Optimization of piperazine-derived ureas privileged structures for effective –antiadenovirus agents

Article abstract of DOI:10.1016/j.ejmech.2019.111840

In recent years, human adenovirus (HAdV) infections have shown a high clinical impact in both immunosuppressed and immunocompetent patients. The research into specific antiviral drugs for the treatment of HAdV infections in immunocompromised patients cons

Full text of DOI:10.1016/j.ejmech.2019.111840

Journal Pre-proof
Optimization of piperazine-derived ureas privileged structures for effective
antiadenovirus agents
Sarah Mazzotta, José Antonio Marrugal Lorenzo, Margarita Vega-Holm, Ana Serna-
Gallego, Jaime Álvarez-Vidal, Judith Berastegui-Cabrera, José Pérez del Palacio,
Caridad Díaz, Francesca Aiello, Jerónimo Pachón, Fernando Iglesias-Guerra, José
Manuel Vega-Pérez, Javier Sánchez-Céspedes
PII:
S0223-5234(19)30992-4
DOI:
https://doi.org/10.1016/j.ejmech.2019.111840
EJMECH 111840
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 2 September 2019
Revised Date: 29 October 2019
Accepted Date: 30 October 2019
Please cite this article as: S. Mazzotta, José.Antonio. Marrugal Lorenzo, M. Vega-Holm, A. Serna-
Gallego, J. Álvarez-Vidal, J. Berastegui-Cabrera, José. Pérez del Palacio, C. Díaz, F. Aiello, Jeró.
Pachón, F. Iglesias-Guerra, José.Manuel. Vega-Pérez, J. Sánchez-Céspedes, Optimization of
piperazine-derived ureas privileged structures for effective antiadenovirus agents, European Journal of
Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2019.111840.
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Graphical Abstract
Optimization of piperazine-derived ureas privileged structures for effective
antiadenovirus agents
a,d†
b†
a,*
Sarah Mazzotta,
José Antonio Marrugal Lorenzo, Margarita Vega-Holm, Ana
b
a
b
Serna-Gallego, Jaime Álvarez-Vidal, Judith Berastegui-Cabrera, José Pérez del
Palacio, Caridad Díaz, Francesca Aiello, Jerónimo Pachón, Fernando Iglesias-
Guerra, José Manuel Vega-Pérez, Javier Sánchez-Céspedes
c
c
d
b,e
a
a
b,*
a
Department of Organic and Medicinal Chemistry, Faculty of Pharmacy, University of Seville,
E-41071 Seville, Spain.
b
Clinical Unit of Infectious Diseases, Microbiology, and Preventive Medicine. Institute of
Biomedicine of Seville (IBiS), University Hospital Virgen del Rocío/CSIC/University
of Seville, Seville, Spain
c
Fundación Medina, Parque Tecnológico de Ciencias de la Salud, E-18016 Granada, Spain.
d
Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, 87036
Arcavacata di Rende (CS) Italy.
e
Department of Medicine, University of Seville, E-41009 Seville, Spain.
E-mail: mvegaholm@us.es
E-mail: jsanchez-ibis@us.es
Corresponding Author
*
M.V.H. Mailing address: Department of Organic and Medicinal Chemistry, Faculty of
Pharmacy, University of Seville, 41071, Seville, Spain. Telephone: +34 954556737, E-
mail: mvegaholm@us.es
*
J.S.C. Mailing address: Institute of Biomedicine of Seville (IBiS), University Hospital
Virgen del Rocío/CSIC/University of Seville, Unit of Infectious Diseases, Microbiology
and Preventive Medicine, Seville, Spain. Telephone: +34 955 923 100, E-mail:
jsanchez-ibis@us.es
Author Contributions
†
S.M., J.A.M.L contributed equally to this work (joint first authors).

Optimization of piperazine-derived ureas privileged
structures for effective anti-adenovirus agents.
a,d†
b†
a,*
Sarah Mazzotta,
José Antonio Marrugal-Lorenzo, Margarita Vega-Holm, Ana
b
a
b
Serna-Gallego, Jaime Álvarez-Vidal, Judith Berastegui-Cabrera, José Pérez del
c
c
d
b,e
Palacio, Caridad Díaz, Francesca Aiello, Jerónimo Pachón, Fernando Iglesias-
a
a
b,*
Guerra, José Manuel Vega-Pérez, Javier Sánchez-Céspedes
a
Department of Organic and Medicinal Chemistry, Faculty of Pharmacy, University of Seville,
Profesor García González 2, E-41071 Seville, Spain.
b
Unit of Infectious Diseases, Microbiology and Preventive Medicine, Institute of Biomedicine
of Seville (IBiS), University Hospital Virgen del Rocío/CSIC/University of Seville, E41013
Seville, Spain.
c
Fundación Medina, Parque Tecnológico de Ciencias de la Salud, E-18016 Granada, Spain.
d
Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, 87036
Arcavacata di Rende (CS) Italy
e
Department of Medicine, University of Seville, E-41009 Seville, Spain.
Keywords: Adenovirus; Antiviral drug; Privileged structures, Thiourea/Urea piperazine
derivatives
ABSTRACT
In recent years, human adenovirus (HAdV) infections have shown a high clinical impact
in both immunosuppressed and immunocompetent patients. The research into specific
antiviral drugs for the treatment of HAdV infections in immunocompromised patients
constitutes a principal objective for medicinal chemistry due to the lack of any specific

secure drug to treat these infections. In this study, we report a small-molecule library
67 compounds) designed from an optimization process of piperazine-derived urea
(
privileged structures and their biological evaluation: antiviral activity and cytotoxicity.
The active compounds selected were further evaluated to gain mechanistic
understanding for their inhibition. Twelve derivatives were identified that inhibited
HAdV infections at nanomolar and low micromolar concentrations (IC from 0.6 to 5.1
5
0
µM) with low cytotoxicity. In addition, our mechanistic assays suggested differences in
the way the derivatives exert their anti-HAdV activity targeting transcription, DNA
replication and later steps in the HAdV replication cycle. Furthermore, eight of the 12
studied derivatives blocked human cytomegalovirus (HCMV) DNA replication at low
micromolar concentrations. The data provided herein indicates that the 12 thiourea/urea
piperazine derivatives studied may represent potential lead compounds for clinical
evaluation and development of new anti-HAdV drugs.

1
. INTRODUCTION
Human adenovirus (HAdV) is a non-enveloped virus with an icosahedral capsid
containing a linear and double-stranded DNA genome of 34−36 kb. This virus has been
classified into more than 60 serotypes grouped into seven subgroups or species (HAdV
A−G) [1]. The high clinical impact of HAdV infections has been well-documented in
immunosuppressed adult patients and children for many years [2-5]. In paediatric
allogenic hematopoietic stem-cell transplant (allo-HSCT) recipients, HAdV infections
occur in frequencies between 3% and 47% with high rates of associated mortality
[
2,6,7]. These infections have frequently been associated with respiratory,
gastrointestinal, and ocular diseases in healthy individuals, and are usually presented as
self-limiting infections and are rarely associated with severe clinical diseases [2].
Although the incidence of HAdV community-acquired pneumonia (CAP) in
immunocompetent individuals appears to be low, with the advances in molecular
techniques of diagnosis, HAdV infections have been found to be increasingly involved
in occasional cases and outbreaks in healthy adults [8-12]. Despite this significant
clinical impact, there are as yet no specific antiviral therapies currently approved for the
treatment of HAdV infections. The non-specific therapeutic options available fail to
present satisfactory results in terms of efficacy and toxicity [13,14]. Therefore, the
development of low-toxic chemotherapy drugs that are effective against adenoviral
infections remains a priority.
Acyclic nucleoside phosphonates (ANPs) represent a key class of antiviral agents. To
overcome their known drawbacks (poor bioavailability and renal toxicity), lipophilic
conjugates were studied. Only brincidofovir, (1) (CMX001), which is a lipidic
conjugate of cidofovir (2) that finished a phase-III clinical trial in 2016 with no reported
results so far (ClinicalTrials.gov Identifier: NCT02087306) and is now being evaluated

for the treatment of serious HAdV infection or disease (ClinicalTrials.gov Identifier:
NCT02596997), represents a potential candidate for use in the treatment of HAdV
infections [15, 16]. Different octadecyloxyethyl (ODE) esters of these phosphonates (3)
were also synthesized to increase bioavailability and antiviral efficacy. Following their
evaluation, they demonstrated significant antiviral activities in A549 cells [17] (Figure
1
).
<
Figure 1>
New types of anti-HAdV agents of a non-nucleoside nature have been discovered that
exhibit an inhibitory effect on HAdV. Compounds of series of 5-amino derivatives of
uracil [18] (4, Figure 2), and compounds from a small library of 1,2,3-triazole-
containing 3-hydroxy-quinazoline-2,4(1H,3H)-diones [19] (5, Figure 2), were identified
as potent and specific in vitro viral inhibitors and may constitute promising candidates
for the development of drugs that are effective against HAdV infections.
<
Figure 2>
As a part of our research focused on obtaining novel small molecules with biological
activities (mainly antimicrobial and antitumoral) [20], in a previous study, we designed,
synthesized, and evaluated the antiviral activity of three generations of new 4-acyl-1-
phenylaminocarbonyl-2-methylpiperazine
and
4-acyl-1-phenylaminocarbonyl-2-
phenylpiperazine derivatives, which present a total of 52 compounds [21]. In this study,
six new 2-phenylpiperazine derivatives are identified that show antiviral activity against
HAdV and HCMV at low micromolar concentrations and present low cytotoxicity (6-
1
1, Figure 3). The mechanisms of action of the selected derivatives are studied, and
their structure–activity relationships are established.
Figure 3>
<

Currently, the main problem for the development of new drugs involves the discovery
of novel lead compounds instead of employing synthesis. The attempts to increase new
drug productivity through creative discovery technologies have fallen short of
producing the satisfactory results. For these reasons, “privileged structure”-guided
scaffold re-evolution/refining has become a primary strategy for the identification of
structurally innovative chemotypes by modifying the central core structure and/or the
side-chain of the existing active compounds [22].
In this context, since compounds 6-11 (based on an innovative piperazine-derived
scaffold) can be considered as potentially strong candidates for the development of a
new class of antiviral compounds, we decided to explore different structural
modification possibilities thereon. Faced with the urgent need for a new therapeutic
alternative, our purpose was to identify optimized anti-HAdV agents in an attempt to
find lead candidates (closely related structures [22b]) that would help to better
characterize their mechanism of antiviral activity. A description is now given of the use
of these piperazine-derived urea privileged structures (with high anti-HAdV activity and
chemical versatility), which were obtained by a short and high-yielding methodology,
for the development and the biological characterization of a collection of 67
compounds. The new compounds were designed while preserving the three
characteristic regions of our original general framework: the piperazine core, the urea
function at N-1, and the amide group at N-4. However, at the same time, these parts are
considered as points for the structural variation to generate chemical diversity.
2
. Results & discussion
Figure 4 shows the actions carried out on the general structure of the hit compounds 6-
1 in order to generate the new derivatives. In Pathway A, the urea function was
replaced by a thiourea function. In Pathway B, the acyl groups at N-4 were exchanged
1

with three different 2-substituted acetyl groups. Finally, in Pathway C, the central core
of 2-substituted piperazine was modified by 2,6-dimethylpiperazine and unsubstituted
piperazine (Figure 4).
<
Figure 4>
The anti-HAdV activity of the new compounds obtained in each route was evaluated
using plaque assays. Furthermore, the evaluation of their effect on cellular viability was
also carried out (determination of CC ) for those compounds that showed a percentage
5
0
of inhibition in the plaque assay >80%, in order to ascertain the safety profile of the
most active compounds. Although cidofovir has not been approved by the health
authorities to treat HAdV infections, it remains the drug of choice for the treatment of
these infections. It was also evaluated in parallel to our compounds, following our own
methodologies.
Route A: Replacement of the urea function with a thiourea function.
We have carried out this action on the basis of the structure of two compounds found in
our previous work, 12 and 13 (Figure 5), which showed great anti-HAdV activity (94%
of the HAdV infection in the plaque assay) but also resulted in being significantly
cytotoxic, with CC values of 26.3 and 31.4 µM, respectively.
50
<
Figure 5>
The analogues of these compounds were prepared by preserving the methyl piperazine
core, the thiourea function at nitrogen 1, and the phenylamine group (Scheme 1, Table
1
). In order to generate chemical diversity, two points of variation are considered in
their structure: (1) different acyl functions located at the N-4: urethane (as in compound
2) or different 2-substitued acetyl derived amides, obtained firstly by an isosteric
1
replacement (CH instead of oxygen) to give 2-tert-butylacetyl derivatives and secondly
2
by the modification of the substituent at the 2 position of the acetyl moiety, to obtain 2-

cyclohexylacetyl and 2-phenylacetylderivatives; (2) the substituents on the phenyl ring
of the thiourea function at N-1 (groups with different electronic properties)
Compounds 20-50 were obtained following the previously described short and high-
yielding synthetic methodology that involved two reactions, by employing 2-methyl
piperazine as the starting material [21] (Scheme 1, Table 1)
<
Scheme 1>
From this screening, we have found 12 compounds that presented HAdV5-GFP plaque-
formation inhibition >80% and showed low cytotoxicity (CC >100 µM) (Table 1).
5
0
Related to the group at para position on the phenyl group, NO , Cl, CN, CF and F are
2
3
common substituents in the active compounds (compounds 21, 22, 35, 36, 37, 38, 39,
3, 44, 45 and 46). The 3,5-bistrifuoromethyl group (not previously evaluated) is
4
present in two active compounds (42 and 50). Except for one case (compound 41), all of
them present electron-withdrawing groups. In terms of the type of the acyl group at N-4,
none of the 2-tert butyl acetyl derivatives (27-34) gave plaque-formation inhibition
>80%, while the highest number of active compounds were detected among those
compounds having 2-cyclohexylacetyl moiety (35-39, 41 and 42).
Those compounds with HAdV inhibition infection >90% in the plaque assay and
CC >100 µM were selected for further evaluation in order to obtain mechanistic
50
understanding for their inhibitory activity; this selection included a total of seven
compounds (22, 37, 38, 41, 43, 45 and 50).
<
Table 1>
At this point, in order to increase the number and structural diversity of thiourea
analogues, we decided to incur two different modifications on the structure of the
selected seven thiourea derivatives. Firstly, during the development of our general

scaffold, we had previously observed that a benzofurane-2-carbonyl group at N-4
instead of a tert-butoxycarbonyl group improved the antiviral activity and preserved
high CC50 in most of the compounds [21]. We therefore decided to incorporate this
2 3
group at N-4 thereby preserving p-NO , p-CN, p-F and p-CF substituted phenyl amine
thiocarbonyl group at N-1. Secondly, since our initial prototypes (6-11) were based on a
phenyl piperazine backbone, the preparation of the 2-phenyl piperazine derived
analogues was a natural evolution for our seven selected thiourea derivatives.
In order to prepare 4-benzofurane-2-carbonyl-2-methylpiperazine analogues (56-59)
and 2-phenyl piperazine derivatives (60-67), the appropriate monoacyl 2-substituted
piperazine derivatives (51-55) were firstly synthetized (Scheme 1, Table 2).
As for the biological evaluation of these analogues, Table 2 shows the percentage of
inhibition obtained for the plaque assay and the results of the cellular viability
evaluation (CC ) for compounds 56−67. Compounds 56-59 showed high inhibition in
5
0
the plaque assay (100%), but all were highly cytotoxic; this is the same profile as
compound 13 (Figure 5), which possesses a benzoyl group at N-4. In both types of
thiourea derivatives there is an aromatic amide group at N-4. However, the previously
described 4-benzofurane-2-carbonyl 2-methyl piperazine urea derivatives, showed
slightly lower activity (% of plaque-formation inhibition values between 70-94%) and
presented low cytotoxicity [21]. Clearly this modification in our thioureas did not lead
to potentially interesting analogues.
However, related to 2-phenyl piperazine derivatives (compounds 60-67), all of them
excepting 61, 62 and 67, did not show a high percentage of plaque-formation inhibition,
with respect to their methyl analogues (22, 38, 41, 43 and 45). Only compounds 61, 62
and 67 presented anti-HAdV activity (80% inhibition in the plaque assay for compound
6
2 and 100% for compounds 61 and 67). It is important to notice that 61 was
synthesized as an analogue of the 2-methyl piperazine derivative 34, which inhibited

HAdV plaque formation in 4.8%. In this case, the presence of the phenyl group
improved the antiviral activity profile. From this group, only compound 67 was selected
for further studies (CC > 100 µM).
5
0
<
Table 2>
Route B: Exchange the acyl groups at N-4 with three different 2-substituted acetyl
groups. From our results from Route A, the presence of 2-substituted acetyl groups at
N-4 improved the profile of compound 12 and obtained analogues with high activity
and without cytotoxicity (the selected seven thioureas). The subsequent modification in
our prototypes (6-11) was the preparation of the 2-susbtituted acetyl-2-phenylpiperazine
derivatives. Compounds 7, 10, and 11 (4-NO , 2-NO and 2-Cl,5-CF derivatives,
2
2,
3
respectively) with lowest IC values (2.1, 2.5, and 1.1 µM, respectively) [21] were
5
0
chosen as models to incur this structural modification. Scheme 2 shows the synthesis of
compounds 68-74 (Table 3).
<
Scheme 2>
Compounds 68-74 were obtained in a high yield and submitted to biological assays
Table 3). Another two 2-phenyl piperazine derivatives were easily prepared in order to
(
evaluate the relevance of the benzofurane-2-carbonyl amide group and the urea function
in the biological activity of our scaffold. These derivatives were designed as analogues
of prototype 11. Compounds 75 (diamide) and 76 (diurea), shown in Figure 6, were
synthesized directly from 2-phenyl piperazine as precursor with an excess of the
reactive agent (acyl chloride or isocyanate). These compounds were evaluated using the
aforementioned biological approaches and compared to the biological data of the
prototypes (Table 3).
<
Figure 6>

<
Table 3>
The first seven compounds from this route (68-74) were active, with a percentage of
plaque-formation inhibition >80% for six of them. In terms of safety both tert-
butylacetyl derivatives, p- and o-nitro (68 and 69), presented little cytotoxicity, as well
as the p-NO cyclohexylacetyl derivative (70). Compound 75 gave a very low % of
2
plaque-formation inhibition; however, compound 76, the analogue diurea derivative of
prototype 11, showed high inhibition (90.4%), similar to that of 11. The urea function
seems to be crucial for the viral-replication inhibition.
From this group of derivatives, compounds 71, 73 and 76 (percentage of plaque-
formation inhibition >90% and CC >100 µM) were selected for further biological
5
0
evaluations.
Pathway C: The central core of 2-substituted piperazine was exchanged with 2,6-
dimethylpiperazine and unsubstituted piperazine.
Analogues of compounds 6-11 were prepared based on a 2,6-dimethylpiperazine or
piperazine central ring instead of the 2-phenyl piperazine ring. The rationale behind this
structural modification involved the evaluation of the effect of the presence of an
additional substituent in the piperazine core and of the use of an unsubstituted ring. The
other parts of the general structure were preserved, with the additional introduction of
two electron-donators groups (Me, OMe) as substituents at para position of the phenyl
ring of the urea function. The benzofurane-2-carbonyl monoamide derivative 78 was
prepared in the same way as other monoamides, and, by means of reaction with
isocyanates, compounds 79-85 were obtained in high yields (Scheme 3, Table 4)
<
Scheme 3>

Piperazine analogues of 7, 10, and 11 were first prepared from 1-Boc-piperazine (86) by
reaction with appropriate isocyanates (obtaining compounds 87-89), and secondly,
through the deprotection of the Boc group by treatment with CF COOH, followed by
3
reaction with benzofurano-2-carbonyl chloride, compounds 90 and 91 were produced
(Scheme 4, Table 4).
<
Scheme 4>
Finally, in the same way as for those based on a phenylpiperazine core (75 and 76), the
benzofurane-2-carbonyl diamide derivative (92) and the diurea derivative (93) were
prepared directly from piperazine (Figure 6). Table 4 shows biological data of the core-
modified analogues 79-85, 87-93
<
Table 4>
Among the 2,6-dimetilpiperazine urea derivatives, only two compounds (80 and 82)
showed antiviral activity (percentage of plaque-formation inhibition 81.1% and 72.2
respectively), although not as high as that of the models 8 and 10 (percentage of plaque-
formation inhibition > 90%,). They also showed low cytotoxicity. In general terms, this
central backbone failed to provide any improvement over the prototypes. Among the
piperazine derivatives, however, we have two compounds with a percentage of plaque-
formation inhibition >90% and CC >100 µM (87 and 89). Both compounds were Boc
50
derivatives; one with p-NO phenyl urea group (87) and the other with 2-Cl-5-CF -
2
3
disubstituted phenyl urea (89), analogues to compounds 6 and 11 respectively. In our
previous work, the 2-phenyl piperazine analogue to 89, with a 2-Cl-5-CF -disubstituted
3
phenyl urea at N-1 and a Boc group at N-4, showed 100% plaque-formation inhibition

but since its CC was lower than 100 µM, it was not selected for further studies. The
50
presence of a benzofurane-2-carbonyl group did not yield active compounds (90 and
1), which is in contrast to those with a phenyl piperazine as central core for which the
9
replacement of Boc group with a benzofurane-2-carbonyl group led to the current
prototypes (7-11) [21]. Both dibenzofurane-2-carbonyl derivative (92) and the diurea
derivative (93) also presented low antiviral activity. In the case of diurea 93, its
percentage of plaque-formation inhibition was lower than that for compound 76 (33.9%
vs 90.4%). All of these piperazine derivatives presented low cytotoxicity.
Since these derivatives belong to an innovative class of achiral derivatives of our
privileged structure, compounds 87 and 89 were selected for further biological studies.
Compounds for further analysis were selected based on their antiviral activity in the
plaque assay (>90% inhibition at 10 µM concentration and IC values) and on their
5
0
cytotoxicity (CC >100 µM). A total of twelve compounds from the three pathways
5
0
described below were selected in order to obtain some mechanistic understanding for
their inhibitory activity. Compound 76 was not included in this selection (IC 7.0±1.0
5
0
µM, SI 28.7).
The twelve selected derivatives blocked HAdV infection in a dose-dependent manner,
and presented IC values from 0.6 µM to 5.1 µM (Table 5, Supplementary Information
5
0
Figure S68). Compared with the compounds selected from the previous library of 4-
acyl-1-phenylaminocarbonyl-2-phenylpiperazine derivatives, all of which fall under a
European patent (Number of application: EP16382073.1.; Name of the Invention:
Piperazine derivatives as antiviral agents with increased therapeutic activity; year of
application: 2016), this new library shows slight optimization in activity since the
molecules reached anti-HAdV activity at the nM range in two (17%) cases (600 nM for
3
8 and 700 nM for 67) and eight (67%) of the cases showed IC < 2.5 µM. In the
50

precursor library, the lowest values of IC registered were in the µM range, 33 % of
5
0
which were < 2.5 µM. Previously reported IC values for cidofovir are significantly
5
0
higher than those shown by any of these phenyl- and methylpiperazine urea/thiourea
derivatives [23, 24]. Following our own methodologies, the IC value of cidofovir for
5
0
HAdV5 was 24.06±5.9 µM, which is also significantly higher than the values obtained
for the piperazine derivatives studied (Table 5)
<
Table 5>
These selected derivatives presented a better safety profile than did the original
compounds of the 4-acyl-1-phenylaminothiocarbonyl-2-methylpiperazine derivatives 12
and 13, and presented a similar range to that of the six hit compounds selected in our
previous work, with CC50 values between 104.3-210.4 µM vs 130.8-199.8 µM,
respectively [21].
The anti-HAdV effect of these derivatives was subsequently evaluated using a virus
burst assay to measure their effectiveness in blocking the production of new virus
particles. Treatment with the compounds was associated with overall reductions in virus
yield between 9.3-fold and 1,690-fold (Table 5). These values lay in the same range as
those for the prototypes 6-11, with the exception of compound 67, which presented an
extraordinary reduction of HAdV yield of 1,690-fold.
Insights into their antiviral mechanism of action
Impact on HAdV entry
As for the mechanism of action presented by these new derivatives, we also observed
significant differences compared to the previous library. After the initial attachment of
HAdV particles to cellular receptors by the fibre, αvβ3 or αvβ5 integrins act as co-
receptors by binding to a highly conserved motif in the penton base, which is an

essential interaction for virus internalization by clathrin-mediated endocytosis [25, 26].
Once inside the endosome, the partial decapsidation of viral particles and the
subsequent exposition of several viral proteins, specifically the membrane lytic protein
VI, trigger the endosomolysis. The virus then escapes to the cytoplasm [27] where it is
then transported via microtubules to the nuclear envelope and anchored to a nuclear
pore to deliver its genome into the nucleus [28-30]. We examined whether any of these
selected compounds inhibited any of the steps in the HAdV entry that would reflect in
the number of HAdV genomes that reach the host nucleus after a synchronized infection
[
14, 31]. Thus, we demonstrated that nine out of the twelve selected compounds did not
seem to block any step of the cell entry pathway up to the HAdV DNA delivery to the
nucleus. Human adenovirus genomes accumulated normally at the nucleus of the host
cell (Supplementary Information, Figure S69), just as with the previous 4-acyl-1-
phenylaminocarbonyl-2-phenylpiperazine derivatives [21]. However, for compounds
7
1, 73, and 89, a significant inhibition of the HAdV genome accumulation at the
nucleus of the host cell were observed, suggesting that at least part of the inhibitory
activity of these compounds reaches its target soon after HAdV enters the cell.
Impact on HAdV DNA replication
Once HAdV DNA, along with protein VII [29], enters the cell nucleus, then the
transcription occurs of the HAdV immediate early gene E1A by the cellular RNA
polymerase II, which is a prerequisite for the subsequent expression of the early genes
E1B, E2, and E4 and for HAdV DNA replication [32]. Our results showed a significant
inhibition of HAdV DNA replication by compounds 41, 43, 45, 50, and 87 (Figure 7A).
These compounds may interact directly to block the activity of viral proteins involved in
HAdV DNA replication, such as HAdV DNA polymerase. Alternatively, these
compounds may interfere with a previous step during the transcription of the immediate

early gene E1A. Figure 7B shows how compound 50 significantly inhibited the
transcription of the immediate early HAdV gene E1A. Prior to HAdV DNA replication,
the transcription of E1A by cellular RNA polymerase II takes place from the E1A
promoter [32]. The E1A protein is necessary both for the subsequent expression of the
early genes E1B, E2, E3, and E4 from different promoters and for HAdV DNA
replication. α-Amanitin, a toxin derived from the mushroom Amanita phalloides, can
interact and block the RNA polymerase II activity [33, 34]. Like α-amanitin, compound
5
0 displays dose-dependent inhibitory activity for the generation of new HAdV viral
particles [33]. The concentrations of compound 50 and α-amanitin necessary to inhibit
HAdV transcription lie within a similar range, (between 1-30 µM) [33, 34]. However,
α-amanitin shows cytopathological effects (CPE) at concentrations as low as 5 µM [33].
Given the phenotypic similarities presented by these two molecules, it would be
possible that compound 50 could block the host RNA polymerase II, since we also
detected the inhibition of CMV infection in the presence of this compound, whose
transcription is also mediated by the host RNA polymerase II [35]. Other scenarios may
account for our finding that compound 50 interferes with the HAdV early transactivator,
E1A. Compound 50 may also strengthen the DNA-pVII complex or interfere with any
of the proteins associated with the repression/activation of the E1A transcription.
Since compounds 41, 43, 45, and 87 did not block transcription of the E1A gene, they
may be interfering directly with a protein involved in HAdV DNA replication. As a
proof of concept of the possible role of HAdV and CMV DNA polymerases as targets
for these compounds, we investigated whether the presence of compounds 41 and 43
affected the in vitro amplification efficiency of the DNA polymerase from
bacteriophage Phi29, which is a closely-related DNA polymerase belonging to the same
family of DNA polymerases (family B). As can be observed in Figure 7D, compound

4
1 showed significant inhibition of the Phi29 DNA polymerase activity, which suggests
that its preferential target may be the HAdV and CMV DNA polymerases.
Figure 7>
<
Compounds 22, 37, 38 and 67 did not show any inhibition of the HAdV genome
accessibility to the nucleus nor of the HAdV de novo DNA synthesis. The mechanism
of action for these compounds may be related to later steps in the HAdV replicative
cycle, such as assembly, maturation, and even release of the new viral particles. Further
studies are needed to clarify the specific mechanisms for the inhibition of HAdV
infection by these seven compounds. Since they have shown high variability regarding
their mechanism of action, the compounds could be useful as a tool to unravel the
complex events involved in the HAdV replicative cycle after HAdV entry.
The twelve derivatives presented here target different steps in the HAdV replicative
cycle: inhibition of early steps during HAdV entry into the cells, inhibition of the
transcription of the E1A gene, interference with the viral DNA polymerase and other
not fully characterized mechanisms interfering with HAdV DNA replication and
interference with later steps after HAdV DNA replication.
Synergistic activity of the selected derivatives
Since we identify different mechanisms of action for the selected compounds we
hypothesized that their combination will improve their antiviral activity blocking HAdV
infection at lower concentrations due to their synergistic effect. In order to assess this
hypothesis, three representative derivatives of each mechanism described were selected
to perform a combination study based on the Chou-Talalay method for drug
combination using the CalcuSyn software [36, 37]. The three derivatives that presented
different mechanisms of action in later stages after the arrival of the HAdV genomes
into nucleus were: compound 38 as a compound acting in later stages after DNA

replication; compound 41 as an inhibitor of the HAdV DNA replication process (HAdV
DNA polymerase); and compound 50 as an inhibitor of the E1A transcription (all of
them are 4-acyl-1-phenylaminothiocarbonyl-2-methylpiperazine derivatives). The ratio
for each combination was selected based on the IC values for each derivative. The
5
0
data for all the combinations showed good conformity to the mass-action law (r ranged
from 0.937 to 0.967) (Table 6). The combination of these three derivatives was
classified as synergistic when they were combined at the different ratios and at all the
inhibitory levels assayed; specifically this activity was classified as very strong
synergism for the combination 38+41 (1:4), targeting HAdV DNA polymerase activity
(
41) and later steps after HAdV DNA replication (38) at all three levels of inhibition
IC , IC and IC ), and for the combination 38+41+50 (1:4:8) at the IC , including in
(
5
0
75
90
90
this combination compound 50 targeting the gen E1A transcription. For the 41+50 (1:2)
combination the IC and IC levels of inhibition were classified as strong synergism.
5
0
75
Finally, the combinations 41+50 (1:2) at IC , 38+50 (1:8) at all the levels of inhibition
9
0
and 38+41+50 (1:4:8) at the IC50 and IC75 levels of inhibition were classified as
synergism.
The existence of different mechanisms for the antiviral activity of these three
compounds is this way supported by the significant combinatory index values obtained
using the CalcuSyn software for all the combinations. These derivatives showed a
synergistic to very strong synergistic activity when combined in pairs or including all
three compounds at all the levels of inhibition assayed.
<
Table 6>
Anti-HCMV activity of 4-acyl-1-phenylaminothiocarbonyl-2-methylpiperazine
derivatives

The following step explored the possible inhibitory activity of the selected compounds
on HCMV DNA replication since other antiviral compounds have shown broad activity
against multiple dsDNA viruses including HCMV and HAdV [21, 38]. Quantification
of total HCMV DNA 72 hours after the infection of MRC-5 cells revealed: significant
differences between samples treated with compounds 41, 50, and 87; and differences
not statistically significant for compounds 43, 45, 71, 73, and 89, and those treated with
the same volume of DMSO, five of which had previously shown inhibition of the
HAdV DNA replication (Figure 7C).
Study of the stability of the derivatives in hamster serum
Determination of the stability of new chemical entities in serum is important as
compounds (with the exception of pro-drugs where rapid conversion in serum is
desirable), which rapidly degrade in serum, generally show poor in vivo efficacy [39].
We assessed the stability of the compounds in Syrian hamster serum as a previous step
to the evaluation of their efficacy and safety in the Syrian hamster model of HAdV
infection [16]. This assay would allow us to prioritize the anti-HAdV compounds with
the structural motifs most resistant to degradation by serum proteins and those
compounds with the lowest binding to serum proteins, which will potentially show
better in vivo efficacy. The four derivatives that presented the best SI (>100) were
included in the evaluation (22, 38, 43, and 67, with different modes of action to block
HAdV infection, see below) together with compound 50 due to its activity as an
inhibitor of the E1A transcription (again all of these five selected compounds are 4-
acyl-1-phenylaminothiocarbonyl-2-methylpiperazine derivatives). Only compounds 22
and 38 (tert-butoxycarbonyl and 2-cyclohexylacetyl derivatives respectively) showed a
significant degradation after two hours of incubation in the Syrian hamster serum, with
percentages of remaining compound <70%. Compounds 43 and 67 showed no
degradation after the incubation time with percentages of remaining compound of

1
06.7±3.8 and 100.0±9.2 respectively, whilst compound 50 showed a percentage of
remaining compound of 92.8% (the three of them, 2-phenylacetyl derivatives)
Supplementary Information, Table S70).
(
In silico prediction of physicochemical properties: drug-likeness evaluation
During the drug discovery and development processes of new molecules with the final
goal to identify those with the best chance to become an effective drug for patients,
apart from efficacy and toxicity, many failures are related to pharmacokinetics, i.e., the
distribution of the compound in the organism [40]. In this sense, the early estimation of
ADME behaviors (Absorption, Distribution, Metabolism and Excretion) has decreased
the fraction of global pharmacokinetics related failures in later phases of development.
Although there are different routes of drug administration, oral dosing is highly
preferred for the patient’s comfort and compliance. Bioavailability is highly
multifactorial, but is primarily driven by gastrointestinal absorption. The concept of
drug-likeness (to assess qualitatively the chance for a molecule to become an oral drug
with respect to bioavailability), established from the analyses of the physiochemical
properties or/and structural features of drug candidates, has been widely used to rule out
compounds with properties most probably incompatible with an acceptable
pharmacokinetics profile. There are different rule-based filters, with diverse ranges of
properties inside of which the molecule is defined as drug-like [41].
Some physicochemical properties of the 12 selected compounds have been predicted
using free online software (http://www.molinspiration.com) to assess their compliance
with the Lipinski's rule of five [42].

As shown in Table S71 (Supporting Information) eleven out of twelve conformed well
to the Lipinski's rule of five. Only compound 67 failed with two violations, LogP and
Molecular Weight values exceeded the accepted ranges.
A second web tool (SwissADME, freely accessible at http://www.swissadme.ch) was
also used to evaluate pharmacokinetics and drug-likeness of these small molecules (data
no shown) [43]. Excepting compound 67 (which showed the same two Lipinski’s rules
violations), the rest of the compounds conformed again to both Lipinski's and Veber’s
rules. In addition, the displayed Bioavailability Radar (a rapid appraisal of drug-
likeness based on six physicochemical properties: lipophilicity, size, polarity, solubility,
flexibility and saturation that define the suitable physicochemical space for oral
bioavailability) predicted only compound 67 as no orally bioavailable (too lipophilic,
insoluble and high sized). According to the white of the BOILED-egg [41, 43], the
human gastrointestinal absorption predicted was high for all compounds, except for
compound 67.
2
. Conclusions
The twelve selected piperazine-derived thioureas/ureas presented here showed
significant anti-HAdV activity within nanomolar and low micromolar ranges with low
cytotoxicity. The strengthening of their antiviral activity in combination supports the
existence of different mechanisms of action. These molecules target different steps in
the HAdV replicative cycle: i) blockage of the HAdV genome accessibility to the
nucleus (71, 73 and 89) targeting early steps after HAdV enters the cell, ii) inhibition of
the transcription of the E1A gene (50), iii) interference with HAdV DNA replication
(41, 43, 45, and 87) and specifically in case of compound 41 interfering with the viral
DNA polymerase, iv) and other not fully characterized mechanisms interfering with
later steps after HAdV DNA replication (22, 37, 38 and 67). Although further studies

are needed for the full characterization of the mechanisms of action of these molecules,
these could also provide great insights towards a better understanding of the HAdV
replication cycle.
Herein we have identified new mechanisms of action respect to the prototypes used for
the generation of this new library of compounds, which were targeting HAdV
replication and later steps. The selected compounds also improve the antiviral activity
of the ones they come from, showing anti-HAdV activity at nanomolar concentrations.
Based on their mechanisms of action and their antiviral activity and serum stability,
compounds 22, 41, 43, 50, 67 and 73 have been selected for further in vivo studies in the
immunosuppressed Syriam Hamster model of HAdV infections towards improving our
knowledge regarding their efficacy and safety as the first step in their development as
therapeutic alternatives for the treatment of HAdV infections.
4
4
. Experimental Section
.1 Chemistry. General Chemistry Methods. All reagents, solvents, and starting
materials were obtained from commercial suppliers and used without further
purification. The crude reaction mixtures were concentrated under reduced pressure by
removing the organic solvents in a rotary evaporator. Reactions were monitored by thin
layer chromatography (TLC) using Kieselgel 60 F254 (E. Merck) plates and UV detector
for visualization. Flash column chromatography was performed on Silica Gel 60 (E.
Merck). All reported yields are of purified products. Melting points were obtained on a
Stuart Melting Point Apparatus SMP 10 and are uncorrected. Mass spectra were
recorded on a Micromass AUTOSPECQ mass spectrometer: EI at 70 eV and CI at 150
eV, HR mass measurements with resolutions of 10,000. FAB mass spectra were
recorded using a thioglycerol matrix. NMR spectra were recorded at 25 ºC on a Bruker
1
13
AV500 spectrometer at 500 MHz for H and 125 MHz for C. COSY, DEPT, HSQC,

and NOESY experiments were performed to assign the signals in the NMR spectra. The
chemical shifts (δ) reported are given in parts per million (ppm) on the
δ scale relative
1
to TMS, and the coupling constants (J) are in hertz (Hz). H chemical shift values (δ)
are referenced to the residual nondeuterated components of the NMR solvents (δ = 2.54
1
3
ppm for DMSO, δ = 7.26 ppm for CDCl ). The C chemical shifts (δ) are referenced to
3
CDCl (central peak, δ = 39.5 ppm for DMSO and 77.16 ppm for CDCl ) as the internal
3
3
standard. The spin multiplicities are reported as s (singlet), d (doublet), t (triplet), q
quadruplet), m (multiplet), or br s (broad singlet).. The purity of final compounds was
(
evaluated by C, H and N analysis. The purity of all the final compounds was confirmed
to be ≥95% by combustion.
General Procedure 1. Chemoselective N-acylation reaction of 2-Substituted
piperazines or 2,6-Disubstituted piperazine (16-19, 51-55, 78). 2-Substituted
piperazine or 2,6-Disubstituted piperazine (5.0 mmol) was dissolved in dry
dichloromethane (100 mL) and cooled to 0 ºC. A solution of the appropriate acylating
agent (5.0 mmol) in dichloromethane (20 mL) was added dropwise in 30 minutes, and
then pyridine (7.5 mmol). The reaction mixture was kept into an ice-water bath with
stirring 12 hours and left at room temperature until TLC showed that all the starting
material had reacted. The reaction mixture was evaporated to dryness to obtain the
corresponding monoacylderivative. Column chromatography gave the pure compound
in high yield.
1
-tert-Butoxycarbonyl-3-methylpiperazine (16) [21]. The product was obtained as a
syrup and purified by column chromatography using dichloromethane-methanol (15:1)
+
1
as eluent (750 mg, 75% yield). MS (CI): m/z 201 (20%) [M+H] . H NMR (500 MHz,
DMSO-d6) 3.75–3.71 (m, 2H), 2.85–2.82 (m, 1H), 2.75–2.69 (m, 1H), 2.60–2.54 (m,
H), 2.39–2.34 (m, 1H), 1.41 (s, 9H), 0.96 (d, J = 6.3 Hz, 3H). C NMR (125 MHz,
δ
13
3

DMSO-d6)
δ 154.5, 79.3, 51.2, 50.5, 45.5, 44.4, 28.6, 19,3. HRMS (m/z): calcd for
+.
C H N O 200.1528 [M] ; found 200.1525.
10
20
2
2
1
-(3,3-Dimethylbutanoyl)-3-methylpiperazine (17). The product was obtained as a
syrup and purified by column chromatography using ethyl acetate-methanol (3:1) as
+
1
eluent (792 mg, 80% yield). MS (CI): m/z 199 (100%) [M+H] . H NMR (500 MHz,
CDCl3) 4.57 (d, J = 12.4 Hz, 1H), 3.85 (m, 1H), 3.52–3.08 (m, 2H), 3.06–2.62 (m,
δ
1
3
3
5
2
H), 2.23 (s, 2H), 1.33 (br s, 3H), 1.02 (s, 9H). C NMR (125 MHz, CDCl )
δ
170.3,
3
1.4, 45.8, 44.6, 44.5, 39.3, 31.5, 30.0, 17.3. HRMS (m/z): calcd for C H N ONa
11
22
2
+
22.1624 [M+Na] ; found 222.1619.
1
-(2-Cyclohexylacetyl)-3-methylpiperazine (18). The product was obtained as a syrup
and purified by column chromatography using ethyl acetate-methanol (4:1) as eluent
1
(
1.1 g, 97% yield). H NMR (500 MHz, DMSO-d )
δ
4.26 (d, J = 11.7 Hz, 1H), 3.76 (d,
6
J = 11.4 Hz, 1H), 3.05–2.88 (m, 2H), 2.70–2.58 (m, 3H), 2.26–2.14 (m, 3H), 1.75–1.60
13
(
m, 6H), 1.30–1.10 (m, 3H), 1.05–0.91 (m, 5H). C NMR (125 MHz, DMSO-d ) δ
6
1
69.7, 52.0, 50.8, 50.3, 47.7, 45.4, 44.8, 41.0, 34.4, 32.7, 25.8, 25.6, 18.7, 18.4. HRMS
+
.
(
m/z): calcd for C H N O 225.1961 [M+H] ; found 225.1964.
13 25 2
3
-Methyl-1-(2-phenylacetyl)piperazine (19). The product was obtained as a syrup and
purified by column chromatography using ethyl acetate-methanol (4:1) as eluent (1.1 g,
1
9
1
1
1
1
8% yield). H NMR (500 MHz, DMSO-d )
δ
7.33–7.22 (m, 5H), 4.25 (d, J = 12.7 Hz,
6
H), 3.09–2.62 (m, 3H), 2.58–2.53 (m, 1H), 2.49–2.34 (m, 1H), 1.75-1.60 (m, 6H),
13
.03, 0.99 (2s, J = 6.4 Hz, 3H). C NMR (125 MHz, DMSO-d )
δ 168.7, 135.9, 135.8,
6
28.9, 128.8, 128.3, 128.2, 126.3, 51.5, 50.5, 50.1, 47.1, 45.0, 44.6, 44.2 40.6, 18.1,
+
7.7. HRMS (m/z): calcd for C H N O 219.1492 [M+H] ; found 219.1486.
1
3
19
2

1
-(Benzofuran-2-carbonyl)-3-methylpiperazine (51). The product was obtained as a
solid and purified by column chromatography using dichloromethane-methanol (40:1)
1
as eluent (903 mg, 74% yield), mp 101–103 ºC. H NMR (500 MHz, DMSO-d )
δ 7.7–
6
7
2
1
6
.5 (m, 5H), 4.47 (br s, 2H), 3.10 (d, J = 11.4 Hz, 1H), 2.94–2.86 (m, 2H), 1.97 (br s,
1
3
H), 1.13 (d, J = 5.0 Hz, 3H). C NMR (125 MHz, DMSO-d )
δ
159.8, 154.6, 149.1,
6
27.0, 126.4, 123.6, 122.2, 111.9, 111.8, 51.1, 46.1, 19.4. Anal. Calcd C H N O : C,
1
4
16
2
2
8.55; H, 6.99; N, 11.42. Found: C, 68.32; H, 6.62; N, 11.22.
1
-tert-Butoxycarbonyl-3-phenylpiperazine (52) [21].The product was obtained as a
syrup and purified by column chromatography using hexane−ethyl acetate (2:1) as
+
eluent (864 mg, 66% yield), mp 103−105 °C. MS (CI): m/z 263 (100%) [M+H] . 1H
NMR (500 MHz, CDCl ) δ 7.4−7.3 (m, 5H), 4.05 (br s, 2H), 3.70 (dd, J = 2.4 Hz, J =
3
1
9
4
2
0.5 Hz, 1H,), 3.07 (m, 1H), 2.9−2.8 (m, 2H), 2.72 (br s, 1H), 1.90 (br s, 1H), 1.47 (s,
1
3
H). C NMR (125 MHz, CDCl ) δ 154.8, 141.5, 128.5, 127.8, 127.0, 79.7, 60.3, 51.5,
3
+
6.1, 43.4, 28.5. HRMS (m/z): calcd for C H N O 263.1754 [M+H] ; found
15
23
2
2
63.1748.
1
-(3,3-Dimethylbutanoyl)-3-phenylpiperazine (53). The product was obtained as a
syrup and purified by column chromatography using ethyl acetate-methanol (20:1) as
+
1
eluent (1.0 g, 77% yield). MS (CI): m/z 261 (100%) [M+H] . H NMR (500 MHz,
DMSO-d6) 7,45–7,27 (m, 5H), 4,44–4.39 (m, 1H), 3,94–3,85 (m, 1H), 3,58 (dd, J =
.3 Hz, J = 10.3 Hz, 1H), 3,31 (br s, 1H), 3,11–2,99 (m, 2H), 2,91–2,68 (m, 1H), 2,61–
δ
2
2
δ
4
1
3
,57 (m, 1H), 2,31–2,20 (m, 2H), 1,02, 1,00 (2s, 9H). C NMR (125 MHz, DMSO-d )
6
169.3, 169.2, 142.2, 141.9, 128.2, 128.1, 127.4, 127.3, 127.0, 126.9, 60.1, 59.4, 53.5,
8.1, 46.4, 45.9, 45.5, 43.8, 43.6, 41.1, 40.1, 30.9, 29.8. HRMS (m/z): calcd for
+
C H N O 261.1961 [M+H] ; found 261.1956.
16
25
2

1
-(2-Cyclohexylacetyl)-3-phenylpiperazine (54). The product was obtained as a syrup
and purified by column chromatography using ethyl acetate-methanol (30:1) as eluent
+
1
(
815 mg, 57% yield). MS (CI): m/z 287 (100%) [M+H] . H NMR (500 MHz, CDCl ) δ
3
7
3
1
.40–7.27 (m, 5H), 4.67–4.63 (m, 1H), 3.84–3.79 (m, 1H), 3.69–3.67 (m, 1H), 3.26–
.02 (m, 2H), 2.91–2.83 (m, 1H), 2.59–2.54 (m, 1H), 2.29–2.16 (m, 2H), 2.10–1.91 (m,
1
3
H), 1.85–1.65 (m, 5H), 1.33–1.21 (m, 2H), 1.18–1.10 (m, 1H), 1.02–0.92 (m, 2H). C
170.9, 141.3, 141.1, 128.7, 128.5, 128.2, 127.8, 127.0,
26.9, 61.4, 60.2, 53.6, 48.8, 46.7, 46.3, 46.1, 41.8, 40.8, 40.7, 35.3, 35.2, 33.5, 33.4,
NMR (125 MHz, CDCl3)
δ
1
3
+
3.3, 26.3, 26.2. HRMS (m/z): calcd for C H N O 287.2118 [M+H] ; found 287.2111.
1
8
27
2
3
-Phenyl-1-(2-phenylacetyl)piperazine (55). The product was obtained as a syrup and
purified by column chromatography using ethyl acetate-methanol (100:1) as eluent (784
1
mg, 56% yield). ). H NMR (500 MHz, CDCl )
δ 7.39–7.12 (m, 10H), 4.68–4.59 (m,
3
1
H), 3.83–3.62 (m, 3H), 3.29–3.13 (m, 1H), 3.11–2.92 (m, 2H), 2.84–2.56 (m, 2H),
13
2
.18 (br s, 1H). C NMR (125 MHz, CDCl ) δ 169.5, 169.4, 141.0, 140.8, 135.3, 135.1,
3
1
28.9, 128.7, 128.6, 128.5, 128.0, 127.9, 127.0, 126.9, 126.8, 60.8, 60.0, 53.8, 48.9,
+
4
6.5, 46.2, 45.9, 42.0, 41.4, 41.2. HRMS (m/z): calcd for C H N O 281.1648 [M+H] ;
1
8
21
2
found 281.1644.
1
-(Benzofuran-2-carbonyl)-3,5-dimethylpiperazine (77). The product was obtained
as a syrup and purified by column chromatography using dichloromethane-methanol
1
(
20:1) as eluent (942 mg, 73% yield). H NMR (500 MHz, CDCl )
δ
7.76 (d, J = 7.8
3
Hz, 1H), 7.68 (dd, J = 8.1 Hz, J = 0.6 Hz, 1H), 7.48–7.44 (m, 1H), 7.41 (s, 1H), 7.37–
13
7
.33 (m, 1H), 4.46–4.10 (m, 2H), 2.98–2.75 (m, 3H), 1.16–0.94 (m, 6H). C NMR (125
MHz, CDCl3)
δ
158.6, 153.9, 148.2, 126.7, 126.4 123.6, 122.4, 111.8, 110.7, 54.9, 18.2.
+
HRMS (m/z): calcd for C H N O 259.1441 [M+H] ; found 259.1444.
1
5
19
2
2

General Procedure 2. Synthesis of the thiourea / urea derivatives (12, 20–50, 56-67,
6
8-74, 79-85, 87-91). To a solution of the monoacyl derivative (16–19, 51–55, 78 and
6) (0.75 mmol) in dry dichloromethane (10 mL) was added the corresponding
8
isothiocyanate/isocyanate (0.9 mmol). The reaction mixture was stirred at room
temperature until TLC showed that all the starting material had reacted. The reaction
mixture was evaporated to dryness. The compounds were purified by flash
chromatography on silica gel using the appropriate eluent. For compounds 75 and 92
1
.8 mmol of the appropriate isocyanate were employed.
4
-tert-Butoxycarbonyl-2-methyl-1-[(4-nitrophenyl)aminothiocarbonyl]piperazine
(
12) [21]. The product was obtained as a solid and purified by column chromatography
using hexane-ethyl acetate (2:1) as eluent (234 mg, 82% yield), mp 174–176 ºC. MS
+
1
(
FAB): m/z 403 (95%) [M+Na] . H NMR (500 MHz, DMSO-d )
δ
9,72 (br s, 1H),
.16–8.11 (m, 2H), 7.61–7.59 (m, 2H), 5.14–5.05 (m, 1H), 4.43–4.35 (m, 1H), 4.00–
.86 (m, 1H), 3.83–3.75 (m, 1H), 3.22–3.12 (m, 1H), 3.06–2.93 (m, 1H), 1.44 (s, 9H),
6
8
3
1
1
4
1
1
3
.21 (d, J = 6.7 Hz, 3H). C NMR (125 MHz, DMSO-d )
δ
181.3, 154.5, 147.7, 142.4,
6
23.9, 122.9, 79.2, 51.8, 43.1, 27.9, 26.8, 14.7. HRMS (m/z): calcd for C H N O SNa
17
24
4
4
+
03.1410 [M+Na] ; found 403.1405. Anal. Calcd C H N O S: C, 53.67; H, 6.36; N,
17
24
4
4
4.73; S, 8.43. Found: C, 53.60; H, 6.58; N, 14.56; S, 8.27.
4
-tert-Butoxycarbonyl-1-[(4-chlorophenyl)aminothiocarbonyl]-2-methylpiperazine
(
20). The product was obtained as a solid and purified by column chromatography using
hexane-ethyl acetate (2:1) as eluent (202 mg, 73% yield), mp 143–144 ºC. MS (FAB):
+
1
m/z 392 (100%) [M+Na] . H NMR (500 MHz, CDCl )
δ
7.30 (d, J = 8.4 Hz, 2H),
.16–7.33 (m, 3H), 5.13–4.76 (m, 1H), 4.45–3.79 (m, 3H), 3.39–3.32 (m, 1H), 3.21 (dd,
J = 3.5 Hz, J = 13.5 Hz, 1H), 3.12–2.91 (m, 1H), 1.48 (s, 9H), 1.28 (d, J = 6.7 Hz, 3H).
3
7
1
3
C NMR (125 MHz, CDCl3)
δ 183.3, 155.0, 138.5, 131.0, 129.8, 129.2, 126.9, 125.4,

8
0.5, 52.4, 43.8, 28.4, 15.1. HRMS (m/z): calcd for C H ClN O SNa 392.1170
17 24 3 2
+
[
M+Na] ; found 392.1163. Anal. Calcd C H ClN O S: C, 55.20; H, 6.54; N, 11.36; S,
17 24 3 2
8
.67. Found: C, 55.48; H, 6.67; N, 11.44, S, 9,01.
4
-tert-Butoxycarbonyl-1-[(4-cyanophenyl)aminothiocarbonyl]-2-methylpiperazine
(
21). The product was obtained as a solid and purified by column chromatography using
hexane-ethyl acetate (3:1) as eluent (205 mg, 76% yield), mp 132–134 ºC. MS (FAB):
+
1
m/z 383 (100%) [M+Na] . H NMR (500 MHz, CDCl )
δ
7.61 (d, J = 8.4 Hz, 2H), 7.32
3
(d, J = 8.4 Hz, 2H), 7.22 (br s, 1H), 5.18–4.86 (m, 1H), 4.40–3.73 (m, 4H), 3.44–3.37
(m, 1H), 3.26–3.19 (m, 1H), 3.14–2.90 (m, 1H), 1.48 (s, 9H), 1,32 (d, J = 6.8 Hz, 3H).
1
3
C NMR (125 MHz, CDCl3)
δ
182.5, 154.9, 144.0, 133.2, 126.5, 122.7, 118,6. 80.5,
+
5
2.8, 44.1, 28.4, 15.3. HRMS (m/z): calcd for C H N O SNa 383.1512 [M+Na] ;
1
8
24
4
2
found 383.1500. Anal. Calcd C H N O S: C, 59.97; H, 6.71; N, 15.54; S, 8.90. Found:
18
24
4
2
C, 60.03; H, 6.81; N, 15.20; S, 9.15.
4
-tert-Butoxycarbonyl-1-[(4-fluorophenyl)aminothiocarbonyl]-2-methylpiperazine
22). The product was obtained as a solid and purified by column chromatography using
hexane-ethyl acetate (3:1) as eluent (209 mg, 79% yield), mp 74–76 ºC. MS (FAB): m/z
(
+
1
3
7
3
76 (100%) [M+Na] . H NMR (500 MHz, CDCl )
δ
7.22–7.14 (m, 2H), 7.10 (s, 1H),
3
.06–7.02 (m, 2H), 5.14–4.75 (m, 1H), 4.51–3.69 (m, 3H), 3.41–3.32 (m, 1H), 3.24–
1
3
.16 (m, 1H), 3.14–2.75 (m, 1H), 1.47 (s, 9H), 1.28 (d, J = 6.8 Hz, 3H). C NMR (125
MHz, CDCl3)
δ
183.6, 155.0, 135.9, 126.6, 115,9. 80.4, 52.3, 43.6, 28.4, 15.1. HRMS
+
(
m/z): calcd. for C H FN O SNa 376.1465 [M+Na] ; found 376.1456. Anal. Calcd
1
7
24
3
2
C H FN O S: C, 57.77; H, 6.84; N, 11.89; S, 9.07. Found: C, 58.00; H, 6.82; N,
17
24
3
2
1
1.68; S, 9.48.