Computational studies on the anastrozole and letrozole, effective chemotherapy drugs against breast...

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 122 (2014) 142–152

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Spectrochimica Acta Part A: Molecular andBiomolecular Spectroscopy

journal homepage: www.elsevier .com/locate /saa

Computational studies on the anastrozole and letrozole, effectivechemotherapy drugs against breast cancer

1386-1425/$ - see front matter � 2013 Elsevier B.V. All rights reserved.http://dx.doi.org/10.1016/j.saa.2013.11.028

⇑ Corresponding author. Fax: +90 462 325 3196.E-mail address: [email protected] (H.T. Akçay).

Hakki Türker Akçay a,⇑, Riza Bayrak b

a Department of Chemistry, Faculty of Art and Science, Recep Tayyip Erdogan University, 53100 Rize, Turkeyb Department of Chemistry, Faculty of Art and Science, Sinop University, 57000 Sinop, Turkey

h i g h l i g h t s

� Geometric optimization andvibrational investigation.� Calculations of chemical shift of the

title compounds using DFT method.� Natural bond orbital analysis of the

title compounds.� Determination of global and local

reactivity parameters of the titlecompounds.

g r a p h i c a l a b s t r a c t

a r t i c l e i n f o

Article history:Received 13 September 2013Received in revised form 5 November 2013Accepted 6 November 2013Available online 16 November 2013

Keywords:DFTAnastrozoleLetrozoleLocal reactivityNMR

a b s t r a c t

In this paper, computational studies were carried out on anastrozole and letrozole, chemotherapy drugsused against breast cancer. Optimization and frequency calculations were performed at B3LYP/6–31G (d)basis set and vibrational frequencies were assignment. Single point calculations were performed at DFTmethod with a hybrid functional B3LYP/6–311G (d, p) basis set. Theoretical NMR data were obtained atDFT method with a hybrid functional B3LYP/6–311G++ (2d, p) with GIAO (Gauge-Independent AtomicOrbital). IEF-PCM method was used as solvation model. NBO calculations were performed by the samebasis set and calculation method with single point calculation. Global and localized reactivity parame-ters; fukui indices (f) chemical hardness (g), softness (S), chemical potential (l), electronegativity (v)and electrophilicity index (x) were calculated. All computational parameters were compared with theexperimental results obtained from the literature.

� 2013 Elsevier B.V. All rights reserved.

Introduction

Breast cancer, the most common type of cancer among womenis an important part of cancer-related death. It is known that theprogression of breast cancer is related to the hormone estrogen.Previous studies showed that the concentration of 17b-estradiol(E2) in breast tumor can be ten-fold higher than those in plasma.Breast cancer cells require estrogen to progress thus, blocking ofthe estrogen synthesis can prevent to progression of the cancer.

Aromatase, in other words cytochrome P450 is an enzyme havinga function of estradiol E2 synthesis from cholesterol. Estradiol isthe most biologically active estrogen. Thus, inhibition of aromatasecan provide treatment of the breast cancer [1,2].

Aromatase inhibitors are classified as steroidal- (type I) and non-steroidal-type (type II). Type II inhibitors have heterocyclic azolemoiety binding to the heme–iron in aromatase. Anastrozole andletrozole are important type (II) aromatase inhibitors used in thetreatment of breast cancer. These compounds having 1,2,4-triazolemoieties coordinate the heme–iron of cytochrome P450. In addition,benzonitrile substituted anastrozole and letrozole mimic the struc-ture of enzyme’s natural substrate androstenedione. Anastrozole

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H.T. Akçay, R. Bayrak / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 122 (2014) 142–152 143

and letrozole are marked trade name with Arimidex and Femararespectively. Its known that letrozole are more effective thananastrozole in binding with active site of aromatase [3–11].

The aim of this study was to examine the relationship betweeninhibition mechanism of the aromatase enzyme and electronicproperties of anastrozole and letrozole. For this purpose, determi-nation of structural parameters, vibrational investigations andNMR studies were done computationally and results wereconfirmed by comparing with the experimental data obtained fromliterature [12,13]. Then HOMO and LUMO energies, electrostaticpotential map, global and local reactivity parameters were calcu-lated and were studied the effect of these parameters on inhibitionmechanism of the aromatase enzyme.

Computational details

The crystal structure of anastrozole was determined previously[14]. All experimental results; FT-IR, NMR data, were obtained atliterature [12,13]. The geometric optimization study was per-formed by using its crystal structure parameters. For geometricoptimization of letrozole (II), initial geometry was obtainedsemi-emprical PM3 method. The geometric optimization of twocompounds were performed at DFT (density functional theory) cal-culations with a hybrid functional B3LYP (Becke’s three parameterhybrid functional using the LYP correlation functional) 6–31G (d)[15,16] were performed with the Gaussian 03 W [15]. Vibrationalfrequencies were calculated at same basis set with optimizationand obtained vibrational frequencies scaled by 0.9614 [17].Absence of imaginary frequency indicated the structures were inglobal minimum. Vibrational assignments and other computa-tional visualizations were performed by using Gauss-view molecu-lar visualization software [16]. Vibrational assignments werecompared with literature [12,13]. Single point energies of the opti-mized structures were calculated DFT calculations with a hybridfunctional B3LYP/6–311G(d,p) basis set. 1H and 13C chemical shiftcalculations were studied at B3LYP/6311++G (2d, p) basis set withGIAO (Gauge-Independent Atomic Orbital). IEF-PCM method wasused as solvation model and chloroform was used as solvent. Com-putational NMR results were compared with literature [12,13].NBO calculations were performed by using NBO 3.1 program asimplemented in Gaussian 03 W [15].

Results and discussion

Molecular geometry of the compounds

The molecular geometries of the two compounds are in Csgroup symmetry. For anastrozole, X-ray structural parameterswere used for geometric optimization. Geometrical parameters ofthe compounds were listed in Table S1. Optimized structures of

Fig. 1. Optimized geometries of

the compounds were shown in Fig. 1. Letrozole is a compoundsimilar with anastrozole as structural properties, so bond parame-ters of the letrozole obtained from geometric optimization com-pared with those of the crystal structure of the anastrozole. Ascan be seen in Table S1, all geometric parameters agreed with eachother.

Assignments of the vibrational modes of the compounds

The theoretical vibrational frequencies of the compounds werecalculated using B3LYP methods with 6-31G(d) basis set and werecompared with the experimental results in literature [12,13]. Thevibration bands assignments have been made using Gauss-Viewmolecular visualization program [16]. The frequency values com-puted at the calculation level contain known systematic errors.Therefore, we have used the scaling factor value of 0.9614 forB3LYP [17]. Some theoretical and experimental vibrational dataare shown in Tables 1 and 2. As can be seen in Figs. 2 and 3, thetheoretical vibrational frequencies have good correlation with cor-responding experimental results. Correlation between the experi-mental and vibrational of the anastrozole and letrozole wereshown in Fig. 4.

CAH vibrationsThe aromatic structures have characteristic CAH stretching

vibrations at 3000–3100 cm�1 range. Similarly, the vibrationalfrequencies calculated at the range of 3152–3031 cm�1 and3165–3068 cm�1 belongs to anastrozole and letrozole aromaticCH stretching vibrations (t) respectively [18–20]. Experimentalaromatic CH stretching vibrations of anastrozole and letrozole ob-tained from literature [12,13] were observed at 3120–3050 cm�1

and 3101, 2985 cm�1 respectively.Aromatic CH in plane bending vibrations were observed at the

range of 1300–1000 and out of plane bending vibrations were ob-served at the range of 675–1000 cm�1. For anastrozole; CAH inplane bending vibrations were calculated at the range of1449–1108 cm�1 and observed at 1387, 1368, 1272, 1195, 1160,1151 cm�1. The vibrations calculated between 932–697 cm�1 andobserved at 896, 893, 875, 790, 763, 713 cm�1 were assignedCAH out of plane bending vibrations. All experimental CAH vibra-tions were good agreement with the theoretical results. For letroz-ole; aromatic CH in plane bending vibrations calculated at therange 1328–1031 cm�1 and observed at 1270, 1200, 1139,1003 cm�1 experimentally. Aromatic CH out of plane bendingvibrations were calculated at the range of 947–537 cm�1 andobserved at 955–555 cm�1 experimentally [18,21,22].

CH3 vibrationsGenerally CH stretching vibrations in CH3 units locate at the

lower frequency region than those of aromatic ring and asymmet-ric stretching vibrations are at higher frequencies than the

anastrozole and letrozole.

Table 1Some selected vibrational assignments of anastrozole.

Non-scaled (cm�1) Scaled (cm�1) Exp. [12] (cm�1) Int. (km/mol) Assignment

3279 3153 3101 4 t CH Triazol3273 3146 2.3 t CH Triazol3210 3086 3.3 t CHsym phenyl3210 3086 5.8 t CHasym phenyl3205 3081 0.7 t CH phenyl3152 3030 14 tasym CH33151 3029 13.4 tasym CH33148 3027 11.1 tasym CH33146 3025 18.8 tasym CH33145 3023 35.4 tasym CH33140 3019 4.6 tasym CH33140 3019 32 tasym CH33136 3015 2.5 tasym CH33131 3010 4.5 tasym CH23084 2965 2985 15.7 tsymCH23070 2951 12.6 tsym CH33066 2948 13.9 tsym CH33065 2947 12.7 tsym CH33063 2944 11.5 tsym CH32358 2267 5.7 t C„N2356 2265 2235 5.9 t C„N1656 1592 18.6 t C@C1655 1591 1606 26.7 t C@C1560 1500 1500 61.3 t C@N triazole1538 1479 9.7 CH3 deformation1537 1477 8.5 CH3 deformation1533 1473 1475 7.6 CH3 deformation1529 1470 9.1 CH3 deformation1517 1458 4.3 CH3 deformation1516 1457 3.5 CH3 deformation1516 1457 1.8 CH3 deformation1513 1454 0.3 CH3 deformation1508 1449 14.4 t C@C + b CH phenyl + b CH2scisorring

1504 1446 2.3 b CH phenyl + b CH2scisorring

1483 1426 1387 19.3 t C@C + b CH phenyl1475 1418 1368 16.2 b CH triazole + t C = N + b CH2scisorring

1449 1393 1.5 CH3 deformation1448 1392 2.7 CH3 deformation1427 1372 4.5 CH3 deformation1426 1371 3.2 CH3 deformation1405 1351 11.3 b CH triazole + c CH2wagging + t C@N triazole1392 1339 38.3 b CH triazole + c CH2wagging + t C@N triazole1372 1319 0.9 t C@C + b CH phenyl1353 1301 8.7 b CH2twisting + t NAN triazole + t CAN triazole1331 1280 3.6 b CH phenyl1316 1265 1272 34.7 c CH2wagging + t CAN + b CH triazole1307 1257 4.1 b CH phenyl + t CAC + c CH2wagging + c CH31257 1208 34.1 b CH phenyl + t CAC + c CH2wagging + c CH31240 1192 1195 33 b CH triazole + t CAC1238 1190 3.2 b CH phenyl + b CH triazole + c CH31230 1183 7.7 c CH3 + b CH phenyl1219 1171 1160 19.6 b CH phenyl1191 1145 5.1 b CH2twisting + b CH triazole1175 1129 1151 28 b CH triazole1173 1128 4 b CH phenyl1166 1121 4.3 b CH phenyl + c CH3 + b CH2twisting

1156 1111 1.7 t CAC + c CH3wagging

1153 1108 0.6 b CH phenyl + c CH3rocking

1053 1012 0.05 c CH3rocking

1050 1009 0.4 c CH3rocking

1037 997 1012 38.1 c CH2rocking + b triazole ring1018 979 0.5 b CACAC phenyl981 943 3.6 c CH3rocking

974 936 924 9.6 c CH2rocking + b triazole ring969 932 4.6 c CH phenyl + c CH2rocking

960 922 2.4 c CH3rocking + c CH phenyl957 920 0.1 c CH3rocking + c CH phenyl956 919 1.4 c CH3rocking

949 912 0.4 c CH3rocking + c CH phenyl933 896 1.6 c CH phenyl928 892 896 6 c CH phenyl906 871 883 4.1 c CH phenyl895 860 4.8 c CH triazole888 853 875 7.8 c CH phenyl CH + c CH2rocking

144 H.T. Akçay, R. Bayrak / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 122 (2014) 142–152

Table 1 (continued)


839 807 790 10.7 c triazole ring out of plane bending805 774 1.3 c CH3780 750 763 22.7 c CH phenyl + c CH2rocking + c triazole ring747 718 3.5 c CH phenyl725 697 713 5.7 c CH phenyl695 668 679 20.1 c triazole ring669 643 664 13.1 c triazole ring + c phenyl ring658 632 634 3.1 c triazole ring + c phenyl ring634 609 3.3 c triazole ring + c phenyl ring623 599 1.5 c triazole ring + c phenyl ring

Table 2Some selected vibrational assignments of letrozole.


3292 3165 0.5 t CH Triazol3279 3152 3120 6.1 t CH Triazol3228 3103 2.1 tsym CH phenyl3227 3103 0.9 tsym CH phenyl3225 3101 3.5 tsym CH phenyl3223 3099 3.7 tsym CH phenyl3214 3090 1.7 tasym CH phenyl3212 3088 2.1 tasym CH phenyl3197 3074 4.2 tasym CH phenyl3191 3068 3050 7.1 tasym CH phenyl3081 2962 2980 3.8 t CH aliphatic2352 2261 21.6 t C„N2351 2260 2232 32.3 t C„N1670 1605 4.5 t C@C1666 1602 1608 13.9 t C@C1619 1556 2.8 t C@C1614 1552 1.3 t C@C1557 1496 1503 51.1 t C@N + t C@C1556 1496 16.5 t C@N + t C@C1555 1495 17.2 t C@C1474 1417 6.7 t C@N + b CH aliph.1458 1402 1434 12.6 t C@C + b CH aliph.1451 1395 1408 18 t C@C + b CH aliph1402 1348 15 t C@N + b CH aliph.1381 1328 12.7 b CH aliph. + b CH phenyl1365 1312 4.9 b CH aliph. + b CH phenyl1346 1294 2.2 b CH phenyl1342 1290 1.2 b CH phenyl1333 1282 4.3 b CH aliph. + t C@C1322 1271 1270 51.3 b CH triazole + t CAN1303 1253 18 b CH + b CH aliph.1239 1191 1200 23.1 b CH triazole + b CH phen + b CH aliph.1237 1189 3.9 b CH triazole + b CH phen1234 1187 5.7 b CH triazole + b CH phen1224 1177 7.9 b CH triazole + b CH phen + b CH aliph.1220 1173 2.4 b CH phen + b CH aliph. + t NAN triazole1216 1169 1.9 b CH phen + b CH aliph. + t NAN triazole1212 1165 1.5 b CH phen + b CH aliph.1196 1150 0.2 b CH phen + b CH aliph.1170 1125 1139 25 b CH triazole + t NAN triazole1152 1108 5.6 b CH phen1148 1103 1.7 b CH phen1042 1001 8 b CACAC phenyl1041 1001 4.5 b CACAC phenyl1031 991 1003 36.4 b CH triazole + b triazole ring985 947 0.3 c CH phenyl980 942 0.2 c CH phenyl979 941 0.2 c CH phenyl974 936 2.3 c CH phenyl974 936 955 8.7 c CH phenyl + b triazole ring901 867 5.5 c CH triazole896 861 8.9 c CH phenyl883 849 868 9.9 c CH phenyl871 837 858 10.1 c CH phenyl861 828 1 c CH phenyl857 824 1.3 c CH phenyl851 818 8.4 c CH triazole + c CH phenyl

(continued on next page)


Fig. 2. Computational and experimental vibrational spectra of the anastrozole (a) experimental [12], (b) computational (B3LYP/631G(d)).

Table 2 (continued)


838 806 831 40.5 c CH phenyl811 780 789 38.1 c CH phenyl777 747 6.3 c CH phenyl + c C@C phenyl766 737 5.9 c CH phenyl + c C@C phenyl733 705 2.8 c phenyl ring719 691 3.7 c phenyl ring694 667 677 17.7 c CNC triazole665 639 657 7.4 c CNC triazole652 627 2.7 c CNC triazole583 561 567 14.5 c phenyl ring + c CCN nitrile578 556 555 15.8 c phenyl ring + c CCN nitrile560 538 0.7 c phenyl ring + b CN nitrile559 537 0.1 c phenyl ring + c CCN nitrile


Fig. 3. Experimental and computational vibrational spectra of letrozole (a) experimental [13] (b) computational (B3LYP/631G(d)).


symmetric vibrations. Aliphatic asymmetric CH stretching vibra-tions were calculated at the range of 3030–3015 cm�1 and sym-metric vibrations were calculated at the range of 2951–2944 cm�1. Experimental data were agreed with the computa-tional results.

Phenyl substituted CH3 deformation bands observe between at1465–1440 cm�1 and 1390–1370 cm�1. In this study, CH3 defor-mation bands were calculated at the range of 1479–1454 cm�1

and 1393–1371 cm�1. Usually phenyl substituted CH3 rockingvibrations are observed at about 1050 cm�1. Computationally,CH3 rocking vibrations were calculated between at 1108 and912 cm�1 [21].

CH2 vibrationsFor anastrozole; aliphatic CH2 symmetrical stretching vibra-

tions were calculated as 3010 cm�1 and asymmetrical stretching

vibration at were calculated at 2965 cm�1. Generally CH2 deforma-tion vibrations were observed in low frequency region as scissor-ing, wagging, and twisting modes. CH2 scissoring vibrations werecalculated at 1449, 1446, 1368 cm�1 and observed at 1368 cm�1.Wagging and twisting modes were assigned at 1351, 1339, 1265,1257, 1208 cm�1 and 1301, 1145, 1121 cm�1 respectively. CH2

rocking vibrations were calculated at 1037, 974, 969 cm�1

[21,23,24].

C„N vibrationsGenerally, C„N stretching vibrations are observed at about

2250 cm�1 [25,26]. In our study, C„N stretching vibrations ofanastrozole and letrozole were observed at 2235 and 2232 cm�1

respectively and computationally, these modes were calculatedas sharp bands at 2267 and 2265 cm�1 for anastrozole and sharpbands at 2261 cm�1 and 2260 cm�1 for letrozole.

Fig. 4. Correlation between experimental and calculated vibrational data of thecompounds.


Ring vibrationsTriazole C@N stretching vibrations usually appear between at

1500–1300 cm�1.In this study, C@N stretching vibrations occurdue to triazole ring. Experimentally, C@N stretching vibrations ofanastrozole and letrozole assigned at 1500 and 1503 cm�1. Theo-retically, anastrozole and letrozole vibrational frequencies ofC@N were at 1500, 1418, 1351, 1339 cm�1 and 1496, 1417,1348 cm�1 respectively [8].

Table 31H and 13C NMR chemical shifts of the anastrozole.

Atom Experimental [12] B3LYP/6311++G(2dp)

C22 29.03 26.6628C17 30.6995C18 32.7676C21 34.1804C19 37.26 42.2517C15 45.1996C12 53.03 56.4164C11 122.11 126.785C7 123.73 130.585C16 124.23 131.64C9 132.597C20 132.794C6 136.61 144.711C14 148.862C10 143.24 150.652C8 143.44 151.665C13 152.5 158.982

Aromatic C@C stretching vibrations are observed at 1625–1430 cm�1. In this study, the frequencies were calculated at1592, 1591, 1449, 1426, 1319 cm�1 for anastrozole and 1605,1602, 1556, 1552, 1496, 1402, 1395 cm�1 for letrozole. Experimen-tally, anastrozole and letrozole C@C stretching vibrations wereobserved at 1606, 1387 cm�1 and 1608, 1503, 1434, 1408 cm�1

respectively. The CACAC in plane bending mode of benzene arecalculated at 979 cm�1 for anastrozole and at 1001 cm�1 for letroz-ole. The CANAC triazole in plane bending ring vibrations werecalculated at 991, 936 cm�1 (1003, 955 cm�1 experimentally) forletrozole and 997, 936 cm�1 (1012, 924 cm�1 experimentally) foranastrozole. Out of plane bending phenyl ring vibrations were ob-served at the range of 900–500 cm�1. In our study, CH out of planebending vibrations of anastrozole and letrozole were observed atthe range of 932–697 cm�1 and 947–737 cm�1. All computationalfrequencies are in good agreement with the literature [27–29].

NMR spectra of the compounds

NMR is one of the most useful technique the identification ofthe compounds. Thanks to its high accuracy, impurities of the com-pounds are also determined by this technique, thus this techniqueplay an important role with together chromatographic techniquesin drug industry.

NMR calculations with DFT methods give acceptable resultscompared with the experimental results. The geometry of a mole-cule has an important effect on the consistent chemical shift. Inthis study, 1H and 13C chemical shift calculations were studied atB3LYP/6311++G (2d, p) basis set with GIAO (Gauge-IndependentAtomic Orbital). As solvation model, IEF-PCM method was usedand chloroform was chosen as solvent. Computational and experi-mental 1H and 13C chemical shifts are listed in Tables 3 and 4 [30].

The electron withdrawing and electron donating groups haveconsiderable effect on chemical shift of a group in 1H NMR spec-trum. Generally, protons attached to sp3 hybridized carbonsobserve between at 0 and 2 ppm and aromatic protons resonateat the range of 7–8 ppm. In 13C NMR spectrometry, sp3 hybridizedcarbons resonate between at 10–90 ppm and sp2 hybridized car-bons resonate between at 100 and 220 ppm. Aromatic carbonsare observed at the range of 100–170 ppm and sp hybridized nitrilecarbons resonated at 110–130 ppm [24,31,32].

For anastrozole; 12 aliphatic protons resonated between at1.9648 and 1.2634 ppm theoretically and at 1.75 ppm asexperimentally. Aromatic protons resonated at the range of

Atom Experimental [12] B3LYP/6311++G(2dp)

H24 7.5 8.3484H29 8.15 8.1962H28 8 8.1429H23 7.3 8.1527H25 7.4121H26 5.4 5.7017H27 5.1362H28 1.75 1.9648H29 1.946H40 1.8951H37 1.8947H34 1.8748H41 1.8481H31 1.7774H32 1.738H33 1.7193H35 1.6717H30 1.3722H36 1.2634

Table 41H and 13C NMR chemical shifts of the letrozole.

Atom Experimental [13] B3LYP/6311++G(2dp) Atom Experimental [13] B3LYP/6311++G(2dp)

C3 66 72.75 H33 8.07 8.22C22 113.29 114.89 H13 7.30 8.20C12 118.04 H14 8.12C26 117.82 126.94 H23 7.28 8.12C25 127.00 H24 8.07C17 128.9 132.85 H32 8.09 7.86C16 134.84 H9 7.72 8.06C7 135.98 H11 7.22C6 137.82 H19 7.70 7.77C10 132.91 139.82 H21 6.85C8 140.81 H4 6.81 7.33C18 140.88C20 141.47C5 141.76 150.80C2 143.68 151.28C15 153.05 153.37C1 159.72


7.3–8.15 ppm theoretically and at the range of 7.4–8.3 ppm asexperimentally. H26 and H27 protons resonated at 5.4 ppm exper-imentally and theoretically chemical shifts of the H26 and H27protons calculated at 5.7017 and 5.1362 ppm. In anastrozole, sp3

hybridized aliphatic carbons were observed at 26.66, 30.70,32.77, 34.18, 42.25, 45.20, 56.41 ppm computationally and at29.03, 37.26, 53.03 ppm experimentally. C16 and C20 nitrile car-bons were calculated at 131.64 and 132.79 ppm respectively andobserved at 124.23 ppm experimentally. All sp2 hybridized carbonsresonated at the range of 122.11–152.50 ppm experimentally andat the range of 126.79 – 158.98 ppm theoretically. 1H NMR and13C NMR spectra of anastrozole were shown in Figs. S1 and S2.The experimental 1H NMR and 13C NMR data of anastrozole wereobtained from literature and agreement with computational re-sults [12,13].

For letrozole; only one aliphatic proton resonate at 6.81 exper-imentally and 7.33 ppm computationally. All of the other protonshave hybridization of sp2 and resonated at the aromatic region in1H NMR spectrum. In 13C NMR, aliphatic sp3 hybridized C3 carbonresonated at 66 ppm experimentally and 72.75 ppm theoretically.Nitrile groups hybridized sp resonated at 117.82 ppm experimen-tally and at 126.94 and 127.00 ppm theoretically as expected.Other carbons hybridized sp2 resonated at the range of113.29 – 153.05 ppm experimentally and 114.89 – 153.37 ppmtheoretically. 1H NMR and 13C NMR spectra of letrozole are shownin Figs. S3 and S4. The experimental 1H NMR and 13C NMR data ofletrozole were obtained from literature are in agreement withcomputational results [12,13].

NBO analysis

Natural bond orbital (NBO) analysis is an effective method todescribe the inter- and intra-molecular bonding, conjugation andcharge transfer phenomenon. The charge transfer from donor (i)to acceptor (j) orbitals results from increasing stabilization energy.The second-order Fock matrix was used the determination of thedonor–acceptor interactions.

The stabilization energy (E2) gained from this interactionscalculated as;

E2 ¼ DEij ¼F2ði;jÞ

ej � ei

where qi is the donor orbital occupancy, ei and ej diagonal elementsand F(i,j) is the diagonal NBO Fock matrix element. The larger the E2

value expands the conjugation of the whole system. The

hyperconjugative interaction energy was calculated from second-order perturbation approach [33–35]. In this study, NBO calcula-tions were performed at B3LYP/631++G(d, p) basis set. Orbital inter-action and second order perturbation theory energy analysis ofanastrozole and letrozole were shown in Table S2.

The interaction between r(CAC) and r� (CAC) orbitals causethe intra-molecular charge transfer provided the extra stabilizationenergy to system. While the electron density of the r orbitaldecreases, the electron density of the anti-bonding r� orbital in-creases as a result of charge transfer between the donor and accep-tor orbitals. As can be seen in Table S2, LP? r� and LP ? p� chargetransfers provided the highest stabilization energy to system.

For anastrozole LP (N1) ? p� (N2AC13), LP (N1) ? p* (N3AC14)transitions provided extra stabilization energy to the triazole rings.Another important interaction, r ? r� observed as r (N2AC13)? r� (N1AC12), r (C10AC19) ? r� (N5AC20), r (C15AC17) ? r�

(N4AC16) gained 5.27, 4.53, 4.41 kJ mol�1 energy to the moleculerespectively. p ? p* interactions are important as well as LP ? r�

interactions in terms of stabilization energy. The interactionof p (N2AC13) ? p� (N3AC14), p (C6AC7) ? p� (C8AC9), p(C8AC9) ? p� (C10AC11) provide 10.58, 20.2, 21.16 kJ mol�1 theinteraction stabilization energy respectively.

In letrozole LP (N29) ? p� (C1AN30), LP (N29) ? p� (C2AN31)and provide the extra stabilization energy to the triazole rings. An-other important interaction, r ? r� observed as r (C1AN30) ? r�

(C3AN29), r (C1AN31) ? r� (C2AH32), r (C12AC25) ? r�

(C25AN28) gained 5.11, 5.69, 5.09 kJ mol�1 energy to the moleculerespectively. p ? p* interactions are important as well as LP ? r�

interactions in terms of stabilization energy. The interaction of p(C2AN31) ? p� (C1AN30), p (C5AC7) ? p� (C10AC12), p(C10AC12) ? p� (C6AC8), p (C15AC17) ? p� (C20AC22) provide31.19, 21.48, 19.67, 22.23 kJ mol�1 the interaction stabilizationenergy, respectively.

Chemical reactivity

Single point calculation and global reactivityHOMO (highest occupied molecular orbital) and LUMO (lowest

unoccupied molecular orbital) give importing formation aboutchemical reactivity of a compound. Nucleophilic and electrophilicreactions were governed by HOMO and LUMO respectively. HOMOis responsible for donating an electron, while LUMO behaves anelectron acceptor. In addition, the energy gap between HOMOand LUMO determines the electronic transition, optical polarizabil-ity, chemical hardness and softness and kinetic stability of amolecule [24]. In this study, single point calculations were studied

Fig. 5. Some frontier orbitals of (a) anastrozole, (b) letrozole.

Fig. 6. Electrostatic potential map of anastrozole and letrozole (isovalue: 0.004).


at B3LYP/631++G(d, p) basis set and in addition to gas phase,IEF-PCM method was used as solvation model and water, ethanoland chloroform were chosen as solvents. Some molecule orbitalsof anastrozole and letrozole were shown in Fig. 5. As can be seenin Fig. 5, HOMO and HOMO � 1 located at all over the molecule,while LUMO and LUMO + 1 located on phenyl rings in both anas-trozole and letrozole. The polarity of a molecule is related with di-pole moment. The molecules having higher dipole moment valueshow the higher chemical reactivity [36].

Electronic properties of anastrozole and letrozole were calcu-lated according to Koopmans’ theorem. Ionization potential as-signed IP is equal to negative value of HOMO energy andelectron affinity assigned EA is equal to negative value of LUMO en-ergy. Global parameters like chemical hardness (g), softness (S),chemical potential (l), electronegativity (v) and electrophilicityindex (x) were calculated according to literature [24]. Global reac-tivity parameters were shown in Table S3. The energy values ofHOMOs and total SCF energies in both anastrozole and letrozoleare quite similar. This is remarkable, because anastrozole andletrozole bind the cytochrome P450 using their HOMO electrons.This similarity supports to generate new aromatase inhibitors hav-ing approximate HOMO energies. There were no other similaritiesbetween the global reactivity parameters of anastrozole andletrozole.

�v ¼ l ¼ IPþEA2 g ¼ IP�EA

2

S ¼ 12g x ¼ l2

2g

Local reactivityMolecular electrostatic potential (MEP) maps are good tools to

take information about reactive sites of molecules [37,38]. In thisstudy, MEP maps of anastrozole and letrozole were calculated atsame basis set used in their single point calculations. While the re-gion of negative charge was shown deep red color, positive onewas shown deep blue color. As can be seen in Fig. 6, negativecharge regions on the structures located on nitrogen atoms of tri-azole and of nitrile groups. According to these charge distributions,both anastrozole and letrozole must have three different nucleo-philic centers complexing with iron–heme of cyotochrome P450,but previous studies stated that these molecules complexed withiron–heme group of enzyme, using only nitrogen atoms of triazole

moieties [3–6,10,11]. Nitrogen atoms of nitrile groups did notcontribute the complexation, due to their sp hybridization withneighbor carbon atoms probably. Fukui functions were used toexplain the difference in known inconsistent results.

Local reactivity is an important parameter for the determina-tion of the most reactive sites of the molecule against the electro-philic and nucleophilic attack. Fukui function is used to determinenucleophilicity and electrophilicity properties of atom by atom in amolecule. For this purpose, the mulliken charges of cationic andanionic forms of the molecule is calculated and fukui functions(f�j ; fþj ), local softness (Sþk ; S�k ) and local electrophilicity(xþk ; x�k ) values were calculated using these charge values. Fukuifunctions and reactivity parameters are calculated using followingequations [24,39–42].

f�j ¼ qjðNÞ � qjðN � 1Þfþj ¼ qjðN þ 1Þ � qjðNÞxþk ¼ xfþj x�k ¼ xf�jSþk ¼ sfþj S�k ¼ sf�jDf ¼ fþj � f�j

In our study, local reactivity parameters were calculated at B3LYP/631++G(d, p) basis set and some atomic fukui (f�j ; fþj ), local electro-philicity (xþk ; x�k ), local softness (Sþk ; S�k ) values were listed inTable 5. Df is a good parameter for determining the properties ofnucleophilicity and electrophilicity. If Df < 0, the site have nucleo-philic property and Df > 0, the site have electrophilic property. Ascan be seen in Table 5, N2 and N3 atoms were nucleophilic center

Table 5Fukui functions and local reactivity parametrers of some atoms in anastrozole and in letrozole.

Atoms fþj f�j Df Sþk S�k xþk x�k

AnastrozoleN1 0.017 0.002 0.0150 0.003 0.0003 0.055 0.005N2 0.008 0.031 �0.0227 0.001 0.0051 0.027 0.103N3 0.033 0.047 �0.0133 0.006 0.0077 0.111 0.155N4 0.044 0.039 0.0051 0.007 0.0064 0.147 0.130N5 0.054 0.054 0.0003 0.009 0.0088 0.180 0.178C16 0.003 0.001 0.0023 0.001 0.0001 0.011 0.003C20 0.014 0.010 0.0039 0.002 0.0017 0.048 0.035

LetrozoleC25 0.0698 0.0504 0.0193 0.0134 0.0097 0.3547 0.2564C26 0.0556 0.0577 �0.0021 0.0107 0.0111 0.2828 0.2934N27 0.0670 0.0726 �0.0056 0.0128 0.0139 0.3403 0.3690N28 0.0796 0.0659 0.0137 0.0152 0.0126 0.4046 0.3350N29 �0.0142 0.0061 �0.0203 �0.0027 0.0012 �0.0724 0.0309N30 0.0061 0.0240 �0.0179 0.0012 0.0046 0.0312 0.1220N31 0.0275 0.0432 �0.0157 0.0053 0.0083 0.1398 0.2194

Fukui functions (f�j ; fþj ) and local softness (Sþk ; S�k ) and local electrophilicity (xþk ; x�k ).


and C16 and C20 atoms are electrophilic center in anastrozole. Inletrozole, N29, N30, N31 atoms in triazole moieties were nucleo-philic centers and C25, C26 atoms in nitrile groups are electrophiliccenters. Therefore, aromatase inhibitors, anastrozole and letrozolewith triazole moiety having nucleophilic properties bind to iron–heme in enzyme complex.

Conclusion

Consequently, the computational studies of anastrozole andletrozole, aromatase inhibitors, were performed at DFT – B3LYPmethod. All experimental data were obtained from literature. Opti-mized structural parameters were compared experimental X-raycrystallographic data obtained from literature. Vibrationalfrequency calculation and vibrational assignments were done insame basis set used in optimization. Both in structural parametersand vibrational results are good agreement with the experimentalresults. Another important parameter NMR for structural analysisof the compounds were performed computationally at DFT –B3LYP method and at 6–311G++(2d, p) basis set. The results ofcomputational NMR data were good agreement with the literature.In addition, this study focused on the electron density and reactiv-ity parameters of the compounds to better understand the inacti-vation mechanism of cytochrome P450. When forming a complexwith cytochrome P450, anastrozole and letrozole act as nucleo-philes, giving a pair of electron from HOMO. Therefore, the energyof HOMO is important factor in forming inhibitor-enzyme com-plex. In our study, HOMO energy of anastrozole and letrozole arecalculated as -7.534 and -7.734 eV respectively. The closeness ofthe HOMO energies of anastrozole and letrozole is important interms of generating new inhibitors. Although each structures havethree nucleophilic centers, only triazole group complexed with ac-tive site of cyotochrome P450. Once investigation of local reactiv-ity; it was seen that nitrogen atoms in triazole groups havenucleophilic character, while carbon atoms of nitrile groups haveelectrophilic character. This situation supported that nucleophilicattack to active site of cytochrome P450 conducts from triazolenitrogens of anastrozole and letrozole.

Appendix A. Supplementary material

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.saa.2013.11.028.

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