Indian Journal of Chemistry Vol. 43A, October 2004, pp. 2043-2048

Ab initio calculation on the effect of substituents in the alkylation mechanism by nitrogen mustards at N -7 of guanine

P K Bhattacharyya & C Medhi *

Chemistry Department, Gauhati University, P.O. Gauhati University, Guwahati 781 014, Assam, India Email: chitrani@satyam.net.in

Received 1 October; revised 19 July 2004

Ab initio calculations of some DNA binding drugs, nitrogen mustards, have been performed for studying the effect of substituents in the alkylation reaction . The aziridinium ion of different drugs having different substituents has been studied. The substituents do not affect the formation of aziridinium ion of most drugs but for bendamustine and some model drugs of nitrogen mustards with subst ituents, ethyl and ethoxy groups, the energies of forming the aziridinium ion are high. The geometrical parameters of the aziridinium ions of drugs remain almost the same. However in bendamustine and some other derivatives of nitrogen mustards, distortion of aziridinium rings are observed. The results do not basically encourage the modification drugs with various substituents in the process of designing new drugs of enhanced alkylating ability. Unlike other drugs, bendamustine and some derivatives may generate carbonium ion in the intermediate state of alkylation .

DNA is an important target of most alkylating age­nts1 ·5. It has been known that the nitrogen mustards bind at N-7 of guanine, while the binding affinities are somewhat related to drug activity s-s. On this basis, it is expected that the anticancer properties should relate to the N-7 affinities of nitrogen mustards. Thus, in designing new nitrogen mustard, the drugs that possess strong cross-linking ability in the GC rich region of DNA are importane 4

·8-

11 . The selectivity for guanine rich region by nitrogen mustards is understood indirectly as the requirement for potent drugs that draw attention in drug discovery.

In the rational drug design, another useful approach for analyzing the drug action is focused on the reactivity of aziridinium ion9

. Thus, one can look into the mechanisms that could give important criteria of investigating the observed differences in the biological properties of nitrogen mustards. The reactivity of aziridinium ions may differ with substitutions at N-4 position, in the sense that the aromatic substituent as in bendamustine may not produce similar aziridinium ion as that of aliphatic substituent8

d. At best, such changes in the substituents could affect the alkylation reaction of nitrogen mustards. From the experimental point of view, both the aliphatic and aromatic substituents have little difference in the selectivity for guanine N-7 but acquire variation of biological propcrties8

.11 . Also, the

variation of electronic effect from an aliphatic and an

aromatic substituent in the nitrogen mustards cannot be ignored and subsequently are expected to affect the formation of aziridinium ion in the intermediate step of alkylation8

c.

Alternatively, the alkylation reaction can be understood from the ability to form aziridinium ion or from the activity of aziridinium ion to inhibit the N-7 of guanine of DNA. Suitable models of drugs can be chosen for studying the general influence of substituents on aziridinium ring before analyzing the interaction with N-7 of guanine. Although the formation of aziridinium ring is favorable in nitrogen mustards, it is not exactly known whether the alkylation occurs through direct attack of aziridinium ring at N-7 of guanine or af~er ring opening8

. Hence, the theoretical study on the effect of substituents during aziridinium ring formation of various nitrogen mustards is important.

Methodology In this study we have carried out calculations using

Hartree Fock /6-31 G* basis set to determine the stable aziridinium ions (intermediates) of different drugs 17

.

The optimum geometry of the intermediate is determined by optmuzmg all the geometrical parameters of aziridinium ring. To do that initially the geometries of the starting tricyclic ring were arbitrarily chosen without considering the relaxation in the ground state structure of other geometrical parameters of drug

2044 INDIAN J CHEM, SEC A, OCTOBER 2004

and subsequently optimized to get new structure of least energy only. After the most stable geometry of aziridinium ring was obtained, the differences between the energies of ground state nitrogen mustard and aziridinium ion was evaluated( £1£).

L1E= Eaz - Eg , where Eaz and Eg are the energies of aziridinium ion and drug in the ground state. Employing the same procedure the calculations were carried out for all the drugs. The polarization due to solvent effect on aziridinium ring was studied using SCRF-dipole(c~78 for water) adopted in Gaussian programme code. Accordingly, the molecular volumes of all drugs were first estimated before computing the solvent induced charge distribution in aziridinium ion.

Result and Discussion The aziridinium ions of fifteen substituted nitrogen

mustards are studied to analyze the reactivity of these intermediates during alkylation reaction (Fig.l). We have computed the stabilities of aziridinium rings of these drugs (Fig. 2, Table 1). There are noticeable variations in the stabilities of aziridinium ions. The reason for analyzing such stability is to evaluate the energetic of alkylation at N-7 of guanine. Similarly the stabilities of aziridinium ions of some clinically used nitrogen mustards of different anticancer properties are also studied. In fact, among the known drugs, mustine to tryptophane (Fig. 2), the energies of forming aziridinium ions are almost the same except in some drugs like bendamustine and model 4 in which the energies are slightly large (Table 1).

Based on these observations, it is obvious that the energy barrier of forming aziridinium ring may be related to its ability for direct attack at N-7 of guanine(Fig. 3). The narrow changes in the stabilities of aziridinium rings of these drugs indicate that substituents usually do not have much influence in the intermediate reaction step, in particular, the aziridinium ring. In the case of bendamustine, the energy of forming this intermediate is high (Table 1). It indicates less ability of generating aziridinium ion and subsequently the chances of direct alkylation by this intermediate are unlikely. Thus the effect of substituents in the alkylation mechanism through aziridinium ring is less for this drug.

Besides studying the comparative stabilities, we further analyzed the geometrical parameters of aziridinium ions(Table 2). In spite of the electronk effect from small to medium sized substituents, the geometries of the aziridinium ions of these drugs remain the same. Though the geometrical parameters

R = -CH(CH:JCH2CH3

R=- C(C~)3

R=-OCH2C~

R=.-0

R=-Q-cH3

R=-o-OCH:J R=-o-NH2 R = -oCH,CQOH

NitrO!Jen Mustard

Substituent1

Substituent2

Substituent3

Substituent4

Substituent5

Substituent6

Substituent?

Substituent8

Substituent9

R • -Q-CH,COOH Subslil\Jenl10

NH2

R • -Q-CH,COOH Substituent11

CH3

R,. -o-NHCOCH3 Substituent12

R =. -o-CH(OH)COOH Substituent12

R "' -o-CH(OC~)COOH Substituent14

Ra -o-NHC~ Substituent15

Fig. !-Nitrogen mustards with different substituents

of the aziridinium 1;ngs corresponding to model nitrogen mustards do not show great influence due to the substituents, remarkable changes in the orientation of substituent with respect to the rings are observed (Fig.4a and 4b ). The orientation of aromatic substituent with respect to aziridinium ring is different from that of aliphatic substituent. So, it is possible for

BHATT ACHARYYA & MEDHI: AB INITIO CALCULATION ON N1TROGEN MUSTARDS 2045

R~-Q)

--0--.<CH(NH~COOH

R~ ~.} N

Nitrogen Mustards

Mustine

Melphlan

Chlorambucil

Bendamustine

Chloranphazine

T richloromethi ne

Tryptophane

Fig. 2---Ciinically used nitrogen mustards

Table !-The computed stabilities of aziridinium ions for different drugs

Drug t!E (kcalmor 1)

Substituent I 179.93777 Substituent 2 176.53052 Substituent 3 180.48996 Substituent 4 214.40555 Substituent 5 180.20131 Substituent 6 178.73299 Substituent 7 178.42553 Substituent 8 177.32116 Substituent 9 179.57383 Substituent 10 179.19106 Substituent 11 179.07812 Substituent 12 176.55562 Substituent 13 179.49225 Substituent 14 181.65080 Substituent 15 176.28580 Mus tine 183.60229 Melphlan 177.89217 Chlorambucil 175.94696 Benda mustine 235.87184 Chloranphazine 177.06388 Trichloromethine 193.65461 Tryptophane 177.11470

step I

c r1 ~'3

I 4 { y· R_ ~·(.ic,

Cti,CH,a 3 2 1

Drug In ground state. Azlrldlnlum ton Intermediate

l step II

CH,CH,O R-N< CH,CH, 0

"'-.N____)l__ (<•>)l, NH

N NA.NH2

I DNA Chain

Drug-11uantne adduct

Fig. 3--Aikylation mechanism of nitrogen mustards mustards at N-7 of guanine

the nitrogen mustards to form a saturated aziridinium ring, which cannot be induced further by substituents. Therefore, the insignificant relaxation of geometrical parameters of aziridinium ions due to electronic effect of different substituents may be an indication of forrning a saturated ring structure. On the other hand, the electronic effect of the substituent may induce the aziridinium ring to different orientations.

In order to rationalize the mechanism of direct attack at N-7 of guanine by aziridinium ring we have computed the Mulliken net charges on the atomic centers of aziridinium ions of these drugs. We envisaged little unsymmetrical distribution of charge densities on C6 and C7(ring carbons)(Table 3). However, the small variation of net charges on C6 and C7 may lead to different mechanisms. The reaction was analyzed to confirm the predominance of aziridinium ion intermediate rather than generating the carbonium ion intermediate in the reaction pathway. We observed unequal net charges on C6 and C7 of aziridinium ion for bendamustine that might be useful for explaining the cationic mechanism involving carbonium ion in the intermediate state8

• Similarly, some model drugs(models 1 and 4 ) also possess unequal charge distribution on these carbon atoms as compared to those of other drugs. If we examine the instance of N-7 attack by aziridinium ion, direct interaction either with C7 or C6 of aziridinium ion should take place and further polarization on these atoms may form carbonium ion before interacting with N-7 of guanine. This cationic mechanism is expected in bendamustine, and models 1 and 4, since the aziridinium ions of these drugs acquire more unequal charge distribution than

2046 INDIAN J CHEM, SEC A, OCTOBER 2004

Table 2-The computed geometrical parameters of the aziridiniurn ions of different drugs

Drug riCA) r2 (A) r3(A) 8(467) <1>(5467) (deg) (de g)

Substituent 1 1.5105 1.4891 1.5101 60.52 !07.58 Substituent 2 1.5105 1.4872 1.5!09 60.53 !08.19 Substituent 3 1.5128 1.4873 1.5080 60.34 !06.43 Substituent 4 1.5118 1.4923 1.5153 60.58 102.16 Substituent 5 1.5095 1.4897 1.5076 60.35 !08.70 Substituent 6 1.5092 1.4897 1.5073 60.34 !08.33 Substituent 7 1.5095 1.4894 1.5075 60.35 !08.90 Substituent 8 1.5094 1.4894 1.5074 60.35 !08.85 Substituent 9 1.5092 1.4898 1.5072 60.34 109.94 Substituent !0 1.5092 1.4898 1.5073 60.34 !09.90 Subslituent II 1.5090 1.4898 1.5071 60.33 108.99 Substituent 12 1.5091 1.4895 1.5070 60.33 !08.93 Substituent 13 1.5094 1.4897 1.5074 60.34 108.97 Substituent 14 1.5096 1.4897 1.5076 60.35 108.96 Substituent 15 1.5091 1.4894 1.4873 60.34 108.43 Mustine 1.5070 1.4913 1.5085 60.41 !09.10 Melphlan 1.5144 1.4885 1.5085 60.30 108.90 Chlorambucil 1.5093 1.4897 1.5071 60.34 108.45 Bendamustine 1.5085 1.4896 1.5069 60.34 108.93 Chloranphazine 1.5090 1.4897 1.5070 60.33 108.96 Trichlorornethine 1.5082 1.4904 1.5090 60.42 109.56 Tryptophane 1.5086 1.4896 1.5066 60.33 108.95

(a) Cl' (b)

Cl' c---H

.:-

"H c--H

//f fl "H H' H' H c \ H\_/H H-C\ / /

,,, ·,,

;6:(~" Hh/N-\~"""" C\H H

H-C \ H-e~\ \ H \H H H

Fig. 4--Conformation of the intermediate of nitrogen mustard [(a)-Aromatic substituent; (b)Aliphatic substi tuent]

those of other drugs. In general, the alkylating ability by C6 or C7 of aziridinium rings cannot be differentiated due to the narrow differences in the Mulliken net charges on these carbon atoms. The net charges on these atoms in both aziridinium rings and ground state structures of nitrogen mustards are not drastically changed despite the strong electronic effect from substituents which might be due to the simultaneous withdrawal of electron densities by

chloride ions(Table 3). Hence, the approach of designing different model drugs of nitrogen mustard by using different substituents at N4 may not enhance alkylating abilities.

Again, another method for differentiating these drugs is the conformations of substituents with respect to aziridinium ring at the intermediate reaction path. In this case we have noted distinct changes in the conformations of the rings for different substituents;

BHATT ACHARYYA & MEDHJ: AB INITIO CALCULATION ON NITROGEN MUSTARDS 2047

the conformations of aromatic and aliphatic substituents vary significantly, some substituent might obstruct the approach of the aziridinium ring towards N-7 of guanine and in turn facilitate polarization of C6 and C7(Figs. 4a and 4b). The distortion of aziridinium rings is significant in drugs with aliphatic groups and the substituents lie in the orientation opposite to the aziridinium ring. In this case, direct interaction of C6 or C7 with N-7 is not hindered. These observations support the experimental results of the favored alkylation mechanism of nitrogen

mustard only through aziridinium ion9·1

1.16

• However, the aromatic substituents lie at the sterically hindered position in which aziridinium ring plane flip with the plane of aromatic substituent(Fig. 4a). In order to probe such changes in the conformations with respect to substituents, we have analyzed the net charges on the other 2-chloroethylene side chain (Fig. 3). Table 4 shows the variation of the Mulliken net charges on the atoms of another 2-chloroethylene side chain in the ground and the intermediate states. We noted significant variation of Mulliken net

Table 3--The computed Mulliken net charges on N4, C6 and C7 of drugs in the ground and intermediate states in absence of solvent field

Drug Ground state Intermediate state

N4 C6 C7 N4 C6 C7

Mustine -0.265 -0.013 -0.057 -0.184 -0.011 -0.008 Melphlan -0.275 -0.010 -0.056 -0.188 -0.008 -0.008 Chlorambucil -0.275 -0.010 -0.056 -0.188 -0.009 -0.012 Bendamustine -0.275 -0.010 -0.056 -0.187 -0.011 -0.012 Substituent I -0.281 -0.010 -0.057 -0.188 -0.134 -0.009 Substituent 2 -0.277 -0.013 -0.056 -0.190 -0.014 -0.016 Substituent 3 -0.285 -0.014 -0.054 -0.192 -0.016 -0.020 Substituent 4 -0.168 -0.023 -0.055 -0.102 -0.007 -0.014 Substituent 5 -0.277 -0.011 -0.053 -0.188 -0.009 -0.012 Substituent 6 -0.277 -0.011 -0.053 -0.188 -0.009 -0.012 Substituent 7 -0.277 -0.011 -0.053 -0.187 -0.010 -0.012 Substituent 8 -0.277 -0.011 -0.053 -0.187 -0.010 -0.012 Substituent 9 -0.277 -0.011 -0.053 -0.188 -0.009 -0.012 Substituent 10 -0.276 -0.011 -0.053 -0.188 -0.009 -0.012 Substituent II -0.277 -0.011 -0.053 -0.188 -0.009 -0.012 Substituent 12 -0.277 -0.011 -0.053 -0.187 -0.009 -0.013 Substituent 13 -0.277 -0.011 -0.053 -0.188 -0.009 -0.012 Substituent 14 -0.277 -0.011 -0.053 -0.188 -0.009 -0.012 Substituent 15 -0.277 -0.011 -0.053 -0.186 -0.010 -0.013

Table 4-The computed Mulliken net charge densities on the atoms of the other chloroethyl branch ( in free molecule and in the aziridinium ion)

Charges on the atoms in

Drug Ground state Intermediate state

Cl C2 C3 Cl C2 C3

Substituent I -0.189 -0.067 -0.012 -0.105 -0.072 +0.004 Substituent 2 -0.193 -0.055 -0.015 -0.108 -0.071 +0.000 Substituent 3 -0.196 -0.052 -0.018 -0.110 -0.071 -0.00 I Substituent 4 -0.186 -0.054 -0.019 -0.102 -0.072 +0.006 Substituent 5 -0.188 -0.055 -0.012 -0.108 -0.071 +0.008 Substituent 6 -0.189 -0.054 -0.012 -0.109 -0.070 +0.008 Substituent 7 -0.189 -0.054 -0.011 -0.110 -0.070 +0.008 Substituent 8 -0.190 -0.054 -0.012 -0.111 -0.070 +0.008 Substituent 9 -0.188 -0.055 -0.012 -0.109 -0.071 +0.008 Substituent 10 -0.188 -0.054 -0.012 -0.109 -0.070 +0.008 Substituent II -0.189 -0.055 -0.012 -0.110 -0.070 +0.008 Substituent 12 -0.190 -0.054 -0.012 -0.112 -0.070 +0.008 Substituent 13 -0.188 -0.055 -0.012 -0.109 -0.070 +0.008 Substituent 14 -0.187 -0.055 -0.012 -0.109 -0.071 +0.008 Substituent 15 -0.190 -0.054 -0.012 -0.112 -0.069 +0.008

2048 INDIAN J CHEM, SEC A, OCTOBER 2004

Table 5----Computed net charges on N4, C6 and C7 of drugs in the ground and in~ermediate (aziridinium ring) states in presence of solvent field

Drug In the ground state In the aziridinium ion

N4 C6 C7 N4 C6 C7

Substituent 1 -0.574 -0.189 -0.192 -0.579 -0.190 -0.190 Substituent 2 . -0.578 -0.195 -0.188 -0.580 -0.194 -0.189 Substituent 3 -0.593 -0.190 -0.193 -0.596 -0.188 -0.194 Substituent 4 -0.256 -0.177 -0.190 -0.262 -0.175 -0.197 Substituent 5 -0.628 -0.187 -0.192 -0.634 -0.186 -0.191 Substituent 6 -0.628 -0.187 -0.191 -0.634 -0.186 -0.191 Substituent 7 -0.628 -0.187 -0.192 -0.635 -0.186 -0.192 Substituent 8 -0.627 -0.187 -0.192 -0.631 -0.186 -0.191 Substituent 9 -0.629 -0.188 -0.192 -0.639 -0.185 -0.191 Substituent I 0 -0.632 -0.187 -0.193 -0.637 -0.187 -0.193 Substituent 11 -0.630 -0.187 -0.191 -0.638 -0.187 -0.191 Substituent 12 -0.627 -0. 187 -0.192 -0.634 -0.187 -0.191 Substituent 13 -0.630 -0.187 -0.191 -0.639 -0.187 -0.191 Substituent 14 -0.630 -0.187 -0.191 -0.640 -0.186 -0.191 Substituent 15 -0.626 -0. 187 -0.191 -0.631 -0.186 -0.191

charges on Cll, C2 and C3 in these two states and the results indicate that substitution at N4 induces the side chain without serious effect on the aziridinium ring, resulting in significant change in the orientation of substituents(Fig. 4a and 4b). The observation is likely to address another factor for the variation of alkylating abilities of various nitrogen mustards.

The solvent effect in the polarization of aziridinium ring may be considered as another specific context useful for better understanding of the formation of carbonium ion(Tables 4 and 5). As we can see that the charge distribution at C6 and C7 shows narrow differences in most of the drugs, but in some drugs, the net charges on these atoms a1e unequal . Thus, we have studied the solvent effect on aziridinium ion by using solvent SCRF method. The polarization on C6 and C7 due to solvent dielectric field are observed, which is perhaps a significant evidence for conforming the generation of carbonium ion(Table 5). There are some differences between the net charges on the C6, C7 and N4 of the ground and the intermediate states in presence of solvent field. Hence, not only the steric effect due to conformational disposition of substituent but also the solvent field may be taken as an important factor for understanding the mechanism of alkylation through either carbonium ion or aziridinium ring.

Acknowledgement CM acknowledges CSIR, New Delhi, for research

grant

References 1 Kohn K W, Hartley J A & Mattes W B, Nucleic Acid Res,

24( 1987) 1053 L

2 Chun E H L, Gonzales L, Lewis F S, Jones J & Rutman R J, Cancer Res,29( 1969) 1184.

3 Bernardi G, Olofssan B, Filipski J, Zerial M, Salinas J, Cuny G, Meunier-Rotival M & Rodier F, Science,228( 1985)953.

4 Mattes W B, Hartley J A & Kohn K W, Nucleic Acid Res, 14( 1986) 2971.

5 Engkvist 0, Astrand P & Karlstom G, Chem Rev, l00(2000) 4087.

6 Hartley J A, Forrow S M, Souhami R L, Biochemistry, 29 ( 1990)2985.

7 Kohn K W, in Molecular Aspect of Anticancer Dntg Action, edited by S J Neidle & M J Waring (Macmillan Press, London) 1980.

8 (a) Prakash A S, Denny W A, Gourdie T A, Yalu K K, Woodgate P D, Wakein L PG, Biochemistry, 29(1990)9799; (b) Millis K K, Colvin M E, Shulman-Roskes E M, Ludeman S M, Colvin 0 M & Gamcsik M P, J Med Chem, 38(1995) 2166; (c) Alcami M, Mo 0 & Yanez M, J Am Chem Soc, 115(1993)11074.

9 Hamza A, Broch H, Yasilescu D, J Biomol Stntct Dyn, 6(1996)915.

10 Broch H, Viani R, Vasilescu D, lnt J Quant Chem, 1(1991) 119. 11 Gibson N W, Hartley J A, Strong J M & Kohn K W, Cancer

Res,46( 1986)553. 12 Harshman K D & Dervan P V, Nucleic Acid Res,13(1985)4825. 13 Kohn K W, in Molecular Aspect of Anticancer Dntg Action,

edited by S J Neidle & M J Waring(Macmillan Press, London) 1980,pp. 133.

14 Zakrewska K & Pullman B, J Biomol Stmct Dyn,3(1985)437. 15 Broggini M, Ponti M, Ottolenghi S, D' Incalci M, Mongelli N &

Mantovari R, Nucleic Acid Res, 17(1989) 1051. 16 Hovinen J, Pettersson-Femholm T, Lahti M & Vilpo J, Chem

Res Toxicol,11(1998),1377. 17 Frisch M J, Trucks G W, Head-Gordon M, Gill P M V,

Wong M W, Foresman J B, Johnson B G, Schlegel H B, Robb M A, Replogle E S, Gomperts R, Andres J L, Raghavachari K, Brinkley J S, Gonzalez C, Martin R L, Fox D L, Defrees, D J, Baker J, Stewart J P, Poplc J A, GAUSSIAN 94 (Gaussian Inc, Pittsburgh. PA).