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Chemistry and Molecular Biology in the Search for New LHRH Antagonists.

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Chemistry and Molecular Biology in the Search for New LHRW Antagonists
Bernhard Kutscher,* Michael Bernd, Thomas Beckers, Emmanuel E. Polymeropgulos,
and Jiirgen Engel
Dedicated to Proj>ssor Herrherr Offermanns on [he occusion of his 60th birrhdujl
Hormones--and in particular, the sex
the first growth factors discovered to be involuntary
helpers of cancer. Female breast cancer
and male prostate cancer are the best
known examples of tumors acknowledged to be hormone-dependent. A look
at cancer statistics shows that breast
cancer is still the most frequent cancer in
women; in men, prostate cancer plays a
similarly dominant role with increasing
age. Shutting down the main production
site of the sex hormmes estrogen and
testosterone either by removing the
ovaries or by castration is a well-known
and often effective therapy; however.
these procedures can be problematic due
to the concomjttant psychological
stress. Modern hormone therapy for advanced breast cancer and prostate cancer attempts to spare the patient such
irreversible operative procedures for as
1. Introduction
The releasing hormone, gonadorelin (GnKH; synonymous
with LH KH, luteinizing hormone-releasing hormone or gonadolibcrin). together with its specific receptor, plays a central
role in neuroendwrinology.“I The decapeptidc LH’RH, discovercd by A . V. Schally et al. and R. Guillemin et al.,‘’] is formed
in the cell bodies1 of hypothalamic neurons and is secreted in
pulses into the blood stream. Ultimately it stimulates secretion
of the sex-specifia hormones in the testes and ovarics. Intensive
research has revealed that specific receptors for LHRH and
synthetic analogues are also present in the pituitary gland and
other tissues (for example, tumor cells)f31and organs.
Three concepts,for therapeutic application have emerged. The
first is the restoration of normal physiology by administration
of LHRH by infusion pump to promote fertility in men and
women who are infertile due tedcfective endogenous LHKH
secrction Second, long-lasting LHRH agonists arc used in a
depot form. which bring about desensitization of the pituitary
receptors and thus interpupt the signal cascade. This results in a
biochemical ”castration”, which opens up new therapeutic possibilities for hormone-dependent diseases such as prostate canccr, breast cancer, anbendometriosis. Although superagonists
long as possible, by using hormone antagonists. such as the LHRH antagonists, which hinder deployment of the
hormone itself and thus :ts growthpromoting activity.
conformation analysis
LHRH antagonists pcptidomimctics
receptors tumor therapy
are generally well tolerated, they .have the disadvantage that
hormone secretion (estrogen. testosterone) I S initially stimulated
before the depletion of receptors o r “down” regulation can take
This has led to
place, and thus the illness temporarily wor~ens.’~’
the developmcnt of the third concept: use of LHRH antagon i s t ~ . ‘ ~At’ present, about 5000 LHRH analogues have been
synthesized worldwide and tested in vitro or in vivo.IL1 ‘1 Whilst
LHKH agonists have been on the market for about len years
(see Section 2). the LNRH antagonists that have been developed furthest arc still in clinical testing (Figurc I).!’’
1.1 Prof B Kurschea. Dr Mr Bernd. Dr T Bcckers. Dr E E Polymeropoulos.
Prof J t n g c l
AST.4 Medica AG. Konzernforschung
WeismullcrrtrJrw 45. D-60315Frankfurt (Germany)
Fax. Int. code -(69)4001-2628
c-mail Ur.Rcrnhard K
Chrm Inr Ed. E ~ g l19Y7. 36. 2148 2161
Figure 1. Anfilumor actrvlty of LHRH agorliscs dml antapoists in man
FSII = follicle-sumdating hormone. ACTH = adrrhocartmtropc hamdm .
0 W I k Y - V C H Verlag GmbH. D-69451 Wcinhcim. 1997
05300833;97/3620-2149 S 17 50
B. Kutscher et al.
Complemetary to the reviews on LHRH agonists and antagonists which are already available,” - ‘I this contribution covers not only the current developments in synthesis, but in addition, the establishment of the human receptor for specific
characterization of substances, and presents methods and progress in the search for, and development of, peptidomimetics.
2. LHRH Peptides as Therapeutic Agents
2.1. Agonists
The first years after the discovery of the gondarelins were
marked by the search for more active agonists, since their therapeutic potential as, for example, antitumor agents or in gynecology, was apparent.[’* Such “superagonists” bring about a
very effective reversible inhibition of the release of steriodal sex
hormones. The exchange of glycine6 of the native LHRH for
other, always D-configured, amino acids, is common to all modern superagonists; some have a C-terminal ethylamide (buserelin, leuprorelin) or azaglycinamide (goserelin) residue instead of
glycinamide. Eight to ten amino acids of the LHRH sequence
are conserved in all clinically relevant superagonists ; by exchange at a maximum of two positions, the biological activity,
or hormone suppression in tumor patients, can be increased by
a factor of up to 100 on subcutaneous a p p l i ~ a t i o n . [ ~ . ” ~ ‘ ~ ~
Table 1 summarizes the most important derivatives currently on
the market.
Buserelin’” (Profact, Suprecur), leuprorelin’” (Carcinil,
Enatone), triporelin‘” (Decapeptyl) and goserelin’” (Zoladex)
(trade names in Germany) are the products on the market with
the highest turnover; worldwide, a turnover of about two billion
Bernhard Kutscher, born in 1957,
completed his chemistry studies in
1984 at the Universitat Frankfurt
(Germany) with a doctorate under
the supervision of Prof. H . Kessler
in the field of peptide synthesis. He
then joined Degussa as laboratory
manager (Laborleiter) in the research department “Pharmazeutische Chemie”. Since 1987 he has
M. Bernd
B. Kutscher
J. Engel
T. Beckers
E. E. Polymeropoulos
headed the Department “Chemische
Forschung Wirkstoffe” of ASTA Medica. Today he is responsiblefor the Department “Chemische Forschung”. In 1992 he was
appointed lecturer at the Technische Universitat Miinchen and became Honorary Professor there in 1996. His fields of research
include the synthesis of peptides, phospholipids and heterocycles, automation of synthesis, and combinatiorial chemistry.
Michael Bernd, born in 1954, completed his doctorate in Chemistry with Professor H . Kessler at the Universitat Frankfurt in
1983. In 1984 he became Laborleiter in the Department “Organische Forschung” of Degussa and in 1989 moved to ASTA
Medica where he is responsiblefor peptide synthesis in the Department “Chemische Forschung Tumor
Thomas Beckers, born in 1961, studied Biology specializing in Biochemistry at the Universitat Frankfurt. His diploma was
completed at the Max-Planck-Institut fur Biophysik in Frankfurt. After graduating with a doctorate under Prof. J. Engels,
Institut fur Organische Chemie, Universitat Frankfurt, in the field of Molecular Biology and a postdoctorate at the Massachusetts General HospitallHarvard Medical School in the research group of Dr. B. Seed, Boston ( U S A ) ,he joined the department
for cancer research at ASTA Medica in the field of tumor research.
Emmanuel E. Polymeropoulos, born in Athens in 1943, obtained his doctorate in Physical Chemistry from Rutgers University,
New Jersey ( U S A ) . From 1914 to 1980 he was scientific assistant in the research group of Prof. H. Kuhn at the Max-Planck-Institut fur biophysikalische Chemie Gottingen (Germany) and thereafter as scientific assistant in the research group of Prof. J.
Brickmann at the Institut fur Physikalische Chemie der Technischen Hochschule Darmstadt (Germany) till 1984. After one and
a half years at the Department of Physics of the research institute “Demokritos” in Athens he joined ASTA Medica in 1986,
where he is responsiblefor computer applications in the Department “Chemische Forschung ”.
Jiirgen Engel, born in 1945, completed his engineering studies at the Naturwissenschaftlich-technische Akademie in Isny
(Germany) and began his studies in Chemistry at the Technische Hochschule Braunschweig (Germany) in 1969. He ended these
studies in 1979 with a doctorate at the Institut fur Organische Chemie der Universitat Braunschweig with Prof. H. H . Inhoffen.
From 1976 to 1981 he was Laborleiter in the Department “Chemische Forschung Wirkstoffe”at ASTA Medica, in 1981 head
of research coordination, and in addition was promoted to head of the “Chemische Forschung Wirkstoffe”in 1982. Since 1981
he has given a series of lectures at the Universitat Regensburg, where he completed his habilitation in 1985, and became Professor
in 1990. In May 1993 he was appointed Honorary Professor at the Universitat Dresden. Since f 996 he has been chairman of
the Fachgruppe Medizinische Chemie of the Gesellschaft Deutscher Chemiker.
Angew. Chem. In!. Ed. Engl. 1997, 36, 2148-2161
LHKH Antagonis1.s
Table 1. Structure of LHRH, and the LHRH agonists on the market (given are only those amino acid residues that are different in LHRH)
Name (Co )
Buserelin (Hoechst)
Nafarelin (Syntex)
Leuprorelin (Abbott. Takeda)
Goserelin (Zeneca)
Histrelin (Ortho)
Triptorelin (Ferring)
[a] Glp
= pyroglutamic
German marks with active compound formulations corresponding to these peptides was achieved in 1994; of this,
over half belonged to the firms Abbott and Takeda with
leuprorelin. A total market volume of about 2.4 billion German
marks across all indications is expected at the end of the
century." 2bl
Annual production of peptide pharmaceuticals is of the magnitude of less than 100 kg for buserelin and significantly over
100 kg for the market leader, leuprorelin. At this order of magnitude, these substances are only made by classical organic
preparative synthesis (fragment condensation in solution). The
firm Hoechst, for example, synthesizes the nonapeptide buserelin from the units Glp-His, Trp-Ser-Tyr, and D-Ser(t Bu)-LeuArg-Pro-NHEt; the tri- and tetrapeptide units are coupled to
form the corresponding C-terminal heptapeptide, and then the
N-terminal dipeptide is condensed with this to form the complete sequence. Control of the physicochemical process parameters, such as concentrations, precipitations, separations, reaction temperature profiles, and purification techniques, is
important for successful peptide synthesis on a technical scale,
since there is naturally a significant difference to those required
for laboratory scale synthesis.[13a1
arginine or isopropyllysine. Excellent documentation of the
stepwise optimization can be found in many classical review
Antagonists of the second generation caused formation of
temporary edemas of the face and extremities in animal experiments, due in part to massive histamine release by mast cell
degranulation. Cyanosis and respiratory impairment were also
observed.[16] The cause of these intolerable side effects is
thought to be the combination of D-arginine at position 6 with
the three aromatic amino acids at the N-terminus of the seq ~ e n c e . ~For
' ~ ] the desired biological antagonist potency, an
R-configured basic amino acid is necessary at position 6; A. V.
Schally et al. achieved the breakthrough to highly active antagonists free of side effects with the derivatives SB-75 (INN:
cetrorelix) and SB-88, by incorporating hydrophilic, nonbasic
amino acids with side-chain carbamoyl functions (D-citrulline6
(D-Cit6), D-homocitruhe6) .I1
The biological activity of such sequences that are free of significant anaphylactic potential is limited exclusively to R-Configured amino acids at position 6; peptides with L-citrulline6 are
practically inactive in vivo due to rapid enzymatic degradation
(Figure 2).["]
2.2. Antagonists
Common to the intrinsic activity of all superagonists is the
initial temporary stimulation of gonadotrophin release. Soon
after the use of highly active agonists became an established
therapy, a search began for corresponding antagonists, which
do not bring about an initial hormone release, to avoid this
therapeutically counterproductive effect. A final big hurdle for
clinical use of highly active anatagonists was the inherent anaphylactic potential of these peptides.
Starting from the sequence of native LHRH, the individual
positions of the peptide chain were examined in rapid succession
for their contribution to biological activity. Particular attention
was paid to side effects. The most effective early improvements
in antagonistic activity were achieved by using D-Phe' instead of
histidine, by D-amino acids at position 6 instead of Gly6, and the
exchange of C-terminal glycine for D-Ala". Further stepwise
optimization led to the sequence scheme now usual for all modern antagonists of D-Na11-D-CpaZ-D-Pa13(Nal = 2-naphthylalanine, Cpa = Phe(CCI), Pal = 3-pyridylalanine) as hydrophobic cluster, plus a D-configured aromatic or aliphatic, yet
hydrophilic, aminocarboxylic acid6, and the C-terminal hydrophilic sequence X x ~ ~ - P r o ~ - D - A l where
a ' ~ , Xxx is either
A n g m . Chem. Inf. 0 1 . Engl. 1997, 36, 2148-2161
Figure 2. Stability of [D-Cit6]SB-75(cetrorelix; red) and [~-Cit~]SB-75
(yellow) under the influence of human serum (HPLC peptide content x[%] over 60 h).
Today, cetrorelix is manufactured exclusively by classical
fragment condensation, on a kilogram scale. Two synthesis
strategies, worked out in detail and optimized over several
years, proceed via either the N-terminal tripeptide (D-Na1'-DCpa2-~-Pa13)
or the C-terminal tripeptide (Args-Pro9-~-Ala'o)
and the complementary heptapeptide (Ser4-D-A1aLo) or
(Nal' -Len7) to the protected decapeptide with tert-butyl sidechain protection; the hydrochloric acid deprotection is followed
by final purification by preparative HPLC. The C- and N-terminal functionalization of the acetylated decapeptide amide, nec2151
B. ..Kutscher
et al.
essary for biological activity and to avoid rapid enzymatic
nosyl-D-serine6,that has improved water solubility; however, its
degradation, is introduced at the level of the terminal tripeptide,
biological activity is possibly insufficient for clinical use as an
by acetylation of the free a-amino group of naphthylalanine’
antitumor agent.[34*351 The firm Schering is also developing
with acetylhydroxysuccinimide, or saponification of the resultpeptide antagonists, using nonproteinogenic amino acids, such
ing alanine methyl ester in alcoholic a m m o r ~ i a . [ ’ ~ ~ * ~ I
as &-dialkylated lysine or benzodiazepine aminocarboxylic
Folkers et al. were able to improve active antagonists succesacids.[36.3 7 1 ZK-157348 contains N-6-morpholinolysine at the
sively by consistent and systematic modifications both at the
positions 6 (D-Morlys) and 8 (L-Morlys); biological data have
relevant sequence positions and the side-chain substituents; the
not been published.
most successful were complex substitutions at the positions 5, 6
An interesting method for the manufacture of orally adminisand, in part, 8. Starting from antide, a highly potent but poorly
tered LHRH antagonists is being pursued by the Australian
water-soluble decapeptide with nicotinoyl-substituted lysine5,
firm Biotech, who are concerned with the synthesis of conjuor with D-lySine6 (Scheme l), the analogues [PicLys5,~- gates of antide and vitamin B,, . The derivatives have similar in
(6ANic)Lys6]antide ( = nictide where pic = picolinyl, 6ANic =
vitro and in vivo activity to antide; however, they have much
6-aminonicotinoyl), [D-3-Qal1,c-PzACAla5,~-PicLys6,ArgS]better aqueous solubility and can thus be administered parenterantide ( = argtide where Qal = 3-(3-quinolyl)alanine, c-PzAC =
all^.[^^*^'] Scheme 1 shows the relevant derivatives and known
cis-3(4-pyrazinylcarbonyl-aminocyclohexyl), and [PiCLySs,Dcandidates for development.
(PicSar)Lys6]antide ( = sartide where Sar = sarcosine (NThe search for peptide and peptidomimetic structures of the
methylglycine)) were synthesized and their biological activity
next (that is, the fourth) generation with improved pharmacocharacterized.“’- 231
logic activity continues undiminished worldwide. In addition to
Flouret et al. have reported over 104 analogues of antide, the
the classical indication of hormone-dependent tumor treatment,
clinical use of which is impaired by unfavorable solubility charthe LHRH antagonists can also be used to treat nonmalignant
acteristics. They describe the exchange of the antide-typical
tissue abnormalities, for example, uterine myoma or benign
nicotinoyl group for a variety of other acyl substituents, and
prostate hyperplasia (BPH), with good prospects of success. In
exchange of lysine for other short-chained amino acids such as
modern reproductive medicine, LHRH antagonists have signif2,3-diaminopropionic acid or 2,4-diaminobutyric acid, and the
icant advantages over the agonists used exclusively until now.
biological activity in relation to these modification^.^'^ Xiao et
Today, the highly active decapeptides SB-88 and SB-75 (INN:
al. have synthesized a number of active antagonists (Tx series)
c e t r ~ r e l i x ) [are
~ ~used
in various laboratories as lead structures
with unnatural amino acids at positions 5 and 6; the most active
for the discovery of stable derivatives with good aqueous solubility and long-lasting in vivo activity. For peptide antagonists,
sequence Tx-44 contains the amino acid 4-(morpholinomethy1)one looks for cetrorelix analogues with modifications particuphenylalanine (Mop) derived from phenylalanine instead of tyrosine5.[24- 261
larly at positions 5, 6, and 7. Nonproteinogenic amino acids,
An important contribution was made by Rivier et al., whose
D-alkyl derivatives and amino acids with specifically altered side
optimization led to the decapeptide “azaline” with novel modichains are introduced at these positions. Characteristic substitufications at positions 5 and 6, where aminotriazole-subsituted
tion patterns of such modifications of cetrorelix analogues are
explained in Figure 3, with D-lysine6 as an example.
p-aminophenylalanine or lysine are positioned!27. 1‘’ Azaline B
The best derivatives with the highest receptor affinities have
is probably one of the most active antagonists presently availso far been obtained by suitable modifications of the &-amino
able worldwide. The current status of work on the “Azaline B
function of D-lysine6 with polar, hydrophilic carboxylic acids.
family” is documented in a review by R i ~ i e r . ’ ~ ~ ’
With T-148, Schally et al. found a sequence that has bisacetylAn interesting strategy for discovery of antagonists with good
2,3-diaminopropionic acid bound through an amide linkage to
water solubility was published by Roeske et aLr301Decapeptide
and that is highly active
sequences with side chain modifications of D - G ~ uor~ D-LYS~ the &-aminofunction of ~-lysine~-SB-75
in vitro. Sugar units of the D-glucopyranuronic acid type as Dderivatives were synthesized with strongly polar and hydrophilic
lysine6 side chain also give highly active antagonists with longgroups such as taurine. The strongest antiovulatory activity
lasting hormone suppression in v ~ v o . [431
~ ~Insertion
of a-amino
(AOA) was found on substitution Of D-LyS6 with gulonic acid or
carboxylic acids at positions of the peptide scaffold other than
D - G ~ usubstitution
with tris(hydroxymethy1)aminomethane
position 6 also brings about stronger receptor affinity in certain
(Tris; 75 and 50% AOA, respectively, 1 pg, rat model).
cases, but has not yet achieved increased activity in v ~ v o . [ ~ ~ ]
Deghenghi published a highly active decapeptide sequence
D-24308, an L-Cpa2 analogue of antarelix is a spectacular
with minimal histamine release and good water solubility. This
example of how reversal of chirality at a sensitive position on the
structure, known as antarelix, differs from SB-75 (cetrorelix) in
molecular scaffold, which has a characteristic sequence of optithat it has homocitrulline6 instead of citrulline6 and isopropycally active centers, can bring about a dramatic loss of activity
llysine’ instead of a r g i ~ ~ i n e ~321
By using Lys(iPr)’, residual
(Scheme 1). With exchange of D-Cpa2 for L-Cpa2, the biological
potential for histamine release can be further reduced.
activity is completely lost.
The firm Organon is developing the antagonist ganirelix (RSThe work of Haviv et al. with restrictions, cyclizations and
26306) under licence from the firm Syntex; this is a decapeptide
sequence t r ~ n c a t i o n s [ ~is~ a- ~good
~ ] example of intensive rewith novel alkyl-modified D- and L-homoarginine units at posisearch on effective antagonists. Abbott’s A-76154 is an octapeptions 6 and 8.[331The development of a similar forerunner
tide antagonist with LHRH receptor affinity of a similar order
derivative called detirelix (Arg8-ganirelix) was suspended by
of magnitude to that of active decapeptides such as A-75998 or
Syntex. With ramorelix (HOE 013), the firm Hoechst (HMR) has
a peptide antagonist with a sugar -amino acid unit o-a-L-rham’3
21 52
Angew. Chem. Int. Ed. Engl. 1997,36,2148-2161
LHKH Antagonists
P h w MU
Ceuordix (S6-75
ASTA Medlcs)
Phaa I
Antide ( h a Serono)
Qanlrdlx (Symex
H.-b Roche)
Phaea II
A-75898 (Abbott-Takeda),
Aullne B (Salk Indude)
Antarellx (ASTA)
Detirelix (Syntex)
1 x 4 (Tianlln Inst.
Fam. Plan.)
ZK 157348 (&hering)
58-86 (AWi Medlca)
NIII-OlU (OrlhO)
A-76154 (Abbott)
Scheme 1. Structures of LHRH antagonists (cetrorelix, the compound furthest along in clinical tests, serves as basis). hArg(Et,) =
homoarginine(NG,NG-diethyl),Anis = anisol, Aph = p-aminophenylalanine, FPS = 4-fluorophenylpropionic acid, Hci = hornocitrulline,
Mop = morpholinomethylphenylamine, Morlys = N-morpholinolysine, Rha = rhamnosyl.
3. Structure and Conformation
Modeling of peptide systems such as LHRH as a means to
explain the biologically active conformation began in the 1970s
with the development of potential functions to examine enXxx = Tyr or other amino acids
ergetic and entropic terms that contribute to the stabilization of
Yyy = Leu or other amino acids
the conformation of a peptide chain in solution.[53.541 The first
Z u = Arg or Lys(iPr)
investigations of LHRH indicated that there was no preR = carboxylic acids
ferred conformation in s o l ~ t i o n . [ ”’z 6~0 1 ~ Monahan et al. explained the significant difference in both in vitro and in vivo
Example :T-148
activity between [Ala6]LHRH and [D-AI~~ILHRH
by the forAc-~-Nal-D-Cpa-D-Pal-Ser-Tyr-~-Lys(Ac~Dap)-Leu-Arg-Pro-D-Ala-NH2
mation of a B-turn in the Ser4-Tyr5-Gly6-Leu7 segment of
Figure 3. Side chain variation in (D-Lys6)-cetrorelix analogues. Ac,Dap =
LHRH.[561This assumption was supported by empirical conforN2,N3-diacetyl-2,3-diaininopropionic
mational analyses of LHRH and analogous compounds by Momany (Figure 4).[57.581 Substitution of the segment Tyr5-Gly6Leu7-Arg*by a sterically hindered fi-lactam[611as a model of a
Molecular restrictions, such as head-to-tail cyclization, can be
pII’-turn resulted in an LHRH-analogous compound that was
accompanied by improved stability and duration of activity in
more potent than LHRH both in vitro and in vivo.
peptides and also in heterocyclic lead structures, if the restricMolecular dynamics (MD) simulation calculations pertion includes the bioactive conformation (matched core) .[5 21
formed by Struthers et al.r62a.b1 supported the presence of a PI1
The same applies to dimerization and partial dimerization
turn and indicated spatial separation of the hydrophobic side
through binding of suitable (partial) sequences, for example on
chains (Trp3, Tyr’, Leu7), which possibly interact with a hydrothe Lys6 side chain, and also for cyclization from the C-terminus
phobic region of the receptor.1561These calculations led to the
to the middle of the molecule. A multitude of cyclopeptides with
LHRH sequences have been synthesized which confirm this (see
synthesis of the cyclic LHRH antagonist cyclo[A3.4-Pro’-~Schemes 3 and 4). Their conformational properties are disCpaZ-~-Trp3-Ser4-Tyr5-Trp6-N-Me-Leu7-Arg8-Pro9-~-Al
cussed in Section 3.
(I, Scheme 2), which, however, binds more weakly to the recepH
Angew. Chem. hi.Ed. Engl. 1997,36,2148-2161
B. Kutscher et al.
carried out (Scheme 3) .[651 All three cyclopeptides have significantly weaker binding than the linear derivatives (see Section 5b). NMR investigations in methanol and DMSO did not
give any indication of preferred conformations for these cyclized
compounds; thus no further insight into receptor-binding conformations was
By 2D-NMR investigations of
LHRH in various solvents1671and NMR investigations of
cetrorelix in DMSO, trifluoroethanol (TFE), H,O, and H,O/
SDS (SDS = sodium dodecylsulfate) ,[681 preferred extended
conformations were found for LHRH, and evidence was found
for a possible fold in the TyrS-Arg8 segment of cetrorelix that,
however, bears no resemblance to a p1I'-turn.
The search for active, conformationally restricted LHRH antagonists was continued by Dutta et al.[69*701
By targeted cyclization of linear peptides, a range of cyclic derivatives was
obtained, in which the side chains of the amino acids at positions 5 and 8 formed part of the ring system. The most potent
compound of this group was Ac-~-Cpa'-D-cpa'-D-Trp~-Ser~Figure 4. Conformation of LHRH, reproduced according to data in Refs. [57,58].
(II), which
showed threefold stronger ovulatory inhibition
than the corresponding linear compound. As a
result of conformational analysis of this antagonist and its linear analogue, bicyclic LHRH antagonists were obtained in which the amino acids 5
and 8, and also both termini, were bridged.r7'] The
examination of this compound led to structures in
which a P-turn was present between positions 3
and 6 (Figure 5). Since the in vivo results were
generally less favorable for the bicyclic peptides
than for monocyclic 5 - 8 bridged derivatives, this
was an indication that the free end groups play an
important role in binding to the receptor.
Use of a novel starting point for design of
LHRH antagonists was attempted by Struthers
based on analysis of MD simulations on
I,'73Jthey suggested a receptor- binding conformation (supported by NMR investigations) for
LHRH antagonists. However, in vitro results gave
a KDvalue greater than 100 nM for binding to the
LHRH receptor, which was much higher than the
KD value of the corresponding linear compound
Scheme 2. Structures of cyclized LHRH analogues, whose conformations have been elucidated.
(KD= 0.21 nM). These findings suggest that 1) the
free N- or C-terminus of the linear derivative plays
an important role in receptor binding, and 2)
tor than LHRH itself. This compound has two p I I ' - t ~ r n s , [ ~ ~ ]fitting of the cyclic antagonist to the receptor binding pocket
which are formed with the help of an internal network of hydrowas suboptimal.
gen bonds. This stabilizing effect was also examined by the
By optimization, the antagonist Ac-A3s4Pro'-~-Fpa2-~-Trp3isosteric substitution of -CH,NH- instead of -CONH- at all
amino acid positions of the antagonist [N-Ac-D-Nal', D-Phe', ',
Fpa = 4-fluorophenylalanine) was obtained, in which the side
chains of Dap4 and Aspg are bridged. The substance has a
D-Arg6, Phe7, D-AI~"]-LHRH, except for position 9.[64a1
substitutions brought about a reduction or even absence of anbinding constant of KD= 55 nM. The compound A ~ - A ~ . ~ - p r o ' D- Fpa' -D-Trp3-cyclo(Asp4-Tyr5- ~ - ( 2 ) N a-1Leu7
~ -Arg8-Pro9tiovulatory activity in biological tests. Such an interpretation
Dpr")-NH, (Dpr = 2,3-diaminopropionic acid, IV)[74*7s1was
assumes that these modifications do not lead to changed binding
to the receptor. However, it is known that amide bonds of a
synthesized by bridging amino acids 4 and 10, whereby a pI1'turn in segment 5-6-7-8 is stabilized by two hydrogen bridges.
substrate are also involved in receptor
The influence of incorporation of L-Tic (Tic = tetrahydroisoTo gain a better understanding of the interaction between
quinoylalanine) at various positions of the linear antagonist
LHRH antagonists and the human receptor, a head-to-tail
cyclization of cetrorelix to cyclic cetrorelix ("cyclorelix") , of
D-Ala'O-NH, was also examined, with molecular mechanics calantrarelix to cycloantrarelix, and of SB-88 to cyclo-SB-88 was
Angew. Chem. Int. Ed. Engl. 1997,36, 2148-2161
LHKH Antagonists
ated agonists were less active than the
corresponding nonmethylated compounds. All Tyr5(N-Me) derivatives of
antagonists had surprisingly good
aqueous solubility. In particular, for
the sequence A c - ~ - ( 2 ) N a l ' - ~ - C p a ~ - ~ (3)PaI3 - Ser4 - Tyr5(N - Me) - D - Lys( N ~ c- )Leu7
~ - Lys(iPr)' -Pro9-D - Ala' ONH, (A-75998), the conformational
change was crucial for both solubility
and binding affinity to the receptor.
On N-methylation, the ratio of cis/
trans isomers is changed and hydrogen-bridge formation is not possible,
which leads to poorer association or
aggregation. In addition, a hydrogen
bridge, which normally contributes to
stabilization of the BII-turn, is broken
by the methylation. A further developScheme 3 Cyclized andlogues of cetrorelix and structurally related compounds (code numbers)
ment was attempted by Liff et al., who
tried to bring about targeted changes
in the conformation of LHRH-analogous compounds by
methylation of C, atom centers.[791However, such substitutions
have not resulted in any significant conformational changes.
Comprehensive conformational analyses of native LHRH
and of LHRH agonists and antagonists were performed by
Nikiforovich and Marshall.[8o*''I A high flexibility was established for LHRH, in agreement with previous NMR investigations on linear peptides of corresponding chain length. Due to
the function of Gly as a D-amino acid, the fragment Tyr5-Gly6Leu7-Arg' has a preferred conformation, yet without forming a
marked PII-turn. Calculations indicate different conformations
for agonists and antagonists in segment 5-8. However, the geometry of the N-terminal tripeptide was similar for agonists and
A further attempt to determine the "biologically active conformation" of LHRH and analogues was made with NMR and
MD investigations on the conformationally restricted bicyclic
antagonist bicyclo(4-10/5-8)[Ac-(2)Na11-~-Cpa2-~-Trp3-Asp4Glu5-~-Arg6-Leu7-Lys8-Prog-Dpr10]-NH,
(V).[821The existence of a pII'-turn in segment 5-8 (D-Arg at i + l position)
could be shown here. The additional 5-8 ring closure changed
the conformation of the 4- 10 bridge in the original monocyclic
only slightly.
Figure 5. Conformation of the antagonist biCyClo(l-10/5-8)(-D-Cpa'-o-Cpa2-DNMR investigations on a series of cyclized LHRH antagoTrp3-Ser4-cyclo(Glu5-~-Arg6-Le~7-Lyss)-Pro9-~-Ala1o-),
reproduced according
to the data in Ref. [71].
nists and their linear analogues (best cyclic antagonist: AC-D-
c ~ l a t i o n s . [Substitution
of D-Tic at position 6 seems to contribute to the maintenance of a PI1'-turn. It is known that a
D-amino acid flanked by L-amino acids is a strong p1I'-turn
inducer. The D-amino acid then occupies the i+ 1 position. DPro, which is the natural counterpart of D-Tic, exerts a particularly strong effect.[76b1Further mimetic models for PII-turns
were discussed by Rizo and Gierasch in a review article on
theoretical considerations of conformationally restricted peptides.[771
N-methylation of a-amide bonds in LHRH a g o n i s t ~ [and
antagonists[451has the surprising effect of converting some agonists into antagonists. In the majority of cases, the N-methylAngew. Chrm. Int. Ed. Engl. 1997,36, 2148-2161
D-Ala'O-NH,, VI) resulted in a structure with a p-turn present
in segment 3-6, as in Refs. [71, 831. In addition, the side chains
1 and 10 were now in direct proximity to one another, due to the
existence of a network of hydrogen bridges. These cyclic compounds showed higher potency in the antiovulation assay
(AOA) than the corresponding linear peptides.
Recent results on putative binding conformations of LHRH
antagonists were obtained by Rizo et al. in a study of the bicyclic
antagonist bicyclo(4-l0/5,5'-8)(Ac-~-(2)NaI'-~-cpa~-~-Pa~~Asp4-Glu5(Gly)-~-Arg6-Leu7-Dbu8-Prog-Dpr10-NH,)
Dbu = 2,4-diaminobutyric acid)
This antagonist has a
pII-turn at amino acids 5-6, a pI'-turn at 6-7 and a PII-like
B. Kutscher et al.
turn between Pro9 and the side chain of Dpr”. Comparison
with the results in Ref. 1821 clearly shows that 4-10/5-8 bicyclic
structures-despite double restrictiondan adopt many conformations.
Until now, cyclized LHRH agonists and antagonists have not
shown any significantly better biological activity than linear
derivatives. It is clear that the termini must be free to take part
in binding to the receptor. Although NMR investigations and
M D simulations indicate the existence of a 8-turn in segment
5-8 or 3-6, the high flexibility of oligopeptides remains the
main problem in the search for the biologically active conformation and in optimization of structure-activity relationships. It is
becoming more and more obvious that new methods are needed
to solve this problem.
4. Receptor Assays
4.1. The Pituitary LHRH Receptor-A Representative
of the Rhodopsin-Like G-Protein-Coupled Family
of Serpentine Receptors
Although the hypothalamic peptide luteinizing hormonereleasing hormone was isolated and its sequence determined in
1971 by A. V. Schally and coworkers,[’. 851 the corresponding
cDNA was not successfully cloned until 1984. This coded for the
precursor protein of 92 amino acids, from which the decapeptide is formed by proteolytic processing.t861Almost another ten
years of intensive research passed until in 1992, various research
groups managed to clone the high affinity receptor for LHRH
from the murine gonadotrophic aT3-I cell line.[87.881
This immortalized murine pituitary cell line shows relatively high
LHRH receptor expression, which enabled isolation of the
cDNA by expression cloning in oocytes of Xenopus
Based on the murine LHRH receptor cDNA
sequence, the homologous cDNA genes of human, rat, cow, and
sheep receptors were subsequently
The homology
of the murine receptor to that of the rat is 97% and to the
human receptor 89 %. The pituitary high-affinity receptor
for LHRH belongs to the
rhodopsin-like family of Gprotein-coupled 7-helix receptors, which are also described
as serpentine receptors (a
review is given in Iismaa
et alJ9’]). Further representatives of this family are the
receptors for the peptides
thyrotropinreleasing hormone (TRH) ,
cholecystokinin (CCK), neurokinins A and B, and Substance P.[991The cDNA of the
human LHRH receptor codes
and that the “signature sequence” typical for many G-proteincoupled receptors is changed from DRY‘40 to DRS’40.[‘001
Complementary to the LHRH cDNA, the chromosomal gene of
the human receptor, composed of three exons and two introns,
LHRH induces dose-dependent synwas recently
thesis of the “second messenger” ~-myo-inositol-l,4,5-triphosphate (InsP,) and a biphasic rise in the intracellular calcium
ICaz’] both in cultivated gonadotrophic pituitary cells and in
the aT3-3 cell line (Figure 6).[’OZ1 Since these signals could not
be inhibited by pertussis toxin, participation of the G i and Go
subfamily of trimeric G-proteins could be excluded.r1031Recent
work on the gonadotrophic otT3-1 cell line shows, however, that
signal transmission following activation of the receptor takes
place through a G protein of the subtype G,/G,, and is thus
coupled by phospholipase C subtype p (PLCB) to the inositol
phospholipid signal pathway.[104’The heterologously expressed
LHRH receptor is also functional in recombinant cell systems,
that is, stimulation with agonists leads, after G-protein coupling, to synthesis of InsP, .L105,931
4.2. The Human LHRH ReceptorCloning, Heterologous Expression and
Recombinant Systems for Testing LHRH Antagonists
Cloning of the human receptor is of great importance for
discovery and characterization of potent LHRH antagonists for
clinical application, and this has become possible with the development of relevant, simple test systems. In the past, an initial in
vitro characterization of peptide LHRH antagonists was done
with classical receptor-ligand binding tests with iodinated LHRH
derivatives, which mostly use crudely purified membrane fractions from the rat pituitary.[106slo7]Since a species-dependent
binding of LHRH derivatives has been reported by various authors, this was a pragmatic method although not without problem~.‘~’* - 091 In particular, species-specific binding might be
the case for nonpeptide receptor ligands, since these probably
for a protein of 328 amino
acids. It is striking that the receptor has practically no c terminal cytoplasmic domain
(only two amino acid residues)
activation of protein kinase C
and gene activation
Figure 6. Simplified representation of the signal transduction pathway of the LNRH receptor. DAG = diacylglycerol.
Angew. Chem. In{. Ed. Engl. 1997, 36, 2148-2161
LHRH Antagonisls
interact with nonconserved amino acids outside the peptidebinding pocket of the receptor protein. This was shown for the
neurokinin-1 receptor, for example, with Substance P as endogenous ligand and chemically very diverse, antagonistic, nonpeptide compounds." lo' Taking this into consideration, the
human LHRH receptor was thus stably expressed in different
mammalian cell lines such as murine L cells. The individual cell
clones overexpressing the receptor were isolated, and a pharmacological characterization/validation of the test system was performed with receptor-ligand binding tests on intact cells under
physiological conditions, with the authentic hormone and relevant LHRH superagonists and antagonist^.^'^^^ A multitude of
further structurally diverse peptide LHRH antagonists were
tested with this system in displacement binding experiments
with ['2SI]-cetrorelix. The results for both clinically relevant and
cyclized peptides are discussed in Section 5 (see also Figure 8).
In addition to the determination of binding affinity, expressed
as the dissociation constant KD[nM], the functional characterization of a ligand is also of importance. In early work, cultivated
primary cell cultures from, for example, chicken pituitary, were
used for this purpose, and agonist-stimulated or antagonist-inhibited release of luteinizing hormone (LH) was measured in a
radioimmunoassay (RIA) .11091 This reliable but complicated
method has been simplified by determination of intracellular
messenger substances such as InsP, in permanent cell lines or,
alternatively, in suitable reporter gene test systems. In Figure 7a, detection of InsP, following stimulation with LHRH
and with the superagonist [D-Trp6]LHRH is shown as an example of the use of recombinant cell lines overexpressing the
human LHRH receptor. The agonist-stimulated signal is dosedependently reduced to the level in unstimulated cells, using an
antagonist such as cetrorelix (Figure 7b).r'05J
At present, selected peptides are being tested in vivo by measuring the testosterone concentration in male rat serum. Here, a
single subcutaneous application of a potent LHRH antagonist
leads to persistent testosterone suppression. According to our
current knowledge, the activity in animals generally correlates
well with the binding affinity determined in vitro on human
receptors. In cases where no in vivo activity was observed despite high binding affinity, the peptide probably had pharmacokinetic characteristics that played a decisive role. In the future, transgenic animals will also be very important for in vivo
5. Comparative Studies
5.1. Comparison of Clinically Relevant Antagonists
The binding affinity and also the functional characteristics of
certain, in part clinically relevant, peptide LHRH antagonists
have been tested on the human LHRH receptor. The antagonists tested were cetrorelix (SB-75, D-20761), SB-88 (D-20455),
antide (Serono), antarelix (D-23234), ganirelix (Organon/
D-24598), and a truncated-sequence antagonist from Abbott,
(ProNHEt)-A-76154 (D-24710). The binding affinity was determined as the dissociation constant KD [nM] with suitable displacement binding tests with ['251]-cetrorelix as labeled ligand
and various concentrations of the peptide to be tested (Figure 8
and Table 2). Cetrorelix showed the highest affinity for the human receptor, followed by its analogue SB-88.
Selected peptide antagonists were tested with a functional
assay in the cell line used for the radioligand binding tests (see
Figure 7b). Here, inhibition of InsP, synthesis induced by 10 nM
m Cetrorellxl D-20761
Antarelixl D-23234
0 Ganlrelixl D-24598
Figure 7. Functional characterization of peptide LHRH antagonists. a) Stimulation of InsP, synthesls by LHRH or by the superagonists [o-Trp6]LHRH
(triporelin/decapeptyl) in murine L cells, which overexpress the human LHRH
receptor. The InsP, concentration is given for 4 x 106cells; cells not stimulated with
peptide are shown as control. b) Inhibition of stimulation by 10 nM LHRH by
various antagonists (1 nM and 10 nM). The cellular InsP, concentration c(InsP,),,,
["A]is given relative to the maximum InsP, concentration specifically induced by
10 nM LHRH (100% corrt2sponds to 7.63 pmol and 11.68 pmol InsP,, respectively,
per 4 x 1 O6 cells) .
Angew. Chem. Inr. Ed. En$
Cycbrellxl D-23620
Figure 8. Binding affinity of selected peptide LHRH antagonists. Dose-response
curves of competitive binding tests with ['251]-cetrorelixas labeled ligand are given
for cetrorelix (KD = 0.18 nM), antarelix ( K , = 0.26 nM), ganirelix (KD= 0.45 nM),
and cyclorelix (KD= 9.81 nM). Evaluation of many experiments and a comparjson
of the binding affinities are shown in Tables 2 and 3.
B. Kutscher et al.
Table 2. Binding affinity of clinically relevant peptide LHRH receptor antagonists[a].
Binding affinity KD[nM]
Cetrorelix (D-20761)
SB88 (D-20455)
Antarelix (D-23234)
Antide (Bachem)
Ganirelix (D-24598)
[Pro8-NHEt]A-76154 (D-24710)
3.610k0.89 (4)
0.202+0.03 (10)
0.209i0.002 (2)
0.233 i0.04 (3)
0.360f0.13 (3)
0.405 0.04 (2)
2.371 k0.69 (2)
[a] The binding affinity of the peptide LHRH antagonists was determined in a
displacement binding test with [1251]cetrorelixas labeled ligand. The dose-response
curves and the dissociation constants KOfCT"(n = number of experiments, in parentheses) were calculated with the program EBDA/Ligand 3.0[133]. The binding
affinity of the authentic LHRH ligand is shown for comparison.
acids of the human LHRH receptor were positioned on the
seven transmembrane helices of bacteriorhodopsin by means of
homology modeling. The extracellular turns were modeled in
the same way with data from the Brookhaven Protein
The orientation of the helices was fixed by hydropathy studies.
Following energy minimization of the entire model with the
AMBER force field, an MD simulation was performed over
100 ps. The model structure was stable under the conditions of
this simulation; the relative orientation of the seven helices to
each other changed little. In addition, the proline-containing
helices formed the bend typical for such structures (Figure 9).
This model could give information about the biologically active
conformation of LHRH agonists and antagonists and thus facilitate the way to optimization of structure-activity relationships.
LHRH was measured at antagonist concentrations of 1 nM and
10 nM. In this test, cetrorelix is also the most effective inhibitor
of secondary messenger formation.
5.2. Comparison of Cyclized Antagonistic
LHRH Derivatives
Several cyclized peptide LHRH antagonists were tested for
binding affinity to the human LHRH receptor: cyclized
cetrorelix ("cyclorelix", D-23620), cycloantarelix (D-24236),
cyclo-SB-88 (D-24307) and partially cyclized cetrorelix ("halfcyclorelix", D-24418). The binding affinities, calculated as dissociation constants KD [nM], are summarized in Table 3. All
cyclopeptide analogues of the potent antagonists show significantly weaker binding than the linear precursors.
Table 3. Binding affinity of cyclized peptide LHRH receptor antagonists [a]
Binding affinity KD[nM]
Cyclorelix (D-23620)
Cyclo-Antarelix (D-24236)
Cyclo-SB 88 (D-24307)
Halbcyclorelix (D-24418)
13.64+ 1.69
24.74f 10.57
10.41 kO.27
[a] The binding affinity of the cyclized LHRH antagonists was determined in a
displacement binding test with [1251]cetrorelixas labeled ligand. In each case two
experiments were performed. The dose-response curves and the dissociation constants KDf CT" (n = number of experiments, here n = 2) were calculated with the
program EBDA/Ligand 3.0[133].
6. LHRH-Receptor Modeling
It has not yet been possible to determine the structure of a
receptor coupled to a G-protein. Knowledge of the molecular
structure and the functionality of the receptor has only been
obtained by targeted mutagenesis experiments." - ' I 3 ] Molecular modeling offers the opportunity to obtain an approximate,
three-dimensional structure of a receptor. Hoflack et al. have
suggested a model for binding and activation of the gonadothyrotrophin hormone at the receptor.[' 141 A modcl of the LHRH
receptor was outlined by Kiihne et al.,[l'sl which is based on the
well-known crystal structure of bacteriorhodopsin[l 16] and on
material published on bovine rhodopsin.["
The amino
Figure 9. Energy-minimized structure of the human LHRH receptor model.
7. The Search for Peptidomimetics
for the LHRH Receptor
The search for peptidomimetic substances to replace peptides
as active substances is being undertaken worldwide; prominent
examples are angiotensin I1 antagonists such as Losartan, and
(substituted) benzodiazepinones as high affinity ligands for the
somatostatin receptor. Literature on the endothelin receptor
antagonists (Ro 46-2005, L-744453), growth hormone analogues (MK-0677), CCK antagonists (MK-329), bradykinin B,
receptor antagonists (Win-64338), and Substance P antagonists
(CP-99994) is rapidly expanding.["'- lZ4] Intensive efforts are
being made particularly in the area of fibrinogen receptor antagonists, to replace the native Arg-Gly-Asp sequence ("RGD")
and to obtain active substances that can be administered orally.r1251
For some years, increased efforts have also been made to find
substances with affinity to the LHRH receptor that do not have
Angew. Chem. Int. Ed. Engl. 1997,36,2148-2161
LHRH Antagonists
the characteristic substance-specific properties and also disadvantages of peptides (short half-life, lack of bioavailability), yet
have a high binding affinity. Ideally, such substances should be
able to be administered orally, be sufficiently stable in the organism, and possess favorable pharmacological parameters
comparable to peptide antagonists. Clinical investigations of the
influence of the antimycotic ketoconazole on prostate cancer
and testosterone suppression indicate that it may have a LHRHmimetic effect.['26. '271 Further investigations by the firm Abbott showed weak antagonistic activity both in vitro and in vivo
for ketoconazole and modified analogues (Scheme 4);however,
the biological activity of such derivatives is most probably not
mediated mainly by LHRH antagonism.r128*
hormone-dependent tumors.['31' 331 A lead structure from the
latter substance class is currently in extensive pharmacological
ASTA Medica is also developing substances with potential as
antagonistic LHRH mimetics. Based on our work with sequence-truncated and cyclized peptides, both short-chain cyclopeptides and low molecular weight mimetics based on condensed heterocyclenes are being synthesized and evaluated. This
strategy is confirmed by recent publications from the firm
Takeda, who report cyclopentapeptides with affinity to the human LHRH receptor of the order of IC,, = 0.07 pM.'1341 The
search for lead structures is facilitated and further accelerated
by the use of compound libraries and high-throughput screening
8. Conclusions and Outlook
Ketoconazolepartialskeleton with dipeptide unit
Scheme 4. LHRH mimetics based on the ketoconazole partial structure [128,129].
A multitude of publications on stabilization and modification
of peptide sequences (pseudo-peptides) with the help of mimetic
structures, for example, the use of N-alkyl amino acids, cyclization and sequence truncation without loss of activity, originate
from Abbott,151 . 4 5 . 7 8 . 1301
Complex, highly substituted nitrogen heterocycles such as
those published by the firm McNeillab (Scheme 5) seem to have
LHRH antagonistic potential.[1351In patent literature, Takeda
describe benzodiazepines, benzodiazepinones, heterocyclic
benzo-substituted alkylamines, and recently, thienopyridine
carboxylic acid derivatives as LHRH receptor antagonists
with receptor-binding inhibition at submicromolar concentrations, suitable amongst other uses as antitumor agents for
We would like to thank H . Kessler, Technische Universitat
Munchen, for many N M R spectroscopic investigations and for
suggestions during drafting of the
manuscript, and A . f? Schally, Tulane Universitv. New Orleans. for the introduction to
the topic of endocrinology and his constant
support. The cooperation with H . Michel and
M . Reilander, M P I Frankfurt, and with G.
Krause and R. Kuhne, Forschungsinstitut fur
molekulare Pharmakologie Berlin, in the
field of the human LHRH receptor was supported by a project of the Bundesministerium
fur Bildung und Forschung ( B M B F No.
Received: November 4, 1997 [A 196IEl
Takeda, EP 679642 A1
_ I ,
McNeil, US-A 4678784
LHRH agonists are well-established, high-turnover pharmaceuticals with the main area of application in tumor therapy and
gynecology. Despite an early start to the search and intensive
worldwide research activity, no LHRH antagonists are on the
market today; cetrorelix, with its improved selectivity, high stability, and long-lasting action, is in an advanced stage of development and thus has the best chance of being the first LHRH
antagonist to be launched.
With the help of molecular biology, human receptor assays
have been established and functional tests developed, so that
targeted optimization and characterization of clinically relevant
antagonists can follow. Intensive conformational analyses on
both linear and cyclized antagonists have not yet delivered any
strategy for enhancing the activity or bioavailability. Despite
advanced formulation technologies, the goal of a simple, economical, easily administrable form has not yet been adequately
achieved in this research area. Assuming similar activity characteristics, peptidomimetics are, in general, superior with respect to cost of manufacture and method of application to
the peptide derivatives on which they are based; with the
available methods and lead structures, the search for these
pharmacologically important substances will certainly be intensified in the future, so that the fourth generation of LHRH
derivatives will meet the challenges of modern pharmaceutical
Takeda. WO 95128405
Scheme 5 . Peptidomimetic LHRH antagonists [131,132].
Angew. Chem. Ini. Ed. E<zgl. 1997, 36, 2148-2161
German version: Angen,. Chem. 1997. 109,2240-2254
Translated by Dr. J. Cooper, Frankfurt (Germany)
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