Enantiomeric 4-Acylamino-6-alkyloxy-2 Alkylthiopyrimidines As Potential A3Adenosine Receptor Antagonists: HPLC Chiral Resolution and Absolute Configuration Assignment by a Full Set of Chiroptical Spectroscopy

Article abstract of DOI:10.1002/chir.22599

The chiral separation of enantiomeric couples of three potential A₃adenosine receptor antagonists: (R/S)-N-(6-(1-phenylethoxy)-2-(propylthio)pyrimidin-4-yl)acetamide (1), (R/S)-N-(2-(1-phenylethylthio)-6-propoxypyrimidin-4-yl)acetamide (2), and

Full text of DOI:10.1002/chir.22599

CHIRALITY 28:434–440 (2016)
Enantiomeric 4-Acylamino-6-alkyloxy-2 Alkylthiopyrimidines As
Potential A3 Adenosine Receptor Antagonists: HPLC Chiral Resolution
and Absolute Configuration Assignment
by a Full Set of Chiroptical Spectroscopy
DANIELA ROSSI,1 RITA NASTI,1 ANNAMARIA MARRA,1 SILVIA MENEGHINI,1,2 GIUSEPPE MAZZEO,2 GIOVANNA LONGHI,2
MAURIZIO MEMO,2 BARBARA COSIMELLI,3 GIOVANNI GRECO,3 ETTORE NOVELLINO,3 FEDERICO DA SETTIMO,4
4
4
2
*
1**
CLAUDIA MARTINI, SABRINA TALIANI, SERGIO ABBATE, AND SIMONA COLLINA
1Dipartimento di Scienze del Farmaco, Università di Pavia, Pavia, Italy
2Dipartimento di Medicina Molecolare e Traslazionale, Università di Brescia, Brescia, Italy
3Dipartimento di Farmacia, Università di Napoli “Federico II”, Napoli, Italy
4Dipartimento di Farmacia, Università di Pisa, Pisa, Italy
ABSTRACT
The chiral separation of enantiomeric couples of three potential A3 adenosine re-
ceptor antagonists: (R/S)-N-(6-(1-phenylethoxy)-2-(propylthio)pyrimidin-4-yl)acetamide (1), (R/
S)-N-(2-(1-phenylethylthio)-6-propoxypyrimidin-4-yl)acetamide (2), and (R/S)-N-(2-(benzylthio)-
6-sec-butoxypyrimidin-4-yl)acetamide (3) was achieved by high-performance liquid chromatog-
raphy (HPLC). Three types of chiroptical spectroscopies, namely, optical rotatory dispersion
(ORD), electronic circular dichroism (ECD), and vibrational circular dichroism (VCD), were ap-
plied to enantiomeric compounds. Through comparison with Density Functional Theory (DFT)
calculations, encompassing extensive conformational analysis, full assignment of the absolute
configuration (AC) for the three sets of compounds was obtained. Chirality 28:434–440,
2016. © 2016 Wiley Periodicals, Inc.
KEY WORDS: A3 adenosine receptor antagonists; chiral HPLC; optical rotatory dispersion
(ORD); electronic circular dichroism (ECD); vibrational circular dichroism
(VCD); Density Functional Theory (DFT); absolute configuration assignment
In mammals, adenosine acts as a transmitter by binding to
four subtypes of G-protein-coupled-receptors (GPCRs),
namely, the A1, A2A, A2B, and the A3 adenosine receptors
(ARs).1,2 Activation of ARs by agonists, such as adenosine
itself or synthetic ligands, leads to different intracellular
events starting with inhibition (A1 and A3) or stimulation
(A2A and A2B) of adenylate cyclase, stimulation of phospholi-
pase C (A1, A2B, and A3), activation of potassium channels,
and inhibition of calcium channels (A1).2 Different from A1,
7.5 nM (Ia) and 525 nM (Ib) towards A3 AR and excellent
selectivity over A1 and A2A ARs.
Recently, we synthesized the chiral pyrimidines 1-3
(Fig. 1), formally derived from previously reported pyrimi-
dines Ia and Ib by insertion of a methyl group on the exocy-
clic carbon atom attached to the oxygen or the sulfur
(unpublished results). Specifically, 1 is a methyl derivative
of Ia, whereas 2 and 3 are methyl derivatives of Ib. We rea-
soned that the new compounds, bearing branched substitu-
ents, more lipophilic, and less flexible with respect to their
desmethyl counterparts, might be endowed with improved
potency and selectivity as A3 AR antagonists. As a conse-
quence, a stereogenic center is present in compounds 1-3.
To study the influence of chirality on the putative A3 AR an-
tagonist properties of structures 1-3, we performed the chiral
resolution of racemic 1-3 and assigned the absolute configu-
ration to pure enantiomers. These tasks were achieved by
using chiral high-performance liquid chromatography
(HPLC) and chiroptical spectroscopy methods combined
with Density Functional Theory (DFT) calculations. In detail,
on the basis of our previous experience we resolved enantio-
meric 1-3 via enantioselective HPLC on chiral stationary
phases (CSPs), this approach being an effective way for both
the analytical and the preparative separations of chiral
A
2A, and A2B ARs, A3 AR exhibits significant differences in pri-
mary amino acid sequence homology (74%) between human
and rat.3 A3 AR can be found in human lungs, liver, kidneys,
heart, brain, uterus, eyes, and testes,4 where it is involved in
a variety of physiological processes, including cell cycle
regulation and cell growth.5 Given its key role, A3 AR is
considered an attractive target for pharmacological therapies
by medicinal chemists.6,7 A3 AR agonists may be useful
as cardioprotective and cerebroprotective agents, as
antiinflammatory and immunosuppressive agents, as cyto-
statics and chemoprotective compounds in cancer therapy.
A3 AR antagonists might be employed for the acute treatment
of stroke, for glaucoma, and also as antiasthmatic and
antiallergic drugs. The aforementioned potential clinical
applications of A3 AR ligands have been recently reviewed
by Borea et al.6 Recently, some of us have been involved in
the synthesis and biological evaluation of novel classes of an-
tagonists at the human A3 AR.8–11 Among those investigated,
4-acylamino-6-alkyloxy-2 alkylthiopyrimidines of general for-
mula I stand out for ease of synthesis (Scheme 1), drug like-
ness, potency, and selectivity for the target receptor.10 In this
class, compounds Ia and Ib (Scheme 1) exhibit Ki values of
© 2016 Wiley Periodicals, Inc.
*Correspondence to: Prof. S. Collina, Dipartimento di Scienze del Farmaco,
Università di Pavia, Viale Taramelli 12, 27100 Pavia, Italy. E-mail: simona.
collina@unipv.it; S. Abbate, Dipartimento di Medicina Molecolare
e
Traslazionale, Università di Brescia, Brescia, Italy. E-mail: sergio.abbate@unibs.it
Received for publication 4 December 2015; Accepted 1 March 2016
DOI: 10.1002/chir.22599
Published online 20 April 2016 in Wiley Online Library
(wileyonlinelibrary.com).

HPLC CHIRAL RESOLUTION AND ABSOLUTE CONFIGURATION ASSIGNMENT
435
Scheme 1. Synthesis of 4-acylamino-6-alkyloxy-2-alkylthiopyrimidines (I). Reagents and conditions: (i)methyl iodide or (phenyl)alkylbromide, NaOH 1M; (ii)
methyl iodide or (substituted-phenyl)alkyl bromide, anhydrous DMF, excess K2CO3; (iii) acetic or propionic anhydride, catalytic conc. H2SO4.
Fig. 1. Structures of the compounds studied in this work.
compounds.12–14 We carried out the absolute configuration
(3,5-dimethyl-phenylcarbamate), amylose tris[(S)-α-methylbenzylcarbamate],
cellulose tris(3,5-dimethylphenyl carbamate), and cellulose tris-(4-
methylbenzoate), respectively. A Hamilton (Reno, NV) syringe (syringe
volume: 2.5 mL; loop: 2 mL) was employed for (semi)-preparative chiral
resolutions. Chromatogram acquisitions and elaborations were performed
using the Borwin PDA and Borwin Chromatograph software.
(AC) assignment study of 1-3 resolved enantiomers by a full
set of chiroptical spectroscopies and comparison with calcu-
lated spectra obtained by ab initio methods (DFT-).15–17 Thus,
optical rotatory dispersion (ORD), electronic circular dichro-
ism (ECD), and vibrational circular dichroism (VCD) spectra
were recorded and compared to the calculated ones. The
combined use of the three techniques is particularly advised
for the AC assigned of flexible molecules, like drugs and nat-
Compound Characterization
Optical rotation values were measured on a Jasco photoelectric polar-
imeter DIP 1000 using a 0.5 dm cell and a sodium and mercury lamp
ural products, which had been treated previously by the use
of separate forms of chiroptical methods.18–30 Overall, this ar-
(λ = 589 nm, 435 nm, 405 nm); sample concentration values (c) are given
in 10-2g mL-1. Optical data are reported in Supplementary Table SI-2.
ticle describes the work carried out to separate the enantio-
meric couples for 1-3 and to assign their absolute
configurations in order to understand how chirality may influ-
Nuclear magnetic resonance spectra were recorded on a Varian (Palo
Alto, CA) Inova 500 spectrometer operating at 500 MHz for the proton
and 125 MHz for the carbon (compound 1) or on a Varian Mercury 400
spectrometer operating at 400 MHz for the proton and 100 MHz for the
carbon (compounds 2 and 3) in DMSO-d6 solution.
ence their interaction with human A3 AR.
(+)-R-N-[6-(1-Phenylethoxy)-2-(propylsulfanyl)pyrimidin-4-yl]acetamide
[(+)-R-1]: 1H-NMR: 10.70 (bs, 1H, NH); 7.39–7.32 (m, 5H, H-Ar); 7.14 (s,
1H, H-5); 6.12 (q, 1H, J = 6.2, OCH) 2.96 (m, 2H, SCH2); 2.07 (s, 3H,
CH3); 1.60-1.54 (m, 5H, CH2 and CH3); 0.94 (t, 3H, J = 7.3, CH3).13C-
NMR: 170.5; 169.6; 169.1; 158.7; 142.3; 128.4; 125.6; 89.8; 73.9; 31.7;
24.0; 24.0; 22.8; 22.4; 13.2.
(-)-S-N-[6-(1-Phenylethoxy)-2-(propylsulfanyl)pyrimidin-4-yl]acetamide
[(-)-S-1]: the 1H-NMR spectrum is identical to that of the correspond-
ing enantiomer.
MATERIALS AND METHODS
Chiral Chromatographic Resolution
Chromatographic resolution was carried out at room temperature on a
Jasco (Tokyo, Japan) system consisting of a PU-1580 pump, 851-AS
autosampler, and MD-1510 Photo Diode Array (PDA) detector. In order
to identify the best conditions to be properly scaled up, analytical screen-
ing was performed on the following columns: Chiralpak AD
(250 mm × 4.6 mm, 5 μm), Chiralpak AS-H (250 mm × 4.6 mm, 5 μm),
Chiralcel OD-H (150 mm × 4.6, 5 μm), and Chiralcel OJ-H
(150 mm × 4.6 mm, 5 μm) from Daicel Chemical Industries (Tokyo, Ja-
pan). The chiral stationary phases of these columns were amylose tris
(+)-R-N-{2-[(1-Phenylethyl)sulfanyl]-6-propoxypyrimidin-4-yl}acetamide
[(+)-R-2]: 1H-NMR: 10.73 (bs, 1H, NH); 7.48–7.44 (m, 2H, H-Ar); 7.36-
7.31 (m, 2H, H-Ar); 7.27-7.23 (m, 1H, H-Ar); 7.08 (s, 1H, H-5); 5.00 (q,
Chirality DOI 10.1002/chir

436
ROSSI ET AL.
1H, J = 7.1, SCH); 4.29–4.17 (m, 2H, OCH2); 2.09 (s, 3H, CH3); 1.72-1.62
(m, 5H, CH2 and CH3); 0.94 (t, 3H, J = 7.4, CH3). 13C-NMR: 170.5; 170.0;
169.1; 158.6; 143.0; 128.5; 127.2; 127.1; 89.5; 68.0; 43.5; 24.1; 22.3; 21.6;
10.3.
(-)-S-N-{2-[(1-Phenylethyl)sulfanyl]-6-propoxypyrimidin-4-yl}acetamide
[(-)-S-2]: the 1H-NMR spectrum is identical to that of the correspond-
ing enantiomer.
(+)-R-N-[2-(Benzylsulfanyl)-6-(butan-2-yloxy)pyrimidin-4-yl]acet-
amide [(+)-R-3]: 1H-NMR: 10.73 (bs, 1H, NH); 7.44–7.41 (m, 2H, H-
Ar); 7.34–7.29 (m, 2H, H-Ar); 7.27-7.24 (m, 1H, H-Ar); 7.07 (s, 1H, H-
5); 5.15-5.06 (m, 1H, OCH); 4.39 (s, 2H, SCH2); 2.09 (s, 3H, CH3);
1.70-1.52 (m, 2H, CH2); 1.22 (d, 3H, J = 6.2, CH3); 0.86 (t, 3H, J = 7.4,
CH3). 13C-NMR: 170,5; 169.7; 169.1; 158.6; 137.9; 128.7; 128.4; 127.0;
89.9; 73.8; 33.9; 28.2; 24.1; 19.1; 9.5.
(-)-S-N-[2-(Benzylsulfanyl)-6-(butan-2-yloxy)pyrimidin-4-yl]acetamide [(-)-S-
3]: the 1H-NMR spectrum is identical to that of the corresponding
enantiomer.
RESULTS AND DISCUSSION
Chiral Resolution
The approach we followed for obtaining enantiopure 1-3 is
chiral chromatographic resolution (HPLC). This methodol-
ogy is suitable both for analytical and preparative purposes
and is particularly useful in the early stage of the drug discov-
ery processes, when small-scale separation is needed and
both enantiomers have to be investigated.
As there is currently no way to predict which column–
mobile phase combination will provide a separation of the de-
sired product, the development process generally involves
screening a number of chiral stationary phases and potential
mobile phases in a systematic scheme. Based on our experi-
ence, for primary HPLC screening we normally use the sets
of columns and mobile phases shown in Table SI-1. If this ini-
tial screening is not successful, typically we move to a second-
ary screen, where the lesser-used columns and solvents are
employed, again in a similar process. Accordingly, to find a
baseline separation quickly, in this work the polysaccharide-
based CSPs of Table SI-1 were initially screened. Moreover,
the design of the experiments took into account the good sol-
ubility of 1-3 in alcohols. For this reason the first set of exper-
iments was performed on the considered columns, eluting
with just methanol.14
As a result of a first screening with pure methanol, it was
found that Chiralcel OJ-H (4.6 mm × 150 mm, 5 μm) lead to rel-
atively short retention times, high enantioselectivity, and high
resolution for compound 2 (α = 3.3, Rs = 6.7) and 3 (α = 1.9,
Rs = 4.1). Accordingly, these experimental conditions were se-
lected for the scale-up to (semi)-preparative scale. As regards
compound 1, a very poor resolution was obtained (α = 1.33,
Rs = 0.3) and therefore it was necessary to extend the screen-
ing also to different mobile phases (Table SI-3). Baseline sep-
aration, high enantioselectivity, and resolution were obtained
eluting with heptane/ethanol 95/5 (v/v) (α = 1.5, Rs = 2.4).
Chromatographic profiles of racemates, together with experi-
mental conditions, are given in Fig. 2 and Table 1.
The developed analytical methods were suitably trans-
ferred to the semipreparative scale, employing a Chiralcel
OJ-H column (10 mm x 250 mm, 5 μm). In this way batches
of about 40 mg of racemic 1-2 and of 25 mg of racemic 3 were
processed, affording enantiomeric 1-3 in good overall yields
(81.8%, 93.1%, and 97.2%, respectively) (Fig. SI-1). The optical
rotatory power of the obtained enantiomers, their enantio-
meric excess, determined by HPLC and recovery yields are
summarized in Table 2. To assign the absolute configuration
of the enantiomers of 1-3 we performed ORD analysis and for
1 and 2 we recorded ECD and VCD spectra. The identity of
the resolved enantiomers 1-3 was confirmed by 1H-NMR
analysis.
ORD Curves, ECD, and VCD Spectra
The DIP 1000-type photoelectric polarimeter from Jasco was used for
[α] measurements, which were carried out at room temperature using
sodium and mercury sources at three different wavelengths (589, 435,
405 nm) and CG3-50 cell (3.5 mm I.D. × 50 mm pathlength). ORD values
were recorded both in chloroform and in methanol for 2 and 3 and just
in methanol for 1. In all cases ORD measurements were carried out for
the first eluted enantiomer at all available wavelengths. The ORD and
samples concentration values are reported in Table SI-2.
UV and ECD spectra were obtained with a Jasco 815SE apparatus in
the range 350–180 nm, using 0.1 mm pathlength quartz cuvettes under
the following conditions: 0.5 s integration time, 200 nm/min scan speed,
1 nm bandpass, 10 accumulations. UV/ECD spectra were recorded in
acetonitrile for all samples (2.4 · 10-3 M and 3.6 · 10-3 M for 1, first
eluted and second eluted enantiomers respectively; 3.02 · 10-3 M and
3.32 · 10-3 M for 2, first eluted and second eluted enantiomer respec-
tively; for 3 no reliable UV/ECD spectrum was taken, for paucity of
compounds).
IR and VCD spectra were collected in the range from 2000 to 850 cm-1
with a Jasco FVS6000 FTIR instrument equipped with a liquid N2-cooled
MCT detector. A 200 μm pathlength BaF2 cell was used with the follow-
ing conditions: 2000 accumulations, 4 cm-1 resolution. IR/VCD spectra
were recorded in CCl4 solution for 2 and 3 (0.068 M and 0.070 M for 2,
first eluted and second eluted compound, respectively; for 3 no reliable
VCD data were recorded, see above) and in deuterated methanol solution
for 1 (0.040 M both eluted compounds).
Computational Detail
Preliminary conformational analysis was performed for (R)-1-3 with
the MMFF94s molecular mechanics (MM) force field: 44 conformers
for 1, 55 for 2 and 43 for 3 were found below 10 Kcal/mol. These sets
of conformers were fully optimized at the B3LYP/6-31G* level using the
Gaussian09 package.31 Conformers within 2 kcal/mol in relative free
energy with respect to the most stable one were treated at the
B3LYP/TZVP level of theory. Calculated free energies were used to
determine the Boltzmann population factor of the conformers at
298.15 K.
VCD spectra were calculated for 1 and 2 at the same B3LYP/TZVP
level. The IEF-PCM approach was also employed in order to optimize
and calculate VCD spectra of deuterated species of 1 (in N-H bond) in
CD3OD solution. For 2 no deuteration was adopted. Calculations of
ORD and ECD spectra were performed by the TD-DFT approach with
allowance of 30 excited states using the Gaussian Coulomb-attenuated
CAM-B3LYP functional and aug-cc-pVDZ as basis set. Calculated VCD
spectra were simulated with the Gaussian package31 assigning 8 cm-1
Lorentzian bands shape to all vibrational transitions. ECD bands were
assumed to have Gaussian line shape with 0.2 eV bandwidth. Theoretical
ORD, ECD, and VCD spectra were obtained as weighted averages by the
Boltzmann population factors.
Analysis of ORD Data
Let us begin our analysis from compound 2, which was re-
vealed to be simple to interpret from the computational point
of view. The 13 calculated conformers of (R)-2 with popula-
tion factors above 2% exhibit positive OR values at three differ-
ent wavelengths and show the same ORD trend (see Fig. SI-2,
middle panel). The weighted average theoretical ORD curve
shows the same magnitude, but opposite sign as compared
with the measured curves of the first eluted enantiomer
(Fig. 3, middle panel). From this we deduce that the second
eluted enantiomer has (R)-AC. Unlike for 2, the calculated
Chirality DOI 10.1002/chir

HPLC CHIRAL RESOLUTION AND ABSOLUTE CONFIGURATION ASSIGNMENT
437
Fig. 2. Analytical chromatographic profiles for (R/S)-1, (R/S)-2, and (R/S)-3 (from left to right) obtained with Chiralcel OJ-H (4.6 mm × 150 mm, 5 μm); eluent
heptane/ethanol 95/5 (v/v) for 1 and 100% MeOH for 2 and 3; flow rate: 1 mL/min; conc.: 1 mg/mL; injection volume: 10 μL; detection at 250, 240, and 254 nm,
respectively.
TABLE 1. (Semi)-preparative resolution of racemic 1-3 on a Chiralcel OJ-H column (10 mm x 250 mm, 5 μm)
Compound
Eluent
tA (min)
tB (min)
Amount processed (mg)
Concentration (mg/mL)
Number of cycles
1
2
3
n-heptane/EtOH (95:5 v/v)
MeOH
MeOH
13.87
12.24
12.79
20.25
32.32
22.15
38
40
25
2.0
5.0
5.0
10
4
3
Flow rate: 4.0 mL/min; injection volume: 2.0 mL.
TABLE 2. Chiroptical characteristics (OR, ee, and amount) of the separated enantiomers for 1-3
22
Compound
λ
C
½aꢀλ (MeOH)
eea
Isolated amount(mg)
Yield (%)
(+)-1
(-)-1
(+)-2
(-)-2
(+)-3
(-)-3
435
435
589
589
435
435
0.1%
0.1%
0.2%
0.2%
0.5%
0.5%
+40.4
-40.4
99.9%b
99.9%b
99.9%c
99.9%c
99.9%c
99.9%c
15.2
15.9
18.5
18.7
12.0
12.3
40.0
41.8
46.3
46.8
48.0
49.2
-214.7
+214
+56.2
-56.4
aDetermined on Chiralcel OJ-H (4.6 mm x 150 mm, dp 5 μm) and eluting with
bn-heptane 95/5 EtOH (v/v) or
c100% methanol.
Fig. 3. Comparison of average calculated (CAM-B3LYP/aug-cc-pVDZ on DFT/B3LYP/TZVP input geometries) ORD curves for (R)-1-3 (green line) and exper-
imental (solid blue line, methanol, and dotted blue line, chloroform) for the first eluted couple of compounds 1-3. In the calculated (R)-3 ORD curve average the
conformer in Figure SI-3 is excluded (see text and Fig. SI-2).
(R)-1 ORD curves do not present the same sign and trend for
the 15 conformers above 3% population (Fig. SI-2, left panel);
the calculated values appear a bit too large with respect to ex-
periments. Anyway, the weighted average calculated ORD
curve matches the trend and the sign of the experimental
one, which indicates that the first eluted enantiomer has
(R)-AC (Fig. 3, left panel). Figure SI-2 shows that for good
prediction of the ORD curves all calculated conformers are
necessary, even those with the wrong sign. For (R)-3, we
noticed that the ORD curves of the 15 calculated conformers
with population above 0.8% (Fig. SI-2, right panel) present the
same trend except for one of them reported in Figure SI-3
(conformer 3 g).
Even if its population factor is rather low (4.4%), the calculated
values of [α] at three λ are very high; so this conformer nega-
tively influences the trend of the average calculated ORD curve.
If conformer 3g is removed, the calculated ORD improves con-
siderably as compared to experiment. Such conformer differs
from the other ones by the thio-phenyl group being distorted
in such a way as to be closer to the central pyrimidine moiety.
However, similar to compound 1, ORD analysis suggests that
the first eluted enantiomer of 3 has (R)-configuration.
Chirality DOI 10.1002/chir

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ROSSI ET AL.
Fig. 4. Comparison of average calculated (CAM-B3LYP on DFT/B3LYP/TZVP input geometries) ECD spectra for (R)-1-2 (green line) and experimental ones
(blue line, first eluted, and red line, second eluted).
Analysis of ECD Spectra
PCM model, which allows taking into account the solvent in-
fluence. The obtained spectra were similar to the ones calcu-
lated in gas phase. Thus, theoretical ECD spectra of (R) and
(S) enantiomers of 1 and 2 are in agreement with the
experimental ones of the first eluted enantiomer of 1 and 2,
respectively. These results are in agreement with those
obtained performing ORD experiments and therefore the
AC was undoubtedly assigned (Fig. 4). In the case of the first
eluted enantiomer of 3, the ECD spectra registered had a
poor signal-to-noise ratio and therefore they were not
considered.
Calculated UV absorption spectra of (R)-1 and (R)-2 exhibit
five and, respectively, three important bands which nicely
correlate with all the experimental ones (Fig. SI-4). For both
1 and 2 the experimental ECD spectra of the two pairs of en-
antiomers are excellent mirror images of each other (Fig. SI-
5): the ECD experimental spectra of 1 and 2 are in good
agreement with the calculated average ECD spectra (Fig. 4).
Compared to (R)-2, the ECD spectrum of (R)-1 has the same
strong positive 190 nm feature, while the central broad feature
at 230 nm is negative in 1, and positive in 2. Finally, at about
280 nm, a positive feature is present in both molecules, in 1
being weak and broad, while in 2 being a strong shoulder
of a 250 nm band. For 1, the calculated average ECD spec-
trum is in good agreement with the experimental one; the
small features at high wavelengths are calculated at shorter
wavelengths compared to those used for the experiment.
For 2, the situation is slightly worse. To improve the quality
of ECD and UV calculated spectra of (R)-2 we applied the
Analysis of VCD Spectra
Since VCD for compound 1 was run in CD3OD, where it
shows good solubility, we ran a calculation for (R)-1 with N-
H replaced by N-D: the average IR calculated spectrum fits
rather well the experimental one, especially for the two peaks
at ~1565–1544 cm-1 and the two peaks at ~1435–1407 cm-1
(Fig. 5, left panel). VCD experimental spectra present few
Fig. 5. Comparison of experimental IR and VCD spectra (blue, first eluted enantiomer, and red, second eluted enantiomer) of 1-2 with calculated (green) IR and
VCD average spectra for (R)-1-2. 1 and 2 experimental spectra are the semi-difference between enantiomers. 1 refers to solution in CD3OD, while data for 2 was
taken in CCl4.
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HPLC CHIRAL RESOLUTION AND ABSOLUTE CONFIGURATION ASSIGNMENT
439
Fig. 6. 3D-structure of the most populated conformers for molecules 1-3 with (R)-configuration calculated in vacuo.
significant bands that are matched by calculations, in sign
even if not 100% in magnitude. This may be related to the flex-
ibility of the molecule. The negative VCD band of the first
eluted enantiomer, at 1567 cm-1, can be associated with the
calculated one at 1558 cm-1, originated by the aromatic C = C
and C = N stretching (Fig. 5), while the IR peak around
1693 cm-1 can be related to C = O stretching. VCD confirms
the same conclusion arrived at by the ORD and ECD tech-
niques. We endeavored to measure IR and VCD spectra of
1 first eluted enantiomer in CCl4 solution, getting as close
as possible to the 0.04 M concentration (1 is not very soluble
in CCl4). The results are given in Figure SI-6 and a CD3OD
solution, namely, the (R)-1 enantiomer corresponds to the
first eluted sample. For 2 the whole IR calculated spectrum
provides good prediction of all the experimental peaks, while
only few VCD experimental bands appear (Fig. 5, right panel)
and are reproduced in sign and magnitude by the average cal-
culated spectra of (R)-2. Molecular flexibility is so important
that the VCD spectrum of one conformer is cancelled by the
VCD of another one. The AC is the same predicted by ORD
and ECD analyses. This assignment was made on the basis
of the first three bands (IR peaks: 1427, 1284, and 1210 cm-1,
respectively), which can be attributed to CH2 wagging
(1422 cm-1) and C* bending motions (1285 and 1220 cm-1), re-
spectively. The whole IR spectra is reported in Figure SI-7.
The IR and VCD spectra recorded in other solvents (CDCl3
and CS2) did not give rise to results. Regarding the VCD spec-
tra of (–)-3, a poor signal-to-noise ratio was obtained, as
already evidenced in the case of ECD spectra.
feasible if a systematic investigation of possible conformers
had not been conducted. Indeed, in order to establish AC,
one has to carry out a detailed and reliable conformational
analysis aimed at understanding the arrangement of the sub-
stituents in the different conformers. In Figure 6 we report
the five most populated conformers for 1 and the three most
populated conformers for 2 and 3 (see also Table SI-4). All in-
vestigated conformers present the amide group R” bound to
the pyrimidine ring, always in the same position and on the
same plane.
CONCLUSION
In the present article we have described the isolation of 1-3
pure enantiomers and the assignment of their absolute configu-
ration in order to investigate the role of chirality on the potential
A3 AR antagonist properties of these compounds. To the best of
our knowledge, pharmacological enantioselectivity of A3 AR
antagonists has been previously investigated only by Jacobson
and colleagues.32
An enantioselective HPLC procedure using a Chiralcel OJ-H
column was successfully applied to the enantioresolution of
1-3, obtaining both enantiomers with an enantiomeric excess
of about 99.9% and in amounts suitable for absolute configura-
tion assignment and biological investigation. In line with emerg-
ing works, we employed a full set of chiroptical spectroscopies
to determine the AC of the resolved enantiomers.18,25,29 In
detail, AC of enantiomeric 1-3 was assigned by comparing ex-
perimental and calculated ORD curves and, in the case of
enantiomeric 1 and 2, also comparing ECD and VCD spectra
with the DFT calculated ones. It has to be underlined that all
the applied techniques gave rise to converging conclusions.
However, this behavior is not universal and VCD sometimes
Chirality DOI 10.1002/chir
Conformational Analysis
The previously discussed comparison of experimental and
calculated ORD, ECD, and VCD spectra would not have been

440
ROSSI ET AL.
arrives at safer conclusions.18,25,33 To sum up, the first eluted en-
antiomers are (R)-1, (S)-2, and (R)-3, and the second eluted
ones are (S)-1, (R)-2, and (S)-3, respectively. Overall, this
work provided enantiomerically pure 1-3 currently being
employed by us to study the influence of chirality on the interac-
tion of this class of compounds with human A3 AR. The results
of these efforts will be reported in due course.
15. Nafie LA. Vibrational optical activity, principles and applications. New
York; John Wiley & Sons: 2011.
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tool for biomolecular structural determination. Fresenius J Anal Chem
2000;366:727–734.
17. Abbate S, Lebon F, Longhi G, Morelli CF, Ubiali D, Speranza G. Vibrational and
electronic circular dichroism spectroscopies and DFT calculations for the as-
signment of the absolute configuration of hydroxysubstituted 2-tetralols. RSC
Adv 2012;2:10200–10208.
18. Polavarapu PL. A single chiroptical spectroscopic method may not be able
to establish the absolute configurations of diastereomers: dimethylesters
of hibiscus and garcinia acids. J Phys Chem A 2011;115:5665–5673.
19. Mazzeo G, Santoro E, Andolfi A, Cimmino A, Troselj P, Petrovic AG,
Superchi S, Evidente A, Berova N. Absolute configurations of fungal and
plant metabolites by chiroptical methods. ORD, ECD, and VCD studies
on phyllostin, scytolide, and oxysporone. J Nat Prod 2013;76:588–599.
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ACKNOWLEDGMENTS
We thank CINECA, via Raffaello Sanzio 4, 20090 Segrate, MI,
Italy, for granting us computer time.
SUPPORTING INFORMATION
Additional supporting information may be found in the
online version of this article at the publisher’s web-site.
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Chirality DOI 10.1002/chir