Synthesis of CJ-15,208, a novel κ-opioid receptor antagonist

Article abstract of DOI:10.1016/j.tetlet.2010.07.086

The tryptophan isomers of the cyclic tetrapeptide CJ-15,208, reported to be a kappa opioid receptor (KOR) antagonist [Saito, T.; Hirai, H.; Kim, Y. J.; Kojima, Y.; Matsunaga, Y.; Nishida, H.; Sakakibara, T.; Suga, O.; Sujaku, T.; Kojima, N. J. Antibiot. (

Full text of DOI:10.1016/j.tetlet.2010.07.086

Tetrahedron Letters 51 (2010) 5020–5023
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Synthesis of CJ-15,208, a novel j-opioid receptor antagonist
Nicolette C. Ross a, , Santosh S. Kulkarni a,à, Jay P. McLaughlin b, , Jane V. Aldrich a,
*
a Department of Medicinal Chemistry, The University of Kansas, Lawrence, KS 66045, USA
b Department of Psychology, Northeastern University, Boston, MA 02115, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The tryptophan isomers of the cyclic tetrapeptide CJ-15,208, reported to be a kappa opioid receptor (KOR)
antagonist [Saito, T.; Hirai, H.; Kim, Y. J.; Kojima, Y.; Matsunaga, Y.; Nishida, H.; Sakakibara, T.; Suga, O.;
Sujaku, T.; Kojima, N. J. Antibiot. (Tokyo) 2002, 55, 847–854.], were synthesized to determine the trypto-
phan stereochemistry in the natural product. A strategy was developed to select linear precursor peptides
that favor cyclization using molecular modeling, and optimized cyclization conditions are reported. The
Received 7 June 2010
Accepted 15 July 2010
Available online 21 July 2010
Keywords:
Cyclic tetrapeptide
Cyclization
optical rotation of the
L-Trp isomer is consistent with that of the natural product. Unexpectedly both iso-
mers exhibit similar nanomolar affinity for KOR.
Ó 2010 Elsevier Ltd. All rights reserved.
Molecular modeling
Kappa opioid receptor (KOR) antagonists have demonstrated
promising activity in animal models as antidepressants, anxiolytics,
and therapeutics for cocaine and opiate abuse.1 Thus, there is con-
siderable interest in developing new KOR antagonists as potential
therapeutic agents. Several nonpeptide selective KOR antagonists
(i.e., norbinaltorphimine (norBNI), 50-guanidinonaltrindole (GNTI),
and (3R)-7-hydroxy-N-[(1S)-1-[[(3R,4R)-4-(3-hydroxyphenyl)-3,4-
dimethyl-1-piperidinyl]methyl]-2-methylpropyl]-1,2,3,4-tetrahy-
dro-3-isoquinolinecarboxamide (JDTic)) have been identified and
used as pharmacological tools to study KOR.1a,2 However, all these
compounds exhibit exceptionally long antagonist activity, lasting
for weeks to more than a month after a single injection,1a a profile
which could potentially limit their use as therapeutic agents. In
contrast, peptide antagonists are expected to exhibit shorter dura-
tions of activity in vivo as a result of metabolism by proteases.3
While linear peptides are often metabolized too rapidly for use
in vivo, cyclization can increase metabolic stability and improve
pharmacokinetic properties.3,4
information about the spatial requirements necessary for the inter-
action of ligand functional groups with the target binding site.
Saito et al.6 reported the isolation and characterization of the
cyclic tetrapeptide CJ-15,208 (Fig. 2) from the fermentation broth
of the fungus Ctenomyces serratus ATCC 15502. In binding assays this
peptide was reported to preferentially bind to KOR with an IC50
value of 47 nM and antagonized the KOR agonist asimadoline in
the electrically-stimulated rabbit vas deferens.6 Thus, this peptide
provides a novel lead for the development of new KOR antagonists.
Saito et al. elucidated the structure of CJ-15,208 using high reso-
lution fast atom bombardment mass spectrometry and NMR to
establish the sequence and cyclic structure of this peptide.6 This
cyclic tetrapeptide consists of a proline, a tryptophan, and two
phenylalanine residues (Fig. 2). While amino acid analysis and HPLC
of chiral derivatives established the identity and stereochemistry of
the Pro and Phe residues, the stereochemistry of the Trp residue was
not determined due to destruction during acid hydrolysis.6 While
there is a brief reference to the L-Trp isomer in a conference proceed-
Head-to-tail cyclic peptides represent a unique class of peptides
that exhibit biological activity at a broad range of targets. A num-
ber of cyclic tetrapeptides with diverse biological activities have
been obtained from different natural sources, including the cyto-
toxic chlamydocin and immunosuppressant FR 235222 (Fig. 1).5
Constrained cyclic peptides have substantially fewer degrees of
freedom than linear peptides and therefore may provide valuable
ing,7 no details on the synthesis were provided nor was evidence
presented to establish whether this isomer was the natural product.
Therefore, we undertook the synthesis of both isomers of CJ-15,208
varying in the stereochemistry of the Trp residue (Fig. 2) to conclu-
sively identify the natural product and to develop synthetic
schemes that may be applied to the synthesis of future analogs.8
Difficulties have often been encountered during the synthesis of
cyclic tetrapeptides due to the constrained 12-membered ring
structure of the product. Most cyclic tetrapeptide natural products
contain at least one turn-inducing residue such as Pro, an N-substi-
* Corresponding author. Tel.: +1 785 864 2287; fax: +1 785 864 5326.
E-mail address: jaldrich@ku.edu (J.V. Aldrich).
tuted amino acid, Gly, or Aib (a-aminoisobutyric acid) that favors a
folded conformation and is essential for cyclization.9 The cycliza-
tion reaction still often leads to the formation of the cyclic octapep-
tide, which in some cases is the predominant product.10 The amino
Present address: Torrey Pines Institute for Molecular Studies, Port St. Lucie, FL
34987, USA.
à
Present address: Syngene Int. Ltd, Biocon Park, Bangalore, India.
0040-4039/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2010.07.086

N. C. Ross et al. / Tetrahedron Letters 51 (2010) 5020–5023
5021
Table 1
O
O
Analysis of the conformations of the linear peptides by molecular modeling
Linear sequence
-Trp isomer
Hydrogen bonds
Conformationa
Distanceb (Å)
O
O
O
O
L
O
N
HN
O
N
HN
Phe-Trp-Phe-
Trp-Phe- -Pro-Phe
Phe- -Pro-Phe-Trp
-Pro-Phe-Trp-Phe
D
-Pro
—
2
1
Extended
c-Turn
b-Turn
Extended
11.5
6.5
3.4
D
D
NH HN
O
O
NH
O
HN
O
—
11.9
D
NH
D
-Trp isomer
-Trp-Phe-
-Trp-Phe- -Pro-Phe
-Pro-Phe- -Trp
-Pro-Phe-D-Trp-Phe
Phe-
D
D
-Pro
—
2
Extended
10.4
2.9
c
-Turn
D
D
Chlamydocin
Trapoxin B
Phe-
D
D
2
—
c
-Turn
3.1
11.6
OH
Extended
D
a
Preliminary assignment based on hydrogen bonding patterns.
The distance was measured from the carboxy terminal carbonyl carbon to the
O
O
N
O
b
O
amino terminal nitrogen.
O
N
HN
N
HN
O
NH HN
O
O
NH HN
O
O
OCH3
we initiated a molecular modeling study with the four possible lin-
ear precursors for both tryptophan isomers of CJ-15,208
(Table 1).14
The results of the molecular modeling suggested which linear
sequences were likely to be compatible with cyclization. The con-
formation of the linear precursor peptide plays a very important
role in successful cyclizations, and intramolecular hydrogen bonds
can stabilize a folded conformation that is favorable for cycliza-
Apicidin
FR235222
Figure 1. Cyclic tetrapeptide natural products.
tion.4 Analysis of the linear sequence Phe-Trp-Phe-
D-Pro with D-
proline at the C-terminus suggested that it preferentially existed
in an extended conformation with a distance of about 11 Å be-
tween the amino and carboxy termini (Table 1), which is consistent
with our inability to obtain the cyclic tetrapeptide from this linear
precursor. In contrast, analysis of the linear peptides with proline
at the Ci+1 or Ci+2 position resulted in folded conformations with
the amino and carboxy termini in closer proximity. A similar anal-
O
O
N
HN
HN
NH
O
O
NH
ysis of the possible linear sequences containing
formed. Based on this analysis cyclization of the linear sequences
Phe- -Pro-Phe-Trp for the -Trp peptide and -Trp-Phe- -Pro-Phe
or Phe- -Pro-Phe- -Trp for the -Trp isomer was expected to yield
the required cyclic tetrapeptides.
D-Trp was also per-
D
L
D
D
CJ-15,208
Figure 2. Cyclic tetrapeptide natural product CJ-15,208.
D
D
D
The linear peptide precursors were synthesized on the 2-chloro-
trityl resin (Scheme 1). Attachment of the first amino acid to the
resin was performed using a twofold excess of amino acid and DIEA
(N,N-diisopropylethylamine) (Scheme 1).15 The synthesis of the
linear peptides was performed using Fmoc-protected amino acids
and standard coupling and deprotection protocols. Previously
Mou and Singh reported that the protection of the indole nitrogen
of Trp in the precursor of apicidin was necessary for the successful
cyclization using the pentafluorophenyl ester method.11c Therefore
the indole nitrogen of the Trp residue in our linear precursor pep-
tides was initially protected with the Boc-protecting group.8 This
proved unnecessary using the cyclization conditions described be-
low, however, and the cyclic peptides were subsequently synthe-
sized using Fmoc-Trp without side chain protection (Scheme 1).
The crude linear peptides were P98% pure by analytical HLPC fol-
lowing cleavage from the resin with 1% trifluoroacetic acid (TFA) in
dichloromethane (DCM).15
acid sequence of the linear precursor and the method of activation
of the carboxy terminus also have significant influence on the re-
sults of the cyclization reaction.10a,11
Initially the syntheses of the isomers of CJ-15,208 were at-
tempted using a similar strategy to that described for trapoxin
B12 based on the similarity in backbone structure. The cyclization
reaction was performed using the linear sequences Phe-Trp-Phe-
D
-Pro and Phe-D-Trp-Phe-D-Pro with D-proline at the C-terminus.
Despite a variety of attempts using different reaction conditions
and coupling reagents, including the pentafluorophenyl ester
methodology reported by Schmidt et al.,9a,11b the desired cyclic tet-
rapeptides were not observed.
The sequence of the precursor linear peptide can have a marked
influence on the success of the cyclization reaction.11a In cyclic tet-
rapeptides such as CJ-15,208 there are four different linear se-
quences that can be used to attempt cyclization. However, not all
of the linear sequences may give the desired cyclic tetrapeptide
in high yield, with some sequences yielding little or no desired
product.11a,b Also which linear sequences will successfully cyclize
differs for different cyclic tetrapeptides.11a,b
Molecular modeling and conformational analysis can aid in
identifying linear precursor peptides that may adopt conforma-
tions that can undergo cyclization.13 Therefore we utilized molec-
ular modeling to choose linear sequences for cyclization to
increase the probability of cyclic tetrapeptide formation. Thus,
The cyclization of the linear peptide Phe-D-Pro-Phe-Trp was
performed in solution. The conditions used for the cyclization
had a marked influence on the results of this reaction, in terms
of the formation of the dimeric cyclic octapeptide versus the de-
sired cyclic tetrapeptide. Highly dilute conditions minimize the
formation of the cyclic octapeptide.12 Also the use of the azabenzo-
triazole-derived uronium reagent HATU (2-(1H-7-azabenzotriazol-
1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate) has been
reported to facilitate a difficult cyclization and decrease cyclodi-
merization compared to benzotriazole-derived coupling agents,10a

5022
N. C. Ross et al. / Tetrahedron Letters 51 (2010) 5020–5023
Scheme 1. Synthesis of the cyclic tetrapeptide containing L-Trp.
most likely due to neighboring group assistance by the ring nitro-
gen. When the cyclization of Phe- -Pro-Phe-Trp was performed in
merization of the C-terminal residue that occurred during cycliza-
tion under the conditions described above.
D
DCM/MeCN, however, even in dilute solution with slow addition of
the linear peptide, the cyclic octapeptide was obtained in addition
The optical rotation of the
matched the reported value for the natural product (
(c 0.05, DMSO), lit.6
D = ꢀ64.0° (c 0.05, DMSO)), while the optical
rotation of the isomer containing -Trp had the opposite sign
D = +19.8° (c 0.05, DMSO)). Surprisingly both cyclic tetrapeptides
exhibited nanomolar affinity for KOR (Ki ( SEM) = 21.2 5.6 and
46.9 16.1 nM for the -Trp and -Trp isomers, respectively), in
L
-Trp cyclic tetrapeptide closely
a
D = ꢀ63.6°
to the desired cyclic tetrapeptide. The rate of addition of Phe-
D
-Pro-
a
Phe-Trp also affected the yields of the cyclic tetrapeptide versus
cyclic octapeptide; even in dilute solution the rapid addition of
the linear peptide resulted in the cyclic octapeptide predominat-
ing, even when DMF (N,N-dimethylformamide) was used as the
solvent.16 Based on these results and procedures reported previ-
ously for the other cyclic tetrapeptides,10a,12 optimized conditions
for the cyclization were developed which involved the slow addi-
tion of the linear peptide in DMF over 12 h to a dilute solution of
HATU and DIEA in DMF.17 Mass spectrometry and HPLC analysis
of the isolated product indicated the formation of the desired cyclic
D
(a
L
D
competition radioligand-binding assays using mouse brain mem-
branes and [3H]U69,593 as the radioligand). These results are sim-
ilar to those recently reported for the
Dolle et al.21
D-Trp isomer of CJ-15,208 by
In conclusion, we successfully synthesized the two isomers of
the cyclic tetrapeptide CJ-15,208 containing - and -tryptophan.
L
D
tetrapeptide cyclo[Phe-
D
-Pro-Phe-Trp]18 with minimum formation
Using the molecular modeling approach described here appropriate
linear sequences were chosen for cyclization. The synthesis of the
L-Trp isomer still proved challenging, however, because of the ten-
of the cyclic octapeptide (<5%) under these conditions.
The same optimized conditions were also used to cyclize
D-Trp-
Phe-
analyzed by mass spectrometry and HPLC.19 In contrast to the
-Trp peptide, cyclization of -Trp-Phe- -Pro-Phe yielded minimal
cyclic octapeptide even under conditions where the linear -Trp
precursor yielded almost exclusively the cyclic dimer. Thus the
second -amino acid in the -Trp peptide facilitated the folding
of the linear precursor into a conformation that favors cyclization
to give the cyclic tetrapeptide, making the synthesis of this cyclic
peptide more straight forward than the L-Trp isomer.
D
-Pro-Phe to give the
D
-Trp isomer of CJ-15,208, which was
dency of the linear precursor to dimerize during cyclization. Opti-
mized cyclization conditions were identified that minimized the
formation of the cyclic octapeptide and maximized the yield of
L
D
D
L
the desired cyclic tetrapeptide. The optical rotation of the L-Trp iso-
mer agrees with the optical rotation reported for the natural prod-
uct CJ-15,208.6 Unexpectedly, both compounds exhibit nanomolar
affinity for KOR and thus can serve as excellent leads for further
structural modification. Such studies are currently underway in
our laboratory. Cyclization using a linear peptide that is preselected
based on molecular modeling as described here will assist in the
synthesis of analogues of these and other cyclic tetrapeptides.
D
D
One concern during head-to-tail cyclizations is epimerization of
the C-terminal residue. Azabenzotriazole reagents such as HATU
minimize epimerization of the C-terminal residue of peptides dur-
ing activation and coupling.20 The
L
-Trp and
tides had very different retention times on HPLC (tR = 26.0 and
35.5 min, respectively), and the -Trp peptide was not detected
during cyclization of Phe- -Pro-Phe-Trp, suggesting minimum epi-
D
-Trp cyclic tetrapep-
Acknowledgments
D
This research was supported by NIDA grant R01 DA018832.
The authors thank Robert Drake of the KU Mass Spectrometry
D

N. C. Ross et al. / Tetrahedron Letters 51 (2010) 5020–5023
5023
11. (a) Pastuszak, J.; Gardner, J. H.; Singh, J.; Rich, D. H. J. Org. Chem. 1982, 47,
2982–2987; (b) Schmidt, U.; Beutler, U.; Lieberknecht, A. Angew. Chem., Int. Ed.
Engl. 1989, 28, 333–334; (c) Mou, L.; Singh, G. Tetrahedron Lett. 2001, 42, 6603–
6606; (d) Meutermans, W. D.; Bourne, G. T.; Golding, S. W.; Horton, D. A.;
Campitelli, M. R.; Craik, D.; Scanlon, M.; Smythe, M. L. Org. Lett. 2003, 5, 2711–
2714.
Laboratory for his assistance with the ESI spectra and Wendy
Hartsock for her assistance with HPLC and for preparation of the
manuscript.
Supplementary data
12. Taunton, J.; Collins, J. L.; Schreiber, S. L. J. Am. Chem. Soc. 1996, 118, 10412–
10422.
13. (a) Cavelier-Frontin, F.; Pepe, G.; Verducci, J.; Siri, D.; Jacquier, R. J. Am. Chem.
Soc. 1992, 114, 8885–8890; (b) Besser, D.; Olender, R.; Rosenfeld, R.; Arad, O.;
Reissmann, S. J. Pept. Res. 2000, 56, 337–345.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.tetlet.2010.07.086.
14. The linear precursors were constructed and minimized using the Tripos force
field in SYBYL7.0 (Tripos, Inc., St. Louis, MO). Electrostatic charges were
generated using the Gasteiger and Marsili method within SYBYL; an 8Å non-
bonded cut-off was employed and a dielectric constant of 4 was applied for
References and notes
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McLaughlin, J. P. AAPS J. 2009, 11, 312–322.
2. Aldrich, J. V.; Vigil-Cruz, S. C.. In Burger’s Medicinal Chemistry & Drug Discovery;
Abraham, D. J., Ed.; John Wiley & Sons: New York, 2003; Vol. 6, pp 329–481.
3. Aldrich, J. V.; Patkar, K. A.; McLaughlin, J. P. Proc. Natl. Acad. Sci. U.S.A. 2009, 106,
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minimizations.
A three-step minimization protocol, consisting of steepest
decent gradient (100 iterations), conjugate gradient (10,000 iterations) and
then a BFGS gradient minimization with convergence criterion of 0.001 kcal/
mol, was utilized to obtain convergence.
15. Chan, W. C.; White, P. D. Fmoc Solid Phase Peptide Synthesis:
Approach; Oxford University Press: New York, 2000. pp. 41–76.
A Practical
4. Gilon, C.; Halle, D.; Chorev, M.; Selinger, Z.; Byk, G. Biopolymers 1991, 31, 745–
750.
16. The yields of cyclic tetrapeptide and cyclic octapeptide measured by HPLC
analysis following rapid addition were 28% and 44%, respectively.
17. The crude linear peptide (50 mg, 0.072 mmol) in DMF (10 mL) was added
dropwise at a rate of 1.6 mL/h (using a KD Scientific single infusion syringe
5. (a) Closse, A.; Huguenin, R. Helv. Chim. Acta 1974, 57, 533–545; (b) Itazaki, H.;
Nagashima, K.; Sugita, K.; Yoshida, H.; Kawamura, Y.; Yasuda, Y.; Matsumoto,
K.; Ishii, K.; Uotani, N.; Nakai, H.; Terui, A.; Yoshimatsu, S.; Ikenishi, Y.;
Nakagawa, Y. J. Antibiot. (Tokyo) 1990, 43, 1524–1532; (c) Singh, S. B.; Zink, D.
L.; Polishook, J. D.; Dombrowski, A. W.; Darkin-Rattray, S. J.; Schmatz, D. M.;
Goetz, M. A. Tetrahedron Lett. 1996, 37, 8077–8080; (d) Mori, H.; Urano, Y.;
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6. Saito, T.; Hirai, H.; Kim, Y. J.; Kojima, Y.; Matsunaga, Y.; Nishida, H.; Sakakibara,
T.; Suga, O.; Sujaku, T.; Kojima, N. J. Antibiot. (Tokyo) 2002, 55, 847–854.
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J. A., Eds.; EDK: Paris, France, 2001; pp 271–272.
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pump) to
a solution of HATU (46 mg, 0.12 mmol) and DIEA (0.10 mL,
0.58 mmol) in DMF (150 mL) over 6 h. After 6 h a second portion of HATU
(46 mg, 0.12 mmol) was added to the reaction in one portion, and additional
linear peptide (50 mg, 0.072 mmol) in DMF (10 mL) was added dropwise at a
rate of 1.6 mL/h as described above. The reaction was then allowed to stir at
room temperature for an additional 12–24 h, and the solvent evaporated.
Following dissolution in EtOAc or DCM, the solution was washed (with 2 N
citric acid, sat bicarbonate and brine), dried (MgSO4), and the solvent was
removed to give the crude cyclic peptide.
18. ESI-MS (M+Na)+ = 600.2566 (calcd 600.2587); HPLC: tR = 26.0 min (30–70%
aqueous MeOH over 40 min starting after 1 min, flow rate 0.8 mL/min).
19. ESI-MS (M+Na)+ = 600.2345 (calcd 600.2587); HPLC: tR = 35.5 min.
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A. L.; DeHaven, R. N.; Carroll, P. J. Bioorg. Med. Chem. Lett. 2009, 19, 3647–3650.
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