Mimicking a proline tripeptide with pyrazolidines and a cyclopentane linker

Article abstract of DOI:10.1016/j.tetlet.2015.09.063

In this work the five-step assembly of a peptidomimetic structurally resembling Prolyl-Prolyl-Proline tripeptide is reported. Proline units are mimicked by two pyrazolidine rings connected to trans-1,2-cyclopentanedicarboxylic acid. The Pro-Pro-Pro mimetic resembles a tri-l-proline unit but with increased lipophilicity and structural constraints imposed by the linker.

Full text of DOI:10.1016/j.tetlet.2015.09.063

Accepted Manuscript
Mimicking a Proline Tripeptide with Pyrazolidines and a Cyclopentane Linker
Peter Vertesaljai, Iryna O. Lebedyeva, Alexander A. Oliferenko, Xin Qi, Junjie
Fu, David A. Ostrov, Abdullah M. Asiri, C. Dennis Hall, Alan Katritzky
PII:
S0040-4039(15)30104-0
DOI:
http://dx.doi.org/10.1016/j.tetlet.2015.09.063
TETL 46735
Reference:
Tetrahedron Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
13 July 2015
8 September 2015
16 September 2015
Please cite this article as: Vertesaljai, P., Lebedyeva, I.O., Oliferenko, A.A., Qi, X., Fu, J., Ostrov, D.A., Asiri, A.M.,
Dennis Hall, C., Katritzky, A., Mimicking a Proline Tripeptide with Pyrazolidines and a Cyclopentane Linker,
Tetrahedron Letters (2015), doi: http://dx.doi.org/10.1016/j.tetlet.2015.09.063
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Graphical Abstract
Mimicking a proline tripeptide with pyrazolidines
and a cyclopentane linker
Peter Vertesaljai, Iryna O. Lebedyeva, Xin Qi, Alexander A. Oliferenko, Junjie Fu, David A. Ostrov, Abdullah M.
Asiri, C. Dennis Hall, and the late Alan Katritzky

Tetrahedron Letters
journal homepage: www.elsevier.com
Mimicking a Proline Tripeptide with Pyrazolidines and a Cyclopentane Linker
Peter Vertesaljaia, Iryna O. Lebedyevaa, b, Alexander A. Oliferenkoc, Xin Qid, Junjie Fud, David A. Ostrove, Abdullah
M. Asiri,f C. Dennis Halla,∗and the late Alan Katritzkya
a Center for Heterocyclic Compounds, Department of Chemistry, University of Florida, Gainesville, FL 32611-7200, USA
b Department of Chemistry and Physics, Georgia Regents University, 1120 15th Street SCI W3005, Augusta, GA 30912, USA
c EigenChem Technologies Inc., Alachua, FL 32615
d Department of Medicinal Chemistry, College of Pharmacy, University of Florida, Gainesville, FL 32611-7200, USA
eDepartment of Pathology, Immunology and Laboratory Medicine, College of Medicine, University of Florida, Gainesville, FL 32611-7200, USA
f Center of Excellence For Advanced Materials Research , King Abdulaziz University, Jeddah 21589, Saudi Arabia
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
In this work the five-step assembly of a peptidomimetic structurally resembling Prolyl-Prolyl-
Proline tripeptide is reported. Proline units are mimicked by two pyrazolidine rings connected to
trans-1,2-cyclopentanedicarboxylic acid. The Pro-Pro-Pro mimetic resembles a tri-L-proline unit
but with increased lipophilicity and structural constraints imposed by the linker.
Available online
2015 Elsevier Ltd. All rights reserved.
Keywords:
Peptide
Heterocycles
Peptidomimetic
Proline
Coupling
Corresponding author. Tel: (352) 392-0554; fax: (352) 392-9199; e-mail: charlesdennishall@gmail.com

Introduction
Figure 2. Reported aza-mimetics 5-9 of L-proline unit.
Several peptidomimetics of type 7 with the aza-Pro at different
positions have been reported and suggest that in contrast to the
natural proteins, the presence of aza-Pro in the chain prevents the
formation of a β-turn for the following residue.14 Recently
Peptides play an increasingly important role in the treatment
of diabetes, cancer, metabolic, cardiovascular, and infectious
diseases.1 Based on the fact that the FDA approved six peptides
in 2012 (lucinactant, peginesatide, pasireotide, carfilzomib,
linaclotide, and teduglutide), peptide and peptidomimetic
therapeutic compositions have spurred additional efforts in drug
discovery.2
reported aza-prolines
8 and 9 synthesized by 5-exo-dig
cyclization (favored over 6-endo-dig) expanded the range to
optically active aza-prolines.15
In this work a new approach towards the cis-trans-cis Pro-
Pro-Pro triplet has been developed by combining two
pyrazolidine rings with trans-DL-1,2-cyclopentanedicarboxylic
acid. In this way, L-proline is mimicked by the aza-proline units
as well as the cyclopentane ring.
L-Proline is often found at the end of an α helix or in protein
turns or loops and is known to change amino acid triplets to the
cis-trans-cis configuration.3 The Met97TyrProProProTyr102 motif
of T-lymphocyte-associated protein 4 (CTLA-4) and CD28
sequences is critical in mediating interaction with B7 membrane
protein. Mutagenesis experiments indicate that the polyproline
motif is essential for the binding of CG152, CD28, and ICOS
receptors to their respective ligands.4,5 The cis-trans-cis
configuration of the three consecutive proline residues in CTLA-
4 and their relatively low B factors, suggests that these proline
residues play crucial roles in binding B7.4 Constrained unnatural
amino acids 1 and 2 (Figure 1), showing structural features
similar to proline, have been used to explore the capabilities of β-
turn scaffolds.6 In a library of proline mimetics 2, the R2
substituents were diversified and the five-membered ring was
constrained as the 1,2,3-triazole moiety.7 Cyclohexane-1,2-
dicarboxylic acid and 2-aminocyclohexanecarboxylic acid are
proline isosteres in thrombin inhibitors.8 Proline mimetics
Synthesis of the N-Boc-N-Cbz di-protected hydrazine 10
proceeded first by mono-protection of hydrazine hydrate with
Boc-anhydride, followed by protection of the remaining amino
group with Cbz. N,N’-Diprotected hydrazine 11 was then
cyclized to form the pyrazolidine ring by reaction with1,3-
dibromopropane and sodium hydride in DMF to give 12 in 78%
yield (Scheme 1).
Scheme 1. Assembly of N,N’-diprotected pyrazolidine ring 12.
containing
cyclopentane-1,2-dicarboxylic
acid
3
and
The Boc group of N,N’-diprotected intermediate 12 was
deprotected by 2N HCl/MeOH solution to give hydrochloride 13
cyclopentene-1,5 dicarboxylic acid 4 have been prepared and
crystallized with thrombin to study the interaction with the in quantitative yield. N-Cbz-protected pyrazolidine 13 was
binding site of these thrombin inhibitors.9
coupled with trans-DL-1,2-cyclopentanedicarboxylic acid 14 using
standard EDCI/HOBt-mediated coupling to give Cbz-protected
mimetic tri-proline residue 15. Intermediate 15 was deprotected in
situ to give the final dipyrazolidine system 16 in 89% yield. Thus,
a tri-proline mimic was synthesized in five steps, namely, ring
assembly, coupling, protection and deprotection.
The peptidomimetic 16 was analyzed computationally to
compare it with the native triprolyl scaffold. For computational
purposes, the C-terminus was removed from Pro-Pro-Pro to make
it comparable with 16. Molecular structures were drawn with
ChemBioDraw Ultra 12 and optimized with PC Model using the
MMX force field. Physical properties listed in Table 1 were
derived from the same force field. Although rather similar to
triprolyl in terms of the steric energy and heat of formation,
compound 16 shows higher strain due to the conformationally
restricted N-N bonds of the pyrazolidine moieties. As indicated by
the dipole moments, peptidomometic 16 is significantly less polar
than the triprolyl scaffold, which translates into higher
lipophilicity. This enhances pharmacokinetic properties such as
absorption and distribution.
Figure 1. Reported mimetics of L-proline.
Peptidomimetics with aza-moieties are known to be more
resistant to protease cleavage.10,11 Aza-Pro
5 has been
successfully incorporated into solid-phase organic synthesis
providing stable aza-Pro-containing peptidomimetics.12 Aza-Pro
6, was included in SAR studies of small molecules which mimic
the FKBR binding protein.13

Scheme 2. Synthesis of trans-DL-cyclopentane-1,2-diylbis(pyrazolidin-1-ylmethanone) 16.
Dr. M. C. A. Dancel (University of Florida) for HRMS
analyses.
The ability of proline to form cis-peptide bonds and undergo cis-
trans isomerization has been well-studied.6 The conformations
17
of poly-proline peptides have been calculated16,
and it was
References and notes
suggested that the trans-cis conversion of poly-proline chain may
occur not only at the terminus but also in the middle of the
1. Du, A. W.; Stenzel, M. H. Biomacromolecules 2014, 15, 1097.
2. Albericio, F.; Kruger, H. G. Future Med. Chem. 2012, 4, 1527.
3. Georgiades, J. A.; Schroeder-Georgiades, I. M. In Role of proline
rich peptides in cellular communication mechanisms and treatment
of diseases; Google Patents, 2011.
19
chain.18,
X-ray analysis of several aza-proline containing
peptides revealed that because of steric hindrance, both nitrogen
atoms are not co-planar. Reduced electronic conjugation in the
two aza-Pro adjacent amide groups explains the longer amide
bond distances and the weak proton-accepting character of the
two pyrazolidine nitrogens. The absolute configuration of both
aza-Pro-nitrogens depends on the chemical nature of the
sequence but in all cases the aza-Pro residue assumes the same
three-dimensional structure and causes folding opposite to that
induced by proline.20
4. Ostrov, D. A.; Shi, W.; Schwartz, J. C.; Almo, S. C.; Nathenson, S.
G. Science (New York, N.Y.) 2000, 290, 816.
5. International Journal of Peptides 2012, 2012, 14.
6. Trabocchi, A.; Cini, N.; Menchi, G.; Guarna, A. Tetrahedron Lett.
2003, 44, 3489.
7. Rodríguez-Borges, J. E.; Gonçalves, S.; do Vale, M. L.; García-
Mera, X.; Coelho, A.; Sotelo, E. J. Comb. Chem. 2008, 10, 372.
8. Semple, J. E.; Rowley, D. C.; Brunck, T. K.; Ripka, W. C. Bioorg.
Med. Chem. Lett. 1997, 7, 315.
9. Noteberg, D.; Branalt, J.; Kvarnstrom, I.; Linschoten, M.; Musil, D.;
Nystrom, J. E.; Zuccarello, G.; Samuelsson, B. J. Med. Chem. 2000,
43, 1705.
10. Tal-Gan, Y.; Freeman, N. S.; Klein, S.; Levitzki, A.; Gilon, C. Chem.
Biol. Drug Des. 2011, 78, 887.
11. Melendez, R. E.; Lubell, W. D. J. Am. Chem. Soc. 2004, 126, 6759.
12. Freeman, N. S.; Tal-Gan, Y.; Klein, S.; Levitzki, A.; Gilon, C. J.
Org. Chem. 2011, 76, 3078.
13. Wilkinson, D. E.; Thomas Iv, B. E.; Limburg, D. C.; Holmes, A.;
Sauer, H.; Ross, D. T.; Soni, R.; Chen, Y.; Guo, H.; Howorth, P.;
Valentine, H.; Spicer, D.; Fuller, M.; Steiner, J. P.; Hamilton, G. S.;
Wu, Y.-Q. Bioorg. Med.Chem. 2003, 11, 4815.
Table 1. Comparison of calculated physical properties
Property
Trans isomer 16 Triprolyl
Steric energy, kcal/mol
Strain energy, kcal/mol
36.1
23.8
33.2
17.7
88.7
5.46
1.37
Heat of formation, kcal/mol 87.6
Dipole moment, D
Log P (calc.)
3.97
4.43
14. Lecoq, A.; Boussard, G.; Marraud, M.; Aubry, A. Tetrahedron Lett.
1992, 33, 5209.
15. 15 Bouvet, S.; Moreau, X.; Coeffard, V.; Greck, C. J. Org. Chem.
2013, 78, 427.
16. 16 Dasgupta, B.; Chakrabarti, P.; Basu, G. FEBS Lett. 581, 4529.
17. 17 Zhang, W.-J.; Berglund, A.; Kao, J. L. F.; Couty, J.-P.;
Gershengorn, M. C.; Marshall, G. R. J. Am. Chem. Soc. 2003, 125,
1221.
18. 18 Tanaka, S.; Scheraga, H. A. Macromolecules 1974, 7, 698.
19. 19 Zhao, J.; Siu, C.-K.; Shi, T.; Hopkinson, A. C.; Siu, K. W. M. J.
Phys. Chem. B 2009, 113, 4963.
In conclusion, a novel mimetic of L-Pro-L-Pro-L-Pro 16
consisting of two pyrazolidine moieties linked by amide bonds
to a cyclopentane ring in the trans configuration has been
synthesisized in five steps from N,N’-diprotected hydrazine.
Mono N-terminus deprotection of the product followed by
coupling with trans-DL-1,2-cyclopentanedicarboxylic acid and
deprotection of the remaining secondary amino group, resulted
in a di-aza mimic of the tri-proline peptide. Molecular
structures of the original Pro-Pro-Pro tripeptide and the di-aza
mimic were studied using molecular mechanics, and it was
concluded that the non-polar cyclopentane ring of 16 increases
the lipophilicity of the peptidomimetic, while the pyrazolidine
moieties instil conformational constraints that induce turn.
20. 20 AndrÉ, F.; Vicherat, A.; Boussard, G. U. Y.; Aubry, A.;
Marraud, M. J. Pept. Res. 1997, 50, 372.
21. 21 Jablonski, J. J.; Basu, D.; Engel, D. A.; Geysen, H. M. Bioorg.
Med. Chem. 2012, 20, 487.
22. 22 East, S. P.; Ayscough, A.; Toogood-Johnson, I.; Taylor, S.;
Thomas, W. Bioorg. Med. Chem. Lett. 2011, 21, 4032.
23. 23 Brazier, J. B.; Cavill, J. L.; Elliott, R. L.; Evans, G.; Gibbs, T. J.
K.; Jones, I. L.; Platts, J. A.; Tomkinson, N. C. O. Tetrahedron 2009,
65, 9961.
Acknowledgments
Preparation of tert-butyl hydrazinecarboxylate 10: Hydrazine hydrate (113
mg, 2.26 mmol, 2.26 eq.) was dissolved in IPA (5 mL) and cooled down to
0 °C. Di-tert-butyl carbonate (174 mg, 1 mmol, 1 eq.) in IPA (1 mL) was
added drop-wise and the mixture was stirred for 2 hours. The solvent was
then evaporated and the residue was dissolved in DCM, dried over MgSO4,
and filtered. Evaporation of the solvent gave tert-butyl
We thank the University of Florida and the Kenan Foundation
for support with this project. This paper was also funded in
part by generous support from King Abdulaziz University,
under grant No D-006/431. The authors, therefore,
acknowledge the technical and financial support of KAU. The
authors are grateful to Mr. Z. Wang for helpful discussions and
1
hydrazinecarboxylate 10 as a white semi-solid (128 mg, 97 %). H NMR

(300 MHz, CDCl3) δ 6.73 (s, 1H), 3.83 (s, 2H), 1.46 (s, 9H); 13C NMR (75
MHz, CDCl3) δ 158.2, 80.1, 28.2.21
Preparation of 1-benzyl 2-(tert-butyl) hydrazine-1,2-dicarboxylate 11:
Tert-butyl hydrazinecarboxylate 10 (132 mg, 1 mmol) was dissolved in dry
DCM and cooled down to -78 °C. Benzyl chloroformate (20.5 mg 1.2
mmol) was added drop-wise followed by Na2CO3 (106 mg, 1 mmol). The
reaction mixture was allowed to r. t. and stirred overnight. Filtration,
evaporation of solvents, and recrystallization from hexanes/EtOAc 50:1
gave 1-benzyl 2-(tert-butyl) hydrazine-1,2-dicarboxylate 11 as a white
solid (226 mg, 85%), mp 75.0−77.0 °C, lit. 72–74 oC. 1H NMR (300 MHz,
CDCl3) δ 7.34 (s, 5H), 6.80 (s, 1H), 6.49 (s, 1H), 5.16 (s, 2H), 1.46 (s, 9H);
13C NMR (75 MHz, CDCl3) δ 156.9, 155.9, 135.8, 128.7, 128.5, 128.4,
81.9, 67.8, 28.3.22
Preparation of 1-benzyl 2-(tert-butyl) pyrazolidine-1,2-dicarboxylate
12: 1-Benzyl 2-(tert-butyl) hydrazine-1,2-dicarboxylate 11 (266 mg, 1
mmol, 1 eq.) was dissolved in anhydrous DMF (10 mL) and cooled down
to 0°C. Sodium hydride 60% dispersion in mineral oil (84 mg, 2.1 mmol,
2.1 eq.) was added and the mixture was stirred for 30 minutes under
nitrogen atmosphere. 1,3-Dibromopropane (0.11 mL, 1.05 mmol, 1.05 eq.)
was added to the mixture and it was stirred overnight at room temperature.
The mixture was then acidified with saturated citric acid solution and
extracted three times with diethyl ether. The combined organic layers were
washed with citric acid solution three times, dried over MgSO4, filtered
and the solvent was evaporated. Purification by column chromatography
(hexanes-EtOAc 3:1) gave 1-benzyl 2-(tert-butyl) pyrazolidine-1,2-
dicarboxylate 12 as a colorless oil (238 mg, 78 %). 1H NMR (300 MHz,
CDCl3) δ 7.42–7.24 (m, 5H), 5.18 (d, J = 31.3 Hz, 2H), 3.92 (d, J = 23.3
Hz, 2H), 3.39–3.07 (m, 2H), 2.09–1.95 (m, 2H), 1.42 (s, 9H); 13C NMR
(75 MHz, CDCl3) δ 156.7, 156.2, 136.2, 128.4, 128.0, 127.8, 81.4, 67.6,
46.7, 46.2, 28.0, 25.6.22
Preparation of benzyl pyrazolidine-1-carboxylate hydrochloride 13: 1-
benzyl 2-(tert-butyl) pyrazolidine-1,2-dicarboxylate (310 mg, 1 mmol, 1
eq.) was dissolved in 2N HCl/MeOH and it was stirred at r. t. for 24 hours.
Evaporation of the solvent followed by recrystallization from ether gave
benzyl pyrazolidine-1-carboxylate hydrochloride 13 as a colorless oil (503
mg, 86 %); 1H NMR (300 MHz, CD3OD) δ 7.61–7.25 (m, 5H), 5.28 (d, J =
1.7 Hz, 3H), 3.77 (td, J = 6.9, 1.9 Hz, 2H), 3.63 (td, J = 7.0, 1.9 Hz, 2H),
2.38 (pd, J = 6.8, 1.8 Hz, 2H); 13C NMR (75 MHz, CD3OD) δ 136.7,
129.9, 129.8, 129.7, 70.3, 47.6, 25.7.23
Preparation
of
trans-DL-cyclopentane-1,2-diylbis(pyrazolidin-1-
ylmethanone) 16: Benzyl pyrazolidine-1-carboxylate hydrochloride 13
(534 mg, 2.2 mmol), trans-DL-1,2-cyclopentanedycarboxylic acid 14 (158
mg, 1 mmol), EDCI (341 mg, 2.2 mmol), and 1H-benzo[d][1,2,3]triazol-1-
ol hydrate (368 mg, 2.4 mmol) were dissolved in dry THF (20 mL) and
stirred at 0 °C. TEA (0.3 mL, 2.1 mmol) was added to the mixture, which
was then stirred at 0 °C for 30 minutes. After that, it was allowed to r. t.
and stirred overnight. After the solvent was evaporated, the residue was
dissolved in EtOAc, washed with water and brine, dried over MgSO4, and
the solvent was evaporated again. Without further purification, the crude
intermediate 15 was deprotected using standard hydrogenation conditions
(H2/Pd/C, r. t. in 10 mL of MeOH). After filtration and evaporation of the
solvent, the crude product was purified by preparative HPLC. Product 16
is a colorless oil (236 mg, 89 %). 1H NMR (600 MHz, D2O) δ 3.70 – 3.57
(m, 1H), 3.55–3.46 (m, 1H), 3.39 (t, J = 7.5 Hz, 3H), 3.11 (q, J = 9.0 Hz,
1H), 3.03–2.83 (m, 4H), 2.09 (p, J = 6.9 Hz, 1H), 2.06–1.93 (m, 5H),
1.79–1.66 (m, 2H), 1.66–1.50 (m, 2H); 13C NMR (150 MHz, D2O) δ 174.9,
174.6, 170.8, 170.5, 47.3, 47.3, 46.8, 46.3, 46.3, 46.2, 46.1, 45.8, 45.6,
45.5, 44.9, 44.8, 29.9, 29.7, 29.5, 26.8, 26.1, 24.8, 24.7. HRMS
(C13H22N4O2) Calcd: 266.17, Found: 267.3
Supplementary Material
1H and 13C spectra of all novel compounds listed in
experimental section. This material is available free of
charge
electronically