COMPOUNDS FOR USE IN THE TREATMENT OF ACUTE INTERMITTENT PORPHYRIA

Abstract

The invention provides compounds of formula (I), their pharmaceutically acceptable salts and prodrugs thereof for use in preventing, inhibiting or treating a disease caused by a mutation in the gene coding for hydroxymethylbilane synthase, in particular for preventing, inhibiting or treating acute intermittent porphyria: (I) wherein: A is selected from N and CR.sup.10 (wherein R.sup.10 is H, —NO.sub.2, C.sub.1-6 haloalkyl or —C(O)R.sup.17 in which R.sup.17 is H or C.sub.1-6 alkyl); Z is selected from N and CR.sup.9 (wherein R.sup.9 is H, halogen (e.g. F, Cl, Br or I) or —OR.sup.16 in which R.sup.16 is H, C.sub.1-6 haloalkyl, or optionally substituted C.sub.1-6 alkyl); L is selected from —CH.sub.2—, —C(O)—, —CH(OH)—, —C(O)—NR′—, and —NR′—C(O)— (wherein R′ is H or C.sub.1-3 alkyl, e.g. —CH.sub.3); R.sup.1 is H; R.sup.2 is selected from H, halogen (e.g. F, Cl, Br or I), —NR.sup.11R.sup.12 (wherein R.sup.11 and R.sup.12 are independently selected from H and C.sub.1-6 alkyl or, together with the nitrogen atom to which they are attached, form a 5- or 6-membered saturated ring), and —OR13 (wherein R.sup.13 is H or C.sub.1-6 alkyl); R.sup.3 is selected from H, —CH.sup.2OH and —C(O)R.sup.14 (wherein R.sup.14 is H or C.sub.1-6 alkyl); R.sup.4 is selected from H, halogen (e.g. F, Cl, Br or I) and —OR.sup.15 (where R.sup.15 is H or C.sub.1-6 alkyl); R.sup.5 is selected from H and C.sub.1-6 alkyl; R.sup.6 is selected from H, —NO.sub.2 and halogen (e.g. F, Cl, Br or I); R.sup.7 is H; and R.sup.8 is selected from H, C.sub.1-6 alkyl, and halogen (e.g. F, Cl, Br or I); or wherein: R.sup.7 and R.sup.8 together with the intervening ring carbon atoms form an unsaturated ring, preferably an aryl ring.

##STR00001##

Claims

1. A method of prevention or treatment of a disease caused by a mutation in the gene coding for hydroxymethylbilane synthase, said method comprising the step of administering to a patient in need thereof a pharmaceutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt or prodrug thereof: ##STR00063## wherein: A is selected from N and CR.sup.10 (wherein R.sup.10 is H, —NO.sub.2, C.sub.1-6 haloalkyl or —C(O)R.sup.17 in which R.sup.17 is H or C.sub.1-6 alkyl); Z is selected from N and CR.sup.9 (wherein R.sup.9 is H, halogen or —OR.sup.16 in which R.sup.16 is H, C.sub.1-6 haloalkyl, or optionally substituted C.sub.1-6 alkyl); L is selected from —CH.sub.2—, —C(O)—, —CH(OH)—, —C(O)—NR′—, and —NR′—C(O)— (wherein R′ is H or C.sub.1-3 alkyl); R.sup.1 is H; R.sup.2 is selected from H, halogen, —NR.sup.11R.sup.12 (wherein R.sup.11 and R.sup.12 are independently selected from H and C.sub.1-6 alkyl or, together with the nitrogen atom to which they are attached, form a 5- or 6-membered saturated ring), and —OR.sup.13 (wherein R.sup.13 is H or C.sub.1-6 alkyl); R.sup.3 is selected from H, —CH.sub.2OH and —C(O)R.sup.14 (wherein R.sup.14 is H or C.sub.1-6 alkyl); R.sup.4 is selected from H, halogen and —OR.sup.15 (where R.sup.15 is H or C.sub.1-6 alkyl); R.sup.5 is selected from H and C.sub.1-6 alkyl; R.sup.6 is selected from H, —NO.sub.2 and halogen; R.sup.7 is H; and R.sup.8 is selected from H, C.sub.1-6 alkyl, and halogen; or wherein: R.sup.7 and R.sup.8 together with the intervening ring carbon atoms form an unsaturated ring.

2. The method according to claim 1, wherein R.sup.2 is selected from H, halogen, and —OR.sup.13 (wherein R.sup.13 is H or C.sub.1-6 alkyl).

3. The method according to claim 1, wherein R.sup.3 is selected from H and —C(O)H.

4. The method according to claim 1, wherein R.sup.4 is selected from H, —OH and Cl.

5. The method according to claim 1, wherein R.sup.6 is selected from H and halogen.

6. The method according to claim 1, wherein R.sup.8 is selected from H, halogen and —CH.sub.3.

7. The method according to claim 1, wherein R.sup.7 and R.sup.8 together with the intervening ring carbon atoms form an unsaturated ring.

8. The method according to claim 1, wherein R.sup.9 is selected from H, halogen and —OR.sup.16 (wherein R.sup.16 is H, —CF.sub.3 or —CH.sub.3).

9. The method according to claim 1, wherein R.sup.10 is selected from —NO.sub.2 and —CF.sub.3.

10. The method according to claim 1, wherein the compound is a compound of general formula (IV), or a pharmaceutically acceptable salt or prodrug thereof: ##STR00064## wherein R.sup.1 to R.sup.10 are as defined in claim 1.

11. The method according to claim 1, wherein the compound is a compound of general formula (V), or a pharmaceutically acceptable salt or prodrug thereof: ##STR00065## wherein R.sup.1 to R.sup.10 are as defined in claim 1.

12. The method according to claim 1, wherein the compound is selected from the following and their pharmaceutically acceptable salts and prodrugs: ##STR00066## ##STR00067##

13-24. (canceled)

25. The method according to claim 1, wherein the disease is acute intermittent porphyria.

26. The method according to claim 1, wherein R.sup.2 is selected from H, —OCH.sub.3, —OH and Cl.

27. The method according to claim 1, wherein R.sup.6 is selected from H and Cl.

28. The method according to claim 1, wherein R.sup.8 is selected from H and Cl.

29. The method according to claim 7, wherein the unsaturated ring is a 5- or 6-membered carbocyclic ring.

30. The method according to claim 7, wherein the unsaturated ring is an aryl ring.

31. The method according to claim 30, wherein the aryl ring is an optionally substituted phenyl ring.

32. The method according to claim 1, wherein R.sup.9 is selected from H, Cl, —OCF.sub.3 and —OCH.sub.3.

Description

[0134] The invention will now be described in more detail in the following non-limiting Examples and with reference to the accompanying figures, in which:

[0135] FIG. 1 shows the protection of compound BG-1 against limited tryptic proteolysis of WT-HMBS. (A) SDS PAGE showing the effect of the indicated compound (84 μM and 2% DMSO) with HMBS. Std, low molecular weight standards; Control n.t., no trypsin added; Control DMSO, HMBS with 2% DMSO and trypsin; BG-1, HMBS with 2% DMSO, trypsin and compound BG-1. (B) Quantification of the lowest 31.5 kDa band relative to the full-length HMBS at 42.5 kDa. **p<0.01 for differences compared to the DMSO control, calculated by two-sample student's t-test for equal variance.

[0136] FIG. 2 shows western blotting and immunoquantification of the relative amount of HMBS in cell lysates from WT-HMBS stably transfected HepG2 cells treated with either compound BG-1 (A) or compound BG-2 (B) at the indicated concentrations. DMSO (2%) was included in all samples. Representative blots are shown, and the histograms below represent the quantification of the relative HMBS levels (n=3), using GAPDH as the protein loading control.

[0137] FIG. 3 shows the schematic protocol followed in the mice trials. Female compound heterozygote Hmbs-deficient T1/T2.sup.−/− mice were kept on normal diet and given 10 or 20 mg/kg/day (trial T1/T2-A and T1/T2-B, respectively) of either the desired compound or DMSO, by oral gavage, for 12 consecutive days. Biochemical acute attack was induced by intraperitoneal injection of phenobarbital days 10-12. Urine was collected on day 1 and 10-12, and blood samples were collected before—and livers were harvested after—sacrifice.

[0138] FIG. 4 shows the effect of the compound BG-1 in Hmbs-deficient mice (trial T1/T2-A). One group of Hmbs T1/T2.sup.−/− mice (n=6) were orally treated for 12 days with 10 mg/kg/day of compound BG-1. On day 10, 11 and 12 they were induced by phenobarbital. A control group was given 10% DMSO, and likewise induced with phenobarbital. On days 1 and 10-12 urine was collected and pooled from each group before measurement. (A,B) Bars represent porphyrin precursor ALA (A) and PBG (B) in urine from control group (white) and compound BG-1 (blue).

[0139] FIG. 5 shows concentration-dependent SPR with HMBS immobilized to sensor chip. (A) No apparent concentration for half-maximal binding (S.sub.0.5)-value was obtained for compound BG-1 with SPR. The data from the Octet measurements (A; inset) provided an S.sub.0.5=83±7. (B) An S.sub.0.5=63±3 μM was obtained, also using sigmoidal fitting, from the binding isotherm for compound BG-2.

[0140] FIG. 6 shows the effect on ALA/PBG excretion of the compound BG-1 and compound BG-2 in Hmbs-deficient mice (trial T1/T2-B). Two groups of Hmbs T1/T2.sup.−/− mice (n=6 in each group) were treated for 12 days with 20 mg/kg/day of either compound BG-1 or compound BG-2. I.p. injection of phenobarbital was given on days 10-12 to induce the heme biosynthesis, and thus precipitation of biochemical acute attack. A control group was given 10% DMSO and likewise induced with phenobarbital. Urine was collected on day 1 and 10-12, and livers were harvested after sacrifice. Protein levels were measured in liver lysates by western blot quantification. (A,B) Urine from the mice was pooled for each group and bars represent porphyrin precursors ALA (A) and PBG (B) treated with compound BG-1 (blue) and compound BG-2 (green). Control group is shown in white. (C) Scatter plots (circles) with mean (line) representing HMBS protein levels in mice livers treated with compound BG-1 (blue), and compound BG-2 (green). *p<0.05 for differences compared to the corresponding control (10% DMSO without compound; white circles), calculated by unpaired two-tailed t-test. (D) Scatter plot (circles) with mean (line) showing the enzymatic activity in liver tissue. **p<0.01 and ****p<0.0001 for differences compared to the corresponding control (10% DMSO without compound), calculated by unpaired two-tailed t-test. (E,F) The relative concentrations of ALA (E) and PBG (F) were measured in liver tissue extracts after treatment with compound BG-1 (blue) and compound BG-2 (green). *p<0.05 for differences compared to the corresponding control (10% DMSO without compound, white), calculated unpaired two-tailed t-test)

EXAMPLES

General Procedures

Expression and Purification of HMBS Proteins:

[0141] WT-HMBS was expressed and purified to apparent homogeneity as previously described (Bustad et al., Bioscience reports 2013; 33(4)). The protein was further purified by size exclusion chromatography with a Superdex™ 75 10/300 GL column (GE Healthcare) in 20 mM HEPES, 150 mM NaCl, pH 8.2 and stored as aliquots in liquid N2 until use.

[0142] The enzyme used in the binding assays using the Octet RED96 was expressed using a new construct with an N-terminal 6×HIS affinity tag and a TEV protease cleavage site. Full-length HMBS was cloned into pET-28a(+)-TEV vector and transformed into BL21 (DE3) cells for expression. Expression was done in Terrific Broth medium with IPTG induction. Cells were cultured 16 h at 20° C. with 220 rpm shaking. After harvesting, the cells were lysed with sonication, and standard affinity purification was performed using Ni-NTA affinity matrix. Protein was eluted with 20 mM HEPES, 150 mM NaCl (gel filtration buffer), pH 8 supplemented with 400 mM imidazole. Affinity tag was cleaved overnight and removed with passing the protein through Ni-NTA. The protein was further purified by size exclusion chromatography with a Superdex™ 75 10/300 GL or 16/60 PG column (GE Healthcare, Chicago, Ill.) in gel filtration buffer, pH 8.0, and stored as aliquots in liquid N2 until use.

Enzymatic Activity Assay of Recombinant HMBS:

[0143] The standard enzymatic activity of recombinant WT-HMBS was assayed at 37° C. as reported previously (Bustad et al., Bioscience reports, 2013; 33(4)). Compounds were added at a concentration of 84 μM. Absorbance of uroporphyrinogen I was determined at 405 nm. The enzyme activity in the presence of the compounds was normalized relative to DMSO-control (relative activity).

[0144] The effect of the compounds on the stability of HMBS activity was assayed by pre-incubating the HMBS (4-5 μg) in 50 mM HEPES pH 8.2, 84 μM compound and 2% DMSO for 20 min at 70° C., and then placed on ice for 5 min. The enzymatic activity at 37° C. was subsequently measured as reported previously (Bustad et al., Bioscience reports 2013; 3 3(4)). Controls without compound but with equal concentration of DMSO were included. The remaining activity in the presence of compounds was normalized relative to DMSO-control (relative activity). K.sub.m and V.sub.max were determined using an increasing concentration of PBG (3.125-1000 μM), and the kinetic parameters were obtained by non-linear curve fitting to Michaelis-Menten enzyme kinetics using GraphPad Prism version 8.2.0 for Windows, GraphPad Software, La Jolla Calif. USA, www.graphpad.com.

Limited Proteolysis by Trypsin:

[0145] Limited proteolysis by trypsin was performed at 37° C. in 20 mM HEPES, 150 mM NaCl, 2% DMSO, pH 8.2, with 0.15 μg/μl HMBS in the absence (DMSO-control) or presence of 84 μM compound and 2% DMSO. The proteolysis was initiated by adding 1 μg/ml TPCK-treated trypsin (Sigma-Aldrich). After 30 min, aliquots were removed and transferred to Laemmli loading buffer containing 2 μg/ml soybean trypsin inhibitor. Samples resolved by electrophoresis with 10% Mini-Protean® TGX™ gels (Bio-Rad Laboratories, Inc.) were analyzed using the Image Lab™ software (Bio-Rad Laboratories, Inc.). The unpaired t-test (two tailed) was performed using GraphPad Prism.

Transfection of HepG2 Cells and Growth in the Presence of Compounds:

[0146] The human hepatoma HepG2 cells were obtained from Leibniz-Institut DSMZ—Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH. Cells were maintained in RPMI 1640, GlutaMAX™ (Thermo Fisher Scientific) medium supplemented with 10% heat-inactivated fetal bovine serum and 1% penicillin-streptomycin (Thermo Fisher Scientific) in a humidified incubator with 5% CO.sub.2 at 37° C. HMBS cDNA was inserted into the pcDNA3.1(+) cloning vector (Thermo Fisher Scientific). The HepG2 cells were then transfected with the pcDNA3.1(+) vector containing HMBS using FuGENE®HD Transfection Reagent (Promega, Madison, Wis.) according to the manufacturer's recommendations. Stably transfected clones were selected for resistance to the neomycin analogue G418 (Thermo Fisher Scientific). WT-HMBS transfected HepG2 cells (2×10.sup.6) were seeded and grown for 22 h before compounds were added to final concentrations of 0, 40, 84, 120 and 168 μM in 2% DMSO. Cells were harvested after 24 h and analyzed as described below.

Surface Plasmon Resonance (SPR):

[0147] Surface plasmon resonance experiments for the estimation of the concentration of compound at half-maximal binding (S.sub.0.5) were performed using a Biacore T200 (GE Healthcare) instrument at 25° C. 150 μg/ml WT-HMBS in 10 mM sodium acetate pH 4.5 was immobilized onto a CM5-S sensor chip through amine-coupling chemistry and PBS containing 0.05% surfactant P20 as running buffer, reaching immobilization levels ˜15,000. The baseline was equilibrated for 1-2 h, before the compounds were assayed in a concentration-dependent manner (0-200 μM), using running buffer with 5% DMSO and 30 μl/min flow rate. Contact and dissociation time was 60 s, with a final wash after 50% DMSO injection. Blank immobilization, solvent correction and negative control (assay buffer) were included for the analysis of the sensorgrams using the Biacore T200 Evaluation software v2.0. The allosteric sigmoidal curve fitting was performed using GraphPad Prism.

Octet RED96:

[0148] Octet RED96 system (ForteBio Biologics by Molecular Devices, LLC., San Jose, Calif.) with super streptavidin (SSA) biosensors was used as an additional method for determining the K.sub.d-value for the binding of the compounds. Loading HMBS to the SSA sensors required biotinylation, which was carried out at room temperature mixing 1.5 molar excess of NHS-ester biotinylation reagent (EZ-Link™ NHS-PEG4-Biotin, Thermo Fisher Scientific) to protein. After 30 minutes excess of biotin was removed using Zeba spin desalting column (Thermo Fisher Scientific) and a gel filtration buffer was changed to reaction buffer, PBS-P+(GE Healthcare) supplemented with 5% DMSO. Sensors were loaded with 5 μg/ml of biotinylated HMBS, reaching 6 nm surface thickness. Triplicates for the concentration series of the compounds were measured, and double reference subtraction was applied for data analysis based on the steady-state kinetics with equilibrium binding signal (Req) using ForteBio Data analysis 9.0. The allosteric sigmoidal curve fitting was performed using GraphPad Prism.

Animal Studies:

[0149] The compound heterozygote Hmbs-deficient T1/T2.sup.−/− mouse model (Lindberg et al., Nature genetics, 1996; 12(2):195-9) was utilized in the two sets of animal studies performed, T1/T2-A and T1/T2-B: i) In the T1/T2-A study mice (2-4 months old, 16-22 g) were given 10 mg/kg/day of compound BG-1. ii) In the T1/T2-B study, mice (2-3 months old, 17-22 g) were treated with 20 mg/kg/day of either compound BG-1 or compound BG-2. The mice in both groups were given phenobarbital (Gardenal®) at 100 mg/kg through i.p. injection on days 10-12 of the study. Urine from these mice was collected day 1 before start of treatment, and each day of phenobarbital injection (day 10, 11 and 12). The protocol is presented in FIG. 3.

[0150] Compounds were dissolved in 10% DMSO and all studies included treatment groups with 6 mice in each, including a control group given only 10% DMSO. The compounds or DMSO alone were administered for twelve consecutive days and the mice were sacrificed 30 min after the last dose of compound or phenobarbital. The mice were anaesthetized by i.p. injection of tribromoethanol (3 mg/kg), blood samples collected on EDTA by retro-orbital puncture and livers harvested and flash frozen in liquid N2 before storage at −80° C.

[0151] Pooled urinary porphyrin precursor levels (ALA and PBG) were analyzed by sequential ion-exchange chromatography using the ALA/PBG by Column Test (Bio-Rad Laboratories, Inc.) according to the manufacturer's recommendations.

Cell and Tissue Sampling:

[0152] HepG2 cells were washed in ice-cold PBS before 10 min lysis on ice with cold RIPA buffer (25 mM Tris-HCl, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS (Cell Signaling Technologies, Danvers, Mass.) and cOmplete protease inhibitor cocktail (Roche Diagnostics)). Lysates were centrifuged (10,000 g, 15 min) and supernatants were removed and stored at −80° C. until further use. Frozen liver tissue was homogenized in 50 mM Tris-HCl, pH 7.4, 100 mM KCl, 1 mM DTT, 0.2 mM PMSF, 1 mM benzamidine, 1 mM EDTA and 1 tablet/10 ml cOmplete ULTRA protease inhibitor cocktail (Roche Diagnostics) using TissueLyser II (Qiagen, Venlo, Netherlands). The extracts were clarified by centrifugation at 14,000 g for 20 min at 4° C., and supernatants were stored at −80° C.

Enzymatic Activity Assay of HMBS in Liver Tissue:

[0153] The crude liver homogenates were passed through Zeba spin desalting columns (Thermo Fisher Scientific) to remove small molecules <2000 Da. 50 mM HEPES pH 8.2 was used as equilibration buffer. 25 μl filtrated homogenate (400-500 μg total protein) was added to 110 μl sample mix (50 mM HEPES, pH 8.2, 1% Triton™ X-100) and incubated for 10 min at 37° C. before adding PBG (1 mM). The reaction was stopped after 1 h by adding ice-cooled 100% TCA to a final concentration of 25%, incubated at room temperature (RT) for 10 min and centrifuged at 10,000 g for 10 min. The absorption was measured in the supernatant at A409 with baseline correction at A380 using NanoDrop™ 2000c spectrophotometer (Thermo Fisher Scientific). The activity of HMBS was expressed as relative activity in the compound-treated cell compared to DMSO-controls. A blank sample was prepared for each homogenate. The unpaired t-test (two tailed) was performed using GraphPad Prism.

Quantitative Detection of HMBS in Cell Lysate and Tissue by Immunoblot:

[0154] Cell lysate samples (20 μg total protein) were separated by electrophoresis and subsequently transferred to a PVDF transfer membrane (Bio-Rad). Membranes were blocked for 1 hour at RT with 5% non-fat dry milk (Bio-Rad Laboratories) in Tris-buffered saline (TBS; 20 mm Tris-HCl, 140 mM NaCl pH 7.4), containing 0.1% Tween® (0.1% TBS-T). Immunoblotting was carried out with 1:1,000 anti-HMBS primary Ab (H300; Santa Cruz Biotechnology, Dallas, Tex.) in 0.1% TBS-T, overnight at 4° C. Subsequently the membranes were washed extensively in 0.1% TBS-T followed by 1 h incubation at RT with HRP-conjugated goat-anti-rabbit IgG secondary Ab (Bio-Rad) 1:5,000 dilution. Anti-GAPDH (Abcam, Cambridge, UK) was used as loading control. Chemiluminescence of secondary Ab-HRP conjugates was elicited using Luminata™ Crescendo Western HRP Substrate (Merck Millipore, Burlington, Mass.), imaged with Gel Doc™ XR+ (Bio-Rad) and quantified using Image Lab software (Bio-Rad).

[0155] Liver homogenates (5 μg total protein/lane) were loaded onto 10% Mini-Protean® TGX™ gels (Bio-Rad) and separated with Tris/Glycine/SDS electrophoresis buffer (Bio-Rad). Trans-Blot® Turbo™ Transfer Starter System (Bio-Rad) was used to transfer the proteins onto Immun-Blot® low fluorescence PVDF membranes (Bio-Rad). The membranes were then blocked with TBS containing 1% Tween® 20 (1% TBS-T) and 3% BSA for 1 h. HMBS was probed with 1:2,000 monoclonal mouse anti-HMBS (H-11, Santa Cruz Biotechnology), together with 1:1,000 rabbit anti-actin (Sigma-Aldrich), in 1% TBS-T, 3% BSA overnight, 4° C. Alexa Fluor 647 conjugated donkey-anti-mouse and Alexa Fluor 488 conjugated donkey-anti-rabbit (both Thermo Fisher Scientific) were used as secondary antibodies and incubated in 1:1,000 dilutions in 0.1% TBS-T for 1 h. Each step was followed by extensive wash with 0.1% TBS-T. Fluorescence detection was performed using G-Box Chemi-XRQ (Syngene Synoptics, Cambridge, UK) with filters UV06 and 705 nm for AF-488 and AF-647, respectively, and the band intensities of HMBS relative to the loading control (actin) were determined using ImageJ (Schneider et al., Nature methods 2012; 9(7): 671-5). Plotting and the two-tailed unpaired t-test were performed using GraphPad Prism version 8.2.0.

Determination of Compound and Metabolites in Liver Tissue Samples:

[0156] One volume of homogenized liver tissue was mixed with two volumes of acetonitrile:MetOH (1:1, v/v) and centrifuged. High performance liquid chromatography/tandem mass spectrometry (HPLC-MS/MS) was used to determine the concentration of compound BG-1, ALA and PBG in the supernatants. Analyses of samples were conducted by the Bioanalytical Laboratory personnel at Enamine/Bienta (Bienta/Enamine Ltd Biology Services, Kiev, Ukraine, www.bienta.net). The unpaired t-test (one tailed) was performed using GraphPad Prism version 8.2.0.

Statistics:

[0157] Results are presented as mean±SD, except for relative data where relative mean is presented with error of propagation. Statistical comparisons were done using two-sample student's t-test for equal variance, and statistically significance was defined as p<0.05 or lower, as specified in the text. All statistical analyses and plotting of data were performed in GraphPad Prism 8.2.0.

NMR spectroscopy:

[0158] .sup.1H NMR spectra were recorded at 400 MHz on a Bruker Avance III NMR spectrometer. Samples were prepared in deuterated chloroform (CDCl.sub.3) or dimethylsulphoxide (DMSO-d.sub.6) and the raw data were processed using the ACD NMR software.

UPLC-MS Analysis:

[0159] LCMS analysis was conducted on a Waters Acquity UPLC system consisting of an Acquity i-Class Sample Manager-FL, Acquity i-Class Binary Solvent Manager and Acquity i-Class UPLC Column Manager. UV detection was achieved using an Acquity i-Class UPLC PDA detector (scanning from 210 to 400 nm), whereas mass detection was achieved using an Acquity QDa detector (mass scanning from 100-1250 Da; positive and negative modes simultaneously). A Waters Acquity UPLC BEH C18 column (2.1×50 mm, 1.7 μm) was used to achieve the separation of the analytes.

[0160] Samples were prepared by dissolving (with or without sonication) into 1 ml of a 1:1 (v/v) mixture of MeCN in H.sub.2O. The resulting solutions were filtered through a 0.2 μm syringe filter before being submitted for analysis. All of the solvents (including formic acid and 36% ammonia solution) used were used as the HPLC grade. Four different analytical methods were used, the details of which are presented below.

[0161] Acidic run (2 min): 0.1% v/v Formic acid in water [Eluent A]; 0.1% v/v Formic acid in MeCN [Eluent B]; Flow rate 0.8 ml/min; injection volume 2 μl and 1.5 min equilibration time between samples.

TABLE-US-00001 Time (min) Eluent A (%) Eluent B (%) 0.00 95 5 0.25 95 5 1.25 5 95 1.55 5 95 1.65 95 5 2.00 95 5

[0162] Acidic run (4 min): 0.1% v/v formic acid in water [Eluent A]; 0.1% v/v formic acid in MeCN [Eluent B]; Flow rate 0.8 ml/min; injection volume 2 μl and 1.5 min equilibration time between samples.

TABLE-US-00002 Time (min) Eluent A (%) Eluent B (%) 0.00 95 5 0.25 95 5 2.75 5 95 3.25 5 95 3.35 95 5 4.00 95 5

[0163] Basic run (2 min): 0.1% ammonia in water [Eluent A]; 0.1% ammonia in MeCN [Eluent B]; Flow rate 0.8 ml/min; injection volume 2 μl and 1.5 min equilibration time between samples.

TABLE-US-00003 Time (min) Eluent A (%) Eluent B (%) 0.00 95 5 0.25 95 5 1.25 5 95 1.55 5 95 1.65 95 5 2.00 95 5

[0164] Basic run (4 min): 0.1% ammonia in water [Eluent A]; 0.1% ammonia in MeCN [Eluent B]; Flow rate 0.8 ml/min; injection volume 2 μl and 1.5 min equilibration time between samples.

TABLE-US-00004 Time (min) Eluent A (%) Eluent B (%) 0.00 95 5 0.25 95 5 2.75 5 95 3.25 5 95 3.35 95 5 4.00 95 5

Example 1—Synthesis of (4-chloro-3-(trifluoromethyl)phenyl)(phenyl)methanone

[0165] ##STR00025##

[0166] To a stirred solution of 4-bromo-1-chloro-2-(trifluoromethyl)benzene (314 mg, 1.21 mmol) in THF (6.0 ml) at −78° C. was added a solution of n-BuLi in hexanes (1.6 M, 0.58 ml, 1.45 mmol), and the resulting mixture was stirred at −78° C. for 30 min. A solution of N-methoxy-N-methylbenzamide (200 mg, 1.21 mmol) in THF (0.5 ml) was added to the reaction, and the resulting mixture was allowed to warm to room temperature with stirring over 60 min. The mixture was quenched by the addition of a saturated aqueous solution of ammonium chloride (10 ml) and extracted with DCM (2×10 ml). The combined organics were dried over sodium sulphate, filtered and evaporated to dryness to give a residue. Purification by flash column chromatography over silica gel (Biotage) eluting with a gradient of EtOAc (0 to 10%) in hexanes gave the desired product as a white solid (64 mg, Y=18%).

[0167] UPLC-MS (Acidic Method, 4 min): rt=2.22 min, m z=n.d. [M+H].sup.+

[0168] .sup.1H NMR (400 MHz, Chloroform-d) δ 8.15 (d, J=2.0 Hz, 1H), 7.91 (dd, J=8.3, 2.1 Hz, 1H), 7.81-7.74 (m, 2H), 7.69-7.61 (m, 2H), 7.57-7.48 (m, 2H).

Example 2—Synthesis of (6-chloropyridin-3-yl)(phenyl)methanone

[0169] ##STR00026##

[0170] This compound was prepared according to the standard procedure for the formation of ketones via the addition of an organolithium species to a Weinreb amide in Example 1, using 5-bromo-2-chloropyridine (150 mg, 0.91 mmol) and N-methoxy-N-methylbenzamide (175 mg, 0.91 mmol). The desired product was isolated as a green oil (71.3 mg, Y=36%)

[0171] UPLC-MS (Acidic Method, 4 min): rt=1.67 min, m z=218 [M+H].sup.+

[0172] .sup.1H NMR (400 MHz, Chloroform-d) δ 8.77 (d, J=2.4, 0.8 Hz, 1H), 8.10 (dd, J=8.3, 2.4 Hz, 1H), 7.84-7.77 (m, 2H), 7.68-7.62 (m, 1H), 7.56-7.50 (m, 2H), 7.50-7.46 (m, 1H).

Example 3—Synthesis of (4-chloro-3-nitrophenyl)(phenyl)methanol

[0173] ##STR00027##

[0174] Sodium borohydride (72.3 mg, 1.91 mmol) was added in a single portion to a stirred solution of (4-chloro-3-nitrophenyl)(phenyl)methanone (200 mg, 0.76 mmol) in methanol (5 ml) at 0° C., and the resulting mixture was warmed to room temperature with stirring for 16 h. The mixture was quenched by the addition of a saturated aqueous solution of ammonium chloride (10 ml) and evaporated to dryness to give a residue. Purification by flash column chromatography over silica gel (Biotage) eluting with a gradient of EtOAc (0 to 50%) in hexanes gave the desired product as a white solid (105 mg, Y=52%).

[0175] UPLC-MS (Acidic Method, 4 min): rt=1.80 min, m z=n.d. [M+H].sup.+

[0176] .sup.1H NMR (400 MHz, Chloroform-d) δ 7.95 (d, J=1.8 Hz, 1H), 7.53-7.46 (m, 2H), 7.41-7.29 (m, 5H), 5.86 (d, J=3.1 Hz, 1H), 2.39 (d, J=3.2 Hz, 1H).

Example 4—Synthesis of 4-benzyl-1-chloro-2-nitrobenzene

[0177] ##STR00028##

[0178] A stirred solution of (4-chloro-3-nitrophenyl)(phenyl)methanone (250 mg, 0.95 mmol), triethylsilane (0.99 ml, 6.2 mmol) and boron trifluoride diethyl etherate (0.79 ml, 6.42 mmol) was heated at 65° C. for 2 h. The mixture was quenched by the addition of a saturated aqueous solution of sodium bicarbonate (10 ml) and extracted with EtOAc (3×10 ml). The combined organics were dried over sodium sulphate, filtered and evaporated to dryness to give a residue. Purification by flash column chromatography over silica gel (Biotage) eluting with a gradient of EtOAc (0 to 30%) in hexanes gave the desired product as a white solid (209 mg, Y=89%).

[0179] UPLC-MS (Acidic Method, 4 min): rt=2.14 min, m z=n.d. [M+H].sup.+

[0180] .sup.1H NMR (400 MHz, Chloroform-d) δ 7.67 (d, J=2.1 Hz, 1H), 7.43 (d, J=8.2 Hz, 1H), 7.35-7.29 (m, 3H), 7.27-7.22 (m, 1H), 7.19-7.13 (m, 2H), 4.01 (s, 2H).

Example 5—Synthesis of 4-chloro-N-methyl-3-nitro-N-phenylbenzamide

[0181] ##STR00029##

[0182] A stirred solution of 4-chloro-3-nitro-N-phenylbenzamide (222 mg, 0.8 mmol) in DMF (2 Ml) was treated with sodium hydride (60% dispersion, 39 mg, 0.96 mmol), and the resulting mixture was stirred for 30 min. Iodomethane (60 ml, 0.96 mmol) was added to the reaction, and the resulting mixture was stirred for 16 h. The reaction was quenched by the addition of a saturated aqueous solution of ammonium chloride (20 ml) and extracted with EtOAc (3×20 ml). The combined organics were dried over sodium sulphate, filtered and evaporated to dryness to give a residue. Purification by flash column chromatography over silica gel (Biotage) eluting with a gradient of EtOAc (0 to 50%) in hexanes gave the desired product as a white solid (74 mg, Y=32%).

[0183] UPLC-MS (Acidic Method, 4 min): rt=1.79 min, m z=277 [M+H].sup.+

[0184] .sup.1H NMR (400 MHz, DMSO-d6) δ 7.96 (s, 1H), 7.63 (d, J=8.4 Hz, 1H), 7.50 (d, J=8.4 Hz, 1H), 7.36-7.18 (m, 6H), 3.38 (s, 3H).

Example 6—Synthesis of (4-chloro-3-nitrophenyl)(2,6-dimethylphenyl)methanone

[0185] ##STR00030##

[0186] Activated manganese(IV) oxide (178 mg, 2.05 mmol) was added to a solution of (4-chloro-3-nitrophenyl)(2,6-dimethylphenyl)methanol (120 mg, 0.41 mmol) in dichloromethane (10 mL) at ambient temperature, and the resulting mixture was stirred at ambient temperature for 16 h. The reaction mixture was filtered through a plug of Celite, washing with DCM. The combined filtrate was evaporated to dryness to give the crude product as a residue. Purification by flash column chromatography over silica gel (Biotage) eluting with a gradient of EtOAc (0 to 5%) in hexanes gave the desired product as a white solid (45 mg, Y=38%).

[0187] UPLC-MS (Acidic Method, 2 min): rt=1.28 min, m z=n.d. [M+H].sup.+

[0188] .sup.1H NMR (400 MHz, Chloroform-d) δ 8.24 (d, J=2.0 Hz, 1H), 7.90 (dd, J=8.4, 2.0 Hz, 1H), 7.66 (d, J=8.4 Hz, 1H), 7.30 (t, J=7.7 Hz, 1H), 7.12 (d, J=7.6 Hz, 2H), 2.12 (s, 6H).

Example 7—Synthesis of (4-chloro-3-nitrophenyl)(4-(trifluoromethoxy)phenyl)methanone

[0189] ##STR00031##

[0190] Activated manganese(IV) oxide (125 mg, 149 mmol) was added to a solution of (4-chloro-3-nitrophenyl)(4-(trifluoromethoxy)phenyl)methanol (100 mg, 0.29 mmol) in dichloromethane (10 ml) at ambient temperature, and the resulting mixture was stirred at ambient temperature for 16 h. The reaction mixture was filtered through a plug of Celite, washing with DCM. The combined filtrate was evaporated to dryness to give the crude product as a residue. Purification by flash column chromatography over silica gel (Biotage) eluting with a gradient of EtOAc (0 to 30%) in hexanes gave the desired product as a white solid (60 mg, Y=60%).

[0191] UPLC-MS (Acidic Method, 4 min): rt=2.20 min, m z=n.d. [M+H].sup.+

[0192] .sup.1H NMR (400 MHz, Chloroform-d) δ 8.27 (d, J=2.0 Hz, 1H), 7.94 (dd, 1H), 7.89-7.82 (m, 2H), 7.72 (d, J=8.3 Hz, 1H), 7.39-7.35 (m, 2H).

Example 8—Synthesis of (4-chloro-3-nitrophenyl)(4-(trifluoromethoxy)phenyl)methanol

[0193] ##STR00032##

[0194] A solution of (4-(trifluoromethoxy)phenyl)magnesium bromide in THF (0.5 M, 11.0 ml, 5.5 mmol) was added dropwise to a stirred solution of 4-chloro-3-nitrobenzaldehyde (1.0 g, 5.39 mmol) in anhydrous THF (40 ml) at −78° C., and the resulting mixture was allowed to warm to ambient temperature with stirring for 16 h. The mixture was quenched by the addition of a saturated aqueous solution of ammonium chloride (10 ml) and extracted with EtOAc (2×10 ml). The combined organics were dried over sodium sulphate, filtered and evaporated to dryness to give a residue. Purification by flash column chromatography over silica gel (Biotage) eluting with a gradient of EtOAc (0 to 30%) in hexanes gave the desired product as a pale yellow oil (1.1 g, 69%).

[0195] UPLC-MS (Acidic Method, 2 min): rt=1.22 min, m z=346 [M−H].sup.+

[0196] .sup.1H NMR (400 MHz, Chloroform-d) δ 7.94 (d, J=1.9 Hz, 1H), 7.55-7.45 (m, 2H), 7.39 (d, J=8.6 Hz, 2H), 7.25-7.18 (m, 2H), 5.88 (s, 1H), 2.41 (s, 1H).

Example 9—Synthesis of (4-chloro-3-(trifluoromethyl)phenyl)(4-(trifluoromethoxy) phenyl)methanone

[0197] ##STR00033##

[0198] Activated manganese(IV) oxide (469 mg, 5.4 mmol) was added to a solution of (4-chloro-3-(trifluoromethyl)phenyl)(4-(trifluoromethoxy)phenyl)methanol (400 mg, 1.08 mmol) in dichloromethane (6.2 mL) at ambient temperature, and the resulting mixture was stirred at ambient temperature for 16 h. The reaction mixture was filtered through a plug of Celite, washing with DCM. The combined filtrate was evaporated to dryness to give the crude product as a residue. Purification by flash column chromatography over silica gel (Biotage) eluting with a gradient of EtOAc (0 to 20%) in hexanes gave the desired product as a white solid (93 mg, Y=24%).

[0199] UPLC (4 min, acidic): rt=2.46 min, no mass ionisation, 100% purity by UV.

[0200] .sup.1H NMR (400 MHz, Chloroform-d) δ 8.13 (d, J=2.1 Hz, 1H), 7.92-7.80 (m, 3H), 7.66 (d, J=8.3 Hz, 1H), 7.40-7.31 (m, 2H).

[0201] 19F NMR (DMSO-d6) δ: −62.33, −67.60

Example 10—Synthesis of (4-chloro-3-nitrophenyl)(4-methoxyphenyl)methanol

[0202] ##STR00034##

[0203] A solution of 4-methoxyphenyl)magnesium in THF (0.5 M, 11.0 ml, 5.5 mmol) was added dropwise to a stirred solution of 4-chloro-3-nitrobenzaldehyde (1.0 g, 5.39 mmol) in anhydrous THF (40 mL) at −78° C., and the resulting mixture was allowed to warm to ambient temperature with stirring for 16 h. The mixture was quenched by the addition of a saturated aqueous solution of ammonium chloride (10 ml) and extracted with EtOAc (2×10 ml). The combined organics were dried over sodium sulphate, filtered and evaporated to dryness to give a residue. Purification by flash column chromatography over silica gel (Biotage) eluting with a gradient of EtOAc (0 to 30%) in hexanes gave the desired product as a pale yellow oil (1.1 g, 67%).

[0204] UPLC (4 min, acidic): rt=1.81 min, no mass ionisation, 100% purity by UV.

[0205] .sup.1H NMR (DMSO-d6) δ: 8.03 (d, J=1.9 Hz, 1H), 7.70 (d, J=8.4 Hz, 1H), 7.63 (ddd, J=8.4, 2.1, 0.6 Hz, 1H), 7.33-7.25 (m, 2H), 6.92-6.84 (m, 2H), 6.16 (d, J=3.5 Hz, 1H), 5.77 (d, J=3.1 Hz, 1H), 3.72 (s, 3H).

Example 11—Synthesis of 5-(2-chlorobenzyl)-2-hydroxybenzaldehyde

[0206] ##STR00035##

[0207] A solution of 2-chlorobenzylzinc chloride in THF (0.5 M, 6.5 mL, 3.23 mmol) was added dropwise to a stirred suspension of 5-bromo-2-hydroxybenzaldehyde (500 mg, 2.49 mmol), SPhos (20 mg, 0.05 mmol), palladium(II) acetate (5.6 mg, 0.025 mmol) in anhydrous THF (2.5 mL) at 0° C., and the resulting mixture was allowed to warm to ambient temperature while stirring for 16 h. The mixture was quenched by the addition of a saturated aqueous solution of ammonium chloride (10 ml) and extracted with EtOAc (2×20 ml). The combined organics were dried over sodium sulphate, filtered and evaporated to dryness to give a residue. Purification by flash column chromatography over silica gel (Biotage) eluting with a gradient of EtOAc (0 to 10%) in hexanes gave the desired product as a colourless oil (250 mg, 41%).

[0208] UPLC (4 min, acidic): rt=2.08 min, no mass ionisation, 100% purity by UV.

[0209] .sup.1H NMR (Chloroform-d) δ: 10.89 (s, 1H), 9.81 (s, 1H), 7.42-7.33 (m, 2H), 7.32 (d, J=2.3 Hz, 1H), 7.25-7.12 (m, 3H), 6.92 (d, J=8.5 Hz, 1H), 4.07 (s, 2H).

Example 12—Synthesis of (3-nitro-4-(pyrrolidin-1-yl)phenyl)(4-(2-(pyrrolidin-1-yl) ethoxy)phenyl)methanone

[0210] ##STR00036##

[0211] To a solution of 4-(2-bromoethoxy)phenyl)(4-chloro-3-nitrophenyl)methanone (60 mg, 0.16 mmol) in anhydrous DMF (4.0 ml) was added K.sub.2CO.sub.3 (320 mesh, 86 mg, 0.62 mmol) and pyrrolidine (0.026 ml, 0.31 mmol), and the resulting suspension was stirred at 85° C. for 16 h. The reaction mixture was cooled to ambient temperature and partitioned between EtOAc (10 ml) and water (10 ml). The organic phase was separated and the aqueous phase washed with EtOAc (10 ml). The combined organic phases were dried over sodium sulphate, filtered and evaporated to dryness to give the crude product as an oil. Purification by flash column chromatography over silica gel (Biotage) eluting with a gradient of methanol (0 to 20%) in DCM gave the desired product as a solid (55 mg, Y=86%).

[0212] UPLC (4 min, acidic): rt=1.49 min. ESI(+)=410.3, 100% purity by UV

[0213] .sup.1H NMR (Chloroform-d) δ: 8.13 (d, J=2.2 Hz, 1H), 7.84 (dd, J=9.0, 2.2 Hz, 1H), 7.72-7.64 (m, 2H), 6.96-6.88 (m, 2H), 6.87 (d, J=9.0 Hz, 1H), 4.24 (t, J=5.6 Hz, 2H), 3.29-3.21 (m, 4H), 3.02 (t, J=5.6 Hz, 2H), 2.78 (s, 4H), 2.02-1.91 (m, 4H), 1.89-1.81 (m, 4H).

Example 13—Synthesis of 5-[(2-chlorophenyl)methyl]-2-hydroxy-3-(trifluoromethyl) benzaldehyde

[0214] ##STR00037##

[0215] To an oven-dried two-neck round bottom flask under a nitrogen atmosphere was added 5-bromo-2-hydroxy-3-(trifluoromethyl)benzaldehyde (500 mg, 1.86 mmol, 1.0 eq.), SPhos (15.3 mg, 0.037 mmol, 0.02 eq.), palladium(II) acetate (4.2 mg, 0.019 mmol, 0.01 eq.) and anhydrous THF (5.0 mL). The resulting orange solution was cooled to 0° C. before the dropwise addition of 2-chlorobenzyl zinc chloride (0.5 M in THF, 4.8 mL, 2.42 mmol, 1.3 eq.). The reaction was allowed to warm slowly to room temperature and stirred for 18 h. The reaction mixture was re-cooled to 0° C., quenched with saturated aqueous ammonium chloride solution (20 mL) and extracted with EtOAc (2×50 mL). The organic phase was separated, dried over sodium sulfate, filtered and concentrated in vacuo. The crude material was dry loaded on silica gel and purified by flash column chromatography (Biotage, 25 g Si, gradient elution 0-10% EtOAc/hexane) to afford the title compound (173 mg, 93% purity by UPLC) as a pale yellow semi-solid. A portion (50 mg) of this material was taken directly into the next step (see Example 14) and the remainder (120 mg) was further purified by prep-HPLC (C18, gradient 0-95% MeCN/H.sub.2O+NH.sub.4OH) and freeze dried to afford the title compound (38.0 mg, 0.12 mmol, 7%) as an off-white powder.

[0216] 120 mg of the material was further purified by prep-HPLC (C18, gradient 0-95% MeCN/H.sub.2O+NH.sub.4OH) and freeze dried to afford the title compound (38.0 mg, 0.12 mmol, 7%) as an off-white powder.

[0217] UPLC MS (Acidic Method, 4 min): rt 2.29 min, ES.sup.− m/z 313.1 [M-1]−, >99% purity by UV.

[0218] .sup.1H NMR (400 MHz, DMSO) δ 10.07 (s, 1H), 11.48 (s, 1H), 7.80 (dd, J=17.1, 2.3 Hz, 2H), 7.46 (dd, J=7.5, 1.8 Hz, 1H), 7.42-7.37 (m, 1H), 7.31 (dtd, J=14.9, 7.4, 1.8 Hz, 2H), 4.14 (s, 2H). 97% purity.

[0219] .sup.19F NMR (376 MHz, DMSO) δ −60.99.

Example 14—Synthesis of 4-(2-chlorobenzyl)-2-(hydroxymethyl)-6-(trifluoromethyl) phenol

[0220] ##STR00038##

[0221] To a cooled (0° C.) solution of 5-[(2-chlorophenyl)methyl]−2-hydroxy-3-(trifluoromethyl) benzaldehyde prepared in accordance with Example 13 (50.0 mg, 0.16 mmol, 1.0 eq.) in methanol (5.0 mL) was added sodium borohydride (12.0 mg, 0.32 mmol, 2.0 eq.) and the reaction was stirred at room temperature for 3 h. The reaction was concentrated in vacuo and the residue was dissolved in EtOAc (30 mL) and washed sequentially with water (30 mL) and brine (30 mL). The organic layer was dried over sodium sulfate, filtered and concentrated in vacuo. The resultant residue was purified by prep-HPLC (C18, gradient 0-95% MeCN/H.sub.2O+NH.sub.4OH) and freeze dried to afford the title compound (11.0 mg, 0.04 mmol, 22%) as an off-white powder.

[0222] UPLC MS (Acidic Method, 4 min): rt 2.04 min, ES.sup.− m/z 315.1 [M-1]−, >99% purity by UV.

[0223] .sup.1H NMR (400 MHz, DMSO) δ 7.44 (dd, J=7.6, 1.6 Hz, 1H), 7.37-7.22 (m, 5H), 4.57 (s, 2H), 4.03 (s, 2H).

[0224] .sup.19F NMR (376 MHz, DMSO) δ −60.29.

Example 15—Activity and Proteolysis Assay

[0225] An enzymatic assay was performed to investigate the effect of compound BG-1 on the activity and conformational stability of HMBS. HMBS activity of the purified enzyme was measured in the absence and presence of compound BG-1 (at 84 μM) at standard conditions (37° C.) but also after pre-incubation at 70° C. for 20 min based on the high thermal stability of WT-HMBS. The results are presented in Table 1 below.

[0226] Limited tryptic proteolysis was also applied to compound BG-1. Proteolysis of WT-HMBS provided three major bands with relative content 34.5±0.3%, 14.7±0.8% and 50.9±0.5%, corresponding to remaining full-length HMBS (˜42.5 kDa), and two fragments of ˜41.0 kDa and ˜31.5 kDa, respectively (FIG. 1). Compound BG-1 exhibited protection against proteolysis (see FIG. 1). The effect of this compound at 84 μM on the steady-state enzyme kinetics of HMBS was then calculated (Table 2). The results showed a reduction in both K.sub.M and V.sub.max, which agreed with mixed inhibition indicating a preferential binding to the substrate-bound complex.

TABLE-US-00005 TABLE 1 The effect of compound BG-1 on the activity, conformational stability, and limited tryptic proteolysis of HMBS Relative Protection Compound activity, Relative activity, against tryptic ID ΔT.sub.m.sup.a (° C.) standard.sup.b pre-inc. at 70° C.sup.c proteolysis.sup.d CTRL — 1.00 1.00 — BG-1 1.6 0.98 ± 0.06 1.01 ± 0.06 ++** .sup.aThe thermal upshift values at y = 0.5 in scaled fluorescence curves (ΔT.sub.m) monitored by DSF. The average compound concentration in DSF screening was 122 μM (2% DMSO). .sup.bActivity assay performed at standard conditions, with 100 μM PBG at 37° C., 84 μM compound and 2% DMSO, which was added in all controls. .sup.cAssay including pre-incubation of HMBS with compound at 70° C., and subsequent standard activity assay, with 100 μM PBG at 37° C., 84 μM compound and 2% DMSO. .sup.dSymbols: ++, 10% remaining full-length HMBS relative to DMSO. **p < 0.01 for significant protection against tryptic proteolysis compared with the DMSO control sample, calculated by two-sample student's t-test for equal variance.

TABLE-US-00006 TABLE 2 Steady-state enzyme kinetic parameters of HMBS in the presence of compound BG-1. V.sub.max.sup.a K.sub.M(PBG).sup.a (nmol/ ID Structure (μM) min/mg) BG-1 [00039] 69 ± 4* 56 ± 1* .sup.aEffect of the compound on the enzyme kinetic parameters for HMBS activity, measured at fixed compound concentration (84 μM in 2% DMSO) and variable PBG (0-1 mM) at 37° C., and fitted to Michaelis-Menten kinetics. The K.sub.M (PBG) and V.sub.max values for the DMSO control of WT-HMBS were 86 ± 5 μM and 61 ± 2 nmol/min/mg, respectively. *P ≤ 0.05, for significant difference compared with the values for DMSO control sample, calculated by unpaired two-tailed t-test.

Example 16—Effect of Compound BG-1 in Human Hepatoma HepG2 Cells

[0227] The stabilizing and potential PC effect of compound BG-1 was investigated in HepG2 cells over-expressing HMBS by analyzing the compound concentration effect on the steady-state levels of the enzyme (see FIG. 2A). Quantitative western immunoblotting revealed an increasing relative amount of HMBS with increasing concentration of compound BG-1.

Example 17—ALA Excretion in Hmbs-Deficient Mice (Trial T1/T2 A)

[0228] The compound heterozygote Hmbs-deficient T1/T2.sup.−/− mouse model for AIP, which allows monitoring the level of precursors ALA and PBG in urine after phenobarbital induction, was used. One group of six mice was given 10 mg/kg/day (trial denoted T1/T2-A) of compound BG-1 for 12 days. A control group of six mice, treated with only DMSO was also included. To induce the heme synthesis, phenobarbital (Gardenal®) was given during the last three days of the study (see FIG. 3). No apparent toxicity in the treated mice was detected as assessed by normal behavior and organ appearances.

[0229] Hmbs-deficient T1/T2.sup.−/− mice do not show elevated excretion of urinary ALA and PBG until induction of biochemical acute attacks (Lindberg et al., Nature genetics. 1996; 12(2):195-9), and indeed a rapid increase in urinary ALA and PBG was seen for the non-treated control mice by day 11 and even higher by day 12, following the administration of phenobarbital (white bars, FIG. 4A,B). The treatment with 10 mg/kg/day did not cause any significant change in HMBS protein levels or activity in either erythrocytes or liver, compared to the non-treated Hmbs-deficient mice in the T1/T2-A trial. However, a slight decreasing tendency in urinary levels of ALA, but not PBG, was observed for compound BG-1 treatment by day 12 (blue bars, FIG. 4B) compared to non-treated mice, indicating that a higher compound concentration may result in an increased metabolite level correction. Similarly, compound analogues with higher affinity might increase the effect due to more efficient dose-dependent effect in vivo. No toxic effect was registered for this compound.

Example 18—Effect of Analogues of Compound BG-1 on the Thermal Stability, Proteolysis and Activity of HMBS

[0230] Compounds BG-2, BG-3 and BG-4 were tested on recombinant WT-HMBS using DSF and tryptic proteolysis. In cells, BG-2 increased the HMBS protein levels similarly to BG-1 (see FIGS. 2A and 2B), and enzyme kinetic analyses showed a weak mixed inhibitory effect (see Table 3).

TABLE-US-00007 TABLE 3 The effect of the compounds on the ΔT.sub.m measured by DSF, the limited tryptic proteolysis and the activity of HMBS ΔT.sub.m.sup.b Protection against K.sub.M(PBG).sup.d V.sub.max.sup.d ID Mw (° C.) tryptic proteolysis.sup.c (μM) (nmol/min/mg) CTRL — — — 86 ± 5 61 ± 2  BG-2 261.66 4.9 +* 84 ± 8 54 ± 2** [00040] BG-3 257.24 6.6 +/− — — [00041] BG-4 293.7  4.7 +/− — — [00042] .sup.bThe thermal upshift values (ΔT.sub.m) monitored by DSF. The average compound concentration in DSF screening was 122 μM (2% DMSO). .sup.cSymbols: +/−, ±2%; +, >4%. .sup.dEffect of the compounds on the enzyme kinetic parameters for HMBS activity, measured at fixed compound concentration (84 μM with 2% DMSO) and variable PBG (0-1 mM) at 37° C., and fitted to Michaelis-Menten kinetics. *P < 0.05 and **p < 0.01, for significant difference compared with the values for DMSO control sample, calculated by unpaired two-tailed t-test.

Example 19—Surface Plasmon Resonance (SPR) and Octet RED96 Studies

[0231] The binding of compounds BG-1 and BG-2 to HMBS was analyzed by surface plasmon resonance (SPR) and response units from concentration-dependent steady state measurements were analyzed assuming a 1:1 binding model. Compound BG-1 showed some unspecific binding, and an accurate S.sub.0.5 value could not be obtained. The interaction between compound BG-1 and HMBS was therefore further studied with Octet RED96 system with super streptavidin (SSA) biosensors. For the loading of the sensors, the protein needed to be biotinylated but no alteration of the buffer conditions was required. The data analysis using double reference subtraction accounts for non-specific binding and minimizes the well-based and sensor variability. The analyses with Octet provided an S.sub.0.5 value of 83±7 μM for compound BG-1, obtained by fitting the data to a (sigmoidal) binding isotherm with saturable concentration dependence (FIG. 5A (inset)). For compound BG-2, SPR allowed the measurement of good concentration-dependent binding data, which also yielded a sigmoidal binding curve, providing a S.sub.0.5 value of 63±3 μM (FIG. 5B).

Example 20—Preventive Effect on ALA/PBG Excretion in Hmbs-Deficient Mice (Trial T1/T2-B)

[0232] In a second animal trial using Hmbs-deficient T1/T2.sup.−/− mice, denoted T1/T2-B (n=6 in each group), the chaperone potential of compounds BG-1 and BG-2 at higher concentration (20 mg/kg/day) was investigated. The experimental setup was as for T1/T2-A (see FIG. 3). The experimental set up was otherwise identical, with n=6 in each treatment group and a control group (n=6) receiving DMSO instead of the compound. The effect of the compounds was, as in trial T1/T2-A, monitored by measuring urinary excretion of ALA and PBG. Both compound BG-1 and BG-2 reduced the urinary ALA and PBG excretion, and the latter to almost half of the value in the control group (FIG. 6A,B).

[0233] Quantitative western immunoblotting of liver tissue revealed a pronounced increase (2-fold) in steady-state levels of HMBS in the presence of the compounds BG-1 and BG-2 (FIG. 6C).

[0234] The effect of treatment with compound BG-1 and BG-2 on hepatic HMBS activity was measured in the liver homogenates, resulting in significantly increased enzyme activity in mice treated with both compounds as compared with the control group (FIG. 6D). Furthermore, the relative concentrations of ALA and PBG, as well as of compound accumulated in liver, which were found to be decreased for the mice treated with compound BG-1 (p<0.05 and p=0.053, respectively, compared with control mice; FIG. 4E,F).

Example 21—Octet RED Studies on Other Compounds

[0235] Octet RED (Forte Bio) was used for screening binding and determining the K.sub.D values of various compounds. OctetRED is based on the technique called Bio-layer interferometry (BLI). It measures changes in an interference pattern generated from visible light reflected from an optical layer and a biolayer containing proteins of interest. In the assay the target protein is biotinylated and coupled as a layer on the optic probe (Super Strepavidin). The concentration series was from 6.25 to 500 μM.

[0236] For the analysis method of K.sub.D determination, steady state analysis was used, where the response from steady state of the association phase was used for determining the binding curve for the analyte. Buffer for the analysis was selected to PBS-P+5% DMSO.

TABLE-US-00008 TABLE 4 K.sub.D values for the binding of the indicated compounds to HMBS, measured by Octet. K.sub.D Structure (μM) [00043] 90 [00044] 40 42 160 140 400 mM range (no saturation with 500 μM) mM range (no saturation with 500 μM) [00045] mM range (no saturation with 500 μM) [00046] mM range (no saturation with 500 μM) [00047] mM range (no saturation with 500 μM) [00048] 7 [00049] 110 [00050] mM range (no saturation with 500 μM) [00051] 300 [00052] 200 [00053] mM range (no saturation with 500 μM) [00054] 140 [00055] 9.6 [00056] >200 [00057] 190 [00058] >200

Example 22—Further Octet RED Studies

[0237] Octet RED96 (Forte Bio) was used for screening binding and determining the K.sub.D values for certain compounds. In the assay the target protein (WT-HMBS) was biotinylated and coupled as a layer on the optic probe. The concentration series for the tested compounds were from 3.1 to 150 μM, dissolved in PBS-P+5% DMSO.

[0238] For the analysis method of K.sub.D determination, steady state analysis was used in which the response from steady-state of the association phase was used for determining the binding curve for the analyte. K.sub.D values were calculated using sigmoidal fitting. The buffer for the analysis was selected to PBS-P+5% DMSO.

TABLE-US-00009 TABLE 5 K.sub.D values for the binding of the indicated compounds to HMBS, measured by Octet. Soluble in DMSO Solubility in K.sub.D Structure (100%) buffer Binding (μM) [00059] Yes 384 μM Yes 83 [00060] Yes  52 μM Yes 9.6 [00061] Yes Yes Yes 9.3 [00062] Yes Yes Yes 90

Example 23—Activity Measurements In Vitro

[0239] Activity measurements were carried out using TECAN plate reader for absorbance measurements. Activity measurements were based on measuring the light absorbance at 405 nm for determining the concentration of formed product, preuroporphyrinogen. Standard curve for measurements was measured using absorbance of known concentrations for Uroporphyrin I dihydrochloride, the cyclic derivative of preuroporphyrinogen. Two concentrations of the compound of Example 14, i.e. 5 and 50 μM in 2.5% DMSO were used. Protein was incubated 30 min in 37° C. with the compound, and mixed with MIX-buffer (50 mM HEPES pH 8, 2.5% DMSO) on Corning 96 well plate (clear bottom, half area, black). The solutions were pre-warmed to 37° C. prior to reaction. Reaction was started by adding PBG and after 5 min the reaction was stopped by adding the STOP solution (5 M HCl and 0.1% p-Benzoquinone mixed 1:1). Wells were covered and protected from light during the whole reaction. Results show activation of HMBS for the compound of Example 14 when compared to HMBS with 5% DMSO only.