Synthesis, CoMFA analysis, and receptor docking of 3,5-diacyl-2,4- dialkylpyridine derivatives as selective A3 adenosine receptor antagonists

706

J . Med. Chem. 1999, 42, 706-721

Syn th esis, CoMF A An a lysis, a n d Recep tor Dock in g of

3,5-Dia cyl-2,4-Dia lk ylp yr id in e Der iva tives a s Selective A₃Ad en osin e Recep tor

An ta gon ists

An-Hu Li, Stefano Moro, Nancy Forsyth, Neli Melman, Xiao-duo J i, and Kenneth A. J acobson*

Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes, Digestive and Kidney

Diseases, National Institutes of Health, Bethesda, Maryland 20892-0810

Received September 29, 1998

3,5-Diacyl-2,4-dialkyl-6-phenylpyridine derivatives have been found to be selective antagonists

at both human and rat A₃adenosine receptors (Li et al. J . Med. Chem. 1998, 41, 3186-3201).

In the present study, ring-constrained, fluoro, hydroxy, and other derivatives in this series

have been synthesized and tested for affinity at adenosine receptors in radioligand binding

assays. K_ivalues at recombinant human and rat A₃adenosine receptors were determined using

[

¹²⁵I]AB-MECA (N⁶-(4-amino-3-iodobenzyl)-5′-N-methylcarbamoyladenosine). Selectivity for A₃

adenosine receptors was determined vs radioligand binding at rat brain A₁and A_2Areceptors,

and structure-activity relationships at various positions of the pyridine ring (the 3- and 5-acyl

substituents and the 2- and 4-alkyl substituents) were probed. At the 5-position inclusion of a

â-fluoroethyl (7) or a γ-fluoropropyl ester (26) was favorable for human A₃receptor affinity,

resulting in K_ivalues of 4.2 and 9.7 nM, respectively, while the pentafluoropropyl analogue

was clearly less potent at human A₃receptors. At the 2-, 3-, and 4-positions, fluoro or hydroxy

substitution failed to enhance potency and selectivity at human A₃receptors. Several analogues

were nearly equipotent at rat and human A₃receptors. To further define the pharmacophore

conformationally, a lactam, a lactone, and thiolactones were tested in adenosine receptor

binding. The most potent analogue in this group was compound 34, in which a thiolactone was

formed between 3- and 4-positions and which had a K_ivalue of 248 nM at human A₃receptors.

Using affinity data and a general pharmacophore model for A₃adenosine receptor antagonists

recently proposed, we applied comparative molecular field analysis (CoMFA) to obtain a three-

dimensional quantitative structure-activity relationship for pyridine derivatives, having good

predictability (r²

) 0.873) for compounds in the test set. A rhodopsin-based model of the

pred

human A₃receptor was built, and the pyridine reference ligand 2,3,4,5-tetraethyl-6-phenyl-

pyridine-3-thiocarboxylate-5-carboxylate (MRS 1476) was docked in the putative ligand binding

site. Interactions between receptor transmembrane domains and the steric and the electrostatic

contour plots obtained from the CoMFA analysis were analyzed.

In tr od u ction

diacylpyridines, both previously reported¹⁶and newly

synthesized, through oxidation of the corresponding 1,4-

dihydropyridines, as antagonists of high selectivity for

human A₃receptors. Moderate selectivity for rat A₃

receptors was also present. Fluoro, hydroxy, and other

derivatives in this series, which otherwise have a highly

hydrophobic character, have been synthesized and

tested for affinity at adenosine receptors in radioligand

binding assays. Sterically constrained (bicyclic) ana-

logues were designed and prepared for purposes of

conformational analysis. New pyridine derivatives with

higher affinity and selectivity for A₃receptors and

potentially improved pharmacodynamic properties have

been obtained.

Selective antagonists belonging to a variety of chemi-

cal classes have been reported for adenosine A₃recep-

tors.¹Such antagonists are sought as potential antiin-

flammatory, antiasthmatic, or antiischemic agents.^2-5

The ligand requirements of the A₃receptor are unique

within the class of adenosine receptors,⁶and there are

also pronounced species differences in antagonist

affinity.^7-9Xanthines, such as caffeine and theophylline,

which have provided versatile leads for antagonists at

the other adenosine receptor subtypes, are much less

potent at the A₃receptor; thus, it has been necessary

to use library screening to identify novel leads (Figure

1). 1,4-Dihydropyridines (e.g., MRS 1191, 1),^10-12tri-

azoloquinazolines (e.g., MRS 1220, 2),¹³flavonoids,¹⁴

a

A three-dimensional quantitative structure-activity

relationship (3D-QSAR) for pyridines as human A₃

receptor antagonists was obtained by applying com-

parative molecular field analysis (CoMFA)^17,18and a

triazolonaphthyridine (L-249313, 3),¹⁵pyridines (e.g.,

MRS 1523, 4),¹⁶and isoquinolines (e.g. VUF 8504, 5)¹⁷

have been reported as A₃receptor selective antagonists.

In the present study, we have further explored

structure-activity relationships (SARs) for the 1,3-

previously reported general pharmacophore model.¹⁹

A

model of the human A₃receptor, containing the docked

pyridine reference ligand 2,3,4,5-tetraethyl-6-phenyl-

pyridine-3-thiocarboxylate-5-carboxylate (MRS 1476),¹⁶

was built and analyzed to help interpret these results.

In particular, the steric and the electrostatic contour

* Correspondence to Dr. K. A. J acobson; Chief, Molecular Recog-

nition Section; LBC, NIDDK, NIH; Bldg. 8A, Rm. B1A-19; Bethesda,

MD 20892-0810. Tel: (301) 496-9024. Fax: (301) 480-8422. E-mail:

kajacobs@helix.nih.gov.

10.1021/jm980550w This article not subject to U.S. Copyright. Published 1999 by the American Chemical Society

Published on Web 02/09/1999

Synthesis of 3,5-diacyl-2,4-dialkylpyridines

J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 4 707

Sch em e 1. Synthesis of Substituted

1,4-Dihydropyridines Using the Hantzsch Reaction and

Oxidation to Pyridine Derivatives

Sch em e 2. Synthesis of Cyclic Derivatives 32-35

F igu r e 1. Structures of key A₃adenosine receptor selective

antagonists and agonists. K_ivalues in µM were reported in

refs 12-17.

plots, obtained from the CoMFA analysis of 41 pyridine

derivatives, are included inside the ligand binding

cavity.

Resu lts

Syn th esis. Novel 3,5-diacylpyridine derivatives (7-

35) were synthesized and tested for affinity in radio-

ligand binding assays^20-22at adenosine receptors as

shown in Tables 1 and 2. As in the previous studies,^11,16

pyridine analogues were prepared from the correspond-

ing dihydropyridines (Scheme 1) as shown.

The Hantzsch condensation, Scheme 1, which in-

volved condensing three components, a â-enaminoester

(36), an aldehyde (37), and a â-ketoester (38), was used

for the 1,4-dihydropyridines (39). The corresponding

pyridines were prepared through oxidation of the dihy-

dropyridines using tetrachloroquinone (40). Monofluoro-

pyridines (7, 14, 19, 21, 24, and 26) were prepared by

fluorinating corresponding hydroxyl-pyridines with DAST

(Schemes 6, 7, and 8 in Supporting Information), and

the synthesis of ring-constrained pyridine analogues is

shown in Scheme 2. A lactone pyridine, 32, thiolactone

pyridines, 34 and 35, and a lactam pyridine, 33b, were

prepared through the deprotection-intramolecular trans-

esterification of the precursor pyridine derivatives. A

two-step route through acylation of Meldrum’s acid

(43),²³followed by opening of the ring with an alcohol

or thiol and decarboxylation was adopted for preparing

â-ketoesters or â-ketothioesters 38 (Scheme 3), needed

as precursors for the 1,4-dihydropyridines. Hydroxy-

lated â-ketoester 46 was prepared by a transesterifica-

tion reaction. Acid chlorides 42b and 42c were prepared

as shown in Scheme 4. Thiols 50, 51, and 52 were

prepared from selective protection of hydroxylated thiols

(Scheme 4). Synthesis of protected aldehydes (37c-g)

708 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 4

Li et al.

Synthesis of 3,5-diacyl-2,4-dialkylpyridines

J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 4 709

Ta ble 2. Affinities of

4-Phenylethynyl-6-phenyl-1,4-dihydropyridine Derivatives in

Radioligand Binding Assays at Rat A₃Receptors

a

compd

K_i(nM) rA₃

1420 ( 190

rA₁/rA₃

28

rA₃/hA₃

45

1

(MRS 1191)

4

113 ( 12

410 ( 48

140

6.0

21

(MRS 1523)

6

(MRS 1476)

>100

7

600 ( 116

183 ( 33

260 ( 24

495 ( 29

156 ( 16

900 ( 76

512 ( 39

4260 ( 680

2580 ( 250

19

42

76

17

81

140

22

<1

27

1.9

15

53

2.1

10

14

20

21

24

26

31

34

9.0

12

>23

6.4

a

Displacement of specific [¹²⁵I]AB-MECA binding at rat A₃

receptors stably expressed in CHO cells^16,22(n ) 3-5).

Sch em e 3. Synthesis of â-Keto Esters

is summarized in Scheme 5. â-Enaminoesters (36) were

made by refluxing the corresponding â-ketoesters (38i-m

and 46) and ammonium acetate in ethanol (Scheme 9

in Supporting Information). Yields and characterization

of the pyridine derivatives tested as A₃receptor antago-

nists are described in Table 3.

P h a r m a cology. The pyridine analogues previously

reported as A₃receptor selective antagonists, being

devoid of functionality on the substituent alkyl groups,

were highly hydrophobic. In an attempt to probe the

electronic requirements of binding and also to introduce

potentially water-solubilizing groups, we have intro-

duced various functional groups in the analogues.

710 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 4

Li et al.

Ta ble 3. Yields and Analysis of Pyridine Derivatives

Sch em e 4. Synthesis of Protected Intermediate Acid

Chlorides and Thiols

no.

formula

₂₁H₂₄FNO₃S

₂₉H₂₈N₂O₅S‚0.25H₂O

₃₀H₃₀N₂O₅S

₂₆H₃₄ClNO₃S

₂₃H₂₉NO₄S

₃₀H₃₅NO₄S

₂₃H₂₈FNO₃S

₂₄H₂₉NO₄S₂‚0.55C₇H₈

₂₃H₂₉NO₄S

₂₈H₃₇NO₅S‚0.4H₂O

₂₇H₃₅NO₅S

₂₃H₂₈FNO₃S‚0.8C₇H₈

₂₃H₂₆F₃NO₃S

₂₄H₃₀FNO₃S‚1.1C₇H₈

₂₂H₂₇NO₄S

₂₄H₂₉NO₄S₂‚0.1C₇H₈

₂₃H₂₈FNO₃S‚0.8C₇H₈

₂₃H₂₉NO₄S‚0.2C₇H₈

₂₃H₂₈FNO₃S

₂₃H₂₄F₅NO₃S

₂₃H₂₈FNO₃S

₂₃H₂₈FNO₃S‚0.15C₇H₈

₂₃H₂₈FNO₃S

₂₄H₃₂INO₃S‚0.73I₂

₂₀H₂₁NO₄

₂₀H₂₄N₂O₃

₂₀H₂₂N₂O₃‚2C₇H₈‚H₂O

₂₀H₂₁NO₃S‚1.5H₂O

₂₀H₂₁NO₃S‚1.0H₂O

analysis

yield (%)

7

8

9

C

HRMS^e

C, H, N

H, N, C^a

C, H, N

HRMS^c

C, H, N

HRMS^d

C, H, N

C, H, N^b

C, H, N

50

56

72

56

57

54

88

37

52

8

22

73

16

77

40

68

83

86

41

66

80

85

92

22

45

55

24

98

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33a

33b

34

35

Sch em e 5. Synthesis of Protected Intermediate

Aldehydes

a

Elemental analysis for compound 22, C: calcd, 65.81; found,

b

67.67. Elemental analysis for compound 33, N: d, 5.18; found,

4.18. ^cThis compound was shown to be pure on analytic TLC (silica

gel 60; 250 µm; EtOAc-petroleum ether ) 10:90 (v/v)): compound

26, R_f) 0.49. EI calcd for C₂₃H₂₈FNO₃S (M⁺): 417.1774; found,

d

417.1780. This compound was shown to be pure on analytic TLC

(silica gel 60; 250 µm; EtOAc-petroleum ether ) 10:90 (v/v)):

compound 30, R_f) 0.62. EI calcd for C₁₂H₂₃FNO₃(M⁺- SEt):

e

356.1662; found, 356.1667. This compound was shown to be pure

on analytic TLC (silica gel 60; 250 µm; EtOAc-petroleum ether

) 10:90 (v/v)): compound 7, EI calcd for C₂₁H₂₄FNO₃S (M⁺):

389.1461; found, 389.1423.

more potent than the 4-ethyl analogue, 6, at A₁recep-

tors and less potent at A₃receptors. The next higher

phthaloylamino homologue, 9, was less potent at all

subtypes. The effects on receptor affinity of homologa-

tion of the alkyl groups of compound 6 were explored.

Enhanced human A₃affinity was observed for the

5-propyl ester, 10, but not for the 4-propyl derivative,

4, or for the 3-hexylthio ester-6-(3-chlorophenyl) ana-

logue, 11. Nevertheless, additional analogues were

based on the structure of 4 rather than 10 due to its

noted high affinity at rat A₃receptors.¹⁶

The 2-ethyl group of compound 4 was modified in the

form of â-hydroxyethyl, benzyloxyethyl, and acetylthio-

ethyl substituents in compounds 12-15. The hydroxy

group of 12 greatly diminished affinity at human A₃

receptors, while a fluoro group at the same position (14)

greatly increased affinity. The acetyl thioester of 15 was

tolerated better in A₃receptor binding than the bulky

benzyloxy group of 13.

Compound 16, containing a â-hydroxyethylthio ester

at the 3-position, was 3-fold less potent at A₃receptors

than the corresponding 3-ethylthio ester, 4. The pres-

ence of a bulky tetrahydropyranyl group and a dioxolane

group in 17 and 18, respectively, further decreased

affinity at adenosine receptors. The introduction of

fluorine at the 3-thioester group in compounds 19-21

failed to enhance potency and selectivity at human A₃

Compound 6, a tetraethyl analogue, which was among

the most potent at human A₃receptors in the previous

study (Table 1), was substituted with a â-fluoroethyl

ester at the 5-position resulting in compound 7, which

displayed a 5-fold enhancement of affinity at human A₃

receptors (K_ivalue of 4.2 nM). At the 4-position, steric

bulk was introduced. A phthaloylamino analogue, 8, was

Synthesis of 3,5-diacyl-2,4-dialkylpyridines

J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 4 711

Ta ble 4. Observed and Predicted Receptor Binding Affinity

Values of the Compounds Forming the A₃Test Set

receptors, although the â-trifluoroethyl analogue, 20,

was comparable in affinity to 4. Introduction of fluoro

(24), hydroxy (22, 25), or thioester (23) groups at the

4-position failed to enhance potency/selectivity.

compd

pK_i, mM (obsvd)

pK_i, mM (pred)^a

10

24

27

32

35

8.08

7.22

6.35

<4.5

<4.0

7.92

7.45

6.55

4.35

3.98

At the 5-position propyl ester, a monofluoro substitu-

tion in compound 26 was favorable for A₃affinity

(similar to compound 4 above), while the pentafluoro

analogue, 27, was clearly less potent at A₃receptors.

a

Values predicted by the CoMFA model.

Fluoro substitution of the 6-phenyl group at ortho

(28), meta (29), and para (30) positions produced only

minor changes in the adenosine receptor affinity, while

quaternization at the 1-position in the methyl pyri-

dinium salt, 31, resulted in moderate selectivity for

human A₃receptors, although affinity was greatly

diminished.

Ta ble 5. Statistic of the Calibration CoMFA Model

human A₃

41

number of compounds

principal components^a

3

b

r²

0.667

0.968

141.67

<0.001

0.931

0.571

0.429

0.185

0.022

cv

r²

c

F_test

To further define the pharmacophore conformation-

ally, various ring-constrained analogues, 32-35, were

tested in adenosine receptor binding. The most potent

analogue in this group was compound 34 (K_ivalue of

248 nM at human A₃receptors), in which a six-

membered thiolactone was formed between the 3- and

4-positions. The homologous compound containing an

ester instead of thioester, 32, and an analogue contain-

ing a thiolactone formed between 2- and 3-substituents,

35, were inactive at human A₃receptors. A seven-

membered lactam, 33b, was 10-fold less potent than 34

in binding to human A₃receptors. An acyclic amide

derivative, 33a , to which 33b may be compared, was

prepared and found to be inactive at human A₃recep-

tors.

p value

d

r²

bs

steric contribution

electrostatic contribution

SEP^e

std dev^f

a

b

Minimum

s

) 2.0. Standard error of prediction (cross-

validated) ) (PRESS/(n - c - l))^1/2, n ) number of rows, c )

number of components. ^cRatio of r²explained to unexplained )

r²/(l - r²). r²_bs) r²after bootstrapping. ^eCross-validated r²after

d

leave-one-out procedure: r²_cv) (SD - PRESS)/SD, SD ) Y_actual

-

Y

_mean)². PRESS ) ∑(Y_predicted- Y_actual). For further explaination

of these mathematical formulas, see ref 40. ^fThe std dev column

belongs with the bootstrapping r².

Binding affinities at recombinant rat A₃receptors

indicated that compounds 10, 14, and 21 were g40-fold

subtype selective (Table 2). The N-methyl pyridinium

derivative, 31, although weaker in binding, was mod-

erately selective for rat A₃vs A₁receptors. The ratio of

K_ivalues at rat A₃vs human A₃receptors varied greatly,

from <1 (compound 14) to 140 (compound 7), indicating

specific interspecies differences in pyridine recognition.

Compound 21 was nearly equipotent at rat and human

A₃receptors.

Molecu la r Mod elin g. 1. CoMF A. CoMFA 3D-QSAR

methodology was applied to the data from A₃binding

assay as a means of identifying the structural features

of pyridine derivatives (present study and ref 16)

responsible for affinity. A CoMFA analysis of flavonoid

derivatives as A₃receptor antagonists has already been

reported.²⁴

F igu r e 2. Fitted vs measured pK_ivalues for the CoMFA

analysis of human A₃training set. The model was derived

using three principal components yielding a cross-validated

r²value of 0.667: (9) bulky substituents at the 4-position of

the pyridine ring; (b) fluoro substituents at the 5-position ester

group.

PLS³⁹was used in conjunction with cross-validation

to obtain the optimal number of components to be used

in the subsequent analyses. PLS analysis based on least-

electrostatic contributions ) 0.429. These parameters

are provided in Table 5.

squares fit gave a correlation with a cross-validated r²

cv

The CoMFA-derived QSAR of pyridine derivatives

exhibited a good cross-validated correlation, indicating

that it was highly predictive. Cross-validation provides

information concerning the predictive ability of the

QSAR data set by minimizing the occurrence of chance

correlations in the QSAR model. The high bootstrapped

r²value and a small standard deviation suggest a high

degree of confidence in the analysis. Fitted vs measured

pK_ivalues for the CoMFA analysis of the human A₃

training set are shown in Figure 2.

of 0.667, with the maximum number of components set

equal to 3 (maximum number of components set equal

to 2, 4, or 5 gave unreliable cross-validated r²_cve 0.500)

and the cross-validation groups set equal to the number

of observations.⁴⁰The non-cross-validated PLS analysis

was repeated with the optimum number of components,

as determined by the cross-validated analysis, to give

an r²of 0.968. To obtain statistical confidence limits,

cv

the non-cross-validated analysis was repeated with 10

bootstrap groups, which yielded an r²of 0.931 (optimum

number of components was 3), SEP ) 0.185, standard

deviation ) 0.022, steric contribution ) 0.571, and

Compounds 10, 24, and 27 (test set) were used to

evaluate the predictive power of this CoMFA model. As

in the calibration step, a good predictive ability with

712 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 4

Li et al.

Ta ble 6. Observed and Calculated Receptor Binding Affinity Values of the Compounds Forming the A₃Training Sets

Compounds Synthesized in the Present Study

compd

pK_i, µM (obsvd)

pK_i, µM (calcd)

compd

pK_i, µM (obsvd)

pK_i, µM (calcd)

4

6

7

7.72

7.67

8.37

6.61

8.08

6.66

6.96

6.71

7.29

7.26

7.74

7.08

7.92

7.78

8.04

6.73

7.92

7.02

6.86

6.48

7.16

7.30

7.62

7.19

22

23

24

25

26

27

28

29

30

31

33b

34

6.58

6.91

7.22

6.77

8.01

6.35

7.64

7.54

7.51

5.59

5.70

7.60

7.04

6.78

7.45

6.88

7.82

6.85

7.52

7.55

7.49

5.95

6.05

7.75

8

10

11

13

15

16

19

20

21

Compounds from Ref 16

compd

R₂

R₃

R₄

R₅

R₆

pK_i, µM (obsvd)

pK_i, µM (calcd)

65

66

67

68

69

70

71

72

73

74

75

76

77

78

79

80

81

82

83

84

85

86

87

CH₃

CH₂CH₃

OCH₂CH₃

OCH₂CH₂CH₃

CH₃

CH₂CH₃

CH₂Ph

CH₂CH₃

CH₂Ph

Ph

5.35

6.67

6.75

7.37

6.78

6.71

5.58

6.11

5.55

5.56

5.62

5.41

6.92

5.89

6.73

7.87

5.47

7.80

8.10

7.48

7.77

7.46

6.84

5.87

6.85

6.86

7.22

6.65

6.79

6.03

6.41

5.63

5.80

5.48

5.47

6.92

5.80

6.88

7.97

5.88

7.80

8.10

7.63

7.81

7.59

6.99

OCH₂CH₃

SCH₂CH₃

SCH₂CH₂OCH₃

SCH₂CH₃

OCH₂CH₃

OH

SCH₂CH₃

SCH₂CH₂CH₃

SCH₂CH₃

CH₂CH₃

CH₂CH₂CH₃

CH₂CH₃

CH(OCH₃)₂

Ph-CHdCH- (trans)

Ph-CtC-

CH₃

CH₂CH₃

CH₂Ph

cyclobutyl

cyclopentyl

Ph

CH₂CH₃

CH₂CH₂OH

CH₂CH₃

CH₂CH₂CH₃

CH₂CH₃

Ph

3-Cl-Ph

cyclopentyl

Ph

3-Cl-Ph

Ph

CH₂CH₂CH₃

(CH₂)₂OCH₃

(CH₂)₃CH₃

cyclobutyl

Ph

an r²

) 0.873 for the compounds in the test set was

substituents in this position (see compounds 6, 68, and

84, Table 6) enhance affinity. The two ester groups at

3- and 5-positions are also surrounded by green poly-

hedra, suggesting that the presence of alkyl substituents

increase A₃receptor affinity (see compounds 10 and 21,

Table 1). Another important region of bulk tolerance is

found around the meta position of the phenyl ring at

the C-6 position of pyridine. In fact, a m-chloro deriva-

tive 80 displayed a favorable affinity. In contrast, the

region of space around the C-4 position of the pyridine

moiety is contained within a yellow polyhedron, sug-

gesting that bulky substituents are not tolerated by the

receptor at this position. Both styryl and phenylpro-

pargyl derivatives (see compounds 73 and 74, Table 6)

are among the less active compounds. Substituents of

high electron density were also predicted to be accepted

in the side chain of both ester groups at the 3- and

5-positions (red polyhedron in Figure 3B). Accordingly,

at the 5-position a γ-fluoropropyl or â-fluoroethyl ester

was favorable for A₃affinity, and at the 3-position fluoro

substitution was tolerated (see compounds 19 and 20,

Table 1).

pred

obtained. Table 4 shows that the affinities of all the

examined compounds were predicted within 0.25 log

unit across a range of 2.00 log units (Table 6). An

additional test (compounds 32 and 35) was used to

better explore the predictability of our CoMFA model.

The model predicted accurately that these cyclic deriva-

tives would be very weak antagonists at the human A₃

receptor.

The coefficients corresponding to each sampled field

point in the resulting correlation equation were graphi-

cally contoured. Contours corresponding to the steric

(green and yellow) and electrostatic (blue and red) fields

are plotted together with compound 6 in Figure 3A and

3B, respectively. The polyhedra describe the regions of

space where the steric and the electrostatic fields are

predicted by the CoMFA model to have the greatest

effect on binding affinity. The yellow and the blue

polyhedra correspond to regions of the field that are

predicted to decrease the A₃receptor affinity, whereas

the green and the red regions are predicted to increase

binding affinity.

As shown in Figure 3A, a green contour around the

C-2 position of pyridine moiety suggests that bulky

2. MRS 1476 Bin d in g Site on th e Hu m a n A₃

Recep tor . We have recently developed a model of the

Synthesis of 3,5-diacyl-2,4-dialkylpyridines

J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 4 713

helical axes is roughly 10 Å, consistent with a common

interhelical contact distance.¹⁹The interhelical angles,

measured between the principal axes of adjacent helices,

range between -150 to 170° for antiparallel, and 10 to

25° for the parallel helices. This is typical of a 3-4 type

helix-helix contact associated with optimal interactions

between nearly parallel aligned helices.¹⁹After cross-

docking, each helix maintained almost the same position

and tilt angle found in the published rhodopsin 2D

electron density map.^27,28Moreover, in the cross-docked

model, TM3, TM4, TM5, TM6, and TM7 were rotated

clockwise 10°, 0°, 10°, 5°, and 10°, respectively, about

their transmembrane axes with respect to the ligand-

free receptor model. The energy of the cross-docked MRS

1476-receptor complex structure is about 65 kcal/mol

lower with respect to the starting structure.

As previously reported, the recognition of MRS 1476

(6)^16,19seems to occur in the upper region of the helical

bundle. The steric and the electrostatic contour plots

obtained from the CoMFA analysis without cross-

validation have been included in the MRS 1476 binding

site of the human A₃receptor (Figure 3C). This repre-

sentation emphasizes the putative microscopic interac-

tions between ligand and receptor. The pyridine moiety

of 6 is most favorably oriented perpendicular to the

plane of the lipid bilayer with the carbonyl of the 5-ester

group in proximity to Ser275 (TM7) and the carbonyl

of the 3-ester group close to Asn250 (TM6). Two

important hydrophilic interactions are hydrogen bonds

between the two ester groups and these two polar amino

acids. The TM7 backbone is close to the 4-position of

the pyridine ring. A strong steric control around the

4-position of the pyridine structure is suggested. Two

hydrophobic pockets are likely present around the

2-position, where an alkyl side chain would bind (Leu90,

TM3 and Phe182, TM5), and around the 6-position

where the phenyl ring would bind (Leu90, TM3 and

Ile186, TM5).

Discu ssion

We have identified subtle structural modifications of

pyridines which lead to enhanced potency or selectivity

at A₃receptors. Fluoro derivatives, 7 and 26, are the

most potent pyridine derivatives in the present study.

The inclusion of hydroxyl groups for potentially enhanc-

ing water solubility failed to increase A₃receptor

affinity.

Furthermore, the present set of analogues has allowed

us to more effectively model the pyridine pharmaco-

phore and its fit in the putative binding site of the

receptor. Recently, we have published a general phar-

macophore map for human A₃receptor antagonists¹⁹

derived by a combination of ab initio quantum mechan-

ical calculations, electrostatic potential map comparison,

and the steric and electrostatic alignment (SEAL)

method. This general pharmacophore features the 3-po-

sition carbonyl group in the “down” position,¹⁶which

was confirmed experimentally in the present study

through the synthesis of ring-constrained analogues.

The conformationally constrained lactam 33b, in which

the 3-carbonyl was “down”, bound to the A₃receptor

with micromolar affinity, while the acyclic amide ana-

logue 33a was inactive. The thiolactone 34, in which

the 3-carbonyl was “down”, bound to the A₃receptor

F igu r e 3. (A, top) CoMFA steric STDEV*COEFF contour plot

from the analysis based on the A₃receptor 3D-QSAR without

cross-validation. Compound 6 shown inside the field. Favoring

activity: green, bulky group (contribution level 80%); yellow,

less bulky. (B, middle) CoMFA electrostatic STDEV*COEFF

contour plots from the analysis based on the A₃receptor 3D-

QSAR without cross-validation. Compound 6 shown inside the

field. Favoring activity: blue, positive charge (contribution

level 70%); red, negative charge. (C, bottom) Side view of the

6-A₃complex model. The side chains of the important residues

in proximity (e5 Å) to the docked pyridine molecule are

highlighted and labeleδ: Leu90 (TM3), Phe182 (TM5), Ser242

(TM6), Ser247 (TM6), Asn250 (TM6), Ser271 (TM7), His272

(TM7), Ser275 (TM7). The steric and the electrostatic contour

plots, obtained from the CoMFA analysis, are included into

ligand binding cavity.

human A₃receptor, using rhodopsin as a template, by

adapting a facile method to simulate the reorganization

of the native receptor structure induced by the ligand

coordination (cross-docking procedure).²⁵Details of the

model building are given in the Experimental Section.

Like other G protein-coupled receptor models,²⁶the

length of the membrane-spanning region is about 40 Å.

The interhelical distance between the pairs of adjacent

714 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 4

Li et al.

sulfonate (PPTS), diethylaminosulfur trifluoride (DAST), so-

dium acetate, hydrazine, and sodium thiomethoxide were

purchased from Aldrich (St. Louis, MO). 5-(2-Hydroxylethyl)

2-ethyl-4-ethyl-3-(ethylsulfanylcarbonyl)-6-phenylpyridine-5-

carboxylate (59) and MRS1523 (4) were prepared by our

previous method.¹⁶All other materials were obtained from

commercial sources.

Proton nuclear magnetic resonance spectroscopy was per-

formed on a Varian GEMINI-300 spectrometer, and all spectra

were obtained in CDCl₃. Chemical shifts (δ) relative to

tetramethylsilane are given. Chemical-ionization (CI) mass

spectrometry was performed with a Finnigan 4600 mass

spectrometer, and electron-impact (EI) mass spectrometry with

a VG7070F mass spectrometer at 6 kV. Elemental analysis

was performed either by Atlantic Microlab Inc. (Norcross, GA)

or by Galbraith Laboratories, Inc. (Knoxville, TN).

Tw o-Step P r ocess for P r ep a r a tion of P yr id in e De-

r iva tives: Ha n tzsch Con d en sa tion a n d Su bsequ en t Oxi-

d a tion (Sch em e 1). Ha n tzsch Con d en sa tion : Equimolar

amounts (0.5-1.0 mmol) of the appropriate â-enaminoester

(36), aldehyde (37), and â-ketoester (38) were dissolved in 2-5

mL of absolute ethanol (for preparing compound 27, 2,2,3,3,3-

pentafluoropropanol was used instead of ethanol). The mixture

was sealed in a Pyrex tube and heated, with stirring, to 80 °C

for 18-24 h. After the mixture cooled to room temperature,

the solvent was evaporated, and the residue was purified by

preparative TLC (silica 60; 1000 or 2000 mm; Analtech,

Newark, DE; petroleum ether-ethyl acetate (4:1-9:1)). These

1,4-dihydropyridines (39) were directly used in the next step

without further purification.

Oxid a tion of 1,4-Dih yd r op yr id in es in to Cor r esp on d -

in g P yr id in e Der iva tives. Equimolar amounts of the 1,4-

dihydropyridines (39, ∼0.2 mmol) and tetrachloro-1,4-benzo-

quinone (40) in THF (2-4 mL) were mixed and refluxed

overnight. After the mixture cooled to room temperature, the

solvent was removed, and the residue was purified by prepara-

tive TLC (silica 60; 1000 mm; Analtech, Newark, DE; petro-

leum ether-ethyl acetate (9:1-19:1)) to give the desired

products.

5-Eth yl 2,4-Dieth yl-3-(eth ylsu lfa n ylca r bon yl)-6-p h en -

ylp yr id in e-5-ca r boxyla te (7). ¹H NMR δ: 1.24 (t, J ) 7.8

Hz, 3 H), 1.34 (t, J ) 7.8 Hz, 3 H), 1.41 (t, J ) 7.8 Hz, 3 H),

2.73 (q, J ) 7.8 Hz, 2 H), 2.87 (q, J ) 7.8 Hz, 2 H), 3.14 (q, J

) 7.8 Hz, 2 H), 4.23 (m, 2 H), 4.31 (t, J ) 4.8 Hz, 1 H), 4.39 (t,

J ) 4.8 Hz, 1 H), 7.41-7.44 (m, 3 H), 7.59-7.61 (m, 2 H). MS

(CI/NH₃): m/z 390 (M⁺+ 1, base). MS (EI): m/z 389 (M⁺), 360

(M⁺- Et), 329 (MH⁺- EtS, base), 300 (M⁺- COSEt).

5-Eth yl 2-Eth yl-4-(2-p h th a lim id oeth yl)-3-(eth ylsu lfa n -

ylca r bon yl)-6-p h en ylp yr id in e-5-ca r boxyla te (8). ¹H NMR

δ: 1.02 (t, J ) 7.8 Hz, 3 H), 1.32 (t, J ) 7.8 Hz, 3 H), 1.46 (t,

J ) 7.8 Hz, 3 H), 2.88 (q, J ) 7.8 Hz, 2 H), 3.09 (t, J ) 6.6 Hz,

2H), 3.22 (q, J ) 7.8 Hz, 2 H), 3.98 (t, J ) 6.9 Hz, 2 H), 4.24

(q, J ) 7.8 Hz, 2 H), 7.41-7.44 (m, 3 H), 7.62 (m, 2 H), 7.70-

7.72 (m, 2 H), 7.82-7.85 (m, 2 H). MS (CI/NH₃): m/z 517 (M⁺

+ 1, base).

5-Eth yl 2-Eth yl-4-(3-p h th a lim id op r op yl)-3-(eth ylsu l-

fa n ylca r bon yl)-6-p h en ylp yr id in e-5-ca r boxyla te (9). ¹H

NMR δ: 0.90 (t, J ) 7.8 Hz, 3 H), 1.31 (t, J ) 7.8 Hz, 3 H),

1.33 (t, J ) 7.8 Hz, 3 H), 2.03 (m, 2 H), 2.71 (m, 2 H), 2.84 (q,

J ) 7.8 Hz, 2 H), 2.98 (q, J ) 7.8 Hz, 2 H), 3.74 (t, J ) 6.9 Hz,

2 H), 3.98 (t, J ) 7.8 Hz, 2 H), 7.38-7.41 (m, 3 H), 7.54-7.57

(m, 2 H), 7.72-7.74 (m, 2 H), 7.86-7.88 (m, 2 H). MS (CI/

NH₃): m/z 546 (M⁺+ 2).

with moderate affinity, while another thiolactone, 35,

in which the 3-carbonyl was “up”, was inactive.

Based on the proposed pharmacophore map, we

hypothesize that the receptor binding properties of

different A₃antagonist derivatives are due to recogni-

tion at a common region inside the receptor binding site

and, consequently, a common electrostatic potential

profile. To test this hypothesis computationally, we built

a model of the human A₃receptor, into which the

pyridine MRS 1476 (6) was docked. Complementary

interactions between the ligand and the receptor were

evident, such as hydrogen bonding with Ser247 (TM7),

an interaction with Asn250 (TM6), and the hydrophobic

interaction with the region around Leu90 (TM3), Phe182

(TM5), and Ile186 (TM5). Perhaps the most critical

interactions were between the carbonyl of the 5-ester

group and Ser275 (TM7) and between the carbonyl of

the 3-ester group and Asp250 (TM6). In particular, we

have already reported¹⁹that the region of negative

electrostatic potential, that in the present set of pyr-

idines is located around the carbonyl of the ester at the

5-position, is present in all the known A₃antagonist

classes, such as adenines, xanthines, triazoloquinazo-

lines, flavonoids, thiazolopyridines, and 6-phenyl-1,4-

dihydropyridines. A strong steric control is suggested

around the 4-position of pyridine structures, at which

bulky substituents are not well tolerated, and which in

our model is very close to the pyridine 4-position. In

contrast, in the 1,4-dihydropyridines^10-12bulky substit-

uents at the 4-position enhance the A₃affinity. As we

have previously proposed,¹⁹changing the C₄-hybridiza-

tion from sp³to sp², corresponding to the transformation

of 1,4-dihydropyridine to pyridine, would change the

C₅-C₄-R₄angle from 68.1° to 0.2°. In this way, the 1,4-

dihydropyridine 4-position substituent is positioned

between TM6 and TM7. Moreover, as presented in this

paper, the introduction of polar substituents, such as a

fluorine atom, in the side chain of both ester groups in

3- and 5-position increases the ligand affinity.

To better analyze the steric and the electrostatic

requirements of the A₃receptor binding cavity, we

decided to include the steric and the electrostatic

contour plots obtained from the CoMFA analysis inside

the MRS 1476 binding site. As shown in Figure 3C, the

proposed pharmacophore model fits very well within the

transmembrane binding cleft.

In conclusion, pyridine derivatives have served as

structural scaffolds for enhancement of selectivity at

human A₃receptors and as important modeling probes

for the study of the human A₃receptor binding site and

interspecies differences.

Exp er im en ta l Section

Syn th esis. Ma ter ia ls a n d In str u m en ta tion . 2-Mercap-

toethanol, 3-mercapto-1-propanol, 3-mercapto-1,2-propanediol,

2,2,2-trifluoroethanethiol, hexanethiol, ethanethiol, thionyl

chloride, benzyl alcohol, thiolacetic acid, â-propiolactone (47),

acrolein, acrylic acid, 1-propanol, 1,3-propanediol, 2,2,3,3,3-

pentafluoropropanol, ammonium acetate, ethyl benzoyl acetate

(45), 3,4-dihydro-2H-pyran, 3-amino-1-propanol, 1,4-butane-

diol, 4-aminobutyraldehyde diethylacetal (55), N-carbethoxy-

phthalimide (53), tetrachloro-1,4-benzoquinone (40), 2,2-

dimethyl-1,3-dioxane-4,6-dione (43), aldehydes (37, except for

37c-g), all acid chlorides (42, except 42b and 42c, obtained

by the reaction of the precursor acids with thionyl chloride),

pyridinium chlorochromate (PCC), pyridinium p-toluene-

5-P r op yl 2-Eth yl-4-eth yl-3-(h exylsu lfa n ylca r bon yl)-6-

1

(3-ch lor op h en yl)p yr id in e-5-ca r boxyla te (11). H NMR δ:

0.72 (t, J ) 7.8 Hz, 3 H), 0.92 (t, J ) 6.9 Hz, 3 H), 1.23 (t, J )

7.8 Hz, 3 H), 1.34 (t, J ) 7.8 Hz, 3 H), 1.31-1.36 (m, 4 H),

1.39-1.53 (m, 4 H), 1.71 (m, 2 H), 2.72 (q, J ) 7.8 Hz, 2 H),

2.86 (q, J ) 7.8 Hz, 2 H), 3.14 (t, J ) 6.9 Hz, 2 H), 4.04 (t, J

) 6.9 Hz, 2 H), 7.32-7.41 (m, 2 H), 7.49 (m, 1 H), 7.63 (s, 1

H). MS (CI/NH₃): m/z 476 (M⁺, base).

P r ep a r a tion of P yr id in e 12 by Deben zyla tion of Com -

p ou n d 13.⁵¹A mixture of a benzyloxy pyridine, 13 (81 mg,

0.16 mmol), boron trifluoride diethyl etherate (82 mg, 0.576

Synthesis of 3,5-diacyl-2,4-dialkylpyridines

J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 4 715

mmol), and ethanethiol (0.5 mL) was stirred at room temper-

ature overnight. The reaction mixture was poured into water

and extracted with ether (3 × 20 mL). The organic phases were

combined, washed with water, dried over MgSO₄, and evapo-

rated, leaving a crude material which was purified with

preparative TLC (silica 60; 1000 mm; Analtech, Newark, DE;

petroleum ether-ethyl acetate (4:1)) to give 38 mg of hydroxyl-

pyridine, 12 (yield: 57%).

5-P r op yl 2-(2-Hyd r oxyleth yl)-4-p r op yl-3-(eth ylsu lfa n -

ylcar bon yl)-6-ph en ylpyr idin e-5-car boxylate (12). ¹H NMR

δ: 0.65 (t, J ) 7.8 Hz, 3 H), 0.95 (t, J ) 7.8 Hz, 3 H), 1.40 (m,

2 H), 1.41 (t, J ) 7.8 Hz, 3 H), 1.62 (m, 2 H), 2.68 (m, 2 H),

3.07 (t, J ) 6.0 Hz, 2 H), 3.14 (q, J ) 7.8 Hz, 2 H), 3.99 (t, J

) 6.9 Hz, 2 H), 4.05 (t, J ) 6.0 Hz, 2 H), 7.42-7.45 (m, 3 H),

7.57-7.59 (m, 2 H). MS (CI/NH₃): m/z 416 (M⁺+ 1), 354 (M⁺

- SEt, base).

H), 7.21 (t, J ) 6.0 Hz, 1 H), 7.38 (m, 1 H), 7.48 (m, 1 H). MS

(CI/NH₃): m/z 418 (M⁺+ 1, base).

5-P r op yl 2-E t h yl-4-p r op yl-3-(et h ylsu lfa n ylca r b on yl)-

1

6-(m -flu or op h en yl)p yr id in e-5-ca r boxyla te (29). H NMR

δ: 0.71 (t, J ) 7.8 Hz, 3 H), 0.95 (t, J ) 7.8 Hz, 3 H), 1.34 (t,

J ) 7.8 Hz, 3 H), 1.42 (t, J ) 7.8 Hz, 3 H), 1.47 (m, 2 H), 1.64

(m, 2 H), 2.65 (m, 2 H), 2.86 (q, J ) 7.8 Hz, 2 H), 3.13 (q, J )

7.8 Hz, 2 H), 4.02 (t, J ) 6.9 Hz, 2 H), 7.10 (m, 1 H), 7.37 (m,

3 H). MS (CI/NH₃): m/z 418 (M⁺+ 1, base).

5-P r op yl 2-E t h yl-4-p r op yl-3-(et h ylsu lfa n ylca r b on yl)-

6-(p-flu or op h en yl)p yr id in e-5-ca r boxyla te (30). ¹H NMR

δ: 0.71 (t, J ) 7.8 Hz, 3 H), 0.95 (t, J ) 7.8 Hz, 3 H), 1.33 (t,

J ) 7.8 Hz, 3 H), 1.42 (t, J ) 7.8 Hz, 3 H), 1.44 (m, 2 H), 1.62

(m, 2 H), 2.65 (m, 2 H), 2.85 (q, J ) 7.8 Hz, 2 H), 3.13 (q, J )

7.8 Hz, 2 H), 4.01 (t, J ) 6.9 Hz, 2 H), 7.09-7.15 (m, 2 H),

7.58-7.63 (m, 2 H). MS (EI): m/z 416 (M⁺- 1), 356 (M⁺

-

SEt, base), 330 (M⁺- CO₂Pr).

5-P r op yl 2-(2-Ben zyloxylet h yl)-4-p r op yl-3-(et h ylsu l-

1

fa n ylca r bon yl)-6-p h en ylp yr id in e-5-ca r boxyla te (13). H

5-P r op yl 2-Eth yl-4-(2-tetr a h yd r op yr a n yloxyleth yl)-3-

(e t h ylsu lfa n ylca r b on yl)-6-p h e n ylp yr id in e -5-ca r b oxy-

NMR δ: 0.66 (t, J ) 7.8 Hz, 3 H), 0.95 (t, J ) 7.8 Hz, 3 H),

1.38 (t, J ) 7.8 Hz, 3 H), 1.41 (m, 2 H), 1.63 (m, 2 H), 2.67 (m,

2 H), 3.13 (q, J ) 7.8 Hz, 2 H), 3.20 (t, J ) 7.8 Hz, 2 H), 3.95

(t, J ) 6.9 Hz, 2 H), 3.97 (q, J ) 7.8 Hz, 2 H), 4.54 (s, 2 H),

7.25-7.31 (m, 5 H), 7.38-7.42 (m, 3 H), 7.54-7.58 (m, 2 H).

MS (CI/NH₃): m/z 506 (M⁺+ 1), 444 (M⁺- SEt).

5-Eth yl 2-(2-Acetylth ioeth yl)-4-p r op yl-3-(eth ylsu lfa n -

ylcar bon yl)-6-ph en ylpyr idin e-5-car boxylate (15). ¹H NMR

δ: 0.95 (t, J ) 6.9 Hz, 3 H), 1.01 (t, J ) 6.9 Hz, 3 H), 1.41 (t,

J ) 6.9 Hz, 3 H), 1.63 (m, 2 H), 2.32 (s, 3 H), 2.67 (m, 2 H),

3.11-3.18 (m, 4 H), 3.37 (q, J ) 6.6 Hz, 2 H), 4.11 (q, J ) 6.9

Hz, 2 H), 7.42-7.45 (m, 3 H), 7.59-7.63 (m, 2 H). MS (CI/

NH₃): m/z 460 (M⁺+ 1, base).

1

la te (41). H NMR δ: 0.63 (t, J ) 7.8 Hz, 3 H), 1.34 (t, J )

7.8 Hz, 3 H), 1.37 (m, 2 H), 1.42 (t, J ) 7.8 Hz, 3 H), 1.47-

1.81 (m, 6 H), 2.88 (q, J ) 7.8 Hz, 2 H), 3.08 (t, J ) 7.8 Hz, 2

H), 3.14 (q, J ) 7.8 Hz, 2 H), 3.46 (m, 1 H), 3.63 (m, 1 H), 3.77

(m, 1 H), 3.89 (m, 1 H), 3.98 (t, J ) 6.9 Hz, 2 H), 4.58 (t, J )

3.9 Hz, 1 H), 7.41-7.45 (m, 3 H), 7.58-7.61 (m, 2 H). MS (CI/

NH₃): m/z 486 (M⁺+ 1, base).

5-(3-Hyd r oxylp r op yl) 2-Eth yl-4-p r op yl-3-eth ylsu lfa n yl-

ca r bon yl-6-p h en ylp yr id in e-5-ca r boxyla te (60). ¹H NMR

δ: 0.95 (t, J ) 7.8 Hz, 3 H), 1.34 (t, J ) 7.8 Hz, 3 H), 1.42 (t,

J ) 7.8 Hz, 3 H), 1.61 (m, 2 H), 1.83 (m, 2 H), 2.66 (m, 2 H),

2.86 (q, J ) 7.8 Hz, 2 H), 3.14 (q, J ) 7.8 Hz, 2 H), 3.82 (t, J

) 6.9 Hz, 2 H), 4.10 (t, J ) 6.9 Hz, 2 H), 7.41-7.44 (m, 3 H),

7.57-7.60 (m, 2 H). MS (CI/NH₃): m/z 416 (M⁺+ 1).

5-P r op yl 2-Eth yl-4-p r op yl-3-(2-tetr a h yd r op yr a n yloxyl-

e t h ylsu lfa n ylca r b on yl)-6-p h e n ylp yr id in e -5-ca r b oxy-

1

la te (17). H NMR δ: 0.66 (t, J ) 7.8 Hz, 3 H), 0.95 (t, J )

5-P r op yl 2-Eth yl-4-(3-tetr a h yd r op yr a n yloxylp r op yl)-

3-(et h ylsu lfa n ylca r b on yl)-6-p h en ylp yr id in e-5-ca r b oxy-

7.8 Hz, 3 H), 1.34 (t, J ) 7.8 Hz, 3 H), 1.39 (m, 2 H), 1.52-

1.83 (m, 8 H), 2.67 (m, 2 H), 2.87 (q, J ) 7.8 Hz, 2 H), 3.40 (t,

J ) 6.0 Hz, 2 H), 3.54 (m, 1 H), 3.71 (m, 1 H), 3.92 (m, 2 H),

3.98 (t, J ) 6.9 Hz, 2 H), 4.70 (t, J ) 3.0 Hz, 1 H), 7.40-7.46

(m, 3 H), 7.59-7.62 (m, 2 H). MS (CI/NH₃): m/z 500 (M⁺+ 1).

5-P r opyl 2-Eth yl-4-pr opyl-3-((2,2-dim eth yl-1,3-dioxolan -

4-yl)m et h ylsu lfa n ylca r b on yl)-6-p h en ylp yr id in e-5-ca r -

1

la te (62). H NMR δ: 0.67 (t, J ) 7.8 Hz, 3 H), 1.34 (t, J )

7.8 Hz, 3 H), 1.41 (t, J ) 7.8 Hz, 3 H), 1.42 (m, 2 H), 1.54-

1.96 (m, 8 H), 2.78 (m, 2 H), 2.87 (q, J ) 7.8 Hz, 2 H), 3.13 (q,

J ) 7.8 Hz, 2 H), 3.41 (m, 1 H), 3.50 (m, 1 H), 3.73 (m, 1 H),

3.84 (m, 1 H), 3.99 (t, J ) 6.9 Hz, 2 H), 4.59 (t, J ) 3.0 Hz, 1

H), 7.40-7.43 (m, 3 H), 7.60-7.62 (m, 2 H). MS (CI/NH₃): m/z

500 (M⁺+ 1, base).

1

boxyla te (18). H NMR δ: 0.66 (t, J ) 7.8 Hz, 3 H), 0.95 (t,

J ) 7.8 Hz, 3 H), 1.34 (t, J ) 7.8 Hz, 3 H), 1.38 (s, 3 H), 1.39

(m, 2 H), 1.47 (s, 3 H), 1.62 (m, 2 H), 2.66 (m, 2 H), 2.86 (q, J

) 7.8 Hz, 2 H), 3.38 (m, 2 H), 3.75 (dd, J ) 6.0, 8.7 Hz, 1 H),

3.98 (t, J ) 6.9 Hz, 2 H), 4.16 (dd, J ) 6.0, 8.7 Hz, 1 H), 4.39

(m, 1 H), 7.41-7.44 (m, 3 H), 7.59-7.62 (m, 2 H). MS (CI/

NH₃): m/z 486 (M⁺+ 1, base).

5-P r op yl 2-Eth yl-4-p r op yl-3-(3-tetr a h yd r op yr a n yloxyl-

p r op ylsu lfa n ylca r b on yl)-6-p h en ylp yr id in e-5-ca r b oxy-

1

la te (63). H NMR δ: 0.66 (t, J ) 7.8 Hz, 3 H), 0.94 (t, J )

7.8 Hz, 3 H), 1.33 (t, J ) 7.8 Hz, 3 H), 1.39 (m, 2 H), 1.54-

1.86 (m, 8 H), 2.04 (m, 2 H), 2.66 (m, 2 H), 2.85 (q, J ) 7.8 Hz,

2 H), 3.24 (m, 2 H), 3.52 (m, 2 H), 3.88 (m, 2 H), 3.98 (t, J )

6.9 Hz, 2 H), 4.61 (t, J ) 3.9 Hz, 1 H), 7.40-7.44 (m, 3 H),

7.59-7.61 (m, 2 H). MS (CI/NH₃): m/z 514 (M⁺+ 1, base).

5-P r op yl 2-Eth yl-4-p r op yl-3-(2,2,2-tr iflu or oeth ylsu lfa n -

ylcar bon yl)-6-ph en ylpyr idin e-5-car boxylate (20). ¹H NMR

δ: 0.66 (t, J ) 7.8 Hz, 3 H), 0.97 (t, J ) 6.9 Hz, 3 H), 1.33 (t,

J ) 7.8 Hz, 3 H), 1.41 (m, 2 H), 1.63 (m, 2 H), 2.65 (m, 2 H),

2.84 (q, J ) 7.8 Hz, 2 H), 3.98 (t, J ) 6.9 Hz, 2 H), 4.45 (q, J

) 7.8 Hz, 2 H), 7.41 (m, 3 H), 7.61 (m, 2 H). MS (CI/NH₃): m/z

466 (M⁺- 1), 384 (M⁺- SCH₂CF₃, base).

5-P r op yl 2-Eth yl-4-(2-a cetylth ioeth yl)-3-(eth ylsu lfa n -

ylcar bon yl)-6-ph en ylpyr idin e-5-car boxylate (23). ¹H NMR

δ: 0.65 (t, J ) 7.8 Hz, 3 H), 1.38 (m, 2 H), 1.40 (t, J ) 7.8 Hz,

3 H), 1.46 (t, J ) 7.8 Hz, 3 H), 2.34 (s, 3 H), 3.03-3.12 (m, 6

H), 3.28 (q, J ) 7.8 Hz, 2 H), 4.11 (t, J ) 6.9 Hz, 2 H), 7.46-

7.57 (m, 3 H), 8.09 (m, 2 H). MS (CI/NH₃): m/z 460 (M⁺+ 1).

5-(2,2,3,3,3-P en ta flu or op r op yl) 2-Eth yl-4-p r op yl-3-(eth -

ylsu lfan ylcar bon yl)-6-ph en ylpyr idin e-5-car boxylate (27).

¹H NMR δ: 0.95 (t, J ) 7.8 Hz, 3 H), 1.35 (t, J ) 7.8 Hz, 3 H),

1.42 (t, J ) 7.8 Hz, 3 H), 1.58-1.65 (m, 2 H), 2.65 (m, 2 H),

2.88 (q, J ) 7.8 Hz, 2 H), 3.15 (q, J ) 7.8 Hz, 2 H), 4.38 (t, J

) 12.6 Hz, 2 H), 7.42-7.44 (m, 3 H), 7.53-7.56 (m, 2 H). MS

(CI/NH₃): m/z 490 (M⁺+ 1, base).

Gen er a l P r oced u r e F or P r ep a r in g Mon oflu or o-p yr -

id in es: By F lu or in a tin g Cor r esp on d in g Hyd r oxyl-p yr -

id in es w ith DAST. Under N₂, hydroxyl-pyridine (59, 60, 25,

64, 16, or 12, ∼0.05 to ∼0.3 mmol) was dissolved in anhydrous

CH₂Cl₂(1-3 mL) and the solution was cooled to -78 °C. Two

equivalents of DAST were then added by syringe. The reaction

was stirred at -78 °C for 0.5 h before the temperature was

allowed to rise to room temperature. The stirring was contin-

ued until the reaction was complete (by TLC). The reaction

mixture was carefully hydrolyzed upon addition of two drops

of water. The organic phase was separated, and the aqueous

phase was extracted with ether. The organic phases were

combined and dried with anhydrous MgSO₄. After the solvent

was removed, the residue was purified by preparative TLC

(silica 60; 1000 or 2000 mm; Analtech, Newark, DE; petroleum

ether-ethyl acetate (4:1-19:1)) to provide pure monofluoro-

pyridines (7, 14, 19, 21, 24, and 26).

5-P r op yl 2-E t h yl-4-p r op yl-3-(et h ylsu lfa n ylca r bon yl)-

6-(o-flu or op h en yl)p yr id in e-5-ca r boxyla te (28). ¹H NMR

δ: 0.73 (t, J ) 7.8 Hz, 3 H), 0.95 (t, J ) 7.8 Hz, 3 H), 1.32 (t,

J ) 7.8 Hz, 3 H), 1.39 (m, 2 H), 1.42 (t, J ) 7.8 Hz, 3 H), 1.62

(m, 2 H), 2.73 (m, 2 H), 2.85 (q, J ) 7.8 Hz, 2 H), 3.14 (q, J )

7.8 Hz, 2 H), 3.97 (t, J ) 6.9 Hz, 2 H), 7.11 (t, J ) 9.0 Hz, 1

5-(2-F lu or oeth yl) 2-Eth yl-4-eth yl-3-(eth ylsu lfa n ylca r -

bon yl)-6-p h en ylp yr id in e-5-ca r boxyla te (7). ¹H NMR δ:

1.24 (t, J ) 7.8 Hz, 3 H), 1.34 (t, J ) 7.8 Hz, 3 H), 1.41 (t, J )

7.8 Hz, 3 H), 2.73 (q, J ) 7.8 Hz, 2 H), 2.87 (q, J ) 7.8 Hz, 2

H), 3.14 (q, J ) 7.8 Hz, 2 H), 4.23 (m, 2 H), 4.31 (t, J ) 4.8 Hz,

1 H), 4.39 (t, J ) 4.8 Hz, 1 H), 7.41-7.44 (m, 3 H), 7.59-7.61

716 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 4

(m, 2 H). MS (CI/NH₃): m/z 390 (M⁺+ 1, base). MS (EI): m/z

Li et al.

J ) 7.8 Hz, 3 H), 1.38 (m, 2 H), 1.63 (m, 2 H), 2.67 (t, J ) 6.9

Hz, 2 H), 2.87 (t, J ) 6.9 Hz, 2 H), 3.26 (t, J ) 6.9 Hz, 2 H),

3.98 (t, J ) 6.9 Hz, 2 H), 4.52 (t, J ) 6.9 Hz, 2 H), 7.42 (m, 3

H), 7.59-7.62 (m, 2 H). MS (EI): m/z 434 (MH⁺+ NH₄), 387

(MH⁺- Et), 372 (M⁺- Pr), 354 (M⁺- SEt).

5-P r op yl 2-Eth yl-4-p r op yl-3-(3-h yd r oxylp r op ylsu lfa n -

ylcar bon yl)-6-ph en ylpyr idin e-5-car boxylate (64). ¹H NMR

δ: 0.62 (t, J ) 7.8 Hz, 3 H), 1.03 (t, J ) 7.8 Hz, 3 H), 1.30 (m,

2 H), 1.34 (t, J ) 7.8 Hz, 3 H), 1.35 (m, 2 H), 1.90 (m, 2 H),

2.82 (m, 2 H), 2.87 (q, J ) 7.8 Hz, 2 H), 3.06 (q, J ) 7.8 Hz, 2

H), 3.68 (t, J ) 6.0 Hz, 2 H), 3.93 (t, J ) 6.9 Hz, 2 H), 7.37-

7.45 (m, 3 H), 7.66-7.69 (m, 2 H). MS (CI/NH₃): m/z 382 (M⁺

- OEt), 354 (M⁺- CO₂Pr), 324 (M⁺- COSEt).

P r ep a r a tion of P yr id in iu m Sa lt 31 by Qu a ter n a r y

Am in a tion of Com p ou n d 4 w ith Iod om eth a n e. A mixture

of 4 (MRS 1523, 40 mg, 0.1 mmol) and iodomethane (71 mg,

0.5 mmol) in 4 mL of anhydrous methanol was sealed in a

Pyrex tube and heated at 80 °C for 2 days. After the mixture

cooled to room temperature, the solvent and excess MeI were

removed under reduced pressure, leaving a deep red tar. The

tar was washed with hexanes many times to remove the

unreacted MRS 1523, and 12 mg of the desired product (31)

was afforded (yield: 22%).

389(M⁺), 360 (M⁺- Et), 329 (MH⁺- EtS, base), 300 (M⁺

-

EtSCO).

5-P r op yl 2-(2-F lu or oeth yl)-4-p r op yl-3-(eth ylsu lfa n yl-

ca r bon yl)-6-p h en ylp yr id in e-5-ca r boxyla te (14). ¹H NMR

δ: 0.66 (t, J ) 8.1 Hz, 3 H), 0.95 (t, J ) 8.1 Hz, 3 H), 1.40 (m,

2 H), 1.42 (t, J ) 8.1 Hz, 3 H), 1.62 (m, 2 H), 2.67 (t, J ) 7.8

Hz, 2 H), 3.15 (q, J ) 7.8 Hz, 2 H), 3.24 (t, J ) 6.9 Hz, 1 H),

3.30 (t, J ) 6.9 Hz, 1 H), 3.99 (t, J ) 6.9 Hz, 2 H), 4.83 (t, J )

6.9 Hz, 1 H), 4.99 (t, J ) 6.9 Hz, 1 H), 7.41-7.43 (m, 3 H),

7.59 (m, 2 H). MS (CI/NH₃): m/z 418 (M⁺+ 1, base).

5-P r op yl 2-Eth yl-4-p r op yl-3-(2-flu or oeth ylsu lfa n ylca r -

bon yl)-6-p h en ylp yr id in e-5-ca r boxyla te (19). ¹H NMR δ:

0.66 (t, J ) 7.8 Hz, 3 H), 0.95 (t, J ) 7.8 Hz, 3 H), 1.34 (t, J )

7.8 Hz, 3 H), 1.39 (m, 2 H), 1.64 (m, 2 H), 2.66 (m, 2 H), 2.85

(q, J ) 7.8 Hz, 2 H), 3.43 (t, J ) 6.0 Hz, 1 H), 3.50 (t, J ) 6.0

Hz, 1 H), 3.98 (t, J ) 6.6 Hz, 2 H), 4.59 (t, J ) 6.0 Hz, 1 H),

4.74 (t, J ) 6.0 Hz, 1 H), 7.42 (m, 3 H), 7.61 (m, 2 H). MS

(CI/NH₃): m/z 418 (M⁺+ 1, base).

5-P r op yl 2-Eth yl-4-p r op yl-3-(3-flu or op r op ylsu lfa n yl-

ca r bon yl)-6-p h en ylp yr id in e-5-ca r boxyla te (21). ¹H NMR

δ: 0.66 (t, J ) 7.8 Hz, 3 H), 0.94 (t, J ) 7.8 Hz, 3 H), 1.34 (t,

J ) 7.8 Hz, 3 H), 1.39 (m, 2 H), 1.62 (m, 2 H), 2.08-2.20 (m,

2 H), 2.65 (m, 2 H), 2.85 (q, J ) 7.8 Hz, 2 H), 3.26 (t, J ) 6.9

Hz, 2 H), 3.98 (t, J ) 6.9 Hz, 2 H), 4.51 (t, J ) 6.0 Hz, 1 H),

4.67 (t, J ) 6.0 Hz, 1 H), 7.40-7.44 (m, 3 H), 7.59-7.62 (m, 2

H). MS (CI/NH₃): m/z 432 (M⁺+ 1, base).

5-P r op yl 2-Eth yl-4-(3-flu or op r op yl)-3-(eth ylsu lfa n yl-

ca r bon yl)-6-p h en ylp yr id in e-5-ca r boxyla te (24). ¹H NMR

δ: 0.66 (t, J ) 7.8 Hz, 3 H), 1.34 (t, J ) 7.8 Hz, 3 H), 1.40 (m,

2 H), 1.41 (t, J ) 7.8 Hz, 3 H), 1.98-2.13 (m, 2 H), 2.82 (m, 2

H), 2.88 (q, J ) 7.8 Hz, 2 H), 3.14 (q, J ) 7.8 Hz, 2 H), 3.99 (t,

J ) 6.9 Hz, 2 H), 4.37 (t, J ) 6.0 Hz, 1 H), 4.53 (t, J ) 6.0 Hz,

1 H), 7.41-7.43 (m, 3 H), 7.59-7.62 (m, 2 H). MS (CI/NH₃):

m/z 418 (M⁺+ 1, base).

5-(3-F lu or op r op yl) 2-Eth yl-4-p r op yl-3-(eth ylsu lfa n yl-

ca r bon yl)-6-p h en ylp yr id in e-5-ca r boxyla te (26). ¹H NMR

δ: 0.95 (t, J ) 7.8 Hz, 3 H), 1.36 (t, J ) 7.8 Hz, 3 H), 1.42 (t,

J ) 7.8 Hz, 3 H), 1.65 (m, 2 H), 1.73 (m, 2 H), 2.66 (m, 2 H),

2.87 (q, J ) 7.8 Hz, 2 H), 3.13 (q, J ) 7.8 Hz, 2 H), 3.95 (t, J

) 6.0 Hz, 1 H), 4.11 (t, J ) 6.0 Hz, 1 H), 4.14 (t, J ) 6.0 Hz,

2 H), 7.42 (m, 3 H), 7.58 (m, 2 H). MS (CI/NH₃): m/z 418 (M⁺

+ 1, base). HRMS: calcd for C₂₃H₂₈FNO₃S, 417.1774; found,

417.1780.

Gen er a l P r oced u r e F or Dep r otectin g THP -P r otected

P yr id in es.⁴³A mixture of THP-protected pyridine (17, 41, 62,

or 63, ∼0.1 to ∼0.4 mmol) and a catalytic amount of PPTS

(10%) was stirred and refluxed in absolute ethanol (5-10 mL)

until the reaction was complete (by TLC). After the mixture

cooled to room temperature, the solvent was removed, and the

residue was chromatographed on preparative TLC (silica 60;

1000 or 2000 mm; Analtech, Newark, DE; petroleum ether-

ethyl acetate (4:1-9:1)) to give the hydroxyl-pyridines (16, 25,

or 64). However, when the similar condition was applied to

THP-pyridine 41, besides the expected product 22, the lactone

compound 32 was also obtained through an intramolecular

transesterification (Scheme 2).

5-P r op yl 2-Eth yl-4-p r op yl-3-(2-h yd r oxyleth ylsu lfa n yl-

ca r bon yl)-6-p h en ylp yr id in e-5-ca r boxyla te (16). ¹H NMR

δ: 0.66 (t, J ) 7.8 Hz, 3 H), 0.95 (t, J ) 7.8 Hz, 3 H), 1.34 (t,

J ) 7.8 Hz, 3 H), 1.39 (m, 2 H), 1.62 (m, 2 H), 2.68 (m, 2 H),

2.86 (q, J ) 7.8 Hz, 2 H), 3.53 (t, J ) 6.0 Hz, 2 H), 3.92 (t, J

) 6.0 Hz, 2 H), 3.98 (t, J ) 6.0 Hz, 2 H), 7.41 (m, 3 H), 7.60

(m, 2 H). MS (CI/NH₃): m/z 416 (M⁺+ 1, base).

1-Meth yl-2-eth yl-4-p r op yl-3-(eth ylsu lfa n ylca r bon yl)-5-

p r op yloxylca r bon yl-6-p h en ylp yr id in iu m Iod id e (31). ¹H

NMR δ: 0.67 (t, J ) 7.8 Hz, 3 H), 1.04 (t, J ) 7.8 Hz, 3 H),

1.40 (m, 2 H), 1.48 (t, J ) 7.8 Hz, 3 H), 1.55 (t, J ) 7.8 Hz, 3

H), 1.74 (m, 2 H), 2.17 (s, 3 H), 2.87 (t, J ) 7.8 Hz, 2 H), 3.27

(q, J ) 7.8 Hz, 2 H), 3.42 (q, J ) 7.8 Hz, 2 H), 4.05 (t, J ) 7.8

Hz, 2 H), 7.62-7.74 (m, 5 H). MS (CI/NH₃): m/z 558 (M⁺

+

NH₄), 525 (M⁺- 1 - Me), 414 (M⁺- I), 369 (M⁺- 1 - I -

Me-Et).

La cton e (32). ¹H NMR δ: 0.64 (t, J ) 7.8 Hz, 3 H), 1.37 (t,

J ) 7.8 Hz, 3 H), 1.38 (m, 2 H), 3.12 (t, J ) 6.0 Hz, 2 H), 3.35

(q, J ) 7.8 Hz, 2 H), 4.03 (t, J ) 6.9 Hz, 2 H), 4.49 (t, J ) 6.0

Hz, 2 H), 7.45-7.47 (m, 3 H), 7.65-7.67 (m, 2 H). MS (CI/

NH₃): m/z 340 (M⁺+ 1).

5-Eth yl 2-Meth yl-4-eth yl-3-(N-eth yla m in oca r bon yl)-6-

p h en ylp yr id in e-5-ca r boxyla te (33a ). N-Ethyl acetoacet-

amide¹²(129 mg, 1.0 mmol), propionaldehyde (116 mg, 2.0

mmol), and ethyl 3-amino-3-phenyl-2-propenoate (191 mg, 1.0

mmol) were dissolved in 2 mL of absolute ethanol. The mixture

was sealed in a Pyrex tube and heated, with stirring, to 90 °C

overnight. After the mixture cooled to room temperature, the

solvent was evaporated, and the residue was purified by

preparative TLC (silica 60; 1000 mm; Analtech, Newark, DE;

petroleum ether-ethyl acetate (2:1)) to give 167 mg of dihy-

dropyridine. To a solution of this dihydropyridine in 4 mL THF

was added tetrachloro-1,4-benzoquinone (40, 120 mg), and the

mixture was refluxed overnight. After the mixture cooled to

room temperature, the solvent was removed, and the residue

was purified by preparative TLC (silica 60; 1000 mm; Analtech,

Newark, DE; petroleum ether-ethyl acetate (4:1)) to give 91

mg (yield: 55%) of compound 33a. ¹H NMR δ: 0.97 (t, J ) 7.8

Hz, 3 H), 1.24 (t, J ) 7.8 Hz, 3 H), 1.27 (t, J ) 7.8 Hz, 3 H),

2.60 (s, 3 H), 2.72 (q, J ) 7.8 Hz, 2 H), 3.53 (m, 2 H), 4.07 (q,

J ) 7.8 Hz, 2 H), 5.85 (t, J ) 2.0 Hz, 1 H), 7.39-7.42 (m, 3 H),

7.52-7.55 (m, 2 H). MS (CI/NH₃): m/z 341 (M⁺+ 1, base).

P r ep a r a tion of La cta m P yr id in e 33b (Sch em e 2). A

mixture of phthalimide pyridine 9 (57 mg, 0.105 mmol) and

anhydrous hydrazine (20 mg, 0.628 mmol) was stirred in 2

mL of absolute ethanol overnight at room temperature and

then refluxed for 2 h. After the mixture cooled to room

temperature, the solvent was removed, and the residue was

separated using preparative TLC (silica 60; 1000 mm; Anal-

tech, Newark, DE; petroleum ether-ethyl acetate (2:1)) to give

10 mg of the desired lactam (yield: 24%).

5-P r op yl 2-Eth yl-4-(2-h yd r oxyleth yl)-3-(eth ylsu lfa n yl-

ca r bon yl)-6-p h en ylp yr id in e-5-ca r boxyla te (22). ¹H NMR

δ: 0.59 (t, J ) 7.8 Hz, 3 H), 1.31 (m, 2 H), 1.35 (t, J ) 7.8 Hz,

3 H), 1.41 (t, J ) 7.8 Hz, 3 H), 2.90 (q, J ) 7.8 Hz, 2 H), 3.00

(t, J ) 6.9 Hz, 2 H), 3.15 (q, J ) 7.8 Hz, 2 H), 3.88 (t, J ) 6.9

Hz, 2 H), 3.95 (t, J ) 6.9 Hz, 2 H), 7.41-7.46 (m, 3 H), 7.58-

7.60 (m, 2 H). MS (CI/NH₃): m/z 340 (M⁺- SEt).

1

La cta m (33b). H NMR δ: 1.01 (t, J ) 7.8 Hz, 3 H), 1.33

(t, J ) 7.8 Hz, 3 H), 2.05 (m, 2 H), 2.85 (t, J ) 6.9 Hz, 2 H),

3.07 (t, J ) 7.8 Hz, 2 H), 3.15 (m, 2 H), 4.13 (q, J ) 7.8 Hz, 2

H), 6.31 (m, 1 H), 7.43 (m, 3 H), 7.61-7.63 (m, 2 H). MS (CI/

NH₃): m/z 356 (M⁺+ NH₄), 339 (M⁺+ 1).

5-P r op yl 2-Eth yl-4-(3-h yd r oxylp r op yl)-3-(eth ylsu lfa n -

ylcar bon yl)-6-ph en ylpyr idin e-5-car boxylate (25). ¹H NMR

δ: 0.66 (t, J ) 7.8 Hz, 3 H), 0.95 (t, J ) 7.8 Hz, 3 H), 1.34 (t,

P r ep a r a tion of Th iola cton es 34 a n d 35 (Sch em e 2).⁴⁴

Under N₂, at room temperature, to a stirred solution of 23 or

Synthesis of 3,5-diacyl-2,4-dialkylpyridines

J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 4 717

15 (50 mg, 0.109 mmol) in anhydrous methanol (5 mL) was

added solid sodium thiomethoxide (7.6 mg, 0.109 mmol). The

reaction reached completion (TLC) after stirring for 30 min.

The solvent was removed, and the residue was purified by

preparative TLC (silica 60; 1000 or 2000 mm; Analtech,

Newark, DE; petroleum ether-ethyl acetate (9:1)) to give 38

mg of 34 (yield: 98%) and 38 mg of 35 (yield: 98%).

3,4-Th iola cton e (34). ¹H NMR δ: 0.66 (t, J ) 7.8 Hz, 3

H), 1.33 (t, J ) 7.8 Hz, 3 H), 1.40 (m, 2 H), 2.71-3.06 (m, 4

H), 4.02 (t, J ) 7.8 Hz, 2 H), 7.41-7.43 (m, 3 H), 7.59-7.61

(m, 2 H). MS (CI/NH₃): m/z 374 (M⁺+ NH₄), 356 (M⁺+ 1).

(t, J ) 7.8 Hz, 1 H), 7.70-7.73 (m, 2 H), 7.83-7.85 (m, 2 H).

MS (CI/NH₃): m/ z 309 (M⁺+ NH₄), 217 (M⁺- OEt - Et,

base).

A mixture of 3-phthaloyl-4-aminobutyraldehyde diethylac-

etal (56, 8.30 g, 28.5 mmol) and 1 N HCl (56 mL) in acetone

(60 mL) was heated under reflux for 2 h. Acetone was removed,

and the residue was extracted with ether (3 × 80 mL). The

organic phase was washed with water, dried over MgSO₄,

filtered, and the solvent removed by evaporation. The residue

was column chromatographed (silica gel, petroleum ether-

ethyl acetate (9:1)). ¹H NMR (CDCl₃/TMS) δ: 2.03 (m, 2 H),

2.54 (t, J ) 6.9 Hz, 2 H), 3.75 (t, J ) 6.9 Hz, 2 H), 7.70-7.76

(m, 2 H), 7.83-7.88 (m, 2 H), 9.78 (s, 1 H). MS (CI/NH₃): m/ z

217 (M⁺, base).

P r ep a r a t ion of â-Acet ylt h iop r op ion a ld eh yd e (37e)

(Sch em e 5).⁴⁷The same condition (for acid 49) was applied

to prepare â-acetylthiopropionaldehyde (37e) from thiolacetic

acid (2.13 g, 28 mmol) and acrolein (1.12 g, 1.34 mL, 20 mmol).

The desired aldehyde 37e (2.12 g) was obtained, yield: 80%.

¹H NMR (CDCl₃/TMS) δ: 2.34 (s, 3 H), 2.81 (t, J ) 6.9 Hz, 2

H), 3.12 (t, J ) 6.9 Hz, 2 H), 9.76 (s, 1 H). MS (CI/NH₃): m/ z

150 (M⁺+ NH₄, base), 133 (M⁺+ 1).

P r ep a r a tion of THP -P r otected Ald eh yd es 37f a n d 37g

(Sch em e 5).⁴³A solution of 1,3-propanediol or 1,4-butanediol

(30 mmol) and 3,4-dihydro-2H-pyran (30 mmol) in dry dichlo-

romethane (25 mL) was cooled to 0 °C. Under stirring, PPTS

(0.754 g, 3 mmol) was carefully added and allowed to react at

the same temperature for 30 min before raising the temper-

ature to room temperature. The reaction was then stirred for

4 h, the PPTS was filtered off, and the solvent was removed.

Preparative TLC of the residue (silica gel, petroleum ether-

ethyl acetate (4:1)) gave THP-protected alcohols, 57 (yield:

63%) or 58 (yield: 42%).

2,3-Th iola cton e (35). ¹H NMR δ: 0.96 (t, J ) 6.9 Hz, 3

H), 1.00 (t, J ) 6.9 Hz, 3 H), 1.61 (m, 2 H), 2.65 (m, 2 H), 3.00

(t, J ) 6.6 Hz, 2 H), 3.12 (t, J ) 6.6 Hz, 2 H), 4.11 (q, J ) 6.9

Hz, 2 H), 7.42-7.44 (m, 3 H), 7.59 (m, 2 H). MS (CI/NH₃): m/z

356 (M⁺+ 1, base).

Gen er a l P r oced u r e for P r ep a r a tion of â-Am in o-r,â-

u n sa tu r a ted ester s (36) (Sch em e 9). A â-ketoester (38i-

m , 46, 3 mmol) and ammonium acetate (4.5 mmol) were mixed

in 5 mL of absolute ethanol and refluxed at 80 °C for 24 h.

The solvent was removed, and the residue was chromato-

graphed by preparative TLC to give the desired compounds

in moderate yields.

(3-H yd r oxylp r op yl) 3-Am in o-3-p h en yl-2-p r op en oa t e

1

(36c). H NMR δ: 1.86-1.93 (m, 2 H), 3.71 (t, J ) 6.0 Hz, 2

H), 4.32 (t, J ) 6.0 Hz, 2 H), 4.97 (s, 1 H), 7.39-7.46 (m, 3 H),

7.53-7.56 (m, 2 H). MS (CI/NH₃): m/z 240 (MH⁺+ NH₄), 222

(M⁺+ 1, base).

(2,2,3,3,3-P en ta flu or op r op yl) 3-Am in o-3-p h en yl-2-p r o-

p en oa te (36d ). ¹H NMR δ: 4.60 (t, J ) 12.6 Hz, 2 H), 5.03 (s,

1 H), 7.44-7.49 (m, 3 H), 7.55-7.57 (m, 2 H). MS (CI/NH₃):

m/z 313 (M⁺+ NH₄), 296 (M⁺+ 1, base).

P r op yl 3-Am in o-3-(2-flu or op h en yl)-2-p r op en oa te (36e).

¹H NMR δ: 0.97 (t, J ) 7.8 Hz, 3 H), 1.69 (m, 2 H), 4.08 (t, J

) 6.9 Hz, 2 H), 4.91 (s, 1 H), 7.10-7.21 (m, 2 H), 7.39 (m, 1

H), 7.49 (m, 1 H). MS (CI/NH₃): m/z 224 (M⁺+ 1, base).

P r op yl 3-Am in o-3-(3-flu or op h en yl)-2-p r op en oa te (36f).

¹H NMR δ: 0.98 (t, J ) 7.8 Hz, 3 H), 1.69 (m, 2 H), 4.09 (t, J

) 6.9 Hz, 2 H), 4.97 (s, 1 H), 7.10-7.43 (m, 4 H). MS

(CI/NH₃): m/z 224 (M⁺+ 1, base).

F or 57. ¹H NMR (CDCl₃/TMS) δ: 1.53-1.60 (m, 4 H), 1.68-

1.90 (m, 4 H), 3.53 (m, 1 H), 3.61 (m, 1 H), 3.80 (t, J ) 6.0 Hz,

2 H), 3.87 (m, 1 H), 3.95 (m, 1 H), 4.60 (s, 1 H). MS (CI/NH₃):

m/ z 178 (M⁺+ NH₄, base).

F or 58. ¹H NMR (CDCl₃/TMS) δ: 1.53-1.86 (m, 10 H), 3.44

(m, 1 H), 3.51 (m, 1 H), 3.68 (t, J ) 6.9 Hz, 2 H), 3.78-3.90

(m, 2 H), 4.62 (t, J ) 3.0 Hz, 1 H). MS (CI/NH₃): m/ z 192 (M⁺

+ NH₄, base), 175 (M⁺+ 1).

Oxid a tion .⁴⁸A mixture of alcohol 57 or 58 (1 equiv), PCC

(2 equiv), and sodium acetate (0.4 equiv.) in 10-30 mL of

dichloromethane was stirred at room temperature for 2 h.

Ether (30 mL) was then added, and the reaction was filtered.

After the solvent was removed, the residue was purified by

preparative TLC (silica 60; 2000 mm; Analtech, Newark, DE;

petroleum ether-ethyl acetate (9:1)) to give THP-protected

aldehyde, 37f, in 83% yield or 37g in 81% yield.

P r opyl 3-Am in o-3-(4-flu or oph en yl)-2-pr op en oa te (36g).

¹H NMR δ: 0.97 (t, J ) 7.8 Hz, 3 H), 1.66-1.73 (m, 2 H), 4.08

(t, J ) 6.9 Hz, 2 H), 4.94 (s, 1 H), 7.07-7.13 (m, 2 H), 7.52-

7.57 (m, 2 H). MS (CI/NH₃): m/z 224 (M⁺+ 1, base).

P r ep a r a tion of 3-P h th a lim id op r op ion a ld eh yd e (37c)

(Sch em e 5).⁴⁵N-Carbethoxy-phthalimide (53, 4.384 g, 20

mmol), 3-amino-1-propanol (1.502 g, 20 mmol), and triethyl-

amine (2.024 g, 20 mmol) were dissolved in THF (30 mL) and

allowed to react for 6 h. The reaction mixture was evaporated,

and the residue was purified by column chromatography (silica

60; petroleum ether-ethyl acetate (4:1)) to give 4.08 g of

3-phthalimido-1-propanol 54 (yield: 99%). ¹H NMR (CDCl₃/

TMS) δ: 1.85-1.93 (m, 2 H), 3.63 (m, 2 H), 3.87 (t, J ) 6.9

Hz, 2 H), 7.73-7.75 (m, 2 H), 7.86-7.88 (m, 2 H). MS (CI/

NH₃): m/ z 223 (M⁺+ NH₄, base), 206 (M⁺+ 1).

F or 37f. ¹H NMR (CDCl₃/TMS) δ: 1.53-1.80 (m, 6 H), 2.70

(dt, J ) 6.0, 1.8 Hz, 2 H), 3.51 (m, 2 H), 3.85 (m, 2 H), 4.63 (t,

J ) 3.9 Hz, 1 H), 9.83 (t, J ) 1.8 Hz, 1 H). MS (CI/NH₃): m/ z

176 (M⁺+ NH₄, base).

F or 37g. ¹H NMR (CDCl₃/TMS) δ: 1.54-1.88 (m, 6 H), 1.95

(m, 2 H), 2.55 (t, J ) 6.6 Hz, 2 H), 3.42 (m, 1 H), 4.49 (m, 1 H),

3.75-3.86 (m, 2 H), 4.57 (t, J ) 3.0 Hz, 1 H), 9.80 (s, 1 H). MS

(CI/NH₃): m/ z 190 (M⁺+ NH₄, base).

Oxid a tion .⁴⁶A mixture of 3-phthalimido-1-propanol (54,

1.026 g, 5 mmol), PCC (2.156 g, 10 mmol), and silica gel (2.156

g) in 30 mL of dichloromethane was stirred at room temper-

ature for 2 h. Ether (30 mL) was then added, and the reaction

was filtered. After the solvent was removed, the residue was

purified by preparative TLC (silica 60; 2000 mm; Analtech,

Newark, DE; petroleum ether-ethyl acetate (4:1)) to give 0.629

Syn t h esis of â-Ket oest er s 38 via Meld r u m ’s Acid s

(Sch em e 3).²³The preparation of S-ethyl 3-oxothiovalerate

(38a ) is provided as an example. 2,2-Dimethyl-1,3-dioxane-

4,6-dione (43, 0.721 g, 5 mmol) and propionyl chloride (0.509

g, 5.5 mmol) were dissolved in 10 mL of dry CH₂Cl₂. At 0 °C,

0.81 mL (0.791 g, 10 mmol) of pyridine (in the cases of aromatic

acid chlorides, using 4-(dimethylamino)pyridine instead of

pyridine) was then added dropwise. The reaction temperature

was kept at 0 °C for 1 h and then raised to room temperature

for an additional 1 h. The reaction mixture was washed with

1 N HCl (10 mL) and water (5 mL) and then dried with

anhydrous MgSO₄. Removal of the solvent left the desired

product (44a ), which was directly used for the next reaction

without purification.

1

g of the aldehyde 37c (yield: 62%). H NMR (CDCl₃/TMS) δ:

2.88 (t, J ) 6.9 Hz, 6 H), 3.85 (t, J ) 6.9 Hz, 2 H), 7.72-7.74

(m, 2 H), 7.81-7.87 (m, 2 H), 9.83 (s, 1 H). MS (EI): m/ z 203

(M⁺), 175 (MH⁺- CHO), 160 (M⁺- CH₂CHO, base).

P r ep a r a t ion of 3-P h t h a lim id ob u t yr a ld eh yd e (37d )

(Sch em e 5).⁴⁵The same conditions (for 54) were used for

preparing phthalimidoacetal 56 from N-carbethoxyphthalim-

ide (53) and 4-aminobutyraldehyde diethylacetal (55). The

yield for 56 was 95%. ¹H NMR (CDCl₃/TMS) δ: 1.19 (t, J )

6.9 Hz, 6 H), 1.65-1.70 (m, 2 H), 1.72-1.77 (m, 2 H), 3.49 (m,

2 H), 3.62 (q, J ) 6.9 Hz, 2 H), 3.72 (t, J ) 6.9 Hz, 2 H), 4.52

Compound 44a (670 mg, 3.35 mmol) and ethanethiol (0.621

g, 10 mmol) were mixed in 10 mL of toluene. This mixture

was heated at 80 °C in a flask with an effective reflux

718 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 4

Li et al.

condenser for 24 h. The solvent and excess ethanethiol were

removed, and the residue was purified by TLC (silica 60,

petroleum ether-ethyl acetate (9:1)) to give the desired

product 282 mg, yield: 53%.

P r ep a r a tion of â-Ben zyloxylp r op ion yl Ch lor id e (42b)

(Sch em e 4).⁴⁹â-Propiolactone (47, 2.16 g, 30 mmol) and

benzyl alcohol (19.5 g, 80 mmol) were stirred at 75 °C for 24

h. After the mixture cooled to room temperature, 60 mL of 1

N NaOH was carefully added to the reaction mixture, which

was stirred for 1 h and then extracted with dichloromethane

(3 × 40 mL). The aqueous phase was acidified with 80 mL of

1 N HCl, extracted with ether (3 × 80 mL), dried over MgSO₄,

and filtered. Removal of the solvent left a viscous oil (48), 4.91

1

S-Eth yl 3-Oxoth iova ler a te (38a ). H NMR δ: 1.07 (t, J

) 6.9 Hz, 3 H), 1.28 (t, J ) 6.9 Hz, 3 H), 2.58 (q, J ) 6.9 Hz,

2 H), 2.94 (q, J ) 6.9 Hz, 2 H), 3.66 (s, 2 H). MS (CI/NH₃):

m/ z 178 (M⁺+ NH₄), 161 (M⁺+ 1).

1

S-Hexyl 3-Oxoth iova ler a te (38b). H NMR δ: 0.88 (t, J

1

g, yield: 91%. H NMR (CDCl₃/TMS) δ: 2.67 (t, J ) 6.9 Hz, 2

) 6.9 Hz, 3 H), 1.07 (t, J ) 7.8 Hz, 3 H), 1.26-1.41 (m, 6 H),

1.59 (m, 2 H), 2.58 (q, J ) 7.8 Hz, 2 H), 2.92 (t, J ) 6.9 Hz, 2

H), 3.66 (s, 2 H). MS (CI/NH₃): m/ z 234 (M⁺+ NH₄, base),

217 (M⁺+ 1).

H), 3.76 (t, J ) 6.9 Hz, 2 H), 4.56 (s, 2 H), 7.34 (m, 5 H). MS

(CI/NH₃): m/ z 180 (M⁺).

Acid 48 (4.90 g, 27 mmol) and thionyl chloride (6.42 g, 54

mmol) were mixed carefully under N₂while cooling in an ice

bath. After the gas evolution stopped, the mixture was refluxed

for 2 h. The excess thionyl chloride was removed by vacuum

leaving compound 42b, which was used directly in the next

step.

P r ep a r a tion of â-Acetylth iop r op ion yl Ch lor id e (42c)

(Sch em e 4).⁵⁰Under ice-cooling conditions, 3 mL (42 mmol)

of thiolacetic acid was added very carefully to acrylic acid (2.16

g, 30 mmol). After the mixture was stirred and cooled for 1 h,

the temperature was allowed to rise to 25 °C, and stirring

continued overnight. Removing all volatile materials gave 2.36

g of the acid 49, yield: 53%.

S-(2,2,2-Tr iflu or oeth yl) 3-Oxoth iova ler a te (38c). ¹H

NMR δ: 1.09 (t, J ) 7.8 Hz, 3 H), 2.58 (q, J ) 7.8 Hz, 2 H),

3.64 (q, J ) 9.9 Hz, 2 H), 3.76 (s, 2 H). MS (CI/NH₃): m/ z 232

(M⁺+ NH₄, base), 214 (M⁺+ 1).

S-(2-Tetr a h yd r op yr a n yloxyleth yl) 3-Oxoth iova ler a te

1

(38d ). H NMR δ: 1.07 (t, J ) 7.8 Hz, 3 H), 1.53-1.84 (m, 6

H), 2.58 (q, J ) 7.8 Hz, 2 H), 3.18 (t, J ) 6.9 Hz, 2 H), 3.49-

3.62 (m, 2 H), 3.68 (s, 2 H), 3.82-3.90 (m, 2 H), 4.63 (m, 1 H).

MS (CI/NH₃): m/ z 278 (M⁺+ NH₄), 261 (M⁺+ 1).

S-(3-Tetr a h yd r op yr a n yloxylp r op yl) 3-Oxoth iova ler a te

1

(38e). H NMR δ: 1.07 (t, J ) 7.8 Hz, 3 H), 1.52-1.86 (m, 6

H), 1.91 (m, 2 H), 2.58 (q, J ) 7.8 Hz, 2 H), 3.04 (t, J ) 7.8 Hz,

2 H), 3.46 (m, 2 H), 3.67 (s, 2 H), 3.82 (m, 2 H), 4.58 (t, J ) 3.0

Hz, 1 H). MS (CI/NH₃): m/ z 292 (M⁺+ NH₄, base).

To prepare â-acetylthiopropionyl chloride (42c), the same

condition (for 42b) was adopted using acid 49.

P r ep a r a t ion of THP -P r ot ect ed Th iols 50 a n d 51

(Sch em e 4).⁴³A solution of 2-mercaptoethanol or 3-mercapto-

1-propanol (30 mmol) and 3,4-dihydro-2H-pyran (30 mmol) in

dry dichloromethane (25 mL) was cooled to 0 °C. While the

mixture was being stirred, 3 mmol of PPTS was carefully

added, and the reaction mixture was left at the same temper-

ature for 30 min before returning to room temperature. The

reaction was then stirred for 4 h, the PPTS was filtered off,

and the solvent was removed. Chromatography of the residue

(silica gel, petroleum ether-ethyl acetate (9:1)) gave the

desired thiol 50 (yield: 60%) or 51 (yield: 81%).

S-((2,2-Dim eth yl-1,3-d ioxola n -4-yl)m eth yl) 3-Oxoth io-

va ler a te (38f). H NMR δ: 1.08 (t, J ) 6.9 Hz, 3 H), 1.34 (s,

1

3 H), 1.43 (s, 3 H), 2.57 (q, J ) 6.9 Hz, 2 H), 3.15 (dd, J ) 6.0,

3.0 Hz, 2 H), 3.67 (m, 1 H), 3.70 (s, 2 H), 4.08 (m, 1 H), 4.26

(m, 1 H). MS (CI/NH₃): m/ z 264 (M⁺+ NH₄, base), 247 (M⁺

+ 1).

1

S-Eth yl 3-Oxoth io(ben zyloxylva ler a te) (38g). H NMR

δ: 1.27 (t, J ) 7.8 Hz, 3 H), 2.84 (t, J ) 6.9 Hz, 2 H), 2.92 (q,

J ) 7.8 Hz, 2 H), 3.70 (s, 2 H), 3.75 (t, J ) 6.9 Hz, 2 H), 4.51

(s, 2 H), 7.33 (m, 5 H). MS (CI/NH₃): m/ z 284 (M⁺+ NH₄,

base), 267 (M⁺+ 1).

1

F or 50. H NMR (CDCl₃/TMS) δ: 1.52-1.88 (m, 6 H), 1.72

1

S-Eth yl 3-Oxoth io(a cetylth iova ler a te) (38h ). H NMR

(t, J ) 3.9 Hz, 1 H), 2.73 (dt, J ) 6.9, 3.9 Hz, 2 H), 3.49-3.60

(m, 2 H), 3.83-3.93 (m, 2 H), 4.65 (t, J ) 3.0 Hz, 1 H). MS

(CI/NH₃): m/ z 180 (M⁺+ NH₄).

δ: 1.27 (t, J ) 7.8 Hz, 3 H), 2.32 (s, 3 H), 2.91 (q, J ) 7.8 Hz,

2 H), 2.94 (t, J ) 6.9 Hz, 2 H), 3.07 (t, J ) 6.9 Hz, 2 H), 3.66

(s, 2 H). MS (CI/NH₃): m/ z 252 (M⁺+ NH₄, base), 235 (M⁺

+

1

F or 51. H NMR (CDCl₃/TMS) δ: 1.39 (t, J ) 7.8 Hz, 1 H),

1).

1.52-1.59 (m, 4 H), 1.68-1.83 (m, 2 H), 1.86-1.95 (m, 2 H),

2.65 (dt, J ) 6.9, 7.8 Hz, 2 H), 3.50 (m, 2 H), 3.85 (m, 2 H),

4.59 (t, J ) 3.9 Hz, 1 H). MS (CI/NH₃): m/ z 194 (M⁺+ NH₄),

177 (M⁺+ 1).

(2,2,3,3,3-P en ta flu or op r op yl) Ben zoyl Aceta te (38j). ¹H

NMR δ: 4.12 (s, 2 H), 4.64 (t, J ) 12.6 Hz, 2 H), 7.43-7.66

(m, 3 H), 7.92-7.98 (m, 2 H). MS (CI/NH₃): m/ z 314 (M⁺

NH₄, base), 297 (M⁺+ 1).

+

P r ep a r a tion of Th iol 52 (Sch em e 4). 3-Mercapto-1,2-

propanediol (2.16 g, 20 mmol), PPTS (0.503 g, 2 mml), and

anhydrous MgSO₄(2 g) were dissolved in 20 mL of dry acetone.

The reaction mixture was stirred at room temperature for 2

days. Solid materials were filtered off. The filtrate was

evaporated, and the residue was separated with preparative

TLC (silica 60; 2000 mm; Analtech, Newark, DE; petroleum

ether-ethyl acetate (9:1)) to give 2.01 g of thiol 52 (yield: 68%).

¹H NMR (CDCl₃/TMS) δ: 1.37 (s, 3 H), 1.44 (s, 3 H), 1.48 (t, J

) 7.8 Hz, 1 H), 2.61 (m, 1 H), 2.75 (m, 1 H), 3.77 (m, 1 H),

4.09-4.23 (m, 2 H).

P r op yl o-F lu or oben zoyl Aceta te (38k ). ¹H NMR δ: 0.90

(t, J ) 7.8 Hz, 3 H), 1.64 (m, 2 H), 4.00 (s, 2 H), 4.12 (t, J )

6.9 Hz, 2 H), 7.13 (m, 1 H), 7.25 (m, 1 H), 7.57 (m, 1 H), 7.91

(m, 1 H). MS (CI/NH₃): m/ z 242 (M⁺+ NH₄, base), 225 (M⁺

+ 1).

P r op yl m -F lu or oben zoyl Aceta te (38l). ¹H NMR δ: 0.90

(t, J ) 7.8 Hz, 3 H), 1.64 (m, 2 H), 3.98 (s, 2 H), 4.12 (t, J )

6.9 Hz, 2 H), 7.31 (m, 1 H), 7.47 (m, 1 H), 7.65 (d, J ) 6.9 Hz,

1 H), 7.73 (d, J ) 6.9 Hz, 1 H). MS (CI/NH₃): m/ z 242 (M⁺

NH₄, base).

+

P r op yl p-F lu or oben zoyl Aceta te (38m ). ¹H NMR δ: 0.90

(t, J ) 7.8 Hz, 3 H), 1.61-1.72 (m, 2 H), 3.98 (s, 2 H), 4.12 (t,

J ) 6.9 Hz, 2 H), 7.11-7.19 (m, 2 H), 7.97-8.02 (m, 2 H). MS

(CI/NH₃): m/ z 242 (M⁺+ NH₄, base), 225 (M⁺+ 1).

Molecu la r Mod elin g. All calculations were performed on

a Silicon Graphics Indigo 2 R8000 workstation.

The MRS 1476 model was constructed using the “Sketch

Molecule” of SYBYL 6.4.2.²⁹Semiempirical molecular orbital

calculations were done using the AM1 Hamiltonian²¹as

implemented in MOPAC 6.0³⁰(keywords: PREC, GNORM )

0.1, EF, MMOK if necessary).

To prepare compound 46, a transesterification reaction was

used. Ethyl benzoyl acetate (45, 1.92 g, 10 mmol) and 1,3-

propanediol (0.761 g, 10 mmol) in toluene (10 mL) were heated

with stirring for 24 h. The solvent was removed, and the

residue was column chromatographed (silica 60, petroleum

ether-ethyl acetate (3:1)) to give 1.67 g of the desired product,

yield: 75%.

Hyd r oxylp r op yl Ben zoyl Aceta te (46). ¹H NMR δ: 1.87

(m, 2 H), 3.69 (t, J ) 6.0 Hz, 2 H), 4.03 (s, 2 H), 4.34 (t, J )

6.0 Hz, 2 H), 7.43-7.52 (m, 2 H), 7.58-7.64 (m, 1 H), 7.92-

7.96 (m, 2 H). MS (CI/NH₃): m/ z 240 (M⁺+ NH₄, base), 223

(M⁺+ 1).

The three-dimensional human A₃receptor model was built

and optimized using SYBYL 6.4.2 and Macromodel 5.0,³²

respectively, based on the approach described by Moro et al.²⁵

Briefly, the seven transmembrane helical domains were

identified with the aid of Kyte-Doolittle hydrophobicity³³and

33

E_minisurface probability parameters. The helices were built

and energy minimized for each transmembrane sequence. The

minimized helices were then grouped together to form a helical

bundle matching the overall characteristics of the electron

Synthesis of 3,5-diacyl-2,4-dialkylpyridines

J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 4 719

density map of rhodopsin. The helical bundle was energy-

minimized using the AMBER³⁴all-atom force field, until the

root mean square value of the conjugate gradient (CG) was

<0.1 kcal/mol/Å. A fixed dielectric constant of 4.0 was used

throughout these calculations.

CoMF A F ield Ca lcu la t ion s a n d R egr ession Tech -

n iqu es. The electrostatic and steric fields were sampled along

a three-dimensional lattice encompassing all molecules in each

receptor data set. The lattice consisted of 9261 sample points

based on a 1.0 Å lattice spacing with boundaries extending

2.0 Å beyond the largest structure in all directions. The 2.00,

1.50, and 1.25 Å lattice spacing was found not to improve the

CoMFA results. The lattice points within the union volume of

the superimposed structures were dropped. The probe used

to calculate the CoMFA fields consisted of an sp³carbon atom

with a +1 charge and a van der Waals radius of 1.52 Å. The

steric and electrostatic fields were calculated separately for

each molecule using a Lennard-J ones 6-12 potential and a

Coulombic potential with a 1/r distance-dependent dielectric.

The steric and electrostatic energies were truncated at 30 kcal/

mol. The field values corresponding to the 9261 sample points

for each molecule, together with binding affinity data, were

stored in a SYBYL Molecular Spreadsheet to facilitate statisti-

cal analysis.

Partial least-squares (PLS) regression analysis³⁹was per-

formed on the pyridine data sets using a subset of the CoMFA

field sample points falling within a standard deviation of e1.0

kcal/mol. The steric and the electrostatic fields were scaled to

equalize their weighting in the CoMFA models (SYBYL

command “scaling CoMFA_std”). PLS was performed using

cross-validation to evaluate the predictive ability of the CoMFA

models.⁴⁰The optimal number of latent variables was derived

from the cross-validation equation having the lowest standard

error, and a significance level of g99.5% was estimated using

the stepwise F-test. Bootstrap analysis⁴⁰of the data set was

used to evaluate the statistical confidence limits of the results.

A s value of 2.0 was adopted for both the cross-validated and

non-cross-validated analysis. The s values of 1.0 or 0.5 did not

significantly change the calculated r².

The MRS 1476 structure was rigidly docked into the helical

bundle using graphical manipulation with continuous energy

monitoring (Dock module of SYBYL). The manually docked

local energy minimized receptor-ligand complexes were sub-

jected to an additional conjugate gradient minimization run

of 300 steps. Partial atomic charges for the ligands were taken

from the MOPAC output files. A fixed dielectric constant of

4.0 was used throughout the docking calculations. We have

recently introduced the cross-docking procedure to obtain

energetically refined structures of GPCR-ligand complexes.²⁵

We applied this technique to predict the structure of the MRS

1476-A₃receptor complex. Cross docking allows possible

ligand-induced rearrangements of the 7TM bundle to be

explored by sampling 7TM conformations in the presence of

the docked ligands. Small translations and rotations were

applied to each helix relative to its original position until a

new lower energy geometry was obtained. These manual

adjustments were followed by 25 ps of molecular dynamics

(MD module of Macromodel) performed at a constant temper-

ature of 300 K using a time step of 0.001 ps and a dielectric

constant of 4.0. This procedure was followed by another

sequence of CG energy minimization to a gradient threshold

of <0.1 kcal/mol/Å. Energy minimization of the complexes was

performed using the AMBER all-atom force field in Macro-

Model.

CoMF A An a lysis. CoMFA¹⁸is a three-dimensional QSAR

method that operates on a set of ligands that have been

superimposed to reflect their anticipated common binding

orientation. CoMFA models describe the extent to which the

change in magnitude of the electrostatic and steric fields as a

function of compound, sampled as a function of spatial position

around the compound set, accounts for the variance in

measured biological activity. The CoMFA study was performed

using SYBYL 6.4.2,²⁹and quantum calculations used through-

out this study were performed using MOPAC (Ver. 6.0)³¹and

Gaussian 94.³⁵

Initial PLS analyses were performed in conjunction with the

cross-validation (leave-one-out method) option to obtain the

optimal number of components to be used in the subsequent

analyses of the data set. The PLS analysis was repeated with

the number of cross-validation groups set to zero. The optimal

number of components was designated as that which yielded

the highest cross-validated r²values in the non-cross-validated

(conventional) analyses. The final PLS analysis with 10

bootstrap groups and the optimal number of components was

performed on the complete data set.

Da ta Set a n d Molecu la r Su p er p osition . A total of 41

pyridine derivatives were included in the training set used to

generate the CoMFA model for A₃receptors (see Tables 1 and

6). The synthesis and binding affinity constants of 65-87

pyridine derivatives were reported in detail previously.¹⁶

The corresponding calibration equation (resulting from the

simultaneous contribution of all the observations) was derived

after the optimal dimensionality of each receptor model was

established, by PLS analysis and cross-validation. The calibra-

tion equation with latent variables was then converted to the

original parametric space represented by probe-ligand inter-

action energies. A 3D-QSAR was therefore derived whose

coefficients were associated with statistically significant lattice

locations. CoMFA coefficient contour maps were generated by

interpolation of the pairwise products between the 3D-QSAR

coefficients and the standard deviations of the associated

energy variables.

The pyridine moiety and the two ester groups at 3- and

5-positions are believed to be a key determinant of binding

interactions of pyridine derivatives. Therefore, the ligands in

this study were superimposed on both the pyridine nucleus

and the carbonyl of the 3- and 5-ester groups by fitting a

minimum energy conformation of the compound 6 (reference

structure). The low-energy conformer of compound 6 was

predicted in a previous study.¹⁶All the other compounds were

fitted by a least squares algorithm to the reference structure

so as to maximally align their substituents with the corre-

sponding substituents of the reference structure. The substit-

uents at the meta position of the 6-phenyl ring were aligned

using the best fit inside the hydrophobic cavity of the human

A₃receptor, as reported.¹⁹The fitted conformations of each

compound were fully minimized using MOPAC (AM1 Hamil-

tonian, keywords: PREC, GNORM)0.01, EF).

Atom ic Ch a r ge Ca lcu la tion s. Partial atomic charges are

required for calculating electrostatic fields in CoMFA. Partial

atomic charges for compound 6 were calculated at the semiem-

pirical level using the MNDO,³⁶AM1, and PM3³⁷Hamiltonians

of MOPAC. The results of each method were compared to those

obtained using the RHF/6-31G(*)//RHF/3-21G(*) ab initio level

of Gaussian 94. The charges derived from electrostatic poten-

tials³⁸and calculated using AM1 were found to agree best with

the ab initio charges. Therefore, partial atomic charges for all

the pyridine derivatives were calculated using the AM1

Hamiltonian.

Th e Test Set. The test set consisted of three molecules (10,

24, and 27, see Table 4). These structures were chosen to

maximize a uniform sampling of biological activity. All pre-

dicted activities for the test set molecules were calculated using

the optimized CoMFA model. The results of the non-cross-

validated calibration model on the test sets are summarized

in Table 5.

“P r ed ictive” r ²Va lu es. The “predictive” r²

was based

pred

only on molecules not included in the training set and is

defined as explained by Marshall and co-workers.⁴¹

P h a r m a cology. Ra d ioliga n d Bin d in g Stu d ies. Binding

of [³H]R-N⁶-phenylisopropyladenosine ([³H]R-PIA) to A₁recep-

tors from rat cerebral cortex membranes and of [³H]-2-[4-[(2-

carboxyethyl)phenyl]ethylamino]-5′-N-ethylcarbamoyladeno-

sine ([³H]CGS 21680) to A_2Areceptors from rat striatal

membranes was performed as described previously.^20,21

720 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 4

Li et al.

Adenosine deaminase (3 units/mL) was present during the

preparation of the brain membranes, in a preincubation of 30

min at 30 °C, and during the incubation with the radioligands.

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Binding of [¹²⁵I]N⁶-(4-amino-3-iodobenzyl)-5′-N-methylcar-

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[

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All nonradioactive compounds were initially dissolved in

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where the amount of DMSO never exceeded 2%.

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man GF/B filters, using a Brandell cell harvester (Brandell,

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Abbr evia tion s

[

¹²⁵I]AB-MECA, [¹²⁵I]N⁶-(4-amino-3-iodobenzyl)-5′-N-

methylcarbamoyladenosine; CGS 21680, 2-[4-[(2-car-

boxyethyl)phenyl]ethyl-amino]-5′-N-ethylcarbamoyladeno-

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comparative molecular field analysis; DMAP, N,N-

dimethylaminopyridine; DMSO, dimethyl sulfoxide;

DAST, diethylaminosulfur trifluoride; DPPA, diphen-

ylphosphoryl azide; EDAC, 1-ethyl-3-(3-dimethylami-

nopropyl)carbodiimide; HEK cells, human embryonic

kidney cells; IB-MECA, N⁶-(3-iodobenzyl)-5′-N-methyl-

carbamoyladenosine; K_i, equilibrium inhibition con-

stant; MRS 1191, 3-ethyl 5-benzyl 2-methyl-6-phenyl-

4-phenylethynyl-1,4-(()-dihydropyridine-3,5-dicarbox-

ylate; MRS 1476, 2,3,4,5-tetraethyl-6-phenylpyridine-

3-thiocarboxylate-5-carboxylate; MRS 1523, 2,3-ethyl-

4,5-dipropyl-6-phenylpyridine-3-thiocarboxylate-5-car-

boxylate; PCC, pyridinium chlorochromate; PLS, partial

least squares; PPTS, pyridinium p-toluenesulfonate;

R-PIA, R-N⁶-phenylisopropyladenosine; SAR, structure-

activity relationship; SEAL, steric and electrostatic

alignment; TBAF, tetrabutylammonium fluoride; THP,

tetrahydropyranyl; TM, transmembrane helical domain;

Tris, tris(hydroxymethyl)aminomethane.

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J M980550W

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Synthesis, CoMFA analysis, and receptor docking of 3,5-diacyl-2,4- dialkylpyridine derivatives as selective A3 adenosine receptor antagonists

DOI: 10.1021/jm980550w

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