Indian Journal of Biochemistry & Biophysics Vol. 37, August 2000, pp. 268-272

Molecular structure-activity relationship study of some non-steroidal anti­inflammatory agents using electrostatic potential mapping

C Gopi Mohan· and PC Mishrat

t Department o f Physics, Banaras Hindu Uni versity, Varanas i 22 1 005 , India

Received 4 October 1999; revised /3 March 2000

A series of 6, 11 -dihydro-11-oxod ibenz[b,e]oxepin-2-acetic acids (DOAA) which are known to be ant i-infl ammatory agents were studied. The geometri es of some of the molecules obtained from X-ray crystall ography were used in the calcul ations as such while the geometries of their deri vati ves were obtained by local, part ial geometry opti mization arou nd the sites of substitution employing the AM I method, keeping the remaining parts o f the geometries the same as those in the parent molecules. Molecular electrostatic potential (MEP) mapping was performed for the molecul es using optimi zed hybridi zati on d ispl acement charges (HOC) co mbined wi th Lowdin charges, as thi s charge distri bution has been shown earlier to yield near ab initio quali ty resul ts. A good correlation has been found between the MEP values near the oxygen atoms of the hydroxyl groups of the carboxy groups of the molecules and their anti-in fla mmatory activities. The result is broadly in agreement with the model proposed earlier by other authors regarding the structure-acti vi ty relationshi p fo r other simil ar molecules.

Non-stero idal anti -inflammatory drugs are commonl y used in the treatment of infl ammation, pain and fever'. These drugs ac t by preventing the producti on of prostaglandins2

. Prostaglandins are produced from fatty ac ids by cyclooxygenase pathway and work like hormones and control a number of processes involving the nervous system, blood pressure etc . Therefore, inhibition of the cyclooxygenase enzyme acti vity can be a major step towards bl ocking the production of prostaglandinsH. Usage of anti­inflammatory drugs such as pyrazolones, fenamates and salicylates fo r long periods of time causes side effects such as gas tro intestinal irritation, bone marrow suppress ion and also effects the centra l nervous system. Inhibiti on of the fatty ac id cycloxygenase enzyme by aspirin and indomethacin acting as anti ­inflammatory d rugs has been known5

. Many other non-stero idal anti -inflammatory drugs which do not produce the above me ntioned effects have been developed, e .g. ibuprofen, naproxen, ketoprofen, tolmetin , phenylbutazone etc. A series of other non­stero idal anti- inflammatory drugs belong ing to the class of 6, 11-dihydro-ll-oxodibenz[b,e]oxepin-2-acetic ac ids (DOAA) have a lso been deve loped3.6. One of the molecules of thi s class, namely 6, 11-d ihydro- 11-oxodibenz[b,e]oxepin-2-acet ic ac id

* Present address of the author fo r correspondence: Molecular Biophys ics Uni t, Indi an Institute of Science, Bangalore 560 0 12, India

(oxepac) (molecule 1) is about e leven times more active than indomethac in3

·6

-8

. Further, thi s molecule (1) is structurall y similar to indomethacin and has been in use as an effective anti-inf lammatory drug3

·6

.

M olecular e lectrostatic potential (MEP) mapping is particularl y suitable where e lectrostatic interactions play a dominant ro le9

-20. T he present work deals with

a series of DOAA molecules3 with the ai m to inves tigate the re lationship of their an ti- inflammatory activ ities with their MEP features.

Method of calculations The MEP values near di ffe rent electrophilic sites of

the molecules studied were obtained usi ng the optimi zed hybridi zation di splacement charges (HDC) combined Lowdin charges. We have shown that the combination of HDC and Lowd in c harges d istributed continuously in three di mensi on spherica lly symmetrically reproduces near ab initio qual ity MEP maps '4-2o

T he MEP V(r ) at a po int r due to the nuc le i located at Ra and a continuous electronic c harge di stribut ion p(r ') is given by

V(r ) = NL:, ZaliRa- rl- p(r )dr ' / lr ' -rl ... ( I)

where N is the total number of nuclei and Za is the charge at nucleus a. We may represent the fi rst and second terms on the right hand s ide of equation (I) by V"(r ) and y et (r ) which would stand fo r nuclear and e lectronic contributions to V(r ) respectively.

MOHAN & MISHRA: MOLECULAR STUDY OF NON-STEROIDAL ANTI-INFLAMMATORY AGENTS 269

It is well known that MEP maps computed using point charges do not reproduce most of the topological features correctly 10

'13 .This shortcoming of

MEP maps can be overcome by considering continuously distributed electronic charges. Accordingly, the total electronic charges located at different points in molecules can be distributed continuously in three dimension in the forms given by squares of the corresponding Slater ns valence atomic orbitals where the value of n (principal quantum number 1, 2, 3 etc.) corresponds to the valence shell of the atom under consideration. The expression for y et (r) for n= I, 2 and 3 and the definition of HOC are available in the literature 14

•17

• A parametrically optimized approach for computing HOC has been developed to get near ab initio quality MEP maps of molecules . There are two parameters K and <; involved in this procedure for each atom as explained in the literature 14

-17 and its optimized values are

presented in Table I. Geometries of some of the anti-inflammatory drugs

such as 6, 11-dihydro-11-oxodibenz[b,e ]-oxepin-2-acetic acid (molecule 1) its ethyl derivative (molecule 2) and methyl derivative (molecule 4) have been determined by X-ray crystallograph/ and these geometries were used in the calculations as such. The geometries of the other molecules which are derived by substitutions in the molecules for which X-ray crystallographic geometries are available, were

Table !--Optimized values of the parameters K and c; for HDC of different non-hydrogenic atoms

Atom K (a.u)

c Na

Nh

N'

0.30

0.45

0.85

0.10

MNDO AMI

1.625 1.625( 1.625)

1.600 1.600(1.950)

1.350 1.480( 1.950)

2.220 2.360(1.950)

od o.8o 1.100 I.I00(2.275l

o c 0.80 1.400 1.580(2.275)

o r 0.25 1.900 2.470(2.275)

p 0.70 0.930 1.550(2.600)

s11 1.21 1.300 1.250( 1.817)

si 1.2 1 I.o5o 1.180(1.817)

si o.86 1.000 1.280( 1.817)

Clk 0.83 0.930 1.750(2.033) aNitrogen of N02 group, NO group or N2 molecule; hN itrogen of pyridine; ' Nitrogen of NH2 (or NH) group ; dOxygen of N02 (or NO) group; 00xygen of carbonyl group; roxygen of hydroxyl group; gFluorine atom; 11Sulfur of SH group; ;Sulfur atom in ring; iSulfur of C=S group; kChlorine atom. The values given in parentheses. obtained from Slater's rules, were used fo r the charges located on the atomic sites22

__ _

obtained by local geometry optimization around the site of substitution, including the substituents, employing the AMI method21

. This approach appears to be better than that in which one can fully optimize the geometries theoretically, since it would minimise the possibility of geometry related questions regarding reliability of the results.

Results and Discussion

The molecular structures of the molecules studied are shown in Fig. I . The net electronic charges in the unit of magnitude of electronic charge lei at the non­hydrogenic atomic and HOC sites for one of the molecules i.e. molecule 1, taken as a representative case, are presented in Table 2. The substitutions R and X5 in Fig. I are shown in Table 3. The distance of HOC from the corresponding atom are as follows: 0.23A for carbon atoms, O.llA for the oxygen atom of a hydroxyl group and 0 .37 A for the oxygen atom of a carbonyl group. We find that HOC are all negative while the charges at the carbon and the oxygen atomic si tes are usually positive or small negative. The atomic sites are usually positively charged as a significant amount of negative charge is shifted from each of them as HOC. The total net Lowdin charges for any atom would be obtained by adding the corresponding charges given in the two columns in Table 2. The non-hydrogen atoms which . are associated with a HOC each, for example for molecule 1 (Table 2), may be put in three different categories; (i) Oxygen atoms of hydroxyl groups i.e. 0 5 and 0 20 for which the magnitudes of HOC are 1.30 and 1.34 lei respectively, (ii) Oxygen atoms of carbonyl groups i.e. 01 8 and 0, 9 for which the magnitude of HOC are 0.40 and 0.39 lei respectively, and (iii) Carbon atoms in approximately sp2 or sp3

Fig. !-Molecular skeleton of the parent molecule 6, 11-dihydro-11 -oxodibenz[b,e] oxepin-2-acetic acid . [The substi tution site X5

and the 0 2u-R group are shown in boxes. The substituents are given in Table 3].

270 INDIAN J. BIOCHEM. BIOPHYS., VOL. 37, AUGUST 2000

valence states, the magnitude of HOC associated with them varying widely between about -0.05 and -0.40 le i (Table 2). These resul ts suggest that the lone pair charges on the oxygen atoms are usually much less than the conventional value of 2 and that carbon atoms may also be associated with significant

Table 2-Net charges at different non-hydrogenic atoms of molecule 1 in unit of the magnitude of electronic charge

Atomic/HOC Charge at Charge at site" atomic site HDC site

cl 0.287 -0.320

c 2 -0.053 -0.049

c3 0.208 -0.273

c4 0.136 -0.307

Os 1.082 -1 .299

Cr, 0.415 -0.399

C1 0.151 -0.274

CR 0.161 -0.267

c~ 0.137 -0.271

clll 0.226 -0.306

C11 -0.003 -0. 120

C12 0.057 -0.136

Cu 0.486 -0.168

cl4 -0.049 -0.152

C1s 0.250 -0. 124

elf> 0.173 -0.274

cl7 0.584 -0. 284

01 x 0.107 -0.400

019 0.001 -0.391

0 20 1.050 -1.343

"Atomic numbering scheme is shown in Fig. I

amounts of HOC, depending on the local electronic environments.

The deepest MEP minima were found to lie near the X5 site in all the cases except in molecule 5 where a sulfur atom is substituted in place of an oxygen atom (Table 3). In this case (molecule 5), the minimum MEP value near X5 (i.e. S5) has a much smaller magnitude than those in other cases while the molecule has a fairly high anti-inflammatory activity (Table 3). Thus the MEP minimum near this site (X5)

does not seem to be related to activities of the molecules. An examination of the MEP values near X5 (i.e. 0 5) in the other mol ecules vis-a-vis their potencies and absence of any sati sfac tory correlation between the two sets of quantities also supports this conclusion. The minimum MEP values near the 01 8

and 0 19 atoms also do not corre late sati sfactorily with the anti-inflammatory activities of the molecules (Table 3). Therefore, the 0 18 and 0 19 s ites also do not appear to be involved electrostatically e.g. through hydrogen bonding, in the action of the molecules (Table 3).

The computed MEP maps of four molecules of the class i.e. molecules 1, 3, 7 and 8 in the respective 0 20C 17C 16 planes are presented in Fig. 2. These maps show a MEP minimum located near the corresponding 0 20 atom each along with the MEP minima near the other electrophilic sites. A significant decrease in the anti-inflammatory activity is found in going from molecule 1 to molecule 10 (Table 3)1 and there is a simi lar qualitative variation in the magnitudes of the minimum MEP values near 0 20 . Gund and Shen have suggested the presence of a carboxy group which does

Table 3-Structures, anti-intl ammatory act ivities of the 6, 11-dihydro- 11-oxodibenz[b ,e]oxepin 2-acetic acids and minimum electrostatic potential values near electronegative sites

Substitutions at" -log.,EDso MEP near the site (kJ/mol)

C1, Cx, Xs 0 2u-R Xs OIK 0 19 0 20

c9 and clll

H 0 OH 5.20 -264.89 -223.59 -246.87 -253.93

H 0 OCH2CH3 5. 16 -261.96 -221.54 -248.71 -250.80

8-CI 0 OH 4.58 -255.44 -214.56 -243. 19 -247.87

H 0 OCH, 4.5 1 -257.99 -211.13 -229.56 -247.75

H s OH 4.36 -10.45 -236.59 -257.86 -250.13

7-CI 0 OH >4.35 -258.32 -212.89 -246.45 -248.88

9-CI 0 OH <4.30 -256.02 -213.22 -242.89 -245.49

10-CH3 0 OH <4.30 -267.35 -2 14.52 -248.50 -242.44

8-0CH3 0 OH 4.23 -268.98 -232.20 -253 .89 -240.35

9-F 0 OH 3.44 -255.69 -2 11.80 -242.40 -232.26

"See Fig. I , for ato mic numbering scheme.

MOHAN & MISHRA: MOLECULAR STUDY OF NON-STEROIDAL ANTI-INFLAMMATORY AGENTS 271

(a l

• -253 ·4 ... -212.5

X -247·9 .. -205 ·5

" -245·5 A -205 ·4 • -146 .5

--122· 7 --- -61.4

" -242·4 A -200·5 • -140· 5

--121 . 2 --- -so.s

Fig. 2-MEP maps (kJ/mol) of mol ecules: (a) 6, 11 -dihydro-11-oxodibenz[b ,e] oxepin-2-acetic acid (molecule 1); (b) 6,11-dihydro-11-oxo-8-chloro-dibenz[b,e] oxepin-2-acetic acid (molecule 3), (c) 6, 11 -dihydro- ll-oxo-9-choloro-dibenz[b,e] oxepin-2-acetic acid (molecule 7), and (d) 6, 11-dihydro-11-oxo-1 0-methyl-dibenz[b,e] oxepin-2-acetic acid (molecu le 8) , obtained using HOC-corrected Lowdin (AM I) charges.

not lie in the ring plane, as an important structural requirement for high anti-inflammatory activity of the arylacetic acid molecules e .g. indomethacin, pirprofen and 6-chloro-5-cyclo-hexylindan-1-(S)-carboxylic acid etc.4 and the same may be applicable in the present case also. The series of molecules studied here also have a non planar carboxy group each . The dihedral angles between the C1CzC3 and C1 6C170zo planes (Fig. 1) are found to vary between 53 and I 17 deg. for the different active molecules (Table 3) . For the most active molecule i.e. molecule 1, this dihedral angle has a value close to 53 deg. Thus the present MEP results suggest that one of the structural requirements included in the Gund and Shen model4

would also be applicable to the present class of molecules, namely a non-planar carboxy group each .

255·00 .---------------,

0 E

....... .., ~

Q. w ::>!:

250.25

0

4.05

0 0

0

4.55

-loge Ed 50

0

0

5.05

Fig. 3--Yariation of the magnitudes of minimum MEP values (kJ/mol) with the anti-infl ammatory activi ties (logcED50) of the molecules along with the least squares fitted straight line.

Thus the oxygen atom 0 20 of the hydroxyl group is likely to be electrostatically involved e.g. through hydrogen bonding in the action of drugs. The interaction relating to the inhibition of the cyclooxygenase enzyme by the DOAA molecules has been proposed earlier to involve hydrogen bonding3

.4 .

A least squares fitting between the -logeEdso (potency) and IMEPI (kJ/mol) values near the Ozo site (Table 3) gave the equation

MEP= a( -logeEdso) + b

where a=8.43 and b=210.9 (kJ/mol). The variation of MEP with -logeEd50 is presented in Fig. 3 along with the least squares fitted straight line. The correlation coefficient in this case was found to be 0.85.

Molecules 3, 6 and 7 are derived from molecule 1 by substitution of a chlorine atom at the 8, 7 or 9 position respectively. These molecules are less active than 1, their activities being in the order 3>6>7. When a fluorine atom is substituted at position 9 in place of chlorine, the molecular activity drops down further drastically (Table 3). Substitution of a methoxy group at position 8 or that of a methyl group at position I 0 also decreases molecular activity appreciably. Thus none of the different substitutions made in the benzene moiety in molecule 1 enhances molecular activity. It appears that this benzene moiety as such has the optimum shape, size and electronic property for interaction with the receptor cyclooxygenase.

Conclusion There is a good correlation between the

experimentally observed anti-inflammatory activities and the computed MEP values near the oxygen atoms

272 INDIAN J. BIOCHEM. BIOPHYS., VOL. 37, AUGUST 2000

of the hydroxyl groups of the carboxy groups of the DOAA molecules studied here. Thus the hydroxyl group oxygen atom which lies appreciably away from the ring plane appears to be involved in interaction with the enzyme cycloxygenase through hydrogen bonding. These results are broadly in agreement with the model proposed by other authors earlier for the arylacetic acid series of anti-inflammatory drug molecules.

Acknowledgement One of the author (PCM) is thankful to CSIR (New

Delhi) and UGC (New Delhi) for financial support. CGM thanks Department of Biotechnology (DBT­India) for financial assistance in the form of a Research Associateship.

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