Note: Descriptions are shown in the official language in which they were submitted.
~119099
ANTI-ALLERGIC COMPOSITIONS OF 4,9-DIHYDRO-
4,9-DIOXO-lH-NAPHTHO r2,3-d~ TRIAZOLES
This application is a divisional of Applicant's copending
application Serial No. 315,389, filed October 31, 1978.
This invention relates the use of certain a, 9-dihydro-
4,9-dioxo-lH-naphthoL2,3-d] -triazoles in the treatment of allergic
diseases, to pharmaceutical compositions comprising these compounds,
to certain novel compounds within this class and to a process for
their operation.
It is generally recognised that certain cells e.g. mast
cells are activated by antibody-antigen combinations and release
substances such as histamine and SRS-A, which mediate an allergic
response. We have discovered that 4,9-dihydro-4,9-dioxo-lH-naphthO
~,3-d~ -triazoles of formula (I):-
1 ~ H
R ~ ¦ (I)
R4 O
and pharmaceutically acceptable salts thereof wherein Rl, R2, R3,
and R4 which may be the same or different, represent hydrogen,
halogen, nitro, lower alkyl or lower alkoxy, or any adjacent two
of Rl and R4 taken together represent an alkylene group containing
from 3 to 5 carbon atoms or a 1,4-buta-1,3-dienylene group, inhibit
this type of antigen-induced response in mammals, and are therefore
of value in the prophylaxis of diseases in
'~``~. i
t~ ~119()99
~j. .
which the symptoms are controlled by mediators of the allergic response.
Examples of such diseases include bronchial asthma, rhinitis, hayfever and
allergic eczema.
Not all the compounds of the class are novel:
4,9-dihydro-4,9-dioxo-lH-naphtho C2,3-~ triazole is known, and listed below
are a number of literature references each of which discloses a different
method by which the compound may be prepared.
K. Fries, R. Walter and K. Schilling, Annalen 576 248 (1935). L.F.
Fieser and E.L. Martin, J. Amer. Chem. Soc. 57 1844 (1935). W.L. Mosby and
M.L. Silva, J. Chem. Soc. 1003 (1965).
In addition, there are disclosed in U.S. Patent No. 3,294,812 issued
December 27, 1966 to American Cyanamid Company, compounds of the general
formula (I) wherein Rl and R4 are as defined previously and R2 and R3
represent hydrogen.
It has never been reported or suggested in the literature tilat the
above compounds would have any type of pharmacological activity.
Accordingly in its broadest aspect, the use of a compound of formula
(I) as defined above or a pharmaceutically acceptable salt thereof as an
antiallergic agent is provided by this invention.
By lower alkyl and lower alkoxy, in this specification we mean such
groups containing up to six carbon atoms.
In formula (I) examples of suitable lower alkyl groups include
metnyl, ethyl and n-propyl.
Examples of suitable lower alkoxy groups include methoxy, ethoxy, and
n-propoxy.
Examples of suitable halogens include fluorine and chlorine.
The alkylene groups which any two of Rl to R4 may together
represent are propylene, butylene and pentylene.
-- 2 --
~119099
~ ere compounds of formula (I) are high]y substituted, it is
appreciated that substituents Rl to R4 are selected for steric
compatability.
The triazole moiety of the compounds of formula (I) has an acidic
hydrogen, and accordingly may form salts. Examples of pharmaceutically
acceptable salts falling within the scope of this invention include aluminum,
alkali metal and alkaline earth metal salts such as the sodium, potassium and
magnesium salt; and salts with organic bases such as amines or amino compounds
including physiologically active amines such as (-) ephedrine. The sodium and
ephedrine salts (-) are preferred.
Compounds of general formula (I) in which at least one of Rl to
R~ is other than hydrogen, are novel (with the aforementioned exception) and
accordingly in a further aspect the invention provides a compound of formula
(I):- H
\ ~ ~ N
/ ~ N /
3 (I)
R4 o
and pharmaceutically acceptable salts thereof wherein Rl, R2, R3 and
R4 which may be the same or different, represent hydrogen, halogen, nitro,
lower alkyl or lower alkoxy, or any adjacent two of Rl to R4 taken
together represent an alkylene group containing from 3 to 5 carbon atoms or a
1,4-buta-1,3-dienylene group, provided that Rl to R4 do not all represent
hydrogen.
-- 3 --
~119099
~,.,~....
Within this group of novel compounds there are a variety of
sub-groups. One group is that in which at least one of Rl to R4 is
hydrogen and the remainder are as previously defined. An example of such a
compound is 4,9-dihydro-6,7-dimethyl-5-nitro-4,9-dioxo-lH-naphtho {2,3-~ -
triazole. A further group is one where two of Rl to R4 are hydrogen and
the remainder are as previously defined. An example of such a compound is 4,9-
dihydro-5,6-dimethyl-4,9-dioxo-lH-naphtho C ,3-d~ -triazole. One preferred sub-
~roup of compounds of formula (I) is that in which
- 3a -
.. 1~19099
Rl and R4 are hydrogen and R2 and R3, which may be the
same or different, represent methyl, ethyl or n-propyl.
An example of one such compound is:-
4,9-dihydro-6,7-dimethyl-4,9-dioxo-lH-naphtho-[2,3-d]-
triazole, which together with its sodium salt, constitutes
the preferred embodiment of this invention.
The compounds of formula (I) above and pharma-
ceutically acceptable salts thereof may be prepared by
a number of different methods.
The first such method comprises oxidizing a
naphthotriazole of formula (II):-H
IRl
R2 ~ N\
R3 ~ N//N (II)
wherein Rl, R2, R3 and R4 are as defined with reference
to formula (I) with a powerful oxidizing agent, and
thereafter optionally converting the compound of formula
(I) into a pharmaceutically acceptable salt.
Suitable oxidizing agents include chromium
trioxide and chromic acid. The method is carried out in
a manner analogous to that described by K. Fries, R.
Walter and K. Schilling, Annalen 576, 248,(1935); for
the preparation of 4,9-dihydro-4,9-dioxo-lH-naphtho-
[2,3-d]-triazole.
A second method comprises deamination of a 2-
amino-naphtho-triazole (III):-
Rl O
~ \
R3 ~ ~N NH2
R4 0
~ 9o99
wherein Rl, R2, R3, and R4 are as defined with reference
to formula (I) above, with nitrous acid, and thereafter
optionally converting the compound of formula (I) into
a pharmaceutically acceptable salt thereof.
The method is carried out in a manner analogous
to that described by W.L. Mosby and M.L. Silva, J.
Chem. Soc., 1003, (1965), for the preparation of
4,9-dihydro-4,9-dioxo-lH-naphtho[2,3-d]-triazole.
However, we have found that compounds of formula
(I) above are most conveniently made by a process which
comprises reacting a compound of formula (IV):-
R2 ~ NH2 (IV)
R3 ~ ~ NHR
wherein Rl, R2, R3 and R4 are as defined with reference
to formula (I) and R is hydrogen or an acyl group, with
nitrous acid, and thereafter optionally converting the
compound of formula (I) into a pharmaceutically acceptable
salt.
Suitable acyl groups include benzoyl and lower
alkanoyl. Fxamples of suitable lower alkanoyl groups are
acetyl, propionyl and butyryl.
The nitrous acid is most suitably generated in situ
from an alkali metal nitrite and an acid.
Most suitably the alkali metal nitrite is sodium
nitrite, and the acid is a mineral acid such as hydrochloric
acid.
The reaction is carried out in a solvent which
is inert to the reagents and products. Examples of such
solvents include water and acetic acid.
We have found water to be most convenient.
1~19099
The reaction should be carried out at room
temperature or below i.e. preferably between 0C and 25C.
The preparation of the compounds (I) from the
quinone intermediates (IV) is facilitated by reducing
S the quinone to the corresponding hydroquinone 9 ( IVa):-
Rl OH
2 ~ (IVa~
R3 HR
~4 OH
wherein R and Rl to R4 are as defined with reference to
formula (IV) above and reacting the hydroquinone (IVa)
with nitrous acid. These are generally more soluble in
acid media than the quinones. The reduction may be
carried out using any standard method for reducing
quinones to hydroquinones. We have found sodium dithionite
to be a most convenient reducing agent for this purpose.
The hydroquinones (IVa) oxidize to the parent quinone
during the reaction with nitrous acid.
Naphthotriazoles of this invention having at
least one nitro substituent may be prepared by direct
nitration of an appropriate triazole.
In a further aspect the invention provides a
process for preparing a compound of formula (Ia):-
1~ N (la)
and pharmaceutically acceptable salts thereof; wherein
~119099
r.--. .~
`::
Rl, R2, R3 and R4 which may be the same or differentrepresent hydrogen, halogen, nitro, lower alkyl and lower
alkoxy, or any adjacent two of Rl to R4 taken together
represent an alkylene group containing from 3 to 5
carbon atoms or a 1,4-buta-1,3-dienylene group, provided
that at least one of Rl to R4 represent nitro which process
comprises nitrating a compound of formula (Ib):-
R ~ (Tb)
wherein Rl, R2, R3 and R4, which may be the same ordifferent, represent hydrogen, halogen, nitro, lower
alkyl or lower alkoxy, or any adjacent
two of Rl to R4 taken together represent an alkylene
group containing from 3 to 5 carbon atoms or a 1,4-
buta-1,3-dienylene group7 provided that at least one of
Rl to R4 represents hydrogen.
The reaction is carried out with a conventional
nitrating agent under conventional reaction conditions.
For convenience we choose to carry out the reaction with
fuming nitric acid and concentrated sulphuric acid.
Equally, the nitration step could be carried out with
other standard reagents.
The choice of temperature at which the reaction
is carried out is dependent upon the reactivity and
sensitivity of the starting materials. Thus compounds
which do not decompose to any significant extent may be
nitrated rapidly at high temperatures for example up
to 120C, 100C being generally convenient. More
sensitive compounds require more prolonged reaction times
3~119099 h'-.
at lower tempexatures. ~he time for which a reaction
should be allowed to proceed depends upon the starting
materials, nitrating agent and temperature but may be
determined by routine procedures e.g. by following the
course of the reaction by thin layer chromatography.
The synthetic route to the intermediates (IV)
is summarised in the following scheme in which
R, Rl, R2, R3 and R4 are as defined with reference to
formula (IV) above, and ~ is chlorine or bromine and R
is an acyl group as discussed above with reference to
(IV).
:~119099
R Rl O
Rz
R / ~R
( V ) \~,
( IV)
lll9V99
r~
r~
_ 10 --
The intermediate (IV) is prepared by displacement of
halogen from the corresponding 2-acylamino-3-halonaphtho-
1,4-quinone (V) using ammonia and where desired thereafter
removing the acyl group.
The reaction is carried out by passing dry
ammonia into a solution of the compound (V) in a high
boiling aprotic organic solvent which is inert to the
reagent and products. Examples of such solvents include
nitrobenzene, N,N-dimethylforrnamide and hexamethyl-
phosphoramide. Preferably the solvent is nitrobenzene.
Suitably the reaction is carried out at elevated temperatures
(i.e. 80 - 180C).
The 2-acylamino-3-halonaphtho-1,4-quinones (V)
are prepared by reacting an active ~-acylating derivative
of benzoic or a lower alkanoic acid R -OH with a 2-amino-3-
halonaphthoquinone (VI). Suitable N-acylating derivatives
include the acid halide and the acid anhydride. This
reaction is generally carried out in the absence of
solvent in the presence of an excess of the N-acylating
derivative. If desired, the reaction may be carried out
in a polar organic solvent which is inert to the reagents
and products. Examples of such solvents include pyridine
and acetic acid.
The reaction is also suitably carried out at
moderate temperature i.e.less than 100C, room temperature
being most convenient. In order to facilitate the reaction,
a small amount of mineral acid may be added as a
catalyst.
The 2-amino-3-halo-1,4-naphthoquinones (VI)
are prepared by reacting the corresponding 2,3-dihalo-
1,4-naphthoquinone (VII) with ammonia. This reaction is
performed by passing dry ammonia into a solution of the
compound (VII) in a high boiling aprotic organic solvent
which is inert to the reagents and products, as described
for the conversion of (V) to (IV) above.
` 1119099
Preferably the solvent is nitrobenzene. Suitably
the reaction is carried out at elevated temperatures
i.e. 80 - 180C.
The 2,3-dihalo-1,4-naphthoquinones (VII) are
prepared from the corresponding naphthoquinones by a
conventional halogenation reaction.
In order to use compounds of formula (I) or
salts thereof for medical purposes, they are formulated
in accordance with standard pharmaceutical practice as
pharmaceutical compositions.
The invention further provides pharmaceutical
compositions comprising a compound of formula (I) above
or a pharmaceutically acceptable salt thereof together
with a pharmaceutically acceptable carrier.
Compounds of formula (I) may be administered
topically or systemically. In accordance with usual
pharmaceutical procedure, the active material will be
purified so as to contain minimum amounts of by-products
or other impurities.
Topical formulations for administration to the
skin include lotions and creams. Topical formulations
for administration to the respiratory tract include
solutions for application via a nebulizer or as an aerosol,
snuffs and microfine insufflatable powders.
The active ingredient in an insufflatable powder has a
small particle size i.e. less than 50 microns and preferably
less than 10 microns. The active material is co-presented
with a solid carrier such as lactose which has a particle
size of less than 50 microns. Insufflatable compositions
in particular will be rendered substantially free of
microbial contaminant.
Systemic administration may be achieved by rectal,
oral or parenteral administration. A typical suppository
formulation comprises the active compound with a binding
and or lubricating agent such as gelatin or cocoa butter
or other low melting vegetable waxes or fats. Typical
1119~99
- 12 -
parenteral compositions comprise a solution or suspension
of the active material in a sterile aqueous carrier or
parenterally acceptable oil.
Compounds of formula (I) which are active when
given orally may be compounded in the form of syrups,
tablets, capsules and lozenges. A syrup formulation
will generally consist of a suspension or solution of
the compound in a suitable liquid carrier such as
ethyl alcohol, glycerine or water with a flavouring or
colouring agent. Where the composition is in the form
of a capsule, the solid in granular form optionally with
a binding agent is encapsulate in an edible shell e.g.
of gelatin. Where the composition is in the form of
a tablet, any suitable pharmaceutical carrier routinely
used for preparing solid formulations may be used.
Examples of such carriers include magnesium stearate,
starch, lactose, glucose, sucrose, rice flour and chalk.
Preferably the composition is in unit dose form such as
a pill, capsule or metered aerosol so that *he patient
may administer to himself a single dose.
Where appropriate, small amounts of anti-asthmatics
and bronchodilators for example sympathomimetic amines
such as isoprenaline, isoetharine, salbutamol, phenylephrine
and ephedrine; xanthine derivatives such as theophylline
and aminophylline; and corticosteroids such as prednisolone
and adrenal stimulants such as ACTH may be included.
As is common practice, the compositions will usually be
accompained by written or printed directions for use in
the medical treatment concerned, in this case as an anti-
allergic agent for treatment of, for example, asthma,hay-fever or rhinitis.
The following examples illustrate the invention.
1119099
- 13 -
Example 1
a). 2-Amino-3-bromo-6,7-dimethyl-1,4-naphthoquinone
Dry ammonia was passed through a refluxing
solution of 2,3-dibromo-6,7-dimethyl-1,4-naphthoquinone
(109; mp 237 - 238C) in dry nitrobenzene (65ml) for
30 mins. and the orange solid which separated on cooling
was filtered off and washed with light petroleum. After
removal of ammonium bromide with water, 6.96 g. (86%)
of material of mp 228C was obtained. Recrystallisation
from glacial acetic acid gave analytically pure material
of the same melting point.~ max(mull) 3400, 3300, 1680,
1610, 1585, 1570 cm 1. ~ (DMS0) 2.35 (6H,s); 7.35
(2H, exchangeable broad singlet); 7.74 (2H,s). (Found;
C, 51.50; H, 3.38; N, 4.79; C12HlOBrN02 requires; C,
51.45; H, 3.60; N, 5.00~0).
b). 2-Acetamido-3-bromo-6,7-dimethyl-1,4-naphthoquinone
10 g. of 2-amino-3-bromo-6,7-dimethyl-1,4-
naphthoquinone were dissolved in acetic anhydride
(lOOml) containing a few drops of concentrated sulphuric
acid and the yellow acetyl derivative which separated
was filtered off and dried to give 9.0 g of crude
product. Recrystallisation from chloroform-petrol gave
7.75g (67%) of material of mp 252C. ~ max(mull) 3210,
1685, 1665, 1600 cm ; ~(DMS0) 2.11(3H,s); 2.38 (6H,s);
7.80 (lH,s); 7.85 (lH,s), 1 low field exchangeable proton
(~ound; C, 50.98; H, 3.76; N, 4.30; C14H12BrN03 requires;
C, 52.03; H, 3.74; N, 4,33%),
c). 2-Acetamido-3-amino-6,7-dimethyl-1,4-naphthoquinone
Dry ammonia was passed through a stirred refluxing
solution of 2-acetamido-3-bromo-6,7-dimethyl-1,4-
naphthoquinone (7.5g) in nitrobenzene (50ml) for 1 hr.
and the mixture cooled. The red solid which separated
was filtered off, washed well with ether-petrol and
then water to give 4.46g. (77%) of material of mp
1119()99
- 14 -
212 - 216C. Recrystallisation from ethanol raised the
melting point to 216C, ~max(mull), 3400, 3270, 1675,
1665, 1630 cm . ~ (DMS0) 2.05 (3H,s); 2.35 (6H,s);
6.64 (2H, exchangeable s); 7.71(2H,s); 9.00 (lH,
exchangeable s.) (Found; C, 65.07; H, 5.43; N, 10.66;
C14H14N203 requires; C, 65.10; H, 5.46; N, 10.85%).
d). 4.9-dihvdro-6,7-dimethyl-4,9-dioxo-lH-naphtho
[2,3-d]-triazole
Sodium dithionite (2.5g) in water (lOml) was
added to 2-acetamido-3-amino-6,7-dimethyl-1,4-naphtho-
quinone (1.68g) in water (150ml) and the stirred suspension
left for 1 hr. The white hydroquinone which formed was
filtered off and added to 2N HCl (50ml). The solution
was cooled to 0C and a solution of sodium nitrite (2g)
in water (20ml) was added over 2 hrs. The suspension
which formed was stirred overnight at room temperature
and the white product filtered off to give 1.34g (84%)
of material of mp 262C (dec) which was shown to be a
monohydrate, ~max(mull) 3590, 3420, 1700, 1665, 1595 cm
After recrystallisation from acetone-water and drying
under vacuum at 107C, the anhydrous material was isolated,
~max(mull) 3120, 1695, 1675, 1595 cm 1. ~(DMS0)
2.22 (6H,s); 7.56 (2H,s); 1 low field broad exchangeable
proton. (~ound; C, 63.20; H, 3.96; N, 18.33; C12HgN302
requires C, 63.44, H, 3.99; N, 18.50%).
` ~19~)99
,._
- 15 _
Example 2
a). 2-Amino-3-bromo-6(7)-methyl-1,4-naphthoquinone
Dry ammonia was passed through a stirred, refluxing
solution of 2,3-dibromo-6-methyl-1,4-naphthoquinone (12.3g,
mp. 192 C) in dry nitrobenzene (75ml) for 1 hr. The
red solid which separated on cooling was filtered off,
washed well with petroleum ether (bp. 40 - 60) and water
and recrystallised from glacial acetic acid to give 7.5 g.
(75%) of material of mp 173C. (Found; C, 49.78; H,
3-23; N, 5.08; CllH8BrN02 requires; C, 49.65; H, 3.03;
N, 5.26%).
b). 2-Acetamido-3-bromo-6(-7)-methyl-1,4-naphthoquinone
. . . ~
Acetylation of 2-amino-3-bromo-6(7)-methyl-1,4-
naphthoquinone (7.59) as described in Example lb afforded
6.09 (70~0) of the 2-acetamido derivative of mp 192 - 193C.
(Found; C, 49.51; H, 3.23; Br, 25.59; N, 4.16; C13HlOBrN03
requires; C, 50.67; H, 3.27; Br, 25.93; N, 4.54%)
c). 2-Acetamido-3-amino-6(7)-methyl-1,4-naphthoquinone
Dry ammonia was passed through a stirred re-
fluxing solution of 2-acetamido-3-bromo-6(7)-methyl-1,4-
naphthoquinone (6g) in nitrobenzene (50ml) for 1 hr.
and the product worked up as in example lc to give 1.7g
(40~o) of title compound of mp (EtOH) 200 - 201C. (Found;
C, 63.80; H, 4.92; N, 11.16; C13H12N203 requires; C,
63.93; H, 4.95; N, 11.47%).
d). 4.9-Dihydro-4,9-dioxo-6-methyl-lH-naphthor2l3-d
triazole
. .
1.6g of 2-acetamido-3-amino-6(7)-methyl-1,4
naphthoquinone was converted into the title triazole by
the procedure outlined in example ld. Yield 0.759 (51%)
mp (aqueous acetone) 235 (dec). (Found; C, 57.25; H,
3.77; N, 18.00; CllH7N302.H20 requires; C, 57.07; H,
3.92; N, 18.27%).
1119099
- 16 -
Example 3
a) 2,3-Dibromo-6-methoxynaphtho-1,4-quinone
Bromine (25.4g) was added to a stirred mixture
of 6-methoxynaphtho-1,4-quinone (15.19, 0.08 mole) and
anhydrous sodium acetate (13.3g) in chloroform (125ml)
and the mixture stirred at room temperature for 3 days.
After filtration the resulting liquid was evaporated
to a dark solid, 23~2g (84%) which after recrystallization
from petroleum ether (b.p. 60-80C) had mp 146-148C.
(Found; C, 38.33; H, 1.70; Br, 46.72; CllH6Br203
requires; C, 38.19; H, 1.75; Br, 46.19%).
b) 2-Amino-3-bromo-6(7)-methoxynaphtho-1,4-quinone
Dry ammonia was passed through a stirred, re-
fluxing solution of 2,3-dibromo-6-methoxynaphtho-1,4-
quinone (9.79, 0.028 mole) in N,N-dimethylformamide
(80ml) for 45 min. and the cooled mixture poured into
water. The red solid which separated was filtered off,
washed well with water and dried to give 7.34g (93%)
of product which had mp. (EtOH/H20) 184 C (dec).
c) 2-Acetamido-3-bromo-6(7)-methoxynaphtho-1,4-quinone
Concentrated sulphuric acid (2 drops) was
added to a suspension of 2-amino-3-bromo-6(7)-methoxy-
naphtho-1,4-quinone (3.6g, 0.0125 mole) in acetic
anhydride (30ml) and the dark mixture stirred for 15
min. After pouring into water an olive green solid
separated which was filtered off and recrystallized
from ethanol containing charcoal to give 3.4g (84%)
of a yellow solid of mp 229 C. (Found; C, 48.76; H,
2-90; N, 4.46; C13HlOBrN04 requires; C, 48.17; H, 3.11;
N, 4.32%).
1~19099
- 17 -
d) 2-Acetamido-3-amino-6(7)-methoxynaphtho-1,4-quinone
-
Dry ammonia was passed for 1 hr. through a
stirred, refluxing solution of 2-acetamido-3-bromo-6(7)-
methoxynaphtho-1,4-quinone (5.19; 0.0158 mole) in
nitrobenzene (50ml) and the cooled solution filtered free of
inorganic ~aterial and concentrated in vacuo. The red
solid which separated was filtered off and washed with
ether to give 2.59 (61%) of material of mp 97C (dec).
e) 4,9-Dihvdro-4~9-dioxo-6-methoxv-lH-naphtho r 2 3-dl-
triazole
To a stirred suspension of 2-acetamido-3-
amino-6(7)-methoxynaptho-1,4-quinone (2.59; 0.0097 mole)
in water (200ml) was added a solution of sodium dithionite
(49) in water (20ml) in one portion. After a further
1 hr. the precipitated hydroquinone was filtered off
and suspended in 2N hydroxhloric acid (70ml) at 0C.
A solution of sodium nitrite (39) in water (20ml) was
added over 90 mins. and the mixture stirred overnight.
The precipitated triazole was filtered off and re-
crystallized from acetone/water to give 0.889 (38%)
of derivative of mp 238C (dec). (Found; C, 53.40;
H, 3.75; N, 17.01; CllH7N303. H20 requires; C, 53.38;
H, 3.67; N, 17.10~o).
1119099
.. .
Example 4
4,9-Dihydro-4,9-dioxo-5-nitro-lH-naphtho-[2,3-d]-triazole
4,9-Dihydro-4,9-dioxo-lH-naphtho-[2,3-d]-
triazole (5g, 0.025 mole) was dissolved in a mixture of
concentrated sulphuric acid (25ml) and fuming nitric
acid (50ml). The resulting solution was heated on a steam
bath for 10-15 mins and then poured onto ice (250 g).
When all the ice had melted the solid was filtered off,
washed with water (3 x 20ml) and dried to yield a bright
yellow solid (4.469, 74%), mp 227-35 (dec). Cl NMR
showed this to consist of a mixture of the 5- and
6-isomers in the approximate ratio 9:1 Multiple
recrystallization from acetone and then ethyl acetate
yielded a yellow solid, mp 244-6 (dec) (0.640g, 10.4%)
consisting of the title compound containing<5% of the
6 - isomer. (Found; C, 49.47; H, 1.48; N, 22.86;
CloH4N404 requires; C, 49.20; H, 1.64; N, 22.95%).
`` 1119099
-- 19 --
Example 5
a) 2-Bromo-6-fluoro-3-hydroxynaphtho-1,4-quinone:
A solution of bromine (12.59), in chloroform
(SOml) was added to a stirred suspension of 6-fluoro-3-
hydroxynaphtho-1,4-quinone (12.5g; 0.065 mole) in
chloroform (50ml) over 15 mins. During the addition the
mixture became a clear dark red solution. Stirring
was continued for a further 4 hours and a yellow solid
which had been deposited as crystals was filtered off,
washed with ice-cold chloroform and dried. Yield
13.959 (80~o) ~ mp 183C (CHC13).
b) 2-Amino-3-chloro-6(7)-fluoronaphtho-1,4-quinone:
-
A solution of 2-bromo-6-fluoro-3-hydroxynaphtho-
1,4-quinone (13.93g; 0.052 mole) in dry benzene (200ml)
was refluxed with thionyl chloride (6.89, 0.057 mole)
and D.M.F. (3 drops) for 3 hours. The dark red solution
produced was cooled and evaporated to yield an orange
solid which was recrystallised from chloroform. Analysis
showed this solid to be a mixture of 2,3-dichloro-6-
fluoronaphtho-1,4-quinone and 2-bromo-3-chloro-6(7)-
fluoronaphtho-1,4-quinone.
Dry ammonia was passed through a stirred solution
of the above mixture (5.89) in nitrobenzene (75ml)
at room temperature for 3 hours. The mixture was filtered
and the dark red filtrate was diluted with anhydrous ether.
Dark red crystals were deposited. The crystals were
collected, washed with anhydrous ether and dried. Yield
3.59 mp 167 (d). Mass spec. M 224.9991 corresponding
10 5 2
` ~ 11~09g
- 20 -
c) 2-Acetamido-3-chloro-6(7)-fluoronaphtho-1,4-quinone
Concentrated sulphuric acid (2 drops) was added
to a suspension of 2-amino-3-chloro-6(7)-fluoronaphtho-
1,4-quinone (3.59; 0.0155 mole) in acetic anhydride
(25ml). An immediate reaction took place and a yellow
crystalline product was formed which was recrystallised
from ethyl acetate. Yield 2.19 (51%); mp 225-7 (EtOAc).
d) 2-Acetamido-3-amino-6(7)-fluoronaphtho-1,4-quinone
. .
Dry ammonia was passed for 75 mins. through a
stirred solution of 2-acetamido-3-chloro-6(7)-
fluoronaphtho-1,4-quinone (2g; 0.0079 mole) in nitro-
benzene (20ml) at 110C. A dark red solid crystallised
in the mixture. This was filtered off, extracted with
chloroform, the extracts were combined and the solvent
was evaporated to yield a red solid that was washed with
dry ether. The product had mp 218-220.
e) 4.9-Dihydro-4,9-dioxo-6-fluoro-lH-naphthor2,3-dl-
triazole
To a stirred suspension of 2-acetamido-3-amino-
6(7)-fluoronaphtho-1,4-quinone (l. Og, 0.004 mole) in
water (150ml) was added a solution of sodium dithionite
(2.5g) in water (lOml) in one portion. After a further
1 hour the precipitated hydroquinone was filtered off
and suspended in 2N HCl (50ml) at 5C. A solution of
sodium nitrite (1.59) in water (15ml) was added over
30 mins. and the mixture stirred overnight. The
precipitated triazole was filtered off and recrystallised
from acetone/water to give 0.559 [63%] of derivative of
mp 233 (dec), Vmax(mull) 1690, 1600cm ; M 217.0296,
10 4 3 2-
Example 6
4,9-Dihydro-6,7-dimethyl-4,9-dioxo-5-nitronaphtho[2,3-d]-
triazole
4,9-Dihydro-6,7-dimethyl-4,9-dioxonaphthotriazole (0.4g,
0.0018 mole) was dissolved in a mixture of concentrated
sulphuric acid (4ml ) and fuming nitric acid (8 ml).
The solution was heated on a steam bath for two hours
and then poured onto ice (200~). The iced mixture so
obtained was left to stand overnight. The resulting
solid was filtered off, washed with water (2 x 50ml)
and dried to give a pale yellow solid (380mg, 80~o) of
mp 260-3. This was recrystallized from ethanol to
yield pale yellow crystals of mp 263-266 C (dec), vmax
(mull) 3500, 3430, 1690cm 1, ~ (DMS0) 2.24 (3H, s);
2.55 (3H,s); 8.18 (lH, s), 1 mid field broad exchangeable
proton. (~ound; C, 53.10; H, 2.59; N, 20.56; C12H8N404
requires; C, 52.95; H, 2.96; N, 20.58%).
,ii9o99
- 22 -
Example 7
(-)-Ephedrine salt of 4,9-Dihydro-6~7-dimethvl-4.9-dioxo-
naphtho[2,3-d]-~ -triazole
A solu-tion of 4,9-dihydro-6,7-dimethyl-4,9-
dioxo-naphtho[2,3-d]-V-triazole (454mg, 0.002 mole)
in methanol (20ml) was added to a solution of (-)ephedrine
(330mg, 0.002 mole) in methanol (lOml) in one portion.
After standing for 15 mins. the solution remained clear
although the colour had darkened. The solvent was
removed _ vacuo and the brown solid recrystallized from
chloroform/petrol (60-80) to give 550mg (63%) of the
a~ove named salt, mp 252 C, vTax(mull) 3350-2400 broad,
1665, 1600, 1390, 1260, 980cm . ~ (DMS0) 0.97 (3H,
d~; 2.36 (6H, s); 2.54 (lH, quartet); 2.70 (3H,s);
5.14 (lH, d); 7.42 (5H, s); 7.88 (3H, s) and exchangeables.
(Found; C, 64.18; H, 6.46; N, 13.28; C22H24N403H20
requires; C, 64.45; H, 6.39; N, 13.65%).
~9099
..
- 23 -
Example 8
4,9-Dihydro-4,9-dioxo-lH-naphtho[2,3-d]-triazole
. .
A solution of sodium dithionite (2.5g.) in water
(lOml.) was added to a stirred suspension of 2-
acetylamino-3-amino-1,4-naphthoquinone (1.5g.) in water
(150ml) and the mixture stirred at room temperature
until the solid had decolourized (1 hr.). The solid
was filtered off, dissolved in water (25ml.) containing
concentrated hydrochloric acid (3 ml), decolourized
with charcoal and diluted to 50 ml. by addition of water.
A solution of sodium nitrite (lOml of 10%) was added
to cooled (0C) stirred solution and after 1 hr. at 0
followed by 2 hrs. at room temperature the precipitated
off-white solid was filtered off. Recrystallisation from
lS acetone-water gave 0.62g. (44%) of the title compound
of mp 250C (dec) (Lit. mp. 240-245C Fieser and Martin
loc. cit.), ~ max(mull) 1690 cm . (Found; C, 60.05;
H, 2.81; N, 20.90; CloH5N302 requires; C, 60-30; H,
2.53; N, 21.01%).
~ ~i9099
- 24 -
Example 9
Ammonium Salt of 4,9-Dihvdro-4,9-dioxo-5-nitro-naphtho
~2,3-dltriazole
4,9-dihydro-4,9-dioxo-5-nitronaphthotriazole
(0.24~9, 0.001 mole) was suspended in ammonia solution
(1:1 water/880 ammonia) and warmed on a steam bath for
30 minutes. The mixture became reddish and was cooled
to room temperature The insoluble solid was filtered
off and dried to yield 0.21g (80%) of a pink solid whose
NMR spectrum showed it to be the title compound (d .
DMS0, typical 5-substitution pattern multiplet ~ 8.0-
8.5; broad exchangeable peak 5.7,4H). The compound had
no melting point but charred at circa 235C. (~ound; C,
45.51; H, 2.44; N, 26.18; CloH7N504 requires; C,
45.98; H, 2.70; N, 26.81).
. ,, : .
: - :
~ ~;90g9
Example 10
Sodium Salt of 4~9-Dihvdro-6~?~dimethyl-4~9-dioxonaphtho
~2.3-dltriazole
a) A suspension of the parent triazole (1.33g) in
water (50ml) was treated with 2.5N sodium hydroxide
solution until the pH of the solution was 6.5. The
resulting yellow solution was filtered and evaporated
to dryness. The residual yellowish solid was re-
crystallized from ethanol to yield a buff solid which
turned yellow on drying. Yield 1.433g mp 360.
Anhydrous material was obtained by drying in vacuum
at 110 over phosphorus pentoxide when the material
looses its crystallinity (Found; Na:8.89%, C12H8N302
Na requires; 9.23%).
b) 4,9-Dihydro-6,7~dimethyl-4,9-dioxonaphtho[2,3-d~
triazole (l.Og) was suspended in water (50 ml) and the
- pH brought to 6.8 by the addition of 5N sodium hydroxide
solution. The resulting solution was evaporated to
dryness in vacuo . The residue was shaken with ethanol
(20 ml) and again evaporated to dryness, this ethanol
treatment was repeated. The solid was now dissolved in
a minimum of boiling ethanol and the resulting solution
cooled to 5C. A few needles slowly formed, these were
filtered off, rinsed with ethanol and dried to yield
0.21g of bright yellow needles. The NMR spectrum of
this material (in d DMSO) contained a peak corresponding
to lOH (after substracting the solvent contribution)
which exchanged with D20. Microanalysis was also
consistant with the presence of a pentahydrate (Found:
C, 42.91, H, 5.14, N, 12.41, C12H8N302Na.5H20 requires
C, 42.48, H, 5.35, N, 12.39).
~,~,9~9
- 26 -
c) Crude 4,9-dihydro-6,?-dimethyl-4,9-dioxonaphtho
[2,3-d]triazole sodium salt was prepared as in b)
above. The residue from evaporation of the solution for
the third time was dried at 0.2mm/Hg for about 5
minutes and then dissolved in the minimum of boiling
methanol. On cooling bright yellow needles were deposited
from the solution, these were filtered off and dried.
NMR suggests these are anhydrous.
~ "
~119099
Passive Cutaneous Anaphyla~is (PCA)
_
Serum containing heat labile homocytotropic
antibody was raised in rats to crystallized ovalbumin XOA
by the method of Mota (I. Mota, Immunology, 7,681
[1964]) using Bordettela pertussis vaccine as adjuvant.
Passive cutaneous anaphylaxis (PCA) was carried
out by a method based on that of Ovary and Bier, (A.
Ovary and O.G. Bier, Proc. Soc. Exp. Biol. Med. Bl, 584,
[1952]) as modified by Goose and Blair. (Immunology 16,
749 (1969).
Male Wistar rats of 250 - 3009. were given
0.1 ml. of each of six twofold serial dilutions of pooled
antiserum in 0.9% saline injected intradermally into
separate sites on their shaved backs. Later (72 hrs.)
the animals were challenged by intravenous injection
of 0.3 ml. of a 1% solut on of ovalbumin in an isotonic
solution of saline buffered with 0 ~5M, pH 7.2,
Sorenson Buffer (PBS), mixed with 0.2 ml. of a 5% solution
of Pontamine Sky Blue (6BX C.I. 24410, Raymond A. Lamb,
London) in isotonic saline. The rats were killed after
20 min and the diameter of the blue wheals at the antibody
injection sites was measured on the outer surface of the
skin. The starting dilution of the serum was adjusted
so that there was no response, after challenge, at the
injection site of the highest dilution and a maximum
resp~nse at the lowest dilutions. Typically six twofold
serial dilutions of the serum from 1/4 to 1/128 were
used.
Compounds were tested for their ability to reduce
the diameter of the wheals at those intradermal sites
which in control animals gave less than maximum response.
Each dose of the compound was administered to six rats
at a measured time prior to intravenous challenge with
ovalbumin. Control groups of six rats were given the
same volume of carrier fluid (0.2ml, 1009 1) at the same
o
~9o99
- 28 -
time prior to challenge.
The results were calc~lated as followss % inhibition
of PCA = 100 (1 - a/b) where a : the sum of the diameters
of the wheals produced in the test animal at the sites
of antibody dilutions as used iJ'. control groups and b =
the mean sum of the diameters oi the wheals produced in
the control group of animals at those antibody sites
where at least five out of six of the animals gave less
than maximum response. A typical variation in the
control group of animals was SEM+ 6%.
. . ._ Carrier Time Dose% Inhib
Example Route
Fluid ~mins)mq/kq~ PCA
_ .... _ .
2(d~S.C. PBS 10 20 73
. NWaHCO~ 30 20 57
3(e)S.C. WBiSth 10 20 61
NaHC0~ 30 20 34
4 S.C. wBitSh 10 10 44
NaHC03 30 10 19
5(e)S.C. with 10 20 90
NaHC03 30 20 27
.
6 S.C. wBitSh 10 1 66
NaCH03 30 1 28
8 S.C. wBitSh 5 1 39
_ NaCH03 5 2 82
,, .. :
- 29 -
Time between administration of compound and antigen
challenge.
Acute toxicity: no toxic symptoms were observed with
any of the compounds while carrying out the tests
described above.
Toxicity: Oral LD50 of the compound of example l(d)
in Charles River Sprage Dawley CD strain Male Rats:
980 mgkg . LD50 data confidence limits : 95%, 548 -
1,413 mgkg 1.
- :
