**3. Perindopril: polymorphs and hydrates3**

Perindopril, 2-methylpropane-2-amine-(2S,3aS,7aS)-1-[(2S)-2-[[(1S)-1-ethoxy-carbonyl-butyl] amino]propanoyl]octahydro-1H-indole-2-carboxylic acid, is an antihypertensive drug that acts through the inhibition of angiotensin converting enzyme (ACE), a zinc metalloenzyme involved in the control of blood pressure. It is effective in the treatment and prevention of several medical conditions, such as reducing blood pressure, reversing abnormalities of vascular structure and function in patients with essential hypertension, congestive heart failure, post-myocardial infarction and diabetic nephropathy87-91. Perindopril along with ramipril were associated with lower mortality than most other ACE inhibitors92. Besides the antihypertensive properties, it also comprises vasculoprotective and antithrombotic effects, playing a favourable role in terms of cardiovascular morbidity93-99.

This API is, in fact, an acid-ester prodrug that is converted into the active diacid perindoprilat by hydrolysis promoted by the liver esterases after administration93, 100. It is orally administered in the form of tablets containing its 1:1 salts with erbumine (*tert*butylamine) (Aceon®) or L-arginine (Coversyl®)43, 101. The perindopril L-arginine salt is equivalent to perindopril erbumine (Figure 18) but it is more stable and therefore it can be distributed to all the climatic zones without the need for specific packaging101.

Over the last years, several forms of perindopril erbumine have been disclosed and several patents have been filed mainly based on their typical powder XRPD patterns44, 45, 102-105. Perindopril erbumine is known to exist in several polymorphic forms46, 48, 102, 103, 105-107, as well as mono-, di- and sesqui-hydrated forms, characterized by XRPD, vibrational spectroscopy and thermal analysis methods47, 108. Also amorphous compositions have been patented42 as well as a perindopril tosylate form109.

Some of the different pharmacological and adverse effects exerted by ACE inhibitors may depend on the different phisicochemical (solubility, lipophilicity, acidity) and pharmacokinetic (absorption, protein binding, half-life and metabolic disposition) properties but also on their ability to penetrate and bind tissue sites110. Theoretical studies on p*K*a, lipophilicity, solubility, absorption and polar surface of ACE inhibitors, including perindopril, and its active metabolite, perindoprilat, have been reported111. In 2009, Remko presented theoretical calculations of molecular structure and stability of the arginine and erbumine salts of perindopril43.

<sup>3</sup> Adapted with permission from First Crystal Structures of the Antihypertensive Drug Perindopril Erbumine: A Novel Hydrated Form and Polymorphs α and β, Vânia André, Luis Cunha-Silva, M. Teresa Duarte, and Pedro Paulo Santos, *Crystal Growth & Design*, 2011, *11* (9), pp 3703–3706, DOI: 10.1021/cg200430z. Copyright (2011) American Chemical Society".

Perindopril, 2-methylpropane-2-amine-(2S,3aS,7aS)-1-[(2S)-2-[[(1S)-1-ethoxy-carbonyl-butyl] amino]propanoyl]octahydro-1H-indole-2-carboxylic acid, is an antihypertensive drug that acts through the inhibition of angiotensin converting enzyme (ACE), a zinc metalloenzyme involved in the control of blood pressure. It is effective in the treatment and prevention of several medical conditions, such as reducing blood pressure, reversing abnormalities of vascular structure and function in patients with essential hypertension, congestive heart failure, post-myocardial infarction and diabetic nephropathy87-91. Perindopril along with ramipril were associated with lower mortality than most other ACE inhibitors92. Besides the antihypertensive properties, it also comprises vasculoprotective and antithrombotic effects,

This API is, in fact, an acid-ester prodrug that is converted into the active diacid perindoprilat by hydrolysis promoted by the liver esterases after administration93, 100. It is orally administered in the form of tablets containing its 1:1 salts with erbumine (*tert*butylamine) (Aceon®) or L-arginine (Coversyl®)43, 101. The perindopril L-arginine salt is equivalent to perindopril erbumine (Figure 18) but it is more stable and therefore it can be

Over the last years, several forms of perindopril erbumine have been disclosed and several patents have been filed mainly based on their typical powder XRPD patterns44, 45, 102-105. Perindopril erbumine is known to exist in several polymorphic forms46, 48, 102, 103, 105-107, as well as mono-, di- and sesqui-hydrated forms, characterized by XRPD, vibrational spectroscopy and thermal analysis methods47, 108. Also amorphous compositions have been

Some of the different pharmacological and adverse effects exerted by ACE inhibitors may depend on the different phisicochemical (solubility, lipophilicity, acidity) and pharmacokinetic (absorption, protein binding, half-life and metabolic disposition) properties but also on their ability to penetrate and bind tissue sites110. Theoretical studies on p*K*a, lipophilicity, solubility, absorption and polar surface of ACE inhibitors, including perindopril, and its active metabolite, perindoprilat, have been reported111. In 2009, Remko presented theoretical calculations of molecular structure and stability of the arginine and

3 Adapted with permission from First Crystal Structures of the Antihypertensive Drug Perindopril Erbumine: A Novel Hydrated Form and Polymorphs α and β, Vânia André, Luis Cunha-Silva, M. Teresa Duarte, and Pedro Paulo Santos, *Crystal Growth & Design*, 2011, *11* (9), pp 3703–3706, DOI:

Fig. 17. pH dependent stability of **5**. [Ester derivative reported in83]

playing a favourable role in terms of cardiovascular morbidity93-99.

distributed to all the climatic zones without the need for specific packaging101.

**3. Perindopril: polymorphs and hydrates3**

patented42 as well as a perindopril tosylate form109.

10.1021/cg200430z. Copyright (2011) American Chemical Society".

erbumine salts of perindopril43.

Fig. 18. Chemical diagram of perindopril erbumine salt.

Careful searches in the literature and in the Cambridge Structural Database112 revealed that, although this API is known since 1981, until very recently only the crystal structure of perindoprilat, the pharmacologically active compound, had been determined in 199193. In 2011, Remko and co-workers41 unveiled the crystal structure of perindopril erbumine dehydrate.

Also in 2011, during a polymorphic screening of perindopril erbumine, the molecular structures of its α and β polymorphs45, 113 have been determined by SCXRD as well as an unprecedented hydrated form of formula (C4H12N)(C19H31N2O5)1.25H2O40, 114. Elemental and Karl-Fischer analyses confirmed the water contents of the three forms, that were were fully characterized by XRPD, vibrational spectroscopy (ATR-FT-IR and FT-Raman) and thermal analysis methods (TGA, DSC and HSM)40. Furthermore, stability, solubility and dissolution profile studies were performed.

The crystal packing of polymorphic forms α and β show similar hydrogen bonding interactions involving the perindopril and the erbumine ions. Perindopril anions interact with erbumine cations in an extended NH···O hydrogen bonding network leading to a supramolecular structure with the moieties organized in a double-chain arrangement. Each erbumine cation connects with three perindopril anions *via* the amine moiety: two of them are in the same chain whereas the other perindopril belongs to the opposite chain where the positioning of the anions in their respective chains, it is possible to notice that they assume antiparallel orientations i.e., perindopril anions of one chain are rotated of 180º relatively to the anions in the adjacent chain. Consequently two related types of �� �(6) synthons are formed in both chains that are connected among them by �� �(2) motifs.

The NH···O hydrogen bond distances are within the ranges of 2.707 - 2.803 Å and 2.738 - 2.788 Å in α and β forms, respectively. These double-chains do not establish classical hydrogen bonds among them neither in α nor β forms.

The new 1:1:1.25 hydrated form crystallizes with a triclinic symmetry, in the P1 chiral space group. This hydrated form was obtained both by solution and by LAG, which, as previously said, has several advantages not only in the preparation process, where equally yield and

Novel Challenges in Crystal Engineering: Polymorphs

Double-chain array formed by <sup>2</sup>

diagnosing the presence of NH and NH3

bonds, by the presence of resolved peaks.

and New Crystal Forms of Active Pharmaceutical Ingredients 85

Fig. 20. Crystalline packing of the novel hydrated form of perindopril erbumine (1:1:1.25):

a b Fig. 21. (a) DSC and (b) TGA pattern for all the forms of perindopril erbumine discussed. bands (Figure 21). In particular the strong bands in the range of 3200-2600 cm-1 of the FT-Raman spectra are attributed to the υs(C–H) and υs(N–H) stretching vibrational modes

respectively. The strong bands around 1642, 1569 cm-1 and 1387 cm-1 (observed in both the FT-IR and FT-Raman spectra) are assigned to the υs(COO-) and υas(COO-) respectively, confirming the deprotonation of the carboxylic acid group. Contrasting with the FT-IR spectra of forms α and β, the spectrum of the hydrated form in the 3200-2600 cm-1 range reflects the presence of crystallization water molecules involved in well defined hydrogen

The combination of data obtained from DSC, TGA and HSM indicates that the novel hydrated form is stable until approximately 80°C, temperature at which a peak is observed in the DSC (Figure 21.a), a smooth mass loss is detected in the TGA (Figure 21.b) and bubbles start to appear in the HSM. The water loss occurs from this temperature until

<sup>1</sup> *D* (2) motifs is highlighted in blue40.

+ groups in the perindopril and erbumine cation,

<sup>2</sup> *C* (6)and <sup>1</sup>

purity are obtained, but also in an environmental context115-119. Its asymmetric unit consists of two crystallographic independent perindopril anions, two erbumine cations and 2.5 water molecules. The CO distances in the carboxylate moiety and the location of the three hydrogen atoms in the amine moieties from the electron density map confirmed the presence of the salt.

The chiral centers in both perindopril crystallographic independent anions of the hydrated form exhibit the (S) configuration, corresponding exactly to the same configuration of the starting form α as well as of form β, what is important to assure the pharmacological activity of the API (Figure 18). The main conformational differences between these crystallographic independent anions are noted in the –CH2CH2CH3 terminal groups (torsion angles of - 58.2(4)° *vs* 175.1(9)°). The crystal packing of this hydrated form is very similar to the one described for polymorphic forms α and β, involving similar hydrogen bonding interactions between the perindopril and the erbumine ions (Figure 19). The NH···O hydrogen bond distances are within the ranges of 2.75 - 2.781 Å. The main difference between this hydrate and the polymorphs previously described is that while the double-chains do not establish classical hydrogen bonds among them neither in α nor β forms, in the hydrated form water molecules play an important role by linking adjacent chains through interactions between two crystallographically independent perindopril anions *via* the carbonyl group of one [OW···OC=O distance of 2.717Å] and the amine moiety of the other [NN-H···OW distance of 2.430Å]. Water molecules lie in the free spaces arising from the supramolecular arrangement described (Figure 19) and interact through cooperative OW–H···OW hydrogen bonds forming trimeric water clusters [O···O distances in the cluster: 2.644, 2.687 and 2.932 Å] (Figures 19 and 20). Vibrational spectroscopy (FT-IR and FT-Raman) studies support the structural features

unveiled by SCXRD data which are reflected in the spectra through a number of diagnostic

Fig. 19. Crystal packing of the novel hydrated form of perindopril erbumine (1:1:1.25): (a) supramolecular arrangement with the perindopril anions and erbumine cations organized in double-chains; H bonds represented as blue dashed lines; water molecules were omitted for clarity; (b) detailed hydrogen bonding within the water cluster. Only hydrogen atoms involved in hydrogen bonding are shown, with exception of water molecules for which no hydrogen atoms are displayed40.

purity are obtained, but also in an environmental context115-119. Its asymmetric unit consists of two crystallographic independent perindopril anions, two erbumine cations and 2.5 water molecules. The CO distances in the carboxylate moiety and the location of the three hydrogen atoms in the amine moieties from the electron density map confirmed the

The chiral centers in both perindopril crystallographic independent anions of the hydrated form exhibit the (S) configuration, corresponding exactly to the same configuration of the starting form α as well as of form β, what is important to assure the pharmacological activity of the API (Figure 18). The main conformational differences between these crystallographic independent anions are noted in the –CH2CH2CH3 terminal groups (torsion angles of - 58.2(4)° *vs* 175.1(9)°). The crystal packing of this hydrated form is very similar to the one described for polymorphic forms α and β, involving similar hydrogen bonding interactions between the perindopril and the erbumine ions (Figure 19). The NH···O hydrogen bond distances are within the ranges of 2.75 - 2.781 Å. The main difference between this hydrate and the polymorphs previously described is that while the double-chains do not establish classical hydrogen bonds among them neither in α nor β forms, in the hydrated form water molecules play an important role by linking adjacent chains through interactions between two crystallographically independent perindopril anions *via* the carbonyl group of one [OW···OC=O distance of 2.717Å] and the amine moiety of the other [NN-H···OW distance of 2.430Å]. Water molecules lie in the free spaces arising from the supramolecular arrangement described (Figure 19) and interact through cooperative OW–H···OW hydrogen bonds forming trimeric water clusters [O···O distances in the cluster: 2.644, 2.687 and 2.932 Å] (Figures 19 and 20). Vibrational spectroscopy (FT-IR and FT-Raman) studies support the structural features unveiled by SCXRD data which are reflected in the spectra through a number of diagnostic

a b Fig. 19. Crystal packing of the novel hydrated form of perindopril erbumine (1:1:1.25): (a) supramolecular arrangement with the perindopril anions and erbumine cations organized in double-chains; H bonds represented as blue dashed lines; water molecules were omitted for clarity; (b) detailed hydrogen bonding within the water cluster. Only hydrogen atoms involved in hydrogen bonding are shown, with exception of water molecules for which no

presence of the salt.

hydrogen atoms are displayed40.

Fig. 20. Crystalline packing of the novel hydrated form of perindopril erbumine (1:1:1.25): Double-chain array formed by <sup>2</sup> <sup>2</sup> *C* (6)and <sup>1</sup> <sup>1</sup> *D* (2) motifs is highlighted in blue40.

Fig. 21. (a) DSC and (b) TGA pattern for all the forms of perindopril erbumine discussed.

bands (Figure 21). In particular the strong bands in the range of 3200-2600 cm-1 of the FT-Raman spectra are attributed to the υs(C–H) and υs(N–H) stretching vibrational modes diagnosing the presence of NH and NH3 + groups in the perindopril and erbumine cation, respectively. The strong bands around 1642, 1569 cm-1 and 1387 cm-1 (observed in both the FT-IR and FT-Raman spectra) are assigned to the υs(COO-) and υas(COO-) respectively, confirming the deprotonation of the carboxylic acid group. Contrasting with the FT-IR spectra of forms α and β, the spectrum of the hydrated form in the 3200-2600 cm-1 range reflects the presence of crystallization water molecules involved in well defined hydrogen bonds, by the presence of resolved peaks.

The combination of data obtained from DSC, TGA and HSM indicates that the novel hydrated form is stable until approximately 80°C, temperature at which a peak is observed in the DSC (Figure 21.a), a smooth mass loss is detected in the TGA (Figure 21.b) and bubbles start to appear in the HSM. The water loss occurs from this temperature until

Novel Challenges in Crystal Engineering: Polymorphs

*Discovery Today*, 13, (2008):440.

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[4] O. Almarsson and M. J. Zaworotko, Crystal engineering of the composition of

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approximately 120°C. At 164ºC melting and decomposition take place. TGA for forms α and β reveals that there is no mass loss before 120°C, confirming the absence of water in both these forms.

The new 1:1:1.25 hydrate has shown to be as stable on shelf as form α for eighteen months and water slurry experiments revealed that it as a thermodynamically stable form. It has also shown to have a similar dissolution profile (Figure 22) as the commercially available drug and to be slightly more soluble in water than the α form40.

Fig. 22. Dissolution profile for the 1:1:1.25 hydrated form.

A probable reason for this is the enhanced stability provided by the presence of the water molecules linking the erbumine-perindopril double chains. Analysis of crystal structure has again proven to be quite important for the establishment of the intermolecular interactions responsible for the supramolecular arrangement and thus the physicochemical properties of APIs.

### **4. Concluding remarks**

Over the last two decades crystal engineering, a key tool for the design of new crystal forms, has made possible the synthesis of novel pharmaceutical materials as well as molecular level control of crystallization and phase transformations. Advances in crystal engineering and supramolecular chemistry invite us to consider new perspectives and perhaps definitions of the various solid-state forms that the same and/or different molecules may adopt in terms of molecular assemblies and architectures.

Pharmaceutical co-crystals have proven to offer potential benefits of superior efficacy, solubility and stability in drug formulation. It seems reasonable to assert that co-crystal approaches should be considered routinely as part of a broader set of form and formulation explorations to achieve the best possible drug products. Although the interest in co-crystals and polymorphs and their utility is obvious, identifying and implementing an efficient discovery and control method remains a challenge.

#### **5. Acknowledgements**

The authors acknowledge Fundação para a Ciência e a Tecnologia, MCTES, Portugal, for funding the Project POCI/QUI/58791/2004, PEst-OE/QUI/UI0100 /2011, and the Ph.D. Grant SFRH/40474/2007 (V.A.).
