PROCESS FOR THE MANUFACTURE OF (2S,3S,4S,5R,6S)-3,4,5-TRIHYDROXY-6-(((4AR,10AR)-7-HYDROXY-1-PROPYL-1,2,3,4,4A,5,10,10A-OCTAHYDROBENZO[G]QUINOLIN-6-YL)OXY)TETRAHYDRO-2H-PYRAN-2-CARBOXYLIC ACID

Abstract

The present invention relates to a process for manufacturing (2S,3S,4S,5R,6S)-3,4,5-trihydroxy-6-(((4aR,10aR)-7-hydroxy-1-propyl-1,2,3,4,4a,5,10,10a-octahydrobenzo[g]quinolin-6-yl)oxy)tetrahydro-2H-pyran-2-carboxylic acid with the formula (Id) below and pharmaceutically acceptable salts thereof

##STR00001##

The compound of formula (Id) is a prodrug of a catecholamine for use in treatment of neurodegenerative diseases and disorders such as Parkinson's Disease.

The invention also relates to a new intermediate of said process.

Claims

1.-15. (canceled)

16. A process for the preparation of compound (Id) ##STR00042## or a pharmaceutically acceptable salt thereof, comprising deprotecting compound (A3) to obtain compound (Id), or a pharmaceutically acceptable salt thereof, according to the reaction scheme below: ##STR00043##

17. The process of claim 16, comprising deprotecting compound (A3) by contacting compound (A3) with a nucleophilic reagent to obtain compound (Id), or a pharmaceutically acceptable salt thereof.

18. The process according to claim 17, further comprising the step of isolating compound (Id), or a pharmaceutically acceptable salt thereof.

19. The process according to claim 17, wherein said nucleophilic reagent is selected from potassium hydroxide, potassium cyanide, and sodium hydroxide.

20. The process according to claim 17, wherein said deprotection takes place in a mixture of methanol and water.

21. The process according to claim 17, wherein compound (Id) is obtained as a potassium salt of compound (Id), and wherein potassium hydroxide or potassium cyanide is used as nucleophilic reagent.

22. The process according to claim 17, wherein compound (Id) is obtained as a potassium salt of compound (Id), and wherein potassium hydroxide is used as nucleophilic reagent.

23. The process according to claim 17, wherein compound (Id) is obtained as a sodium salt of compound (Id), and wherein sodium hydroxide is used as nucleophilic reagent.

24. The process according to claim 19, wherein compound (Id) is obtained in a solution, and the process further comprises neutralizing the solution with a strong acid.

25. The process according to claim 24, wherein the strong acid is HCl.

26. The process according to claim 17, wherein compound (Id) is obtained as (2S,3S,4S,5R,6S)-3,4,5-trihydroxy-6-(((4aR,10aR)-7-hydroxy-1-propyl-1,2,3,4,4a,5,10,10a-octahydrobenzo[g]quinolin-6-yl)oxy)tetrahydro-2H-pyran-2-carboxylic acid heptahydrate.

27. The process according to claim 21, further comprising formulating the potassium salt of compound (Id) into a solid oral dosage form.

28. The process according to claim 22, further comprising formulating the potassium salt of compound (Id) into a solid oral dosage form.

29. The process according to claim 23, further comprising formulating the sodium salt of compound (Id) into a solid oral dosage form.

30. The process according to claim 24, further comprising formulating compound (Id), or a pharmaceutically acceptable salt thereof, into a solid oral dosage form.

31. The process according to claim 26, further comprising formulating (2S,3S,4S,5R,6S)-3,4,5-trihydroxy-6-(((4aR,10aR)-7-hydroxy-1-propyl-1,2,3,4,4a,5,10,10a-octahydrobenzo[g]quinolin-6-yl)oxy)tetrahydro-2H-pyran-2-carboxylic acid heptahydrate compound (Id) into a solid oral dosage form.

32. The process according to claim 17, wherein compound (A3) is prepared using the following step reacting compound (A2) with (2S,3S,4S,5R,6R)-2-(methoxycarbonyl)-6-(2,2,2-trichloro-1-iminoethoxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate to obtain compound (A3) according to the reaction scheme below ##STR00044## wherein said reaction takes place in an aprotic solvent in the presence of a Lewis acid.

33. The process according to claim 32, further comprising the step of isolating compound (A3).

34. The process according to claim 32, wherein said aprotic solvent is dichloromethane or benzotrifluoride.

35. The process according to claim 32, wherein said aprotic solvent is dichloromethane, and said Lewis acid is boron trifluoride diethyl etherate.

36. The process according to claim 32, wherein said aprotic solvent is benzotrifluoride, and said Lewis acid is boron trifluoride diethyl etherate.

Description

BRIEF DESCRIPTION OF FIGURES

[0076] FIG. 1: PK profiles in Wistar rats obtained after oral dosing according to Example 7. Profiles are based on mean plasma concentrations from 3 subjects for each compound.

[0077] X-axis: time (hours); Y-axis: plasma concentration of Compound (1) (μg/mL) obtained after dosing of the following compounds 0: compound (Ia); A: compound (Ib); +: compound (Id).

[0078] FIGS. 2-3: Locomotor activity time-course (FIG. 2) and total distance travelled (FIG. 3) following treatment with vehicle (H.sub.2O, p.o.), or compound (Id) (10, 30, 100 or 300 μg/kg, p.o.) and compared to standard-of-care (SoC) treatments: apomorphine (APO, 3 mg/kg, s.c.), pramipexole (PPX, 0.3 mg/kg, s.c.). Animals were dosed at t=60 minutes after a 60-minutes habituation period in test chambers, and activity was monitored for 350 minutes thereafter.

[0079] Data was evaluated by use of a Kruskal-Wallis test with Dunn's Multiple Comparisons test, resulting in an overall P-value of <0.0001.

[0080] FIG. 2: X-axis: time (min); Y-axis: Distance travelled (cm) ±SEM/5-minute-bins FIG. 3: Y-axis: Total distance travelled (cm) ±SEM. Significance levels for post-hoc comparisons (relative to the vehicle group) are indicated: *<0.05, **<0.01, ***<0.001, ****<0.0001.

[0081] FIGS. 4-5: Relationships between plasma concentrations of compound (Id) and compound (I) and hyperactivity induced by compound (Id) (100 μg/kg, p.o.) (FIG. 4) and the corresponding relationship between plasma apomorphine concentrations and hyperactivity induced by apomorphine (3 mg/kg, s.c.) (FIG. 5).

[0082] X-axis time (min); Y-axis left: Distance travelled (cm) ±SEM/5-minute-bins; Y-axis right (FIG. 4): plasma concentration of compound (I) (μg/mL); Y axis right (FIG. 5): plasma concentration of apomorphine (ng/mL).

[0083] ∇: Distance traveled (cm).Math.plasma concentration.

[0084] FIG. 6: Conversion of compound (Id) to compound (I) in rat (FIG. 6A) and human (FIG. 6B) hepatocytes.

[0085] X-axis time (min); Y-axis: concentration of compound (I) (μg/mL).

[0086] FIG. 7: Conversion of compound (Id) in rat (FIG. 7A) and human (FIG. 7B) whole blood.

[0087] X-axis time (min); Y-axis: concentration of compound (I) (μg/mL).

DETAILED DESCRIPTION OF THE INVENTION

[0088] The present invention relates to a process for manufacturing the compound (2S,3S,4S,5R,6S)-3,4,5-trihydroxy-6-(((4aR,10aR)-7-hydroxy-1-propyl-1,2,3,4,4a,5,10,10a-octahydrobenzo[g]quinolin-6-yl)oxy)tetrahydro-2H-pyran-2-carboxylic acid with the formula (Id) below and salts thereof

##STR00013##

[0089] The compound of formula (Id) is a prodrug of (4aR,10aR)-1-Propyl-1,2,3,4,4a,5,10,10a-octahydro-benzo[g]quinoline-6,7-diol [compound (I)] which is a dual D1/D2 agonist with in vitro data listed in Table 2.

[0090] The inventors have observed that compound (I) is conjugated in rat and human hepatocytes to sulfate and glucuronide derivatives including compound (Id). The conjugates have shown to be converted to compound (I) by conjugation and de-conjugation in the body.

[0091] Glucuronide and sulfate derivatives are commonly known to be unstable in the intestine. The derivatives are formed as highly polar and soluble metabolites to facilitate the elimination of compounds from the body and are consequently easily excreted. For example, in bile duct cannulated rats, glucuronide and sulfate conjugates are often found in bile while their de-conjugate (i.e. the parent compound) is found in faeces. The back-conversion of glucuronide and sulfate conjugates in the intestine to the parent compound which is then sometimes subsequently reabsorbed, is known as part of the enterohepatic re-circulation process. As mentioned earlier, oral dosing of phenethyl catecholamines, such as apomorphine, has generally proven unsuccessful due to low bioavailability. Likewise, compound (I) suffers from low oral bioavailability (Liu et al., Bioorganic Med. Chem. (2008), 16: 3438-3444). With this in mind and considering the instability of glucuronide and sulfate conjugates in the gastrointestinal tract, it would not be expected that oral dosing of glucuronide conjugates of compound (I) can be used to achieve sufficient plasma exposure of the compound.

[0092] The principle of applying glucuronide derivatives as prodrugs for oral delivery has been explored for retinoic acid (Goswami et al., J. Nutritional Biochem. (2003) 14: 703-709) and for morphine (Stain-Texier et al., Drug Metab. and Disposition (1998) 26 (5): 383-387). Both studies showed very low exposure levels of the parent compounds after oral dosing of the derivatives. Another study suggests the use of budenoside-ß-D-glucuronide as a prodrug for local delivery of budenoside to the large intestine for treatment of Ulcerative Colitis based on poor absorption of the prodrug itself from the intestinal system (Nolen et al., J. Pharm Sci. (1995), 84 (6): 677-681).

[0093] Nevertheless, surprisingly, it has been observed that oral dosing of compound (Id) which has been identified as a metabolite of compound (I) in rats and minipigs provides a systemic exposure of compound (I) in plasma, suggesting the usefulness of said compound as an orally active prodrug of compound (I).

[0094] The plasma profile of compound (I) resulting from oral dosing of compounds (Ia) and (Ib) and compound (Id) to Wistar rats according to Example 7 are shown in FIG. 1. For all the compounds, the doses were corrected by molecular weight to equal a dose of 300 μg/kg of compound (Ib) corresponding to 287 μg/kg of compound (I). The inventors have found that oral dosing of compounds (Ia) and (Ib) to Wistar rats results in early and high peak concentrations of compound (I). Such high peak concentrations are in humans likely to be associated with dopaminergic side effects such as for example nausea, vomiting and light headedness. In contrast, dosing of the compound (Id), results in a slower absorption rate avoiding rapid peak concentrations accompanied by a sustained exposure of compound (I) in plasma. Additionally, the plasma exposure of compound (I) in Wistar rats is maintained throughout 24 hours although the obtained AUC of compound (I) is generally lower than the AUC obtained after dosing of compound (Ib). However, since the peak concentrations of compound (I) which are expected to drive the side effects are lower, higher doses might be administered of the compound (Id) to potentially achieve higher overall plasma concentrations of compound (I) compared to what is achievable from dosing compounds (Ia) and (Ib). When investigating PK properties of compound (Ic), the inventors found that the plasma concentrations of compound (I) were extremely low, leaving compound (Ic) unsuitable as a prodrug of compound (I) for oral administration and confirming that the oral bioavailability demonstrated for the compound of formula (Id) was highly unpredictable. PK parameters for the PK studies in Wistar rats are listed in Table 3.

[0095] In vivo conversion of compound (Id) to compound (I) has also been observed by after oral dosing of compound (Id) in minipigs.

[0096] Bioconversion of compound (Id) in human is supported by the Experiments of Example 4a and Example 4b indicating conversion to the compound of formula (I) in rat and human hepatocytes and in rat and human blood (FIGS. 6 and 7).

[0097] Thus, in conclusion, the compound of formula (Id) is useful as an orally active prodrug of compound (I) and has been observed in rats to provide a PK profile avoiding the peak C.sub.max observed for the known prodrugs (Ia) and (Ib) and providing a significantly higher AUC of compound (I) than compound (Ic).

[0098] Compound (Id) has further been explored in the rat locomotor activity assay according to Example 8. The assay demonstrated a dopaminergic effect obtained after oral administration of compound (Id) c.f. FIGS. 2, 3 and 4. The fact that the compound of formula (Id) possesses no in vitro dopaminergic activity c.f. example 5 and table 2, further indicates that the effect of compound (Id) in the rat locomotor activity assay is obtained by conversion of compound (Id) to compound (I).

[0099] Finally, an important issue associated with the prior art compound (Ib) is that this compound is an agonist of the 5-HT2B receptor. Since 5-HT2B receptor agonists have been linked to pathogenesis of valvular heart disease (VHD) after long term exposure, such compounds are not suitable for use in the treatment of chronical diseases (Rothman et al., Circulation (2000), 102: 2836-2841; and Cavero and Guillon, J. Pharmacol. Toxicol. Methods (2014), 69: 150-161). Thus, a further advantage of compound (Id) is that the compound is not a 5-HT2B agonists c.f. example 6 and Table 2.

[0100] The compound of formula (Id) is useful in the treatment of neurodegenerative diseases and disorders such as Parkinson's disease and/or other conditions for which treatment with a dopamine agonist is therapeutically beneficial. The compound, being suitable for oral administration has the potential of providing a new treatment paradigm in Parkinson's Disease.

[0101] The invention provides a scalable synthesis of compound (Id). A key step is a direct glucuronide coupling reaction on compound (A2) using (2S,3S,4S,5R,6R)-2-(methoxycarbonyl)-6-(2,2,2-trichloro-1-iminoethoxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate as the coupling donor. The invention also comprises a deprotection step utilizing sodium hydroxide in methanol/water thereby avoiding the use of for example toxic KCN. The overall process starting from compound (I) is illustrated in brief in scheme 4 below.

##STR00014##

[0102] A process for the preparation of compound (I) to be used in step 1) has been disclosed in WO 2009/026934. WO2019/101917 discloses a process for preparation of the compound A2 and compound (Id).

[0103] Table 1 below provide an overview of the compounds (A2) and (A3) which are intermediates with the following respective compound names:

TABLE-US-00001 TABLE 1 Overview of intermediates Abbreviated name Chemical Name Structure drawing (A2) (4aR,10aR)-1-propyl-7- ((triisopropylsilyl)oxy)- 1,2,3,4,4a,5,10,10a- octahydrobenzo[g]quinolin-6-ol [00015] (A3) (2S,3S,4S,5R,6S)-2- (methoxycarbonyl)-6- (((4aR,10aR)-1-propyl-7- ((triisopropylsilyl)oxy)- 1,2,3,4,4a,5,10,10a- octahydrobenzo[g]quinolin-6- yl)oxy)tetrahydro-2H-pyran- 3,4,5-triyl triacetate [00016]

[0104] The reactant triisopropylsilyl chloride, used in step 1), can be purchased at Sigma-Aldrich (CAS Number: 13154-24-0).

[0105] The reactant (2S,3S,4S,5R,6R)-2-(methoxycarbonyl)-6-(2,2,2-trichloro-1-iminoethoxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate, used in step 2), can be purchased at Sigma-Aldrich (CAS Number: 92420-89-8).

In Step 1) Compound (I) is Selectively Protected with a Triisopropylsilyl (TIPS) Protection Group to Afford the Compound (A2)

[0106] ##STR00017##

[0107] Compound (1) is reacted with triisopropylsilyl chloride in an aprotic solvent in the presence of a base. The inventors found that performing the reaction in an aprotic solvent such as dichloromethane (CH.sub.2Cl.sub.2), sulfolane or methyl-isobutylketone (MIBK) in the presence of a base such as N,N-diisopropylethylamine (DIPEA) or triethylamine resulted in a high conversion and selectivity. High conversion was observed when using 4-5 eq. DIPEA and performing the reaction at room temperature.

[0108] In one embodiment of the invention, step 1 is performed using dichloromethane (CH.sub.2Cl.sub.2) as solvent.

[0109] In another embodiment of the invention, step 1 is performed using sulfolane as solvent.

[0110] In yet another embodiment of the invention, step 1 is performed using methyl-isobutylketone (MIBK) as solvent.

In Step 2) Compound (A2) is Coupled with (2S,3S,4S,5R,6R)-2-(methoxycarbonyl)-6-(2,2,2-trichloro-1-iminoethoxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate

[0111] ##STR00018##

[0112] The reaction takes place in an aprotic solvent, preferably dichloromethane or benzotrifluoride, in the presence of a Lewis acid, preferably boron trifluoride diethyl etherate.

In Step 3) Compound (A3) is Deprotected Using a Suitable Nucleophilic Reagent to Afford Compound (Id) or a Pharmaceutically Acceptable Salt Thereof

[0113] ##STR00019##

[0114] The deprotection takes place in a solvent, for example a mixture of methanol (MeOH) and water, in the presence of a suitable nucleophilic reagent, for example a base, preferably a hydroxide base such as potassium hydroxide (KOH) or sodium hydroxide (NaOH).

[0115] In one embodiment, step 3) takes place in the presence of a solvent, such as a mixture of methanol (MeOH) and water.

[0116] In one embodiment of the invention, step 3 takes place using one or more suitable nucleophilic reagents, such as for example a hydroxide base and NH.sub.4F. More specifically, step 3 may take place using a combination of NH.sub.4F and potassium hydroxide (KOH) or sodium hydroxide (NaOH).

[0117] In a specific embodiment, step 3 takes place using potassium hydroxide (KOH) and NH.sub.4F.

Embodiments of the Invention

[0118] In the following, embodiments of the invention are disclosed. The first embodiment is denoted E1, the second embodiment is denoted E2 and so forth.

[0119] E1. A process for the preparation of compound (Id) with the formula below

##STR00020## [0120] from compound (I) with the formula below

##STR00021## [0121] wherein said process comprises the following step [0122] 2) reacting compound (A2) with (2S,3S,4S,5R,6R)-2-(methoxycarbonyl)-6-(2,2,2-trichloro-1-iminoethoxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate to obtain compound (A3) according to the reaction scheme below

##STR00022## [0123] wherein said reaction takes place in an aprotic solvent in the presence of a Lewis acid.

[0124] E2. A process for the manufacturing of compound (A3) below comprising the following step [0125] 2) reacting compound (A2) with (2S,3S,4S,5R,6R)-2-(methoxycarbonyl)-6-(2,2,2-trichloro-1-iminoethoxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate to obtain compound (A3) according to the reaction scheme below

##STR00023## [0126] wherein said reaction takes place in an aprotic solvent in the presence of a Lewis acid.

[0127] E3. The process according to any of embodiments 1-2, wherein said aprotic solvent used in step 2) is dichloromethane.

[0128] E4. The process according to any of embodiments 1-3, wherein said Lewis acid used in step 2) is boron trifluoride diethyl etherate.

[0129] E5. The process according to any of embodiments 1-4, wherein said aprotic solvent is dichloromethane and said Lewis acid is boron trifluoride diethyl etherate.

[0130] E6. The compound of formula (A3) below

##STR00024## [0131] or a salt thereof.

[0132] E7. Use of a compound according to embodiment 6, in a process for the manufacture of the compound of formula (Id).

[0133] E8. A process for the preparation of compound (Id) with the formula below

##STR00025## [0134] from compound (I) with the formula below

##STR00026## [0135] wherein said process comprises the following step [0136] 3) deprotecting compound (A3) by contacting compound (A3) with a base to obtain compound (Id) according to the reaction scheme below

##STR00027##

[0137] E9. The process according to any of embodiments 1 and 3-5 wherein step 2) is followed by the following step [0138] 3) deprotecting compound (A3) by contacting compound (A3) with a base to obtain compound (Id) according to the reaction scheme below

##STR00028##

[0139] E10. The process according to any of embodiments 8-9, wherein said base used in step 3) is selected from potassium hydroxide and sodium hydroxide.

[0140] E11. The process according to any of embodiments 8-10, wherein said deprotection takes place in a mixture of methanol and water.

[0141] E12. The process according to any of embodiments 1-5 and 9-11, wherein compound (A2) has been obtained by the following step [0142] 1) reacting compound (I) with triisopropylsilyl chloride to obtain compound (A2) according to the reaction scheme below

##STR00029## [0143] wherein the reaction takes place in an aprotic solvent in the presence of a base.

[0144] E13. The process according to embodiment 12, wherein said aprotic solvent used in step 1) is dichloromethane.

[0145] E14. The process according to any of embodiments 12-13, wherein said base used in step 1) is N,N-diisopropylethylamine (DIPEA).

[0146] E15. The process according to any of embodiments 12-14, wherein said aprotic solvent is dichloromethane and said base is N,N-diisopropylethylamine (DIPEA).

[0147] E16. The process according to any of embodiments 14-15, wherein said N,N-diisopropylethylamine (DIPEA) is present in an amount of 4-5 eq. relative to compound (I).

[0148] E17. The process according to any of embodiments 14-16, wherein said N,N-diisopropylethylamine (DIPEA) is present in an amount of about 4.6 eq. relative to compound (I).

[0149] E18. A process for the preparation of compound (Id) from compound (I); [0150] wherein said process comprises [0151] step 2) according to any of embodiments 1 and 3-5; followed by [0152] step 3) according to any of embodiments 8 and 10-11; [0153] wherein compound A2 used in step 2) has been obtained by [0154] step 1) according to any of embodiments 12-17.

[0155] E19. The compound (Id) with the formula below

##STR00030## [0156] obtained by the process according to any of embodiments 1, 3-5 and 8-18.

[0157] E20. The process according to any one of embodiments 1, 3 to 5, 8, 10 to 11, and 12 to 17, wherein the process comprising an additional step of formulating compound Id into a solid oral dosage form.

[0158] Items [0159] The following items serve to describe the invention and embodiments thereof.

[0160] Item 1. A process for the preparation of compound (Id) with the formula below, or a pharmaceutically acceptable salt thereof

##STR00031## [0161] from compound (I), with the formula below

##STR00032## [0162] wherein said process comprises the following step [0163] 2) reacting compound (A2) with (2S,3S,4S,5R,6R)-2-(methoxycarbonyl)-6-(2,2,2-trichloro-1-iminoethoxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate to obtain compound (A3) according to the reaction scheme below

##STR00033## [0164] wherein said reaction takes place in an aprotic solvent in the presence of a Lewis acid.

[0165] Item 2. A process for the manufacturing of compound (A3) below comprising the following step [0166] 2) reacting compound (A2) with (2S,3S,4S,5R,6R)-2-(methoxycarbonyl)-6-(2,2,2-trichloro-1-iminoethoxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate to obtain compound (A3) according to the reaction scheme below

##STR00034## [0167] wherein said reaction takes place in an aprotic solvent in the presence of a Lewis acid.

[0168] Item 3. The process according to any one of items 1-2, wherein step 2) comprises a step of isolating compound (A3).

[0169] Item 4. The process according to any one of items 1-3, wherein said aprotic solvent is dichloromethane or benzotrifluoride.

[0170] Item 5. The process according to any of items 1-4, wherein said aprotic solvent is dichloromethane and said Lewis acid is boron trifluoride diethyl etherate.

[0171] Item 6. The process according to any of items 1-4, wherein said aprotic solvent is benzotrifluoride and said Lewis acid is boron trifluoride diethyl etherate.

[0172] Item 7. A compound of formula (A3) below

##STR00035## [0173] or a salt thereof.

[0174] Item 8. Use of a compound according to item 7, in a process for the manufacture of the compound of formula (Id) or a pharmaceutically acceptable salt thereof.

[0175] Item 9. Compound (A3) directly obtained by the process according to any one of items 2-6.

[0176] Item 10. A process for the preparation of compound (Id) with the formula below

##STR00036## [0177] or a pharmaceutically acceptable salt thereof, [0178] from compound (I) with the formula below

##STR00037## [0179] wherein said process comprises the following step [0180] 3) deprotecting compound (A3) by contacting compound (A3) with a nucleophilic reagent to obtain compound (Id), or a pharmaceutically acceptable salt thereof according to the reaction scheme below

##STR00038##

[0181] Item 11. The process according to any one of items 1-6 wherein said process comprise a step 3) as defined below [0182] 3) deprotecting compound (A3) by contacting compound (A3) with a nucleophilic reagent to obtain compound (Id), or a pharmaceutically acceptable salt thereof according to the reaction scheme below.

##STR00039##

[0183] Item 12. The process according to any one of items 10-11, wherein said deprotection takes place in a mixture of methanol and water.

[0184] Item 13. The process according to any one of items 10-12, wherein said nucleophilic reagent used in step 3) is selected from potassium hydroxide, potassium cyanide, and sodium hydroxide.

[0185] Item 14. The process according to any one of items 10-13, wherein step 3) comprises the step of isolating compound (Id), or a pharmaceutically acceptable salt thereof.

[0186] Item 15. The process according to any one of items 13-14, wherein compound (Id) is obtained as a potassium salt of compound (Id), and wherein potassium hydroxide or potassium cyanide is used as nucleophilic reagent in step 3).

[0187] Item 16. The process according to any one of items 13-15, wherein compound (Id) is obtained as a potassium salt of compound (Id), and wherein potassium hydroxide is used as nucleophilic reagent in step 3).

[0188] Item 17. The process according to any one of items 10-14, wherein compound (Id) is obtained as a sodium salt of compound (Id), and wherein sodium hydroxide is used as nucleophilic reagent in step 3).

[0189] Item 18. The process according to any one of items 10-14, wherein a solution obtained in step 3) comprising compound (Id) is subsequently neutralized with a strong acid.

[0190] Item 19. The process according to item 18, wherein the strong acid is HCl.

[0191] Item 20. The process according to any one of items 18-19 wherein compound (Id) is obtained as (2S,3S,4S,5R,6S)-3,4,5-trihydroxy-6-(((4aR,10aR)-7-hydroxy-1-propyl-1,2,3,4,4a,5,10,10a-octahydrobenzo[g]quinolin-6-yl)oxy)tetrahydro-2H-pyran-2-carboxylic acid heptahydrate.

[0192] Item 21. The process according to any one of items 1-6 and 10-20, wherein compound (A2) has been obtained by the following step [0193] 1) reacting compound (I), or a salt thereof with triisopropylsilyl chloride to obtain compound (A2) according to the reaction scheme below

##STR00040## [0194] wherein the reaction takes place in an aprotic solvent in the presence of a base.

[0195] Item 22. The process according to item 21, wherein said aprotic solvent is dichloromethane, sulfolane or methyl-isobutylketone.

[0196] Item 23. The process according to any one of items 21-22, wherein said aprotic solvent is dichloromethane.

[0197] Item 24. The process according to any one of items 21-22, wherein said aprotic solvent is sulfolane.

[0198] Item 25. The process according to any one of items 21-22, wherein said aprotic solvent is methyl-isobutylketone.

[0199] Item 26. The process according to any one of items 21-25, wherein said base is N,N-diisopropylethylamine or triethylamine.

[0200] Item 27. The process according to item 21, wherein said aprotic solvent is dichloromethane and said base is N,N-diisopropylethylamine.

[0201] Item 28. The process according to any one of items 26-27, wherein said N,N-diisopropylethylamine (DIPEA) is present in an amount of 4-5 equivalents relative to the amount of compound (I).

[0202] Item 29. The process according to any one of items 21-28, wherein step 1) comprises a step of isolating compound (A2).

[0203] Item 30. A process for the preparation of compound (Id), or a pharmaceutically acceptable salt thereof, from compound (I);

[0204] wherein said process comprises [0205] step 2) according to any one of items 1 and 3-6; followed by [0206] step 3) according to any one of items 11-20; [0207] wherein compound (A2) used in step 2) has been obtained by [0208] step 1) according to any one of items 21-29.

[0209] Item 30. The compound (Id) with the formula below

##STR00041## [0210] or a pharmaceutically acceptable salt thereof [0211] obtained by the process according to any of items 1, 3-6, 11-20 and 21-29.

[0212] Item 31. The process according to any one of items 1 and 3-6, 11-20, 21-29, wherein the process comprises an additional step of formulating compound (Id), or pharmaceutically acceptable salt thereof into a solid oral dosage form.

[0213] All references, including publications, patent applications and patents, cited herein are hereby incorporated by reference in their entirety and to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety (to the maximum extent permitted by law).

[0214] Headings and sub-headings are used herein for convenience only and should not be construed as limiting the invention in any way.

[0215] The description herein of any aspect or aspect of the invention using terms such as “comprising”, “having,” “including” or “containing” with reference to an element or elements is intended to provide support for a similar aspect or aspect of the invention that “consists of”, “consists essentially of” or “substantially comprises” that particular element or elements, unless otherwise stated or clearly contradicted by context (e.g., a composition described herein as comprising a particular element should be understood as also describing a composition consisting of that element, unless otherwise stated or clearly contradicted by context).

[0216] The use of any and all examples, or exemplary language (including “for instance”, “for example”, “e.g.”, “such as” and “as such”) in the present specification is intended merely to better illuminate the invention and does not pose a limitation on the scope of invention unless otherwise indicated.

[0217] It should be understood that the various aspects, embodiments, items, implementations and features of the invention mentioned herein may be claimed separately, or in any combination.

[0218] The present invention includes all modifications and equivalents of the subject-matter recited in the claims appended hereto, as permitted by applicable law.

Experimental Section

[0219] Preparation of the Compound of Formula (Id) and Intermediates

[0220] NMR Methods

[0221] QNMR (600 MHz):

TABLE-US-00002 1) Relaxation delay   40 sec 2) Acquisition time 3.76 sec 3) Time domain 64k 4) Size 32k 5) Dummy scans 4 6) Scans 8 7) Pulse   30 deg LC-MS methods

[0222] method A: LC-MS were run on Waters Aquity UPLC-MS consisting of Waters Aquity including column manager, binary solvent manager, sample organizer, PDA detector (operating at 254 nM), ELS detector, and TQ-MS equipped with APPI-source operating in positive ion mode.

[0223] LC-conditions: The column was Acquity UPLC BEH C18 1.7 μm; 2.1×150 mm operating at 60° C. with 0.6 ml/min of a binary gradient consisting of water+0.05% trifluoroacetic acid (A) and acetonitrile/water (95:5)+0.05% trifluoroacetic acid.

[0224] Gradient (linear):

TABLE-US-00003 0.00 min  10% B 3.00 min 100% B 3.60 min  10% B Total run time: 3.6 minutes

[0225] Method B: LC-MS were run on Agilent 1260 HPLC consisting of column comp, Binary pump, Hip sample, and Single Q-MS equipped with ESI-source operating in positive ion mode.

[0226] LC-conditions: Column: lnertsustain AQ-C18 HP 3.0 μm; 3.0×50 mm operating at 35° C. with 1.2 ml/min of a binary gradient consisting of water+0.05% trifluoroacetic acid (A) and acetonitrile+0.05% trifluoroacetic acid (B).

[0227] Gradient (linear):

TABLE-US-00004 0.00 min  0% B 3.00 min 95% B 4.00 min 95% B Total run time: 4.0 minutes

[0228] LC-MS Method C:

[0229] Instrument: Shimadzu LCMS-2020

[0230] Column: Phenomenex Kinetex EVO C18, 100×2.1 mm, 2.6 μm, ULC-016, UV-Vis Detector: 190-800 nm, Flow rate: 0.5 ml/min, Mobile Phase A: H.sub.2O+0.1% HCOOH, Mobile Phase B: acetonitrile

[0231] Gradient (linear):

TABLE-US-00005  1.00 min  2% B 10.00 min 90% B 13.00 min 90% B 13.10 min  2% B Total run time: 13.1 minutes

[0232] Preparative HPLC method A:

[0233] Column: AQ gel, UV Detector: 210 nm, flow rate: 1 L/min, Mobile Phase A: Water (0.05% NH.sub.4HCO.sub.3), Mobile Phase B: acetonitrile.

[0234] Gradient (linear):

TABLE-US-00006 0.00 min  5% B 30.0 min 30% B Total run time: 30.0 minutes

[0235] Preparative HPLC method B:

[0236] Column: RP-C18, 360 g column, Flow rate: 150 ml/min, UV Detector wavelength: 210 nm. Mobile Phase A: water, Mobile Phase B: acetonitrile

[0237] Gradient (linear):

TABLE-US-00007 0.00 min  5% B 4.00 min 30% B Total run time: 4.0 minutes

[0238] Quantitative HPLC:

[0239] Column: Phenomenex Synergi Polar RP, 150×4.6 mm×4.0 μm, Thermo-Dionex Ultimate 3000 Pump, Autosampler, Column compartment, Variable Wavelength Detector, Flow rate: 1 ml/min, UV Detector wavelength: 210 nm. Mobile Phase A: water-acetonitrile 98:2+0.1% trifluoroacetic acid, Mobile Phase B: acetonitrile+0.1% trifluoroacetonitrile.

[0240] Gradient (linear):

TABLE-US-00008 0.00 min  2% B 6.00 min 90% B 9.00 min 90% B 9.50 min  2% B 15.0 min  2% B Total run time: 15.0 minutes

Example 1: Preparation of Compound (A2) (Step 1)

Example 1a

[0241] A 1 L three necked-flask was charged with 15 g (50.4 mmol, 1 eq.) HCl salt of compound (I), 450 ml dry dichloromethane, 40.4 ml (232 mmol) N,N-diisopropylethylamine (DIPEA) and 20.5 ml (96 mmol, 1.9 eq.) triisopropylsilyl chloride. The mixture was stirred at 20-25° C. under inert atmosphere. After 48 hours, the reaction mixture was cooled down to 0-5° C. and saturated NH.sub.4Cl solution was added (300 ml). The mixture was stirred for 10 minutes and then the phases were separated. The organic layer was washed with deionized water (2×150 ml), dried on Na.sub.2SO.sub.4 and evaporated, affording compound (A2) (27.8 g). Used directly in the next step (example 2a).

[0242] LC-MS (method A): retention time (RT)=2.71 min, [M+H].sup.+=418.2 m/z.

Example 1b

[0243] Into a 3 L three-necked round-bottom flask purged and maintained with an inert atmosphere of nitrogen, was placed HCl salt of compound (I) (68 g, 228 mmol), dichloromethane (1.8 L), N,N-diisopropylethylamine (DIPEA) (83.6 g) and triisopropylsilyl chloride (135.7 g, 704.0 mmol). The resulting solution was stirred for 2 days at 25° C. The reaction was then quenched by the addition of 1000 mL of NH.sub.4Cl. The resulting solution was extracted with dichloromethane (2×1 L) and the organic layers combined and concentrated under vacuum. The residue was purified using silica gel column chromatography (eluent: ethyl acetate/petroleum ether (1:1)). This afforded compound (A2) (78 g) as an oil. Used directly in the next step (example 2b).

[0244] LC-MS (method B): RT=1.606 min, [M+H].sup.+=418 m/z

[0245] .sup.1H NMR (CDC.sub.3, ppm): δ 6.64 (d, J=8.2 Hz, 1H), 6.49 (d, J=8.2 Hz, 1H), 3.11 (dd, J=15.5, 5.0 Hz, 1H), 2.97 (dd, J=17.5, 5.0 Hz, 1H), 2.80-2.50 (m, 3H), 2.23 (dd, J=17.5, 11.5 Hz, 1H), 1.95 (d, J=13.0 Hz, 1H), 1.80-1.65 (m, 3H), 1.41-1.23 (m, 3H), 1.16-1.03 (m, 33H, including TIPS impurity), 0.91 (t, J=7.5 Hz, 3H).

Example 2: Preparation of Compound (A3) (Step 2)

Example 2a

[0246] A 500 ml three-necked flask equipped with CaCl.sub.2 tube was charged with compound (A2) (8.7 g, 21 mmol), anhydrous dichloromethane (260 mL) and (2S,3S,4S,5R,6R)-2-(methoxycarbonyl)-6-(2,2,2-trichloro-1-iminoethoxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate (20 g, 42 mmol). The solution was cooled down to 0-5° C. and boron trifluoride diethyl etherate (5.2 mL, 42 mmol) was added dropwise. The reaction mixture was allowed to warm to room temperature and stirred overnight. The reaction mixture was poured into ice cold saturated solution of NaHCO.sub.3 (770 mL). After 10 minutes stirring the phases were separated and the aqueous phase was extracted with dichloromethane (235 mL). The combined organic phase was dried on Na.sub.2SO.sub.4 and evaporated to dryness to give 27.9 g crude product as an oil.

[0247] The crude material was purified by normal phase silica gel column chromatography affording compound (A3) (first experiment: 7.2 g, >90% purity (Quantitative HPLC) (second experiment 2.2 g, ˜80% purity (Quantitative HPLC).

[0248] LC-MS (method C): RT=8.33 min, [M+H].sup.+=418.4 m/z

[0249] .sup.1H NMR: (CDCl.sub.3, ppm): δ 6.98 (d, J=8.5 Hz, 1H), 6.84 (d, J=8.5 Hz, 1H), 5.38-5.30 (m, 3H), 5.12 (d, J=6.0 Hz, 1H), 4.26-4.17 (m, 1H), 3.77 (s, 3H), 3.18 (dd, J=16.0, 5.0 Hz, 1H), 3.10-2.96 (m, 2H), 2.86-2.70 (m, 1H), 2.31 (s, 3H), 2.15-2.00 (m, 10H), 1.91 (d, J=13.0 Hz, 1H), 1.55 (q, J=7.5 Hz, 2H), 1.35-1.20 (m, 3H), 1.16-1.04 (m, 1H), 1.01-0.90 (m, 24H).

Example 2b

[0250] Into a 3 L three-necked round-bottom flask purged and maintained with an inert atmosphere of nitrogen, was placed compound (A2) (60.0 g, 144 mmol, 1.0 eq), dichloromethane (1.2 L) and (2S,3S,4S,5R,6R)-2-(methoxycarbonyl)-6-(2,2,2-trichloro-1-iminoethoxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate (351.3 g, 733.9 mmol). Then boron trifluoride diethyl etherate (150 g, 1.25 eq) was added dropwise at room temperature. The resulting solution was stirred for 2 days at 25° C. The reaction mixture was filtered and the filtrate was concentrated under vacuum. The residue was applied onto a silica gel column (eluent: ethyl acetate/petroleum ether (1:10)) affording compound (A3) (75 g) of as a solid.

[0251] LC-MS (method B): RT=3.531 min. [M+H].sup.+=720 m/z

[0252] .sup.1H NMR: (CDCl.sub.3, ppm): δ 6.98 (d, J=8.5 Hz, 1H), 6.84 (d, J=8.5 Hz, 1H), 5.38-5.30 (m, 3H), 5.12 (d, J=6.0 Hz, 1H), 4.26-4.17 (m, 1H), 3.77 (s, 3H), 3.18 (dd, J=16.0, 5.0 Hz, 1H), 3.10-2.96 (m, 2H), 2.86-2.70 (m, 1H), 2.31 (s, 3H), 2.15-2.00 (m, 10H), 1.91 (d, J=13.0 Hz, 1H), 1.55 (q, J=7.5 Hz, 2H), 1.35-1.20 (m, 3H), 1.16-1.04 (m, 1H), 1.01-0.90 (m, 24H).

Example 3: Preparation of Compound (Id) (Step 3)

Example 3a (Using KOH)

[0253] Into a 10 L three-necked round-bottom flask purged and maintained with an inert atmosphere of nitrogen, was placed compound (A3) (75 g, 102 mmol), methanol (4 L), and water (375 mL). This was followed by the addition of potassium hydroxide (28.7 g), NH.sub.4F (3.8 g) at 0° C. The resulting solution was stirred overnight at 25° C. The resulting solution was neutralized with 1 N HCl (˜200 mL, pH adjusted to 7.1) and concentrated under reduced pressure to afford a 250 mL solution. The solution was purified by preparative HPLC (method A) affording compound (Id) (40 g) as a solid. The afforded compound (Id) is obtained as a heptahydrate of compound (Id).

[0254] LC-MS (method B): RT=1.902 min, [M+H].sup.+=438.3 m/z.

[0255] .sup.1H NMR (300 MHz, D.sub.2O): δ 6.83 (d, J=8.5 Hz, 1H), 6.74 (d, J=8.5 Hz, 1H), 4.74 (d, J=7.5 Hz, 1H), 3.59-3.54 (m, 2H), 3.54-3.45 (m, 3H) 3.36-3.13 (m, 4H), 3.08-2.99 (m, 2H), 2.72 (dd, J=14.5, 12.0 Hz, 1H), 2.27 (dd, J=17.5, 11.5 Hz, 1H), 1.95 (t, J=15.0 Hz, 2H), 1.88-1.68 (m, 3H), 1.68-1.58 (m, 1H), 1.31 (dq, J=13.5, 3.5 Hz, 1H), 0.91 (t, J=7.5 Hz, 3H).

Example 3b (Comparative Example Using KCN)

[0256] In a three-necked flask 6.1 g (8.2 mmol) compound (A3) was dissolved in 260 ml MeOH/water (12:1) mixture and treated with 10.0 g KCN (19 eq.) at 0° C. After addition, the reaction mixture was stirred at room temperature. After 16 hours the reaction mixture was filtered to remove the insoluble inorganic salts. The filtrate was evaporated to dryness to give 15.2 g crude compound (Id). The crude product was purified by preparative HPLC (method B) affording compound (Id) (2.8 g) as a solid. The afforded compound (Id) is obtained as a potassium salt of compound (Id).

[0257] LC-MS (method C): RT=4.17 min, [M+H].sup.+=438.3 m/z.

[0258] In vitro and in vivo characterization of compound (Id)

Example 4a: Conversion of the Compound of Formula (Id) in Rat and Human Hepatocytes

[0259] Compound (Id) was incubated at 1 μg/mL with hepatocytes from human or rat suspended in DMEM (Dulbecco's Modified Eagle Medium) with HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) at pH 7.4. The cell concentration at incubation was 1×10.sup.6 viable cells/mL. The incubations were performed in glass tubes at 37° C. with a total incubation volume of 3.5 mL and with duplicate incubations for each test item. The 3.5 mL of hepatocyte suspension was equilibrated for 10 minutes in a water bath set to 37° C. where after the incubations were initiated by adding 3.5 μL of a stock solution of the test item in DMSO (Dimethyl sulfoxide) and gently inverting the tubes. The final solvent concentration in the incubations was 0.1% DMSO. Samples of 600 μL were withdrawn from the incubations at the pre-determined time points of 0.25, 5, 15, 30 and 60 minutes after ensuring homogeneity of hepatocyte suspensions. The withdrawn volume was added to 1 mL Nunc cryotubes on wet ice containing 60 μL of ice-cold ascorbic acid (100 mg/mL) and 30 μL of ice cold 100 mM saccharic acid 1.4-lactone in 0.5 M citric acid. The tubes were mixed and 35 μL of a solution of ice cold 20% formic acid was added. The tubes were mixed thoroughly and stored at −80° C. awaiting analysis. Analysis method and Instrumentation used for analysis of (I) from dosing compound (Id) was the one described in Example 7 below in the section “Instrumentation used for analysis of compound (I) from dosing of compound (Ic) and (Id).”

[0260] FIG. 6 indicates a time dependent conversion to compound (I) from (Id) in both rat and human hepatocytes.

Example 4b: Conversion of the Compound of Formula (Id) in Fresh Rat and Human Blood

[0261] Conversion of (Id) in human blood (average of 3 donors) and rat blood (average of 45 donors) to (I) was shown in fresh blood at 37° C. spiked with 1 μg/mL of (Id). (I) was measured at 0, 5, 15, 30 and 60 minutes in isolated plasma. Analysis method and Instrumentation as described in Example 7 below in the section “Instrumentation used for analysis of compound (I) from dosing of compounds (Ic) and (Id).”

[0262] FIG. 7 indicates a time dependent conversion to compound (I) from (Id), in both rat and human blood.

Example 5: Dopamine Agonist Activity

[0263] Dopamine D1 Receptor Agonism

[0264] Dopamine D1 receptor agonism was measured using a HTRF cAMP from CisBio using the protocol developed by HD Biosciences (China). Briefly, the assay is a homogeneous time resolved-fluorescence resonance energy transfer (HTRF) assay that measures production of cAMP by cells in a competitive immunoassay between native cAMP produced by cells and cAMP-labeled with XL-665. A cryptate-labeled anti-cAMP antibody visualizes the tracer. The assay was performed in accordance with instructions from manufacturer.

[0265] Test compounds were added to wells of microplates (384 format). HEK-293 cells expressing the human D1 receptor were plated at 1000 cells/well and incubated 30 minutes at room temperature. cAMP-d2 tracer was added to wells and followed by addition of Anti-cAMP antibody-cryptate preparation and incubated for 1 hour at room temperature in dark. HTRF cAMP was measured by excitation of the donor with 337 nm laser (the “TRF light unit”) and subsequent (delay time 100 microseconds) measurement of cryptate and d2 emission at 615 nm and 665 nm over a time window of 200 microseconds with a 2000 microseconds time window between repeats/100 flashes). HTRF measurements were performed on an Envision microplate reader (PerkinElmer). The HTRF signal was calculated as the emission-ratio at 665 nm over 615 nm. The HTRF ratio readout for test compounds was normalized to 0% and 100% stimulation using control wells with DMSO-solvent or 30 μM dopamine. Test compound potency (EC.sub.50) was estimated by nonlinear regression using the sigmoidal dose-response (variable slope) using Xlfit 4 (IDBS, Guildford, Surrey, UK, model 205).

y=(A+((B−A)/(1+((C/x){circumflex over ( )}D))))

where y is the normalized HTRF ratio measurement for a given concentration of test compound, x is the concentration of test compound, A is the estimated efficacy at infinite compound dilution, and B is the maximal efficacy. C is the EC.sub.50 value and D is the Hill slope coefficient. EC.sub.50 estimates were obtained from an independent experiment and the logarithmic average was calculated.

[0266] Dopamine D2 Receptor Agonism

[0267] Dopamine D2 receptor agonism was measured using a calcium mobilization assay protocol developed by HD Biosciences (China). Briefly, HEK293/G15 cells expressing human D2 receptor were plated at a density of 15000 cells/well in clear-bottomed, Matrigel-coated 384-well plates and grown for 24 hours at 37° C. in the presence of 5% CO.sub.2. The cells were incubated with calcium-sensitive fluorescent dye, Fluo8, for 60-90 minutes at 37° C. in the dark. Test compounds were prepared at 3-fold concentrated solution in 1×HBSS buffer with Ca.sup.2+ and Mg.sup.2+. Calcium Flux signal was immediately recorded after compounds were added from compound plate to cell plate at FLIPR (Molecular Devices). The fluorescence data were normalized to yield responses for no stimulation (buffer) and full stimulation (1 μM of dopamine) of 0% and 100% stimulation, respectively. Test compound potency (EC.sub.50) was estimated by nonlinear regression using the sigmoidal dose-response (variable slope) using Xlfit 4 (IDBS, Guildford, Surrey, UK, model 205).

y=(A+((B−A)/(1+((C/x){circumflex over ( )}D))))

where y is the normalized ratio measurement for a given concentration of test compound, x is the concentration of test compound, A is the estimated efficacy at infinite compound dilution, and B is the maximal efficacy. C is the EC.sub.50 value and D is the Hill slope coefficient. EC.sub.50 estimates were obtained from independent experiment and the logarithmic average was calculated.

Example 6: 5-HT2B Agonist Activity and Binding Assay

[0268] 5-HT2B Agonist Activity Assay

[0269] Evaluation of the agonist activity of compounds (I), (Ia), (Ib), (Ic) and (Id) at the human 5-HT2B receptor was performed by Eurofins/Cerep (France) measuring the compound effects on inositol monophosphate (IP1) production using the HTRF detection method. Briefly, the human 5-HT2B receptor was expressed in transfected CHO cells. The cells were suspended in a buffer containing 10 mM Hepes/NaOH (pH 7.4), 4.2 mM KCl, 146 mM NaCl, 1 mM CaCl.sub.2, 0.5 mM MgCl.sub.2, 5.5 mM glucose and 50 mM LiCl, then distributed in microplates at a density of 4100 cells/well and incubated for 30 minutes at 37° C. in the presence of buffer (basal control), test compound or reference agonist. For stimulated control measurement, separate assay wells contained 1 μM 5-HT. Following incubation, the cells were lysed and the fluorescence acceptor (fluorophen D2-labeled IP1) and fluorescence donor (anti-IP1 antibody labeled with europium cryptate) were added. After 60 minutes at room temperature, the fluorescence transfer was measured at lambda (Ex) 337 nm and lambda (Em) 620 and 665 nm using a microplate reader (Rubystar, BMG). The IP1 concentration was determined by dividing the signal measured at 665 nm by that measured at 620 nm (ratio). The results were expressed as a percent of the control response to 1 μM 5-HT. The standard reference agonist was 5-HT, which was tested in each experiment at several concentrations to generate a concentration-response curve from which its EC.sub.50 value is calculated as described above for dopamine functional assays.

[0270] 5-HT2B Binding Assay

[0271] Evaluation of the affinity of compound (Id) for the human 5-HT2B receptor was determined in a radioligand binding assay at Eurofins/Cerep (France). Membrane homogenates prepared from CHO cells expressing the human 5HT2B receptor were incubated for 60 minutes at room temperature with 0.2 nM [1251](±)DOI (1-(4-iodo-2, 5-dimethoxyphenyl)propan-2-amine) in the absence or presence of the test compound in a buffer containing 50 mM Tris-HCl (pH 7.4), 5 mM MgCl.sub.2, 10 μM pargyline and 0.1% ascorbic acid. Nonspecific binding is determined in the presence of 1 μM (±)DOI. Following incubation, the samples were filtered rapidly under vacuum through glass fiber filters (GF/B, Packard) presoaked with 0.3% polyethyleneimine (PEI) and rinsed several times with ice-cold 50 mM Tris-HCl using a 96-sample cell harvester (Unifilter, Packard). The filters were dried and counted for radioactivity in a scintillation counter (Topcount, Packard) using a scintillation cocktail (Microscint 0, Packard). The results are expressed as a percent inhibition of the control radioligand specific binding. The standard reference compound was (±)DOI, which was tested in each experiment at several concentrations to obtain a competition curve from which its IC.sub.50 is calculated.

TABLE-US-00009 TABLE 2 In vitro activities for the compounds of formula (I), (Ia), (Ib), (Ic) and (Id) obtained according to Examples 5 and 6 Com- D1 EC.sub.50 D2 EC.sub.50 5-HT2B EC.sub.50 pound (nM)/E.sub.max (nM)/E.sub.max (nM)/E.sub.max Parent (I) 3.3/99% 1.3/91% 2900 nM/50% compound Prodrugs in the (Ia) >1000 >1000 >6000 nM, 58% @ state of the art 30 μM (Ib) >1000 46 nM/100% 3.8 nM/79% (Ic) nd nd −5% @ 10 μM Compound (Id) 2700/98% 1100/92% −25% @ 10 μM* obtained by the invention *indicate binding affinity (% inhibition of control, specific binding at concentration indicated) nd: not determined

Example 7: PK Experiments in Rats

[0272] For all the experiments, blood samples of approximately 0.68 mL were drawn from the tail or sublingual vein and put into K.sub.3EDTA tubes that had been pre-cooled and prepared with stabilizing solution consisting of 80 μL ascorbic acid and 40 μL 100 mM D-saccharic acid 1,4 lactone in water. The tubes were inverted gently 6-8 times to ensure thorough mixing and then placed in wet ice. The collecting tube was placed in wet ice for up to 30 minutes until centrifugation. Once removed from the wet ice the centrifugation was initiated immediately. Immediately after end of centrifugation the samples were returned to wet ice. Three sub-samples of 130 μL plasma were transferred to each of three appropriately labelled cryotubes containing 6.5 μL pre-cooled formic acid (20%) (the tubes were pre-spiked and stored refrigerated prior to use). The tube lid was immediately replaced and the plasma solution was thoroughly mixed by inverting gently 6-8 times. The samples were stored frozen at nominally −70° C. within 60 minutes after sampling. Centrifugation conditions at 3000 G for 10 minutes at 4° C. Plasma was placed on water-ice following collection. Final storage at approximately −70° C.

[0273] Plasma samples were analyzed by solid phase extraction or direct protein precipitation followed by UPLC-MS/MS. MS detection using electrospray in the positive ion mode with monitoring of specific mass-to-charge transitions for compound (I) using internal standards for correcting the response. The concentration-time data was analyzed, using standard software using appropriate noncompartmental techniques to obtain estimates of the derived PK parameters.

[0274] Instrumentation Used for Analysis of Compound (I) from Dosing Compound (Ia):

[0275] Mass spectrometer (LC-MS/MS) Waters Acquity-Sciex API 5000. Analytical column Waters BEH UPLC Phenyl 100×2.1 mm column, 1.7 μm particle size. Mobile phase A: 20 mM ammonium formate (aq)+0.5% formic acid. Mobile phase B: Acetonitrile. Gradient run from 95/5% to 2/98 in 6.1 min. Flow rate 0.5 mL/min. MRM monitoring (multiple reaction monitoring) of test item and the added analytical standards Dosing and blood sampling: Han Wistar rats were supplied by Charles River Laboratories, Sulzfeld, Germany. An artificial, automatically controlled, light and dark cycle of 12 hours was maintained. The rats received a standard laboratory diet from Brogaarden (Altromin 1324 pellets). The rats had unrestricted access to the diet. During the study (a 4-week toxicity study) the rats received once daily doses of (Ia) orally by gavage. From rats given 300 μg/kg (Ia), blood samples from 3 male satellite animals were collected on the following time points at Day 29: 0.5, 1, 2, 4, 6, 8, 12 and 24 hours after dosing.

[0276] Instrumentation Used for Analysis of Compound (I) from Dosing of Compound (Ib):

[0277] Mass spectrometer (LC-MS/MS) Waters Acquity-Sciex API 5000. Analytical column Waters BEH UPLC Phenyl 100×2.1 mm column, 1.7 μm particle size. Mobile phase A: 20 mM ammonium formate (aq)+0.5% formic acid. Mobile phase B: Acetonitrile. Gradient run from 95/5% to 2/98 in 6.1 min. Flow rate 0.5 mL/min. MRM monitoring of test item and the added analytical standards.

[0278] Dosing and blood sampling: Han Wistar rats were supplied by Charles River Laboratories, UK. An artificial, automatically controlled, light and dark cycle of 12 hours was maintained. The rats received a standard laboratory diet (Teklad 2014C Diet.). The rats had unrestricted access to the diet. During the study (a 26-week toxicity study) the rats received once daily doses of (Ib) orally by gavage. From rats given 300 μg/kg (Ib), blood samples from 3 male satellite animals were collected on the following time points at day 182: 0.5, 1, 2, 4, 8 and 24 hours after dosing.

[0279] Instrumentation used for analysis of compound (I) from dosing of compounds (Ic) and (Id): Mass spectrometer (LC-MS/MS) Waters Acquity-Waters Xevo TQ-S. Analytical column Acquity BEH C18 100×2.1 mm, 1.7 μm. Mobile phase A: 20 mM NH.sub.4—Formate+0.2% formic acid. Mobile phase B: Acetonitrile+0.2% formic acid. Gradient run from 95/5% to 5/95% in 11.0 min. Flow rate 0.3 mL/min. MRM monitoring of test item and the added analytical standards.

[0280] Dosing and blood sampling for compound (Id): Han Wistar rats were supplied by Charles River Laboratories, Wiga GmbH, Germany. An artificial, automatically controlled, light and dark cycle of 12 hours was maintained. The rats received a standard laboratory diet from Brogaarden (Altromin 1324 pellets). The rats had unrestricted access to the diet. Male Han Wistar rats were dosed a single oral gavage administration of compound (Id) orally by gavage. Rats were given 633 μg/kg of compound (Id), blood samples from 3 male animals were collected on the following time points at Day 1: 1, 2, 4, 6, 8, and 24 hours after dosing.

[0281] Dosing and blood sampling for compound (Ic): Han Wistar rats were supplied by Envigo, UK. An artificial, automatically controlled, light and dark cycle of 12 hours was maintained. The rats received a standard laboratory diet Teklad 2014C. The rats had unrestricted access to the diet. Male Han Wistar rats were dosed a single oral gavage administration of (Ic). Rats were given 494 μg/kg (Ic). Blood samples from 3 male animals were collected on the following time points at Day 1: 1, 2, 4, 6, 8, and 24 hours after dosing Instrumentation used for analysis of apomorphine: Mass spectrometer (UPCLC-MS/MS) Waters Acquity I-Class-Waters Xevo TQ-S. Analytical column Acquity HSS T3 C18 50×2.1 mm, 1.8 μm. Mobile phase A: 10 mM NH.sub.4—Formate 0.2% formic acid:acetonitril (95:5). Mobile phase B: 10 mM NH.sub.4—Formate 0.2% formic acid:acetonitril (5:95). Gradient run from 95/5% to 5/95% in 2.40 minutes. Flow rate 0.3 mL/min. MRM detection of test items and the added analytical standards.

[0282] Dosing and blood sampling for Apomorphine: Animals for the study were as described in Dosing and blood sampling for compound (Id). Additionally, rats were administered a single dose of apomorphine subcutaneously. From rats administered 3000 μg/kg (apomorphine), blood samples from 3 male animals were collected on the following time points at Day 1: 0.25, 0.5, 1, 1.5, 2, 3, 5 and 7 hours SC administration after dosing.

TABLE-US-00010 TABLE 3 PK parameters for (4aR,10aR)-1-Propyl--1,2,3,4,4a,5,10,10a-octahydro- benzo[g]quinoline-6,7-diol (compound (I)) after oral dosing of 0.300 mg/kg (Ia), 0.300 mg/kg (Ib), 0.633 mg/kg of TFA salt of compound (Id) and 494 μg/kg (Ic) to Wistar rats according to Example 7 Exposure T.sub.max C.sub.max AUC.sub.0-24 t.sub.1/2 at 24 h compound (hour) (pg/mL) (pg * h/mL) (hour) (pg/mL) Prodrugs in (Ia) 1.0 3160 13600 4.09  48 ± 26 the state of (Ib) 0.5 4990 31000 N/A 147 ± 28 the art (Ic) 1.0 14 104 N/A N/A Compound (Id) 4.0 1350 15500 6.8 208 ± 89 obtained by the invention

Example 8: PK/PD of Compound (Id)/Compound (I) in Rat Hyperactivity Assay

[0283] Animals

[0284] In total, 206 male CD rats (Charles River, Germany) weighing 200-250 grams (165-190 grams upon arrival) were used in the study. Animals were housed at a standard temperature (22±1° C.) and in a light-controlled environment (lights on from 7 am to 8 μm) with ad libitum access to food and water. The experiment described below was performed in accordance with the standard operating procedures of Charles River Discovery Research Services Finland Ltd. and in accordance with the national Animal Experiment Board of Finland (Elainkoelautakunta, ELLA) authority on animal testing.

[0285] Locomotor Activity Testing, Open Field

[0286] The test device is a square Plexiglass-arena (measuring 40×40×40 cm), in which the movement paths of the rats are recorded by an activity monitor (Med. Associates Inc.). Before the test period is initiated, rats are habituated to their test cage for 60 minutes. Upon completion of habituation, animals were treated with either compound or vehicle and placed back into the open field apparatus. The main test parameter measured is ambulatory distance (recorded in 5-minute segments). Overall time of measurement after receiving initial treatment was 360 minutes. Total follow up period in the study was 420 min, including 60 min of habituation.

[0287] Results

[0288] Oral administration of compound (Id) was assessed in the rat locomotor activity assay, and this functional readout was then correlated to plasma concentrations of compound (I).

[0289] Apomorphine and pramipexole were also concomitantly tested in this assay as comparators (i.e. known standard-of-care (SoC) in the Parkinson's Disease field), and plasma concentration was analyzed for apomorphine.

[0290] As shown in FIG. 2, compound (Id) (10 to 300 μg/kg, p.o.) increases locomotor activity with an effect starting approximatively 2 hours post-administration (around the 180-minute time point) and lasting until the end of recording (at the 415-minute time point). In contrary, the hyperactivity induced by apomorphine (3 mg/kg, s.c.) is immediate but short-lasting as the effect is gone 1.5 hours. post administration (at the 150-minute time point). Pramipexole (0.3 mg/kg, s.c.) also induces an increase in activity, but its effect appears about 1 hour post administration and is gone 2.5 hours later (at the 270-minute time point). The total distance travelled as seen in FIG. 3 demonstrates a significantly increased activity for both compound (Id) and the two comparators tested, and this effect is the one that is to be expected from dopamine agonists.

[0291] In parallel with the locomotor activity assessment, plasma samples were taken from satellite animals at 6 different time points (1.5, 2, 3, 4, 5 & 7 hours post-dose for animals treated with compound (Id)). Pharmacokinetic analysis demonstrates that the behavioural effects of compound (Id) (100 μg/kg, p.o.) correlate with the plasma concentrations of compound (I) (see FIG. 4), demonstrating that the behavioural effect of compound (Id) is driven by Compound (I) rather than by Compound (Id) itself. The corresponding exposure analysis of apomorphine (at 1.25, 1.5, 2, 3, 5 & 7 hours post-dose) resulted in a correlation between plasma concentrations of apomorphine and hyperactive behaviour (see FIG. 5).

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