Crystallographic and NMR investigation of ergometrine and methylergometrine, two alkaloids from claviceps purpurea

Article abstract of DOI:10.3390/molecules25020331

Ergometrine and methylergometrine are two alkaloids that are used as maleate salts for the prevention and control of postpartum hemorrhage. Although the two molecules have been known for a long time, few and discordant crystallographic and NMR spectroscopic data are available in the literature. With the aim of providing more conclusive data, we performed a careful NMR study for the complete assignment of the ¹H, ¹³C, and ¹⁵N NMR signals of ergometrine, methylergometrine, and their maleate salts. This information allowed for a better definition of their conformational equilibria. In addition, the stereochemistry and the intermolecular interactions in the solid state of the two maleate salts were deeply investigated by means of single-crystal X-ray diffraction, showing the capability of these derivatives to act as both hydrogen-bond donors and acceptors, and evidencing a correlation between the number of intermolecular interactions and their different solubility.

Full text of DOI:10.3390/molecules25020331

molecules
Article
Crystallographic and NMR Investigation of
Ergometrine and Methylergometrine, Two Alkaloids
from Claviceps Purpurea
Fiorella Meneghetti 1 , Patrizia Ferraboschi 2 , Paride Grisenti 3, Shahrzad Reza Elahi 2,
Matteo Mori 1 and Samuele Ciceri 2,
*
1
Department of Pharmaceutical Sciences, University of Milan, via L. Mangiagalli, 25, 20133 Milano, Italy;
fiorella.meneghetti@unimi.it (F.M.); matteo.mori@unimi.it (M.M.)
Department of Medical Biotechnology and Translational Medicine, University of Milan, Via Saldini 50,
20133 Milano, Italy; patrizia.ferraboschi@unimi.it (P.F.); shahrzad.rezaelahi@gmail.com (S.R.E.)
Chemical-Pharmaceutical Consulting and IP Management, Viale Giovanni da Cermenate 58, 20141 Milano,
Italy; grisenti.paride60@gmail.com
2
3
*
Correspondence: samuele.ciceri@guest.unimi.it; Tel.: +39-02-50316052
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 11 December 2019; Accepted: 10 January 2020; Published: 14 January 2020
Abstract: Ergometrine and methylergometrine are two alkaloids that are used as maleate salts for the
prevention and control of postpartum hemorrhage. Although the two molecules have been known
for a long time, few and discordant crystallographic and NMR spectroscopic data are available in the
literature. With the aim of providing more conclusive data, we performed a careful NMR study for
the complete assignment of the 1H, 13C, and 15N NMR signals of ergometrine, methylergometrine,
and their maleate salts. This information allowed for a better definition of their conformational
equilibria. In addition, the stereochemistry and the intermolecular interactions in the solid state of the
two maleate salts were deeply investigated by means of single-crystal X-ray diffraction, showing the
capability of these derivatives to act as both hydrogen-bond donors and acceptors, and evidencing a
correlation between the number of intermolecular interactions and their different solubility.
Keywords: ergot; 9,10-unsaturated ergoline; alkaloids; 15N NMR; 13C NMR; 1H NMR; X-ray analysis;
oxytocic activity
1. Introduction
The fungus Claviceps purpurea (commonly known as ergot) is a plant pathogen that produces a
family of toxic alkaloids endowed with pharmacological properties [1,2]. These ergot alkaloids are
derivatives of the tetracyclic ergoline
substituent at the 8-position.
1
(Figure 1) and can be chemically classified by the nature of the
8
9
10
11
7
D
H
12
N
13
14
6
5
A
C
4
16
15
3
B
N
2
H
1
1
Figure 1. Structure of ergoline (1).
Molecules 2020, 25, 331; doi:10.3390/molecules25020331
www.mdpi.com/journal/molecules

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From a therapeutic point of view, the d-lysergic acid amides are in a prominent position among
the ergot alkaloids. The d-lysergic acid carries a carboxyl group at the 8 position (cis with respect
2
β
to the H-5) and a 9–10 double bond (Figure 2). The presence of the double bond is responsible for
the easy and spontaneous isomerization of the carboxyl substituent at the C-8 stereocenter, leading
to mixtures of d-lysergic and d-isolysergic acid
two stereocenters are present, namely at the 5 and 8 positions. The stereocenter at the 5 position
presents an R configuration, while the substituent at the 8 position can be either in the -configuration
(trans with respect to the H-5) or in the -configuration (cis with respect to the H-5). The -isomer is
-isomers are usually endowed with a
3. In the tetracyclic moiety of compounds 2 and 3,
α
β
α
usually indicated by the prefix iso- or by the ending –inine. The
significant biological activity.
β
Among the d-lysergic derivatives, the natural amide ergometrine (ergonovine)
4 and the
semisynthetic methylergometrine (methylergonovine) (Figure 2) are endowed with uterotonic
5
activity, and for this reason, their maleate salts are administered in the third stage of labor for the
prevention of postpartum hemorrhage [3,4].
Although ergometrine
starting from the lysergic azide, was published in 1943 [
studies are reported in the literature. In two 2017 Chinese patents [
ergometrine are reported but not assigned; in 1974 [
], the assigned 13C NMR (60 MHz) data were
reported. In the case of ergometrine maleate, the XRD and the single-crystal X-ray diffraction [10] are
4
was isolated in 1932 [5] and the first synthesis of methylergometrine
5,
6
], only a few spectroscopic and crystallographic
1
7,8], the H NMR data of the
4
9
reported, and only the 1H NMR (400 MHz) characterization is available [11]. Methylergometrine
5 was
analyzed by 1H NMR (values not assigned) in a 2014 WO patent [12]; the corresponding maleate was
characterized by XRD, IR, and DSC in a 2015 Chinese patent [13] and by synchrotron powder diffraction
data [14]. These incomplete (and sometimes contradictory) NMR and crystallographic analyses
prompted us to gather new and more conclusive data for the two amides,
epimerization of these compounds, we decided to expand our investigations to their maleate salts,
, as well. Indeed, ergometrine and methylergometrine are always obtained as mixtures of epimers,
4
and
5
. Considering the easy
6
and
7
4
5
while the corresponding maleate salts can be obtained as pure β-isomers by simple crystallization.
In a continuing effort to fully characterize molecules that exert important therapeutic
properties [15–17], a complete NMR (1H, 13C, and 15N) characterization of compounds
4, 5, 6,
and
and
7
7
was carried out. Moreover, a detailed crystallographic investigation was performed on
in order to explore their conformational features. In addition, we carefully analyzed the
6
hydrogen-bonding interactions of these molecules, as the earlier report did not account for their
supramolecular aggregation.
Figure 2. Structures of d-lysergic (2), d-isolysergic acid (3), ergometrine (4), methylergometrine (5),
ergometrine maleate (6), and methylergometrine maleate (7).

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2. Results and Discussion
Ergometrine
4
and methylergometrine
5
were obtained by treatment of the corresponding
commercially available maleates,
Materials and Methods section.
6 and 7, with sodium hydrogen carbonate as described in the
2.1. NMR Spectroscopy
The NMR study was carried out on
administered derivatives). Compounds
and
4
and
5 and on their maleate salts 6 and 7 (the clinically
4
and
5
were dissolved in DMSO-d6, while compounds
6
1
7
were dissolved in D2O. Although in both solvents the H NMR spectra of
6
and
7
present
broadened signals and a slight concentration dependence of chemical shifts, D2O was chosen in
these cases due to more resolved NMR spectra. 1H, 13C, and 15N resonances (Tables 1–3) were
unambiguously assigned combining information from 1D and 2D NMR (COSY, NOESY, HSQC, 1H-13C
HMBC, and 1H-15N HMBC) experiments.
Table 1. 1H NMR chemical shifts (ppm) a and coupling constants (Hz) b of compounds 4–7.
1H
4 (DMSO-d6)
5 (DMSO-d6)
6 (D2O)
7 (D2O)
H-1
H-2
10.69 (brs)
7.02 (brs)
2.44 (overlapped)
3.46 (dd, J = 14.9, 5.7)
3.00–2.93 (m)
10.70 (brs)
7.02 (brs, partially overlapped)
2.45 (overlapped)
−
−
7.15 (brs)
7.16 (brs)
H-4α
H-4β
H-5β
H-7β
H-7α
H-8α
H-9
2.89 (brt, J = 13.1)
3.69 (overlapped)
3.92 (br)
3.47 (brt, 10.8)
3.77 (br)
2.91 (brt, J = 13.1)
3.73 (overlapped)
3.99 (br)
3.49 (brt, J = 11.7)
3.79 (br)
3.46 (dd, J = 14.7, 5.5)
3.00–2.93 (m)
2.50 (overlapped with DMSO) 2.50 (overlapped with DMSO)
3.00 (dd, J = 11.0, 5.0)
3.40–3.36 (m, overlapped)
6.34 (brs)
3.02 (dd, J = 11.2, 5.3)
3.47–3.42 (m, overlapped)
6.35 (brs)
3.83 (br)
6.46 (brs)
3.89 (overlapped)
6.49 (brs)
7.03 (brdd, J = 8.2, 0.8,
partially overlapped)
7.06 (tripletoid m)
7.18 (dd, J = 7.4, 0.9)
2.45 (s)
H-12
7.04 (overlapped)
7.22 (d, J = 7.3)
7.23 (d, J = 7.1)
H-13
H-14
N-CH3
CH
7.06 (tripletoid m)
7.18 (dd, J = 6.9, 1.1)
2.44 (s)
7.26 (tripletoid m)
7.42 (d, J = 7.8)
3.11 (s)
4.10-4.02 (m)
7.27 (tripletoid m)
7.43 (d, J = 7.6)
3.13 (s)
3.86–3.76 (m)
1.05 (d, J = 6.6)
−
3.71–3.62 (m)
0.85 (t, J = 7.4)
1.39–1.26 (m)
1.66–1.53 (m)
3.37–3.30 (m)
3.42–3.38 (m)
4.68 (t, J = 5.7)
7.69 (d, J = 8.5)
3.94–3.84 (m)
0.95 (t, J = 7.1)
1.54–1.42 (m)
1.73–1.62 (m)
3.63 (dd, J = 11.7, 6.6)
3.73 (dd, J = 11.5, 4.6)
−
CH3
1.21 (d, J = 6.9)
CHaCH3
CHbCH3
CHaOH
CHbOH
CH2OH
CONH
CH=CH
maleate
−
−
−
3.32–3.25 (m)
3.43–3.36 (m)
4.72 (brs)
3.61 (dd, J = 11.5, 6.6)
3.71 (dd, J = 11.7, 6.0)
−
7.76 (d, J=7.8)
−
−
−
−
6.21 (s)
6.23 (s)
a Assignments from 1H–1H COSY, NOESY, and HSQC data. b Coupling constants were obtained by direct inspection
of the spectra. Error in the measured 1H-1H coupling constants was ± 0.5 Hz.
Table 2. 13C NMR chemical shifts (ppm) a of compounds 4–7.
13C
4 (DMSO-d6) 5 (DMSO-d6)
6 (D2O)
7 (D2O)
C-2
C-3
C-4
119.2
109.0
26.8
119.2
109.0
26.8
121.5
106.4
24.8
121.5
106.4
24.7
C-5
62.6
62.6
62.8
62.7
C-7
C-8
55.5
42.8
55.7
42.9
54.1
41.4
54.1 (overlapped)
41.5
C-9
120.3
135.1
127.4
111.0
122.2
109.7
133.8
125.8
120.4
135.0
127.5
111.0
122.3
109.7
133.9
125.8
118.7
132.6
125.1
113.2
124.0
112.2
134.3
125.7
118.7
132.6
125.2
113.2
124.0
112.2
134.3
125.7
C-10
C-11
C-12
C-13
C-14
C-15
C-16

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Table 2. Cont.
13C
4 (DMSO-d6) 5 (DMSO-d6)
6 (D2O)
7 (D2O)
N-CH3
CH
CH3
CH2CH3
CH2OH
CH=CH
maleate
COOH
CONH
43.4
46.5
17.2
−
43.4
52.1
10.5
23.7
63.0
42.1
48.3
16.5
−
42.0
54.2 (overlapped)
10.3
24.0
63.8
64.4
65.0
−
−
134.9
134.9
−
171.2
a
−
171.5
171.8
171.6
172.4
171.7
Assignments from HSQC and HMBC data.
Table 3. 15N NMR chemical shifts (ppm) a of compounds 4–7.
4 (DMSO-d6)
5 (DMSO-d6)
6 (D2O)
7 (D2O)
N-1
N-6
CONH
−251.1 (128.4)
−340.3 (39.2)
−255.5 (124.0)
−251.0 (128.5)
−340.2 (39.3)
−258.1 (121.4)
−253.3 (126.2)
−332.8 (46.7)
−248.3 (131.2)
−253.3 (126.2)
−332.7 (46.8)
−251.4 (128.1)
Assignments from 1H-15N HMBC data using nitromethane as the external reference (CH3NO2
δ = 0.00 ppm).
a
In parenthesis 15N chemical shifts referred to ammonia (δNH3 = −379.5 ppm respect to CH3NO2).
The proton assignments were accomplished using the general knowledge of chemical shift
dispersion with the aid of the COSY, HSQC, and NOESY experiments. Starting from the characteristic
resonance of H-9 (6.34, 6.35, 6.46, and 6.49 ppm for
the resonances of all the other protons, especially the resonance of H-8. The interpretation of the
1H NMR spectra of compounds
and was further facilitated by the resonances of exchangeable
protons H-1, CH2OH, and CONH.
The NOESY experiments were performed to integrate structural data with stereochemical
information. Despite the poorly resolved 2D spectra of NOESY experiments obtained for and
4, 5, 6, and 7, respectively), we were able to assign
4
5
6
7
due to the broad nature of the D-ring proton signals, some information about the stereochemistry
and conformation of the D-ring was collected by observing the NOE correlation of the methyl group
bound to the nitrogen atom at the 6 position. This methyl group interacts sterically with H-5
H-7 , H-7 , and H-4 , indicating—as previously described by Kidric et al. [18] for 9,10-unsatured
ergolines dissolved in solvents such as DMSO and water—that the D-ring was preferentially in its D1
8R half-chair conformation. The very weak NOE correlation of N–CH3 with H-8 and H-4 could
β,
β
α
β
α
α
be explained by the existence of a conformational equilibrium in solution between D1 8R and D2 8R
half-chair conformations (Figure 3). When the D-ring is in its D2 8R configuration, the methyl group is
in the
α position, which results in a feeble NOE effect. The existence of this conformational equilibrium
in aqueous solution also explains the broad nature of the C- and D-ring proton signals.
Figure 3. Possible conformations of ergoline D-ring in solution.
The analysis of NOESY spectra of the free bases,
4
and
5, gave us a clear picture of the spatial
interactions of protons at the 4 position with H-5 and of protons at the 7 position with H-8
β
α
. The NOE

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correlations of H-5
β
(3.00–2.93 ppm) with one of the two protons at the 4 position (3.46 ppm) and of
H-8
) allowed us to unequivocally identify H-4
between the H-8 and H-5 protons was a clue, but not an absolute proof, of their position on opposite
faces of the D-ring. When N-CH3 (2.44 ppm for ; 2.45 ppm for ) was irradiated, NOE enhancement
was observed for H-5 , suggesting that the analyzed and in DMSO-d6 were in their D1 8R half-chair
α
(3.40–3.36 ppm) with one of the two protons at the 7 position (3.00 ppm for
4 and 3.02 ppm for
5
β
and H-7 protons. The absence of spatial interactions
α
4
5
β
4
5
conformation. The CH, CH2, and CH3 carbon atoms were assigned based on chemical shift analysis
and confirmed by the HSQC experiment. In particular, the predicted deshielding of C-5 and C-7 was
observed, both near the nitrogen atom in position 6, further confirming the right 1H NMR assignment
of the protons bound to these carbon atoms. In the 13C NMR spectra of compounds
6 and 7, the broad
nature and low intensity of the C-4, C-8, N-CH3, C-7, C-5, and C-9 signals can be explained by the
previously reported conformational equilibrium between the D1 8R and D2 8R half-chair conformations.
The quaternary carbon atoms were unambiguously assigned using the information obtained from
1H-13C HMBC experiments (Table 4). Following this approach, the 13C resonance assignments of
were in accordance with the previously reported ones [9].
4
Table 4. Long range couplings of hydrogen with quaternary carbon atoms observed in the HMBC
spectra of compounds 4–7.
HMBC (C→H)
4 and 5 (DMSO-d6)
Quaternary Carbon
6 and 7 (D2O)
C-3
H-4α, H-4β, H-2, H-1
H-13, H-12, H-5, H-4α, H-4β
H-13, H-12, H-9
H-14, H-13, H-2, H-1
H-14, H-12, H-4β, H-2, H-1
H-4α, H-2
H-13, H-12, H-4α
H-13, H-9
H-13, H-12, H-2
H-14, H-12, H-4α, H-2
C-10
C-11
C-15
C-16
1H-15N HMBC experiments were performed in order to assign the resonances of the three
chemically different nitrogen atoms in the examined compounds and allowed us to confirm their
hypothesized structure. For compounds
H-2, H-14, and H-13, the amidic nitrogen (
protons present on the amidic moiety, and curiously but not unexpectedly, N-6 (
332.7 ppm for ) couples very weakly only with H-4 , which gives a well-resolved signal in the
1H NMR spectrum. For compounds
and , the indole nitrogen N-1 ( 251.1 ppm for 251.0 ppm
for ) couples with H-1, H-2, H-14, and H-13, the amidic nitrogen ( 255.5 ppm for 258.1 ppm for
340.3 ppm for 340.2 ppm for ) couples
6
and
7
, the indole nitrogen N-1 (
−
253.3 ppm) couples with
) couples with the
332.8 ppm for 6;
−
248.3 ppm for
6;
−
251.4 ppm for
7
−
−
7
α
4
5
−
4;
−
5
−
4;
−
5)
couples with the protons of the amidic moiety, and N-6 (
with N-CH3, H-7β, and H-5.
−
4
;
−
5
2.2. X-ray Analysis
Since the structure adopted by a given compound upon crystallization could exert a profound
effect on the solid-state properties of the system, we decided to perform a crystallographic analysis of
derivatives
6 and 7. Their X-ray molecular structures are presented, as ORTEP views [19], in Figure 4.

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Figure 4. ORTEP drawings [19] of
6 (left) and 7 (right), with the arbitrary atom numbering (ellipsoids
are at 40% probability and H atoms are represented as spheres of arbitrary radii).
The maleate salt of ergometrine, compound 6, crystallized in the orthorhombic P212121 system, as
previously reported in the literature [20], while the maleate salt of methylergometrine
7 crystallized in
a different space group, namely the monoclinic P21 [14].
The dianionic maleate group bridges two neighboring alkaloid molecules, whose conformation is
mainly determined by a central rigid core, consisting of an indole plane connected to a six-membered
ring and a tetra-hydro-pyridine. Since the absolute configuration of C5 of the starting material
was known, it was possible to determine by X-ray diffraction the relative configuration of C8 in
the two crystal structures. The single crystal diffraction data unambiguously confirmed that the
substituent at the 8-position is equatorially oriented (cis with respect to H-5), and with respect to an
absolute configuration R at the 5-position, this relative orientation means the R configuration of C8.
The cores of the two molecules are closely related: the ring C of the central skeleton is in a slightly
distorted envelope conformation, as indicated by the puckering parameters [21] QT (total puckering
amplitude) = 0.3803(1)Å,
whereas ring D adopts a distorted chair conformation, with puckering coordinates [21] QT = 0.5208
(1)Å, and QT = 0.5170 (1)Å, , which is in agreement with the
= 126.1(1)◦ for = 127.7(1)◦ for
θ 6, and QT = 0.3630(1)Å, θ 7,
(torsion angle) = 53.8(1)◦ for = 51.8(1)◦ for
θ
6
θ
7
previously reported structures. The conformation of the tetracyclic system is also conserved in the
ergometrinine crystal structure [22], which is the biologically inactive isomer of 4.
The crystal lattice of both structures is dominated by hydrogen bonds (Figure 5); in 6, in particular,
the negatively charged carboxylate groups of the anions are engaged with a hydrogen belonging to the
positively charged amino groups of the cations; moreover, bifurcated hydrogen bonds linking O1 with
N1 and O2 of symmetry-related ergometrine molecules contribute to stabilize the crystals. In addition,
C
π
-H···O contacts between neighboring anions give rise to the formation of molecular chains along the
a-axis of the unit cell, further consolidating the crystal packing. In , we revealed some differences
with respect to the previously reported structure [14]: in particular, the absence of the intermolecular
interaction between the amidic oxygen and the NH of ring B and the presence of C
7
π
-H···O contacts
between neighboring anions, which give rise to the formation of molecular chains along the b-axis of
the unit cell.

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Figure 5. The dashed lines show the intermolecular contacts of
6
(above), viewed about along the
a-axis, and of 7 (below), viewed about along the b-axis (only the hydrogens involved in the interactions
are shown).
The hydrogen bond geometry of
6 and 7, summarized in Table 5, evidenced the capacity of these
derivatives to act as both hydrogen-bond donors and acceptors.
Moreover, the crystallographic analysis suggests that the lower solubility of 6 in the crystallization
mixture (1:1 acetone/ethanol) is caused by the presence of an extensive system of hydrogen bonding.
Therefore, the higher solubility of may be correlated to the reduced number of intermolecular
interactions found in the solid state, leading to a decreased crystal lattice energy. The less soluble
crystallizes in a compact structure as prisms, while the poor-quality crystals of (obtained from a
7
6
7
1:1 ethanol/water crystallization mixture) are assembled in transparent platelets, showing a reasonable
correlation between the different morphological aspect of the crystals and their solubility.

Molecules 2020, 25, 331
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Table 5. Hydrogen-bonds geometry of 6 and 7 (arbitrary atom-numbering scheme used in Figure 4).
6
7
H···A
D···A
D-H···A
H···A
D···A
D-H···A
H-Bond
Distance (Å) Distance (Å)
Angle (◦)
Distance (Å) Distance (Å)
Angle (◦)
N2-H···O3
N1-H···O2 I
C22-H···O6 II
C21-H···O4 II
C21-H···O6 II
O2-H···O1 II
N1-H···O1 III
N3-H···O5 IV
1.8(1)
2.9(1)
2.7(1)
2.8 (1)
2.7(1)
2.2(1)
2.1(1)
2.1(1)
I
2.7(1)
3.5(1)
3.3(1)
3.7(1)
3.4(1)
2.8(1)
2.8(1)
2.9(1)
176(1)
140(1)
134(1)
171(1)
132(1)
131(1)
155(1)
163(1)
1.8(1)
3.2(1)
3.3(1)
3.3(1)
168(1)
-
-
2.7(1) V
130(1)
-
-
2.6 (1) V
133(1)
-
-
-
-
2.1(1) VI
2.(1)
172(1)
z; V x, y + 1, z;
1
1
1
1
1
1
Equivalent positions:
−
x, y − /2
,
/2 − z; II
x
−
1, y, z; III
1
−
x, y − /2
,
/2 − z; IV
x
− /2
,
/2 − y,
−
VI
1
−x, y + /2, 1 − z.
3. Materials and Methods
3.1. Chemistry
Ergometrine maleate and methylergometrine maleate, all reagents and solvents were purchased
from Sigma-Aldrich (Merck, Kenilworth, NJ, USA).
3.1.1. Ergometrine 4 from the Corresponding Maleate 6
To a suspension of commercial ergometrine maleate (50 mg, 0.11 mmol) in ethyl acetate (10 mL),
a saturated aqueous sodium bicarbonate solution (10 mL) was added. After stirring at room temperature
in the dark for 30 min, the layers were separated. The aqueous phase was extracted with ethyl acetate
(2
×
10 mL). The collected organic phases were dried over sodium sulfate and filtered. After solvent
removal under reduced pressure, a white solid was obtained (35 mg, 0.11 mmol, 96%).
[α]D20 = −17.9◦ (c = 1; pyridine) lit. [6] −16◦
(1)
3.1.2. Methylergometrine 5 from the Corresponding Maleate 7
Methylergometrine 5 was obtained following the previously described procedure for ergometrine 4,
starting from commercial methylergometrine maleate (50 mg, 0.11 mmol). A white solid was obtained
(34 mg, 0.10 mmol, 91%).
[α]D20 = −49.8◦ (c = 0.4; pyridine) lit. [6] −45◦
(2)
3.2. NMR Spectroscopy
NMR spectra were recorded on a Bruker AVANCE 500 spectrometer (Bruker, Billerica, MA, USA)
equipped with a 5 mm broadband inverse (BBI) detection probe with field z-gradient operating at
500.13, 125.76, and 50.69 MHz for 1H, 13C, and 15N respectively. NMR spectra were recorded at 300 K
for compounds
4 and 5 in DMSO-d6 (isotopic enrichment 99.9 atom % D), for their maleate salts,
6
and , in D2O (isotopic enrichment 99.9 atom % D) solution. The data were collected and processed
7
by XWIN-NMR software (version 3.5, Bruker, Billerica, MA, USA) running on a PC with Microsoft
Windows 7. The samples (10 mg) were dissolved in the appropriate solvent (0.75 mL) in a 5 mm NMR
1
tube. The acquisition parameters for 1D were as follows: H spectral width of 5000 Hz and 32 K data
points providing a digital resolution of ca. 0.305 Hz per point, relaxation delay 10 s; 13C spectral
width of 29,412 Hz, and 64 K data points providing a digital resolution of ca. 0.898 Hz per point,
relaxation delay 2 s. The experimental error in the measured 1H-1H coupling constants was
±
0.5 Hz.
) of the 1H NMR and 13C NMR spectra are reported in ppm using the central peak of
DMSO-d6 signals (2.50 ppm for 1H; 39.52 ppm for 13C) for compounds
and and using methanol as
Chemical shifts (
δ
4
5

Molecules 2020, 25, 331
9 of 11
external reference in the spectra recorded in D2O (signals of CH3OH in D2O: 3.34 ppm for 1H; 49.50 ppm
for 13C). Methylergometrine maleate D2O solution showed a pH of 4.5, while ergometrine maleate D2O
solution showed a pH of 4.2. The splitting pattern abbreviations are as follows: s, singlet; d, doublet;
t, triplet; q, quartet; m, multiplet; and br, broad signal. For two-dimensional experiments, standard
Bruker microprograms using gradient selection (gs) were applied. Gs-COSY-45 and phase sensitive
gs-NOESY experiments were acquired with 512 t1 increments; 2048 t2 points; and a spectral width
of 11.0 ppm. The gs-NOESY experiments were performed with a mixing time of 0.800 s on samples
degassed under a flush of argon in a screwcap sample tube. There were not significant differences
in the results obtained at different mixing times (0.5–1.5 s). The acquisition data for gs-HSQC and
gs-HMBC experiments were acquired with 512 t1 increments, 2048 t2 points, and a spectral width of
11.0 ppm for 1H and 200 ppm for 13C. Delay values were optimized for 1JC,H 140 Hz and nJC,H 8.0 Hz.
1
For H-15N HMBC experiments, nitromethane was used as the external reference, referencing the
resonance of its 15N at 0.00 ppm. Samples concentration: 10 mg/0.75 mL corresponding to 41, 39, 30,
and 29 mM for 4, 5, 6, and 7, respectively. The acquisitions were performed setting an acquisition time
of 0.5 s, a delay between scans of 10 s, a 1JN,H value of 90.0 Hz, and a nJN,H value of 1.9 Hz. This last
parameter was set after several attempts between 1 and 10 Hz. Total 1H-15N HMBC experimental time:
64 h.
The NMR spectra are available in the Supplementary Materials section.
3.3. X-ray Crystallography
Crystals of
temperature. Poor-quality crystals of
6
were obtained as pale-yellow prisms from a 1:1 acetone/ethanol solution at room
were obtained, after many attempts, as colorless elongated
7
platelets from a 1:1 ethanol/water solution at room temperature. All experiments were carried out in
the dark to avoid degradation.
Diffraction data for the crystals of
6 and 7 were collected with a Bruker-AXS CCD-based
three-circle diffractometer, working at ambient temperature with graphite-monochromatized Mo-K
X-ray radiation (λ = 0.7107 Å).
α
X-ray diffraction data in the
θ
range 2–25◦ were collected acquiring three sets of 600 bidimensional
CCD frames with the following operative conditions: omega rotation axis, scan width 0.3◦, acquisition
time 40 s, sample-to-detector distance 60 mm, and phi angle fixed at three different values (0◦, 120◦,
240◦) for the three different sets.
Omega-rotation frames were processed with the SAINT software [23] for data reduction (including
intensity integration, background, Lorentz, and polarization corrections) and for the determination of
accurate unit-cell dimensions, which were obtained by least-squares refinement of the positions of
2023 independent reflections with I > 10σ(I) in the θ
range 2–19◦. Absorption effects were empirically
evaluated by the SADABS software [24], and absorption correction was applied to the data (0.87 and
0.99 min and max transmission factor). The structures were solved by direct methods (SIR-14) [25] and
2
completed by iterative cycles of full-matrix least squares refinement on F0 and DF synthesis using the
WingX.v2014.1 [26] program. The hydrogen atoms bonded to carbon were included at geometrically
calculated positions and refined using a riding model. Uiso(H) is defined as 1.2 Ueq of the parent
carbon atoms for the phenyl and methylene residues and 1.5 Ueq of the parent carbon atoms for the
methyl group. Crystallographic data has been deposited with the Cambridge Crystallographic Data
Centre under CCDC deposition numbers 1970319 (6) and 1970320 (7). Copies of this information
may be obtained free of charge from the Director, CCDC, 12 Union Road, Cambridge CB2 1EY, UK
(fax: +44-1223-336033; e-mail: deposit@ccdc.cam.ac.uk or www:http://www.ccdc.cam.ac.uk).
Crystal data for 6: C23H27N3O6, Mr = 441.47 g/mol, Orthorhombic, Space group P212121,
a = 5.7139(7) Å, b = 12.3104(1) Å, c = 33.2683(4) Å, V = 2340(1) Å3, Z = 4, Dcalc = 1.253 Mg/m3, F(000) = 936,
R = 0.057 (reflections collected/unique = 4113/2921), wR2 = 0.146, T = 294(2)K, and GOF = 1.026.
The reflections were collected in the range 1.22◦ ≤ θ ≤ 25.05◦ (limiting indices =
−
6
≤
h
≤
6,
−
14
≤
k
≤
14,

Molecules 2020, 25, 331
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−
39
≤
l
≤
39) employing a 0.30
×
0.20
×
0.07 mm crystal. The residual positive and negative electron
densities in the final map were 0.303 and −0.176 Å−3
.
Crystal data for 7: C24H29N3O6, Mr = 455.5 g/mol, Monoclinic, Space group P21, a = 10.844 (1) Å,
b = 5.702 (6) Å, c = 21.029 (2) Å,
β
= 91.469(2) Z = 2, V = 1299.2(4) Å3, Dcalc = 1.164 Mg/m3, F(000) = 484,
R = 0.056 (reflections collected/unique = 4577/3576), wR2 = 0.1607, T = 294(2)K, and GOF = 1.052.
The reflections were collected in the range 0.969◦ ≤ θ ≤ 25.04◦ (limiting indices =
−
12
≤
h
≤
12,
−
6
≤
k
≤
6,
−
25
≤
l
≤
25) employing a 0.30
×
0.08
×
0.03 mm crystal. The residual positive and negative electron
densities in the final map were 0.486 and −0.296 Å−3
.
3.4. Optical Rotatory Power
The values of optical rotations were registered on a polarimeter (mod. 241, PerkinElmer, Waltham,
MA, USA) in a 1 dm path length cell at 20 ◦C, setting the wavelength at 589 nm.
4. Conclusions
Although ergometrine and methylergometrine have been known for over 70 years and they
have been used as antihemorrhagic agents for a long time, few and sometimes contradictory NMR
and crystallographic data are available in the literature. In this work, we provide the complete
assignment of 1H, 13C, and 15N NMR signals of ergometrine, methylergometrine, and their maleate
salts together with a solid-state analysis of ergometrine maleate and methylergometrine maleate.
The new and more accurate X-ray and NMR data will contribute to widen the available information
about conformational equilibria and hydrogen-bonding interactions, which could prove useful for the
study of other ergot alkaloids.
Supplementary Materials: The following are available online, The NMR spectra (1H, 13C, COSY, HSQC,
1H-13C HMBC, 1H-15N HMBC and NOESY) of compounds 4–7 are available online.
Author Contributions: Conceptualization of the work: P.F. and P.G.; synthesis: S.R.E.; crystallographic analysis:
F.M. and M.M.; NMR analysis: S.C.; writing—original draft preparation, review and editing: F.M., P.F. and S.C.;
supervision: P.F. All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 4–7 are available from the authors.
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2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).