Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
~'~3~i662
PHENETHANOLAMINES AND USES THEREOF
.
This invention relates generally to anti-inflam-
matory, topically applied nonsteroidal compositions
and to their uses and relates more specifiaally to
such compositions having as active ingredient B2-
adrenergic agonist(s).
This invention also relates to heterocyclic car-
bon compounds of the indole series having an amino
substituent and relates to drug bio-afecting and body-
treating processes employing these compounds.
Applicants emphasize that although there are at
least hundreds (perhaps thousands) of B2-agonists
known in the art, only salbutamol has been disclosed
as having any topical anti-inflammatory activity. It
is believed that no structure-to-activity relationship
for predicting topical anti-inflammatory activity is
known in the art at this time. The art area is very
unpredictable.
Inflammation is exhibited by most skin diseases.
A variety of inflammatosy skin diseases and conditions
(including chronic and acute types) has resulted in an
ongoing search for anti-inflammatory drugs.
i~3566Z
The introduction of steroids provided the derma-
tologist with a class of anti-inflammatory agents that
are therapeutically active against a wide spectrum of
inflammatory skin diseases. However, the effect of
steroids in many inflammatory conditions, particularly
in those of a chronic nature, is only palliative and
requires extended use. And such extended use of
steroids also results in various adverse effects,
including atrophy of skin, striae, telangiectasia,
steroid acne, and adrenal suppression, especially in
children. Additionally, in various chronic inflamma-
tory skin diseases, the termination of steroid therapy
has led to the reappearance of inflammatory symptoms
and sometimes with increased intensity. In response
to the drawbacks of using steroids, over the last 20
years many new nonsteroidal anti-inflammatory agents
(i.e., NSAIA) have been developed for use in various
diseases, including rheumatic diseases. These com-
pounds generally appear to be free of some of the
adverse effects of steroids, especially tissue atro-
pby, adrenal suppression, and other less severe re-
bound effects.
one class of compounds included within the group
of NSAIA is a group of compounds that are prostagland-
in synthetase inhibitors. These materials are gener-
ally active in reducing UVB-induced erythema (i.e.,
erythema induced by ultraviolet light) in guinea pigs;
but the materials are only slightly active or are in-
active in other tests relating to dermatitis, includ-
ing the croton oil and the oxazolone ear edema assays
further described in the examples below. Therefore,
other classes of nonsteroidal compounds with topical
anti-inflammatory activity are of interest.
~Z~3566~
B adrenergic agonists tincluding ~1 and B2
agonists) are compounds which have been proposed to
act through the stimulation of adenylate cyclase,
resulting in the conversion of adenosine triphosphate
(i.e., ATP) to cyclic 3',5',-adenosine monophosphate
(i.e., C-AMP). See, for example, R. J. Brittain, et
al, Adv. Drug Res. 5, 197, 1970. The walls of essen-
tially all nucleated mammalian cells contain the
enzyme adenylate cyclase, which is stimulated by vari-
ous compounds including prostaglandin E and B-adrener-
gic drugs.
Adenylate cyclase activity has been reported to
be present in human and animal epidermis. Disorders
in adenylate cyclase activity and in C-AMP levels have
been reported in proliferative skin diseases ~uch as
eczema, psoriasis, epidermolytic hyperkeratosis and
lamellar ichthyosis.
In short, B agonists are a class of compounds
which stimulate the adrenergic system of the human
body.
Materials which are classified as Bl agonists
are B agonists which selectively react with the B
receptors and elicit cardiac stimulation.
Materials which are classified as B2 agonists
selectively react with the B2 receptors which are
present in the smooth muscles of the blood vessels and
bronchi; these materials elicit bronchodilation and
vasodilation.
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~Z35~
In V. S. Patent 4,323,575 to G. ~ones, April 6,
1982, disubstituted catecholamines (which may or may
not be ~2 agonists) having topical anti-in~lammatory
activity are disclosed.
In U.S. ~atent 3,341,584 to Larsen et al sulfon-
anilides having the general formula I are disclosed.
~ Ik- ~ ~ormula
X
As disclosed in that patent, the ~ulfonanilides of
fo:mula I, wherein Z i9 C~O~, are pharmacologically
active phenethanolamines having actions which either
resemble the effects of the adrenal medullary hormones
or adrenergic naurotransmitters or oppose the effects
of the adrenal medullary hormones or adrenergic neuro-
transmitters. Alkyl and aryl-sulfonamido nuclearly
substituted phenalkanolamines have useful pharmacolog-
ic effects, suiting them variously as vasopressors,
vasodepressors, analgesics, bronchodilators, -recep-
tor stimulants, B-receptor stimulants, a -receptor
blocking agents, B-receptor blocking agents, papa-
verine-like smooth muscle depressants, or anti-inflam-
matory agents useful in controlling or preventing ana-
phylaxis.
Anaphylaxis is defined in the McGraw-Hill
Dictionary of Scientific and Technical Terms, Second
Edition, 1978, as hypersensitivity following parenter-
al injection of an antigen, whereln local or systemic
- 4 -
-~3
~Z3SÇ~Z
allergenic reaction occurs when the antigen is rein-
troduced after a time lapse. Topical is defined to be
"local or designed for local application" and that
term is so used in this application. Therefore,
because anaphylaxis and topical inflammations are dif-
ferent conditions physiologically, a drug which is
useful in treating one of these condi~ions is general-
ly not useful in treating the other condition.
In U.S. Patent 3,801,631, to Comer et al.,
patented April 2, 1974, 2-hydroxy-5'-[1-hydroxy-2-(2-
methyl-l-phenyl-2-propylamino)ethyl]methanesulfon-
anilide, called zinterol (which is included within the
broad genus of sulfonic acid amides disclosed in U.S.
Patent No. 3,341,584 cited above) is disclosed.
Zinterol was there described as a potent anotexigenic
agent, as an orally active bronchodilator, and as
having analgesic activity.
In the article "Adrenergic Sulfonanilides. 4.
Centrally Active ~-Adrenergic Agonists", D. L. Temple
et al, Journal of Medicinal Chemistry, Vol. 19, No. 5,
Pgs. 626-633 (1976), zinterol (compound No. 43) is
described as a potent anorexiant and as a narcotic
antagonist.
Additionally, in U.S. Patent 3,919,424 and in
U.S. Patent 3,993,776, further description of the uses
of zinterol is given.
Salbutamol is a B2 agonist. This material was
described in R. Seely et al, Proc. Soc. Exp. Biol.
Med. 159, 223 (1978) a~ being useful as a topical
anti-inflammatory agent.
. .
~;~356~iZ
The synthesis of salbutamol is described in Drugs
of the Future IV, 629 (1979). There, s~lbutamol is
indicated as being useful as an anti-inflammatory
agent when applied locally. It is further stated that
salbutamol given orally in the control of asthma com-
pares favorably with related drugs. A mechanism for
the action of salbutamol is proposed. (See page 631
of the reference.)
A 1980 publication by Saiichirou Seo et al,
"Inhibition of Adjuvant Arthritis by Salbutamol and
Aminophylline," European J. of Pharmacology, 63,
267-274, 1980, describes inhibition of swelling in the
paws of mice by injections of combinations of sal-
butamol and aminophylline.
Other materials showing some structural similari-
ty to zinterol and having topical anti-inflammatory
activity are disclosed in U.S. Patent 4,323,575 to
Jones. These materials may or may not be ~-agonists
and only testing would determine whether they are.
In U.S. Patent 4,088,756 to Voorhees, other
~-agonists which may or may not have anti-inflammatory
activity are disclosed.
However, as further described below, which ~2-
agonists will be effective topical anti-inflammatory
agents cannot be predicted with any reasonable degree
of certainty. After mllch experimentation, applicants
found that nearly all ~2 agonists they tested for
such activity were either ineffective, highly toxic,
or both.
~Z356~;2
Therefore, despite what has been known in the
prior art, there is a continuing need for non-steroid-
al anti-inflammatory drugs which exhibit consistently
good anti-inflammatory activity and which are nontoxic.
A very large body of prior art exists for
fl-adrsnergic agonist compounds of the catechol-type
phenethanolamine series.
Larsen, et al., U.S. Patent 3,341,584 cited above
broadly disclosed catechol type phenethanolamines
wherein one of the phenyl ring hydroxy groups was
replaced with sulfonic acid amido thereby giving
compounds with B-adrenergic biological activity.
Robinson, U.S. Patent 2,908,691, patented
October 13, 1959, disclosed a broad group of hydroxy-
phenalkylaminoalkylindoles specifically described as
having various effects on the central nervous system
as well as acting as antisecretory agents, effective
in reducing gastric acidity. The most relevant
compound of this reference would seem to be 3-(2-[2-
hydroxy-2-(3,4-dihydroxyphenyl)ethylamino]propyl)indole
tartrate. This compound was prepared as Example 7 in
the reference.
An object of this invention i9 a material which
when placed into a suitable vehicle provides a compo-
sition which when topically applied reduces the amount
of topical inflammation of a mammal.
Another object of this invention i5 a composition
in the form of an ointment, cream, lotion or other
formulation to be topically applied to a mammal so as
1~356~
to reduce or hinder the development of skin
inflammation.
A further object of this invention is a method of
using a compound (or compounds, in a mixture) for the
purpose of reducing topical inflammation of mammals.
The above-described objects are satisfied by the
compositions of the present invention, which comprise:
(a) An amount effective to produce a topical
anti-inflammatory effect of at least one
compound (or pharmaceutically acceptable
salt(s) or solvate(s) thereof) selected from
the group consisting of compounds having the
general formula II R
? ~H-C~ A- c-B~
, f~
wherein R and R are independently H or
a lower alkyl group, provided that R and
R cannot both be H, M is either ~, a
phenyl group, or an indole group of formula
(a)
~ (~),
3566Z
A is ~ CH2-)n in which n i9 the integer
0, 1, or 2, and B ls (-CH2)m ln which m
i8 the $nte~er 0, 1, or 2, R is either
-O~ or -OCO ~ C~3, and R i8 either
-N~_~02-CH3 or -oCO ~ 3
excluding ~he compounds of formula II
wherein collectively R is -CH3 and R
is H or -CH3; M is an indole group; A is
the group (-CH2-)n, in which n is 0; B
is the group (-CH2-)m, in which m is 0;
R is -OH and R ls -NH-SO2-CH3; and
(b) A compatible, topically acceptable vehicle
for combining said ite~ (a) given above therewith.
In a pr~ferred ~apect of th~ lnventlon, Rl and
R ar~ both ~ethyl groups ~nd ~ and n nr~ both 0. A
preferred compound for use in the methods and composi-
tion~ of the invention is the compound of formula II
wherein n is 0, m is 0, Rl is -CH3, R is -CH3,
M is phenyl, R i5 -OH, ~nd R is -N~-SO2-CH3.
This compound is known as zinterol (referred to
here~nafter as compound III).
Another preferred compound for use in the methods
and composition-~ of the invention i8 the compound of
forDula II wherein n is 0, m is 0, sl is -C~3,
R is H, M i~ an indole group, R is -O~, and R
is -N~-SO2-C~3, ~hich compound i~ hereinafter
referred to as Compound IV.
Yet another preferred compound for use in the
methods and oompositions of the invention iq the com-
pound of formula I~ wherein n i~ 0, m i8 O, Rl and
R are both -CH3, M is an indole group, R i9
-OH, and R is -NH S02-CH3, which compound is
hereinafter reerred to as Compound V or azazinterol.
~'~`~
_ 9 _
~3St;6Z
A still further preferred compound for use in the
methods and compositions oP the invention is the com-
pound of formula II wherein n is 0, m is 0, R and
R are both -CH3, M is hydrogen, and R and R
are both -OCO ~ CH3. That compound is hereinafter
referred to as bitolterol and is commercially avail-
able for use in treating allergies but has not been
known previously to be useful for treating topical
in1ammations.
In another aspect of the invention, a method for
reducing topical inflammation in mammals comprises:
applying a compound of formula II topically to the
mammal so that localized (as opposed to systemic)
activity against topical inflammation results.
Further, according to tbe invention, a composi-
tion to be topically applied to reduce the amount of
topical inflammation of mammals comprises at least one
compound of formula II present in a nontoxic amount
sufficient to reduce inflammation and present in a
pharmaceutically acceptable carrier material or
materials, wherein A, 8, R , R , R , R and M
are as described above.
In another preferred aspect of the invention, a
composition to be topically applied comprises at least
one compound selected from the group consisting of
zinterol, compound IV, compound V, and bitolterol, at
least one compound of which is present in an amount
sufficient to reduce inflammation but insufficient to
be toxic and present in a pharmaceutically acceptable
carrier.
~ 10 -
~Z3566Z
It is emphasized that the term "topical" as used
throughout this document means local or designed for
locai application to produce a local effect with
preferably no concomitant systemic effect. Thus, the
compounds to be used in the methods and compositions
of the invention can be applied in any of a variety of
ways, provided that they are not injected or swallow-
ed. They can be applied, for example, cutaneously,
nasally, vaginally, rectally, otically, and buccally.
They will be used with a dermatogically acceptable
vehicle preferably chosen such that systemic absorp-
tion of the active ingredient is hindered or reduced.
This ir.vention also concerns antiasthmatic agents
which are bronchodilators and potent yet selective
inhibitors of smooth muscle contraction. The poten-
cies and selectivities of these agents in inhibiting
smooth muscle contraction caused by antigen-induced
release of chemical mediators has been demonstrated in
pharmacological tests utilizing immunized guinea pig
tracheal rings. These agents include compound IV and
compound V and their pharmaceutically acceptable
solvates and salts thereoi, and the invention includes
their utilization as antiasthmatic agents.
The compound(s) which are to be placed into a
vehicle so as to provide a composition(s) suitable for
topical use as an anti-inflammatory preparation(s) in
mammals are the compounds of formula II, recited
above, (or pharmaceutically acceptable salts and sol-
vates thereof), wherein M is either a phenyl group, or
an indole group or hydrogen, wherein A is (-CH2-)n
and wherein n equals 0, 1, or 2; wherein B is
(-C~2)m and wherein m is 0, 1, or 2; wherein R
. .
~Z35~i6Z
and R are independently ~ or a lower alkyl group,
provided that R and R cannot both be H; wherein
R is either -OH or -OCO ~ C~3; and wherein R
is either -NH-SO2-CH3 or -OCO-~ C~3.
Applicants wish to emphasize that they tested
many ~2 adrenergic agonists (all of which are
analogs of zinterol). Of approximately 45 such com-
pounds, only four had consistently high topical anti-
inflammatory activity without apparent toxicity in
tests which are described in the examples below. The
remainder of the compounds, on the other hand, exhi-
bited either toxicity when applied topically to the
test animals, ineffective and/or inconsistent anti-
inflammatory activity, or both.
The compound(s) to be placed into a vehicle so as
to provide a composition suitable for topical use as
an anti-inflammatory preparation in mammals are
prepared in the following manner.
The preparation of zinterol is described in
detail in U.S. Patent No. 3,801,631 to William T.
Comer et al, ~2'-hydroxy-5'-[1-hydroxy-2-(2-methyl-1-
phenyl-2-propylamino)ethyl]methanesul~onanilide and
Its Salts~.
As used herein, ~e stands for a methyl group. A
detailed description of the preparation of compounds
IV and V is the following. Compound IV and V can be
prepared by selecting from two general methods. $he
first synthetic method shown hereinafter/
.. .~ , .
.
`` ~2356~2
Method 1
H H2 ~ j Compound IV
H2N ~ catalyst
- ~. :.. :
.. ,~ . .. .
~b)
VIII
involves reductive amination of an indolylcarbonyl
compound with an appropriate phenethanolamine. Choice
of reagents and conditions for reductive aminations
are well known to those skilled in the art. In
general, the reaction is carried out by shaking a
solution of the appropriate carbonyl compound and
phenolic amine in a solvent such as a lower alkanol,
e.g. methanol, in the presence of a hydrogenation
catalyst, e.g. a noble metal catalyst such as platinum
oxide, in a hydrogen atmosphere. As an alternative,
the reaction could also be carried out stepwise by
first forming the condensation product of the carbonyl
compound and the phenolic amine and then conducting
the hydrogenation as a separate operation.
A variation of synthetic method 1 entails
nucleophilic displacement by the phenolic amine on an
indolylalkyl halide or an equivalent. This i9 shown
below as ~ethod lA.
- 13 -
~ . .. . .. ...
3S6~
Meth~d lA
" ~ Compound IY (if R=H)
(VIII) or
N Compound V (if R=Me)
tc)
wherein X is a typical leavin~ group such as halide,
tosylate, etc. Again, choice of reaction conditions
and reagents for nucleophilic displacement reactions
are well known and would be familiar to one practiced
in the chemical arts.
The second process which can be used for
preparation of compound IV or compound V is shown
below as general synthetic method 2. This general
method can also be used for the pteparation of
compound V as shown.
Method 2
~<R'd2 r~J~
tX) tIX) Compound IV (if R~H)
or
Compound V ~ if R=Me)
- 14 -
~Z3566~2
This process comprise~ alkylation of the phenolic
bromoketone by the appropriate indolylalkylamine
followed by reduction df the carbonyl group to a
secondary alcohol. In practice, the phenolic OH group
is protected during the nucleophilic displacement
reaction. This is done to prevent participation by
the phenolic group in nucleophilic attack of its own
thereby giving unwanted ether byproducts. Generally,
the protection is done via a benzyl group which is
subsequently removed by catalytic reduction.
These general synthetic methods have been incor-
porated into the actual synthetic schemes used to
produce compounds IV and V. ~hese specific schemes
are outlined below.
Scheme A: Preparation of Compound IV
! e 8r
3r ~ C6~1~R4 ~ > C ~ ~ N~52C~3
IX
Q 1 EtOH
~Cl
OH HCl
H2~o~s2c~3 ~eo~ J~,~SO2CH3
VIII XI
Pe2 1 ~ - 15 -
~35~Z
011
X2 ~ Pd/C
~eOH
R ~ ~
o - N2 ' :,
2) NaB~
XII, R=H ~eO~
R O
NH2 Br ~ ~HcH22h~3
X, R=H IX'
Scheme A outlines the preparation of compound IV.
Two pathways are depicted, both beginning with one of
the bromoketones, IX and IX'. In the upper pathway IX
is reacted with hexamethylenetetramine tC6H12N4)
to yield a quaternary salt which is converted to the
aminoketone XI followed by catalytic hydrogenation to
the phenethanolamine VIII. Reductive alkylation of
3-indolylacetone with VIII affoxds the subject compound
IV as shown. ~he lower and preferred pathway proceeds
via nucleophilic attack of the indolylamine X or IX'
(the O-benzyl analog of IX) followed by borohydride
reduction to give the benzyl-blocked phenolic group
intermediate (XII: R~) as shown which iq in turn
catalytically reduced to the desired end product.
- 16 -
~Z3S6~Z
Scheme B: Preparation of Compound V
BrJ~S02C~31) ~ x
2) NaBH4
X: R=Me ~_ ' EtOH ,
R ~ O~
N~S02C~3
H tXII: R=Me )
Pt2 1 ~e
X2
OH
~lSOzCH3
Compound V
-- 17 --
,,
6~Z
Scheme B depicts the preparation of compound V
utilizing essentially the same pathway as shown in the
lower part of Scheme A above. In Scheme 8 the
appropriate indolylamine is reacted wlth IX', and the
resulting adduct is reduced with borohydride to give
the protected phenolic compound (XII: R=Me) as shown
which is then converted via catalytic hydrogenation to
the desired subject compound compound V.
These two synthetic schemes, supra., will be
exemplified in greater detail hereinbelow. Intermedi-
ate compounds utilized in these syntheses are either
available commercially, e.g. 3-indolyl acetone; or as
described in the chemical li~erature such as the
references cited in the Background of the ~nvention
section hereinabove.
The preparation of bitolterol is discussed in
U.S. Patent 4,138,581.
For medicinal use, the pharmaceutically accept-
able solvates and salts are those complexes in which
the solvent, metal cation or acid anion does not
contribute significantly to toxicity or pharmacologic-
al activity of the organic drug ion. The sulfonamido
group is the acidic function utilized in metal salt
formation. Examples of metal salts include the
sodium, potassium, calcium, magnesium, aluminum and
zinc salts. ~etal and acid addition salts are
obtained, respectively, either by reaction o~ the
selected compound with a ~uitable metallic base to
form a metal salt or with an organic or inorganic acid
to form an acid addition salt, preferably by contact
- 18 -
~ ~.
1~3566Z
in solution, or by any of the standard methods detail-
ed in the literature and available to any practitioner
skilled in the art. Examples of useful organic acids
are carboxylic acids such as maleic acid, acetic acid,
tartaric acid, propionic acid, fumaric acid, isethion-
ic acid, succinic acid, pamoic acid, cyclamic acid,
pavalic acid, and the like. Useful inorganic acids
are hydrohalide acids (such as HCl, HBr, HI), sulfuric
acid, phosphoric acid, and the like.
Solvates as used herein are complexes comprising
an organic drug molecule and a solvent moiety of
formula ROH, wherein R most commonly is hydrogen or a
Cl or C4 alkyl group. The most common solvate is
the hydrate.
It is also to be understood that the compounds of
the present invention include all the optical isomer
forms, that is, mi~tures of enantiomers, e.g., racemic
modifications as well as the individual enantiomers
and diastereomers. The individual optical isomers of
the phenethanolamine class of compounds of which the
instant compounds are members, have most generally
been obtained by one of four basic methods. These
are: 1) the fractional recrystallization of chiral
acid salt derivatives; 2) derivatization with a
chiral organic reagent, resolution, and regeneration
of the original compound in optical isomer form; 3)
synthesis of the single optical isomer using chiral
intermediate~; and 4) column chromatography utilizing
chiral stationary phases. The application of these
various methods are well known to practitioners in the
art.
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1;~3566;~
The compounds recited above which are to be
placed into a vehicle so as to provide compositions
suitable for topical use as anti-inflammatory prepara-
tions in mammals can be placed into the following
vehicles. The resulting mixtures are pharmaceutical
preparations of the invention. The vehicle can be any
nontoxic material or mixture of materials which is
suitable for use in preparing pharmaceutically accept-
able ointments, salves, lotions, sprays, suppositories
and other similar medicaments. The vehicle, addition-
ally, will be chosen so that it preferably hinders or
reduces systemic absorption of the active material(s)
and it should not react with the active ingredient(s)
described above. Additionally, the active ingredi-
ent(s) should be both soluble in the vehicle and
should be released by the vehicle topically. Fur-
thermore, the mixtures so formed will preferably be
stable over an extended period of time, for example on
the order of months or years.
The active ingredient(s) will generally be dis-
solved into a component of the vehicle. For example,
zinterol hydrochloride is both water soluble and
soluble at least to some extent in various organic
materials. For topical applications to the skin,
because there is both an aqueous phase and a non-
aqueous phase in the ~kin, both water soluble and oil
soluble portions of the vehicle will permeate the
skin. However, for topical use, one would use some
organic phase in the vehicle (for example, petrolatum
or mineral oil).
Vehicles for carrying active ingredients into the
skin, for example, creams, lotions, gels, ointments,
- 20 -
.. .
:~356i~Z
suppositories, and sprays, as well as methods of pre-
paration thereof, are well known in the art. In the
present invention, at least one active ingredient will
be dissolved in a portion of the vehicle in which it
is soluble, and the resulting mixture ~7ill then be
mixed in any suitable way with the remaining ingredi-
ents of the vehicle.
The relative amount of vehicle to be mixed with
active ingredient(s) (i.e., with the compounds des-
cribed above) in forming the mixtures of the invention
will be selected depending upon the solubility of the
active ingredient(s) in the vehicle. However, it is
believed that the optimal concentration is generally
the saturation point. For zinterol hydrochloride,
however, the optimal concentration thereof in a cream
vehicle was found to be 0.2 w/v percent, although up
to 0.7 w/v percent thereof will dissolve in creams.
The mixtures of the invention will be administer-
ed in the following way. Based upon the tests des-
cribed in the examples below, the mixtures of the
invention prepared from active ingredient(s) in suit-
able vehicle should be applied as soon as possible
after the skin has come into contact with the
material(s) tha~ caused the inflammation being treated.
The mixtures of the invention will b~ applied
directly to the area of inflammation to produce a
localized effect. Although in salbutamol (discussed
above) a systemic effect was noted, none was found in
preliminary tests done on the materials used in this
invention. It is an advantage to have no ~ystemic
- 21 -
`~ ~ 2 3 S 6 6 Z
effect and to have minimal absorption of these
materials.
Additionally, biological testing of compounds IV
and V demonstrates that they possess intrinsic bron
chodilator action and they are able to reverse antigen
induced tracheal contraction~ This contractile res-
ponse to antigen has been characterized as consisting
of an initial spasm caused by release of preformed
histamine followed by a sustained contraction due to
the release of newly synthesized SRS-A (slow reacting
substance of anaphylaxis) (c~: Brockl.ehurst: The
Release of ~ist.amine and Formation of a Slow-Reacting
Substance (SRS-A) Durlng Anaphylactic Shock, Journal of
Physiologyl 151:416-435, 1960). The ability of the
subject compounds to inhibit the contractile response
mediated by SRS-A with significantly greater inhi-
bitory potency compared with the released histamine
contractile response demonstrates an advantage in
electivity for the subject compounds which would make
them particularly useful as antiasthmatic agents. The
utility of compounds IV and V in this regard can be
demonstrated in various pharmacologial tests which
include inhibi~ion of methacholine-induced broncho-
spasm in rats, and inhibition of smooth muscle con-
traction caused by antigen-induced release of chemical
mediators in tracheal rings isolated from immunized
guinea pigs. This latter method has been adapted
from Adams and Lichtenstein: In Vitro Studies of
Antigen induced Bronchospasm: Effective Antihistamine
and SRS-A Antagonist on Response of Sen~itized Guinea
Pig and ~uman Airways to Antigen. Journal of
Immunol., 122:555-562, ~1979).
- 22 -
`~;~!;~ . .
s~
For use as antiasthmatics, therapeutic processes
of this invention comprise systemic administration, by
both oral and parenteral routés as well as by inhala-
tion of an effective, nontoxic amount of compound IV
or compound V or a pharmaceutically acceptable salt
thereof. An effective amount is construed to mean a
dose which exerts the desired pharmacological acti-
vity, such as those stated hereinabove without undue
toxic side effects when administered to a mammal in
need of such treatment. Dosage will vary, according
to the subject and route of administration selected,
with an expected range of about 0.1 mcg to 100 mg/kg
body weight of a compound of Formula IV or V or a ~harma-
ceutical acceptable acid addition salt thereof
generally providing the desired therapeutic effect.
Compounds IV and V can be formulated according to
conventional pharmaceutical practice to provide
pharmaceutical compositions of unit dosage form
comprising, for example, tablets, capsules, powders,
granules, emulsions, suspensions, and the like. These
preparations contain the active ingredient, usually in
admixture with nontoxic pharmaceutical excipients, to
give solid dosage forms or as a solution, suspension,
or emulsion to give a liquid preparation. It is
understood that other standard pharmaceutical prac-
tices also apply such as the addition of sweetening
and flavoring agents or use of binders, etc. Further,
the compositions may also contain other absorbing
agents, stabilizing agents, wetting agents and buffers.
Additionally, liquid preparations of compounds IV
and V may be used for administration by inhalation
given, for example, by nebulization. The instant
- 23 -
. ~ . .
356~
compounds can also be administered as a powder for
insufflation, consisting of a blend of inert powder
ingredients admixed with an appropriate amount of the
instant compound of appropriate particle size, admi-
nistered by a powder in~ufflation device. Generally,
one part micronized drug is blended with 50 parts USP
lactose having appropriate microbial properties. This
blend is encapsulated for use in a suitable insuf-
flation device. Prior to use, the capsule must be
punctured or opened to allow release of the powder
blend.
EXAMPLES
In examples 1-4, the following types of tests
~i.e., models) on animals were used. These were ~1)
croton oil-induced ear edema in mice, (2) oxazolone-
induced ear edema in mice, and (3) UVB-induced
erythema in guinea pigs.
Example 1
In the croton oil assay, (which is a standard
test, which is fully dejcribed in Tonelli et al.,
Endocrinolo~Y, vol. 77, pp. 625-634, 1965), topical
application of four ~ croton oil in ethanol (v/v~ to
the ears of mice causes intercellular edema, vasodila-
tion, and polymorphonuclear leucocyte infiltcation
into the dermis, leading to an increase over normal
- 24 -
:
?
3566~
ear wei~ht of about 70 to 100%. The inflammatory res-
pon~e is nearly maximal by 6 hours. In the croton oil
tests, four volume ~ croton oil in ethanol ~as applied
to the inner aspect of both ears of each test mouse,
and various test materials in vehicle systems ~ere
applied to the outer aspect of the ears immediately
following croton oil application. Control animals
were exposed either to croton oil alone or to croton
oil followed by the vehicle alone.
Six hours after exposure to croton oil and/or
test material, animals were sacrificed; and punch
biopsies of the ears were weighed and compared to the
respective vehicle control.
Compounds were tested in simple solutions,
including dimethylacetamide/acetone/ethanol (i.e.,
D~AC/A/E v/v 40/30/30) and N-methyl pyrrolidone/
ethanol (NMP/E v/v 50/50). Comparative controls were
chosen based on their known activity in each of the
three above-described animal assays and included in
all three tests (in Examples 1, 2 and 3) hydrocorti-
sone valerate (HCV) in the croton oil and oxazolone
assays, indomethacin (which is a potent ASPIRIN-like
nonsteroidal anti-inflammatory agent) in the UVB test,
and salbutamol (a B2 agonist, discussed above in the
aackground of the Invention).
The percent inhibition of induced mouse ear edema
(or erythema) for each of the three models (in
Examples 1, 2 and 3) is calculated:
Control Ear Weight - Test Ear Weight
Control Ear Weigh~ X100
* Trade mark for acetylsalicylic acid (ASA)
- 25 -
~3566Z
The croton oil assay appeared to be more sensi-
tive to steroidal anti-inflammatory agents than to
ASPIRIN-like nonsteroidal anti-inflammatory agents.
Unexpectedly, unlike the ASPIRIN-like nonsteroidal
anti-inflammatory agents, the B2-agonists used in
this invention were effective in reducing the croton
oil-induced inflammation.
The anti-inflammatory activities of approximately
45 ~2-adrenergic agonists were evaluated in the
croton oil-induced mouse ear edema assay (which pro-
duces acute dermatitis); and the more active compounds
were subsequently tested in the oxazolone-induced
mouse ear edema assay (which produces contact allergic
dermatitis) and in the UVB-induced erythema assay in
guinea pigs.
Out of the group of approximately 45 compounds
which included zinterol and analogs thereof, four com-
pounds (one of which was zinterol) demonstrated high
topical cutaneous anti-inflammatory activity in the
croton oil assay at 1.6 w/v % (weight/total volume
ethanol+test material). These four compounds were
subsequently tested topically at other concentrations
in the croton oil assay and were also tested topically
in the oxazolone assay and in the UVB test. In these
subsequent ~ests, zinterol appeared to be the most
consistently active compound.
Given below in Table I are the results of zin-
terol and the controls salbutamol and ~CV, at 1.6 w/v%
and 0.2 w/v% in the croton oil assay in each oi two
solvent systems. Also included in Table I is data for
bitolterol, a commercially available B2 agonist
* Trade mark for acetylsalicylic acid (ASA)
- 26 -
~356~;2
which has previously been used as an anti-allergy com-
pound but which has not previously been known for
utility as topically active against cutaneous inflam-
mations. A direct comparison of bitolterol and zin-
terol was made. Both exhibited similar topical anti-
inflammatory activity.
The results in Table I show that in the croton
oil assay, ~interol at 1.6 w/v percent and at 0.2 w/v
percent and bitolterol at 1.6 w/v percent all showed
good to moderate reductions in ear edema and were
equivalent to or slightly less effective than hydro-
cortisone valerate (i.e., ~CV) but were more effective
than salbutamol~
Example 2
Oxazolone-induced contact sensitization in mice
is characterized by edema and cellular infiltration,
primarily of the monocyte type, with close to 100%
increase in the mouse ear weight. ~This model is
fully described in N. J. Lowe et al., British J. of
Dermatoloqy, vol. 96, pp. ~33-438, 1977. In this
model, ~est materials were applied topically to the
outer aspect of the challenged ear of each test animal
im,lediately following the challenge application of
oxazolone to the inner aspect of the ear. The animals
were sacrificed at 8 or 24 hours after treatment; and
punch biopsies of the ears were weighed and compared
to controls which were challenged as described above
and exposed to the vehicle alone~
~`~``.;
.~, .
1;~35~
Table I
% Inhibition
of Croton Oil-Induced ~ouse Ear Edema
in Two Vehiclesl
~-7r 5~'TI~8;77EIOH~ In N~P/EToH3
Compound l.~ wT X ~ vX 1.6 w/vX 0.2 w/v%
Zinterol 69a, 50b 54h,34i 69m, 63n 92r, sgs
81C, 66d 29j~6k 700~ 48P 56t, 44u
63e, 48f 201 73q 57v, 3gx
45~ ~4Y~ 61~
Bitolterol 61 42X, 25Y
34Z
Salbutamo14 26a~ ob _34j*,_45k 54m, 4gn
23, OP
37q
HCV 78C, 59d 25i, 38k 70n~ 71q 71r, 625
16e, 48f 341, 64t, 67U
599 73v
i 1 Each value is the mean of 10 to 15 animals. Approximately 10
to 35X variability is observed in this test.
2 Dimethylacetamide/Acetone/Ethanol tV/Y3 40/40/30)
3 N-methyl 2-pyrrolidone/Ethano~ (v/v, 50/50).
4 Tested in Ethanol/H~O (50/50) due to so~ubility limitations~
a to Z Yalues witn the same alphabetical superscript were
obserYed in the same experiment.
* The minus sign indicates no inhibition, but rather
potentiation, of the inflammation.
28
lZ356fi;~
The oxazolone assay appeared to be sensitive to
steroidal anti-inflammatory drugs and relatively in-
sensitive to nonsteroidal anti-inflammatory drugs.
Again, unexpectedly, unlike the ASA-like nonstero-
idals tsuch as indomethacin), the ~2-agonists showed
topical anti-inflammatory activity.
In Table II, the results of tests on percent of
inhibition of oxazolone-induced edema in mouse ears
using various concentrations of zin~erol, salbutamol,
or HCV as active ingredient are given for oxazolone in
three solvent systems of DMAC/acetone/ethanol (v/v
40:30:30).
From the results in Table II, in the oxazolone
assay, one can observe that zinterol at 3 and 1.6
weight percent showed slight reduction in ear edema
with no dose-related effect and was equal to
salbutamol but slightly less active than ~CV.
Compounds in Tables I and II can be compared
directly.
Example 3
Another series of tests were run for the sake of
completeness, although it was not expected that ~2-
agonists ~which are vasodilators) would show resul~s
comparable to the ASA-like nonsteroidal agents
(which are not vasodilators). In the UVB tes~,
cutaneous erythema is induced in guinea pigs. This
test is a standard test widely u3ed for testing anti-
inflammatory agents and i8 fully described in R. F.
- 29 -
! ,. J
1~356~2
Table II
% Inhibition X Inhibition
of Oxazolone-Induçed of Oxazolone-Induced
Edema in ~ouse Earl-2 Edema in Mouse Ear2~3
Concentration Concentration
Compound _0.2 w/v% 1.6 w/v% ~ ~ 7~ 7V~-
Zinterol 22a~ _17b 48C~ 17d 35h, 30i 391
4le 14i, ~3k
Salbutamol _la, _gb 2c, od9 21h, 281 311
HCY 16a, 16b 36C, 27d 38h, 8i 391
31J, 42k
1 Above agents tested in DMAC/acetone/ethanol ~v/Y 40:30:30).
2 Each value is the mean of 10 to 15 animals. Approximately 20
to 35X variability is observed in this test.
3 Above agents tested in N-methylpyrrolidone:ethanol (v/v 1:1).
a to 1 Values with the same alphabetical superscript were
observed in the same experiment.
- 30 -
~3~6~2
Swingle, ~Evaluation for Anti-Inflammatory Activity",
in Anti-Inflammatorv Agents, vol. 2, ed. by Scherrer
and Whitehouse, pp. 34-122, London: Academic Press,
1974. In the UVB model, the test material was applied
topically to the irradiated sites immedia~ely following
exposure to UVB. Erythema was scored on a O to 4 scale,
3 and 6 hours after irradiation.
In Table III, the percent changes in UVB-induced
erythema in guinea pigs at 3 and 6 hours after treat-
ment with zinterol are given, along with results of
treatment with salbutamol and indomethacin.
As shown by the resul~s in Table III for the UVB
assay, zinterol at 3 and 1.6 weight percent showed
slight to moderate activity with no consistent dose
effect and was highly variable. Similar effects were
seen with salbutamol. However, indomethacin at 1
weight percent showed good to very good activity on a
consistent basis.
The results given above in Tables I, II and III
are summarized below in Table IV.
Based upon the summary in Table IV, one can
validly conclude that, at the same concentrations,
zinterol appears to be almost as effective a HCV and
more effective than salbutamol in the croton oil
assay. Therefore, zinterol is a promising candidate
for reducing anti-inflammatory activity in hu~ans,
based upon the data disclosed above. Zinterol is
expected to be devoid of many side efiects which are
exhibited by the current steroid therapy.
- 31 -
1235~6Z
Table III
% Change in UVB - Induced Erythema
in Guinea Pig at 3 and 6 Hours after Treatmentl
Drug Concentration tX)2 Drug Concentrat;on ~%)3
Time (Hr.~ _
Compound ~ 3 6 3 6
Zinterol 0 13 NT* 9 26 48 18
37 56 75 25
9 19
. 27 +9
Salbutamol4 9 +4 +39 ~39 42 32
74 71 45 25
Indomethacin 72 43 100 74
4 48 93 85
82 5~ 91 87
1 All test material applied immediately post-irradiation.
Test material prepared in Dimethyl acetamide/acetone/ethanol
(v/v, 40/30~30) and given as w/v~.
Test material prepared in N-methyl 2-pyrrolidoneJethanol (v/v,
50/SO and given as w/v%.
4 Tested in Ethanol/H20 (50/50) or N~P/H20 (50/50) due to
solubility limitations.
*Not tested due to solubility 1imits at 1.6%.
- 32
~Z3S662
Table IV
Topical Anti-Inflammatory Activity of Zinterol
in 3 Animal Models
(Salbutamol, HCV and Indomethacin Tested as Comparative Controls)
Croton Oil Assay Oxazolone Assay UV-g Assay
(Edema) (Edema) - _ (Erythema)
Zinterol ++~ + +
Salbutamol + ~ +
HCV ++~ NT~
Indomethacin + + +~+
Anti-Inflammatory Activity and (range of % inhibition)
+ = Slight (30-44), +~ = moderate (45-59) and ~++ = high ( 60%)
lNT = Not tested.
. .
~Z35662
~xample g
In further testing, two analogs of zinterol, com-
pounds IV and V, were tested and were found to show
anti-inflammatory activity comparable to that of
zinterol.
In these tests, the anti-inflammatory effect of
these three ~2 agonists when applied topically to
the croton oil-induced ear edema in mice was inves-
tigated. In these tests, 50 ~1 of 4 weight percent
croton oil in ethanol was applied to the inner aspects
of the right and the left ears of Swiss albino mice,
followed immediately by 25 ~1 of suspensions of 0.02,
0.2, and 0.8 weight percent o~ compound IV and com-
pound V in N-methylpyrrolidone/Ethyl alcohol (i.e.,
NMP/ETOH) applied to the outer aspect of each ear.
Croton oil treated and nontreated control groups were
included. These control groups are included in the
data as described hereinbefore.
Six hours later, the animals were sacrificed with
C2 gas, and a 5/16 inch punch biopsy of each ear
was taken and weighed immediately.
The anti-inflammatory effects for the test agents
were assessed by a comparison of the biopsy weights of
the test and control groups.
The results of three studies for various concen-
trations of test material in NMP/ETOH are given below
in Table V and show that both compound IV and compound
V are comparable to ~interol in reducing the mouse ear
edema.
_ 34 -
~Z3S~62
Table V
X Reduction of Ear Edema ~eight as
Compared to Control Ears + S.D.
Test ~aterial
Concentration (%)* Compound IY Compound V Zinterol
0.02 10.6 29~9 14.2
25.6 23.6
Mean + S.D. 18.1+10.6 18.g+6.6
0.2 32.9 53.5 39.5
48.7 66.2
49.4 46.6
Mean ~ S.D. 43.7+9.3 50.8+13.8
0.8 61.6 69.9 68.2
53.4 56.6
Medn + S.D. 57.5+5.8 62.4+8.2
* Neight/Yolume
.
. . ~ata i.`~en on the samë ~in~ in Tables III and Y were
. ........... .... . . . .. . . . . .
`- obta~ned in the sam~ experiment, an~ therefore a direct
comparison is shown.
.. ...... .. . . .
1~35~Z
It will be appreciated that the compounds of
formula II can be formulated into a wide variety of
formulations by standard means well known to those
skilled in the art. Such formulations include for
example nasal sprays (one spray of which may be pre-
pared, for example, with trichloromonofluoromethane,
dichlorodifluoromethane, and oleic acid), rectal sup-
positories, vaginal suppositories, ointments, creams,
gels, and lotions.
The methods of preparation of compounds IV and V
and their biological actions will appear more fully
from a consideration of the following examples and
appended claims which are given for the purpose of
illustration only and are not to be con~trued as
limiting the invention in sphere or scope. In the
following examples, used to illustrate the forPgoing
synthetic processes, temperatures are expressed in
degrees Celsius and melting points are uncorrected.
The nuclear magnetic resonance (NMR) spectral charac-
teristics refer to chemical shifts ~) expressed as
parts per million (ppm) vereus tetramethylsilane (TMS)
as reference standard. The relative area reported for
the various shifts and the proton NMR spectral data
corresponds to the number of hydrogen atoms of a
particular functional type in the molecule. The
nature of the shifts as to multiplicity i reported as
broad singlet (bs), singlet (s), multiplet (m), or
doublet (d). Abbreviations employed are DMSO-d6
(deuterodimethylsulfoxide), CDC13 (deuterchloroorm)
and are otherwise conventional. The lnfrared tIR)
spectral descriptions include only absorption wave
numbers (cm ) having functional grouR identifica-
tion value. The IR determinations were employed using
r~
- ~3L235~16Z
potassium b~omide (~Br) as diluent. The elemental
analyses are seported as percent by weight.
Example 5
5'-(2-Amino-l-hydroxyethyl)-2'-hydroxy-
methanesulfonanilide Hydrochloride(VIII)
~ o a stirred solution of hexamethylenetetramine
(27.4 9, 0.19 mole) in 650 mL chloroform was added in
portions 5'-bromoacetyl-2'-hydroxymethanesulfonanilide
(IX; 40.0 9, 0.13 mole). The resyl~ing suspension was
refluxed for 16-18 hrs, cooled to room temperature,
and then filtered. The solid obtained was washed with
chloroform and dried in air to give 56.6 9 (97.3~) of
quaternary salt product, m.p. 16~-167.
This solid was dissolved in 400 mL ethanol and
treated with 65 mL conc. HCl. Refluxing the resulting
solution for several minutes caused the initiation of
precipitation in the hot solution. The mixture was
chilled to aid in completion of precipitation. The
solid, isolated by filtration, was washed by beiny
stirred in water and refiltered. Recrystallization of
this material from methanol-isopropyl ether gave a
solid product, 5'-glycyl-2'-hydroxymethanesulfon-
anilide hydrochloride, m.p. 219-221.
A portion (7.3 9. 0.026 mole) of this aminoketone
hydrochloride and 10~ palladium-on-carbon (2.0 g) were
suspended in 200 mL of hot 90 ethanol. Thi~ warm
suspension was reduced by shaking under 60 psi hydro-
gen on a Parr hydrogenation apparatus. Following
- 37 -
~35662
12-14 hrs of shaking, the hydrogenation mixture was
removed from the Parr apparatus and the catalyst
removed by filtration. The filtrate was concentrated
in vacuo to a white solid residue which was suspended
in an isopropyl alcohol-isopropyl ether medium and
refiltered to give a quantitative yield of ~III as the
hydrochloride salt, m.p. 179-180 (dec.).
Example 6
2'-Hydroxy-5'~ hydroxy-2-[3-t3-indolyl)-2-
pro~xlamino~ethYl)methanesulfonanilide (Compound IV)
The hydrochloride salt of VIII (5.65 g., 0.02
mole), prepared above, was converted to the base by
suspension in 150 mL of ethanol followed by treatment
wi~h 2.0 mL of 10.0 N NaOH added dropwise with stir-
ring. The original solid dissolved with concommitant
precipitation of NaCl. The NaCl was removed by fil-
tration and the filtrate was concentrated in vacuo to
a white solid residue which was dissolved in 100 mL of
methanol. A reductive alkylation was carried out by
adding glacial acetic acid (1.2 g., 0.02 mole) and
3-indolylacetone (3.5 g, 0.02 mole), diluting the
resulting solution to 150 mL with additional ethanol
and then adding 0.2 9 PtO2. This suspension was
hydrogenated under 60 psi hydrogen until hydrogen
uptake ceased (approximately 4 hrs). The suspension
was removed from the hydrogenation apparatus, filter-
ed, and the filtrate concentrated ln vacuo to an oily
residue which was dissolved in warm methanol treated
with several drops of glacial acetic acid. Dilution
with ethyl ether and stireing while chilling allowed
- 38 -
~3S~6Z
collection by filtration of a solid which was washed
with methanol to given 4.7 9 of the acetate salt of
compound IV, m.p. 196.5-197 tdec.).
This material was dissolved in a minimal amount
of hot dimethylformamide, filtered and diluted ~7ith an
equal amount of water to yield a precipitate which was
isolated by filtration giving the free base of
compound IV, m.pO 198-199 ~dec.).
nal. Calcd- for C20H25N3O4S: C, 59.53;
H, 6.24; N, 10.41; S, 7.94. Found: C, 59,96; ~,
6.15; N, 10.65; S, 8.05.
NMR (D~SO-d6): 0.98 ~3,m); 2.75 (5,m); 2.93
(3,s); 4.52 (l,m); 6.00 t4,bs); 7.15 (8,m).
IR (KBr): 750, 1125, 1150, 1240, 1280, 1325,
1500, 1610 and 2940 cm
Example 7
Alternate Preparation of Compound IV
A solution of 3-(2-aminopropyl)indole (11.0 g,
0.06 mole) in 730 mL acetonitrile was stirred under a
nitrogen atmosphere as 5'-bromoacetyl-2'-benzyloxy-
methanesulfonanilide (12.5 9, 0.03 mole) was added in
a single portion. After stirring at room temperature
for 0.5 hr, a cold solution of sodium borohydride (4.8
g, 0.126 mole~ in 219 mL me~hanol was added at a fast
dropwise rate. Progress of the reaction was followed
by disappearance of the bromoketone spot on thin layer
.
' ~ .
" :lZ3S66Z
chromatography. Additional sodium borohydride is
sometimes necessary for complete extinction of the
aminoketone starting material. When reaction was
complete, the solvent was removed in vacuo and the
residue suspended in 0.5 liter of H2O and treated
with 4N NaOH to bring about complete solution. This
solution was washed well with ether and then the pH
was adjusted with acetic acid to pH 8. The resulting
mixture was extracted with methylene chloride, the
extracts combined and dried (MgS04) and then concen-
trated in vacuo to give 14.5 9 of residual gum.
This gum may be purified by chromatographing on a
silica gel column eluting with chloroform-methanol-
ammonium hydroxide (90:10:1) to yield 11.6 g of the
benzyloxy derivative of compound IV.
The O-benzyl protecting group was removed by
hydrogenating a mixture of the O-benzyl derivative of
compound IV (11.5 g, 0.02 mole) and 1.8 g of 10%
palladium-on-carbon tmade wet with absolute ethanol)
in 820 mL methanol in a Parr low pressure apparatus.
Upon completion of hydrogen take-up, the reduction
mixture was filtered and the solid washed with addi-
tional methanol. All methanol portions were combined
and concentrated to a small volume (approximately 100
mL) and upon standing a white solid gradually preci-
pitated. The solid was isolated by filtration, washed
with methanol and dried in air to give 5.6 9 material,
m.p. 197-198 (61%~. This material was dissolved in
40 mL of hot dimethylformamide, filtered and 45 mL
H2O added to the filtrate. Trituration of this
solution induced crystallization. Another 10 mL H2O
was added and the mixture was chilled in an ice bath
- 40 -
:~35~6;2
following which the solid was isolated by filtration
and washed well with H2O. Drying in air provided 5
g of compound IV, m.p. 197-200 (dec.).
Example 8
N-(2-Hydroxy-5-[1-hydroxy-2-([2-(lH-
indol-3-yl)-l,l-dimethylethyl]amino)ethyl]
phenyl)methanesulfonamide (Compound V)
A solution of 2-~2-amino-2-methylpropyl)indole
(X, R=Me; 37.7 9! 0.2 mole) and triethylamine (10.1 g.
0.1 mole) in 1.2 liter of dioxane which had been dis-
tilled over sodium metal, was stirred under a nitrogen
atmosphere as 5'-bromoacetyl-2'-benzyloxymethane-
sulfonanilide ~39.8 g, 0.1 mole) was added. The
resulting mixture was left stirring for 8-12 hr at
approximately 25 under the nitrogen atmosphere. The
reaction mixture was filtered, removing some solid
precipitate, and the filtrate was treated with a cold
solution of sodium borohydride (15 9, 0.4 mole) in l
liter of absolute ethanol. The borohydride solution
was added dropwise to the stirred reaction filtrate.
Progress of ~he reaction was followed by disappearance
of the bromoketone spot on thin layer chromatography.
Additional sodium borohydride is sometimes necessary
for complete extinction of the aminoketone starting
material. When reaction was complete~ the solvent was
removed ln vacuo and the residue was dissolved in 0.2N
NaOH and washed with ether. The pH of this solution
was then adjusted with acetic acid to p~ 8 and this
resulting mixture was extracted with methylene
chloride, the extracts combined and dried (MgSO4) an
"` ~Z35~
then concentrated in vacuo to give the crude O-ben~yl
derivative-of compound V as a residual gum.
The O-benzyl protecting group was removed by
hydrogenating a mixture of the O-benzyl derivative of
compound V (3.25 g, 0.006 mole) and 1.0 g of
Pd(OH)z/C (Pearlman catalyst) in 100 mL methanol in
a Parr low-pressure hydrogenation apparatus. Upon
completion of hydrogen take-up, the reduction mixture
~as filtered and the solid suspended in water and lN
HCl added with warming so that product dissolved. The
insoluble catalyst was removed by filtration and the
acidic filtrate was made basic (pH 8) with ammonium
hydroxide. The resulting precipitate was isolated by
filtration, washed with water, and dried in air to
give a nearly quantitative yield of product (compound V)
as the monohydrate, m.p. 211-212.
21 27 3 4 2
57.92; H, 6.72; N, 9.65; H2O, 4.14. Found: C,
58.05; H, 6.68; N, 9.64; H2O, 3.63.
NMR (DMSO-d6): 1.01 (3,s); 1.05 (3,s); 2.75
(4,m); 2.92 (3,s); 4.49 (l,m); 5.30 (6,bs); 7.12 (8,m).
IR (KBr): 740, 1010, 1115, 1125, 1235, 1280,
1460, 1500, 1600, and 1610 cm 1.
The monohydrate product, obtained above, was con-
verted to the hydrochloride hydrate by dissolution in
dilute HCl followed by concentration in vacuo to a
solid foam, m.p. 105-125. Calcd. for C21H27N3O4S-HCl-H2O
C, 53.44; H, 6~41; N, 8.91; H2O, 3.82. Found: C,
53.19; H, 6.37; N, 8.92; ~2' 3.64.
- 42 -
lZ3566~
NMR ~DMSO-d6): 1.28 (6,s); 2.95 (3,s); 1.17
(4,m); 3.80 (l,bs); 4.90 (l,m); 7.02 (4,m); 7.25
(3,m); 7.60 (l,m); 8.50 (l,bs); 8.78 (l,bs); 9.30
(l,bs); 10.00 (l,bs); 11.10 (l,bs).
IR (KBr): 750, 960, llS0, 1295, 1320, 1400,
1460, 1515, and 1620 cm
- 43 -
