BACKGROUND TO THE INVENTION

Globally, cancer affects 14 million people and accounts for one fifth of deaths in both developing and developed countries. The World Health Organization (WHO) reports that one out of three people will be diagnosed and treated for some form of life-threatening cancer in their life time, making cancer one of the leading causes of death after heart disease. Incidences of most cancers increase with age and these numbers continue to rise with increasing life expectancy. In sub-Saharan Africa, prostate and lung cancer is most prevalent in males; breast and cervical cancer are the most common types to affect women. Cervical and esophageal cancers are among the most common cancers affecting Southern Africans and do not generally present symptoms until a late stage. Hence, diagnosis is likely only made after the cancer has spread or is at an advanced stage. According to reports from CANSA (Cancer Association of South Africa), cervical cancer kills more women in Southern Africa than any other form of cancer, except for breast cancer. As such, it is important to focus on the detection and treatment of these high incidence cancers and their impact in a Southern African setting. Conventionally, there are four approaches to cancer treatment after diagnosis: surgery, chemotherapy, radiotherapy and palliative care. Radiotherapy or radiation therapy (RT) can be an effective treatment, especially for localized or solid cancers and about half of cancer patients receive radiation as a curative or palliative treatment. Radiotherapy uses high energy radiation to control cancer cell growth by damaging DNA within the cancer cell. If the DNA of a cancer cell is sufficiently damaged, the cells are unable to replicate as usual and the growth of the tumor is inhibited. Different types of irradiation, such as X-rays, gamma rays, electron beams, protons, and/or charged particles are irradiated at the tumor site either from outside (external-beam radiotherapy) or inside (internal radiotherapy) the body. However, although RT alone or combined with other modalities is effective for treating cancer globally, some cancers are not responding. This could be due to the development of radiation resistance but it depends also on the cancer type, location and an early diagnosis is important. Therefore, attempts to combine RT with cellular and molecular targeted biological modalities which determine the sensitivity or resistance to ionizing radiation is an unmet need. Secondly, new targeted therapies are urgently needed that only kill cancer cells without inducing normal tissue toxicity. Thirdly, new imaging probes with a higher specificity and appropriate for early cancer detection are needed as early detection is often the key to surviving any form of cancer. These objectives can be achieved through the radiolabeling of small molecule inhibitors, which can be used for the detection as well as the treatment of specific tumor types.

It is an object of the present invention to provide a new radiolabeled pharmaceutical compound for the early detection and treatment of cancer, in particular cervical and esophageal cancer.

SUMMARY OF THE INVENTION

According to the present invention there is provided a hydroxamate metalloprotease inhibitor compound for use in a method of diagnosing or treating cancer, inflammatory diseases or Alzheimer’s disease, comprising a zinc-chelating N-hydroxamate moiety radiolabeled with a radionuclide, the zinc-chelating N-hydroxamine moiety, having the general structure: wherein:

R ₁ is an alkyl group, preferably a short alkyl chain such as methyl or ethyl;

R ₂ is an alkyl, alkenylene, alkynylene, or aryl group, such as 3-phenyl -1 - propyl, alkenylene, or alkynylene;

R ₃ is H or lower alkyl; R ₄ is an alkyl, alkenylene, alkynylene, or aryl group, such as tert-butyl; alkenylene, alkynylene, or arylene;

R ₅ is H or lower alkyl; and

R ₆ is an alkyl group, preferably a short alkyl chain such as methyl.

The radionuclide is preferably incorporated into an aryl group such as a phenyl ring on the R ₂ moiety, typically in the para- position.

The hydroxamate metalloprotease inhibitor is typically GI254023X ((2R,3S)- 3-(Formylhydroyamino)-2-(3-phenyl-1 -propyl)butanoic Acid [( 1 S)-2,2- Dimethyl-1 -(methylcarbamoyl)-1 -propyl]amide) and derivatives thereof.

In the case where the compound is for use in a method of diagnosing cancer, inflammatory diseases or Alzheimer’s disease, the radionuclide may be a diagnostic radionuclide selected from ^99m Tc, ¹⁸⁸ Re, ¹⁸⁶ Re, ¹⁵³ Sm, ⁶⁷ Ga, ⁶⁸ Ga, ¹¹¹ In, ⁵⁹ Fe, ⁶³ Zn, ⁵² Fe, ⁴⁵ Ti, ⁶⁰ Cu, ⁶¹ Cu, ⁶⁷ Cu, ⁶⁴ Cu, ⁶² Cu, ¹⁹⁸ Au, ¹⁹⁹ Au, ^195m Pt, ^{191 m} Pt, ^{193 m} Pt, ^{117 m} S n , ⁸⁹ Zr ^{1 77} Lu , 1 ⁸ F ⁷⁴ Br, ⁷⁵ Br, ⁷⁶ Br, ⁷⁷ Br, ⁸² Br, ⁸⁵ Br and ¹²³ | .

In the case where the compound is for use in a method of treating cancer, inflammatory diseases or Alzheimer’s disease, the radionuclide may be a therapeutic radionuclide selected from ¹⁸⁸ Re, ¹⁸⁶ Re, ¹⁵³ Sm, ¹⁶⁶ Ho, ⁹⁰ Y, ⁸⁹ Sr, ¹¹¹ In, 1 ⁵³ Gd, ²²⁵ Ac, ²¹² Bi, ²¹³ Bi, ²¹¹ At, ⁶⁰ Cu, ⁶¹ Cu, ⁶⁷ Cu, ⁶⁴ Cu, ⁶² Cu, ¹⁹⁸ Au, ¹⁹⁹ Au ^195m Pt ^193m Pt ¹⁹⁷ Pt ^{117 m} S n ¹⁰³ Pd ^103m Rh ¹⁷⁷ Lu ²²³ Ra ²²⁴ Ra ²²⁷ Th ³² P ¹⁶¹ Tb, ³³ P, ¹²⁴ l, ¹²⁵ l, ¹³¹ l, ²⁰³ Pb, ²⁰¹ TI, ¹¹⁹ Sb, ^58m Co, ⁷⁴ Br, ⁷⁵ Br, ⁷⁶ Br, ⁷⁷ Br, ⁸⁰ Br, ⁸² Br, ⁸⁵ Br and ¹⁶¹ Ho.

Radiolabeled compounds of the invention may be used in targeted radionuclide therapy wherein a patient is treated with a compound of the invention comprising a diagnostic radionuclide to identify the presence of a cancer or disease, followed by treatment with a compound of the invention comprising a therapeutic radionuclide to treat said cancer or disease. Typical radionuclides are isotopes of I and Br. For example, this therapy could be achieved by injecting a patient with a compound of the invention radiolabeled with diagnostic radionuclide ¹²³ l and identifying a tumor or disease using SPECT imaging or ¹²⁴ l for PET imaging, and then exchanging the radionuclide with a therapeutic radionuclide such as ¹²⁵ l or ¹³¹ l and injecting said patient for therapy.

The cancer may be cervical or esophageal cancer.

In another embodiment of the invention, the cancer is glioblastoma.

The invention also covers methods of medical treatment using the compounds and therapies described above.

The invention further covers methods of producing a radiolabeled hydroxamate metalloprotease inhibitor described above.

The radiolabeled compound may be synthesized by the direct radio- iodination on the aromatic ring of said compound or a precursor using S _E Ar aromatic substitution chemistry methodology with N-chlorosuccinimide in trifluoroacetic acid.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is UV chromatographs of the compounds ¹²³ I-SN-31 1 , l-SN-311 and SN-31 1 ;

Figure 2 is a graph showing the Uptake of [ ¹²³ l]Nal and ¹²³ I-SN-311 in HeLa and WHCO5 cells as a function of Bq activity per well as a function of cell number; and

Figure 3 is a graph showing Total Uptake in WHCO5 and HeLa cells of ¹²³ l- SN-311.

DESCRIPTION OF PREFERRED EMBODIMENTS

The aim of the invention is to synthesize a new theranostic radiopharmaceutical. The theranostic approach in nuclear medicine couples diagnostic imaging and therapy using the same molecule or at least very similar molecules, which are either radiolabeled with a different isotope or administered in different dosages. A theranostic agent needs to have two functions:

1. Diagnostic: to find a specific molecular target which is present and (over)expressed in a certain type of cancer, that can be quantified and located.

2. Therapeutic: delivering a targeted radiation treatment to the same molecular target (over)expressed by the cancer cells.

The premise for this theranostic approach is that you only treat what you can visualize and that the efficacy of therapy can be quantified and monitored. Theranostic radiopharmaceuticals are therefore a useful tool in the development of personalized medicine approaches where the therapy is tailor made for the individual patient. In this context, it is very important to select a drug target that is only present or overexpressed on a tumor cell, such as a specific receptor, compared to healthy cells throughout the body. Secondly, a drug needs to be synthesized that binds to this specific cancer target. If this is the case, radiolabeling the drug with an imaging or a therapeutic radioisotope could enable to image the tumor or deliver a radiation dose that can kill the tumor cells, respectively.

A preferred compound is the compound GI254023X ((2R,3S)-3- (Formylhydroyamino)-2-(3-phenyl-1 -propyl)butanoic Acid [(1 S)-2,2- Dimethyl-1 -(methylcarbamoyl)-1 -propyl]amide) from GlaxoSmithKline (GSK) described in W02000/0012083, the content of which is incorporated herein by reference.

GI254023X has three stereogenic centers, as well as the characteristic N- formyl hydroxylamine functionality. The latter can occur in either E- or Z- configurations which, together with the three centers of chirality, make four stereogenic elements that result in eight possible diastereomers of the inhibitor as racemates (sixteen stereoisomers in total).

The alkyl substituents are preferred at position A (hereon referred to as carbon A), while the substituent at position B (hereon referred to as carbon B) tolerates both alkyl and aryl groups for binding in the hydrophobic pocket of the enzyme. In addition, S-stereogenicity orientation is preferred at position C, ensuring the substituent faces away from the binding pocket of the enzyme and points towards the solvent. Finally, a short alkyl chain, such as a methyl group, is favored as the N-alkyl substituent of the secondary amide at the C-terminus.

According to the present invention, the radionuclide is incorporated into the phenyl ring of the side chain at stereo-center B.

The structure below shows modified GI254023X labelled with the radionuclide ¹²³ Ι Radiolabeling approaches/methodologies included in this invention

1. SN-311 + ¹²³ l (diagnostic isotope)

In one embodiment of the invention, the radiolabeled compound is achieved by the direct radio-iodination on the aromatic ring of the precursor, which is referred to as SN-31 1 . The synthesized compound GI254023X (syn-) differs only in its relative stereochemistry to GI254023X (anti-). Primarily studies indicated very little difference between the two for the ADAM 10 receptor uptake, with in vitro tests on the radiolabeled compound confirming cell binding affinity as expected. Synthetic challenges were encountered during the synthesis, which primarily emanated from a failure to reproduce the stereoselective alkylation of methyl-3-hydroxybutyrate with cinnamyl bromide in the original GI254023X synthesis (W02000012083). This resulted in the adoption of a strategy to use an Evans syn-aldol reaction to accomplish the synthesis of C-2/C-3 ‘syn’ variants (see the Figure above) of GI254023X: SN-254 and SN-311 . However, only SN-311 was taken into the radiolabeling studies, due to its improved biological activity. ADAM10 biological activity was measured for SN-31 1 and EC5o values in cervical (HeLa) and esophageal (WHCO5) cancer cell lines were found to fall within the 95% confidence interval of that of GI254023X. This indicated that the C- 2/C-3 relative stereochemistry in GI254023X is not biologically determining and that SN-311 could be radiolabeled with ¹²³ L

1.1 Synthesis of compounds SN-254 and SN-311

• SN-254 synthesis

Scheme 1 : Reagents and Conditions: (a) SOCI ₂ , 80 °C, 96%; (b) (S)-4- benzyloxazolidin-2-one, n-BuLi, THF, -78 - 0 °C, 79%; (c) i) Bu2B(OTf), Et3N, 0 °C ii) CH3CHO, -78 - -10 °C, 94%; (d) LiOH, H ₂ O ₂ , H ₂ O:THF, 0 °C, 97%; (e) O-benzylhydroxylamine, DCC, HOBt, DMAP, DCM, 0 °C, 94%; (f) i) MsCI, pyridine, 0 °C ii) K ₂ CO ₃ , acetone, reflux, 58%; (g) 1 M NaOH, dioxane, rt, 89%; (h) formic acetic anhydride, pyridine, THF, rt, 78%; (i) L-tert-leucine N- methyl amide, DCC, HOBt, DMAP, DCM, 0 °C - rt, 78%; (j) Pd/C, H ₂ , MeOH, rt, 95%.

(S)-4-Benzyloxazolidin-2-one was coupled to 2 to give the N-acyl oxazolidinone derivative, 3. The boron-mediated stereoselective aldol reaction of 3 produced 4 with C-2/C-3 syn-relative stereochemistry and (2S, 3R) -absolute stereochemistry. The oxazolidinone auxiliary was removed and the resultant acid coupled to O-benzylhydroxylamine to generate 6. Following this, 6 was subjected to a two-step sequence involving mesylation followed by base-mediated intramolecular cyclisation to generate the β- lactam, 7. Thereafter, the lactam ring was hydrolyzed to produce acid, 8, which was N-formylated with formic acetic anhydride to synthesize 9. Afterwards, 9 was reacted with L-tert-leucine N-methyl amide to produce amide, 10. Finally, hydrogenolysis of 10 produced SN-254 in 10 linear steps, with (2S,3S)-stereochemistry.

Scheme 2: Reagents and Conditions: (a) SOCI ₂ , 80 °C, 95%; (b) (R)-4- benzyloxazolidin-2-one, n-BuLi, THF, -78 - 0 °C, 94%; (c) i) Bu ₂ B(OTf), Et3N, 0 °C ii) CH3CHO, -78 - -10 °C, 93%; (d) LiOH, H ₂ O ₂ , H ₂ O:THF, 0 °C, 70%; (e) O-benzylhydroxylamine, DCC, HOBt, DMAP, DCM, 0 °C, 70%; (f) i) MsCI, pyridine, 0 °C ii) K ₂ CO ₃ , acetone, reflux, 68%; (g) 1 M NaOH, dioxane, rt, 75%; (h) formic acetic anhydride, pyridine, THF, rt, 82%; (i) L-tert-leucine N- methyl amide, DCC, HOBt, DMAP, DCM, 0 °C - rt, 64%; (j) Pd/C, H ₂ , MeOH, rt, 93%.

(F?)-4-Benzyloxazolidin-2-one was coupled to 12 to give the N-acyl oxazolidinone derivative, 13, which underwent a boron-mediated stereoselective aldol reaction to produce 14. The oxazolidinone auxiliary was removed and the resultant acid coupled with O-benzylhydroxylamine to produce 16. Thereafter, 16 was subjected to mesylation and base-mediated intramolecular cyclisation to generate the β-lactam, 17. Subsequently lactam ring was hydrolyzed to produce acid, 17, which underwent N-formylation reaction using formic acetic anhydride to afford, 19. Then, 19 was coupled with L-tert-leucine N-methyl amide to yield amide, 20, with subsequent hydrogenolysis producing SN-311 in 10 linear steps, with (2R,3R)- stereochemistry.

1.2 Iodination of SN-311 (l-SN-311)

Scheme 3: Synthesis of l-SN-311. Reagents and conditions: (a) AgOTf, l ₂ , DCM, rt, 58%. l-SN-311 was produced by reacting iodine with SN-311 and silver tritiate in a S _E Ar reaction (via l ⁺ ). The product was isolated as a brown oil in 58% yield.

1.3 Radioiodination of SN-311 ( ¹²³ I-SN-311 )

• Synthesis

Scheme 4: Synthesis of ¹²³ I-SN-311. Reagents and conditions: (a) [ ¹²³ l]Nal, NCS, TFA, NaOH, 65 - 70 °C.

The radiolabeling was also achieved using an S _E Ar reaction by adding [ ¹²³ l]Nal in dilute NaOH solution to a solution of SN-311 and N- chlorosuccinimide in trifluoroacetic acid, with heating of the mixture for approximately 2.5 hours at 65 - 70 °C. The radiolabeled material was isolated on a reversed phase C18 Sep-Pak mini-cartridge column after preparation of the column using 5 mL of de-ionized water followed by 6 mL ethanol.

The HPLC chromatographs (UV detector) are shown in Fig. 1 , where it was determined that ¹²³ l was incorporated on the aromatic ring of compound SN- 311 , by virtue of a peak with the same retention time in both the ‘cold’ and radiolabeled samples at 9.6 minutes. From the retention times of traces B and C, it could be concluded that substitution in the radiolabeled run was predominantly para- as in the silver triflate/iodine method (verified by 1H NMR spectroscopy). The radiolabeled material was then immediately added to a solution of 0.15 M phosphate-buffered saline at pH 7.4, which was done to safely transport the radioactive material to the cell cultures for evaluation in cancer cell lines. 2. Radioiodination of GI254023X

Scheme 5: Synthesis of ¹²³ l-GI254023X. Reagents and conditions: (a) [ ¹²³ l]Nal , NCS, TFA, NaOH, 65 - 70 °C. 1 ²³ l-GI254023X was synthesized using [ ¹²³ l]Nal in dilute NaOH with N- chlorosuccinimide in trifluoroacetic acid

3. GI254023X + theranostic isotope

As mentioned above, the goal is not only to develop an imaging agent but a theranostic agent, which includes also a therapeutic radioisotope. The idea is that when ¹²³ l-GI254023X SPECT imaging is able to identify the presence of ADAM 10 in the tumor, the patient would be a candidate for targeted ADAM 10 radionuclide therapy. This therapy could be achieved by subsequently injecting radiolabeled GI254023X but now exchanging the diagnostic radionuclide ( ¹²³ l) with a therapeutic one: ¹²⁵ l (therapy) or ¹³¹ l (therapy). A further aspect of the invention is the use of this radiolabeled theranostic ADAM 10 inhibitor for other clinical applications, such as other types of cancers (next to glioblastoma, cervical and esophageal cancer that are described in the pre-clinical investigation below) and perhaps even Alzheimer’s disease ¹²³ l-GI254023X and ¹³¹ l-GI254023X could be a very interesting theranostic application, particularly for glioblastoma. In the following sections, the first potential clinical application of the invention will be described in more detail with reference to preliminary results of pre-clinical investigations. ADAM10 biological activity was conducted on SN-311 with EC ₅₀ values in cervical (HeLa) and esophageal (WHCO5) cancer cell lines found to fall within the 95% confidence interval of that of GI254023X. This indicated that the C-2/C-3-relative stereochemistry in GI254023X is not biologically determining and that SN-31 1 could be radiolabeled with ¹²³ l. Radioactively- labelled compound ¹²³ I-SN311 with the y-emitter ¹²³ l was synthesized as a potential radiodiagnostic, following the direct iodination of compound SN-311 with [ ¹²³ l]Nal . ¹²³ l-SN-31 1 was successfully synthesized using [ ¹²³ l]Nal in dilute NaOH solution with N-chlorosuccinimide in trifluoroacetic acid using S _E Ar aromatic substitution chemistry methodology. The substitution regioselectivity was para- on the aromatic ring, with the rest of the molecule left intact as determined by HPLC comparison with a synthesized ‘cold’ equivalent, the latter carefully evaluated by 1H and 13C NMR spectroscopies.

Cellular uptake studies demonstrated that the radiolabeled compound ( ¹²³ l- SN-311 ) was taken up into cervical cancer cell lines. ¹²³ I-SN-311 was proposed to be used as a radiopharmaceutical for the early diagnosis of cervical cancer. A further possibility is to exchange the diagnostic radionuclide with a therapeutic isotope. (Iodine: 1-123 for SPECT, 1-124 for PET; 1-131 and 1-125 for therapy). ¹²³ l-GI254023X was synthesized using [ ¹²³ l]Nal in dilute NaOH with N- chlorosuccinimide in trifluoroacetic acid using S _E Ar aromatic substitution chemistry methodology. All in vitro radiobiology studies thus far have been carried out using ¹²³ l-GI254023X.

As mentioned, not only detection is possible, but also treatment by exchanging the diagnostic radionuclide with a therapeutic one using the following pairs. 1-123 for SPECT, 1-124 for PET, 1-131 and 1-125 for therapy. Isotopes of Br can also be used.

A further aspect of the invention is the use of the radiolabeled inhibitor, ¹²³ l- GI254023X in other applications in theranostic. The proposed new application will use ¹²³ l-GI254023X as a radiopharmaceutical in other types of cancers (only cervical cancer is being investigated in concepts 1 and 2 above). ¹²³ l-GI254023X and potentially ¹³¹ l-GI254023X can be used in the diagnosis and treatment of glioblastoma.

First pre-clinical results on the radiolabeled compound

Cervical Cancer

In order to test the biological activity of the radiolabeled compound in vitro, two cell lines were chosen as esophageal (WHCO5) and cervical (HeLa) cancer cell lines (using [ ¹²³ l]Nal as the control) and the radioactivity uptake was measured using a bore-hole or scintillation counter. The method involved administration of the ¹²³ l radiolabeled ADAM10 inhibitor, which was diluted in complete cell growth medium and added to the cultures to give a final concentration of 3 μCi per well or 0.3 μCi per well depending on the assay. The well plates were incubated for one hour in a humidified CO ₂ incubator at 37 °C. The measurements were performed with a ¹²³ l Multi Channel Analyzer, and during the incubation time, a calibration and counting efficiency determination was carried out on the channel.33

After the one-hour incubation period, the growth medium was aspirated, leaving the adherent cell monolayers intact and the cell cultures rinsed twice with cold PBS to remove any residual extracellular radioactivity. The cell monolayers were lysed with 1 mL 1 M NaOH, and 1 mL of each lysed suspension was transferred to a clean test tube. Radioactive counts and corrections applied for different cell numbers per well are listed in Table 1. The data is presented as an average of triplicates and was corrected for ¹²³ l decay using Eff Corr as the correcting factor, which considers the half-life and decay of the radionuclide over a specific time interval. Table 1 : Uptake of [ ¹²³ l]Nal and ¹²³ l-46 (3 mCi) in HeLa and WHCO5 cancer cells

Figure 2 presents the activity (Bq) per well as a function of cell number and is a graphical representation of data listed in Table 1 . Results are the mean +/- SEM of experiments performed in triplicate. For both cell lines, no appreciable increase in the activity of [ ¹²³ l]Nal with cell number was observed, whereas with the inhibitor (green and blue circles), there was a significant uptake, particularly in the WHCO cell line.

Free [ ¹²³ l]Nal showed minimal uptake in both cells lines, although the base line readings were higher in WHCO5 cells as compared to HeLa cells (Table 1 and Fig. 8). There is a clear uptake of ¹²³ I-SN-311 in both HeLa and WHCO5 cancer cell lines, with both showing a linear increase in uptake with an increase in the number of cells. The faster-growing WHCO5 cells show more uptake of radioactivity, and this corresponds to the literature, which reports that faster growing cell lines have an increased uptake of substances.34

A second set of readings were made using only 0.3 μCi per well, the principle reason for this was to reduce the influence of non-specific binding to the plastic of each well. The data is summarized in Figure 3. Data represents an average and standard deviation of 12 replicates using 0.3 μCi of ¹²³ I-SN-311 and 200000 cells. Again, the faster growing WHCO5 cells showed significantly higher uptake of the radiolabeled compound (127.63 Bq) compared to the slower growing HeLa cells (81.76 Bq).

The similar and comparable radiolabeling results of GI254023X and SN-31 1 further demonstrated that the stereochemical configurational attributes were not crucial for uptake. Importantly, the uptake studies have demonstrated that the radiolabeled compound ( ¹²³ I-SN-31 1 ) was taken up into HeLa and WHCO5 cancer cells.

Glioblastoma

The synthesized radiopharmaceutical has potential as theranostic agent for growth inhibition of glioblastoma (GB), as well as for GB tumor imaging. GB comprise high-grade gliomas (HGG), and is the most aggressive and most common malignant brain tumor in adults, with a high mortality and morbidity. ADAM 10 promotes glioma migration and invasion and has been identified as a promising prognostic factor. Expression of ADAM 10 was confirmed in 22- 64% of GB specimens while it was absent in the normal brain. ADAM10 inhibition has been shown to boost an immune response against GB-initiating cells and to sensitize GB cells to therapy.