Synthesis of tricyclic 1,3-oxazin-4-ones and kinetic analysis of cholesterol esterase and acetylcholinesterase inhibition

8270

J. Med. Chem. 2005, 48, 8270-8288

Synthesis of Tricyclic 1,3-Oxazin-4-ones and Kinetic Analysis of Cholesterol

Esterase and Acetylcholinesterase Inhibition

Markus Pietsch and Michael Gu¨tschow*

Pharmaceutical Institute, University of Bonn, Kreuzbergweg 26, D-53115 Bonn, Germany

Received August 31, 2005

A series of thieno[1,3]oxazin-4-ones and thieno[1,3]thiazin-4-ones were synthesized and

investigated as inhibitors of the R/â hydrolases cholesterol esterase (CEase) and acetylcho-

linesterase (AChE). The introduction of a cycloaliphatic five- or six-membered ring fused at

the thiophene was favorable for CEase inhibition. Such compounds were analyzed as true

alternate substrate inhibitors. 6,7-Dihydro-2-(dimethylamino)-4H,5H-cyclopenta[4,5]thieno-

[2,3-d][1,3]oxazin-4-one (33) exhibited a K_ivalue of 630 nM and excelled in its low susceptibility

to CEase-catalyzed degradation. Compound 33 and its analogues did not inhibit AChE. The

introduction of a tetrahydropyrido ring with bulky hydrophobic substituents at the basic

nitrogen provided inhibitors of AChE which were completely inactive toward CEase. 7-Benzyl-

5,6,7,8-tetrahydro-2-(N-3,4-dimethoxybenzyl-N-methylamino)-4H-pyrido[4′,3′:4,5]thieno[2,3-d]-

[1,3]oxazin-4-one (21) had the IC₅₀value of 330 nM for AChE inhibition. A residual enzymatic

activity at an infinite inhibitor concentration and thus a catalytically active ternary enzyme-

substrate-inhibitor complex was concluded. To specify kinetic parameters of inhibition, a new

method was derived to characterize selected thieno[1,3]oxazin-4-ones as hyperbolic mixed-type

inhibitors of AChE.

Introduction

strongly dependent on bile salt activation.¹⁶CEase

mainly originated from pancreatic acinar cells and

lactating glands of higher mammals and released into

the circulation was also found in the liver, in macro-

phages, endothelial cells, and eosinophiles.¹⁶Once

secreted into the lumen of the duodenum, pancreatic

CEase, together with other pancreatic lipolytic enzymes

and preduodenal lipase, acts to complete the digestion

of dietary lipids.^12,13

On the basis of a proposed model, CEase has a dual

effect on cholesterol absorption by virtue of both the

hydrolysis of dietary cholesterol esters and lecithin.

Thus, CEase contributes to the formation of lysolecithin-

rich micelles, which act as vesicles for an efficient

absorption of free cholesterol.¹⁷It has been reported that

cholesterol uptake into Caco-2 cells, an enterocyte

model, from lecithin-containing micelles was accelerated

by CEase.¹⁸

The role of CEase has been suggested to extend

beyond that of hydrolyzing dietary lipids. It enhanced

sterol transfer between small unilamellar phospholipid

vesicles in vitro¹⁹and effected plasma lipoprotein me-

tabolism.¹⁶Several lines of evidence indicated a role of

vascular CEase in atherosclerosis. CEase may be pro-

atherogenic since it facilitated the conversion of the

larger and less atherogenic low-density lipoprotein

particles to smaller and more atherogenic lipoprotein

subspecies.^16,20The catalytic activity of CEase led to a

high degree of unesterified cholesterol within the core

of low-density lipoproteins. Such modified lipoprotein

species resembled aortic unilamellar and multilamellar

lipid particles that accumulate in the extracellular

spaces of atherosclerotic lesions.^21-23CEase was also

shown to act synergistically with sphingomyelinase to

promote cholesterol crystal nucleation and aggregation

Cholesterol esterase (CEase, EC 3.1.1.13) and acetyl-

cholinesterase (AChE, EC 3.1.1.7) belong to the R/â

hydrolase family.^1,2This protein family is characterized

by the R/â hydrolase fold, a structural motive consisting

of a mostly parallel, eight-stranded â sheet surrounded

on both sides by R helices. Although their members are

related by divergent evolution, the R/â hydrolase fold

is able to provide a stable scaffold with spatial positions

for the active site components, that are well conserved

in all enzymes of the family.^2-5CEase and AChE

contain a catalytic triad composed of serine, histidine

and an acidic amino acid (CEase: Asp, AChE: Glu), that

are positioned in very short loops beyond the end of

strands five, eight and seven, respectively.^1,3Like serine

proteases, CEase and AChE utilize an acylation-

deacylation mechanism. Both the acylation and deacy-

lation stages transit tetrahedral intermediates that are

stabilized by a bi- or tripartite oxyanion hole.^1,3,4,6,7

While there are similarities in the tertiary structure and

the catalytic mechanism, CEase and AChE differ in the

substrate specificity as well as in their physiological

functions and their role in pathological processes.

The three-dimensional architecture of CEase is known

from several crystal structures.^8-11CEase is a less

specific lipolytic enzyme. The activation by primary bile

salts is required for the hydrolysis of a broad spectrum

of substrates, including lipophilic compounds with long-

chain fatty acid moieties such as cholesterol esters,

triacylglycerols, phospholipids, and ceramides.^12-16The

hydrolysis of water-soluble substrates such as esters of

short-chain fatty acids or lysophospholipids is not

* To whom correspondence should be addressed (phone: +49 228

732317; fax: +49 228 732567; e-mail: guetschow@uni-bonn.de).

Published on Web 11/12/2005

Tricyclic 1,3-Oxazin-4-ones

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 26 8271

of low-density lipoproteins. Both events are regarded

to be important in atherosclerosis.²⁴In contrast, CEase

may be antiatherogenic by facilitating the selective

uptake of high-density lipoprotein-associated cholesterol

esters by liver cells and thus reducing cholesterol

accumulation in peripheral tissues. Furthermore, the

enzyme might reduce the atherogenicity of oxidized low-

density lipoproteins by decreasing its lysophosphatidyl-

choline and ceramide content.^16,25-28

Much attention has focused on the inhibition of CEase

as a potential target particularly for the development

of hypocholesterolemic agents. Several classes of mech-

anism-based inhibitors have been described including

boronic and borinic acids, aryl haloketones, aryl phos-

phates and phosphonates as well as aryl and cholesteryl

carbamates with the carbamates being the most inten-

sive studied inhibitors of CEase.^29-32Substituted 6-chloro-

2-pyrones are representatives of a further class of potent

CEase inhibitors.^33,346-Chloro-3-(1-ethyl-2-cyclohexyl)-

2H-pyran-2-one was found to inhibit the hydrolysis of

cholesteryl oleate in vivo, thereby limiting the bioavail-

ability of cholesterol derived from cholesterol esters. As

a result, the appearance of cholesterol in serum and

liver of hamsters was reduced in a dose-dependent

manner.¹⁷

In a previous paper, we investigated the inhibition

kinetics of the reaction of some fused 1,3-oxazin-4-ones

and 1,3-thiazin-4-ones with bovine pancreatic CEase.³⁵

Certain thieno[1,3]oxazin-4-ones acted as alternate

substrate inhibitors being hydrolyzed by CEase under

steady-state conditions via an acylation-deacylation

mechanism, similar to the interaction of 3,1-benzoxazin-

4-ones and analogous thieno[1,3]oxazin-4-ones with

serine proteases.^36-412-Diethylamino-6,7-dihydro-4H,5H-

cyclopenta[4,5]thieno[2,3-d][1,3]oxazin-4-one was shown

to significantly inhibit the CEase-catalyzed cleavage of

cholesterol esters in isolated human high-density lipo-

proteins. In that study, matrix-assisted laser desorption

and ionization time-of-flight mass spectrometry (MALDI-

TOF MS) was used to investigate the extracts of human

lipoproteins after treatment with CEase and to monitor

the effects of the inhibitor.⁴²

AChE is a serine hydrolase⁴³that exhibits a broad

substrate specificity for various amides and esters

including acyl homologues and N-demethylated ana-

logues of acetylcholine. Specificity of AChE is highest

for acetylselenocholine, acetylthiocholine (ATCh), and

acetylcholine;⁴⁴it hydrolyzes acetylcholine faster than

other choline esters and presents little activity on

butyrylcholine.⁴⁵

fibrils formed by Aâ alone.^53,54AChE was found to be

associated with amyloid plaques and neurofibrillary

tangles in Alzheimer’s disease (AD) and may contribute

to their development.^55-57

AD is a slowly progressive dementia paralleled by

selective neuronal cell death, which is probably caused

by Aâ fibrils or oligomers.^58,59In addition, the ‘cholin-

ergic hypothesis’ was focused on the loss of cholinergic

neurotransmission in AD.⁶⁰This has provided the

rationale for the current major therapeutic approach to

AD, the inhibition of the catalytic function of AChE,

thereby increasing the bioavailability of acetylcholine

at the synaptic cleft and improving the cholinergic

neurotransmission as well as the cognitive functions.^61,62

Tacrine (Cognex), donepezil (Aricept), galanthamine

(Reminyl) and rivastigmine (Exelon) are currently ap-

proved AChE inhibitors for the treatment of AD.⁶³

Beside its central role in the therapy of AD, AChE has

been targeted in treatments for myasthenia gravis,

glaucoma, obstipation, and spasmolysis and to antago-

nize muscle relaxation in anesthesiology.⁶⁴

While the catalytic function is associated to the active

site of AChE, the peripheral anionic site (PAS) modu-

lates the ‘nonclassical’ activities of the enzyme.⁶⁵The

PAS was shown to be involved in an accelerated Aâ fibril

formation, and thus, low molecular weight compounds

that bind to PAS as well as a PAS-directed monoclonal

antibody did not only inhibit the catalytic activity but

also prevented the amyloidogenic effect of AChE.^66,67On

the other hand, AChE inhibitors that primarily interact

with the active site had no effect on amyloid polymer-

ization.⁶³The PAS, situated at the surface of AChE, is

connected with the active site by a deep and narrow

gorge (around 20 Å long), lined by fourteen highly

conserved aromatic residues. The active site is located

at the bottom of the gorge and consists of the esteratic

site, where hydrolysis proceeds via the catalytic triad

Ser200-His440-Glu327 (Torpedo californica AChE num-

bering),⁶⁸as well as the anionic site with the indole ring

of the conserved Trp84, that has been shown to interact

with the quaternary ammonium group of acetylcholine

via a cation-π interaction.^46,68Another conserved aro-

matic residue, Phe330, is also involved in this interac-

tion. Similarly, the principal element of PAS is the

indole of Trp279.⁶⁹

Considering the role of the PAS relative to its involve-

ment in the processes of AD, the development of dual

site inhibitors of AChE, e.g. acyloxybiphenyl carba-

mates,⁷⁰donepezil and AP2238,⁷¹is driven by the fact

that they may simultaneously alleviate the cognitive

deficit in AD due to inhibition of the catalytic activity

and avoid the assembly of Aâ by interacting with the

PAS.

On the basis of these considerations and previous

results,³⁵we have prepared a series of thieno[1,3]oxazin-

4-ones and thieno[1,3]thiazin-4-ones and investigated

their ability to inhibit bovine pancreatic CEase as well

as AChE from Electrophorus electricus. The interaction

of selected compounds with the target esterases was

kinetically characterized. As it is demonstrated in the

results section, two concepts, the alternate substrate

inhibition of CEase and a dual site inhibition of AChE,

have been implemented in this study.

The physiological role of AChE, the hydrolytic de-

struction of the neurotransmitter acetylcholine at cho-

linergic synapses, is necessary for the temporal control

of cholinergic transmission. Besides this ‘classical’ func-

tion, AChE was reported to exhibit several ‘nonclassical’

activities.^45,46The enzyme was suggested to act as an

adhesion protein in synaptic development and mainte-

nance⁴⁶as well as to be involved in neurite growth,⁴⁷

hematopoiesis and osteogenesis.⁴⁸In addition to these

physiological functions, AChE has been shown to ac-

celerate the pathophysiological assembly of the amy-

loid-â (Aâ) peptide into amyloid fibrils in vitro^49,50

and in vivo,^51,52with complexes of AChE and Aâ

displaying an enhanced neurotoxicity in comparison to

8272 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 26

Pietsch and Gu¨tschow

Scheme 1^a

Scheme 4^a

^aReagents: (a) RNCO, pyridine, 70 °C; (b) TFA, trifluoroacetic

anhydride, 0-25 °C; (c) H₂O, NaHCO₃, 0 °C.

Scheme 2^a

^aReagents: (a) p-nitrophenyl chloroformate, pyridine, Cl(CH₂)₂Cl,

0 °C; (b) HNR¹R², Cl(CH₂)₂Cl, 0 °C (14, 18), or 0-25 °C (15-17);

(c) TFA, trifluoroacetic anhydride, 0-25 °C (19, 20, 22, 23), or TFA,

0 °C, then trifluoroacetic anhydride, CH₂Cl₂, 0-4 °C (21); (d) H₂O,

NaHCO₃, 0 °C (19, 20, 22, 23), or CH₃OH/H₂O, NaHCO₃, 0 °C

(21).

^aReagents: (a) phenyl isothiocyanate, C₂H₅OH, 80 °C (6, R )

Ph), or benzoyl isothiocyanate, CH₃COCH₃, room temperature (6,

R ) Bz was used in the next step without purification); (b) TFA,

0-25 °C; (c) H₂O, NaHCO₃, 0 °C; (d) HgO, CH₂Cl₂, room temper-

ature.

Scheme 5^a

Scheme 3^a

^aReagents: (a) dichloromethylene dimethylammonium chloride,

1 M ethereal HCl, Cl(CH₂)₂Cl, reflux; (b) n-hexane, reflux (33, 35,

and 36), or C₂H₅OH/H₂O, 0 °C (34), or H₂O, Na₂CO₃, 0 °C (37-

40).

Scheme 6^a

^aReagents: (a) trichloroacetyl isocyanate, THF, 0-10 °C; (b)

CH₃OH, THF, 60 °C; (c) TFA, 0-25 °C; (d) trifluoroacetic anhy-

dride, TFA, 0-25 °C; (e) CH₃OH/H₂O, NaHCO₃, 0 °C.

Results and Discussion

Synthesis. The Gewald thiophene synthesis^72,73al-

lowed for a facile synthetic entry to the classes of thieno-

[2,3-d][1,3]oxazin-4-ones and thieno[2,3-d][1,3]thiazin-

4-ones. Tertiary butyl ester (Schemes 1-5) or ethyl ester

(Scheme 6) of 2-aminothiophene-3-carboxylates were

used as starting materials to prepare the final thieno-

[2,3-d][1,3]oxazin-4-ones and thieno[2,3-d][1,3]thiazin-

4-ones, respectively. The requisite aminothiophenes 1

and 26-32 (Scheme 5) were synthesized from the

appropriate ketone, sulfur, and tert-butyl cyanoacetate

in the presence of morpholine by an one-pot thiolation-

heterocyclization reaction. To afford 25 (Scheme 5), the

Knoevenagel adduct of cyclopentanone and tert-butyl

cyanoacetate was prepared and subsequently treated

with sulfur in the presence of diethylamine.

^aReagents: (a) benzoyl isothiocyanate, CH₃COCH₃(43) or

RNCS, C₂H₅OH (44-46), reflux; (b) 94% H₂SO₄, room temperature

- 100 °C (47 from 43), or concd H₂SO₄, room temperature (48-

50, 51 from 43); (c) H₂O, NaOH, 0 °C (47, 49-51), or H₂O,

NaHCO₃, 0 °C (48).

propyl isocyanate or cyclohexyl isocyanate in pyridine

to yield ureas 2 and 3, respectively. Subsequent treat-

ment with a mixture of trifluoroacetic acid and tri-

fluoroacetic anhydride resulted in deesterification and

cyclocondenstaion, to obtain 4 and 5.

The preparation of 2-phenylamino- and 2-benzoyl-

amino-substituted thienoxazinones is shown in Scheme

2-Alkylamino-substituted thienoxazinones (Scheme 1)

were synthesized according to the procedure of Hallen-

bach et al.⁷⁴Aminothiophene 1 was reacted with iso-

Tricyclic 1,3-Oxazin-4-ones

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 26 8273

2. The thiourea derivative 6 was easily obtained from

aminothiophene 1 and phenyl isothiocyanate. Depro-

tection of 6 with trifluoroacetic acid followed by cycliza-

tion using yellow mercury(II) oxide in dichloromethane³⁹

at room temperature furnished the final product 7.

Compound 8 was similarly prepared without isolating

the thiourea intermediate.

The synthetic route to the 2-aminothienoxazin-4-one

12 is outlined in Scheme 3. Aminothiophene 1 was

converted to urea 9 by the action of trichloroacetyl

isocyanate. Cleavage of the trichloroacetyl residue with

methanol yielded 10, which was reacted with trifluoro-

acetic acid to obtain the 2-ureidothiophene-3-carboxylic

acid 11. Ring closure to 12 was then performed upon

the action of trifluoroacetic anhydride.

The synthesis of 2-secondary-amino-substituted thien-

oxazinones 19-23 was accomplished by a novel route

shown in Scheme 4. The urethane hydrochloride 13 was

easily obtained by the reaction of the aminothiophene

1 with p-nitrophenyl chloroformate in the presence of

pyridine. Nucleophilic attack of secondary amines at the

activated urethane carbonyl readily afforded ureas 14-

18, which were subsequently cyclized to the desired

thienoxazinones.

2-Dimethylamino-substituted thienoxazinones 33-41

(Scheme 5) were synthesized by a facile one-step route

according to Player et al.⁷⁵Aminothiophenes 1 and 25-

32 were reacted with dichloromethylene dimethylam-

monium chloride in the presence of ethereal hydrochlo-

ric acid to form the hydrochlorides of the desired

products. Treatment of the hydrochlorides with boiling

n-hexane (33, 35, and 36) or 50% C₂H₅OH (34), purifi-

cation by column chromatography (41), or addition of a

saturated sodium carbonate solution followed by column

chromatography (37-40) yielded the thienoxazinones

33-41.

acids and thieno[2,3-d]pyrimidin-2,4-diones, respec-

tively. Benzoylamino derivatives 8 and 51 (Table 4) are

the most acidic substances of the present series, and

hence, no conversion was observed at pH 11 over a

period of 24 h. Both the alkaline hydrolysis of thieno-

[1,3]oxazin-4-ones and the process by which they inhibit

CEase involve an attack of a nucleophilic oxygen upon

the C-4 carbonyl carbon. Structural features promoting

enzyme inhibition over chemical reactivity are crucial

for the design of heterocyclic alternate substrate inhibi-

tors.

Inhibition of CEase and AChE. The kinetic pa-

rameters of the inhibition of CEase and AChE by 36

compounds were determined spectrophotometrically and

are given in Tables 1-4. CEase inhibition was assayed

in the presence of p-nitrophenyl butyrate (pNPB) as a

chromogenic substrate. K_ivalues toward CEase were

obtained from measurements in the presence of a single

concentration or at various concentrations of the inhibi-

tor, [I], in consideration of the substrate concentration

and a formal competitive type of inhibition.

The activity of AChE was determined in a coupled

assay with the substrate ATCh and 5,5′-dithio-bis-(2-

nitrobenzoic acid) (DTNB). Active inhibitors of AChE

were used at five or six different concentrations. Plots

of the rates versus [I], however, did not become asymp-

totic to the x-axis. A residual activity at infinite con-

centration of the inhibitor, v_[I]f∞, had to be considered.

The value of v_[I]f∞at a defined substrate concentration

can be obtained from a plot of the rates versus [I] and

nonlinear regression according to eq 1,

(v₀- v_[I]f∞

)

v )

+ v_[I]f∞

(1)

[I]

1 +

(

)

IC₅₀

The preparation of thienothiazin-4-ones is shown in

Scheme 6. In contrast to the synthetic routes to afford

the thienoxazinones via tert-butyl precursors (Schemes

1-5), the thienothiazinones 47-51 were prepared by a

route using the ethyl 2-aminothiophenecarboxylate 42

as starting material. Treatment with isothiocyanates

gave the thiourea derivatives 43-46. Ring closure to

47-51 was then performed upon the action of concen-

trated sulfuric acid, according to previously reported

procedures.^76,77

where v₀is the velocity in the absence of the inhibitor.

The IC₅₀value calculated by this equation corresponds

to the concentration of the inhibitor which reduces the

rate of the enzyme-catalyzed reaction to a velocity (v₀

- v_[I]f∞)/2 + v_[I]f∞, i.e. the velocity half between v₀and

v_[I]f∞. Data v_[I]f∞as relative velocities with respect to

v₀, as well as IC₅₀values are given in Tables 1-4. To

judge the potency of inhibitors of AChE mainly IC₅₀but

also the values v_[I]f∞were taken into account.

Inhibition of the target enzymes by 2-diethylamino-

substituted oxazinones is shown in Table 1. Different

residues R⁵and R⁶were introduced or formed together

with two thiophene carbons a further fused ring. Polar

groups, such as CONH₂in compound 55 or the basic

tetrahydropyridine unit in 19, were unfavorable for an

inhibition of CEase. In contrast, nonpolar moieties,

particularly when forming a bridged ring system, re-

sulted in potent inhibition of CEase. K_ivalues less than

2 µM were determined for compounds with cycloaliphat-

ic five- and six-membered rings. The methyl-branched

derivative 58 was found to be similarly active as 56 and

57, which have already been described in a previous

study.³⁵When the 5-isopropyl residue in 53 was con-

nected with the thiophene C-6 carbon by an ethylene

bridge, the enhanced rigidity promoted a 4-fold increase

in potency (53 versus 58). However, ring closure of 54

with a methylene unit also resulted in pronounced

inhibition (54 versus 56), an effect that can rather be

Alkaline Hydrolysis. Stability of the compounds

was measured spectrophotometrically in aqueous buffer,

pH 11.0, at 25 °C. Alkaline hydrolysis rate constants of

the thieno[1,3]oxazin-4-ones as well as the thieno[1,3]-

thiazin-4-ones were determined by the disappearance

of the long-wavelength ultraviolet chromophore that

followed the first-order exponential decay. The decade

-

logarithms of the second-order rate constant, log k_OH

,

were calculated and outlined in Tables 1-4. In com-

parison to thienoxazinones with a disubstituted 2-amino

group, compounds 4, 5, and 7 bearing a monosubstituted

-

2-amino moiety showed an increased stability (log k_OH

e -1.90) which might arise from deprotonation at the

2-amino substituent.^78,79Resonance stabilization of the

negative charge in thieno[1,3]oxazin-4-ones reduces the

electrophilicity at C-4 carbon. As described,^38,39aqueous

alkaline hydrolysis proceeds by hydroxide attack at C-4

to produce the corresponding ureidothiophenecarboxylic

8274 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 26

Pietsch and Gu¨tschow

Table 1. Inhibition of Bovine CEase and AChE from Electrophorus electricus^a

CEase

AChE

b

R⁵

Me

i-Pr

Me

R⁶

K_i(µM)

15

6.4

4.3

>50

IC₅₀(µM)

>50

v_[I]f∞

log k_OH(M^-1s^-1

)

-

compd

52

53

54

55

56

57

58

59

60

19

H

-

-1.14^c

-1.27^c

-1.63^c

-0.40^d

-1.03^c

-1.59^d

-1.41^c

-1.42^c

-1.13^c

-1.35

H

>50

Me

CONH₂

-(CH₂)₃-

0.58 ( 0.05^e

1.86 ( 0.05^e

1.64 ( 0.05

6.0

-(CH₂)₄-

-CH(Me)(CH₂)₃-

-(CH₂)₅-

-CHdCH-CHdCH-

-CH₂CH₂N(Bn)CH₂-

7.5

>50

2.08 ( 0.07

0.061 ( 0.004

^aValues with standard error were calculated from duplicate experiments at five or six inhibitor concentrations; those without standard

error are mean values of duplicate inhibition experiments at a single inhibitor concentration. ^bValues of v_[I]f∞were calculated relative

to v₀. ^cData from ref 39. ^dData from ref 38. ^eData from ref 35.

Table 2. Inhibition of Bovine CEase and AChE from Electrophorus electricus^a

CEase

AChE

R¹

R²

K_i(µM)

IC₅₀(µM)

v_[I]f∞

log k_OH(M^-1s^-1

)

-

compd

61

34

57^c

62

63

H

i-Pr

Me

Et

>50

-

-0.49^b

-1.24

-1.59^b

-0.79

-1.08^b

Me

Et

2.15 ( 0.19

1.86 ( 0.05^d

18

5.7

Me

cyclohexyl

-(CH₂)₂O(CH₂)₂-

^aValues with standard error were calculated from duplicate experiments at five or six inhibitor concentrations; those without standard

error are mean values of duplicate inhibition experiments at a single inhibitor concentration. ^bData from ref 38. ^cData are also noted in

Table 1. ^dData from ref 35.

Table 3. Inhibition of Bovine CEase and AChE from

attributed to an enhancement in lipophilicity than in

rigidity. A further variation in the size of the fused

cycloaliphatic moiety diminished activity as in case of

59 bearing a seven-membered ring.

Electrophorus electricus^a

Among the compounds outlined in Table 1, inhibition

of AChE was determined only for the tetrahydropyrido

derivative 19. Compound 19 is different from the other

2-diethylamino derivatives in Table 1 in that it bears a

bulky benzyl residue attached at a basic nitrogen. This

finding prompted us to design a series of analogues of

the parent 19 to further elucidate structure-activity

relationships of tetrahydropyrido-anellated thieno[1,3]-

oxazinones as inhibitors of AChE. The corresponding

results are presented below (Table 3, Table 4).

The tetramethylene bridge of 57 was then maintained

and different amino groups were introduced at position

2 (Table 2). Dimethylamino substitution in 34 provided

an additional potent inhibitor of CEase. The increased

steric requirement of the cyclohexyl methylamino group

was unfavorable (62 versus 34) as well as the polar

connection of the ethyl rests of 57 as achieved in the

morpholino derivative 63. As expected from the afore-

mentioned results, such tetramethylene compounds did

not inhibit AChE.

CEase

AChE

-

log k_OH

b

compd n

X

K_i(µM)

IC₅₀(µM)

v_[I]f∞

(M^-1s^-1

)

33 0 CH₂

0.63 ( 0.03 >50

2.15 ( 0.19 >50

-

-1.13

-1.24

-1.00

-1.05

34^c1 CH₂

35

36

37

38

39

40

41

1 O

1 S

>50

5.76 ( 0.23 >50

1 N-Me >50

1 N-Et >50

1 N-i-Bu >50

1 N-Bn >50

1 N-Bz >50

38.9 ( 1.2

10.5 ( 0.5

0.076 ( 0.006 -0.98

0.064 ( 0.006 -0.99

0.36 ( 0.01 0.043 ( 0.002 -1.16

0.89 ( 0.02 0.066 ( 0.003 -0.97

35.6 ( 1.7

0.266 ( 0.010 -0.79

^aValues with standard error were calculated from duplicate

experiments at five or six inhibitor concentrations; those without

standard error are mean values of duplicate inhibition experiments

at a single inhibitor concentration. ^bValues of v_[I]f∞were calcu-

lated relative to v₀. ^cData are also noted in Table 2.

contraction to the fused cyclopentene-containing ana-

logue increased CEase inhibition. The resulting inhibi-

tor 33 exhibited a K_ivalue of 630 nM. The 7-methylene

group of 34 was exchanged for sulfur or oxygen. The

increasing polarity of the carbon-sulfur and carbon-

oxygen bonds was still tolerated in the former case 36

but gave an inactive tetrahydropyran derivate 35. Basic

tetrahydropyridines 37-40 confirmed this tendency.

Our next approach was to attain structural diversity

in the ring fused to the thiophene nucleus of thieno-

[1,3]oxazinones (Table 3). The 2-dimethylamino moiety,

as it occurs in 34, was retained unchanged. In ac-

cordance to the data from the 2-diethylamino series, ring

Tricyclic 1,3-Oxazin-4-ones

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 26 8275

Table 4. Inhibition of Bovine CEase and AChE from Electrophorus electricus^a

CEase

AChE

b

R¹

R²

X

K_i(µM)

IC₅₀(µM)

v_[I]f∞

log k_OH(M^-1s^-1

)

-

compd

12

47

4

48

5

49

7

50

8

51

40^d

19^e

20

21

22

23

H

Me

Et

Me

H

O

S

O

S

O

S

O

S

>50

5.90 ( 0.20

1.20 ( 0.05

0.74 ( 0.44

0.58 ( 0.03

0.056 ( 0.006

0.045 ( 0.004

0.544 ( 0.035

0.137 ( 0.006

0.806 ( 0.008

0

0.550 ( 0.018

0.213 ( 0.012

0.064 ( 0.012

-1.47

-2.25

-1.97

-2.18

-2.42

-2.15

-1.90

-1.70

i-Pr

cyclohexyl

Ph

Bz

Me

-

2.82 ( 0.22

0.94 ( 0.24

1.03 ( 0.07

2.86 ( 0.21

5.2

0.89 ( 0.02

2.08 ( 0.07

0.84 ( 0.07

0.33 ( 0.01

0.77 ( 0.07

1.32 ( 0.13

c

O

S

-

c

-

O

0.066 ( 0.003

0.061 ( 0.004

0.212 ( 0.008

0.042 ( 0.004

0.059 ( 0.011

0.085 ( 0.014

-0.97

-1.35

-0.51

-1.24

-0.82

-1.06

Et

Bn

3,4-(OMe)₂-Bn

-(CH₂)₂O(CH₂)₂-

-(CH₂)₂N(Bn)(CH₂)₂-

^aValues with standard error were calculated from duplicate experiments at five or six inhibitor concentrations; those without standard

error are mean values of duplicate inhibition experiments at a single inhibitor concentration. ^bValues of v_[I]f∞were calculated relative

to v₀. ^cNo conversion was observed after 24 h. ^dData are also noted in Table 3. ^eData are also noted in Table 1.

These tertiary amines were inactive toward CEase as

the nonbasic, but polar benzamide 41. The following

structure-activity relationships could be deduced from

the data shown in Tables 1-3, (i) a dimethylamino or

diethylamino group at position 2 and (ii) a cycloaliphatic

five- or six-membered ring fused at the thiophene were

favorable for CEase inhibition. Accordingly, 14 other

derivatives that did not fulfill these requirements were

inactive (Table 4).

From the data in Table 3, significant structure-

activity relationships could also be ascertained for AChE

inhibition. A nitrogen at position 7 was necessary to

obtain anticholinesterases (33-36 versus 37-41), but not

sufficient for strong inhibition which required a lipo-

philic substituent at a basic nitrogen. The isobutyl and

the benzyl compound (39 and 40) showed IC₅₀values

toward AChE in the nanomolar range. Noteworthy, the

extension of the methyl rest in 37 by an isopropyl or a

phenyl group in 39 or 40, respectively, increased inhibi-

tory potency by 2 orders of magnitude.

N-Benzyl substitution at the tetrahydropyrido unit of

tricyclic thieno[1,3]oxazinones was found to be advanta-

geous for AChE inhibition (19, Table 1; 40, Table 3).

This substructure was therefore maintained whereas

the 2-amino substituent was varied and the ring oxygen

was replaced by sulfur in certain cases (Table 4). In

general, the representatives of the so defined structure

exhibited AChE inhibiting potency. The differences

between the NH₂-, primary-amino and secondary-amino

derivatives were rather small. The exchange of the ring

oxygen for sulfur did not substantially alter the IC₅₀

values. The residual activity at infinite inhibitor con-

centration (v_[I]f∞) was found to be lowered in case of the

thieno[1,3]thiazin-4-ones (12, 4, 5, 7, X ) O, versus 47-

50, X ) S). When one methyl group of the dimethyl-

amino compound 40 was replaced by benzyl, the same

IC₅₀value but an increased residual activity was

determined (40 versus 20). However, exchange of one

methyl group in 40 for 3,4-dimethoxybenzyl led to the

most active AChE inhibitor of the present series (40

versus 21). Compound 21 had an IC₅₀value of 330 nM

and showed the lowest residual activity of all compounds

investigated.

Kinetic Characterization of the Inhibition of

CEase. In a previous study we have demonstrated that

thieno[1,3]oxazin-4-ones have the capability to act as

alternate substrate inhibitors of CEase, provided that

the enzyme-catalyzed consumption of the inhibitor

occurs under steady-state conditions. Due to the ac-

cumulation of an enzyme-inhibitor complex, the trans-

formation followed a zero-order kinetics. An essential

conclusion that was derived from the general kinetic

model is the concurrence of the K_ivalue of a given

inhibitor and the separately determined Michaelis

constant, K_m^I, of its enzymatic consumption.³⁵

The kinetic characterization of the new CEase inhibi-

tor 33 was done as follows. Five different concentrations

of 33 were used, and the rates were plotted against the

inhibitor concentrations (Figure 1). A value K_i(1 +

[S]/K_m^S) ) 1.78 µM was obtained, and a value K_i) 0.63

µM (Table 3) could be calculated from the respective

substrate concentration, [S], and a value of 110 µM for

the Michaelis-Menten constant, K_m^S, of the chromoge-

nic substrate pNPB. To reveal the type of inhibition,

reactions were measured in the presence of different

substrate concentrations and the data were analyzed

according to the Hanes-Woolf method (Figure 2). The

[S]/v versus [S] plot (Figure 2a) showed parallel lines

indicating a formal competitive type of inhibition. The

secondary plot of the intercepts versus [I] and a linear

regression gave K_i) 0.68 µM (Figure 2b). This value

was in accordance with the calculated one (Table 3), and

thus the competitive mode was confirmed and the

inhibitor could be characterized as active site-directed.

Next, the enzyme-catalyzed consumption of 33 was

examined and a general kinetic model for alternate

substrate inhibition via acyl enzymes was used.³⁵The

enzymatic turnover of compound 33 at an initial con-

8276 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 26

Pietsch and Gu¨tschow

Figure 1. Inhibition of CEase by compound 33. Plot of the

rates versus [I] for the inhibition of CEase by compound 33 in

100 mM sodium phosphate, 100 mM NaCl, pH 7.0 with 6 mM

TC, 6% acetonitrile and 10 ng/mL CEase. Initial substrate

concentration was 200 µM pNPB. Rates were obtained by

linear regression of the progress curves (not shown) and are

mean values of duplicate experiments. The solid line was

drawn using the best-fit parameters from a fit according to

an equation of competitive inhibition, which gave K_i(1 +

[S]/K_m^S) ) 1.78 ( 0.08 µM. The insert is a Dixon plot to show

the linearity.

Figure 3. Kinetics of the CEase-catalyzed hydrolysis of

compound 33. Reaction was performed in 100 mM sodium

phosphate, 100 mM NaCl, pH 7.0 with 6 mM TC, 6% aceto-

nitrile, and an adjusted activity of CEase. Initial concentration

of compound 33, [I₀], was 25 µM. (a) Depletion of compound

33 is illustrated by monitoring UV/vis-spectra at 10 min-

intervals. The arrow indicates the initial spectrum. (b) Hy-

drolysis of compound 33 was followed at 350 nm. Control

reaction in the absence of CEase is shown to demonstrate the

stability of compound 33. (c) Plot ([I₀] - [I])/t versus (ln[I₀] -

ln[I])/t, where [I₀] is the initial concentration of the inhibitor

33 and [I] is the inhibitor concentration at the time t. The

values for [I] at t ) 200-250 min based on the data shown in

part b. Linear regression gave a negative slope of 0.65 ( 0.02

Figure 2. Inhibition of CEase by compound 33 in the presence

of different concentrations of the chromogenic substrate pNPB.

(a) Hanes-Woolf plot using mean values of rates from

duplicate experiments in 100 mM sodium phosphate, 100 mM

NaCl, pH 7.0 with 6 mM TC, 6% acetonitrile and 10 ng/mL

CEase. Concentrations of compound 33 were as follows: open

circles, [I] ) 0; full circles, [I] ) 4 µM; open squares, [I] ) 8

µM; full squares, [I] ) 12 µM. Linear regression gave values

for vertical intercepts. (b) Plot of vertical intercepts versus the

concentrations of compound 33. Linear regression according

to equation, intercept ) [K_m^S[I]/(K_iV_max^S)] + (K_m^S/V_max^S), gave

a slope K_m^S/(K_iV_max^S) ) 13.1 ( 0.2 min, that corresponds to a

value K_i) 0.68 ( 0.01 µM.

I

µM that corresponds to K_mand an intercept of V_max) 0.086

( 0.001 µM min^-1

.

boxylic acid as the product. Its formation can easily be

explained by a nucleophilic attack of the active site

serine at the C4-carbon of 33 to give an intermediate

acyl-enzyme which subsequently undergoes hydrolytic

cleavage. A similar conversion of the thieno[1,3]oxazin-

4-one 56 to the corresponding 2-(3,3-diethylureido)-

thiophene-3-carboxylic acid has been shown previ-

ously.³⁵The time-dependent decrease of the concentration

of 33 monitored at a fixed wavelength is depicted in

Figure 3b. The reaction followed zero-order kinetics

until the hydrolysis was nearly finished. These data

were used for a plot (Figure 3c) according to an

integrated form of the Michaelis-Menten equation.

centration of 25 µM was investigated in the presence of

a high CEase concentration (∼730-fold higher compared

with the inhibition experiments in the presence of

pNPB). Spectral changes were monitored in 10 min

intervals (Figure 3a). The final spectra revealed the

corresponding 2-(3,3-dimethylureido)thiophene-3-car-

Tricyclic 1,3-Oxazin-4-ones

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 26 8277

Table 5. Kinetic Parameters of the CEase-Catalyzed Turnover

of Thieno[1,3]oxazin-4-ones

Scheme 7. Kinetic Model for the Interaction of

Hyperbolic Mixed-Type Inhibitors with AChE

I

K_m

(µM)

V_max

(µM min^-1

V_max^I/K_m

(min^-1

compd

n

X

R¹R²

)

33^a

56^b

35

0

1

CH₂

O

Me Me

Et Et

0.65 ( 0.02 0.086 ( 0.001 0.13

0.65 ( 0.05 0.533 ( 0.003 0.82

Me Me >25

>0.033^c

>0.030^c

0.0013

0.0012

40

N-Bn Me Me >25

^aAn molar extinction coefficient ꢀ ) 9.84 mM^-1cm^-1was used

7. This model represents the general situation where

the substrate, S, and the inhibitor, I, bind to the enzyme,

E, at different sites, thus allowing for the formation of

both an enzyme-inhibitor complex, EI, as well as an

enzyme-substrate-inhibitor complex, ESI. The corre-

sponding dissociation constants of ES and EI are K_Sand

K_i, respectively. The dissociation constants of the ter-

nary complex, ESI, are RK_Sand RK_i. Since the overall

equilibrium constant for the formation of ESI must be

the same regardless of a path via ES or via EI, the same

factor R has to be included in the model.^80,81The complex

ESI is still functional with a decreased rate of product

formation governed by the catalytic constant, âk_P. This

type of inhibition was referred to as hyperbolic mixed-

type inhibition, as the shape of a reciprocal velocity, 1/v,

versus [I] plot is hyperbolic,^80,82and the factor R is finite

and different from 1. In the case of linear inhibition with

â ) 0, the kinetic analysis using the Lineweaver-Burk

plot, or the similar Hanes-Woolf plot, and correspond-

ing secondary plots provides the parameters of enzyme

inhibition. However, a more complex situation arises for

hyperbolic inhibition with â * 0. For example, with

Lineweaver-Burk analysis, the common replots are

hyperbolic and a further reciprocal replot has to be done

for linearization.⁸⁰Baici has ingeniously presented the

specific velocity plot to overcome these difficulties.⁸¹We

intended to use the well-established Hanes-Woolf plot,

[S]/v versus [S], which is preferable to the double

reciprocal Lineweaver-Burk plot, 1/v versus 1/[S], since

the Hanes-Woolf plot exhibit less variation in ac-

curacy.^83,84Thus, a new alternative algorithm was

derived to calculate kinetic parameters of hyperbolic

mixed-type inhibition of AChE.

I

to determine K_mand V_max

were defined according to V_max> [I₀] V_max^I/K_m

. .

^bꢀ ) 9.97 mM^-1cm^{-1 c}The limits

I

.

Values for maximum velocity, V_max^I, and for the Michae-

lis constant, K_m^I, of the enzymatic consumption of the

I

inhibitor could be obtained, and V_max^I/K_mcould there-

with be calculated (Table 5). The enzyme-catalyzed

conversion was similarly inspected for compounds 35

and 40 (Table 5) with an equal CEase activity adjusted

toward pNPB prior to the experiments. Compound 56

was reinvestigated³⁵in order to obtain a V_max^Ivalue that

could be compared with those of the other compounds.

Thieno[1,3]oxazinones 33 and 56 could be classified

as true alternate substrate inhibitors of CEase based

on the concurrence of the K_iand K_m^Ivalues. Compound

I

33 had a K_iof 0.63 µM (Table 3) and a K_mof 0.65 µM

(Table 5), 56 exhibited a K_iof 0.58 µM (Table 1) and a

K_m^Iof 0.65 µM (Table 5). Although both compounds are

equally potent as inhibitors of CEase, the new thieno-

[1,3]oxazinone 33 is superior in terms of resistance to

I

enzymatic degradation. The relevant parameter is V_max

being 6-fold lower in the case of 33. It can be concluded

that the acyl-enzyme, E-I, derived from the reaction

of CEase with 33 undergoes an accordingly decelerated

hydrolytic deacylation. The alternate substrate mode of

CEase inhibition by 33 and 56 can analogously be

supposed for the other active compounds of the present

series (K_i< 20 µM; Tables 1-3).

When incubating the tetrahydropyrano derivative 35

and the tetrahydropyrido derivative 40, respectively,

with the same amount of CEase, a very slow consump-

tion was observed. The reaction was monitored over 66

h and followed a first-order kinetics over the entire time

course. The first-order rate constants that correspond

The Michaelis-Menten equation valid for the general

type of inhibition as depicted in Scheme 7 is as follows.

I

to V_max^I/K_mwere obtained by nonlinear regression

V_max[S]

v )

(2)

(Table 5). Compounds 35 and 40 are very poor sub-

strates of CEase. Their V_max^I/K_m^Ivalues are 2 orders of

magnitude lower than that of 33. To evaluate compound

35 and 40 as alternate substrate inhibitors, the reaction

order of the enzymatic turnover is meaningful. Even at

the initial concentration of 25 µM, the first-order kinet-

ics indicated that an accumulation of an enzyme-

inhibitor complex did not occur. Likewise it can be

[I] + K_i

[I] + RK_i

RK_S

+ [S]

(

)

(

)

â[I] + RK_i

At infinitely high inhibitor concentrations, the product

is exclusively formed from ESI with a rate constant âk_P.

Under this conditions, the rate, v_[I]f∞, at a given

substrate concentration is defined by eq 3.

I

reasoned that compounds 35 and 40 have a K_mvalue

âV_max[S]

higher than 25 µM. Their affinity to bind to CEase is

low, and this conclusion is in accordance with their

failure to affect CEase in the inhibition assay (K_i> 50

µM, Table 3).

v_[I]f∞

)

(3)

RK_S+ [S]

Subtractive combination of eqs 2 and 3 gave eq 4,

which was simplified for the cases â * 0, [I] ) 0 (eq 5),

â ) 0, [I] * 0 (eq 6), and â ) 0, [I] ) 0 (eq 7). With â )

0, the rate v_[I]f∞becomes zero. Those portion of the

Kinetic Characterization of the Inhibition of

AChE. A kinetic model for the inhibition of acetylcho-

linesterase was considered that is outlined in Scheme

8278 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 26

Pietsch and Gu¨tschow

Table 6. Kinetic Parameters for the Inhibition of AChE by

Anticholinesterases

product formation that results only from the decay of

ES is named v_â)0

.

(R - â)K_S+ (1 - â)[S]

RK_S+ [S]

v - v_[I]f∞

)

(

)

V_max[S]

(4)

[I]

RK_i

K 1 +

+ [S] 1 +

(

)

S

(

)

(

)

K_i

(R - â)K_S+ (1 - â)[S] V_max[S]

compd

X

IC₅₀(µM)

K_i(µM)

R

â

v₀- v_[I]f∞

)

(5)

(6)

(

)(

39

40

N-i-Bu 0.36 ( 0.01

N-Bn 0.89 ( 0.02

0.66 ( 0.02

1.70 ( 0.12

0.33 0.054

0.37 0.076

RK_S+ [S]

K_S+ [S]

tacrine^a

-

0.027 ( 0.001 0.025 ( 0.001 1.48 0

2.20 ( 0.15

galanthamine^a

1.56 ( 0.07

4.82 0

V_max[S]

v_â)0

)

^av_[I]f∞was set to zero.

[I]

RK_i

K_S1 +

+ [S] 1 +

(

)

(

)

K_i

V_max[S]

v₀)

(7)

K_S+ [S]

Equation 8 is another expression for v_â)0 that can

be calculated from eqs 4-7.

(v - v_[I]f∞

)

V_max[S]

[I]

v₀)

(8)

[I]

RK_i

(v₀- v_[I]f∞

)

K_S1 +

+ [S] 1 +

(

)

(

)

K_i

Figure 4. Inhibition of AChE by compound 39. Plot of the

rates versus [I] for the inhibition of AChE by compound 39 in

100 mM sodium phosphate, 100 mM NaCl, pH 7.3 with 350

µM DTNB, 6% acetonitrile, and 0.033 U/ml AChE. Initial

substrate concentration was 500 µM ATCh. Rates were

obtained by linear regression of the progress curves (not

shown) and are mean values of duplicate experiments. A

nonlinear regression according to eq 1, gave IC₅₀) 0.36 ( 0.01

µM and v_[I]f∞) 0.00134 ( 0.00005 min^-1, that corresponds to

0.043 ( 0.002 relative to v₀. The insert is a Dixon plot to show

the linearity.

Equation 8 allows for an easy access to the analysis

of the data following the common Hanes-Woolf method,

using the values v, v₀, v_[I]f∞, and [S] (eq 9).

(v₀- v_[I]f∞)[S]

(v - v_[I]f∞)v₀

K_S

[S]

V_max

[I]

RK_i

)

1 +

+

1 +

(9)

(

)

(

V_max

Replots of the slopes (eq 10) and intercepts (eq 11) of

eq 9 versus [I] give values for RK_iand K_i; the value R

can be calculated from the ratio of RK_iand K_i.

enzyme-inhibitor interaction, it should be determined

whether the inhibitor binds to the enzyme-substrate

complex, ES; with a greater affinity than to the free

enzyme, E, or vice versa. The preference of the inhibitor

to bind to ES is reflected in values R < 1 (Scheme 7)

that indicate mixed type inhibition with a pronounced

uncompetitive component. A higher affinity of the

inhibitor to E, and thus values R > 1, corresponds to

mixed type inhibition with a more competitive charac-

ter. Moreover, K_ivalues being independent of the

substrate concentration should be made available.

Actually, the kinetic analysis of the hyperbolic mixed-

type inhibition of exemplary compounds was accom-

plished using the new methodology derived above. The

basic thieno[1,3]oxazin-4-ones 39 and 40 as well as

tacrine and galanthamine were selected (Table 6). The

inhibition of AChE by the isobutyl derivative 39 is

illustrated in Figure 4. An IC₅₀value of 360 nM and a

relative residual activity v_[I]f∞of 0.043 was obtained.

Most of the active compounds gave similar plots, rate

versus inhibitor concentration, and the parameters are

noted in Tables 1-4. From observed residual activities

v_[I]f∞and thus â values different from zero, the occur-

rence of a ternary ESI complex could be predicted

(Scheme 7). For those compounds, a purely competitive

1

V_max

slope )

[I] +

(10)

(11)

V_maxRK_i

K_S

intercept )

[I] +

V_maxK_i

V_max

The constant â is accessible via eq 3 and eq 7.

Linearization of these equations according to the Hanes-

Woolf plot give eq 12 and eq 13, respectively. The value

of â can be calculated as the quotient of the slopes of eq

13 and eq 12.

RK_S

[S]

)

+

(12)

(13)

v_[I]f∞âV_maxâV_max

K_S

[S]

v₀

[S]

)

+

V_maxV_max

As noted above, most of the AChE inhibitors of the

present series exhibited residual activity at infinite

concentration. The rates v_[I]f∞, relative to v₀, are listed

in Tables 1-4. From this it follows that â (Scheme 7)

takes a value different from zero. To elucidate the

Tricyclic 1,3-Oxazin-4-ones

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 26 8279

type of inhibition and thus a relevant interaction with

the esteratic site of AChE could be excluded.

Next, the dependence of the rates on the concentration

of tacrine and galanthamine was analyzed. The non-

linear regression gave more accurate results when v_[I]f∞

was set to zero. It could therefore be concluded for both

inhibitors, that the ESI complex, if formed at all, is not

catalytically active. The further kinetic analysis for

tacrine and galanthamine followed the model of linear

mixed-type inhibition⁸⁰with â ) 0 (Scheme 7).

The determination of the factor R (Table 6) was

performed to estimate the portion of competitive and

uncompetitive inhibition, respectively. According to eq

9, a plot of (v₀- v_[I]f∞) [S]/((v - v_[I]f∞) v₀) versus [S]

and the respective secondary plots allow for the calcula-

tion of K_iand R. The procedure for compound 39 is

depicted in Figures 5a and 5b, and was similarly applied

to compound 40. Values R ) 0.33 and R ) 0.37 for 39

and 40, respectively, were obtained. In the case of

tacrine and galanthamine, where v_[I]f∞was set to zero,

eq 9 can be simplified to that of the common Hanes-

Woolf plot, [S]/v versus [S]. Corresponding analysis gave

a factor R of 1.48 for tacrine and of 4.82 for galan-

thamine.

The factor â corresponds to the relative residual

velocity v_[I]f∞at infinite substrate concentration (eq 3),

i.e. the ratio between the maximum velocity of the

productive decay of ESI and ES (Scheme 7). The

determination of factor â for compound 39 is illustrated

in Figure 5c, showing the plots [S]/v₀versus [S] and

[S]/v_[I]f∞versus [S]. Values of â for the inhibitors 39

and 40 (0.054 and 0.076, respectively) were obtained by

linear regression according to eqs 12 and 13. The data

v_[I]f∞as noted in Table 3 are necessarily somewhat

lower. Taken together, the two basic tetrahydropyrido

derivatives 39 and 40 form ternary ESI complexes with

a restricted catalytic efficiency. Their activity is less

than 8% but still significant.

The R values indicated that both 39 and 40 are mixed-

type inhibitors of AChE with a pronounced uncompeti-

tive character (Table 6). The constant K_ithat responds

to competitive inhibition was calculated to be 0.66 µM

(39) and 1.70 µM (40), respectively. The constant of the

uncompetitive inhibition, RK_i, was 0.22 µM (39) and 0.63

µM (40), respectively. These parameters imply IC₅₀

values between K_iand RK_i, closer to the latter value.

This was indeed the case for inhibitors 39 and 40 (Table

6).

The R values for tacrine and galanthamine, on the

other hand, demonstrate a pronounced competitive

mode for tacrine (R ) 1.48) that was even stronger in

the case of galanthamine (R ) 4.82). These findings were

in agreement with literature data. On AChE from

Electrophorus electricus, an R value of 1.88 (K_i) 20.4

nM) was determined for tacrine⁸⁵and purely competi-

tive inhibition (K_i) 660 nM) was reported for galan-

thamine.⁸⁶The kinetic behavior reflects structural

information derived from crystal structures of AChE

complexed with tacrine⁶⁹and galanthamine.^87,88Both

anticholinesterases occupied the anionic binding site

stacking against the indole ring of Trp84 (Torpedo

californica AChE numbering)⁶⁸to form a favorable π-π

interaction. While galanthamine did not interact with

Phe330, tacrine was sandwiched between the aromatic

Figure 5. Inhibition of AChE by compound 39 in the presence

of different concentrations of the substrate ATCh. (a) Modified

Hanes-Woolf plot using mean values of rates from duplicate

experiments in 100 mM sodium phosphate, 100 mM NaCl, pH

7.3 with 350 µM DTNB, 6% acetonitrile, and 0.033 U/mL

AChE. Concentrations of compound 39 were as follows: open

circles, [I] ) 0; full circles, [I] ) 1 µM; open squares, [I] ) 2

µM; full squares, [I] ) 4 µM; open triangles, [I] ) 6 µM. Linear

regression according to eq 9 gave values for slopes and vertical

intercepts. (b) Plot of 10²× slopes (full circles) and vertical

intercepts (open triangles) versus the concentrations of com-

pound 39. Linear regression according to eq 10 gave a slope

10²/(RK_iV_max) ) 9.26 ( 0.29 µM^-1min, that corresponds to a

value RK_i) 0.22 ( 0.01 µM. Linear regression according to

eq 11 gave a slope K_m/(K_iV_max) ) 17.9 ( 0.5 min, that

corresponds to a value K_i) 0.66 ( 0.02 µM. The ratio of RK_i

and K_igave a value R ) 0.33. (c) Hanes-Woolf plot using mean

values of v₀(open circles) and v_[I]f∞(full squares) from

duplicate experiments. Linear regression according to eq 13

gave a slope 10^-2/V_max) 0.204 ( 0.002 min for the plot 10^-2

×

[ATCh]/v₀versus [ATCh], and a linear regression according

to eq 12 gave a slope 10^-3/(â V_max) ) 0.375 ( 0.038 min for the

plot 10^-3× [ATCh]/v_[I]f∞versus [ATCh]. Thus, a value â )

0.054 could be calculated.

rings of Trp84 and Phe330 and additionally fixed

through direct cation-π interactions on the residues of

8280 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 26

Pietsch and Gu¨tschow

the anionic binding site.⁸⁹Tacrine did not occupy the

immediate vicinity of the catalytic serine, whereas

galanthamine participated in an hydrogen bond with

the active site Ser200.^69,88These arrangements provided

some rationale for the stronger competitive behavior of

galanthamine compared to tacrine.

of thienoxazinones served as an electrophile to mimic

the scissile ester bond of substrates. Those compounds

that underwent an enzyme-catalyzed steady-state hy-

drolysis were characterized as alternate substrate in-

hibitors. Kinetic parameters of the enzymatic turnover

that assess the inhibitor’s effectiveness are K_mand V_max

,

both with preferably low values. In this regard, the new

inhibitor 33 meets the criteria of an efficient alternate

substrate inhibitor of CEase. Moreover, 33 and its

analogues are distinguished by high chemical stability.

On the other hand, in the case of AChE it was

supposed that the lactone unit of thienoxazinones did

not interact with the serine of the catalytic triad, and

the inhibitors were oriented along the active site gorge.

This assumption based on structural resemblance to

known anticholinesterases and, more important, on the

kinetic behavior of the active thienoxazinones. Catalyti-

cally competent ESI complexes with varying activities

were detected and a stronger uncompetitive component

was observed for selected compounds. To specify the

parameters of inhibition, a new method was derived

that let us kinetically characterize certain thieno[1,3]-

oxazin-4-ones as hyperbolic mixed-type inhibitors of

AChE. Structural modifications of the efficient inhibitor

21 are currently under investigation in our laboratories.

Such approaches include the opening of the tetrahydro-

pyridine ring to achieve a higher flexibility of the basic

moiety.

The kinetic analyses as well as the structural simi-

larities between known inhibitors and the pyridothieno-

[1,3]oxazinones led us to suspect the orientation of our

inhibitors in the active-site gorge of AChE. The benzyl-

substituted tetrahydropyridine derivatives, being almost

all active, resembled donepezil and AP2238⁷¹with

respect to the benzyl substituent, its attachment to a

basic nitrogen and the carbonyl group in a similar

distance. The crystal structure of AChE (Torpedo cali-

fornica) complexed with donepezil⁹⁰showed the inhibi-

tor oriented along the axis of the gorge, extending from

the anionic site to the PAS. The interaction occurred

via aromatic π-π stacking between the benzyl moiety

of donepezil and Trp84 as well as the indanone ring and

Trp279. The charged piperidine nitrogen made a cat-

ion-π interaction with Phe330 at the midpoint of the

gorge. The carbonyl interacted with the aromatic rings

of Phe331 and Phe290, and indirectly via a water

molecule with the amide bond of Phe288. Donepezil did

not directly interact with residues of the catalytic

triad.⁹⁰Similar contacts stabilized the complex of

AP2238 with human AChE as concluded from docking

studies. The coumarin ring interacted with the PAS and

the carbonyl group established an H-bond interaction

with a phenylalanine backbone amide group.⁷¹Both

donepezil and AP2238 have been described as mixed-

type inhibitors.^71,85Noteworthy these substances ex-

celled as inhibitors of the AChE-induced Aâ aggregation,

a feature which was present only to a minor extent in

the case of tacrine.^49,71

Experimental Section

General Methods and Materials. Melting points were

determined on a Gallenkamp capillary melting point ap-

paratus and a Bu¨chi 510 oil bath apparatus, and are not

corrected. Compounds 6 and 44 were prepared using the Bu¨chi

Glas Uster autoclave ‘TinyClave’. Thin-layer chromatography

was performed on Merck aluminum sheets, silica gel 60 F₂₅₄

.

Preparative column chromatography was performed on Merck

silica gel 60, 70-230 mesh. ¹³C NMR spectra (125 MHz) and

¹H NMR spectra (500 MHz) were recorded on a Bruker

Advance DRX 500 spectrometer in CDCl₃at 25 °C or in DMSO-

d₆at 30 °C. ¹³C NMR signals were assigned on the basis of

DEPT-135 spectra and ¹³C/¹H correlation experiments (HSQC).

Chemical shifts (δ) are reported in ppm. Spin multiplicities

are indicated by the following symbols: s (singlet), br s (broad

singlet), d (doublet), br d (broad duplet), dd (doublet of doublet),

t (triplet), br t (broad triplet), tt (triplet of triplet), q (quartet),

sept (septet), and m (multiplet). IR spectra were measured

with a Perkin-Elmer 1600 series FTIR spectrophotometer.

Mass spectra (70 eV) were obtained on a MS-50 A.E.I.

spectrometer under electron impact ionization (EI). Elemental

analyses were performed with a Vario EL apparatus. UV

spectra and spectrophotometric assays were done on a Varian

Cary 50 Bio UV/vis spectrometer and a Varian Cary 100 Bio

UV/vis spectrometer, both with a cell holder equipped with a

constant temperature water bath. CEase from bovine pancreas

(71.5 units/mg), ATCh, DTNB, sodium taurocholate (TC),

pNPB, and tacrine hydrochloride hemihydrate were obtained

from Sigma (Steinheim, Germany). AChE from Electrophorus

electricus (1044 U/mg and 1276 U/mg) and anhydrous DMSO

were purchased from Fluka (Deisenhofen, Germany). Galan-

thamine hydrobromide was from Calbiochem (Bad Soden,

Germany). Compounds 26,³⁸42,⁹¹43,⁷⁶45, 46,⁷⁷52, 53, 55,

56, 58-60,³⁹54, 57, and 61-63³⁸were prepared as described

elsewhere. All ¹H NMR and ¹³C NMR data can be found in

the Supporting Information.

It is tempting to assume that the benzyl-substituted

tetrahydropyridine derivatives of our study (e.g. 40)

bind similarly to the active site gorge of AChE. An

orientation toward the PAS and away from the esteratic

site would be in accordance with the kinetic data of the

enzyme-inhibitor interaction. In view of the structures

of donepezil and AP2238, a dimethoxy phenyl moiety

was introduced into the 2-substituent of our tricyclic

skeleton to facilitate a contact to the PAS. The resulting

derivative 21 proved to be the most potent AChE

inhibitor of the present series.

Conclusions

Thirty six thieno[1,3]oxazin-4-ones and thieno[1,3]-

thiazin-4-ones were evaluated in vitro as inhibitors of

bovine pancreatic CEase as well as AChE from Elec-

trophorus electricus, two members of the R/â-hydrolase

family. Although the heterocyclic compounds of this

series possess structural similarity, selective inhibitors

of both esterases were identified. A crucial feature for

specific interaction with AChE was the presence of a

basic nitrogen in the ring fused to the thienoxazinone.

When this moiety was excised or replaced by a meth-

ylene group, selectivity was reversed and inhibitors of

CEase were attained, in principle, without further

structural changes. The kinetic analyses revealed a

generally different mode of inhibition toward the two

target enzymes. In the case of CEase, the lactone unit

tert-Butyl 2-Amino-6-benzyl-4,5,6,7-tetrahydrothieno-

[2,3-c]pyridine-3-carboxylate (1). A mixture of N-benzyl-

4-piperidone (9.46 g, 50 mmol), sulfur (1.6 g, 50 mmol), tert-

butyl cyanoacetate (7.06 g, 50 mmol), and C₂H₅OH (25 mL)

was treated dropwise with morpholine (4.35 g, 50 mmol) at

Tricyclic 1,3-Oxazin-4-ones

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 26 8281

45 °C over 5 min. After being stirred for 5 h at 45 °C, the

mixture was diluted with H₂O (200 mL) and extracted with

ether (4 × 100 mL). The organic layer was washed with H₂O

(4 × 50 mL) and dried (Na₂SO₄). The solvent was removed in

vacuo, and the crude product was recrystallized from n-hexane

to yield 1 (14.8 g, 86%) as yellowish crystals: mp 115-118

°C. Anal. (C₁₉H₂₄N₂O₂S) C, H, N.

tert-Butyl 6-Benzyl-4,5,6,7-tetrahydro-2-(3-isopropyl-

ureido)thieno[2,3-c]pyridine-3-carboxylate (2). A mixture

of 1 (6.89 g, 20 mmol), isopropyl isocyanate (3.92 g, 46 mmol),

and dry pyridine (40 mL) was stirred at 70 °C for 66 h under

argon atmosphere and kept at room temperature for 7 h. It

was poured into ice-water (600 mL), and the precipitate was

collected by filtration, washed with H₂O, dried, and recrystal-

lized from CH₃OH to yield 2 (4.32 g, 49%) as yellowish

crystals: mp 96-98 °C. Anal. (C₂₃H₃₁N₃O₃S × 0.5 H₂O) C, H,

N.

tert-Butyl 6-Benzyl-2-(3-cyclohexylureido)-4,5,6,7-tet-

rahydrothieno[2,3-c]pyridine-3-carboxylate (3). A mix-

ture of 1 (6.89 g, 20 mmol), cyclohexyl isocyanate (5.76 g, 46

mmol), and dry pyridine (40 mL) was stirred at 70 °C for 66 h

under argon atmosphere, kept at room temperature for 7 h,

and filtered. The filtrate was poured into ice-water (600 mL)

and extracted with ethyl acetate (2 × 150 mL). The organic

layer was washed with H₂O (2 × 300 mL) and dried (Na₂SO₄).

The solvent was removed in vacuo, and the crude product was

extracted with boiling n-hexane (3 × 300 mL). After cooling

of the extractant, the precipitate was collected by filtration,

combined with the residue of the extraction, and recrystallized

from CH₃OH to yield 3 (5.56 g, 55%) as yellowish crystals: mp

106.5-108.5 °C. Anal. (C₂₆H₃₅N₃O₃S × CH₃OH) C, H, N.

7-Benzyl-5,6,7,8-tetrahydro-2-phenylamino-4H-pyrido-

[4′,3′:4,5]thieno[2,3-d][1,3]oxazin-4-one (7). Compound 6

(2.40 g, 5 mmol) was added in portions over 20 min to

trifluoroacetic acid (7 mL) at 0 °C. After being stirred for

additional 30 min at 0 °C and for 1.5 h at room temperature,

the mixture was poured into chilled saturated aqueous

NaHCO₃(120 mL). The precipitate was collected by filtration,

washed with H₂O, and dried to obtain a yellowish solid (3.14

g). It was dissolved in boiling dry CH₂Cl₂(150 mL), cooled to

room temperature, and filtered. After adding yellow HgO (2.56

g, 11.80 mmol) to the filtrate, the mixture was stirred at room

temperature for 48 h. Silica gel (5.3 g) was added and the

mixture was stirred for 2 min. The inorganic material was

filtered off and washed with dry CH₂Cl₂(75 mL). The wash

was combined with the filtrate, and the solvent was removed

in vacuo to yield 7 (0.34 g, 16%) as a yellowish solid: mp 196-

197 °C (ethyl acetate/n-hexane, 1:4, decomp); HRMS m/z calcd

for C₂₂H₁₈N₃O₂S (M⁺- H), 388.1120; found, 388.1129. Anal.

(C₂₂H₁₉N₃O₂S × 0.3 CH₃COOC₂H₅) C, H, N.

2-Benzoylamino-7-benzyl-5,6,7,8-tetrahydro-4H-pyrido-

[4′,3′:4,5]thieno[2,3-d][1,3]oxazin-4-one (8). Ammonium

thiocyanate (1.00 g, 13.10 mmol) was dissolved in boiling dry

CH₃COCH₃(8 mL), filtered, and cooled to 0 °C. After dropwise

addition of benzoyl chloride (1.63 g, 11.6 mmol), the mixture

was refluxed for 5 min. The precipitate was filtered off and

washed with dry CH₃COCH₃(6 mL). The filtrate and the wash

were combined and added to a solution of 1 (2.00 g, 5.80 mmol)

in dry CH₃COCH₃(12 mL). After being stirred for 2 h at room

temperature, the precipitate was collected by filtration, washed

with cold CH₃COCH₃, and dried to obtain a yellowish solid

(2.54 g). It was added in portions over 20 min to trifluoroacetic

acid (6 mL) at 0 °C. After being stirred for additional 40 min

at 0 °C and for 2 h at room temperature, the mixture was

poured into chilled saturated aqueous NaHCO₃(100 mL). The

precipitate was collected by filtration, washed with H₂O, and

dried to obtain a yellowish solid (2.54 g). It was dissolved in

dry CH₂Cl₂(110 mL), yellow HgO (1.95 g, 9 mmol) was added,

and the mixture was stirred at room temperature for 48 h.

Silica gel (4 g) was added and the mixture was stirred for 2

min. The inorganic material was filtered off and washed with

dry CH₂Cl₂(55 mL). After combination of the wash with the

filtrate and the removal of the solvent in vacuo, the residue

was recrystallized from ethyl acetate/n-hexane (1:2) to yield 8

(0.18 g, 7%) as a yellowish solid: mp 143-146 °C (decomp);

HRMS m/z calcd for C₂₃H₁₈N₃O₃S (M⁺- H), 416.1069; found,

416.1075. Anal. (C₂₃H₁₉N₃O₃S) C, H, N.

tert-Butyl 6-Benzyl-2-(3-trichloroacetylureido)-4,5,6,7-

tetrahydrothieno[2,3-c]pyridine-3-carboxylate (9). Com-

pound 1 (3.44 g, 10 mmol) was dissolved in dry THF (15 mL)

and the solution was cooled to 0 °C. After adding cold

trichloroacetyl isocyanate (4.33 g, 23 mmol), the reaction

mixture was stirred at 0 °C for 30 min and at 7-10 °C for 2.5

h under argon atmosphere. Purification by column chroma-

tography (petrol ether/ethyl acetate, 2:1, performed at 8 °C)

and removal of the solvent in vacuo at 7-10 °C yielded 9 (4.77

g, 90%) as a greenish solid: mp 179-182 °C (decomp).

tert-Butyl 6-Benzyl-4,5,6,7-tetrahydro-2-ureidothieno-

[2,3-c]pyridine-3-carboxylate (10). Compound 9 (10.21 g,

19.16 mmol) was dissolved in a mixture of dry CH₃OH (25 mL)

and dry THF (25 mL) and the solution was stirred at 60 °C

for 5.5 h. After removal of the solvent in vacuo the crude

product was recrystallized from CH₃OH to obtain 10 (2.37 g,

32%) as yellowish crystals: mp 224-225 °C (decomp). Anal.

(C₂₀H₂₅N₃O₃S) C, H, N.

7-Benzyl-5,6,7,8-tetrahydro-2-isopropylamino-4H-py-

rido[4′,3′:4,5]thieno[2,3-d][1,3]oxazin-4-one (4). A mixture

of trifluoroacetic anhydride (1.47 g, 7 mmol) and trifluoroacetic

acid (6 mL) was stirred at 0 °C under argon atmosphere.

Compound 2 (2.15 g, 4.90 mmol) was added in portions over

30 min. Stirring was continued at 0 °C for 30 min and at room-

temperature overnight. The mixture was poured into chilled

saturated aqueous NaHCO₃(100 mL). The precipitate was

collected by filtration, washed with H₂O, and dried. The filtrate

was extracted with ethyl acetate (2 × 100 mL). The organic

layer was washed with H₂O (2 × 100 mL) and saturated

aqueous NaCl (100 mL) and dried (Na₂SO₄). After removal of

the solvent in vacuo, the residue was combined with the

precipitate and recrystallized twice from ethyl acetate/n-

hexane (3:4) with the addition of silica gel (8.5 g) to yield 4

(0.47 g, 27%) as a white solid: mp 192-193 °C (decomp);

HRMS m/z calcd for C₁₉H₂₀N₃O₂S (M⁺- H), 354.1276; found,

354.1276. Anal. (C₁₉H₂₁N₃O₂S × 0.25 H₂O) C, H, N.

7-Benzyl-2-cyclohexylamino-5,6,7,8-tetrahydro-4H-py-

rido[4′,3′:4,5]thieno[2,3-d][1,3]oxazin-4-one (5). According

to the procedure outlined above, 3 (2.35 g, 4.68 mmol) was used

in place of 2. The mixture was poured into chilled saturated

aqueous NaHCO₃(120 mL). The precipitate was collected by

filtration, washed with H₂O, and dried. Boiling ethyl acetate

(400 mL) and silica gel (23 g) were added, and the mixture

was refluxed and filtered. After adding n-hexane (300 mL) and

silica gel (19 g) to the filtrate, it was refluxed and filtered.

The solvent was removed in vacuo, and the residue was

recrystallized from ethyl acetate/n-hexane (2:3) with the addi-

tion of silica gel (7.5 g) to yield 5 (0.15 g, 8%) as a white solid:

mp 189-192 °C (decomp); HRMS m/z calcd for C₂₂H₂₄N₃O₂S

(M⁺- H), 394.1589; found, 394.1585. Anal. (C₁₉H₂₁N₃O₂S ×

0.5 H₂O) C, H, N.

6-Benzyl-3-carboxy-4,5,6,7-tetrahydro-2-ureidothieno-

[2,3-c]pyridin-6-ium Trifluoroacetate (11). Compound 10

(1.16 g, 3 mmol) was added in portions over 30 min to

trifluoroacetic acid (4 mL) at 0 °C under argon atmosphere.

After being stirred in an open flask equipped with a drying

tube at room temperature for additional 1.5 h, the trifluoro-

acetic acid was removed in vacuo at room temperature. The

residue was dissolved in dry CH₂Cl₂(5 mL) and n-hexane (5

mL) was added to obtain an oily precipitate. After keeping at

-18 °C for 4 days, the precipitate was consolidated. It was

tert-Butyl

6-Benzyl-4,5,6,7-tetrahydro-2-(3-phenyl-

thioureido)thieno[2,3-c]pyridine-3-carboxylate (6). A mix-

ture of 1 (3.44 g, 10 mmol), phenyl isothiocyanate (3.79 g, 28

mmol), and dry C₂H₅OH (4 mL) was stirred in an autoclave

at 80 °C for 1.5 h under argon atmosphere. After keeping the

reaction mixture at room-temperature overnight, the precipi-

tate was collected by filtration, washed with cold C₂H₅OH, and

dried to obtain 6 (4.52 g, 94%) as a yellowish solid: mp 190-

191 °C (decomp). Anal. (C₂₆H₂₉N₃O₂S₂) C, H, N.

8282 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 26

Pietsch and Gu¨tschow

collected by filtration, washed with n-hexane, and dried to

obtain 11 (1.61 g, 84%) as a white solid: mp 153-156 °C

(decomp). Anal. (C₁₆H₁₇N₃O₃S × 2.7 CF₃COOH) C, H, N.

(Na₂SO₄). The solvent was removed in vacuo, and the crude

product was recrystallized from CH₃OH to yield 16 (1.31 g,

60%) as yellow crystals: mp 141-142 °C. Anal. (C₃₀H₃₇N₃O₅S)

C, H, N.

2-Amino-7-benzyl-5,6,7,8-tetrahydro-4H-pyrido[4′,3′:

4,5]thieno[2,3-d][1,3]oxazin-4-one (12). Compound 11 (0.8

g, 1.25 mmol) was dissolved in trifluoroacetic acid (4 mL) and

stirred at 0 °C under argon atmosphere. A solution of tri-

fluoroacetic anhydride (0.38 g, 1.79 mmol) in trifluoroacetic

acid (2 mL) was added dropwise over 15 min. Stirring was

continued at 0 °C for 1 h and at room-temperature overnight.

The trifluoroacetic acid and the trifluoroacetic anhydride were

removed in vacuo at room temperature. The residue was

dissolved in dry CH₂Cl₂(4 mL), and n-hexane (4 mL) was

added to obtain an oily precipitate. After keeping at -18 °C

for 6 days, the precipitate was consolidated. It was collected

by filtration, washed with n-hexane, and dried. The crude

product was dissolved in a mixture of CH₃OH (60 mL) and

ice-water (180 mL) and filtered. The acidic filtrate (pH 3-4)

was adjusted to pH 7 by dropwise addition of saturated

aqueous NaHCO₃. The precipitate was collected by filtration,

washed with cold H₂O, and dried to obtain 12 (0.21 g, 51%) as

a yellowish solid: mp 184.5-185 °C (decomp); HRMS m/z calcd

for C₁₆H₁₄N₃O₂S (M⁺- H), 312.0807; found, 312.0803. Anal.

(C₁₆H₁₅N₃O₂S × 0.7 H₂O) C, H, N.

tert-Butyl 6-Benzyl-4,5,6,7-tetrahydro-2-[(4-morpholi-

nylcarbonyl)amino]thieno[2,3-c]pyridine-3-carboxy-

late (17). Compound 13 (2.18, 3.84 mmol) was suspended in

dry Cl(CH₂)₂Cl (100 mL) and stirred at 0 °C under argon

atmosphere. A solution of morpholine (3.66 g, 42 mmol) in dry

Cl(CH₂)₂Cl (20 mL) was added dropwise over 30 min. After

being stirred at 0 °C for 2 h and at room temperature for 1.5

h, the reaction mixture was washed with H₂O (5 × 200 mL)

and saturated aqueous NaCl (100 mL) and dried (Na₂SO₄). The

solvent was removed in vacuo, and the crude product was

recrystallized from CH₃OH to yield 17 (1.37 g, 78%) as yellow

crystals: mp 204.5-207.5 °C (decomp). Anal. (C₂₄H₃₁N₃O₄S)

C, H, N.

tert-Butyl 6-Benzyl-2-[(4-benzyl-1-piperazinylcarbon-

yl)amino]-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-car-

boxylate (18). Compound 13 (2.18 g, 3.84 mmol) was sus-

pended in dry Cl(CH₂)₂Cl (60 mL) and stirred at 0 °C under

argon atmosphere. A solution of 1-benzylpiperazine (2.47 g,

14 mmol) in dry Cl(CH₂)₂Cl (10 mL) was added dropwise over

10 min. After being stirred at 0 °C for 1.5 h, the reaction

mixture was diluted with Cl(CH₂)₂Cl (40 mL), washed with

H₂O (6 × 200 mL) and saturated aqueous NaCl (200 mL) and

dried (Na₂SO₄). The solvent was removed in vacuo, and the

crude product was recrystallized from CH₃OH to yield 18 (1.41

g, 66%) as yellowish crystals: mp 127.5-129.5 °C. Anal.

(C₃₁H₃₈N₄O₃S × 0.35 CH₃OH) C, N; H: calcd, 7.12, found, 6.71.

6-Benzyl-3-tert-butoxycarbonyl-4,5,6,7-tetrahydro-2-

(4-nitrophenoxycarbonylamino)thieno[2,3-c]pyridin-6-

ium Chloride (13). A mixture of 1 (4.13 g, 12 mmol), dry

pyridine (1.66 g, 21 mmol), and dry Cl(CH₂)₂Cl (150 mL) was

stirred at 0 °C. A solution of p-nitrophenyl chloroformate (2.58

g, 12.8 mmol) in dry Cl(CH₂)₂Cl (15 mL) was added dropwise

over 35 min. After being stirred at 0 °C for 1.5 h, the reaction

mixture was kept at -18 °C overnight. The precipitate was

collected by filtration, washed with dry CH₂Cl₂, and dried to

obtain 13 (3.82 g, 56%) as white crystals: mp 169-172 °C

(decomp). Anal. (C₂₆H₂₇N₃O₆S × HCl × 0.25 CH₂Cl₂) C, H, N.

7-Benzyl-2-diethylamino-5,6,7,8-tetrahydro-4H-pyrido-

[4′,3′:4,5]thieno[2,3-d][1,3]oxazin-4-one (19). A mixture of

trifluoroacetic anhydride (0.88 g, 4.2 mmol) and trifluoroacetic

acid (3 mL) was stirred at 0 °C under argon atmosphere.

Compound 14 (1.33 g, 2.95 mmol) was added in portions over

30 min. Stirring was continued at 0 °C for 30 min and at room-

temperature overnight. The mixture was poured into chilled

saturated aqueous NaHCO₃(70 mL) and extracted with ethyl

acetate (2 × 100 mL). The organic layer was washed with H₂O

(2 × 50 mL) and saturated aqueous NaCl (50 mL) and dried

(Na₂SO₄). After removal of the solvent in vacuo, the residue

was recrystallized from n-hexane to yield 19 (0.86 g, 79%) in

two modifications, as yellow crystals and a yellow solid, which

were combined: mp 116-117 °C; HRMS m/z calcd for

C₂₀H₂₂N₃O₂S (M⁺- H), 368.1433; found, 368.1441. Anal.

(C₂₀H₂₃N₃O₂S) C, H, N.

7-Benzyl-2-(N-benzyl-N-methylamino)-5,6,7,8-tetrahy-

dro-4H-pyrido[4′,3′:4,5]thieno[2,3-d][1,3]oxazin-4-one (20).

A mixture of trifluoroacetic anhydride (0.59 g, 2.80 mmol) and

trifluoroacetic acid (3.5 mL) was stirred at 0 °C under argon

atmosphere. Compound 15 (0.98 g, 2 mmol) was added in

portions over 30 min. Stirring was continued at 0 °C for 30

min and at room-temperature overnight. The mixture was

poured into chilled saturated aqueous NaHCO₃(90 mL) and

extracted with ethyl acetate (2 × 100 mL). The organic layer

was washed with H₂O (2 × 50 mL) and saturated aqueous

NaCl (50 mL) and dried (Na₂SO₄). After removal of the solvent

in vacuo, the residue was recrystallized from n-hexane to yield

20 (0.63 g, 75%) as a white solid: mp 156-158 °C; HRMS m/z

calcd for C₂₄H₂₂N₃O₂S (M⁺- H), 416.1433; found, 416.1434.

Anal. (C₂₄H₂₃N₃O₂S × 0.25 H₂O) C, H, N.

tert-Butyl 6-Benzyl-2-(3,3-diethylureido)-4,5,6,7-tet-

rahydrothieno[2,3-c]pyridine-3-carboxylate (14). Com-

pound 13 (3.28 g, 5.78 mmol) was suspended in dry Cl(CH₂)₂Cl

(90 mL) and stirred at 0 °C under argon atmosphere. A

solution of diethylamine (1.54 g, 21 mmol) in dry Cl(CH₂)₂Cl

(15 mL) was added dropwise over 20 min. After being stirred

at 0 °C for 30 min, the reaction mixture was diluted with

Cl(CH₂)₂Cl (100 mL), washed with H₂O (5 × 300 mL) and

saturated aqueous NaCl (300 mL), and dried (Na₂SO₄). The

solvent was removed in vacuo, and the crude product was

recrystallized from CH₃OH to yield 14 (1.46 g, 56%) as dark

yellow crystals: mp 141-143 °C. Anal. (C₂₄H₃₃N₃O₃S × 0.25

CH₃OH) C, H, N.

tert-Butyl 6-Benzyl-2-(3-benzyl-3-methylureido)-4,5,6,7-

tetrahydrothieno[2,3-c]pyridine-3-carboxylate (15). Com-

pound 13 (2.84 g, 5 mmol) was suspended in dry Cl(CH₂)₂Cl

(70 mL) and stirred at 0 °C under argon atmosphere. A

solution of N-benzyl-N-methylamine (4.24 g, 35 mmol) in dry

Cl(CH₂)₂Cl (30 mL) was added dropwise over 20 min. After

being stirred at 0 °C for 1.5 h and at room temperature for

additional 1.5 h, the reaction mixture was kept at - 18 °C

overnight. The precipitate was filtered off and washed with

dry Cl(CH₂)₂Cl (80 mL). After combination of the organic

layers, they were washed with H₂O (7 × 200 mL) and

saturated aqueous NaCl (200 mL) and dried (Na₂SO₄). The

solvent was removed in vacuo, and the crude product was

recrystallized from CH₃OH to yield 15 (1.64 g, 67%) as yellow

crystals: mp 153-154 °C. Anal. (C₂₈H₃₃N₃O₃S) C, H, N.

tert-Butyl 6-Benzyl-2-[3-(3,4-dimethoxybenzyl)-3-me-

thylureido]-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-

carboxylate (16). Compound 13 (2.27 g, 4 mmol) was

suspended in dry Cl(CH₂)₂Cl (60 mL) and stirred at 0 °C under

argon atmosphere. A solution of N-(3,4-dimethoxybenzyl)-N-

methylamine (2.34 g, 14 mmol) in dry Cl(CH₂)₂Cl (10 mL) was

added dropwise over 20 min. After being stirred at 0 °C for

1.5 h and at room temperature for 1 h, the precipitate was

filtered off and washed with dry Cl(CH₂)₂Cl (80 mL). After

combination of the organic layers, they were washed with H₂O

(7 × 200 mL) and saturated aqueous NaCl (200 mL) and dried

7-Benzyl-5,6,7,8-tetrahydro-2-(N-3,4-dimethoxybenzyl-

N-methylamino)-4H-pyrido[4′,3′:4,5]thieno[2,3-d][1,3]-

oxazin-4-one (21). Compound 16 (1.10 g, 2 mmol) was added

in portions over 30 min to trifluoroacetic acid (3 mL), which

was stirred at 0 °C under argon atmosphere. After being

stirred for additional 2 h at 0 °C, the mixture was washed with

cold n-hexane (2 × 5 mL) and a cold mixture of dry CH₂Cl₂

(20 mL) and n-hexane (30 mL). The oily residue was dried in

vacuo, dissolved in dry CH₂Cl₂(2 mL), and stirred at 0 °C

under argon atmosphere. A solution of trifluoroacetic anhy-

dride (0.59 g, 2.80 mmol) in dry CH₂Cl₂(4 mL) was added

dropwise over 15 min. Stirring was continued at 0 °C for 1 h,

and the reaction mixture was kept at 4 °C overnight. After

Tricyclic 1,3-Oxazin-4-ones

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 26 8283

drying in vacuo, the residue was dissolved in cold CH₃OH (30

mL) and poured into ice-water (60 mL). The mixture (pH 2-3)

was adjusted to pH 8 by dropwise addition of saturated

aqueous NaHCO₃. The precipitate was collected by filtration,

washed with water, dried, and purified by column chroma-

tography (CH₂Cl₂/ethyl acetate, 4:1) followed by recrystalliza-

tion from ethyl acetate/n-hexane (1:9) to yield 21 (0.08 g, 8%)

as a yellowish solid: mp 157-159 °C (decomp, crystal conver-

sion to yellow crystals at 120 °C); HRMS m/z calcd for

C₂₆H₂₆N₃O₄S (M⁺- H), 476.1644; found, 476.1638. Anal.

(C₂₆H₂₇N₃O₄S × 0.75 H₂O) C, H, N.

being stirred for 5 h at 65 °C, the mixture was diluted with

0.4 M acetic acid (80 mL) and extracted with ether (1 × 65

mL and 3 × 15 mL). The organic layer was washed with H₂O

(3 × 20 mL) and dried (Na₂SO₄). The solvent was removed in

vacuo, and the crude product was recrystallized from n-hexane

to yield 27 (1.98 g, 86%) as yellow crystals: mp 95-96 °C.

Anal. (C₁₂H₁₇NO₃S) C, H, N.

tert-Butyl 2-Amino-4,7-dihydro-5H-thieno[2,3-c]thio-

pyran-3-carboxylate (28). A mixture of tetrahydrothiopyran-

4-one (0.93 g, 8 mmol), sulfur (0.26 g, 8 mmol), tert-butyl

cyanoacetate (1.13 g, 8 mmol), and C₂H₅OH (10 mL) was

treated dropwise with morpholine (0.70 g, 8 mmol) at 60 °C

over 5 min. After being stirred for 5 h at 60 °C, the mixture

was diluted with 0.4 M acetic acid (80 mL) and extracted with

ether (1 × 65 mL and 3 × 15 mL). The organic layer was

washed with H₂O (3 × 20 mL) and dried (Na₂SO₄). The solvent

was removed in vacuo, and the crude product was recrystal-

lized from n-hexane to yield 28 (1.60 g, 74%) as yellowish

crystals: mp 91-93 °C. Anal. (C₁₂H₁₇NO₂S₂) H, N; C: calcd,

53.11; found, 53.59.

tert-Butyl 2-Amino-4,5,6,7-tetrahydro-6-methylthieno-

[2,3-c]pyridine-3-carboxylate (29). A mixture of N-methyl-

4-piperidone (1.13 g, 10 mmol), sulfur (0.32 g, 10 mmol), tert-

butyl cyanoacetate (1.41 g, 10 mmol), and C₂H₅OH (10 mL)

was treated dropwise with morpholine (0.87 g, 10 mmol) at

45 °C over 5 min. After being stirred for 5 h at 45 °C, the

mixture was diluted with H₂O (30 mL) and extracted with

ether (1 × 65 mL and 4 × 15 mL). The organic layer was

washed with H₂O (3 × 20 mL) and dried (Na₂SO₄). The solvent

was removed in vacuo, and the crude product was recrystal-

lized from n-hexane to yield 29 (2.1 g, 78%) as yellowish

crystals: mp 134 °C. Anal. (C₁₃H₂₀N₂O₂S) C, H, N.

tert-Butyl 2-Amino-6-ethyl-4,5,6,7-tetrahydrothieno-

[2,3-c]pyridine-3-carboxylate (30). Compound 30 was pre-

pared by the foregoing procedure, but using N-ethyl-4-

piperidone (1.27 g, 10 mmol) in place of N-methyl-4-piperidone.

The crude product was recrystallized from n-hexane to

yield 30 (1.7 g, 60%) as yellow crystals: mp 74 °C. Anal.

(C₁₄H₂₂N₂O₂S) C, H, N.

7-Benzyl-5,6,7,8-tetrahydro-2-(morpholin-4-yl)-4H-py-

rido[4′,3′:4,5]thieno[2,3-d][1,3]oxazin-4-one (22). A mix-

ture of trifluoroacetic anhydride (0.59 g, 2.8 mmol) and

trifluoroacetic acid (2 mL) was stirred at 0 °C under argon

atmosphere. Compound 17 (0.92 g, 2 mmol) was added in

portions over 30 min. Stirring was continued at 0 °C for 30

min and at room-temperature overnight. The mixture was

poured into chilled saturated aqueous NaHCO₃(50 mL) and

extracted with ethyl acetate (2 × 200 mL). The organic layer

was washed with H₂O (2 × 100 mL) and saturated aqueous

NaCl (100 mL) and dried (Na₂SO₄). After removal of the

solvent in vacuo, the residue was recrystallized from n-hexane

to yield 22 (0.25 g, 33%) as yellow crystals: mp 154-155.5

°C; HRMS m/z calcd for C₂₀H₂₀N₃O₃S (M⁺- H), 382.1225;

found, 382.1229. Anal. (C₂₀H₂₁N₃O₃S) C, H, N.

7-Benzyl-2-(4-benzylpiperazin-1-yl)-5,6,7,8-tetrahydro-

4H-pyrido[4′,3′:4,5]thieno[2,3-d][1,3]oxazin-4-one (23). A

mixture of trifluoroacetic anhydride (0.59 g, 2.8 mmol) and

trifluoroacetic acid (3 mL) was stirred at 0 °C under argon

atmosphere. Compound 18 (1.09 g, 1.96 mmol) was added in

portions over 1 h. Stirring was continued at 0 °C for 30 min

and at room-temperature overnight. The mixture was poured

into chilled saturated aqueous NaHCO₃(70 mL) and extracted

with ethyl acetate (2 × 100 mL). Insoluble material was

filtered off and washed with ethyl acetate (2 × 50 mL). After

combination of the organic layers, they were washed with H₂O

(2 × 100 mL) and saturated aqueous NaCl (100 mL) and dried

(Na₂SO₄). The solvent was removed in vacuo, and the residue

was recrystallized from n-hexane to yield 23 (0.07 g, 8%) as

yellow crystals: mp 149-150.5 °C; HRMS m/z calcd for

C₂₇H₂₇N₄O₂S (M⁺- H), 471.1885; found, 471.1885. Anal.

(C₂₇H₂₈N₄O₂S × 0.25 H₂O) C, H, N.

tert-Butyl 2-Amino-4,5,6,7-tetrahydro-6-(2-methylpro-

pyl)thieno[2,3-c]pyridine-3-carboxylate (31). According to

the procedure outlined above, N-(2-methylpropyl)-4-piperidone

(1.55 g, 10 mmol) was used in place of N-methyl-4-piperidone.

The crude product was dissolved in boiling n-hexane and

filtered. Removal of the solvent in vacuo yielded 31 (2.94 g,

95%) as a bay oil. A sample for elemental analysis was

prepared by dissolving 31 in dry Cl(CH₂)₂Cl, adding 4 M HCl

solution in 1,4-dioxane, and ether. The mixture was kept at

-18 °C and the hydrochloride of 31 was filtered off (mp > 120

°C, decomp). Anal. (C₁₆H₂₆N₂O₂S × 1.4 HCl) C, H, N.

tert-Butyl R-Cyano-R-cyclopentylidene-acetate (24).

Following a similar methodology,⁹²cyclopentanone (18.8 g, 224

mmol) and tert-butyl cyanoacetate (25.3 g, 179 mmol) were

dissolved in dry toluene. Glacial acetic acid (3.00 g, 50 mmol)

and ammonium acetate (2.00 g, 26 mmol) were added, and the

mixture was refluxed for 15 h using a Dean-Stark water trap.

After cooling, the reaction mixture was washed with half-

saturated aqueous NaCl (4 × 30 mL) and dried (Na₂SO₄).

The volume was reduced in vacuo, and the mixture was kept

at -72 °C. The precipitate was collected by filtration to

obtain 24 (16.2 g, 44%) as brownish crystals: mp 93-94 °C

(C₂H₅OH), lit. mp 87-88 °C.⁹³Anal. (C₁₂H₁₇NO₂× 0.1

C₂H₅OH) C, H, N.

tert-Butyl 2-Amino-6-benzoyl-4,5,6,7-tetrahydrothieno-

[2,3-c]pyridine-3-carboxylate (32). A mixture of N-benzoyl-

4-piperidone (0.55 g, 2.7 mmol), sulfur (0.09 g, 2.7 mmol), tert-

butyl cyanoacetate (0.38 g, 2.7 mmol), and C₂H₅OH (30 mL)

was treated dropwise with morpholine (0.24 g, 2.7 mmol) at

45 °C over 5 min. After being stirred for 15 h at 45 °C, the

white precipitate was collected by filtration. The filtrate was

diluted with H₂O (30 mL) and extracted with ether (1 × 70

mL and 2 × 15 mL). The organic layer was washed with H₂O

(2 × 50 mL) and dried (Na₂SO₄). The solvent was removed in

vacuo to obtain an additional amount of 32 (total yield 0.82 g,

82%): mp 210-217 °C (decomp). Anal. (C₁₉H₂₂N₂O₃S) C, H,

N.

6,7-Dihydro-2-(dimethylamino)-4H,5H-cyclopenta[4,5]-

thieno[2,3-d][1,3]oxazin-4-one (33). Compound 25 (1.00 g,

4.18 mmol) was dissolved in dry Cl(CH₂)₂Cl (15 mL), and

dichloromethylene dimethylammonium chloride (0.68 g, 4.18

mmol) was added. After being stirred for 5 min at room

temperature, the reaction mixture was cooled to 0 °C, treated

with 1 M ethereal HCl (0.9 mL), and refluxed for 1 h. After

cooling, the precipitate was collected by filtration, washed with

cold Cl(CH₂)₂Cl, and dried. The crude product was recrystal-

lized from n-hexane to yield 33 (0.44 g, 45%) as white

tert-Butyl

2-Amino-5,6-dihydro-4H-cyclopenta[b]-

thiophene-3-carboxylate (25). A mixture of compound 24

(4.15 g, 20 mmol), sulfur (0.64 g, 20 mmol), and C₂H₅OH (25

mL) was treated dropwise with diethylamine (1.46 g, 20 mmol)

at 60 °C over 5 min. After being stirred for 10 h at 60 °C, and

84 h at room temperature, the mixture was diluted with 0.4

M acetic acid (120 mL) and extracted with ether (1 × 75 mL

and 4 × 30 mL). The organic layer was washed with H₂O (4 ×

20 mL), dried (Na₂SO₄), and the solvent was removed in vacuo.

The crude product was purified by 2-fold column chromatog-

raphy (petrol ether/ethyl acetate, 10:1) to yield 25 (2.05 g, 43%)

as yellow crystals: mp 68-72 °C. Anal. (C₁₂H₁₇NO₂S) C, H,

N.

tert-Butyl 2-Amino-4,7-dihydro-5H-thieno[2,3-c]pyran-

3-carboxylate (27). A mixture of tetrahydropyran-4-one (0.90

g, 9 mmol), sulfur (0.29 g, 9 mmol), tert-butyl cyanoacetate

(1.27 g, 9 mmol), and C₂H₅OH (10 mL) was treated dropwise

with morpholine (0.78 g, 9 mmol) at 65 °C over 5 min. After

8284 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 26

Pietsch and Gu¨tschow

crystals: mp 143-144 °C; HRMS m/z calcd for C₁₁H₁₂N₂O₂S

with H₂O (200 mL) and dried (Na₂SO₄). The solvent was

removed in vacuo, and the residue was purified by 2-fold

column chromatography (petrol ether/CH₃COCH₃/CH₃OH, 5:3:

2) and recrystallization from n-hexane to yield 38 (0.09 g,

6%) as yellowish crystals: mp 95-96.5 °C; HRMS m/z calcd

for C₁₃H₁₇N₃O₂S (M⁺), 279.1041; found, 279.1039. Anal.

(C₁₃H₁₇N₃O₂S × 0.25 H₂O) C, H, N.

(M⁺), 236.0620; found, 236.0616. Anal. (C₁₁H₁₂N₂O₂S) C, H,

N.

5,6,7,8-Tetrahydro-2-(dimethylamino)-4H-benzo[4,5]-

thieno[2,3-d][1,3]oxazin-4-one (34). Compound 26 (1.00 g,

9 mmol) was dissolved in dry Cl(CH₂)₂Cl (25 mL), and

dichloromethylene dimethylammonium chloride (1.46 g, 9

mmol) was added. The reaction mixture was cooled to 0 °C,

treated with 1 M ethereal HCl (1.8 mL), and refluxed for 1 h.

After cooling, the precipitate was collected by filtration, washed

with cold Cl(CH₂)₂Cl, and dried to obtain 34 × HCl (0.72 g,

43%). An analytical sample was obtained by recrystallization

from 50% C₂H₅OH to yield 34 as a white solid: mp 165-166

°C, lit. mp 156-157 °C;⁷⁵HRMS m/z calcd for C₁₂H₁₄N₂O₂S

(M⁺), 250.0776; found, 250.0778. Anal. (C₁₂H₁₄N₂O₂S × HCl)

C, H, N.

5,8-Dihydro-2-(dimethylamino)-4H,6H-pyrano[4′,3′:4,5]-

thieno[2,3-d][1,3]oxazin-4-one (35). Compound 27 (1.28 g,

5 mmol) was dissolved in dry Cl(CH₂)₂Cl (30 mL), and

dichloromethylene dimethylammonium chloride (0.81 g, 5

mmol) was added. After being stirred for 30 min at 50 °C, the

reaction mixture was cooled to 0 °C, treated with 1 M ethereal

HCl (1.8 mL), and refluxed for 1 h. After removal of the solvent

in vacuo, the residue was treated with boiling n-hexane (250

mL). The suspension was refluxed for 30 min and filtered. The

filtrate was cooled and the precipitate was collected by

filtration to obtain 35 (0.25 g, 20%) as white crystals: mp 194-

196 °C (decomp); HRMS m/z calcd for C₁₁H₁₂N₂O₃S (M⁺),

252.0569; found, 252.0572. Anal. (C₁₁H₁₂N₂O₃S) H, N; C: calcd,

52.37; found, 52.93.

2-Dimethylamino-5,8-dihydro-4H,6H-thiopyrano[4′,3′:

4,5]thieno[2,3-d][1,3]oxazin-4-one (36). Compound 28 (1.36

g, 5 mmol) was dissolved in dry Cl(CH₂)₂Cl (15 mL), and

dichloromethylene dimethylammonium chloride (0.81 g, 5

mmol) was added. After being stirred for 15 min at room

temperature, the reaction mixture was cooled to 0 °C, treated

with 1 M ethereal HCl (1.1 mL), and refluxed for 1 h. After

keeping the reaction mixture for 45 min at 4 °C, the precipitate

was collected by filtration, washed with cold Cl(CH₂)₂Cl, and

dried. The crude product was treated with boiling n-hexane

(100 mL). The suspension was refluxed for 40 min and filtered.

The filtrate was cooled and the precipitate was collected by

filtration to obtain 36 (0.64 g, 48%) as white crystals: mp 174-

175 °C; HRMS m/z calcd for C₁₁H₁₂N₂O₂S₂(M⁺), 268.0340;

found, 268.0330. Anal. (C₁₁H₁₂N₂O₂S₂) H, N; C: calcd, 49.23;

found, 48.71.

5,6,7,8-Tetrahydro-7-methyl-2-(dimethylamino)-4H-py-

rido[4′,3′:4,5]thieno[2,3-d][1,3]oxazin-4-one (37): General

Procedure for Thieno[2,3-d][1,3]oxazin-4-ones 37, 38,

and 40. Compound 29 (1.34 g, 5 mmol) was dissolved in dry

Cl(CH₂)₂Cl (70 mL), and dichloromethylene dimethylammo-

nium chloride (0.81 g, 5 mmol) was added. After being stirred

for 1 h at room temperature and 1 h at 50 °C, the reaction

mixture was cooled to 0 °C, treated with 1 M ethereal HCl

(6.1 mL), stirred for 1 h at 0 °C, and refluxed for 3.5 h. After

cooling, the precipitate was collected by filtration, washed with

cold Cl(CH₂)₂Cl, and dried. The crude product was dissolved

in ice-water (650 mL) and filtered. The acidic filtrate (pH 3-4)

was adjusted to pH 10 by dropwise addition of saturated

aqueous Na₂CO₃and extracted with ethyl acetate (1 × 250

mL and 2 × 500 mL). The organic layer was washed with H₂O

(200 mL) and dried (Na₂SO₄). The solvent was removed in

vacuo, and the residue was purified by column chromatography

(petrol ether/CH₃COCH₃/CH₃OH, 5:3:2) to yield 37 (0.34 g,

26%) as yellowish crystals: mp 166-169 °C (decomp); HRMS

m/z calcd for C₁₂H₁₅N₃O₂S (M⁺), 265.0885; found, 265.0887.

Anal. (C₁₂H₁₅N₃O₂S) C, H; N: calcd, 15.84; found, 15.39.

5,6,7,8-Tetrahydro-2-(dimethylamino)-7-(2-methyl-

propyl)-4H-pyrido[4′,3′:4,5]thieno[2,3-d][1,3]oxazin-4-

one (39). Compound 31 (1.55 g, 5 mmol) was dissolved in dry

Cl(CH₂)₂Cl (70 mL), and dichloromethylene dimethylammo-

nium chloride (0.81 g, 5 mmol) was added. After being stirred

for 1 h at room temperature and 1 h at 50 °C, the reaction

mixture was cooled to 0 °C, treated with 1 M ethereal HCl

(6.1 mL), stirred for 1 h at 0 °C, and refluxed for 3.5 h. The

solvent was removed in vacuo; the residue was dissolved in

ice-water (700 mL) and filtered. The acidic filtrate (pH 4-5)

was adjusted to pH 10 by dropwise addition of saturated

aqueous Na₂CO₃and extracted with ethyl acetate (2 × 250

mL and 2 × 500 mL). The organic layer was washed with H₂O

(2 × 250 mL) and dried (Na₂SO₄). The solvent was removed

in vacuo, and the residue was purified by column chromatog-

raphy (ethyl acetate) and recrystallization from n-hexane to

yield 39 (0.29 g, 19%) as yellow crystals: mp 117-118 °C;

HRMS m/z calcd for C₁₅H₂₁N₃O₂S (M⁺), 307.1354; found,

307.1352. Anal. (C₁₅H₂₁N₃O₂S) H, N; C: calcd, 58.61; found,

59.06.

7-Benzyl-5,6,7,8-tetrahydro-2-(dimethylamino)-4H-py-

rido[4′,3′:4,5]thieno[2,3-d][1,3]oxazin-4-one (40). This com-

pound was prepared from 1 (1.72 g, 5 mmol). The crude

product was dissolved in ice-water (800 mL) and filtered.

The acidic filtrate (pH 3-4) was adjusted to pH 10 by drop-

wise addition of saturated sodium carbonate solution and

extracted with ethyl acetate (2 × 200 mL and 2 × 300 mL).

The organic layer was washed with H₂O (3 × 300 mL) and

dried (Na₂SO₄). The solvent was removed in vacuo, and the

residue was purified by column chromatography (petrol ether/

ethyl acetate, 2:1) to yield 40 as yellowish crystals. A second

fraction from the chromatographic purification was recrystal-

lized from n-hexane to obtain an additional amount of the

product (total yield 0.47 g, 28%): mp 147-148 °C; HRMS m/z

calcd for C₁₈H₁₉N₃O₂S (M⁺), 341.1198; found, 341.1184. Anal.

(C₁₈H₁₉N₃O₂S) C, H, N.

7-Benzoyl-5,6,7,8-tetrahydro-2-(dimethylamino)-4H-

pyrido[4′,3′:4,5]thieno[2,3-d][1,3]oxazin-4-one (41). Com-

pound 32 (1.08 g, 3 mmol) was dissolved in dry Cl(CH₂)₂Cl

(100 mL), and dichloromethylene dimethylammonium chloride

(0.49 g, 3 mmol) was added. After being stirred for 1.5 h at

room temperature and 1 h at 50 °C, the reaction mixture was

cooled to 0 °C, treated with 1 M ethereal HCl (1.8 mL), stirred

for 30 min at 0 °C, and refluxed for 3.5 h. After removal of the

solvent in vacuo, the crude product was purified by column

chromatography (ethyl acetate) to obtain 41 (0.30 g, 27%) as

a yellowish solid: mp 159-162 °C (n-hexane, decomp); HRMS

m/z calcd for C₁₈H₁₇N₃O₃S (M⁺), 355.0991; found, 355.0995.

Anal. (C₁₈H₁₇N₃O₃S × 0.5 H₂O) C, H, N.

Ethyl 6-Benzyl-4,5,6,7-tetrahydro-2-(3-isopropylthio-

ureido)thieno[2,3-c]pyridine-3-carboxylate (44). A mix-

ture of 42 (6.33 g, 20 mmol), isopropyl isothiocyanate (2.83 g,

28 mmol), and dry C₂H₅OH (8 mL) was stirred in an autoclave

at 80 °C for 18 h under argon atmosphere. After cooling,

further isopropyl isothiocyanate (2.83 g, 28 mmol) was added,

and the reaction mixture was stirred at 80 °C for 23 h. After

keeping at room-temperature overnight, the precipitate was

collected by filtration, washed with cold C₂H₅OH, and dried

to obtain 44 (7.13 g, 78%) as a salmon solid: mp 114.5-117

°C (CH₃OH). Anal. (C₂₁H₂₇N₃O₂S₂× 1.2 CH₃OH) C, N; H:

calcd, 7.03; found, 6.61.

2-Amino-7-benzyl-5,6,7,8-tetrahydro-4H-pyrido[4′,3′:

4,5]thieno[2,3-d][1,3]thiazin-4-one (47). Compound 47 was

prepared from 43 as described, mp 161-163 °C.⁷⁶

7-Benzyl-5,6,7,8-tetrahydro-2-isopropylamino-4H-py-

rido[4′,3′:4,5]thieno[2,3-d][1,3]-thiazin-4-one (48). A mix-

ture of compound 44 (2.09 g, 5 mmol) and concentrated H₂SO₄

7-Ethyl-5,6,7,8-tetrahydro-2-(dimethylamino)-4H-py-

rido[4′,3′:4,5]thieno[2,3-d][1,3]oxazin-4-one (38). This com-

pound was prepared from 30 (1.41 g, 5 mmol). The crude

product was dissolved in ice-water (700 mL) and filtered. The

acidic filtrate (pH 4) was adjusted to pH 10 by dropwise

addition of saturated aqueous Na₂CO₃, and extracted with

ethyl acetate (3 × 500 mL). The organic layer was washed

Tricyclic 1,3-Oxazin-4-ones

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 26 8285

(10 mL) was kept at room temperature for 3 days and poured

into a mixture of saturated aqueous NaHCO₃(500 mL) and

ice (500 g). The precipitate was collected by filtration, washed

with H₂O, and dried to obtain 48 (1.67 g, 90%) as a yellow

solid: mp 193.5-197 °C (CH₃OH, decomp); HRMS m/z calcd

for C₁₉H₂₀N₃OS₂(M⁺- H), 370.1048; found, 370.1041. Anal.

(C₁₉H₂₁N₃OS₂) C, H, N.

7-Benzyl-2-cyclohexylamino-5,6,7,8-tetrahydro-4H-py-

rido[4′,3′:4,5]thieno[2,3-d][1,3]thiazin-4-one (49). Com-

pound 49 was prepared from 45 as described, mp 204-205

°C.⁷⁷

Similarly, the inhibition of CEase by compound 33 (4-12 µM)

was studied in the presence of different substrate concentra-

tions (100-600 µM).

CEase-Catalyzed Turnover of the Heterocyclic Com-

pounds. The enzymatic conversion of the compounds was

followed spectrophotometrically at 25 °C. Into a cuvette

containing assay buffer, 500 µL of TC solution, 50 µL of

acetonitrile, and 10 µL of an inhibitor solution were added,

thoroughly mixed, and incubated for 5 min at 25 °C. The

reaction was initiated by adding a volume (59.8-61.3 µL) of a

CEase solution (122 µg/mL). This volume was adjusted by the

determination of a 1:6250 dilution that converts pNPB (200

µM) with a rate of 18 µM/min. A molar extinction coefficient,

ꢀ ) 7.67 mM^-1cm^-1, for p-nitrophenol at pH 7.0 was used for

this purpose. The entire volume was 1 mL containing the

following concentrations: 6 mM TC, CEase (adjusted activity),

6% acetonitrile, and 25 µM inhibitor. Reactions were analyzed

by monitoring UV/vis spectra in fixed time intervals or by

following the time course at 350 nm. The molar extinction

coefficient at 350 nm for each analyzed compound was

determined separately in duplicate experiments at 10 different

concentrations (5-50 µM). Control experiments to prove the

stability of the compounds were performed by adding 100 mM

sodium phosphate buffer, pH 7.0, instead of the enzyme

solution.

7-Benzyl-5,6,7,8-tetrahydro-2-phenylamino-4H-pyrido-

[4′,3′:4,5]thieno[2,3-d][1,3]thiazin-4-one (50). Compound 50

was prepared from 46 as described, mp 147-149 °C.⁷⁷

2-Benzoylamino-7-benzyl-5,6,7,8-tetrahydro-4H-pyrido-

[4′,3′:4,5]thieno[2,3-d][1,3]thiazin-4-one (51). A mixture of

compound 43 (1.44 g, 3 mmol) and concentrated H₂SO₄(6 mL)

was kept at room temperature for 2 days and poured into ice-

water (200 mL). The solution was adjusted to pH 8-9 by

dropwise addition of 2.5 N aqueous NaOH (70 mL) and 5%

aqueous Na₂CO₃(2.5 mL). The precipitate was collected by

filtration, washed with H₂O, dried, and recrystallized from

ethyl acetate to obtain 51 (0.38 g, 29%) as yellow crystals: mp

217-220 °C (decomp); HRMS m/z calcd for C₂₃H₁₉N₃O₂S₂(M⁺),

433.0919; found, 433.0910. Anal. (C₂₃H₁₉N₃O₂S₂) C, H, N.

AChE Inhibition Assay. AChE inhibition was assayed

spectrophometrically at 25 °C according to the method of

Ellman et al.⁹⁵Assay buffer was 100 mM sodium phosphate,

100 mM NaCl, pH 7.3. A stock solution of AChE (100 U/mL)

in assay buffer was kept at 0 °C. A 1:30 dilution was prepared

immediately before starting the measurement. ATCh (10 mM)

and DTNB (7 mM) were dissolved in assay buffer and kept at

0 °C. Galanthamine, tacrine, and compounds 49 and 51 were

dissolved in a 1:1 mixture of 0.1 M HCl and acetonitrile. Stock

solutions of all other inhibitors were prepared in acetonitrile.

Thieno[1,3]oxazin-4-ones 33-36 and 52-63, as well as thieno-

[1,3]thiazin-4-one 51 were analyzed at a single concentration

(5-50 µM). IC₅₀values were calculated using the equation IC₅₀

) [I]/(v₀/v - 1), where v₀and v are the rates in the absence

and presence of inhibitor. For galanthamine, tacrine, and the

thieno[1,3]oxazin-4-ones 4, 5, 7, 8, 12, 19-23, and 37-41, as

well as the thieno[1,3]thiazin-4-ones 47-50 at least five

different inhibitor concentrations were used. Progress curves

were characterized by a linear steady-state turnover of the

substrate and values of a linear regression were fitted accord-

ing to eq 1. Into a cuvette containing 830 µL of assay buffer,

50 µL of the DTNB solution, 50 µL of acetonitrile, 10 µL of an

inhibitor solution, and 10 µL of an AChE solution (3.33 U/ml)

were added and thoroughly mixed. After incubation for 15 min

at 25 °C, the reaction was initiated by adding 50 µL of the

ATCh solution. Inhibition studies with galanthamine, tacrine,

and compounds 49 and 51 were performed under the same

conditions, using 825 µL of buffer and 55 µL of acetonitrile.

Concentrations were as follows: 500 µM ATCh (0.91 × K_m),

350 µM DTNB, 0.033 U/mL AChE, 6% acetonitrile, and

different inhibitor concentrations. Uninhibited enzyme activity

was determined by adding acetonitrile instead of the inhibitor

solution. Determination of Michaelis constant for the substrate

ATCh was done at 11 different ATCh concentrations (50-2500

µM) to give a value K_m) 550 ( 40 µM. The rates of AChE-

catalyzed ATCh hydrolysis were corrected by those of the

nonenzymatic hydrolysis of ATCh as determined by using 10

µL of assay buffer, instead of the enzyme solution. Progress

curves were monitored at 412 nm over 5 min. Similarly, the

inhibition of AChE by galanthamine (0.76-8 µM), tacrine

(0.02-0.1 µM), 39 (1-6 µM), and 40 (2-6 µM) was studied in

the presence of different substrate concentrations (250-1000

µM).

Kinetic Analysis of the Nonenzymatic Hydrolysis.

Alkaline hydrolysis was followed spectrophotometrically at a

fixed wavelength (thieno[1,3]oxazin-4-ones 4, 5, 7, 8, 12, 19-

23, 33-41, and 62 ) 325-338 nm and thieno[1,3]thiazin-4-

ones 47-51 ) 367 nm), by monitoring the disappearance of

the compounds in 50 mM CAPS, pH 11.0, at 25 °C. Stock

solutions of the inhibitors were prepared in DMSO; the final

inhibitor concentration was 2.5-20 µM and the final DMSO

concentration was 5%. Curves were analyzed as first-order

reactions.

CEase Inhibition Assay in the Presence of a Chro-

mogenic Substrate. CEase inhibition was assayed spectro-

photometrically at 25 °C.^30,94Assay buffer was 100 mM sodium

phosphate, 100 mM NaCl, pH 7.0. A stock solution of CEase

(2.44 mg/mL) in 100 mM sodium phosphate buffer, pH 7.0,

was 1:20 diluted with the same buffer and kept at 0 °C for 4

h. A 1:122 dilution was done immediately before starting the

measurement. TC (12 mM) was dissolved in assay buffer and

kept at 25 °C. Compounds 49 and 51 were dissolved in a 1:1

mixture of 0.1 M HCl and acetonitrile. Stock solutions of all

other inhibitors and of pNPB (20 mM) were prepared in

acetonitrile. Thieno[1,3]oxazin-4-ones 4, 5, 7, 8, 12, 19-23, 35,

37-41, 52-55, and 59-63, as well as thieno[1,3]thiazin-4-

ones 47-51 were analyzed at a single concentration (5-50

µM). K_ivalues were calculated using the equation K_i(1 +

[S]/K_m^S) ) [I]/(v₀/v - 1), where v₀and v are the rates in the

absence and presence of inhibitor. For thieno[1,3]oxazin-4-ones

33, 34, 36, and 58, at least five different inhibitor concentra-

tions were used. Progress curves were characterized by a linear

steady-state turnover of the substrate, and values of a linear

regression were fitted to an equation of competitive inhibition

to obtain K_i(1 + [S]/K_m^S) values. Into a cuvette containing 430

µL of assay buffer, 500 µL of the TC solution, 40 µL of

acetonitrile, 10 µL of the pNPB solution, and 10 µL of an

inhibitor solution were added and thoroughly mixed. After

incubation for 5 min at 25 °C, the reaction was initiated by

adding 10 µL of the enzyme solution (1 µg/mL). Inhibition

studies with the compounds 49 and 51 were performed under

the same conditions, using 425 µL of buffer and 45 µL of

acetonitrile. Concentrations were as follows: 200 µM pNPB

(1.82 × K_m^S),³⁵6 mM TC, 10 ng/mL CEase, 6% acetonitrile,

and different inhibitor concentrations. Uninhibited enzyme

activity was determined by adding acetonitrile instead of the

inhibitor solution. The rates of CEase-catalyzed pNPB hy-

drolysis were corrected by those of the nonenzymatic hydroly-

sis of pNPB as determined by using 10 µL of 100 mM sodium

phosphate buffer, pH 7.0, instead of the enzyme solution.

Progress curves were monitored at 405 nm over 6 min.

Acknowledgment. The authors thank C. M. Gonza´-

lez Tanarro for analyzing AChE inhibition by galan-

thamine and tacrine and are grateful to the Graduate

College 677 for financial support.

8286 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 26

Pietsch and Gu¨tschow

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Supporting Information Available: Kinetic analysis of

the inhibition of CEase, elemental analysis data, IR data, UV

1

data, H NMR data, ¹³C NMR data, and MS (EI) data of the

synthesized compounds. This material is available free of

charge via the Internet at http://pubs.acs.org.

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JM0508639

Article Doi

Synthesis of tricyclic 1,3-oxazin-4-ones and kinetic analysis of cholesterol esterase and acetylcholinesterase inhibition

DOI: 10.1021/jm0508639

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Full text of DOI:10.1021/jm0508639

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