The use of quantum chemical indices in the interpretation of biological activity of some substituted uracils

Journal of Molecular Structure (Theochem), 306 (1994) 321-327 0166-1280/94/%07.00 0 ‘1994 - Elsevier Science B.V. All rights reserved

321

The use of quantum chemical indices in the interpretation of biological activity of some substituted uracils Pauls G. Abdul-Ahad Petroleum

Research Centre, P.O. Box 10039, Jadiriyah,

Baghdad, Iraq

(Received 12 October 1993; accepted 19 October 1993) Abstract Calculations are performed on a series of 40 uracils substituted in positions 1, 5 and 6 using a semiempirical molecular orbital (MO) method with complete neglect of differential overlap (CND0/2). These compounds are potent inhibitors of uridine phosphorlyase from 256 rat tumours. Approximate ground state electronic properties are obtained for each molecule studied as well as the electrostatic potential. First- and second-order interaction energies for 14 hypothetical point charges around the drug molecule are calculated. These points provide some information on the molecule as seen by either another molecule or by a surface Relationships between reported antitumour activity expressed as p(C,e), the negative logarithm or the molar concentration required to achieve 50% inhibition of enzyme activity, and the calculated chemical indices are formulated by performing multiple regression analyses. The best correlations obtained between the biological activity p(C,s) and the electronic indices are considered in a mechanistic context. These equations can be used to predict the activity of untested molecules.

Introduction Computer-aided design can be used in many diverse industries and technologies. The major benefits are the ability to evaluate more designs. The use of computer techniques to design new molecules, particularly in the pharmaceutical application of drug design, is of great interest. Molecules can be joined in three-dimensional space or built from three-dimensional fragments. The spatial distribution and Cartesian coordinates for a molecule can be determined from standard bond lengths and bond angles. These atomic coordinates can be transferred into a quantum chemical program to obtain molecular energies and other chemical indices. Several semiempirical molecular orbital methods are available for calculation of quantum chemical indices, such SSDI 0166-1280(93)03593-V

as, complete

neglect of differential overlap (CND0/2), intermediate neglect of differential overlap (INDO), minimum intermediate neglect of differential overlap (MIND0/3) and neglect of diatomic differential overlap (NDDO). To utilize these technologies in drug design, knowledge of the structure and mechanism of action of an enzyme is necessary to design compounds having optimal binding properties. This has to rely on the use of the aforementioned semiempirical methods to find the structural requirements for good binding and competitive enzyme inhibitors [l]. The inhibition caused by the drug can be reversible or irreversible. Reversible inhibition can be characterized by an equilibrium between the enzyme and the inhibitory drug. The inhibition is irreversible when it increases with time, provided that the inhibitory drug is

322

present in excess; the efficiency of the drug is then expressed not by an equilibrium constant but by a rate constant, which determines the fraction of the enzyme inhibited in a given period of time by a certain concentration of the inhibitor. There are two enzymes that start the detoxification of 5-fluoro-2-deoxyuridine (FUDR) by cleavage to 5-fluorouracil (FU) [2]. These are thymidine phosphorylase and uridine phosphorylase. Walker 256 rat tumour contains an FUDR cleaving enzyme that is only uridine phosphorylase [3]. Thymidine phosphorylase is an enzyme that catalyzes phosphorolysis of the nucleoside linkage of pyrimidine 2deoxy-nucleosides such as thymidine with formation of 2-deoxy a-D-ribofuranose l-phosphate and the pyrimidine. This enzyme has been isolated from a variety of plant, animal and bacterial sources. Some excellent inhibitors have been introduced for the inhibition of uridine phosphorylase from Walker 256 rat tumour. Inhibition of this enzyme by l-, 5and &substituted uracils has been reported [4-61. By a proper choice of substituent and its position on uracil, inhibitors of the Walker 256 FUDR phosphorylase have been found that complex 800-fold better than the substrate FUDR [4]. To design an effective and active site-directed irreversible inhibitor of uridine phosphorylase that utilizes the hydrophobic bonding region, it is useful to know the types of hydrophobic moieties that will give maximum interaction with the enzyme. Furthermore, binding of uracil could be made more effective when substituted with electron withdrawing groups at the l-, 5- and 6-positions which increase the acidity of uracil. The best inhibitors can be obtained by combining both phenomena, that is, hydrophobic interactions and increased acidity [7]. With a view to clarifying these assumptions, the present work employs semiempirical molecular orbital (MO) methods in an attempt to interpret the mechanism of action and inhibition of Walker 256 FUDR phosphorylase by l-, 5- and 6-substituted uracils. Therefore, it is necessary to investigate what type of substituted uracil might

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give a strong hydrophobic interaction with the Walker 256 uridine phosphorylase which cleaves FUDR to FU. Experimental

Semiempirical MO calculations were performed on 40 uracils substituted in positions 1, 5 and 6 using the complete neglect of differential overlap (CND0/2-3R) program (QCPE 261). Calculated electronic properties are: the net atomic charge QX; the atomic polarizability o [8]; the frontier electron density FE,, FN, [9] which indicates the susceptibility of the site x to nucleophilic attack; and the energy of binding of atom x to the rest of the molecule EBIND,, for each centre x in the molecule. The bond energies E(x-y) are obtained for each pair of bonded atoms x and y, using the energy breakdown procedure [lo]; and the transition energy A which is the difference between the frontier orbital energies (EHOMO and ELEMO) was also calculated. This energy is relevant to reactivity if the molecule reacts in its first excited state (activation energy for the reaction). EHOMO measures the electron donating ability while ELEMO measures the electron accepting ability. These energy levels are very important in determining the ease of formation of charge transfer complexes between the drug and receptor. The first- and second-order interaction energies, Vi and V,, of a point charge placed at a given site in the molecular environment are calculated. Vi is the electrostatic potential calculated by using method III of Ref. 11. V, is calculated by an uncoupled Hartree-Fock perturbation procedure [ 121. All numerical calculations were performed on an IBM 4341 computer.

Method

The spatial distribution and Cartesian coordinates of all atoms are determined. These atomic coordinates were transferred into a quantum

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+

Fig. 1. The numbering scheme of uracil and positions hypothetical point charges.

of

chemical program [ 13,141 to obtain the molecular orbital charge densities. These results may then be used for the calculation of other molecular properties. The atom numbering scheme used in the present study is given in Fig. 1. The starting point geometry of uracil is obtained from Ref. 15. This geometry is optimized by using program GEOMIN (QCPE 312) with the CND0/2 option to obtain a more reliable conformation of the molecules in solution. Standard bond lengths

Fig. 2. Electrostatic contour map for uracil (all values are in kilocalories per mole).

and bond angles [16] are used for the substituent groups. Conformational flexibility is of particular importance for the drug-receptor model. Hypothetical point charges around drug molecules provide information on the molecule as seen by

-1 %‘ksal/m@l

Fig. 3. Electrostatic

solid map for uracil viewed from the direction at 15” to the horizontal.

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Table 1 Observed biological No.

activity and calculated chemical indixes for l-, S- and 6-substituted uracils Substituent

P(C50)

I

FE(N3)

(ev)

(ev) 0.064060

H

2.5376

0.10210

6-NH2 I-CH3

2.3979 2.4436

0.10064 0.10297

5

6-CF3 5-F

2.4814 3.1135

0.10209 0.10162

6 7

5-Br 5-NO2

8 9

1-n-C3H, I-n-C,Hs

3.6020 4.8239 2.8239

0.10316 0.10426 0.10387

3.4559

10 11

l-n-CsH,, 6-NHC,Hs

3.6020 2.7212

0.10360 0.10414

12

6-OCsHs S-NH&H5

2.0915

2 3 4

13 14

3.0969 3.7212 5.2757

15

5-OCsHs 5-CH&Hs

16 17

5-SCsHs

5.2839

5-NHCH&H,

18 19

5-CH2C6Hs, 5-CH$sH,,

6-CF3 I-CHs

3.5686 3.1804

20 21 22 23

5-CH&H,, 6-CH$sH, 6-CHzC6Hs,

3-CH30

6-CH&sH,,

3-N02,

24 25 26 27 28 29 30 31 32

6-NI-QHs, 6-NHCsH3,

5-Br 5-Br

0.023959 0.020086 0.089636 0.012329 0.002942 0.063442 0.021290 0.000880

0.10115

0.014983 0.023524

0.10101 0.10170

0.005683 0.000846

0.10161 0.09712

0.000779 0.260946

0.10327 0.10172

0.074915 0.001422

0.10249

5.2076

0.10361

0.047730 0.006214

5.7212 2.3565 3.4814 3.8239

0.10011 0.10205

0.207383 0.005421

0.10302 0.10285 0.10175

0.000153 0.000549 0.028614 0.021034

2,3-Q 2,3-(Cf.&),

3.3979 3.2218

6-NHCsH3; 2,6-(CH3)r 6-NHCH2CsHs

2.4559

0.10181 0.06020 0.10196 0.10274

6-NHCH&sHs, 6-NHCH2C6H4,

5-Br 2-Cl

3.3979 3.2596 3.6197

6-NHCH&H‘,, 6-NHCH2CsH4,

3-Cl 4-Cl

3.4814 3.3016

0.10194 0.10193 0.10193

0.020466 0.021643 0.001164 0.022792 0.023714 0.023678 0.069882

33

I-CH&Hs l-CH2C6H4,

3-OH

3.8239 3.7447

0.10351 0.10347

34 35 36

I-CH2CsH4, l-CH2CsH4, I-CH2CsH4,

3-NO2 3-Cl 2-CH3

3.1487 4.1366 4.7447

0.10330 0.10340 0.10353

0.051731 0.081398 0.06269 1 0.064113

37 38

l-CH2C6H4, l-CH2C6H4, l-CH2C6H4,

3-CHs 4-CH3 3-CHsO

4.5850 3.4317

0.10352 0.10357

0.060021 0.053877

1-CHzCsHs,

3,5(CH3)2

4.2218 4.7958

0.10348 0.10363

0.047971 0.061788

39 40

either another molecule or by a surface. Molecular probes can be moved around a chemical to map out its surface. Fourteen points in the molecular environment relevant to the possible receptor binding site are chosen. Five points are displaced by 1 k~ from each of 0, and 0s in the different

directions perpendicular to the plane of uracil. Two points are displaced by 1 A from each of C2 and N3 in the direction perpendicular to the plane of uracil. These points are thought to represent a receptor group in the vicinity of C2 and N3 because of the sensitivity of the drug

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407)

Q0-b)

EN-I-b)

A

W

(e)

WI

@W

0.07023 0.07003 0.06992

0.14420

-0.74790 -0.74810

14.595

0.07073

0.15019

-0.74780 -0.74800

0.07057 0.07072 0.07172

0.15222 0.14971

-0.74830 -0.74770

0.16006 0.14152

-0.74840 -0.74770

0.06983

0.14127 0.14354

14.478 14.275 13.972 14.050 13.215 12.790 14.249

0.13663

-0.74762

11.228

0.06987

0.14194 0.13916

-0.74765 -0.74793

14.202 14.575

0.07026 0.06993

0.14430 0.14357

-0.74827 -0.74779

13.787 12.886

0.14722

-0.74795

12.904

0.07734 0.14757 0.14364

-0.74338

7.4154 11.999 12.898

0.06930 0.06970

0.07014 0.06720 0.07060 0.06991 0.07066 0.06984 0.06851 0.06997 0.07040 0.07057 0.07011 0.06996

0.14856 0.14269 0.11718 0.14127 0.14538 0.14838 0.14269 0.14059

0.06996 0.06998 0.07046 0.07002

0.14063 0.14086 0.14661 0.14133

0.07004 0.07004

0.14176 0.14184 0.14436

0.07137 0.07150 0.07119 0.07131 0.07135 0.07142 0.07133 0.07150 0.07142

0.14473 0.14751 0.14590 0.14422 0.14416 0.14393 0.14468 0.14394

activity to the substituent in the para position. The precise positions and the coordinate system are indicated in Fig. 1. The H axis is out of the plane. The electrostatic potential contour and solid maps are plotted for uracil using method III of Ref. 11, with CND0/2 data. These maps are shown in Figs. 2 and 3 respectively.

-0.74700 -0.74783 -0.74772 -0.74750 -0.74358 -0.74795 -0.74770 -0.74782 -0.74810 -0.74807 -0.74807 -0.74806 -0.74788 -0.74808 -0.74809 -0.74809 -0.74749 -0.74749 -0.74742 -0.74746 -0.74750 -0.74749

13.278 13.663 12.840 13.592 12.399 11.932 13.475 14.339 14.33 1 14.277 12.940 13.938 14.094 13.882 13.475 13.275 13.158 13.525 13.350

-0.74748

13.353 13.158

-0.74750 -0.74749

13.220 13.392

Results and discussion Semiempirical calculations were used to study the structure-activity relationships of a series of 40 uracils substituted in positions 1, 5 and 6. These are potent inhibitors of FUDR phosphorylase from Walker 256 rat tumour. The

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experimental data are obtained from Refs. 3 and 4. The biological activities are expressed as p(C,,), the negative logarithm of the molar concentration required to achieve 50% inhibition of enzyme activity and are given in Table 1. The calculated chemical indices are also given in Table 1. Statistically significant regressions are obtained with p(C,,) as inhibitory potency for a series of substituted uracils. Simple and two-variable regression analyses are used to provide an empirical link between the observed p(C,,,) values and the indices presently calculated. The most significant (best) relationships are obtained by searching the regression of inhibitive potency on each single and all pair contributions. The likelihood of random correlation of regressions increases with the number searched, therefore extreme caution was exercised in the interpretation of the statistical parameters

1171. The three best simple regressions with p(C,,) as the dependent variable, having significance level above 99.9%. were: p(C,,) = 547(f116.2)E(N3-Hs)

+ 413.0(f86.8) (1)

for which r = 0.607; s = 0.72; F(1,38) = 22.2; p(C,,) = 9.403(f2.3)FE(N3)

+ 3.198(fO.l)

(2)

for which r = 0.545; s = 0.76; F( 1,38) = 16.07; p(C,,) = -0.385(fO.l)A

+ 8.718(f1.3)

(3)

for which r = 0.512; s = 0.78; F(1,38) = 14.07. The three best two-variable regression equations are obtained with p(Cs,) as the dependent variable. All regressions are significant at the 99.9% level. p(C,,) = 292.6(f82.7)a(N1) + 746.0(&l 16.2)E(N2-Hg) + 531.3(+83.1)

(4)

for which

r =

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0.726; s = 0.63; F(2,37) = 20.7;

p(C,,) = 497.5(f142.5)a(07) + 788.9(&123.3)E(N3-Hs) + 558(f86.9) for which

r =

(5)

0.724; s = 0.63; F(2,37) = 20.48,

p(C,,) = 50.53(*16.3)Q(Hs) + 1063.0(&197.3)E(N,-Hs) + 791 .O(f145.5)

(6)

for which r = 0.705; s = 0.65; F(2,37) = 18.36; where r is the correlation coefficient, s is the standard error of estimate and F is the variance ratio. The bond energy E(Ns-Hs) which appears with positive coefficient in regressions (l), (4) and (5) is very interesting. Any reduction of stability of this bond is likely to be brought about by withdrawal of electrons from this region. This may make it more reactive in certain cases by providing an electron-positive region for a coulombic interaction with the enzyme. It might also increase the susceptibility of the bond to nucleophilic attack. The appearance of frontier electron density in regression (2) indicates that N3 may adopt a donor role. The appearance of Q(Hs) in regression (6) is readily explained in terms of electrostatic interaction. The appearance of A with a negative coefficient in regression (3), indicates that the greater the ease of electron excitation, the higher the drug activity. The electrostatic potential contour and solid maps, plotted for uracil, are given in Figs. 2 and 3 respectively. The maps indicate that electrophilic attack might occur readily at O7 as well as 0s. The main features revealed by these maps are a region extending around the relatively electropositive portions, and the negative potential attractive to cations near the two oxygen atoms. The potential well in the vicinity of the oxygens is shown in Fig. 3. This well is resolved into two minima, of depth -100 and -120 kcalmol-‘. Contours are placed at intervals of 25 kcalmol-’ , though confined to

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the electropositive region. Figure 2 shows these minima in three dimensions as looked at from the x-y plane. The viewing direction is 15” downward from the horizontal. Two hypothetical point charges Pt, and Pt, out of 14 in the plane of atoms O&-N3 and out of the one plane of atoms C2-N3-C4, of bond length 1 w and bond angles 90” and 109” respectively, have a significant correlation coefficient of 0.704. These regressions were obtained between drug activity, p(C,s), with the calculated Vt, V, and the three components of the electrical field. The significance levels are 99.9%. These points are relevant to possible binding sites. References W. Shive and C.G. Skinner, in H. Hochste and D. Quastel (Eds.), Metabolic Inhibitors, Vols. 1 and 2, Academic Press, New York, 1963. B.R. Baker, J. Med. Chem., 10 (1956) 297. M. Zimmerman, Biochem. Res. Commun., 16 (1964) 600.

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