Synthesis and anti-acetylcholinesterase properties of novel β- And γ-substituted alkoxy organophosphonates

Article abstract of DOI:10.1016/j.bmcl.2013.02.010

Activated organophosphate (OP) insecticides and chemical agents inhibit acetylcholinesterase (AChE) to form OP-AChE adducts. Whereas the structure of the OP correlates with the rate of inhibition, the structure of the OP-AChE adduct influences the rate at

Full text of DOI:10.1016/j.bmcl.2013.02.010

Bioorganic & Medicinal Chemistry Letters 23 (2013) 2048–2051
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and anti-acetylcholinesterase properties of novel
b- and c-substituted alkoxy organophosphonates
S. Kaleem Ahmed, Yamina Belabassi, Lakshmi Sankaranarayanan, Chih-Kai Chao, John M. Gerdes,
⇑
Charles M. Thompson
Department of Biomedical and Pharmaceutical Sciences, The University of Montana, Missoula, MT 59812, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Activated organophosphate (OP) insecticides and chemical agents inhibit acetylcholinesterase (AChE) to
form OP-AChE adducts. Whereas the structure of the OP correlates with the rate of inhibition, the struc-
ture of the OP-AChE adduct influences the rate at which post-inhibitory reactivation or aging phenomena
Received 18 November 2012
Revised 21 January 2013
Accepted 1 February 2013
Available online 13 February 2013
occurs. In this report, we prepared a panel of b-substituted ethoxy and c-substituted propoxy phospho-
noesters of the type p-NO2PhO-P(X)(R)[(O(CH2)nZ] (R = Me, Et; X = O, S; n = 2, 3; Z = halogen, OTs) and
examined the inhibition of three AChEs by select structures in the panel. The b-fluoroethoxy methylphos-
phonate analog (R = Me, Z = F, n = 2) was the most potent anti-AChE compound comparable (ki
ꢀ6 Â 106 MÀ1 minÀ1) to paraoxon against EEAChE. Analogs with Z = Br, I, or OTs were weak inhibitors
of the AChEs, and methyl phosphonates (R = Me) were more potent than the corresponding ethyl phos-
phonates (R = Et). As expected, analogs with a thionate linkage (P@S) were poor inhibitors of the AChEs.
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
Organophosphates
Inhibition
Electric eel AChE
Recombinant human AChE
Rat brain AChE
Organophosphate (OP) compounds (e.g., diazinon, parathion;
Fig. 1; P@S) have been widely employed as insecticides in which
their mode of toxic action first requires conversion to the more
reactive P@O (oxon) form.1,2 Conversely, OP compounds that con-
tain the oxon functional group and possess a leaving group that is
readily stabilized upon release (e.g., FÀ, CNÀ) are readily reactive
and define the properties present in nerve agents like VX, sarin
(GB) and soman (GD) (Fig. 1).3,4
t
ꢀ2–10 h. Conversely, OP-AChE adducts containing branched
½
alkoxy
groups
like
sarin
(R = CH(CH3)2)
or
soman
(R = CH(CH3)(C(CH3)3) undergo aging to the methyl phosphonate
anion (Y = CH3) in 3–6 h and 2–5 min, respectively, affording little
chance for the OP-adducts to reactivate.8
When the OP-ester component attached to AChE is a small, un-
branched alkoxy group (R = CH2CH3, CH2CH2CH3), these adducts
are known to reactivate slowly and do not undergo appreciable
The mechanism of toxicity of most highly reactive OPs results
from the inhibition of acetylcholinesterase (AChE), specifically
phosphorylating an active site serine to produce an OP-AChE ad-
duct.1,2,4 Formation of OP-AChE adducts diminish or halt the
hydrolysis of the neurotransmitter, acetylcholine (ACh) causing a
surplus of ACh in the neuronal synapse that triggers overexcitation
of nicotinic and muscarinic receptors leading to neurotoxic events.
Once the OP-AChE adduct is formed it can undergo reactivation
(restoration of activity) or aging, which is defined as loss of a phos-
phoester group leading to an oxyanion that is intractable to reacti-
vation (Scheme 1).5–7
aging (t ꢀ36–48 h) suggesting that these OP-AChE adducts are
½
relatively stable.9 Herein, we report the synthesis and anti-AChE
activity of OP compounds designed to form analogs of these stable
OP-AChE adducts, for example, (ZCH2CH2O)(Y)P(O)-AChE and
(ZCH2CH2CH2O)(Y)P(O)-AChE (Y = CH3, CH2CH3) in which Z is a
halogen or tosylate substituent. It was thought that these custom-
ized inhibitors would form OP-AChE adducts in which the Z-substi-
tuent is positioned to influence the adduct stability and post-
inhibitory reaction pathways. Since there are no systematic reports
that prepare AChE-reactive, organophosphate compounds contain-
ing b- or c-substituent phosphoester groups, this study was aimed
The reactivation of OP-AChE adducts occurs by nucleophilic
displacement at phosphorus (by water, oximes), whereas aging is
influenced by branching and/or inductive properties that favor
cationic (proton-assisted) mechanisms. For example, dimethoxy-
phosphoryl-AChE (R = CH3, Y = OCH3) undergoes reactivation
within 1–2 h (or minutes using oxime antidotes) and ages with a
to identify useful synthetic strategies to these structure supported
by preliminary enzymatic evidence demonstrating their potential
as enzyme inhibitors.
The OP targets (4 and 5) were designed and synthesized
(Scheme 2) to include a number of key features. To produce OPs
with useful inhibitory properties, a p-nitrophenol ester was in-
stalled as a leaving group. To examine the role, if any, of the alkyl
group on the inhibition mechanism, both methyl and ethyl phos-
phonates were considered as targets. Both phosphonothionate
⇑
Corresponding author. Tel.: +1 406 243 4643; fax: +1 406 243 5227.
E-mail address: charles.thompson@umontana.edu (C.M. Thompson).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.02.010

S. K. Ahmed et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2048–2051
2049
NO2
end, twenty novel OP structures were produced with the most
notable variation occurring in the alkoxy b, c-substituents as F,
Br, I and OTs.
S(O)
S(O)
CO2Et
CO2Et
H3CH2CO
H3CH2CO
H3CO
H3CO
P
P
S
O
Compounds 4a–j and 5a–j were first synthesized (Scheme 2)
starting from the corresponding phosphonyl chlorides 1a
(R = CH3) and 1b (R = CH3CH2). Reaction with p-nitrophenol (PNP)
in base affords the bis(4-nitrophenoxy) alkylphosphonates (2a–b)
in good yield 87–91%.11 To convert 2a–b to the 4-nitrophenyl,
hydrogen alkylphosphonic acids, LiOH, NaOH, KOH, Ba(OH)2,
Ca(OH)2 were examined. Most conditions formed the hydrolyzed
diacids or mixtures of hydrolysis products11 except LiOH (0.5 M
aq) with CH3CN that led to monoesters 3a–b in moderate yield
51–54%.12
malathion
(insecticide)
parathion
(insecticide)
CH3
H3C CH3
H3C
O
O
O
P
F
C
CH O
H3C CH
H3CH2CO
P Me
P
F
Me
O
Me
SCH2CH2N(iPr)2
H3C
(VX)
(sarin: GB)
(soman: GD)
Figure 1. Representative OP insecticides and nerve agents.
Monoesters 3a–b were converted to the target compounds 4a–j
by reaction of the corresponding haloalcohol or tosyl alcohol in the
presence of a coupling reagent, dicyclohexylcarbodiimide (DCC) or
benzotriazol-1-yl-oxy-tris-(dimethylamino)-phosphonium hexa-
fluorophosphate (BOPÁPF6). Both coupling reactions were success-
ful; however, the use of DCC afforded higher yields.13 Coupling of
monoesters 3a–b to the alcohols was also attempted using Mitsun-
obu conditions or water soluble carbodiimides, however, neither
worked.14,15 Oxons 4a–j were treated with Lawesson’s reagent to
form the corresponding phosphonothionates 5a–j in 60–65%
yield.16
A more straightforward route to the target OPs 4a–j and 5a–j
was undertaken by attempting to directly introduce the Z substitu-
ent by nucleophilic displacement of a leaving group on the ester
side chain (Scheme 3). For example, the bromoethyl ester 4c was
treated with silver tosylate to form 4f in 48% yield. We next at-
tempted to prepare the 2-fluoro derivative 4a using tetrabutyl
ammonium fluoride (TBAF; 1.0 M aq THF; 10 min) and the yield
was low (13%). We attributed this low product yield to competing
nucleophilic substitution at phosphorus and formation of a P–F
bond as confirmed by the appearance of a dF–P in the 31P
(J = 1074 Hz). To circumvent the P–F bond formation problem,
which results in a toxic product, we examined the TBAF reaction
using the thionate (P@S) 5f as starting material, which owing to
OH
water or
oxime
O
P
Y
O
P
AChE
O
Y
R
kreact
ser
X
O R
reactivated
O
OH
ser
cholinesterase
AChE
AChE
O
O-
P
ser
kaging
phosphorylated
acetylcholinesterase
O
Y
cholinesterase
(AChE)
(OP-AChE adduct)
AChE
ser
"aging"
Scheme 1. OP-inhibition of AChE and subsequent reactivation and aging reactions.
(P@S) and phosphonate oxon (P@O) analogs were prepared. Phos-
phonothionates are expected to be less reactive toward nucleo-
philic displacement than oxons, and therefore likely to be poor
inhibitors of AChE.10 However, the thionate P@S bond reduces
reactivity at the phosphorus center allowing functional group
transformations to more readily proceed elsewhere on the mole-
cule. Substituted, ethoxy and n-propoxy phosphoester groups were
considered to evaluate the ease of substituent installation and
potentially examine the contribution of inductive effects on inhibi-
tion. Additionally, if certain alkoxy substituents (Z) are leaving
groups, the alkylation of AChE residues residing in proximity to
the active site serine will be examined in a future study. To this
stronger pp–dp overlap, would be less reactive toward nucleo-
philes at phosphorus. When 5f was treated with TBAF, fluoride
ion preferably displaced the OTs leaving group rather than the p-
nitrophenol ester moiety in 10 min to afford 5a in 33% yield. This
yield was acceptable because there was no evidence of P(S)–F bond
formation. The b-fluoroethoxy thionate 5a was treated with m-
CPBA to obtain 4a in 58% yield.17–19 In sum, nucleophilic displace-
ment of a side chain leaving group (Z) works with phosphonothio-
nates as substrates but poorly with phosphonates leading to the
reaction occurring preferentially at phosphorus producing hazard-
ous phosphonofluoridates. All compounds were characterized by
1H, 13C, 31P, 19F NMRs and HRMS data.23–26
O
P
O
R
OH
O
P
NO2
R
P
O
O
R
Cl
O
(i)
(ii)
Cl
NO2
1a-b
NO2
3a-b
a: R = CH3
b: R = C2H5
2a-b
Many of the new compounds were screened for their inhibitory
potency against EEAChE (Table 1), and five were assessed in detail
for their anti-cholinesterase activity. Owing to variations in the
S
P
O
O
R
O
(CH2)n Z
R
P
O
(CH2)n
Z
O
(iii)
(iv)
O
O
O
PNPO
PNPO
F
(ii)
P
Z
P
F
P
F
NO2
and
NO2
O
O
O
13%
H3C
H3C
H3C
5a-j
4a
4a-j
4c:Z = Br
(i)
4f: Z = OTs
a: R = CH3, n = 2, Z = F
b: R = C2H5, n = 2, Z = F
c: R = CH3, n = 2, Z = Br
d: R = CH3, n = 3, Z = Br
e: R = CH3, n = 2, Z = I
f: R = CH3, n = 2, Z = OTs
g: R = C2H5, n = 2, Z = I
h: R =C2H5, n = 3, Z = I
i: R =C2H5, n = 2, Z = Br
j: R =C2H5, n = 3, Z = Br
(iii)
58%
S
S
PNPO
PNPO
OTs
(ii)
33%
P
P
F
O
O
H3C
H3C
5f
5a
Scheme 2. (i) p-NO2PhOH, Et3N, CH2Cl2, 0 °C to rt, 4 h; (ii) 0.5 M LiOH, CH3CN, rt,
1 h; (iii) Z(CH2)nOH, DCC or BOPÁPF6, CH2Cl2, rt, 24 h; (iv) Lawesson’s reagent,
toluene, 80 °C, 3 h.
Scheme 3. Alternate route to target organophosphates: (i) TsOÀAg+, CH3CN, 65 °C,
4 h; (ii) 1.0 M TBAF in THF, rt, 10 min; (iii) m-CPBA, CH2Cl2, rt, 30 min.

2050
S. K. Ahmed et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2048–2051
Table 1
As found for EEAChE, compound 4a was a 100-fold more potent
inhibitor of rHAChE than the ethyl analog 4b. To our surprise, the
methylphosphonate 4a and ethyl phosphonate analog 4b showed
comparable inhibition values (ꢀ106 MÀ1 minÀ1) against RBAChE
suggesting that this enzyme is less sensitive to the added occlusion
and inductively electron-donating properties of the ethyl group at
phosphorus. Compounds 4a and 4b were 25- to 100-fold less
potent inhibitors of RBAChE than paraoxon by their respective
concentration-dependent analyses.
As noted, the (CH3)(CH3CH2O)P(O)–serine adduct neither reacti-
vates nor ages rapidly, which may be due to a combination of
inductive and steric effects that reduce reactivity at the phospho-
rus atom and also reduce the propensity to form cation-like inter-
mediates thought to underscore the aging mechanism. Since the
phosphylation of AChE inhibition by compounds 4a, 4b, 4c, 4f,
and 5c occur with concomitant loss of the p-nitrophenoxy group,
the resulting OP-AChE adducts closely resemble that formed from
VX, namely, the (CH3)(CH3CH2O)P(O)–serine adduct, which differs
only in the retained b-substituent.22 For example, reaction of AChE
Inhibition rate constants (ki MÀ1 minÀ1) for 4a, 4b, 4c, 4f and 5c against EEAChE,
rHAChE and RBAChE
Compd
Method EEAChE
rHAChE
RBAChE
4a
CD
TD
CD
TD
CD
TD
TD
5.90 0.15 Â 106 7.51 0.21 Â 106 6.11 0.25 Â 106
2.50 0.50 Â 106 1.40 0.59 Â 107 6.16 0.28 Â 106
5.52 0.09 Â 104 6.16 0.16 Â 104 1.16 0.06 Â 106
8.64 2.20 Â 104 1.02 0.05 Â 105 3.77 0.48 Â 106
4b
4c
4f
5c
8.11 0.29 Â 104 nd
1.14 0.03 Â 105 nd
1.17 0.14 Â 104 nd
1.96 0.05 Â 106 nd
nd
nd
nd
Paraoxon CD
TD
1.58 0.22 Â 108
3.39 0.14 Â 107
EEAChE: electric eel acetylcholinesterase; rHAChE: recombinant human acetyl-
cholinesterase; RBAChE: rat brain acetylcholinesterase. All ki values (mean SEM)
were determined from 2 to 4 different experiments at 25 °C. nd: Not determined;
CD: concentration-dependent; TD: time-dependent. All rate constants (ki) were
determined by Ellman assay.21
inhibitor solubility and reactivity, the bimolecular inhibition con-
stants (ki) for select compounds were determined using two kinetic
methods: (a) a set inhibitor concentration [I] incubated with AChE
for various time points (time-dependent) and (b) variable [I] incu-
bated with AChE and measured at a set time point (concentration-
dependent20) kinetics (Table 1). Compounds 4a and 4b differ only
in the alkyl-P bond as methyl- and ethyl phosphonates, respec-
tively, and were examined by concentration- and time-dependent
methods. Compounds 4a, 4c and 4f are all methyl phosphonates
that vary in the b-ethyl substituent Z and were examined by con-
centration or time-dependent kinetics. Paraoxon was also exam-
ined as a control.
First, the difference between methyl- and ethyl phosphonates
was examined. The fluoroethoxy methyl phosphonate 4a was
found to be a 30- to 100-fold better inhibitor of EEAChE than the
corresponding ethyl phosphonate 4b as determined by both time-
and concentration-dependent kinetic methods. The inhibitory po-
tency of 4a was ki ꢀ5 Â 106 MÀ1 minÀ1 toward EEAChE, which is
comparable to the highly potent inhibitor, paraoxon.
with 4a results in phosphylation at ser-200 to produce a
(Me)(FCH2CH2O)P(O)–serine adduct.
In summary, a series of b- and
c-substituted alkoxy meth-
ylphosphonates were successfully prepared. The synthetic path-
way is highlighted by a new approach that allows selective
nucleophilic displacement at a phosphoester side chain rather than
at a reactive phosphorus center. The enzymatic analyses demon-
strated that the b-fluoroethoxy analog 4a was the best inhibitor
of acetylcholinesterases of those tested, and comparable in
strength to paraoxon. The kinetic analyses also revealed two note-
worthy results. First, the size of the alkoxy group substituent does
not adversely affect the inhibitor strength. Second, the phosphono-
thionate analog 5c was shown to possess anti-cholinesterase activ-
ity whereas P@S structures are normally inactive. The data
produced in this study now makes possible advanced studies to
further elucidate the mechanism of reactivation and aging by
alkylphosphonates bearing substituted esters.
Acknowledgments
Changing from 2-fluoroethoxy 4a to the 2-bromoethoxy meth-
ylphosphonate 4c led to a 70-fold decrease in inhibitory potency.
This large difference in inhibition is somewhat surprising in light
of the relatively small inductive and steric differences between F
and Br that would be expected. However, additional or unforeseen
steric considerations that affect the inhibitory potency other than
those indirectly influencing the phosphorus atom cannot be
excluded.
Replacing halogen substituents with a tosylate 4f afforded a 50-
fold weaker inhibitor of EEAChE than the corresponding fluoro ana-
log 4a but comparable to the bromo analog 4c. This result suggests
that the larger tosylate group did not adversely affect the inhibi-
tion of EEAChE, whereas altering the phosphonate from methyl
to ethyl played as significant role in reducing the reactivity at
phosphorus.
None of the phosphonothionates (5: P@S) were inhibitors of
EEAChE (ki <102 MÀ1 minÀ1) except 5c that blocked enzyme activ-
ity at only sevenfold less than that of the corresponding oxon
(P@O) (5: P@S). Typically, oxons are 100-fold more potent antich-
olinesterases than the corresponding thionates.10
Since the methyl b-fluoroethoxy phosphonate 4a results in an
OP-AChE adduct that closely resembles inhibition by the nerve
agent VX, the concentration- and time-dependent inhibition of
two mammalian acetylcholinesterases, recombinant human and
rat brain, was also examined. For comparison, the ethylphospho-
nate analog 4b and paraoxon were also studied. In general, the
methyl b-fluoroethoxyphosphonate 4a was a marginally more po-
tent an inhibitor of the mammalian enzymes than that of EEAChE.
This research was supported by R21 NS072079 (CMT/JMG), the
Core Laboratory for Neuromolecular Production P30 NS055022
(CMT), and NS058229 (ATERIS Technologies LLC).
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CDCl3) d/ppm 31.14; HRMS Calcd for chemical formula C7H8NO5P 217.0140.
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25. General procedure for the synthesis of 2/3-haloalkylnitrophenyl alkylphosphonates
and 2-(methyl (4-nitrophenoxy)phosphoryloxy)ethyl-4-ethylbenzene sulfonate
(4a–j): To 4-nitrophenyl hydrogen alkylphosphonate (0.5 mmol) in dry
CH2Cl2 (5 mL) was added the 2/3-haloalcohol or 2-hydroxyethyl 4-
methylbenzenesulfonate (0.5 mmol) and DCC (0.9 mmol) at rt with stirring
for 24 h. The reaction mixture was filtered through filter paper to remove DCU,
the filtrate diluted with CH2Cl2 (50 mL), washed with DI water (3 Â 50 mL),
and the CH2Cl2 layer dried (Na2SO4). Filtration of Na2SO4 and removal of the
solvent yielded the crude product that was purified over silica using 6:4 EtOAc/
hex to afford the product. 2-Fluoroethyl 4-nitrophenyl methylphosphonate (4a; a
colorless sticky mass) (76.3 mg; 58%): 1H NMR (500 MHz, CDCl3) d 8.85 (d,
J = 9.27 Hz, 2H), 7.40 (d, J = 9.27 Hz, 2H), 4.50–4.67 (m, 2H), 4.24–4.47 (m, 2H),
1.75 (d, J = 17.85 Hz, 3H); 13C NMR (500 MHz, CDCl3) d/ppm 155.14, 144.75,
125.89, 121.11, 82.72, 81.35, 12.21 (d, JCP = 144.15 Hz); 31P NMR (500 MHz,
CDCl3) d/ppm 29.33; 19F NMR (500 MHz, CDCl3) d/ppm À224.47; HRMS Calcd
for chemical formula C9H11NFO5P 263.0359. Found: 264.0434 [(M+H)+].
26. General procedure for synthesis of O-2/3-haloalkyl O-4-nitrophenyl
alkylphosphonothioates and 2-(methyl(4-nitrophenoxy)phosphorothioyloxy)-
ethyl-4-methylbenzene sulfonate (5a–j): Compound 4a–j (0.2 mmol) taken up
in 3 mL of dry toluene was added Lawesson’s reagent (0.1 mmol) and the
reaction brought to reflux for 3 h after which the reaction mass was filtered,
washed with 2 mL CHCl3, and the filtrate directly loaded on preparative TLC
plate using 1:3, ethyl acetate/hexanes to obtain the pure product. O-2-
22. Millard, C. B.; Koellner, G.; Ordentlich, A.; Shafferman, A.; Silman, I.; Sussman, J.
L. J. Am. Chem. Soc. 1999, 121, 9883.
23. General procedure for synthesis of bis(4-nitrophenyl) alkylphosphonate (2a–b): To
the alkyl phosphonyl dichloride (11.3 mmol) was added 4-nitrophenol
(22.5 mmol) in CH2Cl2 (20 mL). The mixture was cooled in an ice-water bath
and triethylamine (TEA; 45.1 mmol) in dry CH2Cl2 (5 mL) was added drop wise
with stirring. TEA–HCl formed as the mixture stirred for 4 h at rt whereupon
the reaction mixture was poured into 100 mL of ice cold water and extracted
with CH2Cl2 (2 Â 100 mL). The combined organic layers were dried over
anhydrous Na2SO4, filtered and the solvent removed to yield crude bis(4-
nitrophenyl) alkylphosphonate that was purified on a short silica column using
3:7, ethyl acetate/hexanes. Bis(4-nitrophenyl)methylphosphonate (2a)
isolated as a pale yellow solid (3.49 g, 91%); 1H NMR (500 MHz, CDCl3) d/
ppm 8.22 (d, J = 9.29 Hz, 4H), 7.37 (d, J = 9.29 Hz, 4H), 1.95 (d, J = 17.71 Hz, 3H);
13C NMR (500 MHz, CDCl3) d/ppm 154.53, 145.08, 125.88, 121.11, 12.62 (d,
JCP = 144.25 Hz); 31P NMR (500 MHz, CDCl3) d/ppm 25.34; HRMS Calcd for
C
13H11N2O7P 338.0304. Found: 339.0307 [(M+H)+]. See: Ghanem, E.; Li, Y.; Xu,
C.; Raushel, F. M. Biochemistry 2007, 46, 9032.
24. General procedure for the synthesis of 4-nitrophenyl hydrogen alkylphosphonates
(3a–b): To the bis(4-nitrophenyl) alkylphosphonate (7.4 mmol) in CH3CN
(28.8 mL) was added 0.5 M LiOH (28.8 mL) drop wise using
a pressure
equalizing funnel over 20 min and stirred at rt for 1 h. The CH3CN was
removed under reduced pressure, and the aqueous solution extracted with
CH2Cl2 (3 Â 250 mL) to remove p-nitrophenol. The aqueous phase was then
acidified with 3 N HCl to pH 0.5 and extracted with CH2Cl2 (3 Â 250 mL). The
organic phases were combined, and concentrated to give a crude semisolid that
was re-crystallized using 10% EtOAc, 90% n-pentane. 4-Nitrophenyl hydrogen
methylphosphonate (3a) was isolated as a crystalline light brown solid (0.86 g,
54%); 1H NMR (500 MHz, CDCl3) d/ppm 8.20 (d, J = 9.13 Hz, 2H), 7.32 (d,
Fluoroethyl-O-p-nitrophenyl methyphosphonothioate (5a; isolated as
a
semisolid) (35.7 mg; 64%): 1H NMR (500 MHz, CDCl3) d 8.26 (d, J = 9.27 Hz,
2H), 7.34 (d, J = 9.27 Hz, 2H), 4.49–4.66 (m, 2H), 4.24–4.48 (m, 2H), 2.11 (d,
J = 15 Hz, 3H); 13C NMR (500 MHz, CDCl3) d 154.99, 145.01, 125.43, 122.41,
82.70, 81.33, 66.35, 22.67 (d, JCP = 460 Hz); 31P NMR (500 MHz, CDCl3) d 96.15;
19F NMR 500 MHz, CDCl3) d À224.29; HRMS Cacld for C9H11FNO4PS 279.0130.
Found: 280.0128 [(M+H)+].