Endothelin receptor antagonists

The present invention relates to dihydroisoindole compounds, pharmaceutical compositions containing these compounds, and their use as endothelin receptor antagonists.

This application is a 371 of PCT/US95/07193 filed Jun. 6, 1995. 
FIELD OF THE INVENTION 
The present invention relates to novel dihydroisoindole derivatives, 
pharmaceutical compositions containing these compounds and their use as 
endothelin receptor antagonists. 
Endothelin (ET) is a highly potent vasoconstrictor peptide synthesized and 
released by the vascular endothelium. Endothelin exists as three isoforms, 
ET-1, ET-2 and ET-3. Unless otherwise stated "endothelin" shall mean any 
or all of the isoforms of endothelin!. Endothelin has profound effects on 
the cardiovascular system, and in particular, the coronary, renal and 
cerebral circulation. Elevated or abnormal release of endothelin is 
associated with smooth muscle contraction which is involved in the 
pathogenesis of cardiovascular, cerebrovascular, respiratory and renal 
pathophysiology. Elevated levels of endothelin have been reported in 
plasma from patients with essential hypertension, acute myocardial 
infarction, subarachnoid hemorrhage, atherosclerosis, and patients with 
uraemia undergoing dialysis. 
In vivo, endothelin has pronounced effects on blood pressure and cardiac 
output. An intravenous bolus injection of ET (0.1 to 3 nmol/kg) in rats 
causes a transient, dose-related depressor response (lasting 0.5 to 2 
minutes) followed by a sustained, dose-dependent rise in arterial blood 
pressure which can remain elevated for 2 to 3 hours following dosing. 
Doses above 3 nmol/kg in a rat often prove fatal. 
Endothelin appears to produce a preferential effect in the renal vascular 
bed. It produces a marked, long-lasting decrease in renal blood flow, 
accompanied by a significant decrease in GFR, urine volume, urinary sodium 
and potassium excretion. Endothelin produces a sustained antinatriuretic 
effect, despite significant elevations in atrial natriuretic peptide. 
Endothelin also stimulates plasma renin activity. These findings suggest 
that ET is involved in the regulation of renal function and is involved in 
a variety of renal disorders including acute renal failure, cyclosporine 
nephrotoxicity, radio contrast induced renal failure and chronic renal 
failure. 
Studies have shown that in vivo, the cerebral vasculature is highly 
sensitive to both the vasodilator and vasoconstrictor effects of 
endothelin. Therefore, ET may be an important mediator of cerebral 
vasospasm, a frequent and often fatal consequence of subarachnoid 
hemorrhage. 
ET also exhibits direct central nervous system effects such as severe apnea 
and ischemic lesions which suggests that ET may contribute to the 
development of cerebral infarcts and neuronal death. 
ET has also been implicated in myocardial ischemia (Nichols et al. Br. J. 
Pharm. 99:597-601, 1989 and Clozel and Clozel, Circ. Res., 65:1193-1200, 
1989) coronary vasospasm (Fukuda et al., Eur. J. Pharm. 165:301-304, 1989 
and Luscher, Circ. 83:701, 1991) heart failure, proliferation of vascular 
smooth muscle cells, (Takagi, Biochem & Biophys. Res. Commun.; 
168:537-543, 1990, Bobek et al., Am. J. Physiol. 258:408-C415, 1990) and 
atherosclerosis, (Nakaki et al., Biochem. & Biophys. Res. Commun. 
158:880-881, 1989, and Lerman et al., New Eng. J. of Med. 325:997-1001, 
1991). Increased levels of endothelin have been shown after coronary 
balloon angioplasty (Kadel et al., No. 2491 Circ. 82:627, 1990). 
Further, endothelin has been found to be a potent constrictor of isolated 
mammalian airway tissue including human bronchus (Uchida et al., Eur J. of 
Pharm. 154:227-228 1988, LaGente, Clin. Exp. Allergy 20:343-348, 1990; and 
Springall et al., Lancet, 337:697-701, 1991). Endothelin may play a role 
in the pathogenesis of interstitial pulmonary fibrosis and associated 
pulmonary hypertension, Giard et al., Third International Conference on 
Endothelin, 1993, p. 34 and ARDS (Adult Respiratory Distress Syndrome), 
Sanai et al., Supra, p. 112. 
Endothelin has been associated with the induction of hemorrhagic and 
necrotic damage in the gastric mucosa (Whittle et al., Br. J. Pharm. 
95:1011-1013, 1988); Raynaud's phenomenon, Cinniniello et al., Lancet 
337:114-115, 1991); Crohn's Disease and ulcerative colitis, Munch et al., 
Lancet, Vol. 339, p. 381; Migraine (Edmeads, Headache, Feb. 1991 p 127); 
Sepsis (Weitzberg et al., Circ. Shock 33:222-227, 1991; Pittet et al., 
Ann. Surg. 213:262-264, 1991), Cyclosporin-induced renal failure or 
hypertension (Eur. J. Pharmacol., 180:191-192, 1990, Kidney Int, 
37:1487-1491, 1990) and endotoxin shock and other endotoxin induced 
diseases (Biochem, Biophys. Res. Commun., 161:1220-1227, 1989, Acta 
Physiol. Scand. 137:317-318, 1989) and inflammatory skin diseases. (Clin 
Res. 41:451 and 484, 1993) and macular degeneration. 
Endothelin has also been implicated in preclampsia of pregnancy. Clark et 
al., Am. J. Obstet. Gynecol. March 1992, p. 962-968; Kamor et al., N. Eng. 
J. of Med., Nov. 22, 1990, p. 1486-1487: Dekker et al., Eur J. Ob. and 
Gyn. and Rep. Bio. 40 (1991) 215-220; Schiff et al., Am. J. Obstet. 
Gynecol. Feb. 1992, p. 624-628; diabetes mellitus, Takahashi et al., 
Diabetologia (1990) 33:306-310; and acute vascular rejection following 
kidney transplant, Watschinger et al., Transplantation Vol. 52, No. 4, pp. 
743-746. 
Endothelin stimulates both bone resorption and anabolism and may have a 
role in the coupling of bone remodeling. Tatrai et al. Endocrinology, Vol. 
131, p. 603-607. 
Endothelin has been reported to stimulate the transport of sperm in the 
uterine cavity, Casey et al., J. Clin. Endo and Metabolism, Vol. 74, No. 
1, p. 223-225, therefore endothelin antagonists may be useful as male 
contraceptives. Endothelin modulates the ovarian/menstrual cycle, 
Kenegsberg, J. of Clin. Endo. and Met., Vol. 74, No. 1, p. 12, and may 
also play a role in the regulation of penile vascular tone in man, Lau et 
al., Asia Pacific J. of Pharm., 1991, 6:287-292 and Tejada et al., J. 
Amer. Physio. Soc. 1991, H1078-H1085. Endothelin also mediates a potent 
contraction of human prostatic smooth muscle, Langenstroer et al., J. 
Urology, Vol. 149, p. 495-499. 
Thus, endothelin receptor antagonists would offer a unique approach toward 
the pharmacotherapy of hypertension, pulmonary, hypertension, renal 
failure, ischemia induced renal failure, sepsis-endotoxin induced renal 
failure, prophylaxis and/or treatment of radio-contrast induced renal 
failure, acute and chronic cyclosporin induced renal failure, 
cerebrovascular disease, myocardial ischemia, angina, heart failure, 
asthma, atherosclerosis, macular degeneration, Raynaud's phenomenon, 
ulcers, sepsis, migraine, glaucoma, endotoxin shock, endotoxin induced 
multiple organ failure or disseminated intravascular coagulation, 
cyclosporin-induced renal failure and as an adjunct in angioplasty for 
prevention of restenosis, diabetes, preclampsia of pregnancy, bone 
remodeling, kidney transplant, male contraceptives, infertility and 
priaprism and benign prostatic hypertrophy. 
SUMMARY OF THE INVENTION 
This invention comprises dihydroisoindole derivatives represented by 
Formula (I) and pharmaceutical compositions containing these compounds, 
and their use as endothelin receptor antagonists which are useful in the 
treatment of a variety of cardiovascular and renal diseases including but 
not limited to: hypertension, acute and chronic renal failure, 
cyclosporine induced nephrotoxicity, stroke, cerebrovascular vasospasm, 
myocardial ischemia, angina, heart failure and atherosclerosis, and as an 
adjunct in angioplasty for prevention of restenosis and for benign 
prostatic hypertrophy. 
This invention further constitutes a method for antagonizing endothelin 
receptors in an animal, including humans, which comprises administering to 
an animal in need thereof an effective amount of a compound of Formula 
(I). 
DETAILED DESCRIPTION OF THE INVENTION 
The compounds of this invention are represented by structural Formula (I) 
##STR1## 
wherein: 
R.sub.1 is --X(CH.sub.2).sub.n Ar or --X(CH.sub.2).sub.n R.sub.8 or 
R.sub.2 is hydrogen, Ar or C.sub.1-4 alkyl; 
P.sub.1 is tetrazole, SO.sub.2 NR.sub.7 R.sub.11, CONR.sub.7 SO.sub.2 
R.sub.11, or (CH.sub.2).sub.s R.sub.8 ; 
R.sub.3 and R.sub.5 are independently hydrogen, R.sub.11, OH, C.sub.1-8 
alkoxy, S(O).sub.q R.sub.11, N(R.sub.6).sub.2, Br, F, I, Cl, CF.sub.3, 
NHCOR.sub.6, R.sub.11 CO.sub.2 R.sub.7, --X--R.sub.9 --Y, or 
--X(CH.sub.2).sub.n R.sub.8 wherein each methylene group within 
--X(CH.sub.2).sub.n R.sub.8 may be unsubstituted or substituted by one or 
two --(CH.sub.2).sub.n Ar groups; 
R.sub.4 is hydrogen, R.sub.11, OH, C.sub.1-5 alkoxy, S(O).sub.q R.sub.11, 
N(R.sub.6).sub.2, --X(R.sub.11), Br, F, I, Cl or NHCOR.sub.6 wherein the 
C.sub.1-5 alkoxy may be unsubstituted or substituted by OH, methoxy or 
halogen; 
R.sub.6 is independently hydrogen or C.sub.1-4 alkyl; 
R.sub.7 is independently hydrogen, C.sub.1-10 alkyl, C.sub.2-10 alkenyl or 
C.sub.2-8 alkynyl, all of which may be unsubstituted or substituted by one 
or more OH, N(R.sub.6).sub.2, CO.sub.2 R.sub.12, halogen or XC.sub.1-5 
alkyl; or R.sub.7 is (CH.sub.2).sub.n Ar; 
R.sub.8 is hydrogen, R.sub.11, CO.sub.2 R.sub.7, CO.sub.2 C(R.sub.11).sub.2 
O(CO)XR.sub.7, PO.sub.3 (R.sub.7).sub.2, SO.sub.2 NR.sub.7 R.sub.11, 
NR.sub.7 SO.sub.2 R.sub.11,CONR.sub.7 SO.sub.2 R.sub.11, SO.sub.3 R.sub.7, 
SO.sub.2 R.sub.7, P(O)(OR.sub.7)R.sub.7, CN, --CO.sub.2 (CH.sub.2).sub.m 
C(O)N(R.sub.6).sub.2, C(R.sub.11).sub.2 N(R.sub.7).sub.2, 
C(O)N(R.sub.6).sub.2 or tetrazole; 
R.sub.9 is (CH.sub.2).sub.n, C.sub.1-10 alkylene, C.sub.2-10 alkenylene or 
phenylenyl, all of which may be unsubstituted or substituted by one or 
more OH, N(R.sub.6).sub.2, COOH, halogen, or R.sub.9 may be &gt;C.dbd.O or 
XC.sub.1-5 alkyl; 
R.sub.11 is hydrogen, Ar, C.sub.1-8 alkylene, C.sub.2-8 alkenylene, 
C.sub.2-8 -alkynylene, all of which may be unsubstituted or substituted by 
one or more OH, CH.sub.2 OH, N(R.sub.6).sub.2 or halogen; 
R.sub.12 is hydrogen, C.sub.1-6 alkyl, C.sub.2-6 alkenyl or C.sub.2-7 
alkynyl; 
X is (CH.sub.2).sub.n, O or NR.sub.6 ; 
Y is CH.sub.3 or X(CH.sub.2).sub.n Ar; 
Ar is: 
##STR2## 
naphthyl, indolyl, pyridyl, thienyl, oxazolidinyl, oxazolyl, thiazolyl, 
isothiazolyl, pyrazolyl, triazolyl, tetrazolyl, imidazolyl, 
imidazolidinyl, thiazolidinyl, isoxazolyl, oxadiazolyl, thiadiazolyl, 
morpholinyl, piperidinyl, piperazinyl, pyrrolyl, or pyrimidyl; all of 
which may be unsubstittited or substituted by one or more R.sub.3 or 
R.sub.4 groups; 
A is C.dbd.O, or C(R.sub.6).sub.2 !.sub.m ; 
B is --CH.sub.2 -- or--O--; 
Z.sub.1 and Z.sub.2 are independently hydrogen, C.sub.1-8 alkyl, C.sub.2-8 
alkenyl, C.sub.2-8 alkynyl, OH, C.sub.1-8 alkoxy, S(O).sub.q C.sub.1-8 
alkyl, N(R.sub.6).sub.2, Br, F, I, Cl, NHCOR.sub.6, --X--R.sub.9 --Y, 
--X(CH.sub.2).sub.n R.sub.8, phenyl, benzyl or C.sub.3-6 cycloalkyl 
wherein the C.sub.1-8 alkyl, C.sub.2-8 alkenyl or C.sub.2-8 alkynyl may be 
optionally substituted by COOH, OH, CO(CH.sub.2).sub.n CH.sub.3, 
CO(CH.sub.2).sub.n CH.sub.2 N(R.sub.6).sub.2, or halogen; or Z.sub.1 and 
Z.sub.2 together may be --O--A--O-- on contiguous carbons; 
Z.sub.3 is Z.sub.1 or --X--R.sub.9 --Y; 
q is zero, one or two; 
n is an integer from 0 to six; 
s is an integer from one to six; 
m is 1, 2 or 3; and the dotted line indicates the optional presence of a 
double bond; provided P.sub.1 is not (CH.sub.2).sub.s NH.sub.2 ; 
or a pharmaceutically acceptable salt thereof. 
Also included in the invention are pharmaceutically acceptable salt 
complexes. 
All alkyl, alkenyl, alkynyl and alkoxy groups may be straight or branched. 
The term "halogen" is used to mean iodo, fluoro, chloro or bromo. Alkyl 
groups may be substituted by one or more halogens up to perhalogenation. 
The compounds of the present invention may contain one or more asymmetric 
carbon atoms and may exist in racemic and optically active form. All of 
these compounds and diastereoisomers are contemplated to be within the 
scope of the present invention. 
The present invention provides compounds of formula (I) above, 
##STR3## 
which can be prepared by reacting a suitably substituted orthobromobenzoic 
acid such as (2) 
##STR4## 
with two equivalents of n-Butyllithium in a solvent such as THF under 
argon at -78.degree. C. followed by the addition of an acid chloride of 
formula (3) 
##STR5## 
wherein Z.sub.1, Z.sub.2 and Z.sub.3 are as defined in formula I, in a 
suitable solvent such as THF to provide a compound of formula (4) 
##STR6## 
Treatment of compound (4) with a suitable reducing agent such as Zn in 
acetic acid provides a phenyl substituted phthalide of formula (5) 
##STR7## 
Treatment of (5) with an organomagesium compound of formula (6) 
EQU R.sub.1 MgBr (6) 
wherein R.sub.1 is as defined in formula I, in a suitable solvent such as 
ether at reflux provides, after acid treatment, compounds of formula (7) 
##STR8## 
Oxidation of (7) under appropriate conditions such as with Jones reagent 
provides compounds of formula (8) 
##STR9## 
Isoindoles of formula (10) can be obtained by treatment of (8), with a 
primary amine of formula (9) 
EQU H.sub.2 NQ (9) 
wherein Q is hydrogen or (CH.sub.2).sub.s R.sub.8, in a suitable solvent 
such as ethanol at reflux; in the presence of sodium borohydride in 
ethanol. 
##STR10## 
Reduction of an isoindole of formula (10) under the appropriate conditions 
such as Zn(Cu) in hot acetic acid or with hydrogen gas under pressure in 
the presence of a suitable catalyst such as 10% palladium on charcoal 
affords compounds of formula (11). 
##STR11## 
Compounds of formula (11) may be convened to those of formula (I) by 
acylation or alkylation of Q to P.sub.1 as appropriate. 
With appropriate manipulation and protection of any chemical 
functionalities, synthesis of the remaining compounds of the Formula (I) 
is accomplished by methods analogous to those above. 
In order to use a compound of the Formula (I) or a pharmaceutically 
acceptable salt thereof for the treatment of humans and other mammals it 
is normally formulated in accordance with standard pharmaceutical practice 
as a pharmaceutical composition. 
Compounds of Formula (I) and their pharmaceutically acceptable salts may be 
administered in a standard manner for the treatment of the indicated 
diseases, for example orally, parenterally, sub-lingually, transdermally, 
rectally, via inhalation or via buccal administration. 
Compounds of Formula (I) and their pharmaceutically acceptable salts which 
are active when given orally can be formulated as syrups, tablets, 
capsules and lozenges. A syrup formulation will generally consist of a 
suspension or solution of the compound or salt in a liquid carrier for 
example, ethanol, peanut oil, olive oil, glycerine or water with a 
flavouring or colouring agent. Where the composition is in the form of a 
tablet, any pharmaceutical carrier routinely used for preparing solid 
formulations may be used. Examples of such carriers include magnesium 
stearate, terra alba, talc, gelatin, agar, pectin, acacia, stearic acid, 
starch, lactose and sucrose. Where the composition is in the form of a 
capsule, any routine encapsulation is suitable, for example using the 
aforementioned carriers in a hard gelatin capsule shell. Where the 
composition is in the form of a soft gelatin shell capsule any 
pharmaceutical carrier routinely used for preparing dispersions or 
suspensions may be considered, for example aqueous gums, celluloses, 
silicates or oils and are incorporated in a soft gelatin capsule shell. 
Typical parenteral compositions consist of a solution or suspension of the 
compound or salt in a sterile aqueous or non-aqueous carrier optionally 
containing a parenterally acceptable oil, for example polyethylene glycol, 
polyvinylpyrrolidone, lecithin, arachis oil, or sesame oil. 
Typical compositions for inhalation are in the form of a solution, 
suspension or emulsion that may be administered as a dry powder or in the 
form of an aerosol using a conventional propellant such as 
dichlorodifluoromethane or trichlorofluoromethane. 
A typical suppository formulation comprises a compound of Formula (1a-1d) 
or a pharmaceutically acceptable salt thereof which is active when 
administered in this way, with a binding and/or lubricating agent, for 
example polymeric glycols, gelatins, cocoa-butter or other low melting 
vegetable waxes or fats or their synthetic analogues. 
Typical transdermal formulations comprise a conventional aqueous or 
nonaqueous vehicle, for example a cream, ointment, lotion or paste or are 
in the form of a medicated plaster, patch or membrane. 
Preferably the composition is in unit dosage form, for example a tablet, 
capsule or metered aerosol dose, so that the patient may administer to 
themselves a single dose. 
Each dosage unit for oral administration contains suitably from 0.1 mg to 
500 mg/Kg, and preferably from 1 mg to 100 mg/Kg, and each dosage unit for 
parenteral administration contains suitably from 0.1 mg to 100 mg, of a 
compound of Formula (I) or a pharmaceutically acceptable salt thereof 
calculated as the free acid. Each dosage unit for intranasal 
administration contains suitably 1-400 mg and preferably 10 to 200 mg per 
person. A topical formulation contains suitably 0.01 to 1.0% of a compound 
of Formula (I). 
The daily dosage regimen for oral administration is suitably about 0.01 
mg/Kg to 40 mg/Kg, of a compound of Formula (I) or a pharmaceutically 
acceptable salt thereof calculated as the free acid. The daily dosage 
regimen for parenteral administration is suitably about 0.001 mg/Kg to 40 
mg/Kg, of a compound of the Formula (I) or a pharmaceutically acceptable 
salt thereof calculated as the free acid. The daily dosage regimen for 
intranasal administration and oral inhalation is suitably about 10 to 
about 500 mg/person. The active ingredient may be administered from 1 to 6 
times a day, sufficient to exhibit the desired activity. 
No unacceptable toxicological effects are expected when compounds of the 
invention are administered in accordance with the present invention. 
The biological activity of the compounds of Formula (I) are demonstrated by 
the following tests: 
I. Binding Assay 
A) Membrane Preparation (Rat cerebellum or kidney cortex) 
Rat cerebellum or kidney cortex were rapidly dissected and frozen 
immediately in liquid nitrogen or used fresh. The tissues, 1-2 g for 
cerebellum or 3-5 g for kidney cortex, were homogenized in 15 mls of 
buffer containing 20 mM Tris HCl and 5 mM EDTA, pH 7.5 at 4.degree. C. 
using a motor-driven homogenizer. The homogenates were filtered through 
cheesecloth and centrifuged at 20,000 x g for 10 minutes at 4.degree. C. 
The supernatant was removed and centrifuged at 40,000 xg for 30 minutes at 
4.degree. C. The resulting pellet was resuspended in a small volume of 
buffer containing 50 mM Tris, 10 mM MgCl.sub.2, pH 7.5; aliquotted with 
small vials and frozen in liquid nitrogen. The membranes were diluted to 
give 1 and 5 micrograms of protein for each tube for cerebellum and kidney 
cortex in the binding assay. 
Freshly isolated rat mesenteric artery and collateral vascular bed were 
washed in ice cold saline (on ice) and lymph nodes were removed from along 
the major vessel. Then, the tissue was homogenized using a polytron in 
buffer containing 20 mM Tris and 5 mM EDTA, pH 7.5 at 4.degree. C. in 15 
ml volume for .about.6 gm of mesenteric artery bed. The homogenate was 
strained through cheesecloth and centrifuged at 2,000 xg for 10 min. at 
4.degree. C. The supernatant was removed and centrifuged at 40,000 xg for 
30 min. at 4.degree. C. The resulting pellet was resuspended as explained 
above for cerebellum and kidney cortex. Approximately 10 micrograms of 
membrane protein was used for each tube in binding experiments. 
B) CHO Cell Membrane Preparation 
CHO cells stably transfected with human ET.sub.A and ET.sub.B receptors 
were grown in 245 mmx 245 mm tissue culture plates in Dulbecco's modified 
Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS). The 
confluent cells were washed with DPBS (Dulbecco's phosphate buffered 
saline) containing protease inhibitor cockatil (5 mM EDTA, 0.5 mM PMSF, 5 
ug/ml leupeptin, and 0.1 U/ml aprotinin) and scraped in the same buffer. 
After centrifugation at 800 xg, the cells were lysed by freezing in liquid 
nitrogen and thawing on ice followed by homogenization (30 times using 
glass dounce homogenizer) in lysis buffer containing 20 mM Tris HCl, pH 
7.5 and the protease inhibitor cocktail. After an initital centrifugation 
at 800 xg for 10 min to remove unbroken cells and nuclei, the supernatants 
were centrifuged at 40,000 xg for 15 min and the pellet was resuspended in 
50 mM Tris HCl, pH 7.5 and 10 mM MgCl.sub.2 and stored in small aliquots 
at -70.degree. C. after freezing in liquid N.sub.2. Protein was determined 
using BCA method and bovine serum albumin as the standard. 
C) .sup.125 I!ET-1 Binding Protocol 
.sup.125 I!ET-1 binding to membranes from rat cerebellum (2-5 mg 
protein/assay tube) or kidney cortex (3-8 micrograms protein/assay tube) 
or CHO cell membranes (containing 4-6 and 1-2 micrograms of membrane 
protein for ET.sub.A and ET.sub.B receptors, respectively) were measured 
after 60 minutes incubation at 30.degree. C. in 50 mM Tris HCl, 10 mM 
MgCl.sub.2, 0.05% BSA, pH 7.5 buffer in a total volume of 100 microliters. 
Membrane protein was added to tubes containing either buffer or indicated 
concentration of compounds. .sup.125 I!ET-1 (2200 Ci/mmol) was diluted in 
the same buffer containing BSA to give a final concentration of 0.2-0.5 nM 
ET-1. Total and nonspecific binding were measured in the absence and 
presence of 100 nM unlabelled ET-1. After the incubation, the reactions 
were stopped with 3.0 ml cold buffer containing 50 mM Tris and 10 mM 
MgCl.sub.2, pH 7.5. Membrane bound radioactivity was separated from free 
ligand by filtering through Whatman GF/C filter paper and washing the 
filters 5 times with 3 ml of cold buffer using a Brandel cell harvester. 
Filter papers were counted in a gamma counter with an efficiency of 75%. 
IC.sub.50 's for the compounds of this invention range from 0.01 nm to 50 
uM. 
II. In Vitro Vascular Smooth Muscle Activity 
Rat aorta are cleaned of connective tissue and adherent fat, and cut into 
ring segments approximately 3 to 4 mm in length. Vascular rings are 
suspended in organ bath chambers (10 ml) containing Krebs-bicarbonate 
solution of the following composition (millimolar): NaCl, 112.0; KCl, 4.7; 
KH.sub.2 PO.sub.4, 1.2; MgSO.sub.4, 1.2; CaCl.sub.2, 2.5; NaHCO.sub.3, 
25.0; and dextrose, 11.0. Tissue bath solutions are maintained at 
37.degree. C. and aerated continuously with 95% O.sub.2 /5% CO.sub.2. 
Resting tensions of aorta are maintained at 1 g and allowed to equilibrate 
for 2 hrs., during which time the bathing solution is changed every 15 to 
20 min. Isometric tensions are recorded on Beckman R-611 dynographs with 
Grass FT03 force-displacement transducer. Cumulative 
concentration-response curves to ET-1 or other contractile agonists are 
constructed by the method of step-wise addition of the agonist. ET-1 
concentrations are increased only after the previous concentration 
produces a steady-state contractile response. Only one 
concentration-response curve to ET-1 is generated in each tissue. ET 
receptor antagonists are added to paired tissues 30 min prior to the 
initiation of the concentration-response to contractile agonists. 
ET-1 induced vascular contractions are expressed as a percentage of the 
response elicited by 60 mM KCl for each individual tissue which is 
determined at the beginning of each experiment. Data are expressed as the 
mean .+-.S.E.M. Dissociation constants (K.sub.b) of competitive 
antagonists were determined by the standard method of Arunlakshana and 
Schild. The potency range for compounds of this invention range from 0.1 
nM to 50 mm. 
The following examples are illustrative and are not limiting of the 
compounds of this invention.

EXAMPLE 1 
Preparation of (1RS 
,3RS)-3-(2-Carboxymethoxy-4-methoxy)phenyl!-1-(3,4-methylenedioxyphenyl) 
-5-(prop-1-yloxy) (1H,3H-dihydro-isoindol-2-yl)!acetic acid, 
trifluoroacetate salt. 
a) Benzyl 3-(Prop-1-yloxy)benzoyl acetate 
To a solution of benzyl alcohol (0.197 ml, 1.91 mmol), 
4-dimethylaminopyridine (0.077 g, 0.64 mmol) in dry toluene (5 ml) under 
argon was added methyl 3-(prop-1-yloxy)benzoyl acetate (0.300 g, 1.27 
mmol) in toluene (0.5 ml). The reaction was allowed to stirred at reflux 
for 24 h. On cooling the mixture was then quenched with saturated ammonium 
chloride and partitioned with ethyl acetate. The combined organic extracts 
was washed successively with: water, brine and dried (MgSO.sub.4). After 
removing the solvent under reduced pressure, flash chromatography of the 
residue (silica gel, 3:7 ethyl acetate:hexane) afforded the title compound 
as a yellow oil (0.320 g, 80%). 
b) Benzyl 
3-(3,4-methylenedioxyphenyl)-2-3-prop-1-yloxy)-benzoyl!propenoate 
To a solution of 3,4-(methylenedioxy)benzaldehyde (4.81 g, 32.1 mmol), 
benzyl 3-(Prop-1-yloxy)benzoyl acetate (10.0 g, 32.1 mmol) in benzene (50 
ml) was added piperidine (0.31 ml, 3.21 mmol) followed by acetic acid (10 
drops). The reaction was allowed to stir at reflux equipped with a dean 
stark apparatus for 2 h. Upon removal of the solvent a yellow oil was 
obtained. Recrystallization from ethyl acetate/hexane afforded the title 
compound as an off white solid (9.8 g, 69%). 
c) 
Benzyl-(1RS,2SR)-(3,4-methylenedioxyphenyl)-5-prop-1-yloxy-3-oxo-indane-2- 
carboxylate 
A solution of benzyl 
3-(3,4-methylenedioxyphenyl)-2-3-prop-1-yloxy)-benzoyl!propenoate (9.3 g, 
20.9 mmol) in trifloroacetic acid (40 ml) was stirred at room temperature 
for 1.5 h. The solvent was removed and the resulting residue was dissolved 
in ethyl acetate washed successively with: water, 5% sodium bicarbonate 
and brine. The organic extract was dried (MgSO.sub.4) and removal of the 
solvent under reduced pressure gave the title compound as a yellow oil 
(9.8 g, quantitative). 
d) 1-(3,4-Methylenedioxyphenyl)-5-prop-1-yloxy-3-oxo-indane 
To a solution of benzyl-(1RS 
,2SR)-(3,4-methylenedioxyphenyl)-5-prop-1-yloxy-3-oxo-indane-2-carboxylate 
(8.2 g, 18.4 mmol) in warm acetic acid (130 ml), was added under argon 10% 
Pd/C (4.1 g) followed by 1,4-cyclohexadiene (17.4 ml, 185 mmol). The 
reaction was exothermic and gas evolution was observed. The reaction was 
allowed to stir under argon at room temperature for 2 h. The mixture was 
filtered through a pad of celite and concentrated in vacuo. Flash 
chromatography of the residue (silica gel, 3:7 ethyl acetate:hexane) 
afforded the title compound as a pinkish solid (5.08 g, 93%). 
e) 3-(3,4-Methylenedioxyphenyl)-6-prop-1-yloxy inden-1-one 
A solution of 1-(3,4-methylenedioxyphenyl)-5-prop-1-yloxy-3-oxo-indane (2.7 
g, 9.06 mmol), 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (2.45 g, 10.87 
mmol) in 1,4-dioxane (80 ml) was stirred at reflux for 2 h. The solvent 
was removed under reduced pressure and the resulting residue was dissolved 
in methylene chloride and filtered. The filtrate was concentrated and 
flash chromatography (silica gel, 3:7 ethyl acetate:hexane) afforded the 
title compound as a red solid (1.48 g, 55%). 
f) 
(1RS)-1-(2-Benzyloxy-4-methoxy)phenyl!-1-hydroxy-3-(3,4-methylenedioxyphe 
nyl)-6-prop-1-yloxy-1H-indene 
To a solution of 3-(3,4-methylenedioxyphenyl)-6-prop-1-yloxy inden-1-one 
(2.2 g, 7.4 mmol) in THF (50 ml) at 0.degree. C. under argon, was added 
dropwise a freshly prepared solution of 2-benzyloxy-4-methoxyphenyl 
magnesium bromide (7.07 g, 22.3 mmol) in a 1:1 mixture of THF:Et.sub.2 O 
(30 ml, total). The reaction was stirred at 0.degree. C. for 20 minutes. 
The mixture was then quenched with 1N HCl and extracted with ethyl 
acetate. The organic extract was then washed with water, brine, and dried 
(MgSO.sub.4). After removing the solvent under reduced pressure, flash 
chromatography of the residue (silica gel, 3:7 ethyl acetate:hexane) 
afforded the title compound as off white crystals (1.77 g, 46%). 
g) 
1-(2-Benzyloxy-4-methoxybenzoyl)-2-(3,4-methylenedioxybenzoyl)-5-prop-1-yl 
oxybenzene 
To a solution of 
(1RS)-1-(2-Benzyloxy-4-methoxy)phenyl!-1-hydroxy-3-(3,4-methylenedioxyphe 
nyl)-6-prop-1-yloxy-1H-indene (0.100 g, 0.19 mmol) in a mixture of 1:1:1 
carbron tetrachloride:acetonitrile:water (18 ml, total) was added 
ruthenium (III) chloride hydrate (0.010 g, 0.048 mmol) followed by sodium 
periodate (0.071 g, 0.32 mmol) under argon. The reaction was stirred at 
room temperature for 1.5 h. A 1:1 mixture of starting material and desired 
product was observed after 1.5 h. To the reaction was added an additional 
amount of ruthenium (III) chloride hydrate and sodium periodate (0.010 g 
and 0.071 g respectively), and stirring continued at room temperature for 
20 minutes. The mixture was filtered through a pad of celite and the 
filtrate was partitioned between ethyl acetate and water. The combined 
organic extract was washed successively with: water, brine and dried 
(MgSO.sub.4). After removing the solvent under reduced pressure, flash 
chromatography of the residue (silica gel, 3:7 ethyl acetate:hexane) 
afforded the title compound as a yellow oil (0.050 g, 50%). 
h) 
1-(2-Hydroxy-4-methoxybenzoyl)-2-(3,4-methylenedioxybenzoyl)-5-prop-1-ylox 
ybenzene 
To a solution of 
1-(2-benzyloxy-4-methoxybenzoyl)-2-(3,4-methylenedioxybenzoyl)-5-prop-1-yl 
oxybenzene (0.437 g, 0.84 mmol) in a mixture 1:1 ethyl acetate:hexane (20 
ml) was added 10% Pd/C (0.040 g) and the mixture was shaken under hydrogen 
atmosphere at 60 psi for 24 h. The reaction mixture was filtered through a 
pad of celite and the filtrate dried (MgSO.sub.4). After removing the 
solvent under reduced pressure, flash chromatography of the residue 
(silica gel, 3:7 ethyl acetate:hexane) afforded the title compound as a 
pale yellow oil (0.270 g, 72%). 
i) 
(1RS)-1-Hydroxy-1-(2-hydroxy-4-methoxy)phenyl!-3-(3,4-methylenedioxypheny 
l-6-prop-1-yloxy-1H-isoindole 
A bomb was charged with 
1-(2-hydroxy-4-methoxybenzoyl)-2-(3,4-methylenedioxybenzoyl)-5-prop-1-ylox 
ybenzene (0.270 g, 0.608 mmol), cooled to -78.degree. C. (acetone/dry ice 
bath), and liquid ammonia (15 ml) was added and the vessel was sealed. The 
reaction was heated at 65.degree. C. reaching a steady pressure of 300 psi 
for 24 h. The pressure was slowly released after the mixture had cooled to 
room temperature, and the resulting residue was dissolved in ethyl 
acetate. After removing the solvent under reduced pressure, flash 
chromatography of the residue (silica gel, 1:1 ethyl acetate:hexane) 
afforded the title compound as a yellow oil (0.200 g, 76%). 
j) 
(1RS,3RS)-3-(2-Hydroxy-4-methoxy)phenyl!-1-(3,4-methylenedioxyphenyl)-5-p 
rop-1-yloxy-isoindoline 
To a flask containing dry ether (2 ml) was added with stirring aluminum 
chloride (0.037 g, 0.28 mmol) at 0.degree. C. under argon, followed by 1M 
lithium aluminum hydride in THF (0.28 ml, 0.28 mmol). The mixture was 
stirred at 0.degree. C. for 15 minutes, then to it was rapidly added 
(1RS)-1-Hydroxy-1-(2-hydroxy-4-methoxy)phenyl!-3-(3,4-methylenedioxypheny 
l-6-prop-1-yloxy-1H-isoindole (0.043 g, 0.10 mmol). After stirring for 20 
minutes at 0.degree. C. the reaction was quenched with water, 15% sodium 
hydroxide, and water. The mixture was extracted with ethyl acetate and the 
combined organic extract was washed successively with: water, brine and 
dried (MgSO.sub.4). After removing the solvent under reduced pressure, 
flash chromatography of the residue (silica gel, 3:7 ethyl acetate:hexane) 
afforded the title compound as a pale yellow solid (0.012 g, 28%). 
k) Methyl trifluoromethylsulfonyloxyacetate 
To a solution of triflic anhydride (3.7 ml, 20.0 mmol) in methylene 
chloride (10 ml) at -5.degree. C. under argon, was added dropwise over 30 
minutes a solution of methyl glycolate (1.8 g, 20.0 mmol), pyridine (1.55 
ml, 20.0 mmol), and anhydrous methylene chloride (5 ml). The reaction was 
allowed to stir at between 0.degree.-5.degree. C. for 1.5 h. The mixture 
was then washed with water several times. The organic extract was then 
washed with water, brine, and dried (MgSO.sub.4). After removing the 
solvent under reduced pressure, flash chromatography of the residue 
(silica gel, methylene chloride) afforded the title compound as a 
colorless oil (3.0 g, 68%). 
l) 
Methyl(1RS,3RS)-3-(2-hydroxy-4-methoxy)phenyl!-1-(3,4-methylenedioxypheny 
l)-5-prop-1-yloxy-(1H,3H-dihydroisoindol-2yl)acetate 
To a solution of 
(1RS,3RS)-3-(2-hydroxy-4-methoxy)phenyl!-1-(3,4-methylenedioxyphenyl)-5-p 
rop-1-yloxy isoindoline (0.060 g, 0.14 mmol) in methylene chloride (3 ml) 
was added triethylamine (0.040 ml, 0.28 mmol) followed by methyl 
trifluoromethlysulfonyloxyacetate (0.065 g, 0.28 mmol) under argon. The 
reaction was stirred at room temperature for 24 h. The mixture was then 
partitioned between 1N HCl and ethyl acetate. The organic extract was 
washed successively with water, brine and dried (MgSO.sub.4). After 
removing the solvent under reduced pressure, flash chromatography of the 
residue (silica gel, 2:8 ethyl acetate:hexane) afforded the title compound 
as a colorless oil (0.040 g, 57%). 
m) 
Methyl(1RS,3RS)-3-(2-carbomethoxymethoxy-4-methoxy)phenyl!-1-(3,4-methyle 
nedioxyphenyl)-5-prop-1-yloxy-(1H,3H-dihydroisoindo-2-yl)acetate 
To a solution of 
methyl(1RS,3RS)-3-(2-hydroxy-4-methoxy)phenyl!-1-(3,4-methylenedioxypheny 
l)-5-prop-1-yloxy (1H,3H-dihydroisoindol-2yl)acetate (0.040 g, 0.08 mmol) 
in dry DMF (0.5 ml) was added potassium carbonate (0.11 g, 0.8 mmol) under 
argon. The mixture was stirred at room temperature for 20 minutes, then 
ethyl bromoacetate (0.012 ml, 0.1 mol) added and stirring continued for 24 
h. The reaction was quenched with 1N HCl and extracted with ethyl acetate. 
The combined organic extract was washed successively with water, brine and 
dried (MgSO.sub.4). After removing the solvent under reduced pressure, 
flash chromatography of the residue (silica gel, 3:7 ethyl acetate:hexane) 
afforded the title compound as a colorless oil (0.030 g, 64%). 
n) 
(1RS,3RS)-3-(2-Carboxymethoxy-4-methoxy)phenyl!-1-(3,4-methylenedioxyphen 
yl)-5-prop-1-yloxy-(1H,3H-dihydroisoindol-2-yl)acetic acid 
To a solution of 
Methyl(1RS,3RS)-3-(2-carbomethoxymethoxy-4-methoxy)phenyl!-1-(3,4-methyle 
nedioxyphenyl)-5-prop-1-yloxy-(1H,3H-dihydroisoindol-2-yl)acetate (0.030 g, 
0.052 mmol) in warm isopropanol (2 ml) was added 6N sodium hydroxide 
(0.043 ml, 0.26 mmol). The reaction was stirred at reflux for 2 h. The 
mixture was then quenched with 3N HCl and extracted with ethyl acetate. 
The combined organic extract was washed successively with water, brine and 
dried (MgSO.sub.4). After removing the solvent under reduced pressure, 
purification of the resulting residue by reversed phase HPLC (1:1 
acetonitrile:water, and 1% TFA) afforded the title compound as a white 
solid (0.020 g, 71%). MS (ESI) m/e 536 M+H!.sup.+ ; mp: 154.degree.-158 
.degree. C. (dec.); Anal. (C.sub.31 H.sub.30 F.sub.3 NO.sub.11.3.5H.sub.2 
O) calcd. C, 41.95; H, 3.73; N, 1.66: found: C, 42.14; H, 3.82; N, 1.33. 
EXAMPLE 2 
Preparation of 
(1RS,3RS)-3-(4-Methoxyphenyl)-1-(3,4-methylenedioxyphenyl)-(1H,3H-dihydroi 
soindol-2yl)acetic acid 
a) 2-Hydroxy-4'-methoxyacetophenone 
To a mixture of aluminum chloride (22.5 g, 0.169 mol) and chlorobenzene (50 
ml) was added anisole (18 g, 0.166 mol) at 10.degree. C. Keeping the 
temperature below 25.degree. C., o-methoxybenzoyl chloride (28 g, 0.164 
mol) was added dropwise to the reaction. The solution was stirred for 30 
minutes at room temperature, then it was heated on a steam bath for 2 h. 
After cooling to 0.degree.-5.degree. C. in an ice bath the mixture was 
treated with 10% HCl (100 ml) and stirred at room temperature for 18 h. 
The product was extracted with ether (3.times.100 ml) and the combined 
organic extract was washed with 5% sodium hydroxide (3.times.50 ml). The 
combined aqueous extract was acidified with concentrated HCl and extracted 
with ether (3x). The combined organic extract was washed with water and 
dried (MgSO.sub.4). Removal of the solvent under reduced pressure gave the 
title compound as an amber oil (22.9 g, 61%). 
b) 2-Hydroxy-4'-methoxyacetophenone 3,4-methylenedioxybenzoylhydrazone 
To a solution of 3,4-methylenedioxybenzhydrazine (4.89 g, 27.1 mmol), 
2-Hydroxy-4'-methoxyacetophenone (6.19 g, 27.1 mmol) in isopropanol (150 
ml) was added sodium acetate (1 g), and the mixture was stirred at reflux 
for 24 h, after which only a small amount of product had formed. An 
additional amount of sodium acetate (1 g) was added to the reaction and 
stirring at reflux continued for 24 h during which time a precipitate 
formed. The precipitate was filtered while hot to afford the title 
compound as a solid (3.8 g, 36%). 
c) 1-(4-Methoxybenzoyl)-2-(3,4-methylenedioxybenzoyl)benzene 
To a solution of 2-Hydroxy-4'-methoxyacetophenone 
3,4-methylenedioxybenzoylhydrazone (3.83 g, 9.82 mmol) of in acetic acid 
(40 ml) was added iodobenzene diacetate (6.32 g, 19.6 mmol) and the 
suspension was stirred at room temperature for 24 h and then stirred at 
reflux for 3 h. The mixture was cooled, then partitioned between ethyl 
acetate and water. The combined organic extract was washed successively 
with: water, brine and dried (MgSO.sub.4). After removing the solvent 
under reduced pressure, flash chromatography of the residue (silica gel, 
3:7 ethyl acetate:hexane) afforded the title compound as an off white 
solid (2.4 g, 68%). 
d) 
(1RS)-1-Hydroxy-1-(4-methoxyphenyl)-3-(3,4-methylenedioxyphenyl)-1H-isoind 
ole 
A bomb was charged 
1-(4-methoxybenzoyl)-2-(3,4-methylenedioxybenzoyl)benzene (1.0 g, 2.8 
mmol) was cooled to -78.degree. C. (acetone/dry ice bath), and liquid 
ammonia (20 ml) was added and the vessel was sealed. The reaction was 
heated at 70.degree. C. reaching a steady pressure of 300 psi for 24 h. 
The pressure was slowly released after the mixture had cooled to room 
temperature, and the brown residue was partitioned between ethyl acetate 
and water. The organic extract was acidified with 3N HCl. The resulting 
aqueous layer (containing the hydrochloride salt) was washed with ethyl 
acetate, then basified by treatment with concentrated ammonium hydroxide 
and extracted with ethyl acetate. The combined organic extract was 
successively washed with water brine and dried (MgSO.sub.4). Removal of 
the solvent under reduced pressure gave an isomeric mixture of the title 
compound as a brown foam (0.900 g, 90%). 
e) (1RS,3RS)-3-(4-Methoxyphenyl)-1-(3,4-methylenedioxyphenyl)isoindoline 
To a flask containing dry ether (2 ml) was added with stirring aluminum 
chloride (0.037 g, 0.28 mmol) at 0.degree. C. under argon, followed by 1M 
lithium aluminum hydride in THF (0.28 ml, 0.28 mmol). The mixture was 
stirred at 0.degree. C. for 5 minutes, then to it was rapidly added 
(1RS)-1-Hydroxy-1-(4-methoxyphenyl)-3-(3,4-methylenedioxyphenyl)-1H-isoind 
ole (0.050 g, 0.14 mmol), followed by dry THF (2 ml). After stirring for 10 
minutes at 0.degree. C. the reaction was quenched with water, 15% sodium 
hydroxide, and water. The mixture was extracted with ethyl acetate and the 
combined organic extract was successively washed with: water, brine and 
dried (MgSO.sub.4). After removing the solvent, flash chromatography of 
the residue (silica gel, 3:7 ethyl acetate:hexane) afforded the title 
compound as a yellow solid (0.030 g, 45%). MS (ESI) m/e 346 M+H!.sup.+ ; 
mp: 123.degree.-125.degree. C. 
f) 
Methyl(1RS,3RS)-3-(4-methoxyphenyl)-1-(3,4-methylenedioxyphenyl)-(1H,3H-di 
hydroisoindol-2yl)acetate 
To a solution of 
(1RS,3RS)-1-(4-methoxyphenyl)-3-(3,4-methylenedioxyphenyl)isoindoline 
(0.140 g, 0.41 mmol) in methylene chloride (3 ml) was added triethylamine 
(0.085 ml, 0.61 mmol) followed by methyl trifluoromethlysulfonyloxyacetate 
(0.065 g, 0.28 mmol) under argon. The reaction was stirred at room 
temperature for 2.5 h. The mixture was then partitioned between ethyl 
acetate and water. The combined organic extract was washed successively 
with: water, brine and dried (MgSO.sub.4). After removing the solvent 
under reduced pressure, flash chromatography of the residue (silica gel, 
3:7 ethyl acetate:hexane) afforded the title compound as a oil (0.130 g, 
76%). 
g) 
(1RS,3RS)-3-(4-Methoxyphenyl)-1-(3,4-methylenedioxyphenyl)-(1H,3H-dihydroi 
soindol-2yl)acetic acid 
To a solution of 
Methyl(1RS,3RS)-3-(4-methoxyphenyl)-1-(3,4-methylenedioxyphenyl)-(1H,3H-di 
hydroisoindol-2yl)acetate (0.130 g, 0.31 mmol) in isopropanol (3 ml) was 
added 6M sodium hydroxide (0.151 mmol). The reaction was stirred at reflux 
for 24 h. The organic solvent was removed and the aqueous layer was 
acidified with 3N HCl and extracted with ethyl acetate. The organic 
extract was washed successively with: water, brine and dried (MgSO.sub.4). 
Removal of the solvent under reduced pressure gave the title compound as a 
colorless oil (0.100 g, 81%). MS (ESI) m/e 404 M+H!.sup.+ ; Anal. 
(C.sub.24 H.sub.21 NO.sub.5.) calcd. C, 71.45; H, 5.25; N, 3.47: found: C, 
71.44; H, 5.17; N, 3.42; .sup.1 H NMR (400 MHz, CDCl.sub.3) .delta.7.35 
(d, J=8.7 Hz, 2H), 7.17 (d, J=8.7 Hz, 2H), 6.95-6.80 (mm, 7H), 5.94 (s, 
2H), 5.34 (s, 2H), 3.81 (s, 3H), 3.47 (s, 2H). 
EXAMPLE 3 
Formulations for pharmaceutical use incorporating compounds of the present 
invention can be prepared in various forms and with numerous excipients. 
Examples of such formulations are given below. 
Inhalant Formulation 
A compound of formula I (1 mg to 100 mg) is aerosolized from a metered dose 
inhaler to deliver the desired amount of drug per use. 
______________________________________ 
Tablets/Ingredients Per Tablet 
______________________________________ 
1. Active ingredient 40 mg 
(Cpd of Formula I) 
2. Corn Starch 20 mg 
3. Alginic acid 20 mg 
Sodium alginate 20 mg 
5. Mg stearate 1.3 mg 
2.3 mg 
______________________________________ 
Procedure for Tablets: 
Step 1 Blend ingredients No. 1, No. 2, No. 3 and No. 4 in a suitable 
mixer/blender. 
Step 2 Add sufficient water portion-wise to the blend from Step 1 with 
careful mixing after each addition. Such additions of water and mixing 
until the mass is of a consistency to permit its converion to wet 
granules. 
Step 3 The wet mass is converted to granules by passing it through an 
oscillating granulator using a No. 8 mesh (2.38 mm) screen. 
Step 4 The wet granules are then dried in an oven at 140.degree. F. 
(60.degree. C.) until dry. 
Step 5 The dry granules are lubricated with ingredient No. 5. 
Step 6 The lubricated granules are compressed on a suitable tablet press. 
Parenteral Formulation 
A pharmaceutical composition for parenteral administration is prepared by 
dissolving an appropriate amount of a compound of formula I in 
polyethylene glycol with heating. This solution is then diluted with water 
for injections Ph Eur. (to 100 ml). The solution is then steriled by 
filtration through a 0.22 micron membrane filter and sealed in sterile 
containers.