Synthesis and testing of trifluoromethyl-containing phosphonate-peptide conjugates as inhibitors of serine hydrolases

Article abstract of DOI:10.1016/j.bmcl.2011.09.030

A modification of novel fluorinated organophosphorous compounds containing terminal alkyne group by different azidopeptides via Cu(I)-catalyzed click chemistry has been described. The inhibitor activity of trifluoromethyl- containing methylphosphonates an

Full text of DOI:10.1016/j.bmcl.2011.09.030

Bioorganic & Medicinal Chemistry Letters 21 (2011) 7216–7218
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Bioorganic & Medicinal Chemistry Letters
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Synthesis and testing of trifluoromethyl-containing phosphonate–peptide
conjugates as inhibitors of serine hydrolases
Nadezhda V. Sokolova a, Valentine G. Nenajdenko a, , Vladimir B. Sokolov b, Olga G. Serebryakova b,
⇑
Galina F. Makhaeva b
a Department of Chemistry, Moscow State University, Leninskie Gory 1, Moscow 119991, Russia
b Institute of Physiologically Active Compounds Russian Academy of Sciences, Severny proezd 1, Chernogolovka 142432, Russia
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 5 July 2011
Revised 29 August 2011
Accepted 10 September 2011
Available online 16 September 2011
A modification of novel fluorinated organophosphorous compounds containing terminal alkyne group by
different azidopeptides via Cu(I)-catalyzed click chemistry has been described. The inhibitor activity of
trifluoromethyl-containing methylphosphonates and their peptide-conjugates towards acetylcholines-
terase, butyrylcholinesterase, and carboxylesterase has been investigated. It was shown that the incorpo-
ration of peptide fragments significantly modulates the esterase profile of starting methylphosphonates.
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Serine hydrolases
Organophosphorous compounds
Inhibitors
Peptides
Click chemistry
The design and synthesis of novel, effective, low toxicity, and
selective inhibitors of serine hydrolases such as acetylcholinester-
ase (EC 3.1.1.7, AChE), butyrylcholinesterase (EC 3.1.1.8, BChE), and
carboxylesterase (EC 3.1.1.1, CaE) is of considerable interest for
medicinal and agricultural chemistry. One of the major problems
of mechanism-based inhibitors is the selectivity of their action.
Cross-reactivity with related enzymes often limits the practical
application of the molecule because of undesired inhibition which
may lead to serious side effects.
Recently, we reported the synthesis and anti-esterase activity of
fluorinated organophosphorous compounds having original fluo-
rine-containing leaving groups. Furthermore, in some cases we dis-
closed high selectivity of such compounds towards CaE.1
ideal linkers in the processes of conjugation or decoration of bio-
logically active molecules.6
Herein, we present the modification of phosphorylated hexa-
fluoroisopropanol and methyl trifluorolactate by peptide frag-
ments via Cu(I)-catalyzed click chemistry to obtain new selective
inhibitors of serine hydrolases. For this aim, we synthesized organ-
ophosphorous inhibitors (OPI) of serine hydrolases containing ter-
minal alkyne group from propargyl methyl phosphonite and
hexafluoroacetone or methyl trifluoropyruvate by the Abramov
reaction with the subsequent phosphonate–phosphate rearrange-
ment. The corresponding azidopeptides are available by the Ugi
reaction7 based on chiral isocyanoazides.8 Finally, we obtained no-
vel CF3-containing phosphonate–peptide conjugates linked via
1,2,3-triazole moiety using Cu(I)-catalyzed Huisgen 1,3-dipolar
cycloaddition reaction.
The synthetic route used for the preparation of starting phos-
phonates 3a,b containing terminal alkyne groups is shown in
Scheme 1. For this aim, methyl dichlorophosphine was treated
with an excess of propargyl alcohol (2 equiv) at À30 °C afforded
propargyl methyl phosphonite 1 in 65% yield. On the next stage,
propargyl methyl phosphonite 1 was exothermically reacted with
trifluoromethyl carbonyl compounds—hexafluoroacetone or
methyl trifluoropyruvate to form phosphinate intermediates 2a,b.
Finally, the subsequent phosphinate–phosphonate rearrange-
ment of intermediates 2a,b in the presence of catalytic amounts
of triethylamine allowed us to obtain previously unknown racemic
phosphonates 3a,b in 77% (3a) and 82% (3b) yields, respectively.
The incorporation of peptide fragments into the molecules of
organophosphorous inhibitors (OPI) may dramatically change the
esterase profile of these compounds2 and lead to the increasing
of their selectivity towards BChE and CaE.
The Cu(I)-catalyzed azide–alkyne coupling (‘click’ reaction)3 be-
came one of the modern and valuable tools in the area of biocon-
jugation chemistry.4 1,2,3-Triazole moieties can be considered as
bioisosteres of amide bonds which are extremely stable under
physiological conditions, can form hydrogen bonds, and are not
toxic.5 Due to its structural features 1,2,3-triazoles can serve as
⇑
Corresponding author. Tel.: +7 495 939 2276; fax: +7 495 932 8846.
E-mail address: nen@acylium.chem.msu.ru (V.G. Nenajdenko).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.09.030

N. V. Sokolova et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7216–7218
7217
R
Cl
P
OH
O
OH
H
CF3C(O)R
b
a
O
2 eq.
P
F3C
P
H3C
Cl
O
CH3
O
1, 65%
CH3
2a, b
R
c
O
O
F3C
P
O
CH3
3a,b
R = CF3 (3a), 77%; C(O)OMe (3b), 82%
Scheme 1. Reagents and conditions: (a) À30 °C, 0.5 h; (b) THF, 20 °C, 1 h; (c) Et3N, reflux, 1 h.
R1
N3
R5 COOH
NC
R2
O
4a: R1 = Me; R2,R3 = Me; R4 = DMB; R5
4b: R1 = i-Pr; R2,R3 = H; R4 = Bn; R5
=
, 55%
NHBoc
+
R4 NH2
R3
=
NHBoc, 60%
a
4c: R1 = H; R2,R3 = Me; R4 = Bn; R5 = CF3, 30%
4d: R1 = Me; R2,R3 = H; R4 = Bn; R5 = CF3, 60%
4e: R1 = i-Pr; R2,R3 = H; R4 = Bn; R5 = CF3, 50%
R4
N
O
R1
R5
N3
N
H
4f: R1
=
; R2,R3 = H; R4 = Bn; R5 = CF3, 60%
R3 R2
O
4a-f
Scheme 2. Reagents and conditions: (a) MeOH, rt, 24 h.
The starting azidopeptides 4a–f were prepared in good yields
via the Ugi reaction of chiral isocyanoazides with carbonyl com-
pounds, amines and Boc-protected amino acids or trifluoroacetic
acid (Scheme 2).9 These compounds bearing the azide function
can be conjugated with other biologically active molecules via click
chemistry.
Having in hands the corresponding sets of CF3-containing meth-
ylphosphonates 3 and azidopeptides 4 we investigated a bioconju-
gation process. Thus, heating the educts with Cu(II)/sodium
ascorbate in a biphasic mixture of CH2Cl2–H2O provided the de-
sired products 5a–h in good yields as a mixture of diastereomers
(Scheme 3).
The inhibitor activity of CF3-containing methylphosphonates
3a–b and CF3-containing phosphonate–peptide conjugates 5a–h
was evaluated using human erythrocyte AChE, horse serum BChE,
and pig liver CaE (‘Sigma’, USA). The inhibitor activity was charac-
terized by the value of IC50—concentration of inhibitor that is nec-
essary for the reducing of the enzymatic activity to 50% at the
standard conditions (phosphate buffer pH 7.5, 25 °C, 12 min incu-
bation). The results of the inhibitory potencies and selectivities of
obtained compounds to CaE versus BChE are collected in Table 1.
The starting methylphosphonates 3a,b inhibit AChE with IC50
6.50 Â 10À5 and 4.36 Â 10À5 M correspondingly. They show more
specificity to CaE in comparison with BChE. CaE selectivity of 3a
is higher than that of 3b that agrees with our previous data for
O,O-dialkylphosphates.1
BChE. It should be noted that anti-CaE inhibitor activity and selec-
tivity to CaE depends on the structure of peptide fragment. Conju-
gates 5c,f,h are the most effective CaE inhibitors with selectivity to
CaE versus BChE of 12, 13, and 11, respectively. The less active to
CaE conjugate 5g also shows selectivity to CaE (C/B = 13) due to
its lower activity against BChE. Compounds 5f,g contain the same
peptide fragment but 5f (R = CF3) is 2.5 times more active to CaE
than 5g (R = CO(O)Me). On the other hand, the phosphonate 3a
(R = CF3) is less active to CaE than 3b (R = CO(O)Me). Consequently,
the inhibitor activity of the starting phosphonates 3 can be con-
trolled by the introduction of peptide fragments.
Human carboxylesterase 1 (hCE1) is an endobiotic-processing
serine hydrolase that exhibits relatively broad substrate specific-
ity.11 The active site cavity of hCE1 is large (ꢀ1,300 Å3 in volume)
and is lined predominantly by hydrophobic amino acids, with the
exception of residues in the catalytic triad. A structural comparison
of the active site of hCE1 with that of hAChE bound to the Alzhei-
mer’s drug tacrine established that hCE1 has a considerably larger
and more featureless pocket in comparison with small AChE active
site cavity, suitable for nonspecific substrate binding.12 This differ-
ence in sizes of active sites explains the preference of rather large
phosphonate–peptide conjugates to CaE versus AChE. Thus, incor-
poration of peptide fragments significantly changes the esterase
profile of the starting methylphosphonates and along with the
structure of the trifluoromethyl-containing leaving groups deter-
mines the selectivity of conjugates to CaE.
The incorporation of peptide fragments significantly changes
the esterase profile of the starting methylphosphonates. We found
that conjugates 5a–h are weak inhibitors of BChE and have no
inhibitor activity towards AChE. These results can be explained
by distinct differences in the volume of BChE and AChE active site
gorge: the available estimated volume of the BChE active site gorge
(ꢀ500 Å3) is more than 1.5 times greater than that of AChE
(ꢀ300 Å3).10
Carboxylesterases are responsible for the metabolism of differ-
ent therapeutically useful drugs. The use of selective inhibitors of
these proteins might be valuable in modulating the efficacy of such
agents.13 The lack of the reactivity of the obtained phosphonate–
peptide conjugates with AChE indicates to their low acute toxicity
and makes the most active compounds promising candidates for
therapeutic use.
In summary, we described the conjugation of fluorinated organ-
ophosphorous compounds containing terminal alkyne group 3a,b
with azidopeptides 4a–f via the Cu(I)-catalyzed Huisgen 1,3-dipolar
cycloaddition reaction. It was shown that the incorporation of
Phosphonate–peptide conjugates 5a,b,e are weak inhibitors of
CaE and do not show inhibitor preference between BChE and
CaE. Other compounds more effectively inhibit CaE compared to

7218
N. V. Sokolova et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7216–7218
R4
R5
R
O
: R = CF3; R1 = Me; R2,R3 = Me; R4 = DMB; R5
5a
=
, 56%
O
N
R1
O
F3C
P
NHBoc
O
R3
O
N
H
R2
CH3
: R = CF3; R1 = i-Pr; R2,R3 = H; R4 = Bn; R5
=
, 40%
N3
5b
NHBoc
4a-f
3a,b
1
; R2,R3 = H; R4 = Bn; R5 = CF3, 45%
5c
5d
5e
: R = CF3; R
=
a
: R = CF3; R1 = H; R2,R3 = Me; R4 = Bn; R5 = CF3, 45%
: R = CF3; R1 = Me; R2,R3 = H; R4 = Bn; R5 = CF3, 46%
: R = CF3; R1 = i-Pr; R2,R3 = H; R4 = Bn; R5 = CF3, 57%
R4
N
R5
O
R1
R
O
N
N
R3
N
5f
5g
N
H
R2
O
P
: R = C(O)OMe; R1 = i-Pr; R2,R3 = H; R4 = Bn; R5 = CF3, 65%
O
F3C
O
CH3
5a-h
5h: R = C(O)OMe; R1 = i-Pr; R2,R3 = H; R4 = Bn; R5
=
, 55%
NHBoc
Scheme 3. Reagents and conditions: (a) sodium ascorbate (40%), CuSO4Á5H2O (10%), CH2Cl2–H2O (10:1), 40 °C, 1 h.
Table 1
Inhibitor activity of phosphonates 3, 5 against AChE, BChE and CaE (IC50, M) and their selectivity to CaE versus BChE (C/B) (pH 7.5, 25 °C, t = 12 min)
Compounds
IC50, M
C/B
AChE
BChE
CaE
3a
3b
5a
5b
5c
5d
5e
5f
(6.50 0.43) Â 10À5
(4.36 0.28) Â 10À5
No inhibiton
No inhibiton
No inhibiton
No inhibiton
No inhibiton
No inhibiton
No inhibiton
(2.49 0.13) Â 10À4
(2.89 0.19) Â 10À5
(7.77 0.74) Â 10À4
(5.95 0.58) Â 10À4
(7.02 0.61) Â 10À4
(7.46 0.73) Â 10À4
(2.18 0.19) Â 10À4
(1.10 0.12) Â 10À3
(2.00 0.21) Â 10À3
(9.60 0.88) Â 10À4
(2.31 0.19) Â 10À5
(6.06 0.60) Â 10À6
(8.25 0.57) Â 10À4
(7.85 0.80) Â 10À4
(6.39 0.59) Â 10À5
(1.45 0.11) Â 10À4
(1.82 0.14) Â 10À4
(8.58 0.78) Â 10À5
(1.58 0.13) Â 10À4
(7.93 0.76) Â 10À5
11
5
1
1
11
5
1
13
13
12
5g
5h
No inhibiton
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peptide fragments significantly changes the esterase profile of start-
ing methylphosphonates and yields selective inhibitors of CaE that
have no inhibitor activity towards AChE. This approach can be
applied for the focused design and synthesis of peptide-containing
irreversible inhibitors of various serine hydrolases.
Supplementary data
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Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.09.030.
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