Design, synthesis and biological evaluation of novel benzothiadiazine derivatives as potent PI3Kδ-selective inhibitors for treating B-cell-mediated malignancies

Article abstract of DOI:10.1016/j.ejmech.2019.03.005

A series of 24 benzothiadiazine derivatives with structural novelty were designed, synthesized and biologically evaluated as PI3Kδ-selective inhibitors. As a consequence of the structure-activity relationship (SAR) study, compounds 63 and 71 were identifi

Full text of DOI:10.1016/j.ejmech.2019.03.005

Accepted Manuscript
Design, synthesis and biological evaluation of novel benzothiadiazine derivatives as
potent PI3Kδ-selective inhibitors for treating B-cell-mediated malignancies
Xiaodong Ma, Jun Wei, Chang Wang, Dongyan Gu, Yongzhou Hu, Rong Sheng
PII:
S0223-5234(19)30216-8
DOI:
https://doi.org/10.1016/j.ejmech.2019.03.005
EJMECH 11175
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 11 January 2019
Revised Date: 27 February 2019
Accepted Date: 2 March 2019
Please cite this article as: X. Ma, J. Wei, C. Wang, D. Gu, Y. Hu, R. Sheng, Design, synthesis and
biological evaluation of novel benzothiadiazine derivatives as potent PI3Kδ-selective inhibitors for
treating B-cell-mediated malignancies, European Journal of Medicinal Chemistry (2019), doi: https://
doi.org/10.1016/j.ejmech.2019.03.005.
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Graphical abstract
Design, Synthesis and Biological Evaluation of Novel Benzothiadiazine
Derivatives as Potent PI3Kδ-selective Inhibitors for Treating B-cell-mediated
Malignancies
Xiaodong Ma a, b, c, Jun Wei a, Chang Wang a, Dongyan Gu a, Yongzhou Hu a, Rong Sheng a, *

ACCEPTED MANUSCRIPT
Design, Synthesis and Biological Evaluation of Novel Benzothiadiazine Derivatives as Potent
PI3Kδ-selective Inhibitors for Treating B-cell-mediated Malignancies
Xiaodong Ma a, b, c, Jun Wei a, Chang Wang a, Dongyan Gu a, Yongzhou Hu a, Rong Sheng a,
*
a.
ZJU-ENS Joint Laboratory of Medicinal Chemistry, Zhejiang Province Key Laboratory of Anti-Cancer Drug Research,
College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China
b. School of Pharmacy, Anhui University of Chinese Medicine, Hefei 230031, China
c. Department of Medicinal Chemistry, Anhui Academy of Chinese Medicine, Hefei 230031, China
Abstract: A series of 24 benzothiadiazine derivatives with structural novelty were designed,
synthesized and biologically evaluated as PI3Kδ-selective inhibitors. As a consequence of the
structure-activity relationship (SAR) study, compounds 63 and 71 were identified with single-digit
nanomolar IC50 values against PI3Kδ and submicromolar GI50 values against human malignant B-cell
line SU-DHL-6. Furthermore, chiral resolution of the key amine intermediate of these two compounds
was performed to achieve corresponding enantiomers. In subsequent biological evaluation, S-63 (IC50:
4.6 nM) and S-71 (IC50: below 0.32 nM) demonstrated comparable and superior PI3Kδ inhibitory
activity, respectively, to that of idelalisib. Additionally, both S-63 (GI50: 33.2 nM) and S-71 (GI50: 15.9
nM) exerted enhanced anti-proliferative activity against the SU-DHL-6 cell line than that of idelalisib.
Moreover, both S-63 and S-71 exhibited excellent PI3Kδ selectivity. In the further in vivo
pharmacokinetic (PK) study, S-63 displayed a good plasma exposure and an acceptable oral
bioavailability of 29.2%. By virtue of its biological performance, S-63 merits further development as a
potential therapeutic agent for battling B-cell-mediated malignancies.
Key words: Benzothiadiazine derivatives; PI3Kδ-selective inhibitors; SAR; Chiral resolution; PK
study
* Corresponding author: Rong Sheng
ZJU-ENS Joint Laboratory of Medicinal Chemistry, Zhejiang Province Key Laboratory of Anti-Cancer
Drug Research, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China
E-mail: shengr@zju.edu.cn
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1. Introduction
Aberrant phosphoinositide 3-kinase (PI3K) signaling is implicated in a myriad of pathological
conditions [1-5]. PI3K, the lipid kinase transforming phosphatidylinositol 4, 5-bisphosphate (PIP2) to
the second messenger phosphatidylinositol 3, 4, 5-trisphosphate (PIP3), comprises several classes,
among which class I PI3Ks have been widely accepted as targets for battling human malignancies [6-8].
Owing to the differentiation in the catalytic subunit and biological function, Class I PI3Ks can be
further divided into four highly homologous subtypes, PI3Kα, PI3Kβ, PI3Kγ and PI3Kδ [9].
Predominantly enriched in leukocytes, and essential for B-cell proliferation and function, PI3Kδ
provides a unique opportunity for therapeutic intervention in B-cell-mediated malignancies,
autoimmune disorders, and inflammatory diseases [10-13]. In comparison, PI3Kα and PI3Kβ are
ubiquitously expressed and vital for embryonic development, insulin action, as well as platelet
aggregation [9, 14-16]. Considering these characteristics, the application of pan-class I PI3K inhibitors
for treating leukocyte-mediated conditions may be compromised by limited tolerability due to the
concomitant suppression of other subtypes irrelevant to disease initiation and progression [17, 18].
Hence, the past decade has witnessed an increasing investment in PI3Kδ-selective inhibitors
specifically ablating PI3Kδ signaling without interfering with the biological function of the other three
class I PI3K isoforms [19-24].
Idelalisib (Zydelig) 1 is the first-in-class PI3Kδ-selective inhibitor approved for treating chronic
lymphocytic leukemia (CLL), follicular lymphoma (FL), and small lymphocytic lymphoma (SLL) in
2014 [25, 26]. Duvelisib 2, a δ-weighted PI3Kδ/γ dual inhibitor [27], has also been approved recently
for curing hematopoietic malignancies. Several other candidates, as exemplified by acalisib 3,
AMG-319 4, tenalisib 5 and umbralisib 6 (https://www.pharmacodia.com/cn), have progressed to
clinical trials as remedies for B or T cell-related malignancies (Figure 1). With respect to the binding
mode, these advanced PI3Kδ inhibitors may be defined as ATP-competitive allosteric inhibitors and
are capable of inducing a hydrophobic selectivity pocket between residues Trp760 and Met752, which
is not present in the apo structure of the enzyme [28, 29].
Despite differentiated chemical structures, these allosteric inhibitors, also termed as the
propeller-shaped inhibitors, harbor approximately the same pharmacophore, featuring a bicyclic
heteroaromatic core, a six-membered aryl group directly attached to it, as well as a hinge binder (HB)
tethered to it via a short spacer [30] (Figure 1). Among these, the six-membered aryl moiety and HB
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assume a perpendicular conformation to the heteroaromatic core that is embedded in the selectivity
pocket. The spacer between the bicyclic heteroaromatic core and HB, commonly containing an amino
linker and a chiral carbon, is vital for inducing the specificity pocket. In addition to the PI3Kδ
inhibitors characterized by this pharmacophore, recent medicinal chemistry efforts have also
culminated in propeller-shaped inhibitors with nonfused aromatic core, and nonpropeller-shaped
inhibitors [31].
Figure 1. The approved or clinically investigated PI3Kδ-selective inhibitors.
Built upon our insight into the typical propeller-shaped PI3Kδ-selective inhibitors, especially the
successful exploration of 2, 4 and 5 via bioisosterism, we envisioned that replacement of quinazolinone
of idelalisib with the benzothiadiazine template may be a feasible approach to obtaining novel
PI3Kδ-selective inhibitors (Figure 2). To explore the structure-activity relationships (SARs) of these
benzothiadiazines, different R1, R2 and R3 substituents were investigated to obtain optimal moieties.
Also, for some compounds, the aminopurine was replaced by its ring-opening surrogate 4,
6-diamino-5-carbonitrile pyrimidine, or reversed with its N9 tethered to the chiral carbon. Through
careful modification on N-2 phenyl moiety, C-8 replacement, C-3 short spacer, and hinge binder, both
compounds 63 and 71 were identified with attractive PI3Kδ inhibitory activity and anti-proliferative
efficacy. In addition, chiral resolution of the key amine intermediate towards the preparation of
representative compounds (63 and 71) was performed to pave the way for revealing the impact of
absolute configuration on PI3Kδ inhibitory activity and specificity. Based on the in vitro biological
evaluation, S-63, the most promising benzothiadiazine derivative throughout the series, was further
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investigated for in vivo PK profiles. We herein communicate our recent efforts leading to a novel
structural series of benzothiadiazine derivatives as PI3Kδ-selective inhibitors.
Figure 2. The design rationale of target benzothiadiazine derivatives.
2. Results and Discussion
2.1. Chemistry
The synthetic route to target benzothiadiazine derivatives is displayed in Scheme 1. 1,
2-difluoro-3-nitrobenzene 7 or 1, 2-dichloro-3-nitrobenzene 8 was converted into corresponding
benzenesulfonyl chloride 9 or 10, respectively, according to a reported procedure [32]. The resulting
products or 2-nitrobenzenesulfonyl chloride 11 were subsequently condensed with various aromatic
amines to afford sulfonamides 12−19. The nitro moiety of the sulfonamides was then reduced by
NaBH4, and the produced amines were converted to C-3 alkyl substituted benzothiadiazines 20−31
after condensation with orthoester and intramolecular cyclization [33]. Next, α-bromination of the
derived products yielded 32−43 [34, 35], which were subjected to a two-step Gabriel synthesis to
generate the key amine intermediates 44−54. Ultimately, 44−54 reacted with 6-chloro-9H-purine,
6-chloro-2-fluoro-9H-purine, 2,6-dichloro-9H-purine or 4-amino-6-chloropyrimidine-5-carbonitrile to
produce 55−72 as the target compounds [18, 21, 36]. Additionally, the treatment of bromides 32−34, 37,
39 and 40 with adenine in the presence of K2CO3 gernerated target compounds 73−78.
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Scheme 1. Reagents and conditions: (a) (1) benzyl mercaptan, K2CO3, DMF, rt; (2) Cl2, DCM/H2O,
0 °C; (b) corresponding aromatic amine, pyridine, DCM, 0 °C to rt; (c) (1) NaBH4, NiCl2 (hexahydrate),
MeOH/DCM, 0 °C to rt; (2) corresponding orthoester, 130 °C, N2; (3) Ph2O, 250 °C, N2 (As for 30 and
31, intramolecular cyclization occurred during the treatment with orthoester, and this step was omitted);
(d) Br2, AcONa, AcOH, 40 °C; (e) (1) phthalimide, K2CO3, DMF, 40 °C; (2) hydrazine hydrate (85%),
EtOH, 80 °C, N2; (f) 6-chloro-9H-purine, 6-chloro-2-fluoro-9H-purine, 2,6-dichloro-9H-purine or
4-amino-6-chloropyrimidine-5-carbonitrile, DIPEA, t-BuOH, 80 °C, N2; (g) adenine, K2CO3, DMF, rt.
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2.2. SAR Investigation
According to the design rationale summarized in Figure 2, 24 benzothiadiazines were prepared and
biologically evaluated for the PI3Kδ inhibitory activity. The experimental data of compounds bearing
aminopurine as HB and those bearing 4, 6-diamino-5-carbonitrile pyrimidine or reversed aminopurine
as HB are displayed in Table 1 and Table 2, respectively. Among them, 9 compounds, including
63−65, 68, 69, 71−73 and 78, exhibited potent PI3Kδ inhibitory activity with IC50 values below 100
nM. In particular, the purine derivative 63 and 4, 6-diamino-5-carbonitrile pyrimidine derivative 71,
both with C-8 chloro and C-3 ethyl substitution, exerted remarkable PI3Kδ inhibitory activity with
respective IC50 values of 9.8 and 3.2 nM.
Table 1. PI3Kδ inhibitory activity of target compounds bearing aminopurine as HB.
Cpd.
55
R1
R2
H
H
H
H
H
H
H
F
R3
Me
Me
Me
Et
R4
H
H
H
H
H
H
H
H
H
H
F
PI3Kδ (IC50, nM)
Ph
344
1999
>3000
>3000
>3000
>3000
921
4-F-Ph
56
3-CF3-Ph
57
3-CF3-Ph
58
4-OCF3-Ph
Et
59
3, 4-di-OCH3-Ph
Et
60
3-Py
Ph
Me
Et
61
>3000
9.8
62
Ph
Cl
Cl
H
H
F
Me
Et
63
Ph
81
64
Ph
Me
Me
Et
92
65
Ph
Cl
F
1202
383
66
Ph
67
14
PI-103
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Table 2. PI3Kδ inhibitory activity of target compounds bearing 4, 6-diamino-5-carbonitrile pyrimidine
or reversed aminopurine as HB.
Cpd.
68
R1
R2
R3
W
PI3Kδ (IC50, nM)
Ph
H
Me
15
Ph
Ph
F
F
Me
Et
35
141
3.2
69
70
71
72
73
74
75
76
77
Ph
Cl
Cl
H
H
H
H
H
F
Me
Et
Ph
12
Ph
Me
Me
Et
68
4-F-Ph
4-F-Ph
4-OCF3-Ph
3-Py
Ph
628
>3000
>3000
623
Et
Me
Me
54
14
78
PI-103
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From the biological data presented in Table 1 and 2, some valuable SARs can be deduced. Firstly,
the N-2 phenyl moiety was optimum and substitution at the phenyl was detrimental to PI3Kδ inhibitory
activity. In addition, replacing the N-2 phenyl moiety with pyridinyl weakened the enzymatic activity
by approximately 3-fold (61 versus 55). When comparing 58 (IC50 > 3000 nM) to 59 (IC50 > 3000 nM)
or 60 (IC50 > 3000 nM), it became evident that neither the 4-OCF3 nor 3, 4-di-OCH3 substitution
boosted PI3Kδ inhibitory activity. Secondly, the introduction of a chloro-substituent at the C-8 position
significantly improved PI3Kδ inhibitory activity. As for compounds with aminopurine as HB, C-8
chlorinated compound 63 (IC50 = 9.8 nM) was 35-fold more potent than its C-8 unsubstituted
counterpart 55, and C-8 chlorinated compound 64 (IC50 = 81 nM) was over 35-fold more active than
C-8 fluorinated counterpart 62 (IC50 > 3000 nM). Similar results can also be observed for
benzothiadiazines with 4, 6-diamino-5-carbonitrile pyrimidine as HB, e.g. 71 versus 68, 71 versus 69,
and 72 versus 70. Thirdly, HB had an important impact on PI3Kδ inhibitory activity. Replacing
aminopurine with 4, 6-diamino-5-carbonitrile pyrimidine can dramatically improve PI3Kδ inhibitory
activity, as illustrated by 68 versus 55, 70 versus 62, 71 versus 63, and 72 versus 64. The reversion of
the aminopurine also enhanced PI3Kδ inhibitory activity, as demonstrated by 73 versus 55, 74 versus
56, and 77 versus 61. Finally, compounds with a C-3 ethyl substituent were superior to their
counterparts with a C-3 propyl substituent in PI3Kδ inhibitory activity, as shown by 63 versus 64, 69
versus 70, and 71 versus 72.
Table 3. The anti-proliferative activity of selected compounds against SU-DHL-6 cell line.
Cpd.
63
SU-DHL-6 (GI50, nM)
Cpd.
71
SU-DHL-6 (GI50, nM)
770
2193
4689
3501
1217
10511
187
1675
23147
7203
64
72
65
73
68
78
69
7.9
Paclitaxel
70
Compounds with PI3Kδ IC50 values below 200 nM were evaluated for their anti-proliferative activity
against the human malignant B-cell line SU-DHL-6. In general, over half of them exhibited favorable
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anti-proliferative efficacy with GI50 values ranging from low micromolar to submicromolar level, and
the cellular activity was consistent with the enzymatic activity (Table 3). Compounds 63 and 71, the
two most potent PI3Kδ inhibitors throughout this series, also exerted remarkable anti-proliferative
efficacy with respective GI50 values of 770 and 187 nM against the SU-DHL-6 cell line.
2.3. Chiral Resolution
Figure 3. The chiral resolution of the key amine intermediate 53 and the result for X-ray single crystal
diffraction of S-63.
Upon SAR investigation stated above, two benzothiadiazine derivatives 63 and 71 were identified
with promising in vitro potency, both characterized by single-digit nanomolar PI3Kδ inhibitory activity
and submicromolar anti-proliferative efficacy. Therefore, we subsequently undertook the chiral
resolution of 53, the key amine intermediate towards the preparation of compounds 63 and 71, which
would pave the way to reveal how the absolute configuration affected PI3Kδ inhibitory activity and
subtype specificity (Figure 3). Through our investigation on different chiral resolving reagents, S or
R-mandelic acid was identified to be optimum. Both the R-mandelic acid salt of 53-1 and S-mandelic
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acid salt of 53-2 were attained in high de value (over 95%) after recrystallization from ethyl acetate
(EA). After basifying the R-mandelic acid salt of 53-1, the free amine was converted into the
corresponding purine derivative S-63, the absolute configuration of which was determined via X-ray
single crystal diffraction. Accordingly, the absolute configurations of 53-1 and 53-2 were determined as
S and R, respectively. On the basis of this, we prepared R-63, S-71 and R-71 for further biological
evaluation.
2.4. In Vitro Activity and Subtype Selectivity of Optically Pure Compounds
Table 4. In vitro activity of the optically pure compounds.
PI3Kδ
SU-DHL-6
Cpd.
R5
(IC50, nM) a
(GI50, nM) a
4.6
62
33.2
190
S-63
R-63
S-71
＜0.32
15.9
7.0
1.8
70.3
124
R-71
Idelalisib
a. The data were obtained from separated experiment from corresponding one performed for the
racemates.
The successful resolution of the key amine intermediate 53 prompted us to investigate the PI3Kδ
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inhibitory potency and subtype specificity of the optically pure compounds, including S-63, R-63, S-71
and R-71 (Table 4). Consequently, S-63 and R-71, with respective IC50 values of 4.6 and 7.0 nM,
exhibited comparable PI3Kδ inhibitory activity to that of idelalisib. In particular, S-71 demonstrated
potent PI3Kδ inhibitory activity with IC50 value below 0.32 nM, which was superior to that of idelalisib.
Importantly, the impact of absolute configuration on enzymatic activity was clearly observed according
to the obtained biological data, and the S-enantiomers (S-63 and S-71) displayed dramatically
improved enzymatic potency compared to that of corresponding R-enantiomers (R-63 and R-71). In
addition to the favorable enzymatic activity, both S-63 and S-71, with respective GI50 values of 33.2
and 15.9 nM, exhibited enhanced anti-proliferative activity than that of idelalisib.
The absolute configuration also had a considerable influence on subtype specificity, as illustrated by
the superior PI3Kδ specificity of S-63 and S-71 to that of the corresponding enantiomers R-63 and
R-71 over the other class I PI3K subtypes (Table 5). Among them, S-63 exhibited comparable
δ-subtype selectivity to that of idelalisib, while S-71 displayed a more favorable specificity.
Table 5. Subtype specificity data of optically pure compounds.
S-63
(IC50, nM)
4.6
R-63
(IC50, nM)
62
S-71
(IC50, nM)
﹤0.32
R-71
(IC50, nM)
7.0
Idelalisib
(IC50, nM)
1.8
Kinases
PI3Kδ
PI3Kα
3637
>5000
996
>5000
2164
(791-fold)a
766
(>81-fold)a
3990
(>3113-fold)a
55
(>714-fold)a
169
(1202-fold)a
154
PI3Kβ
PI3Kγ
(167-fold)a
156
(64-fold)a
1905
(>172-fold)a
100
(24-fold)a
112
(85-fold)a
63
(34-fold)a
(31-fold)a
(>313-fold)a
(16-fold)a
(35-fold)a
a. Selectivity fold
2.5. Pharmacokinetics (PKs)
By virtue of the favorable PI3Kδ inhibitory activity and specificity, S-63 was selected for further PK
evaluation in Sprague−Dawley (SD) Rats with idelalisib used as the reference. Despite its better PI3Kδ
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inhibitory activity and specificity, S-71 exhibited a remarkably lower AUC value than that of S-63 in
the preliminary PK evaluation (Data not shown) and was not investigated extensively. As shown in
Table 6, S-63 displayed a favorable plasma exposure (AUC0-t = 2009±993 h ng/mL) and an acceptable
oral bioavailability (F% = 29.2±14.7). Moreover, the clearance, volume distribution, and elimination
half-life were comparable to those of idelalisib.
Table 6. The PK property of selected compound S-63.
Administration
Cmax
T1/2
(h)a
AUC0-t
Vss
CL
Cpd.
Route and
Dosage
F%a
(ng/mL)a
(h ng/mL)a
(L/kg)a
(mL/kg/min)a
iv (2 mg/kg)
po (10 mg/kg)
iv (2 mg/kg)
po (10 mg/kg)
_
0.518±0.121
1.20±0.256
1400±446
2009±993
1396 ± 298
3191±802
0.853±0.0854
25.8±9.67
29.2±14.7
45.8±11.5
S-63
643±413
_
_
_
24.5±4.83
_
0.519 ± 0.091
1.03±0.0541
0.884±0.044
_
Idelalisib
2317±1075
a. Data were shown as mean ± SD (n=3).
2.6. Molecular Docking
Figure 4. The molecular docking of S-63 (a) and S-71 (b) into the PI3Kδ active site.
The molecular docking analysis of S-63 and S-71 was carried out to elucidate their possible binding
modes with PI3Kδ active site (Figure 4). Consequently, the purine moiety of S-63 was engaged in
H-bond contacts with residues Glu826 and Val828 in the hinge region, while the benzothiadiazine
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template inserted deeply into the allosteric specificity pocket with Trp760 on one side and Met752 on
the other side (Figure 4a). Similarly, S-71 formed two H-bonds with residues in the hinge region,
while the benzothiadiazine core occupied the allosteric selectivity pocket (Figure 4b). These results of
molecular simulation may account for the PI3Kδ inhibitory potency and the subtype selectivity of S-63
and S-71.
3. Conclusions
In summary, we have designed and synthesized a series of benzothiadiazine-based PI3Kδ-selective
inhibitors via bioisosterism. Among these compounds, 63 and 71 displayed remarkable PI3Kδ
inhibitory activity (IC50s: below 10 nM), along with promising anti-proliferative potency against the
B-cell leukemia SU-DHL-6 cells (GI50s: below 1 µM). We next performed the chiral resolution of the
key amine intermediate towards their synthesis and prepared corresponding enantiomers. In subsequent
biological evaluation, S-63 and S-71 demonstrated comparable and superior PI3Kδ inhibitory activity,
respectively, to that of idelalisib. Additionally, both compounds exerted enhanced anti-proliferative
potency against the SU-DHL-6 cells compared to that of idelalisib. In terms of PI3Kδ selectivity, S-63
and S-71 were comparable and superior to idelalisib, respectively. Furthermore, the S-configuration
was more beneficial to both PI3Kδ inhibitory activity and specificity than R-configuration. In further in
vivo PK study, S-63 displayed a good plasma exposure and an acceptable oral bioavailability of 29.2%.
Owing to its favorable biological performance, S-63 merits further development as a potential
therapeutic agent for treating B-cell-mediated malignancies.
4. Experimental section
4.1. Chemistry
All the reagents and solvents were purchased from common commercial suppliers. If necessary,
purification was carried out prior to use. Melting points were uncorrected and determined on a Büchi
1
B-540 apparatus. H and 13CNMR spectra were recorded on a Bruker Avance III 500 (500 MHz) or
Bruker Avance 400 II (400MHz) spectrometer in the indicated solvent. ESI-MS were obtained by
Bruker Esquire-LC-00075 spectrometer, and HRMS were recorded on an Agilent 6224 TOF LC/MS
spectrometer. Flash column chromatography was performed using silica gel (200–300 mesh). HPLC
was performed using an Agilent 1200 system with UV detection at 254 nm, eluting with a binary
solvent system A and B [A: MeOH; B: H2O with 0.12% ammonium acetate (W/V)]. Analytical purity
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of all target compounds was shown in the supplementary material. Chiral HPLC was performed on a
Shimadzu LC-2010C system with UV detection at 254 nm, eluting with a binary solvent system C and
D (C: i-PrOH; D: hexane).
4.1.1. General procedure for intermediates 9 and 10
2-Fluoro-6-nitrobenzenesulfonyl chloride 9 and 2-chloro-6-nitrobenzenesulfonyl chloride 10 were
prepared according to
a reported procedure from 1, 2-difluoro-3-nitrobenzene 7 and 1,
2-dichloro-3-nitrobenzene 8, respectively, as light yellow solids.
4.1.2. General procedure for intermediates 12-19
To a solution of aniline (23 mL, 249 mmol, 1.1 eq) and pyridine (22 mL, 271 mmol, 1.2 eq) in
anhydrous DCM (200 mL) was added a solution of 2-nitrobenzenesulfonyl chloride (50.0 g, 226 mmol,
1.0 eq) in anhydrous DCM (200 mL) dropwise at 0 ºC. The resultant mixture was stirred at room
temperature for 0.5 h and then quenched with dilute hydrochloric acid (1 N). After filtration, the filtrate
was washed with brine, dried over anhydrous Na2SO4, and concentrated in vacuo. The residue was
ultimately subjected to recrystallization with EtOH to provide N-phenyl-2-nitrobenzenesulfonamide 12
as a light yellow solid. Yield 83%; ESI-MS: m/z = 279 [M+H]+. Intermediates 13−19 were prepared in
a procedure similar to that described for 12.
N-(4-fluorophenyl)-2-nitrobenzenesulfonamide (13) Light yellow solid; yield 76%; ESI-MS: m/z =
297 [M+H]+.
N-(3-(trifluoromethyl)phenyl)-2-nitrobenzenesulfonamide (14) Light yellow solid; yield 73%;
ESI-MS: m/z = 347 [M+H]+.
N-(4-(trifluoromethoxy)phenyl)-2-nitrobenzenesulfonamide (15) Light yellow solid; yield 82%;
ESI-MS: m/z = 363 [M+H]+.
N-(3,4-dimethoxyphenyl)-2-nitrobenzenesulfonamide (16) Light yellow solid; yield 89%; ESI-MS:
m/z = 339 [M+H]+.
N-(pyridin-3-yl)-2-nitrobenzenesulfonamide (17) The crude product was purified by flash column
chromatography using EA/petroleum ether (PE) (1:2) as the eluent. Light yellow solid; yield 70%;
ESI-MS: m/z = 280 [M+H]+.
N-phenyl-2-fluoro-6-nitrobenzenesulfonamide (18) The crude product was purified by flash column
1
chromatography using EA/PE (1:12‒2:9) as the eluent. Light yellow solid; yield 68%; H NMR (500
MHz, DMSO-d6): δ 11.14 (s, 1H), 7.93–7.85 (m, 1H), 7.78 (d, 8.0 Hz, 1H), 7.73 (t, 9.0 Hz, 1H), 7.29 (t,
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8.0 Hz, 2H), 7.16–7.02 (m, 3H); ESI-MS: m/z = 297 [M+H]+.
N-phenyl-2-chloro-6-nitro-benzenesulfonamide (19) The crude product was purified by flash column
1
chromatography using EA/PE (1:12‒2:9) as the eluent. Light yellow solid; Yield 63%; H NMR (500
MHz, DMSO-d6): δ 11.14 (s, 1H), 7.91–7.85 (m, 2H), 7.81 (t, 8.0 Hz, 1H), 7.29–7.24 (m, 2H), 7.10–
7.06 (m, 3H); ESI-MS: m/z = 313 [M+H]+.
4.1.3. General procedure for intermediates 20-31
.
To a solution of 12 (25.0 g, 89.9 mmol, 1.0 eq) and NiCl2 6H2O (42.8 g, 180 mmol, 2.0 eq) in DCM
(300 mL) and MeOH (100 mL) was added NaBH4 (13.7 g, 360 mmol, 4.0 eq) carefully at 0 ºC. The
resultant mixture was stirred at room temperature for 0.5 h and the solvent was removed under reduced
pressure. The residue was then extracted with EA before filtration, and the filtrate was washed
successively with H2O and brine. After being dried over anhydrous Na2SO4, the organic layer was
concentrated in vacuo to afford a clear and viscous oil that was slowly solidified. Yield 91%.
A solution of the newly afforded product (10.0 g, 40.3 mmol, 1.0 eq) in triethylorthopropionate (16
mL, 80.6 mmol, 2.0 eq) was stirred at 130 ºC under N2 atmosphere. After TLC indicated the total
conversion, removal of EtOH formed during the reaction and the excessive triethylorthopropionate
under reduced pressure gave a viscous oil. Ph2O (30 mL) was then added and the resultant solution
boiled at 250 ºC under N2 atmosphere for 4 h. Subsequently, it was totally cooled and kept at −10 ºC
overnight. PE was added to the mixture, which was then stirred at room temperature. Following
filtration, the precipitate was washed successively with PE and MeOH to afford
3-ethyl-2-phenyl-2H-benzo[e][1,2,4]thiadiazine 1,1-dioxide 20 as a pale solid. Yield 94% (for two
steps); 1H NMR (500 MHz, DMSO-d6): δ 7.95 (dd, 1.5 Hz, 8.0 Hz, 1H), 7.88–7.82 (m, 1H), 7.67–7.63
(m, 1H), 7.62–7.56 (m, 4H), 7.53–7.48 (m, 2H), 2.33 (q, 7.5 Hz, 2H), 1.11 (t, 7.5 Hz, 3H); ESI-MS:
m/z = 287 [M+H]+. Intermediates 21−31 were prepared in a procedure similar to that described for 20.
For 21−29, the reaction mixture of the intramolecular cyclization was directly subjected to flash
column chromatography utilizing EA/PE (1:7) as the eluent to afford the product. As for 30 and 31, the
intramolecular cyclization occurred when the reduced product of 19 was treated with corresponding
orthoester. After removal of EtOH and the excessive orthoester, the residue was purified by flash
column chromatography utilizing EA/PE (1:7) as the eluent to afford the product.
3-Ethyl-2-(4-fluorophenyl)-2H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (21) White solid; yield 84%
1
(for three steps); H NMR (500 MHz, DMSO-d6): δ 7.96 (d, 8.0 Hz, 1H), 7.88–7.82 (m, 1H), 7.65 (d,
15

ACCEPTED MANUSCRIPT
8.0 Hz, 1H), 7.63–7.56 (m, 3H), 7.43 (t, 8.5 Hz, 2H), 2.33 (q, 7.0 Hz, 2H), 1.11 (t, 7.0 Hz, 3H);
ESI-MS: m/z = 305 [M+H]+.
2-(4-Fluorophenyl)-3-propyl-2H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (22) White solid; yield 80%
(for three steps); ESI-MS: m/z = 319 [M+H]+.
3-Ethyl-2-(3-(trifluoromethyl)phenyl)-2H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (23) White solid;
yield 79% (for three steps); 1H NMR (500 MHz, DMSO-d6): δ 8.00–7.95 (m, 3H), 7.91–7.81 (m, 3H),
7.66 (d, 9.5 Hz, 1H), 7.60 (t, 9.5 Hz, 1H), 2.33 (q, 9.0 Hz, 2H), 1.11 (t, 9.0 Hz, 3H); ESI-MS: m/z =
355 [M+H]+.
3-Propyl-2-(3-(trifluoromethyl)phenyl)-2H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (24) White solid;
yield 73% (for three steps); 1H NMR (500 MHz, DMSO-d6): δ 8.00–7.92 (m, 3H), 7.89–7.81 (m, 3H),
7.66 (d, 9.5 Hz, 1H), 7.60 (t, 9.5 Hz, 1H), 2.30 (t, 9.0 Hz, 2H), 1.71–1.55 (m, 2H), 0.83 (t, 9.0 Hz, 3H);
ESI-MS: m/z = 369 [M+H]+.
3-Propyl-2-(4-(trifluoromethoxy)phenyl)-2H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (25) White solid;
1
yield 84% (for three steps); H NMR (500 MHz, DMSO-d6): δ 7.96 (d, 7.5 Hz, 1H), 7.86 (t, 8.0 Hz,
1H), 7.71–7.64 (m, 3H), 7.62–7.55 (m, 3H), 2.32 (t, 7.5 Hz, 2H), 1.67–1.59 (m, 2H), 0.85 (t, 7.5 Hz,
3H); ESI-MS: m/z = 385 [M+H]+.
2-(3,4-Dimethoxyphenyl)-3-propyl-2H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (26) White solid;
yield 78% (for three steps); 1H NMR (500 MHz, DMSO-d6): δ 7.94 (dd, 1.5 Hz, 7.5 Hz, 1H), 7.86–7.79
(m, 1H), 7.62 (d, 8.0 Hz, 1H), 7.59–7.56 (m, 1H), 7.12 (d, 8.5 Hz, 1H), 7.06 (dd, 2.5 Hz, 8.5 Hz, 1H),
7.01 (d, 2.5 Hz, 1H), 3.84 (s, 3H), 3.76 (s, 3H), 2.34 (t, 7.5 Hz, 2H), 1.70–1.61 (m, 2H), 0.86 (t, 7.5 Hz,
3H); ESI-MS: m/z = 361 [M+H]+.
3-Ethyl-2-(pyridin-3-yl)-2H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (27) White solid; yield 62% (for
1
three steps); H NMR (500 MHz, DMSO-d6): δ 8.78 (dd, 1.5 Hz, 5.0 Hz, 1H), 8.72 (d, 2.5 Hz, 1H),
8.07–8.03 (m, 1H), 7.98 (dd, 1.0 Hz, 8.0 Hz, 1H), 7.89–7.85 (m, 1H), 7.69–7.60 (m, 3H), 2.34 (q, 7.5
Hz, 2H), 1.12 (t, 7.5 Hz, 3H); ESI-MS: m/z = 288 [M+H]+.
3-Ethyl-8-fluoro-2-phenyl-2H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (28) White solid; yield 78%
1
(for two steps); H NMR (500 MHz, DMSO-d6): δ 7.90–7.83 (m, 1H), 7.63–7.58 (m, 3H), 7.57–7.52
(m, 2H), 7.51–7.43 (m, 2H), 2.30 (q, 7.5 Hz, 2H), 1.10 (t, 7.5 Hz, 3H); ESI-MS: m/z = 305 [M+H]+.
8-Fluoro-2-phenyl-3-propyl-2H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (29) White solid; yield 81%
1
(for two steps); H NMR (500 MHz, DMSO-d6): δ 7.89–7.81 (m, 1H), 7.63–7.58 (m, 3H), 7.55–7.51
16

ACCEPTED MANUSCRIPT
(m, 2H), 7.50–7.44 (m, 2H), 2.28 (t, 7.0 Hz, 2H), 1.65–1.57 (m, 2H), 0.82 (t, 7.5 Hz, 3H); ESI-MS: m/z
= 319 [M+H]+.
8-Chloro-3-ethyl-2-phenyl-2H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (30) White solid; yield 78%
(for two steps); 1H NMR (500 MHz, DMSO-d6): δ 7.84–7.76 (m, 1H), 7.70–7.50 (m, 7H), 2.28 (q, 9.0
Hz, 2H), 1.09 (t, 9.0 Hz, 3H); ESI-MS: m/z = 321 [M+H]+.
8-Chloro-2-phenyl-3-propyl-2H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (31) White solid; yield 80%
(for two steps); 1H NMR (500 MHz, DMSO-d6): δ 7.80 (t, 8.5 Hz, 1H), 7.66 (dd, 1.0 Hz, 8.0 Hz, 1H),
7.63–7.57 (m, 4H), 7.55–7.50 (m, 2H), 2.67 (t, 7.5 Hz, 2H), 1.65–1.57 (m, 2H), 0.82 (t, 7.5 Hz, 3H);
ESI-MS: m/z = 335 [M+H]+.
4.1.4. General procedure for intermediates 32-43
To a solution of 20 (6.00 g, 20.9 mmol, 1.0 eq) and AcONa (2.41 g, 29.3 mmol, 1.4 eq) in AcOH (40
mL) were carefully added Br2 (1.50 mL, 29.3 mmol, 1.4 eq), and the resultant mixture was stirred at 40
ºC till TLC demonstrated total conversion of 20. After the reaction mixture was cooled, water was
added to quench the reaction and the deposit collected by filtration. The solution of the filter cake in
DCM was then washed with saturated Na2S2O3 solution, brine, dried over Na2SO4, and concentrated in
vacuo.
The
residue
was
subjected
to
recrystallization
using
EA
to
afford
3-(1-bromoethyl)-2-phenyl-2H-benzo[e][1,2,4]thiadiazine 1,1-dioxide 32 as a white solid. Yield 91%;
1H NMR (500 MHz, DMSO-d6): δ 7.99 (dd, 1.5 Hz, 8.0 Hz, 1H), 7.94–7.88 (m, 1H), 7.75 (dd, 1.0 Hz,
8.0 Hz, 1H), 7.68 (dt, 1.0 Hz, 8.0 Hz, 1H), 7.65–7.42 (m, 5H), 4.55 (q, 6.5 Hz, 1H), 1.93 (t, 6.5 Hz, 3H);
ESI-MS: m/z = 365 [M+H]+. Intermediates 33−43 were prepared in a procedure similar to that
described for 32. The crude product was purified by flash column chromatography utilizing EA/PE
(1:10) as the eluent.
3-(1-Bromoethyl)-2-(4-fluorophenyl)-2H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (33) White solid;
yield 83%; 1H NMR (500 MHz, DMSO-d6): δ 7.99 (dd, 1.0 Hz, 8.0 Hz, 1H), 7.95–7.87 (m, 1H), 7.75
(d, 8.0 Hz, 1H), 7.72–7.54 (m, 3H), 7.45 (t, 8.0 Hz, 2H), 4.59 (q, 6.5 Hz, 1H), 1.93 (t, 6.5 Hz, 3H);
ESI-MS: m/z = 383 [M+H]+.
3-(1-Bromopropyl)-2-(4-fluorophenyl)-2H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (34) White solid;
yield 85%; 1H NMR (500 MHz, DMSO-d6): δ 8.00 (dd, 1.0 Hz, 7.5 Hz, 1H), 7.95–7.87 (m, 1H), 7.73
(d, 7.5 Hz, 1H), 7.70–7.67 (m, 1H), 7.61 (s, 2H), 7.46 (t, 7.5 Hz, 2H), 4.30 (q, 7.0 Hz, 1H), 2.42–2.33
(m, 1H), 2.18–2.09 (m, 1H), 0.94 (t, 7.0 Hz, 3H); ESI-MS: m/z = 397 [M+H]+.
17

ACCEPTED MANUSCRIPT
3-(1-Bromoethyl)-2-(3-(trifluoromethyl)phenyl)-2H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (35)
1
White solid; yield 79%; H NMR (500 MHz, DMSO-d6): δ 8.02–7.97 (m, 3H), 7.92 (t, 9.5 Hz, 2H),
7.85 (t, 9.5 Hz, 1H), 7.76 (d, 10.5 Hz, 1H), 7.69 (d, 9.5 Hz, 1H), 4.58 (q, 8.0 Hz, 1H), 1.94 (d, 8.0 Hz,
3H); ESI-MS: m/z = 433 [M+H]+.
3-(1-Bromopropyl)-2-(3-(trifluoromethyl)phenyl)-2H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (36)
White solid; yield 79%; 1H NMR (500 MHz, DMSO-d6): δ 8.03–7.99 (m, 2H), 7.96–7.82 (m, 4H), 7.75
(d, 10.5 Hz, 1H), 7.69 (t, 9.5 Hz, 1H), 4.29 (t, 9.0 Hz, 1H), 2.44–2.31 (m, 1H), 2.18–2.07 (m, 1H), 0.94
(t, 9.0 Hz, 3H); ESI-MS: m/z = 447 [M+H]+.
3-(1-Bromopropyl)-2-(4-(trifluoromethoxy)phenyl)-2H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (37)
1
White solid; yield 84%; H NMR (500 MHz, DMSO-d6): δ 8.00 (d, 9.5 Hz, 1H), 7.91 (t, 9.5 Hz, 1H),
7.83–7.52 (m, 6H), 4.32 (t, 9.0 Hz, 1H), 2.43–2.31 (m, 1H), 2.21–2.07 (m, 1H), 0.94 (t, 9.0 Hz, 3H);
ESI-MS: m/z = 463 [M+H]+.
3-(1-Bromopropyl)-2-(3,4-dimethoxyphenyl)-2H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (38) White
solid; yield 93%; 1H NMR (500 MHz, DMSO-d6): δ 7.99 (dd, 1.5 Hz, 7.5 Hz, 1H), 7.92–7.86 (m, 1H),
7.71 (dd, 1.0 Hz, 8.5 Hz, 1H), 7.68–7.65 (m, 1H), 7.31–6.88 (m, 3H), 4.33 (t, 7.5 Hz, 1H), 3.84 (s, 3H),
3.74 (s, 3H), 2.43–2.33 (m, 1H), 2.14–2.08 (m, 1H), 0.93 (t, 7.0 Hz, 3H); ESI-MS: m/z = 439 [M+H]+.
3-(1-Bromoethyl)-2-(pyridin-3-yl)-2H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (39) White solid; yield
76%; ESI-MS: m/z = 366 [M+H]+.
3-(1-Bromoethyl)-8-fluoro-2-phenyl-2H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (40) White solid;
yield 89%; 1H NMR (500 MHz, DMSO-d6): δ 7.97–7.87 (m, 1H), 7.71–7.45 (m, 7H), 4.51 (q, 6.5 Hz,
1H), 1.91 (d, 6.5 Hz, 3H); ESI-MS: m/z = 383 [M+H]+.
3-(1-Bromopropyl)-8-fluoro-2-phenyl-2H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (41) White solid;
yield 86%; ESI-MS: m/z = 397 [M+H]+.
3-(1-Bromoethyl)-8-chloro-2-phenyl-2H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (42) White solid;
yield 92%; 1H NMR (500 MHz, DMSO-d6): δ 7.62–7.57 (m, 3H), 7.56–7.50 (m, 4H), 7.48 (dd, 2.0 Hz,
7.0 Hz, 1H), 4.38 (q, 7.0 Hz, 1H), 1.96 (d, 6.5 Hz, 3H); ESI-MS: m/z = 399 [M+H]+.
3-(1-Bromopropyl)-8-chloro-2-phenyl-2H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (43) White solid;
yield 87%; 1H NMR (500 MHz, DMSO-d6): δ 7.86 (t, 8.5 Hz, 1H), 7.75 (dd, 1.0 Hz, 8.0 Hz, 1H), 7.68
(dd, 1.0 Hz, 8.0 Hz, 1H), 7.65–7.42 (m, 5H), 4.19 (t, 7.5 Hz, 1H), 2.40–2.31 (m, 1H), 2.15–2.06 (m,
1H), 0.92 (d, 7.0 Hz, 3H); ESI-MS: m/z = 413 [M+H]+.
18

ACCEPTED MANUSCRIPT
4.1.5. General procedure for intermediates 44-54
To a solution of 32 (7.86 g, 21.6 mmol, 1.0 eq) and phthalimide (3.18 g, 21.6 mmol, 1.0 eq) in
anhydrous DMF (30 mL) was added anhydrous K2CO3 (4.47 g, 32.4 mmol, 1.5 eq). The resultant
mixture was stirred at 40 ºC for 6 h, and H2O was added under agitation to quench the reaction. After
filtration, the solution of the filter cake in DCM was washed successively with H2O and brine, dried
over anhydrous Na2SO4, and concentrated in vacuo. To the residue, EtOH (100 mL) and hydrazine
hydrate (85%, 2.6 mL, 43.2 mmol, 2.0 eq) were added. The resultant mixture was stirred at 80 ºC under
N2 atmosphere till TLC demonstrated total conversion of the substrate. After the reaction mixture was
completely cooled, the precipitate was filtered and the filtrate was concentered in vacuo. To the residue
EA was added, and the undissolved component was filtered. The filtrate was subsequently washed
successively with NaOH solution (0.5 N) and brine, dried over anhydrous Na2SO4, and concentrated in
vacuo. Final flash column chromatography utilizing EA/PE/TEA (60:60:1) as the eluent afforded
3-(1-aminoethyl)-2-phenyl-2H-benzo[e][1,2,4]thiadiazine 1,1-dioxide 44 as a light yellow oil that was
slowly solidified. Yield 52% (for two steps); ESI-MS: m/z = 302 [M+H]+. Intermediates 45−54 were
prepared in a procedure similar to that described for 44.
3-(1-Aminoethyl)-2-(4-fluorophenyl)-2H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (45) Light yellow
oil; yield 39% (for two steps); ESI-MS: m/z = 320 [M+H]+.
3-(1-Aminoethyl)-2-(3-(trifluoromethyl)phenyl)-2H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (46)
Light yellow oil; yield 32% (for two steps); ESI-MS: m/z = 370 [M+H]+.
3-(1-Aminopropyl)-2-(3-(trifluoromethyl)phenyl)-2H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (47)
Light yellow oil; yield 54% (for two steps); ESI-MS: m/z = 384 [M+H]+.
3-(1-Aminopropyl)-2-(4-(trifluoromethoxy)phenyl)-2H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (48)
Light yellow oil; yield 50% (for two steps); ESI-MS: m/z = 400 [M+H]+.
3-(1-Aminopropyl)-2-(3,4-dimethoxyphenyl)-2H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (49) Light
yellow oil; yield 55% (for two steps); ESI-MS: m/z = 376 [M+H]+.
3-(1-Aminoethyl)-2-(pyridin-3-yl)-2H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (50) Light yellow oil;
yield 34% (for two steps); ESI-MS: m/z = 303 [M+H]+.
3-(1-Aminoethyl)-8-fluoro-2-phenyl-2H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (51) Light yellow oil;
yield 37% (for two steps); ESI-MS: m/z = 320 [M+H]+.
3-(1-Aminopropyl)-8-fluoro-2-phenyl-2H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (52) Light yellow
19

ACCEPTED MANUSCRIPT
1
solid; yield 53% (for two steps); H NMR (500 MHz, DMSO-d6): δ 7.91–7.82 (m, 1H), 7.66–7.56 (m,
3H), 7.55–7.45 (m, 4H), 3.14–3.07 (m, 1H), 2.03 (brs, 2H), 1.74–1.63 (m, 1H), 1.47–1.37 (m, 1H),
0.76 (t, 7.5 Hz, 3H); ESI-MS: m/z = 334 [M+H]+.
3-(1-Aminoethyl)-8-chloro-2-phenyl-2H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (53) Light yellow
oil; yield 40% (for two steps); ESI-MS: m/z = 336 [M+H]+.
3-(1-Aminopropyl)-8-chloro-2-phenyl-2H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (54) Light yellow
solid; yield 56% (for two steps); 1H NMR (500 MHz, DMSO-d6): δ 7.81 (t, 8.0 Hz, 1H), 7.67 (dd, 1.0
Hz, 8.0 Hz, 1H), 7.64 (dd, 1.0 Hz, 8.5 Hz, 1H), 7.61–7.56 (m, 3H), 7.55–7.46 (m, 2H), 3.14–3.02 (m,
1H), 2.00 (brs, 2H), 1.76–1.61 (m, 1H), 1.47–1.35 (m, 1H), 0.77 (t, 7.5 Hz, 3H); ESI-MS: m/z = 350
[M+H]+.
4.1.6.
General procedure for target compounds 55-72
To the solution of 44 (200 mg, 0.664 mmol, 1.1 eq) in t-BuOH (4 mL) were added
6-chloro-9H-purine (93 mg, 0.604 mmol, 1.0 eq) and DIPEA (149 µL, 0.906 mmol, 1.5 eq). The
resultant mixture was stirred at 80 ºC under N2 atmosphere for 8 h and concentrated in vacuo. To the
residue was added DCM, and the mixture was washed successively with saturated NaHCO3 solution,
dilute hydrochloric acid (0.5 N) and brine. The organic layer was dried over anhydrous Na2SO4 and
concentrated in vacuo. Flash column chromatography utilizing EA/PE (1:1–3:1) and EA/AcOH (80:1)
as the eluent afforded a light yellow oil. It was then dissolved in DCM, and the resulting solution was
washed successively with saturated NaHCO3 solution, brine, dried over anhydrous Na2SO4, and
concentrated
in
vacuo
to
afford
3-(1-((9H-purin-6-yl)amino)ethyl)-2-phenyl-2H-benzo[e][1,2,4]thiadiazine 1,1-dioxide 55 as a light
yellow solid. Yield 28%; 1H NMR (500 MHz, DMSO-d6): δ 12.96 (s, 0.7H), 12.10 (s, 0.3H), 8.31–8.09
(m, 2H), 7.94 (d, 7.5 Hz, 2H), 7.80 (t, 7.5 Hz, 1H), 7.68–7.50 (m, 7H), 4.91–4.71 (m, 1H), 1.48 (d, 7.0
Hz, 3H); 13C NMR (125 MHz, DMSO-d6): δ 158.79, 153.40, 152.38, 142.09, 139.70, 134.82, 132.52,
130.65, 130.46, 130.11, 129.86, 129.73, 128.24, 128.19, 127.09, 121.95, 48.54, 18.83; ESI-HRMS: m/z
calcd for C20H17N7O2S [M+H]+ 420.1243, found 420.1235; HPLC: tR = 15.19 min, flow rate 1.2
mL/min, DiamonsilTM C18 5µ 4.6×200 mm, rt, eluent A-75%, eluent B-25%.
Compound 56-72 were prepared in a manner similar to that described for 55. As for compounds
68-72, the products were precipitated from the reaction mixture and obtained by filtration without
further treatment.
20

ACCEPTED MANUSCRIPT
3-(1-((9H-purin-6-yl)amino)ethyl)-2-(4-fluorophenyl)-2H-benzo[e][1,2,4]thiadiazine 1,1-dioxide
(56) Light yellow solid; yield 21%; 1H NMR (500 MHz, CDCl3): δ 8.36 (s, 1H), 8.05 (s, 1H), 7.90 (dd,
1.5 Hz, 8.0 Hz, 1H), 7.71 (t, 7.5 Hz, 1H), 7.64 (d, 7.5 Hz, 1H), 7.60–7.46 (m, 3H), 7.28–7.18 (m, 2H),
7.12–7.03 (m, 1H), 5.21–4.94 (m, 1H), 1.61 (d, 7.0 Hz, 3H); 13C NMR (125 MHz, DMSO-d6): δ 163.22
(d, JC-F = 247.5 Hz), 158.64, 152.29, 144.01, 142.06, 134.88, 132.93, 128.25 (d, JC-F = 8.75 Hz), 126.93,
122.71 (d, JC-F = 8.75 Hz), 122.01, 121.74, 118.87, 117.00 (d, JC-F = 22.5 Hz), 116.61 (d, JC-F = 22.5
Hz), 114.47, 48.47, 18.77; ESI-HRMS: m/z calcd for C20H16FN7O2S [M+H]+ 438.1148, found
438.1146; HPLC: tR = 7.67 min, flow rate 1.0 mL/min, COSMOSIL 5C18-MS-II column (4.6ID×250
mm), rt, eluent A-65%, eluent B-35%.
3-(1-((9H-purin-6-yl)amino)ethyl)-2-(3-(trifluoromethyl)phenyl)-2H-benzo[e][1,2,4]thiadiazine
1,1-dioxide (57) Light yellow solid; yield 25%; 1H NMR (500 MHz, DMSO-d6): δ 12.95 (s, 1H), 8.17
(s, 2H), 8.07–7.88 (m, 4H), 7.84 (t, 7.0 Hz, 2H), 7.77–7.67 (m, 1H), 7.66–7.55 (m, 2H), 4.93–4.81 (m,
1H), 1.50 (d, 7.0 Hz, 3H); ESI-HRMS: m/z calcd for C21H16F3N7O2S [M+H]+ 488.1117, found
488.1122; HPLC: tR = 7.87 min, flow rate 1.0 mL/min, COSMOSIL 5C18-MS-II column (4.6ID×250
mm), rt, eluent A-70%, eluent B-30%.
3-(1-((9H-purin-6-yl)amino)propyl)-2-(3-(trifluoromethyl)phenyl)-2H-benzo[e][1,2,4]thiadiazine
1,1-dioxide (58) Light yellow solid; yield 40%; 1H NMR (500 MHz, DMSO-d6): δ 12.96 (s, 1H), 8.18
(s, 1H), 8.11–7.70 (m, 8H), 7.68-7.54 (m, 2H), 4.82–4.55 (m, 1H), 2.11–1.85 (m, 2H), 0.86 (t, 6.5 Hz,
3H); 13C NMR (125 MHz, DMSO-d6): δ 157.14, 154.33, 153.81, 151.91, 149.29, 141.95, 140.46,
139.86, 139.15, 134.99, 134.47, 133.37, 131.39, 130.63 (q, JC-F = 32.5 Hz), 128.47, 127.27, 127.09,
123.82 (q, JC-F = 270 Hz), 121.94, 53.96, 25.95, 11.09; ESI-HRMS: m/z calcd for C22H18F3N7O2S
[M+H]+ 502.1273, found 502.1277; HPLC: tR = 10.56 min, flow rate 1.0 mL/min, COSMOSIL
5C18-MS-II column (4.6ID×250 mm), rt, eluent A-70%, eluent B-30%.
3-(1-((9H-purin-6-yl)amino)propyl)-2-(4-(trifluoromethoxy)phenyl)-2H-benzo[e][1,2,4]thiadiazine
1
1,1-dioxide (59) Light yellow solid; yield 47%; H NMR (500 MHz, DMSO-d6): δ 12.98 (s, 0.85H),
12.12 (s, 0.15H), 8.30 (s, 0.3H), 8.22–8.04 (m, 1.7H), 7.95 (d, 7.0 Hz, 2H), 7.82 (t, 7.0 Hz, 1H), 7.78–
7.66 (m, 2H), 7.65–7.44 (m, 4H), 4.85–4.56 (m, 1H), 2.08–1.80 (m, 2H), 0.86 (t, 7.0 Hz, 3H);
ESI-HRMS: m/z calcd for C22H18F3N7O3S [M+H]+ 518.1222, found 518.1214; HPLC: tR = 13.16 min,
flow rate 1.0 mL/min, COSMOSIL 5C18-MS-II column (4.6ID×250 mm), rt, eluent A-70%, eluent
B-30%.
21

ACCEPTED MANUSCRIPT
3-(1-((9H-purin-6-yl)amino)propyl)-2-(3,4-dimethoxyphenyl)-2H-benzo[e][1,2,4]thiadiazine
1
1,1-dioxide (60) Light yellow solid; yield 40%; H NMR (500 MHz, CDCl3): δ 8.30 (s, 1H), 8.03 (s,
1H), 7.89 (dd, 1.5 Hz, 7.5 Hz, 1H), 7.73–7.61 (m, 2H), 7.51–7.44 (m, 1H), 7.17–6.76 (m, 3H), 5.28–
5.12 (m, 1H), 3.96 (s, 3H), 3.79 (s, 3H), 2.17–2.06 (m, 1H), 1.96–1.86 (m, 1H), 0.98 (t, 7.0 Hz, 3H);
13C NMR (125 MHz, DMSO-d6): δ 157.88, 153.76, 152.29, 150.55, 149.53, 146.74, 142.08, 139.65,
134.72, 128.32, 128.21, 128.10, 127.11, 124.56, 122.81, 121.97, 113.62, 112.04, 56.16, 56.07, 53.71,
26.22, 11.01; ESI-HRMS: m/z calcd for C23H23N7O4S [M+H]+ 494.1610, found 494.1607; HPLC: tR =
9.68 min, flow rate 1.0 mL/min, COSMOSIL 5C18-MS-II column (4.6ID×250 mm), rt, eluent A-60%,
eluent B-40%.
3-(1-((9H-purin-6-yl)amino)ethyl)-2-(pyridin-3-yl)-2H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (61)
Light yellow solid; yield 19%; 1H NMR (500 MHz, DMSO-d6): δ 12.96 (s, 0.8H), 12.01 (s, 0.2H), 8.77
(s, 1H), 8.66 (s, 1H), 8.29 (s, 0.2H), 8.18–8.10 (m, 1.8H), 8.08 (s, 2H), 7.97 (dd, 1.0 Hz, 8.0 Hz, 1H),
7.85–7.80 (m, 1H), 7.65–7.47 (m, 3H), 4.84–4.74 (m, 1H), 1.51 (d, 7.0 Hz, 3H); ESI-HRMS: m/z calcd
for C19H16N8O2S [M+H]+ 421.1195, found 421.1197; HPLC: tR = 6.91 min, flow rate 1.0 mL/min,
COSMOSIL 5C18-MS-II column (4.6ID×250 mm), rt, eluent A-60%, eluent B-40%.
3-(1-((9H-purin-6-yl)amino)propyl)-8-fluoro-2-phenyl-2H-benzo[e][1,2,4]thiadiazine
1,1-dioxide
(62) White solid; yield 46%; 1H NMR (500 MHz, DMSO-d6): δ 12.99 (s, 0.8H), 12.16 (s, 0.2H), 8.37–
8.02 (m, 2H), 7.98–7.72 (m, 2H), 7.70–7.52 (m, 5H), 7.46 (t, 9.0 Hz, 1H), 7.36 (d, 7.5 Hz, 1H), 4.77–
4.53 (m, 1H), 2.04–1.73 (m, 2H), 0.81 (t, 7.0 Hz, 3H); 13C NMR (125 MHz, DMSO-d6): δ 158.76,
156.29 (d, JC-F = 255 Hz), 153.96, 152.33, 150.40, 143.90, 139.70, 135.91 (d, JC-F = 10.00 Hz), 131.75,
130.92, 130.13, 124.59, 120.53, 119.33, 115.78 (d, JC-F = 12.50 Hz), 114.82 (d, JC-F = 20.00 Hz), 54.21,
25.91, 11.12; ESI-HRMS: m/z calcd for C21H18FN7O2S [M+H]+ 452.1305, found 452.1303; HPLC: tR
= 6.43 min, flow rate 1.0 mL/min, COSMOSIL 5C18-MS-II column (4.6ID×250 mm), rt, eluent
A-70%, eluent B-30%.
3-(1-((9H-purin-6-yl)amino)ethyl)-8-chloro-2-phenyl-2H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (63)
1
Pale solid; yield 23%; H NMR (500 MHz, DMSO-d6): δ 12.97 (s, 0.8H), 12.06 (s, 0.2H), 8.29 (s,
0.2H), 8.20 (s, 0.2H), 8.17–8.10 (m, 1.6H), 8.02 (brs, 1H), 7.74 (t, 8.0 Hz, 1H), 7.70–7.61 (m, 3H),
7.60–7.50 (m, 3H), 7.48–7.43 (m, 1H), 4.79–4.65 (m, 1H), 1.47 (d, 7.0 Hz, 3H); 1H NMR (500 MHz,
CDCl3): δ 8.33 (s, 1H), 7.97 (s, 1H), 7.63–7.47 (m, 7H), 7.42 (d, 6.0 Hz, 1H), 6.79 (brs, 1H), 5.28–4.91
(m, 1H), 1.56 (d, 5.0 Hz, 1H); 13C NMR (125 MHz, DMSO-d6): δ 159.21, 152.33, 144.26, 139.70,
22

ACCEPTED MANUSCRIPT
135.07, 131.98, 131.04, 130.89, 130.14, 130.03, 129.66, 127.98, 127.94, 127.89, 125.22, 119.27, 48.54,
18.17; ESI-HRMS: m/z calcd for C20H16ClN7O2S [M+H]+ 454.0853, found 454.0851; HPLC: tR = 7.74
min, flow rate 1.2 mL/min, COSMOSIL 5C18-MS-II column (4.6ID×250 mm), rt, eluent A-65%,
eluent B-35%.
3-(1-((9H-purin-6-yl)amino)propyl)-8-chloro-2-phenyl-2H-benzo[e][1,2,4]thiadiazine
1,1-dioxide
(64) White solid; yield 51%; 1H NMR (500 MHz, CDCl3): δ 13.87 (brs, 1H), 8.35 (s, 1H), 8.05 (s, 1H),
7.69–7.36 (m, 8H), 6.88 (d, 7.0 Hz, 1H), 5.20–4.97 (m, 1H), 2.14–2.07 (m, 1H), 1.95–1.86 (m, 1H),
0.98 (t, 7.0 Hz, 3H); 13C NMR (125 MHz, DMSO-d6): δ 158.31, 153.25, 152.24, 144.14, 140.53,
135.09, 132.07, 130.93, 130.08, 129.81, 129.77, 128.30, 127.99, 127.87, 125.35, 121.69, 54.14, 25.92,
11.07; ESI-MS: m/z = 468 [M+H]+; HPLC: tR = 10.75 min, flow rate 1.2 mL/min, COSMOSIL
5C18-MS-II column (4.6ID×250 mm), rt, eluent A-65%, eluent B-35%.
3-(1-((2-Fluoro-9H-purin-6-yl)amino)ethyl)-2-phenyl-2H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (65)
Pale solid; yield 46%; 1H NMR (500 MHz, DMSO-d6): δ 13.15–12.93 (m, 0.8H), 12.12 (s, 0.2H), 8.71
(d, 4.5 Hz, 0.7H), 8.43–8.23 (m, 0.6H), 8.14 (s, 0.7H), 7.95 (d, 7.0 Hz, 1H), 7.86–7.76 (m, 1H), 7.67–
7.43 (m, 7H), 4.84–4.65 (m, 1H), 1.49 (d, 6.0 Hz, 3H); 13C NMR (125 MHz, DMSO-d6): δ 158.61 (d,
JC-F = 202.5 Hz), 157.87, 155.16, 151.55, 142.01, 134.88, 132.44, 132.38, 130.65, 130.30, 130.07,
129.72, 128.33, 128.30, 127.07, 121.99, 48.76, 18.53; ESI-HRMS: m/z calcd for C20H16FN7O2S
[M+H]+ 438.1148, found 438.1146; HPLC: tR = 7.28 min, flow rate 1.2 mL/min, COSMOSIL
5C18-MS-II column (4.6ID×250 mm), rt, eluent A-65%, eluent B-35%.
3-(1-((2-Chloro-9H-purin-6-yl)amino)ethyl)-2-phenyl-2H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (66)
Pale solid; yield 38%; 1H NMR (500 MHz, DMSO-d6): δ 13.14 (brs, 1H), 8.68 (s, 0.8H), 8.46–8.12 (m,
1.2H), 8.05–7.90 (m, 1H), 7.80 (t, 7.0 Hz, 1H), 7.74–7.64 (m, 2H), 7.63–7.28 (m, 5H), 4.83–4.61 (m,
1H), 1.48 (d, 6.0 Hz, 3H); 13C NMR (125 MHz, DMSO-d6): δ 158.06, 152.88, 142.01, 140.35, 135.15,
134.88, 133.07, 132.36, 130.69, 130.30, 130.14, 129.70, 128.29, 127.08, 125.00, 121.99, 48.67, 18.30;
ESI-HRMS: m/z calcd for C20H16ClN7O2S [M+H]+ 454.0853, found 454.0854; HPLC: tR = 7.77 min,
flow rate 1.2 mL/min, COSMOSIL 5C18-MS-II column (4.6ID×250 mm), rt, eluent A-70%, eluent
B-30%.
8-Fluoro-3-(1-((2-fluoro-9H-purin-6-yl)amino)propyl)-2-phenyl-2H-benzo[e][1,2,4]thiadiazine
1,1-dioxide (67) Pale solid; yield 42%; 1H NMR (500 MHz, CDCl3): δ 8.00 (s, 1H), 7.68–7.44 (m, 7H),
7.40 (d, 8.5 Hz, 1H), 7.17 (t, 8.5 Hz, 1H), 7.11 (d, 8.0 Hz, 1H), 4.99–4.87 (m, 1H), 2.15–2.02 (m, 1H),
23

ACCEPTED MANUSCRIPT
1.97–1.84 (m, 1H), 0.97 (d, 7.0 Hz, 3H); 13C NMR (125 MHz, DMSO-d6): δ 158.58 (d, JC-F = 204 Hz),
158.02, 157.33, 156.65 (d, JC-F = 273 Hz), 155.73, 155.29, 144.58, 143.84, 140.15, 135.98 (d, JC-F
=
8.25 Hz), 131.59, 130.90 (d, JC-F = 21.25 Hz), 130.26, 130.10, 124.66 (d, JC-F = 2.50 Hz), 115.93 (d,
JC-F = 20.0 Hz), 54.67, 25.51, 11.35; ESI-HRMS: m/z calcd for C21H17F2N7O2S [M+H]+ 470.1211,
found 470.1227; HPLC: tR = 7.86 min, flow rate 1.0 mL/min, COSMOSIL 5C18-MS-II column
(4.6ID×250 mm), rt, eluent A-70%, eluent B-30%.
4-Amino-6-((1-(1,1-dioxido-2-phenyl-2H-benzo[e][1,2,4]thiadiazin-3-yl)ethyl)amino)pyrimidine-5-
carbonitrile (68) Yield 63%; 1H NMR (500 MHz, DMSO-d6): δ 7.96 (dd, 1.5 Hz, 7.5 Hz, 1H), 7.93 (s,
1H), 7.87–7.81 (m, 1H), 7.69 (d, 7.0 Hz, 1H), 7.64–7.50 (m, 7H), 7.32 (brs, 2H), 4.73–4.65 (m, 1H),
1.40 (d, 7.0 Hz, 3H); 13C NMR (125 MHz, DMSO-d6): δ 170.03, 167.26, 165.12, 163.24, 147.28,
140.23, 137.73, 135.94, 135.65, 135.44, 133.62, 133.61, 132.40, 127.32, 121.05, 73.97, 54.32, 23.83;
ESI-HRMS: m/z calcd for C20H17N7O2S [M+H]+ 420.1243, found 420.1241; HPLC: tR = 7.38 min,
flow rate 1.0 mL/min, COSMOSIL 5C18-MS-II column (4.6ID×250 mm), rt, eluent A-65%, eluent
B-35%.
4-Amino-6-((1-(8-fluoro-1,1-dioxido-2-phenyl-2H-benzo[e][1,2,4]thiadiazin-3-yl)ethyl)amino)pyri
1
midine-5-carbonitrile (69) White solid, yield 58%; H NMR (500 MHz, DMSO-d6): δ 7.93 (s, 1H),
7.89–7.82 (m, 1H), 7.70 (d, 7.0 Hz, 1H), 7.63–7.58 (m, 2H), 7.57–7.53 (m, 3H), 7.51–7.47 (m, 1H),
7.43 (d, 8.0 Hz, 1H), 7.31 (brs, 2H), 4.72–4.58 (m, 1H), 1.40 (d, 7.0 Hz, 3H); 13C NMR (125 MHz,
DMSO-d6): δ 164.72, 161.87, 159.76, 158.68, 156.38 (d, JC-F = 255 Hz), 143.87, 136.99 (d, JC-F
=
10.00 Hz), 131.53, 130.91, 130.10, 124.63 (d, JC-F = 2.50 Hz), 115.70, 115.64 (d, JC-F = 13.75 Hz),
114.82 (d, JC-F = 18.75 Hz), 68.64, 49.16, 18.44; ESI-HRMS: m/z calcd for C20H16FN7O2S [M+H]+
438.1148, found 438.1149; HPLC: tR = 7.36 min, flow rate 1.0 mL/min, COSMOSIL 5C18-MS-II
column (4.6ID×250 mm), rt, eluent A-65%, eluent B-35%.
4-Amino-6-((1-(8-fluoro-1,1-dioxido-2-phenyl-2H-benzo[e][1,2,4]thiadiazin-3-yl)propyl)amino)pyri
1
midine-5-carbonitrile (70) White solid, yield 69%; H NMR (500 MHz, DMSO-d6): δ 7.92 (s, 1H),
7.90–7.83 (m, 1H), 7.57 (s, 5H), 7.54–7.48 (m, 2H), 7.43 (d, 8.0 Hz, 1H), 7.33 (brs, 2H), 4.59–4.52 (m,
1H), 1.94–1.84 (m, 2H), 0.76 (t, 7.5 Hz, 3H); 13C NMR (125 MHz, DMSO-d6): δ 164.71, 162.51,
159.81, 157.91, 156.30 (d, JC-F = 255 Hz), 143.74, 136.03 (d, JC-F = 10.0 Hz), 131.65, 130.96, 130.77,
130.18, 124.63 (d, JC-F = 2.50 Hz), 115.76 (d, JC-F = 13.75 Hz), 115.70, 114.97 (d, JC-F = 20.00 Hz),
68.78, 54.53, 25.42, 10.93; ESI-HRMS: m/z calcd for C21H18FN7O2S [M+H]+ 452.1305, found
24

ACCEPTED MANUSCRIPT
452.1318; HPLC: tR = 10.23 min, flow rate 1.0 mL/min, COSMOSIL 5C18-MS-II column (4.6ID×250
mm), rt, eluent A-65%, eluent B-35%.
4-Amino-6-((1-(8-chloro-1,1-dioxido-2-phenyl-2H-benzo[e][1,2,4]thiadiazin-3-yl)ethyl)amino)pyri
1
midine-5-carbonitrile (71) White solid, yield 62%; H NMR (500 MHz, DMSO-d6): δ 7.91 (s, 1H),
7.76 (t, 8.0 Hz, 1H), 7.66–7.63 (m, 2H), 7.59–7.56 (m, 2H), 7.55–7.47 (m, 4H), 7.28 (brs, 2H), 4.69–
4.56 (m, 1H), 1.38 (d, 6.5 Hz, 3H); 13C NMR (125 MHz, DMSO-d6): δ 164.71, 161.88, 159.76, 158.32,
144.10, 135.17, 131.84, 130.91, 130.86, 130.06, 129.79, 128.02, 127.95, 125.18, 115.71, 68.64, 49.13,
18.38; ESI-HRMS: m/z calcd for C20H16ClN7O2S [M+H]+ 454.0853, found 454.0850; HPLC: tR
=
10.09 min, flow rate 1.0 mL/min, COSMOSIL 5C18-MS-II column (4.6ID×250 mm), rt, eluent A-65%,
eluent B-35%.
4-Amino-6-((1-(8-chloro-1,1-dioxido-2-phenyl-2H-benzo[e][1,2,4]thiadiazin-3-yl)propyl)amino)pyri
1
midine-5-carbonitrile (72) White solid; yield 71%; H NMR (500 MHz, DMSO-d6): δ 7.92 (s, 1H),
7.81 (t, 8.0 Hz, 1H), 7.69 (d, 7.5 Hz, 1H), 7.61–7.47 (m, 7H), 7.33 (brs, 2H), 4.59–4.49 (m, 1H), 1.93–
1.81 (m, 2H), 0.76 (t, 7.0 Hz, 3H); 13C NMR (125 MHz, DMSO-d6): δ 164.70, 162.52, 159.81, 157.55,
143.98, 135.21, 131.97, 130.92, 130.78, 130.13, 129.91, 128.02, 127.91, 125.34, 115.71, 68.78, 54.47,
25.36, 10.90; ESI-HRMS: m/z calcd for C21H18ClN7O2S [M+H]+ 468.1009, found 468.1001; HPLC: tR
= 9.37 min, flow rate 1.0 mL/min, COSMOSIL 5C18-MS-II column (4.6ID×250 mm), rt, eluent
A-70%, eluent B-30%.
4.1.7. General procedure for target compounds 73-78
To a solution of 32 (60 mg, 0.165 mmol, 1.0 eq) and adenine (22 mg, 0.165 mmol, 1.0 eq) in
anhydrous DMF (1 mL) was added anhydrous K2CO3 (23 mg, 0.165 mmol, 1.0 eq). The resultant
mixture was stirred at room temperature for 6 h, and H2O was added under agitation to quench the
reaction. After filtration, the solution of the filter cake in DCM was washed successively with H2O and
brine, dried over anhydrous Na2SO4, and concentrated in vacuo. The residue was subjected to flash
column
chromatography
utilizing
EA
as
the
eluent
to
afford
3-(1-(6-amino-9H-purin-9-yl)ethyl)-2-phenyl-2H-benzo[e][1,2,4]thiadiazine 1,1-dioxide 73 as a white
1
solid. Yield 65%; H NMR (500 MHz, DMSO-d6): δ 8.15 (s, 1H), 7.99–7.92 (m, 2H), 7.84 (t, 8.0 Hz,
1H), 7.64 (t, 7.5 Hz, 1H), 7.57 (d, 8.0 Hz, 1H), 7.53–7.42 (m, 3H), 7.34 (brs, 2H), 7.21 (s, 2H), 5.41 (q,
7.0 Hz, 1H), 1.78 (d, 7.0 Hz, 3H); 13C NMR (100 MHz, DMSO-d6): δ 158.28, 154.37, 152.84, 149.49,
141.45, 139.57, 135.01, 131.94, 130.74, 130.31, 129.60, 129.03, 128.72, 127.12, 121.99, 118.56, 51.26,
25

ACCEPTED MANUSCRIPT
18.91; ESI-HRMS: m/z calcd for C20H17N7O2S [M+H]+ 420.1243, found 420.1243; HPLC: tR = 7.11
min, flow rate 1.0 mL/min, COSMOSIL 5C18-MS-II column (4.6ID×250 mm), rt, eluent A-60%,
eluent B-40%.
Compound 74-78 were prepared in a procedure similar to that described for 73.
3-(1-(6-Amino-9H-purin-9-yl)ethyl)-2-(4-fluorophenyl)-2H-benzo[e][1,2,4]thiadiazine 1,1-dioxide
(74) White solid; yield 47%; 1H NMR (500 MHz, DMSO-d6): δ 8.15 (s, 1H), 8.02–7.93 (m, 2H), 7.87
(t, 8.0 Hz, 1H), 7.75–7.53 (m, 3H), 7.45–7.07 (m, 5H), 5.44 (q, 7.0 Hz, 1H), 1.79 (d, 7.0 Hz, 3H); 13C
NMR (100 MHz, DMSO-d6): δ 163.20 (d, JC-F = 247 Hz), 156.30, 154.16, 152.82, 149.41, 141.42,
139.38, 135.07, 132.02 (d, JC-F = 9.00 Hz), 129.06, 128.78, 128.01, 126.99, 122.05, 118.53, 117.24 (d,
JC-F = 22.0 Hz), 51.21, 19.01; ESI-HRMS: m/z calcd for C20H16FN7O2S [M+H]+ 438.1148, found
438.1155; HPLC: tR = 7.99 min, flow rate 1.0 mL/min, COSMOSIL 5C18-MS-II column (4.6ID×250
mm), rt, eluent A-60%, eluent B-40%.
3-(1-(6-Amino-9H-purin-9-yl)propyl)-2-(4-fluorophenyl)-2H-benzo[e][1,2,4]thiadiazine
1,1-dioxide (75) White solid; yield 62%; 1H NMR (500 MHz, DMSO-d6): δ 8.20 (s, 1H), 8.02–7.93 (m,
2H), 7.89 (t, 7.5 Hz, 1H), 7.73–7.63 (m, 2H), 7.50–7.01 (m, 6H), 5.25 (dd, 5.0 Hz, 9.5 Hz, 1H), 2.43–
2.34 (m, 1H), 2.31–2.21 (m, 1H), 0.74 (t, 7.0 Hz, 3H); 13C NMR (100 MHz, DMSO-d6): δ 163.20 (d,
JC-F = 247 Hz), 156.29, 153.48, 152.82, 149.95, 141.42, 139.56, 135.06, 132.01 (d, JC-F = 8.00 Hz),
129.15, 128.84, 128.08 (d, JC-F = 2.00 Hz), 127.13, 122.00, 118.31, 117.27 (d, JC-F = 24.0 Hz), 56.43,
26.78, 10.75; ESI-HRMS: m/z calcd for C21H18FN7O2S [M+H]+ 452.1305, found 452.1303; HPLC: tR
= 11.06 min, flow rate 1.0 mL/min, COSMOSIL 5C18-MS-II column (4.6ID×250 mm), rt, eluent
A-60%, eluent B-40%.
3-(1-(6-Amino-9H-purin-9-yl)propyl)-2-(4-(trifluoromethoxy)phenyl)-2H-benzo[e][1,2,4]thiadiazine
1
1,1-dioxide (76) White solid; yield 55%; H NMR (500 MHz, DMSO-d6): δ 8.15 (s, 1H), 7.98 (d, 7.5
Hz, 1H), 7.95–7.83 (m, 2H), 7.74 (d, 7.5 Hz, 1H), 7.69 (t, 7.0 Hz, 1H), 7.58–6.88 (m, 6H), 5.28 (t, 7.0
Hz, 1H), 2.45–2.35 (m, 1H), 2.32–2.19 (m, 1H), 0.74 (d, 7.0 Hz, 3H); 13C NMR (100 MHz, DMSO-d6):
δ 156.31, 153.15, 152.78, 149.89, 149.62, 141.41, 139.30, 135.15, 131.61, 130.76, 129.30, 128.93,
127.22, 122.62, 121.99, 120.34 (q, JC-F = 256 Hz), 118.23, 56.40, 26.85, 10.66; ESI-HRMS: m/z calcd
for C22H18F3N7O3S [M+H]+ 518.1222, found 518.1230; HPLC: tR = 8.90 min, flow rate 1.0 mL/min,
COSMOSIL 5C18-MS-II column (4.6ID×250 mm), rt, eluent A-70%, eluent B-30%.
3-(1-(6-Amino-9H-purin-9-yl)ethyl)-2-(pyridin-3-yl)-2H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (77)
26

ACCEPTED MANUSCRIPT
White solid; yield 52%; 1H NMR (500 MHz, DMSO-d6): δ 8.63 (d, 4.5 Hz, 1H), 8.47 (s, 1H), 8.14 (s,
1H), 7.99 (d, 7.5 Hz, 1H), 7.95 (s, 1H), 7.89 (t, 7.5 Hz, 1H), 7.76 (brs, 1H), 7.71–7.63 (m, 2H), 7.48
(brs, 1H), 7.25 (s, 2H), 5.46 (q, 6.5 Hz, 1H), 1.81 (d, 6.5 Hz, 3H); 13C NMR (100 MHz, DMSO-d6): δ
156.30, 153.70, 152.89, 151.37, 149.58, 149.32, 141.36, 139.25, 137.22, 135.23, 129.27, 129.15,
128.93, 127.00, 125.03, 122.06, 118.51, 51.29, 18.93; ESI-HRMS: m/z calcd for C19H16N8O2S [M+H]+
421.1195, found 421.1195; HPLC: tR = 9.61 min, flow rate 1.0 mL/min, COSMOSIL 5C18-MS-II
column (4.6ID×250 mm), rt, eluent A-50%, eluent B-50%.
3-(1-(6-Amino-9H-purin-9-yl)ethyl)-8-fluoro-2-phenyl-2H-benzo[e][1,2,4]thiadiazine 1,1-dioxide
1
(78) White solid; yield 48%; H NMR (500 MHz, DMSO-d6): δ 8.16 (s, 1H), 7.99 (s, 1H), 7.91–7.82
(m, 1H), 7.65–7.29 (m, 7H), 7.25 (s, 2H), 5.39 (q, 7.0 Hz, 1H), 1.78 (d, 7.0 Hz, 3H); 13C NMR (100
MHz, DMSO-d6): δ 156.27, 156.21 (d, JC-F = 255 Hz), 155.05, 152.85, 149.47, 143.25, 139.58, 136.12
(d, JC-F = 10.0 Hz), 131.10 (d, JC-F = 5.0 Hz), 130.35, 130.16, 125.10, 118.58, 115.79, 115.66, 115.61
(d, JC-F = 20.0 Hz), 51.39, 18.89; ESI-HRMS: m/z calcd for C20H16FN7O2S [M+H]+ 438.1148, found
438.1145; HPLC: tR = 6.95 min, flow rate 1.0 mL/min, COSMOSIL 5C18-MS-II column (4.6ID×250
mm), rt, eluent A-60%, eluent B-40%.
4.1.8. The procedure for optically pure compounds S-63, R-63, S-71 and R-71
To a solution of 53 (1.00 g, 3.0 mmol, 1.0 eq) in EtOH (14 mL) was added a solution of R-mandelic
acid (0.46 g, 3.0 mmol, 1.0eq) in EtOH (7 mL) dropwise at room temperature. The resultant mixture
was stirred for 0.5 h, and white solid was precipitated during this period. Then, EA (7 mL) was added
slowly, and the resultant mixture was allowed to stand at −10 °C for 4 h. After filtration, the filter cake
was washed with pre-cooled EA, dried in vacuo, and subjected to recrystallization with EA to afford
R-mandelic acid salt of S-53 (de = 99 %) as the white crystal. Yield 34%; mp 173–175 °C; ESI-MS:
m/z = 336 [M+H]+; Chiral HPLC: tR = 15.0 min, flow rate 0.5 mL/min, Welch Ultimate Cellud-Y
column (4.6×250 mm, 5 µM), 15 °C, eluent C-40%, eluent D-60%.
The S-mandelic acid salt of R-53 (de = 95 %) was obtained in a similar procedure to that for
R-mandelic acid salt of S-53 as the white crystal. Yield 42%; mp 174–175 °C; ESI-MS: m/z = 336
[M+H]+; Chiral HPLC: tR = 11.5 min, flow rate 0.5 mL/min, Welch Ultimate Cellud-Y column
(4.6×250 mm, 5 µM), 15 °C, eluent C-40%, eluent D-60%.
The S-mandelic acid salt of R-53 and R-mandelic acid salt of S-53 were basified successively with
NaOH solution (0.5 N) to afford R-53 and S-53, respectively. The optical purity of R-53 and S-53 was
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analyzed by chiral HPLC. R-53: ee = 95%; tR = 11.5 min; S-53: ee = 99%; tR = 15.0 min; Chiral HPLC:
flow rate 0.5 mL/min, Welch Ultimate Cellud-Y column (4.6×250 mm, 5 µM), 15 °C, eluent C-40%,
eluent D-60%.
Compounds S-63, R-63, S-71 and R-71 were prepared in a procedure similar to that described for
corresponding racemates.
(S)-3-(1-((9H-purin-6-yl)amino)ethyl)-8-chloro-2-phenyl-2H-benzo[e][1,2,4]thiadiazine 1,1-dioxide
(S-63) 1H NMR (500 MHz, CDCl3): δ 8.37 (s, 1H), 8.03 (s, 1H), 7.64–7.49 (m, 7H), 7.44 (dd, 1.0 Hz,
7.5 Hz, 1H), 7.00 (brs, 1H), 5.21–4.96 (m, 1H), 1.59 (d, 7.0 Hz, 3H); ESI-HRMS: m/z calcd for
C20H16ClN7O2S [M+H]+ 454.0853, found 454.0840; HPLC: tR = 9.16 min, flow rate 1.0 mL/min,
COSMOSIL 5C18-MS-II column (4.6ID×250 mm), rt, eluent A-65%, eluent B-35%. The title
compound can be obtained as single crystal by slow evaporation from a solution of the pure compound
in a mixture of EtOH and EA at room temperature.
(S)-3-(1-((9H-purin-6-yl)amino)ethyl)-8-chloro-2-phenyl-2H-benzo[e][1,2,4]thiadiazine 1,1-dioxide
1
(R-63) H NMR (500 MHz, CDCl3): δ 8.36 (s, 1H), 8.03 (s, 1H), 7.63–7.49 (m, 7H), 7.44 (d, 7.5 Hz,
1H), 6.96 (brs, 1H), 5.19–4.98 (m, 1H), 1.59 (d, 6.5 Hz, 3H); ESI-HRMS: m/z calcd for
C20H16ClN7O2S [M+H]+ 454.0853, found 454.0841; HPLC: tR = 7.88 min, flow rate 1.2 mL/min,
COSMOSIL 5C18-MS-II column (4.6ID×250 mm), rt, eluent A-65%, eluent B-35%.
(S)-4-amino-6-((1-(8-chloro-1,1-dioxido-2-phenyl-2H-benzo[e][1,2,4]thiadiazin-3-yl)ethyl)amino)py
rimidine-5-carbonitrile (S-71) 1H NMR (500 MHz, CDCl3): δ 8.11 (s, 1H), 7.66–7.44 (m, 8H), 6.53 (d,
7.5 Hz, 1H), 5.47 (s, 2H), 4.96–4.86 (m, 1H), 1.49 (d, 7.0 Hz, 3H); ESI-HRMS: m/z calcd for
C20H16ClN7O2S [M+H]+ 454.0853, found 454.0835; HPLC: tR = 10.32 min, flow rate 1.0 mL/min,
COSMOSIL 5C18-MS-II column (4.6ID×250 mm), rt, eluent A-65%, eluent B-35%.
(R)-4-amino-6-((1-(8-chloro-1,1-dioxido-2-phenyl-2H-benzo[e][1,2,4]thiadiazin-3-yl)ethyl)amino)p
1
yrimidine-5-carbonitrile (R-71) H NMR (500 MHz, CDCl3): δ 8.10 (s, 1H), 7.66–7.41 (m, 8H), 6.57
(d, 7.5 Hz, 1H), 5.56 (s, 2H), 4.95–4.86 (m, 1H), 1.49 (d, 6.5 Hz, 3H); ESI-HRMS: m/z calcd for
C20H16ClN7O2S [M+H]+ 454.0853, found 454.0864; HPLC: tR = 10.29 min, flow rate 1.0 mL/min,
COSMOSIL 5C18-MS-II column (4.6ID×250 mm), rt, eluent A-65%, eluent B-35%.
4.2. Class I PI3Ks Biochemical Assay
ADP-Glo Luminescent Assay (Promega) was performed for evaluating the inhibitory activities
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