Glycolipids and pharmaceutical compositions thereof for use in therapy

Abstract

The invention may provide, in part, compounds for use as antiproliferative, chemotherapeutic, antiviral, cell sensitising or adjuvant agents, and pharmaceutical compositions including the compounds. The compounds may be for use in treating diseases and disorders related to cell proliferation such as cancer, or in treating diseases and disorder which are linked to aberrant control of protein synthesis, such as cancer, viral infection, muscle wasting, autistic spectrum disorders, Alzheimer's disease, Huntingdon's disease and Parkinson's disease.

Claims

1. A compound having the structure ##STR00043## or a pharmaceutically acceptable salt thereof.

2. A pharmaceutical composition comprising the compound of claim 1, or a pharmaceutically acceptable salt thereof, together with a pharmaceutically acceptable carrier, diluent or excipient.

3. A nutraceutical composition comprising the compound of claim 1, or a pharmaceutically acceptable salt thereof together with a nutraceutically acceptable carrier, diluent or excipient.

4. A kit comprising the compound of claim 1, or a pharmaceutically acceptable salt thereof, and an anti-cancer agent, wherein the anti-cancer agent is provided in a form suitable for, and/or with instructions for, administration in a daily dosage which is significantly reduced compared to the dosage of the anti-cancer agent if administered alone.

5. A method for the treatment of a subject having a disease or condition selected from the group consisting of cancer, autistic spectrum disorders, Alzheimer's disease, Parkinson's disease, Huntington's disease, muscle wasting and viral infection, the method comprising administering a compound of claim 1 to the subject.

6. The method of claim 5, wherein the compound is selected from the group consisting of an inhibitor of protein translation, a chemotherapeutic agent, a cell sensitising agent, an antiproliferative agent, an antiviral agent and an adjuvant.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) Embodiments of the present invention will now be described, purely by way of example, with reference to the accompanying drawings, in which:

(2) FIG. 1—illustrates the specific chemical structure of a compound of Formula II which is an example of compound of Formula I and Ia.

(3) FIG. 2—demonstrates that a compound of Formula II inhibits translation and protein synthesis. Established techniques such as polysome profiling were used to show a reduction in the number of polyribosomes in neuroblastoma cells after treatment with a compound of Formula II. Polysome peaks from left to right show increasing number of ribosomes associated with mRNA. This peaks at 3 ribosomes after treatment with a compound of Formula II showing rapid and reproducible perturbation of translation. Cells were treated for 20 minutes with a compound of Formula II then harvested and prepared as described in materials and methods. The supernatants were loaded onto 10-50% sucrose gradients and spun for 2 hrs at 38,000 RPM. Gradients were visualised using a UV detector. A reduction in the amount of polyribosomes is observed after treatment with a compound of Formula II.

(4) FIG. 3a—shows that treatment of SH-SY5Y cells with a compound of Formula II (referred to as “active”) results in the selective inhibition of amyloid precursor protein APP 5′UTR luciferase reporter relative to a Renilla luciferase reporter construct containing a short unstructured 5′UTR. SH-SY5Y cells were simultaneously co-transfected with APP-5′UTR firefly luciferase construct and a Renilla control vector. Luciferase levels were assayed using a Glomax Luminometer and Stop-n-Glo luciferase reagents (standard procedure throughout). The data presented represents 8 biological repetitions and use as the active a semi refined compound of Formula II. Levels of compound were estimated based on amounts of purified compound obtained from fresh tomato tissue.

(5) FIG. 3b—shows the result of experiments in which neuroblastoma cells were transfected with a firefly luciferase translation reporter 24 hours before treatment—cells were then treated for 4 hrs with either a compound of Formula II (active) or 1 μM Hippuristanol. Graphs represent 4 biological repetitions per treatment—2 independent treatments of a compound of Formula II. Inhibition of translation reporter activity by a compound of Formula II is equivalent to treatment with 1 μM Hippuristanol (proven inhibitor of eIf4A) alone for both experiments.

(6) FIG. 3c—shows the results of experiments in which cells were transfected with a firefly luciferase translation reporter 24 hours before treatment—cells were then treated for 4 hrs with Hippuristanol (1 μM) or Hippuristanol plus a compound of Formula II (active). Graphs represent 4 biological repetitions per treatment. Hippuristanol and Hippuristanol plus a compound of Formula II result in significant inhibition (p=0.01 and 0.009 respectively). No difference is observed between Hippuristanol and Hippuristanol plus a compound of Formula II.

(7) FIG. 3d—shows that treatment with a compound of Formula II (referred to as “active”) selectively reduces the levels of firefly luciferase reporter activity dependant on 5′UTR sequence. The 5′UTRs of genes which negatively associate with the progression of Alzheimer's disease—amyloid precursor protein (APP) and beta secretases (BACE) are all inhibited by treatment with a compound of Formula II, whereas the equivalent reporter levels of housekeeping genes actin and thioredoxin (TXN) are not inhibited. The 5′UTR of cancer associated epidermal growth factor receptor (EGFR) is also selectively inhibited by treatment with a compound of Formula II. Neuroblastoma cells were transfected 24 hours prior to treatment with a compound of Formula II. After treatment, cells were prepared as described in the materials and methods. Each experiment represents between 6 and 8 biological repetitions.

(8) FIG. 4a—shows the results of experiments in which fast growing breast cancer cells MCF7 were treated for 96 hours with a compound of Formula II only. Growth of MCF7 cell lines were slowed by the treatment with a compound of Formula II. Experiment represents 6 biological repetitions, Error=S.E.M.

(9) FIG. 4b—shows the results of experiments in which fast growing breast cancer cells MDA-MB-231 were treated for 96 hours with a compound of Formula II only. Growth of MDA-MB-231 cell lines were slowed by the treatment with a compound of Formula II. Experiment represents 6 biological repetitions, Error=S.E.M.

(10) FIG. 4c—shows the results of experiments in which slow growing SKOV3 ovarian cancer cells were treated for 96 hours with a compound of Formula II only. Growth of SKOV3 cell lines were slowed by the treatment with a compound of Formula II at higher doses. Experiment represents 6 biological repetitions, Error=S.E.M.

(11) FIG. 4d—shows the results of experiments in which slow growing A549 lung carcinoma cancer cells were treated for 96 hours with a compound of Formula II plus a very low dose Cisplatin™ (1 μM). Cisplatin™ resistant A549 lung cancer cells were sensitised to treatment with a compound of Formula II in combination with 1 μM Cisplatin™—a complete kill is achieved at higher doses of a compound of Formula II.

(12) FIG. 4e—shows the results of experiments in which slow growing SH-SY5Y neuroblastoma cancer cells were treated for 96 hours with a compound of Formula II plus low dose Cisplatin™. Cisplatin™ resistant A549 lung cancer cells were sensitised to treatment with a compound of Formula II in combination with 2.5 μM Cisplatin™—a complete kill is achieved at higher doses (10 μg) of a compound of Formula II.

(13) FIG. 4f—shows the results of experiments in which slow growing SKOV-3 ovarian cancer cells were treated for 96 hours with a compound of Formula II plus low dose Cisplatin™. Cisplatin™ resistant SKOV-3 ovarian cancer cells were sensitised to treatment with a compound of Formula II in combination with 2.5 μM Cisplatin™ (p=0.003) (right hand bar in the figure), no effect is observed from treatment with equivalent levels of a compound of Formula II alone (left hand bar in the figure) or Cisplatin™ alone.

(14) FIG. 5—shows that a compound of Formula II (the active) selectively inhibits the translation of genes known to excacerbate the symptoms of autistic spectrum disorders. Experiments conducted using a published luciferase reporter system (Gkogkas et al Nature 2013, 493:371-7) demonstrate that treatment with Formula II selectively reduces the translation of the longer more structured 5′ untranslated region (reporter 1) of the gene neuroligin 1 relative to neuroligin 2. Selective inhibition of neuroligin 1 protein levels has been demonstrated to restore the normal excitation/inhibition ratio and rectifies the social behaviour deficits observed in an autism mouse model (see Gkogkas et al, Nature 2013, 493:371-7). The mechanism of action and level of activity of Formula II are consistent with and comparative to a proven translational inhibitor extracted from a rare coral species (Hippuristanol—a proven inhibitor of eIf4A). In this experiment 1 μM of Hippuristanol and 1.3 μM of Formula I was used

(15) FIG. 6—shows that treatment with a compound of Formula II (active) inhibits the growth of the chemoresistant cancer cell line A549, lung carcinoma. Cells were treated with a range of doses of active for either 48 or 96 hrs. Each data point is representaive of at least 4 biological repetitions. Data is reproducable with different cultures of A549 cells, in two different laboratories at Nottingham (a) Biosciences and (b) Cancer Biology and efficacy has been demonstrated using standard techniques e.g. WST-1 (a) and MTT (b) and stably transfected luciferase cells (not shown).

(16) FIG. 7—shows that treatment with a low dose of a compound of Formula II (active) sensitises chemo-resistant A549 lung carcinoma cells to very low levels of Cisplatin (2 μM). Cells were treated with either 2 μM Cisplatin™ alone, c. 1 μg active alone or c. 1 μg active in combination with 2 μM Cisplatin™. A WST-1 cell proliferation assay was performed 96 hrs after treatment. Experiment represents 4 independent biological repetitions. A significant increase in the efficacy of Cisplatin™ is observed when treated in combination with the compound of Formula II.

(17) FIG. 8—shows the effect of a compound of Formula II (which is exemplary of Formula I and Ia) on chemo-resistant primary canine histocytic sarcoma tumour cells biopsied from a 7 yr old retriever. Cells were cultured for 6 days with a single dose treatment of Cisplatin™ (10 μM) or a combination of Cisplatin™ plus a compound of Formula II (active). Images were taken after 6 days treatment and are representative of three independently treated wells. Each image (40× magnification) represents the majority of the well area and is of an equivalent area in each photograph. Similar results were observed with the active in combination with carboplatin (2 μM dose).

(18) FIG. 9a—shows the effect of the treatment of A549 lung carcinoma cells with a synthetic molecule of Formula II. The data shown demonstrates that cell growth is inhibited by Formula II in a dose dependent manner. A WST-1 cell proliferation assay was performed 72 hrs after treatment. Experiment represents 4 independent biological repetitions.

(19) FIG. 9b—shows that treatment of chemo-resistant A549 lung carcinoma cells with a synthetic molecule of Formula II sensitises the cells to very low level doses of Cisplatin (2 μM). Cells were treated with either 2 μM or 10 μM Cisplatin™ alone, or 10 μg synthetic molecule Formula II in combination with 2 μM Cisplatin™. A WST-1 cell proliferation assay was performed 72 hrs after treatment. Experiment represents 4 independent biological repetitions. A 5-fold increase in the efficacy of Cisplatin™ is observed when treated in combination with the synthetic Formula II.

(20) FIG. 9c—shows that treatment of chemo-resistant A549 lung carcinoma cells with a chemically synthesised acetyl derivative of Formula II sensitises the cells to very low level Cisplatin™ (2 μM). Cells were treated with either 2 μM, 5 μM or 10 μM Cisplatin™ alone, 50 μg synthetic acetyl derivative molecule or 30 μg in combination with 2 μM Cisplatin™. A WST-1 cell proliferation assay was performed 72 hrs after treatment. Experiment represents four independent biological repetitions. No effect was detected after treatment with 50 μg synthetic acetyl derivative, however an 8-fold increase in the efficacy of Cisplatin™ is observed when treated in combination with 30 μg synthetic acetyl derivative and 2 M Cisplatin™.

(21) FIG. 10—Compound 46, NLGN Translation reporter assay. FIG. 10 shows that a compound of Formula 46 selectively inhibits the translation of genes known to exacerbate the symptoms of autistic spectrum disorders. Experiments conducted using a published luciferase reporter system (Gkogkas et al 2013. Nature, 493:371-7) demonstrate that treatment with Formula II selectively reduces the translation of construct containing the 5′ untranslated region (reporter 1) of the gene neuroligin 1 relative to neuroligin 2. Selective inhibition of neuroligin 1 protein levels has been demonstrated to restore the normal excitation/inhibition ratio and rectifies the social behaviour deficits observed in an autism mouse model (see Gkogkas et al, 2013. Nature, 493:371-7). FIG. 10B shows that inhibition of translation at this dose for this length of time is independent of the anti-proliferative activity of the molecule.

(22) FIG. 11—Natural molecule plus cisplatin compared to Hippuristanol plus cisplatin. FIG. 11 shows that treatment of chemo-resistant A549 lung carcinoma cells with either a synthetic molecule of Formula II or the known inhibitor of eIF4A are both anti-proliferative. When used in combination, the sensitizing effects to very low level doses of Cisplatin (2 μM) of the synthetic molecule of Formula II is equivalent to hippuristanol. The ratio between anti-proliferative activity to chemo sensitizing activity is also equivalent.

(23) FIG. 12—CrPV assay—Natural molecule targets eIF4A. Treatment with 20 μM of the synthetic natural molecule selectively inhibits cap dependant translation. Treatment with Formula II selectively reduces the translation of the firefly luciferase gene relative to the renilla gene, which is downstream of the eIF4A independent CrPV IRES.

(24) FIG. 13—Dose curves of compounds 46, 99 and 123. Shows that treatment with synthetic derivatives of the compound of Formula II (46, 99 and 123) inhibits the growth of the chemoresistant cancer cell line A549, lung carcinoma in a dose dependant manner. Cells were treated with a range of doses of active for 96 hrs. Each data point is representative of at least 4 biological repetitions and error=s.e.m).

(25) FIG. 14—Cisplatin™ combination experiments—Compounds 46, 99 and 123. Shows that treatment with synthetic derivatives of the compound of Formula II (either 46, 99 and 123) sensitizes chemoresistant cancer cell line A549, lung carcinoma to low dose cisplatin. Cells were treated with a range of doses of active in combination with a range of doses of Cisplatin™ for 96 hrs. Each data point is representative of at least 4 biological repetitions and error=s.e.m).

(26) FIG. 15—shows structures of compounds 46, 99 and 123.

(27) FIG. 16—shows that treatment with a range of synthetic derivatives of the compound of Formula I and Ia has antiproliferative and chemosensitizing sensitizes effects which are linked to structure. Cells were treated with a range of different derivatives at three different doses (20 μM, 40 μM, 80 μM) of active. To determine sensitizing effects, additional experiments were also conducted in combination with 2 μM Cisplatin™ for 96 hrs. Each data point is representative of at least 4 biological repetitions and error=s.e.m).

(28) FIG. 17—shows that treatment with a range of synthetic derivatives of the compound of Formula I and Ia has antiproliferative and chemosensitizing sensitizes effects which are linked to structure, and effects are additive or synergistic with Cisplatin™. Cells were treated with a range of different derivatives at three different doses (20 μM, 40 μM, 80 μM) of active. To determine sensitizing effects, additional experiments were also conducted in combination with 2 μM Cisplatin™ for 96 hrs. Each data point is representative of at least 4 biological repetitions and error=s.e.m).

EXAMPLES

(29) A Compound of Formula I Inhibits Protein Synthesis

(30) Polysome Profiling

(31) By using polysome ribosome profiling a compound of Formula I, as exemplified in these experiments by the compound of Formula II (FIG. 1), is demonstrated to be an inhibitor of protein synthesis as shown by profiling of the number of ribosomes associated with mRNA in the presence and absence of the compound (FIG. 2). Standard sucrose density polysome profiling techniques demonstrate that treatment with a compound of Formula II reduces the average numbers of ribosomes per message in cultured human cells. The number of ribosomes is indicative of the translation of an mRNA and synthesis of the protein encoded by the mRNA, and treatment with the compound of Formula II decreases the number of ribosomes per message thereby reducing global protein synthesis.

(32) Use of the Compound(s) for the Inhibition of eIF4A

(33) By using a well characterised luciferase based reporter assay it was further determined that this class of molecule functions as a protein synthesis inhibitor via targeting the helicase eIF4A. The cricket paralysis virus RNA contains a well-documented internal ribosomal entry site (CrPV IRES); this internal ribosomal entry site does not require eIF4A for active translation (Bordeleau et al, 2006 Nature Chemical Biology, 2: 213-220). Cap-dependent (eIF4A dependant) translation (firefly luciferase signal), but not CrPV IRES-dependent translation (Renilla luciferase signal), was inhibited after 3 hours treatment with the Synthetic version of the natural molecule. Since the lack of a requirement for eIF4A for CrPV translation is well documented (e.g. Bordeleau et al, 2006 Nature Chemical Biology, 2: 213-220) this data further demonstrates inhibition is selective and provides evidence that the target is the translation initiation factor eIF4A.

(34) Reporter Assays

(35) Reporter assays were used to demonstrate that the compound of Formula II is an inhibitor of protein synthesis. Firefly/renilla luciferase reporter experiments conducted using cultured human cell lines show that the compound of Formula II is a selective and facile inhibitor of protein synthesis (schematics of the reporter constructs are included in FIGS. 3a, 3d and 5). The compound of Formula II is shown to selectively decrease the levels of a reporter construct containing a long structured 5′UTR upstream of a firefly luciferase gene, but to have little effect on a co-transfected renilla luciferase reporter construct containing a short unstructured 5′UTR (FIGS. 3a and 5).

(36) The degree of translation inhibition is shown to be equivalent to that of a known inhibitor of translation, hippuristanol (FIGS. 3b and 5). Comparative structured 5′UTR firefly luciferase reporter experiments conducted using either hippuristanol or a compound of Formula II show the inhibition of reporter levels is equivalent. Co-treatment with a compound of Formula II and hippuristanol (FIG. 3c) shows no additive inhibitor effects providing further evidence that both molecules are acting on the same target, this may be the translation complex helicase protein eIF4A.

(37) Firefly luciferase reporter experiments conducted with the 5′UTRs of genes which negatively associate with disease demonstrate that inhibition is both selective and relevant to the treatment of disease such as Alzheimer's disease, cancer and autistic spectrum disorders by selectively altering the translation of select transcripts while the translation of housekeeping or cytoprotective genes remains unaffected (FIGS. 3d and 5). After treatment with the compound of Formula II the levels of translation of a reporter construct containing the 5′UTR of amyloid precursor protein, which is processed into toxic amyloid the major constituent of amyloid plaques in Alzheimer's disease, is inhibited relative to equivalent control treatments. Similar significant inhibition (p=0.005) of a construct containing the 5′UTR of the epidermal growth factor receptor gene (EGFR), a gene whose expression and levels of proteins negatively associate with cancer progression and survival is also observed after treatment. This data also supports the model of selective inhibition.

(38) The data also supports the use of compounds of Formula I for the treatment of diseases such as Alzheimer's disease, cancer and autistic spectrum disorders.

(39) Use of a Compound of Formula I in the Treatment of Cancer

(40) A compound of Formula I or Ia, as exemplified by the compound of Formula II, may be used alone in the treatment of cancer as demonstrated by its ability to act as an antiproliferative agent when used as a treatment in isolation (FIGS. 4a, b, c, 5 and 6). As cancer cells recruit the protein synthesis machinery to drive proliferation, this presents an attractive target for therapy. It is well established that rapidly growing tumour cell lines require relatively higher levels of protein synthesis than normal cells—treatment of rapidly proliferating breast cancer cell lines (MCF-7 and MDA-MD-231) with the compound of Formula II dramatically limits the proliferation of these cell types (FIG. 4a, 4b). Equivalent treatments of slow growing cell lines e.g. SKOV3 ovarian cancer cells using a compound of Formula II (FIG. 4c) shows some slowing of proliferation.

(41) Similar results are seen with A549 lung cancer cells (FIG. 6 and FIG. 9a). Similar results were seen with the compound of Formula II when purified from a natural source (FIG. 6) and with a chemically synthesised compound of Formula II (FIG. 9a).

(42) A compound of Formula I or Ia, as exemplified by the compound of Formula II, may also be used in combination with other chemotherapeutic agents for the treatment of cancer. The compound of Formula I or Ia may sensitise cells to the chemotherapeutic agents thereby reducing the dose of chemotherapeutic agent needed. This is particularly advantageous as chemotherapeutic agents can be toxic and particularly difficult for patients to tolerate. The side effects of chemotherapeutic agents at the doses currently required are in some cases so severe that the use of potentially effective drugs is precluded.

(43) Known inhibitors of protein synthesis such as hippuristanol have proven potent anti-cancer properties when used in combination with chemotherapeutic agents such as Cisplatin™ or Doxorubicin™. However hippuristanol is naturally found in coral to is scarce and expensive to obtain, furthermore it is very difficult and expensive to synthesise. The data presented here demonstrates that a compound of Formula I or Ia, exemplified by the compound of Formula II, can be used as an adjuvant in combination with chemotherapeutic agents to enhance cell death. In particular this combination has a potent effect at slowing proliferation or killing cancer cells. Slow growing and difficult to treat tumour cell types such as A549 lung cancer cells, SH-SY5Y neuroblastoma or SKOV-3 cancer cells are all sensitised by exposure to Formula II to very low doses of Cisplatin™ (FIGS. 4d, 4e, 4f and 7)—a complete kill can be achieved in both A549 and SH-SY5Y cells after a single dose treatment with g quantities of a compound of Formula II in combination with 1 μM or 2.5 μM treatments of Cisplatin™. A similar effect is observed when rapidly growing tumour cell lines or primary tumour cells (FIG. 8) are treated with a compound of Formula II and chemotherapeutics such as Cisplatin™. In this example primary tumour cells were isolated from a dog and then in vitro exposed to the compound of Formula II and Cisplatin™. The results show that when treated with only Cisplatin™ many cancer cells remain, however when treated with Cisplatin™ and the compound of Formula II substantially all tumour cells were killed. Not visible in the images reproduced here but visible under the microscope, it can be seen that white blood cells which were transferred with the tissue sample were still alive after the Cisplatin™ and Formula II treatment. This demonstrates the adjuvant properties of a compound of Formula I, more specifically that compounds of Formula I can sensitise cancer cells to the effects of chemotherapeutic agents.

(44) The results in FIG. 9b demonstrate that a chemically synthesised compound of Formula II is also effective as an anticancer agent alone, and as an agent to sensitise cancer cells to other chemotherapeutic agents. Previously discussed data was obtained used a compound of Formula II isolated from tomatoes.

(45) The results shown in FIG. 11 demonstrate that the relative anti-proliferative effects of treatment with a chemically synthesised compound of Formula II is comparable in efficacy to treatment with the known inhibitor hippuristanol. The relative activity as an agent to sensitise cells to cisplatin treatment is also equivalent at this dose.

(46) The results in FIG. 9c demonstrate that a chemically synthesised acetyl derivative of a compound of Formula II is also effective as an agent to sensitise cancer cells to other chemotherapeutic agents, in this particular example to Cisplatin™. The acetyl derivative of Formula II used in this study is illustrated below:

(47) ##STR00036##

(48) FIG. 13 demonstrates that chemically synthesised derivatives of Formula II (Compounds 46, 99 and 123) are also effective in a dose dependant manner as an anticancer agent alone, and can act as an agent to sensitise cancer cells to other chemotherapeutic agents (FIG. 14).

(49) Use of the compound(s) for the treatment of autism.

(50) Direct evidence is provided that inhibiting eIF4A represents a new route to treating ASD based on the data presented by Gkogkas et al. Nature 2013, 493:371-7. Firefly/renilla luciferase reporter experiments conducted using cultured human cell lines show that eIF4A is a viable therapeutic target for the treatment of ASD and that eIF4A1 inhibition using either hippuristanol or compound of the synthetic version of the natural molecule and synthetic derivatives (data also shown for 46) result in the selective inhibition of NLGN1 translation.

(51) Treatment with the compound of Formula II (and compound 46) (FIG. 5 and FIG. 10) selectively decrease the firefly luciferase signal from a reporter construct containing the NLGN1 5′UTR upstream of a firefly luciferase gene. The translation of luciferase reporters downstream of the NLGN1 5′UTR has been proven to be dependent on the activity of the translation initiation complex eIF4F (Gkogkas et al Nature 2013, 493:371-7)—a complex containing the helicase eIF4A. Equivalent treatment had little effect on the signal generated from either a co-transfected renilla luciferase reporter control or cells transfected with an equivalent construct containing the NLGN2 5′UTR upstream of a firefly luciferase gene (FIG. 5 and FIG. 10).

(52) The level of translation inhibition of NLGN1 is shown to be equivalent to that induced by a known inhibitor of eIF4A, hippuristanol. This data further demonstrates that the compound acts to target the translation initiation complex and also provides proof that the translation of NLGN1 is relatively more dependent on the activity of eIF4A in comparison with NLGN2. Data also shows that the inhibitory effects observed are not due to the anti-proliferative activity of the compound at this dose and treatment time.

(53) Materials and Methods

(54) Production of the Compound of Formula II The compound of Formula II is a glycoglycerol lipid, the synthesis of such compounds is well known. The skilled man could readily make the compound of Formula II, or the an acetyl derivative thereof, by following the reaction mechanism described in Manzo, E.; Letizia Ciavatta, M.; Pagano, D.; Fontana, A. Tetrahedron Lett. 2012, 53, 879.

(55) Alternatively the compound of Formula II may be recovered from plant materials, for example tomatoes. Tomatoes were grown under standard glass house, harvested and snap frozen in liquid nitrogen. Tissue was ground under liquid nitrogen to form a powder, mixed with 2 volumes of MeOH (wt/vol) and heated at 50° C. for 10 minutes. This mixture was then centrifuged at 4000 RPM to pellet cellular debris and the supernatant transferred to a clean tube. The MeOH was then partitioned into a chloroform phase, and the chloroform layer then dried down to yield a pellet.

(56) The crude extract was adsorbed onto chromatography grade silica gel and dry-loaded onto a silica gel flash chromatography column. The products were eluted with a gradient of 0-20% methanol in dichloromethane, and fractions were collected and tested for biological activity. The active fractions were evaporated in vacuo to give an oil (155 mg). Further purification was performed by batch-wise reverse phase HPLC (Varian Prostar; Polaris 5 micron C18-A column (250 mm×10 mm); gradient elution 80% H2O 20% MeCN to 0% H2O100% MeCN following the following method: 80% H2O 20% MeCN 2 min; 0% H2O 100% MeCN 20 min; 0% H2O 100% MeCN 48 min; 80% H2O 20% MeCN 50 min). The active fractions (eluting at 30 min) were collected and evaporated in vacuo to give the active molecule whose NMR (1H and 13C), HRMS and IR data confirmed it to be the structure shown in FIG. 1.

(57) Cell Culture Conditions

(58) Cells were cultured and maintained using standard conditions as described on the American Type Culture Collection Web page (see ATCC for details http://www.lgcstandards-atcc.org) in appropriate media e.g. Dulbecco's Modified Eagle's Medium (DMEM) or Roswell Park Memorial Institute medium (RPMI) (Sigma) supplemented with 10% FCS, and 1% Penicillin/Streptomycin (Life Technologies).

(59) Polysome Profiling

(60) Polysome profiles were obtained using sucrose density centrifugation. Briefly one 15 cm plate of cultured Neuroblastoma cells (SH-SY5Y) were grown per treatment to a confluency of 70%. Cells were then treated with either active or equivalent DMSO vehicle control for 20 min. Cells were harvested, lysed and loaded onto sucrose gradients then centrifuged at 38,000 RPM for 2 hours (as described in Bottley et al, 2010). Gradients were fractionated and polysome profiles determined through a continuous monitoring at absorbance 260 nm (described previously Johannes et al. 1999).

(61) Transient Transfection Conditions and Luciferase Reporter Constructs

(62) Experiments conducted using Firefly luciferase reporter plasmids containing the 5′ untranslated regions (UTRs) of the genes amyloid precursor protein (APP), thioredoxin (TXN) were conducted with reagents and materials described by Bottley et al, 2010. Experiments conducted using Firefly luciferase reporter plasmids containing the 5′ UTRs of the genes EGFR, BACE1 and Actin were conducted with reagents and materials described by Webb, 2012 (http://etheses.nottingham.ac.uk/2724/). Firefly luciferase reporter plasmids containing the 5′ untranslated regions (UTRs) of the genes Neuroligin 1 and Neuroligin 2 were a kind gift from Professor Nahum Sonenberg (McGill) and used as described by Gkogkas et al, Nature 2013, 493:371-7.

(63) Cells were transfected using FuGene 6 (Roche) following the manufacturer's instructions. The activities of firefly and renilla luciferase in lysates prepared from transfected cells were measured using a commercially available Luciferase reporter assay system (Promega) and light emission was measured over a 10 sec interval using a TECAN luminometer. For each experiment described, data was obtained from a minimum of at least 3 biological repetitions per treatment.

(64) Cell Proliferation Experiments

(65) Prior to treatment cells were cultured to an appropriate confluency in 96 well tissue culture plates (Fisher). Cells remained either supplemented with fresh media or treated with fresh media containing active or an equivalent volume of DMSO (vehicle control). Where used, Cisplatin™ was diluted to a stock concentration in Dimethylformamide (DMF), then handled as per the manufacturer's instructions. To determine relative cell viability, reagents WST-1 (Roche) or MTT (Sigma) were used as per the manufacturer's instructions and absorbance at 450 nm measured using a Victor plate reader (Perkin Elmer).

(66) Primary Canine Tumour Cell Experiments.

(67) Biopsy tissue was removed from knee, abdomen and skin of a 7 year old dog. Cells harvested from the canine source were confirmed through histological evaluation to be histiocytic sarcoma tumour cells. Samples were fragmented prior to collagenase treatment in controlled conditions at 37° C. for 3 hours. Cells were then sedimented by low speed centrifugation and resuspended in selective culture media using proprietary methods and materials developed by Petscreen Ltd. Experiments were performed in 96 well tissue culture plates with a minimum of three biological repetitions per treatment.

(68) Production of the Compounds of Formula I and La in FIGS. 16 and 17

(69) The compounds of Formula I and Ia in FIGS. 16 and 17 are synthetic variants of the compound of Formula II. For example, they may use glucose or mannose sugar units rather than galactose and they may use a central linker unit that has an additional CH.sub.2 group.

(70) The synthesis of glycoglycerol lipids and the like is well known and it is within the skilled person's ability to modify known reaction techniques for synthesising glycoglycerol lipids to produce the compounds of Formula I and Ia in FIGS. 16 and 17 (which are compounds 99, 218, 139, 184, 123, 180, 124, 159, 38, 215, 146, 122, 119, 62, 120, 46, 61, 57, 60, 56, 154, and 58, which are also shown in the description above).

(71) Specifically, the compounds of Formula I and Ia in FIGS. 16 and 17 were each made by following the reaction mechanism described in Manzo, E.; Letizia Ciavatta, M.; Pagano, D.; Fontana, A. Tetrahedron Lett. 2012, 53, 879.

(72) This synthesis is a versatile and simple procedure based on trichloro-acetimidate methodology and the use of peracetate sugar substrates. The chemical strategy allows stereoselective preparation of lipid derivatives, and other related derivatives, of sugars such as galactose and glucose and mannose. The synthetic approach is designed to obtain enantiomerically pure regio- and stereo-isomers including derivatives containing poly-unsaturated fatty acids.

(73) In essence, the synthesis recognises that glycoglycerol lipids such as:

(74) ##STR00037##

(75) can be derived from the starting materials:

(76) ##STR00038##

(77) The required variations on these starting materials to achieve the compounds in FIGS. 16 and 17 can be readily seen by the skilled person; e.g. a different sugar unit, a linker unit with an additional CH.sub.2 group, a choice of R and R1 groups.

(78) The manufacture of each of the compounds in FIGS. 16 and 17 was therefore based on following steps from that known synthetic route (shown schematically below) and with selection of the appropriate starting materials/reagents to provide the appropriate sugar unit, R and R1 groups and linker unit therebetween.

(79) ##STR00039##

(80) The majority of the compounds made and illustrated in FIGS. 16 and 17 are directly based on this synthesis, with the difference solely lying in the choice of sugar, and whether it is protected or not, and the choice of R and R′ groups. For example, compounds 123, 180, 124, 38, 122, 119, 62, 120, 61, 57, 60, 56 and 58.

(81) Synthetic Route to Compound 159

(82) The synthetic route to 159 followed an identical route to that used for all other esters mentioned in the above Tetrahedron Letters paper by Manzo, E et al, with the only difference being that diphenyl acetic acid was used instead of a fatty acid to provide the R and R′ groups.

(83) Synthetic Route to Compound 139

(84) ##STR00040##

(85) Preparation of Ketone A

(86) The ketone A (step 1 above) was synthesised from galactose according to: A Cavezza, C. Boulle, A. Guéguiniat, P. Pichaud, S. Trouille, L. Ricard, M. Dalko-Csiba, Bioorganic & Medicinal Chemistry Letters 2009, 19, 845-849.

(87) Preparation of Ketone B

(88) To a stirred suspension of the known ketone A (1.81 g, 8.1 mmol) in dichloromethane (8 mL) and pyridine (4.90 mL, 60.0 mmol) at OC was added acetic anhydride (4.72 mL, 4.72 mmol) drop wise. The resulting reaction mixture was warmed to room temp and stirred overnight (ca 16 hours). The reaction was poured in to water and extracted with dichloromethane (3×50 mL), the combined organic phase were washed with 3M HCl (3×50 mL), sat NaHCO.sub.3(50 mL), brine (50 mL), dried over MgSO4 and evaporated, to afford a gum which was purified by silica gel chromatography (1:1 to 0:1 Petrol:Et2O) to afford the tetra acetate ketone B (2.87 g, 7.43 mmol, 57%) as a pale yellow solid.

(89) Preparation of Alcohol C

(90) To a stirred solution of the ketone B (420 mg, 1.08 mmol) in THF (10 mL) at −78° C. was added MeMgBr (1.4M, 1.85 mL, 2.6 mmol) drop wise. The resulting solution was stirred at −78° C. for 4 hours. The reaction was quenched by the addition of sat. ammonium chloride solution (20 mL) and extracted with EtOAc (3×25 mL), the combined organic phase were washed with brine (25 mL), and dried over MgSO4 and evaporated, to afford a gum which was purified by silica gel chromatography (1:1 to 0:1 Petrol:EtOAc) to afford the alcohol C (133 mg, 0.328 mmol, 30.5%) as a colourless solid.

(91) Preparation of Ester D

(92) DCC coupling according to a slightly modified procedure reported in Tetrahedron Lett. 2012, 53, 879.

(93) To a stirred solution of the alcohol C (126 mg, 0.31 mmol) in dichloromethane (6 mL) at room temp under argon was added linolenic acid (94.5 mg, 0.34 mmol), dicyclohexylcarbodiimide (70.6 mg, 0.34 mmol) and DMAP (8.4 mg, 0.068 mmol), the reaction mixture was stirred overnight (ca 16 hours) at room temp. The reaction was cooled to −20° C., and filtered, the filtrated was and evaporated under reduced pressure and the mixture was purified by silica gel chromatography (8:1 to 4:1 Petrol:EtOAc) to afford the ester D (115 mg, 0.173 mmol, 55.8%) as a colourless oil.

(94) Preparation of Compound 139

(95) Deprotection according to the procedure reported in Tetrahedron Lett. 2012, 53, 879.

(96) To a stirred solution of the ester D (105 mg, 0.158 mmol) in aq. ethanol (85%) (5 mL) at 44° C. was added hydrazine mono-hydrate (63 μL, 1.26 mmol), the reaction mixture was stirred at 44° C. for 4 hours. The solvent was removed under a stream of nitrogen and the residue was purified by silica gel chromatography 10:1 dichloromethane:MeOH) to afford the compound 139 (38 mg, 0.077 mmol, 48%) as a colourless oil.

(97) Synthetic Route to Compounds 99, 218, 184, 215 and 46

(98) The modified linker unit as used in compounds 99, 218, 184, 215 and 46 (where there is an additional CH.sub.2 within the linker unit) as compared to the linker unit illustrated in the Tetrahedron Letters reaction scheme above) is not commercially available; however it is a known compound, whose synthesis is reported in the following papers: C. Iwata, N. Maezaki, K. Hattori, M. Fujita, Y. Moritani, Y. Takemoto, T. Tanaka, T. Imanishi, Chemical and Pharmaceutical Bulletin, 1993, 41, (2), 339-345 R. Schillera, L. Tichotováa, J. Pavlíka, V. Buchtab, B Melicharc, I. Votrubad, J. Kuneša, M. Špuláka, M. Poura, Bioorganic & Medicinal Chemistry Letters, 2010, 20, (24), 7358-7360 H. A. Bates, J. Farina, M. Tong, J. Org. Chem., 1986, 51 (14), 2637-2641.

(99) The linker unit was therefore synthesised according to the known methodology, before being used in the Tetrahedron Letters reaction scheme.

(100) To illustrate this, the synthetic route to compound 46 is set out below:

(101) ##STR00041##

(102) This synthesis illustrates the straightforward nature of the modifications needed to the reaction scheme from the above Tetrahedron Letters paper by Manzo, E et al to synthesise compounds having an altered linker unit.

(103) It will be noted that this route is almost identical to that described in the Tetrahedron Letters paper but it does differ in Step 4 where a modified alcohol is used to modify the linker unit. The preparation of this modified alcohol is given in J. Org. Chem. 1986, 51, 2637 (it is structure 14 in that paper).

(104) The adaptations to the above synthetic route to compound 46 that would be required to reach the compounds 99, 218, 184 and 215 (which also include the modified linker unit) are easily apparent. The differences lie in the choice of sugar, and whether it is protected or not, and the choice of R and R′ groups.

(105) Synthetic Route to Compounds 146 and 154

(106) ##STR00042##

(107) Preparation of Compound 146

(108) To a stirred solution of the commercially available galactose bis-acetonide (260 mg, 1.00 mmol) in dichloromethane (10 mL) at room temp under argon was added linolenic acid (278 mg, 1.0 mmol), dicyclohexylcarbodiimide (206 mg, 1.0 mmol) and DMAP (24 mg, 0.2 mmol), the reaction mixture was stirred overnight (ca 16 hours) at room temp. The reaction was cooled to −20° C., and filtered, the filtrated was and evaporated under reduced pressure and the mixture was purified by silica gel chromatography (8:1 to 2:1 Petrol:Et2O) to afford the compound 146 (438 mg, 0.84 mmol, 84%) as a colourless oil.

(109) Preparation of Compound 154

(110) To a stirred solution of the compound 146 (106 mg, 0.20 mmol) in DCM (1 mL) at 0° C. was added trifluoroacetic acid (1 mL), and the reaction was stirred for 12 hours. The reaction was evaporated under reduced pressure and the residue was purified by silica gel chromatography (10:1 DCM:MeOH) to afford compound 154 as a mixture of a anomers (60 mg, 0.136 mmol, 68%) as a colourless oil.