6,6′-Dimethyl-1,1′,5,5′-tetraaza-6,6′-bi(bicyclo[3.1.0]hexane): synthesis and investigation of molecular structure by quantum-chemical calculations, NMR spectroscopy and X-ray diffraction analysis

A new diaziridine derivative with two bicyclic diaziridine-containing moieties in one molecule, 6,6′-dimethyl-1,1′,5,5′-tetraaza-6,6′-bi(bicyclo[3.1.0]hexane) (BiDiMDAH), has been synthesized for the first time. Its molecular structure has been investigated in the gas phase by means of quantum-chemical (QC) calculations, in CDCl3 solution by 1D and 2D NMR spectroscopy and in the solid state by the X-ray diffraction (XRD) technique. It was found by QC calculations that BiDiMDAH molecule in a free state presents a single conformer of C2 symmetry with trans orientation of the methyl groups about two carbons connecting diaziridine cycles. The 5-memebered rings in BiDiMDAH molecule has been found to be essentially planar. In order to explain conformational preferences of BiDiMDAH, natural bond orbitals (NBO) and atoms in molecules (AIM) analyses have been performed. According to the XRD data, BiDiMDAH crystallizes in space group P21/c with molecular structure resembling the structure obtained by the means of QC calculations for the lowest energy form. However, the relative arrangement of the methyl groups of the solid BiDiMDAH molecule is similar to that of the one of the transition states. The data of NMR spectroscopy has revealed that only one conformer is present in CDCl3 solution in agreement with the data of QC and XRD studies. The gaseous standard enthalpy of formation of BiDiMDAH has been estimated to be 106.1±1.2 kcal/mol by means of G4 theory.

Earlier we studied the molecular structures and peculiarity of conformational behavior of bicyclic diaziridine derivatives-1,5-diazabicyclo[3.1.0]hexane 1 (DABH) by the gas electron diffraction (GED) method [7] and 6.6′-bis(1,5diazabicyclo[3.1.0]hexane) 2-by the X-ray diffraction (XRD) [1] and the gas electron diffraction (GED) [4] methods (Fig. 1). It was shown that the bicyclic fragment of these compounds exists exclusively as a boat conformation of C s symmetry at 20°C and the Natural Bond Orbital (NBO) analysis showed that the most important stabilization factor in the boat conformation is the n(N) → σ*(C-C) anomeric effect. We also investigated the molecular structure of 6,6-dimethyl-1,5-diazabicyclo[3.1.0.]hexane 3 by the GED and the XRD methods [3]. The introduction of two methyl substituents to the carbon atom of diaziridine cycle of compound 3 makes the pyrazolidine moiety of this substance almost planar. The obtained results demonstrate great influence of substituents on the conformation of the DABH system.
The direction of action on the central nervous system of diaziridine derivatives also depends on the type of substituents in the diaziridine ring [16]. In particular, it was found that the neurotropic activity of diaziridine derivatives increases when the second diaziridine ring is introduced [15]. Recently, we have studied the molecular structure of tetramezine [1,2-bis-(3,3dimethyldiaziridin-1-yl)ethane], containing two diaziridine cycles in one molecule, by the GED method together with vibrational spectroscopy and quantum chemical calculations [12]. Herein the results of theoretical and experimental investigations of molecular structure of 6,6′-dimethyl-1,1′,5,5′-tetraaza-6,6′bi(bicyclo[3.1.0]hexane) (BiDiMDAH) (4), containing two DABH cycles in one molecule, by the means of QC calculations, NMR spectroscopy and XRD analysis are presented.

General remarks
BiDiMDAH was synthesized for the first time in the N. D. Zelinsky Institute of Organic Chemistry. All starting materials were purchased from ACROS catalog and were used as purchased. The structure of BiDiMDAH was elucidated with the help of IR spectrum, high-resolution mass spectrum (HRMS), mass spectrum under electron impulse (EI), NMR spectra, and XRD diffraction analysis. The IR spectrum was recorded on InfraLUM FT-801 and Bruker "Alpha" spectrometer in the range 400-4000 cm −1 (resolution 2 cm −1 ). HRMS was recorded on a Bruker micro TOF-QTM spectrometer with electrospray ionization (ESI). All measurements were performed in a positive (+MS) ion mode (interface capillary voltage: 4500 V) with scan range m/z: 50-3000. External calibration of the mass spectrometer was performed with Electrospray Calibrant Solution (Fluka). A direct syringe injection was used for all analyzed solutions in MeCN (flow rate: 3 μL min −1 ). Mass spectrum EI were recorded using a Finnigan MAT INCOS-50 instrument. All NMR spectra were recorded on Bruker AV-600 spectrometer equipped with a Zgradient broadband observe probe at room temperature. The X-ray diffraction experiment for a single crystal of BiDiMDAH was carried out on a "Bruker APEX-II CCD" diffractometer at 120 K during data collection.

Synthesis of BiDiMDAH
The solution of 2,3-butanedione (1.76 ml, 20 mmol) in 5 ml H 2 O was added dropwise to the solution of 1,3diaminopropane (3.02 ml, 40 mmol) in 50 ml H 2 O at stirring and at the temperature of 0-10°С. Then the aqueous solutions 15% HCl and 12% NaOCl were added dropwise to the reaction mixture to adjust pH=11.0. The reaction mixture was stirred at 0-5°С and pH value not exceeding 11.5-12.0 (which was supported by addition of 20% aqueous solution NaOH when it was necessary) for 12 h. Then the temperature was raised to 20°С, and the reaction mixture was saturated with NaCl and extracted with CHCl 3 (50 ml × 3). The organic layer was dried over K 2 CO 3 , the solvent was evaporated, and the residue was recrystallized from acetone. Yield 0.41 g (11%), colorless solid, mp = 187-188°C. The IR and mass spectra of BiDiMDAH are deposited in the ESI.

NMR experiments
A 25-mg portion of the sample was dissolved in 0.6 mL of CDCl 3 . The 1 H and 13 C chemical shifts (δ), for which values are reported in ppm, were calibrated against the residual protons (7.27 ppm) and carbon atoms (77.00 ppm) of the CDCl 3 . The coupling constants, J, are given in Hz. Multiplicities are indicated by s (singlet), d (doublet), and q (quartet). The 1D spectra of BiDiMDAH are presented in the ESI as Fig. S1 and

XRD experiments
Single crystals of C 10 H 18 N 4 were grown from acetone. The structure was solved with Olex2 [49] and Olex2.solve [50] program software, applying Charge Flipping and refined with the XL [51] refinement package using Least Squares minimization. The conditions of the X-ray diffraction experiment and the refined cell constants for BiDiMDABH are listed in    Table S4, anisotropic displacement parameters as  Table S5, and hydrogen atom coordinates as Table S6, respectively. For XRD atom numbering, see Fig. S7 of the ESI.

Results and discussion
According to the data of QC computations, a free molecule of BiDiMDAH exists as single conformation I of C 2 symmetry with trans orientation of the methyl groups on the C-C bridge connecting two diaziridine cycles (Fig. 3). BiDiMDAH species with cis orientation of the methyl groups were found to be transition states (TS3 and TS4, see Fig. 3). The TS4 has a similar molecular structure to the TS3 but differs in the orientation of one of the methyl groups which is rotated around the NC-CH 3 bond by about 60°in comparison to TS3. The TS3 and TS4 are more than 15 kcal mol −1 higher in energy than the main conformer I (see Table 2).
The instability of the TS3 and TS4 may be connected with unfavorable electrostatic interactions of nitrogen atoms (the corresponding NPA charges were calculated to be~−0.4 e).
In order to obtain a qualitative measure of overall atom-atom electrostatics, NBO Natural Columbic Energy (NCE) concept Among BiDiMDAH species with trans orientation of the methyl groups, two low-lying transition states were found (TS1 and TS2, see Fig. 3). The structures of these transition states differ from those of conformer I in the orientation of the methyl groups (see Fig. 3). Thus, in the TS1, one of the methyl groups is rotated around the corresponding NC-CH 3 bond by about 60°in comparison with the lowest energy conformation (I) and in the TS2 the both methyl groups are rotated around the NC-CH 3 bond by  about 60°in comparison with the lowest energy conformation (I). The rotational analysis around one of the NC-CH 3 bonds of conformer I is presented in Fig. 4. The instability of the TS1 and TS2 may be connected with repulsions of the hydrogen atoms of the methyl groups and the hydrogen atoms of the five-membered rings.
The computed geometrical parameters of a stable BiDiMDAH conformation at B3LYP/6-31G(d,p), B3LYP/ cc-pVTZ, and MP2/cc-pVTZ levels of theory and the experimental values obtained by the XRD diffraction for the solid state are given in Table 3.
The average values of BiDiMDAH bond lengths and bond angles obtained by QC calculations are similar to those for solid state (see Table 3); the computed values of torsion angles are in most cases particularly the same as that in the crystal. However, there is a noticeable difference within the 5memberred ring: the values of the N5-N1-C2-C3 and the C2-C3-C4-N5 torsion angles are by about 8°and 11°bigger in the crystal than in a free molecule. The greatest differences between the QC and the XRD molecular structures are observed in the positions of the hydrogen atoms of both methyl groups (confer Fig S7 of the ESI). In the crystal phase, both methyl groups are rotated around the corresponding NC-CH 3 bonds by about 50°in comparison with the lowest energy conformation I (see Table 3). Moreover, the structure of BiDiMDAH in the solid state resembles the structure of one of the low-lying transition states of a free molecule, TS2, which is by about 2 kcal mol −1 higher than the main conformer (see Table 2). It can be noted that the crystal structure is stabilized mainly by short contacts, which realize between the H atoms of the methyl groups and the N atoms of the neighboring molecules.
According to the results of QC computations and XRD analysis, the essentially planar skeletons of the 5-ring moieties are observed. This fact may be caused by the two possible tendencies. The first tendency is connected with the n(N)→σ*(C-C) and the n(N) →σ*(C-H) anomeric effects, stabilizing the boat conformation of the 6membered rings. Thus, the lone pair of the N1 atom interacts with the C2-C3 and the C2-H16 antibonding orbitals (n(N1)→σ * (C2-C3 has E(2)=3.6 kcal mol −1 (Fig. 5) and the n(N1) → σ * (C2-H16) interaction has E(2) = 4.3 kcal mol −1 ) and the lone pair of the N5 atom interacts with the C3-C4 and the C4-H20 antibonding orbitals (n(N5) → σ * (C3-C4) has E(2) = 3.6 kcal mol −1 and the n(N5) → σ * (C4-H20) interaction has E(2) = 4.3 kcal mol −1 ). The second tendency is the steric repulsion of the H21 atom of the methyl group and the H17, the H19, and the H15 atoms of the 5-membered ring. Thus, NBO steric analysis reveals the corresponding pairwise steric exchange energies: 2.3 kcal mol −1 for the C7-H21 and the C3-H17 bonds, 1.6 kcal mol −1 for the C7-H21 and the C4-H19 bonds, and 1.2 kcal mol −1 for the C7-H21 and the C2-H15 bonds, respectively. The interatomic distance between H21 and H17 is equal to 2.06 Å according to B3LYP/6-31G(d,p), 2.08 Å according to B3LYP/ cc-PVTZ, and 2.00 Å according to MP2/cc-PVTZ levels of theory. That is a little bit less than the sum of the two atomic Wan der Waals radii [52] or natural atomic Wan der Waals radii of the H atoms [43]. However, AIM computations did not reveal a critical point between the corresponding H atoms as well as natural bond critical point analysis [53] applied as implemented into NBO 7.0 program. Therefore, it seems that the steric repulsion of the H atoms is the leading force, which causes the flattering of the 5-membered ring in order to achieve the minimal H…H distance which the sum of the Wan der Waals radii permit.
The structure of BiDiMDAH was compared with the structures of similar compounds (see Table 4). The largest difference was found for the value of the torsion angle N5-N1-C2-C3 in BiDiMDAH and compounds 1 and 2. Compounds 1 Fig. 4 Potential function for the internal rotation of the CH 3 group about the C6-C7 bond in conformer I and 2 lack the methyl group and subsequently the repulsion of the corresponding hydrogen atoms as in BiDiMDAH, leading to the flattening of the 5-membered ring, is missing. It is worth to notice that the N5-N1-C2-C3 torsion angle in BiDiMDAH and compound 3 is practically the same (the 5-membered ring moiety of compound 3 is also nearly flat). This is probably connected with the presence of the methyl groups in compound 3 and the corresponding H…H repulsions as supposed for BiDiMDAH molecule.
The analysis of the 1 H and 13 C 1D NMR spectra of BiDiMDAH showed that in solution, the compound exists only in a single conformation (i.e., there are clear peaks of only one compound, Fig. S1 and S2, for 1 H and 13 C 1D NMR spectra, respectively).
The data of { 1 H-13 C}HSQC and { 1 H-1 Н}gNOESY 2D spectra helped to enable all protons and carbon atoms to be assigned together with their inter relationships. The results of the heteronuclear correlation NMR { 1 H-13 C}HSQC and { 1 H-13 C}HMBC spectra are presented in Table 5. These spectra show how far away each proton was found from a particular carbon atom, respectively, through one or 2-3 bonds.  The 2D NMR spectrum { 1 H-1 H}gNOESY shows the spatial arrangement of protons relative to each other ( Table 6, Fig.  S5 of the ESI). Protons H15, H17, and H19 have cross-peaks with proton H21 (Fig. 6).
The detected cross-peaks of proton H17 with proton H21 (nuclear Overheuser effect) confirms of the endo-position of the methyl groups at the C ring atom in the diaziridine moiety.
Thus, the results of 1D and 2D NMR spectra reveal that in CDCl 3 solution BiDiMDAH molecule exists only in a single conformation and the distances between H21 proton of the methyl group and the protons H17 of the ring moiety are small enough to produce nuclear Overheuser effect. This fact supports that 6-membered rings of BiDiMDAH do not adopt a chair conformation, but rather a flattened boat form is preferred.

Conclusions
In summary, a new bicyclic diaziridine derivative 6,6′-dimethyl-1,1′,5,5′-tetraaza-6,6′-bi(bicyclo[3.1.0]hexane) (BiDiMDAH) was synthesized for the first time and its molecular structure was studied by QC calculations, NMR spectroscopy, and XRD analysis. The molecular structures of BiDiMDAH obtained by the means of QC computations and the XRD analysis were found to be quite similar with trans orientation of the methyl groups and an essentially planar 5-ring moieties. It has been shown that one of the low-energy transition states of a free molecule has such relative arrangement of methyl groups which is  observed in the crystalline BiDiMDAH. Two competing effects were found to define the resulting molecular structure of BiDiMDAH: the hyperconjugation effect supporting a boat conformation of the both 6-membered rings, and the effect of steric repulsion of the terminal hydrogen atoms of the methyl groups with the corresponding hydrogen atoms of 5-membered cycles, leading to a planarity of 5-membered ring moieties. The results obtained for the CDCl 3 solution by NMR method are in agreement with those obtained by means of QC computations.