WO2020035782A1

WO2020035782A1 - Molecular probes for analysis of metabolism of glycolysis inhibitors prodrugs and synthesis methods therefore

Info

Publication number: WO2020035782A1
Application number: PCT/IB2019/056853
Authority: WO
Inventors: Donald Picker
Original assignee: Dermin Sp. Z O.O.
Priority date: 2018-08-13
Filing date: 2019-08-13
Publication date: 2020-02-20

Abstract

The present invention relates to compounds according to structure (I) whereby one of R1 to R3 is -OCOCD3 and the other two are either hydroxyl or OAc, their synthesis and their use in for the analysis of the metabolism of glycolysis inhibitors.

Description

Molecular probes for analysis of metabolism of glycolysis inhibitors prodrugs and synthesis methods therefore

D e s c r i p t i o n

The present invention relates to the field of glycolysis inhibitors and their produgs, especially to the metabolism of such glycolysis inhibitors in the body.

Glycolysis inhibitors are of high interest in clinical research and the pharmaceutical field due to their potential use as therapeutics. Potential indications include anti -diabetes (e.g. acarbose and miglitol) or anti-viral (e.g. oseltamivit or zanamivir) applications.

The metabolism of these inhibitors in the human body, however, is inter alia vital for the usage of those inhibitors as potential drugs and therefore there is a constant need for compounds which are useful in the analysis of the metabolism of glycolysis inhibitors as well as synthesis methods for those compounds.

It is therefore an object to provide such compounds, especially compounds which are deuterated at predefinied moieties, and synthesis methods therefor. This object is met by compounds according to Claim 1 of the present invention. Accordingly, compounds according to structure (I) are provided

whereby one of R¹ to R³ is -OCOCD₃ and the other two are either hydroxyl or OAc.

It has been shown that these compounds are valuable in the analysis of the metabolism of glycolysis inhibitors, especially due to their defined deuteration pattern.

According to a preferred embodiment of the present invention, at least one of R¹ to R³ is hydroxyl.

According to a preferred embodiment of the present invention, either R¹ or R³ is -OCOCD3.

The present invention furthermore relates to a synthesis method for compounds according to the present invention, involving the steps of a) reacting a suitable precursor compound with a silylating agent to obtain a monosilyl- protected precursor compound b) optional protecting of further hydroxy groups of the compound obtained in step a) by orthogonal protective groups c) detachment of the silyl protective group

d) acetylation of the deprotected group to introduce a -CO-CD₃ group e) optional deprotection of the protected hydroxy groups in step b)

It has been shown that by doing so it is possible to synthesize the inventive compounds in a straightforward fashion. Another advantage of the inventive synthesis method is the introduction of the (precious) deuteriated group at a late stage of the synthesis. It is especially preferred that a sterically demanding silyl group is used in step a), especially if R¹ is to contain the deuteriated group. Silyl groups with t-butyl and/or aryl groups are especially preferred, most preferred is TBDMS (t-Butyldimetylsilyl). These silyl groups are known to prefer primary hydroxy groups over secondary or tertiary group, so that a high selectivity in the silylation can be reached.

In step b) especially benzyl and/or substituted benzyl groups are preferred due to their orthogonality with silyl groups. Step e) can then subsequentially occur using hydrogenation, preferably under Pd catalysis. Step c) can be performed using fluorides, with TBAF (Tertbutylammonium fluoride) especialyl preferred.

In step d) especially per-deuterated acetyl anhydride and/or deuterated acyl halides, especially deuterated acyl chloride are preferred, with per-deuterated acetyl anhydride especially preferred.

It goes without saying that further synthesis steps can be performed between any of the steps a) to e). For example the hydroxy group at C-l (the glycosidylic hydroxy group) can be inserted by hydration of a double bond between C-l and C-2 (i.e. the precursor compound(s)

include glucals and/or glucal derivatives). Subsequently, according to a preferred

embodiment, the precursor compound in step a) is a glucal or glucal derivative and the method includes a step f) which can be performed after step d) or e) f) hydration of the double bond between C-l and C-2

Step f) can be performed by first adding aqueos solution of H-Hal (with Cl and Br especially preferred), followed by neutralization, preferably by carbonate, hydrogen carbonate and/or phosphate solution, wich hydrogen carbonate solution especially preferred.

The present invention furthermore relates to the use of the inventive compounds for the analysis of the metabolism of glycolysis inhibitors.

The present invention furthermore relates to an analysis method for the metabolism of glycolysis inhibitors including the step of subjecting the inventive compounds in an assay.

The assay can be in vitro or in vivo.

The aforementioned components, as well as the claimed components and the components to be used in accordance with the invention in the described embodiments, are not subject to any special exceptions with respect to their size, shape, material selection and technical concept such that the selection criteria known in the pertinent field can be applied without limitations.

Additional details, characteristics and advantages of the object of the invention are disclosed in the subclaims and the following description of the examples. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are intended to provide further explanation of the present invention as claimed.

1. Synthesis of 3-0-trideuteroacetyl-2-deoxy-D-glucose (9)

1.1. Synthesis of 3,6-di-O-acetyl-D-glucal (2)

Tri-O-acetyl-D-glucal (1) (10 g, 36.7 mmol) suspension in phosphate buffer (pH 6) (100 mL) and acetonitrile (10 mL) was prepared and Amanolipase (10 g) was added. The reaction mixture was stirred overnight at room temperature. The reaction mixture was then filtered through Celite, and filtrate was extracted with ethyl acetate (3 x 75 mL). Combined extracts were dried with NaiSCri. Drying agent was filtered off and solvent was evaporated to dryness. Crude product was purified by short column chromatography using hexanes/ethyl acetate gradient for elution. Fractions contained product were pooled together and evaporated to dryness to give 7.4 g of 3,6-di-O-acetyl-D-glucal (yield 87.5%). Ή NMR spectra were in the agreement with the literature data.

1.2. Synthesis of 3,6-di-0-acetyl-3-0-tert-butyldimethylsilyl-D-glucal (3)

Obtained in previous step 3,6-di-O-acetyl-D-glucal (2) (7.4 g, 32.1 mmol) was dissolved in dichloromethane (100 mL). Imidazole (4g, 60 mmol) followed by tert-butyldimethylsilyl chloride (5.8 g, 38.5 mmol) were added and the reaction mixture was stirred at room temperature. Progress of the reaction was monitored using TLC method. After reaction was completed the reaction mixture was diluted with dichloromethane (100 mL), washed with

water (3 x 100 mL), and then dried over NaiSCri. Drying agent was filtered off and solvent was evaporated to dryness to give crude 3,6-di-0-acetyl-3-0-tert-butyldimethylsilyl-D-glucal (Yield quantitative) which without further purification was used in the next step. The structure of the product was confirmed by Ή NMR method and compared with literature.

1.3. Synthesis of 3-O-tert-butyldimethylsilyl-D-glucal (4)

Crude 3,6-di-0-acetyl-3-0-tert-butyldimethylsilyl-D-glucal (3) (~32 mmol) was dissolved in methanol (100 mL). Solution of 1N sodium methoxide in methanol (1 mL, 1 mmol) was added and the reaction mixture was stirred at room temperature until substrate was fully deacetylated (TLC). After reaction was completed acetic acid (1 mmol) was added, and the reaction mixture was stirred at room temperature for additional 10 min. then solvent was evaporated to dryness. 3-O-tert-butyldimethylsilyl-D-ghical was obtained in quantitative yield with purity that allowed to proceed to the next step without further purification. Ή NMR spectra of the product was acquired and compare with the literature.

1.4. Synthesis of 3,6-di-0-benzyl-3-0-tert-butyldimethylsilyl-D-glucal (5)

Crude 3-O-tert-butyldimethylsilyl-D-glucal (4) (~32 mmol) was dissolved in DMF (50 mL), and obtained solution was cooled to -20°C. Sodium hydride (60% suspension in mineral oil) (2.8 g, 70.4 mmol) was added followed by benzyl bromide (8.4 mL, 70.4 mmol). The reaction mixture was stirred at -20°C for 30 min., then cooling bath was removed and stirring was continued and temperature was allowed to rise to ambient. After reaction was completed (TLC analysis), the reaction mixture was diluted with hexanes (150 mL) and then water (100 mL) was added dropwise to neutralize excess of NaH. After 30 min. of vigorous stirring solution layers were separated. Organic layer was washed with water until neutral, and dried with NaiSOu Drying agent was filtered off and solvent was evaporated to dryness to give crude 3,6-di-0-benzyl-3-0-tert-butyldimethylsilyl-D-glucal (Yield quantitative) which

without further purification was used in the next step. The structure of the product was confirmed by Ή NMR method and compared with literature.

1.5. Synthesis of 3,6-di-O-benzyl-D-glucal (6)

Crude 3,6-di-0-benzyl-3-0-tert-butyldimethylsilyl-D-glucal (5) (3.8 g, 8.6 mmol) was dissolved in THF (38 mL). Tetra-butylammonium fluoride (2.25 g, 8.6 mmol) was added and the reaction mixture was stirred at room temperature. After reaction was completed (TLC) solvent was evaporated to dryness and ethyl acetate (60 mL) was added. Obtained solution was washed with water (3 x 50 mL) then dried over Na2S04. Drying agent was filtered off and solvent was evaporated to dryness to give crude 3,6-di-O-benzyl-D-glucal which without further purification was used in the next step. The structure of the product was confirmed by Ή NMR method and compared with literature.

1.6. Synthesis of 3,6-di-0-benzyl-3-0-trideuteroacetyl-D-glucal (7)

3,6-di-O-benzyl-D-glucal (6) (2.8 g, 8.6 mmol) was dissolved in dichloromethane (28 mL). Pyridine (0.7 mL) followed by per-deuterated acetyl anhydride (0.81 mL, 8.6 mmol) were added and the reaction mixture was stirred at room temperature overnight. After reaction was completed the reaction mixture was diluted with DCM (50 mL) and washed with 10% Na2C03 water solution, then with water until neutral and dried over Na2S04. Drying agent was filtered off and solvent was evaporated to dryness, and product was purified by column chromatography using hexanes/ethyl acetate gradient for elution. Fractions contained product were pooled together and evaporated to dryness to give 2.73 g of 3,6-di-0-benzyl-3-0- trideuteroacetyl-D-glucal (Yield 85.3%)

¾ NMR (CDCb, d, ppm), 7.38 - 7.22 (m, 10 H, Harom), 6.44 (dd, 1H, J = 6.0 Hz, J = 1.2 Hz), 5.43 (ddd, 1H, J = 6.2 Hz, J = 2.7 Hz, J = 1.1 Hz), 4.75 (dd, 1H, J = 6.1 Hz, J = 2.9 Hz), 4.70 (d, 1H, J = 11.5 Hz), 4.66 (d, 1H, J = 8.3 Hz), 4.59 (d, 1H, J = 8.3 Hz), 4.56 (d, 1H, J =

11.5 Hz), 4.09 (ddd, 1H, J = 8.7 Hz, J = 4.5 Hz, J = 3.1 Hz), 3.93 (dd, 1H, J = 8.7 Hz, J = 6.3 Hz), 3.83 (dd, 1H, J = 10.8 Hz, J = 4.5 Hz), 3.75 (dd, 1H, J = 10.8 Hz, J = 3.1 Hz)

1.7. Synthesis of 3,6-di-0-benzyl-3-0-trideuteroacetyl-2-deoxy-D-glucose (8)

3.6-di-0-benzyl-3-0-trideuteroacetyl -D-glucal (7) (2.73 g, 7.35 mmol) was dissolved in THF (27 mL). 48% HBr water solution (1 mL, 8.8 mmol) was added and the reaction mixture was stirred at room temp until all substrate was consumed (TLC). Reaction mixture was diluted with water (30 mL), then NaHCCb (8.8 mmol, 0.74 g) was added, and the reaction mixture was stirred additional 15 min. Ethyl acetate (50 mL) was added and layers were separated. Organic layer was washed with water (2 x 25 mL) and dried over Na2S04. Drying agent was filtered off and solvent was evaporated to dryness, and product was purified by column chromatography using hexanes/ethyl acetate gradient for elution. Fractions contained product were pooled together and evaporated to dryness to give 1.8 g of 3,6-di-0-benzyl-3-0- trideuteroacetyl-2-deoxy-D-glucose (Yield 62.9%).

1.8. Synthesis of 3-0-(trideuteroacetyl-2-deoxy-D-glucose (9)

3.6-di-0-benzyl-3-0-trideuteroacetyl-2-deoxy-D-glucose (8) (1.8 g, 4.6 mmol), was dissolved in THF (20 mL). Pd(OH)2/C (20% wet, Degussa) (290 mg) was added and the mixture was hydrogenated using Paar apparatus (p = 45 psi) for 1.5 h, then reaction mixture was filtered through Celite. Filtrate was evaporated to dryness, and product was purified by column chromatography using DCM/Methanol gradient for elution Fractions contained product were pooled together and evaporated to dryness to give 0.87g of 3-0-trideuteroacetyl-2-deoxy-D- glucose (Yield 90 %).

2. Synthesis of 6-0-trideuteroacetyl-2-deoxy-D-glucose (16)

2.1. Synthesis of 6-O-tert-butyldimethylsilyl-D-glucal (11)

D-Glucal (10) (7.85 g, 53.7 mmol) was dissolved in DMF (20 mL). Imidazole (6.1 g, 90 mmol) was added and obtained solution was cooled to 0°C. Tert-butyldimethylsilyl chloride (8.9 g, 59.1 mmol) was added and the reaction mixture was stirred at 0°C overnight.

After reaction was completed the reaction mixture was diluted with ethyl acetate (100 mL), washed with water (3 x 100 mL), and then dried over NaiSCri. Drying agent was filtered off and solvent was evaporated to dryness and product was separated using column

chromatography using hexanes/ethyl acetate gradient for elution. Fractions contained product were pooled together and evaporated to dryness to give 8.7 g of 6-O-tert-butyldimethylsilyl- D-glucal (Yield 62 %). The structure of the product was confirmed by Ή NMR method and compared with literature .

2.2. Synthesis of 3,4-di-0-benzyl-6-0-tert-butyldimethylsilyl-D-glucal (12)

6-O-tert-butyldimethylsilyl-D-glucal (11) (4.5 g, 17.3 mmol) was dissolved in DMF (45 mL), and obtained solution was cooled to -20°C. Sodium hydride (60% suspension in mineral oil) (1.52 g, 38 mmol) was added followed by benzyl bromide (4.5 mL, 38 mmol). The reaction mixture was stirred at -20°C for 30 min., then cooling bath was removed and stirring was continued when temperature was allowed to rise to ambient. After reaction was completed

(TLC), the reaction mixture was diluted with hexanes (120 mL) and water (100 mL) was added dropwise to neutralize excess of NaH. After 30 minut of vigorous stirring layers were separated. Organic layer was washed with water until neutral, and dried with Na2S04. Drying agent was filtered off and solvent was evaporated to dryness and product was separated using column chromatography using hexanes/ethyl acetate gradient for elution. Fractions contained product were pooled together and evaporated to dryness to give 5.5 g of 3,4-di-0-benzyl-6-0- tert-butyldimethylsilyl-D-glucal (Yield 72.2 %). The structure of the product was confirmed by Ή NMR method and compared with literature. The structure of the product was confirmed by Ή NMR method and compared with literature.

2.3. Synthesis of 3,4-di-O-benzyl-D-glucal (13)

3.4-di-0-benzyl-6-0-tert-butyldimethylsilyl-D-glucal (12) (5.5 g, 12.5 mmol) was dissolved in THF (55 mL). Tetra-butylammonium fluoride (3.3 g, 12.5 mmol) was added and the reaction mixture was stirred at room temperature. After reaction was completed (TLC) solvent was evaporated to dryness and ethyl acetate (100 mL) was added. Obtained solution was washed with water (3 x 50 mL) then dried over NaiSOu Drying agent was filtered off and solvent was evaporated to dryness to give crude 3,6-di-O-benzyl-D-glucal (Yield:

quantitative), which without further purification was used in the next step. The structure of the product was confirmed by Ή NMR method and is in the agreement with literature data.

2.4. Synthesis of 3,4-di-0-benzyl-6-0-trideuteroacetyl -D-glucal (14)

3.4-di-O-benzyl-D-glucal (13) (4.3 g, 13.2 mmol) was dissolved in dichloromethane (43 mL). Pyridine (2.2 mL) followed by per-deuterated acetyl anhydride (1.24 mL, 13.2 mmol) were added and the reaction mixture was stirred at room temperature overnight. After reaction was completed the reaction mixture was diluted with DCM (50 mL) and washed with 10%

Na2C03 water solution, then with water until neutral and dried over NarSOu Drying agent was filtered off and solvent was evaporated to dryness, and product was purified by column

chromatography using hexanes/ethyl acetate gradient for elution. Fractions contained product were pooled together and evaporated to dryness to give 4.4 g of 3,4-di-0-benzyl-6-0- trideuteroacetyl -D-glucal (Yield 89.8%)

¾ NMR (CDCb, d, ppm), 7.40 - 7.28 (m, 10 H, Harom), 6.40 (dd, 1H, J = 6.2 Hz, J = 1.2 Hz), 4.95 (dd, 1H, J = 6.1 Hz, J = 2.7 Hz), 4.86 (d, 1H, J = 11.3 Hz), 4.67 (d, 2H, J = 11.1 Hz), 4.59 (d, lH, J = 11.1 Hz), 4.41 (dd, 1H, J = 12.1 Hz, J = 2.9 Hz), 4.35 (dd, 1H, J = 12.1 Hz, J = 5.2 Hz), 4.23 (ddd, 1H, J = 6.0 Hz, J = 2.6 Hz, J = 1.3 Hz), 4.10 (ddd, 1H, J = 8.5 Hz, J = 5.2 Hz, J = 2.9 Hz), 3.77 (dd, 1H, J = 8.5 Hz, J = 6.1 Hz)

2.5. Synthesis of 3,4-di-0-benzyl-6-0-trideuteroacetyl-2-deoxy-D-glucose (15)

3.4-di-0-benzyl-6-0-trideuteroacetyl-D-glucal (14) (4.4 g, 11.8 mmol) was dissolved in THF (44 mL). 48% HBr water solution (1 mL, 8.8 mmol) was added and the reaction mixture was stirred at room temp until all substrate was consumed (TLC). Reaction mixture was diluted with water (50 mL), then NaHCCb (8.8 mmol, 0.74 g) was added, and the reaction mixture was stirred additional 15 min. Ethyl acetate (100 mL) was added and layers were separated. Organic layer was washed with water (2 x 50 mL) and dried over Na2S04. Drying agent was filtered off and solvent was evaporated to dryness, and product was purified by column chromatography using hexanes/ethyl acetate gradient for elution. Fractions contained product were pooled together and evaporated to dryness to give 4.1 g of 3,4-di-0-benzyl-6-0- trideuteroacetyl-2-deoxy-D-glucose (Yield 89.1 %)

2.6. Synthesis of 6-0-trideuteroacetyl-2-deoxy-D-glucose (16)

3.4-di-0-benzyl-6-0-trideuteroacetyl-2-deoxy-D-glucose (15) (4.1 g, 10.5 mmol) was dissolved in THF (50 mL). Pd(OH)2/C (20% wet, Degussa) (530 mg) was added and the mixture was hydrogenated using Paar apparatus (p = 45 psi) for 1.5 h, then reaction mixture was filtered trough Celite. Filtrate was evaporated to dryness, and product was purified by column chromatography using DCM/Methanol gradient for elution Fractions contained

product were pooled together and evaporated to dryness to give 2.1 g of 6-O-trideuteroacetyl- 2-deoxy-D-glucose (Yield 96 %)

¾ NMR (MeOD-3, d, ppm) 5.33 (d, 1H, J = 3.0 Hz), 4.91 (dd, 1H, J = 9.8 Hz, J = 2.0 Hz), 4.36 (dd, 1H, J = 12.2 Hz, J = 2.2 Hz), 4.30 (d, 2H, J = 3.5 Hz), 4.22 (dd, 1H, J = 12.1H_z, J = 5.3 Hz), 4.00 - 3.85 (m, 1H), 3.53 (ddd, 1H, J = 9.8 Hz, J = 5.3 Hz, J = 2.2 Hz), 3.37 (dd, 1H,

J = J = 9.6 Hz), 3.31 (dd, J = J = 10.2 Hz), 2.24 (ddd, J = 12.5 Hz, J = 5.0 Hz, J = 2.0 Hz),

2.10 (ddd, 1H, J = 13.4 Hz, J = 5.0 Hz, J = 1.0 Hz), 1.67 (ddd, 1H. J = 11.9 Hz, J = 9.8 Hz, J = 3.6 Hz), 1.49 (ddd, 1H, J = 12.1 Hz, J = 9.8 Hz, J = 12.1 Hz)

a:b ratio ~l : l

3. Synthesis of 6-0-acetyl-3-0-trideuteroacetyl-2-deoxy-D-glucose (23)

3.1. Synthesis of 4-0-benzyl-6-tert-butyldimethylsilyl-D-glucal (18)

4-O-Benzyl-D-glucal (17) (2.65 g, 11.2 mmol) was dissolved in dichloromethane (26 mL). Imidazole (1.26 g, 18.45 mmol) followed by tert-butyldimethylsilyl chloride (1.86 g, 12.3 mmol) were added and the reaction mixture was stirred at room temperature. Progress of the reaction was monitored using TLC method. After reaction was completed the reaction mixture was diluted with dichloromethane (100 mL), washed with water (3 x 100 mL), and then dried over NaiSCL. Drying agent was filtered off and solvent was evaporated to dryness and product was purified by column chromatography using hexanes/ethyl acetate gradient for

elution. Fractions contained product were pooled together and evaporated to dryness to give

3.3 g of 4-0-benzyl-6-tert-butyldimethylsilyl-D-glucal (Yield 87.8%). The structure of the product was confirmed by Ή NMR method and compared with literature.

3.2. Synthesis of 4-0-benzyl-6-tert-butyldimethylsilyl-3-0-trideuteroacetyl-D-glucal (19)

4-0-benzyl-6-tert-butyldimethylsilyl-D-glucal (3.3 g, 9.9 mmol) was dissolved in

dichloromethane (30 mL). Pyridine (1.4 mL) followed by per-deuterated acetyl anhydride (1.15 mL, 11.8 mmol) were added and the reaction mixture was stirred at room temperature overnight. After reaction was completed the reaction mixture was diluted with DCM (100 mL) and washed with 10% NaiCCh water solution, then with water until neutral and dried over NaiSCri. Drying agent was filtered off and solvent was evaporated to dryness, and product was purified by column chromatography using hexanes/ethyl acetate gradient for elution. Fractions contained product were pooled together and evaporated to dryness to give

3.4 g of 4-0-benzyl-6-tert-butyldimethylsilyl-3-0-trideuteroacetyl-D-glucal (Yield 86.7%)

¾ NMR (CDCb, d, ppm), 7.36 - 7.28 (m, 5 H, Harom), 6.41 (dd, 1H, J = 6.1 Hz, J = 1.3 Hz), 5.41 (ddd, 1H, J = 6.4 Hz, J = 3.0 Hz, J = 1.7 Hz), 4.73 (s, 2H), 4.71 (dd, 1H, J = 6.0 Hz, J = 2.9 Hz), 4.0 - 3.8 (m, 4H), 0.91 (s, 9H), 0.08 (s, 6H)

3.3. Synthesis of 4-0-benzyl-3-0-trideuteroacetyl-D-glucal (20)

4-0-benzyl-6-tert-butyldimethylsilyl-3-0-trideuteroacetyl-D-glucal (3.4 g, 8.6 mmol) was dissolved in THF (35 mL). Tetra-butylammonium fluoride (2.24 g, 8.6 mmol) was added and the reaction mixture was stirred at room temperature. After reaction was completed (TLC) solvent was evaporated to dryness and ethyl acetate (100 mL) was added. Obtained solution was washed with water (3 x 50 mL) then dried over Na2S04. Drying agent was filtered off and solvent was evaporated to dryness product was purified by column chromatography using hexanes/ethyl acetate gradient for elution. Fractions contained product were pooled together

and evaporated to dryness to give 1.6 g of 4-0-benzyl-3-0-trideuteroacetyl-D-glucal (Yield 66.1%)

¾ NMR (CDCb, d, ppm), 7.36 - 7.28 (m, 5 H, Harom), 6.41 (dd, 1H, J = 6.0 Hz, J = 1.4 Hz), 5.46 (ddd, 1H, J = 6.7 Hz, J = 2.5 Hz, J = 1.5 Hz), 4.76 (dd, 1H, J = 6.0 Hz, J = 2.6 Hz), 4.74 (d, lH, J = 11.6 Hz), 4.70 (d, 1H, J = 11.6 Hz), 4.0 - 3.84 (m, 4H), 1.81 (dd, 1H, J = 6.8 Hz,

H = 6.4 Hz)

3.4. Synthesis of 6-0-acetyl-4-0-benzyl-3-0-trideuteroacetyl-D-glucal (21)

4-0-benzyl-3-0-trideuteroacetyl-D-glucal (1.6 g, 5.7 mmol) was dissolved in

dichloromethane (20 mL). Pyridine (0.7 mL) followed by acetyl chloride (0.45 mL, 6.26 mmol) were added and the reaction mixture was stirred at room temperature. After reaction was completed (TLC) the reaction mixture was diluted with DCM (100 mL) and washed with 10% NaiCCb water solution, then with water until neutral and dried over NaiSCri. Drying agent was filtered off and solvent was evaporated to dryness, and product was purified by column chromatography using hexanes/ethyl acetate gradient for elution. Fractions contained product were pooled together and evaporated to dryness to give 1.76 g of 6-0-acetyl-4-0- benzyl-3-O-trideuteroacetyl-D-glucal (Yield 95.5%)

¾ NMR (CDCL, d, ppm), 7.40 - 7.28 (m, 5 H, Harom), 6.42 (dd, 1H, J = 6.1 Hz, J = 1.1 Hz), 5.45 - 5.39 (m, lH), 4.8 l (dd, 1H, J = 6.1 Hz, J = 3.1 Hz), 4.72 (d, 1H, J = 11.6 Hz), 4.64 (d, lH, J = 11.6 Hz), 4.40 (dd, 1H, J = 12.1 Hz, J = 3.2 Hz), 4.32 (dd, 1H, J = 12.1 Hz, J = 5.3 Hz), 4.16 (ddd, 1H, J = 8.1 Hz, J = 5.3 Hz, J = 3.2 Hz), 3.81 (dd, 1H, J = 8.1 Hz, J = 6.0 Hz), 2.07 (s, 3H)

3.5. Synthesis of 6-0-acetyl-4-0-benzyl-3-0-trideuteroacetyl-2-deoxy-D-glucose (22)

6-0-acetyl-4-0-benzyl-3-0-trideuteroacetyl-D-glucal (1.76 g, 5.4 mmol) was dissolved in THF (20 mL). 48% HBr water solution (0.61 mL, 5.4 mmol) was added and the reaction mixture was stirred at room temp until all substrate was consumed (TLC). Reaction mixture

was diluted with water (20 mL), then NaHCC (5.4 mmol, 0.45 g) was added, and the reaction mixture was stirred additional 15 min. Ethyl acetate (50 mL) was added and layers were separated. Organic layer was washed with water (2 x 50 mL) and dried over Na2S04. Drying agent was filtered off and solvent was evaporated to dryness, and product was purified by column chromatography using hexanes/ethyl acetate gradient for elution fractions contained product were pooled together and evaporated to dryness to give 1.18 g of 6-O- acetyl -4-0-benzyl-3-0-trideuteroacetyl-2-deoxy-D-glucose (Yield 64.1%)

3.6. Synthesis of 6-0-acetyl-3-0-trideuteroacetyl-2-deoxy-D-glucose (23)

6-0-acetyl-4-0-benzyl-3-0-trideuteroacetyl-2-deoxy-D-glucose (1.18 g, 3.5 mmol) was dissolved in THL (18 mL). Pd(OH)2/C (20% wet, Degussa) (160 mg) was added and the mixture was hydrogenated using Paar apparatus (p = 45 psi) for 1 h, then reaction mixture was filtered trough Celite. filtrate was evaporated to dryness, and product was purified by column chromatography using DCM/Methanol gradient for elution fractions contained product were pooled together and evaporated to dryness to give 0.85 g of 6-0-acetyl-3-0- trideuteroacetyl-2-deoxy-D-glucose (Yield 97 %) (L25-98-2)

¾-NMR (DMSO-d6, d, ppm), 6.47 (d, 1H, J = 4.0 Hz), 5.33 (d, 1H, J = 6.1 Hz), 5.14 (dd,

1H, J = J = 3.0 Hz), 5.0 (ddd, 1H, J = 11.5 Hz, J = 9.2 Hz, J = 5.0 Hz), 4.25 (dd, 1H, J = 11.8 Hz, J = 1.8 Hz), 4.06 (dd, 1H, J = 11.7 Hz, J = 5.9 Hz), 3.86 (ddd, 1H, J = 7.6 Hz, J = 5.8 Hz, J = 1.8 Hz), 3.32 - 3.31 (m, 1H), 2.03 (s, 3H), 1.91 (dd, 1H, J = 12.3 Hz, J = 5.1 Hz), 1.55 - 1.44 (m, 1H); a isomer only

4. Synthesis of 3-0-acetyl-6-0-trideuteroacetyl-2-deoxy-D-glucose (28)

4.1. Synthesis of 4-0-benzyl-6-tert-butyldimethylsilyl-3-0-acetyl-D-glucal (24) 4-0-benzyl-6-tert-butyldimethylsilyl-3-0-acetyl-D-glucal (2.3 g, 6.8 mmol), was dissolved in dichloromethane (23 mL). Pyridine (1 mL) followed by acetyl anhydride (0.8 mL, 8.2 mmol) were added and the reaction mixture was stirred at room temperature. After reaction was completed (TLC) the reaction mixture was diluted with DCM (50 mL) and washed with 10% NaiCCh water solution, then with water until neutral and dried over NaiSCri. Drying agent was filtered off and solvent was evaporated to dryness, and product was purified by column chromatography using hexanes/ethyl acetate gradient for elution. Fractions contained product were pooled together and evaporated to dryness to give 2.25g of 4-0-benzyl-6-tert- butyldimethylsilyl-3-O-acetyl-D-glucal (Yield 83.8%). The structure of the product was confirmed by Ή NMR method and compared with literature.

4.2. Synthesis of 3-0-acetyl-4-0-benzyl-D-glucal (25)

4-0-benzyl-6-tert-butyldimethylsilyl-3 -O-acetyl -D-glucal (2.25 g, 5.7 mmol) was dissolved in THF (20 mL). Tetra-butylammonium fluoride (1.5 g, 5.7 mmol) was added and the reaction mixture was stirred at room temperature. After reaction was completed (TLC) solvent was

evaporated to dryness and ethyl acetate (50 mL) was added. Obtained solution was washed with water (3 x 50 mL) then dried over Na2S04. Drying agent was fdtered off and solvent was evaporated to dryness product was purified by column chromatography using hexanes/ethyl acetate gradient for elution. Fractions contained product were pooled together and evaporated to dryness to give 1.2 g of 3 -O-acetyl -4-0- benzyl-D-glucal (Yield 75.6%). The structure of the product was confirmed by Ή NMR method and compared with literature.

4.3. Synthesis of 3-0-acetyl-4-0-benzyl-6-0-trideuteroacetyl-D-glucal (26)

3-0-acetyl-4-0-benzyl-D-glucal (1.2 g, 4.3 mmol) was dissolved in dichloromethane (12 mL). Pyridine (0.76 mL) followed by per-deuterated acetyl anhydride (0.445 mL, 4.7 mmol) were added and the reaction mixture was stirred at room temperature overnight. After reaction was completed the reaction mixture was diluted with DCM (30 mL) and washed with 10% Na2C03 water solution, then with water until neutral and dried over NarSOu Drying agent was filtered off and solvent was evaporated to dryness, and product was purified by column chromatography using hexanes/ethyl acetate gradient for elution. Fractions contained product were pooled together and evaporated to dryness to give 1.31 g of 3 -O-acetyl -4-0-benzyl-6-0- trideuteroacetyl-D-glucal (yield 94.2%)

4.4. Synthesis of 3-0-acetyl-4-0-benzyl-6-0-trideuteroacetyl-2-deoxy-D-glucose (27)

3-0-acetyl-4-0-benzyl-6-0-trideuteroacetyl-D-glucal (1.3 g, 4.05 mmol) was dissolved in THF (13 mL). 48% HBr water solution (0.46 mL, 4.05 mmol) was added and the reaction mixture was stirred at room temp until all substrate was consumed (TLC). Reaction mixture was diluted with water (20 mL), then NaHC03 (4.05 mmol, 0.34 g) was added, and the reaction mixture was stirred additional 15 min. Ethyl acetate (30 mL) was added and layers were separated. Organic layer was washed with water (2 x 20 mL) and dried over NarSOu Drying agent was filtered off and solvent was evaporated to dryness, and product was purified by column chromatography using hexanes/ethyl acetate gradient for elution. Fractions

contained product were pooled together and evaporated to dryness to give 0.82 g of 3-0- acetyl-4-0-benzyl-6-0-trideuteroacetyl-2-deoxy-D-glucose (Yield 59.4 %)

4.5. Synthesis of 3-0-acetyl-6-0-trideuteroacetyl-2-deoxy-D-glucose (28)

3-0-acetyl-4-0-benzyl-6-0-trideuteroacetyl-2-deoxy-D-glucose (0.82 g, 2.4 mmol) was dissolved in THF (15 mL). Pd(OH)2/C (20% wet, Degussa) (150 mg) was added and the mixture was hydrogenated using Paar apparatus (p = 45 psi) for 1 h, then reaction mixture was filtered trough Celite. Filtrate was evaporated to dryness, and product was purified by column chromatography using DCM/Methanol gradient for elution Fractions contained product were pooled together and evaporated to dryness to give 0.57 g of 3-0-acetyl-6-0- trideuteroacetyl-2-deoxy-D-glucose (Yield 95 %) (F25-99-2)

¾-NMR (DMSO-d6, d, ppm), 6.47 (dd, 1H, J = 4.0 Hz, J = 1.4 Hz), 5.33 (d, 1H, J = 6.1 Hz), 5.17 dd, 1H, J = J = 3.1 Hz), 5.03 (ddd, 1H, J = 11.2 Hz, J = 9.3 Hz, J = 5.lHz), 4.25 (dd, 1H, J = 11.7 Hz, J = 1.8 Hz), 4.09 (dd, 1H, J = 11.7 Hz, J = 5.8 Hz), 3.89 (ddd, 1H, J = 9.7 Hz, J = 5.8 Hz, J = 1.8 Hz), 3.30 (ddd, 1H, J = 9.6 Hz, J = 9.5 Hz, J = 8.1 Hz), 2.02 (s, 3H), 1.94 (ddd, 1H, J = 12.5 Hz, J = 5.1 Hz, J = 1.1 Hz), 1.56 - 1.44 (m, 1H)

The particular combinations of elements and features in the above detailed embodiments are exemplary only; the interchanging and substitution of these teachings with other teachings in this and the patents/applications incorporated by reference are also expressly contemplated.

As those skilled in the art will recognize, variations, modifications, and other implementations of what is described herein can occur to those of ordinary skill in the art without departing from the spirit and the scope of the invention as claimed. Accordingly, the foregoing description is by way of example only and is not intended as limiting. In the claims, the word "comprising" does not exclude other elements or steps, and the indefinite article "a" or "an" does not exclude a plurality. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measured cannot be used to advantage. The invention's scope is defined in the following claims and the

equivalents thereto. Furthermore, reference signs used in the description and claims do not limit the scope of the invention as claimed.

Claims

C l a i m s

1. Compounds according to structure (I)

2. Compunds according to claim 1, whereby at least one of R¹ to R³ is hydroxyl

3. Compounds according to claim 1 or 2, whereby either R¹ or R³ is -OCOCD₃.

4. A synthesis method for the compounds according to any of the claims 1 to 3, involving the steps of: a) reacting a suitable precursor compound with a silylating agent to obtain a monosilyl-protected precursor compound b) optinal protecting of further hydroxy groups of the compound obtained in step a) by orthogonal protective groups c) detachment of the silyl protective group

5. The method of claim 4, whereby a sterically demanding silyl group is used in step a)

6. The method of claim 4 or 5, whereby a silyl group comprising aryl or t-butyl moieties is used in step a)

7. The method of any of the claims 4 to 6, whereby the silyl group in step a) is TBDMS (t-Butyldimetylsilyl) .

8. The method of any of the claims 4 to 7, whereby in step b) benzyl and/or substituted benzyl groups are used.

9. The method of any of the claims 4 to 8, thereby in step c) TBAF (Tertbutylammonium fluoride) is used

10. The method of any of the claims 4 to 9, whereby in step d) per-deuterated acetyl anhydride and/or deuterated acyl halides are used.

11. The method of any of the claims 4 to 10, whereby the precursor compound in step a) is a glucal or glucal derivative and the method includes a step f) which can be performed after step d) or e).

12. The method according to claim 11, whereby step f) is performed by first adding

aqueos solution of H-Hal (with Cl and Br especially preferred), followed by neutralization.

13. The method according to claim 12, whereby neutralization is performed using

carbonate, hydrogen carbonate and/or phosphate solution.

14. Use of a compound according to any of the claims 1 to 3 for compounds for the analysis of the metabolism of glycolysis inhibitors.

15. An analysis method method for the metabolism of glycolysis inhibitors including the step of subjecting a compound according to any of the claims 1 to 3 in an assay.

16. The method according to claim 15, whereby the assay is in vivo.

17. The method according to claim 16, whereby the assay is in vitro.