Synthesis of constrained analogues of cholecystokinin/opioid chimeric peptides

Article abstract of DOI:10.1016/j.tetlet.2006.01.096

In our ongoing research on the synthesis of constrained analogues of CCK/opioid chimeric peptides, a bicyclic dipeptide mimetic for Nle-Asp was designed and synthesized. Starting from β-allyl substituted aspartic acids, the terminal double bond was oxidiz

Full text of DOI:10.1016/j.tetlet.2006.01.096

Tetrahedron Letters 47 (2006) 2233–2236
Synthesis of constrained analogues of
cholecystokinin/opioid chimeric peptides
John M. Ndungu,a James P. Cain,a Peg Davis,b Shou-W. Ma,b Todd W. Vanderah,b
Josephine Lai,b Frank Porrecab and Victor J. Hrubya,*
aDepartment of Chemistry, University of Arizona, Tucson, AZ 85721, USA
bDepartment of Pharmacology, University of Arizona, Tucson, AZ 85724, USA
Received 14 December 2005; revised 17 January 2006; accepted 18 January 2006
Available online 13 February 2006
Abstract—In our ongoing research on the synthesis of constrained analogues of CCK/opioid chimeric peptides, a bicyclic dipeptide
mimetic for Nle-Asp was designed and synthesized. Starting from b-allyl substituted aspartic acids, the terminal double bond was
oxidized resulting in spontaneous cyclization to form racemic hemiaminals. Allylation of the hemiaminals afforded 5-allyl substi-
tuted proline analogues, which on oxidation, Horner–Emmons olefination, asymmetric hydrogenation, and bicyclization afforded
bicyclic dipeptide mimetics for Nle-Asp. Constrained CCK/opioid peptide analogues containing bicyclic dipeptide mimetics for
Nle-Gly, Nle-Asp, and homoPhe-Gly were then synthesized and analyzed at both the CCK and opioid receptors.
Ó 2006 Elsevier Ltd. All rights reserved.
Our group has recently been involved in the design and
synthesis of chimeric peptides that interact with both the
H
N
H
N
CCK and opioid receptors.1 Constrained analogues of
these peptides have been synthesized via disulfide and
lactam cyclizations.2 To further explore the topographi-
cal requirements for interaction of these peptides with
receptors, bicyclic dipeptide mimetics for Nle-Gly (1a,
1b, 1c), and homoPhe-Gly 2 have been designed and
synthesized (Fig. 1).3 In this letter, the design and syn-
thesis of indolizidinone bicyclic dipeptide mimetics for
Nle-Asp 3 and the synthesis of peptides containing bicy-
clic dipeptide mimetics are discussed. The peptides were
tested at both the CCK and opioid receptors.
S
S
BocHN
BocHN
BocHN
O
CO2Me
O
CO2Me
1a
1b
Ph
H
N
H
S
S
BocHN
CbzHN
N
O
CO2Me
O
CO2Me
2
1c
The synthesis of the indolizidinone type of bicyclic
dipeptide mimetics has been reported by a number of
authors.4 In our lab, we have developed the synthesis
of these mimetics from analogues of pyroglutamic acid.5
The target compound can be obtained from lactam
cyclization of dehydroamino acids derived from Hor-
ner–Emmons olefination of allyl substituted proline
analogues.
CO2Me
CO2Bn
N
O
3
Figure 1. Bicyclic dipeptide mimetics.
Alkylation of aspartic acid with different electrophiles
has been reported by our group3,6 and other authors.7
Alkylation with allyl bromide in the presence of lithium
bis(trimethylsilyl)amide (LHMDS) and HMPA resulted
in the formation of two b-allyl substituted aspartic acids
in a total yield of 57% and a ratio of 4:1 in favor of the
(2S,3R)-5a isomer (Scheme 1).
*
Corresponding author. Tel.: +1 520 621 6332; fax: +1 520 621
8407; e-mail: hruby@u.arizona.edu
0040-4039/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2006.01.096

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J. M. Ndungu et al. / Tetrahedron Letters 47 (2006) 2233–2236
When osmylation was used for the oxidation of 8a, the
1. LHMDS / HMPA
BocHN
BocHN
CO2Me
dehydroamino acid was obtained in 71% yield (Scheme
3). Oxidation of compound 8b by ozonolysis followed
by Horner–Emmons olefination gave the desired com-
pound and an unidentified compound in a total yield
of 38% and a ratio of 1:1. The reaction was optimized
by changing the protocol to oxidation by osmylation
followed by olefination to give the desired dehydro-
amino acid 9b in 35% yield.
2. Allyl bromide
-42 oC
CO2Bn
4
55-57%
BocHN
+
CO2Me
CO2Bn
5a
CO2Me
CO2Bn
5b
Scheme 1. Alkylation of aspartic acid with allyl bromide.
Osmylation and Horner–Emmons olefination of 8c gave
the dehydroamino acid 9c in 40% yield. Asymmetric
hydrogenation of the dehydroamino acids 9a, 9b, and
9c gave the saturated analogues, which after deprotec-
tion of the Boc group were cyclized by heating in pyri-
dine at 50 °C for 4 days to give the bicyclic dipeptide
mimetics 3a, 3b, and 3c, respectively.
HO
Boc
CO2Me
MeOH
p-TsOH
O3
BocHN
N
CO2Me
CO2Bn
5a
DMS
CO2Bn
6
The three bicyclic dipeptide mimetics were then charac-
terized by NOE measurements (Fig. 3). A strong NOE
value was observed for H3 and H6 in compounds 3b
and 3c (4.2% and 4.0%, respectively) signifying a cis
relationship between the two hydrogens. For compound
3a, the NOE value between H3 and H6 was relatively
low (1.9%) due to the trans relationship between the
hydrogens. The cis relationship between H8 and H9 in
3a and 3b resulted in a strong NOE value of 4.8%
(2S, 3R)-5a
(2S, 3S)-5b
CO2Me
CO2Bn
BF3.OEt2
MeO
Boc
CO2Me
N
H2C=CHCH2TMS
Et2O
N
Boc
CO2Bn
(2S, 3R, 5S)-8a
(2S, 3R, 5R)-8b
(2S, 3S, 5R)-8c
7
Scheme 2. Synthesis of d-allyl-substituted proline analogues.
MeO2C
CO2Me
CO2Bn
CO2Me
CO2Bn
When 5a was subjected to ozonolysis, the resultant alde-
hyde spontaneously cyclized to the racemic hemiaminal
6 (Scheme 2). The hydroxyl group was then methylated
and the resultant compound 7 reacted with BF3ÆOEt2
and allyl trimethyl silane at ꢀ78 °C!rt to give com-
pounds 8a and 8b in 48% yield and a ratio of 1:1. When
the minor isomer 5b was subjected to ozonolysis and
allylation, only compound 8c was obtained in 51% yield.
a
b
CbzHN
N
N
Boc
Boc
(2S, 3R, 5S)-8a
(2S, 3R, 5R)-8b
(2S, 3S, 5R)-8c
(2S, 3R, 5S)-9a
(2S, 3R, 5R)-9b
(2S, 3S, 5R)-9c
CO2Me
CO2Bn
N
c,d,e
CbzHN
O
The structure of compound 8a was confirmed by X-ray
crystallography (Fig. 2).
(2S, 3R, 5S, 8S)-3a
(2S, 3R, 5R, 8S)-3b
(2S, 3S, 5R, 8S)-3c
Compound 8a was then subjected to ozonolysis and the
resultant aldehyde subjected to a Horner–Emmons olef-
ination8 to give the dehydroamino acid 9a in 58% yield.
Scheme 3. Synthesis of Nle-Gly bicyclic dipeptide mimetic. Reagents
and conditions: (a) OsO4, NaIO4; (b) (MeO)2POCH(NHCbz)CO2Me,
DBU; (c) (S,S)-COD-EtDUPHOSRh(I)OTf, H2; (d) 20% TFA/DCM,
(e) pyridine, 50 °C.
2.3%
4.2%
1.9%
H6
N
H6
N
8H
4.8%
CO2Me
H9
CO2Bn
8H
4.8%
CO2Me
H9
CO2Bn
3H
3H
CbzHN
CbzHN
O
O
3b
3a
4.0%
H6
8H
1.9%
CO2Me
H9
CO2Bn
3H
N
CbzHN
O
3c
Figure 2. X-ray crystal structure of compound 8a.
Figure 3. NOE data for Nle-Asp bicyclic dipeptide mimetics.

J. M. Ndungu et al. / Tetrahedron Letters 47 (2006) 2233–2236
2235
compared with the weak value of 1.9% observed for
compound 3c.
and the Na-Boc group deprotected using TFA. The ami-
no group was then Fmoc-protected. For compound 3a,
both the carboxyl and amino groups were deprotected
by hydrogenation and the amino group Fmoc-pro-
tected. Fmoc/t-Bu solid phase peptide synthesis method
was used for the synthesis of the peptides. The peptides
JMN1-5 (Fig. 4) were consequently synthesized and
analyzed at both the CCK and opioid receptors.
To introduce the bicyclic dipeptide mimetics 1a, 1b, 1c,
and 2 into the peptides, the methyl ester was hydrolyzed
HO
H
S
H
When the peptides were evaluated at the opioid recep-
tors they showed weak activity at both the d- and l-opi-
oid receptors (Table 1). Peptide JMN2, however,
showed low micromolar binding affinity for both the
d- and l-opioid receptors. The only peptide that showed
an agonist effect at the opioid receptors is peptide JMN5
though it had low binding affinity.
N
O
H2N
H2N
H2N
N
H
N
H
N
NH2
NH2
NH2
O
O
N
H
O
N
H
O
O
O
CO2H
N
JMN1
H
HO
H
N
At the CCK receptors, peptides JMN1-4 showed low
micromolar binding affinities and biological activities
at the CCK-B receptors (Table 2). This can be attributed
to the C-terminal tetrapeptide (Trp-Nle-Asp-Phe), which
has been shown to be the minimum sequence required for
activity at CCK-B receptors. Introduction of a bicyclic
dipeptide mimetic for Nle-Asp (see JMN6) led to loss
of activity at the CCK receptors possibly due to interfer-
ence with the tetrapeptide unit. Substitution of D-Trp for
Trp in JMN4 to give JMN5 led to loss of activity at the
CCK receptors even though there was improved activity
at the opioid receptors. However, unlike in most other
CCK peptides where substitution of D-Trp leads to
antagonistic properties,1 JMN5 retained agonist proper-
ties. The peptides had relatively low binding profile at the
CCK-A receptors and showed no agonist biological
activity.
S
H
N
O
H
N
H
N
O
O
N
H
O
N
H
O
O
O
CO2H
N
H
JMN2
HO
H
N
S
H
N
O
H
N
H
N
O
O
N
H
O
N
H
O
O
O
CO2H
N
H
JMN3
HO
In conclusion, novel bicyclic dipeptide mimetic contain-
ing CCK/opioid chimeric peptides was synthesized and
evaluated at both the CCK and opioid receptors. Pep-
tides JMN1-5 were active at the CCK-B receptors while
JMN5 that contained a D-Trp showed weak opioid
activity. To discover peptides that will have agonist
properties at the opioid receptors and antagonist prop-
erties at the CCK-B receptors, more analogues of these
peptides will need to be synthesized. Our first target
H
S
H
N
N
O
O
H2N
H
H
N
N
NH2
O
O
N
N
H
H
O
O
O
CO2H
NH
JMN4
HO
H
Table 1. Biological evaluation at the opioid receptors
S
H
GTP bindinga
hDOR rMOR
EC50 (nM) EC50 (nM) IC50 (nM) IC50 (nM)
Competitionb
N
Drug
N
O
O
H2N
H
H
N
N
NH2
O
O
hDOR rMOR
N
N
H
H
O
O
O
CO2H
SNF9007 n/d
n/d
1800
n/d
NA
NA
NA
770
880
250
74
1850
337
1700
1400
2700
1500
5200
1000
9300
220
3500
8600
8100
3100
NH
RSA501
JMN1
JMN2
JMN3
JMN4
JMN5
JMN6
1000
JMN5
n/d
NA
NA
NA
460
NA
HO
H
N
O
O
O
OH
O
H
H
N
N
H
N
H2N
N
N
H
H
NH2
O
O
O
O
n/d = not determined, NA = no activity at 10ꢀ5 M.
[
b Competitive binding assays against radiolabeled [3H]DPDPE at
hDOR and [3H]DAMGO at rMOR. hDOR and rMOR were
expressed from CHO cell lines.
NH
a
35S]GTP-c-S binding assay.
JMN6
Figure 4. Novel bicyclic dipeptide mimetic containing CCK/opioid
chimeric peptides.

2236
J. M. Ndungu et al. / Tetrahedron Letters 47 (2006) 2233–2236
Table 2. Biological evaluation at CCK receptors
Drug
Functional analysisa
Binding affinityb (ki, nM)
CCK-A agonist activityc
hCCK-A
EC50 (nM)
hCCK-B
EC50 (nM)
hCCK-A
[
hCCK-B
125I] CCK8
125I] CCK8
[
SNF9007
RSA501
JMN1
JMN2
JMN3
JMN4
JMN5
JMN6
n/d
790
NA
NA
NA
NA
NA
NA
n/d
3100
4.1
6.9
2.3
1.9
160
NA
3300
140
740
1200
2400
810
2.1
14
70
100
16
11
1400
>10,000
n/d
None
None
None
None
None
None
None
1800
>10,000
n/d = not determined, NA = no activity at 10ꢀ5 M.
a Phosphoinositide (PI) hydrolysis assay in hCCK-A and hCCK-B receptors in HEK cell lines.
b Competition against [125I] CCK8 (sulfated) in hCCK-A and hCCK-B receptors in HEK cell lines in the presence of naloxone.
c Contraction of isolated tissue relative to initial contraction with KCl in the presence of naloxone in GPI/LMMP.
2. Unpublished work.
would be the substitution of D-Trp for Trp on all the
peptides, which may lead to improved opioid activity
as with JMN5. A combination of D-Trp4 and NMeNle5
may also lead to improved activities at both the opioid
and CCK receptors and possibly this may impart
CCK antagonistic properties.
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Acknowledgments
This work is supported by grants from the US Public
Health Service DA 12394 and DA 06284.
Supplementary data
6. Ndungu, J. M.; Gu, X.; Gross, D. E.; Ying, J.; Hruby, V. J.
Tetrahedron Lett. 2004, 45, 3245–3247.
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.tetlet.
2006.01.096.
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