Eur. J. Med. Chem.

23 (1988) 183-188 183

0 Elsevier, Paris

Original paper

Synthesis of indolo[3,2-clquinolines and indolo[3,2-dlbenzazepines

and their interaction with DNA
El-Sayed IBRAHIM, Anita M. MONTGOMERIE, Andrew H. SNEDDON, George R. PROCTOR* and Brian GREEN?

Departments of Chemistry and Biochemistry, University of Strathclyde, Glasgow Gl IXL, Scotland, U.K.
(Received July 10, 1987, accepted November 24, 1987)

Summary - A number of indolo[3,2-clquinolines and tetrahydroindolo[3,2-d]-1-benzazepines were synthesised by

Fischer indolisation of ketones including 7-chloro-1,2,3,4-tetrahydroquinol-4-one and 8-chloro-2,3,4,5-tetrahydro-l-benz-
azepin-5-one with several hydrazines. Some of the planar indolo[3,Zc]quinoline derivatives were shown by their effects
on DNA supercoiling to form intercalation complexes with DNA and to inhibit the synthesis of macromolecules in cultured
KB cells. Non-planar tetrahydroindolo[3,2-d]-I-benzazepines did not form intercalation complexes with DNA under the
same conditions. 3a, b, d inhibited the synthesis of macromolecules in cultured KB cells, 3a being the most effective. No
significant anti-tumor activity was detected for 3a, b, 4a, e in the NIH screening program.

R&urn6 - Synth&se d’indoles[3,2-c]quinolCines et d’indolo[3,2-d]benzaz&pines et etude de leur interaction avec I’ADN.

Des indolo[3,2-c]quinole’ines et des te’trahydroindolo[3,2-d]-I-benzazepines ont et.& synthe’tise’espar l’indolisation de Fischer
de c&ones (par exemple, chloro-7 te’trahydro-1,2,3,4 quinolone-4 et chloro-8 te’trahydro-2,3,4,5 benz-I azepinone-5) avec
des hydrazines. Les e#ets des derives plans sur le ctsupercoiling>>de I’ADN (SV 40) laissent supposer que ces mole’cules s’inter-
talent entre les paires de bases de I’ADN. Les derives 3a, b, d inhibent la synthtse des macromole’cules dans les cellules KB
en culture, 3a &ant le plus efficace. -Aucun des derives 3a, b, k, e n’a mantfeste’ une activite’ anti-tumorale significative.
indoloquinolines / indolobenzazepines / DNA / supercoiling

Introduction Chemistry

Drugs based on 4-amino-7-chloroquinoline (such as, chloro-
quine and amodiaquine, 1 and 2) play a key role in present-
day management of malaria [ 1, 21. The suggestion that such
drugs may be active against other diseases with a malarial
vector, such as filariasis [3] has made it useful to synthesise
new types of derivatives and also to look for other potential
biological activities.
Marquez et al. [4, 51 first synthesised the indolo[3,2-cl-
quinoline 3a on the basis that larger, flat analogues of
amodiaquine would show more effective intercalative
binding to DNA and therefore potentially superior anti-
malarial activity. As an extension of that work, we have
synthesised the tetrahydroindolo[3,2-d]-1-benzazepine anal-
ogue 4a with a view to potential dehydrogenation to large
planar molecules such as 5. A number of other compounds
with these two ring systems has also been prepared and
examined for potential biological activity.
Indolo[3,2-clquinolines were made by Fisher indolisation
of the chlorotetrahydroquinolone 6 with various hydrazines
(7c, SC, 9c, 10~). Most of the latter were made by adaptations
of literature methods; while they were identified spectro-
scopically and yielded the desired products (e.g., 3), chemical
analyses were unsatisfactory and usually their mass spectra
failed to reveal the molecular ion. The indolo[3,2-clquinolines
3 were obtained, however, in tolerable yield as the hydro-
chloride salts, from which the free bases could be isolated.
All of the latter products were fully characterised.
Tetrahydrohydroindolo[3,2-d]-l-benzazepines 4 were
obtained by reacting the tetrahydro-1-benzazepin-5-one
(11, R1 = R2 = H) with substituted hydrazines. The
ketone was made from the previously reported N-tosyl-
ketone (11, RI = tosyl, R2 = H) now made in improved
yield. Both the indolo-1-benzazepines and their hydro-
chlorides were fully characterised. Nearly all attempts to

“Author to whom correspondence should be addressed: Pure and Applied Chemistry, University of Strathclyde, Cathedral Street, Glasgow Gl IXL,
Scotland, U.K.
1:Deceased March, 1988.
184

dehydrogenate tetrahydroindolo[3,2-cl-l-benzazepines were It is noteworthy that, even for the non-planar derivatives
unsuccessful; however active manganese dioxide did yield 4a, c, d, for which intercalation would seem implausible,
in one case a low yield of a material tentatively formulated the number of potential binding sites (v - 0.27) is much
as 13. This is worthy of further study. less than the one per base pair expected for non-specific
external binding. Molecular model studies suggest the
possibility of an AT-specific interaction in which the posi-
R es&s and Discussion tively charged dialkylamino group interacts with the DNA
phosphate and the indolo nitrogen interacts with the free
Potential biological activity 2-0~0 group of the DNA thymine forming a hydrogen
bond. Such AT-specific binding is supported by the obser-
In a preliminary report [6], we confirmed the earlier obser- vation that poly(dAT) has more potential binding sites
vation [4] that 3a binds readily to DNA. Binding to DNA for compounds such as 4a under the standard conditions
of these compounds was analysed by following the changes than does calf thymus or salmon sperm DNA (55-56x
in the absorption or fluorescence spectrum of the compound A + T) while M. luteus DNA (28% A + T) reacts very
during titration with increasing concentrations of DNA weakly and this reaction shows the salt sensitivity typical
171. The spectral changes, which were of the same general of external binding modes [6]. Such AT-restricted binding
type (bathochromic shift and depression of absorbance) should give a v value of - 0.28 for calf thymus DNA as
for all compounds listed in Table I, were analysed by found for 4a, d (Table I), whereas for poly(dAT) itself,
Scatchard plots, which are only valid for non-interacting the v value approaches the 0.5 expected for one binding
binding sites [8]; the characteristics of the principal binding site per base pair. While the charged dialkylamino substituent
observed are given in Table I. is, as expected, the dominant one in the external binding
of the 4 series, the presence of a polar chloro group also
favours the AT-selective external binding of these non-
Table I. Characteristics of major DNA-binding. planar derivatives; its importance for intercalative binding
Compd. 10M5 x KA Bound/DNA Intercalation of the parent 4-aminoquinoline (chloroquine) series is
(M-9 phosphate well-established [lo].
v Since the basic binding data did not discriminate clearly
between the non-planar (4 series) and planar derivatives
1 4.9 0.13 (3 series), a direct method of establishing whether or not
3aa 2.2 0.24 I intercalation occurs was used. DNA is locally unwound
3b
3a 4.9
2.8 0.20
0.43 1 when a molecule intercalates and this affects the overall
3f” 1.5 0.54 superhelical density of a closed circular DNA molecule [l I].
4a 1.9 0.27 The compounds were therefore examined for their ability
4ac 0.36 0.43 to induce DNA supercoiling upon removal during electro-
4c 0.5 0.26
4ad 0.08 0.27 - phoresis from closed circular DNA molecules which had
4e negligible binding been relaxed enzymatically while they were still bound ]12].
The results in Fig. 1 show that DNA molecules become
From absorption and fluorescence changes of compounds during supercoiled (hence run faster on gels) after being relaxed in
titration with calf thymus DNA. Reactions were carried out in 0.01 M the presence of compounds 3a, b, c, d but not the non-
sodium chloride/Tris or sodium phosphate buffer, pH 7.5. planar benzazepine derivatives 4a, b at similar concen-
“In 0.2 M NaCl (complex precipitates in low salt).
bin 0.01 M acetate, pH 4.0 (insoluble at neutral pH). trations.
CBinding to poly(dAT). Although not apparent from simple binding data at
dBy equilibrium dialysis. moderate ionic strength, intercalative binding to DNA
of 3b is clearly shown to occur by this direct method,
consistent with observations on the members of the planar
series. 13 is beginning to show the same effects as 3a, b
Of the different types of binding found, one, presumably at the lower concentrations that could be used. (The range
external, becomes saturated at roughly one ligand molecule of compounds that could be tested was limited by solubilities
per base pair (Y - 0.5). The others show a restricted range or in some cases by precipitation of DNA complexes.
of sites (V < 0.27). Some compounds also appeared to affect the topoisomerase
It is not surprising to find binding with a restricted activity, giving rise to a high proportion of nicked (relaxed)
number of potential sites in the case of planar molecules DNA molecules, a well known effect of intercalating agents
(such as 3a, d and 13) with v < 0.23 ; this would be expected D31).
from Scatchard plots [S] if the compounds were intercalating
into the DNA structure as Marquez et al. [4] suggested
for 3a. The binding of 3f was studied at pH 4.0, where Effects on cultured KB cells
the DNA is ordered but protonated on the cytosine residues
[9] so the binding may be rendered less specific in this The effects of the intercalating derivatives on the synthesis
modified DNA, whereas the spectral method used for of macromolecules by human (KB) cells in vitro was also
3b only reveals external binding at this salt concentration. examined. 3a, at a concentration of 4 x lop4 M caused
Fig. 1. Effects of compounds on supercoiling of closed circular DNA. 0.254.4 pg of partially relaxed SV40 DNA was further relaxed with 1.5 units
topoisomerase 1 in the presence of varying concentrations of each test compound and the mixture was then electrophoresed in 0.8 % agarose gels.
Removal of intercalated molecules from the relaxed DNA during electrophoresis causes the resulting underwound DNA to become supercoiled
and thus run ahead of the relaxed DNA. Where test compounds were present during relaxation is indicated above the relevant tracks, the arrow
showing the direction of increased concentration [from 1.7 x 10-e M to 7 x 10-5 M (3b, 4b) ; 8 x 10-e M to 8 x 10-S M (3a, 4a) ; 2.3 x 10-S M
to 0.94 x 10-C M (14)l. S = supercoiled DNA (untreated) ; I = DNA used for the test incubations (partially relaxed) ; 0 = control (no test
compounds during’ the final relaxation) ; R = .fully relaxed DNA.

a rapid and marked inhibition of RNA synthesis, over
95% inhibition occurring within 10 min: this result with
intact human cells is consistent with observations on isolated
bacterial RNA polymerase [4]. The effect on DNA synthesis
was rather smaller and protein synthesis was least affected,
the inhibition being less than 50% after 1 h (Fig. 2). The
dimethyl analogue 3b was less active in this system, with
no effect on RNA synthesis over the first 20 min and a
reduction of only a third after 60 min. 3d was least effective,
giving only 20% inhibition of uridine incorporation in
1 h. The effects were confirmed by sucrose gradient centri-
fugation, which showed that synthesis of all sizes of RNA
was essentially completely abolished by 3a, whereas 3d
caused only partial inhibition, with more of the label being
found in smaller RNA species.
The results confirm the importance of the planarity
of the molecule for effective interaction with DNA and
for biological effectiveness of these extended aminoquinoline
derivatives. None of the compounds tested by in vivo
screening (3a, b; $a, e) showed useful anti-tumour activity,

0 20 40 60 20 40 60
TIME (mid TIME (mid
Fig. 2. Effects of compounds 3a, b on DNA, RNA and protein synthesis in cultured KB cells. A. Total incorporation of tritiated precursor. Open
symbols: control cells; solid symbols: incubated with compound 3a for the time indicated; 0 : [3H]uridine; n : [3H]leucine. B. Incorporation
of precursors by treated cells as a percentage of that for the control cells. 0: [sH]uridine plus 3a; H : [sH]thymidine plus 3a; A : [3H]leucine
plus 3a; x : [sH]uridine plus 3b.
186

one problem being toxicity towards the host micea. Com- OMe
pound 3a also failed to show significant activity against
P. berghei in tests in the mouseb. Nonetheless the results
do suggest that the properties of the target molecule 5
should be of great interest.

Experimental protocols
Chemical synthesis
Melting points were obtained on a Gallenkamp melting point apparatus
in open capillaries and are uncorrected. Infrared spectra were recorded
on a Perkin-Elmer 397 spectrometer.
Mass spectra were determined on an A.E.I. (Kratos) MS9 spectro-
meter fitted with a mass soectrometrv services solid state console
and using GEC-905 computer system fdr data capture and processing.
1H NMR spectra were recorded on a Perkin-Elmer R.14 (100 MHz)
or R.32 (9b MHz) instrument. All spectra were determined using 0
deuteriochloroform as the solvent, unless otherwise stated, and all
chemical shifts are quoted on the scale with tetramethylsilane as the
internal standard. Column chromatography was performed using
M.F.C. grade silica gel, supplied by Hopkin and Williams Ltd. Thin- CIJlxl = ’ N
laver chromatoeraohv (TLC1 was carried out on 5 x 20 cm ulates H ii
3 4 6
cdated with Kiiseigei & type 60 (Merck), whereas preparative-TLC
was performed on Kieselgel HP254 (Merck). Analyses indicated by a. Rl=OMe, Rz=CHzNEtz a. Rl=Cl, Rz=OMe, Rs=CHsNEtz
the symbols of the elements or functions were within 5 0.4% of the b. Rl=OMe, Rz=CHzNMez b. Rl=Cl, Rs=OMe, RS=CHzNMez
theoretical values (*indicates values within f 0.5% of the theoretical c. Rl=H, Rz=CHzNEtz c. Rl=CI, Rz=H, RS=CHzNEtz
one). d. Rl=H, Rz=CHsNMez d. Rl=H, Rz=OMe, R3=CHzNEtz
e. Rl=OMe, Rz=H e. Rl=Cl, Rz=Rs=H
S-Chloro-2,3,4,5-tetrahydro-I-benzazepin-Sane 11, R1 = R2 = H f. Rl=Rz=H f. Rl=Rz=Rs=H
N-Tosyl-2,3,4,5-tetrahydro-l-benzazepin-5-one (6 g, 0.017 mol) was
dissolved in a mixture of concentrated sulphuric acid (30 ml) and Scheme 1.
glacial acetic acid (45 ml) heated to 80°C and stirred for 4 h. The
reaction mixture was poured into ice/concentrated hydrochloric acid OMe Ok?
oCH2N~12 oCHsNM” oCH2Nb ock’2N’Ac2
and extracted with chloroform. The aqueous layer was then basified
with sodium hydroxide solution (lo%), and again extracted (CHCIJ),
dried (Na2S04) and evaporated, leaving the product (2.5 g, 74.4x),
mp: 104oC. v,,&nujol) 3320 cm-l (N-H), 1645 cm-l (C=O). Anal. R R R R
(CIOH~OCINO) C, H, N. 7 x 9 IO
2-Diethylaminomethyl-4-nitroanisole 7a a. R=NOZ a. R=NOz a. R=N02 a. R=NOZ
2-Chloromethyl-4-nitroanisole [18] (10 g, 0.050 mol) in dry benzene b. R=NHz b. R=NHz b. R=NHz b. R=NHz
(300 ml) was added dronwise to an excess of diethvlamine (150 ml) c. R=NHNHz c. R=NHNHz c. R=NHNH2 c. R=NHNH2
with stiiring. This was rekuxed for 3 h, after which t;me diethilamine:
hydrochloride was removed by filtering when cool. The filtrate was
evaporated under reduced pressure, leaving a residue (11.8 g) which
was dissolved in hydrochloric acid (100 ml, 2 N) and extracted with
ether. Drying and evaporation yielded starting material (4.3 g). The
aqueous layer was basified (NaOH) and extracted again with ether,
yielding the desired product (6.0 g). a Meo$$R’ 4
The hydrochloride was formed by dropping ethanolic hydrogen
chloride onto the oil until a white solid appeared. This was crystallised Cl A
from ethanol and had mp : 178oC (lit. 178oC [19]). Anal. RI’
(cdhsN203*HCl) C, H, N. vmax 1595, 1615 cm-l 6 8.55 (2H, d, II I2 13
aryl H); 7.5 (lH, d, aryl H); 4.8 (6H, q, N(CH&H&); 4.2 (2H, a. Rl=Rz=H
-0CH3); 3.7 (2H, d, -CHzNEtz); 1.7 (4H, t, N(CHzCH&). b. Rl=OMe, Rz=CHzNEts
znal. (C12H1sN203) C, H, N. Scheme 2.
2-Dimethvlaminomethvl-4-nitroanisole Sa was made similarly, the
hydrochldride had mp- 222OC.
Anal. (CIOH&INZO~) C, H, N. Frye base
6 8.05-8.28 (2H, m, aryl H); 6.94 (lH, d, aryl H); 3.93 (3H, s, OMe); Similarlv 3-dimethvlaminomethvlnitrobenzene 1Oa free base Anal.
(C9H~2N&) C, H, .*N. 6 7.~k.25 (4H, m, aryl l?); 3.51 (2H, s,
3.55 (lH, s, OCHZ) and 2.3 (6H, s, 2 x CH3).
Also made thus was 3-diethylaminomethylitrobenzene 9a, the -CHs-); 2.27 (6H, s, 2 x CH3). The hydrochloride had mp: 193-
hydrochloride Anal. (CllH17ClN202) C, H, N. Free base 6 7.4-8.4 196oC. Anal. (C~H1&1Nz0~) C, H, N.
(4H, m, aryl H); 3.65 (2H, s, -CH2-); 2.55 (4H, q, 2 x CHZCHS); 5-Amino-2-methoxy-N,N-diethylbenzylamine 7b
1.05 (6H, t, 2 x CH3CH.z). The anisole (7a, 20 g, 0.084 mol), ethanol (300 ml) and platinum
oxide (100 mg) were hydrogenated at 3 atmospheres pressure, until
the uptake of hydrogen ceased. The reaction mixture was filtered
“These data are the results of screening performed under the auspices through Kieselguhr and the filtrate evaporated to give an oil (16.7 g,
of the Developmental Therapeutics Program, Division of Cancer 95.6%) which was distilled in a Kugelrohr tube (155-160°C/0.3 mm).
Treatment. N.C.I., Bethesda. MD. U.S.A. 6 6.8 (3H, m, aryl H) ; 3.7 (3H, s, -0CH3) ; 3.5 (2H, s, --CHsNEtz) ;
bData fro& screening program; Walt& Reed Army Institute of Research, 3.4 (2H, s, -NHz, exch.); 2.6 (4H, q, N(CHYCH&); 1.1 (6H, t,
Washington, DC., U.S.A. N(CHYCH&). Anal. (C~ZHZONZO) C, H, N.
5-Amino-2-methoxy-N,N-dimethylbenzylamine 86 was made by catalytic t, 2 x CH~CHZ-). Anal. (C~aHa6~~ClNa0) C, H, N.
(PtOz) reduction of the appropriate nitro compound @a) in ethanolic The following were nreuared similarlv.
hydrogen chloride solution. The free base had bp: 140°C/0.1 mm. b. 3- Chloro-I~-dimethyla~inomethyl-6~7-dihydro-9-methoxy-indolo-
MW: (isothermal/MeOH) 168; M+ 180.1245. C~OHXNZO MW: [3,2-d]-benzazepine 46. The hydrochloride had mp: 24OoC (dec).
180.1263. Anal. *C, H, N. 6 6.4-6.72 (3H, m, aryl H); 3.7 (3H, s, Anal. (C2oH~ClNa0.3 HChHaO) C, H, N. Free base mp: > 340°C.
OMe); 3.45 (2H, br. s., exch., NHz); 3.38 (2H, s, -CHz); 2.25 (6H, Found M+ 357.1456 (16.6%. 355.1483 (51.5X). CzoHzz37C1N30
s, 2 x CHa). The dihydrochloride hadmp : 242OC. Anal.(CloHlsClzNzO- requires 357.1422; C&~aa@!lNaO requires 355:1451.
C, H, N, Cl. Reduction in neutral solution led to partial conversion
c. 3-Chloro-IO-diethylaminometlzyl-6, 7-dihydro-indolo-[3,2-dlbenzazepine
into dimeric products.
Similarly : 3-diethylaminomethylaniline 9b bp : 14OoC/O.l mm. Anal. le. The dihydrochloride hydrate separated from the reaction mixture.
(CiiHlaNz) C, H, N. The dihydrochloride Anal. (CirHzoClzNa) C, H, N. Anal. (CziH~4ClN3.2 HClmH20) C, H, N. Free base found Mf 355.1645
Also 3-dimethylaminomethylaniline lob bp : 13OoC/O.l mm. Anal. (10.9x), 353.1674 (33.9%). C~iH24~~ClNa requires 355.1629, C21H24-
(CgHraNZ) C, *H, N. 6 6.5-7.2 (4H, m, aryl H); 3.5-3.7 (2H, br. s., 35ClNa requires 353.1659.
exch., NH%) ; 3.32 (2H, s, -CHz-); 2.24 (6H, s, 2 x CH3). d. IO-Diethylaminomethyl-6,7-dihydro-9 -methoxyindolo [3,2-d] benzaze-
pine 4d. Free base mp : 95-96oC from ethanol. Anal. (CZZHZ~N~O *
Attempted preparation of Shydrazino-2-methoxy-N,N-diethylbenzyl- HaO) C, H, N. The hydrochloride had mp: 176-178OC. Anal. (CZZ-
amine 7c cf. [4, 5, 201 Ha7NaO -3 HCl *HzO) C, H, N.
The benzylamine (7b, 4 g, 0.019 mol), in concentrated hydrochloric e. 3-Chloro-6.7-dihvdroindolo13.2-dl-I-benzazeoine 4e. The free base
acid (25 ml). was cooled to O°C and sodium nitrite (2.32 g, 0.016 mol) (recrystallised from chloroform), mp: 212OC. M+ 270.0727, 268.0748.
in water (5’ml) was added slowly with vigourous stirring. After a CsHlaClNn reauires M 270.0738. 268.0767. 6 7.2-6.6 (8H. m. 7 arvl
few min. sulnhamic acid was added in small uortions until the reaction Hfexch., NH) ;A4.2 (lH, br., s., e&h., -NH-); 3.4 (2Hj t, --dHa-j;
with starch iodine paper remained positive.-Stannous chloride (14 g, 3.1 (2H, t, -CH2-). Anal. (Cr6HisClN~) C, H, N. The hydrochloride
0.1 mol) in concentrated hydrochloric acid (7.5 ml) was added drop- crystallised from ethanolic hydrogen chloride and had mp: 249-
wise at OOC. The mixture was stirred for 2 h at O’C, then filtered. 251°C. Anal. (CnjH14ClaNz) “C, H, N required 9.2; found 8.5.
The solid was dissolved in sodium hvdroxide solution (25X) and f. 6,7-Dihydroindolo[3,2-d]-I-benzazepine 4J Free base (from ethanol)
extracted with ether, which was dried (NazS04) and evaporated,
yielding the product (3.6 g, 83.9%). bp: 14OoC/O.l mm. 6 7.2-6.4 mp: 168-170°C. M+ 234.1145 Ci6H14N2 requires M 234.1157. 6
8.05 (lH, s, exch., -NH-); 7.4-6.65 (SH, m, aryl H); 3.85 (lH,
(4H, m, aryl H); 3.7 (3H, m, -0CHa); 3,6 (2H, d, CHzNEtz); 2.55 s, exch., -NH-); 3.5 (2H, t, -CHa-) ; 3.2 (2H, t, -CH2-). Anal.
(4H, q, -N(CHZCH&); 1.1 (6H, t, -N(CH&H&). Anal. un- C, H, N. The hydrochloride had mp : 215-220%. Anal. (Ci6H14Na,HCl)
satisfactory. Repeated attempts failed to produce purer material.
All other hydrazines gave similar results. C, H, N.
The following indoloquinoline derivatives were also made by analo- 5-Hydroxydibenz(b,h)-1,6-diazaazulene 13
gous 14, 51 procedures from the appropriate crude hydrazines and
ketone 6a. 6,7-Dihydroindolo[3,2-d]-1-benzazepine (0.66 g). manganese dioxide
[21] (5.7 g) and toluene (150 ml) were refluxed and stirred for 24 h.
b. 3-Chloro-9-dimethylaminomethyl-8-methoxy-llH-indolo[3,2-c]quino- Flash chromatography on silica (Merck 9385), elution (4% ethanol/
line 35. Dihydrochloride hydrate mp: 280°C (dec.). Anal. (Cn,HzzCla- chloroform) gave product (98 mg), mp: > 310°C as a dark powder
N302) C, H, N. Free base found M+ 341.1106 (9 %), 339.1145 (34.8 %). from CHCL/MeOH. M+ 246.0765. c1&0N20 requires M 246.0793.
Ci9Hls*3X!1Na0 requires 341.1109, C19Hl~~~ClNa0 requires 339.1138 vmaX(nujol) 2600-3200 cm-l (-OH). Anal. (CniHioNaG) *C, H, N.
c. 3- Chloro-9-diethylaminomethyl-1IH - indolo [3,2 - c] quinoline 3c.
Free base recrystallised from ethanol as yellow prisms, mp: 285OC.
M+ 339.1328 -(5.9 %), 339.1350 (17.8%): CZCIH&IN~ requires M Biological methods
339.1316, 337.1346. Anal. C, H, N.
Except where indicated, nucleic acids were purchased from Sigma
d. 3- Chloro-9-dimethylaminomethyl- IIH- indolo [3,2-c] quinoline 3d.
Chemical Co., Poole, Dorset, U.K. Some calf thymus DNA was
Dihydrochloride hydrate, mp: > 300°C. M+ 311.1001 (5.5 %), 309.1033 obtained from Worthington. BRL (Gibco) nrovided the closed circular
(29.2%). C~oHr6ClN3.2 HCl*HzO requires M 311.1003, 309.1033. DNA (SV40-90 % sup&helical form 1) and the topoisomerase (type 1,
Anal. C, H, N. calf thymus). The agarose used was Seakem LE (Miles Scientific).
e. 3-Chloro-8-methoxy-IlH-indolo[3,2-clquinoline hydrochloride hemi- [aH]Thymidine (47.5 Ci/mmol), [sH]uridine (29 Ci/mmol) and [3H]-
hydrate 3e. mp: > 250°C. Anal. (CnrH1iClN20 *HCl *t/2 HzO) leucine (51 Ci/mmol) were supplied by Amersham International.
C, H, N. Test compounds were usually examined as the hydrochlorides
f. 3-Chloro-IIH-indolo[3,2-clquinoline hydrochloride hydrate 3f because of their greater solubility. All such solutions were shielded
mn : 280-282°C. M+ 252.0416. CrsHaClNz*HChHaO. Anal. C, H, N. from light and w&e freshly prepared from the solid, though in a few
6 iDtO> 7.3-7.5 (4H, m, aryl H); 6.9-7.2 (3H, m, aryl H); 5.05 cases diluted (ethanolic) stock solutions were used.
(lH, s, 6-H); 4.27 (lH, s, exch., NH). Absorption spectra were measured on a Pye-Unicam SP8-100
spectrophotometer and fluorescence measurements on a Perkin-
g. 2-Methoxy-llH-indolo[3,2-clquinoline 12a. Free base, Elmer 3000 fluorescence spectrometer.
280-285°C. M+ 248.0942. C16Hr2N20 requires M 248.0950. Atil! For work involving closed circular DNA, all reaction tubes, buffers
C, H, N. and micropipette tipswere autoclaved to ensure that they were free of
b. 9-Diethylaminomethyl-2,8-dimethoxy- IlH - indolo [3,2-c] quinoline 12b. nuclease activity.
The free base crystallised from chloroform as a yellow powder mp:
230-233oC. M+ 363.1941. CzzH2sNaO~ requires M 363.1941. Carbon DNA-binding experiments
analyses were consistently low (2-3%) presumably due to traces Non-covalent binding was analysed [7] by measuring the absorbance
of chloroform. Anal. *N. or fluorescence of the test compound at fixed wavelengths in the
presence of varying concentrations of calf thymus DNA, poly(dAT)
3-Chloro-IO-diethy~aminomethyl-Y-methoxy-indolo[3,2-d]-benzazepine 4a. etc. in 10 mM Tris buffer containing the required concentration of
Chloroaminoketone (6a, 1 g), hydrazine (7c, 1.5 g), ethanol (20 ml) NaCl or in sodium phosphate buffer at pH 7.5. The major binding
and concentrated hydrochloric acid (5 ml) were refluxed together species was determined from Scatchard plots [S].
for 24 h. The hvdrochloride formed was treated with sodium hvdroxide
solution; the base liberated was filtered, washe’d with water and dried. Detection of intercalation
Upon recrystallisation from ethanol, it had mp: 74-75OC. Mf 383.1788. The procedure is based on that used by Wiesehahn and Hearst [12]
C~zH26~~ClN30 requires 383.1764. 6 7.7 (lH, s, exch. NH); 7.3-7.0 except that, to allow drug-induced changes to become readily apparent
(3H, m. arvl H) ; 6.7-6.5 (2H, m, aryl H) ; 4.3 (lH, s, exch., NH); independently of inhibition of topoisomerase action, the closed circular
3.8 -(3H, s; Oh&i) ; 3.6 (2H, s; -Cl&--N) ; 3.4’ (2H, t, -CHz-j ; DNA was first relaxed before the drug was added.
3.05 (2H, t, -CH2); 2.6-2.4 (4H, q, 2 x CHzCH3); 1.1-0.9 (6H, SV40 DNA was largely relaxed by incubation for 1 h at 37OC with
DNA topoisomerase 1. The relaxed DNA was then evenly distributed &ference$
into a series of Eppendorf tubes (0.254.4 pug/tube). Ligand was
added in varying concentrations to the test series, followed, after .
a brief incubation, by a further 1.5 units of topoisomerase with appro-
priate buffer (total vol. lo-20 ~1). After further incubation at 37oC
for l-3 h, the mixture was electrophoresed in 0.8% agarose gels
at 100 V per gel. The gels were then stained in ethidium bromide
solution (50 pg/l) to allow the DNA bands to be detected on a trans-
illuminator (302 nm).
Effects on cultured cells
Human-derived KB cells (Flow Laboratories) were cultured in Earle’s 6 Montgomerie A. M., Proctor G. R. & Green B. (1979) Biochem.
minimum essential medium, with Earle’s salts and bicarbonate, sup-
plemented with 5% newborn calf serum. Test compounds, in filter-
sterilized solution, radioactive precursor’s etc. were added directly
8
to the cultures in 24-well tissue culture plates. Cells were incubated
with the appropriate labelled precursor for lo-120 min in the presence 9 Zimmer C., Luck G. & Venner H. (1968) Biopolymers 6, 563-574
and absence of the compound being tested. At the relevant times, 10
the medium was replaced with medium containing excess unlabelled 11
precursor and incubated for 10 mm, after which the cells were washed 12
with fresh medium. The cells were then trypsinised and collected
on Whatman GFjC glass fibre discs, presoaked in saturated phosphate. 13
Each sample was washed repeatedly with ice cold 10% trichloroacetic
acid containing unlabelled precursor, followed by absolute ethanol. 14
The dried discs were finally counted in a Packard Tricarb liquid scin-
tillation spectrometer. 15
16
17
Acknowledgments
18
This investigation was supported in part by the Cancer Research
Campaign. 19
We thank Marion Cameron for skilled technical assistance and
also acknowledge the contributions of a number of undergraduate 20
students, especially Jacqueline Armour and Ann Stirling. 21
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