707
BISQUATERNARY AiVTITUMOR AGENTS
July 1937
TABLE Ia
Qy3"rn R
k
I
%-
r-Calcd,
R'
Alp,
oc
C
H
N
I
S
C
Found, %I€ K
7
S
6 2 . 3 5 . 0 6 . 9 7 . 9 62.0 4 . 9 7 . 0 326 dec 7 . 5 61.4 5 . 4 61.8 5 . 4 299 dec 62.6 5 . 7 7 . 3 62.7 3 . 9 282 der 64.6 5.9 7.2 64.1 6 . 0 284 dec 64.6 6 . 2 6 . 9 64.4 6 . 0 302 dec 65.7 6 . 4 6 . d 63.7 6 . 4 302 dec 61.6 .i. 4 7 . 1 6 1 . 5 R.4 282 dec 62.4 #5. 8 6 . 9 62.0 ,3.6 274 dec 63.4 6 . 2 6 . 5 63.6 6 . 4 282 dec 67.4 3.7 15.1 67.3 4 . 1 14.6 297-299 60.0 4 . 9 7.3 60.3 5 . 1 322 dec 60.1 5 . 3 7.0 60.1 5.4 312 dec 61.5 5 . 5 6 . 8 61.3 5 . 6 308 dec 62.8 5.7 so2 306 dec 6 . 7 63.1 5 . 9 60.7 5 . 4 7 . 4 60.4 5 , 6 NH2 300 dece 62.8 5.6 XH2 270 dec" 7 . 3 62.4 5 . 7 62.8 5 . 9 SH2 280 dece 7.0 62.4 6 . 0 62.9 6 . 2 6 . 7 62.5 6 . 1 NHz 275 dece a The anion used is p-toluenesulfonate. * Free base. Ri relative to internal standard; see Experimental according to our experimental procedure against the tumor system increase in life span of 25-507c, +; S O - l O O ~ , Experimental Sectioii for full details). e With previous darkening and sintering H H H H H H H H II YO2 NO, NO? NO2
R D ~L1210d
8.1 7.5 7.4 7.4 6.7 6,s; 7.4 7.1 6.4
0.61 0.68 0.80 0.82 0.8T 0.92 0.78 0.87 0.93
zt
+
+ + zt -
+ -
7 . 4 0.81 6 . 9 0.91 6 . 9 1.00 6 . 8 1.08 7 . 1 0.74 6.9 0.83 6 . 9 0.94 i 6.8 1.0j + Section. d Resiilts 10070, (iee
+
+;
++
TABLE110
E
R R
n
Mp,'C
Formula
3* 229-230 CnHzoNaOa C41H44N40&.H20 ClH, 3 264-265 CajHo2Na08S?.Hr0 (CH2)3CH3 3 258-259 238-240 C4qHmN408Sz (CHs)5CH3 3 4* 250-251 C2aHl2N402 CJL 4 262-264 C4zHaJaOsS2 (CHl)~CHp 4 264 dec C4&4s40&& 276 dec CaOH62N408Sl (CH2),CH3 4 a-d See corresponding footnotes in Table I.
R
P C a l c d , %-----C H N
71 61 62 65 72 63 64 6.5
1 3 9 6 3 1 6 8
showing maximum biological activity can a comparison of structural types be made. Then it appears that biological activity is determined to a greater extent by structural features rather than by physical properties. The quaternizing function may carry substituents, and the resultant molecules show antileukemic eff ectiveriess if, presumably, the lipophilic-hydrophilic properties are in the correct range [e.g., I (Table I), R' = H; R = (CHz)20CH3]. Examination of a series of related molecules where the terephthaloyl backbone was replaced by flexible aliphatic dicarboxylic acid (11, Table 11) of approximately the same linear dimensions and in which a range of quaternizing functions were used showed no activity. Similar results were obtained when aralkyldicarboxylic acids were used (I11 and IV, Table 111). However, the isosteric series where pyridine-2,5dicarboxyl replaced terephthaloyl contained active members (V, Table 111), but activity did not appear to be as high as in the parent series.
6.2 5.8 6.3 6.7 -5.6 5.8 6.4 6.9
S
14.4 8.0 7.5 7.1 14.1 7.0
8.0 7.5 7.0
%--
---Found. C
H
N
71.3 60.9 62.7 65.5 72.1 63.0 64.4 65.7
5.9 5.6 6.4 6.9 5.9 6.0 6.5 7.0
14.2
14.2 6.7
S
Roc
8.1 7.4 7.3
0.91 0.98 1.06
-
7.8 7.7 7.3
0.96 1.03 1.12
-
L1210d
-
-
I n a series of nitro-substituted derivatives (I, R' = NO2, Table I), no activity was observed although the corresponding primary amines (I, R' = NH2, Table I) were active. In an attempt to examine the effectof reducing charge separation the quaternary salts from tere- and isophthaloyl derivatives of 5- and 7-aminoquinoline were examined (VI-X, Tables I11 and IV), but no active compounds were found. Conversely, when intercharge separation was increased by a variety of means, augmented activity resulted. Both the cinnamoyl series (XI, Table V) and the phenylquinolines (XII, Table V) contained active members. Also, certain of the phenoxyacetic derivatives (XIII, Table V) were active although at a reduced level; this lower activity may be a consequence of a departure from planarity about the ether-methylene linkage. Since the bismethyl quaternary salt (XII, R = CH,) was active while the corresponding ethyl derivative (XII, R = CZHJ was inactive, it was assumed that
Yol. 10
360°. dnal. Calcd for C ~ ~ H I R N0.5(CH3)2NCHO: ~OQ. 72.6; H, 4.8; N, 13.9. Found: C, 72.8; H , 4.9; S, 13.7. Crystallization from phenol gave a sample, mp >360". Anal. Calcd for C 2 ~ H d 4 0 2 : C, 74.6; H , 4 . 3 ; S, 13.4. Found: 74.4; H, 4 . 5 ; K, 13.3. Most bis bases, prepared by similar methods, were obtained i t i virtually quantitative yield. Only wheii the acid was siih-
c,
stitiited did the yield.: drop, biit generally they were bettei, th:iii 6 5 5 . 1Iairy of these compounds failed t o melt belo\\. :Xii13. Solveiits for crystallization are limited; occasiotially 1-biit:uiol coiild be iised brit more normally, pyridine, phetiol, l ) A l F , Nmethylpyrrolidoiie, or dimethyl riilfoxide (I)1ISO) n n s i ~ s r i l . Cryhtallization from I)NF often yields colvates frcim .rvhirh i t i-. extremely difliciilt to remove the solverit hy dryii~giir C U C U O . The bases were dried a t 100" i v a r i ~ r i m )for : t i i d ) Quaternizati0n.-Iii all cases alkyl p-tolueiie used as quateriiizing agents. The following exemplifies 1 l i t , general method. N,N'-(6-G)uitiolyl)tereplithalamide( 1.0 g ) \v:idissolved in boilitig IjLlF ( 1 0 rnl), the sulutioii was c.oolrtl t o 140°, arid met.hyl p-tolueriesulforiate ( 4 molar proport iotis) w:is added iri oiie portioii. The solutioii was then heated :It 140 131' for 30 miii. The riiixlrire was cooled well atid the vi' salt was collected. The solid was suspended iii hoiliiig w : i t c s t ' (100 ml) and etharrol sloaly was added iintil soliition w:i> V I ) J J I plete. The soliitioii was filtered and sodium p-tolue~res~ilfo~rati~ (10 g ) was added to the hot solutioii arid the mixture was cooled slowly, the crystallirie salt slowly qeparatirig i i i a piire c~)~iditiiiii. mp 326" dec. Difficulties were sometimes experienced in purifying quateriiai'y salts prepared from the longer rhaiii alkyl p-toluerieiulfoti~it~~.. This was traced to the competing elimiiiatiori reactiori givirtg rise to free p-toluenesulforiic acid which was bound by the hete1.r)cyclic base. T o remove unqimterriized bases the >ample 11 et e recrystallized f r u m aqueous solvetits in the iisual way M ith t l w illclusion of 2 molar porportions of pyridine. The more iiisol~il)lc bases liberated from their salts by the pyridirie were filt.eri:tl f r o i i i the hot solutioii.
BISQUATERNARY ANTITUMOR AGENTS
July 1967
711
TABLE VI0 m \N+/
\ C/
H
\
O
d
23 7 -
hlp,
oc
>360 330-335 310-312 325-326
C
Formula
Calcd,
R-N,
Yo
H
76.6 4.7 CaoHzzNJ& C ~ ~ H ~ Z N ~ O ~ S Z62.9 . ~ H Z O5 . 3 5.6 C ~ S H ~ ~ X ~HzO ~ O ~ S63.6 L. CjoHjoN4OaSz.0.5HtO 6 6 . 2 5.7
1 \ N/
/ O 0N H \
---
N
R
--
S
11.9 7.3 7.1 7.0
Found, % N
C
H
76.8 63.1 63.4 66.4
5.1 5.6 5.5 5.7
11.9
4 9 5.3 5.7 6.0
11.8
4.9 5.4 5.4 5,7
11.8
m , 0o-c +
\
/NHCO\
/CONH\
/
6
Rr
6.7 6.9 6.9
0.69 0.81 0.93
+ ++ ++
7.1 7.2 6.9
0.75 0.84 0.88
++ ++ ++
7.8 6.8 6.9
0.81 0,96 0.99
-
L1210d
\ + ,N-R
mi h CH3 C,Hj (CHzj2CH3
>360 333-343 23G233 337-339
C3OHzZN402 C4eH42N40&.3.5HzO C48H46N40882.3HzO CjnHjoNa0&.2.5HzO
76.6 61.0 62.3 63.6
4.7 5.5 5.7 5.9
11.9 7.1 6 9 6.8
76.2 61.0 62.4 63.7
R
m
a-d
>360 305-308 29G294 290-291 See corresponding footnotes in Table I.
Either DRIF or N-methylpyrrolidone was used as solvent for quaternization. The latter gave better results when solubility problems were encountered. Care must be taken that all of the base is in solution before adding the alkyl p-toluenesulfonate. The alkyl p-toluenesulfonat,es should be shaken with sufficient dry lIgC03 to neutralize free acid before use. If the quaternary salt does not separate from the reaction mixture it may be precipitated with et8heror dry acetone. The salts were crystallized from aqueous sodium p-toluenesiilfonate with the addition of methanol, ethanol, or DMF. Occasionally the anhydrous form of the salt could be crystallized from 1-butanol-methanol mixtures. The quaternary salts as crystallized from aqueous solvents were invariably hydrated. For analysis samples have been dried i n vacuo over silica gel a t room temperatures. Attempts to dry thoroughly a t elevated temperatures gave extremely hygroscopic samples and in some cases a loss of crystallinity. Melting points have been determined on the samples dried and ready for analysis and are really decomposition points of either the hydrate or the quaternary salt and are dependent on the rate of heating. Careful attention t o detail is necessary to reproduce the same melting point for different samples prepared a t different times. Melting points have been determined on an Electrothermal melting point apparatns with the makers-supplied, stem-corrected thermometer and with a 2"/min heating rate from 20' below the melting point. Paper chromatography is a superior index of purity to melting point and compounds listed have been purified, where possible, to give only one spot. Chromatography.-The solvent used was the top phase from a mixture of 1-butanol (4 vol.) and 2% aqueous sodium p-toluenesulfonate ( 3 vol.), the paper being Whatman No. 1. The quaternary salts were applied as their p-toluenesulfonate salts in phenol or aqueous DhIF. It' is important that the applied spots are not completely dried on the paper, otherwise the salts crystallize on the surface of the paper and tail badly or fail to move. Development was horizontal and the quaternary salts were located either by their fluorescence or by spraying with Dragendorff's reagent. 3,8-Diamino-5-methyl-6-phenylphenanthridinium p toluenesulfonate (dimidium p-toluenesulfonate) was used as a convenient colored internal standard having an Rf value in the median range (Rf0.69). All Rr values were taken in reference to dimidium as one and are quoted as RDvalues. Reduction of Nitro Quaternary Salts.-The nitro salts, prepared by t'he standard methods outlined, were reduced in aqueous ethanol with freshly prepared Fe(OH)2essentially by the method
11.9 7.6 6.9 7.0
76.2 65.5 62.6 65.5
used for t,he reduction of nitrophenanthridinium quaternary salts.1' Terephthalic Acid Monobenzyl Ester.-Terepht'halic acid ( 15 g) and a solution of KOH (12 g) in HzO (150 ml) were heated together until solution was complete. The pH of the solution was adjusted to 9 with dilut,e HCl. Ethanol (100 ml) and benzyl chloride (10.8 ml) were added and the mixture was heated under reflux for 2 hr. A solution of KHCOI (10 g) in HzO (50 ml) was added to the thoroughly cooled reaction mix and the oily layer was separated by gravity filtration. The filtrate was acidified (concentrated HC1) and shaken with ethyl acetate (500 ml), and precipitated terephthalic acid was removed by filtration. The organic layer was separated, washed with saturated NaCl, dried (NazSOa), and evaporated. The solid crystallized from aqueous ethanol as silky needles, mp 179-180" (7.0 9). Anal. Calcd for C15H120a: C, 70.3; H, 4.7. Found: C, 70.1; H, 4.9.
4'-(2-PyridyI)-4''-(3-pyridyl)terephthalanilide.-Terephthalic acid monobenzyl ester ( 5 g) was suspended in benzene (20 ml) and pyridine (1.6 ml) and SOCL (20 ml) was added. The mixture was refluxed for 10 min and evaporated in zlacuo, and the residue was extracted with boiling benzene. Evaporation of the ext,racts gave the acid chloride; needles from petroleum ether (4G6Oo),mp 29-30' (4.1 9). The acid chloride was immediately added to a solution of 3-(4-aminophenyl)pyridine (2.6 g) in dry pyridine (20 ml) and the mixture was heated on the Hz0 bath for 2 hr. Precipitation with H2O afforded the crude amide ester which was washed well with 2 ",OH and dried. Crystallization from methanol gave pure material (5.7 gj, mp 194-193". Anal. Calcd for CZ6HeO~203:C, 76.5; H, 4.9; N, 6.9. Found: C, 76.2; H, 5.3; N, 6.8. This ester (5 g ) was hydrolyzed by suspending i i i boiling methanol (100 ml) and adding 2 N aqueous KOH (50 ml); after 10 min of boiling the solution was cooled and HzO (150 ml) was added. The solution was filtered and the filtrate was adjusted to pH 6 with acetic acid. The precipitated acid crystallized from DhIF-methanol; mp 330-331'. Anal. Calcd for C19H14Nz03: C, 71.7; H, 4.4; N, 8.8. Found: C, 71.5; H, 4.8; N, 8.8. A mixture of this acid (2.1 gj and 2-(4-aminophenyl)pyridine (1.17 g) in pyridine (20 ml) was stirred a t 0" while PCl, (0.35 ml) was slowly added. After 1 hr a t O", the mixture was heated in a (17) L. P. Wdls and J. Whittaker, J Chem. Soc., 41 (1950)
C;. J . ATWELLAND H . F. CAIN
712
C'orllp
