New triazolothiadiazole and triazolothiadiazine derivatives as kinesin Eg5 and HIV inhibitors: Synthesis, QSAR and modeling studies

Article abstract of DOI:10.1515/znb-2014-0162

A new series of fused 1,2,4-triazoles, namely 6-aryl-3-(furan-2-yl)-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazoles 3a-h and 4a-f as well as 6-aryl-3-(furan-2-yl)- 7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazines 5a-h, were synthesized by the condensation of 4-amin

Full text of DOI:10.1515/znb-2014-0162

Z. Naturforsch. 2015; 70(1)b: 47–58
Imtiaz Khana, Shahid Hameed*, Najim A. Al-Masoudib,*, Nabeel A. Abdul-Reda
and Jim Simpson
New triazolothiadiazole and triazolothiadiazine
derivatives as kinesin Eg5 and HIV inhibitors:
synthesis, QSAR and modeling studies
Abstract: A new series of fused 1,2,4-triazoles, namely
6-aryl-3-(furan-2-yl)-[1,2,4]triazolo[3,4-b][1,3,4]thia-
1 Introduction
diazoles 3a–h and 4a–f as well as 6-aryl-3-(furan-2-yl)-
Uncontrolled proliferation in cancer cells is dependent on
7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazines 5a–h, were
mitosis – a highly regulated process in cell division. The
synthesized by the condensation of 4-amino-5-(furan-2-
mitotic spindle, therefore, is a validated target in cancer
yl)-4H-1,2,4-triazole-3-thiol (2) with substituted aromatic
chemotherapy [1]. The multicomponent mitotic spindle is
acids and phenacyl bromides, respectively. The structures
responsible for accurate segregation of duplicated chro-
of the newly synthesized compounds were established
mosomes into their daughter cells in eukaryotes during
using spectroscopic analysis, while that of 3e was con-
cell division [2]. The spindle formation is essentially
firmed independently by a single-crystal X-ray structure
driven by microtubule-associated proteins and molecular
determination. The compounds were evaluated for their
motors, cytoplasmic dynein, and kinesins from various
antiviral activity against the replication of HIV-1 and HIV-2
subfamilies [3].
in MT-4 cells using an MTT assay. In a docking study, 4b
Small molecules of natural as well as synthetic origin
interacted with several amino acids in the reverse tran-
that target the microtubule cytoskeleton have been proven
scriptase (RT) binding site of HIV-1. Some new analogues
invaluable for investigating the mechanisms of cell divi-
were selected for evaluation of their Eg5 inhibitory activity
sion. Well-known antiproliferative agents, such as vinca
using an in vitro malachite green ATPase assay. The QSAR
alkaloids, colcimid, paclitaxel, and epothilones, that
of these new analogues was studied as well.
modulate the microtubule polymerization, interfere with
the polymerization–depolymerization process by alter-
Keywords: anti-HIV activity; kinesin Eg5 inhibitors;
ing microtubule dynamics resulting in extended mitotic
molecular modeling study; NNRTIs; triazolothiadiazines;
arrest and cell death [4]. Microtubule disrupters have been
triazolothiadiazoles.
proven effective chemotherapeutics. However, the toxicity
of microtubule disrupters imposes limitations to their use
DOI 10.1515/znb-2014-0162
Received July 25, 2014; accepted August 29, 2014
as anticancer agents [5], as microtubules are essential for
many cellular functions.
More recently, new mitotic targets such as kinesins
have attracted great interest worldwide as potential can-
didates for new generation antiproliferative drugs [6, 7].
a
Present address: School of Chemistry, University of Nottingham,
There are roughly 14 classes of kinesin motors with the
functions ranging from vesicular transport to cell divi-
sion. Most prominent of these is the kinesin spindle
protein (KSP), also known as Eg5, essential for the forma-
tion and separation of bipolar spindles during human cell
division [8]. Eg5 is a homotetrameric motor formed by the
antiparallel arrangement of two dimers [9] and has the
ability to crosslink antiparallel microtubules emanating
from the two centrosomes at G2/M. Through its plus-end-
directed microtubule-based motor activity, Eg5 is capable
of generating outward forces that contribute to the sepa-
ration of two centrosomes, a process that is essential for
University Park, Nottingham, NG7 2RD, UK.
b
Present address: Am Tannenhof 8, 78457 Konstanz, Germany.
*Corresponding authors: Shahid Hameed, Department of
Chemistry, Quaid-i-Azam University, Islamabad-45320, Pakistan,
e-mail: shameed@qau.edu.pk; and Najim A. Al-Masoudi,
Department of Chemistry, College of Science, University of Basrah,
Basrah, Iraq, e-mail: najim.al-masoudi@gmx.de
Imtiaz Khan: Department of Chemistry, Quaid-i-Azam University,
Islamabad-45320, Pakistan
Nabeel A. Abdul-Reda: Department of Chemistry, College of
Education, University of Qadisiya, Qadisiya, Iraq
Jim Simpson: Department of Chemistry, University of Otago,
P. O. Box 56, Dunedin 9054, New Zealand
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ꢀꢀꢀꢁ
48ꢀ ꢀI. Khan et al.: New triazolothiadiazole and triazolothiadiazine derivatives as kinesin Eg5 and HIV inhibitors
the successful establishment of a bipolar spindle during synthesis of new analogues of these compounds with evalu-
mitosis [3].
Anticancer drug screening and chemical biologi-
ation of their anti-HIV and kinesin Eg5 inhibitory activity.
cal approaches have identified a range of structurally
diverse KSP inhibitors that have been found to cause cell
cycle arrest, differentiation and/or apoptosis of tumor
cells. Over the past decade, the first generation of KSP
2 Results and discussion
inhibitors, including monastrol [10], (S)-trityl-l-cysteine 2.1 Chemistry
(STLC) [11], N-(4-acetyl-5-methyl-5-phenyl-4,5-di-
hydro-1,3,4-thiadiazol-2-yl)acetamide (K858) [12], In the present work, 4-amino-5-(furan-2-yl)-4H-[1,2,4]
[(5R,11aS)-2-benzyl-5-(3-hydroxyphenyl)-6H-1,2,3,5,11,11a- triazole-3-thiol (2), prepared previously from the avail-
hexahydroimidazo[1,5-b]-β-carboline-1,3-dione (Trans-24) able hydrazide 1 in two steps following a reported proce-
[13], Ispinesib [14], and MK-0731 [15], have been reported. dure [20], has been selected as a key intermediate for the
On the other hand, HIV-1 reverse transcriptase (HIV-1 synthesis of the target compounds. Treatment of 2 with
RT) that catalyzes several steps in the replication of HIV substituted benzoic acids in the presence of phosphoryl
has been identified as an attractive target for the devel- chloride furnished the triazolo-thiadiazole derivatives
opment of anti-AIDS drugs. The rapid emergence of drug 3a–h in 71–79ꢀ% yield, while similar treatment with sub-
resistance has increased the need for more diverse anti- stituted phenoxyacetic acids afforded the analogues 4a–f
HIV agents, with either new structures or mechanisms of in 70–80ꢀ% yield (Scheme 1).
action [16, 17]. Consequently, there is a need to develop
The structures of 3a–h and 4a–f were confirmed by
1
novel, selective, potent, safe and inexpensive antiviral the H NMR spectra, which exhibited additional signals
agents that are also effective against mutant strains of HIV. for the protons of the newly introduced aromatic ring.
In continuation of our ongoing studies on triazolothia- The methylene protons in compounds 4a–f appeared as
diazoles and triazolothiadiazines [18, 19], we report here the singlets in the region δꢀꢀ= ꢀꢀ5.68–5.53 ppm. The aromatic
N
N
N
N
2 steps
i
SH
CONHNH2
S
N
N
O
O
O
NH2
N
R1
1
2
3a–h
ii
iii
N
N
N
N
S
S
N
N
N
O
O
R2
N
O
4a–f
5a–h
R3
R3
4-OCH3C6
H4
R1
R2
Entry
Entry
4a
Entry
2-OH
4-OH
2-CH3
4-CH3
4-OCH3
4-F
5a
H4
3a 2-F,4-Cl-C6H3
4b
4c
3b
5b 4-Cl-C6
H4
3-Cl-4-F-C6H3
3c 3-furanyl
5c
4-F-C6
4d
4e
3d
5d 4-CH3-C6H4
2-CH
3-3-furanyl
3e
3f
5e
H
4-F-C6 4CH2
biphen-4-yl
4f
5f
H2
-C6
2-Cl-4,5-F2
2-pyrrolyl
naphthalen-l-yl
4-NO2-C6H4
3g
3h
5g
5h
H
H
4-OC2 5C6 4CH2
3,4-Cl2-C6H3
Scheme 1ꢂReagents and conditions: (i) ArCO2H, POCl3, reflux, 6–7 h; (ii) ArOCH2CO2H, POCl3, reflux, 6–7 h; (iii) PhC(ꢀ= ꢀO)CH2Br, EtOH, reflux,
6–7 h.
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ꢁꢀꢀꢀ
I. Khan et al.: New triazolothiadiazole and triazolothiadiazine derivatives as kinesin Eg5 and HIV inhibitorsꢂ
ꢂ49
Fig. 1ꢂMolecular structure of 6-(4-fluorobenzyl)-3-(furan-2-yl)-[1,2,4]triazolo[3,4-b][1,3,4] thiadiazole (3e) in the crystal. Displacement ellip-
soids are drawn at the 50ꢁ% probability level, H atoms as spheres with arbitrary radius.
protons of the phenoxy and furan rings have been fully
2.2 Bioactivities
assigned (cf. Experimental Section). In the 13C NMR
spectra of 3a–h and 4a–f, C-3 atoms of the triazolothiadia- 2.2.1 In vitro anti-kinesin Eg5 activity
zole ring resonated in the range δꢀꢀ= ꢀꢀ154.5–145.8 ppm, while
the C-6 signals were observed at δꢀꢀ= ꢀꢀ172.7–169.4 ppm. The Antimitotic agents that have been used so far in cancer
resonances at δꢀꢀ= ꢀꢀ163.7–155.6 ppm were assigned to C-8 of treatment, such as taxanes and vinca alkaloids, perturb
the fused nucleus. The structure of 3e was independently the tubulin polymerization/depolymerization, cause
confirmed by a single-crystal X-ray diffraction analysis mitotic arrest and subsequent cell death [22]. However,
(Fig. 1 and Experimental Section).
these drugs produce serious side effects because microtu-
Treatment of 2 with various substituted phenacyl bro- bules also have essential intracellular functions in non-
mides afforded 6-aryl-3-(furan-2-yl)-7H-[1,2,4]triazolo[3,4- dividing cells [5]. A new alternative approach to prevent
b][1,3,4]thiadiazines 5a–h in 73–81ꢀ% yield. The structural mitotic-spindle formation is the inhibition of proteins,
1
build-up of these compounds was assigned from the H such as the mitotic motors (kinesins) that interact with
1
and 13C NMR spectra. In the H NMR spectra of 5a–h, the microtubules and cause mitotic arrest [3].
signals at δꢀꢀ= ꢀꢀ4.63–4.43 ppm were assigned to the CH2
Monastrol (MA) has been reported as one of the anti-
protons of the thiadiazine moiety. In the 13C NMR spectra, mitotic agents that arrest the cells in mitosis by specifi-
the resonances at δꢀꢀ= ꢀꢀ23.1–23.6 ppm were assigned to the cally inhibiting Eg5 [10], a member of the kinesin 5 family.
methylene carbon atoms of the thiadiazine ring. Com- Like many enzyme inhibitors, monastrol might be sub-
pound 5h was selected for further NMR studies, since its strate-competitive, inhibiting the ATP hydrolysis cycle of
gradient HMBC spectrum [21] revealed three couplings Eg5 by directly competing with ATP [3, 10] or microtubule
3
of CH2 protons (δHꢀꢀ= ꢀꢀ4.46 ppm): a J coupling to C-8 of the binding. Alternatively, monastrol might inhibit the motor
triazolothiazdiazine ring at δCꢀꢀ= ꢀꢀ154.8 ppm, a 2JC,H coupling domain allosterically, either by inhibiting ATP hydroly-
with C-6 of the same ring at δCꢀꢀ= ꢀꢀ162.9 ppm as well as 3J cou- sis or by uncoupling partner head interactions to inhibit
pling with C-1′ of the aromatic ring at δCꢀꢀ= ꢀꢀ133.9 ppm. Addi- motor but not ATPase activity [23].
tionally, a 3JC,H coupling between 3-H of the furan moiety at
Compounds 3a, 3c, 3f, 4a, 4d, 5b, and 5g were screened
δHꢀꢀ= ꢀꢀ7.22 ppm and C-3 of the triazole ring at δCꢀꢀ= ꢀꢀ112.8 ppm for their inhibition of Eg5 activity using an in vitro malachite
was observed (Fig. 2).
green ATPase assay (enzyme-coupled assay) [24]. Compound
4a matched the selection criteria of a selective inhibitor of
Eg5 in this assay in comparison to monastrol. It showed an
ATPase inhibition value of 40ꢀ% at 100 μm concentration
(Fig. 3). From the data, it appears that a hydroxyl group at
position 2 of the aromatic ring is important for inhibition.
1
H
N
N
S
8
6
N
3
H
H
7
O
N
1'
2.2.2 In vitro anti-HIV activity
l
C
Compounds 3a–h, 4a–f and 5a–h were evaluated for
their in vitro anti-HIV-1 (strain IIIB) and anti-HIV-2 (strain
ROD) activity and monitored by the inhibition of the
l
C
Fig. 2ꢂJC,H correlations in the HMBC NMR spectrum of 5h.
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ꢀꢀꢀꢁ
50ꢀ ꢀI. Khan et al.: New triazolothiadiazole and triazolothiadiazine derivatives as kinesin Eg5 and HIV inhibitors
120%
2 replication in cell cultures with EC50 of ꢀ> ꢀ 1.51, ꢀ> ꢀ 0.89
and ꢀ> ꢀ 0.67 μm, respectively, but no selectivity was wit-
nessed (SI ꢀ< ꢀ 1). However, implantation of halogen (e.g.,:
F, Cl) or hydroxyl groups in 2 and 4 positions of the
phenyl ring considerably increased the anti-HIV activ-
ity, in comparison to the effectiveness of other functional
groups. This inhibitory activity can be shown also with
the triazolo[3,4-b][1,3,4]thiadiazine-substituted naph-
thalene moiety (e.g., 5f).
100%
80%
60%
40%
20%
0%
DMSO MA 3a
3c
3f
4a
4d
5b
5g
only 80 µM
2.2.3 Molecular modeling analysis
Fig. 3ꢂIn vitro ATPase activity of mitotic kinesin Eg5 of some tria-
zolothiadiazine derivatives.
The molecular modeling studies of 3a, 4a and 5f were
performed as HIV-1 reverse transcriptase (HIV-1 RT)
inhibitors using the software package sybyl 6.5 (TRIPOS
virus-induced cytopathic effect in the (MT-4) cells, based Assoc., St Louis, MO, USA) running on a Silicon Graph-
on an MTT assay [25]. The results are summarized in ics Octane 10 000 workstation. Conformational ener-
Table 1, in which the data for nevirapine (BOE/BIRG587) gies were calculated through the molecular mechanics
[26] and azidothymidine (DDN/AZT) [27] are included for TRIPOS force field [28]. Full geometry optimizations
comparison. Compounds 3e, 4b and 5f were found to be were realized with the semiempirical molecular orbital
the only compounds from the series inhibiting HIV-1 and method AM1.
Table 1ꢂIn vitro anti-HIV-1a and HIV-2b activity of triazolothiadizole and triazolothiadiazine derivatives 3a–h, 4a–f and 5a–h.
c
c
d
Entry
EC50 (ꢁμg mL–1
)
EC50 (ꢁμg mL–1
)
CC50 (ꢁμg mL–1
)
SIe (IIIB)
SIe (ROD)
3a
ꢀ> ꢀ47.53
ꢀ> ꢀ78.03
ꢀ> ꢀ67.53
ꢀ> ꢀ37.05
ꢀ> ꢀ1.51
ꢀ> ꢀ66.25
ꢀ> ꢀ59.15
ꢀ> ꢀ21.68
ꢀ> ꢀ11.75
ꢀ> ꢀ0.89
ꢀ> ꢀ79.48
ꢀ> ꢀ88.50
ꢀ> ꢀ30.98
ꢀ> ꢀ89.10
ꢀ> ꢀ1.29
ꢀ> ꢀ47.53
ꢀ> ꢀ78.03
ꢀ> ꢀ67.53
ꢀ> ꢀ37.05
ꢀ> ꢀ1.51
ꢀ> ꢀ66.25
ꢀ> ꢀ59.15
ꢀ> ꢀ21.68
ꢀ> ꢀ11.75
ꢀ> ꢀ0.89
ꢀ> ꢀ79.48
ꢀ> ꢀ88.50
ꢀ> ꢀ30.98
ꢀ> ꢀ89.10
ꢀ> ꢀ1.29
47.53
78.03
67.53
37.05
1.51
66.25
59.15
21.68
11.75
0.89
79.48
88.50
30.98
89.10
1.29
ꢀ< ꢀ1
ꢀ< ꢀ1
ꢀ< ꢀ1
ꢀ< ꢀ1
3b
3c
ꢀ< ꢀ1
ꢀ< ꢀ1
3d
ꢀ< ꢀ1
ꢀ< ꢀ1
3e
ꢀ< ꢀ1
ꢀ< ꢀ1
3f
ꢀ< ꢀ1
ꢀ< ꢀ1
3g
3h
4a
ꢀ< ꢀ1
ꢀ< ꢀ1
ꢀ< ꢀ1
ꢀ< ꢀ1
ꢀ< ꢀ1
ꢀ< ꢀ1
4b
4c
ꢀ< ꢀ1
ꢀ< ꢀ1
ꢀ< ꢀ1
ꢀ< ꢀ1
4d
ꢀ< ꢀ1
ꢀ< ꢀ1
4e
ꢀ< ꢀ1
ꢀ< ꢀ1
4f
ꢀ< ꢀ1
ꢀ< ꢀ1
5a
ꢀ< ꢀ1
ꢀ< ꢀ1
5b
5c
ꢀ> ꢀ71.28
ꢀ> ꢀ31.45
ꢀ> ꢀ11.56
ꢀ> ꢀ5.13
ꢀ> ꢀ71.28
ꢀ> ꢀ31.45
ꢀ> ꢀ11.56
ꢀ> ꢀ5.13
71.28
31.45
11.56
5.13
ꢀ< ꢀ1
ꢀ< ꢀ1
ꢀ< ꢀ1
ꢀ< ꢀ1
5d
ꢀ< ꢀ1
ꢀ< ꢀ1
5e
ꢀ< ꢀ1
ꢀ< ꢀ1
5f
ꢀ> ꢀ0.67
ꢀ> ꢀ0.67
0.67
ꢀ< ꢀ1
ꢀ< ꢀ1
5g
5h
Nevirapine
AZT
ꢀ> ꢀ83.20
ꢀ> ꢀ50.38
0.050
ꢀ> ꢀ83.20
ꢀ> ꢀ50.38
ꢀ> ꢀ4.00
83.20
50.38
ꢀ> ꢀ4.00
ꢀ> ꢀ25
ꢀ< ꢀ1
ꢀ< ꢀ1
ꢀ< ꢀ1
ꢀ< ꢀ1
ꢀ> ꢀ80
ꢀ> ꢀ11 363
ꢀ< ꢀ1
0.0022
0.00094
ꢀ> ꢀ26 596
aAnti-HIV-1 activity measured on strain IIIB. bAnti-HIV-2 activity measured with strain ROD. cCompound concentration required to achieve
50 % protection of MT-4 cells from HIV-1 and 2-induced cytopathogenic effect. dCompound concentration that reduces the viability of
mock-infected MT-4 cells by 50ꢁ%. eSelectivity index (CC50/EC50).
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I. Khan et al.: New triazolothiadiazole and triazolothiadiazine derivatives as kinesin Eg5 and HIV inhibitorsꢂ
ꢂ51
Table 2ꢂBinding energies, Ki, and inhibition constant values of 3a–g, 4a–f and 5a–g at Tꢁꢀ= ꢀꢁ298.5 K.
Compd.
Binding energya
Ki (ꢁμm)
Intermolecular
energya
Internal
energya
Torsional
energya
Unbound
external energya
3a
3b
3c
3d
3e
3f
3g
4a
4b
4c
4d
4e
4f
–7.07
–7.24
–6.90
–6.84
–6.95
–6.92
–6.25
–8.61
–9.60
–7.47
–7.29
–6.97
–6.98
–7.38
–7.52
–7.19
–7.41
–8.85
–9.83
–7.14
6.58
4.89
–7.69
–7.53
–7.2
–7.11
–7.41
–7.18
–6.57
–8.5
–8.13
–7.9
–7.81
–7.7
–7.47
–7.69
–7.81
–7.48
–7.7
–9.43
–10.43
–7.72
–0.53
–0.28
–0.15
–0.27
–0.51
–0.4
0.6
0.3
0.3
0.3
0.6
0.3
0.3
0.89
0.89
0.6
0.6
0.89
0.6
0.6
0.3
0.3
0.3
0.6
0.6
0.6
–0.55
–0.27
–0.16
–0.24
–0.37
–0.37
–0.26
–0.19
–0.37
–0.33
–0.34
–0.38
–0.28
–0.39
–0.35
–0.33
–0.35
–0.67
–0.9
8.73
9.63
8.05
8.52
26.23
0.4892
4.81
–0.24
–1.19
–0.39
–0.49
–0.41
–0.55
–0.39
–0.41
–0.37
–0.34
–0.37
–0.68
–0.9
3.36
4.56
7.79
7.65
5a
5b
5c
5d
5e
5f
3.9
3.07
5.35
3.67
0.3276
0.0618
5.85
5g
–0.42
–0.41
aThe energy values are in kcal mol–1.
The prospective ligands were ranked according to the arene-rich subpocket surrounded by the aromatic side
highest binding energy of the best conformers. Thus, the chains of Tyr179, Tyr186, and Trp227 residues and appar-
binding energy score for the 3a–g, 4a–f, and 5a–g series ently developing π-π stacking interactions with the three
ranged from –6.25 to –9.83 kcal mol–1 (Table 2), indicat- residues. The triazolothiadiazole backbone is located in
ing selectivity and potency profiles of these derivatives to the middle of the binding pocket, anchoring the oxygen
bind the active site of HIV-RT pocket, especially those car- atom of the furan ring in a favorable position for hydro-
rying hydroxyaryl residues (e.g., 4b). Compound 4b has gen bonding with the Lys102 of the reverse transcriptase
been selected to show its binding to the enzyme pocket. (RT) enzyme, in addition to the same bonding between
As shown in Fig. 4, the phenyl ring of 4b fitted into an the amino group of Lys101 with the hydroxyl group of the
Tyr186
N
N
Tyr179
OH
O
S
H
N
O
N
O
Val188
H
O
N
Val106
H
Lys102
H
Lys101
H2N
N
Trp227
H
Fig. 4ꢂCompound 4b shows three hydrogen bonds: Lys102 with the oxygen of the furan ring, Lys101 with the OH group of the phenyl group
and Tyr186 with the oxygen atom of the benzyl group. In addition, hydrophobic interactions between of the phenyl moiety of 4b and Tyr179,
Tyr186 as well as Trp227 of reverse transcriptase (RT) enzyme residues were observed.
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ꢀꢀꢀꢁ
52ꢀ ꢀI. Khan et al.: New triazolothiadiazole and triazolothiadiazine derivatives as kinesin Eg5 and HIV inhibitors
Table 4ꢂCalculated and predicted EC50 of 4a–f.
benzyl moiety. Overall, the combination of hydrophobic
interactions and π stacking appears to govern the binding
of 4b with HIV RT (binding energy –9.83 kcal mol–1).
Compd.
EC50 calcd.
EC50 predicted
Residuals
4a
4b
4c
4d
4e
4f
11.750000
0.890000
79.480000
88.500000
30.980000
89.100000
11.745702
0.893131
79.484749
88.494036
30.980246
89.102137
0.004298
–0.003131
–0.004749
0.005964
–0.000246
–0.002137
2.2.4 Quantitative structure-activity relationship
The binding scores for 3a, 4b, and 5f were congruent with
their anti-HIV activity. A good correlation between the
predicted binding free energies and the experimentally
observed inhibitory activities (EC50) (Tables 2 and 3) sug-
gests that the identified binding conformations of these
inhibitors are reliable. The results of docking studies
provide an insight into the pharmacophoric structural
EC50 = 221.046349884 × X5 + 10629.588732628 × X9
+ 2508.141746599 × X15 − 752.566106747 × X34
− 22049.514189079
requirements for the HIV-1 RT inhibitory activity of this where:
class of molecules.
X5: H: AlogP
For an understanding of the quantitative structure- X9: L: Balaban index JX
activity relationships, a statistical analysis using a X15: R: Kappa-3
genetic function approximation (GFA) method [29] was X34: AK: Chi (3): path (valence modified).
made. First, a study table was belted and presented in
Table 2. Second, a correlation matrix by using Fried-
man LOF measure [30] (Table 3) was derived, and finally
3 Conclusion
In conclusion, we report the in vitro inhibition of kinesin
Eg5 via the ATPase activity assay as well as the anti-HIV
activity. Compound 4b, as the only compound of the tria-
zolothiadiazine series, showed moderate inhibition of
ATPase activity (40ꢀ%) at 100 μm. In addition, compounds
3e, and 5f inhibited the replication of HIV-1 and HIV-2
in cell cultures, but the compounds need to be further
pursued for toxicity and pharmacokinetics.
regression parameters were obtained. Regarding this
method, various statistical measures can be adapted to
calculate the fitness of a GFA model during the evolution
process.
Table 3 shows the GFA analysis which gives a
summary of the input parameters used for the calculation.
Also, it reports whether the GFA algorithm converged in a
specified number of generations. The Friedman’s lack-of-
fit (LOF) score in Table 3 evaluates the QSAR model. The
lower the LOF, the less likely it is that the GFA model will
fit the data. The significant regression is given by the F
test, the higher the value, the better is the model. Using
this method, compounds 4a–f were selected for studying
the predicted equation for EC50 calculation (Table 4).
4 Experimental
4.1 General
Melting points were measured on a Stuart melting point
apparatus (SMP3) in open capillaries and are uncorrected.
IR spectra were recorded on an FTS 3000 MX, Bio-Rad
Merlin (Excalibur model) spectrophotometer. NMR
spectra were recorded either on a 300- or 400-MHz spec-
trometer (Avance III; Bruker, Reinstetten, Germany), using
the residual solvent signal for calibration of the spectrum.
Heteronuclear assignments were verified by HMBC spec-
troscopy experiments. The Rf values were determined by
employing Merck 60 F254 precoated silica gel plates; the
eluent was CHCl3-MeOH (9:1). Product spots were visu-
alized under UV light at 254 and 365 nm. The elemental
Table 3ꢂValidation table of the genetic function approximation.
Friedman LOF
4.95ꢁꢀ× ꢀꢁ10–4
1.00
R2
Adjusted R2
1.00
Cross validated R2
0.999983
yes
Significant regression
Significance-of-regression F value
Critical SOR F value (95ꢁ%)
Replicate points
2.229275ꢁꢀ× ꢀꢁ107
225.749641
0
Computed experimental error
Lack-of-fit points
0.00
1
Min expt. error for non-significant LOF (95ꢁ%)
0.004866
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ꢂ53
analysis was performed on a Leco CHNS-932 Elemental 3-(Furan-2-yl)-6-(furan-3-yl)-[1,2,4]triazolo[3,4-b]
Analyzer (Leco, St. Joseph, MI, USA).
[1,3,4]thiadiazole (3c)ꢁFrom furan-3-carboxylic acid
(123 mg).Yield: 186 mg (72ꢀ%), light-brown solid, m.p.: 177–
178ꢀ°C, Rfꢀꢀ= ꢀꢀ0.72.ꢀ–ꢀIR (neat): νꢀꢀ= ꢀꢀ3021 (Ar-H), 1621 (Cꢀ= ꢀN),
4.2 General procedure for the preparation of 1591, 1518 (2ꢀꢀ× ꢀꢀCꢀ= ꢀC) cm–1. – H NMR ([D6]DMSO): δꢀꢀ= ꢀꢀ8.03
1
(d, 1H, J4,5furanꢀꢀ= ꢀꢀ1.5 Hz, H5furan), 8.00 (s, 1H, H2furan), 7.36 (m,
1,2,4-triazolo[3,4-b][1,3,4]thiadiazoles
2H, 3-Hfuranꢀ+ꢀ5-Hfuran), 7.17 (d 1H, J4,5furanꢀꢀ= ꢀꢀ1.1 Hz, 4-Hfuran),
3a–h and 4a–f
6.80 (dd, 1H, J3,4furanꢀꢀ= ꢀꢀ3.6 Hz, J4,5furanꢀꢀ= ꢀꢀ1.5 Hz, 4-Hfuran) ppm.
A mixture of 2 (182 mg, 1.00 mmol) and a substituted aro-
–
13C NMR ([D6]DMSO): δꢀꢀ= ꢀꢀ172.7 (C-6), 160.2 (C-8), 153.6
matic acid (1.10 mmol) in POCl3 (5 mL) was heated under (C-2furan), 146.5 (C-3), 145.8 (C-5furan), 140.5 (C-5furan), 139.6
reflux for 6–7 h. The reaction mixture was slowly poured (C-2furan), 135.1 (C-3furan), 112.6 (C-4furan), 112.2 (C-5furan), 108.7
into crushed ice with stirring and neutralized with solid (C-3furan) ppm. – C11H6N4O2S (258.26): calcd. C 51.16, H 2.34,
sodium bicarbonate. The solid product obtained was fil- N 21.69; found C 50.97, H 2.15, N 21.57.
tered, washed with cold water, and recrystallized from
ethanol to give the desired products.
3-(Furan-2-yl)-6-(2-methylfuran-3-yl)-[1, 2,4]
triazolo[3,4-b][1,3,4]thiadiazole (3d)ꢁFrom 2-meth-
6-(4-Chloro-2-fluorophenyl)-3-(furan-2-yl)-[1,2,4] ylfuran-3-carboxylic acid (139 mg). Yield: 214 mg (78ꢀ%),
triazolo[3,4-b][1,3,4]thiadiazole (3a)ꢀFrom 4-chloro- light-yellow solid, m.p.: 190–191ꢀ°C, Rfꢀꢀ= ꢀꢀ0.71. – IR (neat):
2-fluorobenzoic acid (191 mg). Yield: 228 mg (71ꢀ%), νꢀꢀ= ꢀꢀ3096 (Ar-H), 1620 (Cꢀ= ꢀN), 1592, 1521 (2ꢀꢀ× ꢀꢀCꢀ= ꢀC) cm–1.
1
light-yellow solid, m.p.: 257–258ꢀ°C, Rfꢀꢀ= ꢀꢀ0.73. – IR (neat): – H NMR ([D6]DMSO): δꢀꢀ= ꢀꢀ8.02 (d, 1H, J4,5furanꢀꢀ= ꢀꢀ1.5 Hz,
νꢀꢀ= ꢀꢀ3066 (Ar-H), 1606 (Cꢀ= ꢀN), 1574, 1514 (2ꢀꢀ× ꢀꢀCꢀ= ꢀC), 1204 5-Hfuran), 7.80 (d, 1H, J4′,5′furanꢀꢀ= ꢀꢀ2.0 Hz, 5′-Hfuran), 7.28 (d,
1
(C-F), 1024 (C-Cl) cm–1. – H NMR ([D6]DMSO): δꢀꢀ= ꢀꢀ8.05 1H, J3,4furanꢀꢀ= ꢀꢀ3.3 Hz, 3-Hfuran), 7.02 (d, 1H, J4′,5′furanꢀꢀ= ꢀꢀ2.0 Hz,
(d, 1H, J4,5furanꢀꢀ= ꢀꢀ1.2 Hz, 5-Hfuran), 7.90 (d, 1H, J3,Fꢀꢀ= ꢀꢀ7.5 Hz, 4′-Hfuran), 6.80 (dd, 1H, J3,4furanꢀꢀ= ꢀꢀ3.3 Hz, J4,5furanꢀꢀ= ꢀꢀ1.5 Hz,
3-Harom.), 7.62 (d, 1H, J5,6ꢀꢀ= ꢀꢀ8.4 Hz, 6-Harom.), 7.42 (d, 1H, 4-Hfuran), 2.14 (s, 3H, CH3) ppm. – 13C NMR ([D6]DMSO):
J
3,4furanꢀꢀ= ꢀꢀ3.6 Hz, 3-Hfuran), 7.36 (d, 1H, J5,6arom.ꢀꢀ= ꢀꢀ8.4 Hz, 5-Harom.), δꢀꢀ= ꢀꢀ171.5 (C-6), 161.3 (C-8), 155.0 (C-2furan), 153.5 (C-2afuran),
6.82 (dd, 1H, J3,4furanꢀꢀ= ꢀꢀ3.6 Hz, J4,5furanꢀꢀ= ꢀꢀ1.2 Hz, 4-Hfuran) ppm. 145.8 (C-5afuranꢀ+ꢀC-3), 140.5 (C-5furan), 139.5 (C-3afuran), 112.2
13C NMR ([D6]DMSO): δꢀꢀ= ꢀꢀ170.9 (C-6), 163.7 (C-8), 157.4 (d, (C-4furan), 112.0 (C-4afuran), 110.3 (C-3furan), 14.3 (CH3) ppm. –
–
J
C2,Fꢀꢀ= ꢀꢀ248 Hz, C-2arom.-F), 154.9 (C-2furan), 149.7 (C-3), 141.9 C12H8N4O2S (272.28): calcd. C 52.93, H, 2.96, N 20.58; found
(C-5furan), 137.8 (d, J4,Fꢀꢀ= ꢀꢀ20 Hz, C-4arom.), 134.6 (d, J6,Fꢀꢀ= ꢀꢀ22 Hz, C 52.81, H 2.88, N 20.41.
C-6arom.), 130.6 (C-5arom.), 127.5 (d, JC1,Fꢀꢀ= ꢀꢀ120 Hz, C-1arom.), 117.2
(d, JC3,Fꢀꢀ= ꢀꢀ121 Hz, C-3arom.), 112.5 (C-4furan), 108.7 (C-3furan) ppm. 6-(4-Fluorobenzyl)-3-(furan-2-yl)-[1,2,4]triazolo[3,4-b]
– C13H6ClFN4OS (320.73): calcd. C 48.68, H 1.89, N 17.47; [1,3,4]thiadiazole (3e)ꢁFrom 2-(4-(fluorophenyl)acetic
found C 48.51, H 1.74, N 17.59.
acid (154 mg). Yield: 228 mg (76ꢀ%), light-brown solid,
m.p.: 150–151ꢀ°C, Rfꢀꢀ= ꢀꢀ0.68. – IR (neat): νꢀꢀ= ꢀꢀ3064 (Ar-H),
6-(3-Chloro-4-fluorophenyl)-3-(furan-2-yl)-[1,2,4] 1621 (Cꢀ= ꢀN), 1592, 1521 (2ꢀꢀ× ꢀꢀCꢀ= ꢀC), 1138 (C-F) cm–1. – 1H NMR
triazolo[3,4-b][1,3,4]thiadiazole (3b)ꢁFrom 3-chloro- ([D6]DMSO): δꢀꢀ= ꢀꢀ8.01 (d, 1H, J4,5furanꢀꢀ= ꢀꢀ1.2 Hz, 5-Hfuran), 7.52–
4-fluorobenzoic acid (191 mg). Yield: 234 mg (73ꢀ%), 7.47 (m, 2H, 2-Harom.ꢀ+ꢀ6-Harom.), 7.25–7.19 (m, 3H, 3-Harom.ꢀ+ꢀ6-
off-white solid, m.p.: 263–264ꢀ°C, Rfꢀꢀ= ꢀꢀ0.75.ꢀ–ꢀIR (neat):
νꢀꢀ= ꢀꢀ3062 (Ar-H), 1625 (Cꢀ= ꢀN), 1597, 1525 (2ꢀꢀ× ꢀꢀCꢀ= ꢀC), 1221 4-Hfuran), 4.51 (s, 2H, CH2) ppm. – 13C NMR ([D6]DMSO):
(C-F), 1012 (C-Cl) cm–1.ꢀ–ꢀ1H NMR ([D6]DMSO): δꢀꢀ= ꢀꢀ8.37 δꢀꢀ= ꢀꢀ171.4 (C-6), 163.7 (C-8), 157.5 (d, JC4′,Fꢀꢀ= ꢀꢀ249 Hz, C-4arom.
Harom.ꢀ+ꢀ3-Hfuran), 6.78 (dd, 1H, J3,4furanꢀꢀ= ꢀꢀ3.3 Hz, J4,5furanꢀꢀ= ꢀꢀ1.5 Hz,
-
(d, 1H, JH2,Fꢀꢀ= ꢀꢀ6.6 Hz, 2-Harom.), 8.04 (d, 1H, J4,5furanꢀꢀ= ꢀꢀ1.2 Hz, F), 154.5 (C-2furan), 145.8 (C-3), 140.6 (C-5furan), 132.0 (C-1arom.),
5-Hfuran), 7.73–7.67 (m, 1H, 6-Harom.ꢀ+ꢀ5-Harom.), 7.49 (d, 1H, 131.8 (d, JC2,4,Fꢀꢀ= ꢀꢀ21 Hz, C-2arom.ꢀ+ꢀC-6arom.), 116.2 (JC2,4,Fꢀꢀ= ꢀꢀ118 Hz,
J
3,4furanꢀꢀ= ꢀꢀ3.6 Hz, 3-Hfuran), 6.82 (dd, 1H, J3,4furanꢀꢀ= ꢀꢀ3.6 Hz, C-3arom.ꢀ+ꢀC-5arom.), 112.6 (C-4furan), 111.9 (C-3furan), 36.7 (CH2)
4,5furanꢀꢀ= ꢀꢀ1.2 Hz, 4-Hfuran
ppm.ꢀ–ꢀ13C NMR ([D6]DMSO): ppm. – C14H9FN4OS (300.31): calcd. C 55.99, H 3.02,N 18.66;
found C 55.84, H 2.92, N 18.50.
J
)
δꢀꢀ= ꢀꢀ169.9 (C-6), 163.7 (C-8), 156.8 (d, JC4,Fꢀꢀ= ꢀꢀ250 Hz, C-4arom.
F), 153.9 (C-2furan), 149.8 (C-3), 141.8 (C-5furan), 133.5 (C-1arom.),
-
130.7 (d, JC2′,Fꢀꢀ= ꢀꢀ22 Hz, C-2arom.), 130.5 (d, JC6,Fꢀꢀ= ꢀꢀ22 Hz, C-6arom.), 6-(2-Chloro-4,5-difluorophenyl)-3-(furan-2-yl)-[1,2,4]
127.5 (d, JC3,Fꢀꢀ= ꢀꢀ123 Hz, C-3arom.), 117.0 (d, JC5,Fꢀꢀ= ꢀꢀ120 Hz, triazolo[3,4-b][1,3,4]thiadiazole (3f)ꢁFrom 2-chloro-
C-5arom.), 112.8 (C-4furan), 107.9 (C-3furan) ppm.ꢀ–ꢀC13H6ClFN4OS 4,5-difluorobenzoic acid (211 mg). Yield: 254 mg (75ꢀ%),
(320.73): calcd. C 48.68, H 1.89, N, 17.47; found C 48.76, H light-yellow solid, m.p.: 226–227ꢀ°C, Rfꢀꢀ= ꢀꢀ0.64. – IR (neat):
1.76, N 17.56.
νꢀꢀ= ꢀꢀ3058 (Ar-H), 1622 (Cꢀ= ꢀN), 1591, 1515 (2ꢀꢀ× ꢀꢀCꢀ= ꢀC), 1182 (C-F),
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54ꢀ
ꢀI. Khan et al.: New triazolothiadiazole and triazolothiadiazine derivatives as kinesin Eg5 and HIV inhibitors
1
1020 (C-Cl) cm–1. – H NMR ([D6]DMSO): δꢀꢀ= ꢀꢀ8.36 (m, 1H, 5.48 (s, 1H, OH). – 13C NMR ([D6]DMSO): δꢀꢀ= ꢀꢀ167.2 )C-6),
4-Harom.), 8.11 (m, 1H, 6-Harom.), 8.04 (d, J4,5furanꢀꢀ= ꢀꢀ1.0 Hz, 161.2 (C-8), 154.9 (C-2furan), 149.7 (C-1arom.), 145.4 (C-OH), 144.8
5-Hfuran), 7.43 (d, 1H, J3,4furanꢀꢀ= ꢀꢀ3.6 Hz, 3-Hfuran), 6.81 (dd, (C-3), 141.9 (C-5furan), 128.3, 127.5 (C-4arom.ꢀ+ꢀC-5arom.), 117.2
1H, J3,4furanꢀꢀ= ꢀꢀ3.6 Hz, J4,5furanꢀꢀ= ꢀꢀ1.8 Hz, 4-Hfuran) ppm. – 13C (C-6arom.ꢀ+ꢀC-3arom.), 112.1 (C-4furan), 109.8 (C-3furan), 65.5 (CH2)
NMR ([D6]DMSO): δꢀꢀ= ꢀꢀ170.9 (C-6), 163.6 (C-8), 158.2 (2ꢀꢀ× ꢀꢀd, ppm. – C14H10N4O3S (314.32): calcd. C 53.50, H 3.21, N 17.82;
J
C4,F4ꢀꢀ= ꢀꢀ249 Hz, JC4′,F5ꢀꢀ= ꢀꢀ118 Hz, C-4arom.-F), 156.8 (C-2furan), found C 53.37, H 3.07, 17.69.
151.3 (C-3), 147.7 (2ꢀꢀ× ꢀꢀd, JC5,F5ꢀꢀ= ꢀꢀ247 Hz, JC5,F4ꢀꢀ= ꢀꢀ119 Hz, C-5arom.
-
F),134.5 (C1arom.), 129.1 (d, JC2,F4ꢀꢀ= ꢀꢀ25 Hz, C-2arom.), 121.0 4-((3-(Furan-2-yl)-[1,2,4]triazolo[3,4-b][1,3,4]thiadi-
(m, C-3arom.ꢀ+ꢀC-6arom.), 112.1 (C-4furan), 110.6 (C-3furan) ppm. azol-6-yl)methoxy)phenol (4b)ꢁFrom 2-(4-hydroxy-
– C13H5ClF2N4OS (338.32): calcd. C 46.10, H 1.49, N 16.54; phenoxy)acetic acid (185 mg). Yield: 232 (74ꢀ%), off-white
found C 45.97, H 1.32, N 16.59.
solid, m.p.: 174–175ꢀ°C, Rfꢀꢀ= ꢀꢀ0.75. – IR (neat): νꢀꢀ= ꢀꢀ3115 (OH),
3029 (Ar-H), 1622(Cꢀ= ꢀN), 1546, 1498 (2ꢀꢀ× ꢀꢀCꢀ= ꢀC) cm–1. – H
1
3-(Furan-2-yl)-6-(1H-pyrrol-2-yl)-[1,2,4]triazolo[3,4-b] NMR ([D6]DMSO): δꢀꢀ= ꢀꢀ8.02 (br s, 1H, 5-Hfuran), 7.46–7.42 (m,
[1,3,4]thiadiazole (3g)ꢁFrom 1H-pyrrol-2-carboxylic acid 4H, Harom.), 7.20 (d, 1H, J3,4furanꢀꢀ= ꢀꢀ3.3 Hz, 3-Hfuran), 6.78 (dd,
(122 mg). Yield: 203 mg (79ꢀ%), black solid; m.p.: 223–
J3,4furanꢀꢀ= ꢀꢀ3.6 Hz, J4,5furanꢀꢀ= ꢀꢀ1.5 Hz, 4-Hfuran), 5.62 (s, 2H, CH2),
224ꢀ°C, Rfꢀꢀ= ꢀꢀ0.66. – IR (neat): νꢀꢀ= ꢀꢀ3119 (N-H), 3054 (Ar-H), 5.44 (s, 1H, OH) ppm. – 13C NMR ([D6]DMSO): δꢀꢀ= ꢀꢀ166.1 )
1621 (Cꢀ= ꢀN), 1581, 1518 (2ꢀꢀ× ꢀꢀCꢀ= ꢀC) cm–1. – 1H NMR ([D6]DMSO): C-6ꢀ+ꢀC-8), 154.8 (C-2furan), 149.7 (C-1arom.), 145.4 (C-OH), 144.9
δꢀꢀ= ꢀꢀ12.55 (s, 1H, NH), 8.02 (d, 1H, J4,5furanꢀꢀ= ꢀꢀ1.5 Hz, 5-Hfuran), 7.22 (C-3), 142.9 (C-5furan), 129.7, 129.2 (Carom.), 112.1 (C-4furan), 109.8
(m, 2H, 3-Hfuranꢀ+ꢀ5-Hpyrrole), 7.02–6.94 (m, 2H, 4-Hfuranꢀ+ꢀ3-Hpyr- (C-3furan), 65.3 (CH2) ppm. – C14H10N4O3S (314.32): calcd. C
role), 6.79 (dd, 1H, J3,4pyrroleꢀꢀ= ꢀꢀ3.6 Hz, J4,5furanꢀꢀ= ꢀꢀ1.5 Hz, 4-Hpyrrole
ppm. – 13C NMR ([D6]DMSO): δꢀꢀ= ꢀꢀ169.4 (C-6), 159.6 (C-8),
154.5 (C-2furanꢀ+ꢀC-3), 138.7 (C-5furan), 127.8 (C-2pyrrole), 122.5 3-(Furan-2-yl)-6-(2-tolyloxymethyl)-[1,2,4]triazolo[3,4-
(C-5pyrrole), 115.3 (C-4furanꢀ+ꢀC-4pyrrole), 107.1 (C-3furanꢀ+ꢀC-3pyrrol b][1,3,4]thiadiazole (4c)ꢁFrom 2-(tolyloxy)acetic acid
)
53.50, H 3.21, N 17.82; found C 53.59, H 3.11, N 17.74.
)
ppm. – C11H7FN5OS (257.27): calcd. C 51.35, H 2.74, N 27.22; (183 mg). Yield: 234 mg (75ꢀ%), off-white solid, m.p.: 186–
found C 51.23, H 2.58, N 27.39.
187ꢀ°C, Rfꢀꢀ= ꢀꢀ0.76. – IR (neat): νꢀꢀ= ꢀꢀ3041 (Ar-H), 1605 (Cꢀ= ꢀN),
1
1589, 1516 (2ꢀꢀ× ꢀꢀCꢀ= ꢀC) cm–1. – H NMR ([D6]DMSO): δꢀꢀ= ꢀꢀ8.02
6-(4-Ethoxybenzyl)-3-(furan-2-yl)-[1,2,4]triazolo[3,4- (d, 1H, J4,5furanꢀꢀ= ꢀꢀ1.5 Hz, 5-Hfuran), 7.32 (br s, 1H, 3-Hfuran), 7.23–
b][1,3,4]thiadiazole (3h)ꢁFrom 2-(4-(ethoxyphenyl) 7.17 (m, 2H, 3-Harom.ꢀ+ꢀ4-Harom.), 7.12 (d, 1H, Jꢀꢀ= ꢀꢀ7.8 Hz, 6-Harom.),
acetic acid (198 mg). Yield: 273 mg (77ꢀ%), light-brown 6.94 (t, 1H, Jꢀꢀ= ꢀꢀ7.8 Hz, 5-Harom.), 6.79 (dd, 1H, J3,4furanꢀꢀ= ꢀꢀ3.6 Hz,
solid, m.p.: 158–159ꢀ°C, Rfꢀꢀ= ꢀꢀ0.70. – IR (neat): νꢀꢀ= ꢀꢀ3071
J4,5furanꢀꢀ= ꢀꢀ1.5 Hz, 4-Hfuran), 5.61 (s, 2H, CH2), 2.25 (s, 3H, CH3)
1
(Ar-H), 1611 (Cꢀ= ꢀN), 1585, 1527 (2ꢀꢀ× ꢀꢀCꢀ= ꢀC) cm–1. – H NMR ppm. – 13C NMR ([D6]DMSO): δꢀꢀ= ꢀꢀ169.6 (C-6ꢀ+ꢀC-8), 155.6
([D6]DMSO): δꢀꢀ= ꢀꢀ8.01 (d, 1H, J4,5furanꢀꢀ= ꢀꢀ1.2 Hz, 5-Hfuran), 7.34 (C-1arom.), 154.1 (C-2furan), 145.9 (C-3), 140.5 (C-5furan), 131.3,
(d, 2H, J2,3ꢀꢀ= ꢀꢀJ6,5ꢀꢀ= ꢀꢀ8.7 Hz, 2-Harom.ꢀ+ꢀ6-Harom.), 7.22 (d, 1H, 126.6, 122.3 (Carom.), 112.7 (C-4furan), 112.6 (C-6arom.), 111.9
J
3,4furanꢀꢀ= ꢀꢀ3.3 Hz, 3-Hfuran), 6.93 (d, 2H, 3-Harom.ꢀ+ꢀ5-Harom.), 6.78 (C-3furan), 65.4 (CH2), 16.4 (CH3) ppm. – C15H12N4O2S (312.35):
(dd, 1H, J3,4furanꢀꢀ= ꢀꢀ3.3 Hz, J4,5furanꢀꢀ= ꢀꢀ1.2 Hz, 4-Hfuran), 4.42 (s, 2H, calcd. C 57.68, H 3.87, N 17.94; found C 57.52, H 3.99, N 17.81.
CH2), 4.01 (q, 2H, Jꢀꢀ= ꢀꢀ6.9 Hz, CH2), 1.31 (t, 3H, Jꢀꢀ= ꢀꢀ6.9 Hz,
CH3) ppm. – 13C NMR ([D6]DMSO): δꢀꢀ= ꢀꢀ172.4 (C-8), 158.5 3-(Fur an-2-yl)- 6-(4-tolylox ymethyl)-[1, 2 ,4]
(C-6),154.5 (C-2furanꢀ+ꢀC-OEt), 145.8 (C-3), 140.6 (C-5furan), 131.0 triazolo[3,4-b][1,3,4]thiadiazole (4d)ꢁFrom 2-(toly-
(C-2arom.ꢀ+ꢀC-6arom.), 127.4 (C-1arom.), 115.3 (C-3arom.ꢀ+ꢀC-5arom.), loxy)acetic acid (183 mg). Yield: 228 mg (73ꢀ%), off-white
112.6 (C-4furan), 111.9 (C-3furan), 63.5 (OCH2), 36.8 (CH2), 15.1 solid, m.p.: 191–192ꢀ°C, Rfꢀꢀ= ꢀꢀ0.75. – IR (neat): νꢀꢀ= ꢀꢀ3039
1
(CH3) ppm. – C17H14N4O3S (354.38): calcd. C 57.62, H 3.98, N (Ar-H), 1611 (Cꢀ= ꢀN), 1588, 1508 (2ꢀꢀ× ꢀꢀCꢀ= ꢀC) cm–1. – H NMR
15.81; found C 57.81, H 3.79, N 15.62.
([D6]DMSO): δꢀꢀ= ꢀꢀ8.02 (d, 1H, J4,5furanꢀꢀ= ꢀꢀ1.2 Hz, 5-Hfuran), 7.22
(d, 1H, J3,4furanꢀꢀ= ꢀꢀ3.7 Hz, 3-Hfuran), 7.15 (d, 2H, Jꢀꢀ= ꢀꢀ8.4 Hz,
2-((3-(Furan-2-yl)-[1,2,4]triazolo[3,4-b][1,3,4]thiadia- 3-Harom.ꢀ+ꢀ5-Harom.), 7.02 (d, 2H, Jꢀꢀ= ꢀꢀ7.8 Hz, 2-Harom.ꢀ+ꢀ6-Harom.),
zol-6-yl)methoxy)phenol (4a)ꢁFrom 2-(2-hydroxyphe- 6.79 (dd, 1H, J3,4furanꢀꢀ= ꢀꢀ3.7 Hz, J4,5furanꢀꢀ= ꢀꢀ1.5 Hz, 4-Hfuran), 5.56
noxy)acetic acid (185 mg). Yield: 232 mg (70ꢀ%), off-white (s, 2H, CH2), 2.24 (s, 3H, CH3) ppm. – 13C NMR ([D6]DMSO):
solid, m.p.: 180–181ꢀ°C, Rfꢀꢀ= ꢀꢀ0.74. – IR (neat): νꢀꢀ= ꢀꢀ3147 (OH), δꢀꢀ= ꢀꢀ169.3 (C-6ꢀ+ꢀC-8), 155.4 (C-1arom.), 154.1 (C-2furan), 145.9
1
3035 (Ar-H), 1618 (Cꢀ= ꢀN), 1551, 1508 (2ꢀꢀ× ꢀꢀCꢀ= ꢀC) cm–1. – H (C-3), 140.4 (C-5furan), 131.5, 130.5 (C-3arom.ꢀ+ꢀC-5arom.), 115.4
NMR ([D6]DMSO): δꢀꢀ= ꢀꢀ8.02 (br s, 1H, 5-Hfuran), 7.49–7.40 (m, (C-2arom.ꢀ+ꢀC-6arom.), 112.6 (C-4furan), 111.9 (C-3furan), 65.4 (CH2),
4H, Harom.), 7.22 (d, 1H, J3,4furanꢀꢀ= ꢀꢀ3.6 Hz, 3-Hfuran), 6.79 (dd, 1H, 20.6 (CH3) ppm. – C15H12N4O2S (312.35): calcd. C 57.68, H
J3,4furanꢀꢀ= ꢀꢀ3.6 Hz, J4,5furanꢀꢀ= ꢀꢀ1.8 Hz, 4-Hfuran), 5.68 (s, 2H, CH2), 3.87, N 17.94; found C 57.60, H 3.72, N 17.85.
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ꢁꢀꢀꢀ
I. Khan et al.: New triazolothiadiazole and triazolothiadiazine derivatives as kinesin Eg5 and HIV inhibitorsꢂ
ꢂ55
3-(Furan-2-yl)-6-((4-methoxyphenoxy)methyl)- 1510 (2ꢀꢀ× ꢀꢀCꢀ= ꢀC) cm–1. – 1H NMR ([D6]DMSO): δꢀꢀ= ꢀꢀ8.05 (d, 2H,
[1,2,4]triazolo[3,4-b][1,3,4]thiadiazole
(4e)ꢁFrom Jꢀꢀ= ꢀꢀ8.7 Hz, 2-Harom.ꢀ+ꢀ6-Harom.), 7.99 (d, 1H, J4,5furanꢀꢀ= ꢀꢀ1.5 Hz,
2-(4-methoxyphenoxy)acetic acid (200 mg). Yield: 5-Hfuran), 7.24 (d, 1H, J3,4furanꢀꢀ= ꢀꢀ2.7 Hz, 3-Hfuran), 7.14 (d, 2H,
253 mg (77ꢀ%), off-white solid, m.p.: 148–149ꢀ°C, Jꢀꢀ= ꢀꢀ8.7 Hz, 3-Harom.ꢀ+ꢀ5-Harom.), 6.77 (dd, 1H, J3,4furanꢀꢀ= ꢀꢀ2.7 Hz,
Rfꢀꢀ= ꢀꢀ0.72. – IR (neat): νꢀꢀ= ꢀꢀ3026 (Ar-H), 1620 (Cꢀ= ꢀN), 1592,
J4,5furanꢀꢀ= ꢀꢀ1.5 Hz, 4-Hfuran), 4.43 (s, 2H, CH2), 3.86 (s, 3H, CH3)
1
1505 (2ꢀꢀ× ꢀꢀCꢀ= ꢀC) cm–1. – H NMR ([D6]DMSO): δꢀꢀ= ꢀꢀ8.02 (d, ppm. – 13C NMR ([D6]DMSO): δꢀꢀ= ꢀꢀ162.9 (C-6ꢀ+ꢀC-OMe),
1H, J4,5furanꢀꢀ= ꢀꢀ1.2 Hz, 5-Hfuran), 7.22 (d, 1H, J3,4furanꢀꢀ= ꢀꢀ3.3 Hz, 156.2 (C-8ꢀ+ꢀC-2furan), 145.7 (C-3), 142.4 (C-5furan), 130.0
3-Hfuran), 7.06 (d, 2H, Jꢀꢀ= ꢀꢀ7.5 Hz, 2-Harom.ꢀ+ꢀ6-Harom.), (C-2arom.ꢀ+ꢀC-6arom.), 125.7 (C-1arom.), 115.1 (C-3arom.ꢀ+ꢀC-5arom.),
6.90 (d, 2H, Jꢀꢀ= ꢀꢀ7.5 Hz, 3-Harom.ꢀ+ꢀ5-Harom.), 6.78 (dd, 1H, 113.6 (C-4furan), 112.6 (C-3furan), 56.1 (OCH3), 23.1 (CH2) ppm.
J
3,4furanꢀꢀ= ꢀꢀ3.3 Hz, J4,5furanꢀꢀ= ꢀꢀ1.2 Hz, 4-Hfuran), 5.53 (s, 2H, CH2), – C15H12N4O2S (312.35): calcd. C 57.68, H 3.87, N 17.94; found
3.70 (s, 3H, CH3) ppm. – 13C NMR ([D6]DMSO): δꢀꢀ= ꢀꢀ169.4 C 57.53, H 3.66, N 17.85.
(C-6ꢀ+ꢀC-8), 154.9 (C-2furan), 154.1 (C-OMe), 151.4 (C-1arom.),
145.9 (C-3), 140.4 (C-5furan), 116.7 (C-2arom.ꢀ+ꢀC-6arom.), 115.2 6-(4-Chlorophenyl)-3-(furan-2-yl)-7H-[1, 2,4]
(C-3arom.ꢀ+ꢀC-5arom.), 112.6 (C-4furan), 111.9 (C-3furan), 66.0 (CH2), triazolo[3,4-b][1,3,4]thiadiazine (5b)ꢁFrom 4-chlo-
55.8 (OMe) ppm. – C15H12N4O3S (328.35): calcd. C 54.87, H rophenacyl bromide (280 mg). Yield: 260 mg (79ꢀ%),
3.68, N 17.06; found C 54.71, H 3.48, N 16.92.
light-yellow solid, m.p.: 231–232ꢀ°C, Rfꢀꢀ= ꢀꢀ0.66. – IR (neat):
νꢀꢀ= ꢀꢀ3037 (Ar-H), 2910, 2824 (2ꢀꢀ× ꢀꢀC-H), 1591 (Cꢀ= ꢀN), 1562, 1515
6-((4-Fluorophenoxy)methyl)-3-(furan-2-yl)-[1,2,4] (2ꢀꢀ× ꢀꢀCꢀ= ꢀC), 1076 (C-Cl) cm–1. – 1H NMR ([D6]DMSO): δꢀꢀ= ꢀꢀ8.70
triazolo[3,4-b][1,3,4]thiadiazole (4f)ꢁFrom 2-(4-fluo- (br s, 2H, 2-Harom.ꢀ+ꢀ6-Harom.), 7.99 (d, 1H, J4,5furanꢀꢀ= ꢀꢀ1.8 Hz,
rophenoxy)acetic acid (187 mg). Yield: 253 mg (80ꢀ%), 5-Hfuran), 7.67 (br s, 2H, 3-Harom.ꢀ+ꢀ5-Harom.), 7.22 (d, 1H,
light-brown solid, m.p.: 164–165ꢀ°C, Rfꢀꢀ= ꢀꢀ0.77. – IR (neat):
νꢀꢀ= ꢀꢀ3019 (Ar-H), 1625 (Cꢀ= ꢀN), 1552, 1503 (2ꢀꢀ× ꢀꢀCꢀ= ꢀC), 1203 (C-F)
J
3,4furanꢀꢀ= ꢀꢀ3.6 Hz, 3-Hfuran), 6.77 (dd, 1H, J3,4furanꢀꢀ= ꢀꢀ3.6 Hz,
J
4,5furanꢀꢀ= ꢀꢀ1.8 Hz, 4-Hfuran), 4.45 (s, 2H, CH2) ppm. – 13C NMR
cm–1. – 1H NMR ([D6]DMSO): δꢀꢀ= ꢀꢀ8.02 (d, 1H, J4,5furanꢀꢀ= ꢀꢀ1.5 Hz, ([D6]DMSO): δꢀꢀ= ꢀꢀ163.1 (C-6), 155.6 (C-8ꢀ+ꢀC-2furan), 145.8
5-Hfuran), 7.22–7.13 (m, 5H, Harom.ꢀ+ꢀ3-Hfuran), 6.78 (dd, 1H, (C-3), 142.2 (C-5furan), 137.4 (C-4arom.-Cl), 132.6 (C-1arom.), 129.9
J
3,4furanꢀꢀ= ꢀꢀ3.6 Hz, J4,5furanꢀꢀ= ꢀꢀ1.5 Hz, 4-Hfuran), 5.59 (s, 2H, CH2) (C-3arom.ꢀ+ꢀC-5arom.), 129.7 (C-2arom.ꢀ+ꢀC-6arom.), 113.6 (C-4furan),
ppm. – 13C NMR ([D6]DMSO): δꢀꢀ= ꢀꢀ168.8 (C-6), 159.4 (C-8), 112.6 (C-3furan), 23.3 (CH2) ppm. – C14H9ClN4OS (316.77):
155.3 (d, JC4,Fꢀꢀ= ꢀꢀ249 Hz, C-4arom.-F), 154.2 (C-1arom.), 153.9 calcd. C 53.08, H 2.86, N 17.69; found C 52.96, H 2.74, N 17.56.
(C-2furan), 145.9 (C-3), 140.4 (C-5furan), 117.2 (d, JC3,5,Fꢀꢀ= ꢀꢀ118 Hz,
C-3arom.ꢀ+ꢀC-5arom.), 116.6 (d, JC2,6,Fꢀꢀ= ꢀꢀ20 Hz, C-2arom.ꢀ+ꢀC-6arom.), 6-(4-Fluorophenyl)-3-(furan-2-yl)-7H-[1, 2,4]
112.6 (C-4furan), 111.9 (C-3furan), 65.9 (CH2) ppm. – C14H9FN4O2S triazolo[3,4-b][1,3,4]thiadiazine (5c)ꢁFrom 4-fluo-
(316.31): calcd. C 53.16, H 2.87, N 17.71; found C 53.22, H 2.70, rophenacyl bromide (260 mg). Yield: 231 mg (77ꢀ%),
N 17.56.
colorless solid, m.p.: 248–249ꢀ°C, Rfꢀꢀ= ꢀꢀ0.69. – IR (neat):
νꢀꢀ= ꢀꢀ3049 (Ar-H), 2912, 2843 (2ꢀꢀ× ꢀꢀC-H), 1599 (Cꢀ= ꢀN),
1556, 1511 (2ꢀꢀ× ꢀꢀCꢀ= ꢀC), 1223 (C-F) cm–1. – 1H NMR ([D6]
DMSO): δꢀꢀ= ꢀꢀ8.14 (m, 2H, 2-Harom.ꢀ+ꢀ6-Harom.), 7.99 (d, 1H,
4.3 General procedure for the synthesis of
1,2,4-triazolo[3,4-b][1,3,4]thiadiazines
5a–h
J
4,5furanꢀꢀ= ꢀꢀ1.8 Hz, 5-Hfuran), 7.46 (m, 2H, 3-Harom.ꢀ+ꢀ5-Harom.), 7.23
(d, 1H, J3,4furanꢀꢀ= ꢀꢀ3.6 Hz, 3-Hfuran), 6.77 (dd, 1H, J3,4furanꢀꢀ= ꢀꢀ3.6 Hz,
4,5furanꢀꢀ= ꢀꢀ1.8 Hz, 4-Hfuran), 4.46 (s, 2H, CH2) ppm. – 13C NMR
J
A mixture of 2 (182 mg, 1.00 mmol) and a substituted ([D6]DMSO): δꢀꢀ= ꢀꢀ166.5 (d, JC4,Fꢀꢀ= ꢀꢀ249 Hz, C4arom.-F), 163.2
phenacyl bromide (1.20 mmol) was heated under reflux (C-6), 155.7 (C-8ꢀ+ꢀC-2furan), 145.7 (C-3), 142.2 (C-5furan), 130.7
in abs. ethanol (10 mL) for 6–7 h. The reaction mass was (C-1arom.), 130.3 (d, JC2,6,Fꢀꢀ= ꢀꢀ18 Hz, C-2arom.ꢀ+ꢀC-6arom.), 116.9
poured into crushed ice and neutralized with NaHCO3. The (d, JC3,5,Fꢀꢀ= ꢀꢀ123 Hz, C-3arom.ꢀ+ꢀC-5arom.), 113.6 (C-4furan), 112.9
solid product obtained was filtered, washed with water, (C-3furan), 23.4 (CH2) ppm. – C14H9FN4OS (300.31): calcd. C
dried, and recrystallized from EtOH to afford the desired 55.99, H 3.02, N 18.66; found C 55.84, H 2.90, N 18.51.
products.
3-(Furan-2-yl)-6-(p-tolyl)-7H-[1,2,4]triazolo[3,4-b]
3-(Furan-2-yl)-6-(4-methoxyphenyl)-7H-[1,2,4] [1,3,4]thiadiazine (5d)ꢁFrom 4-methylphenacyl bromide
triazolo[3,4-b][1,3,4]thiadiazine (5a)ꢁFrom 4-meth- (255 mg). Yield: 219 mg (74ꢀ%), light-orange solid, m.p.:
oxyphenacyl bromide (275 mg). Yield:253 mg (81ꢀ%), 190–191ꢀ°C, Rfꢀꢀ= ꢀꢀ0.69. – IR (neat): νꢀꢀ= ꢀꢀ3016 (Ar-H), 2917, 2837
yellow solid, m.p.: 214–215ꢀ°C, Rfꢀꢀ= ꢀꢀ0.62. – IR (neat): (2ꢀꢀ× ꢀꢀC-H), 1606 (Cꢀ= ꢀN), 1584, 1509 (2ꢀꢀ× ꢀꢀCꢀ= ꢀC) cm–1. – 1H NMR
νꢀꢀ= ꢀꢀ3015 (Ar-H), 2912, 2857 (2ꢀꢀ× ꢀꢀC-H), 1604 (Cꢀ= ꢀN), 1556, ([D6]DMSO): δꢀꢀ= ꢀꢀ7.99 (d, 1H, J4,5furanꢀꢀ= ꢀꢀ1.5 Hz, 5-Hfuran), 7.96 (d,
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56ꢀ ꢀI. Khan et al.: New triazolothiadiazole and triazolothiadiazine derivatives as kinesin Eg5 and HIV inhibitors
2H, Jꢀꢀ= ꢀꢀ8.1 Hz, 2-Harom.ꢀ+ꢀ6-Harom.), 7.40 (d, 2H, Jꢀꢀ= ꢀꢀ8.1 3-Hz, (d, 2H, Jꢀꢀ= ꢀꢀ8.7 Hz, 3-Harom.ꢀ+ꢀ5-Harom.), 8.29 (d, 2H, Jꢀꢀ= ꢀꢀ8.7 Hz,
arom.ꢀ+ꢀ5-Harom.), 7.23 (d, 1H, J3,4furanꢀꢀ= ꢀꢀ3.3 Hz, 3-Hfuran), 6.77 2-Harom.ꢀ+ꢀ6-Harom.), 8.01 (d, 1H, J4,5furanꢀꢀ= ꢀꢀ1.5 Hz, 5-Hfuran), 7.24
(dd, 1H, J3,4furanꢀꢀ= ꢀꢀ3.3 Hz, J4,5furanꢀꢀ= ꢀꢀ1.5 Hz, 4-Hfuran), 4.43 (s, 2H, (d, 1H, J3,4furanꢀꢀ= ꢀꢀ3.3 Hz, 3-Hfuran), 6.77 (dd, 1H, J3,4furanꢀꢀ= ꢀꢀ3.3 Hz,
CH2), 2.40 (s, 3H, CH3) ppm. – 13C NMR ([D6]DMSO): δꢀꢀ= ꢀꢀ164.2 4,5furanꢀꢀ= ꢀꢀ1.5 Hz, 4-Hfuran), 4.53 (s, 2H, CH2) ppm. – 13C NMR
H
J
(C-6), 156.5 (C-8ꢀ+ꢀC-2furan), 146.0 (C-3), 142.9 (C-5furan), 140.9 ([D6]DMSO): δꢀꢀ= ꢀꢀ163.1 (C-6), 154.9 (C-8ꢀ+ꢀC-2furan), 149.7
(C-4arom.-CH3), 130.9 (C-1arom.), 130.2 (C-2arom.ꢀ+ꢀC-6arom.), 128.1 (C-4arom.-NO2ꢀ+ꢀC-3), 142.2 (C-5furan), 140.6 (C-1arom.), 129.5
(C-3arom.ꢀ+ꢀC-5arom.), 113.5 (C-4furan), 112.7 (C-3furan), 23.3 (CH2), (C-2arom.ꢀ+ꢀC-6arom.), 124.6 (C-3arom.ꢀ+ꢀC-5arom.), 113.9 (C-4furan),
21.5 (CH3) ppm. – C15H12N4OS (296.35): calcd. C 60.79, H 113.0 (C-3furan), 23.5 (CH2) ppm. – C14H9N5O3S (327.32): calcd.
4.08, N 18.91; found C 60.62, H 4.18, N 18.74.
C 51.37, H 2.77, N 21.40, found C 51.26, H 2.58, N 21.47.
6-([1,1′-Biphenyl]-4-yl)-3-(furan-2-yl)-7H-[1,2,4] 6-(3,4-Dichlorophenyl)-3-(furan-2-yl)-7H-[1,2,4]
triazolo[3,4-b][1,3,4]thiadiazine (5e)ꢁFrom 4-phenyl- triazolo[3,4-b][1,3,4]thiadiazine (5h)ꢁFrom 2,4-dichlo-
phenacyl bromide (330 mg). Yield: 276 mg (77ꢀ%), yellow rophenacyl bromide (320 mg). Yield: 267 mg (76ꢀ%),
solid, m.p.: 228–229ꢀ°C, Rfꢀꢀ= ꢀꢀ0.64. – IR (neat): νꢀꢀ= ꢀꢀ3034 off-white solid, m.p.: 261–262ꢀ°C, Rfꢀꢀ= ꢀꢀ0.72. – IR (neat):
(Ar-H), 2915, 2851 (2ꢀꢀ× ꢀꢀC-H), 1605 (Cꢀ= ꢀN), 1551, 1517 (2ꢀꢀ× ꢀꢀCꢀ= ꢀC) νꢀꢀ= ꢀꢀ3020 (Ar-H), 2908, 2851 (2ꢀꢀ× ꢀꢀC-H), 1685 (Cꢀ= ꢀN), 1548,
1
1
cm–1. – H NMR ([D6]DMSO): δꢀꢀ= ꢀꢀ8.16 (d, 2H, Jꢀꢀ= ꢀꢀ8.4 Hz, 1515 (2ꢀꢀ× ꢀꢀCꢀ= ꢀC), 1017 (C-Cl) cm–1. – H NMR ([D6]DMSO):
2-Harom.ꢀ+ꢀ6-Harom.), 8.01 (d, 1H, J4,5furanꢀꢀ= ꢀꢀ1.5 Hz, 5-Hfuran), 7.92 δꢀꢀ= ꢀꢀ8.29 (d, 1H, J4,5furanꢀꢀ= ꢀꢀ1.5 Hz, 5-Hfuran), 8.04 (br s,
(d, 2H, Jꢀꢀ= ꢀꢀ8.4 Hz, 3-Harom.ꢀ+ꢀ5-Harom.), 7.79 (d, 2H, Jꢀꢀ= ꢀꢀ7.2 Hz, 2H, 5-Harom.ꢀ+ꢀ6-Harom.), 7.90 (s, 1H, 2-Harom.), 7.22 (d, 1H,
3′-Harom.ꢀ+ꢀ5′-Harom.), 7.53 (d, 2H, Jꢀꢀ= ꢀꢀ7.2 Hz, 2′-Harom.ꢀ+ꢀ6′-Harom.),
7.43 (t, 1H, Jꢀꢀ= ꢀꢀ7.2 Hz, 4-Harom.), 7.23 (d, 1H, J3,4furanꢀꢀ= ꢀꢀ3.6 Hz,
J
J
3,4furanꢀꢀ= ꢀꢀ3.6 Hz, 3-Hfuran), 6.78 (dd, 1H, J3,4furanꢀꢀ= ꢀꢀ3.6 Hz,
4,5furanꢀꢀ= ꢀꢀ1.5 Hz, 4-Hfuran), 4.46 (s, 2H, CH2) ppm. – 13C NMR
3-Hfuran), 6.79 (dd, 1H, J3,4furanꢀꢀ= ꢀꢀ3.3 Hz, J4,5furanꢀꢀ= ꢀꢀ1.5 Hz, 4-Hfuran), ([D6]DMSO): δꢀꢀ= ꢀꢀ162.9 (C-6), 154.8 (C-8), 149.7 (C-2furan),
4.50 (s, 2H, CH2) ppm. – 13C NMR ([D6]DMSO): δꢀꢀ= ꢀꢀ163.6 (C-6), 142.7 (C-5furan), 134.7 (C-4arom.-Cl), 133.9 (C-3arom.-Clꢀ+ꢀC4arom.
-
156.2 (C-8ꢀ+ꢀC-2furan), 145.8 (C-3), 144.1 (C-4arom.), 142.3 (C-5furan), C1), 130.1 (C-2arom.), 129.5 (C-5arom.), 128.3 (C-2arom.), 112.8
140.9 (C-1arom.), 129.6 (C-4arom.), 128.8, 128.7 (C-2arom.ꢀ+ꢀC-6arom.), (C-3furan), 113.7 (C-4furan), 23.6 (CH2) ppm. – C14H8Cl2N4OS
127.8, 127.4 (C-3arom.ꢀ+ꢀC-5arom.), 113.8 (C-4furan), 112.5 (C-3furan), (351.21): calcd. C 47.88, H 2.30, N 15.95; found C 47.65, H
23.3 (CH2) ppm. – C20H14N4OS (358.42): calcd. C 67.02, H 3.94, 2.36, N 15.84.
N 15.63; found C 66.91, H 3.82, N 15.64.
3-(Furan-2-yl)-6-(naphthalen-1-yl)-7H-[1, 2,4] 4.4 Crystal structure determination of 3e
triazolo[3,4-b][1,3,4]thiadiazine (5f)ꢁFrom 2-bromo-
2′-acetonaphthone (299 mg). Yield: 242 mg (73ꢀ%), Data were collected on a Bruker APEXII Kappa CCD sin-
yellow solid, m.p.: 219–220ꢀ°C, Rfꢀꢀ= ꢀꢀ0.68. – IR (neat): gle-crystal diffractometer equipped with a graphite mono-
νꢀꢀ= ꢀꢀ3056 (Ar-H), 2915, 2842 (2ꢀꢀ× ꢀꢀC-H), 1605 (Cꢀ= ꢀN), 1596, chromator. The structure was solved by Direct Methods
1
1512 (2ꢀꢀ× ꢀꢀCꢀ= ꢀC) cm–1. – H NMR ([D6]DMSO): δꢀꢀ= ꢀꢀ8.67 (br and refined by full-matrix least-squares procedures on F2
s, 1H, 2-Harom.), 8.19 (m, 1H, 4-Harom.), 8.18 (m, 2H, Harom.), with the program shelxs-97 [31] and titan [32]. Hydro-
8.03 (m, 2H, Harom.ꢀ+ꢀ5-Hfuran), 7.67 (m, 2H, Harom.), 7.30 (d, gen atoms were placed in idealized positions, and their
1H, J3,4furanꢀꢀ= ꢀꢀ3.3 Hz, 3-Hfuran), 6.80 (dd, 1H, J3,4furanꢀꢀ= ꢀꢀ3.3 Hz, displacement parameters were refined isotropically with
J
4,5furanꢀꢀ= ꢀꢀ1.8 Hz, 4-Hfuran), 4.60 (s, 2H, CH2) ppm. – 13C NMR Ueqꢀꢀ= ꢀꢀ1.2–1.5 Ueq(C), while other nonhydrogen atoms were
([D6]DMSO): δꢀꢀ= ꢀꢀ162.8 (C-6), 156.8 (C-8ꢀ+ꢀC-2furan), 145.9
(C-3), 142.4 (C-5furan), 134.8 (C-8aarom.), 132.9 (C-4aarom.),
129.9 (C-1arom.), 129.5 (C-4arom.), 129.4, 128.9, 128.2, 127.7, 123.6
(Carom.), 113.5 (C-4furan), 112.7 (C-3furan), 23.2 (CH2) ppm. –
C18H12N4OS (332.38): calcd. C 65.04, H 3.64, N 16.86; found
C 64.98, H 3.75, N 16.69.
Table 5ꢂHydrogen bond geometry of 3e (Å and deg)a.
D–H···A
d(D–H)
d(H···A)
d(D···A)
ꢀ< ꢀ(DHA)
C(8)–H(8B)···O(1)ii
C(14)–H(14)···N(1)ii
C(6)–H(6)···F(1)iii
C(8)–H(8B)···O(1)iv
C(11)–H(11)···N(2)v
C(7)–H(7)···F(1)vi
0.99
0.95
0.95
0.99
0.95
0.95
2.59
2.67
2.61
2.54
2.51
2.63
3.386(2)
3.385(2)
3.140(2)
3.410(2)
3.382(2)
3.223(2)
137.7
132.1
115.2
146.5
151.8
120.8
3-(Furan-2-yl)-6-(4-nitrophenyl)-7H-[1,2,4]triazolo[3,4-
b][1,3,4]thiadiazine
(5g)ꢁFrom
4-nitrophenacyl
bromide (293 mg). Yield: 245 mg (75ꢀ%), yellow solid,
m.p.: 270–271ꢀ°C, Rfꢀꢀ= ꢀꢀ0.67. – IR (neat): νꢀꢀ= ꢀꢀ3031 (Ar-H),
aSymmetry codes: (ii) x, y+1, z; (iii) –x+1/2, –y+1/2, z–1/2; (iv) –x+1,
2931, 2856 (2ꢀꢀ× ꢀꢀC-H), 1599 (Cꢀ= ꢀN), 1574, 1513 (2ꢀꢀ× ꢀꢀCꢀ= ꢀC),
1469, 1344 (2ꢀꢀ× ꢀꢀNO2) cm–1. – H NMR ([D6]DMSO): δꢀꢀ= ꢀꢀ8.42 –y, –z+1; (v) x–1/2, y+1/2, –z+3/2; (vi) x, –y, z–1/2.
1
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I. Khan et al.: New triazolothiadiazole and triazolothiadiazine derivatives as kinesin Eg5 and HIV inhibitorsꢂ
ꢂ57
Pyruvate
L-Lactate
O-
H
+
2O
O-
O
O
Mg ATP
ATPase
(Eg5)
HO
O
Pyruvate
kinase
NAD
(260 nm)
NADH
(340 nm)
OH
O
Mg ADP
+
-
2O3
PO4 -
PO
PEP
Absorbance read at 340 nm
NADH (proportional to ATPase activity
Displays consumption of
Fig. 5ꢂATPase activity screening assay.
refined with anisotropic displacement parameters. Table 4.6 In vitro anti-HIV assay
5 shows the hydrogen bond geometry of 3e.
Crystal structure dataꢁC14H9FN4OS, Mrꢀꢀ= ꢀꢀ300.31, Evaluation of the antiviral activity of compounds 3a–h,
light-brown plates, crystals dimensionꢀꢀ= ꢀꢀ0.47ꢀꢀ× ꢀꢀ0.45ꢀꢀ× ꢀꢀ0.19 4a–f and 5a–h against the HIV-1 strain (IIIB) and the HIV-2
mm3, orthorhombic, space group Pbca, aꢀꢀ= ꢀꢀ13.699(3) strain (ROD) in MT-4 cells was performed using an MTT
bꢀꢀ= ꢀꢀ9.328(2) cꢀꢀ= ꢀꢀ20.497(4) Å, Zꢀꢀ= ꢀꢀ8, Vꢀꢀ= ꢀꢀ2619.3(10) Å3. assay as described previously [25]. In brief, stock solutions
D
calcd.ꢀꢀ= ꢀꢀ1.52 mg cm–3, μ(MoK )ꢀꢀ= ꢀꢀ0.3 mm–1, F(000)ꢀꢀ= ꢀꢀ1232 e, (10 times final concentration) of test compounds were
Tꢀꢀ= ꢀꢀ90(2) K. MoKα radiation,αλꢀꢀ= ꢀꢀ0.71073 Å, 28565 collected added in 25-μL volumes to two series of triplicate wells to
refls. (hkl–17/14, ꢀ ꢀ11, ꢀ ꢀ25), 2639uniquerefls. (Rintꢀꢀ= ꢀꢀ0.0685); allow simultaneous evaluation of their effects on mock-
190 refined parameters. R1/wR2ꢀꢀ= ꢀꢀ0.0464/0.0969 (all data), and HIV-infected cells at the beginning of each experi-
GOFꢀꢀ= ꢀꢀ1.022, Δρfin (max/min)ꢀꢀ= ꢀꢀ0.23/–0.31 e Å–3.
ment. Serial 5-fold dilutions of test compounds were made
CCDC 990263 contains the supplementary crystallo- directly in flat-bottomed 96-well microtiter trays using a
graphic data for this paper. These data can be obtained Biomek 3000 robot (Beckman instruments). Untreated
free of charge from the Cambridge Crystallographic Data control, HIV-, and mock-infected cell samples, were
Centre viawww.ccdc.cs.ac.uk/data_request/cif.
included for each sample. HIV-1(IIIB) [33] or HIV-2 (ROD)
[34] stock (50 μL) at 100–300 CCID50 (50ꢀ% cell culture
infectious dose) or culture medium was added to either of
the infected or mock-infected wells of the microtiter tray.
Mock-infected cells were used to evaluate the effect of test
4.5 Anti-kinesin Eg5 assay
The ATPase activity of the Eg5 motor domain was meas- compound on uninfected cells in order to assess the cyto-
ured using the malachite green assay as described earlier toxicity of the test compounds. Exponentially growing
[24]. The reactions were performed in a buffer (80 mm MT-4 cells [35] were centrifuged for 5 min at 1000 rpm,
Pipes, pHꢀꢀ= ꢀꢀ6.8; 1 mm EGTA, 1 mm MgCl2, 0.1 mgmL–1 BSA, and the supernatant was discarded. The MT-4 cells were
1 mm taxol) supplemented with Eg5 (48 nm) fusion protein resuspended at 6ꢀꢀ× ꢀꢀ105 cells per mL, and 50-μL volumes
and microtubules (200 nm). Ten minutes after compound were transferred to the microtiter tray wells. Five days
addition, reactions were started by the addition of ATP after infection, the viability of the mock- and HIV-infected
(50 mm) and incubated at r.t. for 7 min. The reactions cells was examined spectrophotometrically.
were stopped by adding perchloric acid (444 mm, Fluka),
and the color reaction was started by adding the devel- Acknowledgments: We thank Prof. C. Pannecouque of
oper solution [1 m HCl (Sigma), 33 mm malachite green Rega Institute for Medical Research, Katholieke Univer-
(Sigma), 775 mm ammonium molybdate tetrahydrate siteit Leuven, Belgium, for the anti-HIV test and Prof.
(Sigma)]. After 20 min, the absorbance at 610 nm was T. U. Mayer of Biology Department, University of Kon-
measured using a plate reader (Victor2, Perkin-Elmer). stanz, Germany for the facilities for measurement of the
The IC50 values were determined in three independent in vitro kinesin Eg5 inhibitory activity. Ms. A. Frimel of the
experiments for each compound. Fig. 5 shows the ATPase Chemistry Department, University of Konstanz, Germany,
activity screening assay.
is acknowledged for the 2D NMR experiments.
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58ꢀ
ꢀI. Khan et al.: New triazolothiadiazole and triazolothiadiazine derivatives as kinesin Eg5 and HIV inhibitors
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