Efficient methods for the synthesis of α-aminophosphonate fluoroalkyl esters

Article abstract of DOI:10.1016/j.tetlet.2013.01.039

Two novel synthetic methods for the preparation of bis(trifluoroethyl) esters of α-aminophosphonic acids are presented. Preliminary results on the application of the compounds synthesized as inhibitors of serine proteases are also reported. Structures originating from α-aminoalkylphosphonate diaryl esters represent a novel class of serine protease inactivators.

Full text of DOI:10.1016/j.tetlet.2013.01.039

Tetrahedron Letters 54 (2013) 1566–1568
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Tetrahedron Letters
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Efficient methods for the synthesis of a-aminophosphonate fluoroalkyl esters
⇑
´
´
Marcin Skorenski, Józef Oleksyszyn, Marcin Sienczyk
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Division of Medicinal Chemistry and Microbiology, Faculty of Chemistry, Wrocław University of Technology, Wybrzeze Wyspianskiego 27, 50-370 Wrocław, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
Two novel synthetic methods for the preparation of bis(trifluoroethyl) esters of a-aminophosphonic acids
are presented. Preliminary results on the application of the compounds synthesized as inhibitors of serine
Received 19 October 2012
Revised 2 January 2013
Accepted 10 January 2013
Available online 16 January 2013
proteases are also reported. Structures originating from
a novel class of serine protease inactivators.
a-aminoalkylphosphonate diaryl esters represent
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
Bis(2,2,2-trifluoroethyl) phosphonic esters
Aminophosphonates
a-Amidoalkylation reaction
Serine protease inhibitors
Diaryl esters of a-aminoalkylphosphonate acids and their pep-
CF3
CF3
R
R
tidyl derivatives are well known inhibitors of serine proteases.1,2
The mechanism of their action involves the nucleophilic attack of
catalytic serine on the inhibitor phosphorus atom. Recent studies
have focused on balancing the electrophilic potential of the phos-
phorus atom through the introduction of different substituents
onto the ester ring structure.3–6 This has led to the development
of inhibitors displaying superior selectivity and specificity of action
toward particular serine proteases.7 In addition, this class of com-
O
HOCH2CF3, KF
18-crown-6
OPh
Cbz
Cbz
N
H
P
N
H
P
OPh
O
O
O
<15%
Scheme 1. Synthesis of
a-aminoalkylphosphonate bis(2,2,2-trifluoroethyl) esters
through diphenyl ester transesterification.11,12
pounds is relatively easy to synthesize through
a-amidoalkylation
The previously reported synthetic method for the preparation of
-aminoalkylphosphonate bis(2,2,2-trifluoroethyl) esters em-
of a triaryl phosphite with benzyl carbamate and an appropriate
aldehyde.8 Despite the great potential and utility of
a-amin-
a
oalkylphosphonate diaryl esters,7,9 a major disadvantage is their
poor solubility in aqueous media which limits their practical appli-
cation in biological systems. One strategy to at least partially over-
come such a limitation is the incorporation of solubility-enhancing
groups into the inhibitor structure.10
ployed the transesterification of
a-aminoalkylphosphonate
diphenyl esters11 (Scheme 1), which was based on the Szewczyk
procedure.12 Unfortunately, we have found this method inade-
quate due to the low synthetic yields (<15%), difficulties in final
product purification, and the necessity to use a large excess of
fluorinated alcohols.
Herein, we present a slightly different approach. We have
replaced the aryl ester rings with 2,2,2-trifluoroethyl esters. This
modification resulted in increased water solubility of the synthe-
sized derivatives in comparison to their parent compounds and,
more importantly, due to the presence of the fluorine atoms within
the ethyl esters, the electrophilicity of the phosphorus atom was
high enough to be susceptible to nucleophilic attack of a protease
catalytic serine residue. Although the preliminary results indicated
a decrease in inhibitory potency in comparison to aromatic esters,
In the present Letter, we describe two novel methods for
-aminoalkylphosphonate bis(2,2,2-trifluoroethyl) ester synthesis
a
(Scheme 2). The first approach (Scheme 2, Route A) employs
amidoalkylation of tris(2,2,2-trifluoroethyl) phosphite with benzyl
carbamate and an aldehyde. Briefly, tris(2,2,2-trifluoroethyl)
phosphite was prepared from phosphorus trichloride and 2,2,2-tri-
fluoroethanol under reflux,13 and was used directly in the
amidoalkylation reaction with benzyl carbamate and an aldehyde
in acetic acid. This is the first example of Cbz-protected
nevertheless, bis(2,2,2-trifluoro)ethyl esters of a-aminophosphon-
ic acids represent an interesting class of irreversible serine
a
-aminoalkylphosphonate dialkyl ester synthesis via trialkyl phos-
phite amidoalkylation, the reaction characteristic for diaryl ester
derivative preparation, which was initially postulated by Birum
when 2-chloroethyl phosphite underwent amidoalkylation with
urea and an appropriate aldehyde.14 The second approach (Scheme
protease inhibitors.
⇑
Corresponding author. Tel.: +48 71 320 2439; fax: +48 71 320 2427.
E-mail address: marcin.sienczyk@pwr.wroc.pl (M. Sien´ czyk).
0040-4039/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetlet.2013.01.039

´
1567
M. Skorenski et al. / Tetrahedron Letters 54 (2013) 1566–1568
"A"
"B"
CF3
O
O
H
O
O
CF3
reflux, 3 h
F3C
O
P
t-BuOH
CH2Cl2
P
PCl3
+
+ PCl3
CF3
HO
CF3
HO
O
CF3
CF3
Cbz-NH2
RCHO
Cbz-NH2
RCHO
AcOH
reflux
4 h
Ac2O
TFA
r.t., 24 h
CF3
CF3
O
R
O
O
N
H
P
O
O
1-9
Scheme 2. Synthesis of
a
-aminoalkylphosphonate bis(trifluoroethyl) esters.
Table 1
Synthesis of
Table 2
The solubility of
a
-aminoalkylphosphonate bis(trifluoroethyl) esters
a-aminoalkylphosphonate bis(trifluoroethyl) esters and their
corresponding diphenyl parent compounds in PBS and human plasma
CF3
O
R
Compound
Solubility (mM)
PBS, pH 7.4
O
O
N
H
P
Human plasma
O
CF3
O
1
1.9
0.13
1.9
0.13
1DPPa
Compound
R
Isolated yield (%)
Route A
2
1.1
0.15
1.1
0.15
Route B
2DPPa
1
2
3
4
5
6
7
8
9
–CH2Ph
–CH3
–CH(CH3)2
–CH2CH(CH3)2
–CH(CH3)CH2CH3
–(CH2)2SCH3
–CH2CH2Ph
18
11
28
36
31
12
31
16
40
31
25
31
57
41
22
56
29
45
3
0.9
0.2
0.9
0.2
3DPPa
4
5.1
0.3
5.1
0.3
4DPPa
5
3.2
0.6
5.4
0.6
5DPPa
–CH2CH3
–CH2CH2CH3
6
0.9
0.3
0.9
0.3
6DPPa
7
1.8
0.09
1.8
0.09
7DPPa
8
5.4
0.9
5.4
0.9
2, Route B) relies on the classical method used for
a-amin-
8DPPa
oalkylphosphonate dialkyl ester synthesis.15 Thus, phosphonic
dichloride was initially prepared from phosphorus trichloride in
the presence of tert-butyl alcohol, which was then reacted with
2,2,2-trifluoroethanol to give bis(2,2,2-trifluoroethyl) phosphite.16
Next, bis(2,2,2-trifluoroethyl) phosphite was dissolved in acetic
anhydride followed by the addition of benzyl carbamate, trifluoro-
acetic acid, and an aldehyde.
9
5.3
0.14
5.3
0.14
9DPPa
11
1.2
0.4
1.2
0.4
11DPPa
a
DPP—corresponding diphenyl ester analogue.
Applying both methods, we synthesized a series of Cbz-pro-
tected phosphonic analogues of natural and unnatural amino acids
in reasonable yields (Table 1). When the synthesis followed Route
B, slightly higher yields were noted ranging from 22% [Cbz-Met-
P(OCH2CF3)2, 6] to 57% [Cbz-LeuP(OCH2CF3)2, 4].
In order to synthesize peptidyl derivatives of bis(2,2,2-trifluoro-
ethyl) esters of -aminophosphonic acids, the Cbz-protecting
group was first removed using a 33% solution of HBr in acetic acid
and the resulting hydrobromide salt of the -aminophosphonate
a
a
To examine the solubility of the resulting compounds, either in
PBS buffer or human plasma, we applied a spectrophotometric as-
say as described previously (Table 2).4 Briefly, a serial dilution of
the compounds under analysis was prepared in PBS or freshly iso-
lated human plasma and the absorbance at 620 nm was measured
with reference to the medium used in the assay. In order to deter-
mine the increase in solubility of the fluoroalkylphosphonate, the
solubility of the corresponding phosphonic diphenyl esters (1–9
DPP, Table 2) was examined in parallel. As shown, the solubility
of the fluoroalkyl phosphonic inhibitors was significantly increased
in comparison to the parent aromatic structures, both in PBS and
plasma, ranging from 3-times (6 vs 6DPP) to 38-times (9 vs 9DPP).
bis(2,2,2-trifluoroethyl) ester was coupled to Boc-Val-Pro-OH
using HBTU in the presence of DIPEA as a coupling reagent
(Scheme 3).
The inhibitory activity of the compounds synthesized was
evaluated using chymotrypsin as the model enzyme. The rates of
chymotrypsin inhibition (3 nM, Calbiochem) were measured in
100 mM HEPES, 500 mM NaCl, and pH 7.5 containing 9% DMSO
using a fluorogenic substrate (Suc-Ala-Ala-Pro-Phe-AMC, 5
lM,
Ex. 350 nm, Em. 460 nm). The calculated Michaelis constant (KM)
was 70 lM. The observed rate of inhibition was determined by
the progress curve method as described previously.17,18 For
compounds that showed >50% of chymotrypsin inhibition at a

´
1568
M. Skorenski et al. / Tetrahedron Letters 54 (2013) 1566–1568
O
CF3
CF3
CF3
R
O
R
O
Boc-Val-Pro-OH
HBTU, DIPEA
HBr/AcOH
O
CF3
CF3
O
O
R
N
H
O
O
N
H
P
O
HBr*H2N
P
O
N
O
O
CF3
O
N
H
P
O
O
1,3,4
1a,3a,4a
10
11
12
R = -CH2Ph
R = -CH2CH(CH3)2
R = -CH(CH3)2
Scheme 3. Synthesis of a-aminoalkylphosphonate bis(trifluoroethyl) ester peptidyl derivatives.
In summary, we have reported efficient methods for the
synthesis of bis(2,2,2-trifluoroethyl) esters of -aminophosphonic
acids and their peptidyl derivatives. In addition, we have evaluated
their ability to block irreversibly the proteolytic activity of
chymotrypsin.
Table 3
a
Inhibition of chymotrypsin by
and their peptidyl derivatives
a
-aminoalkylphosphonate bis(trifluoroethyl) esters
Chymotrypsin
Compound
Ki (
lM)
k2/Ki (Mꢀ1sꢀ1
)
1
4
10
11
12
Cbz-PheP(OCH2CF3)2
770
353
54
67
NI
4.50
6.86
11.09
9.75
NI
Acknowledgment
Cbz-LeuP(OCH2CF3)2
Boc-Val-Pro-PheP(OCH2CF3)2
Boc-Val-Pro-LeuP(OCH2CF3)2
Boc-Val-Pro-ValP(OCH2CF3)2
This project was financed by the Wroclaw University of
Technology Statute Funds S10156/Z0313.
NI—no inhibition was observed after 30 min of compound incubation with
chymotrypsin at 37 °C.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.tetlet.2013.01.
039.
500 lM concentration, we examined the kinetic parameters (kobs,
Ki, and k2/Ki). The preliminary data (Table 3) indicate that all the
inhibitors displayed lower potency of action against chymotrypsin,
References and notes
in comparison to
highest activity was shown by Boc-Val-Pro-LeuP(OCH2CF3)2 (11)
with a k2/Ki value of 9.75 Mꢀ1 sꢀ1 (Ki = 67
M). The obtained data
a-aminophosphonate diaryl esters, where the
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serine proteases inhibitors are now in progress.