Solid-phase synthesis of tetrahydropyridazinedione-constrained peptides

Article abstract of DOI:10.1021/ol5026684

The design and solid-phase synthesis of tetrahydropyridazine-3,6-dione (Tpd) peptidomimetics derived from backbone-aminated peptides is reported. The described protocol features the synthesis of chiral α-hydrazino acids suitable for chemoselective incorporation into growing peptide chains. Acid-catalyzed cyclization to form the Tpd ring during cleavage affords the target peptidomimetics in good yield and purity. The scope of Tpd incorporation is demonstrated through the synthesis of constrained peptides featuring nucleophilic/electrophilic side chains and sterically encumbered α-substituted hydrazino acid residues. (Chemical Equation Presented).

Full text of DOI:10.1021/ol5026684

Letter
pubs.acs.org/OrgLett
Solid-Phase Synthesis of Tetrahydropyridazinedione-Constrained
Peptides
Chang Won Kang,† Sujeewa Ranatunga,† Matthew P. Sarnowski,‡ and Juan R. Del Valle*,†,‡
†Drug Discovery Department, H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida 33612, United States
‡Department of Chemistry, University of South Florida, Tampa, Florida 33620, United States
S
* Supporting Information
ABSTRACT: The design and solid-phase synthesis of tetrahydropyr-
idazine-3,6-dione (Tpd) peptidomimetics derived from backbone-
aminated peptides is reported. The described protocol features the
synthesis of chiral α-hydrazino acids suitable for chemoselective
incorporation into growing peptide chains. Acid-catalyzed cyclization to
form the Tpd ring during cleavage affords the target peptidomimetics in
good yield and purity. The scope of Tpd incorporation is demonstrated
through the synthesis of constrained peptides featuring nucleophilic/electrophilic side chains and sterically encumbered α-
substituted hydrazino acid residues.
eptide backbone tethering strategies have proven useful for
Pthe elucidation of potential bioactive conformations and
for enhancing peptide stability. The Freidinger−Veber lactam1
and related structures1c,2 represent important examples of cyclic
constraints suitable for conformational scanning of linear
peptides. These motifs often retain the native composition of
the peptide backbone while restricting rotation about the ω and
ψ bonds across a dipeptide subunit. Given the resurgence of
peptides as lead structures for chemical probe and drug
discovery, there remains a need for new constraints to
interrogate local conformation. The ability to readily
incorporate covalent tethers using solid-phase peptide synthesis
(SPPS) techniques will greatly enhance their utility in
structure−activity relationship studies.
In an effort toward synthetically accessible rigidified peptides,
Figure 1. Design of Tpd-constrained peptides.
we targeted a tetrahydropyridazine-3,6-dione (Tpd) scaffold
derived from an N-aminated aspartyl dipeptide precursor
(Figure 1).3 The peptide backbone amino substituent would
provide not only a nucleophilic handle for intramolecular
cyclization but also an additional site for derivitization or
potential hydrogen-bonding interactions. Engaging the pro-R
Cα substituent as the complementary tethering site affords a
Tpd constraint favoring the ψ torsion typical of a β-strand,
whereas tethering via the pro-S site would favor a −120° ψ
dihedral angle reminiscent of a type II′ β-turn. Thus, changing
the stereochemistry of the presumed aspartate precursor could
provide access to distinct conformational probes.
In contrast to peptoid (N-alkyl glycine oligomer) synthesis,
the assembly of peptides bearing substituents on both Nα and
Cα remains a considerable challenge. The synthesis of peptide
tertiary amide (PTA) libraries on solid support was recently
reported based on a submonomer approach.4 The on-resin
syntheses of some backbone aminated peptides have also been
described, though these have so far been limited to N-amino
glycine-containing structures (“azapeptoids”)5 or to natural
products harboring piperazic acid derivatives.6 To the best of
our knowledge, there exists no general methodology for
preparing Cα-substituted N-amino peptide derivatives by
conventional SPPS.
To assess the feasibility of hydrazino acid acylation, we first
screened a variety of coupling reagents for the reaction between
the known esters 17 and Fmoc-protected aspartate derivatives
in solution (Scheme 1). Even with the less hindered N-amino
glycine ester 1a, most common condensation reagents failed to
effectively promote dipeptide formation (entries 1−3 and 6−
8). The use of HATU gave moderate yields of 2a but afforded
none of the desired product when N-amino alanine derivative
1b was employed as the substrate (entries 4 and 5).
Preactivation of Fmoc-D-Asp(tBu)-OH or Fmoc-D-Asp(Me)-
OH as the mixed anhydride5c gave good yields of 2a−d;
Received: September 9, 2014
Published: October 8, 2014
© 2014 American Chemical Society
5434
dx.doi.org/10.1021/ol5026684 | Org. Lett. 2014, 16, 5434−5437

Organic Letters
Letter
Scheme 1. Solution Phase Synthesis of Tpd Dipeptide
Mimics
Scheme 2. SPPS of Tpd-Gly Peptidomimetics
diastereomeric mixtures of Tpd peptidomimetics following
cleavage. After ruling out potential racemization of the D-Asp
chiral center, we confirmed the configurational instability of the
intermediate chiral α-bromoacetamide under the conditions
required for efficient SN2 displacement (Scheme 3). Reaction of
Scheme 3. Attempted Submonomer-Based SPPS of Cα,Nα-
Disubstituted Variants
a
All reactions carried out at rt for 24 h with 2−3 equiv of carboxylic
b
acid and coupling reagent, and excess base. Thionyl chloride was
used to generate Fmoc-(D)Asp(Me)-Cl. The isolated acid chloride was
reacted with the hydrazo ester derivatives in the presence of collidine.
however, lower conversions were again observed with the α-
substituted coupling partner 1b (entries 9−12). Yields
improved with in situ generation of the Fmoc-protected
amino acid chloride using triphosphgene. Optimal yields
(>80%) and cleaner reactions were obtained when the acid
chloride was preformed in the presence of thionyl chloride and
isolated prior to condensation (entries 15 and 16).8 We found
Fmoc-Asp(Me)-Cl to be a remarkably shelf-stable solid that
showed no appreciable erosion of enantiopurity during short-
term storage or coupling at elevated temperatures.9 Formation
of the tetrahydropyridazinedione ring was achieved in moderate
yield using DIC/DIEA/DMAP following Boc and tert-butyl
ester deprotection of 2a. Alternatively, ring closure was effected
via acidolysis of 2c and subsequent heating in toluene to give 3
in 53% yield.
We next explored a submonomer approach to prepare a
model peptidomimetic containing a Tpd-Gly subunit on solid
support. Analogous to well-established methods for peptoid
synthesis,10 we employed α-bromoacetic acid as a building
block and carried out subsequent SN2 displacement with tert-
butyl carbazate (Scheme 2). Condensation with 3 equiv of
Fmoc-D-Asp(Me)-Cl in the presence of 9 equiv of collidine (50
°C, 1 h × 3) was followed by standard peptide elongation and
cleavage from the resin with 95:5 TFA/H2O. Under these
conditions, acidolysis was attended by Tpd ring closure to give
peptidomimetic 5 as the major product in 23% overall yield
after RP-HPLC purification.
6a with 2 M tert-butyl carbazate in DMF at 50 °C followed by
acidic cleavage from the resin afforded a 1.2:1 diastereomeric
mixture of products (7), as judged by LCMS (acetone was
added to the cleavage cocktail to provide more well-resolved
hydrazone derivatives). The limitations of a bromoacetamide
submonomer protocol for Tpd synthesis were further high-
lighted in our attempt to prepare the corresponding N-amino
phenylalanine derivative on solid support. Incubation of 6b
with tert-butyl carbazate gave rise to cinnamide 8 as the major
product. Presumably, the competing elimination pathway
would also complicate the synthesis of other Tpd-Xaa
dipeptides capable of forming extended conjugated systems
(Xaa = Tyr, Trp, His, Asp/Asn).
To circumvent these issues, we opted to incorporate Boc-
protected hydrazino acid building blocks into growing peptide
chains. Chiral α-hydroxy esters 9 were prepared by
diazotization of the corresponding D-amino acids11 followed
by esterification (Scheme 4). Installation of the tert-butyl
carbazate group via the triflate and subsequent saponification
gave acids 11a−f. Remarkably, solutions of 11 in 0.5 M aq. HCl
partitioned readily into ethyl acetate, suggesting that the α-
In the course of extending the submonomer approach to Cα-
substituted (nonglycine) variants, we observed the formation of
5435
dx.doi.org/10.1021/ol5026684 | Org. Lett. 2014, 16, 5434−5437

Organic Letters
Letter
Scheme 4. Synthesis of Chiral α-Hydrazino Acids and Cα-
Table 1. Tpd Peptidomimetics Prepared by SPPS
Substituted Tpd Peptidomimetics
In summary, we have described the efficient synthesis of
cyclic N-amino peptide derivatives for conformational scanning
of bioactive lead structures. Chiral α-hydrazino acid building
blocks were synthesized in solution and chemoselectively
incorporated on solid phase. Cleavage from the resin and
concomitant ring closure gave rise to rigidified peptidomi-
metics. Notably, our methodology allows for the synthesis of
Tpd-constrained peptides bearing various native and sterically
hindered side chains. Given that this protocol is operationally
simple and amenable to combinatorial synthesis, we anticipate
the Tpd motif will find broad application as a probe of local
peptide conformation. Efforts toward structurally defined and
biologically active N-amino peptide derivatives are currently
underway in our laboratory.
amino group is not easily protonated even at low pH. Previous
failed acylation attempts (see Scheme 2) confirmed the poor
nucleophilicity of this nitrogen and prompted us to explore
chemoselective amidation at the C-terminus. Reaction of 11a
with H-Phe-OMe in the presence of HATU afforded the
desired N-amino dipeptide 12 in 84% yield without any
detectable racemization or self-condensation of the α-hydrazino
acid.12 Encouraged by this result, we reacted 5 equiv of 11a
with resin-bound Tyr in the presence of 5 equiv of HATU, and
10 equiv of DIEA in DMF at 50 °C to give intermediate 13.
Elaboration of the model peptide and tandem cleavage/
cyclization as described above gave diastereomerically pure
Tpd-containing tetrapeptide mimic 14 in 27% overall yield
following RP-HPLC purification.13
ASSOCIATED CONTENT
* Supporting Information
■
S
Full experimental details and copies of NMR and LCMS
spectra for all new compounds. This material is available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
■
*E-mail: juan.delvalle@moffitt.org.
To demonstrate the broad utility of our solid-phase protocol,
we carried out the synthesis of a variety of Tpd-containing
peptidomimetics as shown in Table 1. The described
methodology is tolerant of both D- and L-Tpd subunits, and
the six-membered ring closure is not adversely affected by the
presence of other electrophilic or nucleophilic side chains
within the peptide. In addition, hindered α-hydrazino acids
such as N-amino-Ile, -Val, and -Leu can be readily incorporated.
Analysis of the crude HPLC traces for various Tpd
peptidomimetics revealed that the principle impurity is the
trifluoroacetylated N-amino peptide. However, this byproduct
is typically a minor component of the crude mixture (<15%)
and is readily removed during preparative RP-HPLC
purification. Only in the case of N-amino serine-derivative 21
did we observe inefficient ring closure. Although we were able
to isolate the desired Tpd derivative in low yield, LCMS
revealed the major product to be the uncyclized N-amino
peptide. The nucleophilicity of Nβ appeared to be generally
lower in this case, as the trifluoroacetylated byproduct was also
conspicuously absent from the crude mixture.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We gratefully acknowledge the National Institutes of Health
(R21CA167215) and the Anna Valentine Cancer Fund (FIG
award) for financial support. Additional support of the
Chemical Biology Core Facility was provided by the NIH
through the Moffitt Cancer Center support grant
(P30CA076292).
REFERENCES
■
(1) (a) Freidinger, R. M.; Veber, D. F.; Perlow, D. S.; Brooks, J. R.;
Saperstein, R. Science 1980, 210, 656. (b) Aube, J. Synthetic Routes to
Lactam Peptidomimetics In Advances in Amino Acid Mimetics and
Peptidomimetics, Vol. 1; Abell, A., Ed.; JAI Press Ltd.: London, 1997;
pp 193−232. (c) Perdih, A.; Kikelj, D. Curr. Med. Chem. 2006, 13,
1525. (d) Freidinger, R. M.; Perlow, D. S.; Veber, D. F. J. Org. Chem.
1982, 47, 104.
5436
dx.doi.org/10.1021/ol5026684 | Org. Lett. 2014, 16, 5434−5437

Organic Letters
Letter
(2) For additional reviews and selected examples, see: (a) Hanessian,
S.; McNaughton-Smith, G.; Lombart, H. G.; Lubell, W. D. Tetrahedron
1997, 53, 12789. (b) Cluzeau, J.; Lubell, W. D. Peptide Science 2005,
80, 98. (c) Khashper, A.; Lubell, W. D. Org. Biomol. Chem. 2014, 12,
5052. (d) Scott, W. L.; Martynow, J. G.; Huffman, J. C.; O’Donnell, M.
J. J. Am. Chem. Soc. 2007, 129, 7077. (e) Scott, W. L.; Alsina, J.;
Kennedy, J. H.; O’Donnell, M. J. Org. Lett. 2004, 6, 1629. (f) Jamieson,
A. G.; Boutard, N.; Beauregard, K.; Bodas, M. S.; Ong, H.; Quiniou,
C.; Chemtob, S.; Lubell, W. D. J. Am. Chem. Soc. 2009, 131, 7917.
(g) Ecija, M.; Diez, A.; Rubiralta, M.; Casamitjana, N.; Kogan, M. J.;
Giralt, E. J. Org. Chem. 2003, 68, 9541. (h) Kumar, S.; Flamant-Robin,
C.; Wang, Q.; Chiaroni, A.; Sasaki, N. A. J. Org. Chem. 2005, 70, 5946.
(i) Doan, N.-D.; Hopewell, R.; Lubell, W. D. Org. Lett. 2014, 16, 2232.
(j) Palomo, C.; Aizpurua, J. M.; Benito, A.; Miranda, J. I.; Fratila, R.
M.; Matute, C.; Domercq, M.; Gago, F.; Martin-Santamaria, S.;
Linden, A. J. Am. Chem. Soc. 2003, 125, 16243. (k) Proulx, C.; Lubell,
W. D. Org. Lett. 2012, 14, 4552. (l) Sicherl, F.; Cupido, T.; Albericio,
F. Chem. Commun. 2010, 46, 1266. (m) Robl, J. A.; Cimarusti, M. P.;
Simpkins, L. M.; Weller, H. N.; Pan, Y. Y.; Malley, M.; DiMarco, J. D.
J. Am. Chem. Soc. 1994, 116, 2348. (n) Sun, H.; Martin, C.; Kesselring,
D.; Keller, R.; Moeller, K. D. J. Am. Chem. Soc. 2006, 128, 13761.
(3) For examples of bicyclic variants and Tpd homologues, see:
(a) Rudolphi, K.; Gerwin, N.; Verzijl, N.; van, d. K. P.; van, d. B. W.
Osteoarthritis Cartilage 2003, 11, 738. (b) Chen, M. H.; Goel, O. P.;
Hyun, J. W.; Magano, J.; Rubin, J. R. Bioorg. Med. Chem. Lett. 1999, 9,
1587. (c) Dolle, R. E.; Prasad, C. V. C.; Prouty, C. P.; Salvino, J. M.;
Awad, M. M. A.; Schmidt, S. J.; Hoyer, D.; Ross, T. M.; Graybill, T. L.;
Speier, G. J.; Uhl, J.; Miller, R.; Helaszek, C. T.; Ator, M. A. J. Med.
Chem. 1997, 40, 1941. (d) Handy, E. L.; Totaro, K. A.; Lin, C. P.;
Sello, J. K. Org. Lett. 2014, 16, 3488. (e) Hobbs, C. J.; Bit, R. A.;
Cansfield, A. D.; Harris, B.; Hill, C. H.; Hilyard, K. L.; Kilford, I. R.;
Kitas, E.; Kroehn, A.; Lovell, P.; Pole, D.; Rugman, P.; Sherborne, B.
S.; Smith, I. E. D.; Vesey, D. R.; Walmsley, D. L.; Whittaker, D.;
Williams, G.; Wilson, F.; Banner, D.; Surgenor, A.; Borkakoti, N.
Bioorg. Med. Chem. Lett. 2002, 12, 1365. (f) Robl, J. A.; Sun, C.-Q.;
Simpkins, L. M.; Ryono, D. E.; Barrish, J. C.; Karanewsky, D. S.; Asaad,
M. M.; Schaeffer, T. R.; Trippodo, N. C. Bioorg. Med. Chem. Lett. 1994,
4, 2045.
route in Scheme 4 afforded 14 with >20:1 dr as judged by LCMS and
NMR (see Supporting Information).
(4) Gao, Y.; Kodadek, T. Chem. Biol. 2013, 20, 360.
(5) (a) Cabezas, E.; Satterthwait, A. C. J. Am. Chem. Soc. 1999, 121,
3862. (b) Kanta Sarma, B.; Yousufuddin, M.; Kodadek, T. Chem.
Commun. 2011, 47, 10590. (c) Liu, F.; Stephen, A. G.; Adamson, C. S.;
Gousset, K.; Aman, M. J.; Freed, E. O.; Fisher, R. J.; Burke, T. R., Jr.
Org. Lett. 2006, 8, 5165. (d) Sarma, B. K.; Kodadek, T. ACS Comb. Sci.
2012, 14, 558.
(6) (a) Williams, P. D.; Bock, M. G.; Tung, R. D.; Garsky, V. M.;
Perlow, D. S.; Erb, J. M.; Lundell, G. F.; Gould, N. P.; Whitter, W. L.;
Hoffman, J. B.; Kaufman, M. J.; Clineschmidt, B. V.; Pettibone, D. J.;
Freidinger, R. M.; Veber, D. F. J. Med. Chem. 1992, 35, 3905.
(b) Oelke, A. J.; France, D. J.; Hofmann, T.; Wuitschik, G.; Ley, S. V.
Nat. Prod. Rep. 2011, 28, 1445.
(7) (a) Acherar, S.; Salaun, A.; Le Grel, P.; Le Grel, B.; Jamart-
Gregoire, B. Eur. J. Org. Chem. 2013, 2013, 5603. (b) Bonnet, D.;
Margathe, J. F.; Radford, S.; Pflimlin, E.; Riche, S.; Doman, P.; Hibert,
M.; Ganesan, A. ACS Comb. Sci. 2012, 14, 323.
(8) Carpino, L. A.; Cohen, B. J.; Stephens, K. E.; Sadat-Aalaee, S. Y.;
Tien, J. H.; Langridge, D. C. J. Org. Chem. 1986, 51, 3732.
(9) The integrity of the Fmoc-Asp(Me)-Cl chiral center was
established by synthesis and analysis of the (S,S) and (R,S)
diastereomers of 2d by NMR and HPLC. See the Supporting
Information for details.
(10) Zuckermann, R. N.; Kerr, J. M.; Kent, S. B. H.; Moos, W. H. J.
Am. Chem. Soc. 1992, 114, 10646.
(11) Deechongkit, S.; You, S.-L.; Kelly, J. W. Org. Lett. 2004, 6, 497.
1
(12) The diastereomeric purity of 12 was verified by H NMR (see
Supporting Information).
(13) Synthesis of 14 using the bromoacetamide submonomer
approach yielded a mixture of diastereomers following cleavage. The
5437
dx.doi.org/10.1021/ol5026684 | Org. Lett. 2014, 16, 5434−5437