Synthesis and evaluation of homodimeric GnRHR antagonists having a rigid bis-propargylated benzene core

Article abstract of DOI:10.1016/j.bmc.2008.01.054

The fact that GPCRs might function in a dimeric fashion is currently well accepted. For GnRHR, a GPCR that regulates gonadotropin release, there is evidence that the receptor also functions as a dimer. We here describe the design and synthesis of a set of dimeric GnRHR antagonists in order to understand the interaction of dimeric ligands to the receptor and to address the question whether GnRHR dimerization is a prerequisite for signalling. Biological evaluation of the compounds shows no discrimination between monomeric and dimeric-ligands in respect to binding affinities, however, the dimeric ligands appear to have different functional properties.

Full text of DOI:10.1016/j.bmc.2008.01.054

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 3744–3758
Synthesis and evaluation of homodimeric GnRHR
antagonists having a rigid bis-propargylated benzene core
Kimberly M. Bonger,a, Richard J. B. H. N. van den Berg,a, Annemiek D. Knijnenburg,a
Laura H. Heitman,b Ad P. IJzerman,b Julia Oosterom,c Cornelis M. Timmers,d
Herman S. Overkleefta,* and Gijsbert A. van der Marela,*
aDepartment of Bio-organic Synthesis, Leiden Institute of Chemistry, Gorlaeus Laboratories,
Leiden University, PO Box 9502, 2300 RA Leiden, The Netherlands
bDivision of Medicinal Chemistry, LACDR, Gorlaeus Laboratories, Leiden University,
PO Box 9502, 2300 RA Leiden, The Netherlands
cDepartment of Molecular Pharmacology, N.V. Organon, PO Box 20, 5340 BH, Oss, The Netherlands
dDepartment of Medicinal Chemistry, N.V. Organon, PO Box 20, 5340 BH, Oss, The Netherlands
Received 16 October 2007; revised 23 January 2008; accepted 30 January 2008
Available online 2 February 2008
Abstract—The fact that GPCRs might function in a dimeric fashion is currently well accepted. For GnRHR, a GPCR that regulates
gonadotropin release, there is evidence that the receptor also functions as a dimer. We here describe the design and synthesis of a set of
dimeric GnRHR antagonists in order to understand the interaction of dimeric ligands to the receptor and to address the question
whether GnRHR dimerisation is a prerequisite for signalling. Biological evaluation of the compounds shows no discrimination between
monomeric and dimeric ligands in respect to binding affinities, however, the dimeric ligands appear to have different functional
properties.
ꢀ 2008 Elsevier Ltd. All rights reserved.
1. Introduction
production, rationalizing their therapeutic use in, for in-
stance, prostate cancer.
The Gonadotropin Releasing Hormone Receptor
(GnRHR) belongs to the family of membrane bound
G-Protein Coupled Receptors (GPCRs). The GnRHR
is located in anterior pituitary cells and plays an impor-
tant role in the reproductive system. Stimulation of the
receptor with GnRH, a decapeptidic agonist, initiates
the release of the gonadotropins Luteinizing Hormone
(LH) and Follicle Stimulating Hormone (FSH). FSH
and LH in turn induce follicle stimulation and ovulation
in females and stimulate steroidogenesis in both males
and females. GnRHR antagonists have found wide-
spread use in Controlled Ovarian Stimulation (COS)
protocols for IVF treatment. By inhibiting the gonadal
axis in males, GnRHR antagonists suppress androgen
Several research reports point towards the existence of
GnRHR dimers as the active species involved in GnRHR
signaling.1 GPCR dimerisation and/or oligomerisation2
is a well-recognized phenomenon which may be capital-
ized upon in the development of more active or specific
(ant)agonists.3,4 However, to date there are no dimeric
GnRHR ligands known, with the exception of the dimeric
antibody complexed peptide agonist with which the pos-
sible relevance of GnRHR dimerisation was demon-
strated for the first time.5 With the aim to develop
effective dimeric GnRHR (ant)agonists as research tools
to investigate GnRHR signaling, we recently reported
our results in the preparation and evaluation of a library
of homodimeric compounds (A, Fig. 1) based on the imi-
dazopyrimidinone GnRHR antagonist 1.6 The library
was constructed by modification of antagonist 17 with
an acetylene function and connecting modified ligand 2
to a set of bis-azide functionalized hydrophilic polyethyl-
ene glycol spacers by a 1,3-dipolar cycloaddition. As ref-
erence compounds, a set of monomeric ligands were
prepared in which the bis-azide polyethylene glycol
Keywords: G-protein coupled receptor; Dimerisation; Bivalent ligand;
Gonadotropin releasing hormone receptor; Imidazopyrimidinone;
Antagonist; Allosteric interaction.
*
Corresponding authors. Tel.: +31 71 527 43 42; fax: +31 71 527 43
07; e-mail addresses: h.s.overkleeft@chem.leidenuniv.nl; marel_g@
chem.leidenuniv.nl
Both authors contributed equally to this work.
0968-0896/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.01.054

K. M. Bonger et al. / Bioorg. Med. Chem. 16 (2008) 3744–3758
3745
O
OH
O
NH
N
O
O
N
O
N
N
O
O
O
N
F
O
F
N
O
O
O
N
H
N
H
N
O
N
O
N
N
F
F
N
H
N
H
N
H
N
H
N
N
F
F
O
GnRHR-Ant
GnRHR-Ant
3
1
2
GnRHR-Ant
OH
Previously:
1,3-dipolar azide-alkyne
cycloaddition
N
N
N3
N3
GnRHR-Ant
+
N
N
N
A
GnRHR-Ant
GnRHR-Ant
O
n
N
O
n
2
n = 0, 1, 2, 3, 4
This report:
Peptide Coupling
Conditions
NH2
O
O
H
H
X
N
N
X
H
N
H
O
GnRHR-Ant
N
H
N
GnRHR-Ant
B
X
X
N
GnRHR-Ant
3
+
H2N
H
O
O
OH
O
O
X = Amino Acid
Figure 1. Overview of previously reported library (A) and current library (B) of dimeric GnRHR antagonists.
spacers were substituted on one site with the acetylene
functionalized ligand. Our library of compounds did not
contain a dimeric species with either a significantly en-
hanced GnRHR binding affinity or a significantly en-
hanced functional (ant)agonistic activity. Although one
might conclude from these results that GnRHR dimerisa-
tion is not a prerequisite for signaling, at least in our sys-
tem, we deem it more likely that we have not hit upon the
correct dimeric ligand design. Many permutations are
possible, and one specific alteration in the design that
we address here is the replacement of the flexible, hydro-
philic polyethylene glycol linker system by the rigid,
hydrophobic benzene-based scaffolds as in B depicted in
Figure 1. We thought it of interest to investigate if the thus
imposed spatial constraint in the orientation of the phar-
macophores exerts an effect on the binding and functional
activity of the ligands.
2. Synthesis
The strategy for the preparation of our second-genera-
tion library is outlined in Scheme 1. N-Boc-glycine (A)
was coupled to propargyl amine to afford acetylene 4A
OH
BocHN
O
A
i
H
H
N
H
H
N
III, ii
I, ii
N
N
BocHN
RHN
RHN
NHR
O
O
O
O
7A R = Boc
8A R = H
iii
5A R = Boc
iii
4A
6A R = H
iv
iv
H
N
H
N
H
N
O
N
O
O
N
N
H
H
N
H
O
N
N
O
O
N
N
O
N
F
O
O
O
O
O
N
N
O
O
O
N
O
N
O
O
N
H
N
H
N
H
N
H
N
H
N
H
N
F
N
F
F
F
IIIA
IA
F
Scheme 1. Representative route of synthesis. Reagents and conditions: (i) Isobutyl chloroformate, NMM, propargylamine, DCM, ꢀ20 ꢁC to rt 18 h,
76%. (ii) CuI, Pd(PPh3)4, pyrrolidine, DMF, 18 h, 66% for 5A, 99% for 7A. (iii) TFA/DCM; 1:1; v/v, 1% TIS, 18 h, HPLC purification, then (iv)
pharmacophore 3, BOP, DiPEA, DMF, 18 h, HPLC purification.

3746
K. M. Bonger et al. / Bioorg. Med. Chem. 16 (2008) 3744–3758
in 76% yield. Sonogashira cross coupling (CuI,
Pd(PPh3)4, pyrrolidine and DMF) of 4A to phenyl
iodide (I) gave compound 5A in 66% yield. After Boc
removal (TFA/DCM and 1% TIS) and HPLC purifica-
tion of the resulting ammonium salt compound IA was
prepared by condensation with pharmacophore 3 (pre-
pared as described in Ref. 6 under the agency of BOP
and DiPEA in DMF. The HPLC purification of 6A
proved necessary, since direct condensation of crude
6A after Boc removal proceeded sluggishly and in low
yield. Reaction of 4A with 1,3-diiodobenzene (III) gave
bis-propargyl benzene 7A. Now, Boc-removal, HPLC
purification and condensation with 2 equivalents of car-
boxylate 3 gave bifunctional ligand IIIA. In this fashion,
a 4 · 7 compound library was assembled, using the four
iodo benzene derivatives I–IV and the seven amino acids
A–G (Fig. 2), leading to seven mono-functional
compounds (IA–G), seven ortho-disubstituted benzene
derivatives (IIA–G), seven meta-disubstituted benzene
derivatives (IIIA–G) and seven para-disubstituted ben-
zene derivatives (IVA–G). All synthesis routes pro-
ceeded in an efficiency compared to that of the
examples outlined in Scheme 1 (see Section 4 for details).
All target compounds are depicted in Figure 3.
substituted scaffolds (series IV). Exceptions are dimeric
ligands with spacer G, which show comparable binding
affinities in all cases, but reduced affinities compared to
the monomeric ligand IG.
For the functional assay we made use of CHO cells sta-
bly transfected with the GnRHR and equipped with the
NFAT luciferase reporter gene. These cells were stimu-
lated with a submaximal (EC80) concentration of the
agonist GnRH in the presence of several concentrations
of the test compounds. Antagonistic activity was de-
tected as a decrease of the luminescence signal upon
addition of the luciferase substrate. The IC50 values of
the mono functionalized compounds IA–G and the
antagonistic effects at 3 lM and 10 lM of all com-
pounds (IA–G, IIA–G, IIIA–G and IVA–G) are listed
in Table 2.
The ability of the monomeric ligands to antagonize
GnRHR signaling, expressed as IC50 values, are in good
agreement with the binding affinity (Ki) observed in the
radioligand displacement assay (Table 1). However,
compounds IB and IC, the linker system of which is
derived from the amino acids alanine (B) or valine (C)
show a reduced effect (Emax) compared to the other
monomeric ligands. Most of the bifunctional ligands
show a concentration dependent inhibition of GnRH-in-
duced luminescence. However, full inhibition at the
highest concentration tested (10 lM) was never
observed, suggesting that the potency of the dimeric
ligands is relatively modest. Six compounds (that is com-
pounds IID, E and G, IIIG and IVD and E) show over
50% antagonism at 3 lM. These compounds all hold
longer spacers (that is amino-butanoate D, -hexanoate
E and -pipecolate G).
2.1. Biological evaluation
All newly synthesized compounds were tested on their
ability to bind to the GnRH receptor and to antagonize
GnRH-mediated signaling. The results are summarized
in Tables 1 and 2. The binding affinity of the compounds
was measured by monitoring their ability to displace the
radioactive GnRHR agonist [125I]triptorelin from plas-
ma membranes of GnRHR-expressing Chinese Hamster
Ovary (CHO) cells. As it is evident from Table 1 most
monomeric compounds bind to GnRHR with higher
affinity than their corresponding dimeric compounds.
An exception is dimeric ligand IVB for which a 3-fold
increase was observed compared to the monomeric
counterpart IB.
The literature on the antibody-mediated dimerisation of
GnRHR antagonistic peptides, with agonists as a
result,8 led us to investigate whether our bifunctional
molecules possess agonistic activity. Additional assays
performed with all compounds in an agonistic set-up,
that is, when tested alone in the luciferase reporter gene
assay, did not provide any actives (data not shown).
In general, the compounds with alanine (B) or valine (C)
moieties possess lower binding affinity compared to the
other compounds. Also, the ortho- and meta-substituted
aromatic scaffolds (that is, series II and III, respectively)
show slightly reduced affinity compared to the para-
The discrepancy in binding and functional properties of
the dimeric compounds prompted us to perform addi-
O
OH
N
Scaffold:
O
N
F
O
O
O
N
O
:
GnRHR-Ant
I
I
I
N
H
N
H
OH
N
F
I
I
I
I
3
I
II
III
IV
OH
OH
OH
OH
OH
Spacers:
BocHN
BocHN
BocHN
BocN
BocHN
O
O
O
O
O
A
B
C
D
BocN
OH
OH
BocHN
O
O
F
E
G
Figure 2. Aryl iodides and amino acids employed in the construction of the library.

K. M. Bonger et al. / Bioorg. Med. Chem. 16 (2008) 3744–3758
3747
Similar:
GnRHR-Ant
O
O
O
H
N
O
H
O
O
O
H
N
N
N
H
N
N
H
GnRHR-Ant
GnRHR-Ant
GnRHR-Ant
O
O
O
H
O
O
O
IIIA
IA
H
N
O
H
H
N
N
GnRHR-Ant
N
H
GnRHR-Ant
N
N
H
H
O
IIIB
IB
H
N
O
H
N
H
N
GnRHR-Ant
GnRHR-Ant
N
H
N
H
GnRHR-Ant
GnRHR-Ant
N
H
O
O
O
O
IIIC
IIID
IIIE
IC
O
H
N
O
O
H
N
H
N
N
H
GnRHR-Ant
N
H
GnRHR-Ant
N
O
H
ID
O
H
N
O
O
H
N
H
N
GnRHR-Ant
N
H
N
H
GnRHR-Ant
N
H
GnRHR-Ant
O
O
O
O
O
IE
H
H
N
H
N
GnRHR-Ant
GnRHR-Ant
N
N
GnRHR-Ant
GnRHR-Ant
GnRHR-Ant
GnRHR-Ant
N
N
O
O
O
N
O
N
IIIF
IF
O
O
O
N
H
H
N
H
N
N
O
IG
O
O
IIIG
Figure 3. Structure of monovalent ligands IA–G and dimeric ligands IIIA–G. Structure of dimeric ligands IIA–G and IVA–G.
tional experiments. We examined the effects of two of
our ligands on an entire concentration–effect curve of
the peptide agonist triptorelin. Thus we recorded tripto-
relin curves in the absence and in the presence of two
concentrations (2 and 10 lM) of selected compounds
ID and IVD (Fig. 4).
tide binding site. In order to exclude the possibility that
the reduced efficacy of our compounds is due to cytotox-
icity we determined cell viability by a trypan blue exclu-
sion experiment. We found that cell viability always
exceeded 95% after 4 h of incubation in the absence
(control) or presence of 10 lM of a relevant subset of
our compounds (see Section 4).
As is evident from the curves shown in Figure 4, both
monomeric- and dimeric-ligand ID and IVD show a
rightward shift of the dose–response curve, a clear indi-
cation of the antagonism of the peptide agonist triptore-
lin. Compound IVD shows less antagonistic potency
against triptorelin compared to the monomer, which is
in agreement with the outcome of the functional assay
as depicted in Table 2. However, the maximal efficacy
(Emax) of triptorelin is also decreased, to the same
amount in the presence of both monomeric ligand ID
and dimeric ligand IVD. This result might indicate that
our compounds also possess non-competitive character-
istics, such as binding to an allosteric binding site that
may be close to, or partially overlapping with the pep-
3. Discussion
In our study, dimeric ligands derived from ligand l show
interesting biological properties compared to the mono-
meric counterparts. While the binding properties of the
dimeric ligands and monomeric ligands were in the same
range, some different functional properties were
observed for the dimeric ligands. For example, dimeric
ligand IVD and monomeric ligand ID share similar
binding affinities in the displacement assay, whereas a
decrease in antagonistic potency for dimeric ligand
IVD is observed. A similar trend was observed for

3748
K. M. Bonger et al. / Bioorg. Med. Chem. 16 (2008) 3744–3758
O
O
O
O
H
N
H
N
O
GnRHR-Ant
H
N
N
H
GnRHR-Ant
GnRHR-Ant
GnRHR-Ant
N
H
N
H
O
O
O
O
O
O
O
H
IVA
IVB
N
GnRHR-Ant
GnRHR-Ant
GnRHR-Ant
N
H
IIA
IIB
O
O
H
N
H
N
O
H
N
GnRHR-Ant
N
H
N
H
N
H
O
O
H
N
N
H
O
O
H
N
H
N
O
O
GnRHR-Ant
H
N
N
H
GnRHR-Ant
GnRHR-Ant
N
H
N
H
O
O
O
O
H
N
IVC
IVD
IVE
GnRHR-Ant
GnRHR-Ant
N
H
O
IIC
IID
IIE
O
O
O
H
N
H
N
H
N
N
H
GnRHR-Ant
N
GnRHR-Ant
N
H
H
O
O
O
H
N
GnRHR-Ant
N
H
O
O
O
H
N
H
N
O
O
GnRHR-Ant
H
N
GnRHR-Ant
GnRHR-Ant
N
H
N
H
N
H
O
O
O
O
O
H
N
GnRHR-Ant
N
H
O
O
O
O
H
N
O
GnRHR-Ant
GnRHR-Ant
N
GnRHR-Ant
GnRHR-Ant
N
N
N
H
GnRHR-Ant
GnRHR-Ant
N
H
N
O
H
N
IVF
IIF
O
O
O
N
O
H
N
O
N
N
H
GnRHR-Ant
N
H
O
N
H
IVG
GnRHR-Ant
O
N
N
O
IIG
Fig. 3 (continued).
dimeric ligands. For example, compounds IID, E, G,
IIIG, IVD and E, all bearing longer spacers compared
to the other compounds, are more potent than the other
dimeric compounds. This observation suggests that
dimeric ligands in this series with a larger interpharma-
cophoric distance will exhibit enhanced antagonistic
properties.
Table 1. Binding affinities (Ki values) of monomeric GnRHR antag-
onists IA–IG and dimeric GnRHR antagonists IIA–G, IIIA–G, and
IVA–G
Ki (lM) SEM
1
0.004 0.0006
I
II
III
IV
A
B
C
D
E
F
0.17 0.01
1.6 0.23
1.0 0.26
3.0 1.7
1.1 0.27
5.3 0.26
2.2 0.37
0.53 0.26
0.48 0.04
0.72 0.05
0.60 0.04
0.62 0.009
0.74 0.11
0.51 0.002
Additional assays to support evidence for an allosteric
interaction of the dimeric ligands to the GnRH receptor
show that addition of dimeric ligand IVD to the peptide
agonist triptorelin reduced both potency and efficacy of
the peptide (Fig. 4). However, the monomeric counter-
part ID affected the dose-response curve to the same ex-
tent. Recently, a distinct, that is, non-GnRH peptide,
binding site was reported for a different set of heterocy-
clic GnRHR antagonists.9 It is conceivable that our
compounds bind to the receptor in a similar fashion as
the reported non-peptidic antagonists.
0.77 0.08
0.58 0.003
0.38 0.12
0.26 0.06
0.14 0.05
3.3 0.46
0.84 0.003
0.70 0.05
0.85 0.04
0.50 0.23
0.82 0.008
0.93 0.01
0.93 0.09
0.40 0.02
G
dimeric ligand IVB, which show a 3-fold increase in
binding affinities compared to monomeric ligand IB
and a reduced antagonistic potency in the functional
assay.
From the functional assay, a general correlation is
observed between spacer length and potency of the
The fact that dimeric ligands derived from antagonist 1
do not show enhanced pharmacological properties com-

K. M. Bonger et al. / Bioorg. Med. Chem. 16 (2008) 3744–3758
3749
Table 2. Antagonistic activities (IC50) of monomeric GnRHR antagonist (IA–G) and % inhibition of dimeric GnRHR antagonist IIA–G, IIIA–G,
and IVA–G at 3 and 10 lM concentration
IC50 (lM) SEM
1
0.076 0.027
II
I
III
IV
IC50
(lM) SEM
% E at
3 lM
% E at
10 lM
% E at
3 lM
% E at
10 lM
% E at
3 lM
% E at
10 lM
% E at
3 lM
% E at
10 lM
A
B
C
D
E
F
0.46 0.009
1.1 0.07
92
63
64
97
97
87
83
4
5
9
5
1
5
4
95
61
60
96
99
89
82
3
2
4
4
1
6
2
37 11
73 12
57 7
43 13
20
9
9
14
9
8
34
42
31
9
7
0
59
52
34
6
3
6
13
0
2
18 17
28 17
0.52 0.09
0.68 0.15
0.44 0.07
0.56 0.03
0.46 0.01
12
8
43 15
57
58 13
67
86 12
73
96
95
3
2
40
44 16
31
68 13
1
3
68 15
85 12
67
65 12
48
52 12
54
46
35
3
9
5
5
21
58
7
6
42 14
70
7
8
G
2
5
Figure 4. Curve signatures of peptide-agonist triptorelin in the absence and presence of 2 and 10 lM of monomeric ligand ID (left) and dimeric
ligand IVD (right).
pared to the monomeric counterparts do not allow us to
distinguish between different modes of binding of biva-
lent ligands to the GnRHR. Recently, in a similar study
targeting the serotonin 5-HT4 receptor dimer specifically
with bivalent ligands which are constructed in a similar
fashion as the library described in this report, no dis-
crepancy was observed in monomeric- and dimeric-li-
gands in respect to binding properties. In the
functional assay, most dimeric ligands lost the agonistic
character of the monomeric reference compound.10 Yet,
the authors show a conformational change of 5-HT4R
dimers with bioluminescence resonance energy transfer
(BRET) when subjecting the dimeric partial agonist to
the receptor thus suggesting a simultaneous interaction
of the two pharmacophores of the bivalent ligands to
the receptor dimer. The dimeric ligands we present here
are highly reminiscent to those reported in the 5-HT4
study when considering the nature and size of the linker
systems. It is therefore not unlikely that our dimeric
compounds behave in a similar fashion, that is, simulta-
neous binding to a GnRHR dimer but without a phar-
macological effect. However, further studies are
required to establish the validity of this hypothesis.
ent mode of binding of the dimeric ligands compared to
the monomeric counterparts. To establish whether
dimerisation of GnRH receptor is a prerequisite for sig-
naling or ligand binding, more research is needed. For-
tunately, our synthetic strategy is flexible with respect to
the nature of the linker system and the pharmacophore.
Furthermore, the diiodobenzene scaffold can be easily
modified to, for instance triiodobenzene or other oli-
go-iodoaryl systems, allowing the preparation of tri-
and tetravalent compounds. Future work along these
lines combined with the development of a new library
based on a homo- and hetero-dimeric ligands containing
a different antagonist are now conducted in our
laboratory.
4. Experimental
4.1. GnRHR luciferase reporter gene assay
Chinese Hamster Ovary, CHO-K1, cells with stable
expression of the human Gonadotropin Releasing
Hormone Receptor (GnRH-R) and Nuclear Factor
Activated T-cell luciferase reporter gene were grown to
80–90% confluence in culture medium consisting of
Dulbecco’s Modified Eagle’s Medium (DMEM) supple-
mented with 10% w/v fetal bovine serum, 100 U/ml
penicillin and 100 lg/ml streptomycin and 400 lg/ml
geniticin. On the day of the assay, cells were washed
twice with phosphate buffered saline and then harvested
with cell dissociation solution. Cells were resuspended in
assay medium consisting of DMEM supplemented with
1 mg/L insulin and 5 mg/L apo-transferrin and 3% v/v
DMSO. Then, 10 ll cell suspension containing 7500 cells
In conclusion, we have developed a strategy for the
preparation of a set of dimeric ligands containing hydro-
phobic, rigid linker systems to target the GnRH recep-
tor. The results described in this paper concerning
binding and functional properties do not provide us fur-
ther information to establish the interaction of dimeric
ligands to GnRHR. Combination of the binding and
functional antagonistic properties and the reduction of
the maximal effect of the peptide agonist triptorelin in
the presence of our compounds might indicate an differ-

3750
K. M. Bonger et al. / Bioorg. Med. Chem. 16 (2008) 3744–3758
was added to each well of a 384-well white culture plate.
Thereafter, 10 ll of test compound was added at 10 con-
centrations ranging from final concentration of 10 lM
to 0.3 nM with half log intervals. Compounds were
allowed to preincubate with cells for 30 min followed
by addition of 10 ll agonist GnRH at a final concentra-
tion of 3 nM which produces approximately 80% of the
maximal effect (EC80) when given alone. After 4 h stim-
ulation, 15 ll of lucliteꢂ was added to each well for
detection of luciferase protein and plates were left at
room temperature for 1 h in the dark. Finally, the lumi-
nescence signal was quantified on the TopCountꢂ
Microplate Scintillation and Luminescence Counter.
by rapid filtration through Whatman GF/B filters pre-
soaked with 0.25% PEI for 1 h using a Brandel har-
vester. Filters were subsequently washed three times
with ice-cold wash buffer (50 mM Tris–HCl, pH 7.4,
supplemented with 2 mM MgCl2 and 0.05% BSA). Fil-
ter-bound radioactivity was determined in a c-counter.
All data were analyzed using the non-linear regression
curve-fitting program GraphPad Prism v. 4 (GraphPad
Software Inc., San Diego, CA, USA). Inhibitory binding
constants (Ki values) were derived from the IC50 values
according to Ki = IC50/(1 + [C]/Kd) where [C] is the con-
centration of the radioligand and Kd its dissociation
constant.12 The Kd value (1.1 nM) of [125I]triptorelin
was obtained by computer analysis of saturation curves
(data not shown). All values obtained are means of at
least two independent experiments performed in
duplicate.
4.2. Radioligand binding assays
Ganirelix was provided by Organon (Oss, The Nether-
lands).
[
125I]Triptorelin (specific activity 2200 Ci
mmolꢀ1) was purchased from Perkin Elmer Life Sci-
ences B.V. (Groningen, The Netherlands). CHO cells
stably expressing the human GnRH receptor were
kindly provided by Organon (Oss, The Netherlands).
All other chemicals and cell culture materials were
obtained from standard commercial sources.
4.3. Cytotoxicity
CHOhGnRH_luc cells were seeded on 5-cm diameter
plates in assay medium in the absence (control) or pres-
ence of 10 lM of test compounds. As relevant com-
pounds in this toxicity assay we selected the following:
ID–IVD, IVA and IVE. The cells were incubated for
4 h at 37 ꢁC. Thereafter the cells were harvested using
0.5 ml trypsol and resuspended in 2 ml of PBS. Subse-
quently the number of viable cells was determined by
trypan blue exclusion, where a trypan blue solution
(0.8% (w/v) in PBS) was added to an equal amount of
cell suspension. The proportion of live cells was deter-
mined by counting in a hemocytometer.
CHO (Chinese hamster ovary) cells expressing the wild-
type human GnRH receptor were grown in Ham’s F12
medium containing 10% bovine calf serum, streptomy-
cin (100 lg mLꢀ1), penicillin (100 IU mLꢀ1) and G418
(0.4 mg mLꢀ1) at 37 ꢁC in 5% CO2. The cells were sub-
cultured twice weekly at a ratio of 1:20. For membrane
preparation the cells were subcultured 1:10 and trans-
ferred to large 14-cm diameter plates. For membrane
preparation the cells were detached from the plates by
scraping them into 5 mL PBS, collected and centrifuged
at 1400g (3000 rpm) for 5 min. Pellets derived from 30
plates were pooled and resuspended in 30 mL of ice-cold
50 mM Tris–HCl buffer supplemented with 2 mM
MgCl2, pH 7.4. An UltraThurrax was used to homoge-
nize the cell suspension. Membranes and the cytosolic
fraction were separated by centrifugation at 100,000g
(31,000 rpm) at 4 ꢁC for 20 min. The pellet was resus-
pended in 10 mL of the Tris buffer and the homogeniza-
tion and centrifugation steps were repeated. Tris buffer
(10 mL) was used to resuspend the pellet and the mem-
branes were stored in 500 lL aliquots at ꢀ80 ꢁC. Mem-
brane protein concentrations were measured using the
BCA (bicinchoninic acid) method.11
4.4. Chemical procedures
Reactions were executed at ambient temperatures unless
stated otherwise. All moisture sensitive reactions were
performed under an argon atmosphere. All solvents were
removed by evaporation under reduced pressure. Reac-
tions were monitored by TLC analysis using silica gel
coated plates (0.2 mm thickness) and detection by
254 nm UV-light or by either spraying with a solution of
(NH4)6Mo7O24 · 4H2O
(25 g/L)
and
(NH4)4Ce
(SO4)4 · 2H2O (10 g/L) in 10% sulfuric acid followed by
charring at ꢁ150 ꢁC. Column chromatography was per-
formed on silica gel (40–63 lm). NMR spectra were re-
corded on
a
200/50.1 MHz, 300/75.1 MHz, 400/
100 MHz, 500/125 MHz or 600/150 MHz spectrometer.
Chemical shifts are given in ppm (d) relative to tetrameth-
ylsilane as internal standard. Coupling constants (J) are
given in Hz. All presented 13C-APT spectra are proton
decoupled. For LC/MS analysis, a HPLC-system (detec-
tion simultaneously at 214 and 254 nm) equipped with
an analytical C18 column (4.6 mmD · 250 mmL, 5 l par-
ticle size) in combination with buffers A: H2O, B: MeCN
and C: 0.5% aq TFA and coupled to a mass instrument
with an electronspray interface (ESI) was used. For RP-
HPLC purifications, an automated HPLC system
equipped with a semi-preparative C18 column (5 lm
On the day of the assay membrane aliquots containing
20 lg protein were incubated in a total volume of
100 lL assay buffer (50 mM Tris–HCl, pH 7.4, supple-
mented with 2 mM MgCl2 and 0.1% BSA) at 22 ꢁC for
45 min. Displacement experiments were performed
using five concentrations of competing ligand in the
presence of 30,000 cpm [125I]triptorelin. Non-specific
binding was determined in the presence of 1 lM Ganir-
elix and represented approximately 20% of the total
binding. Total binding was determined in the presence
of buffer and was set at 100% in all experiments, whereas
non-specific binding was set at 0%. Incubations were ter-
minated by dilution with ice-cold Tris–HCl buffer. Sep-
aration of bound from free radioligand was performed
˚
C18, 10 A, 150 · 21.2 mm) was used. The applied buffers
were A: 5% MeCN/H2O + 0.1% TFA and B: MeCN.
High resolution mass spectra were recorded on a mass

K. M. Bonger et al. / Bioorg. Med. Chem. 16 (2008) 3744–3758
3751
spectrometer equipped with an electronspray ion source
in positive mode (source voltage 3.5 kV, sheath gas flow
10%, capillary temperature 275 ꢁC) with resolution
R = 100,000 at m/z 400. The high resolution mass spec-
trometer was calibrated prior to measurements with a cal-
ibration solution (caffeine, MRFA, Ultramark 1621).
4.4.1.5. N-e-tert-Boc-e-amino hexanoic acid-propargyla-
mide (4E). Yield: 2.41 g (8.99 mmol, 90%). Rf = 0.60
(MeOH/DCM, 1:9, v/v). ESI-MS (m/z) calcd for
C14H24N2O3 + H+: 269.2, obsd 269.1. 1H NMR
(200 MHz, CDCl3): d 5.77 (br t, 1H), 4.56 (br t, 1H), 4.05
(dd, 2H, J = 5.5 Hz, J = 2.6 Hz), 3.11 (q, 2H,
J = 6.6 Hz), 2.24–2.17 (m, 3H), 1.44 (s, 9H), 1.74–1.34
(m, 6H). 13C NMR (50 MHz, CDCl3/CD3OD): d 173.3,
156.3, 79.5, 79.2, 71.0 (5· C), 39.9, 35.7, 29.3, 28.7 (4·
CH2), 28.2 (3· CH3), 26.0, 24.9 (2· CH2).
4.4.1. General procedure for coupling of amino acids with
propargylamine (4A–4G). Isobutyl chloroformate
(1.43 mL, 11 mmol) was added to a cooled (ꢀ20 ꢁC) solu-
tion of amino acid (10 mmol) and N-methylmorpholine
(1.52 mL, 14 mmol) in DCM (50 mL). After stirring for
1 h, propargylamine (0.96 mL, 14 mmol) was added.
The reaction mixture was warmed to room temperature
over a period of 2 h and subsequently stirred for 16 h.
The mixture was successively washed with 1 M HCl
(50 mL) and 10% aq NaHCO3 (50 mL). The organic layer
was dried (Na2SO4), filtered and concentrated. The resi-
due was dissolved in EtOAc and triturated with petro-
leum ether to afford titled product as white crystals.
4.4.1.6. N-a-tert-Boc-proline propargylamide (4F).
Yield: 1.71 g (6.79 mmol, 68%). Rf = 0.55 (MeOH/
DCM, 1:9, v/v). ESI-MS (m/z) calcd for C13H20N2
1
O3 + H+: 253.2, obsd 253.0. H NMR mixture of rota-
mers (200 MHz, CDCl3): d 7.35 (br s, 1H), 6.25 (br s,
1H), 4.26 (br s, 1H), 4.02 (br s, 2H), 3.35 (br s, 2H), 2.23
(s, 1H), 2.13 (br s, 2H), 1.88 (br s, 2H), 1.46 (br s, 9H).
13C NMR of rotamers (50 MHz, CDCl3): d 171.9, 80.3,
79.3, 71.2 (4· C), 46.8, 28.7 (2· CH2), 28.3 (3· CH3),
24.2, 23.6 (2· CH2).
4.4.1.1. N-a-tert-Boc-glycine propargylamide (4A).
Yield: 1.60 g (7.55 mmol, 76%). Rf = 0.60 (EtOAc). ESI-
MS (m/z) calcd for C10H16N2O3 + H+: 213.1, obsd
4.4.1.7. N-d-tert-Boc-4-amino cyclohexanoic acid-a-
propargylamide (4G). Yield: 0.97 g (3.65 mmol, 37%).
Rf = 0.60 (MeOH/DCM, 1:9, v/v). ESI-MS (m/z) calcd
for C14H22N2O3 + H+: 267.2, obsd 267.0. 1H NMR
(200 MHz, CDCl3): d 5.65 (s, 1H), 4.25–4.02 (m, 2H),
4.05 (dd, 2H, J = 5.1 Hz, J = 2.6 Hz), 2.74 (br t, 2H,
J = 11.0 Hz), 2.24 (t, 1H, J = 2.6 Hz), 2.33–2.18 (m,
1H), 1.85–1.73 (m, 2H), 1.73–1.58 (m, 2H), 1.45 (s, 9H).
13C NMR (50 MHz, CDCl3): d 174.1, 154.5, 79.5, 71.3
(4· C), 43.0 (CH2), 42.6 (CH), 28.9, 28.3 (2x CH2), 28.3
(3· CH3).
1
213.0. H NMR (200 MHz, CDCl3): d 6.59 (br t, 1H),
5.25 (br t, 1H), 4.07 (dd, 2H, J = 5.5 Hz, J = 2.6 Hz),
3.82 (d, 2H, J = 5.8 Hz), 2.24 (t, 1H, J = 2.6 Hz), 1.46 (s,
9H). 13C NMR (50 MHz, CDCl3): d 169.2, 156.0, 89.8,
80.4, 71.7 (5· C), 29.1 (CH2), 28.3 (3· CH3).
4.4.1.2. N-a-tert-Boc-alanine propargylamide (4B).
Yield: 1.94 g (8.55 mmol, 86%). Rf = 0.55 (MeOH/
DCM, 1:19, v/v). ESI-MS (m/z) calcd for C11H18N2
O3 + H+: 227.1, obsd 227.0. 1H NMR (200 MHz,
CDCl3): d 6.59 (br t), 5.00 (br d, 1H), 4.28–4.12 (m, 1H)
4.05 (dd, 2H, J = 5.5 Hz, J = 2.6 Hz), 2.22 (t, 1H,
J = 2.6 Hz), 1.45 (s, 9H), 1.35 (d, 3H, J = 6.9 Hz). 13C
NMR (50 MHz, CDCl3): d 172.4, 155.5, 80.2, 79.3, 71.5
(5· C), 49.9 (CH), 29.1 (CH2), 28.3 (3· CH3), 18.3
(CH3).
4.4.2. General procedure for coupling of propargylamide
functionalized amino acids with iodobenzene (affording
5A–5G). In separate flasks, a solution of the propargyl
functionalized amino acid (4A–G, 1.0 mmol), iodoben-
zene (1.5 mmol, 111lL) in pyrrolidine/DMF (1/4: v/v),
a solution of CuI (0.1 mmol, 19.1 mg) in DMF (2 mL)
and a solution of Pd(PPh3)4 (0.05 mmol, 57.8 mg) in
DMF (4 mL) were flushed with argon for 1 h in an
ultrasonic bath. To the alkyne solution were added
subsequently the CuI and the Pd(PPh3)4 solutions
and the mixture were stirred for 18 h under inert
atmosphere. The volatiles were removed and the crude
product dissolved in MeOH/DCM (1/9, v/v, 50 mL)
and washed with water (3· 50 mL) and 10% aq NaH-
CO3 (50 mL). The organic layer was dried with
Na2SO4 and concentrated. The crude material was
purified by automated silica gel column chromatogra-
phy (35–65% 1:10; MeOH/DCM; v/v in petroleum
ether).
4.4.1.3. N-a-tert-Boc-valine propargylamide (4C).
Yield: 2.52 g (9.92 mmol, 99%). Rf = 0.65 (MeOH/
DCM, 1:9, v/v). ESI-MS (m/z) calcd for C13H22N2
O3 + H+: 255.2, obsd 255.1. 1H NMR (200 MHz,
CDCl3): d 6.40 (br t, 1H), 5.04 (br d, 1H), 4.05 (dd, 2H,
J = 5.5 Hz, J = 2.2 Hz), 3.92 (dd, 1H, J = 8.9 Hz,
J = 6.4 Hz), 2.22 (t, 1H, J = 2.6 Hz), 2.15 (m, 1H), 1.45
(s, 9H), 0.97 (d, 3H, J = 6.6 Hz), 0.93 (d, 3H, J =
7.0 Hz). 13C NMR (50 MHz, CDCl3): d 171.6, 155.9,
79.9, 79.3, 71.4 (5· C), 59.8, 31.0 (2· CH), 28.9 (CH2),
28.3 (3· CH3), 17.9, 19.1 (2· CH3).
4.4.1.4. N-c-tert-Boc-c-amino butaric acid-propargyla-
mide (4D). Yield: 1.68 g (7.00 mmol, 70%). Rf = 0.50
(MeOH/DCM, 1:19, v/v). ESI-MS (m/z) calcd for
C12H20N2O3 + H+: 241.2, obsd 240.9. 1H NMR
(200 MHz, CDCl3): d 6.39 (br t, 1H), 4.73 (br t, 1H),
4.05 (dd, 2H, J = 5.1 Hz, J = 2.9 Hz), 3.18 (q, 2H,
J = 6.6 Hz), 2.27–2.21 (m, 3H), 1.88–1.75 (m, 2H), 1.44
(s, 9H). 13C NMR (50 MHz, CDCl3): d 173.2, 156.7,
89.3, 79.3, 70.9 (5· C), 39.4, 32.9 28.6, (3· CH2), 28.1
(3· CH3), 25.8 (CH2).
4.4.2.1. N-a-tert-Boc-(3-phenylprop-2-yn-1-amide)-gly-
cine (5A). Yield: 189 mg (0.66 mmol, 66%). Rf = 0.65
(EtOAc). ESI-MS (m/z) calcd for C16H20N2O3 + H+:
1
288.1, obsd 289.0. H NMR (500 MHz, CDCl3): d 7.39
(m, 2H), 7.28 (m, 3H), 7.17 (br t, 1H), 5.69 (s, 1H), 4.28
(d, 2H, J = 4.5 Hz), 3.88 (s, 2H), 1.43 (s, 9H). 13C NMR
(125 MHz, CDCl3): d 169.5, 156.1 (2· C), 131.5, 128.1
(2· CH), 122.3 (C), 84.4, 83.1 (2· C), 80.1 (C), 43.9, 29.7
(2· CH2), 28.0 (3· CH3).

3752
K. M. Bonger et al. / Bioorg. Med. Chem. 16 (2008) 3744–3758
4.4.2.2. N-a-tert-Boc-(3-phenylprop-2-yn-1-amide)-ala-
nine (5B). Yield: 298 mg (0.99 mmol, 99%). Rf = 0.70
(s, 1H), 4.26 (d, 2H, J = 5.0 Hz), 4.12 (br s, 2H), 2.72
(br s, 2H), 2.30 (m, 1H), 1.80 (m, 2H), 1.65 (m, 2H),
1.45 (s, 9H). 13C NMR (125 MHz, CDCl3): d 174.1,
154.5 (2· C) 131.5, 128.1 (2· CH), 122.3, 84.7, 83.1,
79.5 (4· C), 42.9 (CH2), 42.8 (CH), 29.8, 28.3 (2· CH2),
27.4 (3· CH3).
(EtOAc). ESI-MS (m/z) calcd for C17H22N2O3 + H+:
1
302.2, obsd 303.0. H NMR (500 MHz, CDCl3): d 7.39
(m, 2H), 7.27 (m, 3H), 7.09 (s, 1H), 5.42 (d, 1H,
J = 7.0 Hz), 4.30 (br s, 1H), 4.26 (d, 2H, J = 4.5 Hz),
1.40 (s, 9H), 1.37 (d, 3H). 13C NMR (125 MHz, CDCl3):
d 172.5, 155.5 (2x C), 131.5, 128.1 (2· CH), 122.5, 84.6,
83.1, 79.9 (4· C), 49.8 (CH), 29.7 (CH2), 28.2 (3· CH3),
18.3 (CH3).
4.4.3. General procedure for coupling of propargylic
spacers 4A–G with 1,3-diiodobenzene (affording 7A–G),
1,2-diiodobenzene (affording 9A–G) and 1,4-diiodobenzene
(affording 11A–G). In separate flasks, a solution of the
propargyl functionalized amino acid (4A–G, 0.90 mmol),
the desired diiodobenzene (0.30 mmol, 91.1 mg) and pyr-
rolidine (1.80 mmol, 147 lL) in DMF (3 mL), a solution
of CuI (0.06 mmol, 11.5 mg) in DMF (1 mL) and a
solution of Pd(PPh3)4 (0.03 mmol, 34.7 mg) in DMF
(1 mL) were was flushed with argon for 1 h in an ultra-
sonic bath. To the alkyne solution were added subse-
quently the CuI and the Pd(PPh3)4 solutions and the
mixture were stirred for 18 h under argon atmosphere.
The volatiles were removed and the crude product dis-
solved in MeOH/DCM (1/9, v/v, 50 mL) and washed with
water (3· 10 mL) and 10% aq NaHCO3 (10 mL). The or-
ganic layer was dried with Na2SO4 and concentrated. The
crude material was purified by automated silica gel col-
umn chromatography (35–65% 1:10; MeOH/DCM; v/v
in petroleum ether).
4.4.2.3. N-a-tert-Boc-(3-phenylprop-2-yn-1-amide)-valine
(5C). Yield: 322 mg (0.98 mmol, 98%). Rf = 0.80 (EtOAc).
ESI-MS (m/z) calcd for C19H26N2O3 + H+: 330.2, obsd
1
331.0. H NMR (500 MHz, CDCl3): d 7.56 (s, 1H), 7.37
(m, 2H), 7.27 (m, 3H), 5.64 (d, 1H, J = 9.5 Hz), 4.34 (dd,
1H, J = 17.5 Hz, J = 5.5 Hz), 4.13 (d, 2H, J = 4.5 Hz),
2.10–2.06 (m, 1H), 1.42 (s, 9H), 0.99 (d, 3H, J = 7.0 Hz),
0.96 (d, 3H, J = 7.0 Hz). 13C NMR (125 MHz, CDCl3)
d
171.8 (C), 155.9 (C), 131.5, 128.0, (2· CH), 122.5, 84.7,
82.8, 79.5 (4· C), 59.6, 31.2 (2· CH), 29.4 (CH2), 28.2 (3·
CH3), 19.0, 18.1 (2· CH3).
4.4.2.4. N-a-tert-Boc-(3-phenylprop-2-yn-1-amide)-ami-
no butaric acid (5D). Yield: 205 mg (0.65 mmol, 65%).
Rf = 0.55 (EtOAc). ESI-MS (m/z) calcd for C18H24N2
O3 + H+: 316.2, obsd 217.0. 1H NMR (500 MHz, CDCl3):
d 7.41 (m, 2H), 7.29 (m, 3H), 7.23 (s, 1H), 5.25 (s, 1H), 4.23
(s, 2H), 3.13 (t, 2H, J = 4.0 Hz), 2.25 (t, 2H, J = 7.0 Hz),
1.83–1.78 (m, 2H), 1.43 (s, 9H). 13C NMR (125 MHz,
CDCl3): d 172.9, 155.6 (2· C), 131.5, 128.2 (2· CH),
122.5, 84.7, 82.9, 79.3 (4· C), 39.5, 29.6, 25.9, 33.1 (4·
CH2), 28.2 (3· CH3).
4.4.3.1. N,N0-a,a0-di-tert-Boc-(3,30(1,3-phenylene)diprop-
yn-1-amide)-glycine (7A). Yield: 148 mg (0.30 mmol, 99%).
Rf = 0.50 (EtOAc). ESI-MS (m/z) calcd for C26H34N4O6,
1
+ H+: 499.3, obsd 499.5. H NMR (200 MHz, CDCl3): d
7.45–7.16 (m, 4H), 7.07 (br t, 2H), 5.57 (br t, 2H), 4.25 (d,
4H, J = 5.1 Hz), 3.86 (d, 4H, J = 5.1 Hz), 1.44 (s, 18H).
13C NMR (100 MHz, CDCl3): d 170.0, 156.2 (2· C),
131.5, 127.9 (2x CH), 125.4, 88.9, 81.2, 79.9 (4· C), 44.0,
29.6 (2· CH2), 28.2 (3· CH3).
4.4.2.5. N-a-tert-Boc-(3-phenylprop-2-yn-1-amide)-ami-
no hexanoic acid (5E). Yield: 332 mg (0.97 mmol, 97%).
Rf = 0.70 (EtOAc). ESI-MS (m/z) calcd for C20H28N2
O3 + H+: 344.2, obsd 343.1. 1H NMR (500 MHz,
CDCl3): d 7.38 (m, 2H), 7.28 (m, 3H), 7.15 (s, 1H), 5.12
(s, 1H), 4.23 (d, 2H, J = 4.0 Hz), 3.06 (t, 2H,
J = 4.0 Hz), 2.22 (t, 2H, J = 7.5 Hz), 1.65 (m, 2H), 1.49
(m, 2H), 1.43 (s, 9H), 1.32 (m, 2H). 13C NMR
(125 MHz, CDCl3): d 173.3, 155.3 (2· C), 131.4, 128.0
(2· CH), 122.4, 84.7, 82.7, 79.0 (4· C), 39.9, 35.7, 29.5,
29.2 (4· CH2), 28.1 (3· CH3), 25.9, 24.9 (2x CH2).
4.4.3.2. N,N0-a,a0-di-tert-Boc-(3,30(1,3-phenylene)diprop-
2-yn-1-amide)-alanine (7B). Yield: 128 mg (0.24
mmol, 81%). Rf = 0.70 (EtOAc). ESI-MS (m/z) calcd
for C28H38N4O6 + H+: 527.4, obsd 527.4. 1H NMR
(400 MHz, CDCl3): d 7.68 (br s, 2H), 7.65–7.20 (m,
4H), 5.61 (br d, 2H), 4.41–4.29 (m, 2H), 4.24 (d, 4H,
J = 4.4 Hz), 1.43 (s, 18H), 1.37 (d, 6H, J = 6.9 Hz). 13C
NMR (100 MHz, CDCl3): d 172.8, 155.5 (2· C),
131.9, 128.4 (2· CH), 122.7, 85.4, 82.0, 79.8 (4· C),
49.8 (CH), 29.7 (CH2), 28.2 (3· CH3), 18.4 (CH3).
4.4.2.6. N-a-tert-Boc-(3-phenylprop-2-yn-1-amide)-pro-
line (5F). Yield: 302 mg (0.92 mmol, 92%). Rf = 0.55
(EtOAc). ESI-MS (m/z) calcd for C19H24N2O3 + H+:
328.2, obsd 329.1. 1H NMR of rotamers (500 MHz,
CDCl3): d 7.39 (m, 2H), 7.27 (m, 3H), 6.63 (br s, 1H),
4.35 (br d, 1H), 4.24 (br s, 2H), 3.40 (br d, 2H), 2.25
(br dd, 2H), 1.84 (br d, 2H), 1.45 (s, 9H). 13C NMR of
rotamers (125 MHz, CDCl3): d 172.2, 155.3 (2· C),
131.4, 128.0 (2· CH), 122.3, 84.7, 82.9, 80.2 (4· C),
60.8 (CH), 46.8, 29.5 (2· CH2), 28.1 (3· CH3), 24.4,
23.1 (2· CH2).
4.4.3.3. N,N0-a,a0-di-tert-Boc-(3,30(1,3-phenylene)diprop-
2-yn-1-amide)-valine (7C). Yield: 179 mg (0.30 mmol,
99%). Rf = 0.83 (1:10; MeOH/DCM; v/v). ESI-MS (m/
1
z) calcd for C32H46N4O6 + Na+: 605.3, obsd 605.6. H
NMR (400 MHz, CDCl3) d 7.40–7.18 (m, 4H), 7.02
(br s, 2H), 5.36 (br d, 2H), 4.24 (d, 4H, J = 4.4 Hz),
4.06–3.98 (m, 2H), 2.18–2.06 (m, 2H), 1.43 (s, 18H),
0.99–0.94 (m, 12H). 13C NMR (100 MHz, CDCl3) d
171.8, 156.0 (2· C), 134.8, 131.3, 128.2 (3· CH), 122.9,
85.3, 82.4, 79.8 (4· C), 59.9 (CH), 31.0 (CH3), 29.7
(CH2), 28.3 (3· CH3), 19.2 (CH).
4.4.2.7. N-a-tert-Boc-(3-phenylprop-2-yn-1-amide)-ami-
no cyclohexanoic acid (5G). Yield: 323 mg (0.94 mmol,
94%). Rf = 0.85 (EtOAc). ESI-MS (m/z) calcd for
C20H26N2O3 + H+: 342.2, obsd 343.0. 1H NMR
(500 MHz, CDCl3): d 7.39 (m, 2H), 7.28 (m, 3H), 6.43
4.4.3.4. N,N0-a,a0-di-tert-Boc-(3,30(1,3-phenylene)diprop-
2-yn-1-amide)-amino butaric acid (7D). Yield: 165 mg

K. M. Bonger et al. / Bioorg. Med. Chem. 16 (2008) 3744–3758
3753
(0.30 mmol, 99%). Rf = 0.30 (EtOAc). ESI-MS (m/z)
calcd for C30H42N4O6 + H+: 555.9, obsd 555.3. 1H
NMR (400 MHz, CDCl3): d 7.69–7.21 (m, 4H), 6.77
(br s, 2H), 4.84 (br s, 2H), 4.25 (d, 4H, J = 5.2 Hz),
3.21–3.09 (m, 4H), 2.35–2.20 (m, 4H), 1.88–1.75 (m,
4H), 1.44 (s, 18H). 13C NMR (100 MHz, CDCl3): d
172.7, 156.4 (C), 134.7, 131.8, 128.4 (3· CH), 122.8,
85.6, 81.9, 79.1 (4· C), 39.7, 33.2, 29.7 (CH2), 28.3 (3·
CH3), 26.1 (CH2).
4.00 (m, 2H), 1.41 (s, 18H), 1.37 (t, 6H). 13C NMR
(100 MHz, CDCl3): d 173.3, 155.7 (2· C), 131.6, 127.9
(2· CH), 125.6, 88.9, 81.3, 79.9 (4· C), 49.9 (CH), 29.9
(CH2), 28.2 (3· CH3), 18.4 (CH3).
4.4.3.10. N,N0-a,a0-di-tert-Boc-(3,30(1,2-phenylene)diprop-
2-yn-1-amide)-valine (9C). Yield: 164 mg (0.28 mmol, 94%).
Rf = 0.75 (1:1; Tol/EtOAc; v/v). ESI-MS (m/z) calcd for
C32H46N4O6 + H+: 583.3, obsd 583.4. 1H NMR (400 MHz,
CDCl3): d 7.75 (br s, 2H), 7.37–7.34 (m, 2H), 7.22–7.20 (m,
2H), 5.64 (br s, 2H), 4.48 (dd, 2H, J = 17.6 Hz, J = 5.8 Hz),
4.13–4.02 (m, 4H), 2.09–2.01 (m, 2H), 1.41 (s, 18H), 0.93 (d,
12H, J = 6.8 Hz). 13C NMR (100 MHz, CDCl3): d 172.4,
156.1 (2· C), 131.5, 127.7 (2· CH), 125.5, 88.9, 80.9, 79.5
(4· C), 60.0, 31.0 (2· CH), 29.6 (CH2), 28.2 (3· CH3), 19.2,
18.4 (2· CH3).
4.4.3.5. N,N0-a,a0-di-tert-Boc-(3,30(1,3-phenylene)diprop-
2-yn-1-amide)-amino hexanoic acid (7E). Yield: 179 mg
(0.29 mmol, 98%). Rf = 0.45 (EtOAc). ESI-MS (m/z) calcd
for C34H50N4O2 + H+: 611.9, obsd 611.4. 1H NMR
(400 MHz, CDCl3): d 7.42–7.19 (m, 4H), 7.01 (t, 2H,
J = 5.2 Hz), 4.89 (br t, 2H), 4.24 (d, 4H, J = 5.2 Hz),
3.12–3.02 (m, 4H), 2.28–2.19 (m, 6H), 1.70–1.58 (m, 6H),
1.52–1.40 (m, 22H), 1.38–1.27 (m, 4H). 13C NMR
(100 MHz, CDCl3): d 172.7, 156.0 (2· C), 134.7, 131.3,
128.3 (3x CH), 122.8, 85.9, 81.9, 78.9 (4· C), 40.2, 36.0,
29.6 (3· CH2), 28.3 (3· CH3), 26.2, 25.1 (2· CH2).
4.4.3.11. N,N0-a,a0-di-tert-Boc-(3,30(1,2-phenylene)diprop-
2-yn-1-amide)-amino butaric acid (9D). Yield: 118 mg
(0.21 mmol, 71%). Rf = 0.35 (EtOAc). ESI-MS (m/z) calcd
for C30H42N4O6 + H+: 555.9, obsd 555.4. 1H NMR
(200 MHz, CDCl3): d 7.69 (s, 2 H), 7.40–7.20 (m, 4H),
5.18 (s, 2H), 4.27 (d, 4H, J = 5.5 Hz), 3.18–3.12 (m, 4H),
2.38–2.24 (m, 4H), 1.90–1.74 (m, 4H), 1.43 (s, 18H). 13C
NMR (50 MHz, CDCl3): d 173.1, 156.4 (2· C), 131.5,
127.9 (2· CH), 125.5, 89.3, 81.0, 79.0 (4· C), 39.6, 33.2,
29.8 (3· CH2), 28.3 (3· CH3), 26.2 (CH2).
4.4.3.6. N,N0-a,a0-di-tert-Boc-(3,30(1,3-phenylene)diprop-
2-yn-1-amide)-proline (7F). Yield: 172 mg (0.30 mmol,
99%). Rf = 0.45 (EtOAc). ESI-MS (m/z) calcd for C32H42
N4O6 + H+: 579.4, obsd 579.2. 1H NMR (300 MHz,
CDCl3): d 7.37–7.15 (m, 4H), 6.43 (br s, 1H), 4.19 (br m,
6H), 3.42 (br m, 4H), 2.15 (br m, 4H), 1.83 (br m, 4H),
1.41 (s, 9H). 13C NMR (75 MHz, CDCl3): d 171.8, 155.8
(2· C), 134.6, 131.4, 128.1 (3· CH), 122.7, 85.4, 82.1,
80.5 (4· C), 60.5 (CH), 46.7, 30.5, 29.3 (3· CH2), 28.2
(3· CH3), 24.0 (CH2).
4.4.3.12. N,N0-a,a0-di-tert-Boc-(3,30(1,2-phenylene)diprop-
2-yn-1-amide)-amino hexanoic acid (9E). Yield: 181 mg
(0.30 mmol, 99%). Rf = 0.43 (1:10; MeOH/DCM; v/v).
ESI-MS (m/z) calcd for C34H50N4O2 + H+: 611.9, obsd
1
611.4. H NMR (200 MHz, CDCl3): d 7.73 (br t, 2H),
4.4.3.7. N,N0-a,a0-di-tert-Boc-(3,30(1,3-phenylene)diprop-
2-yn-1-amide)-amino cyclohexanoic acid (7G). Yield:
133 mg (0.22 mmol, 73%). Rf = 0.55 (EtOAc). ESI-MS
(m/z) calcd for C34H46N4O6 + H+: 607.5, obsd 607.4.
1H NMR (400 MHz, CDCl3): d 7.40 (s, 1H), 7.31–7.20
(m, 3H), 6.87 (t, 2H, J = 2.4 Hz), 4.21 (d, 4H,
J = 5.2 Hz), 4.20–4.02 (br m, 4H), 2.80–2.65 (br m,
4H), 2.40–2.30 (m, 2H), 1.88–1.73 (m, 4H), 1.72–1.58
(m, 4H), 1.41 (s, 18H). 13C NMR (75 MHz, CDCl3): d
174.2, 154.6 (2· C), 143.8, 131.4, 128.3 (3· CH), 122.8,
85.7, 82.1, 79.6 (4· C), 43.1, 42.8, 29.7, 28.4 (4· CH2),
28.3 (3· CH3).
7.40–7.20 (m, 4H), 5.21 (br t, 2H), 4.04 (d, 4H,
J = 5.1 Hz), 3.06 (t, 4H, J = 5.5 Hz), 2.30–2.15 (m, 4H),
1.72–1.60 (m, 4H), 1.43 (s, 18H), 1.43–1.23 (m, 8H). 13C
NMR (50 MHz, CDCl3/CD3OD): d 173.6, 156.3 (2· C),
131.5, 127.8 (2 · CH), 125.3, 89.0, 80.8, 79.0 (4· C), 39.9,
35.6, 29.4, 29.2 (4· CH2), 28.1 (3· CH3), 25.9, 25.0 (2·
CH2).
4.4.3.13. N,N0-a,a0-di-tert-Boc-(3,30(1,2-phenylene)diprop-
2-yn-1-amide)-proline (9F). Yield: 170 mg (0.29 mmol, 98%).
Rf = 0.35 (EtOAc). ESI-MS (m/z) calcd for C32H42N4
1
O6 + H+: 579.4, obsd 579.4. H NMR (200 MHz, CDCl3):
d 7.39–7.20 (m, 4H), 7.05 (br s, 2H), 6.50 (br s, 2H), 4.35–
4.20 (br m, 4H), 4.20–4.05 (br m, 2H), 3.56–3.30 (br m,
4H), 2.40–2.05 (br m, 4H), 2.05–1.82 (br m, 4H), 1.42 (s,
18H). 13C NMR (50 MHz, CDCl3): d 172.5, 131.6 (2·
CH), 125.4, 89.0, 80.5, 80.3 (4· C), 60.0 (CH), 46.9, 29.7,
29.3 (3· CH2), 28.2 (3· CH3), 23.9 (CH2).
4.4.3.8. N,N0-a,a0-di-tert-Boc-(3,30(1,2-phenylene)diprop-
2-yn-1-amide)-glycine (9A). Yield: 148 mg (0.30 mmol,
99%). Rf = 0.40 (EtOAc). ESI-MS (m/z) calcd for
C26H34N4O6 + H+: 499.4, obsd 499.3. 1H NMR
(400 MHz, CDCl3): d 7.66 (br s, 2H), 7.37–7.34 (m,
2H), 7.24–7.20 (m, 2H), 5.97 (br t, 2H), 4.29 (d, 4H,
J = 5.4 Hz), 3.88 (d, 4H, J = 5.2 Hz), 1.44 (s, 18H). 13C
NMR (100 MHz, CDCl3): d 170.0, 156.2 (2· C), 131.5,
127.9 (2· CH), 125.4, 88.9, 81.2, 79.9 (4· C), 44.0,
29.8 (2· CH2), 28.2 (3· CH3).
4.4.3.14. N,N0-a,a0-di-tert-Boc-(3,30(1,2-phenylene)diprop-
2-yn-1-amide)-amino cyclohexanoic acid (9G). Yield:
144 mg (0.24 mmol, 79%). Rf = 0.70 (EtOAc). ESI-MS
(m/z) calcd for C34H46N4O6 + H+: 607.5, obsd 607.3.
1H NMR (300 MHz, CDCl3): d 7.67–7.62 (m, 2H),
7.50–7.46 (m, 2H), 4.25 (d, 4H, J = 5.4 Hz), 4.23–4.03
(m, 4H), 2.85–2.62 (m, 4H), 2.55–2.37 (m, 2H), 1.85–
1.61 (m, 8H), 1.45 (s, 18H). 13C NMR (50 MHz,
CDCl3): d 174.7, 154.5 (2· C), 131.9, 128.3 (2· CH),
125.6, 89.5, 80.9, 79.3 (4· C), 43.1 (CH2), 40.6 (CH),
29.6 (CH2), 28.2 (3· CH3), 27.7 (CH2).
4.4.3.9. N,N0-a,a0-di-tert-Boc-(3,30(1,2-phenylene)diprop-
2-yn-1-amide)-alanine (9B). Yield: 157 mg (0.3 mmol, 99%).
Rf = 0.75 (EtOAc). ESI-MS (m/z) calcd for C28H38N4
O6 + H+: 527.4, obsd 527.4. 1H NMR (400 MHz, CDCl3):
d 7.45 (br s, 2H), 7.39–7.34 (m, 2H), 7.24–7.20 (m, 2H),
5.67 (d, 2H, J = 7.3 Hz), 4.37 (d, 4H, J = 5.1 Hz), 4.30–

3754
K. M. Bonger et al. / Bioorg. Med. Chem. 16 (2008) 3744–3758
4.4.3.15. N,N0-a,a0-di-tert-Boc-(3,30(1,4-phenylene)diprop-
(m, 2H), 1.85–1.68 (m, 4H), 1.30 (s, 18H). 13C NMR
(100 MHz, DMSO-d6): d 172.4, 153.2 (2· C), 131.5 (4·
CH), 122.3, 90.2, 80.8, 78.4 (4· C), 59.7 (CH), 46.4,
30.3, 28.4 (3x CH2), 27.9 (3· CH3), 23.6 (CH2).
2-yn-1-amide)-glycine (11A). Yield: 148 mg (0.30 mmol,
99%). Rf = 0.70 (EtOAc). ESI-MS (m/z) calcd for C26H34
N4O6, + H+: 499.3, obsd 499.5. 1H NMR (400 MHz,
CDCl3): d 7.25 (s, 4H), 7.09 (br t, 2H), 5.71 (br t, 2H),
4.24 (d, 4H, J = 5.2 Hz), 3.85 (s, 4H), 1.44 (s, 18H). 13C
NMR (100 MHz, CDCl3): d 169.5, 156.2 (2· C), 131.5
(4· CH), 122.5, 86.5, 82.6, 80.2 (4· C), 44.2, 29.7 (2·
CH2), 28.3 (3· CH3).
4.4.3.21. N,N0-a,a0-di-tert-Boc-(3,30(1,3-phenylene)-
diprop-2-yn-1-amide)-amino cyclohexanoic acid (11G).
Yield: 120 mg (0.20 mmol, 66%). Rf = 0.45 (EtOAc).
ESI-MS (m/z) calcd for C34H46N4O6 + H+: 607.5, obsd
607.5. 1H NMR (300 MHz, CDCl3): d 7.28 (s, 4H), 6.19
(t, 2H, J = 4.9 Hz), 4.24 (d, 4H, J = 5.1 Hz), 4.13–4.08
(m, 4H), 2.70 (t, 4H, J = 12.0 Hz), 2.31–2.22 (m, 2H),
1.81–1.77 (m, 4H), 1.69–1.55 (m, 4H), 1.42 (s, 18H). 13C
NMR (75 MHz, CDCl3): d 174.0, 154.5 (2· C), 131.5
(4· CH), 122.5, 86.7, 82.7, 79.6 (4· C), 43.0, 42.9, 29.8,
28.4 (4· CH2), 28.3 (3· CH3).
4.4.3.16. N,N0-a,a0-di-tert-Boc-(3,30(1,4-phenylene)diprop-
2-yn-1-amide)-alanine (11B). Yield: 91.5 mg (0.17 mmol,
58%). Rf = 0.70 (EtOAc). ESI-MS (m/z) calcd for
C28H38N4O6 + H+: 527.4, obsd 527.2. 1H NMR
(300 MHz, CDCl3): d 7.42 (s, 2H), 7.29 (s, 4H), 5.58
(br d, 2H), 4.31–4.15 (m, 6H), 1.43 (s, 18H), 1.38 (d,
6H, J = 7.2 Hz). 13C NMR (100 MHz, CDCl3): d
172.4, 155.6 (2· C), 131.5 (4· CH), 122.5, 86.5, 82.7,
80.2 (4· C), 49.9 (CH), 29.8 (CH2), 28.2 (3· CH3),
18.2 (CH3).
4.4.4. General procedure for coupling of pharmacophore 3
with monomeric spacers 5A–G affording IA–G. Boc pro-
tected compounds 5A–G were subjected to a solution of
1:1 DCM/TFA v/v + 1% TIS for 18 h. The volatiles were
evaporated and the compounds were purified with semi-
preperative HPLC system (0 to 40% B). Accordingly,
20 lmol of amine (6A–G) was dissolved in 100 lL of
DMF and added to a solution containing pharmaco-
phore 36,7 (30 lmol, 13.4 mg), BOP (39 lmol, 17.6 mg)
and DiPEA (120 lmol, 20.4 lL) in 300 lL DMF. The
reaction mixture was stirred at rt for 16 h and diluted
with a mixture of DCM and MeOH (9:1; v/v, 20 mL).
The organic layer was successively washed with water
(3· 10 mL), 10% aq NaHCO3 (3· 10 mL) and brine
(1· 20 mL). The organic layer was dried (Na2SO4), fil-
tered and concentrated. The crude product was purified
on a semi-preperative HPLC system (40 to 60% B) and
lyopholized to obtain the title compounds as amorphous
solids.
4.4.3.17. N,N0-a,a0-di-tert-Boc-(3,30(1,4-phenylene)diprop-
2-yn-1-amide)-valine (11C). Yield: 140 mg (0.23 mmol,
77%). Rf = 0.75 (1:10; MeOH/DCM; v/v). ESI-MS (m/
1
z) calcd for C32H46N4O6 + Na+: 605.3, obsd 605.6. H
NMR (400 MHz, CDCl3): d 7.30 (s, 4H), 6.70 (s, 2H),
5.26 (br d, 2H), 4.32–4.19 (m, 4H), 4.04–3.96 (m, 2H),
2.20–2.02 (m, 2H), 1.44 (s, 18H), 1.01–0.90 (m, 12H).
13C NMR (100 MHz, CDCl3): d 171.5, 156.0 (2· C),
131.5 (4· CH), 122.5, 86.4, 82.8, 79.9 (4· C), 59.9
(CH), 30.9 (CH3), 29.8 (CH2), 28.2 (3· CH3), 19.2 (CH).
4.4.3.18. N,N0-a,a0-di-tert-Boc-(3,30(1,4-phenylene)-
diprop-2-yn-1-amide)-amino butaric acid (11D). Yield:
135 mg (0.23 mmol, 78%). Rf = 0.45 (EtOAc). ESI-MS
(m/z) calcd for C30H42N4O6 + Na+: 577.8, obsd 577.4.
1H NMR (400 MHz, DMSO-d6): d 7.38 (s, 4H), 6.80
(br s, 2H), 4.10 (d, 4H, J = 5.2 Hz), 2.93–2.89 (m, 4H),
2.12–2.05 (m, 4H), 1.66–1.53 (m, 4H), 1.36 (s, 18H).
13C NMR (150 MHz, CDCl3): d 172.9, 156.8 (2· C),
132.8 (4· CH), 123.6, 90.4, 82.2, 78.7 (4· C), 56.1,
33.8, 29.8 (3· CH2), 29.5 (3· CH3), 26.9 (CH2).
4.4.4.1. Compound IA. Yield after RP-HPLC purifica-
tion: 3.0 mg (3.6 lmol, 12%). LC/MS analysis: Rt
7.00 min (gradient 30–90% B) and Rt 3.75 min (gradient
50–90% B). ESI-MS (m/z) calcd for C46H44F2N8O6 + H+:
1
842.9, obsd 843.5. H NMR (600 MHz, DMSO): d 8.82
(s, 1 H), 8.54 (s, 1H), 8.36 (t, 1H, J = 5.5 Hz), 7.93 (t,
1H, J = 5.7 Hz), 7.61 (d, 2H, J = 8.4 Hz), 7.47–7.33 (m,
9H), 7.21–7.06 (m, 6H), 6.15 (t, 1H, J = 5.5 Hz), 5.54
(s, 2H), 5.31 (t, 1H, J = 5.2 Hz), 4.26 (m, 4H), 4.10 (d,
2H, J = 5.4 Hz), 3.63–3.61 (m, 4H), 3.32–3.30 (m, 2H),
3.13–3.09 (m, 2H), 1.30 (t, 3H, J = 7.1 Hz), 1.08 (t, 3H,
J = 7.2 Hz). HRMS m/z calcd for C46H44F2N8O6 + H+:
843.34246, obsd 843.34254.
4.4.3.19. N,N0-a,a0-di-tert-Boc-(3,30(1,3-phenylene)-
diprop-2-yn-1-amide)-amino hexanoic acid (11E). Yield:
64 mg (0.11, 35%). Rf = 0.40 (EtOAc). ESI-MS (m/z)
calcd for C34H50N4O2 + H+: 611.8, obsd 611.3. 1H
NMR (400 MHz, DMSO-d6):
d
8.30 (t, 2H,
J = 5.2 Hz), 7.37 (s, 4H), 5.75 (t, 2H, J = 2.4 Hz), 4.09
(t, 4H, J = 2.8 Hz), 2.86 (q, 4H, J = 6.8 Hz), 2.07 (t,
4H, J = 7.6 Hz), 1.51–1.43 (m, 4H,), 1.35 (s, 18H, tBu
Boc), 1.36–1.31 (m, 4H), 1.23–1.16 (m, 4H). 13C NMR
(150 MHz, DMSO-d6): d 171.9, 155.5 (2· C), 131.5 (4·
CH), 122.2, 89.2, 80.9, 77.2 (4· C), 35.0, 29.2, 28.5 (3·
CH2), 28.2 (3x CH3), 25.9, 24.8 (2· CH2).
4.4.4.2. Compound IB. Yield after RP-HPLC purifica-
tion: 4.3 mg (5.0 lmol, 17%). LC/MS analysis: Rt
7.37 min (gradient 30–90% B) and Rt 4.23 min (gradient
50–90% B). ESI-MS (m/z) calcd for C47H46F2N8O6 + H+:
1
856.9, obsd 857.5. H NMR (600 MHz, DMSO): d 8.82
4.4.3.20. N,N0-a,a0-di-tert-Boc-(3,30(1,3-phenylene)-
diprop-2-yn-1-amide)-proline (11F). Yield: 172 mg
(0.30 mmol, 99%). Rf = 0.40 (EtOAc). ESI-MS (m/z)
calcd for C32H42N4O6 + H+: 579.4, obsd 579.1. 1H
NMR (400 MHz, DMSO-d6): d 8.42 (t, 2H, J = 2.8 Hz),
7.35 (m, 4H), 4.12 (d, 4H, J = 5.6 Hz), 4.05–4.02 (m,
2H), 3.43–3.31 (m, 4H), 3.30–3.20 (m, 2H), 2.17–2.01
(s, 1H), 8.53 (s, 1H), 8.37 (t, 1H, J = 5.3 Hz), 7.86 (d,
1H, J = 7.8 Hz), 7.57 (d, 2H, J = 8.4 Hz), 7.48–7.42 (m,
4H), 7.35–7.30 (m, 5H), 7.28–7.05 (m, 6H), 6.68 (br s,
1H), 5.54 (s, 2H), 5.31 (t, 1H, J = 5.1 Hz), 4.36 (d, 1H,
J = 13.2 Hz), 4.29–4.22 (m, 4H), 4.08 (d, 2H,
J = 1.9 Hz), 3.68–3.53 (m, 4H), 3.12–3.06 (m, 2H), 1.33
(t, 3H, J = 7.1 Hz), 1.25–1.20 (m, 3H), 1.03 (t, 3H,

K. M. Bonger et al. / Bioorg. Med. Chem. 16 (2008) 3744–3758
3755
J = 7.2 Hz). HRMS m/z calcd for C47H46F2N8O6 + H+:
857.35811, obsd 857.35817.
4.4.4.7. Compound IG. Yield after RP-HPLC purifica-
tion: 2.0 mg (2.2 lmol, 7%). LC/MS analysis: Rt 7.41 min
(gradient 30–90% B) and Rt 4.27 min (gradient 50–90%
B). ESI-MS (m/z) calcd for C50H50F2N8O6 + H+: 897.0,
obsd 897.7. 1H NMR (600 MHz, DMSO): d 8.84 (s,
1H), 8.51 (s, 1H), 8.27 (t, 1H, J = 3.6 Hz), 7.59 (d, 2H,
J = 9.0 Hz), 7.43–7.35 (m, 8H), 7.21–7.12 (m, 7H), 6.15
(t, 1H, J = 3.6 Hz), 5.55 (s, 2H), 5.31 (t, 1H,
J = 5.0 Hz), 4.38–4.26 (m, 4H), 4.12–3.98 (m, 3H),
3.62–3.55 (m, 3H), 3.30–3.23 (m, 1H), 3.14–3.08 (m,
3H), 2.52–2.49 (m, 1H), 2.23–2.20 (m, 2H), 1.55–1.52
(m, 1H), 1.30 (t, 3H, J = 7.2 Hz), 1.29–1.17 (m, 3H),
1.05 (t, 3H, J = 7.2 Hz). HRMS m/z calcd for
C50H50F2N8O6 + H+: 897.38941, obsd 897.38932.
4.4.4.3. Compound IC. Yield after RP-HPLC purifica-
tion: 5.1 mg (5.8 lmol, 19%). LC/MS analysis: Rt
8.02 min (gradient 30–90% B) and Rt 4.82 min (gradient
50–90% B). ESI-MS (m/z) calcd for C49H50F2N8O6 + H+:
1
885.0, obsd 885.6. H NMR (600 MHz, DMSO): d 8.80
(s, 1H), 8.66 (s, 1H), 8.45 (t, 1H, J = 5.4 Hz), 7.88 (m,
1H), 7.60 (d, 2H, J = 9.0 Hz), 7.46–7.07 (m, 15H), 6.27
(s, 1 H), 5.54 (s, 2H), 5.31 (t, 1H, J = 4.9 Hz), 4.38 (d,
1H, J = 13.2 Hz), 4.28–4.20 (m, 3H), 4.14–4.03 (m, 4H),
3.69–3.58 (m, 4H), 3.20 (d, 2H, J = 16.2 Hz), 3.11–3.08
(m, 2H), 1.85–1.83 (m, 1H), 1.30 (t, 3H, J = 7.1 Hz),
1.05 (t, 3H, J = 7.2 Hz), 0.72 (d, 3H, J = 6.6 Hz), 0.65
(d, 3H, J = 6.6 Hz). HRMS m/z calcd for
C49H50F2N8O6 + H+: 885.38941, obsd 885.38886.
4.4.5. General procedure for coupling of pharmacophore 3
with dimeric spacers 7A–G, 9A–G or 11A–G affording
IIIA–G, IIA–G or IVA–G. Boc protected compounds
7A–G, 9A–G or 11A–G were subjected to a solution of
1:1 DCM/TFA v/v + 1% TIS for 18 h. The volatiles were
evaporated and the compounds were purified with semi-
preparative HPLC system (0–30% B). Accordingly,
15 lmol of amine (8A–G, 10A–G or 12A–G) was dis-
solved in 100 lL of DMF and added to a solution con-
taining pharmacophore 36,7 (33 lmol, 14.8 mg), BOP
(39 lmol, 17.6 mg) and DiPEA (120 lmol, 20.4 lL) in
300 lL DMF. The reaction mixture was stirred at rt
for 16 h and diluted with a mixture of DCM and MeOH
(9:1; v/v, 20 mL). The organic layer was successively
washed with water (3· 10 mL), 10% aq NaHCO3 (3·
10 mL) and brine (1· 20 mL). The organic layer was
dried (Na2SO4), filtered and concentrated. The crude
product was purified on a semi-preparative HPLC sys-
tem (40–60% B) and lyopholized to obtain the title com-
pounds as amorphous solids.
4.4.4.4. Compound ID. Yield after RP-HPLC purifi-
cation: 5.8 mg (6.7 lmol, 22%). LC/MS analysis: Rt
7.15 min (gradient 30–90% B) and Rt 3.71 min (gradient
50–90% B). ESI-MS (m/z) calcd for C48H48F2N8O6,
1
+ H+: 871.0, obsd 871.7. H NMR (600 MHz, DMSO):
d 8.79 (s, 1H), 8.84 (s, 1H), 8.27 (t, 1H, J = 3.6 Hz), 7.64
(m, 1H, J = 3.6 Hz), 7.59 (d, 2H, J = 8.4 Hz), 7.45 (d,
2H, J = 8.4 Hz), 7.39–7.28 (m, 7H), 7.19–7.07 (m, 6H),
6.49 (br s, 1H), 5.53 (s, 2H), 5.31 (t, 1H, J = 4.8 Hz),
4.32–4.26 (m, 4H), 4.10–4.07 (m, 2H), 3.54 (s, 2H),
3.12–3.00 (m, 4H), 2.90 (q, 2H, J = 6.0 Hz), 2.13–2.05
(m, 2H), 1.52–1.49 (m, 2H), 1.31 (t, 3H, J = 7.2 Hz),
1.05 (t, 3H, J = 7.2 Hz). HRMS m/z calcd for
C48H48F2N8O6, + H+: 871.37376, obsd 871.37354.
4.4.4.5. Compound IE. Yield after RP-HPLC purifica-
tion: 5.3 mg (5.9 lmol, 20%). LC/MS analysis: Rt
7.52 min (gradient 30–90% B) and Rt 4.42 min (gradient
50–90% B). ESI-MS (m/z) calcd for C50H52F2N8O6 + H+:
4.4.5.1. Compound IIA. Yield after RP-HPLC purifica-
tion: 1.83 mg (1.1 lmol, 8%). LC/MS analysis: Rt
8.43 min (gradient 30–90% B) and Rt 5.74 min (gradient
50–90% B). ESI-MS (m/z) calcd for C86H82F4N16
O12 + H+: 1607.9, obsd 1607.8. 1H NMR (600 MHz,
DMSO-d6): d 8.81 (s, 2H), 8.56 (s, 2H), 8.36 (br t, 2H),
7.92 (br t, 2H), 7.63–7.55 (m, 4H), 7.52–7.28 (m, 12H),
7.21–7.02 (m, 12H), 6.17 (s, 2), 5.53 (s, 4H), 5.31 (t, 2H,
J = 4.8 Hz), 4.32–4.22 (m, 8H), 4.18–4.12 (m, 4H),
3.59–3.56 (m, 8H), 3.18–3.08 (m, 8H), 1.29 (t, 6H,
J = 7.0 Hz), 1.05 (t, 6H, J = 7.2 Hz). HRMS m/z
calcd for C86H82F4N16O12 + 2H+: 804.31899, obsd
804.31909.
1
899.0, obsd 899.8. H NMR (600 MHz, DMSO): d 8.80
(s, 1H), 8.55 (s, 1H), 8.27 (t, 1H, J = 3.6 Hz), 6.85 (br s,
1H), 7.59 (d, 2H, J = 8.4 Hz), 7.44 (d, 2H, J = 8.4 Hz),
7.39–7.32 (m, 7H), 7.18–7.06 (m, 6H), 6.15 (t, 1H,
J = 6.0 Hz), 5.53 (s, 2H), 5.31 (t, 1H, J = 5.4 Hz), 4.33
(s, 2H), 4.28 (q, 2H, J = 7.2 Hz), 4.09–4.07 (m, 2H),
3.54 (s, 2H), 3.13–3.00 (m, 4H), 2.79 (q, 2H,
J = 6.6 Hz), 2.09–2.02 (m, 2H), 1.50–1.39 (m, 2H),
1.30–1.13 (m, 7H), 1.06 (t, 3H, J = 7.2 Hz). HRMS m/z
calcd for C50H52F2N8O6 + H+: 899.40506, obsd
899.40559.
4.4.4.6. Compound IF. Yield after RP-HPLC purifica-
tion: 7.03 mg (8.0 lmol, 27%). LC/MS analysis: Rt
7.56 min (gradient 30–90% B) and Rt 4.21 min (gradient
50–90% B). ESI-MS (m/z) calcd for C49H48F2N8O6 + H+:
4.4.5.2. Compound IIB. Yield after RP-HPLC purifica-
tion: 2.09 mg (1.3 lmol, 9%). LC/MS analysis: Rt
8.80 min (gradient 30–90% B) and Rt 6.25 min (gradient
50–90% B). ESI-MS (m/z) calcd for C88H86F4N16
O12 + H+: 1635.8, obsd 1636.9. 1H NMR (600 MHz,
DMSO-d6): d 8.79 (s, 2H), 8.55 (s, 2H), 8.32 (br s, 2H),
7.86 (br s, 2H), 7.62–7.28 (m, 16H), 7.19–7.02 (m, 12H),
6.16 (br s, 2H), 5.53 (s, 4H), 5.31 (t, 2H, J = 4.8 Hz),
4.36–4.30 (m, 2H), 4.28–4.22 (m, 8H), 4.18–4.07 (m,
4H), 3.66–3.55 (m, 4H), 3.18–3.05 (m, 8H), 1.32–1.27
(m, 12H), 1.05 (m, 6H, J = 7.2 Hz). HRMS m/z
calcd for C88H86F4N16O12 + 2H+: 816.33464, obsd
818.33529.
1
883.0, obsd 883.4. H NMR (600 MHz, DMSO): d 8.81
(s, 1H), 8.64 (s, 1H), 8.39 (t, 1H, J = 3.6 Hz), 7.60 (d,
2H, J = 9.0 Hz), 7.45–7.03 (m, 15H), 6.30 (t, 1H,
J = 6.0 Hz), 5.56 (s, 2H), 5.31 (t, 1H, J = 4.8 Hz), 4.38
(br d, 1H), 4.30–4.25 (m, 2H), 4.12–4.04 (m, 4H), 3.70–
3.59 (m, 4H), 3.12–3.06 (m, 4H), 1.90–1.85 (m, 1H),
1.72–1.65 (m, 1H), 1.62–1.52 (m, 2H), 1.29 (t, 3H,
J = 7.2 Hz), 1.04 (t, 3H, J = 7.2 Hz). HRMS m/z
calcd for C49H48F2N8O6 + H+: 883.37376, obsd
883.37334.

3756
K. M. Bonger et al. / Bioorg. Med. Chem. 16 (2008) 3744–3758
4.4.5.3. Compound IIC. Yield after RP-HPLC purifica-
50–90% B). ESI-MS (m/z) calcd for C94H94F4N16
O12 + H+: 1715.9, obsd 1716.1. 1H NMR (600 MHz,
DMSO-d6): d 8.81 (s, 2H), 8.49 (s, 2H), 8.27 (t, 2H,
J = 3.6 Hz), 7.59 (d, 4H, J = 9.0 Hz), 7.50–7.39 (m, 4H),
7.38–7.32 (m, 6H), 7.20–7.07 (m, 14H), 6.13 (t, 2H,
J = 5.4 Hz), 5.55 (s, 4H), 4.34–4.24 (m, 8H), 4.13–4.08
(m, 4H), 4.03 (br d, 2H), 3.66–3.58 (m, 6H), 3.28–3.25
(m, 1H), 3.17–3.07 (m, 3H), 2.56–2.50 (m, 2H), 2.28–2.17
(m, 4H), 2.53 (br d, 2H), 1.29 (t, 6H, J = 7.2 Hz), 1.28–
1.20 (m, 6H), 1.05 (t, 6H, J = 7.2 Hz). HRMS m/z calcd
for C94H94F4N16O12 + 2H+: 858.36594, obsd 858.36542.
tion: 1.50 mg (0.9 lmol, 6%). LC/MS analysis: Rt
9.54 min (gradient 30–90% B) and Rt 7.38 min (gradient
50–90% B). ESI-MS (m/z) calcd for C92H94F4N16
O12 + H+: 1691.9, obsd 1693.1. 1H NMR (600 MHz,
DMSO-d6): d 8.80 (s, 2H), 8.66 (s, 2H), 8.45 (br s, 2H),
7.78 (br s, 2H), 7.60 (d, 4H, J = 9.0 Hz), 7.53–7.05 (m,
24H), 6.12 (s, 2H), 5.54 (s, 4H), 5.31 (t, 2H,
J = 5.4 Hz), 4.38 (br d, 2H), 4.31–4.02 (m, 12), 3.68–
3.55 (m, 4H), 3.17–3.05 (m, 8H), 1.89–1.82 (m, 2H),
1.28 (t, 6H, J = 7.2 Hz), 0.97 (t, 6H, J = 7.2 Hz), 0.73
(br d, 6H), 0.65 (br d, 6H). HRMS m/z calcd for
C92H94F4N16O12 + 2H+: 846.36594, obsd 846.36597.
4.4.5.8. Compound IIIA. Yield after RP-HPLC purifica-
tion: 3.32 mg (2.1 lmol, 14%). LC/MS analysis: Rt
8.02 min (gradient 30–90% B) and Rt 4.90 min (gradient
50–90% B). ESI-MS (m/z) calcd for C86H82F4N16
O12 + H+: 1607.7, obsd 1607.8. 1H NMR (600 MHz,
DMSO-d6): d 8.81 (s, 2H), 8.56 (s, 2H), 8.36 (br t, 2H),
7.97–7.90 (m, 2H), 7.61 (d, 4H, J = 8.4 Hz), 7.46–7.32
(m, 12H), 7.20–7.15 (m, 12H), 6.17 (br t, 2H), 5.53 (s,
4H), 5.31 (t, 2H, J = 3.6 Hz), 4.27–4.25 (m, 8H), 4.17–
4.08 (m, 4H), 3.66–3.58 (m, 8H), 3.18–3.04 (m, 8H), 1.30
(t, 6H, J = 7.2 Hz), 1.05 (t, 6H, J = 7.1 Hz). HRMS m/z
calcd for C86H82F4N16O12 + 2H+: 804.31899, obsd
804.31900.
4.4.5.4. Compound IID. Yield after RP-HPLC purifica-
tion: 1.26 mg (0.8 lmol, 5%). LC/MS analysis: Rt 8.57 min
(gradient 30–90% B) and Rt 5.88 min (gradient 50–90% B).
ESI-MS (m/z) calcd for C90H90F4N16O12 + H+: 1663.8,
1
obsd 1663.9. H NMR (600 MHz, DMSO-d6): d 8.79 (s,
2H), 8.52 (s, 2H), 8.27 (br t, 2H), 7.68–7.58 (m, 6H, 7.48–
7.38 (m, 6H), 7.35–7.30 (m, 2H), 7.20–7.12 (m, 10H),
7.08–7.01 (m, 6H), 6.15 (br t, 2H), 5.52 (s, 4H), 5.31 (t,
2H, J = 5.4 Hz), 4.32–4.26 (m, 7H), 4.15–4.10 (m, 5H),
3.56–3.49 (m, 4H), 3.15–3.02 (m, 8H), 2.93–2.88 (m, 4H),
1.97–1.90 (m, 4H), 1.57–1.50 (m, 4H), 1.28 (t, 6H,
J = 7.8 Hz), 1.05 (t, 6H, J = 7.2 Hz). HRMS m/z calcd
for C90H90F4N16O12 + 2H+: 832.35029, obsd 832.35010.
4.4.5.9. Compound IIIB. Yield after RP-HPLC purifica-
tion: 1.13 mg (0.7 lmol, 5%). LC/MS analysis: Rt 8.62 min
(gradient 30–90% B) and Rt 5.71 min (gradient 50–90% B).
ESI-MS (m/z) calcd for C88H86F4N16O12 + H+: 1635.8,
4.4.5.5. Compound IIE. Yield after RP-HPLC purifica-
tion: 1.42 mg (0.8 lmol, 6%). LC/MS analysis: Rt
8.89 min (gradient 30–90% B) and Rt 6.48 min (gradient
50–90% B). ESI-MS (m/z) calcd for C94H98F4N16
O12 + H+: 1719.9, obsd 1721.1. 1H NMR (600 MHz,
DMSO-d6): d 8.80 (s, 2H), 8.55 (s, 2H), 8.30 (br s, 2H),
7.80 (br s, 2H), 7.59 (br d, 4H), 7.50–7.48 (m, 8H), 7.37–
7.30 (m, 2H), 7.38–7.03 (m, 14H), 6.15 (br t, 2H), 5.53 (s,
4H), 5.31 (t, 2H, J = 5.4 Hz), 4.37–4.22 (m, 8H), 4.16–
4.11 (m, 4H), 3.60–3.49 (br s, 4H), 3.15–2.97 (m, 8H),
2.82–2.73 (m, 4H), 2.12–2.05 (m, 4H), 1.48–1.40 (m, 4H),
1.30–1.14 (m, 14H), 1.05 (t, 6H, J = 6.6 Hz). HRMS m/z
calcd for C94H98F4N16O12 + 2H+: 860.38159, obsd
860.38187.
1
obsd 1636.9. H NMR (600 MHz, DMSO-d6): d 8.84 (s,
2H), 8.51 (s, 2H), 8.29 (t, 2H, J = 4.8 Hz), 7.59 (d, 4H,
J = 9.0 Hz), 7.43–7.35 (m, 12H), 7.21–7.12 (m, 12H), 6.15
(t, 2H, J = 4.8 Hz), 5.55 (s, 4H,), 5.31 (t, 2H,
J = 5.10 Hz), 4.40–4.25 (m, 10H), 4.12–3.99 (d, 3H),
3.68–3.55 (m, 4H), 3.30–3.20 (m, 4H), 3.16–3.07 (m, 4H),
1.32–28 (m, 12H), 1.05 (t, 6H, J = 7.2 Hz). HRMS m/z
calcd for C88H86F4N16O12 + 2H+: 818.33464, obsd
818.33554.
4.4.5.10. Compound IIIC. Yield after RP-HPLC purifi-
cation: 3.56 mg (2.1 lmol, 14%). LC/MS analysis: Rt
9.61 min (gradient 30–90% B) and Rt 7.04 min (gradient
50–90% B). ESI-MS (m/z) calcd for C92H94F4N16
O12 + H+: 1691.9, obsd 1693.1. 1H NMR (600 MHz,
DMSO-d6): d 8.82 (s, 2H), 8.55 (s, 2H), 8.49 (br t, 2H),
7.75 (d, 2H, J = 9.0 Hz), 7.60 (d, 4H, J = 9.0 Hz), 7.48–
7.40 (m, 4H), 7.39 (d, 4H, J = 9.0 Hz), 7.36–7.07 (m,
16H), 6.14 (br t, 2H), 5.54 (s, 4H), 5.31 (t, 2H,
J = 3.6 Hz), 4.37 (br d, 2H), 4.30–4.05 (m, 8H), 3.62 (dd,
4H, J = 63.6 Hz, J = 19.8 Hz), 3.20 (d, 4H, J = 16.8 Hz),
3.15–3.04 (m, 8H), 1.87–1.78 (m, 2H), 1.29 (t, 6H,
J = 7.1 Hz), 1.04 (t, 6H, J = 7.1 Hz), 0.71 (d, 6H, J =
6.6 Hz), 0.63 (d, 6H, J = 6.6 Hz). HRMS m/z calcd for
C92H94F4N16O12 + 2H+: 846.36594, obsd 846.36585.
4.4.5.6. Compound IIF. Yield after RP-HPLC purifica-
tion: 1.55 mg (0.9 lmol, 6%). LC/MS analysis: Rt
9.76 min (gradient 30–90% B) and Rt 7.51 min (gradient
50–90% B). ESI-MS (m/z) calcd for C92H90F4N16
O12 + H+: 1687.8, obsd 1687.4. 1H NMR (600 MHz,
DMSO-d6): d 8.78 (s, 2H), 8.52 (s, 2H), 8.28 (t, 2H),
7.62 (d, 1H, J = 8.4 Hz), 7.57 (d, 4H, J = 8.4 Hz), 7.48–
7.39 (m, 6H), 7.35 (d, 4H, J = 8.4 Hz), 7.30–7.19 (m,
6H), 7.18–7.05 (m, 4H), 7.03–6.95 (m, 3H), 6.15 (t, 2H,
J = 5.4 Hz), 5.53 (s, 4H), 5.31 (t, 2H), 4.35–4.28 (m.
2H), 4.24 (q, 4H, J = 6.6 Hz), 4.15–4.02 (m, 5H), 3.67–
3.40 (m, 8H), 3.15–3.05 (m, 4H), 2.99 (s, 4H), 1.92–1.80
(m, 2H), 1.72–1.63 (m, 2H), 1.62–1.42 (m, 4H), 1.28 (m,
6H, J = 6.6 Hz), 1.04 (t, 6H, J = 7.2 Hz). HRMS m/z
calcd for C92H90F4N16O12 + 2H+: 844.35029, obsd
844.34997.
4.4.5.11. Compound IIID. Yield after RP-HPLC purifi-
cation: 3.35 mg (2.0 lmol, 14%). LC/MS analysis: Rt
8.08 min (gradient 30–90% B) and Rt 5.01 min (gradient
50–90% B). ESI-MS (m/z) calcd for C90H90F4N16
O12 + H+: 1663.8, obsd 1665.0. 1H NMR (600 MHz,
DMSO-d6): d 8.81 (s, 2H), 8.57 (s, 2H), 8.29 (br t, 2H),
7.67 (br t, 2H), 7.60 (d, 4H, J = 9.0 Hz), 7.50–7.30 (m,
4.4.5.7. Compound IIG. Yield after RP-HPLC purifica-
tion: 2.87 mg (1.7 lmol, 11%). LC/MS analysis: Rt
9.72 min (gradient 30–90% B) and Rt 7.56 min (gradient

K. M. Bonger et al. / Bioorg. Med. Chem. 16 (2008) 3744–3758
3757
16H), 7.25–7.05 (m, 12H), 6.19 (br t, 2H), 5.53 (s, 4H), 5.31
(br t, 2H), 4.36–4.28 (m, 7H), 4.16–4.04 (m, 5H), 3.60–3.49
(m, 4H), 3.15–3.05 (m, 8H), 2.92–2.76 (m. 4), 2.18–2.12 (m,
4H), 1.56–1.48 (m, 4H), 1.30 (t, 6H, J = 7.08 Hz), 1.04 (t,
4.4.5.16. Compound IVB. Yield after RP-HPLC purifi-
cation: 2.01 mg (1.2 lmol, 8%). LC/MS analysis: Rt
8.65 min (gradient 30–90% B) and Rt 5.82 min (gradient
50–90% B). ESI-MS (m/z) calcd for C88H86F4N16
O12 + H+: 1635.8, obsd 1637.2. 1H NMR (600 MHz,
DMSO-d6): d 8.79 (s, 2H), 8.52 (s, 2H), 8.31 (t, 2H,
J = 3.6 Hz), 7.60 (d, 4H, J = 9.0 Hz), 7.50–7.38 (m, 12H),
7.20–7.05 (m, 12H), 6.13 (t, 2HJ = 4.2 Hz), 5.53 (s, 4H),
5.31 (t, 2H, J = 3.6 Hz), 4.37–4.24 (m, 2H), 4.18–4.06 (m,
8H), 4.08 (d, 4H), 3.69–3.55 (m, 4H), 3.17–3.06 (m, 8H),
1.32–1.27 (m, 12H), 1.05 (t, 6H, J = 7.1 Hz). HRMS m/z
calcd for C88H86F4N16O12 + 2H+: 818.33464, obsd
818.33469.
6H,
J = 7.14 Hz).
HRMS
m/z
C90H90F4N16O12 + 2H+: 832.35029, obsd 832.34990.
calcd
for
4.4.5.12. Compound IIIE. Yield after RP-HPLC purifi-
cation: 2.53 mg (1.5 lmol, 10%). LC/MS analysis: Rt
8.61 min (gradient 30–90% B) and Rt 5.78 min (gradient
50–90% B). ESI-MS (m/z) calcd for C94H98F4N16
O12 + H+: 1719.9, obsd 1720.1. 1H NMR (600 MHz,
DMSO-d6): d 8.80 (s, 2 H), 8.54 (s, 2H), 8.30 (br t, 2H),
8.26 (br t, 2H), 7.58 (d, 4H, J = 8.4 Hz), 7.50–7.28 (m,
12H), 7.22–7.20 (m, 16H), 6.16 (br t, 2H), 5.53 (s, 4H),
5.31 (br t, 2H), 4.33 (s, 4H), 4.27 (q, 4H, J = 6.6 Hz),
4.12–4.00 (m, 4H), 3.58–3.50 (m, 4H), 3.15–2.98 (m,
8H), 2.82–2.75 (m, 4H), 2.25–1.94 (m, 4H), 1.52–1.38
(m, 4H), 1.30 (t, 6H, J = 6.6 Hz), 1.22–1.12 (m, 8H),
1.05 (t, 6H, J = 7.1 Hz). HRMS m/z calcd for
C94H98F4N16O12 + 2H+: 860.38159, obsd 860.38145.
4.4.5.17. Compound IVC. Yield after RP-HPLC purifi-
cation: 1.31 mg (0.8 lmol, 5%). LC/MS analysis: Rt
9.77 min (gradient 30–90% B) and Rt 7.36 min (gradient
50–90% B). ESI-MS (m/z) calcd for C92H94F4N16
O12 + H+: 1691.9, obsd 1693.3. 1H NMR (600 MHz,
DMSO-d6): d 8.80 (s, 2H), 8.52 (s, 2H), 8.48–8.42 (m,
2H), 7.72 (d, 2H, J = 9.0 Hz), 7.60 (d, 4H, J = 9.0 Hz),
7.48–7.02 (m, 24H), 6.11 (t, 2H, J = 4.2 Hz), 5.54 (s, 4H),
5.31 (t, 2H, J = 3.6 Hz), 4.38 (br d, 2H), 4.30–4.06 (m,
12H), 3.60 (dd, 4H, J = 63.6 Hz, J = 19.8 Hz), 3.22–3.05
(m, 8H), 1.85–1.78 (m, 2H), 1.29 (t, 6H, J = 6.6 Hz), 1.04
(t, 6H, J = 7.2 Hz), 0.71 (d, 6H, J = 6.6 Hz), 0.63 (d, 6H,
J = 6.6 Hz). HRMSm/z calcdforC92H94F4N16O12 + 2H+:
846.36594, obsd 846.36576.
4.4.5.13. Compound IIIF. Yield after RP-HPLC purifi-
cation: 1.59 mg (0.9 lmol, 6%). LC/MS analysis: Rt
8.93 min (gradient 30–90% B) and Rt 6.43 min (gradient
50–90% B). ESI-MS (m/z) calcd for C92H90F4N16
O12 + H+: 1687.8, obsd 1689.0. 1H NMR (600 MHz,
DMSO-d6): d 8.82 (s, 2H), 8.60 (s, 2H), 8.35 (br s, 2H),
7.90 (m, 2H), 7.60 (d, 4H, J = 9.0 Hz), 7.49–7.06 (m,
24H), 6.21 (br t, 2H), 5.56 (s, 4H), 5.31 (t, 2H,
J = 4.8 Hz), 4.38 (br d, 2H), 4.34–4.22 (m, 4H), 4.12–4.00
(m, 5H), 3.69–3.60 (m, 4H), 3.56 (s, 4H), 3.16–3.05 (m,
4H), 1.95–1.52 (br m, 8H), 1.28 (t, 6H, J = 7.2 Hz), 1.04
(t, 6H, J = 7.2 Hz). HRMS m/z calcd for C92H90F4
N16O12 + 2H+: 844.35029, obsd 844.35087.
4.4.5.18. Compound IVD. Yield after RP-HPLC purifi-
cation: 2.44 mg (1.5 lmol, 10%). LC/MS analysis: Rt
7.95 min (gradient 30–90% B) and Rt 4.92 min (gradient
50–90% B). ESI-MS (m/z) calcd for C90H90F4N16
O12 + H+: 1663.8, obsd 1665.1. 1H NMR (600 MHz,
DMSO-d6): d 8.79 (s, 2H), 8.54 (s, 2H), 8.26 (t, 2H,
J = 5.4 Hz), 7.64 (t, 2H, J = 5.4 Hz), 7.60 (d, 4H,
J = 9.0 Hz), 7.49–7.39 (m, 14H), 7.20–6.97 (m, 10H),
6.17 (t, 2H, J = 5.4 Hz), 5.53 (s, 4H), 5.31 (br t, 2H),
4.32–4.25 (m, 8H), 4.15–4.05 (m, 6H), 3.53 (s, 4H),
3.13–3.02 (m, 8H), 2.90–2.86 (m. 4H), 2.03–1.98 (m,
4H), 1.56–1.49 (m, 4H), 1.30 (t, 6H, J = 7.2 Hz), 1.04 (t,
6H, J = 7.2 Hz). HRMS m/z calcd for C90H90F4
N16O12 + 2H+: 832.35029, obsd 832.35036.
4.4.5.14. Compound IIIG. Yield after RP-HPLC puri-
fication: 2.57 mg (1.5 lmol, 10%). LC/MS analysis: Rt
8.50 min (gradient 30–90% B) and Rt 6.03 min (gradient
50–90% B). ESI-MS (m/z) calcd for C94H94F4N16
O12 + H+: 1715.9, obsd 1717.1. 1H NMR (600 MHz,
DMSO-d6): d 8.82 (s, 2H), 8.69 (s, 2H), 8.26 (s, 2H),
7.59 (d, 4H, J = 8.4 Hz), 7.51–7.35 (m, 12H), 7.25–7.06
(m, 12H), 6.15 (t, 2H, J = 5.4 Hz), 5.55 (s, 4H), 5.31
(br t, 2H), 4.37–4.22 (m, 8H), 4.15–3.97 (m, 6H), 3.67–
3.56 (m, 6H), 3.28–3.23 (m, 2H), 2.58–2.50 (m, 2H),
2.29–2.12 (m, 4H), 1.78–1.70 (m, 2H), 1.58–1.50 (m,
2H), 1.31–1.13 (m, 8H), 1.05 (t, 6H, J = 6.6 Hz). HRMS
m/z calcd for C94H94F4N16O12 + 2H+: 858.36594, obsd
858.36643.
4.4.5.19. Compound IVE. Yield after RP-HPLC purifi-
cation: 2.04 mg (1.2 lmol, 8%). LC/MS analysis: Rt
9.25 min (gradient 30–90% B) and Rt 6.76 min (gradient
50–90% B). ESI-MS (m/z) calcd for C94H98F4N16
O12 + H+: 1719.9, obsd 1720.1. 1H NMR (600 MHz,
DMSO-d6): d 8.80 (s, 2H), 8.54 (s, 2H), 8.27 (br t, 4H),
7.58 (d, 4H, J = 9.0 Hz), 7.55–7.28 (m, 12H), 7.21–7.05
(m, 12H), 6.15 (t, 2H), 5.54 (s, 4H), 5.33 (s, 4H), 4.28 (q,
4H, J = 6.6 Hz), 4.08 (d, 4H, J = 4.8 Hz), 3.56–3.52 (m,
8H), 3.14–3.09 (m, 4H), 3.00 (s, 4H), 2.71–2.65 (m, 4H),
2.04 (t, 4H, J = 9.0 Hz), 1.43–1.38 (m, 4H), 1.31 (t, 6H,
J = 7.2 Hz), 1.26–1.15 (m, 8H), 1.05 (t, 6H, J = 7.2 Hz).
HRMS m/z calcd for C94H98F4N16O12 + 2H+: 871.37256,
obsd 871.37218.
4.4.5.15. Compound IVA. Yield after RP-HPLC purifi-
cation: 2.06 mg (1.3 lmol, 9%). LC/MS analysis: Rt
7.94 min (gradient 30–90% B) and Rt 5.08 min (gradient
50–90% B). ESI-MS (m/z) calcd for C86H82F4
1
N16O12 + H+: 1607.7, obsd 1608.0. H NMR (600 MHz,
DMSO-d6): d 8.79 (s, 2H), 8.52 (s, 2H), 8.32 (br t, 2H),
7.91 (br t, 2H), 7.61 (d, 8H, J = 8.4 Hz), 7.48–7.32 (m,
12H), 7.22–7.05 (m, 12H), 6.14 (br t, 2H), 5.54 (s, 4H),
5.32–4.23 (m, 6H), 4.18–4.05 (m, 8H), 3.63–3.58 (m, 8H),
3.18–3.06 (m, 8H), 1.30 (t, 6H, J = 7.2 Hz), 1.05 (t, 6H,
J = 7.2 Hz).HRMSm/zcalcd forC86H82F4N16O12 + 2H+:
804.31899, obsd 804.31867.
4.4.5.20. Compound IVF. Yield after RP-HPLC purifi-
cation: 2.60 mg (1.5 lmol, 10%). LC/MS analysis: Rt
9.14 min (gradient 30–90% B) and Rt 6.39 min (gradient
50–90% B). ESI-MS (m/z) calcd for C92H90F4N16

3758
K. M. Bonger et al. / Bioorg. Med. Chem. 16 (2008) 3744–3758
O12 + H+: 1687.8, obsd 1688.9. 1H NMR (600 MHz,
DMSO-d6): d 8.81 (s, 2H), 8.51 (s, 2H), 8.27 (br s, 2H),
7.60 (d, 4H, J = 9.0 Hz), 7.49–7.08 (m, 20H), 6.16 (br s,
2H), 5.56 (s, 4H), 5.31 (t, 2H), 4.39–4.32 (m, 2H), 4.30–
4.23 (m, 4H), 4.18–4.00 (m, 5H), 3.70–3.58 (m, 4H), 3.56
(s, 4H), 3.15–3.05 (m, 4H), 2.05–1.96 (m, 2H), 1.95–1.80
(m, 2H), 1.72–1.68 (m, 2H), 1.66–1.50 (m, 2H), 1.29 (t,
6H, J = 7.2 Hz), 1.04 (t, 6H, J = 7.2 Hz). HRMS m/z
calcd for C92H90F4N16O12 + 2H+: 844.35029, obsd
844.35040.
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4.4.5.21. Compound IVG. Yield after RP-HPLC purifi-
cation: 2.04 mg (1.2 lmol, 8%). LC/MS analysis: Rt
9.09 min (gradient 30–90% B) and Rt 6.61 min (gradient
50–90% B). ESI-MS (m/z) calcd for C94H94F4N16
O12 + H+: 1715.9, obsd 1716.2. 1H NMR (600 MHz,
DMSO-d6): d 8.82 (s, 2H), 8.50 (s, 2H), 8.26 (s, 2H), 7.59
(d, 4H, J = 7.8 Hz), 7.57–7.32 (m, 12H), 7.31–7.06 (m,
12H) 6.15 (s, 2H), 5.55 (s, 4H), 5.31 (t, 2H), 4.51–4.23 (m,
8H), 4.18–4.09 (m, 6H), 3.62–3.58 (m, 6H), 3.28–3.20 (m,
2H), 3.18–3.08 (m, 8H), 2.60–2.52 (m, 2H), 2.30–2.12 (m,
4H), 1.65–1.50 (m, 4H), 1.49–1.39 (m, 2H), 1.38–1.23 (m,
8H), 1.05 (t, 6H, J = 7.1 Hz). HRMS m/z calcd for
C94H94F4N16O12 + 2H+: 858.36594, obsd 858.36579.
Acknowledgments
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99/33831, 1999.
Financial support from the Council for Chemical Sci-
ences of The Netherlands Organization for Scientific Re-
search (CW-NWO) is gratefully acknowledged. The
assistance of Koen Thal and Saskia Verkaik with the
preparative HPLC system is greatly appreciated. We
further acknowledge Maurice R. van Loosbroek for per-
forming the GnRHR Luciferase reporter gene assay.
8. Conn, P. M.; Rogers, D. C.; Stewart, J. M.; Niedel, J.;
Sheffield, T. Nature 1982, 296, 653–655.
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