17978 Phys. Chem. Chem. Phys., 2011, 13, 17978–17986 This journal is c the Owner Societies 2011

Cite this: Phys. Chem. Chem. Phys., 2011, 13, 17978–17986

Testing the limits of sensitivity in a solid-state structural investigation by

combined X-ray powder diffraction, solid-state NMR, and molecular

modellingw

Xenia Filip, Gheorghe Borodi and Claudiu Filip*

Received 9th June 2011, Accepted 17th August 2011

DOI: 10.1039/c1cp21878f

A solid state structural investigation of ethoxzolamide is performed on microcrystalline powder

by using a multi-technique approach that combines X-ray powder diffraction (XRPD) data

analysis based on direct space methods with information from 13C(15N) solid-state Nuclear

Magnetic Resonance (SS-NMR) and molecular modeling. Quantum chemical computations of the

crystal were employed for geometry optimization and chemical shift calculations based on the

Gauge Including Projector Augmented-Wave (GIPAW) method, whereas a systematic search in

the conformational space was performed on the isolated molecule using a molecular mechanics

(MM) approach. The applied methodology proved useful for: (i) removing ambiguities in the

XRPD crystal structure determination process and further refining the derived structure solutions,

and (ii) getting important insights into the relationship between the complex network of

non-covalent interactions and the induced supra-molecular architectures/crystal packing patterns.

It was found that ethoxzolamide provides an ideal case study for testing the accuracy with which

this methodology allows to distinguish between various structural features emerging from the

analysis of the powder diffraction data.

Introduction

Numerous practical demands, e.g. from the pharmaceutical

industry, for rapid structural characterization in the solid

state requires viable alternatives to the conventional single

crystal diffraction techniques, especially when single crystals

of sufficient size and quality cannot be grown, or become

prohibitively slow to. Studies within this area have been

facilitated by the remarkable progress made in the field of

powder and in silico structural characterization methods such

as X-ray powder diffraction (XRPD), solid-state nuclear

magnetic resonance (SS-NMR), and molecular modeling in

extended periodic systems. Either separately, or in various

combinations, they have already been demonstrated as very

powerful tools in various applications, including crystal struc-

ture determination on powders.

Crystal structure can in principle be solved based solely on

well established XRPD protocols1–3 but, with lower success

rates, confidence levels, and accuracy, compared to single

crystal diffraction. In principle, all of these quality factors

can be improved if complementary information obtained from

other sources is supplied. For instance, the incorporation of

SS-NMR parameters sensitive to changes in crystal packing

and molecular conformation into the XRPD structure deter-

mination process is regarded as a promising approach, because

it has the effect that a correct solution is reached more reliably

and in a shorter time. Employing structural degrees of freedom

like torsion angle restraints,4 inter-molecular distances,5

or 1H–1H proximities6,7 represents the most direct approach

to crystal structure determination on powders by combined

XRPD and SS-NMR, but its widespread use is not possible

yet due to efficiency reasons (the need for isotopic labeling),

or due to the limited 1H spectral resolution. As such, the

vast majority of the applications reported so far in the case of

organic compounds rely on 13C(15N) cross-polarization magic

angle spinning (CP-MAS) NMR spectra recorded on natural

abundance samples.8–32 The information extracted from the

corresponding chemical shifts can be used both before the

XRPD search procedure is started, e.g., to identify the number

of molecules in the asymmetric unit, different solid forms,

presence of structural and/or dynamical disorder in the lattice,

etc., and after the protocol has been completed, for instance to

National Institute for R&D of Isotopic and Molecular Technologies,400293 Cluj, Romania. E-mail: cfilip@itim-cj.ro;Fax: +40 264 420042w Electronic supplementary information (ESI) available: summaryreports for Rietveld refinement of the Etho_dA(0) and Etho_dA(2) crystalstructure models are provided in the corresponding .pdf files; forillustrating specific structural changes under first-principles geometryoptimization, representative crystallographic information files (.cif)are provided for the Etho_dA(0), Etho_dA(1), and Etho_dA(2) modelsdiscussed in the main text. See DOI: 10.1039/c1cp21878f

PCCP Dynamic Article Links

www.rsc.org/pccp PAPER

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This journal is c the Owner Societies 2011 Phys. Chem. Chem. Phys., 2011, 13, 17978–17986 17979

validate the result by comparing the measured chemical shifts

with those computed on the proposed crystal structure.

In the present work, this strategy was considered for the struc-

tural investigation of 6-ethoxy-1,3-benzothiazole-2-sulfonamide

(ethoxzolamide). Since multiple structure solutions emerged

from the analysis of the powder X-ray data, a comprehensive

study was carried out on an ensemble of trial models built by

varying experimental as well as processing parameters. In this

context, the incorporation of complementary information

from 13C(15N) SS-NMR and molecular modeling by ab initio

and molecular mechanics (MM) methods turned out to be

extremely useful not only for reducing the observed ambiguities,

but also for further refining the XRPD structure solution, such

as to meet combined constraints provided by the diffraction

and NMR data, and for characterizing the complex network of

non-covalent interactions in relation with the induced supra-

molecular arrangements and crystal packing patterns.

Ethoxzolamide is one of the first carbonic anhydrase inhi-

bitors (CAIs) used as an active pharmaceutical ingredient

(API) in drugs prescribed for the treatment of edema due to

congestive heart failure, for drug-induced edema, or as an anti-

glaucoma agent.33 However, to the best of our knowledge, the

crystal structure of ethoxzolamide was not reported so far in

specialized structural databases, most probably due to difficulties

in growing it as a single crystal of sufficient quality. Thus, besides

its methodological importance for comprehensive structural

characterization on organic polycrystalline materials, the present

study contributes also to enlarging the structural base of the

CA inhibitors currently on the market. In addition, although

not widely utilized nowadays, ethoxzolamide is the lead mole-

cule used in the design of the second generation of CAIs, such

as dorzolamide,34 and brinzolamide.35 From this perspective,

the results reported here may be useful for future studies aimed

at developing new inhibitors with increased selectivity to

certain CA isosomes.

Experimental

Ethoxzolamide at 98% purity was purchased from commercial

sources and used as received.

X-Ray powder diffraction and solid-state NMR

X-Ray powder diffraction data were recorded with a Bruker

D8 Advance powder diffractometer using Cu-Ka1 radiation

(l = 1.54056 A). The y–2y Bragg–Brentano configuration

geometry and incident beam Ge (111) monochromator were

used. The sample was ground to a fine homogeneous powder

using an agate pestle and mortar set and mounted in a sample

holder. Two distinct measurements were performed at room

temperature within a 2y range of 3.5–451 in steps of 0.011, and

0.0051.

Solid-state 13C(15N) NMR spectra of ethoxzolamide were

recorded at 150.9 and 60.3 MHz 13C and 15N Larmor frequen-

cies with a Bruker AVANCE III-600 MHz spectrometer oper-

ating at room temperature. Standard CP-MAS experiments

were performed in both cases: at 12 kHz spinning frequency

and 1.5 ms contact time for the 13C spectrum, whereas these

parameters have been set to 7 kHz and 6 ms when acquiring the15N spectrum. A reasonable S/N ratio was achieved by averaging

6000 transients in the former case, and 15000 in the latter, with a

recycle delay of 4 s. The 13C(15N) CP-MAS spectra of ethoxzol-

amide are calibrated relative to the 13CH3 line in TMS (tetra-

methylsilane), and the 15NO2 line in nitromethane, through an

indirect procedure which uses adamantane, and respectively the aform of L-glycine, as external standards.

Computational methods

All the computational tasks, ranging from XRPD structure

determination, to calculations based on MM and ab initio

methods, were performed using an integrated software pack-

age, Accelrys Materials Studios (MS) suite.36 It provides

the advantage of increased productivity offered by common

task management and portability of intermediate results, while

keeping at the same time a high accuracy level due to the

powerful simulation/modeling algorithms implemented within

each module.

The crystal structure determination from the XRPD data

was performed with MS Reflex Plus. The crystal lattice para-

meters were determined first by indexing the diffraction patterns:

the solution with the best confidence level was obtained with

the DICVOL algorithm, whereas TREOR, ITO and X-Cell

methods systematically provided multiple solutions that could

not be sorted out according to their associated figures of

merit. Next, an automated search for the most probable space

group based on the systematic absences of Bragg reflections in

the experimental powder pattern was carried out, followed by

the Pawley refinement of the peak profiles. A pseudo-Voight

profile function was considered and a set of characteristic

pattern and sample parameters (separately, with and without

the lattice parameters included) were simultaneously refined

until the value of the associated figures of merit reached their

minima, Rwp B 0.06 (Rp B 0.04). Subsequently, the ethoxzol-

amide molecular structure was built from scratch using the

MS editing tool, its geometry was then optimized with MS

Forcite Plus, and used as a starting model to determine the

preliminary crystal structure solution. For this purpose, a

direct-space search method based on the Simulated Annealing

algorithm was employed by considering nine degrees of free-

dom: six related to the molecular position and orientation,

whereas the remaining three are the independent flexible

torsion angles of the ethoxozolamide molecule shown in

Fig. 1 (N1–C9–S2–N2, C2–O1–C3–C4, C1–C2–O1–C3). To

build relevant statistics, the solutions were systematically

searched by applying a close contact penalty function of which

weighted contribution was varied from 0.5 to 0.9 (in steps of

0.1) in distinct sessions, each comprising five search runs, with

700 000 steps/run. In all cases, the best fit between the simu-

lated and experimental powder patterns was obtained with an

Rwp = 0.18–0.2 (Rp = 0.11–0.12) and taken as the input

crystal structure model for the final Rietveld refinement stage.

First-principles geometry optimization and NMR chemical-

shift calculations were performed by using the MS CASTEP

and MS NMR CASTEP tools. CASTEP implements density

functional theory (DFT) within a generalized gradient approxi-

mation and the planewave pseudopotential approach.37 All

calculations used the PBE exchange–correlation functional

and ultrasoft pseudopotential. The XRPD crystal structure

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solutions have been optimized prior to shielding calculations

in two different ways: (i) the positions of the heavy atoms and

unit cell parameters were kept fixed at their diffraction derived

values, but the hydrogen atoms were allowed to move (the

H-relaxed case), and (ii) by fixing only the unit cell parameters

and allowing the positions of all atoms to be optimized—the

all-relaxed case, respectively. A comparison of the average

forces remaining on the atoms after geometry optimization

was carried out by using a maximum planewave cutoff energy

of 610 eV. When constrained, the heavy atoms were still

affected by average forces (given as Cartesian components)

of B0.08 eV A�1 (C), B0.7 eV A�1 (N), B1 eV A�1 (O) and

B1.6 eV A�1 (S) as compared to B0.003 eV A�1 (H),

indicating that further relaxation was necessary. When also

the heavy atoms were relaxed, average forces of a similar order

of magnitude were determined for all atomic species, ranging

between 0.001 eV A�1 (H) and 0.006 eV A�1 (N).

The NMR shielding parameters were computed by using

the Gauge-Including Projector Augmented-Wave (GIPAW)

method.38 For calculation of the shielding tensors a plane-

wave basis set was employed with a cutoff energy of 610 eV,

with integrals taken over the Brillouin zone by using a

Monkhorst–Pack grid of minimum sample spacing 0.04 A�1,

and ultrasoft pseudopotentials generated on the fly.

MM classical simulations were performed using the MS

Forcite Plus for geometry optimization with respect to minima

in the potential energy surface of an isolated molecule. In all

calculations, the interactions between atoms are taken into

account through an ab initio forcefield named COMPASS

(Condensed-phase OptimizedMolecular Potentials for Atomistic

Simulation Studies).39 An optimized combination of the stee-

pest descent, ABNR (Adjusted Basis set Newton–Raphson),

and quasi-Newton algorithms, was used for geometry optimi-

zation by considering the following convergence criteria:

energy, 10�5 kcal mol�1; maximum force, 0.001 kcal mol�1 A�1;

and maximum displacement, 10�5 A.

The conformational analysis of ethoxzolamide was perfor-

med starting from an isolated molecule extracted from the

XRPD crystal structure model obtained after geometry-

optimization with MS CASTEP. A grid was first constructed

in the conformational space by varying the C1–C2–O1–C3,

C2–O1–C3–C4 and N1–C9–S2–N2 flexible torsion angles in

steps of 101, between �1801 and 1801. Using the COMPASS

forcefield, a systematic search was carried out next by relaxing

all of the generated 46 656 molecular conformations with

respect to these particular torsion angles until the closest local

minimum in the total energy surface was reached in each case.

Results and discussion

Analysis of the XRPD data

Two crystal lattice solutions were obtained from the powder

pattern acquired in steps of 0.011: they correspond to mono-

clinic unit cells having slightly different lattice parameters,

namely, a = 24.1578 A, b = 4.7224 A, c = 21.9671 A, b =

114.621, V = 2278.4 A3, and a = 24.9890 A, b = 4.7235 A,

c = 21.9729 A, b = 118.461, V = 2280.1 A3. The former

emerged with an improved figure of merit from a new diffrac-

tion pattern recorded with smaller steps (0.0051), provided

that the number of analyzed reflections is sufficiently large

(exceeding 35). The lattice parameters were almost identical in

the two cases (variations at the third decimal), except a, for

which a value of 24.1839 A was extracted from the second

diffraction pattern. Given the results of indexing, the first

crystal lattice solution was selected in the end for subsequent

structure elucidation. The most probable space group was

found to be A2/n, which corresponds to eight molecules in the

unit cell if atoms are set in general positions.

The reduced precision in setting the periodicity along the

a crystallographic axis was further investigated during the

XRPD crystal structure determination process (see Computa-

tional methods). For this purpose, a comparative study has

been performed over an ensemble of twenty crystal structure

models, which was built by considering five distinct weighting

coefficients for the applied close contact penalty function, two

distinct powder patterns, and two distinct cases with respect to

the lattice parameters derived from indexing: (i) they are kept

constant throughout the all refinement stages, and (ii) simul-

taneously refined together with the other pattern and sample

specific parameters.

Comparing the structures with optimized lattice parameters

during Pawley refinement, the search for preliminary structure

models, and Rietveld refinement, a da variation of B0.15 A

was found, which is indeed much larger than the average

variations db(dc) B 0.02 A of the b and c lattice parameters:

this suggests that the observed uncertainty in extracting the

a value most probably has a sample related cause, possibly a

certain amount of structural disorder affecting characteristic

distances along this crystallographic axis. Including also the

rest of the structures into the analysis, it could be also

concluded that the optimization of the lattice parameters does

not necessarily improve the quality of the fit, because all twenty

crystal structure models were obtained in the end with almost

the same figures of merit, Rwp = 0.13–0.14 (Rp B 0.11): hence,

very similar crystal packing patterns, supra-molecular arrange-

ments, and associated non-covalent interactions are expected

Fig. 1 Molecular conformations of ethoxzolamide that differ by the

positioning of the –NH2 moiety with respect to the benzothiazole ring,

namely, the up (etho_u) and down (etho_d) conformations. The atom

labelling scheme employed in the present study is also shown.

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throughout the entire ensemble, with only slight fluctuations

of characteristic structural parameters. Similarities have been

indeed found, but, surprisingly, also marked differences, which

are briefly described below.

Differences occur both at the molecular and supra-molecular

level. The former is represented by two distinct molecular con-

formations around the C9–S2 bond—see Fig. 1, where they are

labeled by Etho_u (up) and Etho_d (down), in direct corres-

pondence with the location of the –NH2 moiety relative to

the planar benzothiazole group, when S2 points towards the

observer. The latter is related to the exact values of the

N1–C9–S2–N2 torsion angle in the Etho_u/Etho_d conforma-

tions: in particular, two distinct supra-molecular arrangements

involving the sulfonamide group are established depending

on whether these values are close to B701/�701 (Fig. 2A), or

to B551/�551 (Fig. 2B), respectively. As seen in these figures,

they differ through the hydrogen bonding patterns formed

by the –NH2 moiety. Corresponding to the above emphasized

differences, the crystal structures in the ensemble can be

grouped into four distinct classes of models, hereafter called

Etho_uA, Etho_uB, Etho_dA, and Etho_dB, respectively.

Structural features that are common to all of the analyzed

structures are represented by the supra-molecular arrange-

ments illustrated in Fig. 2C and D, and also by the three

distinct crystal packing patterns shown in Fig. 3, namely:

(a) quasi-planar strands of ethoxzolamide molecules disposed

parallel along their major axes, but with alternate orientations

and slightly displaced with respect to each other along the

vertical direction; (b) vertical stacking of the planar strands

with a characteristic inter-stack separation defined by the b

lattice parameter; (c) adjacent strands making an angle y of

about 451 between them along the horizontal direction, which

finally results in a zigzag type of spatial arrangement on going

from one stack of planar strands to another.

The blue area depicted in Fig. 3a, which serves as a guide for

the eye, does not actually embed benzothiazole rings: in

reality, these rings are slightly shifted above, and below, this

plane, most probably to accommodate neighboring ethoxzol-

amide molecules in their tendency toward the formation of

the N2–H� � �N1 and C1–H� � �O2(3) pairs of hydrogen bonds

emphasized by the dotted black lines in Fig. 2A–C. The –NH2

nitrogen (N2) may have an important contribution also to

the stacking arrangement shown in Fig. 3b, via the establish-

ment of N–H� � �O hydrogen bonds with one (or both, in the B

arrangement) oxygen atoms of the neighboring molecule in

the stack. In addition, the formation of stacks appears to be

favored by driving the ethoxy group in a conformation where

one of the –CH2 hydrogens comes sufficiently close to the

phenyl ring in the upper or, depending on the u/d conforma-

tion, lower strand such as to couple with it through C–H� � �pinteractions (dotted gray lines in Fig. 2A and B). Finally, the

fact that adjacent strands do not stay confined within the same

layer along the horizontal direction, but have the tendency

to get tilted with respect to each other, is most probably a

combined effect of the C4–H� � �O1 hydrogen bonding (see

Fig. 2D) and van der Waals interactions.

Fig. 2 Supra-molecular arrangements identified within the investigated ensemble of XRPD crystal structure models of ethoxzolamide,

together with the proposed hydrogen bonding patterns (black dotted lines) and C–H� � � p (gray dotted lines) interaction responsible for their

formation.

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Quantum mechanical geometry optimization

Given the good correlation between the proposed non-covalent

interactions network and the associated molecular superstructures/

crystal packing patterns, one can conclude that the above

analysis of the powder diffraction data provides an overall

realistic picture of the actual ethoxzolamide crystal structure.

Though the accuracy at atomic scale is still limited due to

the employed structure determination approach, in particular

besides the three independent flexible angles mentioned in

the Computational methods section, all the other parameters

(bond lengths, bond angles, and the remaining torsion angles),

were kept rigid at their values found in the MM optimized

ethoxzolamide molecular structure.

To correct this, first-principles geometry optimization was

carried out on representative crystal structure models, one for

each of the distinct classes mentioned above, and then the

optimized structures obtained under all-relaxed conditions—

see Computational methods for details, were employed as

inputs in a second Rietveld refinement run. As also shown in

a previous work,40 the quality of the fit did improve by applying

this procedure, leading to Rwp values in the range 0.09–0.1, that

is, to figures of merit commonly considered sufficiently small

for a reliable crystal structure solution on powders. The best fit

of the diffraction pattern (Rwp = 0.092) was obtained on the

Etho_dA crystal structure model—see Fig. 4. For this particular

model, detailed reports of the Rietveld refinement performed

before, and after, geometry optimization are presented in

the ESI.

Changes have been observed also with respect to the

characteristic structural parameters of the D–H� � �A hydrogen

bonds depicted in Fig. 2. They are presented in Table 1, which

shows a comparison between the H� � �A (acceptor) distances

measured on three distinct instances of the Etho_dA crystal

structure model, each labeled by an (i) superscript that corres-

pond to the non-optimized structure, i = 0, the geometrically

optimized one, i = 1, and the model obtained after running

the second Rietveld refinement on the optimized structure,

i = 2, respectively. The crystallographic information files (cif)

for these three models are also provided in the ESI.w The

results of a similar analysis are listed in Table 2 for the

hydrogen bonds found in the Etho_uB crystal structure model.

As a common feature, it is observed that the relative

strengths are reversed after geometry optimization for two

types of hydrogen bonding interactions, namely: (i) the pairs

of N2–H� � �N1 and C1–H� � �O2 bonds, which act in opposite

directions upon each molecule to stabilize the quasi-planar

arrangement shown in Fig. 3a, and (ii) the N2–H� � �O2(O3)

bonds, which couple adjacent molecules within a stack (Fig. 3b).

The non-covalent interactions between neighboring stacks

are in turn much less affected, as results from analyzing the

corresponding C4–H� � �O1 hydrogen bond. Although not

explicitly listed in Tables 1 and 2, the distances between the

–CH2 proton and the closest carbon in the phenyl ring invol-

ved in the C–H� � �p interactions (Fig. 2A and B) do also change,

from about 2.6 to 2.9 A, thus weakening this interaction in the

geometrically optimized models.Fig. 3 The three distinct crystal packing patterns identified in the

crystal lattice of ethoxzolamide—see the description in the text.

Fig. 4 Rietveld plot for ethoxzolamide, where the measured pattern

is represented with symbols, the calculated pattern with the red line,

and the difference pattern with the blue line, respectively.

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All of the analyzed distances suffer a new variation after the

second Rietveld refinement was applied upon the optimized

structures. This is to be understood in terms of the different

constraints employed during structure determination in the

two cases: energy/forces minimization at 0 K, for geometry

optimization, vs. the best fit of an XRPD powder pattern

recorded at room temperature, and also within error limits

possibly enhanced by the assumed lattice disorder, for Rietveld

refinement, respectively.

Summarizing, the hydrogen bonding patterns obtained for

both Etho_dA and Etho_uB can be considered quite realistic

with respect to their characteristic structural parameters.41

Even though the dispersion of these parameters over the

compared structures, in particular the H� � �N1 distance, as

well as the Rwp figure of merit, are slightly smaller in the case

of the Etho_dA model compared with Etho_uB, they still

cannot be taken as clear evidence for the fact that the A

supra-molecular arrangement would provide a better picture

for the actual ethoxzolamide structure. Stronger arguments

for such a conclusion are provided instead by a comparison

with the data reported in literature on similar sulfonamide

containing compounds:43–51 specifically, in all of the analyzed

cases it was found that the hydrogen bonding pattern around

the –NH2 moiety corresponds to the supra-molecular arrange-

ment in Fig. 2A.

Experimental vs. computed13C(

15N) NMR chemical shifts

In the 13C and 15N CP-MAS spectra shown in Fig. 5 the nine

chemically distinct carbon and two nitrogen sites in the ethoxzol-

amide molecule are represented by single resonances, which

means that the title compound has only one molecule in the

asymmetric unit, and also that the sample is not a mixture of

different crystalline forms, nor does it contain impurities at

detectable concentrations. This information obtained from

a simple inspection of the spectra has been exploited during

the crystal structure determination from the powder X-ray

data. Slightly broader resonances were obtained for ethoxzol-

amide by about 30% in the case of the 13C NMR lines, and

by about 50% for the –NQnitrogen line, compared with the

corresponding 13C(15N) NMR lines measured in L-glycine

under the same experimental conditions. Most probably, this

correlates with an increased lattice disorder (either conforma-

tional, or motional) in ethoxozolamide relative to the quite

rigid crystal packing of the L-glycine molecules, though it is

also clear that the associated effects are not significant at least

for the ethoxy-benzothiazole molecular fragment. The same

cannot be concluded about the sulfonamide head-group of

the molecule, given the much broader –NH2 line in the 15N

CP-MAS spectrum (B6 ppm FWHH, which is almost ten

times larger than the linewidth in L-glycine), and also its

asymmetric lineshape (see the inset in Fig. 5). This is consistent

with a chemical shift distribution of the N2 nitrogen, possibly

induced by small variations of sulphonamide-related local

structural parameters across the lattice.

The experimental 13C NMR chemical shift values, dex(13C),are compared in Table 3 with the shifts dcalc(13C) computed on

the same Etho_dA and Etho_uB crystal structure models

discussed above—the (0) and (1) superscripts correspond here

to geometry optimization of only protons (H-relaxed case),

and of all atoms (all-relaxed case), respectively. For this

purpose, the isotropic shielding constants s calculated as

described in Computational methods were transformed to

chemical shifts relative to TMS by using the relationship

d = sref � s, with the reference values sref of B167 ppm

(H-relaxed cases), andB168 ppm (all-relaxed cases), extracted

from the linear fit of the computed shielding constants against

experimental shifts.24

The root mean squared deviation (RMSD) between dex anddcalc in the all-relaxed cases falls within a typical variation

range of 2–4 ppm reported in the literature,24,42 whereas much

Table 1 Characteristic H� � �A distances (in A) within the listedhydrogen bonds measured on the three distinct instances of theEtho_dA model, as described in the text

D–H� � �A Etho_dA(0) Etho_dA(1) Etho_dA(2)

N2–H� � �N1 2.45 1.91 2.09N2–H� � �O3 2.55 1.90 1.83C1–H� � �O2 2.12 2.61 2.61C4–H� � �O1 2.52 2.52 2.51

Table 2 Characteristic H� � �A distances (in A) within the listedhydrogen bonds measured on the three distinct instances of theEtho_uB model, as described in the text

D–H� � �A Etho_uB(0) Etho_uB(1) Etho_uB(2)

N2–H� � �N1 2.69 2.15 2.74N2–H� � �O2 2.72 2.16 2.25N2–H� � �O3 2.53 2.20 2.29C1–H� � �O2 2.40 2.70 2.57C4–H� � �O1 2.72 2.67 2.40

Fig. 5 The upper and lower figures show the 13C and 15N CP-MAS

spectra of ethoxzolamide—in both cases the lines with an asterisk

indicate spinning sidebands. The inset shows an enlargement of the

area around the –NH2 NMR line.

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17984 Phys. Chem. Chem. Phys., 2011, 13, 17978–17986 This journal is c the Owner Societies 2011

larger values (B10 ppm) are obtained in the H-relaxed

cases. This basically confirms the major conclusion of the

previous section, that the XRPD crystal structure models are

significantly improved under first-principles geometry optimi-

zation. To obtain further insights into the optimized structure,

a detailed analysis of the 13C chemical shift differences D =

dcalc � dex in the H-relaxed cases was performed in terms of

local structural changes undergone by the optimized ethoxzol-

amide relative to its non-optimized analogue—see for instance,

the Etho_dA(0) and Etho_dA(1) models in the ESI.w It was foundthat the most prominent structural changes, in particular, the

deviation from perfect planarity of the benzothiazole ring

(including its attached C9–S2, and C3–O1 chemical bonds)

obtained after geometry optimization, and the biggest bond

length variations of 0.11 A (C6–C7), and 0.09 A (C9–S1), are

indeed well correlated with the large D values obtained for the

corresponding carbon sites, that is, all carbons except C4, C5,

C2, and C1.

Among these, C1 deserves special attention as an important

part of its calculated chemical shift deviation most likely has

not a structure-related origin, but can rather be explained in

the light of recent findings24 that the GIPAWmethod does not

preserve a uniform linear dependence between calculated and

measured 13C shifts across the entire variation range. Conse-

quently, the low- and high-ppm values are generally under-

and over-estimated when a single linear fit is considered,24,52

but, as shown by Brown and co-workers,7 this effect can be

counteracted by extracting different sref values for distinct

spectral windows. Since there is only one resonance in the

low-ppm region of the ethoxzolamide 13C CP-MAS spectrum,

we could not apply this procedure in practice: alternatively,

employing the average value sref B 173 ppm found in ref. 6

for the methyl resonances, a value of about 15 ppm can be now

estimated for the C1 chemical shift, which this time agrees very

well with the experiment.

The computed 13C chemical shifts show quite reduced, if any,

sensitivity to structural features that differentiate the four distinct

classes of crystal models under study, most probably because

these features refer primarily to the sulfonamide head-group of

ethoxzolamide , and not also to its ethoxy-benzothiazole frag-

ment, of which structural parameters, including inter-molecular

contacts, are almost conserved throughout the entire ensemble.

In this context, it is not surprising that marked differences

between the 15N shielding constants computed on the A and B

supra-molecular arrangements were found for the nitrogen sites,

i.e., scalc(N1) = �80.6 ppm and scalc(N2) = 124.1 ppm in the

case of A models, and scalc(N1) = �101.2 ppm and scalc(N2) =

128.5 ppm for the B models, respectively. In the both cases,

however, a distinction between the u/d conformations was still

not possible.

The shielding differences observed between these two models

are consistent with local structural changes undergone by their

corresponding hydrogen bonding patterns. In particular, the

largest variation of about 30 ppm obtained for the N1 nitrogen

correlates with a N1� � �H distance change from 1.91 to 2.15 A

(see Tables 1 and 2), whereas the smaller scalc difference of

4.4 ppm obtained for N2 can be explained by the reduced N–H

bond length variations (B 0.01 A) within –NH2, and possibly

also by the fact that the most significant structural changes

around this moiety, e.g., the distinct N–H� � �O bonding motifs

found for the two models, occur at larger length scales. It can

be concluded from here that the simultaneous presence of the

A and B arrangements in the sample (for instance, a mixture

of polymorphs) is completely excluded based on the above

theoretical results. Furthermore, it is easy to see from a com-

parison with the experiment that the chemical shift difference

between the two 15N resonances in the spectrum, Dd = �Ds,calculated in the A case, Ddcalc(15N) B 205 ppm, is much

closer to the experimental result, Ddex(15N) = 210 ppm, than

the corresponding value of about 230 ppm obtained on B type

models. This clearly indicates the Etho_A crystal structure

model, in either u or d conformation, as providing the most

faithful representation for the real ethoxzolamide structure in

solid state. The result basically coincides with the conclusion of the

previous section, but now derived on a solid experimental basis.

Conformational analysis

According to the discussion above, an N–H bond length

variation in the order of a few tenths of A may explain the

observed –NH2 line broadening in the 15N CP-MAS spectrum,

and also the fact that the N1 shielding is much less affected by

the accompanying change of the H� � �N1 distance. In principle,

such structural changes could be produced by a certain degree

of conformational disorder around the C9–S2 bond: this

leads to a N1–C9–S2–N2 torsion angle distribution, possibly

followed by small H–N2–H bond angle readjustments such

as to accommodate the changes induced upon the opposing

N2–H� � �N1 and N2–H� � �O2 hydrogen bonding interactions.

In any case, the N1–C9–S2–N2 torsion angle distribution

around an equilibrium value should be rather small; otherwise,

the corresponding variations in the N2–H� � �N1 hydrogen

bond parameters would affect the N1 chemical shift to a larger

extent than that observed experimentally.

To verify this hypothesis, a conformational analysis was

performed at the MM level of theory upon an isolated ethoxzol-

amide molecule having structural parameters that correspond

to the Etho_dA(1) crystal structure model—see Computational

methods. The systematic grid search over the considered

values of the three flexible torsion angles yields a study table

in which all of the relaxed conformations are associated with

their corresponding local minima in the total energy surface.

Table 3 Experimental vs. computed 13C chemical shifts on thespecified crystal structure models of ethoxzolamide (see the descriptionin the text). The last line shows the RMSD with respect to theexperimental shifts

C site dex(13C) [ppm]dcalc(13C) [ppm]

Etho_dA(0) Etho_dA(1) Etho_uB(0) Etho_uB(1)

C1 15.8 10.2 9.9 11.9 11.5C2 66.0 69.1 66.1 69.8 66.9C8 101.0 113.2 100.1 113.1 99.2C4 122.0 118.4 121.5 117.3 120.1C5 125.0 123.7 124.7 123.8 127.2C7 137.5 126.8 140.7 127.2 140.0C6 145.6 127.4 146.2 127.7 148.6C3 160.5 171.1 161.7 171.3 161.2C9 164.5 177.1 167.2 175.6 163.6

RMSD 10.2 2.4 9.6 2.3

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This journal is c the Owner Societies 2011 Phys. Chem. Chem. Phys., 2011, 13, 17978–17986 17985

This association is illustrated in Fig. 6 separately for each of

the scanned torsion angles. As can be seen from these graphs,

the behavior observed for the torsion angles in the ethoxy

group is in sharp contrast with that obtained in the case of the

N1–C9–S2–N2 torsion angle. Whereas the former are tightly

confined around well defined values over the entire energy

variation range, the latter shows a symmetric, and quite broad,

distribution (for instance, with �501 around 01 for the lowest

conformational energies). The result obtained here is consis-

tent with an increased conformational freedom around the

C9–S2 bond in isolated ethoxzolamide molecules. Combined

with the results reported in the previous sections, this tendency

seems to be preserved to a certain extent also in the crystalline

environment. However, considering the structural restrictions

imposed by the combined effect of intra- and inter-molecular

forces, the N1–C9–S2–N2 torsion angle is expected to vary

within a narrower range in the solid state, and around a

different value than shown in Fig. 6. This assumption agrees

well with the proposed interpretation of the 15N SS-NMR

data, thus providing an additional theoretical support for the

major conclusions derived from the present work.

Conclusions

A structural investigation of ethoxzolamide has been performed

on microcrystalline powder by applying a multi-technique

approach that combines XRPD, 13C(15N) SS-NMR, and mole-

cular modelling by both ab initio and MM methods. Lattice

disorder, finally identified as conformational in origin, makes a

standard XRPD structure determination procedure difficult to

apply because it provides multiple crystal structure solutions,

which cannot be sorted out neither on the basis of their Rwp fit

factors, nor from a detailed analysis of the identified supra-

molecular arrangements and crystal packing patterns. In this

context, ethoxzolamide represents an ideal example for study-

ing, in particular, the added contribution of SS-NMR and first-

principles geometry optimization for removing ambiguities,

and improving characteristic quality factors of the retained

structural model, and in general for testing the sensitivity with

which solid-state structural investigations can be carried out on

polycrystalline samples by the applied methodology.

The major results from this point of view can be summarized

as follows: (i) reliable crystal structure solutions that satisfy

combined constraints derived from fitting the powder X-ray

and SS-NMR data were determined; (ii) ‘‘the degeneracy’’ with

respect to the possible supra-molecular arrangements around

the sulphonamide head-group of the molecule was removed;

(iii) the uncertainty in setting the exact periodicity along the a

crystallographic axis, as well as in determining a unique struc-

ture solution, from the XRPD data analysis was found to

originate from an increased conformational freedom around

the C9–S2 bond. However the resolution with respect to the u/d

conformations shown in Fig. 1 could not be achieved, possibly

due to the high local symmetry along the S2–N2 bond axis, not

only at a molecular-, but also at a supra-molecular level. In

conclusion, the multi-technique analysis in the present work

revealed that the structure models Etho_dA(2) and Etho_uA(2)

provide the most faithful representation of the actual ethoxzol-

amide crystal structure, and basically sets the sensitivity limits

of the employed methodology in solving this particular struc-

ture on powder.

Acknowledgements

This work was supported by CNCS-UEFISCDI, project

PN2-IDEI 872/2008, and ANCS, project POSCCE ID536.

We gratefully acknowledge the Data Center of INCDTIM

Cluj for providing computing facilities. The authors also thank

Dr. Crina Cismas for useful discussions.

Notes and references

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a conformational analysis performed at the MM level of theory by a procedure described in the Computational methods section.

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