Journal of Medicinal Chemistry /
r/
@ Copyright VOLUME
1969 by the American Chemical Society
12, T\-0. 2
MARCH
Potential Antitumour Agents.
1969
X. Risquaternary Salts
B. F. CAIN,G. J . ATWELL,AND R. S.SEELYE Cancer Chcmothempy I d o r a t o r y , Cornwall Geriatric Hospital, A irckland, LYewZealand Received M a y 9, 1968 Revised Manuscript Received September 27, 1968 I t is suggested that the biological activity of a variety of cationic agents against experimental leukemias and trypanosomal species is dependent on (a) lipophilic-hydrophilic balance of the agents, ( b ) charge separation, ( e ) sufficient "contact" binding, this requirement varying with the biological test systemoinvolved, ( d ) a close over-all approach to planarity, and (e) capacity to fit a curved site of approximately 40 A in diameter. That migration of these materials may be transport mediated is discussed. I t is shown that a site equivalent to the minor groove in a helical polynucleotide would match the structural requirements.
Earlier papers' have described the experimental antileukemic effectiveness of a series of bisquaternary ammonium heterocycles and have shown that there is a marked dependence of biological activity on the lipophilic-hydrophilic balance of the molecules. A convenient relative measure of this balance of physical properties consisted of the R f values of the drugs in an immiscible solvent mixture.2 The range of physical properties allowing demonstrable activity against the 1,1210 leukemia is very small. For example, in the S,S1-(6-quinoly1)terephthalamide quaternary salts (I) only the n-propyl, n-butyl, and n-amyl homologs show unequivocal activity; many examples of the sharp cut off in this type of agent have been presented.' It has also been shown that the R f values of optimum members of structurally divergent series are extremely similar.'P2 We dram attention to the fact that the biologically active cationic drugs listed in Table I have a similar balance of lipophilic-hydrophilic properties when examined by partition chromatographic method^.^ The agents listed in Table I have been selected by screening programs for activity against Trypanosoma rhodesiense or the L1210 leukemia. Discussion has been limited to those cationic drugs whose pk values are greater than Where active transport mechanisms do not function it is generally accepted that the rate of drug translocation is intimately connected with partition properties (lipophilic-hydrophilic and, as a conse(1) P a r t I X : B. F. Cain, G. J. Atwell, a n d R. iY.Seelye, J. M e d . Chem., 11, 963 (1968), and references therein t o earlier papers. (2) G . J. Atwell and B. F. Cain, ibid., 10, 706 (1967). (3) T h e reason for using dimidium (XXXV, R = CHa) as a n internal chromatographic standard2 can now be seen. (4) An admirable review of t h e current status of trypanosoma1 chemotherapy is presented by F. Hawking, E z p t l . Chemotherapy, 1, 131 (1963); 4, 398 (1966). ( 5 ) E. J. Ariens, M o l . Pharmacal., 7 (1964). (6) W. Kunz, Progr. D r u g Res., 10, 360 (1966). (7) C. Hansch and S. N . Anderson, J . M e d . C h e m . , 10, 745 (19671, and earlier papers quoted therein. (8) E . J. Ariens, Progy. D r u g Res., 10, 429 (1966).
quence, fully ionized drugs do not penetrate cellular membranes at very high rates. Thus, a cumulation of cellular barriers through which an agent must pass can effectively exclude fully ionized drugs.g Consequently, it appears remarkable that the bisonium members of Table I are able to influence disease processes considerably removed from the site of administration. Evidence has been presented that the phthalanilide1° I1 will pass even the blood-brain barrier.13 The illustration that the inhibitory effects of the phthalanilides are proportional to the intracellular concentration of a drug-lipid ~ o m p l e x , ~ *and , l ~ not extracellular free drug concentration, strongly suggests that a transport mechanism is operative. Evidence has been presented that a novel class of phospholipid is involved in this drug-lipid complex.16 Involvement of a lipid carrier mechanism with quaternary ammonium salts has been described previously. l 7 - l 9 If the cationic drugs listed in Table I pass cellular (9) T h e blood-brain barrier offers a n excellent example of this; compare ref 8. (10) T h e name phthalanilide in relation t o experimental antitumor agents has come t o cover a. diverse group of compounds hearing amidinium functions. Examples 11-XI show t h e principle members pertinent to our arguments. Full details of structural types are given in ref 11 a n d 12. While not agreeing with this terminology it is convenient t o use in place of t h e cumbersome systematic nomenclature. (11) R. Hirt, "Chemotherapy of Cancer," P1. -1.Plattner, Ed., Elsevier Publishing Co., Amsterdam, 1964, p 228. (12) J. H. Burchenal, ref 11, p 233. (13) W.I. Rogers, I. M. York, and C. J. Kensler, Cancer Chemotherapy Rept., 19, 67 (1962). (14) D. IT. Yesair, W. I. Rogers, P . E. Baronowsky, I. Wodinsky, P. S. Thayer. and C. J. Kensler, Cancer Res., 27, 314 (1967). (15) D. W. Yesair, F. A . Kolner. W.I. Rogers, P. E. Baronowsky, and C. J. Kensler, ibid., 26, 202 (1966). (16) D. IT. Yesair, W. I. Rogers, J. T. Funkhouser, and C. J. Kensler, J. L i p i d Res., '7, 492 (1966). (17) R . R . Levine, Proceedings of the First International Pharmacological Meeting, Stoc'cholm, 1961. Vol. 4, Hoghen, E d . , Pergamon Press, Oxford, 1962. (18) R. R. Levine and E. \V. Pelikan, J. Pharmacal. E z p t l . Therap., 131, 319 (1961). (19) R. R. Levine and A. F. Spencer, Biochem. Phnrmacol., 8, 248 ( 1 9 6 1 ) .
199
\'(ll. I "
IS
S
SI
SI1
SI11
ST\'
S \'
s \-r
I.
s
20 1
No.
SSI
XSII
XXIII
\
0.s4
+k
0.S9
+
NH,
24.3
24
+k
0.83 0.91 1.04
-
19..i
-
XXV
0.91
++'
28
XXVI
0.90
+
XSVII
0.91
+"
12.5
SSVIII
0.93
++"
19.5
XXIX
0.96
+j
26
XSS
0.9.5
-j
28.
XXXI
0.94
+'
26
XXXII
0.93 0.94 1.04
ti +'
0 . !I0
-"
+
1.00 1.02
Zth +h
+ +
SSIVO
SSSIII
S.5
g
17 20.5
y
27
g
SXXlV
xxxv
XSXVI CH,
S.*5
sS S V l I 1
0 .'3x
+I'
1.01
+'I
NH,
I CH
sI,
h . .i
SLII
I?
20
++ tt
SIJIlI
Is
.-I
+
I{r relative t l J dimiclium (SSSV, 1: = CI13);see ref 2. t' Airelative gauge of :ic.iivity ;tg:tiiici the 1,1210 1r~tkemi:i: i i o :rc.tivilj., : Uneqiiivocal activii y agaiiist Trgpnnosoirru rhodcaimsc denoted hy ; W(J iiirreaie iu life span 2 5 - ~ 5 0 ~ ; , : 50-100r;, ; 100c;, f. ref 4. d Charge separatioiis niea,sui,ed from Coir1ald model.: isidc infra,). Iti anridiiriiini wmpouiids rharge is takeir io be riiidFvav l)et,weeti the two nitrogen atoms c.omprisitrg the remiittiit yysiem. Charge in the qriaterii:rry halts is lakeii t o lie at thc c~iiateriiar iiitrogeii a t t p ; 110 allow:iiice frir delocalizatiuii has heen made. Where several geonietriid iwmers :ire possible, t.hat lyiiig c~lt~aest. io ciirve of 2G.L radius has beeii considered (see discwsioii). See ref 24 fot, a further !i>t of charge parntioiis ti, hlightly differelit i*i,ilc,ri;i. It has been stated that rertaiu phthalaiiilide~have t paiiosomal activity," but a s far :i> wts l{efereirce 2 . Refereiires 11, 12, 24. :ire aware full detail:: of theje test? have i i o t beeti p~iblished. Tests uilder comparable coiidit.iot ill this laboratory. See J. 11. Bir~,c.hthi d , M. S. Lyman, J. K . Purple, V. Culey, S . Smith, and E. Bucholz, Cancw Chenzothcrap)j Kept., 19, 19 (I962), for details of tests of hiimidiiirn (XXXV, It = C2Hn)aiid isometamidiiim ( X X S V I I ) against 1,1210 aiid further murine leukemiah. Berenil (SIV)i r i o t i i ' harids has ocac*asioiiallygiven *tatistically significant life esteiision with the 1,1210 biit activity is of it11 extremely low order. 1 l3iologiixl data of thir paper. liefereiice '27. E Referelice 3 0 . ''&Referenre 28. 1' A . I h r s e r , ExpcJric9n!iu, 23, 178 11967), gives the t)ac:kgroiuitl leading to the development of this i*on1poiiiid. E. Alihich pre.xiited details of the srreeiiiiig results at ihe Niiith Iiiternatioiial C:tiic~)i. Persoiial ci~nim~iiiic~at~ioii from I>r. J. 1,. II:irt~ve!l, CCNSC, Rethe&, Nd. 1' I,. I:. Duv:rll, Cn~acc~r Chi,mo(:(Ingress, Tokyo, 1966. I,. I t . Diivall, ibid., 23, 63 (1962). (hcrrrp!/ L'rpt., 23, 61 (1962).
*
1:
+
+
+
Q
hixriery a p1ioy)holipid complex, i t hecome, ea+ier to uiiderit:ind the di$tributiori of these positively charged niat crinls through tl-ic tihyuw. The lack of paralleli5m 1,rt\v.ccn in t'ifi0 and in o experiments ha< already led Siv:k, tt d,?" to .;tat(> that "Cell permeability to thc phthalariilideh 11i:iy be o i i ( ' of' the most import:int factors responsible for the efic:tcy of some phthalariilides and the irieffectiveriehb of others that are .tiaiirtiir:tlly 4nlil:\r "
I*'roiii t he :~l)oucciiscussioiis it :~ppciirs likely t II:L< thc obscrvcd tlcpciitlciice on lipophilic~-hytlro~~liilic properties of thesc drugs is either :issoci:Lted with t 11c binding of the drugs t o the phospholipid carricr, movcbmerit of the drug t o :ire:~s where t,lict phospholipid occurs, arid ,/'or iiiigrntioii of the drug-lip id coin plttx .!!I (21) Drug diatri1,ution could lie viaualized as f o l l o i n . (a) F'ree c h i & i r i o v t ~ s process throuah a necessarily limit,ed number c)f r-ellular harriers to a site where interaction v i t h tile lipid eomponeni raiit.. 11Ia1-e. ( I > ) 1)rrig-lipiil w m y l e s m o v e s t o thr site 01 action. >LIovrmi.rii < ) f ilrug-lipid complex could lie passive and depend on the l i ~ o p l ~ i l i c - h y d r o j ~ l i i l i ~ ~ ilalance of The complex ( a n d hence tile drua) o r artive when r-dlulnr transjmrt irior~lianislns~ w i i l dlie invvkril.
1)sa partition-dependent
March 1969
BISQUATERSARY S.4LTS -45:~Yl'ITCMOIt AGESTS
Examination of the retention or efflux of a phthalanilide-lipid complex in sensitive and resistant tumor cells22 suggests that drug effectiveness is dependent on time of intracellular residence. Thus, selectivity of action could be intimately related to transport. If a common transport-mediated distribution is accepted and this operates over the diverse set of compounds listed in Table I, differing i n number of charges, charge separation, and other structural features, there can be little specificity of the carrier system except a requirement for a cationic chargez3 and possibly a particular lipophilic-hydrophilic balance.23 Regardless of the mode of distribution, the lipophilic-hydrophilic balance of these cationic agents is a prime determinant for biological activity. While the agents listed in Table I have similar partition properties and biological activity, it was felt, in the early stages of this investigation, that some further evidence of generic relationship was required. Reference to Table I s h o w there is a close relationship among the quaternary salts we have (cf. I, XIX, XXV) and the phthalanilides (cf. 11-XI). The screening from a very large array of phthalanilides leave no doubt that there is a similar relationship between biological activity and physical properties to that observed in the quaternary salts. This can be seen, for example, in the changes in biological activity on successive S-alkylation from I11 to VI and the homologation of the quaternary salts represented by I. The Rfvalues for active members of the phthalanilides that we have been able to examine (11, V, VIII-XI) support this thesis. However, attention has been drawn to the requirement for an Samidinium substituent in the phthalanilides in order to obtain antileukemic activity,"jz4 and that these, therefore, represent a class of compound different from the usual bisamidines (XII-XVII). On the basis of our simple partition hypothesis, the alkyl substituent on these molecules merely serves to adjust the lipophilichydrophilic balance into the critical range required for activity. To obtain proof of this point we first examined a compound containing a diazoamino function (XVIII) where an amide had previously been used (XIX). This compound (XVIII) proved to be active against the 1,1210 leukemia, demonstrating that the diaxoamino function is compatible with activity. Comparison of the R f values of the methyl quaternary salts of XVIII and XIX shows that replacement of the amide function by diazoamino causes an increase in Rf approximating that observed between bismethyl and bisethyl quaternary salts in one series (XIX, R = CHJ and GH;), i.e., equivalent to an increase due to two methylene groups. Accordingly. the diazoamino compound X X , bearing an unwbstituted amidine function, was prepared and shown to be convincingly active against the L1210. T h u s , one major dL*ffer.ence between the phthalandicles and the trypanosoma1 bisamarlines i s a more hydrophilic ( 2 2 ) D. W. Yesair and C. Ho-Fook. Cancer Res., 28, 314 (1968). (23) T h e presumption t h a t cationic charge is a necessary requirement follox\-s from t h e anionic nature of t h e phospholipid carrier. W e make t h e assumption a t thia point in our argument t h a t there is little specificity in t h e carrier. I t should I I remembered ~ tliroughout t h e remainder of t h e discussion t h a t t h e s t r u c t u r e a c t i v i t y relationships derived might apply t o t h e transport system if this had specific requirements. (24) L. Lee Bennett, J r . , Progr. E z p . Tumor ZZea., 7 , 259 (1065).
203
amide backbone in the former which requires a compensatoyy adjustment of the hydrophilic-lipophilic balance by N substitution. It is thus not surprising to find that the phthalanilides are stated to have trypanosomal a ~ t i v i t y . Attempts ~~ to prepare phthalanilides with more lipophilic functions in place of amide but retaining a Az-imidazoline as basic function has given rise to inactive materials as expected from this thesis.1f11f24S substitution in the trypanosomal bisamidines (XII-XVII) has proved dystherapeutic : 2 6 such substitution would be expected to produce more lipophilic and hence less active materials. Since a guanidine function is somewhat more lipophilic than an amidine, it was possible to construct an amide-linked molecule (XXI) with an unsubstituted guanidine function active against the L1210 leukemia. This is in contrast to the usual S-alkylation considered necessary in bisguanidine members of the phthalariilides (cf. VI1 and VIII). Experience with a guanylhydrasonez7 as basic terminus has shown this to be more lipophilic than either an amidine or a guanidine. The preparation of L1210 active hybrid species containing one quaternary salt and one guanylhydrazorie function (XXII, XXIII) leaves little doubt that the bisguanylhydrazones and the bisquaternary salts should be considered as analogs and, hence, from the preceeding arguments, also ac analogs of the amidines and guanidines. With small molecules such as methylglyoxal bisguanylhydrazone (XXVI), which has partition figures close to that of peak members of other series (Table I), replacement of the methyl group by higher alkyl or aryl functions or substitution of the guanidine with S-alkyl groups would be expected to lead to more lipophilic and therefore less active agents. This is in fact the The structure-activity relationships in the phthalanilides led to the hypothesis that there is an apparent dependence on intercharge separation, highest activity being observed with a separation of close to 20 With the recognition of the critical importance of the lipophilic-hydrophilic balance in this area, we were able to construct a series of bisquaternary salts whose whose activity showed a more flexible dependence on intercharge ~ e p a r a t i o n . ~The ~ inactive bisquaternary salts (XXIV) appeared to be ?nomalous in that the intercharge separation (19.5 A) corresponds glosely to that in the active phthalanilides (19-19.5 A) and the partition properties appear to be in the correct range (Table I). However, extension of XXIV by one p-aminobenzoate unit gave XXV which proved highly active against L1210 even though the charge separation (26 A) differed markedly from that in the p h t h a l a n i l i d e ~ . ~This ~ led to the realization that the members of the bisquaternary salts and the phthalanilides, highly active against the L1210, have three essentially coplanar rings between the two basic func( 2 5 ) Quoted in ref 12. .Is far a s me are aware full details of thesetests have not been published. ( 2 6 ) J. S . Ashley, H. J. Barber, A . J . E n i n s , G. Newberry. and A. D. H. Self, J . Chem. Soc., 103 (1942). (27) Part VIII: G. J. Atwell, B. F. Cain, and R . X. Seelye, J . X e d . Chem.. 11, 690 (1968). ( 2 8 ) E. Mihich, Cancer Res., 23, 1375 (1963). (29) E. G. Podrebarac, W. H. Nyberg, F. A . French, and C. C. Clienq, J . .Wed. Chem., 6, 283 (1963). (30) G. J. Atwell. E. F. Cain, a n d 11. S . Seelye, i h i d . , 11, 2 Y j (1968).
March 1969
BISQUATERSAHY SALTb 4 8 L ~ l N T I T U J I O H, i G E S T s
anthridines such as iwmetamidium (XXXVII) 48 or prothidium (XXXVIII) could equally well reside in a site equivalent to the minor groove of a DKA helix or with the phenanthridine nucleus intercalated between the base pairs with the second basic function protruding and matching to a phosphate. It could well be that such charged species may reside in the narrow groove until such time as the central stack of purinepyrimidine pairs opens because of the demands of some metabolic sequence. The phenanthridine moiety could then i n t e r ~ a l a t e . ~ ~ The slot-like nature of the minor groove in DNA helices is such that only drugs of very narrow cross section could enter. This could explain the requirement for a close approach to planarity commented o n ear1ier.l I t is believed that the details given in this paper should allow planned synthesis of trypanosomal and experimental antileukemic agents. We present two successful examples, X L I I and XLIII, active against L1210, predicted from our site model concept. X L I I uses three charges so that the lipophilic-styryl system could be brought into the correct range of hydrophilic-lipophilic properties. The use of three charges affords sufficient electrostatic binding so that three rings of contact need not be provided. Although active this compound could not be claimed to be strikingly so. XIJII is a novel variant predicted from the model which shows an extremely high order of activity. Further successful examples will be reported in due course. z1
Experimental Section Where analyses are indicated only by symbols of the elements, analytical results obtained for these elements were within 10.4910 of the theoretical values. Analyses were performed by L)r. A. L). Campbell, Microchemical Laboratory, University of Otago, Dunedin, S e w Zealand. AIelting points were determiiied on ail Electrothermal melting point apparatus using the maker'ssupplied stem corrected thermometer. A heating rate of 2"/min from 20" below the melting point was used. The symmetrical bis bases listed in Table I1 were prepared from the substituted terephthaloyl chloride and 3-(p-aminobenzamido)pyridine in diethylene glycol dimethyl ether as detailed earlier.ao Methods for quaternization, paper chromatography, etc., have been described adequately.2 Intermediates not previously described in the literature follow. 1-Methyl-4-( p-aminophenyl )pyridinium p-Toluenesu1fonate.4-(p-Nitrophenyl)pyridine (1 g) was dissolved in P h N 0 2 ( 2 ml) at 165". Methyl p-toluenesiilfoiiate (1.5 nil) was added to the hot solution, there was an exothermic reaction, and the quaternary salt Crystallized from the hot solution. The salt was collected after cooling, washed (EttO), and dried. I t proved to be excessively soluble in H20 and since paper chromatography2 showed the material to be homogeneoils it' was reduced to the amiiie 11-ithout further purification. Reduction with Fe dust by the previously described method8" gave the amine which separated from HLO containing sodium p-toluenesulfoiiate as pale yellow
(48) It has been postulated t h a t a small proportion of pseudobase in homidium (XXXV,R = CzHa) m a y facilitate distribution of this type of drug: T. I. Watkins. J . Chem. Soc., 3059 (1952). However, such arguments can not be extended t o t h e discharged variants X X X V I I and X X X V I I I , a n d the amount of neutral sgecies in these would be vanishingly small. (49) There in a n apgarent t'amilial relationship hetween t v o new antitllmnr agents linrovered 17s t h e C C S S C screening prorram tSSSIS a n d S L ) and tryuaflavin ( S S S V I ) and homidium ( S S S V , R = CzIIa). It m a y Le profitable t o examine X X S I X and X L for (a) intercalation into D N A and (b) trypanosomal activity. These molecules could then serve as t h e basis for preyaraLiun of a family uf drug3 related t u yrothidiuin ( X X X V I I I ) a n d isumelaruidiuiri ( X X X V I I ) .
205
TABLE I1
c 0 !Ill )360 326-327 >360 929-330 >860
('~Hzs~nOa (hH4sNsOlasz. I . 5Hz0 C~HZ~NRO~ CsoHisNaOinS?.1 . 5 H z 0
XSIX SXX XXX YXXI XLII YLII YLII XLIII XLIII XLIII XLIII a
('Hsh n
('H3b U
CHS" CH3a CzHsC a CH3' C>HnC CHs(CHz)zC
Free base.
b
61-62 806-807
804-305 324-325 214-216 202-204 189-190
C36fb6?i604
CsiHsiNaOsSa CaaHaoIsNa CsaHssISs CwHziNsO CwHziI2N10 CaiHaiIzNsO CsaHwNsIzO.O.5H20
Anion p-toluenesulfonate.
Anion iodide.
prisms (1.19 g, 677,), mp 261-262'. Anal. ( C I Q H ~ ~ N ~ O ~ S ) C. H. S. '4-[p-( p-Nitrobenzamido)phenyl] pyridine was prepared by intei action of p-nitrobenzoyl chloride and 4-(p-aminophenyl)pyridine in pyridine solution. The free base separated from solution in L)hIF-H20 as yellow needles, mp 286-287". Anal. ( C I ~ H ~ J V ~C, O ZH,) N. l-Methyl-4-[p-(p-nitrobenzamido)phenyl]pyridinium p-Toluenesu1fonate.-A solution of the corresponding base (1.55 g) in 1-methyl-2-pyrrolidone ( 5 ml) was heated to 160" and methyl p-toluenesulfonate (2 ml) was added. After 10 min a t 160" the solution was cooled. The crystalline quaternary salt was washed well (EtZO), dried, and then crystallized from XIeOH-H20;
l-Methyl-4-[p-(p-aminobenzamido)phenyl]pyridinium p toluenesulfonate was prepared by Fe dust reduction of the corresponding nitro compound by the method described earlier.3O The amine separated from l\leOH-H20, containinrr sodium p-toluenesulfonate as pale yellow prisms, mp 298-299". Anal. (CdLzNsO&). C,. H,. S. XVII1.--4 suspension of the preceding amino-substituted quaternary salt (1.894 g) in H20 (25 ml) and HC1 (1.6 ml) was diazotized by slow addition of a solution of ?r'aN02 (0.39 g) in H20 (2 ml), the temperature being maintained below 2'. T h e solution was stirred for 0..5 hr, saturated aqueous sulfamic acid (1 ml) was added, and the solution was stirred for a further 5 min. An ice-cold solution of l-methyl-4-( p-aminopheny1)pyridiiiirim p-tcil~iencsiilfonat,e(1.56 g) in 1320 (10 ml) was then stirred in followed immediately by cold satiirated NaOAc solution (21 nil). T h e reaction was stirred in the ice bath for a further 0.5 hr, then crude product precipitated by the addition of sodium p-toluenesulfonate (15 9). The precipitated salt was collected, washed well with cold aqueous sodium p-toluenesulfonate (lo%), and crystallized from ;LIeOH-H20 containing sodium p-toluenesulfonate, the temperature not being raised above 60'. Several further recrystallizations gave material homogeneous when examined by paper chromatography.% Pure compound separated from hIeOH-H20 as orange needles (3.03 g), mp 196" dec. Anal. ( C ~ ~ H ~ ~ N SC,OH, Z SS.~ ) .4 sample of the compound on warming with Cu2C123 HCl readily evolved N p , confirming the correctness of formulation as a diazoamino compound.60 XX.--il suspension of l-methgl-3-( p-[p-( p-aminophenylcarbamoy1)benzamidol benzamido pyridinium p-toluenesulfonate (3.18 g)27 in H20-MeOH (30:20 ml) containing HC1 (4 ml) was diazotized by drnpwise addition of a solution of S a X O z (0.43 g) in H 2 0 (3 ml) to the vigorously stirred suspension, the temperature being maintained below 2". The mixture was stirred for a further 13 min after complete dissolution. Saturated aqueous sulfamic acid (1 ml) was added, the solution was stirred for a further 5 min, and then p-aminobenzamidine monohydrochloride (1 g) in H20 (5 ml) was added followed immediately by cold saturated NaOAc 125 ml). The viscous mixture was stirred for a further 0.5 hr when sodium p-toluenesulfonate (20 g) was added. After 0.5 hr the precipitated salt was collected, wPn>hed well with ire-water and crystallized repeatedly from ~ l e O I T - ~ 1 2( d0o i 1 t : t i i 1 i i i g sodiiini ~-foliieriesulfc,Ilrttrat below 60'. \Then hoinvgeiieous by paper chromntogruphy2 the y e l h p -
SS
SSIS 1-12 1i(i I .iS
SXSI I :iI I .-I6
SSSI
148 130
SLII
I .-I6 I42
t ~~liietiesiilfiiiiatesalt had nip ISgc dec. Llrrul. ( ( l a a : I I d & S 2 . 0.,iIi20)C, IT, h-,H. A sample on warming with CII?CL3 A- IlCl evolved K2.:" XLI1.--i\ niistiire of 2-(p-aniiiit~.t~r~l)-G-a~niiioyuiiioline (2.61 g), anhy-droiis p-toliienesulfonic w i d ( 5 . 2 g), X-pyridyl--1pyridiniiim chloride hydrochloride (5.7 g), and dry phenol (15 g) was heated under reflux for 1 hr. After removal of phenol Iiy stram distillation w i d e base was precipitated with excess NaOtI. I t was siispended in I I r O (7.3 nil) and HBr was added t o t,he boiling sii>pension until soliltion was c.omplete. .1genel'oiw quaiit it?. of tlectrloriziiig charcoal was added to t,he soliltion and boiliiig cciiitiriued for 3 min. Addition of NaBr (20 g ) t o the hot filtered h i i l i i t i ( i n , followed by slow cooling, afforded the trihydrobromide : I - 1irilli:iiit red needles. The salt was repeatedly crystallized
