Small-Molecule APOBEC3G DNA Cytosine Deaminase Inhibitors Based on a 4-Amino-1,2,4-triazole-3-thiol Scaffold

Article abstract of DOI:10.1002/cmdc.201200411

APOBEC3G (A3G) is a single-stranded DNA cytosine deaminase that functions in innate immunity against retroviruses and retrotransposons. Although A3G can potently restrict Vif-deficient HIV-1 replication by catalyzing excessive levels of G→A hypermutation,

Full text of DOI:10.1002/cmdc.201200411

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DOI: 10.1002/cmdc.201200411
Small-Molecule APOBEC3G DNA Cytosine Deaminase
Inhibitors Based on a 4-Amino-1,2,4-triazole-3-thiol
Scaffold
Margaret E. Olson,[a] Ming Li,[b] Reuben S. Harris,[b] and Daniel A. Harki*[a]
APOBEC3G (A3G) is a single-stranded DNA cytosine deaminase
that functions in innate immunity against retroviruses and ret-
rotransposons. Although A3G can potently restrict Vif-deficient
HIV-1 replication by catalyzing excessive levels of G!A hyper-
mutation, sublethal levels of A3G-catalyzed mutation may con-
tribute to the high level of HIV-1 fitness and its incurable prog-
nosis. To chemically modulate A3G catalytic activity with the
goal of decreasing the HIV-1 genomic mutation rate, we syn-
thesized and biochemically evaluated a class of 4-amino-1,2,4-
triazole-3-thiol small-molecule inhibitors identified by high-
throughput screening. This class of compounds exhibits low-
micromolar (3.9–8.2 mm) inhibitory potency and remarkable
specificity for A3G versus the related cytosine deaminase, APO-
BEC3A. Chemical modification of inhibitors, A3G mutational
screening, and thiol reactivity studies implicate C321, a residue
proximal to the active site, as the critical A3G target for this
class of molecules.
Introduction
Since the discovery of acquired immune deficiency syndrome
(AIDS) and the identification of human immunodeficiency virus
(HIV), the causative retrovirus of AIDS, a wealth of biochemical
and immunological investigation has fueled the development
of more than 25 antiretrovirals and the introduction of highly
active antiretroviral therapy (HAART).[1,2] Although these thera-
pies have slowed the global AIDS epidemic and drastically pro-
longed the life expectancy of HIV-1-positive patients, the immi-
nent development of drug resistance and the toxic side effects
associated with HAART prompt continued efforts toward the
discovery of new therapeutics with unique mechanisms of
action.[3]
virus replication in A3G-expressing cells. The APOBEC3–Vif in-
teraction is therefore essential for HIV-1 infection and provides
a novel and minimally explored target in HIV-1 drug discovery.
Current models hypothesize that A3G restricts HIV-1 replica-
tion by incorporating into budding viral particles by an RNA–
Gag interaction, hijacking transport with the particle until
a target cell is infected, and inhibiting viral cDNA synthesis via
both deamination-independent[7] and deamination-dependent
mechanisms.[4,5,8–10] Deamination-independent inhibition of
HIV-1 replication is predicted to occur through direct A3G in-
teraction with viral genomic RNA, causing physical blockage to
HIV-1 reverse transcriptase progression. The prominent mecha-
nism by which A3G restricts Vif-deficient HIV-1 replication,
however, is through C!U deamination events on minus-
strand viral DNA that become immortalized as G!A hypermu-
tations in the plus (genomic) strand. Such extensive mutation
ultimately renders the virus non-infective.
Research over the past decade has elucidated a previously
unknown mechanism of host–virus interaction between
a family of retrovirus restriction factors found in human host
cells, namely APOBEC3D, APOBEC3F, APOBEC3G, and APO-
BEC3H, and the virally encoded virion infectivity factor (Vif).[4–6]
APOBEC3G (apolipoprotein B mRNA editing enzyme, catalytic
polypeptide-like 3G; A3G), a single-stranded (ss)DNA cytosine-
to-uracil (C!U) deaminase and the archetypal member of this
family, is a potent restrictor of Vif-deficient HIV-1 replication.
Wild-type virus, however, uses Vif to nucleate the formation of
an E3 ubiquitin ligase complex that degrades A3G and enables
The potent anti-HIV-1 activity of A3G has recently sparked
efforts to discover inhibitors of Vif, agonists of A3G, and mole-
cules that mask the A3G–Vif interaction surface.[11–17] Small mol-
ecules that antagonize Vif reinstate the innate antiviral activity
of A3G by boosting G!A hypermutation, accomplishing lethal
mutagenesis. Inhibitor design strategies to increase the HIV-
1 mutation rate can be defined as therapy by hypermutation.[18]
Conversely, there is strong evidence to support the hypothe-
sis that HIV-1 exploits A3G through modulation by Vif. By
taking advantage of the pro-mutational capacities of A3G, HIV-
1 can accomplish an advantageous level of sublethal mutation
to enable viral fitness.[18–21] Sequenced genomes from HIV-1-
positive patients support this model by exhibiting considerable
A3G-dependent mutation patterns despite the ability of Vif to
trigger A3G degradation.[22,23] Thus, genetic variation attributa-
ble to A3G likely contributes to the characteristically high mu-
[a] M. E. Olson, Prof. Dr. D. A. Harki
Department of Medicinal Chemistry, University of Minnesota – Twin Cities
717 Delaware Street S.E., Minneapolis, MN 55414 (USA)
E-mail: daharki@umn.edu
[b] Dr. M. Li, Dr. R. S. Harris
Department of Biochemistry, Molecular Biology & Biophysics
University of Minnesota – Twin Cities
321 Church Street, Minneapolis, MN 55455 (USA)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201200411.
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tation rate of HIV-1, its ability to evade immune clearance
mechanisms, and its rapid evolution of resistance to antiretro-
viral therapies. Therefore, A3G-catalyzed mutation may be es-
sential for HIV-1 to achieve its unprecedented level of viral fit-
ness, and current therapies and vaccination strategies may
benefit from dampening A3G activity, as HIV-1 evolution would
be predicted to slow. Our strategy to decrease the HIV-1 muta-
tion rate by inhibiting A3G can be classified as therapy by hy-
pomutation.[18]
We recently described the development of a fluorescence-
based high-throughput screening (HTS) assay that was used to
identify small-molecule inhibitors of A3G.[24] In our previous
study, screening of 1280 pharmacologically active compounds
(LOPAC, Sigma) demonstrated the A3G-specific inhibitory ca-
pacities of catechol-containing scaffolds, including our proto-
type A3G antagonist MN30 (IC50 =9.1 mm, PubChem CID-
5353329). Mutational screening, structural analysis, and mass
spectrometry identified C321, located proximal to the active
site, as the binding site for these catechol-based A3G inhibi-
tors. To identify additional A3G-specific inhibitors with unique
chemotypes, we screened over 325000 compounds (Sanford–
Burnham Medical Research Institute, NIH MLPCN collection)
using our fluorescence-based A3A/A3G DNA cytosine deamina-
tion assay.[25] A class of 4-amino-1,2,4-triazole-3-thiols and
structurally related analogues were identified and developed
as A3G inhibitors based on MLS-0036803, a HTS hit renamed
MN256.0105 (1) in-house (Figure 1). Scaffolds identical or anal-
ogous to 1 have been previously investigated for fungicidal,[26]
antioxidant,[27] cytotoxic,[28] antimicrobial,[28] and anti-osteoar-
thritic[29] applications.
Scheme 1. Reagents and conditions: a) HCO2H(aq) or AcOH, reflux (to dryness),
95%–quant. (for 4 and 5); b) (EtO)3CH or (EtO)3CCH3, 608C–reflux, 34–69%
(for 6 and 7); c) p-BrC6H4CHO or p-MeOC6H4CHO, AcOH, reflux, 82–84%.
at reflux to yield heterocycle 5. The highest yields were accom-
plished by introducing an empty Dean–Stark trap to remove
excess acetic acid by distillation upon conclusion of the reac-
tion, as opposed to the previously reported method of concen-
tration in vacuo.[30] Imine formation by reaction with para-ani-
saldehyde under acidic conditions delivered 8 (83%, two
steps).
Chemical modification of the 4-amino-1,2,4-triazole-3-thiol
heterocycle 4 was pursued to evaluate the necessity of the
thiol. As shown in Scheme 1, the thiol moiety of congeners 4
and 5 was replaced by a hydroxy group to deliver 6 and 7.
These scaffolds were prepared by treating carbohydrazide (3)
with excess triethylorthoformate (for 6) or triethylorthoacetate
(for 7) to achieve the core 4-amino-1,2,4-triazole-3-ol heterocy-
cles in 69 and 34% yields, respectively.
We were also interested in evaluating scaffolds similar to
1 that were not subject to imine hydrolysis to evaluate the ef-
fects of the additional aromatic functionality on A3G inhibitory
potency. The synthesis of non-hydrolysable amide 9 was ac-
complished in moderate yield through treatment of amine 5
with benzoyl chloride in dioxane at reflux (Scheme 2). We addi-
tionally pursued thiol protection methodologies to further
gauge the necessity of a free sulfhydryl group for biological
potency. For this investigation, we synthesized conjugated
thiadiazole 10 and S-alkylated benzyl analogue 11. Compound
10 was prepared by treatment of 5 with para-methoxybenzo-
nitrile and aqueous phosphoric acid at elevated temperatures
(47% yield). Benzyl protection of 5 was carried out by reaction
Figure 1. Structure of A3G inhibitor MN256.0105 (1) and the frequency at
which the 4-amino-1,2,4-triazole-3-thiol substructure was observed in A3G
hits from high-throughput screening.
Results and Discussion
Synthesis of 4-amino-1,2,4-triazole-3-thiol analogues
To elucidate the mechanism of inhibition for the 4-amino-1,2,4-
triazole-3-thiol class of screening hits and to reconfirm the
original lead molecule, MN256.0105 (1) was synthesized
(Scheme 1). 4-Amino-1,2,4-triazole-3-thiol (4) was obtained in
high yield by reaction of thiocarbohydrazide (2) in aqueous
formic acid at reflux. Recrystallization from ethanol yielded
pure 4 in 95% yield. Compound 1 was then obtained in 82%
yield by condensation of 4 with para-bromobenzaldehyde in
excess acetic acid. Analogue 8 was similarly synthesized in two
steps, first by reaction of thiocarbohydrazide with acetic acid
Scheme 2. Reagents and conditions: a) BzCl, dioxane, reflux, 44%; b) p-
MeOC6H4CN, H3PO4(aq), reflux, 47%; c) BnBr, NaH, DMF, RT, 14%.
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with benzyl bromide, in yields consistent with previous reports,
to afford compound 11.[31]
for either molecule (Table 1). Furthermore, evaluation of thiol-
alkylated scaffolds 10 and 11 revealed no A3G inhibition, fur-
ther demonstrating the necessity of the free thiol.
In addition to screening against wild-type A3G, compounds
1 and 4–11 were tested for inhibitory activity against the relat-
ed cytosine deaminase, APOBEC3A (A3A), to gauge inhibitor
specificity. The primary structure of A3A is 65% identical and
73% similar to the A3G C-terminal catalytic domain. Remarka-
bly, analogues 1 and 4–11 all failed to inhibit A3A activity at
doses up to 100 mm, despite the inherent reactivity of the thiol
functionality. To begin querying if molecules of this class can
exhibit activity in cells, we screened 1 and 4–11 for inhibitory
activity against A3G in HEK293T cell lysates. Unfortunately, no
measureable amounts of A3G inhibition were detected for any
of the molecules screened. Consequently, the reactive thiol
moiety may be unavailable for A3G inhibition in the complex
environment of cell lysates.
Biological evaluation of synthetic analogues
Synthesized small molecules were evaluated for A3G inhibition
with our fluorescence-based deamination assay.[24] Using full-
length human A3G purified from HEK293T cells and an ssDNA
oligonucleotide functionalized with a 5’-6-FAM fluorophore
and a 3’-TAMRA quenching molecule, deamination efficiency
was quantified by measured fluorescence. In the absence of in-
hibitor, deamination of the target cytosine to uracil (C!U) is
followed by uracil DNA glycosylase catalyzed excision of U and
sodium hydroxide catalyzed phosphodiester backbone cleav-
age, which releases the 6-FAM fluorophore from the TAMRA
quench. Dose–response curves were generated to determine
IC50 values for synthesized analogues (Table 1).
Reactivity studies of APOBEC3G inhibitors with cysteine-like
substrates
Table 1. APOBEC3G inhibition by compounds 1 and 4–11.
Candidate Inhibitor
IC50 [mm][a]
The known reactivity properties of thiols with cysteine residues
prompted our evaluation of this scaffold to form disulfide
bonds under biological conditions. Amide analogue 9, which
contains a free thiol, was dissolved in DMSO, diluted with PBS
buffer (pH 7.4) and treated with excess cysteamine. Reaction
aliquots were removed at the following time points: 0 min,
30 min, 1 h, and 12 h, and were analyzed by reversed-phase
(RP)-HPLC for the presence of new peaks. Complete disappear-
ance of the starting material was observed within 30 min yield-
ing one new product, which was characterized as disulfide 12
by mass spectrometry (Figure 2). Therefore, molecules of this
class that contain free thiols are capable of intercepting other
thiols and forming disulfide bonds, which may confer their pri-
mary mechanism of enzyme inhibition.
1
4
5
6
7
8
9
10
11
4.3ꢀ1.1
No Activity
6.1ꢀ1.1
No Activity
No Activity
3.9ꢀ1.2
8.2ꢀ1.2
No Activity
No Activity
[a] Assays performed in triplicate; values are the meanꢀSD.
Synthesized 1 exhibited A3G inhibitory activity equivalent to
that of the HTS material, verifying results obtained from pre-
liminary A3G inhibition (IC50 =4.3ꢀ1.1 mm versus IC50 =2.0 mm,
respectively). Structurally related analogue
8
(IC50 =3.9ꢀ
Mutation studies identify C321 as the inhibitor binding site
1.2 mm) demonstrated equipotent anti-A3G activity to HTS hit
1 (Table 1). As a result, we hypothesized that small modifica-
tions to the phenyl and triazole rings have little effect on A3G
inhibition.
Because free thiols are required for A3G inhibition by mole-
cules of this class, the surface cysteine residues of A3G were in-
vestigated as potential inhibitor binding sites. Previous work
has demonstrated that cysteine 321 (C321) of the catalytic A3G
domain is an inhibitor binding site for covalent modification
by small molecules.[24] Employing this methodology, a triple
mutant construct A3G 2K3A, which has three surface Cys!Ala
substitutions (namely C243A, C321A, and C356A), and a single
mutant A3G C321A were tested in parallel with wild-type
enzyme against thiols 1 and 8 using the previously described
deamination assay.[24] Importantly, in the context of full-length
A3G, these specific substitutions have no effect on localization,
deamination, oligomerization, or HIV-1 Vif-deficient restriction
capabilities.[24,32,33]
Based on previous work with MN30,[24] we hypothesized that
the thiol functionalities of 1 and 8 garnered the A3G inhibitory
activity. Moreover, the suspected hydrolytic susceptibility of
Schiff bases 1 and 8 under biological conditions prompted us
to test the 4-amino-1,2,4-triazole-3-thiol precursors 4 and 5 as
A3G inhibitors. Surprisingly, 4 did not display any inhibitory ac-
tivity at 100 mm, whereas the related 5-methyl analogue 5
(IC50 =6.1ꢀ1.1 mm) was found to be nearly equipotent to its
parent scaffold 8 (IC50 =3.9ꢀ1.2 mm). Incorporation of a non-
hydrolysable linker, yielding 9, maintained inhibition (IC50 =
8.2ꢀ1.2 mm), demonstrating that pendent aromatic functional-
ities on the exocyclic amine have little effect on the potency of
A3G inhibition by molecules of this class.
We found that the mutant enzymes were only partially re-
sistant to compounds 1 and 8, with recovered deamination ef-
ficiency between 19–46% (Figure 3). Interestingly, deaminase
activity was not fully recovered relative to DMSO control or the
previously reported catechol-based covalent inhibitor MN30.[24]
To demonstrate the necessity of the thiol component of
these scaffolds, hydroxy-substituted analogues 6 and 7 were
tested against purified A3G. No inhibitory activity was detected
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Conclusions
High-throughput screening and analogue synthesis have iden-
tified a class of 4-amino-1,2,4-triazole-3-thiols that inhibit the
DNA cytosine deaminase A3G. Counter-screening of the small-
molecule analogues against related APOBEC3 family member
A3A demonstrated marked specificity for A3G inhibition de-
spite the reactivity of the inhibitors to sulfur nucleophiles. Re-
placement or protection of thiol moieties completely abrogat-
ed their inhibitory capabilities, providing evidence that a free
thiol is a key structural feature of this class of inhibitors. A
combination of mutagenesis studies and HPLC assays indicate
that the inhibitory activity of this class of molecules is accom-
plished through disulfide bond formation with C321, a residue
adjacent to the A3G active site. These findings further support
our hypothesis that covalent modification to C321 causes con-
formational change to the A3G protein, where Y315 shifts and
sterically blocks substrate DNA cytosine residues from entering
the A3G active site.[24]
Mutant screening potentially implicates a second mecha-
nism of action based on the inability of the C321A mutant to
fully recover deamination capabilities in comparison with
DMSO control. We hypothesize that the 4-amino-1,2,4-triazole-
3-thiol scaffold may engage the catalytic zinc atom in A3G,
conferring a secondary mechanism of enzyme inhibition, al-
though more evidence is needed to support this theory.
Figure 2. a) 4-Amino-1,2,4-triazole-3-thiol (9) and cysteamine were shaken in
aqueous PBS (pH 7.4) at 378C. Aliquots were analyzed by RP-HPLC at t=0,
30, 60 min, and 12 h. b) Reaction at t=0 min (top) and t=12 h (bottom);
peak 12 was isolated and characterized by HRMS-ESI+ m/z [M+Na]+ calcd:
332.0616, found: 332.0629.
Experimental Section
General synthesis
Reagents were purchased from Sigma–Aldrich, Alfa Aesar, or Acros
and were used without additional purification. Bulk solvents were
obtained from Fisher Scientific, and anhydrous solvents were pur-
chased from Sigma–Aldrich. Reactions were performed under an
atmosphere of dry N2 unless otherwise noted. Silica gel chroma-
tography was performed on a Teledyne-Isco Combiflash Rf-200 in-
strument using Redisep Rf Gold High-Performance silica gel col-
umns (Teledyne-Isco) or self-packed columns with SiliaFlash 60 ꢁ
silica gel (SiliCycle). HPLC analyses were performed on an Agilent
1200 series instrument equipped with a diode array detector and
a Zorbax SB-C18 column (4.6ꢂ150 mm, 3.5 mm, Agilent Technolo-
gies) or a Zorbax SB-AQ column (4.6ꢂ150 mm, 3.5 mm, Agilent
Technologies) for analytical-scale analysis. Compounds used in bio-
logical testing were ꢁ98% pure, as determined by two-wave-
length HPLC analysis (l 254 and 215 nm), except for compounds 4
and 8, which were 92 and 93% pure, respectively. NMR spectrosco-
py was performed with a Bruker Avance instrument operating at
400 MHz (for 1H) and 100 MHz (for 13C) at ambient temperature.
Chemical shifts are reported in parts per million and are normal-
ized to internal solvent peaks or (CH3)4Si (d=0 ppm). Exchangeable
Figure 3. Potency of MN30 (Supporting Information figure S1), 1, and 8
against wild-type A3G and mutants A3G 2K3A and A3G C321A. The mean
and SD of triplicate deaminase assays with 50 mm compound are shown rela-
tive to control (DMSO only); N.D.: not detectable.
Such results imply that these thiol-containing inhibitors may
also function, at least in part, through a second inhibitory
mechanism. Initial hypotheses derived from the field of transi-
tion-metal catalysis suggest that the 4-amino-1,2,4-triazole-3-
thiol scaffold chelates divalent metals.[34–37] A3G, which con-
tains a catalytic zinc atom, may therefore suffer a decrease in
deaminase efficiency in the presence of zinc-chelating mole-
cules; however, additional experiments are necessary to vali-
date this possibility.
1
protons (thiols, alcohols, and amines) were verified by H NMR D2O
exchange experiments. High-resolution mass spectrometry (HRMS)
data were collected in positive-ion mode on a Bruker BioTOF II in-
strument.
4-Amino-1,2,4-triazole-3-thiol (4): Prepared as previously de-
scribed.[38] 1H NMR ([D6]DMSO): d=13.63 (s, 1H, SH), 8.45 (s, 1H),
5.68 ppm (s, 2H, NH2); 13C NMR ([D6]DMSO): d=166.0, 142.3 ppm;
HRMS-ESI+ m/z [M+Na]+ calcd for C2H4N4S: 139.0054, found:
139.0045.
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4-[(4-Bromobenzylidene)amino]-1,2,4-triazole-3-thiol (1): p-Bro-
mobenzaldehyde (0.349 g, 1.89 mmol) was added to a solution of
4-amino-1,2,4-triazole-3-thiol (4, 0.219 g, 1.89 mmol) in AcOH
(8.0 mL) at room temperature. The reaction was then heated at
reflux. After 2 h, the reaction was cooled and poured into ice
water. The resulting precipitate was collected, washed with H2O,
and recrystallized from hot, absolute EtOH to yield compound 1 as
a pale-yellow solid (0.438 g, 82%): 1H NMR (CDCl3): d=10.47 (s,
1H), 8.08 (s, 1H), 7.73 (d, J=2.0 Hz, 2H), 7.63 ppm (d, J=2.0 Hz,
2H); 13C NMR (CDCl3): d=158.83, 158.78, 140.9, 132.4, 131.2, 130.0,
127.3 ppm; HRMS-ESI+ m/z [M+Na]+ calcd for C9H7BrN4S:
304.9472, found: 304.9468.
6-(4-Methoxyphenyl)-3-methyl-1,2,4-triazolo[3,4-b]-
[1,3,4]thiadiazole (10): p-Methoxybenzonitrile (0.100 g, 0.75 mmol)
and 5 (0.098 g, 0.75 mmol) were suspended in aqueous H3PO4
(5 mL) and heated at reflux. After 5 h, the mixture was diluted with
excess H2O, neutralized with aqueous NaOH (40% w/v) to pH 7–8,
and concentrated in vacuo to yield compound 10 as a pale-yellow
1
powder (0.087 g, 47%): H NMR (CDCl3): d=7.83 (d, J=8.0 Hz, 2H),
7.03 (d, J=8.0 Hz, 2H), 3.90 (s, 3H), 2.76 ppm (s, 3H); 13C NMR
(CDCl3): d=166.2, 163.2, 153.1, 144.9, 129.0, 122.1, 115.0, 55.8,
10.6 ppm; HRMS-ESI+ m/z [M+Na]+ calcd for C12H11N3OS:
269.0473, found: 269.0468.
3-(Benzylthio)-5-methyl-1,2,4-triazol-4-amine (11): Prepared as
previously described.[31] 1H NMR (CDCl3): d=7.45–7.43 (m, 2H),
7.36–7.30 (m, 3H), 4.60 (s, 2H), 2.37 ppm (s, 3H); 13C NMR
([D6]DMSO): d=158.7, 152.0, 137.1, 128.7, 127.7, 127.1, 34.4,
10.8 ppm; HRMS-ESI+ m/z [M+H]+ calcd for C10H12N4S: 221.0861,
found: 221.0871.
4-Amino-5-methyl-1,2,4-triazole-3-thiol (5): A mixture of thiocar-
bohydrazide (2, 2.60 g, 24.5 mmol) in AcOH (5.0 mL) in a 100-mL
round-bottom flask was heated at reflux into an empty Dean–Stark
trap. Because product formation is kinetically rapid, this process
served to remove excess acid which enhanced reaction yields. The
reaction was allowed to proceed until product formed as a white
precipitate and all acid was removed. The residual solid was re-
moved from the flask with H2O, filtered, and recrystallized from
hot, aqueous EtOH to yield compound 5 as a white powder
(3.16 g, 99%): 1H NMR ([D6]DMSO): d=13.39 (s, 1H, SH), 5.51 (s,
2H, NH2), 2.11 ppm (s, 3H, CH3); 13C NMR ([D6]DMSO): d=165.37,
149.08, 10.37 ppm; HRMS-ESI+ m/z [M+Na]+ calcd for C3H6N4S:
153.0211, found: 153.0213.
Biological assays
Expression and purification of APOBEC3A, APOBEC3G, APOBEC3G
2K3A and APOBEC3G C321A: A3G, A3A, A3G 2K3A and A3G C321A
were expressed and purified as previously described.[24]
DNA deaminase assays: The DNA deaminase assay was performed
as previously described with the ssDNA oligomer 5’-(6-FAM)-AAA-
TAT-TCC-CTA-ATA-GAT-AAT-GTG-A-(TAMRA)-3’.[24] Deaminase assays
with mutant A3G were performed with 50 mm compound,
0.0675 mm A3G, 0.33 mm ssDNA, and excess uracil DNA glycosylase
(UDG). None of the synthesized compounds inhibited UDG in the
context of the in vitro assay.
4-Amino-1,2,4-triazol-3-ol (6): Prepared as previously described.[39]
1H NMR ([D6]DMSO): d=11.52 (s, 1H, OH), 7.80 (s, 1H), 5.26 ppm
(2H, NH2); 13C NMR ([D6]DMSO): d=154.2, 139.3 ppm; HRMS-ESI+
m/z [M+Na]+ calcd for C2H4N4O: 123.0283, found: 123.0292.
4-Amino-5-methyl-1,2,4-triazol-3-ol (7): Carbohydrazide (3,
0.500 g, 5.550 mmol) was suspended in triethylorthoacetate (1 mL),
heated from 60 to 908C over 45 min, and then held at reflux. After
2 h, the reaction was cooled, concentrated in vacuo, and the crude
solid was recrystallized from hot, absolute EtOH to yield compound
7 as a white crystalline solid (0.214 g, 34%): 1H NMR ([D6]DMSO):
d=11.23 (s, 1H, OH), 5.10 (s, 2H, NH2), 2.07 ppm (s, 3H); 13C NMR
([D6]DMSO): d=154.3, 145.4, 10.7 ppm; HRMS-ESI+ m/z [M+H]+
calcd for C3H6N4O: 115.0614, found: 115.0615.
HPLC assays for disulfide formation: A solution of 9 (10.0 mg,
0.040 mmol) in DMSO (100 mL) was diluted with 1ꢂ aqueous PBS
(5 mL, pH 7.4) and treated with cysteamine (0.684 g, 4.03 mmol,
100 equiv). The solution was then shaken at 378C. Aliquots of the
reaction mixture were taken at the following time points: 0 min,
30 min, 60 min, and 12 h. These reaction aliquots were analyzed by
analytical RP-HPLC. The HPLC analytical method (Zorbax SB-C18
4.6ꢂ150 mm, 3.5 mm column, Agilent Technologies; flow rate:
1.0 mLminꢂ1) involved isocratic 10% CH3CN in 0.1% (v/v) aqueous
CF3CO2H (0–2 min), followed by linear gradients of 10!85%
CH3CN (2–24 min) and 85!95% CH3CN (24–26 min). The major
peak 12 (Figure 2) was isolated, concentrated in vacuo, and charac-
terized by mass spectrometry; HRMS-ESI+ m/z [M+Na]+ calcd for
C12H15N5OS2: 332.0616, found: 332.0629.
4-[(4-Methoxybenzylidene)amino]-5-methyl-1,2,4-triazole-3-thiol
(8): p-Anisaldehyde (93 mL, 0.800 mmol) was added to a solution of
3 (0.100 g, 0.768 mmol) in AcOH (3.5 mL) at room temperature.
The reaction was heated at reflux for 2 h, then cooled and poured
into ice water. The resulting precipitate was collected and washed
with H2O to yield compound 8 as a pale-yellow solid (0.160 g,
1
84%) without further purification: H NMR ([D6]DMSO): d=13.67 (s,
1H), 9.71 (s, 1H), 7.86 (d, J=8.0 Hz, 2H), 7.10 (d, J=8.0 Hz, 2H),
3.85 (s, 3H), 2.32 ppm (s, 3H); 13C NMR (CDCl3): d=163.3, 162.4,
161.5, 149.8, 130.8, 125.2, 114.6, 55.6, 11.3 ppm; HRMS-ESI+ m/z
[M+Na]+ calcd for C11H12N4OS: 271.0630, found: 271.0617.
Acknowledgements
We acknowledge financial support from a University of Minneso-
ta Innovation Grant (to D.A.H. and R.S.H.), the NIH
(P01GM091743 to R.S.H.), and startup funds from the Department
of Medicinal Chemistry and College of Pharmacy (to D.A.H.).
M.E.O thanks the NIH for a Chemistry–Biology Interface Predoc-
toral Traineeship (T32-GM08700) and Dr. Lyle and Sharon Bighley
and the College of Pharmacy for a Bighley Graduate Fellowship.
N-(3-Thio-5-methyl-1,2,4-triazol-4-yl)benzamide (9): A solution of
3 (0.100 g, 0.770 mmol) in dioxane (5 mL) was treated with benzoyl
chloride (100 mL, 0.770 mmol) and heated at reflux for 24 h. The re-
action mixture was cooled to room temperature and concentrated
in vacuo. SiO2 purification (gradient 40!100% EtOAc in hexanes)
gave compound 9 as a white solid (80.0 mg, 44%): 1H NMR
([D6]DMSO): d=13.73 (s, 1H, SH), 11.78 (s, 1H, NH), 8.01–7.98 (m,
2H), 7.71–7.57 (m, 3H), 2.20 ppm (s, 3H); 13C NMR ([D6]DMSO): d=
167.0, 165.4, 149.9, 132.9, 130.9, 128.8, 127.9, 10.0 ppm; HRMS-ESI+
m/z [M+Na]+ calcd for C11H11N3OS: 257.0473, found: 257.0288.
Keywords: antiviral agents · APOBEC3G · drug discovery ·
heterocycles · hypomutation
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2013, 8, 112 – 117 116

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