New insights in the structure-activity relationships of 2-phenylamino-substituted benzothiopyrano[4,3-d]pyrimidines as kinase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2018.03.013

Inhibition of angiogenesis via blocking vascular endothelial growth factor receptor (VEGFR) signaling pathway emerged as an established approach in anticancer therapy. So far, many monoclonal antibodies and ATP-competitive small molecule inhibitors have been clinically validated and approved. In this study, structure-activity relationships (SAR) within the 2-phenylamino-substituted benzothiopyrano[4,3-d]pyrimidine class of kinase inhibitors were further refined by the synthesis and biological evaluation of new compounds 1–21 featuring different substitution patterns on the pendant phenyl moiety, combined with H, OCH₃, or Cl at 8-position. Most compounds showed a promising human kinase insert domain receptor (KDR) inhibition profile, with IC₅₀ values in the submicromolar/low nanomolar range, and promising antiproliferative activity on human umbilical vein endothelial cells (HUVECs) as well as on a panel of three human tumor cell lines. The angio-kinase selectivity profile was assessed for the most promising compound 16 against a set of six human kinases. Finally, computational studies allowed clarifying at molecular level the interaction pattern established by the compounds with KDR, highlighting key stable cation-π interactions, and thus providing the basis for further designing novel inhibitors.

Full text of DOI:10.1016/j.ejmech.2018.03.013

Accepted Manuscript
New insights in the structure-activity relationship of 2-phenylamino-substituted
benzothiopyrano[4,3-d]pyrimidines as kinase inhibitors
Silvia Salerno, Aída Nelly García-Argáez, Elisabetta Barresi, Sabrina Taliani,
Francesca Simorini, Concettina La Motta, Giorgio Amendola, Stefano Tomassi,
Sandro Cosconati, Ettore Novellino, Federico Da Settimo, Anna Maria Marini, Lisa
Dalla Via
PII:
S0223-5234(18)30248-4
10.1016/j.ejmech.2018.03.013
EJMECH 10278
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 26 January 2018
Revised Date: 28 February 2018
Accepted Date: 2 March 2018
Please cite this article as: S. Salerno, Aí.Nelly. García-Argáez, E. Barresi, S. Taliani, F. Simorini, C.
La Motta, G. Amendola, S. Tomassi, S. Cosconati, E. Novellino, F. Da Settimo, A.M. Marini, L.D. Via,
New insights in the structure-activity relationship of 2-phenylamino-substituted benzothiopyrano[4,3-
d]pyrimidines as kinase inhibitors, European Journal of Medicinal Chemistry (2018), doi: 10.1016/
j.ejmech.2018.03.013.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
New insights in the structure-activity relationship of 2-phenylamino-substituted
benzothiopyrano[4,3-d]pyrimidines as kinase inhibitors.
b
a
a
Silvia Salerno , Aída Nelly García-Argáez , Elisabetta Barresi , Sabrina Taliani a,*, Francesca
c
c,*
a
a
c
Simorini , Concettina La Motta , Giorgio Amendola , Stefano Tomassi , Sandro Cosconati
Ettore Novellino d, Federico Da Settimo a, Anna Maria Marini a, Lisa Dalla Via b.
,
a Dipartimento di Farmacia, Università di Pisa, Via Bonanno 6, 56126 Pisa, Italy.
b Dipartimento di Scienze del Farmaco, Università di Padova, Via Marzolo 5, I-35131 Padova, Italy
c DiSTABiF, Università della Campania Luigi Vanvitelli, Via Vivaldi 43, 81100 Caserta, Italy.
d
Dipartimento di Farmacia, Università di Napoli “Federico II”, Via D. Montesano 49, 80131
Napoli, Italy.
1

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Abstract
Inhibition of angiogenesis via blocking vascular endothelial growth factor receptor (VEGFR)
signaling pathway emerged as an established approach in anticancer therapy. So far, many
monoclonal antibodies and ATP-competitive small-molecule inhibitors have been clinically
validated and approved. In this study, structure-activity relationships (SAR) within the 2-
phenylamino-substituted benzothiopyrano[4,3-d]pyrimidine class of kinase inhibitors were further
refined by the synthesis and biological evaluation of new compounds 1-21 featuring different
substitution patterns on the pendant phenyl moiety, combined with H, OCH3, or Cl at 8-position.
Most compounds showed a promising human kinase insert domain receptor (KDR) inhibition
profile, with IC50 values in the submicromolar/low nanomolar range, and promising
antiproliferative activity on human umbilical vein endothelial cells (HUVECs) as well as on a panel
of three human tumor cell lines. The angio-kinase selectivity profile was assessed for the most
promising compound 16 against a set of six human kinases. Finally, computational studies allowed
clarifying at molecular level the interaction pattern established by the compounds with KDR,
highlighting key stable cation-π interactions, and thus providing the basis for further designing
novel inhibitors.
Keywords: benzothiopyranopyrimidines, KDR kinase, antiproliferative activity, kinase inhibitors.
Abbreviations: A-431, epidermoid carcinoma cell line; DMEDA, N,N'-dimethylethylendiamine;
HeLa, cervix adenocarcinoma cell line; h, human; HUVEC, human umbilical vein endothelial cell;
KDR, human kinase insert domain receptor; MSTO-211H, biphasic mesothelioma cell line; SAR,
structure-activity relationships; TKR, tyrosine kinase receptor; VEGF, vascular endothelial growth
factor; VEGFR, vascular endothelial growth factor receptor.
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1. Introduction
Conventional chemotherapeutics have achieved an efficacy plateau against most solid malignancies
and exhibit serious side effects. Advances in understanding of tumor biology have led to the
development of a novel class of anticancer drugs targeting specific pathways involved in growth,
progression and spread of cancer, termed “targeted therapy” [1,2].
Angiogenesis is a pivotal process implicated in tumor growth and metastatic dissemination. In
healthy individuals, angiogenesis is strictly limited to short and sporadic time periods such as
development, reproduction, and wound healing [3]. However, in the setting of tumor growth, the
interplay between co-option of the existing vasculature and tumor angiogenesis becomes an
important mechanism for solid tumors to proliferate and metastasize beyond the limits of passive
diffusion [4,5]. During pathologic angiogenesis, the balance of pro- and anti-angiogenic factors is
shifted to sustained production of proangiogenic factors, leading to the formation of new blood
vessels that support the tumor [3].
The vascular endothelial growth factor (VEGF) pathway represents one of the most important
positive modulators of angiogenesis, and, within the VEGF family (VEGF-A, VEGF-B, VEGF-C,
VEGF-D, VEGF-E), VEGF-A (also termed simply VEGF) is thought to be the most critical in both
physiological and pathological angiogenesis [6].
Various members of the VEGF family have similar abilities to interact with a set of highly
homologous tyrosine kinase receptors (TKRs): VEGFR-1 (Flt-1) and VEGFR-2 (human KDR or
murine Flk-1), largely expressed in endothelial cells, and primarily involved in angiogenesis [7,8].
Therefore, considering the role of the VEGF pathway in angiogenesis, antiangiogenic strategies
often involve the use of small molecule tyrosine kinase inhibitors (TKIs) to target one or more of
the VEGFRs [9,10]. These TKIs compete with ATP binding in the catalytic domain of the tyrosine
kinases, inhibiting kinase activity, thereby preventing activation of downstream signaling cascades.
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A number of small-molecule kinase inhibitors targeting primarily VEGFR (e.g. vatalanib, cediranib,
axitinib, sorafenib, pazopanib, Chart 1) have been approved or are in development for clinical use
[11-13]. In particular, a significant number of VEGFR-2 small molecule inhibitors characterized by
a variously substituted pyrimidine or 1,2,4-triazine ring, either isolated or fused to form different
heterocyclic structures, have been investigated during the last decade and some of them (e.g.,
RO4383596 [14], TKI-28 [15], BMS-690514 [16], AZD2932 [17], Chart 2) are in clinical trials for
the treatment of different tumors [18,19].
Chart 1. VEGFR Inhibitors clinically approved or in development for the treatment of solid
tumors.
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Chart 2. Pyrimidine- and 1,2,4-triazine-based VEGFR inhibitors in clinical trials.
In this context, we recently reported the synthesis and the biological evaluation of novel
antiangiogenic agents featuring the benzothiopyrano[4,3-d]pyrimidine scaffold decorated with a
pendant benzylamino or phenylamino moiety (A and B, respectively, Chart 3) [20], whose design
was based on the structures of some anilino-substituted pyrimidines reported as inhibitors of protein
kinases [21]. In particular, compounds bearing a phenyl-amino side group at the 2-position of the
tricyclic system (B, Chart 3) emerged as the most interesting ones, many of these displaying a
promising inhibitory profile on both human kinase insert domain receptor (KDR) and human
umbilical vein endothelial cells (HUVECs), with IC50 and GI50 values, respectively, in the low
micromolar range [20]. All in all, these studies suggested that the new 2-anilino substituted
heteropolycyclic moiety could be worth of further structural investigation.
According to our initial molecular docking studies [20], the benzothiopyranopyrimidine scaffold is
inserted in the hydrophobic ATP cleft of the enzyme active site. Here, the ligand would be able to
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establish a double H bond with its nitrogen at 3-position and the adjacent aniline NH at 2-position
with the backbone NH and CO of Cys919, respectively. Moreover, according to our previous work,
while the ligand aniline NH would be critical for ligand binding, bulky substituents at 8-position
have a detrimental effect.
A close inspection of our already published theoretical interaction model [20] also reveals the
presence of Lys838 and Phe1047 residues that could provide further interaction points. In principle,
contacts with these latter residues could be further enhanced by designing new
benzothiopyranopyrimidine analogues with potentially higher inhibitory activities.
Based on all these considerations, with the aim of further investigating the structure-activity
relationships (SAR) of these 2-anilino-substituted benzothiopyranopyrimidines for the development
of novel kinase inhibitors acting as antiangiogenic agents, we designed, synthesized and
biologically evaluated new compounds, featuring different substitution patterns on the pendant
phenyl moiety, combined with an H, OCH3, or Cl at 8-position (1-21, Chart 3). The groups on the
pendant phenyl ring were selected taking into account literature data about small molecules kinase
inhibitors, featuring meta- or poly-substituted aniline or phenyl moieties [22-24].
Chart 3. Structures of previously reported (A, B) [20], and newly synthesized
benzothiopyranopyrimidines (1-21).
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The ability of the new synthesized benzothiopyranopyrimidines 1-21 to inhibit the kinase activity of
the KDR was determined by a fluorimetric assay designed to detect and characterize kinase
inhibitors. In addition, the antiproliferative activity of the compounds on HUVECs and on a panel
of human tumor cell lines (HeLa, A-431, and MSTO-211H) was evaluated. For the most promising
compound 16, the angio-kinase selectivity profile was assessed against a set of six human kinases.
Finally, molecular docking calculations allowed clarifying at molecular level the proposed
interaction pattern established by the compounds with KDR.
2. Results and Discussion
2.1. Chemistry
The intermediates in the synthesis of target compounds 1-21 were the 2-aminopyrimidino
derivatives 24a-c, which were prepared by a previously reported two-steps procedure (Scheme 1)
[20]. Briefly, reaction of 7-substituted-2,3-dihydrobenzo[3',2':5,6]thiopyrano-4(4H)-ones 22a-c
with an excess of dimethylformamide dimethylacetal (DMF DMA) in refluxing toluene furnished
the 3-dimethylaminomethylene derivatives 23a-c [20,25]. Intermediates 23a-c were then treated
with guanidine hydrochloride, in the presence of sodium ethoxide in refluxing ethanol, to obtain the
2-aminopyrimidino derivatives 24a-c, with good yields [20]. Finally, compounds 24a-c were
allowed to react with the commercially available appropriately substituted phenyliodide in
anhydrous dioxane, under nitrogen atmosphere and in the presence of K2CO3, N,N'-
dimethylethylendiamine and CuI, affording the crude 1-21, which were purified by flash
chromatography, using petroleum ether 60-80 °C/ethyl acetate 5:5 as the eluting system for all the
compounds.
All the target derivatives showed high purity and the proposed structures were confirmed on the
basis of 1H-NMR, 13C-NMR, and HMRS data.
Scheme 1
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1: X = H, R3 = OCH3, R4 = R5 = H
2: X = H, R3 = Cl, R4 = R5 = H
3: X = H, R3 = Br, R4 = R5 = H
4: X = H, R3 = F, R4 = R5 = H
5: X = H, R3 = CF3, R4 = R5 = H
11: X = Cl, R3 = OCH3, R4 = R5 = H
12: X = Cl, R3 = Cl, R4 = R5 = H
13: X = Cl, R3 = Br, R4 = R5 = H
14: X = Cl, R3 = F, R4 = R5 = H
15: X = Cl, R3 = CF3, R4 = R5 = H
6: X = OCH3, R3 = OCH3, R4 = R5 = H 16: X = H, R3 = R4 = OCH3, R5 = H
7: X = OCH3, R3 = Cl, R4 = R5 = H
8: X = OCH3, R3 = Br, R4 = R5 = H
9: X = OCH3, R3 = F, R4 = R5 = H
10: X = OCH3, R3 = CF3, R4 = R5 = H
17: X = H, R3 = R4 = R5 =OCH3
18: X = R3 = R4 = OCH3, R5 = H
19: X = R3 = R4 = R5 = OCH3
20: X = Cl, R3 = R4 = OCH3, R5 = H
21: X = Cl, R3 = R4 = R5 = OCH3
Reagents and conditions: (i) dimethylformamide dimethylacetal (DMF DMA), refluxing toluene,
(ii) guanidine hydrochloride, EtONa, refluxing ethanol; (iii) 3-substituted, 3,4-dimethoxy or 3,4,5-
trimethoxy phenyliodide, K2CO3, N,N'-dimethylethylendiamine (DMEDA), CuI, dioxane.
2.2. Biological evaluation
2.2.1. Effect on KDR kinase activity
The ability of 1-21 to inhibit the KDR kinase activity was determined on a recombinant human
kinase insert domain receptor, by using a fluorimetric assay.
Table 1 lists the activity of benzothiopiranopyrimidines 1-21, together with that of representative
compounds from series B (2a-i). for comparison purposes. Semaxanib was taken as reference
compound because of its selectivity in inhibiting VEGFR-1 and -2 and VEGF-dependent
endothelial proliferation as compared with other well known tyrosine kinase inhibitors [26]. The
results are expressed as inhibition percentage at 7 µM concentration or as IC50 values for the most
active compounds.
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An interdependent effect on KDR inhibition of the meta-substituent on the pendant anilino phenyl
ring and the substitution pattern at the 8-position of the tricyclic scaffold can be observed for
compounds 1-15. Specifically, within the 8-unsubstituted subset (compounds 1-5), the shift of the
methoxy group from para (2b) to meta position (1) produces a gain in inhibitory efficacy against the
target protein. In general, the replacement of the methoxy with Cl, Br or CF3 groups (2, 3 and 5,
respectively) decreases drastically the activity. The sole exception to this trend is represented by the
fluorine-substituted ligand 4 that shows a 52% of inhibition, which is slightly higher with respect to
that of by semaxanib, but still lower than the activitiy displayed by the methoxy-substituted
compound 1. Interestingly, most derivatives from the 8-methoxy subset (6-10) show good to high
inhibitory efficacy, with percentage values of inhibition comparable or higher than that of the
reference. In this case, the most active compound 8 features the 3′-bromo-8-methoxy substitution
pattern and notably, it is able to induce an almost complete inhibition at the considered
concentration. Conversely, the presence of a chlorine (7) or a fluorine (9) yields 3′-substituted
compounds showing inhibitory efficacy significantly lower with respect to 8 and comparable to that
of semaxanib. The insertion of a chlorine at 8 position gives the subset 11-15 that shows an
inhibitory trend similar to those of the unsubstituted 1-5. In particular, the presence of a methoxy
group or a fluorine atom at the meta (3') position enables an effect on KDR. In this case, for both
compounds the percentage of inhibition is comparable to that of semaxanib. Otherwise, Cl, Br or
CF3 groups render the 8-chloro-substituted benzothiopiranopyrimidines unable to induce any effect
on the kinase activity of the enzyme.
Noteworthy, very interesting results regarding the role of the different substituents on KDR
inhibition were obtained by the insertion of two methoxy groups in R3 and R4 on the phenyl ring of
the side chain and by the presence or the absence of the same group in 8 (compounds 16-21).
Actually, the 8-unsubstituted and 8-Cl derivatives (16, 17, 20, 21), are the most active of the whole
series probably due to electronic effects that favor charge transfer interactions with the target
protein. In contrast, for the 8-OCH3 substituted derivatives 18 and 19, an inhibitory effect is
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observed, probably due to an excessive dimension of the whole molecule that determines its
relocation in the enzyme active site and, consequently, the loss of crucial interactions with the
protein, in agreement with previous findings [20].
The activity of the best performing derivatives was also confirmed by the determination of the IC50
values (Table 1). The results highlighted for all the most active compounds 1, 8, 16, 17, 20 and 21 a
very interesting capacity to inhibit the enzyme, with IC50 values ranging from 0.11 to 1.6 µM
significantly lower with respect to the values obtained for both the most active of the previously
reported benzothiopyranopyrimidines (2b) and the reference compound (semaxanib, IC50 = 12.9
µM). Of note, 16 and 17 (3′,4′-dimethoxy- and 3′,4′,5′-trimethoxy-substituted, respectively)
displayed a 24- and 10-fold increase in KDR inhibitory activity (expressed as IC50 value) with
respect to their parent compound 2b, further supporting the beneficial effect produced by the
methoxy groups.
Table 1. KDR enzyme inhibitory activity and HUVEC growth inhibition of derivatives 1-21 and
semaxanib as reference compound.
KDR
IC50 (µM)b
8.20
KDR
(%)a
54
HUVEC
GI50 (µM)
2.40
X
R3
R4
R5
cpd
2ad
2bd
2cd
2dd
2ed
2fd
H
H
H
H
H
H
H
H
H
OCH3
Cl
H
H
H
H
H
H
67
2.70
0.45
H
<20
35
>50
7.30
OCH3
OCH3
OCH3
H
>50
4.30
OCH3
Cl
39
17.50
>50
0.74
<20
>20
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2gd
2hd
2id
1
Cl
Cl
H
H
H
OCH3
Cl
H
H
<20
<20
61
>50
>50
5.60
0.97
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
0.62
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
0.11
0.27
n.d.
n.d.
0.73
1.60
12.90
>20
16.40
0.35
0.80
7.13
7.00
4.51
5.02
2.49
11.19
1.89
11.48
4.55
14.17
16.51
7.11
18.23
10.46
1.50
1.99
10.82
3.71
2.53
2.54
Cl
H
H
H
OCH3
Cl
H
H
75
H
H
H
<20
<20
52
2
H
Br
H
H
3
H
F
H
H
4
H
CF3
OCH3
Cl
H
H
<20
62
5
OCH3
OCH3
OCH3
OCH3
OCH3
Cl
H
H
6
H
H
40
7
Br
H
H
93
8
F
H
H
35
9
CF3
OCH3
Cl
H
H
21
10
11
12
13
14
15
16
17
18
19
20
21
H
H
44
Cl
H
H
<20
<20
42
Cl
Br
H
H
Cl
F
H
H
Cl
CF3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
-
H
H
<20
97
H
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
-
H
H
OCH3
H
81
OCH3
OCH3
Cl
<20
46
OCH3
H
87
Cl
OCH3
-
73
semaxanibd
-
40
13.60
a
b
Percentage of kinase inhibition obtained at 7 µM of the test compound. IC50 values represent
c
the concentration that induces the 50% reduction in enzyme activity. GI50 values represent the
concentration (µM) of compound able to produce 50% cell death with respect to the control
culture. d Data from ref. [20].
2.2.2. Growth inhibition on HUVEC cells
The new benzothiopyranopyrimidine derivatives 1-21 were evaluated for their effect on cell growth
of HUVEC cells.
The results, expressed as GI50 values, i.e. concentration (µM) able to induce 50% cell death with
respect to a control culture, are shown in Table 1. Semaxanib was used as reference compound.
After a 72h exposure, the majority of the newly synthesized benzothiopyranopyrimidines exert a
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detectable antiproliferative effect on HUVEC cells, with GI50 values ranging in the low micromolar
range (from 0.80 to 18.2 µM). Interestingly, a comparison between the antiproliferative effect
exerted by 1-21 and their ability to inhibit the kinase activity of KDR highlighted a noteworthy
positive correlation. Indeed, among the newly investigated benzothiopyranopyrimidine derivatives
1-21, the compounds that resulted more potent in KDR inhibition (1, 8, 16, 17, 20, 21) also showed
low GI50 values. In particular, for the most potent benzothiopyranopyrimidine 16 both the IC50 and
GI50 values are several folds lower than the corresponding values recorded for semaxanib (see Table
1).
2.2.3. Antiproliferative activity on human tumor cell lines.
The antiproliferative activity of benzothiopyranopyrimidine derivatives 1-21 against a panel of
human tumor cell lines, HeLa (cervix adenocarcinoma), A-431 (epidermoid carcinoma) and MSTO-
211H (biphasic mesothelioma), was evaluated to define their cell proliferation profile.
The results, expressed as GI50 values, are shown in Table 2 and semaxanib was taken into
consideration as reference. The obtained results highlighted for the majority of the newly
synthesized derivatives evident cytotoxic effects, as demonstrated by the GI50 values in the
micromolar range. Cell growth evaluation revealed again a positive effect attributable to a poly-
methoxy substitution on the pendant phenyl ring: molecules from the subseries 16-21 are highly
cytotoxic derivatives, with the exception of 18. Interestingly, 19 proved to be one the most
antiproliferative compound, despite its low percentage of KDR inhibition, suggesting that its
activity is probably mediated by interactions with other molecular targets.
Overall, the results shown in Table 2 appear roughly in accordance with those of Table 1,
suggesting the activity of benzothiopyranopyrimidine derivatives is mainly mediated by KDR
inhibition, although interference with additional biological targets and/or kinases cannot be
excluded.
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Table 2. Cell growth inhibition of derivatives 1-21 and semaxanib as reference compound.
GI50 (µM)a
X
R3
R4
R5
cpd
HeLa
6.03±1.81
>20
A-431
MSTO-211H
0.80±0.14
6.10±1.44
7.67±1.26
2.06±1.04
11.8±2.5
2.57±0.87
5.60±1.27
2.20±0.30
13.0±1.3
9.04±0.52
6.83±1.36
7.78±1.26
8.50±1.14
>20
H
H
OCH3
Cl
H
H
H
H
9.43±0.21
10.4±1.8
10.6±1.6
9.76±0.68
13.5±2.1
2.10±0.36
8.25±2.72
4.77±0.42
15.5±1.4
6.24±0.42
16.8±1.6
>20
1
2
H
Br
H
H
>20
3
H
F
H
H
14.0±2.1
>20
4
H
CF3
OCH3
Cl
H
H
5
OCH3
OCH3
OCH3
OCH3
OCH3
Cl
H
H
2.80±0.44
11.9±1.8
2.93±0.51
10.3±1.6
6.02±0.46
>20
6
H
H
7
Br
H
H
8
F
H
H
9
CF3
OCH3
Cl
H
H
10
H
H
11
Cl
H
H
>20
12
Cl
Br
H
H
>20
17.0±1.4
18.2±1.5
10.0±0.6
2.32±0.41
1.92±0.31
6.06±1.16
1.75±0.44
2.23±0.71
4.27±0.50
37±3
13
Cl
F
H
H
20
14
Cl
CF3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
-
H
H
>20
3.18±0.28
1.63±0.35
1.08±0.19
>20
15
H
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
-
H
1.50±0.29
1.26±0.17
18.8±1.2
0.63±0.15
3.42±0.44
3.46±0.45
38±1
16
H
OCH3
H
17
OCH3
OCH3
Cl
18
OCH3
H
0.92±0.13
4.40±1.15
3.40±1.10
27±2
19
20
Cl
OCH3
-
21
-
semaxanib
a
GI50 values represent the concentration (µM) of compound able to produce 50% cell
death with respect to the control culture.
2.2.4 Effect on human kinases
To investigate the possibility that other kinases, apart from KDR, could be affected by the new
benzothiopyranopyrimidine derivatives, the selectivity profile of compound 16, the most active
compound in inhibiting KDR, was determined. For this purpose, the effect of 16 was further
assessed against a set of six human (h) kinases, i.e. AurA/Aur2 kinase, CDC2/CDK1, EGFR kinase,
PDGFRβ kinase, RAF-1 kinase and Src kinase, which are reported to be mainly involved in cell
proliferation. The obtained results suggest 16 as a multi-target inhibitor, because, besides KDR, it is
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able, analogously to what previously reported for the parent compound 2b [20], to inhibit to a
significant extent also Aurora (AurA/Aur2) kinase, a nuclear serine/threonine protein kinase
involved in many functions related to mitosis (Figure 1, Tables S1a, and S1b) [27]. Moreover, a
relevant inhibitory effect occurs toward the non-receptor tyrosine kinase Src. The multiplicity of
kinase targets of 16 could explain the ability of this compound to inhibit along with HUVEC cells
also a number of other human tumor cell lines.
Figure 1. Percentage of inhibition of kinase activity in the presence of compound 16.
2.3. Molecular Modeling.
To elucidate the possible reasons behind the displayed activity of the newly designed compounds,
molecular docking studies were performed. The calculations were executed with the AutoDock Vina
software [28], employing an ensemble approach, which has been proven to be a successful way to
accurately predict the correct ligand binding poses [29-31] in the case of pronounced protein
plasticity upon ligand binding. Namely, the most active compound, 16, was docked to all the
available crystal structure of VEGFR2 with a resolution below 2 Å (see molecular modeling
methods for PDB codes). The docked poses were visually inspected for correct geometries.
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According to these simulations 16 was predicted to form the most stable complex with the
published VEGFR2 crystal having PDB code 3EWH [32] as indicated by the calculated binding
free energy (∆GVINA= -8.7 kcal/mol). In this structure, the activation loop of the enzyme adopts the
inactive DFG-out conformation.
Interestingly, the 16 docked pose is consistent with our previous findings on the parent compound
2b [20]. The benzothiopyranopyrimidine scaffold is well positioned in the ATP binding site to
establish a double H-bond with the backbone NH and CO of Cys919 on the hinge region through its
nitrogen at position 3 and the adjacent exocyclic NH at position 2 (Figure 2a). The tricyclic system
engages favorable contacts with the hydrophobic cleft which comprises Leu840, Val848, Ala866,
Val899, and Leu1035. Moreover, charge transfer interactions are established with Phe918 and
Phe1047. Of note, the ligand pendant anilino phenyl ring is oriented outwards of the binding pocket
and is stabilized by a cation-π interaction with the Lys838 side-chain.
Figure 2. (a) Binding mode of compound 16, within the VEGFR-2 active site (PDB code 3EWH).
The ligand is represented as green sticks while the protein as orange sticks and ribbons. H-bonds are
depicted as dashed yellow lines. The picture was rendered with the UCSF Chimera software [33].
(b) Electrostatic potentials of compounds 5 and 16 mapped onto their electron density, with a scale
15

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of −20.0 (red) to 35.0 kcal/mol (blue).
The occurrence of such an interaction would explain the influence of the substitution pattern of the
pendant aniline ring on the VEGFR inhibition activity. Indeed, electron-donating substituents
generally increase the negative electrostatic potential of the aromatic ring, thereby enhancing the
charge transfer interaction [34]. This should account for the relatively high inhibition potential
displayed by compounds 1, 16, 17, 20, 21 featuring one, two or three methoxy groups. On the other
hand, the insertion on the phenyl ring of electron-withdrawing substituents should weaken the
cation-π interaction and is therefore generally detrimental for the inhibitory potency. This should
explain why congeners featuring Cl, F, Br, and CF3 (2, 3, 4, 5) show low inhibition towards the
enzyme. The influence of the substitution pattern on the electron-availability of the pendant anilino
moiety is clearly evidenced in Figure 2b where the electrostatic potentials, calculated through ab
initio calculations, of an inactive congener (5) and of the most active of the series (16) are reported.
In general, substitutions at position 8 are not well tolerated, probably due to the limited extension of
the cleft, which does not enable the allocation of bulky substituents, with the sole exception to this
trend being the submicromolar inhibitory activity displayed by compound 8.
3. Conclusions
To further investigate the SAR of the 2-anilino-substituted benzothiopyranopyrimidine class of
kinase inhibitors for the development of novel antiangiogenic agents, a library of novel derivatives
featuring different substitution patterns on the pendant phenyl moiety, combined with H, OCH3, or
Cl at 8-position, was synthesized and biologically evaluated.
Many compounds showed a promising KDR inhibition profile, with IC50 values (determined only
for those derivatives displaying the highest inhibition percentages) in the submicromolar/low
micromolar range (from 0.11 to 1.6 µM), significantly lower with respect to those obtained for both
the most active of the previously reported benzothiopyranpyrimidines (2b) and the reference
16

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compound (semaxanib, IC50 = 12.9 µM). The most interesting results in terms of KDR inhibition
were obtained by the insertion of two or three methoxy groups on the pendant phenyl ring combined
with 8-H and 8-Cl substitution (16, 17, 20, 21), with 16 being the best performing compound of the
whole series.
SAR data were rationalized by the creation of a theoretical ligand/enzyme complex. Visual
inspection of such a complex suggest that electron-donating substituents (such as methoxy) on the
aniline ring would enhance the charge transfer interaction with the target protein thereby producing
a beneficial effect on KDR inhibition activity.
All the new benzothiopyranopyrimidine derivatives were evaluated for their effect on cell growth of
human umbilical vein endothelial primary cells (HUVECs), and a panel of human tumor cell lines,
HeLa (cervix adenocarcinoma), A-431 (epidermoid carcinoma) and MSTO-211H (biphasic
mesothelioma). The majority of these compounds exert evident cytotoxic effects (GI50 values in the
low micromolar range), revealing again a positive effect ascribable to a poly-methoxy substitution
on the pendant phenyl ring.
Finally, selectivity profile of compound 16 has been assessed against a set of six human kinases
(AurA/Aur2 kinase, CDC2/CDK1, EGFR kinase, PDGFRβ kinase, RAF-1 kinase and Src kinase) to
investigate the possibility that other kinases could be involved in its biological activity. Actually,
the obtained results suggested 16 as a multi-target inhibitor, analogously to what previously
reported for its parent compound 2b [20].
Taken together, these data corroborate the 2-anilino-substituted benzothiopyrano moiety as an
interesting structure inside the field of kinase-targeted antitumor agents, providing the platform for
further structural optimization aimed to obtain increasingly potent TK inhibitors.
4. Experimental protocols
4.1.Chemistry
17

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1
The uncorrected melting points were determined using a Reichert Köfler hot-stage apparatus. H
13
NMR spectra were recorded on a Bruker AVANCE 400 (1H, 400 MHz, C, 100 MHz) in DMSO-
d6 (internal standard tetramethylsilane). The coupling constants (J) are given in Hertz. Accurate
molecular weights of compounds were confirmed by ESI mass spectrometry using a Q Exactive
Orbitrap LC−MS/MS (Thermo Fisher Scientific, Waltham, MA, USA). Analytical TLC have been
carried out on Merck 0.2 mm precoated silica gel aluminium sheets (60 F-254). Silica gel 60 (230–
400 mesh) was used for column chromatography. Purity of the target inhibitors 1-21 was
determined using a Shimadzu LC-20AD SP liquid chromatograph equipped with a DDA Detector
set at 204 nm and 280 nm (column C18 (250 mm x 4.6 mm, 5 µm, Shim-pack)); the mobile phase,
delivered at isocratic flow, consisted of methanol (80%) and water (20%) and a flow rate of 1.0
mL/min; all the compounds showed percent purity values of >95%. The combustion analyses of
compounds 1-21 are indicated by symbols, and the analytical results are within ± 0.4% of the
theoretical values. Reagents, starting materials, and solvents were purchased from commercial
suppliers and used as received. 2,3-Dihydrobenzo[3',2':5,6]thiopyrano-4(4H)-one 22a and 7-
methoxy-2,3-dihydrobenzo[3',2':5,6]thiopyrano-4(4H)-one 22b were commercially available. The
following compounds were obtained according to methods previously described: 7-chloro-2,3-
dihydrobenzo[3',2':5,6]thiopyrano-4(4H)-one
dihydrobenzo[3',2':5,6]thiopyran-4(4H)-one
22c
23a,
[25];
3-Dimethylaminomethylen-2,3-
7-Methoxy-3-dimethylaminomethylen-2,3-
dihydrobenzo[3',2':5,6]thiopyran-4(4H)-one 23b and 7-Chloro-3-dimethylaminomethylen-2,3-
dihydrobenzo[3',2':5,6]thiopyran-4(4H)-one 23c [25]; 2-Amino-5H-benzo[3',2':5,6]thiopyrano[4,3-
d]pyrimidine 24a, 2-Amino-8-methoxy-5H-benzo[3',2':5,6]thiopyrano[4,3-d]pyrimidines 24b and
[2-Amino-8-chloro-5H-benzo[3',2':5,6]thiopyrano[4,3-d]pyrimidine 24c [20,25].
4.1.1. General synthetic procedures for 2-Anilino-5H-benzo[3',2':5,6]thiopyrano[4,3-d]pyrimidines
1-21
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The appropriate 2-amino-5H-benzo[3',2':5,6]thiopyrano[4,3-d]pyrimidine 22a-c (0.417 mmol) was
added, at room temperature, under nitrogen atmosphere, to 0.079 g of CuI (0.417 mmol) and 0.115
g of anhydrous K2CO3 (0.833 mmol). Then, the appropriate substituted aryl iodide (0.694 mmol),
0.04 mL of DMEDA (0.417 mmol) and 2.5 mL of dioxane were added. The reaction mixture was
heated at 100 °C and allowed to stir for 24 h. After cooling, the reaction mixture was added with 2.5
mL of concentrated NH3 and with a saturated solution of NaCl (10-15 mL). The mixture was
extracted with ethyl acetate. The organic phase was dried (MgSO4) and evaporated under reduced
pressure giving a residue which was purified by flash chromatography using petroleum ether 60-
80°C/ethyl acetate 5:5 as the eluting system, to give compounds 1-21.
4.1.1.1. 2-(3-Methoxyanilino)-5H-benzothiopyrane[4,3-d]pyrimidine (1). Yield: 30%; m. p. 136-
137 °C; 1H NMR (400 MHz, DMSO-d6): δ 3.77 (s, 3H), 4.03 (s, 2H), 6.54 (dd, Jmin = 1.8 Hz, Jmax
=
8.0 Hz, 1H), 7.20 (t, J = 8.0 Hz, 1H), 7.32-7.45 (m, 4H), 7.63 (t, J = 2.0 Hz, 1H), 8.31 (d, J = 7.2
Hz, 1H), 8.48 (s, 1H), 9.70 (s, 1H) ppm; 13C NMR (100 MHz, DMSO-d6): δ 25.92, 54.88, 104.23,
106.81, 111.06, 115.22, 126.19, 126.97, 127.97, 129.24, 131.22, 132.12, 136.60, 141.82, 156.48,
157.69, 159.31, 159.55 ppm; HRMS (ESI) m/z calculated for C18H16N3OS ([M+H]+) 322.10141,
found: 322.10098. Anal. C18H15N3OS (C, H, N).
4.1.1.2. 2-(3-Chloroanilino)-5H-benzothiopyrane[4,3-d]pyrimidine (2). Yield: 30%; m. p. 159-160
1
°C; H NMR (400 MHz, DMSO-d6): δ 4.04 (s, 2H), 6.99 (dd, Jmin = 1.4 Hz, Jmax = 8.0 Hz 1H),
7.33 (t, J = 8.2 Hz, 1H), 7.37-7.47 (m, 3H), 7.72 (dd, Jmin = 1.4 Hz, Jmax = 8.0 Hz, 1H), 8.06
13
(t, J = 2.2 Hz, 1H), 8.29 (d, J = 8.0 Hz, 1H), 8.53 (s, 1H), 9.95 (s, 1H) ppm; C NMR (100
MHz, DMSO-d6): δ 25.90, 115.81, 116.90, 117.77, 120.69, 126.23, 126.96, 127.99, 130.15,
131.32, 131.95, 132.96, 136.68, 142.16, 156.46, 157.84, 159.01 ppm; HRMS (ESI) m/z calculated
for C17H13ClN3S ([M+H]+) 326.05187, found 326.05103. Anal. C17H12ClN3S (C, H, N).
4.1.1.3. 2-(3-Bromoanilino)-5H-benzothiopyrane[4,3-d]pyrimidine (3). Yield: 30%; m. p. 178-179
°C; 1H NMR (400 MHz, DMSO-d6): δ 4.03 (s, 2H), 7.11 (dd, Jmin = 2.0 Hz, Jmax = 7.6 Hz, 1H),
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7.26 (t, J = 8.0 Hz, 1H), 7.37-7.40 (m, 1H), 7.43-7.44 (m, 2H), 7.74 (dd, Jmin = 1.2 Hz,
Jmax = 8.4 Hz, 1H), 8.21 (t, J = 2.0 Hz, 1H), 8.28 (d, J = 7.6 Hz, 1H), 8.51 (s, 1H), 9.91 (s,
1H) ppm; 13C NMR (100 MHz, DMSO-d6): δ 25.89, 115.78, 117.27, 120.66, 121.52, 123.57,
126.20, 126.95, 127.97, 130.45, 131.31, 131.94, 136.67, 142.29, 156.48, 157.80, 158.97 ppm;
HRMS (ESI) m/z calculated for C17H13BrN3S ([M+H]+) 370.00136, 371.99931, found 370.00084,
371.99873. Anal. C17H12BrN3S (C, H, N).
4.1.1.4. 2-(3-Fluoroanilino)-5H-benzothiopyrane[4,3-d]pyrimidine (4). Yield: 35%; m. p. 141-
1
143°C; H NMR (400 MHz, DMSO-d6): δ 4.04 (s, 2H), 6.76 (dt, Jmin = 2.2 Hz, Jmax = 8.4 Hz,
1H), 7.30-7.46 (m, 4H), 7.56 (dd, Jmin = 1.8 Hz, Jmax = 8.0 Hz, 1H), 7.85 (td, Jmin = 2.2 Hz,
Jmax = 12.4 Hz, 1H), 8.28 (d, J = 7.2 Hz, 1H), 8.52 (s, 1H), 9.96 (s, 1H) ppm; 13C NMR (100
2
2
MHz, DMSO-d6): δ 25.91, 104.96 (d, JCF = 27 Hz), 107.41 (d, JCF = 22 Hz), 114.33, 115.81,
126.29, 126.97, 128.01, 130.10, 131.32, 131.99, 136.67, 142.54, 156.46, 157.88, 159.07, 162.34 (d,
1JCF = 239 Hz) ppm; HRMS (ESI) m/z calculated for C17H13FN3S ([M+H]+) 310.08142, found
310.08051. Anal. C17H12FN3S (C, H, N).
4.1.1.5. 2-(3-Trifluoromethylanilino)-5H-benzothiopyrane[4,3-d]pyrimidine (5). Yield: 50%; m. p.
154-157 °C; 1H NMR (400 MHz, DMSO-d6): δ 4.05 (s, 2H), 7.29 (d, J = 7.6 Hz, 1H), 7.37-7.39 (m,
1H), 7.44-7.46 (m, 2H), 7.54 (t, J = 7.8 Hz, 1H), 7.96 (d, J = 8.4 Hz, 1H), 8.29 (d, J = 7.6 Hz, 1H),
8.48 (s, 1H), 8.54 (s, 1H), 10.09 (s, 1H) ppm; 13C NMR (100 MHz, DMSO-d6): δ 25.88, 114.38 (q,
3
1
3JCF = 4.0 Hz), 115.89, 117.28 (q, JCF = 4.0 Hz), 122.00, 124.40, (q, JCF = 271 Hz), 126.15,
2
126.90, 127.98, 129.33 (q, JCF = 31 Hz), 129.63, 131.37, 131.94, 136.72, 141.41, 156.65, 157.75,
159.00 ppm; HRMS (ESI) m/z calculated for C18H13F3N3S ([M+H]+) 360.07823, found 360.07759.
Anal. C18H12F3N3S (C, H, N).
4.1.1.6. 8-Methoxy-2-(3-methoxyanilino)-5H-benzothiopyrane[4,3-d]pyrimidine (6). Yield: 30%; m.
p. 137-138 °C; 1H NMR (400 MHz, DMSO-d6): δ 3.76 (s, 3H), 3.84 (s, 3H), 4.02 (s, 2H), 6.53
(dd, Jmin = 2.4 Hz, Jmax = 7.8 Hz, 1H), 6.95-6.99 (m, 2H), 7.19 (t, J = 8.1 Hz, 1H), 7.33 (d,
20

ACCEPTED MANUSCRIPT
J = 8.6 Hz, 1H), 7.61 (s, 1H), 8.25 (d, J = 8.8 Hz, 1H), 8.41 (s, 1H), 9.62 (s, 1H) ppm; 13C
NMR (100 MHz, DMSO-d6): δ 26.23, 54.98, 55.57, 111.32, 112.10, 113.86, 115.21, 116.71,
118.65, 121.78, 124.09, 127.87, 130.43, 131.13, 138.27, 153.31, 159.99, 160.87, 164.81 ppm;
HRMS (ESI) m/z calculated for C19H18N3O2S ([M+H]+) 352.11197, found 352.11124. Anal.
C19H17N3O2S (C, H, N).
4.1.1.7. 8-Methoxy-2-(3-chloroanilino)-5H-benzothiopyrane[4,3-d]pyrimidine (7). Yield: 30%; m.
1
p. 139-140 °C; H NMR (400 MHz, DMSO-d6): δ 3.83 (s, 3H), 4.03 (s, 2H), 6.96-7.00 (m,
3H), 7.32 (t, J = 8.0 Hz, 1H), 7.72 (dd, Jmin = 1.2 Hz, Jmax = 8.4 Hz, 1H), 8.05 (t, J = 2.0
13
Hz, 1H), 8.22 (d, J = 8.8 Hz, 1H), 8.45 (s, 1H), 9.87 (s, 1H) ppm; C NMR (100 MHz,
DMSO-d6): δ 26.15, 55.62, 112.29, 112.98, 114.65, 116.83, 117.69, 120.58, 124.66, 128.66,
130.15, 132.96, 138.61, 142.28, 155.97, 157.88, 158.95, 161.42 ppm; HRMS (ESI) m/z calculated
for C18H15ClN3OS ([M+H]+) 356.06244, found 356.06189. Anal. C18H14ClN3OS (C, H, N).
4.1.1.8. 8-Methoxy-2-(3-bromoanilino)-5H-benzothiopyrane[4,3-d]pyrimidine (8). Yield: 30%; m.
1
p. 126-127 °C; H NMR (400 MHz, DMSO-d6): δ 3.83 (s, 3H), 4.02 (s, 2H), 6.93-6.99 (m,
2H), 7.11 (d, J = 7.6 Hz, 1H), 7.26 (t, J = 8.0 Hz, 1H), 7.69-7.78 (m, 2H), 8.20-8.25 (m,
1H), 8.44 (s, 1H), 9.84 (s, 1H) ppm; 13C NMR (100 MHz, DMSO-d6): δ 26.11, 55.59, 112.08,
112.75, 114.24, 119.87, 120.85, 124.48, 124.79, 128.34, 128.86, 138.41, 139.57, 141.89, 155.91,
157.88, 159.06, 161.35 ppm; HRMS (ESI) m/z calculated for C18H15BrN3OS ([M+H]+) 400.01192,
402.00987, found 400.01126, 402.00894. Anal. C18H14BrN3OS (C, H, N).
4.1.1.9. 8-Methoxy-2-(3-fluoroanilino)-5H-benzothiopyrane[4,3-d]pyrimidine (9). Yield: 30%; m.
1
p. 152-153 °C, H NMR (400 MHz, DMSO-d6): δ 3.82 (s, 3H), 4.03 (s, 2H), 6.75 (t, J = 7.6
Hz, 1H), 6.98-7.00 (m, 2H), 7.31-7.34 (m, 1H), 7.54 (d, J = 7.2 Hz, 1H), 7.82-7.85 (m,
1H); 8.23 (d, J = 7.6 Hz, 1H), 8.44 (s, 1H), 9.87 (s, 1H) ppm; 13C NMR (100 MHz, DMSO-
2
2
d6): δ 26.22, 55.57, 104.61 (d, JCF = 25 Hz), 106.82 (d, JCF = 21 Hz), 112.14, 114.28, 119.88,
124.47, 124.72, 126.14, 128.23, 128.81, 138.43, 139.62, 155.876, 157.79, 159.04, 161.03 (d, 1JCF
=
21

ACCEPTED MANUSCRIPT
242 Hz) ppm; HRMS (ESI) m/z calculated for C18H15FN3OS ([M+H]+) 340.09199, found
340.09127. Anal. C18H14FN3OS (C, H, N).
4.1.1.10.
8-Methoxy-2-(3-trifluoromethylanilino)-5H-benzothiopyrane[4,3-d]pyrimidine
(10).
1
Yield: 35%; m. p. 149-150 °C; H NMR (400 MHz, DMSO-d6): δ 3.84 (s, 3H), 4.04 (s, 2H),
6.94 (d, J = 8.4 Hz, 1H), 7.00 (s, 1H), 7.28 (d, J = 7.2 Hz, 1H), 7.53 (t, J = 7.6 Hz, 1H),
7.97 (d, J = 7.6 Hz, 1H), 8.23 (d, J = 8.4 Hz, 1H), 8.44 (s, 1H), 8.47 (s, 1H), 10.01 (s, 1H)
3
ppm; 13C NMR (100 MHz, DMSO-d6): δ 26.12, 56.26, 112.28, 112.88, 114.31 (q, JCF = 4.0 Hz),
114.75, 117.16 (q, 3JCF = 4.0 Hz), 121.93, 124.41 (q, 1JCF = 271 Hz), 124.63, 128.57, 129.31 (q, 2JCF
= 31 Hz), 129.62, 138.66, 141.51, 156.13, 157.79, 158.94, 161.45 ppm; HRMS (ESI) m/z
calculated for C19H15F3N3OS ([M+H]+) 390.08879, found 390.08798. Anal. C19H14F3N3OS (C, H,
N).
4.1.1.11. 8-Chloro-2-(3-methoxyanilino)-5H-benzothiopyrane[4,3-d]pyrimidine (11). Yield: 35%;
1
m. p. 166-167 °C; H NMR (400 MHz, DMSO-d6): δ 3.77 (s, 3H), 4.07 (s, 2H), 6.54 (dd, Jmin
=
2.2 Hz, Jmax = 7.8 Hz, 1H), 7.20 (t, J = 8.2 Hz, 1H), 7.32 (dd, Jmin = 1.2 Hz, Jmax = 8.0 Hz, 1H),
7.46 (dd, Jmin = 2.2 Hz, Jmax = 8.6 Hz, 1H), 7.57-7.59 (m, 2H), 8.27 (d, J = 8.4 Hz 1H), 8.49 (s,
13
1H), 9.77 (s, 1H) ppm; C NMR (100 MHz, DMSO-d6): δ 25.90, 54.89, 104.27, 106.95, 111.11,
114.83, 126.28, 127.21, 128.45, 129.25, 130.92, 135.76, 138.92, 141.69, 156.68, 156.87, 159.30,
159.53 ppm; HRMS (ESI) m/z calculated for C18H15ClN3OS ([M+H]+) 356.06244, found
356.06201. Anal. C18H14ClN3OS (C, H, N).
4.1.1.12. 8-Chloro-2-(3-chloroanilino)-5H-benzothiopyrane[4,3-d]pyrimidine (12) Yield: 35%; m.
p. 220-1221 °C; 1H NMR (400 MHz, DMSO-d6): δ 4.06 (s, 2H), 6.99 (dd, Jmin = 1.8 Hz, Jmax = 7.8
Hz, 1H), 7.32 (t, J = 8.0 Hz, 1H), 7.45 (dd, Jmin = 2.4 Hz, Jmax = 8.4 Hz, 1H), 7.57 (d, J = 2.0 Hz,
1H), 7.71 (dd, Jmin = 2.4 Hz, Jmax = 8.4 Hz, 1H), 7.99 (t, J = 2.0 Hz, 1H), 8.24 (d, J = 8.4 Hz, 1H),
13
8.52 (s, 1H), 9.95 (s, 1H) ppm; C NMR (100 MHz, DMSO-d6): δ 25.88, 115.44, 116.95, 117.83,
120.80, 126.32, 127.24, 128.46, 130.16, 130.75, 132.95, 135.88, 139.00, 142.03, 156.66, 157.04,
22

ACCEPTED MANUSCRIPT
159.01 ppm; HRMS (ESI) m/z calculated for C17H12Cl2N3S ([M+H]+) 360.01290, found 360.01217.
Anal. C17H11Cl2N3S (C, H, N).
4.1.1.13. 8-Chloro-2-(3-bromoanilino)-5H-benzothiopyrane[4,3-d]pyrimidine (13). Yield: 45%; m.
1
p. 212-213 °C; H NMR (400 MHz, DMSO-d6): δ 4.07 (s, 2H), 7.12 (dd, Jmin = 1.8 Hz, Jmax = 7.4
Hz, 1H), 7.26 (t, J = 8.0 Hz, 1H), 7.44 (dd, Jmin = 2.4 Hz, Jmax = 8.4 Hz, 1H), 7.57 (d, J = 2.0 Hz,
1H), 7.75 (dd, Jmin = 1.2 Hz, Jmax = 8.4 Hz, 1H), 8.15 (t, J = 1.8 Hz, 1H), 8.24 (d, J = 8.4 Hz, 1H),
13
8.52 (s, 1H), 9.93 (s, 1H) ppm; C NMR (100 MHz, DMSO-d6): δ 25.87, 115.14, 116,62, 117.32,
120.73, 123.68, 124.81, 126.28, 127.13, 128.36, 130.45, 132.64, 135.73, 138.86, 139.01, 142.17,
158.97 ppm; HRMS (ESI) m/z calculated for C17H12BrClN3S ([M+H]+) 403.96238, 405,96034,
found 403.96274, 405.95975. Anal. C17H11BrClN3S (C, H, N).
4.1.1.14. 8-Chloro-2-(3-fluoroanilino)-5H-benzothiopyrane[4,3-d]pyrimidine (14). Yield: 30%; m.
p. 190-193 °C; 1H NMR (400 MHz, DMSO-d6): δ 4.08 (s, 2H), 6.77 (dt, Jmin = 2.0 Hz, Jmax = 8.4
Hz, 1H), 7.30-7.36 (m, 1H), 7.48 (dd, Jmin = 2.0 Hz, Jmax = 8.4 Hz, 1H), 7.53-7.56 (m, 1H),
7.59 (d, J = 2.0 Hz, 1H), 7.80 (td, Jmin = 2.4 Hz, Jmax = 9.4 Hz, 1H), 8.26 (d, J = 8.4 Hz, 1H),
8.53 (s, 1H), 10.00 (s, 1H) ppm; 13C NMR (100 MHz, DMSO-d6): δ 25.89, 105.18 (d, 2JCF = 26
2
Hz), 106.81 (d, JCF = 24 Hz), 114.37, 115.43, 126,39, 127.26, 128.47, 130.79, 134.60, 135.87,
1
139.00, 142,51, 156.66, 157.07, 159.13, 162.14 (d, JCF = 239 Hz) ppm; HRMS (ESI) m/z
calculated for C17H12ClFN3S ([M+H]+) 344.04245, found 344.04197. Anal. C17H11ClFN3S (C, H,
N).
4.1.1.15. 8-Chloro-2-(3-trifluoromethylanilino)-5H-benzothiopyrane[4,3-d]pyrimidine (15). Yield:
1
35%; m. p. 165-170 °C; H NMR (400 MHz, DMSO-d6): δ 4.08 (s, 2H), 7.29 (d, J = 7.6 Hz, 1H),
7.42 (dd, Jmin = 2.1 Hz, Jmax = 8.6 Hz, 1H), 7.53 (t, J = 7.6 Hz, 1H), 7.57 (d, J = 2.0 Hz, 1H), 7.98
13
(d, J = 8.0 Hz, 1H), 8.24 (d, J = 8.4 Hz, 1H), 8.38 (s, 1H), 8.54 (s, 1H), 10.11 (s, 1H) ppm; C
NMR (100 MHz, DMSO-d6): δ 25.85, 114.44 (q, 3JCF = 4.0 Hz), 115.52, 117.36 (q, 3JCF = 4.0 Hz),
1
2
122.04, 124.35 (q, JCF = 271 Hz), 126.20, 127.23, 128.36, 129.32 (q, JCF = 30 Hz), 129.64,
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130.72, 135.91, 139.04, 141.28, 156.81, 156.95, 158.99 ppm; HRMS (ESI) m/z calculated for
C18H12ClF3N3S ([M+H]+) 394.03926, found 394.03883. Anal. C18H11ClF3N3S (C, H, N).
4.1.1.16. 2-(3,4-Dimethoxyanilino)-5H-benzothiopyrane[4,3-d]pyrimidine (16). Yield: 40%; m. p.
148-149 °C; 1H NMR (400 MHz, DMSO-d6): δ 3.72 (s, 3H), 3.78 (s, 3H), 4.01 (s, 2H), 6.91 (d, J =
8.8 Hz, 1H), 7.26 (dd, Jmin = 2.2 Hz, Jmax = 8.6 Hz, 1H), 7.36-7.43 (m, 3H), 7.63 (d, J = 1.2 Hz, 1H),
13
8.31 (d, J = 7.6 Hz, 1H), 8.44 (s, 1H), 9.51 (s, 1H) ppm; C NMR (100 MHz, DMSOd6): δ 25.94,
55.29, 55.87, 104.27, 110.54, 112.37, 114.59, 126.12, 126.95, 127.93, 131.14, 132.22, 134.34,
136.56, 143.61, 148.53, 156.54, 157.55, 159.42 ppm; HRMS (ESI) m/z calculated for C19H18N3O2S
([M+H]+) 352.11197, found 352.11067. Anal. C19H17N3O2S (C, H, N).
4.1.1.17. 2-(3,4,5-Trimethoxyanilino)-5H-benzothiopyrane[4,3-d]pyrimidine (17). Yield: 30%; m.
1
p. 176-178 °C; H NMR (400 MHz, DMSOd6): δ 3.63 (s, 3H), 3.79 (s, 6H), 4.03 (s, 2H), 7.30 (s,
2H), 7.35-7.38 (m, 1H), 7.43-7.44 (m, 2H), 8.34 (d, J = 7.2 Hz, 1H), 8.47 (s, 1H), 9.58 (s, 1H) ppm;
13C NMR (100 MHz, DMSO-d6): δ 25.92, 55.61 (2C), 60.13, 96.30, 114.99 (2C), 126.07, 126.91,
127.96, 131.24, 131.99, 132.19, 136.63, 136.78, 152.65 (2C), 156.64, 157.50, 159.26 ppm; HRMS
(ESI) m/z calculated for C20H20N3O3S ([M+H]+) 382.12254, found 382.12198. Anal. C20H19N3O3S
(C, H, N).
4.1.1.18. 8-Methoxy-2-(3,4-dimethoxyanilino)-5H-benzothiopyrane[4,3-d]pyrimidine (18). Yield:
35%; m. p. 204-205 °C; 1H NMR (400 MHz, DMSO-d6): δ 3.69 (s, 3H), 3.76 (s, 3H), 3.78 (s, 3H),
3.97 (s, 2H), 6.77 (dd, Jmin = 2.2 Hz, Jmax = 8.6 Hz, 1H), 6.85 (dd, Jmin = 2.4 Hz, Jmax = 8.8 Hz, 1H),
6.91-6.93 (m, 2H), 7.02 (d, J = 2.4 Hz, 1H), 7.79 (d, J = 8.8 Hz, 1H), 8.23 (s, 1H), 9.52 (s, 1H)
ppm; 13C NMR (100 MHz, DMSO-d6): δ 25.92, 55.52, 55.57, 60.01, 111.73, 112.00, 114.87,
119.56, 126.04, 127.84, 132.12, 133.00, 137.99, 138.03, 146.54, 148.68, 151.64, 156.61, 157.48,
159.08 ppm; HRMS (ESI) m/z calculated for C20H20N3O3S ([M+H]+) 382.12254, found 382.12178.
Anal. C20H19N3O3S (C, H, N).
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4.1.1.19. 8-Methoxy-2-(3,4,5-trimethoxyanilino)-5H-benzothiopyrane[4,3-d]pyrimidine (19). Yield:
30%; m. p. 166-168 °C; 1H NMR (400 MHz, DMSO-d6): δ 3.62 (s, 3H), 3.74 (s, 6H), 3.79 (s, 3H),
4.01 (s, 2H), 6.94 (dd, Jmin = 2.4 Hz, Jmax = 8.8 Hz, 1H), 6.99 (d, J = 2.4 Hz, 1H), 7.28 (s, 2H), 8.27
(d, J = 8.4 Hz, 1H), 8.40 (s, 1H), 9.49 (s, 1H) ppm; 13C NMR (100 MHz, DMSO-d6): δ 25.93,
55.28, 55.54 (2C), 60.03, 96.28, 114.92 (2C), 126.00, 126.82, 127.93, 131.14, 132.02, 132.14,
136.56, 136.71, 151.97 (2C), 156.58, 157.45, 159.22 ppm; HRMS (ESI) m/z calculated for
C21H22N3O4S ([M+H]+)412.13310, found 412.13274. Anal. C21H21N3O4S (C, H, N).
4.1.1.20. 8-Chloro-2-(3,4-dimethoxyanilino)-5H-benzothiopyrane[4,3-d]pyrimidine (20). Yield:
35%; m. p. 180-183 °C; 1H NMR (400 MHz, DMSO-d6): δ 3.75 (s, 3H), 3.78 (s, 3H), 4.04 (s, 2H),
6.90 (d, J = 8.8 Hz, 1H), 7.23 (dd, Jmin = 2.2 Hz, Jmax = 9.0 Hz, 1H), 7.44 (dd, Jmin = 1.8 Hz, Jmax
=
8.6 Hz, 1H), 7.55-7.60 (m, 2H), 8.27 (d, J = 8.4 Hz, 1H), 8.44 (s, 1H), 9.53 (s, 1H) ppm; 13C NMR
(100 MHz, DMSO-d6): δ 25.92, 55.32, 55.85, 104.33, 110.64, 112.35, 114.18, 122.31, 126.20,
127.16, 128.42, 131.02, 134.18, 135.65, 138.86, 143.69, 148.54, 156.71, 159.41 ppm; HRMS (ESI)
m/z calculated for C19H17ClN3O2S ([M+H]+) 386.07300, found 386.07244. Anal. C19H16ClN3O2S
(C, H, N).
4.1.1.21. 8-Chloro-2-(3,4,5-trimethoxyanilino)-5H-benzothiopyrane[4,3-d]pyrimidine (21). Yield:
1
30%; m. p. 162-165 °C; H NMR (400 MHz, DMSOd6): δ 3.63 (s, 3H), 3.79 (s, 6H), 4.06 (s, 2H),
7.26 (s, 2H), 7.45 (dd, Jmin = 1.8 Hz, Jmax = 8.2 Hz, 1H), 7.58 (d, J = 2.0 Hz, 1H), 8.3 (d, J = 8.4 Hz,
13
1H), 8.48 (s, 1H), 9.62 (s, 1H) ppm; C NMR (100 MHz, DMSOd6): δ 25.93, 55.26, 55.63 (2C),
96.31, 114.95 (2C), 126.03, 126.86, 127.91, 131.18, 132.01, 132.15, 136.58, 136.72, 152.63 (2C),
156.65, 157.47, 159.24 ppm; HRMS (ESI) m/z calculated for C20H19ClN3O3S ([M+H]+) 416.08356,
found 416.08287. Anal. C20H18ClN3O3S (C, H, N).
4.2. Biological evaluation
4.2.1.
Inhibition growth assay.
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HeLa (human cervix adenocarcinoma cells) and A-431 (skin carcinoma squamous cells) were
grown in Nutrient Mixture F-12 [HAM] (Sigma Chemical Co.) and Dulbecco's modified Eagle's
medium (Sigma Chemical Co.) respectively. MSTO-211H (human biphasic mesothelioma cells)
were grown in RPMI 1640 (Sigma Chemical Co.) supplemented with 2.4 g/L Hepes, 0.11 g/L Na-
pyruvate. 10% Heat-inactivated fetal calf serum (Invitrogen), 100 U/mL penicillin, 100 mg/mL
streptomycin and 0.25 mg/mL amphotericin B (Sigma Chemical Co.) were added to the media.
HUVECs (human umbilical vein endothelial cells) were purchased from Lonza Ltd. and maintained
as recommended. The cells were cultured at 37 °C in a moist atmosphere of 5% carbon dioxide in
air.
Cells (3 x 104) were seeded into each well of a 24-well cell culture plate. After incubation for 24 h,
various concentrations of the test agents were added to the complete medium and cells were
incubated for a further 72 h. A trypan blue assay was performed to determine cell viability.
Cytotoxicity data were expressed as GI50 values, i.e., the concentration of the test agent inducing
50% reduction in cell number compared with control cultures.
4.2.2.
KDR activity assay.
The KDR kinase activity was assessed by using the Z’-LYTE-Tyr1 Peptide assay kit (Invitrogen),
according to the procedures recommended by the manufacturer. Briefly, assays were performed in a
total of 60 μL in 96-well plates (Optiplate-96, black, Perkin Elmer) using fluorescence resonance
energy transfer technology. A Tyr1 substrate (coumarin-fluorescein double labeled peptide) at 2.0
ꢀM was incubated for 1 h at room temperature with 0.7 μg/mL KDR (Life Technologies), 10 μM
ATP, and test agents at room temperature in kinase buffer. Then, 15.0 ꢀL of the development
reagent were added to each well and samples were incubated for further 1 hour. At the end, 15 ꢀL
of the stop reagent were added and fluorescence signal was detected by using a Victor 1460
Multilabel Counter (Perkin Elmer). The fluorescence emission was read at 445 nm and 520 nm
(excitation wavelength = 400 nm) and the percentage of phosphorylation was determined as
indicated by the manufacture.
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4.2.3. In vitro kinases enzymatic inhibition.
The screening of compound 16 on a set of 6 human kinases (AurA/Aur2 kinase, CDC2/CDK1,
EGFR kinase, PDGFRb kinase, RAF-1 kinase, Src kinase) was performed by CEREP (France),
according to the company's standard operating procedures (Figure 1, Tables S1a-S1b).
4.3. Molecular Modeling
The latest version of the docking software AutoDock Vina (version 1.1.2) [28] in conjunction with
the graphical user interface AutoDockTools (ADT) [35] was used to perform the ensemble docking
calculations. AutoDock Vina docking performances and accuracy are assured by a Lamarckian
algorithm and an empirical binding free energy force field. The VEGFR-2 X-ray structures used for
the experiment had the following PDB codes: 1YWN, 2P2H, 2XIR, 3BE2, 3EWH, 3VHE, 3VNT,
3VO3, 3WZD, 3WZE, 4AG8, and 4ASE. The 3D structures were prepared using the Protein
Preparation Wizard of the Maestro suite (Schrödinger Release 2017-2) [36], which assigns bond
orders, adds hydrogen atoms, deletes water molecules and generates the appropriate protonation
states. Maestro was also employed to superimpose all the enzyme structures to the 1YWN structure.
The 2D Sketcher tool of Maestro was used to build compound 16. The ligand protonation and
tautomeric states, as well as its geometry, were optimized through LigPrep, part of the Maestro
suite. Afterwards, both the ligand and the receptors had to be converted to the pdbqt format. The
latter is very similar to a standard pdb but it includes ′Q′ (partial charges) and ′T′ atom types. Each
atom has one line, and special keywords are used if some are required to be flexible in the docking
calculations. Preparing the structures involves ensuring that its atoms are assigned the correct atom
types, adding Gasteiger charges if necessary, merging non-polar hydrogens, detecting aromatic
carbons, and setting up the ′torsion tree′ in the case of ligands. Therefore, the python scripts
prepare_receptor4.py and prepare_ligand4.py, part of ADT, were employed applying the standard
settings. After the preparation, the docking was performed using the default settings of Vina, setting
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the midpoint of the putative binding site as the center of the box. The length of the box was fixed at
23 Å for the X, Y and Z coordinates.
The electrostatic potentials of compounds 5 and 16 were calculated by means of the Jaguar suite
within the Schrödinger package (Schrödinger Release 2017-2) [37] and mapped onto their electron
density. The isovalue of 0.0002 electron/Bhor3 was chosen for the definition of the density surface,
while the electrostatic potential was computed using the 6-31G* basis set with a scale of −20.0 (red)
to 35.0 kcal/mol (blue).
AUTHOR INFORMATION
Corresponding Authors
Prof. Sabrina Taliani *e-mail: sabrina.taliani@unipi.it, Prof. Sandro Cosconati *e-mail:
sandro.cosconati@unicampania.it
Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval to
the final version of the manuscript.
Funding: This work was supported by the University of Pisa (PRA Project: PRA_2017_51).
Appendix A. Supplementary data.
Supplementary data related to this article for publication on line.
References
[1] T.A. Baudino, Targeted Cancer Therapy: The Next Generation of Cancer Treatment, Curr.
Drug. Discov. Technol. 12 (2015) 3-20.
[2] C. Brown, Targeted therapy: An elusive cancer target, Nature 537 (2016) S106-8.
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