NO.
2a b :la h 4n
hbbr
71
DBE Me-IIBE: .\Ie2-DBE
I., t5a b 6a h
-ra
OH-DBE OH-Me-DBE OH-lIe?DRb>
h 8a
b 9a b
819
FLUORESCENT ALKYLATISG AGENTS
September 1069
C1-DBE Cl-JIe-DBE
Isomer
cis trans cis trans cis trans cis trans cis trans cis trans cis trans cis trans
H
R' H
H
405-407 437-439
)le
H
I1
1Ie
Me
II
>500 >e500 308-310
H
H
R
It"
AIp,
CHZCHZOH
JIe
H
CH2CHzOH
Me
3 Ie
CHZCH2OH
H
H
CHZCHZC1
3le
H
CHiCHZC1
OC"
343-34.5 268-270 278-280 285-286 298-300 295-297 303-30.5 205-207 228-230 144-146 1i5-177
JIe CHa CHlCHZCI CI-Me2-DBE trans b 300-302 a See full name in Experimental Section. * Nelting points were obtained on the free base i n each case. cwrected and were obtained on a Mel-Temp lab device.
Analyses
C, H, N N C, H, N N C, H, K S
c, H
S
c, H
C, H, S C, H, S C, H, S
c, H
N ?J N
10a
fore considered to be potentially useful fluorescent alkylating agents. I n this type of compound, the two haloalkyl groups are not on the same S atom and, therefore, may be active independent of each other in contrast to the S mustards of the H S , type. Electron-donating CH, groups in the benzene rings should be expected to enhance the biological activity by increasing the electron density of the Iv atom and the reactivity of the halogen atoms. The present paper reports the synthesis of these compounds, the study of their fluorebcence behavior, and a preliminary study of the interaction of one of them with HeLa cells in vivo as a demonstration of the usefulness of such methods. The synthetic scheme is illustrated in Chart I. The reaction of an o-phenylenediamine with maleic acid gave predominantly cis-bisbenzimidazole (2a, 3a, 4a) (DBE). If malic acid was used in the presence of polyphosphoric acid, mainly trans-bisbenzimidazole (2b) resulted. The resulting bisbenzimidazole was then treated with ethylene chlorohydrin in the presence of S a O H to yield the hydroxyethylbisbenzimidazole., (5-7). The hydrochlorides of the corresponding chloroethylbisbenzimidazole could then be converted to the corresponding free bases (8-10) with SaHCO1. The compounds thus prepared are listed in Table I. The trans isomer of most cis-traus pairs has a higher melting point than the cis isomer. This difference may be due in part to a facile intermolecular H bonding in the trans .tructure, a t this later found support in our ir study. I n the preparation of D B E (2), Me-DBE (3), and 3\Le2-DBE (4), the yield was higher for the tra?is isomer than the cis isomer. An examination of a molecular model of the cis isomer suggested that there teric crowding due to the presence of benzimidazolyl groups on the same side of the double bond. As a result, a-(2-benzimidazolyl)-/3-(aniline-2-carboxamido)-
?;
The melting points are 1111
ethylene was found to be present in the reaction mixture as the major by-product. Some of the bisbenzimidazoles were submitted for antitumor screening at CCNSC. DBE showed no antitumor activity for L1210 at a nontoxic dose. All other compounds qhowed some activity in 9-IiB cells, but no structure-activity relationship could be derived because of insufficient data. The preparation of the dihaloalkylbisbenzimidaaoles was facilitated by the reaction of a bisbenzimidazole with S a S H 2to form the 2 S a salt, followed by reaction with ethylene chlorohydrin to form the dihydroxyethyl derivative. This dihydroxy compound was then treated with SOCl, to form the dichloroethylbisbenaimidazole. This procedure as applied to bisbenzimidazoles is still in the investigative stage.3 In most caqes, the trans isomer shows higher fluorescence maxima and more intense fluorescence than the corresponding cis isomer. I n general, the introduction of alkyl or substituted-alkyl groups shifts the fluorescence maximum to longer wavelengths. In preliminary work on the ability of trans-Cl*\le-DBE (9b) to alkylate HeLa cells iii uiuo, alkylation appeared to have taken place when the sites of alkylation could be seen by the fluorescence of the alkylating agent. In different ztages of alkylation fluorescence appeared a t first (2-6 hr) in the cytoplasm and later on (8-24 hr) in the nucleuq. The czs compound was less efficient showing relatively low fluorescence and ease of quenching during microscopic observation. While extensive nuclear debrii containing fluorescent material could be seen, there mere observed numerous colonie5 of fluorescent-nuclei-containing HeLa cells that suggested that these cells were alive during the fixation qtep with formaldehyde. -11~0, these fluo(3) T. Seki, AI. Sasazima. and I-.\Vatanabe, YnkugaktL Zaasht, 86, 962 (1056).
I900) 225.0 (ah) (2800),272.5 (8:300), 350.0 (3340), 367.5 (4630), .3 (:&io) ).0 (sh) (WOO), 270.0 (Y700), 278.8 (9700), 282.5 (9700)) 288.7 (10,400), 335.0 16050), 355.0 (6470), 375.0 (4840), :497.5 (3550), 427.5 (7M) 225.0 (sh) (11,600), 357.5 (22,6OO), 375.0 (30,100), 395.0 (21,700) 245.0 123,.500), 275.0 (15,600), 282.5 (18,700), 355.0 141,800), 370.0 (48,000), 392.5 (sh) (25,600) 24.5.0 iyh) (21,500), 275.0 (sh) (15,600), 282.5 (18,300), X55.0 (41,000), 370.0 (39,300), 392.5 (sh) (22,400) 282.5 (ah) (22,600), 252.5 (sh) (14,500), 865.0 (40,900), ::X0.0 (43,000), 387.5 (sh) (27,700) 225.0 (sh) (30,400), 250.0 (hh) (14,400), 370.0 (73,000), :j!)o,0 (51,500) 245.0 (sh) [10,800), 282.5 (17,800), 288.7 (19,800), 3 6 2 3 ish) (29,600), 380.0 (34,200), 402.j (sh) (22,400) 2:jO.O (nh) (30,500),245.0 (sh) (20,700), 282.5 (sh) (12,800), 290.0 ( 1 4 ~ 0 0 ) 365.0 ~ (sh) (53,200), 380.0 (61,400), 4012.5 (sh) (41,300) 243.8 (19,800), 273.8 (15,900), 280.0 (17,700), 348.8 128,000), 366.3 (31,400), 385.0 (ah) (19,500) 227.5 (ah) (18,000), 245.0 (sh) (12,800), 275.0 (sh) (7000), 282.5 (i880),3350.0 (35,700), 365.0 (39,800), 387.5 (sh) (25,600) 217.5 1430), 230.0 (yh) ( i 5 5 ) , 270.0 (2790) 227.5 (sh) (46,300), 250.0 (sh) (27,600), 272.5 (sh) (16,600) 290.0 (10,$)00), 372.5 (>100,000), :390.0 (sh) (73,100)
227.5 (sh) (12,300), 290.0 (sh) (4520), 377.5 ( 2 7 , 7 0 0 ) , :i95.0 (sh) (21,100) 811 spectra were obtained for 9.3 X 10-6 &I soliitioiia of bisberiaimidazole in EtOH on a Beckman DB-G gratiiig spectrophot,ometer.
precipitate of crude OH-DBE formed which was collected. This material was dissolved in EtOH (25 ml), the resulting solution was decolorized with charcoal, and the filtrate was allowed to stand a t room temperature. The precipitate of IVa was collected i i i two fractious by filtration aiid allowed to dry in the Further recryst,alair. I t had my 263-267", yield 0.18 g (15 Iizatioii from EtOH gave material melting at 268-2770'. All i~cinipoiindsin this series tenaciously held onto solvent of crystallizatioii. cis-a-( 2- [ 1-( 2-Chloroethyl jbenzimidazolyl] )+-( 2-benzimidazolyl jethylene (Sa, CI-DBE).-The preparation of 7 s was adapted from a procedure by Schmuta and Kilnale7 for the preparation of 2-chloromethyl-3-(~-chloroethyl)benzimidazolefrom the correhponding hydroxy derivative. All other chloroethyl derivatives were prepared similarly (Table I). Compound 5a (0.1 g, 2.3 X mole) was stirred with 20 ml of CHClr with protection from moisture. h solutioii of 2 ml (3.32 g, 0.028 mole) of SOC1: in 10 nil of CHCl, was then added slowly with stirring. Stirring was continued aiid the mixture was refluxed for 3 hr. Upon cooling a large excess of anhydrous Et&) was added. A flocculeiit yellow precipitate of Sa formed as a hydrochloride which was collected, washed (EtzO),and stored in a dessicat,or over CaSO,. The yield was 0.09 g (96.8%), mp 1635-1i5°. The hydrochloride was converted to its free base In IIzO with saturated NaHCO3, mp 197-200" aftec air drying. Recrystallization from EtOH gave small yellow crystals melting :it 205-207', and agaiii coiitaiiiiiig 1 mole of Et011 as solveiit of cryst.allii.ation. ~______ ( i )G. Schmutz and F. Kunzle. H e h . Chim. Acta, 39, 1144 (1956).
s21
Uv and Visible Spectra Study.-Uv and visible spectra of the bisbenzimidazoles were obtained oii a Beckmaii DB-G grating spectrophotometer as 9.3 X 10-6 M solutions in EtOH (Table I1 j. One woiild expect the trans isomer to have a greater A., arid correspoiidirig c th:iii t'he cis cumpoiitid, particularly for the longer waveleiigth baiids. This patterii is borne out for most conipouiids by their IIV and visible spectra, except L\le-L)BE (3a,b) a i d 0H-I)BI.; (5a,b).* Some of the bisbeiiziniidazoles , wed a doublet absorption maxima aroliiid 280 mp chuacter c of benziniidazolez;, 5i6)niethSlbeiizimiciazole, or 5,6-dimeth!.lbeiiziniida~ole.~ In theve cases the cis compoimd often showed niore inteiise absorption t,han the trans conipoutid for these absorptioii bauds. Ir Spectra Study.-Ir spectra for the bisbeiiximidazoles were cibtaiiied in KBr pellets usirig a Perkiii-Elmer 137 NaCl spectrophotometer. The ir data gave coiitributiiig evidenre to the assignment of cis aiid trans forms. The trans compounds all had a stronger absorption at, 970-960 cm-l (C-H out,-of-plarie deformation) compared to the cis isomer. Fluorescence Spectra Study.-Fliioresceiice spectra of the bisbeiiximidazoles were determined oil a11 Aniiiico-Bowinaii spectrophotofluoronieter as 4.7 X JI solutions iii Et011 (Table I11).
Excitation max. Config
cis trans cis trans cis lrans
cis trans
cis
lmns cis trans cis trans cis t 1.u ns
m p (re1 intensity)
370 353 377 :E5 385 383
(0,300) (8.9) (9.15) (1 . 4 ) h (1.79) (7.75) :380(7,65) 380 (14.4) :190 (8.80) 378 (17. .5) 3% (8.40) 393 (15.1) 377 (5.18) 375 ( 8 . 2 5 ) 377 (0.111) 370 ( 2 9 ,2)
Fluorescence max, mfi (re1 intensity)
42.3 425 435 435 443 445 443 445
455 440
443 455 435 435 440 443
(0.254) (13.4)
(8 2 5 ) (1.67)b (1.56) (7 . 00) (7 . 2 5 ) (13.5) (8.0.5) (ltj.9) ( 9 . 00) (15.1) ( 4 . 5 6j (7 .98) (0.118) (28.0)
... 450 ( 6 . 5 7 ) ti,ans 385 (6.58) a All spectra were obtained for 4.7 X 1 0 - 6 Jf wliit,ioris of bisbeiizimidazole i n EtOIi o i l an Aniiricci-Howrrnaii spectrophotofliiorometer. The aiionialy here may be cine to 5'- arid B'-diniethyl isomers.
Titration of Bisbenzimidazo1es.-This procedure was b a d oii a method by Fritz and Hammond.1" HCiO, (70-72c;) (8.5 nil) was mixed with 200 ml of .4rOH arid 20 nil of Ac2O. The solution was allowed to stand overnight to permit complete reaction of AcsO with the HzO present. This solution was then diluted to 1 1. with AcOH to make a solution -0.1 A' with respect to HCIOI. Of this solution 10 ml was diluted to 1 1. m-it,h .lcOFI to make a (8)For these bisbenzimidazoles, isomerization must h a r e taken place in solution readily. T h e resonance hydrid of tlie ground state m a y h a r e contributions from various resonance forms. Resonance forma of the l a t t r r type have a single bond a t the place ~rliereisomerization takes d a c e and contributions from these forms should decrease t h e barrier t o rotarion around this bond a n d increase t h e possibility for isomerization. A similar explanation has been set forch t o explain anomalous results in connection with some cis and trans stilbene compounds [M. Calvin and R. E. Buckles, J. Am. Chem. SOC.,6.2, 3324 (194O)l. (9) K. Hofmann, "The Chwniatry of l i e i ~ r ~ y y v l(i~ru i n p ~ i i i n d sIiuiclaiult. : and its Derivatives," Part I , Interrcience t'iibliuliers, Inc., X e n York, 1;. Y., 1953, p 2-53. (IO) J. S. Fritz and 0.S. fiammond. "Quantitative Organic -\nalysis," John Wiley & Sons, Inc., Ken. l-ork. K. Y., 1957, pp 263-266.
