Journal of Medicinal Chemistry 0 Copyright
1971 by the American Chemical Society
FEBRUARY 1971
VOLUME14, No. 2
Cestocidal Activity of 4-Alkoxy-1-naphthamidines against Dog and Cat Tapeworms ROBERT B. BURROWS,* The Wellcome Research Laboratories, Burroughs Wellcome & Co. Inc., Research Triangle Park, North Carolina 27709
C. J. HATTON, The Wellcome Veterinary Research Station, Frant, Sussex, England
WILLIAM G. LILLIS,AND GEORGE R. HUNT The Wellcome Research Laboratories, Burroughs Wellcome & Co. Inc., Tuckahoe, New York 10707 Received August 6, 1970 Structure-activity relationships in a series of 39 naphthamidines tested against 4 species of dog and cat tapeworms have been evaluated. The compounds fell into 4 groups that varied in activity and toxicity. The group containing those compounds in which R’ was either n-Pr or i-Pr and OR ranged from pentyloxy to decyloxy was the most active and next to the least toxic. Since most members of this group are equal to or better than bunamidine hydrochloride, further comparative studies are in order.
A number of years after a preliminary paper on the 4-alkoxy-1-naphthamidines’ was published some members of the series were found active against the mouse pinworm, Syphacia obvelata,2aand against tapeworms in mice, dogs, cats, and sheep.2b The activity proved to be better against tapeworms than against pinworms and the most active compound a t that time was selected for extensive veterinary investigation. This compound (Table I, 18) was given the name bunamidine hydrochloride and has been used extensively throughout the ivorld since then. Over 20 additional naphthamidines have been made and tested against 2 tapeworms of mice (Hymenolepis nana, Oochoristica symmetyica) and against 4 species in dogs and cats (Dipylidium caninum, Hyclatiyera taeniaeformis, Spirometra mansonoicles, Taenia pisifoymis). The results of the mouse work have been published3and the present paper will report the results of treatiug dogs and cats in America and dogs in England. The 39 naphthamidines selected for the dog and cat studies have the basic structure I’lzb in which R ranges from CH, to CI3H2,and R’ from C2H5t o C8H17. The objective was t o test each of the naphthamidines active against mouse tapeworms against 4 dog and cat
* To whom
I
OR
I
cestodes, to determine what structure-activity relationships existed, to evaluate the whole series or smaller groups, and to find out if any compounds appeared to be superior to bunamidine hydrochloride.
Materials and Methods A (Tuckahoe). Selection of Animals.-All stray dogs and cats purchased by the Laboratory were examined for eggs with the ZnSO, flotation technique and for proglottids by examining the pan beneath the wire bottom of the cages. An animal positive for any species of tapeworm was selected for trial and was kept in an individual cage during the test. Dogs were given canned dog food and H20 and cats, canned cat food and milk. Infections.-All infections were natural, except for Spirometra mansonoides. Although on occasion we have found this species in cats and have carried out the entire life cycle in the laboratory, most of the infections used in this series were obtained from spargana given to us.( Dose.-Throughout these trials a uniform dose of 50 mg/kg was used, except for a few cases in which the shortage of drug made it necessary to reduce the level to 25 mg/kg. Gaps in Table I indicate that the supply of drug was exhausted before all species had been tested.
correspondence should be addressed.
( 1 ) E . Lorz and R . Baltzly, J . Amer. Chem. S o c . , 7 3 , 93 (1951). (2) (a) .\I. Harfenist, J . .Wed. Chem., 6 , 361 (1963); (b) R. Baltzly, R . B. B u r r o w , 11.Harfenist, K. A . Fuller, J. E. D. Keeling, 0. D. Standen, C . J. H a t t o n , V J. Nunns, D. A . Rames, B. D . Blood, V. ILloya, and J. L. Lelij-
veld, Nature (London), 206, 408 (1965). (3) M . Harfenist, R . B. B u r r o w , R . Baltzly, E. Pedersen, G. R. H u n t , S.Gurbaxani, J. E. D . Keeling, a n d 0. D. Standen, J . M e d . Chem., 13, 97 (1970).
(4) Spargana were donated by D r . J. F. Mueller, State University of New York, Upstate Medical Center, Syracuse, N. Y.
87
88 Journal of Medicinal Chemistry, i97i, V o l . 14,*To. f
Procedure.--After ail animal was dosed, it' was observed frequently for evidence of side effects, which were recorded. Each fecal specimen was collected aiid Tyashed through a sieve, the sieve coriteiitj were poured a little a t a time into a blackbottomed pan, aiid searched for worms, or fragments, using :t magnifier-illumiiiator. All fiiidings of any species of parasite were recorded. After 4s hr the animal wa> necropsied. all remaining worms were collected arid counted, aiid with the aid of the magiiifier-illuminator the entire intestinal wall wah examiiied for attached scolices. Where possible, the percentage of worms killed was compiited. Since tapewornis disintegrate rapidly after death, particularly when remaining iii the intestine for some hoiirq, 1110 signs of dead tapeworms were found in m:tiiy animals. In ail earlier study of one of the iiaphthantidinea,5 the average tapen-orm burden of a large number of iintreated animals n-ah used a,>a basis for estimating percentage of elimination of worms in treated animals. This procedure was followed throughout most of the trials doiie with the large group of iiaphthamidiiies. Iii the last fourth of the dogs and cats we used the PAF sedirneiit:rt i o i l techiiique6 on fecal specimen, of all treated i~iiimal., ciiice it iiidicated whether or not the drug had activity. All specimeiis passed daring the first 24 hr after dosing were esamiiied with the PAF techiiiqiie as n-ell as by checking the hieve contents iii a black-bottomed pan. If a large number of egg> and calcareous bodies were .seeii in the sediment, that was an indication of activity a i d at iiecropsy it could be determined whether scolices or ahort portion> remained attached. If few or no eggs or calcareous bodies were seen iii the sediment., the lack of activity could be coiifirmed Then the necropsy was done. :lii occasioiial drug caiised qiiick di>iiitegratioii of worm. aiid destruction of eggs, so t h a t oiily fragmented eggs aiid ca1c:ireoii~ bodies are foiiiid. Piilike the other zpecie-. of tapeworm,*, eggs of Dipyiidiuni canznum were rarely foulid i n the sediment of the P.\F procediire. -1fter the saliiie-EtrO combination wiiq .piui down, the wpernataiii wa,G poured into a >mall beaker, which was set aaide for iiboiit 1 h r for most of the Et20 to ev;iporate. The contenti n-ere then -wirled and poiired into a ceiitrifiige tnhe, more saliiie n-n> added to the 12-ml mark, the tiihe was centrifiiged, the sipernatant. was poured off, and a few drop, of d i n e were added t o the sedimeiit. The aedimeiit was mixed. niid a drop ~ v a -placed oii a slide aiid examined for D . ccmi'n~iiii egg.. Calcareous bodiea from D.cnninum are more likely I!) he foiiiid in the P-IF hedimelit, whereas the egg. themselveb itre recovered more readily from the hedimelit of the supernatztiit. I3 (Frantj.-The procedure5 followed are eswitiallj. iho-e i i s d iii earlier ~ o r with k buiiiimidiiie.' Infections.-The iiifectioii,: of I'ueni'a piszj'oor.nzis, thc orily apecies ii.:ed, were experimeiital, Gravid proglottid> w ? r e obtaiiied from dogs, groiiiid lightly in a mortar, a little formaliii \$-a>added t o inhibit bacterial growh, and the stispelision was -et a4de at room temp for at least 2 day:. Each rabbit wa:, giveii orally :?ii :tq suspen>ioii corit,aiiiiiig approximately 40,000 egg.. After nbout 14 week, t,he rabbits were iiecropsied, aiid the cyst? Lvere i,emoved aiid pooled. Puppies (6-3 week- oldr were given a driiik of protein hydrolpate8 t o stimulate galtric st.; i i i a gelatin capsule. secretion, niid then were gi Mature tapeworms recover procedure ranged from 41) to looc;. The piippie.; 11 individual cages, with wire meah fiaoi.', ahoiit G w er iiifection, and a check was made for egg> or proglottid> iii fecw. When found po-itive, they lyere iised in esperimeiit.. Dose.-JThere suficietit contpoiuid was available, each tirug wa': tefted a t severs1 differeiit levels, generally 100, .io, 2 5 , 12.5, and 6.25 mg of base/kg, iinless prior strtdies 011 mice had iiidicxated that the higher levels might he too toxic for dog?. In ii few case- the highest level tested was 25 mg of base,:kg. The activitj- iricorporated i i i the chart wa,q that obtained with the SO m g of base 'ky level, uiile+ indicated otherwise. Procedure.-For 7 2 hr after dosing all feces were collected aiid examiiied for proglottids a i d eggs. Since the majority of worms qiiickly di-iiitegrated after death, clearance could rarely he esprewxl i n percentage. After 10 days the dogs were iiecrop i e d , the entire iiitestine was slit. open, and the content. were
i )R. 13. Burroirs a n d W ,G . Lillis, d m e r . J . 1-d.Res., 27, 1381 (19661 ifi) R . R.Burrows, Amer. J . Clin. Pulhol., 48, 342 (1967). ti) (a) C . J. Hatton, I'et. H e c . , 77, 408 (1965): (13) ibid., 81, 10-1 i l O H 7 1 . (81 PROTOGEST.
BCRROWS, et aE. washed into a 44-mesh sieve. All material retained by the sieve was searched for worms or fragments.
Results The data from these trials are presented in Table I. Dipylidium was found to be the most sensitive to these compounds, with 29 of 35 trials (83%) resulting in complete elimination. Tae?iia \vas the most resistant and the 2 strains were fourid to be quite different, only 5 of 36 trials (14%) with the American strain giving perfect results compared n-ith 12 of 28 (43%) with the English strain. Hyclatiyei.a, 24 of 37 (E%), and Spirometra, 24 of 39 (62%)) were intermediate and fairly close to each other in response. -1 large chart of squares was made to arrange the compounds in systematic order and to place the test results in their appropriate positions. The systematic arrangement of the various compounds is shown in Figure 1. When the test results were studied on the chart, it n.as found that the compounds fell into 4 groupb, with >light similarit! of activity on both sides of the group interfaces. These 4 groups (AI-D)are outlined on Figurc' 1 and each encloses the compounds of the group. summar? of the results obtained in each group is presented in Table 11. Group A had less than one-fifth of the trials rebulting in coniplete elimination and the average percentage of clearance was 32. Group D, :It the other end of the series, was somewhat better, with less than half the trials being perfect and an average percentage of 40. Group B, which contains buntimidine (18) ~ v a better 1 than either of the others, with io% of the trials being perfect and the average clearance being However, in group C every trial run against Dzpglulzum, Hgdatzgem, aiid Spiromelra resulted in complete n-orm removal, the majority of TapIU infections were eliminated and the overall clearance for the group LI as 91%. Complete removal of the Englibh strain of T . p s i f o r m z s occurred only in Groups B aiid C. but the .Imericari \train responded completely only to 3 compounds in group c. Five compounds of group C (11, 12, 30, 32, 34) removed all tapen-orms of each animal tested (Table I). Tn-o (11, 12) were not tested against the English strain of T . pzszjoi-iizis and 1 (32) was not used against Hyclatigera or the 'Imerican Tae/riu because of insufficient compound. dcute oral toxicities in mice have been done on most of these compound^,^ and the information was extracted from the files for the present use. The range and (average) number of mg/kg po for the 4 groups are: group &I,400-5000 (1730); group B, 200-730 (425) ; group c, 180-1200 ( E O ) ; and group D, 250-1200 (835). Group -In a s the least toxic arid the least active. Group D u-as about tn-ice as toxic as A, but somewhat more active. Of the 2 active groups, C was more effective arid less toxic than €3. The 5 best compounds, all in group C, h:id these IIDjO'sPO: 11,250;12, 500; 30, 400; 32, i 5 0 : aiid 34, >1000. This information suggest5 that 34 is half as toxic as 18 (LDSO540)3and more active. Of the 186 animals used for these trials, 33 vomited, 3 cats died, arid several dogs had some diarrhea. (Since
iGz.
: Y ) Our thanks t o R. V . Fanelli a n d B. R. Sezesny, Pharmacology Department, T h e \Vellcome Research Laboratories, Tuckahoe, N. Y.. who determined the acute toxicities.
Journal of Medicinal Chemistry, 1971, Vol. 14, N o . 2 89
4-ALKOXY-1-NAPHTHAMIDINE CESTOCIDES
TABLE I ACTIVITYOF
THE
VARIOUSNAPHTHAMIDINES, AT 50 MG/KQ,
AQAINST
-% Compd
1 2 3 4 5 6 6a
7 8 9 11 12 13 14 15 16 17 18 19 20 23 24 25
Dc"
Ht
0 100 100 100 100 50 100 100 100 90 100 100 100 100 100
100 100 0 100 50 75 100 100 33 0 100 100 100 0 0 0 100 100 100 0 100 100 100 0 100 100 100
100 100 95 8 100 100 100 100 100 100 100 100 100 100 100 100 100 100
27
29 30 31 32 33 34 35 36 37 38 39 40 41 42 43
0
100 100 100 100 0 100 100 50
100 100
0
FOUR CESTODES OF Doas tapeworms eliminated-SWl
0 50 0 100 0 0 100 100 50 0 100 100 100 100 0 50 100 100 100 25 100 100 100 0 100 100 100 100 33 100 100 100 0 100 100 100 0 100 0
AND
CATS
Tp-A
0
14 0
0 33 0 Ob
50 0 0 100 100
Tp-E
73 0 50b 0 0 0 100 100 0 50"
50b 4 50 0 0 50 1 0 100 81 0 0 0 100 0 0 100 14 0 0 0 0 0 0
0 0
100 100
Ob Ob 100 lOOb 100b 100 0 100
100 100 100 Ob 50b
0
Summary 24/39 5/36 12/28 Cleared/treated 29/35 24/37 22 53 Av yo worms eliminated 90 70 67 a Abbreviations for tapeworms: Dc, Dipylidium caninum; Ht, Hydatigera taeniaeformis; S m , Spirometra mansonoides; T p , Taenia pisiformis; A, American, E, English. b Dosed at 25 mg/kg rather than a t 50. c Dosed at 100 mg/kg rather than at 50.
emesis, 36% in dogs and 25% in cats.
TABLE I1 EFFECTIVENESS OF
AGAINST
-Group Speciesa
Dc Ht Sm Tp-A Tp-E
A-
C/Tb
%
7/11 3/12 1/12 0/12 0/11 11/58
77 38 23 8 16 32
However, this
VARIOUSGROUPSOF NAPHTHAMIDINES apparently had little effect on the activity of the drugs, THE DIFFERENT SPECIES O F CESTODES since 14 of 17 group C animals that vomited were freed
THE
-Group
C/T
B%
-Group
C/T
C-
%
-Group
C/T
D%
9/10 99+ 10/10 100 3/4 75 9/10 90 9/9 100 3/6 58 9/10 93 10/10 100 417 57 0/9 12 5/9 66 0/6 0 6/8 81 6/7 86 0/2 25 Total 33/47 76 40/45 91 10/25 40 See footnote a , Table I. C/T,animals cleared/animals
treated; yo,av per cent of worms removed from group treated.
a few animals had infections with 2 species of tapeworms and since a few experiments were repeated, the number of trials (Table 11) and the number of animals used are not the same). Twenty-one per cent of the 98 dogs and 14% of the 88 cats vomited. Vomition was less in groups A and B, being 13% for dogs and 9% for cats. Group C, which had the best activity, had the most
of tapeworms and had an average clearance of 88%) which compares favorably with the 26 of 28 freed and 93% clearance in those that did not vomit. I n group D, 33% of the dogs and 14% of the cats vomited. The cats purchased for use were strays that were not used to cages, and it was not unusual for 1 or more of each dozen to be found dead in the cage at some time during the first week. Those that survived the first week generally adjusted to cage life. All 3 cats that died were dosed within a few days of arrival, in no case did other animals receiving the same drug die, and 2 of the 3 drugs were among the least toxic, with LDjOIs PO of over 1000 mg/lrg. These deaths are not blamed on the drugs. Several dogs in America and several in England had diarrhea, but this was not attributed to the drugs. Often changes of diet and/or habitat may cause a tem-
HUFFMAN, et ai.
90 Journal of Xediciizal Chemzstry, 1971, Vol. 1.4, S o . 2
species, one articlei0 dealt with the effect of 8 naphthamidines against Echinococcua gt.anulosus in Llrgentine dog. -111 8 of these compound5 fell into group3 and B,(ihad nioderate activity, 2 had good activity, and bunmiidine hydrochloride was considered the better of the 2 good ones. H a t t o i tested ~ ~ ~ 3 different salts of bunsmidine: hydrochloride, h? droxynaphthoate, and p-chlorobenzenesulfonate. He reported that the hydroxynaphthoate produced fev er side effects than either of the others, but 11 as rathei inactive when given on an empty stomach. JVhen niiietl n i t h iood i t WLS quite active. On the other hand, Burron 5 and I,illis5 found the hydrochloride more effective against Y'. pzsiformzs nhen given on un empt!- stomach than when given at or near mealtime. The m0.t eff ectivc group of riaphthamidinez niade to date are thuw iii group C. which contains the conipounds in n hich 012 ranges from pent! lox! to decyloxy and K'is either Ic-Pr or 2-1'1'. Differences betivecn conipound5 having j r ~ l ' r jor~ ( ~ - 1 ' r )'ire ~ minor mid ncit consistently t:iwrable to either. S o v tlixt tlic most active group of the naphthamidincs a p p e ' ~tu tie pinpointed. heveral other probleinz must be at taclml. Thebe iiiyolve additional trials :it oarious (lose les-el> of most of the compounds of the grciup. thc relation oi dosagc time to feeding time, the teating oi diffcrent particle hizes, the preparation of the hydrou! nnphthottte d t of tlie better compounds. and the evaluation of .evcr,d diff pr ent type< of formulatiom of the bettcr d r u p . A\
C,IlI
c
If,l
('411
cjrIC.iT (
I
1
j 17 11
12
C
18 13 6
20 15
14
a
B ~
rrs
7
8
16 9 5 3 1
6 4 2
porary looseness, regardless of whether the dog had been treated or not. The majority of dogs in thede trials had normal bowel niovenients or were somewhat constipated.
Discussion hfter bunamidine hydrochloride was selected as the best of the naphthaniidines niade prior to 1963, a nuniher of papers have appeared concerning its activity against 14 species of tapeworms of mice, dogs, cats, sheep, horses, and poultry. These papers are listed in a prior a r t i ~ l e . ~I n addition t o the various papers dealing with the effect of bunamidine on different cestode
(10) 1 3 . 11. 13louil. T'. l l o y a , and J . I.. Lelijvelil, BuIl. It*. If. U . , 39, G i (1!468).
Syntheses of Halogenated Phenanthrene Amino Alcohols as Antimalarials' JAXES T. TRAXLER, LEROY0. KRBECHEK, RICHARD R. RITER,RICHARD G. WAGNER, AND CLARENCE
w-.HCFF3iA4N*
Internatzonai Xlinerals and Ckemzcal Corporatzon, Growth Scicnccs Ccnic r , Lzbertywlie, Illinots 60048 Received Jwne i5, 1970
h series of phenanthrene-1-amino alcohols has been prepared a i d evaluated against Plasmodium berghei iu mice. The target compounds contain halogen at the 2, 6, 6,7, 6,7,9, or 10 positions, and were made through sequeiices begiiiiiing with chlorobenzene, o-dichlorobenzene, methyl 1-phenaiithrylate, or methyl 3,4-dihydro-lphenanthrylate. Some of these halogen-substituted phenanthrene-1-amino alcohols showed moderate curative activity against P. berghei in mice.
As part of the current U. S. Army Research Program on Malaria, we undertook the syntheses of phenanthrene aminomethylmethanols. The aim was activity against the drug-resistant strain of Plasinocliuin falcipamm. I n a similar program during World War 11, a considerable number of phenanthrene aminomethylmethanols had been prepared, and some showed considerable activity against other types of malaria.2 Most of the earlier
*
T o whom correspondence should be addressed. (1) This is contribution KO. 819 from the Army Research Program on Slalaria. The investigation was conducted under Contract S o . D.4D.41768-C-8025-P-901, U. S. Army Medical Research and Development Command, Report AD862199L. 12) G . R . Coatney, X', C . Cooper, S . B.Eddy, and J. Greenberg, "Surv e y of Antimalarial .igents," Public Health Monograph S o . 9, U. 5 . Government Printing Office, Wasliington, D. C., 1953.
phenanthrene derivatives had the dialkylamino alcohol group at the 3 or 9 position, a few were located a t the 2 position, and one3 was found4 to be at the 1 position. We have described our earlier syntheses of basic phenanthrenemethanols with the dialkylamino alcohol side chain attached at the 1 and at the 4 position, and with H, C1, or Br at the 9 or 10 p ~ s i t i o n . ~We now report the syntheses of phenanthrene-1-amino alcohols substituted with C1 or Br at the 2 , 6, or 6 and 7 positions, and with C1, Br, or H at the 9 or 10 positions. ( 3 ) J . Schultz, 111. .i. Goldlierg, E . P. Ordas, and G . Carsch, J . O r g . Chetn., 11, 329 (1946). (4) L.0. Rrbechek, R . R. Riter, R . G. Wagner, and C. \T, Huffman, J . M e d . Chem., 13, 234 (1970).
