GB2512863A

GB2512863A - Medicinal applications of arylated hydroxy-tetronic acids

Info

Publication number: GB2512863A
Application number: GB1306402.7A
Authority: GB
Inventors: Pornthip Lattmann; Padijarethakkal N Balaram
Original assignee: Individual
Current assignee: Individual
Priority date: 2013-04-09
Filing date: 2013-04-09
Publication date: 2014-10-15
Also published as: GB201306402D0

Abstract

A compound of formula (I), (II), (III) or (IV) or a salt thereof: wherein Sub is selected from hydrogen and a substituent, for use in the treatment of a disease selected from cancer, cachexia, weight loss and pain. The preferred cancers to be treated are gastrointestinal (GI) cancers, especially colon cancer. The cachexia, weight loss or pain to be treated may be associated with cancer. The preferred compounds of formula (I) include those wherein Sub is selected from 4-fluorophenyl, 4-methylphenyl, n-hexyl, 5-meta-nitrophenyl, 5-paratrifluorophenyl and phenyl. The preferred compounds of formula (II) include those wherein Sub is selected from 4-methoxyphenyl and phenyl. The preferred compound of formula (IV) is that wherein Sub is phenyl.

Description

Medicinal applications of arylated hydroxy-tetronic acids Based on our analysis, a full general screening programme was performed in no time with a dozen of molecules and a small, but smart combinatorial library derived from a 11011 -toxic template.

General screening programme, in vivo based [ Anticancer] [ Antivfral] 1 fl Glucose pai! Etbolism Cholesterol The classical drug concept based on animal experiments The best and most promising approach is to use mice as cancer models and we initiated screening by this way.

Cancer is complex and after initial positive anti-neoplastic effects in vivo. we tested 2(5)-furanone containing templates in vitro to confirm the transfer of results from mice into a panel of human cell lines.

Ascorbic acid is a non-toxic template and arylated tetronic acids served as lead structure of versatile derivatives of this non-toxic temp'ate.

In our invention the glycol side chain was replaced by arylated moieties. The aryl group and the reducing properties, associated with the 4-hydroxy group, are essential for their anti-cancer and other medicinal properties.

The present invention is focussed on ana'ogues of vitamin C and the scope of the invention is described by examples only.

Generic scope A compound of formula (I) or (II) or IlL IV and salts thereof SubO SubNH2 Su H 5 Sub stands for a moiety, one or 2 substituents independently connected to the 5-position (5a and Sb) wherein Sub is selected from hydrogen, a halogen, a substituted or unsubstituted cyclic and heterocyclic moiety, phenyl. substituted phenyl, aryl, hetero aryl substituted or unsubstituted, linear or branched alkyl, alkyloxy, alkylcarbonyl, alkyloxycarbonyl, alkenyl. alkenyloxy. ailcenylcarbonyl. and sulphur equivalents of said oxy, carbonyl and oxycarbonyl moieties.

Aromatic groups may be heterocyclic, cyclic or acycfic and which may optionally be substituted by alkyl, alkoxy, or halo; when taken together with the N-atom to which they are bonded, may form an N-containing saturated, unsaturated or partially unsaturated ring system comprising 3 to 10 ring atoms selected from C, N and 0.

One or 2 substituents, as defined above, are attached to the 5 -position and both are varied independently.

I

Most preferred example are:

OH

HO OH F F3C

Example pFPhTA Example TFMPhTA

HO OH

HO OHOHOH 0 0

Example TI-I PTA

Example HTA

HO OH

OH NH2

Example PTA, phenyl hydroxytetronic acid Example PTI, phenyl tetronimide p S.--S. S' Or U

S S

-I-.-S S. F F3CC x N555 0 F3C m F p

FC

List of substituents attached combinatodcally to the template TA, TIM and THA Nole: Subsiiiuenls may be on Ihe oriho, niela and pam position.

SYNTHETIC LIBRARY

{ ;c HO_J} TA T/ THA fl)'( \

H

F

Flow chart: Principle of constructed libraiy of biologica'ly active molecules, of which typical representative example were fully characterised, in order to fully verify the identity of the bio-rnolecules.

Full chemical diversity includes given examples.

Formation of agents illustrated for the example PTA HODH NH2 Synthesis of tetronic acids from tetronjindes by hydrolysis.

preparation of tetroninñcles by aminonolysis The tetronic acids, such as phenyltetronic acid are formed from the corresponding phenyltetronimides by using TFA, (trifluoroacetic acid) in THF (tetrahydrofuran).

Ammonia treatment of the acids such as phenyltetronic acid gives the tetronimides.

HO OH HO OH

Oxidation o6H Reversible oxidation of PTA and reduction of tetrahydroxy-tiuranone TA and here substituted tetronic acids were easily converted by standard oxidising agents into the corresponding TI'TTA, substituted tetra-hydroxy tetronic acids.

Oxidation was performed by air and reduction was performed by using standard reducing agents, such as hydrogen sulfide and sulfites.

This method of formation may be applied to the entire series of agents given by

examples.

Obvious'y, the addition of water, hydratisation, onto the ketone furnished the tetra hydroxy-derivatives. This finding is analogue to cifioraihydrate, which occurred in this form and not as trichioroacetaldehyde.

SYNTHETIC SECTION SELECTED EXAMPLES

of the designed and synthesised combinatorial library Prepared by using Building block TA, TIM, THA and the salt of TIM, which is attached to a substituent in the 5-position as outlined in the synthetic schemes.

Example 1

3,4-Dihydroxy-5-phenyt-furan-2(SH)-one. PTA PTA was prepared by the OXIX process or as described by Wittiak et al.

HOOH

Yield = 40%.; R1 (50% Ether, 50% Petrol ether 40-60) = 0.38; Mol. Weight: 192.2; Mo]. Formula: C10H804; MS (APCI(-D: 191 (M-l) m/z. H NMR (DMSO-d6) 250MHz: 8 = 11.50-11.31 (br s, H, CH-C-OH), 8.82-8.57 (br s, H, C-C-OH), 7.61-6.99 (s, 5H, Ar-H), 5.72 (s, CH) ppm. 3C NMR (DMSO-d5) 250MHz: 8 = 169.5 (C=O), 152.1 (Cu-c-OH), 146.1 (Ar-C), 144.5 (C-c-OH). 129.3, 128.5. 127.3 (Ar-C). 78.1 (CU) p.p.m. IR (KBr-disc) u max: 3369, 2925. 2852, 1760, 1672, 1492, 1450, 1258, 1127, 1009, 796, 747, 692 crn1.

Example 2

3,4-Dihydroxy-5-p-Fluorophenyl-furan-2(SH)-one. FPTA

Example 3

3,4-Dihydroxy-5-m-nitrophenyt-furan-2(5H)-one. mNPTA

Example 3

3,4-Dihydroxy-5-p-trifluoro-phenyl-furan-2(511)-one.

Example 4

3,4-Dihydroxy-5-hexyl-furan-2(5I-I)-one.

Example 5

3,3,4,4-Tetrahydroxy-5-phenyl-dihydro-furan-2-one

Example 6

3,3,4,4-Tetrahydroxy-5-p-fluoro-phenyl-phenyl-dihydro-furan-2-one

Example 7

5-Amino-4-hydroxy-2-phenyt-furan-3-one, PTIM PTA was reacted with ammonia in methanol at RT for 2 days. 2/3 of the solvents was removed and the desired molecule formed brownish crystals in the cold. 0 o

IIC..( "o" Yield = 60%.; m.p. 173-177 "C, R (50% toluene, 49% butanol, 1% acetic acid) = 0.54; Mo]. Weight: 191.2; Mol. Formula: C10I-I9NO. MS (APCI(-)): 190 (M-1) m/z. I-I NMR (DMSO-d6) 250MHz: 5 = 7.90-7.70 (hr s, 2H, NH2), 7.49-7.15 (rn. 5H, Ar-H), 7.10 (s, H, 0-CU) ppm. 13C NMR (DMSO-d6) 250Mhz: 5 = 182.1 (C=0), 172.1 (C-NH2). 137.2 (Ar-C), 128.5 (2 x Ar-C), 127.4 (2 x Ar-C). 123.9 (Ar-C). 111.6 (C-OH), 82.1 0-CH) p.p.m. IR (KBr-disc) u max: 3410, 2991, 2701, 1705, 1641, 1362, 1178, 1009, 708 cal'.

Example 8

5-Amiiio-4-hydroxv-2-(4-methoxy-phenvl)-furan-3-one. pMeOTIM Yield= 51%. Rf(50% toluene, 49% butanol, 1% acetic acid)= 0.20; Mol. Weight: 221.2; Mol. Formula: CjjI-I11NO4; MS (APCI(-)): 220 (M-1) m/z. 1-1 NMR (DMSO-d) 250MHz: 5 = 7.95-7.69 (hr s, 3H, NH2 + OH), 7.35-7.10 (d. 2H, 2 x Ar-U, J = 8.1 Hz), 6.94-6.79 (d, 2H, 2 x Ar-H. J = 8.1 Hz), 5.39 (s. 0-Cl-I). 3.81 (s, 0-Cl-I3) p.p.rn.

3C NMR (DMS0-d)250MHz: ö= 187.9 (=o), 177.8 (CH-O-C, Ar-C), 164.5 (C-NH2), 135.5 (Ar-C), 133.2 (2 x Ar-C), 118.9 (C-OH), 116.8 (2 x Ar-C), 87.9 (O-cH), 60.5 CH2) ppm. JR (KBr-disc) u max: 3346, 3099, 2839, 2675, 2592, 1709, 1609, 1544, 1397. 1263, 1024, 972, 864. 691. 795, 648, 591 cm1.

Example 9

3-hydroxy-5-(4-inethoxy-cyclohexa-2,5-dienylidene)-4-oxo-4,5-dihydro -furan-2-yl-ammonium, pMeOTIMHCI 0.5 g (2.3 rnrnol) 2-imino-5-(4-methoxy-phenyl)-2,5-dihydro-furan-3,4-diol was added to 2 ml 8% HC1 and gently heated for 2 minute. The yellow mixture was allowed to cool and put in the fridge overnight. Yellow crystals formed in solution.

which were ffltered and washed with small amounts cold water before being dried under a stream of argon gas. A yield of 49% was given.

Yield = 49%; Ri (50% toluene, 49% butanol, 1% acetic acid) = 0.81; Mol. Weight: 222.2; Mol. Formula: CjjHiN04; MS (APCI(+)): 222 (M+) mlz. H NMR (DMSO-d) 250MHz: 6 = 9.55-9.50 (br s, 3H, NH3), 8.51-8.26 (br s, H, OH), 8.03-7.78 (d, 2H, 2 x O-CH-Cl-I, J = 9.1 Hz), 7.11-6.85 (d, 2H, 2 x C=C-Cl-I, J = 9.1 Hz), 6.53 (s, H, 0-Cl-I), 3.81 (s, H, 0-Cl-I3) ppm. 13C NMR (DMSO-d6) 250Mhz: 6 = 188.8 (C=0), 167.2 (CNH), 159.5 (O-C=C), 132.1 (O-C=C), 127.1 (0-CH-CH, 2 x C) 119.8 (C=C-CH, 2 xC), 114.5 (C-OH), 99.1 (CH-0-Cl-13), 55.1 (CH3) ppm. IR (KBr-disc) u max: 3344, 3146, 2835, 2360, 1704, 1613, 1527, 1458, 1393. 1255, 1178, 953, 854, 806, 586 ciii'.

CANCER EVALUATION

In vivo test: Allograft study Method A MAC 16/13 cancer cell line was used in this test. 5 mice served as control and 5 mice were treated with the test agent. The day the tumour was transplanted was counted as day zero, the tumour was allowed to settle into the flank to which it was transplanted and the agent was given orally after 1 2 days. The treatment was performed for 11 days and the results are outlined.

Total body weight (g) 28 - -s--treated with PTA -0--untreated (control) 26 -S24 20- 18 - 16 -I I I I I 0 2 4 6 8 10 12 Time after treatment (days) VIA: 300mg /kg Results & discussion The tumour burns off energy and the animals thse weight, significantly less in the treated group (300mg/kg). Weight loss is the main cause of suffering in patients and death in UI cancers.

MAC 16 is a solid mouse tumour originated from the colon. It was transplanted into the flank of the animal. In contrast to the MAC 13 tumour, the MAC 16 tumour is resistant to allcylating agents and both are sensitive to 5-EU treatment.

As the same anti-neoplastic activity was observed for both murine colon cell lines, the biological effect of the present invention is not due the action as an ailcylating agent.

Cyclophosphanilde is only active on the MAC 13 and not the MAC 16 cell line, 5-FU is active on both cell lines, but the effect is weak and experimentally and clinically inefficient. In a clinical setting 5-PU is as bad as the effect found in mice in vivo for colon cancer.

Volume of tumor (mm3) 1400 - 1200 --treated with PTA -0--untreated (control) -1000 -Co

E I: :

Time after treatment (days) PTA: 300mg/kg Anti-inflammatory agents such as ibuprofen and diclophenac failed in terms of anti-cancer activity against these colon cancer cell lines and this was confiirned even at the highest, maximum tolerated doses. The present examples, therefore must have additional mechanisms responsible for their anticancer activity.

A recent patent disclosed inhibition of alkylated tetronic acids as heparinase inhibitiors and this is one possible mechanism in the array of cancer pathways.

PTA is a potent PLA2 inhibitor and this is considered one of the underlying biological mechanisms, which is known. PLA2 inhibitors are not in use as anticancer agents.

The tumour progression is ongoing, but significantly less under treatment resulting in a much-delayed death of cancer. Mice were sacrificed without treatment after 3 weeks and 8 weeks with treatment of 300 mg/kg PTA orally.

Cancer study of FPTA Total Body Weight treated with FPTA -0--Untreated Time after treatment (days) FPTA (50mg/kg dose) Structure activity relationships Testing of analogues of the lead structure resulied in a manifold more potent agent EPTA and the effects are outlined for the chemoresistent MAC 16 cell line (MAC 13 is not shown).

Donor substituents. such as methoxy-groups increase the reducing properties of the agents. The fluorine atom, an electron withdrawing substituent, reduced the reducing properties of the agents -EPTA compared with PTA-, but the anticancer properties of EPTA are 6 times higher compared to the PTA derivative.

If the anticancer effect was linked to anti-oxidant properties, the SAR would be opposite.

Therefore, the anticancer effect is not due to antioxidant properties, but possibly due to the sum and synergistic effects all inhibitory activities in the correct composition.

By testing the molecules in vivo, the properties were discovered within months and no modelling, but a classical drug design guided us in the screening programme.

Volume of Tumor (mm3) 700 S treated with FPTA -0--untreated E 500

E

S o U)

E

0* I I I I I 0 2 4 6 8 10 12 Time after treatment (days) FI'l'A 50mg/kg Treatment was stopped at day 9 and tumour growth immediately went up, thus nicely showing the potent anti-neoplastic effect of FPTA on the MAC 16 murine colon cell line.

Survival studies Untreated, within 3 weeks 100% of all animals died, treated orally with 50mg/kg FPTA 76% survived. When the tumour was removed after 10 days and treatment continued for I month with 50mg/kg animah 5 out of 6 mice Uved for 300 days.

Toxicity No acute toxicity was observed for a dose of PTA at 5000 mg/kg in mice and rats.

Long term toxicity A group of 6 mice fed on PTA, which was added to the food, reproduced normally and the offspring appeared and behaved norma'ly.

in vitro tests -Cytotoxicity assays In vitro tests, using the MTT toxicity assay, inhibition was observed in various cell lined and the results were outlined below.

Methods Cytotoxicity assays using munne carcinoma cell lines (MAC 13 and MAC 16).

The culture media used was RPMI i640 containing hepes, glutamine, antibiotics containing 5% fetal calf serum for MACI3 cells and 10% fetal calf serum for MACI6 cells. On day 0 media, from 250 ml flasks containing either 70% confluent MAC 13 or MACI6 cells, were poured off and the cells were washed with 10 ml PBS. 5 ml of versene was added to the flasks for three minutes. The detached cells were pipetted into plastic universals and spun down for 5 minutes at 1100rpm. The media were poured off and 10 ml of fresh culture media were added to the cells. Cells were counted by the trypan blue exclusion method using a plastic Kova counting chambers. The MAC 13 and MACIÔ cells were resuspended in appropnate volumes and were seeded at 0.5 x io and 2. iO4i 200 p1 respectively in flat-bottomed 96 well plates. Depending on solubiity, test compounds were dissolved in water, alcohol or dimethyl sulphoxide (DMSO) to give stock solutions of 100 mM (iO-IM). Dilution series from l0' M to i09 were made so that each compound was tested at six concentrations and in triplicate.

5-Fluoro-undine (5-EU), a known anticancer agent, was used as a control and tested in the 20-0.02 jiM range. Plates were then incubated at 37°C, 5% CO2 for three days.

Compounds were tested on at least two separate occasions. On day three 20 p1 of 3-(4,5-dirnethylthiazol-2-yl)-2.5-diphenyl-tetrazolium bromide (MTT) (7.5mg Mfl /m of PBS) was added to each well and plates were allowed to incubate for a further 2 hours. 120 pA of culture supernatant was carefufly removed from each well, with a pipetter, and 100 pA of acidified isopropanol containing 10% Triton-XiOO was then added to each well. Plates were agitated for 10 minutes at 800 rpm on a plate shaker. Foflowing this solubilisation step all plates were then read, within 15 minutes, on an Anthos AW200 plate reader at 540 nm with a reference wavelength of 590 nm.

The same assay format was used with the following cdl lines without any modifications with Glivec (iniatinib) as standard.

Anticancer activity -in vitro cytotoxcity Results The best inhibitors in vivo were the tetronic acids PTA and FPTA and in the following the in vitro results on humane cell lines are displayed.

Example PTA

Examp'e FPTA The anfi-neoplastic profile is the overall result, the overall sum of a variety of targeted activities.

MM PACk Miapaca is a human pancreatic carcinonia cell line, it was tested as described for the MAC 13/16 cancer cell lines and selected data are outlined below.

Log10 concentration (uM) Log10 concentration (uM)

PTA PTIM

PTA and PTIM were tested in vitro in human cell lines and an 1C50 about 12 uM / 20.7 uM was determined. The tyrosine kinase inhibitor Gleevec gave an TCSO of 2OuM and served as standard. The same range of inhibition was observed for the murine MAC13/MAC16 cell lines.

PTA is in vivo active (in vitro 1C50 =l2uM). PTIM (in vitro 1C50=2luM) was found inactive in nilce due to its physical properties combining a poor solubility of PTIM in water and most organic solvents (very low bioavailability).

FTPA is 6 times more potent in vivo and about 20 times more potent in vitro with an 1C50 around luM for UI as well as brain cancer cell lines.

Unlike the parent hydroxy tetronic acid, the methoxy -TA showed no anticancer activity, possible due to chemical instability. The methoxy derivative has with the strongest reducing potential the highest antioxidant properties. 100

C -50

--Log10 concentration (uM) MeOPTA MeOTIM Anti-cancer activity of the present invention is clearly only partly measurable in vitro, the main inhibition is due to the inhibition of cell aggregation and possible inhibition of angiogenesis.

AGS -AUS is a human stomach cancer cell line and was selected to confirm activity within the entire UI system.

PTA showed an 1C50 of 12 uM against the AGS cell line, while FTPA showed and IC 50 of 0.8 uM.

PANC -The PANC cell line is a human pancreatic cell line and the use against pancreatic cancer is particularly beneficial as the 5 year survival time is as low as 1%.

The 1C50 for PTA against the PANC cell line was 30 uM. compared with the standard Uleevec of 60 uM in our experiment.

U373MG -The anticancer activity of the agents is not limited to UI cancers. The glioblastoma cell line U373MG, a brain cancer cell line, was determined for PTA about ii uM during the same study.

Analgesic activity The analgesic activity was determined initially for the series and for FPTA the results of the tailflick test are outlined below.

Hot plate test Method Male ICR mice were tested for the reaction time by placing on a hot plate that was thermostatically maintained at 50°C, with a four-wall plexiglass container to confine the animals on the hot plate, as described earlier (Lattrnann et al., 2009). The time that each mouse spent on the hot plate until it licked or jumped in response to pain was recorded as the reaction time. Cut-off time of the test was set at 30 seconds in order to prevent tissue damage. The reaction time of each animal was recorded as the basal reaction time. After being orally fed with either water, test molecule or tramadol, the reaction time of each animal at 10, 30, 60 and 90 mm after treatment were determined (shown 60 mm).

Results Tail Flick Test C Mean: 7.63 Mean 21 78 Mean. 16.58 Mean 21 1 * 0: _ V/ISA r

_____

WA 1 _ * s'gn'f'cmnt differenl from 5% DM50 sigF1ifican different from Tra'nadol 40mg/kg Tramadol was used as standard (bar 2) and 5 mg/kg EPTA resulted in a significant increase of response time (bar 3). The 20 mg/kg EPTA administered PU (bar 4) was found equivalent to the 40mg 1kg tramadol dose given IP.

Cancer and GI cancers are associated with pain and the additional effect of FPTA is particular useful, even if the mode of action is unknown.

Analgesic activity -Formalin test Methods Formaliii solution (1%) was injected in rats (group of 10) into the peritoneal cave of the animal and the effects of licking and biting in response to the induced pain was observed in 2 phases.

Results In addition to the hot plate pain model, the formalin test was performed and the results for FPTA are outlined in the table below.

Groups No. of licks/bites in the First No. of licks/bites in the Second Phase (0-5 mm.) Phase (20-25 mm.) Control 131.25±23.0 103.6±11.46 (Vehicle) FPTA (5mg/kg, 23.25±13.40 25.5±0.11 po) FPTA (20 34.25±11.60 12.12±1.66 mg/kg, po) Morphine (5 33.60±3.55 2.10±0.23 mg/kg, i.p.) The taNe shows the effect of EPTA (5 and 20 mg/kg) on the number of licks/bites in the formalin induced pain in rats.

Licking is a response to pain and FPTA reduced the number of licks from 131 to 34, which is a comparable analgesic effect of morphine. An additional effect was observed in the 21l phase, which is typical for the analgesic effect of anti-inflammatory agents. Both effects will contribute clinically to the overall analgesic activity.

Most interestingly, the arylated furanone EPTA displayed a good analgesic activity in particular in the first phase phase of observation, which is typical for centrally acting agents such as opiates. The effect is possibly due to a couple of molecular targets and synergistic effects.

Claims

CLAIMSGeneric scope A compound of formula (1) or (11) or Ill, IV and sails thereof SubO SubNH2 Sub H H Sub Iv wherein Sub is independently attached to the 5a and 5b position and is selected from hydrogen, a halogen, a substituted orunsubstituted cycfic and heterocyclic moiety, phenyl, substituted phenyl, aryl, hetero aryl substituted or unsubstituted, linear or branched alkyl. alkyloxy, alkylcarbonyl. alkyloxycarbonyl, alkenyl, alkenyloxy.alkenylcarbonyl, and sulphur equivalents of said oxy, carbonyl and oxycarbonyl moieties, aromatic groups may be heterocyclic, cyclic or acyclic and which may optionally be substituted by alkyl, alkoxy, or halo; when taken together with the N-atom to which they are bonded, may form an N-containing saturated, unsaturated or partially unsaturated ring system comprising 3 to 10 ring atoms s&ected from C, N and 0.Sub stands for substituents, a moiety connected at the 3-position, of which 5a and Sb are independently varied around the displayed template. Oxigen is numbered I position.I The general use of a compound, as disclosed by examples, but not limited to the specified examples, as anti-cancer agents.
2 The use of template I, II, Ill, IV as anti-cancer agents.
3 The use of template 1, II, Ill. IV as anti-cancer agents in particular for GI cancers.
4 The use of template I, II, Ill. IV as anti-cancer agents in particular for GI cancers most prefened for colon cancer.
The use of template I, II, Ill, IV as agent against cachexia.
6 The use of template I, II, III, Pt as agent against weight loss in cancers.
7 The general use of template 1, IL Ill, IV as agent against weight loss.
8 The use of template I, II, III, Pt in the management of pain.
9 The use of template I, II, Ill, IV in the management of cancer pain.
The use of template I, II, III, Pt in the management of other conditions related to cancer.
II The use of these agents of template 1,11, III, IV as anticancer agents in the treatment of proliferative diseases and their prevention.
12 The use of these agents of template I. H, III, IV to potentiate anti-cancer properties of standard anti-cancer agents.
13 The use of the agents of template I, II. III, Pt to stop and inhibit the spreading of cancers and to delay cancer progression.