Change in the mode of inhibition of acetylcholinesterase by (4- nitrophenyl)sulfonoxyl derivatives of conformationally constrained choline analogues

Article abstract of DOI:10.1021/tx970019o

A chiral, five-step synthesis of 2-(hydroxymethyl)-2,4- dimethylmorpholine (12) from (R)- and (S)-2-methylglycidols gives an overall yield of 63%. Morpholines (R)- and (S)-12 are converted into 2-(azidomethyl)- 2,4-dimethylmorpholine (15) via 2,4-dimethyl-2-[[(4-nitrophenyl)sulfonoxy]- methyl]morpholine (14). The tertiary morpholines 12, 14, and 15 are quaternarized to afford 2-(hydroxymethyl)-2,4,4-trimethylmorpholinum iodide (2), 2,4,4-trimethyl-2-[[(4-nitrophenyl)-sulfonoxy]methyl]morpholinium iodide (3), and 2-(azidomethyl)-2,4,4-trimethylmorpholinium iodide (4), respectively, which all inhibit acetylcholinesterase (ACHE). These morpholinium inhibitors are compared with conformationally constrained aryl hemicholinium ACHE inhibitors. Enantiomers of 2 and 4 are reversible competitive inhibitors of ACHE, with values of K(i) = 360 ± 30 μM for (S)- 2, 650 ± 90 μM for (R)-2, 450 ± 70 μM for (S)-4, and 560 ± 30 μM for (R)-4, respectively. Enantiomers of 3 are noncompetitive inhibitors of AChE with values of K(i) = 19.0 ± 0.9 μM for (S)-3 and 50 ± 2 μM for (R)-3, respectively. AChE shows a 2-fold chiral discrimination in the case of inhibition by 2 and 3. Inhibition also changes from competitive to noncompetitive when (3-hydroxyphenyl)-N,N,N-trimethylammonium iodide (18) [K(i) = 0.21 ± 0.06 μM; Lee, B. H., Stelly, T. C., Colucci, W. J., Garcia, J. G., Gandour, R. D., and Quinn, D. M. (1992) Chem. Res. Toxicol. 5, 411- 418] is converted into [3-[(4-nitrophenyl)sulfonoxy]-phenyl]-N,N,N- trimethylammonium iodide (5), K(i) = 6.0 ± 0.5 μM. These results indicate that the 4-nitrobenzenesulfonyl group controls the mode of inhibition.

Full text of DOI:10.1021/tx970019o

Chem. Res. Toxicol. 1998, 11, 19-25
19
Ch a n ge in th e Mod e of In h ibition of Acetylch olin ester a se
by (4-Nitr op h en yl)su lfon oxyl Der iva tives of
Con for m a tion a lly Con str a in ed Ch olin e An a logu es
Prashant S. Savle,† Rohit A. Medhekar,‡ Ella L. Kelley,‡,§ J erome G. May,
Steven F. Watkins, Frank R. Fronczek, Daniel M. Quinn,‡ and
Richard D. Gandour*,†
Department of Chemistry, Virginia Tech, Blacksburg, Virginia 24061-0212,
Department of Chemistry, The University of Iowa, Iowa City, Iowa 52242, Department of Chemistry,
Southern University, Baton Rouge, Louisiana 70813, and Department of Chemistry,
Louisiana State University, Baton Rouge, Louisiana 70803-1804
Received February 10, 1997X
A chiral, five-step synthesis of 2-(hydroxymethyl)-2,4-dimethylmorpholine (12) from (R)- and
(S)-2-methylglycidols gives an overall yield of 63%. Morpholines (R)- and (S)-12 are converted
into 2-(azidomethyl)-2,4-dimethylmorpholine (15) via 2,4-dimethyl-2-[[(4-nitrophenyl)sulfonoxy]-
methyl]morpholine (14). The tertiary morpholines 12, 14, and 15 are quaternarized to afford
2-(hydroxymethyl)-2,4,4-trimethylmorpholinum iodide (2), 2,4,4-trimethyl-2-[[(4-nitrophenyl)-
sulfonoxy]methyl]morpholinium iodide (3), and 2-(azidomethyl)-2,4,4-trimethylmorpholinium
iodide (4), respectively, which all inhibit acetylcholinesterase (AChE). These morpholinium
inhibitors are compared with conformationally constrained aryl hemicholinium AChE inhibitors.
Enantiomers of 2 and 4 are reversible competitive inhibitors of AChE, with values of Ki ) 360
( 30 µM for (S)-2, 650 ( 90 µM for (R)-2, 450 ( 70 µM for (S)-4, and 560 ( 30 µM for (R)-4,
respectively. Enantiomers of 3 are noncompetitive inhibitors of AChE with values of Ki )
19.0 ( 0.9 µM for (S)-3 and 50 ( 2 µM for (R)-3, respectively. AChE shows a 2-fold chiral
discrimination in the case of inhibition by 2 and 3. Inhibition also changes from competitive
to noncompetitive when (3-hydroxyphenyl)-N,N,N-trimethylammonium iodide (18) [Ki ) 0.21
( 0.06 µM; Lee, B. H., Stelly, T. C., Colucci, W. J ., Garcia, J . G., Gandour, R. D., and Quinn,
D. M. (1992) Chem. Res. Toxicol. 5, 411-418] is converted into [3-[(4-nitrophenyl)sulfonoxy]-
phenyl]-N,N,N-trimethylammonium iodide (5), Ki ) 6.0 ( 0.5 µM. These results indicate that
the 4-nitrobenzenesulfonyl group controls the mode of inhibition.
site (2, 4). Inhibitors that bind only in the active site
show competitive inhibition, while those that bind only
in the peripheral site show noncompetitive inhibition.
In tr od u ction
Acetylcholinesterase (AChE)1 mediates the chemical
transmission of nerve impulses in the body. AChE, a
serine esterase, catalyzes the hydrolysis of the neu-
rotransmitter acetylcholine by a well-precedented acyl
enzyme mechanism (1, 2). Inhibitors of AChE serve
The X-ray crystal structure of Torpedo californica
AChE reveals an active site, that possesses a hydrogen-
bonded catalytic triad, consisting of Ser200, His440, and
Glu327, which is located at the bottom of a deep and
narrow gorge (5). As in the serine proteases, Ser200 and
His440 function as nucleophilic and general base cataly-
sis, respectively (2, 6). The active site of AChE accom-
modates a wide range of ligands (6). Photoaffinity
labeling studies assign Trp279 to the peripheral site,
which is located ∼14 Å from the active site. The
peripheral site recognizes quaternary ammonium ions
that inhibit enzymatic activity (7). X-ray structures of
bisquaternary compounds bound to AChE show ligands
binding at the peripheral site by interaction with the
π-electrons of the Trp279.
many roles in mental wellness, pest control, and chemical
warfare. Designing AChE inhibitors dates back to Sted-
man (3) in 1929, and many types of inhibitors abound
(2). Some of the strongest reversible inhibitors bind to
two sites on the enzyme, the active site and a peripheral
Racemic hemicholinium compounds 1 inhibit AChE (8).
Hemicholiniums exist in open and cyclic forms (9),
although the equilibrium favors the cyclic morpholinium
structure with the hydroxyl group axial and the R group
equatorial. Morpholiniums provide a conformationally
constrained molecular skeleton on which to anchor other
groups to explore the stereochemical topography of the
active site. Nonracemic 2,2-disubstituted N,N-dimeth-
† Virginia Tech.
‡ The University of Iowa.
§ Southern University.
Louisiana State University.
X Abstract published in Advance ACS Abstracts, December 1, 1997.
1
Abbreviations: AChE, acetylcholinesterase; DTNB, 5,5′-dithiobis-
(2-nitrobenzoic acid); TBDMS-Cl, tert-butyldimethylsilyl chloride;
DMAP, 4-(dimethylamino)pyridine; KH, potassium hydride; TBAF,
tetrabutylammonium fluoride; MTPA, R-methoxy-R-(trifluorometh-
yl)phenylacetic acid; DCC, 1,3-dicyclohexylcarbodiimide.
S0893-228x(97)00019-2 CCC: $15.00 © 1998 American Chemical Society
Published on Web 01/19/1998

20 Chem. Res. Toxicol., Vol. 11, No. 1, 1998
Savle et al.
tion between the lines. 13C chemical shifts are also expressed
in ppm relative to the solvent chemical shift. Assignments of
the 1H and 13C NMR signals were made by comparison with
similar compounds and using 2D 13C-1H correlation and 2D
1H COSY experiments. Infrared spectra, reported in cm-1, were
recorded as thin films on KBr cells. FAB MS samples were
prepared by suspending in glycerol. Elemental analyses were
performed by Atlantic Microlabs of Norcross, GA. The optical
rotations were recorded in a 10- × 100-mm cell.
ylmorpholinium compounds 2-4 suit our goal for explo-
ration as the individual enantiomers can be synthesized.
Unless otherwise noted, materials were obtained from com-
mercial sources and used without further purification. THF was
distilled from sodium-benzophenone ketyl. Diethyl ether was
distilled from sodium. Triethylamine and nitromethane were
distilled from CaH2 and stored over Linde molecular sieves type
3 Å. CH2Cl2 was distilled over CaH2. MeOH was distilled over
a small amount of Mg. Solutions were dried over MgSO4 and
concentrated by rotary evaporation, unless specified otherwise.
(R )-1-[[(t er t -Bu t yld im e t h ylsilyl)oxy]m e t h yl]-1-m e t h -
yloxir a n e, (R)-7 (16). To a stirred solution of (S)-2-methyl-
glycidol (5.6 g, 63 mmol) and anhydrous NEt3 (7.1 g, 70 mmol)
in dry CH2Cl2 (50 mL) was added a solution of tert-butyldi-
methylsilyl chloride (TBDMS-Cl) (10.1 g, 66.8 mmol) in dry
CH2Cl2 (20 mL) dropwise at room temperature. After stirring
for 4 h at room temperature, the cloudy solution was filtered
through Celite. The filtrate was washed with saturated NaH-
CO3 solution (1 × 20 mL) and brine (1 × 25 mL). The organic
phase was dried and concentrated. The residue was purified
by flash chromatography (5% EtOAc-hexanes) to afford 12.7 g
(98%) of (R)-7 as a colorless oil. TLC Rf ) 0.2 (15% ether-
hexanes, silica). 1H NMR (400 MHz): 0.06 (s, 3H), 0.07 (s, 3H),
0.89 (s, 9H), 1.34 (s, 3H), 2.59 (d, J app ) 5.2 Hz, 1H), 2.75 (d,
In itia l Ra tion a le. We based our choice for examining
enantiomers 2-4 as AChE inhibitors on various ideas.
We envisioned that the hydroxyl group in 2 could form
hydrogen bonds to His440 and the enantiomers could
show strong stereoselectivity. We thought that the azido
group in 4 could potentially serve as a photolabeling
group to show where the enantiomers bound in the active
site. We decided to assay 3 because we could make it
readily, and we could rationalize that the sulfonoxyl
group in 3 could serve as a transition-state analogue with
the nitrophenyl ring interacting strongly with the walls
of the gorge leading to the active site. As with many
cases in research, our initial rationale for assaying 2 and
4 produced modest results. To our delight, assays of 3
produced a discovery.
In this report, we describe the chiral syntheses and
structures of the R and S enantiomers of 2-4 and the
inhibition of AChE-catalyzed hydrolysis of acetylthiocho-
line by them. The stereoisomers of 2 and 4 show
competitive inhibition, while those of 3 show noncompeti-
tive inhibition. We also describe the noncompetitive
inhibition by [3-[(4-nitrophenyl)sulfonoxy]phenyl]-N,N,N-
trimethylammonium iodide, 5.
Syn th esis. Interest is increasing in the 2-morpholi-
nylmethyl group for its neuropharmacological and gas-
trokinetic properties (10, 11). There are no reports on
the synthesis of nonracemic 2,2-disubstituted morpho-
lines, only chiral syntheses of 2-substituted, 2,6-disub-
stituted (12), and 2,3,6- (13), 2,2,6- (14), and 2,5,5- (15)
trisubstituted analogues, which are unsuitable for pre-
paring 2-4. 2-Substituted morpholine is synthesized by
opening nonracemic benzyl glycidyl ether with 2-amino-
ethylhydrogen sulfate and then cyclization with sodium
hydroxide to give the morpholine in 37-55% yield (10,
11). This protocol in our hands, however, fails to produce
any 2,2-disubstituted morpholine from benzyl 2-meth-
ylglycidyl ether, because side products form more rapidly
than the desired product. A three-step approach, opening
a protected 2-methylglycidol with N-methylaminoethanol
followed by transformation of the unprotected primary
alcohol into a leaving group and then intramolecular
nucleophilic substitution, gives the desired 2,2-disubsti-
tuted morpholine.
J app ) 4.8 Hz, 1H), 3.60 (d, J app ) 11.2 Hz, 1H), 3.65 (d, J app
)
11.2 Hz, 1H). 13C NMR (100 MHz): -5.5, 17.9, 18.2, 25.8, 51.4,
56.9, 66.5. IR (neat): 1102, 1462, 3048. Anal. Calcd for
C10H22O2Si: C, 59.37; H,10.97. Found: C, 59.31; H, 10.92.
[R]23 -3.7° (c 5.8, CHCl3).
D
Da ta for (S)-7: found C, 59.48; H, 11.12. [R]23 +3.8° (c 2.3,
D
CHCl3).
(R)-[3-[(ter t-Bu tyld im eth ylsilyl)oxy]-2-h yd r oxy-2-m eth -
ylp r op yl](2-h yd r oxyeth yl)m eth yla m in e, (R)-8. A solution
of (R)-7 (3.7 g, 18.4 mmol) and 2-(methylamino)ethanol (1.4 g,
18.4 mmol) in MeOH (60 mL) was stirred for 12 h at 50 °C. The
reaction mixture was concentrated to yield a pale yellow residue,
which was purified by column chromatography on neutral
alumina, eluting with 5% MeOH-EtOAc to give (R)-8 as a
colorless oil, 4.4 g (87%). TLC Rf ) 0.3 (10% MeOH-EtOAc,
neutral alumina). 1H NMR (270 MHz): 0.06 (s, 6H), 0.9 (s, 9H),
1.13 (s, 3H), 2.37 (d, J app ) 14 Hz, 1H), 2.39 (s, 3H), 2.56 (d,
J app ) 14 Hz, 1H), 2.64-2.67 (m, 2H), 2.8 (br, 1H), 3.44 (br s,
2H), 3.61 (t, J app ) 5.2 Hz, 2H). 13C NMR (100 MHz): -5.5,
18.2, 22.9, 25.8, 45.1, 59.6, 61.5, 63.5, 68.3, 72.9. IR (neat):
3458. MS m/z (CI): 278 (M+ + 1), 246 (M+ - CH3OH), 220 (M+
- tert-C4H9). Anal. Calcd for C13H31O3NSi: C, 56.28; H,11.27;
N, 5.05. Found: C, 56.12; H, 11.21; N, 4.98. [R]23 -0.44° (c
D
4.1, CHCl3).
Da ta for (S)-8: found C, 56.20; H, 11.14; N, 4.94. [R]23
D
+0.40° (c 2.1, CHCl3).
(R)-[3-[(ter t-Bu tyld im eth ylsilyl)oxy]-2-h yd r oxy-2-m eth -
ylp r op yl](2-ch lor oeth yl)m eth yla m in e, (R)-9. To a solution
of (R)-8 (2.01 g, 7.02 mmol) and NEt3 (1.8 g, 18.1 mmol) in dry
CH2Cl2 (10 mL) at room temperature was added tosyl chloride
(TsCl) (1.5 g, 7.7 mmol) in dry CH2Cl2 (10 mL) dropwise in 30
min. The reaction mixture was stirred for 5 h and then diluted
with CH2Cl2 (20 mL). The solution was washed with saturated
NaHCO3 (1 × 20 mL) and brine (1 × 25 mL). The extract was
dried and concentrated to afford a dark brown oil, which was
chromatographed on neutral alumina eluting with 1:1 hexanes-
EtOAc to afford (R)-9 as a pale yellow oil (1.8 g, 89%). TLC Rf
) 0.35 (1:3 hexanes-EtOAc, neutral alumina). 1H NMR (270
Exp er im en ta l P r oced u r es
Gen er a l Meth od s. Uncorrected melting points were mea-
sured on a digital melting point apparatus equipped with
multistage ramping rates from 0.1 to 10.0 °C/min. 1H NMR
spectra were recorded at 200, 270, and 400 MHz. 13C NMR
spectra were recorded at 100 MHz. Unless noted otherwise, all
NMR spectra were recorded in CDCl3. Proton chemical shifts
are expressed in ppm downfield from internal TMS. Coupling
constants (expressed as J app in Hz) were the observed separa-
MHz): 0.04 (s, 6H), 0.89 (s, 9H), 1.09 (s, 3H), 2.26 (d, J app
)
13.8 Hz, 1H), 2.39 (s, 3H), 2.62 (d, J app ) 13.8 Hz, 1H), 2.79-
2.85 (m, 2H), 3.0 (br s, 1H), 3.36 (d, J app ) 9.5 Hz, 1H), 3.42 (d,

(4-Nitrophenyl)sulfonoxyl Choline Analogues
Chem. Res. Toxicol., Vol. 11, No. 1, 1998 21
J app ) 9.5 Hz, 1H), 3.54 (t, J app ) 6.6 Hz, 2H). 13C NMR (67.5
MHz): -5.5, 18.1, 22.9, 25.8, 42.1, 44.6, 61.2, 63.4, 68.1, 72.1.
IR (CH2Cl2): 3385. MS m/z (CI): 296 (M+ + 1), 260 (M+ + 1 -
Cl), 238 (M+ - tert-C4H9). Anal. Calcd for C13H30O2NClSi: C,
52.85; H, 10.24; N, 4.74. Found: C, 52.81; H, 10.19; N, 4.65.
(3 × 5 mL). The pale yellow solid was dried under vacuum to
give 0.25 g (84%). The crude product was recrystallized initially
from acetone followed by two recrystallizations from MeOH-
Et2O, which resulted in a constant optical rotation. Colorless
crystals for X-ray analysis were obtained from MeOH by vapor
diffusion with Et2O. Mp: 155.3-155.8 °C. 1H NMR (400 MHz,
CD3OD, TMS): 1.42 (s, 3H), 3.34 (s, 3H), 3.40 (s, 3H), 3.44-
3.53 (m, 6H), 4.11-4.17 (m, 1H), 4.18-4.23 (m, 1H). 13C NMR
(100 MHz, CD3OD, TMS): 20.4, 52.1, 55.4, 55.5, 59.9, 64.1, 66.6,
72.6. IR (KBr): 3346. MS (FAB): 164 (M - I-). Anal. Calcd
for C8H18NO2I: C, 33.45; H, 6.32; N, 4.88. Found: C, 33.54; H,
[R]23 -7.3° (c 1, CHCl3).
D
Da ta for (S)-9: found C, 52.61; H, 10.04; N, 4.61. [R]23D +7.1°
(c 0.5, CHCl3).
(R)-[3-[(ter t-Bu tyld im eth ylsilyl)oxy]-2-h yd r oxy-2-m eth -
y lp r o p y l][2-[[(4-m e t h y lp h e n y l)s u lfo n y l]o x y ]e t h y l]-
m eth yla m in e, (R)-10. To a solution of (R)-8 (2.01 g, 7.02
mmol), 4-(dimethylamino)pyridine (DMAP) (0.040 g, 0.31 mmol),
and NEt3 (1.82 g, 18.1 mmol) in dry CH2Cl2 (10 mL) at room
temperature was added TsCl (1.5 g, 7.7 mmol) in dry CH2Cl2
(10 mL) dropwise in 30 min. The reaction mixture was stirred
for 5 h and then diluted with CH2Cl2 (20 mL). The solution
was washed with saturated NaHCO3 (1 × 20 mL) and brine (1
× 25 mL). The extract was dried and concentrated to afford a
dark brown oil, which was used without purification for the
cyclization. Attempts to purify the crude tosylate by chroma-
tography resulted in cyclization to morpholine as seen from the
NMR. 1H NMR (crude, 400 MHz): 0.04 (s, 6H), 0.89 (s, 9H),
1.1 (s, 3H), 2.26 (d, J app ) 13.8 Hz, 1H), 2.39 (s, 3H), 2.62 (d,
J app ) 13.8 Hz, 1H), 2.79-2.85 (m, 2H), 2.8 (s, 3H), 3.0 (br s,
1H), 3.42 (d, J app ) 9.5 Hz, 1H), 3.64 (d, J app ) 9.5 Hz, 1H), 4.2
(t, J app ) 6.6 Hz, 2H), 7.35 (d, J app ) 9.1 Hz, 2H), 7.65 (d, J app
) 9.1 Hz, 2H). 13C NMR (crude, 100 MHz): -5.5, 18.1, 20.1,
23.9, 25.8, 44.8, 56.9, 61.2, 63.4, 68.1, 71.8, 127.3, 129.7, 134.7,
143.5.
(R)-2-[[(ter t-Bu tyld im eth ylsilyl)oxy]m eth yl]-2,4-d im eth -
ylm or p h olin e, (R)-11. A solution of (R)-9 (2.1 g, 7.2 mmol) in
dry THF (30 mL) was treated with potassium hydride (KH) (0.46
g, 1.32 g of 35 wt % mineral oil dispersion, 12 mmol) for 3 h.
THF was removed under reduced pressure, and the dark reddish
brown residue was dissolved in EtOAc (40 mL). The solution
was washed with saturated NaHCO3 (1 × 20 mL) and brine (1
× 25 mL). The extract was dried and concentrated to afford a
red-dark brown oil, which was chromatographed on neutral
alumina eluting with 9:1 hexanes-EtOAc to afford (R)-11 as a
colorless oil (1.8 g, 92%). TLC Rf ) 0.15 (95:5 hexanes-EtOAc,
neutral alumina). 1H NMR (400 MHz): 0.05 (s, 6H), 0.89 (s,
9H), 1.23 (s, 3H), 2.17 (d, J app ) 10.8 Hz, 1H), 2.22 (s, 3H), 2.27
(d, J app ) 11 Hz, 1H), 2.25-2.29 (m, 1H), 2.33-2.39 (m, 1H),
3.49 (d, J app ) 9.2 Hz, 1H), 3.63 (d, J app ) 9.2 Hz, 1H), 3.69-
3.81 (m, 2H). 13C NMR (100 MHz): -5.4, 20.5, 22.9, 25.9, 46.7,
55.5, 61.4, 61.5, 67.4, 73.7. IR (neat): 1164, 1461, 1623. MS
m/z (EI): 259 (M+), 202 (M+ - tert-C4H9). Anal. Calcd for
6.31; N, 4.81. [R]23 +3.2° (c 0.8, MeOH).
D
Da ta for (S)-2: found C, 33.44; H, 6.28; N, 4.76. [R]23
-
D
3.2° (c 2.8, MeOH).
(R)-2,4-Dim eth yl-2-[[(4-n itr op h en yl)su lfon oxy]m eth yl]-
m or p h olin e, (R)-14. To a solution of (R)-12 (0.40 g, 3.0 mmol)
and NEt3 (0.84 g, 8.3 mmol) in dry CH2Cl2 (15 mL) at room
temperature was added 4-nitrobenzenesulfonyl chloride (0.67
g, 3.0 mmol) in dry CH2Cl2 (10 mL) dropwise in 30 min. The
reaction mixture was stirred for 8 h and then washed with
saturated NaHCO3 (2 × 20 mL) and brine (1 × 25 mL). The
solution was dried and concentrated to afford a dark red-brown
oil, which was chromatographed on neutral alumina eluting
with 10% EtOAc in hexanes to afford (R)-14 as a yellow oil,
which solidified overnight (0.60 g, 70%). Recrystallization from
CH2Cl2-hexanes gave (R)-14 as pale yellow needles. Mp:
187.9-188.6 °C dec. TLC Rf ) 0.2 (5:1 hexanes-EtOAc, neutral
alumina). 1H NMR (400 MHz): 1.20 (s, 3H), 2.08 (d, J app ) 11.6
Hz, 1H), 2.16 (s, 3H), 2.20-2.25 (m, 2H), 2.32 (d, J app ) 11.6
Hz, 1H), 3.55-3.60 (m, 1H), 3.62-3.67 (m, 1H), 4.10 (d, J app
)
10 Hz, 1H), 4.30 (d, J app ) 10 Hz, 1H), 8.13 (d, J app ) 9.2 Hz,
2H), 8.41 (d, J app ) 9.2 Hz, 2H). 13C NMR (100 MHz): 21.2,
46.3, 54.7, 60.7, 61.5, 71.7, 72.8, 124.3, 129.3, 141.8, 150.7. IR
(neat): 1486. MS m/z (EI): 330 (M+). Anal. Calcd for
C
13H18N2O6S: C, 47.26; H, 5.50; N, 8.48. Found: C, 46.99; H,
5.54; N, 8.45. [R]23 -11.5° (c 5.6, CHCl3).
D
Da ta for (S)-14: found C, 47.19; H, 5.44; N, 8.40. [R]23
D
+11.3° (c 1.2, CHCl3).
(R)-2,4,4-Tr im eth yl-2-[[(4-n itr op h en yl)su lfon oxy]m eth -
yl]m or p h olin iu m Iod id e, (R)-3. To a solution of (R)-14 (0.090
g, 0.30 mmol) in dry CH3NO2 (1 mL) was added CH3I (0.12 g,
0.82 mmol). The mixture was stirred for 36 h at room temper-
ature. The solution was evaporated to dryness, and the residue
was washed with dry Et2O (3 × 5 mL). The pale yellow solid
was recrystallized twice from acetone and Et2O (0.12 g, 92%).
Mp: 155.9-156.3 °C. 1H NMR (400 MHz, CD3OD, TMS): 1.41
(s, 3H), 3.27 (s, 3H), 3.33 (s, 3H), 3.35-3.50 (m, 4H), 3.90-3.97
(m, 1H), 4.05-4.12 (m, 1H), 4.21 (d, J app ) 10 Hz, 1H), 4.24 (d,
C
13H29O2NSi: C, 56.69; H, 10.62; N, 5.09. Found: C, 56.74; H,
10.49; N, 4.90. [R]23 -2.3° (c 0.1, CHCl3).
D
J app ) 10 Hz, 1H), 8.20 (d, J app ) 9.2 Hz, 2H), 8.51 (d, J app
)
Da ta for (S)-11: found C, 56.81; H, 10.55; N, 4.96. [R]23
+2.2° (c 0.21, CHCl3).
D
9.2 Hz, 2H). 13C NMR (100 MHz, CD3OD, TMS): 21.3, 53.5,
53.6, 56.9, 61.0, 64.7, 72.4, 74.6, 125.8, 128.0, 130.9, 142.7. IR
(KBr): 1512. Anal. Calcd for C14H21N2O6SI: C, 35.59, H, 4.48,
(R)-2-(Hyd r oxym eth yl)-2,4-d im eth ylm or p h olin e, (R)-12.
A solution of (R)-11 (3.0 g, 11 mmol) in THF (10 mL) was treated
with solid Bu4N+F- (TBAF) (4.5 g, 17.3 mmol) for 8 h at room
temperature. THF was carefully removed under reduced pres-
sure, and the residue was purified on neutral alumina eluting
with CH2Cl2 to yield (R)-12 as a colorless volatile liquid (1.6 g,
91%). Bp: 102-104 °C. TLC Rf ) 0.2 (1:3 hexanes-EtOAc,
neutral alumina). 1H NMR (400 MHz): 1.26 (s, 3H), 2.22 (d,
J app ) 11.6 Hz, 1H), 2.24 (s, 3H), 2.31-2.42 (m, 2H), 2.53 (d,
N, 5.93. Found: C, 35.65; H, 4.59; N, 6.05. [R]23 -12.9° (c
D
0.25, MeOH).
F or (S)-3: found C, 35.61; H, 4.66; N, 6.11. [R]23 +12.7° (c
D
0.13, MeOH).
(R)-2-(Azid om eth yl)-2,4-d im eth ylm or p h olin e, (R)-15. A
solution of (R)-14 (0.40 g, 1.0 mmol) and NaN3 (3.4 g, 52 mmol)
in DMF (5 mL) and water (1 mL) was heated at 70 °C for 72 h.
The reaction mixture was poured in water (30 mL) and extracted
into Et2O (3 × 15 mL). The ethereal extract was washed with
saturated NaHCO3 (2 × 20 mL) and brine (1 × 25 mL). The
extract was dried and concentrated to afford a yellow oil. The
crude product was purified by chromatography on neutral
alumina eluting with 5% EtOAc in hexanes to afford (R)-15 as
a colorless volatile liquid (0.13 g, 65%). TLC Rf ) 0.3 (5:1
hexanes-EtOAc, neutral alumina). 1H NMR (400 MHz): 1.26
(s, 3H), 2.13 (d, J app ) 11.2 Hz, 1H), 2.22 (s, 3H), 2.31-2.40 (m,
2H), 2.34 (d, J app ) 11.2 Hz, 1H), 3.25 (d, J app ) 12.8 Hz, 1H),
3.53 (d, J app ) 12.8 Hz, 1H), 3.77 (dd, J app ) 5.2, 4.8 Hz, 2H).
13C NMR (100 MHz): 21.7, 46.5, 54.9, 55.9, 61.5, 61.6, 73.4. IR
J app ) 11.6 Hz, 1H), 3.59 (d, J app ) 11.2 Hz, 1H), 3.74 (d, J app
)
11.2 Hz, 1H), 3.78-3.83 (m, 1H), 3.94-4.01 (m, 1H). 13C NMR
(100 MHz): 21.5, 46.4, 54.9, 61.5, 62.1, 68.3, 73.0. IR (CH2-
Cl2): 3358. MS m/z (EI): 145 (M+). HRMS: calcd for C7H15O2N1-
145.1103, found 145.1102. [R]23 -2.9° (c 1.1, CHCl3).
D
Da ta for (S)-12: HRMS calcd for C7H15O2N1145.1103, found
145.1107. [R]23 +2.1° (c 0.13, CHCl3).
D
(R)-2-(Hydr oxym eth yl)-2,4,4-tr im eth ylm or ph olin iu m Io-
d id e, (R)-2. To a solution of (R)-12 (0.15 g, 1.0 mmol) in dry
CH3NO2 (2 mL) was added CH3I (0.29 g, 2.0 mmol). The flask
was kept under dark for 5 h at room temperature. The solution
was decanted, and the precipitate was washed with dry Et2O

22 Chem. Res. Toxicol., Vol. 11, No. 1, 1998
Savle et al.
(neat): 2107. MS m/z (EI): 170 (M+). HRMS (CI): calcd for
scans. Refinement was done by full-matrix least-squares, with
neutral-atom scattering factors and anomalous dispersion cor-
rections. All non-hydrogen atoms were refined anisotropically.
For both structures, the OH hydrogen atom was refined isotro-
pically, while the other H atoms were placed in calculated
positions. For (S)-2, R ) 0.035, Rw ) 0.039 for 2976 observa-
tions; for (R)-2, R ) 0.034, Rw ) 0.036 for 3214 observations.
Refinement with reversed absolute configurations yielded R )
0.037, Rw ) 0.041 for the S isomer and R ) 0.038, Rw ) 0.043
for the R isomer; thus both absolute configurations were proven.
C7H15ON4 171.1247, found 171.1245. [R]23 -0.8° (c 0.48,
D
CHCl3).
F or (S)-15: HRMS (CI) calcd for C7H15ON4 171.1247, found
171.1249. [R]23 +0.7° (c 0.26, CHCl3).
D
(R)-2-(Azid om et h yl)-2,4,4-t r im et h ylm or p h olin iu m Io-
d id e, (R)-4. To a solution of (R)-15 (0.050 g, 0.3 mmol) in dry
CH3NO2 (1 mL) was added CH3I (0.12 g, 0.87 mmol). The
mixture was stirred for 24 h at room temperature. The solution
was evaporated to dryness, and the residue was washed with
dry Et2O (3 × 5 mL). The pale white solid was recrystallized
four times from MeOH and Et2O to yield colorless, shiny plates
(0.080 g, 90%). Mp: 128.2-128.7 °C. 1H NMR (400 MHz, CD3-
OD, TMS): 1.48 (s, 3H), 3.33 (s, 3H), 3.38 (s, 3H), 3.41-3.58
(m, 6H), 4.07-4.18 (m, 1H), 4.19-4.21 (m, 1H). 13C NMR (100
MHz, CD3OD, TMS): 22.1, 53.4, 56.9, 57.1, 58.7, 61.3, 65.7, 74.2.
IR (KBr): 2116. MS (FAB): 164 (M - I-). Anal. Calcd for
C8H17N4OI: C, 30.76; H, 5.49; N, 17.95. Found: C, 30.89; H,
Molecu la r Mod elin g. PCMODEL (version 4.0) (17) calcula-
tions with the default dielectric (1.5) and dielectric ) 80 were
performed on 72 unique conformations of 2. These conforma-
tions included the nine possible staggered conformations for
O-C-CH2-O-H for chair and three unique twist-boat con-
formations of the ring. Axial and equatorial positions for
hydroxymethyl were included for all ring conformations.
Molecular modeling studies (18) on (R)- and (S)-2-AChE
complexes were done by placing (R)- and (S)-2 in the same
position as edrophonium (19) in the crystal structure of the 19-
AChE complex and then minimizing the whole structure.
5.56; N, 17.90. [R]23 -4.1° (c 2.8, MeOH).
D
F or (S)-4: found C, 30.88; H, 5.53; N, 17.84. [R]23 +3.8° (c
D
3.4, MeOH).
[3-[(4-Nit r op h en yl)su lfon oxy]p h en yl]-N,N,N-t r im et h -
yla m m on iu m Iod id e, 5. To a solution of 3-(dimethylamino)-
phenol (2.21 g, 15.8 mmol), DMAP (0.02 g), and NEt3 (3.22 g,
31.6 mmol) in dry CH2Cl2 (10 mL) at room temperature was
added 4-nitrobenzenesulfonyl chloride (3.71 g, 16.6 mmol) in dry
CH2Cl2 (10 mL) dropwise in 30 min. The reaction mixture was
stirred for 8 h and then washed with saturated NaHCO3 (1 ×
20 mL) and brine (1 × 25 mL). The solution was dried and
concentrated to afford a dark red-brown oil, which was chro-
matographed on silica gel eluting with CH2Cl2 to afford a red
oil, which solidified overnight. TLC Rf ) 0.2 (CH2Cl2, silica).
Recrystallization from acetone-ether gave reddish brown needles
(4.8 g, 93%), which were converted into 5 by using a similar
procedure to that described for the synthesis of 3. Mp: 171.1-
171.8 °C dec (acetone-Et2O). 1H NMR (400 MHz, CD3OD): 3.7
(s, 9H), 7.35 (dd, J app ) 10, 2 Hz, 1H), 7.69 (t, J app ) 8.4 Hz,
1H), 7.86 (dd, J app ) 2.4, 2 Hz, 1H), 7.97 (dd, J app ) 8, 2.4 Hz,
1H), 8.18 (d, J app ) 9.2 Hz, 2H), 8.47 (d, J app ) 9.2 Hz, 2H). 13C
NMR (100 MHz, CD3OD) 57.8, 116.9, 120.7, 125.7, 125.9, 131.5,
133.2. IR (KBr): 1505, 1434. Anal. Calcd for C15H17N2O5SI:
C, 38.79; H, 3.69; N, 6.04. Found: C, 38.65; H, 3.74; N, 5.88.
En zym a tic Assa ys. (A) Ma ter ia ls. Sodium phosphate
buffer components were reagent grade and used without further
purification. Reactions were run in 0.08 M sodium phosphate
buffer at pH 7.21. Grade V-S AChE (EC 3.1.1.7) from Electro-
phorus electricus was obtained as a lyophilized powder from
Sigma Chemical Co. (St. Louis, MO), and prior to use the
enzyme was dissolved in the phosphate buffer that contained
0.1 N NaCl. Acetylthiocholine chloride and 5,5′-dithiobis(2-
nitrobenzoic acid) (DTNB) were also obtained from Sigma
Chemical Co. and used as received.
(B) Kin etic Mea su r em en ts. AChE activity was measured
at 25.0 ( 0.1EC by the coupled assay method described by
Ellman et al. (19). Time courses were followed at 412 nm and
were fit to the integrated Michaelis-Menten equation to obtain
values of Km and Vmax, as described by Lee et al. (8). The initial
substrate concentration was 0.5 mM. Inhibition was character-
ized by running reactions in the absence of inhibitor (control)
and in the presence of at least four fixed inhibitor concentra-
tions. Controls and inhibition reactions were run at least in
duplicate.
Deter m in a tion of Op tica l P u r ities of (R)- a n d (S)-12. A
solution of morpholine alcohol 12 (0.050 g, 0.30 mmol) in dry
EtOAc (1 mL) was added to a stirred mixture of (R)-(+)-MTPA
(0.070 g, 0.30 mmol), DCC (0.070 g, 0.30 mmol), and DMAP
(0.005 g, 0.5 mmol) in EtOAc (2 mL) under nitrogen. The
mixture was stirred for 2 h at room temperature and then
filtered to remove the precipitate. The precipitate was washed
with EtOAc (2 × 5 mL). The filtrate was concentrated to give
a pale yellow solid, which was purified by alumina preparative
TLC eluting with 20% EtOAc-hexanes, collecting a wide UV
active band to yield Mosher’s ester 13. The optical purities were
determined by the average of integration of the signals for C-2
methyl, N-methyl, and CH2OMTPA. TLC Rf ) 0.25 (5:1
hexanes-EtOAc, neutral alumina). Mosher ester of (R)-12, 1H
NMR (400 MHz): 1.21 (s, 3H), 2.07 (d, J app ) 11.2 Hz, 1H), 2.18
(s, 3H), 2.31-2.40 (m, 2H), 2.37 (d, J app ) 11.2 Hz, 1H), 3.58 (s,
3H), 3.65-3.75 (m, 1H), 3.80-3.87 (m, 1H), 4.27 (d, J app ) 12.8
Hz, 1H), 4.59 (d, J app ) 12.8 Hz, 1H), 7.35-7.68 (m, 5H). Mosher
ester of (S)-12, 1H NMR (400 MHz): 1.18 (s, 3H), 2.07 (d, J app
Sch em e 1. Syn th esis of Mor p h olin e Alcoh ols
Resu lts
Our synthesis (Scheme 1) started with commercially
available (R)- and (S)-2-methylglycidols, 6, having optical
purity of about 90%. After protection of the hydroxyl
group of 6 with TBDMS (16), the epoxides reacted with
N-methyl-2-aminoethanol to give 8. Amino diol 8 when
treated with tosyl chloride and triethylamine gave the
chloro compound 9 in a quantitative yield, instead of the
expected primary tosylate 10. Adding DMAP (20) to the
reaction gave 10 exclusively. We presumed that in the
absence of DMAP, chloride displaced tosylate via an SN2
mechanism to give 9. Treatment of 9 with potassium
) 11.2 Hz, 1H), 2.20 (s, 3H), 2.31-2.40 (m, 2H), 2.37 (d, J app
)
11.2 Hz, 1H), 3.56 (s, 3H), 3.68-3.74 (m, 1H), 3.79-3.85 (m,
1H), 4.35 (d, J app ) 12.8 Hz, 1H), 4.48 (d, J app ) 12.8 Hz, 1H),
7.35-7.68 (m, 5H). The ee values for (R) and (S)-12 were 89.0
( 0.4% and 91.5 ( 0.3%, respectively.
X-r a y Exp er im en ta l. X-ray diffraction data for both enan-
tiomers were collected on an Enraf-Nonius CAD4 diffractometer
equipped with Mo KR radiation and a graphite monochromator.
Two octants of data were collected for each. Data reduction
included corrections for background, Lorentz polarization, and
absorption effects. Absorption corrections were based on ψ

(4-Nitrophenyl)sulfonoxyl Choline Analogues
Chem. Res. Toxicol., Vol. 11, No. 1, 1998 23
Sch em e 2. Syn th esis of Qu a ter n a r y Mor p h olin iu m
Iod id es
respectively. This torsion angle in (2S,6S)-16 and (2S,6S)-
17 is also gauche, but it is anti in (2S,6R)-16 and (2S,6R)-
17. These differences are due to intermolecular hydrogen
bonding. For (S)-2, there is a weak hydrogen bond
between O1-H and an iodide with an O1-I distance of
3.747(4) Å.
2. Solu tion NMR. The similarity in chemical shifts
(at 400 MHz) of the morpholinium ring protons at C-5
and C-3 has thwarted assignment of individual reso-
nances. The axial and equatorial N-methyl groups show
a chemical shift difference of approximately 0.05 ppm.
Our attempts to assign them and the rest of the axial or
equatorial protons in all three compounds using COSY,
NOE difference, 2D-NOESY, ROESY, and HETCOR
experiments have met with little success.
3. Molecu la r Mech a n ics. To understand how these
inhibitors might bind to AChE, we need to analyze the
preferred conformations. Using PCMODEL (17) at both
the default dielectric and a dielectric ) 80, we find that
chair conformers predominate. Surprisingly, twist-boat
conformations account for 14% of the total population.
In the 86% of the population that are chair conformers,
an equatorial hydroxymethyl group is preferred over an
axial one (51:35). The calculated most stable conforma-
tion, which is only 24% of the population, is the same
conformation observed in the solid state. The lack of
reliable parameters for sulfonoxy and azido makes cal-
culations on 3 and 4, respectively, unfeasible at this time.
We are studying the conformational properties of 2
with other computational methods to confirm and ex-
plain this unusually large percentage of twist-boat
conformers.
hydride in THF closed the ring to yield 11. Removal of
the TBDMS group of 11 furnished 12 in quantitative
yield.
The optical purity of 12 was measured by converting
it into a Mosher ester, 13 (not shown). Quaternarization
of 12 gave 2, which was recrystallized to give a sample
with constant rotation and a sharp melting point. Single-
crystal X-ray analysis confirmed the absolute configura-
tions of both enantiomers of 2.
Morpholinyl alcohol 12 was converted into nosylate 14
(Scheme 2). Quaternarization of 14 gave 3 in 92% yield.
Treatment of 14 with 50-fold excess of sodium azide in
DMF at 70 °C for 72 h gave the azidomorpholine 15 in a
moderate yield. Nucleophilic displacement at this neo-
pentyl center required a long reaction time. Compound
15 was subsequently quaternarized to the azidomorpho-
linium iodide 4.
In h ibition of ACh E. 1. Com p etitive In h ibition .
Compounds 2 and 4 are reversible competitive AChE
inhibitors; that is, as inhibitor concentration [I] increases,
Km increases but Vmax remains relatively constant. In
this case V/K ()Vmax/Km) is a decreasing nonlinear
function of increasing inhibitor concentration [I], as
described in eq 1:
Ki
(V/K)I ) (V/K)0
(1)
Ki + [I]
(V/K)I and (V/K)0 are V/K values in the presence and
absence of inhibitor, and Ki is the dissociation constant
of the reversible EI complex. The adjustable parameters
(V/K)0 and Ki were calculated by fitting data to eq 1 by
nonlinear least-squares procedures. Figure 2 shows such
a fit for inhibition of AChE by (S)-2. Compound (S)-2
inhibits 1.8-fold more strongly than (R)-2, but 4 shows
virtually no difference between the two enantiomers
(Table 1).
2. Non com p etitive In h ibition . Compounds 3 and
5 show linear noncompetitive inhibition. The agreement
between Ki calculated by fitting V/ K versus [I] data
to eq 1 and Ki′ calculated by fitting V versus [I] data to
eq 2:
F igu r e 1. ORTEP drawings of (S)- and (R)-2.
Str u ctu r e. 1. Cr ysta l. In the crystal (Figure 1) the
morpholinium ring adopts a chair conformation with the
hydroxymethyl group in an equatorial position. The bond
distances and angles for (R)- and (S)-2 are similar to
those of the carnitine analogues (14) 16 and 17. The only
V0
Vi )
(2)
[I]
1 +
Ki
provides an additional indication of linear noncompetitive
inhibition. In this case, both V/ K and V ()Vmax) are
decreasing nonlinear functions of increasing [I]. This
type of inhibition occurs when the inhibitor binds at the
significant difference is in the torsion angle of the
hydroxymethyl group. The O-C-CH2-OH torsion angle
is gauche, 62.8(4)° and -62.6(5)°, in (R)- and (S)-2,

24 Chem. Res. Toxicol., Vol. 11, No. 1, 1998
Savle et al.
Ta ble 2. Com p a r ison of In h ibition of ACh E
inhibitor
Ki, µM
1a , R ) H, X ) Cl
560 ( 50a
550 ( 20a
220 ( 50a
1630 ( 70a
3690 ( 90a
360 ( 30b
19.0 ( 0.9b
0.21 ( 0.06a
6.0 ( 0.5b
1b, R ) CH3, X ) Br
1c, R ) p-C6H4-NH2, X ) Br
1d , R ) p-C6H4-NO2, X ) Br
1e, R ) p-C6H4-CN, X ) Br
(S)-2
(S)-3
18
5
a
b
Reference 8. This work.
Discu ssion
Strong stereoselectivity, our initial rationale for as-
saying 2-4, does not occur. The lack of stereoselectivity
for the azide 4 is especially disappointing and excludes
further experiments with these analogues. Below, we
discuss the modest stereoselectivity of 2. The discovery
in this study is the noncompetitive inhibition by 3
compared to the competitive inhibition by 2 and 4.
Furthermore, 3 is 10-fold more active than 2 or 4. The
noncompetitive inhibition by 5 supports the conclusion
that the (4-nitrophenyl)sulfonoxyl group controls the
mode of inhibition. Comparing these results with other
inhibitors underscores this conclusion.
F igu r e 2. Fit of V/ K versus inhibitor concentration of (S)-2 to
eq 1 (see text). The inhibition constant, Ki, calculated from
nonlinear least-squares method, is 360 ( 30 µM.
Com p a r ison w ith Hem ich olin iu m s. Both 2 and 4
have Ki values similar to those of the racemic alkyl
hemicholiniums 1a ,b (Table 2). All aryl hemicholiniums,
with 1c as the best, show competitive inhibition. As 1c
is open and closed (9), AChE may stabilize one form over
the other. If AChE stabilizes the closed form, comparison
of 1c with 3 suggests that changing OH and aryl to CH3
and CH2OSO2-aryl, respectively, results in a change from
competitive to noncompetitive inhibition. Furthermore,
(R)- and (S)-3 inhibit significantly better than aryl
hemicholiniums, especially 1d , where R ) p-C6H4-NO2.
The comparison here, however, is not equivalent; 3
inhibits in the peripheral site, while 1d inhibits in the
active site. For competitive inhibition, electron-rich aryl
hemicholinium groups inhibit better than electron-poor.
Whether the reverse is true for noncompetitive inhibition
by the arylsulfonoxyl group remains to be tested.
Com p a r ison of Mor p h olin iu m s a n d Ar ylt r i-
m eth yla m m on iu m s. The strong AChE inhibitor (3-
hydroxyphenyl)trimethylammonium iodide (18; Table 2),
an analogue of edrophonium, 19, has three carbon atoms
between the (CH3)3N+ and the OH as 2, but 18 (Ki ) 0.21
µM) (8) is over 1700-fold more potent than (S)-2. Com-
pound 18 (pKa ) 8.2) should be a better hydrogen-bond
donor than (S)-2 (estimated pKa ∼ 16). The acceptor is
an imidazolyl nitrogen (pKa ) 6.3) in the active site. The
closer the match in pKa between the donor and the
acceptor, i.e., ∆pKa ∼ 0, the stronger the hydrogen bond.
The X-ray structure of the 19-AChE complex shows that
the quaternary nitrogen of 19 is positioned next to the
indole ring of Trp84, while the m-hydroxyl group of 19
is positioned between His440 and Ser200, thus forming
hydrogen bonds with two of the three members of the
catalytic triad (21).
F igu r e 3. Fit of V/ K versus inhibitor concentration of (S)-3 to
eq 1. The inhibition constant, Ki, calculated from nonlinear least-
squares method, is 19.0 ( 0.9 µM. Inset: Plot of 1/Vmax versus
inhibitor concentration of (S)-3 shows a linear noncompetitive
inhibition.
Ta ble 1. E. electr icu s ACh E In h ibition by Mor p h olin iu m s
a t 25 ( 0.1 °C a n d p H 7.21
Ki, µM
configuration
configuration
inhibitor, mode
at C-2: S
at C-2: R
2, reversible competitive
3, noncompetitive
4, reversible competitive
360 ( 30
19.0 ( 0.9
450 ( 70
650 ( 90
50 ( 2
560 ( 30
peripheral site, does not compete with the substrate at
the active site, and has no effect on Km. Figure 3 shows
a plot of Vmax/Km against [I], where I ) (S)-3, and a plot
(inset) of 1/Vmax against [I], which indicates a linear
noncompetitive inhibition. Both (R)- and (S)-3 show
linear noncompetitive inhibition; (S)-3 is 2.5-fold more
active than (R)-3. Compound 5 is 3-fold more active than
(S)-3.
Our molecular modeling studies done by replacing 19
with (R)- and (S)-2 in the 19-AChE complex reveal that
such a hydrogen bond is only possible with (S)-2, where
the quaternary nitrogen could interact favorably with
Trp84. This can explain the 1.8-fold stereochemical
preference for (S)-2.

(4-Nitrophenyl)sulfonoxyl Choline Analogues
Chem. Res. Toxicol., Vol. 11, No. 1, 1998 25
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In addition, the 4-nitrobenzenesulfonyl group levels the
potency of these two classes of AChE inhibitors. Inhibitor
5 is 3-fold more potent than (S)-3, which is a marked
contrast to the 1700-fold enhancement of 18 compared
to (S)-2. In the case of 5 compared to 18, the Ki decreases
28-fold. Loss of the strong hydrogen bond between 18
and His440 reduces the potency. The 4-nitrobenzene-
sulfonyl group controls the mode of inhibition and ap-
parently the affinity of (S)-3 and 5 for AChE.
Why do 3 and 5 bind to the peripheral site and not the
active site? (3-Hydroxyphenyl)trimethylammonium io-
dide and the methylsulfonoxy derivative, strong competi-
tive inhibitors of AChE, have values of Ki ) 0.21 (8) and
0.4 (22) µM, respectively. The 4-nitrophenyl hemicho-
linium 1d is also a competitive inhibitor. Therefore,
either a sulfonoxyl group or a 4-nitrophenyl group is not
sufficient individually to change the mode of inhibition.
The combination of both moieties appears as essential
for changing the mode of inhibition. We do not know at
this point whether any other arylsulfonoxyl derivatives
of 3 and 5 will be noncompetitive inhibitors. No matter
what the explanation, the change in the mode of inhibi-
tion provides a lead to design more analogues.
Su m m a r y
We have achieved a chiral, five-step synthesis of
2-(hydroxymethyl)-2,4-dimethylmorpholine from (R)- and
(S)-2-methylglycidols in an overall yield of 63%. Mor-
pholinium salts, 2, 3, and 4 inhibit AChE. Enantiomers
of 2 and 4 are reversible competitive inhibitors of AChE,
whereas enantiomers of 3 are noncompetitive inhibitors
of AChE. A competitive AChE inhibitor (18) when
converted into the 4-nitrobenzenesulfonyl derivative
yields a noncompetitive inhibitor, 5, but with reduced
potency. In 2 and 3, a small amount of chiral discrimi-
nation is seen. The inhibition results provide leads for
elaborating the design to produce more potent chiral
inhibitors. The successful strategy of connecting moieties
that bind concurrently at both the active and the periph-
eral sites (4) will guide our future efforts.
(14) Sun, G., Savle, P. S., Gandour, R. D., Nic a’ Bha´ırd, N., Ramsay,
R. R., and Fronczek, F. R. (1995) Syntheses, structures, and
enzymatic evaluations of conformationally constrained, analogue
inhibitors of carnitine acetyltransferase: (2R,6R)-, (2S,6S)-,
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trimethylmorpholinium. J . Org. Chem. 60, 6688-6695.
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Substituted morpholine-2S-acetic acid derivatives: Sch 50911 and
related compounds as novel GABAB antagonists. Bioorg. Med.
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Org. Chem. 58, 825-826.
(17) Gajewski, J . J ., and Gilbert, K. E. (1992) PCMODEL Molecular
Modeling Package, version 4.0, Serena Software, P.O. Box 3076,
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(18) SYBYL Molecular Modeling Package, version 6.1 (1994) Tripos,
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Ack n ow led gm en t. R.D.G. thanks the National In-
stitute of Health for support of this work through Grant
GM42016. We thank the reviewers for their valuable
comments and suggestions.
(21) Sussman, J . L., Harel, M., and Silman, I. (1993) Three-
dimensional structure of acetylcholinesterase and of its complexes
with anticholinesterase drugs. Chem. Biol. Interact. 87, 187-197.
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Refer en ces
(1) Rosenberry, T. L. (1975) Acetylcholinesterase. Adv. Enzymol.
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