Synthesis and anticholinesterase activity of some new fluorogenic analogues of organophosphorus nerve agents

Article abstract of DOI:10.1016/j.jfluchem.2006.07.017

Eighteen new fluorogenic analogues of organophosphorus nerve agents were synthesised and characterised. They included analogues of tabun, sarin, cyclosarin, soman, VX, and Russian VX, with the 7-oxy-4-methylcoumarin or 7-oxy-4-(trifluoromethyl)coumarin leaving group. These analogues inhibited acetylcholinesterase (AChE) effectively in vitro and therefore have potential as tools for the identification of novel organophosphatases in biological systems. Analogues of VX and Russian VX with the 7-amino-4-methylcoumarin group, although poor AChE inhibitors, may have utility for screening enzyme libraries for phosphoramidases capable of cleaving P-N bonds.

Full text of DOI:10.1016/j.jfluchem.2006.07.017

Journal of Fluorine Chemistry 127 (2006) 1554–1563
www.elsevier.com/locate/fluor
Synthesis and anticholinesterase activity of some new fluorogenic
analogues of organophosphorus nerve agents
*
Christopher M. Timperley , Krystal E. Casey, Stuart Notman, David J. Sellers,
Nancy E. Williams, Nichola H. Williams, Gareth R. Williams
Defence Science and Technology Laboratory, Porton Down, Salisbury, Wiltshire SP4 0JQ, UK
Received 1 May 2005; received in revised form 27 June 2006; accepted 24 July 2006
Available online 8 August 2006
Abstract
Eighteen new fluorogenic analogues of organophosphorus nerve agents were synthesised and characterised. They included analogues of tabun,
sarin, cyclosarin, soman, VX, and Russian VX, with the 7-oxy-4-methylcoumarin or 7-oxy-4-(trifluoromethyl)coumarin leaving group. These
analogues inhibited acetylcholinesterase (AChE) effectively in vitro and therefore have potential as tools for the identification of novel
organophosphatases in biological systems. Analogues of VX and Russian VX with the 7-amino-4-methylcoumarin group, although poor AChE
inhibitors, may have utility for screening enzyme libraries for phosphoramidases capable of cleaving P–N bonds.
# 2006 Elsevier B.V. All rights reserved.
Keywords: Acetylcholinesterase; Fluorogenic probes; 7-hydroxy-4-methylcoumarin; 7-hydroxy-4-(trifluoromethyl)coumarin; Organophosphorus nerve agent;
Phosphotriesterase
1. Introduction
regeneration of enzyme through attack by water is slow,
producing an irreversibly inhibited AChE unable to hydrolyse
acetylcholine.
Organophosphorus (OP) compounds include pesticides and
military nerve agents (NAs). The first NA, tabun, was
discovered in 1936 and refinement of its structure led to the
more toxic relatives, sarin, soman and cyclosarin (Scheme 1)
[1]. These compounds are volatile liquids that pose mainly a
vapour hazard. Two NAs with greater toxicity, VX and Russian
VX, were developed during the Cold War. They are low-
volatility liquids that pose mainly a skin contact hazard unless
released as an aerosol.
Quantifying turnover of molecules through biological
compartments such as enzymes is aided by the use of non-
fluorescent (fluorogenic) substrates that produce a fluorescent
motif after reaction with specific molecules [3]. Fluorogenic
substrates have been developed for targeting enzymes that can
hydrolyse organophosphorus compounds: coumarin phos-
phates1 for protein phosphatases [4–9] and 7-(diethylpho-
sphoryl)-6,8-difluoro-4-methylumbelliferone (DEPFMU) [10]
for anti-NA enzymes (Scheme 1). Hydrolysis of DEPFMU
produces 7-hydroxy-6,8-difluoro-4-methylcoumarin [10–12], a
blue fluorescent dye with good photophysical properties [13]
such as a high quantum yield (F = 0.89).2 DEPFMU has been
used to target human paraoxonases that might be useful in the
prophylaxis and treatment of organophosphorus insecticide and
NAs inhibit acetylcholinesterase (AChE), an enzyme which
controls nerve impulse transmission, by hydrolysing acetylcho-
line (a neurotransmitter) to acetic acid and choline [2]. The
active-site serine residue attacks the carbonyl carbon of
acetylcholine to form a covalent acetyl-enzyme intermediate.
The enzyme is regenerated after attack by water and release of
acetic acid. NAs mimic acetylcholine and phosphylate the
serine residue, losing either cyanide, fluoride or the N,N-
dialkylaminoethanethiolate group. This step is fast, but
1
Coumarin is the trivial name for 2H-chromen-2-one, sometimes called
umbelliferone as toxic derivatives were once isolated from plants of the family
Umbelliferae.
2
Quantum yield (F) is the probability of luminescence occurring, expressed
by the ratio of the number of photons emitted by the luminescing species to the
number of photons absorbed.
* Corresponding author. Tel.: +44 1980 613566; fax: +44 1980 613834.
E-mail address: cmtimperley@dstl.gov.uk (C.M. Timperley).
0022-1139/$ – see front matter # 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.jfluchem.2006.07.017

C.M. Timperley et al. / Journal of Fluorine Chemistry 127 (2006) 1554–1563
1555
Scheme 1. Structures of NAs, the pesticide paraoxon, and the fluorgenic probe
7-(diethylphosphoryl)-6,8-difluoro-4-methylumbelliferone (DEPFMU).
Fig. 1. Coumarin nerve agent analogues prepared previously [15].
nerve agent poisonings [14]. However, we believe it is too
dissimilar in structure to the NAs to allow optimal searching for
anti-NA enzymes.
These coumarins are commercially available and quantum
yields (F) and wavelengths of maximum absorption (abs) and
emission (em) are known (Fig. 2) [13,16].
To overcome these structural issues, we recently designed
NA analogues based on the pesticides coumaphos 1a and
coroxon 2a (Fig. 1) having the 3-chloro-7-oxy-4-methylcou-
marin leaving group (a) [15]. With the exception of tabun
analogue 3a which did not inhibit AChE, analogues 4a–8a were
0.7–34 times less potent inhibitors of AChE than the parent
NAs and retained the same relative inhibition potency (Russian
VX > cyclosarin > VX > sarin > soman). LD50 values for
The ability of the new NA analogues to inhibit bovine
erythrocycte AChE is also described. The aim was to assess
which new fluorogenic analogues were suitable for biological
applications involving fluorescence detection and to compare
the effect on AChE inhibition in changing from a CH3 to a
CF3 substituent in the coumarin leaving group. No studies
appear to have been published on the reactivity of AChE
towards organophosphorus substrates bearing fluorinated
groups remote from the phosphorus centre. The main effect
should be an increase in size and hydrophobicity in going
from a hydrocarbon to a fluorocarbon substituent, although
the possibility of the latter fluorine–hydrogen bonding with a
residue close to the active site cannot be ignored. As
fluorocarbon materials, with few exceptions, are man-made,
there has been no evolutionary pressure for many enzymes to
evolve specificity towards them, and therefore study of
their interaction with enzymes should reveal information
which will cast further light on the mechanism of enzyme
catalysis. The study described here was partly driven by a
curiosity to see how well a complex biochemical system
cyclosarin (0.06 mg kgꢀ1) and its analogue 8a (0.4 mg kgꢀ1
)
upon subcutaneous administration to guinea pigs displayed a
similar ratio [15]. The NA analogues 1a–8a were suitable
substrates for phosphotriesterase (PTE) from Pseudomonas
diminuta and a mammalian serum paraoxonase (PON1), and
catalytic efficiencies (kcat/KM) ranged across 108 Mꢀ1 sꢀ1. All
were hydrolysed by wild-type PTE. However, wild-type PON1
hydrolysed only the non-bulky analogues efficiently [15].
In this paper, we have extended the range of fluorogenic NA
analogues available for biological studies to include derivatives
of 7-hydroxy-4-methylcoumarin (b), 7-hydroxy-4-(trifluoro-
methyl)coumarin (c) and 7-amino-4-methylcoumarin (d).
Fig. 2. Fluorescent coumarin leaving groups.

1556
C.M. Timperley et al. / Journal of Fluorine Chemistry 127 (2006) 1554–1563
2. Results and discussion
2.1. 7-Hydroxycoumarins
Analogues 1b–8b and 1c–8c were prepared by treating the
respective phosphorus chlorides [15] with the sodium salts of
the hydroxycoumarins in tetrahydrofuran (Fig. 3). Conversion
usually exceeded 90% (as indicated by GC–MS analysis of
crude reaction mixtures) but isolated yields were low due to
decomposition during chromatography on silica gel; chroma-
tography was necessary to obtain the products in sufficient
purity (99%) for AChE inhibition studies.
First-orderratecoefficientsofAChEinhibition,obtainedfrom
a graph of log10 percentage of enzyme activity remaining versus
time (data not shown), were plotted against inhibitor concentra-
tion. Second-order rate coefficients of inhibition (ki) were then
calculated from the gradient of the best straight line produced.
Errorvaluesassociatedwiththekivalueswerecalculatedfromthe
deviationsfromthebeststraightlineinthegraphoffirst-orderrate
coefficient of inhibition versus inhibitor concentration (Table 1).
Studies by other researchers have shown that coumarin
phosphates have high affinity for AChEs from various animal
species [17,18], the phosphorus moiety binding to the active site
and the coumarin group binding to the peripheral anionic site
Fig. 3. New fluorogenic nerve agent analogues.
(AChE) could respond to the structural change 4-CH3 ! 4-
CF3 in the coumarin leaving group, and to see whether this
simple change might affect its ability to hydrolyse such
compounds.
Table 1
Second-order rate coefficients (ki) for inhibition of bovine erythrocyte AChE (pH 7.4, 37 8C)
Compound
Structure
ki (Mꢀ1 minꢀ1
)
Approximate
Pesticide or
nerve agent
ki (Mꢀ1 minꢀ1
pesticide or
nerve agenta
)
ki ratio
a:b
b:c
1
2
3
4
5
6
NA
4.0 ꢁ 0.5 ꢂ 103
2.3 ꢁ 0.5 ꢂ 105
1.2 ꢁ 0.2 ꢂ 103
8.7 ꢁ 0.7 ꢂ 104
2.0 ꢁ 0.5 ꢂ 104
1.7 ꢁ 0.2 ꢂ 106
1.0 ꢁ 0.2 ꢂ 102
1.6 ꢁ 0.3 ꢂ 103
1.1 ꢁ 0.5 ꢂ 103
13.3 ꢁ 0.4 ꢂ 104
2.9 ꢁ 0.3 ꢂ 104
6.1 ꢁ 0.9 ꢂ 105
–
40:1
144:1
1:1
Coumaphos
Coroxon
Tabun
NA
2.1 ꢁ 0.2 ꢂ 104
NA
1:11
–
2.1 ꢁ 0.2 ꢂ 104
3.2 ꢁ 0.1 ꢂ 105
6.6 ꢁ 2.2 ꢂ 106
7.4 ꢁ 1.3 ꢂ 105
2.7 ꢁ 0.6 ꢂ 107
2.2 ꢁ 0.2 ꢂ 105
2.2 ꢁ 0.4 ꢂ 104
2.1 ꢁ 0.5 ꢂ 106
3:1
1:1
1:1
1:2
VX
1:2
Sarin
3:1
Russian VX
7
5.7 ꢁ 1.2 ꢂ 104
1.8 ꢁ 0.1 ꢂ 106
2.3 ꢁ 0.5 ꢂ 105
3.2 ꢁ 0.1 ꢂ 106
4.8 ꢁ 0.7 ꢂ 104
6.3 ꢁ 0.7 ꢂ 105
1:4
1:2
5:1
5:1
Soman
4.1 ꢁ 1.0 ꢂ 104
1.2 ꢁ 1.0 ꢂ 107
8
Cyclosarin
a
Data taken from Ref. [15]. NA = no activity detected. X = leaving group (a), (b) or (c).

C.M. Timperley et al. / Journal of Fluorine Chemistry 127 (2006) 1554–1563
1557
[18,19]. We found that potencies with respect to different
substitution patterns at phosphorus followed the order expected
from consideration of stereoelectronic effects of the substituents
[15]. Thus, thiono analogues 1a–c were worse inhibitors than
their oxo counterparts 2a–c, and the tabun analogues 3a–c were
worseinhibitorsthantheVXanalogues4a–c (replacingMe2Nby
Me rendered the phosphorus atom more electropositive and less
hindered, and thus more responsive to nucleophilic attack).
Compounds with coumarin (a) were equal or less potent
inhibitorsthan thosewith coumarin (b), with theexceptionofVX
analogue 4a which was 2.5 times more potent than analogue 4b.
Compounds with coumarin (b) were also generally better
inhibitors than analogues with coumarin (c), perhaps due a poor
fit of the trifluoromethyl group in the peripheral site.
Considerable controversy surrounds the issue of the size of
the CF3 group. It is clearly larger than a CH3 group but how much
larger is a matter of debate and depends to an extent on the
method used to measure steric bulk. The CF3 group is estimated
from computational studies to approximate an ethyl group [20],
but Taft [21], Charton [22] and Meyer [23] calculations imply
that it is the size of the s-butyl group, i-butyl group, or
intermediate between a methyl and ethyl group, respectively.
Whereas coumaphos 1a and tabun analogue 3a did not inhibit
AChE, the corresponding analogues 1b and 1c, and 3b and 3c,
did inhibit the enzyme. The reason for this is unclear—the
leaving groups (a–c) might be expected to depart with equal
facility due to their electronic similarity. It is possible that a
chlorine atom in the 3-position of the coumarin ring exerts an
unfavourable steric effect that affects its fit in the peripheral site.
The most active inhibitors were the analogues of Russian
VX (6a–c) and cyclosarin (8a–c). Values of ki measured for
cyclosarin in AChEs from different animal species exceed those
measured for other well-known nerve agents [24–26]. The high
activity of cyclosarin and its coumarin derivatives towards
bovine AChE might be due to a good fit of the cyclohexyl group
into the active site [15].
nucleophilicity of the coumarin amino group, which rendered
coupling problematic, and partial decomposition of the
products during chromatography on silica gel. We were unable
to isolate the corresponding analogues of 7-amino-4-(trifluor-
omethyl)coumarin, an even less nucleophilic species.
Due to the poor leaving ability of the aminocoumarin group,
compounds 9 and 10 were poor inhibitors of AChE, and their ki
values were much lower than those found for their hydro-
xycoumarin counterparts 4b and 6b.
2.3. Discussion
Although analogues 1b–8b and 1c–8c could be used to
search for novel organophosphatases, their AChE ki values did
not mirror those found for the NAs (unlike the original
analogues 1a–8a whose ki values did). Because of this, and the
greater quantum yield of 7-hydroxy-3-chloro-4-methylcou-
marin compared to the other coumarins (Fig. 2), analogues 1a–
8a are perhaps the most useful for applications involving
detection of the fluorescent leaving group.
However, one compound in the new series – tabun analogue
3b – has promise. Unlike 3a, which was found to be a poor
substrate for AChE (no noticeable inhibition), analogue 3b was
found to be a moderately good inhibitor. It may therefore prove
useful for identifying anti-tabun enzymes. In addition,
aminocoumarins 9 and 10 are potentially useful for the
discovery of phosphoramidases capable of cleaving P–N bonds.
Althoughacutetoxicitydataarelacking,analogues1b–8band
1c–8c are likely to be less toxic than analogues 1a–8a and the
parent NAs. Comparative toxicity data are available only for
coroxon 2a and its analogue 2b whose oral LD50s in rats are 10
and 29 mg kgꢀ1 respectively [27]. In this case, replacing
coumarin (a) with coumarin (b) lowered toxicity threefold. As
the dermal toxicity of coumaphos is low compared with other
organophosphoruspesticides[28]andmuchlowerthanthatofthe
NAs, compounds 1b–8b and 1c–8c are not expected to have high
percutaneous toxicity. In terms of their ability to inhibit AChE,
they were generally much worse than the parent NAs (except
analogue 7b whose ki exceeded that of soman; refer to Table 3).
2.2. 7-Aminocoumarins
Aminocoumarin analogues of VX and Russian VX, 9 and
10, were prepared by treating the respective phosphorus
chlorides [15] with 7-amino-4-methylcoumarin, triethylamine
and a catalytic amount of 4-dimethylaminopyridine (DMAP) in
tetrahydrofuran (Fig. 4). Yields were low due to the poor
3. Conclusions
The NA analogues could be used to screen for hydrolytic
enzymes in single cells and in cell arrays using electropora-
tion [29]. Image-based methods could be used to quantify a
large number of wells in a single detection procedure [30].
Large libraries of >107 mutant PTEs or PONs expressed in
bacterial cells can be screened by encapsulating single cells
in microdroplets in water-in-oil-in-water double emulsions
and sorting the droplets [31] using fluorescence activated cell
sorting (FACS).3 Large libraries can also be screened by in
3
Fluorescence activated cell sorting (FACS) is a flow cytometry application
in which live fluorescent cells are excited at a specific wavelength and then
sorted into physically separated sub-populations on the basis of their fluores-
cence emission.
Fig. 4. New fluorogenic nerve agent analogues with an aminocoumarin sub-
stituent.

Table 2
Physical and spectroscopic data for the 7-oxy-4-methylcoumaryl analogues 1b–8b (NMR data measured in CDCl3)
Compound
Structure
1H NMR d, J (Hz)
13C NMR d, J (Hz)
31P NMR, d
1b (mp 35–36 8C)
7.58 (1H, d, J 8.5, 5-H), 7.19 (1H, t, J 1.8, 8-H),
7.12–7.18 (1H, m, 6-H), 6.26 (1H, q, J 1.2, 3-H),
4.27 (4H, dq, J 10.1 and 7.0, 2ꢂ OCH2), 2.43
(3H, d, J 1.2, 4-CH3), 1.39 (6H, dt, J 7.1
and 0.8, 2ꢂ CH3)
160.5 (s, C O), 154.3 (s, 9-C), 153.2 (d, J 7.5,
7-C), 151.9 (s, 4-C), 125.5 (s, 5-C), 117.5 (d, J 5.2,
6-C), 117.3 (s, 10-C), 114.3 (s, 3-C), 109.7
(d, J 5.2, 8-C), 65.4 (d, J 5.8, 2ꢂ OCH2),
18.7 (s, 4-CH3), 15.9 (d, J 7.5, 2ꢂ CH3)
+63.2
2b (liquid)
7.61 (1H, d, J 8.3, 5-H), 7.17–7.28 (2H, m, 6-H
and 8-H), 6.25 (1H, q, J 1.2, 3-H), 4.35–4.20 (4H, m,
2ꢂ OCH2), 2.45 (3H, d, J 1.2, 4-CH3), 1.39 (6H, td,
J 7.1 and 1.0, 2ꢂ CH3)
160.2 (s, C O), 154.1 (s, 9-C), 152.9
(d, J 6.3, 7-C), 151.8 (s, 4-C), 125.7
(s, 5-C), 116.9 (s, 10-C), 116.2 (d, J 5.2,
6-C), 113.8 (s, 3-C), 108.2 (d, J 5.8, 8-C),
64.8 (d, J 6.3, 2ꢂ OCH2), 18.4 (s, 4-CH3),
15.8 (d, J 6.9, 2ꢂ CH3)
160.7 (s, C O), 154.5 (s, 9-C), 153.9 (d,
J 6.2, 7-C), 152.2 (s, 4-C), 125.8 (s, 5-C),
116.8 (s, 10-C), 116.7 (d, J 4.8, 6-C),
113.9 (s, 3-C), 108.6 (d, J 5.8, 7-C), 63.3
(d, J 5.8, OCH2), 36.7 (d, J 3.8, N(CH3)2),
18.8 (s, 4-CH3), 16.2 (d, J 6.7, CH3)
ꢀ6.3
+6.6
3b (mp 80–81 8C)
7.57 (1H, d, J 8.7, 5-H), 7.27–7.20 (1H, m, 6-H), 7.18
(1H, s, 8-H), 6.23 (1H, s, 3-H), 4.24–4.13 (2H, m, OCH2),
2.77 (6H, d, J 10.3, N(CH3)2), 2.43 (3H, s, 4-CH3),
1.38 (3H, t, J 7.0, CH3)
4b (mp 82–83 8C)
7.58 (1H, d, J 8.5, 5-H), 7.26–7.20 (1H, m, 6-H), 7.21–7.17
(1H, m, 8-H), 6.25 (1H, q, J 1.2, 3-H), 4.34–4.11 (2H, m,
OCH2), 2.43 (3H, d, J 1.2, 4-CH3), 1.69 (3H, d, J 17.6, P-CH3),
1.35 (3H, t, J 7.1, CH3)
160.4 (s, C O), 154.4 (s, 9-C), 153.0
(d, J 8.1, 7-C), 151.8 (s, 4-C), 125.8
(s, 5-C), 117.0 (s, 10-C), 116.9 (d,
J 4.6, 6-C), 114.0 (s, 3-C), 108.9 (d,
J 5.2, 8-C), 62.8 (d, J 6.9, OCH2), 18.6
(s, 4-CH3), 16.3 (d, J 6.3, CH3), 11.5
(s, J 145.4, P-CH3)
+28.7
5b (mp 43–44 8C)
7.58 (1H, d, J 8.6, 5-H), 7.24 (1H, ddd, J 8.6, 2.4 and 1.2, 6-H),
7.20 (1H, dd, J 2.4 and 1.3, 8-H), 6.25 (1H, q, J 1.3, 3-H),
4.93–4.76 (1H, m, OCH), 2.43 (3H, d, J 1.3, 4-CH3), 1.67
(3H, d, J 17.6, P-CH3), 1.38 (3H, d, J 6.2, CH3),
1.31 (3H, d, J 6.2, CH3)
160.6 (s, C O), 154.5 (s, 9-C), 153.2
(d, J 8.1, 7-C), 152.0 (s, 4-C), 125.9
(s, 5-C), 117.1 (d, J 4.6, 6-C), 117.1
(s, 10-C), 114.1 (s, 3-C), 109.1 (d,
J 4.6, 8-C), 72.1 (d, J 6.9, CH), 24.1
(d, J 4.6, CH3), 23.9 (d, J 4.6, CH3),
18.8 (s, 4-CH3), 12.3 (d, J 145.4, P-CH3)
+27.6
6b (liquid)
7.52–7.60 (2H, m, 2ꢂ 5-H), 7.15–7.31 (4H, m, 2ꢂ [6-H and 8-H]),
6.25 (2H, br. s, 2ꢂ 3-H), 4.49–4.28 (2H, m, 2ꢂ OCH), 2.43
(6H, s, 2ꢂ 4-CH3), 1.68 (3H, d, J 17.6, P-CH3), 1.67 (3H, d,
J 17.6, P-CH3), 1.35 (3H, d, J 6.3, CH3), 1.19 (3H, d, J 6.5, CH3),
0.94 (9H, s, C(CH3)3), 0.90 (9H, s, C(CH3)3)
160.5 (s, 2ꢂ C O), 154.4 (s, 2ꢂ 9-C), 153.2
(d, J 8.1, 7-C), 153.2 (d, J 7.5, 7-C), 151.9
(s, 2ꢂ 4-C), 125.7 (s, 2ꢂ 5-C), 117.2 (d, J 4.0,
6-C), 117.0 (s, 2ꢂ 10-C), 116.9 (d, J 5.2, 6-C),
114.1 (s, 3-C), 114.0 (s, 3-C), 109.1 (d, J 5.2, 8-C),
109.0 (d, J 5.2, 8-C), 83.0 (d, J 7.5, OCH), 82.6
(d, J 7.5, OCH), 35.0 (d, J 6.9, 2ꢂ C(CH3)3), 25.6
(s, C(CH3)3), 25.5 (s, C(CH3)3), 18.7 (s, 2ꢂ 4-CH3),
17.0 (s, 2ꢂ CH3), 12.7 (d, J 146.0, P-CH3), 11.8
(s, d, J 147.1, P-CH3)
28.3/27.4a

C.M. Timperley et al. / Journal of Fluorine Chemistry 127 (2006) 1554–1563
1559
vitro compartmentalization (IVC) [32] where single genes are
transcribed and translated in vitro in aqueous microdroplets
in double emulsions, which are sorted using FACS to select
genes encoding enzymes with improved catalytic activity
[33]. The NA analogues may also be applicable to high-
throughput screening of novel biological hydrolytic enzymes
or catalysts using conventional techniques or microfluidic
devices [34].
4. Experimental
4.1. Materials and analytical equipment
7-Hydroxy-4-methylcoumarin was obtained from Lancaster
Synthesis (Morecambe, UK), 7-hydroxy-4-(trifluoromethyl)-
coumarin from Apollo Scientific (Stockport, UK) and 7-amino-
4-methylcoumarin and other reagents from Sigma–Aldrich
(Gillingham, UK). Triethylamine was distilled from CaH2 and
stored over CaH2. Anhydrous solvents were used: tetrahy-
drofuran (THF) was distilled from sodium/benzophenone.
Organophosphorus precursors were prepared as described
previously [15]. Thin layer chromatography was performed on
˚
MK6F silica gel 60 A plates (Whatman, USA) with detection
by UV light (l = 254 nm). Sorbsil C30 40/60 silica was used for
column chromatography. Melting points were determined on an
Electrothermal apparatus and are uncorrected. NMR spectra
were obtained on a JEOL Lambda 500 instrument (operating at
500 MHz for 1H, 125 MHz for 13C, and 202 MHz for 31P
spectra) or a JEOL Lambda 300 instrument (operating at
1
300 MHz for H, 75 MHz for 13C, and 121.5 MHz for 31P
spectra) as solutions in CDCl3, with internal reference SiMe4
1
for H and 13C, and external (MeO)3P (d 141.3 ppm) for 31P
spectra.
Safety warning. The compounds described have signifi-
cant anticholinesterase activity and must be handled
with care in an efficient fume cupboard. They must not be
allowed to come into contact with skin. Rubber gloves
must be worn and glassware decontaminated in aqueous
bleach.
4.2. LC–MS and LC–MSn experiments
4.2.1. Material
Ammonium formate was purchased from Fluka (Gilling-
ham, UK) and HPLC grade methanol from Aldrich (Gilling-
ham, UK). Water from a Milli-Q system (Millipore, UK) was
used for dilutions and the mobile phase.
4.2.2. LC conditions
A Thermo Finnigan Surveyor LC system was fitted with a
˚
C18 column (150 mm ꢂ 2 mm i.d., 5 mm, 100 A) from
Columbus (Phenomenex, Macclesfield, UK). A linear gradient
elution was performed at a flow rate of 0.2 ml minꢀ1, with a
mobile phase of 20 mM ammonium formate in water (solvent
A) and 20 mM ammonium formate in methanol (solvent B).
The gradient was 5% B (0–5 min), 5–90% B (5–10 min), held at
90% B (10–15 min), returned to the initial composition 5% B

Table 3
Physical and spectroscopic data for the 7-oxy-4-(trifluoromethyl)coumarin analogues 1c–8c (NMR data measured in CDCl3)
Compound
Structurea
1H NMR d, J (Hz)
13C NMR d, J (Hz)
31P NMR d
1c (mp 57–58 8C)
7.69–7.77 (1H, m, 5-H), 7.28 (1H, dd, J 2.3 and
1.6, 8-H), 7.22 (1H, ddd, J 8.9, 2.3 and 1.3, 6-H),
6.76 (1H, s, 3-H), 4.28 (4H, dq, J 10.1 and 7.1,
2ꢂ OCH2), 1.40 (6H, td, J 7.1 and 1.0, 2ꢂ CH3)
158.5 (s, C O), 155.1 (s, 9-C), 154.1 (d, J 6.9, 7-C),
141.1 (q, J 33.1, 4-C), 126.4 (s, 5-C), 121.4 (q, J 275.8,
CF3), 118.4 (d, J 5.2, 6-C), 114.9 (q, J 5.8, 3-C), 110.7
(s, 10-C), 110.1 (d, J 5.2, 8-C), 65.6 (d, J 5.8, 2ꢂ OCH2),
15.9 (d, J 7.5, 2ꢂ CH3)
+62.8
2c (liquid)
7.68–7.76 (1H, m, 5-H), 7.33–7.23 (2H, m, 6-H
and 8-H), 6.76 (1H, s, 3-H), 4.35–4.19 (4H, m,
2ꢂ OCH2) and 1.40 (6H, td, J 7.1 and 1.1, 2ꢂ CH3)
158.5 (s, C O), 154.2 (d, J 5.2, 7-C),
ꢀ6.4
141.1 (q, J 33.0, 4-C), 126.7 (s, 5-C),
121.4 (q, J 275.7, CF3), 117.4 (d, J 4.6, 6-C),
114.9 (q, J 4.6, 3-C), 109.1 (d, J 4.0, 8-C), 65.2
(d, J 5.2, 2ꢂ OCH2), 16.1 (d, J 5.8, 2ꢂ CH3)
3c (mp 37–38 8C)
7.70 (1H, dd, J 8.7 and 1.7, 5-H), 7.24–7.31 (2H, m, 6-H
and 8-H), 6.74 (1H, s, 3-H), 4.25–4.13 (2H, m, OCH2),
2.78 (6H, d, J 10.4, N(CH3)2), 1.39 (3H, td, J 7.1
and 0.9, CH3)
158.8 (s, C O), 155.5 (s, 9-C), 155.0 (d, J 6.7,
7-C), 141.3 (q, J 33.1, 4-C), 126.6 (s, 5-C),
121.5 (q, J 275.9, CF3), 117.6 (d, J 5.3, 6-C),
114.6 (q, J 5.4, 3-C), 110.2 (s, 10-C), 109.2
(d, J 5.3, 8-C), 63.5 (d, J 5.8, OCH2), 36.7
(d, J 3.8, N(CH3)2), 16.2 (d, J 6.7, CH3)
+6.6
4c (mp 68–69 8C)
7.71 (1H, dd, J 8.7 and 1.8, 5-H), 7.34–7.30 (1H, m, 8-H), 158.5 (s, C O), 155.4 (s, 9-C), 154.1 (d, J 8.1, 7-C),
+29.0
7.25–3.32 (1H, m, 6-H), 6.75 (1H, s, 3-H), 4.37–4.10
(2H, m, OCH2), 1.71 (3H, d, J 17.7, P-CH3),
1.36 (3H, t, J 7.1, CH3)
141.1 (q, J 33.0, 4-C), 126.7 (s, 5-C), 117.9 (d, J 4.6,
6-C), 121.4 (q, J 275.8, CF3), 114.8 (q, J 5.8, 3-C),
110.5 (s, 10-C), 109.5 (d, J 5.2, 8-C), 63.0 (d, J 6.3,
OCH2), 16.3 (d, J 6.3, CH3), 11.7 (d, J 144.8, P-CH3)
5c (liquid)
7.70 (1H, dd, J 8.6 and 1.7, 5-H), 7.33–7.28 (1H, m, 8-H), 158.7 (s, C O), 155.5 (s, 9-C), 154.3 (d, J 7.5, 7-C), 141.3
+27.9
7.24–7.33 (1H, m, 6-H), 6.74 (1H, s, 3-H), 4.74–4.92
(1H, m, OCH), 1.68 (3H, d, J 17.7, P-CH3), 1.38 (3H, d,
J 6.2, CH3), 1.31 (3H, d, J 6.2, CH3)
(q, J 33.5, 4-C), 126.8 (s, 5-C), 121.5 (q, J 276, CF3), 118.0
(d, J 4.6, 6-C), 114.9 (q, J 5.8, 3-C), 110.5 (s, 10-C), 109.7
(d, J 4.6, 8-C), 72.4 (d, J 6.3, OCH), 24.1 (d, J 5.2, CH3),
24.0 (d, J 4.0, CH3), 12.5 (d, J 146.0, P-CH3)
6c (liquid)
7.66–7.75 (2H, m, 2ꢂ 5-H), 7.34–7.31 (2H,
158.6 (s, 2ꢂ C O), 155.4 (s, 2ꢂ 9-C), 154.5 (d, J 7.5, 7-C),
154.3 (d, J 8.1, 7-C), 141.2 (q, J 33.3, 2ꢂ 4-C), 126.6 (s,
2ꢂ 5-C), 118.0 (d, J 4.6, 6-C), 117.9 (d, J 4.6, 6-C), 121.4
(q, J 275.4, 2ꢂ CF3), 114.8 (q, J 5.5, 3-C), 114.7 (q, J 5.5, 3-C),
110.4 (s, 10-C), 110.3 (s, 10-C), 109.7 (d, J 4.6, 8-C), 109.5
(d, J 5.2, 8-C), 83.3 (d, J 7.5, OCH), 82.8 (d, J 8.1, OCH),
35.0 (s, C(CH3)3), 34.9 (s, C(CH3)3), 25.5 (s, 2ꢂ C(CH3)3),
17.0 (s, 2ꢂ CH3), 12.8 (d, J 146.0, P-CH3), 11.9 (d, J 147.1,
P-CH3)
+28.5 and 28.4b
m, 2ꢂ 8-H), 7.35–7.27 (2H, m, 2ꢂ 6-H), 6.75
(2H, s, 2ꢂ 3-H), 2ꢂ 4.49–4.32 (2H, m, 2ꢂ OCH), 1.70
(3H, d, J 17.6, P-CH3), 1.69 (3H, d, J 17.6, P-CH3),
1.35 (3H, d, J 6.3, CH3), 1.21 (3H, d, J 6.5, CH3), 0.95
(9H, s, C(CH3)3), 0.90 (9H, s, C(CH3)3)

C.M. Timperley et al. / Journal of Fluorine Chemistry 127 (2006) 1554–1563
1561
(15–20 min) and then equilibrated at 5% B (20–25 min).
Samples were dissolved in methanol and injected (10 ml) at a
concentration of 10 mg mlꢀ1
.
4.2.3. MS conditions
MS analysis was performed using a Finnigan Deca XP+
ion trap MS system fitted with an atmospheric pressure
ionization (API) interface, operated in positive APCI mode.
The discharge current was set at 2 mA, capillary temperature
at 150 8C and vaporizer at 400 8C. The sheath gas flow was
maintained at 60 (arbitrary units) and the auxiliary gas at 20
(arbitrary units). Mass spectra were collected over a scan
range of m/z 50–500 with source CID set between 15 and
20 V. MS2 product ion spectra of protonated molecular ions
(ꢁ2 m/z) were obtained with the ion trap collision energy set
between 25 and 35%, activation Q at 0.25, and the activation
time at 60 ms.
4.3. Measurement of AChE ki values
4.3.1. Materials
Bovine erythrocyte AChE (EC 3.1.1.7) and acetylcholine
iodide (AChI) were obtained from Sigma Chemicals (Dorset,
UK) and standard solutions of NaOH from BDH Laboratory
Supplies (Leicester, UK). Deionised water was used to make
the solutions and for washing the reaction vessel, electrode,
stirrer and delivery tubes.
4.3.2. Apparatus
Inhibition experiments were performed at 37 8C and pH 7.4
using a Metrohm automatic titration apparatus to pH-stat the
reaction. The apparatus comprised a 713 pH Meter, a 614
Impulsomat and two 765 Dosimats (one containing 10 mM
NaOH solution to maintain the pH at 7.4, the other containing
10 mM AChI solution to maintain substrate concentration). The
reaction was monitored using a Kipp and Zonen y ꢀ t pen
recorder. A Haake DC10 was used to control the water bath
temperature and circulate water (at 37 8C) to the jacket
surrounding the reaction vessel.
4.3.3. Method
AChE was dissolved in 0.1 M NaCl-containing pH 7.4
phosphate buffer (5 ꢂ 10ꢀ3 M) to a specific activity of
5 mM units mlꢀ1. The solution was stored in the refrigerator
when not in use. AChI solutions (100 and 10 mM) and
standard NaOH were made up and used throughout the week;
they were refrigerated when not in use. Experiments were
performed at 37 8C in water with an ionic strength of
100 mM in NaCl. The reaction vessel initially contained
5 ml NaCl solution (0.1 M) and 0.5 ml AChE solution
(5 mM units mlꢀ1). AChI (0.10 ml of 10ꢀ1 M) was then
added, giving a total AChI concentration of 1.8 ꢂ 10ꢀ3 M.
The pH-stat was set to maintain the reaction at pH 7.4 by
adding NaOH solution (0.01 M). The rate of addition was
monitored on a y ꢀ t pen recorder. AChI solution (10ꢀ2 M)
was added at the same rate to maintain the substrate concen-
tration. This process was allowed to run for approximately

1562
C.M. Timperley et al. / Journal of Fluorine Chemistry 127 (2006) 1554–1563
Table 4
LC–MS data for R1R2P(X)O-coumarins 1b–8b and 1c–8c and R1R2P(X)NH-coumarins 9 and 10, including m/z ratios with relative abundance in brackets
Compound
R1
R2
X
S
MW
[MH]+
[M + NH4]+
[MH ꢀ R]+
301 (MS2 only)
[MH ꢀ O2R1R2PX]+
–
1ba (RT 19.44 ꢁ 0.05)
EtO
EtO
328
329 (100), 330 (16),
331 (6), 332 (1)
346 (9), 347 (1)
2b (RT 17.55 ꢁ 0.05)
3b (RT 17.48 ꢁ 0.05)
4b (RT 16.62 ꢁ 0.05)
5b (RT 17.29 ꢁ 0.05)
6b (RT 19.02 ꢁ 0.05)
7b (RT 18.04 ꢁ 0.05)
8b (RT 18.77 ꢁ 0.05)
1ca (RT 21.32 ꢁ 0.05)
EtO
EtO
Me2N
Me
O
O
O
O
O
O
O
S
312
311
282
296
338
310
336
382
313 (100), 314 (15)
312 (100), 313 (15)
283 (100), 284 (14)
297 (100), 298 (15)
339 (100), 340 (18)
311 (100), 312 (14)
337 (100), 338 (17)
383 (65), 384 (9),
385 (4), 386 (1)
330 (19), 331 (2)
329 (23), 330 (3)
300 (41), 301 (6)
314 (37), 315 (5)
356 (42), 357 (6)
328 (27), 329 (4)
354 (32), 355 (5)
400 (100), 401 (13)
285 (MS2 only)
284 (MS2 only)
255 (MS2 only)
255 (3)
177 (2)
EtO
177 (1)
EtO
177 (6), 178 (1)
177 (7), 178 (1)
177 (11), 178 (1)
177 (3), 178 (1)
177 (8), 178 (1)
–
i-PrO
PinO
i-BuO
CyO
EtO
Me
Me
255 (17), 256 (2)
255 (1)
Me
Me
255 (8), 256 (1)
355 (MS2 only)
EtO
2c (RT 18.83 ꢁ 0.05)
3c (RT 18.88 ꢁ 0.05)
4c (RT 18.12 ꢁ 0.05)
5c (RT 18.69 ꢁ 0.05)
6c (RT 20.34 ꢁ 0.05)
7c (RT 19.34 ꢁ 0.05)
8c (RT 20.11 ꢁ 0.05)
9 (RT 16.20 ꢁ 0.05)
10 (RT 17.65 ꢁ 0.05)
EtO
EtO
Me2N
Me
O
O
O
O
O
O
O
O
O
366
365
336
350
392
364
390
281
309
367 (57), 368 (8)
366 (74), 367 (10)
337 (32), 338 (9)
351 (100), 352 (14)
393 (11), 394 (2)
365 (44), 366 (6)
391 (39), 392 (7)
282 (100), 283 (14)
310 (100), 311 (17)
384 (100), 385 (14)
383 (100), 384 (13)
354 (100), 355 (12)
368 (61), 369 (8)
410 (100), 411 (14)
382 (100), 383 (12)
408 (100), 409 (14)
299 (72), 300 (8)
327 (64), 328 (8)
339 (MS2 only)
338 (MS2 only)
309 (MS2 only)
309 (13), 310 (2)
309 (14), 310 (2)
309 (2)
–
EtO
–
EtO
–
i-PrO
PinO
i-BuO
CyO
EtO
Me
–
Me
–
Me
–
Me
309 (5), 310 (1)
254 (MS2 only)
254 (MS2 only)
–
Me
176 (15), 177 (2)
176 (24), 177 (3)
i-BuO
Me
RT = retention time in min; Pin = pinacolyl; 12C and 13C isotopes of major m/z are given; relative abundances <1 not included; fragments only observed in MS2 are
marked (MS2 only); – denotes no fragment detected; Denotes sulfur-containing compound (32S and 34S isotopes given).
a
1 min, after which the appropriate phosphorus compound in
solution (0.01–0.1 ml)4 was added. Four different inhibitor
concentrations were investigated.
4.5. O-Ethyl N-(4-methyl-2-oxo-2H-chromen-7-
yl)methylphosphonamidate (9)
A solution of 7-amino-4-methylcoumarin (0.5 g, 2.85 mmol)
andtriethylamine(0.4 ml, 2.86 mmol)inTHF(50 ml)wasadded
dropwise to a stirred solution of ethyl methylphosphonochlor-
idate (0.41 g, 2.86 mmol) in THF (10 ml). A catalytic amount of
DMAP (50 mg) was added and the mixture was heated under
reflux for 7 h under argon. The mixture was allowed to cool to
room temperature. It was concentrated, dissolved in CH2Cl2
(100 ml) and washed with aqueous NaHCO3 (20 ml, 10%, w/v).
The organic layer was separated and dried (MgSO4). Filtration of
the drying agent and evaporation of solvent from the filtrate gave
the crude product. Chromatography on silica gel, eluting with
98:2 then 95:5 CH2Cl2–isopropanol, gave thetitlecompoundas a
glassysyrup(0.2 g, 25%). dH (300 MHz) 7.48 (1H, d, 4JHH 8.5, 8-
H), 6.98–6.92(3H, m, NH, 5-Hand6-H), 6.15(1H,q, 4JHH 1.2, 3-
4.4. Synthesis of analogues (1b–8b) and (1c–8c)
A
solution of the appropriate 7-hydroxycoumarin
(10 mmol) in THF (30 ml) was added via cannula to a stirred
suspension of sodium hydride (0.48 g, 20 mmol) in THF
(20 ml). After addition, the mixture was heated under reflux
for 30 min and then allowed to cool to room temperature. A
solution of the appropriate phosphorus chloride (10 mmol) in
THF (5 ml) was added dropwise via cannula to the 7-
hydroxycoumarin sodium salt suspension stirred at 0–5 8C.
After 12 h, chloroform (55 ml) and water (55 ml) were added.
The mixture was stirred and the two phases separated. The
organic layer was washed with aqueous K2CO3 solution
(55 ml, 0.15 mol dmꢀ3), separated and dried (MgSO4). The
drying agent was filtered off and the filtrate concentrated.
Column chromatography on silica gel, eluting with 1:1
hexane–ethyl acetate (7:3 hexane–ethyl acetate in the case of
7c), gave the fluorogenic analogues in 98% purity as
colourless liquids or white solids. Products were characterised
by multinuclear NMR spectroscopy (Tables 2 and 3) and by
LC–MSn experiments (Table 4).
3
H), 4.21 (1H, ddq, JPH 10.1, 2JHH 7.7 and 3JHH 7.2, CHAHB),
4.01 (1H, ddq, 3JPH 10.1, 2JHH 7.7 and 3JHH 7.2, CHAHB), 2.40
(3H, d, 4JHH 1.2, 4-CH3), 1.70 (3H, d, 2JHP 16.9, P-CH3), 1.36
(3H, t, 3JHH 7.2, CH3). dC (75 MHz) 161.2 (C O), 155.1 (9-C),
152.4 (4-C), 144.3 (6-C), 125.9 (8-C), 114.0 (d, 2JPC 6.9, 7-C),
113.9 (10-C), 112.2 (3-C), 103.8 (d, 4JPC 5.2, 5-C), 60.6 (d, 2JPC
6.9, OCH2), 18.6 (4-CH3), 16.1 (d, 3JPC 6.9, CH3), 12.5 (d, 1JPC
133.2, P-CH3). dP 28.2. CI-MS m/z (rel. abundance) = 282
(M + 1, 60%).
4.6. O-Isobutyl N-(4-methyl-2-oxo-2H-chromen-7-yl)
methylphosphonamidate (10)
4
The compounds investigated are solids that are not very soluble in propan-
A
solution of 7-amino-4-methylcoumarin (0.43 g,
2-ol, the solvent usually used in this procedure. Approximately 50% of the final
solution volume of acetone was used to dissolve the material, which was then
made up using either ethanol or propan-2-ol.
2.45 mmol) and triethylamine (0.38 ml, 2.76 mmol) in THF
(60 ml) was added dropwise to a stirred solution of isobutyl

C.M. Timperley et al. / Journal of Fluorine Chemistry 127 (2006) 1554–1563
1563
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(10 ml). A catalytic amount of DMAP (30 mg) was added
and the mixture was heated under reflux for 9 h under argon.
The mixture was allowed to cool to room temperature. It was
diluted with water, extracted with CH2Cl2 (100 ml) and the
organic extracts were combined and dried (MgSO4). Filtration
and concentration of the filtrate gave crude product (85% pure
by 31P NMR and GC–MS). Chromatography on silica gel,
eluting with ethyl acetate, gave the title compound as a glassy
syrup (0.22 g, 29%). dH (500 MHz) 8.09 (1H, d, 2JPH 5.7, NH),
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6.14 (1H, q, 4JHH 1.2, 3-H), 3.93 (1H, dt, 3JHP 9.7 and 3JHH 6.7,
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2JHP 16.8, P-CH3), 0.96 (3H, d, JHH 6.7, CH3), 0.95 (3H, d,
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(4-C), 144.7 (6-C), 125.6 (8-C), 114.0 (d, 2JPC 7.7, 7-C), 113.6
(10-C), 111.9 (3-C), 103.6 (d, 4JPC 4.8, 5-C), 70.2 (d, 2JPC 6.7,
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Acknowledgements
The authors thank the UK Ministry of Defence for funding
this work and Andrew Griffiths and Luis Brisen˜o-Roa for
interest in the applications of the fluorogenic NA analogues. #
Crown copyright 2006 by permission of HMSO Controller of
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