LMO2 PROTEIN INHIBITORS

Abstract

The present invention relates to compounds of Formula (I) that function as LMO2 activity: Formula (I) wherein R.sub.1, X.sub.1, X.sub.2, X.sub.3, Q, R.sub.2, R.sub.3 and R.sub.4 are each as defined herein. The present invention also relates to processes for the preparation of these compounds, to pharmaceutical compositions comprising them, and to their use in the treatment of proliferative disorders, such as cancer, as well as other diseases or conditions in which LMO2 activity is implicated.

##STR00001##

Claims

1. A compound, or a pharmaceutically acceptable salt, hydrate or solvate thereof, having the structural Formula (I) shown below: ##STR00084## wherein: R.sub.1 is selected from: (i) (1-6C)alkyl which is optionally substituted by one or more R.sub.a; (ii) a group of the formula: ##STR00085## wherein denotes the point of attachment; n is 0 or 1; R.sub.1a and R.sub.1b are selected from hydrogen or methyl; X.sub.4, X.sub.5 and X.sub.6 are selected from C—H, C—R.sub.a or N; (iii) a group of the formula: ##STR00086## wherein denotes the point of attachment; n, R.sub.1a and R.sub.1b are as defined above; Ring B is a saturated or partially unsaturated ring; X.sub.7, X.sub.8 and X.sub.9 are selected from C—H, C—R.sub.a or N if a bond connecting them to an adjacent atom is an unsaturated double bond, or C—H.sub.2, C—HR.sub.a, C—(R.sub.a).sub.2, N—H, N—R.sub.b, S or O if the bonds attaching them to adjacent atoms are single bonds; wherein each R.sub.a is independently selected from (1-4C)alkyl, halo, (1-4C)haloalkyl, (1-4C)haloalkoxy, cyano, nitro, (3-6C)cycloalkyl, (3-6C)cycloalkyl(1-2C)alkyl, phenyl, (CH.sub.2).sub.q1NR.sub.abR.sub.ac, (CH.sub.2).sub.q1OR.sub.ab, (CH.sub.2).sub.q1C(O)R.sub.ab, (CH.sub.2).sub.q1C(O)OR.sub.ab, (CH.sub.2).sub.q1OC(O)R.sub.ab, (CH.sub.2).sub.q1C(O)N(R.sub.ac)R.sub.ab, (CH.sub.2).sub.q1N(R.sub.ac)C(O)R.sub.ab, (CH.sub.2).sub.q1S(O).sub.pR.sub.ab (where p is 0, 1 or 2), (CH.sub.2).sub.q1SO.sub.2N(R.sub.ac)R.sub.1ab, or (CH.sub.2).sub.q1N(R.sub.ac)SO.sub.2R.sub.ab, and wherein: q1 is 0, 1, 2 or 3; R.sub.ab is selected from hydrogen, (1-4C)alkyl, (3-6C)cycloalkyl, (3-6C)cycloalkyl(1-2C)alkyl, aryl, aryl(1-2C)alkyl, heteroaryl, heteroaryl(1-2C)alkyl, heterocyclyl and heterocyclyl(1-2C)alkyl, and wherein R.sub.ab is optionally further substituted by one or more substituent groups independently selected from oxo, (1-4C)alkyl, halo, (1-4C)haloalkyl, (1-4C)haloalkoxy, (1-4C)aminoalkyl, (1-4C)hydroxyalkyl, cyano, nitro, NR.sub.adR.sub.ae, OR.sub.ad, C(O)R.sub.ad, C(O)OR.sub.ad, OC(O)R.sub.ad, C(O)N(R.sub.ae)R.sub.ad, N(R.sub.ae)C(O)R.sub.ad, S(O).sub.pR.sub.ad (where p is 0, 1 or 2), SO.sub.2N(R.sub.ae)R.sub.ad, N(R.sub.ae)SO.sub.2R.sub.ad, or (CH.sub.2).sub.q2NR.sub.adR.sub.ae (where q2 is 1, 2 or 3); wherein R.sub.ad and R.sub.ae are each independently selected from hydrogen or (1-6C)alkyl; and R.sub.ac is selected from hydrogen or (1-2C)alkyl; or where R.sub.ab and R.sub.ac are linked to a common N atom, they may be linked such that, together with the N atom to which they are attached, they form a 5 or 6 membered heteroaryl ring or a 5 to 7 membered heterocyclic ring, each of which is optionally substituted as for R.sub.ab above; wherein R.sub.b is independently selected from (1-4C)alkyl, (1-4C)haloalkyl or —C(O)R.sub.ba, wherein R.sub.ba is selected from (1-4C)alkyl, (3-6C)cycloalkyl, (3-6C)cycloalkyl(1-2C)alkyl, aryl, aryl(1-2C)alkyl, heteroaryl, heteroaryl(1-2C)alkyl, heterocyclyl and heterocyclyl(1-2C)alkyl, and wherein R.sub.ba is optionally further substituted by one or more substituent groups independently selected from oxo, (1-4C)alkyl, halo, (1-4C)haloalkyl, (1-4C)haloalkoxy, (1-4C)aminoalkyl, (1-4C)hydroxyalkyl, cyano, nitro, NR.sub.bdR.sub.be, OR.sub.bd, C(O)R.sub.bd, C(O)OR.sub.bd, OC(O)R.sub.bd, C(O)N(R.sub.be)R.sub.bd, N(R.sub.be)C(O)R.sub.bd, S(O).sub.pR.sub.bd (where p is 0, 1 or 2), SO.sub.2N(R.sub.be)R.sub.bd, N(R.sub.be)SO.sub.2R.sub.bd, or (CH.sub.2).sub.q3NR.sub.bdR.sub.be (where q3 is 1, 2 or 3); wherein R.sub.bd and R.sub.be are each independently selected from hydrogen or (1-6C)alkyl; X.sub.1, X.sub.2 and X.sub.3 are selected from N, N—R.sub.5, O, S and CR.sub.6, wherein R.sub.5 is hydrogen or methyl and R.sub.6 is hydrogen, methyl or halo, with the proviso that at least one of X.sub.1, X.sub.2 and X.sub.3 is selected from N, N—R.sub.5, O and S; Q is a group of the formula: ##STR00087## or —X.sub.12—CH.sub.2—CH.sub.2—NR.sub.7— or —X.sub.13—C(O)—NR.sub.8—; wherein: X.sub.10 and X.sub.11 are selected from N or CH; X.sub.12 and X.sub.13 are selected from NR.sub.9, CH.sub.2, CHR.sub.9 or C(R.sub.9).sub.2; and R.sub.7, R.sub.8 and R.sub.9 are selected from hydrogen or (1-2C)alkyl; R.sub.2 and R.sub.3 are selected from hydrogen or (1-2C)alkyl; R.sub.4 is a phenyl, heteroaryl, or heterocyclyl ring optionally substituted by (1-4C)alkyl, halo, (1-4C)haloalkyl, (1-4C)haloalkoxy, (1-4C)aminoalkyl, (1-4C)hydroxyalkyl, cyano, nitro, NR.sub.4aR.sub.4b, OR.sub.4a, C(O)R.sub.4a, C(O)OR.sub.4a, OC(O)R.sub.4a, C(O)N(R.sub.4b)R.sub.4a, N(R.sub.4b)C(O)R.sub.4a, S(O).sub.pR.sub.4a (where p is 0, 1 or 2), SO.sub.2N(R.sub.4b)R.sub.4a, N(R.sub.4b)SO.sub.2R.sub.4a, or (CH.sub.2).sub.q4NR.sub.4aR.sub.4b (where q4 is 1, 2 or 3); wherein R.sub.4a is selected from hydrogen, (1-4C)alkyl, (3-6C)cycloalkyl, (3-6C)cycloalkyl(1-2C)alkyl, phenyl aryl(1-2C)alkyl, heteroaryl, heteroaryl(1-2C)alkyl, heterocyclyl and heterocyclyl(1-2C)alkyl, and wherein: R.sub.4a is optionally further substituted by (1-4C)alkyl, halo, (1-4C)haloalkyl, (1-4C)haloalkoxy, cyano, nitro, NR.sub.4aaR.sub.4ab, OR.sub.4aa, C(O)R.sub.4aa, C(O)OR.sub.4aa, OC(O)R.sub.4aa, C(O)N(R.sub.4ab)R.sub.4aa, N(R.sub.4ab)C(O)R.sub.4aa, S(O).sub.pR.sub.4aa (where p is 0, 1 or 2), SO.sub.2N(R.sub.4ab)R.sub.4aa, N(R.sub.4ab)SO.sub.2R.sub.4aa, or (CH.sub.2).sub.q5NR.sub.4aaR.sub.4ab (where q5 is 1, 2 or 3) and R.sub.4aa and R.sub.4ab are hydrogen or (1-2C)alkyl; R.sub.4b is selected from hydrogen or (1-2C)alkyl; or R.sub.4a and R.sub.4b are linked to a common N atom, they may be linked such that, together with the N atom to which they are attached, they form a 5 or 6 membered heteroaryl ring or a 5 to 7 membered heterocyclic ring, each of which is optionally substituted as for R.sub.4 above.

2. A compound according to claim 1, or a pharmaceutically acceptable salt, hydrate or solvate thereof, wherein R.sub.1 is selected from: (i) (1-4C)alkyl which is optionally substituted by one or more R.sub.a; (ii) a group of the formula: ##STR00088## wherein denotes the point of attachment; n is 0 or 1; R.sub.1a and R.sub.1b are selected from hydrogen; X.sub.4, X.sub.5 and X.sub.6 are selected from C—H, C—R.sub.a or N; (iii) a group of the formula: ##STR00089## wherein denotes the point of attachment; n, R.sub.1a and R.sub.1b are as defined above; Ring B is a saturated ring; X.sub.7, X.sub.8 and X.sub.9 are selected from C—H; wherein each R.sub.a is independently selected from methyl, halo, (CH.sub.2).sub.q1NR.sub.abR.sub.ac, (CH.sub.2).sub.q1OR.sub.ab, (CH.sub.2).sub.q1C(O)R.sub.ab or (CH.sub.2).sub.q1C(O)OR.sub.ab, and wherein: q1 is 0; R.sub.ab is selected from hydrogen, (1-4C)alkyl, aryl, aryl(1-2C)alkyl, and wherein R.sub.ab is optionally further substituted by one or more substituent groups independently selected from, halo; and R.sub.ac is selected from hydrogen; or where R.sub.ab and R.sub.ac are linked to a common N atom, they may be linked such that, together with the N atom to which they are attached, they form a 5 membered heteroaryl ring or a 5 or 6 membered heterocyclic ring, each of which is optionally substituted as for R.sub.ab above.

3. A compound according to any one of the preceding claims, or a pharmaceutically acceptable salt, hydrate or solvate thereof, wherein R.sub.1 is selected from: (i) a group of the formula: ##STR00090## wherein denotes the point of attachment; n is 0 or 1; R.sub.1a and R.sub.1b are selected from hydrogen; X.sub.4, X.sub.5 and X.sub.6 are selected from C—H, C—R.sub.a or N; (ii) a group of the formula: ##STR00091## wherein denotes the point of attachment; n is 0, R.sub.1a and R.sub.1b are selected from hydrogen Ring B is a saturated ring; X.sub.7, X.sub.8 and X.sub.9 are selected from C—H; wherein each R.sub.a is independently selected from methyl, halo, (CH.sub.2).sub.q1NR.sub.abR.sub.ac, (CH.sub.2).sub.q1OR.sub.ab, (CH.sub.2).sub.q1C(O)R.sub.ab or (CH.sub.2).sub.q1C(O)OR.sub.ab, and wherein: q1 is 0; R.sub.ab is selected from hydrogen, (1-2C)alkyl, phenyl, benzyl, and wherein R.sub.ab is optionally further substituted by one or more substituent groups independently selected from, halo; and R.sub.ac is selected from hydrogen; or where R.sub.ab and R.sub.ac are linked to a common N atom, they may be linked such that, together with the N atom to which they are attached, they form a 5 membered heteroaryl ring or a 5 or 6 membered heterocyclic ring, each of which is optionally substituted as for R.sub.ab above.

4. A compound according to any one of the preceding claims, or a pharmaceutically acceptable salt, hydrate or solvate thereof, wherein R.sub.1 is selected from: ##STR00092## wherein denotes the point of attachment.

5. A compound according to any one of the preceding claims, or a pharmaceutically acceptable salt, hydrate or solvate thereof, wherein X.sub.1, X.sub.2 and X.sub.3 are selected from N, O, S and CR.sub.6, wherein R.sub.6 is hydrogen, methyl or halo, with the proviso that at least one of X.sub.1, X.sub.2 and X.sub.3 is selected from N, O and S.

6. A compound according to any one of the preceding claims, or a pharmaceutically acceptable salt, hydrate or solvate thereof, wherein X.sub.1 is N; X.sub.2 is O or S; and X.sub.3 is CR.sub.6, wherein R.sub.6 is hydrogen.

7. A compound according to any one of the preceding claims, or a pharmaceutically acceptable salt, hydrate or solvate thereof, wherein Q is a group of the formula: ##STR00093## or —X.sub.12—CH.sub.2—CH.sub.2—NR.sub.7— or —X.sub.13—C(O)—NR.sub.8—; wherein: X.sub.10 and X.sub.11 are selected from N; X.sub.12 and X.sub.13 are selected from NRs; and R.sub.7, R.sub.8 and R.sub.9 are selected from hydrogen.

8. A compound according to any one of the preceding claims, or a pharmaceutically acceptable salt, hydrate or solvate thereof, wherein Q is selected from ##STR00094## wherein denotes the point of attachment.

9. A compound according to any one of the preceding claims, or a pharmaceutically acceptable salt, hydrate or solvate thereof, wherein R.sub.2 and R.sub.3 are selected from hydrogen or methyl.

10. A compound according to any one of the preceding claims, or a pharmaceutically acceptable salt, hydrate or solvate thereof, wherein R.sub.2 and R.sub.3 are hydrogen.

11. A compound according to any one of the preceding claims, or a pharmaceutically acceptable salt, hydrate or solvate thereof, wherein R.sub.4 is a phenyl, heteroaryl, or heterocyclyl ring optionally substituted by (1-2C)alkyl, halo, (1-2C)haloalkyl, (1-2C)haloalkoxy, (1-2C)aminoalkyl, (1-2C)hydroxyalkyl, cyano, nitro, NR.sub.4aR.sub.4b, OR.sub.4a, C(O)R.sub.4a, C(O)OR.sub.4a, OC(O)R.sub.4a, C(O)N(R.sub.4b)R.sub.4a, N(R.sub.4b)C(O)R.sub.4a, S(O).sub.pR.sub.4a (where p is 0, 1 or 2), SO.sub.2N(R.sub.4b)R.sub.4a, N(R.sub.4b)SO.sub.2R.sub.4a, or (CH.sub.2).sub.q4NR.sub.4aR.sub.4b (where q4 is 1, 2 or 3); wherein R.sub.4a is selected from hydrogen, (1-2C)alkyl, phenyl aryl(1-2C)alkyl, heteroaryl, heteroaryl(1-2C)alkyl, heterocyclyl and heterocyclyl(1-2C)alkyl, and wherein: R.sub.4a is optionally further substituted by (1-2C)alkyl, halo, (1-2C)haloalkyl, (1-2C)haloalkoxy, cyano, nitro; R.sub.4b is selected from hydrogen or (1-2C)alkyl; or R.sub.4a and R.sub.4b are linked to a common N atom, they may be linked such that, together with the N atom to which they are attached, they form a 5 or 6 membered heteroaryl ring or a 5 to 7 membered heterocyclic ring, each of which is optionally substituted as for R.sub.4 above.

12. A compound according to any one of the preceding claims, or a pharmaceutically acceptable salt, hydrate or solvate thereof, wherein R.sub.4 is a phenyl ring optionally substituted by halo, cyano, nitro, NR.sub.4aR.sub.4b, OR.sub.4a, C(O)R.sub.4a, N(R.sub.4b)C(O)R.sub.4a; wherein R.sub.4a is selected from hydrogen, (1-2C)alkyl, phenyl aryl(1-2C)alkyl; and R.sub.4b is selected from hydrogen.

13. A compound according to any one of the preceding claims, or a pharmaceutically acceptable salt, hydrate or solvate thereof, wherein R.sub.4 is selected from ##STR00095## wherein denotes the point of attachment.

14. A compound of the formula: ##STR00096## wherein R.sub.1, X.sub.1, X.sub.2, X.sub.3, Q, R.sub.2, R.sub.3 and R.sub.4 are each as defined in any one of claims 1 to 13.

15. A compound, or a pharmaceutically acceptable salt, hydrate or solvate thereof, selected from any one of the following: 2-(3-benzyl-2-oxoimidazolidin-1-yl)-N-(4-phenoxyphenyl)oxazole-4-carboxamide (Abd-L6); N-(4-(1H-pyrrol-1-yl)phenyl)-2-(3-(3-chlorobenzyl)-2-oxoimidazolidin-1-yl)oxazole-4-carboxamide (Abd-L7); 2-(3-(4-chlorobenzyl)-2-oxoimidazolidin-1-yl)-N-(3,4-dimethoxyphenyl)oxazole-5-carboxamide (Abd-L8); N-(4-(benzyloxy)phenyl)-2-(3-(3-methoxybenzyl)-2-oxoimidazolidin-1-yl)oxazole-4-carboxamide (Abd-L9); N-(4-(1H-pyrrol-1-yl)phenyl)-2-(3-(3-cyanobenzyl)-2-oxoimidazolidin-1-yl)oxazole-4-carboxamide (Abd-L10); 2-(3-(2-chlorobenzyl)-2-oxoimidazolidin-1-yl)-N-(4-phenoxyphenyl)oxazole-4-carboxamide (Abd-L12); 2-(3-benzyl-2-oxoimidazolidin-1-yl)-N-(4-phenoxybenzyl)oxazole-4-carboxamide (Abd-L13); 2-(3-(2-chlorobenzyl)-2-oxoimidazolidin-1-yl)-N-(4-phenoxybenzyl)oxazole-4-carboxamide (Abd-L14); N-(4-(1H-pyrrol-1-yl)phenyl)-2-(4-(3-methoxybenzyl)piperazin-1-yl)oxazole-4-carboxamide (Abd-L15); N-(4-(benzyloxy)phenyl)-2-(4-(3-methoxybenzyl)piperazin-1-yl)oxazole-4-carboxamide (Abd-L16); N-(4-(benzyloxy)phenyl)-2-(4-(3-methoxybenzyl)piperazin-1-yl)thiazole-4-carboxamide (Abd-L17); N-(4-(1H-pyrrol-1-yl)phenyl)-2-(4-(3-methoxybenzyl)piperazin-1-yl)thiazole-4-carboxamide (Abd-L18); N-(4-(1H-pyrrol-1-yl)phenyl)-2-(4-(4-methoxybenzyl)piperazin-1-yl)oxazole-4-carboxamide (Abd-L19); N-(4-(1H-pyrrol-1-yl)phenyl)-2-(4-(2-methoxybenzyl)piperazin-1-yl)thiazole-4-carboxamide (Abd-L20); 2-(4-(3-methoxybenzyl)piperazin-1-yl)-N-(4-(trifluoromethoxy)phenyl)thiazole-4-carboxamide (Abd-L21); 2-(4-(3-methoxybenzyl)piperazin-1-yl)-N-(6-methoxypyridin-3-yl)thiazole-4-carboxamide (Abd-L22); and 2-(4-(3-methoxybenzyl)piperazin-1-yl)-N-(2-methoxypyrimidin-5-yl)thiazole-4-carboxamide (Abd-L23).

16. A pharmaceutical composition comprising a compound according to any one claims 1 to 15, or a pharmaceutically acceptable salt, hydrate or solvate thereof, in admixture with a pharmaceutically acceptable diluent or carrier.

17. A compound according to any one of claims 1 to 15, or a pharmaceutically acceptable salt or hydrate thereof, for use in therapy.

18. A compound according to any one of claims 1 to 15, or a pharmaceutically acceptable salt, hydrate or solvate thereof, or a pharmaceutical composition according to claim 16, for use in a method of inhibiting cell proliferation, such as the treatment of cancer.

19. The compound or pharmaceutical composition according to claim 18, wherein said cancer is a haematological cancer.

20. A method of treating a proliferative disorder in a patient in need of such treatment, the method comprising administering a therapeutically effective amount of a compound according to any one of claims 1 to 15, or a pharmaceutically acceptable salt, hydrate or solvate thereof, or a pharmaceutical composition according to claim 16.

21. A method of treating cancer in a patient in need of such treatment, the method comprising administering a therapeutically effective amount of a compound according to any one of claims 1 to 15, or a pharmaceutically acceptable salt, hydrate or solvate thereof, or a pharmaceutical composition according to claim 16.

22. The method according to claim 21, wherein said cancer is a haematological cancer.

Description

FIGURES

[0578] FIG. 1: cSPR chemical library screen for LMO2 binding compounds

[0579] (A) The SPR streptavidin chips used for the cSPR screen were channel 1: blank; channel 2: LMO2-ΔLID; channel 3: KRAS; channel 4: LMO2-GS-iDAb. Competitive SPR screening of the PPI-NET compound library (1,500 compounds in total) was done at a single concentration of 150 μM for each compound (Cruz-Migoni et al., 2019). Response levels were normalised by subtracting the response measured against one of the two control reference proteins: LMO2-iDAb LMO2 fusion protein or KRAS and normalised response units (RU.sub.norm). Hit compounds Abd-L1, L2, L3 and L4 are indicated by orange dots. Both LMO2-iDAb and KRAS were used as negative reference proteins. (C) chemical structures and molecular weights (MW) of four hits are shown, together with PPI-NET plate locations (P number) and the designated Antibody-derived (Abd) number. Abd-L1 and Abd-L2 are homologues that differ only by the presence of 1H-pyrrolo[2,3-b]pyridine in Abd-L1 or pyrazolo[1,5-a]pyridine in Abd-L2, both indicated by the red oval. Note: additional amounts of Abd-L2 and Abd-L3 were not available commercially. (D) The LMO2-binding hit compound Abd-L1 was assessed in vitro with NMR waterLOGSY with binding to LMO2-LID or to LMO2-ΔLID comparing the spectra with the proton NMR of the compounds. (E) Caco-2 permeability assay that shows cell import and cell export data for Abd-L1. See also related FIG. 7.

[0580] FIG. 2: Establishing a BRET-based LMO2-iDAb biosensor amenable for a high throughput screening of small molecule libraries

[0581] The BRET2 assay comprises live in-cell generation of signal following interaction of a donor protein (in this case LMO2-RLuc8) and an acceptor protein (in this case GFP.sup.2-anti-LMO2 iDAb) and BRET signal (energy transfer from activated RLuc8 to GFP.sup.2). (A) BRET donor saturation assay with donor LMO2 and different mutant iDAb LMO2 acceptors, iDAb LMO2.sub.dm, LMO2.sub.dm1-dm6. (B) BRET.sub.max and BRET.sub.50 values from the donor saturation curves displayed in A. (C) Western blot data for the expression of the GFP.sup.2-iDAb LMO2 and mutants (using anti-GFP antibody) and expression of LMO2-RLuc8 (with anti-LMO2 antibody). α-tubulin is the loading control. (D) BRET competition assay of LMO2-RLuc8 and the different GFP.sup.2-iDAb LMO2.sub.dmx by expression of a non-relevant control iDAb (anti-RAS (Tanaka et al., 2007); Ctl, white bars) or unmutated iDAb LMO2 (black bars) as competitors. This competition is performed at the lowest dose of competitor (i.e. 0.1 μg). The percentage inhibition by iDAb LMO2 compared to iDAb Ctl is displayed. The iDAb LMO2.sub.dm3 mutant chose for the cell-based screening assay is coloured in blue. Each experiment was performed twice. Where error bars are presented (A, D), they correspond to mean values±standard deviation (SD) of biological repeats. See also related FIGS. 8-10.

[0582] FIG. 3: Cell-based high-throughput screening for inhibitors of LMO2-iDAb LMO2 PPI

[0583] (A) Scheme for the cell-based high throughput screening (HTS) where a diverse chemical library of 10,720 compounds was screened using a BRET cell assay to determine diminution of signal generated by interaction of LMO2-RLuc8 and GFP.sup.2-iDAb.sub.dm3. (B) Scatter plot of the normalised BRET signal from 10,720 compounds tested at 10 μM. 34 compounds (primary hits) caused inhibition of BRET signal below a cut-off of 3 times the SD (minus, −3×SD) of the DMSO BRET signal (Lavoie et al., 2013). Some primary hits are pinpointed in orange. (C, D) Confirmation of inhibition of BRET signal from interaction LMO2 with iDAb LMO2.sub.dm3 (C) and for interaction of LMO2 with unmutated iDAb (D). Eight hits (depicted by blue bars) were confirmed to decrease LMO2-iDAb LMO2.sub.dm3 signal by at least 3×SD of the BRET signal with DMSO control (i.e. DMSO BRET signal±3×SD: 12.2±3.6, threshold set at 8.6 and shown with the dotted line) without affecting LMO2-iDAb LMO2 interaction (i.e. DMSO BRET signal±3×SD: 30.3±3.4, threshold set at 26.9 and shown with the dotted line). P24H7 compound highlighted with a red star is an example of compound that was not pursued further as it affects both iDAb LMO2.sub.dm3 and iDAb LMO2 interaction with LMO2. Experiments in C, D were performed twice. Error bars presented in C, D correspond to mean values±SD of biological repeats. See also related FIG. 11.

[0584] FIG. 4: Structure-activity relationship of antibody derived (Abd) LMO2-binding compounds

[0585] The chemical structures of the hit matter from the BRET screen were examined and a family of compounds identified. (A) Chemical structures of the 8 re-synthesised hits (Abd-L5 to Abd-L12) with their respective molecular weight (MW). (B) Dose response inhibition of LMO2-iDAb LMO2.sub.dm3 interaction by compounds Abd-L5 to Abd-L12 (concentration range: 1, 10, 20 M). The data were obtained from duplicate biological experiments. Error bars correspond to mean values±SD of biological repeats. (C) SAR study of Abd-L9 compound as template. The compound was divided into four substituents (named A-D) that were substituted by various other chemical groups to give new compounds. (D) Structures of representative compounds Abd-L15-L23. The new compounds were tested by BRET assays with LMO2-iDAb LMO2.sub.dm3 interaction. The different SAR-derived compounds are shown with their MW and the percentage of BRET inhibition of the interaction LMO2-iDAb LMO2.sub.dm3 at 20 μM. See also related FIGS. 12 & 13.

[0586] FIG. 5: Abd compounds bind to LMO2 in vitro

[0587] The in vitro binding properties of Abd-L compounds confirmed by waterLOGSY NMR and by photoaffinity labelling. (A-C) WaterLOGSY NMR was carried out to determine Abd-L9 (A), Abd-L10 (B) and Abd-L13 (C) binding to LMO2 fused to the LID of LDB1 (LMO2-LID) or to a shortened version of LID in LMO2-ΔLID. Each of these compounds bind to LMO2-ΔLID (green) and not to LMO2-LID (purple) protein. (D) Chemical structure of Abd-L26 designed for photoaffinity labelling (PAL) to LMO2 protein. This is a compound built on Abd-L15/16 template, with a benzophenone photoreactive moiety, a linker and a biotin tag. (E-G) Pulldown of scFv-LMO2 recombinant protein by Abd-L26 with avidin beads, treated or not with UV light. The protein was either incubated alone (lane 1) or with 20 μM of Abd-L26 (lane 2) without UV treatment. The protein was also incubated with 20 μM Abd-L26 alone (lane 3) or with 100 μM Abd-L9 as competitor (lane 4) treated by UV light. The beads were washed and a Western blot showed the quantity of crosslinked LMO2 on the beads with an anti-biotin (E), anti-LMO2 (F) and an anti-HIS antibody (G).

[0588] FIG. 6: Evaluation of LMO2-binding Abd compound potency in cells

[0589] The potency and specificity of the LMO2 Abd compounds was evaluated in dose response BRET assays (A-C) In BRET assays, Abd-L9, Abd-L10, Abd-L16 and Abd-L22 compounds were assessed in dose inhibition responses for (A) LMO2-iDAb LMO2.sub.dm3, (B) LMO2-LDB1 and (C) LMO2-iDAb LMO2 (unmutated iDAb) BRET interactions. (D) Dose response assays of Abd-L15, Abd-L17, Abd-L18 and Abd-L19 with LMO2-iDAb LMO2.sub.dm3, LMO2-LDB1 and LMO2-iDAb LMO2 BRET interactions. Each experiment was performed twice. Error bars presented in A-D correspond to mean values±SD of biological repeats. See also related FIG. 14.

[0590] FIG. 7 related to FIG. 1: Molecular models of the recombinant proteins used for the cSPR library screen

[0591] (A) LMO2-iDAb LMO2 (PDB ID: 4KFZ), (B) LMO2 only, (C) LMO2-ΔLID and (D) LMO2-LDB1 LID domain fusion protein. LMO2 is shown as a surface representation, the LDB1 LID domain as sticks and iDAb LMO2 in ribbon context with a dotted line representing the linker between LMO2 and iDAb. The LMO2 only, LMO2-LID and LMO2-ΔLID structures are based on the LMO2-LDB1 crystal structure (PDB ID: 2XJY). The N- and C-termini of the proteins are marked for orientation purposes.

[0592] FIG. 8 related to FIG. 2: Establishing a BRET-based LMO2 biosensor

[0593] (A) BRET donor saturation assay with LMO2-RLuc8 as donor and GFP.sup.2-iDAb LMO2, GFP.sup.2-iDAb LMO2.sub.dm and GFP.sup.2-iDAb Ctl (non-relevant anti-RAS iDAb) as acceptors. (B) BRET competition assays between the LMO2-iDAb LMO2 interaction when either iDAb Ctl (grey bars) or iDAb LMO2 (black bars) were used as competitors (the control no competitor (−) is the white bar). (C) BRET competition assay between LMO2-iDAb LMO2.sub.dm interaction and the same competitors as panel B. (D) Western blot analysis of proteins from the BRET competition assay cells shown in panel C. Anti-GFP antibody shows iDAb LMO2.sub.dm expression, anti-LMO2 expression of LMO2-RLuc8 and anti-CMYC antibody shows expression of the competitors. Each experiment was performed twice. When error bars are presented, they correspond to mean values±SD of biological repeats.

[0594] FIG. 9 related to FIG. 2: Localisation of iDAb LMO2 mutations and the corresponding impacted amino acids on LMO2 structure

[0595] The part of LMO2 around the hinge region is shown in grey and in each panel, relevant amino-acids interacting with the iDAb LMO2 are shown in red. (A) Localisation of iDAb LMO2.sub.dm mutations (S55A and T107A) are shown in yellow on the parental iDAb LMO2 structure (cyan) with the affected, interacting LMO2 residue (R109) shown in red on LMO2 structure. (B) Localisation of iDAb LMO2.sub.dm3 mutations (S28G, H31G, S55A, E102A and T107A) are shown in yellow on the parental iDAb LMO2 structure with the affected LMO2 residues shown in red on LMO2 structure. (C) Localisation of iDAb LMO2.sub.dm6 mutations (S28G, H31G, S55A, E102A, S103A and T107A) are shown in yellow on the parental iDAb LMO2 structure with the affected LMO2 residue shown in red on LMO2 structure. LMO2-iDAb LMO2 structure used is PDB 4KFZ. Each panel has a table listing the interacting amino acids of LMO2 and iDAb.

[0596] FIG. 10 related to FIG. 2: DNA and proteins sequences of iDAb LMO2 and its mutants

[0597] (A) DNA and protein sequences of iDAb LMO2. (B-H) DNA and protein sequences of each iDAb LMO2.sub.dm1-LMO2.sub.dm6. The mutated amino acids compared to the parental iDAb LMO2 underlined in brown.

[0598] FIG. 11 related to FIG. 3: BRET-based HTS control interactions

[0599] (A) GFP.sup.2-only and RLuc8-only signal controls from FIG. 3C. (B) GFP.sup.2-only and RLuc8-only signal controls from the BRET experiment displayed on FIG. 3D. Compounds modifying RLuc8 luminescence or intrinsic GFP.sup.2 fluorescence by greater than two-fold were discarded (delimitated with the dotted lines calculated to the DMSO controls) (Lavoie et al., 2013). (C, D) Chemical structures of the 8 confirmed hits from the HTS that are divided into two related subfamilies, the 5 membered ring (C) and 7 membered ring (D). (E) BRET competition assay between the 8 hits and the non-relevant PPI MAX bHLH-CMYC bHLH. Each experiment in (A, B, E) was performed twice. The error bars correspond to mean values±SD of biological repeats.

[0600] FIG. 12 related to FIG. 4: Characterisation of anti-LMO2 Abd compounds

[0601] (A) Dose response effect of the Abd-L5 to Abd-L12 compounds on LMO2-iDAb LMO2 (unmutated) interaction (Abd concentration used: 1, 10, 20 μM). (B) Chemical structures, and their respective molecular weights (MW), of Abd-L13 and Abd-L14, which are two analogues of Abd-L8 and Abd-L21 respectively. (C) Abd-L13 and Abd-L14 were tested by BRET assays with LMO2-iDAb LMO2.sub.dm3 or (D) LMO2-iDAb LMO2 interactions (Abd concentration used: 1, 10, 20 μM). (E) Dose response effect of the Abd-L15 to Abd-L25 compounds on LMO2-iDAb LMO2.sub.dm3 interaction (Abd concentration used: 5, 10, 20 μM). (F) Chemical structures of Abd-L24 and Abd-L25 with their respective molecular weights (MW) and their percentage of BRET inhibition of the interaction LMO2-iDAb LMO2.sub.dm3 at 20 μM. These are two analogues, modified on their positions B and C respectively, which do not affect the BRET interaction LMO2-iDAb LMO2.sub.dm3. Experiments in A, C, D, E were performed twice. Error bars presented in A, C, D, E correspond to mean values±SD of biological repeats.

[0602] FIG. 13 related to FIG. 4: Permeability assays of the anti-LMO2 Abd compounds (A) Parallel artificial membrane permeability assay (PAMPA) with Abd-L9, Abd-L16-18. (B) CaCo-2 permeability assay with Abd-L9 compound.

[0603] FIG. 14 related to FIG. 6: Establishing BRET-based LMO2-natural partner biosensors

[0604] (A) BRET donor saturation assay with RLuc8-LMO2 as donor and full-length TAL1-GFP.sup.2 as acceptor with or without LDB1 and/or E47 co-expression. BRET.sub.max and BRET.sub.50 values are indicated for each BRET pair. (B) BRET donor saturation assay between LMO2-RLuc8 (donor) and full-length GFP.sup.2-LDB1 (acceptor). (C) BRET donor saturation assay with MAX bHLH-RLuc8 as donor and CMYC bHLH-GFP.sup.2 as acceptor. (D-F) BRET competition assays using iDAb Ctl (grey bars), iDAb LMO2 (black bars) as competitors or no competitor (-, white bar) between the following interactions: (D) LMO2-TAL1+E47, (E) LMO2-LDB1 and (F) MAX bHLH-CMYC bHLH. (G, H) BRET dose response assays with LMO2-TAL1+E47 interaction (G) and MAX bHLH-CMYC bHLH interaction (H) with the indicated Abd-L compounds. Each experiment was performed twice. The error bars correspond to mean values±SD of biological repeats.

ANALYTICAL METHODS

Cell Culture

[0605] HEK293T cells were grown in DMEM medium (Life Technologies) and supplemented with 10% FBS (Sigma) and 1% Penicillin/Streptomycin (PS) (Life Technologies). Cells were grown at 37° C. with 5% CO.sub.2.

Molecular Cloning

[0606] The DNA sequences encoding for LMO2-ΔLID (UniProt P25791; residues 26-156 and UniProt Q86U70; residues 334-344, joined by a GS linker), and LMO2-iDAb were cloned into the expression vector pOPINS via the restriction sites KpnI and HindIII, incorporating an AviTag into the 5′ primer. The vector encodes an N-terminal hexa-histidine tag and SUMO tag. The final protein expression constructs encoded for a single fusion protein consisting of His-SUMO-Avi-LMO2-GS-ΔLID and His-SUMO-Avi-LMO2-GS-iDAb. A construct encoding KRAS.sup.G12V.sub.166 with N-terminal His tag and TEV protease cleavage site was modified via PCR to include an AviTag between the TEV site and protein-coding sequence. For the SPR screening, all proteins were prepared with biotin tags.

[0607] iDAb LMO2 mutations were generated by PCR site-directed mutagenesis using pEF-GFP.sup.2-iDAb LMO2.sub.dm as template (Bery et al., 2018) (i.e. iDAb LMO2 S55A/T107A). The following mutations were introduced: iDAb LMO2 S28G/H31G/S55A/T107A, iDAb LMO2 S55A/E102A/T107A, iDAb LMO2 S55A/E102A/S103A/T107A, iDAb LMO2 S28G/H31G/S55A/E102A/T107A, iDAb LMO2 S28G/H31G/S55A/S103A/T107A and iDAb LMO2 S28G/H31G/S55A/E102A/S103A/T107A (FIG. 10).

[0608] LMO2 cDNA was cloned into the pEF-RLuc8-MCS and pEF-MCS-RLuc8 plasmids, MAX bHLH (amino acids 37-102) was inserted into pEF-MCS-RLuc8 plasmid. iDAb LMO2, mutants iDAb LMO2 and full-length LDB1 were cloned into pEF-GFP.sup.2-MCS plasmid and full-length TAL1 and cMYC bHLH (amino acids 354-439) into pEF-MCS-GFP.sup.2 plasmid.

cSPR Screening of PPI-NET Compound Library

[0609] As described previously (Cruz-Migoni et al., 2019) biotinylated LMO2-ΔLID, KRAS and LMO2-iDAb fusion were immobilized on a streptavidin-coated sensor chip SA (GE Healthcare) and the PPI-Net library compounds (comprising 1,500 compounds) were injected over the surface at 150 μM concentration. SPR experiments were carried out, as described, using a Biacore T200 (GE Healthcare) at 10° C. to preserve the protein immobilised on the sensor surface. Control proteins KRAS.sup.G12V.sub.166 and LMO2-iDAb were immobilised at ˜4000 RU and ˜6000 RU respectively to give approximately equimolar immobilisation levels of all three proteins on the sensor surface. Flow cell 1 was blocked by injecting 10 mM biocytin over the surface for 5 minutes at 10 μL.Math.min.sup.−1 and used as the reference channel. Immobilisation was carried out in HEPES running buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 0.005% P20, 5 mM MgCl.sub.2, 10 μM ZnCl.sub.2). Compound solutions were prepared by transferring 1.5 μL PPI-Net stock compounds at 10 mM in 100% DMSO into 96-well plates (Greiner) using a multichannel pipette. 98.5 μL running buffer with 3.5% DMSO was added to yield a solution of 150 μM compound in running buffer with 5% DMSO. Compound solutions were injected over all 4 flow channels for 30 seconds at 30 L.Math.min.sup.−1 and dissociation monitored for 60 seconds. A negative control of running buffer with 5% DMSO was run after every 24 cycles. Data were referenced, solvent corrected and processed using the T200 evaluation software. Data were baseline-corrected using the negative control binding levels as a reference and binding levels measured against LMO2-ΔLID plotted against binding levels measured against the control proteins. Replacement Abd-L1 (PPI-NET identifier P20000560B9) and Abd-L4 (PPI-NET identifier P20000557F5) were purchased from Asinex. Their molecular weights were confirmed by mass spec: Abd-L1 LRMS m/z (ESI.sup.+) 435 [M+H].sup.+; Abd-L2 LRMS m/z (ESI.sup.+) 437 [M+H].sup.+ recorded on an Agilent 6120 spectrometer using solutions of MeOH.

BRET2 Titration Curves and Competition Assays

[0610] For all BRET experiments (titration curves and competition assays) 650,000 HEK293T were seeded in each well of a 6 well plates. After 24 hours at 37° C., cells were transfected with a total of 1.6 μg of DNA mix, containing the donor+acceptor±competitor plasmids, using Lipofectamine 2000 transfection reagent (Thermo-Fisher). In dose response competition experiments, competitors were transfected with the following amount of DNA: 0.1; 0.5 and 1 μg. In single dose competition experiments, competitors were transfected with 0.1 g of DNA. Cells were detached 24 hours later, washed with PBS and seeded in a white 96-well plate (clear bottom, PerkinElmer, Cat #6005181) in OptiMEM no phenol red medium complemented with 4% FBS and cells were incubated for an additional 20-24 hours at 37° C. before the BRET assay reading. A detailed BRET protocol is provided by Bery and Rabbitts, (2019).

Cell Treatment

[0611] Compounds were prepared in 100% DMSO at 10 mM. For BRET competition assays, cells were treated with the indicated compounds at concentration of 1 (or 5), 10 and 20 μM for 22 h. For BRET-based dose response experiments, cells were treated with compounds at concentration of 0.01, 0.1, 1, 4, 10, 25 and 50 μM for 22 h. The compounds were diluted in the BRET medium: OptiMEM no phenol red (Life Technologies) supplemented with 4% FBS and with a final concentration of 0.2% DMSO.

BRET2 Measurements

[0612] BRET2 signal was determined immediately after injection of coelenterazine 400a substrate (10 μM final) to cells (Cayman Chemicals), using a CLARIOstar instrument (BMG Labtech) with a luminescence module. Total GFP.sup.2 fluorescence was detected with excitation and emission peaks set at 405 nm and 515 nm respectively. Total RLuc8 luminescence was measured with the Luminescence 400-700 nm-wavelength filter.

[0613] The BRET signal or BRET ratio corresponds to the light emitted by the GFP.sup.2 acceptor constructs (515 nm±30) upon addition of coelenterazine 400a divided by the light emitted by the RLuc8 donor constructs (410 nm±80). The background signal is subtracted from that BRET ratio using the donor-only negative control where only the RLuc8 fusion plasmid is transfected into the cells. The normalized BRET ratio is the BRET ratio normalized to a negative control (iDAb control or DMSO control) during a competition assay. Total GFP.sup.2 and RLuc8 signals were used to control the protein expression from each plasmid.

Western Blot Analysis

[0614] Cells were washed once with PBS and lysed in SDS-Tris buffer (1% SDS, 10 mM Tris-HCl pH 7.4) supplemented with protease inhibitors (Sigma) and phosphatase inhibitors (Thermo-Fisher). Cell lysates were sonicated with a Branson Sonifier and the protein concentrations determined by using the Pierce BCA protein assay kit (Thermo-Fisher). Equal amounts of protein (20 μg) were resolved on 12.5% SDS-PAGE and subsequently transferred onto a PVDF membrane (GE). The membrane was blocked with 10% non-fat milk (Sigma) in TBS-0.1% Tween20 and incubated overnight with primary antibody at 4° C. After washing the membrane was incubated with HRP conjugated secondary antibody for 1 hour at room temperature (RT, 22° C.). The membrane was washed with TBS-0.1% Tween and developed using Clarity Western ECL Substrate (Bio-Rad) and CL-XPosure films (Thermo-Fisher) or the ChemiDoc XRS+ imaging system (Bio-Rad). Primary antibodies include anti-LMO2 (1/1000, R&D System, Cat #AF2726), anti-GFP (1/500, Santa Cruz Biotechnologies, Cat #sc-9996), anti-biotin (1/1000, CST, Cat #5597S), anti-β-actin (1/5000, Sigma, Cat #A1978) and anti-αtubulin (1/2000, Abcam, Cat #ab4074). Secondary antibodies include anti-CMYC HRP-linked (Novus Biologicals, Cat #NB600-341), anti-mouse IgG HRP-linked (CST), anti-rabbit IgG HRP-linked (CST) and anti-goat IgG HRP-linked (Santa Cruz Biotechnologies).

High Throughput Chemical Screening with LMO2/iDAb LMO2 Mutant BRET Biosensor

[0615] The screen was carried out in 384-well plate format. An in-house library of 10,720 compounds (comprising 6991 compounds from BioFocus and 3729 from ChemBridge) were in 96-well plate format. The library was compressed into 384-well plate format for the HTS purpose. The volume and quantities indicated are for 40 assay 384-well plates. The screen was carried out in duplicate at 10 μM. Two sessions of HTS, containing 5,360 compounds each, were screened in 68 assay plates (34 assay plates in duplicate).

[0616] Before starting, HEK293T cells were seeded into 2×T175 flask. Three days later, the 2×T175 were split into 6×T175. [0617] Day 1: cell seeding: Cells were harvested from 6×T175 flasks at ˜70% confluency. The cells were resuspended in 110 mL of complete DMEM and 120×10.sup.6 inoculated into each of two Corning HYPERFlask M cell culture vessels (Corning, Cat #10030) and 560 mL of medium was added to fill one HYPERFlask. [0618] Day 2: cell transfection with pEF-LMO2-RLuc8 and pEF-GFP.sup.2-iDAb LMO2.sub.dm3: For each HYPERFlask 10 mL of OptiMEM was added together with 19 μg of pEF-LMO2-RLuc8, 37 g of pEF-GFP.sup.2-iDAb LMO2.sub.dm3 and 244 μg of pEF-empty-cyto-myc plasmids. 750 μL of Lipofectamine 2000 was added in 10 mL of OptiMEM, mixed gently. The 10 mL of DNA dilution was added and incubated for 20 minutes. The DNA/Lipofectamine 2000 mix was added in 500 mL of complete DMEM and the medium of the HYPERFlask had been removed. Finally, the medium+transfection mix was carefully poured into the HYPERFlask without creating any bubbles and the flask was filled with medium. [0619] Day 3: cell seeding in 384-well plates: The cells were harvested with 100 mL of trypsin that were added per HYPERFlask, incubated for 2 minutes at 37° C. and the trypsinized cells transferred to a beaker containing 100 mL of complete DMEM. Each flask was washed once with 100 mL of complete DMEM, mixed gently but thoroughly to ensure single cell suspensions (final volume for one flask: 300 mL). 90×10.sup.5 cells were added per 250 mL Corning centrifuge tube (4×250 mL centrifuge tubes were used which was 360×10.sup.6 transfected cells in total) and the cells were centrifuged at 220×g for 5 minutes at RT. Each cell pellet (4 in total) was gently resuspended in 200 mL of Opti-MEM without red phenol+4% FBS+1% PS (hereafter called BRET medium) to a final concentration of 0.45×10.sup.6 of cells/mL. The cells were seeded in white 384-well plates (clear bottom, PerkinElmer, Cat #6007480) with a PerkinElmer Janus liquid handling workstation housed in a Category 2 enclosure (45 μL/well; 20 000 cells). A blank plate was first used to remove any air bubble in the liquid handling workstation. [0620] Day 3: library dilution: 100 μM stock solutions were prepared for each compound in the library (the initial concentration of the library was 10 mM). 150 nL of 10 mM of each compound was added with an Echo Acoustic Dispenser (Labcyte) into 15 μL of BRET medium, giving a final concentration of 100 μM. [0621] Day 3: compounds addition: 1% DMSO was prepared in BRET medium. 5 μL of 1% DMSO solution was dispensed in the columns 1, 2 and 23, 24 as negative controls. Compounds were added to cells in 5 μL (100 μM) in each well using the Perkin Elmer Janus liquid handling workstation (10 μM final concentration, 0.1% DMSO) and the plates Incubated for 20 hours. [0622] Day 4: plate reading: A PHERAstar FSX plate reader (BMG Labtech) was used to read the plates equipped with a BRET2 optic module. The GFP.sup.2 signal of each plate was first measured to assess the relative cell number in each well. After the GFP.sup.2 reading, the bottom of each plate was covered with a white tape. 80 mL of 100 μM BRET substrate (i.e. coelenterazine 400a, Cayman Chemicals) was prepared by dissolving 3 mg of coelenterazine 400a in 32 mL of 100% ethanol and the volume brought to 80 mL by adding 48 mL of BRET medium. BRET reading was carried out by adding 5.5 μL of coelenterazine 400a (final concentration of 10 μM) using injectors and reading the BRET signal of each well. The reading time for one 384-well plate was about 8 minutes, therefore ˜4.5 hours to read 34 plates.

Recombinant Protein Expression

[0623] LMO2-LID protein was expressed in BL21(DE3)-Rosetta2 pLysS cells (Novagen) and HIS-SUMO-AVI-LMO2-ΔLID in Lemo21(DE3) cells (NEB, Cat #2528J). Bacterial cells were cultured at 37° C. in LB medium supplemented with 50 μg.Math.mL.sup.−1 kanamycin and 32 μg.Math.mL.sup.−1 chloramphenicol. At mid-log phase, expression was induced by addition of IPTG to a final concentration of 0.5 mM and supplemented with 0.1 mM ZnCl.sub.2. The cultures were incubated overnight at 18° C. with shaking at 220 rpm. Cells were harvested by centrifugation and resuspended in binding buffer (50 mM HEPES, pH 7.4, 500 mM NaCl, 5% glycerol and 5 mM imidazole). Resuspended pellets were stored at −20° C. Thawed cell pellets were supplemented with complete EDTA-free protease inhibitors at 1× concentration (Roche), 5 μL DNAse per liter of culture volume and MgSO.sub.4 to a final concentration of 2 mM. Cell suspensions were stirred on ice for 15 minutes and lysed using a Constant Systems Cell Disruptor at 23 kpsi, 4° C. Cell extracts were clarified by centrifugation. His-tagged proteins were purified under gravity flow using nickel-Sepharose (GE Healthcare) columns. Bound proteins were washed with 2×50 mL of binding buffer and then 25 mL of wash buffer (50 mM HEPES, pH 7.4, 500 mM NaCl, 5% glycerol and 20 mM imidazole). Bound proteins were eluted with 5 mL of elution buffer (50 mM HEPES, pH 7.4, 500 mM NaCl, 5% glycerol and 50 mM imidazole) and 2×5 mL elution buffer 2 (50 mM HEPES, pH 7.4, 500 mM NaCl, 5% glycerol and 500 mM imidazole). SUMO tags were removed by incubating with SUMO protease overnight at 4° C. Proteins were further purified on a HiLoad 16/60 Superdex 200 Prep Grade column using an ÅKTA Avant system (GE Healthcare) buffered in 10 mM HEPES pH 7.4, 250 mM NaCl. For proteins to be used in SPR experiments, N-terminal AviTags were biotinylated by incubating with BirA enzyme overnight at 4° C. in the presence of 20 mM MgCl.sub.2, 500 μM biotin and 2 mM ATP. Proteins were further purified on a HiLoad 16/60 Superdex 200 Prep Grade column using an ÅKTA Avant system (GE Healthcare) buffered in 10 mM HEPES pH 7.4, 250 mM NaCl and 0.5 mM DTT. KRAS.sub.166.sup.G12V was purified as previously described (Cruz-Migoni et al., 2019), with the addition of the BirA incubation step for biotinylation of the purified protein.

Purification of scFv-LMO2 for PAL Analysis

[0624] For co-expression of recombinant LMO2 and anti-LMO2 scFv, the scFv was cloned into an existing bicistronic expression vector (pRK-His-TEV-VH576-LMO2, Sewell et al 2014). DNA encoding the scFv was amplified by PCR and cloned into the pRK vector to replace the VH576 using NcoI and EcoRI restriction sites. Plasmid DNA was transformed into E. coli C41 (DE3) cells for protein co-expression. A single colony was used to inoculate 50 mL of LB media containing 100 μg.Math.mL.sup.−1 ampicillin which was grown overnight at 37° C., shaking at 225 rpm. The overnight seed culture was diluted 1:100 in 8×1 L of LB containing 100 μg.Math.mL.sup.−1 ampicillin. The cultures were grown at 37° C., shaking at 225 rpm until an OD.sub.600 of 0.6 was reached. ZnSO.sub.4 was added prior to induction to a final concentration of 0.1 mM. Protein expression was induced by the addition of 0.5 mM IPTG (isopropyl 1-thio-beta-D-galactopyranosid) and the cells were incubated overnight at 16° C., shaking at 225 rpm. Cells were harvested by centrifugation at 6000 rpm, for 20 minutes at 4° C. Cell pellets were resuspended in lysis buffer (20 mM Tris pH 8.0, 250 mM NaCl, 20 mM imidazole, 0.1 mM ZnSO.sub.4, 5 mM 2-mercaptoethanol, 5% glycerol) containing EDTA-free protease inhibitor cocktail tablets (Roche, Germany) prior to lysis at 25 kPSI, 4° C. using a cell disruptor system (Constant Systems Ltd, UK). The cell lysate was incubated with DNase I and 2 mM MgCl.sub.2 for 20 minutes at RT before being clarified by centrifugation at 22,000 rpm for one hour at 4° C. LMO2 and anti-LMO2 scFv were co-purified using a 5 mL HisTrap HP column (GE Healthcare, UK) using a 50 mL imidazole gradient from 20 mM to 300 mM. The protein was concentrated to 1.5 mL and purified further by gel filtration using a HiLoad 16/600 Superdex 75 column (GE Healthcare, UK) in 20 mM Tris pH 8.0, 250 mM NaCl, 1 mM DTT. The co-purification of LMO2 and anti-LM02 scFv was verified by standard Western blotting using anti-LMO2 (R&D Systems, AF2726) and anti-His-HRP (Sigma, A7058).

WaterLOGSY NMR

[0625] WaterLOGSY NMR method was used to measure LMO2 ligand interaction (Bataille et al., 2020). WaterLOGSY experiments were conducted at a .sup.1H frequency of 600 MHz using a Bruker Avance spectrometer equipped with a BBI probe. All experiments were conducted at RT. 3 mm diameter NMR tubes with a sample volume of 200 μL were used for all experiments. For LMO2-LID, solutions were buffered using a 10 mM NaPO.sub.4, 250 mM NaCl solution. For LMO2-ΔLID, solutions were buffered using a 10 mM NaPO.sub.4, 50 mM NaCl solution. The sample preparation for measuring ligand binding with LMO2-LID is exemplified as follows; the compound (10 μL of a 10 mM solution in DMSO-d.sub.6) was added to an Eppendorf tube before sequential addition of the appropriate buffer (90 μL), D.sub.2O (20 μL), and the protein (80 μL, 25 μM). The resulting solution was vortexed to mix and transferred to a 3 mm NMR tube prior to the NMR analysis. The sample preparation for measuring ligand binding with LMO2-ΔLID is exemplified as follows; the compound (10 μL of a 10 mM solution in DMSO-d.sub.6) was added to an Eppendorf tube before sequential addition of the appropriate buffer (162 μL), D.sub.2O (20 μL), and the protein (8 μL, 250 μM). The resulting solution was vortexed to mix and transferred to a 3 mm NMR tube prior to the NMR analysis. The sample preparation for checking possible aggregation with the ligand alone is exemplified as follows; the compound (10 μL of a 10 mM solution in DMSO-de) was added to an Eppendorf tube before sequential addition of the appropriate buffer (170 μL) and D.sub.2O (20 μL). The resulting solution was vortexed to mix and transferred to a 3 mm NMR tube prior to the NMR analysis.

PAL Pulldown

[0626] 20 μM of Abd-L26 with or without 100 μM of Abd-L9 (competitor) are added in a final volume of 400 μL of PBS with 40 μg of purified protein of interest (scFv-LMO2). The same samples are prepared for the no UV controls. The samples are incubated for 25 minutes at RT. The samples to be crosslinked are put into ice and under the UV lamp for crosslinking for 1 hour. The no UV controls are kept on ice. During the 1 hour of crosslinking, the agarose monomeric avidin beads (Cat #20228, Thermofisher) are washed twice with PBS. After the crosslink, 20 μL of washed beads are added in all the samples (crosslinked and non-crosslinked) and incubated for 2 hours at 4° C. on a roller. 2 hours later, wash the beads three times with 400 μL of PBS. The samples are finally denatured with 50 μL of 2× loading buffer with BME added directly on the beads (and boiled at 100° C. for 5 minutes) and loaded for a western blot analysis.

CACO-2 and PAMPA Assays

[0627] Caco-2 apparent permeability (Papp) was determined in the Caco-2 human colon carcinoma cell line as described (Bavetsias et al., 2016). Cells were maintained (DMEM with 10% fetal bovine serum, penicillin, and streptomycin) in a humidified atmosphere with 5% CO.sub.2/95% air for 10 days. Cells were plated out onto a cell culture assembly plate (Millipore, UK), and monolayer confluency was checked using a TEER electrode prior to the assay. Media was washed off and replaced with HBSS buffer (pH7.4) containing compound (10 μM, 1% DMSO) in the appropriate apical and basal donor wells. HBSS buffer alone was placed in acceptor wells. In particular instances a specific P-gp inhibitor, LY335979 (5 μM), was added to the HBSS. The Caco-2 plate was incubated for 2 hours at 37° C. Samples from the apical and basolateral chambers were analysed using a Waters (Milford, MA, US) TQ-S LC-MS/MS system. The cell permeability properties of Abd-L compounds was compared to low (nadolol) and high (antipyrine) permeability compounds and a compound with high export (indinavir).

Apparent permeability (Papp) was determined as follows:

[00001]$P_{\mathrm{app}}=\frac{\mathrm{Vr}\left(\mathrm{mL}\right)}{A\left({\mathrm{cm}}^2\right)\times C0\left(\mathrm{\mu M}\right)}\times \mathrm{Rate}\mathrm{of}\mathrm{Diffusion}\left(\mathrm{\mu M}.{\sec }^{-1}\right)$$\begin{array}{c}\mathrm{Where}\mathrm{Vr}=\mathrm{volume}\mathrm{of}\mathrm{receptical}\\ A=\mathrm{surface}\mathrm{area}\mathrm{of}\mathrm{monolayer}\\ C0=\mathrm{Initial}\mathrm{compound}\mathrm{concentration}\mathrm{in}\mathrm{donor}\end{array}$

PAMPA

[0628] The parallel artificial membrane permeability assay (PAMPA) was used to determine compound permeability by passive diffusion. The assay used an artificial membrane consisting of 2% phosphatidyl choline in dodecane (Sigma Aldrich, Dorset, UK). The donor plate was a MultiScreen-IP Plate with 0.45 μm hydrophobe Immobilon-P Membrane (Millipore, UK) and the acceptor plate was a MultiScreen 96-well Transport Receiver Plate (Millipore, UK). The permeability was measured at 3 different pH levels: pH 5, 6.5 and pH 7.4 in buffer containing 1% Bovine Serum Albumin (Sigma Aldrich, Dorset, UK). A 10 mM DMSO stock solution of test compound was used to prepare the 10 μM PAMPA donor solutions and calibration curves in each of the three buffers.

[0629] 6 μL of the membrane solution was added to each well of the donor plate. Buffer donor solutions (200 μL) were added to the appropriate wells of the PAMPA donor plate. 300 L per well of blank PBS (pH 7.4) was added to the PAMPA acceptor plate.

[0630] The donor and acceptor plates were then sandwiched together, covered with a lid and incubated at 30° C. in a humid environment for 16 hours. After the incubation period the plates were removed from the incubator and the sandwich was dismantled. Samples were then transferred into a fresh plate and centrifuged. All sample supernatants were diluted and analysed using a Waters (Milford, MA, US) TQ-S LC-MS/MS system.

[0631] Permeability values (cm/s) were calculated using the following equation:

[00002]$\begin{array}{c}{P}_{\mathrm{app}}=C\times -\ln \left(1-\frac{\left[{\mathrm{drug}}_{\mathrm{acceptor}}\right]}{\left[{\mathrm{drug}}_{\mathrm{equilibrium}}\right]}\right)\\ \mathrm{where}C=\frac{V_D\times V_A}{\left(V_D+V_A\right)\times \mathrm{area}\times \mathrm{time}}\end{array}$$\begin{array}{c}{V}_D=\mathrm{volume}\mathrm{of}\mathrm{donor}\\ V_A=\mathrm{volume}\mathrm{of}\mathrm{acceptor}\\ \mathrm{Area}=\mathrm{surface}\mathrm{area}\mathrm{of}\mathrm{the}\mathrm{membrane}\times \mathrm{porosity}\end{array}$

Results

[0632] In Vitro cSPR Screening of a Chemical Library with an LMO2-iDAb Fusion Protein

[0633] The use of a high affinity intracellular antibody binding to RAS protein in a competitive SPR screening of a chemical library screen has previously been described (Quevedo et al., 2018) and relies on high affinity interaction between antibody and antigen on the SPR chip to select Abd compounds. The same approach was adopted with an intracellular antibody (in the form of an iDAb) binding to the LMO2 protein. Since, in this case, the interaction affinity is the nM range, rather than pM as the anti-RAS, a fusion between LMO2 and the iDAb where the two components were joined in a single polypeptide by a short flexible glycine-serine (GS) linker was used (FIG. 7A compared to the structure of LMO2 from PDB 2XJY in FIG. 7B). LMO2 can also be expressed in E. coli in complex with the LID domain of LIM domain binding 1 (LDB1) as this has been shown to be the only effective way to express soluble LMO2 protein (Ryan et al., 2006) apart for co-expressing with the iDAb. The binding of the iDAb to LMO2 occurs across the LMO2 hinge overlapping to some extent with LDB1 LID binding (Sewell et al., 2014). Consequently, a truncated version of the LID domain was co-expressed with LMO2 (hereafter LMO2-ΔLID) that covers the whole LIM2 of LMO2 but leaves the hinge and LIM1 regions accessible (FIG. 7C compared to the structure of LMO2-LID from PDB 2XJY in FIG. 7D). Therefore, an SPR chip was prepared with the LMO2-iDAb in one channel, LMO2-ΔLID in a second channel and a non-relevant protein, KRAS, was attached to a third channel. The chip format is shown in FIG. 1A. This organization of the SPR channels enabled sub-division of the chemical matter into those that might bind LMO2 in the iDAb binding region (Abd compounds as iDAb surrogates), those that bind LMO2 elsewhere than the LID and iDAb binding regions and those that bind to RAS.

[0634] Among the 1,500 screened compounds, four LMO2-ΔLID Abd hits were identified as having response units (RU) above 10 that did not bind the LMO2-iDAb or KRAS (FIG. 1B). The chemical structures and molecular weights of these are shown in FIG. 1C with the RU data from the screen. The compounds designated Abd-L1 and Abd-L4 were available commercially but Abd-L2 (an analogue of Abd-L1) and Abd-L3 were not. Further characterisation of Abd-L1 was undertaken using an orthogonal assay, 1D NMR waterLOGSY (Dalvit et al., 2001, Bataille et al., 2020) with LMO2-LID and LMO2-ΔLID. Proton peaks showed the polarity shifts caused by interaction with the LMO2-ΔLID whereas limited change was observed with the LMO2-LID protein (FIG. 1D).

[0635] The purpose of compound library screens is to identify chemical matter that can form the basis of drug development and therefore function in cells. Accordingly, the cell permeability properties of Abd-L1 in a CaCo-2 assay compared to low (nadolol) and high (antipyrine) permeability compounds and a compound with high export were assessed (indinavir) (FIG. 1E). The Caco-2 data show that Abd-L1 has low cellular permeability with high efflux in Caco-2 cells (FIG. 1E), which are poor cell-based drug properties.

Establishing a BRET-Based LMO2-iDAb Biosensor for a Small Molecule Screen

[0636] Since the in vitro selection assays clearly do not necessary yield cell permeable compounds, an alternative approach was designed using a cell-based screening method for iDAb surrogates to improve cell properties of chemical hits (i.e. cell uptake, low export, etc). Such a cell-based screen for compounds that inhibit PPIs requires an assay that generates a signal from the PPI but which does not occur via a high affinity interaction because initial chemical hits would be expected to be weak binders. Accordingly, a BRET-based LMO2/iDAb LMO2 biosensor was engineered based on the strategy of RAS biosensors (Bery et al., 2018). Structural data for LMO2-iDAb LMO2 complex (Sewell et al., 2014) was used to optimise the proximity of donor and acceptor moieties. The donor moiety RLuc8 was fused at the carboxy terminal end of LMO2 and the GFP.sup.2 acceptor molecule to the amino terminal end of the iDAb LMO2. The interaction between LMO2-RLuc8 and GFP.sup.2-iDAb LMO2, the lower affinity GFP.sup.2-iDAb LMO2.sub.dm (a dematured iDAb LMO2 (Bery et al., 2018)) or the non-relevant GFP.sup.2-iDAb RAS (Tanaka et al., 2007) (hereafter named iDAb control or iDAb Ctl) was tested by BRET donor saturation assays (FIG. 8A). These data demonstrate that the dematuration mutagenesis has lowered the affinity of the iDAb LMO2.sub.dm since there is 10-fold increase in BRET.sub.50 (an approximation to the relative affinity of the acceptor for the donor protein) of iDAb LMO2.sub.dm compared to iDAb LMO2 (0.44 versus 0.03 respectively, see FIG. 8A). The specificity of these interactions was assessed with a BRET competition assay in which an untagged competitor (iDAb LMO2) or a non-relevant competitor (iDAb Ctl) were expressed with either the BRET pairs LMO2-iDAb LMO2 (FIG. 8B) or LMO2-iDAb LMO2.sub.dm (FIG. 8C). The competitor iDAb LMO2 decreased LMO2-iDAb LMO2 interaction in a dose dependant manner but only to ˜65% at the highest dose of competitor (FIG. 8B). Accordingly, iDAb LMO2 competed the lower affinity iDAb LMO2.sub.dm-LMO2 interaction with a stronger inhibition at its highest dose (˜80%, FIG. 8C) and the expression of these proteins was not altered (FIG. 8D).

[0637] A dematuration method was employed to decrease iDAb affinity based on CDR sequences (Assi et al., 2010) such as it enabled an Alpha-Screen of RAS.sup.G12v-binding compounds and analysis of in vitro derived RAS-binding Abd compounds (Tanaka & Rabbitts, in preparation). Based on the LMO2-iDAb LMO2 structural information (Sewell et al., 2014), we introduced additional mutations on the CDRs of iDAb LMO2.sub.dm that would affect the interaction between key amino acids from the iDAb and LMO2 with alanine or glycine substitution while still retaining specific binding (FIG. 9A-C). Most of the modifications on the iDAb LMO2 affected its binding around the hinge region of LMO2 (FIG. 9A-C). Six additional mutants named iDAb LMO2.sub.dm1-6 (DNA and protein sequences shown in FIG. 10A-H) were constructred and tested in BRET donor saturation assays (FIG. 2A). Each of the iDAb LMO2 mutants (dm1-6) had a decreased BRET.sub.max value (an approximation for the total number of complex LMO2/iDAb and the distance between the donor and the acceptor within the dimer) and an increased BRET.sub.50 value compared to the template iDAb LMO2.sub.dm (FIG. 2B). This suggested an overall decreased affinity of the dematured iDAbs towards LMO2 and the mutations did not affect their expression (FIG. 2C). Finally, a BRET competition experiment was performed with each mutant with the lowest dose of competitor (FIG. 2D) to determine the optimal dematured iDAb for the chemical library screen. The competition data with iDAb LMO2.sub.dm3 showed it was the best mutant as its interaction with LMO2 was almost completely inhibited by iDAb LMO2 (˜90%) while it retained a relatively high BRET signal (FIG. 2D). Therefore, this mutant was selected for a cell-based high throughput screening of small molecules.

High-Throughput Screen for Inhibitors of LMO2-iDAb.SUB.dm3 .Interaction

[0638] The robustness and scalability of the cell-based BRET LMO2-iDAb LMO2.sub.dm3 interaction assay was tested in a high-throughput screen (HTS) to identify compounds that inhibit this interaction. A library of 10,720 small molecules assembled from Biofocus and Chembridge sources were screened. The flowchart of the HTS is described in FIG. 3A. HEK293T cells were transfected on day 1 with plasmids expressing LMO2-RLuc8 and GFP.sup.2-iDAb LMO2.sub.dm3 and 24 hours later, compounds were added to 10 μM and the BRET signals determined after a further 24 hours. Thirty-four primary hits were identified using a cut-off of 3×SD from DMSO controls (Lavoie et al., 2013) (FIG. 3B). These were re-tested using the original BRET assay to confirm inhibition of signal and also using BRET-based interaction assay between LMO2 and unmutated iDAb LMO2 to eliminate compounds binding to the iDAb (FIG. 3C, D). In addition, initial hits affecting RLuc8 luminescence or intrinsic GFP.sup.2 fluorescence by greater than two-fold were not considered further (FIG. 11A, B). This rescreen resulted in eight inhibitors of LMO2-iDAb LMO2.sub.dm3 interaction (FIG. 11C, D). The eight compounds were finally tested with a non-relevant BRET-based interaction assay (MAX bHLH-CMYC bHLH) to provide further confirmation of specific interaction with LMO2 (FIG. 11E). The chemical structures of the selected hits show that the compounds belong to a family that can be divided into two subfamilies due to their chemical similarities. The main difference is the presence of either a 5 or 7 membered ring in each sub-family (FIG. 11C, D, respectively).

Structure-Activity Relationship Study of Abd Compounds

[0639] Samples of the 5-membered ring-containing hits (FIG. 11C) were prepared following literature methods. However, it was found that the core underwent a rearrangement to the 5-membered when attempting to synthesise samples of 7-membered ring-containing compounds (FIG. 11D). Therefore, the corresponding 5-membered ring analogues, which were named Abd-L5 to Abd-L12 were synthesised (FIG. 4A). These analogues were tested in a dose-response BRET assay to verify their ability to bind LMO2 and their potency (FIG. 4B and FIG. 12A). Three of the analogues, Abd-L5, Abd-L8 and Abd-L11, were unable to inhibit LMO2-iDAb LMO2.sub.dm3 interaction (FIG. 4B). The most potent inhibitor, Abd-L9, was used as a template for a structure-activity relationship study (FIG. 4C, D).

[0640] The different moieties of Abd compounds were divided into 4 substituent groups, namely benzyl (position A), imidazolidinone (B), oxazole (C), and aniline (D) (FIG. 4C) and were modified systematically. Representative analogues are shown (Abd-L13 to Abd-L25, FIG. 4D and FIG. 12B-F). Most substituted benzyl groups were found to be well tolerated in position A, the methoxy group could be placed in alpha, meta or para positions on the benzyl ring with minimal effect on the BRET inhibition potency of derivatives (compounds with benzyl modifications (red boxes) and/or aniline (green boxes) modifications are shown in FIG. 4C). It was found that a large array of substituted anilines and benzyl amines were also well tolerated at position D. It is noteworthy that most substituted pyridine-containing compounds did not exhibit activity, which result from the change of basicity in those compounds.

[0641] Modifications to positions B and C had more substantial effect on the potency of the analogues. In position B, any replacement of the imidazolidinone was found to bring a loss of activity (as such pyrimidinone and piperazinones, FIG. 12E, F) apart from the corresponding piperazine (pink box in FIG. 4D). Due to the potential chemical instability of the imidazolidinone, the lower yields and a large number of side-products during synthesis, a piperazine moiety was substituted for further SAR investigations. Those issues were eradicated with the change to the piperazine. In position C, it was found that only the 2,4-substituted-thiazole and 2,4-substituted-oxazole were tolerated as a core and the corresponding 2,5-substituted heterocycles and different heterocycle (such as pyrimidine) led to a loss of activity (see blue box on the position C of the analogues in FIG. 4D and FIG. 12E, F). These data suggest that the B/C positions are important for the interaction of the compounds with LMO2 while position A/D could be modified to add new functional groups.

[0642] Abd-L9 and some analogues were tested in a Parallel Artificial Membrane Permeability Assay (PAMPA) and Abd-L9 in a Caco-2 permeability assays (FIG. 13A, B). This showed the compounds were permeable through a synthetic membrane (PAMPA) or into cells (Caco-2) as would be expected compounds derived from cell-based screens. Abd-L9 showed the best properties in the PAMPA compared to the analogues and a low transport but low efflux ratio in the Caco-2 assay (FIG. 13A, B). These results suggest that while Abd-L9 enters cells with a relatively low efficiency, it is not actively exported from the cells (low efflux ratio).

Abd Compounds Bind LMO2 In Vitro

[0643] The Abd compounds were identified and verified in cell-based assays. LMO2-LID and LMO2-ΔLID were used in waterLOGSY NMR experiments to assess the binding of small molecules with LMO2. One compound from the cSPR screen (Abd-L1, FIG. 1D) and three LMO2 compounds from the cell-based screen were tested (Abd-L9, Abd-L10 and Abd-L13, FIG. 5A-C) and each showed binding to LMO2-ΔLID but not LMO2-LID. Since the difference between the two proteins is the hinge and LIM1, the data concur with cell data that the compounds bind LMO2 and that they bind on the interface restricted to the LIM1 and the hinge region of LMO2.

[0644] These data were further confirmed by using an alternative method: the photoaffinity labelling (PAL), a powerful technique used to study protein-ligand interactions (Smith and Collins, 2015). PAL is the use of a chemical probe that can covalently bind to its target in response to activation by light (Sadakane and Hatanaka, 2006). The extensive SAR data on the LMO2 Abd compounds, suggested attachment sites on the parent ligand. A benzophenone photoreactive group was added in place of the benzyl substituent (position A) and a linker with a biotin tag in position D (FIG. 5D). In order to obtain soluble, recombinant LMO2 protein that has an Abd-L compound-binding site accessible, A phage display screen of scFvs was carried out with LMO2-LID protein antigen and obtained scFv that binds LMO2 and can be co-expressed in E. coli(Miller & Rabbitts, unpublished). By employing the partially purified LMO2-scFv dimer, the PAL technique was performed after inducing crosslinking of a photoaffinity LMO2 PAL compound (designated Abd-L26, that comprises a photo-active substituent and a biotin moiety attached to the compound with a short linker; FIG. 5D) to the scFv-LMO2 complex with UV light for photo-crosslinking. The Abd-L26 in the complex was isolated by interaction of the biotin moiety with avidin beads and the protein analysed by Western blot with either anti-biotin antibody (FIG. 5E), anti-LMO2 antibody (FIG. 5F) or anti-HIS tag (FIG. 5G). The pulldown data show that protein is only crosslinked when the mixture is treated with UV light and we observed a biotin labelled protein (FIG. 5E, lane 3) coincident the size of LMO2 (FIG. 5F). In addition, the recovery of biotinylated LMO2 was inhibited by incubating the protein with Abd-L26 (PAL) in the presence of 5× concentration of Abd-L9 competitor (FIG. 5E, lane 4). The anti-biotin antibody showed that the biotinylated proteins specifically bound to the beads through the PAL Abd-L26 compound while the anti-LMO2 and anti-HIS antibodies show non-specific binding of proteins to the beads. It was noted that the recombinant LMO2 had a tendency to associate non-specifically as well as the scFv with avidin agarose beads used for the pulldown (see lanes 1 & 2, FIG. 5F, G). This is possibly due to partial denaturation of the proteins during the PAL incubation and may explain the apparent partial inability of Abd-L9 to compete the PAL compound (FIG. 5F, lane 4 versus lane 3).

Activity of LMO2 Abd Compounds in Cells

[0645] The specificity and potency of Abd-L compounds in cells by using dose-response BRET assays on different LMO2 PPI was tested. This included LMO2 interaction with the unmutated iDAb and the iDAb.sub.dm3, with its natural partner proteins LDB1 and TAL1 (together with E47) (Wadman et al., 1997) and with a non-relevant control PPI which is the interaction of the bHLH regions of CMYC with MAX. In order to develop the various BRET assays, the direct interaction LMO2 with TAL1 by a BRET donor saturation assay was initially tested (FIG. 14A) but this interaction is weak and gave a high BRET.sub.50 value. Partner proteins involved in the LMO2 complex (Wadman et al., 1997) were added individually and it was found that co-expression of E47, a heterodimerization partner of TAL1, increased the relative affinity of LMO2-TAL1 and the addition of LDB1 gave the strongest binding between LMO2 and TAL1 (FIG. 14A, see decreasing BRET.sub.50 values: from 12.6 to 1). The BRET pairs LMO2-LDB1 (FIG. 14B) were also developed as well as the non-relevant interaction of MAX bHLH with CMYC bHLH (FIG. 14C). Finally, the specificity of these three interactions was tested, with BRET competition assays, by co-expressing non-tagged versions of iDAb Ctl or iDAb LMO2 in the BRET assay cells. iDAb LMO2 inhibited BRET signal from LMO2-TAL1+E47 (FIG. 14D) and from LMO2-LDB1 (FIG. 14E) but not from MAX-CMYC interaction (FIG. 14F).

[0646] The anti-LMO2 Abd compounds were assessed in BRET dose-response assays with the various BRET assays (FIG. 6A-D and FIG. 14G, H). None of the compounds inhibited LMO2-iDAb LMO2.sub.dm3 BRET by more than 40-50%, with the exception of Abd-L22 (˜85%). However, it was found that Abd-L9, Abd-L10 and Abd-L16 had the best IC.sub.50 for the interaction LMO2-iDAb LMO2.sub.dm3 at around 1 μM (FIG. 6A and Table 1) whether or not the compound contained imidazolidinone substituents (Abd-L9 and L10) or a piperazine substituent (Abd-L16). The other compounds tested showed IC.sub.50 values ranging from just over 7 μM to nearly 50 μM for Abd-L19 (FIG. 6A, D and Table 1). When this group of compounds were assayed with LMO2-LDB1 BRET, little effect was observed except Abd-L10 which caused only a small inhibition (35% at the highest concentration of Abd-L10 with an IC.sub.50 of 1.2 μM, Table 1 and FIG. 6B, D). Testing this group of Abd-L compounds with the BRET assays for LMO2-iDAb LMO2 (unmutated iDAb) (FIG. 6C), LMO2-TAL1+E47 (FIG. 14G) or MAX bHLH-CMYC bHLH (FIG. 14H) failed to show any inhibition, even at the highest concentration of compound as used throughout the series of BRET inhibition assays with the exception of Abd-L22 that inhibits LMO2-iDAb LMO2 interaction only at high concentrations (above 25 μM, FIG. 6C).

Intracellular Antibodies as Templates for Drug Discovery

[0647] Intracellular antibody fragments interact with proteins at any antigenic site or where natural partner proteins are involved in PPI. This gives an opportunity using the intracellular antibody to derive compounds that are surrogates for the specific interaction residues with the intracellular antibody. When the intracellular antibody interferes directly with a PPI, rather than employing the natural partner protein, the intracellular antibody can be obtained with very high affinity binding, as shown for selected compounds binding to the RAS proteins (Quevedo et al., 2018) demonstrating that this so-called undruggable target is in fact druggable. The in vitro method, using intracellular antibodies as tools for drug discovery, employs competitive SPR with a chip carrying a target protein with interacting antibody in place (Quevedo et al., 2018). The RAS-binding compounds were successful due to the very high affinity of the anti-RAS antibody limiting loss of antibody-antigen interaction on the SPR chip. As described herein, the similar approach using an anti-LMO2 iDAb was implemented and circumvented the problem of loss of iDAb during the library screen by linking LMO2 and iDAb with a flexible linker. In this way, LMO2-binding chemical matter was identified. Examination of the cell-based properties of one of these compounds showed disadvantageous features.

[0648] Alternatively, in cell-based assays such as the BRET assay described herein, the affinity of the iDAbs for their target is not a limitation. Furthermore, the ability to carry out affinity manipulation on tight binders is facile with antibodies because only the primary sequence identifies the CDRs for mutagenesis in a process called intracellular antibody dematuration (Tanaka & Rabbitts, in preparation). This process does not require structural information and iDAbs of interest with lower binding properties could be directly used in this cell-based approach, which makes this a flexible approach. In addition, cell-based screening is also a more versatile approach as it can be implemented to any protein that is difficult to express, such as LMO2 which has eluded recombinant expression except in co-expression with LDB1 LID (Ryan et al., 2006) or the iDAb (Sewell et al., 2014). Finally, the intrinsic advantage of cell-based assays, in which a signal is generated by the direct interaction of target with iDAb, is that the compounds already have the characteristic of cell entry, which we show here with our LMO2 Abd-L series of compounds.

LMO2 Binding Compounds Derived from a Cell-Based BRET2 Chemical Library Screen

[0649] A cell-based intracellular single domain antibody-guided small molecule selection method as described herein allows the direct identification of compounds that bind at the same region of the iDAb. This has been illustrated using the T cell oncogenic protein LMO2. LMO2 encodes a 18 kDa polypeptide that comprises two zinc-binding LIM domains (Chambers and Rabbitts, 2015). These domains are the interface for binding to class II basic helix-loop-helix (bHLH) transcription factors such as TAL1/E2A and GATA (Wadman et al., 1994). Furthermore, these two DNA-binding complexes are bridged by a scaffolding protein, LDB1 that binds LMO2 on a different interface than the transcription factors (Wadman et al., 1997). An anti-LMO2 iDAb has been characterised that inhibits the tumourigenic function of LMO2 in vivo as it prevents LMO2-dependent tumour growth in a transplantation assay mediated by the disruption of the LMO2-multimeric complex by preventing LDB1 interaction (Tanaka et al., 2011). In detail, the anti-LMO2 intracellular antibody functions as an indirect PPI inhibitor by a new mechanism, which is altering the natural structure of LMO2. The iDAb LMO2 induces a change of conformation between the two LIM domains of LMO2 that is not compatible with the interaction of LDB1 and the transcription factors (Sewell et al., 2014). With the Abd-L compounds selected here, no significant modification of the conformation of LMO2 protein was observed as shown with BRET data employing the TAL1/E47: while the iDAb LMO2 impedes the binding of these proteins with LMO2 (FIG. 14D), the Abd-L compounds did not (FIG. 14G).

[0650] The iDAb was employed to screen a compound library (10K compounds) that bind to LMO2 in the LMO2-iDAb BRET2 cell-based interaction assay. A number of initial hits were obtained and one chemical series of which Abd-L5 to Abd-L12 were the progenitors. Direct binding of compounds Abd-L9, Abd-L10 and Abd-L13 using recombinant LMO2-ΔLID proteins in waterLOGSY NMR was confirmed (FIG. 5A-C). SAR analysis was carried out on the chemical series to determine if the compounds could tolerate larger groups and linkers, and therefore allowing the use of PAL technology. The use of a benzophenone moiety was tested as a photoaffinity label and observed that analogues bearing this group on the right- or the left-hand sides were still active. The linker however could not be located on the left-hand side as this caused loss of activity. A compound was therefore prepared with the benzophenone photoreactive moiety on the piperazine and the biotin linked to the aniline in the para position via a small polyethylene glycol (PEG) chain, generating compound Abd-L26 (FIG. 5D). The cross-linking of Abd-L26 to the LMO2 protein confirmed binding to LMO2 protein (FIG. 5E-F) and this was inhibited by addition of Abd-L9. These data support the conclusion that the chemical series is an intracellular antibody surrogate that bind to LMO2 where the anti-LMO2 iDAb contacts LMO2. The cell-based selection involved competition by the compounds for the interaction of LMO2 with a dematured iDAb and these compounds do not influence the interaction of LMO2 with unmutated iDAb (except Abd-L22 at the highest concentrations) as would be expected from their M interference IC.sub.50. In addition, the compounds do not bind to LMO2-LID fusion whereas they bind to an LMO2-ΔLID fusion (where the LID is truncated for part of the region where the iDAb binds) further defining their binding site on LMO2 as the iDAb interaction region.

Synthesis

[0651] Several methods for the chemical synthesis of heterocyclic carboxamide compounds of the present application are described herein. These and/or other well-known methods may be modified and/or adapted in various ways in order to facilitate the synthesis of additional compounds within the scope of the present application and claims. Such alternative methods and modifications should be understood as being within the spirit and scope of this application and claims. Accordingly, it should be understood that the methods set forth in the following descriptions, schemes and examples are intended for illustrative purposes and are not to be construed as limiting the scope of the disclosure.

[0652] All solvents and reagents were used as supplied (analytical or HPLC grade) without prior purification. Water was purified by an Elix® UV-10 system. Thin layer chromatography was performed on aluminium plates coated with 60 F254 silica. Plates were visualised using UV light (254 nm) or 1% aq. KMnO4. Flash column chromatography was performed on Kieselgel 60M silica in a glass column. NMR spectra were recorded on Bruker Avance spectrometers (AVII400, AVIII 400, AVIIIHD 600 or AVIII 700) in the deuterated solvent stated. The field was locked by external referencing to the relevant deuteron resonance. Chemical shifts (δ) are reported in parts per million (ppm) referenced to the solvent peak. .sup.1H spectra reported to two decimal places, and .sup.13C spectra reported to one decimal place, and coupling constants (J) are quoted in Hz (reported to one decimal place). The multiplicity of each signal is indicated by: s (singlet); br. s (broad singlet); d (doublet); t (triplet); q (quartet); dd (doublet of doublets); td (triplet of doublets); qt (quartet of triplets); or m (multiplet). Low-resolution mass spectra (LRMS) were recorded on an Agilent 6120 spectrometer from solutions of MeOH. Accurate mass measurements were run on either a Bruker MicroTOF internally calibrated with polyalanine, or a Micromass GCT instrument fitted with a Scientific Glass Instruments BPX5 column (15 m×0.25 mm) using amyl acetate as a lock mass, by the mass spectrometry department of the Chemistry Research Laboratory, University of Oxford, UK.; m/z values are reported in Daltons.

General Procedure A: Synthesis of Substituted Imidazolidinones (n=1) and Pyrimidinones (n=2)

[0653] ##STR00030##

[0654] The requisite cyclic urea (1.0 eq.) was dissolved in THF (10 mL) and cooled to 0° C. before portionwise addition of NaH (60% suspension in oil, 1.0 eq.). After 30 min, the suspension was treated with the requisite substituted benzylbromide/chloride (0.9 eq.). The resulting mixture was stirred for 2 h (monitoring by LC-MS and TLC) and warmed to room temperature, before addition of NH.sub.4Cl (sat. aq. sol., 20 mL) and EtOAc (20 mL). The aqueous layer was extracted with EtOAc (2×20 mL), the combined organic phase was washed with water (20 mL), brine (20 mL of saturated aqueous solution of sodium chloride), dried (Na.sub.2SO.sub.4), filtered and concentrated in vacuo (use of a rotary evaporator attached to a diaphragm pump). The crude material was purified on silica gel (5% MeOH in CH.sub.2Cl.sub.2) and the desired compound was obtained as a colourless oil that solidified on standing.

General Procedure B: Synthesis of Substituted Piperazines

[0655] ##STR00031##

[0656] Boc-piperazine (1.1 eq.) was dissolved in MeCN (5 mL) before sequential addition of K.sub.2CO.sub.3 (2.5 eq.) followed by the requisite substituted benzylbromide/chloride (1.0 eq.). The resulting mixture was stirred for 18 h before addition of H.sub.2O/brine (1:1, 20 mL) and EtOAc (20 mL). The aqueous layer was extracted with EtOAc (20 mL), the combined organic phase was dried (Na.sub.2SO.sub.4), filtered and concentrated in vacuo. The crude material was purified on silica gel (10% EtOAc in pentane) and the title compound was obtained as a colourless oil that solidified on standing. The product was dissolved in CH.sub.2Cl.sub.2 (5 mL) before addition of TFA (500 μL). The resulting solution was stirred for 18 h at room temperature and concentrated in vacuo. The compound was used in the next step without further purification.

General Procedure C: Palladium Coupling of Substituted Cyclic Ureas to Chlorooxazoles (X═O) and Chlorothiazoles (X═S)

[0657] ##STR00032##

[0658] The requisite substituted cyclic urea (1.1 eq.), Cs.sub.2CO.sub.3 (3.0 eq.), the ester substituted chloro heterocycle of choice (1.0 eq.) and X-Phos (10% mol) were added sequentially to a microwave vial followed by degassed 1,4-dioxane (2 mL). The suspension was degassed for 5 min with nitrogen before addition of Pd(OAc).sub.2 (5% mol); it was degassed further with nitrogen for another 5 min before the vessel was sealed and the suspension heated to 95° C. for 24 h. The reaction was cooled down, diluted with EtOAc (10 mL) and washed with brine/water (1:1, 10 mL). The organic phase was dried (Na.sub.2SO.sub.4), filtered and concentrated in vacuo. The crude material was purified on silica gel to give the desired compound.

General Procedure D: Synthesis of Substituted Pyperazyl Hererocycles

[0659] ##STR00033##

[0660] The substituted piperazine (1.2 eq.) was dissolved in 1,4-dioxane/N,N-diisopropylethylamine (4:1, 8 mL) before addition of the requisite chloro-heterocycle (1.0 eq.). The solution was stirred at 60° C. for 48 h, cooled to room temperature, diluted with EtOAc (30 mL) and washed with H.sub.2O/brine (1:1, 20 mL). The organic phase was dried (Na.sub.2SO.sub.4), filtered and concentrated in vacuo. The crude material then purified on silica gel to give the title compound.

General Procedure E: Basic Hydrolysis of the Ester Moiety and Subsequent HATU Amide Coupling

[0661] ##STR00034##

[0662] The ester (1.0 eq.) was dissolved in THF/MeOH (4:1) before addition of NaOH (1M aq.) until pH>8. The resulting reaction was stirred for 16 h at room temperature, after which it was acidified with HCl (1M aq.) until pH<5. The solution was concentrated in vacuo and the obtained carboxylic acid was used in the next step without further purification. The acid was dissolved in DMF (2 mL) before sequential addition of N,N-diisopropylethylamine (3.0 eq.), the requisite amine (1.2 eq.) and HATU (1.4 eq.). The resulting solution was stirred for 18 h, diluted with EtOAc (10 mL) and washed with brine/water (1:1, 3×50 mL). The organic phase was dried (Na.sub.2SO.sub.4), filtered and concentrated in vacuo. The crude material was purified on silica gel to give the title compound.

EXPERIMENTAL DATA

Abd-L5:1-benzylimidazolidin-2-one (1)

[0663] ##STR00035##

[0664] Following General Procedure A using 2-imidazolidinone (1.00 g, 11.6 mmol, 1.0 eq.) and benzyl bromide (1.25 mL, 10.4 mmol, 0.9 eq.), the title compound 1 was obtained as a colourless oil that solidified on standing (858 mg, 42%), after purification on silica gel (5% MeOH in CH.sub.2Cl.sub.2).

[0665] m/z LRMS (ESI.sup.+): 177 (100%) [M+H].sup.+.

ethyl 2-(3-benzyl-2-oxoimidazolidin-1-yl)thiazole-5-carboxylate (2)

[0666] ##STR00036##

[0667] Following General Procedure C using cyclic urea 1 (102 mg, 0.578 mmol, 1.1 eq.) and ethyl 2-chlorothiazole-5-carboxylate (100 mg, 0.525 mmol, 1.0 eq.), the title compound 2 was obtained as a yellow oil (125 mg, 72%), after purification on silica gel (3% MeOH in CH.sub.2Cl.sub.2). m/z LRMS (ESI.sup.+): 332 (100%) [M+H].sup.+.

2-(3-benzyl-2-oxoimidazolidin-1-yl)-N-(2-(methylsulfonamido)phenyl)thiazole-5-carboxamide (3) (Abd-L5)

[0668] ##STR00037##

[0669] Following General Procedure E using ester 2 and N-(2-aminophenyl)methanesulfonamide, the title product was obtained as a thick yellow oil that solidified on standing (32 mg, 41%), after two purifications on silica gel (5% MeOH in CH.sub.2Cl.sub.2). m/z LRMS (ESI.sup.+): 472 (100%) [M+H].sup.+. HRMS (ESI.sup.+): calc. for C.sub.21H.sub.22N.sub.5O.sub.4.sup.32S.sub.2[M+H].sup.+ 472.1113. found 472.1120.

Abd-L6: ethyl 2-(3-benzyl-2-oxoimidazolidin-1-yl)oxazole-4-carboxylate (4)

[0670] ##STR00038##

[0671] Following General Procedure C using cyclic urea 1 (110 mg, 0.629 mmol, 1.1 eq.) and ethyl 2-chlorooxazole-4-carboxylate (100 mg, 0.571 mmol, 1.0 eq.), the title compound 4 was obtained as a yellow oil (106 mg, 59%), after purification on silica gel (3% MeOH in CH.sub.2Cl.sub.2). m/z LRMS (ESI.sup.+): 316 (100%) [M+H].sup.+. 2-(3-benzyl-2-oxoimidazolidin-1-yl)-N-(4-phenoxyphenyl)oxazole-4-carboxamide (5) (Abd-L6)

##STR00039##

[0672] Following General Procedure E using ester 4 and 4-phenoxyaniline, the title product was obtained as a thick yellow oil that solidified on standing (27 mg, 52%), after purification on silica gel (5% MeOH in CH.sub.2Cl.sub.2). m/z LRMS (ESI.sup.+): 455 (100%) [M+H].sup.+. HRMS (ESI.sup.+): calc. for C.sub.26H.sub.23N.sub.4O.sub.4 [M+H].sup.+ 455.1719. found 455.1720.

Abd-L7:1-(3-chlorobenzyl)imidazolidin-2-one (6)

[0673] ##STR00040##

[0674] Following General Procedure A using 2-imidazolidinone (500 mg, 5.80 mmol, 1.0 eq.) and 3-chlorobenzyl bromide (685 μL, 5.20 mmol, 0.9 eq.), the title compound 6 was obtained as a colourless oil that solidified on standing (463 mg, 38%), after purification on silica gel (3% MecOH in CH.sub.2Cl.sub.2). m/z LRMS (ESI.sup.+): 211 (100%) [M+H].sup.+.

ethyl 2-(3-(3-chlorobenzyl)-2-oxoimidazolidin-1-yl)oxazole-4-carboxylate (7)

[0675] ##STR00041##

[0676] Following General Procedure C using cyclic urea 6 (132 mg, 0.629 mmol, 1.1 eq.) and ethyl 2-chlorooxazole-4-carboxylate (100 mg, 0.571 mmol, 1.0 eq.), the title compound 7 was obtained as a yellow oil (117 mg, 54%), after purification on silica gel (3% MeOH in CH.sub.2Cl.sub.2). m/z LRMS (ESI.sup.+): 350 (100%) [M+H].sup.+.

N-(4-(1H-pyrrol-1-yl)phenyl)-2-(3-(3-chlorobenzyl)-2-oxoimidazolidin-1-yl)oxazole-4-carboxamide (8) (Abd-L7)

[0677] ##STR00042##

[0678] Following General Procedure E using ester 7 and 4-pyrroleaniline, the title product was obtained as a thick yellow oil that solidified on standing (27 mg, 52%), after purification on silica gel (7% MeOH in CH.sub.2Cl.sub.2).

[0679] m/z LRMS (ESI.sup.+): 462 (100%) [M+H].sup.+. HRMS (ESI.sup.+): calc. for C.sub.24H.sub.21.sup.35ClN.sub.5O.sub.3[M+H].sup.+ 462.1333. found 462.1332.

Abd-L8:1-(4-chlorobenzyl)imidazolidin-2-one (9)

[0680] ##STR00043##

[0681] Following General Procedure A using 2-imidazolidinone (500 mg, 5.80 mmol, 1.0 eq.) and 4-chlorobenzyl bromide (1.07 g, 5.20 mmol, 0.9 eq.), the title compound was obtained as a colourless oil that solidified on standing (463 mg, 38%), after purification on silica gel (5% MeOH in CH.sub.2Cl.sub.2). m/z LRMS (ESI.sup.+): 211 (100%) [M+H].sup.+.

ethyl 2-(3-(4-chlorobenzyl)-2-oxoimidazolidin-1-yl)oxazole-5-carboxylate (10)

[0682] ##STR00044##

[0683] Following General Procedure C using cyclic urea 9 (132 mg, 0.629 mmol, 1.1 eq.) and ethyl 2-chlorooxazole-5-carboxylate (100 mg, 0.571 mmol, 1.0 eq.), the title compound 10 was obtained as a yellow oil (122 mg, 61%), after purification on silica gel (3% MeOH in CH.sub.2Cl.sub.2).

[0684] m/z LRMS (ESI.sup.+): 350 (100%) [M+H].sup.+.

2-(3-(4-chlorobenzyl)-2-oxoimidazolidin-1-yl)-N-(3,4-dimethoxyphenyl)oxazole-5-carboxamide (11) (Abd-L8)

[0685] ##STR00045##

[0686] Following General Procedure E using ester 10 and 3,4-dimethoxyaniline, the title product was obtained as a thick brown oil that solidified on standing (22 mg, 54%), after purification on silica gel (5% MeOH in CH.sub.2Cl.sub.2). m/z LRMS (ESI.sup.+): 457 (100%) [M+H].sup.+. HRMS (ESI.sup.+): calc. for C.sub.22H.sub.2235ClN.sub.4O.sub.5[M+H].sup.+ 457.1279. found 457.1282.

Abd-L9:1-(3-methoxybenzyl)imidazolidin-2-one (12)

[0687] ##STR00046##

[0688] Following General Procedure A using 2-imidazolidinone (1.00 g, 11.6 mmol, 1.0 eq.) and 3-methoxybenzylbromide (1.47 mL, 10.5 mmol, 0.9 eq.), the title compound was obtained as a colourless oil that solidified on standing (1.02 g, 47%), after purification on silica gel (5% MeOH in CH.sub.2Cl.sub.2).

[0689] m/z LRMS (ESI.sup.+): 207 (100%) [M+H].sup.+.

ethyl 2-(3-(3-methoxybenzyl)-2-oxoimidazolidin-1-yl)oxazole-4-carboxylate (13)

[0690] ##STR00047##

[0691] Following General Procedure C using cyclic urea 12 (82 mg, 0.396 mmol, 1.1 eq.) and ethyl 2-chlorooxazole-4-carboxylate (64 mg, 0.360 mmol, 1.0 eq.), the title compound 13 was obtained as a yellow oil (88 mg, 71%), after purification on silica gel (3% MeOH in CH.sub.2Cl.sub.2).

[0692] m/z LRMS (ESI.sup.+): 346 (100%) [M+H].sup.+.

N-(4-(benzyloxy)phenyl)-2-(3-(3-methoxybenzyl)-2-oxoimidazolidin-1-yl)oxazole-4-carboxamide (14) (Abd-L9)

[0693] ##STR00048##

[0694] Following General Procedure E using ester 13 and 4-(benzyloxy)aniline, the title product was obtained as a dark yellow oil that solidified on standing (137 mg, 62%), after purification on silica gel (5% MeOH in CH.sub.2Cl.sub.2).

[0695] m/z LRMS (ESI.sup.+): 499 (100%) [M+H].sup.+. HRMS (ESI.sup.+): calc. for C.sub.28H.sub.27N.sub.4O.sub.5 [M+H].sup.+ 499.1981. found 499.1978.

Abd-L10: 3-((2-oxoimidazolidin-1-yl)methyl)benzonitrile (15)

[0696] ##STR00049##

[0697] Following General Procedure A using 2-imidazolidinone (500 mg, 5.80 mmol, 1.0 eq.) and 3-cyanobenzyl bromide (1.01 g, 5.20 mmol, 0.9 eq.), the title compound was obtained as a colourless oil that solidified on standing (463 mg, 41%), after purification on silica gel (5% MeOH in CH.sub.2Cl.sub.2).

[0698] m/z LRMS (ESI.sup.+): 202 (100%) [M+H].

ethyl 2-(3-(3-cyanobenzyl)-2-oxoimidazolidin-1-yl)oxazole-4-carboxylate (16)

[0699] ##STR00050##

[0700] Following General Procedure C using cyclic urea 15 (126 mg, 0.629 mmol, 1.1 eq.) and ethyl 2-chlorooxazole-4-carboxylate (100 mg, 0.571 mmol, 1.0 eq.), the title compound 16 was obtained as a yellow oil (109 mg, 56%), after purification on silica gel (3% MeOH in CH.sub.2Cl.sub.2). m/z LRMS (ESI.sup.+): 341 (100%) [M+H].sup.+.

N-(4-(1H-pyrrol-1-yl)phenyl)-2-(3-(3-cyanobenzyl)-2-oxoimidazolidin-1-yl)oxazole-4-carboxamide (17) (Abd-10)

[0701] ##STR00051##

[0702] Following General Procedure E using ester 16 and 4-pyrroleaniline, the title product was obtained as a thick yellow oil that solidified on standing (27 mg, 52%), after purification on silica gel (7% MeOH in CH.sub.2Cl.sub.2).

[0703] m/z LRMS (ESI.sup.+): 453 (100%) [M+H].sup.+. HRMS (ESI.sup.+): calc. for C.sub.25H.sub.21N.sub.6O.sub.3 [M+H].sup.+ 453.1675. found 453.1674.

Abd-L11: 2-(3-(4-chlorobenzyl)-2-oxoimidazolidin-1-yl)-N-phenethyloxazole-5-carboxamide (18) (Abd-L11)

[0704] ##STR00052##

[0705] Following General Procedure E using ester 10 and 2-phenylethan-1-amine, the title product was obtained as a thick yellow oil that solidified on standing (18 mg, 49%), after purification on silica gel (5% MeOH in CH.sub.2Cl.sub.2).

[0706] m/z LRMS (ESI.sup.+): 425 (100%) [M+H].sup.+. HRMS (ESI.sup.+): calc. for C.sub.22H.sub.22.sup.35ClN.sub.4O.sub.3[M+H].sup.+ 425.1380. found 425.1382.

Abd-L12: 1-(2-chlorobenzyl)imidazolidin-2-one (19)

[0707] ##STR00053##

[0708] Following General Procedure A using 2-imidazolidinone (500 mg, 5.80 mmol, 1.0 eq.) and 2-chlorobenzyl bromide (680 μL, 5.20 mmol, 0.9 eq.), the title compound was obtained as a colourless oil that solidified on standing (463 mg, 38%), after purification on silica gel (5% MeOH in CH.sub.2Cl.sub.2).

[0709] m/z LRMS (ESI.sup.+): 211 (100%) [M+H].sup.+.

ethyl 2-(3-(2-chlorobenzyl)-2-oxoimidazolidin-1-yl)oxazole-4-carboxylate (20)

[0710] ##STR00054##

[0711] Following General Procedure C using cyclic urea 19 (132 mg, 0.629 mmol, 1.1 eq.) and ethyl 2-chlorooxazole-4-carboxylate (100 mg, 0.571 mmol, 1.0 eq.), the title compound 20 was obtained as a yellow oil (98 mg, 49%), after purification on silica gel (3% MeOH in CH.sub.2Cl.sub.2).

[0712] m/z LRMS (ESI.sup.+): 350 (100%) [M+H].sup.+.

2-(3-(2-chlorobenzyl)-2-oxoimidazolidin-1-yl)-N-(4-phenoxyphenyl)oxazole-4-carboxamide (21) (Abd-L12)

[0713] ##STR00055##

[0714] Following General Procedure E using ester 20 and 4-phenoxyaniline, the title product was obtained as a dark yellow oil that solidified on standing (32 mg, 61%), after purification on silica gel (5% MeOH in CH.sub.2Cl.sub.2).

[0715] m/z LRMS (ESI.sup.+): 489 (100%) [M+H].sup.+. HRMS (ESI.sup.+): calc. for C.sub.26H.sub.22.sup.35ClN.sub.4O.sub.4[M+H].sup.+ 489.1330. found 489.1332.

Abd-L13: 2-(3-benzyl-2-oxoimidazolidin-1-yl)-N-(4-phenoxybenzyl)oxazole-4-carboxamide (22) (Abd-L13)

[0716] ##STR00056##

[0717] Following General Procedure E using ester 4 and (4-phenoxyphenyl)methanamine, the title product was obtained as a thick yellow oil that solidified on standing (17 mg, 42%), after purification on silica gel (5% MeOH in CH.sub.2Cl.sub.2).

[0718] m/z LRMS (ESI.sup.+): 469 (100%) [M+H].sup.+. HRMS (ESI.sup.+): calc. for C.sub.27H.sub.25N.sub.4O.sub.4 [M+H].sup.+ 469.1876. found 469.1874.

Abd-L14: 2-(3-(2-chlorobenzyl)-2-oxoimidazolidin-1-yl)-N-(4-phenoxybenzyl)oxazole-4-carboxamide (23) (Abd-L14)

[0719] ##STR00057##

[0720] Following General Procedure E using ester 20 and (4-phenoxyphenyl)methanamine, the title product was obtained as a dark orange oil that solidified on standing (21 mg, 53%), after purification on silica gel (5% MeOH in CH.sub.2Cl.sub.2).

[0721] m/z LRMS (ESI.sup.+): 503 (100%) [M+H].sup.+. HRMS (ESI.sup.+): calc. for C.sub.27H.sub.2435ClN.sub.4O.sub.4[M+H].sup.+ 503.1486. found 503.1484.

Abd-L15: 1-(3-methoxybenzyl)piperazine (24)

[0722] ##STR00058##

[0723] Following General Procedure B using Boc-piperazine (1.00 g, 5.36 mmol, 1.1 eq.), K.sub.2CO.sub.3 (1.70 g, 12.2 mmol, 2.5 eq.) and 3-methoxybenzylbromide (683 μL, 4.88 mmol, 1.0 eq.) the title compound was obtained as a colourless oil that solidified on standing after purification on silica gel (10% EtOAc in pentane).

[0724] m/z LRMS (ESI.sup.+): 307 (100%) [M+H].sup.+.

[0725] The product was dissolved in CH.sub.2Cl.sub.2 (5 mL) before addition of TFA (500 μL). The resulting solution was stirred for 18 h at room temperature and concentrated in vacuo. The title compound was used in the next step without further purification (987 mg, 98% over two steps).

[0726] m/z LRMS (ESI.sup.+): 207 (100%) [M+H].sup.+.

ethyl 2-(4-(3-methoxybenzyl)piperazin-1-yl)oxazole-4-carboxylate (25)

[0727] ##STR00059##

[0728] Following General Procedure D using piperazine 24 (150 mg, 0.728 mmol, 1.2 eq.) and ethyl 2-chlorooxazole-4-carboxylate (116 mg, 0.661 mmol, 1.0 eq.), the title compound 25 was obtained as a yellow oil (163 mg, 71%), after purification on silica gel (4% MeOH in CH.sub.2Cl.sub.2). m/z LRMS (ESI.sup.+): 346 (100%) [M+H].sup.+.

N-(4-(1H-pyrrol-1-yl)phenyl)-2-(4-(3-methoxybenzyl)piperazin-1-yl)oxazole-4-carboxamide (26)(Abd-L15)

[0729] ##STR00060##

[0730] Following General Procedure E using ester 25 and 4-pyrroleaniline, the title product was obtained as a light brown powder (52 mg, 74%), after purification on silica gel (7% MeOH in CH.sub.2Cl.sub.2).

[0731] m/z LRMS (ESI.sup.+): 458 (100%) [M+H].sup.+. HRMS (ESI.sup.+): calc. for C.sub.26H.sub.28N.sub.5O.sub.3 [M+H].sup.+ 458.2192. found 458.2192.

N-(4-(benzyloxy)phenyl)-2-(4-(3-methoxybenzyl)piperazin-1-yl)oxazole-4-carboxamide (27) (Abd-L16)

[0732] ##STR00061##

[0733] Following General Procedure E using ester 26 and 4-(benzyloxy)aniline, the title product was obtained as a yellow oil that solidified on standing (37 mg, 69%), after purification on silica gel (5% MeOH in CH.sub.2Cl.sub.2). m/z LRMS (ESI.sup.+): 499 (100%) [M+H].sup.+. HRMS (ESI.sup.+): calc. for C.sub.29H.sub.31N.sub.4O.sub.4 [M+H].sup.+ 499.2345. found 499.2345.

Abd-L17 ethyl 2-(4-(3-methoxybenzyl)piperazin-1-yl)thiazole-4-carboxylate (28)

[0734] ##STR00062##

[0735] Following General Procedure D using piperazine 24 (150 mg, 0.728 mmol, 1.2 eq.) and ethyl 2-chlorothiazole-4-carboxylate (126 mg, 0.661 mmol, 1.0 eq.), the title compound 28 was obtained as a yellow oil (212 mg, 89%), after purification on silica gel (4% MeOH in CH.sub.2Cl.sub.2). m/z LRMS (ESI.sup.+): 362 (100%) [M+H].sup.+.

N-(4-(benzyloxy)phenyl)-2-(4-(3-methoxybenzyl)piperazin-1-yl)thiazole-4-carboxamide (29) (Abd-L17)

[0736] ##STR00063##

[0737] Following General Procedure E using ester 28 and 4-(benzyloxy)aniline, the title product was obtained as a yellow oil that solidified on standing (42 mg, 78%), after purification on silica gel (4% MeOH in CH.sub.2Cl.sub.2). m/z LRMS (ESI.sup.+): 515 (100%) [M+H].sup.+. HRMS (ESI.sup.+): calc. for C.sub.29H.sub.31N.sub.4O.sub.3.sup.32S [M+H].sup.+ 515.2117. found 515.2116.

Abd-L18: N-(4-(1H-pyrrol-1-yl)phenyl)-2-(4-(3-methoxybenzyl)piperazin-1-yl)thiazole-4-carboxamide (30) (Abd-L18)

[0738] ##STR00064##

[0739] Following General Procedure E using ester 28 and 4-pyrroleaniline, the title product was obtained as a beige solid (37 mg, 75%), after purification on silica gel (4% MeOH in CH.sub.2Cl.sub.2).

[0740] m/z LRMS (ESI.sup.+): 474 (100%) [M+H].sup.+. HRMS (ESI.sup.+): calc. for C.sub.29H.sub.31N.sub.4O.sub.3.sup.32S [M+H].sup.+ 474.1964. found 474.1965.

Abd-L19: 1-(4-methoxybenzyl)piperazine (31)

[0741] ##STR00065##

[0742] Following General Procedure B using Boc-piperazine (1.00 g, 5.36 mmol, 1.1 eq.), K.sub.2CO.sub.3 (1.70 g, 12.2 mmol, 2.5 eq.) and 4-methoxybenzylbromide (700 μL, 4.88 mmol, 1.0 eq.) the title compound was obtained as a colourless oil that solidified on standing after purification on silica gel (10% EtOAc in pentane). m/z LRMS (ESI.sup.+): 307 (100%) [M+H].sup.+. The product was dissolved in CH.sub.2Cl.sub.2 (5 mL) before addition of TFA (500 μL). The resulting solution was stirred for 18 h at room temperature and concentrated in vacuo. The title compound was used in the next step without further purification (891 mg, 89% over two steps).

[0743] m/z LRMS (ESI.sup.+): 207 (100%) [M+H].sup.+.

ethyl 2-(4-(4-methoxybenzyl)piperazin-1-yl)oxazole-4-carboxylate (32)

[0744] ##STR00066##

[0745] Following General Procedure D using piperazine 31 (150 mg, 0.728 mmol, 1.2 eq.) and ethyl 2-chlorooxazole-4-carboxylate (116 mg, 0.661 mmol, 1.0 eq.), the title compound 32 was obtained as a yellow oil (163 mg, 71%), after purification on silica gel (4% MeOH in CH.sub.2Cl.sub.2). m/z LRMS (ESI.sup.+): 346 (100%) [M+H].sup.+.

N-(4-(1H-pyrrol-1-yl)phenyl)-2-(4-(4-methoxybenzyl)piperazin-1-yl)oxazole-4-carboxamide (33) (Abd-L19)

[0746] ##STR00067##

[0747] Following General Procedure E using ester 32 and 4-pyrroleaniline, the title product was obtained as a brown powder (24 mg, 67%), after purification on silica gel (7% MeOH in CH.sub.2Cl.sub.2).

[0748] m/z LRMS (ESI.sup.+): 458 (100%) [M+H].sup.+. HRMS (ESI.sup.+): calc. for C.sub.26H.sub.28N.sub.5O.sub.3 [M+H].sup.+ 458.2192. found 458.2192.

Abd-L20 1-(2-methoxybenzyl)piperazine (34)

[0749] ##STR00068##

[0750] Following General Procedure B using Boc-piperazine (200 mg, 1.07 mmol, 1.1 eq.), K.sub.2CO.sub.3 (370 mg, 2.68 mmol, 2.5 eq.) and 2-methoxybenzylbromide (195 mg, 0.972 mmol, 1.0 eq.) the title compound was obtained as a colourless oil that solidified on standing after purification on silica gel (10% EtOAc in pentane).

[0751] m/z LRMS (ESI.sup.+): 307 (100%) [M+H].sup.+.

[0752] The product was dissolved in CH.sub.2Cl.sub.2 (5 mL) before addition of TFA (500 μL). The resulting solution was stirred for 18 h at room temperature and concentrated in vacuo. The title compound was used in the next step without further purification (164 mg, 82% over two steps).

[0753] m/z LRMS (ESI.sup.+): 207 (100%) [M+H].sup.+.

ethyl 2-(4-(2-methoxybenzyl)piperazin-1-yl)thiazole-4-carboxylate (35)

[0754] ##STR00069##

[0755] Following General Procedure D using piperazine 34 (100 mg, 0.728 mmol, 1.2 eq.) and ethyl 2-chlorothiazole-4-carboxylate (126 mg, 0.661 mmol, 1.0 eq.), the title compound 28 was obtained as a yellow oil (216 mg, 92%), after purification on silica gel (4% MeOH in CH.sub.2Cl.sub.2). m/z LRMS (ESI.sup.+): 362 (100%) [M+H].sup.+.

N-(4-(1H-pyrrol-1-yl)phenyl)-2-(4-(2-methoxybenzyl)piperazin-1-yl)thiazole-4-carboxamide (36) (Abd-L20)

[0756] ##STR00070##

[0757] Following General Procedure E using ester 35 and 4-pyrroleaniline, the title product was obtained as a yellow oil that solidified on standing (28 mg, 72%), after purification on silica gel (6% MeOH in CH.sub.2Cl.sub.2).

[0758] m/z LRMS (ESI.sup.+): 474 (100%) [M+H].sup.+. HRMS (ESI.sup.+): calc. for C.sub.26H.sub.28N.sub.5O.sub.3 [M+H].sup.+ 474.1864. found 474.1863.

2-(4-(3-methoxybenzyl)piperazin-1-yl)-N-(4-(trifluoromethoxy)phenyl)thiazole-4-carboxamide (37) (Abd-L21)

[0759] ##STR00071##

[0760] Following General Procedure E using ester 28 and 4-(trifluoromethoxy)aniline, the title product was obtained as a dark yellow oil that solidified on standing (28 mg, 76%), after purification on silica gel (4% MeOH in CH.sub.2Cl.sub.2).

[0761] m/z LRMS (ESI.sup.+): 493 (100%) [M+H].sup.+. HRMS (ESI.sup.+): calc. for C.sub.23H.sub.24F.sub.3N.sub.4O.sub.3.sup.32S [M+H].sup.+ 493.1521. found 493.1520.

2-(4-(3-methoxybenzyl)piperazin-1-yl)-N-(6-methoxypyridin-3-yl)thiazole-4-carboxamide (38) (Abd-L22)

[0762] ##STR00072##

[0763] Following General Procedure E using ester 28 and 6-methoxypyridin-3-amine, the title product was obtained as a red oil (23 mg, 71%), after purification on silica gel (4% MeOH in CH.sub.2Cl.sub.2).

[0764] m/z LRMS (ESI.sup.+): 440 (100%) [M+H].sup.+. HRMS (ESI.sup.+): calc. for C.sub.22H.sub.25N.sub.5O.sub.3.sup.32S [M+H].sup.+ 440.1756. found 440.1754.

2-(4-(3-methoxybenzyl)piperazin-1-yl)-N-(2-methoxypyrimidin-5-yl)thiazole-4-carboxamide (39) (Abd-L23)

[0765] ##STR00073##

[0766] Following General Procedure E using ester 28 and 6-methoxypyridin-3-amine, the title product was obtained as a light yellow oil (23 mg, 71%), after purification on silica gel (4% MeOH in CH.sub.2Cl.sub.2).

[0767] m/z LRMS (ESI.sup.+): 441 (100%) [M+H].sup.+. HRMS (ESI.sup.+): calc. for C.sub.21H.sub.2N.sub.6O.sub.3.sup.32S [M+H].sup.+ 441.1709. found 441.1710.

Abd-L24: 1-(3-methoxybenzyl)tetrahydropyrimidin-2(1H)-one (40)

[0768] ##STR00074##

[0769] Following General Procedure A using tetrahydro-2(1H)-pyrimidinone (500 mg, 5.00 mmol, 1.0 eq.), NaH (60% suspension in oil, 134 mg, 5.00 mmol, 1.0 eq.) and 3-methoxybenzylbromide (630 μL, 4.50 mmol, 0.9 eq.), the title compound was obtained as a colourless oil that solidified on standing (564 mg, 57%), after purification on silica gel (5% MeOH in CH.sub.2Cl.sub.2).

[0770] m/z LRMS (ESI.sup.+): 221 (100%) [M+H].sup.+.

ethyl 2-(3-(3-methoxybenzyl)-2-oxotetrahydropyrimidin-1(2H)-yl)oxazole-4-carboxylate (41)

[0771] ##STR00075##

[0772] Following General Procedure C using cyclic urea 40 (132 mg, 0.375 mmol, 1.1 eq.) and ethyl 2-chlorooxazole-4-carboxylate (60 mg, 0.341 mmol, 1.0 eq.), the title compound 41 was obtained as a yellow oil (51 mg, 42%), after purification on silica gel (3% MeOH in CH.sub.2Cl.sub.2). m/z LRMS (ESI.sup.+): 360 (100%) [M+H].sup.+.

N-(4-(1H-pyrrol-1-yl)phenyl)-2-(3-(3-methoxybenzyl)-2-oxotetrahydropyrimidin-1(2H)-yl)oxazole-4-carboxamide (42) (Abd-L24)

[0773] ##STR00076##

[0774] Following General Procedure E using ester 41 and 4-pyrroleaniline, the title product was obtained as a dark yellow oil (12 mg, 41%), after two purifications on silica gel (5% MeOH in CH.sub.2Cl.sub.2).

[0775] m/z LRMS (ESI.sup.+): 472 (100%) [M+H].sup.+. HRMS (ESI.sup.+): calc. for C.sub.26H.sub.26N.sub.5O.sub.4[M+H].sup.+ 472.1985. found 472.1986.

Abd-L25: ethyl 2-(4-(3-methoxybenzyl)piperazin-1-yl)pyrimidine-4-carboxylate (43)

[0776] ##STR00077##

[0777] Following General Procedure D using piperazine 24 (133 mg, 0.645 mmol, 1.2 eq.) and ethyl 2-chloropyrimidine-4-carboxylate (100 mg, 0.538 mmol, 1.0 eq.), the title compound 43 was obtained as a pale yellow solid (178 mg, 93%), after purification on silica gel (4% MeOH in CH.sub.2Cl.sub.2). m/z LRMS (ESI.sup.+): 357 (100%) [M+H].sup.+.

N-(4-(1H-pyrrol-1-yl)phenyl)-2-(4-(3-methoxybenzyl)piperazin-1-yl)pyrimidine-4-carboxamide (44) (Abd-L25)

[0778] ##STR00078##

[0779] Following General Procedure E using ester 43 and 4-pyrroleaniline, the title product was obtained as a beige solid (35 mg, 89%), after purification on silica gel (4% MeOH in CH.sub.2Cl.sub.2). m/z LRMS (ESI.sup.+): 469 (100%) [M+H].sup.+. HRMS (ESI.sup.+): calc. for C.sub.27H.sub.29N.sub.6O.sub.2 [M+H].sup.+ 469.2352. found 469.2353.

Abd-L26 (PAL compound): phenyl(4-(piperazin-1-ylmethyl)phenyl)methanone (45)

[0780] ##STR00079##

[0781] Following General Procedure B using Boc-piperazine (1.00 g, 5.36 mmol, 1.1 eq.), K.sub.2CO.sub.3 (1.70 g, 12.2 mmol, 2.5 eq.) and 4-bromomethylbenzophenone (1.34 g, 4.88 mmol, 1.0 eq.) the title compound was obtained as a colourless oil that solidified on standing after purification on silica gel (8% EtOAc in pentane). m/z LRMS (ESI.sup.+): 381 (100%) [M+H].sup.+. The product was dissolved in CH.sub.2Cl.sub.2 (10 mL) before addition of TFA (1 mL). The resulting solution was stirred for 18 h at room temperature and concentrated in vacuo. The title compound was used in the next step without further purification (1.32 g, 88% over two steps).

[0782] m/z LRMS (ESI.sup.+): 281 (100%) [M+H].sup.+.

Ethyl 2-(4-(4-benzoylbenzyl)piperazin-1-yl)oxazole-4-carboxylate (46)

[0783] ##STR00080##

[0784] Following General Procedure D using piperazine 45 (580 mg, 2.07 mmol, 1.2 eq.) and ethyl 2-chlorooxazole-4-carboxylate (326 mg, 1.86 mmol, 1.0 eq.), the title compound 46 was obtained as a yellow oil (630 mg, 81%), after purification on silica gel (3% MeOH in CH.sub.2Cl.sub.2).

[0785] m/z LRMS (ESI.sup.+): 420 (100%) [M+H].sup.+.

N-(4-aminophenyl)-2-(4-(4-benzoylbenzyl)piperazin-1-yl)oxazole-4-carboxamide (47)

[0786] ##STR00081##

[0787] Following General Procedure E using ester 46 and tert-butyl-4-aminophenylcarbamate, the Boc protected product was obtained as a pale yellow oil that solidified on standing (82 mg, 73%), after purification on silica gel (25% EtOAc in CH.sub.2Cl.sub.2). m/z LRMS (ESI.sup.+): 582 (100%) [M+H].sup.+. The product was dissolved in CH.sub.2Cl.sub.2 (2 mL) before addition of TFA (150 μL). The resulting solution was stirred for 18 h at room temperature and concentrated in vacuo. The title compound was used in the next step without further purification (66 mg, 99%).

[0788] m/z LRMS (ESI.sup.+): 482 (100%) [M+H].sup.+.

benzyl(2-(2-(2-((4-(2-(4-(4-benzoylbenzyl)piperazin-1-yl)oxazole-4-carboxamido)phenyl)amino)-2-oxoethoxy)ethoxy)ethyl)carbamate (48)

[0789] ##STR00082##

[0790] Aniline 47 (60 mg, 0.125 mmol, 1.0 eq.) was dissolved in DMF (2 mL) before sequential addition of N,N-diisopropylethylamine (65 μL, 0.375 mmol, 3.0 eq.), 3-oxo-1-phenyl-2,7,10-trioxa-4-azadodecan-12-oic acid (45 mg, 0.150 mmol, 1.2 eq.) and HATU (67 mg, 0.175 mmol, 1.4 eq.). The resulting solution was stirred for 18 h, diluted with EtOAc (10 mL) and washed with brine/water (1:1, 3×50 mL). The organic phase was dried (Na.sub.2SO.sub.4), filtered and concentrated in vacuo. The crude material was then purified on silica gel (9% MeOH in CH.sub.2Cl.sub.2) to afford the title compound as a yellow oil (72 mg, 76%).

[0791] m/z LRMS (ESI.sup.+): 761 (100%) [M+H].sup.+.

2-(4-(4-benzoylbenzyl)piperazin-1-yl)-N-(4-(2-(2-(2-(5-((3aR,4R,6aS)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)ethoxy)ethoxy)acetamido)phenyl)oxazole-4-carboxamide (49) (Abd-L26)

[0792] ##STR00083##

[0793] Carbamate 48 was dissolved in THE (2 mL) before addition of MeOH (200 μL). The solution was degassed with nitrogen for 5 min before addition of a catalytic amount of Pd/C. The suspension was degassed with nitrogen for a further 5 min before replacing the atmosphere with hydrogen (by mean of a balloon). The reaction was monitored by TLC and MS; after 2 h, the reaction was complete. The balloon was removed and the reaction was passed through a bed of Celite™ using EtOAc and MeOH as an eluent. The product was used in the next step without further purification. The obtained amine (32 mg, 0.050 mmol, 1.0 eq.) was dissolved in DMF (1 mL) before sequential addition of N,N-diisopropylethylamine (26 μL, 0.150 mmol, 3.0 eq.), D-biotin (15 mg, 0.060 mmol, 1.2 eq.) and HATU (27 mg, 0.070 mmol, 1.4 eq.). The resulting solution was stirred for 18 h, diluted with EtOAc (10 mL) and washed with brine/water (1:1, 3×20 mL). The organic phase was dried (Na.sub.2SO.sub.4), filtered and concentrated in vacuo. The crude material was purified on silica gel (10% MeOH in CH.sub.2Cl.sub.2) and further via prep TLC (15% MeOH in CH.sub.2Cl.sub.2) to afford the title compound as a yellow oil (12 mg, 28%).

[0794] m/z LRMS (ESI.sup.+): 853 (100%) [M+H].sup.+.

[0795] While specific embodiments of the invention have been described herein for the purpose of reference and illustration, various modifications will be apparent to a person skilled in the art without departing from the scope of the invention as defined by the appended claims.

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