New insights in the discovery of novel h-MAO-B inhibitors: structural characterization of a series of N-phenyl-4-oxo-4H-chromene-3-carboxamide derivatives

N-(Substituted phenyl)-4-oxo-4H-chromene-3-carboxamides have very similar conformations but show different inhibition activities against h-MAO-B so it may be assumed that the electronic environment provided by the substituents on the phenyl ring is the primary condition for the pharmacological activities displayed by these molecules.


Chemical context
Chromones are a group of natural and synthetic oxygen heterocyclic compounds having a high degree of chemical diversity that is frequently linked to a broad array of biological activities. The chromone-3-(phenyl)carboxamide derivatives, depicted the scheme, have emerged as promising compounds for the management of neurodegenerative diseases such as Alzheimer's and Parkinson's since they display selective inhibition activities against h-MAO-B. Recent data  suggest that the activity and selectivity towards that enzyme is dependent on the nature and position of the substituent located in the exocyclic phenyl ring. When compared with the unsubstituted compound (1), the para substitution in the exocyclic phenyl ring seems to play an important role in the enzymatic interaction: the presence of para-Cl (4c) and -CH 3 (4d) substituents favours the potency while an -OH (4e) substituent has the opposite effect. The data acquired so far point out the importance of a structureactivity relationship study to optimize the potency vs selectivity of this type of inhibitor, namely performing structural and electronic changes in the substituents.

Conformations and intramolecular hydrogen-bond network
The structural analysis confirms that the molecules are 4chromone derivatives with a phenylamide substituent on position number 3 of the pyrone ring. A view of the asymmetric unit of (2b) with the atom-numbering scheme. Displacement ellipsoids are drawn at the 70% probability level.

Figure 2
A view of the asymmetric unit of (3a) with the atom-numbering scheme. Displacement ellipsoids are drawn at the 70% probability level.

Figure 3
A view of the asymmetric unit of (3b) with the atom-numbering scheme. Displacement ellipsoids are drawn at the 70% probability level.

Figure 4
A view of the asymmetric unit of (4a) with the atom-numbering scheme. Displacement ellipsoids are drawn at the 70% probability level.

Figure 5
A view of the asymmetric unit of (4d) with the atom-numbering scheme. Displacement ellipsoids are drawn at the 70% probability level. displacement ellipsoid diagrams with the adopted labelling scheme for (2b), (3a), (3b), (4a), (4d) and (4e), the structurally characterized compounds in this work. As seen, the molecules exhibit an anti conformation with respect to the C-N rotamer of the amide following a pattern given by compound (1), which was previously described by Cagide et al. (2015). Due to the asymmetry of the chromone residue, the anti conformation can assume several geometries depending on the relative position of the carbonyl groups of the chromone ring and the amide group which can be cis or trans related. Compounds (1)-(4) exhibit a trans relation between these bonds as can be seen in Figs. 1 to 6. This molecular conformation allows the establishment of two or three intramolecular hydrogen bonds. Details of the intramolecular hydrogen bonding are given in Tables 2-7. Generally, as seen in the scheme below, there is an intramolecular hydrogen bond involving the amide and the chromone where the amide nitrogen atom acts as donor to the oxo oxygen atom of the chromone ring, forming an S(6) ring; the carboxyl oxygen of the amide acts as acceptor for a weak H interaction with the C-H group located at the ortho position of the phenyl ring, forming another S(6) ring. This hydrogenbonding network probably enhances the planarity of the molecules and may prevent them from adopting some other possible conformations by restraining their geometries. Compounds (2a) and (2b) have substituents located at the ortho position on the benzyl ring with oxygen atoms (methoxy and nitro, respectively) that act as acceptors for the amide nitrogen atom of the carboxamide residue, hence forming a third intramolecular hydrogen bond (see scheme).

Molecular geometries
The values for bond lengths involving the atoms of the carboxamide residue assume the expected ranges for amides with aromatic substituents. The C3-C31 bond ranges from 1.49 to 1.51 Å , which are the typical range values for an Csp 3 -Csp 3 bond (Allen et al., 1987). The C31-O3 bond lengths range from 1.22 to 1.25 Å and the C31-N3 bond lengths are within the 1.33 to 1.37 Å interval, showing the the partial sp 2 character of the amide nitrogen atom attributed to those compounds. Table 1 details selected dihedral angles between the mean planes of aromatic rings, Chr-Phe , between the chromone ring and the amide moiety (the plane defined by atoms O3, C31and N3), Chr-amide , and between the exocyclic phenyl ring and the amide, Phe-amide . Those dihedral angles are primarily due to the rotation of the rings around the C3-C31 and N3-C311 bonds with exception of (3a) that assumes mainly a bent conformation between the rings. The structural analysis of (1) performed previously  revealed that the amide moiety is practically planar with the chromone ring: it makes a dihedral angle of 4.31 (12) with the plane defined by the O, C and N atoms of the amide residue. The loss of planarity for the overall molecule results from the slight twist of the exocyclic phenyl substituent around the amidic N-C bond, which is the main factor affecting the value for the dihedral angle of 9.48 (12) between the best plane of the exocyclic phenyl ring and the O-C-N amidic plane. The dihedral angle between the mean plane of the chromone ring and that of the exocyclic phenyl ring is 10.77 (4) . The Chr-amide dihedral angles for the substituted compounds are A view of the asymmetric unit of (4e) with the atom-numbering scheme. Displacement ellipsoids are drawn at the 70% probability level. Table 1 Selected dihedral angles ( ).
Chr-Phe is the dihedral angle between the mean planes of the chromene and the phenyl ring. Chr-amide is the dihedral angle between the mean planes of the chromone ring and the plane defined by atoms O3, C31 and N3. amide-Phe is the dihedral angle between the mean planes of the phenyl ring and the plane defined by atoms O3, C31 and N3. The suffices A and B for compound (2a) denote the polymeric forms. Basic Conf. denotes the primary shape given by the relative position of the aromatic rings around the carboxamide linkage.
( below 15 for all the compounds, suggesting that the amide moiety is essentially planar with the chromone ring. The strong N3-H3Á Á ÁO4 hydrogen contact may preclude higher rotations around the C3-C31 bond in spite of its Csp 3 -Csp 3 character. The Phe-amide angles present more widely spread values, ranging between 2 and 33 . The substituents with oxygen atoms located at the ortho position on the exocyclic phenyl ring in (2) which, simultaneously, cause steric hindrance and act as acceptors for the hydrogen atom of the amide, thus forming an intramolecular hydrogen bond, suggest that a tricky balance between those two factors allows the formation of several energetically accessible rotated conformations. This fact is especially noticeable in the various conformation polymorphs of (2a). The remaining compounds are not constrained by steric hindrance of the ortho-substituents but they still present a wide range of values for the Phe-amide dihedral angles (between 3 and 24 ). The Chr-Phe values may be used as a measure of the relative positioning of the two aromatic rings which may define the primary conformation for the molecules.
The aromatic rings are usually rotated or co-planar, with exception of (3a) where they are bent with respect to each other. The chromones with halogen substituents assume the most planar conformations, probably related to the typical positive mesomeric effects on the system. Considering the fact that the para-substituent on the exocyclic phenyl ring for chromone-3-phenylcarboxamides has a positive effect on their activity, and the requirement of establishing the factors that can modulate the enzyme-ligand interaction, it can be assumed their h-MAO-B activity is strongly dependent on the electronic environment of the substituent. This is not a preferred conformation that reduces or enhances the activity, so it may be assumed that the electronic environment provided by the substituent is the primary condition for the pharmacological activities displayed by those molecules.

Supramolecular features
The carboxamide H atom is not involved in any intermolecular interaction in any of the compounds.

Figure 7
View of the sheet formed by the interconnection of three C-HÁ Á ÁO hydrogen bonded chains in compound (2b). Hydrogen atoms not involved in the hydrogen bonding have been omitted for clarity.

Figure 8
View of the dimer formed across the inversion centre ( 1 2 , 1 2 , 1 2 ) in (3a). Hydrogen atoms not involved in the hydrogen bonding have been omitted for clarity.
A common feature found for compounds with para substituents, (4a)-(4d) is the formation of a ladder structure composed of molecules propagated by unit axial translations involving intermolecular hydrogen bonds between C2 and O4 of the chromone ring and the C atom located at the ortho position of the exocyclic phenyl ring and the carboxamide O atom. This is also found in (1) and in compound (3b), which has a Br substituent located at the meta position, in which the ladder structure is supplemented by an intermolecular hydrogen bond between C5 and O1 of the chromone moiety. In (4a), the molecules are linked by C2-H2Á Á ÁO4 (x, y À 1, z) and C316-H316Á Á ÁO3 (x, y + 1, z) hydrogen bonds, forming R 2 2 (13) rings structures which are propagated along the b-axis direction by unit translation (Table 5 and Fig. 10). In (4d), the molecules are linked by C2-H2Á Á ÁO4(x + 1, y, z) and C316-H316Á Á ÁO3(x À 1, y, z) hydrogen bonds, forming R 2 2 (13) ring structures which are propagated along the a-axis direction by unit translation (Table 6 and Fig. 11).

Figure 9
View of the two independent ladders formed linked R 2 2 (13) rings which run parallel to the a axis in compound (3b). Hydrogen atoms not involved in the hydrogen bonding have been omitted for clarity. [Symmetry codes (bottom to top): x À 1, y, z; x, y, z; x + 1, y, z.]

Synthesis and crystallization
The compounds were obtained by synthetic strategies described elsewhere (Cagide et al., 2011). Chromone-3carboxamide derivatives were synthesized using chromone-3carboxylic acid as starting material which, after in situ activation with phosphorus(V) oxychloride (POCl 3 ) in dimethylformamide, react with the different substituted anilines. Crystals were recrystallized from ethylacetate forming colourless plates whose dimensions are given in Table 9.

Refinement
Crystal data, data collection and structure refinement details are summarized in Table 9.
In (3b) there are two molecules in the asymmetric unit. The largest difference map peaks are associated with the Br atoms.
In all compounds, H atoms attached to C atoms were treated as riding atoms with C-H(aromatic) = 0.95 Å with U iso (H) = 1.2U eq (C); C-H(methyl), = 0.98 Å with U iso = 1.5U eq (C). In all compounds, the amino H atoms were refined with the exception of (3b) where these atoms were refined as riding atoms with N-H = 0.88 Å with U iso = 1.2U eq (C) and in (4e) in which the positional parameters of the amino and hydroxyl H atoms were refined but their U iso values were View of the ladder formed by the linked R 2 2 (13) rings which run parallel to the a axis in compound (4d). Hydrogen atoms not involved in the hydrogen bonding have been omitted for clarity. [Symmetry codes (bottom to top): x À 1, y, z; x, y, z; x + 1, y, z.]

Figure 12
View of the sheet formed by the interconnection of three C-HÁ Á ÁO hydrogen-bonded chains in compound (4e). Hydrogen atoms not involved in the hydrogen bonding have been omitted for clarity.
Cg1, Cg2 and Cg3(Cg7) are the centroids of the pyrone, of the chromone phenyl and of the carboxamide phenyl rings, respectively. * indicates contacts in which the planes involved are inclined to each other, the perpendicular distance between the planes is an average value and the angle between the planes is given in place of a slippage. Only interplanar interactions with CgÁ Á ÁCg distances less than or equal to 4.0 Å or with angles between the planes of less than 10 are included.  constrained to be U iso (N) = 1.2U eq (N) and U iso (O)b= 1.5U eq (O). The final positions of these atoms were checked in a difference Fourier map, as were the positions of the H atoms in any methyl groups. The quality of the crystals for (4e) was poor and the crystals were twinned. The completeness is 97%. The crystal studied was refined as a two-component twin [twin law: 2-axis (001)