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Accepted Manuscript

Molecular structure of antihypertensive drug perindopril, its active metabolite
perindoprilat and impurity F
M. Remko, J. Bojarska, P. Ježko, W. Maniukiewicz, A. Olczak
PII:
DOI:
Reference:

S0022-2860(12)01150-7
http://dx.doi.org/10.1016/j.molstruc.2012.11.070
MOLSTR 19407

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Journal of Molecular Structure

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Revised Date:
Accepted Date:

23 October 2012
29 November 2012
29 November 2012

Please cite this article as: M. Remko, J. Bojarska, P. Ježko, W. Maniukiewicz, A. Olczak, Molecular structure of
antihypertensive drug perindopril, its active metabolite perindoprilat and impurity F, Journal of Molecular
Structure (2012), doi: http://dx.doi.org/10.1016/j.molstruc.2012.11.070

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Molecular structure of antihypertensive drug perindopril, its active metabolite
perindoprilat and impurity F
M. Remkoa*, J. Bojarskab, P. Ježkoa, W. Maniukiewiczb, A. Olczakb
a

Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Comenius University Bratislava, Odbojárov

10, SK-832 32 Bratislava, Slovakia
b

Institute of General and Ecological Chemistry, Faculty of Chemistry, Lodz University of Technology,

Żeromskiego 116, 90-924 Łódź, Poland

ABSTRACT
The molecular structure of the antihypertensive drug perindopril (2S,3aS,7aS)-1-[(2S)-2[[(2S)-1-ethoxy-1-oxopentan-2-yl]amino]propanoyl]-2,3,3a,4,5,6,7,7a-octahydroindole-2
carboxylic acid), its active metabolite perindoprilat ((2S,3aS,7aS)-1-[(2S)-2-[[(2S)-1carboxybutyl]amino]propanoyl]-2,3,3a,4,5,6,7,7a-octahydroindole-2-carboxylic acid), and
impurity F (ethyl (2S)-2-((3S,5aS,9aS,10aS)-3-methyl-1,4-dioxodecahydropyrazino[1,2a]indol-2(1H)-yl) pentanoate) has been investigated using B3LYP/6-31g(d) and B3LYP/6311+g(d,p) model chemistry. It has been found that solid state conformations of perindoprilat
occur close to, but not actually at minima on the computed gas-phase potential energy
surfaces. Both, neutral and zwitterionic structures of perindopril and perindoprilat have been
investigated. Relative stability of individual ionized species of this drug has been determined.
Water has a remarkable effect on the geometry of the perindopril species studied.
Keywords: Molecular structure, perindopril, perindoprilat, impurity F, ab initio calculations,
solvent effect
*

Corresponding author:

E-mail address: remko@fpharm.uniba.sk

1

5.3a. lowering angiotensin II and potentiating bradykinin [1. Although perindopril was first synthesized (stereoselectively as a single all S enantiomer) in 1982 [17]. 2 .7. Introduction Perindopril (2S.0. ACE inhibitors exhibit also vasculoprotective and antithrombotic activities that play a favorable role in terms of cardiovascular morbidity [711]. 2]. 16]. when we presented the first crystal structures of two tert-butylamine salts (see Cambridge Structural Database Version 5.3aS. ACE inhibitors are effective in reducing blood pressure and improving outcomes in a number of cardiovascular disease states (such as reversing abnormalities of vascular structure and function in patients with essential hypertension. perindopril is observed to be chemically unstable and undergoes degradation in dosage forms to other diacids and diketopiperazines [13].4. Pascard et al. Besides antihypertensive effect. Clinically useful forms of perindopril are its tert-butylamine (perindopril erbumine) or L-arginine (perindopril L-arginine) salts [1].4-dioxodecahydropyrazino [1.10aS)-3-methyl-1. its three dimensional structure was unknown until 2011. known as impurity F in European Pharmacopeia 6.2-a]indol-2(1H)-yl)pentanoate).1.5aS. In 1991. [19] determined configuration and preferential solid-state conformation of perindoprilat ethanol disolvate.3a. diabetic nephropathy) [36]. Perindopril.6.7.3.7aS)-1-[(2S)-2-[[(2S)-1-ethoxy-1-oxopentan-2-yl]amino]propanoyl] 2. is regarded as main degradation product of perindopril. post-myocardial infarction. an acid-ester prodrug. It is worth to note that the geometric parameters of all studied solid structures are in good agreement. Nevertheless. is converted in vivo to perindoprilat ((2S.31. One of them the diketopiperazine ethyl (2S)-2((3S.4. glucuronidation and cyclisation have been identified and analyzed [14].5. Six degradation products of perindopril resulting from hydrolysis.7a-octahydroindole-2-carboxylic acid) is a long-acting angiotensin-converting enzyme (ACE) inhibitor with high tissue ACE affinity.7aS)-1-[(2S)-2-[[(2S)-1-hydroxy-1-oxopentan-2-yl]amino]propanoyl]2.3aS.3. congestive heart failure. These species are known degradation products of perindopril and/or are present as impurities from the route of synthesis [15.6. refcodes IVEGIA and IVEGOG [18] as part of our studies on perindopril and perindoprilat polymorphism. and more recently we have reported structures of two perindoprilat pseudopolymorphs (monohydrate and dimethyl sulfoxide hemisolvate crystallizing as orthorhombic) and also two polymorphs structures (tetragonal and orthorhombic) of its lactam-type degradation product [20-23].7a-octahydroindole-2-carboxylic acid) its active metabolite [12].9aS.

Crystallographic data for all compounds has been deposited with the Cambridge Crystallographic Data Centre as supplementary publication. we carried out optimization calculations in the presence of water. The results of theoretical calculations of the isolated species and solvated structures were compared and discussed with our experimental X-ray crystal data for titled compounds.ccdc. PCM [30-32]. 12 Union Road. Computational procedure Ab initio calculations of the perindopril and its derivatives (Fig.cam. 29]. The perindoprilat was considered in two sets of neutral and ionic complexes. The structures of all gas-phase species were fully optimized at the B3LYP level of theory using 6-31G(d) and 6-311+G(d. Cambridge CB2 1EZ.ac. UK (fax: +44 1223 336 033.ac. X-ray data All full X-ray data and precise description of the crystal structures used in the comparison and in the discussion namely perindoprilat solvates (monohydrate and dimethyl sulfoxide hemisolvate crystallizing as orthorhombic) and two polymorphs (tetragonal and orthorhombic) of its lactam-type degradation product have been published elsewhere [20-23].uk). its biologically active metabolite perindoprilat and impurity F (a degradation product of perindopril). The structures of all condensed-phase (SCRF) species were fully optimized without any geometrical constraint at the B3LYP/6-31G(d) level of theory. In order to evaluate the conformational behavior of these systems in solvent.1. Copies of the data can be obtained free of charge on application to CCDC. e-mail: deposit@ccdc.p) basis sets without any geometrical constraint. Results and discussion 3. molecular structures of perindopril and its degradation product in the isolated state and in aqueous solution. The methodology used in this work is centered on polarized continuum model.uk.Here we report the gas-phase molecular structure of perindopril. www: http://www. 2. Becke3LYP [25-27]) model chemistry [28. Theoretical quantum chemical methods were used for determination of equilibrium geometries of neutral and zwitterionic forms of perindoprilat. Crystallization of the free acid perindopril did not give single crystals suitable for X-ray 3 . 3. 1) were carried out with the Gaussian 09 computer code [24] using density functional theory (DFT.cam.

Φ[C(10)–N(2)–C(11)– C(12). The calculated rotamer around the proline moiety is trans (Φ[C(1)–N(1)–C(9)–O(1) close to 0o).2. Thus. Full geometry optimization carried out using two basis sets of DFT theory. its crystal form is not yet known. Φ[C(10)–N(2)–C(11)–C(17)]. Φ[N(1)–C(9)–C(10)C(16)]. The methyl group of the alanine is in a direction nearly perpendicular to the amide plane (Φ[N(1)–C(9)–C(10)-C(16)] is about 100 – 110o). 34]. Φ[N(2)–C(11)–C(17)-O(3)]. Water has a remarkable effect on the geometry and energy of the studied species. S2 and S3 of the Supporting information. Φ[C(9)–C(10)–N(2)–C(11). Perindopril is delivered to patients in crystalline form. An analysis of the harmonic frequencies at the B3LYP/6311+g(d. respectively.diffraction studies. It is well known that the crystal form of a solid active pharmaceutical ingredient (API) has the potential to impact physicochemical and pharmacokinetic properties of a final dosage form of a drug [35-37]. the graphical interface of Gaussian.2. Perindopril and perindoprilat possess the same proline–alanine functionality. Important geometric parameters of the species studied are given in Tables S1. shows that the increase of the basis set results in only small changes in the equilibrium geometry of the compounds studied. Tables S1 and S2. Φ[C(1)–N(1)– C(9)–C(10). As regards of free acid perindopril. 37]. Continuum-based methods of solvation were used successfully in a variety of problems [33.p) level of theory of the optimized molecules revealed that all the species obtained were in minima. The relative orientation of the alanine moiety and alkyl chains was adopted from previously analyzed experimental structural data of the perindoprilat and degradation products obtained from X-ray crystallography [20-23]. Φ[N(1)–C(9)–C(10)–N(2)]. Water stabilization energies of neutral species are in all cases negative and span a relatively narrow interval of 40 – 50 kJ/mol. The effect of bulk solvent is treated with the PCM solvation method. 3. Screening for polymorphism of 4 . Theoretical calculations 3. 1) used in the DFT calculations were constructed by means of Gauss View. it seems highly probable that its conformation should resemble the conformation determined for perindoprilat. In these molecules the relative molecular orientation is described by eight dihedral angles Φ[C(1)–N(1)–C(9)–O(1). Selected geometric parameters for perindopril and perindoprilat species are given in Tables S1 and S2. crystal and molecular structure of different polymorphic (and pseudopolymorphic) salts of perindopril with erbumine (tert-butylamine) has been determined [18. its solid state structure is still unknown. Molecular structures The initial conformations of the perindopril and its derivatives (Fig.1. However. Alkyl chains were considered in fully extended conformation. However.

Geometry optimization of the isolated zwitterion I is accompanied by intramolecular proton transfer between the -NH2+ group and neighboring negatively charged oxygen atom of the nalkyl chain carboxyl group. this form is. Water molecule directly stabilizes zwitterionic structure via system of N+-H∙∙∙O and OH∙∙∙O=C intermolecular hydrogen bonds [21]. It is thus instructive to study the relative stability of neutral and zwitterionic perindopril species also in the gas phase and/or solvated state. 21]. still by 74. Inserting of perindopril species into water solution reduces their relative stability. More interesting situation is in the case of perindoprilat. The surrounding medium did not shifted the relative stability of perindoprilat species studied. Polar solvent (water) has a remarkable effect on the relative stability of the neutral and zwitterionic species of perindopril. In the solid state zwitterionic form of perindoprilat (zwitterion I) contains a protonated alanine N atom and an anionic carboxylate group at the n-alkyl chain [20]. As regards of perindopril our DFT calculations predict the existence of two stable structures.2 kJ/mol less stable. The zwitterionic form of perindopril can be stabilized in condensed phase (water solution). One of the perindoprilat solvatomorphs investigated crystallizes as a monohydrate [21]. proline and n-alkyl chain carboxyl groups of perindoprilat have been shown that these groups at physiological pH = 7. Theoretical calculations at both levels of the DFT theory of this complex reveal that the zwitterion is also a stable form in the gas-state. This zwitterion represents on the potential energy surface a stable structure with local minimum by 161. 21]. Zwitterions in unit cell of both orthorhombic pseudopolymorphs are held together by rich system of intermolecular hydrogen bonds [20. by comparison with the neutral one. The 5 . Thus. Theoretical calculations of the pKa of both.3 kJ/mol) and the neutral perindopril is preferred over the zwitterionic one. Neutral form of perindopril is more stable by 158 kJ/mol than the corresponding zwitterionic structure.4 are completely ionized [38]. In crystals the zwitterionic form of perindoprilat polymorphs was observed [17.p) method) than the conformation of the neutral perindoprilat. the zwitterionic form converged without an energy barrier to the neutral molecule of perindoprilat. From this point of view we also considered in our calculations zwitterionic structure of perindoprilat containing anionic proline carboxyl group (zwitterion II). Neutral perindoprilat is also stable form in water solution. However. 20. Solvent (water) results in appreciate stabilization of the zwitterion II.perindoprilat and cyclisation product of perindopril – diketopiperazine have been shown that these compounds can exist in several crystalline forms [17.3 kJ/mol higher in energy (B3LYP/6-311+G(d. 20-23] and their 3D structure differs primarily in the n-alkyl chain orientation [21]. Computed relative energy is still quite high (48.

22] and theoretically. An proton transfer from the proline carboxyl to the basic nitrogen atom of alanine is manifested in considerable structural changes of whole molecule (Fig. Perindoprilat Perindoprilat.5 kJ/mol) and positive. Two experimentally described polymorphs of diketopiperazine derivative of perindopril (tetragonal and orthorhombic) served as input data for our extended theoretical calculations.5 kJ/mol).2. the active form of perindopril.7 J/mol K).4 both carboxyl groups are completely ionized [38]. 2). The perhydroindole moiety is in zwitterion slightly rotated out of the plane of the peptide bond (dihedral angle [C(1)–N(1)–C(9)–O(1) is -25o). Diketopiperazine derivatives are important degradation products of several dicarboxylic ACE inhibitors. The molecular structure of the zwitterionic perindopril is quite different. At physiological pH = 7. 3. The molecular geometry of perindopril computed in water solution shows appreciable changes in the region of flexible n-propyl chain and ester moiety. This means that water has little tendency to associate in the gas phase. From this point of view we also examined the molecular structure of perindopril cyclisation product (impurity F. Perindopril Perindopril is an effective ACE inhibitor.1.p) calculated hydrogen bond enthalpy of this complex is negative (-13. Its 3D structure is governed by 9 rotatable bonds [38]. destabilizing.2. As it is shown 6 .1.2. The important geometric parameters are given in Table S1.e.1.B3LYP/6-311+G(d. Fig. The aliphatic part of zwitterion is stabilized by means of a weak intramolecular hydrogen bond of the C=O···H2N+ type. Larger differences (about 1 . The Gibbs energy difference observed between these two polymorphs in vacuo is low (about 1. is chemically dicarboxylic ACE inhibitor.1 kJ/mol) in water solution (B3Lyp/6-31G(d) method) indicating that these polymorphs differ especially by rotating about single bonds. Both B3LYP DFT bond lengths and bond angles of perindopril fit one another to within about 0.5o). i.002 Å for bond lengths and about 1o for bond angles. 3. opposing association. respectively. Hydrogen bond energy was computed as the difference between the total energies of the thermodynamically most stable isolated species.4 kJ/mol) and slightly reduced (1. However. Gibbs energy is large (33. The computed rotamer around the ester carboxyl group in isolated perindopril is syn-clinal (dihedral angle [N(2)–C(11)–C(17)–O(3)] is about 84o) and in water anti-clinal (dihedral angle [N(2)–C(11)–C(17)–O(3)] is 132. 1) both experimentally [21. The entropy term is negative (-157.5o) were observed for dihedral angles.

Unbound perindoprilat may exist in solid state in several zwitterionic polymorphs with protonated alanine nitrogen and anionic carboxyl at the carboxyalkyl carboxylate group [17. 3). In water solution its gas-phase molecular structure changes only slightly. The superposition of the 3-D structures of the perindoprilat monohydrate manifesting the overall difference in experimental solid-state and gas-phase geometries of this species is shown on Fig. As regards of torsion angles. The optimized torsion angles computed at both levels of theory Θ[C(10)N(2)-C(11)-(C17)] and Θ[N(2)-C(11)-C(17)-O(3)] are substantially different (Table S2 of the Supplementary material). Important geometrical parameters of the DFT optimized structure of complex perindoprilat – water are given in Table S3 of the Supplementary material. C-terminal carboxylate anion is stabilized by means of 3 hydrogen bonds with Gln265. The main difference between theoretical and experimental structure is observed in the region of the carboxyalkyl carboxylate group. Tyr504 and Lys495. Selected structural parameters computed using two basis sets for this structure are presented in Table S2 of the Supplementary material.perindoprilat complex displays important intermolecular interactions of perindoprilat with complementary sites of ACE active site (Fig. The carboxyalkyl carboxylate group is more acidic than the C-terminal carboxylate [38] and with protonated basic nitrogen atom of alanine represents a zwitterionic structure stabilized via the system of intermolecular hydrogen bonds of corresponding rests of amino acids (Fig. 21]. Hence. The extension of the basis set in the DFT calculation resulted in only small changes in the bond distances and bond lengths of the perindoprilat. 3). 20.from the analysis of the X-ray data for perindoprilat in the bound state at the angiotensin converting enzyme (ACE) homologue from Drosophila melanogaster (AnCE) (PDB file 2X94) both carboxylic acids are ionized [39]. 4. The possible existence of monoanions of perindoprilat in the isolated state and water solution was 7 . 3). Perindoprilat bound to its protein target exists in the form of monoanion (Fig. 21]. As a starting geometry for DFT calculations we used X-ray structure of two recently discovered new pseudopolymorphs of this drug [20. Theoretically calculated equilibrium gas-phase geometry of isolated perindoprilat represents neutral molecule without intramolecular proton transfer. biologically active conformation of perindopril is a monoanion. PoseView [40] diagram of AnCE . Solvent effect did not changed appreciably geometry of isolated complex (Table S3). the largest changes were observed for the rotation of the carboxyalkyl carboxylate group. In solid state zwitterion may be stabilized by solvate molecule (water). torsion angles [C(10)-N(2)-C(11)-(C17)] and [C(11)-N(2)-C(10)-C(9)] (Table S3 of the Supplementary material).

2. 6). and is closer to the biologically active conformation of bound perindoprilat monoanion (Table S4). The perhydroindole ring of the proline moiety is in both conformers an envelope. 3. However. 4. The calculations showed that the solid-state conformation of this molecule is also preserved in the gas-phase. this 6-membered ring in bound perindoprilat exists as the stable boat form (Fig. Experimentally described two polymorphs of this cyclisation product of perindopril (Fig. 1) were also investigated theoretically. Table S4) of bound perindoprilat and our computed structure for isolated molecule shows that both conformations differ in mutual position of anionic carboxylate groups and flexible n-propyl chain (torsion angles Θ[N(6)– C(7)–C(22)–O(4)]. respectively). 5). Important structural parameters of the impurity F are given in Tables S5 and S6 of the Supplementary material together with the experimental X-ray data for its tetragonal and orthorhombic polymorphs. Perindoprilat is buried deep inside the ACE active site making direct interaction with the Zn(II) ion of the active site [39]. 23]. Solvent water does not appreciably change its computed gas-phase equilibrium geometry. Impurity F Impurity F also shows polymorphism [22. The main difference between calculated 3D structure of both optimized polymorphs are observed for freely rotated n-alkyl groups (Fig. its active metabolite perindoprilat and impurity F using Becke3LYP density functional based theory. Optimized geometry of monoanion of perindoprilat in water solution changed considerably. Conclusions This study reports structural analysis of anihypertensive drug perindopril. Using the theoretical methods the following conclusions can be drawn. Θ[C(16)–N(17)–C(18)–C(24)]. 8 . Θ[N(17)–C(18)–C(24)-O(2)]. Θ[C(16)– N(17)–C(18)–C(19)] and Θ[N(17)–C(18)-C(19)-C(20)]. Table S4 of Supplementary material. The cyclohexane ring in isolated perindoprilat exists in stable chair conformation. strong Zn2+∙∙∙perindoprilat interaction results in considerable structural rearrangement of the drug molecule [41].3. This work yields quantities that may be inaccessible or complementary to experiments. An analysis of the X-ray data ([19].investigated also theoretically.1. Although no detectable structural change of active site was observed. Table S4 also contains structural data for perindoprilat bound to the angiotensin converting enzyme (ACE) homologue from Drosophila melanogaster (AnCE) (PDB code 2X94).

2 kJ/mol less stable.4 kJ/mol) and slightly reduced (1.1 kJ/mol) in water solution (B3Lyp/6-31G(d) method) indicating that these polymorphs differ especially by rotating about single bonds. Total energy difference between the two experimentally described polymorphs of diketopiperazine derivative of perindopril (tetragonal and orthorhombic) in vacuo is low (about 1. Diketopiperazine derivatives (impurity F) are important degradation products of several dicarboxylic ACE inhibitors. Neutral form of perindopril is more stable by 158 kJ/mol than the corresponding zwitterionic structure. For perindopril our DFT calculations predict the existence of two stable structures. this form is. However. 3. Perindoprilat bound to its protein target exists in the form of monoanion. 2. Solvent (water) results in appreciate stabilization of the zwitterion II. Calculations confirm that the zwitterionic form of perindoprilat containing a protonated alanine N atom and an anionic carboxylate group at the n-alkyl chain converged without an energy barrier to the neutral molecule of perindoprilat. However.3 kJ/mol higher in energy (B3LYP/6-311+G(d. The existence of monoanions of perindoprilat in the isolated state and water solution was confirmed also theoretically.p) method) than the conformation of the neutral perindoprilat. Inserting of perindopril species into water solution reduces their relative stability. 9 . by comparison with the neutral one. 4.1.3 kJ/mol) and the neutral perindopril is preferred over the zwitterionic one. Computed relative energy is still quite high (48. second zwitterionic structure of perindoprilat containing anionic proline carboxyl group (zwitterion II) represents on the potential energy surface a stable structure with local minimum by 161. still by 74.

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1.CH3 N N O O COOC2 H5 CH3 H O H Perindopril Hydrolysis CH3 N N O O H Cyclisation N COOH O CH3 O H N H H3C O O CH3 O H CH3 Perindoprilat Impurity F Fig. Pathway of perindopril degradation 12 .

Superimposition of optimized perindopril (green) and its zwitterion (blue) with respect to the amide plane. 2.Fig. 13 .

3. PoseView diagram of complex of perindoprilat in the bound state at the angiotensin converting enzyme (ACE) homologue from Drosophila melanogaster (AnCE) (PDB file 2X94).Fig. 14 .

Superimposition of B3LYP/6-311+G(d. [21]) (red) with respect to the amide plane.Fig. 15 .p) optimized perindoprilat monohydrate (green) and experimentally determined structure (ref. 4. Water molecule was omitted.

5. [19] (red) and B3LYP/6-311+G(d. 16 . For simplicity the hydrogen atoms are omitted.p) optimized perindoprilat (green) with respect to the amide plane. Molecular superimposition of the X-ray structure of perindoprilat.Fig.

6. Molecular superimposition of the B3LYP/6-311+G(d. 17 .p) optimized tetragonal (green) and orthorhombic (violet) polymorphs of impurity F.Fig.

Graphical Abctract .

. ◄It has been found that solid state conformations of perindoprilat occur close to. ◄Perindoprilat bound to its protein target exists in the form of monoanion. but not actually at minima on the computed gas-phase potential energy surfaces.◄Neutral forms of isolated perindopril and perindoprilat are more stable than the corresponding zwitterionic structures. ◄Water has a remarkable effect on the geometry of the perindopril species studied.