A new structure of a serotonin salt: comparison and conformational analysis of all known serotonin complexes

Four serotonin salt structures (serotonin adipate, C₁₀H₁₃N₂O⁺·C₆H₉O₄^-, is a previously unknown structure) were analysed to understand the influence of the anion on serotonin conformation. Hydrogen bonding alone favours a flat conformation, whereas additional stacking inter­actions between ions may possibly account for the nonplanar conformation. Since mol­ecular conformation, stability and biological activity are inter­related, one can consider influencing the chemical and biological properties of serotonin by selecting an appropriate counter-ion for salt formation.

S1. Introduction

Biologically active substances are at the focus of biological, pharmaceutical and chemical research. Serotonin, one of the most common neurotransmitters, is widely studied in relation to its effect on humans at different levels, from cellular up to mental (Mohammad-Zadeh et al., 2008; Seo et al., 2008; Berger et al., 2009). Although serotonin plays a key role in some biological processes, its chemistry and crystallography have not been well studied.

Many biologically active compounds are crystallized with additional components as salts or co-crystals, to improve stability or solubility or to decrease hygroscopicity (Almarsson & Zaworotko, 2004; Libbrecht, 2005; Schultheiss & Newman, 2009; Babu & Nangia, 2011; Brittain, 2012). Serotonin is not an exception. Its low thermal stability in solution can be overcome if a second component, like adipine, is added on crystallization. Serotonin adipate has been used in medical formulations in Russia and Eastern Europe as an analogue of serotonin creatinine sulfate monohydrate, but the crystal structure of the adipate salt remained unknown. Another inter­esting issue is that the conformation of serotonin creatinine sulfate monohydrate is different from that predicted theoretically for a `free' serotonin molecule in the gas phase (Cho­thia, 1969; Pratuangdejkul, Jaudon et al., 2006). It was worth investigating whether the conformation of serotonin in the adipate salt is the same as in creatinine sulfate, remembering that they are pharmacological analogues.

The aim of the present study was to crystallize serotonin adipate, (I), determine its crystal structure and analyse it in a comparison with all the other previously known crystal structures of serotonin (Karle et al., 1965; Thewalt & Bugg, 1972; Aniy et al., 1978), paying special attention to the inter­relation between the crystalline environment of a molecule and the molecular conformation.

S2. Experimental

S2.1. Synthesis and crystallization

Serotonin adipate (purity not less than 98%, according to high-performance liquid chromatography data) was crystallized from an aqueous solution at 313 K. The crystals used in the diffraction experiments were almost transparent, with a light-brown tinge.

S2.2. Refinement

Crystal data, data collection and structure refinement details are summarized in Table 1. All H atoms were located in a difference Fourier map, and their positions and isotropic displacement parameters were refined freely.

S3. Results and discussion

The crystal structure of serotonin adipate (Fig. 1) consists of sheets of molecules, in which chains of serotonin cations with protonated hy­droxy and amino groups, linked via N—H···O hydrogen bonds (Table 2), alternate with chains formed by adipate anions, linked via O—H···O hydrogen bonds (Table 2). Additional O—H···O and N—H···O hydrogen bonds link serotonin cations to adipate anions, so that a two-dimensional network containing inter­connected rings is formed (Fig. 2, Table 2).

The serotonin conformations in four different known crystal structures (including the present structure of serotonin adipate) are compared in Fig. 3. The values of the bond lengths and angles are quite similar in all four structures. The torsion angles are rather close in three of the four compounds, in which the molecules are almost flat, but differ significantly in serotonin picrate, in which the side chain of serotonin is almost orthogonal to the planar bicyclic system (Fig. 3 and Table 3).

To understand the relationship between the molecular conformations and the crystalline environments, the molecular packing in the four structures was compared in relation to inter­molecular inter­actions. We start with discussing the hydrogen bonds, since they most often control crystal structures and the ability of molecules to change conformation. The geometric parameters of the hydrogen bonds are summarized in Table 2. In all four structures the strengths of the hydrogen bonds are classified as moderate; donor–acceptor distances lie in the range 2.7–3.1 Å for N—H···O and O—H···N hydrogen bonds, and in the range 2.5–3.1 Å for O—H···O hydrogen bonds (Fig. 5).

Serotonin oxalate and serotonin adipate not only have similar geometric parameters of their hydrogen bonds but also similar structural motifs, with chains of oxalic and adipic acid molecules, respectively (Fig. 6).

Molecular conformation can be affected also by stacking inter­actions. In general, π–π inter­actions of aromatic systems are possible in all four of these structures, but their strength should depend on the distances between the π-systems and their mutual orientation. The geometric parameters which can be used to characterize the stacking of molecules are defined in the Supplementary materials, and their values are summarized in supplementary Tables S1–S4). The strongest inter­actions are shown in Fig. 7. In the following discussion, the term `centroid' will be taken as referring to the geometric centre of a single cyclic system, e.g. the indole ring contains two such systems, one containing six atoms and the other five.

For serotonin oxalate, only the contact between centroid 1 (Cg1) and centroid 2 (Cg2), with a distance of 4.19 Å, gives any significant contribution to the stacking inter­action, which is not likely to be strong for any pairs of neighbouring molecules, as far as one can judge from the inter­atomic distances (Fig. 7a).

The stacking inter­actions in serotonin adipate have some common features with those of serotonin oxalate, since the rings in neighbouring molecules are almost parallel to each other. The indole rings overlap only partially. All other possible stacking inter­actions do not seem to be significant from an energetic point of view on the basis of their geometric parameters (see Supplementary materials).

Analysis of the stacking inter­actions in serotonin creatinine sulfate is more complicated. Theoretically, if creatinine were capable of adopting the enol form, the orientation and proximity of the aromatic rings would allow a strong π–π inter­action (Fig. 7c). Up to now, there has been no experimental evidence suggesting the existence of the enol form of creatinine in the crystalline state (du Pré & Mendel, 1955; Smith & White, 2001; Goswami et al., 2006)

Karle et al. (1965) noted that the structural data for creatinine sulfate complex are of poor quality, thus `a discussion of individual bonds is not warranted since the refinement process was terminated before completion'. Therefore, using these data, we cannot fully rely on bond lengths to distinguish reliably between the ketone and enol forms. Many other structures containing creatinine (including aromatic systems) are known, and they are invariably composed of the ketone form (du Pré & Mendel, 1955; Smith & White, 2001; Goswami et al., 2006). Therefore, we conclude that π–π inter­actions between the serotonin fragments in creatinine sulfate are likely to be absent. The only possible inter­action is that of the indole π-system of serotonin with the conjugated π-system of the creatinine molecule. This inter­action yields a lower stabilization energy than could be expected for related systems in which π–π stacking inter­actions between aromatic rings exist. From the geometric criteria (see Supplementary materials), it can be assumed that the only serotonin structure that contains a strong π–π stacking inter­action is serotonin picrate, in which the whole indole system is involved in an inter­action with another π-ring. Another point about inter­molecular inter­actions is an additional donor–acceptor inter­action with the indole π-system that can also influence the energy distribution in the crystal structure and complement the stacking inter­action (see Supplementary materials).

Thus, only the structure of serotonin picrate has obvious π–π inter­actions involving the indole π-system, and additionally Y—X(—N═ O)···π inter­actions, while the other three structures have only minor π–π contributions to the overall inter­molecular inter­action.

Additional insight into the inter­actions between serotonin and its immediate crystalline environment can be obtained by analysing the Hirshfeld surfaces (McKinnon et al., 1998; Spackman & McKinnon, 2002) (Fig. 8). It is quite obvious that three out of the four surfaces are quite similar to each other, while the last one, corresponding to another conformation, is significantly different. Some minor differences in the surfaces of serotonin adipate, oxalate and creatinine sulfate are related to the three-dimensional orientation of potentially reactive sites and the curviness of the surface. This can be explained by the difference in quality of the diffraction data.

Qualitative and qu­anti­tative differences in the Hirshfeld fingerprint plots are even more remarkable. The plot for serotonin adipate (bottom row of Fig. 8, leftmost plot) displays characteristics consistent with hydrogen bonding, similar to that in carb­oxy­lic acids: symmetric sharp features point to the lower left quadrant of the plot, the upper one corresponding to the hydrogen-bond donors and the lower one to the acceptors. –NH₃⁺, NH in the indole ring, and OH groups act as donors, while the only acceptor is the OH group (the O atom is an acceptor for another NH₃⁺). The green colour shows that there are many more donor groups than acceptors (blue colour of another band). Another feature are the wings at the lower right (π-acceptor) and upper left (C—H donor) corners of the plot, corresponding to an `aromatic' C—H···π inter­action. The indole ring in serotonin acts as an acceptor for the CH₂ groups of adipic acid which lie above. Moreover, the CH₂ group of ethyl­amine donates its H atom in the C—H···π inter­action with another molecule of serotonin. There are no indications of π–π inter­actions in the region of d_e = d_i = 1.8 Å.

A very similar plot is calculated for serotonin oxalate (bottom row of Fig. 8, second plot from left). The same features characterizing the hydrogen bonding can be observed, namely the two symmetric bands at the lower left corner of the plot. More differences can be seen for the `aromatic' inter­actions: there are no `wings' typical for a C—H···π inter­action, neither is there any evidence of π–π stacking inter­actions. Another difference is the appearance of a significant number of widely spread dots at the upper right corner, which may indicate the presence of `cavities' in the structure, and hence of a non-optimum packing.

The fingerprint plot calculated for serotonin creatinine sulfate (bottom row of Fig. 8, third plot from left) should be considered with care, given that the published data did not contain H-atom coordinates. In this work we calculated the H-atom positions using the Mercury feature `Add missing H-atoms' (Macrae et al., 2008). The unusual shape of the plot seems to be a consequence of the poor structural data. The features related to hydrogen bonding (such as the huge wing in the bottom left) cannot therefore be discussed. Nevertheless, a π–π stacking inter­action is independent of H-atom positions, and the fingerprint shows some evidence of such an inter­action, which cannot be classified as `strong' (Fig. 9).

The fingerprint of serotonin picrate (bottom row of Fig. 8, rightmost plot) gives evidence of a large number of hydrogen-bond donors, so that the colour of the lower left band is red at the position d_e = 1.5 Å and d_i = 1.15 Å [No red visible?]. This can be explained by the existence of additional hydrogen bonds between the NO₂ group of the picrate and the C—H atoms of the indole ring. C—H···π inter­actions are absent. The presence of π–π stacking inter­actions in this structure is also apparent. This can be better visualized by comparing the fingerprint plots of serotonin creatinine sulfate and serotonin picrate, in which C···C contacts are considered as isolated (Fig. 9).

To sum up this analysis of different inter­actions from hydrogen bonds to π–π stacking using different techniques, we should discuss some key features that may cause conformational changes in serotonin. The hydrogen-bond motifs are quite different in almost all these structures, so no clear correlation between molecular structure and hydrogen bonding is observed. C—H···π inter­actions apparently also do not play a key role. However, only one structure [Which?] out of the four has a strong π–π stacking inter­action and this structure shows conformational changes. Thus, on the basis of the current evidence, it looks as though the flat conformation of serotonin may be obtained only in the absence of stacking inter­actions, even when the molecules form a hydrogen-bonded network.

We have not found any clear influence of water molecules on the conformational changes of serotonin in hydrates (serotonin creatinine sulfate monohydrate and serotonin picrate monohydrate), although it is very complicated to divide strictly the contribution of each molecule to the overall hydrogen-bond network and as a result establish its exact role in the whole structure. It should be also mentioned that, according to Karle et al. (1965) and Thewalt & Bugg (1972), these hydrates are stable under ambient conditions.

Another remarkable feature is that the conformation of serotonin in the present serotonin adipate structure differs from the most stable conformation predicted for a single molecule by the adiabatic conformational analysis using quantum chemistry calculations: a `free' serotonin molecule in cationic form should be nonplanar (Cho­thia, 1969; Pratuangdejkul, Jaudon et al., 2006). It also differs from the serotonin conformations in all other known complexes, but the changes are not very significant among the `flat' conformations. We should point out that some differences between serotonin adipate and serotonin creatinine sulfate that are described below can be noteworthy, but do not affect the overall conformation to any great extent.

The most significant difference between the adipate salt and the creatinine sulfate complex of serotonin is the orientation of the OH group (Table 3, ϕ4), which is explained by forming the most energetically favourable hydrogen bond but, as mentioned above, it does not change the overall similarity of these structures. Depending on the values of the two dihedral angles, ϕ1 and ϕ2, serotonin can be in either a +gauche (Gp), -gauche (Gm) or anti (At) conformation (Figs. 3 and 4), and the serotonin conformers will be named hereinafter as, for example, GpAt when ϕ1 corresponds to the +gauche conformation and ϕ2 to the anti conformation. We will name as At only those structures with dihedral angles very close (±2°) to the ideal value of 180°, understanding that these angles can still be quite close to the other angles despite their different names. In this terminology, the serotonin conformation in the adipate salt is AtAt. The corresponding energy should be about 9 kcal mol^-1 (1 kcal mol^-1 = 4.184 kJ mol^-1) greater than that calculated for the most stable GmGp and GpGp conformations predicted for the `free' molecule in the gas phase (Pratuangdejkul, Jaudon et al., 2006). Three other conformations (GpGp, GmGm* and GpAt) were reported for serotonin in different crystalline environments, for creatinine sulfate, picrate and oxalate, respectively. The GmGm and GmGp conformations were presumed to be stabilized by the inter­action of the cationic group of serotonin (NH₃⁺) with the π-system of the indole ring (Pratuangdejkul, Jaudon et al., 2006). Inter­estingly, there are no such inter­actions in the known crystalline forms of serotonin. In the solid state, the conformation seems to be changed solely because of the stacking inter­actions.

We can suppose that the crystalline environment defines the molecular conformation. The calculated conformational energy difference between creatinine sulfate and picrate is only about 2 kcal mol^-1, while the difference in lattice energy is 274 kcal mol^-1 (Caillet et al., 1977). Exact values aside, it is important to note the magnitude of the difference in conformational and lattice energies.

This work has shown the influence of different inter­actions on the molecular conformation of serotonin in the crystalline state. Three main conclusions may be drawn on the basis of the experimental data available so far: (i) the crystalline environment may define the conformation of serotonin molecules; (ii) `flat' (thermodynamically unfavourable) conformations can be stabilized in the crystalline state if hydrogen bonds are the only inter­molecular inter­actions; and (iii) additional stacking and donor–acceptor inter­actions change the molecular conformation dramatically, such that the molecules are no longer flat.

From these results we can suggest an intriguing theory that the intra­molecular geometry of serotonin can be changed by varying the counter-ions in the crystal structures of its salts or molecular complexes. It is worth remembering that forming salts with different anions is a common technique for modifying the substance's biological activity, pharmacological and therapeutic effect, and some physicochemical properties, such as pK_a (Pratuangdejkul, Nosoongnoen et al., 2006). We can also highlight that, according to one hypothesis (Akhrem et al., 1978), the physiological effects of serotonin can be altered by varying its conformation. It is well documented that conformational polymorphs of one-component pharmaceutical crystals show pronounced differences in solubility and biological activity, potentially resulting from the retention of molecular conformation following dissolution, thus affecting subsequent substrate–receptor inter­actions in biochemical pathways (Leonidov, 1997). With an increased understanding of the effects of the crystalline environment on the conformation of biologically active molecules, one can imagine the possibility of explicitly inducing a desired conformation of the target molecule, thus leading to a specific augmentation in bioactivity. This prospect opens an avenue to advanced drug design, keeping these synergetic effects in mind.

It seems very promising to make an attempt at crystallizing serotonin salts with different anions and co-formers and to test the existence of a correlation between crystalline environment, molecular conformation and biological activity for a larger series of salts and complexes.

Related literature

For related literature, see: Akhrem et al. (1978); Almarsson & Zaworotko (2004); Aniy et al. (1978); Babu & Nangia (2011); Berger et al. (2009); Brittain (2012); Caillet et al. (1977); Chothia (1969); Goswami et al. (2006); Karle et al. (1965); Leonidov (1997); Libbrecht (2005); Macrae et al. (2008); McKinnon et al. (1998); Mohammad-Zadeh, Moses & Gwaltney-Brant (2008); Pré & Mendel (1955); Pratuangdejkul, Jaudon, Ducrocq, Nosoongnoen, Guerin, Conti, Loric, Launay & Manivet (2006); Pratuangdejkul, Nosoongnoen, Gurin, Loric, Conti, Launay & Manivet (2006); Schultheiss & Newman (2009); Seo et al. (2008); Smith & White (2001); Spackman & McKinnon (2002); Thewalt & Bugg (1972).

Computing details

Data collection: CrysAlis PRO (Oxford Diffraction, 2008); cell refinement: CrysAlis PRO (Oxford Diffraction, 2008); data reduction: CrysAlis PRO (Oxford Diffraction, 2008); program(s) used to solve structure: SHELXS97 (Sheldrick, 2008); program(s) used to refine structure: SHELXL97 (Sheldrick, 2008); molecular graphics: OLEX2 (Dolomanov et al., 2009), Mercury (Macrae et al., 2008), PLATON (Spek, 2009) and CrystalExplorer (McKinnon et al., 1998; Spackman & McKinnon, 2002); software used to prepare material for publication: OLEX2 (Dolomanov et al., 2009), enCIFer (Allen et al., 2004) and publCIF (Westrip, 2010).

Figures

Fig. 1. The molecular structure of serotonin adipate, showing the atom-labelling scheme. Displacement ellipsoids are drawn at the ??% probability level. [Please complete]

Fig. 2. Fragments of the crystal structure of serotonin adipate, viewed in the bc and ac planes. Hydrogen bonds are shown as dashed lines. [Symmetry codes: (i) x, y + 1, z; (ii) x, y - 1, z; (iii) -x + 1, -y, -z + 1; (iv) x + 1, y, z.]

Fig. 3. Serotonin conformations in different salts, showing overlay pictures of adipate (green in the electronic version of the journal) with (a) oxalate, (b) creatinine sulfate and (c) picrate. The molecules are almost flat and identical in (a) and (b), but differ and are not flat in the case of (c).

Fig. 4. Definition of serotonin atom numbering and dihedral angles. The four torsion angles are defined as ϕ1 (C5—C3—C2—C1), ϕ2 (C3—C2—C1—N1), ϕ3 (C2—C1—N1—H_N11) and ϕ4 (C6—C7—O5—H5).

Fig. 5. Hydrogen-bond-length distribution in serotonin complexes, showing (top) the N···O (and O···N) donor–acceptor distances (Å) and (bottom) the O···O donor–acceptor distances (Å).

Fig. 6. Chains of molecules in (a) serotonin adipate [symmetry codes: (i) x, y + 1, z; (ii) x, y - 1, z] and (b) serotonin oxalate [symmetry codes: (i) -x + 1, -y + 2, -z + 1; (ii) x, y + 1, z; (iii) -x + 1, -y + 3, -z + 1; (iv) x, y - 1, z; (v) -x + 1, -y + 1, -z + 1].

Fig. 7. Stacking interactions in serotonin salts of (a) oxalate [symmetry code: (i) x, y + 1, z], (b) adipate [symmetry codes: (i) -x, -y, -z + 1; (ii) -x + 1, -y, -z + 1], (c) creatinine sulfate [symmetry code: (i) x + 1/2, y - 1/2, z] and (d) picrate [symmetry codes: (i) -x, y - 1/2, -z - 1/2; (ii) -x, y + 1/2, -z - 1/2]. Cg denotes ring centroids (indicated by large spheres). [Added text OK?]

Fig. 8. The molecular conformation, Hirshfeld surfaces and fingerprint plots for the serotonin molecule in various complexes. From left to right: adipate, oxalate, creatinine sulfate and picrate. Each molecule is shown with the Hirshfeld surface mapped with d_norm [coloured between -0.500 (red in the electronic version of the journal) and 1.500 (blue)], where d_norm is the normalized contact distance, which takes the van der Waals radii of the atoms into account.

Fig. 9. Hirshfeld fingerprint plots for the serotonin molecule in complex with (left) creatinine sulfate and (right) picrate. Only C···C interactions are highlighted. The highlighted surface area is 2.8% for creatinine sulfate and 5.8% for picrate salt.

2-(5-Hydroxy-1H-indol-3-yl)ethanaminium 3-carboxypropanoate

Crystal data

C10H13N2O^+·C6H9O4^−	F(000) = 344
Mr = 322.36	322.37
Triclinic, P1	Dx = 1.325 Mg m^−3
a = 7.1582 (3) Å	Mo Kα radiation, λ = 0.7107 Å
b = 10.5984 (7) Å	Cell parameters from 2039 reflections
c = 12.1715 (7) Å	θ = 1.9–26.3°
α = 113.202 (6)°	µ = 0.10 mm^−1
β = 101.233 (4)°	T = 298 K
γ = 98.045 (4)°	Plate, colourless
V = 808.04 (8) Å^3	0.15 × 0.12 × 0.05 mm
Z = 2

Data collection

Oxford Gemini Ultra R diffractometer	3286 independent reflections
Radiation source: Enhance (Mo) X-ray Source	1849 reflections with I > 2σ(I)
Graphite monochromator	Rint = 0.061
Detector resolution: 10.3457 pixels mm^-1	θmax = 26.4°, θmin = 1.9°
ω scans	h = −8→8
Absorption correction: multi-scan (CrysAlis PRO; Oxford Diffraction, 2008)	k = −13→13
Tmin = 0.994, Tmax = 1.000	l = −12→15
10092 measured reflections

Refinement

Refinement on F^2	Primary atom site location: structure-invariant direct methods
Least-squares matrix: full	Secondary atom site location: difference Fourier map
R[F^2 > 2σ(F^2)] = 0.051	Hydrogen site location: difference Fourier map
wR(F^2) = 0.121	All H-atom parameters refined
S = 1.00	w = 1/[σ^2(Fo^2) + (0.0429P)^2] where P = (Fo^2 + 2Fc^2)/3
3286 reflections	(Δ/σ)max < 0.001
296 parameters	Δρmax = 0.16 e Å^−3
0 restraints	Δρmin = −0.20 e Å^−3

Special details

Experimental. CrysAlisPro, Agilent Technologies, Version 1.171.35.15 (release 03-08-2011 CrysAlis171 .NET) (compiled Aug 3 2011,13:03:54) Empirical absorption correction using spherical harmonics, implemented in SCALE3 ABSPACK scaling algorithm.

Geometry. All e.s.d.'s (except the e.s.d. in the dihedral angle between two l.s. planes) are estimated using the full covariance matrix. The cell e.s.d.'s are taken into account individually in the estimation of e.s.d.'s in distances, angles and torsion angles; correlations between e.s.d.'s in cell parameters are only used when they are defined by crystal symmetry. An approximate (isotropic) treatment of cell e.s.d.'s is used for estimating e.s.d.'s involving l.s. planes.

Refinement. Refinement of F² against ALL reflections. The weighted R-factor wR and goodness of fit S are based on F², conventional R-factors R are based on F, with F set to zero for negative F². The threshold expression of F² > σ(F²) is used only for calculating R-factors(gt) etc. and is not relevant to the choice of reflections for refinement. R-factors based on F² are statistically about twice as large as those based on F, and R-factors based on ALL data will be even larger.

Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Ų)

	x	y	z	Uiso*/Ueq
N1	0.2468 (4)	−0.1305 (3)	0.7987 (2)	0.0523 (6)
N2	0.2581 (3)	0.0764 (2)	0.4250 (2)	0.0570 (6)
O1	0.2346 (2)	0.81214 (18)	0.16669 (15)	0.0549 (5)
O2	0.3050 (3)	0.65419 (18)	0.00795 (17)	0.0699 (5)
O3	0.1196 (2)	0.08911 (16)	0.18057 (15)	0.0518 (4)
O4	0.3235 (2)	−0.01023 (16)	0.07900 (15)	0.0524 (4)
O5	0.2448 (3)	0.5954 (2)	0.7695 (2)	0.0704 (6)
C1	0.2592 (3)	0.6863 (2)	0.1048 (2)	0.0414 (5)
C2	0.2307 (4)	0.5868 (2)	0.1627 (2)	0.0445 (6)
C3	0.2515 (4)	0.4404 (2)	0.0852 (2)	0.0444 (6)
C4	0.2290 (4)	0.3395 (2)	0.1442 (2)	0.0464 (6)
C5	0.2498 (4)	0.1940 (2)	0.0616 (2)	0.0450 (6)
C6	0.2288 (3)	0.0849 (2)	0.1118 (2)	0.0392 (5)
C7	0.2618 (4)	−0.1017 (3)	0.6905 (2)	0.0489 (6)
C8	0.2533 (4)	0.0492 (3)	0.7188 (2)	0.0481 (6)
C9	0.2578 (3)	0.0857 (2)	0.6129 (2)	0.0431 (6)
C10	0.2552 (3)	0.2220 (2)	0.6173 (2)	0.0429 (6)
C11	0.2508 (3)	0.3511 (3)	0.7107 (2)	0.0454 (6)
C12	0.2476 (3)	0.4643 (3)	0.6829 (2)	0.0523 (6)
C13	0.2482 (4)	0.4537 (3)	0.5646 (3)	0.0621 (7)
C14	0.2534 (4)	0.3290 (3)	0.4724 (3)	0.0614 (7)
C15	0.2547 (3)	0.2131 (3)	0.4985 (2)	0.0474 (6)
C16	0.2572 (3)	0.0000 (3)	0.4943 (2)	0.0526 (6)
H1	0.261 (3)	0.879 (3)	0.124 (2)	0.080 (8)*
H1A	0.123 (4)	−0.117 (3)	0.821 (3)	0.094 (10)*
H1B	0.249 (3)	−0.232 (3)	0.775 (2)	0.070 (8)*
H1C	0.337 (4)	−0.072 (3)	0.871 (3)	0.099 (11)*
H2	0.236 (4)	0.040 (3)	0.342 (3)	0.101 (11)*
H2A	0.325 (3)	0.631 (2)	0.245 (2)	0.050 (6)*
H2B	0.101 (3)	0.584 (2)	0.181 (2)	0.071 (8)*
H3A	0.156 (3)	0.400 (2)	0.003 (2)	0.051 (6)*
H3B	0.375 (3)	0.448 (2)	0.069 (2)	0.060 (7)*
H4A	0.101 (3)	0.332 (2)	0.162 (2)	0.060 (7)*
H4B	0.328 (3)	0.380 (2)	0.225 (2)	0.070 (8)*
H5	0.254 (4)	0.598 (3)	0.841 (3)	0.088 (11)*
H5A	0.143 (3)	0.154 (2)	−0.021 (2)	0.059 (7)*
H5B	0.370 (3)	0.204 (2)	0.044 (2)	0.067 (8)*
H7A	0.145 (3)	−0.172 (2)	0.618 (2)	0.054 (6)*
H7B	0.382 (3)	−0.123 (2)	0.675 (2)	0.056 (7)*
H8A	0.129 (3)	0.064 (2)	0.748 (2)	0.069 (7)*
H8B	0.359 (3)	0.112 (2)	0.791 (2)	0.056 (7)*
H11	0.250 (3)	0.360 (2)	0.794 (2)	0.051 (6)*
H14	0.249 (3)	0.321 (2)	0.395 (2)	0.062 (7)*
H13	0.238 (3)	0.540 (3)	0.548 (2)	0.067 (7)*
H16	0.252 (3)	−0.104 (3)	0.453 (2)	0.069 (8)*

Atomic displacement parameters (Ų)

	U^11	U^22	U^33	U^12	U^13	U^23
N1	0.0699 (16)	0.0382 (14)	0.0561 (15)	0.0199 (11)	0.0242 (13)	0.0224 (12)
N2	0.0695 (13)	0.0595 (16)	0.0422 (13)	0.0134 (10)	0.0188 (11)	0.0213 (12)
O1	0.0824 (11)	0.0379 (11)	0.0620 (11)	0.0245 (8)	0.0337 (9)	0.0296 (9)
O2	0.1189 (14)	0.0511 (12)	0.0653 (12)	0.0333 (10)	0.0526 (11)	0.0340 (10)
O3	0.0702 (10)	0.0449 (10)	0.0571 (10)	0.0217 (8)	0.0327 (9)	0.0294 (8)
O4	0.0643 (9)	0.0426 (10)	0.0717 (11)	0.0251 (8)	0.0336 (8)	0.0352 (9)
O5	0.1139 (15)	0.0494 (13)	0.0589 (13)	0.0287 (10)	0.0290 (11)	0.0293 (11)
C1	0.0473 (12)	0.0350 (14)	0.0469 (14)	0.0126 (10)	0.0168 (11)	0.0198 (12)
C2	0.0528 (15)	0.0377 (14)	0.0511 (15)	0.0154 (11)	0.0183 (13)	0.0242 (12)
C3	0.0544 (15)	0.0345 (14)	0.0503 (15)	0.0126 (11)	0.0160 (13)	0.0229 (12)
C4	0.0590 (16)	0.0359 (14)	0.0552 (16)	0.0157 (11)	0.0218 (14)	0.0264 (12)
C5	0.0599 (16)	0.0354 (14)	0.0475 (15)	0.0137 (11)	0.0199 (14)	0.0225 (12)
C6	0.0451 (12)	0.0313 (13)	0.0423 (13)	0.0067 (10)	0.0103 (11)	0.0186 (11)
C7	0.0565 (16)	0.0475 (16)	0.0502 (15)	0.0154 (12)	0.0207 (13)	0.0250 (13)
C8	0.0571 (16)	0.0412 (15)	0.0470 (15)	0.0109 (12)	0.0152 (13)	0.0199 (12)
C9	0.0448 (12)	0.0440 (15)	0.0450 (14)	0.0125 (10)	0.0147 (10)	0.0220 (12)
C10	0.0435 (12)	0.0472 (15)	0.0423 (13)	0.0099 (10)	0.0131 (10)	0.0232 (12)
C11	0.0542 (14)	0.0466 (16)	0.0425 (14)	0.0148 (10)	0.0180 (11)	0.0235 (13)
C12	0.0635 (15)	0.0442 (17)	0.0502 (15)	0.0137 (11)	0.0151 (12)	0.0213 (13)
C13	0.0840 (18)	0.0558 (19)	0.0565 (17)	0.0152 (14)	0.0174 (14)	0.0358 (16)
C14	0.0804 (18)	0.064 (2)	0.0460 (16)	0.0101 (13)	0.0168 (14)	0.0328 (16)
C15	0.0506 (13)	0.0493 (16)	0.0423 (14)	0.0103 (10)	0.0130 (11)	0.0203 (13)
C16	0.0583 (15)	0.0474 (18)	0.0494 (16)	0.0117 (11)	0.0161 (12)	0.0180 (14)

Geometric parameters (Å, º)

N1—C7	1.483 (3)	C5—H5A	1.02 (2)
N1—H1A	0.99 (3)	C5—H5B	0.93 (2)
N1—H1B	1.00 (3)	C6—C5	1.508 (3)
N1—H1C	0.90 (3)	C7—H7A	1.02 (2)
N2—C16	1.381 (3)	C7—H7B	0.95 (2)
N2—H2	0.89 (3)	C8—C7	1.510 (3)
O1—H1	1.04 (3)	C8—C9	1.490 (3)
O3—C6	1.245 (2)	C8—H8A	1.03 (2)
O4—C6	1.271 (2)	C8—H8B	0.96 (2)
O5—H5	0.84 (3)	C9—C16	1.368 (3)
O5—C12	1.380 (3)	C10—C9	1.427 (3)
C1—O1	1.305 (3)	C10—C11	1.402 (3)
C1—O2	1.217 (3)	C11—C12	1.371 (3)
C1—C2	1.493 (3)	C11—H11	0.99 (2)
C2—C3	1.511 (3)	C12—C13	1.400 (3)
C2—H2A	0.98 (2)	C13—C14	1.367 (4)
C2—H2B	1.00 (2)	C13—H13	1.02 (2)
C3—H3A	0.98 (2)	C14—H14	0.91 (2)
C3—H3B	0.94 (2)	C15—N2	1.377 (3)
C4—C3	1.512 (3)	C15—C10	1.411 (3)
C4—C5	1.520 (3)	C15—C14	1.385 (3)
C4—H4A	0.98 (2)	C16—H16	1.00 (3)
C4—H4B	0.99 (2)
N1—C7—C8	110.1 (2)	C8—C7—H7A	111.4 (11)
N1—C7—H7A	105.7 (12)	C8—C7—H7B	113.9 (13)
N1—C7—H7B	105.1 (13)	C9—C8—C7	113.9 (2)
N2—C15—C10	107.3 (2)	C9—C8—H8A	111.8 (13)
N2—C15—C14	130.9 (2)	C9—C8—H8B	110.9 (14)
N2—C16—H16	119.2 (15)	C9—C16—N2	109.9 (2)
O1—C1—C2	115.3 (2)	C9—C16—H16	130.8 (15)
O2—C1—O1	121.8 (2)	C10—C9—C8	124.5 (2)
O2—C1—C2	122.9 (2)	C10—C11—H11	120.5 (12)
O3—C6—O4	122.7 (2)	C11—C10—C15	119.0 (2)
O3—C6—C5	120.0 (2)	C11—C10—C9	133.4 (2)
O4—C6—C5	117.3 (2)	C11—C12—O5	122.2 (2)
O5—C12—C13	116.0 (2)	C11—C12—C13	121.8 (2)
C1—O1—H1	112.4 (14)	C12—O5—H5	112 (2)
C1—C2—C3	113.5 (2)	C12—C11—C10	118.4 (2)
C1—C2—H2A	106.7 (12)	C12—C11—H11	121.1 (12)
C1—C2—H2B	108.4 (13)	C12—C13—H13	118.1 (14)
C2—C3—C4	114.2 (2)	C13—C14—C15	118.3 (3)
C2—C3—H3A	109.8 (12)	C13—C14—H14	120.9 (15)
C2—C3—H3B	108.9 (14)	C14—C15—C10	121.8 (2)
C3—C2—H2A	112.2 (12)	C14—C13—C12	120.7 (3)
C3—C2—H2B	111.8 (14)	C14—C13—H13	121.1 (14)
C3—C4—C5	111.8 (2)	C15—N2—C16	108.7 (2)
C3—C4—H4A	109.7 (13)	C15—N2—H2	124 (2)
C3—C4—H4B	108.9 (14)	C15—C10—C9	107.5 (2)
C4—C3—H3A	109.5 (12)	C15—C14—H14	120.7 (16)
C4—C3—H3B	109.4 (14)	C16—N2—H2	126 (2)
C4—C5—H5A	108.4 (12)	C16—C9—C10	106.5 (2)
C4—C5—H5B	108.8 (15)	C16—C9—C8	128.9 (2)
C5—C4—H4A	110.0 (13)	H1A—N1—H1B	108 (2)
C5—C4—H4B	110.4 (13)	H1A—N1—H1C	102 (3)
C6—C5—C4	116.0 (2)	H1B—N1—H1C	112 (2)
C6—C5—H5A	106.4 (12)	H2A—C2—H2B	103.7 (19)
C6—C5—H5B	109.5 (15)	H3A—C3—H3B	104.6 (19)
C7—N1—H1A	112.9 (16)	H4A—C4—H4B	106 (2)
C7—N1—H1B	107.4 (14)	H5A—C5—H5B	107 (2)
C7—N1—H1C	115 (2)	H7A—C7—H7B	110.1 (18)
C7—C8—H8A	106.9 (13)	H8A—C8—H8B	103.9 (19)
C7—C8—H8B	108.9 (13)

Hydrogen-bond geometry (Å, º)

D—H···A	D—H	H···A	D···A	D—H···A
O1—H1···O4^i	1.04 (3)	1.53 (3)	2.569 (2)	172 (2)
N1—H1A···O3^ii	0.99 (3)	1.80 (3)	2.762 (3)	163 (2)
N1—H1B···O5^iii	1.00 (3)	1.80 (3)	2.782 (3)	166 (2)
O5—H5···O2^iv	0.84 (3)	1.82 (3)	2.647 (3)	164 (3)

Symmetry codes: (i) x, y+1, z; (ii) −x, −y, −z+1; (iii) x, y−1, z; (iv) x, y, z+1.