Crystal Structure of the 5-chloro Salicylamides: Three Different Types of the H-bonding Influenced Linear Chain Formation in the Solid State

Three N-substituted 5-chlorosalicylamides (4-chlorophenyl, 2a; benzyl, 2b; phenethyl 2c) differing in the length of the 'linker' between the benzene ring and the amide moiety were prepared in order to compare their supramolecular architecture. The intramolecular NH· · · O(H) hydrogen bond and the intermolecular C=O· · · H–O hydrogen bond were found in the crystal structure of 2a and 2c thus forming an infinite linear chain. Compound 2b had a different arrangement with the intramolecular C=O· · · H–O hydrogen bond and another intermolecular NH· · · O(H) hydrogen forming a linear infinite chain.

Although the antibacterial activity involves multiple mechanisms, these compounds have been shown to be inhibitors of the two-component regulatory systems (TCS) of bacteria [7][8][9].The most recent studies identified them as selective inhibitors of interleukin-12p40 production [10,11] and inhibitors of the protein kinase epidermal growth factor receptor (EGFR PTK) [12] as well as cell permeable inhibitors of poly(ADP-ribose) glycohydrolase (PARG) [13].
The classical approach for the synthesis of N-substituted 2-hydroxybenzamides is based on the refluxing of substituted salicylic acid with appropriate amine (aniline, benzylamine, phenethylamine) in chlorobenzene in the presence of phosphorus trichloride for several hours [14,15].Other synthetic approaches were also reported [16] as microwave assisted synthesis, which reduced total reaction time from several hours to minutes with comparable yields [17].

Results and Discussion
During our research that focused on the antimycobacterial properties of salicylanilide derivatives, we prepared their various O-substituted derivatives such as acetates [18], N-protected amino acid esters [19,20] and carbamates [21].The preparation of the starting salicylanilides was a routine step of the synthetic pathway.Several approaches were tested from which the microwave-assisted synthesis seemed to be optimal (Scheme 1).This procedure is quick and products can be isolated in high yields.Simple crystallization from absolute ethanol is usually used for final purification of the desired salicylamides.

Scheme 1.
Microwave-assisted synthesis of studied salicylamides.In the next study, we focused on investigating the comparison of crystal structures of three different amides of 5-chlorosalicylic acid, namely 5-chloro-2-hydroxy-N-(4-chlorophenyl)benzamide (2a), 5-chloro-2-hydroxy-N-benzylbenzamide (2b) and 5-chloro-2-hydroxy-N-phenethylbenzamide (2c) whose selected structural parameters are summarized in Tables 1 and 2.  As early as 1961, Tamura et al. suggested [22] that polymorphism observed in the crystals of most amides is related to the varied types of molecular arrangement connected by different types of hydrogen bonds.It is also known that the molecules of salicylamides and their thioanalogues can exist in at least two possible configurations due to both intramolecular and intermolecular hydrogen bonds.These two configurations are occasionally labeled as α-and β-form [23], rotameric forms or "open-ring" and "closed-ring" tautomers [24,25] (Figure 1) Sometimes s-cis and s-trans conformers of the α-form are distinguished [26] although according to IUPAC recommendations from 1996 [27] such prefixes should not be applied to N-substituted amides (E/Z-convention is correct).It is widely believed [25] that "closed-ring" tautomers, which are thermodynamically favorable in solution for parent salicylamide [28] as well as for substituted salicylanilides [25,26], are responsible for their inhibition activity mentioned in the introduction.However the situation is completely different in the solid phase.Regardless of substitution on both aromatic rings, all known salicylanilides [29][30][31][32][33][34][35][36][37][38][39] exclusively exist in the "open-ring" β-form.If two stable polymorphs exist [32], then they differ only in their packing patterns and not in their hydrogen bonds patterns.
In the case of compound 2a where the p-chlorophenyl ring is directly attached to the amide nitrogen atom the molecule is nearly planar (dihedral angle between both benzene rings is only 4.96 °) with the intramolecular NH• • • O(H) bond between the amide and hydroxy groups (Figure 2) thus forming an "open-ring" β-form.Another type of the H-bond (Table 2) present within the molecule is the intermolecular contact between the hydroxy group and the C=O group of the adjacent molecule which is perpendicularly oriented towards the first molecule thus forming an infinite linear chain in the solid state (Figure 3).All bond lengths and angles (see Table 1) in 2a are in the usual ranges reported in the literature [40].The structure comparison of 2a with the series of reported salicylanilides [29][30][31][32][33][34][35][36][37][38][39] shows only some small changes mainly in the degree of planarity of compounds and the intramolecular architecture driven by the differences in the types of H-bonding.The second compound 2b containing a methylene linker between the amide nitrogen and the benzene ring is no longer planar but has a bent shape (Figure 4) due to the impossibility of delocalization of a nitrogen lone electron pair into the benzene ring resulting from lack of electronic coupling, and broken by the methylene spacer with saturated bonds.However, the main difference between structures 2a and 2b (except the shape of the molecule) lies in a different type of the hydrogen bond in the molecule.The compound 2b has a "closed-ring" arrangement with an intramolecular hydrogen bond between the carbonyl oxygen atom and the hydroxy group (C=O• • • H-O) similarly as it was observed for N-benzyl-2-hydroxybenzamide [41], N-(3-pyridylmethyl)-2-hydroxybenzamide [42] and even for 2-hydroxy-N-methylbenzamide [43].Another intermolecular NH• • • O(H) bond between the amide and the hydroxy group of 2b then forms a linear infinite chain in which all molecules have the same orientation (Figure 5).The factors that can generally contribute to a preference for one of the two arrangements of hydrogen bonds [32] include bond strength differences between intra-and intermolecular hydrogen bonds, steric demands (bulky substituents may enforce certain arrangement), cooperativity resulting from formation of the hydrogen-bonded chains or networks and competition for a limited number of proton donor and acceptor sites.However for N-substituted 2-hydroxybenzamides there is no obvious reason to expect that the formation of an internal NH• • • O(H) bond would be favored over the formation of an internal C=O• • • HO bond for the above-mentioned reasons.
The ability of an amide to act as a proton acceptor (C=O group is the site of protonation in amides) can be illustrated by the protonation acidity constants for structurally similar N-benzylbenzamide (pK a (BH + ) = −1.83 in water [44]) and for related N-(4-methylphenyl)benzamide (pK a (BH + ) = −2.28 in water [45]).From the negligible difference in these two values it is clear that the acceptor ability of the amide carbonyl group towards protons is almost the same.The difference is much less than one log-unit i.e. less then one order of magnitude.On the other hand, amides behave as very weak N-acids and their dissociation constants were measured mostly in non-aqueous polar aprotic solvents.For example, in DMSO the following values can be found in the literature for N-phenylbenzamide (pK a = 18.77) [46] and for structurally similar benzamide (pK a = 23.35)[47].Further substitution of benzamide nitrogen by an alkyl group (e.g benzyl) must cause an even higher difference in pK a values, which is approximately 5 log-units i.e. around five orders of magnitude.From these values it is clear that N-aryl and N-alkyl substituted 2-hydroxybenzamides strongly differ in their acidity of the amide hydrogen from which it can be concluded that the hydrogen bond donor ability is much poorer for Nbenzyl than for N-phenylsalicylamides.On the other hand the intrinsic hydrogen bond donor ability of the phenolic OH group measured by its pK a value (N-phenylsalicylamide pK a = 9.92 in water [48] and N-methylsalicylamide pK a = 8.03 in water [49]) changes only by less than 2 log units (according to the literature [48]).In conclusion, in the molecule 2a the amide group behaves as much a stronger proton donor than in 2b and provides its hydrogen into a relatively strong hydrogen bond in which oxygen of the OH group acts as a hydrogen bond acceptor.In the molecule 2b such a proton donor ability of the amide group is weaker (ca. 10 5 -times less) and the proton donor ability of the OH group increases approximately 100-times at the same time.This gives rise to the formation of a new type of hydrogen bond with the amide carbonyl group acting as a hydrogen bond acceptor.According to above-mentioned explanation the molecule of 2c should form the same hydrogen bond pattern like in 2b.Available literature [50,51] describing crystal structures of N-substituted salicylamides containing ethylene linker also supports this idea.In fact for 2c we observed intramolecular NH• • • O(H) bonds between the amide and hydroxy groups (Figure 6) with perpendicularly oriented molecules thus forming an infinite chain as in the case of 2a (Figure 7).This unexpected result would be caused by the solvent used for crystal growth.It is known from the literature [52] that the solvent can critically influence the formation of various polymorphic forms.We used relatively low polar chlorobenzene and got "open-ring" β-forms exclusively.Therefore we tried to crystallize the product 2c from polar solvents (ethanol, ethylacetate) but we did not obtain any suitable single crystal for further X-ray study.To the best of our knowledge this is the first case where this type of intramolecular hydrogen bond NH• • • O(H) has been observed in the N-alkyl salicylamides which is normally characteristic for N-aryl salicylamides.

General
All reagents and solvents were purchased from commercial sources (5-chlorosalicylic acid (98%), 4chloroaniline, benzylamine (ReagentPlus 99%), phenethylamine (99%) were purchased from Sigma-Aldrich, Phosphorus trichloride (99%) was purchased from Acros Organics, Chlorobenzene p.a. was obtained from Lach-Ner CZ).Commercial grade reagents were used without further purification.All the melting points were determined on a Melting Point B-545 apparatus (Büchi, Germany) and are uncorrected.The NMR spectra were measured in DMSO-d 6 solutions at ambient temperature on a Bruker Avance III 400 MHz spectrometer (Karlsruhe, Bruker, Germany, 400 MHz for 1 H, 100 MHz for 13 C).The X-ray data were obtained at 150K using Oxford Cryostream low-temperature device on a Nonius KappaCCD diffractometer with MoK radiation ( = 0.71073 Å), a graphite monochromator, and the φ and χ scan mode.

Synthesis
Phosphorus trichloride (30 mmol) was slowly added to a solution or suspension of 5-chloro salicylic acid (60 mmol) and corresponding amine 1a-c (60 mmol) in chlorobenzene (250 mL).The reaction mixture was then heated under reflux in microwave reactor for 20 minutes under constant power (400 W).The hot reaction mixture was then transferred to the Erlenmeyer flask and slowly cooled down to ambient temperature.The solid was collected by filtration and recrystallized from absolute ethanol to give white crystals 2a-c.

Characterization of the Compounds 2a-c
All the compounds were characterized using 1 H and 13 C NMR, as well as melting point of crystallized products.The purity was checked by CHN analyses.We also wanted to compare all data with literature sources.Nevertheless, for the compound 5-chloro-N-(4-chlorophenyl)-2hydroxybenzamide 2a no found NMR characterization was found in the primary literature [53,54], therefore we show the 1 H and 13 C NMR interpretation as well as melting point and elemental analysis below.The analyses of the compound N-benzyl-5-chloro-2-hydroxybenzamide 2b were in good agreement with literature source [55], for this compound we show only the data for elemental analyses, yield and melting point.The characterization of the third investigated molecule (5-chloro-2-hydroxy-Nphenethylbenzamide 2c) was not also clearly shown in the literature [56], this was the reason to show results of the analyses as in the case 2a.

Crystals Growth
Suitable crystals of the investigated compounds were obtained using the following procedure.The reaction mixture was cooled down after MW irradiation, and the precipitated products were collected by filtration.These crystals of crude 2a-2c were dissolved in hot chlorobenzene at 100 °C.The mixture was slowly cooled down to 4 °C with a gradient 0.25 °C per minute.Crystal formed during this procedure were suitable for the X-ray measurement.

X-ray Data Collection and Structure Refinement
The X-ray data for the colorless crystals of 5-chloro-2-hydroxy-N-(4-chlorophenyl)benzamide (2a), 5-chloro-2-hydroxy-N-benzylbenzamide (2b) and 5-chloro-2-hydroxy-N-phenethylbenzamide (2c) were obtained at 150 K using a Oxford Cryostream low-temperature device on a Nonius KappaCCD diffractometer with MoK radiation ( = 0.71073 Å), a graphite monochromator, and the  and  scan mode.Data reductions were performed with DENZO-SMN [57].The absorption was corrected by integration methods [58].Structures were solved by direct methods (Sir92) [59] and refined by full matrix least-square based on F2 (SHELXL97) [60].Hydrogen atoms were mostly localized on a difference Fourier map, however to ensure uniformity of the treatment of the crystal, all hydrogen atoms were recalculated into idealized positions (riding model) and assigned temperature factors H iso (H) = 1.2 U eq (pivot atom) or of 1.5 U eq for the methyl moiety with C-H = 0.97, and 0.93 Å for methylene and hydrogen atoms in aromatic rings moiety, respectively, O-H = 0.82 Å and N-H being 0.86 Å.
Crystallographic data for structural analysis were deposited with the Cambridge Crystallographic Data Centre.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).
Crystallographic data for 2a

Conclusions
The crystal structure of three 5-chloro substituted salicylamide derivatives were determined and registered in the Cambridge Crystallographic Data Centre.Two different types of hydrogen bonds were observed in the investigated structures.In 5-chloro-2-hydroxy-N-(4-chlorophenyl)benzamide (2a) and 5-chloro-2-hydroxy-N-phenethylbenzamide (2c) intramolecular NH• • • O bond between the amide and hydroxy groups were observed.The molecules had an intermolecular contact between the hydroxy group and the C=O group thus forming a linear chain in the solid state.For the 5-chloro-2-hydroxy-Nbenzylbenzamide (2b) an intramolecular bridge between the hydroxy and carbonyl group OH• • • O, and another intermolecular NH• • • O connection between the amide and hydroxy group were observed.The linear solid-state chain of (2b) was formed differently than in the case of compounds 2a and 2c.

Figure 3 .
Figure 3. Packing pattern showing intermolecular H-bonding interactions found in the structure of 2a.

Figure 5 .
Figure 5. Packing pattern showing intermolecular H-bonding interactions found in the structure of 2b.

Figure 7 .
Figure 7. Packing pattern showing intermolecular H-bonding interactions found in the structure of 2c.