methylated derivatives, which in the case of estradiol was identified as 2-methoxyestradiol by paper chromatography. Simple phenols, naphthols, and hydroxyquinolines undergo hydroxylation to catechols. This reaction has also been demonstrated previously in vivo for various simple phenols and hydroxyqinolines.26 In our studies both 4- and 8-hydroxyquinoline are hydroxylated while the 2-hydroxy isomer which exists mainly in the 2-keto form does not react. Various dimethylphenols are active except for 2,6-diniethylphenol in which catechol formation would require replacement of a methyl group by hydroxyl. Siniilarly various dichlorophenols were active, and even 2,4,6-trichlorophenol was converted to a methylated product although both ortho positions are blocked by chloro groups. Since K\’,4DP and glucose-6-phosphate are required, direct methylation of the phenol without an oxidative step is unlikely. Also catechol-0-met hyl t ransf erase involvement is indicated since the formation of the 0-methylated derivative requires magnesium ions and is inhibited by tropolone.12 Direct 0-methylation by the enzynie which 0-methylates 2,6-diiodophenols, described by Toniita, et C L ~ does . , ~ ~not seem likely since their enzyme does not (26) R. I. William8, “Biochemistry of Phenolic Compounds,” J. B. Harborne, Ed., Academic Press Inc., London, 1964, Chapter 6
require magnesium ions. I t s e e m more likely that a replacement of a chloro group by a hydroxyl group, similar to that described by KaufmanZ8for the conversion of p-chloro- or p-fluorophenylalanine to tyrosine, has occurred. Certain other 2,6-dihalophenols also show low activity in our niicrosoinal system. Little correlation of activity with structure was possible for the various monosubstituted phenols (Table VI) studied. Since catechol-0-methyltransferase is present in a large excess and is little influenced by substituent9 so that 0-methylation is observed even in the presence of very bulky ortho substituents such as most of the effects observed are probably related to hydroxylation. Both para- and meta-substituted conipounds were quite active with ortho substitution, often severely reducing activity especially with very bulky groups such as t-butyl or with groups that can interact by hydrogen bonding with the phenol such as in salicylaldehyde. Conipounds sensitive to oxidation such as aiiiinophenols showed low activity, perhaps due to competitive oxidations. (27) K. Tomita, C. &I. C h a , and H. A. Lardy. J . Bior. Chem., 2S9, 1202
(1964).
(28) S. Kaufman, Biochzm. Biophys. A d a , 61, 619 (1961). (29) T. Matsuura, A. Nichinagu, and H. J. Cahnmann, J . Ore. Chem., 27,3620 (1962).
Excretion, Distribution, and Metabolism of Doxapram Hydrochloride ROBERT B. BRUCE,J. E. PITTS,
F R A N K L I N P I N C H B E C K , AND
JACK SEJVYIrlN
Research Laboratories, A . H . Robins Company, Inc., Richmond, Virginia Receibed August 24, 1964 Doxapram hydrochloride, following intravellous injection into the dog, was rapidly metabolieed. No unchanged drug was found in any biological material analyzed. The metabolites formed were distributed throughout the animal body with higher levels occurring in the fat, liver, pancreas, and adrenal glands than in other tissues. Blood levels of the metabolites decreased rapidly during the first hour followed by a much more gradual decline. Large concentrations of the metabolites occurred in the bile. The metabolites were excreted rapidly in the urine during the first 24 hr. Small amounts continued to be excreted up to 120 hr. after injection of doxapram hydrochloride. The primary path of metabolism was through opening of the morpholine ring. A large number of metabolites were formed, two of which were identified as l-ethyl4(2-hydroxyethylaminoethyl)-3,3-diphenyl-Z-pyrrolidinoneand l-ethyl-4-(2-aminoethyl)-3,3-diphenyl-2-pyrrolidinone.
Doxapram hydrochloride’ is the nonproprietary olisni, distribution, and excretion of doxapram. To name for l-ethyl-4-(2-niorpholinoethyl)-3,3-diphenyl- carry out such studies it has been necessary to develop 2-pyrrolidinone hydrochloride (I). This compound is analytical methods suitable for the determination of the drug in the various biological materials. The methods are ultimately based on the oxidation of ‘c6;q CH2CH2hn0 H W doxaprani and doxapram-like materials to benzophenone which is subsequently quantitated froni its I ultraviolet absorption. The methods described, thereC2Hs fore, will determine any material which contains the I diphenylniethyl moiety and which appears in the niaterial finally oxidized. an agent that produces niarked respiratory stimulation and pressor effects in aninials2a,band in huiiians.2C~d Correlation of the results obtained from the “oxidation method” with those found using C14-labeled doxaWhen doxapram is administered to animals under barbiturate-induced sleep, it produces arousal effects2aZc pram indicate that all of the metabolites formed may be determined by the choice of the appropriate proI n this paper we present the results that have been cedure for the “oxidation method.” obtained in studies designed to elucidate the metab(1) C. D. Lunsford, A. D. Cale, Jr., J. W. Ward, B. V. Franko, and H. Jenkins, J . M e d . Chem.. 7, 302 (1964). (2) (a) J. W. W a r d and B. V. Franko, Federation Proe., 21, 325 (1962); (b) W. H. Funderburk and R. 9. Alphin, ibid., 21, 324 (1962); (0) A. J. Wasserman and D. W. Richardson, Clin. Pharmacol. Therap., 4, 321 (1963); (d) H . G. Canter, A m . Rev. Reapiraf. Diseases, 87, 830 (1963).
Experimental Analytical Methods.-Initially, the ultraviolet spectrum of doxapram was considered as a method for quantitation. The
:itrsorptiirii coefficient pri~ved,however, t o be too l(iw to determine the srnall amounts that might be present i n biological materials. This spectrum showed four distinc*t tiiesinia at 2611.5, 264.3, 2*58..5.and 253 mp. Compounds chemically related to doxapram, i.?., those containing the dipheiiylpyrri)litlirioiiie moiety, gave simi1;ir ultraviolet spectra. In some i'ases the masinia were dis1rl:iced. The spectra have proved i i f gre:ii valiie in t h e separntioii :rnd identification of met:rbirlites. The ultraviolet absorption of lieiiziii)heiioiie, i i i contrast t i l that I J ~doxapram, is very strong. The osidatiou of doxapram: iititler proper conditions, should yield this ketone. Attempts V.YIY' made to oxidize with c-hromic ac.icl, hiit ihe results were not t~t~prdiicible.Alkaline permarigana te oxidation i n a boilingwater bath gave consistent results, and tlie qiiaiitity of dosaprani stlded was directly proportional to tlir l~rnzopheiiiirieabsorptiori flilllld,
It. was foiind possible to separate beiizopheiioiie from the cooled ositlut,iori solutioxi by extraction with isooct,ane. This solvent tviiuld not, complet'ely extract the benzophenoue from the alkaline iisiclation mixture. However, if the mistiire were first made acidic with phosphoric acid, estractioii appeared to be complete. 01her acids, sulfuric or hydrochloric,, appeared t,o be oxidized iiuder these condit'ions arid resulted iii ultraviolet ahsirrptioti w1iic.h completely masked the berizophenoiie spectriim. Sirnie s:rmples coiild be oxidized d i r e d y by t,he method dest~rihed. This was ti'tie wheii the Conceittration of the hetizciplietitriie-yieldiriy material was siifficieiitly high, as in the case iif uriric or bile. The limiting factor was ilie capacity of the potnssiitrn permatigaiiate added. Be.c.niise if this, organic solvents itsed fiir extraction miist be reniovetl urmpletely prior to the osidatioii. Tissues were extracted with lieiizeiir or ether, arid the scilveiit was removed from the esira1.t lidore cisidation was carried oiit. The detailed priii~ediir(~+ tisetl i t t these irivestigatious :ire ;is described l)elci\\-. Bile and Urine.--An aliquot of Iiile (0.10 t i i l . ) or urine (0.10-0.30 1111.)was transferred to a 200 X 18 nini. test t,ube having a 24/41 -$ joint (oxidation tube). T o this sample 0.5 nil. of 2 S SaC)H and 5 ml. of .5(.; Ei~IriOIwere added. .\ti air condenser (2.50 X 6 mni.) was coiiriected to the test tithe aiid the sample was hented for I hr. iii i i boiling-water b n t h . The sample was cooled a i i d made acidic by adding 1 nil. of 6 .\ H 3 P 0 , through the air coiideiiser. The cotidenser was removed a t i d 2.0 ml. of Spectrri (:mtle isooctane was added immediately. Tlie contents of the tiihe were mixed for 1 miri. on a I-ortes niiser tlJ extract the benzciirheirorie into the isooctane. The isooctane h y e r was trmsferred t i l a inicrocuvette (1-mi. path leiigth). and its spectrum was dtsteimiined from 250-260 m p . -1base line wvas drawn from 237 l o 2,56 trip arid the peak height was measitred from this line to the ni:isimiim iisiiig a millimeter rider. This lerigth was converted t o c,oriceiitration tiy a predeterniiried f:ictcrr or ciirve prepared from piire doxaprsni under the same roiiditiiins. Blood and Cerebrospinal Fluid.---,% samplc of 5.0nil., or less, i r f I r l i i i r i l or c~erehrospinal fluid was trmsferreti io a 50-nil., glasssirii)pered c*ent,rifuge tiibr c,criitaining 21) nil. ( i f water, I.t) ml.r i f .\' S:iOH, n r t t i 10.0 nil. of herizcwe. 'Tlic, tube \viis shaketi g2ittly fi)r 5 niin. Eniulsions were broken l)y caentrifugatiori. A .i-ml. :iliquot of the rlear tlenxenr l:iyei, \vas transferred to :in osid:itiori tube, :tiid the benzene \viis ev:iporateti in :I 1)oilinpa.:iier I)stli. TOensure coiiiplete rrriiovd if the tienzetie, 2-;3 nil. { i f absoliite et.hanol vias added after tlie c(iiirelits (if the t i i b e :il)peared dry, and the ethaiiol was evaporated i t i h i l i n g witel, with the aid of a stream of air. E'otassiiim prrmaiiganate antl :ilknli mere then added and the priiwdiire n-as carried oiit as described above for ririiie aiid h i k . Other Tissues.--~-Rainples of other tissues, i n quaiitities u p t o .i g., were ground with suffic,ient :inhydrous NasPOr so that they :ippeared dry. The rnisture mis trmsferred to a thimble : i i i t i cstrarted in ii Siistilet apparatus for 4 hr. Nervous tissue, i.c., brain or spinal urd, was estractetl wit11 ethyl ether; other tissues were estrarted with benzene. The precipitate obtained O I I cooling the ether extrarts was separated bl- centrifugation : i t i d dec:tritation. The estravt (benzene or ether) was transferret1 t o a separatiiry funnel and the dosaprani was extracted wit.h t w i IO-mI. portions and then .i nil. of 2 .\- HC1. The HC1 estracts u-ercl c~cini1)inedi n :inother separatory funnel and made alkaline with 6 ml. of 1 0 S SaOH. The alkaline solut,ion was estmcted three times with 5-nil. portions of CHC1,. The lower layers were dried tliroiigh aiihydroiis Sa,SO1 aiid combined in an osidation ttibe. The solverit was evaporated. as described above for lrrtizeiics, and oxidation was carried o u t .
Cerebrospinal fluid (CSF) was obtained by direct puncture into the cisterna magna, and blood was taken from a femoral vein. Saline was infused ( 3 ml./min.) so as to insure a good urine flow. Samples of body fluids were taken prior to and following the i.v. injection of 20 nig.,’kg. of dosapram hydrochloride. The animals were sacrificed by exsanguination, and samples of the following tissues were taken : heart, lung, liver, ,pancreas, kidney, adrenal, spleen, small intestine, large intest,ine, fat, skeletal muscle, spinal cord, mrtes, medulla, and the remainder of the brain.
Results and Discussion Analytical Methods.-The results from the recovery of doxapram, when known amounts were added to samples of various biological materials, is shown in Table I. This table also shows standard deviations obtained from representative tissues. The results of recoveries of fat and brain by the extraction method were somewhat ~ O T Y , probably due to difficulties eiicountered in the extraction procedures. Emulsions and precipitates were formed when benzene was used for the tissue extraction. Isooctane mas used for the extraction of benzophenone from the oxidation media because other solvents that were inimiscible with water and transparent in this ultraviolet region were oxidized to some extent by the permanganate. The use of the “base-line” method for measuring the absorbance was necessary. Control tissues, L e . , tissues having no benzophenone-yielding material present, gave, on occasion, absorbing material. The ultraviolet curve of such material did not show a niaximuni in the region of 230-260 nip, but showed a straight line. The measurement of the absorbance between the base line and the inaxiinum absorbance for benzophenone in millimeters was purely a matter of convenience in this laboratory. The steam distillation procedure offered a number of advantages over the extraction method. The latter method resulted in the determination of only basic materials, whereas all benzophenone-yielding materials were determined by the steam distillation procedure.
I n addition, there was less occasion to lose materials in transferring from one yessel to another. The recoveries obtained by this niethod are also shown in Table I and appear to be satisfactory for the deterinination of total doxaprani and its metabolites in tissues. Urinary Excretion.-The results obtained from the study of urinary excretion of doxapram-related materials are shown in Figure l . This figure gives the average data and variation obtained from the study of 15 dogs by the benzophenone method. Also show1 are data found in two dogs whose excretion was studied through the use of the isotope method. These results indicate that the major portion of the urinary excretion occurs within the first 24 hr. following dosage. An average of approxiniately 33% of the dose was excreted within this time. I n the 48-hr. period shown, approximately 40% of the dose was excreted. The data obtained from one dog using the radioactive compound are shown in the lower curve of this figure. This shows that s l i d e r ainounts of doxaprani materials were determined as compared with the benzophenone method. This might indicate that the pyrrolidinone ring had opened with the loss of the labeled carbon. However, a second dog studied for a 4-hr. period, as shown in the upper curve, would not indicate this. These data are probably within the experimental errors of the two methods of analysis and are not believed to indicate any significant difference. The excretion in several dogs, including the one that received radioactive doxaprani, were studied for 120 hr. The increase in the amount excreted in the 48-120-hr. period accounted for less than 2% of the dose. I n all cases there was some continued excretion during this period. The greatest amount found to be excreted in the urine was 48% of the administered dose. I t was. therefore, apparent that the drug was being excreted through some other niechanisiii or being converted to a form that was not detected by our analytical methods.
50-
d. 40on
-31 3
”-0 30-
20-
Figure 1.--Urinary excretion of doxapram-related materials in 15 dogs. The solid line s h o w the results obtained by chemical analysii and the broken line those obtained with radioisotopeq. Vertical lines indicate the variation in result. of the chemical analyqes.
1(io
Distil-
I.:\tta(,tion method--
lation method
Fecal Excretion.-The fecal exletion was iiot studied as estensirely as was t h c urinary excretion. Th(> chemical method as first usrd \vas not satisfactory Estraction methods ienioved only part of the total doxaprain-relatd materials picwilt. The resiiltb thus obtaiiicd T W I I ~ vai.iablc evcn betn.ren idciitical 5aiiiplcs. Dog iio 18040, 11 hich rcwived labclcd doxapraiii, Jvas studicd i i i t l i i a rcspcct. The rcsult b of this study arc sho\vn iii Tahlc, 11. The major portion of that part excreted in tlic fwes appeared in the. first 24 hr. Some radioartivity roiitinued to appeal t lirough 9-1- hr. following adin iiist rat ion. In the f c w h of this dog 38c& was found. S i n c ~urinary excretion a c ~ ~ i t i t cfor d 31C;. t h e total i t ~ o v c w dmas G F ; of th(1 at1i i i i ri ist c w d do 'l..lBI,J2 11,; F W A LEXCRETIOS O F L ) o x . ~ P R I ~ ~ - C ~ I ? - I ~ EMATERI.LL~ I,.~TEU
Time after adrninistrai ion. iir.
Total doxapramrelated materials cLxcreted, me.
70 of C'? recovered
0-23. 5 4S.S 28.7 22.546.3 1il.1 6.0 46 5-70.5 -1,s 2.8 ;(I, 3-94.0 (! \ 0.5 94,0-120. u IJ 0 'rhia table shows the f e d esciet~ioiifrom a dog t h a ~w:i.adiuiuistered ft single 20-mg./kg. i.v. dose of doxaprarn-Cl'.
March 1965
METABOLISM OF DOXAPRAM
161
Time after dosing, hr.
Figure 2.-Blood levels of doxapram-related materials in 12 dogs. The solid line shows the results obtained by chemical analysis and the broken lines those obtained with radioisotopes. Tertical lines indicate the variation in results of the chemical analyses.
,
/
/
/
/ I
___---/
I
341-62, which received labeled dosapram, was analyzed
4 hr. following administration and found to contain 0.07 mg./lOO nil., confirining that low concentrations were present.
Metabolites.-A number of attempts were made to identify unchanged doxapram in urine and in tissues.
TJrine, from dogs that received i.v. doses of dosapram, was made alkaline and the amines were separated by chloroform extraction. Thin layer chromatography of the concentrated chloroform solution revealed three spots which fluoresced after irradiation and which were positive to permanganate and ninhydrin sprays.
Tlic R , values of these spots did iiot correspond to the hpot obtained from doxapraiii. l o r further identificatioii, the spots were reniovcd and oxidized by the analytical inethod. Each gal-(' typical ultraviolet curves for benzophenone. Such analyses have repratedly been carried out but uncxhanged doxapraiii has never been detected Since relatively large aniouiit s of doxapram-lih iiiatcrials were found in the fat of dogs by the abow analyses and no uiichariged doxapraiii was found in t hc iiriii(1, it was interesting io deteriniiic whether it was present in fat. saiiiple of 53 g. of fat froin dog 26943 (containing 1.:3 nig of dosaprani-like inaterials by analysis) was extracted with chloroforni according to the analytical iiietliod. Thv cstravt was concent rat ed and chroniatographed along with doxapram by tEic thin layer niethod on silica gc.1 using ethanol arid (11 hyl acetate-aiiiiiioiiiiuiii hydrosidc ( 100 : 1 v. 'v.) ab d v e n t s . O w spot coiitaiiiitig dosaprani-like matri.ial> was found i i i the fat (1st ravt. a5 rcvealed by thc itic>tlioclb used for thc abow uriiiv ztiialy\i>. This spot dit1 not rorrcspoiicl t o dosapratii :iiitl t l i p qtruvturc ni tlio miiipound ha5 no1 ticcu dcI('rtiiiii(d. Twa1 i i i ~ i i t of a largo -aiiipl(~oi t)lood i i i thc saiiio i i 1 ; i i i i i w a- that dcscribcd ahovc~a1.o failed to shon- thc' p i ~ c w i i of ~ ~ dox:ipraiii ~ i i i i i i i ( L cviiipoiwnts v,-liichh gavel po-11 ivc niiiliydriii spot aittl $1h i ( 2 l i yielded h c ~ i z o plicvioiic on osidai i o i i IV(W prcw~iii - a i i i p l ~of hlood \ \ a - t a k c i i +N 111111 after dosing f i o i i i a dog that had rcc~ivctlc1o\apiaiii-CL4 .liialysi. of t1iv -aiiiple slion c d 1.17 iiig. 100 1111. of doxapraiii or iiivtabolite,s prcaeiit . . l f t c i centrifuging a porl ion, c~>lls mer(>sc1paratc.d froiii the plasma and eacli I\ a- ztrialyzed. Tlic plasiiia was fouiid to coiitaiii SATT; of the radioactivc niaterial. Repeated washing of the. cells with saline failed t o reniovc all of the radioartive iiiaterial, iiidicating that yoiiie metabolic prodiict iiiust be firmly bound to thc cclls. 111 order to at tctiipt t o deteriiiiiic. what compound iiiight be present i i i the blood, a 180-iid. sample, taken 30 inin. after dosing with d o ~ a p r a i n - C was ~ ~ , dialyzed against pH 7.4, 0.1 J l phosphate buffer. The dialysate \\as evaporated to dryness under reduced pressure, the
-
A~ETABOLISJI
March 1965
alkaline extraction. A large number of metabolites, therefore, appeared to be present. The immediate suspicion was that extraction may have been inconiplete. However, this did not appear to be likely since each extraction was carried until no more appeared in the extracting benzene and since the analytical method can determine as little as 3 y. Lack of coniplete hydrolysis could have accounted for material being present after both alkaline arid acid hydrolysis. These results indicated that at least one of each of the following classes of material was present : an amine, an acid, a “neutral” niaterial, a conjugated amine, and a conjugated acid. Thin layer chroniatography in several solvents on silica gel, as stated above, indicated that three amines were present, none of which was doxapraiii. One of these amines, the one appearing in the largest amount, was isolated as follows: a chloroform solution of the fraction was acidified with concentrated hydrochloric acid and evaporated to dryness. The yellow oil was taken up in a small volume of ethanol and the amine precipitated by adding a large volume of isopropyl ether. The precipitate was filtered off and recrystallized six times from methanol-isobutyl methyl ketone. The resulting material proved to be identical with l-ethyl-4-(2-hydroxyethylaminoethyl)-3,3-diphenyl-2-pyrrolidinone (11). Confirmation was ob( C e H 5 : q
CHiCHzNH CHzCHiOH
I
C2H5
I1
tained from mixture meltiiig points, infrared spectra, t.l.c., and elemental analyses. Two previous reports have been found in the literature concerning the metabolisni of morpholine6a and morpholine derivatives.6b Keither of these indicates opening of the morpholine ring, as was definitely shown by the above results. Reaction of the spots obtained on t.1.c. with ninhydrin showed that one of the spots developed color much more rapidly arid moved more slowly than did the spot corresponding to the above identified secondary amine. This led to the suspicion that the slower moving spot might be the corresponding priniary amine. This amine 1-ethyl-4- (2-aminoethyl)-3,3-diphenyl-2-pyrrolidinone (111)] was synthesized and t.1.c. indicated that the two compounds were the sanie. Acetyl derivatives of each were prepared 011 thin layer plates by spotting each compound, the nietabolic amine having been isolated by t.l.c., and then placing a drop of acetyl chloride on each. The excess acetyl chloride was allowed to evaporate overnight and the chroniatogranis developed with methanol-acetic acid (100 : 1). Both saiiiples gave the sanie Rf value, which was different from that obtained froin the unreacted amine. A larger amount of the mixed metabolic amines was separated by t.1.c. and the amine of interest was eluted from the silica gel with methanol. -4KBr pellet was prepared and its infrared spectrum was compared with a corresponding pellet of the authentic primary amine. These also indicated that the compounds were the same. (6) (a) R. K. Maller and C . Heidelberger, Cancer Res., 17, 296 (1957); (b) A . Englehardt, D Jerchel, H. Weidman, and H. Wick, Arch. E z p t l . Pathol. PhQrmQkd., 236, 10 (1958).
OF
DOXAPRAM
163
(C~H~~SNJCH~CHZ”
I
CiH5 I11
The relative quantities of the three ainines detected were determined from thin layer chroniatograms. The spots mere identified by ninhydrin, removed from the plate, and analyzed, The results indicated that SO% of the aniine fraction occurred as I1 and 25% as 111,with the remainder being the unidentified amine. The fraction obtained by extraction of the acidified urine was also investigated. Thin layer chroinatography on silica gel using ethyl acetate containing 1% acetic acid for developiiieiit revealed five spots that were positive t o perinanganate spray. One spot became fluorescent on irradiation with ultraviolet light. The spots were eluted and analyzed. Only one gave benzophenone on oxidation, indicating the presence of one acidic metabolite, with the diphenylniet hyl moiety unchanged. The presence of the 2-pyrrolidinone structure was indicated by an infrared curve obtained from material eluted from a thin layer chromatogram This curve showed a band at 5.90 I.( which is reported to be typical.‘ Other fractions from the partitioning by solubility have not been investigated. Bile Metabolites.-Bile samples (Table IT) were carried through the extraction procedure as described above for urine. The pattern of distribution appeared to be very similar to that for urine except that a smaller per cent of free amines and a larger per cent of conjugated amines mere present, Xliquots of the free amine fraction were chroniatographed and compared with the corresponding fraction obtained from urine. The chromatogram showed that the same three amines appeared in the bile that were present in the urine: a fourth amine was also present. However, none of these corresponded to unchanged doxapram. The ratios of the aiiiounts of the three amines present, as indicated by analysis, were very siiiiilar to the ratio in urine. Human Urine Metabolites.-Ser era1 samples of urine from patients mho had received doxapram have been analyzed. The results are seen in Table 1’. Some of the administrations were by intravenous drip T.~BLE 1. 1-RIS.XRY
E x c R E T I o s O F I>OX.~l’R.~\r-REL.\TED ~ I . I T E R I I L S IN
Hr-w~-s
mg.
Period of collection, hr.
5,320 2,840 2,000 49,000
0-36 0-40 0-24 0-72
Dose,
Dosapram-materials present
% of mg.
dose
455 244 204 10j4
8.6 8.6 10.2 21.5
and some by injections. Interpretation is, therefore, difficult. The last patient reported was administered 2400, 1500. and 1000 mg. on three consecutive days with urine being collected a t the same time. There was an increase in excretion each day, as would be expected. One of the human urine samples was partitioned by the solubility method as described above. The pattern of distribution is shown in Table IV. Chroniatog-
raphy of the airiirie fraction indicated that the saiiw amines were present as in dog urine. Chroinatography of the acid fraction indicated that the saiiip arid ~va. preseiit. Acknowledgment.-The autliors gratefully ackiiowl-
edge the assistance of other members of the research group at A. H. Robins Co.. in particular, that of 1)). I3eriiard I‘ranko and X s s .Josephiiic. Garhcr for thr :iiiimal wurk a i d Dr. Nerndoii ,Jeiikins for syntlmis 01 coiiipouiids.
A Simple Test Tube Arrangement for Screening Fibrinolytic A4ctivityof Synthetic Organic Compounds1
It has previously been .> a irrant-in-aid of the \\ 5nrnmg Heart 4ssociation. N 1 on Iiaulla “C‘lieinistri r i f llliornbolvsls Iiilman I ibrino7\ me?. Charips C ’Thomas P,~hlisher,Springheid, I11 1961, p
Dugni, “byntiiesii of Organ!< ( orngounds W‘hicli Induce I iliiinol esis, Boulder Colo 1961 14) I\. N. \ o n Iiaulla and K. I . < m i l l’roc. hoc L r p l l Bzoi V e d . , 106, 510 (1961). ii I
atid (for a relatcJ group of coiiipouiids) urea (110 L ~ C t i i r i l y) < t liiourea < clt Iiyliii~ea < allyl-2-thioui*c~a .\h a working hypothr4h it was assiuned that hydrot iopy c~ouldbc the coiiiiiioii d(>iioiiiiriatorfor t h e activity of t hcse colnpol1Ild~. I l l oldcl. to 1est the h y p o t h otliw coinpouiid* with hiioivti hydrotropic activity qiiitc different Ytructiirc- 11CY(’ .;titdid Large asyiiii i i o t r ic* hydrotropic anioiis lil1(~ L’.1-diiiietliylberizeiir~wlfoiiatc and 2-naphttialeiic~ulfoliatebrouglit about a, ,?-fold iiiciease iii activity a\ coinpared to iii(~t1iaii~ liercm large asyniinet ric Iiydrot ropic cat ioiia 1 1 1 ~ ( ~ t t s t r~-y1-propyla1ii1ii01iiiii1i hroniide were cwiipl(1t(.I?ii1cffcctive.j Conipouiidh wit ti a carboxyl group at ( , l i d of the hydrophilic side cliaiii were slightly more active than thc oiies with sulf‘oiiic acid. Thercforc, :it presciit , our scwch for hettrr coiiipounds is coiicciiI iatccl on the f o r i i i c ~group. It has bec11i possible 11) vaiyiiig the s1riicturch iiirthor t o illcrease t tie activity i)y a factor of approsiiuately 40 arid to define SOIIIC atl(lit ional structural requiretuerit s for an active c.oii1poulld (for csan1ple Iliscvission).i 11 is lils~lyt1i:it i i i t cmivc scarell i i i u r c active coiiipouiids \vi11 (L\-c.ntually lead t o syiit het ic tliroiiibolytic drugs. l‘or t l i ( w studies, a yuaiititativc. l ~ u rather t elaborate testiiig proctdure has been lived previously.i ’I’hc iiivestigatioii\ \ v c w Iianipered by the lacl, ill (1uaiiiitativcJ tthztiiig ilicthods zuitablc for ~ C I and, iiiost iiiiportant, suitahle for the m e a d faniiliar with I he t echiiiqw of hlood coagulation aiid fibrinolysis. .I ne\\ , siiiipler nietliod, together n i t 11 ~oiiieinstructive cuiiiples oi 1 est results, is brirfly described iii the 1:sprriliiciital section. It IS hoped that the procedure n-ill c>riablcthe organic chenikt to participate in the quest for I hroinbolytic agents. t l i c b
Experimental Principle. -Huinaii pla5iii:i clot& : L ~ Csuspended :it 37” 1 1 1 I .2fter 24-hr. ~iii~uh,it~i~ii. iif :L compound to be tested
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