A new non-natural arginine-like amino acid derivative with a sulfamoyl group in the side-chain

Article abstract of DOI:10.1007/s00726-009-0267-2

Sulfamoylation of the l-ornithine methyl ester side-chain generates a non-natural arginine isostere which can be coupled with N-Fmoc-l-proline to synthesize analogues which maintain the structural characteristics of the biologically important Pro-Arg dipe

Full text of DOI:10.1007/s00726-009-0267-2

Amino Acids (2010) 38:691–700
DOI 10.1007/s00726-009-0267-2
ORIGINAL ARTICLE
A new non-natural arginine-like amino acid derivative
with a sulfamoyl group in the side-chain
Rosaria De Marco Æ Maria L. Di Gioia Æ
Antonella Leggio Æ Angelo Liguori Æ
Francesca Perri Æ Carlo Siciliano Æ Maria C. Viscomi
Received: 16 December 2008 / Accepted: 19 February 2009 / Published online: 13 March 2009
Ó Springer-Verlag 2009
Abstract Sulfamoylation of the L-ornithine methyl ester
side-chain generates a non-natural arginine isostere which
can be coupled with N-Fmoc-L-proline to synthesize ana-
logues which maintain the structural characteristics of the
biologically important Pro-Arg dipeptide sequence. As a
probe of its biological importance, the sulfamoylated
amino acid derivative was also incorporated as P1 residue
in tripeptide structures matching the C-terminal subse-
quence of fibrinogen. The reported results demonstrate that
the functionalization of L-ornithine side-chain with a neu-
tral sulfamoyl group can generate an arginine bioisostere
which can be used for the synthesis of prototypes of a new
class of human thrombin inhibitors.
EDCI
1-(3-Dimethylaminopropyl)-3-
ethylcarbodiimide hydrochloride
Trifluoroacetic acid
TFA
a-CHCA
DEPT
a-Cyano-4-hydroxy-trans-cynnamic acid
Distorsionless enhancement by polarization
transfer
TOCSY
Total correlation spectroscopy
DQF-COSY Double quantum filtered correlation
spectroscopy
TLC
FCC
Thin layer chromatography
Flash column chromatography
Keywords Ornithine ꢀ Sulfamoyl group ꢀ
Arginine bioisosteres ꢀ Tripeptides ꢀ Thrombin inhibitors ꢀ
Anticoagulants
Introduction
Arginine is a natural amino acid containing a strongly basic
guanidine residue which is protonated at physiological pH
values. Either alone or as a constituent of peptide struc-
tures, arginine plays a leading role in establishing non-
covalent interactions with the active sites of a large number
of enzymes involved in biologically important processes
(Schug and Lindner, 2005; Tyndall et al. 2005; Hadden
et al. 2005; Maryanoff 2004; Sugase et al. 2004; Reddy
et al. 2004; Tung and Weissleder 2003; Kim et al. 2003;
Rockwell et al. 2002; Pellegrini et al. 2002; James et al.
1999; Shearer et al. 1997) and of the eukaryotic 26S pro-
teasome (Groll et al. 2006). Arginine-rich peptide motifs
bound to RNA and DNA strands are also involved in the
natural fate of nucleic acids (Austin et al. 2002). However,
the requirement for arginine as the major determinant for
selective recognition in biological systems is not rigorous.
In fact, protonation of the guanidine residue determines
lack of oral bioavailability and selectivity, and increases
the toxicity of many arginine containing substrates and
Abbreviations
DMF
N,N-Dimethylformamide
Methyl-tert-butyl-ether
MTBE
DMAP
DIEA
HOBt
4-(N,N-Dimethylamino)pyridine
N,N-Diisopropylethylamine
1-Hydroxybenzotriazole
Electronic supplementary material The online version of this
article (doi:10.1007/s00726-009-0267-2) contains supplementary
material, which is available to authorized users.
R. De Marco ꢀ M. L. Di Gioia ꢀ A. Leggio ꢀ A. Liguori (&) ꢀ
F. Perri ꢀ C. Siciliano ꢀ M. C. Viscomi
Dipartimento di Scienze Farmaceutiche,
`
Universita della Calabria, Via P. Bucci, Cubo 15/C,
87036 Arcavacata di Rende (CS), Italy
e-mail: A.Liguori@unical.it
123

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R. De Marco et al.
NHBoc
NHBoc
inhibitors. Replacement of arginine by any other amino
acid in homologous peptide sequences, modification of its
side-chain or guanidine moiety (Kokko et al. 2001) by
introducing non-polar and/or neutral groups of reduced
basicity, and elimination of the cationic site from the side-
chain (Zega et al. 2004; Tapparelli et al. 1993) represent
valuable approaches to afford a series of pharmaceutically
relevant synthetic peptides (Schmuck and Geiger 2005;
CH2N2
CH2Cl2
r.t., 15 min
Fmoc
OH
Fmoc
OMe
O
N
N
H
H
O
1
2
TFA
CH2Cl2
0 °C, 15 min
ˇ ˇ
Isaacs et al. 2006; Peterlin-Masic et al. 2003; Powers et al.
+
-
Method A:
4, DIEA, CH2Cl2
r.t., 2 h; 65%
NH3 CF3CO2
2002; Lee et al. 2000) of greater protease specificity, bio-
availability, inhibition potency, and stability against
proteolysis together with improved binding affinities for
many biologically relevant receptors (Balbo et al. 2003;
Lee et al. 2003; St-Denis et al. 2002; Fischer et al. 2000).
The growing demand for an ideal protease inhibitor has
increased the need for assorted libraries of synthetic com-
pounds. In this context, non-proteinogenic amino acids are
of considerable interest.
6
Method B:
5, DIEA, CH2Cl2
r.t., 16 h; 92%
OMe
FmocHN
3
O
NHSO2NHBoc
OMe
O
S
O
_
N
+
Cl
NHBoc
Boc
S
N
N
Fmoc
O
O
N
H
4
5
Motivated by pharmacological interest, and by the
plethora of biological aspects related to the role of Arg and
of its modified isosteres, we have exploited a facile syn-
thetic access to a new L-ornithine derivative with the aim to
develop a new class of human thrombin inhibitors. We
report the synthesis of the polar and uncharged arginine-
like derivative 6 (Fig. 1), a compound featuring a masked
sulfamoyl group on the d carbon atom of the side-chain in
substitution of the highly basic guanidine residue of natural
arginine. The selected group is a constituent of biologically
active sulfonamides (Schaal et al. 2001; Langenhan et al.
2001; Supuran et al. 2000) and sulfamoyl carbamates
(Dougherty et al. 2005), but among the different moieties
proposed for the chemical functionalization of the L-orni-
thine side-chain (Salemme et al. 1997) no mention can be
found in the literature about the employment of the sulfa-
moyl group. The NHSO2NH2 moiety in the L-ornithine
skeleton could define a particular tetrahedral pharmaco-
phore, surrogate of the guanidine group of arginine,
because it is an efficient H-bonding acceptor (Radkiewicz
et al. 1998) and should confer to compound 6 a weakly
acidic character, due to the possible loss of the SO2NHR
proton (Quan et al. 2003).
O
6
Fig. 1 Synthesis of the amino acid derivative 6
expressed in ppm and calibrated on the resonance fre-
quency of the central line of the solvent signal (CDCl3:
1H, singlet, 7.24 ppm; 13C, triplet, 77.0 ppm. DMSO-d6:
1H, quintet, 2.54 ppm; 13C, septet, 39.5 ppm). Coupling
constants (J) are reported in Hz. 13C-NMR spectra were
recorded at 75 MHz, and 13C-DEPT analyses were
obtained with the WALTZ-16 pulse cpd sequence for the
proton decoupling resolution. Two-dimensional (2D)
1
homonuclear H,1H-TOCSY spectra were obtained using
the MLEV pulse sequence for the isotropic mixing (Bax
and Davis 1985; Braunschweiler and Ernst 1983) with an
80-ms spin lock period. Coupling constant values were
extracted (Rence et al. 1983) from the two-dimensional
homonuclear 1H,1H-DQF-COSY (Derome and William-
son 1990) and the mono-dimensional proton analysis. All
mono- and two-dimensional spectra were recorded at
298 K, and the spectral results were consistent with the
proposed structures. For MALDI MS analysis a 1-lL
portion of a premixed solution of each sample and
a-CHCA (0.3% in TFA) was spotted on the matrix target,
dried at room temperature, and analyzed in the positive
mode. The progress of all reactions was monitored by
TLC using silica gel 60-F254 precoated glass plates, and
UV light (254 nm) or 0.2% ninhydrin in ethanol and
charring as visualizing agent. Kieselgel 60H without
gypsum was used for FCC. The methylene chloride
solution of diazomethane was prepared from N-methyl-
N-nitrosourea using a classical procedure (Leggio 1997).
The concentration of the diazomethane solution (0.66 M)
was obtained by back-titration performed with a standard
Materials and methods
All reagents were used as purchased and without further
purification. All solvents were purified and dried by
standard procedures and distilled prior to use. Melting
points were determined with a Kofler hot-stage apparatus
at atmospheric pressure and are uncorrected. Mono-
dimensional (1D) 1H-NMR spectra were recorded at
300 MHz, by using a dilute solution of each compound in
CDCl3 or DMSO-d6. Chemical shift values (d) are
123

A new non-natural arginine-like amino acid derivative with a sulfamoyl group
693
benzoic acid solution. (CAUTION: diazomethane is
highly toxic; hence, this reagent must be handled care-
fully). Methylene chloride solutions of diazomethane are
stable for long periods if stored on KOH pellets at
-20°C. All reactions were performed using flame-dried
glassware and under an inert atmosphere (dry N2). For in
vitro clotting assays an automatic coagulometer was used
with a maximum coagulation time limit of 500 s for TT
and APTT tests. TT and APTT determinations were
performed with the kits Dade BC Thrombin Reagent and
Dade Actin FSL, respectively.
and finally with a 1:1 MTBE/n-hexane mixture. The
obtained glassy solid was dried under vacuum and imme-
diately subjected to the next reaction step. Rf = 0.11. MS
?
(MALDI) m/z Calcd for C21H25N2O4 369.1810; found:
369.1847 (Dm = ? 10 ppm).
Na-Fmoc-Nd-sulfamoyl(Boc)-L-ornithine
methyl ester (6)
Method A
A solution of the sulfamoyl chloride 4 (0.95 g, 4.62 mmol),
freshly prepared as reported in the literature (Spillane et al.
1998; Kloek and Leschinsky 1976), in dry ethanol-free
CH2Cl2 (3 mL), was added dropwise to a solution of 3
(0.57 g, 1.54 mmol; amount estimated on the basis of a
quantitative transformation of 2 into 3 by acidolysis) in dry
ethanol-free CH2Cl2 (3 mL) containing DIEA (1.18 mL,
6.78 mmol). The resulting mixture was stirred at r.t. and
the progress of the reaction was monitored by TLC
(CHCl3:CH3OH = 95:5). After 2 h, the solvent was
removed under vacuum and the oily residue was solubi-
lised in AcOEt (5 mL). The organic solution was washed
with a 5% aqueous solution of KHSO4 (3 9 5 mL), a 5%
aqueous NaHCO3 (3 9 5 mL) and once with brine (5 mL).
The organic layer was dried over Na2SO4, filtered, and
evaporated to dryness under vacuum. The pale yellow solid
residue was co-evaporated three times with a 1:1 Et2O/
n-hexane mixture to give 6 as a pale yellow glassy solid.
FCC showed pure 6 as a pale yellow powder. Yield: 0.55 g,
65%.
Synthesis of Na-Fmoc-Nd-Boc-L-ornithine
methyl ester (2)
A 0.66 M solution of diazomethane in CH2Cl2 (8.5 mL,
5.6 mmol) was added dropwise to a suspension of 1
(0.73 g, 1.6 mmol) in CH2Cl2 (5 mL). The resulting mix-
ture was stirred at r.t. for 15 min, and the conversion of the
precursor was monitored by TLC (CHCl3:CH3OH = 95:5).
The solvent was removed under reduced pressure condition
and the residue was solubilised in AcOEt (5 mL), and
washed with
a 5% aqueous solution of NaHCO3
(2 9 5 mL) and once with brine (5 mL). The organic
extracts were dried over Na2SO4, filtered and evaporated to
dryness under vacuum. The solid residue was co-evapo-
rated with a 1:1 Et2O/n-hexane mixture and compound 2
was recovered as a white crystalline solid. Yield: 0.72 g,
96%. Mp 149–151°C. Rf = 0.69. 1H-NMR (300 MHz,
CDCl3) d 7.75 (d, J = 7.4 Hz, 2 H), 7.58 (d, J = 7.4 Hz,
2 H), 7.38 (t, J = 7.4 Hz, 2 H), 7.30 (t, J = 7.4 Hz, 2 H),
5.43 (d, J = 8.0 Hz, 1 H), 4.55 (t, J = 6.9 Hz, 1 H),
4.31-4-42 (m, 3 H), 3.74 (s, 3 H), 3.12 (m, 2 H), 1.79-1.94
(m, 1 H), 1.46-1.74 (m; 3 H), 1.42 (s, 9 H) ppm. 13C-NMR
(75 MHz, CDCl3) d 172.8, 156.0, 143.8, 141.3, 128.9,
127.7, 125.1, 119.9, 79.3, 77.5, 67.0, 53.6, 52.5, 47.1, 40.0,
29.8, 28.4, 26.1 ppm. Anal. Calcd for C26H32N2O6: C,
66.65; H, 6.88; N, 5.98. Found: C, 66.86; H, 6.89; N, 5.96.
Method B
Compound 3, obtained from the unblocking of 2 (0.46 g,
1 mmol), and DIEA (0.33 ml, 2 mmol) was added to a
solution of the azanide 6 (0.3 g, 1 mmol) in dry ethanol-
free CH2Cl2 (5 mL). The mixture was stirred at r.t. for
16 h, monitoring the conversion of
3
by TLC
(CHCl3:CH3OH = 95:5). The solvent was removed under
vacuum and the solid residue was solubilised in EtOAc
(10 mL). The organic solution was washed with a 5%
aqueous solution of KHSO4 (3 9 5 mL), a 5% aqueous
solution of NaHCO3 (3 9 5 mL), and once with brine
(5 mL). The organic phase was dried over Na2SO4, filtered
and evaporated to dryness under vacuum to give pure 6 as
pale yellow powder. Yield: 0.49 g, 92%. Mp 119–121°C.
Synthesis of Na-Fmoc-L-ornithine methyl ester
trifluoroacetate (3)
A solution of 2 (0.72 g, 1.54 mmol) in CH2Cl2 (3 mL) was
treated with a solution of TFA in CH2Cl2 (9:1, 5 mL). The
resulting mixture was stirred at 0°C for 15 min. After this
time the consumption of 3 was complete as checked by
TLC (CHCl3:CH3OH = 95:5; compound 3 furnished a
yellow spot to the ninhydrin test), and the volatile com-
ponents of the mixture were removed under vacuum. The
oily residue was co-evaporated with toluene (3 9 5 mL)
1
Rf = 0.56. H-NMR (300 MHz, CDCl3) d 8.01 (s broad,
1 H), 7.74 (d, J = 7.4 Hz, 2 H), 7.58 (d, J = 7.4 Hz, 2 H),
7.38 (t, J = 7.4, 2 H), 7.30 (t, J = 7.4 Hz, 2 H), 5.44
(d, J = 8.3 Hz, 1 H), 5.35 (t, J = 6.16 Hz, 1 H), 4.20-4.43
(m, 3 H), 4.19 (t, J = 6.7 Hz, 1 H), 3.73 (s, 3 H), 3.08
123

694
R. De Marco et al.
(m, 2 H), 1.83-2.00 (m, 1 H), 1.54-1.78 (m, 3 H), 1.46 (s, 9
H) ppm. 13C-NMR DEPT (75 MHz, CDCl3) d 172.8 (CO),
156.1 (CO), 155.9 (CO), 143.8 (C), 141.2 (C), 128.0 (CH),
127.6 (CH), 125.1 (CH), 119.9 (CH), 79.3 (C), 67.0 (CH2),
53.6 (CH), 52.5 (CH3), 47.1 (CH), 40.0 (CH2), 29.7
(CH2), 28.4 (CH3), 26.1 (CH2) ppm. Anal. Calcd for
C26H33N3O8S: C, 57.02; H, 6.07; N, 7.67. Found: C,
57.21; H, 6.09; N, 7.68.
Na-Acetyl-Nd-sulfamoyl(Boc)-L-ornithine
methyl ester (10)
Removal of Fmoc group from 6 by the mercaptoacetic
acid/sodium methoxide reagent system (molar ratio
6/HSCH2CO2H/MeONa 1:5:8)
A solution of 6 (0.23 g, 0.42 mmol) in CH3CN (10 mL)
was reacted with mercaptoacetic acid (0.15 mL, 2.1 mmol)
and sodium methoxide (0.18 g, 3.36 mmol) in CH3OH
(2 mL). TLC monitoring of the reaction mixture showed
complete conversion of 6 after 5.5 h at 50°C. The oily
residue recovered from the work-up of the reaction mixture
(see preparation of compound 8) was immediately acety-
lated with an excess of freshly distilled acetic anhydride, in
a 1:1 mixture of CH2Cl2 (5 mL) and a 10% aqueous
solution of NaHCO3 (5 mL). The biphasic system was
vigorously stirred at r.t., and the reaction was monitored by
TLC (CHCl3:CH3OH = 95:5). After 1 h, the mixture was
concentrated under vacuum and the aqueous residue was
extracted with AcOEt (3 9 5 mL). The organic phase was
Na-Acetyl-Nd-sulfamoyl-L-ornithine methyl ester (8)
Removal of Fmoc group from 6 by the mercaptoacetic
acid/sodium methoxide reagent system (molar ratio
6/HSCH2CO2H/MeONa 1:3:5)
Mercaptoacetic acid (0.09 mL, 1.26 mmol) was added to a
solution of sodium methoxide (0.11 g, 2.1 mmol) in
CH3OH (1 mL) at 0°C. A solution of 6 (0.23 g,
0.42 mmol) in CH3CN (10 mL) was then added and the
resulting mixture was maintained under magnetic stirring
at 50°C. After 3.5 h, TLC (CHCl3:CH3OH = 95:5)
showed the complete conversion of 6. The mixture was
made acidic (pH 5) by adding 0.1 N aqueous solution of
HCl, and extracted with Et2O (3 9 10 mL). The ethereal
extracts were discarded off and the aqueous phase was
made basic (pH 8) with a 10% aqueous solution of
NaHCO3, and then extracted with AcOEt (3 9 10 mL).
The collected organic layers were dried over Na2SO4,
filtered and evaporated to dryness under vacuum to give an
oily residue which was immediately treated with an excess
of freshly distilled acetic anhydride, in a 1:1 mixture of
CH2Cl2 (5 mL) and a 10% aqueous solution of NaHCO3
(5 mL). The biphasic system was vigorously stirred
at r.t., and the reaction was monitored by TLC
(CHCl3:CH3OH = 95:5). After 1 h, the mixture was con-
centrated under vacuum and the aqueous residue was
extracted with AcOEt (3 9 5 mL). The organic phase was
washed with
a 5% aqueous solution of NaHCO3
(3 9 5 mL) and once with brine (5 mL), then dried over
Na2SO4, filtered, and evaporated to dryness under vacuum.
The oily residue was chromatographed to give 10 as the
only reaction product, as pale yellow foam, with the
following yields: 0.14 g, 90%. Rf = 0.65. 1H-NMR
(300 MHz, CDCl3) d 8.28 (s broad, 1 H), 6.54 (d,
J = 7.9 Hz, 1 H), 5.74 (t, J = 6.8 Hz, 1 H), 4.56 (m, 1 H),
3.71 (s, 3 H), 3.05 (m, 2 H), 2.0 (s, 3 H), 1.52-1.96 (m,
4 H), 1.43 (s, 9 H) ppm. 13C-NMR (75 MHz, CDCl3)
d 172.9, 170.6, 150.5, 83.5, 52.5, 51.6, 42.9, 29.5, 28.0,
25.1, 22.9 ppm. Anal. Calcd for C13H25N3O7S: C, 42.50;
H, 6.86; N, 11.44. Found: C, 42.59; H, 6.88; N, 11.46.
Synthesis of dipeptide 12
washed with
a
5% aqueous solution of NaHCO3
A solution of Fmoc-Pro-OH (0.14 g; 0.42 mmol) in dry
ethanol-free CH2Cl2 (2 mL) was treated with HOBt
monohydrate (0.07 g, 0.46 mmol), EDCI (0.1 g,
0.52 mmol), DIEA (0.18 mL, 1.03 mmol), and the result-
ing mixture was stirred at r.t. for 12 h. Compound 9
(0.14 g, 0.42 mmol; amount estimated on the basis of a
complete conversion of 6 in the unblocking step performed
as described for the preparation of 10) solubilised in dry
ethanol-free CH2Cl2 (2 mL) was then added, and the
stirring was maintained for further 12 h. After this time,
TLC (CHCl3:CH3OH = 95:5) showed the complete
consumption of 9. The mixture was then evaporated to
dryness under vacuum and the residue was solubilised with
AcOEt (5 mL), washed with a 5% aqueous solution of
KHSO4 (3 9 5 mL), 5% aqueous solution of NaHCO3
(3 9 5 mL) and once with brine (5 mL), then dried over
Na2SO4, filtered, and evaporated to dryness under vacuum.
The solid residue was purified by FCC to give the acety-
lated compound 8 as pale yellow foam with the following
yields: 0.03 g, 21%. Rf = 0.34. 1H-NMR (300 MHz,
DMSO-d6) d 8.19 (d, J = 8.0 Hz, 1 H), 6.44 (m, 3 H), 4.19
(m, 1 H), 3.62 (s, 3 H), 2.85 (m, 2 H), 1.84 (s, 3 H),
1.37-1.81 (m, 4 H) ppm. 13C-NMR (75 MHz, DMSO-d6) d
172.7, 169.4, 51.7, 41.9, 39.8, 28.3, 25.4, 22.1 ppm. Anal.
Calcd for C8H17N3O5S: C, 35.95; H, 6.41; N, 15.72.
Found: C, 36.05; H, 6.40; N, 15.72.
FCC of the crude material recovered from the reaction
furnished compound 10 as the principal product. Yield:
0.11 g, 68%.
123

A new non-natural arginine-like amino acid derivative with a sulfamoyl group
695
(3 9 5 mL), and once with brine (5 mL). The organic
layer was dried over Na2SO4, filtered and evaporated to
dryness under vacuum to give a solid residue which was
triturated with a 1:4 MTBE/n-hexane mixture. The pre-
cipitate was collected and dried under vacuum. Dipeptide
12 was recovered pure as a yellowish powder. Yield:
the basis of a complete conversion of 12 in the unblocking
step performed as described) solubilised in freshly distilled
DMF (2 mL) was then added dropwise to the mixture, and
the stirring was maintained for further 24 h. After this time,
TLC (CHCl3:CH3OH = 95:5) showed the complete con-
sumption of Boc-D-Phe-OH. The mixture was evaporated
to dryness under vacuum and the solid residue was solu-
bilised with AcOEt (5 mL), washed with a 5% aqueous
solution of KHSO4 (3 9 5 mL), 5% aqueous solution of
NaHCO3 (3 9 5 mL), and once with brine (5 mL). The
organic layer was dried over Na2SO4, filtered and evapo-
rated to dryness under vacuum to give a solid residue
which was triturated with a 1:4 MTBE/n-pentane mixture.
The precipitate was collected and dried under vacuum.
Tripeptide 13 was recovered pure as yellowish powder.
Yield: 0.30 g, 89%. 1H-NMR (300 MHz, CDCl3) 70:30
mixture of rotamers A and B d 7.18-7.41 (m, 6 H), 7.06 (d,
J = 7.1 Hz, 1 H), 6.24 (m, 0.3 H, B), 6.02 (m, 1 H), 5.89
(d, J = 6.9 Hz, 0.7 H, A), 4.52-4.74 (m, 0.3 H, B), 4.50 (m,
0.7 H, A), 4.30-4.48 (m, 0.3 H, B), 4.28 (m, 0.7 H, A), 4.17
(m, 1 H), 3.70-3.90 (m, 1 H), 3.76 (s, 3 H), 3.52 (m, 0.3 H,
B), 3.49 (m, 1 H), 3.28 (m, 0.7 H, A), 2.90-3.16 (m, 0.3 H,
B), 2.67 (m, 0.7 H, A), 1.10-2.15 (m, 8 H), 1.52 (s, 3 H),
1.41 (s, 3 H) ppm. 13C-NMR (75 MHz, CDCl3) 70:30
mixture of the two rotamers A and B d 172.2, 172.0, 171.7,
170.9, 157.03, 155.3, 136.22, 134.7, 129.8, 129.6, 129.4,
129.1, 128.5, 128.0, 127.1, 126.8, 124.3, 120.4, 60.2, 55.7,
53.8, 52.44, 51.9, 51.4, 49.1, 47.2, 43.0, 41.7, 40.4, 39.4,
33.9, 33.3, 33.1, 32.7, 29.6, 28.3, 28.1, 25.6, 25.3, 25.0,
24.7. MS (MALDI) m/z Calcd for C30H48N5O10S?
669.3041; found: 669.3135 (Dm = ? 14 ppm). Anal.
Calcd for C30H47N5O10S: C, 53.80; H, 7.07; N, 10.46.
Found: C, 52.7; H, 7.33; N, 10.04.
1
0.24 g, 88%. H-NMR (300 MHz, CDCl3) 75:25 mixture
of rotamers A and B d 8.43 (s broad, 0.75 H, A), 7.74 (d,
J = 7.4 Hz, 2 H), 7.60 (m, 2 H), 7.52 (s broad, 0.25 H, B),
7.38 (t, J = 7.4 Hz, 2 H), 7.29 (t, J = 7.4 Hz, 2 H), 6.75
(d, J = 8.1 Hz, 0.75 H, A), 6.57 (s broad, 0.25 H, B), 5.46
(t, J = 6.9 Hz, 0.75 H, A), 5.33 (s broad, 0.25 H, B), 4.58
(m, 1 H), 4.41 (m, 3 H), 4.13-4.30 (m, 2 H), 3.71 (s, 3 H),
3.31-3.65 (m, 2 H), 2.90-3.29 (m, 2 H), 2.11 (m, 2 H), 1.91
(m, 2 H), 1.56-1.78 (m, 4 H), 1.43 (s, 2.25 H, A ? B), 1.39
(s, 6.75 H, A ? B) ppm. 13C-NMR (75 MHz, CDCl3)
75:25 mixture of the two rotamers A and B d 172.3, 171.9,
155.7, 150.7, 144.0, 143.7, 141.3, 127.7, 127.1, 125.2,
120.0, 83.5, 67.9, 60.5, 52.5, 51.7, 47.1, 43.1, 31.6, 29.7,
29.5, 28.9, 27.9, 24.8, 24.7 ppm. MS (MALDI) m/z Calcd
for C31H41N4O9S? 644.2523; found: 644,2452 (Dm =
-11 ppm). Anal. Calcd for C31H40N4O9S: C, 57.75; H,
6.25; N, 8.69. Found: C, 57.94; H, 6.27; N, 8.67.
Synthesis of tripeptide 13
Unblocking of 12
A solution of 12 (0.32 g, 0.5 mmol) in CH3CN (10 mL)
was reacted with mercaptoacetic acid (0.18 mL, 2.5 mmol)
and sodium methoxide (0.21 g, 4.0 mmol) in CH3OH
(2 mL). The progress of the reaction was monitored by
TLC (CHCl3:CH3OH = 95:5), showing the complete
conversion of 6 after 5.5 h at 50°C. The mixture was then
made acidic (pH 5) by adding a 0.1 N aqueous solution of
HCl, and extracted with Et2O (3 9 10 mL). The ethereal
extracts were discarded off and the aqueous phase was
made basic (pH 8) with a 10% aqueous solution of
NaHCO3, and then extracted with AcOEt (3 9 10 mL).
The collected organic layers were dried over Na2SO4, fil-
tered and evaporated to dryness under vacuum to give a
glassy pale yellow solid which was used in the next step of
coupling without further purification.
Synthesis of tripeptide trifluoroacetate 14
A solution of 13 (0.30 g; 0.45 mmol) in CH2Cl2 (3 mL)
was treated with a solution of TFA in CH2Cl2 (9:1, 5 mL).
The resulting mixture was stirred at 0°C for 30 min. After
this time the consumption of 13 was complete as checked
by TLC (CHCl3:CH3OH = 95:5; compound 14 furnished a
yellow spot to the ninhydrin test), and the volatile
components of the mixture were removed under vacuum.
The oily residue was co-evaporated with toluene
(3 9 5 mL), absolute EtOH (3 9 5 mL), and triturated
with a 1:2 MTBE/n-pentane mixture. The obtained pre-
cipitate was collected, dried under vacuum and finally
lyophilized to give 14 as a pale yellow glassy solid. Yield:
Coupling
A solution of Boc-D-Phe-OH (0.13 g; 0.5 mmol) in freshly
distilled DMF (2 mL) was treated with EDCI (0.31 g,
0.52 mmol), HOBt monohydrate (0.08 g, 0.55 mmol),
DIEA (0.27 mL, 1.6 mmol), and the resulting mixture was
stirred at r.t. for 2 h. The glassy solid obtained from the
unblocking of 12 (0.21 g, 0.5 mmol; amount estimated on
1
0.25 g, 90%. Rf = 0.10. H-NMR (300 MHz, DMSO-d6/
D2O) d 7.11-7.40 (m, 5 H), 6.50 (s broad, 2 H), 4.02-4-41
(m, 3 H), 3.61 (s, 3 H), 3.4-3.6 (m, 2 H, partially over-
lapped by the water signal), 2.68-3.14 (m, 4 H), 1.34-1.90
123

696
R. De Marco et al.
(m, 8 H) ppm. 13C-NMR (75 MHz, DMSO-d6/D2O) d
172.9, 171.8, 166.8, 134, 94, 130.0, 129.0, 127.9, 59.7, 52.3,
52.1, 49.2, 47.2, 42.5, 37.2, 29.6, 28.6, 26.0, 24.2. MS
(MALDI) m/z Calcd for C20H32N5O6S? 470.2070; found:
470.2014 (Dm = -12 ppm). Anal. Calcd for C22H31N5O5S.
CF3CO2H: C, 45.28; H, 5.53; N, 12.00. Found: C, 44.02; H,
5.68; N, 11.65.
Determination of activated partial thromboplastin
time (APTT)
A second series of samples prepared as described for TT
determinations was treated with the activated partial
thromboplastin time reagent. The coagulometer automati-
cally dispensed the appropriate reagent in the sample of
each tube. The time for the appearance of a fibrin clot was
measured and each value was obtained from a set of three
separate experiments. The same blank used for the control
value of clotting time for TT tests was used to determine a
control value of 31.2 sec (mean value; SD = 1.2; n = 3)
for clotting time in APTT tests. Clotting time values
exceeding the coagulometer limit (500 s) were not
determined.
Clotting assays for tripeptides 13 and 14
Preparation of stock solution
Tripeptides 13 and 14 to be tested were dissolved in DMSO
just prior to use and diluted with PBS buffer (pH 8) to yield
a final 152 mM stock solution for tripeptide 13, and a
337 mM stock solution for tripeptide trifluoroacetate 14.
Results and discussion
The preparation of 6 started from Na-Fmoc-Nd-Boc-L-
ornithine (1), and involved a three-step procedure (Fig. 1).
Methyl ester 2 was obtained by treatment of the starting
precursor 1 with a dichloromethane solution of diazo-
methane (Di Gioia et al. 2003), at room temperature.
Methylation afforded 2 in yields of 96%, and pure enough
to be subjected to the next synthetic step without need for
chromatography. Removal of the Boc group from 2 was
performed by acidolysis at 0°C, using TFA. Conversion of
2 was complete and the resulting salt 3, not isolated, was
immediately subjected to sulfamoylation. The a-amino
function in the side-chain of 3 was finally functionalized
using the sulfamoylating reagent 4 (Fig. 1, Method A), in
the presence of DIEA. Reagent 4 was efficiently prepared
from commercially available chlorosulfonyl isocyanate
(CSI) and tert-butanol in dichloromethane, under the
experimental conditions already described for conventional
routes to this reagent (Spillane et al. 1998; Kloek and
Leschinsky 1976). Although 4 is rapidly formed in a
quantitative yield by this approach, its use in the derivati-
zation of the salt 3 proved to be troublesome. The chloride
4 is very unstable, and it cannot be stored for a long period.
This aspect limits the possibility of an exact stoichiometric
dosage of 4 imposing that the reagent must be prepared
immediately before any sulfamoylation step. Moreover,
under the experimental conditions adopted for the experi-
ment in which 4 is used, the methyl ester 6 can be
recovered as a pure product only after chromatography, and
in yields not exceeding 65%. We found the zwitterionic
azanide 5 to be the optimal reagent for the sulfamoylation
of the trifluoroacetate 3 (Fig. 1, Method B). It can be
obtained in very high yields and purity by a known pro-
cedure (Winum et al. 2001). Since 5 is a stable crystalline
solid, it can also be stored for prolonged periods and easily
Clotting assays
In vitro coagulation assays were performed with pooled
human plasma. Clotting time was measured using an
automatic coagulometer according to the manufacturer’s
instructions. In order to exclude any influence of DMSO in
clotting assays, a blank prepared by mixing the pooled
human plasma (800 lL) and DMSO (200 lL) was sub-
jected to TT and APTT tests. Control values of both the
parameters were not modified with respect to those regis-
tered for a blank prepared by mixing the pooled human
plasma (800 lL) and PBS (200 lL).
Determination of thrombin time (TT)
To each of the tubes containing plasma (800 lL),
increasing amounts (25, 50, 75, 100, 125, and 150 lL;
corresponding to 8.4, 16.8, 25.3, 33.7, 42.1, and
50.5 lmol, respectively) of the stock solutions of tripep-
tide 14 were added. The same protocol was applied to
prepare samples of tripeptide 13. The resulting samples
were diluted to a total volume of 1 mL by adding PBS,
vortexed, and incubated at 37°C for 1 minute, and then
placed on the instrument sample wheel. The coagulometer
automatically dispensed thrombin time reagent in the
sample of each tube. The time for the appearance of a
fibrin clot was measured and each value was obtained
from a set of three separate experiments. Control value of
clotting time for TT tests was determined for three
experiments using a blank prepared by mixing the pooled
human plasma (800 lL) and PBS (200 lL), and was
15.5 s (mean value; SD = 0.6; n = 3). Clotting time
values exceeding the coagulometer time limit (500 s)
were not determined.
123

A new non-natural arginine-like amino acid derivative with a sulfamoyl group
697
NHSO2NHBoc
OMe
dosed for any purpose. The reaction of trifluoroacetate 3
with 5 was performed in the presence of DIEA to generate
the free d-amino group; and a simple work-up of the
reaction mixture allowed the recovery of the methyl ester 6
in yields of 92%, with no need for chromatography. The
use of a base, e.g. DIEA, is strongly recommended: without
the base, compound 6 can be recovered pure only after
column chromatography and in yields not higher than 65%.
DMAP produced during the sulfamoylation did not pro-
voke removal of the base-labile protecting group Fmoc.
The methyl ester 6 was fully characterized by NMR anal-
ysis, and both 1D- and 2D-homonuclear techniques
confirmed the structure proposed for the new arginine-like
NHSO2NHBoc
OMe
Acetylation
Ac
N
H2N
H
O
O
9
10 (90%)
HSCH2CO2H/MeONa
(5:8)
MeCN/MeOH
50 °C, 5.5 h
6
HSCH2CO2H/MeONa
(3:5)
MeCN/MeOH
50 °C, 3.5 h
NHSO2NH2
NHSO2NH2
Acetylation
+
10 (68%)
9 +
derivative 6. The Fmoc group was removed from the
a-amino function of 6 avoiding the use of nitrogenated
species. This was done in order to facilitate the separation
Ac
OMe
O
OMe
N
H2N
H
O
7
8 (21%)
of the unprotected methyl ester from the crude reaction
Fig. 2 Removal of the Fmoc group from 6
mixture. As reported elsewhere (Di Gioia et al. 2004a, b;
Leggio et al. 2000), the Fmoc group can alternatively be
removed from the corresponding methyl esters by reagent
systems composed of AlCl3 and toluene or N,N-dim-
ethylaminoaniline, but acid-labile protecting groups, e.g.
Boc, are not compatible with the experimental conditions
adopted for the Lewis acid-assisted treatment. Fmoc group
was straightforwardly removed by treating 6 with the
reagent system composed of mercaptoacetic acid and
sodium methoxide, upon conditions similar to those
adopted for the removal of the nosyl group from N-meth-
ylated amino acid derivatives (Di Gioia et al. 2003). The
appropriate stoichiometric ratio between the two compo-
nents of the reagent system, referred to one equivalent of 6,
was found to be 5:8. Upon these conditions, treatment of 6
at 50°C for 5.5 h afforded 9 (Fig. 2), which was not sep-
arated and immediately transformed into the corresponding
acetyl derivative 10 by treating the crude material obtained
from the unblocking step with an excess of acetic anhy-
dride. Compound 10 was obtained in yields of 90%, which
was reliable with the complete conversion of the precursor
6 into 9. Removal of the Fmoc masking group was
chemospecific, since both the Boc-protected sulfamoyl
moiety and the methyl ester functionality were not affected
by the treatment. A different composition of the reagent
system mercaptoacetic acid/sodium methoxide, e.g. a 3:5
stoichiometric ratio, gave 10 in lower yields (68%) together
with modest amounts (21%) of 8. The latter compound was
probably formed through the unprotected intermediate
species 7, which could be generated by a concomitant
removal of both the Fmoc and Boc masking groups
(Fig. 2).
determining the absolute specificity and bioavailability of
peptide inhibitors of proteolytic enzymes (Koskinen et al.
2005; Groll et al. 2002; De Tar De Los and Luthra 1977).
In order to evaluate the structural and conformational role
of 6 in short peptide sequences, we prepared the dipeptide
N-Fmoc-L-Pro-L-Orn-(Nd-SO2NHBoc)-OMe
(12)
by
adopting a Fmoc-chemistry protocol usually used for the
solution synthesis of peptides, upon conditions that can
avoid the racemisation of the reaction partners (Romoff
2003). N-Fmoc-L-proline (11) was activated with the sys-
tem HOBt/EDCI/DIEA and allowed to react with 9,
obtained from 6 after removal of the Fmoc group by
treatment with a 5:8 mixture of mercaptoacetic acid and
sodium methoxide as previously described. The only
product obtained from the reaction was dipeptide 12, which
was recovered in yields of 88% after flash column chro-
matography (FCC). 1D- and 2D-homonuclear NMR
analysis of 12 showed resonances which were consistent
with the proposed structure. The 1D 1H spectrum, recorded
in CDCl3 at 298 K, showed the presence of two sets of
rotamers arising from the slow trans/cis isomerization
around the CO–N linkage between the Fmoc group and the
proline ring. Based on the integration of non-overlapping
signals, e.g. those appearing at 6.75 and 6.57 ppm, attrib-
utable to the a-NH proton of the ornithine skeleton
included in the trans form and the same proton in the cis
rotamer, respectively, we calculated a trans/cis ratio of
75:25 for the rotamers. The precise ratio was determined
by the integration of the signals of both the a-NH protons
which were assigned to the respective rotamer in analogy
with the results reported in the literature for small dipep-
tides containing proline as the N-terminal residue
(Sugawara et al. 2001). 2D-homonuclear NMR showed
resonances clearly attributable to the protons belonging to
Since proline occupies a special place among the natural
amino acids, we coupled 9 with N-Fmoc-L-proline. The
sequence Pro–Arg, in fact, is often selected as an ideal
chemical probe in conformational studies, and in
123

698
R. De Marco et al.
the proline and the ornithine skeletons, respectively. In
particular, the cross-signals appearing in the contour plot of
the TOCSY analysis performed in CDCl3 at 298 K con-
firmed the presence of the trans/cis rotamer mixture and
showed correlation patterns typical of the peptide sequence
Pro-Orn. No other signals relative to protons attributable to
any possible diastereomer of 12 appeared in the 1D and 2D
proton spectra. On the other hand, the 1D- and 2D-NMR
analysis of a sample of the crude dipeptide 12, revealed
resonances attributable only to the given structure, con-
firming that, limited to the sensitivity of NMR techniques,
no racemisation of the a-carbon atom present in 6 occurred
during the coupling. Thus, the stereochemistry of the pro-
tected arginine-like derivative 6 is retained both during its
synthetic process and the Fmoc removal, as well as in the
final coupling step.
further lyophilisation in order to remove all the residues of
tert-butanol and any trace of possible co-products generated
1
during the unblocking step. The H NMR spectrum of the
trifluoroacetate salt 14 registered at 298 K in DMSO-d6/D2O
showed exchange of all protons on the N atoms, except for
those of the SO2NH2 moiety, but did not display well
resolved resonances for the other protons. Structure of 14
was definitively attributed by correlation with the corre-
sponding resonances observed in the proton spectrum
obtained for 13 and the molecular weight of the salt was
confirmed by MALDI mass spectrometry.
Both tripeptides 13 and 14 were then subjected to in
vitro coagulation assays in order to evaluate, in a set of
preliminary experiments, their inhibitory effects against
human thrombin. The TT (Thrombin Time), a measure of
the thrombin-fibrinogen reaction in vitro (Jim 1957), and
the activated partial thromboplastin time (APTT), the
parameter used to evaluate the anticoagulant effect on the
thrombin produced by the intrinsic pathway of the coagu-
lation cascade (Proctor and Rappaport 1961), were
determined using pooled human plasma treated with sam-
ples of 13 and 14 at different concentrations. Among the
two tripeptides, 13 did not show appreciable inhibitory
potency in all the performed experiments, while 14
exhibited a good and dose-dependent activity as deter-
mined by the clotting time of human plasma in TT and
APTT tests. In particular, a sample containing 8.4 lmol of
trifluoroacetate 14 prolonged the clotting time of pooled
human plasma to 217.2 s (mean value, SD = 10.2;
n = 3), about three times the control value fibrin clot time
in TT determination, and to 65.4 s (mean value,
SD = 2.5; n = 3), twice the corresponding control value
in APTT measurements. With a more concentrated sample
of 14 (25.3 lmol) a TT value of 493.7 s was recorded,
while APTT was 123.8 s. Another sample containing
33.7 lmol of 14 showed a not detectable TT, with a value
exceeding the coagulometer time limit (500 s), and an
APTT value of 296.8 s was recorded. A not detectable
([500 s) APTT value was also obtained when a sample of
the potential thrombin inhibitor was prepared using
42.1 lmol of tripeptide 14.
In an effort to evaluate the biological role of the sulfa-
moyl group placed in the L-ornithine side-chain, 6 was
finally used as building block in the synthesis of serine-
protease inhibitors. In particular, compound 6 was coupled
with the dipeptide Boc-D-Phe-Pro-OH to prepare tripeptide
analogues of the C-terminal subsequence of fibrinogen, the
natural thrombin substrate. Thrombin, the blood-clotting
enzyme, is a serine protease with trypsin-like specificity
and is able to cleave Arg-Xaa peptide bonds but only in a
very limited number of substrates (Das and Kimball 1995;
Bode et al. 1992). This enzyme has a critical position in the
blood coagulation cascade and thus a central role in the
regulation of haemostasis (Weitz and Crowther 2002;
Hauptmann and Stu¨rzebecher 1999). Moreover, for the
prevention and treatment of thrombosis the control of
thrombin activity is a key target (McDonald 2005) and the
discovery of new classes of inhibitors of this enzyme could
lead to useful drugs for treating thrombotic disorders,
which constitute a serious source of mortality and mor-
bidity in patients worldwide.
Removal of the Fmoc group from 12 was performed as
previously described for 6 using the system composed by
mercaptoacetic acid and sodium methoxide, in the 5:8 stoi-
chiometric ratio. Boc-D-Phe-OH was then coupled to
dipeptide 12, after activation of the free carboxylic group by
the system HOBt/EDCI/DIEA in the presence of DIEA. The
fully protected tripeptide N-Boc-D-Phe-L-Pro-L-Orn-(Nd-
SO2NHBoc)-OMe (13) was recovered pure by FCC in yields
of 88%, and 1H and 13C NMR analysis confirmed the
expected structure. In particular, the proton spectrum regis-
tered in CDCl3 at 298 K showed the presence of a mixture of
rotamers with a trans/cis ratio of 70:30, due to the different
geometries of the peptide bond between the D-Phe and Pro
residues. Acidolysis of 13 by using a solution of TFA in
CH2Cl2 afforded D-Phe-L-Pro-L-Orn-(Nd-SO2NH2)-OMeꢀ
CF3COOH (14) which was obtained by precipitation of the
crude product from a 1:2 MTBE/n-pentane mixture and
Conclusion
In conclusion, we have synthesized the non-natural argi-
nine-like a-amino acid derivative 6 which displays on the
side-chain a masked sulfamoyl moiety. Compound 6 can
easily be coupled with the dipeptide motif D-Phe-Pro to
produce tripeptides of high biological interest. Since the
neutral polar function in the side-chain of 6, at physio-
logical pH values, is certainly capable of H-bonding
through its heteroatoms (Garrett et al. 1964), it could be
123

A new non-natural arginine-like amino acid derivative with a sulfamoyl group
699
in the AlCl3/N,N-dimethylaniline reagent system. Eur J Org
Chem 4437–4441. doi:10.1002/ejoc.200400321
regarded as an efficient anchoring moiety for a wide series of
substrates and inhibitors in their binding with the recognition
Di Gioia ML, Leggio A, Le Pera A, Siciliano C, Sindona G, Liguori A
(2004b) An efficient and highly selective deprotection of
N-Fmoc-a-amino acid and lipophilic N-Fmoc-dipeptide methyl
esters with aluminium trichloride and N,N-dimethylaniline.
J Pept Res 63:383–387. doi:10.1111/j.1399-3011.2004.00104.x
subsites of biological receptors. In particular, rational mod-
ification of the C-terminal tripeptide lead structure found in
fibrinogen by inserting 6 in substitution of the natural argi-
nine at P1 position generates compounds which are active as
thrombin inhibitors. Thus, 6 can really regarded as an argi-
nine mimic and could be strategic for studies on the
importance of the physiological role of different kinds of
proteases, especially in the discovery of promising candi-
dates for future development of clinically useful agents.
The synthesis of the target peptide and the evaluation of
its anticoagulant activity constitute only the initial phase of
a more complex process that involves more experiments
that could subsequently enhance or reduce the interest for
the same product with regard its use in real situations.
´
Dougherty JM, Jimenez M, Hanson PR (2005) Synthesis of cyclic
sulfamoyl carbamates and ureas via ring-closing metathesis.
Tetrahedron 61:6218–6230. doi:10.1016/j.tet.2005.03.140
Fischer MJ, Giese U, Harms CS, Kinnick MD, Lindstrom TD,
McCowan JR, Mest H-J, Morin JM Jr, Mullaney JT, Paal M,
Rapp A, Ru¨hter G, Ruterbories KJ, Sall DJ, Scarborough RM,
Schotten T, Stenzel W, Towner RD, Um SL, Utterback BG,
Wyss VL, Jakubowski JA (2000) Fused bicyclic Gly-Asp b-turn
mimics with potent affinity for GPIIb-IIIa. Exploration of
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doi:10.1016/S0960-894X(00)00008-1
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Acknowledgment The presented work was financially supported by
`
grants of the Ministero dell’Universita e della Ricerca (MIUR, Italy).
Authors gratefully acknowledge Dr. Soluzzo Cavalcanti for all sug-
gestions and precious discussion about the anticoagulant activities.
¨
Groll M, Gotz M, Kaiser M, Weyher E, Moroder L (2006) TMC-95-
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