CA1069899A - Products for combatting cholera - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

Compositions for combatting cholera using novel substituted quinoxalines represented by the formula

Description

'l~hiE :inven~ioll rc]a~es to cc,lnpounds us~ul fo~ ~om~
battincJ cho]era. It a:lr;o rela~e~ to a pl oc~ss ~or p~eparing the compounds.
Cholera is a hi~hly infectious disease caused by a vibri~ organism. Although the infection can be spreacl by person to person contact, the most common source is contaminat~
ed water supplies, the contamination usually arising from sewage containing the organism. Generally, cholera is con-trolled b~ administering suitable dcugs to the person suffer-ing from the disease. However, there are many adv~nt~gefi to treating exposed persons prophylactically and ~rea~iny the contaminated water to prevent or greatly reduce the incidence of the disease.
It is an object of this invention t~ provide compounds, compo~itions containing them, and methods usiny them for com-batting cholera.
It is another object of this invention to provide a method of combatting cholera by prophylactic administra~ion of a substituted quinoxaline.
It is a further object of this invention to provide a method of combatting cholera by the prophylactic administration of a substituted quinoxaline which selectively combats the cholera causing organisms without deleteriously affecting the balance of organisms desired in an environment, e.g., the human biological system, such as, e.g., intestinal flora.
Other obiects will be apparent to those skilled in the art from the disclosure herein.
; It .is the discovery of this in~ention that substituted - ~uinoxalines (hereafter the "C-Compounds") represented by the formula , ~ cb/ ~ 2 - ~

.. . . . . .

.~Oti~38~3 0 N~l~

~ CII=CIi - ~ b~ ~

wherein R is hydrogen or lower alkyl of 1 to 5 carbon atoms, e.g., ethyl, propyl, and the like, are useful for cornbatting cholera-produciny organisms. They are especially use~ul for prophylac~ic administration prior to development of symptoms o~ cholera. The term "lower" as used herein stands for a chain comprising 1-5 carbon atoms.
~ he C-Compounds of the present invention include but are not limited to -C0-1: 2-/~-(2-amino-4-pyrimidinyl)eth2nyl7-quinoxaline 1, 4-dioxide ~;
~0:2: 2-/2-(2-amino-6 methyl~4-pyrimidinyl)ethenyl7-quinoxaline 1,4-dioxide.
These compounds are prepared by reacting, advantayeously in an approximateIy 1:1 mole ratio, quinoxaline -di-N-oxide--2-carboxxaldehyde or its lower alkyl acetal and a compound represented by the formula NH

/~ ' H C-~ ~ R

wherein R has the same meaniny as deine~ above.
.
cb/ 3 .
'. , ' ' ,: , b .t ~
The rc~c-lion ,i.~ adv~lnt~ycou~ly collducted in l~he pre-sence of a s~ro~g ac:id catcll~sk and a suita~],e solvent, e.g,, a lower alkanoic acid ~uch as orrnic aci.d or acetic acid.
The reaction takes place at reaction telnperatures su~ficien~
to effect ~he'reac~,ion and these temperatures can range rom ambient, e.g., O~C. to elevated temper~tures, e.g., ~0C.
or more and preferably, e.g., at from about 25 to 50C. or ~ore. When the reaction is complete, e.g., after abou-t 10 to ~-24 hours, the proc~uct is adv~ntageousl~ recovered by crystalli~-ing it from water.
Catalysts suitahle for the practice of this invention are generally known as strong acids and any strong acid known in the art can be used. Suitable strong acids include b~t are not limited to hydrochloric, hydrobromic, sulfuric, nitric, aryl sulfonic aci,ds, e.g., toluene sulfonic acid, trichloroacetic acid~ etc. The acids are generally used in a rat1o of about 0.5-2 moles of acid per mole of quinoxallrle sta,rting compound.
The quinoxaline di-N~oxide--2-carboxyaldehyde and its acetals used as a starting material for preparing the com pounds of the present invention are known in the art. The acetals can be prepared according to the procedure of Haddadin et al., British Patent 1,305,138, Example ~III. The aldehyde lS described ,in U. S. Patent 3,371,090.
The pyri,midine compounds used as starting materials are similarly known in the art. They are con~nercially avail- ' able and the usual commercial grade is suitable. Preferably they should be of good quality~ free from deleterious material.
The C Compounds of the pre~ent invention are useful for combatting cholera-causi.ng orcJanis'ms, e.g., vibrio organisms.
The compounds are of a low order o~ toxicity and are suit- ~' able for use by oral adrn,ini~tration or prophylactic control .

c~

, -. ' ~o~
o~ choler~, Tlley are advan~ayeous in thclt tlley not only combat cholera causing orcJallisl[ls, bu~ ~hey c~ln selectiv~:ly combat the cholcra-causiny oryani~ms without deleteriously a~fectincJ the b~lance of oryanisms desixed in an environment, e,g., the human biological system.
For oral administration, a C~Cvmpound iS usually compounded in a pharmaceutical unit dosage form such as pill, lozenge, ta~let or capsule with a pharmaceutically-acceptable carrier. Such unit dosage forms, for example, containing from about 50 to about 500 milligrams of a C-Compound, are quite satisfactory and are prepared according to techniques known to those skilled in the art. Thus, these unit dosa~e forms will contain the normal diluents, excepients, lubricating agen~s, and extenders regularly employed in compounding such forms. Exemplary carriers are solids such as lactose and starch and they can be employ-ed in capsules, or tablets in amounts from about 100 to 300 - mg. per dosage unit. In general, the administration of a daily dosage of about 1 to 6 capsules or tablets are suitable.
The preferred daily dosage is from about 0.5 to 3g per day for 3 to 5 days.
The compounds are a]so suitable for use in sterilizing vibrio-organism-containing waters. Typically, above 10, preferably about 30 tc 100, micrograms per milliliter of water can be used.
`~ The invention will be better understood with reference to the followiny examples. It is understood, however, that the examples are intended for illustration only and it is not intended that the invention be l.~mited thereby.
EX~MPI,E 1 To a reaction vessel there was added 15 ml of 99%
ormic acid, 1.15 y of 9~ s~tl~uric acid, 1.09 ~ (0.01 mole) cb,~ 5 .
. ., ' .

9~39~
of 2-~lrnino-4-lncthy] ~yrirn:i(1in~ and ~ 36 g ~0.01 mol~) o~
quinoxalinc--di~ oY~.idc-2~carboxyaldel~yd~ dirnethy].acetal.
The tnixture was hea~ec1 to 45~50 and maintained at that temperature for l0 hours. It was then cooled, diluted with 35 ml of cold water and the pll was adjusted to about 5 with sodium bicarbonate A yellow, crystalline precipitate was ~ormed. It was filtered and washed with water. There was obtained in 64% yield l.8 g o 2-/2-(2-amino-4-pyrimidinyl)-ethenyl7quinoxalirle l,4--dioxide, m.p. 237-239 with decomposition.

The product, designated CO-l for conveniencej was tested against five strains of vibrio cholerae at concentra-tions of l0, 30 and l00 microyrams per milliliter. The results are given in Table l.
Tests were also run to see if the compound was effect-iye against vibrio cholerae El Tor Ogawa 6 in the presence of sewage. Sewage samples were obtained from the sewer sy~tem of the city of Modena, Italy. They were centrifuged to separat:e solids and the supernatant liquid was used in the tests The results are given in Table 2. At l0~g/ml ~ ;
of CO-l, there was no growth of 3 of the organisms after 48 hours, and only marginal growth of the remaining two at l00~ g/ml.

cb/ ~ 6 .. . . . .

9~:3 H . .
h t- I t~ 1- 1 t t 0 ~ I I

~ ~

O ~ ( ~ O ~ A

O lli 10 . o Ql ~' U ~ -- I . ~ ~ ~

~ ~ o o o o o o 4~
O O ~ ~1 ~ r-l (~ Q) O ~ r l r-l ,~ U h E ¦ O

cb/ 7 ' 8~
q~ L c 2 __.
SampleConcerlt~ati.on Effect After of CO-I. 2~ hrs. 4~ hrs. 5 d~s Control ~ +~
Vibrion Se~age - ~~~ -~~ ~~~
Sewa~e ~
Vibrion - -~-~+ +~ +
Se~age ~
Vibri.on 5 ~/ml --- --- ---Sewage V.ibr.ion 10 ~/ml --- --- ---Sewage -~
Vibrion 20 ~/ml Sewage Vibrion 30 ~/ml --- --- ---The compound was tested for acute toxicity by sevexal modes of adminis-tration in four species, namel~7, mice, rat, guinea pig, and rabbit. The compound was found to be of a low order of toxicity. The test results are given below in Tables 3, 4, S and 6.

:' ' ,~ ' ~.cb/ - 8 -.' ' ' ' .

t3~3 r~ 3 ~cute 'roxici.(:y c~:~ CO-:I. in Ferncll~ M.ic~

Dosage, Dead/T~c!at~d l~nimals aft~r mg/kc~_ 1 _ay 2 days 4 days 7 da~s Endope~i1Oneal ~dmini.stratiorl 2000 6/6 . 6/6 1000 6/6 6/~

Esophageal Adrninistration o(x) 0/6 4000 1/12 1~12 (x) By gastric gavage ancl receiving only the vehicle.

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... ~ ' '' ' :

Tahle ~cute 'rOY.iCl~y of CO-l i.n The Gui e }3y ~so ~ cleal ~dmini.~trali.on Dosage, Deacl/Treated within 21 days mg/kg __ _ 500 0/~
1000 1/~
. 2000 5/6 :10 . O (x) _ _____ (x) Onl~ the vehicle was adminlstered.

Table 6 .
Acute Toxic.ity of CO~l in the Rabbit - By Esopha~eal ~dministration Dosage, Dead/Treated Body Weight in g. (m~SE~
mg/kg within 7 days Star-t Termination .
2000 0/2* 2250~2150 2180-21~0 1000 0/4 2037+104.3 1922.5+71.5 : .
0tX) 0/4 2135+ 75 2262~215 20 500 0!2 2000-2100 1650-1550 .
~) Only the vehicle was administered * There were two dead outof seven treated animals, within 4 days. ..
In view of the favorable acute toxicity data, the .
compound was administered orally in sub-acute, but relatively ..
large doses, to mice and rats for 15 days. Data were collected on the e~fec-ts on death rate, we.i.cJht, liver and kiclneys.
28 The data are cJi~ten in Tables 7 and 8, cb/

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r l I .
~rc~ ~ Il') (I) .
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8 ; "~

r a ~ ~ E~ o o~ ~ à
Q .IJ ~D a~ ~ .~s . ~ .
X , ~o ~ ~d X t`l g :)~ O~ ~ ~ a ~ ~ 8 ~ ~

.. Q ~ O ~ O ~h t~ O h a)0 ~ . ~.
,~ ~) ~ ~d ~ E~ ~r~

a ~ h 3 h ~ 5: --' 0 . h O ~O O
~--J ~ ~o 0 ~ ~ 1 .q o ~ ~ ~ ~
rd O ~ O ~
o E- ~r~ r-~
O ~ g cb/ - 12 -~C)f~38~39 Ta~le 8 Sub~cute Toxic;~ of CO~1 in Female ~a-~

Daily Dose: 2 g/kg/day o~ CO-l by c3astric cJaVage for~1 days Oral Treatment Dead/ Body Weight in g (m+SE) Treated Start Terminatlon __ .
Vehicle 2/6 ~x) 200.0~4.1 233.2_5.1 CO-l, 2 g/kg/day 1/6 ~x) 204.1 2.0 210.6_9.6 (x) Death caused by a mistake in esophagus incannalu-tation. This diagnosis was confirmed at the post-mortem examination.

Daily Dose: 2 g/]cg/day of CO~l by gastric g~vage f~r 21 days.

Or~ A~rage Percent We-ght of ~resh Organs {m~SE) Treatment Lun~ Liver Kidneys Vehicle 0.85+0.06 3.45+0.07 0.95+0.04 (3 animals) 1.07+0.09 NS 4.5~+~.10 HS~x) 1.0~ 03 NS
~5 animals) - -(x) Death caused by a mistake in esophagus incannalutation.
This diagnosis was confirmed at the ~ost-mor ern ;
e~amination.

In view of the favorable sub-acute toxicity, the chronic toxicity in female mice was studied. The results are given in Table 9.

, 1~6~8~9 Ira}~
Chronic 'rOxic~it~ in tlle E'elrlale Mouse Daily treatrnent by gastric gavaye for 18 w~eks ~4.5 mo~-ths) a Mortality and Body Weight Oral Treatmentl)ead7 Body Weight in g ~r--~gE) Treated Start~erminat:ion Vehlcle 3/10 28.2~133.0~1.1 CO-l, 500 mg/kg/day 2/10 30.4-J0.9 30~0~0 7 CO-l 250 m /k /day 0/10 27.3~0.5 26.7~0,7 , g g b. Urine excretion. Urine amount excreted by 6 animals in -6 hours Oral Treatment Urine Amount ~ml) ~ - -, Controls 6 -CO-l, 500 mg/kg/da~ 7 CO-l, 250 mg/kg/day 6,5 c. Blood Glucose. Mean values fo~ 6 animals. Blood samples were ~aken 24 hours after the ]ast dose .
Oral Treatment Blo~d Glucose Controls 1.14 CO~l, 500 mg/kg/day 1.06 ~ -CO-l, 250 mg/kg/day 1.10 ~ .
d. SGPT and S~OT. Mean values for 6 animals. Blood sam les were taken 24 hours after the last dose P . _ - .
Oral Treatment Units/ml SGOT SGPT
Controls 125 5 CO-l, 500 mg/kg/day 159 6 29 CO-l, 250 mg~kg/day 118 5 cb/ - 14 -8~

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, 1~9~39 In view o~ the favor~b].e results on cllronic toxicity, a teratogene~lc s~udy was conduc~d wi~h ~ale and female mice and rats. T~,e number o~ youncJ delivered live at birth was comparable with controls. No malformations in either group were observed. The data are glven in Table 10.

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Cvlnpoun(l C0~1 i.f:t ~minister~d to hurnans eY.p~s~d to cholera un(:ler e~ mic condi~:ioll~ .in dos~cJes o~ :~r.om 0.3 to 5 g per pcrson per day for 3-5 days. None of ~he treated persons become in~ected but nurne~ous one~ with whom they are in daily contact do ~ecome i.nfec~e~. It .is determined that C0-1 is effective by oral adminis~ration as a prophylactic agent in p~eventing cholera. The C0-1 is also advantageous in that it select~vely combats, e.y., inhibits growth of cholera-causiny miCrooryanisms~ cholera without deleteriously affecting the oryanisms in the human biological system.

To a reaction vessel there was delivered a solution of 1,15 g of 96% sulfuric acid dissolved in 25 ml acetic acid, 1~23 g ~0.01 mo]e) of 2-amino-4,6-dimethyl pyrimidine and 1.9 ~ ~0.01 mole) of 2-formyl-quinoxaline-di~N-oxide.
The mi.xture was heated at 40C. for 16 hours, then cooled, diluted with water and adjusted ~o pH 5 with sodiurr. bicar--bonate solution. The resulting yellow, crystalline precipi-tate was filtered and washed to give, in 71~ yield, 2.1 g f 2-/2-(2-amino-6-methyl-~-pyrimidinyl)-ethenyl7-quinoxaline 1,4-dioxide, designated a~ C0-2 for convenience. It melted with decomposition at 240C.
The product ~as tested against the five strains of ~ibrio cholerae as described in Example 1. The results are ~i~en ln Table 1. There was no growth after 48 hours of 3 of the organisms at 10 g/ml of C0-2 and only marginal growth of the remaining two at 100 g/ml.
Compound C0-2 is tested for toxicity in the samç
manner as described ~or C0-1 in Example 1. Comparable resul~5 are obtained sho~Jing that the compoun~ is suitable for prophxlaxis.
Compound C0-2 i~ ~dminist~red to hurnans exposed to .

cb/ - 18 -9~
cholera und~r epidernic conditions, in dosac~c~.s o~' rom 0.3 to 5 g per day fox 3 ~o S days, None o the trecltecl pexsons become infected but numerous ones wi.th whom they are in dail~ contact do become infected. It is de~ermined tha~ C0~2 is effective by oral administration as a prophylactic agent in preventing cholera.

Compounds containing, respectively, 6-ethyl, propyl, butyl and penty~ substituents instead of the 6-methyl sub-9 stituent can be similarly usecl as a propllylactic agent.

' c~ 19

Claims (10)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A process for preparing a substituted quinoxaline having the formula in which R is hydrogen or a lower alkyl C1 - C5, comprising reacting in substantially 1:1 mole ratio quinoxaline-di-N-oxide-2-carboxyaldehyde or its lower alkyl acetal and a compound having the formula wherein R is as indicated above, in the presence of a strong acid catalyst.

2. The process of claim 1, wherein the said compound is 2-amino-4-methyl-pyrimidine and the said sub-stituted quinoxaline is 2-{2-(2-amino-4 pyrimidinyl)ethenyl}
quinoxaline 1,4-dioxide.

3. The process of claim 1, wherein the said compound is 2-amino-4,6-dimethyl pyrimidine and the said sub-stituted quinoxaline is 2-{2-t2-amino-6-methyl-4-pyrimidinyl) ethenyl}-quinoxaline 1,4-dioxide.

4. The process of claim 1, 2 or 3, wherein the reaction is carried out in an organic solvent consisting of formic acid or acetic acid.

5. The process o claim 1, 2 or 3, wherein the reaction is carried out at 25-50°C.

6. The process of claim 1, 2 or 3, wherein the proportion of the strong acid is from 0.5 to 2 moles per mole of quinoxaline starting compound.

7. The process of claim 1, 2 or 3, wherein the strong acid is hydrochloric, hydrobromic, sulfuric, nitric, trichloroacetic or an aryl-sulphonic acid.

8. A substituted quinoxaline having the formula:

in which R is hydrogen or a lower alkyl C1 - C5, whenever pre-pared by the process defined in claim 1 or by the obvious chemical equivalent.

9. 2-{2-(2-Amino-4-pyrimidinyl)ethenyl}-quinoxaline 1,4-dioxide, whenever prepared or produced by the process defined in claim 2 or by the obvious chemical equivalent.

10. 2-{2-(2-Amino-6-methyl-4-pyrimidinyl)ethenyl}-quinoxaline 1,4-dioxide, whenever prepared or produced by the process defined in claim 3 or by the obvious chemical equivalent.