Crystallographic and NMR Investigation of Ergometrine and Methylergometrine, Two Alkaloids from Claviceps Purpurea

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.


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 1 (Figure 1) and can be chemically classified by the nature of the substituent at the 8-position.

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 1 (Figure 1) and can be chemically classified by the nature of the substituent at the 8-position.   From a therapeutic point of view, the d-lysergic acid amides are in a prominent position among the ergot alkaloids. The d-lysergic acid 2 carries a carboxyl group at the 8β position (cis with respect 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 3. In the tetracyclic moiety of compounds 2 and 3, 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 α-configuration (trans with respect to the H-5) or in the β-configuration (cis with respect to the H-5). The α-isomer is usually indicated by the prefix iso-or by the ending -inine. The β-isomers are usually endowed with a significant biological activity.
Among the d-lysergic derivatives, the natural amide ergometrine (ergonovine) 4 and the semisynthetic methylergometrine 5 (methylergonovine) (Figure 2) are endowed with uterotonic 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 4 was isolated in 1932 [5] and the first synthesis of methylergometrine 5, starting from the lysergic azide, was published in 1943 [6], only a few spectroscopic and crystallographic studies are reported in the literature. In two 2017 Chinese patents [7,8], the 1 H NMR data of the ergometrine 4 are reported but not assigned; in 1974 [9], the assigned 13 C NMR (60 MHz) data were reported. In the case of ergometrine maleate, the XRD and the single-crystal X-ray diffraction [10] are reported, and only the 1 H NMR (400 MHz) characterization is available [11]. Methylergometrine 5 was analyzed by 1 H 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, 4 and 5. Considering the easy epimerization of these compounds, we decided to expand our investigations to their maleate salts, 6 and 7, as well. Indeed, ergometrine 4 and methylergometrine 5 are always obtained as mixtures of epimers, 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][16][17], a complete NMR ( 1 H, 13 C, and 15 N) characterization of compounds 4, 5, 6, and 7 was carried out. Moreover, a detailed crystallographic investigation was performed on 6 and 7 in order to explore their conformational features. In addition, we carefully analyzed the hydrogen-bonding interactions of these molecules, as the earlier report did not account for their supramolecular aggregation. From a therapeutic point of view, the d-lysergic acid amides are in a prominent position among the ergot alkaloids. The d-lysergic acid 2 carries a carboxyl group at the 8β position (cis with respect 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 3. In the tetracyclic moiety of compounds 2 and 3, 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 α-configuration (trans with respect to the H-5) or in the β-configuration (cis with respect to the H-5). The α-isomer is usually indicated by the prefix iso-or by the ending -inine. The β-isomers are usually endowed with a significant biological activity.
Among the d-lysergic derivatives, the natural amide ergometrine (ergonovine) 4 and the semisynthetic methylergometrine 5 (methylergonovine) (Figure 2) are endowed with uterotonic 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 4 was isolated in 1932 [5] and the first synthesis of methylergometrine 5, starting from the lysergic azide, was published in 1943 [6], only a few spectroscopic and crystallographic studies are reported in the literature. In two 2017 Chinese patents [7,8], the 1 H NMR data of the ergometrine 4 are reported but not assigned; in 1974 [9], the assigned 13 C NMR (60 MHz) data were reported. In the case of ergometrine maleate, the XRD and the single-crystal X-ray diffraction [10] are reported, and only the 1 H NMR (400 MHz) characterization is available [11]. Methylergometrine 5 was analyzed by 1 H 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, 4 and 5. Considering the easy epimerization of these compounds, we decided to expand our investigations to their maleate salts, 6 and 7, as well. Indeed, ergometrine 4 and methylergometrine 5 are always obtained as mixtures of epimers, 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][16][17], a complete NMR ( 1 H, 13 C, and 15 N) characterization of compounds 4, 5, 6, and 7 was carried out. Moreover, a detailed crystallographic investigation was performed on 6 and 7 in order to explore their conformational features. In addition, we carefully analyzed the hydrogen-bonding interactions of these molecules, as the earlier report did not account for their supramolecular aggregation.

Results and Discussion
Ergometrine 4 and methylergometrine 5 were obtained by treatment of the corresponding commercially available maleates, 6 and 7, with sodium hydrogen carbonate as described in the Materials and Methods section.

NMR Spectroscopy
The NMR study was carried out on 4 and 5 and on their maleate salts 6 and 7 (the clinically administered derivatives). Compounds 4 and 5 were dissolved in DMSO-d 6 , while compounds 6 and 7 were dissolved in D 2 O. Although in both solvents the 1 H NMR spectra of 6 and 7 present broadened signals and a slight concentration dependence of chemical shifts, D 2 O was chosen in these cases due to more resolved NMR spectra. 1 H, 13     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 4, 5, 6, and 7, respectively), we were able to assign the resonances of all the other protons, especially the resonance of H-8. The interpretation of the 1 H NMR spectra of compounds 4 and 5 was further facilitated by the resonances of exchangeable protons H-1, CH 2 OH, 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 6 and 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-5β, H-7β, H-7α, and H-4β, 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 D 1 8R half-chair conformation. The very weak NOE correlation of N-CH 3 with H-8 α and H-4 α could be explained by the existence of a conformational equilibrium in solution between D 1 8R and D 2 8R half-chair conformations ( Figure 3). When the D-ring is in its D 2 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.  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 4, 5, 6, and 7, respectively), we were able to assign the resonances of all the other protons, especially the resonance of H-8. The interpretation of the 1 H NMR spectra of compounds 4 and 5 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 6 and 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-5β, H-7β, H-7α, and H-4β, 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 halfchair conformation. The very weak NOE correlation of N-CH3 with H-8 α and H-4 α could be explained by the existence of a conformational equilibrium in solution between D1 8R and D2 8R halfchair 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.  correlations of H-5β (3.00-2.93 ppm) with one of the two protons at the 4 position (3.46 ppm) and of H-8α (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) allowed us to unequivocally identify H-4β and H-7α protons. The absence of spatial interactions 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-CH 3 (2.44 ppm for 4; 2.45 ppm for 5) was irradiated, NOE enhancement was observed for H-5β, suggesting that the analyzed 4 and 5 in DMSO-d 6 were in their D 1 8R half-chair conformation. The CH, CH 2 , and CH 3 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 1 H NMR assignment of the protons bound to these carbon atoms. In the 13 C NMR spectra of compounds 6 and 7, the broad nature and low intensity of the C-4, C-8, N-CH 3 , C-7, C-5, and C-9 signals can be explained by the previously reported conformational equilibrium between the D 1 8R and D 2 8R half-chair conformations. The quaternary carbon atoms were unambiguously assigned using the information obtained from 1 H-13 C HMBC experiments (Table 4). Following this approach, the 13 C resonance assignments of 4 were in accordance with the previously reported ones [9].

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. 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 sixmembered 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, θ (torsion angle) = 53.8(1)° for 6, and QT = 0.3630(1)Å, θ = 51.8(1)° for 7, whereas ring D adopts a distorted chair conformation, with puckering coordinates [21] QT = 0.5208 (1)Å, θ = 126.1(1)° for 6 and QT = 0.5170 (1)Å, θ = 127.7(1)° for 7, which is in agreement with the 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, Cπ-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 7, 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 Cπ-H⋅⋅⋅O contacts between neighboring anions, which give rise to the formation of molecular chains along the b-axis of the unit cell. The maleate salt of ergometrine, compound 6, crystallized in the orthorhombic P2 1 2 1 2 1 system, as previously reported in the literature [20], while the maleate salt of methylergometrine 7 crystallized in a different space group, namely the monoclinic P2 1 [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] Q T (total puckering amplitude) = 0.3803(1)Å, θ (torsion angle) = 53.8(1) • for 6, and Q T = 0.3630(1)Å, θ = 51.8(1) • for 7, whereas ring D adopts a distorted chair conformation, with puckering coordinates [21] Q T = 0.5208 (1)Å, θ = 126.1(1) • for 6 and Q T = 0.5170 (1)Å, θ = 127.7(1) • for 7, which is in agreement with the 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, Cπ-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 7, 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 Cπ-H···O contacts between neighboring anions, which give rise to the formation of molecular chains along the b-axis of the unit cell. 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. Table 5. Hydrogen-bonds geometry of 6 and 7 (arbitrary atom-numbering scheme used in Figure 4).  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 7 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 6 crystallizes in a compact structure as prisms, while the poor-quality crystals of 7 (obtained from a 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. Table 5. Hydrogen-bonds geometry of 6 and 7 (arbitrary atom-numbering scheme used in Figure 4).

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%). 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%).

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 1 H, 13 C, and 15 N respectively. NMR spectra were recorded at 300 K for compounds 4 and 5 in DMSO-d 6 (isotopic enrichment 99.9 atom % D), for their maleate salts, 6 and 7, in D 2 O (isotopic enrichment 99.9 atom % D) solution. The data were collected and processed 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 tube. The acquisition parameters for 1D were as follows: 1 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; 13 C 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 1 H-1 H coupling constants was ±0.5 Hz. Chemical shifts (δ) of the 1 H NMR and 13 C NMR spectra are reported in ppm using the central peak of DMSO-d 6 signals (2.50 ppm for 1 H; 39.52 ppm for 13 C) for compounds 4 and 5 and using methanol as

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.

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 1 H, 13 C, and 15 N 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.