CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of United States Provisional Application

Nos. 61/381,337, filed September 9, 2010; 61/414,061, filed November 16, 2010;

61/419,976, filed December 6, 2010; 61/467, 126, filed March 24, 201 1; and

61/474,040, filed April 1 1, 2011, each incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

[0002] The present invention is directed to the preparation of new aliskiren

intermediates and impurities.

BACKGROUND OF THE INVENTION

[0003] Aliskiren hemifumarate has the chemical name: (2S,4S,5S,7S)-N-(2- carbamoyl-2-methylpropyl)-5-amino-4-hydroxy-2,7-diisopropyl- 8-[4-methoxy-3-(3- methoxypropoxy)phenyl]-octanamide hemifumarate [θ ₃ οϊ½Ν3θ6 · 0.5 C4H4O4] and the following structure:

[0004] Aliskiren hemifumarate is indicated for treatment of hypertension, acting as a renin inhibitor. It is marketed by Novartis as TEKTURNA ^® in a once-daily

formulation. Aliskiren and structurally related compounds are disclosed in U.S. Patent No. 5,559,1 11. Synthesis, pharmacological actions, pharmacokinetics and clinical

studies of aliskiren and structurally related compounds are referred to in Lindsay, K. B.

et ah, J. Org. Chem., Vol. 71, pp 4766-4777 (2006) and in Drugs of the Future, Vol.

26, No.12, pp 1139-1148 (2001). [0005] Preparation of an aliskiren analogue, namely (2S,4S,5S,7S)-7-(4-methoxy-3- (2-(methoxymethoxy)ethyl)benzyl)-5-amino-N-butyl-4-hydroxy-2 -isopropyl-8- methylnonanamide and synthetic intermediates used in the preparation are described in U.S. Patents No. 5,559, 11 1 ("US ' 111") and 5,606,078 ("US Ό78").

[0006] Like any synthetic compound, Aliskiren and its synthetic intermediates can contain extraneous compounds or impurities. These impurities may be, for example, starting materials, by-products of the reaction, products of side reactions, or degradation products. Impurities in Aliskiren, or any active pharmaceutical ingredient ("API"), are undesirable and in extreme cases might even be harmful to a patient being treated with a dosage form containing the API.

[0007] The purity of an API produced in a manufacturing process is critical for commercialization. The U.S. Food and Drug Administration ("FDA") requires that process impurities be maintained below set limits. For example, in its ICH Q7A guidance for API manufacturers, the FDA specifies the quality of raw materials that may be used, as well as acceptable process conditions, such as temperature, pressure, time, and stoichiometric ratios, including purification steps, such as crystallization, distillation, and liquid-liquid extraction. See ICH Good Manufacturing Practice Guide for Active Pharmaceutical Ingredients, Q7A, Current Step 4 Version (November 10, 2000).

[0008] 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. The FDA requires that an API is as free of impurities as possible, so that it is as safe as possible for clinical use. For example, the FDA recommends that the amounts of some impurities be limited to less than 0.1 percent. See ICH Good Manufacturing Practice Guide for Active Pharmaceutical Ingredients, Q7A, Current Step 4 Version (November 10, 2000).

[0009] 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. See Strobel, H.A., et al, Chemical Instrumentation: A Systematic

Approach, 953, 3d ed. (Wiley & Sons, New York 1989). 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."

[0010] The retention time can vary about a mean value based upon the condition of the instrumentation, as well as many other factors. To mitigate the effects such variations have upon accurate identification of an impurity, practitioners often use "relative retention time" ("RRT") to identify impurities. See supra Strobel at 922. 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.

[001 1] 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.

[0012] A reference standard is an "external standard," when a solution of a known concentration of the reference standard and an unknown mixture are analyzed separately using the same technique. See supra Strobel at 924; Snyder, L.R., et al., Introduction to Modern Liquid Chromatography, 549, 2d ed. (John Wiley & Sons, New York 1979). The amount of the impurity in the sample can be determined by comparing the magnitude of the detector response for the reference standard to that for the impurity. See U.S. patent No. 6,333,198, hereby incorporated by reference.

[0013] The reference standard can also be used as an "internal standard," i.e., one that is directly added to the sample in a predetermined amount. When the reference standard is an internal standard, a "response factor," which compensates for differences in the sensitivity of the detector to the impurity and the reference standard, is used to quantify the amount of the impurity in the sample. See supra Strobel at 894. For this purpose, the reference standard is added directly to the mixture, and is known as an "internal standard." See supra Strobel at 925; Snyder at 552.

[0014] The technique of "standard addition" can also be used to quantify the amount of the impurity. This technique is used where the sample contains an unknown detectable amount of the reference standard. In a "standard addition," at least two samples are prepared by adding known and differing amounts of the internal standard. See supra Strobel at 391-393; Snyder at 571-572. The proportion of the detector response due to the reference standard present in the sample can be determined by plotting the detector response against the amount of the reference standard added to each of the samples, and extrapolating the plot to zero. See supra Strobel at 392, Figure 11.4.

SUMMARY OF THE INVENTION

[0015] The present invention relates to new salts of compound VII and to new compound DC, and specifically, compound IX ((2S,4S,5S,7S)-7-(3-amino-2,2- dimethyl-3 -oxopropylcarbamoyl)-4-azido-5 -hydroxy-2-isopropyl- 1 -(4-methoxy-3 -(3 - methoxypropoxy)phenyl)-8-methylnonyl isobutyrate). These compounds can be intermediates in a process for preparing Aliskiren. [0016] The present invention further relates to processes of preparing Aliskiren comprising using each one of the above intermediates, or any combination of the above intermediates.

[0017] The present invention relates also to new compound Imp-VII, and compound Imp-IIF. These compounds can either be by-products, i.e., impurities or can be used as intermediates to prepare Aliskiren. When classified as impurities, they can be used as reference markers or reference standards in the determination of the purity of aliskiren or intermediates of aliskiren, particularly in the herein described process.

BRIEF DESCRIPTION OF THE FIGURES

[0018] Figure 1 provides a powder XRD pattern of amorphous L-Arginine salt of compound VII.

[0019] Figure 2 provides a powder XRD pattern of amorphous compound IX.

[0020] Figure 3 provides a powder XRD pattern of aliskiren isobutyric acid salt.

[0021 ] Figure 4 provides a powder XRD pattern of crystalline compound IX.

[0022] Figure 5 provides a ^l H NMR spectrum of Imp-Ill.

[0023] Figure 6 provides a ¹³ C NMR spectrum of Imp-Ill.

[0024] Figure 7 provides a ^l H NMR spectrum of Imp-V.

[0025] Figure 8 provides a ¹³ C NMR spectrum of Imp-V.

[0026] Figure 9 provides a powder XRD pattern of Aliskiren hemifumarate Form

VIII.

[0027] Figure 10 provides a H NMR spectrum of compound IX.

[0028] Figure 11 provides a C NMR spectrum of compound IX.

[0029] Figure 12 provides a H NMR spectrum of Imp-VII. [0030] Figure 13 provides a ^{1 J} C NMR spectrum of Imp-VII.

DETAILED DESCRIPTION OF THE INVENTION

[0031] As used herein, Aliskiren hemifumarate Form VIII refers to a crystalline Aliskiren hemifumarate characterized by a powder XRD pattern with peaks at 6.0, 7.4, 9.3 and 11.1 ± 0.2 degrees 2-theta, or by the powder XRD as substantially shown in Figure 9 as indicated in PCT Publication No. WO 2009064479.

[0032] A crystal form may be referred to herein as being characterized by graphical data "as depicted in" a Figure. Such data include, for example, powder X-ray diffracto- grams, FTIR spectra, and solid state NMR spectra. The skilled person will understand that such graphical representations of data may be subject to small variations, e.g., in peak relative intensities and peak positions due to factors such as variations in instrument response and variations in sample concentration and purity, which are well known to the skilled person. Nonetheless, the skilled person would readily be capable of comparing the graphical data in the Figures herein with graphical data generated for an unknown crystal form and confirming whether the two sets of graphical data are characterizing the same crystal form or two different crystal forms.

[0033] As used herein, the term "reference standard" refers to a compound that may be used both for quantitative and qualitative analysis of an active pharmaceutical ingredient. For example, the HPLC retention time of the reference standard compound allows a relative retention time to be determined for the active pharmaceutical ingredient, thus making qualitative analysis possible. Furthermore, a known concentration of the reference standard compound in an analytical sample before injection into an HPLC column allows the areas under the HPLC peaks to be compared, thus making quantitative analysis possible.

[0034] As "reference marker" is used in qualitative analysis to identify components of a mixture based upon their position, e.g., in a chromatogram or on a Thin Layer Chromatography (TLC) plate (Strobel pp. 921, 922, 953). For this purpose, the compound does not necessarily have to be added to the mixture if it is present in the mixture. A "reference marker" is used only for qualitative analysis, while a reference standard may be used for quantitative or qualitative analysis, or both. Hence, a reference marker is a subset of a reference standard, and is included within the definition of a reference standard.

[0035] As used herein, unless indicated otherwise, the term "isolated" in reference to the compounds of the present invention corresponds to these compounds that are physically separated from the reaction mixture, in which they are formed

[0036] The present invention relates to new salts of compound VII and to a new compound according to formula IX" , and specifically, Compound IX (as will be shown below). These compounds can be intermediates in a process for preparing Aliskiren. For example, according to the following process (Scheme 1):

s ren

[0037] Compound VII (or its salts) and compound IX ^" can be obtained in a solid form that can be crystallized, and thereby purified. This ability to crystallize a late intermediate in a long synthetic process, especially when those before are all oily, can serve to improve the overall performance of the synthetic process.

[0038] The present invention provides amino acid salts of (2S,4S,5S,7S)-5-azido-4- hydroxy-7-(hydroxy(4-methoxy-3-(3-methoxypropoxy)phenyl)meth yl)-2-isopropyl-8- methylnonanoic acid (referred to herein as compound VII), for example with arginine, lysine or esters of amino acids such as glycine, alanine, phenylalanine, leucine, isoleucine, tyrosine, tryptophan, and valine; in particular, arginine, and more particularly, L-arginine.

[0039] Salts of Compound VII with organic and inorganic bases (such as: lithium salt, potassium salt, barium salt, calcium salt, amine salts such as the dicyclohexyl amine salt and the phenylethyl amine salt) are usually foams or oily materials, and are accordingly difficult to purify, for example by crystallization. However, the salts of the present invention are solids.

[0040] The salt of compound VII with L- Arginine is shown in the structure below:

[0041] The salts of compound VII with amino acids can be prepared, for example, by a process substantially as described in Scheme 2, below:

Li-salt

VII, closed form

VII, opened form

L-Arginine salt of VII (opened form)

Solid compound

[0042] These salts can be used to prepare Aliskiren, for example, by a process comprising preparing the salt of compound VII by the process described above, and further converting it to aliskiren, for example, as in the examples, provided herein.

[0043] The present invention also provides compound according to formula DT :

wherein, Wi and W2 are independently selected from: -H and a suitable hydroxy protecting group; or a diastereomer of said compound. Suitable hydroxy protecting groups include, for example, an ester group, a sulfonate group, a carbonate group, a silyl group and an aryl alkyl group, optionally substituted by 1, 2 or 3 groups selected from C1-C4 alkyl and C1-C4 alkoxy.

[0044] According to some embodiments, the ester group is a radical having the formula -(C=0)R ¹ , such as acetyl, benzoyl, propionyl or isobutyryl; the sulfonate group is a radical having the formula -(S02) ² , such as mesyl, besyl, tosyl, or trifluoro- methylsulfonyl (trifyl); the carbonate group is a radical having the formula -(C=0)OR ³ , and the silyl group is a radical having the formula -SiR ⁴ 3; wherein at each occurrence, R ¹ , R ² and R ³ are independently selected from -Ci-Cio hydrocarbyl, optionally substituted with 1, 2 or 3 substituents independently selected from -OH and halogen; and wherein, at each occurrence, R ⁴ is independently selected from phenyl and Ci-Ce alkyl. Suitable aryl alkyl groups suitable as hydroxy protecting groups include, for example, a benzyl group, optionally substituted by 1, 2 or 3 groups selected from C ₁ -C ₄ alkyl and C1-C4 alkoxy; for example, benzyl, 4-methoxybenzyl or 2,4,dimethoxy- benzyl.

[0045] A hydrocarbyl group is a group, having the recited number of carbon atoms, which is composed entirely of carbon and hydrogen atoms. Suitable -C1-C1 ₀ hydrocarbyl groups in R ¹ , R ² and R ³ include, for example, C1-C7 alkyl groups, such as methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, t-butyl, pentyl, hexyl and heptyl; _, C2-C7 alkenyl groups such as propenyl, butenyl and cyclohexenyl; C3-C7 cycloalkyl groups such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and norbornyl; C3-C7 cycloalkyl- C1-C3 alkyl groups such as cyclopropylmethyl and cyclopentylethyl; C6-C1 ₀ aryl, such as phenyl, naphthyl, tolyl, xylyl, and mesityl; and phenyl C1-C4 alkyl, such as benzyl and phenylethyl.

[0046] Suitable silyl groups include, for example: trimethylsilyl, triethylsilyl, triisopropylsilyl (TIPS), tert-butyldimethylsilyl (TBDMS), tert-butyldiphenylsilyl (TBDPS) and [2-(trimethylsilyl)ethoxy]methyl (SEM).

[0047] Compound IX ^" is also provided in an isolated form, e.g., as a solid.

Compound DC can be in amorphous or crystalline form.

[0048] In particular, the present invention provides compound IX ((2S,4S,5S,7S)-7- (3-amino-2,2-dimethyl-3-oxopropylcarbamoyl)-4-azido-5-hydrox y-2-isopropyl-l-(4- methoxy-3-(3-methoxypropoxy)phenyl)-8-methylnonyl isobutyrate), or any of its diastereomers. Compound IX has the following formula:

[0049] In some embodiments, compound IX is provided in an isolated form, e.g., as a solid. Compound IX can be in amorphous or crystalline form.

[0050] The crystalline form of Compound IX can be characterized by selecting one, two, three, four, five or more characteristic peaks from the diffractogram depicted in Figure 4, that includes peaks at 5.9°, 9.0°, 9.3°, 9.9°, 10.7°, 14.1°, 14.9°, 16.5°, 17.0°, 18.0°, 18.7°, 20.4°, 21.0°, 21.3°, 22.7°, 23.9°, 24.4°, 25.6°, 26.5° and 27.2 ± 0.2° two theta.

[0051] Crystalline Compound IX can be characterized, for example, by data selected from: an XRD powder diffraction pattern having peaks at 9.9°, 14.1°, 18.0°, 21.0°, and 22.7° ± 0.2° two theta; an XRD pattern as shown in Figure 4; and combinations thereof. Crystalline Compound IX can be further characterized by additional peaks at 9.0°, 9.3°, 14.9°, 16.5°, and 17.0° ± 0.2° two theta.

[0052] Compound IX can be used to prepare Aliskiren or salts thereof, for example, by a process comprising preparing compound IX, and converting it to Aliskiren or salts thereof, for example, by a hydrogenation process. The salts of Aliskiren may be formed for example, when the R-C(0)0 group, which is removed during

hydrogenation, reacts with Aliskiren.

[0053] These salts can be used to prepare Aliskiren or aliskiren hemifumarate, or formulations thereof. For example, Aliskiren base can be prepared from the above mentioned Aliskiren salts, by reacting them with a basic aqueous solution. Optionally, the obtained Aliskiren base is further treated with fumaric acid to result with Aliskiren hemifumarate. [0054] Hydrogenation may be carried out at atmospheric pressure or at greater than atmospheric pressure, for example at pressures from about 1 to about 10 atmospheres, or from about 1 to about 5 atmospheres, or from about 1 to about 3 atmospheres. The hydrogenation is typically carried out in the presence of a suitable catalyst. Suitable catalysts include, for example palladium on carbon.

[0055] In a particular embodiment, after the separation of the catalyst from the reaction mixture and the removal of the solvent, the obtained product is dissolved in methylene chloride, and HC1 (gas) is added.

[0056] The Aliskiren obtained after exposing the reaction mixture to HC1 (gas) has a very high purity, for example 99.50 to 99.99%, or 99.80 to 99.99%, or 99.95 to 99.98% as measured by HPLC.

[0057] The present invention relates also to new compound Imp-ΙΙΓ, and compound Imp-VII. These compounds can either be by-products, i.e., impurities or can be used as intermediates to prepare Aliskiren. When classified as impurities, they can be used as reference markers or reference standards in the determination of the purity of aliskiren or intermediates of aliskiren, particularly in the herein described process.

The present invention also provides compound according to formula Imp-

or a diastereomer or salt thereof; wherein, Wi and W2 are independently selected from: -H, and a suitable hydroxy protecting group; and G ¹ and G ² are independently selected from: -H, and an amino protecting group. Suitable hydroxy protecting groups include, for example an ester group, a sulfonate group, a carbonate group, a silyl group and an aryl alkyl group, optionally substituted by 1, 2 or 3 groups selected from C ₁ -C ₄ alkyl and C1-C4 alkoxy. [0059] According to some embodiments, the ester group is a radical having the formula -(C=0)R ¹ , such as acetyl, benzoyl, propenyl or isobutyryl; the sulfonate group is a radical having the formula -(S02) ² , such as mesyl, trifluoromethylsulfonyl (trifyl), besyl or tosyl; the carbonate group is a radical having the formula -(C=0)OR ³ , and the silyl group is a radical having the formula -SiR ⁴ 3; wherein at each occurrence, R ¹ , R ² , and R ³ are independently selected from -Ci-Ciohydrocarbyl, optionally substituted with 1, 2 or 3 substituents independently selected from -OH and halogen; and wherein, at each occurrence, R ⁴ is independently selected from phenyl and Ci-Ce alkyl. Suitable silyl groups useful as hydroxy protecting groups include, for example: trimethylsilyl, triethylsilyl, triisopropylsilyl (TIPS), tert-butyldimethylsilyl (TBDMS), tert-butyldiphenylsilyl (TBDPS) and [2-(trimethylsilyl)ethoxy]methyl (SEM). Suitable aryl alkyl groups suitable as hydroxy protecting groups include, for example, a benzyl group, optionally substituted by 1, 2 or 3 groups selected from C ₁ -C ₄ alkyl and C1-C4 alkoxy; for example, benzyl, 4-methoxybenzyl or 2,4,dimethoxybenzyl.

[0060] Suitable hydrocarbyl groups in a W ¹ or W ² group in a compound according to Formula Imp-ΙΙΓ may be defined in the same way as a hydrocarbyl group occurring in the W ¹ or W ² group of a compound according to Formula IX ^" , as described above. Suitable amino protecting groups include, for example, a group that forms a carbamate group with the nitrogen in the amino group, such as -C(=0)OC ₁ -C ₁ o hydrocarbyl, e.g., -C(=0)OEt, -C(=0)OCH ₃ or -C(=0)OBN (Cbz); a silyl group; an aryl alkyl group, optionally substituted by 1, 2 or 3 groups selected from C ₁ -C ₄ alkyl and C1-C4 alkoxy; and a group that forms a cyclic imide with the nitrogen of the amino group, e.g., a phthalimide or maleimide, optionally substituted by 1, 2, 3 or 4 substituents independently selected from C ₁ -C ₄ alkyl, phenyl, halogen, and nitro.

[0061] Suitable silyl groups include, for example, the silyl groups described above for W ¹ and W ² , e.g., trimethylsilyl, TBDMS and SEM groups.

[0062] Suitable aryl alkyl groups useful as amino protecting groups include, for example, a benzyl group, optionally substituted by 1, 2 or 3 groups selected from C ₁ -C ₄ alkyl and C1-C4 alkoxy; for example, benzyl, 4-methoxybenzyl or 2,4,dimethoxy- benzyl. [0063] Suitable cyclic imide groups for bis protection of the amine include, for example, phthalimide, dichlorophthalimide, tetrachlorophthalimide, 4-nitrophthalimide, diphenylmaleimide and dimethylmaleimide.

[0064] In particular, the present invention provides compound Imp-Ill

((2S,4S,5S,7S)-4-amino-7-(3-amino-2,2-dimethyl-3-oxopropy lcarbamoyl)-5-hydroxy- 2-isopropyl-l-(4-methoxy-3-(3-methoxypropoxy)phenyl)-8-methy lnonyl isobutyrate), or any of its diastereomers, of the following formula:

; or salts thereof.

[0065] In some embodiments, Imp-III is provided in an isolated form, e.g., as a solid. In some embodiments, Imp-III is crystalline.

[0066] The compound Imp-III can be characterized by data selected from: 1H NMR (300 MHz, CDC1 ₃ ), δ/ppm, having peaks at about 0.80-1.00 (m, 12 H); 1.10-1.16 (m, 6H), 1.20-1.23 (m, 6H); 1.30-1.45 (m, 1H), 1.50-1.90 (m, 5H); 2.00-2.60 (m, 10H); 3.06-3.12 (m, 1H); 3.31-3.38 (m, 1H), 3.35 (s, 1H); 3.49-3.51 (m, 1H), 3.55-3.59 (m, 2H); 3.84 (s, 1H); 4.07-4.12 (m, 2H); 5.60-5.75 (m, 2H); 6.38 (broad, 1H); 6.50-6.55 (m, 1H), 6.80-6.85 (m, 3H); ^X H NMR pattern as depicted in figure 5; a ¹³ C NMR pattern as depicted in figure 6; a LCMS/ESI: [M+H] ⁺ =638; and combinations thereof.

[0067] The present invention also provides compound Imp-VII (2S,4S)-N-(3-amino- 2,2-dimethyl-3-oxopropyl)-4-hydroxy-2-isopropyl-4-((2S,4S)-4 -isopropyl-5-(4- methoxy-3-(3-methoxypropoxy)phenyl)pyrrolidin-2-yl)butanamid e of the following formula:

or any of its diastereomers. [0068] Compound Imp-VII, can be characterized by data selected from: 1H NMR (300 MHz, DMSO d-6), δ/ppm, having peaks at about 0.74 (d, J=6.6 Hz, 6H), 0.92- 0.99 (m, 12 H), 1.18-1.32 (m, 1 H), 1.4-1.5 (m, 1H), 1.58-1.72 (m, 2H), 1.72-1.88 (m, 3H), 2.10-2.20 (m, 1 H), 3.05-3.15 (m, 1H), 3.18-3.30 (m, 5H), 3.45-3.52 (m, 2H), 3.60-3.70 (m, 1H), 3.74 (s, 3H), 3.95-4.05 (m, 4H), 4.10-4.20 (m, 2H), 6.75-6.92 (m, 4H), 7.11 (s, 1H), 7.40-7.50 (m, 1H); ^X H NMR pattern as depicted in figure 12; a ¹³ C NMR (75 MHz, DMSO d-6), δ/ppm, having peaks at about 178.3; 174.2; 148.6; 147.9; 133.3; 120.2; 112.1; 111.5; 73.5; 73.2; 68.5; 65.3; 57.8; 55.5; 49.8; 47.6; 46.1; 45.9; 42.4; 30.6; 29.0; 26.4; 23.5; 23.4; 20.8; 20.7; 19.5, 15.7; a ¹³ C NMR pattern as depicted in figure 13; a LCMS/ESI: [M+H] ⁺ = 550; [M+Na] ⁺ = 572, [M+K] ⁺ = 588; and combinations thereof.

[0069] In some embodiments, Imp-VII is provided in an isolated form, e.g., as a solid. In some embodiments, Imp-VII is crystalline.

[0070] The present invention is also directed to a method of using Imp-Ill and/or Imp-VII as a reference marker(s) to analyze the purity of Aliskiren or salts thereof.

[0071] This method comprises: a) providing a reference sample comprising Aliskiren or salts thereof and Imp-Ill and/or Imp-VII; b) analyzing the reference sample by HPLC and determining the relative retention time of Imp-Ill and/or Imp-VII compared to Aliskiren or salts thereof; c) analyzing a sample of Aliskiren or salts thereof by HPLC and determining the relative retention times of the contents of the sample as compared to Aliskiren or salts thereof; and d) comparing the relative retention times calculated in step c) to the relative retention time calculated in step b) for Imp-Ill and/or Imp-VII, wherein if any of the relative retention times calculated in step c) are substantially the same as the relative retention time of Imp-Ill and/or Imp- VII, Imp-Ill and/or Imp-VII are present in the sample of Aliskiren or salts thereof.

[0072] The present invention is also directed to a method of using Imp-Ill and/or Imp-VII as a reference standard(s) to quantify the amount of Imp-Ill and/or Imp-VII in a sample of Aliskiren or salts thereof. [0073] This method comprises: a) measuring by HPLC the area under the peak corresponding to Imp-Ill and/or Imp-VII in a sample of Aliskiren or salts thereof having an unknown amount of Imp-Ill and/or Imp-VII; b) measuring by HPLC the area under a peak corresponding to Imp-Ill and/or Imp-VII in a reference standard comprising a known amount of Imp-Ill and/or Imp-VII; and c) determining the amount of Imp-Ill and/or Imp-VII in the Aliskiren or salts thereof sample by comparing the area calculated in step a) to the area calculated in step b.

[0074] The HPLC analysis may be performed, for example, using a high performance liquid chromatograph having a, Ascentis Express C8 2.7um, 150*2. lmm column and an ultraviolet detector at 230 nm. The sample to be analyzed is dissolved in acetonitrile:water (80%:20%) and gradient eluted through the column with a mixture of water: 0.1% TFA (90: 10), followed by a mixture of acetonitrile: methanol (90:10).

[0075] The present invention also provides A process of analyzing the purity of Aliskiren or a salt thereof comprising providing a reference sample containing

Aliskiren or a salt thereof and a compound according to Formula Imp ΙΙΓ and/or Imp- VII, and determining the relative HPLC retention time of the Formula Imp-ΙΙΓ and/or Imp-VII compound compared to the Aliskiren or salt thereof.

[0076] The present invention further provides a process of quantifying the amount of a compound of Formula Imp-ΙΙΓ and/or Imp-VII in a sample of Aliskiren or a salt thereof comprising:

(a) performing HPLC analysis of a reference sample containing a known amount of a compound according to Formula Imp-IIF and/or Imp-VII and measuring the area under the peak corresponding to the Formula Imp-IIF and/or Imp-VII compound;

(b) performing HPLC analysis of a sample containing Aliskiren or a salt thereof and an unknown amount of the compound of Formula Imp-IIF and/or Imp-VII and measuring the area under the peak corresponding to said compound according to Formula Imp-IIF and/or Imp-VII; and (c) determining the amount of the compound of Formula Imp-ΙΙΓ and/or Imp-VII in the Aliskiren sample by comparing the peak area measured in step (a) to the peak area measured in step (b).

[0077] Compounds according to Formulae Imp-IIF and/or Imp-VIF can be used to prepare Aliskiren or salts thereof, for example, by a process comprising preparing compounds of Formulae Imp-IIF and/or Imp-VIF, and further converting them to Aliskiren or salts thereof, for example, by a hydrogenation process.

[0078] Having thus described the invention with reference to particular preferred embodiments and illustrative examples, those in the art can appreciate modifications to the invention as described and illustrated that do not depart from the spirit and scope of the invention as disclosed in the specification. The examples are set forth to aid in understanding the invention, but are not intended to limit its scope in any way, and should not be so construed.

[0079] XRD analysis was performed on ARL (SCFNTAG) powder X-Ray diffractometer model X'TRA equipped with a solid state detector. Copper radiation of 1.5418 A was used. Scanning parameters: range: 2-40 degrees two-theta; scan mode: continuous scan; step size: 0.05°; and a rate of 3 deg/min.

[0080] Peak positions were determined by using silicon powder as internal standard in an admixture with the sample measured. The position of the silicon (11 1) peak was corrected to be 28.45 degrees two theta. (No correction was performed on the presented diffractograms in the figures.)

[0081] ^X H NMR (300 MHz) and ¹³ C NMR (75 MHz) were recorded on a Bruker 300 MHz spectrometer in CDCI ₃ as a solvent and the chemical shifts were given in δ value with TMS as an internal standard;

[0082] LCMS interpretation was achieved from analysis, performed on Agilent 1100 HPLC connected to Bruker Esquir 6000 spectrometer.

[0083] Impurity profile was check using HPLC method: UPLC

Column & packing: Ascentis Express C8 2.7μηι, 150*2. lmm

Eluent A: 90% Water : 10% Eluent B : 0.1% TFA

Eluent B: 90% Acetonitrile : 10% Methanol

Gradient of Eluent: Time (min) Eluent A (%) Eluent B (%)

0 80 20

6.0 80 20 12 75 25 30 0 100

Stop time: 30 min

Equilibration time: 6 min

Flow: 0.5 ml/min

Detector: 230 nm

Injection volume: 3 μΐ

Diluent ACN: Water 8:2

Column temperature 30°C

Autosampler temperature 15°C EXAMPLES

Example 1 : Preparation of the salt of ( ^, 2S,4S,5SJS -5-azido-4-hydroxy-7-( ^' hvdroxy( ^' 4- methoxy-3-(3-methoxypropoxy)phenyl)methyl)-2-isopropyl-8-met hylnonanoic acid (VII. opened form) with L-Arginine (as a solid intermediate).

[0084] A IN solution of LiOH in water (80 mL) was added dropwise at 0-2 °C to a solution of intermediate VII, (3S,5S)-5-((lS,3S)-l-azido-3-(hydroxy(4-methoxy-3-(3- methoxypropoxy)phenyl)methyl)-4-methylpentyl)-3-isopropyldih ydrofuran-2(3H)-one (16.2 g) in methanol (80 mL). The resulting mixture was stirred for 30 min at 0-2 °C, and then allowed to warm to 25°C (with stirring). The mixture was then stirred at 25 °C for about 2 h, with HPLC in-process control. The methanol was then evaporated at 25 °C / <10 mbar, and the resulting aqueous solution was extracted with toluene. Ethyl acetate (80 mL) and brine (20 mL) were added to the aqueous solution, and the mixture was vigorously stirred. The organic layer, containing the Li-salt, was separated, dried over sodium sulfate, filtered and evaporated to give a brown foam (12.3 g). The foam (7.8 g) was mixed with t-butyl ether (80 mL) and a 10 % aqueous solution of citric acid. This mixture was stirred for about 10 min. The organic layer was separated and evaporated to dryness at 25 °C/ <10 mbar, to provide a yellow oil (8.0 g, VII, opened form). The yellow oil was dissolved in ethanol (100 mL) and treated with a solution of L-Arginine (2.9 g) in water (20 mL). This mixture was stirred for about 40 min at 25 °C. The solvent was then evaporated, and the residue was stripped with absolute ethanol (100 mL). The resulting residue was mixed with acetonitrile (100 mL) to provide a beige precipitate. The acetonitrile was then evaporated to provide the product as a beige powder (9.1 g), in amorphous form.

[0085] The beige powder (1 g) was triturated with acetonitrile (10 mL) for 15 h at 25 °C, and the resulting solid was filtered under a nitrogen atmosphere to give a solid having a melting point of 116.1 °C. Figure 1 provides an X-ray diffractogram of the L- arginine salt of (2S,4S,5S,7S)-5-azido-4-hydroxy-7-(hydroxy(4-methoxy-3-(3- methoxypropoxy)phenyl)methyl)-2-isopropyl-8-methylnonanoic acid. The solid was found to be amorphous.

Example 2: Preparation of ( ^' 2S,4S,5SJS -7-( ^' 3-amino-2,2-dimethyl-3-oxopropyl- carbamoyl)-4-azido-5-hydroxy-2-isopropyl-l-(4-methoxy-3-(3-m ethoxypropoxy)- phenyl)-8-methylnonyl isobutyrate (Compound DO

[0086] (2S,4S)-4-Azido-2-isopropyl-4-((2S,4S)-4-isopropyl-5-oxotetr ahydrofuran- 2-yl)-l-(4-methoxy-3-(3-methoxypropoxy)phenyl)butyl isobutyrate (8.7 g, 0.016 mol) was mixed with 3-amino-2,2-dimethylpropanamide (9.24 g, 0.080 mol). The resulting mixture was heated to 88 °C and the liquid mixture was stirred overnight at this temperature. The reaction was monitored by HPLC. When the reaction was complete, the mixture was diluted with ethyl acetate (100 mL) and cooled to 20 °C. The precipitate formed in the mixture was filtered off, and the filtrate was washed with water (20 mL), and then with brine (20 mLx3), and then dried over sodium sulfate. After filtration and evaporation, (2S,4S,5S,7S)-7-(3-amino-2,2-dimethyl-3- oxopropylcarbamoyl)-4-azido-5-hydroxy-2-isopropyl-l-(4-metho xy-3-(3-methoxy- propoxy)phenyl)-8-methylnonyl isobutyrate was obtained as an off-white solid (10.75 g, 0.016 mol).

[0087] The solid was dissolved in dichloromethane. The solution was filtered through silica gel, the filter cake was washed with a mixture of

dichloromethane/methanol (9: 1) and the filtrate was then evaporated to give a white solid. Figure 2 provides an X-ray diffractogram of (2S,4S,5S,7S)-7-(3-amino-2,2- dimethyl-3 -oxopropylcarbamoyl)-4-azido-5 -hydroxy-2-isopropyl- 1 -(4-methoxy-3 -(3 - methoxypropoxy)phenyl)-8-methylnonyl isobutyrate. The solid was found to be amorphous.

Example 3 : Preparation of Aliskiren from Compound IX.

[0088] A mixture of Compound IX (1 g) and 10% Pd/C (0.3 g) in methanol (30 mL) was hydrogenated at 50 °C and 5 bar for 24 h. The reaction mixture was then filtered through diatomaceous earth (Celite) and concentrated to give a solid residue. The residue was dissolved in ethyl acetate. The solution was washed with an aqueous solution of NaOH (5%), and then concentrated to give aliskiren base (0.8 g).

Example 4: Preparation of Compound VIII from (2S,4S,5SJS -5-azido-4-hydroxy-7-

(hydroxy(4-methoxy-3-(3-methoxypropoxy)phenyl)methyl)-2-i sopropyl-8-methyl- nonanoic acid arginine salt (Compound VII arginine salt),

[0089] The arginine salt (15 g) was dissolved in water (70 mL). MTBE (100 mL) was added to provide a solution, and this mixture was acidified with citric acid to pH 3. The acidified mixture was stirred for 1 h. Then the organic layer was separated, and the aqueous layer was washed with MTBE (50 mL). The organic fractions were combined and concentrated to give an oily residue of (3S,5S)-5-((lS,3S)-l-azido-3-(hydroxy(4- methoxy-3-(3-methoxypropoxy)phenyl)methyl)-4-methylpentyl)-3 -isopropyl- dihydrofuran-2(3H)-one (11.7 g) - compound VII.

[0090] Compound VII (5.74 g) was dissolved in toluene (60 mL). DMAP (0.14 g), triethylamine (1.89 mL) and isobutyric anhydride (2.27 mL) were added to the solution and the resulting mixture was stirred at 20 °C for 1 h. The mixture was then quenched with 10 % aq. citric acid (30 mL). The organic layer was separated, washed with saturated aqueous aHC03 (30 mL) and then with water (30 mL). The toluene was evaporated to give 5.95 g of (2S,4S)-4-azido-2-isopropyl-4-((2S,4S)-4-isopropyl-5- oxotetrahydrofuran-2-yl)- 1 -(4-methoxy-3 -(3 -methoxypropoxy)phenyl)butyl isobutyrate (Compound VIII).

Example 5 : Preparation of Aliskiren from Compound IX.

[0091] A mixture of Compound IX (0.5 g) and 10 % Pd/C (0.1 g) in methanol (40 mL) was hydrogenated at 50 °C and 5 bar for 23 h. A second portion of Pd/C (0.1 g) was added and the mixture was hydrogenated for additional 24 h. The mixture was filtered through Celite and evaporated to give a solid residue. The residue was dissolved in ethyl acetate, and the solution was washed with aq. solution of NaOH (5%) and evaporated to give aliskiren base.

[0092] NMR (CDC1 ₃ , δ): 0.86 (d, J=6.9 Hz, 6H); 0.92 (d, J=6.6 Hz, 3H); 0.93 (d, J=6.6, 3H), 1.22 (s , 6H); 1.12-1.20 (m, 1H) 1.25-1.42 (m, 2H); 1.62-1.72 (m, 3H); 1.77-1.87 (m, 1H); 2.05-2.20 (m, 4H); 2.31-2.35 (m, 1H); 2.45-2.49 (m, 2H); 3.03-3.07 (m, 1H); 3.35 (s, 3H); 3.35-3.50 (m, 2H); 3.58 (t, J=6.3 Hz, 2H); 3.83 (s, 3H); 4.09 (t, J=6.6 Hz); 5.89 (s, 1H); 6.43 (s, 1H); 6.62-6.70 (m, 2H); 6.77-6.82 (m, 1H).

Example 6: Preparation of Aliskiren Isobutyric acid salt.

[0093] A mixture Compound IX (0.5 g) and 10 % Pd/C (0.1 g) in methanol (40 mL) was hydrogenated at 50 °C and 5 bar for 23 h. The second portion of Pd/C (0.1 g) was added and the hydrogenation was continued for 24 h. The mixture was filtered through Celite and evaporated to give a solid residue.

[0094] NMR (CDCI ₃ , δ): 0.8-0.95 (m, 12 H); 1.10 (d, J=7.2 Hz, 3H); 1.09 (d, J=7.2 Hz, 3H); 1.20 (s, 3H); 1.21 (s, 3H); 1.20-1.35 (m, 2H); 1.35-1.45 (m, 2H); 1.65-1.85 (m, 3H); 1.88-1.95 (m, 1H); 2.04-2.12 (m, 2H); 2.32-2.42 (m, 1H); 2.44-2.65 (m, 3H); 3.00-3.10 (m, 1H); 3.36 (s, 3H); 3.35-3.45 (m, 2H); 3.58 (t, J=6.3 Hz, 2H); 3.83 (s, 3H); 4.09 (t, J=6.6 Hz, 2H); 5.89 (s, 1H); 6.43 (s, 1H); 6.62-6.71 (m, 2H); 6.76-6.82 (m, 1H), 7.68-7.80 (broad).

Example 7: Preparation of Aliskiren Isobutyric acid salt.

[0095] Isobutyric acid (0.42 mL) was added to a solution of Aliskiren base (2.5 g) in methanol (15 mL). The mixture was stirred overnight, and the methanol was evaporated to give white solid. Example 8: Crystallization of Compound IX.

[0096] Compound IX was dissolved in acetonitrile (2 mL), the solution was cooled to -30 °C, diluted with diisopropyl ether (DIPE) (20 mL) and pentane (10 mL). The turbid mixture was heated to 20 °C and left to crystallize without stirring. Separated crystals were filtered and washed with pentane. XRD pattern is shown in Figure 4.

Example 9: Preparation of Imp-III ((2S,4S,5S,7S)-4-amino-7-(3-amino-2,2-dimethyl-3- oxopropylcarbamoyl)-5-hydroxy-2-isopropyl-l-(4-methoxy-3-(3- methoxypropoxy)phenyl)-8-methylnonyl isobutyrate).

[0097] To a 3-neck 500 ml round bottom flask, Compound IX (18 g, assay 87.8%) and a mixture of EtOFF/FLO (171 ml EtOH and 9 ml FLO) were added. The mixture was stirred for dissolution. Ammonium formate (4.27 g) was added, followed by addition of Pd/C 10%, Type JM 487 (1.8 g). The reaction mixture was stirred for 2 days, and then the catalyst was filtered off. The filtrate was evaporated to give a yellow oil. The oil was dissolved in dichloromethane (45 mL), and this solution was washed with water (20 mLx4) and brine (20 ml). The organic layer was separated, dried over MgS0 ₄ and evaporated to give 10.4 g of a foamy solid. The solid was purified by chromatography (S1O ₂ , DCM:EtOH (3: 1)), to give the Imp-III (3.91 g) as a mixture of two diastereomers with 98.5% purity.

Example 10: Preparation of Imp-V ((3S,5S,6S)-5-hydroxy-3-isopropyl-6-((S)-2-(4- methoxy-3-(3-methoxypropoxy)benzyl)-3-methylbutyl)piperidin- 2-one).

[0098] To a 500 ml round bottom, one necked flask, containing (3S,5S)-5-((l S,3S)- 3-(3-(3-methoxypropoxy)-4-methoxybenzyl)-l-amino-4-methylpen tyl)-dihydro-3- isopropylfuran-2(3H)-one (7.32 g), 3-amino-2,2-dimethylpropionamide (9.76 g) was added. The reaction mixture was heated to 90°C and left to stir magnetically overnight. The reaction mixture was then cooled to 25 °C and dissolved in MTBE (150 mL), and the resulting solution was stirred with 4N HC1 (60 mL) for 2 hours. The organic layer was then separated, washed with H ₂ 0 (60 ml) and brine, dried over MgSC^ and evaporated to give Imp-V as a foamy semi-solid, (4 g) with a purity of 98.2 %, as a mixture of two diastereomers. Example 1 1 : Preparation of Aliskiren Base.

[0099] tert-Butyl-(3S,5S,6S,8S)-8-(3-amino-2,2-dimethyl-3-oxopropyl carbamoyl)- 6-hydroxy-3-(4-methoxy-3-(3-methoxypropoxy)benzyl)-2,9-dimet hyldecan-5- ylcarbamate (4.0 g, 6.1 mmol) was dissolved at 25 °C in dichloromethane (DCM) (40 ml, 10V). The solution was cooled to 0 - 5 °C and HC1 gas (2.5 g, 0.06 mol) was bubbled through the solution. The reaction mixture was maintained at 0 - 5 °C for an additional 2-3h and monitored by HPLC. When the reaction was complete, as determined via HPLC by disappearance of starting material, a IN NaOH solution was added dropwise to adjust the pH of the mixture to pH 9. The organic layer was then separated and concentrated to provide Aliskiren base as a white foam in a quantitative yield.

Example 12: Crystallization of Aliskiren hemifumarate.

[00100] Fumaric acid (2.14 g) was added to a solution of Aliskiren base (22.64 g) in methanol (226 mL) at 35 °C and the mixture was stirred for about 1 h to a complete dissolution and evaporated. Then, the Aliskiren hemifumarate crude foam (4.97 g) was dissolved in methanol, and the obtained solution was evaporated to the weight 14.91 g. This residual solution was heated to 35 °C, and acetonitrile (112 mL) was added to the solution at 35 °C. The solution was seeded at 35 °C with Aliskiren Hemifumarate (Form VIII, 5 mg), the mixture was cooled to 25 °C (during 2 h), while the

precipitation started at 29 °C. The mixture was stirred at room temperature overnight. The white precipitate was separated by filtration; the cake was washed with a mixture of methanol and acetonitrile (2:22.5, 10 mL) to give Form VIII. The product was dried at 40 °C in a vacuum oven overnight to give Aliskiren hemifumarate, Form VIII, as white solid (3.69 g, 74.25 %).

Example 13: Crystallization of Aliskiren hemifumarate.

[00101] Aliskiren Hemifumarate foam (1.86 g) was dissolved in methanol (50 mL) at 35 °C. The solution was evaporated to the weight 7.44 g and acetonitrile (52.75 mL) was added dropwise to the solution. The mixture was seeded with Form VIII (2 mg) at 35 °C. The mixture was stirred at room temperature overnight. The precipitate was separated by filtration and washed with a mixture of methanol and acetonitrile (4:28, 4 mL) to give Aliskiren Hemifumarate. The product was dried overnight at 40 °C in a vacuum oven to give Aliskiren hemifumarate 1.05 g (56.5 %).

Example 14: Crystallization of Aliskiren hemifumarate.

[00102] Fumaric acid (3.7 g) was added at 25 °C to a solution of Aliskiren base (34.9 g) dissolved in methanol (70 mL) and the reaction mixture was stirred for 3 h.

Acetonitrile (720 mL) was added and the resulting mixture was heated to 60 °C.

The mixture was filtered via filter paper (0.6 micron); the filter was washed with a mixture of acetonitrile (245 mL) and methanol (15 mL). The obtained clear solution was cooled to 35 °C (for 2 h) and seeded with Form VIII (0.77 g). The mixture was stirred at 35 °C for 2 h and cooled stepwise to 10 °C.

[00103] At each cooling step, the reactor jacket was cooled by 5 °C in about 1.5 h and maintained at the obtained temperature for about 1 h. When reaching 10 °C, the slurry was stirred for 20 h at 10 °C. During the whole crystallization process, the slurry was stirred at 150 rpm. The precipitated solids were separated by filtration and the cake was washed with cold acetonitrile (60 mL x 2). The material (34.5 g) was dried at 60 °C/(less then 50 mbar) for 24 h to give Aliskiren hemifumarate (27.3 g), Form VIII.

Example 15: Crystallization of Aliskiren hemifumarate.

[00104] Fumaric acid (1.8 g) was added at 25 °C to a solution of Aliskiren base (16.9 g) dissolved in methanol (42 mL) and the reaction mixture was stirred for 3 h.

Acetonitrile (400 mL) was added, the resulting mixture was filtered via filter paper (0.6 micron) at 25 °C, and the filter was washed with a mixture of acetonitrile (70 mL) and methanol (7 mL); the resulting solution was heated to 60 °C, then cooled

to 35 °C (for 2 h) and seeded with Form VIII (0.4 g). The mixture was stirred at 35 °C for 2 h and cooled stepwise to 10 °C.

[00105] At each cooling step, the reactor jacket was cooled by 5°C in about 1.5 h and maintained at the obtained temperature for about 1 h. When reaching 10°C, the slurry was stirred for 20 h at 10 °C. During the whole crystallization process, the slurry was stirred at 150 rpm. The precipitated solids were separated by filtration; the cake was washed with cold acetonitrile (30 mL x 2). The material (14.0 g) was dried at 60 °C/(less then 50 mbar) for 24 h to give Aliskiren hemifumarate (13.9 g), Form VIII.

Example 16: Preparation of Imp-VII.

[00106] A mixture of Imp-III (10 g) and 66 % sulfuric acid (30 mL) in toluene (50 mL) was stirred at 20 °C (HPLC monitoring). The organic layer was separated, and discarded. The aqueous layer was mixed with Me-THF (100 mL) and the mixture was basified with a ₂ C03 (solid) to pH 8.5. The organic layer was separated, washed to 15 % brine (2x 100 mL), and evaporated to obtain white foam.