^■ INDOLOQUINONg COMPOUNDS'

The present investigation relates to new iπdoloquinone compounds and to methods of preparing them.

M ito mycins ¹ have been known for many years to have strong antibacterial and cytostatic activity. In particular, mitomycin C, which has the formula:-

has been shown to be active against breast, pancreatic and prostatic adeπocarciπomas, and notably against colon, bladder, lung and gastro-iπtestinal cancer. However, clinically, mitomycin C was found to have disadvantages and consequently many derivatives of mitomycin C have been synthesized in attempts to develop more effective antitumour agents with a higher antitumour activity and a lower toxicity than mitomycin C itself.

Among the derivatives of mitomycin C, 7-methoxymitosene of the formula:-

is known to have important antibacterial activity in vitro and in mice ² ,

and this fact prompted the synthesis of a number of related indoloquinones of the general formula^:-

These compounds were found to have antibacterial activity, but none show antitumour activity below the toxic dosis. However the degree of variation in the structure of indoloquinones produced was limited by the method of synthesis used.

The mitomycin antibiotics belong together with the aπthracyclines ⁴ , the aziridiπyl quinoπes^ , streptoπigriπ ⁶ , saframycin^ and mitoxaπthrone^ to the quiπoid antitumour compounds^ , which require reduction to exert their main cytotoxic effects. The following two mechanisms of action may be involved:

A. Redox cycling ¹ ^

B. Bioreductive alkylation ^{1 1} .

A. Redox cycling (Scheme I A). Quiπoid cytostatic agents are transformed by an enzymatic one-electron reduction into their corresponding semiquinone radical anions. Because of its ubiquitous existaπce in many cells and cell compartments NADPH-cytochrome P-450 reductase is regarded as the major enzyme catalyzing these reactions. Depending on the redox potential of the semiquinone radical anioπ, a reaction with oxygen may occur. The superoxide anion formed can undergo a variety of reactions. It spontaneously dis utates to hydrogen peroxide and singlet oxygen ( ¹ θ2).

Ground state dioxygeπ is formed when superoxide dismutase is involved in this step. Superoxide anioπ radicals together with hydrogen peroxide molecules give rise to the formation of cytotoxic hydroxyl radicals in the metal-catalyzed Haber-Weiss cycle.

Scheme I A

The hydroxyl radical, the most reactive oxygen metabolite, is sugested to be responsible for some of the serious damages occurring during redox cycling processes, e.g. lipid peroxidation, enzyme inactivatioπ and DNA cleavage. Further as can be derived from scheme I A, NADPH depletion could also be a critical event. Tumour cells are deficient in enzymes that normally protect the cell against free radical damage and this may explain part of the selective cytotoxicity of the antitumour quiπones^. χ _πe protective mechanism embraces a combined action of either superoxide dismutase and catalase or superoxide dismutase and glutathion peroxidase. ^•

B. Bio reductive alkylation

Several reduced quinoid cytostatic agents are not stable, but decompose into reactive intermediates which can undergo πucleophilic addition reactions with several biologically important nucleophiles.

The concept of "bioreductive alkylation", which applies to the mechanism of action of these antitumour compounds, has been formulated by Lin et al. in 19721 ^{1 a} , on studying the biological activity of derivatives of methyl substituted beπzoquiπoπes and πaphthoquinoπes. The bioreductive activation of quinone containg cytostatic agents in essence comprises the reduction of quinone (& to its dihydroform (fl after which HX is expelled and the α-methyleπe carbαnyl structure (£) generated. The latter form supposedly acts as a Michael acceptor and binds the nucleophiie (Scheme I B).

_5_ _

Scheme 1 B

The efficiency of such compouds can be enhanced by the introduction of additional methyl substituents bearing a leaving group. Upon reductions two or more unsaturated moieties are formed, creating possibilities for the crossliπkiπg of DNA molecules (Scheme ! C). In this way the anti¬ tumour activity of the quiπoid compound will be reinforced.

Scheme 1 C

Bioreductive aikylatiπg indoloquinones have become of increasing importance, since they might act as selective chemotherapeutics for

hypoxic cells ^' ' . These malignant cells, which form part of the slowly growing solid tumours, provide a more efficient reducing environment then do oxygen rich tumour or normal cells. So the reductive activation of the quinone-hydroquiπoπe type may proceed more effectively. The concept of bioreductive alkylation is of potential major significance as a mechanism of action of the mitomycins, anthracycϋπes and aziridiπyl quinones. They are reductively activated by the uptake of both one as well as two electrons.

The molecular mechanism of action of the mitomycin (1) antibiotics has been studied extensively the last five years ^{'1 4} . The reactive intermediates (10.-12.) which arise upon reductive activation are given in Fig. I ^{1 5} - ^{1 6} .

Hi tomyαne

Mitomycin*

Portoramye

Fig.

It has been well established, that mitomycin C (MMC), the prototype of this class of anticancer drugs, either upon chemical or enzymatical reduction, may bind via its C-1 atom covalently to suitable nucleophiles (e.g DNA or DNA) ^{1 5} . It has also been observed, that MMC acts as a bisalkylatiπg agent via its C-1 and C-10 carbon atom ¹ ^. On interaction with DNA crossliπked adducts are formed in this manner. Whether mςno- or bis-alkylation • occurs is strongly dependent on the reducing

/

conditions, the nature of the nucleophiles and the environmental pH. Crossliπking of DNA with protein by reductively activated Mitomycin C has been reported ^{1 7} . Recent studies by Dorr et al. ^{1 8} conducted in mamaiiaπ tumour cell cultures, indicated that mitomycin C caused rapid DNA instrand crosslinks in a dose dependent fashion. Good correlations between DNA crosslinks and cytotox ^' icity have also been reported ^{1 8} ' ^{1 9} . The chemical structures of monofuπctioπally linked adducts obtained upon catalytically hydrogeπation of MMC in the presence of calf thymus DNA were reported by Hashimoto et al. ^15c * _' ^e . Three modified nucleophiles were obtained: C-1 alkylation products of MMC with the 0-6 and N-2 positions of guanine, and the N-6 position on adeπiπe. Pan et al. ²⁰ re¬ ported the alkylation of DNA after anaerobic reductive activation of mitomycin C by NADPH cytochrome P-450 reductase and xanthiπe oxϊdase. The preferential site for monofuπctional binding was found to be the 0-6 position of guanine. This in contrast to the results obtained during catalytically hydrogeπation, showing the influence of the reduction conditions on the site of alkylation of DNA.

The first steps in the molecular activation sequence of MMC comprise its conversion into hydroquiπoπe £ ^{1 1 c} > ²¹ . The latter intermediate is stiucturaiiy related to intermediates arising from the mitoseπes ²² after reductive activation. The driving force for the activation of the C-1 position in preference to the C-10 position in MMC has to be ascribed to the opening of the aziridine ring thereby releasing the strain energy during the formation of quinoπ methide UL Activation of the second electrophilic center (C-10) may take place via one of the following two ways: i. Conversion of the monoquinone methide 10 into bisquinone methide 11 via elimination of the elements of HOCONH£. ii. Nucleophilic trapping of quinone methide 10. and elimination of HOCON H2 from the resulting adduct affording iminium derivative 12,

which may act as both an electrophilic or a nucleophilic trap. Horπemaπn c.s. ^{1 6 a} and recently Kohn as. ²⁸ have presented evidence favouring the imiπium pathway. Finally the dual reactivity of quinone methide 10 has also unequivocally been established ^{1 8a,} 9- ²⁴ . Very recently, M.R. Bachur et al. have demonstrated, using electrochemical techniques, that a one-electron reduction of the drug suffices to activate it ²⁵ . Previously this group ²⁶ and also others ²⁷ have presented unambiguous E.S.R. evi¬ dence for the eπzymeticaliy generated semiquinone metabolite of Mitomycine C. Bachur et al. ²⁶ proposed that mitomycine C undergoes primary molecular activation in the cell under anaerobic conditions via flavoenzyme single electron transfer to the quinone nucleus. The resulting radical anion produces moπofunct -tnal adducts with DNA and other nucleophiles, which can undergo secundary flavoenzyme activation leading to a new anaerobic free radical. Via this secundary activation, moπofunctional adducts may be converted into bifunctional adducts e.g. crossliπked DNA.

A different mechanism operating under aerobic conditions is the formation of reactive oxygen species produced by redox cycling. When Mitomycin C was incubated with supercoiled covalently closed circular (CCC) DNA and a reducing agent in the presence of oxygen it produced single strand breaks ²⁸ . Strand breaking of circular DNA by reduced mitomycin is oxygen-dependent and is inhibited by catalase, by superoxide dismutase and by free radical scavengers. The formation of hydroxyl radicals during reduction of mitomycin C by NADPH cytochrαme P-450 reductase and xanthine oxidase under aerobic conditions has been reported ²⁹ ' ⁸ 0, as well as the formation of both superoxide anion and hydroxyl radicals in tumour cells ⁸ 0. The lethal action of mitomycin C - in particular the toxicity - can be partly correlated to the generation of reactive oxygen species.

References:

1 6d. M. Bean and H.Kohπ, J.Org. Chem., [Q, 293 (1985).

17. R.E. Meyπ, S-F. Jenkins and L.H. Thompson, Cancer Res., 4f, 3510 (1 985).

18. R.T. Dorr, G.T. Bowden, D.S. Alberts and J.D. Liddϋ, Cancer Res., if, 3510 (1985).

1 9. K.A. Kennedy, J.D. Gurl, L. Leondaris and D. Alabaster, Cancer Res., JL 3541 (1985).

20. S.S. Pan, T. Iracki and N.R. Bachur, Mol. Pharmac, ££, 622 (1986). 21 a. S.J. Danishefsky and M. Ciufolini, J. Amer. Chem. Soc, 1 06. 6424 (1984).

21 b. S.J. Danishefsky and M. Egbertsen, J.Amer. Chem. Soc, 1 08. 4648

(1 986). 22a. W.S. Taylor, G. Leadbetter, D.L Fost and W \. Remers, J.Med. Chem., 20 , 138 (1977). 22b. J.C. Hodges and W.A. Remers, J. Med. Chem., 21, 1184 (1981 ). 22c. M.L. Casπer, W.A. Remers and W.T. Bradπer, J. Med. Chem., 2JL 921 ( 1 985).

23. N. Zeiπ and H. Kohπ, J. Amer. Chem. Sod 08. 296 (1986).

24. H. Kohn and N. Zein, J. Amer. Chem. Soc, 105., 4105 (1983). 25. P.A. Andrews, S.S. Pan and N.R. Bachur, J \mer. Chem. Soc, 108. 4158 (1986).

26. S.S. Pan, P.A. Andrews and C.J. Glover, J. Biol. Chem., 25JL (2), 959 (1 984).

27. B. Kalyaπaraman, E. Pere -Reyes and P.R. Mason, Biochim. Biophys. Acta, £3JL 1 19 (1980).

28. J.W. Lown, Mol. Cellular biochem., 5JL 17 (1983).

29. T. Komigama, T. Kikuchi and Y. Suyiera, Biochem. Pharmac, 3J. (22), 3651 (1982).

30. CA. Pritsos and A.C. Sartorelli, Cancer Res., 6_, 3528 (1986).

For the synthesis of mito mycin analog ues having improved antibacterial and cytostatic activity, the present Applicants have been concerned with the following requirements:

(i) The presence of a quinone ring . is.. essential, and by varying the substitueπts of the beπzoquinoπe ring, the reduction potential of the molecule and consequently the selectivity of the bioreductive alkylatiπg cytostatic agent can be directed.

(ii) The analogue has to contain one or two leaving groups attached either to C-1 and/or C-10, or to carbon atoms in vinylogous positions to

C-1 or C-10. (iii) The lipophilicity of the molecule as a whole can be controlled by the introduction of additional substituents.

The present Applicants have developed synthesis methods which can be used to prepare indoloquinones of more varied strucure fulfilling these requirements, and it is an object of the present invention to provide indoloquinones which have improved antibacterial and cytostatic activities at non-toxic dosages.

According to a first aspect of the invention, there are provided indoloquinoπe compounds of the general formula:-

where R2 and R3 are in each case, hydrogen, halogen, an alkyl group (which may be substituted), an alkoxy group or an aryloxy group, an alkylthio group or an arylthio group, an primary or secondary amino group, an hydroxy or an amino group;

R5 is hydrogen, an hydroxy group, an alkoxy group, an alkyl group (which may be substituted), or a carbohydrate moiety; Rg and R7 are in each case hydrogen, halogen, or an alkyl group; R ₈ is a group -CH2X1 , a group -Cθ2" ⁺ , where M ⁺ is a metal ion; a group -Cθ£R 0 _' where R-j g is hydrogen or an alkyl group (which may be substituted) ;a group -CONR'R ", where R ^* and R" are hydrogen or alkyl groups (which may be substituted);

Rg is a group -CR-j -j R^^' ^a Qroup -Cθ2 ^" M ⁺ , where M ⁺ is a metal ion; a group CO2 3, where R-j -j , R-j ⁿ R13 are in each case hydrogen or an alkyl group (which may be substituted); a group -CONR R , where R and R" are in each case hydrogen or an alkyl group (which may be substituted) ;and

Xl and X2 (when present) are hydrogen, or groups selected from -OH, -OR, -OC=OR, -OC0 ₂ R, -OC-ONRR, -SH, SR, -SC-OR, -SC=SR, -SC0 ₂ R, -SC=SOR, -SC=0NRR, -SC-SNRR, and-NRR [where R is hydrogen, an alkyl group (which may be substituted), or an aryl group (which may be substituted)], -OSOR, -OS0 ₂ R and OP(OR) ₂

[where R is hydrogen, an alkyl group (which may be substituted), an aryl group (which may be substituted), or a carbohydrate moiety]; and X- _] and X2 may be the same or different.

According to a further aspect of the invention preferred bio¬ reductive alkylating indoloquiπoπe compounds are compounds of the general formuia:-

where R2 is a methoxy group -OCH3, or a primary or secondary amino group;

R ₃ is hydrogen or a methyl group; R ₅ is a methyl group or a butyl group. X-] and X2 are selected from hydrogen, -OH, -OAc, -OCOOCH3, -CONH _2ι -OCONHCH ₂ CH ₂ Cl, -OCOC ₆ H ₅ , -OCOC ₄ H ₉ , -NHC ₆ H ₅ , and -OCH3 and may be the same or. different. Particularly preferred bioreductive alkylating indoloquinoπe compounds according to the invention are:-

(1 ) 3-Acetoxymethyl-5-methoxy-1 -methyl-2-[1 ^L i-indole-4,7-dione]- prop-β-en-α-yl acetate (Compound E.O. 1)

(1 A) 3-Hydroxymethyl-5-methoxy-1-methyl-2-[1H.-indole-4,7-dione] prop-β-en-α-yl acetate (Compound E.O. 1A)

(1B) 3-Acetoxymethyl-5-methoxy-1-methyl-2-[1H_-indoIe-4,7-dione]- prop-β-en-α-ol (Compound E.O. 1B)

(2) Methyl 5-methoxy-3-methoxycarboπyIoxymethyl-1-methyl-2-

[1H-iπdole-4,7 dioπe] prop-β-en-α-yl carbonate (Compound E.O. 2)

(3) 3-Carbamoyloxymethyl-5-methoxy-1 -methyI-2-[1 i±-iπdoIe-4,7- dioπe]prop-β-en-α-yl carbamate (Compound E.O. 3)

EO.3

(4) 3-Acetoxymethyl-5-aziridino-1 -methyI-2-[1i±-iπdole-4,7- dione] prop-β-eπ-α-yl acetate (Compound E.O. 4)

(4A) 3-Acetoxymethyl-5-aziridiπo-1 -methyl-2-[1 H.-iπdole-4,7- dione] prop-β-en-α-ol (Compound E.O. 4A)

(5) 3-Ac ₈ toxymethyl-5-(2-hydroxyethyl-1-amino)-1 -methyl-2- [1H-iπdole-4,7-dione] prop-β-en-α-yl acetate (Compound E,0. 5)

(6) 3-Acetcxymethyl-5-(2,3-dihydroxypropyl-1 -amiπo)-1 -methyl- 2-[1 ϋ-indole-4,7-dione] prop-β-eπ-α-yl acetate (Compound E.O. 6)

(7) 3-HydroxymethyI-5-methoxy-1 -methy!-2-[1 i±-indole-4,7-dioπe] prop-β-en-α-ol (Compound E.O. 7)

(8) 3-Hydroxymethyl-1 -methyl-5-propyleneamiπo- 2-[1 i±-indole-4,7- dioπe] prop-β-en-α-ol (Compound E.O. 8)

(9) 5-Aziridino-3-hydroxymethyl-1 -methyl-2-[1 H.-indole-4,7- dione]prop-β-en-α-ol (Compound E.O. 9)

(10) 5-Methoxy-3-methoxymethyl-1 -methyl-2-[1 hL-indole-4,7- dioπe]prop-β-eπ-α-yl N-[2-chloro-1-ethyl]carbamate (Compound

(11 ) 3-Hydroxymethyl-5-(4-hydroxypiperidino)-1 -methyl-2-[1 H.-indole -4,7-dioπe]prop-β-eπ-α-ol (Compound E.O-. 11 )

(12) 3-Hydroxymethyl-1 -methyl-5-morpholino-2-[1]i-indoIe- 4,7- dione] prop-β-en-α-ol (Compound E.O. 12)

(13) 3-(N-chloroethyIcarbamoyloxymethyI)-5-methoxy-1 -methyl-

2-[1ϋ-iπdole-4,7-dione] prop-β-en-α-yl N-chloroethyl carbamate (Compound E.O. 13)

(14) 3-Hydroxymethyl-5-pheπylamino-1 -methyl-2-[1 H.-indole- 4,7-dioπe] prop-β-en-α-ol (Compound E.O. 15)

(15) 3-Hydroxymethyl-5-methoxy-1 -butyl-2-[1 H-indole-4 ₎ 7-dione] prop-β-en-α-ol (Compound E.O. 16)

(16) 5-Aziridino-3-hydroxymethyl-1 -butyl-2-[1 H-indole-4,7-dione] prop-β-en-α-ol (Compound E.O. 17)

(17) 1 ,6-Dimethyl-5-hydroxymethyI-5-methoxy-2-[1 ]±-indoIe- 4,7-dione]prop-β-en-α-ol (Compound E.O. 18)

(18) 5-Aziridino-1 ,6-dimethyl-3-hydroxymethyl-2-[1 ]±-indole- 4,7-dione]prop-β-en-α-ol (Compound E.O. 19)

(19) 3-Acetoxymethyl-1 ,6-dimethyl-5-methoxy-2-[1 H_-indole- 4,7-dione]prop-β-en-α-yl acetate (E.O. 33)

(20) 3-Acetoxymethyl-5-aziridino-1 ,6-dimethyI-2-[1 j±-indo!e -4,7-dione]prop-β-en-α-yl acetate (E.O. 35)

(21 ) 3-Benzoxymethyl-5-methoxy-1 -methyl-2-[1K-iπdole-4,7-dione] prop-β-en-α-yl benzoate (Compound E.O. 36)

H ₅

(22) 3-[N-butyIcarbamoy!oxymethy!]-5-methoxy-1 -methyl-2-[1 Hb indole-4,7-dione]prop-β-en-α-yI N-butylcarbamate

(Compound E.O. 37)

(23) 5-Methoxy-1 -methyi-3-[N-phenylcarbamoyloxymethyl]-2-[1 H.- indoIe-4,7-dioπe]prop-β-en-α-yI N-phenylcarbamate (Com¬ pound E.O. 38)

(24) 5-Methoxy-1 -methyl-3-(N-phenyIaminomethyI)-2-[1Jl-indoIe- 4,7-dione]prpp-β-en-α-yl acetate (Compound E.O. 39)

(25) 5-Methoxy-1 ,3-dimethy!-2-[1 E.-indo!e-4,7-dione]prop-β-en-α- acetate (Compound E.O. 41)

(26) 3-Acetoxymethyi-5-[2-(N,N-dimethylamiπo)ethyl-1 -amino]-1 - methyl-2-[1 i±-indole-4,7-dione]prop-β-en-α-yl acetate (Compound E.O. 47)

(27) 3-AcetoxymethyI-5-[2-(N,N-dimethylamino)ethyl-1 -amino]-1 - methyi-2-[1R-indole-4,7-dione]prop-β-en-α-ol (Compound E.O. 48).

(28) 3-Hyd roxy methyl- 1 -methyl-5-[2-pyridylethy 1-1 -ami no]- 2-[1H-indole-4,7-dione]prop-β-en-α-ol (Compound E.O. 51)

(29) 3-Acetoxymethyl-1 -methyl-5-[2-pyridylethyl-1 -amino]-2-[1l±- iπdole-4,7-dione]prop-β-en-α-yl acetate (Compound E.O. 52)

(30) 3-AcetoxymethyI-1 -methyl-5-propyleneamino-2-[1 H_-iπdoIe-4, dione]prop-β-eπ-α-yl acetate (Compound E.O. 53)

(31 ) 5-Ethyla ino-3-hydroxymethyI-1 -methyl-2-[1 H.-iπdoIe-4,7- dioπe]prop-β-eπ-α-ol (Compound E.O. 56)

C ₂

(32) 3-Acetoxymethyl-5-ethylamino-1 -methyI-2-[1 j±-indoie-4,7- dione]prop-β-en-α-yl acetate (Compound E.0.58)

(33) 3-Acetoxymethyl-5-ethy!amino-1 -methyl-2-[1 J±-indole-4,7- dione]prop-β-en-α-ol (Compound E.0.59)

(34) 3-AcetoxymethyI-1 -methyl-5-morphoIino-2-[1 E-iπdole-4,7- dione]prop-β-eπ-α-yl acetate (Compound E.O.60)

(35) 3-Acetoxymethyl-1 -methyl-5-[2-pyridylethy!-1 -amino]-2-[1Ji- indole-4,7-dioπe]prop-β-eπ-α-ol (Compound E.O.62)

(36) O-ethyl 5-methoxy-1 -methyl-2-[1 j±-indole-4,7-dioπe]prop-β-en- α-yl dithiocarbamate (Compound E.O.64)

Of the preferred compounds, it has been found that the compounds. E.0.1 to E.O. 6 and E.O. 8 to E.O. 15 can all be prepared from the com¬ pound E.0.7 in simple ways. Thus the compounds E.0.1 to E.O. 3, E.O. 10 and E.O. 13 can be directly prepared from E.O. 7 by replacing the hydroxy groups X- _| and X2 by the required functional groups, which have good "leaving group" properties. The compounds E.O. 8, E.O. 9, E.O. 11 c d - E.O. 12 can be prepared directly from E.O. 7 by treatment with an excess of the appropriate secondary amine. Finally the compounds E.O. 4, E.O. 5 and E.O. 6 can be prepared by treating the compound E.O. 1 (prepared from E.O. 7 by treatment with acetic anhydride) with an excess of the required primary or secondary amine.

These syntheses, with an indication of the yields obtained for the various compounds, are set out in reaction scheme II, and are illustrated in Examples 2 to 13 below.

The synthesis of compounds E.O. 36 to E.O. 38, E.O. 47, E.O. 48, E.O. 51 to E.O. 53, E.O. 56, E.O. 58, E.O. 59, E.O. 60 and E.O. 62 from either E.O. 1 or E.O. 7 proceeded analogously to the synthesis of the compounds described above. Compounds E.O. 39, E.O. 41 and E.O. 64 have been obtained from indoloquinone E.O. 1 under reductive alkylation conditions, and are illustrated in examples 14 to 16 below.

Indoloquinones E.O. 33 and E.O. 35 have been ultimately obtained from compound E.O. 18.

Selective hydrolysis of one of the acetate groups in the indoloquinones E.O. 1 and E.O. 4 yielded compounds E.O. 1A and E.O. 1 B, and E.O. 4A respectively.

HNRJRJ/ROH

B l Hold

E.0.1 CΛc OΛc 6O-

Yield (96) (E.O.1)

^R 2 HNR _j R ₂ /ROH

E.O.8 70-75

SCHEME π

Yield ⁽ .%)

E.o 85-90 ^•

»<

E.O.5 NHCU ₂ CH ₂ OH 70

E.O.6 ffllCH-CHOHCH ₂ 0H 60-65

The compound E.O.7 can itself be prepared starting from -chlαrαpheπol in accordance with the following reaction scheme :-

Scheme III Contd.

KOH C2HSOH

H _? 0

DDQ / PhCH _j

Scheme III Contd.

The various steps in the synthesis of compound E.O.7 in accordance with this reaction are described in detail in the following Example : Example 1 Synthesis of Compound E.O.7 ( 2J3 )

3-Chloro-4-nitroαheno 1 C∑) a) Nitrαsation of -chlαrαphenαl (I)

The nitrosation of m-chlorophenol (I) was carried out fallowing the method given by Hodgson et al (J. Chem. Soc. 1940, 1270) for the nitrαsation of m-f luorophenαl .

A solution of 265.5 g NaNO- in cone. H ₂ S0 ₄ (2.54 1) was prepared. The addition of NaNO- to cone. H-SO. took place, while stirring and cooling at +5 C. After the addition of crushed ice (1.2 kg) to this solution at a temperature of +5 C m--chlorophenol (227.5 g; 1.77 mole) dissolved in pyridine (400 ml) was introduced. Thereupon the reaction mixture was poured into ice water. The crystals were collected by filtration, washed with water and dried in the air. b) Oxydation of 3-chlαro-4-nitrosophenol (2)

The oxydation of 3-chloro-4-πitrosophenol (2.) to 3- chloro-4-nitrophenol ( ) proceeded as described by Hodgson et al (J. Chem. Soc. 1925, 1579). To a stirred solution of 1.65 kg K ₃ Fe( N) ₆ in 5%

KOH aq. (22 1) was added the crude 3-chlαrα-4-nitrαso- phehol. Stirring was continued for seven days at room temperature. Thereupon the reaction mixture was

/

acidified dropwise with sulphuric acid and extracted with ether. The ethereal extracts were washed with sat. NaCl aq. and dried with MgSO.. Evaporation of the solvent in vacuo yielded 199.5 g (65?ά) of crude 3- chloro-4-nitrophenσl (solid) which was sufficiently pure far the conversion into 3-chloro-4-nitraanisale M.p. 121-122 ^α C. 3-chlαr α-4-nitrαanisαle (4)

In an atmosphere of N_ were added to a stirred suspension of 31 g NaH in 2.2 1 anhydrous THF 199.5 g (1.15 mole) 3- chlαrα-4-nitrophenαl (3_) . This was followed by the addition of dimethy Isulf ate (575 ml). The whole mixture was heated under reflux- during 1.5 h. After cooling, it was poured into a cold (Q°C) diluted NH^QH solution, to destroy the- excess of d ime thylsul fate , and stirred for two hours. The product was isolated by extraction with diethylether . The ethereal extracts were dried with MgSO^ and evaporated at reduced pressure affording 156 g (72.4?«) of 3-chloro-4-nitroanisαle. ¹ H NMR δ(CDCl ₃ ); 8.07 (d, 1H , J = 9.5 Hz, H-5), 7.05 (d, 1H , J=2.5 Hz, H-2), 6.99 (d.d, 1H , J = 9.5 Hz, J=2.5 Hz, H-6), 3.92 (s, 3H, 0CH ₃ ). Ethyl 5-methoxy-2-nitrophenylcyanoacetate (5_)

To a suspension of 44.2 g NaH in DMF (870 ml) was added a solution of ethylcyanoacetate (197.7 g) in anhydrous DMF (162 ml). After the addition the reaction mixture was stirred for 10 minutes. Subsequently 156 g (0.83 mole) 3-chloro-4- nitroanisαle were added. The resulting dark solution was

heated at 50-55 ^α C for 16 h. After cooling, the reaction mixture was poured into cold (0°C) 5% XOH aq.

The solution thus obtained was washed with ether (3x).

Thereupon the solution was acidified with cone. 3 ^Q ?ϊ HCI aq., while cooling by adding crushed ice. During the acidification the colour of the mixture turned from dark red to pale yellow. From the cσllodial solution the product was isolated via an extraction with 1,1,1-tri- chlαrαethane (5x). The- combined organic layers were dried with MgSQ . After evaporation of the solvent, a red oily residue was obtained, which was employed in the next step without further purification.

Yield: 218.5 g (99.5?.: contained only some DMF).

Purification of a sample by flash column chrαmatography using silicagel and CH _? C1 ₂ as eluent afforded a pale yellow oil (yield: 95-100?ά) which crystallised on standing in the refrigerator.

M.p. 44-47°C. IfUCHCl,): 2220 (CΞN) , 1740 (ester C=0) ,

1580 (N0 ₂ )

¹ H NMR δ(CDCl ₃ ): 8.28 (d, IH , J=9 Hz, H-3) , 7.27 (d, IH, J=2.5 Hz, H-6), 7.04 (d.d, IH , J=2.5 Hz and J=9 Hz, H-4) , 5.68 (s, IH, CHCNC0 ₂ C ₂ H ₅ ) , 4.33 (q, 2H , J=7 Hz C0 ₂ CH_ ₂ CH ₃ ), 3.98 (s, 3H, 0CH ₃ ), 1.33 (t, 3H, J=7 Hz C0 ₂ CH ₂ CH ₃ ). An exact mass determination gave 264.0759; ^c i ^H i2 ^N 2°5 ^reα . ^uiι:es 264.0772 (4.9).

Diethyl 5-methoκy-2-nitrophenylmalonate ( 6 )

A solution of 85 g (0.32 mole) ethyl 5-methαxy-2-nitro- phenylcyaπαacetate (5) in a mixture of ethanαl (510 ml)

and was saturated with HCI gas on cooling in ice. The reaction mixture was stirred during two days at room temperature. Thereupon ice water (500 ml) was added and stirring was continued for 24 hrs at room temperature. The crystals, which had been separated were collected by filtration. Recrystallization from aqueous ethanol afforded 85 g (85,4%) of 6 (white crystals).

M.p. 92-93°C. IR(CHC1 ₃ ): 1725 (ester C=0) , 1580 (N0 ₂ ) ♦

^■ " ^■ H NMR δ(CDCl ₃ ): 8.23 (d, IH , 3=10 Hz, H-3), 6.9-7.05 ( , 2H, H-4 and H-6), 5.44 (s, IH, CH. CO^H^, 4.30 (q,

4H, 3=7 Hz, C0 ₂ CH ₂ CH ₃ ), 3.92 (s, 3H , 0CH ₃ ), 1.31 (t, 3H ,

3=7 Hz, C0 ₂ CH ₂ CH_ ₃ ) .

An exact mass determination gave 311.1004; C, H ₁₇ NQ_ requires 311.1004 (0.0). Methyl 3.3-diethoxycarbonyl-2,3-dihydro-5-me hoxy-2- indαleacrylate ( 9_)

a) Catalytic reduction of ( §_) : diethyl 2-amino-5- methox-yphenylmalonate (7_)

Diethyl 5-methαxy-2-nitrophenylmalonate (_6) (20 g, 64.3 mmαl) was reduced with H- a atmospheric pressure using a mixture of toluene (250 ml) and abs. ethanol (15 ml) as solvent and Pt0 ₂ (300 g) as catalyst. After the theoretical amount of hydrogen

was consumed the reaction mixture was filtered through high flow. The filtrate was concentrated in vacua at a bath temperature beneath 30 C. The resulting pale green oily mixture was, because of its instability, employed in the next step without further purification.

IR(CHC1 ₃ ): 3440 and 3350 (NH), 1730 (ester C=0). ¹ H NMR <S(CDC1 ₃ ): 6.7-6.9 (m, 3H , aromatic H's),

4.69 (s, IH, CHC0 ₂ C ₂ H ₅ ), 4.27 (q, 4H , 3=7 Hz, C0 ₂ CH_ ₂ CH ₃ ), 3.77 (s, 3H , 0CH ₃ ), 3.5-4.0 (br s, 2H , NH ₂ ), 1.30 (t, 3H, 3=7 Hz, C0 ₂ CH ₂ CH ₃ ). b) Condensation of die hy1-2-amino-5-me hoxyphenyl- malαnate (J_) with methyl 3-farmylacrylate

The crude diethyl 2-amino-5-me thoxyphenyImalαnate (7_) was dissolved in ethaπol (350 ml). To this solution was added at room temperature methyl 3- formylacrylate (see, Bαhlmanπ et al, Ber. 8_9 (1956) 1276) (7.33 g) dissolved in methanαl (50 ml). The green reaction mixture was stirred for 15 minutes. Intermezzo:

For the determination of the spectral properties of the imine B_, the condensation of diethyl 2-amino-5- methoxyphenylmalonats with methyl 3-formylacrylate was carried out using toluene as solvent. After evaporation of the solvent IR and H NMR spectra of the residue were recorded.

IR (CHC1 ₃ ): 1730 (ester C=0). ¹ H NMR S(CDC1 ₃ ): 8.23 (d, IH, J=9 Hz, N=CH), 7.47 (dd, IH, J=16 Hz and

3=9 Hz, CiH=CHC0 ₇ f1e) , 6.7-7.3 ( , 2H , aromatic H's), 6.41 (d, IH, 3=16 Hz, CHsCHCQ^le) , 5.51 [s , IH , CΕ(C0 ₂ C H ₅ )J , 4.24 (q, 4H, 3=7 Hz, OCh^CH..), 3.84 (s, 3H, OCH ) , 1.27 (t, 6H, 3=7 Hz, OCH^HL.). c) The 1,5-electrαcyclisatiαn reaction

Cyclisation of the i iπe 8_ tα methyl 3, 3-diethoxy- carbonyl-2,3-dihydro-5-me hoxy-2-indoleacryla e (9_) was attained by addition of Zπ(0Ac) ₂ , 2 H ₂ 0 (4.5 g) to the ethanαlic solution of the imine. Stirring was continued for one hour. Thereupon the solvent was removed in vacua. Ta the residue were added: 2N HCI (300 ml) and CH ₇ C1 ₂ (100 ml). The organic layer was separated and the aqueous phase was extracted with CH _? C1 ₇ . The combined organic layers were washed subsequently with 2N HCI, sat. NaHCQ-, aq. and sat. NaCl aq. and dried with MgSQ.. Evaporation of the solvent gave 2 . (98.6% ) of a reddish oil which was employed in the next step without further purification. Purifying of a sample of the residue by flash column chromatography (Si0 ₂ / CH ₂ C1 ₇ / acetone 95/5) afforded a pale yellow oil. IR(CHC1 ₃ ): 3375 (NH, w), 1730 (ester C=Q). ¹ H NMR δ(CDCl ₃ ): 6.6-7.15 ( , 4H, CH^CHCO^e and aromatic H's), 6.17 (dd, IH, 3=15.5 Hz and 3=1 Hz, CH=CHC0 ₂ Me), 5.15 (dd, IH, 3=6.5 Hz and 3=1 Hz, N-CH.) , 4-4.5 (m, 4H, 0CH ₂ ), 3.78 (s, 3H) and 3.73 (s, 3H) (0CH.J and C0 ₂ CH ₃ ), 3.44 (br s, IH, NH_) , 1.31 (t, 3H) and 1.20 (t, 3H) (3=7 Hz, 0CH ₂ CH ₃ ).

An exact mass de er ination gave 377.1452;

^C 19 ^H 23 ^N 1°7 ^rec I ^uires 377.1430 (5.8). Me hyl ιM-ace yl-3.3-die hoxycarbonyl-2 _f -dihvdro-5-methoxy-

2-indoleacrylate ( 10)

The crude indoline 9. ^was dissolved in acetic anhydride (35 ml). This solution was stirred far 1 h at room temperature. Thereupon the acetic anhydride was removed in vacua. The residue (~27 g) was sufficiently pure for further conversions. A sample of this residue was purified by flash column chromatography (SiQ ₇ , CH ₇ C1 ₇ / acetone 95/5). A pale yellow oily product was obtained. IR(CHC1 ₃ ): 1730 (ester C=0), 1655 (N-C=0). ¹ H NMR δ(CDCl ₃ _): 8.07 (br d, IH, 3=7.5 Hz, H-7), 7.13 (d, IH , 3=2.5 Hz, H-4), 6.9-2 (dd, IH , 3=2.5 Hz, 3=7.5 Hz, H-6) , 6.75 (dd, IH, 3=15.5 Hz and 3=7 Hz, CHsCHCO-Mβ) , 6.03 (dd, IH , 3=15.5 Hz and 3=1 Hz, CH=CHC0 ₂ Me) , 5.68 (br d, IH , 3=7 Hz N-C l) , 4-4.5 (m, 4H, OCH^CH--), 3.83 (s, 3H) and 3.72 (s, 3H) (0CH ₃ and CO^H- _j ), 2.25 (s, 3H, NCOC ^), 1.30 (s, 3H) and 1.23 (s, 3H) (C0 ₂ CH ₂ CI± ₃ ) . N-acetyl-3-carboxy-2.3-dihvdro-5-me hoxy-2-indoleacrylic acid (11)

The crude N-acetylindole (KO was dissolved in ethanol (270 ml). On cooling in ice a cold (0 C) solution of KOH (21.6 g) in water (180 ml) was added. Stirring and cooling was continued for 18 h. The reaction mixture was poured into ice water. The resulting solution was extracted with ether (3x) and acidified with 2N HCI.

The indoline acid (_H) was isolated from the aqueous solution by extraction with CHC1, (6x). The combined organic layers were dried over MgSO^. Evaporation of the vαlatiles afforded 18.8 g αf a foam (96?. based on 6).

IR(KBr): 2700-3600 (carboxylic acid OH), 1710 (carboxylic acid C=0). ¹ H NMR (acetone-d _≤ ) : 8.1 (br s, IH, H-7) , 6.8-7.2 ( , 3H, CH^CHCO^e, and H-4, H-6), 6.5-7.5 (br s, 2H, carboxylic acid 0H_) , 5.95 (d.d, IH, 3=15.5 Hz and 3=1.5 Hz, CH=CHC0 ^" ₂ Me), 5.6 (br s, IH, H-2), 4.07 (br s, IH, H-3), 3.81 (s, 3H, 0CH ₃ ), 2.25 (br s, 3H, C0CH. ₃ ) . Methyl N-acetyl-2.3-dihvdro-5-methoxy-3-metho ycarbonyl- 2-indoleacrylate (12)

To a solution of indoline acid (1 (18.8 g) in anhydrous DMF (250 ml) were added subsequently K-CO.- (19.6 g) and dimethylsulfate (52 ml). This mixture was stirred for 4 h at roam temperature. Thereupon it was poured into an excess αf 2N HCI. The indoline ester derivative (12) was isolated by an extraction with 1,1,1-trichlorαethane (4x). The organic extracts were washed with sat. NaCl aq. and dried over MgSO,. After evaporation of the solvent a reddish oil (-20 g: contained some DMF) was obtained, which could be employed in the next step without further purification. A sample of this residue was purified by flash column chromatography (SiO-, CH^Cl^/ cetone 95/5). IR(CHC1 ₃ ): 1730 (ester C=0), 1650 (N-Cs0). ¹ H NMR δ(CDCl ₃ ): 8.13 (br d, IH, 3=8 Hz, H-7) , 6.75-8.05 (m, 3H, H-4, H-6 and CH=CHC0 ₂ Me), 5.96 (dd, IH, 3=15.5 Hz,

CH=CHC0 ₂ Me) _f 5.45 (br d, IH , NCH) , 3.87 (br s, IH , CHC0 ₂ C ₂ H ₅ ), 3.81 (s, 3H) , 3.77 (s, 3H) and 3.73 (s, 3H) (OCH- and CO^H..), 2.24 (s, 3H , NCQCH_ ₃ ) . An exact mass de ermination gave 333.1240; C.-^H _ . ^Q ^ requires 333.1212 (8.4) .

Methyl 5-methαxy-3-methαxycarbαny1-2-iπdoleacrylate (14)

A solution of the crude indoline (1_2) in toluene was heated under reflux with 1.05 eq. of DDQ for 18 h. A greyish precipitate of DDQ H ₂ was farmed, which was removed by filtration. Thereupon the toluene was evaporated in vacuo. A very dark coloured residue containing methyl N-ace tyl-5-methαxy-3-methαxycarbαny1-2- iπdαleacrylate (J was obtained. Intermezzo: A sample αf this residue was submitted to flash column chromatography (Si0 ₂ , CH ₂ Cl ₂ /acε one 95/5) affording a reddish oil which has been charac erized by IR, H NMR and mass spectrometry . IR(CHC1 ₃ ): 1710 (ester C=0). ¹ H NMR δ(CDCl- _j ): 8.25 (d, IH, 3=16 Hz-, Cj±=CHC0 ₂ Me ) , 7.94 (d, IH, 3=9 Hz, H-7),

7.60 (d, IH, 3=2.5 Hz, H-4), 7.02 (dd, 3=9 Hz and 3=2.5 Hz, H-6), 6.22 (d, IH , 3=16 Hz, CH=CHC0 ₂ Me) , 4.98 (s, 3H), 4.91 (s, 3H) and 4.87 (s, 3H) (0CH ₃ and C0 ₂ CH ₃ ), 2.60 (s, 3 H, NC0CH ₃ ). An exact mass determination gave 331.1056; C i γ ^H i7 ^N ι°5 requires 333.1056 (0.0). Purifying this residue via column chromatography using

Al 0 ₃ (basic) and mixtures αf CH ₂ C1 ₂ and acetone (8/Z-> 5/5) as elueπts afforded 11.07 g αf the N-deace ylated indole derivative (_ _) as yellow crystals (59?ά based on 6). M.p. 2Q6-207°C (MeOH). IR(KBr): 3290 (NH, vs), 1690 (ester C=Q) .

¹ H NMR δ(DMSQ-d ₆ ): 12.32 (br s, IH, NH), 8.44 (d, IH, 3=16 Hz, CH=CHC0 ₂ Me), 7.51 (d, IH, 3=2.5 Hz H-4) , 7.42 (d, IH, 3=8.5 Hz, H-7), 6.99 (dd, IH, 3=8.5 Hz and 3=2.5, H-6), 6.78 (d, IH, ) , 3.93 (s, 3H), 3.84 (s, 3H) and 3.81 (s, 3H) (C0 ₂ Me and 0CH ₃ ).

An exact mass de ermination gave 289.0950; C,cH ,0, requires 289.0950 (0.0).

Methyl 5-methoxy-3-methoxycarbonyl-4-n itro-2-indoleacrylate

(il) Tα a solution of 9.2 g (31.8 mmol) of methyl 5-methoxy-3~ methcxycarbαπyl-2-indαleacrylate (.14) in acetic acid (123 ml), cooled in an ice/water bath, a cold (0 C) mixture of fuming nitric acid (16.5 ml) and acetic acid (64 ml) was added. The whole mixture was stirred subsequently for 2.5 h at room temperature. A yellow suspension was obtained, which was poured into ice/water. The crystals were collected by filtration washed with water and dried at 50-6Q°C in vacua. Yield: 9.34 g (88? ₀ '). M.p. 243-245°C (MeOH). IR(KBr): 3290 (indole NH), 1700 (ester C=Q). ^λ r NMR δ(DMS0-d ₆ ): 12.88 (br s, IH, NH), 8.21 (d, IH , 3 = 16.4 Hz, CH=CHC0 ₂ Me) , 7.70 (d, IH , 3 = 9.1 Hz, Ar H), _7#3g (d, IH, J=9.1 Hz, Ar H) , 6.06 (d, IH, J=16.4 Hz, CH=CHC0 ₂ Me), 3.93 (s, 3H), 3.79 (s, 3H), 3.73 (s, 3H).

An exact mass de ermination gave 334.0805: C. _< -H.,N 0 requires

Me hyl 5-mεthoxy-3-methoxycarbcnyl-N-me hy l-4-nitrα-2-indαleacry late (16)

This synthesis was carried out under an atmosphere αf dry „. Tα a stirred suspension of NaH (2.9 g) in dimethylformamide (145 ml) were added 9.34 g (28 mmol) me thy 1 5-methoxy-3-me hαxycarbαnyl-4-πitrα-2-indαle- acrylate (15_) . Thereupon the whale mixture was heated at 45-50 C. When the evolution of H_ had ceased methyliodide (25 ml) was added to the dark red solution. During an additional (1 h) heating of the reaction mixture at 60 C, the colour of the solution turned tα yellow. After cooling this mixture was poured into cαld (Q°C) 10?ι NaHSO^ aq. Yellow crystals separated, which were collected by filtration, washed with water and ethanol, and finally dried in vacuo at 50-60°C. Yield: 9.23 g (953). M.p. 211-213°C. IR(KBr) : 1700 (ester C = Q) . ¹ H NMR δ(DMS0-d _≤ ), 8.06 (d, IH, 3=16.5 Hz Cϋ=CHC0 ₂ Me ) , 7.96 (d, IH, 3=9 Hz, aromatic H), 7.43 (d, IH, 3=9 Hz, aromatic H), 6.68 (d, IH, 3=16.5 Hz CH=CHC0 ₂ Me), 3.93

(s, 6H), 3.83 (s, 3H) and 3.71 (s, 3H). An exact mass determination gave 348.0946; ^c ι≤ ^H ιg ^N 2 ^α 7 requires

348.0957 (3.2). Methyl 4-amino-5-methoxy-3-methoxycarbonyl-N-methyl-2-indoleacrylat e(17)

To a suspension of 9.23 g (26.5 mmol) methyl 5-methoxy-3- methoxycarbonyl-N-methyl-4-nitro-2-indoleacrylate ⁽ .16) in ethanol (745 ml) were added subsequently tin (14.9 g) and 3N HCI (200 ml). This mixture was heated under reflux for 30 minutes.

Thereupon the solution was decanted from the excess of tin and neutralized with sat. aq. NaHCQ,. The red suspension thus obtained was added tα an equal volume of water. The aqueous phase

5 was extracted with CHCl ₃ (5x). The combined organic

₌ — ira-yeTs ^" were washed with sat. aq. NaCl aq. (2x), dried over MgSO, and concentrated at reduced pressure. A red crystalline (7.94 g) residue was obtained which could be employed in the next reaction step without further purification. 10 M.p. 164.5-165.5°C (MeOH, red crystals).

IR(KBr): 3470 and 3350 (Nh^), 1710 (ester C=0). ¹ H NMR δ(DMS0-d ₆ ): 8.08 (d, IH , 3 = 16.5 Hz, CH = CHC0 ₂ He ) , 7.05 (d, IH, 3=8.8 Hz, Ar H), 6.75 (d, IH , 3=8.8 Hz, Ar H), 6.44 (d, IH, 3 = 16.5 Hz, CH = CHC0 ₂ Me ) , 5.83 (br s, 2H , N H_ ₂ ) , 15 3.85 (s, 3H), 3.81 (s, 3H), 3.80 (s, 3H) , 3.77 (s, 3H) . An exact mass determination gave 318.1227; C _j _5 ^H a' 2 ^CI 5 requires 318.1216 (3.5) . Methyl 5-methoχv-3-rπe hoxycarbonyl-N-methyl-4,7-dioxo-2- indαleacrylate (JLj3_)

20 To a solution αf 7.95 g (25 mmol) methyl 4-amino-5- me t hox y-3-me th ox y c ar on yl-N-me hy 1-2 -indole aery la e ( 17) in acetone (1.0 1) was added a solution of Fremy's salt (33.65 g) in a NaH ₂ P0 ₄ /Na ₂ HP0 ₄ buffer (l.o 1, 0.3 M; pH 6). The whole mixture was stirred at room temperature far 1 h.

25 The orange-brown crystals, which had been separated, were collected by filtration, washed with water and methanol, and dried in vacua at 50-60°C, affording 5.75 g of indoloquinαπe (18) . To obtain a second crop the filtrate was extracted with

CH.C1 ₂ (4 ). The combined organic layers were washed subsequently with sat. NaCl aq. and dried with MgSO.. After evaporation of the solvents a dark red residue was obtained, from which by flash column

5 chromatography (Si0 ₇ , CH Cl ₇ /acetone 95/5) a second crop of 1.1 g of indσlαquinαne (.18.) could be isolated.

Total yield: 6.85 g (82J5). M.p. 235-236°C

IR(KBr): 1715 (ester C=Q) , 1680 (quinone C=0) , 1600

( quinone C=0) . Q ¹ H NMR δ(DMS0-d _≤ ): 7.63 (d, IH , 3=16.5 Hz, CH_=CHC0 ₂ Me) , 6.38 (d, IH, 3=16.5 Hz, CHrCiHCO^le) , 5.96 (s, IH , H-6) , 4.05 (s, 3H), 3.84 (s, 3H), 3.81 (s, 3H) and 3.76 (s, 3H) An exact mass determination gave 333.0843; requires 333.0333 (1.5). Methyl 4.7-dihvdroχv-5-metho\v-3-me ho\vcarbonyl-N-me hyl-

2-indαleacrylate ( 19)

Methyl 5-methoxy-3-me oxycarbonyl-N-methyl-4, 7-dio o-2- indoleacrylate (1_8_) (6.64 g, 20 mmol) was dissolved in a mixture of chloroform (600 ml) and ethanol (215 ml). The reduction was carried out on stirring with an aqueous solution (260 ml) of Na _p S_0 (42 g) at room temperature for 30 minutes .

The organic layer was separated and washed with sat. NaCl aq., dried over MgSO and concentrated under reduced pressure. The residue (6.6 g; 99%) was sufficiently pure for the employment in the next reaction step.

IR(KBr) : 3200-3500 (OH) , 1720 (ester C=Q). ^l r\ NMR δ(DM30-d ₆ ): 10.60 (s, IH , OH) , 9.48 (br s, IH , OH) ,

8.04 (d, IH, 3 = 16.5 Hz, CH_=CHC0 ₂ Me ) , 6.53 (s, IH , H-6) ,

6.44 (d, IH, 3=16.5 Hz, CH=CHC0 ₂ Me), 4.06 (s, 3H), 3.90

(s, 3H), 3.83 (s, 3H) and 3.76 (s, 3H). . 3-Hvd ox methyl-5-me o _\ v-l-me hyl-2-lj.H-indole-4,7-dionej rop- β-en- -αl (20 or E.O.7)

To a stirred suspension αf 6.6 g (19.7 mmol) of methyl 4,7-dihydrαxy-5-methαχ.y-3-methαxycarbαnyl-N-methyl-2- Q indαleacrylate (JL_9) in anhydrous ^CH 2 ^C:L 2 (? ^QQ ml) was added under an atmosphere of dry N„ a 1.5 M solution of diisαbutylaluminiumhydride (DIBAL-H) in toluene (119 ml) keeping the temperature below -30 C. The whole mixture was stirred for 2.5 h at 0 C. Thereupon 198 ml IN (O.IM HCI) FeCl_ were added, keeping the temperature at O C on cooling in a Dry-Ice/ethanol bach. The reaction mixture was stirred for 10 in at 0°C and filtered sub _¬ sequently through high flaw. The dark red upper layer of th filter cake was extracted with hot CHC1., (6x). The organic layer of the filtrate and the combined CHC1 ₃ extracts were washed with sat. NaCl aq. (2x) and dried over MgSO.. Evaporation of the volatiles gave 4.4 g of a dark crystalline residue. Flash column chromatography (Si0 ₂ , CH ₂ Cl ₂ /acetαne 7/3) afforded 3.0 g (55?,.) of E.O.7 5 (purple crystals).

M.p. 216-218°C (CH ₂ Cl ₂ /acetoπe 6/4).

IR(KBc): 3100-3600 (OH), 1680 (quinαne C=0 ⁾ , 1600 (quinone C=C) .

^: H NMR δ(CDCl ₃ ): 6.48 (d, IH, 3=16.1 Hz, CH=CHCH ₂ 0H), 6.14 (dt, IH, 3=16.1 Hz and 3=6.4 Hz, CH_=CH ₂ 0H), 5.66 (s, IH, H-6), 4.68 (d, 2H, 3=6.4 Hz, CH=CHCH ₂ 0H), 4.38 (s, 2H,

Ar-CH ₂ 0H), -3.9 (br s, IH, OH), 3.91 (s, 3H), 3.82 (s, 3H). An exact mass determination gave 277.0950; C^H^ O^ requires 277.0950 (0.0) .

Notes IR spectra were recorded on a Perkin-Elmer 257 instrument. ¹ H NMR spectra were taken on Variaπ A-10 and Brucker WM 250 instruments. Chemical shifts are reported as δ values in ppm relative to TMS (δ TMS = 0.0 ppm). All mass spectral data were recorded on an AEi-902 or Varian Mat 711 mass spectrometer. Figures in the parentheses after exact mass determinations give the absolute value between calculated and recorded masses (in ppm). Melting points are uncorrected.

The new bioreductive alkylatiπg indoloquinones of the ^' present invention are believed to act as cytostatic agents to interfere with DNA replication (Scheme IV). Reduction of the beπzoquinoπe ring and consecutive elimination of both leaving groups X leads to a very reactive bisvinylogous ortho-quinon methide (D_). On anchoring , via a Michael addition, both complementary strands of DNA to this compound, crosslinking occurs, resulting in disturbance of the DNA-repIication process.

The reductive alkylation mechanism of the indoloquinones (II) has been studied in more detail. For this purpose a number of these compounds have been subjected to various reduction conditions (Scheme V). Upon treatment of para-indoloquinoπe derivative 2 _ (Ftø with N a2S2C»4 in the presence of a weak external nucieophile [(a para substituted derivative of) amiπobenzeπe] the C-10 adducts (22) were obtained, the acyl substituent at C-1 ' carbon atom stiil being present.

Most significantly, if the reaction was run in the absence of the weak nucieophile but in the presence of Et N the product appeared to be the 3-methyl derivative 23.

Scheme V

The formation of C-10 adducts has also been observed on trapping the reactive intermediates, obtained after Na2 S 2 > 4 reduction of indoloquinones £1 or NHC2H5), with sulphur nucleophiles: potassium ethylxanthate or sodium N.N-diethyl-dithiocarbamate anioπs.

Most understandingly these reactions do not proceed in the absence of

Scheme VI b R, « NHf^H _j

From these results we can conclude that the bioreductive alkylating indoloquinones 21 are exclusively activated at the C-10 carbon atom upon Na2S2U4 reduction. This in contrast to the activation process ob¬ served with the mitomycins, which yielded for the greater part C-1 5 adducts. The mechanism of activation of the C-10 carbon atom has been depicted in Scheme VI. The reactive intermediate 2_6_ can undergo nucleophiiic as well as electrophilic addition reactions.

In addition to the formation of reactive imiπium species reduction experiments with H2/Ptθ2 have provided evidence for the iπtermediacy ιo of quinone methides (27. 28). on trapping them with electrophiles (H ⁺ or D ⁺ ), and indolodihydroquinoπe (29) Under suitable chosen conditions the quinone methides might also undergo nucleophilic addition reactions [Scheme Vil;see E.A. Oostveen and W.N. Speckamp, Mitomycin analogs I. Indoloquinones as (potential) bisalkylating agents, Tetrahedron, £3. ;

₁₅ 255-262 (1987)].

21 21

23.

Scheme VII

The influence of the R2- uinone substituent has been clearly demonstrated upon carrying out the reduction (H2 P tθ 2 ) of indoloquinones 30a and 30 b (Scheme VIII). The elimination of the OAc-group is stimulated by an electron donating substituent, which is in agreement with the (bio) reductive activation mechanism. During the

aα α R _j - OCH _j α R _j OCH, (17%) _ - α. R,« 0CH, 134%) b R, « NHC-H ₅ _21 b. R _j i NHC^ _j 66%) b. fl- i NHCjHs 19 %)

Scheme VIII activation sequence the NHEt group may also function as an internal proton acceptor.

It has also been observed that this role can also be exerted by an external base (Et3lM; See last reference mentioned above). So far it has been assumed that for the formation of the quinone methides and the iminium compounds from indoloquinones (II) a 2 electro n/2H ⁺ reduction sequence is required. It is also possible, however, that one-electron reduced indoloquinones (II) - semiquinone radical anions -decompose to alkylating intermediates (Scheme IX). Clear evidence for the latter activation process has been revealed recently in

Scheme IX

the reduction of MMC and mitosenes {See: P.A. Andrews, S.S. Pan and N.R. Bachur, J.Amer.Chem.Soα, 108. 4158 (1986)}.

The group R2 (formula II) has a strong influence on he half wave reduction potential and consequently on the selectivity of the potential bioreductive alkylating cytostatic agent. Moreover, the lipophiiϊcity of the molecule as a whole is partly determined by the nature of this group. Apart from the methoxy group, alkoxy groups and amino groups, derived from a wide variety of primary and secondary amines can be used. As examples, apart from the amino groups in the preferred compounds, the following groups are suitable: CH3OCH2CH2NH-, HSCH ₂ CH ₂ NH-, C2H5SCH2CH2NH-, 3-pyridylamino-, c-N(CH ₂ CH ₂ )2NCHO, F, Cl, Br, 3-pyrazoyl and

N —

NC y

The ease of the formation of the reactive intermediates C and D from the hydroquinone B in the reaction scheme IV is determined by the leaving group character of the substituents X (X-j and X2) in formula I and II. Apart from the groups used in the compounds E.O. 1 to E.O. 64, the groups -OCONHCH ₃ , -OS0 ₂ CH ₃ , -OSOCH ₃ , -NHAr, -SC=SN(Et) ₂ , and -OSθ2CgH5-p-CH3, may be particularly mentioned.

By following reaction scheme II I, using an appropriately alkyl-substituted alkylbenzene to prepare the compound I, indoloquinones in which R3 is an alkyl group can readily be obtained. In this way, the compound E.O. 18 . can be prepared, in which R2=OCH3; R3=CH3 and X _{1 ss} χ ₂ =OH. This compound can be easily converted into the corresponding 5-aziridino compound E.O. 19.

Where the group R2 is -O-alkyl, various alkyl groups can be introduced using 2-alkyl-5-chloro-4-nitrophenol as substrate.

Substituents R2 different from -O-alkyl and/ or R3 different from H, alkyl, can be introduced in the reaction scheme III by using suitably substituted o-chloronitrobeπzenes.

The N-substitueπt R5 (Formula I) can be an alkyl group other than methyl and this can be effected using the reaction scheme III by treating the indole derivative 1_5_ with R5 X (where X= halogen and R5 = the required alkyl group) in the presence of a suitable base. In this way, the lipophylicity of the molecule as a whole can be varied without considerably affecting the reduction potential. As an example, in order to enhance the lipophylicity of the compounds E.O. 7 and E.O. 9, the compound E.O. 16 in which the N-CH3 grouping of E.O. 7 has been replaced by N-C4Hg, has been prepared in this way and from this compound E.O. 17 which corresponds to the compound E.O. 9 except that the N-substituent (R5) is C Hg, has been prepared in an analogous way to the preparation of E.O. 9 from E.O. 7.

So far as alkyl group substituents Rg, R7, R- _j -] and R-| 2 ^are concerned, these can be introduced in the reaction scheme III in either of the two ways:

(a) before the electrocyciization reaction, by choosing appropriate aldehydes for the condensation reaction with the aniline derivative Z; or

(b) by the introduction of the required Rg to Rg groups after the 1 ,5-electrocyclization into the H3 substituent group (or other Z substituent group) by means of a suitable reaction sequence.

Bioreductive alkylation is possibly, besides redox cycling, one of the underlying mechanisms of cytotoxicity of the compounds of formula (I), in which the groups R3 and Rg are -Cθ2R- _{] Q} and -CO2 13 respectively, or

a group Cθ2~ ⁺ (M ⁺ being a metal ion).

This has been established by catalytic reduction (H ₂ / Pt0 ₂ ) of indoloquinones E.O. 14 and E.O. 57 in EtOD (Scheme X). The cytotoxic intermediate quinone methides ⁽ 34^ have been trapped by deuterium ions.

_σ, fi 50 % D- ιncoro*rαtιθn

_b_ fl i NHC,H _j 75 V. 0- ιncorBerαlon Q. _or _ _fl v

The high cytotoxic activities of indoloquinone E.O. 22 has to be ascribed to the presence of a second (bioreductive)alky(ation center. Bioreductive alkylation experiments using N,N-diethyldithiocarbamate anions as nucleophiles have provided evidence for this additional alkylation center (Scheme XI).

lim* conversion ( %)

N^ SCNIC,H^, / CH _j Cl, ' M«OH / H,0 2-hr 20% S

Scheme XI B No® SCN(C,H _s l, / CH7CI, / M*OH / H,0 / Nα,S,O _t 20 mm 89 %

Preferred compounds of this type, which have been found to have cytostatic activities are the compounds of the general formula:-

where R-, R_ and R ₅ are as in general formula I.

Particularly preferred indoloquinone compounds with the general formula III are:- (37) Methyl 5-methoxy-3-methoxycarbonyl-1 -methyl-2-[1 i±-indole-4,7- dione]acrylate (Compound E.O. 14)

(38) Methyl 5-aziridino-3-methoxycarboπy!-1 -methy!-2-[1 i±-in- dole- 4,7- dionejacryiate (Compound E.O. 22)

(39) Methyl 5-(2,3-dihydroxypropyl-1 -amino)-3-methoxycarbonyl- -1 -methyl-2-[1Jl-indole-4,7-dioπe]acrylate (Compound E.O. 23)

HOCH ₂ CHOHCH ₂ NH

(40) Methyl 5-hydroxy-3-methαxycarboπyl-1 -methyI-2-[1 i±-indoie-4,7- dione]acrylate (Compound E.O. 24)

(41 ) Methyl 3-methoxycarbony!-1 -methyl-5-propyIeneamiπo-2-[1 H.- indole- 4,7-dioπe]acrylate (Compound E.O. 28)

(42) Methyl 5-methoxy-3-methoxycarbonyf-2-[1 H.-indole-4,7-dione] acrylate (Compound E.O. 29)

(43) Methyl 1 ,6-dimethyl-5-methoxy-3-methoxycarbonyl-2-[1H.-iπdoIe-

4,7-dioπe]acrylate (Compound E.O. 32)

(44) Methyl 5-aziridiπo-1 ,6-dimethyl-3-methoxycarbonyl-2-[1 H.- indole-4,7-dione]acrylate (Compound E.O. 34)

(45) Methyl 3-methαxycarbonyl-1 -methyl-5-morphoIino-2-[1 H_- indole-4,7-dione]acryIate (Compound E.O. 55)

(46) Methyl 5-ethylamiπo-3-methoxycarboπyl-1 -methyl-2-[1 H.- indole-4,7-dione]acrylate (Compound E.O. 57)

These compounds, indoloquinone E.O. 29 excluded, can be readily

prepared from the compound IS. (E.O. 14), which is an intermediate in the reaction scheme III given above for the preparation of the compound E.O. 7. Compound E.O. 29 can be synthesized from indole derivative 5_ (scheme III) following reduction (NO2 group) and oxidation pathways (Fremy's salt). Compound E.O. 29 has also been proven to be a suitable starting indoloquinone for the introduction of several Rs-substituents.

Details for the preparation of the compounds E.O. 1 to E.O. 6, and E.O. 8 to E.O. 13, from compound E.O. 7 and their characterization are given in the following examples 2 to 13. Details for the synthesis of the indoloquinones E.O. 39, E.O. 41 and E.O. 64 in the examples ι*» ^{15 and 16} - Details of the preparation of the compound E.O. 22 from compound IS. (E.O. 14) and its characterization is given in example i ^"7 -

Examples 2 to 13, and 14, 15 and i ^β are divided into four groups:- A -Synthesis of compounds E.O. 1 to E.O. 3, E.O. 10 and E.O. 13, in which both hydroxyl groups in E.O. 7 are substituted by functional groups, which possess good leaving group properties;

B -Syntheses of compounds E.O. 4 to E.O. 6, by treatment of compound E.O. 1 with an excess of a primary or secondary amine; and C -Syntheses of compounds E.O. 8, E.O. 9, E.O. 11 and E.O. 12 by treatment of compound E.O. 7 with an excess of a secondary amine; and D -Syntheses of compounds E.O. 39, E.O. 41 and E.O. 64 via reductive alkylation pathways using indoloquinone E.O. 1 as substrate.

Groug Example 2

3-Acetoxymethyl-5-methoxy-1 -methyl-2-π H-iπdole-4.7-d?onβ1- prop-β-en-tt-yl acetate (E.O. 1^

To a solution of 0.24 g (0.87 mmol) of E.O.7 in CH ₂ C1 ₂ (35 ml) were added pyridine (7 ml) and acetic anhydride (5 ml). After stirring for 6 h at room temperature the mixture was poured into ice. The water layer was extracted with CHCl,(3x). The combined organic layers were washed with cold (Q°C) 3N HCI (3x) and sat. NaHCO.. aq. dried over MgSO. and evaporated under reduced pressure. The residue was submitted to flash column chromatography (Si0 ₂ , CH-C^/acetone 95/5), affording 0.205 g ( 65% ) of E.0.1 as orange crystals.

M.p. 167-168°C (MeOH). IR(KBr): 1725 (ester C=0), 1680 (quinone C=0), 1600 (quinone C=C).

^• " ^■ H NMR δ(CDCl ₃ ): 6.49 (dt, IH , J=16.1 Hz, J=1.4 Hz, CH_=CHCH ₂ 0H) , 6.11 (dt, IH, J=16.1 Hz and J=5.8 Hz, CH=C CH ₂ 0H), 5.66 (s, IH, H-6), 5.23 (s, 2H, ArCH_ ₂ ) , 4.74 (dd, 2H, J=5.8 Hz and J=1.4 Hz), 3.93 (s, 3H), 3.80 (s, 3H), 2.10 (s, 3H, C0CH ₃ ) and 2,04 (s, 3H, COCH _j ) An exact mass determination gave 361.1133; ^ a^ιg^ι ^U ₇ requires 361.1105 (7.8).

Ex amp le 3

Methyl 5-methoxy-3-rnethoxycarbonyloxymethyl-l-methyl-2- LlH-indole-4,7--dioneJ oro?-β-en-α-yl carbona'ce (E.O.2)

Tα a chilled solution (-10°C) αf 72 mg (0.26 mraαl) of E.O.7 in a mixture of anhydrous pyridine (12 ml) and anhydrous CH ₂ C1 ₂ (45 ml) was added, while stirring, methyl chlorofαrmate (2.5 ml) dissolved in CH ₂ C1 ₂ (5 ml). During the addition the temperature was kept below -10 C.

Thereupon the reaction mixture was stirred overnight at room temperature. The work-up and purification was essentially the same as described for E.0.1, affording

60 mg (59?ά) E.O.2 (orange crystals).

M.p. 156-157°C (MeOH). IR( Br): 1740 (ester C=0), 1680

(quinone C=0), 1600 (quinone C=C). ¹ H NMR δ(CDCl ₃ ): 6.54 (dt, IH , Jrlό.l Hz and J=1.4 Hz,

Cli=CHCH ₉ 0H) , 6.17 (dt, IH , J=16.1 Hz and J=5.6 Hz, CH=CH_-

CH ₂ 0H), 5.65 (s, IH, H-6), 5.32 (s, 2H, ArCH_ ₂ ), 4.81

(dd, 2H, J-.1.4 Hz and J=5.6 Hz, CH=CHCH_ ₂ ), 3.93 (s, 3H),

3.81 (s, 3H), 3.79 (s, 3H) and 3.77 (s, 3H). An e-xact mass determination gave 393.1097; C,gH.„ -0_ requires 393.1134 (9.4).

Example 4

3-Carbamoyloxymethyl-5-methoxy-l-methyl-2-LlH-indole-4.7- dionej prop -β-en-α-yl carbamate (E.O.3) Tα a chilled solution (0°C) of 0.150 g (0.54 mmol) αf crude E.O.7 in a mixture of anhydrous pyridine (30 ml) and anhydrous CH-C1- (75 ml) was added pheπyl chloro- for ate (6 ml) while stirring and keeping the temperature below 0 C. Thereupon the whole mixture was stirred at

roam temperature for I h. Tho reaction mixture was worked up accαrdinq tα the procedure described for E.0.1 and E.O.2. The crude product obtained after evaporation αf the vαlatiles was purified by flash column chroma- tography (SiO-, CH _? Cl ₂ /acetone 95/5) , yielding 181 mq

(65?.) of phenyl 4, 7-diαxα-5-methαxy-N-methy1-3-phenαxy- carbαnyloxymethy1-2-iπdoleprαp-β -eπ-α-y1 carbonate. The latter product was dissolved in CH-Cl.-, (40 ml). Ammonia gas was passed into this solution, while chilling in a Dry-Ice/ethanol bath for 0.5 h. The Dry-Ice/ethanol bath was removed and the reaction mixture was stirred at room temperature for 2 h. The excess of ammonia was removed by warming on a water bath. Red crystals separated, which were collected by filtration. The crystals were washed with CH_C1 _? and ethanol (abs). After drying in vacua at 5Q°C, 60 mg αf E.O. (47?ά based on E.O.7) were obtained. M.p. > 300°C. IR( Br): 3440, 3380, 3340, 3280 and 3210 (NH ₂ ) , 1720 and 1695 (carbamate C=0), 1675 (quinone C=0) , 1605 (quinone C=C). ¹ H NMR δCDMSO-dg) , 6.68 (d, IH , J=16.1 Hz, CH=CHCH ₂ 0H) , 6.49 (br s, 4H , NH ₂ ), 6.20 (dt,

IH, J=16.1 Hz and J=5.5 Hz, CH=CHCH ₂ 0H) _f 5.86 (s, IH , H-6) ,

5.04 (s, 2H, Ar CH ₂ ) , 4.67 (d, 2H , J=5.5 Hz, CH=CHCH_ ₂ ),

3.92 (s, 3H) , 3.79 (s, 3H). An exact mass determination gave 363.1047; C,gH. ₇ N,0_ requires 363.1066 (5.2). Example 5

3-(N-chloroe thylcarbamoy loxy ethyl )-5-methoxy-l-methyl-2-0--j- indole-4,7-dionelprop-8-en-α-yl N-chloroethy carbamate (E.O.13)

A solution of 94 mg (0.34 mmol) αf E.O.7 in anhydrous

C!I ₇ C1 ₉ (30 ml) was refluxod with N-chlorocthy 1ioocyanate (10 ml) for 48 h. After evaporation of the solvent and the excess of N-chlαrαe hy1isαcyana e in vacua, the residue was submitted to flash column chromatography (Si0 ₂ » CH ₂ Cl ₂ /acetone 95/5) giving 95 mg (60S) of E.O.13 (purple crystals) .

M.p. 171-172°C. IR(KBr): 3300 (NH), 1690 (carbamate C=0) , 1670 (shoulder: quinone C=0), 1600 (quinone C=C). ¹ H NMR δ(CDCl ₃ ): 6.51 (d, IH, J=16.1 Hz, CH=CHCH ₂ 0H) , 6.16 (dt, IH, J=5.4Hz and J=16.1 Hz) , 5.65 (s, IH, H-6) , 5.27 (s, 2H, Ar CH ₉ ), 5.16 (br s, 2H, NH), 3.93 (s, 3H), 3.80 (s, 3.4-3.7 (m, 8H)). Example 6

5-Me thoχv-3-methoxymethyl-1-methyl-2-D-H-indole-4,7-dione] prop- 8-eπ- -yl N-chloroethylcarbamate (E.O.10)

A solution of 90 mg (0.325 mmol) of E.O.7 in anhydrous CH _? C1_ (30 ml) was refluxed with N-chloroethylisocyanate (10 ml) for 48 h. After evaporation of the solvent and the excess of N-chlαroethylisαcyanate in vacuo, the residue was crystallized from methanαl. The orange

.crystals were collected by filtration and dried in vacuo. Yield: 56 mg (48S). M.p. 176-177°C.

IR( Br): 3300 (NH), 1700 (carbamate C=0) , 1675 (quinone C=Q), 1600 (quinone C=C). ¹ H NMR δ(CDCl ₃ ): 6.35-6.55 (m, 2H, CH=CHCH ^" _Z ) , 5.64 (s, IH, H-6), 5.18 (br s, IH, NH), 4.78 (d. 2H, J=3.7 Hz, CH=CHCH ₂ ), 4.61 (s, 2H , ArgCH^), 3.94 (s, 3H), 3.80

It

(s, 3H), 3.45-3.7 ( , 4H , 0NHCH_ ₂ CH_ ₂ C1) , ^' 3.39 (s, 3H ,

CH ₉ OCΠ_ ₃ ) .

Group S

Example 7

3-Ace o yme hyl-5-aziridino- 1-me hyl- 2-p.H-indole-4,7-dione]prop- 8-en- -yl acetate (E.O.4)

A suspension αf 90 mg (0.25 mmol) of E.O.2 in anhydrous methanol was heated with aziridine (3.5 ml) at 40-45 C for 2 h. The residue obtained, after the evaporation of the solvent and the excess aziridine in vacua, was sub- mitted to flash column ch omatography (Si0 ₂ , CH-Cl-/ acetone 7/3), affording 78 mg (845.) αf E.O.4 (reddish crystals ) .

M.p. 195-196°C (MeOH). IR(KBr): 1730 (ester C=0), 1667

(quinone C=0) , 1590 (quinone C=C). H NMR δ(CDCl ₃ ): 6.49 (dt, IH, 3=16.1 Hz and 3=1.4 Hz, CH=CH-CH ₂ ), 6.10

(dt, IH, 3=16.1 Hz and 3=5.8 Hz, CH_=CH-CH ₂ ), 5.80 (s, IH ,

H-6), 5.24 (s, 2H, Ar C£ ₂ ), 4.74 (dd, 2H, 3=1.4 Hz and

3=5.8 Hz), 3.92 (s, 3H , N-CH--), 2.19 (s, 4H , -CH ₂ N), 2.10 and 2.06 (s, 3H, 0C0CH..). An exact mass determination gave 372.1345; ^i α^O^^ό requires 372.1369 (6.5).

Example 8

3-Acetoxymethyl-5-(2-hvdroxyethyl-l-amino)-l-.methyl- 2- lH-indole-4,7-dione] prop-β-en-α-yl acetate (E.O.5) To a solution αf 40 mg (0.111 mmαl) of E.0.1 in anhydrous methanol (60 ml) was added ethaπαlamiπe (1 ml). The whole mixture was stirred for 6 h at room temperature. Thereupon it was poured into water and extracted with

CHC1 ₃ (5x). The combined organic layers were washed with sat. NaCl aq. and dried aver a^SO^. After evaporation of the volatiles the residue was submitted tα flash column chromatography (Si0 ₂ , CH ₂ Cl ₂ /ace tone 7/3).

Yield: 30 mg (70?.) αf purple crystals.

M.p. 198-200°C (MeOH). IR(KBr): 3200-3600 (OH), 3360 (NH), 1710 (ester C=0) , 1650 (quinone C=0), 1595 (quinone C=C). ¹ H NMR δ(CDCl ₃ ): 6.49 (dt, IH , 3=16.1 Hz and 3=1.4 Hz, CH_=CHCH ₂ ), 7.16 (br t, IH , NH) , 6.09 (dt, IH , 3=16.1 Hz and 3=1.4 Hz, CH=CH_CH ₂ ), 5.23 (s, 3H , H-6 and Ar CH_ ₂ ), 4.74 (dd, 2H, 3=5.9 Hz and 3=1.4 Hz, CH=CH-CH_ ₂ ), 3.96 (s, 3H, N-CH ₃ ) , 3.80-3.95 (m, 2H, CH_ ₂ -0), 3.25-3.35 ( , 2H, CH ₉ N), 2.11 and 2.06 (s, 3H, 0C0CH_ ₃ ), 1.79 (br t, IH, OH).

An exact mass determina ion gave 390.1385; ^ I _O ^? _? ^! ! requires 390.1344 (10.5). Example 9 3-Acetoxymethyl-5-C2.3-dihvdroxyDrooyl-l-amino)-l-methyl- -2- |LH-indole-4,7-dione] prop-β-en-α-yl acetate (E.O.6) Tα a solution of 90 mg (0.25 mmol) of E.O.3 in anhydrous methanol (110 ml) was added l-amiπα-2 , 3-dihydroxyprαpane (0.45 g). The whole mixture was heated at 45-50°C for 10 h. The work-up was essentially the same as described for E.O.5. Flash column chromatography (SiQ ₂ , CH ₂ C1 ₂ / acetone 6/4) gave 65 mg (62?ό) of E.O.6 (purple crystals). M.p. 180-181°C (MeOH). IR(KBr): 3100-3600 (NH and OH),

1720 (ester C=0 , 1600 (quinone C=0), 1590 (quinone C=C ⁾ . ^: H NMR δ(CDCl ₃ ): 6.47 (dt, IH , 3=16.0 Hz and 3=1.3 Hz) , 6.26 (br t, IH, NH) , 6.09 (dt, IH , 3=16.0 Hz and 3=5.8 Hz, CH=CHCH ₂ ), 5.21 (s, IH , H-6) , 5.19 (s, 2H , Ar CH_ ₂ ), 4.74 (dd, 2H, 3=5.8 Hz and 3=1.3 Hz), 3.83-4.07 (m, IH ,

CHOH), 3.93 (s, 3H, N-CH _j ), 3.53-3.83 (m, 2H , CH_ ₂ 0H) , 3.10- 3.30 (m, 2H, CH_ ₂ N), 2.79 (d, IH, 3=4.2 Hz, CHOJH) , 2.18 (br t, IH, CH ₂ 0H_), 2.11 and 2.05 (s, 3H , 0C0CH_ ₃ ). Group C Example 10

3-Hvdroxymethyl-ι-methyl- 5-propyleneamino-2-U.H-indole-4,7-dionej prop-β-en-α-ol (E.O.8)

A solution of 55.5 mg (0.20 mmol) of E.O.7 in anhydrous methanol was heated with pr opy lene i ins (1 ml) at 60-65°C for 1 h. After stirring overnight the solvent and the excess of p opy lene imine were removed in vacuo. From the residue obtained indoloquinone E.O.3 was isolated by flash column chromatography (Si0 ₂ , CH ₂ Cl ₂ /ace tone 7/3). Yield: 44 mg (73?.) (red crystals). M.p. 122.5-124°C (MeOH). IR(KBr): 3100-3600 (OH), 1660 (quinone C=0), 1590 (quinone C=C).

¹ H NMR (100 MHz) δ(CDCl ₃ ): 6.49 (dt, IH , 3=1.4 Hz and 3 = 16 Hz, Chl = CHCH ₂ 0H) , 6.13 (dt, IH, 3 = 4.5 Hz and 3 = 16 Hz, CH=CHCH ₂ 0H) , 5,77 (s, IH , H-6), 4.71 (br d, 2H , 3=6.5 Hz, Ar CH ₂ 0H), 4.4 (br s, 2H, CH=CHCH ₂ 0H), 4.25 (br t, IH , 3 = 6.5 Hz, Ar CH^OH) , 3.91 (s, 3H , N-CH.- _j ), 2-2.5 (m, 4H, CH = CHCH ₂ 0H_, CH ₂ , CH-N), 1.43 (d, 3H, 3 = 5 Hz, CHCh^). An exact mass determination gave 302.1294; requires 302.1267 (9) .

Ex amp le 11

5-Aziridlno-3-hvdroxy ^, πethyl- ι-me hyl- 2- Q.H-indole-4,7-dione] prop-β-en-α-ol (E.O.9)

A solution of 80 mg (0.289 mmαl) αf E.O.7 in anhydrous methanol (30 ml) was heated with aziridine (1 ml) at

40-45°C for 1 h. The work-up proceeded analogously to that described for E.O.8, affording, after column chromatography (Si0 ₂ , CH ₂ Cl ₂ /ace tone 6/4) , 58 mg (70S) αf indoloquinone E.O. (purple crystals). M.p. 160-169 ^Q C. IR(KBr): 3100-3600 (OH), 1660 (quinone

C=0) , 1595 (quinone C=C) .

¹ H NMR δ(CDCl ₃ ): 6.4-6.55 (m, IH , CH_=CHCH ₂ ), 6.12 (dt, IH,

3=4.6 Hz and 3=16.0 Hz, CK=CHCH ₂ ) . 5.79 (s, IH, H-6) , 4.68 (d, 2H,

3=7.1 Hz, Ar CH_ ₉ 0H) ^' , 4.38 ( , 2H, CH=CHCH_ ₂ 0H) , 4.13 (t, IH , 3=7.1 Hz, Ar CH ₂ 0H) , 3.90 (s, 3H, N-CH- _j ), 2.20 (s, 4H ,

CH_ ₂ ) , 1.76 (t, IH, 3=5.6 Hz, CH=CHCH ₂ 0H_) .

An exact mass determination gave 288.1082; ^c i 5 ^H ₁ 6 ^lSJ 2°4 requires 288.1110 (9.9).

Example 12 3-Hydroxymethyl-5-(4-hvdroxypioeridino) -l-methyl-2- ClH-indole

-4, 7-dione] prop-β-en-α-ol (E.O.11)

To a solution of 92 mg (0.33 mmol) αf E.O.7 in anhydrous methanol (30 ml) were added 4-hydroxypiρer idine (100 mg), triethylamine (2 ml) and potassium carbonate (7 mg). The whole mixture was refluxed for 16 h. Thereupon the volatiies were removed at reduced pressure. The residue was mixed with silicagel and submitted to flash column chromatography (Si0_, CH-Cl^/ace one 6/4).

Yield: 72 mg ( 63?i ) of indoloquinone E.O.11 (black crys als) .

M.p. 18Q-181°C (MeOH). IR(KBr): 3100-3600 (OH), 1660

(quinone C=0), 1595 (quinone C=C). ^l H NMR δ(DMS0-d.): 6.35-6.6 ( , 2H , CH=CH) 5..50 (s, IH ,

6

H-6), 5.04 (t, IH, 3=5.3 Hz, CH=CHCH ₂ 0H_) , 4.79 (d, IH , 3=4.1 Hz, CH0H_) , 4.72 (t, IH , 3=5.1 Hz, Ar CH^IH) , 4.56 (d, 2H, 3=5.0 Hz, Ar CH_ ₂ 0H), 4.20 ( , 2H , CH=CHCH_ ₂ ), 3.90 (s, 3H, NCH ₃ ), 3.6-3.8 [n , 3H , CHOH, NCH ₂ (a)], 3.0- 3.2 [ , 2H, NCH ₇ (e)J , 1.7-1.9 [m, 2H , CH_ ₂ CH0H (a)] ,

Example 13

3-Hvdroxyme thyl- 1-me thyl-5-morphσ1ino-2-[jLH-indole-4,7-dionel prop-β-en-α-ol (E.O.12) To a solution of 90 mg (0.325 mmαl) of indoloquinone

E.O.7 in anhydrous methanol (30 ml) were added morpholine (86 mq), trie thyla ine (2 ml) and potassium carbonate (10 mg). The- whole mixture was refluxed far 16 h. Viork-up was essentially the same as described for E.O.11. Yield: 56 mg (52?ό) (claret coloured crystals).

M.p. 204-205°C (MeOH) . IR(KBr): 3100-3600 (OH), 1660 (quinone C=0) .

¹ H NMR δ(DMS0-d ₆ ): 6.4-6.6 (m, 2H , CH=CH), 5.50 (s, IH , H-6), 5.05 (t, IH, 3=5.3 Hz, CH=CHCH ₂ 0H_) , 4.71 (t, IH, 3=5.1 Hz, Ar CH ₂ 0H_) , 4.55 (d, 2H , 3=5.0 Hz Ar CH^OH) ,

4.20 (m, 2H, CH=CH_ ₂ 0H) , 3.89 .(s , 3H , NCH ₃ ), 3.72 (m, 4H ,

0-CH ₂ ).

An exact mass de ermination gave 332.1344; C. H _2Q N ₂ 0c requires 332.1372 (8.6).

Compounds E.O.6, E.O.10, E.O.11 and E.O.13 could not be analyzed by Electron Impact Mass Spectrα etry either due tα their low volatility or their instability at high temperatures. In consequence no accurate mass determination of the latter compounds are available.

The field desorption mass spectrum of E.O.6 showed a molecular iαn peak (M ) at m/e 420. The Field desorption mass spectrum of E.O.13 showed very intensive peaks at m/e 487 and 489.

GROUP D Example 14

5-Me hoxy-1-me hy 1-3- ( -phenylaminomethyl ) - 2- Q-H-indole-4 , 7-dione] prop -β-en-α-yl acetate (E.O.39)

To a vigorously stirred solution of 81 mg (0.22 mmol) of E.0.1 and 800 mg of aniline (8.6 mmol) in a mixture of me hylene chloride (24 ml) and methanol (15 ml) was added a solution of Na_S _? 0 (2.4 g) in water (20 ml). Stirring was continued for 2 minutes. Thereupon the organic layer was separated and washed twice with sat. aq. NaCl. After drying over Na_S0 the solution was evaporated under reduced pressure.

The residue was submitted to flash column chromatography using Si0_ as stationary phase and consecutively methylene chloride - to remove the excess of aniline - and a mixture of methylene chloride and acetone (95/5) as eluer.s, affording 71 mg (80 ) of E.O.39 as red crystals.

Mp: 153-154°C (methanol)

IR (KBr): 3350 (NH), 1735 (ester C=0) , 1670 (quinone C=0), 1600

(quinone C=C)

^■ " ^" H NMR 6(CDC1 ₃ ): 7.06-7.13 (m,2H, phenyl-H) , 6.5-6.7 (m, 4H, phenyl-H and -CH=CHCH ₂ ~) , 6.15 (dt, J=16Hz and J=5.8 Hz,

-CH=CHCH ₂ -), 5.64 (s, IH, H-6), 4.88 (br s, IH, NH) , 4.79 (dd, 2H,

J=1.3 Hz and 5.8Hz, -CH=CHCH , 4.37 (s, 2H, ArCH -) , 3.88 (s, 3H) and 3.81 (s, 3H) , 2.12 (s, 3H, -COCH ) .

An exact mass determination gave 394.1527; C__H-_N_0_

2222 2 5 requires 394.1529 (0.5).

Example 15

Methoxy-l,3-dimethyl-2-[lH-indole-4,7-dione] prop-β-en-α-yl acetate (E.O.41)

To a vigorously stirred solution of 86 mg (0.24 mmol) of E.0.1 in a mixture of methylene chloride (18 ml), methanol (9 ml) and triethylamine (3 ml) was added a solution of Na S„0 (1.5 g) in water (15 ml). Stirring was continued for five minutes. Thereupon the organic layer was separated and washed twice with sat. aq. NaCl. After drying over MgSO . the solution was evaporated under reduced pressure.

The residue was submitted to flash column chromatography (SiO-,

CH Cl-/acetone: 95/5), affording 58 mg (80%) of E.O.41 as claret crystals.

Mp: 166-167°C (MeOH). IR (CHC1 ): 1735 (ester C=0) , 1670 (quinone C=0) , 1600 (quinone C=C).

^X H NMR <f (CDC1 ₃ ): 6.51 (d, IH, J=16 Hz, -CH=CHCH ₂ ), 6.10 (dt, IH, J=16 and 6H, -CH=CHCH -) , 5.63 (s, IH, H-6), 4.75 (d, 2H, J=6Hz, -CH=CHCH -), 3.94 (s, 3H) and 3.80 (s, 3H) , 2.39 (s, 3H, Ar-CH ), 2.12 (s, 3H, COCH.). An exact mass determination gave 303.1094; C H.JO. requires 303.1107 (4.2).

Example 16

0-Ethyl 5-methoxy-l-methyI -2- ^" [lH-indole-4,7-dione]prop-β-en-α-yl dithiocarbonate (E.O.64)

To a vigorously stirred solution of 61 mg (0.17 mmol) of E.0.1 and 272 mg (1.7 mmol) of potassium ethylxanthate in a mixture of methylene chloride (18 ml) and methanol (12 ml) was added a solution of Na-S-0 (1.8 g) in water (15 ml). Stirring was continued for ten minutes. Thereupon the organic layer was separated and washed twice with sat. aq. NaCl. After drying over gS0 ₄ the solution was evaporated under reduced pressure. The residue was submitted to flash column chromatography (Si0_, CH_C1 ₂ / ce one 95/5), affording 65 mg (39%) of E.O.64 as a red oil. IR (CHC1-): 1740 (ester C=0) , 1672 (quinone C=0) , 1600 (quinone C=C).

^X H NMR 6 (CDC1-): 6.52 (d, IH, J=16.2 Hz, -CH=CHCH ₂ ), 6.17 (dt, IH, J=16.2 Hz, and J=5.7 Hz, -CH=CHCH ₂ «), 5.65 (s, IH, H-6), 4.76 (dd, 2H, J=5.7 Hz and J»l.l Hz, -CH=CHCH ₂ ) , 4.6-4.8 (m, 4H), 3.93 (s, 3H) and 3.80 (s, 3H), 2.12 (s, 3H, -C0CH ₃ ), 1.42 (t, 3H, J-7.1 Hz, CH ₂ CH ₃ ).

Exarπo le 17 ethyl 5-aziridino-3-methoxycarbonyl-l-methyl-2-Q.H-indole-4,7- dione] acrylate (E.O.22)

A suspension of methyl 5-methαxy-3-methoxycarbony1-N- 5 methyl-4,7-diαxα-2-indoleacrylate (JJ3) (600 mg, 1.8 mmαl) in anhydrous methanol (200 ml) was heated with aziridine (5 ml) at 45-50 C for 5 h. The residue obtained, after the evaporation of the solvent and the excess of aziridine in vacuo, was submitted to flash column 10 chromatography (Si0 ₂ , CH ₂ Cl ₂ /ace one 95/5), affording 570 mg (92?ό) of E.O.22 (orange crystals).

M.p. 2Q4-206°C (MeOH). IR(KBr): 1720 (ester C= ^Q ), 1690

(quinone C=Q), 1590 (quinone C=C).

^X H NMR δ (C0C1 ₃ ): 7.60 (d, IH , J=16.2 Hz, CH=CHC0 ₂ Me), 156.40 (d, IH, 3=16.2 Hz, CHsCHCQ^-le) , 5.85 (s, IH , H-6) ,

4.04 (s, 3H), 3.94 (s, 3H), 3.79 (s, 3H) and 2.21 (s, 4H,

-CH ₂ N).

An exacfcmass determination gave 344.0992; ^c _j 7 ^H i6 ^N 2 ^α requires 344.1008 (4.6). 20 Elemental analysis:

Calculated for ^C ₁₇ ^H ₁₆ ^N 2°6 ^{= C} ' ^{59,29; H} ' ^4*69, Found: C, 59.17; H, 4.68.

Example 1$

3-Hvdroxymethyl-5-methoxy-1 -methyl-2-| ^" 1 H-indole-4.7-dione]prop- β-en- -yl acetate (E.O. 1 A

To a solution of 40 mg (0.11 mmol) of E.O. 1 in acetone (15 ml) wa carefully added 10 N H2SO4 (20 ml). The whole mixture was stirred f

15 mln at room temperature. Thereupon the reaction mixture, containin for the geater part, the indoloquinones E.O. 1 A and E.O. 7, was poured in

a sat. aq. solution of NaHC03- The water layer was extracted with CHCI3

The combined extracts were dried over MgSO^.. The residue obtained after the evaporation of the solvent was submitted to flash column chromatography (Siθ2, C^C^/acetone 95/5), affording 7 mg (20 %) of 5 indoloquinone E.O. 1A (red crystals).

M.p. 168-169° C (MeOH).

IR (KBr): 3360 (OH), 1735 (ester C-O), 1670 (quinone C-O) and 1595

(quinone C-C).

¹ NMR δ (CDCI ₃ ): 6.48 (dt, 1 H, J-16.1 Hz and J-1.4 Hz, CH-CHCH ₂ OAc), 10 6.05 (dt, 1 H, J-16.0 Hz and J=5.8 Hz, CH=CHCH ₂ OH), 5.67 (s, 1 H, H-6), 4.75

(dd, 2H, J=5.8 Hz and J-1.4 Hz, CH=CHCH ₂ ), 4.66 (d, 2H, J-7 Hz, ArCU2").

3.96 (t, 1 H, J=7 Hz, OH), 3.90 (s, 3H), 3.82 (s, 3H) and 2.10 (s, 3H,

An exact mass determination gave: 319.1095; Ci gH - _j yNO g requires 15 319.1056 (0.9).

Example 19

3-Acetoxvmethvl-5-methoxv-1 -methvl-2-M H -indole-4.7-dione1-Droo-

A solution of 100 mg (0.275 mmol) in a mixture of methanol (70 ml)- and 20 NEt3 (1 ml) was refluxed for 35 min. Thereupon the solvent was removed in vacuo. The residue obtained was submitted to flash column chromatography (Siθ2; CH ₂ Cl2 acetone 7/3) affording 61 mg (70 %) of

E.0. 1B.

M.p. 183-185° C (MeOH). 25 IR (KBr): 3480 (OH), 1715 (ester C-O), 1670 (quinone C-O), 1595 (quinone

C-C).

¹ NMR δ (CDCI3): 6.51 (d, 1 H, J-16.1 Hz , Cϋ-CHCH ₂ 0Ac), 6.19 (dt, 1 H,

J-16.1 Hz and J-4.6 Hz, CH-C&CH ₂ OH), 5.65 (s, 1H, H-6) , 5.24 (s, 2H,

ArCH.2-). 4.39 (m, 2H, CH-CHCH_ ₂ ), 3.93 (s, 3H), 3.80 (s, 3H) and 2.04 (s, ^"

3H, -COCH3), 3.93 (t, 1 H, J-5.4 Hz, OH).

An exact mass determination gave: 31 9.1095; C1 5H 1 7N O 5 requires

319.1056 (0.9).

Example 20 ₅ 3-Acetoxymethyl-5-aziridi n o-1 -mβthvl-2-ri H -indole-4.7-dio ne1 prop-β-en-α-ol (E.O. 4A)

A solution of 155 mg (0.42 mmol) E.O. 4 in a mixture of anhydrous methanol (120 ml) and NEt ₃ (2 ml) was heated at 50° C for 1 h.

Thereupon the solution was evaporated. The residue obtained, for the 10 greater part a mixture of indoloquinone E.O. 4A and E.O. 9 was submitted to flash column chromatography (Siθ2; CH ₂ Cl2 acetone 7/3), affording

90 mg (64 %) of compound E.O. 4A (red crystals).

M.p. 193-195° C (MeOH; dec).

IR (KBr): 3360 (OH), 1725 (ester C-O), 1665 (quinone C-O), 1575 (quinone ⁱ s C-C).

¹ NMR δ (CDCI3): 6.51 (dt, 1 H, J-16.1 Hz and J-1.8 Hz, CH=CHCH ₂ OH), 6.18

(dt, 1 H, J-16.1 Hz and J-4.6 Hz, CH=CHCH ₂ OH), 5.79 (s, 1 H, H-6) , 5.25 (s,

2H, ArCH_2-). 4.38 (m, 2H, CH=CHCϋ ₂ ), 3.92 (s, 3H, NCH3), 2.19 (s, 4H,

-CH ₂ N-), 2.06 (s, 3H, -COCH3), 1.78 (t, 1 H, J-5.5 Hz, OH). 20 An exact mass determination gave: 330.1305; C<\ γH -\ £^ 2^5 requires

330.1216 (27).

Example 21

3-Acetoxymethyl-1 ,6-dimethyl-5-methoxy-2-[1 H-indole-4.7-dione]- orop- B-en-cc-vl acetate (E.O. 33) __ The synthesis of this compound from indoloquinone E.O. 18 proceeded

25 similar to that described for indoloquinone E.O. 1 from E.O. 7.

Yield: 85 %; Orange red crystals.

M.p. 185-187° C.

IR (KBr): 1725 (ester C-O), 1660 (quinone C-O), 1600 (quinone C-C).

¹ NMR δ (CDCI3): 6.50 (dt, 1 H, J-16.1 Hz and J- 1.3 Hz, CH_-CHCH ₂ OAc),

6.10 (dt, 1 H, J-16.1 Hz and J-5.8 Hz, CH=CHCH ₂ OH), 5.23 (s, 2H, ArCH ₂ -),

4.74 (dd, 2H, J-5.8 Hz and J-1.3 Hz, CH-CHCH2), 4.00, 3.92, 2.10, 2.05 and

1.94 (s, 3H). 5An exact mass determination gave: 375.1317; C- _| g H 2i N θ 7 requires

375.1318 (0.3)

Example 22

3-Acetoxymethyl-5-aziridi no-1 .6-di methyl-2-ri H -indole-4.7-dio nel prop-β-en-α-vl acetate (E.O. 35) 10 The synthesis of this compound from indoloquinone E.O. 33 proceeded similar to that described for indoloquinone E.O. 4 from E.O. 1.

Yield: 82 %; purple crystals.

M.p. 200-202° C (MeOH).

IR (KBr): 1725 (ester C-O), 1660 (quinone C-O), 1590 (quinone C-C). 15 ¹ NMR δ (CDCI3): 6.51 (dt, 1H, J-16.1 Hz and J-1.3 Hz, 6.11

(dt, 1H, J-16.1 Hz and J-5.8 Hz, CH-CHCH2-).5.26 (s, 2H, ArCH.2-).4.76

(dd, 2H, J-5.8 and J-1.3 Hz, (s, 3H, NCH3), 2.31 (s, 4H,

-CH ₂ N-), 2.12, 2.07 and 2.06 (s, 3H).

An exact mass determination gave: 386.1478; C2o ^H 22 ^N 2°6 requires 20 386.1478 (0.0)

Exam le 2d

3-Benzoxvmethvl-5-methoxv-1 -methvl-2-f1 H -indole-4.7-dione1orop- B-en-ct-vl benzoate (E.O. 6^

A solution of 70 mg (0.253 mmol) of indoloquinone E.O. 7 and 80 mg 25 (0.633 mmol) of benzoylchloride in a mixture of CH2CL2 (anh.; 10 ml) and pyridine (1 ml) was refluxed for 3 h.Thereupon the reaction mixture was cooled, diluted with an additional amount of CH2C L2 and washed with cold aq. 3N HCI (5x). After drying over MgS04 the solvents were removed in vacuo. The residue obtained was submitted to flash column

chromatography (Si0 ₂ ; CH ₂ Cl2/acetone 95/5), affording 52 mg (43 %) of compound E.O. 36 (red crystals).

M.p. 166-168° C (MeOH).

IR (CHCI3): 1715 (ester C-O), 1670 (quinone C-O), 1595 (quinone C-C). 5 ¹ NMR δ (CDCI3): 7.9-8.1 (m, 4H, phenyl-H), 7.2-7.6 (m, 6 H, phenyl-H)

6.67 (dt, 1 H, J-16.1 Hz and J-1.4 Hz, CH=CHCH ₂ -), 6.34 (dt, 1 H, J-16.1 Hz and J-5.6 Hz, CH=CH.CH ₂ -), 5.68 (s, 1 H, H-6) , 5.55 (s, 2H, ArCHg-). 4.99

(dd, 2H, J-5.6 Hz and J-1.4 Hz, CH-CHCH^), 3.97 (s, 3H) and 3.80 (s, 3H-).

FD MS: m/e 485 (M ⁺ ). _? Example 24

3-rN-butylcarbamoyloxymethyl1-5-methoxy-1 -methyl-2-f1 H -indole-4.7- dione)Drop-B-en-o:-yl N-butylcarbamate (E.O. 37)

To a solution of 70 mg (0.253 mmol) in CH2C L2 (15 ml) were added

K2CO3 (1 g) and n-butyl isocyanate (2 ml). The whole was refluxed foor 4 5 h. After cooling the reaction mixture to -room temperature, the excess of

K2CO3 was removed by filtration. The residue, obtained after evaporation of the solvent and the excess of π-butyl isocyanate in vacuo, was sumitted to flash column chromatography (Siθ2; CH C /acetone 95/5), affording 76 mg (63 %) of compound E.O. 37 (red crystals). 0 M.p. 190-191 ° C (MeOH).

IR (CHCI3): 3300 (NH),1685 (br.; carbamate C-O and quinone C-O), 1600

(quinone C-C).

¹ NMR δ (CDCI3): 6.51 (br.d, 1 H, J-16.2 Hz, CH,=CHCH ₂ -), 6.34 (dt, 1 H,

J-16.2 Hz and J-5.4 Hz, CH-Cj±CH ₂ -), 5.65 (s, 1 H, H-6) , 5.24 (s, 2H, 5 ArCE2").4.7-4.8 (br., 2H, NH), 4.73 (br. m, 2H, CH-CHCH2), 3.93 (s, 3H) and 3.80 (s, 3H), 3.1-3.3 (m, 4H, NHCH ₂ -), 1.2-1.6 (m, 8H, -CE2CH2- H3),

0.8-1.0 (m, 6H, -CH2CH3)

FD MS: m/e 475 (M ⁺ )

Example 25

5-M ethoxy-1 -mβthyl-3-fN-phenylcarbamoyloxymethvll-2-ri H-indolβ-

4.7-dionelproo-β-eπ-α-yl N-ohenylcarbamaτe (E.O. 38)

To a solution of 70 mg (0.253 mmol) in CH2C L2 (15 ml) were added 5K2CO3 (1 g) and n-phenyl isocyanate (400 mg). The whole was refluxed foor 17 h. After cooling the reaction mixture to room temperature, the excess of K2CO3 was removed by filtration. The residue, obtained after evaporation of the solvent and the excess of in vacuo, was susupended in methanol. The crystalline product was collected by filtration and finally losubmitted to flash column chromatography (Siθ2 ; CH2Cl2 aceto ne

95/5), affording 85 mg (50 %) of compound E.O. 38 (dark red crystals).

M.p. 177-178° C.

IR (CHCI3): 3300 (NH),1690 (br.; carbamate C-O and quinone C-O), 1600

(quinone C-C). 15 ¹ NMR . δ (C D C I3): 7.2-7.4 (m, 10H, phenyl-H), 6.4-6.6 (br. m, 1 H,

CH=CHCH ₂ -), 6.1 -6.3 (br. m, 1 H, CH=CH.CH ₂ -), 5.65 (s, 1 H, H-6) , 5.30 (s,

2H, ArCU2-).5-0-5.3 (br. m, 2H, NH), 4.3-4.4 (m, 4H, CH_2C-6H ₅ ), 4.7-4.8

(br. m, 2H, CH-CHCH^). 3.91 and 3.80 (s, 3H).

Anal. Calcd for C30H29N3O7: C, 66.29; H, 5.38; N, 7.73. Found: C, 66.38; H, 205.39; N, 7.60.

Example 2$ 2-ri H-indole-4.7-dione]oroo-β-en-o.-yl acetate (E.O. 47) A). 3-hydroxymethyl-5-[2-(N,N-dimethylamino)ethyl-1 -amino]-1 - 25 methyl-2-[1 H-indole-4,7-dione]prop-β-en-α-ol.

A solution of 206 mg (0.744 mmol) of indoloquinone E.O. 7 in a mixture of methanol (60 ml) and H ₂ N C H 2C H ₂ N ( C H3)2 (1 g) was refluxed for 2 h. After the evaporation of the solvent and the excess of the reagent, a crystalline mass was obtained which was employed

in the next step without further purification. B) The synthesis of indoloquinone E.O. 47.

The crude reaction product was dissolved in a mixture of CH2CI2 (28 ml) and pyridine (5.6 ml), containing DMAP (20 mg) and AC2O (4 ml). After stirring for 1 h at room temperature the solvents and the excess of the reagents were removed in vacuo. The residue thus obtained was submitted to flash column chromatography (Siθ2'.

CH ₂ CI /MeOH 8/2), affording 260 mg (84 %) of compound E.O. 47

(purple crystals). M.p. 149-150° C.

IR (KBr): 3310 (NH), 1730 (ester C-O), 1660 (quinone C-O), 1590

(quinone C-C).

¹ NMR δ (CDCI ₃ ): 6.4-6.6 (m, 2H, NH and CH = CHCH ₂ -), 6- ¹⁰ (dt, 1 H,

J-16.1 Hz and J-5.9 Hz, CH=CHCH ₂ -), 5.24 (s, 2H, ArC H_ ₂ -), ⁵ -17 (s, 1 H, H-6), 4.75 (dd, 2H, J-5.9 and J-1.4 Hz, CH=CHCH_ ₂ ). 3.97

(s, 3H, NCH ₃ ), 3.05-3.2 (m, 2H, CH_ ₂ NH), 2.55 [t, 2H,

J-6.1 Hz, -CU2N(CH ₃ ) ₂ ], 2.23 [s, 6H, N(CH ₃ ) ₂ ], 2.11 and 2.06 (s, 3H).

Anal. Calcd for C ₂ ι H ₂ 7N ₃ 0 ₆ : C, 60.42; H, 6.52; N, 10.07. Found: C,

60.37; H, 6.55; N, 10.05. Example 27

3-Acetoxymethyl-5-[2- N.N-dimethylamino ethyl-1 -amino]-1 -methyl- 2 -f1 H-indole-4.7-dione]prθD-β-en-α-ol (E.O. 48)

A solution of 50 mg (0.120 mmol) of indoloquiπoπe E.O. 47 in a mixture of methanol (anh.; 20 ml) and NEts (0.5 ml) was heated for 1 h at 50° C. The residue obtained after the evaporation of the solvents in vacuo was submitted to flash column chromatography (Siθ2; C^C^/MeOH 8/2), affording 25 mg (57 %) of compound E.O. 48 (purple crystals). M.p. 153-155° C. IR (KBr): 3100-3500 (NH and OH), 1730 (ester C-O), 1660 (quinone C-O),

1590 (quinone C-C).

¹ NMR δ (CDCl ₃ ): 6.50 (dt, 1H, 16.1 Hz and 1.5 Hz , CH=CHCH ₂ -), 6.42 (m, 1 H, NH), 6.16 (dt, 1 H, J-16.1 Hz and J-4.7 Hz, CH-CH.CH ₂ -), 5.24 (s, 2H, ArCH.2-). 5.16 (s, 1 H, H-6), 4.37 (dd, 2H, J-4.7 and J-1.6 Hz, CH-CHCU2). 3.96 (s, 3H, NCH3), 3.05-3.2 (m, 2H, CE2 H), 2.55 [t, 2H, J-6.1 Hz, -CH.2N(CH ₃ ) ₂ ], 2.24 [s, 6H, N(CH ₃ ) ₂ ], 2.06 (s, 3H).

Anal. Calcd for C2i H ₂ 7N ₃ 0 ₆ : C, 60.42; H, 6.52; N, 10.07. Found: C, 60.37; H, 6.55; N, 10.05. FD MS: m/e 375 (M ⁺ ). Example 29

3-Hvdroxymethyl-1 -methyl-5-r2-oyridylethyl-1 -amino ^" l-2-ri H -indole-

4.7-dione]Drop-β-en-α-ol (E.O. 51 )

A similar synthesis procedure has been applied as for the synthesis of the precursor of indoloquinone E.O. 47 (Example 26) Yield: 74 %; dark purple crystals.

M.p.: 208-210° C.

IR (KBr): 3100-3500 (NH and OH), 1650 (quinone C-O), 1590 (quinone

C-C).

¹ NMR δ (DMSO-d ₆ ): 8.45-8.55 (m, 1 H, py-H), 7.7-7.85(m, 1 H, py-H), 7.2-7.45 (m, 3H, py-H and NH), 6.4-6.6 (m, 2H, CH=CECH ₂ -), 5.17 (s, 1 H,

H-6), 5.04 and 4.77 (m, 1 H, OH), 4.56 (d, 2H, ArCE.2"). ⁴ -2 ( , 2H,

CH-CHCE2). 3.92 (s, 3H, NCH3), 3.45-3.6 (m, 2H, CE2NH), 3.04 [t, 2H,

FD MS: m/e 367 (M ⁺ ). Example 29

3-Acetoxvmethvl-1 -methvl-5-r2-pvridvlethvl-1 -aminol-2-ri H-indole-

4.7-dionelproo-β-en-oc-vl acetate (E.O. 52^

A similar synthesis procedure has been applied as for the synthesis of indoloquinone E.O. 48 (Example 26)

Yield: 86 %; purple crystals.

M.p.: 156-157° C.

IR (CHCI ₃ ): 3300 (NH), 1730 (ester C-O), 1660 (quinone C-O), 1590

(quinone C-C). ¹ NMR δ (CDCI3): 8.55-8.6 (m, 1 H, py-H), 7.55-7.65 (m, 1 H, py-H), 7.1 -7.2

(m, 2H, py-H ), 6.4-6.6 (m, 2H, CE=CHCH ₂ - and NH ), 6.07 (dt, 1 H, J-16.0

Hz and J-5.9 Hz, CH=CECH ₂ -), 5.24 (s, 1 H, H-6), 5.22 (s, 2H, ArC 2").

4.73 (dd, 2H, J-5.9 and J-1.2 Hz, CH-CHCH ₂ ), 3.96 (s, 3H, NCH3), 3.5-3.6

(m, 2H, C 2NH), 3.09 [t, 2H, J-6.6 Hz, -CE2*Py]. 2.10 and 2.04 (s, 3H,

FD MS: m/e 451 (M ⁺ ).

Example 30

3-Acetoxymethyl-1 -meth l-5-proDyleneamino-2-[1 H-indole-4.7-dione]- proD-β-en-tt-vl acetate (E.O. 53) Indoloquinone E.O. 53 'has been synthesized from compound E.O. 8 using the same synthesis procedure as has been described for compound E.O. 47

(Example 26 B).

Yield: 70 %; purple crystals.

M.p.:143-145°C (MeOH). IR (CHCI3): 1735 (ester C-O), 1665 (quinone C-O), 1585 (quinone C-C).

¹ NMR δ (CDCI3): 6.51 (dt, 1 H, J-16.1 Hz and J-1.3 Hz, CE=CHCH ₂ -), 6.12

(dt, 1 H, J-16.1 Hz and J-5.8 Hz, CH-CECH ₂ -), 5.79 (s, 1 H, H-6), 5.27 (s,

2H, ArCE2"). ⁴ -76 (dd, 2H, J-5.8 and J-1.3 Hz, CH-CHCE.2). 3.94 (s, 3H,

NCH ₃ ), 2.25-2.4 (m, 1 H, CECH3), 2.05-2.20 (m, 6H, -COCH3 and CH ₂ N), 1.42 (d, 3H, J-5.5 Hz, CHCE3).

BIOLOGICAL DATA: (a) In vitro activity experiments

The indoloquinones II (formula II) have been tested in respect of their cytotoxic activity against L1 21 0 cells and R-1 cells (Rhabdomyosarcoma Cells) in a bioliquid assay at the department of oncology of the Free University of Amsterdam (Table I). In addition to this seven compounds (E.O. 1 . E.O. 2. E.O. 4, E.O. 7, E.O. 9, E.O. 16 and E.O. 17) have been tested on 11210 activity in a cloπogenic assay at TNO, Rijswijk. Further, the lowest active doses of the compounds E.O. 1 , E.O. 2, E.O. 4 and E.O. 9 have been determined at TNO Rijswijk, according to the method described by Lamberts et al. [Oncology, 301 (1983)]. (Table II). Eight indoloquinones (E.O. 1 , E.O. 2, E.O. 4, E.O. 4A, E.O. 8, E.O. 9, E.O. 33 and E.O. 35) have been tested in a panel of five human tumour lines (TNO

Rijswijk; Table 111). From the latter group three indoloquinones (E.O 1 , E.O. 4 and E.O 9) have been selected for a cytotoxicity study, involving five slowly growing human tumour lines (University of Freiburg; Table IV) .

(i) Determination of the R-1 activity fbioliαuid assay)

For the determination of the R-1 activity, Rhabdomyosarcoma cells were brought into Falcon multiwells (growth area: 9.6 cm ² ) (. O ⁶ cells per dish) containing 3 ml of Dulbecco's medium supplemented with 10 % Foeto Calf Serum (FCS). When the cells have become attached to the polymeric support forming a mono-layer (after about 16 hours), they were incubated at 37° C in an atmosphere of 5 % C0 in humidified air with the compound to be tested (in the appropriate concentration) dissolved in the same medium. Thereupon the drug solution was removed and the cells were covered with fresh medium. After 48 hours the cells were trypsinized and counted on a Sysmex microcell counter (CC110).

TABLE I

IN VITRO ACTIVITY DATA (RHABDOMYOSARCOMA/L1210) OF THE INDOLOQUINONES II

• ID-50(μg/ml)

COMP. π ₂ R3 «5 1 *2 Hil 1210

E.0.1 OCH ₃ H CH ₃ OAc OAc 0.3 2.3

E.O. 1 A CCH ₃ H CH ₃ CH OAc 3.2 2.3

E.0. 1 B 0CH3 H CH ₃ OAc CH 0.05 0.9

E.O. 2 CCH ₃ H CH ₃ OCCCCH3 CCCCCH3 0.4 0.5

E.O.3 CCH ₃ H CH ₃ CCCNH ₂ OCCNH2 > 10 a

E.0.4 H CH ₃ OAc OAc 0.025 1.6

E.O. 4A H CH ₃ OAc CH 0.02 0.15

E.O.5 NHCH ₂ CH ₂ OH H CH ₃ OAc OAc > 10 > 10

E.O. S NHCH2CHCHCH2CH H CH ₃ OAc OAc > 10 > 10

E.O.7 OCH3 H CH ₃ CH CH 4.2 0.5

E.0. 9 H CH ₃ CH CH 0.003 0.46

E.O. 10 OCH3 H CH ₃ OCH3 OCONHCH ₂ CH ₂ C! >10 > 10 E.0. 11 H CH ₃ CH CH > 10 - > 10

E.0. 12 *o OH

H CH ₃ CH CH > 10 3.3

E.O. 13 OCH3 H CH ₃ OCONHCH ₂ CH ₂ CI 1.1 10

E.O. 15 NHC ₆ H ₅ H CH3 CH CH 0.7 E.0. 16 OCH3 H C ₄ H ₉ CH CH

E.0. 17 H C ₄ H ₉ CH CH -

E.O. 18 OO-h CH3 CH ₃ CH CH 2.2 2.4 E.0. 19 CH ₃ CH3 CH CH 0.1 2.0

E.0. 33 OCH3 CH3 CH ₃ OAc OAc 2.2 3.6 E.0. 35 CH3 CH ₃ OAc OAc 0.3 0.55

-

TABLE I

IN VITRO ACTIVITY DATA (RHABDOMYOSARCOMA/L121Q) OF THE INDOLOQUINONES II. (continued)

ID-50(μg/ml)

COMP. R ₂ R3 RS X, x ₂ Hrl L1210

E.O.36 OCH ₃ H CH ₃ OCOC ₆ H ₅ 2.6 0.7

E.O.37 OCH3 H CH ₃ OCONHC ₄ H ₉ 6.5 > 10

E.O.38 OCH3 H CH ₃ OCONHCgHs 8.5 >10

E.O.39 OCH3 H CH ₃ NHC ₆ H ₅ OAc 1.0 10

E.O.41 OCH3 H CH ₃ H OAc >10 >10

E.O.47 NHCH CH ₂ (CH ₃ )2 H CH ₃ OAc OAc 4.4 >10

E.O.48 NHCH ₂ CH ₂ (CH ₃ )2 H CH ₃ OAc CH >10 >10

E.O.51 H CH ₃ CH CH 4.5 >10 -CH ₂ CH ₂ NH-

E.O.52 H CH ₃ OAc OAc 3.3 >10

-CH ₂ CH ₂ NH-

E.O.53 H CH ₃ OAc OAc 1.4 3.4

Ή CH,

E.O.56 NHC ₂ H ₅ H CH ₃ CH CH >10 5.1

E.O.58 NHC ₂ H ₅ H CH3 OAc OAc >10 >10

E.O.59 NHC ₂ H ₅ H CH ₃ OAc CH >10 >10

E.O.60 H CH ₃ OAc OAc 3.3 >10

•N O V f

E.O.62 H CH ₃ OAc CH >10 >10 ^" V-CHJCHJNH-

E.0.64 CCH3 H CH ₃ SCSN(Et) ₂ OAc - -

MMC NH ₂ 0.03 0.05

Seven indoloquinones - E.O.1 , E.O.2, E.O.4, EO.7, E.0.9, E.O/16, and E.0.17- have been tested on L1210 activity in a donogenic assay at TNO Rijswijk. The ID-values (μg/ ml) amounted respectively: 2.5, 3.7, « 1, 2.2, 0.8, 0.9 and 0.3 μg/ ml.

The para-iπdoloquiπoπes together with mitomycin C were tested on their activity against R-1 cells. The results are given in Table I.

(ii) Determination of LI 21 0 activity (bioliouid assay).

For this purpose, L1210 cells were grown, as suspension, in Falcon multi-wells (growth area: 9.6 cm^) using RPMI supplemented with 15 % FCS and 2-mercapto-ethaπα! (60 μmol) as medium. In this medium, the cells were continuously incubated with the compound to be tested at 37° C in an atmosphere of 5 % CO2 in humidified air for 48 hours. Thereupon they were counted on a Sysmex micrαcell counter (CC110). Test results are also given in Table l.

( i i i) Determination of the L121 Q activity fciσnocenic assay)

The L1210 donogenic assay used was an improved variant of the method described earlier by H. MARTIN et al. [Cancer Chemother.. Rep., 5_L 451 (1967)] and LM. van Putteπ et al. [Cancer Treat. Rep., 6J 373 (1 976)] for the growth into colonies of L1210 cells in a soft agar medium. From a suspension culture - 1 00 L1210 Cells (0.1 ml) were plated into 3.5 mm culture dishes (Falcon), containing 1 ml of soft agar growth medium and the compound to be tested in appropriate concentrations. The soft agar growth medium consisted of Dulbecco's medium supplemented with 1 5.8 % horse serum, 60 μ m o l

2-mercapto-ethaπoi, 20 mg/ml L-asparagine and 0.3 % bacto agar (Difco).

The culture dishes were incubated at 37° C in an atmosphere of 10

% CO2 in humidified air for 8 days. After this period of continuous drug exposure, colonies were counted and dose-effect curves were made. From these ID-50 values were calculated which are also given in Table I.

The in vitro L1210 and R-1 activities of the indoloquinones III

TABLE IA

TN VTT O ACTIVITY OF THE PARA- INDOLOQUINONE DIESTERS

COMP. n ₂ ^■ *3 *5 R-1 LI 210 ει 2

E.O. 14 OCH ₃ H CH3 > 10 0.3 •243

E.O. 29 OCH ₃ H H 4.9 2.2 -2 1

E.O. 32 OCH3 CH ₃ CH ₃ > 10 3.5

E.O. 23 NHCH2CHCHCH2CH H CH ₃ > ιo > 10 -343

E.O. 57 NHC ₂ H _S H CH ₃ 3.2 1.4

E.O. 55 H CH ₃ 8.9 1.4 -240

/

^■ N 0

E.O. 24 CH H CH ₃ > 10 > 10 -299

E.O. 22 H CH ₃ 0.5 0.01 -179

<

E.O. CH ₃ CH ₃ 2.8 2.7 -239

«

E.O. 23 H CH ₃ 5.7 22 -171

CH,

5 ^* T ^β πarv ^β wa e reouαβπ oonπiiats (£1 2} πav* 0»»n anvmmtύ n m« Prtarmacaulicai Lacorαiory αf tπ« University β' Uirβcnt. Only tfι« tint (qumon«)rMuctιon poi ooal has Man given.

(formula III), were also determined in the bioliquid assays as described above. The ID-50 values for these compounds are given in Table IA.

(iv) Determination of lowest active dose (L1210 cells: bioliσuid assa ^y)

The lowest active doses for the compounds E.O. 1 , E.O. 2, E.O. 4 and

E.O. 9 were determined according to the method described by Lamberts et al. [Oncology, 4£L 301 (1983)]. In this method, L1210 cells were grown in a series of wells in a culture medium and in the presence of the compound to be tested, as in the determination of L1210 activity in a

bioliquid assay reported above, but the compound was present in respective wells in a different concentration, the concentrations extending over an appropriate range. After a suitable incubation period, the lowest active dose for the compound in question was determined by comparing the diameter of each of the precipitation spots with that of a control containing only the culture and the cell suspension, a smaller diameter indicating growth inhibition (Table II).

TA8LE II

LOWEST ACTIVE DOSE (LAD) (L1210 ; BIOLIQUID ASSAY)

COMPOUND LAO 1 (πg/mi) LAO 2 ^a (πg/ml)

E.Q. 1 1C24-2048 1C24-2048

E.0.2 1024-2048 1024-2048

E.O. a 32-64 64 E.O. 9 512-1C24 102*

¹ LAO 2 refers to a Suoiicatβ β-Oβπmβπt.

(v) Determination of the in vitro activity aσaiπst a panel of five human tumour lines (TNO Riiswiik: Table III)

Compounds E.O. 1 , E.O. 2, E.O. 4, E.O. 4A, E.O. 8, E.O. 9, E.O. 22, E.O. 33, and- E.O. 35 were tested for their activity against five human tumour cell lines at the Radiobiologicai Institute TNO Rijswijk. The new in vitro prescreen uses human tumour clones; it was set up by Dr. P. Lelieveld at TNO Rijswijk and comprises testing compounds in a bioliquid assay for their cytostatic activity against the following five human tumour lines:

TABLE III

IN VITRO ACTIVITY : PANEL OF 5 HUMAN TUMOUR LINES

(TNO.RIJSWIJK)

A 204 CF-7 T 24 WiDr lgR-37

Dose of drug under test = μg/ml

0.01 0.1 1.0 10 0.01 0.1 1.0 10 0.01 0.1 1 .0 10 0.01 0.1 1.0 10 0.01 0.1 1 0 10

E.O. 1 - - ± + - - - + ± - - - + - - ± +

E.O.2 - - - + - - - + - + - - - + - - - +

E.O.4 - - + + - - ± + + + - - ± + - - + +

E.O.4A - - + + - - ±/+ + + + - - + + - ± + +

E.O.8 - - - ± - - - - - ± - - - -l± - - - +

E.O.9 - + + + - + + + + + + - + + + + + + +

E.O. 33 - - - + - - - ± - + - - - -l± - - - +

E.O. 35 - - - ± /+ - - - ± - ± - - - ± - - ± +

E.O. 22 - - + - - ± + - - ± - - +

MMC - ± + + - - ± + ^■ l± ±lr + - - ± + - ±/+ + +

Cisplatin - - ± + - - - ± ^■ l± + - - - + - - ^■ l± +

Methotrex. ± + + + - - - - -l± ±/+ ±/+ - -

±/+ -l± -l± -l± -l± ± ±

Vinblast. -l± ± ±/+ ±/+ + + + + + + + + +

Vincrist. ± ± ±/+ ±1+ + + + + +

A: A 204 cells; rhabdomyosarcoma

M: CF-7 breast cancer cells, hormone sensilive

T: T 24 cells; bladder carcinoma

W:- WiDr cells; colon lumour

Z: lgR-37 cells melanoma

A: A204 cells, rhabdomyosarcoma

M: MCF-7 breast cancer cells, oestrogen receptor-positive [See: G.J.

Goldenberg and E.K. Froese, Cancer Res., 4^ 5147 (1982)] T: T24 cells, bladder carcinoma W: WiDr cells, colon tumour [See: P. Noguchi et al., In Vitro, 1J .

401 (1979)] 2: IgR 37 ceils, melanoma

To substantiate the new TNO prescreeπ, the following applies: CF-breast cancer cells are routinely used to determine hormone sensitivity

- The WiDr tumour line is presently under investigation as one of the colon tumour cell lines for the new in vitro screening panel at the NCI.

After a continuous exposure to the drugs to be tested and to Adriamycin in ^* 24 well tissue culture clusters, type 3524 (Costar)', the remaining cells are fixed and stained. Using increasing drug concentrations the inhibiting concentration can be estimated qualitatively.

The experiments were carried out as follows:

Cells: the human tumour cells are maintained in Dulbecco's medium supplemented with 10 percent foetal calf serum.

A, T and Z ceil suspensions: 5.10 ⁴ cells/ml

M and W cell suspensions: 10^ cells/ml.

The cells were grafted into 16 mm wells for 48 hours (0.5 ml cell suspension per well was used).

Drugs: the compound to be tested was dissolved in appropriate concentrations in a mixture of Hepes-buffered Hanks' balanced salt

solution and ethanol. The drug solutions (0.05 ml) were added to the different wells. The final ethanol concentration in each well was less than one percent.

The cells were continuously incubated with the compound under test at

₅ 37° in an atmosphere of 10 percent CO2 in humidified air for about 72 hours.

Determination of the inhibiting dose: the cells were fixed and stained with a solution of crystal violet in methaπαl/formaldehyde. Scoring: • no inhibition of cell growth

10 + total cell-kill

High and selective in vitro toxicity effects have been observed for some indoloquinones in the TNO panel of human tumour lines (See: Table III). Adπamycin was used as the reference compound; comparisons have also been made against other cytostatics including mitomycin C.

^"■ 5 TABLE IV

COLONY INHIBITION: IN V TRO, CONTINUOUS DRUG EXPOSURE

TUMOUR TYPE E.O. 9 E.O. 4 E.O. 1

0.01 0.1 1.0 0.001 0.01 0.1 0.01 0.1 1.0

Colon 1 81 • 47 * 9 *— 51 - 56 - se¬ 108 - 37 * 21 1

Colon 2 136 - 20 — 1 *** 86 - 70 - l l ** 71 - 24 — 3 -

Lung 0 — * 0 *— 0 *—► 64 - 8 *** 0 *+* 48 * 4 . ** 0 .

Mammary 38 * 2 *~ 0 *+» 93 - 46 * 0 +*+ 99 - 0 *-** 0 -

Renal 123 - 8 — 0 *→→ 103 - 88 - 41 * NE 68 - 0 -

25 * Inhibition βl colony lormaion trβatatf control (T/C) " T/C: • iSO: * 30-50: ♦♦ 10-30: *~. <10

(vi) Determination of the in vitro activity aoainst five slowly growing human tumour lines (University of Freiburg: Table IV) Three indoloquinones were tested at the University of Freiburg 3 ₀ with respect to their in vitro inhibition of colony formation of the

following five human tumour cell lines: lung-NSCLC, breast, renal and two colon lines.

At a low concentration of 0.01 μg/ml, E.O. 9 and E.O. 4 were active against the lung cell line. At a concentration of 0.1 μg/ml, activity was seen with all three analogues in several lines (Table IV).

(vii) Electrochemical properties of the oara-iπdolσαuinones

Additional evidence for the occurrence of a bioreductive activation

TABLE v

HALVE AVΞ POTENTIALS OF SOME PARA- INDOLOQUINONES

E l.2 fmVI at OH - 3 vs Ag/AgC!

COMPOUND I II III ιv

E.O. -367 -435 -775

E.0. 6 -515 -600 -985

E.0. 7 -367

E.0. 8 -380 -780

E.O. 9 -370 -785

E.O. 11 -430

E.O. 12 .420

mechanism during the cytotoxic action of indoloquinones (II) was obtained via electrochemical studies, which have been carried out at the Pharmaceutical Laboratory of the University of Utrecht. The direct current polarogram of indoloquinone E.O. 7 displays a reduction wave representing the reversible reduction of the benzoquinone nucleus. The DC-polarograms of indoloquinones E.O. 1 and E.O. 6, however, are composed of two or more reduction waves, indicating the formation of new eiectrαchemically reducible compounds during the electro¬ chemical reduction, presumably via the elimination of one or both OAc groups (Table V). Similar waves have been observed upon reducing MMC. The latter results have been affirmed by cyclic voltammetric experiments with compound E.O. 6. The shape and size of the reduction and oxidation wave differ considerably. After the first cycle , however,

TABLE VI

THE INFLUENCE OF THE NATURE OF THE SUBSTITUENTS R2 AND R3 ON THE ID-50 VALUES

ID-50(μg/ml)

CO P. R *3 X ₂ ELL L1210

EO.1 OCH3 H OAc OAc 0.3 2.3

EO.33 OCH3 CH ₃ OAc OAc 2.2 3.6 E.O.60 H OAc OAc 3.3 >10

-N 0

E.0.47 NHCH ₂ CH ₂ (CH ₃ ) ₂ H OAc OAc 4.4 >10 E.O.52 H OAc OAc 3.3 >10

CH ₂ CH ₂ NH-

E.0.58 NHC ₂ H ₅ H OAc OAc >10 >10

E.0.5 NHCH ₂ CH2θH H OAc OAc >10 >10 ε.0.6 NHCH2CHCHCH2OH H OAc OAc >10 >10

E.0.1B OCH3 H OAc OH 0.05 0.9

E.O.48 NHCH ₂ CH ₂ (CH ₃ ) ₂ H OAc CH >10 >10

E.O.62 H OAc CH >10 >10 -CH ₂ CH ₂ NH-

E.0.7 OCH3 H CH CH 4.2 0.5 E.0.18 OCH3 CH ₃ CH CH 2.2 2.4

E.O.12 H CH CH >10 3.8

-N O \ t

E.0.11 CH CH >10 >10

E.O.51 ^• o OH

H CH CH ^' 4.5 >10

CH ₂ CH ₂ NH-

E.0.56 NHC ₂ H ₅ CH CH >10 5.1

'The compounds in the three different series have been arranged according to the increasing electrondonating properties of the substituents R ₂ ^{and **} 3-

the chemically trapped intermediates afford rather reversible vαltammograms.

It was clear from these experiments that a consecutive chemical reaction occurs after the reduction of the quinone ring, which might be important for the activation in vivo.

(vii i) Correlation of structural parameters with in vitro cytotoxicity of the indoloquinones II.

The influence of the nature of the substituents Ro and R on the ID-50 values (Table VH . The following conclusions can be drawn:

Generally the indoloquinones display a different toxicity against the two cell lines.

The activity decreases considerably upon increasing the electron- donating nature of the R and R3 substituents. - The rather high activities (R-1 ) of indoloquinones E.O. 47 and E.O. 52 may be ascribed to an intramolecular assisted proton abstraction by the additional nitrogen atoms in the reductive activation sequence. Presumably a similar process is also partly responsible for the promising activity of a new C-7 M MC analogue, carrying a substituent at carbon atom C-7.

The influence of the nature of the leaving groups X-i and Xo on the

ID-50 values (Table VIIV The main conclusion, which can be drawn from this table is that a reduction of the leaving group abilities of substituents X-j and X2 does increase the 1D-50 values. This effect is the most pronounced for the substituent X- _] at the C-10 carbon atom.

Striking is the rather high R-1 activity observed for compound E.O. 39. Reduction experiments (H2 tθ2) have provided evidence for the

propensity of the one- or two-electron reduced indoloquinone E.O. 39 to decompose into reactive iminium and quinone methide species (Scheme XII).

TABLE VII

THE INFLUENCE OF THE NATURE OF THE LEAVING GROUPS Xi AND X ON THE ID -50 VALUES .

COMP. R- R *1 *2 HJ. 11212.

E.O. 1 OCH ₃ H OAc OAc 0.3 2.3 0 ε.o iA OCH ₃ H CH OAc 3.2 2.3 ε.o. is OCH ₃ H OAc ^' CH 0.05 0.9 ε.o.2 OCH ₃ H OCCCCH3 CCCOCH3 0.4 0.5

E.O. 36 OCH3 H 0C0C ₆ H ₅ 2.6 0.7 ε.O. 37 OCH3 H OCONHC ₄ H _g 6.5 > 10 5 E.O. 33 OCH3 H 0C0ΛJHC ₆ H ₅ 8.5 > 10 ε.o. 13 0CH3 H 0C0NHCH ₂ CH ₂ CI 1.1 10 ε.o. io OCH3 H OCH3 0C0NHCH ₂ CH ₂ CI >10 > 10 ε.o. 1 0CH3 H H OAc > 10 > 10 ε.O. 39 OCH ₃ H NHC ₆ H ₅ OAc 1.0 10

E.O. 39

Scheme XII

The in vitro activities of th ariridiπvt indoloπuinones (Table VIII:

Fig, n, Substitution of the methoxy group at the quinone nucleus by an aziridiπyl group reduces the ID-50 values - especially in the R-1 series - to a great extent. The presence of the additional alkylation center in this group of indoloquinones has been clearly demonstrated on reducing compound E.O. 4 with Na2S θ4 and simultaneous trapping of the reactive intermediate with N,N-diethyldithiocarbamate-anions (Scheme XIII).

TABLE VIII

IN VTTRO ACTIVITY OF THE BIOREDUCTIVE AUCΪLATING AZIRIDINYL INDOLOQUINONES

ID-50{μg/ml)

COMP. R ₂ *3 *1 *2 flil U21Q

E0.4 H H OAC OAc 0.025 1.6

E.0. A H H OAC CH 0.022 0.15

15 E0.9 H H CH CH 0.0034 0.46

E.0. 35 H CH3 OAc OAc 0.32 0.55

E.0. 19 H CH3 CH CH 0.12 2.0

E.0. 53 α+3 H OAc OAc 1.4 3.4

£0.3 CH3 H CH CH 7.8 0.43

20 MMC 0.03 0.05

23. 21

SCHEME XIII

89

The inhibiting doses observed in the R-1 experiments are generally lower than those in the L1210 experiments. Introduction of a methyl group either on one of the carbon atoms of the aziridinyl ring or at the C-6 carbon atom of the indole nucleus, reduces the cytotoxic activities of the indoloquinones considerably.

BIOLOGICAL DATA

(b) In vivo experiments (i) Determination of the acute toxicitv

The acute toxicity experiments were caried out with male C57 Black/Rij X CBA/Rij Fl hybrid mice at TNO, Rijswijk. The drugs were admiπstrated IP as suspensions in carboxymethyl cellulose (2%) as a single dose at the first day. The results are given in Table IX below.

(ϋ) In vivo L1210 ex ^p eriments

The in vivo L1210 experiments were carried out in male Balb/c X DBA.2 Fl hybrid mice at TNO, Rijswijk. In these experiments also, the

TABLE IX

ACUTE TOXICITY AND IN VTVO L1210 EXPERIMENTS {TNO, RUSWUK).

COMPOUND DOSES (MG/KG) %T/C LD-50 (MG/KG)

6.0.1 10-30

10,15,25 100

E.O.2 30-100

30 100

40 113 0 60 100 80 tax.

E.0.4 -30

4.8.12 100 15 tox. 5 20 100 25.30 tox.

E.0.7 >100

100.125.160. 200 89

250 tox. 0 E.O.9 10

2 144

4 100 6.8.10 tox.

E.0.16 80 5 80 89 100 100

E.0.17 50-80

50.60 100

MMC ³ 8 0 4 200 5 175 6 175

* Rβmtri «t al- J. «d Chβrn..28.1β (1983): max. aflaα (% T/C) 124 • 153 (3 - β mo/kα;): i rang* based on numerous determinations.

5drugs were administered IP as suspensions in carboxymethyl cellulose (2%) as a single dose at the first day. The results are also given in Table IX.

(iii) In vivo P388 experiments

These experiments were carried out at the Iπsitutθ Jules Bordθt, ⁽³ Brussels, on CDF, female mice. The mice were inoculated intraperitoπβally (IP) with 1x 10 ⁶ P 388 tumour cells. After 24 hours,

the compounds were administrated as suspensions in "Tween-80" or using saline as vehicle. Six mice were used at each dose of the compound, given on days 1 to 5 and eighteen control mice were injected with saline.

Two indoloquinones (E.O. 4 and E.O. 35) showed reproducible and marginal activity at the optimal dose (T/C approx.: 130 %) [Table X].

TABLE x

IN VTVO P388 EXPERIMENTS (NCI; BRUSSELS)

COMPOUND DOSES (MG/KG) MAX T/C { ) E O 1 7 5 105

E O 2 10 120 E.O •: 10 135 E.O 4A 100 120 E.O. 9 1.5 106 5 E O 25 400 131

(iv) Tumour growth inhibition of human tumour xenoorafts by indo¬ loquinone E.O. 9 in nude mice. Based on the results obtained during the in vitro experiments indoloquinone E.O. 9 was selected for an extended in vivo study. The o activity of this compound has been determined . in four human tumour xenografts transplanted in nude mice.

The xenograft experiments have been carried by Dr. H.H. Fiebig of the

University of Freiburg [Lung-NSCLC (LXGF) line, Renal line, Breast line;

See: H.H. Fiebig et al., Behring Iπst. Mitt., 7__ 343] and by Dr. E. Boven of ₅ the Free Uπiversty Hospital Amsterdam [Ovarian (MRI-H-207) line; See: E.

Boven et al., Cancer Res., jϊ, 86 (1985) and E. Boven et al., Eur. J. Cliπ.

Oncol., 21. 1253, (1985)].

Lung- NSCLC (LXFG) line:

(This is the same line as evaluated in vitro: Table IV). The tumour was transplanted s.c. and E.O. 9 was adminstarted i.v. on days 14 and 21 after transplantation at a dose of 4 and 6 mg/kg. 5 An optimal growth inhibition of 42 % and a specific growth delay of 1.03 was reached (Fig. II).

Renal line (RXF 423):

(This is the same line as evaluated in vitro: Table IV). The tumour was transplanted s.c. and E.O. 9 was administrated i.v. on io days 22 and 29 after transplantation at a dose of 4 and 6 mg/kg. No significant growth inhibition was achieved (Fig. III).

Breast line:

(Due to low take in vivg this is not the same line as evaluated in vitro). The tumour was transplanted s.c. and E.O. 9 was administrated i.v. on isdays 46 and 53 after transplantation at a dose of 4 and 6 mg/kg. An optimal growth inhibition of 51 % was reached (Fig. IV).

Ovarian (MRl-H-207) line:

These experiments have been carried out by Dr. E. Boven of the Free University Hospital, Amsterdam. ?n Materials and method?; Mice: C57B1 athymic (nu/nu) female mice, 8 to 10 weeks of age were used for s.c. bilateral implantation of tumour fragments 2 mm diameter. Treatment: E.O. 9 was dissolved in saline at a concentration of 0.5

mg/ml prior to administration. Drug doses and schedules were derived from studies in experimental murine tumour systems and from orientation studies in non-tumour-bearing nude mice. Treatment was started i.v. when tumours measured 50-150 rnrn^. After randomization a group of 6-7 tumour-bearing mice was treated and a group of 5-6 tumour-bearing mice served as control.

Evaluation of efficacy: Tumours were measured twice weekly in three dimensions. Efficacy was expressed as the mean of relative tumour volume iπ treated animals vs. that iπ control animals x 100 (%T/C). The optimal value was calculated within 35 days after the iast injection. Toxic death (dead mice within two weeks after the last injection) were excluded from the evaluation.

MRi-H-207: An uπdiffereπtiated adenocarcinoma kindly provided by Dr. A. E. Bogden, Worcester MA, with a doubling time of 3-5 days.

TABLE XI

ACTIVITY OF CYTOSTATIC AGENTS IN THE OVARIAN CANCER XENOGRAFT MRI-H-207

COMPOUND DOSE DAYS OF T/C% TOXIC

(MG/KG) TREATMENT DEATHS

E.0. 9 5 0.7 3.8 0/7

E.0.9 6 0.7 2.4 1/7

MMC 5 0.7 CR ^a 1/7

Cisplatm 5 0.8 CR 0/7

Carboplatin 60 0.6 CR 0/6 M 150 0, 2. 4. 6 CR 1/6

CTX 180 0. 15 CR 27

DXR 10 0, 7 CR 1/6

MTX 150 0. 9. 16 47 2/7

5FU 60 0, 6. 13. 20 63 n a: CR • complete remission

Results

The tumour was transplanted i.v. at a dose of 5 and 6 mg/kg q8dx2. An optimal growth inhibition of 98 % was reached (Fig. V; Table XI). Comparitive activity of clinical relevant compounds in ovarian cance are given as well in Table XI.