Synthesis and pharmacological screening of a large library of 1,3,4-thiadiazolines as innovative therapeutic tools for the treatment of prostate cancer and melanoma

Article abstract of DOI:10.1016/j.ejmech.2015.10.023

Antimitotic agents are widely used in cancer chemotherapy but the numerous side effects and the onset of resistance limit their clinical efficacy. Therefore, with the purpose of discovering more selective and efficient anticancer agents to be administered alone or in combination with traditional drugs, we synthesized a large library of 1,3,4-thiadiazoline analogues, maintaining the pharmacophoric structure of an antiproliferative compound known as K858: this is a new inhibitor of kinesin Eg5, able to induce the mitotic arrest in colorectal cancer cells and in xenograft ovarian cancer cells. We screened 103 compounds to assess their antiproliferative activity on PC3 prostate cancer cell line. Two derivatives, compounds 32 (corresponding to K858) and 33, have shown to be the most effective against prostate tumor cells and also towards two melanoma cell lines (SK-MEL-5 and SK-MEL-28) at low micromolar concentrations, confirming the pharmacological activity of this scaffold and revealing the potential role of 1,3,4-thiadiazolines in the management of cancer.

Full text of DOI:10.1016/j.ejmech.2015.10.023

European Journal of Medicinal Chemistry 105 (2015) 245e262
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Synthesis and pharmacological screening of a large library of 1,3,4-
thiadiazolines as innovative therapeutic tools for the treatment of
prostate cancer and melanoma
,
Celeste De Monte a, Simone Carradori b *, Daniela Secci a, Melissa D'Ascenzio c,
Paolo Guglielmi a, Adriano Mollica b, Stefania Morrone d, Susanna Scarpa d,
e
e
e
ꢀ
Anna Maria Agliano , Sabrina Giantulli , Ida Silvestri
a Dipartimento di Chimica e Tecnologie del Farmaco, Sapienza University of Rome, P.le A. Moro 5, 00185 Rome, Italy
b Department of Pharmacy, “G. D'Annunzio” University of Chieti-Pescara, Via dei Vestini 31, 66100 Chieti, Italy
c Target Discovery Institute, University of Oxford, NDM Research Building, Roosevelt Drive, Headington, Oxford, United Kingdom
d Department of Experimental Medicine, Sapienza University of Rome, Viale Regina Elena, 00185 Rome, Italy
e Department of Molecular Medicine, Sapienza University of Rome, Viale Regina Elena, 00185 Rome, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Antimitotic agents are widely used in cancer chemotherapy but the numerous side effects and the onset
of resistance limit their clinical efficacy. Therefore, with the purpose of discovering more selective and
efficient anticancer agents to be administered alone or in combination with traditional drugs, we syn-
thesized a large library of 1,3,4-thiadiazoline analogues, maintaining the pharmacophoric structure of an
antiproliferative compound known as K858: this is a new inhibitor of kinesin Eg5, able to induce the
mitotic arrest in colorectal cancer cells and in xenograft ovarian cancer cells. We screened 103 com-
pounds to assess their antiproliferative activity on PC3 prostate cancer cell line. Two derivatives, com-
pounds 32 (corresponding to K858) and 33, have shown to be the most effective against prostate tumor
cells and also towards two melanoma cell lines (SK-MEL-5 and SK-MEL-28) at low micromolar con-
centrations, confirming the pharmacological activity of this scaffold and revealing the potential role of
1,3,4-thiadiazolines in the management of cancer.
Received 3 July 2015
Received in revised form
8 October 2015
Accepted 11 October 2015
Available online xxx
Keywords:
Antiproliferative activity
Eg5 inhibitors
Melanoma
Prostate cancer
Thiadiazolines
© 2015 Elsevier Masson SAS. All rights reserved.
1. Introduction
formation, cell cycle arrest and cell death [3,4]. Despite their use-
fulness in chemotherapy these drugs are characterized by many
Antimitotic agents represent a very important class of drugs for
the clinical management of cancer. The conventional antimitotic
compounds provoke mitotic arrest and cell death by binding to
tubulin and breaking microtubule dynamics [1,2], for this reason
these drugs are also named microtubule-targeting agents (MTAs).
Microtubules are important for cell division, motility and many
other functions and their activity is regulated by polymerization
dynamics. Vinca alkaloids (vincristine, vinblastine, vinorelbine)
inhibit microtubule polymerization, whereas taxanes (paclitaxel,
docetaxel) stabilize microtubules by promoting polymerization. It
has been shown that these drugs at low concentrations suppress
microtubule dynamics leading to an aberrant mitotic spindle
side effects such as nausea, vomiting, diarrhea, neurotoxicity and
myelotoxicity. Neurotoxicity, due to the fact that microtubules are
involved in neuron axonal transport, is characterized by headache,
dizziness, depression, peripheral neuropathy with numbness and
weakness, cranial neuropathy with jaw pain and vocal cord
dysfunction, autonomic neuropathy with constipation and urinary
retention. On the other hand, myelosuppression is caused by the
antiproliferative effect on rapidly dividing hematopoietic cells and
it is mainly manifested by neutropenia [5]. Furthermore, these
drugs often induce multidrug resistant mutations in tubulin
expression, in mitosis checkpoint proteins and in apoptotic
pathway which lead to therapeutic failure [6e9]. Taking into ac-
count all these considerations new molecules able to target mitosis
without disrupting microtubule dynamics are an important topic of
study [10e13].
* Corresponding author.
E-mail address: simone.carradori@unich.it (S. Carradori).
http://dx.doi.org/10.1016/j.ejmech.2015.10.023
0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.

246
C. De Monte et al. / European Journal of Medicinal Chemistry 105 (2015) 245e262
As regards five membered heterocyclic compounds, 1,3,4-
proapoptotic effects of these thiadiazolines suggest their potential
role in cancer therapy and the possibility of discovering new anti-
cancer drugs to be used alone or in combination with the tradi-
tional antimitotic agents.
thiadiazoles have been widely studied for their potential role in
medicinal chemistry and promising results have been reported
mainly for K858, a thiadiazoline compound inducing cell death in
colorectal and ovarian cancer cells [14,15]. K858 blocks centrosome
separation and induces mitotic arrest as well as the formation of
monopolar in human colorectal cancer cell line, HCT116. Unlike
paclitaxel or vincristine, K858 has no effect on microtubule poly-
merization and preferentially causes cell death in neoplastic cells
with respect to normal cells. Moreover, K858 antitumor activity has
been demonstrated also in human xenograft tumor models (A2780
cell line). K858, as well as the thiadiazoline ARRY-520, selectively
inhibits the kinesin Eg5 ATPase activity [15,16]. Eg5 belongs to
kinesins, a family of motor proteins involved in cytokinesis, intra-
cellular vesicle and organelle transport and mitosis, by unidirec-
tionally moving along microtubules using ATP hydrolysis to
generate motile force for the movement. Eg5 plays a pivotal role in
the separation of the duplicated centrosomes during prophase and
drives bipolar spindle assembly [17]. Inhibition of Eg5 causes
monoastral spindle phenotype and mitotic arrest [18e20]. To
support these results, it has been shown that knockdown of Eg5
mRNA using siRNA leads to inhibit the proliferation of cancer cells
in culture by aberrant mitotic spindle formation and mitotic cell
cycle arrest [21]. Moreover Eg5 is overexpressed in several malig-
nancies such as leukemia, breast, lung, ovarian, bladder and
pancreatic cancers and it is almost no detected in nonproliferative
tissues like neurons, and it is of great interest to be targeted by new
anticancer agents [22e26].
Given our interest in developing new agents for cancer therapy
[27e29] and the various literature data about the antitumor effect
of thiadiazolines, we designed and produced a large library of
compounds that consists of 1,3,4-thiadiazolines (1e102) provided
with aliphatic, cycloaliphatic, aromatic and heteroaromatic sub-
stituents at C5 of this ring and symmetrical amidic/hydrazido
chains at C2 and C4. For comparison with K858, an oxidized thia-
diazole compound 103 was also provided (Scheme 1). The choice of
different substituents was made to explore the chemical space at
C5, highlighting the biological differences after the proposed
chemical modifications. Some moieties were selected from other
published 1,3,4-thiadiazoline compounds with recognized antimi-
totic activity (i.e., ARRY-520). The amidic/hydazido lateral chains
were also designed after insertion of a methylene unit (56e101)
and halogenation with chlorine (55) and fluorine (102) atoms.
We screened all these compounds against PC3 prostate cancer
cell line, confirming the antiproliferative activity of some de-
rivatives. Successively, the most active molecules were also shown
to be effective against two melanoma cell lines (SK-MEL-5 e SK-
MEL-28) at low micromolar concentrations. The three best de-
rivatives were also evaluated for their ability to inhibit Eg5 kinesin
activity and to induce apoptosis. The antiproliferative and
2. Results and discussion
2.1. Chemistry
Different carbonyl compounds reacted with thiosemicarbazide
in ethanol with catalytic amounts of acetic acid. The resulting thi-
osemicarbazone intermediates were cyclized into 1,3,4-
thiadiazolines by reaction with several symmetrical anhydrides
(acetic, propionic, dichloroacetic, pentafluoropropionic) used as
solvents. The oxidation of the thiadiazoline compound 31 was
accomplished by KMnO4 in acetic acid to yield the corresponding
thiadiazole 103 [30]. All the synthesized products were washed
with n-hexane and petroleum ether and purified by column chro-
matography before characterization by spectroscopic methods and
elemental analysis. Compounds 32 and 87 had already been char-
acterized in a previous work and their spectroscopic data are in
accordance with the literature [31].
2.2. Pharmacological evaluation
First, the synthesized compounds were dissolved in DMSO and
then culture media or PIPES buffer was used to obtain 1 mM stock
concentration.
(0e100 M) were conducted in order to identify the times (0e96 h)
and the optimal concentrations. Cells were treated with 10 M of
different compounds for 72 h in MTT (3-[4,5-dimethylthiazol-2-yl]-
2,5-diphenyltetrazolium bromide) assay, with 5 M of different
compounds for Fluorescence-Activated Cell Sorting (FACS) analysis
Preliminary
doseeresponse
experiments
m
m
m
and 0.1, 0.5 and 1
inhibition test.
mg/mL of each compound were used for kinesin
We evaluated the effects of the different K858 analogues on PC3,
a hormone-insensitive highly metastatic human prostate adeno-
carcinoma cell line, because several studies were focused on the
identification of novel compounds with improved efficacy for
prostate cancer management taking advantage of the interest in
kinesin inhibitors [32,33]. We performed an intensive screening
exposing PC3 cells to six different concentrations of compounds
1e103 (0e100
mM) for 24, 48 and 72 h Fig. 1 shows the anti-
proliferative effects of the best active compounds within our 1,3,4-
thiadiazolidine scaffold (28, 32/K858, 33, 39, 41, 43, 55, 66 and 88)
by using MTT test on PC3 cells.
From these results we can observe that a proper functionaliza-
tion of the 1,3,4-thiadiazoline nucleus is requested to display
promising antiproliferative activity. Compound 32 (K858), our
reference lead compound, was also active against PC3 cell line thus
Scheme 1. Chemical structures of the K858-based thiadiazolines for the development of new antitumoral agents.

C. De Monte et al. / European Journal of Medicinal Chemistry 105 (2015) 245e262
247
100,00
150%
100%
50%
0%
100μM
90,00
50μM
80,00
10μM
70,00
5μM
1μM
60,00
1μM
10μM
28
32
33
0.5μM
50,00
39
0,1 μg/mL
41
43
100μM
45
55
64
66
40,00
88
0,5 μg/mL
1 μg/mL
30,00
Fig. 1. Antiproliferative activity of the best K858 analogues (28, 32/K858, 33, 39, 41, 43,
45, 55, 64, 66 and 88) on human prostate cancer cell line, PC3, evaluated by MTT assay.
Cells were treated with the selected compounds for 72 h. The results are expressed as
percent of viable cells respect to control. The values are presented as the mean of at
least three independent assays.
20,00
10,00
0,00
32
33
55
compounds
confirming its importance in the therapy of cancer. Some analogues
in which the aromatic portion of K858 was substituted with 4-acetyl
(39) or 2,5-difluoro (41, with the same substituent of ARRY-520)
presented a higher inhibitory activity. Moreover, the bioisosteric
replacements of the aryl ring with a thiophene (28) or a pyridin-2-yl
(43) maintained the biological activity. As regards to the methyl
group at C5 of K858, its substitution with an ethyl moiety in some
derivatives (33, 66 and 88) improved the antiproliferative potency.
Acetic lateral chains are generally preferred but could be extended
(66 and 88) or halogenated (55) to obtain promising and active
compounds against PC3 cells. Finally, the concurrent presence at C5
of the thiadiazoline ring of two (cyclo)aliphatic chains (1e24) was
not essential to display such kind of biological activity.
Fig. 3. Inhibition of kinesin activity with different concentration (0.1, 0.5 and 1 mg/mL)
of compounds 32, 33 and 55. The result shows the percent of remaining ATPase activity
after compounds treatment. The values are presented as the mean of at least three
independent assays.
viability cells detected by trypan blue with respect to control while
25% and 18% resulted, respectively, for compounds 32 and 33 (5 mM).
Inhibition of kinesin Eg5 motor action was also evaluated in vitro
for the most active compounds. As expected, compounds 32, 33 and
55 showed ability to inhibit KSP enzymatic activity by using ATPase
assay. This inhibition was dose-dependent and compound 33 may
be considered even more effective than 32/K858 (Fig. 3).
Considering their cytotoxic effects, we further analyzed the ef-
fects of 32 and 33 on cell cycle distribution and apoptotic rate of
different cell lines (PC3, SK-MEL-5 and SK-MEL-28) (Fig. 4).
To verify that the antiproliferative effect displayed by various
compounds was not limited to PC3 cells, we next carried out
treatments on cells derived from another tumor, such as melanoma
cell lines: SK-MEL-5 and SK-MEL-28 (Fig. 2) at three different con-
Cells were treated with 5 mM of each compound because higher
concentration did not allow a cell survival for a long time such 96 h.
FACS analyses by using propidium bromide DNA staining showed
an increasing percentage of cells in G2/M, as detectable after 24 h
treatment with each compound. The accumulation of cells with 4N
DNA is persisting for at least 96 h and it is consistent with an arrest
in mitosis. Arrest on cell cycle progression has been validated also
by RT-PCR analyses. In different experiments we obtained a
decreased expression of cyclin D1 on PC3 as well as on SK-MEL cells
centrations (5, 10 and 50 mM), because of the interesting results
reported in a Phase II study with another kinesin inhibitor on
metastatic or recurrent malignant melanoma [34]. We chose SK-
MEL-5 and SK-MEL-28 for their different p53 expression, wild
type and mutated, respectively. The results are the media of at least
three experiments: K858 (compound 32) and compound 33 (sub-
stitution of the methyl group with an ethyl group at C5) exerted an
interesting antiproliferative activity against these two melanoma
lines. Other compounds (45, 55 and 64) displayed a discrete bio-
logical activity but inferior than the parent compound. Both MTT
and trypan blue (Fig. S1, Supporting information) assays showed
that several compounds have an interesting antitumor activity in
human prostate and melanoma cell lines. Among them, compound
33 showed a comparable effect with respect to 32, which corre-
sponds to K858. By using the same experimental conditions, the
treatment of PC3 with docetaxel (100 nM) over 72 h showed 32% of
treated with 5 mM 32 or 5 mM 33 for 24 h compared to untreated
cells (Fig. 4C). It is known that cyclin D1 is often overexpressed in a
variety of cancers and is associated with tumorigenesis and
metastasis. Several mitogenic and transformation signaling cas-
cades converge in fact in cyclin D, which acts as a mediator during
cell-cycle progression. Among the three cyclin D isoforms (D1, D2,
and D3), cyclin D1 overexpression is most frequently associated
with human cancer [35,36]. Cyclin D1 is rarely mutated, but its
Fig. 2. Antiproliferative activity of the best active compounds (32/K858, 33, 45, 55 and 64) on human melanoma cell lines, SK-MEL-5 (left) and SK-MEL-28 (right), evaluated by MTT
assay. Cells were treated with the selected compounds for 72 h. The results are expressed as percent of viable cells respect to control. The values are presented as the mean of at least
three independent assays.

248
C. De Monte et al. / European Journal of Medicinal Chemistry 105 (2015) 245e262
Fig. 4. Cell cycle analysis of different cell lines treated with 5
of 32 and 33 for 24 h (C).
mM of 32 and 33 for 24 h (A) and 96 h (B). Expression of GADPH (left) and Cyclin D (right) of cells incubated with 5 mM
overexpression confers a selective growth advantage and hence
acts as a driver of neoplastic disease [37].
layer chromatography was carried out on SigmaeAldrich® silica gel
on TLA aluminum foils with fluorescent indicator 254 nm. Visuali-
zation was carried out under ultra-violet irradiation (254 nm). NMR
spectra were recorded on a Bruker AV400 (1H: 400 MHz, 13C:
101 MHz). Coupling constants J are valued in Hertz (Hz). Chemical
3. Conclusion
shifts are expressed as
d units (parts per millions), based on
There is great interest in developing more potent kinesin in-
appearance rather than interpretation, and are referenced to the
residual non deuterated solvent peak. IR spectra were recorded on a
FT-IR PerkineElmer SpectrumOne equipped with ATR system. Ab-
sorption maxima (nmax) are reported in wavenumbers (cmꢀ1). All
melting points were measured on a Stuart® melting point apparatus
SMP1, and are uncorrected. Temperatures are reported in ꢁC. Where
given, systematic compound names are those generated by Chem-
Draw Ultra 8.0 following IUPAC conventions.
hibitors for clinical applications able to improve side effect profiles
over taxanes. We herein identified
a novel series of 1,3,4-
thiadiazoline analogues that induce potent mitotic arrest as puta-
tive kinesin inhibitors. Their antitumor activity is showed at low
concentration and is detectable on different cell types (PC3, SK-
MEL-5 and SK-MEL-28). In particular, derivate 33 displayed signif-
icant antitumor activity with respect to its parent compound 32/
K858. The introduction of an ethyl moiety at C5 position of the
thiadiazoline ring was effective in enhancing the anti-mitotic ef-
fects and improving inhibition of kinesin activity. Further experi-
ments have at the moment been carrying out in order to better
define apoptosis pathway induced by these compounds.
4.2. Chemistry
4.2.1. Synthesis of the thiosemicarbazone intermediates
Different carbonyl compounds were reacted with thio-
semicarbazide (1.0 eq.) in ethanol (50 mL) with catalytic amounts of
acetic acid for 12e72 h at room temperature. The mixture was
filtered and the solid washed with petroleum ether and n-hexane
and purified by column chromatography (ethyl acetate:hexane) to
obtain the corresponding thiosemicarbazone in high yields
(75e99%). Characterization data for new thiosemicarbazone in-
termediates are also reported below.
4. Experimental protocols
4.1. General
Solvents were used as supplied without further purification.
Where mixtures of solvents are specified, the stated ratios are vol-
ume:volume. Reagents and standard drug (docetaxel) were used
directly as supplied by SigmaeAldrich® Italy. Column chromatog-
raphy was carried out using SigmaeAldrich® silica gel (high purity
grade, pore size 60 Å, 230e400 mesh particle size). Analytical thin-
4.2.2. 1-(Hexan-3-ylidene)thiosemicarbazide
White powder, mp 51e53 ꢁC, 90% yield; 1H NMR (400 MHz,

C. De Monte et al. / European Journal of Medicinal Chemistry 105 (2015) 245e262
249
DMSO-d6):
d
0.86e1.06 (m, 6H, 2 ꢂ CH3), 1.37e1.48 (m, 1H, CH2),
and 46 the mixture was poured on ice and extracted with chloro-
form (3 ꢂ 25 mL). The organics were reunited, dried over sodium
sulfate and concentrated in vacuo. The products were purified by
chromatography (ethyl acetate:petroleum ether) before character-
ization by spectroscopic methods.
1.49e1.56 (m, 1H, CH2), 2.15e2.30 (m, 2H, CH2), 2.34e2,40 (m, 2H,
CH2), 7.45 (bs, 1H, NH2, D2O exch.), 8.04 (bs, 1H, NH2, D2O exch.),
10.06 (bs, 1H, NH, D2O exch.); 13C NMR (101 MHz, DMSO-d6):
d
10.33 (CH3CH2C), 14.15 (CH3CH2CH2), 19.50 (CH3CH2CH2), 22.76
(CH3CH2C), 38.10 (CH3CH2CH2), 158.36 (C]N), 179.00 (C]S).
4.2.7. N-(4-Acetyl-5,5-dimethyl-4,5-dihydro-1,3,4-thiadiazol-2-yl)
acetamide (1)
4.2.3. 1-(1-(4-Acetylphenyl)ethylidene)thiosemicarbazide
Yellow powder, mp 221e225 ꢁC, 82% yield; 1H NMR (400 MHz,
White powder, mp 198e201 ꢁC, 62% yield; 1H NMR (400 MHz,
DMSO-d6):
d
2.32 (s, 3H, CH3), 2.60 (s, 3H, CH3), 7.94 (d, J ¼ 8.4 Hz,
CDCl3):
d
1.98 (s, 6H, 2 ꢂ CH3), 2.18 (s, 3H, CH3), 2.19 (s, 3H, CH3),
2H, benzene), 8.05 (bs, 1H, NH2, D2O exch.), 8.09 (d, J ¼ 8.4 Hz, 2H,
9.07 (bs, 1H, NH, D2O exch.); 13C NMR (101 MHz, CDCl3):
d
23.04
benzene), 8.39 (bs, 1H, NH2, D2O exch.), 10.35 (bs, 1H, NH, D2O
(NHCOCH3), 23.92 (NCOCH3), 28.82 (2 ꢂ CH3C), 76.81 (CH3CCH3),
144.33 (C]N), 169.77 (NC]O), 170.20 (NHC]O). Anal. Calcd for
C8H13N3O2S: C, 44.63; H, 6.09; N, 19.52. Found: C, 44.34; H, 6.28; N,
19.82.
exch.); 13C NMR (101 MHz, DMSO-d6):
d 14.42 (CH3C]N), 27.28
(CH3C]O), 127.23 (2 ꢂ CH-benzene), 128.52 (2 ꢂ CH-benzene),
137.24 (C-benzene), 142.33 (C-benzene), 146.97 (C]N), 179.62 (C]
S), 198.02 (C]O).
4.2.8. N-(4-Acetyl-5-ethyl-5-methyl-4,5-dihydro-1,3,4-thiadiazol-
2-yl)acetamide (2)
4.2.4. 1-(1-(2,5-Difluorophenyl)ethylidene)thiosemicarbazide
White powder, mp 156e160 ꢁC, 91% yield; 1H NMR (400 MHz,
White powder, mp 157e162 ꢁC, 60% yield; 1H NMR (400 MHz,
CDCl3):
d
2.31 (s, 3H, CH3), 6.42 (bs, 1H, NH2, D2O exch.), 7.06e7.09
CDCl3):
3H, CH3), 2.18 (s, 3H, CH3), 2.19 (s, 3H, CH3), 2.53e2.62 (m, 1H, CH2),
8.62 (bs, 1H, NH, D2O exch.); 13C NMR (101 MHz, CDCl3):
9.25
d
1.01 (t, J ¼ 7.2 Hz, 3H, CH3), 1.84e1.93 (m, 1H, CH2), 1.98 (s,
(m, 2H, benzene), 7.21e7.24 (m,1H, benzene), 7.31 (bs,1H, NH2, D2O
exch.), 8.79 (bs, 1H, NH, D2O exch.). 13C NMR (101 MHz, DMSO-d6):
d
d
17.71 (CH3C]N), 116.50 (CH-benzene), 117.65 (CH-benzene),
(CH3CH2), 23.02 (NHCOCH3), 23.85 (NCOCH3), 28.14 (CH3C), 32.54
(CH2), 81.97 (CH3CCH2), 144.32 (C]N), 169.70 (NC]O), 170.03
(NHC]O). Anal. Calcd for C9H15N3O2S: C, 47.14; H, 6.59; N, 18.33.
Found: C, 46.92; H, 6.38; N, 18.52.
118.08 (CH-benzene), 128.51 (C-benzene), 145.11 (C-benzene),
155.75 (C-benzene), 159.69 (C]N), 179.75 (C]S).
4.2.5. General synthesis of 1,3,4-thiadiazolines 1e103
The resulting thiosemicarbazone intermediates were converted
into 1,3,4-thiadiazolines by reaction with several symmetrical an-
hydrides (acetic, propionic, dichloroacetic, pentafluoropropionic)
used as the solvent. The oxidation of compound 31 with KMnO4 in
acetic acid, adding after 1 h H2O and H2O2 30%, gave the corre-
sponding thiadiazole 103 (Scheme 2). The products were purified
by chromatography (ethyl acetate:petroleum ether) before char-
acterization by spectroscopic methods.
In general, in the IR spectrum (neat) derivatives 1e103 showed a
band at 3212-3147 cmꢀ1 due to the stretching of the amide NeH, at
1707-1693 due to the stretching of C]O bond and at 1660e1600 for
the stretching of C]N.
4.2.9. N-(4-Acetyl-5-methyl-5-propyl-4,5-dihydro-1,3,4-thiadiazol-
2-yl)acetamide (3)
White powder, mp 140e141 ꢁC, 68% yield; 1H NMR (400 MHz,
CDCl3):
d
0.93 (t, J ¼ 7.2 Hz, 3H, CH3), 1.25e1.34 (m, 1H, CH2),
1.57e1.64 (m, 1H, CH2), 1.83e1.91 (m, 1H, CH2), 1.96 (s, 3H, CH3),
2.18 (s, 3H, CH3), 2.19 (s, 3H, CH3), 2.43e2.51 (m, 1H, CH2), 9.04 (bs,
1H, NH, D2O exch.); 13C NMR (101 MHz, CDCl3):
d 13.80 (CH3CH2),
18.33 (CH3CH2), 23.02 (CH3C), 23.89 (NHCOCH3), 28.29 (NCOCH3),
41.83 (CH2CH2C), 81.16 (CH3CCH2), 144.37 (C]N), 169.66 (NC]O),
170.03 (NHC]O). Anal. Calcd for C10H17N3O2S: C, 49.36; H, 7.04; N,
17.27. Found: C, 49.64; H, 7.29; N, 17.04.
4.2.10. N-(4-Acetyl-5,5-diethyl-4,5-dihydro-1,3,4-thiadiazol-2-yl)
acetamide (4)
4.2.6. General synthesis of compounds 1e54
The thiosemicarbazone intermediate (1.0 eq.) was added to a
stirring solution of acetic anhydride (5.0 eq.) and the reaction
mixture was stirred for 2e72 h at 90 ꢁC. The mixture was poured on
ice and the resulting suspension was filtered and washed with
water, petroleum ether and diethyl ether. As regards compounds 42
White powder, mp 141e146 ꢁC, 69% yield; 1H NMR (400 MHz,
CDCl3):
2.18 (s, 3H, CH3), 2.20 (s, 3H, CH3), 2.54e2.63 (m, 2H, CH2), 8.61 (bs,
1H, NH, D2O exch.); 13C NMR (101 MHz, CDCl3):
8.82 (2 ꢂ CH3CH2),
23.00 (2 ꢂ CH3CH2), 23.68 (NHCOCH3), 31.91 (NCOCH3), 87.13
d
1.02 (t, J ¼ 7.2 Hz, 6H, 2 ꢂ CH3), 1.78e1.87 (m, 2H, CH2),
d
Scheme 2. Synthesis of the thiadiazolines 1e102 and thiadiazole 103. Reagents and conditions: (i) EtOH, CH3COOH (cat.), rt.; (ii) reflux; (iii) CH3COOH, KMnO4, 0 ꢁC; (iv) H2O, H2O2,
rt.

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C. De Monte et al. / European Journal of Medicinal Chemistry 105 (2015) 245e262
(CH2CCH2), 144.38 (C]N), 169.72 (NC]O), 169.90 (NHC]O). Anal.
Calcd for C10H17N3O2S: C, 49.36; H, 7.04; N,17.27. Found: C, 49.14; H,
6.87; N, 17.55.
4.2.16. N-(4-Acetyl-5-methyl-5-pentyl-4,5-dihydro-1,3,4-
thiadiazol-2-yl)acetamide (10)
Light yellow powder, mp 98e103 ꢁC, 62% yield; 1H NMR
(400 MHz, CDCl3):
d
0.87 (t, J ¼ 6.6 Hz, 3H, CH3), 1.27e1.31 (m, 5H,
CH2), 1.56e1.58 (m, 1H, CH2), 1.87e1.94 (m, 1H, CH2), 1.96 (s, 3H,
CH3), 2.18 (s, 6H, 2 ꢂ CH3), 2.45e2.52 (m, 1H, CH2), 8.80 (bs, 1H, NH,
4.2.11. N-(4-Acetyl-5-isobutyl-5-methyl-4,5-dihydro-1,3,4-
thiadiazol-2-yl)acetamide (5)
D2O exch.); 13C NMR (101 MHz, CDCl3):
d 13.98 (CH2CH3), 22.50
White powder, mp 157e162 ꢁC, 65% yield; 1H NMR (400 MHz,
(CH3CCH2CH2), 23.04 (NHCOCH3), 23.87 (CH3CH2), 24.68 (NCOCH3),
28.31 (CH3C), 31.48 (CH3CH2CH2), 39.64 (CH3CCH2), 81.47
(CH3CCH2), 144.15 (C]N), 169.39 (NC]O), 169.94 (NHC]O). Anal.
Calcd for C12H21N3O2S: C, 53.11; H, 7.80; N, 15.48. Found: C, 52.89;
H, 7.99; N, 15.29.
CDCl3):
d
0.93 (d, J ¼ 6.8 Hz, 3H, CH3), 0.99 (d, J ¼ 6.8 Hz, 3H, CH3),
1.87e1.96 (m, 2H, CH2), 1.98 (s, 3H, CH3), 2.15 (s, 3H, CH3), 2.18 (s,
3H, CH3), 2.45e2.51 (m, 1H, CH), 8.31 (bs, 1H, NH, D2O exch.); 13C
NMR (101 MHz, CDCl3):
d 22.87 (CH3CHCH3), 23.05 (CH3C), 24.15
(NHCOCH3), 24.65 (CH3CH), 25.68 (CH3CH), 29.31 (NCOCH3), 47.06
(CH2), 80.92 (CH3CCH2), 144.57 (C]N), 169.69 (NC]O), 170.17
(NHC]O). Anal. Calcd for C11H19N3O2S: C, 51.34; H, 7.44; N, 16.33.
Found: C, 51.65; H, 7.20; N, 16.17.
4.2.17. N-(4-Acetyl-5-butyl-5-ethyl-4,5-dihydro-1,3,4-thiadiazol-2-
yl)acetamide (11)
White powder, mp 48e50 ꢁC, 44% yield; 1H NMR (400 MHz,
CDCl3):
d
0.90 (t, J ¼ 7.2 Hz, 3H, CH3), 1.01 (t, J ¼ 7.4 Hz, 3H, CH3),
4.2.12. N-(4-Acetyl-5-methyl-5-butyl-4,5-dihydro-1,3,4-thiadiazol-
2-yl)acetamide (6)
1.23e1.37 (m, 3H, CH2), 1.55e1.65 (m, 1H, CH2), 1.79e1.87 (m, 2H,
CH2), 2.18 (s, 3H, CH3), 2.19 (s, 3H, CH3), 2.48e2.60 (m, 2H, CH2),
White powder, mp 146e150 ꢁC, 63% yield; 1H NMR (400 MHz,
8.54 (bs, 1H, NH, D2O exch.); 13C NMR (101 MHz, CDCl3):
d 8.72
CDCl3): d 0.90e0.94 (m, 3H, CH3), 1.26e1.37 (m, 3H, CH2), 1.55e1.60
(CH3CH2C), 14.02 (CH3CH2CH2), 22.48 (CH3CH2CH2), 22.96
(NHCOCH3), 23.68 (NCOCH3), 26.65 (CH3CH2CH2CH2), 32.04
(CH3CH2C), 38.88 (CH3CH2CCH2), 86.35 (CH2CCH2), 144.43 (C]N),
169.73 (NC]O), 169.86 (NHC]O). Anal. Calcd for C12H21N3O2S: C,
53.11; H, 7.80; N, 15.48. Found: C, 53.38; H, 7.57; N, 15.64.
(m, 1H, CH2), 1.91e1.96 (m, 1H, CH2), 1.98 (s, 3H, 3H, CH3), 2.18 (s,
3H, CH3), 2.19 (s, 3H, CH3), 2.48e2.55 (m, 1H, CH2), 8.08 (bs, 1H, NH,
D2O exch.); 13C NMR (101 MHz, CDCl3):
d 13.99 (CH2CH3), 22.43
(CH3CH2), 23.01 (NHCOCH3), 23.88 (CH3CH2CH2), 27.13 (NCOCH3),
28.30 (CH3C), 39.44 (CH3CCH2), 81.33 (CH3CCH2), 144.34 (C]N),
169.95 (NC]O), 169.96 (NHC]O). Anal. Calcd for C11H19N3O2S: C,
51.34; H, 7.44; N, 16.33. Found: C, 51.17; H, 7.63; N, 16.69.
4.2.18. N-(4-Acetyl-5-hexyl-5-methyl-4,5-dihydro-1,3,4-thiadiazol-
2-yl)acetamide (12)
White powder, mp 57e58 ꢁC, 71% yield; 1H NMR (400 MHz,
4.2.13. N-(4-Acetyl-5-ethyl-5-propyl-4,5-dihydro-1,3,4-thiadiazol-
2-yl)acetamide (7)
CDCl3):
d
0.86 (t, J ¼ 7.2 Hz, 3H, CH3), 1.25e1.28 (m, 7H, CH2),
White powder, mp 134e136 ꢁC, 58% yield; 1H NMR (400 MHz,
1.55e1.57 (m, 1H, CH2), 1.87e1.90 (m, 1H, CH2), 1.95 (s, 3H, CH3),
2.18 (s, 3H, CH3), 2.20 (s, 3H, CH3), 2.47e2.51 (m, 1H, CH2), 9.51 (bs,
DMSO-d6):
d
0.87e0.92 (m, 6H, 2 ꢂ CH3), 1.16e1.20 (m, 1H, CH2),
1H, NH, D2O exch.); 13C NMR (101 MHz, CDCl3):
d 14.02 (CH3CH2),
1.52e1.56 (m, 1H, CH2), 1.70e1.77 (m, 2H, CH2), 2.02 (s, 3H, CH3),
2.11 (s, 3H, CH3), 2.37e2.47 (m, 2H, CH2), 11.40 (bs, 1H, NH, D2O
22.55 (CH3CCH2CH2), 22.97 (NHCOCH3), 23.86 (CH3CH2CH2), 24.95
(NCOCH3), 28.30 (CH3C), 28.97 (CH3CH2CH2CH2), 31.65
(CH3CH2CH2) 39.63 (CH3CCH2), 81.20 (CH3CCH2), 144.49 (C]N),
169.74 (NC]O), 169.99 (NHC]O). Anal. Calcd for C13H23N3O2S: C,
54.71; H, 8.12; N, 14.72. Found: C, 54.97; H, 7.85; N, 14.41.
exch.); 13C NMR (101 MHz, DMSO-d6):
d 8.91 (CH3CH2C), 14.07
(CH3CH2CH2), 17.89 (CH3CH2CH2), 22.92 (NHCOCH3), 23.96
(NCOCH3), 31.73 (CH3CH2C), 41.15 (CH3CH2CH2), 85.26 (CH2CCH2),
142.93 (C]N), 168.31 (NC]O), 169.50 (NHC]O). Anal. Calcd for
C
11H19N3O2S: C, 51.34; H, 7.44; N, 16.33. Found: C, 51.59; H, 7.21; N,
16.11.
4.2.19. N-(1-Acetyl-4-thia-1,2-diazaspiro[4.4]non-2-en-3-yl)
acetamide (13)
White powder, mp 178e183 ꢁC, 83% yield; 1H NMR (400 MHz,
4.2.14. N-(4-Acetyl-5-methyl-5-(2-methylprop-1-enyl)-4,5-
dihydro-1,3,4-thiadiazol-2-yl)acetamide (8)
CDCl3):
d 1.63e1.70 (m, 2H, cyclopentane), 1.92e1.96 (m, 2H,
White powder, mp 161e165 ꢁC, 65% yield; 1H NMR (400 MHz,
cyclopentane), 2.02e2.07 (m, 2H, cyclopentane), 2.18 (s, 3H, CH3),
2.19 (s, 3H, CH3), 2.92e2.99 (m, 2H, cyclopentane), 8.03 (bs, 1H, NH,
CDCl3):
3H, CH3), 2.19 (s, 3H, CH3), 5.61 (s, 1H, CH¼), 9.22 (bs, 1H, NH, D2O
exch.); 13C NMR (101 MHz, CDCl3):
23.34 (NHCOCH3), 26.69 (NCOCH3), 30.79 (CH3C), 76.14 (CH3CCH¼),
126.06 (CH¼), 137.98 (C¼), 145.21 (C]N), 168.51 (NC]O), 169.86
(NHC]O). Anal. Calcd for C11H17N3O2S: C, 51.74; H, 6.71; N, 16.46.
Found: C, 51.95; H, 6.50; N, 16.69.
d 1.69 (s, 3H, CH3), 1.74 (s, 3H, CH3), 1.98 (s, 3H, CH3), 2.17 (s,
D2O exch.); 13C NMR (101 MHz, CDCl3):
d
22.97 (2 ꢂ CH2-cyclo-
pentane), 24.13 (NHCOCH3), 24.81 (NCOCH3), 39.92 (2 ꢂ CH2-
cyclopentane), 85.14 (C-cyclopentane), 144.99 (C]N), 169.69 (NC]
O), 169.76 (NHC]O). Anal. Calcd for C10H15N3O2S: C, 49.77; H, 6.27;
N, 17.41. Found: C, 49.56; H, 6.04; N, 17.25.
d
18.59 (CH3C¼), 22.78 (CH3C¼),
4.2.20. N-(1-Acetyl-6-methyl-4-thia-1,2-diazaspiro[4.4]non-2-en-
3-yl)acetamide (14)
4.2.15. N-(4-Acetyl-5-(but-3-enyl)-5-methyl-4,5-dihydro-1,3,4-
thiadiazol-2-yl)acetamide (9)
White powder, mp 222e225 ꢁC, 73% yield; 1H NMR (400 MHz,
Light yellow powder, mp 159e163 ꢁC, 73% yield; 1H NMR
CDCl3):
d
1.04e1.05 (d, J ¼ 6.8 Hz, 3H, CH3), 1.16e1.21 (m, 1H,
(400 MHz, CDCl3):
d
1.96e1.99 (m, 1H, CH2), 2.02 (s, 3H, CH3),
cyclopentane), 1.63e1.70 (m, 1H, cyclopentane), 1.85e1.87 (m, 2H,
cyclopentane), 2.07e2.14 (m, 1H, cyclopentane), 2.17 (s, 3H, CH3),
2.20 (s, 3H, CH3), 3.18e3.29 (m, 2H, cyclopentane), 8.07 (bs, 1H, NH,
2.04e2.07 (m, 1H, CH2), 2.18 (s, 6H, 2 ꢂ CH3), 2.30e2.39 (m, 1H,
CH2), 2.61e2.68 (m, 1H, CH2), 4.95e5.06 (m, 2H, CH2¼), 5.75e5.85
(m, 1H, CH¼), 8.78 (bs, 1H, NH, D2O exch.); 13C NMR (101 MHz,
D2O exch.); 13C NMR (101 MHz, CDCl3):
d 14.03 (CH3CH-cyclo-
CDCl3):
d
23.01 (NHCOCH3), 23.86 (NCOCH3), 28.42 (CH2CH2CH¼),
pentane), 20.14 (CH2-cyclopentane), 23.13 (NHCOCH3), 24.48
(NCOCH3), 30.63 (CH2-cyclopentane), 37.64 (CH2-cyclopentane),
39.06 (CH-cyclopentane), 91.57 (C-cyclopentane), 144.35 (C]N),
169.28 (NC]O), 169.90 (NHC]O). Anal. Calcd for C11H17N3O2S: C,
51.74; H, 6.71; N, 16.46. Found: C, 51.96; H, 6.53; N, 16.65.
29.30 (CH3C), 38.65 (CCH2CH2), 80.67 (CH3CCH2), 115.22 (CH2¼),
136.99 (CH¼), 144.38 (C]N), 169.79 (NC]O), 170.11 (NHC]O).
Anal. Calcd for C11H17N3O2S: C, 51.74; H, 6.71; N, 16.46. Found: C,
51.59; H, 6.42; N, 16.22.

C. De Monte et al. / European Journal of Medicinal Chemistry 105 (2015) 245e262
251
4.2.21. N-(1-Acetyl-7-methyl-4-thia-1,2-diazaspiro[4.4]non-2-en-
3-yl)acetamide (15)
cyclohexane), 3.17e3.25 (m, 1H, cyclohexane), 7.96 (bs, 1H, NH, D2O
exch.); 13C NMR (101 MHz, CDCl3):
17.10 (CH3CH-cyclohexane),
d
White powder, mp 176e181 ꢁC, 78% yield; 1H NMR (400 MHz,
21.88 (NHCOCH3), 23.12 (NCOCH3), 24.59 (2 ꢂ CH2-cyclohexane),
33.12 (2 ꢂ CH2-cyclohexane), 35.70 (CH-cyclohexane), 84.65 (C-
cyclohexane), 144.59 (C]N), 169.64 (NC]O), 170.17 (NHC]O).
Anal. Calcd for C12H19N3O2S: C, 53.51; H, 7.11; N, 15.60. Found: C,
53.30; H, 7.42; N, 15.82.
CDCl3):
d
1.06e1.08 (d, J ¼ 6.4 Hz, 3H, CH3), 1.58e1.62 (m, 1H,
cyclopentane), 1.66e1.72 (m, 1H, cyclopentane), 1.82e1.84 (m, 1H,
cyclopentane), 2.01e2.06 (m, 1H, cyclopentane), 2.09e2.14 (m, 2H,
cyclopentane), 2.18 (s, 3H, CH3), 2.19 (s, 3H, CH3), 3.08e3.20 (m, 1H,
cyclopentane), 8.11 (bs, 1H, NH, D2O exch.); 13C NMR (101 MHz,
CDCl3):
d
19.39 (CH3CH-cyclopentane), 22.95 (NHCOCH3), 24.15
4.2.26. N-(1-Acetyl-7,7-dimethyl-4-thia-1,2-diazaspiro[4.5]dec-2-
en-3-yl)acetamide (20)
(NCOCH3), 34.05 (CH2-cyclopentane), 39.40 (CH-cyclopentane),
42.03 (CH2-cyclopentane), 48.44 (CH2-cyclopentane), 84.86 (C-
cyclopentane), 145.10 (C]N), 169.52 (NC]O), 169.72 (NHC]O).
Anal. Calcd for C11H17N3O2S: C, 51.74; H, 6.71; N, 16.46. Found: C,
51.47; H, 6.99; N, 16.28.
White powder, mp 180e185 ꢁC, 88% yield; 1H NMR (400 MHz,
CDCl3):
d 1.00 (s, 3H, CH3), 1.07 (s, 3H, CH3), 1.28e1.31 (m, 1H,
cyclohexane), 1.35e1.38 (m, 1H, cyclohexane), 1.65e1.67 (m, 2H,
cyclohexane), 1.91e1.95 (m, 1H, cyclohexane), 2.03e2.07 (m, 1H,
cyclohexane), 2.19 (s, 6H, 2 ꢂ CH3), 2.74e2.82 (m, 1H, cyclohexane),
3.22e3.25 (m, 1H, cyclohexane), 7.90 (bs, 1H, NH, D2O exch.); 13C
4.2.22. N-(1-Acetyl-4-thia-1,2-diazaspiro[4.5]dec-2-en-3-yl)-
acetamide (16)
NMR (101 MHz, CDCl3):
d 19.75 (CH2-cyclohexane), 23.13
White powder, mp 227e232 ꢁC, 84% yield; 1H NMR (400 MHz,
(NHCOCH3), 25.18 (NCOCH3), 26.34 (CH3CCH3), 32.02 (CH3C-cyclo-
hexane), 33.98 (CH3C-cyclohexane), 35.79 (CH2-cyclohexane), 37.78
(CH2-cyclohexane), 46.33 (CH2-cyclohexane), 82.96 (C-cyclo-
hexane), 145.43 (C]N), 169.63 (NC]O), 170.46 (NHC]O). Anal.
Calcd for C13H21N3O2S: C, 55.10; H, 7.47; N, 14.83. Found: C, 54.84;
H, 7.28; N, 15.09.
CDCl3):
d 1.25e1.32 (m, 1H, cyclohexane), 1.40e1.49 (m, 2H, cyclo-
hexane), 1.62e1.67 (m, 1H, cyclohexane), 1.82e1.85 (m, 2H, cyclo-
hexane), 2.04e2.07 (m, 2H, cyclohexane), 2.17 (s, 6H, 2 ꢂ CH3),
2.93e2.98 (m, 2H, cyclohexane), 8.27 (bs, 1H, NH, D2O exch.); 13C
NMR (101 MHz, CDCl3):
d
23.20 (2 ꢂ CH2-cyclohexane), 24.56
(NHCOCH3), 24.63 (NCOCH3), 24.76 (CH2-cyclohexane), 36.00
(2 ꢂ CH2-cyclohexane), 85.21 (C-cyclohexane), 144.15 (C]N),
169.20 (NC]O), 170.07 (NHC]O). Anal. Calcd for C11H17N3O2S: C,
51.74; H, 6.71; N, 16.46. Found: C, 51.52; H, 6.55; N, 16.19.
4.2.27. N-(1-Acetyl-6-tert-butyl-4-thia-1,2-diazaspiro[4.5]dec-2-
en-3-yl)acetamide (21)
White powder, mp 158e163 ꢁC, 75% yield; 1H NMR (400 MHz,
CDCl3):
d
1.02 (s, 9H, 3 ꢂ CH3), 1.28e1.38 (m, 1H, cyclohexane),
4.2.23. N-(1-Acetyl-6-methyl-4-thia-1,2-diazaspiro[4.5]dec-2-en-
3-yl)acetamide (17)
1.51e1.59 (m, 2H, cyclohexane), 1.71e1.74 (m, 2H, cyclohexane),
1.95e1.99 (m, 2H, cyclohexane), 2.11 (s, 3H, CH3), 2.19 (s, 3H, CH3),
2.90e2.98 (m, 2H, cyclohexane), 8.02 (bs, 1H, NH, D2O exch.); 13C
White powder, mp 227e230 ꢁC, 92% yield; 1H NMR (400 MHz,
CDCl3):
d
0.93e0.95 (d, J ¼ 6.8 Hz, 3H, CH3), 1.40e1.49 (m, 2H,
NMR (101 MHz, CDCl3): d 23.23 (CH2-cyclohexane), 23.99 (CH2-
cyclohexane), 1.63e1.71 (m, 2H, cyclohexane), 1.79e1.82 (m, 1H,
cyclohexane), 2.11e2.15 (m, 2H, cyclohexane), 2.17 (s, 6H, 2 ꢂ CH3),
2.90e2.94 (m, 1H, cyclohexane), 2.97e3.04 (m, 1H, cyclohexane),
cyclohexane), 25.08 (NHCOCH3), 26.15 (NCOCH3), 29.03 (3 ꢂ CH3C),
29.40 ((CH3)3C), 34.89 (CH2-cyclohexane), 40.73 (CH2-cyclo-
hexane), 48.68 (CH-cyclohexane), 87.70 (C-cyclohexane), 144.40
(C]N), 169.54 (NC]O), 170.39 (NHC]O). Anal. Calcd for
8.18 (bs, 1H, NH, D2O exch.); 13C NMR (101 MHz, CDCl3):
d 15.82
(CH3CH-cyclohexane), 16.56 (CH2-cyclohexane), 23.32 (NHCOCH3),
24.64 (NCOCH3), 25.13 (CH2-cyclohexane), 29.50 (CH2-cyclo-
hexane), 32.78 (CH2-cyclohexane), 36.86 (CH-cyclohexane), 90.87
(C-cyclohexane), 143.31 (C]N), 169.59 (NC]O), 170.85 (NHC]O).
Anal. Calcd for C12H19N3O2S: C, 53.51; H, 7.11; N, 15.60. Found: C,
53.83; H, 6.89; N, 15.91.
C15H25N3O2S: C, 57.85; H, 8.09; N, 13.49. Found: C, 58.03; H, 7.80; N,
13.25.
4.2.28. N-(1-Acetyl-4-thia-1,2-diazaspiro[4.6]undec-2-en-3-yl)-
acetamide (22)
White powder, mp 220e221 ꢁC, 81% yield; 1H NMR (400 MHz,
CDCl3):
d 1.55e1.58 (m, 6H, cycloheptane), 1.82e1.84 (m, 2H,
4.2.24. N-(1-Acetyl-7-methyl-4-thia-1,2-diazaspiro[4.5]dec-2-en-
3-yl)acetamide (18)
cycloheptane), 2.13e2.15 (m, 2H, cycloheptane), 2.17 (s, 3H, CH3),
2.19 (s, 3H, CH3), 2.84e2.90 (m, 2H, cycloheptane), 8.91 (bs, 1H, NH,
White powder, mp 201e206 ꢁC, 75% yield; 1H NMR (400 MHz,
D2O exch.); 13C NMR (101 MHz, CDCl3):
d
23.12 (2 ꢂ CH2-cyclo-
CDCl3):
d
0.93e0.95 (d, J ¼ 6.8 Hz, 3H, CH3), 1.42e1.49 (m, 1H,
heptane), 23.84 (NHCOCH2CH3), 24.28 (NCOCH2CH3), 27.53
(2 ꢂ CH2-cycloheptane), 40.00 (2 ꢂ CH2-cycloheptane), 87.40 (C-
cycloheptane), 144.39 (C]N), 169.60 (NC]O, NHC]O). Anal. Calcd
for C12H19N3O2S: C, 53.51; H, 7.11; N, 15.60. Found: C, 53.77; H, 7.38;
N, 15.32.
cyclohexane), 1.53e1.66 (m, 3H, cyclohexane), 1.80e1.84 (m, 1H,
cyclohexane), 1.93e2.11 (m, 2H, cyclohexane), 2.18 (s, 6H, 2 ꢂ CH3),
2.62e2.68 (m, 1H, cyclohexane), 2.80e2.90 (m, 1H, cyclohexane),
8.67 (bs, 1H, NH, D2O exch.); 13C NMR (101 MHz, CDCl3):
d 22.17
(CH2-cyclohexane), 23.02 (CH3CH-cyclohexane), 24.11 (NHCOCH3),
24.61 (NCOCH3), 31.54 (CH-cyclohexane), 33.32 (CH2-cyclohexane),
35.29 (CH2-cyclohexane), 44.05 (CH2-cyclohexane), 84.53 (C-
cyclohexane), 144.69 (C]N), 169.76 (NC]O), 170.16 (NHC]O).
Anal. Calcd for C12H19N3O2S: C, 53.51; H, 7.11; N, 15.60. Found: C,
53.24; H, 7.39; N, 15.37.
4.2.29. N-(1-Acetyl-4-thia-1,2-diazaspiro[4.7]dodec-2-en-3-yl)
acetamide (23)
White powder, mp 198e204 ꢁC, 85% yield; 1H NMR (400 MHz,
DMSO-d6):
d 1.44e1.55 (m, 6H, cyclooctane), 1.61e1.68 (m, 1H,
cyclooctane), 1.78e1.82 (m, 2H, cyclooctane), 2.02 (s, 3H, CH3),
2.05e2.06 (m, 1H, cyclooctane), 2.09 (s, 3H, CH3), 2.32e2.34 (m, 1H,
cyclooctane), 2.66e2.68 (m, 1H, cyclooctane), 2.86e2.92 (m, 2H,
cyclooctane), 11.43 (bs, 1H, NH, D2O exch.); 13C NMR (101 MHz,
4.2.25. N-(1-Acetyl-8-methyl-4-thia-1,2-diazaspiro[4.5]dec-2-en-
3-yl)acetamide (19)
White powder, mp 205e207 ꢁC, 87% yield; 1H NMR (400 MHz,
DMSO-d6):
d
23.05 (NHCOCH3), 23.37 (NCOCH3), 23.88 (2 ꢂ CH2-
CDCl3):
d
0.90e0.92 (d, J ¼ 6.8 Hz, 3H, CH3), 1.14e1.17 (m, 1H,
cyclooctane), 24.73 (CH2-cyclooctane), 27.55 (2 ꢂ CH2-cyclooctane),
36.88 (2 ꢂ CH2-cyclooctane), 86.75 (C-cyclooctane), 143.05 (C]N),
168.57 (NC]O), 169.58 (NHC]O). Anal. Calcd for C13H21N3O2S: C,
55.10; H, 7.47; N, 14.83. Found: C, 55.42; H, 7.69; N, 14.56.
cyclohexane), 1.58e1.63 (m, 2H, cyclohexane), 1.68e1.71 (m, 1H,
cyclohexane), 1.77e1.87 (m, 2H, cyclohexane), 2.02e2.05 (m, 1H,
cyclohexane), 2.17 (s, 3H, CH3), 2.18 (s, 3H, CH3), 2.98e3.06 (m, 1H,

252
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4.2.30. N-(4-Acetyl-5-cyclohexyl-5-methyl-4,5-dihydro-1,3,4-
thiadiazol-2-yl)acetamide (24)
4.2.35. N-[4-Acetyl-5-(5-chlorothiophen-2-yl)-5-methyl-4,5-
dihydro-1,3,4-thiadiazol-2-yl]acetamide (29)
Light brown powder, mp 206e210 ꢁC, 93% yield; 1H NMR
Brown powder, mp 152e155 ꢁC, 57% yield; 1H NMR (400 MHz,
(400 MHz, DMSO-d6):
d
0.85e0.94 (m, 1H, cyclohexane), 1.02e1.19
DMSO-d6):
6.96e6.98 (m, 2H, thiophene), 11.72 (bs, 1H, NH, D2O exch.); 13C
NMR (101 MHz, DMSO-d6): 22.85 (NHCOCH3), 24.17 (NCOCH3),
27.30 (CH3C), 76.13 (CH3C), 124.74 (C3H-thiophene), 126.85 (C5-
thiophene), 128.85 (C4H-thiophene), 142.57 (C2-thiophene), 147.88
(C]N), 168.52 (NC]O), 170.02 (NHC]O). Anal. Calcd for
d 2.05 (s, 3H, CH3), 2.15 (s, 3H, CH3), 2.27 (s, 3H, CH3),
(m, 4H, cyclohexane), 1.41e1.43 (m, 1H, cyclohexane), 1.58e1.61 (m,
1H, cyclohexane), 1.71e1.74 (m, 3H, cyclohexane), 1.85 (s, 3H, CH3),
1.88e1.90 (m, 1H, cyclohexane), 2.00 (s, 3H, CH3), 2.08 (s, 3H, CH3),
d
11.40 (bs, 1H, NH, D2O exch.); 13C NMR (101 MHz, CDCl3):
d 23.15
(NHCOCH3), 24.02 (NCOCH3), 25.69 (CH2-cyclohexane), 25.88
(CH2e (CH2-cyclohexane)), 26.15 (CH2-cyclohexane), 27.04 (CH2-
cyclohexane), 27.35 (CH3C), 28.32 (CH2-cyclohexane), 44.20 (CH-
cyclohexane), 86.38 (CH3C), 144.03 (C]N), 169.18 (NC]O), 169.63
(NHC]O). Anal. Calcd for C13H21N3O2S: C, 55.10; H, 7.47; N, 14.83.
Found: C, 54.91; H, 7.22; N, 14.60.
C11H12ClN3O2S2: C, 41.57; H, 3.81; N, 13.22. Found: C, 41.89; H, 4.02;
N, 13.04.
4.2.36. N-[4-Acetyl-5-methyl-5-(thiophen-3-yl)-4,5-dihydro-1,3,4-
thiadiazol-2-yl]acetamide (30)
White powder, mp 222e225 ꢁC, 86% yield; 1H NMR (400 MHz,
DMSO-d6):
d 2.03 (s, 3H, CH3), 2.16 (s, 3H, CH3), 2.26 (s, 3H, CH3),
4.2.31. N-[4-Acetyl-5-(furan-2-yl)-4,5-dihydro-1,3,4-thiadiazol-2-
yl]acetamide (25)
7.06e7.07 (m, 1H, thiophene), 7.38e7.39 (m, 1H, thiophene),
7.48e7.50 (m, 1H, thiophene), 11.61 (bs, 1H, NH, D2O exch.); 13C
Brown powder, mp 177e180 ꢁC, 44% yield; 1H NMR (400 MHz,
NMR (101 MHz, DMSO-d6):
d 22.93 (NHCOCH3), 24.24 (NCOCH3),
DMSO-d6):
d 2.04 (s, 3H, CH3), 2.14 (s, 3H, CH3), 6.30e6.31 (d,
27.55 (CH3C), 76.36 (CH3C), 121.16 (C2H-thiophene), 126.31 (C5H-
thiophene), 127.25 (C4H-thiophene), 142.83 (C3-thiophene), 145.42
(C]N), 168.31 (NC]O), 169.84 (NHC]O). Anal. Calcd for
J ¼ 4.0 Hz, 1H, furan), 6.38e6.39 (m, 1H, furan), 6.89 (s, 1H, thia-
diazoline), 7.59 (bs, 1H, furan), 11.75 (bs, 1H, NH, D2O exch.); 13C
NMR (101 MHz, DMSO-d6):
d 22.31 (NCOCH3), 22.98 (NHCOCH3),
C11H13N3O2S2: C, 46.62; H, 4.62; N, 14.83. Found: C, 46.45; H, 4.92;
59.75 (CH), 107.65 (C3H-furan), 111.11 (C4H-furan), 143.61 (C5H-
furan), 146.32 (C]N), 151.95 (C2-furan), 167.84 (NC]O), 170.01
(NHC]O). Anal. Calcd for C10H11N3O3S: C, 47.42; H, 4.38; N, 16.59.
Found: C, 47.64; H, 4.19; N, 16.83.
N, 14.99.
4.2.37. N-(4-Acetyl-4,5-dihydro-5-phenyl-1,3,4-thiadiazol-2-yl)
acetamide (31)
White powder, mp 221e228 ꢁC, 84% yield; 1H NMR (400 MHz,
4.2.32. N-[4-Acetyl-5-(furan-2-yl)-5-methyl-4,5-dihydro-1,3,4-
thiadiazol-2-yl]acetamide (26)
DMSO-d6):
d 2.04 (s, 3H, CH3), 2.21 (s, 3H, CH3), 6.83 (s, 1H, thia-
diazoline), 7.24e7.26 (m, 2H, benzene), 7.28e7.31 (m, 1H, benzene),
Light brown powder, mp 159e163 ꢁC, 71% yield; 1H NMR
7.34e7.38 (m, 2H, benzene), 11.76 (bs, 1H, NH, D2O exch.); 13C NMR
(400 MHz, DMSO-d6):
3H, CH3), 6.37e6.39 (m, 2H, furan), 7.59 (s, 1H, furan), 11.63 (bs, 1H,
NH, D2O exch.); 13C NMR (101 MHz, DMSO-d6):
22.87 (NCOCH3),
d 2.02 (s, 3H, CH3), 2.12 (s, 3H, CH3), 2.17 (s,
(101 MHz, DMSO-d6):
d 22.36 (NHCOCH3), 23.02 (NCOCH3), 66.35
(CH), 125.56 (2 ꢂ CH-benzene), 128.52 (CH-benzene), 129.16
(2 ꢂ CH-benzene), 141.87 (C-benzene), 146.50 (C]N), 167.89 (NC]
O), 169.90 (NHC]O). Anal. Calcd for C12H13N3O2S: C, 54.74; H, 4.98;
N, 15.96. Found: C, 54.91; H, 5.15; N, 16.17.
d
23.99 (NHCOCH3), 26.00 (CH3C), 73.58 (CH3C), 106.87 (C3H-furan),
111.14 (C4H-furan), 142.27 (C5H-furan), 143.22 (C]N), 153.98 (C2-
furan), 168.17 (NC]O), 169.93 (NHC]O). Anal. Calcd for
C
11H13N3O3S: C, 49.43; H, 4.90; N, 15.72. Found: C, 49.67; H, 5.12; N,
4.2.38. N-(4-Acetyl-5-ethyl-4,5-dihydro-5-phenyl-1,3,4-thiadiazol-
2-yl)acetamide (33)
15.50.
White powder, mp 216e218 ꢁC, 37% yield; 1H NMR (400 MHz,
4.2.33. N-[4-Acetyl-5-(thiophen-2-yl)-4,5-dihydro-1,3,4-
thiadiazol-2-yl]acetamide (27)
DMSO-d6):
d
1.03 (t, J ¼ 7.0 Hz, 3H, CH3), 2.01 (s, 3H, CH3), 2.22 (s,
3H, CH3), 2.39e2.41 (m, 1H, CH2), 2.81e2.88 (m,1H, CH2), 7.26e7.38
Light brown powder, mp 197e200 ꢁC, 72% yield; 1H NMR
(m, 5H, benzene), 11.62 (bs, 1H, NH, D2O exch.); 13C NMR (101 MHz,
(400 MHz, DMSO-d6):
d 2.05 (s, 3H, CH3), 2.14 (s, 3H, CH3),
DMSO-d6):
d 9.24 (CH3CH2), 22.92 (NHCOCH3), 23.97 (NCOCH3),
6.92e6.95 (m, 1H, thiophene), 7.05e7.06 (m, 1H, thiophene), 7.11 (s,
1H, thiadiazoline), 7.43e7.46 (m, 1H, thiophene), 11.79 (bs, 1H, NH,
29.41 (CH3CH2), 83.99 (CH3CH2C), 124.93 (2 ꢂ CH-benzene), 127.78
(CH-benzene), 128.97 (2 ꢂ CH-benzene), 142.72 (C-benzene),
145.03 (C]N), 168.15 (NC]O), 169.84 (NHC]O). Anal. Calcd for
D2O exch.); 13C NMR (101 MHz, DMSO-d6):
d 22.27 (NHCOCH3),
23.03 (NCOCH3), 61.96 (CH), 125.77 (C5H-thiophene), 126.68 (C3H-
thiophene), 127.29 (C4H-thiophene), 145.06 (C2-thiophene), 146.67
(C]N), 167.73 (NC]O), 169.98 (NHC]O). Anal. Calcd for
C14H17N3O2S: C, 57.71; H, 5.88; N, 14.42. Found: C, 57.98; H, 6.07; N,
14.21.
C
10H11N3O2S2: C, 44.59; H, 4.12; N, 15.60. Found: C, 44.24; H, 3.90;
4.2.39. N-(4-Acetyl-4,5-dihydro-5,5-diphenyl-1,3,4-thiadiazol-2-
yl)acetamide (34)
N, 15.83.
Yellow powder, mp 196e198 ꢁC, 43% yield; 1H NMR (400 MHz,
4.2.34. N-[4-Acetyl-5-methyl-5-(thiophen-2-yl)-4,5-dihydro-1,3,4-
thiadiazol-2-yl]acetamide (28)
DMSO-d6):
benzene), 11.76 (bs, 1H, NH, D2O exch.); 13C NMR (101 MHz, DMSO-
d6):
benzene), 128.28 (4 ꢂ CH-benzene), 128.46 (4 ꢂ CH-benzene),
140.91 (C]N), 143.19 (2 ꢂ C-benzene), 168.04 (NC]O), 170.08
(NHC]O). Anal. Calcd for C18H17N3O2S: C, 63.70; H, 5.05; N, 12.38.
Found: C, 63.97; H, 4.84; N, 12.18.
d 2.03 (s, 3H, CH3), 2.20 (s, 3H, CH3), 7.32e7.39 (m, 10H,
White powder, mp 199e203 ꢁC, 89% yield; 1H NMR (400 MHz,
d
22.87 (NHCOCH3), 24.30 (NCOCH3), 85.69 (C), 128.10 (2 ꢂ CH-
DMSO-d6):
d
2.02 (s, 3H, CH3), 2.13 (s, 3H, CH3), 2.31 (s, 3H, CH3),
6.91e6.93 (m, 1H, thiophene), 7.04e7.05 (m, 1H, thiophene),
7.45e7.47 (m, 1H, thiophene), 11.67 (bs, 1H, NH, D2O exch.); 13C
NMR (101 MHz, DMSO-d6):
d 22.90 (NHCOCH3), 24.29 (NCOCH3),
28.02 (CH3C), 76.43 (CH3C), 124.76 (C5H-thiophene), 126.50 (C3H-
thiophene), 127.19 (C4H-thiophene), 142.50 (C2-thiophene), 149.15
(C]N), 168.41 (NC]O), 169.96 (NHC]O). Anal. Calcd for
4.2.40. N-[4-Acetyl-4,5-dihydro-5-methyl-5-(2-nitrophenyl)-1,3,4-
thiadiazol-2-yl]acetamide (35)
C
11H13N3O2S2: C, 46.62; H, 4.62; N, 14.83. Found: C, 46.94; H, 4.45;
Yellow powder, mp 199e201 ꢁC, 35% yield; 1H NMR (400 MHz,
N, 14.61.
DMSO-d6):
d 2.05 (s, 3H, CH3), 2.09 (s, 3H, CH3), 2.20 (s, 3H, CH3),

C. De Monte et al. / European Journal of Medicinal Chemistry 105 (2015) 245e262
253
7.55e7.72 (m, 4H, benzene), 11.75 (bs, 1H, NH, D2O exch.); 13C NMR
(101 MHz, DMSO-d6): 22.86 (NHCOCH3), 23.48 (NCOCH3), 28.97
(CH3C), 75.76 (CH3C), 125.07 (CH-benzene), 127.55 (CH-benzene),
129.63 (CH-benzene), 132.62 (CH-benzene), 134.64 (C-benzene),
143.81 (C]N), 148.33 (C-benzene), 168.18 (NC]O), 170.07 (NHC]
O). Anal. Calcd for C13H14N4O4S: C, 48.44; H, 4.38; N,17.38. Found: C,
48.21; H, 4.55; N, 17.60.
(NCOCH3), 61.05 (CH), 113.81 (CH-benzene), 116.86 (CH-benzene),
118.11 (CH-benzene), 130.38 (C-benzene), 146.15 (C]N), 155.18 (d,
J ¼ 243.61 Hz, C-benzene), 158.65 (d, J ¼ 243.51 Hz, C-benzene),
168.21 (NC]O), 170.09 (NHC]O). Anal. Calcd for C12H11F2N3O2S: C,
48.16; H, 3.70; N, 14.04. Found: C, 47.85; H, 3.98; N, 14.21.
d
4.2.46. N-[4-Acetyl-5-(2,5-difluorophenyl)-4,5-dihydro-5-methyl-
1,3,4-thiadiazol-2-yl]acetamide (41)
4.2.41. N-[4-Acetyl-4,5-dihydro-5-methyl-5-(3-nitrophenyl)-1,3,4-
thiadiazol-2-yl]acetamide (36)
White powder, mp 191e194 ꢁC, 91% yield; 1H NMR (400 MHz,
CDCl3):
6.95e7.05 (m, 3H, benzene), 9.03 (bs, 1H, NH, D2O exch.); 13C NMR
(101 MHz, DMSO-d6): 22.76 (NHCOCH3), 23.64 (NCOCH3), 27.73
d 2.07 (s, 3H, CH3), 2.28 (s, 3H, CH3), 2.38 (s, 3H, CH3),
Yellow powder, mp 232e233 ꢁC, 61% yield; 1H NMR (400 MHz,
DMSO-d6):
d
2.04 (s, 3H, CH3), 2.21 (s, 3H, CH3), 2.34 (s, 3H, CH3),
d
7.67e7.71 (m, 1H, benzene), 7.87e7.89 (m, 1H, benzene), 8.12e8.17
(CH3C), 75.24 (CH3C), 114.12 (CH-benzene), 116.47 (CH-benzene),
118.46 (CH-benzene), 132.16 (C-benzene), 143.20 (C]N), 155.94 (d,
J ¼ 247.35 Hz, C-benzene), 158.19 (d, J ¼ 205.23 Hz, C-benzene),
168.05 (NC]O), 170.02 (NHC]O). Anal. Calcd for C13H13F2N3O2S: C,
49.83; H, 4.18; N, 13.41. Found: C, 50.02; H, 4.42; N, 13.19.
(m, 2H, benzene), 11.77 (bs, 1H, NH, D2O exch.); 13C NMR (101 MHz,
DMSO-d6):
d 22.87 (NHCOCH3), 23.98 (NCOCH3), 26.64 (CH3C),
78.12 (CH3C), 119.74 (CH-benzene), 122.92 (CH-benzene), 130.67
(CH-benzene), 132.05 (CH-benzene), 142.91 (C]N), 146.31 (C-
benzene), 148.30 (C-benzene), 168.37 (NC]O), 170.07 (NHC]O).
Anal. Calcd for C13H14N4O4S: C, 48.44; H, 4.38; N, 17.38. Found: C,
48.69; H, 4.12; N, 17.10.
4.2.47. N-[4-Acetyl-5-(pyridin-2-yl)-4,5-dihydro-1,3,4-thiadiazol-
2-yl]acetamide (42)
Light brown powder, mp 205e211 ꢁC, 40% yield; 1H NMR
4.2.42. N-[4-Acetyl-4,5-dihydro-5-methyl-5-(4-nitrophenyl)-1,3,4-
thiadiazol-2-yl]acetamide (37)
(400 MHz, DMSO-d6): d 2.03 (s, 3H, CH3), 2.22 (s, 3H, CH3), 6.79 (s,
1H, thiadiazoline), 7.26e7.33 (m, 2H, pyridine), 7.78e7.81 (m, 1H,
pyridine), 8.52e8.53 (m, 1H, pyridine), 11.75 (bs, 1H, NH, D2O
Yellow powder, mp 221e226 ꢁC, 77% yield; 1H NMR (400 MHz,
DMSO-d6):
d
1.83 (s, 3H, CH3), 2.00 (s, 3H, CH3), 2.10 (s, 3H, CH3),
exch.); 13C NMR (101 MHz, DMSO-d6):
d 22.34 (NHCOCH3), 22.96
7.45 (d, J ¼ 8.8 Hz, 2H, benzene), 8.01 (d, J ¼ 8.8 Hz, 2H, benzene),
(NCOCH3), 67.42 (CH), 120.04 (C5H-pyridine), 123.69 (C3H-pyri-
dine), 137.75 (C4H-pyridine), 145.69 (C]N), 150.07 (C6H-pyridine),
159.10 (C2-pyridine), 168.09 (NC]O), 169.93 (NHC]O). Anal. Calcd
for C11H12N4O2S: C, 49.99; H, 4.58; N, 21.20. Found: C, 49.72; H,
4.24; N, 21.45.
11.58 (bs, 1H, NH, D2O exch.); 13C NMR (101 MHz, DMSO-d6):
d
22.79 (NHCOCH3), 23.87 (NCOCH3), 26.67 (CH3C), 78.12 (CH3C),
124.08 (2 ꢂ CH-benzene), 126.56 (2 ꢂ CH-benzene), 142.79 (C]N),
146.93 (C-benzene), 151.11 (C-benzene), 168.31 (NC]O), 170.04
(NHC]O). Anal. Calcd for C13H14N4O4S: C, 48.44; H, 4.38; N, 17.38.
Found: C, 48.72; H, 4.09; N, 17.04.
4.2.48. N-[4-Acetyl-5-methyl-5-(pyridin-2-yl)-4,5-dihydro-1,3,4-
thiadiazol-2-yl]acetamide (43)
4.2.43. N-[4-Acetyl-5-(4-acetylphenyl)-5-methyl-4,5-dihydro-
1,3,4-thiadiazol-2-yl]acetamide (38)
White powder, mp 185e188 ꢁC, 76% yield; 1H NMR (400 MHz,
DMSO-d6):
d 2.00 (s, 3H, CH3), 2.18 (s, 3H, CH3), 2.26 (s, 3H, CH3),
Light orange powder, mp 224e226 ꢁC, 60% yield; 1H NMR
7.25e7.28 (m, 1H, pyridine), 7.35e7.37 (d, J ¼ 8.0 Hz, 1H, pyridine),
(400 MHz, DMSO-d6):
d 2.02 (s, 3H, CH3), 2.20 (s, 3H, CH3), 2.30 (s,
7.75e7.80 (m, 1H, pyridine), 8.50e8.51 (m, 1H, pyridine), 11.64 (bs,
3H, CH3), 2.58 (s, 3H, CH3), 7.50 (d, J ¼ 7.2 Hz, 2H, benzene), 7.93 (d,
1H, NH, D2O exch.); 13C NMR (101 MHz, DMSO-d6):
d 22.87
J ¼ 7.2 Hz, 2H, benzene), 11.72 (bs, 1H, NH, D2O exch.); 13C NMR
(NHCOCH3), 23.99 (NCOCH3), 26.39 (CH3C), 80.07 (CH3C), 119.56
(C5H-pyridine), 122.90 (C3H-pyridine), 137.55 (C4H-pyridine),
142.46 (C]N), 149.12 (C6H-pyridine), 161.30 (C2-pyridine), 168.50
(NC]O), 169.88 (NHC]O). Anal. Calcd for C12H14N4O2S: C, 51.78; H,
5.07; N, 20.13. Found: C, 51.94; H, 4.82; N, 20.39.
(101 MHz, DMSO-d6):
d 22.85 (NHCOCH3), 24.02 (NCOCH3), 26.82
(COCH3), 27.10 (CH3C), 78.57 (CH3C), 125.34 (2 ꢂ CH-benzene),
128.94 (2 ꢂ CH-benzene), 136.20 (C-benzene), 142.73 (C]N),
148.90 (C-benzene), 168.27 (NC]O), 169.94 (NHC]O), 197.65
(COCH3). Anal. Calcd for C15H17N3O3S: C, 56.41; H, 5.37; N, 13.16.
Found: C, 56.24; H, 5.11; N, 12.89.
4.2.49. N-[4-Acetyl-5-(pyridin-3-yl)-4,5-dihydro-1,3,4-thiadiazol-
2-yl]acetamide (44)
4.2.44. 4-(5-Acetamido-3-acetyl-2-methyl-2,3-dihydro-1,3,4-
thiadiazol-2-yl)phenyl acetate (39)
White powder, mp 180e185 ꢁC, 61% yield; 1H NMR (400 MHz,
CDCl3):
d 2.19 (s, 3H, CH3), 2.22 (s, 3H, CH3), 6.85 (s, 1H, thiadia-
White powder, mp 180e185 ꢁC, 46% yield; 1H NMR (400 MHz,
zoline), 7.32e7.36 (m, 1H, pyridine), 7.69e7.71 (d, J ¼ 8.0 Hz, 1H,
CDCl3):
d 2.08 (s, 3H, CH3), 2.27 (s, 3H, CH3), 2.30 (s, 3H, CH3), 2.39
pyridine), 8.56e8.57 (m, 1H, pyridine), 8.64 (s, 1H, pyridine), 10.37
(s, 3H, CH3), 7.08 (d, J ¼ 8.4 Hz, 2H, benzene), 7.45 (d, J ¼ 8.4 Hz, 2H,
(bs, 1H, NH, D2O exch.); 13C NMR (101 MHz, DMSO-d6):
d 22.28
benzene), 8.65 (bs, 1H, NH, D2O exch.); 13C NMR (101 MHz, CDCl3):
(NHCOCH3), 23.00 (NCOCH3), 64.32 (CH), 124.31 (C5H-pyridine),
133.61 (C3-pyridine), 137.26 (C4H-pyridine), 146.41 (C]N), 147.32
(C6H-pyridine), 149.74 (C2H-pyridine), 168.04 (NC]O), 170.02
(NHC]O). Anal. Calcd for C11H12N4O2S: C, 49.99; H, 4.58; N, 21.20.
Found: C, 50.25; H, 4.80; N, 21.02.
d
21.16 (OCOCH3), 22.48 (NHCOCH3), 23.80 (NCOCH3), 26.74 (CH3C),
84.87 (CH3C), 121.51 (2 ꢂ CH-benzene), 126.41 (2 ꢂ CH-benzene),
140.28 (C-benzene), 144.52 (C]N), 150.06 (C-benzene), 169.43
(NC]O), 169.54 (OCOCH3), 170.23 (NHC]O). Anal. Calcd for
C
15H17N3O4S: C, 53.72; H, 5.11; N, 12.53. Found: C, 53.44; H, 4.85; N,
12.20.
4.2.50. N-[4-Acetyl-5-methyl-5-(pyridin-3-yl)-4,5-dihydro-1,3,4-
thiadiazol-2-yl]acetamide (45)
4.2.45. N-[4-Acetyl-5-(2,5-difluorophenyl)-4,5-dihydro-1,3,4-
thiadiazol-2-yl]acetamide (40)
White powder, mp 228e231 ꢁC, 37% yield; 1H NMR (400 MHz,
CDCl3):
d 2.08 (s, 3H, CH3), 2.24 (s, 3H, CH3), 2.40 (s, 3H, CH3),
White powder, mp 190e194 ꢁC, 95% yield; 1H NMR (400 MHz,
7.32e7.35 (m, 1H, pyridine), 7.80e7.82 (d, J ¼ 8.0 Hz, 1H, pyridine),
CDCl3):
zoline), 6.95e7.03 (m, 3H, benzene), 8.55 (bs, 1H, NH, D2O exch.);
13C NMR (101 MHz, DMSO-d6):
22.26 (NHCOCH3), 22.88
d 2.15 (s, 3H, CH3), 2.31 (s, 3H, CH3), 6.81 (s, 1H, thiadia-
8.54e8.55 (m, 1H, pyridine), 8.74 (s, 1H, pyridine), 10.03 (bs,1H, NH,
D2O exch.); 13C NMR (101 MHz, DMSO-d6):
d
22.89 (NHCOCH3),
d
24.01 (NCOCH3), 26.62 (CH3C), 77.44 (CH3C), 123.82 (C5H-pyridine),

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C. De Monte et al. / European Journal of Medicinal Chemistry 105 (2015) 245e262
133.06 (C4H-pyridine), 139.60 (C3-pyridine), 142.82 (C]N), 146.57
(C6H-pyridine), 148.88 (C2H-pyridine), 168.36 (NC]O), 169.98
(NHC]O). Anal. Calcd for C12H14N4O2S: C, 51.78; H, 5.07; N, 20.13.
Found: C, 51.52; H, 5.38; N, 19.92.
123.57 (CH-naphthalene), 126.03 (CH-naphthalene), 126.64 (CH-
naphthalene), 127.14 (CH-naphthalene), 128.79 (CH-naphthalene),
129.00 (CH-naphthalene), 129.36 (C-naphthalene), 134.16 (C-
naphthalene), 136.27 (C-naphthalene), 147.57 (C]N), 168.13 (NC]
O), 170.00 (NHC]O). Anal. Calcd for C16H15N3O2S: C, 61.32; H, 4.82;
N, 13.41. Found: C, 61.03; H, 4.65; N, 13.29.
4.2.51. N-[4-Acetyl-5-(pyridin-4-yl)-4,5-dihydro-1,3,4-thiadiazol-
2-yl]acetamide (46)
Yellow powder, mp 212e217 ꢁC, 64% yield; 1H NMR (400 MHz,
4.2.56. N-[4-Acetyl-5-methyl-5-(naphthalen-1-yl)-4,5-dihydro-
1,3,4-thiadiazol-2-yl]acetamide (51)
DMSO-d6):
d 2.01 (s, 3H, CH3), 2.21 (s, 3H, CH3), 6.80 (s, 1H, thia-
diazoline), 7.21e7.23 (d, J ¼ 8.0 Hz, 2H, pyridine), 8.52e8.54 (d,
White powder, mp 190e195 ꢁC, 86% yield; 1H NMR (400 MHz,
J ¼ 8 Hz, 2H, pyridine), 11.83 (bs, 1H, NH, D2O exch.); 13C NMR
CDCl3):
d 1.79 (s, 3H, CH3), 2.22 (s, 3H, CH3), 2.46 (s, 3H, CH3),
(101 MHz, DMSO-d6):
d
22.23 (NHCOCH3), 22.96 (NCOCH3), 65.00
7.40e7.44 (m, 1H, naphthalene), 7.48e7.50 (m, 2H, naphthalene),
7.62e7.64 (d, J ¼ 7.6 Hz, 1H, naphthalene), 7.82e7.84 (d, J ¼ 8.0 Hz,
1H, naphthalene), 7.88e7.90 (m, 1H, naphthalene), 7.99e8.01 (m,
1H, naphthalene), 9.49 (bs, 1H, NH, D2O exch.); 13C NMR (101 MHz,
(CH), 120.35 (C3H-pyridine, C5H-pyridine), 146.31 (C]N), 149.94
(C4-pyridine), 150.62 (C2H-pyridine, C6H-pyridine), 168.16 (NC]O),
170.07 (NHC]O). Anal. Calcd for C11H12N4O2S: C, 49.99; H, 4.58; N,
21.20. Found: C, 50.19; H, 4.32; N, 21.54.
DMSO-d6):
d 23.04 (NHCOCH3), 23.48 (NCOCH3), 29.92 (CH3C),
78.33 (CH3C), 123.89 (CH-naphthalene), 125.00 (CH-naphthalene),
125.24 (CH-naphthalene), 125.85 (CH-naphthalene), 126.37 (CH-
naphthalene), 129.83 (CH-naphthalene), 129.93 (CH-naphthalene),
130.23 (C-naphthalene), 134.68 (C-naphthalene), 136.16 (C-naph-
thalene), 144.17 (C]N), 167.41 (NC]O), 170.05 (NHC]O). Anal.
Calcd for C17H17N3O2S: C, 62.36; H, 5.23; N, 12.83. Found: C, 62.67;
H, 5.09; N, 12.59.
4.2.52. N-[4-Acetyl-5-methyl-5-(pyridin-4-yl)-4,5-dihydro-1,3,4-
thiadiazol-2-yl]acetamide (47)
White powder, mp 223e227 ꢁC, 45% yield; 1H NMR (400 MHz,
DMSO-d6):
d 2.01 (s, 3H, CH3), 2.20 (s, 3H, CH3), 2.24 (s, 3H, CH3),
7.32e7.34 (d, J ¼ 6.0 Hz, 2H, pyridine), 8.53e8.55 (d, J ¼ 6.0 Hz, 2H,
pyridine), 11.72 (bs, 1H, NH, D2O exch.); 13C NMR (101 MHz, DMSO-
d6):
d 22.86 (NHCOCH3), 23.93 (NCOCH3), 26.16 (CH3C), 77.68
(CH3C), 119.97 (C3H-pyridine, C5H-pyridine), 142.73 (C]N), 150.42
(C2H-pyridine, C6H-pyridine), 152.43 (C4-pyridine), 168.36 (NC]O),
170.06 (NHC]O). Anal. Calcd for C12H14N4O2S: C, 51.78; H, 5.07; N,
20.13. Found: C, 51.49; H, 5.35; N, 20.44.
4.2.57. N-[4-Acetyl-5-methyl-5-(naphthalen-2-yl)-4,5-dihydro-
1,3,4-thiadiazol-2-yl]acetamide (52)
White powder, mp 182e187 ꢁC, 58% yield; 1H NMR (400 MHz,
DMSO-d6):
d 2.03 (s, 3H, CH3), 2.22 (s, 3H, CH3), 2.39 (s, 3H, CH3),
7.52e7.55 (m, 3H, naphthalene), 7.85e7.95 (m, 4H, naphthalene),
4.2.53. N-[4-Acetyl-5-methyl-5-(pyrazin-2-yl)-4,5-dihydro-1,3,4-
thiadiazol-2-yl]acetamide (48)
11.67 (bs, 1H, NH, D2O exch.); 13C NMR (101 MHz, DMSO-d6):
d
22.97 (NHCOCH3), 24.20 (NCOCH3), 27.02 (CH3C), 79.23 (CH3C),
White powder, mp 210e212 ꢁC, 58% yield; 1H NMR (400 MHz,
123.16 (CH-naphthalene), 124.01 (CH-naphthalene), 126.82 (CH-
naphthalene), 126.94 (CH-naphthalene), 127.82 (CH-naphthalene),
128.70 (CH-naphthalene), 128.86 (CH-naphthalene), 132.59 (C-
naphthalene), 132.93 (C-naphthalene), 141.27 (C-naphthalene),
142.91 (C]N), 168.28 (NC]O), 169.93 (NHC]O). Anal. Calcd for
DMSO-d6):
8.56 (s, 1H, pyrazine), 8.61 (s, 1H, pyrazine), 8.77 (s, 1H, pyrazine),
11.74 (bs,1H, NH, D2O exch.); 13C NMR (101 MHz, DMSO-d6):
22.85
d 2.04 (s, 3H, CH3), 2.20 (s, 3H, CH3), 2.29 (s, 3H, CH3),
d
(NHCOCH3), 23.75 (NCOCH3), 25.89 (CH3C), 78.40 (CH3C), 141.95
(C5H-pyrazine), 142.50 (C3H-pyrazine), 143.68 (C6H-pyrazine),
143.78 (C]N), 156.74 (C2-pyrazine), 168.59 (NC]O), 170.04 (NHC]
O). Anal. Calcd for C11H13N5O2S: C, 47.30; H, 4.69; N, 25.07. Found: C,
47.04; H, 4.93; N, 24.81.
C17H17N3O2S: C, 62.36; H, 5.23; N, 12.83. Found: C, 62.10; H, 5.44; N,
13.07.
4.2.58. N-[4-Acetyl-5-methyl-5-(2-oxo-2H-chromen-3-yl)-4,5-
dihydro-1,3,4-thiadiazol-2-yl]acetamide (53)
4.2.54. N-[4-Acetyl-5-(1,3-benzodioxol-5-yl)-4,5-dihydro-1,3,4-
thiadiazol-2-yl]acetamide (49)
White powder, mp 247e249 ꢁC, 74% yield; 1H NMR (400 MHz,
CDCl3):
d 2.13 (s, 3H, CH3), 2.26 (s, 3H, CH3), 2.42 (s, 3H, CH3),
White powder, mp 132e137 ꢁC, 61% yield; 1H NMR (400 MHz,
7.30e7.33 (m, 2H, chromene), 7.50e7.54 (m, 2H, chromene), 7.56 (s,
DMSO-d6):
d
2.02 (s, 3H, CH3), 2.17 (s, 3H, CH3), 5.98 (s, 2H, CH2-
1H, chromene), 8.12 (bs, 1H, NH, D2O exch.); 13C NMR (101 MHz,
benzodioxole), 6.70e6.73 (m, 2H, benzodioxole), 6.77 (s, 1H, thia-
diazoline), 6.84e6.89 (m, 1H, benzodioxole), 11.72 (bs, 1H, NH, D2O
DMSO-d6): d 22.87 (NHCOCH3), 23.73 (NCOCH3), 27.29 (CH3C),
76.32 (CH3C), 116.26 (CH-chromene), 118.96 (C-chromene), 125.19
(CH-chromene), 127.48 (CH-chromene), 129.55 (C-chromene),
132.55 (CH-chromene), 138.07 (CH-chromene), 144.06 (C]N),
153.18 (C-chromene), 158.22 (OC]O), 168.55 (NC]O), 169.94
(NHC]O). Anal. Calcd for C16H15N3O4S: C, 55.64; H, 4.38; N, 12.17.
Found: C, 55.47; H, 4.14; N, 11.94.
exch.); 13C NMR (101 MHz, DMSO-d6):
d 22.40 (NHCOCH3), 23.02
(NCOCH3), 66.22 (CH), 101.68 (CH2-benzodioxole), 106.12 (CH-
benzodioxole), 108.66 (CH-benzodioxole), 119.04 (CH-benzodiox-
ole), 135.89 (C-benzodioxole), 146.49 (C]N), 147.50 (C-benzodiox-
ole), 147.97 (C-benzodioxole), 167.80 (NC]O), 169.87 (NHC]O).
Anal. Calcd for C13H13N3O4S: C, 50.81; H, 4.26; N, 13.67. Found: C,
51.05; H, 4.50; N, 13.91.
4.2.59. N-[4-Acetyl-5-methyl-5-(ferrocen-2-yl)-4,5-dihydro-1,3,4-
thiadiazol-2-yl]acetamide (54)
4.2.55. N-[4-Acetyl-5-(naphthalen-1-yl)-4,5-dihydro-1,3,4-
thiadiazol-2-yl]acetamide (50)
Orange powder, mp 207e212 ꢁC, 84% yield; 1H NMR (400 MHz,
DMSO-d6):
d 1.95 (s, 3H, CH3), 2.05 (s, 3H, CH3), 2.22 (s, 3H, CH3),
White powder, mp 232e236 ꢁC, 58% yield; 1H NMR (400 MHz,
4.17 (s, 6H, ferrocene), 4.25 (s, 1H, ferrocene), 4.30 (s, 1H, ferrocene),
DMSO-d6):
d 1.99 (s, 3H, CH3), 2.32 (s, 3H, CH3), 7.14e7.16 (d,
4.42 (s, 1H, ferrocene), 11.46 (bs, 1H, NH, D2O exch.); 13C NMR
J ¼ 7.2 Hz, 1H, naphthalene), 7.44e7.48 (t, J ¼ 7.4 Hz, 1H, naph-
thalene), 7.53 (s,1H, thiadiazoline), 7.57e7.61 (m, 2H, naphthalene),
7.87e7.89 (d, J ¼ 8.0 Hz, 1H, naphthalene), 7.97e7.99 (d, J ¼ 7.6 Hz,
1H, naphthalene), 8.11e8.13 (d, J ¼ 7.6 Hz, 1H, naphthalene), 11.78
(101 MHz, DMSO-d6): d 23.03 (NHCOCH3), 24.21 (NCOCH3), 27.76
(CH3C), 68.09 (CH-ferrocene), 68.48 (CH-ferrocene), 69.01 (CH-
ferrocene), 69.50 (5 ꢂ CH-ferrocene), 70.01 (CH-ferrocene) 78.95
(CH3C), 89.68 (C-ferrocene), 142.27 (C]N), 167.45 (NC]O), 169.71
(NHC]O). Anal. Calcd for C17H19FeN3O2S: C, 53.00; H, 4.97; N,10.91.
Found: C, 52.82; H, 5.21; N, 11.14.
(bs, 1H, NH, D2O exch.); 13C NMR (101 MHz, DMSO-d6):
d 22.48
(NHCOCH3), 23.02 (NCOCH3), 64.26 (CH), 121.55 (CH-naphthalene),

C. De Monte et al. / European Journal of Medicinal Chemistry 105 (2015) 245e262
255
4.2.60. Synthesis of 2,2-dichloro-N-[4-(2,2-dichloroacetyl)-4,5-
4.2.65. N-(5,5-Diethyl-4-propanoyl-4,5-dihydro-1,3,4-thiadiazol-
2-yl)propanamide (59)
Light yellow powder, mp 50e52 ꢁC, 51% yield; 1H NMR
(400 MHz, CDCl3):
J ¼ 7.6 Hz, 3H, CH3), 1.23 (t, J ¼ 7.6 Hz, 3H, CH3), 1.79e1.88 (m, 2H,
CH2), 2.40 (q, J ¼ 7.6 Hz, 2H, CH2), 2.53 (q, J ¼ 7.6 Hz, 2H, CH2),
2.57e2.66 (m, 2H, CH2), 8.13 (bs, 1H, NH, D2O exch.); 13C NMR
dihydro-5-methyl-5-phenyl-1,3,4-thiadiazol-2-yl]acetamide (55)
The thiosemicarbazone intermediate (1.0 eq.) was added to a
stirring solution of dichloroacetic anhydride (5.0 eq.) and the re-
action mixture was stirred for 72 h at 90 ꢁC. The mixture was
poured on ice and the resulting suspension was filtered and washed
with water, petroleum ether and diethyl ether to obtain compound
55 as an orange powder (89% yield); mp 205e210 ꢁC; 1H NMR
d
1.02 (t, J ¼ 7.4 Hz, 6H, 2 ꢂ CH3), 1.12 (t,
(101 MHz, CDCl3):
d
8.71 (2 ꢂ CH3CH2), 9.01 (NHCOCH2CH3), 9.37
(400 MHz, DMSO-d6):
CH), 7.19e7.26 (m, 5H, benzene), 12.57 (bs, 1H, NH, D2O exch.); 13
NMR (101 MHz, DMSO-d6): 26.28 (CH3C), 66.10 (NHCOCHCl2),
d
2.17 (s, 3H, CH3), 6.39 (s,1H, CH), 6.81 (s,1H,
(NCOCH2CH3), 29.05 (NHCOCH2CH3), 29.26 (NCOCH2CH3), 31.90
(2 ꢂ CH3CH2), 87.36 (CH2CCH2), 143.47 (C]N), 173.15 (NC]O),
173.19 (NHC]O). Anal. Calcd for C12H21N3O2S: C, 53.11; H, 7.80; N,
15.48. Found: C, 53.44; H, 7.53; N, 15.65.
C
d
67.01 (NCOCHCl2), 80.75 (CH3C), 125.05 (2 ꢂ CH-benzene), 128.65
(CH-benzene), 129.15 (2 ꢂ CH-benzene), 142.41 (C]N), 144.80 (C-
benzene), 160.31 (NC]O), 163.79 (NHC]O). Anal. Calcd for
4.2.66. N-[5-Methyl-5-(2-methylpropyl)-4-propanoyl-4,5-dihydro-
1,3,4-thiadiazol-2-yl]propanamide (60)
C
13H11Cl4N3O2S: C, 37.61; H, 2.67; N, 10.12. Found: C, 37.30; H, 2.48;
N, 9.89.
White powder, mp 70e73 ꢁC, 77% yield; 1H NMR (400 MHz,
CDCl3):
d
0.94 (d, J ¼ 6.0 Hz, 3H, CH3), 1.00 (d, J ¼ 6.4 Hz, 3H, CH3),
4.2.61. General synthesis of compounds 56e101
1.11 (t, J ¼ 7.4 Hz, 3H, CH3), 1.24 (t, J ¼ 7.4 Hz, 3H, CH3), 1.93e1.95 (m,
1H, CH2), 1.96e1.98 (m, 1H, CH), 1.99 (s, 3H, CH3), 2.40e2.45 (m, 1H,
CH2), 2.49e2.52 (m, 4H, 2 ꢂ CH2), 8.15 (bs, 1H, NH, D2O exch.); 13C
The thiosemicarbazone intermediate (1.0 eq.) was added to a
stirring solution of propionic anhydride (5.0 eq.) and the reaction
mixture was stirred for 2e72 h at 90 ꢁC. The mixture was poured on
ice and the resulting suspension was filtered and washed with
water, petroleum ether and diethyl ether. As regards compounds 57,
59, 62, 68e70, 72e74, 76e79, and 94, the mixture was poured on
ice and extracted with chloroform. The organics were reunited,
dried over sodium sulfate and concentrated in vacuo. The products
were purified by column chromatography (ethyl acetate:petroleum
ether) before characterization by spectroscopic methods.
NMR (101 MHz, CDCl3):
(CH3CHCH3), 24.57
d 9.04 (NHCOCH2CH3, NCOCH2CH3), 22.93
(2 ꢂ CH3CH),
25.72
(CH3C),
29.32
(NHCOCH2CH3, NCOCH2CH3), 47.15 (CH2), 81.30 (CH3CCH2), 143.66
(C]N), 173.08 (NC]O), 173.30 (NHC]O). Anal. Calcd for
C
13H23N3O2S: C, 54.71; H, 8.12; N, 14.72. Found: C, 54.49; H, 7.83; N,
14.45.
4.2.67. N-(5-Butyl-4,5-dihydro-5-methyl-4-propionyl-1,3,4-
thiadiazol-2-yl)propionamide (61)
4.2.62. N-(5,5-Dimethyl-4-propanoyl-4,5-dihydro-1,3,4-thiadiazol-
2-yl)propanamide (56)
White powder, mp 72e76 ꢁC, 78% yield; 1H NMR (400 MHz,
DMSO-d6):
d
0.87 (t, J ¼ 7.2 Hz, 3H, CH3), 0.99e1.02 (m, 6H, 2 ꢂ CH3),
White powder, mp 127e133 ꢁC, 57% yield; 1H NMR (400 MHz,
1.11e1.15 (m, 1H, CH2), 1.25e1.32 (m, 2H, CH2), 1.44e1.49 (m, 1H,
CH2), 1.86 (s, 3H, CH3), 2.30e2.37 (m, 4H, 2 ꢂ CH2), 2.41e2.48 (m,
2H, CH2), 11.36 (bs, 1H, NH, D2O exch.); 13C NMR (101 MHz, DMSO-
CDCl3):
d
1.12 (t, J ¼ 7.4 Hz, 3H, CH3), 1.23 (t, J ¼ 7.4 Hz, 3H, CH3), 1.99
(s, 6H, 2 ꢂ CH3), 2.41 (q, J ¼ 7.6 Hz, 2H, CH2), 2.51 (q, J ¼ 7.6 Hz, 2H,
CH2), 8.06 (bs, 1H, NH, D2O exch.); 13C NMR (101 MHz, CDCl3):
d6): d 9.29 (NHCOCH2CH3), 9.53 (NCOCH2CH3), 14.32 (CH2CH3),
d
9.08 (NHCOCH2CH3), 9.10 (NCOCH2CH3), 28.74 (NHCOCH2CH3),
22.40 (CH3CH2), 27.12 (CH3CH2CH2), 28.48 (CH3C), 28.70
(NHCOCH2CH3), 28.94 (NCOCH2CH3), 39.05 (CH3CCH2), 80.37
(CH3CCH2), 143.00 (C]N), 171.70 (NC]O), 173.16 (NHC]O). Anal.
Calcd for C13H23N3O2S: C, 54.71; H, 8.12; N, 14.72. Found: C, 54.55;
H, 8.29; N, 15.01.
29.05 (NCOCH2CH3), 29.25 (2 ꢂ CH3C), 77.13 (CH3CCH3), 143.61 (C]
N), 173.41 (NC]O, NHC]O). Anal. Calcd for C10H17N3O2S: C, 49.36;
H, 7.04; N, 17.27. Found: C, 49.05; H, 7.22; N, 16.99.
4.2.63. N-(5-Ethyl-5-methyl-4-propanoyl-4,5-dihydro-1,3,4-
thiadiazol-2-yl)propanamide (57)
4.2.68. N-(5-Ethyl-4,5-dihydro-5-propyl-4-propionyl-1,3,4-
thiadiazol-2-yl)propionamide (62)
White powder, mp 110e113 ꢁC, 47% yield; 1H NMR (400 MHz,
CDCl3):
d
1.01 (t, J ¼ 7.4 Hz, 3H, CH3), 1.12 (t, J ¼ 7.6 Hz, 3H, CH3), 1.23
Light yellow oil, 44% yield; 1H NMR (400 MHz, DMSO-d6):
(t, J ¼ 7.6 Hz, 3H, CH3), 1.85e1.94 (m, 1H, CH2), 1.99 (s, 3H, CH3), 2.41
(q, J ¼ 7.6 Hz, 2H, CH2), 2.48e2.52 (m, 2H, CH2), 2.53e2.65 (m, 1H,
CH2), 8.06 (bs, 1H, NH, D2O exch.); 13C NMR (101 MHz, CDCl3):
d
0.87e0.90 (m, 6H, 2 ꢂ CH3), 1.01 (t, J ¼ 7.4 Hz, 6H, 2 ꢂ CH3),
1.14e1.20 (m, 1H, CH2), 1.51e1.58 (m, 1H, CH2), 1.70e1.77 (m, 2H,
CH2), 2.32 (q, J ¼ 7.6 Hz, 2H, CH2), 2.42e2.49 (m, 4H, 2 ꢂ CH2), 11.35
d
9.06 (CH3CH2), 9.17 (NHCOCH2CH3), 9.24 (NCOCH2CH3), 28.13
(bs, 1H, NH, D2O exch.); 13C NMR (101 MHz, Methanol-d4):
d 8.06
(CH3C), 29.11 (NHCOCH2CH3), 29.33 (NCOCH2CH3), 32.57 (CH2),
82.30 (CH3CCH2), 143.37 (C]N), 173.06 (NC]O), 173.25 (NHC]O).
Anal. Calcd for C11H19N3O2S: C, 51.34; H, 7.44; N, 16.33. Found: C,
51.55; H, 7.62; N, 16.09.
(CH3CH2C), 8.51 (NHCOCH2CH3), 8.80 (NCOCH2CH3), 13.31
(CH3CH2CH2), 17.69 (CH3CH2CH2), 28.65 (NHCOCH2CH3), 28.92
(NCOCH2CH3), 31.73 (CH3CH2C), 41.11 (CH3CH2CH2), 85.95
(CH2CCH2), 143.75 (C]N), 173.19 (NC]O), 173.30 (NHC]O). Anal.
Calcd for C13H23N3O2S: C, 54.71; H, 8.12; N, 14.72. Found: C, 54.98;
H, 8.31; N, 15.10.
4.2.64. N-(5-Methyl-4-propanoyl-5-propyl-4,5-dihydro-1,3,4-
thiadiazol-2-yl)propanamide (58)
White powder, mp 74e75 ꢁC, 75% yield; 1H NMR (400 MHz,
4.2.69. N-[5-Methyl-5-(2-methylprop-1-en-1-yl)-4-propanoyl-4,5-
dihydro-1,3,4-thiadiazol-2-yl]propanamide (63)
CDCl3):
d
0.95 (t, J ¼ 7.0 Hz, 3H, CH3),1.12 (t, J ¼ 7.2 Hz, 3H, CH3),1.23
(t, J ¼ 7.4 Hz, 3H, CH3), 1.26e1.28 (m, 1H, CH2), 1.59e1.61 (m, 1H,
CH2), 1.87e1.92 (m, 1H, CH2), 1.98 (s, 3H, CH3), 2.41e2.43 (m, 2H,
CH2), 2.48e2.52 (m, 3H, CH2), 8.25 (bs, 1H, NH, D2O exch.); 13C NMR
Light yellow powder, mp 105e108 ꢁC, 78% yield; 1H NMR
(400 MHz, CDCl3):
3H, CH3), 1.71 (s, 3H, CH3), 1.76 (s, 3H, CH3), 1.99 (s, 3H, CH3), 2.42 (q,
d
1.12 (t, J ¼ 7.4 Hz, 3H, CH3), 1.23 (t, J ¼ 7.4 Hz,
(101 MHz, CDCl3):
d
9.05 (NHCOCH2CH3), 9.19 (NCOCH2CH3), 13.76
J ¼ 7.6 Hz, 2H, CH2), 2.52 (q, J ¼ 7.6 Hz, 2H, CH2), 5.65 (s, 1H, CH¼),
(CH3CH2), 18.28 (CH3CH2), 28.24 (CH3C), 29.11 (NHCOCH2CH3),
29.29 (NCOCH2CH3), 41.88 (CH2CH2C), 81.46 (CH3CCH2), 143.50 (C]
N), 173.14 (NC]O, NHC]O). Anal. Calcd for C12H21N3O2S: C, 53.11;
H, 7.80; N, 15.48. Found: C, 52.89; H, 8.02; N, 15.27.
8.37 (bs, 1H, NH, D2O exch.); 13C NMR (101 MHz, CDCl3):
d 9.05
(NHCOCH2CH3), 9.31 (NCOCH2CH3), 18.63 (CH3C¼), 26.68 (CH3C¼),
28.74 (CH3C), 29.17 (NHCOCH2CH3), 30.75 (NCOCH2CH3), 76.44
(CH3CCH¼), 126.31 (CH¼), 137.69 (C¼), 144.29 (C]N), 171.70 (NC]

256
C. De Monte et al. / European Journal of Medicinal Chemistry 105 (2015) 245e262
O), 173.29 (NHC]O). Anal. Calcd for C13H21N3O2S: C, 55.10; H, 7.47;
N, 14.83. Found: C, 54.84; H, 7.26; N, 15.11.
CDCl3):
d
1.12 (t, J ¼ 7.4 Hz, 3H, CH3), 1.23 (t, J ¼ 7.6 Hz, 3H, CH3),
1.66e1.69 (m, 2H, cyclopentane), 1.94e1.98 (m, 2H, cyclopentane),
2.03e2.08 (m, 2H, cyclopentane), 2.40 (q, J ¼ 7.6 Hz, 2H, CH2), 2.53
(q, J ¼ 7.6 Hz, 2H, CH2), 2.90e2.98 (m, 2H, cyclopentane), 8.17 (bs,
4.2.70. N-[5-(But-3-en-1-yl)-5-methyl-4-propanoyl-4,5-dihydro-
1,3,4-thiadiazol-2-yl]propanamide (64)
1H, NH, D2O exch.); 13C NMR (101 MHz, CDCl3):
d 9.08
Light yellow powder, mp 57e60 ꢁC, 99% yield; 1H NMR
(NHCOCH2CH3), 9.12 (NCOCH2CH3), 24.97 (2 ꢂ CH2-cyclopentane),
29.23 (NHCOCH2CH3), 29.31 (NCOCH2CH3), 40.11 (2 ꢂ CH2-cyclo-
pentane), 85.34 (C-cyclopentane), 144.21 (C]N), 172.82 (NC]O),
173.32 (NHC]O). Anal. Calcd for C12H19N3O2S: C, 53.51; H, 7.11; N,
15.60. Found: C, 53.35; H, 6.85; N, 15.81.
(400 MHz, CDCl3):
d
1.12 (t, J ¼ 7.4 Hz, 3H, CH3), 1.23 (t, J ¼ 7.4 Hz,
3H, CH3), 1.93e1.97 (m, 1H, CH2), 2.00 (s, 3H, CH3), 2.06e2.07 (m,
1H, CH2), 2.34e2.37 (m, 1H, CH2), 2.39e2.44 (m, 2H, CH2),
2.48e2.54 (m, 2H, CH2), 2.63e2.70 (m, 1H, CH2), 4.96e4.98 (m, 1H,
CH2¼), 5.02e5.07 (m, 1H, CH2¼), 5.77e5.87 (m, 1H, CH¼), 8.20 (bs,
1H, NH, D2O exch.); 13C NMR (101 MHz, CDCl3):
d
9.05
4.2.75. N-(1-(1-Oxopropyl)-6-methyl-4-thia-1,2-diazaspiro[4.4]
non-2-en-3-yl)propanamide (69)
(NHCOCH2CH3), 9.23 (NCOCH2CH3), 28.41 (CH2CH2CH¼), 29.15
(CH3C), 29.33 (NHCOCH2CH3, NCOCH2CH3), 38.73 (CCH2CH2), 81.09
(CH3CCH2), 115.16 (CH2¼), 137.08 (CH¼), 143.33 (C]N), 173.00
(NC]O), 173.28 (NHC]O). Anal. Calcd for C13H21N3O2S: C, 55.10; H,
7.47; N, 14.83. Found: C, 55.32; H, 7.69; N, 14.57.
White powder, mp 90e95 ꢁC, 37% yield; 1H NMR (400 MHz,
CDCl3):
d
1.05 (d, J ¼ 6.4 Hz, 3H, CH3), 1.12 (t, J ¼ 7.4 Hz, 3H, CH3),
1.23 (t, J ¼ 7.4 Hz, 3H, CH3), 1.62e1.68 (m, 2H, cyclopentane),
1.85e1.88 (m, 2H, cyclopentane), 2.09e2.14 (m, 1H, cyclopentane),
2.41 (q, J ¼ 7.6 Hz, 2H, CH2), 2.51e2.57 (m, 2H, CH2), 3.23e3.29 (m,
2H, cyclopentane), 7.92 (bs, 1H, NH, D2O exch.); 13C NMR (101 MHz,
4.2.71. N-(5-Methyl-5-pentyl-4-propanoyl-4,5-dihydro-1,3,4-
thiadiazol-2-yl)propanamide (65)
CDCl3):
d 9.07 (NHCOCH2CH3), 9.21 (NCOCH2CH3), 13.96 (CH3CH-
White powder, mp 92e99 ꢁC, 69% yield; 1H NMR (400 MHz,
cyclopentane), 20.19 (CH2-cyclopentane), 29.36 (NHCOCH2CH3),
29.63 (NCOCH2CH3), 30.67 (CH2-cyclopentane), 37.71 (CH2-cyclo-
pentane), 39.02 (CH-cyclopentane), 91.57 (C-cyclopentane), 143.83
(C]N), 173.13 (NC]O), 173.21 (NHC]O). Anal. Calcd for
DMSO-d6):
d
0.85 (t, J ¼ 6.8 Hz, 3H, CH3), 0.98e1.03 (m, 6H,
2 ꢂ CH3), 1.14e1.16 (m, 1H, CH2), 1.24e1.27 (m, 4H, 2 ꢂ CH2),
1.47e1.51 (m, 1H, CH2), 1.77e1.84 (m, 1H, CH2), 1.86 (s, 3H, CH3),
2.30e2.48 (m, 5H, CH2), 11.35 (bs, 1H, NH, D2O exch.); 13C NMR
C13H21N3O2S: C, 55.10; H, 7.47; N, 14.83. Found: C, 55.38; H, 7.73; N,
(101 MHz, DMSO-d6):
d
9.30 (NHCOCH2CH3), 9.55 (NCOCH2CH3),
14.64.
14.21 (CH2CH3), 22.43 (CH3CCH2CH2), 24.50 (CH3CH2), 28.50
(CH3C), 28.69 (NHCOCH2CH3), 28.95 (NCOCH2CH3), 31.36
(CH3CH2CH2), 39.16 (CH3CCH2), 80.38 (CH3CCH2), 142.99 (C]N),
171.72 (NC]O), 173.14 (NHC]O). Anal. Calcd for C14H25N3O2S: C,
56.16; H, 8.42; N, 14.03. Found: C, 56.34; H, 8.25; N, 14.31.
4.2.76. N-(1-(1-Oxopropyl)-7-methyl-4-thia-1,2-diazaspiro[4.4]
non-2-en-3-yl)propanamide (70)
Yellow oil, 42% yield; 1H NMR (400 MHz, CDCl3):
d 1.08 (d,
J ¼ 6.4 Hz, 3H, CH3),1.12 (t, J ¼ 7.6 Hz, 3H, CH3),1.23 (t, J ¼ 7.4 Hz, 3H,
CH3), 1.60e1.72 (m, 1H, cyclopentane), 1.83e1.85 (m, 1H, cyclo-
pentane), 2.00e2.03 (m, 1H, cyclopentane), 2.09e2.13 (m, 2H,
cyclopentane), 2.39e2.45 (m, 2H, CH2), 2.50e2.55 (m, 2H, CH2),
2.82e2.84 (m, 1H, cyclopentane), 3.08e3.15 (m, 1H, cyclopentane),
4.2.72. N-(5-Butyl-5-ethyl-4-propanoyl-4,5-dihydro-1,3,4-
thiadiazol-2-yl)propanamide (66)
Light yellow powder, mp 97e101 ꢁC, 82% yield; 1H NMR
(400 MHz, CDCl3):
d
0.93 (t, J ¼ 7.0 Hz, 3H, CH3), 1.02 (t, J ¼ 7.0 Hz,
8.46 (bs, 1H, NH, D2O exch.); 13C NMR (101 MHz, CDCl3):
d 9.01
3H, CH3), 1.13 (t, J ¼ 7.4 Hz, 3H, CH3), 1.24 (t, J ¼ 7.2 Hz, 3H, CH3),
1.33e1.37 (m, 2H, CH2), 1.61e1.67 (m, 2H, CH2), 1.81e1.89 (m, 2H,
CH2), 2.39e2.44 (m, 2H, CH2), 2.50e2.56 (m, 2H, CH2), 2.58e2.65
(m, 2H, CH2), 8.06 (bs, 1H, NH, D2O exch.); 13C NMR (101 MHz,
(NHCOCH2CH3), 9.11 (NCOCH2CH3), 19.24 (CH3CH-cyclopentane),
29.11 (NHCOCH2CH3), 29.22 (NCOCH2CH3), 32.69 (CH2-cyclo-
pentane), 34.14 (CH2-cyclopentane), 39.60 (CH2-cyclopentane),
48.42 (CH-cyclopentane), 85.02 (C-cyclopentane), 144.32 (C]N),
172.78 (NC]O), 173.50 (NHC]O). Anal. Calcd for C13H21N3O2S: C,
55.10; H, 7.47; N, 14.83. Found: C, 54.80; H, 7.20; N, 14.59.
CDCl3):
d 8.64 (CH3CH2C), 9.03 (NHCOCH2CH3), 9.41 (NCOCH2CH3),
14.01 (CH3CH2CH2), 22.46 (CH3CH2CH2), 26.61 (CH3CH2CH2CH2),
29.08 (NHCOCH2CH3), 29.25 (NCOCH2CH3), 32.00 (CH3CH2C), 38.93
(CH3CH2CCH2), 86.53 (CH2CCH2), 143.59 (C]N), 173.13 (NC]O),
173.22 (NHC]O). Anal. Calcd for C14H25N3O2S: C, 56.16; H, 8.42; N,
14.03. Found: C, 55.91; H, 8.70; N, 13.82.
4.2.77. N-[1-(1-Oxopropyl)-4-thia-1,2-diazaspiro[4.5]dec-2-en-3-
yl]propanamide (71)
Light brown powder, mp 110e115 ꢁC, 67% yield; 1H NMR
(400 MHz, CDCl3):
d
1.11 (t, J ¼ 7.4 Hz, 3H, CH3), 1.24 (t, J ¼ 7.6 Hz,
4.2.73. N-(5-Hexyl-5-methyl-4-propanoyl-4,5-dihydro-1,3,4-
thiadiazol-2-yl)propanamide (67)
3H, CH3), 1.29e1.33 (m, 1H, cyclohexane), 1.41e1.51 (m, 2H, cyclo-
hexane), 1.65e1.69 (m, 1H, cyclohexane), 1.83e1.87 (m, 2H, cyclo-
hexane), 2.05e2.08 (m, 2H, cyclohexane), 2.41 (q, J ¼ 7.6 Hz, 2H,
CH2), 2.51 (q, J ¼ 7.6 Hz, 2H, CH2), 2.95e3.03 (m, 2H, cyclohexane),
White powder, mp 50e55 ꢁC, 87% yield; 1H NMR (400 MHz,
CDCl3):
d
0.88 (t, J ¼ 6.8 Hz, 3H, CH3), 1.12 (t, J ¼ 7.6 Hz, 3H, CH3),1.23
(t, J ¼ 7.6 Hz, 3H, CH3), 1.26e1.28 (m, 1H, CH2), 1.29e1.32 (m, 6H,
CH2), 1.53e1.60 (m, 1H, CH2), 1.89e1.96 (m, 1H, CH2), 1.98 (s, 3H,
CH3), 2.41 (q, J ¼ 7.6 Hz, 2H, CH2), 2.48e2.55 (m, 3H, CH2), 8.14 (bs,
7.98 (bs, 1H, NH, D2O exch.); 13C NMR (101 MHz, CDCl3):
d 9.15
(NHCOCH2CH3, NCOCH2CH3), 24.53 (2 ꢂ CH2-cyclohexane), 24.76
(CH2-cyclohexane), 29.37 (NHCOCH2CH3), 29.69 (NCOCH2CH3),
35.93 (2 ꢂ CH2-cyclohexane), 85.07 (C-cyclohexane), 143.72 (C]N),
173.32 (NC]O), 173.39 (NHC]O). Anal. Calcd for C13H21N3O2S: C,
55.10; H, 7.47; N, 14.83. Found: C, 55.35; H, 7.16; N, 14.50.
1H, NH, D2O exch.); 13C NMR (101 MHz, CDCl3):
d 9.06
(NHCOCH2CH3), 9.27 (NCOCH2CH3), 14.00 (CH3CH2), 22.53
(CH3CCH2CH2), 24.91 (CH3CH2CH2), 28.27 (CH3C), 28.96
(CH3CH2CH2CH2), 29.14 (NHCOCH2CH3), 29.27 (NCOCH2CH3), 31.65
(CH3CH2CH2) 39.65 (CH3CCH2), 81.48 (CH3CCH2), 143.59 (C]N),
173.23 (NC]O, NHC]O). Anal. Calcd for C15H27N3O2S: C, 57.48; H,
8.68; N, 13.41. Found: C, 57.24; H, 8.93; N, 13.19.
4.2.78. N-[1-(1-Oxopropyl)-6-methyl-4-thia-1,2-diazaspiro[4.5]
dec-2-en-3-yl] propanamide (72)
White powder, mp 90e94 ꢁC, 34% yield; 1H NMR (400 MHz,
CDCl3):
d
0.95 (d, J ¼ 6.4 Hz, 3H, CH3), 1.11 (t, J ¼ 7.4 Hz, 3H, CH3),
4.2.74. N-[1-(1-Oxopropyl)-4-thia-1,2-diazaspiro[4.4]non-2-en-3-
yl]propanamide (68)
1.23 (t, J ¼ 7.6 Hz, 3H, CH3), 1.47e1.62 (m, 1H, cyclohexane),
1.65e1.67 (m, 1H, cyclohexane), 1.79e1.80 (m, 1H, cyclohexane),
1.82e1.84 (m, 1H, cyclohexane), 1.95e2.04 (m, 1H, cyclohexane),
Pink powder, mp 71e75 ꢁC, 49% yield; 1H NMR (400 MHz,

C. De Monte et al. / European Journal of Medicinal Chemistry 105 (2015) 245e262
257
2.12e2.15 (m, 1H, cyclohexane), 2.29e2.31 (m, 1H, cyclohexane),
4.2.82. N-[1-(1-Oxopropyl)-6-tert-butyl-4-thia-1,2-diazaspiro[4.5]
dec-2-en-3-yl] propanamide (76)
Yellow powder, mp 111e112 ꢁC, 42% yield; 1H NMR (400 MHz,
CDCl3):
J ¼ 7.6 Hz, 3H, CH3), 1.28e1.32 (m, 1H, cyclohexane), 1.55e1.62 (m,
2H, cyclohexane), 1.71e1.75 (m, 2H, cyclohexane), 1.94e2.00 (m,
2H, cyclohexane), 2.35e2.50 (m, 4H, 2 ꢂ CH2), 2.94e3.02 (m, 2H,
cyclohexane), 7.83 (bs, 1H, NH, D2O exch.); 13C NMR (101 MHz,
2.38e2.58 (m, 4H, 2 ꢂ CH2), 2.93e3.07 (m, 1H, cyclohexane),
3.58e3.59 (m, 1H, cyclohexane), 8.01 (bs, 1H, NH, D2O exch.); 13C
NMR (101 MHz, CDCl3):
d
9.07 (NHCOCH2CH3), 9.28 (NCOCH2CH3),
d
1.02 (s, 9H, 3 ꢂ CH3), 1.10 (t, J ¼ 7.4 Hz, 3H, CH3), 1.25 (t,
16.44 (CH3CH-cyclohexane), 19.67 (CH2-cyclohexane), 24.66 (CH2-
cyclohexane), 27.27 (CH2-cyclohexane), 29.41 (NHCOCH2CH3),
29.73 (NHCOCH2CH3), 32.76 (CH2-cyclohexane), 41.34 (CH-cyclo-
hexane), 90.41 (C-cyclohexane), 143.37 (C]N), 173.09 (NC]O),
174.32 (NHC]O). Anal. Calcd for C14H23N3O2S: C, 56.54; H, 7.79; N,
14.13. Found: C, 56.28; H, 7.96; N, 14.41.
CDCl3):
d 8.87 (NHCOCH2CH3), 9.02 (NCOCH2CH3), 23.95 (CH2-
cyclohexane), 26.11 (CH2-cyclohexane), 29.05 (3 ꢂ CH3C), 29.42
((CH3)3C), 29.49 (NHCOCH2CH3), 30.08 (NCOCH2CH3), 34.92 (CH2-
cyclohexane), 40.67 (CH2-cyclohexane), 48.79 (CH-cyclohexane),
88.15 (C-cyclohexane), 143.48 (C]N), 172.96 (NC]O), 173.30
(NHC]O). Anal. Calcd for C17H29N3O2S: C, 60.14; H, 8.61; N, 12.38.
Found: C, 59.87; H, 8.44; N, 12.16.
4.2.79. N-[1-(1-Oxopropyl)-7-methyl-4-thia-1,2-diazaspiro[4.5]
dec-2-en-3-yl] propanamide (73)
Light brown oil, 41% yield; 1H NMR (400 MHz, CDCl3):
d 0.94 (d,
J ¼ 8.0 Hz, 3H, CH3), 0.99e1.03 (m, 1H, cyclohexane), 1.08 (t,
J ¼ 7.6 Hz, 3H, CH3), 1.21 (t, J ¼ 7.6 Hz, 3H, CH3), 1.43e1.67 (m, 3H,
cyclohexane), 1.81e1.84 (m, 1H, cyclohexane), 1.93e2.06 (m, 2H,
cyclohexane), 2.38e2.45 (m, 2H, CH2), 2.47e2.53 (m, 2H, CH2),
2.64e2.71 (m, 1H, cyclohexane), 2.84e2.92 (m, 1H, cyclohexane),
4.2.83. N-[1-(1-Oxopropyl)-4-thia-1,2-diazaspiro[4.6]undec-2-en-
3-yl]-propanamide (77)
Light yellow powder, mp 60e65 ꢁC, 60% yield; 1H NMR
(400 MHz, CDCl3):
d
1.12 (t, J ¼ 7.6 Hz, 3H, CH3), 1.24 (t, J ¼ 7.6 Hz,
8.56 (bs, 1H, NH, D2O exch.); 13C NMR (101 MHz, CDCl3):
d
9.09
(NHCOCH2CH3), 9.11 (NCOCH2CH3), 22.10 (CH2-cyclohexane), 24.17
(CH-cyclohexane), 29.39 (CH3CH-cyclohexane), 29.69
3H, CH3), 1.56e1.68 (m, 6H, cycloheptane), 1.86e1.89 (m, 2H,
cycloheptane), 2.15e2.20 (m, 2H, cycloheptane), 2.41 (q, J ¼ 7.6 Hz,
2H, CH2), 2.50 (q, J ¼ 7.6 Hz, 2H, CH2), 2.89e2.95 (m, 2H, cyclo-
heptane), 7.84 (bs, 1H, NH, D2O exch.); 13C NMR (101 MHz, CDCl3):
(NHCOCH2CH3), 31.58 (NCOCH2CH3), 33.23 (CH2-cyclohexane),
35.30 (CH2-cyclohexane), 44.07 (CH2-cyclohexane), 84.99 (C-
cyclohexane), 143.86 (C]N), 173.17 (NC]O), 173.37 (NHC]O).
Anal. Calcd for C14H23N3O2S: C, 56.54; H, 7.79; N, 14.13. Found: C,
56.76; H, 7.47; N, 14.30.
d
9.09 (NHCOCH2CH3, NCOCH2CH3), 23.92 (2 ꢂ CH2-cycloheptane),
27.70 (2 ꢂ CH2-cycloheptane), 29.38 (NHCOCH2CH3, NCOCH2CH3),
40.00 (2 ꢂ CH2-cycloheptane), 87.59 (C-cycloheptane), 143.64 (C]
N), 172.76 (NC]O), 173.08 (NHC]O). Anal. Calcd for C14H23N3O2S:
C, 56.54; H, 7.79; N, 14.13. Found: C, 56.75; H, 7.50; N, 14.34.
4.2.80. N-[1-(1-Oxopropyl)-8-methyl-4-thia-1,2-diazaspiro[4.5]
dec-2-en-3-yl] propanamide (74)
4.2.84. N-[1-(1-Oxopropyl)-4-thia-1,2-diazaspiro[4.7]dodec-2-en-
3-yl]propanamide (78)
Yellow powder, mp 74e81 ꢁC, 30% yield; 1H NMR (400 MHz,
Light brown oil, 54% yield; 1H NMR (400 MHz, CDCl3):
d 1.11 (t,
CDCl3):
d
0.91 (d, J ¼ 8.0 Hz, 3H, CH3), 1.10 (t, J ¼ 7.6 Hz, 3H, CH3),
J ¼ 7.6 Hz, 3H, CH3), 1.23 (t, J ¼ 7.6 Hz, 3H, CH3), 1.51e1.65 (m, 7H,
cyclooctane), 1.73e1.78 (m, 1H, cyclooctane), 1.85e1.91 (m, 2H,
cyclooctane), 2.16e2.22 (m, 2H, cyclooctane), 2.38e2.52 (m, 4H,
2 ꢂ CH2), 3.02e3.08 (m, 2H, cyclooctane), 8.38 (bs, 1H, NH, D2O
1.12e1.14 (m, 1H, cyclohexane), 1.23 (t, J ¼ 7.6 Hz, 3H, CH3),
1.45e1.51 (m, 1H, cyclohexane), 1.60e1.64 (m, 1H, cyclohexane),
1.77e1.86 (m, 2H, cyclohexane), 1.99e2.05 (m, 1H, cyclohexane),
2.39e2.45 (m, 2H, CH2), 2.47e2.55 (m, 2H, CH2), 2.67e2.71 (m, 1H,
cyclohexane), 3.00e3.08 (m, 1H, cyclohexane), 3.19e3.27 (m, 1H,
cyclohexane), 8.26 (bs, 1H, NH, D2O exch.);13C NMR (101 MHz,
exch.); 13C NMR (101 MHz, CDCl3):
d 9.05 (NHCOCH2CH3), 9.11
(NCOCH2CH3), 23.30 (2 ꢂ CH2-cyclooctane), 23.84 (2 ꢂ CH2-cyclo-
octane), 27.36 (CH2-cyclooctane), 29.33 (NHCOCH2CH3), 29.44
(NCOCH2CH3), 36.96 (2 ꢂ CH2-cyclooctane), 88.12 (C-cyclooctane),
143.93 (C]N), 173.08 (NC]O), 173.27 (NHC]O). Anal. Calcd for
CDCl3):
d 9.05 (NHCOCH2CH3), 9.06 (NCOCH2CH3), 21.90 (CH3CH-
cyclohexane), 29.43 (2 ꢂ CH2-cyclohexane), 30.40 (NHCOCH2CH3),
31.12 (NCOCH2CH3), 33.17 (2 ꢂ CH2-cyclohexane), 35.71 (CH-cyclo-
hexane), 85.14 (C-cyclohexane), 143.67 (C]N), 172.96 (NC]O),
173.29 (NHC]O). Anal. Calcd for C14H23N3O2S: C, 56.54; H, 7.79; N,
14.13. Found: C, 56.37; H, 8.02; N, 13.92.
C15H25N3O2S: C, 57.85; H, 8.09; N, 13.49. Found: C, 58.09; H, 7.82; N,
13.60.
4.2.85. N-[4-(1-Oxopropyl)-5-cyclohexyl-5-methyl-4,5-dihydro-
1,3,4-thiadiazol-2-yl]propanamide (79)
White powder, mp 127e131 ꢁC, 58% yield; 1H NMR (400 MHz,
4.2.81. N-[1-(1-Oxopropyl)-7,7-dimethy-4-thia-1,2-diazaspiro[4.5]
dec-2-en-3-yl] propanamide (75)
CDCl3):
d
1.06e1.08 (m, 1H, cyclohexane), 1.13 (t, J ¼ 7.4 Hz, 3H,
CH3), 1.15e1.17 (m, 1H, cyclohexane), 1.24 (t, J ¼ 7.6 Hz, 3H, CH3),
1.28e1.35 (m, 2H, cyclohexane), 1.52e1.59 (m, 2H, cyclohexane),
1.66e1.69 (m, 1H, cyclohexane), 1.78e1.81 (m, 3H, cyclohexane),
2.01 (s, 3H, CH3), 2.41 (q, J ¼ 7.6 Hz, 2H, CH2), 2.52 (q, J ¼ 7.6 Hz, 2H,
CH2), 2.68e2.72 (m, 1H, cyclohexane), 7.80 (bs, 1H, NH, D2O exch.);
Yellow powder, mp 125e130 ꢁC, 79% yield; 1H NMR (400 MHz,
CDCl3):
d
0.99 (s, 3H, CH3), 1.06 (s, 3H, CH3), 1.10 (t, J ¼ 7.4 Hz, 3H,
CH3), 1.23 (t, 3H, J ¼ 7.6 Hz, CH3), 1.28e1.32 (m, 1H, cyclohexane),
1.35e1.37 (m, 1H, cyclohexane), 1.63e1.66 (m, 2H, cyclohexane),
1.90e1.94 (m, 1H, cyclohexane), 2.01e2.04 (m, 1H, cyclohexane),
2.41 (q, J ¼ 7.6 Hz, 2H, CH2), 2.52 (q, J ¼ 7.6 Hz, 2H, CH2), 2.75e2.77
(m,1H, cyclohexane), 3.27 (d, J ¼ 13.6 Hz,1H, cyclohexane), 8.03 (bs,
13C NMR (101 MHz, CDCl3):
d 9.05 (NHCOCH2CH3), 9.19
(NCOCH2CH3), 25.64 (CH2-cyclohexane), 25.88 (CH2-cyclohexane),
26.14 (CH2-cyclohexane), 27.07 (CH2-cyclohexane), 27.24 (CH3C),
28.32
1H, NH, D2O exch.); 13C NMR (101 MHz, CDCl3):
d
9.07
(CH2-cyclohexane),
29.29
(NHCOCH2CH3),
29.40
(NHCOCH2CH3), 9.24 (NCOCH2CH3), 19.68 (CH2-cyclohexane), 26.29
(CH3CCH3), 29.27 (CH3C-cyclohexane), 30.12 (CH3C-cyclohexane),
31.97 (NHCOCH2CH3), 33.92 (NCOCH2CH3), 35.70 (CH2-cyclo-
hexane), 37.66 (CH2-cyclohexane), 46.34 (CH2-cyclohexane), 83.15
(C-cyclohexane), 144.54 (C]N), 173.35 (NC]O), 173.65 (NHC]O).
Anal. Calcd for C15H25N3O2S: C, 57.85; H, 8.09; N, 13.49. Found: C,
57.60; H, 8.27; N, 13.74.
(NCOCH2CH3), 44.16 (CH-cyclohexane), 86.46 (CH3C), 143.37 (C]
N), 172.84 (NC]O), 172.89 (NHC]O). Anal. Calcd for C15H25N3O2S:
C, 57.85; H, 8.09; N, 13.49. Found: C, 57.50; H, 8.36; N, 13.17.
4.2.86. N-[4-(1-Oxopropyl)-5-(furan-2-yl)-4,5-dihydro-1,3,4-
thiadiazol-2-yl]propanamide (80)
Light yellow powder, mp 181e186 ꢁC, 52% yield; 1H NMR

258
C. De Monte et al. / European Journal of Medicinal Chemistry 105 (2015) 245e262
(400 MHz, CDCl3):
d
1.12 (t, J ¼ 7.4 Hz, 3H, CH3), 1.20 (t, J ¼ 7.6 Hz,
4.2.91. N-[4-(1-Oxopropyl)-5-methyl-5-(thiophen-3-yl)-4,5-
dihydro-1,3,4-thiadiazol-2-yl]propanamide (85)
3H, CH3), 2.35e2.45 (m, 2H, CH2), 2.50e2.61 (m, 2H, CH2),
6.29e6.30 (m, 2H, furan), 6.91 (s, 1H, thiadiazole), 7.33 (s, 1H,
furan), 8.37 (bs, 1H, NH, D2O exch.); 13C NMR (101 MHz, DMSO-d6):
White powder, mp 135e136 ꢁC, 87% yield; 1H NMR (400 MHz,
DMSO-d6):
d
0.97e1.02 (m, 6H, 2 ꢂ CH3), 2.26 (s, 3H, CH3), 2.33 (q,
d
9.26 (NHCOCH2CH3, NCOCH2CH3), 27.44 (NCOCH2CH3), 28.73
J ¼ 7.6 Hz, 2H, CH2), 2.53e2.57 (m, 2H, CH2), 7.04e7.05 (m, 1H,
thiophene), 7.37e7.38 (m, 1H, thiophene), 7.48e7.50 (m, 1H, thio-
phene), 11.55 (bs, 1H, NH, D2O exch.); 13C NMR (101 MHz, DMSO-
(NHCOCH2CH3), 59.86 (CH), 107.49 (C3H-furan), 111.08 (C4H-furan),
143.55 (C5H-furan), 146.27 (C]N), 152.04 (C2-furan), 171.07 (NC]
O), 173.54 (NHC]O). Anal. Calcd for C12H15N3O3S: C, 51.23; H, 5.37;
N, 14.94. Found: C, 51.08; H, 5.56; N, 15.17.
d6):
d 9.40 (NHCOCH2CH3, NCOCH2CH3), 27.53 (NHCOCH2CH3),
28.68 (NCOCH2CH3), 28.93 (CH3C), 76.35 (CH3C), 121.04 (C2H-thio-
phene), 126.27 (C5H-thiophene), 127.17 (C4H-thiophene), 142.85
(C3-thiophene), 145.55 (C]N), 171.46 (NC]O), 173.40 (NHC]O).
Anal. Calcd for C13H17N3O2S2: C, 50.14; H, 5.50; N, 13.49. Found: C,
50.29; H, 5.72; N, 13.26.
4.2.87. N-[4-(1-Oxopropyl)-5-(furan-2-yl)-5-methyl-4,5-dihydro-
1,3,4-thiadiazol-2-yl]propanamide (81)
Light brown powder, mp 105e110 ꢁC, 95% yield; 1H NMR
(400 MHz, CDCl3):
d
1.11 (t, J ¼ 7.6 Hz, 3H, CH3), 1.20 (t, J ¼ 7.6 Hz,
3H, CH3), 2.32 (s, 3H, CH3), 2.37 (q, J ¼ 7.6 Hz, 2H, CH2), 2.57 (q,
J ¼ 7.6 Hz, 2H, CH2), 6.32e6.34 (m, 2H, furan), 7.37 (s, 1H, furan),
4.2.92. N-(4,5-Dihydro-5-phenyl-4-propionyl-1,3,4-thiadiazol-2-
yl)propionamide (86)
8.47 (bs, 1H, NH, D2O exch.); 13C NMR (101 MHz, CDCl3):
d
8.98
White powder, mp 140e145 ꢁC, 73% yield; 1H NMR (400 MHz,
(NHCOCH2CH3), 9.10 (NCOCH2CH3), 26.00 (NCOCH2CH3), 28.94
(NHCOCH2CH3), 28.98 (CH3C), 74.31 (CH3C), 106.74 (C3H-furan),
110.62 (C4H-furan), 142.44 (C5H-furan), 143.15 (C]N), 153.26 (C2-
furan), 172.99 (NC]O), 173.42 (NHC]O). Anal. Calcd for
DMSO-d6):
d
0.98e1.06 (m, 6H, 2 ꢂ CH3), 2.35 (q, J ¼ 7.6 Hz, 2H,
CH2), 2.55e2.61 (m, 2H, CH2), 6.83 (s, 1H, thiadiazoline), 7.23e7.25
(m, 2H, benzene), 7.29e7.31 (m, 1H, benzene), 7.34e7.36 (m, 2H,
benzene), 11.71 (bs, 1H, NH, D2O exch.). 13C NMR (101 MHz, DMSO-
C
13H17N3O3S: C, 52.86; H, 5.80; N, 14.23. Found: C, 53.02; H, 5.98; N,
d6):
d
9.24 (NHCOCH2CH3), 9.37 (NCOCH2CH3), 27.51
14.48.
(NHCOCH2CH3), 28.75 (NCOCH2CH3), 66.43 (CH), 125.51 (2 ꢂ CH-
benzene), 128.45 (CH-benzene), 129.14 (2 ꢂ CH-benzene), 142.01
(C-benzene), 146.51 (C]N), 171.16 (NC]O), 173.48 (NHC]O). Anal.
Calcd for C14H17N3O2S: C, 57.71; H, 5.88; N, 14.42. Found: C, 57.49;
H, 5.52; N, 14.71.
4.2.88. N-[4-(1-Oxopropyl)-5-(thiophen-2-yl)-4,5-dihydro-1,3,4-
thiadiazol-2-yl]propanamide (82)
Light brown powder, mp 158e160 ꢁC, 84% yield; 1H NMR
(400 MHz, DMSO-d6):
d
1.00e1.05 (m, 6H, 2 ꢂ CH3), 2.34e2.40 (m,
2H, CH2), 2.52e2.56 (m, 2H, CH2), 6.95e6.97 (m, 1H, thiophene),
7.06e7.07 (m,1H, thiophene), 7.13 (s,1H, thiadiazole), 7.45e7.46 (m,
1H, thiophene), 11.75 (bs, 1H, NH, D2O exch.); 13C NMR (101 MHz,
4.2.93. N-(5-Ethyl-5-phenyl-4-propionyl-4,5-dihydro-1,3,4-
thiadiazol-2-yl)propionamide (88)
White powder, mp 205e207 ꢁC, 85% yield; 1H NMR (400 MHz,
DMSO-d6):
d
9.25
(NHCOCH2CH3,
NCOCH2CH3),
27.44
DMSO-d6):
d
0.97e1.04 (m, 9H, 3 ꢂ CH3), 2.32 (q, J ¼ 7.6 Hz, 2H,
(NHCOCH2CH3), 28.73 (NCOCH2CH3), 62.07 (CH), 125.62 (C5H-
thiophene), 126.57 (C3H-thiophene), 127.27 (C4H-thiophene),
145.19 (C2-thiophene), 146.65 (C]N), 170.98 (NC]O), 173.55
(NHC]O). Anal. Calcd for C12H15N3O2S2: C, 48.46; H, 5.08; N, 14.13.
Found: C, 48.20; H, 5.32; N, 14.35.
CH2), 2.39e2.41 (m, 1H, CH2), 2.57e2.65 (m, 2H, CH2), 2.85e2.90
(m, 1H, CH2), 7.24e7.38 (m, 5H, benzene), 11.55 (bs, 1H, NH, D2O
exch.); 13C NMR (101 MHz, DMSO-d6):
d 9.17 (CH3CH2), 9.29
(NHCOCH2CH3), 9.59 (NCOCH2CH3), 28.67 (NHCOCH2CH3), 28.86
(NCOCH2CH3), 29.36 (CH3CH2), 83.88 (CH3CH2C), 124.89 (2 ꢂ CH-
benzene), 127.72 (CH-benzene), 128.94 (2 ꢂ CH-benzene), 142.72
(C-benzene), 145.18 (C]N), 171.34 (NC]O), 173.41 (NHC]O). Anal.
Calcd for C16H21N3O2S: C, 60.16; H, 6.63; N, 13.16. Found: C, 59.91;
H, 6.44; N, 13.37.
4.2.89. N-[4-(1-Oxopropyl)-5-methyl-5-(thiophen-2-yl)-4,5-
dihydro-1,3,4-thiadiazol-2-yl]propanamide (83)
White powder, mp 115e120 ꢁC, 73% yield; 1H NMR (400 MHz,
Aceton-d6):
d
0.77 (t, J ¼ 7.6 Hz, 3H, CH3), 0.88 (t, J ¼ 7.6 Hz, 3H,
CH3), 2.16 (s, 3H, CH3), 2.22e2.29 (m, 4H, 2 ꢂ CH2), 6.68e6.70 (m,
1H, thiophene), 6.85e6.86 (m, 1H, thiophene), 7.15e7.16 (d,
J ¼ 4 Hz, 1H, thiophene), 10.23 (bs, 1H, NH, D2O exch.); 13C NMR
4.2.94. N-[4-(1-Oxopropyl)-5-(pyridin-2-yl)-4,5-dihydro-1,3,4-
thiadiazol-2-yl]propanamide (89)
Light yellow powder, mp 155e156 ꢁC, 35% yield; 1H NMR
(101 MHz, DMSO-d6):
d
9.35 (NHCOCH2CH3, NCOCH2CH3), 28.01
(400 MHz, CDCl3):
d
1.15 (t, J ¼ 7.6 Hz, 3H, CH3), 1.20 (t, J ¼ 7.4 Hz,
(NHCOCH2CH3), 28.66 (NCOCH2CH3), 29.04 (CH3C), 76.47 (CH3C),
124.61 (C5H-thiophene), 126.41 (C3H-thiophene), 127.11 (C4H-
thiophene), 142.50 (C2-thiophene), 149.31 (C]N), 171.57 (NC]O),
173.50 (NHC]O). Anal. Calcd for C13H17N3O2S2: C, 50.14; H, 5.50; N,
13.49. Found: C, 49.92; H, 5.22; N, 13.65.
3H, CH3), 2.36e2.43 (m, 2H, CH2), 2.59e2.66 (m, 2H, CH2), 6.91 (s,
1H, thiadiazoline), 7.22e7.26 (m, 2H, pyridine), 7.67e7.71 (m, 1H,
pyridine), 8.52 (bs, 1H, NH, D2O exch.), 8.60e8.61 (m, 1H, pyridine);
13C NMR (101 MHz, CDCl3):
d 8.75 (NHCOCH2CH3), 8.84
(NCOCH2CH3), 27.55 (NHCOCH2CH3), 28.95 (NCOCH2CH3), 68.08
(CH), 120.40 (C5H-pyridine), 123.45 (C3H-pyridine), 137.47 (C4H-
pyridine), 146.29 (C]N), 149.50 (C6H-pyridine), 158.24 (C2-pyri-
dine), 172.75 (NC]O), 173.31 (NHC]O). Anal. Calcd for
4.2.90. N-[4-(1-Oxopropyl)-5-(5-chlorothiophen-2-yl)-5-methyl-
4,5-dihydro-1,3,4-thiadiazol-2-yl]propanamide (84)
Yellow powder, mp 120e123 ꢁC, 47% yield; 1H NMR (400 MHz,
C13H16N4O2S: C, 53.41; H, 5.52; N, 19.16. Found: C, 53.19; H, 5.83; N,
CDCl3):
d
1.11 (t, J ¼ 7.4 Hz, 3H, CH3),1.23 (t, J ¼ 7.4 Hz, 3H, CH3), 2.38
19.33.
(s, 3H, CH3), 2.40e2.44 (m, 2H, CH2), 2.54 (q, J ¼ 7.6 Hz, 2H, CH2),
6.74 (d, J ¼ 4.0 Hz, 1H, thiophene), 6.89 (d, J ¼ 4.0 Hz, 1H, thio-
phene), 8.18 (bs, 1H, NH, D2O exch.); 13C NMR (101 MHz, CDCl3):
4.2.95. N-[4-(1-Oxopropyl)-5-methyl-5-(pyridin-2-yl)-4,5-
dihydro-1,3,4-thiadiazol-2-yl]propanamide (90)
d
8.94 (NHCOCH2CH3), 9.07 (NCOCH2CH3), 27.49 (NHCOCH2CH3),
White powder, mp 136e141 ꢁC, 73% yield; 1H NMR (400 MHz,
29.05 (NCOCH2CH3), 29.17 (CH3C), 76.96 (CH3C), 124.04 (C3H-thio-
phene), 125.55 (ClC5-thiophene), 130.80 (C4H-thiophene), 142.97
(C2-thiophene), 146.47 (C]N), 173.06 (NC]O), 173.23 (NHC]O).
Anal. Calcd for C13H16ClN3O2S2: C, 45.14; H, 4.66; N, 12.15. Found: C,
45.38; H, 4.90; N, 12.41.
DMSO-d6):
d
0.97e1.03 (m, 6H, 2 ꢂ CH3), 2.28 (s, 3H, CH3),
2.32e2.35 (m, 2H, CH2), 2.56e2.60 (m, 2H, CH2), 7.27e7.30 (m, 1H,
pyridine), 7.36 (d, J ¼ 8.0 Hz, 1H, pyridine), 7.79 (t, J ¼ 7.4 Hz, 1H,
pyridine), 8.52e8.53 (m, 1H, pyridine), 11.58 (bs, 1H, NH, D2O
exch.); 13C NMR (101 MHz, DMSO-d6):
d 9.32 (NHCOCH2CH3,

C. De Monte et al. / European Journal of Medicinal Chemistry 105 (2015) 245e262
259
NCOCH2CH3), 26.35 (NHCOCH2CH3), 28.64 (NCOCH2CH3), 28.74
4.2.100. N-[4-(1-Oxopropyl)-5-methyl-5-(pyrazin-2-yl)-4,5-
(CH3C), 80.05 (CH3C), 119.53 (C5H-pyridine), 122.84 (C3H-pyridine),
137.51 (C4H-pyridine), 142.53 (C]N), 149.07 (C6H-pyridine), 161.44
(C2-pyridine), 171.67 (NC]O), 173.48 (NHC]O). Anal. Calcd for
dihydro-1,3,4-thiadiazol-2-yl]propanamide (95)
White powder, mp 152e155 ꢁC, 70% yield; 1H NMR (400 MHz,
DMSO-d6):
d
0.97e1.02 (m, 6H, 2 ꢂ CH3), 2.28 (s, 3H, CH3), 2.34 (q,
C
14H18N4O2S: C, 54.88; H, 5.92; N, 18.29. Found: C, 55.04; H, 6.11; N,
J ¼ 7.6 Hz, 2H, CH2), 2.55e2.60 (m, 2H, CH2), 8.55e8.56 (m, 1H,
pyrazine), 8.60e8.61 (m, 1H, pyrazine), 8.74 (s, 1H, pyrazine), 11.68
17.97.
(bs, 1H, NH, D2O exch.); 13C NMR (101 MHz, DMSO-d6):
d 9.24
(NHCOCH2CH3, NCOCH2CH3), 25.84 (NHCOCH2CH3), 28.53
(NCOCH2CH3), 28.62 (CH3C), 78.37 (CH3C), 141.97 (C5H-pyrazine),
142.55 (C3H-pyrazine), 143.61 (C6H-pyrazine), 143.74 (C]N),
156.86 (C2-pyrazine), 171.74 (NC]O), 173.62 (NHC]O). Anal. Calcd
for C13H17N5O2S: C, 50.80; H, 5.57; N, 22.78. Found: C, 50.65; H,
5.89; N, 22.99.
4.2.96. N-[4-(1-Oxopropyl)-5-(pyridin-3-yl)-4,5-dihydro-1,3,4-
thiadiazol-2-yl]propanamide (91)
Light yellow powder, mp 159e162 ꢁC, 71% yield; 1H NMR
(400 MHz, DMSO-d6):
d
0.99e1.05 (m, 6H, 2 ꢂ CH3), 2.36 (q,
J ¼ 7.6 Hz, 2H, CH2), 2.54e2.65 (m, 2H, CH2), 6.89 (s, 1H, thiadia-
zoline), 7.39e7.42 (m, 1H, pyridine), 7.65 (d, J ¼ 8.0 Hz, 1H, pyri-
dine), 8.49e8.51 (m, 2H, pyridine), 11.78 (bs,1H, NH, D2O exch.); 13
C
4.2.101. N-[4-(1-Oxopropyl)-5-(1,3-benzodioxol-5-yl)-4,5-dihydro-
1,3,4-thiadiazol-2-yl]propanamide (96)
NMR (101 MHz, DMSO-d6):
d 9.30 (NHCOCH2CH3, NCOCH2CH3),
27.41 (NHCOCH2CH3), 28.70 (NCOCH2CH3), 64.35 (CH), 124.35 (C5H-
pyridine), 133.54 (C3-pyridine), 137.41 (C4H-pyridine), 146.40 (C]
N), 147.25 (C6H-pyridine), 149.69 (C2H-pyridine), 171.26 (NC]O),
173.59 (NHC]O). Anal. Calcd for C13H16N4O2S: C, 53.41; H, 5.52; N,
19.16. Found: C, 53.24; H, 5.36; N, 18.94.
White powder, mp 81e87 ꢁC, 54% yield; 1H NMR (400 MHz,
DMSO-d6):
d
1.02e1.14 (m, 6H, 2 ꢂ CH3), 2.27e2.45 (m, 2H, CH2),
3.30e3.39 (m, 2H, CH2), 6.01 (s, 2H, CH2-benzodioxole), 6.67e6.91
(m, 3H, benzodioxole, 1H, thiadiazoline), 11.70 (bs, 1H, NH, D2O
exch.); 13C NMR (101 MHz, DMSO-d6):
d 9.21 (NHCOCH2CH3), 9.32
(NCOCH2CH3), 27.49 (NHCOCH2CH3), 28.72 (NCOCH2CH3), 66.35
(CH), 101.67 (CH2-benzodioxole), 106.08 (CH-benzodioxole), 108.62
(CH-benzodioxole), 119.03 (CH-benzodioxole), 136.03 (C-benzo-
dioxole), 146.49 (C]N), 147.49 (C-benzodioxole), 147.98 (C-benzo-
dioxole), 171.05 (NC]O), 173.44 (NHC]O). Anal. Calcd for
4.2.97. N-[4-(1-Oxopropyl)-5-methyl-5-(pyridin-3-yl)-4,5-
dihydro-1,3,4-thiadiazol-2-yl]propanamide (92)
White powder, mp 159e164 ꢁC, 52% yield; 1H NMR (400 MHz,
CDCl3):
d
1.07 (t, J ¼ 7.6 Hz, 3H, CH3), 1.18 (t, J ¼ 7.4 Hz, 3H, CH3),
2.34e2.38 (m, 2H, CH2), 2.40 (s, 3H, CH3), 2.55e2.57 (m, 2H, CH2),
7.31e7.34 (m, 1H, pyridine), 7.80 (d, J ¼ 8.0 Hz, 1H, pyridine),
8.54e8.55 (m, 1H, pyridine), 8.73 (s, 1H, pyridine), 9.64 (bs, 1H, NH,
C15H17N3O4S: C, 53.72; H, 5.11; N, 12.53. Found: C, 53.90; H, 5.42; N,
12.78.
D2O exch.); 13C NMR (101 MHz, CDCl3):
d 8.84 (NHCOCH2CH3), 8.95
4.2.102. N-[4-(1-Oxopropyl)-5-(naphthalen-1-yl)-4,5-dihydro-
1,3,4-thiadiazol-2-yl]propanamide (97)
(NCOCH2CH3), 26.34 (NHCOCH2CH3), 28.92 (NCOCH2CH3), 29.08
(CH3C), 77.54 (CH3C), 123.52 (C5H-pyridine), 133.71 (C4H-pyridine),
139.42 (C3-pyridine), 143.32 (C]N), 146.12 (C6H-pyridine), 148.03
(C2H-pyridine), 172.40 (NC]O), 173.40 (NHC]O). Anal. Calcd for
White powder, mp 180e183 ꢁC, 92% yield; 1H NMR (400 MHz,
CDCl3):
d
1.11 (t, J ¼ 7.4 Hz, 3H, CH3), 1.26 (t, J ¼ 7.6 Hz, 3H, CH3), 2.28
(q, J ¼ 7.6 Hz, 2H, CH2), 2.74e2.80 (m, 2H, CH2), 7.24 (d, J ¼ 7.2 Hz,
1H, naphthalene), 7.41 (t, J ¼ 7.6 Hz,1H, naphthalene), 7.51e7.57 (m,
2H, naphthalene), 7.59 (s, 1H, thiadiazoline), 7.80 (d, J ¼ 8.0 Hz, 1H,
naphthalene), 7.88e7.93 (m, 2H, naphthalene), 8.23 (bs, 1H, NH,
C
14H18N4O2S: C, 54.88; H, 5.92; N, 18.29. Found: C, 54.61; H, 5.76; N,
18.07.
D2O exch.); 13C NMR (101 MHz, DMSO-d6):
d 9.24 (NHCOCH2CH3),
4.2.98. N-[4-(1-Oxopropyl)-5-(pyridin-4-yl)-4,5-dihydro-1,3,4-
thiadiazol-2-yl]propanamide (93)
9.51 (NCOCH2CH3), 27.57 (NHCOCH2CH3), 28.73 (NCOCH2CH3),
64.23 (CH), 121.46 (CH-naphthalene), 123.58 (CH-naphthalene),
126.01 (CH-naphthalene), 126.61 (CH-naphthalene), 127.10 (CH-
naphthalene), 128.81 (CH-naphthalene), 128.95 (CH-naphthalene),
129.34 (C-naphthalene), 134.15 (C-naphthalene), 136.40 (C-naph-
thalene), 147.59 (C]N), 171.36 (NC]O), 173.55 (NHC]O). Anal.
Calcd for C18H19N3O2S: C, 63.32; H, 5.61; N, 12.31. Found: C, 63.15;
H, 5.93; N, 12.11.
Orange powder, mp 165e168 ꢁC, 31% yield; 1H NMR (400 MHz,
DMSO-d6):
d
1.00 (t, J ¼ 7.6 Hz, 3H, CH3), 1.05 (t, J ¼ 7.4 Hz, 3H, CH3),
2.34 (q, J ¼ 7.6 Hz, 2H, CH2), 2.56e2.69 (m, 2H, CH2), 6.83 (s, 1H,
thiadiazoline), 7.24 (d, J ¼ 6.0 Hz, 2H, pyridine), 8.56 (d, J ¼ 6.0 Hz,
2H, pyridine), 11.98 (bs, 1H, NH, D2O exch.); 13C NMR (101 MHz,
DMSO-d6):
d 9.21 (NHCOCH2CH3), 9.32 (NCOCH2CH3), 27.35
(NHCOCH2CH3), 28.70 (NCOCH2CH3), 65.02 (CH), 120.31 (C3H-pyr-
idine, C5H-pyridine), 146.36 (C]N), 150.10 (C4-pyridine), 150.62
(C2H-pyridine, C6H-pyridine), 171.38 (NC]O), 173.71 (NHC]O).
Anal. Calcd for C13H16N4O2S: C, 53.41; H, 5.52; N, 19.16. Found: C,
53.62; H, 5.84; N, 19.37.
4.2.103. N-[4-(1-Oxopropyl)-5-methyl-5-(naphthalen-1-yl)-4,5-
dihydro-1,3,4-thiadiazol-2-yl]propanamide (98)
White powder, mp 183e185 ꢁC, 88% yield; 1H NMR (400 MHz,
CDCl3):
d
1.00 (t, J ¼ 7.4 Hz, 3H, CH3), 1.21 (t, J ¼ 7.6 Hz, 3H, CH3),
4.2.99. N-[4-(1-Oxopropyl)-5-methyl-5-(pyridin-4-yl)-4,5-
dihydro-1,3,4-thiadiazol-2-yl]propanamide (94)
2.34e2.40 (m, 2H, CH2), 2.49 (s, 3H, CH3), 2.52e2.56 (m, 2H, CH2),
7.43e7.48 (m, 3H, naphthalene), 7.65 (d, J ¼ 7.6 Hz, 1H, naphtha-
lene), 7.83 (d, J ¼ 8.4 Hz, 1H, naphthalene), 7.87e7.89 (m, 1H,
naphthalene), 8.03e8.06 (m, 1H, naphthalene), 8.21 (bs, 1H, NH,
Light yellow powder, mp 126e130 ꢁC, 55% yield; 1H NMR
(400 MHz, CDCl3):
d
1.12 (t, J ¼ 7.4 Hz, 3H, CH3),1.19 (t, J ¼ 7.6 Hz, 3H,
CH3), 2.36 (s, 3H, CH3), 2.38e2.42 (m, 2H, CH2), 2.60 (q, J ¼ 7.6 Hz,
2H, CH2), 7.36 (d, J ¼ 6.0 Hz, 2H, pyridine), 8.63 (d, J ¼ 5.6 Hz, 2H,
pyridine), 9.84 (bs, 1H, NH, D2O exch.); 13C NMR (101 MHz, Meth-
D2O exch.); 13C NMR (101 MHz, DMSO-d6):
d 9.26 (NHCOCH2CH3),
9.37 (NCOCH2CH3), 28.45 (NHCOCH2CH3), 28.75 (NCOCH2CH3),
29.86 (CH3C), 78.35 (CH3C), 124.06 (CH-naphthalene), 125.05 (CH-
naphthalene), 125.23 (CH-naphthalene), 125.85 (CH-naphthalene),
126.25 (CH-naphthalene), 129.83 (CH-naphthalene), 129.84 (CH-
naphthalene), 130.27 (C-naphthalene), 134.69 (C-naphthalene),
136.22 (C-naphthalene), 144.21 (C]N), 170.59 (NC]O), 173.62
(NHC]O). Anal. Calcd for C19H21N3O2S: C, 64.20; H, 5.95; N, 11.82.
Found: C, 64.02; H, 6.14; N, 12.04.
anol-d4):
d 7.99 (NHCOCH2CH3), 8.06 (NCOCH2CH3), 24.99
(NHCOCH2CH3), 28.37 (NCOCH2CH3), 28.51 (CH3C), 77.81 (CH3C),
120.22 (C3H-pyridine, C5H-pyridine), 143.41 (C]N), 149.17 (C2H-
pyridine, C6H-pyridine), 153.25 (C4-pyridine), 172.86 (NC]O),
173.72 (NHC]O). Anal. Calcd for C14H18N4O2S: C, 54.88; H, 5.92; N,
18.29. Found: C, 54.59; H, 5.68; N, 18.54.

260
C. De Monte et al. / European Journal of Medicinal Chemistry 105 (2015) 245e262
4.2.104. N-[4-(1-Oxopropyl)-5-methyl-5-(naphthalen-2-yl)-4,5-
dihydro-1,3,4-thiadiazol-2-yl]propanamide (99)
4.2.108. Synthesis of N-(5-phenyl-1,3,4-thiadiazol-2-yl)acetamide
(103)
White powder, mp 168e174 ꢁC, 66% yield; 1H NMR (400 MHz,
Potassium permanganate (2.0 eq.) was added portion wise at
0 ꢁC to a stirring suspension of compound 31 (1.0 eq.) in acetic acid
(25 mL). After 1 h H2O (12 mL) and H2O2 30% (1.1 eq.) were added
and the reaction mixture was stirred for 15 h at 0 ꢁC. The mixture
was poured on ice and extracted with chloroform (3 ꢂ 30 mL). The
organics were reunited, dried over sodium sulfate and concen-
trated in vacuo. Purification by column chromatography on silica gel
(ethyl acetate:petroleum ether 1:1) gave compound 103 as a white
powder (80% yield); mp 284e286 ꢁC; 1H NMR (400 MHz, DMSO-
CDCl3):
d
1.05 (t, J ¼ 7.6 Hz, 3H, CH3), 1.14 (t, J ¼ 7.4 Hz, 3H, CH3), 2.19
(q, J ¼ 7.6 Hz, 2H, CH2), 2.50 (s, 3H, CH3), 2.65 (q, J ¼ 7.6 Hz, 2H, CH2),
7.46e7.49 (m, 2H, naphthalene), 7.57e7.60 (m, 1H, naphthalene),
7.78e7.83 (m, 4H, naphthalene), 8.60 (bs, 1H, NH, D2O exch.); 13C
NMR (101 MHz, DMSO-d6):
d
9.36 (NHCOCH2CH3), 9.48
(NCOCH2CH3), 27.00 (NHCOCH2CH3), 28.76 (NCOCH2CH3), 28.95
(CH3C), 79.22 (CH3C), 123.21 (CH-naphthalene), 124.03 (CH-naph-
thalene), 126.77 (CH-naphthalene), 126.90 (CH-naphthalene),
127.81 (CH-naphthalene), 128.67 (CH-naphthalene), 128.83 (CH-
naphthalene), 132.61 (C-naphthalene), 132.96 (C-naphthalene),
141.40 (C-naphthalene), 142.96 (C]N), 171.44 (NC]O), 173.51
(NHC]O). Anal. Calcd for C19H21N3O2S: C, 64.20; H, 5.95; N, 11.82.
Found: C, 64.43; H, 5.74; N, 11.59.
d6):
2H, benzene), 12.65 (bs, 1H, NH, D2O exch.); 13C NMR (101 MHz,
DMSO-d6):
d 2.23 (s, 3H, CH3), 7.52e7.53 (m, 3H, benzene), 7.94e7.95 (m,
d
22.91 (NHCOCH3), 127.34 (2 ꢂ CH-benzene), 129.80
(2 ꢂ CH-benzene),130.67 (CH-benzene),131.00 (C-benzene),158.86
(C]N), 162.12 (NHC]O), 169.15 (C). Anal. Calcd for C10H9N3OS: C,
54.78; H, 4.14; N, 19.16. Found: C, 54.49; H, 4.36; N, 19.40.
4.2.105. N-[4-(1-Oxopropyl)-5-methyl-5-(2-oxo-2H-chromen-3-
yl)-4,5-dihydro-1,3,4-thiadiazol-2-yl]propanamide (100)
4.3. Cell lines
Light green powder, mp 224e226 ꢁC, 86% yield; 1H NMR
(400 MHz, CDCl3):
d
1.13 (t, J ¼ 7.6 Hz, 3H, CH3), 1.20 (t, J ¼ 7.4 Hz,
All cell lines used in our experiments were obtained from
Interlab Cell Line Collection (ICLC) (Istituto Nazionale per la Ricerca
sul Cancro, Genoa, Italy). PC3, an androgen-independent human
prostate cell line, was grown in DMEM (Euroclone, Life Science
Division, GB, Pero, Italy). SK-MEL-5 e SK-MEL-28 human melanoma
cells were grown in DMEM supplemented with Non Essential
Amino Acids (NEAA) and Na pyruvate. All media were supple-
3H, CH3), 2.38e2.40 (m, 2H, CH2), 2.42 (s, 3H, CH3), 2.60e2.64 (m,
2H, CH2), 7.31e7.34 (m, 2H, chromene), 7.51e7.55 (m, 2H, chro-
mene), 7.59 (s,1H, chromene), 8.15 (bs,1H, NH, D2O exch.); 13C NMR
(101 MHz, DMSO-d6):
d 9.33 (NHCOCH2CH3, NCOCH2CH3), 27.35
(CH3C), 28.42 (NHCOCH2CH3), 28.63 (NCOCH2CH3), 76.23 (CH3C),
116.23 (CH-chromene), 118.97 (C-chromene), 125.12 (CH-chro-
mene), 127.57 (CH-chromene), 129.50 (C-chromene), 132.48 (CH-
chromene), 138.05 (CH-chromene), 143.99 (C]N), 153.17 (C-chro-
mene), 158.11 (OC]O), 171.65 (NC]O), 173.51 (NHC]O). Anal.
Calcd for C18H19N3O4S: C, 57.89; H, 5.13; N, 11.25. Found: C, 57.64; H,
4.90; N, 11.02.
mented with 100 U/mL penicillin, 100 mg/mL 250 streptomycin and
10% fetal bovine serum (Euroclone, Life Science Division, GB, Pero,
Italy). Cells were maintained in a tissue culture incubator at 37 ꢁC,
5% CO2. The cells were plated and after 24 h each compound was
added. Each investigated compound was dissolved in dimethyl
sulfoxide (DMSO; Sigma, USA) to get a concentration of 1 M, and
then successive dilutions were made in the culture medium.
4.2.106. N-[4-(1-Oxopropyl)-5-methyl-5-(ferrocen-2-yl)-4,5-
dihydro-1,3,4-thiadiazol-2-yl]propanamide (101)
Yellow powder, mp 170e173 ꢁC, 69% yield; 1H NMR (400 MHz,
4.4. Cells proliferation assay
DMSO-d6):
d
1.14 (t, J ¼ 7.4 Hz, 3H, CH3), 1.32 (t, J ¼ 7.4 Hz, 3H, CH3),
2.50 (s, 3H, CH3), 2.63e2.65 (m, 4H, 2 ꢂ CH2), 4.46 (s, 6H, ferrocene),
Cells were seeded at the density of 5 ꢂ 103/well in 96-wells flat
4.54 (s,1H, ferrocene), 4.60 (s,1H, ferrocene), 4.72 (s,1H, ferrocene),
plate in culture medium in triplicate. Following 24 h culture, 10 mM
11.68 (bs, 1H, NH, D2O exch.); 13C NMR (101 MHz, DMSO-d6):
d
9.31
of different compounds under investigation were added in tripli-
cate. After 72 h cells were then incubated with MTT (3-[4,5-
dimethylthiazol-2-yl]-2,5-diphenyltetrazolium) (SigmaeAldrich,
Saint Louis, Missouri, USA) for 4 h. Formed crystals were dissolved
(NHCOCH2CH3), 9.37 (NCOCH2CH3), 27.72 (NHCOCH2CH3), 28.67
(NCOCH2CH3), 28.78 (CH3C), 68.09 (CH-ferrocene), 68.55 (CH-
ferrocene), 68.98 (CH-ferrocene), 69.51 (5 ꢂ CH-ferrocene), 70.11
(CH-ferrocene) 78.99 (CH3C), 89.54 (C-ferrocene), 142.27 (C]N),
170.65 (NC]O), 173.30 (NHC]O). Anal. Calcd for C19H23FeN3O2S: C,
55.21; H, 5.61; N, 10.17. Found: C, 55.02; H, 5.82; N, 9.94.
in 100 mL of DMSO and further incubated for 15 min. The absor-
bance was measured in a plate reader spectrophotometer (Lab-
system Multiskan MS), using a test wavelength of 540 nm and a
reference wavelength of 690 nm. Cells incubated with culture
medium alone represented the controls, wells containing medium
alone represented the blanks.
4.2.107. Synthesis of 2,2,3,3,3-pentafluoro-N-[5-methyl-4-
(2,2,3,3,3-pentafluoropropanoyl)-5-phenyl-4,5-dihydro-1,3,4-
thiadiazol-2-yl]propanamide (102)
The thiosemicarbazone intermediate (1.0 eq.) was added to a
stirring solution of pentafluoropropionic anhydride (5.0 eq.) and
the reaction mixture was stirred for 72 h at 90 ꢁC. The mixture was
poured on ice and extracted with chloroform (3 ꢂ 30 mL). The
organics were reunited, dried over sodium sulfate and concen-
trated in vacuo. Purification by column chromatography on silica gel
(ethyl acetate:petroleum ether 1:2) gave compound 102 as a white
powder (84% yield); mp 123e125 ꢁC; 1H NMR (400 MHz, CDCl3):
4.5. Cell viability assay
Cytotoxicity was tested by the trypan blue dye exclusion. Briefly,
exponentially growing cells were suspended in a concentration of
1 ꢂ 105/mL in 24-well microplate and 24 h later the compound was
added (10 mM). Cells alone or cells in diluted DMSO represent the
control. After different incubation times (24, 48 and 72 h) cells were
harvested, diluted (1:1) with a solution 0.4% Trypan blue stain and
counted by an optical microscope in a Neubauer chamber. Cell
viability was determined and expressed as percent of control cell
growth. Cell viability in control samples was always 95e98%.
d
2.48 (s, 3H, CH3), 7.36e7.49 (m, 5H, benzene), 9.03 (bs, 1H, NH,
D2O exch.); 13C NMR (101 MHz, DMSO-d6):
d
25.69 (CH3C), 81.88
(CH3C), 125.21 (2 ꢂ CF2CF3, 2 ꢂ CH-benzene), 128.90 (CH-benzene),
129.23 (2 ꢂ CH-benzene, 2 ꢂ CF2CF3), 141.45 (C]N), 147.59 (C-
benzene), 153.82 (NC]O), 158.29 (NHC]O). Anal. Calcd for
4.6. Flow cytometry analysis
C
15H9F10N3O2S: C, 37.12; H, 1.87; N, 8.66. Found: C, 36.91; H, 1.55; N,
8.93.
Flow cytometry was used to detect quantitatively apoptotic rate

C. De Monte et al. / European Journal of Medicinal Chemistry 105 (2015) 245e262
261
and the distribution of cell cycle. After 0, 24 and 96 h of incubation
with 5 M of different compounds, both adherent and floating cells
received no payment in preparation of this manuscript.
m
were collected in ice-cold phosphate buffer (PBS) and then
Acknowledgments
1.5 ꢂ 106 were fixed in 2 mL ethanol 70%, and then incubated 1 h at
4 ꢁC. Cellular pellet was dissolved in 0.5 mL of 5
mg/mL propidium
This work was supported by “Ateneo 2012” project (Prof. D.
Secci) and Filas (research project nꢁ ASR2, Regione Lazio, Italy).
iodide and 1 mg/mL RNase A in PBS. The stained cells were incu-
bated at room temperature for 30 min in the dark. The DNA content
of the cells was analyzed by FACS Calibur flow cytometry using the
CellQuest analysis program in order to detect the distribution of
subpopulation through the cell cycle. The DNA content in the sub
G1 population was considered to represent apoptotic cells.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2015.10.023.
4.7. Reverse transcriptase polymerase chain reaction (rt-pcr) assay
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Conflict of interest
The authors state no conflict of interest and that they have

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