Synthesis and biological activity of N-terminal lipidated and/or fluorescently labeled conjugates of astressin as corticotropin releasing factor antagonists

Article abstract of DOI:10.1016/j.bmc.2004.07.035

This report describes the synthesis of eight N-terminally modified astressin analogs and their biochemical evaluation as corticotropin releasing factor (CRF) antagonists. The lipidated astressin derivatives were tested on rat CRF receptor type 1 and 2α an

Full text of DOI:10.1016/j.bmc.2004.07.035

Bioorganic & Medicinal Chemistry 12 (2004) 5099–5106
Synthesis and biological activity of N-terminal lipidated and/or
fluorescently labeled conjugates of astressin as
corticotropin releasing factor antagonists
b
a
Dirk T. S. Rijkers,a, Jack A. J. den Hartog and Rob M. J. Liskamp
*
aDepartment of Medicinal Chemistry, Utrecht Institute for Pharmaceutical Sciences, Faculty of Pharmaceutical Sciences,
Utrecht University, PO Box 80082, 3508 TB Utrecht, The Netherlands
bSolvay Pharmaceuticals, Research Laboratories, C.J. van Houtenlaan 36, 1381 CP Weesp, The Netherlands
Received 23 March 2004; accepted 16 July 2004
Available online 11 August 2004
Abstract—This report describes the synthesis of eight N-terminally modified astressin analogs and their biochemical evaluation as
corticotropin releasing factor (CRF) antagonists. The lipidated astressin derivatives were tested on rat CRF receptor type 1 and 2a
and were found to be active as CRF antagonists (rCRFR1: pA2 = 7.5–8.3; rCRFR2a: pA2 = 7.5–9.0) with nearly equal activities as
compared to unmodified astressin (rCRFR1: pA2 = 8.3 0.09; rCRFR2a: pA2 = 8.7 0.08).
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
which describe the membrane passage of lipopep-
tides.14,15
Transport of peptide hormones over phospholipid mem-
branes is considered as rather difficult mainly as a result
of their size and highly polar nature.1,2 In the current lit-
erature several strategies have been described for mem-
brane passage of large and polar biomolecules (e.g.,
peptide, proteins, and nucleic acids).3 One strategy for
peptide delivery through biological membranes involves
the covalent coupling of a nontransportable peptide to a
transportable vector peptide.4–8 Among these are the
nuclear transcription activator protein (Tat) encoded
by HIV type 1,9 the Antennapedia homeodomain-
derived peptide penetratin,10 polyarginine-based
oligomers,11 and guanidine-rich peptoids12 or oligo-
carbamates.13 A major limitation of such peptide chime-
ras in case of neuropeptides is the unpredictable
influence of the vector peptide on receptor binding and
(in)activation. However, lipidation of a peptide is a
well-accepted approach to enhance peptide-membrane
interaction and several examples exist in the literature,
Corticotropin releasing factor (CRF) is a peptide amide
of 41 amino acids, which stimulates the release of
adrenocorticotropic hormone (ACTH), b-endorphin
and other pro-opiomelanocortin (POMC) derived prod-
ucts from the anterior pituitary gland and acts within
the brain to modulate a wide range of stress responses.16
Overexpression of CRF is related to psychiatric illnesses
such as major depression and anxiety disorders.17 There-
fore the development of antagonists is of high interest to
obtain insight into the physiological role of CRF in the
body response to stress. With respect to this, approaches
to increase the ability of these antagonists to penetrate
into the desired brain regions, for example, by lipida-
tion, are especially interesting.
The first peptide-based CRF antagonist, a-helical
CRF(9–41),18 formed the basis for an intensive search
for better CRF antagonists, which ultimately led to
the discovery of astressin {cyclo(30–33)[D-Phe12,
Nle21,38, Glu30, Lys33]hCRF(12–41)}.19,20 We decided
to synthesize a series of lipidated astressin conjugates for
future membrane transport studies. First, for these con-
jugates the influence of the N-terminal modification on
CRF antagonistic potency was studied since derivatiza-
tion of a neuropeptide, either at its N/C-terminus or
at residues within the peptide sequence (e.g., serine/
Abbreviations: hCRF: human corticotropin releasing factor; rCRF: rat
corticotropin releasing factor; dans: dansyl; Nle: norleucine; pal: pal-
mitoyl; succ: succinimidyl
Keywords: Antagonists; Peptides and polypeptides; Peptide conjugates;
Solid phase synthesis.
*
Corresponding author. Tel.: +31-30-253-6916/7275; fax: +31-30-253-
6655; e-mail: d.t.s.rijkers@pharm.uu.nl
0968-0896/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.07.035

5100
D. T. S. Rijkers et al. / Bioorg. Med. Chem. 12 (2004) 5099–5106
cysteine), can be to a large extent decisive for its biolog-
ical activity. We found that lipidation of the a-amino
functionality did not alter the potency of astressin as
CRF antagonist.
check the completion of the allyl deprotection and in or-
der to verify that a premature loss of the Fmoc group
had not occurred, a small amount of resin was treated
with trifluoroacetic acid (TFA). Indeed, the Na-Fmoc-
protected linear astressin molecule (6) was found as
the major product according to HPLC and electrospray
ionization mass spectrometry (ESI MS, m/z, found:
3803.90; calcd: 3804.48). Ring closure was carried out
by treatment with benzotriazol-1-yl-oxy-tris-(dimethyl-
amino)phosphonium hexafluorophosphate (BOP)/N-hy-
droxybenzotriazole (HOBt)25 in the presence of N,N-
diisopropylethylamine (DIPEA) in N-methylpyrro-
lidone (NMP). Formation of the lactam bridge was mon-
itored by the Kaiser test26 and was completed after 16h.
2. Results and discussion
2.1. Synthesis
Our intention was to modify the N-terminal a-amino
functionality of astressin with three small hydrophobic
moieties leading to 5b–d, three fatty acid derivatives of
different length, and degree of saturation leading to
5e–g and two lipidated amino acid residues affording
5h–i as are shown in Figure 1. The latter lipidated amino
acids were inspired by the work of Asakuma et al.21 and
Pakalns et al.22 The dansyl moiety in derivatives 5d and
5i was used as a fluorescent probe to monitor the process
of membrane translocation of the corresponding astres-
sin conjugates.
After ring closure, a small portion of the resin was re-
moved and treated with TFA to yield an Fmoc-pro-
tected astressin analog (5c). The remainder of the resin
was treated with 20% piperidine in NMP to remove
the Fmoc group. Subsequently, the resin was divided
into eight portions (one control peptide, seven modified
peptides) and in each portion the N-terminal a-amino
group was modified according to procedures shown in
Table 1.
For a rapid access to these astressin conjugates, our pre-
viously described23 synthesis of astressin was adjusted as
follows (Scheme 1). It was decided that the lactam
bridge had to be introduced before modification of the
N-terminus. Therefore, the a-amino functionality of
the terminal D-Phe12 residue was temporarily protected
with the orthogonal 9-fluorenylmethyloxycarbonyl
(Fmoc) group. It was expected that this group was stable
under the conditions used for allyl deprotection and ring
closure. Allyl-based protecting groups of the glutamic
acid30- and lysine33 side chains could be removed with-
in 2h in the presence of Pd(0) with phenylsilane as scav-
enger24 and was a rapid and very clean reaction. To
Acetylation was complete within 15min using the stand-
ard capping reaction conditions. Coupling of Fmoc-
Ahx-OH (Ahx: 6-aminohexanoic acid) was easy and
after deprotection subsequent coupling of the fluores-
cent dansyl group was performed in the presence of
the mild base N-methylmorpholine (NMM) to avoid di-
substitution as was found in the presence of the stronger
base DIPEA.27 The Kaiser test showed false positive
colored resin beads during the coupling of the dansyl
moiety due to the acidic character of the sulfonamide
Figure 1. Chemical structures of the N-terminal astressin modifications: (a) none, free N-terminus (H); (b) acetyl (Ac); (c) 9-fluorenylmethyloxy-
carbonyl (Fmoc); (d) N-dansyl-6-aminohexanoyl (Dans-Ahx); (e) tetradecanoyl (Myr); (f) eicosanoyl (Ara); (g) cis-9-octadecenoyl (oleic); (h)
(PalO)2Gluinv-Succ-OH; (i) (PalO)Lysinv(Dans)-Succ-OH.

D. T. S. Rijkers et al. / Bioorg. Med. Chem. 12 (2004) 5099–5106
5101
Scheme 1. Schematic overview of the synthesis of the lipo-astressin conjugates.
Table 1. Acylation conditions of the compounds synthesized in this study
N-terminal modification
Compound
Acylation conditions
Cleavage
5a None, free N-terminus (H)
5b Acetyl (Ac)
5c Fluorenylmethyloxycarbonyl (Fmoc) Fmoc-astressin
Astressin
Ac-astressin
––
A
A
A
A
Ac2O/DIPEA/HOBt in NMP, 15min
Directly obtained from 2
(i) Fmoc-Ahx-OH/HBTU/HOBt in NMP, 45min
5d N-dansyl-6-aminohexanoyl
(Dans-Ahx)
Dans-Ahx-astressin
(ii) 20% piperidine in NMP, 30min
(iii) Dansylchloride/NMM in DCE, 16h
Myristic acid/BOP/HOBt/DIPEA in
NMP/DCE 1:1, 16h
5e Tetradecanoyl (Myr)
5f Eicosanoyl (Ara)
Myr-astressin
A
A
B
Ara-astressin
Arachidic acid/BOP/HOBt/DIPEA in
NMP/DCE 1:2, 16h
5g cis-9-Octadecenoyl (Oleic)
5h (PalO)2Gluinv-Succ-OH
5i (PalO)Lysinv(Fmoc)-Succ-OH
Oleic-astressin
Oleic acid/BOP/HOBt/DIPEA in
NMP/DCE 1:1, 16h
8/BOP/HOBt/DIPEA in
(PalO)2Gluinv-Succ-astressin
A
A
NMP/DCE 1:1, 16h
(PalO)Lysinv(Dans)-Succ-astressin (i) 11/BOP/HOBt/DIPEA in
NMP/DCE 1:1, 16h
(ii) 20% Piperidine in NMP, 30min
(iii) Dansylchloride/NMM in DCE, 16h
A: TFA/H2O/EDT/TIS 85:8.5:4.5:2 v/v/v/v; B: TFA/H2O/EDT 90:5:5 v/v/v.
hydrogen compared to the NH of a normal amide bond.
Therefore, the bromophenol blue test (BPB), which was
more reliable in this case was used.28 The use of Dans-
Ahx-OH as a building block is not recommended, since
protonation of the tertiary amine of the dansyl function-
ality hampered aqueous work-up. The fatty acid deriva-
tives were coupled with BOP/HOBt in the presence of
DIPEA. To prevent precipitation of the activated esters,
a mixture of NMP/1,2-dichloroethane (DCE) 1:1 v/v
was used. Coupling of the fatty acids was monitored
by the Kaiser test and was found to be complete after
16h. Synthesis of the lipophilic glutamic acid derivative
8 and lysine derivative 11 were carried out analogously
the procedure of Asakuma et al.21 and Pakalns et al.22
(Scheme 2). As was mentioned above, functionalization
of the e-amino group of 11 with the dansyl moiety was
carried out on the solid support.
The fatty acid astressin conjugates were deprotected
and detached from the resin by treatment with

5102
D. T. S. Rijkers et al. / Bioorg. Med. Chem. 12 (2004) 5099–5106
TFA/H2O/1,2-ethanedithiol
(EDT)/triisopropylsilane
Compounds 5a–i were tested for their CRF antagonistic
activity on two rat CRF receptor subtypes 1 and 2a.
(TIS) 85:8.5:4.5:2 v/v/v/v. To avoid reduction of the
double bond in the oleic acid residue of 4g, TIS was
not used as scavenger and TFA/H2O/EDT 90:5:5 v/v/v
was used instead.
2.2. Biological activity
Results of the biological activity studies are given in
Table 2. The astressin conjugates 5a–i were evaluated
for their functional antagonistic activity (pA2)29,30 on
the rat CRF receptor type 1 and 2a (rCRFR1, respec-
tively, rCRFR2a). The functional assay in this study
used a cloned rat CRFR1 and CRFR2a in a LVIP cell
line furnished with a cAMP-dependent reporter gene
for b-galactosidase. Compounds 5a–i were tested for
antagonistic properties assessing their ability to attenu-
ate CRF-induced (against 1nM CRF in rCRFR1;
against 10nM CRF in rCRFR2a) expression of b-galac-
tosidase using Schild analysis. The given pA2 values
represent the mean value of six independent determina-
tions.
It can be concluded that these synthetic procedures effi-
ciently and successfully resulted in several N-terminally
modified astressin derivatives (5a–i). The overall yields
in all these cases were moderate (33–64%) and according
to HPLC and MS analysis, the crude products consisted
mainly of the desired astressin conjugates. We also tried
to acylate the N-terminus with 1-adamantyl isocyanate,
cholic acid and cis-4,7,10,13,16,19-docosahexaenoic acid
but the synthesis of these conjugates failed mainly due to
the intrinsic vulnerability of these N-terminal modifica-
tions with respect to the chosen synthesis/deprotection
strategy, since the peptides could only be obtained after
a final TFA-treatment.
Scheme 2. Schematic overview of the synthesis of the lipidated amino acid residues.
Table 2. Biophysical and biological data of the astressin derivatives
(M + H)+
calcd
(M + H)+
found
pA2
rCRFR1
pA2
rCRFR2a
a
a
˚
HPLC C4 300A
˚
HPLC CN 100A
Compound
5a
5b
5c
5d
5e
5f
Astressin
Ac-astressin
Fmoc-astressin
3564.23
3606.27
3786.47
3910.68
3774.59
3858.75
3828.68
4242.28
4249.19
3565.40
3606.21
3786.03
3909.95
3775.70
3858.65
3828.08
4242.10
4249.98
27.12
31.18
21.05
22.03
22.08
22.62
23.93
24.62
24.36
26.15
25.51
8.3 0.09
8.3 0.13
7.8 0.03
8.5 0.05
8.0 0.07
7.9 0.04
7.5 0.07
7.3 0.03
7.7 0.03
8.7 0.08
9.0 0.1
28.93
32.33
8.2 0.02
8.9 0.04
8.2 0.07
8.1 0.04
7.8 0.06
7.5 0.03
8.0 0.03
Dans-Ahx-astressin
Myr-astressin
Ara-astressin
39.34
43.29
5g
5h
5i
Oleic-astressin
(PalO)2Gluinv-Succ-astressin
(PalO)Lysinv(Dans)-Succ-astressin
40.66
Not eluted
Not eluted
a The pA2 value is the mean value of six independent determinations.

D. T. S. Rijkers et al. / Bioorg. Med. Chem. 12 (2004) 5099–5106
5103
Compounds 5b–i were found to be active as CRF antag-
onists with nearly equal potency of (unmodified) astres-
sin (5a). Generally, CRF antagonistic activity was found
to be independent of the type of N-terminal acylation.
These results agree well with our earlier observations
that N-terminal truncation of astressin resulted in active
CRF antagonists.31 These lipidated CRF antagonists
were designed with the purpose to obtain compounds
capable of passage of membrane barriers, for example,
present in the central nerve system, since CRF receptors
are predominantly localized here. However, in view of
earlier results obtained with myristoylated peptides15 it
is also very likely that in addition to binding extracellu-
larly, the astressin conjugates have penetrated into cells
or at least are present in cellular membranes. Appar-
ently, this did not interfere with their CRF antagonistic
action. Fluorescent microscopy studies using, for exam-
ple, dansylated derivatives 5d and 5i might shed light on
this issue and will be carried out in due course.
tive to CDCl3 (77.0ppm). The 13C NMR spectra were re-
corded using the attached proton test (APT) sequence.
The optical rotations were measured at 20ꢁC using a Jas-
co P-1010 polarimeter. Melting points were measured on
a Buchi Schmelzpunktbestimmungsapparat (according
¨
to Dr. Tottoli) and are uncorrected. Combustion analy-
ses were done by Kolbe Mikroanalytisches Labor (Mul-
¨
heim an der Ruhr, Germany).
3.2. Peptide synthesis
3.2.1. Peptide assembly. The linear sequence of (fully
protected) astressin (Fmoc-D-Phe-His(Trt)-Leu-Leu-
Arg(Pbf)-Glu(OtBu)-Val-Leu-Glu(OtBu)-Nle-Ala-Arg-
(Pbf)-Ala-Glu(OtBu)-Gln(Trt)-Leu-Ala-Gln(Trt)-Glu-
(OAll)-Ala-His(Trt)-Lys(Aloc)-Asn(Trt)-Arg(Pbf)-Lys-
(Boc)-Leu-Nle-Glu(OtBu)-Ile-Ile-NH-RinkAmide-Argo-
Gel) was synthesized as described earlier.23,31 The allyl
protective groups were removed by swirling the resin
in DCE with phenylsilane (25equiv) in the presence of
3equiv tetrakistriphenylphosphine palladium(0) under
an argon atmosphere for 2h. The resin was extensively
washed with DCM (2 · 2min), NMP (2 · 2min), 0.5%
DIPEA/NMP (3 · 5min), 0.02M diethyldithiocarbamic
acid sodium salt in NMP (3 · 15min), NMP
(5 · 5min), and DCM (3 · 3min), respectively, to
remove the Pd catalyst.33 Ring closure between the
unprotected side chains of glutamic acid and lysine
was achieved on the resin with 3equiv BOP/HOBt in
the presence of 9equiv DIPEA in NMP for 16h. Then,
the resin was washed with NMP (3 · 2min), DCM
(3 · 2min), and NMP (3 · 2min). Deprotection and ring
closure reaction were monitored by the Kaiser test.26
After removal of the N-terminal Fmoc functionality
by treatment with 20% piperidine in NMP (3 · 8min),
the resin was washed with NMP (5 · 2min) and DCM
(5 · 2min) and subsequently dried in a vacuum desicca-
tor.
3. Experimental
3.1. Materials
Na-9-Fluorenylmethyloxycarbonyl (Fmoc) protected
amino acids, and N-hydroxybenzotriazole (HOBt) were
purchased from Advanced ChemTech Europe. The side
chain protecting groups were chosen as: allyl (All) for
glutamic acid, allyloxycarbonyl (Aloc) for lysine, tert-bu-
tyl (tBu) for glutamic acid, tert-butyloxycarbonyl (Boc)
for lysine, 2,2,4,6,7-pentamethyl-dihydrobenzofuran-5-
sulfonyl (Pbf) for arginine, and trityl (Trt) for aspara-
gine, glutamine, and histidine. Fmoc-Glu(OAll)-OH,
Fmoc-Lys(Aloc)-OH, Fmoc-6-aminohexanoic acid
(Fmoc-Ahx-OH), and Fmoc-D-Phe-OH were obtained
from Neosystem Laboratoire. The coupling reagents, 2-
(1H-benzotriazol-1-yl)-1,1,3,3,-tetramethyluronium hexa-
fluorophosphate (HBTU) and benzotriazol-1-yl-oxy-
tris-(dimethylamino)phosphonium hexafluorophosphate
(BOP) were purchased from Richelieu Biotechnologies.
Peptide grade N-methylpyrrolidone (NMP), dichloro-
methane(DCM),1,2-dichloroethane(DCE)andtrifluoro-
acetic acid (TFA) and HPLC-grade acetonitrile, hexane
and methyl tert-butyl ether (MTBE) were from Biosolve.
3.2.2. N-terminal modification. Each portion of dry resin
(160mg, 0.02mmol) was suspended in NMP (3 · 2min)
and DCE (3 · 2min) to swell the resin before the rea-
gents were added. The coupling and deprotection reac-
tions were monitored by the Kaiser test.26 The
coupling reaction of dansylchloride was monitored by
the bromophenol blue test.28 If necessary, the coupling
reaction was repeated once to achieve complete derivati-
zation.
Piperidine,
N,N-diisopropylethylamine
(DIPEA),
N-methylmorpholine (NMM), triethylamine (TEA), 4-
(N,N-dimethylamino)pyridine (DMAP), succinic anhy-
dride, dansylchloride, and tetrakistriphenylphosphine
palladium(0) were obtained from Acros Organics. Triiso-
propylsilane (TIS) and 1,2-ethanedithiol (EDT) were
supplied by Merck. Phenylsilane, myristic acid, arachidic
acid, oleic acid, and hexadecanol were obtained from
Fluka. N,N0-diisopropylcarbodiimde (DIC) was from
Aldrich. All other chemicals were at least analytical rea-
gent grade. Rf values were determined by thin layer chro-
matography (TLC) on Merck precoated silicagel 60F254
plates. Spots were visualized by UV-quenching, ninhy-
drin or Cl2/TDM.32 1H NMR spectra were recorded on
a Varian G-300 (300.1MHz) spectrometer and chemical
shifts are given in ppm (d) relative to TMS. 13C NMR
spectra were recorded on a Varian G-300 (75.5MHz)
spectrometer and chemical shifts are given in ppm rela-
3.2.2.1. Ac-astressin (4b). The resin was treated with
a solution of 0.5M Ac2O, 0.125M DIPEA, and
0.015M HOBt in NMP (2mL) for 15min. Then, the
resin was washed with NMP (5 · 2min) and DCM
(5 · 2min) and subsequently dried on air.
3.2.2.2. Dans-Ahx-astressin (4d). The resin was trea-
ted with Fmoc-Ahx-OH (85mg, 0.24mmol, 12equiv),
HBTU (91mg, 0.24mmol, 12equiv), HOBt (37mg,
0.24mmol, 12equiv), and DIPEA (126lL, 0.72mmol,
36equiv) in NMP (2mL) for 45min at room tempera-
ture. Then, the resin was washed with NMP
(3 · 2min), DCM (3 · 2min), and NMP (3 · 2min)
and the Fmoc group was removed by treatment with

5104
D. T. S. Rijkers et al. / Bioorg. Med. Chem. 12 (2004) 5099–5106
20% piperidine in NMP (2mL, 3 · 10min) subsequently
followed by washing the resin with NMP (5 · 2min) and
DCM (5 · 2min). Finally, dansylchloride (65mg,
0.24mmol, 12equiv) dissolved in DCE (2mL) was added
to the resin followed by NMM (79lL, 0.72mmol,
36equiv). After a reaction time of 16h at room temper-
ature, the resin was filtered and washed with NMP
(5 · 2min) and DCM (5 · 2min) and subsequently dried
on air.
for 16h at room temperature. Then, the resin was fil-
tered and washed with NMP (3 · 2min), DCM
(3 · 2min), and NMP (3 · 2min) and subsequently trea-
ted with 20% piperidine in NMP (2mL, 3 · 10min) to
remove the Fmoc group, followed by washing steps with
NMP (5 · 2min) and DCM (5 · 2min). To this resin,
dansylchloride (65mg, 0.24mmol, 12equiv) dissolved
in DCE (2mL) was added to the resin followed by
NMM (79lL, 0.72mmol, 36equiv). After a reaction
time of 16h at room temperature, the resin was filtered
and washed with NMP (5 · 2min) and DCM
(5 · 2min) and subsequently dried on air.
3.2.2.3. Myr-astressin (4e). Myristic acid (55mg,
0.24mmol, 12equiv) was dissolved in NMP (1mL) and
BOP (106mg, 0.24mmol, 12equiv), HOBt (37mg,
0.24mmol) were added followed by the addition of
DCE (1mL). The clear solution was transferred to the
resin and DIPEA (126lL, 0.72mmol, 36equiv) was
added. The reaction mixture was shaken for 16h at
room temperature. Then, the resin was filtered and
washed with NMP (5 · 2min) and DCM (5 · 2min)
and subsequently dried on air.
3.2.3. Deprotection and cleavage from the resin. Resins
4a–f and 4h–i were treated with TFA/H2O/EDT/TIS
85:8.5:2:4.5 v/v/v/v and resin 4g was treated with TFA/
H2O/EDT 90:5:5 v/v/v for 3h at room temperature.
The peptides were precipitated with MTBE/hexane 1:1
v/v at ꢀ20ꢁC and finally lyophilized from acetonitrile/
H2O 1:1 v/v. The obtained yields were: 5a: 64%, 5b:
56%, 5c: 35%, 5d: 56%, 5e: 50%, 5f: 39%, 5g: 49%, 5h:
33%, and 5i: 40%.
3.2.2.4. Ara-astressin (4f). Arachidic acid (75mg,
0.24mmol, 12equiv) was dissolved in NMP (1mL) and
BOP (106mg, 0.24mmol, 12equiv), HOBt (37mg,
0.24mmol) were added followed by the addition of
DCE (1mL). The clear solution was transferred to the
resin and DIPEA (126lL, 0.72mmol, 36equiv) was
added. To prevent premature precipitation of the acti-
vated fatty acid an additional amount of DCE (1mL)
was added. The reaction mixture was shaken for 16h
at room temperature. Then, the resin was filtered and
washed with NMP (5 · 2min) and DCM (5 · 2min)
and subsequently dried on air.
3.2.4. Tos-OHÆH-Glu(OC16H33)-OC16H33 (7). Glutamic
acid (7.4g, 50mmol), hexadecanol (24.2g, 100mmol),
and p-toluenesulfonic acid monohydrate (10.5g,
55mmol) were suspended in toluene (300mL) and re-
fluxed for 16h in a Dean–Stark apparatus. Subsequently,
the clear and colorless reaction mixture was evaporated
to dryness. Pure 7 was obtained after crystallization
from aceton in 47% yield (17.9g). Rf (DCM/MeOH
1
98:2 v/v): 0.19; mp: 63ꢁC; [a]D +2.5 (c 1, CHCl3); H
NMR (CDCl3): d = 0.88 (t, 6H), 1.26 (br s, 52H), 2.18
(m, 2H), 2.34 (s, 3H), 2.46 (br m, 2H), 3.96–4.09 (m,
5H), 7.11/7.14–7.73/7.75 (dd, 4H), 8.29 (br s, 3H); 13C
NMR (CDCl3): d = 14.1, 21.3, 22.7, 25.2, 25.7, 25.9,
28.2, 28.5, 29.3, 29.4, 29.4, 29.6, 29.7, 29.7, 31.9, 52.4,
64.9, 66.6, 126.1, 128.8, 140.1, 141.4, 168.9, 172.2. Anal.
calcd for C44H81NO7S: C, 68.80; H, 10.63; N, 1.82; S,
4.17. Found: C, 68.72; H, 10.60; N, 1.86; S, 4.08; ESI
MS m/z calcd for C37H74NO4: ([M ꢀ Tos-OH] + H)+,
596.97; found: ([M ꢀ Tos-OH] + H)+, 596.65.
3.2.2.5. Oleic-astressin (4g). Oleic acid (76lL,
0.24mmol, 12equiv) was dissolved in NMP (1mL) and
BOP (106mg, 0.24mmol, 12equiv), HOBt (37mg,
0.24mmol) were added followed by the addition of
DCE (1mL). The clear solution was transferred to the
resin and DIPEA (126lL, 0.72mmol, 36equiv) was
added. The reaction mixture was shaken for 16h at
room temperature. Then, the resin was filtered and
washed with NMP (5 · 2min) and DCM (5 · 2min)
and subsequently dried on air.
3.2.5. Succinimidyl-Gluinv(OC16H33)-OC16H33 (8). Com-
pound 7 (15g, 19.6mmol) was dissolved in CHCl3/THF
1:1 v/v (150mL), and TEA (4.08mL, 29.3mmol,
1.5equiv) followed by succinic anhydride (2.9g,
29.3mmol, 1.5equiv) were added. After stirring for
16h at room temperature, the reaction mixture was
evaporated in vacuo. The residue was crystallized from
aceton in 93% yield (12.6g). Rf (DCM/MeOH 98:2
v/v): 0.08; mp: 59–61ꢁC; [a]D +8.9 (c 1, CHCl3); 1H
NMR (CDCl3): d = 0.88 (t, 6H), 1.26 (br s, 52H), 1.62
(m, 4H), 1.97–2.17 (dm, 2H), 2.55 (m, 2H), 2.66 (m,
2H), 3.06/3.09–3.11/3.14 (dd, 2H), 4.05/4.12 (dt, 4H),
4.59 (m, 1H), 6.82 (d, 1H); 13C NMR (CDCl3):
d = 8.4, 14.1, 22.7, 25.8, 25.9, 27.5, 28.5, 28.5, 29.2,
29.3, 29.3 29.49, 29.5, 29.6, 29.6, 29.7, 30.3, 30.6, 31.5,
31.9, 45.0, 51.7, 64.9, 65.7, 172.0, 172.5, 172.9, 176.9.
Anal. calcd for C41H77NO7: C, 70.75; H, 11.15; N,
2.01. Found: C, 70.68; H, 11.24; N, 2.08; ESI MS m/z
calcd for C41H78NO7: (M + H)+, 696.58; found:
(M + H)+, 697.35.
3.2.2.6. (PalO)2Gluinv-Succ-astressin (4h). Compound
8 (167mg, 0.24mmol, 12equiv) was dissolved in NMP
(1mL) and BOP (106mg, 0.24mmol, 12equiv), HOBt
(37mg, 0.24mmol) were added followed by the addition
of DCE (1mL). The clear solution was transferred to the
resin and DIPEA (126lL, 0.72mmol, 36equiv) was
added. The reaction mixture was shaken for 16h at
room temperature. Then, the resin was filtered and
washed with NMP (5 · 2min) and DCM (5 · 2min)
and subsequently dried on air.
3.2.2.7. (PalO)Lysinv (Dans)-Succ-astressin (4i). Com-
pound 11 (166mg, 0.24mmol, 12equiv) was dissolved in
NMP (1mL) and BOP (106mg, 0.24mmol, 12equiv),
HOBt (37mg, 0.24mmol) were added followed by the
addition of DCE (1mL). The clear solution was trans-
ferred to the resin and DIPEA (126lL, 0.72mmol,
36equiv) was added. The reaction mixture was shaken

D. T. S. Rijkers et al. / Bioorg. Med. Chem. 12 (2004) 5099–5106
5105
3.2.6. Boc-Lys(Cbz)-OC16H33 (9). Boc-Lys(Cbz)-OH
(3.8g, 10mmol), hexadecanol (2.4g, 10mmol), and
DMAP (122mg, 1mmol, 0.1equiv) were dissolved in
DCM (100mL). The solution was cooled on ice and
DIC (2.2mL, 14mmol, 1.4equiv) was added portion-
wise. The reaction mixture was stirred for 1h at 0ꢁC fol-
lowed by 16h at room temperature. Subsequently, the
turbid reaction mixture was filtered and the solvent
was removed under reduced pressure. The residue was
redissolved in EtOAc (150mL) and subsequently
washed with H2O, 1N KHSO4, H2O, 5% NaHCO3,
and brine (3 · 50mL each). The EtOAc layer was dried
(Na2SO4), filtered, and evaporated in vacuo. The residue
was crystallized from aqueous aceton in 88% yield
(5.3g). Rf (DCM/MeOH 98:2 v/v): 0.50, Rf (DCM/
MeOH 94:6 v/v): 0.88; mp: 55–59ꢁC; [a]D +3.5 (c 0.99,
moved by palladium on activated carbon (10% Pd) in
a hydrogen atmosphere. After 2h, the reaction mixture
was filtered over Hyflo and the filtrate was evaporated
to dryness. The residue was suspended in CH3CN/H2O
1:1 v/v (30mL) and DIPEA was added to obtain a pH
value of 8.5–9. Fmoc-ONSu (229mg, 0.7mmol) dis-
solved in CH3CN was added in one portion and the
pH was adjusted to pH8 by adding DIPEA. After stir-
ring for 2h the reaction mixture became almost clear
and 1N KHSO4 was added until a pH of 2 was reached.
The turbid reaction mixture was extracted with EtOAc
(3 · 20mL) and the organic layer was washed with 1N
KHSO4 and brine (twice 20mL), dried (Na2SO4), fil-
tered, and evaporated in vacuo. The residue was crystal-
lized from aceton in 66% yield (312mg). Rf (DCM/
MeOH 98:2 v/v): 0.05, Rf (DCM/MeOH 94:6 v/v):
0.34; mp: 110–113ꢁC; [a]D +32.1 (c 1, CHCl3); 1H
NMR (CDCl3): d = 0.88 (t, 3H), 1.25 (br s, 26H),
1.38–1.64 (br m, 7H), 1.90 (m, 1H), 2.37–2.97 (br m,
4H), 3.15 (m, 2H), 4.13 (dd, 2H), 4.22 (m, 1H), 4.41
(dd, 2H), 4.58–4.67 (dm, 1H), 5.17/6.48 (dm, 1H),
6.35/6.38–6.54/6.56 (dd, 1H), 7.31–7.40 (dt, 4H), 7.56–
7.77 (dd, 4H); 13C NMR (CDCl3): d = 14.1, 21.5, 22.1,
22.7, 25.8, 28.2, 28.5, 29.2, 29.3, 29.5, 29.6, 29.6, 29.7,
30.8, 31.41, 31.9, 32.5, 40.6, 47.0, 51.6, 52.0, 65.7, 66.7,
67.5, 112.0, 125.0, 127.0, 127.7, 141.3, 143.9, 158.6,
171.8, 172.5, 176.8. Anal. calcd for C41H60N2O7:
C, 71.07; H, 8.73; N, 4.04. Found: C, 70.85; H, 8.68;
N, 3.95; ESI MS m/z calcd for C41H61N2O7:
1
CHCl3); H NMR (CDCl3): d = 0.88 (t, 3H), 1.26 (br
s, 26H), 1.43 (s, 9H), 1.12–1.80 (m, 8H), 3.18 (m, 2H),
4.11 (t, 2H), 4.26 (m, 1H), 4.83 (br s, 1H), 5.10 (br s,
3H), 7.35 (s, 5H); 13C NMR (CDCl3): d = 14.1, 22.4,
22.7, 25.8, 28.3, 28.5, 29.2, 29.3, 29.5, 29.56, 29.6, 29.7,
31.9, 32.5, 40.6, 53.2, 65.5, 66.6, 79.8, 128.1, 128.5,
136.5, 156.4, 172.8. Anal. calcd for C35H60N2O6: C,
69.50; H, 10.00; N, 4.63. Found: C, 69.44; H, 10.15;
N, 4.56; ESI MS m/z calcd for C35H60N2O6Na:
(M + Na)+, 627.44; found: (M + Na)+, 627.50,
([M ꢀ C5H8O2] + H)+, 505.50.
3.2.7. Succinimidyl-Lysinv(Cbz)-OC16H33 (10). Com-
pound 9 (1.0g, 1.7mmol) was dissolved in TFA/DCM
1:1 v/v (20mL) and stirred for 1h at room temperature.
The reaction mixture was evaporated under reduced
pressure and coevaporated with DCM (3 · 20mL) to re-
move any residual TFA. The resulting oil was dissolved
in CHCl3/THF 1:1 v/v (20mL). This solution was neu-
tralized with TEA and subsequently, TEA (350lL,
2.5mmol, 1.5equiv) followed by succinic anhydride
(250mg, 2.5mmol, 1.5equiv) were added. After stirring
for 16h at room temperature, the reaction mixture was
evaporated in vacuo. The residue was acidified with
1N KHSO4 and extracted into EtOAc (3 · 20mL).
The organic layer was washed with 1N KHSO4 and
brine (twice 50mL), dried (Na2SO4), filtered, and subse-
quently evaporated at reduced pressure. The residue was
crystallized from aceton in 78% yield (782mg). Rf
(DCM/MeOH 98:2 v/v): 0.06, Rf (DCM/MeOH 94:6
(M + H)+,
(M + Na)+, 715.60.
693.45;
found:
(M + H)+,
693.60,
3.3. Peptide purification and peptide characterization
HPLC runs were performed on a Gilson HPLC work-
station. The crude peptides (30–100mg) were dissolved
in a minimum amount of 0.1% TFA in H2O and loaded
on a preparative HPLC column (Alltech Adsorbo-
˚
sphereꢂXL C4, 10lm particle size, 300A pore size, l:
250mm, id: 22mm). The peptides were eluted with a
flow rate of 10.0mL/min using a linear gradient of buffer
B (20–90% in 80min) in buffer A (buffer A: 0.1% TFA in
H2O, buffer B: 0.1% TFA in CH3CN/H2O 8:2 v/v). Pep-
tide purity was analyzed by analytical HPLC on an All-
˚
tech AdsorbosphereꢂXL C4 (5lm particle size, 300A
pore size, l: 250mm, id: 4.6mm) column and on a Merck
1
˚
v/v): 0.32; mp: 98–101ꢁC; [a]D +36.1 (c 1, CHCl3); H
LiChroSpher CN (5lm particle size, 100A pore size, l:
NMR (CDCl3): d = 0.88 (t, 3H), 1.25 (br s, 26H),
1.39–1.91 (m, 8H), 2.54–2.92 (m, 4H), 3.16 (m, 2H),
4.12 (t, 2H), 4.63 (m, 1H), 5.09/5.14 (dd, 5H), 6.35 (d,
1H), 6.54 (m, 1H), 7.35 (s, 5H); 13C NMR (CDCl3):
d = 14.1, 21.6, 22.1, 22.6, 25.8, 28.4, 29.1, 29.3, 29.5,
29.5, 29.6, 29.6, 30.6, 31.3, 31.9, 32.4, 40.6, 40.6, 51.7,
52.0, 65.7, 67.0, 67.3, 127.9, 128.0, 128.1, 128.5, 136.0,
136.4, 156.7, 158.5, 171.8, 172.5, 176.0, 176.8. Anal.
calcd for C34H56N2O7: C, 67.52; H, 9.33; N, 4.63.
Found: C, 67.34; H, 9.38; N, 4.52; ESI MS m/z calcd
for C34H57N2O7: (M + H)+, 605.42; found: (M + H)+,
605.55, (M + Na)+: 627.55.
250mm, id: 4.6mm) column at a flow rate of 1.0mL/
min using a linear gradient of buffer B (20–90% in
40min) in buffer A (buffer A: 0.1% TFA in H2O; buffer
B: 0.1% TFA in CH3CN/H2O 8:2 v/v). Purity was deter-
mined to be 95% or higher. The peptides were character-
ized by mass spectrometry. Electrospray ionization mass
spectrometry (ESI MS) and LC ESI MS was performed
on a Shimadzu LC MS-QP8000 single quadrupole
bench-top mass spectrometer operating in a position
ion mode (deflector voltage was set on 50V, which pro-
duces predominantly (M + nH)n+ ions with little evi-
dence of fragmentation for this type of peptides).
Samples (typically 30lL) were injected into a moving
solvent (200lL/min; 0.1% TFA in H2O) using Shimadzu
LC-10AD HPLC pumps and loaded onto an Alltech
3.2.8. Succinimidyl-Lysinv(Fmoc)-OC16H33 (11). Com-
pound 10 (410mg, 0.7mmol) was dissolved in THF
(20mL). The benzyloxycarbonyl functionality was re-
˚
AdsorbosphereꢂXL C4 (5lm particle size, 300A pore

5106
D. T. S. Rijkers et al. / Bioorg. Med. Chem. 12 (2004) 5099–5106
4. Pooga, M.; Hallbrink, M.; Zorko, M.; Langel, U. FASEB
J. 1998, 67, 12.
5. Rousselle, C.; Clair, P.; Lefauconnier, J.-M.; Kaczorek,
M.; Scherrmann, J.-M.; Temsamani, J. Mol. Pharmacol.
2000, 57, 679.
size, l: 150mm, id: 2.1mm) column using a linear gra-
dient of acetronitrile (0–90%) in 0.1% TFA in H2O over
30min. The mass of each analog was measured and the
observed (M + nH)n+ values were correlated with the
calculated average (M + nH)n+ values using MacBio-
Spec (Sciex Instruments, Thornhill, Ontario, Canada).
6. Du, C.; Yao, S.; Rojas, M.; Lin, Y.-Z. J. Pept. Res. 1998,
51, 235.
7. Schwarze, S. R.; Ho, A.; Vocero-Akbani, A.; Dowdy,
S. F. Science 1999, 285, 1569.
3.4. Biological testing
8. Derossi, D.; Chassaing, G.; Prochiantz, A. Trends Cell
Biol. 1998, 8, 84.
Rat CRF antagonistic activity (expressed as pA2 values)
was assayed by measuring the reduced induction of
b-galactosidase in response to rat CRF in a stable
CRF receptor 1, or receptor 2a expressing LVIP2.0Zc
cell line that contain an exogenous cAMP-responsive
b-galactosidase receptor gene product.29 Formation of
cAMP was stimulated with rat CRF (rCRFR1: 1nM;
rCRFR2a: 10nM) for 3h. The increase of cAMP re-
sulted in an increase in the production of b-galacto-
sidase capable of hydrolysis of the chromogenic
substrate o-nitrophenyl-b-D-galactopyranoside resulting
in a yellow color that was measured at 405nm.30 Anta-
gonistic activity can be assessed after a 30min preincu-
bation with putative antagonists and subsequent
incubation with CRF for 3h (rCRFR1: 1nM;
rCRFR2a: 10nM). Antagonistic potency is defined as
the concentration of antagonist at which the agonist
concentration needs to be doubled to arrive at the same
effect as in the absence of antagonist. This pA2 value was
calculated from a Schild analysis. Schild analysis was
done by measuring CRF-induced b-galactosidase
expression in absence or presence of three different
`
9. Vives, E.; Biodin, P.; Lebleu, B. J. Biol. Chem. 1997, 272,
16010.
10. Derossi, D.; Joliot, A. H.; Chassaing, G.; Prochiantz, A.
J. Biol. Chem. 1994, 269, 10444.
11. Futaki, S.; Suzuki, T.; Wohashi, T.; Yagami, T.; Tanaka,
S.; Ueda, K.; Sugiura, Y. J. Biol. Chem. 2001, 276, 5836.
12. Wender, P. A.; Mitchell, D. J.; Pattabiraman, K.; Pelkey,
E. T.; Steinman, L.; Rothbard, J. B. Proc. Natl. Acad. Sci.
U.S.A. 2000, 97, 13003.
13. Wender, P. A.; Rothbard, J. B.; Jessop, T. C.; Kreider,
E. L.; Wylie, B. L. J. Am. Chem. Soc. 2002, 124, 13382.
14. Resh, M. D. Biochim. Biophys. Acta 1999, 1451, 1.
15. Eichholtz, T.; de Bont, D. B. A.; de Widt, J.; Liskamp, R.
M. J.; Ploegh, H. L. J. Biol. Chem. 1993, 268, 1982.
16. Vale, W.; Spiess, J.; Rivier, C.; Rivier, J. Science 1981, 213,
1394.
17. Behan, D. P.; Grigoriadis, D. E.; Lovenberg, T.; Chal-
mers, D.; Heinrichs, S.; Liaw, C.; De Souza, E. B. Mol.
Psychiatr. 1996, 1, 265.
18. Rivier, J.; Rivier, C.; Vale, W. Science 1984, 224, 889.
19. Gulyas, J.; Rivier, C.; Perrin, M.; Koerber, S. C.; Sutton,
S.; Corrigan, A.; Lahrichi, S. L.; Craig, A. G.; Vale, W.;
Rivier, J. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 10575.
20. Miranda, A.; Lahrichi, S. L.; Gulyas, J.; Koerber, S. C.;
Craig, A. G.; Corrigan, A.; Rivier, C.; Vale, W.; Rivier, J.
J. Med. Chem. 1997, 40, 3651.
antagonist-peptide
concentrations
(10ꢀ5–10ꢀ8 M).
Antagonist-induced right-ward shifts of CRF-stimula-
tion is a measure of antagonist potency and can be cal-
culated as pA2 = log(DR ꢀ 1) ꢀ log[antagonist]; where
DR (dose ratio) is defined as the ratio between the
EC50 of CRF (pEC50 [CRFR1]: 9.4; pEC50 [CRFR2a]:
8.9) in presence of a specified antagonist concentration,
compared with control EC50 values. The given pA2 val-
ues represent the mean value of six independent determi-
nations.
21. Asakuma, S.; Okada, H.; Kunitake, T. J. Am. Chem. Soc.
1991, 113, 1749.
22. Pakalns, T.; Haverstick, K. L.; Fields, G. B.; McCarthy,
J. B.; Mooradian, D. L.; Tirrell, M. Biomaterials 1999,
20, 2265.
23. Rijkers, D. T. S.; den Hartog, J. A. J.; Liskamp, R. M. J.
Biopolymers 2002, 63, 141.
´
24. Dessolin, M.; Guillerez, M.-G.; Thieriet, N.; Guibe, F.;
Loffet, A. Tetrahedron Lett. 1995, 36, 5741.
25. Castro, B.; Dormoy, J. R.; Evin, G.; Selve, C. Tetrahedron
1975, 1219.
26. Kaiser, E.; Colescott, R. L.; Bossinger, C. D.; Cook, P. I.
Anal. Biochem. 1970, 34, 595.
Acknowledgements
We would like to thank Dr. J. Frankena and Dr. E.
Ronken for their contributions to this work. This re-
search was financed by a strategic alliance between Sol-
vay Pharmaceuticals, the Ministry of Economic Affairs
and Utrecht University, The Netherlands.
27. Monnee, M. C. F.; Marijne, M. F.; Brouwer, A. J.;
Liskamp, R. M. J. Tetrahedron Lett. 2000, 41, 7991.
ˇ
28. Krchnak, V.; Vagner, J.; Safar, P.; Lebl, M. Collect.
ˇ ´
Czech. Chem. Commun. 1988, 53, 2542.
´
´ˇ
29. Ko¨nig, M.; Mahan, L. C.; Marsh, J. W.; Fink, J. S.;
Brown, M. J. Mol. Cell. Neurosci. 1991, 2, 331.
30. Liaw, C. W.; Grigoriadis, D. E.; De Souza, E. B.;
Oltersdorf, T. J. Mol. Neurosci. 1994, 5, 83.
31. Rijkers, D. T. S.; Kruijtzer, J. A. W.; van Oostenbrugge,
M.; Ronken, E.; den Hartog, J. A. J.; Liskamp, R. M. J.
ChemBioChem 2004, 5, 340.
32. von Arx, E.; Faupel, M.; Bruggen, M. J. Chromatogr.
1976, 120, 224.
33. Kates, S. A.; Daniels, S. B.; Albericio, F. Anal. Biochem.
1993, 212, 303.
References and notes
1. Pardridge, W. M. Pharm. Sci. Technol. Today 1999, 2, 49.
2. Temsamani, J.; Scherrmann, J.-M.; Rees, A. R.; Kacz-
orek, M. Pharm. Sci. Technol. Today 2000, 3, 155.
3. Lindgren, M.; Hallbrink, M.; Prochiantz, A.; Langel, U.
Trends Pharmacol. Sci. 2000, 21, 99.