Sulfur incorporation generally improves Ricin inhibition in pterin-appended glycine-phenylalanine dipeptide mimics

Article abstract of DOI:10.1016/j.bmcl.2013.10.017

Several 7-aminoamido-pterins were synthesized to evaluate the electronic and biochemical subtleties observed in the 'linker space' when N-{N-(pterin-7-yl)carbonylglycyl}-l-phenylalanine 1 was bound to the active site of RTA. The gylcine-phenylalanine dipeptide analogs included both amides and thioamides. Decarboxy gly-phe analog 2 showed a 6.4-fold decrease in potency (IC₅₀ = 128 μM), yet the analogous thioamide 7 recovered the lost activity and performed similarly to the parent inhibitor (IC₅₀ = 29 μM). Thiourea 12 exhibited an IC₅₀ nearly six times lower than the oxo analog 13. All inhibitors showed the pterin head-group firmly bound in their X-ray structures yet the pendants were not fully resolved suggesting that all pendants are not firmly bound in the RTA linker space. Calculated log P values do not correlate to the increase in bioactivity suggesting other factors dominate.

Full text of DOI:10.1016/j.bmcl.2013.10.017

Bioorganic & Medicinal Chemistry Letters 23 (2013) 6799–6804
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Sulfur incorporation generally improves Ricin inhibition
in pterin-appended glycine-phenylalanine dipeptide mimics
Paul A. Wiget, Lawrence A. Manzano, Jeff M. Pruet, Grace Gao, Ryota Saito, Arthur F. Monzingo,
⇑
Karl R. Jasheway, Jon D. Robertus, Eric V. Anslyn
Department of Chemistry and Biochemistry, University of Texas at Austin, 1 University Station A1590, Austin, TX 78712, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Several 7-aminoamido-pterins were synthesized to evaluate the electronic and biochemical subtleties
Received 26 August 2013
Revised 3 October 2013
Accepted 7 October 2013
Available online 12 October 2013
observed in the ‘linker space’ when N-{N-(pterin-7-yl)carbonylglycyl}-
the active site of RTA. The gylcine–phenylalanine dipeptide analogs included both amides and thioam-
ides. Decarboxy gly-phe analog 2 showed a 6.4-fold decrease in potency (IC50 = 128 M), yet the analo-
gous thioamide recovered the lost activity and performed similarly to the parent inhibitor
(IC50 = 29 M). Thiourea 12 exhibited an IC50 nearly six times lower than the oxo analog 13. All inhibitors
showed the pterin head-group firmly bound in their X-ray structures yet the pendants were not fully
resolved suggesting that all pendants are not firmly bound in the RTA linker space. Calculated logP values
do not correlate to the increase in bioactivity suggesting other factors dominate.
Ó 2013 Published by Elsevier Ltd.
L-phenylalanine 1 was bound to
l
7
l
Keywords:
Pterin
Thioamide
Dipeptide mimic
Ricin inhibitors
RTA
SAR
As recently as April 2013 the biotoxin ricin has been employed
as a potential weapon against high-ranking government officials
including US Senator Roger Wickers and President Barack Obama.1
Ricin toxicity can occur from aerosol exposure in addition to a
due to the modification of the three dimensional space the mole-
cule occupies. These changes can be due to (amongst others) the
addition or reduction of steric bulk, conformational restrictions,
and/or hydrogen bonding preferences.6
wealth of other methods at doses below 1
l
g/kg body weight. Ricin
Highly specific interactions can be mapped and optimized with-
in classes of molecules by carefully controlling the modifications in
a drug candidate by keeping the overall sterics and degrees of free-
dom the same or highly similar. One way this has been achieved is
by amide bond translocation within drug candidates. Coppola et al.
found that reversing the order of the amide in one such substrate
could produce the dramatic effect of having no activity to 33% inhi-
has already seen use as a weapon, and due to the ease of castor oil
procurement and toxin extraction, the threat persists.2
The cytotoxin ricin is a prototypical type 2 ribosome inactivat-
ing protein found in castor beans.3 Ricin Toxin A (RTA), the A chain
of the cytotoxin Ricin, catalyzes adenosine depurination, and is an
optimal candidate for inhibitor Structure–Activity Relationship
(SAR) studies due to its ease of handling and known and reproduc-
ible crystal structure.4 Additionally, we have previously reported
numerous pterin-based RTA inhibitors as potential anti-Ricin
agents via structure-based design and virtual screening.5 The pter-
in heterocycle is not only consistent in showing activity with a
multitude of pendants attached, but also with rare exception deliv-
ers firmly resolved X-ray crystallographic data.
bition of their target protein at 10 lM. Translocation has also
shown varying effects on the stability of candidates in microsomes,
increasing t1/2, by a factor of 3.7
Conversion of amides to thioamides has been used frequently to
alter the native conformation of peptides by modifying the hydro-
gen bond strength and rotational barriers of the molecule or drug
candidate. Thioamides have been found to be compatibly stable
Structure–Activity Relationships (SAR) correlate the biological
response of a specific process to the varying of functional groups
of a biomolecule (i.e., protein mutation or modification), or to par-
ticipating substrates such as drug candidates, co-factors, etc. As
with proteins, subtle changes in drug candidate structure can dras-
tically change drug efficacy, uptake, binding affinity, toxicity, etc.
in such tertiary structures as b-sheets and a-helicies, even at
elevated temperatures. Their incorporation into organocatalysts
has seen improvements of selectivity factors (s) by an order of
magnitude.8 The geometry, determined by both spectroscopic9
and computational10 methods show shorter N–C(S) bond lengths,
longer C–S bond lengths, and higher amide rotational barriers,
suggesting a greater degree of N–C double bond character. In drug
candidates thioamide incorporation has produced dramatic
changes in KD, log p, and hydrolysis rate, thus changing biological
⇑
Corresponding author.
E-mail address: anslyn@austin.utexas.edu (E.V. Anslyn).
0960-894X/$ - see front matter Ó 2013 Published by Elsevier Ltd.
http://dx.doi.org/10.1016/j.bmcl.2013.10.017

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P. A. Wiget et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6799–6804
O
N
was a Gly-Phe appended pterin 115 (Fig. 1). In the space of the RTA
protein that joins the pterin binding site and a secondary binding
site referred to as the ‘linker space’, tyrosine 80 is available for
N
N
O
CO2H
HN
H2N
H
N
N
hydrogen bond donation and p-stacking. Previously reported sub-
H
O
strates exhibited hydrogen bonding with Tyr 80 but the mode, nat-
ure, and optimum location of the hydrogen bond acceptor
remained unclear. Additionally, Trp221 provides an edge-to-face16
interaction with the benzene ring of 1. In order to explore the sub-
tleties of the linker-space between the specificity pocket and the
secondary binding pocket through substrate structure–activity
relationships, herein we report a series highly similar (n + m = 3)
of ‘Inside’ and ‘Outside’ amide- and thioamide-appended pterin
analogs of 1 (see Fig. 2). Additionally, as proteolytic stability is of
concern in peptide-based drug candidates, we chose to decarboxyl-
ate all inhibitors to circumvent this familiar drug-candidate weak-
ness.17 Therefore, the inhibitors are designed to probe the linker
space via amide transposition, inversion, and through conversion
to the thioamide in comparison to the parent compound 1.
The syntheses of ‘Inside’ aminoamides began with carbonyl-
diimidizole (CDI) facilitated amide coupling to the appropriate
Boc-protected aminoacid18 (such that n + m = 3 to maintain gly-
phe homology). Deprotection and ether/DCM precipitation of the
pure aminoamide salt afforded the desired gly-phe analogs. Simi-
larly, mono-Boc protected diamines19 were reacted with either
1
IC50 = 20mM
Figure 1. N-{N-(Pterin-7-yl)carbonylglycyl}-L-phenylalanine.
activity.11 Urea or thiourea substitution has been shown to resolve
the ambiguity in cytokine response of the parent amide molecule
and produce CD1d agonists.12 Hence, incorporating ureas or thio-
ureas is yet another way of easily modifying the conformational
degrees of freedom and hydrogen bonding ability within potential
drug candidates.
Although vaccines and cellular trafficking inhibitors have pro-
ven to be viable Ricin countermeasures, our previously reported
competitive active-site inhibitors have shown increased suc-
cess.13–15 Our work has shown that both synthetic and peptide side
chains appended on the pterin ring system have accessed new
binding modes not predicted by models, shown a preference for
hydrophobic chains, and improved our understanding of the two
primary binding pockets.14,15 Of the most highly active compounds
amide
inversion
S
remove
O
N
O
CO2H
HN
H
1
N
H2N
N
N
N
H
O
edge-to-face
electronic
effects
amide
transposition
O
N
O
N
N
N
Y
HN
H2N
HN
H2N
H
N
H
H
R
R
m
m
N
N
N
N
N
n
n
H
O
O
Y
"Outside" amide
"Inside" amide
n+m=3
Y = O or S
Figure 2. Proposed variations of 1.
"Inside" aminoamides
O
O
m
m
CDI
then Ph(CH2)mNH2
BocHN
+H3N
1) H+
Eq.1
BocHN
CO2H
N
N
n
n
H
H
n
2) precipitate
2a, n=1, m=2, 81%
3a, n=2, m=1, 83%
4a, n=3, m=0, 45%
2b, n=1, m=2, 79%
3b, n=2, m=1, 80%
4b, n=3, m=0, 62%
"Outside" aminoamides
H
m
1) H+
H
Ph(CH2)mCO2Cl
m
+H3N
N
Eq. 2
BocHN
NH2
BocHN
N
n
n
n
2) precipitate
O
O
5b, n=2, m=1, 70%
6b, n=3, m=0, 34%
5a, n=2, m=1, 68%
6a, n=3, m=0, 92%
Scheme 1. Synthesis of Inside and Outside Gly-Phe aminoamide analogs.

P. A. Wiget et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6799–6804
6801
O
n+m=3
BocHN
S
S
m
Lawesson's Reagent
1) H+
m
Eq. 1
Eq. 2
m
+H3N
BocHN
N
H
n
N
n
N
n
2) precipitate
THF
H
H
7a, n=1, m=2, quant.
8a, n=2, m=1, quant.
9a, n=3, m=0, 43%
7b, n=1, m=2, 31%
8b, n=2, m=1, 45#%
9b, n=3, m=0, na
n+m=3
BocHN
H
Lawesson's Reagent
m
H
m
H
N
m
N
1) H+
+H3N
N
BocHN
n
n
n
THF
O
2) precipitate
S
S
10a, n=2, m=1, quant.
11a, n=3, m=0, quant.
10b, n=2, m=1, 67%
11b, n=3, m=0, na
NCY
Y
Y
1) H+
BocHN
Eq. 3
BocHN
+H3N
NH2
N
N
N
N
2) precipitate
Y=O,S
H
H
H
H
12a, Y=S, quant
13a, Y=O, quant
12b, Y=S, 83%
13b, Y=O, 44%
Scheme 2. Synthesis of Inside and Outside aminothioamides (Eqs. 1 and 2), aminothiourea and aminourea (Eq. 3) Gly-Phe analogs.
range of deprotection conditions and the compounds repeatedly
decomposed.24
O
O
H2N
H2N
Ar
N
R
N
Thiourea 12b (Scheme 2, Eq. 3) was envisioned in order to gain
the desired analinic hydrogen placement associated with the thio-
carbonyl placement in 9b (Scheme 2, Eq. 1), as well as circumvent
associated Boc deprotection challenges. The necessary ammonium
precursor (12b) was readily obtained in high yield in a one-pot
reaction. The treatment of mono-Boc-protected ethylenediamine
with phenylisothiocyanate in DCM for 30 min, followed by the
addition of anhydrous HCl and ether-induced precipitation pro-
duced the desired compound in good yield with no further need
for purification (Scheme 2, Eq. 3). The urea was also prepared anal-
ogously for comparison, however the yield suffered.
Benzoyl-protected and carboxybenzyl-protected ethylenediam-
ines 14b25 and 15b26 were utilized to examine the effect of chain
length while pyridine and dimethoxybenzene substituted aminoa-
mides 16b27 and 17b28 were synthesized to probe the varying elec-
tronic effects of the edge-to-face interaction observed between 1
and Trp221(Fig. 3).
The syntheses of pterin-7-aminoamide, pterin-7-aminothioamide,
and pterin-7aminourea and thiourea analogs were completed
using 7-methoxycarbonyl-pterin (7-MCP)29 as shown in Scheme 3.
This ester was coupled to the HCl or TFA salt of the desired gly-phe
analog via our previously reported dual function 1,8-diazabicyclo-
undec-7-ene (DBU) process, whereby DBU both solublizes the
reagents in methanol and catalyzes the coupling.15 Basic work-up
followed by neutralization, sonication, and either filtration,
centrafugation, or lyophilyzation, produced the pure compounds
in 30–90% yields as seen in Table 1.
H
H
R = Ph, 14b
R = OBn, 15b
Ar = 2-pya, 16b
Ar = 3,4-DMPb, 17b
Figure 3. Aminoamides synthesized to probe the effect of varying chain length and
the edge-to-face interaction. a2-py = 2-pyridyl, b3,4-DMP = 3,4-dimethoxyphenyl.
O
N
O
N
N
N
N
N
2eq. DBU, MeOH
HN
H2N
HN
H2N
OMe
NHR
then RNH
2
O
O
Scheme 3. Coupling of Gly-Phe analogs with 7-CMP.
benzoylchloride (m = 0) or phenylacetylchloride (m = 1) and subse-
quently deprotected. Precipitation produced the desired ‘Outside’
aminoamide Gly-Phe analogs (Scheme 1, Eq. 2).
The desired thioamide series faced a number of synthetic chal-
lenges. The insolubility of pterins20 in common thionating solvents
such as DCM or THF, as well as the regiocontrol concerns associ-
ated with the multiple amide carbonyls in the pterin heterocycle
required the thionation step to preceed aminoamide-pterin cou-
pling.21 Unfortunately, the thionation of the free aminoamides
and Fmoc-protected aminoamides was ultimately unsuccessful.
We were concerned about the viability of amine protection as
the tert-butylcarbamates due to the necessity of acid-induced
deprotection, as hydrolysis of thioamides is well precedented un-
der acidic conditions.22 To our delight, all but two Boc-protected
aminoamides were successfully thionated using Lawesson’s
reagent,23 and deprotected with anhydrous acid in moderate yield
(Scheme 2, Eqs. 1 and 2). However, 9b and 11b were unavailable as
deprotection of the aminothioamides was unsuccessful under a
The complete list of yields and IC50s for the Gly-Phe pterin ana-
logs studied can be seen in Table 1 for comparison. Observation of
the Inside aminoamide series (2–4) showed that decarboxy gly-
cine-phenylalanine analog 2 exhibited RTA inhibition with an
IC50 of 128 lM. This is 6.4 times less potent than the peptidic pter-
in reference 1, simply by removal of the carboxylic acid moiety.
The activity was somewhat recovered via amide transposition by

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P. A. Wiget et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6799–6804
Table 1
Pterin-based RTA inhibitors appended with Gly-Phe analogs
O
N
HN
Entry
Yield (%)
IC50
(lM)
logP30
LipE31
H2N
N
N
O
O
CO2H
1
2
5915
30
2015
128
À0.11 0.62
À0.37 0.54
4.81
4.26
RHN
RHN
RHN
N
H
O
N
H
H
N
3
61
52
0.25 0.56
4.03
O
O
4
5
73
64
76
44
0.65 0.50
0.27 0.55
3.47
4.09
RHN
RHN
RHN
N
H
O
N
H
H
N
6
7
8
85
64
90
47
29
49
0.67 0.46
0.84 0.72
0.73 0.66
3.66
3.70
3.58
O
S
RHN
RHN
N
H
H
N
S
S
S
10
12
13
56
30
82
38
39
0.74 0.68
0.60 0.52
0.15 0.46
3.68
3.81
3.53
RHN
RHN
N
H
N
H
N
H
O
209
RHN
RHN
N
H
N
H
O
14
61
449
0.34 0.50
3.01
N
H
O
O
RHN
15
16
17
87
56
58
28
160
68
0.55 0.29
À0.57 0.70
0.22 0.67
4.00
4.37
3.95
N
H
O
O
RHN
RHN
N
H
N
OMe
OMe
N
H
a single carbon as seen with 3. This compound showed an increase
in IC50 of nearly two and half times to 52 M. Further transposition
showed a slight decrease in activity as seen in 4 (IC50 = 76 M). The
Outside aminoamides 5 and 6 both slightly out-performed their In-
side analogs. They also showed remarkably similar activities;
was recovered by sulfur incorporation into the pendant aminoa-
mide. Thioamide 7 showed an IC50 of 29 M compared to the
20 M reported for 1. The dose–response curve used to calculate
the IC50 value for this compound is given in Figure 4. Conversion
to thioamides had little effect when comparing 3 (52 M) to 8
(49 M) and 5 (44 M)to 10(38 M). Comparing the calculated
logP30 and the resulting Lipophilic Efficiency31 (LipE), no correla-
l
l
l
l
l
44
lM and 47
lM, respectively. Surprisingly, nearly all activity lost
l
l
l
by simple decarboxylation of the gly-phe reference compound 1

P. A. Wiget et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6799–6804
6803
Lastly, we studied variation of the arene electronic effects. Con-
sistent with favorable edge-to-face weak energetics with indole,
the more electron-rich arenes performed better. Compound 17
had the highest level of inhibition amongst the pendants with
the same length, carbonyl position and carbonyl orientation, with
an IC50 of 68
viously mentioned with an IC50 of 128
Gly-Phe analog 16 performed the least effectively among this ser-
ies with an IC50 of 160 M. However, as the majority of inhibitors
lM, followed by decarboxy Gly-Phe analog 2 as pre-
l
M. The pyridine-appended
l
showed little X-ray density beyond the pterin-proximate amide
methylene, a clear explanation of this trend will require further
studies observing SARs focused solely on arene substituents.
In summary, an examination of decarboxylated pterin gly-phe
analogs was performed producing thioamide 7 with an IC50 of
29 lM for the inhibition of the Ricin A-chain. Thiourea 12 was also
found to be effective in the low micromolar range and was superior
to the urea analog. Carboxybenzyl analog 15 showed activity com-
parable to both 1 and 7, however as the H-bond to Tyr 80 was not
unique to this compound, the source of this activity is unclear.
None of the pendants showed sufficient electron density in the
X-ray structures for adequate interpretation. Lipophilic Efficiency
provided little explanation of the observed inhibition. This repre-
sents the groundwork for future SAR studies into RTA inhibitors
based on previously successful peptide-pterin conjugates.
Figure 4. Dose–response curve of 7 versus % RTA activity.
tion to the increased activity is observed suggesting other factors
dominate the bioactivity.
Although 12 clearly out-performed 4, it is not clear as to
whether it is a suitable analog for comparison, as a urea is clearly
not an amide and an additional degree of freedom has been re-
moved. To understand the observed increase in activity, we com-
pared 12 to 13. Not only is 12 five and a half times more potent
than its oxo analog 13, compound 13 is nearly three times less ac-
tive than 4. This suggests that 13 is a poor analog for oxygen-to-
sulfur comparison at this position.
Acknowledgments
Instrumentation and technical assistance for this work were
provided by the Macromolecular Crystallography Facility, with
financial support from the College of Natural Sciences, the Office
of the Executive Vice President and Provost, and the Institute for
Cellular and Molecular Biology at the University of Texas at Austin.
The Berkeley Center for Structural Biology is supported in part by
the National Institutes of Health, National Institute of General
Medical Sciences, and the Howard Hughes Medical Institute. The
Advanced Light Source is supported by the Director, Office of Sci-
ence, Office of Basic Energy Sciences, of the U.S. Department of En-
ergy under Contract No. DE-AC02-05CH11231.
Observing variations across the length of the inhibitor showed a
strong preference for those pendants that linearly incorporated 7
or 8 heavy atoms between the pterin ring and the arene. Although
the cause for the decrease in activity of 14 is not understood, 15
showed remarkable activity. The X-ray of 1532 showed a firmly
bound pendant through the carbamate oxygen, however the ben-
zyl moiety remained unresolved. The structure also showed a weak
hydrogen bond between the N-H of 15 and Tyr 80 with a bond
length of 3.5 Å (see Fig. 5). This intact H-bond cannot be used to
definitively account for the low IC50 as urea 1333 (IC50 = 209
lM)
This work was supported by the National Institutes of Health
(NIH) grant AI 075509 as well as the Welch Foundation (F-1151).
also shows a 3.5 Å H-bond to Tyr 80, although it exists in a different
orientation. Due to poor resolution, compound 13 was fit to a 1:1
mixture of two RTA states. Figure 5 shows 13 equally bound into
two different RTA conformations; one where the H-bond to Tyr
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.
10.017.
80 is intact and another where Tyr 80 is involved in a slipped
stack with the pteridine heterocycle precluding the possibility of
the H-bond to 13.
p-
Figure 5. Comparison of the 3.5 Å H-bonds to Tyr 80 in carbamate 15 (left) and urea 13 (right). Note the two conformations of Tyr in 13-bound RTA.

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P. A. Wiget et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6799–6804
15. Saito, R.; Pruet, J. M.; Manzano, L. A.; Jasheway, K.; Monzingo, A. F.; Wiget, P. A.;
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28. This compound was synthesized analogously to Scheme 1, Eq.
Supplementary data for details).
1 (See
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32. This compound is available in the PDB titled 4MX5. The crystallographic data is
available in the Supplementary data.
33. This compound is available in the PDB titled 4MX1. The crystallographic data is
available in the Supplementary data.