Accepted Manuscript

Molecular structure of antihypertensive drug perindopril, its active metabolite

perindoprilat and impurity F
M. Remko, J. Bojarska, P. Jeko, 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. Jeko, 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. Jekoa, W. Maniukiewiczb, A. Olczakb
a

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

10, SK-832 32 Bratislava, Slovakia

b

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

eromskiego 116, 90-924 d, 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. Introduction

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) is a long-acting angiotensin-converting
enzyme (ACE) inhibitor with high tissue ACE affinity, lowering angiotensin II and
potentiating bradykinin [1, 2]. 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,
congestive heart failure, post-myocardial infarction, diabetic nephropathy) [36]. Besides
antihypertensive effect, ACE inhibitors exhibit also vasculoprotective and antithrombotic
activities that play a favorable role in terms of cardiovascular morbidity [711]. Clinically
useful forms of perindopril are its tert-butylamine (perindopril erbumine) or L-arginine
(perindopril L-arginine) salts [1]. Perindopril, an acid-ester prodrug, is converted in vivo to
perindoprilat ((2S,3aS,7aS)-1-[(2S)-2-[[(2S)-1-hydroxy-1-oxopentan-2-yl]amino]propanoyl]2,3,3a,4,5,6,7,7a-octahydroindole-2-carboxylic acid) its active metabolite [12]. Nevertheless,
perindopril is observed to be chemically unstable and undergoes degradation in dosage forms
to other diacids and diketopiperazines [13]. Six degradation products of perindopril resulting
from hydrolysis, glucuronidation and cyclisation have been identified and analyzed [14].
These species are known degradation products of perindopril and/or are present as impurities
from the route of synthesis [15, 16]. One of them the diketopiperazine ethyl (2S)-2((3S,5aS,9aS,10aS)-3-methyl-1,4-dioxodecahydropyrazino [1,2-a]indol-2(1H)-yl)pentanoate),
known as impurity F in European Pharmacopeia 6.0, is regarded as main degradation product
of perindopril. Although perindopril was first synthesized (stereoselectively as a single all S
enantiomer) in 1982 [17], its three dimensional structure was unknown until 2011, when we
presented the first crystal structures of two tert-butylamine salts (see Cambridge Structural
Database Version 5.31; refcodes IVEGIA and IVEGOG [18] as part of our studies on
perindopril and perindoprilat polymorphism. In 1991, Pascard et al. [19] determined
configuration and preferential solid-state conformation of perindoprilat ethanol disolvate, 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]. It is worth to note that the geometric parameters of all studied solid structures are in
good agreement.

Here we report the gas-phase molecular structure of perindopril, its biologically active
metabolite perindoprilat and impurity F (a degradation product of perindopril). Theoretical
quantum chemical methods were used for determination of equilibrium geometries of neutral
and zwitterionic forms of perindoprilat, molecular structures of perindopril and its degradation
product in the isolated state and in aqueous solution. 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.

2. Computational procedure

Ab initio calculations of the perindopril and its derivatives (Fig. 1) were carried out with the
Gaussian 09 computer code [24] using density functional theory (DFT, Becke3LYP [25-27])
model chemistry [28, 29]. The perindoprilat was considered in two sets of neutral and ionic
complexes. In order to evaluate the conformational behavior of these systems in solvent, we
carried out optimization calculations in the presence of water. The methodology used in this
work is centered on polarized continuum model, PCM [30-32]. 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,p)
basis sets without any geometrical constraint. The structures of all condensed-phase (SCRF)
species were fully optimized without any geometrical constraint at the B3LYP/6-31G(d) level
of theory.

3. Results and discussion

3.1. 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].
Crystallographic data for all compounds has been deposited with the Cambridge
Crystallographic Data Centre as supplementary publication. Copies of the data can be obtained
free of charge on application to CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (fax: +44
1223 336 033; e-mail: deposit@ccdc.cam.ac.uk, www: http://www.ccdc.cam.ac.uk).
Crystallization of the free acid perindopril did not give single crystals suitable for X-ray

diffraction studies. Thus, its solid state structure is still unknown. However, it seems highly
probable that its conformation should resemble the conformation determined for perindoprilat.

3.2. Theoretical calculations

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

perindoprilat and cyclisation product of perindopril diketopiperazine have been shown that
these compounds can exist in several crystalline forms [17, 20-23] and their 3D structure
differs primarily in the n-alkyl chain orientation [21]. In crystals the zwitterionic form of
perindoprilat polymorphs was observed [17, 20, 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.
As regards of perindopril our DFT calculations predict the existence of two stable
structures. Neutral form of perindopril is more stable by 158 kJ/mol than the corresponding
zwitterionic structure. The zwitterionic form of perindopril can be stabilized in condensed
phase (water solution). Polar solvent (water) has a remarkable effect on the relative stability of
the neutral and zwitterionic species of perindopril. Inserting of perindopril species into water
solution reduces their relative stability. Computed relative energy is still quite high (48.3
kJ/mol) and the neutral perindopril is preferred over the zwitterionic one.
More interesting situation is in the case of perindoprilat. Zwitterions in unit cell of both
orthorhombic pseudopolymorphs are held together by rich system of intermolecular hydrogen
bonds [20, 21]. 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].
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. Thus, the zwitterionic form converged without an energy barrier to
the neutral molecule of perindoprilat. The surrounding medium did not shifted the relative
stability of perindoprilat species studied. Neutral perindoprilat is also stable form in water
solution. Theoretical calculations of the pKa of both, proline and n-alkyl chain carboxyl groups
of perindoprilat have been shown that these groups at physiological pH = 7.4 are completely
ionized [38]. From this point of view we also considered in our calculations zwitterionic
structure of perindoprilat containing anionic proline carboxyl group (zwitterion II). This
zwitterion represents on the potential energy surface a stable structure with local minimum by
161.3 kJ/mol higher in energy (B3LYP/6-311+G(d,p) method) than the conformation of the
neutral perindoprilat. Solvent (water) results in appreciate stabilization of the zwitterion II.
However, this form is, by comparison with the neutral one, still by 74.2 kJ/mol less stable.
One of the perindoprilat solvatomorphs investigated crystallizes as a monohydrate [21].
Water molecule directly stabilizes zwitterionic structure via system of N+-HO and OHO=C intermolecular hydrogen bonds [21]. 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. The
5

B3LYP/6-311+G(d,p) calculated hydrogen bond enthalpy of this complex is negative (-13.5

kJ/mol). However, Gibbs energy is large (33.5 kJ/mol) and positive, i.e. destabilizing. The
entropy term is negative (-157.7 J/mol K), opposing association. This means that water has
little tendency to associate in the gas phase. Hydrogen bond energy was computed as the
difference between the total energies of the thermodynamically most stable isolated species.
Diketopiperazine derivatives are important degradation products of several
dicarboxylic ACE inhibitors. From this point of view we also examined the molecular
structure of perindopril cyclisation product (impurity F, Fig. 1) both experimentally [21, 22]
and theoretically. Two experimentally described polymorphs of diketopiperazine derivative of
perindopril (tetragonal and orthorhombic) served as input data for our extended theoretical
calculations. The Gibbs energy difference observed between these two polymorphs in vacuo is
low (about 1.4 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.

3.2.1.1. Perindopril
Perindopril is an effective ACE inhibitor. Its 3D structure is governed by 9 rotatable bonds
[38]. The important geometric parameters are given in Table S1. Both B3LYP DFT bond
lengths and bond angles of perindopril fit one another to within about 0.002 for bond
lengths and about 1o for bond angles. Larger differences (about 1 - 5o) were observed for
dihedral angles. The molecular geometry of perindopril computed in water solution shows
appreciable changes in the region of flexible n-propyl chain and ester moiety, respectively.
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 132.5o). The molecular structure of the zwitterionic
perindopril is quite different. An proton transfer from the proline carboxyl to the basic
nitrogen atom of alanine is manifested in considerable structural changes of whole molecule
(Fig. 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). The aliphatic part of zwitterion
is stabilized by means of a weak intramolecular hydrogen bond of the C=OH2N+ type.
3.2.1.2. Perindoprilat
Perindoprilat, the active form of perindopril, is chemically dicarboxylic ACE inhibitor.
At physiological pH = 7.4 both carboxyl groups are completely ionized [38]. As it is shown

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]. PoseView [40] diagram of AnCE - perindoprilat
complex displays important intermolecular interactions of perindoprilat with complementary
sites of ACE active site (Fig. 3). 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. 3). C-terminal carboxylate anion is stabilized by
means of 3 hydrogen bonds with Gln265, Tyr504 and Lys495. Hence, biologically active
conformation of perindopril is a monoanion. Unbound perindoprilat may exist in solid state in
several zwitterionic polymorphs with protonated alanine nitrogen and anionic carboxyl at the
carboxyalkyl carboxylate group [17, 20, 21]. As a starting geometry for DFT calculations we
used X-ray structure of two recently discovered new pseudopolymorphs of this drug [20, 21].
Theoretically calculated equilibrium gas-phase geometry of isolated perindoprilat represents
neutral molecule without intramolecular proton transfer. Selected structural parameters
computed using two basis sets for this structure are presented in Table S2 of the
Supplementary material. 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. As regards of
torsion angles, the largest changes were observed for the rotation of the carboxyalkyl
carboxylate group. The optimized torsion angles computed at both levels of theory [C(10)N(2)-C(11)-(C17)] and [N(2)-C(11)-C(17)-O(3)] are substantially different (Table S2 of the
Supplementary material). In water solution its gas-phase molecular structure changes only
slightly. In solid state zwitterion may be stabilized by solvate molecule (water). Important
geometrical parameters of the DFT optimized structure of complex perindoprilat water are
given in Table S3 of the Supplementary material. The main difference between theoretical and
experimental structure is observed in the region of the carboxyalkyl carboxylate group, torsion
angles [C(10)-N(2)-C(11)-(C17)] and [C(11)-N(2)-C(10)-C(9)] (Table S3 of the
Supplementary material). 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. 4. Solvent effect did not changed appreciably
geometry of isolated complex (Table S3).
Perindoprilat bound to its protein target exists in the form of monoanion (Fig. 3). The
possible existence of monoanions of perindoprilat in the isolated state and water solution was

investigated also theoretically, Table S4 of Supplementary material. Table S4 also contains

structural data for perindoprilat bound to the angiotensin converting enzyme (ACE)
homologue from Drosophila melanogaster (AnCE) (PDB code 2X94). Perindoprilat is buried
deep inside the ACE active site making direct interaction with the Zn(II) ion of the active site
[39]. Although no detectable structural change of active site was observed, strong
Zn2+perindoprilat interaction results in considerable structural rearrangement of the drug
molecule [41]. An analysis of the X-ray data ([19], 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 [N(6)
C(7)C(22)O(4)], [C(16)N(17)C(18)C(24)], [N(17)C(18)C(24)-O(2)], [C(16)
N(17)C(18)C(19)] and [N(17)C(18)-C(19)-C(20)], respectively). The perhydroindole
ring of the proline moiety is in both conformers an envelope. The cyclohexane ring in isolated
perindoprilat exists in stable chair conformation. However, this 6-membered ring in bound
perindoprilat exists as the stable boat form (Fig. 5). Optimized geometry of monoanion of
perindoprilat in water solution changed considerably, and is closer to the biologically active
conformation of bound perindoprilat monoanion (Table S4).

3.2.1.3. Impurity F
Impurity F also shows polymorphism [22, 23]. Experimentally described two polymorphs of
this cyclisation product of perindopril (Fig. 1) were also investigated theoretically. 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. The calculations showed that the solid-state conformation of this molecule is also
preserved in the gas-phase. Solvent water does not appreciably change its computed gas-phase
equilibrium geometry. The main difference between calculated 3D structure of both optimized
polymorphs are observed for freely rotated n-alkyl groups (Fig. 6).

4. Conclusions
This study reports structural analysis of anihypertensive drug perindopril, its active
metabolite perindoprilat and impurity F using Becke3LYP density functional based theory.
This work yields quantities that may be inaccessible or complementary to experiments. Using
the theoretical methods the following conclusions can be drawn.

1. For perindopril our DFT calculations predict the existence of two stable structures. Neutral
form of perindopril is more stable by 158 kJ/mol than the corresponding zwitterionic
structure. Inserting of perindopril species into water solution reduces their relative
stability. Computed relative energy is still quite high (48.3 kJ/mol) and the neutral
perindopril is preferred over the zwitterionic one.
2. 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, 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 161.3
kJ/mol higher in energy (B3LYP/6-311+G(d,p) method) than the conformation of the
neutral perindoprilat. Solvent (water) results in appreciate stabilization of the zwitterion II.
However, this form is, by comparison with the neutral one, still by 74.2 kJ/mol less stable.
3. Perindoprilat bound to its protein target exists in the form of monoanion. The existence of
monoanions of perindoprilat in the isolated state and water solution was confirmed also
theoretically.
4. Diketopiperazine derivatives (impurity F) are important degradation products of several
dicarboxylic ACE inhibitors. Total energy difference between the two experimentally
described polymorphs of diketopiperazine derivative of perindopril (tetragonal and
orthorhombic) in vacuo is low (about 1.4 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.

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11

CH3
N

N
O

COOC2 H5
CH3

O
H
Perindopril

Hydrolysis

CH3
N

N
O

Cyclisation

COOH
O

CH3

O
H

N
H

H3C

O
O

CH3
O

H
CH3
Perindoprilat

Impurity F

Fig. 1. Pathway of perindopril degradation

12

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

13

Fig. 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).

14

Fig. 4. Superimposition of B3LYP/6-311+G(d,p) optimized perindoprilat monohydrate

(green) and experimentally determined structure (ref. [21]) (red) with respect to the amide
plane. Water molecule was omitted.

15

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

16

Fig. 6. Molecular superimposition of the B3LYP/6-311+G(d,p) optimized tetragonal (green)

and orthorhombic (violet) polymorphs of impurity F.

17

Graphical Abctract

Neutral forms of isolated perindopril and perindoprilat are more stable than the
corresponding zwitterionic structures. 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. Perindoprilat bound to its protein target exists in the form of monoanion.
Water has a remarkable effect on the geometry of the perindopril species studied.