Conformational analysis of E/Z-isomeric pairs of rosuvastatin and its lactonized analogues

Article abstract of DOI:10.1016/j.tet.2013.05.020

Most of the super-statins contain a CC double bond spacer between the heterocyclic and the chiral dihydroxycarboxylic moieties. The known drugs are E-geometric isomers, whereas very little is known about their Z-isomeric analogues. This study explains the unusual resonance line broadening observed in ¹H NMR spectra of Z-isomeric rosuvastatin analogues at room temperature, which originates from dynamic exchange between different conformers. Conformational equilibria and intrinsic preferences of Z-isomeric rosuvastatin analogues provide valuable insight into conformational variability that is important for studying potential interactions within the binding site of the enzyme.

Full text of DOI:10.1016/j.tet.2013.05.020

Tetrahedron 69 (2013) 6262e6268
Contents lists available at SciVerse ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Conformational analysis of E/Z-isomeric pairs of rosuvastatin and its
lactonized analogues
,
,
d
b
,
,d,f,
*
Jan Fabris a b, Damjan Makuc c , Zdenko Casar e, Janez Plavec c
ꢀ
a Cadonic Consultancy Services, LL.C., Cesta na postajo 74, SI-1351 Brezovica pri Ljubljani, Slovenia
Lek Pharmaceuticals, d.d., Sandoz Development Center Slovenia, API Development, Organic Synthesis Department, Kolodvorska 27, SI-1234 Menges,
b
ꢀ
Slovenia
c Slovenian NMR Centre, National Institute of Chemistry, Hajdrihova 19, SI-1000 Ljubljana, Slovenia
d EN-FIST Centre of Excellence, Dunajska cesta 156, SI-1000 Ljubljana, Slovenia
e
ꢀ
ꢀ
Faculty of Pharmacy, University of Ljubljana, Askerceva cesta 7, SI-1000 Ljubljana, Slovenia
f
ꢀ
ꢀ
Faculty of Chemistry and Chemical Technology, University of Ljubljana, Askerceva cesta 5, SI-1000 Ljubljana, Slovenia
a r t i c l e i n f o
a b s t r a c t
Article history:
Most of the super-statins contain a C]C double bond spacer between the heterocyclic and the chiral
dihydroxycarboxylic moieties. The known drugs are E-geometric isomers, whereas very little is known
about their Z-isomeric analogues. This study explains the unusual resonance line broadening observed in
1H NMR spectra of Z-isomeric rosuvastatin analogues at room temperature, which originates from dy-
namic exchange between different conformers. Conformational equilibria and intrinsic preferences of
Z-isomeric rosuvastatin analogues provide valuable insight into conformational variability that is im-
portant for studying potential interactions within the binding site of the enzyme.
Received 22 February 2013
Received in revised form 24 April 2013
Accepted 9 May 2013
Available online 14 May 2013
Keywords:
Conformational analysis
Isomerism
NMR
Ó 2013 Elsevier Ltd. All rights reserved.
Statins
Drugs
1. Introduction
dihydroxycarboxylic moiety. Examples of such compounds are flu-
vastatin, rosuvastatin and pitavastatin. Even though the presence of
Nowadays cardiovascular diseases (CVD) represent one of the
most frequent causes of death worldwide.1 It is well known that
overaccumulation of cholesterol in the organism plays a central role
in CVD.2 The inhibition of enzyme 3-hydroxy-3-methylglutaryl-
coenzyme A reductase (HMGR), which is the rate-limiting enzyme
in the biosynthesis of cholesterol, effectively lowers the concen-
tration of low density lipoproteins in the blood thus reducing the
risk of CVD.3 The most effective HMGR inhibitors are statins and
they represent one of the most valuable therapeutic classes of
compounds in the pharmaceutical sector. Statins were discovered
as fungal metabolites in the 1970s (e.g., mevastatin4 and lova-
statin5) and were soon developed through semi-synthetic ana-
logues (pravastatin6 and simvastatin7) towards fully synthetic
compounds named super-statins (fluvastatin,8 atorvastatin,9 rosu-
vastatin10 and pitavastatin11).12
a double bond enables the formation of two geometric (E and Z)
isomers, it is important to note that all of the commercial super-
statins are E-geometric isomers. In general, in biologically active
compounds E-configuration along C]C double bond is not always
most active or preferred in terms of pharmacodynamic properties.
There are some commercially available drugs and biologically active
compounds with Z-configuration along the exocyclic C]C double
bonds. Examples of such substances are e.g., cis-cefprozil,13 lafuti-
dine,14 many combretastatins15 and prostaglandins16 together with
their synthetic analogues. During the discovery stage of more potent
and efficient super-statins a series of analogues has been prepared
and tested for the inhibition of HMGR. The large efforts included also
some Z-isomeric analogues, which showed only weak in vitro ac-
tivity in some studies17 and a complete loss of activity in the
others.18 Interestingly, literature data highlighting the rationale for
lowactivityof Z-isomeric statins are scarce. Structural mechanism of
inhibition was investigated using X-ray crystal structures of HMGR
complexed with different E-isomeric statins.19 Furthermore, the
kinetic and thermodynamic characterization of binding to HMGR
and inhibition of HMGR by different statins have contributed
important details on molecular mechanism for inhibition.20
Most of the marketed super-statins comprise a C]C double bond
as a spacer between the heterocyclic and the pharmacophoric chiral
* Corresponding author. Tel.: þ386 1 476 0353; fax: þ386 1 476 0300; e-mail
address: janez.plavec@ki.si (J. Plavec).
0040-4020/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tet.2013.05.020

J. Fabris et al. / Tetrahedron 69 (2013) 6262e6268
6263
We have recently elaborated a new synthetic route to rosuvas-
tatin calcium via Wittig coupling reaction21 between phosphonium
salt of an appropriately functionalized heterocyclic moiety and
lactonized statin side chain precursor.22 An interesting side-
product was isolated and characterized as the Z-isomeric 4-O-TBS
protected rosuvastatin lactone Z-1.21 Surprisingly, 1H NMR spec-
trum of this novel rosuvastatin analogue showed broad lines, which
could indicate the presence of different rotational conformers.
Excited by the observation of unexpected conformational proper-
ties of Z-1 in solution at room temperature, we were encouraged to
synthesize two more rosuvastatin analogues Z-2 and Z-3 (Fig. 1).
All three Z-isomeric rosuvastatin derivatives showed broad 1H NMR
resonances.
Scheme 1. Reagents and conditions: (i) (nBu)4NF, AcOH, THF, 0 ꢀC to rt; (ii) NaOH, THF/
H2O, 30 ꢀC then CaCl2 (aq), rt.
as well as from 1He13C heteronuclear correlations in HSQC and
HMBC spectra. The unambiguous 1H and 13C chemical shift as-
signments of novel compounds Z-2 and Z-3 are reported in the
Experimental section.
The comparison of 1H NMR spectra of rosuvastatin E-3 and its
isomeric analogue Z-3 showed NMR resonances with similar
chemical shifts (Fig. 2). However, remarkable differences in signal
line-widths were observed for the two geometric isomers. Z-3
showed broader NMR signals (e.g., Dn1/2z8 Hz for H7) in
comparison to its E-counterpart (Dn1/2z3 Hz).
Fig. 1. Chemical structures and atom numbering of 4-O-TBS protected rosuvastatin
lactone 1, deprotected rosuvastatin lactone
FeC6H4).
2
and rosuvastatin calcium
3
(R¼p-
We hypothesized that at sufficiently low temperature the
intramolecular rotations will slow down on the NMR chemical shift
time scale and resolved signals will be observed enabling charac-
terization of interconverting rotamers. Temperature-dependent
NMR study provided insight into rotational equilibria of Z-iso-
meric analogues in comparison to their E-isomers. The chemical
structures of rosuvastatin analogues with a linker region between
their hydrophobic heterocyclic moieties and chiral dihydrox-
ycarboxylic side chain exhibit several single bonds that could be
conformationally flexible. NMR enabled the localization of rota-
tional degrees of freedom and an evaluation of these preferences
through van’t Hoff analysis. The assessment of the rotational energy
barrier from coalescence temperature was complemented by ab
initio calculations thus giving a comprehensive insight into con-
formational features of isomeric pair of rosuvastatin that are im-
portant for understanding their physical and biochemical
properties.
Fig. 2. 1H NMR (600 MHz) spectra of E-3 (a) and Z-3 (b) dissolved in D2O at 298 K.
Due to the low solubility of rosuvastatin and its derivatives in
water and the need to perform NMR experiments at low temper-
atures different organic solvents were used. Upon cooling of Z-3 in
methanol-d4 to 223 K two sets of sharp and well-resolved signals
were observed in the ratio of 5:1, which were labelled as major (M)
and minor (m) conformers in solution (Fig. 3). Heating of the
sample above the coalescence temperature of ca. 303 K resulted in
a single set of signals (Fig. S1). Similarly, two sets of signals were
observed for Z-isomeric 4-O-TBS protected rosuvastatin lactone Z-1
and deprotected Z-isomeric rosuvastatin lactone Z-2 in acetone-d6
at 223 K. The perusal of 1H NMR spectra at 223 K showed in-
teresting differences in multiplicity of H5 and H7 resonances. The
4JH5eH7 allylic coupling constant with value of 1.1 Hz was observed
for the minor conformer of Z-1 at 223 K in acetone-d6 (Table 1). In
2. Results and discussion
Compounds E-1, E-2, E-3, and Z-1 were prepared according to
the published procedure.21 In addition, Z-1 was deprotected using
tetrabutylammonium fluoride trihydrate and acetic acid to give
Z-2, which was further treated with aqueous NaOH and CaCl2, re-
spectively, to yield Z-isomeric rosuvastatin calcium Z-3 (Scheme 1).
Assignments of 1H and 13C NMR resonances for Z-1, Z-2 and Z-3
were achieved from signal multiplicities, integral values and char-
acteristic chemical shifts from the through-bond correlations in 2D
DQF-COSY spectra, through-space correlations in 2D NOESY spectra
4
contrast, no JH5eH7 coupling constant was observed for the major
conformer in acetone-d6. Well-defined differences in allylic

6264
J. Fabris et al. / Tetrahedron 69 (2013) 6262e6268
suggested anti orientation of H5 and H6 along C5eC6 bond in the
major rotamers and syn orientation in the minor rotamers with W-
geometry along the H5eC5eC6]C7eH7 bonds in the latter. In-
terestingly, only one set of signals was observed for each of E-1, E-2
and E-3. The observed multiplicity pattern of E-isomers is in ac-
cordance with a W-geometry arrangement between H5 and H7
protons, where H5 and H6 protons have an anti orientation across
the C5eC6 bond.
4
The observed differences in the JH5eH7 allylic coupling con-
stants and distinct cross-peak intensities in the NOESY spectra of
the major and minor rotamers along the C5eC6 bond were used for
their identification. The key differences in the volumes of NOESY
cross-peaks were observed between H5 and H7 protons with re-
spect to the protons of fluorophenyl and isopropyl groups of Z-1, Z-
2 and Z-3. In the case of Z-1, the major rotamer showed a very
strong NOESY cross-peak between H7 and the o-fluorophenyl
protons, whereas a weak cross-peak was observed between H5 and
the o-fluorophenyl protons (Fig. 4a). In full support, a strong NOESY
interaction was noted between H7 and the tertiary proton of the
isopropyl group, which exhibits NOESY cross-peak of moderate
intensity with H5 as well. In addition, the orientation of H5 is de-
fined by its NOESY signal with the methyl protons of the isopropyl
group. The above spatial proximities are consistent with an anti
orientation of H5eC5 and H7eC7 bonds observed in the lowest
energy structure of Z-1 as calculated by DFT calculations (Fig. 4a).
In contrast, no NOESY cross-peaks were observed between H5
and the isopropyl protons for the minor rotamer of Z-1. This ob-
servation is in agreement with the DFT optimized structure of Z-1
presented in Fig. 4b. The orientation of H5 away from the isopropyl
group attached to C60 is supported by moderate NOESY cross-peaks
between H5 and o-fluorophenyl protons as well as between H7 and
fluorophenyl, and strong NOESY interaction between H7 and iso-
propyl protons. The conformational features of minor rotamer of Z-
1 are in agreement with the syn orientation of H5 and H6 along the
C5eC6 bond (Fig. 4b).
Fig. 3. 1H NMR (600 MHz) spectra of Z-3 dissolved in methanol-d4 from 223 to 303 K.
Labels M and m denote H7 resonances at
minor conformers, respectively.
d ca. 6.6 ppm corresponding to the major and
Table 1
1He1H coupling constants in different solvents at 223 K
Compound
Major conformer (M)
Minor conformer (m)
3JH5eH6
3JH6eH7
4JH5eH7
3JH5eH6
3JH6eH7
4JH5eH7
Z-1a
Z-2a
Z-3b
E-1a
E-2a
E-3b
10.0
10.2
mc
6.8
7.0
11.2
11.2
11.2
16.1
16.1
15.9
0.0
0.0
0.0
9.2
9.4
mc
d
11.3
11.2
11.7
d
1.1
0.8
<0.6d
d
Higher rotational flexibility and consequently lower mole frac-
tions of minor conformers of Z-2 and Z-3 prevented detailed con-
formational analysis based on NOESY experiments. Nevertheless,
the proposed anti orientation of H5 and H6 along the C5eC6 bond
in the major rotamer and syn orientation in the minor rotamer is in
good agreement with the energetic preferences across C5eC6 bond
of Z-rosuvastatin derivatives (vide infra).
0.9
0.9
d
d
d
6.8
<0.6d
d
d
d
a
Compound dissolved in acetone-d6.
Compound dissolved in methanol-d4.
Broad multiplet was observed.
b
c
d
J was within the line-width of the resonance.
The dynamic equilibrium between the two conformers, M%m,
was evaluated on the basis of their mole fractions, which were
estimated by the integral values of well-resolved H7 proton signals
at five different temperatures in the range from 223 to 263 K
(Fig. 3). As the temperature increased, the population of conformer
m increased with respect to conformer M (Table 2). Thermody-
namic parameters for Z-1, Z-2 and Z-3 were obtained from van’t
Hoff plots (Fig. 5). The slope of the lines in van’t Hoff plots sug-
gested the predominance of enthalpy contributions in controlling
the dynamic equilibria M%m for each of Z-1, Z-2 and Z-3 (Table 2).
The experimental observations on the conformational equilibria
between rotamers M and m were augmented by quantum me-
chanical calculations at B3LYP/6-311þG(d,p) level of theory using
coupling constants between major and minor conformers sug-
gested the presence of two rotamers along the C5eC6 single bond
in Z-1.
4
Similar JH5eH7 coupling constants of 0.8 and 0 Hz were found
for the minor and major rotamers of Z-2, respectively (Table 1).
4
Furthermore, no JH5eH7 coupling constant was observed for the
major rotamer of Z-3 in methanol-d4 at 223 K. Broad multiplet for
H7 signal was observed for the minor rotamer of Z-3 and the cor-
4
responding JH5eH7 allylic coupling constant was within the line-
width of the resonance. Rotations along the C5eC6 and C50eC7
single bonds allow rosuvastatin derivatives Z-1, Z-2 and Z-3 to
adopt several conformations with distinct orientation of dihy-
droxycarboxylic and heterocyclic moieties. The use of the modified
Karplus equation proposed by Garbisch23 allowed an assessment of
the orientation of H5 with respect to H7. The 4JH5eH7 allylic coupling
constants of ca. 1 and 0 Hz are in agreement with torsion angles
[H5eC5eC6eH6] of 0ꢀꢁ30ꢀ and 180ꢀꢁ30ꢀ, respectively. In addi-
tion, the same torsion angle can be estimated from the analysis of
3JH5eH6 coupling constantsdtheir values above 9 Hz suggest24 two
possible orientations along C5eC6 bond with torsion angles of
approximately 0ꢀꢁ30ꢀ and 180ꢀꢁ30ꢀ. Consequently, the observa-
Gaussian 09 program.25 Torsion angle
q [H5eC5eC6eH6] was de-
fined to follow energetic variations induced by reorientation
around the C5eC6 bond in Z-1, Z-2 and Z-3. The relative energy
profile along torsion angle
q
with 30ꢀ resolution shows that the
rotamer with the lowest energy adopts an anti orientation of H5
and H6 across the C5eC6 bond (Fig. 6a). Energy minimization of
anti and syn rotamers of Z-1 in the local energy minima established
by energy profile calculations were performed without any con-
straints; anti rotamer of Z-1 exhibited the lowest energy, while the
syn rotamer showed 1.7 kcal molꢂ1 higher energy. The torsion an-
4
tion of JH5eH7 long-range coupling constants in Z-1, Z-2 and Z-3
gles q
of freely optimized anti and syn rotamers of Z-1 were 179.4ꢀ

J. Fabris et al. / Tetrahedron 69 (2013) 6262e6268
6265
Fig. 4. Freely optimized structures of Z-1 at B3LYP/6-311þG(d,p) level of theory with anti (a) and syn (b) orientations of H5 and H6 protons along the C5eC6 bond. The key NOESY
signals from a NOESY spectrum (mixing time 200 ms, 253 K) are marked with green arrows. Numbers correspond to relative integral volumes with respect to H6eH7 cross-peak,
which is arbitrarily set to 100 units. For clarity tert-butyldimethylsilyl group attached to C3 is not shown.
Table 2
Temperature variation of conformer population and thermodynamic parameters for
dynamic equilibria of Z-1, Z-2 and Z-3
Compound Mole fractions of minor conformers
Thermodynamic
parametersa
223 K 233 K 243 K 253 K 263 K
D
Hꢀ eT
D
Sꢀ
D
Gꢀ
Z-1b
Z-2b
Z-3c
0.246
0.123
0.154
0.266
0.143
0.163
0.278
0.175
0.176
0.299
0.201
0.209
0.307
0.227
0.231
0.90 ꢂ0.55 0.35
2.20 ꢂ1.77 0.43
1.50 ꢂ0.97 0.53
a
Reported in kcal molꢂ1
.
b
c
Compound dissolved in acetone-d6.
Compound dissolved in methanol-d4.
and 48.6ꢀ, respectively. The respective rotational energy barrier of
5.2 kcal molꢂ1 along the torsion angle
q
was found at
q
¼330ꢀ.
Noteworthy, conformational features of energetically optimized
rotamers are in good agreement with NOESY intensities and 4JH5eH7
allylic coupling constants, where rotamer M exhibits anti orienta-
tion and rotamer m corresponds to a syn orientation of H5 and H6
along the C5eC6 bond.
Two minima were found for an anti orientation of H5 and H6
along the C5eC6 bond in Z-2 with low rotational energy barrier of
2.1 kcal molꢂ1 (Fig. 6a). The syn rotamer of Z-2 exhibited signifi-
cantly higher energy with respect to the global minimum
(5.3 kcal molꢂ1). Similarly, the two minima with the low rotational
energy barrier were observed in anti region for Z-3. Rotamer of Z-3
with syn orientation of H5 and H6 along the C5eC6 bond exhibited
higher energy of 3.8 kcal molꢂ1. Z-1, Z-2 and Z-3 showed rotational
energy barriers of 4.9e6.4 kcal molꢂ1 between syn and anti
rotamers along the C5eC6 bonds.
Fig. 5. Van’t Hoff plots for equilibrium M%m of Z-1 (:), Z-2 (-) and Z-3 (O) in
the temperature range from 223 to 263 K. The straight lines are the best fits to the
experimental data using least-square method (Pearson correlation coefficient R2 was
0.9879, 0.9968 and 0.9439 for Z-1, Z-2 and Z-3, respectively). The mean-square de-
viations were below 0.003 with the largest individual deviation below 0.05.
The values of rotational energy barriers along the C5eC6 bonds
in Z-1, Z-2 and Z-3 from ab initio calculations were between 4.9 and
6.4 kcal molꢂ1 (Fig. 6). In contrast, the experimentally determined
rotational energy barriers were much largerdbetween 14.4 and
14.8 kcal molꢂ1 (Table 3). The above discrepancies implied that
another conformationally flexible bond is important for observa-
tion of two rotamers at low temperatures. Since the C6]C7 double
bond is tilted with respect to the aromatic plane and the two sides
of aromatic ring are different, the rotation along the C50eC7 bond
would generate a pair of atropisomers. Further exploration focused
E-isomers exhibit the lowest energies in syn region of E-1, E-2
and E-3 (Fig. 6b). The low rotational energy barrier of ca.
2 kcal molꢂ1 is consistent with a single set of signals observed in 1H
NMR spectra.
The coalescence temperatures enabled estimation of exchange
rates kE and
equation (Table 3).
D
Gz for rotational energy barriers using the Eyring

6266
J. Fabris et al. / Tetrahedron 69 (2013) 6262e6268
Fig. 7. Four major energetically favourable conformers of Z-1 (R¼p-FeC6H4, R1 and R2
represent the lactone moiety) with the corresponding rotational energy barriers across
C50eC7 and C5eC6 bonds.
that were observed in NMR spectra at lower temperatures most
probably correspond to one of the diagonal pairs shown in Fig. 7
((M)-syn and (P)-anti or (M)-anti and (P)-syn), with concerted ro-
tation along two single bonds of Z-1. However, on the basis of J
coupling constants and NOESY experiments it could not be de-
termined, which diagonal pair presented in Fig. 7 is present in the
solution.
Conformational analysis together with thermodynamic prefer-
ences of Z-isomeric rosuvastatin analogues provides insight into
conformational variability that is important for studying potential
interactions within the HMGR binding site. A recent study com-
bining theoretical and experimental methods showed that an in-
hibitory peptide causes ordering of secondary structure of HMGR
upon binding,26, which implies that a wide variety of molecules
could bind to the active site of HMGR and the newly synthesized
Z-isomers of rosuvastatin are not excluded.
Fig. 6. Relative potential energy of Z-1 (-), Z-2 ( ) and Z-3 ( ) in (a) and of E-1 (-),
E-2 ( ) and E-3 ( ) in (b) as a function of the [H5eC5eC6eH6] torsion angle at the
B3LYP/6-311þG(d,p) level of theory.
Table 3
Exchange rates kE and DG
z for rotational energy barriers of Z-1, Z-2 and Z-3a
Compound
Dn (Hz)
kE (sꢂ1
)
DG )
z (kcal molꢂ1
Z-1b
Z-2b
Z-3c
58.0
127.5
91.2
129
283
203
14.8
14.4
14.6
a
The coalescence temperature for Z-1, Z-2 and Z-3 was estimated to be 303 K.
Compound dissolved in acetone-d6.
Compound dissolved in methanol-d4.
b
c
solely on Z-1 because NOESY experiments prevented detailed
conformational analysis of Z-2 and Z-3. Four major energetically
preferred conformations are expected for Z-1 with respect to ro-
tations along C5eC6 and C50eC7 single bonds (Fig. 7).
Torsion angle
f
[C60eC50eC7eH7] was defined and its potential
energy profile was calculated for Z-1 with 30ꢀ resolution at B3LYP/
6-311þG(d,p) level of theory. During the energy profile calculations
the torsion angle q (rotation along C5eC6 bond) was set both to anti
and syn orientations. It turned out that when the torsion angle
q
was set in syn orientation, the rotation along the C50eC7 bond
exhibited very high energy barrier. Relative potential energy profile
of Z-1 as a function of the torsion angle
in anti orientation, showed two minima at
which suggests presence of atropisomers (Fig. 8). Rotational energy
barrier along C50eC7 bond is 12.7 kcal molꢂ1, which is in good
agreement with experimentally determined rotational energy
barrier (Table 3). Two out of the four major possible conformers
f, where torsion angle q was
f
¼60ꢀ and
f
¼240ꢀ,
Fig. 8. Relative potential energy of Z-1 as a function of the
f
[C60eC50eC7eH7] torsion
angle at the B3LYP/6-311þG(d,p) level of theory. The torsion angle
q [H5eC5eC6eH6]
was set to anti orientation. Rotation between
f
¼300ꢀ and
f
¼360ꢀ led to very high
energy.

J. Fabris et al. / Tetrahedron 69 (2013) 6262e6268
6267
3. Conclusion
Note that structure calculations for Z-3 and E-3 were performed on
their acidic forms with COOH groups.
Both Z- and E-isomers of rosuvastatin and its lactonized ana-
logues were prepared and characterized by heteronuclear NMR
spectroscopy. The NMR resonance line broadening in 1H NMR
spectra observed for Z-isomeric rosuvastatin analogues at room
temperature originates from the dynamic exchange between the
two conformers. In contrast, isomers E-1, E-2 and E-3 exhibit a sin-
gle set of narrow NMR resonances. The two conformers of Z-isomers
were distinguished at low temperature, where well-defined differ-
ences in 4JH5eH7 allylic coupling constants between major and minor
conformers suggested the presence of two rotamers of Z-1 along the
C5eC6 single bond. An anti orientation of H5 and H6 along the
C5eC6 bond was assigned to the major rotamer, whereas the minor
rotamer exhibited a syn orientation. Thermodynamic preferences
between the two conformers were assessed with 1H NMR experi-
ments over a temperature range from 223 to 263 K. An increase in
the sample temperature was followed by an increase in the pop-
ulation of the minor conformer. Additionally, two conformers ob-
served in the NMR spectra at lowered temperature most likely
correspond to a pair of atropisomers, where concerted rotation
along both C5eC6 and C50eC7 bonds is supported by experimen-
tally determined as well as by calculated rotational energy barrier.
4.4. Synthetic procedures
4.4.1. Synthesis of N-(4-(4-fluorophenyl)-5-((Z)-2-((2S,4R)-4-
h y d r o x y - 6 - o x o t e t r a h y d r o - 2 H - p y r a n - 2 - y l ) v i n y l ) - 6 -
isopropylpyrimidin-2-yl)-N-methylmethanesulfonamide (Z-2). A so-
lution of tetrabutylammonium fluoride trihydrate (191 mg,
0.61 mmol; 1.75 equiv) and AcOH (95 mL, 1.66 mmol; 4.8 equiv) in
THF (2 mL) was cooled in an ice bath. Then a solution of Z-121
(200 mg, 0.35 mmol) in THF (2 mL) was added. The solution was
left to stir for 22 h at room temperature. The solvent was evaporated
under reduced pressure and the residue was dissolved in EtOAc
(5 mL). The organic layer was washed with water (5 mL), saturated
solution of NaHCO3 (5 mL), brine (5 mL) and water (5 mL), re-
spectively. Combined organic layers were dried over Na2SO4 and
evaporated under reduced pressure. The residue was purified by
column chromatography (silica gel, EtOAceHex, 1:1) to give 89 mg
25
(55% yield) of pure Z-2; mp 188.6 ꢀC (onset), 198.0 ꢀC (peak); [
a]
D
þ126 (c 0.25, MeOH); nmax (KBr) 3444, 2969, 2932, 1735, 1606, 1548,
1511, 1371, 1230, 1155, 962, 769, 567, 521 cmꢂ1
dH (600 MHz, ace-
;
tone-d6) 7.82 (2H, b, 4-FeC6H4), 7.22 (2H, b, 4-FeC6H4), 6.89 (1H, b,
H-7), 5.79 (1H, b, H-6), 4.82 (1H, b, H-5), 4.23 (1H, b, OH), 4.12 (1H, b,
H-3), 3.54 (6H, s, NeCH3 and SO2eCH3), 3.36 (1H, b, CH(CH3)2), 2.55
(1H, dd, J 17.6, 4.1 Hz, H-2a), 2.38e2.32 (1H, m, H-2b),1.56e1.42 (1H,
m, H-4a), 1.30 (3H, d, J 6.5 Hz, CH(CH3)2), 1.26 (3H, d, J 6.5 Hz,
CH(CH3)2), 0.82 (1H, b, H-4b); dC (75 MHz, acetone-d6) 175.9 (C-60),
169.4 (C-1), 164.6 (C-40), 164.4 (d, JCF 248.0 Hz, 4-FeC6H4), 159.3 (C-
20), 135.5 (d, JCF 3.3 Hz, 4-FeC6H4), 133.7 (C-6), 133.2 (d, JCF 8.7 Hz, 4-
FeC6H4), 127.9 (C-7), 120.2 (C-50), 115.9 (d, JCF 21.7 Hz, 4-FeC6H4),
72.9 (C-5), 62.8 (C-3), 42.4 (SO2eCH3), 39.3 (C-2), 34.8 (C-4), 33.7
(NeCH3), 33.5 (CH(CH3)2), 22.0 (CH(CH3)2), 21.4 (CH(CH3)2); HRMS
(ESI): MHþ, found: 464.1647. C22H27FN3O5S requires 464.1650.
4. Experimental section
4.1. General
Reagents and solvents were acquired from commercial sources
and used without further purification. Reactions were monitored
by using analytical TLC plates (Merck; silica gel 60 F254, 0.25 mm),
and compounds were visualized with UV radiation. Silica gel grade
60 (70e230 mesh, Merck) was used for column chromatography.
Melting points were determined with a Mettler Toledo DSC822e
apparatus (heating rate 10 ꢀC/min) and are referred to as onset
values and peak values. Optical rotations were measured on a Per-
kin Elmer 341-series polarimeter; only noteworthy absorptions are
listed. High-resolution mass spectra were obtained with a VG-
Analytical AutospecQ instrument and a Q-TOF Premier instrument.
4.4.2. Synthesis of (3R,5S,Z)-7-(4-(4-fluorophenyl)-6-isopropyl-2-(N-
methylmethylsulfonamido)pyrimidin-5-yl)-3,5-dihydroxyhept-6-
enoate calcium (Z-3). To a solution of Z-2 (139 mg, 0.30 mmol) in
THFewater, 4:1 (5 mL) at 30 ꢀC was added 2 M aqueous NaOH
(161 mL, 0.32 mmol, 1.075 equiv). After 2 h the reaction was finished
4.2. NMR experiments
and THF was evaporated under reduced pressure. To a 2 mL of an
aqueous solution of the sodium salt of Z-isomeric rosuvastatin was
added a solution of CaCl2 (70 mg, 0.60 mmol; 2 equiv) in water
(2 mL). The resulting white precipitation was left to stir at rt for
18 h. Then it was filtered off, washed with water (3ꢃ5 mL) and
dried at 60 ꢀC under vacuum to give 48 mg (64% yield) of pure Z-3;
1H and 13C NMR spectra were acquired on Agilent Technologies
(Varian) VNMRS 600 MHz, Unity Inova 300 MHz and DD2 300 MHz
NMR spectrometers. Sample concentrations used in NMR studies
were ca. 20 mM dissolved in acetone-d6 or methanol-d4. Two-
dimensional homonuclear (COSY, NOESY) and heteronuclear
(HSQC, HMBC) NMR experiments with gradients were used to
structurally elucidate rosuvastatin analogues. 2D NOESY experi-
mentswere performed using a mixing time of 200 ms, which ensures
the operation in the initial linear part of the NOESY buildup curve.
[a
]
25 þ30.8 [c 0.25, MeOH]; nmax (KBr) 3426, 2969, 2932,1605,1549,
D
1511, 1439, 1394, 1370, 1336, 1228, 1155, 963, 771, 575, 521,
508 cmꢂ1
; dH (600 MHz, methanol-d4) 7.79 (2H, b, 4-FeC6H4), 7.17
(2H, b, 4-FeC6H4), 6.64 (1H, b, H-7), 5.62 (1H, b, H-6), 4.53 (2H, b,
OH ꢃ2), 3.84 (1H, b, H-5), 3.79 (1H, b, H-3), 3.55 (3H, s, NeCH3),
3.52 (3H, s, SO2eCH3), 3.38 (1H, b, CH(CH3)2), 2.01 (1H, b, H-2a),
1.96 (1H, b, H-2b),1.30 (3H, b, CH(CH3)2),1.28 (3H, b, CH(CH3)2),1.27
(1H, b, H-4a), 0.25 ppm (1H, b, H-4b); dC (75 MHz, methanol-d4)
181.6 (C-1), 176.8 (C-60), 164.9 (C-40), 164.9 (d, JCF 249.2 Hz, 4-
FeC6H4), 159.4 (C-20), 137.5 (C-6), 135.9 (d, JCF 3.4 Hz, 4-FeC6H4),
133.4 (d, JCF 8.4 Hz, 4-FeC6H4), 124.8 (C-7), 121.2 (C-50), 116.1 (d, JCF
21.8 Hz, 4-FeC6H4), 69.4 (C-3), 68.4 (C-5), 44.8 (C-2), 42.4 (C-4),
42.3 (SO2eCH3), 33.8 (NeCH3 and CH(CH3)2), 22.7 (CH(CH3)2),
22.0 ppm (CH(CH3)2); HRMS (ESI): MHþ, found: 482.1750.
C22H29FN3O6S requires 482.1756.
4.3. Ab initio calculations
Initial structures were generated by Chem3D Pro 10.0 software
and energy minimization at B3LYP/6-311þG(d,p) level was per-
formed without any constraints for anti orientation along the
C5eC6 bond using Gaussian 09.25 Torsion angles
q
[H5eC5eC6eH6] and
energetic changes induced by reorientation. The relative energy
profile of the torsion angles and
were calculated with 30ꢀ res-
olution, where orientations were restrained along
f
[C60eC50eC7eH7] were defined to follow
q
f
[H5eC5eC6eH6] and [C60eC50eC7eH7] torsion angles, re-
spectively, while other degrees of freedom were freely optimized.
Frequency calculations verified that the optimized geometries at
(local) minima were stable points on the potential energy surface.
Acknowledgements
This work was supported by the Slovenian Research Agency
(ARRS, Grant Nos. P1-0242 and J1-4020), EU FP7 projects with

6268
J. Fabris et al. / Tetrahedron 69 (2013) 6262e6268
9. Ferguson, E.; McNally, W.; Bocan, T. M. A.; Uhlendorf, P. D.; Krause, B. R.;
acronyms EAST-NMR (Grant No. 228461) and Bio-NMR (Grant No.
261863). We gratefully acknowledge Dr. I. Gazic Smilovic for valu-
able discussions; L. Kolenc, S. Borisek and M. Borisek for assistance
in some analytical work; Dr. M. Crnugelj, A. Gacesa and M. Friedrich
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for acquiring some NMR spectra; Dr. D. Urankar and Prof. J. Kosmrlj
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the Republic of Slovenia (TIA) for young researcher fellowship (MR-
10/75). Operation part financed by the European Union, European
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Supplementary data
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Supplementary data associated with this article can be found in
the online version. It contains 1H NMR spectrum of Z-3 in metha-
nol-d4 at 328 K, freely optimized structure of Z-2 at the B3LYP/6-
311þG(d,p) level of theory with anti orientation along C5eC6
bond, 1H and 13C NMR spectra for Z-2 and Z-3, Cartesian co-
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