New cyanopeptide-derived low molecular weight thrombin inhibitors

Article abstract of DOI:10.1002/ardp.200300726

Thrombosis is the result of defective regulation of the hemostasis system. This cardiovascular disorder may lead to deep vein thrombosis, myocardial infarction, and stroke. The majority of current drug research is focused on finding inhibitors of thrombin - the global player in hemostasis. In our work, we emphasize investigation of the marine environment to yield new lead structures from marine organisms like blue-green algae (cyanobacteria). This article 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 (2) as a lead structure, we have developed and synthesized new, selective acting inhibitors of thrombin (RA-1001 and RA-1002), which are suitable targets for further structure-activity studies.

Full text of DOI:10.1002/ardp.200300726

372 Radau et al.
Arch. Pharm. Pharm. Med. Chem. 2003, 336, 372–380
Gregor Radaua,
Jana Gebela,
Daniel Rauhb
New Cyanopeptide-Derived Low Molecular Weight
Thrombin Inhibitors
Thrombosis is the result of defective regulation of the hemostasis system.This cardio-
vascular disorder may lead to deep vein thrombosis, myocardial infarction, and stroke.
The majority of current drug research is focused on finding inhibitors of thrombin – the
global player in hemostasis.In our work, we emphasize investigation of the marine envi-
ronment to yield new lead structures from marine organisms like blue-green algae (cy-
anobacteria). This article deals with the design, syntheses, and inhibition tests of new
low molecular weight thrombin inhibitors utilizing cyanopeptides, the secondary metabo-
lites of cyanobacteria with interesting biological activities, as new lead structures. Start-
ing with aeruginosin 98-B (2) as a lead structure, we have developed and synthesized
new, selective acting inhibitors of thrombin (RA-1001 and RA-1002), which are suitable
targets for further structure-activity studies.
a
Institute of Pharmacy,
Ernst-Moritz-Arndt-University
Greifswald, Pharmaceutical/
Medicinal Chemistry,
Friedrich-Ludwig-Jahn-
Str. 17, D-17487 Greifswald,
Germany
Institute of Pharmaceutical
Chemistry,
Philipps-University Marburg,
Marbacher Weg 6,
b
D-35032 Marburg, Germany
Keywords: Cyanobacteria; Cyanopeptides; Peptide syntheses; Rational drug design;
Thrombin inhibitors
Received: September 10, 2002; Accepted: October 22, 2002 [FP726]
DOI 10.1002/ardp.200300726
Introduction
Results and discussion
Cyanobacteria were proved to be reservoirs of compounds
with high biological potency [1]. Especially their secondary
metabolites of peptidic character – the cyanopeptides – ex-
ert a broad spectrum of biological activities, reaching from
antitumor [2] effects to cardioactive [3] and immunosup-
pressive [4] behavior.
Structural considerations
Due to their biological potency, we chose linear cyanopep-
tides from the aeruginosin-family (1-6, Figure 1) [6–10] as
putative lead structures for the design and synthesis of new
selective serine protease inhibitors. Compound 4a is the
most potent secondary metabolite concerning the inhibition
of trypsin, thrombin, and plasmin (IC50 (4a): 0.2/0.04/
0.3 µg/mL), whereas 1 and 2 are almost equipotent inhibi-
tors (IC50: 0.6 and 0.6/7.0 and 10.0/6.0 and 7.0 µg/mL).
A considerable number of non-toxic cyanopeptides inhibit
members of the serine proteases family [5], which play cen-
tral roles in the human organism.Thus, trypsin is essential
for the liberation of important pancreas enzymes, whereas
trypsin-like serine proteases like thrombin, plasmin, factor
Xa, t-PA, and kallikrein, represent the main actors in the in-
terlaced cycles of blood coagulation, fibrinolysis, kinin-kal-
likrein system, and the complement system. A disturbance
of the sensitive, by endogeneous protease inhibitors (ser-
pins) regulated balance caused by malfunctions of one or
more enzymes can result in critical effects on human organ-
ism, e.g.thromboembolic complications like venous and ar-
terial thrombosis, stroke, restenosis, and recurrent myocar-
dial infarction. The development of oral thrombin inhibitors
therefore presents a notable measure for improving the
treatment of the above-mentioned disorders.
X-ray crystallographic data of the cocrystallized trypsin-aer-
uginosin 98-B (2)- complex [11] show that 2 inhibits the en-
zyme neither by formation of a covalent bond, as assumed
for the inhibition mechanism of cyclotheonamide (sponge-
metabolite) [12], nor by the classical mechanism, as well-
known for A90720A (cyanobacterium-metabolite) [13].
Due to the unusual mode of inhibition – there are no interac-
tions between 2 and the catalytic triad of trypsin – we decid-
ed to choose aeruginosin 98-B as a lead structure for the
design of new serine protease inhibitors.Although 4a inhib-
its thrombin much better than 2, the synthesis of the argal
subunit (intramolecular hemiaminal of arginine aldehyde) in
4a seemed to be more difficult and time-consuming than
the synthesis of agmatine (decarboxylated arginine deriva-
tive).
Correspondence: Gregor Radau, Institut für Pharmazie,
Pharmazeutische/Medizinische Chemie, Ernst-Moritz-Arndt-
Universität Greifswald, Friedrich-Ludwig-Jahn-Str.17, D-17487
Greifswald, Germany. Phone: +49 3834 86-4880, Fax:
+49 3834 86-4874, e-mail: radau@pharmazie.uni-greifswald.de.
Containing no L-configurated amino acids, 2 acts rather in
an unconventional mode with specificity and recognition el-
ements of trypsin by employing a series of hydrogen bonds
© 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
0365-6233/08/0372

Arch. Pharm. Pharm. Med. Chem. 2003, 336, 372–380
New cyanopeptide-derived low molecular weight Thrombin inhibitors 373
Figure 1. Linear serine protease inhibitors from Microcystis aeruginosa (1, 2, 3, 5) and Microcystis viridis (4a, 4b, 6).
compared to the inhibition of trypsin.On the other hand an-
other cyanopeptide – aeruginosin 298-A (5, Figure 1) –
shows unexpected interactions with thrombin in the ternary
hirugen-thrombin complex [14]. One of several surprising
interactions is the gentle displacement of the 60 insertion
loops caused by Choi’s six-membered ring. Thus, within
limits thrombin reacts flexible towards structural influences
caused by ligands and increases the flexibility to the reper-
toire of structure-based drug design. In our opinion, the di-
versity and quality of the above described interactions be-
tween aeruginosin 98-B and trypsin justify further investiga-
tions on the design of new low molecular weight serine pro-
tease inhibitors based on 2 as a new lead structure.
Figure 2. Trypsin-aeruginosin 98-B-interactions according
to Sandler et al. [11].
Syntheses
In the following we describe the syntheses of RA-1000, RA-
(Figure 2) [11]. The guanidine group of agmatine reaches
deeply inside the S1-pocket and forms, in addition to two
strong hydrogen bonds to the Asp189-carboxylate side
chain, strong water-mediated interactions to the side chain
of Gln192. D-allo-Ile, which is only in the D-allo configura-
tion free of steric conflicts with the Choi (2-carboxy-6-hy-
droxy-octahydroindole) moiety, interacts as a hydrophobic
element with the Trp215 aryl-binding site. In association
with the neighboring hydroxyphenyllactic acid (Hpla),
D-allo-Ile forms series of hydrogen bonds to the enzyme.
There is one further water-mediated interaction in the
trypsin-complex, namely a hydrogen bond between the
phenolic hydroxyl of Hpla, the amide NH of Cys220, and the
carbonyl oxygen of Ser146.The sulfate moiety protrudes in-
to the solvent in opposite direction to trypsin – however, at
the corresponding place of thrombin it would rise into the
hydrophobic pocket (Tyr60A/Trp60D), a very unfavorable
position.Therefore one can attribute a discriminating role to
the sulfate group that results in poor thrombin inhibition
1001, and RA-1002, which contain some simplified struc-
tural elements compared to 2 (Figure 3). These modifica-
tions of the original lead structure were achieved by (1) the
exchange of Hpla for a 3-(4-hydroxy-phenyl)propionyl par-
tial structure, (2) the formal reduction of Choi into L-proline,
and (3) the use of the guanylated piperazinylethyl ester
(RA-1000) or agmatine (RA-1001) as basic side chains. In
addition, Hpla was replaced by a benzoyl moiety, D-allo-iso-
leucine by L-leucine, and finally the C-terminal side-chain
was shortened by one carbon atom to give RA-1002.The
P1 modifications – the positions P on the inhibitor/substrate
are counted from the point of cleavage and thus have the
same numbering as the enzyme subsites S they occupy
[15] – were performed to examine whether the guanylated
piperazine moiety fits into the S1 pocket, and whether the
piperazinylethyl ester is able to resist hydrolysis.The modifi-
cations in P2 are reasonable, because – as shown in the
above-mentioned X-ray studies – Choi’s six-membered
ring does not contribute to the protease inhibition in any
© 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

374 Radau et al.
Arch. Pharm. Pharm. Med. Chem. 2003, 336, 372–380
Figure 3. Synthetic analogues of aeruginosin 98-B (2).
Scheme 1. Attempted synthesis of piperazine derivative 11.
Scheme 2. Two possible pathways for the synthesis of dipeptide 16.
Scheme 3. Syntheses of compounds RA-1000 and RA-1001.
© 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Arch. Pharm. Pharm. Med. Chem. 2003, 336, 372–380
New cyanopeptide-derived low molecular weight Thrombin inhibitors 375
Scheme 4. Synthesis of compound RA-1002.
way. Since L-proline was employed to occupy P2, it was no
longer necessary to pay attention to collisions between P2
and residues at P3, because prolines spatial claim be-
comes smaller compared to Choi.Thus, L-isoleucine and L-
leucine were set into P3. Finally, Hpla was exchanged for
3-(4-hydroxyphenyl)propionic acid and benzoic acid, re-
spectively.
tion and reaction with S-methylisothiouronium iodide
(SMITU) gave the corresponding guanidine derivatives
RA-1000 and RA-1001, respectively (Scheme 3).
The synthesis of RA-1002 started with benzoylation of L-
leucine methyl ester hydrochloride (Scheme 4).N-Benzoyl-
L-isoleucine methyl ester (24) was saponified with LiOH
(1M) in dimethoxyethane.Both steps were carried out using
alternative procedures, which are less complicated com-
pared to the methods described in the literature [17–18].
The resulting carboxylic acid (25) was transformed into the
dipeptide methyl ester 26 by condensation with proline
methyl ester. Hydrolysis, activation (DIC), and reaction with
mono-Boc-substituted diaminopropane (28) yielded the
appropriate derivative 29.Compounds 19 and 28 were syn-
thesized by analogy with the procedure described by Krap-
cho [19], using an eightfold excess of 1,4-diaminobutane
and 1,3-diaminopropane referred to di-tert-butyl dicarbo-
nate (Boc2O) in order to avoid the formation of bis-Boc-sub-
stituted diaminoalkanes. Compound RA-1002 was synthe-
sized by cleavage of the Boc protecting group and subse-
quent reaction of the resulting TFA salt (30) with S-methyl-
isothiouronium iodide under basic conditions (DIPEA).
The first attempt to synthesize precursor 11 (Scheme 1)
started with the transformation of HPA (3-[4-hydroxyphen-
yl]propionic acid, 7) into its succinimide ester 8, which is
enabled to react with completely unprotected L-isoleucine
to give the appropriate N-acylated amino acid 9 [16].As ex-
pected, 11 was not available through the connection of 9
and 10 without any protection or blockade of the secondary
amino group of piperazine in 10.
Alternatively, 11 was synthesized in a successive synthesis
strategy from the N-terminus to the C-terminus using the
same activating routine (Schemes 2 and 3).Thus, dipeptide
16 was prepared by taking the pathways A and B, respec-
tively (Scheme 2). On the one hand 9 was condensed with
proline methyl ester (12) (route A) leading directly to 15, and
on the other hand 12 was condensed with Boc-L-Ile-OH to
the appropriate dipeptide 13 (route B).Cleavage of the Boc
group afforded 14, which was submitted to the reaction with
the activated ester 8.Hydrolysis of the methyl ester 15 gave
the desired building block 16, which represents the com-
mon starting point in the syntheses of RA-1000 and
RA-1001, respectively.Route A has to be preferred regard-
ing the better over-all yields compared to route B.
Enzyme inhibition tests/Conclusions
The measurements were carried out as described by
Stürzebecher et al. [20]; Ki-values were calculated accord-
ing to Dixon [21] using a linear regression program. As ex-
pected, the guanidine derivatives RA-1001 and RA-1002
are potent thrombin inhibitors with Ki values of 5.6 µM and
8.7 µM, respectively. RA-1001 is the only member of the
synthetic aeruginosin analogues shown in Table 1 that is
able to inhibit trypsin. In contrast to RA-1001, aeruginosin
98-B (2) inhibits trypsin stronger than thrombin.The reason
Typically, the putative inhibitors RA-1000 and RA-1001
were prepared by reaction of 16 with N-Boc-protected 1-(2-
hydroxyethyl)piperazine (17) or with mono-Boc-protected
1,4-diaminobutane (19) under carbodiimide catalysis (di-
isopropyl carbodiimide, DIC). Subsequent Boc deprotec-
© 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

376 Radau et al.
Arch. Pharm. Pharm. Med. Chem. 2003, 336, 372–380
Table 1. Inhibitory data of aeruginosins (1–6) and their synthetic analogues.
No.
Ref.
IC50 (µM)
throm
Ki (µM)
uPA
tryp
plas
trypsin
thrombin
f. Xa
tryptase
1
2
3
4a
4b
5
6
21
30
[6]
[6]
[7]
[8]
[8]
0.87
0.92
5.31
0.31
1.68
1.65
74.89
–
10.16
15.27
4.50
0.06
0.15
0.50
13.22
–
8.70
10.69
6.81
0.46
1.23
---
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
[10]
[9]
–
–
–
–
–
–
–
–
–
–
99.85
–
>1000
>1000
>1000
32
9.0
62
>1000
5.6
8.7
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
–
–
–
–
–
–
–
–
–
–
–
–
RA-1000
RA-1001
RA-1002
>1000
– not determined, --- no inhibition, tryp = trypsin, throm = thrombin, plas = plasmin, uPA = plasminogen activator urokinase, f.
Xa = factor Xa.
for this different behavior may be explained by steric con-
flicts between aeruginosin’s bulky Choi group and the S2
pocket of thrombin, which are less prominent in the proline
moiety of RA-1001. Surprisingly, the synthetic precursor of
RA-1001, primary amine 21, inhibits thrombin in the same
order of magnitude (Ki 9.0 µM).The precursor of RA-1002,
amine 30, shows a Ki value of 62 µM for the inhibition of
thrombin. Obviously, structural features of the positions P4
(hydroxyl function) and P1 (length of guanidinoalkyl chain)
in 21 contribute to its better inhibitory activity.These features
are less strong distinct concerning the inhibition of thrombin
through RA-1001 and RA-1002. None of the four anal-
ogues decreases the activity of plasminogen activator
urokinase (uPA), factor Xa, and mast cell tryptase.RA-1000
lacks any inhibitory activity towards the enzymes in ques-
tion.
would like to thank J. Stürzebecher, Zentrum für Vaskuläre
Biologie und Medizin (Erfurt), Klinikum der Friedrich-Schil-
ler-Universität Jena, for performing the inhibition tests.
Experimental
General
Melting points are not corrected, Mikroheiztisch PHMK 80-2747 F.
Küstner Nachf. KG Dresden. – IR spectra (cm–1, KBr or KBr/film in
case of oils): IR-Spektrometer Perkin-Elmer 1600 series FTIR. –
NMR spectra Bruker DPX 200 (200 MHz), NMR spectra Bruker
DPX 300 (300 MHz), δ (ppm), solvents:CDCl3, DMSO-D6, MeOH-
D4, internal standard: TMS (δ = 0.00 ppm). – Elemental analysis:
Perkin-Elmer Elemental Analyzer 2400 CHN, all compounds gave
satisfactory elemental analyses. – Chromatography:cc:Merck sili-
ca gel 60 (0.063–0.200 mm);tlc:Merck aluminium foils silica gel 60
F254. – Optical rotation ([α]): Polartronic D (Schmidt Haensch
GmbH), determined with Na-D-line (589.3 nm) at 23 °C.– L-Proline
methyl ester hydrochloride (12) and L-leucine methyl ester hydro-
chloride (23) were prepared by using the thionyl chloride method
[22].
In summary, we have developed three equipotent thrombin
inhibitors (21, RA-1001, and RA-1002) based on the struc-
ture of the cyanopeptide aeruginosin 98-B.Moreover, com-
pounds 21, 30, and RA-1002 seem to be selective inhibi-
tors of thrombin.First X-ray studies of RA-1001-trypsin- and
RA-1002-trypsin-complexes confirm an aeruginosin-anal-
ogous binding mode that is worth to be optimized. Further
SAR studies concerning P1, P3, and P4 moieties are cur-
rently in process to increase the inhibitory potency and se-
lectivity of both new lead structures RA-1001 and RA-1002.
Abbreviations of amino acids follow the recommendations of the
IUPAC-IUB Joint Commission on Biochemical Nomenclature:Eur.
J. Biochem. 1984, 138, 9–37. Other abbreviations: Boc: tert.-Butyl-
oxycarbonyl, Boc2O: di-tert.-butyl dicarbonate, DCC: N,N-di-
cyclohexylcarbodiimide, DCU: N,N-dicyclohexylurea, DIC: N,N-di-
isopropylcarbodiimide, DIPEA: diisopropylethylamine, DME: 1,2-
dimethoxyethane, EtOAc: ethyl acetate, HOSu: N-hydroxysuccin-
imide, HPA: 3-(4-hydroxyphenyl)propionic acid, PE: petroleum
ether, rt: room temperature, SMITU: S-methylisothiouronium io-
dide, TFA: trifluoroacetic acid.
Succinimidyl 3-(4-hydroxyphenyl)propionate (8)
Acknowledgements
To an ice-cooled solution of 3-(4-hydroxyphenyl)propionic acid (7,
1.66 g, 10.00 mmol) and HOSu (1.15 g, 10.00 mmol) in EtOAc
(30 mL) a solution of DCC (2.06 g, 10.00 mmol) in EtOAc (15 mL)
Financial support from the Fonds der Chemischen Indust-
rie and the BMBF is gratefully acknowledged.The authors
© 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Arch. Pharm. Pharm. Med. Chem. 2003, 336, 372–380
New cyanopeptide-derived low molecular weight Thrombin inhibitors 377
was added dropwise over a period of 15 min.While stirring for 16 h,
the mixture was allowed to warm up to room temperature.The pre-
cipitated DCU was filtered off, the filtrate was evaporated, and the
resulting crude product was recrystallized from isopropanol.Yield:
2.05 g (78 %) of colorless crystals. – mp: 125–127 °C (i-PrOH)
(129 °C, i-PrOH) [23] – 1H-NMR (200 MHz, DMSO-D6): δ (ppm) =
2.81 (s, 4 H, OSu), 2.82–2.93 (m, 4 H, Ph-CH2-CH2), 6.67 (d, 8.5
Hz, 2H, Harom.), 7.07 (d, 8.5 Hz, 2H, Harom.), 9.22 (s, 1H, OH). – IR
(KBr, cm–1): 3401, 1775, 1743, 1517, 1221, 1071. – C13H13NO5
(263.25). C, H, N.
N-[3-(4-Hydroxyphenyl)propionyl]-L-isoleucyl-L-proline methyl es-
ter (15)
Method A: Compounds 9 (1.10 g, 4.00 mmol) and 12 (0.83 g, 5.00
mmol) were dissolved in EtOAc (30 mL) and cooled to 0 °C.To this
mixture a solution of DCC (0.89 g, 4.30 mmol) in EtOAc (10 mL)
was added dropwise over a period of 15 min.While stirring for 16 h,
the mixture was allowed to warm up to room temperature.The pre-
cipitated DCU was filtered off and the filtrate evaporated; the resi-
due was chromatographed over silica gel (eluent: EtOAc/PE 3:1).
Yield: 1.32 g (84 %) of a colorless, amorphous substance.
N-[3-(4-Hydroxyphenyl)propionyl]-L-isoleucine (9)
Method B:Compound 8 (1.18 g, 4.50 mmol) was dissolved in etha-
nol (20 mL) and slowly added dropwise to a solution of L-isoleucyl-
L-proline methyl ester (14, 1.2 g, 4.90 mmol) in ethanol (10 mL),
which was generated from 13 following the TFA/DIPEA protocol
(see below). The mixture was stirred over night at room tempera-
ture. After evaporation of the solvent, the residue was chromato-
graphed over silica gel (eluent: EtOAc/PE 3:1).
TFA/DIPEA protocol: To a solution of 13 (1.68 g, 4.90 mmol) in
CH2Cl2 (20 mL) TFA (7 mL) was added dropwise. After stirring at rt
for 3 h, the mixture was evaporated in vacuo, dissolved in methanol
and evaporated once again to eliminate residualTFA.The resulting
oil was dissolved in CH2Cl2 and the amino group was released from
its TFA salt by addition of DIPEA (1.90 g, 14.70 mmol) in CH2Cl2
(10 mL). The mixture was evaporated in vacuo; the residue was
dissolved in ethanol (10 mL) and was subjected to the above-
described procedure.
Yield: 0.73 g (48 %) of a colorless, amorphous substance. – mp:
60 °C (PE/EtOAc) – 1H-NMR (300 MHz, CDCl3):δ (ppm) = 0.80 (d,
6.8 Hz, 3 H, Ile-CH-CH3), 0.94 (t, 7.3 Hz, 3 H, Ile-CH2-CH3), 1.00–
1.17 (m, 1 H, Ile-CH2-CH3), 1.29–1.42 (m, 1 H, Ile-CH2-CH3),
1.62–1.77 (m, 1 H, Ile-β-CH), 1.90–2.26 (m, 4 H, Pro-β-CH2-
γ-CH2), 2.36 (t, 7.8 Hz, 2 H, Ph-CH2-CH2), 2.81 (t, 7.8 Hz, 2 H, Ph-
CH2-CH2), 3.50–3.60 (m, 1 H, Pro-N-CH2), 3.70 (s, 3 H, OCH3),
3.83–3.93 (m, 1 H, Pro-N-CH2), 4.41–4.45 (dd, 8.3 Hz + 3.3 Hz,
1H, Pro-α-H), 4.80–4.86 (dd, 9.2 Hz + 5.5 Hz, 1 H, Ile-α-H), 6.24
(d, 9.2 Hz, 1 H, NH), 6.73 (d, 8.5 Hz, 2 H, Harom.), 6.96 (d, 8.5 Hz,
2H, Harom.), 7.51 (s, 1 H, OH). – IR (KBr, cm–1): 3293, 2963, 2932,
2877, 1747, 1627, 1542, 1517, 1448, 1364, 1267, 1219, 1197,
1172, 1100, 1048, 998, 830, 721, 535, 489. – [α] = –102.5 (2 %,
CH2Cl2). – C21H30N2O5 (390.48). C, H, N.
A solution of 8 (1.97 g, 7.50 mmol) in ethanol (30 mL) was added
dropwise to a mixture of L-isoleucine (1.48 g, 11.25 mmol) and
NaHCO3 (1.90 g) in water (22 mL).The mixture was stirred for 16 h
at room temperature and the organic solvent was evaporated.The
remaining aqueous solution was acidified (pH 2) by addition of
conc.HCl and extracted with CH2Cl2 (3 × 15 mL).The combined or-
ganic layers were dried with Na2SO4 and evaporated under re-
duced pressure.The residue was recrystallized from EtOAc.Yield:
1.60 g (76 % ) of a colorless powder. – mp: 122–126 °C (EtOAc) –
1H-NMR (300 MHz, DMSO-D6):δ (ppm) = 0.81 (d, 6.8 Hz, 3 H, Ile-
CH-CH3), 0.81 (t, 7.4 Hz, 3 H, Ile-CH2-CH3), 1.14 (m, 1 H, Ile-CH2),
1.33 (m, 1 H, Ile-CH2), 1.72 (m, 1 H, Ile-β-CH), 2.40 (t, 7.7 Hz, 2 H,
Ph-CH2-CH2), 2.68 (t, 7.7 Hz, 2 H, Ph-CH2-CH2), 4.17 (dd, 8.5 Hz +
6.2 Hz, 1 H, Ile-α-H), 6.64 (d, 8.5 Hz, 2 H, Harom.), 6.98 (d, 8.5 Hz,
2H, Harom.), 7.94 (d, 8.5 Hz, 1 H, Ile-NH), 9.12 (s, 1 H, OH), 12.50 (s,
1H, COOH). – IR (KBr, cm–1): 2500–3400, 3303, 2969, 2939,
1710, 1630, 1548, 1514, 1450, 1226, 823, 729, 693, 592, 530, 436.
– [α] = –11.5 (2 % , MeOH). – C15H21NO4 (279.34). C, H, N.
N-[3-(4-Hydroxyphenyl)propionyl]-L-isoleucyl-L-proline 2-(piper-
azin-1-yl)ethyl ester · TFA (11)
TFA (4 mL) was added to an ice-cooled solution of 0.43 g
(0.73 mmol) of 18 in CH2Cl2 (9 mL). After stirring at rt for 3 h, the
mixture was evaporated in vacuo, dissolved in methanol and evap-
orated once again to eliminate residual TFA.Yield: 0.17 g (39 %) of
a colorless, viscous substance which was subjected to reaction
with SMITU without further purification. C26H40N4O5 (488.62).
Boc-L-Isoleucyl-L-proline methyl ester (13)
Boc-L-Isoleucine (2.40 g, 10.00 mmol) and DCC (2.06 g,
10.00 mmol) were dissolved in ice-cooled EtOAc (20 mL) and
stirred for 2 h. L-Proline methyl ester hydrochloride (12, 2.48 g,
15.00 mmol) was added to an aqueous solution of Na2CO3 (1.70 g
in 5 mL H2O) and stirred for 15 min.The mixture was extracted with
CH2Cl2 (3 × 15 mL), the combined CH2Cl2 layers were dried over
Na2SO4, and the filtrate was evaporated.The resulting oil was dis-
solved in EtOAc (10 mL) and added dropwise to the activated Boc-
L-isoleucine solution at 0 °C.While stirring for 16 h, the mixture was
allowed to warm up to room temperature. The precipitated DCU
was filtered off and the filtrate was evaporated; the residue was
chromatographed on silica gel (eluent:EtOAc/PE 1:4).Yield:2.49 g
(73 %) of a colorless, amorphous substance. – mp: 56–58 °C (PE/
EtOAc) (“oily product” [24]). – 1H-NMR (200 MHz, CDCl3):δ (ppm)
= 0.91 (t, 7.4 Hz, 3 H, Ile-CH2CH3), 1.02 (d, 7.2 Hz, 3 H, Ile-CH-
CH3), 1.10–1.60 (m, 2 H, Ile-CH2CH3), 1.42 (s, 9 H, Boc), 1.60–
2.30 (m, 2 H, Pro-γ-CH2;m, 2H, Pro-β-CH2;m, 1H, Ile-β-CH), 3.72
(s, 3 H, OCH3), 4.29 (m, 1 H, Pro-α-H), 4.54 (m, 1 H, Ile-α-H), 5.15
(d, 7.4 Hz, 1 H, NH). – IR (KBr, cm–1): 3303, 2966, 2933, 2877,
1749, 1707, 1645, 1502, 1435, 1391, 1366, 1316, 1279, 1249,
1173, 1093, 1043, 1019.– [α] = –34.2 (2 %, CH2Cl2).– C17H30N2O5
(342.44). C, H, N.
N-[3-(4-Hydroxyphenyl)propionyl]-L-isoleucyl-L-proline (16)
Methyl ester 15 (1.95 g, 5.00 mmol) was stirred in a mixture of
aqueous LiOH (1 M, 5 mL) and DME (5 mL) at room temperature
for 2 h.The mixture was acidified (pH 4) by addition of aqueous cit-
ric acid (10 %) and extracted with EtOAc (2 × 20 mL). The com-
bined organic layers were dried with Na2SO4 and the filtrate evapo-
rated; the residue was chromatographed over silica gel (eluent:
CH2Cl2/EtOAc/PE/MeOH 10:10:10:1 → CH2Cl2/EtOAc/MeOH
10:10:5). Yield: 1.87 g (quant.) of a colorless, amorphous sub-
stance. – mp: 70 °C – 1H-NMR (300 MHz, MeOH-D4): δ (ppm) =
0.84 (d, 6.7 Hz, 3 H, Ile-CH-CH3), 0.97 (t, 7.7 Hz, 3 H, Ile-CH2-CH3),
0.95–1.10 (m, 1 H, Ile-CH2-CH3), 1.35–1.50 (m, 1 H, Ile-CH2-CH3),
1.70–2.05 (m, 4 H, Pro-β-CH2-γ-CH2), 2.20–2.30 (m, 1 H, Ile-
β-CH), 2.41 (t, 7.4 Hz, 2 H, Ph-CH2-CH2), 2.79 (t, 7.4 Hz, 2 H, Ph-
CH2-CH2), 3.55–3.70 (m, 1 H, Pro-N-CH2), 3.80–3.95 (m, 1 H, Pro-
N-CH2), 4.35–4.45 (m, 2 H, Pro-α-H, Ile-α-H), 6.68 (d, 8.5 Hz, 2 H,
Harom.), 6.97 (d, 8.5 Hz, 2 H, Harom.). – IR (KBr, cm–1): 3304, 2967,
1733, 1613, 1541, 1516, 1454, 1375, 1225, 1102, 1045, 994, 828,
668, 536.– [α] = –80.0 (2 % , MeOH).– C20H28N2O5 (376.46).C, H,
N.
© 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

378 Radau et al.
Arch. Pharm. Pharm. Med. Chem. 2003, 336, 372–380
1-(tert.-Butyloxycarbonyl)-4-(2-hydroxyethyl)piperazine (17)
stirred for an additional hour. A solution of 19 (0.91 g,
4.84 mmol) in CH2Cl2 (70 mL) was added dropwise over a peri-
od of 2 h. While stirring for 2 days, the mixture was allowed to
warm up to room temperature.The solvent was evaporated un-
der reduced pressure and the resulting residue was purified by
column chromatography (silica gel, eluent: CH2Cl2/EtOAc/PE/
MeOH 10:10:10:1). Yield: 0.64 g (26 %) of a colorless sub-
stance. – mp: 130–131 °C – 1H-NMR (300 MHz, CDCl3): δ
(ppm) = 0.80–0.95 (m, 6 H, Ile-CH-CH3 + Ile-CH2-CH3); 1.00–
1.15 (m, 1 H, Ile-CH2-CH3), 1.25–1.60 (m, 4 H, NH-
CH2CH2CH2CH2-NHBoc + m, 1 H, Ile-CH2-CH3), 1.43 (s, 9 H,
Boc), 1.60–1.80 (m, 1 H, Ile-β-H), 1.80–2.15 (m, 3 H, Pro-
β-CH2 + Pro-γ-CH2), 2.20–2.30 (m, 1 H, Pro-β-CH2), 2.41–2.52
(m, 2 H, Ph-CH2-CH2), 2.85 (m, 2 H, Ph-CH2-CH2), 2.95–3.25
(m, 4 H, CH2NHBoc + Pro-CONH-CH2), 3.55–3.65 (m, 1 H,
Pro-N-CH2), 3.75–3.85 (m, 1 H, Pro-N-CH2), 4.36–4.46 (m,
1 H, Pro-α-H), 4.52–4.58 (m, 1 H, Ile-α-H), 5.00 (t, broad, 1 H,
NH-Boc), 6.24 (d, broad, 1 H, Ile-NH), 6.73 (d, 8.3 Hz, 2 H,
Harom.), 6.89 (t, broad, 1 H, Pro-CONH-CH2), 6.99 (d, 8.3 Hz,
2 H, Harom.), 7.42 (s, 1 H, OH). – [α] = –43.61 (c = 2 %, MeOH). –
C29H46N4O6 (546.70). C, H, N.
1-(2-Hydroxyethyl)piperazine (1.30 g, 10.0 mmol) was dissolved in
CH2Cl2 (10 mL), cooled to 0°C, and finally a solution of di-tert-butyl
dicarbonate (Boc2O, 2.40 g, 11.00 mmol) in CH2Cl2 (5 mL) was
added dropwise. After stirring over night the solvent was evaporat-
ed and the residue chromatographed over silica gel (eluent:
EtOAc/PE 3:1 → CH2Cl2/PE/MeOH 10:1:1).Yield:2.20 g (96 %) of
a colorless oil.– 1H-NMR (300 MHz, CDCl3):δ(ppm) = 1.46 (s, 9 H,
t-butyl), 2.46 (t, 5.1 Hz, 4 H, N(CH2CH2)2N-Boc), 2.55 (t, 5.4 Hz,
2H, HO-CH2CH2N), 3.44 (t, 5.1 Hz, 4 H, N(CH2CH2)2N-Boc), 3.63
(t, 5.4 Hz, 2 H, HO-CH2CH2N). – C11H22N2O3 (230.31). C, H, N.
N-[3-(4-Hydroxyphenyl)propionyl]-L-isoleucyl-L-proline 2-[4-
(tert.-butyloxycarbonyl)-piperazin-1-yl]ethyl ester (18)
A solution of DIC (0.56 g, 4.40 mmol) in CH2Cl2 (30 mL) was
added dropwise to a stirred, ice-cooled mixture of 16 (1.65 g,
4.40 mmol) in CH2Cl2 (120 mL) over a period of 20 min and was
stirred for one additional hour. Compound 17 (1.11 g,
4.84 mmol) in CH2Cl2 (70 mL) was added dropwise over a peri-
od of 2 h. While stirring over night, the mixture was allowed to
warm up to room temperature.The solvent was evaporated un-
der reduced pressure and the resulting residue was purified by
chromatography (silica gel, eluent: CH2Cl2/EtOAc/PE/MeOH
10:10:10:1).Yield:1.42 g (55 %) of a colorless substance.– mp:
60 °C – 1H-NMR (300 MHz, CDCl3): δ (ppm) = 0.84 (t, 7.3 Hz,
3 H, Ile-CH2-CH3), 0.95 (d, 6.8 Hz, 3 H, Ile-CH-CH3), 1.00–1.15
(m, 1 H, Ile-CH2-CH3), 1.30–1.45 (m, 1 H, Ile-CH2-CH3), 1.46 (s,
9 H, t-butyl), 1.60–1.75 (m, 1 H, Ile-β-H), 1.85–2.25 (m, 4 H,
1-Amino-4-{N-[3-(4-hydroxyphenyl)propionyl]-L-isoleucyl-L-pro-
lylamino}butane x TFA (21)
TFA (4 mL) was added to an ice-cooled solution of 0.60 g of 20
(1.10 mmol) in CH2Cl2 (10 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 chromatography (CH2Cl2/EtOAc/
MeOH 10:10:5).Yield:0.40 g (66 %) of a colorless, viscous sub-
stance. – 1H-NMR (300 MHz, MeOH-D4): δ (ppm) = 0.90–0.95
(m, 6 H, Ile-CH-CH3 + Ile-CH2-CH3); 1.10–1.20 (m, 1 H, Ile-
CH2-CH3), 1.25–1.35 (m, 1 H, Ile-CH2-CH3), 1.50–1.60 (m, 4 H,
NH-CH2CH2CH2CH2-NH2), 1.70–1.80 (m, 1 H, Ile-β-H), 1.95–
2.05 (m, 3 H, Pro-β-CH2 + Pro-γ-CH2), 2.10–2.20 (m, 1 H,
Pro-β-CH2), 2.45–2.55 (m, 2 H, Ph-CH2-CH2), 2.81 (t, 7.5 Hz,
2 H, Ph-CH2-CH2), 3.10–3.25 (m, 4 H, CH2NH2 + Pro-CONH-
CH2), 3.55–3.65 (m, 1 H, Pro-N-CH2), 3.85–3.90 (m, 1 H, Pro-
N-CH2), 4.35–4.40 (m, 2 H, Pro-α-H + Ile-α-H), 6.67 (d, 8.5 Hz,
2 H, Harom.), 7.00 (d, 8.5 Hz, 2 H, Harom.). – [α] = –52.45 (c =
1.85 %, MeOH). – C26H39F3N4O6 (560.61). C, H, N.
Pro-β-CH2-γ-CH2), 2.30–2.50 (m, 6 H, OCH2CH2N
+
N(CH2CH2)2N-Boc), 2.58–2.64 (m, 2 H, Ph-CH2-CH2), 2.79–
2.85 (m, 2 H, Ph-CH2-CH2), 3.36–3.44 (m, 4 H, N(CH2CH2)2N-
Boc), 3.50–3.60 (m, 1 H, Pro-N-CH2), 3.80–3.90 (m, 1 H, Pro-
N-CH2), 4.24 (t, 5.9 Hz, 2 H, OCH2), 4.41 (dd, 8.3 Hz + 3.5 Hz,
1 H, Pro-α-H), 4.82 (dd, 9.0 Hz + 5.2 Hz, 1 H, Ile-α-H), 6.14 (d,
9.1 Hz, 1 H, NH), 6.73 (d, 8.6 Hz, 2 H, Harom.), 6.98 (d, 8.6 Hz,
2 H, Harom.). – IR (KBr, cm–1): 3293, 2970, 2934, 2876, 1748,
1698, 1628, 1516, 1453, 1366, 1245, 1171, 1130, 1004, 864,
830, 770 534. – [α] = –32.83 (c = 2 %, MeOH). – C31H48N4O7
(588.75). C, H, N.
N-[3-(4-Hydroxyphenyl)propionyl]-L-isoleucyl-L-proline 2-(4-
amidino-piperazin-1-yl)ethyl ester · 2 HI (RA-1000)
1-Guanidino-4-{N-[3-(4-hydroxyphenyl)propionyl]-L-isoleu-
cyl-L-prolylamino}butane · HI (RA-1001)
A solution of 11 (1.45 g, 2.98 mmol) and DIPEA (0.85 g, 6.55
mmol) in CH2Cl2 (40 mL) was stirred at rt for 3 h. After evapora-
tion of the solvent, H2O (10 mL) and SMITU (0.86 g, 3.12 mmol)
were added and the mixture was stirred over night at rt.The sol-
vent was evaporated and the crude product was purified by col-
umn chromatography (CH2Cl2/EtOAc/MeOH 10:10:5). Yield:
0.47 g (24 %) of a colorless, viscous substance. – 1H-NMR
(300 MHz, MeOH-D4): δ (ppm) = 0.90–0.95 (m, 6 H, Ile-CH-
CH3 + Ile-CH2-CH3); 1.10–1.20 (m, 1 H, Ile-CH2-CH3), 1.25–
1.35 (m, 1 H, Ile-CH2-CH3), 1.70–1.80 (m, 1 H, Ile-β-H), 1.95–
2.10 (m, 3 H, Pro-β-CH2 + Pro-γ-CH2), 2.10–2.20 (m, 1 H,
Pro-β-CH2), 2.20–2.40 (m, 6 H, OCH2CH2N + N(CH2CH2)2-
A solution of 21 (350 mg, 0.64 mmol) and DIPEA (86 mg,
0.67 mmol) in CH2Cl2 (10 mL) was stirred at rt for 3 h. After
evaporation of the solvent, H2O (5 mL) and SMITU (146 mg,
0.67 mmol) were added to the residue.The mixture was stirred
over night at rt, the solvent was evaporated, and the crude
product was purified by column chromatography (CH2Cl2/
EtOAc/MeOH 10:10:5).Yield: 0.37 g (94 %) of a colorless, vis-
1
cous substance. – H-NMR (DMSO-D6, 300 MHz): δ (ppm) =
0.73–0.86 (m, 6 H, Ile-CH2-CH3 + Ile-CH-CH3), 0.90–1.10 (m,
1 H, Ile-CH2), 1.10–1.30 (m, 1 H, Ile-CH2), 1.35–1.50 (m, 4 H,
NHCH2CH2CH2, agmatine), 1.60–2.10 (m, 5 H, Pro-β-/γ-CH2 +
Ile-β-CH), 2.36 (t, 7.6 Hz, 2 H, Ph-CH2CH2), 2.65 (t, 7.6 Hz, 2 H,
Ph-CH2CH2), 2.90–3.15 (m, 4 H, CONHCH2 + CH2-guan.), 3.50
(m, 1 H, Pro-δ-CH2), 3.72 (m, 1 H, Pro-δ-CH2), 4.22 (m, 1 H,
Ile-α-H), 4.34 (m, 1 H, Pro-α-H), 6.62 (d, 8.6 Hz, 2 H, Haromat.),
6.96 (d, 8.6 Hz, 2 H, Haromat.), 7.60 (m, 1 H, NH, agmatine), 8.03
(m, 1 H, NH, Ile), 8.84 (s, 5 H, guan.-HI).– [α] = –57.94 (c = 2 %,
MeOH). – C25H41IN6O4 (616.54). C, H, N.
N
guan.), 2.53 (t, 7.5 Hz, 2 H, Ph-CH2-CH2), 2.81 (t, 7.5 Hz, 2 H,
Ph-CH2-CH2), 3.35–3.45 (m, 4 H, N(CH2CH2)2Nguan.), 3.55–
3.65 (m, 1 H, Pro-N-CH2), 3.85–3.90 (m, 1 H, Pro-N-CH2), 4.16
(t, 5.7 Hz, 2 H, OCH2), 4.35–4.40 (m, 2 H, Pro-α-H + Ile-α-H),
6.67 (d, 8.5 Hz, 2 H, Harom.), 7.00 (d, 8.5 Hz, 2 H, Harom.). – [α] =
–18.21 (c = 2 %, MeOH). – C27H44I2N6O5 (786.48). C, H, N.
1-tert.-Butyloxycarbonylamino-4-{N-[3-(4-hydroxyphenyl)pro-
pionyl]-L-isoleucyl-L-prolylamino}butane (20)
N-Benzoyl-L-leucine methyl ester (24)
A solution of DIC (0.56 g, 4.40 mmol) in CH2Cl2 (30 mL) was
added dropwise to a stirred, ice-cooled mixture of 16 (1.65 g,
4.40 mmol) in CH2Cl2 (120 mL) over a period of 20 min and was
To a solution of 1.41 g benzoylchloride (10.00 mmol) in CH2Cl2
(30 mL) a mixture of 1.82 g L-leucine methyl ester hydrochlo-
ride (10.00 mmol) and 2.84 g DIPEA (22.00 mmol) in CH2Cl2
© 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Arch. Pharm. Pharm. Med. Chem. 2003, 336, 372–380
New cyanopeptide-derived low molecular weight Thrombin inhibitors 379
(50 mL) was added over a period of 30 min at rt. The mixture
was stirred for 16 h and the solvent was evaporated to half of
the volume and filtered off from DIPEAxHCl. After evaporation
to dryness the residue was recrystallized from EtOAc/PE.
Colorless crystals; Yield: 1.52 g (61 %) – mp: 101.5–103 °C
(EtOAc/PE) (103–105 °C [25], 102 °C [18]) – 1H-NMR (CDCl3,
300 MHz): δ (ppm) = 0.97 (d, 6.3 Hz, 3 H, CH(CH3)2), 0.99 (d,
6.3 Hz, 3 H, CH(CH3)2), 1.64–1.79 (m, 3 H, CH(CH3)2 + β-CH2),
3.77 (s, 3 H, OCH3), 4.84–4.91 (m, 1 H, α-H), 6.57 (d, 7.9 Hz,
1 H, NH), 7.40–7.53 (m, 3 H, Harom.), 7.79–7.83 (m, 2 H, Harom.).
– IR (KBr, cm–1): 3274, 3064, 2955, 2867, 1746, 1636, 1604,
1579, 1542, 1493, 1469, 1451, 1433, 1351, 1304, 1275, 1228,
1207, 1182, 1087, 1029, 986, 874, 860, 823, 804, 743, 701,
650, 552, 482. – [α] = –21.80 (2 %, MeOH). – C14H19NO3
(249.31). C, H, N.
1-tert.-Butyloxycarbonylamino-3-[(N-benzoyl)-L-leucyl-L-pro-
lyl-amino]propane (29)
0.66 g (5.19 mmol) DIC in CH2Cl2 (30 mL) was added dropwise
to an ice-cooled solution of 1.50 g (4.72 mmol) of 27 in CH2Cl2
(50 mL) over a period of 10 min. After stirring for 30 min, a solu-
tion of 0.91 g (5.19 mmol) 1-amino-3-[(tert-butyloxycarbon-
yl)amino]propane (28) in CH2Cl2 (50 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). Colorless substance; Yield: 1.35 g (60 %) – mp:
57–60 °C – 1H-NMR (300 MHz, CDCl3): δ (ppm) = 1.01 (d, 6.3
Hz, 3 H, CH(CH3)2), 1.02 (d, 6.3 Hz, 3 H, CH(CH3)2), 1.40 (s,
9 H, Boc), 1.51–1.65 (m, 3 H, CH(CH3)2 + Leu-β-CH2), 1.72–
1.81 (m, 2 H, NH-CH2CH2-CH2NHBoc), 2.01–2.08 (m, 3 H,
Pro-β-CH2 + Pro-γ-CH2), 2.32–2.35 (m, 1 H, Pro-β-CH2),
2.96–3.12 (m, 2 H, NH-CH2CH2CH2NHBoc), 3.24–3.30 (q, 6.0
Hz, 2 H, CH2NHBoc), 3.51–3.60 (m, 1 H, Pro-N-CH2), 4.05–
4.11 (m, 1 H, Pro-N-CH2), 4.58–4.61 (m, 1 H, Pro-α-H), 4.73–
4.83 (m, 1 H, Leu-α-H), 5.26 (t, 1 H, broad, NH-Boc), 6.94 (d,
6.3 Hz, 1 H, Leu-NH), 7.16 (t, 1 H, broad, Pro-NH-CH2), 7.40–
7.52 (m, 3 H, Harom.), 7.76–7.79 (m, 2 H, Harom.).– IR (KBr, cm–1):
3325, 3066, 2959, 2933, 2871, 1689, 1636, 1578, 1537, 1490,
1448, 1390, 1365, 1343, 1251, 1171, 712, 694. – [α] = +34.35
(1.95 %, MeOH). – C26H40N4O5 (488.63). C, H, N.
N-Benzoyl-L-leucine (25)
3.00 g of 24 (12.05 mmol) were saponified according to the pro-
cedure in the synthesis of compound 16. Colorless crystals;
Yield:2.77 g (98 %) – mp:105.5–106.5 °C (EtOAc/PE) (106 °C,
EtOAc/PE [26]; 105–106 °C [27]) – 1H-NMR (DMSO-D6,
300 MHz): δ (ppm) = 0.88 (d, 6.3 Hz, 6 H, CH(CH3)2), 0.92 (d,
6.3 Hz, 3 H, CH(CH3)2), 1.54–1.83 (m, 3 H, CH(CH3)2 + β-CH2),
4.41–4.47 (m, 1 H, α-H), 7.44–7.54 (m, 3 H, Harom.), 7.87–7.90
(m, 2 H, Harom.), 8.57 (d, 1 H, 8.0 Hz, NH), 12.50 (s, 1 H, COOH).
– IR (KBr, cm–1): 2850–3350, 3336, 3234, 3072, 2955, 2872,
1731, 1699, 1643, 1623, 1558, 1534, 1491, 1471, 1439, 1418,
1371, 1340, 1269, 1234, 1203, 1172, 1143, 1074, 1027, 929,
866, 800, 716, 690, 584, 536, 487, 470. – [α] = –9.83 (2 %, Me-
OH). – C13H17NO3 (235.29). C, H, N.
1-Amino-3-[(N-benzoyl)-L-leucyl-L-prolyl-amino]propane ·
TFA (30)
TFA (8 mL) was added to an ice-cooled solution of 0.92 g of 29
(1.94 mmol) in CH2Cl2 (20 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 chromatography (CH2Cl2/EtOAc/
MeOH 10:10:5).Yield:0.47 g (49 %) of a colorless, viscous sub-
N-Benzoyl-L-leucyl-L-proline methyl ester (26)
1.26 g (10.00 mmol) DIC in CH2Cl2 (30 mL) was added drop-
wise to an ice-cooled solution of 2.35 g (10.00 mmol) 25 in
CH2Cl2 (100 mL) over a period of 30 min. After stirring for fur-
ther 30 min, a solution of 1.82 g (11.00 mmol) L-proline methyl
ester hydrochloride and 1.56 g (12.10 mmol) DIPEA in CH2Cl2
(40 mL) was added (45 min).The mixture was stirred for 16 h al-
lowing to warm up to rt and – after evaporation of the solvent –
the residue was purified by column chromatography (PE/
EtOAc = 1:1). Colorless, viscous substance; Yield: 3.48 g
(86 %). – 1H-NMR (CDCl3, 300 MHz): δ (ppm) = 0.89–1.07 (m,
6 H, CH(CH3)2), 1.50–1.85 (m, 3 H, CH(CH3)2 + Leu-β-CH2),
1.90–2.30 (m, 4 H, Pro-β-CH2 + Pro-γ-CH2), 3.55–3.65 (m, 1 H,
Pro-N-CH2), 3.70 (s, 3 H, OCH3), 3.85–3.96 (m, 1 H, Pro-N-
CH2), 4.44–4.48 (m, 1 H, Pro-α-H), 5.03–5.16 (m, 1 H, Leu-
α-H), 6.97 (d, 8.4 Hz, 1 H, NH), 7.39–7.49 (m, 3 H, Harom.),
7.77–7.82 (m, 2 H, Harom.). – IR (KBr/film, cm–1): 3307, 3059,
2955, 2871, 1745, 1633, 1579, 1537, 1490, 1437, 1386, 1366,
1343, 1291, 1197, 1174, 711, 693. – [α] = –48.98 (1.18 %,
MeOH). – C19H26N2O4 (346.43). C, H, N.
1
stance. – H-NMR (300 MHz, DMSO-D6): δ (ppm) = 0.92 (d,
6.4 Hz, 3 H, CH(CH3)2), 0.93 (d, 6.4 Hz, 3 H, CH(CH3)2), 1.60–
1.80 (m, 1 H, CH(CH3)2 + m, 2 H, Leu-β-CH2 + m, 2 H, NH-
CH2CH2CH2NH+3), 1.85–1.95 (m, 3 H, Pro-β-CH2 + Pro-γ-CH2),
2.00–2.05 (m, 1 H, Pro-β-CH2), 2.70–2.80 (m, 2 H, NH-
CH2CH2CH2NH+3), 3.05–3.20 (m, 2 H, CH2NH2), 3.50–3.60 (m,
1 H, Pro-N-CH2), 3.75–3.85 (m, 1 H, Pro-N-CH2), 4.20–4.30
(m, 1 H, Pro-α-H), 4.60–4.70 (m, 1 H, Leu-α-H), 7.40–7.60 (m,
3 H, Harom.), 7.65–7.75 (s, 3 H, broad, NH+3), 7.76 (t, 5.8 Hz, 1 H,
NH-CH2CH2CH2NH3+), 7.85–7.95 (m, 2 H, Harom.), 8.65 (d, 6.8
Hz, 1 H, Leu-NH). – [α] = –38.13 (c = 2 %, MeOH). –
C23H33F3N4O5 (502.53). C, H, N.
1-Guanidino-3-[(N-benzoyl)-L-leucyl-L-prolyl-amino]propane ·
HI (RA-1002)
A solution of 30 (200 mg, 0.41 mmol) and DIPEA (57 mg, 0.45
mmol) in CH2Cl2 (10 mL) was stirred at rt for 3 h. After evapora-
tion of the solvent, 146 mg (0.67 mmol) SMITU in a mixture of
H2O/EtOH (5 mL/5 mL) were added to the residue.The mixture
was stirred over night at rt, the solvent was evaporated, and the
crude product was purified by column chromatography
(CH2Cl2/EtOAc/MeOH 10:10:5).Yield: 0.12 g (52 %) of a color-
N-Benzoyl-L-leucyl-L-proline (27)
1.36 g (3.92 mmol) of 26 were saponified according to the pro-
cedure in the synthesis of compound 16. Colorless crystals;
Yield: 1.01 g (81 %) – mp: 89–92 °C – 1H-NMR (DMSO-D6,
300 MHz): δ (ppm) = 0.78–0.95 (m, 6 H, CH(CH3)2), 1.30–2.30
(m, 7 H, Pro-β-CH2-γ-CH2 + CH(CH3)2 + Leu-β-CH2), 3.51–3.60
(m, 1 H, Pro-N-CH2), 3.72–3.81 (m, 1 H, Pro-N-CH2), 4.18–
4.29 (m, 1 H, Pro-α-H), 4.48–4.56 (m, 1 H, Leu-α-H), 7.42–
7.53 (m, 3 H, Harom.), 7.87–7.92 (m, 2 H, Harom.), 8.47 (d, 8.0 Hz,
1 H, NH), 12.53 (s, 1 H, COOH). – IR (KBr, cm–1): 2800-3500,
3324, 3062, 2957, 2872, 1732, 1631, 1578, 1538, 1490, 1450,
1342, 1296, 1221, 1192, 920, 870, 803, 712, 693, 668, 600, 570.
– [α] = –39.66 (2 %, MeOH). – C18H24N2O4 (332.40). C, H, N.
1
less, viscous substance. – H-NMR (300 MHz, DMSO-D6): δ
(ppm) = 0.92 (d, 6.3 Hz, 3 H, CH(CH3)2), 0.93 (d, 6.3 Hz, 3 H,
CH(CH3)2), 1.60–1.95 (m, 9 H, Leu-β-CH2 + Leu-γ-CH + Pro-
β-CH2 + Pro-γ-CH2 + CH2CH2-guan.), 2.70–2.85 (m, 2 H,
CONHCH2), 3.05–3.15 (m, 2 H, CH2-guan.), 3.45–3.65 (m, 1 H,
Pro-δ-CH2), 3.70–3.90 (m, 1 H, Pro-δ-CH2), 4.25 (m, 1 H,
Pro-α-H), 4.65 (m, 1 H, Leu-α-H), 7.37–7.56 (m, 3 H, Haromat.),
7.90 (m, 2 H, Haromat.), 8.33 (t, 1 H, broad, Pro-NH), 8.48 (s, 5 H,
broad, guan.-HI), 8.65 (d, 7.1 Hz, 1 H, Leu-NH). – [α] = –54.38
(c = 2 %, MeOH). – C22H35IN6O3 (558.46). C, H, N.
© 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

380 Radau et al.
Arch. Pharm. Pharm. Med. Chem. 2003, 336, 372–380
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