WO1996018611A2

WO1996018611A2 - Benzophenanthrolines and related fused acridines

Info

Publication number: WO1996018611A2
Application number: PCT/GB1995/002948
Authority: WO
Inventors: Paul William Groundwater; Kevin Richard Solomons; Ali Munawar Munawar
Original assignee: University College Cardiff Consultants Limited
Priority date: 1994-12-16
Filing date: 1995-12-18
Publication date: 1996-06-20
Also published as: WO1996018611A3; AU4266296A; GB9517506D0

Abstract

7-amino-5,6-dihydrobenzo[b][1,7]phenanthroline (compound MAM 102; Figure 1), or related fused acridines, are prepared by the condensation of A and B under dehydration conditions. 7-amino[b][1,7]phenanthroline (compound MAM 58, Figure 2), or similar related fused acridines, are then prepared by dehydrogenation of the condensation product. The compounds are inhibitors of protein tyrosine kinases and can be used in pharmaceutical formulations for the treatment of mammalian carcinomas.

Description

Benzophenanthrolines and Related Fused Acridines

The invention relates to benzophenanthrolines and related fused acridines, and in particular such compounds active as inhibitors of protein tyrosine kinases (typically for use in chemotherapeutic treatment of cancer).

Protein tyrosine kinases (PTKs) are enzymes which catalyse the transfer of phosphates from a donor atom (for example, in adenosinetriphosphate, ATP), to the tyrosine residue of a peptide substrate. PTKs form an integral part of the cell surface receptors of several growth factors, including epidermal growth factor (EGF), platelet-derived growth factor (PDGF) and insulin. The EGF receptor molecule comprises an outer receptor region which specifically binds the EGF, a transmembrane region and a catalytic cytoplasmic receptor domain. PTKs are also the products of several oncogenes and the cellular genes from which they originate (proto-oncogenes). Binding of the appropriate growth factor to the extra cellular domain of the receptor results in receptor activation and leads, ultimately, to cell proliferation via a cascade of PTKs.

In several diseases, such as many carcinomas, atherosclerosis and psoriasis, cells have generally lost the ability to regulate the activity of the PTKs and these enzymes become continuously activated resulting in uncontrolled cell proliferation.

Flavones, such as myricetin, are known to be inhibitors of PTKs. Myricetin has been shown to inhibit the PTK activity of the oncogene product ppl30fbs by competition with ATP, but inhibits the PTK activity of the insulin receptor non-competitively to ATP. Thus, there may be a flavone binding site at, or near the ATP binding site of the oncogene- encoded PTKs but remote from the ATP binding site of the insulin receptor.

We have surprisingly found a new class of compounds, which can function as PTK inhibitors. According to one aspect of the present invention, there are provided benzophenanthrolines and related fused acridines represented by the general formula (I):

where R¹, R² and X each represents H, -OH or a lower alkoxyl group; R³ represents -CH₂-CH₂- or -CH=CH- ; R⁴ represents the atoms necessary to complete an optionally substituted 5- or 6- member heterocyclic aromatic ring; and Z represents -OH or -NH₂.

In preferred embodiments of the invention R⁴ represents -N=CH-CH=CH-,

-CH=CH-O-, or -C(O)-CH=C-O- (where Ar is aryl).

I

Ar

In particularly preferred embodiments of the invention, the compound is a phenanthroline, which may be 7-aminobenzo[b][l ,7]phenanthroline or 7-amino-5,6- dihydrobenzo[b][l ,7]phenanthroline (these both being compounds of formula I in which R\ R² and X are all hydrogen, Z is -NH₂ and R⁴ is -N=CH-CH=CH-).

According to a further aspect of the present invention, there is provided an inhibitor of protein tyrosine kinases, which inhibitor has the general formula (I), for use in the treatment of mammalian carcinomas.

According to another aspect of the present invention, there is provided a pharmaceutical composition, comprising an inhibitor of PTKs of the general formula (I), together with a pharmaceutical carrier, diluent or excipient therefor. Thus, advantageously the pharmaceutical composition may be used for the treatment of carcinomas. Preferably, the composition is in a suitable form to be applied intravenously (for example in a sterile aqueous medium, such as a saline solution). In a preferred embodiment of the invention, the composition may be used in treatment of mammalian carcinomas, such as human carcinomas. According to a further aspect of the present invention there is provided a process for preparing a compound of the formula (I) in which R³ is -CH₂-CH₂- which comprises reacting a compound of formula A with a compound of formula B under conditions permitting removal of water from the condensation reaction.

A B

According to yet a further aspect of the invention there is also provided a process for preparing a benzophenanthroline of the formula (I) in which R³ is -CH=CH- which comprises dehydrogenation of the phenanthroline produced by the process described above. Preferably compound A comprises 5,6,7,8 - tetrahydroquinoline and compound B comprises 2-aminobenzonitrile.

The invention may be more clearly understood from the following description of .an embodiment of the invention, with reference to the following drawings and worked Examples, given by way of illustration only, wherein:

Figure 1 shows chemical formulae of exemplary PTK inhibitors according to the present invention;

Figure 2 is a graphical representation of the response of a MKN 45 cell line to high concentrations of Epidermal Growth Factor (EGF);

Figure 3 is a graphical representation of the response of MKN 45 cells to low concentrations of EGF;

Figure 4 is a graphical representation of the response of a SV40-3T3 cell line to high concentrations of EGF; and

Figure 5 is a tabulated representation of the level of inhibition of spontaneous proliferation of MKN 45 cells by benzo[b][l ,7]phenanthrolines and their effect on MKN 45 cell viability. Example 1

A number of 7-aminobenzo[b][l,7]phenanthrolines were prepared according to the following scheme.

-O, D F

7-Amino-5,6-dihydrobenzo[b][l,7]phenanthroline (MAM 102, Figure 1)

Method A

A mixture of 5,6,7,8-tetr.ahydroquinolin-5-one (1 in which X=H) (1.34g, 9.1mmol), 2-aminobenzonitrile (2 in which R' =R²=H) (1.07g, 9.1mmol), and p- toluenesulfonic acid (0.107g, 0.91mmol) in toluene (50ml) was refluxed for 100 hours with azeotropic removal of water. The toluene was removed, the residue was dissolved in DME (50ml), and sodium amide (0.89g, 22.7mmol) was added. The mixture was refluxed for 3 hours under a nitrogen atmosphere.

The solvent was evaporated and the residue was treated with ammonium chloride solution (10%w/v; 50ml), filtered and recrystallized from methanol to give the title compound (1.5g, 67%), m.p. 108-110°C (resolidifies then melts again at 166-168°C) (Found: C, 72.2; H, 6.1 ; N, 15.63, C₁₆H₁₃N₃.H₂O requires C, 72.4; H, 5.7; N, 15.84%); _^ (Nujo^/cm-¹ 1661, 1613, and 1577; δ_H (360MHz; DMSO-d₆) 3.00 (2H, m, CH₂), 3.08 (2H, m, CH₂), 6.7 (2H, s, NH₂), 7.4 (2H, m), 7.59 (1H, t, J=7.8), 7.83 (1H, d, J=8.4), 8.23 (1H, d, J=8.4), 8.49 (1H, dd, J=4.8,1.6), and 8.67 (1H, dd, J=7.8,1.6); δC (90MHz;DMSO-d₆21.39 (CH₂), 30.25 (CH₂), 108.12 (q), 118.31 (q), 122.38 (CH), 122.45 (CH), 123.9 (CH), 128.7 (CH), 129.2 (CH), 130.5 (q), 132.8 (CH), 147.4 (CH), 148.2 (q), 149.3 (CH), 150.4 (q), and 158.45 (CH); m/z 247 (M⁺, 100%), 246 (65), 124 (65), 123 (8), 110 (10), 109 (5), 102 (3), 96 (6), and 77 (2). Method B

A mixture of 5,6,7,8-tetrahydroquinolin-5-one (1) (0.3g, 2.04mmol), 2- aminobenzonitrile (2) (0.24g, 2.04mmol) and polyphosphoric acid (5g) was heated at 150- 160°C for three hours under nitrogen. The mixture was poured into ice cold water and neutralized with ammonium hydroxide. Recrystallization from methanol gave the title compound (0.153g, 30%). 7-Aminobenzo[b][l,7]phenanthroline (MAM 58, Figure 1)

7-Amino-5,6-dihydrobenzo[b][l,7]phenanthroline (0.25g, 0.94 mmol) and activated manganese dioxide (2.5g) in DMF (25ml) was heated at reflux overnight. The reaction mixture was filtered through a bed of celite. The solvent was evaporated and the solid recrystallized from ethanol to give the title compound (0.1035g, 42%), m.p. 246-248°C (Found: C, 78.4; H, 4.6; N, 16.95. C₁₆H„N₃ requires C, 78.35; H, 4.5; N, 17.1 %); v,^ (Nujoiycπr¹ 3304, 1657, and 1604; δ_H (360MHz; DMSO-d₆) 7.48 (1H, dt, J =7.2,2.0), 7.68- 7.70 (2H m), 7.77 (dt, 1H, J =7.0,2.0), 7.85 (2H, s, NH₂), 8.03 (1H, d, J=7.2), 8.54 (1H, d, J = 10.0), 8.56 (1H, d, J = 10.0), 8.99 (1H, dd, J=4,2.0), and 9.59 (1H, dd, J=8.0,2.0); δ_c (90MHz;DMSO-d₆) 108.62 (q), 115.10 (q), 121.7 (CH), 123.1 (CH), 123.24 (2CH), 125.24 (CH), 126.77 (q), 129.32 (CH), 132.22 (CH), 132.98 (CH), 146.65 (q), 146.65 (q), 147.86 (q), 150.01 (q), 150.28 (CH), and 151.15 (q); m/z 245 (M⁺, 100%), 218 (9), 217 (8), 123 (12), and 96 (13). Example 2

To investigate the action of the inhibitors against carcinoma, two cell lines were used which were both of a transformed nature. The MKN 45 cells were human derived gastric carcinoma cells, and the SV40-3T3 cells were virally transformed (by SV40 virus of mouse fibroblast origin). Cell Culture

Cell lines were kept in continuous culture over the period of the investigation. This required sub-culturing twice weekly into fresh media and continuous monitoring of the cultures in order to meet the demands for cells when required. The MKN 45 cells were grown in RPMI 1640 with 10% fetal calf serum (FCS) supplement, and the SV40-3T3 in Dulbecco modified eagle medium (M.E.M.) supplemented with 10% FCS. As both cell lines adhered to the culture flask and grew in the form of a monolayer rather than in suspension, a dissociation treatment (trypsin 0.05% plus ethylenediamine tetraacetic acid (EDTA)) 0.02% in calcium/magnesium free phosphate buffered saline (PBS) was required for their resuspension. As with all work involving cell culture, it was necessary to apply aseptic techniques throughout the handling of the cells during their subculturing and experimental set up, in order to prevent contamination. Inhibitor and EGF treatment

The inhibitors to be tested, in the form of powder were: 7-amino-5,6-dihydrobenzo[b][l,7]phenanthroline and 7-aminobenzo[b][l,7]phenanthroline. Molecular structures of the compounds tested are shown as MAM 102 and MAM 58 respectively in Figure 1. They were dissolved in DMSO and diluted for their application to the test cultures before the EGF stimulus. This gave the compounds time to pass through the cell membrane and interact with PTK in the cytoplasm before the EGF stimulation could take place. The benzo [b][l ,7] phenanthrolines, were applied at a range of concentrations between 1 and 10 μM. The human recombinant EGF (expressed in S. Cerevisiae) was added to the wells at a range of concentrations between 100 and 0 ng/ml, and then in later tests for the MKN 45 cells at the lower concentrations between 10 and 0 ng/ml, due to sensitivity of the cells to the EGF treatment (in a negative way). Proliferation Assays

To examine the effects of the FCS on the induction of cell proliferation concentrations between 10 and 0.01 % FCS diluted in growth media were tested. Results of these tests indicated for suitable FCS dosages which could be applied to

cultures to support the cells without any proliferation induction. The EGF stimulus was added first at a concentration range between 100 and 1 μM which produced less proliferation at the higher concentrations (i.e. negative response), and therefore subsequent additions were again made at lower concentrations of 10 to 0.1 μM for further tests. The tests were performed in flat welled micro- titre trays, in which each well was inoculated with 100 μL of cell suspension of standard concentration 2 x 10⁵ cells/ml (day 1). The cells were allowed to settle and adhere to the substratum for 24 hrs., and the FCS containing media was then replaced with RPMI/DMEM serum free media (day 2). After a further 48 hrs. the inhibitor was added (am day 4) and left for 4 hrs. to take effect. Then the stimulus was supplied to the cells at the required concentration i.e. EGF, FCS (pm day 4), and the cells were left overnight. Finally 10 μL of ³H thymidine (18.5 KBq) was added to each test well, and incubated for periods of 4 to 7 hrs (a.m.; day 5), after which the cells could be harvested and ³H counts analysed (p.m.; day 5).

At harvesting the supernatant of each well was removed carefully and the remaining extracellular ³H washed off with Dulbecco's phosphate buffered saline (PBS). The labelled cells which still adhered to the floor of the well, were then lysed to release their radioactivity, with 100 μL of 1 molar NaOH (for 30 min), and transferred to the vial. Each well was then rinsed with 150 μL of TX-100 2% and 150 μL of distilled water (x2), respectively and this was then also transferred to the corresponding vial which contained 10 ml of scintillation fluid. After then sealing and mixing the content of each vial, the samples were placed into the LKB scintillation counter programmed to count ³H.

This weekly procedure was continued throughout the investigation and modified where need be, for e.g. testing EGF or FCS with no inhibitor treatments. The more proliferation had occurred in a specific sample, the higher the proportions of ³H detected and the various treatments could thereby be compared.

The first step towards the analysis of the results was to reduce the data counts collected by calculating the averages of all the wells sampled for each separate test. These were usually averages of three to six figures (wells), and provided a clearer overview of the data. The percentages of inhibition of cell proliferation were then calculated for a general analysis of the results. This was done by working out the difference between the count obtained with no inhibitor application (control), and the counts obtained from the application of different inhibitor concentrations for each set of data. Further then, the exact percentages of EGF stimulated proliferation and spontaneous proliferation were calculated, to extract the exact proportions of EGF stimulated inhibition. The exact percentage of proliferation inhibition of specifically EGF stimulated proliferation were also calculated in an attempt to separate out this from spontaneous proliferation inhibition. The standard deviation values calculated for each result obώned was a guide to the variation of the data and therefore giving an indication to its significance. Viability Test

A check on the viability of cells was especially important in these experiments to ensure that lower ³H counts were due to less cell proliferation by inhibition of PTK, and not due to non-specific toxic effects of the inhibitor. Cell viability was therefore checked routinely in all experiments. For the viability tests a common combination of stain was used which was made up of FDA (fluorescein diacetate 5 mg/ml) and ethidium bromide (5 mg/ml in medium) at 1 : 100 and applied to the cell samples at 1 : 1 ratio. The living cells take up the fluorescein and therefore appear green under the fluorescence microscope, whilst the dead cells appear red. Results

The results illustrate the degree of inhibition of proliferation of EGF stimulated cells. The first set of results concerns the response of the cell lines used to EGF alone. Data for FCS alone and combinations of FCS and EGF were also collected but are not presented as these experiments were carried out only to establish the suitable concentrations of FCS for use in tests, without stimulation of cell proliferation (0.01 % FCS). Following on from this, the data recording the action of the inhibitor on spontaneous cell proliferation are presented (i.e. proliferation without specific stimulation), and finally results relating to inhibition of EGF stimulated proliferation are reported. Cell response to FCS and EGF

The results of the experiment designed to test the proliferation response of MKN 45 to EGF are shown in Fig.2. From this it is clear that these cells were not stimulated to proliferate by EGF. It appeared that their proliferation was suppressed by the growth factor, as the wells treated with EGF contained less ³H thymidine than the control wells with no EGF. The MKN 45 cells were sensitive to the growth factor but in a negative correlation, at the concentrations applied and a detrimental effect was noticed when observing the morphology of the cells under phase contrast. The test was therefore repeated using lower EGF concentrations ranging from 10 to 0 ng/ml per well and with the addition of 0.1 % FCS, which has a cooperative effect with the EGF to bring out the full potential of the growth factor (previous work had suggested that a concentration of 0.01 % FCS would be a safe concentration to use, as FCS at such high dilutions does not induce proliferation alone). The results gained from these further tests are summarised in Fig.3, and provide further evidence for the conclusion that the MKN 45 cells are not stimulated to proliferate in response to EGF. This is an indication of the fact that these cells may not have any EGF-receptors expressed on their surface or that receptors which are present are non¬ functional in binding EGF to activate PTK.

From the results of similar tests done with the SV40-3T3 cells, very different observations were made (see Fig.4). These indicated stimulation of the proliferation response, even at the higher concentrations of EGF which inhibited growth of the human cells. The higher growth factor concentration did not have a detrimental effect on the SV40 cells (as was seen for the MKN 45), and rather than stopping their division the cells made use of the EGF to stimulate further proliferation. It was therefore concluded that the transformed mouse cells are stimulated to proliferate by EGF, which indicates the presence of functional EGF-receptors on their surface. jPrQltferptiQn

Figure 5 shows that inhibition occurs with addition of MAM 102 and MAM 58 to MKN 45 cells and that inhibition also increases for these compounds when the concentration of inhibitor is raised from 1 to 10μM.

Claims

Claims •

1. A compound of the general formula (I):

where R', R² and X each represents H, -OH or a lower alkoxyl group; R³ represents -CH₂-CH₂- or -CH=CH- ; R⁴ represents the atoms necessary to complete an optionally substituted 5- or 6- member heterocyclic aromatic ring; and Z represents -OH or -NH₂.

2. A compound according to claim 1, wherein R⁴ represents -CH=CH-O-, -N=CH-CH=CH-, or -C(O)-CH=C-O- (where Ar is aryl) .

I

Ar

3. 7-amino[b][l,7]phenjuιthroline.

4. 7-amino-5,6-dihydrobenzo[b][l,7]phenanthroline.

5. For use in the treatment of mammalian carcinoma, a compound according to any of claims 1 to 4.

6. Use according to claim 5, wherein said carcinoma is human.

7. A pharmaceutical composition comprising a compound according to any of claims 1 to 4, together with a pharmaceutical carrier, diluent or excipient therefor.

8. A pharmaceutical composition according to claim 7, for use in the treatment of mammalian carcinoma.

9. A pharmaceutical composition according to claim 8, wherein said carcinoma is human carcinoma.

10. A process for preparing a compound according to claim 1 in which R³ is -CH₂-CH₂- which comprises reacting a compound of formula A with a compound of formula B

A B under conditions permitting removal of water from the condensation reaction.

11. A process for preparing a compound according to claim 1 in which R³ is

-CH = CH-, which comprises dehydrogenating the product of the process according to claim 10.

12. A process according to claim 10 or 11, wherein compound A is 5,6,7,8- tetrahydroquinoline and compound B is 2-aminobenzonitrile.