The following specification particularly describes the nature of the invention and the manner in which it is to be performed

Synthesis and Characterization of Potential Impurities of Aliskiren and Its Intermediates

INTRODUCTION
The present application relates to impurities and metabolites of Aiskiren. In particular  the present application relates to a process for the preparation  and characterization of potential impurities and metabolites of Aliskiren along with their use as reference markers.
Aliskiren hemifumarate is described chemically as 5(S)-amino-4(S)-hydroxy-2(S) 7(S)-diisopropyl-8-[4-methoxy-3-(3-methoxypropyloxy)phenyl] octanoic acid N-(2-carbamoyl-2 2-dimethylethyl) amide hemifumarate and is represented structurally by Formula I.

Formula 1
Aliskiren is first in a class of drugs called direct rennin inhibitors and is useful in the treatment of primary hypertension. Aliskiren is commercially available in the USA  in the form of its hemifumarate salt  as the active ingredient in TEKTURNA™ products.
U.S. Patent No. 5 559 111 discloses aliskiren  its pharmaceutically acceptable salts. The patent also gives processes for its preparation.
Various processes for the preparation of Aliskiren  its salts and its intermediates have been described in subsequent patents and applications. Most of the references relate to convergent synthetic routes for the preparation of aliskiren wherein two intermediates which are referred to as synthons are prepared separately and coupled together to give aliskiren in the course of entire synthesis.
The product of a chemical reaction is rarely a single compound with sufficient purity to comply with pharmaceutical standards. Side products and by-products of the reaction and adjunct reagents used in the reaction will  in most cases  also be present in the product. At certain stages during processing of an API such as Aliskiren  it must be analyzed for purity  typically  by high performance liquid chromatography ("HPLC") or thin-layer chromatography ("TLC")  to determine if it is suitable for continued processing and  ultimately  for use in a pharmaceutical product. Generally  side products  by-products  and adjunct reagents (collectively "impurities") are identified spectroscopically and/or with another physical method  and then associated with a peak position  such as that in a chromatogram  or a spot on a TLC plate.
International conference on Harmonization (ICH) guidelines recommends identifying and characterizing all impurities that are present at a level of 0.10%. In this context  a comprehensive study is undertaken to synthesize and characterize impurities of Aliskiren.
Information about the different possible impurities and their synthetic routes is a prerequisite for a thorough understanding of the impurity formation pathway of the antihypertensive drug Aliskiren Hemifumarate. Keeping in view this regulatory importance of Aliskiren Hemifumarate impurities  the process-related impurities and metabolites in synthesis of Aliskiren Hemifumarate were identified synthesized and characterized using mass  HRMS  IR  and NMR techniques.
Once a particular impurity has been associated with a peak position  the impurity can be identified in a sample by its relative position in the chromatogram  where the position in the chromatogram is measured in minutes between injection of the sample on the column and elution of the impurity through the detector. The relative position in the chromatogram is known as the "retention time." The RRT of an impurity is calculated by dividing the retention time of the impurity by the retention time of a reference marker. The reference marker may be the API in which the impurity is present  or may be another compound that is either present in or added to the sample. A reference marker should be present in the sample in an amount that is sufficiently large to be detectable  but not in an amount large enough to saturate the column.
Those skilled in the art of drug manufacturing research and development understand that a relatively pure compound can be used as a "reference standard." A reference standard is similar to a reference marker  except that it may be used not only to identify the impurity  but also to quantify the amount of the impurity present in the sample.
During the synthesis of Aliskiren Hemifumarate  we have observed eleven impurities: aliskiren lactone amine  alskiren Schiff base  aliskien ether cleavage impurities-1  2  3  aliskiren mono methyl  N-methyl aliksiren  N-alkylated (synthon-dimer)  aliskiren dihydroxy aliskiren  azide intermediate  aliskiren enatiomer and diasteriomer. The present work describes the synthesis and characterization of these impurities.
All the above impurities (related substances) were detected during the impurity profile study of an antihypertensive drug substance  Aliskiren Hemifumarate
The present inventors have synthesized  and characterized these compounds of Aliskiren which can be used as reference markers for determining the purity of Aliskiren.

SUMMARY
The present application relates to processes for the preparation of impurities and metabolites of Aliskiren  and their use as reference markers.
In one aspect  the present application relates to preparation and purification of the following impurities:
(2S 4S 5S 7S)-N-(3-amino-2-methyl-3-oxopropyl)-5-azido-4-hydroxy-2-isopropyl-7-(4-methoxy-3-(3-methoxypropoxy)benzyl)-8-methylnonanamide fumarate of Formula II (Monomethyl impurity);

Formula II
(2R 4S 5S 7S)-5-amino-N-(3-amino-2 2-dimethyl-3-oxopropyl)-4-hydroxy-2-isopropyl -7-(4-methoxy-3-(3-methoxypropoxy)benzyl)-8-methylnonanamide of Formula III (Diastereomer impurity).

Formula III
(2S 4S 5S 7S)-N-(3-amino-2 2-dimethyl-3-oxopropyl)-4 5-dihydroxy-2-isopropyl-7-(4-methoxy-3-(3-methoxypropoxy)benzyl)-8-methylnonanamide of Formula IV (Dihydroxy impurity).

Formula IV
(2S 4S 5S 7S)-5-amino-N-(3-((3-amino-2 2-dimethyl-3-oxopropyl)amino)-2 2-dimethyl-3-oxopropyl)-4-hydroxy-2-isopropyl-7-(4-methoxy-3-(3-methoxypropoxy)benzyl)-8-methylnonanamide of Formula V (N-alkylated impurity);

Formula V
(2S 4S 5S 7S)-N-(3-amino-2 2-dimethyl-3-oxopropyl)-4-hydroxy-2-isopropyl-7-(4-methoxy-3-(3-methoxypropoxy)benzyl)-8-methyl-5-(propan-2-ylideneamino)nonanamide of Formula VI (sciff`s base impurity);

Formula VI
(2S 4S 5S 7S)-N-(3-amino-2 2-dimethyl-3-oxopropyl)-5-azido-4-hydroxy-2-isopropyl-7-(4-methoxy-3-(3-methoxypropoxy)benzyl)-8-methylnonanamide of Formula VII (Azido derivative);

Formula VII
(2R 4R 5R 7R)-5-amino-N-(3-amino-2 2-dimethyl-3-oxopropyl)-4-hydroxy-2-isopropyl-7-(4-methoxy-3-(3-methoxypropoxy)benzyl)-8-methylnonanamide of Formula VIII (Enatiomer of aliskiren)

Formula VIII
(2S 4S 5S 7S)-N-(3-amino-2 2-dimethyl-3-oxopropyl)-4-hydroxy-2-isopropyl-7-(4-methoxy-3-(3-methoxypropoxy)benzyl)-8-methyl-5-(methylamino)nonanamide of Formula IX (N-methyl aliskiren)

Formula IX
In another aspect  the present application relates to preparation and characterization of metabolites of Aliskiren. In particular  the present application relates to the following metabolites:
(2S 4S 5S 7S)-5-amino-N-(3-amino-2 2-dimethyl-3-oxopropyl)-4-hydroxy-7-(3-hydroxy-4-methoxybenzyl)-2-isopropyl-8-methylnonanamide Formula X 

Formula X
(2S 4S 5S 7S)-5-amino-N-(3-amino-2 2-dimethyl-3-oxopropyl)-4-hydroxy-7-(3-(3-hydroxypropoxy)-4-methoxybenzyl)-2-isopropyl-8-methylnonanamide Formula XI

Formula XI
(2S 4S 5S 7S)-5-amino-N-(3-amino-2 2-dimethyl-3-oxopropyl)-4-hydroxy-7-(4-hydroxy-3-(3-methoxypropoxy)benzyl)-2-isopropyl-8-methylnonanamide Formula XII

Formula XII
(3S 5S)-5-((1S 3S)-1-amino-3-(4-methoxy-3-(3-methoxypropoxy)benzyl)-4-methylpentyl)-3-isopropyldihydrofuran-2(3H)-one Formula XIII

Formula XIII
Yet another aspect of the present application relates to the use of impurities and metabolites described in the application as reference markers or reference standards in determination of the purity of Aliskiren or its salts.

BRIEF DESCRIPTION OF FIGURES
Fig. 1: Relative retention times of the impurities with respect to Aliskiren Hemifumarate  structures.

DETAILED DESCRIPTION
Aliskiren Hemifumarate of Formula 1 has been synthesized via convergent synthesis represented by Scheme 1 with slight modification of reported synthesis (US 7 0009 078 B1). Coupling of Segment-A Grignard reagent with segment–B gives 2. Streoselective bromo lactonization of 2 gives 3. Nucleophilic substitution bromo group of 3 in SN2 manner with azide group gives 4. Condensation of 3-amino-2 2-dimethylpropanamide and 4 gives open azido Aliskiren 5. Reduction of 5 gives Alsikrien Free base 6. Final step is salt formation of 6 with fumaric acid.

Scheme 1
(2S 4S 5S 7S)-N-(3-amino-2-methyl-3-oxopropyl)-5-azido-4-hydroxy-2-isopropyl-7-(4-methoxy-3-(3-methoxypropoxy)benzyl)-8-methylnonanamide fumarate of Formula II (Monomethyl impurity) is a carry-over impurity from 3-amino-2 2-dimethylpropanamide (segment-C). The presence of 3-amino-2-methylpropanamide 9 in segment-C also undergoes lactone aminalysis. The impurity is prepared starting with 2-cyanoacetamide 7 according to Scheme 2. 7 on reaction with DMS in presence of K2CO3 in toluene solvents give 2-methyl cyano acetamide 8  which on catalytic hydrogenation with Raney Ni in methanol yielded 9. This 9 on lactone aminolysis give 10  which on catalytic hydrogenation with Pd/C in methanol yielded Formula II. The electro spray ionization (ESI) mass spectrum of Formula II displayed peaks at m/z 538.0 [(M+H)+] in positive ion mode and as sodium adduct at m/z 560.0 [(M+H)+Na] and LC-MS-MS displayed peaks at m/z 538.3 and daughter ion peaks at m/z 520.5  503.5  486.1  436.7  422.4  404.3 and 9 compound peaks at m/z 117.20. In 1H NMR spectrum of compound Formula 2  corresponding peak of mono-methyl at δ 1.15 ppm is present.

Scheme 2
(2R 4S 5S 7S)-5-amino-N-(3-amino-2 2-dimethyl-3-oxopropyl)-4-hydroxy-2-isopropyl-7-(4-methoxy-3-(3-methoxypropoxy)benzyl)-8-methylnonanamide of Formula III (Diasteriomer) is formed on lactone aminolysis of Compound 4 with segment-C. This is due to lactone aminolysis is a slow chemical conversion  hence during reaction there may be chances for the recimization of isopropyl group present in the lactone. We have enriched the precursor 12 impurity by maintaining the reaction at 100 °C and prolonged maintenance. 12 on hydrogenation with Pd/C in methanol and Methanolic ammonia give the corresponding diasteriomer Formula 3. This compound Formula III is isolated by reverse phase preparative HPLC. The ESI mass spectrum of the compound of Formula III displayed a peak at m/z 552.4 [(M+H)+] in positive ion mode and as sodium ion adduct at m/z 574.3 [(M+H)+Na] and HRMS also supported the elemental count. 1H NMR spectrum of compound Formula III is similar with aliskiren free base.
Scheme 3
2S 4S 5S 7S)-N-(3-amino-2 2-dimethyl-3-oxopropyl)-4 5-dihydroxy-2-isopropyl-7-(4-methoxy-3-(3-methoxypropoxy)benzyl)-8-methylnonanamide of Formula IV (Dihydroxy) is a process related impurity. Route cause for formation of dihydro Formula IV is presence of hydroxyl lactone 14 while lactone aminolysis. The 14 is formed during the bromolactoniztion of cross coupled derivative 2. The 14 is prepared by oxidative cyclization of bromo derivative 3 using hydrogen peroxide  which on lactone aminolysis with segment-C gives desired dihydroxy Formula 4. The ESI mass spectrum of Formula 4 diplayed a peak at m/z 438 [(M+H)+] in positive ion mode and as sodium ion adduct at m/z 451 [(M+H)+Na]. In 1H NMR spectrum of Formula 4  corresponding peaks of amine at δ 7.17  6.58 were absent.

Scheme 4
(2S 4S 5S 7S)-5-amino-N-(3-((3-amino-2 2-dimethyl-3-oxopropyl)amino)-2 2-dimethyl-3-oxopropyl)-4-hydroxy-2-isopropyl-7-(4-methoxy-3-(3-methoxypropoxy)benzyl)-8-methylnonanamide of Formula V (N-alkylated) is a process related impurity. During the lactone aminolysis this impurity is forming and with respect to reaction time this impurity is raising. This impurity is identified by LC-MS-MS. In LC-MS-MS fragmentation pattern is different from the aliskiren. The peak at m/z 117 is appeared for aliskiren where as for this impurity peak appeared at m/z 215. This is possible for the dimer of segment-C. Hence  N-alkylated Formula 5 is prepared by following chemical sequence. Hydrolysis of cyano-ester 16  gives acid 17  which on coupling with segment-C gives cyano-dimer 18. This cyano-dimer 18 on hydrogenation with Raney nickel in methanol gives the segment-C dimer 19  which on reaction with 4 gives 19 in presence of 2-hydroxy pryridine and triethyl amine. The 20 on hydrogenation with Pd/C in methanol gives corresponding N-alkylated Formula 5 derivative. The ESI mass spectrum of Formula 5 displayed a peak at m/z 651 [(M+H)+] in positive ion mode and as sodium ion adduct at m/z 673 [(M+H)+Na]. In 1H NMR spectrum of compound Formula V additional signals at δ 1.03 along with 0.106 provides the information that presence of extra di-methyl groups of corresponding extra segment-C and presence of triplet at δ 7.73 indicates the extra amide bond attached to two hydrogen containing carbon atom.

Scheme 5
(2S 4S 5S 7S)-N-(3-amino-2 2-dimethyl-3-oxopropyl)-4-hydroxy-2-isopropyl-7-(4-methoxy-3-(3-methoxypropoxy)benzyl)-8-methyl-5-(propan-2-ylideneamino) nonanamide of Formula VI (sciff`s base) is not process related impurity and it is carobonyl solvent contaminated impurity. Because aliskiren hemifumarate is highly reactive towards carbonyl compounds particularly with acetone solvent even traces level of acetone can generate the Schiff base impurity Formula VI. Schiff base impurity Formula VI is prepared by refluxing 5 in acetone solvent. The ESI mass spectrum of the compound of Formula VI displayed a peak at m/z 592 [(M+H)+] in positive ion mode and as sodium ion adduct at m/z 614 [(M+H)+Na]. In 1H NMR spectrum of compound Formula VI additional signals at δ 1.10 and 1.23 indicate the presence of dimethyl group in the compound.

Scheme 6
(2S 4S 5S 7S)-N-(3-amino-2 2-dimethyl-3-oxopropyl)-5-azido-4-hydroxy-2-isopropyl-7-(4-methoxy-3-(3-methoxypropoxy)benzyl)-8-methylnonanamide of Formula VII (Azido derivative) is the carry over impurity. Presence of un-reacted azido-derivative 4 in hydrogenation leads to presence of this impurity in the final API.
(2R 4R 5R 7R)-5-amino-N-(3-amino-2 2-dimethyl-3-oxopropyl)-4-hydroxy-2-isopropyl-7-(4-methoxy-3-(3-methoxypropoxy)benzyl)-8-methylnonanamide of Formula 8 (Enatiomer) can be synthesized by taking the racemic mixture of (R/S)-Segment-A and E  R derivative -B compound (enantiomer of C-B) is coupled to get the mixture of cross coupled product 2a. After that the cross coupled product is subjected for halolactonization reaction in presence of NBS and ortho phosphoric acid and obtained the mixture of bromolactonized compounds 3a. Afterwards the bromolactonized compound 3a is subjected for silica gel Coolum chromatogram purification and separated the enantio pure isomer which is used as source for the preparation of aliskiren enantiomer Formula VIII.

Scheme 7

(2S 4S 5S 7S)-N-(3-amino-2 2-dimethyl-3-oxopropyl)-4-hydroxy-2-isopropyl-7-(4-methoxy-3-(3-methoxypropoxy)benzyl)-8-methyl-5-(methylamino)nonanamide of Formula IX (N-methyl aliskiren) is process related impurity. It is forming during the reduction of azide group to amine. The reaction is conducted in methanol solvent hence there is chance of formation of this impurity by Palladium mediated N-methylation. This compound Formula IX was synthesized and is isolated by reverse phase preparative HPLC. The electro spray ionization (ESI) mass spectrum of Formula IX displayed peaks at m/z 565.0 [(M+H)+] in positive ion mode and as sodium adduct at m/z 588.0 [(M+H)+Na]

Ether cleavage imputes Formula X to XII and XIII were the metabolites formed in the synthetic sequence of aliskiren. Formula X to XII were formed during the work-up process of stage-1 where 2 N HCl is used for the quenching of Grignard reagent. Formula XIII is formed due to presence of un-reacted azido lactone compound in the reduction of lactone opened azido compound.
Processes for the preparation of the metabolites can be represented by Scheme 8. The Formula X and XI was isolated from a crude sample (obtained from synthesis) by prepara¬tive HPLC and Formula XII and XIII were synthesized.


Scheme 8

A Waters Model Alliance 2690-separation module equipped with a Waters 996-photo diode array detector was used for the studies. The analysis was carried out on ACE C 18 4.6x 150 mm  3 µm particle size with a mobile phase consisting of A: (degassed buffer) 1.96 grams of solid ortho phosphoric acid and 1.74 grams of tetra n butyl ammonium hydrozen sulphate in mixture of Water: Methanol: Acetonitrile in the ratio of 800: 100: 100 mL. B: 1.96 grams of solid ortho phosphoric acid and 1.74 grams of tetra n butyl ammonium hydrozen sulphate in mixture of Water: Acetonitrile in the ratio of 250:750 mL. Sample was dissolved in diluent of 0.02M solid ortho phosphoric acid in Water: Acetonitrile in the ratio of 800:200 mL. The injection load was 20µl. Gradient elution was used with UV detection at 228 nm at a flow rate of 0.8 ml/min (t(min)/A(v/v)/B(v/v) = 0.01/85/15  4/85/15  25/75/25  40/25/75  50/25/75  51/85/15  65/85/15). The column temperature was main¬tained at 35°C. The data was recorded using Waters Millennium software.
The preparative HPLC system used was a Waters system equipped with W600 quaternary solvent delivery module and Delta prep 2487 dual wavelength UV detector. Data was processed through Waters Empower software. A YMC Aq 10x250mm  10 µm (CRTO06O) column was used for preparative work. The mobile phase employed was solution A as 15.0 mL of trifluoro acetic acid in 15000 mL of milli Q water and solution B as acetonitrile. The gradient program was as follows: t (min)/B (v/v): 0.01/5.0  5.0/5.0  40.0/50.0  42.0/90  42.0/90.0  47.0/5.055.0/5.0. The flow rate was kept at 6 ml/min and the column eluent was monitored at 228 nm. The above mentioned gradient system was used for the isolation of these metabolites. The fractions were collected and pooled. Acetonitrile present in the pooled fractions was evaporated using rotavapor at room temperature. After evaporation of total solvent  aqueous layer kept for lyophilization. The solids obtained from the concentrated fractions were used to generate spectral data.

The prepared and isolated imurities were co-injected with Aliskiren Hemifumarate to confirm the identity of the metabolites based on retention matching. All impurities were well resolved from Aliskiren Hemifumarate and each other. Relative retention times of the impurities with respect to Aliskiren Hemifumarate  structures are shown in Fig. 1

Certain specific aspects and embodiments of the present invention will be explained in more detail with reference to the following examples  which are provided only for purposes of illustration and should not be construed as limiting the scope of the invention in any manner. Reasonable variations of the described procedures are intended to be within the scope of the present invention.

EXAMPLES
Example 1: Preparation of 2-cyanopropanamide (8).
To a solution of 2-cyanoacetamide 7 (20gm  0.23 mol) and toluene (200 ml) is added K2CO3 (39.39 gm  0.28 mol). Then DMS (17.69 gm  0.28 mol) is added slowly drop wise at 25 -35 °C. Then the reaction maintained at 55-60 °C for 6-7 h. After completion the reaction mass quenched using DM water (300 mL) and separated both the layers. Aqueous layer is extracted with ethyl acetate (6 X 100 mL). The solvent is completely distilled under vacuum to obtain 2-cyanopropanamide 8 as crude. (19.5 gm  83.6 % yield  purity by G.C 96.05%).

Example 2: Preparation of 3-amino-2-methylpropanamide (9)
To a solution of 2-cyanopropanamide 8 (7.3 gm  0.074) in MeOH (28.0 mL) is added Raney Nickel (7.3 gm) and 10 % Methanolic ammonia (13.3mL  0.078 mol). Resulted solution is hydrogenated using 7-8 kg/cm2 H2 gas pressure at 55-60 °C for 2-3 h. After completion of reaction the resulted solution is filtered over celite and washed the celite using MeOH (70.0 mL). The obtained mother liquors are distilled under vacuum to obtain as crude. (7.0 gm  90 % yield  purity by G.C 78.32%).

Example 3: Preparation of (2S 4S 5S 7S)-N-(3-amino-2-methyl-3-oxopropyl)-5-azido-4-hydroxy-2-isopropyl-7-(4-methoxy-3-(3-methoxypropoxy)benzyl)-8-methylnonanamide (10)
To a solution of TEA (7.5 mL) and 4 (3.0 gm  0.006 mol)  is added 9 (3.05 gm  0.024 mol) and 2-hydroxypyridine (0.61 gm  0.006 mol). Then the reaction is maintained at 85-90°C for 14-16 hrs. After completion of reaction quenched with 5% NaHCO3 solution (15 mL) and reaction mass is extracted using toluene (4x 15 mL). Resulted organic layer is washed using hot Dm water (2x15mL) twice followed by sat.NaCl solution (2x15 mL). The final organic layer is distilled under vacuum at 60°C to obtain crude 10. (3.3 gm  91.6 % yield)

Example 4: Preparation of (2S 4S 5S 7S)-N-(3-amino-2-methyl-3-oxopropyl)-5-azido-4-hydroxy-2-isopropyl-7-(4-methoxy-3-(3-methoxypropoxy)benzyl)-8-methylnonanamide fumarate of Formula II (Monomethyl aliksiren).

To a solution of 10 (7.0 gm  0.012 mol) in methanol (70.0 mL)  is added 5% Pd-C (0.7 gm) and 10% methanolic ammonia (3.48 gm  0.020 mol). Then the reaction is maintained at 25-35°C under reduced  5-6 kg/cm2 H2 gas pressure for 5-6 hrs. After completion of reaction  reaction mass is filter over celite and washed the bed with MeOH (30.0mL). Resulted mother liquors distilled under vacuum at 40°C to obtain crude. (5.3 gm  80.0 % yield  purity by HPLC 71.0 % (considering both isomers)). The crude (5.30gm) is dissolved in MeOH (30.0 mL) and added fumaric acid (057 gm  0.004 mol). Reaction mass was stirred to form clear solution. Reaction mass distilled at 40°c to obtain crude salt. To the crude salt 1.5:8.5 ratio of MeOH:acetonitrile (25.0 mL) and acetonitrile (125.0 mL) is added and stirred for 8-10 hr for solid isolation. Filtered the solid and washed with acetonitrile (25.0 mL) to obtain 11 as solid. (3.8 gm  65.5 % yield  purity by HPLC 87.0 % (considering both isomers)).

Example 5: Preparation of (2R 4S 5S 7S)-5-amino-N-(3-amino-2 2-dimethyl-3-oxopropyl)-4-hydroxy-2-isopropyl-7-(4-methoxy-3-(3-methoxypropoxy)benzyl)-8-methylnonanamide of Formula III (Diasteriomer).

Part 1. To a solution of TEA (25 mL) and 4 (10.0 gm  0.02 mol)  is added Segment-C (10.6 gm  0.08 mol) and 2-hydroxypyridine (2.0 gm  0.02 mol). Then the reaction is maintained at 95-100°C for 48 hr. After completion of reaction quenched with 5% NaHCO3 solution (50 mL) and reaction mass is extracted using toluene (4 x 50 mL). Resulted organic layer is ished using hot Dm water (2 x 50 mL) twice followed by sat. NaCl solution (2 x 50 mL). The final organic layer is distilled under vacuum at 60°C to obtain crude 12. (3.3 gm  91.6 % yield)
Part 2. To a solution of 12 (10.0 gm  0.0181 mol) in methanol (100 mL)  is added 5% Pd-C (1.0 gm) and 10% methanolic ammonia (5.5 gm  0.032 mol). Then the reaction is maintained at 25-35°C under reduced  5-6 kg/cm2 H2 gas pressure for 5-6 hrs. After completion of reaction  reaction mass is filter over celite and washed the bed with MeOH (30.0mL). Resulted mother liquors distilled under vacuum at 40°C to obtain crude 13. From the crude sample the desired compound is collected by simple reverse phase preparative HPLC. (5.3 gm  80.0 % yield (at crude stage purity by HPLC 18.8% ). After isolation purity by HPLC 82.70%).

Example 6: Preparation of (3S)-5-((1S 3S)-1-hydroxy-3-(4-methoxy-3-(3-methoxypropoxy)benzyl)-4-methylpentyl)-3-isopropyldihydrofuran-2(3H)-one (14)
To a solution of 2 (16.65 gm  mol) and isopropanol (150 mL) is added 2N LiOH solution ( 66.6 mL  mol) at 0 °C over a period of 10 min and the mixture stirred for 1.5 h. to the reaction mixture 2N HCl (100 mL) is added drop wise  and the reaction mixture is stirred for 1 h at 25-35° C. the reaction mixture is diluted with DM water (500 mL) and extracted with tert-butyl methyl ether (3 X 250 mL). the organic layers are ished consecutively with water ( 2 X 500 mL) and concentrated aqueous NaCl solution (200 mL)  dried over sodium sulfate and concentrated on a rotary evaporator to obtain 14 as crude. (11.0gm  75.8 % yield)

Example 7: Preparation of 2S 4S 5S 7S)-N-(3-amino-2 2-dimethyl-3-oxopropyl)-4 5-dihydroxy-2-isopropyl-7-(4-methoxy-3-(3-methoxypropoxy)benzyl)-8-methylnonanamide of Formula IV (Dihydroxy).
To a solution of TEA (12.5 mL) and 14 (5.0 gm  0.01 mol)  is added Segment-C (5.3 gm  0.04 mol) and 2-hydroxypyridine (1.0 gm  0.01 mol). Then the reaction is maintained at 95-100°C for 48hr. After completion of reaction quenched with 5% NaHCO3 solution (50 mL) and reaction mass is extracted using toluene (4 x 50 mL). Resulted organic layer is ished using hot Dm water (2 x 50 mL) twice followed by sat. NaCl solution (2 x 50 mL). The final organic layer is distilled under vacuum at 60°C to obtain Formula 4. (6.0 gm  95.0% yield purity by HPLC 87.0%)

Example 8: Preparation of 2-cyano-2-methylpropanoic acid (17)
To a solution of ethyl 2-cyano-2-methylpropanoate 16 (30.0 gm  0.21 mol) and MeOH:Water (120.0 mL(1:1)) is added LiOH.H2O (10.89 gm  0.26 mol). Resulted reaction mixture is stirred at 25-35 °C for 2-3 h. concentrated the reaction mixture on a rotary evaporator. Aqueous phase is washed with tet-buty methyl ether (2 X 100 mL) and adjuste the pH of aqueous pahse to 2 using 1N HCl solution (25.0 mL). Extracted the aqueous phase with ethyl acetate (2 X 50.0 mL) and dried over sodium sulfate  concentrated the organic phases on a rotary evaporator to obtain 17 as crude. (9.5 gm  79.6 % yield)

Example 9: Preparation of N-(3-amino-2 2-dimethyl-3-oxopropyl)-2-cyano-2-methylpropanamide (18).
To a solution of 17 (15.0 gm  0.132 mol) and acetonitrile (150 mL) is added mixture of acetonitrile (125.0 mL)  CDI (25.8 gm  0.159 mol) at 0-5°C for 10-15 min. resulted reactionmixture is stirred at 25-35° C for 4-5h. Cooled the reaction mixture to 0-5°C and filter the solid. Mother liquors is concentrated on a rotary evaporator. Crude 18 is dissolved in ethyl acetate (50 mL) and stirred for 1-2 h at 0-5 °C. Filtered the solid and washed with ethyl acetate (50mL) to obtain 18 as solid. (15.0 gm  % 53.5% yield)

Example 10: Preparation of 3-amino-N-(3-amino-2 2-dimethyl-3-oxopropyl)-2 2-dimethylpropanamide (19).
To a solution of 18 (15 gm  0.07) in MeOH (150.0 mL) is added Raney Nickel (15.0 gm) and 10 % Methanolic ammonia (12.6 gm  0.074 mol). Resulted solution is hydrogenated using 6-7 kg/cm2 H2 gas pressure at 50-60 °C for 5-6 h. After completion of reaction the resulted solution is filtered over celite and washed the celite using MeOH (15.0 mL). The obtained mother liquors are distilled under vacuum to 19 obtain as crude. The obtained 19 crude was purified by column chromatography. (7.0 gm  92 % yield).

Example 11: Preparation of (2S 4S 5S 7S)-N-(3-((3-amino-2 2-dimethyl-3-oxopropyl)amino)-2 2-dimethyl-3-oxopropyl)-5-azido-4-hydroxy-2-isopropyl-7-(4-methoxy-3-(3-methoxypropoxy)benzyl)-8-methylnonanamide (20).
To a solution of TEA (5.0 mL) and 4 (2.0 gm  mol)  is added 19 (3.7 gm  mol) and 2-hydroxypyridine 0.40 gm  mol). Then the reaction is maintained at 85-90°C for 14-16 hrs. After completion of reaction quenched with 5% NaHCO3 solution (20 mL) and reaction mass is extracted using toluene (4x 20 mL). Resulted organic layer is ished using hot Dm water (2x20mL) twice followed by sat. NaCl solution (2x20 mL). The final organic layer is distilled under vacuum at 60°C to obtain crude 20. (2.8 gm  96.0 % yield)

Example 12: Preparation of (2S 4S 5S 7S)-5-amino-N-(3-((3-amino-2 2-dimethyl-3-oxopropyl)amino)-2 2-dimethyl-3-oxopropyl)-4-hydroxy-2-isopropyl-7-(4-methoxy-3-(3-methoxypropoxy)benzyl)-8-methylnonanamide Formula V (N-alkylated aliskiren)
To a solution of 20 (3.0 gm  mol) in methanol (30.0 mL)  is added 5% Pd-C (0.3 gm) and 10% methanolic ammonia (1.28 gm  0.020 mol). Then the reaction is maintained at 25-35°C under reduced  5-6 kg/cm2 H2 gas pressure for 1-2 hrs. After completion of reaction  reaction mass is filter over celite and ished the bed with MeOH (20.0.0mL). Resulted mother liquors distilled under vacuum at 40°C to obtain crude Formula 5. (2.0 gm  53.5% yield  purity by HPLC 87%).

Example 13: Preparation of (2S 4S 5S 7S)-N-(3-amino-2 2-dimethyl-3-oxopropyl)-4-hydroxy-2-isopropyl-7-(4-methoxy-3-(3-methoxypropoxy)benzyl)-8-methyl-5-(propan-2-ylideneamino)nonanamide of Formula 6 (Sciff`s base)
To as solution of Acetone (30 mL) and 5 (10.0 gm  0.018 mol) is stirred at 35-40 °C for 4-5h. the reaction mixture is concentrated on a rotary evaporator to obtain Formula 6. (10.0 gm  93.4 % yield purity by HPLC 89.5%)

Example 14: Preparation of (2S 7R E)-2-isopropyl-7-(4-methoxy-3-(3-methoxypropoxy)benzyl)-N N 8-trimethylnon-4-enamide (2a).
To magnesium powder (4.72 gm  0.19mol) is added tetrahydrofuran (70.0 ml) at 70° C (Jacket temperature). In a separate RBF prepared a solution of synthon-A (35.0 gm 0.11 mol)  Tetrahydrofuran (70.0 mL)  MeMgCl (7.0 mL) and 1 2-dibromoethane (0.714 mL) (synthon-a solution). this solution around 1% added to above RBF and stirred for 5-01 min at 70 °C (Jacket temaparature). Then 1 2-dibromoethane (0.714 mL) added and observed for exothermicity. After exothermicity added the remaining synthon-a solution is added dropwise over a period of 30-45 minutes at 70°C (Jacket temperature). The mixture is agitated for another 60 minutes under reflux and then cooled down to -5 to 0° C. Then added dropwise over a period of 10 minutes a solution of Synthon-B (23.6 gm 0.116 mol)  N-methylpyr¬rolidone (0.22 gm  0.002)  iron(III) acetylacetonate (0.78 gm 0.002 mol) in 70.0 ml tetrahy¬drofuran at -5 to 0° C and observed for exothermicity. The reaction mixture is agitated for a further 90 minutes at 25-35 °C. cooled the reaction mass to 0-5 °C  add 10% hydrochloric acid (175.0 mL). The mixture is now extracted with Toluene (2x175.0 and 1x 105.0 mL) and the organic phases washed consecutively with 10% hydrochloric acid (105.0 mL) and water (1x175 ml). The combined organic phases are dried with sodium sulfate  filtered and concentrated by evaporation on the Rotavapor. The title compound 2a is obtained as slightly yellowish oil (46.0g  96%  yield  purity by HPLC 77.2% (40.16+37.08))

Example 15: Preparation of (3S)-5-((1R 3S)-1-bromo-3-(4-methoxy-3-(3-methoxypropoxy)benzyl)-4-methylpentyl)-3-isopropyldihydrofuran-2(3H)-one (3a)
To a solution of 2a (45.0 gm  0.08 mol (35.0 gm based on purity)) and tetrahydrofuran (450.0 mL) is added water (4.5 ml) and the mixture cooled to 0-5° C.. Then 10 times 1.3 ml 42.5% o-phosphoric acid and 10 times 1.64 g N-bromosuccinimide are added alternately every 3 minutes. The reaction mixture is agitated for another 90 minutes at 0° C. and then  over a period of 10 minutes  5% sodium hydrogen sulfite solution (225.0 ml) is added at 0-5° C. The mixture is agitated for another 15 minutes at 0° C. and then extracted with Ethyl acetate (3x225 mL). The organic phases are washed consecu¬tively with IN hydrochloric acid (1x225.0 mL)  water (1x225.0)  5% aqueous sodium hydrogencarbonate solution (1x225.0 mL) and brine (1x225.0 mL)  dried with sodium sulfate and concentrated by evaporation on the Rotavapor (50.0gm  100 % yield purity by HPLC 52.35 %  (both isomers 23.88+28.47)). By means of flash chromatography (ethyl acetate/hexane 1:3)  title compound 3a is obtained from the residue as a slightly yellowish oil (22.0 gm  56.0% yield).

Example 16: Preparation of (3S 5S)-5-((1S 3S)-1-azido-3-(4-methoxy-3-(3-methoxypropoxy)benzyl)-4-methylpentyl)-3-isopropyldihydrofuran-2(3H)-one.
A mixture of 3a  (3.0 gm  0.006 mol)  sodium azide (1.87 gm  0.02 mol)  tripropylene glycol (21.6 mL) and 7.2 ml water is agitated for 12 hours at 80° C.then second lot of sodium azide (0.39 gm  0.006 mol)is added and continued the reaction for 12 hours at 80°C.The reaction mixture is cooled to ambient tem¬perature  and dimethylamino-l-propylamine (1.5 ml) is added and the mixture then agitated for another 3 hours at ambient temperature. The reaction mixture is poured onto water (30.0 ml) and extracted with Toluene (1x30.0 and 5x15.0 ml). The organic phases are washed consecutively with 15 ml 0.5 N HC1  15 ml NaHCO3 (5% in water)  water (1x15 ml) and 15.0 ml brine. The organic phases are dried with 3.0 g sodium sulfate  filtered and concentrated by evaporation on the Rotavapor. Title compound 4 is obtained as slightly yellowish oil (2.3 gm  85% yield  purity by HPLC 66.4 % (both isomers12.08+54.32)):

Example 17: Preparation of (2R 4S 5S 7S)-N-(3-amino-2 2-dimethyl-3-oxopropyl)-5-azido-4-hydroxy-2-isopropyl-7-(4-methoxy-3-(3-methoxypropoxy)benzyl)-8-methylnonanamide.
A mixture of the compound obtained in a Example 16 (2.0 gm  0.0043 mol)   3-amino-2 2-dimethylpropionamide (2.01 gm  0.0173 mol)  2-hydroxypyri¬dine (0.41 gm  0.0043 mol) in 5.0 ml triethylamine is agitated over a period of 16 hours at 90° C. The cooled reaction mixture is cooled to ambient temperature and Toluene (10.0 mL) is added. Then reaction mass is quenched with 5 % sodium hydro¬gencarbonate solution (10.0 mL) and separated the organic layer. Reaction mixture is extracted using toluene (3x10.0 mL). The combined organic phases were washed with hot Dm water (2x 10.0 mL) followed by brine (2x10.0 mL). The combined organic phases are dried with 2.0 g sodium sulfate  filtered and concen¬trated on the rotary evaporator. The residue is dried and crude title compound 5a is obtained as an oil (2.1 gm  84% yield  purity by HPLC maojor isomer 49.36 %  minor isomer 9.86 %))

Example 18: Preparation of (2R 4S 5S 7S)-5-amino-N-(3-amino-2 2-dimethyl-3-oxopropyl)-4-hydroxy-2-isopropyl-7-(4-methoxy-3-(3-methoxypropoxy)benzyl)-8-methylnonanamide Formula VIII.
5a crude (1.5 gm  0.0025 mol) is hydrogenated for 6 hours in the presence of 0.15 g Pd/C 5% and Methanolic ammonia (0.88 gm  0.0519 mol) in 45 ml Methanol at ambient temperature using 5-6 kg/cm2 Hydrogen gas pressure. The reaction mixture is filtered and the catalyst washed with Methanol (15.0 mL). The mother liquors are concentrated by evaporation. The title compound 6 is obtained as slightly yellowish oil. (1.5 gm  (Quantitativ eyield)  purity by HPLC 74.0 %). The Formula 8 crude was purified by preparative HPLC technique.

Example 19: Preparation of (2S 4S 5S 7S)-N-(3-amino-2 2-dimethyl-3-oxopropyl)-4-hydroxy-2-isopropyl-7-(4-methoxy-3-(3-methoxypropoxy)benzyl)-8-methyl-5-(methylamino)nonanamide Formula IX (N-methyl aliskiren)
To a solution of 5 (3.0 gm  0.005 mol) in acetonitrile (15.0 ml) is added benzaldehyde (0.69 gm  0.0065 mol). Reaction maintained at 25-35 °C up to complete formation of imine by TLC. Then DMS added (0.40 gm  0.0065mol). Reaction maintained at 50-55 °C up to complete formation of monmethyl aliskiren by TLC. After completion of reaction DM water (15.0 mL) is added and reaction mass extracted using Ethylacetate (2x15.0 mL). The combined organic phases are washed using 5% sodium bicarbonate solution (15.0mL).followed by Dm water (15.0mL)  brine (15.0 mL). Distill of the solvent completely under vacuum at 40°C to obtain the title compound as yellowish oily liquid. To the crude silica (15.0 gm) and dicloromethane (20. 0 ml) is added and stirred for 10 minutes. Reaction mass distilled completely under vacuum at 40 °C. The silica absorbed compound is washed with 50% ethylacetate and hexane mixture (3x50.0 mL). Then resulted silica absorbed compound is extracted in to methanol (3x50.0 mL). The combined organic layers (methanol) are distilled under vacuum to obtain title compound as yellowish oily liquid (3.0 gm  58% yield  and purity after preparative 91.3%).

Example 20: Preparation of (2S 4S 5S 7S)-5-amino-N-(3-amino-2 2-dimethyl-3-oxopropyl)-4-hydroxy-7-(3-hydroxy-4-methoxybenzyl)-2-isopropyl-8-methylnonanamide Formula X
To a solution of 5 (10.0 gm  0.018 mol) in dichloremethane (20 mL) was added slowly drop wise 1M BBr solution (144.4 ml  0.145 mol) for 10-15 minutes at -10 to -5°C. After addition  the reaction mass was maintained for 10-15 min at -10 to -5°C. Reaction was monitored by TLC. After completion of reaction mass it was quenched with 10% Methanolic ammonia solution (ml) and distilled the RM completely. Then 5% Na2CO3 solution was added and the aqueous layer was extracted using using ethyl actate (3x120 mL). The resulted organic layer was distilled at 40°C to obtain crude Formula 10 (8.0 gm  93% yield  purity by HPLC before preparative 30.42%  after preparative 87.68 %)

Example 21: Preparation of (2S 4S 5S 7S)-5-amino-N-(3-amino-2 2-dimethyl-3-oxopropyl)-4-hydroxy-7-(4-hydroxy-3-(3-methoxypropoxy)benzyl)-2-isopropyl-8-methylnonanamide Formula XI.
To a solution of AlCl3 (10.0 gm  0.075 mol) in dichloremethane (20 mL) was added slowly drop wise Ethanethiol (20.0 mL  0.27 mol) for 10-15 minutes at 0-5°C. After addition  the reaction mass was maintained for 10-15 min at 0-5°C. To the above reaction mixture was added 5 (10.0 gm  0.018 mol) in dichloremethane (20 mL) slowly drop wise for 10-15 minutes at 0-5°C. Reaction maintained for 1.5-2 hr at 25-35°C and reaction was monitored by TLC. After completion of reaction mass was quenched in to water and adjusted the pH of reaction mass to 9-10 at 0-5°C using 10% Na2CO3 solution. Then the reaction mass was extracted using ethyl actate (3x120 mL). The resulted organic layer was distilled at 40°C to obtain Formula 11 (2.5 gm  25.0 % yield  purity by HPLC 77.06%).

Example 22: Preparation of (2S 4S 5S 7S)-5-amino-N-(3-amino-2 2-dimethyl-3-oxopropyl)-4-hydroxy-7-(3-(3-hydroxypropoxy)-4-methoxybenzyl)-2-isopropyl-8-methylnonanamide Formula XII.
To a solution of NaI (16.2 gm  0.108 mol) in mixture of acetonitrile and dichloremethane (1:1  120 mL) was added slowly drop wise SiCl4 (13.3 mL  0.108 mol) for 10-15 minutes at 25-35°C. After addition  the reaction mass was maintained for 10-15 min at 25-35°C. To the above reaction mixture was added 5 (12.0 gm  0.0217 mol) in mixture of acetonitrile and dichloremethane (1:1  160 mL) slowly drop wise for 10-15 minutes at 25-35°C. Reaction maintained for 1.5-2 hr at 25-35°C and reaction was monitored by TLC. After completion of reaction reaction mass was quenched in to water and adjusted the pH of reaction mass to 9-10 at 0-5°C using 10% Na2CO3 solution. Then the reaction mass was extracted using ethyl actate (3x120 mL). The resulted organic layer was distilled at 40°C crude formula 12 (10.0 gm  86.0% yield  purity by HPLC before preparative 29.81% and after preparative 94.02%).

Example 23: Preparation of (3S 5S)-5-((1S 3S)-1-amino-3-(4-methoxy-3-(3-methoxypropoxy)benzyl)-4-methylpentyl)-3-isopropyldihydrofuran-2(3H)-one Formula XIII
To a solution of THF (150.0 mL) and 4 (12.0 gm  0.026 mol) was added triethyl amine (2.62 gm  0.026 mol) and 5% Pd-C (1.8 gm). The resulted reaction mass was stirred in a autoclave vessel at 25-35°C using 5-6 kg/cm2 H2 gas pressure up to completion of reaction. Then filtered the reaction mass over hyflow and washed the bed with THF (180.0 mL). Resulting mother liquors distilled completely under vaccum at 40 °C to obtain the title compound as yellowish oily liquid. (10.0 gm  88% yield  purity by HPLC 90 %).

We claim:

1. Use of the impurities of Formula II to Formula IX as reference markers for determining the purity of Aliskiren.
2. Use of the metabolites X to XIII for as reference markers for determining the purity of Aliskiren.