CA2232074A1 - Granulatimide compounds as g2 checkpoint inhibitors - Google Patents

Abstract

Compounds are provided that inhibit the G2 checkpoint in the cell cycle and are useful for sensitizing G1 deficient cancer cells to killing by DNA damaging agents.
Compounds of this invention have the general formula:
wherein W is selected from the group consisting of:

Description

' CA 02232074 1998-03-13 Field of Invention This invention relates to'substances which inhibit arrest of the cell cycle during the G2 phase and which are useful for sensitizing cancer cells to the effect of DNA
damaging agents.
Background Normal cells respond 1.o DNA damage in one of two ways, depending upon their type and the degree of damage. The cells may activate an apoptotic pathway leading to suicide of the cell and its removal or, survival of a damaged cell may be promoted by activating checkpoints that temporarily halt the normal cycle of growth and division to allow time for DNA repair. The checkpoints operate during the G1 phase of the cycle so that DNA is repaired before it is replicated in the S phase; and, during the G2 phase so that DNA is repaired before chromosomes are segregated in the mitosis phase (M).
Few G2 checkpoint inhibitors are known. Two groups have been found: the purine analogues (caffeine, pentoxifylline, 2-aminopurine, 6-dimethylaminopurine), and staurosporine with its derivative UCN-O1 (7-hydroxystaurosporine). In addition, the protein phosphatase inhibitors (o~;adaic acid and fostriecin) can act as G2 checkpoint inhibitors but they also induce premature mitosis in the absence of DNA damage and have tumour-promoting activity.

- ,7 _ Experiments employing cells deficient in the tumour suppressor protein p53 have demonstrated the value of the two groups of G2 checkpoint inhibitors described above, for selectively sensitizing cancer cells. Pentoxifylline has been shown to enhance cispl.atin induced killing of p53- MCF-7 cells 30-fold and radiation induced killing of p53- A549 human lung adenocarcinoma cells 5-fold. Caffeine has been shown to enhance radiation induced killing of p53- mouse embryonic fibroblasts 3-fold and radiation induced killing of p53- A549 human lung adenocarcinoma cells 5-fold. UCN-O1 has been shown to enhance cisplatin induced killing of p53-MCF-7 cells 25-fold. UCN-O1 is active in vitro as a G2 checkpoint inhibitor in the submicromolar range.
Over 50% of human can~~ers exhibit a loss of function of the protein p53. Cells with mutated p53 are unable to activate the G1 checkpoir.~t in response to DNA damage.
However, the G2 checkpoint (although usually weaker than in normal cells) still provides an opportunity to repair the DNA damage before cell division. Inhibition of the G2 checkpoint alone would have little effect on normal or cancer cells. However, G2 checkpoint inhibitors used in combination with a DNA damaging agent would increase the killing of cancer cells which cannot activate the G1 checkpoint.

Summary of The Invention In one aspect, this invention provides novel G2 checkpoint inhibitor compounds of formula I as defined herein, including salts thereof. This invention includes .the naturally occurring compound, granulatimide, in purified or partially purified form (including granulatimide containing extracts taken from naturally occurring sources).
In another aspect, this invention provides the use of compounds of formula I and pharmaceutically acceptable salts thereof, as a cytotoxic agent; and, to inhibit the G2 checkpoint, including: release of cells that are arrested at the G2 checkpoint thereby permitting the cells to proceed to mitosis or to prevent G2 checkpoint arrest in cells (for example, in response to DNA damage). This invention also provides the' use of compounds of formula I
and pharmaceutically acceptable salts thereof, to sensitize cancer cells to the effect~~ of DNA damaging agents and the use of such compounds in the formulation of agents, including medicaments, for potentiating the effect of DNA
damaging agents on cancer cells. This invention also provides the use of compounds of formula I and pharmaceutically acceptable salts thereof, to increase the killing of cancer cells by DNA damaging agents.
This invention provides a method of increasing the killing by DNA damage of cancer cells having G1 checkpoint deficiency, comprising the steps of administering a DNA
damaging agent to said cancer cells thereby damaging DNA of said cells, and administering a G2 checkpoint inhibitor compound to said cells, wherein the G2 inhibitor compound is a compound of formula I, or a pharmaceutically acceptable salt thereof.
Throughout this specification, the term "G2" or G2 phase" means the phase of the cell cycle between the end of DNA synthesis and the beginning of mitosis. A cell in G2 has an interphase nucleus as determined by microscopy, and duplicated DNA (usuall~y determined by flow cytometry).
Throughout this specification, the term "G2 checkpoint inhibitor" means a substance which is capable of releasing a cell from arrest in G2 phase brought about by DNA damage, preventing a cell from arrest in G2 phase in response to DNA damage, or both.
Throughout this specification, the term "DNA damaging agent" means any substance or treatment which induces DNA
damage in a cell, including W irradiation, gamma irradiation, X-rays, alky7_ating agents, antibiotics that induce DNA damage by b_Lnding to DNA, inhibitors of ~topoisomerases and any compound used in chemotherapy which acts by causing DNA damage. Examples of specific compounds are cisplatin, VM-26, and procarbazine.
Brief Description of the Drawinas Figure 1 are graphs showing:
(A) G2 checkpoint inhibition by granulatimide;

_ 5 -(B) granulatimide and y-irradiation kill p53- cells synergistically (MCF-7 mp53 cells seeded at 1000 cells per well in 96-well plates were grown overnight and irradiated with 0 Gy (O), 2 Gy (~), 4 (~) or 6 Gy (~), immediately after irradiation the cells were treated with granulatimide for 16 h, cell surviva~~L was measured using a soluble tetrazolium salt assay (CellTiter96'", Promega), 100% cell survival is defined as the surviving fraction after irradiation alone (6 Gy induced about 80% cell death); and (C) granulatimide and 'y-irradiation do not kill p53' cells synergistically (method identical to (B) except that MCF-'7 cells are used).
Detailed Description The G2 checkpoint inhibitor assay described herein involves treating cancer cells with a DNA damaging agent under conditions whereby arrest of the cells at G2 will be induced. A sample to be tested for G2 checkpoint inhibition activity and an agent which arrests cells in mitosis are applied to this cells, following which it is determined whether the G2 checkpoint is overcome and the cells enter mitosis. Release of the cells from arrest at G2 or prevention of arrest at G2, such that the cells proceed to mitosis, is the indicator of G2 checkpoint inhibition activity.

- Ei -The assay requires use of a cancer cell culture in which the cells are incapable of arrest at the G1 checkpoint in response to DNA damage. The cells may be from any of the numerous cancer cell lines for which it is known that the cells are incapable of arrest at G1. Such cells include cells which are p53 deficient, including:
any cell line with a natural mutation or deletion in the p53 gene which renders p53 inactive; any cell line expressing a viral oncogen.e which disrupts p53 ; any cell line expressing dominant-negative mutant p53 (some mutant p53 proteins inhibit wild-type protein such as in the p53-MCF-7 cells used in the examples herein); and any primary or established cell line derived from p53 "negative transgenic" animals. Specific examples of suitable cancer cells are the following: cancer cells which have incorporated the human papillomavirus type-16 E6 gene, cell lines which are mutant for p53 function including Burkitt's human lymphoma cell line CA46 and the human colon carcinoma cell line HT-29 (CA46 and HT-29 are available from the American Type Culture Collection). Cell lines with a genetic deficiency which disrupts the G1 checkpoint other than by disruption of p53, may also be employed in this assay.
Once a cell line incapable of G1 arrest is chosen, conditions for arresting a majority of the cells at G2 in response to a DNA damaging agent are optimized by determining appropriate culture conditions, incubation time, and type and dosage of DNA damaging agent.

_ .7 _ Preferably, at least 50% o:E the cells in a culture will be arrested at G2 in response to the DNA damaging agent.
Maximizing the proportion of cells in the population which are arrested at G2 will reduce the background signal.
Once the conditions for G2 arrest in the cell culture are established, the assay may be carried out in one of two ways. First, the cells may be arrested at G2 in response to the DNA damaging agent and then treated with the sample to determine whether there: is release from G2 arrest or, the cells may be treated with the sample prior to the time when the majority of the cells would be expected to be arrested at G2 and determine whether the cells are prevented from G2 arrest.
Release from G2 arrest or prevention of G2 arrest is detected by a quantitative determination of the cells which proceed to mitosis. The assay culture is treated with a agent which will arrest such cells in mitosis. Such agents include microtubule depolym~erizing agents that arrest cells in metaphase, such as nocodazole. This will prevent cells from exiting mitosis and entering the next cell cycle.
In the assay, determination of the cells which proceed to mitosis is carried out using any of the known immunological methods by employing antibodies which have specificity for mitotic cells. Monoclonal antibodies demonstrating such specificity are known and include MPM-2 which was raised against mitotic HeLa cells and recognizes phospho-epitopes that are highly conserved in mitotic proteins of all eukaryotic species. Other examples are the monoclonal antibodies recognizing phospho-epitopes in the paired helical filament proteins (PHF) found in brain tissue of patients suffering from Alzheimer's disease as described in: PCT International Application published July 4, 1996 under No. WO 96/20218; and, Vincent, I. et a1.
(1996) "Mitotic Mechanism, in Alzheimer's Disease?" The Journal of Cell Biology, 132:413-425. The examples in this specification make use of the antibody TG-3 described in the latter two reference;, which may be obtained from Albert Einstein College of Medicine of Yeshiva University, Bronx, New York.
TG-3 has a high specificity for mitotic cells. When TG-3 is used in the ELISA assay described herein, a very good signal/noise ratio i~> achieved permitting the ELISA
assay to be easily monitored by optical absorbance.
Dying cells exhibit some characteristics which are similar to mitotic cells. TG-3 does not react with dying cells which prevents apoptotic cells from being counted in the assay of this invention. The efficiency of the assay is not affected by the presence of dying cells.
Immunological method: useful for determination of mitotic cells in this assay include any method for determining antibody-antigen binding, including:
immunocytochemistry (eg. immunofluorescence), flow cytometry, immunoblotting, and ELISA. Several immunological methods are' described in detail in the examples herein as well a:~ in Vincent I. et a1. [supra] .

_ c~ _ Other immunological procedures not described herein are well-known in the art and may be readily adapted for use in this assay. However, high throughput testing of samples may best be achieved by use: of ELISA.
As described in more da_tail in the examples herein, an extract from a Brazilian ascidian tested positively in the assay and analysis of the extract resulted in the identification of the novel compound, granulatimide, shown below.
H
I
TvT
~H
J
is H
Granulatarride Granulatimide compounds having the structure of formula I, are inhibitors c~f the G2 checkpoint and may be used to selectively sensiti;,e cancer cells to the effect of DNA damaging agents thereby potentiating cytotoxity of DNA
damaging agents. Such compounds may be administered in conjunction with DNA damaging agents to increase killing of cancer cells which are deficient in the G1 checkpoint.

- 1.0 -Compounds of this invention have the following formula, including all steroisomers thereof when the compound may exist as stereoisomers:
to Z
I
wherein W is selected from the group consisting of:

(a) (a) Q ~N
(b) ~ (b) QJ
N
(a) (a) ~N
(b) \ Q c~) ~ Q
N' (a) (a) (b) ~ ~ Q
(b) Q ~N
Rt _, N

- 7_1 -and wherein:
R1) is selected from the group consisting of : H; R;
RCO-; ArCo-; and, ArCH2-, wherein Ar is an aromatic substituent selected from the group consisting of:
phenyl, naphthyl, anthracyl, phenanthryl, furan, pyrrole, thiophene, benzofuran, benzothiophene, quinoline, isoquinoline, imidazole, thiazole, oxazole, and pyridine, and Ar may be optionally substituted with R or Z;
R is selected from th.e group consisting of : H; and, a structural fragment having a saturated or unsaturated linear, branched, or cyclic, skeleton containing one to tern carbon atoms, zero to four nitrogen atoms, zero t:o four oxygen atoms, and zero to four sulfur atoms, wherein the carbon atoms may be . optionally substituted with a substituent selected from the group consisting of : -OH; -OR3; -OZCR3, -SH;
- SR3 ; - SOCR3 ; -NHZ ; -NHR3 ; -NH ( R3 ) 2 ; -NHCOR3 ; NRCOR3 ; - I ;
-Br; -Cl; -F; -CN; -COZH; -C02R3; -CHO; -COR3; -CONH2;
-CONHR3; -CON (R3) 2; -COSH; -COSR3; -N02; -S03H; -SOR3;
and -SOZR3, wherein R3 is a linear, branched or cyclic, one to ten carbon :saturated or unsaturated alkyl group;
Z is selected from the group consisting of: -OH; -OR;
-OZCR; -SH; -SR; -SOCR; -NHz; -NHR; -NH (R) z; -NHCOR;
NRCOR; -I; -Br; -C1- -F; -CN- -COzH; -C02R; -CHO; -COR;

- J:2 --CONH2; -CONHR; -CON (R) 2; -COSH; -COSR; -NOZ; -S03H;
-SOR; and, -SOzR;
Q is selected from the group consisting of: NR2; O;
and S , where in R2 = R.~ ; and X and Y are independently selected from the group consisting of: 0; H, OH; and H2.
Preferably, Q is nitrogen. Also preferably, R1 is H or CH3. Also preferably, X and Y are oxygen. Also preferably, Z is hydrogen.
While the examples in this specification describe purification of granulatirnide from a natural source, the compounds of formula I can be synthesized by routine modification of the following synthesis scheme:

~2 O t-BuOK
MeOC(O)C(O)OMe \ NH DMF

2 N Bioorganic and Medicinal 1 Chemistry Letters, 1998, rv P 1 vol 8) 47 Pi TEA
ibid Tf20 B(OH)Z
i~
~N ~% O

N
Tf Palladium catalyzed N ~ coupling P1 P3 ibid and P1 Heterocycles,1998, 48, 11 i) 6n Photocyclization ii) oxidation JACS, 1996, 118, 2825 Remove Protecting Groups N J ~ , _.

- 1.4 -In the preceding scheme, Pl, P2 and P3 are suitable protecting groups. The synthesis begins with an indole compound appropriately substituted by Z, which may be made by methods known in the art.
Alternatively, compounds of formula I can be produced from granulatimide which may be synthesized as described above or obtained from natural sources as described in the Examples. The following :;thematic describes modification of granulatimide to produce derivatives having different X
and Y substituents. They ratio of II to III will be dictated by the choice of protecting groups P1, PZ and P3, which may be (for example) Cbz, R or H. The chemistry is described in Link, et al- (1996) J. American Chemical Society 118:2825, at p. 28:32.
All Z substituents can be added using standard electrophilic substitution chemistry. All R, RCO and ArCH2 groups can be added using base and an appropriate alkyl halide (RX), acyl halide (RCOX), anhydride ((RCO)20) or benzyl halide (ArCH2X) .

- 1!5 -..
'~ > n 0. p, r N
~r N
H
=~U
H
wE~U
4 >
O z +~1 w z~~ N
H
H oc_z ~ z z v z-~
H ~ ,a:
Z w E-~ U
H
w E
C~
rw D
O i;
a~ ~:
O
v ..
a", U
°' a0o v~ n .a o.
o Z
ot-z Compounds of this invention, particularly the salts, are sufficiently water-soluble that the compounds may be administered in vivo in aqz~eous form. A preferred mode of administration is intravenous, with the goal being to achieve a circulating concentration of the compound in the patient of about 10-S - 10-' molar. Pharmaceutically acceptable salts of the compounds of this invention include the hydrochloride salt.
gXAMPL$S
G2 Checkpoint Inhibition Ass ~r MCF-7 mp53 cells were cultured as monolayers in DMEM
supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 50 units/ml penicillin, 50 ~.g/ml streptomycin, 1 mM sodium pyruvate, MEM non-essential amino acids, 1 ~g/ml bovine insulin 1 ~,g/ml hydrocortisone, 1 ng/ml human epidermal growth factor, and 1 ng/ml ~i-estradiol at 37°C in humidified 5% CO2. The ce:Lls were seeded at 10, 000 cells per well of 96-well polystyrene tissue culture plates (Falcon) in 100 ~1 medium. The cells were allowed to grow for 24 hours and then were irradiated with 6.5 Gy using a soCo source (Gammacell 200'x", Atomic Energy Commission of Canada) delivering Y-rays at a dose rate of 1.4 Gy/min.
Extracts from marine organisms at about 10 ~,g/ml (from 1000-fold stocks in DMSO) and 100 ng/ml nocodazole (Sigma, from a 1000-fold stock in DMSO) were added 16 hours after irradiation and the cells were incubated for a further 8 hours. Caffeine at 2 mM (Sigma, from a 100 mM solution in phosphate-buffered saline) was used as a positive control.
After drug treatment, the cell culture medium was removed and the cells were lysed by adding 100 ~1 of ice-cold lysis buffer (1 mM EGTA pH 7.4, 0.2 mM PMSF) and pipeting up-and-down 10 i:imes. The cell lysates were transferred to 96-well Po:lySorp'" plates (Nunc) and dried completely by blowing warm air at about 37°C with a hair dryer positioned about 3 feet above plates. Protein binding sites were blocked by adding 200 ~1 per well of antibody buffer (10 mM Tris HC1 pH 7.4, 150 mM NaCl, 0.1 mM
PMSF, 3% (w/v) dried non-fat milk (Carnation)) for 1 hour at room temperature. This was removed and replaced with 100 ~,1 antibody buffer containing 0.1-0.15 ug/ml TG-3 monoclonal antibody and horseradish peroxidase-labelled goat anti-mouse IgM (Southern Biotechnology Associates) at a dilution of 1/500.
After overnight incubation at 4°C, the antibody solution was removed and tree wells were rinsed 3 times with 200 ul 10 mM Tris HC1 pH T.4, 0.02% Tween 20~". 100 ~1 of 120 mM Na2HP04, 100 mM citric acid, pH 4.0 containing 0.5 mg/ml 2,2'-azino-bis (:3-ethylbenzthiazoline-6-sulfonic acid) and 0.01% hydrogen peroxide as added for 1 hour at room temperature and the plates were read at 405 nm using a BioTek~" plate reader. Positive controls treated with 2 mM caffeine gave absorbance readings of about 1Ø
G2 checkpoint inhibition was detected in extracts from the ascidian Didemnum granulatum (Subphylum Urochordata, Class Ascidiacea) collected from two Brazilian locations:
Arqurpelego do Aroredo and the Sao Sebastino channel.
Fresh collected specimens or specimens frozen on dry ice were homogenized in methanol and filtered. Homegates were concentrated to dryness in vacuo to give a gummy residue.
Small amounts of the residue were dissolved in DMSO for the G2 checkpoint inhibition a;ssay.
Isolation and Characterization of Granulatimide Methanol extracts from the Brazilian ascidian Didemnum granulatum were fractionated by gel filtration on Sephadex LH-20 (elutent: MeOH) followed by reversed phase HPLC
(eluent acetonitrile/0.05% trifluoroacetic acid (1:1)).
This led to the isolation of the novel compound granulatimide and the known alkaloid didemnimide A.
Didemnimide A was identified by.comparison of its NMR and MS data with literature values (H.C. Vervoort, et al.
(1997) J. Org. Chem. 62:1486). NMR experiments identified a disubstituted imidazole cnoiety in granulatimide, leading to the conclusion that it has a bond between C-2 of the indole and C-14 of the imid.azole rings. The presence of an anisoptropic effect in gran.ulatimide and not in didemnimide A indicates that granulatimide is a rigid planar heterocycle, unlike didemnimide A in which the indole and maleimide rings are twisted relative to each other along the C-3 to C-8 bond. The imidazo[4,5a]carbazole heterocyclic aromatic core of granulatimide appears to be without precedent in natural products.

Granulatimide shows half-maximal G2 checkpoint inhibition (ICSO) at 1.8~0.2 ~.M (Fig. lA) . Didemnimide A
shows no activity at concentration of 0.1-30 ~M).
Granulatimide shows mild cytotoxicity, with an ICSO of 40~4 ~.M (Fig. 1B, curve 0 Gy) , well above the ICso for checkpoint inhibition.
G2 checkpoint inhibitors and DNA-damaging agents would be expected to kill p53- cells synergistically. When such cells were exposed to 2, 4 or 6 Gy of 'y-irradiation and to different concentrations of granulatimide for 16 hours, cells died in higher numbers than for the sum of each treatment alone (Fig. 1B), showing that y-irradiation and granulatimide acted synergistically.
The ICso for cytotoxicity of granulatimide was reduced by 5-fold when cells were irradiated with 6 Gy (Fig. 1B).
By comparison, caffeine, the most commonly used G2 checkpoint inhibitor, exhibits an ICSo of 1 mM for G2 checkpoint inhibition, has very little cytotoxicity alone and also acts synergistically with 'y-irradiation.
Irradiation with granulatimide treatment does not kill p53+ cells synergist~_cally, as shown in Figure 1C.
Therefore, granulatimide preferentially kills irradiated p53- (tumour) cells over irradiated p53+ (normal) cells.

Characterization of Granulatimide Granulatimide was isolated as a bright orange solid (L1V (~max~""(E) ) 210 (10, 200) , 231 (10, 600) 280 (6, 550) , 470 (1,870)) that gave a [M + H]'ion at m/z 277.0730 in the HRFABMS appropriate for a. molecular formula of C15H8N402 (calculated mass for C15H9N~OZ, 277.0714) . An NH proton resonance at b 11.11 shows HMBC correlations to carbonyl resonances at d 169.8 a:nd 168.8 and aromatic carbon resonances at b 126.39 and :112.22, confirming the presence of a maleimide substructure in granulatimide. The COSY
spectrum identified a four proton spin system (b 8.51, d, J = 6 Hz (H-4)); 7.43, t, J = 6 Hz (H-6); 7.35, t, J = 6 Hz (H-5) ; 7.67 d, J= 8.07 (H-7) that may be assigned to the H-4 to H-7 protons of an indole residue. Irradiation of a broad proton resonance at b 13.48 induces an NOE only in the aromatic doublet at b '7.68, which assigns the doublet to H-7 and the broad proton resonance to the indole NH.
The absence of a resonance in the 1H NMR spectrum of granulatimide that may be a:~signed to the indole H-2 proton indicates the presence of a substituent at C-2.
A very broad resonance at b 8.10 in the 1H NMR spectrum of granulatimide is assigned to the imidazole H-16 proton and a similarly broad resonance at b 9.12 is assigned to the imidazole NH proton. The broadness of the imidazole resonances is attributed tc~ tautomeric equilibrium of the NH protons. A large chemical shift is observed for the H-4 resonance (b 8.51) in g:ranulatimide relative to the chemical shift observed for ~ the H-4 resonance in - 2:1 -didemnimide A (b 7.07) which is attributed to a neighbouring group effect from the C-9 maleimide carbonyl in granulatimide.
Although various aspects of the present invention have been described in detail, it will be apparent that changes and modification of those aspects described herein will fall within the scope of the appended claims. All publications referred to herein are hereby incorporated by reference.

Claims