Sustainable, three-component, one-pot procedure to obtain active anti-flavivirus agents

Article abstract of DOI:10.1016/j.ejmech.2020.112992

The mosquito-borne viruses belonging to the genus Flavivirus such as Dengue virus (DENV) and Zika virus (ZIKV) cause human infections ranging from mild flu-like symptoms to hemorrhagic fevers, hepatitis, and neuropathies. To date, there are vaccines only for few flaviviruses while no effective treatments are available. Pyridobenzothiazole (PBTZ) derivatives are a class of compounds endowed with a promising broad-spectrum anti-flavivirus activity and most of them have been reported as potent inhibitors of the flaviviral NS5 polymerase. However, synthesis of PBTZ analogues entails a high number of purification steps, the use of hazardous reagents and environmentally unsustainable generation of waste. Considering the promising antiviral activity of PBTZ analogues which require further exploration, in this work, we report the development of a new and sustainable three-component reaction (3CR) that can be combined with a basic hydrolysis in a one-pot procedure to obtain the PBTZ scaffold, thus reducing the number of synthetic steps, improving yields and saving time. 3CR was significantly explored in order to demonstrate its wide scope by using different starting materials. In addition, taking advantage of these procedures, we next designed and synthesized a new set of PBTZ analogues that were tested as anti-DENV-2 and anti-ZIKV agents. Compound 22 inhibited DENV-2 NS5 polymerase with an IC₅₀ of 10.4 μM and represented the best anti-flavivirus compound of the new series by inhibiting DENV-2- and ZIKV-infected cells with EC₅₀ values of 1.2 and 5.0 μM, respectively, that translates into attractive selectivity indexes (SI - 83 and 20, respectively). These results strongly reaffirm PBTZ derivatives as promising anti-flavivirus agents that now can be synthesized through a convenient and sustainable 3CR in order to obtain more potent compounds for further pre-clinical development studies.

Full text of DOI:10.1016/j.ejmech.2020.112992

Journal Pre-proof
Sustainable, three-component, one-pot procedure to obtain active anti-flavivirus
agents
Tommaso Felicetti, Maria Sole Burali, Chin Piaw Gwee, Kitti Wing Ki Chan, Sylvie
Alonso, Serena Massari, Stefano Sabatini, Oriana Tabarrini, Maria Letizia Barreca,
Violetta Cecchetti, Subhash G. Vasudevan, Giuseppe Manfroni
PII:
S0223-5234(20)30964-8
DOI:
https://doi.org/10.1016/j.ejmech.2020.112992
EJMECH 112992
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 29 September 2020
Revised Date: 30 October 2020
Accepted Date: 2 November 2020
Please cite this article as: T. Felicetti, M.S. Burali, C.P. Gwee, K.W. Ki Chan, S. Alonso, S. Massari, S.
Sabatini, O. Tabarrini, M.L. Barreca, V. Cecchetti, S.G. Vasudevan, G. Manfroni, Sustainable, three-
component, one-pot procedure to obtain active anti-flavivirus agents, European Journal of Medicinal
Chemistry, https://doi.org/10.1016/j.ejmech.2020.112992.
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Sustainable, three-component, one-pot procedure to obtain
active anti-flavivirus agents
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Tommaso Felicetti,1 Maria Sole Burali,1 Chin Piaw Gwee,2,3 Kitti Wing Ki Chan,2 Sylvie Alonso,3,4
Serena Massari,1 Stefano Sabatini,1 Oriana Tabarrini,1 Maria Letizia Barreca,1 Violetta Cecchetti,1
Subhash G. Vasudevan,2,3,5* and Giuseppe Manfroni1,*
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1Dipartimento di Scienze Farmaceutiche, Università
̀ degli Studi di Perugia, via del Liceo, 1-06123
Perugia, Italy
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2Program in Emerging Infectious Diseases, Duke-NUS Medical School, Singapore 169857
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3Infectious Diseases Translational Research Programme, Department of Microbiology &
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Immunology, Yong Loo Lin School of Medicine and Immunology Programme, Life Sciences
Institute, National University of Singapore, Singapore 117545
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5Institute for Glycomics, Griffith University, Queensland 4022, Australia;
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ABSTRACT
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The mosquito-borne viruses belonging to the genus Flavivirus such as Dengue virus (DENV) and Zika
virus (ZIKV) cause human infections ranging from mild flu-like symptoms to hemorrhagic fevers, hepatitis,
and neuropathies. To date, there are vaccines only for few flaviviruses while no effective treatments are
available.
Pyridobenzothiazole (PBTZ) derivatives are a class of compounds endowed with a promising broad-
spectrum anti-flavivirus activity and most of them have been reported as potent inhibitors of the flaviviral
NS5 polymerase. However, synthesis of PBTZ analogues entails a high number of purification steps, the use
of hazardous reagents and environmentally unsustainable generation of waste.
Considering the promising antiviral activity of PBTZ analogues which require further exploration, in this
work, we report the development of a new and sustainable three-component reaction (3CR) that can be
combined with a basic hydrolysis in a one-pot procedure to obtain the PBTZ scaffold, thus reducing the
number of synthetic steps, improving yields and saving time. 3CR was significantly explored in order to
demonstrate its wide scope by using different starting materials. In addition, taking advantage of these
procedures, we next designed and synthesized a new set of PBTZ analogues that were tested as anti-DENV-2
and anti-ZIKV agents. Compound 22 inhibited DENV-2 NS5 polymerase with an IC50 of 10.4 µM and
represented the best anti-flavivirus compound of the new series by inhibiting DENV-2- and ZIKV-infected
cells with EC50 values of 1.2 and 5.0 µM, respectively, that translates into attractive selectivity indexes (SI -
83 and 20, respectively). These results strongly reaffirm PBTZ derivatives as promising anti-flavivirus
agents that now can be synthesized through a convenient and sustainable 3CR in order to obtain more potent
compounds for further pre-clinical development studies.
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Keywords: three-component reaction (3CR), antiviral agents, NS5 polymerase, one-pot procedure, Dengue
inhibitors, Zika inhibitors.
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1. Introduction
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The genus Flavivirus, family Flaviviridae consists of over 70 positive-sense single-stranded RNA viruses
that are transmitted by insect (arthropod) vectors, mosquitos (of the genera Aedes and Culex) and ticks, and
these viruses are therefore also classified as arboviruses (arthropod-borne) [1,2]. The most studied
flaviviruses are Zika virus (ZIKV), Dengue virus (DENV), West Nile virus (WNV), Japanese encephalitis
virus (JEV), Yellow fever virus (YFV) and tick-borne encephalitis virus (TBEV) [2]. About 400 million
cases of DENV infections occur annually, 100 million of which develop symptoms ranging from mild flu-
like illness to severe hemorrhagic fever and shock syndrome [3]. Moreover, ZIKV caused a widespread
epidemic leading to congenital Zika syndrome (CZS) in the Americas that raised alarm bells as it was
occurring at the same time as Brazil was preparing to host the 2016 Olympic games [4]. With about 500,000
suspected cases only between 2015 and 2016, ZIKV represents a serious threat for the public health,
especially due to its association with microcephaly in newborns [4]. For these reasons, in 2019 WHO
included DENV and ZIKV in the list of “10 Threats to Global Health” [5]. There are no antiviral drugs
available for these infections and the live-attenuated tetravalent vaccine Dengvaxia (CYD-TDV, Sanofi-
Pasteur), approved in 20 endemic countries, shows limited efficacy with some safety concerns [1].
Therefore, new anti-flavivirus drugs are required and compounds with broad-spectrum anti-flavivirus
activity would be particularly desirable in the perspective to treat cases of flavivirus coinfections that can
impact close to two-thirds of the world’s population.
The flavivirus RNA genome encodes for structural and non-structural proteins having different roles
during the viral life cycle and among them, the RNA-dependent RNA polymerase (RdRp) is particularly
appealing for the development of anti-flavivirus compounds [1,6–9]. The flaviviral RdRp is located at the C-
terminal portion of the NS5 protein and shows a typical right-hand orientation with three subdomains:
fingers, palm and thumb [10,11]. The flaviviral RdRp is essential for the viral replication cycle, is highly
conserved and lacks a eukaryotic homologue, therefore it represents an attractive target for the development
of broad-spectrum anti-flavivirus agents [1,12].
We previously identified and optimized a class of 1H-pyrido[2,1-b][1,3]benzothiazol-1-ones (PBTZs)
which displayed a promising broad-spectrum anti-flavivirus activity by acting within the framework of the
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replication complex [13–16]. This peculiar mode of action (MoA) is characterized by the dual inhibition of
NS5 RdRp and NS3-NS5 protein-protein interaction together with the production of multiple variants in the
3’-untranslated region (UTR) of DENV genome. Indeed, similarly to NS3-NS5 interaction-defective mutants
that showed an impairment in the infectious virus production [17], PBTZ derivatives were also able to
significantly reduce the infectivity of released virions in the second round of infection [14]. Interestingly, by
RNA-Electrophoretic Mobility Shift Assay (REMSA), we also observed the appearance of unbound RNA
following the addition of a PBTZ analogue to NS5/3′ UTR RNA mixture, thus suggesting that PBTZs may
interfere with the NS5-RNA interaction [15]. Our studies demonstrated that the PBTZ class is very
promising and further optimization is desirable to obtain molecules suitable for pre-clinical evaluation.
However, the development of new PBTZ analogues faces several challenging synthetic drawbacks related to
time-consuming procedures, use of hazardous reagents and room temperature instability of some
intermediates.
The synthesis of the PBTZ nucleus was reported for the first time by Kato and colleagues in 1979 and its
preparation entailed a two-step procedure [18]: i) synthesis of intermediate benzothiazole acrylate (2) via
condensation of the in-situ prepared Vilsmeier reagent and benzothiazole acetate (1) in 78% yield and ii)
cyclization via reaction of acrylate (2) with an anhydride, such as acetic anhydride, to give PTBZ derivative
3 in 62% yield (Scheme 1a). Criticisms related to this procedure entail waste production and hazardous
reagent use such as the in-situ preparation of the Vilsmeier reagent from POCl3 and DMF in an excess ratio
(2:1) with respect to the benzothiazole acetate (1), in the first step, and a large excess of the anhydride (20:1)
with respect to the benzothiazole acrylate (2), in the second step.
In order to synthesize anti-HCV PTBZs [19], we applied the synthetic route reported by Kato and
colleagues, and, starting from cyclohexyloxy benzothiazole acetate (4), ethyl 1-oxo-2-phenyl-1H-pyrido[2,1-
b][1,3]benzothiazole ester analogue (6) was obtained in 68% overall yield (Scheme 1b). By applying this
procedure, we sought to overcome the above-mentioned issues, by reducing, in the second step, the amount
of phenylacetic anhydride from 20 to 2 equiv., obtaining compound 6 in good yield (83% yield).
Nevertheless, the synthetic procedure still suffered from other issues, in particular during the first reaction
(indicated in Scheme 1b as “rate-limiting step”): i) the aqueous work-up required the use of a large amount
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of halogenated solvent (CHCl3) to extract the benzothiazole acrylate analogue 5 from water; ii) the reaction
always yielded a side product, never characterized before and resulting in a fluorescent spot under UV light
on thin layer chromatography (TLC), which needed a chromatography purification to isolate the pure
intermediate (5); and iii) compound 5 was poorly stable at room temperature and difficult to be stored over
time.
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Considering the great societal expectation that more environmentally sustainable chemical processes
should be developed, green chemistry has become a mainstream research topic and its 12 principles pave the
way towards a more sustainable chemistry [20]. In this regard, multicomponent reactions (MCRs) are
reactions in which three or more starting materials react to form the desired product, thus being characterized
by atom economy, limited numbers of synthetic steps, simplified-product isolation procedures, time saving
and sustainability [21]. Therefore, the identification of new MCRs, to synthesize more complex scaffolds and
extend the chemical space of the modern organic chemistry in respect of the environment, represents a
relevant and challenging topic.
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In this study, we describe a more convenient procedure for the synthesis of PBTZ core. In particular, we
merged more synthetic steps into one, performing a multicomponent reaction (MCR) (Scheme 1d) aimed at
reducing the reaction time, limiting the use of reagents and matching the principles of the green chemistry
[20,22]. In addition, we performed an optimized synthetic procedure in which the MCR could be coupled
with a basic hydrolysis. Through this one-step procedure, acid PBTZ scaffold was easily obtained and
functionalized with different amino acids affording derivatives able to inhibit the DENV-2 NS5 RdRp and
endowed with a potent anti-flavivirus activity.
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2. Results and discussion
2.1. Planning and optimization of the MCR to achieve the PBTZ key synthon
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The replacement of the Vilsmeier reagent with the N,N-dimethylformamide dimethyl acetal (DMF-DMA)
in the reaction with benzothiazole acetate 4 afforded acrylate intermediate (5) (Scheme 1c) preserving good
yields (85%) and reducing the formation of the fluorescent by-product that was easily removed by treating
the reaction crude with EtOAc. The characterization of the fluorescent product, never characterized until
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now, ethyl 1-oxo-1H-pyrido[2,1-b][1,3]benzothiazole-4-carboxylate (7 - Scheme 1c), suggested us that
PBTZ core could be formed in the same reaction conditions as acrylate formation. Thus, we attempted to
optimize the procedure to obtain PTBZ analogues via a three-component reaction (3CR) between a
benzothiazole acetate, the DMF-DMA and an anhydride (Scheme 1d).
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Scheme 1.
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As initial survey of the 3CR, we used as a template the unsubstituted benzothiazole acetate (1). Thus,
ethyl 2-(benzo[d]thiazol-2-yl)acetate (1), DMF-DMA and phenylacetic anhydride, prepared as previously
reported [14], were mixed in a flask and dipped immediately into a pre-heated oil bath at 110 °C (Table 1,
entry 1). After 6h, although in low yield (8%), the reaction furnished compound 8. No traces of any
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fluorescent spot attributable to a potential by-product derived from the reaction of benzothiazole acetate (1)
and the acrylate intermediate were observed. In addition, since only EtOH was used to crystallize compound
8, the use of solvents such as EtOAc and Et2O and the need of a chromatography purification were avoided
in line with a green approach.
Subsequently, in order to improve the reaction yield, we performed the 3CR by using different reaction
conditions (Table 1, entries 2-12).
We initially evaluated the effect of different ratio between the reactants (entries 2 and 3). The reaction
performed by using 2 equiv. of both DMF-DMA and phenylacetic anhydride (entry 2) was more
advantageous (35% yield). On the other hand, the reaction performed with 3 equiv. of DMF-DMA (entry 3)
led to the formation of 8 less efficiently (12% yield) and of traces of the side fluorescent product (compound
9), close analogue of 7 observed in the previous reactions.
Then, we evaluated the effect of the addition of common catalysts used in organic chemistry such as
AcOH, CuBr2 and CuI (entries 4-6). AcOH was used because it is widely employed as acid catalyst in
cyclization reactions, while CuBr2 and CuI were selected due to the ability of copper to catalyze carbon–
carbon and carbon–heteroatom bond formation. No significant reaction was observed in presence of the
AcOH (entry 4) after 12h. Differently, the presence of CuBr2 or CuI (entries 5 and 6) led to the formation of
the compound 8 after 3h, as detected by TLC. We were unable to remove CuBr2 during the reaction work-up
by using EtOH, while the removal of CuI (entry 6) by filtering the boiling EtOH allowed to recover
compound 8 in 37% yield. Although the presence of CuI as catalyst showed a slight benefit, we focused on
the optimization of the reaction conditions of entry 2 with the aim to respect the atom economy.
Based on the observation that the increase of the equiv. of DMF-DMA was detrimental (entry 3) while a
more efficient reaction was obtained by enhancing the equiv. of phenylacetic anhydride (entry 2), we came
back to study the effect of different ratio between the reactants (entries 7-12). In particular, we investigated
the effect of 1.1 equiv. of DMF-DMA and rising equiv. of anhydride (from 2 to 5 equiv., entries 7-10) and 2
equiv. of DMF-DMA with 4 or 5 equiv. of anhydride (entries 11 and 12). We were pleased to find that, with
the exception of the reaction performed by using 2 equiv. of anhydride, all the reactions furnished compound
8 rapidly (2h), efficiently (from 56 to 71% yield) and without the formation of side product 9. The best
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results were obtained by reacting 1 equiv. of the benzothiazole acetate (1), 1.1 equiv. of DMF-DMA and 3
equiv. of phenylacetic anhydride at 110 °C (entry 8). This 3CR furnished compound 8 after 2h in 71% yield.
Table 1. Optimization of 3CR conditions for 8.a
Ratiob
1:1:1
Yield (8)/(9)c
8%/NDd
Entry
1
Temperature
110 °C
110 °C
110 °C
110 °C
110 °C
110 °C
110 °C
110 °C
110 °C
110 °C
110 °C
110 °C
90 °C
Catalyst
Time (h)
-
6
6
2
1:2:2
-
35%/ND
12%/trace
ND/ND
3
1:3:1.2
1:2:2
-
4
4
AcOH (1 eq)
12
3
5
1:2:2
CuBr2 (1 eq)
ND/ND
6
1:2:2
CuI (1 eq)
3
37%/ND
45%/trace
71%/ND
56%/ND
62%/ND
62%/ND
56%/ND
59%/trace
46%/ND
7
1:1.1:2
1:1.1:3
1:1.1:4
1:1.1:5
1:2:4
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-
-
-
-
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-
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8
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9
2
10
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12
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14
2
2
1:2:5
2
1:1.1:3
2
1:1.1:3
rt
2
aThe reaction was performed on 0.9 mmol scale of 1. bReferred to the ratio between 1:DMF-
DMA:phenylacetic anhydride. cIsolated yields. dND: not detected by TLC.
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In order to undertake a greener approach, we also tried to reduce the temperature of the reaction (entries
13 and 14). Interestingly, both the reactions performed at 90 °C and rt were equally rapid (2h) although less
efficient (59% and 46% yield, respectively) and traces of the side product 9 were observed in the reaction
conducted at 90 °C.
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Of note, the reaction crudes were only treated with EtOH (25 mL) and refluxed, and the target compounds
were then collected as pure solids after precipitations. When traces of the insoluble 9 were detected, filtration
of the boiling EtOH enabled us to collect pure target compounds. To compare the yield of the 3CR using our
optimized conditions (Table 1, entry 8) with the classic two-step procedure, we synthesized 8 starting from
benzothiazole acetate 1, by using the classic method. Initially, benzothiazole acetate 1 (1 equiv.) was reacted
with 3 equiv. of DMF-DMA in dry DMF at 90 °C. After 2h, intermediate benzothiazole acrylate (2) was
isolated in 76% yield by treating the crude with EtOAc and filtering. Then, acrylate 2 was reacted with 2
equiv. of phenylacetic anhydride for 2h at 110 °C and, after treatment with Et2O and following filtration,
target compound 8 was obtained in 53% yield.
These data highlighted significant advantages of the 3CR over the multi-step procedure (MSP): i) it was
more efficient showing a 71% yield with respect to the MSP showing an overall yield of 40%; ii) it was more
rapid (2h of the 3CR vs 4h of the MSP); iii) it required a lower use of solvents. In particular, no solvent was
used in the 3CR and only 25 mL of EtOH were needed for the work-up to obtain pure compound 8. On the
contrary, MSP necessitated the use of solvents such as DMF during the reaction and EtOAc and Et2O during
the work-up to remove the side product 9 and recover compound 8, respectively.
In order to compare the optimized 3CR and MSP by green parameters, indicators commonly applied in
sustainable green chemistry such as atom economy (AE), reaction mass efficiency (RME), process mass
intensity (PMI), simple E-factor (sEF) and complete E-factor (cEF) were evaluated.
The first parameter (AE), regarding the ratio between the molecular weight (MW) of the target compound
and the MW of the starting materials for the used equivalents, shows a similar behavior between MSP (32%)
and 3CR (31%). Conversely, when masses of reactants and products are considered in the RME, differences
between the two procedures starts getting significant (MSP = 13%; 3CR = 22%). To be noted, when solvents
used for the reaction and work-up are taken into account, green parameters (PMI, sEF and cEF) widely
support the use of the 3CR (92.96, 3.50 and 91.96, respectively) with respect to the MSP (292.44, 14.29, and
291.44, respectively). Therefore, 3CR seems more favored than MSP even considering approved green
parameters.
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In a successive step of this study, we decided to investigate the role of both anhydride and DMF-DMA in
the 3CR. Thus, we repeated the reaction by using the optimized conditions and replacing phenylacetic
anhydride with the corresponding ethyl 2-phenylacetate or replacing DMF-DMA with trimethyl
orthoformate. Both reactions did not furnish compound 8 after 2h, while led to the formation of the side
product 9 in traces and in 58% yield, respectively. These data clearly suggested that both the anhydride and
DMF-DMA are required for the positive outcome of the reaction and, in particular, to allow cyclization.
Moreover, the replacement of DMF-DMA with trimethyl orthoformate strongly promoted the formation of
the side product 9.
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2.2. Scope evolution of the 3CR and one-pot procedure
2.2.1. Variation of the anhydride
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Utilizing the optimized conditions, we then studied the scope of the reaction (Table 2). The first step was
to replace the phenylacetic anhydride with the acetic anhydride (10a), firstly used by Kato and colleagues for
the PBTZ formation [18], with the aim to further compare 3CR with MSP. Interestingly, acetic anhydride
10a (entry 1) was a suitable substrate for this reaction, reacting smoothly with 1 and DMF-DMA to give
product 3 in 55% yield after 2h. The same reaction performed through the MSP (entry 2) was both less rapid
(2 + 8h) and efficient (43% yield). In addition, the evaluation of the three key green parameters PMI, sEF
and cEF (Table 2) highlighted the greater sustainability of the 3CR than the MSP.
Subsequently, succinic anhydride (10b) and 3-phenylpropanoic anhydride (10c) were chosen to extend
the scope of the reaction and a set of phenylacetic anhydrides (10d-i) was selected in view of their use for
medicinal chemistry purpose such as functionalization of the phenyl at PBTZ C-2 position.
Anhydrides 10c-i were prepared starting from the corresponding acids according to the literature
protocols [14,23–25], while succinic anhydride (10b) was purchased.
When succinic anhydride 10b was used (entry 3), no significant reaction was observed after 12h.
However, the reaction did not even occur by MSP, showing that compound 11b could not be obtained
regardless of the conditions used. On the contrary, 3-phenylpropanoic anhydride 10c and differently
substituted phenylacetic anhydrides (10d-i) (entries 4-10) afforded target compounds 11c-i in moderate to
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good yields (from 19 to 58%). In particular, 3-phenylpropanoic anhydride 10c furnished the worst results
(entry 4), suggesting that a greater reactivity of the α carbon of the anhydride is required during the reaction.
The substituted phenylacetic anhydrides (10d, 10e, 10g and 10i) afforded target compounds without
traces of by-product 9 (entries 5, 6, 8 and 10), differently from anhydrides 10f and 10h (entries 7 and 9), both
having an ortho substituent that likely impaired the reaction owing to the steric hindrance. However, with the
exception of compound 11c, all the target compounds 11d-i were obtained with acceptable green parameters
(Table 2) and collected as pure solids after crystallization of the reaction crude by EtOH.
Table 2. Scope of 3CR using different anhydrides 10a-i.a
Yield
Compd.
(R)
Time
(h)
Entry
Anhydride
(3, 11b-
PMI
sEF/cEF
i)/(9)b
1
2
3CR
MSP
2
55%/NDc
43%//ND
150.18 3.38/149.18
345.79 14.65/344.79
10a (acetic)
3 R = H
2+8
11b R =
3
3CR
10b (succinic)
12
ND/ND
-
-/-
(-CH2CO2H)
4
5
3CR
3CR
3CR
3CR
3CR
3CR
3CR
10c, R = -CH2-C6H5
10d, R = 4Cl-C6H4
10e, R = 4OMe-C6H4
10f, R = 2F-C6H4
3
2
2
2
2
2
3
19%d/ND
53%/ND
46%/ND
334.76 16.36/333.76
114.23 5.50/113.23
132.99 6.42/131.99
11c
11d
11e
11f
11g
11h
11i
6
7
45%/trace 139.76 6.39/138.76
58%/ND 108.43 4.73/107.43
36%/trace 176.68 8.16/175.68
44%/ND 144.55 6.50/143.55
8
10g, R = 3F-C6H4
10h, R = 2Me-C6H4
10i, R = 3Me-C6H4
9
10
aThe reaction was performed on 0.9 mmol scale of 1. bAll yields related to 3CRs have been calculated after
crystallization by EtOH. cND: not detected by TLC. dThe yield of the reaction has been calculated after
purification by chromatography.
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2.2.2. Variation of the benzothiazole
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Subsequently, we investigated the role of some substituents on the benzothiazole core (Table 3). Thus,
using our optimized conditions, we performed the 3CR starting from C-5 substituted benzothiazole acetate
derivatives 4, 12 and 13 (entries 1-3), prepared according to the literature protocols [19], to obtain the PBTZ
derivatives 6, 14 and 15. The use of cyclohexyloxy benzothiazole acetate 4 was prompted by the need to
develop a suitable procedure for the synthesis of compound 6, a key intermediate useful for the synthesis of
anti-flavivirus PBTZs. Interestingly, the 3CRs showed good yields (ranging from 63% to 80%) and ideal
green parameters, bringing out that also substituted benzothiazole acetate derivatives are suitable substrates
for the 3CR aimed at building substituted PBTZ core.
Table 3. Scope of 3CR using three different benzothiazole acetates (4, 12 and 13).a
Yieldb
Entry
R
Benzothiazole
Compd.
Time (h)
PMI
72.79
96.48
75.12
sEF/cEF
2.82/71.79
3.79/95.48
2.74/74.12
1
2
3
3CR
3CR
3CR
-OCy
-OMe
-Cl
6
2
2
71%
63%
80%
4
6
12
14
13
15
aThe reaction was performed on 0.9 mmol scale of 4, 12 or 13. bAll yields related to 3CRs have been calculated after
crystallization by EtOH.
230
2.3. One-pot procedure: 3CR and in-situ basic hydrolysis
231
232
233
234
235
236
237
Strongly motivated from these good results, we also attempted a one-pot procedure to directly obtain the
corresponding PBTZ acid derivatives 16 and 17 (Scheme 2), key intermediates useful to perform different
kinds of reaction couplings. Indeed, compound 17 represents a key synthon to afford potent anti-flavivirus
PBTZ analogues by amide functionalization at C-4 position. The achievement of the key synthon 17 in a
one-pot procedure would be time saving and may strongly reduce the amount of solvents used for multi-step
reactions and purifications of chemical intermediates. Thus, once performed the 3CR to obtain 6 and 8, we
directly added EtOH and 10% aqueous NaOH in the reaction vessel and after 2h, we observed complete
13

238
239
conversion. Compound 16 and 17 were smoothly recovered following acidification and filtration as a pure
solid in good 65 and 73% overall yields, respectively.
240
Scheme 2.
241
242
3. Design, synthesis and anti-flavivirus evaluation of new PBTZ derivatives
243
244
245
246
247
248
249
250
251
252
253
254
255
256
257
258
259
Based on the promising results of the recently published PBTZ analogue 18 having a β-alanine moiety at
C-4 amide position [15], we decided to investigate the introduction of some chemical modifications on the β-
alanine chain and how those could impact on the antiviral activity of the new derivatives (19-26 – Table 4).
Intriguingly, since the removal of the carboxylic function from the PBTZ C-4 portion led to the loss of RdRp
inhibition while retaining good cell-based antiviral activity, as for derivative 27 [15], we also designed three
different alcohol PBTZ derivatives (28-30).
Synthetic procedures to afford the target compounds 19-26 and 28-30 starting from synthons 6 or 17,
obtained by 3CR and one-pot procedures, have been reported in the Supplementary Material (Schemes S1
and S2). For a first survey of biological evaluation, compounds 19, 21, 22, 24, 25 and 28 having a chiral
centre, were synthesized using racemic amino acids.
All compounds were initially subjected to in vitro DENV-2 de novo initiation and elongation polymerase
assays as previously described [26] employing purified recombinant NS5 full-length protein (Table 4). After
evaluation of IC50 values of RdRp elongation inhibition for those compounds exhibiting an inhibition ≥ 70%
at 30 µM, single point inhibition experiments at 10 µM against both DENV-2 and ZIKV infected Huh7 cells
were performed for all PBTZ analogues. For compounds that were unable to reduce the viral titer of at least
70%, the percentage of inhibition at 10 µM is reported in Table 4, while the active compounds (viral titer
reduction > 70%) were subjected to a dose−response testing from 0.01 to 100 µM in order to calculate the
14

260
261
262
effective concentration that results in 50% reduction in infective virus particles (EC50) for both DENV-2 and
ZIKV. In parallel, toxicity was determined on Huh7 cells to obtain CC50 values, and the selectivity index (SI;
CC50/EC50) was calculated (Table 4).
263
264
265
266
267
268
269
The introduction of a methyl group on the β carbon of the β-alanine portion afforded derivative 19 with a
comparable RdRp inhibition (just below the 70% threshold in elongation inhibition and no de novo initiation
inhibition) as the hit 18 (77.6%). On the other hand, a reduction of the anti-DENV-2 and -ZIKV activity in
cell-based assays was observed, with compound 19 that did not reach in both cases the 70% of viral titer
reduction at 10 µM, differently from 18 showing EC50 values of 3.5 and 1.0 µM, respectively. When the size
of the group on the β carbon was increased (derivatives 20-22), a significant improvement in terms of RdRp
inhibition was obtained. All three derivatives reached almost the 100% inhibition at 30 µM in elongation
270
271
272
273
274
275
276
277
278
279
280
activity and gained RdRp inhibition also in de novo phase (≥70% at 30 µM) with respect to 18 (no significant
inhibition observed). Corresponding IC50 values of the elongation polymerase activity resulted from 1.6 to
3.6-fold better than 18. However, improvements with respect to 18 in the cell-based anti-DENV-2 activity
were observed only for the phenyl derivative 22 (EC50 = 1.2 µM, CC50 = 100 µM, SI = 83).
When shifting the introduction of an alkyl group to the α carbon of the β-alanine portion, compound 23
having a cyclopropyl exhibited a comparable activity to 21 (isopropyl on the β carbon) both in RdRp
inhibition and cell-based anti-DENV-2 activity. Therefore, there are evidences that the introduction of
lipophilic moieties on the β-alanine chain led to an improvement in RdRp inhibition, which was not
translated in an increase in anti-DENV-2 activity with regard to 18, except for compound 22. However, the
introduction of these lipophilic portions significantly impaired anti-ZIKV activity, with only phenyl
derivative 22 reaching the 70% viral titer reduction and showing an EC50 of 5.0 µM (SI = 20), 5-fold higher
281
282
than that of 18 (EC50 = 1.0 µM, SI > 100).
Table 4. DENV-2 RdRp biochemical assay and antiviral (DENV-2 and ZIKV) evaluation in HuH7 cells of compounds
19-26, 28-30 and reference compounds 18, 27 and NITD008.
15

Polymerase assay against
DENV-2 full-length NS5
Infection cell-based assay
EC50 (µM),d(SI)e
Compd
R
Inhibition %a
CC50 (µM)c
HuH7 cells
>100
Elong.
De
IC50 (µM)b
Elong.
DENV-2
ZIKV
novo
18[15]
NDf
22.7
77.6
37.7 ± 1.75
NDf
3.5 ± 0.45 (>30)
1.0 ± 0.44 (>100)
29.0%g
69.3
95.9
92.8
NDf
~100
~100
68.1%g
19
81.5
75.9
23.2 ± 2.60
22.1 ± 0.09
3.1 ±0.38 (32)
8.4 ± 0.48 (12)
10.6%g
47.4%g
20
21
79.3
96.2
10.4 ± 0.09
~100
1.2 ± 0.36 (83)
5.0 ±4.2 (20)
22
68.1
NDf
11.0
96.7
64.9
64.6
17.8 ± 1.14
NDf
~100
NDf
7.5 ± 3.48 (13.3)
65.3%g
15.8%g
26.3%g
23
24
25
NDf
>100
6.4 ± 2.8 (15.6)
8.2 ± 0.08(12.2)
NDf
10
37.2
63.3
NDf
NDf
52.3
86.3
0.9 ± 0.02 (58)
1.9 ± 0.03 (43)
7.3 ± 4.20 (7.1)
1.8 ± 0.68 (43)
26
27[15]
NDf
NDf
36.9
42.7
NDf
NDf
22.2
17.9
1.3 ± 0.13 (17)
5.8 ± 0.81 (3)
11.1 ± 1.13 (2)
7.3 ± 0.49 (2.4)
28
29
30
NDf
42.5
-
NDf
21.7
100
1.1 ± 0.62 (20)
1.0 (100)
8.1 ± 3.13 (2.6)
0.2 (>100)
NITD-
008
-
-
-
a % inhibitory activity against RdRp in de novo (primer independent) and elongation (primer dependent) polymerase
assays at single 30 µM concentration. bElong. IC50 is the concentration that inhibits 50% of RdRp activity in elongation
assay; values represent average ± SE from a single experiment carried out with duplicates. c CC50 is the cytotoxic
concentration that affects 50% of cell viability as determined by CellTiter-Glo Luminescent Assay (Promega). d EC50 is
the effective concentration that inhibits 50% virus infection as determined by plaque assay against DENV-2 EDEN
3295 (GenBank accession: EU081177.1) after 48h treatment and ZIKV H/PF/2013 (GenBank accession: KJ776791.2)
e
after 24h treatment; values represent average ± SE from a single experiment carried out with duplicates. SI is
f
selectivity index calculated as ratio CC50/EC50. ND: not determined due to poor or no activity in the corresponding
assay. g Percentage of viral inhibition at 10µM.
16

283
284
285
286
287
288
289
290
291
292
293
294
295
296
297
298
299
300
301
302
303
304
305
The decrease in the degree of flexibility of the aliphatic side chain, obtained by two different constrains,
furnished the 3-carboxy piperidine derivative 24 and the β-proline derivative 25, both showing a moderate
RdRp elongation inhibition (≈ 65%) while not exhibiting any inhibition of the de novo polymerase activity,
similarly to 18. An opposite trend was instead observed for the two analogues when considering the cell-
based anti-flavivirus activity. Piperidine derivative 24 at 10uM showed a 65.3% of DENV-2 viral titer
reduction but only a 26.3% of reduction for ZIKV. On the other hand, pyrrolidine analogue 25 at 10 µM
reached the 70% of viral titer reduction against both viruses and exhibited EC50 values of 6.4 and 8.2 µM
against DENV-2 and ZIKV, respectively, and a tolerable SI (15.6 and 12.2, respectively).
Interestingly, when carboxylic function of 18 was replaced with an amide portion resulting in the close
analogue 26, we observed the loss of RdRp inhibition but an increase in anti-DENV-2 activity (EC50 = 0.9
µM). However, anti-ZIKV activity (EC50 = 7.3 µM) was about 7-fold less than that of 18. Despite
cytotoxicity evaluation of 26 on Huh7 cells displayed a lower CC50 (52.3 µM) than 18 (>100 µM), the
resulting SI is suitable for anti-DENV-2 activity (58) and slightly under the accepted threshold for anti-ZIKV
activity (7.1).
As expected, derivatives having the alcoholic functionality (28-30) did not show any significant RdRp
inhibition at 30 µM, as their analogue 27 [15], thus confirming the key role of the carboxylic group in the
PBTZ derivatives to inhibit flavivirus RdRp. Regarding the cell-based anti-flavivirus activity of 28-30, low
EC50 values were obtained for all three compounds but the evaluation of CC50 values often yielded unsuitable
SIs (ranging from 2 to 20). Best results were obtained against DENV-2, with compounds 28 and 30
displaying EC50 values of 1.3 (SI = 17) and 1.1 µM (SI = 20), respectively. However, all three derivatives
showed an anti-flavivirus profile worse than 27 (EC50 DENV-2 = 1.9 µM, SI = 43; EC50 ZIKV = 1.8 µM, SI = 43).
Taken together, these results highlighted that the introduction of a methyl group or an increase of the rigidity
on the ethanolic chain of 27 reduced the antiviral activity.
306
5. Conclusions
307
308
In conclusion, we report on the exploration and optimization of the synthesis of the ethyl 1-oxo-1H-
pyrido[2,1-b][1,3]benzothiazole-4-carboxylate and its corresponding acid, a scaffold found on several broad-
17

309
310
311
312
313
314
315
316
317
318
spectrum anti-flavivirus agents. Through a 3CR and a one-pot procedure, both environment-friendly
approaches, we quickly and easily obtained the PBTZ scaffold that was further functionalized to synthesize a
set of eleven PBTZ analogues. Through the evaluation of the NS5 RdRp inhibition and antiviral activity
against DENV-2- and ZIKV-infected cells of the PBTZ derivatives, we identified compound 22, having a
phenyl moiety on the β carbon of the β-alanine chain, as the best compound of the series since endowed with
a potent antiviral activity against both DENV-2 and ZIKV (EC50 = 1.2 and 5.0 µM, respectively) and a
suitable SI (83 and 20, respectively). Worthy mentioning, the amidic derivative 26 showed the best anti-
DENV-2 activity (EC50 = 0.9 µM, SI = 58) among the series of the PBTZ analogues. Lacking the free
carboxylic function, compound 26 did not inhibit the NS5 RdRp, but its very promising activity on DENV-2-
infected cells deserves further investigation to understand its mechanism of action.
319
320
4. Experimental section
4.1. Chemistry
321
322
323
324
325
326
327
328
329
330
331
332
333
334
Unless otherwise indicated, all starting materials were commercially available. Reagents and solvents
were purchased from common commercially suppliers and were used as received. Organic solutions were
dried over anhydrous Na2SO4 and concentrated with a Büchi rotary evaporator under reduced pressure. All
reactions were routinely checked by TLC on silica gel 60F254 (Merck) and visualized by using UV and
iodine. Flash column chromatography separations were carried out on Merck silica gel 60 (mesh 230-400).
Yields are referred to purified products and are not optimized. Melting points were determined in capillary
tubes using a Stuart SMP3. 1H NMR and 13C NMR spectra were recorded at 400 and 101 MHz, respectively,
with a Bruker Advance DRX- 400 instrument. Chemical shifts (δ) are reported in ppm relative to TMS and
calibrated using residual undeuterated solvent as internal reference. Coupling constants (J) are reported in
Hz. Spectra were acquired at 298 K. Data processing were performed with MestReNova software, and the
spectral data are consistent with the assigned structures. Compounds 19-26 and 28-30 were ≥95% pure as
determined by LC/MS using an Agilent 1290 Infinity System machine equipped with DAD detector from
190 to 640 nm. The purity was revealed at 254 nm using a Phenomenex AERIS Widepore C4, 4.6 mm, 100
mm (6.6 lm) with flow rate: 0.85 ml/min; acquisition time: 10 min; gradient: acetonitrile in water containing
18

335
336
337
338
0.1% of formic acid (0 100% in 10 min); oven temperature, 30 C. Peak retention time is given in minutes.
Detection was based on electrospray ionization (ESI) in negative polarity using Agilent 1290 Infinity System
equipped with a MS detector Agilent 6540UHD Accurate Mass Q-TOF. Optical rotatory power was
determined using DIP-360 JASCO digital polarimeter.
339
340
341
342
343
344
345
346
4.1.1. General procedure for the 3CR of compounds 3, 6, 11c-i, 14 and 15 (Method A). In a round-
bottom flask, benzothiazole acetate derivatives (1, 4, 12 and 13) (0.90 mmol), DMF-DMA (0.99 mmol) and
phenyl acetic anhydride or anhydrides 10a-i (2.70 mmol) were added and the mixture was placed in a 110 °C
pre-heated oil bath under stirring. After the disappearance of benzothiazole acetate derivatives by monitoring
TLC, EtOH (25 mL) was added. When traces of 9 were detected by TLC, the boiling EtOH was immediately
filtered and the desired compound was collected after evaporation of the filtrate as pure yellow solids. When
traces of 9 were absent by TLC, after cooling of the reaction mixture, crystallization was allowed, and pure
yellow compounds were collected following filtration.
347
348
349
350
351
352
353
4.1.2. General Procedure for the amidation reaction for compounds 25, 26 and 40-44 (Method B).
Under N2 atmosphere, to a mixture of the appropriate acid (1.0 mmol), and the appropriate amino acid
methyl ester hydrochloride or the desired amine (1.3 mmol) in dry DMSO (15 mL), TBTU coupling reagent
(1.3 mmol), and DIPEA (4.6 mmol) were added. After stirring at room temperature, the reaction mixture was
poured into ice/water and acidified with 2N HCl (pH = 4-5) to give a precipitate which was filtered under
vacuum to give the relative solid which was purified, as described below for each compound, in order to
obtain the desired PBTZ derivative.
354
355
356
357
358
359
360
4.1.3.
1-{[8-(Cyclohexyloxy)-1-oxo-2-phenyl-1H-pyrido[2,1-b][1,3]benzothiazol-4-
yl]carbonyl}pyrrolidine-3-carboxylic acid (25). Following the general procedure (Method B), starting
from acid (17) and using methyl pyrrolidine-3-carboxylate hydrochloride (37), compound 25 was obtained,
after purification by flash column chromatography eluting with CHCl3/MeOH (100 to 97:3), as a yellow
1
solid in 27% yield. Reaction time: 4h; m.p. 118-120 °C. H NMR (400 MHz, [D6]DMSO): δ = 12.71 (brs,
1H, COOH), 8.85 (d, J = 2.3 Hz, 1H, H-9), 8.03 (s, 1H, H-3), 7.88 (d, J = 8.7 Hz, 1H, H-6), 7.72 (d, J = 7.5
Hz, 2H, H-2’ and H-6’), 7.41 (t, J = 7.8 Hz, 2H, H-3’ and H-5’), 7.32 (t, J = 7.3 Hz, 1H, H-4’) 7.19 (dd, J =
19

361
362
363
364
365
366
2.4 and 8.8 Hz, 1H, H-7), 4.41-4.36 (m, 1H, OCH), 3.77-3.66 (m, 4H, pyrrolidine CH2 x 2), 3.10-3.03 (m,
1H, pyrrolidine CH), 2.15-1.92 (m, 2H, pyrrolidine CH2), 1.90 (m, 2H, cyclohexyloxy CH2), 1.70-1.67 (m,
2H, cyclohexyloxy CH2), 1.50-1.23 (m, 6H, cyclohexyloxy CH2 x 3). 13C NMR (101 MHz, [D6]DMSO): δ =
174.41, 165.51, 160.97, 156.55, 151.77, 139.34, 136.86, 135.04, 129.18, 128.48, 127.70, 123.01, 121.74,
120.35, 116.81, 108.18, 107.41, 76.13, 50.45, 47.82, 42.73, 31.66, 28.80, 25.55, 23.42. HPLC: rt 6.344,
HRMS (ESI) calculated for C29H29N2O5S [M+H]+ 517.1796 found 517.1802.
367
368
369
370
371
372
373
374
375
376
377
378
379
4.1.4.
N-(3-amino-3-oxopropyl)-8-(cyclohexyloxy)-1-oxo-2-phenyl-1H-benzo[4,5]thiazolo[3,2-
a]pyridine-4-carboxamide (26). Following the general procedure (Method B), starting from acid 17 and
using β-alaninamide hydrochloride (38), compound 26 was obtained, after purification by flash column
chromatography eluting with CHCl3/MeOH (97:3), as a yellow solid in 25% yield. Reaction time: 4h; m.p.
204-205 °C (d). 1H NMR (400 MHz, [D6]DMSO): δ = 8.84 (d, J = 2.0 Hz, 1H, H-9), 8.72 (t, J = 5.4 Hz, 1H,
CONH), 8.38 (s, 1H, H-3), 7.87 (d, J = 8.7 Hz, 1H, H-6), 7.76 (d, J = 7.6 Hz, 2H, H-2’ and H-6’), 7.43 (t, J =
7.4 Hz, 2H, H-3’ and H-5’), 7.35-7.31 (m, 2H, H-4’ and CONH2 x 1/2), 7.16 (dd, J = 2.1 and 8.7 Hz , 1H, H-
1
7), 6.84 (brs, 1H, CONH2 x /2), 4.37-4.33 (m, 1H, OCH), 3.44 (q, J = 6.65 Hz, 2H, NHCH2CH2CONH2),
2.34 (t, J = 7.1 Hz, 2H, NHCH2CH2CONH2), 1.89 (m, 2H, cyclohexyloxy CH2), 1.68-1.66 (m, 2H,
cyclohexyloxy CH2) 1.46-1.23 (m, 6H, cyclohexyloxy CH2 x 3). 13C NMR (101 MHz, [D6]DMSO): δ =
172.82, 164.31, 161.25, 156.18, 151.34, 138.88, 136.74, 133.86, 129.27, 128.33, 127.69, 123.02, 122.22,
121.02, 116.40, 107.46, 105.84, 75.58, 36.44, 35.44, 31.50, 25.46, 23.41. HPLC: rt 5.976, HRMS (ESI)
calculated for C27H28N3O4S [M+H]+ 490.1800 found 490.1796.
380
381
382
383
384
385
386
4.1.5.
Methyl
3-({[8-(cyclohexyloxy)-1-oxo-2-phenyl-1H-pyrido[2,1-b][1,3]benzothiazol-4-
yl]carbonyl}amino)butanoate (40). Following the general procedure (Method B), starting from acid 17 and
using methyl 3-aminobutanoate hydrochloride (31), compound 40 was obtained, after purification by flash
column chromatography eluting with Cy/EtOAc (8:2), as a yellow solid in 59% yield. Reaction time: 4h;
1
m.p. 178-180 °C. H NMR (400 MHz, [D6]DMSO): δ = 8.85 (d, J = 1.9 Hz, 1H, H-9), 8.76 (t, J = 5.5 Hz,
1H, CONH), 8.38 (s, 1H, H-3), 7.89 (d, J = 8.7 Hz, 1H, H-6), 7.76 (d, J = 7.9 Hz, 2H, H-2’ and H-6’), 7.46-
7.42 (m, 2H, H-3’ and H-5’), 7.36-7.32 (m, 1H, H-4’), 7.18 (dd, J = 1.7 and 8.8 Hz, 1H, H-7), 4.39-4.35 (m,
20

387
388
389
1H, OCH), 3.57 (s, 3H, CO2CH3), 3.51-3.44 (m, 1H, CH3CHCHaHb), 3.36-3.29 (m, 1H, CH3CHCHaHb),
2.79-2.70 (m, 1H, NHCHCH2), 1.91 (m, 2H, cyclohexyloxy CH2), 1.78-1.67 (m, 2H, cyclohexyloxy CH2),
1.56-1.18 (m, 6H, cyclohexyloxy CH2 x 3), 1.08 (d, J = 7.0 Hz, 3H, CH3CHCH2).
390
391
392
393
394
395
396
397
398
4.1.6.
Methyl
3-({[8-(cyclohexyloxy)-1-oxo-2-phenyl-1H-pyrido[2,1-b][1,3]benzothiazol-4-
yl]carbonyl}amino)-3-methylbutanoate (41). Following the general procedure (Method B), starting from
acid 17 and using methyl 3-amino-3-methylbutanoate hydrochloride (32), compound 41 was obtained, after
purification by flash column chromatography eluting with Cy/EtOAc (8:2), as a yellow solid in 39% yield.
Reaction time: 3h; m.p. 176-178 °C. 1H NMR (400 MHz, [D6]DMSO): δ = 8.56 (brs, 1H, H-9), 8.40 (s, 1H,
H-3), 7.99 (s, 1H, CONH), 7.86 (d, J = 8.6 Hz, 1H, H-6), 7.73 (d, J = 7.5 Hz, 2H, H-2’ and H-6’), 7.45-7.41
(m, 2H, H-3’ and H-5’), 7.35-7.32 (m, 1H, H-4’), 7.17 (d, J = 8.2 Hz, 1H, H-7), 4.36-4.28 (m, 1H, OCH),
3.50 (s, 3H, CO2CH3), 2.89 (s, 2H, CH2), 1.94 (m, 2H, cyclohexyloxy CH2), 1.68-1.58 (m, 2H,
cyclohexyloxy CH2), 1.46-1.17 (m, 12H, cyclohexyloxy CH2 x 3 and CH3 x 2).
399
400
401
402
403
404
405
406
407
408
4.1.7.
Methyl
3-({[8-(cyclohexyloxy)-1-oxo-2-phenyl-1H-pyrido[2,1-b][1,3]benzothiazol-4-
yl]carbonyl}amino)-4-methylpentanoate (42). Following the general procedure (Method B), starting from
acid 17 and using methyl 3-amino-4-methylpentanoate hydrochloride (33), compound 42 was obtained, after
purification by flash column chromatography eluting Cy/EtOAc (8:2), as a yellow solid in 77% yield.
Reaction time: 3h; m.p. 162-164 °C. 1H NMR (400 MHz, [D6]DMSO): δ = 8.89 (brs, 1H, H-9), 8.39 (s, 1H,
H-3), 8.29 (d, J = 8.5 Hz, 1H, CONH), 7.87 (d, J = 8.7 Hz, 1H, H-6), 7.29 (d, J = 7.5 Hz, 2H, H-2’ and H-
6’), 7.46-7.42 (m, 2H, H-3’ and H-5’), 7.36-7.32 (m, 1H, H-4’), 7.18-7.16 (m, 1H, H-7), 4.36 (m, 1H, OCH),
4.23-4.22 (m, 1H, NHCHCH2), 3.51 (s, 3H, CO2CH3), 2.68-2.59 (m, 2H, NHCHCH2), 1.88 (m, 2H,
cyclohexyloxy CH2), 1.81 (m, 1H, iPr CH), 1.77-1.68 (m, 2H, cyclohexyloxy CH2), 1.47-1.18 (m, 6H,
cyclohexyloxy CH2 x 3), 0.86 (d, J = 6.7 Hz, 6H, iPr CH3 x 2).
409
410
411
412
4.1.8.
Methyl
3-({[8-(cyclohexyloxy)-1-oxo-2-phenyl-1H-pyrido[2,1-b][1,3]benzothiazol-4-
yl]carbonyl}amino)-3-phenylpropanoate (43). Following the general procedure (Method B), starting from
acid 17 and using methyl 3-amino-3-phenylpropanoate hydrochloride (34), compound 43 was obtained, after
purification by column chromatography eluting with Cy/EtOAc (8:2), as a yellow solid in 60% yield.
21

413
414
415
416
417
418
Reaction time: 4h; m.p. 143-145 °C. 1H NMR (400 MHz, [D6]DMSO): δ = 8.96 (d, J = 8.0 Hz, 1H, CONH),
8.84 (d, J = 2.4 Hz, 1H, H-9), 8.44 (s, 1H, H-3), 7.87 (d, J = 8.8 Hz, 1H, H-6), 7.75-7.72 (m, 2H, Ar-H),
7.48-7.46 (m, 2H, Ar-H), 7.44-7.36 (m, 3H, Ar-H), 7.34-7.29 (m, 2H, Ar-H), 7.24-7.22 (m, 1H, Ar-H), 7.14
(dd, J = 2.4 and 8.7 Hz, 1H, H-7), 5.52-5.46 (m, 1H, NHCHCH2), 4.39-4.35 (m, 1H, OCH), 3.54 (s, 3H,
CO2CH3) 3.01-2.95 (m, 1H, NHCHCHaHb), 2.91-2.86 (m, 1H, NHCHCHaHb), 1.95-1.90 (m, 2H,
cyclohexyloxy CH2), 1.70-1.67 (m, 2H, cyclohexyloxy CH2), 1.51-1.21 (m, 6H, cyclohexyloxy CH2 x 3).
419
420
421
422
423
424
425
426
427
428
4.1.9.
Methyl
1-({[8-(cyclohexyloxy)-1-oxo-2-phenyl-1H-pyrido[2,1-b][1,3]benzothiazol-4-
yl]carbonyl}amino)cyclopropanecarboxylate (44). Following the general procedure (Method B), starting
from acid 17 and using methyl 1-(aminomethyl)cyclopropanecarboxylate hydrochloride (35), compound 44
was obtained, after purification by flash column chromatography eluting with Cy/EtOAc (5:5), as a yellow
1
solid in 16% yield. Reaction time: 3h; m.p. 235-237 °C. H NMR (400 MHz, [D6]DMSO): δ = 8.85 (d, J =
2.3 Hz, 1H, H-9), 8.57 (t, J = 5.2 Hz, 1H, CONH), 8.42 (s, 1H, H-3), 7.87 (d, J= 8.7 Hz, 1H, H-6), 7.74 (d, J
= 7.3 Hz, 2H, H-2’ and H-6’) 7.43 (t, J =7.5 Hz, 2H, H-3’ and H-5’), 7.34 (m, 1H, H-4’), 7.17 (dd, J = 2.3
and 7.2 Hz, 1H, H-7), 4.39-4.35 (m, 1H, OCH), 3.57-3.54 (m, 5H, CO2CH3 and NHCH2), 1.90 (m, 2H,
cyclohexyloxy CH2), 1.70-1.67(m, 2H, cyclohexyloxy CH2) 1.50-1.25 (m, 6H, cyclohexyloxy CH2 x 3),
1.09-1.06 (m, 2H, cyclopropyl CH2), 1.00-0.97 (m, 2H, cyclopropyl CH2).
429
430
431
432
433
434
435
436
437
438
4.1.10.
Ethyl
1-{[8-(cyclohexyloxy)-1-oxo-2-phenyl-1H-pyrido[2,1-b][1,3]benzothiazol-4-
yl]carbonyl}piperidine-3-carboxylate (45). Under N2 atmosphere, to a solution of acid 17 (0.3 g 1.4 mmol)
in dry CH2Cl2, EDCI*HCl (0.32 g, 2.1 mmol), HOBt (0.19 g, 1.4 mmol), DIPEA (0.7 mL, 4.2 mmol) and
ethyl nipecotate 36 (0.2 mL, 1.4 mmol) were subsequently added and the reaction was stirred at room
temperature for 4h. After evaporation of the solvent under vacuum, the reaction mixture was poured into
ice/water and the resulting solid was filtered under vacuum to give a yellow solid in 53% yield. Reaction
time: 4h; m.p. 133-135 °C (d). 1H NMR (400 MHz, [D6]DMSO): δ = 8.83 (d, J = 2.2 Hz, 1H, H-9), 7.87 (d, J
= 8.7 Hz, 1H, H-6), 7.83 (s, 1H, H-3), 7.71 (d, J = 7.4 Hz, 2H, H-2’ and H-6’), 7.42-7.38 (m, 2H, H-3’ and
H-5’), 7.33-7.30 (m, 1H, H-4’), 7.18 (dd, J = 2.3 and 8.8 Hz, 1H, H-7), 4.39-4.35 (m, 1H, OCH), 4.09-4.03
(m, 1H, piperidine CH), 3.98-3.91 (m, 2H, CO2CH2CH3), 3.81-3.78 (m, 1H, piperidine CH), 2.65-2.62 (m,
22

439
440
441
1H, piperidine CH), 1.95-1.93 (m, 3H, cyclohexyloxy CH2 and piperidine CH), 1.70-1.66 (m, 3H,
cyclohexyloxy CH2 and piperidine CH), 1.49-1.20 (m, 10H, cyclohexyloxy CH2 x 3 and piperidine CH2 x 2),
1.07 (t, J = 7.1 Hz, 3H, CO2CH2CH3).
442
443
444
445
446
447
448
449
450
451
452
453
454
4.1.11.
8-(Cyclohexyloxy)-4-[(3-hydroxypiperidin-1-yl)carbonyl]-2-phenyl-1H-pyrido[2,1-
b][1,3]benzothiazol-1-one (28). Following the procedure used for 45 and using 3-hydrossipiperidine
hydrochloride 39 (0.03 g, 0.24 mmol), compound 28 was obtained, after purification by flash column
chromatography eluting with CHCl3/MeOH (99:1), as a yellow solid in 32% yield. Reaction time: 8h; m.p.
167-169 °C. 1H NMR (400 MHz, [D6]DMSO): δ = 8.84 (d, J = 1.8 Hz, 1H, H-9), 7.88-7.86 (m, 2H, H-3 and
H-6), 7.73 (d, J = 7.8 Hz, 2H, h-2’ and H-6’), 7.41 (t, J = 7.6 Hz, 2H, H-3’ and H-5’), 7.31 (t, J = 7.3 Hz,
1H, H-4’), 7.17 (dd, J = 1.9 and 8.7 Hz, 1H, H-7), 4.89 (brs, 1H, OH), 4.40-4.36 (m, 1H, OCH), 3.74 (m, 1H,
piperidine CH), 3.73-3.56 (m, 2H, piperidine CH2), 3.49-3.43 (m, 1H, piperidine CH), 3.15 (m, 1H,
piperidine CH), 1.95-1.90 (m, 2H, cyclohexyloxy CH2), 1.77-1.68 (m, 4H, piperidine CH2 and
13
cyclohexyloxy CH2), 1.55-1.24 (m, 8H, cyclohexyloxy CH2 x 3 and piperidine CH2). C NMR (101 MHz,
[D6]DMSO): δ = 167.26, 160.98, 156.31, 150.77, 139.48, 136.68, 135.82, 129.16, 128.52, 127.75, 123.26,
122.2, 119.31, 116.45, 107.78, 107.27, 75.66, 65.23, 53.02, 45.35, 33.79, 32.77, 25.52, 23.47, 22.50. HPLC:
rt 6.377, HRMS (ESI) calculated for C29H31N2O4S [M+H]+ 503.2004 found 503.2004.
455
456
457
458
459
4.1.12. General procedure for hydrolysis of methyl esters for compounds 19-24. (Method C). A
solution of the appropriate methyl ester compound (40-45) (1 mmol) in 1,4 dioxane and 1N LiOH (5 mmol)
was stirred at room temperature for 3/4h. Then, the reaction mixture was poured into ice/water, acidified
with 2N HCl (pH = 3-4) to give a precipitate that was filtered and purified as described below for each
product.
460
461
462
463
464
4.1.13.
3-({[8-(Cyclohexyloxy)-1-oxo-2-phenyl-1H-pyrido[2,1-b][1,3]benzothiazol-4-
yl]carbonyl}amino)butanoic acid (19). Following the general procedure (Method C), starting from
derivative 40, compound 19 was obtained, after purification by flash column chromatography eluting with
1
CHCl3/MeOH (97:3), as a yellow solid in 50% yield. Reaction time: 3h; m.p.: 198 °C. H NMR (400 MHz,
[D6]DMSO): δ = 12.30 (brs, 1H, CO2H), 8.85 (d, J = 2.3 Hz, 1H, H-9), 8.75 (t, J = 5.8 Hz, 1H, CONH), 8.40
23

465
466
467
468
469
470
471
472
(s, 1H, H-3), 7.88 (d, J = 8.7 Hz, 1H, H-6), 7.75 (d, J = 7.3 Hz, 2H, H-2’ and H-6’), 7.44 (t, J = 7.8 Hz, 2H,
H-3’ and H-5’), 7.33 (m, 1H, H-4’), 7.10 (dd, J = 2.4 and 8.7 Hz, 1H, H-7), 4.39-4-35 (m, 1H, OCH), 3.50-
3.23 (m, 1H, NHCHCHaHb), 3.42-3.23 (m, 1H, NHCHCHaHb), 2.68-2.63 (m, 1H, NHCHCH2), 1.96-1.92 (m,
2H, cyclohexyloxy CH2), 1.70-1.66 (m, 2H, cyclohexyloxy CH2), 1.49-1.23 (m, 6H, cyclohexyloxy CH2 x
3), 1.06 (d, J = 7.1 Hz, CH3). 13C NMR (101 MHz, [D6]DMSO): δ = 176.43, 164.50, 161.27, 156.22, 151.50,
138.91, 136.75, 133.85, 129.30, 128.35, 127.72, 123.06, 122.28, 120.99, 116.43, 107.48, 105.73, 75.60,
52.58, 42.81, 31.52, 25.47, 23.43, 15.42. HPLC: rt 6.574, HRMS (ESI) calculated for C28H29N2O5S [M+H]+
505.1796 found 505.1797.
473
474
475
476
477
478
479
480
481
482
483
484
4.1.14.
3-({[8-(Cyclohexyloxy)-1-oxo-2-phenyl-1H-pyrido[2,1-b][1,3]benzothiazol-4-
yl]carbonyl}amino)-3-methylbutanoic acid (20). Following the general procedure (Method C), starting
from derivative 41, compound 20 was obtained, after purification by flash column chromatography eluting
1
with CHCl3/MeOH (97:3), as a yellow solid in 46% yield. Reaction time: 3h; m.p. 168-170 °C. H NMR
(400 MHz, [D6]DMSO): δ = 12.02 (brs, 1H, CO2H), 8.85 (d, J = 2.2 Hz, 1H, H-9), 8.40 (s, 1H, H-3), 8.04
(brs, 1H, CONH), 7.85 (d, J = 8.6 Hz, 1H, H-6), 7.73 (d, J = 7.3 Hz, 2H, H-2’ and H-6’), 7.43 (t, J = 7.3 Hz,
2H, H-3’ and H-5’), 7.33 (t, J = 6.7 Hz, 1H, H-4’), 7.17 (dd, J = 1.7 and 8.8 Hz, 1H, H-7), 4.39-4.30 (m, 1H,
OCH), 2.79 (s, 2H, CH2), 1.93-1.90 (m, 2H, cyclohexyloxy CH2), 1.70-1.67 (m, 2H, cyclohexyloxy CH2),
1.49-1.17 (m, 12H, cyclohexyloxy CH2 x 3 and CH3 x 2). 13C NMR (101 MHz, [D6]DMSO): δ = 172.70,
164.35, 161.28, 156.22, 151.25, 138.87, 136.85, 134.46, 129.43, 128.32, 127.63, 122.98, 122.15, 121.09,
116.46, 107.49, 106.67, 75.64, 52.66, 43.67, 31.53, 27.47, 25.46, 23.41. HPLC: rt 6.893, HRMS (ESI)
calculated for C29H31N2O5S [M+H]+ 519.1953 found 519.1958.
485
486
487
488
489
490
4.1.15.
3-({[8-(Cyclohexyloxy)-1-oxo-2-phenyl-1H-pyrido[2,1-b][1,3]benzothiazol-4-
yl]carbonyl}amino)-4-methylpentanoic acid (21). Following the general procedure (Method C), starting
from derivative 42, compound 21 was obtained, after purification flash by column chromatography eluting
1
with CHCl3/MeOH (97:3), as a yellow solid in 41% yield. Reaction time: 3h; m.p. 146-148 °C. H NMR
(400 MHz, [D6]DMSO): δ = 12.18 (brs, 1H, CO2H), 8.86 (d, J = 2.4 Hz, 1H, H-9), 8.43 (s, 1H, H-3), 8.33 (d,
J = 8.4 Hz, 1H, CONH), 7.88 (d, J = 8.7 Hz, 1H, H-6), 7.73 (d, J = 7.4 Hz, 2H, H-2’ and H-6’), 7.46-7.42
24

491
492
493
494
495
496
497
(m, 2H, H-3’ and H-5’), 7.36-7.32 (m, 1H, H-4’), 7.18 (dd, J = 2.4 and 8.7 Hz, 1H, H-7), 4.40-4.35 (m, 1H,
OCH), 4.27-4.20 (m, 1H, NHCHCH2), 2.57-2.42 (m, 2H, NHCHCH2), 1.92-1.91 (m, 2H, cyclohexyloxy
CH2), 1.85-1.80 (m, 1H, iPr CH ), 1.71-1.56 (m, 2H, cyclohexyloxy CH2), 1.50-1.24 (m, 6H, cyclohexyloxy
CH2 x 3), 0.86 (d, J = 6.8 Hz, 6H, iPr CH3 x 2). 13C NMR (101 MHz, [D6]DMSO): δ = 173.34, 136.88,
161.28, 156.23, 151.57, 138.94, 136.84, 133.83, 129.39, 128.36, 127.69, 122.99, 122.39, 121.12, 116.44,
107.51, 105.93, 75.66, 52.24, 36.62, 32.24, 31.53, 25.46, 23.42, 19.22, 19.07. HPLC: rt 6.951, HRMS (ESI)
calculated for C30H33N2O5S [M+H]+ 533.2109 found 533,2111.
498
499
500
501
502
503
504
505
506
507
508
509
510
511
4.1.16.
3-({[8-(Cyclohexyloxy)-1-oxo-2-phenyl-1H-pyrido[2,1-b][1,3]benzothiazol-4-
yl]carbonyl}amino)-3-phenylpropanoic acid (22). Following the general procedure (Method C), starting
from derivative 43, compound 22 was obtained, after purification by flash column chromatography eluting
1
with CHCl3/MeOH (97:3), as a yellow in 58% yield. Reaction time: 3h; m.p. 155-158 °C. H NMR (400
MHz, [D6]DMSO): δ = 12.19 (brs, 1H, CO2H), 9.07 (d, J = 7.7 Hz 1H, CONH), 8.81 (d, J = 2.4 Hz, 1H, H-
9), 8.47 (s, 1H, H-3), 7.87 (d, J = 8.8 Hz, 1H, H-6), 7.76-7.71 (m, 2H, H-2’ and H-6’), 7.46 (t, J = 7.4 Hz,
2H, H-3’ and H-5’), 7.41-7.36 (m, 3H, H-4’, H-2’’ and H-6’’), 7.34-7.29 (m, 2H, H-3’’ and H-5’’), 7.24-7.20
(m, 1H, H-4’’), 7.17 (dd, J = 2.4 and 8.8 Hz, 1H, H-7), 5.48-5.39 (m, 1H, NHCHCH2), 4.39-4.34 (m, 1H,
OCH), 2.91-2.79 (m, 1H, NHCHCHaHb), 2.76-2.75 (m, 1H, NHCHCHaHb), 1.91-1.90 (m, 2H,
cyclohexyloxy CH2), 1.74-1.68 (m, 2H, cyclohexyloxy CH2), 1.49-1.21 (m, 6H, cyclohexyloxy CH2 x 3). 13C
NMR (101 MHz, [D6]DMSO): δ = 172.20, 163.59, 161.28, 156.24, 151.83, 143.04, 138.91, 136.82, 133.78,
129.42, 128.77, 128.42, 127.77, 127.47, 126.99, 123.06, 122.54, 120.96, 116.46, 107.42, 105.52, 75.61,
50.60, 40.99, 31.51, 25.46, 23.43. HPLC: rt 6.964, HRMS (ESI) calculated for C33H31N2O5S [M+H]+
567.1953 found 567.1960.
512
513
514
515
516
4.1.17.
1-[({[8-(Cyclohexyloxy)-1-oxo-2-phenyl-1H-pyrido[2,1-b][1,3]benzothiazol-4-
yl]carbonyl}amino)methyl]cyclopropanecarboxylic acid (23). Following the general procedure (Method
C), starting from derivative 44, compound 23 was obtained, after purification by column chromatography
1
eluting with CHCl3/MeOH (97:3), as a yellow solid in 19% yield. Reaction time: 3h; m.p. 250-253 °C. H
NMR (400 MHz, [D6]DMSO): δ = 12.07 (brs, 1H, CO2H), 8.83 (d, J = 2.4 Hz, 1H, H-9), 8.58 (t, J = 5.3 Hz,
25

517
518
519
520
521
522
523
524
1H, CONH), 8.43 (s, 1H, H-3), 7.87 (d, J = 8.7 Hz, 1H, H-6), 7.75-7.73 (m, 2H, H-2’ and H-6’), 7.43 (t, J =
7.8 Hz, 2H, H-3’ and H-5’), 7.35-7.31 (m, 1H, H-4’), 7.15 (dd, J = 2.3 and 8.8 Hz, 1H, H-7), 4.38-4.32 (m,
1H, OCH), 3.53 (d, J = 5.2 Hz, 2H, CH2), 1.92-1.89 (m, 2H, cyclohexyloxy CH2), 1.69-1.66 (m, 2H,
cyclohexyloxy CH2), 1.42-1.16 (m, 6H, cyclohexyloxy CH2 x 3), 1.04-1.01 (m, 2H, cyclopropryl CH2), 0.94-
0.91 (m, 2H, cyclopropyl CH2). 13C NMR (101 MHz, [D6]DMSO): δ = 175.83, 164.68, 161.28, 156.19,
151.60, 138.92, 136.81, 134.05, 129.38, 128.33, 127.67, 123.01, 122.27, 121.03, 116.42, 107.43, 105.76,
75.59, 42.14, 31.50, 25.46, 23.42, 23.29, 13.77. HPLC: rt 6.697, HRMS (ESI) calculated for C29H29N2O5S
[M+H]+ 517.1796 found 517.1800.
525
526
527
528
529
530
531
532
533
534
535
536
537
4.1.18.
1-{[8-(Cyclohexyloxy)-1-oxo-2-phenyl-1H-pyrido[2,1-b][1,3]benzothiazol-4-
yl]carbonyl}piperidine-3-carboxylic acid (24). Following the general procedure (Method C), starting from
derivative 45, compound 24 was obtained, after purification by flash column chromatography eluting with
1
CHCl3/MeOH (97:3), as a yellow solid in 44% yield. Reaction time: 3h; m.p. 119-120 °C. H NMR (400
MHz, [D6]DMSO): δ = 12.21 (brs, 1H, CO2H), 8.83 (d, J = 1.8 Hz, 1H, H-9), 7.87 (d, J = 8.7 Hz, 1H, H-6),
7.79 (s, 1H, H-3), 7.71 (d, J = 7.4 Hz, 2H, H-2’ and H-6’), 7.40 (t, J = 7.5 Hz, 2H, H-3’ and H-5’), 7.31 (t, J
= 7.2 Hz, 1H, H-4’), 7.18 (dd, J = 1.9 and 8.6 Hz, 1H, H-7), 4.37-4.29 (m, 1H, OCH), 4.08-4.05 (m, 1H,
piperidine CH), 3.81-3.72 (m, 1H, piperidine CH), 3.23-3.16 (m, 2H, piperidine CH2), 2.51-2.46 (m, 1H,
piperidine CH), 1.93 (m, 3H, cyclohexyloxy CH2 and piperidine CH), 1.66 (m, 4H, cyclohexyloxy CH2 and
piperidine CH2), 1.47-1.23 (m, 7H, cyclohexyloxy CH2 x 3 and piperidine CH). 13C NMR (101 MHz,
[D6]DMSO): δ = 174.73, 167.15, 160.98, 156.33, 150.91, 139.50, 136.67, 135.61, 129.20, 128.53, 127.80,
123.26, 121.90, 119.29, 116.46, 107.79, 107.06, 75.67, 47.59, 45.85, 41.14, 31.55, 27.23, 25.53, 24.52,
23.48. HPLC: rt 6.485, HRMS (ESI) calculated for C30H31N2O5S [M+H]+ 531.1953 found 531.1959.
538
539
540
541
542
4.1.19.
8-(Cyclohexyloxy)-N-[(1R)-2-hydroxy-1-methylethyl]-1-oxo-2-phenyl-1H-pyrido[2,1-
b][1,3]benzothiazole-4-carboxamide (29). A mixture of starting derivative 6 (0.3 g, 0.67 mmol) and (R)-2-
aminopropan-1-ol 46 (2 mL) was heated at reflux in neat conditions for 5h. Then, the reaction mixture was
poured into ice/water and acidified with 2N HCl (pH = 5) to give a precipitate which was filtered under
vacuum. After purification by flash column chromatography eluting with CHCl3/MeOH (98:2), compound
26

1
543
544
545
546
547
548
549
550
551
552
29 was obtained as a yellow solid in 39% yield. Reaction time: 4h; m.p. 139-140 °C. H NMR (400 MHz,
[D6]DMSO): δ = 8.86 (d, J = 2.4 Hz, 1H, H-9), 8.44 (s, 1H, H-3), 7.91 (d, J = 8.7 Hz, 1H, H-6), 7.76-7.74
(m, 2H, H-2’ and H-6’), 7.44 (t, J = 7.7 Hz, 2H, H-3’ and H-5’), 7.34-7.32 (m, 1H, H-4’), 7.17 (dd, J = 2.4
and 8.8 Hz, 1H, H-7), 4.76 (t, J = 5.8 Hz, 1H, CHCH2OH), 4.39-4.35 (m, 1H, OCH), 4.07-3.99 (m, 1H,
CHCH2OH), 3.48-3.40 (m, 2H, CHCH2OH), 1.92-1.90 (m, 2H, cyclohexyloxy CH2), 1.69-1.67 (m, 2H,
cyclohexyloxy CH2), 1.50-1.26 (m, 6H, cyclohexyloxy CH2 x 3), 1.11 (d, J = 6.7 Hz, 3H, CH3).13C NMR
(101 MHz, [D6]DMSO): δ = 163.83, 161.30, 156.17, 151.42, 138.89, 136.81, 134.05, 129.38, 128.33,
127.67, 122.99, 122.30, 121.11, 166.42, 107.45, 105.99, 75.59, 64.71, 47.76, 31.50, 25.45, 23.41, 17.51.
HPLC: rt 6.430, HRMS (ESI) calculated for C27H29N2O4S [M+H]+ 477.1847 found 477.1847. [α]D = - 0.036
(0.5 % p/v, DMSO).
553
554
555
556
557
558
559
560
561
562
563
564
4.1.20.
8-(cyclohexyloxy)-N-[(1S)-2-hydroxy-1-methylethyl]-1-oxo-2-phenyl-1H-pyrido[2,1-
b][1,3]benzothiazole-4-carboxamide (30). Following procedure reported above for compound 29 and using
(S)-2-aminopropan-1-ol 47 (2 mL), compound 30 was obtained, after purification by flash column
chromatography CHCl3/ MeOH (98:2), as a yellow solid in 11% yield. Reaction time: 4h; m.p. 139-140 °C.
1H NMR (400MHz, MeOD): δ = 8.83 (d, J = 2.4 Hz, 1H, H-9), 8.28 (s, 1H, H-3), 7.76-7.74 (m, 2H, H-2’
and H-5’), 7.62 (d, J = 8.7 Hz, 1H, H-6), 7.42-7.36 (m, 2H, H-3’ and H-5’), 7.33-7.30 (m, 1H, H-4’), 7.05
(dd, J = 2.4 and 8.7 Hz, 1H, H-7), 4.35-4.29 (m, 1H, OCH), 4.19-4.13 (m, 1H, CHCH2OH), 3.62-3.53 (m,
2H, CHCH2OH), 2.00-1.97 (m, 2H, cyclohexyloxy CH2), 1.78-1.75 (m, 2H, cyclohexyloxy CH2), 1.57-1.32
(m, 6H, cyclohexyloxy CH2 x 3), 1.23 (d, J = 6.8 Hz, 3H, CH3). 13C NMR (101 MHz, DMSO-d6): δ 164.71,
161.84, 156.43, 151.52, 138.50, 136.38, 133.52, 128.76, 127.67, 127.11, 122.94, 121.53, 120.47, 116.35,
106.79, 105.96, 75.53, 64.64, 47.62, 31.26, 25.23, 23.15, 15.74. HPLC: rt 6.425, HRMS (ESI) calculated for
C27H29N2O4S [M+H]+ 477.1848 found 477.1848. [α]D = + 0.036 (0.5 % p/v, DMSO).
565
566
4.2. Biological part
4.2.1 Cell lines and viruses
27

567
568
569
570
BHK-21 cells (baby hamster kidney fibroblast; ATCC) were grown in RPMI 1640 medium (Gibco)
o
supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) penicillin/streptomycin (P/S) at 37 C
in 5% CO2. HuH-7 cells (human hepatocarcinoma; ATCC) were cultured in DMEM medium (Gibco) with
4.5g/L glucose, 10% FBS and 1% P/S at 37oC in 5% CO2.
571
572
573
574
575
DENV-2 EDEN3295 (GenBank accession: EU081177) was obtained from the Early Dengue infection
and outcome (EDEN) study [27]. The ZIKV strain H/PF/2013 (GenBank accession: KJ776791.2) was a gift
from Cécile Baronti at Aix Marseille Université. These viruses used in this study were grown in C6/36 cells,
titered in BHK-21 cells and stored at -80oC. Institutional approval has been granted by Duke-NUS Medical
School to perform experiments with ZIKV.
576
4.2.2 in vitro NS5 polymerase assays
577
578
579
580
581
582
583
584
585
586
587
588
The de novo initiation/elongation assay was performed as previously described [15,17]. Briefly, the
initiation assay reaction comprised 100 nM DENV-2 NS5, 100 nM in vitro transcribed DENV-2 5’UTR-
3’UTR RNA, 20 µM ATP, 20 µM GTP, 20 µM UTP, 5 µM Atto-CTP (Trilink Biotechnologies), in a volume
of 15 µL of reaction buffer comprising 50 Mm Tris-HCL, Ph7.5, 10 Mm KCl, 1mM MgCl2, 0.3 mM MnCl2,
0.001% Triton X-100 and 10 µM cysteine. The elongation assay reaction comprised of 100 nM DENV-2
NS5, 100 nM U30-3’ UTR (Integrated DNA Technologies), 3 µM Atto-ATP, in a volume of 15 µL of
reaction comprised of 50 mM Tris-HCL, pH 7.5, 10 mM KCl, 0.5 mM MnCl2, 0.001% Triton and 10 µM
cysteine. All reactions were incubated at 37°C for 1 h and 10 µL of STOP buffer (200 mM NaCl, 25 mM
MgCl2, 1.5 M DEA, pH10; Promega) with 25 nM calf intestine alkaline phosphatase was added to the wells
to stop the reactions. The plate was centrifuged briefly at 1200 rpm, followed by incubation at RT for 60
mins and the release AttoPhos was monitored by reading on a Tecan machine at excitationmax and
emissionmax wavelengths 422 nm and 566 nm respectively.
589
4.2.3 Cell cytotoxicity test
590
591
HuH-7 cells were seeded in a 96-well white opaque plate (Grenier) at a density of 2 × 104. Compounds
were diluted to the indicated concentrations in the medium and incubated with cells for 48h at 37
.
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