Evaluation of Substituted 1,2,3-Dithiazoles as Inhibitors of the Feline Immunodeficiency Virus (FIV) Nucleocapsid Protein via a Proposed Zinc Ejection Mechanism

Article abstract of DOI:10.1002/cmdc.201600260

A diverse library of 5-thieno-, 5-oxo-, and 5-imino-1,2,3-dithiazole derivatives was synthesized and evaluated for efficacy against the feline immunodeficiency virus (FIV) as a model for HIV in cells. Several diverse compounds from this series displayed n

Full text of DOI:10.1002/cmdc.201600260

DOI: 10.1002/cmdc.201600260
Communications
Evaluation of Substituted 1,2,3-Dithiazoles as Inhibitors of
the Feline Immunodeficiency Virus (FIV) Nucleocapsid
Protein via a Proposed Zinc Ejection Mechanism
Christopher R. M. Asquith,[a, b] Lidia S. Konstantinova,[c, d] Tuomo Laitinen,[e] Marina L. Meli,[b]
Antti Poso,[e] Oleg A. Rakitin,[c, d] Regina Hofmann-Lehmann,[b] and Stephen T. Hilton*[a]
A diverse library of 5-thieno-, 5-oxo-, and 5-imino-1,2,3-dithia-
zole derivatives was synthesized and evaluated for efficacy
against the feline immunodeficiency virus (FIV) as a model for
HIV in cells. Several diverse compounds from this series dis-
played nanomolar activity and low toxicity, representing a po-
tential new class of compounds for the treatment of FIV and
HIV.
transcription,[6,7] promotion of dimerization, packing and or-
ganization of protein–RNA complexes within freshly created vi-
rions.[8–10] The NCp is an attractive protein target, as it has
been shown to be mutation resistant with inhibition yielding
noninfectious virions.[6,11] It contains a conserved double zinc
finger peptide unit C-X2-C-X4-H-X4-C (CCHC) that is found in
nearly all retroviruses with the exception of spumaviruses,[7] in-
cluding HIV-1/2,[12,13] FIV,[14] simian immunodeficiency virus
(SIV),[15] equine infectious anemia virus (EIAV),[16] amongst
others.[17] The development of an effective agent that could
target one or both of these zinc fingers of the nucleocapsid
protein would render the virus inert, as deletion or modifica-
tion of either zinc finger leads to virus inactivation.[18,19]
The continual discovery of novel therapeutics against various
viral and bacterial strains is essential due to their ability to
adapt and become resistant to current treatments. The human
immunodeficiency virus (HIV) is a case in point, with an array
of drugs used to target multiple points in the viral life cycle,
but the continual development of resistance by the virus has
continued to erode their efficacy.[1] HIV, which results in the de-
velopment of acquired immunodeficiency syndrome (AIDS),
has caused over 25 million deaths worldwide, with over 34 mil-
lion people currently infected with HIV.[2] Amongst non-primate
lentiviruses such as HIV-1 and HIV-2, only the feline immunode-
ficiency virus (FIV) causes a similar compromised immune
system as that observed in humans,[3] with several FIV strains
also displaying central nervous system involvement and analo-
gous AIDS-type disease progression.[4,5]
Two different approaches have been used toward the devel-
opment of nucleocapsid inhibitors: the first, based on small
molecules that compete with binding of the substrate RNA nu-
cleic acid chain has met with some success.[20,21] The second
approach is based on the irreversible ejection of the structural
Zn2+ ion, and has been more productive. Key compounds with
this mechanism of action are exemplified by compounds 1–
8.[22–29] Common functionalities include a disulfide bridge and
more recently a diselenide bridge[30] and/or a stable electroni-
cally deficient core/functional group(s) (Figure 1). As part of
a longer-term program focusing on the development of novel
anti-FIV/HIV agents that target the NCp via zinc abstraction, we
explored the development of novel compound classes by
using FIV as a surrogate model for HIV; the results of our inves-
tigations are reported herein.
The short basic nucleic acid binding nucleocapsid protein
(NCp) of HIV and FIV is an underexplored antiviral target with
no clinically used drugs, despite its involvement at multiple
points of the viral replication cycle. These include annealing of
lys
the cellular primer tRNA3 to the primer binding site in reverse
[a] Dr. C. R. M. Asquith, Dr. S. T. Hilton
School of Pharmacy, Faculty of Life Sciences, University College London,
London, WC1N 1AX (UK)
E-mail: s.hilton@ucl.ac.uk
[b] Dr. C. R. M. Asquith, Dr. M. L. Meli, Prof. R. Hofmann-Lehmann
Clinical Laboratory & Center for Clinical Studies, Vetsuisse Faculty, University
of Zurich, 8057 Zurich (Switzerland)
[c] Dr. L. S. Konstantinova, Prof. O. A. Rakitin
Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences,
Moscow, 119991 (Russian Federation)
[d] Dr. L. S. Konstantinova, Prof. O. A. Rakitin
Nanotechnology Education and Research Center, South Ural State
University, Lenina Ave. 76, Chelyabinsk 454080 (Russian Federation)
[e] Dr. T. Laitinen, Prof. A. Poso
School of Pharmacy, Faculty of Health Sciences, University of Eastern
Finland, Kuopio, 70211 (Finland)
The ORCID identification number(s) for the author(s) of this article can
be found under http://dx.doi.org/10.1002/cmdc.201600260.
Figure 1. Previously reported zinc abstractors 1–8.
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To develop novel therapeutic agents against the NCp of FIV,
we used an in silico homology model derived from HIV-1 and
EIAV nucleocapsid proteins to explore heteroatom-rich disul-
fide-containing compounds, by building on our previous
knowledge relating to bis[1,2]dithiolo[1,4]thiazines and bis[1,2]-
dithiolopyrrole derivatives[31] and tetrathiocine derivatives.[32]
The 1,2,3-diathiazole core caught our interest, as it has been
shown to have a broad biological activity profile, including an-
tibacterial,[33–35] anticancer,[37–38] antifungal/herbicidal,[39–43] and
anti-melanin activities.[44]
commonly reported with the Appel salt chemistry, this could
present an issue relating to the ability of the chloride to act as
a potential leaving group in the final compound.
The syntheses of novel 4- and 5-substituted-1,2,3-dithiazoles
bearing a thioketone, oxo, imino or ethyl 2-cyano-2-acetate yli-
dene functionality at C5 were performed using the reaction
between substituted ethanone oximes and sulfur monochlor-
ide according to a one-pot protocol, generating an in situ pre-
functionalized dithiazolium salt intermediate before treatment
with a selected nucleophile (Scheme 1).[48,37]
The 1,2,3-dithiazole scaffold is stable and has the potential
to be substituted at the C4 and C5 positions, while containing
an electrophilic disulfide bond. Substitutions were made to ex-
plore the propensity of the disulfide to react with the cysteine
thiolates of the NCp model. Investigation of the alignment and
electronic distribution of the 1,2,3-dithiazole core led to
a small selection of structurally diverse compounds, which
were computationally modeled using density functional theory
(DFT), and a small selection of compounds were then synthe-
sized and tested (Figure 2).
Scheme 1. Synthesis of dithiazole derivatives. Reagents and conditions:
a) S2Cl2/pyridine, CH3CN, Ar, À5 to 08C, 15 min; b) thioacetamide, 0 to 258C,
CH3CN, Ar, 2 h; c) formic acid, CH3CN, Ar, 30 min at 08C, 1 h at reflux;
d) ethyl 2-cyanoacetate, pyridine, 258C, CH2Cl2, 5 h; e) aniline/benzyl amine,
CH3CN, Ar, 30 min at 08C, followed by pyridine.
The ethanone oximes 9a–g were prepared according to
published procedures in quantitative yields by heating a mix-
ture of the corresponding ketones or aldehydes with excess
hydroxylamine hydrochloride with sodium acetate in methanol
at reflux.[49] The oximes were subsequently suspended in aceto-
nitrile and reacted with disulfur dichloride, with pyridine
added dropwise to form the related dithiazolium salt inter-
mediate, which was then treated with the appropriate nucleo-
philic source.
Figure 2. Virtual screening was based on both molecular docking (left) and
DFT molecular orbital calculations (right). Molecular docking was used to de-
termine if the screened compound is able to achieve a suitable docking
pose. The DFT calculations were based on the docking poses and thus vali-
dated whether the frontier orbitals of the reactants are geometrically opti-
mally located (HOMO orbitals are marked with a red surface, and the LUMO
orbitals are of the blue-colored 1,2,3-dithiazole scaffold), (Schrçdinger Maes-
tro).
There are a number of methods for the formation of 4-
chloro-1,2,3-dithiazolo-5-thione, including the use of hydrogen
sulfide in acetonitrile[46] or 2-cyanothioacetamide,[50] but we
chose to use thioacetamide for its ease of use and low cost.
Thioacetamide was added after preparation of the dithiazole
salt derivative, and this selectively gave compounds 10a–e in
good yields. 5-Oxodithiazoles were synthesized by replacing
thioacetamide with formic acid to produce 11 a–c. 5-Iminodi-
thiazoles were prepared in a similar fashion by substituting thi-
oacetamide with aniline to give 12a–d. Compound 13 was
produced by treatment of the 9a dithiazole salt derivative
with ethyl cyanoacetate; although modeling indicated that this
compound was not optimal, we wanted to investigate if
having a polar head group such as this cyano ester derivative
would be tolerated.
A series of selected 1,2,3-dithiazoles containing a disulfide
bridge and our supporting mechanistic rationale demonstrate
the potential of this heterocyclic system. These heterocycles
are underreported in the literature due to the fact that C5-sub-
stituted derivatives are a relative synthetic challenge.[45] To
date, 1,2,3-dithiazole chemistry has centered around the 4,5-di-
chloro-1,2,3-dithiazolium chloride salt (Appel salt) and the sus-
ceptibility of attack by nucleophiles at the S1, S2, C4, and C5
atoms.[46] We anticipated that these synthetic mechanisms to
access the core could also play a role in the zinc abstraction
mechanism.[47] Although chloride substitution at C5 is more
Compounds 10–13 were then screened for toxicity in vitro
against feline kidney cells and tested for antiviral efficacy in vi-
tro against a chronically infected feline lymphoid cell line. Bio-
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logical testing of the compounds was based on a dual ap-
proach, the first aimed at identifying any compounds with
nonspecific toxicity at three higher concentrations (1–100 mm)
in a short MMT cell viability assay[51] over 24 h with Crandell
Rees feline kidney cells (CrFK).[52] The second provided an en-
hanced longer-term cytotoxicity screen and an anti-FIV profile
at six concentrations (1 nm to 100 mm) over seven days, using
an IL-2-independent feline lymphoblastoid cell line (FL-4).[53]
FL-4 cells infected with FIV were exposed to the compounds
over a period of seven days and sampled every day and at
each of six concentrations to determine the extent of viral rep-
lication/suppression. Viral RNA was isolated from cell culture
supernatants using the MagNA Pure LC System by the Total
Nucleic Acid Isolation Kit (Roche Applied Science). Viral loads
were determined by quantitative real-time reverse transcrip-
tion polymerase chain reaction (RT-qPCR).[54] The remaining
cells were subjected to an MTT cell viability assay to rule out
any toxicity effects, to validate that the RT-qPCR result is not
caused by nonspecific toxicity, and to provide a therapeutic
index for each compound.
not necessarily be truly representative of an in vivo system. Im-
portantly, the overall results show that even with prolonged
exposure to these potentially zinc abstracting agents, there is
a good therapeutic index, which suggests that zinc abstraction
is not intrinsically tied to cytotoxicity. The toxicity does not
show any correlation with the activity displayed by the most
active compounds such as 10a and 11 b. While 12a also dis-
plays good activity and represents an improvement on previ-
ous work within our group, the therapeutic index (CC50/EC50)
still requires further improvement.
After the initial promising result with 10a we looked to
expand a small cluster of compounds around the main scaffold
and alter structural features of the core compound to increase
the therapeutic index. However, with 10b–e, we found there
to be a decrease in activity relative to 10a, with 10d and 10e
showing unfavorable therapeutic indexes in comparison with
10a, combined with lower overall activity. The slightly in-
creased EC50 value of 10c could be due to limited cell penetra-
tion relative to the other counterparts, or the fact that sulfur is
less electronegative than oxygen.[55] This drop in activity was
not observed with the switch to the oxygen analogue 11 b
which was almost as potent as 10a, but in displaying in-
creased toxicity, the corresponding therapeutic index was
lower. We expected a boost from a nitro group substitution
(11 c and 12c) but this modification led to an increase in toxici-
ty with no material potency gain. The consistent substituent
across each series was the para-methoxyphenyl group, which
was used to screen the ’head group’. Whilst the results of
these compounds were quite consistent, they did not demon-
strate improved potency, but displayed increased toxicity over
The results obtained demonstrate that activity was observed
at varying potencies across each series with nanomolar activi-
ties as observed with 10a (Table 1). Toxicity was generally light
when observed in the initial screening with CrFK cells with 24
hours exposure. This is generally a good indication of the level
of toxicity that would be observed if there was a clearance
mechanism in the FL-4 assay. There appears to be a roughly
tenfold increase in toxicity when looking at the results for the
FL-4 assay relative to the CrFK data, but this has to be taken in
the context of chronic exposure in an in vitro assay and would
Table 1. Results from the 1,2,3-dithiazole scaffold FIV screening.
Compd
X
R
CrFK [%][a]
CC50 [mm][b]
EC50 [mm][b]
TI[c]
cLogP[d]
10a
10b
10c
10d
10e
11 a
11 b
11 c
12a
12b
12c
12d
S
S
S
S
S
O
O
O
N-Ph
N-Ph
N-Ph
N-Ph
phenyl
>100
96.43
>100
74.1
>100
39.24
42.17
3.04
0.023
0.456
2.66
0.9044
0.1773
0.193
>4000
3.53
3.45
3.67
4.3
3.38
2.16
2.38
1.98
5.84
5.76
5.58
4.24
4-methoxyphenyl
4-fluorophenyl
2-benzofuranyl
2-thiophenyl
4-methoxyphenyl
4-fluorophenyl
4-nitrophenyl
phenyl
86.1
15.8
6.7
57.2
6.02
17.1
211.6
>2500
>100
>100
87.45
75.66
66.78
92.51
76.64
40.84
63.52
6.26
7.49
4.53
0.025
0.4325
0.5888
0.1398
0.5001
0.0665
14.5
12.7
32.4
19.5
424.2
4-methoxyphenyl
4-nitrophenyl
ethyl ester
9.74
28.21
13
4-fluorophenyl
90.33
3.24
0.2397
13.5
3.36
14
15
16
S
–
–
phenyl
AZT
raltegravir
71.11
>100
>100
0.681
>100
>100
0.392
5.31
0.01
1.7
18.8
>10000
4.79
À0.16
1.16
[a] Percent viability; compound concentration of 10 mm on CrFK cells for 24 h. [b] Geometric mean, each concentration tested in triplicate after 7 days as
a difference of an untreated DMSO control FL-4 cells with <10% error. [c] Therapeutic index: CC50/EC50, which is the ratio of toxicity to activity. [d] Calculat-
ed using ChemBioDraw Ultra 14.
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10a in this in vitro system. Interestingly, an increase in cLogP
beyond 5 did not significantly inhibit the activity or affect the
solubility of compounds 12a–c. Compounds 12d and 13 were
investigated to understand if disruption to the core structure
by removal of sulfur or oxygen would be tolerated; although
activity was observed, there was not a significant improvement
over 10a, due to an increased metabolic liability from the
ethyl ester. The activity of azidothymidine (AZT, 15) and ralte-
gravir (16) are consistent with previous reports in FIV/HIV.[56,57]
The focus of this work builds on the idea that the disulfide
bridge is pivotal to the activity of the compounds against the
NCp target. We also aimed to demonstrate that the disulfide is
essential for activity by synthesizing a disrupted 1,2,3-dithia-
zole system with a disulfide bond by treatment of 10a with
benzylamine (Scheme 2),[58,59] to give 1,2,5-thiadiazolothione
Figure 3. Proposed mechanism of action of 1,2,3-dithiazole for modification
of the NCp homology model. A) Summary of the mechanism of zinc ejection
from NCp/NCp7. B) A Zn2+-coordinating cysteine thiol(ate) reacts with the
disulfide of the 1,2,3-dithiazole core to generate a transient intermediate di-
sulfide. The disulfide then rearranges to form an intramolecular protein di-
sulfide with consequent decrease in zinc ion affinity. The ejected zinc ion (or
part Zn2+ complex) can then complex with a second 1,2,3-dithiazole core
(reduced) to form a stable complex.
Scheme 2. Reaction of 5H-1,2,3-dithiazole-5-thiones 10a to form 14. Re-
agents and conditions: a) benzyl amine, THF, Ar, 3 h, RT.
14. However, we found the utility of this compound to be lim-
ited by toxicity, after prolonged cellular exposure. The EC50
closely mapped to the CC50 with a resulting therapeutic index
of less than 2; this meant that this was not a definitive way to
demonstrate if the disulfide bridge is essential or not. This ob-
servation may or may not support our hypothesis that the di-
sulfide bridge is essential for the activity observed, but was
not conclusive.
a kinetic argument whereby the energy barrier for zinc ejection
is favored and, in addition, that the formation of a stable zinc
complex is potentially thermodynamically favored. When the
requisite disulfide bridge in the inhibitor is interrupted in com-
pound 14, this disruption to the zinc finger can no longer
occur, and abstraction of zinc by our proposed mechanism is
not available to this compound, leading to the observed limit-
ed activity (Figure 4).
With the activities observed, our proposed mechanism of
action of this compound class involves modification of the
zinc-binding sites on the NCp of FIV which we determined by
using DFT calculations. Support for this idea comes from previ-
ous NMR and MS studies on HIV NCp7 that have shown ob-
servable formation of protein–zinc thiol(ate) complexes and
covalent modifications.[25] We reasoned that 1,2,3-dithiazole-
mediated zinc ion abstraction ejection also occurs via an analo-
gous mechanism to known zinc binding/disrupting com-
pounds in which a zinc-binding cysteinyl thiol(ate) reacts with
the disulfide of the core to generate a transient protein–DTA
disulfide (Figure 3).[25,36,60] This can then rearrange to form an
intramolecular protein disulfide with a consequent decrease in
zinc ion affinity. The ejected zinc ion (or zinc 1,2,3-dithiazole
complex) can then potentially complex with a second (re-
duced) 1,2,3-dithiazole core to form a stable complex which is
analogous to work previously reported on the epidithiodiketo-
piperazine class of natural products.[60–62]
The sub-micromolar potencies of the compounds described
in this study combined with the lower toxicities show a step
change in progression toward the development of useful can-
didate compounds for targeting the FIV/HIV nucleocapsid pro-
tein. The low toxicity and high activity of the initial hit 10a led
Observations from our calculations show that it is unlikely
that thermodynamic parameters drive the observed kinetics.
Once disulfide exchange occurs between the zinc finger and
the inhibitor, the zinc finger in the protein is no longer able to
re-form, and equilibrium to regenerate the structure is unable
to be established. We therefore propose this mechanism using
Figure 4. Global reactivity was estimated by calculation of the Fukui frontier
molecular orbitals of 10–14 and the propensity of the protein to undergo
zinc abstraction. Fukui f+ negative visualized using iso-values 0.0015 (white)
and 0.005 (green), (Schrçdinger Maestro).
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the cells were lysed with methanol (200 mL) to reveal a bright-
purple formazan product.[51] The methanol–formazan absorbance
was determined at 570 nm (BioTek Synergy HT plate reader with
KC4 software, Cambridge systems), and values expressed as 50%
cytotoxic concentration (CC50).
us to believe that improved compounds with enhanced char-
acteristics were possible. Many avenues of development exist,
such as substitution at the para position of the pendant
phenyl ring to prevent P450 metabolism while still maintaining
activity. We demonstrated that 10a strikes a good overall bal-
ance between activity and toxicity. With the quantum calcula-
tions demonstrating the viability of zinc ejection, this opens
the possibility of our investigations with the aim of generating
lead structures with more favorable profiles. While we have en-
hanced the repertoire of the 5-thieno-, 5-oxo-, 5-imino-1,2,3-di-
thiazole class of compounds, the hope is that this work will
allow us to generate a pre-clinical candidate that can treat
both FIV and HIV in an in vivo setting, and this will be reported
in due course.
FIV viral loading studies: The potential antiviral effect against FIV
was tested in a cell culture assay using an IL-2-independent feline
FL-4 lymphoblastoid cell line.[53] The compounds were added at
concentrations ranges in triplicate as described above. The cell su-
pernatant (100 mL) was removed, frozen at À208C and replaced
daily for 7 days. To determine the “long-term” cytotoxicity, on
day 7 the cell culture supernatant was replaced with phenol-red-
free medium (80 mL), and the cells were subjected to MTT assay as
before. The day 7 cell culture supernatants were centrifuged in
a table-top centrifuge (10000 g) at maximal speed for 2 min to
pellet cells and debris. The cell culture supernatants of each tripli-
cate were pooled and frozen immediately at À208C until analysis.
Total nucleic acids (TNA) were extracted using the MagnaPure LC
TNA extraction kit (Roche, Basel, Switzerland) from 100 mL pooled
solution according to the manufacturer’s instructions. FIV RNA was
quantitated by RT-qPCR as described for FIV, using DSMO and un-
treated cells as controls.[54] Results were expressed as 50% effective
concentrations (EC50) determined by a sigmoid dose–response
curve at 50% inhibition, with sigmoid dose–response curve fitting
using GraphPad Prism version 6.0 (GraphPad Inc., La Jolla, CA, USA)
and an RNA standard.
Experimental Section
Modeling
Molecular docking: A quantum polarized molecular docking study
for a number of compounds (Figure 2) was conducted, which was
similar to that previously generated using a homology model,[31]
with the version used in this study based on HIV-1 PDB ID: 2JZW
(NCp7)[63] and EIAV PDB ID: 2BL6 (NCp11) as a model for the FIV
NCp.[16] The inhibitory mechanism of compounds is assumed to
begin with coordination of compounds against more the accessi-
ble zinc finger motif of the model (Figure 3). Our previous homolo-
gy modeling and docking studies suggest that inhibitory activity
can be partly explained by means of facile coordination of com-
pounds close to the more accessible zinc finger structure.
Chemistry
General: Melting points were determined on a Kofler hot-stage ap-
paratus and are uncorrected. IR spectra were recorded on a Spe-
cord M-80 instrument in KBr pellets. 1H NMR were recorded on
a Bruker WM 300 spectrometer (300 MHz), and 13C NMR spectra
were recorded on a Bruker AM300 (75.5 MHz) in CDCl3. J values are
given in hertz. Mass spectra were recorded on a Finnigan MAT
INCOS 50 instrument using electron impact ionization. Elemental
analyses were performed on PerkinElmer 2400 Elemental Analyser.
DFT molecular orbital calculations: The zinc finger structure is
not well described by means of molecular-mechanics-based molec-
ular docking; hence we used Jaguar DFT calculations for the dock-
ing-pose-derived model. The geometry of zinc finger cysteine and
histidine residues were constrained to their initial geometry using
Cartesian constraints to connector carbon atoms. B3LYP of theory
and MSV basis set were used for geometry optimizations in the
gas phase. Reactivities of selected compounds were separately es-
timated by unconstrained gas-phase geometry optimizations
(B3LYP/6-31g**) followed by single-point Fukui frontier molecular
orbital calculations (visualized in Figure 4; Schrçdinger Inc., New
York, NY, USA).
General procedure A: 4-Phenyl-5H-1,2,3-dithiazole-5-thione
(10a): Pyridine (0.243 mL, 3.0 mmol) was added dropwise at À5 to
08C to a stirred solution of (E)-1-phenylethan-1-one oxime 9a[47]
(135 mg, 1.0 mmol) and sulfur monochloride (0.160 mL, 2.0 mmol)
in acetonitrile (10 mL) under an inert atmosphere of argon. The
mixture was stirred at 08C for 15 min, and thioacetamide (83 mg,
1.1 mmol) was added in one portion. The resulting mixture was
stirred at RT for 2 h, filtered, and the solvents were evaporated
under reduced pressure. The residue was purified by column chro-
matography (Silica gel Merck 60, light petroleum and then light pe-
troleum/CH2Cl2 mixtures) to afford compound 10a as a brown
solid (155 mg, 73%): mp: 95–1008C. Anal. calcd for C8H5NS3 (%): C
Biology
Initial short cytotoxicity assay: Crandell Reese feline kidney (CrFK)
cells[52] were cultured in 96-well plates (TPP, Trasadingen, Switzer-
land) at a density of 10000 cells per well, in complete medium
[RPMI 1640 medium (Sigma–Aldrich, Buchs, Switzerland) containing
10% fetal calf serum (FCS), 1% v/v (200 mm) glutamine, and 1% v/
v antibiotic/antimycotic (Ab/Am) (all three from Gibco Life Technol-
ogies, Zug, Switzerland)]. The antiviral compounds were dissolved
in 2% DMSO (Sigma–Aldrich, Buchs, Switzerland) to make a 1m
stock solution. The compounds were 10-fold serially diluted with
complete medium. Six dilutions of the compounds (100 mm to
1 nm) were added in triplicate to the CrFK cells (200 mL) and incu-
bated for 24 h (378C, 5% CO2). The medium was removed and re-
placed with phenol-red-free medium (180 mL) and 3-(4,5-dime-
thylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; 20 mL,
4 mgmLÀ1; Sigma–Aldrich, Buchs, Switzerland), and cells were incu-
bated at 378C for 4 h. The medium was removed by vacuum and
1
45.47, H 2.39, N 6.63, found (%): C 45.55, H 2.43, N 6.70; H NMR
(300 MHz, CDCl3): d=7.36 (3H, m, Ph), 7.47 ppm (2H, m, Ph);
13C NMR (75.5 MHz, CDCl3): d=128.0, (2CH, Ph), 129.4 (2CH, Ph),
130.5 (CH, Ph), 131.3, 179.3 (2sp2 tertiary C), 208.0 ppm (C=S); MS
(EI, 70 eV), m/z (%): 211 (100) [M]+, 135 (95), 103 (30); IR (KBr): n˜ =
3056, 2920 (CÀH), 2852, 2356, 2336, 1436, 1132 (C=S), 604,
499 cmÀ1 (SÀS).[37]
4-(4-Methoxyphenyl)-5H-1,2,3-dithiazole-5-thione (10b): (E)-1-(4-
Methoxyphenyl)ethan-1-one oxime 9b[47] (165 mg, 1 mmol) was
treated according to general procedure A to afford compound
10b as pale-brown crystals (73 mg, 30%): mp: 68–718C. Anal.
calcd for C9H7NOS3 (%): C 44.79, H 2.92, N 5.80, O 6.63, found (%):
1
C 44.95, H 3.08, N 6.04; H NMR (300 MHz, CDCl3): d=3.87 (3H, s,
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CH3); 6.98 (2H, d, 2CH, J=8.8 Hz), 8.98 ppm (2H, d, 2CH, J=
8.8 Hz); 13C NMR (75.5 MHz, CDCl3): d=55.4 (CH3), 113.5 (2CH, Ar),
123.9 (2CH, Ar), 131.1, 161.4, 167.3 (3Cquat), 208.4 ppm (C=S); MS
(EI, 70 eV), m/z (%): 241 (28) [M]+, 210 (15), 197 (4), 133 (100); IR
(KBr): n˜ =2924 (CÀH), 2852, 1600, 1516, 1280, 1144, 1036, 828, 752,
4-(4-Fluorophenyl)-5H-1,2,3-dithiazole-5-one (11b): (E)-1-(4-Fluo-
rophenyl)ethan-1-one oxime 9c[47] (153 mg, 1 mmol) was treated
according to general procedure B to afford compound 11 b as
yellow crystals (107 mg, 50%): mp: 70–728C. Anal. calcd for
C8H4FNOS2 (%): C 45.06, H 1.89, F 8.91, N 6.57, O 7.50, found (%): C
1
700 cmÀ1 [48]
.
44.89, H 1.98, N 6.37; H NMR (300 MHz, CDCl3): d=7.15 (2H, t, J=
8.5, Ar), 8.19 ppm (2H, t, J=8.5, Ar); 13C NMR (75.5 MHz, CDCl3):
d=115.8 (2CH, d, J=23.0 Hz, Ar), 130.1 (2CH, d, J=7.0 Hz, Ar),
127.1, 153.8, 164.3 (CÀF, s, J=251.0 Hz), (3sp2 C), 190.1 ppm (C=O);
MS (EI, 70 eV), m/z (%): 213 (14) [M]+, 185 (14), 121 (40); IR (KBr):
n˜ =3072 (CÀH), 1920, 1676 (C=O), 1596, 1512, 1408, 1272, 1164,
4-(4-Fluorophenyl)-5H-1,2,3-dithiazole-5-thione (10c): (E)-1-(4-
Fluorophenyl)ethan-1-one oxime 9c[47] (153 mg, 1 mmol) was treat-
ed according to general procedure A to afford compound 10c as
brown crystals (92 mg, 40%): mp: 148–1528C. Anal. calcd for
C8H4FNS3 (%): C 41.90, H 1.76, N 6.11, found (%): C 41.83, H 1.89, F
804, 696 cmÀ1 [59]
.
1
8.29, N 6.11; H NMR (300 MHz, CDCl3): d=7.15 (2H, t, J=8.80, Ar),
7.98 ppm (2H, m, Ar); 13C NMR (75.5 MHz, CDCl3): d=115.4, 130.2
(sp2 tertiary C), 131.7 (4CH, Ar), 150.9, 140.6, 165.7 (3sp2 tertiary C),
208.0 ppm (C=S); MS (EI, 70 eV), m/z (%): 229 (78) [M]+, 209 (10),
185(12), 153 (89); IR (KBr): n˜ =2924 (CÀH), 2856, 1888, 1600, 1508,
4-(4-Nitrophenyl)-5H-1,2,3-dithiazole-5-one (11c): (E)-1-(4-Nitro-
phenyl)ethan-1-one oxime 9 f[47] (153 mg, 1 mmol) was treated ac-
cording to general procedure B to afford compound 10c as yellow
crystals (84 mg, 35%): mp: 155–1568C. Anal. calcd for C8H4N2O3S2
(%): C 39.99, H 1.68, N 11.66, found (%): C 40.31, H 1.81, N 11.62, O
1408, 1272, 1136, 852, 792, 680 cmÀ1 [37]
.
1
19.98, S 26.69; H NMR (300 MHz, CDCl3): d=8.33 (2H, d, J=8.8 Hz,
4-(1-Benzofuran-2-yl)-5H-1,2,3-dithiazole-5-thione (10d): (E)-1-
(Benzofuran-2-yl)ethan-1-one oxime 9d[47] (175 mg, 1 mmol) was
treated according to general procedure A to afford compound
10d as red crystals (138 mg, 55%): mp: 132–1348C. Anal. calcd for
C10H5NOS3 (%): C 47.79, H 2.01, N 5.57, O 6.37, found (%): C 47.92,
Ar), 8.40 ppm (2H, d, J=8.8 Hz, Ar); 13C NMR (75.5 MHz, CDCl3): d=
129.0 (2CH, Ar), 133.9 (2CH, Ar), 141.7, 153.4, 158.7 (3sp2 C),
195.5 ppm (C=O); MS (EI, 70 eV), m/z (%): 240 (5) [M]+; IR (KBr): n˜ =
3100 (CÀH), 1667 (C=O), 1652, 1600, 1512, 1408, 1348, 1300, 1272,
860, 796, 672 cmÀ1 [59]
.
1
H 2.23, N 6.76; H NMR (300 MHz, CDCl3): d=7.32 (1H, m, Bzf), 7.44
(1H, m, Bzf), 7.62 (1H, d, J=8.1, Bzf), 7.73 (1H, d, J=7.3, Bzf),
8.50 ppm (1H, s, Bzf); 13C NMR (75.5 MHz, CDCl3): d=110.7, 111.4,
122.9, 123.6, 127.1 (5CH, Bzf), 132.9, 140.8, 147.6, 168.7 (4sp2 terti-
ary C), 205.2 ppm (C=S); MS (EI, 70 eV), m/z (%): 251 (23) [M]+, 175
(59), 143 (58); IR (KBr): n˜ =1648, 1612, 1560, 1348, 1260, 1164,
General procedure C: N-[(5Z)-4-Phenyl-5H-1,2,3-dithiazol-5-ylide-
ne]aniline (12a): Pyridine (0.243 mL, 3 mmol) was added dropwise
at À5 to 08C to a stirred solution of (E)-1-phenylethan-1-one oxime
9a[47] (135 mg, 1 mmol) and sulfur monochloride (0.160 mL,
2 mmol) in acetonitrile (10 mL) under an inert atmosphere of
argon. The mixture was stirred at 08C for 15 min, whereupon ani-
line (1 mmol) was added, and the mixture stirred at 08C for
30 min, followed by the addition of pyridine (0.162 mL, 2 mmol).
The reaction mixture was filtered, and solvents were evaporated
under reduced pressure. The residue was purified by column chro-
matography (Silica gel Merck 60, light petroleum and then light pe-
troleum/CH2Cl2 mixtures) to afford compound 12a as bright-yellow
crystals (149 mg, 55%): mp: 73–768C. Anal. calcd for C14H10N2S2
(%): C 62.19, H 3.73, N 10.36, found (%): C 62.25, H 3.68, N 10.62;
1H NMR (300 MHz, CDCl3): d=7.19 (3H, m, Ph), 7.48 (5H, m, Ph),
8.22 ppm (2H, m, Ph); 13C NMR (75.5 MHz, CDCl3): d=119.0, 125.6,
128.1, 129.0, 129.8, 130.3 (10CH, Ar), 132.6, 153.1, 159.2, 165.4 ppm
(4sp2 tertiary C); MS (EI, 70 eV), m/z (%): 270 (96) [M]+; IR (KBr): n˜ =
3048 (CÀH), 1604, 1598, 1500 (C=N), 1280, 633, 488 cmÀ1 (SÀS).[37]
1036, 828, 748, 700 cmÀ1 [37]
.
4-Thien-2-yl-5H-1,2,3-dithiazole-5-thione (10e): (E)-1-(Thiophen-2-
yl)ethan-1-one oxime 9e[63] (141 mg, 1 mmol) was treated accord-
ing to general procedure A to afford compound 10e as red crystals
(54 mg, 25%): mp: 89–928C. Anal. calcd for C6H3NS4 (%): C 33.16, H
1.39, N 6.44, found (%): C 33.28, H 1.54, N 6.14; HRMS Anal. calcd
1
for C6H3NS4: 216.9148, found: 216.9151; H NMR (300 MHz, CDCl3):
d=7.14 (1H, m, Th), 7.53 (1H, d, J=5.1 Hz, Th), 8.36 ppm (1H, d,
J=4.4 Hz, Th); 13C NMR (75.5 MHz, CDCl3): d=127.2, 130.7, 131.7
(3CH, Th), 133.9, 164.0 (2sp2 tertiary C), 205.6 ppm (C=S); MS (EI,
70 eV), m/z (%): 217 (73) [M]+, 141 (100), 109 (53); IR (KBr): n˜ =
3100, 2920 (CÀH), 2852, 1520, 1412, 1364, 1276, 1140, 984, 824,
784 cmÀ1 [37]
.
General procedure B: 4-(4-Methoxyphenyl)-5H-1,2,3-dithiazole-
5-one (11a): Pyridine (0.243 mL, 3 mmol) was added dropwise at
À5 to 08C to a stirred solution of (E)-1-(4-methoxyphenyl)ethan-1-
one oxime 9 f[47] (165 mg, 1 mmol) and sulfur monochloride
(0.160 mL, 2 mmol) in acetonitrile (10 mL) under argon. The mix-
ture was stirred at 08C for 15 min followed by addition of formic
acid (0.189 mL, 5 mmol), and the resulting mixture was stirred at
08C for 30 min and heated at reflux for 1 h, filtered, and the sol-
vent was evaporated under reduced pressure. The residue was pu-
rified by column chromatography (Silica gel Merck 60, light petro-
leum and then light petroleum/CH2Cl2 mixtures) to afford com-
pound 11 a as yellow crystals (146 mg, 65%): mp: 59–628C. Anal.
calcd for C9H7NO2S2 (%): C 47.98, H 3.13, N 6.22, O 14.20, found
N-[(5Z)-4-(4-Methoxyphenyl)-5H-1,2,3-dithiazol-5-ylidene]-N-phe-
nylamine (12b): (E)-1-(4-Methoxyphenyl)ethan-1-one oxime 9b[64]
(135 mg, 1 mmol) was treated according to general procedure C to
afford compound 12b as yellow crystals (81 mg, 27%): mp: 84–
858C. Anal. calcd for C15H12N2OS2 (%): C 59.98, H 4.03, N 9.33, O
1
5.33, found (%): C 60.21, H 4.22, N 9.32; H NMR (300 MHz, CDCl3):
d=3.88 (3H, s, CH3), 6.98 (2H, d, J=9.5 Hz, Ar), 7.20 (3H, m, Ar),
7.46 (2H, d, J=8.8 Hz, Ar), 8.25 ppm (2H, d, J=8.8 Hz, Ar); 13C NMR
(75.5 MHz, CDCl3): d=55.3 (CH3), 113.5 (2CH, Ar), 119.0 (2CH, Ar),
129.8 (2CH, Ar), 130.6 (2CH, Ar), 125.3 (CH, Ar); 125.6, 153.1, 158.4,
161.2, 165.8 ppm (5 sp2 C); MS (EI, 70 eV), m/z (%): 300 (25) [M]+,
167 (71), 133 (100); IR (KBr): n˜ =3088, 2956 (CÀH), 2836, 1600,
1508, 1484, 1284, 1172, 752, 692 cmÀ1
.
1
(%): C 48.12, H 3.31, N 6.48; H NMR (300 MHz, CDCl3): d=3.87 (3H,
s, CH3) 6.97 (2H, d, J=8.8 Hz, Ar), 8.15 ppm (2H, d, J=9.5 Hz, Ar);
13C NMR (75.5 MHz, CDCl3): d=55.5 (CH3), 114.1 (2CH, Ar), 129.6
(2CH, Ar), 123.8, 154.4, 161.8 (3sp2 C), 190.5 ppm (C=O); MS (EI,
70 eV), m/z (%): 225 (27) [M]+, 197 (7), 165 (5), 133 (100); IR (KBr):
n˜ =3064 (CÀH), 3008, 2920, 2840, 1656 (C=O), 1576, 1508, 1248,
N-[(5Z)-4-(4-Nitrophenyl)-5H-1,2,3-dithiazol-5-ylidene]-N-phenyl-
amine (12c): (E)-1-(4-Nitrophenyl)ethan-1-one oxime 9 f[47]
(180 mg, 1 mmol) was treated according to general procedure C to
afford compound 12c as orange crystals (85 mg, 27%): mp: 171–
1728C. Anal. calcd for C8H4N2O2S3 (%): C 53.32, H 2.88, N 13.32, O
10.15, found (%): C 53.48, H 2.88, N 13.32; 1H NMR (300 MHz,
1180, 832, 684 cmÀ1 [59]
.
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CDCl3): d=7.23 (3H, m, Ph), 7.48 (2H, m, Ph), 8.31 (2H, d, J=
8.3 Hz, Ar), 8.49 ppm (2H, d, J=8.3 Hz, Ar); 13C NMR (75.5 MHz,
CDCl3): d=124.2, 128.6, 135.3, 135.4, 131.0 (9CH, Ar), 143.5, 153.4,
157.9, 162.5, 164.0 ppm (5sp2 tertiary C); MS (EI, 70 eV), m/z (%):
315 (13) [M]+, 167 (54); IR (KBr): n˜ =1612, 1600, 1580, 1576, 1492,
Acknowledgements
The authors are grateful to Bloomsbury Colleges—University of
London, University College London, University of Zurich, Biocenter
Finland/DDCB, and the Russian Science Foundation (grant 15-13-
10022) for financial support toward the goals of our work. We
also thank the University of Zurich and the Center for Clinical
Studies at the Vetsuisse Faculty for use of their facilities and the
CSC-IT Center for Science Ltd. (Finland) for allocation of compu-
tational resources.
1276, 1076, 832, 756, 696 cmÀ1 [37]
.
Ethyl (5Z)-5-(phenylimino)-5H-1,2,3-dithiazole-4-carboxylate
(12d): Ethyl (E)-2-(hydroxyimino)propanoate 9g[65] (131 mg,
1 mmol) was treated according to general procedure C to afford
compound 12d as a red oil (154 mg, 58%): Anal. calcd for
C11H10N2O2S2 (%): C 49.61, H 3.78, N 10.52, found (%): C 49.88, H
1
4.02, N 10.42; H NMR (300 MHz, CDCl3): d=1.43 (3H, t, J=7.3 Hz,
CH3), 4.38 (2H, q, J=7.3 Hz, CH2), 7.16 (3H, m, Ph), 7,46 ppm (2H,
m, Ph); 13C NMR (75.5 MHz, CDCl3): d=14.2 (CH3), 63.0 (CH2), 119.3
(2CH, Ph), 126.4 (CH), 129.8 (2CH, Ph), 139.4, 152.1, 153.3,
160.4 ppm (4sp2 tertiary C); MS (EI, 70 eV), m/z (%): 266 (40) [M]+,
167 (80); IR (KBr): n˜ =3076, 2960 (CÀH), 2924, 2852, 1744 (C=O),
Keywords: FIV · HIV-1 · homology models · nucleocapsid
protein · zinc abstraction · zinc ejection
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1700 (C=O), 1584, 1540, 1484, 1368, 1216, 1144, 740, 676 cmÀ1 [37]
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Ethyl (Z)-2-cyano-2-(4-(4-fluorophenyl)-5H-1,2,3-dithiazol-5-ylide-
ne)acetate (13): Sulfur monochloride (0.32 mL, 4 mmol) and pyri-
dine (0.48 mL, 6 mmol) were successively added dropwise to a solu-
tion of (E)-1-(4-fluorophenyl)ethan-1-one oxime (9c; 153 mg,
1 mmol) in CH2Cl2 (15 mL) at À78C under argon. The reaction mix-
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cetate (1.13 g, 10 mmol) in acetonitrile (10 mL) was added drop-
wise at À158C, and the resulting mixture was stirred for 1 h. Pyri-
dine (0.32 mL, 4 mmol) was added dropwise at À158C, the temper-
ature steadily increased to room temperature, and the solvent was
evaporated under reduced pressure. The residue was purified by
column chromatography (Silica gel Merck 60, light petroleum and
then light petroleum/CH2Cl2 mixtures) to afford compound 13 as
orange crystals (49 mg, 16%): mp: 188–1938C. Anal. calcd for
C13H9FN2O2S2 (%): C 50.64, H 2.94, F 6.16, N 9.09, found (%): C
1
50.49; H 3.10; N 9.12; H NMR (300 MHz, CDCl3): d=1.38 (3H, t, J=
7.3 Hz, CH3), 4.37 (2H, q, J=7.3 Hz, CH2), 7.21 (2H, t, J 8.5, Ar),
7.46 ppm (2H, m, Ar); 13C NMR (75.5 MHz, CDCl3): d=14.3 (CH3),
63.2 (CH2), 116.1 (2CH), 131.5 (2CH), 90.3, 113.1, 128.6, 162.3, 163.1,
166.3, 167.5 ppm (7sp2 C); MS (EI, 70 eV), m/z (%): 308 (62) [M]+,
235 (100), 153 (22); IR (KBr): n˜ =2928 (CÀH), 2204 (CN), 1664, 1520,
1444, 1280, 1104, 824, 792, 700 cmÀ1
.
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amine (0.109 mL, 1 mmol) was added to a solution of 4-phenyl-5H-
1,2,3-dithiazole-5-thione (10a; 0.106 mg, 0.5 mmol) in THF (4 mL)
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Merck 60, light petroleum, and then light petroleum/CH2Cl2 mix-
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1
9.85, S 22.55, found (%): C 63.45, H 4.39, N 10.05, S 22.68; H NMR
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130.5 (CH, Ar), 133.0 (sp2 C), 133.2 (sp2 C), 160.5 (sp2 C), 177.4 ppm
(C=S); MS (EI, 70 eV), m/z (%): 284 (25) [M]+, 251 (13); IR (KBr): n˜ =
3064, 2928 (CÀH), 2852, 1496, 1456, 1428, 1344, 1332, 1292, 1208,
1028, 848, 772, 756, 708, 696 cmÀ1 [59]
.
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Received: May 20, 2016
Revised: July 21, 2016
Published online on && &&, 0000
&
ChemMedChem 2016, 11, 1 – 9
www.chemmedchem.org
8
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!

COMMUNICATIONS
Antiviral 1,2,3! A diverse library of
1,2,3-dithiazole derivatives were synthe-
sized and tested against feline immuno-
deficiency virus (FIV) in cells using den-
sity functional theory to determine
a degree of suitability in a simplified nu-
cleocapsid homology model. The com-
pounds displayed nanomolar activity
and relatively low toxicity and represent
a new class of compounds for the treat-
ment of FIV and HIV. This work demon-
strates the versatility of the of 1,2,3-di-
thiazole scaffold, adding antiviral capaci-
ties to the portfolio of biological activi-
ties already known for this compound
class.
C. R. M. Asquith, L. S. Konstantinova,
T. Laitinen, M. L. Meli, A. Poso,
O. A. Rakitin, R. Hofmann-Lehmann,
S. T. Hilton*
&& – &&
Evaluation of Substituted 1,2,3-
Dithiazoles as Inhibitors of the Feline
Immunodeficiency Virus (FIV)
Nucleocapsid Protein via a Proposed
Zinc Ejection Mechanism
ChemMedChem 2016, 11, 1 – 9
www.chemmedchem.org
9
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
These are not the final page numbers! ÞÞ