic layer, washing it with water, concentrating the 
organic layer and purifying it by flash column chromatography using a 
silica gel column. Compounds of the formula II are obtained by the 
procedure reported in the literature (Acta. Chim. Sinica. 37, 129 (1979)). 
The compounds of formula I may be administered in different manners, 
preferably perorally or parenterally, in doses ranging from 2.5 to 100 
mg/kg of body weight. As antimalarial drugs, dosage unit forms such as 
dragees or capsules for oral administration or solutions and suspensions 
respectively for injections, each containing 100 to 400 mg of active 
substance, are preferred. Such dosage units are administered once to three 
times daily depending upon the condition of the patient. 
For oral administration, there may be used in particular tablets, dragees, 
capsules, powders or granules which contain the active substance together 
with the usual carriers, adjuvants and/or excipients such as starch, 
cellulose powder, talcum, magnesium stearate, sugar, gelatin, calcium, 
carbonate, finely divided silicic acid, carboxymethyl cellulose or similar 
substances. 
For parenteral administration, in particular for intramuscular injections, 
there may be used sterile suspensions, for example, oily suspensions 
prepared with the use of sesame oil, vegetable oil, castor oil or 
synthetic triglycerides, optionally with the simultaneous use of surface 
active substances such as sorbitan fatty acid esters. Furthermore, there 
may also be used aqueous suspensions prepared, for example, with the use 
of ethoxylated sorbitan fatty acid esters, optionally with the addition of 
thickeners such as polyethylene glycol or carboxymethyl cellulose. 
BIOLOGICAL EVALUATION METHODOLOGY 
The evaluation of blood-schizontocidal activity "28-day test" described by 
Rather and Fink [W. H. O. Report on the Scientific Working Group on the 
Chemotherapy in Malaria, TDR/Chemal 3-rd Review, 85.3, Geneva, Jun. 3-5, 
1985 and references contained therein] was followed. 
Mice: All experiments were carried out in random bred male and female Swiss 
mice obtained from the Hoechst India Limited breeding house at Mulund, 
Bombay. The animals were free from Eperythrozoon coccoides. The animals 
received food pellets and water ad lib and were kept at 22-25.degree. C. 
room temperature. 
Parasite: Plasmodium berghei K-173 strain drug-sensitive and P. berghei 
(NS) moderately resistant to chloroquine were obtained from London School 
of Hygiene and Tropical Medicines. The strains produce lethal infection at 
1.times.10.sup.7 parasitized red blood cells per mouse when inoculated 
intraperitoneally. 
Administration of compounds: The compounds were administered orally or 
subcutaneously as per methods described by Rather and Fink [W. H. O. 
Report on the Scientific Working Group on the Chemotherapy in Malaria, 
TDR/Chemical 3rd Review, 85.3, Geneva, Jun. 3-5, 1985 and references 
contained therein]. 
compounds of the invention were homogenized in double refined Kardi oil or 
peanut oil or corn oil with one or two drops of polyoxyethylenesorbitan 
monooleate ((R)Tween-80, Sigma Chanicallo, England) and such suspensions 
were used for subcutaneous inoculation in mice. The drugs were 
administered for 5 days. 1st dosing was done within 2 hours of infection 
(D+0), followed by D+1, D+2, D+3 and D+4. 
Observation of the treated mice: The blood smears were prepared at 
different intervals from D+4 and continued up to D+28. Blood smears were 
drawn from the terminal end of the trail and stained in Giemsa. Mice which 
were free from P. berghei on D+28 were considered as completely cured. 
Results obtained with compounds of Formula I of the invention are listed in 
Table 2. 
TABLE 2 
__________________________________________________________________________ 
Antimalarial Activity 
##STR8## 
ACTIVITY 
No. of 
No. of 
ROUTE OF 
animals 
animals 
ADMINIS- 
cured/ 
cured/ 
DOSE 
TRATION .times. 
treated 
treated 
R.sub.1 
R.sub.2 R.sub.3 mg/kg 
5 D + 7 
D + 28 
__________________________________________________________________________ 
H CH.sub.3 COCH.sub.3 (.beta.) 
5.0 s.c. 6/6 6/6 
H CH.sub.2 OCH(CH.sub.3).sub.2 
CH.sub.2 OCH(CH.sub.3).sub.2 
(.alpha.) 
5.0 s.c. 6/6 4/6 
H CH.sub.3 C CH (.beta.) 
5.0 s.c. 6/6 6/6 
CH.sub.3 
CH.sub.3 C CH (.beta.) 
5.0 s.c. 6/6 6/6 
CH.sub. 3 
C.sub.2 H.sub.5 
C CH (.beta.) 
5.0 s.c. 6/6 4/6 
(.beta.) 
10.0 
s.c. 6/6 4/6 
H H 
##STR9## (.beta.) 
10.0 
s.c. 6/6 4/6 
H H 
##STR10## (.alpha. + .beta.) 
50.0 
s.c. 8/8 8/8 
H H 
##STR11## (.beta.) 
5.0 s.c. 5/5 5/5 
H H 
##STR12## (.beta.) 
5.0 s.c. 6/6 5/6 
Artemisinin 100.0 
p.o. 4/10 
0/10 
50.0 
p.o. 0/6 0/6 
20.0 
s.c. 4/6 0/6 
10.0 
s.c. 0/6 0/6 
__________________________________________________________________________ 
ANTIAMOEBIC EVALUATION METHODOLOGY 
In vitro tests were carried out in Jones medium using a polyaxenic culture, 
the strain of Entamoeba histolytica was BY 80 isolated in Bombay in 
Hoechst Pharmaceuticals in 1980. The inoculum used was 3.times.10.sup.4 
amoebae per tube. The derivatives under test were dissolved in DMSO and 
serial dilution tests were carried out in the same medium. Microscopical 
examination was made after 48 hours of incubation at 37.degree. C. The 
minimum inhibitory concentration (MIC) was determined by observing the the 
amoebae under microscope for physical damage (lysis or immobility) of the 
trophozoites. Controls with and without 0.2% DSMO were run to compare the 
growth of amoebae with the treated group. Meronidazole was used as a 
standard drug. 
As a typical representative example, Compound Nos. 5 and 7 in Table 1 when 
tested as described above showed antiamoebic activity at a MIC 
concentration of 200 ug/ml. 
The following examples illustrate the invention but do not limit the scope 
of the invention.

EXAMPLE 1 
3.alpha.,12.alpha.-Epoxy-3,4,5,5a.alpha.,6,7,8,8a.alpha.,9,10,12.beta.,12a- 
dodecahydro-10.beta.-(1-acetylethoxy) 
-3.beta.,6.alpha.,9.beta.-trimethylpyrano)4,3-j) (1-2)benodioxepin 
To a solution of dihydroquinghaosu (0.10 g, 0.0004 m) and 
3-hydroxy-2-butanone (0.2 g, 0.002 m) in 15 ml chloroform was added 
BF.sub.3 etherate (3 drops) at 0.degree. C., and after the addition, the 
mixture was slowly brought to room temperature and stirred for 6 h. The 
reaction mixture was then diluted with water and the organic layer was 
separated, washed thoroughly with water, dried over anhydrous sodium 
sulphate and concentrated to obtain the residue, as an oil which was 
purified by flash column chromatography over silica gel using petroleum 
ether: ethyl acetate (8.7:1.3) as eluants. Concentration of the first few 
fractions gave the pure 
3.alpha.,12.alpha.-Epoxy-3,4,5,5a.alpha.,6,7,8,8a.alpha.,9,10,12.beta.12a- 
dodecahydro-10.beta.-(1-acetylethoxy)-3.beta., 6.alpha., 
9.beta.-trimethyl-pyrano[4,3-j] [1,2]benzodioxepin as an oil in 33% yield. 
EXAMPLE 2 
3.alpha.,12.beta.,Epoxy-3,4,5,5a.alpha.,6,7,8,8a.alpha.,9,10,12.beta.,12a-d 
oecahydro-10.alpha.-[1,2-bis(isopropoxy) 
-propyl-2]oxy-3.beta.,6.alpha.,9.beta.-trimethylpyrano[4,3j][1,2]benzodiox 
epin. 
To a solution of dihydroquinghaosu (0.05 g 0.002 m) and 
1,3-bis(isopropoxy)propan-2-ol (0.05 g, 0.0003 m) in 15 ml chloroform was 
added BF.sub.3 etherate (2 drops at 0.degree. C., and after the addition, 
the mixture was stirred in an ice bath for an additional 15 minutes. The 
reaction mixture was then diluted with water, dried over anhydrous sodium 
sulphate and concentrated to obtain the residue, as an oil which was 
purified by flash column chromatigraphy over silica gel using petroleum 
ether: ethyl acetate (8.5:1.5) as eluants to give 
3.alpha.,12.alpha.-Epoxy-3,4,5,5a.alpha., 
6,7,8,8a.alpha.,9,10.alpha.,12.beta.,12a-dodecahydro-10.alpha.-[1,3-bis(is 
opropoxy)-propyl-2]oxy-3.beta.6.alpha., 9.beta.-trimethyl pyrano 
[4,3-j][1,2]benzodoxepin as an oil in 39% yield. 
Following the procedure described in the above example, the compounds 
reported in Table 1 were prepared similarly using an appropriate 
nucleophile in place of 1,3-bis(isopropoxy)propan-2-ol. 
EXAMPLE 3 
5-[(3.alpha.,12.alpha.-Epoxy-3,4,5,5a.alpha.,6,7,8,8a.alpha.,9.10,12.beta., 
12a-dodecahydro-3.beta.,6.alpha., 
9.beta.-trimethylpyrano(4,3-j)(1,2)-benzodioxepin)-10-oxymethyl]-3-(4-carb 
oxyl phenyl) isoxazole. 
The solution of 
5-[(3.alpha.,12.alpha.-Epoxy-3,4,5,5a.alpha.,6,7,8,8a.alpha.,9, 
10,12.beta.,12a-dodecahydro-3.beta.,6.alpha.,9.beta.-trimethylpyrano[4,3-j 
][1, 2]benzodioxepin)-10-oxymethyl]-3-[4-carboethoxyphenyl]isoxazole (1.150 
g, 1.0003 m) in 16 ml 2.5% methanolic potassium hydroxide was allowed to 
stand for a day. The reaction mixture was concentrated, and the residue 
was dissolved in water. The aqueous solution was then acidified with 
acetic acid, and the separated solid was filtered to obtain 
5-[3.alpha.,12.alpha.-epoxy-3,4,5a.alpha.,6,7,8, 
8a.alpha.,9,10,12.beta.,12a-dodecahydro-3.beta.,6.alpha.,9.beta.-trimethyl 
pyrano[4,3-j][1,2]benzodioxepin)-10-oxymethyl]-3-(4-carboxyphenyl)isoxazole 
which was recrystallized from methylene chloride/petroleum ether, m.p 
150-152.degree. C. in 55% yield.