Design and Synthesis of Novel mu-delta

Heterodimer Selective Antagonists

Lisa

1
Daconta ,

Keith

2
Olsen ,

and Dr. Victor

3
Hruby

Department of Chemistry and Biochemistry, Department of Pharmacology, University of Arizona

1. Swell Polystrene-Wang-Lys(Mtt)-FMOC resin in DMF & DCM

2. Deprotect FMOC group on Lys backbone with 10% Pip / 5%
DBU in 1:1 DMF:NMP
3. Washes (DMF, DCM, THF, NMP) for 1 min at 3 times each
4. 3 equivalent coupling to appropriate amino acid with 0.25M
HOAt solution in 3:1 NMP:DCM and 3 equv of DIC (2 hrs)

4. Deprotect FMOC group on the coupled amino acid

with 10% Pip / 5% DBU in 1:1 DMF:NMP
5. Washes (DCM, THF, NMP) for 2-4min at 3 times each
6. 3 equv coupling to appropriate aa
* Perform 2 equv double coupling with 2:2:4 solution of
PyBOP:HOAt:DIPEA in NMP for D1Nal, Phe, Pro, and
Tyr
6. Repeat steps 4-6 until mu construct (blue) + linker is
complete

3.
4.
5.
6.

Figure 9: Dose-Response Curve of select antagonists through the testing

of DORCHO cells in the presence of 100 nM [D-Ala]Deltorphin-II (a known
agonist to illicit a response) along with a known antagonist (Naloxone) as a
positive control for inhibition5.

Concentrate peptide under vigorous Argon bubbling

Precipitate peptide in ice cold ether, decant ether
Dry peptide under vacuum
Purify via HPLC or obtain mass via Mass-Spec

Delta
Table 2: IC50 and IMAX of DOR GTPgS Antagonist Activity in the presence of 100 nM [DAla]Deltorphin-II5

1287

Figure 4: Optimized procedure for KO302-18

(major product mass indicated)
1451

Figure 3: Optimized procedure for KO301-18

(major product mass indicated)

Heterodimer complex is upregulated

after morphine treatment, resulting in
greater tolerance for the opioid drug
than MOR or DOR separately

Generating a selective MORDOR

heteromer antagonist provides a useful
pharmacological tool and potential
therapeutic for opioid induced
tolerance.
Figure 1: A pictorial illustration of the Mu (MOR) and Delta (DOR) receptors as monomers
(top) and the Mu-Delta (MORDOR) heterodimer (bottom), and the pharmacological
responses of either state5.

GOAL: Identify a lead compound that

acts as a selective Mu-Delta
heteromer antagonist.

Deprotection Trials: Deprotections using 20%

Piperidine and 5% DBU in DMF along with couplings
using 0.25M HOAt with DIC in 3:1 NMP:DCM (or
second coupling solution where applicable) produced
the greatest yield.
Optimized Synthesis: Both syntheses returned a high
percent yield and a high purity of desired peptide for
the use of 10% Pip / 5% DBU as deprotection
solution with couplings using 0.25M HOAt with DIC in
3:1 NMP:DCM (or second coupling solution where
applicable)

1591

Figure 7: Deprotection trial (20% Pip / 5% DBU) in DMF

Figure 6: Deprotection trial (20% Piperidine) in DMF

Figure 2: A demonstration of a heterobivalent antagonist binding only to the respective

monomer when the mu and delta monomers are too far apart (top) and the same
heterobivalent ligand binding preferentially to the heterodimer MORDOR complex
(below)5.

DOR antagonist is preserved in the MOR-18-DOR scaffold.

The best deprotection solution for FMOC removal is 20%
Piperidine and 5% DBU in 1:1 NMP:DMF
The optimized synthesis demonstrated that a deprotection
solution of 10% Piperidine and 5% DBU was efficient
For bulkier amino acid groups, it is optimal to perform 2
coupling steps. The first is the general coupling solution and
the second solution is composed of 2:2:4
PyBOP:HOAt:DIPEA in NMP.
Future studies include assessing antagonist activity at MOR
and the MORDOR heteromer

540

1287

Mu

1200

Some Analgesia
Some Tolerance
Affective Components

This ligand design

maintains the antagonist
activity of DOR, indicating
promising preliminary
results.

1. Shrink resin by washing thrice at 1 minute each (THF, DMF,

DCM, ether, and MeOH)
2. Cleave peptide from resin with 95% TFA, 1% TIS, and 2%
water for 2.5 hours

Figure 5: Deprotection trial (1% DBU) in DMF

Analgesia
Tolerance
Addiction
Constipation

The DOR pharmacophore

maintains and potentiates
antagonist activity at the
Delta Opioid Receptor
(DOR)

1. Deprotect the (Mtt) group on the Lysine sidechain with

2% TFA in DCM for 50 total minutes
2. Washes (DCM, THF, NMP)
3. 3 equivalent coupling to appropriate amino acid

* 2 equv double couple for Tic and Tyr aa with 2:2:4 solution of
PyBOP:HOAt:DIPEA in NMP
5. Washes (DMF, DCM, THF, NMP)
6. Deprotect FMOC group on the coupled amino acid with 10%
Pip / 5% DBU in 1:1 DMF:NMP
7. Repeat steps 3-6 until delta construct (red) + linker is complete
* Cap Tyrosine with 1:1:18 Acetic Anhydride:DIPEA:DMF (15 min)

1294

Clinical opioid overdoses (i.e. oxycodone, hydrocodone,

methadone, and/or fentanyl) kill 78 Americans every
day8. Furthermore, the increasing dependency and addiction
to opioid drugs in order to treat chronic pain (pain enduring for
greater than 3 months) is a national epidemic. Recent studies
suggest that different opioid subtypes may physically interact
via heterodimerization constituting distinct GPCR signaling
units. The mu (MOR) and delta (DOR) receptors have been
found to dimerize in vivo, creating heterodimer complexes that
are proposed to be involved in the developing opioid drug
tolerance during chronic pain pathology treatments4. Based
upon research conducted by Portoghese et al., these dimeric
GPCR complexes (such as the mu-delta heterodimer) can be
targeted with bivalent ligands, which could result in significant
therapeutic advantages for the treatment of chronic pain and
potential reduction of addiction; however, no selective
antagonistic ligands have been reported7. Therefore, we report
the design and synthesis of a heterobivalent mu-delta ligand
(MOR-18-DOR) which tethers a mu to a delta antagonist
pharmacophore separated by an 18-atom spacer.

KO302-18: (BOC)Tyr(tBu)-Tic-Gly-Ava-Lys(Mtt)-Gly-D1Nal-Phe-Pro-(tBu)Tyr(BOC)

Figure 8: Deprotection trial (20% Pip / 5% DBU) in NMP

Protein by Method

Percent Yield

Crude
Purity

1.
2.
3.
4.
5.
6.

Optimized KO301-18

97%

~50%

Optimized KO302-18

95%

~50%

7.
8.

Deprotection (1% DBU in DMF) KO302-18

<10%

<10%

Deprotection (20% Pip in DMF) KO302-18

<10%

<10%

Deprotection (20% Pip/5% DBU in DMF) KO302-18

22%

<10%

Deprotection (20% Pip/5% in NMP) KO302-18

<10%

<10%

Table 1: Percent yield and crude purity results of deprotection trials and optimized
syntheses for the creation of KO301-18 and KO302-185

CBC Spring 2016 Graduate, University of Arizona (lisadaconta@email.arizona.edu)

CBC Hruby Group PhD Graduate Researcher (keitholson@email.arizona.edu)
Regents Professor Emeritus of Chemistry and Biochemistry (hruby@email.arizona.edu)
Massotte, D. "In Vivo Opioid Receptor Heteromerization: Where Do We Stand?" Wiley Online Library. British Journal of Pharmacology, 1 July 2014. Web. 11
Apr. 2016.
Olsen, Keith. Development of Selective MOR-DOR Heterodimer Antagonists UA Science (2015) Print.
Ong, Edmund W., and Catherine M. Cahill. "MolecularPerspectives for Mu/delta Opioid Receptor Heteromers as Distinct, Functional Receptors." Cells. MDPI, 3
March 2014. Web. 17 Dec. 2015.
Shonberg, Jeremy, Peter J. Scammells, and Ben Capuano. "Design Strategies for Bivalent Ligands Targeting GPCRs." ChemMedChem 6.6 (2011): 963-74.
Web.
"Understanding the Epidemic." Centers for Disease Control and Prevention. Centers for Disease Control and Prevention, 14 Mar. 2016. Web. 11 Apr. 2016.

The author would like to thank the University of Arizona and the Hruby Group for
providing all necessary resources and facilities throughout the investigation. Vast
thanks are also extended to Mr. Keith Olsen for extensive aid in all areas of inquiry
and guidance.