Conformational comparative analysis of 2′,3′-dideoxythymidine analogues by molecular mechanics calculations (chem-x ) and by semiempirical methods (AM1)

Journal of

MOLECULAR STRUCTURE

ELSEVIER

Journal of Molecular Structure 350 (1995) 147-160

Conformational comparative analysis of 2',31-dideoxythymidine analogues by molecular mechanics calculations (CHEM-X)and by semiempirical methods (AM 1) D. Galisteo*, J.A. L6pez Sastre, H. Martinez Garcia, R. Nufiez Miguel Departamento de Quimica Organica, Universidad de Valladolid, 47011 Valladolid, Spain

Received20 September 1994

Abstract

The conformations of 2t,3Ldideoxynucleoside analogues with known antiviral activity have been studied by molecular mechanics (MM) and molecular orbital (MO) calculations. In accordance with results already reported, the parameters P and 0m were found by the AM 1 method, which differed from the experimental results. By MM calculations, however, the results obtained corresponded with those expected. Hence, MM calculations provided adequate geometries for those compounds which may be also studied by MO calculations in order to determine their electrostatic properties. The geometrical and electrostatic data were compared with the experimental results obtained by crystallographic analysis and by IH NMR.

I. Introduction

Antivial activity is demonstrated by many pyrimidine nucleosides [1], especially by some members of the series of 2~,3~-dideoxynucleosides (ddN), such as 3~-azido-3~-deoxythymidine (AZT) which was found to be a selective inhibitor of the reverse transcriptase of the human immunodeficiency virus (HIV) in cell cultures [2], and thus it has been extensively studied as a potential drug in the treatment of AIDS. Little has been reported on the mechanism of inhibition of HIV-1 by AZT. Some authors believe that phosphorylation of these nucleosides through the nucleoside quinase [2] is the first step in the process. Due to the side effects caused by AZT, considerable attention has also * Corresponding author.

been given to the development of new related drugs that may be effective against the AIDS virus and, at the same time, with low, if any, toxicity. In order to design computationally powerful antiviral agents, a conformational comparative analysis of 2r,3Ldideoxynucleoside analogues has been carried out by AM1 and by molecular mechanics (MM) calculations (CHEM-X) (see Fig. 1). These compounds were chosen based on the assumption that conformational properties might be associated with antiviral activity, thus conformational and electrostatic characteristics of the natural substrate thymidine, 1, and AZT, 2, such as activity patterns, were studied and later compared with the characteristics found in other compounds. The MM2 (87) program was applied to thymidine derivatives by Yates [3,4]. The AM1 method was

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148

D. Galisteo et al./Journal of Molecular Structure 350 (1995) 147-160

O

HN /~.,..N ~

used by da Motta Neto to examine the effect of the conformational changes in the quality of the AZT molecule in its ground state, including solvation effects and their implications over the antiviral activity of this kind of compound [6]. Conformations of AZT were described semiempirically by Sabio and Topiol [7] who, for the conformations they were describing, studied the possible bonds to be formed by hydrogen bridge. Using AM 1 and M M calculations, this research analyses the conformational properties of the 2',3'dideoxynucleoside analogues and their electrostatic properties, and later compares this with the geometrical and electrostatic characteristics experimentally reported, such as the crystallographic structures collected by the Cambridge data base [10] and the 1H N M R data.

/ CH3

0

R

1 - R= OH 2 - R= Na 3-R=F 4 - R= CN 5 - R= NH2

2. M e t h o d s

Fig. 1. Structure of 2',3'-dideoxynucleosideanalogues.

i

The glycosidic link (X), the rotation round the exocyclic bond C 4 ' - C 5 t, the dihedral angle H5'O5'C5'C4' (~b) and the puckering of the

4

H3C~c/C4~N H

jr Ho '

i

HO., I NL °

/, C3'

C2'

I

R

1.-R=OH 2.-R= N3 3.-R=F 4.-R= CN 5.- R = NH2

///N ct N N -/

Fig. 2. Schematicdiagram of the structureof 2',3'-dideoxypyrimidinenucleosideswith the numberingschemeusedin the text and tables (consistent with the recommendationsof IUPAC-IUB [21,22]).

D. Galisteo et al./Journal of Molecular Structure 350 (1995) 147-160

furanose ring were studied in order to determine the geometric optimizations and to carry out the conformational analysis. In the particular case of AZT, the rotation round the bond C3'-N~ was also considered (see Fig. 2). All geometry optimizations were performed with complete relaxation of all structural parameters. The structures 1-5 were modelled using the CHEM-X [9] program (the conformational analysis option). All molecular orbital (MO) calculations reported herein employed the AM1 semiempirical Hamiltonian within the MOPACv. 6.00 [8] program on a VAX computer. Furthermore, after each optimization, a force constant was calculated to verify whether the optimized structures were indeed local minima (no imaginary frequencies) or transition states (one imaginary frequency).

3. Results and discussion Fig. 2 shows the schematic diagram of the structure of 2~,3'-dideoxypyrimidine nucleosides. The compounds have been characterized by four

149

conformational degrees of freedom, namely the torsional angle of the glycosidic link N1 (thymine)-Cl'(furan) (X), the exocyclic link C4'(furan)-C5'(methylene) (O, the bond C5'(methylene)-O5'(hydroxyl) (~b), and the puckering of the furanose ring, as shown in Fig. 2. A fifth degree of freedom, the rotation round the bond C3'(furan)-N~(azide), was also studied in the case of AZT. The furanose ring in the different conformations was described on the basis of pseudorotation, thus determining the conformation of every furanose ring using two parameters, namely the "phase angle" of pseudorotation, P, and the degree of pucker, 0m, which are calculated from the endocyclic torsion angle in sugar [11]. An increasing order was chosen to present energy and optimized geometries. Only conformations in which the relative energy is 2.5 kcal mol -l higher than the global minimum were characterized on the assumption that populations with lower-energy conformations are smaller than 0.5%. Tables 1 and 2 show MM and AM1 calculations on thymidine, 1. Within the energy range

Table 1 Summary of MM calculations on thymidine, 1 Conformer

Energy (kcal mol-1)

X

(

1-1A 1-2A 1-3A 1-4A 1-5A I-6A 1-7A 1-8A 1-9A

0.00 0.13 0.13 0.35 0.43 0.43 0.48 0.79 0.94 1.11 1.18 1.18 1.27 1.30 1.38 1.45 1.67 1.74 2.37 2.38

- 160.95 170.39 - 175.26 179.24 - 176.83 - 173.97 - 170.73 - 156.71 - 176.25 - 173.70 - 173.83 - 173.70 - 179.73 -151.44 169.90 - 150.80 43.83 39.00 36.21 175.49

51.32 -53.87 59.67 -55.07 - 173.99 49.78 176.72 68.05 - 176.79 -48.00 - 56.49 -48.00 - 175.78 -175.27 - 175.33 53.56 -62.96 -57.90 - 168.23 47.46

1-10h 1-11A 1-12A 1-13A 1-I4A 1-15A

1-16h 1-17A 1-18A 1-19A

1-20A

~

C1'-C4'

178.74 -69.39 172.95 -64.40 -58.36 172.60 -58.17 71.75 -62.41 -51.90 -61.84 - 51.90 -60.17 -53.09 -57.93 -96.52 -73.11 -69.23 -61.80 -91.05

X, C 2 N 1 C I ' O 4 r ; ~', o 5 r c 5 ' c 4 ~ c 3 ' ; ~, H5~O5'C5~C4~; C 1 ~ - C 4 ' , C 1 ' C 2 ' C 3 ' C 4 t '

33.00 -34.93 38.63 -30.43 36.09 -33.69 33.99 32.27 26.10 33.45 35.68 33.45 -29.08 -33.75 -34.52 -32.90 -32.15 -36.00 34.70 -32.90

P

0m

12.71 182.98 1.25 145.17 15.06 166.03 19.42 33.14 44.95 18.15 7.17 6.90 142.67 167.11 184.95 176.67 168.67 167.28 19.82 189.81

33.83 34.94 38.61 37.03 37.38 34.73 36.05 38.57 36.88 35.15 36.99 35.96 36.60 34.67 34.63 32.96 32.74 36.91 36.89 33.39

150

D. Galisteo et al./Journal o f Molecular Structure 350 (1995) 147-160

Table 2 Summary of AM 1 calculations on thymidine, 1 Conformer

Energy (kcal mol-l )

X

~

q~

1-1B 1-2B I-3B 1-4B 1-5B 1-6B

0.00 0.21 0.49 0.51 0.96 0.98

-96.96 80.69 -143.41 -171.15 - 107.28 -97.30

1-7B

1.18

- 105.24

1-8B 1-gB 1-10B I-liB 1-12B 1-13B 1-14B 1-15B 1-16B 1-17B 1-18B 1-19B 1-20B 1-21B 1-22B

1.28 1.31 1.39 1.54 1.71 1.73 1.85 1.98 2.20 2.60 2.89 3.00 3.29 3.46 3.66 3.68 4.89

80.81 - 107.48 - 100.78 -170.28 -172.71 - 134.72 - 176.94 88.72 80.01 78.82 -107.54 - 179.87 2.81 - 104.39 -174.05 81.20 -176.31

- 175.54 - 178.73 173.51 -178.57 - 1.23 -34.93 68.22 -77.49 35.45 -77.46 -78.64 -59.20 -74.78 40.28 61.99 -71.89 -65.53 34.51 43.16 - 176.82 - 108.05 -67.01 - 84.66 172.93

-55.41 -57.04 -47.37 -49.10 -76.03 -60.74 65.94 61.18 176.52 54.50 56.02 -59.74 -62.47 62.95 -65.92 -62.37 78.35 163.48 62.14 -66.56 - 167.08 -174.19 169.73 165.44

- 163.94

82.31

- 139.43

172.83

1-23B

Cryst. Opt. (MO) Cryst. Opt. (MM) Cryst.a

13.69

C1'-C4'

9.64 8.51 -5.98 5.27 8.38 7.91

P

0m

-48.93 -54.30 -45.98 -68.06 -61.79 -62.19

14.61 14.56 19.43 14.19 17.77 16.93

15.04

4.71

15.05

10.16 0.18 11.40 9.65 -3.22 - 12.72 3.29 13.50 -3.55 9.92 10.64 8.69 1.76 1.16 -5.91 - 1.05 5.01

-51.17 -87.71 -36.86 -54.14 259.34 206.04 -70.70 -45.85 254.56 -47.37 -20.96 -37.97 -81.32 -85.14 252.06 266.02 -75.84

16.27 5.01 14.25 16.56 17.29 14.14 9.98 19.38 13.15 14.62 11.35 11.04 11.93 14.15 19.16 14.40 20.44

- 175.06

-34.29

159.34

36.66

- 151.86

- 36.93

187.57

37.22

a Ref. [28]. a f o r e m e n t i o n e d , 20 d i f f e r e n t c o n f o r m a t i o n s w e r e

a n d three trans c o n f o r m e r s were found. The rest

l o c a t e d t h r e e o f t h e s e 20 e x h i b i t s y n c o n f i g u r a t i o n s (1-17A, 1-18A, 1-19A) b e t w e e n t h y m i d i n e a n d

are - g c o n f o r m e r s .

the furanose ring, thus f a v o u r i n g the anti c o n f o r -

o f these are within the relative energy range afore-

m a t i o n in t h i s c o m p o u n d . B o t h t h e g a u c h e a n d t h e

mentioned. Four of these exhibit a syn configura-

B y M O c a l c u l a t i o n s , 23 m i n i m a w e r e f o u n d ; 16

trans forms were observed for the dihedral angle

tion b e t w e e n the t h y m i d i n e a n d the f u r a n o s e ring,

C 4 ' - C 5 ' (if). F o r t h e d i h e d r a l a n g l e H51C5~C4'C3', h o w e v e r , o n l y o n e + g c o n f o r m e r (1-8A, T a b l e 1)

w i t h C 2 N 1 C 1 ' O 4 1 (X) in t h e 8 0 . 0 1 - 8 8 . 7 2 ° r a n g e . For the

12 a n t i c o n f o r m e r s , t h e d i h e d r a l a n g l e

Table 3 Coupling constants (Hz) in 2'-deoxythymidine, 1

Ref. [16] Calculated (MM) Calculated (MO)

Jl '2-Jl '2"

J2'3'-J2"3'

.]3'4'

J4'5'-.]4'5"

%S

13.6a 4.9/4.2 7.3/3.3

10.9a 4.7/5.5 4.4/8.0

3.9 4.4 3.4

3.6/5.1 4.3/4.7 4.0/7.0

63.55 52.69 68.22

a Average value determined from unresolved peak signals.

D. Galisteo et al./Journal of Molecular Structure 350 (1995) 147-160

151

Table 4 S u m m a r y of M M c a l c u l a t i o n on A Z T , 2 Conformer

Energy (kcal m o l 1)

2-1A 2-2A 2-3A 2-4A 2-5A 2-6A 2-7A 2-8A 2-9A 2-10A 2-11A 2-12A 2-13A 2-14A 2-15A 2-16A 2-17A 2-18A 2-19A 2-20A 2-21A 2-22A 2-23A 2-24A 2-25A 2-26A 2-27A 2-28A 2-29A 2-30A 2-31A 2-32A 2-33A 2-34A 2-35A 2-36A 2-37A 2-38A 2-39A 2-40A 2-41A 2-42A 2-43A 2-44A 2-45A 2-46A 2-47A 2-48A 2-49A

0.00 0.03 0.14 0.16 0.27 0.37 0.56 0.59 0.62 0.63 0.65 0.68 0.74 0.74 0.80 0.86 0.88 0.90 0.91 0.99 1.01 1.07 1.07 1.34 1.36 1.48 1.49 1.51 1.52 1.54 1.56 1.64 1.65 1.83 1.93 1.97 2.02 2.05 2.07 2.13 2.16 2.17 2.20 2.24 2.31 2.31 2.31 2.36 2.37

X

-168.73 - 168.76 - 168.74 -168.74 - 168.75 70.33 70.34 -168.75 - 1018.03 - 1018.03 70.37 -168.77 -168.74 70.31 - 1018.02 -168.77 -168.74 - 168.74 70.29 -168.74 70.33 70.31 70.34 - 1018.09 -1018.03 70.29 - 108.99 - 168.77 70.25 - 1018.00 70.34 - 168.74 - 108.99 -168.80 - 168.79 -168.79 18.95 -168.75 70.30 -1018.01 -168.75 -168.80 70.20 -168.74 10.09 -168.75 70.30 -108.99 -168.77

~

-60.32 -60.31 518.99 1718.69 60.00 -60.33 518.99 1718.69 -60.31 1718.70 1718.67 -60.31 1718.69 -60.33 60.00 -60.31 1718.69 60.00 -60.33 60.00 518.99 518.99 1718.67 - 60.31 -60.31 -60.33 60.00 -60.32 518.99 1718.69 1718.67 518.99 60.00 1718.70 1718.69 -60.31 -60.34 1718.69 1718.67 1718.70 -60.31 60.00 -60.33 60.00 518.98 1718.69 1718.68 1718.69 -60.31

(

-49.75 -418.98 -418.97 -418.94 -50.00 -418.55 -418.97 70.18 70.21 70.22 -418.94 70.17 -418.97 -418.98 70.19 -1618.94 -1618.93 70.15 -1618.93 -1618.96 -50.00 -1618.95 -1618.92 -418.98 -418.55 -418.90 -418.97 -418.90 -418.93 -418.96 -418.97 -418.92 - 50.00 -1618.86 -418.90 -1618.86 -418.96 -1618.90 -418.90 -418.97 -1618.91 -1618.88 -1618.91 -1618.93 -418.97 70.14 -1618.90 70.18 70.13

4~

C1'-C4'

-174.02 - 54.07 - 174.01 -174.02 - 54.07 -174.02 - 174.02 -174.16 65.84 65.84 -174.02 -174.16 -54.08 - 54.07 65.83 -54.18 -54.17 - 174.16 -54.18 -54.17 -54.07 -54.18 -54.18 - 54.07 -174.01 65.99 - 174.01 65.99 66.00 - 174.01 -54.07 65.99 - 54.07 65.88 65.99 65.88 - 174.01 -174.11 65.99 -54.08 -174.12 65.88 -174.12 -174.12 - 174.01 -54.21 -174.11 -174.16 -54.21

18.42 18.43 18.47 18.47 18.47 18.33 18.38 18.46 18.40 18.40 18.37 18.42 18.47 18.33 18.40 18.43 18.47 18.46 18.34 18.47 18.38 18.38 18.37 18.40 18.40 18.34 18.44 18.43 18.38 18.43 18.37 18.47 18.44 18.48 18.48 18.43 18.29 18.47 18.37 18.43 18.43 18.47 18.34 18.47 18.34 18.46 18.37 18.43 18.43

X = C 2 N 1 C 1 ' O 4 ' ; c = N f l N a C 3 ' C 2 ' ; ( = O 5 ' C 5 ' C 4 ' C 3 ' ; q~ = H 5 ' O 5 ' C 5 ' C 4 ' ; C 1 ' - C 4 ' = C 1 ' C 2 ' C 3 ' C 4 ' .

P

6.35 6.45 6.45 6.15 6.48 6.21 6.45 6.21 6.45 6.05 6.21 6.21 6.05 6.12 6.35 6.05 6.48 6.45 6.21 6.45 6.45 6.21 6.35 6.05 6.48 6.21 6.45 6.21 6.45 6.05 6.35 6.02 6.02 6.35 6.15 6.48 6.78 6.45 6.21 6.45 6.45 6.02 6.35 6.68 6.05 6.48 6.21 6.45 6.21

0m

18.51 18.52 18.52 18.51 18.42 18.61 18.52 18.61 18.52 18.50 18.61 18.61 18.50 18.61 18.51 18.50 18.42 18.52 18.61 18.52 18.52 18.61 18.51 18.50 18.42 18.61 18.52 18.61 18.52 18.50 18.51 18.60 18.60 18.51 18.51 18.42 18.43 18.52 18.61 18.52 18.52 18.60 18.51 18.43 18.50 18.42 18.61 18.52 18.61

D. Galisteo et al./Journal of Molecular Structure 350 (1995) 147-160

152

Table 5 Summary of A M 1 calculation on AZT, 2 Conformer

Energy (kcal mol-1)

X

~

~

~

C1'-C4'

P

0m

2-1B 2-2B 2-3B 2-.4B 2-5B 2-6B 2-7B 2-8B 2-9B 2-10B 2-11B 2-12B 2-13B 2-14B 2-15B 2-16B 2-17B 2-18B 2-19B 2-20B 2-21B 2-22B 2-23B 2-24B 2-25B 2-26B 2-27B AZT-A Cryst. a AZT-A Opt. (MO) AZT-A Opt. (MM) AZT-B Cryst. a AZT-B Opt. (MO) AZT-B Opt. (MM)

0.00 0.52 0.53 1.02 1.07 1.10 1.35 1.52 1.89 1.93 1.94 2.01 2.07 2.24 2.26 2.28 2.33 2.42 2.46 2.48 2.50 3.37 3.86 4.05 4.15 5.52 5.62 67.57

-107.06 80.27 -98.31 -173.18 -151.76 -141.62 -125.45 80.47 -101.43 -171.83 -120.63 -107.54 -111.28 -155.00 70.24 73.87 87.88 -179.79 82.89 - 108.64 -166.08 94.12 1.67 81.44 -99.85 -175.17 -115.84 -124.40

-60.47 -62.75 -62.91 -59.35 -58.83 -60.45 -57.20 -62.11 -61.04 -57.76 -59.36 -60.30 -59.59 -58.84 -61.18 -61.77 -60.07 -58.59 -61.06 60.71 -58.69 -58.67 -64.91 -67.32 61.96 -171.24 52.26 177.59

70.90 -178.18 -174.99 -178.45 157.11 173.99 18.54 -76.08 -76.82 -77.86 39.87 40.64 36.18 -63.75 24.04 36.81 63.82 44.79 169.57 68.35 37.38 79.33 -179.86 -86.91 -40.38 -57.92 -164.50 50.94

66.00 -57.93 -56.50 -51.55 40.59 -45.78 -78.95 64.25 60.41 60.72 -78.11 172.03 -178.48 -61.90 68.93 56.39 -68.41 62.72 45.67 66.93 60.73 57.71 -67.25 161.93 -59.19 86.82 56.79 -129.08

18.03 11.10 13.72 14.87 -15.78 -15.48 22.10 11.97 14.70 17.22 -20.23 11.26 -11.53 -18.24 -17.55 13.18 15.53 10.30 9.56 19.75 -14.20 23.08 11.84 7.89 14.34 19.63 20.15 -32.37

5.46 -44.95 -34.33 -38.67 215.59 202.95 -12.60 -44.98 -26.44 -35.46 194.61 -13.89 200.88 214.78 165.71 29.41 -39.77 -32.51 -58.70 11.32 216.04 -16.16 -40.85 -61.08 -35.76 -26.21 0.46 175.07

18.08 15.68 16.59 19.08 19.43 16.83 22.65 16.96 16.42 21.12 20.87 11.64 12.31 22.16 18.16 15.15 20.17 12.21 18.48 20.19 17.56 24.04 15.60 16.33 17.62 21.85 20.10 35.52

4.57

-113.15

171.41

36.09

159.23

18.39

-3.13

18.43

-

-135.13

170.25

58.70

178.39

-33.52

162.35

35.50

33.08

-173.55

176.22

173.39

90.68

-29.30

215.41

35.95

4.56

-145.39

174.68

151.08

36.43

-13.60

208.79

15.52

-175.33

178.98

176.47

64.96

-35.37

188.77

35.82

a Ref. [14].

between rings ranges from 180.06 to 263.04 (-96.96°). The global minimum exhibits an anti configuration between the thymidine and the furan ring with a dihedral angle C2N1Cl/O4 t of -96.96 ° . This conformation, which is called "high-anti", is seldom found in the pyrmidine nucleosides [23]. The trans configuration and the two gauche forms (except the conformer 1-9B,

176.52 °, which only exhibits a trans configuration, as shown in Table 2) were observed in the conformers in which a minimum energy for the dihedral angle H5~O5~C51C4 was found. This situation changes if the full set of conformers is considered, the conformers with relative energy higher than 2.5 kcal mo1-1 prefer trans configurations for the dihedral angle H5tC5~C4'C3 ~.

D. Galisteo et al./Journal of Molecular Structure 350 (1995) 147-160

The crystallographic structural data shown in Table 2 do not correspond to any minimum-energy conformer. M M and M O calculations were then used in order to determine whether the conformations achieved were similar to any minimum-energy conformations. The optimized crystallographic structure conformations obtained using both calculations were not analogues to any minimum-energy conformations. Using M M calculations (ClaEM-X), the crystal structure optimization did not agree with the results already obtained for thymidine [3]. Using (MM2(87) [4], however, the results agreed. The conformer 1-14A (Table 1) shows the highest similarity to its crystallographic structure conformation, although the dihedral angle H5'C5~C4~C3 ~ differs from -151.86 ° for the crystallographic structure to -53.09 ° for the structure obtained by M M calculations. Without optimization, the crystallographic structure energy obtained by the MOPAC program was 13.69 kcal mo1-1 higher than the minimum energy, decreasing to 4.89 kcal mol -~ after optimization. P (phase angle) and 0m (amplitude) values obtained by M M calculations were similar to the 3-pyrimidines' puckering values (P = 0-30 °, C3 rendo and P = 150-180 °, C2'-endo, 0m = 35-45°), although those values calculated by M O calculations were different (P ranges from - 2 0 ° to - 8 5 ° , only one conformer reaches 4.71°). Table 3 shows the coupling constants, calculated using the 3JHH [5] program, in 2~-deoxythymidine. The following equation [17] was used to relate the coupling constants to the percentage of S character

of the furanose ring: %S = 100 ×

J1'2' (J1 '2' '[- J3'4')

As shown in Table 3, the calculated coupling constant values were rather distant from the experimental ones. The S forms predominated in both the calculated and experimental coupling constants; nevertheless, it can be seen that the percentage of S character obtained was similar, whether using the M O calculations or the solution data. Using M M calculations in the analysis of 3~azido-3'-deoxythymidine 2, 49 conformers were found. Most of them exhibit preferentially anti conformations between the base and the sugar ring (Table 4). Of these 49 conformers, 32 have their azido group pointing down (N/~N~C3'C2' = - 6 0 and 60 °) and 17 of them have their azido group pointing sideways (N~N~C3'C2' = 180°). This observation is interesting because there is a trend of focussing attention on the latter set since this is the conformation found by X-ray analysis of A Z T crystals [14]. Either gauche or trans forms were found in all the dihedral angles O5'C5'C4'C3' studied. The results obtained by M O calculations coincided with data already reported by Sabio and Topiol [7] (see Table 5). Two independent crystallographic molecules A Z T - A and AZT-B [12-15] were found using X-ray methods. They were optimized by M M calculations (CHEM-X) and by M O calculations (AM1). As in the case of thymidine, the use of theoretical methods in the analysis of crystallographic structures did not produce

Table 6 Coupling constants (Hz) in AZT

Ref. [17] Ref. [18] Ref. [18]b Ref. [19] Calculated (MM) Calculated (MO)

J1 '2'-Jl '2"

J2'3'-J2"3'

J3'4'

J4'5'-']4'5"

%S

66.43a 6.4/6.7 13.0c 13.0c 6.5/2.5 7.1/3.4

7.2/5.2 13.0c 12.8c 6.5/8.5 6.4/8.3

4.85 4.9 5.5 5.5 5.7 5.5

4.1/3.9 3.5/4.6 4.6/3.5 5.3/6.9 5.3/3.9

57.00 56.63 54.17 54.17 53.28 56.35

a Average value determined from unresolved peak signals. b Coupling constants in D20. c Only the sums JHl'2' + Jm'2" and JH2'3' + JH2"3' are obtained.

153

D. Galisteo et al./Journal of Molecular Structure 350 (1995) 147-160

154

Table 7 S u m m a r y o f M M c a l c u l a t i o n o n F-ddT, 3 Conformer

Energy (kcal m o l - 1)

X

(

~b

C 1 ' - C4'

3-1A 3-2A 3-3A 3-4A 3-5A 3-6A 3-7A 3-8A 3-9A 3-10A 3-11A 3-12A 3-13A 3-14A 3-15A

0.00 0.07 0.17 0.20 1.27 1.27 1.29 1.35 1.37 1.43 1.47 1.49 1.56 1.61 1.64

-170.79 - 171.65 -178.06 -175.01 - 172.52 - 180.00 -177.42 - 144.03 - 141.15 -175.09 - 178.83 -178.72 -170.62 - 164.69 -179.27

48.72 49.68 53.52 48.54 -57.04 50.16 -57.57 57.16 65.95 179.24 - 73.49 -175.88 -63.07 -68.65 -65.03

179.02 - 175.81 -175.73 -171.33 -63.53 74.48 -59.77 90.19 72.04 -56.78 66.98 -57.85 -178.21 67.92 179.40

37.32 36.26 37.62 37.91 35.59 36.32 36.88 32.84 32.85 36.77 38.50 38.15 33.12 32.21 38.66

0m

P

2.49 7.09 -1.83 7.76 17.29 21.1 11.74 24.69 33.88 11.38 12.65 10.60 25.18 33.62 12.64

37.34 36.58 37.62 38.25 37.28 38.91 37.69 36.10 39.63 37.54 39.46 38.86 36.58 38.67 39.66

X = C 2 N 1 C 1 ' O 4 ' ; ( = O 5 ' C 5 ' C 4 ' C 3 ' ; 4~ = H 5 ' O 5 ' C 5 ' C 4 ' ; C 1 ' - C 4 ' = C 1 ' C 2 ' C 3 ' C 4 ' .

geometries which were similar to minimum-energy conformations. With respect to the puckering of the furanose ring, P and 0m were calculated by M M and MO calculations, resulting in values which were contrary to those expected [11]. On the basis of P (phase angle), three different kinds of conformation were obtained by MO calculations: C3'endo/C2'-exo ( P = 0 - 3 0 ° ) , Cl'-endo ( P ~ 300 °) and C3'exo/C4'-endo (P ~ 215°). The third conformation is much less commonly observed in nucleosides in general but is frequently found in the structures of the very potent anti-HIV compounds in which 0m = 15-25 °. In all the structures located by M M calculations, P ~ 6 ° (C3'-endo/C2'-exo) and 0m = 18-42-18.61 °, thus forming geometries having more planar furanose rings than MO ones. Table 6 shows experimental and calculated coupling constants in AZT and the percentage of S character, observing an equilibrium between S forms (C2'-endo) and N forms (C3'-endo). As shown in Table 7, 15 conformers were found within the established energy limit using M M calculations in the analysis of 3'-fluorine-3'deoxythymidine, 3, which, in vivo, is considered to be as effective as AZT against the AIDS virus, although with less severe secondary effects [12]. These conformers exhibit anti conformations for

the glycosidic link, and gauche and trans forms for the dihedral angles 05'C5'C4'C3' and H5'O5'C4'C3'. A total of 20 energy minima were found by MO calculations; 16 of these were below 2.5 kcal mol -l (see Table 8). Six of them exhibit syn conformations between the thymidine and the furanose ring with a dihedral angle C2N1C1'O4' ranging from 69.79 ° to 87.40 °, whereas ten of them exhibit anti conformations, with their dihedral angle between rings ranging from 180.71 ° to 263.77 ° (-96.23°). The global energy minimum exhibits anti conformations between the thymidine and the furanose ring with the dihedral angle C2N1C1'O4' reaching -105.44 °. The trans state and the two gauche forms for the dihedral angle O5'C5'C4'C3' were found whereas for the dihedral angle H5'O5'C5'C4', only the two gauche forms were found in the low-energy conformers, except for the 3-9B conformer which exhibits a trans form (170.07 °) as shown in Table 8. Once Tables 7 and 8 were compared, some conclusions were draw: (i) for the energy-minimum conformations, obtained by M M calculations, the dihedral angle q~ is preferentially trans whereas this form was not observed for the energy-minimum conformations obtained by MO calculations, with the exception of3-9B; (ii) this situation changed after considering the complete set of conformers

155

D. Galisteo et al./Journal of Molecular Structure 350 (1995) 147-160 Table 8 Summary of AM 1 calculations on F-ddT, 3 C 1 ' - C4'

0m

4.22 -45.96 -38.75 -61.61 -51.68 113.58 -48.97 -53.81

15.44 14.96 15.00 17.25 16.77 9.50 16.45 16.43

Energy (kcal mol 1)

X

(

3-1B 3-2B 3-3B 3-4B 3-5B 3-6B 3-7B 3-8B 3-9B 3-10B 3-11B 3-12B 3-13B 3-14B 3-15B 3-16B 3-17B 3-18B 3-19B 3-20B F-ddTA a Cryst. F-ddTA Cryst. Opt. (MO) F-ddTA Cryst. Opt. (MM) F-ddTB a Cryst. F-ddTB Cryst. Opt. (MO) f-ddTB Cryst. Opt. (MM)

0.00 0.17 0.78 1.14 1.16 1.42 1.46 1.52 1.68 1.78 1.91 1.98 2.09 2.14 2.44 2.45 3.33 3.85 3.92 5.18 86.55

- 105.44 79.39 -98.48 -170.71 -173.64 69.79 80.65 -96.23 80.39 87.40 82.22 - 174.40 - 179.29 -172.76 -101.60 81.53 -176.32 - 176.49 96.74 -138.50

68.78 -178.56 -175.33 157.24 -177.76 29.96 -76.96 -44.94 37.50 -73.78 64.02 164.54 -61.36 42.46 -78.38 -78.29 -75.86 -63.41 174.46 54.14 50.20

-64.01 -65.77 39.16 -63.92 64.25 58.82 56.65 174.11 -170.44 167.99 - 179.19 154.3

6.65 15.00 9.74 3.33 8.96 11.66 12.35 5.28 -0.29 3.36 20.08 -34.90

-61.83 -38.41 -57.52 -78.72 -40.91 -50.70 -32.27 -68.16 269.04 - 53.46 -40.59 163.51

14.19 19.14 18.06 16.86 11.91 18.47 14.67 14.25 17.87 16.46 26.47 36.40

1.68

-107.90

37.52

169.86

8.70

-21.60

9.37

-

- 138.44

58.38

179.45

-34.06

160.77

36.11

37.52

-159.58

52.86

155.32

-31.24

169.21

31.76

1.68

-107.68

37.49

169.65

8.72

-21.78

9.37

-

- 154.87

60.98

179.60

- 33.60

170.88

34.03

- 107.94

4~

P

Conformer

67.25 -56.83 -55.25 42.69 -49.70 64.12 64.00 -65.15 170.07

15.39 10.44 11.74 8.19 10.37 -3.85 10.78 9.75 8.59

-21.64

9.25

a Ref. [291.

obtained by MO calculations, since conformers with energy 2.5 kcal mo1-1 higher than the global minimum exhibited preferentially a trans form for the dihedral angle H5'C5'C4'CY (~b). Table 9 shows the coupling constants and percentage of S character in F-ddT, 3. The coupling constants calculated by the 3JHHprogram did not agree with the experimental values, the S forms predominating in the experimental values and in the S character calculated from coupling constants, J, found by MO calculations. This did not

happen when the percentage of S character was calculated by MM. The puckering of the ring was also studied by MO calculations to obtain the conformations for F-ddT, obtaining similar results to those for AZT. In the forms obtained by optimizations of MM calculations, the P and 0m values obtained were considered normal for nucleosides (P = 0-35 °, 0 m = 35-45°). The Y-cyano-Y-deoxythymidine compound (CN-ddT) 4, might be regarded as anti-HIV since the cyano group is a good isoster of both the azido

D. Galisteo et al./Journal o f Molecular Structure 350 (1995) 147-160

156

Table 9 Coupling constants (Hz) in F-ddT

Ref. [26] Ref. [17] Calculated (MM) Calculated (MO)

Jl '2'-Jl '2"

J2'3'-,]2 "3

J3 '4'

J4'5'-J4"5'

%S

9.1/5,7 9.25 4.8/1,7 7.4/3,5

5.3/1.5 5.7/7.1 4.4/7.4

1.5 0 6.1 4.1

4.3/4.3 1.5/3.2 5.3/3.8

85.85 > 99 44.04 64.35

and the hydroxyl groups owing to the similarity among some of their stereoelectronic properties. Unfortunately, this compound lacks antiviral activity [20]. Table 10 shows a summary of M M calculations on CN-ddT; the 13 conformers shown are within the established energy limit. All of them, with the exception of 4-8A, exhibit anti configurations for the glycosidic link. The two gauche forms were observed for the bond C4~-C5 ~ and the two gauche forms and the trans configuration were found for the dihedral angle H5'05'C5'C4'. By MO calculations, 20 minima were located. Of these 20, 14 are within the relative energy limit as shown in Table 11. Five of these 14 exhibit a syn configuration between the thymidine and the furanose ring with a dihedral angle of C2N1ClrO4 ~ in the interval ranging from 73.32 ° to 81.27 ° . In the nine anti conformers, the dihedral angle between anti rings ranged from 188.45 to 260.49 ° (-99.51°). The trans state and the two gauche forms were observed for the dihedral angle O5~C5'C4'C3,

whereas only the two gauche forms between the minimum-energy conformers were found for the dihedral angle H5rO5tC5~C4 t with the exception of the 4-8B conformer which exhibits a trans conformation as shown in Table 9. Once the complete set of conformers was considered, the situation changed since the conformers with energy of 3 kcal mo1-1 higher than the global minimum exhibited preferentially a trans form for the dihedral angle H5rC51C4~C3 ~. Table 12 shows the coupling constants and percentage of S character in 4. A higher percentage of S forms in the geometries obtained by MO calculations is evident. Again, the puckering of the furanose ring was calculated by MO calculations obtaining similar results to those obtained for AZT. By M M calculations, the P and 0m values obtained were considered normal for nucleosides (P = 0-35 °, 0m = 35--45°). The 3t-amino-3t-deoxythymidine compound, 5, was analysed by M M calculations. This compound is HIV-inactive at subcytotoxic

Table 10 Summary of MM calculations on CN-ddT, 4 Conformer

Energy (kcal mol 1)

X

4-1A 4-2A 4-3A 4-4A 4-5A 4-6A 4-7A 4-8A 4-9A 4-10A 4-11A 4-12A 4-13A

0.00

- 173.81

0.12 0.20 0.66 0.68 0.90 1.41 1.84 1.85 1.87 1.88 2.29 2.30

- 144.32 -173.77 - 176.89 -172.77 - 177.00 - 175.08 68.32 - 115.76 -121.50 - 176.70 - 171.74 - 176.81

ff

49.28 40.28 -55.10 49.15 -56.11 177.48 -60.65 -51.86 62.26 48.89 63.49 -49.48 178.54

q~

C 11-C4t

P

0m

174.81 178.21 -71.17 94.96 179.35 -60.07 86.03 - 179.93 70.37 163.46 -90.59 -68.56 62.94

28.17

12.54

34.50 25.96 30.75 21.61 30.57 24.21 20.55 25.50 27.16 27.37 21.04 29.24

26.27 18.06 20.96 18.54 17.28 18.76 22.40 19.31 19.05 9.76 24.02 15.55

28.89 38.47 27.35 32.88 22.78 32.05 25.56 22.17 27.02 28.78 27.80 22.99 30.31

D. Galisteo et al./Journal of Molecular Structure 350 (1995) 147-160

157

Table 11 Summary of AM 1 calculations on CN-ddT, 4 Conformer

Energy (kcal mo1-1)

X

(

4~

C 1'-C4'

P

0m

4-1B 4-2B 4-3B 4-4B 4-5B 4-6B 4-7B 4-8B 4-9B 4-10B 4-11B 4-12B 4-13B 4-14B 4-15B 4-16B 4-17B 4-18B 4-19B 4-20B

0.00 0.12 0.88 1.19 1.23 1.29 1.67 1.71 1.77 1.79 1.83 2.01 2.06 2.28 2.79 2.88 3.21 3.89 4.01 4.95

- 106.12 79.11 -99.51 73.32 -174.15 -171.55 80.86 - 109.78 79.96 -95.70 -120.68 81.27 - 179.94 - 174.99 -173.88 -100.95 80.91 - 176.92 -176.72 97.42

67.22 - 178.74 -175.59 31.18 -178.17 155.66 -77.80 38.09 -76.33 -48.07 19.55 168.25 42.60 -62.81 -79.57 -82.14 -77.16 -63.95 174.22 52.22

67.87 -57.73 -56.11 60.46 -51.29 43.61 68.52 168.01 -66.37 -68.94 -85.64 44.39 64.00 -66.42 63.43 62.06 175.37 - 169.81 166.99 179.43

17.20 11.85 13.37 12.87 13.66 11.62 11.01 12.81 8.72 9.93 16.99 10.98 13.14 4.66 11.69 12.08 8.18 1.61 5.40 21.78

6.89 -39.75 -31.22 26.27 -41.8 -51.51 -48.97 -9.09 -54.25 -53.05 -19.38 -51.92 -26.16 -74.05 -51.76 -34.92 -56.96 -84.80 -75.36 -34.84

17.33 15.35 15.67 14.39 18.38 18.64 16.76 12.96 14.89 16.47 18.02 17.83 14.59 17.10 18.90 14.76 15.04 17.65 21.36 26.56

X = C2N1C1'O4'; ( = O5'C5'C4'CY; 0 = H5'O5'C5PC4'; C 1 ' - C 4 ' = C1'C2'C3 'C4r.

concentrations but active when phosphorylated as NH2-dTTP [24]. A total of 27 conformers were found within the minimum-energy interval, as shown in Table 13. All of them exhibit anti configurations for the glycosidic link, with trans and -4-gauche forms for the dihedral angles O5/C5/C4'C3 ' and H5'O5tC41C3 I. By MO calculations, 16 local minima were found. Of these, 13 exhibit anti configurations whereas three of them show syn configurations between the thymidine base and the furanose ring (C2N1CI'C2' = 8188°). Two gauche forms and the trans configuration were found for the rest of the dihedral angles. Analysing the ring conformation by MO calculations, the conformers obtained had P values of approximately 300 ° (Cl~-endo, 1E), which is uncommon for this type of compound, and 0m

values corresponding to rings more planar than expected. By MM calculations, however, both P and 0m values were as expected: P ranged from 0 ° to 36 ° and 0m = 34-40 ° which are considered normal values for nucleosides. Table 14 shows the coupling constants in NHz-ddT, 5, and the percentage of S character. In order to obtain the relationship between the stereochemistry and the electronic properties of the compounds, the charge distribution was calculated in thymidine, 1, in AZT, 2, in F-ddT, 3 (all active analogues), in CN-ddT, 4, and in NH2ddT, 5 (both inactive compounds) by the semiempirical method neglect of diatomic differential overlap (NDDO [25]). Tables 15 and 16 show the results obtained. Although the geometry of the global local minimum found for every compound by MM

Table 12 Coupling constants (Hz) and % S character in CN-ddT

Calculated (MM) Calculated (MO)

Jl '2'-J1 '2"

J2'3'-']2"3'

J3'4'

J 4 ' 5 ' - J4'5 "

%S

6.0/2.3 7.2/3.2

7.5/7.4 7.1/8.2

7.7 5.9

2.7/4.3 5.3/3.6

43.80 54.96

D. Galisteo et al./Journal of Molecular Structure 350 (1995) 147-160

158

Table 13 Summary of MM calculations on NH2-ddT, 5 Conformer

Energy (kcal mol- l )

X

~

(


Clt-C4 '

P

0m

5-1A 5-2A 5-3A 5-4A 5-5A 5-6A 5-7A 5-8A 5-9A 5-10A 5-11A 5-12A 5-13A 5-14A 5-15A 5-16A 5-17A 5-18A 5-19A 5-20A 5-21A 5-22A 5-23A 5-24A 5-25A 5-26A 5-27A

0.00 0.09 0.11 0.29 0.32 0.34 0.38 0.41 0.53 0.53 0.69 0.74 0.78 0.81 0.85 0.88 0.90 0.94 1.04 1.27 1.55 1.56 1.64 1.84 1.91 2.00 2.02

-170.36 -171.34 -173.82 -174.06 -171.06 -170.19 178.63 -161.82 -170.64 -157.67 -152.54 174.77 -171.31 -172.41 -149.94 -164.90 -146.83 -173.73 -134.13 171.96 -148.69 169.21 -177.01 175.88 -179.57 -172.41 -176.46

158.68 158.05 158.58 156.72 154.67 150.20 160.79 153.66 156.86 157.84 148.62 157.97 150.52 150.89 149.94 153.07 150.84 160.67 150.26 157.46 153.06 152.43 156.79 159.47 160.59 152.06 154.11

50.69 52.81 56.38 -55.97 174.64 -69.81 -172.96 178.76 -56.92 -58.49 55.30 -178.45 -69.48 -62.47 -52.87 72.14 -58.40 49.64 61.61 -61.32 55.96 55.16 173.20 -168.68 -170.70 -176.53 -179.86

-173.78 177.23 175.19 71.33 -58.23 69.84 -55.56 -59.60 -60.84 73.59 173.58 -58.85 -175.56 -61.65 -64.77 78.20 -177.88 -86.98 66.94 177.21 -87.77 68.17 51.96 178.71 66.28 -175.60 53.3221

38.03 37.56 38.04 36.19 34.01 29.18 40.10 32.79 36.47 36.98 28.33 37.31 29.54 30.28 29.78 33.21 30.01 39.78 29.52 36.63 32.53 32.48 36.13 38.88 40.04 31.08 33.46

5.18 1.98 3.77 10.62 20.20 38.11 3.99 29.96 5.64 13.77 33.48 18.27 35.14 33.36 30.49 25.94 33.92 -0.56 39.38 17.58 21.10 10.37 11.30 6.81 -0.19 38.52 30.36

38.16 37.62 38.08 36.83 36.23 37.11 40.20 37.86 36.68 38.09 33.93 39.28 36.07 36.28 34.58 36.92 36.15 39.80 38.45 38.39 34.84 33.04 36.81 39.18 40.00 39.75 38.83

X = C2NIC1~O4';

= N3'C3'C2'C1'; ¢ = O5'C5'C4'C3'; ~b = H5'O5'C5'C4'; C 1 ' - C 4 ' = C1'C2'C3'C4'.

calculations differed from the one found by MO calculations, the atomic net charges over the corresponding atoms were similar, observing more variation in the charges in the C3' substituted centres. Contrary to results already reported [25], it cannot be concluded that the presence of a negative net charge over the substituting atom in the carbon C3' is in accord with anti-HIV activity, since in this study negative charges were also

found for the inactive compounds CN-ddT, 4, and NHz-ddT, 5.

4. Conclusions M M calculations and MO calculations exhibit different conformations of minimum energy. Both of them, nevertheless, show a preference for the anti conformation of the glycosidic link.

Table 14 Coupling constants (Hz) in NH2-ddT, 5

Ref. [24] Calculated (MM) Calculated (MO)

Jl ' 2 ' - J l / 2 "

J2'3'-J2"3'

J3'4'

J4'5'-J4'5"

%S

4.5/7.3 5.3/1.9 7.1/2.9

7.8/7.4 7.8/6.8 7.7/7.8

7.1 7.8 6.5

2.9/4.8 4.1/4.8 4.9/4.2

38.87 40.45 52.21

D. Galisteo et al./Journal of Molecular Structure 350 (1995) 147-160 Table 15 Calculated atomic net charges (e-) in 3'-substituted thymidines for the global minimum localized by MO Atom

3'-OH

3'-F

3'-NH 2

3'-N 3

3'-CN

C2 N1 CI' C2' C3' C4' O1' C5' 05' X(C3')

0.4029 -0.2796 0.1491 -0.2315 0.0102 -0.0627 -0.2810 -0.0258 -0.3158 -0.3044

0.4026 -0.2813 0.1563 -0.2276 0.0362 -0.0461 -0.2808 -0.0285 -0.3643 -0.1534

0.4017 -0.2798 0.1585 -0.2386 -0.0537 -0.0123 -0.2884 -0.0263 -0.3339 -0.3337 0.1496 0.1583

0.4023 -0.2807 0.1538 -0.2129 -0.0517 -0.0333 -0.2835 -0.0275 -0.3311 -0.2747 0.2126 -0.0263

0.4026 -0.2815 0.1541 -0.1885 -0.0461 -0.0084 -0.2828 -0.0302 -0.3316 -0.1451 -0.0185

Considering the puckering of the furanose ring, the geometries obtained by MM calculations have 0m and P values (amplitude and phase angle respectively) in accordance with the experimental data. When the percentage of S character was calculated from the coupling constants however, the S forms predominated in all cases with the exception of the compound NHz-ddT, 5, in which an excess of population N-type solution (60:40) was observed experimentally. The percentages of S character calculated by MM calculations and MO calculations were in accord with the experimental values, particularly with the values obtained from the coupling constants of the geometries calculated by MO calculations.

159

Not only one minimum-energy conformation to which a majority population corresponded but a series of them were located within the energy range of 2.5 kcal. On the basis of the theoretical methods employed, the existence of a negative atomic net charge over the substituting atom bonded to of C3' was not associated with antiHIV activity since there are inactive compounds with a similar charge. For example, the net charge on the fluorine atom of F-ddT, 3 (which is active, -0.1534), and the net charge on the carbon atom of the CN group of CN-ddT, 4 (also inactive, -0.1451) are similar. Some previous research [23,27] has concluded that very planar geometries are obtained for the furanose ring when semiempirical methods are used (0 m = 15-30°). In this research, the AZT molecule, 1, is an exception since the geometries for the furanose ring (0m ~ 18°) obtained by MM calculations (CrtEM-X) were different from those obtained by semiempirical methods (AM1) which produced more planar furanose rings (0m = 20°).

Acknowledgements Thanks are due to Junta de Castilla y Leon for financial support and to Pedro A. Fuertes for the English translation.

References Table 16 Calculated atomic net charges (e-) in 3'-substituted thymidines for the global minimum localized by MM Atom

3'-OH

3'-F

3'-NH 2

3'-N 3

3'-CN

C2 NI CI' C2' C3' C4' O1' C5' O5' X(C3 t)

0.4078 -0.2818 0.1762 -0.2431 0.0105 -0.0026 -0.2536 -0.0178 -0.3419 -0.3116

0.4059 -0.2840 0.1778 -0.2397 0.0410 0.0018 -0.2517 -0.0268 -0.3416 -0.1697

0.4055 -0.2809 0.1797 -0.2496 -0.2496 0.0362 -0.2575 -0.0175 -0.3413 -0.3491 0.1404 0.1590

0.4054 -0.2895 0.1809 -0.2140 -0.0486 0.0243 -0.2708 -0.0222 -0.3179 -0.2657 0.2085 -0.0375

0.4062 -0.2807 0.1808 -0.2005 -0.0289 0.0418 -0.2564 -0.0278 -0.3400 -0.1556 -0.0293

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