Silver(I)-Promoted conversion of thioamides to amidines: Divergent synthesis of a key series of vancomycin aglycon residue 4 amidines that clarify binding behavior to model ligands

Article abstract of DOI:10.1021/ja302808p

Development of a general Ag(I)-promoted reaction for the conversion of thioamides to amidines is disclosed. This reaction was employed to prepare a key series of vancomycin aglycon residue 4 substituted amidines that were used to clarify their interaction with model ligands of peptidoglycan precursors and explore their resulting impact on antimicrobial properties.

Full text of DOI:10.1021/ja302808p

Communication
pubs.acs.org/JACS
Silver(I)-Promoted Conversion of Thioamides to Amidines: Divergent
Synthesis of a Key Series of Vancomycin Aglycon Residue 4 Amidines
That Clarify Binding Behavior to Model Ligands
Akinori Okano, Robert C. James, Joshua G. Pierce, Jian Xie, and Dale L. Boger*
Department of Chemistry and Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines
Road, La Jolla, California 92037, United States
S
* Supporting Information
removal of the lone pair repulsion between the vancomycin
residue 4 carbonyl and D-Ala-D-Lac ester oxygens,12 we
ABSTRACT: Development of a general Ag(I)-promoted
reaction for the conversion of thioamides to amidines is
disclosed. This reaction was employed to prepare a key
series of vancomycin aglycon residue 4 substituted
amidines that were used to clarify their interaction with
model ligands of peptidoglycan precursors and explore
their resulting impact on antimicrobial properties.
reported [Ψ[C(NH)NH]Tpg4]vancomycin aglycon (4)13
in a search for improved dual binding affinities and
antimicrobial activities (Figure 1). Amidine 4 displayed
he glycopeptide antibiotics are the most important class of
T
drugs used in the treatment of resistant bacterial
infections, including those caused by methicillin-resistant
Staphylococcus aureus (MRSA).1 After more than 50 years of
clinical use, the emergence of resistant Gram-positive
pathogens including vancomycin-resistant Enterococci (VRE)
and vancomycin-resistant Staphylococcus aureus (VRSA)
presents a serious public health problem at a time few new
antibiotics are being developed.2 This has led to renewed
interest in the search for additional effective treatments for
resistant pathogens that display the durability of vancomycin,
including the development of new derivatives of the
glycopeptide antibiotics.3,4 Discovered at Eli Lilly, vancomycin
(1) was disclosed in 19565 and introduced into the clinic in
1958 although its structure was not established until nearly 30
years later.6 With the emergence of MRSA, it has become the
drug of last resort for the treatment of such resistant bacterial
infections.1
The glycopeptide antibiotics inhibit bacterial cell wall
synthesis by binding the precursor peptidoglycan peptide
terminus D-Ala-D-Ala.7,8 In the two most prominent resistant
phenotypes (VanA and VanB), this precursor is remodeled to
D-Ala-D-Lac, incorporating an ester in place of the amide in the
natural ligand.9 Synthesis of lipid intermediate I and II,
containing the D-Ala-D-Ala termini, continues but vancomy-
cin-resistant bacteria sense the antibiotic challenge10 and
initiate a late stage remodeling from D-Ala-D-Ala to D-Ala-D-
Lac to avoid the antibiotic action. The binding affinity of
vancomycin for the altered ligand is reduced (1000-fold),
resulting in a corresponding loss in antimicrobial activity (1000-
fold). Thus, efforts to redesign the vancomycin binding pocket
for its use against vancomycin-resistant bacteria must target
compounds that not only bind D-Ala-D-Lac but also maintain
binding to D-Ala-D-Ala.
Figure 1. Vancomycin aglycon residue 4 modifications and proposed
dual binding behavior of the amidine 4.
effective, balanced binding affinity for both model ligands at a
level that is within 2- to 3-fold that exhibited by vancomycin
aglycon for D-Ala-D-Ala. Accurately reflecting these binding
properties, 4 exhibited potent antimicrobial activity (MIC =
0.31 μg/mL, VanA E. faecalis) against VRE, being equipotent to
the activity that vancomycin displays against sensitive bacterial
strains. Although this represents a single atom exchange in the
antibiotic (O→NH) to counter a corresponding single atom
Following an initial success with [Ψ[CH2NH]Tpg4]-
vancomycin aglycon (3)11 to achieve this dual binding by the
Received: March 22, 2012
Published: May 8, 2012
© 2012 American Chemical Society
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Communication
Figure 2. Residue 4 substituted amidines.
Figure 3. Amidine formation.
exchange in the cell wall precursors of resistant bacteria (NH→
O), the modified antibiotic also maintains vancomycin’s ability
to bind the unaltered peptidoglycan D-Ala-D-Ala by virtue of its
apparent ability to serve as either a H-bond donor (for D-Ala-D-
Lac) or H-bond acceptor (for D-Ala-D-Ala). Whereas the former
entails binding of the expectedly protonated amidine (pKa =
12.5), the latter requires binding of the unprotonated amidine.
Herein, we report the synthesis of a key series of substituted
amidines designed to clarify their protonation state when
bound to model ligands and explore additional questions on the
potential behavior of such derivatives (Figure 2). Since selective
modification of vancomycin at the residue 4 site is not yet
possible, a divergent14 total synthesis based on our efforts
targeting the naturally occurring aglycons15−19 was designed
that proceeds through an intermediate capable of late-stage
diversification. The approach incorporated a residue 4
thioamide, which could be selectively modified at the final
stage of the divergent synthesis. In these studies, we found that
the thioamide 5 could be selectively converted to the amidine 4
in a single step using a previously unexamined AgOAc-
promoted reaction with NH3 in MeOH. Importantly, this
reaction was successful (50−85%) on a fully functionalized and
deprotected vancomycin aglycon.13
Because of the magnitude of the effort involved, the survey
herein was conducted on the advanced synthetic intermediate 9
bearing the residue 4 thioamide, but with a C-terminus
hydroxymethyl group in place of the carboxylic acid. This
intermediate is available in 22 versus 26 steps, and its
derivatives, including the amidine 10, exhibit binding and in
vitro antimicrobial properties indistinguishable from the
corresponding vancomycin aglycon derivatives.13b
The first of the substituted amidines that we were especially
interested in targeting was the N-methylamidine 11. Un-
expectedly, efforts to convert thioamide 9 to 11 using AgOAc
and MeNH2−MeOH under the reaction conditions used to
prepare 4 and 10 were not successful. As a result, the various
parameters of this reaction were examined first using the
simpler substrate 15 (Figure 3).20,21
Like the reaction with 9, attempts to convert 15 to 17 using
MeNH2 (2 M in MeOH) and AgOAc (2−10 equiv) in MeOH
were not especially successful. More surprisingly, we also found
that AgOAc (3 equiv) in NH3−MeOH was not as effective in
converting 15 to the parent amidine 16 although 15 is rapidly
consumed.22 This led to an examination of a series of
alternative Ag(I) salts. These studies revealed that the more
reactive Ag(I) salts including AgBF4 and AgOCOCF3 were
effective at promoting the conversion of 15 to the parent
amidine 16 (83%), the N-methylamidine 17 (93%, 1:1 E:Z), or
the N,N-dimethylamidine 18 (82%) in good yields in MeOH at
room temperature (Figure 3). Moreover, these conditions were
successful in converting the residue 4 thioamide in 9 to the N-
methylamidine 11 as an inseparable or equilibrating 1:1 mixture
of E/Z isomers (5 equiv of AgBF4, 2 M MeNH2 in MeOH, 25
°C, 30 min), Figure 2.
Extension of the methodology to the preparation of the N-
hydroxyamidine (amidoxime) 19 upon reaction of 15 with
hydroxylamine is summarized in Figure 4. AgOAc proved
modestly effective at promoting the formation of 19 in MeOH,
whereas the more reactive Ag(I) salts resulted in further
reaction of the product amidoxime 19, leading to liberation of
the N-hydroxyamidoxime and thioamide cleavage. This
cleavage reaction of thioamide 15 was suppressed by running
the reaction in less polar and aprotic solvents where 19 was
isolated in excellent yields. Generation of 12, requiring the use
of a protic solvent (MeOH), provided the easily handled
residue 4 amidoxime as a single E-isomer.
Similar observations were made in the preparation of the Boc
protected N-aminoamidine (amidrazone) 20 upon reaction of
15 with BocNHNH2 (Figure 4). Due to the high nucleophil-
icity of BocNHNH2, most Ag(I)-promoted reactions led to
double addition and cleavage of the thioamide. Short reaction
times (5 min) with AgBF4 (5 equiv) and limiting the amount of
BocNHNH2 (2 equiv, MeOH, 73%) or the use of aprotic,
nonpolar solvents suppressed the overreaction and provided 20
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Figure 6. Dual binding of N-methylamidine 11. Effective binding to D-
Ala-D-Lac must entail the protonated amidine.
assessment was conducted with a sample composed of either an
inseparable or equilibrating 1:1 mixture of E/Z isomers, the
results still indicate that the substitution of the amidine with a
small methyl group is sufficient to significantly diminish its
binding and antimicrobial properties. Whereas it is difficult to
infer details about the protonation state of an amidine when
binding D-Ala-D-Ala, the comparison of 11 with 10 support
expectations that it must be the protonated amidine that binds
D-Ala-D-Lac. Unlike 10, the unprotonated state of 11 would be
incapable of H-bonding to the ligand and suffers a further
destabilizing lone pair/lone pair interaction, Figure 6.
The behavior of N-cyanoamidine 14, which cannot be
protonated (pKa = 1), proved even more interesting. Although
its affinities and activity were reduced relative to the amide 8,
the relative behavior of 8 and 14 was identical and distinct from
those of the amidines 10 and 11 (Figure 2). Like the amide 8,
N-cyanoamidine 14 bound D-Ala-D-Ala much more effectively
than D-Ala-D-Lac, which it failed to bind (≥120-fold).
Accordingly, 14 lacked antimicrobial activity against VanA
VRE (MIC > 40 μg/mL) but remained active against
vancomycin-sensitive S. aureus (MIC = 10 μg/mL) at a level
consistent with its affinity for D-Ala-D-Ala. Moreover, this
affinity for D-Ala-D-Ala was found to be roughly equivalent to
that of N-methylamidine 11, albeit 20-fold less than the parent
amide 8 or amidine 10. The inability of the unprotonated
amidine 14 to bind D-Ala-D-Lac confirms that the effective D-
Ala-D-Lac binding of the parent amidine 10 and N-
methylamidine 11 must entail binding of the protonated
amidines, replacing the destabilizing lone pair repulsion with a
stabilizing electrostatic interaction and weak reverse H-bond.
Similarly, the comparable binding affinities of the unprotonated
cyanoamidine 14 and the N-methylamidine 11 with D-Ala-D-Ala
indicate both bind in their unprotonated state, accepting a H-
bond from the linking amide in the bound ligand (Figure 7).
Figure 4. Amidoxime and amidrazone formation.
Figure 5. N-Cyanoamidine formation.
in good yields. Such problems were less significant with 9,
where the residue 4 thioamide is sterically hindered. The well
behaved Boc protected precursor to the amidrazone 13 was
isolated in good yield as a single isomer.
The amine anticipated to be the most challenging was
cyanamide, due to its lower nucleophilicity (Figure 5).
Remarkably, use of AgOAc (5 equiv) in MeOH led to rapid
conversion of 15 to N-cyanoamidine 21 (30 equiv of H2NCN,
10 min, 85%). Extending this reaction to the preparation of the
vancomycin aglycon N-cyanoamidine using AgOAc (5 equiv)
provided 14 as a single isomer whose properties were
consistent with the Z-configuration or equilibration to (Z)-14
under the assay conditions. The conversion of the thioamide 15
to the N-cycanoamidine 21 could also be conducted in aprotic
solvents (THF > CH3CN > DMF). The further inclusion of
Et3N (10 equiv) gave rise to a reaction that was complete in
minutes and provided superb yields of 21 (91−94%).
Figure 7. N-Cyanoamidine 14 behavior paralleling that of amide 8. D-
Ala-D-Ala (and lack of D-Ala-D-Lac) binding represents unprotonated
amidine binding.
The results of the examination of the amidines 11−14 are
summarized in Figure 2. N-Methylamidine 11 proved to be 30
to 50 times less effective than the parent amidine 10 at
binding23 the model D-Ala-D-Ala and D-Ala-D-Lac ligands 6 and
7, respectively, but 11 bound both with near equal affinities.
Accordingly, it was found to be active against VanA VRE (MIC
= 20 μg/mL), albeit being 60-fold less potent than 10 precisely
in line with its relative binding characteristics. Although the
The amidoxime 12 and amidrazone 13 were important to
examine for an additional reason. Both possess the potential for
covalent attachment to bound D-Ala-D-Lac. Unlike the well-
behaved physical properties of its N-Boc precursor, the
amidrazone 13 obtained upon N-Boc deprotection (TFA, 25
°C, 12 h) proved unmanageable to work with. It was found to
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Journal of the American Chemical Society
Communication
(2) James, R. C.; Pierce, J. G.; Okano, A.; Xie, J.; Boger, D. L. ACS
Chem. Biol. 2012, 7, DOI: 10.1021/cb300007j.
(3) Malabarba, A.; Nicas, T. I.; Thompson, R. C. Med. Res. Rev. 1997,
17, 69.
be insoluble in both protic (buffer, H2O, and MeOH) and polar
aprotic solvents (DMSO), preventing its true assessment in
binding or antimicrobial assays where it proved ineffective
(Figure 2). Even prolonged incubation of suspensions of 13
with D-Ala-D-Lac in the binding assay buffer (>4 months) failed
to provide evidence of either reaction with the ligand (ester
amidation) or ligand hydrolysis. In contrast, the amidoxime 12
was well behaved and easy to characterize. It was isolated as a
single isomer, which we assigned as the E-isomer because of a
potential stabilizing H-bond from the amide NH linking
residues 3 and 4. Consistent with this assignment, both its
binding and antimicrobial activity are reduced ≥200-fold
relative to the parent amidine 10 (Figure 2). Prolonged
incubation of 12 with D-Ala-D-Lac in the binding assay buffer
(>6 months) also failed to provide evidence of either reaction
with the ligand (transesterification)24 or ligand hydrolysis.
Despite the lower activity of the 12, it still represents a
derivative class that merits future consideration as an in vivo
antimicrobial agent. Its well behaved physical properties as an
unprotonated amidine (pKa = 6 vs 12.5), facilitating its
absorption and permeability, as well as its likely rapid in vivo
reduction to the active amidine suggest such amidoximes
should continue to be examined in work going forward.25
Complementary to the studies detailed herein, the parent
amidines 4 and 10 were shown to display dipeptide ligand
binding selectivities and affinities that were identical to those of
the corresponding amides 2 and 8, confirming that they (1)
bind such ligands in the same manner and (2) are subject to the
same structural recognition features that dominate the
vancomycin interaction with D-Ala-D-Ala. This eliminated the
possibility that the amidines may be interacting with the ligands
in a unique manner.13 With the development of a single step
Ag(I)-promoted reaction applicable to amines with a wide
range of nucleophilicities, the divergent synthesis of a series of
substituted amidines from a common residue 4 thioamide was
conducted. The resulting amidines were used to define
additional details of their interaction with model ligands,
indicating that it requires the unprotonated amidine to bind D-
Ala-D-Ala and the protonated amidine to bind D-Ala-D-Lac.
(4) (a) Sussmuth, R. D. ChemBioChem 2002, 3, 295. (b) Walker, S.;
̈
Chen, L.; Helm, J.; Hu, Y.; Rew, Y.; Shin, D.; Boger, D. L. Chem. Rev.
2005, 105, 449. (c) von Nussbaum, F.; Brands, M.; Hinzen, B.;
Weigand, S.; Habich, D. Angew. Chem., Int. Ed. 2006, 45, 5072.
̈
(5) McCormick, M. H.; Stark, W. M.; Pittenger, G. E.; Pittenger, R.
C.; McGuire, J. M. Antibiot. Annu. 1955−1956, 606.
(6) Harris, C. M.; Kopecka, H.; Harris, T. M. J. Am. Chem. Soc. 1983,
105, 6915.
(7) Perkins, H. R. Pharmacol. Ther. 1982, 16, 181.
(8) Williams, D. H.; Williamson, M. P.; Butcher, D. W.; Hammond,
S. J. J. Am. Chem. Soc. 1983, 105, 1332.
(9) Bugg, T. D. H.; Wright, G. D.; Dutka−Malen, S.; Arthur, M.;
Courvalin, P.; Walsh, C. T. Biochemistry 1991, 30, 10408.
(10) (a) Koteva, K.; Hong, H.-J.; Wang, X. D.; Nazi, I.; Hughes, D.;
Naldrett, M. J.; Buttner, M. J.; Wright, G. D. Nat. Chem. Biol. 2010, 6,
327. (b) Hong, H.-J.; Hutchings, M. I.; Buttner, M. J. Adv. Exp. Med.
Biol. 2008, 631, 200.
(11) Crowley, B. M.; Boger, D. L. J. Am. Chem. Soc. 2006, 128, 2885.
(12) McComas, C. C.; Crowley, B. M.; Boger, D. L. J. Am. Chem. Soc.
2003, 125, 9314.
(13) (a) Xie, J.; Pierce, J. G.; James, R. C.; Okano, A.; Boger, D. L. J.
Am. Chem. Soc. 2011, 133, 13946. (b) Xie, J.; Okano, A.; Pierce, J. G.;
James, R. C.; Stamm, S.; Crane, C. M.; Boger, D. L. J. Am. Chem. Soc.
2012, 134, 1284.
(14) Boger, D. L.; Brotherton, C. E. J. Org. Chem. 1984, 49, 4050.
(15) Boger, D. L. Med. Res. Rev. 2001, 21, 356.
(16) (a) Boger, D. L.; Miyazaki, S.; Kim, S. H.; Wu, J. H.; Loiseleur,
O.; Castle, S. L. J. Am. Chem. Soc. 1999, 121, 3226. (b) Boger, D. L.;
Miyazaki, S.; Kim, S. H.; Wu, J. H.; Castle, S. L.; Loiseleur, O.; Jin, Q.
J. Am. Chem. Soc. 1999, 121, 10004.
(17) (a) Boger, D. L.; Kim, S. H.; Miyazaki, S.; Strittmatter, H.;
Weng, J.-H.; Mori, Y.; Rogel, O.; Castle, S. L.; McAtee, J. J. J. Am.
Chem. Soc. 2000, 122, 7416. (b) Boger, D. L.; Kim, S. H.; Mori, Y.;
Weng, J.-H.; Rogel, O.; Castle, S. L.; McAtee, J. J. J. Am. Chem. Soc.
2001, 123, 1862.
(18) Crowley, B. M.; Mori, Y.; McComas, C. C.; Tang, D.; Boger, D.
L. J. Am. Chem. Soc. 2004, 126, 4310.
(19) (a) Garfunkle, J.; Kimball, F. S.; Trzupek, J. D.; Takazawa, S.;
Shimamura, H.; Tomishima, M.; Boger, D. L. J. Am. Chem. Soc. 2009,
131, 16036. (b) Shimamura, H.; Breazzano, S. P.; Garfunkle, J.;
Kimball, F. S.; Trzupek, J. D.; Boger, D. L. J. Am. Chem. Soc. 2010, 132,
7776. (c) Breazzano, S. P.; Boger, D. L. J. Am. Chem. Soc. 2011, 133,
18495.
(20) (a) Shibuya, I.; Taguchi, Y.; Tsuchiya, T.; Oishi, A.; Katoh, E.
Bull. Chem. Soc. Jpn. 1994, 67, 3048. (b) Cacchi, S.; La Torre, F.;
Misiti, D. Chem. Ind. 1978, 669. (c) Corey, E. J.; Boger, D. L.
Tetrahedron Lett. 1978, 5.
(21) (a) Marchand-Brynaert, J.; Moya-Portuguez, M.; Huber, I.;
Ghosez, L. J. Chem. Soc., Chem. Commun. 1983, 818. (b) Sauve, G.;
Rao, V. S.; Lajoie, G.; Belleau, B. Can. J. Chem. 1985, 63, 3089.
(22) In MeOH and in the absence of a reacting amine, treatment of
15 with AgOAc (3 equiv) rapidly provides the corresponding O-
methylimidate (>90%). However, subsequent treatment of the in situ
generated O-methylimidate with NH3 (7 M in MeOH) fails to provide
16, indicating that it is not an intermediate en route to the amidine.
(23) Nieto, M.; Perkins, H. R. Biochem. J. 1971, 124, 845.
(24) Simanenko, Y. S.; Prokop’eva, T. M.; Belousova, I. A.; Popov, A.
F.; Karpichev, E. A. Theor. Exp. Chem. 2001, 37, 288.
(25) Clement, B. Drug Metab. Rev. 2002, 34, 565.
ASSOCIATED CONTENT
* Supporting Information
■
S
Experimental details are provided. This material is available free
of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
boger@scripps.edu
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We gratefully acknowledge the support of the National
Institutes of Health (CA041101) and Skaggs Institute for
Chemical Biology. A.O. is a JSPS Fellow (2010−2011), R.C.J. is
a Skaggs Fellow (2006−2012), and J.G.P. was the recipient of
an NIH postdoctoral fellowship (CA144333, 2009−2011).
REFERENCES
■
(1) (a) Glycopeptide Antibiotics; Nagarajan, R., Ed.; Marcel Dekker:
New York, 1994. (b) Kahne, D.; Leimkuhler, C.; Lu, W.; Walsh, C. T.
Chem. Rev. 2005, 105, 425.
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