Synthesis and characterization of enantiomerically pure cis- and trans-3-fluoro-2,4-dioxa-9-aza-3-phosphadecalin 3-oxides as acetylcholine mimetics and inhibitors of acetylcholinesterase

Article abstract of DOI:10.1002/hlca.201000457

The title compounds, the P(3)-axially and P(3)-equatorially substituted cis- and trans-configured 9-benzyl-3-fluoro-2,4-dioxa-9-aza-3-phosphadecalin 3-oxides (=9-benzyl-3-fluoro-2,4-dioxa-9-aza-3-phosphabicyclo[4.4.0]decane 3-oxides=7-benzyl-2-fluorohexah

Full text of DOI:10.1002/hlca.201000457

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Helvetica Chimica Acta – Vol. 94 (2011)
Synthesis and Characterization of Enantiomerically Pure cis- and trans-3-
Fluoro-2,4-dioxa-9-aza-3-phosphadecalin 3-Oxides as Acetylcholine Mimetics
and Inhibitors of Acetylcholinesterase
by Piergiorgio Lorenzetto, Michael Wꢀchter, and Peter Rꢁedi*
Organisch-chemisches Institut der Universitꢀt Zꢁrich, Winterthurerstrasse 190, CH-8057 Zꢁrich
(phone: þ 41-44-8215579; e-mail: peru@oci.uzh.ch)
Dedicated to Professor Heinz Heimgartner on the occasion of his 70th birthday
The title compounds, the P(3)-axially and P(3)-equatorially substituted cis- and trans-configured 9-
benzyl-3-fluoro-2,4-dioxa-9-aza-3-phosphadecalin 3-oxides (¼ 9-benzyl-3-fluoro-2,4-dioxa-9-aza-3-phos-
phabicyclo[4.4.0]decane 3-oxides ¼ 7-benzyl-2-fluorohexahydro-4H-1,3,2-dioxaphosphorino[4,5-c]pyri-
dine 2-oxides) were prepared (ee > 99%) and fully characterized (Schemes 2 and 4). The absolute
configurations were deduced from that of their precursors, the enantiomerically pure ethyl 1-benzyl-3-
hydroxypiperidine-4-carboxylates and 1-benzyl-3-hydroxypiperidine-4-methanols which were unambig-
uously assigned. Being configuratively fixed and conformationally constrained phosphorus analogues of
acetylcholine, the title compounds represent acetylcholine mimetics and are suitable probes for the
investigation of molecular interactions with acetylcholinesterase. As determined by kinetic methods, all
of the compounds are moderate irreversible inhibitors of the enzyme.
1. Introduction. – In the course of our research project concerning the synthesis of
configuratively fixed and conformationally constrained organophosphates as inhibitors
of acetylcholinesterase (AChE) and related serine hydrolases (e.g., chymotrypsin), we
have reported on the preparation and characterization of the racemic P(3)-axially and
P(3)-equatorially substituted cis- and trans-2,4-dioxa-9-aza- (I), cis- and trans-2,4-
dioxa-8-aza- (II), and cis- and trans-2,4-dioxa-7-aza-3-phosphadecalin 3-oxides (III)
[1]. The first enantiomerically pure 2,4-dioxa-3-phosphadecalin 3-oxides (IV, L ¼ F [2],
L ¼ 2,4-dinitrophenoxy [3] and the corresponding (1,5,5-2H3)-isomers [4]) have been
presented, too (Scheme 1). Being P-analogues of acetylcholine (7-aza- and 9-aza
isomers) or g-homoacetylcholine mimetics (8-aza isomers), the compounds are
considered to be suitable probes for the investigation of molecular interactions with
the enzyme, such as the recognition conformation of acetylcholine (¼2-(acetyloxy)-
N,N,N-trimethylethanaminium; ACh) [5] and the stereochemical course of the
inhibition reaction [3][4]. In this report, we discuss the synthesis and full character-
ization of the first enantiomerically pure 9-benzyl-3-fluoro-2,4-dioxa-9-aza-3-phospha-
decalin 3-oxides (¼ 9-benzyl-3-fluoro-2,4-dioxa-9-aza-3-phosphabicyclo[4.4.0]decane
3-oxides ¼ 7-benzyl-2-fluorohexahydro-4H-1,3,2-dioxaphosphorino[4,5-c]pyridine 2-
oxides) 14 and 15 representing type-I inhibitors. In this context, we also give a full
account on the absolute configurations and the chiroptical data of their presursors,
ethyl trans- and cis-1-benzyl-3-hydroxypiperidine-4-carboxylates ((þ)- and (ꢀ)-2 and
(þ)- and (ꢀ)-3, resp.) and trans- and cis-1-benzyl-3-hydroxypiperidine-4-methanols
ꢂ 2011 Verlag Helvetica Chimica Acta AG, Zꢁrich

Helvetica Chimica Acta – Vol. 94 (2011)
747
((þ)- and (ꢀ)-8 and (þ)- and (ꢀ)-9, resp.) as partly presented in a previous report to
clear fundamental structural inconsistencies in the current literature [6]1).
Scheme 1a)
2. Synthesis and Characterization of the Precursors. – 2.1. Ethyl (þ)- and (ꢀ)-trans-
and (þ)- and (ꢀ)-cis-1-Benzyl-3-hydroxypiperidine-4-carboxylates ((þ)-2 and (ꢀ)-2
and (þ)-3 and (ꢀ)-3, resp.). 2.1.1. General. Reduction of ethyl 1-benzyl-3-oxopiper-
idine-4-carboxylate (1) with NaBH4 gave the ethyl (ꢁ)-trans- and (ꢁ)-cis-1-benzyl-3-
hydroxypiperidine-4-carboxylates ((ꢁ)-2 and (ꢁ)-3, resp.; Scheme 2). The resulting
four hydroxy esters could be separated directly by anal. HPLC on Chiralcelꢀ OD-H
1
)
In [6], the discussion has been restricted to the characterization of the cis-hydroxy esters (þ)- and
(ꢀ)-3 and its consequences in terms of a structural revision.

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Helvetica Chimica Acta – Vol. 94 (2011)
749
(Fig. 1,a; Table 1). Although the separation satisfied analytical purposes, the enantio-
merically pure (þ)- and (ꢀ)-trans- and (þ)- and (ꢀ)-cis-3-hydroxy esters ((þ)- and (ꢀ)-
2 and (þ)- and (ꢀ)-3, resp.) were prepared more efficiently by enantioselective
reductions (see below) [7].
Fig. 1. HPLC Traces (Chiralcelꢄ OD-H (5 m), hexane/iPrOH 12 :1) of the trans- and cis-ethyl 1-benzyl-3-
hydroxypiperidine-4-carboxylates 2 and 3, respectively, after reduction of ethyl 1-benzyl-3-oxopiperidine-
4-carboxylate (1) under various conditions: a) NaBH4; b) Bakersꢁ yeast; c) [Ru{(þ)-(R)-binap}Cl(-
cym)]Cl/H2; d) [Ru{(ꢀ)-(S)-binap}Cl(cym)]Cl/H2
2.1.2. Biological Reduction. According to an established protocol [8], the
piperidinone 1 was reduced with bakersꢃ yeast under nonfermenting conditions to
afford (ꢀ)-2 (6%, [a]D ¼ ꢀ23.1, ee 95%) and (þ)-3 (37%, [a]D ¼ þ41.2, ee 82%) after
chromatography (SiO2) [6].
2.1.3. Stereoselective Hydrogenations. To have access to all four stereoisomers of
ethyl 1-benzyl-3-hydroxypiperidine-4-carboxylate, i.e., to (þ)- and (ꢀ)-2 and (þ)- and

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Helvetica Chimica Acta – Vol. 94 (2011)
Table 1. Stereochemical Outcome of the Reduction of Ethyl 1-Benzyl-3-oxopiperidine-4-carboxylate (1)
under Various Conditions: Anal. HPLC Parameters and Chiroptical Properties of the trans- and cis-Ethyl
1-Benzyl-3-hydroxypiperidine-4-carboxylates 2 and 3
c
Reducing agent
NaBH4
Product
k’a)
ee [%]b)
[a]D
)
(ꢁ)-2
1.33, 1.71
2.19, 2.75
1.33
2.19
1.33
2.75
1.71
2.19
–
–
95
82
–
–
(ꢁ)-3
Bakersꢃ yeast
(ꢀ)-2 ((3S,4R))
(þ)-3 ((3R,4R))
(ꢀ)-2 ((3S,4R))
(ꢀ)-3 ((3S,4S))
(þ)-2 ((3R,4S))
(þ)-3 ((3R,4R))
ꢀ 23.1
þ 41.2
[Ru{(þ)-(R)-binap}Cl(cym)]Cl, H2
[Ru{(ꢀ)-(S)-binap}Cl(cym)]Cl, H2
67 (> 99)d)
49 (> 99)d)
74 (> 99)d)
41 (> 99)d)
ꢀ 24.9d)
ꢀ 56.3d)
þ 25.2d)
þ 56.6d)
a) k’ ¼ Retention factor; HPLC: Chiralcelꢀ OD-H (5 m, 250 ꢂ 4.6 mm), hexane/iPrOH 12 : 1, flow-rate
1 ml/min, and Rs > 2. b) Determined according to area-%. c) c ¼ 1, CHCl3. d) After purification by
preparative HPLC; Chiralcelꢀ OD (10 m, 250 ꢂ 20 mm), hexane/EtOH 15 : 1, flow-rate 5 ml/min, and
Rs > 2.
(ꢀ)-3, the 3-oxo ester 1 was catalytically hydrogenated with both [Ru{(þ)-(R)-
binap}Cl(cym)]Cl and [Ru{(ꢀ)-(S)-binap}Cl(cym)]Cl (binap ¼ [1,1’-binaphthalene]-
2,2’-diylbis[diphenylphosphine], cym ¼ p-cymene ¼ 1-methyl-4-(1-methylethyl)ben-
zene; see Exper. Part) according to [9] (Scheme 2, Table 1). Contrary to the biological
reduction, the reactions proceeded with low diastereoselectivity (de ca. 10% in favor of
the cis-isomers 3, Fig. 1). Reaction with [Ru{(þ)-(R)-binap}Cl(cym)]Cl and chromato-
graphic separation (SiO2) of the diastereoisomers yielded (ꢀ)-2 (22%, ee 74%) and
(ꢀ)-3 (27%, ee 49%), and the analogous treatment with [Ru{(ꢀ)-(S)-binap}Cl-
(cym)]Cl gave (þ)-2 (20%, ee 81%) and (þ)-3 (30%, ee 41%). Final chromatographic
purification (Chiralcelꢀ OD) afforded the enantiomerically pure 3-hydroxy esters (þ)-
and (ꢀ)-2 and (þ)- and (ꢀ)-3 (Scheme 1, Table 1).
Although the hydrogenation conditions were optimized, the enantiofacial differ-
entiation remained inefficient. This fact is explained by competitive directing effects
[10] of the ester and the amine group present in 1/1’2) that result in a set of coexistent
equilibria and variably chelated species, e.g., A, A’, B, and B’ (Scheme 3). The
assumption was verified when the benzyl group was replaced by electron-withdrawing
substituents at the N-atom [7]. Catalytic hydrogenation of the derivatives 4 – 7
(Scheme 3) resulted in significantly increased ee values in the cis series, whereas those
of the trans series remained almost unaffected. After exhaustive experimental studies,
the highest enantioselectivity was observed with ethyl 1-(4-bromobenzoyl)-3-oxopi-
peridine-4-carboxylate (7)3): Hydrogenation of 7 in the presence of [Ru{(þ)-(R)-
binap}Cl(cym)]Cl yielded (ꢀ)-2 (ee 66%) and (ꢀ)-3 (ee 97%), and treatment with
1
According to the H- and 13C-NMR spectra, the enol form 1’ is predominant [7][11].
2
)
)
3
The tosyl (4), 4-nitrobenzoyl (5), and 2,4-dinitrobenzoyl (6) derivatives were less suited (eecis
< 75%, eetrans < 60%). Moreover, no satisfactory HPLC system for the separation of the
corresponding resulting hydroxy esters could be elaborated, and the by-products due to the
concomitant reduction of the SO2 and NO2 groups, respectively, could not be completely avoided.
For details, see [7].

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[Ru{(ꢀ)-(S)-binap}Cl(cym)]Cl gave (þ)-2 (ee 66%) and (þ)-3 (ee 83%) [7]. However,
since all attempts to improve this still unsatisfactory result were not successful, the
straightforward procedure with the 1-benzyl derivative 1 presented above was applied.
In particular, the enantiomerically enriched fractions could be separated by prep.
HPLC much more efficiently than the racemic mixtures.
Scheme 3a)
2.1.4. Absolute Configurations. As previously evidenced in detail [6], the (þ)-
(3R,4R)- and the (ꢀ)-(3S,4S)-configuration, respectively, were assigned to the cis-3-
hydroxy esters (þ)- and (ꢀ)-3 by means of the high-field 1H-NMR Mosher method [12]
((R)- and (S)-MTPA esters 10a, 10b, 11a, and 11b, in Scheme 2). Since the general
applicability of this procedure to such piperidinols and -diols was confirmed [13]4), the
4
)
It was not assured a priori that the Mosher method is reliably applicable to such N-heterocyclic
compounds [6][13]. However, detailed differential analyses of the respective d(S) – d(R) values
allowed also assignments of the absolute configurations of bis-MTPA esters of hydroxypiper-
idinemethanols [13].

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Helvetica Chimica Acta – Vol. 94 (2011)
absolute configurations of the trans-3-hydroxy esters 2 could be established, too.
Esterification of (þ)- and (ꢀ)-2 with (þ)-(S)-MTPA-Cl (¼(þ)-(S)-a-methoxy-a-
(trifluoromethyl)benzeneacetyl chloride) afforded the (R)-esters 8a and 9a, and the
corresponding (S)-esters 8b and 9b were isolated after reaction of (þ)- and (ꢀ)-2 with
(ꢀ)-(R)-MTPA-Cl (Scheme 2). From the d(S) – d(R) values in the 1H-NMR spectra of
the 8a/9b and the 9a/9b couples, the (3R)-configuration for (þ)-2 and the (3S)-
configuration for (ꢀ)-2 could be unambiguously assigned (see Exper. Part).
These results confirm the stereochemical outcome of the biological reduction and
fully corroborate the considerations on the [RuII(binap)]-catalyzed hydrogenations of
related carbonyl compounds. The stereochemistry and the stereoselectivity of
NAD(H)-dependent oxidoreductions are well investigated [14]. Bakersꢃ yeast is a
representative of E3-type enzymes [15] that exhibit pro-(R)-H/re-face selectivity, and it
shows pronounced cis diastereoselectivity in the reduction of cyclic ketones. Therefore,
the main product of the biological reduction of the 3-oxopiperidine-4-carboxylate 1 is
(3R,4R)-3 as predicted [8]5). The absolute configurations of the stereoisomeric 1-
benzyl-3-hydroxypiperidine-4-carboxylates (þ)- and (ꢀ)-2 and (þ)- and (ꢀ)-3 (Ta-
ble 1) are fully consistent with the empirically predicted face selectivity of the
asymmetric H-transfer to functionalized ketones [9][17][18] and the dynamic kinetic
resolution of 2-substituted 3-oxocarboxylic esters [18][19]. According to the very large
number of experimental results, it can be concluded that [Ru{(ꢀ)-(S)(binap)}] has
predominantly si-face and [Ru{(þ)-(R)(binap)}] re-face selectivity, irrespective of the
bulkiness and the character of the substituents that only affect the ee values.
2.2. 1-Benzyl-3-hydroxypiperidine-4-methanols 12 and 13. The enantiomerically
pure diols, the trans-configured (þ)-(3R,4R)- and (ꢀ)-(3S,4S)-1-benzyl-3-hydroxypi-
peridine-4-methanols ((þ)-12 and (ꢀ)-12, resp.) and the cis-configured (þ)-(3R,4S)-
and (ꢀ)-(3S,4R)-1-benzyl-3-hydroxypiperidine-4-methanols ((þ)-13 and (ꢀ)-13, resp.)
were obtained after reduction of the corresponding ethyl 1-benzyl-3-hydroxypiper-
idine-4-carboxylates (þ)- and (ꢀ)-2 and (þ)- and (ꢀ)-3, respectively, with LiAlH4
(Scheme 2). The absolute configurations follow directly from their precursors.
3. Synthesis and Characterization of the 9-Benzyl-3-fluoro-2,4-dioxa-9-aza-3-
phosphadecalins. – 3.1. 2,4-Dioxa-9-aza-phosphadecalins 14 and 15. The enantiomeri-
cally pure trans-9-benzyl-3-fluoro-2,4-dioxa-9-aza-3-phosphadecalin 3-oxides 14
(Scheme 3) were prepared from (þ)- or (ꢀ)-12 by reaction with POCl2F and
chromatographic separation of the resulting P(3)-epimer mixture (axial/equatorial
ca. 1:1) into the pure axial ((ꢀ)-14a and (þ)-14a) and equatorial epimers ((ꢀ)-14b and
(þ)-14b)). Similarly, starting from (þ)- or (ꢀ)-13, the cis-9-benzyl-3-fluoro-2,4-dioxa-
9-aza-3-phosphadecalin 3-oxides ((þ)-15a and (ꢀ)-15a, and (þ)-15b and (ꢀ)-15b) were
5
)
The argumentation needs a remark: Although the definition of re and si is strictly based on the
priority rules [16], it has been shown that rather the bulkiness and the hydrophobic characteristics of
the carbonyl substituents determine the stereochemical course of biological reductions [14][15].
Hence, the synonyms RL (large) and RS (small) are more adequate. According to these
considerations, C(4) is assigned as RL and C(2) as RS in 1 and the ꢅbiologicalꢃ re-face is accurately
specified as si, see also [8].

Helvetica Chimica Acta – Vol. 94 (2011)
753
obtained (Scheme 4). The NMR data of the 2,4-dioxa-9-aza-3-phosphadecalins 14 and
15 (see Exper. Part) exhibit the same essential features as the type-IV congeners
(Scheme 1) [2 – 4]. In particular, the 31P-NMR spectra confirm the relative config-
uration at the P-atom, the double-chair conformations of the axial epimers 14a and 15a,
and distorted conformations [1 – 4][20] of the 2,4-dioxa-3-phospha moiety of the
equatorial epimers (14b and 15b)6). Due to the strongly electronegative F-substituent,
Scheme 4a)
a) P(O)Cl2F, pyridine, 08. b) CC (SiO2, hexane/Et2O).
a) [a]D in CHCl3 (c ¼ 1.00), ee > 99%.
6
)
Generally, the 31P-NMR resonance of the axial epimer is shifted upfield with respect to the
equatorial one, and the chemical-shift difference (Dd ¼ deq ꢀ dax) is > 0, and its magnitude is
inversely proportional to the electronegativity of the substituent at the P-atom. However, the cyclic
phosphorofluoridates of the cis-series of the type I – IV compounds (Scheme 1, L ¼ F) display Dd <
0, a fact that is only explained by significant conformational changes. The magnitude of the 3J(P,H)
1
in the H-coupled 31P-NMR is indicative of the conformation of the 2,4-dioxa-3-phospha moiety:
Diagnostically relevant values for the axial epimers are 3J(P,HeqꢀC(5)) ꢃ 25 Hz and
3
3J(P,HaxꢀC(5)) ꢃ 0, whereas the equatorial ones display 3J(P,HaxꢀC(5)) ꢃ J(P,HeqꢀC(5)) ꢃ 10 –
15 Hz. Hence, the axial epimers exhibit a d-type and the equatorial ones a m-type splitting pattern
(see [20]).

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Helvetica Chimica Acta – Vol. 94 (2011)
the chemical-shift difference (Dd ¼ deq – dax) is small in the trans-couple 14a/14b (Dd ¼
þ0.6 ppm) and even negative in the cis-couple 15a/15b (Dd ¼ ꢀ0.4 ppm).
3.2. Conformations of 14 and 15 in Solution. As discussed [1 – 4] and directly
evidenced [20], the anomeric (stereoelectronic) effect is the predominant parameter
that determines the conformation of the 3-substituted 2,4-dioxa-3-phosphadecalin 3-
oxides6). In the 3-axially substituted 3-fluoro-3-phosphadecalins 14a and 15a, both the
steric and the anomeric effects act in the same direction. As can be deduced from the
31P-NMR chemical shifts and vicinal couplings (3J(P,H))6), these compounds adopt the
neat double-chair conformation (C-1)7)8) (Scheme 5).
Scheme 5
However, in the equatorial epimers 14b and 15b, the steric and the stereoelectronic
effects are opposite. Although the chair conformation is sterically favored, the
7
)
)
The short terms for the conformations (IUPAC convention) were introduced in [20]: C ¼ chair, B ¼
boat, E ¼ envelope, TB ¼ twist-boat.
8
There is no direct evidence for the completely inverted C-2 conformation. Hence, we assume that
the strong anomeric preference prevents this interconversion.

Helvetica Chimica Acta – Vol. 94 (2011)
755
anomeric preference of the F-substituent to move into an axial position results in
nonchair conformations such as boat or twist-boats (i.e., B, TB-1, and TB-27);
Scheme 5). According to the vicinal-coupling data (3J(P,HꢀC(1)) ꢃ 0, 3J(P,Haxꢀ
C(5)) ¼ 3J(P,HeqꢀC(5)) ¼ 10.5 Hz), and on the basis of our recent detailed conforma-
tional studies on the type-III [20 – 22] and type-IV congeners [21][22] (Scheme 1), we
conclude by analogy that the trans-configured epimers 14b exist in an equilibrium
mixture of C-1 and TB-2 (Scheme 5)9).
The flexibility of the cis-decalin system and the P(3)-equatorial F-substituent
render the situation significantly more complex, and additional conformations must be
envisaged for the cis-configured equatorial epimers 15b. In particular, the bicyclic
system can undergo complete ring inversion to yield the prominent C-2 arrangement
(Scheme 5), which seems to be favored by the anomeric effect, the only drawback being
the steric effect of the N-benzyl group10). With regard to the experimental
3J(P,HꢀC(1)) ¼ 3J(P,HaxꢀC(5)) ¼ 16.5 Hz, and 3J(P,HeqꢀC(5)) ¼ 7.5 Hz, none of the
conformations depicted in Scheme 5 can be excluded, nor can any be assigned. Hence,
we assume that 15b exists in solution as a complex mixture of C-1/C-2, and/or TB-1/TB-
2, and most likely, also B and envelope E have to be considered11). A full account of
our detailed conformational studies on the 3-fluoro-2,4-dioxa-3-phosphadecalin 3-
oxides (types I – IV) will be presented later [22].
3.3. Crystallographic Analysis of (þ)-15a12). The structure of (þ)-15a was solved
and refined successfully. The compound in the crystal was enantiomerically pure, and
the absolute configuration of the molecule was determined independently by the
diffraction experiment. The refinement of the absolute structure parameter (see Exper.
Part) confidently confirmed that the refined coordinates (Table 3) represent the true
enantiomorph with the expected (1R,3S,6S) configuration (Fig. 2). This result
independently corroborates the configurational assignment of the precursor (þ)-ethyl
(3R,4R)-1-benzyl-3-hydroxypiperidine-4-carboxylate ((þ)-3) by means of the high-
1
field H-NMR Mosher method and its reliability [6]. As discussed above, the six-
membered ring containing the P-atom has an undistorted chair conformation with the
F-atom in the axial position, and the N-benzyl group occupies the sterically favored
equatorial position.
Since 3J(P,HꢀC(1)) ꢃ 0, conformations B and TB-1 are excluded. The 31P-NMR spectra are well
resolved, and coalescence phenomena are not observed, i.e., the interconversion C-1 > TB-2 is fast
on the NMR time scale. In contrast to [20 – 22], the conformational assignments are tentative and
not corroborated by variable-temperature NMR experiments nor X-ray crystallographic analyses.
According to crystal structures of 7-benzyl-3-fluoro-2,4-dioxa-7-aza-3-phosphadecalin 3-oxides
(type III, Scheme 1) [20][21] and because of the lack of anomeric preferences, the piperidine moiety
is assumed to adopt a chair conformation in solution with the N-benzyl group in an equatorial
position; see also Fig. 2.
9
)
10
)
11
12
)
)
As discussed for 14b, the conformational assignments are tentative, and the interconversions must
be fast on the NMR time scale.
The full data set is summarized in Table 3 (see Exper. Part). CCDC-802270 contains supplementary
crystallographic data. These can be obtained free of charge from the Cambridge Crystallographic
Data Centre, via www.ccdc.cam.ac.uk/data_request/cif.

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Helvetica Chimica Acta – Vol. 94 (2011)
Fig. 2. Molecular structure of (þ)-15a ((1R,3S,6S)). Trivial numbering; 50% probability ellipsoids.
4. Enzyme Kinetics. – 4.1. General. From the array of existing methods to evaluate
kinetic data13), we rely in this report on the straightforward one according to [27]. In
this approach, only the overall process is considered (Scheme 6), and the various
imprecisions associated with such a simplified view are taken into account. Generally,
the bimolecular rate constant (association constant ka) is considered to be the most
reliable parameter to indicate the inhibitory power of an organophosphate for an
esterase. The most simple approach for evaluating ka is based on the assumption that
the inhibitor I and the esterase E react directly to form the irreversibly phosphorylated
active center EꢀI (Scheme 6, mechanism a), and intermediate reactions are insignif-
icant. A more detailed approach involves a preceding reversible step, resulting in the
formation of an associative enzymeꢀinhibitor complex E · I* followed by an irrever-
sible phosphorylation step. The reversible step depends on the affinity of the inhibitor
for the active site and is governed by the dissociation (or affinity) constant KD, and the
irreversible-phosphorylation constant kp (Scheme 6, mechanism b). The latter param-
eters are related to the ꢅoverall inhibitory potencyꢃ by the expression ki ¼ kp/KD, where
ki might be considered as a ꢅbimolecular reaction constantꢃ. However, being a
13
)
Although apparently a simple theme, the reaction of an enzyme with a substrate and/or inhibitor to
yield products or a modified (inhibited) enzyme species is a highly complex cycle with multiple
equilibria and accordingly complex rate parameters. As a matter of fact, there exists no analytical
solution for the exact description of the general case [23][24]. Hence, many procedures for data
analyses are applied in the current literature, ranging from rather simplified ones [27] to highly
sophisticated experimental and mathematical treatments, see, e.g., [25]. However, being customized
to the individual problems, none is generally applicable. Therefore, after a primary, still partial
attempt [24], we have presented an integrated approach with a reappraisal of kinetic mechanisms
and diagnostic methods [26]. In this context, first results with selected (þ)- and (ꢀ)-3-fluoro-2,4-
dioxa-3-phosphadecalin 3-oxides have been reported [2][21][26]. Because the kinetic experiments
and the data evaluation for the (þ)- and (ꢀ)-9-benzyl-3-fluoro-2,4-dioxa-9-aza-3-phosphadecalin 3-
oxides 14 and 15 were performed earlier than the recent findings [26], the analysis of the assay data
follows the procedure according to [24].

Helvetica Chimica Acta – Vol. 94 (2011)
757
Scheme 6
combination of an equilibrium and a rate parameter, it is different from a real rate
constant [27]. In particular, the treatment is applicable when the inhibition reaction
follows first-order kinetics.
4.2. Data Analysis and Results. The inhibition studies were based on the Ellman
assay [28]. The hydrolysis of the substrate acetylthiocholine (¼2-(acetylthio)-N,N,N-
trimethylethanaminium; ATC) was monitored photometrically at 412 ꢁ 2 nm (libera-
tion of the bis-anion of 5-mercapto-2-nitrobenzoic acid). Determination of the
apparent rate constants kobs from the progress curves and the mathematical evaluation
of the dependence kobs ¼ f([I]) to evaluate the inhibition parameters (KD, ka, ki, and
kp) were performed according to [24]. The mathematical equations (Eqns. 1 – 3)
underlying to Scheme 6, and further details are summarized in the Exper. Part. For all of
the 8 stereoisomeric, enantiomerically pure (þ)- and (ꢀ)-9-benzyl-3-fluoro-2,4-dioxa-
9-aza-3-phosphadecalins 14 and 15, the plot (kobs ¼ f([I])) exhibited a linear depend-
ence. Hence, they inhibited the enzyme irreversibly according to mechanism a that is
only governed by the association constant (ka)14). The experimental results are
summarized in Table 2.
Table 2. Kinetic Data of the Inhibition of AChE with the Enantiomerically Pure 9-Benzyl-3-fluoro-2,4-
dioxa-9-aza-3-phosphadecalin 3-oxides (þ)- and (ꢀ)-14, and (þ)- and (ꢀ)-15. P(O)F(iPr)2 as reference.
ka [mꢀ1sꢀ1
]
Mechanism
ka [mꢀ1sꢀ1
]
Mechanism
(þ)-14a
(ꢀ)-14a
(þ)-14b
(ꢀ)-14b
12.1 ꢁ 0.6
23.8 ꢁ 1.1
10.7 ꢁ 0.7
14.2 ꢁ 0.9
a
a
a
a
(þ)-15a
8.6 ꢁ 0.4
8.2 ꢁ 0.4
7.6 ꢁ 0.6
8.4 ꢁ 0.7
a
a
a
a
a
(ꢀ)-15a
(þ)-15b
(ꢀ)-15b
P(O)F(OiPr)2
242 ꢁ 9
14
)
Mechanism a is equal to mechanism 1A in the general approach [26].

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Helvetica Chimica Acta – Vol. 94 (2011)
Compared to diisopropyl phosphorofluoridate (P(O)F(OiPr)2) that is generally
used as a very reliable standard reference, the type-I compounds are rather moderate
inhibitors of AChE. They are significantly weaker than the type-IV congeners, and, in
particular, do not display the pronounced diastereo- nor enantioselectivity as was
observed with the latter [2][21][29]. The only exception is the couple (þ)- and (ꢀ)-14a
with the (3S)-configured (þ)-14a being the most potent inhibitor of the type-I
series15). Due to its trans-axial arrangement, we suggest that (þ)-14a probably
approximates a recognition conformation of ACh16). However, the rather scarce result
does not show a consistent pattern allowing a reliable rationalization with respect to a
structureꢀactivity relationship17).
Remarks. – The hydrolysis of ACh catalyzed by AChE is a nearly diffusion-
controlled, highly complex process, and all steps induce conformational changes of both
the enzyme and the substrate. In view of this complexity, our reported experimental
results do not allow reliable conclusions with respect to the effective inhibition
mechanism (active site, peripheral or other conformatively induced irreversible binding
at another nucleophilic site) of the investigated 9-benzyl-3-fluoro-2,4-dioxa-9-aza-3-
phosphadecalin 3-oxides 14 and 15. Although these compounds are regarded as the
virtual ACh mimetics, their inhibitory power did not meet our expectations.
Concerning the recognition conformation18), recent investigations [32] clearly indicate
that no decisive conformation of a substrate (ACh or inhibitor) can be assigned without
knowing the respective situation of the current state of the proceeding of the respective
reaction. Moreover, as our compounds differ from the natural substrate and have
additional structural features, in particular the N-benzyl group, that significantly could
influence both the steric demand and the basicity of the compound, the situation is even
more complicated, and conclusions of evidencing a recognition conformation from the
inhibitory data is only speculative.
The authors are indebted to PD Dr. A. Linden, head of the X-ray department of our institute, for the
high-quality X-ray crystallographic analysis. The financial support of the project by the Swiss National
Science Foundation is gratefully acknowledged.
15
)
This result is consistent with the finding that the (SP)-configuration seems to be preferred by serine
hydrolases [30], and it agrees with our experience with the 8 stereoisomeric (þ)- and (ꢀ)-3-fluoro-
2,4-dioxa-3-phosphadecalin 3-oxides (type IV) [2][3][21][29].
16
17
)
)
It is fairly well established already by anterior work [31] that the most populated conformation of
ACh is gauche with a torsion angle V between ca. 150 and 1808 (Scheme 1).
Although the data were evaluated by the simplified method [24], they are considered to be reliable.
Comparison of the data of the (þ)- and (ꢀ)-3-fluoro-2,4-dioxa-3-phosphadecalin 3-oxides (type IV)
that have been evaluated by either [24] and [26] did not exhibit fundamental differences, and the
kinetic data were consistent on the whole [2][21][29]. However, the proceeding according to [26]
enables subtle individual mechanistical refinements.
Many previous investigations suggest the existence of a physiologically active (recognition)
conformation of ACh [31 – 35]. A comprehensive set of structural snapshots of the steps leading to
the intermediates of catalysis and the potential regulation by substrate binding to various allosteric
sites at the enzyme surface was presented recently [35]. It convincingly demonstrates that the ACh
positioned in the active site adopts a relaxed trans,gauche conformation, consistent with the most
stable conformation of ACh seen by crystallography [36] and NMR spectroscopy [37], whereas ACh
bound in the peripheral anionic site adopts a nonrelaxed trans,trans conformation [35].
18
)

Helvetica Chimica Acta – Vol. 94 (2011)
759
Experimental Part
1. General. See [1][4][6]. For the particular precautions in preparing and handling the organo-
phosphates, see [1]. Enantioselective hydrogenations with {(þ)-(1R)- and (ꢀ)-(1S)-[1,1’-binaphthalene]-
2,2’-diylbis[diphenylphosphine-kP]}chloro[(1,2,3,4,5,6-h]-1-methyl-4-(1-methylethyl)benzene]ruthen-
ium(1þ) chloride ([Ru{(þ)-(R)-binap}Cl(cym)]Cl and [Ru{(ꢀ)-(S)-binap}Cl(cym)]Cl, resp.; Fluka
14800 and 14801, resp.) were performed in a high-pressure reactor (Parr 452 HC2) equipped with a
Teflonꢄ vessel. The MTPA derivatives were prepared with (ꢀ)-(R)- and (þ)-(S)-a-methoxy-a-
(trifluoromethyl)benzeneacetyl chloride ((ꢀ)-(R)- and (þ)-(S)-MTPA-Cl, resp.; Fluka 65363 and
6536, resp., ChiraSelect). CC ¼ Column chromatography. Anal. HPLC: Pharmacia-LKB HPLC pump
2248, Hewlett-Packard-HP-1040M diode-array detection system, data handling with a Hewlett-Packard-
HP-Chemstation for LC, revision A.04.02; column Chiralcelꢀ OD-H (5 m, 250 ꢂ 4.6 mm; Daicel Chemical
Industries, Ltd.); eluent hexane/iPrOH 12 :1; flow rate 1 ml/min at r.t.; ldet 220 nm. Prep. HPLC: Applied-
Biosystems-400 solvent-delivery system, Applied-Biosystems-783A programmable absorbance de-
tector; column Chiralcelꢀ OD (10 m, 250 ꢂ 20 mm); eluent hexane/EtOH 15 :1; flow rate 5 ml/min
at r.t.; ldet 254 nm. Determination of ee: based on the integration of the peak areas of the anal.
HPLC separations (a ¼ 1.26, Rs > 2). NMR Assignments: based on extensive 2D-NMR experiments (see
[1]).
2. Ethyl 3-hydroxy-1-(phenylmethyl)piperidine-4-carboxylates (þ)- and (ꢀ)-2 and (þ)- and (ꢀ)-3.
2.1. (ꢁ)-Ethyl trans- and cis-3-Hydroxy-1-(phenylmethyl)piperidine-4-carboxylate (ꢁ)-2 and (ꢁ)-3, resp.,
and HPLC Separation of the Enantiomers. To a soln. of ethyl 3-oxo-1-(phenylmethyl)piperidine-4-
carboxylate hydrochloride (1 · HCl; 551 mg, 1.85 mmol) and anh. Na2CO3 in anh. Et2O (20 ml), NaBH4
(49 mg, 1.28 mmol) was added in portions, and the mixture was stirred at r.t. for 1 h. Workup and
continuous extraction with Et2O yielded (ꢁ)-2/(ꢁ)-3 as a yellowish oil (330 mg, 86%). Separation of the
diastereoisomers was effected by CC (SiO2, CH2Cl2/MeOH 98 :2), but the four components of the
mixture could be separated directly by anal. HPLC, too: (ꢀ)-2 (k’ ¼ 1.33), (þ)-2 (k’ ¼ 1.71) (a ¼ 1.29,
RS ¼ 1.9); (þ)-3 (k’ ¼ 2.19), (ꢀ)-3 (k’ ¼ 2.75) (a ¼ 1.26, RS ¼ 2.0) (Table 1). Although the separation
satisfied the anal. purposes, the enantiomerically pure compounds were prepared more efficiently by
enantioselective hydrogenations, separation of the diastereoiomers, and final purification by prep.
HPLC.
2.2. (þ)- and (ꢀ)-2 (trans isomers) and (þ)- and (ꢀ)-3 (cis isomers). The enantiomerically pure
compounds were obtained as explicitly described in [6]: Bakersꢃ yeast reduction of 1 afforded (ꢀ)-2
([a]D ¼ ꢀ23.1, ee 95%) and (þ)-3 ([a]D ¼ þ42.2, ee 82%).
Hydrogenation of 1 with ([Ru{(ꢀ)-(S)-binap}Cl(cym)]Cl, gave (þ)-2 ([a]D ¼ þ25.2, ee > 99%) and
(þ)-3 ([a]D ¼ þ56.6, ee > 99%). The analogous procedure with ([Ru{(þ)-(R)-binap}Cl(cym)]Cl
afforded (ꢀ)-2 ([a]D ¼ ꢀ24.9, ee > 99%) and (ꢀ)-3 ([a]D ¼ ꢀ56.3, ee > 99%).
(þ)-Ethyl (3R,4S)-3-Hydroxy-1-(phenylmethyl)piperidine-4-carboxylate ((þ)-2): Colorless viscous
oil. Rf (CH2Cl2/MeOH 98 :2) 0.19. [a]D ¼ þ25.2 (CHCl3, c ¼ 1.00). IR (film): 3439s, 3086m, 3062m, 3028s,
2979s 2941vs, 2805s, 2765s, 1731vs, 1466s, 1453s, 1445s, 1395s, 1367s, 1320s, 1278vs, 1183vs, 1135s, 1089vs,
1037vs, 990s, 949m, 910m, 880m, 859m, 813w, 787w, 746vs, 700vs. 1H-NMR (600 MHz, CDCl3): 7.33 – 7.24
(m, PhCH2); 4.18 (q, 3J ¼ 7.1, MeCH2); 3.96 (td, 3J(3,2ax) ¼ 3J(3,4) ¼ 9.1, 3J(3,2eq) ¼ 4.2, HꢀC(3)); 3.53,
3
4
3.525 (AB, 2J ¼ 13.2, PhCH2); 3.16 (br. s, OH); 2.99 (ddd, 2J ¼ 10.9, J(2eq,3) ¼ 4.2, J(2eq,6eq) ¼ 1.2,
HeqꢀC(2)); 2.77 (dtd, J ¼ 11.2, J(6eq,5ax) ¼ 3J(6eq,5eq) ¼ 3.8, J(6eq,2eq) ¼ 1.2, HeqꢀC(6)); 2.26 (ddd,
2
3
4
3J(4,3) ¼ 9.1, 3J(4,5ax) ¼ 11.2, 3J(4,5eq) ¼ 4.4, HꢀC(4)); 2.06 (td, 2J ¼ 3J(6ax,5ax) ¼11.1, 3J(6ax,5eq) ¼ 2.9,
2
3
3
2
HaxꢀC(6)); 2.04 – 1.95 (m, dd- and dt-like, J ꢃ J ꢃ 11, J ꢃ 3, HaxꢀC(2), HeqꢀC(5)); 1.73 (br. qd, J ꢃ
3J(5ax,4) ꢃ J(5ax,6ax) ꢃ 11, 3J(5ax,6eq) ꢃ 3, HaxꢀC(5)); 1.27 (t, 3J ¼ 7.1, MeCH2). 13C-NMR (75.4 MHz,
3
CDCl3): 174.2 (CO); 137.9 (C(1’)); 128.9 (C(3’), C(5’)); 128.2 (C(2’), C(6’)); 127.0 (C(4’)); 67.9 (C(3));
62.5 (PhCH2); 60.6 (MeCH2); 58.7 (C(2)); 52.1 (C(6)); 48.8 (C(4)); 26.4 (C(5)); 14.1 (MeCH2). CI-MS
(NH3): 264 (100, [M þ H]þ), 263 (12, Mþ), 245 (15, [M ꢀ H2O]þ).
(ꢀ)-Ethyl (3S,4R)-3-Hydroxy-1-(phenylmethyl)piperidine-4-carboxylate ((ꢀ)-2): [a]D ¼ ꢀ24.9
(CHCl3, c ¼ 1.00). All other data: identical with those of (þ)-2.
(þ)-Ethyl (3R,4R)-3-Hydroxy-1-(phenylmethyl)piperidine-4-carboxylate ((þ)-3): Colorless powder.
M.p. ca. 358. Rf (CH2Cl2/MeOH 98 :2) 0.26. [a]D ¼ þ56.6 (CHCl3, c ¼ 1.00). All other data: see [6].

760
Helvetica Chimica Acta – Vol. 94 (2011)
(ꢀ)-Ethyl (3S,4S)-3-Hydroxy-1-(phenylmethyl)piperidine-4-carboxylate ((ꢀ)-3): [a]D ¼ ꢀ56.3
(CHCl3, c ¼ 1.00). All other data: identical with those of (þ)-3, see [6].
3. (R)- and (S)-MTPA Esters for the Determination of the Absolute Configuration. The trans-3-
hydroxy ester (þ)- or (ꢀ)-2 (40 mg, 0.15 mmol) was dissolved in dry pyridine (250 ml) and treated with
(þ)-(S)-MTPA-Cl (43 ml, 0.23 mmol, 1.5 equiv.) at r.t. for 24 h under Ar. Evaporation and chromato-
graphic purification (SiO2, CH2Cl2/MeOH 98 :2) of the crude product afforded the (R)-MTPA esters 8a
or 9a, resp. Analogously, (þ)- or (ꢀ)-2, resp., were treated with (ꢀ)-(R)-MTPA-Cl to yield the (S)-
MTPA esters 8b or 9b, resp. All MTPA esters were isolated in pure form as colorless, viscous oils: 8a
(64 mg, 89%), 8b (63 mg, 88%), 9a (55 mg, 76%), and 9b (58 mg, 81%).
(R)- and (S)-MTPA Esters of (þ)-2: Ethyl (3R,4S)-1-(phenylmethyl)-3-[(2R)-3,3,3-trifluoro-2-
1
methoxy-1-oxo-2-phenylpropoxy]piperidine-4-carboxylate ((R)-MTPA Ester; 8a): H-NMR (600 MHz,
CDCl3): 7.43 (d-like, 3J ¼ 8), 7.31 – 7.17 (m, Ph); 5.37 (td, 3J(3,2ax) ¼ 3J(3,4) ¼ 9.7, 3J(3,2eq) ¼ 4.6,
3
2
5
HꢀC(3)); 3.93 (q, J ¼ 7.1, MeCH2); 3.56, 3.40 (AB, J ¼ 13.3, PhCH2); 3.43 (d, J(Me,F) ¼ 1.1, MeO);
3.15 (ddd, 2J ¼ 10.2, 3J(2eq,3) ¼ 4.4, J(2eq,6eq) ¼ 1.0, HeqꢀC(2)); 2.67 (br. dt, 2J ¼ 12.3, 3J(6eq,5ax) ꢃ
4
3J(6eq,5eq) ꢃ 4, HeqꢀC(6)); 2.46 (ddd, J(4,3) ¼ 9.7, J(4,5ax) ¼ 11.3, J(4,5eq) ¼ 4.7, HꢀC(4)); 2.10 (dd,
3
3
3
2J ¼ 10.4, J(2ax,3) ¼ 9.7, HaxꢀC(2)); 1.97 (td, J ¼ 3J(6ax,5ax) ¼ 11.2, J(6ax,5eq) ¼ 2.8, HaxꢀC(6)); 1.87
3
2
3
(br. dq, 2J ꢃ 11, 3J(5eq,4) ꢃ J(5eq,6ax) ꢃ J(5eq, 6eq) ꢃ 4, HeqꢀC(5)); 1.72 (br. qd, 2J ꢃ J(5ax,4) ꢃ
3J(5ax,6ax) ꢃ 11, 3J(5ax,6eq) ꢃ 4, HaxꢀC(5)); 1.07 (t, 3J ¼ 7.1, MeCH2). 13C-NMR (75.4 MHz, CDCl3):
172.4 (COOEt); 165.4 (CO of MTPA); 137.3 (C(1’)); 132.1 (C(1’’)); 129.4 (C(3’), C(5’)); 128.6 (C(3’’),
3
3
3
1
C(5’’)); 128.1 (C(2’), C(6’), C(2’’), C(6’’)); 127.6 (C(4’)); 127.0 (C(4’’)); 123.2 (q, J(C,F) ¼ 288.2, CF3);
2
85.1 (q, J(C,F) ꢃ 40, PhC(MeO)(CF3)CO); 71.6 (C(3)); 62.4 (PhCH2); 60.8 (MeCH2); 55.6 (MeO);
55.2 (C(2)); 51.8 (C(6)); 44.1 (C(4)); 22.8 (C(5)); 13.9 (MeCH2). 19F-NMR (564.5 MHz, CDCl3): ꢀ 72.21
(s, CF3). CI-MS (NH3): 497 (100, [M þ NH4]þ), 479 (13, Mþ), 263 (12, [M ꢀ MTPA]þ), 245 (17, [M þ
H ꢀ MTPA ꢀ H2O]þ).
Ethyl (3R,4S)-1-(Phenylmethyl)-3-[(2S)-3,3,3-trifluoro-2-methoxy-1-oxo-2-phenylpropoxy]piperi-
1
3
dine-4-carboxylate ((S)-MTPA Ester; 8b): H-NMR (600 MHz, CDCl3): 7.40 (d-like, J ¼ 7), 7.39 – 7.15
(m, Ph); 5.37 (td, 3J(3,2ax) ¼ 3J(3,4) ¼ 9.8, 3J(3,2eq) ¼ 4.6, HꢀC(3)); 4.03 (q, 3J ¼ 7.1, MeCH2); 3.53, 3.41
(AB, 2J ¼ 13.2, PhCH2); 3.42 (d, 5J(Me,F) ¼ 1.2, MeO); 3.11 (ddd, 2J ¼ 10.6, 3J(2eq,3) ¼ 4.5,
4J(2eq,6eq) ¼ 1.2, HeqꢀC(2)); 2.77 (br. dt, 2J ¼ 11.3, 3J(6eq,5ax) ꢃ J(6eq,5eq) ꢃ 3, HeqꢀC(6)); 2.46
3
(ddd, 3J(4,3) ¼ 9.8, 3J(4,5ax) ¼ 11.8, 3J(4,5eq) ¼ 4.5, HꢀC(4)); 1.96 – 1.86 (m, HaxꢀC(2), HeqꢀC(5),
HaxꢀC(6)); 1.75 (br. qd, 2J ꢃ J(5ax,4) ꢃ J(5ax,6ax) ꢃ 11, 3J(5ax,6eq) ꢃ 4, HaxꢀC(5)); 1.13 (t, 3J ¼ 7.1,
MeCH2)). 13C-NMR (75.4 MHz, CDCl3): 172.4 (COOEt); 165.4 (CO of MTPA); 137.3 (C(1’)); 132.1
(C(1’’)); 129.5 (C(3’), C(5’)); 128.8 (C(3’’), C(5’’)); 128.2 (C(2’), C(6’), C(2’’), C(6’’)); 127.3 (C(4’)); 127.2
3
3
1
2
(C(4’’)); 123.2 (q, J(C,F) ¼ 288.2, CF3); 85.1 (q, J(C,F) ꢃ 40, PhC(MeO)(CF3)CO); 72.2 (C(3)); 62.1
(PhCH2); 60.8 (MeCH2); 55.3 (MeO); 55.1 (C(2)); 51.2 (C(6)); 46.4 (C(4)); 27.4 (C(5)); 13.9 (MeCH2).
19F-NMR (564.5 MHz, CDCl3): ꢀ 72.07 (s, CF3). CI-MS (NH3): 497 (100, [M þ NH4]þ), 479 (15, Mþ),
263 (9, [M ꢀ MTPA]þ), 245 (18, [M þ H ꢀ MTPA ꢀ H2O]þ).
Dd(1H) ¼ d(S) – d(R) (in Hz)19): HꢀC(3) Dd ¼ 0, HaxꢀC(2) Dd ¼ ꢀ90, HeqꢀC(2) Dd ¼ ꢀ24, MeCH2
Dd ¼ þ60, and MeCH2 Dd ¼ þ36 ! (3R)-configuration. Dd(19F) ¼ d(S) – d(R) (in Hz): CF3 Dd ¼
þ79 ! (3R)-configuration.
(R)- and (S)-MTPA Ester 9a and 9b of (ꢀ)-2. Being enantiomeric compounds, 9a and 8b (9a ¼ ent-
8b) as well as 9b and 8a (9b ¼ ent-8a) exhibited identical NMR spectra, only the sign of the Dd were
inverted. Dd(1H) ¼ d(S) – d(R) (in Hz)19): HꢀC(3) Dd ¼ 0, HaxꢀC(2) Dd ¼ þ90, HeqꢀC(2) Dd ¼ þ24,
MeCH2, Dd ¼ ꢀ60, and MeCH2 Dd ¼ ꢀ36 ! (3S)-configuration. Dd(19F) ¼ d(S) – d(R) (in Hz): CF3
Dd ¼ ꢀ79 ! (3S)-configuration.
19
)
In contrast to the MTPA derivatives 10a/10b and 11a/11b of the cis-congeners (þ)- and (ꢀ)-3 [6],
the 1H-NMR signals were well resolved. For unambiguous additional comparisons, also MTPA
esters starting from (ꢁ)-2, and from enantiomerically enriched (þ)- and (ꢀ)-2 (ee > 50%) were
analyzed. The data of the respective diastereoisomer pairs were consistent in every respect and
showed the relative displacements as expected [12]. Since 8a ¼ ent-9b and 8b ¼ ent-9a, 8a
(2’R,3R,4S) and 9b (2’S,3S,4R) as well as 8b (2’S,3R,4S) and 9a (2’R,3S,4R) have identical NMR
spectra.

Helvetica Chimica Acta – Vol. 94 (2011)
761
The same procedure was adopted for the derivatization of the cis-3-hydroxy esters (þ)- and (ꢀ)-3
affording the (R)-MTPA esters 10a and 11a, resp., and the (S)-MTPA esters 10b and 11b, resp. For exper.
details, the full data set, and the unambiguous assignment of the absolute configuration of the cis-3-
hydroxy esters (þ)- and (ꢀ)-3, see [6].
4. 3-Hydroxy-1-(phenylmethyl)piperidine-4-methanols (þ)- and (ꢀ)-12 (trans isomers) and (þ)- and
(ꢀ)-13 (cis isomers). To a cooled soln. (ca. 08) of (þ)- or (ꢀ)-2 (350 mg, 1.33 mmol) in anh. THF (20 ml),
LiAlH4 (100 mg, 2.66 mmol) was slowly added in portions. After 30 min at 08, the mixture was refluxed
for 2 h. Continuous extraction with Et2O, workup, and CC (SiO2, Et2O) afforded (þ)-12 (195 mg, 66%)
or (ꢀ)-12 (190 mg, 65%), resp. as colorless crystals. Analogously, the reduction of (þ)- or (ꢀ)-3 (400 mg,
1.52 mmol) afforded (þ)-13 (235 mg, 70%) or (ꢀ)-13 (240 mg, 71%), resp.
(þ)-(3R,4R)-3-Hydroxy-1-(phenylmethyl)piperidine-4-methanol ((þ)-12): Colorless crystals. M.p.
111 – 1128. [a]D ¼ þ6.3 (CHCl3, c ¼ 1.00). All other data: identical with those of (ꢁ)-12 as specified in [1].
(ꢀ)-(3S,4S)-3-Hydroxy-1-(phenylmethyl)piperidine-4-methanol ((ꢀ)-12): [a]D ¼ ꢀ6.6 (CHCl3, c ¼
1.00). All other data: identical with those of (þ)-12 (and (ꢁ)-12 [1]).
(þ)-(3R,4S)-3-Hydroxy-1-(phenylmethyl)piperidine-4-methanol ((þ)-13). Colorless crystals. M.p.
82 – 848. [a]D ¼ þ11.4 (CHCl3, c ¼ 1.00). All other data: identical with those of (ꢁ)-13 as specified in [1].
(ꢀ)-(3S,4R)-3-Hydroxy-1-(phenylmethyl)piperidine-4-methanol ((ꢀ)-13). [a]D ¼ ꢀ10.9 (CHCl3,
c ¼ 1.00). All other data: identical with those of (þ)-13 (and (ꢁ)-13 [1]).
5. trans- and cis-9-Benzyl-3-fluoro-2,4-dioxa-9-aza-3-phosphadecalin 3-Oxides (¼2-Fluorohexahy-
dro-7-(phenylmethyl)-4H-1,3,2-dioxaphosphorino[4,5-c]pyridine 2-Oxides) (þ)- and (ꢀ)-14a, (þ)- and
(ꢀ)-14b, (þ)- and (ꢀ)-15a, and (þ)- and (ꢀ)-15b, resp. To a cooled soln. (08) of (þ)-12 (113 mg,
0.87 mmol) in Et2O (3 ml) in a glove box (N2 atmosphere), anh. pyridine (125 ml (123 mg), 1.55 mmol)
and a cooled soln. (08) of POCl2F [38] (110 ml (173 mg), 1.26 mmol, ca. 1.5 equiv.) in Et2O (1 ml) were
added with a syringe, and the mixture was kept for ca. 2 min at 08 when the mixture was withdrawn and
quickly passed through SiO2 (pH 5.6 [4], Et2O). The precipitated pyridinium salt in the reaction flask
was thoroughly sonicated for 5 min with Et2O, the combined solns. were gently evaporated (N2 stream),
and the residue was subjected to CC (SiO2, pH 5.6 [4], hexane/Et2O 4 :1): (ꢀ)-14a (63 mg, 37%) and (ꢀ)-
14b (63 mg, 37%).
Applying the identical procedure, the following compounds were prepared from the different
starting diols (each 100 mg): from (ꢀ)-12, (þ)-14a (54 mg, 36%) and (þ)-14b (57 mg, 38%); from (þ)-13,
(þ)-15a (51 mg, 34%) and (þ)-15b (25 mg, 17%); from (ꢀ)-13, (ꢀ)-15a (71 mg, 47%) and (ꢀ)-15b
(56 mg, 37%); in all cases the axial epimer was less polar.
(þ)-(1S,3R,6S)-9-Benzyl-3-fluoro-2,4-dioxa-9-aza-3-phosphadecalin 3-Oxide ( ¼ (þ)-(2R,4aS,8aS)-
2-Fluorohexahydro-7-(phenylmethyl)-4H-1,3,2-dioxaphosphorino[4,5-c]pyridine 2-Oxide; (þ)-14a):
Colorless viscous oil. Rf (Et2O) 0.18. [a]2D5 ¼ þ11.2 (c ¼ 1.00, CHCl3). IR (CHCl3): 2951m, 2928m,
2816m, 2774m, 1952w, 1877w, 1825w, 1602w, 1495m, 1468m, 1454m, 1444m, 1334s, 1306m, 1160m, 1070s,
1096s, 1072s, 1045s, 1009s, 982s, 933m, 899s, 882s, 858m. 1H-NMR (600 MHz, CDCl3): 7.27 – 7.19 (m,
PhCH2); 4.30 (A of ABX-P, 2J ¼ 11.2, 3J(5eq,P) ¼ 24.3, 3J(5eq,6) ¼ 4.4, HeqꢀC(5)); 4.24 (td, 3J(1,6) ¼
3J(1,10ax) ¼ 10.9, 3J(1,10eq) ¼ 4.2, HꢀC(1)); 4.10 (B of ABX-P, 2J ¼ 3J(5ax,6) ¼ 11.2, HaxꢀC(5)); 3.53,
2
2
3
4
3.51 (AB, J ¼ 13.1, PhCH2); 3.11 (ddd, J ¼ 10.5, J(10eq,1) ¼ 4.2, J(10eq,8eq) ¼ 1.5 (HeqꢀC(10)); 2.85
(dquint.-like, 2J ¼ 11.5, 3J(8eq,7ax) ꢃ J(8eq,7eq) ¼ 4.3, 4J(8eq,10eq) ¼ 1.5, HeqꢀC(8)); 2.09 (t, 2J ¼
3
3J(10ax,1) ¼ 10.5, HaxꢀC(10)); 2.02 (ddd, 2J ¼ 11.5, 3J(8ax,7ax) ¼ 12.4, 3J(8ax,7eq) ¼ 4.0, HaxꢀC(8));
1.92 (X of ABX-P, qt-like, w1/2 ꢃ 25, HꢀC(6)); 1.59 (dq, 2J ¼ 12.4, 3J(7eq,6) ꢃ J(7eq,8ax) ꢃ J(7eq,8eq) ꢃ
4, HeqꢀC(7)); 1.23 (qd, 2J ¼ 3J(7ax,6) ¼ 3J(7ax,8ax) ¼ 12.4, 3J(7ax,8eq) ¼ 4.3, HaxꢀC(7)). 13C-NMR
(150.9 MHz, CDCl3): 137.3 (C(1’)); 128.8 (C(3’), C(5’)); 128.3 (C(2’), C(6’)); 127.4 (C(4’)); 80.64 (d,
2J(1,P) ¼ 6.4, C(1)); 73.7 (d, 2J(5,P) ¼ 7.5, C(5)); 62.1 (PhCH2); 56.6 (d, 3J(10,P) ¼ 11.9, C(10)); 51.7
3
3
(C(8)); 39.6 (d, J(6,P) ¼ 6.3, C(6)); 24.3 (C(7)). 31P-NMR (121.4 MHz, CDCl3): ꢀ 16.5 (dd, J(P,F) ¼
1008, 3J(P,HeqꢀC(5)) ¼ 24.3). 19F{1H}-NMR (282.4 MHz, (CDCl3): ꢀ 86.1 (d, 1J(F,P) ¼ 1010). EI-MS:
285 (9, Mþ), 194 (12, [M ꢀ PhCH2]þ), 171 (21), 160 (26), 91 (100, PhCHþ2 ), 65 (24).
3
1
(ꢀ)-(1R,3S,6R)-9-Benzyl-3-fluoro-2,4-dioxa-9-aza-3-phosphadecalin 3-Oxide ((ꢀ)-14a): [a]D25
ꢀ10.6 (c ¼ 1.00, CHCl3). All other data: identical with those of (þ)-14a.
¼
(þ)-(1S,3S,6S)-9-Benzyl-3-fluoro-2,4-dioxa-9-aza-3-phosphadecalin 3-Oxide ((¼2-Fluorohexahy-
dro-7-(phenylmethyl)-4H-1,3,2-dioxaphosphorino[4,5-c]pyridine 2-Oxide (þ)-14b): Colorless prisms.

762
Helvetica Chimica Acta – Vol. 94 (2011)
M.p. 88 – 928. Rf (Et2O) 0.09. [a]2D5 ¼ þ9.6 (c ¼ 1.00, CHCl3). IR (CHCl3): 2950m, 2930m, 2815m, 2773m,
1952w, 1878w, 1820w, 1711w, 1602w, 1494m, 1474m, 1468m, 1454m, 1332s, 1302m, 1159m, 1103m, 1053s,
1013s, 980s, 897s, 879s. 1H-NMR (600 MHz, CDCl3): 7.36 – 7.24 (m, PhCH2); 4.50 – 4.37 (A of ABX-P and
m, not resolved, HeqꢀC(5)20), HꢀC(1)); 4.21 (B of ABX-P, 2J ¼ 3J(5ax,P) ¼ 3J(5ax,6) ¼ 10.5, 4J(5ax,F) ¼
4.0, HaxꢀC(5))20); 3.59, 3.585 (AB, 2J ¼ 13.2, PhCH2); 3.22 (ddd, 2J ¼ 10.6, 3J(10eq,1) ¼ 4.6,
3
4J(10eq,8eq) ¼ 1.5, HeqꢀC(10)); 2.91 (dquint.-like, 2J ¼ 11.6, 3J(8eq,7ax) ꢃ J(8eq,7eq) ¼ 4.0,
4J(8eq,10eq) ¼ 1.5, HeqꢀC(8)); 2.15 (X of ABX-P, qt-like, w1/2 ꢃ 25, HꢀC(6)); 2.13 – 2.03 (m, HaxꢀC(8),
HaxꢀC(10)); 1.70 (dq, 2J ¼ 12.3, 3J(7eq,6) ꢃ J(7eq,8ax) ꢃ J(7eq,8eq) ꢃ 4, HeqꢀC(7)); 1.33 (qd, 2J ¼
3J(7ax,6) ¼ 3J(7ax,8ax) ¼ 12.3, 3J(7ax,8eq) ¼ 4.3, HaxꢀC(7)). 13C-NMR (150.9 MHz, CDCl3): 137.1
(C(1’)); 128.8 (C(3’), C(5’)); 128.3 (C(2’), C(6’)), 127.4 (C(4’)); 80.2 (d, 2J(1,P) ¼ 5.3, C(1)); 73.6 (d,
3
3
3
3
2J(5,P) ¼ 7.2, C(5)); 62.0 (PhCH2); 56.9 (d, J(10,P) ¼ 8.9, C(10)); 51.5 (C(8)); 38.4 (d, J(6,P) ¼ 13.1,
C(6)); 24.3 (C(7)). 31P-NMR (121.4 MHz, CDCl3): ꢀ 15.9 (dt, 1J(P,F) ¼ 1002, 3J(P,HaxꢀC(5)) ¼
3J(P,HeqꢀC(5)) ¼ 10.5)20). 19F{1H}-NMR (282.4 MHz, (CDCl3): ꢀ 69.4 (d, J(F,P) ¼ 1000). EI-MS: 285
1
(7, Mþ), 194 (6, [M ꢀ PhCH2]þ), 171 (15), 160 (18), 91 (100, PhCHþ2 ), 65 (10).
(ꢀ)-(1R,3R,6R)-9-Benzyl-3-fluoro-2,4-dioxa-9-aza-3-phosphadecalin 3-Oxide ((ꢀ)-14b): [a]D25
ꢀ9.6 (c ¼ 1.00, CHCl3). All other data: identical with those of (þ)-14b.
¼
(þ)-(1R,3S,6S)-9-Benzyl-3-fluoro-2,4-dioxa-9-aza-3-phosphadecalin 3-Oxide ( ¼ (þ)-(2S,4aS,8aR)-
2-Fluorohexahydro-7-(phenylmethyl)-4H-1,3,2-dioxaphosphorino[4,5-c]pyridine 2-Oxide; (þ)-15a):
Colorless prisms. M.p. 75 – 778. Rf (Et2O) 0.13. [a]2D5 ¼ þ18.6 (c ¼ 1.00, CHCl3). IR (CHCl3): 2953s,
2812s, 2768s, 1955w, 1877w, 1816w, 1758w, 1601w, 1586w, 1495m, 1495s, 1454s, 1444s, 1371s, 1337s, 1272s,
1168s, 1125s, 1092s, 1075s, 1026s, 981s, 881s. 1H-NMR (600 MHz, CDCl3): 7.33 – 7.24 (m, PhCH2); 4.70 (m,
w1/2 ꢃ 6, HꢀC(1)); 4.56 (A of ABX-P, 2J ¼ 11.4, 3J(5ax,P) ¼ 2.5, 3J(5ax,6ax) ꢃ 0, HaxꢀC(5))20)21); 4.27 (B
of ABX-P, J ¼ 11.4, J(5eq,P) ¼ 24.8, J(5eq,6) ¼ 1.2, HeqꢀC(5))20); 3.58, 3.55 (AB, J ¼ 13.5, PhCH2);
2
3
3
2
3.18 (dt, J ¼ 13.0, J(10eq,1) ¼ 4J(10eq,8eq) ¼ 2.1, HeqꢀC(10)); 3.00 (dquint.-like, J ¼ 12, J(8eq,7ax) ꢃ
3J(8eq,7eq) ꢃ 4.5, 4J(8eq,10eq) ¼ 2.1, HeqꢀC(8)); 2.29 (qd, 2J ¼ 3J(7ax,8ax) ¼ 12.0, 3J(7ax,8eq) ¼ 4.3,
HaxꢀC(7)); 2.26 (ddd, 2J ¼ 13.0, 3J(10ax,1) ¼ 1.8, 4J(10ax,P) ¼ 5.9, HaxꢀC(10)); 2.12 (td, 2J ¼
3J(8ax,7ax) ¼ 11.7, 3J(8ax,7eq) ¼ 2.4, HaxꢀC(8)); 1.77 (X of ABX-P, dquint.-like, w1/2 ꢃ 25, HꢀC(6));
1.56 (m, dq-like, w1/2 ꢃ 25, HeqꢀC(7)). 13C-NMR (150.9 MHz, CDCl3): 137.5 (C(1’)); 128.7 (C(3’), C(5’));
128.2 (C(2’), C(6’)); 127.1 (C(4’)); 78.5 (d, 2J(1,P) ¼ 7.2, C(1)); 73.1 (d, 2J(5,P) ¼ 7.1, C(5)); 61.9 (PhCH2);
56.4 (d, 3J(9,P) ¼ 9.1, C(10)); 51.7 (C(8)); 34.3 (d, 3J(6,P) ¼ 5.6, C(6)); 22.8 (C(7)). 31P-NMR (121.4 MHz,
CDCl3): ꢀ 16.5 (dd br. d, 1J(P,F) ¼ 1006, 3J(P,HeqꢀC(5)) ¼ 24.8, 4J(P,HaxꢀC(10)) ¼ 5.9, 3J(P,HaxꢀC(6)) ꢃ
2)20). 19F{1H}-NMR (282.4 MHz, (CDCl3): ꢀ 86.2 (d, 1J(F,P) ¼ 1007). EI-MS: 285 (16, Mþ), 194 (5, [M ꢀ
PhCH2]þ), 160 (61), 147 (27), 118 (26), 91 (100, PhCHþ2 ), 80 (27).
2
3
2
3
(ꢀ)-(1S,3R,6R)-9-Benzyl-3-fluoro-2,4-dioxa-9-aza-3-phosphadecalin 3-Oxide ((ꢀ)-15a): [a]D25
¼
ꢀ19.7 (c ¼ 1.00, CHCl3). All other data: identical with those of (þ)-15a.
(þ)-(1R,3R,6S)-9-Benzyl-3-fluoro-2,4-dioxa-9-aza-3-phosphadecalin 3-Oxide ( ¼ (þ)-(2R,4aS,
8aR)-2-Fluorohexahydro-7-(phenylmethyl)-4H-1,3,2-dioxaphosphorino[4,5-c]pyridine
2-Oxide;
(þ)-15b): Colorless plates. M.p. 95 – 988. Rf (Et2O) 0.07. [a]2D5 ¼ þ12.1 (c ¼ 1.00, CHCl3). IR (CHCl3):
2953s, 2930s, 2813s, 2787s, 2768s, 1954w, 1876w, 1816w, 1737w, 1602w, 1586w, 1495m, 1454s, 1443s, 1370s,
1329s, 1093s, 1076s, 1012s, 984s, 949s, 886s. 1H-NMR (600 MHz, CDCl3): 7.36 – 7.24 (m, PhCH2); 4.80 (m,
not resolved, w1/2 ꢃ 30, HꢀC(1)); 4.53 (A of ABX-P, 2J ¼ 11.4, 3J(5ax,P) ¼ 16.5, 3J(5ax,6) ¼ 3.5,
HaxꢀC(5))20); 4.39 (B of ABX-P, 2J ¼ 11.4, 3J(5eq,P) ¼ 16.5, 3J(5eq,6) ¼ 4.8, HeqꢀC(5))20); 3.58 (s,
PhCH2); 2.84 (m, dt-like, 2J ¼ 11.8, HeqꢀC(8)); 2.70 (m, td-like, 2J ¼ 11.8, HeqꢀC(8))); 2.44 (m, w1/2 ꢃ 20,
CH2(10)); 2.30 (X of ABX-P, m, w1/2 ꢃ 30, HꢀC(6)); 1.82 (m, q-like, w1/2 ꢃ 15, CH2(7)). 13C-NMR
(150.9 MHz, CDCl3): 137.2 (C(1’)); 128.7 (C(3’), C(5’)); 128.3 (C(2’), C(6’)); 127.3 (C(4’)); 79.2 (2J(1,P) ¼
2
3
7.7, C(1)); 69.7 (d, J(5,P) ¼ 6.4, C(5)); 62.2 (PhCH2); 53.6 (C(10)); 49.3 (C(8)); 32.8 (d, J(6,P) ¼ 8.1,
20
)
The descriptors ꢅaxꢃ and ꢅeqꢃ for CH2(5) are based on their relative positions in the chair
conformation of the 2,4-dioxa-3-phospha moiety. As discussed (see Scheme 5), the conformation is
rather a twist-boat (TB-2) than a chair in the P(3)-equatorially substituted compounds. For reasons
of simplicity, the notation ꢅaxꢃ and ꢅeqꢃ is maintained. HaxꢀC(5) is alwayas cis to HꢀC(1) and
HeqꢀC(5) trans to HꢀC(1), see [20].
1
)
Assigned by H{31P} and 31P/1H-correlated spectra (HMQC).
21

Helvetica Chimica Acta – Vol. 94 (2011)
763
C(6)); 24.3 (C(7)). 31P-NMR (121.4 MHz, CDCl3): ꢀ 16.9 (dtd, 1J(P,F) ¼ 997, 3J(P,HꢀC(1)) ¼
3J(P,HaxꢀC(5)) ¼ 16.5, 3J(P,HeqꢀC(5)) ¼ 7.5 )20). 19F{1H}-NMR (282.4 MHz, (CDCl3): ꢀ 76.9 (d,
1J(F,P) ¼ 999). EI-MS: 285 (16, Mþ), 194 (24, [M ꢀ PhCH2]þ), 160 (51), 147 (19), 118 (23), 91 (100,
PhCHþ2 ), 80 (32).
(ꢀ)-(1S,3S,6R)-9-Benzyl-3-fluoro-2,4-dioxa-9-aza-3-phosphadecalin 3-Oxide ((ꢀ)-15b): [a]D25
ꢀ12.4 (c ¼ 1.00, CHCl3). All other data: identical with those of (þ)-15b.
¼
5. X-Ray Crystal-Structure Determination of (þ)-15a12). All measurements were conducted at low-
temperature by means of a Nonius-KappaCCD area-detector diffractometer [39] with graphite-
monochromated MoKa radiation (l 0.71073 ꢆ) and an Oxford-Cryosystems-Cryostream-700 cooler. The
unit-cell constants and an orientation matrix for data collection were obtained from a least-squares
refinement of the setting angles of 28171 reflections in the range 48 < 2q < 508. The mosaicity was
1.033(2)8. A total of 222 frames were collected by using w scans with k offsets, 100-s exposure time, a
rotation angle of 2.08 per frame, and a crystal-detector distance of 30.0 mm.
Data reduction was performed with HKL DENZO and SCALEPACK [40]. The intensities were
corrected for Lorentz and polarization effects, and an absorption correction based on the multi-scan
method [41] was applied. The space group was determined from the systematic absences, packing
considerations, a statistical analysis of intensity distribution, and the successful solution and refinement of
the structure. Equivalent reflections, other than Friedel pairs, were merged. Data collection and
refinement parameters are compiled in Table 3. A view of the molecule is depicted in Fig. 2.
The structure was solved by direct methods with SIR92 [42], which revealed the positions of all non-
H-atoms. There are two symmetry-independent molecules in the asymmetric unit. Both have the same
configuration and very similar conformations. The atomic coordinates of the two molecules were tested
carefully for a relationship from a higher-symmetry space group with the program PLATON [43], but
none could be found. The non-H-atoms were refined anisotropically. All of the H-atoms were placed in
geometrically calculated positions and refined with a riding model where each H-atom was assigned a
fixed isotropic displacement parameter with a value equal to 1.2Ueq of its parent atom. Refinement of the
structure was carried out on F2 by using full-matrix least-squares procedures, which minimized the
function Sw(F2o ꢀ Fc2 )2. The weighting scheme was based on counting statistics and included a factor to
downweight the intense reflections. Plots of Sw(F2o ꢀ Fc2 )2 vs. F/Fc(max) and resolution showed no
unusual trends. A correction for secondary extinction was applied. Refinement of the absolute structure
parameter [44] yielded a value of 0.04(8), which confidently confirms that the refined coordinates, as
listed in Table 3, represent the true enantiomorph. Neutral-atom-scattering factors for non-H-atoms were
taken from [45], and the scattering factors for H-atoms were taken from [46]. Anomalous dispersion
effects were included in Fc [47]; the values for f’ and f’’ were those of [48]. The values of the mass
attenuation coefficients were those of [49]. All calculations were performed with the SHELXL97
program [50], and the crystallographic diagram was drawn with ORTEPII [51].
6. Enzyme Kinetics. 6.1. General. Acetylcholinesterase (AChE) from Electrophorus electricus
(electric eel), Sigma C-2888, type V-S, lyophilized powder ꢄ 1000 units/mg protein; acetylthiocholine
(ATC), Fluka 01480 BioChemika; 5,5’-dithiobis[2-nitrobenzoic acid] (DTNB, Ellmans reagent [28]),
Fluka 43760 BioChemika; diisopropyl fluorophosphate (¼diisopropyl phosphorofluoridate; DFP),
Fluka 38399 purum; H2O, Fluka, 95304 (HPLC quality); MeCN, Fluka 00695 puriss., abs. (over molecular
sieves); NaCl, Fluka 71378 BioChemika MicroSelect; NaH2PO4 · H2O, Merck 6346 p.a.; Na2HPO4 · 2 H2O,
Merck 6580 p.a.; Pluronic F-68, Sigma P-1300 (lot 88 H1000); Tris buffer, Fluka 93349 BioChemika
MicroSelect. Volumes ꢅ 1 ml were measured accurately with microliter pipettes Socorex S þ (100 –
1000 ml) and Socorex Autoclavable Calibra 822 (10 – 100 ml, 1 – 10 ml), volumes < 1 ml by diluting in
volumetric flasks. pH Determinations: Knick Portamess 762 Calimatic; electrode, Mettler InLab 423 S7;
calibration, Mettler standard buffers pH 4.01 and pH 7.00; electrolyte, Mettler standard, 3m KCl sat. with
AgCl; measurements at 228, accuracy ꢁ 0.02 pH units. Enzyme kinetic progress curves and photometric
titration of the active sites of AChE: Hewlett-Packard-8452A diode-array spectrophotometer with HP
89532A UV-VIS software (revision A.00.00). Curve fitting and data analyses were performed with
OriginPro 7.5G SR3 v.7.5853 (www.originlab.com).
6.2. Phosphate Buffer pH 7.00. Soln. A: Na2HPO4 · 2 H2O (4.45 g), NaCl (1.46 g), and Pluronic F-68
(25 mg) were dissolved in H2O (250 ml). Soln. B: NaH2PO4 · H2O (6.90 g), NaCl (2.92 g), and Pluronic

764
Helvetica Chimica Acta – Vol. 94 (2011)
Table 3. Crystallographic Data of (þ)-15a
Crystallized from
Empirical formula
Mr
hexane/acetone
C13H17FNO3P
285.25
2qmax [8]
Transmission factors
(min; max)
50
0.788; 0.985
Crystal color, habit
Crystal dimensions
[mm]
Temperature [K]
Crystal system
Space group
Z
Reflections for
cell determination
2q Range for
cell determination [8]
Unit cell parameters:
a[ꢆ]
colorless, prism
Total reflections
20334
4866
0.10 ꢂ 0.15 ꢂ 0.28 measured
Symmetry-independent
reflections
Rint
160(1)
monoclinic
P21 (#4)
4
0.068
Reflections with I > 2s(I) 4201
Reflections used in
refinement
Parameters refined;
restraints
Final R(F) (I > 2s(I)
reflections)
wR(F2) (all data)
Weights
4866
28171
345; 1
0.0361
4 – 50
6.3402(3)
4.6194(7)
15.0597(6)
98.441(2)
1380.8(1)
600
0.0887
b[ꢆ]
c[ꢆ]
b[8]
w ¼ [s2(F2o ) þ (0.0431P)2 þ 0.1989P]ꢀ1
where P ¼ (F2o þ 2Fc2 )/3
1.047
Goodness of fit
Secondary extinction
coefficient
Final Dmax/s
D1 (max; min) [e ꢆꢀ3
s(d(CꢀC)) [ꢆ]
V [ꢆ3]
F(000)
0.015(2)
Dx [g cmꢀ3
]
1.372
0.214
0.001
m(MoKa) [mmꢀ1
]
]
0.17; ꢀ 0.20
Scan type
w
0.003 – 0.005
F-68 (25 mg) were dissolved in H2O (500 ml). The solns. were adjusted to pH 7.00 ꢁ 0.02 by mixing. Prior
to use, the buffer was filtered (TRP syringe filter, max. 0.5 MPa, PES-membrane 0.22 m, gamma-
sterilized, free of pyrogens).
6.3. AChE Solution. AChE (1 mg) was dissolved in the phosphate buffer pH 7.00 (1 ml), thoroughly
mixed, and stored at 58 (stock soln.). To the phosphate buffer (5 ml), AChE stock soln. (5 ml) was added
and thoroughly mixed. The soln. (1 ml) was diluted to 10 ml with phosphate buffer. Such a soln. could be
used for the determination of Km and one assay series.
6.4. ATC and DTNB Solutions. ATC (22.6 mg) was dissolved in phosphate buffer (1 ml; ! 78 mm)
and thoroughly mixed. The soln. was freshly prepared prior to use and stored at 08 (ice bath). DTNB
(15 mg) was dissolved in phosphate buffer (2 ml; ! 19 mm) and well mixed prior to use.
6.5. Inhibitor Solution (representative example). The respective 3-substituted 3-phosphadecalin
(3.5 mg) was dissolved in abs. MeCN (200 ml). From this stock soln., a dilution series was prepared to
yield five different concentrations. The total volume of each assay was 25 ml.
6.6. Determination of Km. Prior to each assay series, the Michaelis constant (Km) was determined
under the assay conditions (instead of an inhibitor, MeCN (25 ml) was added). The linear increase of the
absorption was monitored at 412 ꢁ 2 nm at five different concentrations of the substrate (ATC) ([S] ¼
[ATC] from 100 – 500 mm). The kobs values were treated according to zeroth-order kinetics (v ¼ const-
ant). Km was calculated from the resulting straight line of the plot 1/kobs ¼ f(1/[S]); Km was always in the
range of 160 mm.
6.7. Ellman-Assay [28]. In a polystyrene cell (4 ml, d ¼ 1 cm), phosphate buffer pH 7.00 (2 ml),
DTNB soln. (100 ml), and ATC soln. (20 ml) were mixed and thermostatted at 258. Then inhibitor soln. (x
ml, known [I], x ꢅ 25 ml), and MeCN ((25 ꢀ x) ml) were added. At t ¼ 0, the AChE soln. (1 ml) was added
and the mixture gently mixed for 5 s. After 10 s, the monitoring of the absorption at 412 ꢁ 2 nm (liberated
bis-anion of 5-mercapto-2-nitrobenzoic acid) automatically started, and 600 data points were collected

Helvetica Chimica Acta – Vol. 94 (2011)
765
for 10 min at various concentrations of the inhibitor. As in the Km determinations, the total volume was
3.15 ml and the concentration of the substrate [S] ¼ 497 mm. Per inhibitor, at least five measurements with
different inhibitor concentrations were performed, the smallest one being ca. 1/5 – 1/10 of the largest one.
The largest concentration sufficient for the determination of P(O)F(OiPr)2 (DFP) was ca. 160 mm.
6.8. Data Analysis [24]. The integrated rate equation describing product generation (monitored by
the absorbance A) and the apparent rate constants kobs is given by Eqn. 1. It is fitted (R > 0.999) to
progress curves recorded at fixed [S] and variable [I] (primary plot, A ¼ f(t)) to obtain a series of kobs
values and their standard errors (SE). The inhibition parameters are obtained from the secondary plots
(kobs ¼ f([I])) that result from weighted (SEꢀ2) linear or nonlinear regression according to Eqns. 2 or 3.
The analysis of these plots enables a differentiation between the inhibition mechanisms: kobs depends
linearly upon the inhibitor concentration for mechanism a and hyperbolically for mechanism b. For
mechanism a, the ka values are calculated according to Eqn. 2, its slope kobs/[I] is obtained from the linear
regression. The decisive plot for mechanism b is doubly reciprocal (1/kobs ¼ f(1/[I]), Eqn. 3), and the KD
and kp values are calculated by linear regression, the slope being (KD/kp)(1 þ [S]/Km) and the intercept 1/
kp. The overall inhibitory potency ki is expressed by kp/KD (see Scheme 6).
À
kobs
½Iꢇ
Á
vz
kobs
obs ꢆ t
A ¼
1 ꢀ eꢀk
(1)
(2)
(3)
ꢀ
ꢁꢀ
ꢁꢀ
ꢁ
½Sꢇ
ka ¼
1 þ
Km
ꢀ
ꢁꢀ
ꢁ
1
kobs
KD
kp
½Sꢇ
1
½Iꢇ
1
kp
¼
1 þ
ꢀ
Km
6.9. Results. For all eight stereoisomeric 3-fluoro-2,4-dioxa-9-aza-3-phosphadecalinoxides 14 and 15,
the secondary plot (kobs ¼ f([I])) exhibited a linear dependence. This behavior indicates that the
inhibition follows mechanism a for all compounds.
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Received December 16, 2010