The synthesis and bioloqical properties of a 142-methylpyridin-4-yl ) olivacine derivative

Starting from 2-(6-methoxy-I -methyl-9H-carbazol-2-y1)ethylamine and 2methylisonicotinic acid, 9-hydroxy-5,6-dimethyl-I -(2-methylpyridin-4-y1)-6Hpyrido[4,3-blcarbazole (5) was obtained. The new compound showed significant cytostatic activity for cultured L1210 cells and no inhibition of growth of the E. coli 056 strain was observed. The bactericidal activity of normal human serum against E. coli 056 was not affected by the examined compound 5 and its isomer 4.


Introduction
The high in vitro anticancer activity of the natural alkaloids ellipticine 1 and its isomer olivacine l a (Fig. 1) provokes great interest in modifying their structure to obtain new derivatives with a more advantageous therapeutic index.
Recently, our further modifications of the structure of olivacine have led to new derivatives, such as 2 -4, which are presented in Fig. 2  This pyrido[4,3-blcarbazole derivative 5 was obtained according to Scheme 1 below.

Results and Discussion
The starting compound, 2-(6-methoxy-I -methyl-9H-carbazol-2-yl)ethylamine (6), has already been described [6].It was allowed to react with a mixed anhydride of 2-methylisonicotinic acid and cyclization of the resulting amide 7 with phosphorus oxychloride in boiling toluene gave 9-methoxy-5-methyl-1-(2-methylpyridin-4-yl)-  1, all products were more active than olivacine, depending on the kind of substituent at position 1 in the main heterocyclic system, the analogues (4 and 5) of the predecessor 2 display in vitro cytostatic activity of the same order.
The complement system plays a key role in the defence against Tab. 2. Growth of the E. coli 056 strain in YP in the presence of olivacine derivatives 4 and 5 *c.f.u., colony-forming units The results presented in Table 2 show no inhibitory effect of olivacine derivatives 4 and 5 on the multiplication of E. coli 056 strain.The number of bacterial cells growing in the presence or absence of the examined compounds was nearly equal.
A similar situation was observed in the investigation of anticomplement activity of derivatives 4 and 5.
The results shown in Table 3 give no indication that olivacine derivatives 4 and 5 influence the level of activation and cytolytic effect of complement of normal bovine serum.These data indicate the possibility of using these olivacine derivatives as cytostatic drugs in the therapy of tumor diseases.The results also indicate that the olivacine derivatives show higher cytostatic activity than olivacine.

Experimental
Melting points were determined on a Kofler apparatus and were uncorrected; H NMR spectra were recorded on a Tesla BS 587 A at 80 MHz or on a Bruker 300 at 300.14 MHz, using TMS as the internal standard.Column chromatography was carried out on silica gel (Merck Kieselgel 100).All of the newly obtained compounds were analyzed for C, H, and N, and the analytical results were within k0.4% of the theoretical values.

-M e t h o x y -5 -m e t h y W ( 2 -m e t h y l p y r l d i n ~ carbazole (8)
The amide 7 (747 mg, 2 mmol) was dissolved in boiling toluene (70 ml) and treated drop-wise with phosphorous oxychloride (5 ml).Reflux was continued for a 12 h period, and evaporation under reduced pressure afforded a residue which was taken up in water (IOOml), basified to pH 9-10 with concentrated aqueous ammonia, and extracted with methylene chloride.Evaporation of the solvent provided a solid residue, which was recrystallized from ethanol to give yellow crystals (320 mg, 45%

Preparation of bacterial culture
The E. coli 056 strains were growth in YP broth overnight, and then bacterial cells of the early exponential growth phase were transferred to fresh YP and incubated at 37°C for I h .After incubation, the bacterial cells were centrifuged (4000 rpm for 20 min) and resuspended in 0.8% NaCI.

Effect of olivacine derivatives on growth of bacteria
The bacteria in 0.8% NaCl were mixed with the DMSO solution of olivacine derivatives and incubated at 37°C in a water bath After 0, 60, and 180 min.probes were collected, diluted, and plated on nutrient agar plates for 18 h at 37°C.The number of colony-forming units (c.f.u.) at time 0 was taken as 100%.

Bactericidal activity of normal bovine serum (NBS)
The bactericidal activity of NBS was determinated according to the procedure of Doroszkiewicz et al. as described previously [9].Briefly, the bacterial suspensions in saline were mixed with 12.5% NBS alone or with olivacine derivatives in a suitable concentration.Bacteria with serum were incubated in a water bath at 37°C and samples were collected at 0, 60 and 180 min., diluted, and cultured on nutrient agar plates for 18 h at 37°C.The number of colony-forming units (c.f.u.) at time 0 was taken as 100%.

Fig. 1 .Fig. 2 .Fig. 3 .
Fig. 1.The structure of ellipticine (1) and its isomer olivacine (la) microorganisms, and the bactericidal activity of serum depends on many different factors.The 0-specific side chains of lipopolysaccharides (LPS) and outer membrane proteins (OMPs) play a decisive role in this phenomenon [4,5,8].The aim of this study was to determine and compare the influence of olivacine 5 on the growth of bacteria E. coli 056 and compare the level of bactericidal activity of NBS against the E. coli 056 strain.