Structure Re-determination of LASSBio-294 – a cardioactive compound of the

N-acylhydrazone class – using X-ray powder diffraction data

Fanny N. Costaa, Fabio Furlan Ferreira

b, Tiago F. da Silva

c,d, Eliezer J. Barreiro

c,d, Lídia M.

Limac,d

, Delson Braza, Regina C. Barroso

e

a Laboratório de Instrumentação Nuclear/COPPE, Universidade Federal do Rio de Janeiro, C.P. 68.509, CEP

21941-972, Rio de Janeiro, RJ, Brazil.

b Centro de Ciências Naturais e Humanas (CCNH), Univerisdade Federal do ABC (UFABC), Av. dos Estados,

5001, CEP 09210-580, Santo André, SP, Brazil.

c Laboratório de Avaliação e Síntese de Substâncias Bioativas (LASSBio), Faculdade de Farmácia, Universidade

Federal do Rio de Janeiro, RJ, Brazil.

d Programa de Pós-Graduação em Química, Instituto de Química, Universidade Federal do Rio de Janeiro, Cidade

Universitária, Rio de Janeiro, RJ, Brazil

e Instituto de Física, Universidade do Estado do Rio de Janeiro (UERJ), CEP 20550-900, Rio de janeiro, RJ, Brazil

Many N-acylhydrazone derivatives synthetized in LASSBio® cannot be prepared as

single crystals of sufficient size and/or quality for structure determination to be carried out using

single crystal X-ray diffraction techniques. This article highlights the opportunity for

determining crystal structures of this class of compounds directly from powder diffraction data.

For this task, the crystal structure of LASSBio-294 was re-determined by means of conventional

X-ray powder diffraction data and so, compared with the crystal structure already determined for

single crystal data. LASSBio-294 is a cardioactive compound of the N-acylhydrazone class,

which can become part of the therapeutic interventions designed to decrease exertional fatigue,

and, consequently, improve the quality of life of patients suffering from chronic heart failure. Its

final crystal structure was refined by means of the Rietveld method (Rietveld, 1967; 1969). This

drug crystallizes in a monoclinic (P21/c) space group, with unit cell parameters a = 11.3413(3)

Å, b = 12.3573(4) Å, c = 9.0158(3) Å, β = 89.821(2)°, V = 1263.55(7) Å3, Z = 4, Z´ = 1 and calc

= 1.4419(1) g cm-3

. The goodness-of-fit indicator and R-factors were, respectively: χ2 = 1.203,

RBragg = 0.696%, Rwp = 5.59%, Rexp = 4.65% and Rp = 4.18%. The molecules in LASSBio-294

are H-bonded along the c-axis involving the atoms N(3)–H(8)···O(4).

Key words: N-acylhydrazone, X-ray powder diffractometry, crystal structure determination,

Rietveld method.

0885-7156/2013/28(S2)/S2/1/$18.00 ©2013 JCPDS-ICDD S491S491 Vol. 28, No.S2, September 2013.

I. Introduction

Medicinal Chemistry studies the molecular reasons for the action of drugs in order to

describe the relationship between chemical structure and pharmacological activity, ranking the

different functional contributions (Wermuth, 1996; Nic et al., 2007; Barreiro and Fraga, 2008). It

includes the structural design of new substances that have useful pharmacotherapeutic properties,

able to represent new chemical entities, candidates for prototypes of new drugs to safe use (Lima and

Barreiro, 2005; Viegas-Júnior et al., 2007; Lombardino and Lowe, 2004). This complex task involves

multiple factors responsible for the desired therapeutic response and the resulting of complexity

biological systems, covering a range of concepts from different disciplines and cannot be

performed randomly (Barreiro et al., 2002; Duarte et al., 2007; Barreiro et al., 1997; Evans et al., 1988).

Among the factors involved in the elucidation of the steps to describe the relationship

between chemical structure and pharmacological activity of a prototype of a new drug can be

included the study of atomic-level structure.

The majority of active pharmaceutical ingredients (API) can exist in different solid-state

forms. These solid forms can differ widely in their physicochemical, mechanical, and

biopharmaceutical properties, and thus can influence the quality, safety, and efficacy of the drug

product (Grant, 1999; Haleblian and McCrone, 1969). These properties include particle shape,

size, particle size distribution, surface area, porosity, and surface morphology. A comprehensive

understanding of these properties is essential to understand the behavior of API and drug product

performance (Brittain et al., 1991). For this task a variety of physical techniques

(crystallography, spectroscopy, thermal analysis and microscopy) are useful for characterizing

the solid forms of pharmaceuticals (Yu et al., 1998).

Due to the fact that polycrystalline forms are detected by X-ray diffraction, the powder

diffraction has played a central role in structural physics, chemistry and materials science over

the past twenty-five years. Today, structures of much higher complexity have become accessible

to powder diffractionists (Cheetham, 2006). X-ray powder diffractometry is a powerful

technique for the identification of crystalline solid phases. Every crystalline solid phase has a

unique X-ray powder diffraction (XRPD) pattern, which can form the basis for its identification.

In a powder mixture, each crystalline phase produces its pattern independently of the other

constituents in the mixture. The technique is unique, since it combines absolute specificity with a

high degree of accuracy (Hull, 1919; Shell, 1963).

Fanny N. Costa et al. S492S492 Vol. 28, No.S2, September 2013.

The use of X-rays for determination of three-dimensional atomic arrangements in crystal

structures of organic and inorganic compounds is an important stage in the design of new

substances as well as in the development of the new procedures for the synthesis of the already

known compounds (Chernyshev, 2001). The single crystal X-ray diffraction is the most powerful

approach for the determination of structural information at the atomic level. Nevertheless, it is

important to recall that the requirement for a single crystal sample of appropriate size and quality

imposes a natural limitation on the scope of this technique. Unfortunately, many crystalline

solids can be prepared only as microcrystalline powders, and therefore cannot be studied using

single crystal X-ray diffraction techniques. The most direct approach is to use X-ray powder

diffraction data, although it is important to recognize that the process of carrying out structure

determination from powder diffraction data is substantially more challenging than structure

determination from single crystal diffraction data (Harris and Cheung, 2004). The availability of

reliable procedures for determining crystal structures directly from X-ray powder diffraction data

has the potential to make considerable impact in structural sciences. For this reason, much

research activity in recent years has been devoted to the development and application of new

techniques for carrying out structure determination directly from powder diffraction data, and

has led to significant advances in the scope and power of techniques in this field (Menden, 1998;

Harris et al., 2001; Langford and Louër, 1996; Florence et al., 2005; David et al., 2006a).

The methodology of structure determination from X-ray diffraction data has been

employed as a technique able to define the relative configuration of the compounds, which is

directly related to the biological activity. This technique can be applied to identify polymorphic

phases, to characterize possible intermolecular and intramolecular interactions beyond providing

information about conformational freedom, flatness and also it can be used to distinguish the

correct orientation of the structural groups of the molecule. The physicochemical properties and

the pharmacokinetics characteristics can be understood from this information (Harris, 2002).

A bioactive compound of the N-acylhydrazone (NAH) class (named LASSBio-294)

(Barreiro, 2002; Sudo et al., 2001; Silva et al., 2002; Sudo et al., 2006; Kümmerle et al., 2009)

was synthesized and characterized in LASSBio (Laboratório de Avaliação e Síntese de

Substâncias Bioativas) of the Federal University of Rio de Janeiro (UFRJ). It was shown to

produce a cardioinotropic effect and vasodilatation (Silva et al., 2005). This novel

thienylhydrazone compound (2-thienylidene) 3,4-methylenedioxybenzoylhydrazine, herein

Structure Re-determination of LASSBio-294 – a cardioactive compound S493S493 Vol. 28, No.S2, September 2013.

referred to as LASSBio-294, was initially obtained as part of a program of synthesis of novel

anti-inflammatory leads with an N-acylhydrazone scaffold (Figueiredo et al., 2000).

Subsequently, this compound was identified as a bioisoster of a family of pyridazinone

compounds that increase cyclic adenosine 3’5’ cyclic monophosphate (AMP) levels by inhibiting

selectively the cyclic AMP-specific, low-Km phosphodiesterase 4 (PDE4) (Piaz et al., 1997). A

study was undertaken to investigate the effects of this thienylhydrazone in the contractile

properties of single skeletal muscle fibers of the frog. In addition to demonstrating that

LASSBio-294 has positive inotropic effects in single fibers of phasic muscles of the frog, the

results also showed that the compound reduces fatigue development and accelerates recovery of

maximal tetanic force after fatigue has developed. Some lines of evidence suggest that the effects

of LASSBio-294 on skeletal muscle inotropism and fatigue can be accounted for by

phosphodiesterase inhibition adenosinergic receptors improving the quality of life of patients

suffering from chronic heart failure (LeJemtel et al., 1986) and other physiopathological

conditions in which skeletal muscle dysfunctions are associated with decreased levels of

cytosolic cyclic AMP (Gonzalez-Serratos et al., 2001).

Many N-acylhydrazone (NAH) derivative analogues, synthesized in LASSBio®, planned

by structural modification of the lead compound LASSBio-294 do not create crystals suitable for

single crystal X-ray diffraction. Therefore, the compound LASSBio-294 was elected for our

study as reference crystal structure for the purpose of evaluating the accuracy of the structures

solved using the powder diffraction approach. Since the crystal structure of this compound was

previously determinated (Kümmerle et al., 2009) using single crystal diffraction, the aim of this

work is to demonstrate that through successful re-determination of crystal structure of LASSBio-

294 directly from powder diffraction data we will have the opportunity for determining crystal

structures of other NAH derivatives, unavailable for the study by means of single crystal and

better understanding of pharmacological profiles of these compounds.

In this work, X-ray powder diffraction data and a simulated annealing algorithm

implemented in the DASH software program (David et al., 2006b) were used to re-determine the

crystal structure of LASSBio-294, on the basis of previous procedures (Ferreira et al., 2010;

Ferreira et al., 2011). Thus, a Rietveld refinement of the final crystal structure was conducted

using the Topas Academic v.5 (Coelho et al., 2011) software program providing a satisfactory fit.

Fanny N. Costa et al. S494S494 Vol. 28, No.S2, September 2013.

II. Experimental

A. Materials and Methods

Applying classic and typical strategies of medicinal chemistry to design new molecular

architecture, LASSBio-294 compound was planned as a simplified analogue of pyridazinone

scaffold, known as phosphodiesterase (PDE) inhibitor compounds. Besides that, the inclusion of

the 1,3-benzodioxolyl ring in LASSBio-294 coming from the recognition of attractive natural

biofores in Brazilian abundant natural product, safrole. In addition, the inclusion of the 2-thienyl

ring attached to the imine moiety of NAH, resulted on a novel thienylhydrazone compound (2’-

thienylidene)3,4-methylenedioxybenzoylhydrazine, herein named LASSBio-294 (Barreiro and

Fraga, 2008; Kümmerle et al., 2009).

Briefly, according to the procedure described previously by Lima et al. (2000), LASSBio-294

was synthesized from the starting material safrole (4-allyl-1,2-methylenedioxybenzene). The first

step in the synthesis consisted of a base-catalyzed isomerization of safrole that yielded isosafrole.

By oxidative cleavage, isosafrole was converted into piperonal. Then, by using the Yamada’s

oxidative procedure, piperonal was converted in the methyl 3,4-methylenedioxybenzoate.

Subsequently, the key 3,4-methylenedioxyhydrazine was obtained by means of nucleophilic

substitution of the ester with hydrazine hydrate in ethanol at reflux. Finally, LASSBio-294 was

obtained by condensing the hydrazine derivative with equimolar amounts of thiophene-2-

carboxaldehyde in ethanol by using hydrochloric acid as a catalyst (Gonzalez-Serratos et al., 2001).

The XRPD pattern of LASSBio-294, which was gently hand-grinded in a pestle and

mortar then passed through a 45-µm sieve, was collected on a Panalytical X’Pert Pro

diffractometer operating at 40 kV and 40 mA. Measurements were carried out with Cu Kα

radiation (λ = 1.5418 Å) and diffraction patterns were collected in the 2θ region ranging from 4º

to 50º, with step scans of 0.01º and counting time of 40 s step-1

. Diffraction data were recorded

by a Real Time Multiple Strip (RTMS) X´Celerator detector using a scattered beam graphite

monochromator, a divergence slit of 1º, a Soller slit of 0.04 rad and an antiscattering slit of 3.9 mm.

Structure Re-determination of LASSBio-294 – a cardioactive compound S495S495 Vol. 28, No.S2, September 2013.

III. Results and discussion

A. Indexing and structure determination

The DASH software program (David et al., 2006b) was used for indexing the powder

pattern as well as for the crystal structure determination of LASSBio-294 using conventional X-

ray powder diffraction data. Data from 5.500º to 49.996º (2θ), leading to a real-space resolution

of 1.8242 Å, were used. A Bayesian high-pass filter (David and Sivia, 2001) implemented in

DASH was employed to estimate the background. The first 20 reflections were individually

fitted and used in the indexing procedure by means of the DICVOL91 (Boultif and Louër, 1991)

routine (within DASH), thus yielding the following values for a monoclinic cell: a = 11.3328 Å,

b = 12.3460 Å, c = 9.0151 Å, β = 89.78° and V = 1261.34 Å3. After the unit-cell parameters have

been roughly evaluated, a Pawley fit (Pawley, 1981) was performed and, by using a Bayesian

statistical analysis procedure (Markvardsen et al., 2001), the space group was determined as

being P21/c. The unit-cell volume was calculated by taking into account the individual average

volumes of the atoms from volume increments (Hofmann, 2002). The expected cell volume of

1256.32 Å3 is similar to the value found in the indexing procedure (1261.34 Å

3) and suggested

four molecules per unit cell (Z = 4) and one molecule in the asymmetric unit (Z´ = 1). The

integrated intensities as well as their correlations were extracted by means of a Pawley

refinement, which converged to a χ2 value of 2.25.

The values found via DASH were then used in conjunction with the aforementioned

chemical structure in the process of crystal structure determination by means of a simulated

annealing algorithm (Aarts and Korst, 1991; van Laarhoven and Aarts, 1992). Twenty runs (on a

total of 2.00×107 movements) of the simulated annealing process were globally optimized and

the best result was then considered in the final refinement of the structure. During the simulated

annealing process, ten parameters (three describing the positional coordinates, four, of which

three are independent, describing the molecular orientation and three flexible torsion angles) were

allowed to vary (David et al., 2006b).

Generally, the visual assessment of the goodness of fit between the experimental and

simulated powder pattern and the ratio ‘profile 2/Pawley

2’, which should be, in most cases,

less than 5 (David et al., 2006b), are good indicative of a successful structure solution. Although

in the present case the ratio ‘profile 2/Pawley

2’ (16.19/2.25 7.2) was a little bit greater than

Fanny N. Costa et al. S496S496 Vol. 28, No.S2, September 2013.

5, the best solution found in the simulated annealing runs was considered in the Rietveld

refinement (Rietveld, 1967; 1969) process using the Topas Academic v.5 software program

(Coelho et al., 2011). A reasonable visual fitting as well as statistical values and chemical

structure were obtained. Figure 1 shows the molecular structure of LASSBio-294 with some

H-bonds displayed by cyan lines.

Figure 1. Molecular structure of LASSBio-294 displaying some H-bonds (cyan lines). The symmetry codes are: (i)

.

B. Structure refinement

The background was fitted using a 20-term Chebyschev polynomial. The peak asymmetry

was fitted by the simple axial divergence model of Cheary and Coelho (Cheary and Coelho,

1998a; 1998b). The peak profiles were modeled by the Double-Voigt approach (Balzar, 1993)

with anisotropic peak profiles adjusted using spherical harmonics (Järvinen, 1993) as well as

preferred orientation of the crystals. Bond distances and angles as well as soft planar restraints on

rings were restrained to distances and angles mean values obtained from the MOGUL

Cambridge Structure Databank System (Bruno et al., 2004). Isotropic atomic displacements

(Biso) were constrained to be equal for all non-hydrogen atoms. For hydrogen atoms, Biso values

were constrained to be 1.2 times larger than those of the respective atoms to which they are

connected. Their factional coordinates were refined restraining the distances between them and

the atoms they are connected according to values found in the literature for X-ray diffraction data

that were then modified in Mercury (Macrae et al., 2008) accordingly: C–H = 0.96 Å, N–H =

0.87 Å and O–H = 0.99 Å. The final refined values for the unit-cell parameters after the Rietveld

fit were: a = 11.3413(3) Å, b = 12.3573(4) Å, c = 9.0158(3) Å, β = 89.821(2)°, V = 1263.55(7)

Å3, Z = 4, Z´ = 1 and calc = 1.4419(1) g cm

-3. The goodness-of-fit indicator and R-factors (Toby,

1 12 2

, ,x y z

Structure Re-determination of LASSBio-294 – a cardioactive compound S497S497 Vol. 28, No.S2, September 2013.

2006), RBragg, Rwp, Rexp and Rp were, respectively: χ2 = 1.203, RBragg = 0.696%, Rwp = 5.59%, Rexp

= 4.65% and Rp = 4.18%. Figure 2 displays the final Rietveld refinement.

Figure 2. Final Rietveld refinement of the LASSBio-294 sample. The 2θ region starting at 30º was magnified by a factor of 10 for

better visualization of the Bragg peaks.

C. Structure description, as result from powder data

The crystal structure of LASSBio-294 is comprised by four formula units per unit cell (Z = 4),

accommodating one molecule in the asymmetric unit (Z´ = 1), as represented in Figure 3.

Figure 3. Unit cell representation of LASSBio-294 along the b-axis displaying some hydrogen bonds (cyan lines) between the

atoms N(3)–H(8)···O(4) thus forming a network of molecular aggregates in LASSBio-294 along the c-axis.

Within the unit cell the molecules are held together by short hydrogen bonds between atoms

N(3)–H(8)···O(4) (D–H = 0.871(6) Å, H···A = 2.072(7) Å, D···A = 2.860(7) Å and D–H···A =

Fanny N. Costa et al. S498S498 Vol. 28, No.S2, September 2013.

150.2(6)º; where “D” and “A” are, respectively, hydrogen donor and acceptor, as represented by

cyan lines in Figure 3 (powder data), thus forming a network of molecular aggregates along the c-axis.

The values found by single crystal diffraction were: N(1)–H(10)···O(1) (D–H = 0.876 Å, H···A

= 2.006 Å, D···A = 2.829 Å and D–H···A = 156.1º). The possible complementary π-stacking

interactions between N-acylhydrazone scaffold with thiophenyl ring (C(13) – C(21) = 3.534 Å)

and also N-acylhydrazone scaffold with 1,3-benzodioxoly ring (C(13) – O(17) = 3.386 Å) are

shown in Figure 4.

Figure 4. Unit cell representation of LASSBio-294 displaying some π-stacking interactions (magenta lines) between the atoms

C(13) – C(21) and C(13) – O(17) as well as its distances (Å).

It seems these interactions contribute for the organization of the space arrangement in the unit

cell. The geometry of the crystal structure was validated by means of a Mogul check. All bond

distances and angles are in accordance to the corresponding values found in the CSD database

(Allen, 2002). Also, in order to check the consistency of the obtained results for space group

choice, unit cell parameters, bond distances, angles and torsions, the PLATON software program

was used (Spek, 2003). The ADDSYM routine did not detect any obvious extra crystallographic

symmetry. CCDC ID: 907702 contains the supplementary crystallographic data for this paper.

These data can be obtained free of charge from the Cambridge Crystallographic Data Centre via

www.ccdc.cam.ac.uk/data_request/cif.

Crystal data as well as details of the structure determination are displayed in Table I.

Structure Re-determination of LASSBio-294 – a cardioactive compound S499S499 Vol. 28, No.S2, September 2013.

Table I. Details from the Rietveld refinement of the crystal structure of LASSBio-294.

Chemical formula C13H10N2O3S

Formula weight (g mol-1) 274.30

Crystal system Monoclinic Space group P21/c (No. 14)

a, b, c (Å) 11.3413(3), 12.3573(4), 9.0158(3)

β (°) 89.821(2) Volume (Å3) 1263.55(7)

Z, Z´ 4, 1

calc (g cm-3) 1.4419(1)

μ (mm-1) 2.344

T (K) 298

Data collection Diffractometer X´Celerator Pro CuKα sealed tube

Monochromator graphite (diffracted beam)

Wavelength (Å) 1.54178

2 range (°) 4 – 50

Step size (°) 0.01

Time per step (s) 40 Refinement

Number of data points 4450

Number of contributing reflections 225

Number of restraints 79 Number of refined parameters 96

Rexp (%) 4.646

Rwp (%) 5.590 RBragg (%) 0.697

χ2 1.203

d-DW 1.507

Structural atomic parameters obtained from the Rietveld refinement are shown in Table II.

Table II. Final coordinates and equivalent isotropic displacement parameters (Uiso = Biso/82) of

all atoms for LASSBio-294 crystal structure.

Atom x/a y/b z/c Biso (Å2)

S(22) 0.5533(4) -0.0214(3) -0.2819(5) 0.0879(2)

O(4) 0.7561(8) 0.3409(5) -0.2424(6) 0.0879(2) O(17) 0.9471(4) 0.5005(4) 0.3390(5) 0.0879(2)

O(19) 0.9261(4) 0.6756(4) 0.2463(5) 0.0879(2)

N(3) 0.7310(7) 0.2444(4) -0.0355(5) 0.0879(2) N(7) 0.6817(7) 0.1600(4) -0.1132(6) 0.0879(2)

C(1) 0.7749(4) 0.3304(3) -0.1122(4) 0.0879(2)

C(2) 0.8144(3) 0.4273(3) -0.0209(4) 0.0879(2) C(5) 0.8021(3) 0.5333(4) -0.0757(4) 0.0879(2)

C(6) 0.8638(2) 0.4104(4) 0.1205(4) 0.0879(2)

C(9) 0.8385(2) 0.6231(4) 0.0094(5) 0.0879(2) C(11) 0.8982(2) 0.4994(3) 0.2008(5) 0.0879(2)

C(13) 0.6730(8) 0.0699(4) -0.0466(6) 0.0879(2)

C(15) 0.8861(3) 0.6020(3) 0.1473(5) 0.0879(2) C(18) 0.6203(6) -0.0247(3) -0.1133(5) 0.0879(2)

C(20) 0.9713(9) 0.6126(4) 0.3686(7) 0.0879(2)

C(21) 0.6217(6) -0.1285(4) -0.0534(6) 0.0879(2) C(25) 0.5687(7) -0.2058(4) -0.1485(6) 0.0879(2)

C(27) 0.5372(10) -0.1601(4) -0.2818(6) 0.0879(2)

H(8) 0.7337(7) 0.2433(4) 0.0610(5) 0.1055(3) H(10) 0.7685(3) 0.5447(4) -0.1721(4) 0.1055(3)

H(12) 0.8719(2) 0.3387(4) 0.1602(4) 0.1055(3) H(14) 0.7020(8) 0.0648(4) 0.0531(6) 0.1055(3)

H(16) 0.8298(2) 0.6957(4) -0.0267(5) 0.1055(3)

H(23) 0.9347(9) 0.6343(4) 0.4601(7) 0.1055(3) H(24) 1.0549(9) 0.6231(4) 0.3753(7) 0.1055(3)

H(26) 0.6552(6) -0.1459(4) 0.0413(6) 0.1055(3)

H(28) 0.5561(7) -0.2803(4) -0.1231(6) 0.1055(3) H(29) 0.5092(10) -0.2008(4) -0.3652(6) 0.1055(3)

Fanny N. Costa et al. S500S500 Vol. 28, No.S2, September 2013.

Table III and Table IV display some selected bond lengths and angles, respectively, from powder

and single crystal structure solutions.

Table III. Some selected bond lengths from powder and single crystal structure solutions.

Powder Single crystal

Bonds Lengths (Å) Bonds Lengths (Å)

S(22)–C(18) 1.702(7) S(1)–C(2) 1.709(2) S(22)–C(27) 1.724(6) S(1)–C(3) 1.701(3)

O(4)–C(1) 1.201(7) O(1)–C(6) 1.229(2)

O(17)–C(11) 1.366(6) O(2)–C(11) 1.417(3) O(17)–C(20) 1.437(7) O(2)–C(12) 1.377(2)

O(19)–C(15) 1.354(6) O(3)–C(10) 1.376(3)

O(19)–C(20) 1.445(8) O(3)–C(11) 1.419(3) N(3)–N(7) 1.376(8) N(1)–N(2) 1.387(2)

N(3)–C(1) 1.361(7) N(1)–C(6) 1.350(2)

N(7)–C(13) 1.269(7) N(2)–C(1) 1.274(2) C(1)–C(2) 1.521(5) C(1)–C(2) 1.446(3)

C(2)–C(6) 1.410(5) C(2)–C(5) 1.381(3)

C(2)–C(5) 1.407(6) C(3)–C(4) 1.334(5) C(5)–C(9) 1.412(6) C(4)–C(5) 1.411(4)

C(6)–C(11) 1.374(6) C(6)–C(7) 1.482(3)

C(9)–C(15) 1.382(6) C(7)–C(13) 1.402(2) C(11)–C(15) 1.364(5) C(7)–C(8) 1.392(3)

C(13)–C(18) 1.445(7) C(8)–C(9) 1.393(3)

C(18)–C(21) 1.392(6) C(9)–C(10) 1.360(3) C(21)–C(25) 1.419(8) C(10)–C(12) 1.379(3)

C(25)–C(27) 1.376(8) C(12)–C(13) 1.361(3)

Table IV. Some selected bond angles from powder and single crystal structure solutions.

Powder Single crystal

Bonds Angles (°) Bonds Angles (°)

C(18)–S(22)–C(27) 91.3(3) C(2)–S(1)–C(3) 91.46(14) C(11)–O(17)–C(20) 104.9(4) C(11)–O(2)–C(12) 105.81(17)

C(15)–O(19)–C(20) 105.2(4) C(10)–O(3)–C(11) 105.82(16)

N(7)–N(3)–C(1) 118.8(4) N(2)–N(1)–C(6) 118.46(14) N(3)–N(7)–C(13) 117.1(5) N(1)–N(2)–C(1) 115.66(15)

O(4)–C(1)–N(3) 121.0(5) N(2)–C(1)–C(2) 120.26(17)

N(3)–C(1)–C(2) 116.6(3) C(1)–C(2)–C(5) 126.12(19) O(4)–C(1)–C(2) 119.8(4) S(1)–C(2)–C(1) 122.42(15)

C(1)–C(2)–C(6) 119.4(3) S(1)–C(2)–C(5) 111.46(16)

C(1)–C(2)–C(5) 120.9(3) S(1)–C(3)–C(4) 112.5(2) C(5)–C(2)–C(6) 119.7(4) C(3)–C(4)–C(5) 113.4(2)

C(2)–C(5)–C(9) 120.8(3) C(2)–C(5)–C(4) 111.2(2)

C(2)–C(6)–C(11) 118.2(4) O(1)–C(6)–N(1) 122.03(17) C(5)–C(9)–C(15) 117.2(4) N(1)–C(6)–C(7) 116.44(15)

C(6)–C(11)–C(15) 121.9(4) O(1)–C(6)–C(7) 121.52(16) O(17)–C(11)–C(15) 110.8(4) C(6)–C(7)–C(13) 122.73(16)

O(17)–C(11)–C(6) 127.3(4) C(8)–C(7)–C(13) 120.25(16)

N(7)–C(13)–C(18) 123.0(6) C(6)–C(7)–C(8) 117.02(16) C(9)–C(15)–C(11) 122.3(4) C(7)–C(8)–C(9) 121.74(18)

O(19)–C(15)–C(9) 126.8(4) C(8)–C(9)–C(10) 116.75(19)

O(19)–C(15)–C(11) 110.9(4) O(3)–C(10)–C(9) 128.46(18) C(13)–C(18)–C(21) 125.4(5) C(9)–C(10)–C(12) 121.85(18)

S(22)–C(18)–C(13) 122.6(4) O(3)–C(10)–C(12) 109.69(16)

S(22)–C(18)–C(21) 112.0(4) O(2)–C(11)–O(3) 108.92(19) O(17)–C(20)–O(19) 108.0(5) O(2)–C(12)–C(10) 109.76(16)

C(18)–C(21)–C(25) 112.4(5) O(2)–C(12)–C(13) 127.61(17)

C(21)–C(25)–C(27) 111.3(5) C(10)–C(12)–C(13) 122.63(17) S(22)–C(27)–C(25) 112.4(5) C(7)–C(13)–C(12) 116.76(17)

C(18)–S(22)–C(27) 91.3(3) C(2)–S(1)–C(3) 91.46(14)

Structure Re-determination of LASSBio-294 – a cardioactive compound S501S501 Vol. 28, No.S2, September 2013.

On the basis of an analysis on potential hydrogen interactions performed with PLATON

it was found that atom N(3) is the potential donor of the acceptor atom O(4). The bonds

involving the atoms N(3)–H(8)···O(4) form a network of molecular aggregates in LASSBio-294

along the c-axis, as shown in Figure 3 (in case of single crystal data the network is formed by the

bonds involving atoms N(1)–H(10)···O(1) and N(1)–H(10)···N(2)).

The torsion angle involving the atoms N(3)–C(1)–C(2)–C(5) was –146.4(5)°, being

limited by –120° and –180°. Under this condition, the molecule is arranged in a –anti-clinal (ac)

conformation. The torsion angles involving the atoms C(1)–N(3)–N(7)–C(13), atoms N(7)–

C(13)–C(18)–C(21) and atoms N(7)–N(3)–C(1)–C(2) were, respectively, 160.2(7)°, 171.4(8)°

and 172.7(6)°, all of them being limited by 150° and 180° (for single crystal data the respective

torsion angles were: C(6)–N(1)–N(2)–C(1) = –166.95(17)°, N(2)–C(1)–C(2)–C(5) = –168.3(2)°

and atoms N(2)–N(1)–C(6)–C(7) = –178.25(15)°). In this case, the molecule adopted a +anti-

periplanar (ap) conformation for all torsion angles.

The 5-membered ring system composed by S(22)–C(18)–C(21)–C(25)–C(27) atoms

shows that the highest deviation from the least-squares plane passing through this ring is

0.050(10) Å for C(27) (root mean square error (r.m.s.e.) of contributing atoms is 0.006 Å; the

same analysis performed for single crystal data revealed the highest deviation from the least-

squares plane passing through this ring is 0.006(2) Å for C(5) and r.m.s.e was 0.002 Å). The

other 5-membered ring system, composed by O(17)–O(19)–C(11)–C(15)–C(20) atoms, indicates

that the highest deviation from the least-squares plane passing through this ring is 0.024(10) Å

for C(20) (root mean square error (r.m.s.e.) of contributing atoms is 0.005 Å). Also, the 6-

membered ring system, composed by C(2)–C(5)–C(6)–C(9)–C(11)–C(15) atoms, indicates that

the highest deviation from the least-squares plane passing through this ring is 0.002(3) Å for

C(6) (root mean square error (r.m.s.e.) of contributing atoms is 0.004 Å; the same analysis

performed for single crystal data revealed the highest deviation from the least-squares plane

passing through this ring is 0.007(2) Å for C(7) and r.m.s.e was 0.002 Å) demonstrating that the

rings of the LASSBio-294 molecule are almost planar.

The consistency of the crystal structure geometry was evaluated by a calculation of

intramolecular bond distances and angles using MOGUL, resulting that neither bond distances

nor angles were found as “unusual”. The criteria to confirm the molecular geometry in Mogul

was based on the choice of exact fragments in the CSD database – if the number of exact

Fanny N. Costa et al. S502S502 Vol. 28, No.S2, September 2013.

fragments were less than 15 to bonds, angles and rings, and 40 to torsion angles, the molecular

geometry would be reported as “unusual”.

D. Comparison between crystal structure of LASSBio-294 determined by single

crystal and by powder diffraction

The crystal structure of this compound was previously described in 2009 by Kümmerle et

al. There, X-ray analysis of LASSBio-294 and other three NAH single crystals obtained from an

ethanol saturated solution was carried out to best understand the influence of the methyl group

for the bioprofile of those NAH derivatives. Data pertaining to the X-ray crystallographic

determination of LASSBio-294 by means of single crystal X-ray diffraction was deposited with

CCDC ID: 707596.

Figure 5 shows the solution for LASSBio-294 obtained from powder data (in colors –

present work) overlaid on the single crystal structure (orange) (Kümmerle et al., 2009). This

comparison presents a small difference between these structures but which can be explained by

the slightly variations on bond lengths, bond angles and torsion angles, for example. From a

chemical point of view the structures are quite similar. The relative configuration of E about the

imine double bond was determined in both cases and the preferential flat conformation (amide

hydrogen anti-periplanar to the carbonyl oxygen), in the solid-phase, was also observed. As also

shown in Figure 5, both crystal structures exhibit the orientation of sulfur, of thienyl ring, in the

same direction of carbonyl oxygen.

Figure 5. Overlay of the crystal structure of LASSBio-294 determined directly from powder diffraction data (in colors) and single

crystal diffraction data (orange) (Kümmerle et al., 2009).

Structure Re-determination of LASSBio-294 – a cardioactive compound S503S503 Vol. 28, No.S2, September 2013.

IV. Conclusion

Considering the step of condensation at synthetic route could produce a diasteroisomeric

ratio (E/Z), the X-ray powder diffraction is no doubt a powerful technique providing three-

dimensional structure of the molecule, thus allowing the characterization of the relative

configuration E, of the imine double bond. This information corroborate with results obtained by

single crystal X-ray diffraction (Kümmerle et al., 2009).

The packaging of unit cell was also observed as a result of intermolecular H-bonds involving

peptide bond of the N-acylhydrazone function arranged alternately as well as possible π-stacking

complementary interactions. The high conformational constraint or near planarity of the

compound was also exemplified by the measured dihedral angles. Finally, heteroatoms of the N-

acylhydrazone function and sulfur of thienyl ring orientations in the same direction provides

regions of high electron density permitting interactions with other entities, for example

macromolecules.

The X-ray powder diffraction was successful applied in the crystal structure

determination of LASSBio-294 demonstrating the technique can be applied to solve the

structures of other compounds of NAH class, since many derivatives cannot be prepared as a

crystal suitable for single crystal X-ray diffraction studies. In this context, the three-dimensional

arrangement information accessed by means of this technique contribute for better understanding

the full pharmacodynamic profiles and physicochemical properties of this class of compounds.

Acknowledgments

We would like to thank the Brazilian agencies from financial support and fellowships:

CNPq (# 305186/2012-4; # 477296/2011-4); INCT-INOFAR (CNPq #573.564/2008-6;

FAPERJ E-26/170.020/2008), FAPESP (# 2008/10537-3). We also thank the CBPF (BR) for

experimental support.

Aarts, E. and Korst, J. (1991). Simulated Annealing and Boltzmann Machines: a Stochastic Approach to

Combinatorial Optimization and Neural Computing (John Wiley & Sons, Chichester, UK), 2nd ed., p. 284.

Allen, F. H. (2002). “The Cambridge Structural Database: a quarter of a million crystal structures

and rising,” Acta Crystallogr., Sect. B: Struct. Sci., 58, 380-388.

Fanny N. Costa et al. S504S504 Vol. 28, No.S2, September 2013.

Balzar, D. (1993). “X-Ray-diffraction line broadening - Modeling and applications to high-Tc

Superconductors,” J. Res. Natl. Inst. Stand. Technol. 98, 321-353.

Barreiro, E. J. (2002). “Estratégia de Simplificação Molecular no Planejamento Racional de

Fármacos: A Descoberta de Novo Agente Cardioativo,” Quím. Nova 25, 1172-1180.

Barreiro, E. J., Albuquerque, M. G., Sant´ana, C. M. R., Rodrigues, C. R. and Alencastro, R. B.

(1997). “Modelagem molecular: uma ferramenta para o planejamento racional de

fármacos na Química Medicinal,” Quím. Nova 20, 300-310.

Barreiro, E. J. and Fraga, C. A. M. (2008). Química Medicinal - As Bases Moleculares da Ação

dos Fármacos. (Artmed, Porto Alegre), 2nd ed., p. 243.

Barreiro, E. J Fraga, C. A. M., Miranda, A. I. P. and Rodrigues, C. R.

(2002). “Química medicinal de n-acilidrazonas: novos compostos-protótipos de fármacos

analgésicos, antiinflamatórios e anti-trombóticos,” Quím. Nova 25, 129-148.

Boultif, A. and Louër, D. (1991). “Indexing of powder diffraction patterns for low-symmetry

lattices by the successive dichotomy method,” J. Appl. Crystallogr. 24, 987–993.

Brittain, H. G., Bogdanowich, S. J., Bugay, D. E., DeVincentis, J., Lewen, G., and Newman, A. W.

(1991). “Physical characterization of pharmaceutical solids,” Pharm. Res., 8, 963-973.

Bruno, I. J., Cole, J. C., Kessler, M., Luo, J., Motherwell, W. D. S., Purkis, L. H., Smith, B. R.,

Taylor, R., Cooper, R. I., Harris, S. E. and Orpen, A. G. (2004). “Retrieval of

Crystallographically-Derived Molecular Geometry Information,” J. Chem. Inf. Comput. Sci. 44,

2133-2144.

Cheary, R. W. and Coelho, A. A. (1998a). “Axial divergence in a conventional X-ray powder

diffractometer. I. Theoretical foundations,” J. Appl. Crystallogr. 31, 851-861.

Cheary, R. W. and Coelho, A. A. (1998b). “Axial divergence in a conventional X-ray powder

diffractometer. II. Realization and evaluation in a Fundamental-Parameter profile fitting

procedure,” J. Appl. Crystallogr. 31, 862-868.

Cheetham, A. K. (2006). Structure determination from powder diffraction data: an overview

(International Union of Crystallography / Oxford Science Publications, New York), 1st ed., p. 337.

Chernyshev, V. V. (2001). “Structure determination from powder diffraction,” Russ. Chem.

Bull. Int. Ed. 50, 2273-2292.

Coelho, A. A., Evans, J., Evans, I., Kern, A. and S. Parsons (2011). “The TOPAS symbolic

computation system,” Powder Diffr. 26, s22-s25.

David, W. I. F., Shankland, K., McCusker, L. B. and Baerlocher, C. (2006a). Structure

determination from powder diffraction data. (Oxford University Press, New York), 1st

ed., p. 337 and references therein.

Structure Re-determination of LASSBio-294 – a cardioactive compound S505S505 Vol. 28, No.S2, September 2013.

David, W. I. F., Shankland, K., van de Streek, J., Pidcock, E., Motherwell, W. D. S. and Cole, J. C.

(2006b). “DASH: a program for crystal structure determination from powder diffraction

Data,” J. Appl. Crystallogr. 39, 910-915.

David, W. I. F. and Sivia, D. S. (2001). “Background estimation using a robust Bayesian

Analysis,” J. Appl. Crystallogr., 34, 318-324.

Duarte, C. D., Barreiro, E. J. and Fraga, C. A. M. (2007). “Privileged Structures: A Useful

Concept for the Rational Design of New Lead Drug Candidates,” Mini Rev. Med. Chem. 7, 1108-

1119.

Evans, B. E., Rittle, K. E., Bock, M. G., Dipardo, R. M., Freidinger, R. M., Whitter, W. L., Lundell,

G. F., Veber, D. F., Anderson, P. S., Chang, R. S. L., Lotti, V. J., Cerino, D. J., Chen, T. B.,

Kling, P. J., Kunkel, K. A., Springer, J. P. and Hirshfieldt, J. (1988). “Methods for Drug Discovery:

Development of Potent, Selective, Orally Effective Cholecystokinin Antagonists,” J. Med. Chem.

31, 2235-2246.

Ferreira, F. F., A. C., Antonio, Rosa, P. C. P. and Paiva-Santos, C. O. (2010). “Crystal structure

determination of mebendazole form A using high-resolution synchrotron X-ray powder

diffraction data,” J. Pharm. Sci. 99, 1734-1744.

Ferreira, F. F., Trindade, A. C., Antonio, S. G. and Paiva-Santos, C. O. (2011). “Crystal structure

of propylthiouracil determined using high-resolution synchrotron X-ray powder

diffraction,” CrystEngComm 13, 5474 - 5479.

Figueiredo, J. M., Camara, C. A., Amarante, E. G., Miranda, A. L. P., Santos, F. M., Rodrigues, C. R.,

Fraga, C. A. M. and Barreiro, E. J. (2000). “Design and synthesis of novel potent antinociceptive

agents: N-acetylimidazolyl N-acylhydrazone derivatives,” Bioorg. Med. Chem. 8, 2243-2248.

Florence, A. J., Shankland, N., Shankland, K., David, W. I. F., Pidcock, E., Xu, X., Johnston, A.,

Kennedy, A. R., Cox, P. J., Evans, J. S. O., Steele, G., Cosgrove, S. D. and C. S. Frampton

(2005). “Solving molecular crystal structures from laboratory X-ray powder diffraction data with

DASH: the state of the art and challenges,” J. Appl. Crystallogr, 38, 249-259.

Gonzalez-Serratos, H., Chang, R., Pereira, E. F. R., N. G. Castro, Aracava, Y., Melo, P. A., Lima, P.

C., Fraga, C. A. M., Barreiro, E. J. and E. X. Albuquerque (2001). “A novel

thienylhydrazone, 2-thienylidene3,4-methylenedioxybenzoylhydrazine, increases inotropism and

decreases fatigue of skeletal muscle,” J. Pharmacol. Exp. Ther. 299, 558-566.

Grant, D. J. W. (1999). “Theory and origin of polymorphism”, in Polymorphism in pharmaceutical

solids, edited by H. G. Brittain (New York, Marcel Dekker), Vol. 95, pp. 1-33.

Fanny N. Costa et al. S506S506 Vol. 28, No.S2, September 2013.

Haleblian, J. K. and McCrone, W. J. (1969). “Pharmaceutical applications of polymorphism,” J.

Pharm. Sci. 58, 911-929.

Harris, K. D. M. (2002). “Structure determination of molecular materials from powder

diffraction data,” Curr. Opin. Solid St. Mat. Sci. 6, 125-130.

Harris, K. D. M. and Cheung, E. Y. (2004). “How to determine structures when single crystals

cannot be grown: opportunities for structure determination of molecular materials using powder

diffraction data,” Chem. Soc. Rev. 33, 526-538.

Harris, K. D. M., Tremayne, M. and Kariuki, B. M. (2001). “Contemporary advances in the use of

powder X-ray diffraction for structure determination.” Angew. Chem. Int. Ed., 40, 1626-1651.

Hofmann, D. W. M. (2002). “Fast estimation of crystal densities,” Acta Crystallogr., Sect. B:

Struct. Sci. 58, 489-493.

Hull, A. W. (1919). “A New Method of Chemical Analysis,” J. Am. Chem. Soc. 41, 1168-1175.

Järvinen, M. (1993). “Application of symmetrized harmonics expansion to correction of the

preferred orientation effect,” J. Appl. Crystallogr. 26, 525-531.

Kümmerle, A. E., Raimundo, J. M., Leal, C. M., da Silva, G. S., Balliano, T. L., Pereira, M. A., de

Simone, C. A., Sudo, R. T., Zapata-Sudo, G., Fraga, C. A. M. and E. J. Barreiro

(2009). “Studies towards the identification of putative bioactive conformation of potent

vasodilator arylidene N-acylhydrazone derivatives,” Eur. J. Med. Chem. 44, 4004-4009.

Langford, J. I. Louër, D. (1996). “Powder Diffraction,” Rep. Prog. Phys., 59, 131-234.

LeJemtel, T. H., Gumbardo D,, Chadwick, B., Rutman, H. I. and Sonnenblick, E. H. (1986).

“Milrinone for long-term therapy of severe heart failure: clinical experience with special

reference to maximal exercise tolerance,” Circulation 73, III213–III218.

Lima, L. M. and Barreiro, E. J. (2005). “Bioisosterism: A Useful Strategy for Molecular

Modification and Drug Design,” Curr. Med. Chem. 12, 23- 49.

Lima, P. C., Lima, L. M., Silva, K. C., Léda, P. H., Miranda, A. L., Fraga, C. A. and Barreiro, E. J.

(2000). “Synthesis and analgesic activity of novel N-acylarylhydrazones and isosters, derived

from natural safrole,” Eur. J. Med. Chem. 35, 187-203.

Lombardino, J. G. and Lowe, J. A. (2004). “The role of the medicinal chemist in drug discovery

– then and now,” Nat. Rev. Drug Discov. 3, 853-862.

Macrae, C. F., I. J. Bruno, I. J., Chisholm, J. A., Edgington, P. R., McCabe, P., Pidcock, E., Rodriguez-

Monge, L., Taylor, R., van De Streek, J. and Wood, P. A. Wood (2008). “Mercury CSD 2.0– new

features for the visualization and investigation of crystal structures,” J. Appl. Crystallogr. 41,

466-470.

Markvardsen, A. J., David, W. I. F., Johnson, J. C. and Shankland, K. (2001). “A probabilistic

Structure Re-determination of LASSBio-294 – a cardioactive compound S507S507 Vol. 28, No.S2, September 2013.

approach to space-group determination from powder diffraction data,” Acta Crystallogr., Sect. A:

Found. Crystallogr. 57, 47-54.

Menden, A. (1998). “Crystal Structure Solution from Powder Diffraction Data – State of the Art

and Perspectives,” Croat. Chem. Acta 71, 615-633.

Nic, M., J. Jirat and B. Kosata (2007). IUPAC Compendium of Chemical Terminology - The

Gold Book. (Blackwell Scientific Publications, Oxford), 2nd ed., p. 1622.

Pawley, G. S. (1981). “Unit-cell refinement from powder diffraction scans,” J Appl.

Crystallogr. 14, 357-361.

Piaz, V. D., Giovannoni, M. P., Castellana, C., Palacios, J. M., Beleta, J., Domenech, T. and Segarra,

V. (1997). “New heterocyclic fused pyridazinones as potent and selective phosphodiesterase

IV inhibitors,” J. Med. Chem. 40, 1417–1421.

Rietveld, H. M. (1967). “Line profiles of neutron powder-diffraction peaks for structure

Refinement,” Acta Crystallogr. 22, 151-152.

Rietveld, H. M. (1969). “A Profile Refinement Method for Nuclear and Magnetic Structures,” J.

Appl. Crystallogr. 2, 65-71.

Shell, J. W. (1963). “X-Ray and Crystallographic Applications in Pharmaceutical Research II,”

J. Pharm. Sci. 52, 24-29.

Silva, A. G., G. Zapata-Sudo, A. E. Kummerle, C. A. M. Fraga, E. J. Barreiro and R. T. Sudo

(2005). “Synthesis and vasodilatory activity of new N-acylhydrazone derivatives,

designed as LASSBio-294 analogues,” Bioorg. Med. Chem. Lett. 13, 3431-3437.

Silva, C. L. M., F. Noël and E. J. Barreiro (2002). “Cyclic GMP-dependent vasodilatory

properties of LASSBio-294 in rat aorta,” Br. J. Pharm. 135, 293-298.

Spek, A. L. (2003). “Single-crystal structure validation with the program PLATON,” J. Appl.

Crystallogr. 36, 7-13.

Sudo, R. T., E. X. Albuquerque, E. J. Barreiro, Y. Aracava, W. M. Cintra, P. A. Melo, F. G.

Noël, G. Z. Sudo, C. L. M. Silva, N. G. Castro, P. D. Fernandes, C. A. M. Fraga and A.

L. P. Miranda (2006). Thienylhydrazon with digitalis-like properties (positive inotropic effects).

Appl. Nr. 10/070,328, US Patent 7,091,238 B1, University of Maryland.

Sudo, R. T., G. Zapata-Sudo and E. J. Barreiro (2001). “The new compound, LASSBio- 294, increases

the contractility of intact and saponin-skinned cardiac muscle from wistar rats,” Br. J. Pharm.

134, 603- 613.

Toby, B. H. (2006). “R factors in Rietveld analysis: How good is good enough?,” Powder Diffr.

21, 67-70.

van Laarhoven, P. J. M. and E. H. L. Aarts (1992). Simulated Annealing: Theory and

Fanny N. Costa et al. S508S508 Vol. 28, No.S2, September 2013.

Applications (Kluwer Academic Publishers, Dordrecht, Holland), 4th ed., p. 198.

Viegas-Júnior, C., A. Danuello, V. S. Bolzani, E. J. Barreiro and C. A. M. Fraga (2007).

“Molecular hybridization: a useful tool in the desing of new drug prototypes,” Curr. Med. Chem.

14, 1829-1852.

Wermuth, C. G. (1996). The Practice of Medicinal Chemistry (Academic Press: New York),

1st ed., p. 968.

Yu, L., S. M. Reutzel and G. A. Stephenson (1998). “Physical characterization of polymorphic

drugs: an integrated characterization strategy,” Pharm. Sci. Tech. Today 1, 118-127.

Structure Re-determination of LASSBio-294 – a cardioactive compound S509S509 Vol. 28, No.S2, September 2013.