Optimized 5-membered heterocycle-linked pterins for the inhibition of Ricin Toxin A

Article abstract of DOI:10.1021/ml300099t

The optimization of a series of pterin amides for use as Ricin Toxin A (RTA) inhibitors is reported. On the basis of crystallographic data of a previous furan-linked pterin, various expanded furans were synthesized, linked to the pterin, and tested for inhibition. Concurrently, heteroanalogues of furan were explored, leading to the discovery of more potent triazole-linked pterins. Additionally, we discuss a dramatic improvement in the synthesis of these pterin amides via a dual role by diazabicycloundecene (DBU). This synthetic enhancement facilitates rapid diversification of the previously challenging pterin heterocycle, potentially aiding future medicinal research involving this structure.

Full text of DOI:10.1021/ml300099t

Letter
pubs.acs.org/acsmedchemlett
Optimized 5-Membered Heterocycle-Linked Pterins for the Inhibition
of Ricin Toxin A
Jeff M. Pruet,‡ Ryota Saito,*,‡,† Lawrence A. Manzano, Karl R. Jasheway, Paul A. Wiget, Ishan Kamat,
Eric V. Anslyn,* and Jon D. Robertus*
Department of Chemistry and Biochemistry, University of Texas at Austin, 1 University Station A1590, Austin, Texas 78712, United
States
S
* Supporting Information
ABSTRACT: The optimization of a series of pterin amides for
use as Ricin Toxin A (RTA) inhibitors is reported. On the basis
of crystallographic data of a previous furan-linked pterin, various
expanded furans were synthesized, linked to the pterin, and
tested for inhibition. Concurrently, heteroanalogues of furan
were explored, leading to the discovery of more potent triazole-linked pterins. Additionally, we discuss a dramatic improvement
in the synthesis of these pterin amides via a dual role by diazabicycloundecene (DBU). This synthetic enhancement facilitates
rapid diversification of the previously challenging pterin heterocycle, potentially aiding future medicinal research involving this
structure.
KEYWORDS: Pterin, Ricin inhibitors, DBU, RTA, heterocycles
icin is a cytotoxin that is easily isolated from castor beans
and castor oil byproducts.1 It is categorized as a type II
moving toward this second binding pocket, which could
improve inhibitor binding affinity and target specificity.
Another preliminary observation reported for this furan-linked
pterin was that while the furan ring appeared held in place via
hydrogen bonding to the Tyr-80 residue of RTA, the 4 Å
distance between the heteroatoms indicated only a weak
interaction. It thus follows that, by optimizing the interaction
between Tyr-80 and the heterocycle, we should achieve
superior inhibition. To accomplish this task, we investigated
heteroanalogues of furan: thiophene, imidazole, and triazole.
Pterins are notorious for their insolubility, severely hindering
the ease of derivitization.9 For this reason, functionalization of
the pterin was held to the last step. Our initial method for
forming the amide required suspension of the pterin methyl
ester (1) in methanol in the presence of the amine at elevated
temperatures (Scheme 1). Coupling directly to the carboxylic
R
ribosome inactivating protein, which contains a catalytic A
chain with N-glycosidase activity and a lectin B chain assisting
cellular uptake.2 The A chain of ricin (RTA) has been well
characterized, and its mechanism of action is well understood.3
RTA depurinates a specific adenosine on rRNA, thus inhibiting
protein synthesis and resulting in cell death, with toxic doses as
low as 0.1−1 μg/kg, depending on the mode of admin-
istration.4 The ease in which it is acquired, low toxic dose, and
lack of an antidote make ricin a notable chemical weapons
threat. A number of antiricin agents have been reported, though
many are thought to act by disrupting cellular trafficking, and
their binding target is not clear.5 The highly evolved specificity
pocket of RTA makes the discovery of small molecule active-
site inhibitors quite challenging. To date, there have been
limited reports of RTA inhibitors showing activity below the
micromolar range. Cyclic nucleotides acting as transition state
analogues have shown nanomolar range Kd, but these were
active only at low pH.6
a
Scheme 1. Synthesis of Heteroanalogues of Furanylpterin
Recently we reported on the utility of 7-substituted pterins,
based on 7-carboxypterin (7CP), as RTA inhibitors, which
showed IC50 values as low as 210 μM in a luciferase translation-
based assay.7 This was seen as a significant improvement over
previous pterin-based inhibitors, which had activity at or above
600 μM.8 Within this series, we reported a furan-linked pterin
amide having an IC50 of 380 μM. While this was inferior to
7CP, X-ray analysis of the binding mode indicated the furan
ring bridging the gap between the primary binding pocket and a
nearby secondary pocket. In its natural state, the primary
binding pocket binds the targeted adenosine while the second
pocket binds an adjacent guanosine in the RNA sequence.6a We
postulated that furans derivatized at position 5 would fill space
a
Full structure shown in Table 1.
Received: April 23, 2012
Accepted: May 29, 2012
Published: May 29, 2012
© 2012 American Chemical Society
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Letter
acid, as is common with amide bond formation, was not a viable
option, as the solubility restrictions of 7CP were incompatible
with typical activating agents and coupling conditions. The
thiophene and imidazole-linked pterins (2−4) were synthesized
from the respective amines (Scheme 1).
Scheme 2. (a) Synthesis of Expanded Furan Derivatives. (b)
Conjugation of Amines to 7-Methoxycarbonylpterin
When these structures were soaked into preformed RTA
crystals, and analyzed crystallographically, the pterin was clearly
observed bound into the specificity pocket, but there was
limited electron density for the pendent. The IC50 values,
obtained in the luciferase assay, were consistent with results
seen for the parent furan compound, with only a slight
improvement in activity. However, as crystallographic data only
showed the pterin rigidly bound, the activity likely only stems
from interactions with the pterin alone. Furthermore, as the 5-
membered rings are not held in place, compounds 2−4 were
not seen as prime candidates for rationally designed
optimization.
Turning our attention to triazole, we synthesized 4-
aminomethyltriazole and condensed it with the pterin ester 1.
Compound 5 was tested for inhibition and was not only
superior to the simple furan-linked pterin but also more potent
than 7CP, with an IC50 of 70 μM.
The crystal structure of the RTA·5 complex showed the full
density of the ligand and also revealed a firm hydrogen bond to
Tyr-80, with a 2.8 Å distance between heteroatoms, as seen in
Figure 1.
proposed DBU attacking the ester, activating the carbonyl in
situ, thus expediting aminolysis.
Upon adding DBU to the suspension of 1 in methanol, we
quickly discovered an additional benefit of DBU, as the pterin
fully dissolved. The solubility of unfunctionalized pterin in
methanol is unprecedented. To investigate the nature of this
solubility, we precipitated with dioxane, whereupon NMR of
the yellow solid showed a 1:1 mixture of pterin to DBU,
indicative of an organic salt. The pKa of the lactam NH of
pterin has been reported as ∼8, within the range of DBU
deprotonation.11 This solubility allowed reactions to be run at
higher concentrations, decreasing the amount of amine
required. With the DBU first acting to form an organic salt, 2
equiv of DBU were added to the pterin ester, in a minimal
amount of methanol and 2 equiv of amine. Monitoring the
reaction at room temperature revealed after 4 h the reaction
had gone to completion. Isolation of the desired product was
achieved by addition of aqueous hydrochloric acid, providing
the product as a precipitate in acceptable yield. The proposed
role of DBU in the amidation reaction is illustrated in Figure S1
(see the Supporting Information).
We screened other amine basestriethylamine, Hunig’s
base, and DABCOand found none showed the remarkable
improvement in solubility or rate acceleration.12 This supports
the proposed nucleophilic character for DBU being crucial, as
the tertiary amines could not proceed by such a mechanism.
Proceeding with the improved synthesis, we synthesized the
expanded furan linked pterins 9−11 and tested them for RTA
inhibition. Compounds 9 and 10 showed diminished activity
relative to the lead furan compound. Additionally, the X-ray
data for the RTA·inhibitor complexes only showed density for
the pterin ring, indicating disordered and uncompensated
binding of the pendent. Compound 11, however, showed a
remarkable improvement in activity in the luciferase assay. At
∼30 μM, it was nearly an order of magnitude more potent than
7CP. This was a very encouraging result and justified our
design of expanded furans. Surprisingly, the X-ray data of the
bound inhibitor revealed a disordered binding of the pendent,
but strong density displayed for the pterin ring alone.
Figure 1. X-ray structure of 5 bound into RTA. Hydrogen bonds to
the protein are all between 2.6 and 2.9 Å. Density is from a 1.9 Å
OMIT map contoured at 3σ. A water molecule is shown as a labeled
sphere.
While this result shows great potential for triazoles, we still
wished to optimize the lead furan-linked pterin via construction
of expanded 2-aminomethylfurans substituted at the 5 position,
hoping to bring the furan into the range of activity seen for the
simple triazole 5.
We began with the synthesis of the desired expanded furans
(6−8), starting from commercially available ethyl 5-chlor-
omethyl-2-furancarboxylate, as shown in Scheme 2a.
Our initial method of amidation gave acceptable yields with
commercially available/simple amines, which can be used in
large excess but became impractical with more complex
synthetic amines, as seen by the low yield of compound 9
(Scheme 2b). Seeking to improve the conversion of ester to
amide, we took note of the work by Vaidyanathan et al., who
used DBU as a mild catalyst for amidation of esters.10 They
Given the synthetic effort required for the expanded furans,
compared with a simple “click chemistry”13 approach for
triazoles, and the positive results for compound 5, focus was
shifted from furan to expanded triazole inhibitors. Any number
of azides can be reacted with propargyl amine to provide an
expanded triazole to be further reacted with the pterin ester
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(1). However, to facilitate rapid diversification, we investigated
an alternative synthetic route to the triazoles. Rather than fully
synthesizing the heterocycle prior to condensation with the
pterin, we first synthesized the propargyl-linked pterin amide
(12), to react with various azides in parallel. In the absence of
DBU, compound 12 failed to react. However, first isolating 12
as the highly soluble DBU salt allowed for synthesis of various
triazoles via traditional “click” conditions. Compounds 13−20
were synthesized in this manner (Scheme 3).
Table 1. Summary of Results
a
Scheme 3. Synthesis of New Triazole-Linked Pterin
a
Full structures are shown in Table 1.
These structures were chosen to probe the effect of the
electron density of the aromatic side-chain, and the length of
the linker (Table 1). Screening the newly synthesized triazole-
linked pterins revealed many of them performed quite poorly.
All compounds with benzyl substituents showed lackluster
IC50’s in the 400−500 μM range. The pyridyl functionalized
triazole gave better results, with an IC50 of roughly 110 μM.
While less active than the parent triazole (5), this compound is
still superior to 7CP and all other previously published pterin
inhibitors. The only structure more potent than 5 was
compound 19, with an IC50 of 15 μM. The X-ray data for
the RTA·19 complex showed the binding of the ligand was still
somewhat disordered, as there was strong density for the pterin
ring but only limited density for the triazole ring and possible
density observed for the heavy sulfur atom (see the Supporting
Information). This indicates the triazole ring is relatively fixed
in position, but the methylene spacer to the thiophenol adopts
multiple geometries. Furthermore, the benzene ring of the
thiophenol is freely rotating. It is possible that the free rotation
has displaced trapped water molecules from the active-site,
giving rise to favorable entropic effects, compensating the
limited enthalpically favorable interactions. This hypothesis is
partially supported by the disappearance of two water
molecules previously observed in the space near the disordered
thiophenol. We are currently investigating ITC experiments
which may further elucidate the nature of the protein−ligand
interaction. Compound 19 is currently one of the most potent
RTA inhibitors to date.
A representative dose−response curve from the luciferase
assay for compound 19 is shown in Figure 2. RTA activity was
determined through normalization of luciferase counts with and
without inhibitor. Percent RTA activity is plotted as a function
of inhibitor concentration (see Supporting Information for
experimental details).
In summary, we have synthesized three expanded furans to
optimize a previous furan-linked pterin. Toward this goal, we
achieved success with compound 11. In the process, we
improved our synthetic strategy through the use of DBU, which
gave remarkable solubility to a notoriously insoluble hetero-
cycle. We screened various heteroanalogues of furan, leading to
improved interaction with RTA through a triazole-linked pterin
(5). With the beneficial solubility, we rapidly constructed a
library of new pterins via click chemistry. While the small
triazole library only identified a single potent inhibitor (19), the
ease of diversification highlights a significant advance in pterin
functionalization. The use of the DBU salt of 12 for rapid
diversification should represent an important contribution to
medicinal chemistry, as this is a readily available, stable source
of pterin that can be easily incorporated into a range of
biologically and medicinally relevant compounds.
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Letter
action. Proteins 1991, 10 (3), 270−8. (c) Montfort, W.; Villafranca, J.
E.; Monzingo, A. F.; Ernst, S. R.; Katzin, B.; Rutenber, E.; Xuong, N.
H.; Hamlin, R.; Robertus, J. D. The 3-Dimensional Structure of Ricin
at 2.8-A. J. Biol. Chem. 1987, 262 (11), 5398−5403. (d) Mlsna, D.;
Monzingo, A. F.; Katzin, B. J.; Ernst, S.; Robertus, J. D. Structure of
recombinant ricin A chain at 2.3 A. Protein Sci. 1993, 2 (3), 429−35.
(4) Miller, D. J.; Ravikumar, K.; Shen, H.; Suh, J. K.; Kerwin, S. M.;
Robertus, J. D. Structure-based design and characterization of novel
platforms for ricin and shiga toxin inhibition. J. Med. Chem. 2002, 45
(1), 90−8.
(5) (a) Wahome, P. G.; Bai, Y.; Neal, L. M.; Robertus, J. D.; Mantis,
N. J. Identification of small-molecule inhibitors of ricin and shiga toxin
using a cell-based high-throughput screen. Toxicon 2010, 56, 313−23.
(b) Stechmann, B.; Bai, S. K.; Gobbo, E.; Lopez, R.; Merer, G.;
Pinchard, S.; Panigai, L.; Tenza, D.; Raposo, G.; Beaumelle, B.;
Sauvaire, D.; Gillet, D.; Johannes, L.; Barbier, J. Inhibition of
retrograde transport protects mice from lethal ricin challenge. Cell
2010, 141 (2), 231−42.
(6) (a) Ho, M. C.; Sturm, M. B.; Almo, S. C.; Schramm, V. L.
Transition state analogues in structures of ricin and saporin ribosome-
inactivating proteins. Proc. Natl. Acad. Sci. U. S. A. 2009, 106 (48),
20276−81. (b) Chen, X.-Y.; Berti, P. J.; Schramm, V. L. Transition-
State Analysis for Depurination of DNA by Ricin A-Chain. J. Am.
Chem. Soc. 2000, 122 (28), 6527−6534.
Figure 2. Compound 19 versus RTA. RTA activity is plotted as a
function of inhibitor concentration and fit to a hyperbolic decay
function.
ASSOCIATED CONTENT
* Supporting Information
■
S
Experimental procedures and compound characterizations,
proposed DBU mechanism, X-ray data for compound 19,
additional dose response curves, and details for the in vitro
assay. This material is available free of charge via the Internet at
http://pubs.acs.org.
(7) Pruet, J. M.; Jasheway, K. R.; Manzano, L. A.; Bai, Y.; Anslyn, E.
V.; Robertus, J. D. 7-Substituted pterins provide a new direction for
ricin A chain inhibitors. Eur. J. Med. Chem. 2011, 46, 3608−3615.
(8) Yan, X.; Hollis, T.; Svinth, M.; Day, P.; Monzingo, A. F.; Milne,
G. W. A.; Robertus, J. D. Structure-based identification of a ricin
inhibitor. J. Mol. Biol. 1997, 266 (5), 1043−1049.
(9) (a) Waring, P. The Synthesis of 6-Aminomethyl-5,6,7,8-
Tetrahydropterin. Aust. J. Chem. 1988, 41 (5), 667−676. (b) Taylor,
E. C.; Ray, P. S. Pteridines. 52. Convenient Synthesis of 6-
formylpterin. Synth. Commun. 1987, 17 (16), 1865−1868. (c) Pruet,
J. M.; Robertus, J. D.; Anslyn, E. V. Acyl radical insertion for the direct
formation of new 7-substituted pterin analogs. Tetrahedron Lett. 2010,
51 (18), 2539−2540.
AUTHOR INFORMATION
Corresponding Author
*R.S.: tel, +81-47-472-1926; e-mail, saito@chem.sci.toho-u.ac.
jp. E.V.A.: tel, +1 512 471 0068; fax, +512 471 7791; e-mail,
anslyn@austin.utexas.edu. J.D.R.: tel, +1 512 471 3175; e-mail,
jrobertus@mail.utexas.edu.
■
Present Address
†Department of Chemistry, Toho University Miyama, Funaba-
shi, Japan.
Author Contributions
‡These authors contributed equally to this work.
(10) Price, K. E.; Larrivee-Aboussafy, C.; Lillie, B. M.; McLaughlin, R.
W.; Mustakis, J.; Hettenbach, K. W.; Hawkins, J. M.; Vaidyanathan, R.
Mild and efficient DBU-catalyzed amidation of cyanoacetates. Org.
Lett. 2009, 11 (9), 2003−6.
Notes
The authors declare no competing financial interest.
(11) Brown, D. J. Pteridines; Wiley: New York, 1988; pp xxvii, 730.
(12) Of the amines tested, DBU is the most basic. There is enough
difference in the basicity of DBU and DABCO that the latter may not
deprotonate the pterin to a large enough extent to fully form the
organic salt. However, the remaining bases are all similar in strength,
with protonated DBU and Hunig’s base only differing by 0.5 pKa units.
Thus, all are capable of forming the organic salt, yet only DBU should
form a salt “greasy” enough to dissolve in methanol. Nevertheless,
much of the utility of DBU stems from the in situ activation of the
ester, which would not be possible with the three tertiary amines. pKa's
for the four bases, among others, can be found at: www.cem.msu.edu/
∼reusch/OrgPage/basicity.htm.
ACKNOWLEDGMENTS
■
This work was supported by National Institutes of Health
(NIH) Grant AI 075509, by Robert A. Welch Foundation
Grant F1225, and by the College of Natural Sciences support to
the Center for Structural Biology. Assistance for this work was
provided by the Macromolecular Crystallography Facility, with
financial support from the College of Natural Sciences, the
Office of the Executive Vice President and Provost, the Institute
for Cellular and Molecular Biology, the University of Texas at
Austin.
(13) Meldal, M.; Tornoe, C. W. Cu-catalyzed azide-alkyne
cycloaddition. Chem. Rev. 2008, 108 (8), 2952−3015.
ABBREVIATIONS
RTA, ricin toxin A; PTA, pteroic acid; 7CP, 7-carboxy pterin;
DBU, 1,8-diazabicyclounde-7-cene
■
REFERENCES
■
(1) Franz, D. Textbook of Military Medicine: Aspects of Chemical and
Biological Warfare; 1997.
(2) (a) Lord, J. M.; Roberts, L. M.; Robertus, J. D. Ricin: structure,
mode of action, and some current applications. FASEB J. 1994, 8 (2),
201−8. (b) Robertus, J. The structure and action of ricin, a cytotoxic
N-glycosidase. Semin. Cell Biol. 1991, 2 (1), 23−30.
(3) (a) Monzingo, A. F.; Robertus, J. D. X-ray analysis of substrate
analogs in the ricin A-chain active site. J. Mol. Biol. 1992, 227 (4),
1136−45. (b) Ready, M. P.; Kim, Y.; Robertus, J. D. Site-directed
mutagenesis of ricin A-chain and implications for the mechanism of
591
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