New cyanopeptide-derived low molecular weight inhibitors of trypsin-like serine proteases

Article abstract of DOI:10.1002/ardp.200300765

This paper deals with the design, syntheses, and inhibition tests of new low molecular weight thrombin inhibitors utilizing cyanopeptides, the secondary metabolites of cyanobacteria with interesting biological activities, as new lead structures. Starting with aeruginosin 98-B (1) as a lead structure, we have developed and synthesised new, selective acting inhibitors of serine proteases (RA-1005 and RA-1009), which are suitable targets for further structure-activity studies.

Full text of DOI:10.1002/ardp.200300765

300 Radau et al.
Arch. Pharm. Pharm. Med. Chem. 2003, 336, 300–309
Gregor Radau,
Sonja Schermuly,
Alexandra Fritsche
New Cyanopeptide-Derived Low Molecular Weight
Inhibitors of trypsin-like Serine Proteases
This paper deals with the design, syntheses, and inhibition tests of new low molecu-
lar weight thrombin inhibitors utilizing cyanopeptides, the secondary metabolites of
cyanobacteria with interesting biological activities, as new lead structures. Starting
with aeruginosin 98-B (1) as a lead structure, we have developed and synthesised
new, selective acting inhibitors of serine proteases (RA-1005 and RA-1009), which
are suitable targets for further structure-activity studies.
Institute of Pharmacy,
Ernst-Moritz-Arndt-University
Greifswald,
Pharmaceutical/Medicinal
Chemistry, Greifswald,
Germany
Keywords: Cyanobacteria; Cyanopeptides; Peptide syntheses; Rational drug de-
sign; Thrombin inhibitors
Received: January 7, 2003; Accepted: April 1, 2003 [FP 765]
DOI 101002/ardp.200300765 *
X-ray studies of RA-1001-trypsin- and RA-1002-trypsin-
complexes confirm an aeruginosin-analogous binding
Introduction
Contemporary trends in drug discovery from natural
sources favour investigations of the marine environment
to yield numerous, often highly complex, chemical com-
pounds. A considerable number of non-toxic cyanopep-
tides – the secondary metabolites of cyanobacteria
(blue-green algae) – inhibit members of the serine pro-
teases family [1] which play central roles in the human
organism. Thrombin and factor Xa represent the main
actors in the interlaced cycles of blood coagulation and
fibrinolysis. A disturbance of the sensitive balance
caused by malfunctions of enzymes can result in throm-
boembolic complications like venous and arterial throm-
bosis, stroke, restenosis, and recurrent myocardial in-
farction. The development of oral thrombin inhibitors
therefore presents a notable measure for improving the
treatment of the above mentioned disorders.
mode according to the findings described by Sandler et
al. [10].There is a lack of any close interactions of struc-
tural elements of aeruginosin 98-B and its synthetic
analogues with trypsin’s catalytic triad (His57, Asp102,
Ser195) – especially concerning residue Ser195. This
fact is remarkable, because Ser195 usually attacks the
carbonyl group in position P1 of the substrate as a nucle-
ophil (“acylation mechanism”, for details see [8] and
[10]).Therefore, cyanopeptides of the aeruginosin family
as well as their synthetic analogues inhibit trypsin in a
non-classical manner. Solely, interactions of positions
P1 to P4 with subsites S1 to S4 contribute to the inhibito-
ry power. Agmatines guanidinyl group in the RA-1001-
trypsin-complex reaches deeply inside the S1-pocket
forming a salt bridge to the Asp189-carboxylate side
chain.The amide moiety of proline interacts with Gln192
and Ser214. The 3-(4-hydroxyphenyl)propionyl-L-iso-
leucyl core in RA-1001 seems to be too flexible to con-
tact with any moiety of trypsin’s S3/S4 subsites, what is
expressed by a low electron density in the X-ray struc-
ture. Compound RA-1002 shows the same interactions
with S1/S2 subsites of trypsin like RA-1001.Additionally,
the L-leucine unit shows interactions with the backbone
amino acid Gly216.The aromatic residues in both inhibi-
tors show no close contact to the enzyme.These findings
motivated us to work further on the optimization of the
structure/activity relationship of synthetic aeruginosin
analogues.In the course of our continuing studies on the
development of serine proteases inhibitors we focused
our attention on the optimization of the P1-P4 moieties.
Results and discussion
Structural considerations
Until now, fourteen members of the aeruginosin family
have been identified [2–7]. In previous reports, we de-
scribed the syntheses of the thrombin inhibitors RA-
1001 and RA-1002 as well as their precursors (RA-1003
and RA-1004) utilizing aeruginosin 98-B – a secondary
metabolite from cyanobacterium Microcystis aeruginosa
– as the starting lead structure (Figure 1) [8–9]. Com-
pounds RA-1001, RA-1002, and RA-1003 are equipo-
tent thrombin inhibitors (Table 1). Moreover, RA-1002,
RA-1003, and RA-1004 seem to be selective inhibitors
of thrombin [8].
Now we report on the synthesis of five new inhibitors
(RA-1005 – RA-1009) which were modified in positions
P1, P2, P3, and P4 – compared with the lead structures
RA-1001 and RA-1002, respectively. RA-1005 was
modified in P2; the L-proline was exchanged for glycine
what results in a more flexible conformation. In RA-1006
Correspondence: Gregor Radau, Institute of Pharmacy,
Ernst-Moritz-Arndt-University Greifswald, Pharmaceutical/
Medicinal
Chemistry,
Friedrich-Ludwig-Jahn-Str.
17,
D-17487 Greifswald, Germany. Phone: +49 3834 864880, Fax:
+49 3834 864874, e-mail: radau@pharmazie.uni-greifswald.de
© 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
0365-6233/06–07/0300 $ 17.50+.50/0
* In der gedruckten Version war der DOI falsch. Richtig muss es heißen: DOI 10.1002/ardp.200300765

Arch. Pharm. Pharm. Med. Chem. 2003, 336, 300–309
New Cyanopeptide-derived Thrombin Inhibitors 301
Figure 1. Aeruginosin 98-B (1) from Microcystis aeruginosa and synthetic analogues RA-1001 – RA-1009.
Table 1. Inhibitory data of synthetic analogues of aeruginosin 98-B.
Cp.
Ki (µM)
trypsin
thrombin
uPA
plasm
f. Xa
tryptase
RA-1001
RA-1002
RA-1003
RA-1004
RA-1005
RA-1006
RA-1007
RA-1008
RA-1009
32
5.6
8.7
9.0
62
400
>1000
>1000
>1000
>1000
>1000
–
–
–
–
–
>1000
>1000
>1000
>1000
>1000
>1000
140
>1000
>1000
>1000
>1000
>1000
>1000
>1000
54
>1000
>1000
>1000
–
–
–
–
–
–
>1000
>1000
5.4
160
>1000
10.6
>1000
–
>1000
>1000
>1000
>1000
(19 %)a
–
uPA = plasminogen activator urokinase, plasm = plasmin, f. Xa = factor Xa, – = not determined.
a
inhibition at conc. (inhibitor) of 150 µM.
a 4-methylbenzoyl unit was used in P4 instead of the
benzoyl group in RA-1004, and L-leucine was ex-
changed for L-isoleucine in P3 of RA-1007. In RA-1008
the phenylmethanesulfonyl moiety was introduced into
P4 and the length of the basic side chain in P1 was ex-
tended by one carbon atom compared with RA-1002. In
RA-1009 we chose a cyclic guanidine equivalent in posi-
tion P1.
hydrochloride under DCC-catalysis and basic conditions
(DIPEA: diisopropylethylamine; DCC: N,NЈ-dicyclohexyl-
carbodiimide). After hydrolysis of the dipeptide ethyl es-
ter (LiOH/DME: 1,2-dimethoxyethane) the resulting acid
4 was connected with the basic side-chain – the mono-
Boc-substituted 1,4-diaminobutane (6) (Boc: tert-butyl-
oxycarbonyl). This step of the synthesis route was the
limiting factor in each route of this type. The mono-Boc-
substituted diaminoalkanes 6 and 7 were synthesized in
analogy to the procedure described by Krapcho [11] in
high yields using an eightfold excess of 1,4-diaminobu-
tane and 1,3-diaminopropane referred to di-tert-butyl di-
carbonate (Boc2O) in order to avoid the formation of bis-
Boc-substituted diaminoalkanes (Scheme 2). Finally,
the Boc protecting group was cleaved by means of
Syntheses
The synthesis of RA-1005 starts with the synthesis of
3-(4-hydroxyphenyl)propionyl-L-isoleucine (2) which
was described previously [8] (Scheme 1). Compound 2
reacts in a high-yielded reaction with glycine ethyl ester

302 Radau et al.
Arch. Pharm. Pharm. Med. Chem. 2003, 336, 300–309
Scheme 1. Synthesis of RA-1005.
Scheme 2. Syntheses of basic building blocks.
trifluoroacetic acid (TFA) to yield the desired target mole-
cule RA-1005.
In general terms, the right and most successful synthetic
route is very difficult to predict.The synthesis of RA-1007
for example was more effective following the procedure
described in scheme 4 compared with the latter proce-
dure.In the preparation of RA-1007 the central dipeptide
scaffold was synthesised first and the N-terminal ben-
zoyl moiety and the basic C-terminal side chain were in-
troduced in subsequent steps. N-Boc-L-isoleucine was
esterificated with N-hydroxysuccinimide (HOSu) under
DCC catalysis to its succinimidyl ester 17, which suc-
cessfully reacts with completely unprotected L-proline to
give the appropriate N-acylated dipeptide 18. Upon Boc
cleavage (TFA/dichloromethane), the completely unpro-
tected L-Ile-L-Pro (19) reacted with succinimidyl ben-
zoate with a high yield to the benzoyl derivative 20 under
alkaline conditions.The C-terminal basic side chain was
introduced into Bz-L-Ile-L-Pro (20) according to the pro-
cedure described in the synthesis of RA-1006.
In contrast to the synthesis of RA-1005 the benzoylation
of L-leucine methyl ester hydrochloride was the first step
in the preparation of RA-1006 (Scheme 3). N-(4-Methyl-
benzoyl)-L-isoleucine methyl ester (12) was saponified
with LiOH (1M) in dimethoxyethane (DME). Both steps
were carried out using alternative procedures which are
less complicated compared to the methods described in
the literature [12, 13]. The resulting carboxylic acid (13)
was transformed into the dipeptide methyl ester 14 by
condensation with proline methyl ester. Hydrolysis, acti-
vation (DIC), and reaction with mono-Boc-substituted di-
aminopropane (7) yielded the appropriate derivative 16.
Boc cleavage was realized in a TFA/dichloromethane
mixture and closed this very successful synthesis route
to the desired RA-1006.

Arch. Pharm. Pharm. Med. Chem. 2003, 336, 300–309
New Cyanopeptide-derived Thrombin Inhibitors 303
Scheme 3. Synthesis of RA-1006.
Scheme 4. Synthesis of RA-1007.
Compound RA-1008 was prepared in an analogous
manner as described for the synthesis of RA-1006
(Scheme 5).Thus, L-leucine methyl ester hydrochloride
was acylated by phenylmethanesulfonyl chloride to the
appropriate sulfonamide 22.Treatment of the methyl es-
ter with LiOH (1M)/DME yielded the amino acid 23.Cou-
pling of 23 with L-proline methyl ester hydrochloride us-
ing DIC (N,NЈ-diisopropylcarbodiimide) and DIPEA in
dichloromethane and subsequent saponification of the
intermediate product 24 (LiOH/DME) afforded the N-sul-
fonated dipeptide acid 25. In contrast to the previously
reported syntheses, 25 was condensed with bis-tert-
butyloxycarbonyl-agmatine (11) (DIC/dichloromethane)
to the Boc-protected precursor of RA-1008 (26). Treat-
ment of 26 with TFA/dichloromethane gave the guani-
dine derivative RA-1008 in satisfactory yields.
Bis-Boc-agmatine (11) was prepared via two short syn-
theses (Scheme 2).Using the first route, the Boc protect-
ing groups were introduced twice in thiourea by means of
di-tert-butyl dicarbonate – upon deprotonation of thiou-
rea by sodium hydride [14]. Bis-Boc-thiourea (9) was
subjected to the nucleophilic attack of 1,4-diaminobu-
tane to give the desired bis-Boc-agmatine (11) [15]. In a
second route, S-methylisothiouronium sulfate was
acylated by di-tert-butyl dicarbonate in a two-phase-mix-
ture under alkaline conditions to the bis-Boc-substituted
derivative 10 [16, 17], which reacted with 1,4-diaminobu-
tane to 11 [18].

304 Radau et al.
Arch. Pharm. Pharm. Med. Chem. 2003, 336, 300–309
Scheme 5. Syntheses of RA-1008 and RA-1009.
Compound RA-1009 was obtained in very low yield by
condensation of the synthetic precursor of RA-1002, Bz-
L-Leu-L-Pro-OH (27) [8], with N-(2-pyrimidinyl)-1,4-di-
aminobutane (8) – a cyclic guaninidine derivative, which
was synthesised according to the synthesis of N-(2-pyri-
midinyl)-1,3-diaminopropane [19].
The P2 moiety of RA-1005 consists of glycine instead of
L-proline. This modification causes a better conforma-
tional flexibility, which leads to a loss of selectivity. Com-
pound RA-1005 inhibits thrombin only weakly, but reacts
stronger with trypsin – constrained conformations in po-
sition P2 seem to be essential for a selective thrombin
inhibition.This is a further example for the fact that effec-
tive inhibition of thrombin depends on restricted confor-
mations. Introducing a methylene group into the phenyl
ring in P4 of RA-1004 leads to RA-1006 which inhibits
thrombin certainly slightly weaker than RA-1004, but se-
lectively at least. Exchanging L-leucine for L-isoleucine
in P3 (RA-1007) changes the inhibitory selectivity to-
wards factor Xa. The reasons for this surprising and in-
teresting result have to be clearified by further investiga-
tions. Compound RA-1008 shows a satisfying inhibition
of thrombin, but in addition a stronger inhibition of
trypsin, too. Comparing the structural features of RA-
1008 with those of RA-1001, the loss of selectivity
seems to accompany the chain length of four carbon at-
oms (agmatine) in P1.The phenylmethanesulfonyl moie-
ty in P4 may contribute to the stronger inhibition of
trypsin. Compound RA-1009 shows only a weak inhibi-
tion of trypsin (19 % at an inhibitor concentration of
150 µM).The pyrimidine core in P1 does not seem to fit
as good as agmatine or noragmatine into the S1 subsite
of trypsin-like serine proteases.
Enzyme inhibition tests/Conclusions
The measurements were carried out as described by
Stürzebecher et al. [20]; Ki-values were calculated ac-
cording to Dixon [21] using a linear regression program.
Compounds RA-1001 and RA-1002 are potent inhibi-
tors of thrombin (Ki 5.6 µM and 8.7 µM, respectively). In
contrast to RA-1001, aeruginosin 98-B (1) inhibits
trypsin stronger than thrombin (IC50 0.6 and 10.0 µg/mL
[2]). The reason for the differing behaviour may be ex-
plained by steric conflicts between aeruginosin’s bulky
Choi (2-carboxy-6-hydroxy-octahydroindole) group in
position P2 and the S2 pocket of thrombin (Tyr60A/
Trp60D-loop) which are less prominent in the proline
moiety of RA-1001.Surprisingly, the synthetic precursor
of RA-1001, primary amine RA-1003, inhibits thrombin
in the same order of magnitude (Ki 9.0 µM). None of the
four analogues (RA-1001 – RA-1004) decreases the ac-
tivity of plasminogen activator urokinase (uPA), factor
Xa, and human mast cell tryptase.

Arch. Pharm. Pharm. Med. Chem. 2003, 336, 300–309
New Cyanopeptide-derived Thrombin Inhibitors 305
In summary, we have developed four equipotent
thrombin inhibitors (RA-1001, RA-1002, RA-1003, and
RA-1008) based on the structure of the cyanopeptide
aeruginosin 98-B (1). In addition, compounds RA-1002,
RA-1003, RA-1004, and RA-1006 act as selective inhib-
itors of thrombin, whereas RA-1001 and RA-1008 also
inhibit trypsin. Compound RA-1007 was found to be a
selective inhibitor of blood coagulation factor Xa, where-
as RA-1006 inhibits thrombin. Further investigations on
structure/activity relationships – especially concerning
the tranformation of the primary amine derivatives in ap-
propriate guanidine derivatives – are still under way, to
complete the results of this study.
(11.83 mmol) and 1.68 g DIPEA (13.01 mmol) in dichlorometh-
ane (50 ml) was added dropwise (20 min). While stirring for
16 h, the mixture was allowed to warm up to room temperature.
The precipitated DCU was filtered off, the filtrate was evaporat-
ed, and the resulting crude product was purified by column
chromatography (EtOAc/PE/CH2Cl2 1:1:1).Yield: 3.32 g (85 %)
of a colourless, amorphous substance. – mp: 98–100 °C – 1H-
NMR (200 MHz, CDCl3): δ (ppm) = 0.78–0.90 (m, 6 H, Ile-CH2-
CH3 + Ile-CH-CH3), 0.90–1.25 (m, 1 H, Ile-CH2-CH3), 1.27 (t,
7.2 Hz, 3 H, OCH2CH3), 1.30–1.55 (m, 1 H, Ile-CH2-CH3),
1.60–2.00 (m, 1 H, Ile-β-H), 2.45 (dt, 2 H, Ph-CH2-CH2), 2.83 (t,
2 H, Ph-CH2-CH2), 3.91–4.02 (m, 2 H, Gly-α-H), 4.19 (q,
7.2 Hz, 2 H, OCH2CH3), 4.32 (t, 1 H, Ile-α-H), 6.45 (d, 8.8 Hz,
1H, Ile-NH), 6.72 (d, 8.4 Hz, 2 H, Harom.), 6.88 (t, 1 H, Gly-NH),
6.97 (d, 8.4 Hz, 2 H, Harom.). – IR (KBr, cm–1): 3291, 3083, 2964,
2933, 2875, 1741, 1637, 1546, 1514, 1449, 1371, 1299, 1217,
1101, 1024, 826, 686, 533, 477. – [α] = –31.83 (c = 2 %, Me-
OH). – C19H28N2O5 (364.45). C, H, N.
Acknowledgements
N-[3-(4-Hydroxyphenyl)propionyl]-L-isoleucyl-glycine (4)
Financial support from the Fonds der Chemischen In-
dustrie and the BMBF is gratefully acknowledged. The
authors would like to thank J. Stürzebecher, Zentrum für
Vaskuläre Biologie und Medizin (Erfurt), Klinikum der
Friedrich-Schiller-Universität Jena, for performing the in-
hibition tests.
Ethyl ester 3 (2.86 g, 7.85 mmol) was stirred in a mixture of
aqueous LiOH (1 M, 8 mL) and DME (10 mL) at room tempera-
ture for 2 h. The mixture was acidified (pH 4) by addition of
aqueous citric acid (10 %) and extracted with EtOAc (2 ×
30 mL). The combined organic layers were dried with Na2SO4
and the filtrate evaporated; the residue was chromatographed
over silica gel (eluent: CH2Cl2/EtOAc/PE/MeOH 10:10:10:1).
Yield: 2.04 g (77 %) of a colourless, amorphous substance. –
mp: 61–63 °C – 1H-NMR (300 MHz, DMSO-D6): δ (ppm) =
0.75–0.82 (d + t, 6 H, Ile-CH2-CH3 + Ile-CH-CH3), 0.95–1.10 (m,
1 H, Ile-CH2), 1.30–1.40 (m, 1 H, Ile-CH2), 1.63–1.73 (m, 1 H,
Ile-β-H), 2.30–2.50 (m, 2 H, Ph-CH2-CH2), 2.64–2.72 (m, 2 H,
Ph-CH2-CH2), 3.64–3.73 (dd, 17.3 Hz + 5.8 Hz, 1 H, Gly-α-H),
3.74–3.82 (dd, 17.3 Hz + 5.8 Hz, 1 H, Gly-α-H), 4.20 (m, 1 H,
Ile-α-H), 6.63 (d, 8.4 Hz, 2 H, Harom.), 6.98 (d, 8.4 Hz, 2 H,
Experimental
General
Melting points are not corrected, Mikroheiztisch PHMK
80-2747 (F. Küstner Nachf. KG, Dresden, Germany) – IR spec-
tra (cm–1, KBr or KBr/film in case of oils): IR-Spektrometer Per-
kin-Elmer 1600 series FTIR (Perkin-Elmer-Deutschland,
H
arom.), 7.82 (d, 9.0 Hz, 1 H, NH), 8.21 (t, 5.9 Hz, 1 H, Gly-NH),
9.09 (s, 1 H, OH), 12.47 (s, 1 H, COOH). – IR (KBr, cm–1):
2500–3400, 3306, 3098, 2965, 2934, 2877, 1731, 1646, 1541,
1515, 1453, 1376, 1220, 1102, 1041, 828, 668, 536. – [α] =
–29.93 (c = 1.96 %, MeOH). – C17H24N2O5 (336.39). C, H, N.
Rodgau, Germany).
– NMR spectra Bruker DPX 200
(200 MHz), NMR spectra Bruker DPX 300 (300 MHz) (Bruker,
Rheinstetten, Germany), δ (ppm), solvents: CDCl3, DMSO-D6,
MeOH-D4, internal standard:TMS (δ = 0.00 ppm). – Elemental
analysis:Perkin-Elmer Elemental Analyzer 2400 CHN, all com-
pounds gave satisfactory elemental analyses.- Chromatogra-
phy: cc: Merck silica gel 60 (0.063–0.200 mm); tlc: Merck alu-
minium foils silica gel 60 F254 (E. Merck, Darmstadt, Germany).
– Optical rotation ([α]):Polartronic D (Schmidt Haensch GmbH,
Berlin, Germany), determined with Na-D-line (589.3 nm) at
23 °C. – L-Proline methyl ester hydrochloride (12) and L-leu-
cine methyl ester hydrochloride (23) were prepared by using
the thionyl chloride method [22].
1-(tert-Butyloxycarbonylamino)-4-{N-[3-(4-hydroxyphenyl)pro-
pionyl]-L-isoleucyl-glycylamino}butane (5)
0.76 g (6.04 mmol) DIC in CH2Cl2 (35 mL) was added dropwise
to an ice-cooled solution of 1.85 g (5.49 mmol) of 4 in CH2Cl2
(60 mL) over a period of 15 min. After stirring for 30 min, a solu-
tion of 1.14 g (6.04 mmol) 1-amino-4-[(tert-butyloxycarbo-
nyl)amino]butane (6) in CH2Cl2 (60 mL) was added (15 min).
The mixture was stirred for 16 h allowing to warm up to rt and –
after evaporation of the solvent – the residue was purified
by column chromatography (CH2Cl2/PE/EtOAc/MeOH
10:10:10:1). Colourless substance;Yield: 0.60 g (22 %) – mp:
Abbreviations of amino acids follow the recommendations of
the IUPAC-IUB Joint Commission on Biochemical Nomencla-
ture [23]. Other abbreviations: Boc: tert-Butyloxycarbonyl,
Boc2O:di-tert-butyl dicarbonate, DCC:N,N-dicyclohexylcarbo-
diimide, DCU:N,N-dicyclohexylurea, DIC:N,NЈ-diisopropylcar-
bodiimide, DIPEA: diisopropylethylamine, DME: 1,2-dimeth-
oxyethane, EtOAc: ethyl acetate, HOSu: N-hydroxysuccin-
imide, PE:petroleum ether, rt:room temperature,TFA:trifluoro-
acetic acid.
1
130–131 °C – H-NMR (300 MHz, DMSO-D6): δ (ppm) = 0.79
(m, 6 H, Ile-CH-CH3 + Ile-CH2-CH3), 1.30–1.45 (s + m, 14 H,
Boc + NH-CH2CH2CH2CH2NHBoc + Ile-CH2-CH3), 1.62–1.72
(m, 1 H, Ile-β-H), 2.30–2.50 (m, 2 H, Ph-CH2-CH2), 2.67 (m,
2 H, Ph-CH2-CH2), 2.85–2.95 (m, 2 H, CH2NHBoc), 3.30–3.10
(m, 2 H, Gly-CONH-CH2), 3.55–3.75 (2× dd, 16.5 Hz + 6.0 Hz
bzw.5.5 Hz, 2 H, Gly-α-H), 4.04 (t, 7.7 Hz, 1 H, Ile-α-H), 6.64 (d,
8.4 Hz, 2 H, Harom.), 6.75 (t, 5.4 Hz, 1 H, NH-Boc), 6.99 (d, 8.4
Hz, 2 H, Harom.), 7.65 (t, 5.6 Hz, 1 H, Gly-CONH-butyl), 7.96 (d,
7.7 Hz, 1 H, Ile-NH), 8.18 (t, 5.8 Hz, 1 H, Gly-NH), 9.09 (s, 1 H,
OH). – IR (KBr, cm–1): 3298, 3082, 2964, 2934, 2873, 1686,
1630, 1542, 1516, 1452, 1366, 1286, 1244, 1170, 828, 694,
534. – [α] = –28.84 (c = 2 %, MeOH) – C26H42N4O6 (506.65). C,
H, N.
N-[3-(4-Hydroxyphenyl)propionyl]-L-isoleucyl-glycine ethyl ester
(3)
An ice-cooled solution of 3.00 g N-[3-(4-Hydroxyphenyl)propio-
nyl]-L-isoleucine (10.75 mmol) in dichloromethane (50 mL)
was added to a solution of 2.22 g DCC (10.75 mmol) in dichlo-
romethane (20 mL) over a period of 30 min.After stirring for one
hour a mixture of 1.65 g glycine ethyl ester hydrochloride

306 Radau et al.
Arch. Pharm. Pharm. Med. Chem. 2003, 336, 300–309
1-Amino-4-{N-[3-(4-hydroxyphenyl)propionyl]-L-isoleucyl-gly-
cylamino}butane · TFA (RA-1005)
1H-NMR (DMSO-D6, 300 MHz): δ (ppm) = 0.87 (d, 6.3 Hz, 3 H,
CH(CH3)2), 0.92 (d, 6.3 Hz, 3 H, CH(CH3)2), 1.57–1.77 (m, 3 H,
CH(CH3)2 + β-CH2), 2.35 (s, 3 H, CH3), 4.39–4.45 (m, 1 H, α-H),
7.27 (d, 8.2 Hz, 2 H, Harom.), 7.79 (d, 8.2 Hz, 2 H, Harom.), 8.47 (d,
8.0 Hz, 1 H, NH), 12.35 (s, broad, 1 H, COOH).– IR (KBr, cm–1):
2850-3350, 3329, 3043, 2959, 2870, 1728, 1637, 1612, 1532,
1412, 1386, 1231. – [α] = –7.43 (c = 1.93 %, MeOH). –
C14H19NO3 (249.31). C, H, N.
TFA (3 mL) was added to an ice-cooled solution of 0.51 g of 5
(1.00 mmol) in CH2Cl2 (15 mL). After stirring at rt for 4 h, the
mixture was evaporated in vacuo, dissolved in methanol, and
evaporated once again to eliminate residual TFA. The remain-
ing residue was purified by column chromatography (CH2Cl2/
EtOAc/MeOH 10:10:5).Yield:0.22 g (44 %) of a colourless, vis-
1
cous substance. – H-NMR (300 MHz, DMSO-D6): δ (ppm) =
N-(4-Methylbenzoyl)-L-leucyl-L-proline methyl ester (14)
9.16 (s, 1 H, OH), 8.21 (t, 5.8 Hz, 1 H, Gly-NH), 7.98 (d, 7.6 Hz,
1 H, Ile-NH), 7.73–7.80 (s, broad, 4 H, Gly-CONH-butyl + NH+3),
6.98 (d, 8.4 Hz, 2 H, Harom.), 6.64 (d, 8.4 Hz, 2 H, Harom.), 4.03 (t,
7.6 Hz, 1 H, Ile-α-H), 3.55–3.75 (2× dd, 16.4 Hz + 6.1 Hz bzw.
5.4 Hz, 2 H, Gly-α-H), 3.00–3.10 (m, 2 H, Gly-CONH-CH2),
2.75–2.80 (t, 2 H, CH2NH3+), 2.68 (t, 7.6 Hz, 2 H, Ph-CH2-CH2),
2.35–2.45 (m, 2 H, Ph-CH2-CH2), 1.65–1.72 (m, 1 H, Ile-β-H),
1.40–1.55 (m, 5 H, NH-CH2CH2CH2CH2NH+3 + Ile-CH2-CH3),
0.76–0.80 (m, 6 H, Ile-CH-CH3 + Ile-CH2-CH3). – [α] = –22.94
(c = 2 %, MeOH) – C23H35F3N4O6 (520.54). C, H, N.
0.48 g (3.80 mmol) DIC in CH2Cl2 (15 mL) was added dropwise
to an ice-cooled solution of 0.86 g (3.45 mmol) 13 in CH2Cl2
(35 mL) over a period of 30 min.After stirring for further 30 min,
a solution of 0.63 g (3.80 mmol) L-proline methyl ester hydro-
chloride and 0.54 g (4.17 mmol) DIPEA in CH2Cl2 (15 mL) was
added (40 min). The mixture was stirred for 16 h allowing to
warm up to rt and – after evaporation of the solvent – the resi-
due was purified by column chromatography (PE/EtOAc 1:1).
Colourless crystals; Yield: 1.05 g (85 %). – mp: 144–147 °C –
1H-NMR (CDCl3, 300 MHz): δ (ppm) = 0.94 (d, 6.4 Hz, 3 H,
CH(CH3)2), 1.05 (d, 6.6 Hz, 3 H, CH(CH3)2), 1.50–1.65 (m, 3 H,
CH(CH3)2 + Leu-β-CH2), 1.85–2.25 (m, 4 H, Pro-β-CH2 + Pro-
γ-CH2), 2.39 (s, 3 H, CH3), 3.56–3.61 (m, 1 H, Pro-δ-CH2), 3.69
(s, 3 H, OCH3), 3.74–3.78 (m, 1 H, Pro-δ-CH2), 4.45–4.55 (m,
1 H, Pro-α-H), 5.05–5.15 (m, 1 H, Leu-α-H), 6.89 (d, 8.7 Hz,
1 H, NH), 7.22 (d, 8.2 Hz, 2 H, Harom.), 7.70 (d, 8.2 Hz, 2 H,
N-(2-Pyrimidinyl)-1,4-diaminobutane (8)
13.84 g of DAB (157.30 mmol) were added to 6.00 g of 2-chlo-
ropyrimidine (52.40 mmol) and stirred at 100 °C for 6 h. After
cooling to rt H2O (30 mL) was added, the mixture was stirred for
16 h, and additional H2O (70 mL) was added. To the yellowish
solution 4.20 g NaOH (105.00 mmol) in H2O (20 mL) were add-
ed slowly and the solution was stirred for 1 h.The mixture was
extracted with dichloromethane (3 × 100 mL), the combined or-
ganic layers were dried with Na2SO4 and evaporated im vacuo.
The resulting residue was recrystallized from PE/EtOAc.Yield:
2.47 g (28 %) of a colourless crystals. – mp: 66–67 °C – 1H-
NMR (DMSO-D6, 300 MHz): δ (ppm) = 1.32 (s, broad, 2 H,
NH2), 1.50–1.60 (m, 2 H, CH2CH2-CH2CH2NH2), 1.60–1.70 (m,
2 H, CH2CH2CH2CH2NH2), 2.74 (t, 7.0 Hz, 2 H, CH2-NH2), 3.42
(q, 6.8 Hz, 2 H, NH-CH2), 5.47 (s, broad, 1 H, NH), 8.26 (d,
4.8 Hz, 2 H, H-4/6pyim.), 6.50 (t, 4.8 Hz, 1 H, H-5pyim.). – IR (KBr,
cm–1): 3257, 3108, 2937, 1596, 1512, 1463, 1418, 1370, 1348,
1278, 1230, 1183, 1113, 1088, 1072, 944, 802, 791, 736, 643,
617, 513, 408. – C8H14N4 (166.23). C, H, N.
H
arom.). – IR (KBr/Film, cm–1): 3323, 2955, 2871, 1749, 1633,
1537, 1436, 1196. – [α] = –42.87 (1.5 %, MeOH). – C20H28N2O4
(360.45). C, H, N.
N-(4-Methylbenzoyl)-L-leucyl-L-proline (15)
Methyl ester 14 (0.94 g, 2.60 mmol) was stirred in a mixture of
aqueous LiOH (1 M, 5 mL) and DME (10 mL) at room tempera-
ture for 2 h. The mixture was acidified (pH 4) by addition of
aqueous citric acid (10 %) and extracted with EtOAc (2 × 20
mL).The combined organic layers were dried with Na2SO4 and
the filtrate evaporated; the residue was chromatographed over
silica gel (eluent: CH2Cl2/EtOAc/PE/MeOH 10:10:10:1).Yield:
1
0.62 g (69 %) of a colourless, viscous substance. – H-NMR
(DMSO-D6, 300 MHz): δ (ppm) = 0.90 (d, 6.5 Hz, 3 H,
CH(CH3)2), 1.00 (d, 6.6 Hz, 3 H, CH(CH3)2), 1.30–1.50 (m, 3 H,
CH(CH3)2 + Leu-β-CH2), 1.65–1.95 (m, 4 H, Pro-β-CH2 + Pro-
γ-CH2), 2.35 (s, 3 H, CH3), 3.50–3.65 (m, 1 H, Pro-δ-CH2),
3.70–3.85 (m, 1 H, Pro-δ-CH2), 4.45–4.65 (m, 1 H, Pro-α-H),
5.00–5.20 (m, 1 H, Leu-α-H), 7.26 (d, 8.1 Hz, 2 H, Harom.), 7.81
(d, 8.1 Hz, 2 H, Harom.), 8.60 (2 d, 1 H, NH), 12.45 (s, broad, 1 H,
COOH). – IR (KBr, cm–1): 3446, 2800-3500, 3339, 2959, 2872,
1721, 1616, 1386, 1222. – [α] = –37.66 (c = 2 %, MeOH). –
C19H26N2O4 (346.43). C, H, N.
N-(4-Methylbenzoyl)-L-leucine methyl ester (12)
To a solution of 3.09 g 4-methylbenzoylchloride (20.00 mmol) in
EtOAc (100 mL) a mixture of 3.63 g L-leucine methyl ester hy-
drochloride (20.00 mmol) and 5.81 g DIPEA (45.00 mmol) in
EtOAc/dichloromethane (170 mL/40 mL) was added over a pe-
riod of 40 min at rt.The mixture was stirred for 16 h and the sol-
vent was evaporated to half of the volume and filtered off from
DIPEA · HCl.After evaporation to dryness the residue was puri-
fied by column chromatography (PE/EtOAc 5:1).Yield: 4.06 g
(77 %) of colourless crystals. – mp: 80–81 °C. – 1H-NMR
(CDCl3, 300 MHz): δ (ppm) = 0.98 (d, 6.0 Hz, 6 H, CH(CH3)2),
1.63–1.78 (m, 3 H, CH(CH3)2 + β-CH2), 2.39 (s, 3 H, CH3), 3.76
(s, 3 H, OCH3), 4.82–4.90 (m, 1 H, α-H), 6.54 (d, 8.1 Hz, 1 H,
NH), 7.23 (d, 8.3 Hz, 2 H, Harom.), 7.70 (d, 8.3 Hz, 2 H, Harom.). –
IR (KBr, cm–1): 3293, 3066, 3028, 2958, 2360, 1752, 1630,
1545, 1246, 1162.– [α] = –15.50 (c = 2 %, MeOH).– C15H21NO3
(263.34). C, H, N.
1-(tert-Butyloxycarbonylamino)-3-[N-(4-methylbenzoyl)-L-leu-
cyl-L-prolyl-amino]propane (16)
0.20 g (1.58 mmol) DIC in CH2Cl2 (10 mL) was added dropwise
to an ice-cooled solution of 0.50 g (1.44 mmol) of 15 in CH2Cl2
(15 mL) over a period of 10 min. After stirring for 30 min, a solu-
tion of 0.27 g (1.58 mmol) 1-amino-3-[(tert-butyloxycarbo-
nyl)amino]propane (7) in CH2Cl2 (20 mL) was added (15 min).
The mixture was stirred for 16 h allowing to warm up to rt and –
after evaporation of the solvent – the residue was purified
by column chromatography (CH2Cl2/PE/EtOAc/MeOH
10:10:10:1). Colourless, viscous substance; Yield: 0.36 g
(50 %) – 1H-NMR (300 MHz, CDCl3): δ (ppm) = 1.01 (2× d,
6.3 Hz, 6 H, CH(CH3)2), 1.40 (s, 9 H, Boc), 1.50–1.65 (m, 4 H,
N-(4-Methylbenzoyl)-L-leucine (13)
Methyl ester 12 (1.44 g, 5.47 mmol) was stirred in a mixture of
aqueous LiOH (1 M, 6 mL) and DME (15 mL) at room tempera-
ture for 2 h. The mixture was acidified (pH 4) by addition of
aqueous citric acid (10 %) and extracted with EtOAc (2 ×
20 mL). The combined organic layers were dried with Na2SO4
and the filtrate evaporated; the residue was chromatographed
over silica gel (eluent: CH2Cl2/EtOAc/PE/MeOH 10:10:10:1).
Yield: 1.03 g (76 %) of colourless crystals. – mp: 124–126 °C –
Pro-β-CH2
+
Pro-γ-CH2), 1.70–1.75 (m, 5 H, NH-
CH2CH2CH2NHBoc + CH(CH3)2 + Leu-β-CH2), 2.40 (s, 3 H,
CH3), 2.95–3.17 (m, 2 H, NH-CH2CH2CH2NHBoc), 3.23–3.29
(m, 2 H, CH2NHBoc), 3.80–3.90 (m, 1 H, Pro-δ-CH2), 3.50–

Arch. Pharm. Pharm. Med. Chem. 2003, 336, 300–309
New Cyanopeptide-derived Thrombin Inhibitors 307
L-Isoleucyl-L-proline (19)
3.60 (m, 1 H, Pro-δ-CH2), 4.07–4.09 (m, 1 H, Pro-α-H), 4.70–
4.77 (m, 1 H, Leu-α-H), 5.27 (t, broad, 1 H, NH-Boc), 6.85 (d,
6.9 Hz, 1 H, Leu-NH), 7.00 (t, broad, 1 H, Pro-NH-CH2), 7.23 (d,
8.2 Hz, 2 H, Harom.), 7.67 (d, 8.2 Hz, 2 H, Harom.). – [α] = -32.89
(c = 2 %, MeOH) – C27H43N4O5 (503.67). C, H, N.
TFA (8 mL) was added to an ice-cooled solution of 3.09 g (9.42
mmol) of 18 in CH2Cl2 (20 mL). After stirring at rt for 3 h, the mix-
ture was evaporated in vacuo, dissolved in methanol and evapo-
rated once again to eliminate residual TFA. The colourless, vis-
cous substance was subjected to reaction with succinimidyl ben-
zoate without further purification. C13H21F3N2O5 (342.31).
1-Amino-3-[N-(methylbenzoyl)-L-leucyl-L-prolyl-amino]propane
· TFA (RA-1006)
TFA (3 mL) was added to an ice-cooled solution of 0.32 g of 16
(0.63 mmol) in CH2Cl2 (15 mL). After stirring at rt for 4 h, the
mixture was evaporated in vacuo, dissolved in methanol and
evaporated once again to eliminate residual TFA. The remain-
ing residue was purified by column chromatography (CH2Cl2/
EtOAc/MeOH 10:10:5).Yield:0.15 g (46 %) of a colourless, vis-
cous substance. – 1H-NMR (CDCl3, 300 MHz): δ (ppm) = 1.03
(2× d, 6.3 Hz, 6 H, CH(CH3)2), 1.50–1.65 (m, 5 H, Pro-β-CH2 +
N-Benzoyl-L-isoleucyl-L-proline (20)
A solution of succinimidyl benzoate (1.38 g, 6.28 mmol) in etha-
nol (30 mL) was added dropwise to a mixture of L-isoleucyl-L-
proline (19, 3.22 g, 9.42 mmol) and NaHCO3 (1.60 g) in water
(20 mL). The mixture was stirred for 16 h at room temperature
and the organic solvent was evaporated. The remaining aque-
ous solution was acidified (pH 2) by addition of conc. HCl and
extracted with CH2Cl2 (3 × 15 mL).The combined organic layers
were dried with Na2SO4 and evaporated under reduced pres-
sure. The residue was purified by column chromatography.
Pro-γ-CH2
+
CH(CH3)2), 1.70–1.85 (m, 4 H, NH-
CH2CH2CH2NHBoc + Leu-β-CH2), 2.40 (s, 3 H, CH3), 2.83–
2.87 (m, 2 H, NH-CH2CH2-CH2NH2), 3.20–3.35 (m, 2 H,
CH2CH2CH2NH2), 3.60–3.70 (m, 1 H, Pro-δ-CH2), 3.95–4.05
(m, 1 H, Pro-δ-CH2), 4.39–4.42 (m, 1 H, Pro-α-H), 4.70–4.73
(m, 1 H, Leu-α-H), 7.30 (d, 8.3 Hz, 2 H, Harom.), 7.78 (d, 8.3 Hz,
2 H, Harom.). – [α] = –41.23 (c = 2 %, MeOH) – C24F3H35N4O5
(516.55). C, H, N.
1
Yield: 1.82 g (58 %) of a colourless, viscous substance. – H-
NMR (DMSO-d6, 300 MHz): δ (ppm) = 0.84 (t, 7.3 Hz, 3 H, Ile-
CH2-CH3), 0.97 (d, 6.8 Hz, 3 H, Ile-CH-CH3), 1.10–1.20 (m, 1 H,
Ile-CH2-CH3), 1.50–1.60 (m, 1 H, Ile-CH2-CH3), 1.80–2.20 (m,
5 H, Pro-β-CH2 + Pro-γ-CH2 + Ile-β-H), 3.55–3.65 (m, 1 H,
Pro-δ-CH2), 3.90–4.00 (m, 1 H, Pro-δ-CH2), 4.21–4.28 (m, 1 H,
Pro-α-H), 4.50–4.56 (m, 1 H, Ile-α-H), 7.42–7.52 (m, 3 H,
Harom.), 7.87–7.88 (m, 2 H, Harom.), 8.59 (d, 8.2 Hz, 1 H, NH),
12.30 (s, broad, 1 H, COOH). – IR (KBr, cm–1): 2800-3500,
3316, 2964, 2931, 2877, 1719, 1629, 1534, 1451, 1189, 694. –
[α] = –70.18 (c = 2 %, MeOH). – C18H24N2O4 (332.40). C, H, N.
N-(tert-Butyloxycarbonyl)-L-isoleucine N-succinimidyl ester (17)
To an ice-cooled mixture of 4.80 g N-[tert-butyloxycarbonyl]-L-
isoleucine (hemihydrate, 20.00 mmol) and 2.30 g HOSu
(20.00 mmol) in EtOAc (50 mL) a solution of 4.12 g DCC
(20.00 mmol) in EtOAc (20 mL) was added dropwise (30 min).
While stirring for 16 h, the mixture was allowed to warm up to
room temperature.The precipitated DCU was filtered off, the fil-
trate was evaporated, and the resulting crude product was re-
crystallized from i-PrOH.Yield:5.91 g (90 %) of colourless crys-
tals. – mp: 87–88 °C (i-PrOH) (lit.: 92–93 °C, i-Pr2O, [24]) – 1H-
NMR (CDCl3, 300 MHz,):δ (ppm) = 0.97 (t, 7.4 Hz, 3 H, Ile-CH2-
CH3), 1.05 (d, 6.7 Hz, 3 H, Ile-CH-CH3), 1.20–1.36 (m, 1 H, Ile-
CH2-CH3), 1.46 (s, 9 H, Boc), 1.52–1.67 (m, 1 H, Ile-CH2-CH3),
1.92–2.05 (m, 1 H, Ile-β-H), 2.84 (s, 4 H, OCH2CH2O), 4.60–
4.65 (m, 1 H, α-H), 5.06 (d, 8.7 Hz, 1 H, NH). – IR (KBr, cm–1):
3371, 3313, 2970, 2936, 2879, 1810, 1784, 1741, 1698, 1529,
1508, 1457, 1425, 1392, 1368, 1308, 1252, 1208, 1166, 1079,
1046, 1014, 910, 873, 813, 648. – C15H24N2O6 (328.37). C, H,
N.
1-(tert-Butyloxycarbonylamino)-3-(N-benzoyl-L-isoleucyl-L-pro-
lyl-amino)propane (21)
0.21 g (1.61 mmol) DIC in CH2Cl2 (10 mL) was added dropwise
to an ice-cooled solution of 0.49 g (1.46 mmol) of 20 in CH2Cl2
(15 mL) over a period of 10 min. After stirring for 30 min, a solu-
tion of 0.28 g (1.58 mmol) 1-amino-3-[(tert-butyloxycarbo-
nyl)amino]propane (7) in CH2Cl2 (20 mL) was added (15 min).
The mixture was stirred for 16 h allowing to warm up to rt and –
after evaporation of the solvent – the residue was purified
by column chromatography (CH2Cl2/PE/EtOAc/MeOH
10:10:10:1). Colourless, viscous substance; Yield: 0.27 g
(38 %) – 1H-NMR (300 MHz, CDCl3): δ (ppm) = 0.91 (t, 7.5 Hz,
3 H, Ile-CH2-CH3), 1.03 (d, 6.6 Hz, 3 H, Ile-CH-CH3), 1.43 (s,
9 H, Boc), 1.55–1.70 (m, 4 H, NH-CH2CH2CH2NHBoc + Ile-
CH2-CH3), 1.85–2.00 (m, 4 H, Pro-β-CH2 + Pro-γ-CH2 + Ile-
β-H), 2.15–2.25 (m, 1 H, Pro-β-CH2), 3.00–3.35 (m, 4 H,
CH2NHBoc + NH-CH2CH2CH2NHBoc), 3.65–3.75 (m, 1 H,
Pro-δ-CH2), 3.80–3.95 (m, 1 H, Pro-δ-CH2), 4.45–4.55 (m, 1 H,
Pro-α-H), 4.85–4.90 (m, 1 H, Ile-α-H), 5.31 (t, broad, 1 H, NH-
Boc), 6.91 (d, 9.0 Hz, 1 H, Ile-NH), 7.02 (t, broad, 1 H, Pro-NH-
CH2), 7.40–7.51 (m, 3 H, Harom.), 7.79–7.82 (m, 2 H, Harom.).– [α]
= –43.79 (1.18 %, MeOH). – C26H40N4O5 (488.63). C, H, N.
N-(tert-Butyloxycarbonyl)-L-isoleucyl-L-proline (18)
A solution of 5.91 g of 17 (18.02 mmol) in EtOH (30 mL) and ac-
etone (10 mL) was added dropwise into a mixture of 3.11 g L-
proline (27.03 mmol) and 4.50 g NaHCO3 in H2O (60 mL) within
30 min. The mixture was stirred for 16 h at rt and the organic
solvents were evaporated.The remaining aqueous phase was
acidified with conc.HCl (pH 2) and extracted with dichlorometh-
ane (3 × 50 mL). The combined organic layers were dried with
Na2SO4 and evaporated. The crude product was purified by
column chromatography (EtOAc/PE/CH2Cl2 3:2:1). Yield:
5.22 g (88.0 %) of a colourless, viscous substance. – 1H-NMR
(300 MHz, CDCl3): δ (ppm) = 0.89 (t, 6.8 Hz, 3 H, Ile-CH2-CH3),
0.97 (d, 6.7 Hz, 3 H, Ile-CH-CH3), 1.08–1.22 (m, 1 H, Ile-CH2-
CH3), 1.43 (s, 9 H, Boc), 1.55–1.63 (m, 1 H, Ile-CH2-CH3),
1-Amino-3-(N-benzoyl-L-isoleucyl-L-prolyl-amino)propane ·TFA
(RA-1007)
TFA (3 mL) was added to an ice-cooled solution of 0.23 g of 21
(0.47 mmol) in CH2Cl2 (15 mL). After stirring at rt for 4 h, the
mixture was evaporated in vacuo, dissolved in methanol, and
evaporated once again to eliminate residual TFA. The remain-
ing residue was purified by column chromatography (CH2Cl2/
EtOAc/MeOH 10:10:5).Yield:0.12 g (51 %) of a colourless, vis-
1.70–1.80 (m, 1 H, Ile-β-H), 1.95–2.25 (m, 4 H, Pro-β-CH2
+
Pro-γ-CH2), 3.60–3.72 (m, 1 H, Pro-δ-CH2), 3.78–3.88 (m, 1 H,
Pro-δ-CH2), 4.26–4.32 (m, 1 H, Leu-α-H), 4.57–4.61 (m, 1 H,
Pro-α-H), 5.30 (d, 9.5 Hz, 1 H, NH), 7.18 (s, broad, 1 H, COOH).
– IR (KBr, cm–1): 2600–3500, 3436, 2971, 2934, 2878, 1708,
1636, 1516, 1454, 1392, 1367, 1315, 1251, 1170, 1091, 1044,
1020, 913, 860, 778, 664, 605. – [α] = –85.30 (c = 2.11 %, Me-
OH) – C16H28N2O5 (328.41). C, H, N.
1
cous substance. – H-NMR (300 MHz, MeOH-d4): δ (ppm) =
0.95 (t, 7.4 Hz, 3 H, Ile-CH2-CH3), 1.07 (d, 6.8 Hz, 3 H, Ile-CH-
CH3), 1.55–1.70 (m, 4 H, NH-CH2CH2CH2NH2 + Ile-CH2-CH3),
1.95–2.05 (m, 4 H, Pro-β-CH2 + Pro-γ-CH2 + Ile-β-H), 2.10–
2.25 (m, 1 H, Pro-β-CH2), 2.88 (m, 2 H, NH-CH2CH2CH2NH2),

308 Radau et al.
Arch. Pharm. Pharm. Med. Chem. 2003, 336, 300–309
3.15–3.40 (m, 2 H, CH2NH2), 3.65–3.75 (m, 1 H, Pro-δ-CH2),
4.05–4.15 (m, 1 H, Pro-δ-CH2), 4.35–4.37 (m, 1 H, Pro-α-H),
4.65–4.68 (m, 1 H, Ile-α-H), 7.40–7.55 (m, 3 H, Harom.), 7.80–
7.85 (m, 2 H, Harom.). – IR (KBr, cm–1): 3442, 2927, 1630, 1578,
1540, 1489, 1384, 1316, 714. – [α] = -61.8 (c = 2 %, MeOH) –
C23H33F3N4O5 (502.53). C, H, N.
784, 731, 700, 632, 544. – [α] = – 42.12 (c = 2 %, MeOH) –
C19H28N2O5S (396.51). C, H, N.
N-(Phenylmethanesulfonyl)-L-leucyl-L-proline (25)
Methyl ester 24 (0.90 g, 2.27 mmol) was stirred in a mixture of
aqueous LiOH (1 M, 5 mL) and DME (10 mL) at rt for 2 h. The
mixture was acidified (pH 4) by addition of aqueous citric acid
(10 %) and extracted with EtOAc (2 × 25 mL).The combined or-
ganic layers were dried with Na2SO4 and the filtrate evaporat-
ed; the residue was chromatographed over silica gel (CH2Cl2/
EtOAc/PE/MeOH 10:10:10:1). Yield: 0.53 g (62 %) of colour-
less crystals. – mp: 177–179 °C – 1H-NMR (DMSO-d6, 300
MHz): δ (ppm) = 0.85 (d, 6.6 Hz, 6 H, CH(CH3)2), 1.30–1.40 (m,
2 H, Leu-β-H), 1.65–1.80 (m, 1 H, CH(CH3)2), 1.78–1.95 (m,
3 H, Pro-β-CH2 + Pro-γ-CH2), 2.10–2.20 (m, 1 H, Pro-β-CH2),
3.30–3.60 (m, 2 H, Pro-δ-CH2), 3.85–3.95 (m, 1 H, Leu-α-H),
4.20–4.30 (m, 1 H, Pro-α-H), 4.24 (s, 2 H, Ph-CH2SO2), 7.30–
7.45 (m, 6 H, NH + Harom.), 12.33 (s, broad, 1 H, COOH). – IR
(KBr, cm–1): 3211, 2958, 2916, 2873, 1719, 1613, 1454, 1430,
1325, 1258, 1154, 1128, 1092, 938, 812, 784, 698, 606, 547,
528, 471. – [α] = –68.83 (c = 2 %, MeOH) – C18H26N2O5S
(382.48). C, H, N.
N-(Phenylmethanesulfonyl)-L-leucine methyl ester (22)
To a solution of 5.72 g phenylmethanesulfonyl chloride (30.00
mmol) in dichloromethane (100 mL) a mixture of 5.46 g L-leu-
cine methyl ester hydrochloride (30.00 mmol) and 8.51 g
DIPEA (66.00 mmol) in dichloromethane (150 mL) was added
over a period of 45 min at rt.The mixture was stirred for 16 h and
after evaporation to dryness the residue was purified by column
chromatography (PE/EtOAc 5:1).Yield: 5.45 g (61 %) of a col-
ourless, amorphous substance. – mp: 79–80 °C – 1H-NMR
(CDCl3, 300 MHz): δ (ppm) = 0.87 (d, 6.6 Hz, 3 H, CH(CH3)2),
0.90 (d, 6.6 Hz, 3 H, CH(CH3)2), 1.40–1.57 (m, 2 H, β-H), 1.61–
1.78 (sept., 6.6 Hz, 1 H, CH(CH3)2), 3.73 (s, 3 H, OCH3), 3.88–
3.96 (td, 8.8 Hz und 5.8 Hz, 1 H, α-H), 4.23 (d, 13.8 Hz, 1 H, Ph-
CH2SO2), 4.30 (d, 13.8 Hz, 1 H, Ph-CH2SO2), 4.74 (d, 9.0 Hz,
1 H, NH), 7.34–7.44 (m, 5H, Harom.). – IR (KBr, cm–1): 3260,
2958, 2868, 1726 (C=O), 1637, 1495, 1486, 1434, 1409, 1352,
1337, 1325, 1272, 1229, 1201, 1148, 1129, 1096, 989, 948,
910, 824, 783, 702, 609, 547, 524, 474. – [α] = – 30.9 (c = 2 %,
MeOH). – C14H21NO4S (299.39). C, H, N.
1-[N2,N3-Bis(tert-butyloxycarbonyl)guanidino]-4-(N-phenyl-
methanesulfonyl-L-leucyl-L-prolyl-amino)butane (26)
178 mg (1.38 mmol) DIC in CH2Cl2 (5 mL) was added dropwise
to an ice-cooled solution of 479 mg (1.25 mmol) 25 in CH2Cl2
(10 mL) over a period of 10 min. After stirring for 30 min, a solu-
tion of 497 mg (1.51 mmol) 11 in CH2Cl2 (10 mL) was added
(10 min). The mixture was stirred for 16 h allowing to warm up
to rt and – after evaporation of the solvent – the residue was pu-
rified by column chromatography (PE/EtOAc 1:1).Yield: 0.35 g
(40 %) of colourless crystals. – mp: 64–66 °C – 1H-NMR
(CDCl3, 300 MHz): δ (ppm) = 0.85 (d, 6.6 Hz, 3 H, CH(CH3)2),
0.87 (d, 6.7 Hz, 3 H, CH(CH3)2), 1.20–1.30 (m, 2 H, Leu-β-H),
1.40–1.65 (m, 22 H, 2× Boc + NH-CH2CH2CH2CH2Nguan.),
1.80–2.10 (m, 3 H, Pro-β-CH2 + Pro-γ-CH2), 2.15–2.25 (m, 1 H,
Pro-β-CH2), 2.95–3.05 (m, 1 H, Pro-δ-CH2), 3.15–3.25 (m, 1 H,
Pro-δ-CH2), 3.30–3.40 (m, 2 H, NH-CH2CH2CH2CH2Nguan.),
3.75–3.85 (m, 2 H, NH-CH2CH2CH2CH2Nguan.), 4.10–4.25 (m,
2 H, Pro-α-H + Ph-CH2), 4.31 (d, 13.6 Hz, 1 H, Ph-CH2), 4.35–
4.45 (m, 1 H, Leu-α-H), 5.45–5.50 (d, broad, 1 H, Leu-NH),
6.80 (t, broad, 1 H, Pro-NH), 7.30–7.45 (m, 5 H, Harom.), 8.28 (s,
broad, 1 H, NHguan.), 11.45 (s, broad, 1 H, NHguan.). – IR (KBr,
cm–1): 3340, 2968, 2933, 1720, 1638, 1576, 1456, 1415, 1367,
1331, 1252, 1157, 1133, 1052, 1027, 780, 698, 546. – [α] =
–32.66 (c = 2 %, MeOH) – C33H54N6O8S (694.88). C, H, N.
N-(Phenylmethanesulfonyl)-L-leucine (23)
Methyl ester 22 (3.62 g, 12.10 mmol) was stirred in a mixture of
aqueous LiOH (1 M, 12 mL) and DME (20 mL) at room temper-
ature for 2 h. The mixture was acidified (pH 4) by addition of
aqueous citric acid (10 %) and extracted with EtOAc (2 ×
50 mL). The combined organic layers were dried with Na2SO4
and the filtrate evaporated; the residue was chromatographed
over silica gel (CH2Cl2/EtOAc/PE/MeOH 10:10:10:1). Yield:
1.76 g (51 %) of a colourless, amorphous substance. – mp:
129–132 °C – 1H-NMR (DMSO-d6, 300 MHz):δ (ppm) = 0.85 (d,
6.6 Hz, 6 H, CH(CH3)2), 1.45 (t, 7.2 Hz, 2 H, β-H), 1.66 (sept.,
6.6 Hz, 1 H, CH(CH3)2), 3.75 (dt, 8 Hz, 1 H, α-H), 4.25 (d,
13.8 Hz, 1 H, Ph-CH2SO2), 4.34 (d, 13.8 Hz, 1 H, Ph-CH2SO2),
7.33–7.41 (m, 5 H, Harom.), 7.57 (d, 8.6 Hz, 1 H, NH), 12.70 (s,
broad, 1 H, COOH). – IR (KBr, cm–1): 3174, 2956, 2870, 1744,
1467, 1403, 1313, 1231, 1201, 1147, 1121, 1091, 1015, 966,
937, 915, 847, 790, 735, 698, 683, 607, 568, 524, 464. – [α] =
–10.0 (c = 2 %, MeOH) – C13H19NO4S (285.36). C, H, N.
N-(Phenylmethanesulfonyl)-L-leucyl-L-proline methyl ester
(24)
1-Guanidino-4-(N-phenylmethanesulfonyl-L-leucyl-L-prolyl-
amino)butane · TFA (RA-1008)
1.10 g (8.72 mmol) DIC in CH2Cl2 (20 mL) was added dropwise
to an ice-cooled solution of 2.26 g (7.93 mmol) 23 in CH2Cl2
(90 mL) over a period of 45 min.After stirring for further 30 min,
a solution of 1.44 g (8.72 mmol) L-proline methyl ester hydro-
chloride and 1.24 g (9.59 mmol) DIPEA in CH2Cl2 (90 mL) was
added (60 min). The mixture was stirred for 16 h allowing to
warm up to rt and – after evaporation of the solvent – the resi-
due was purified by column chromatography (PE/EtOAc 5:1).
TFA (1 mL) was added to an ice-cooled solution of 0.24 g of 26
(0.34 mmol) in CH2Cl2 (10 mL). After stirring at rt for 5 h, the
mixture was evaporated in vacuo, dissolved in methanol and
evaporated once again to eliminate residual TFA. The remain-
ing residue was purified by column chromatography (PE/
CH2Cl2/EtOAc/MeOH 10:10:10:1).Yield: 0.14 g (65 %) of a col-
ourless, viscous substance.– 1H-NMR (DMSO-d6, 300 MHz):δ
(ppm) = 0.80 (2× d, 6.5 Hz, 6 H, CH(CH3)2), 1.30–1.60 (m, 7 H,
NH-CH2CH2CH2CH2Nguan. + Leu-β-H + CH(CH3)2), 1.70–2.05
(m, 4 H, Pro-β-CH2 + Pro-γ-CH2), 3.00–3.10 (m, 1 H, Pro-
δ-CH2), 3.10–3.60 (m, 5 H, NH-CH2CH2CH2CH2Nguan. + Pro-
δ-CH2), 3.85–3.90 (m, 1 H, Ile-α-H), 4.05–4.10 (m, 1 H, Pro-
α-H), 4.15–4.30 (m, 2 H, Ph-CH2), 7.25–7.40 (m, 5 H, Haromat.),
7.45 (m, 1 H, NH-CH2CH2CH2CH2Nguan.), 7.90 (s, broad, 1 H,
NH, Leu), 8.40 (s, broad, 3 H, guanidine), 8.66 (s, broad, 1 H,
guanidine). – IR (KBr/Film, cm–1): 3391, 2964, 1676, 1560,
1458, 1315, 1209, 1142, 844, 801, 723, 699, 604, 546, 520.
1
Yield: 0.91 g (29 %) of a colourless, viscous substance. – H-
NMR (CDCl3, 300 MHz): δ (ppm) = 0.90 (d, 6.6 Hz, 3 H,
CH(CH3)2), 0.91 (d, 6.6 Hz, 3 H, CH(CH3)2), 1.30–1.45 (m, 2 H,
Leu-β-H), 1.80–2.00 (m, 4 H, CH(CH3)2 + Pro-β-CH2 + Pro-
γ-CH2), 2.10–2.25 (m, 1 H, Pro-β-CH2), 3.02–3.10 (m, 1 H,
Pro-δ-CH2), 3.30–3.37 (m, 1 H, Pro-δ-CH2), 3.72 (s, 3 H,
OCH3), 3.80–3.90 (m, 1 H, Pro-α-H), 4.16 (d, 13.9 Hz, 1 H, Ph-
CH2), 4.32 (d, 13.9 Hz, 1 H, Ph-CH2), 4.43–4.47 (m, 1 H, Leu-
α-H), 5.13 (d, 9.1 Hz, 1 H, NH), 7.33–7.46 (m, 5 H, Harom.). – IR
(KBr/Film, cm–1): 3291, 2964, 2872, 2669, 1743, 1644, 1563,
1455, 1435, 1404, 1364, 1321, 1170, 1131, 1095, 1025, 931,

Arch. Pharm. Pharm. Med. Chem. 2003, 336, 300–309
New Cyanopeptide-derived Thrombin Inhibitors 309
– [α] = –43.51 (c = 2 %, MeOH) – C25F3H39N6O6S (608.68). C,
[8] G. Radau, J. Gebel, D. Rauh, Arch. Pharm. Pharm. Med.
Chem. 2003, in press.
H, N.
1-[(Pyrimidin-2-yl)amino]-4-(N-benzoyl-L-leucyl-L-prolyl-ami-
no)butane (RA-1009)
[9] G. Radau, D. Rauh, Bioorg. Med. Chem. Lett. 2000, 8,
779–781.
116 mg (0.90 mmol) DIC in CH2Cl2 (5 mL) was added dropwise
to an ice-cooled solution of 285 mg (0.90 mmol) 27 in CH2Cl2
(10 mL) over a period of 10 min. After stirring for 30 min, a solu-
tion of 164 mg (0.99 mmol) 8 in CH2Cl2 (10 mL) was added
(10 min). The mixture was stirred for 16 h allowing to warm up
to rt and – after evaporation of the solvent – the residue was pu-
rified by column chromatography (PE/CH2Cl2/EtOAc/MeOH
10:10:10:1).Yield: 72 mg (17 %) of a colourless, viscous sub-
stance.– 1H-NMR (CDCl3, 300 MHz):δ (ppm) = 0.90 (d, 6.6 Hz,
3 H, CH(CH3)2), 1.04 (d, 6.6 Hz, 3 H, CH(CH3)2), 1.55–1.65 (m,
[10] B. Sandler, M. Murakami, J. Clardy, J. Am. Chem. Soc.
1998, 120, 595–596.
[11] A. P. Krapcho, C. S. Kuell, Synth. Commun. 1990, 20,
2559–2564.
[12] M. Bergmann, L. Zervas, J. Biol. Chem. 1936, 113, 341–
357.
[13] P. Karrer, W. Kehl, Helv. Chim. Acta 1930, 13, 50–63.
[14] E. J. Iwanowicz, M. A. Poss, J. Lin, Synth. Commun. 1993,
23, 1443–1445.
3 H, CH(CH3)2 + Leu-β-H), 1.90–2.05 (m, 3 H, Pro-β-CH2
+
NH-
Pro-γ-CH2), 2.05–2.30 (m, 5 H, Pro-β-CH2
+
[15] A. Expósito, M. Fernández-Suárez, T. Iglesias, L. Muñoz,
CH2CH2CH2CH2Nguan.), 3.55–3.65 (m, 1 H, Pro-δ-CH2), 3.80–
3.90 (m, 1 H, Pro-δ-CH2), 3.85–4.00 (m, 4 H, NH-
CH2CH2CH2CH2NHguan.), 4.20–4.30 (m, 1 H, Pro-α-H), 4.50–
4.60 (m, 1 H, Leu-α-H), 4.60–4.70 (m, 1 H, NH-
R. Riguera, J. Org. Chem. 2001, 66, 4206–4213.
[16] R. J. Bergeron, J. S. McManis, J. Org. Chem. 1987, 52,
1700–1703.
CH2CH2CH2CH2NHguan.),
5.05–5.15
(m,
1 H,
NH-
CH2CH2CH2CH2NHguan.), 6.90 (d, 8.2 Hz, 1 H, Leu-NH), 7.45–
7.65 (m, 4 H, Harom.), 7.70–7.90 (m, 4 H, Harom.). – C26H36N6O3
(480.60). C, H, N.
[17] G. Delle Monache, B. Botta, F. Delle Monache, R. Espinal,
S. C. De Bonnevaux, C. De Luca, M. Botta, F. Corelli, M.
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Bonnevaux, C.De Luca, M.Botta, F.Corelli, A.Tafi, R.Sac-
co, G. Delle Monache, J. Med. Chem. 2001, 44, 2950–
2958.
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