A convenient synthesis of new α-aminoalkylphosphonates, aromatic analogues of arginine as inhibitors of trypsin-like enzymes

Article abstract of DOI:10.1016/j.tetlet.2004.08.021

A simple and efficient protocol for the synthesis of new N-protected α-aminoalkylphosphonic diphenyl esters - aromatic analogues of arginine - is presented. The crucial, guanylating step was achieved using S-ethyl-N,N′-di(Boc)isothiourea in chloroform and

Full text of DOI:10.1016/j.tetlet.2004.08.021

Tetrahedron
Letters
Tetrahedron Letters 45 (2004) 7251–7254
A convenient synthesis of new a-aminoalkylphosphonates,
aromatic analogues of arginine as inhibitors of trypsin-like enzymes
Marcin Sienczyk* and Jozef Oleksyszyn
Institute of Organic Chemistry, Biochemistry and Biotechnology, Wroclaw University of Technology,
Wybrzeze Wyspianskiego 27, 50-370 Wroclaw, Poland
Received 7 July 2004; revised 28 July 2004; accepted 4 August 2004
Available online 19 August 2004
Abstract—A simple and efficient protocol for the synthesis of new N-protected a-aminoalkylphosphonic diphenyl esters––aromatic
analogues of arginine––is presented. The crucial, guanylating step was achieved using S-ethyl-N,N0-di(Boc)isothiourea in chloro-
form and in the presence of Et3N and HgCl2. Deprotection of the derivatives obtained was performed usingtrifluoroacetic acid
in CH2Cl2 or hydrogenolysis over Pd/C. The products are potent inhibitors of trypsin.
ꢀ 2004 Elsevier Ltd. All rights reserved.
Trypsin-like serine proteases compose a family of
enzymes with a wide range of activity. They are known
to be involved in many physiological states and patho-
logical disorders. Their uncontrolled activity is very haz-
ardous and often leads to serious diseases like
emphysema, cystic fibrosis or cancer development and
progression. For example, the key enzyme in tumour
growth and metastasis is urokinase plasminogen activa-
tor (uPA) belonging to the trypsin-type serine protease
family. Under normal physiological conditions this
enzyme plays an essential role in angiogenesis processes
but its uncontrolled activity results in enormous levels of
active plasmin, which facilitates movement of cancer
cells.1
was reported.6 However, the yields of most steps were
low and some yields of final products did not exceed
5%. Here we report a simple and more efficient method
for the synthesis of a-aminophosphonic diphenyl esters
as aromatic analogues of arginine.7
The overall synthetic approach is outlined in Scheme 1.
In the first step, an amidoalkylation reaction was
applied for the synthesis of Cbz-N-protected a-amino-
phosphonate diphenyl ester derivatives.8 These com-
pounds were obtained as racemic mixtures upon
condensation of 3-nitrobenzaldehyde (derivatives a) or
4-nitrobenzaldehyde (derivatives b), benzyl carbamate
and triphenyl phosphite. Compound 1a was obtained
in 71% yield and 1b in 64% yield.9 Deprotection of
1a–b using33% HBr in acetic acid gave the correspond-
inghydrobromide salts of the appropriate a-amino-
phosphonates, ready to apply in future planned
peptide synthesis.10
To date numerous phosphonate- and peptidyl-phospho-
nate diphenyl esters have been used as effective and
selective inhibitors of serine proteases,2 but only a few
papers describe the synthesis of phosphonate analogues
of arginine,3 lysine4 or their mimetics.5 After presenta-
tion of our preliminary results on the synthesis of such
analogues as inhibitors of uPA at the Polish–Austrian–
German–Hungarian–Italian Joint Meeting on Medicinal
Chemistry (Cracow, October 2003) and duringthe
preparation of this manuscript, an alternative synthesis
of phosphonic analogues and their peptide derivatives
For the reduction of nitro derivatives 1a–b, anhydrous
SnCl2 (5equiv) dissolved in water (20equiv), as a reduc-
tive agent in refluxing ethyl acetate was applied. The
desired aromatic analogues 2a and 2b possessingan
aromatic amine were obtained in quantitative yields as
pale yellow solids, which turned brown upon exposure
to light.11 Under these conditions we did not observe
removal of the Cbz group.
Keywords: a-Aminoalkylphosphonates; Arginine mimetics; Trypsin-
like enzyme inhibitors.
The Boc-protected guanidine derivatives 3a–b were
obtained
using
S-ethyl-N,N0-di(Boc)isothiourea
(1.1equiv) in chloroform as guanylating agent, Et3N
*
Correspondingauthor. Tel.: +48 71 320 40 27; fax: +48 71 328 40
64; e-mail: marcin.sienczyk@pwr.wroc.pl
0040-4039/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2004.08.021

7252
M. Sienczyk, J. Oleksyszyn / Tetrahedron Letters 45 (2004) 7251–7254
NH2
NO2
NO2
i
ii
1a - 70%
1b - 64%
2a > 99%
2b > 99%
Cbz
OPh
OPh
Cbz
OPh
OPh
N
P
N
P
O
H
H
H
O
O
iii
1a-b
2a-b
3a - 84%
3b - 72%
H
H
N
+
N
N
H2N
NH2
N
Boc
Boc
Boc
Boc
v
NH
NH
NH
iv
5a - 68%
5b - 76%
4a - 67%
4b - 70%
Cbz
OPh
OPh
Cbz
OPh
OPh
OPh
OPh
N
P
N
H
P
H2N
P
H
O
O
O
5a-b
3a-b
4a-b
Scheme 1. Preparation of compounds 3a and 3b. The conditions are: (i) P(OPh)3, benzyl carbamate, AcOH, 80ꢁC; (ii) SnCl2/H2O, AcOEt, reflux; (iii)
S-ethyl-N,N0-di(Boc)-isothiourea, Et3N, HgCl2, CHCl3; (iv) TFA, CH2Cl2; (v) H2/Pd–C.
(3equiv) and HgCl2 (1.2equiv) as promoter.12 The reac-
tion was monitored by TLC until the startingmaterial
had disappeared. Compounds 3a and 3b were obtained
in 84% and 72% yields, respectively. The guanidine
derivatives could be additionally recrystallized from a
mixture of CH2Cl2/hexane (1:2) to give white crystals
of 3a and pearl-white crystals of 3b.
of arginine at the C-terminal. The typical procedure is
outlined in Scheme 2. Compound 1a was converted into
hydrobromide salt 6 in 95% yield using33% HBr in
acetic acid. Couplingof 6 with Cbz-amino acids using
DCC/HOBt yielded the desired phosphonopeptides 7
in yields exceeding50%. Treatment of 7 with SnCl2/
H2O in refluxingAcOEt reduced the nitro rgoup of
the aromatic ringto afford amines 8. We did not observe
removal of the Cbz protection duringthe reduction of
the phosphonopeptides. Further crystallization from n-
hexane gave pure derivatives. Guanidinylation using S-
ethyl-N,N0-di(Boc)-isothiourea in chloroform afforded
crystalline, solid products 9. Finally, removal of the
Boc groups was accomplished using TFA/CH2Cl2 (1:1).
At this stage two possible methods for deprotection
were possible. Hydrogenolysis over Pd/C gives the
desired a-aminophosphonates with a free a-amino
group (5a and 5b obtained in 68% and 76% yields,
respectively), whereas Boc-deprotection of the guanidine
group can be achieved by the application of trifluoro-
acetic acid (50% solution in CH2Cl2) to yield the TFA
salts of 4a and 4b obtained in 67% and 70% yields,
respectively.
The inhibitory activity of the compounds described
above towards trypsin was determined usinga chromo-
genic assay.13 As mentioned in a recent paper,6 these
compounds show slow-bindinginhibitory behaviour.
The enzyme and the inhibitor were incubated for
A similar strategy was applied for the synthesis of sev-
eral di- and tripeptides with the aromatic analogue
NO2
NO2
NO2
i
ii
iii
95%
O
50 - 80%
>95%
H
OPh
OPh
OPh
OPh
Cbz
OPh
OPh
N
Br- +H3N
P
Cbz
N
H
P
N
H
P
O
R
O
O
1a
6
7
H
N
+
N
H2N
NH2
Boc
Boc
NH2
NH
NH
iv
>70%
v
>65%
O
O
O
H
H
N
H
OPh
OPh
N
OPh
OPh
OPh
OPh
N
Cbz
N
H
P
Cbz
N
H
P
Cbz
N
P
H
R
O
R
O
R
O
8
9
10
Scheme 2. Preparation of phosphonopeptides. R-Amino acid side chain. The conditions are: (i) HBr/AcOH; (ii) Cbz-amino acid, DCC, HOBt, Et3N,
DMF; (iii) SnCl2/H2O, AcOEt, reflux; (iv) S-ethyl-N,N0-di(Boc)-isothiourea, Et3N, HgCl2, CHCl3; (v) TFA, CH2Cl2.

M. Sienczyk, J. Oleksyszyn / Tetrahedron Letters 45 (2004) 7251–7254
7253
Table 1. Inhibitory activity of new a-aminophosphonate diphenyl
esters, aromatic analogues of arginine towards trypsin
2a: White, pale yellow solid, mp 120ꢁC [Found C, 66.51;
H, 4.97; N, 5.80; P, 6.32. C27H25N2O5P requires C, 66.39;
H, 5.16; N, 5.73; P, 6.34%]; 31P NMR (CDCl3): 15.72 (s);
1H NMR (CDCl3): 3.88 (s, 2H), 5.09 (dd, J = 12.2,
28.7Hz, 2H), 5.48 (dd, J = 9.9, 21.8Hz, 1H), 5.93 (d,
J = 7.7Hz, 1H), 6.62–7.38 (m, Ar–H, 19H); 13C NMR
(CDCl3): 52.9 (d, J = 158.7Hz), 67.51, 115.00 (d,
J = 7.2Hz), 115.65, 118.63 (d, J = 6.64Hz), 120.51 (dd,
J = 4.3, 6.4Hz), 125.35 (d, J = 4.0Hz), 128.22, 128.28,
128.56, 129.69 (d, J = 8.1Hz), 129.84, 135.18, 136.03,
146.51, 150.14 (d, J = 9.5Hz), 155.55 (d, J = 9.0Hz).
3a: White crystals, mp 86-88ꢁC [Found C, 62.11; H, 6.21;
N, 7.45; P, 4.40. C38H43N4O9P requires C, 62.46; H, 5.93;
N, 7.67; P, 4.24%]; 31P NMR (CDCl3): 15.22 (s); 1H NMR
(CDCl3): 1.43 (s, 9H), 1.49 (s, 9H), 5.07 (dd, J = 12.2,
33.45Hz, 2H), 5.55 (dd, J = 9.8, 22.3Hz, 1H), 6.04 (d,
J = 3.1Hz, 1H), 6.89–7.73 (m, Ar–H, 19H), 10.38 (s, 1H),
11.60 (s, 1H); 13C NMR (CDCl3): 28.17, 52.73 (d,
J = 160.0Hz), 67.56, 79.75, 83.87, 120.53 (d, J = 4.1Hz),
121.56, 122.71, 124.50, 125.38 (d, J = 4.1Hz), 128.27,
128.56, 129.50, 129.73 (d, J = 3.6Hz), 134.86, 136.00,
137.44, 150.15, 153.36, 155.44, 161.40, 163.4.
Compound
IC50 (lM)
4a
4b
0.122
13
10min at room temperature before the addition of sub-
strate. Our observation proved that longer preincuba-
tion times do not seem to change the IC50 values.
Surprisingly, we have noticed that compound 4a is a
more active inhibitor of trypsin than of uPA
(IC50 = 1.6lM, Ref. 6, Table 1).
Additionally, our preliminary results showed that these
compounds are potent inducers of apoptosis in human
cancer cell lines; these results will be published sepa-
rately in due course.
In conclusion, we have presented an alternative route
for the synthesis of novel protected a-aminophosphonic
diphenyl esters, aromatic mimetics of arginine, which
appears to be more efficient if compared to current
methods presented in the literature. The compounds
obtained are potent inhibitors for trypsin-like enzymes
even as racemic mixtures. The new challenge in this area
is the synthesis of optically pure a-aminophosphonates
of Cbz-ArgP(OPh)2, Cbz-HomoArgP(OPh)2, Cbz-(3-
GuPhg)P(OPh)2, Cbz-(4-GuPhg)P(OPh)2. We are cur-
rently at advanced stages in the synthesis of pure
enantiomers of these phosphonic analogues of arginine.
1b: White solid, mp 160–161ꢁC [Found C, 62.58; H, 4.48;
N, 5.48; P, 6.04. C27H23N2O7P requires C, 62.55; H, 4.47;
N, 5.40; P, 5.97%];31P NMR (CDCl3): 13,66 (s); 1H NMR
(CDCl3): 5.09 (q, J = 12.1Hz, 2H), 5.65 (dd, J = 9.0,
23.4Hz, 1H), 6.13 (dd, J = 5.7, 8.8Hz, 1H), 6.91–8.19 (m,
Ar–H, 19H); 13C NMR (CDCl3): 52.60 (d, J = 155.93Hz),
67.89, 120.22 (dd, J = 4.3, 11.8Hz), 123.94, 125.79,
128.27, 128.58 (d, J = 9.7Hz), 129.05 (d, J = 5.6Hz),
129.92, 135.67, 141.65, 147.96, 149.78 (d, J = 9.1Hz),
155.45.
2b: White, pale yellow solid, mp 176–178ꢁC [Found C,
66.23; H, 5.25; N, 5.68; P, 6.32. C27H25N2O5P requires C,
66.39; H, 5.16; N, 5.73; P, 6.34%]; 31P NMR (DMSO):
1
17.38 (s); H NMR (DMSO): 5.02 (dd, J = 12.5, 26.3Hz,
2H), 5.13 (s, 2H), 5.28 (dd, J = 10.1, 20.1Hz, 1H), 6.47–
7.30 (m, Ar–H, 19H), 8.61 (d, J = 10.1Hz, 1H); 13C NMR
(DMSO): 52.97 (d, J = 159.3Hz), 66.52, 114.06, 120.84 (t,
J = 4.6Hz), 121.07, 125.63 (d, J = 6.6Hz), 128.37 (d,
J = 3.5Hz), 128.84, 129.87 (d, J = 6.3Hz), 130.26,
137.24, 149.24, 150.58 (q, J = 11.0Hz), 156.44 (d,
J = 8.5Hz).
3b: White, pearl crystals, 117–118ꢁC [Found C, 59.48; H,
6.50; N, 7.17; P, 4.05. C38H43N4O9P · 2H2O requires C,
59.52; H, 6.18; N, 7.31; P, 4.05%]; 31P NMR (CDCl3):
15.39 (s); 1H NMR (CDCl3): 1.50 (s, 9H), 1.53 (s, 9H),
5.10 (dd, J = 12.2, 25.0Hz, 2H), 5.53 (dd, J = 9.7, 22.0Hz,
1H), 5.79 (d, J = 6.9Hz, 1H), 6.90–7.64 (m, Ar–H, 19H),
10.36 (s, 1H), 11.60 (s, 1H); 13C NMR (CDCl3): 28.16 (d,
J = 7.2Hz), 52.41 (d, J = 154.0Hz), 67.59, 79.75, 83.87,
120.49 (t, J = 3.3Hz), 122.44, 125.40, 128.27 (d,
J = 5.7Hz), 128.58, 128.80 (d, J = 8.5Hz), 129.74,
130.36, 135.96, 137.31, 150.01 (d, J = 11.6Hz), 155.41,
153.43, 159.57, 163.49.
References and notes
1. Duffy, M. J. Curr. Pharm. Des. 2004, 10, 39–49.
2. (a) Oleksyszyn, J.; Powers, J. C. Biochemistry 1991, 30,
485–493; (b) Oleksyszyn, J.; Boduszek, B.; Kam, C.-M.;
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37, 3969–3976.
3. Wang, C.-L. J.; Taylor, T. L.; Mical, A. J.; Spitz, S.;
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Hamilton, R.; Walker, B. J.; Walker, B. Tetrahedron Lett.
1993, 34, 2847–2850.
5. Cheng, L.; Goodwin, C.; Scully, M. F.; Kakkar, V. V.;
Claeson, G. Tetrahedron Lett. 1991, 32, 7333–7336.
6. Joossens, J.; Van der Veken, P.; Lambeir, A.-M.; August-
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7. This work is the subject of patent application (P368474).
8. Oleksyszyn, J.; Subotkowska, L.; Mastalerz, P. Synthesis,
1979, 985–986.
10. Duringpeptide synthesis we discovered that the hydro-
bromide salt of the p-nitro derivative is highly unstable in
alkaline medium––even addition of Et3N prevents peptide
formation. The Cbz-protected derivative is more stable,
but after 12h in weak-alkaline solution (DMSO–Et3N) we
observed, by 31P NMR monitoring, formation of a
degradation product. This abnormal behaviour and
probable mechanism leadingto this instability, is currently
the subject of additional investigations.
11. Wiesner, J.; Wißner, P.; Dahse, H.-M.; Jomaa, H.;
Schlitzer, M. Bioorg. Med. Chem. Lett. 2001, 9, 785–792.
12. (a) Cucha, S.; Costa, M. B.; Napolitano, H. B.; Lariucci,
C.; Vencato, I. Tetrahedron 2001, 57, 1671–1675; (b) Kim,
K. S.; Qian, L. Tetrahedron Lett. 1993, 34, 7677–7680.
9. 1H, 13C NMR and 31P NMR spectra were recorded at
300.13, 75.47 and 121.50MHz, respectively.
1a: White crystals, mp 150ꢁC [Found C, 62.36; H, 4.70; N,
5.30; P, 5.80. C27H23N2O7P requires C, 62.55; H, 4.47; N,
5.40; P, 5.97%]; 31P NMR (CDCl3): 17.31 (s); 1H NMR
(CDCl3): 5.12 (q, J = 12.1Hz, 2H), 5.67 (dd, J = 8.8,
23.2Hz, 1H), 6.16 (dd, J = 6.0, 9.0Hz, 1H), 6.94–8.35 (m,
Ar–H, 19H); 13C NMR (CDCl3): 52.36 (d, J = 155.8Hz),
67.91, 120.20 (dd, J = 4.4, 12.4Hz), 122.96 (d, J = 6.1Hz),
123.56, 125.75 (d, J = 3.0Hz), 128.28, 128.57 (d,
J = 11.1Hz), 129.83, 129.92, 134.19, 135.65, 136.72,
148.44, 150.07, 155.6.

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M. Sienczyk, J. Oleksyszyn / Tetrahedron Letters 45 (2004) 7251–7254
13. The inhibitory effects of inhibitors on the enzymatic
activity of trypsin from bovine pancreas were evaluated
using N-a-benzoyl-L-arginine 4-nitroanilide as a chromo-
genic substrate. The change of absorbance was measured
at 410nm at room temperature (Biochrom 4060). The
assay buffer used was HEPES 0.1M (pH = 7.5) containing
0.01M CaCl2. The final concentration was 0.15lM for
trypsin and 100lM for its substrate.