Synthesis of novel 7-azaindole derivatives containing pyridin-3-ylmethyl dithiocarbamate moiety as potent PKM2 activators and PKM2 nucleus translocation inhibitors

Article abstract of DOI:10.1016/j.ejmech.2019.03.003

Multiple lines of evidence have indicated that pyruvate kinase M2 (PKM2) is upregulated in most cancer cells and it is increasingly recognized as a potential therapeutic target in oncology. In a continuation of our discovery of lead compound 5 and SAR study, the 7-azaindole moiety in compound 5 was systematically optimized. The results showed that compound 6f, which has a difluoroethyl substitution on the 7-azaindole ring, exhibited high PKM2 activation potency and anti-proliferation activities on A375 cell lines. In a xenograft mouse model, oral administration of compound 6f led to significant tumor regression without obvious toxicity. Further mechanistic studies revealed that 6f could influence the translocation of PKM2 into nucleus, as well as induction of apoptosis and autophagy of A375 cells. More importantly, compound 6f significantly inhibited migration of A375 cells in a concentration-dependent manner. Collectively, 6f may serve as a lead compound in the development of potent PKM2 activators for cancer therapy.

Full text of DOI:10.1016/j.ejmech.2019.03.003

Accepted Manuscript
Synthesis of novel 7-azaindole derivatives containing pyridin-3-ylmethyl
dithiocarbamate moietyas potent PKM2 activators and PKM2 nucleus translocation
inhibitors
Bin Liu, Xia Yuan, Bo Xu, Han Zhang, Ridong Li, Xin Wang, Zemei Ge, Runtao Li
PII:
S0223-5234(19)30214-4
DOI:
https://doi.org/10.1016/j.ejmech.2019.03.003
EJMECH 11173
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 28 December 2018
Revised Date: 19 February 2019
Accepted Date: 1 March 2019
Please cite this article as: B. Liu, X. Yuan, B. Xu, H. Zhang, R. Li, X. Wang, Z. Ge, R. Li, Synthesis of
novel 7-azaindole derivatives containing pyridin-3-ylmethyl dithiocarbamate moietyas potent PKM2
activators and PKM2 nucleus translocation inhibitors, European Journal of Medicinal Chemistry (2019),
doi: https://doi.org/10.1016/j.ejmech.2019.03.003.
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ACCEPTED MANUSCRIPT
Synthesis of novel 7-azaindole derivatives containing
pyridin-3-ylmethyl dithiocarbamate moietyas potent
PKM2 activators and PKM2 nucleus translocation
inhibitors
Graphical abstract

ACCEPTED MANUSCRIPT
Synthesis of novel 7-azaindole derivatives containing
pyridin-3-ylmethyl dithiocarbamate moiety as potent
PKM2 activators and PKM2 nucleus translocation
inhibitors
,a
,a
Bin Liu† , Xia Yuan† , Bo Xua, Han Zhanga, Ridong Lia,b, Xin Wanga, Zemei Gea *,
Runtao Lia *
a
State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences,
Peking University, Beijing 100191, China.
b Institute of Systems Biomedicine, School of Basic Medical Sciences, Peking University, Beijing
100191, China.
† These authors contributed equally.
* Corresponding authors; email: zmge@bjmu.edu.cn; lirt@bjmu.edu.cn.
Abstract
Multiple lines of evidence have indicated that pyruvate kinase M2 (PKM2) is
upregulated in most cancer cells and it is increasingly recognized as a potential
therapeutic target in oncology. In a continuation of our discovery of lead compound 5
and SAR study, the 7-azaindole moiety in compound 5 was systematically optimized.
The results showed that compound 6f, which has a difluroethyl substitution on the
7-azaindole ring, exhibited high PKM2 activation potency and anti-proliferation
activities on A375 cell lines. In a xenograft mouse model, oral administration of
compound 6f led to significant tumor regression without obvious toxicity. Further
mechanistic studies revealed that 6f could influence the translocation of PKM2 into
nucleus, as well as induction of apoptosis and autophagy of A375 cells. More
importantly, compound 6f significantly inhibited migration of A375 cells in a
concentration-dependent manner. Collectively, 6f may serve as a lead compound in
the development of potent PKM2 activators for cancer therapy.
Keywords: PKM2; Structure-Activity Relationships; Nuclear Translocation;
Apoptosis; Autophagy
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1. Introduction
Cancer is currently the leading cause of death globally [1-3]. Over the past decades,
cancer therapeutic drugs via various anti-cancer mechanisms are approved [4-6].
Recently, targeting cancer metabolism has become a valid strategy in the development
of new tumor treatments [7-9]. In 2017, FDA approved Enasidenib (AG-221), an
inhibitor of mutant isocitrate dehydrogenase 2 (IDH2) protein, for the treatment of
relapsed or refractory acute myeloid leukemia [10].
In addition to IDH, the pyruvate kinase M2 (PKM2) has also emerged as a critical
regulator in cancer cell metabolism. In the 1920s, Otto Warburg found that most
cancer cells produce energy by a high rate of glycolysis followed by lactic acid
fermentation in the cytoplasm, despite the availability of oxygen, known as Warburg
Effect [11]. Pyruvate kinase (PK) regulates the final rate-limiting step of glycolysis
and converts phosphoenolpyruvate (PEP) to pyruvate while phosphorylating ADP to
ATP. Although, there are four isoforms of PK in mammalian cells [12-13], named
PKL, PKR, PKM1 and PKM2, only PKM2 is necessary for cancer metabolism and
tumor growth [14-19]. This finding is further supported by the research that when
PKM2 was replaced with PKM1, the constitutively active PKM1 delayed xenograft
tumor growth [20]. Moreover, PKM1 as a fully active tetramer acts only to maximize
the efficiency of glycolysis. Unlike PKM1, PKM2 acts as a key regulator in
maintaining the balance between the energy needs (tetramer) and building block needs
for increasing biomass (dimer) [21-23]. It has been demonstrated that restoring the
activity of PKM2 (dimer) to PKM1-like levels (tetramer) may cause cancer cells to
revert to a metabolic state like that of normal cells [24]. Therefore, fixation of PKM2
in its active tetrameric form by activators may have the therapeutic potential for the
treatment of cancer.
Up till now, several PKM2 activators have been reported [25-37]. In 2010, two
PKM2 activators N,N’-diarylsulfonamide (1) [27] and thieno[3,2-b]pyrrole[3,2-d]-
pyridazinone (2) [29] were first reported (Fig. 1). Structural studies revealed that
these two activators bind PKM2 at the subunit interaction interface, which is different
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from that of the endogenous activator fructose-1,6-bisphosphate (FBP) [26]. In
addition, another interesting scaffold quinoline sulfonamide 3, which adopted a
unique and different binding mode from compound 1 or 2, was also discovered by
Dang’s group [31]. In spite of these discoveries, none of these compounds had an
inhibitory effect on tumor cell proliferation, unless the tumor cell assay was
performed under hypoxic condition [26, 30] or in models where non-essential amino
acids such as serine were stripped [21, 31]. More recently, while our paper was in
preparation, Chen and co-workers reported a natural product micheliolide 4 (MCL)
which could irreversibly activate PKM2 and suppress the growth of leukemia cells
through reducing its nuclear translocation [38]. Our groups recently have also
reported
a
novel 7-azaindole derivative containing pyridin-3-ylmethyl
dithiocarbamate moiety 5 as a potent PKM2 activator, which was obtained by
screening our in-house compound library [39]. Interestingly, this series of activators
could strongly inhibit cellular growth in tumor cell lines, while maintaining low
toxicities towards non-cancer cell lines. Moreover, compound 5 has high selectivity
towards PKM2 versus other PK isoforms, such as PKM1 and PKR. In this work, we
report the optimization of compound 5 and investigation of its unique mechanism in
regulating PKM2 nuclear translocation. Our efforts led to the discovery of compound
6f as a promising PKM2 activator with both in vitro and in vivo antitumor activity.
Fig. 1. Structures of representative PKM2 activators
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2. Results and Discussion
2.1. Optimization strategy
In order to understand the binding mechanism for guidance of optimization, the
molecular docking of compound 5 with PKM2 was performed using the co-crystal
structure of 3 (also called NZT, PDB:4G1N), as we anticipate these two molecules
may share a similar binding mode [31, 39]. As shown in Fig. 2, compound 5 could
align well with NZT, and the azaindole moiety is caged among residues Phe26,
Leu27, Met30 and Tyr390 from chain A. The pyridinyl group is packed between
residues Phe26 and Leu 394 in chain B. Moreover, the 7-nitrogen in the azaindole
ring forms a hydrogen bond with Tyr390A. The carbonyl group in the compound is
found to interact with the side chain of Lys311B through hydrogen bonding, which
highly simulates the function of the same group of NZT. Furthermore, there is a π-π
staking interaction between the 7-azaindole ring and Phe26A.
We had previously demonstrated that the pyridin-3-ylmethanamine moiety provides
several advantages such as improved PKM2 potency (Fig. 2A) and selectivity [32,
39]. Therefore, we elected to keep this core structure intact. In addition, the docked
model showed that there seems little space for the modification on the
pyridin-3-ylmethanamine ring (Fig. 2B). Based on the docking results and the
preliminary SAR from HTS, the optimization for lead compound 5 was focused on
the azaindole ring of the molecule and was divided into three parts.
Substitution of 1-nitrogen of 7-azaindole ring
Although, 1-nitrogen of 7-azaindole ring forms a hydrogen bond with Ala388 (Fig.
2A), we believe that this hydrogen bond interaction is dispensable, and there are some
room here for substituent groups. The modified compounds may be still active when
the interaction is blocked by the appropriate groups.
A
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F26
Y390
L353
A388
K311
B
Q393
E397
Fig. 2. A. Predicted binding mode of compound 5 with PKM2 (cartoon). NZT is shown in green
sticks and compound 5 is shown in pink sticks. Key residues are shown in purple sticks. Yellow
dotted lines are hydrogen bond interactions; B. Possible clash residues in red circle.
Introduction of substitutions on 7-azaindole ring
The docking results revealed that there are some spaces inside the pocket formed
between PKM2 and the 7-azaindole scaffold, and the inner pocket might fit a bulkier
group for enhanced hydrophobic interactions. We therefore planned to introduce
substitutions, especially hydrophobic groups, to the 4th-6th position of 7-azaindole ring
for increased binding affinity.
Replacing 7-azaindole ring with 7-azaindole-like heterocyclic rings
We explored some 7-azaindole-like heterocyclic rings that may show higher
binding potency to PKM2 than 7-azaindole.
2.2. SAR investigation and anti-proliferation activities
All the synthesized compounds were firstly evaluated for the cell-free PKM2
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activation activity via a previously reported assay [39]. These compounds with higher
PKM2 activation activity were further examined for their anticancer activities.
Various substitutions were introduced onto the 1-nitrogen of 7-azaindole ring to
form the corresponding compounds 6a-6f. As shown in Table 1, all the target
compounds exhibited moderate to excellent PKM2 activation activity (2.50 - 4.56 µM)
except compound 6e. This result shows that our initial prediction is reasonable,
though the PKM2 activation potency is slightly decreased comparing with the lead 5
(AC50 = 1.0 µM). 1-Phenyl substituted compound (6d, AC50 = 2.50 µM) exhibited
higher activity than 1-thiophene substituted compound (6e, AC50 > 20 µM), which
demonstrated that different substituents on the 1st position had different effects on the
activity. Notably, when the 1-methyl group of 6a was replaced by 1,1-difluoroethyl,
the corresponding compound 6f (R1 = -CH2CF2H) not only exhibited better activity
(AC50 = 2.67 µM), but also showed the highest activation (149%). Therefore,
compound 6f was also docked into the above model (Fig. 3), as can be seen,
1,1-difluoroethyl group could be well adapted in the binding pocket without large
steric clashes. This preliminary exploration resulted in some satisfying outcome and
verified that the hydrogen bond interaction with A388 is not absolutely required.
Fig. 3. Predicted binding mode of compound 6f (orange sticks) with PKM2 (cartoon). Key
residues are shown in purple sticks. Yellow dotted lines are hydrogen bond interactions;
For the substitution modification on 7-azaindole ring, the 4 or 5-halogen substituted
analogues (6h, AC50 = 0.61 µM; 6l, AC50 = 0.18 µM) greatly enhanced the activity of
PKM2. The possible reason may be the increased hydrophobic interactions based on
the docking results. Moreover, the electron-withdrawing effect of the halogen may
also contribute to the stronger hydrogen bond interaction between 7-nitrogen and
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Y390. Unfortunately, when 7-azaindole was substituted with other hydrophobic
groups at the 4th or 5th position (6i-6k, 6m), the activity was significant decreased,
suggesting that large groups on these positions had detrimental effect. 6-Substituted
7-azaindole derivatives with halogen or methyl (6n, 6o) led to complete loss of
binding affinity with PKM2 (AC50 > 20 µM) in comparison with the unsubstituted
derivative 5.
Table 1. In vitro PKM2 activation activity of 7-azaindole derivatives
a
AC50
/
Compd.
R1
R2
Max (%)
µMb
5
6a
6b
6c
6d
6e
6f
H
H
H
1.00
4.35
3.93
4.56
2.50
NAc
2.67
1.14
0.61
NA
113
114
93
Me
cyclo-Bu
H
cyclo-Pr
H
42
1-Ph
H
77
2-Thiophenyl
H
NA
149
122
83
-CH2CF2H
H
6g
6h
6i
H
H
H
H
H
H
H
H
H
4-Cl
4-Br
4- cyclo-Pr
4-Ethyne
4-Ph
5-Cl
5-Cl-Ph
6-Cl
6-Me
NA
24
6j
>20
2.17
0.18
> 20
NA
6k
6l
28
71
6m
6n
6o
NA
NA
NA
NA
a AC50 = half maximal active concentration; b Data are expressed as the mean of at least two determinations; c NA
= Not active on PKM2;
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In order to further explore the structure-activity relationships, we moved 7-nitrogen
from compound 5 to 8-nitrogen to afford compound 15a and replaced 8-carbon in
compound 5 with nitrogen atom to obtain compound 15b. Comparing with compound
5 (Table 2), the PKM2 activation potency of 15a was completely abolished (AC50 >
20 µM). However, the PKM2 activation potency of 15b was greatly improved by
about 4 to 5 times (AC50 = 0.22 µM), though the maximum response was moderate
(Max = 44%). Encouraged by the excellent PKM2 activation activity of 15b, we then
synthesized compound 15c by introducing phenyl onto 2nd-position of 15b. We found
that 15c (AC50 = 0.45 µM, Max = 148%) did not only maintain the activation potency,
but also significantly increase the maximal activity. The docking results showed that
2-phenyl could make additional π-π staking interactions with Phe26B (Figure S1).
These results suggested that the nitrogen at 7-position is very important for activity
and the introduction of substitute at 2-position may improve the maximal response
activity.
Table 2. In vitro PKM2 activation activity of heterocycle derivatives
AC50/
µMa
Max
(%)
Compd.
X
Y
R
5
N
C
N
N
C
N
N
N
H
H
1.00
>20
0.22
0.45
113
23
15a
15b
15c
H
44
Ph
148
a Data are expressed as the mean of at least two determinations;
Compounds with higher PKM2 activation activity were subsequently evaluated
for their inhibitory activities against PKM2 high expression cell lines, H1299 (human
lung cancer), HCT116 (human colon cancer) and A375 (human melanoma caner). As
illustrated in Table 3, all of them showed robust tumor inhibition, especially on A375
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cell line, and their IC50 values ranged from 3.86 to 0.21 µM. Several 7-azaindole
analogues (6a, 6c, 6f and 6g) were more potent than the lead compound 5. Among
them, 6f was the best one with IC50 values of 2.29, 0.71, and 0.28 µM against H1299,
HCT116 and A375 cell lines, respectively. Comparing the anti-proliferation activities
of compounds 5 with 15b, it is clear that the pyrazolopyridazine scaffold was also a
good starting point for further optimization.
Table 3. Inhibition of cell growth of H1299, HCT116 and A375 cell lines
Antiproliferation / IC50 (µM)a
Compd.
H1299
3.68 ± 0.40
2.09 ± 0.07
4.20 ± 1.68
9.51 ± 0.41
10.03 ± 1.53
2.28 ± 0.08
2.50 ± 0.35
>10
HCT116
A375
5
6a
6b
6c
3.09 ± 0.97
0.94 ± 0.11
3.52 ± 0.25
0.84 ± 0.10
5.36 ± 0.47
0.71 ± 0.01
2.06 ± 0.45
4.90 ± 1.13
6.16 ± 0.51
1.52 ± 0.18
9.08 ± 0.69
1.03 ± 0.07
0.29 ± 0.10
1.34 ± 0.01
0.21 ± 0.01
3.86 ±1.58
0.28 ± 0.09
0.41 ± 0.08
1.99 ± 0.40
3.38 ± 0.24
0.33 ± 0.08
2.77 ± 0.36
6d
6f
6g
6h
6l
8.87 ± 0.52
1.76 ± 0.91
>10
15b
15c
a Data are average of three independent experimental measurements and expressed as Mean ±SD
Finally, we selected 6f for further investigation owing to its high PKM2
activation potency (highest activation) and excellent anti-proliferation activity. First,
the toxicities of 6f on non-cancer cells were tested. As shown in Table 4, it showed
low toxicities on Helf (human embryonic lung fibroblast) and C2C12 (mouse
myoblast) cell lines. Second, due to the many structural similarities between
compound 6f and B-raf kinase inhibitor vemurafenib, 6f was also tested on the effect
against serine-threonine kinase B-raf in a single-point binding at 10 µM (Table 4). It
displayed low inhibition (41%) compared with vemurafenib, confirming that
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compound 6f was not targeting B-raf. Finally, based on its high PKM2 potency,
excellent inhibition of tumor cell growth and low toxicity towards normal cells,
compound 6f was further profiled in vivo.
Table 4. The toxicity on non-cancer cell lines and inhibition activities against B-Raf
kinases of 6f.
IC50/µMa
10 µM
Compd.
inhibition rate
on B-Raf (%)
Helf
C2C12
>20 (inhibition rate
>20 (inhibition rate
41
10% at 20 µM）
46% at 20 µM）
6f
NDb
ND
95
Vemurafenib (VE)
a Data are obtained for at least two determinations; b Not determined.
2.3. Preliminary in vivo antitumor efficacy of 6f in the A375 xenograft model
In order to assess the in vivo antitumor efficacy of compound 6f, an A375 xenograft
nude mice model was established, with vemurafenib as the positive control
(Vemurafenib: an approved drug for the management of late-stage or unresectable
melanoma). Tumor-bearing mice were treated orally with 6f at doses of 50 mg/kg (ig,
bid) for 14 consecutive days, meanwhile vemurafenib was administered at 12.5 mg/kg
(ig, bid). As shown in Fig. 4A, 6f could significantly suppress tumor growth. In
addition, no significant body weight loss was observed in the groups, indicating that
this treatment was well tolerated (Fig. 4B). These primary results revealed that 6f
exhibited significant anticancer activity with relatively low toxicity.
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Fig. 4. 6f inhibits tumor growth in vivo. (A) Tumor growth curves in mice receiving the respective
treatments by tumor volume. (B) Average body weight of mice. **, P<0.01; ***, P<0.001.
2.4. Synthetic Chemistry
Scheme 1. General synthetic routes for compounds 6 and 15. Reagents and conditions: (a) (HCHO)n,
o
CF3COOH·(iPr)2NH, DMF, 90 C, 24 h; (b) 3-(aminomethyl)pyridine, CS2, TEA, DMF, rt, 6 h; (c) AlCl3, DCM,
then NaOH(1N).
All the new derivatives were synthesized following the general synthetic routes
shown in Scheme 1. Acetylation of substituted 7-azaindole derivatives (7) gave the
3-acetyl-7-azaindole derivatives (8). Treatment of acetoxylated heterocyles (8 and 16)
with paraformaldehyde catalyzed by diisopropylammonium trifluoroacetate afforded
the intermediates acryloyl heterocyles (9 and 17), which were further treated with
dithiocarbamates generated from CS2 and 3-(aminomethyl)pyridine to yield the
corresponding target compounds (6 and 15), respectively. Therefore, the key steps
were the preparations of starting materials of substituted 7-azaindole analogues (7a-7o)
and acetoxylated heterocycles (16a-16c).
Preparation of 7a-7o
1-Substituted 7-azaindole 7a-7f could be achieved via a SN2 or Ulman C-N
11

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coupling reactions (Scheme 2A). The formation of 7f could not occur via the
Friedel-Crafts reaction due to the influence of 1,1-difluoroethyl at 1-N position.
Therefore, 8f was prepared from the N-alkylation of acetylated 7-azaindole 5b
(Scheme 2A). Alternatively, 4- or 5- substituted derivatives（7i-7k）were prepared
through Sukuzi or Sonogashira reactions of commercially available 4-Br-7-azaindole
7h (or 5-Cl-7-azaindole, 7l) with corresponding boronic acid or TMS protected
acetylene (Scheme 2B). For the synthesis of 7-azaindole scaffold 7n and 7o,
7-azaindole 5a was oxidized by m-CPBA to give 7-azaindole N-oxide 13a, which was
treated with acid halides to give the intermediate 13b. Acyl groups are readily
removed under basic conditions to get 7n [40]. Protection of indole NH as
sulfonamide (7n to 14a), followed by palladium catalyzed Negishi cross-coupling
with MeZnCl was carried out to introduce the 6-methyl group in 7-azaindole (14b)
[41]. After the protecting group was removed, intermediate 7o was obtained smoothly
(Scheme 2C). Other substituted 7-azaindoles were commercially available.
Preparation of 16a-16c
The synthetic routes for the acetylated heterocycles 16a-16c are depicted in
Scheme 3 according to the published papers [42-43]. For the construction of
pyrazolo[1,5-a]pyridinyl 16a, 1-aminopyridinium iodide (19a) was reacted with
3-butyn-2-one through 1,3-dipolar cycloaddition under basic conditions to give the
target molecule. Alternatively, pyridazine (18) was treated with hydroxylamine
O-sulfonic acid to afford the intermediate 19b, which was further converted into
16b-16c by the same routes.
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A
O
(a)
(b)
N
R1
7a: R1 = Me,
N
N
N
H
7b: R1 = cyclo-Bu
(d)
O
5b
N
N
N
N
N
N
H
5a
7c
CH2CF2H
8f
(a)
(d)
N
N
(c)
CH2CF2H
N
R1
N
7f
7d: R1 = Ph
7e R1 = 2-Thiophene
Br
B
(f)
(e)
(g)
N
N
Ts
N
N
N
N
H
Ts
7i
11b
11a
TMS
Br
(i)
(h)
N
N
N
N
N
N
H
H
H
7j
12
7h
Ph
(j)
Cl
N
N
H
7k
Cl
(j)
N
N
H
N
N
H
7l
7m
C
(g)
(l)
(k)
N
N
Cl
N
Cl
N
N
N
O
H
H
O
Ph
13b
13a
7n
(m)
N
N
H
5a
(o)
(n)
N
S
Cl
N
O
O
N
S
Me
N
O
O
N
Me
N
H
Ph
Ph
14b
14a
7o
Scheme 2. Synthesis of key intermediates 7a-7o and 8f. Reagents and conditions: (a) NaH, THF, 0℃ or Cs2CO3,
DMF, 70℃; (b) cyclo-PrB(OH)2, Cu(OAc)2, Bipy, Na2CO3, DCE; (c) PhI or 2-I-thiophene, CuI, K3PO4,
Ethane-1,2-diamine, 1,4-dioxane, reflux; (d) AlCl3, DCM, then NaOH(1N); (e) TosCl, TBHS, NaOH(aq), DCM; (f)
13

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cyclo-PrB(OH)2, Pd(OAc)2, X-Phos, Cs2CO3, toluene/H2O; (g) 4N NaOH, MeOH; (h) TMS−ethyne, CuI,
Pd(PPh3)Cl2, TEA; (i) 1N NaOH, MeOH; (j) Pd(PPh3)4, Cs2CO3, dppf, 1,4-dioxane/H2O, ArB(OH)2; (k) m-CPBA,
EtOAc; (l) PhCOCl, HMDS, toluene; (m) PhSO2Cl, NaOH(aq), TBHS, DCM; (n) Pd(PPh3)4, MeZnCl, THF, 80℃;
(o) 2N NaOH(aq), EtOH, reflux.
Scheme 3. Synthesis of key intermediates 16a-16c. Reagents and conditions: (a) KOH, K2CO3, DMSO; (b)
KHCO3, KOH, H2O.
2.5. Compound 6f reduces the nuclear translocation of PKM2
Recently, Yang et al. has demonstrated that the nuclear function of PKM2
contributes to cell proliferation via EGFR activation pathway [44-46]. Furthermore,
when PKM2 fails to translocate to the nucleus, EGF-promoted Warburg effect could
be impaired, suggesting that the antitumor activity of some PKM2 activators may also
arise from inhibition of PKM2’s nuclear function [47-48]. Sizemore et al. also found
that PKM2 could regulate homologous recombination mediated DNA double-strand
break repair through its nuclear accumulation [49]. More recently, Li’s group found
that compound 5 (MCL) could activate PKM2, inhibit the acetylation of lysine433,
and finally influence the translocation of PKM2 into nucleus [38]. To demonstrate
whether compound 6f could inhibit the translocation of PKM2, an
immunofluorescence assay was performed with compound NZT as the control. Fig.
5A revealed that only 6f treatment for 24 h resulted in retardation of the nuclear
accumulation of PKM2 in A375 cells in a concentration-dependent manner. The
percentage of immunofluorescence intensity (cytoplasma) in 6f 2.0 µM group was
significantly lower than that in CTR group (Fig. 5B, p < 0.05). Because most cells
died after treatment with 4.0 µM 6f for 24 h, a 12 h immunofluorescence assay was
performed. As shown in Figure S2, an obvious reduction of the nuclear translocation
of PKM2 was also observed after treatment with 6f. All of these results demonstrated
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that 6f could effectively reduce PKM2 nuclear translocation, and further suppress
tumorigenesis. Inhibition of PKM2 nuclear translocation may be one of the key
reasons why 6f not only exhibited high PKM2 activity, but also showed robust
anti-proliferation effect on tumor cells both in vitro and in vivo (Other unknown
activities of 6f may also be contributing to the effects on tumor growth.).
A
Hochest
Cytoplasma
Merge
CTR
NZT
2.0 µM
6f
1.0 µM
6f
2.0 µM
B
15

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Fig. 5. 6f inhibits PKM2 translocation into the nucleus. (A) High content screening
immunofluorescence microscopy was performed to analyze localization of PKM2 in A375 cells
after treatment with NZT or 6f for 24 h; PKM2 was labeled with antibody, DNA was visualized
with Hoechst (blue) (B) Average immunofluorescence intensity; a) Intensity in nucleus; b)
Intensity in cytoplasm; c) Intensity of cells. *, P<0.05.
2.6. Compound 6f could induce cellular apoptosis and autophagy
Since PKM2 regulates the apoptotic activity and inhibits autophagy [50-52],
which contribute to the development of malignant cancer, compound 6f might induce
cellular apoptosis and promote autophagy. The annexin-V/PI double staining assay
was applied to determine the number and stage of apoptotic A375 cells after treated
with 6f using flow cytometry. As shown in Fig. 6A, treatment of A375 cells with
compound 6f for 24 h led to a concentration-dependent apoptosis, and the percentages
of apoptotic cells were 12.0%, 21.0% and 28.3%, respectively. Specially, when
treated with 6f at 2.0 and 4.0 µM, the numbers of apoptotic cells were remarkably
higher than that of the control group (P < 0.05). Since the transition of LC3-I to
LC3-II is an imperative step in autophagy and the number of LC3B puncta represents
the number of autophagosomes, we utilized immunofluorescence staining (high
content screening) to analyze whether autophagy was involved in the process of cell
death induced by 6f (Fig. 6B). Treatment of A375 cells with 6f resulted in substantial
increase in LC3B fluorescence intensities compared to DMSO control (Fig. 6C),
consistent with other related indicators, such as LC3B spot area. Thus, it
demonstrated that 6f could also promote cellular autophagy in
a
concentration-dependent manner.
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2.7. Effects of compound 6f on tumor cell migration
Recent studies have shown that PKM2 is associated with the migration and evasion
of cancer cells [52]. The effects of compound 6f on tumor cell migration were
assessed using living cell high content screening workstations. As displayed in Fig.
7A and 7B, after treatment with compound 6f for 24 h, both the migration speed and
accumulated distance of A375 cells were significantly inhibited in a
concentration-dependent manner. These results demonstrated that 6f could efficiently
exhibit antimetastatic potential against cancer cells. Meanwhile, the proliferation rate
of A375 cells could also be determined, which is in accordance with the previously
determined IC50 (Fig. 7C).
3. Conclusion
A series of derivatives of 5 were designed and synthesized to optimize the
7-azaindole moiety. Several compounds were found to show potent PKM2 activation
activity and high inhibitory activity on tumor cell lines. A representative compound 6f
was selected for further study. Compound 6f exhibited high proliferation inhibitory
activity in vitro, and significant anticancer activity in vivo. The preliminary
mechanism studies showed that 6f effectively inhibited the translocation of PKM2
into the nucleus, which may be one of the key mechanisms for its role in inhibiting
proliferation. The results also showed that compound 6f could induce cellular
apoptosis, promote autophagy, and strongly inhibit the migration of A375 cells in a
concentration-dependent manner. The pharmacokinetics and safety studies of 6f are
currently underway.
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A
CTR
1.0 µM
2.0 µM
4.0 µM
B
Hochest
Cytoplasma
Merge
CTR
NZT
2.0 µM
6f
2.0 µM
C
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Fig. 6. (A) 6f induced apoptosis in A375 cells. A375 cells were harvested after treatment with
various concentrations of 6f or DMSO for 24 h; (B) Immunofluorescence staining of A375 cells
showing the appearance of autophagosomes. Immunofluorescence staining of A375 cells treated
with DSMO or 6f for 24 h, LC3B was labeled with antibody (green), and DNA was visualized
with Hoechst (blue); (C) Average immunofluorescence intensity or areas; a) LC3B intensity in
cytoplasma; b) LC3B spot areas;. *, P<0.05; **, P<0.01.
Fig. 7. (A) The migration speed of A375 cells after treatment with 6f; (B) The accumulated
distance of A375 cells after treatment with 6f; (C) Proliferation rates. *, P<0.05; **, P<0.01; ***,
P<0.001.
4. Experimental section
4.1. General
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Unless otherwise stated, the chemical starting materials were purchased from J&K,
TCI, Innochem, Ark or Arcos and used without purification. The metal-catalysts were
purchased from J&K, and solvents were used without pre-purification. All of the
reactions were monitored by thin layer chromatography (TLC) on GF254 silica gel
plates. Melting points were determined on X4 microscope and measured as
1
uncorrected values. H NMR and 13C NMR spectra were recorded on Bruker
AVANCE Ⅲ 400 MHz and 100 MHz spectrometer respectively in CDCl3 or
DMSO-d6. NMR chemical shifts are reported as values (ppm) relative to internal
tetramethylsilane and splitting patterns are designated as: s =singlet, d = doublet, t =
triplet, q = quartet, m = multiplet. HRMS of the products were carried out on Brucker
Apex IV FTMS (Sample (about 0.1 mg) was dissolved in appropriate solvents
(CH3CN or MeOH) and detected the positive cationic signal.).
Cell Lines and Culture: H1299, HCT116 and A375 were purchased from ATCC.
All the cell lines were maintained in appropriate medium as the manufacturer
suggested. HCT116 and A375 were cultured in DMEM (H1299, RPMI 1640) medium
(Sigma-Aldrich) supplemented with 10% fetal bovine serum (Gibco BRL, USA), in a
humidified incubator at 37 °C and 5% CO2/95% air.
4.2. General procedure for the synthesis of compounds 6a-6o and 15a-15c
A solution of heteroaryl ketones (5.0 mmol), paraformaldehyde (10.0 mmol) with
diisopropylammonium trifluoroacetate (5.0 mmol) in DMF (15 mL) was heated to
90℃ for 24 h. After the completion of the reaction monitored by TLC (PE/EA = 8/1),
the reaction mixture was cooled down to room temperature and water (40 mL) was
added. The mixture was then extracted with EtOAc (20 mL×3), and washed with
saturated NaCl (aq) for three times. The organic phase was dried with anhydride
sodium sulfate and concentrated under reduced vacuum. The crude product (9a-9o,
17a-17c) was directly used in next step without further purification.
To a solution of pyridin-3-ylmethanamine (5.0 mmol) in DMF (15 mL) was added
Et3N (5.1 mmol, 0.71 mL) and CS2 (5.0 mmol, 0.30 mL). The reaction mixture was
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continuously stirred for 15 min, then the crude intermediate from 9a-9o (or 17a-17c)
were added respectively, and the resulting mixture was stirred for 12 h at room
temperature. Water (50 mL) was added and the mixture was extracted with ethyl
acetate (15 mL×3). The combined organic phase was washed with saturated NaCl (aq)
for three times, dried over anhydride sodium sulfate and concentrated under reduced
vacuum. The residue was further purified by column chromatography on silica gel
using PE/EA as eluent to afford 6a-6o and 15a-15c. Further recrystallized from hot
EtOAc/PET to give crystals of the target compounds.
4.2.1.
3-(1-Methyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-3-oxopropyl
(pyridin-3-ylmethyl)carbamodithioate (6a)
1
White solid, m. p. 150.9-151.8℃, yield: 47%, two steps. H NMR (400 MHz,
DMSO-d6) δ 10.47 (t, J = 5.5 Hz, 1H), 8.57 (s, 1H), 8.54 – 8.41 (m, 3H), 8.38 (dd, J =
4.7, 1.4 Hz, 1H), 7.69 (d, J = 7.8 Hz, 1H), 7.36 (dd, J = 7.7, 4.9 Hz, 1H), 7.30 (dd, J =
7.8, 4.7 Hz, 1H), 4.85 (d, J = 5.6 Hz, 2H), 3.87 (s, 3H), 3.54 (t, J = 6.8 Hz, 2H), 3.29
13
(t, J = 6.9 Hz, 2H). C NMR (100 MHz, DMSO-d6) δ 197.4, 192.6, 149.1, 148.4,
148.0, 144.1, 137.8, 135.5, 133.0, 129.7, 123.5, 118.4, 117.9, 113.2, 47.1, 38.2, 31.5,
29.2. HRMS m/z (ESI) calcd for C18H19N4OS2 (M+H)+: 371.0995, found 371.1004.
4.2.2.
3-(1-Cyclobutyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-3-oxopropy
l(pyridin-3-ylmethyl)carbamodithioate (6b)
1
White solid, m. p. 141.3-142.4℃, yield: 32%, two steps. H NMR (400 MHz,
DMSO-d6) δ 10.50 (t, J = 5.5 Hz, 1H), 8.84 (s, 1H), 8.53 (d, J = 1.6 Hz, 1H), 8.51 –
8.43 (m, 2H), 8.36 (dd, J = 4.7, 1.5 Hz, 1H), 7.70 (d, J = 7.8 Hz, 1H), 7.37 (dd, J =
7.7, 4.8 Hz, 1H), 7.29 (dd, J = 7.8, 4.7 Hz, 1H), 5.33 (p, J = 8.8 Hz, 1H), 4.87 (d, J =
5.5 Hz, 2H), 3.57 (t, J = 6.8 Hz, 2H), 3.36 (t, J = 6.8 Hz, 2H), 2.86 – 2.55 (m, 2H),
2.46 (dd, J = 6.8, 3.5 Hz, 2H), 2.13 – 1.65 (m, 2H). 13C NMR (100 MHz, DMSO-d6) δ
198.0, 193.4, 149.6, 148.9, 147.9, 144.4, 136.0, 135.1, 133.4, 130.4, 124.0, 119.1,
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118.7, 114.3, 48.7, 47.6, 38.9, 30.6, 29.7, 15.0. HRMS m/z (ESI) calcd for
C21H23N4OS2 (M+H)+: 411.1308, found 411.1311.
4.2.3.
3-(1-Cyclopropyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-3-oxopropyl
(pyridin-3-ylmethyl)carbamodithioate (6c)
1
White solid, m. p. 147.7-149.1℃, yield: 49%, two steps. H NMR (400 MHz,
DMSO-d6) δ 10.49 (t, J = 5.5 Hz, 1H), 8.52 (d, J = 1.7 Hz, 1H), 8.50 (s, 1H), 8.49 –
8.43 (m, 2H), 8.38 (dd, J = 4.7, 1.6 Hz, 1H), 7.69 (dd, J = 6.0, 1.8 Hz, 1H), 7.36 (dd, J
= 7.8, 4.8 Hz, 1H), 7.30 (dd, J = 7.9, 4.7 Hz, 1H), 4.86 (d, J = 5.6 Hz, 2H), 3.73 (dq, J
= 7.4, 4.1 Hz, 1H), 3.55 (dd, J = 12.9, 5.9 Hz, 2H), 3.30 (t, J = 6.9 Hz, 2H), 1.34 –
13
0.87 (m, 4H). C NMR (100 MHz, DMSO-d6) δ 197.5, 192.9, 149.1, 148.9, 148.4,
144.2, 136.0, 135.5, 133.0, 129.8, 123.5, 118.8, 118.5, 113.2, 47.1, 38.3, 29.3, 27.3,
6.1. HRMS m/z (ESI) calcd for C20H21N4OS2 (M+H)+: 397.1151, found 397.1159.
4.2.4.
3-Oxo-3-(1-phenyl-1H-pyrrolo[2,3-b]pyridin-3-yl)propyl
(pyridin-3-ylmethyl)carbamodithioate (6d)
White solid, m. p. 119.1-121.1℃, yield: 54%, two steps. 1H NMR (400 MHz, CDCl3)
δ 8.93 (s, 1H), 8.62 (d, J = 7.8 Hz, 1H), 8.43 (m, 3H), 8.24 (s, 1H), 7.72 (d, J = 7.8 Hz,
2H), 7.64 (d, J = 7.6 Hz, 1H), 7.54 (t, J = 7.7 Hz, 2H), 7.41 (t, J = 7.4 Hz, 1H), 7.27 –
7.22 (m, 1H), 7.22 – 7.09 (m, 1H), 4.90 (d, J = 5.1 Hz, 2H), 3.66 (t, J = 6.4 Hz, 2H),
13
3.40 (t, J = 6.5 Hz, 2H). C NMR (100 MHz, CDCl3) δ 198.8, 193.7, 149.3, 148.9,
148.1, 145.2, 137.1, 136.2, 134.5, 132.5, 131.2, 129.7, 127.9, 124.6, 123.7, 119.4,
119.3, 115.9, 48.1, 39.6, 29.9. HRMS m/z (ESI) calcd for C23H21N4OS2 (M+H)+:
433.1151, found 433.1155.
4.2.5.
3-Oxo-3-(1-(thiophen-2-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)propyl (pyridin-3-ylmethyl)
carbamodithioate (6e)
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1
Light yellow solid, m. p. 157.2-158.5℃, yield: 22%, two steps. H NMR (400 MHz,
DMSO-d6) δ 10.51 (t, J = 5.4 Hz, 1H), 8.53 (s, 1H), 8.47 (t, J = 3.6 Hz, 2H), 8.25 (d, J
= 3.8 Hz, 1H), 8.13 (d, J = 7.7 Hz, 1H), 7.94 (d, J = 4.3 Hz, 1H), 7.70 (d, J = 7.8 Hz,
1H), 7.60 (d, J = 4.3 Hz, 1H), 7.36 (dd, J = 7.7, 4.8 Hz, 1H), 7.30 (dd, J = 7.8, 4.8 Hz,
1H), 6.86 (d, J = 3.8 Hz, 1H), 4.86 (d, J = 5.5 Hz, 2H), 3.54 (t, J = 6.7 Hz, 2H), 3.47 –
13
3.29 (m, 2H). C NMR (100 MHz, DMSO-d6) δ 197.2 191.2, 149.1, 148.4, 146.5,
145.9, 143.7, 135.6, 135.5, 132.9, 132.8, 129.9, 127.2, 123.5, 121.5, 118.0, 114.8,
104.6, 47.1, 37.7, 29.1. HRMS m/z (ESI) calcd for C21H19N4OS3 (M+H)+: 439.0716,
found 439.0721.
4.2.6.
3-(1-(2,2-Difluoroethyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)-3-oxopropyl (pyridin-3-
ylmethyl)carbamodithioate (6f)
1
White solid, m. p. 135.3-136.6℃, yield: 43%, two steps. H NMR (400 MHz,
DMSO-d6) δ 10.49 (t, J = 5.3 Hz, 1H), 8.60 (s, 1H), 8.49 (dd, J = 14.1, 6.7 Hz, 3H),
8.41 (d, J = 4.6 Hz, 1H), 7.70 (d, J = 7.7 Hz, 1H), 7.49 – 7.28 (m, 2H), 6.51 (m, 1H),
5.00 – 4.74 (m, 4H), 3.56 (t, J = 6.7 Hz, 2H), 3.35 (d, J = 3.8 Hz, 2H). 13C NMR (100
MHz, DMSO-d6) δ 197.4, 192.9, 149.1, 148.4, 147.9, 144.5, 137.1, 135.5, 132.9,
130.1, 123.5, 118.9, 117.6, 114.4, 113.7, 47.1, 45.8 (t, JC-F = 26 Hz), 38.5, 29.0.
HRMS m/z (ESI) calcd for C19H19F2N4OS2 (M+H)+: 421.0963, found 421.0965.
4.2.7.
3-(4-Chloro-1H-pyrrolo[2,3-b]pyridin-3-yl)-3-oxopropyl
(pyridin-3-ylmethyl)carbamodithioate (6g)
1
White solid, m. p. 158.9-160.0℃, yield: 26%, two steps. H NMR (400 MHz,
DMSO-d6) δ 12.84 (s, 1H), 10.55 (d, J = 45.4 Hz, 1H), 8.59 (s, 1H), 8.52 (s, 1H), 8.47
(d, J = 3.1 Hz, 1H), 8.24 (d, J = 4.4 Hz, 1H), 7.69 (d, J = 7.4 Hz, 1H), 7.43 – 7.33 (m,
1H), 7.30 (d, J = 5.1 Hz, 1H), 4.85 (d, J = 3.7 Hz, 2H), 3.53 (t, J = 6.0 Hz, 2H), 3.47 –
13
3.29 (m, 2H). C NMR (100 MHz, DMSO-d6) δ 197.6, 191.4, 150.5, 149.1, 148.5,
144.7, 136.0, 135.9, 135.5, 133.0, 123.6, 119.3, 115.4, 115.3, 47.1, 39.4, 29.4. HRMS
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m/z (ESI) calcd for C17H16ClN4OS2 (M+H)+: 391.0449, found 391.0450.
4.2.8.
3-(4-Bromo-1H-pyrrolo[2,3-b]pyridin-3-yl)-3-oxopropyl
(pyridin-3-ylmethyl)carbamodithioate (6h)
1
White solid, m. p. 165.7-167.0℃, yield: 30%, two steps. H NMR (400 MHz,
DMSO-d6) δ 12.79 (s, 1H), 10.47 (s, 1H), 8.51 (m, 3H), 8.14 (d, J = 5.0 Hz, 1H), 7.69
(d, J = 7.6 Hz, 1H), 7.48 (d, J = 5.0 Hz, 1H), 7.36 (dd, J = 7.3, 4.9 Hz, 1H), 4.86 (d, J
= 5.3 Hz, 2H), 3.53 (t, J = 6.6 Hz, 2H), 3.34 (t, J = 6.6 Hz, 2H). 13C NMR (100 MHz,
DMSO-d6) δ 197.6, 191.5, 150.0, 149.1, 148.4, 144.3, 135.7, 135.5, 133.0, 124.5,
123.5, 122.9, 117.5, 115.4, 47.1, 39.7, 29.4. HRMS m/z (ESI) calcd for
C17H16BrN4OS2 (M+H)+: 434.9943, found 434.9948.
4.2.9.
3-(4-Cyclopropyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-3-oxopropyl (pyridin-3-ylmethyl)
carbamodithioate (6i)
1
White solid, m. p. 177.5-178.2℃, yield: 18%, two steps. H NMR (400 MHz,
DMSO-d6) δ 12.47 (s, 1H), 10.46 (t, J = 5.2 Hz, 1H), 8.52 (s, 2H), 8.47 (d, J = 4.4 Hz,
1H), 8.13 (d, J = 5.0 Hz, 1H), 7.69 (d, J = 7.7 Hz, 1H), 7.36 (dd, J = 7.6, 4.8 Hz, 1H),
6.59 (d, J = 5.1 Hz, 1H), 4.86 (d, J = 5.4 Hz, 2H), 3.63 (dd, J = 8.8, 3.9 Hz, 1H), 3.54
(t, J = 6.6 Hz, 2H), 3.38 (t, J = 6.7 Hz, 2H), 1.08 (d, J = 8.0 Hz, 2H), 0.81 (d, J = 4.4
Hz, 2H). 13C NMR (100 MHz, DMSO-d6) δ 197.7, 192.6, 149.4, 149.1, 148.7, 148.4,
144.5, 135.9, 135.4, 132.9, 123.5, 116.8, 116.4, 111.3, 47.0, 38.9, 29.8, 13.1, 11.4.
HRMS m/z (ESI) calcd for C20H21N4OS2 (M+H)+: 397.1151, found 397.1158.
4.2.10.
3-(4-Ethynyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-3-oxopropyl
(pyridin-3-ylmethyl)carbamodithioate (6j)
1
White solid, m. p. 186.1-188.8℃, yield: 20%, two steps. H NMR (400 MHz,
DMSO-d6) δ 12.71 (s, 1H), 10.49 (s, 1H), 8.86 – 8.42 (m, 3H), 8.28 (s, 1H), 7.70 (s,
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1H), 7.33 (d, J = 24.5 Hz, 2H), 4.85 (s, 2H), 4.59 (s, 1H), 3.52 (s, 2H), 3.35 (s, 2H).
13C NMR (100 MHz, DMSO-d6) δ 197.6, 191.5, 149.7, 149.1, 148.4, 143.7, 135.5,
133.0, 123.5, 123.3, 122.8, 116.1, 115.7, 109.5, 88.4, 82.0, 47.1, 39.0, 29.3. HRMS
m/z (ESI) calcd for C19H17N4OS2 (M+H)+: 381.0838, found 381.0838.
4.2.11.
3-Oxo-3-(4-phenyl-1H-pyrrolo[2,3-b]pyridin-3-yl)propyl
(pyridin-3-ylmethyl)carbamodithioate (6k)
1
White solid, m. p. 204.5-205.7℃, yield: 30%, two steps. H NMR (400 MHz,
DMSO-d6) δ 12.63 (s, 1H), 10.46 (t, J = 5.2 Hz, 1H), 8.50 (d, J = 11.8 Hz, 3H), 8.36
(d, J = 4.6 Hz, 1H), 7.68 (d, J = 7.4 Hz, 1H), 7.35 (dt, J = 11.4, 6.5 Hz, 6H), 7.15 (d, J
13
= 4.8 Hz, 1H), 4.84 (d, J = 5.3 Hz, 2H), 3.36 (s, 2H), 3.17 (t, J = 6.7 Hz, 2H). C
NMR (100 MHz, DMSO-d6) δ 197.6, 191.5, 150.1, 149.1, 148.4, 144.1, 143.7, 140.0,
135.5, 134.6, 133.0, 128.5, 127.5, 123.5, 119.1, 116.5, 114.0, 47.1, 39.2, 29.7. HRMS
m/z (ESI) calcd for C23H21N4OS2 (M+H)+: 433.1151, found 433.1164.
4.2.12.
3-(5-Chloro-1H-pyrrolo[2,3-b]pyridin-3-yl)-3-oxopropyl
(pyridin-3-ylmethyl)carbamodithioate (6l)
1
White solid, m. p. 162.7-163.8℃, yield: 31%, two steps. H NMR (400 MHz,
DMSO-d6) δ 12.75 (s, 1H), 10.47 (s, 1H), 8.59 (s, 1H), 8.52 (s, 1H), 8.47 (d, J = 4.3
Hz, 1H), 8.43 (s, 1H), 8.33 (s, 1H), 7.69 (d, J = 7.5 Hz, 1H), 7.56 – 7.24 (m, 1H), 4.85
13
(s, 2H), 3.54 (t, J = 6.5 Hz, 2H), 3.32 (t, J = 6.5 Hz, 2H). C NMR (100 MHz,
DMSO-d6) δ 197.4, 193.2, 149.1, 148.4, 147.2, 142.5, 136.1, 135.5, 133.0, 128.4,
125.1, 123.5, 118.5, 114.2, 47.1, 38.3, 29.2. HRMS m/z (ESI) calcd for
C17H16ClN4OS2 (M+H)+: 391.0449, found 391.0456.
4.2.13.
3-(5-(4-Chlorophenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)-3-oxopropyl
(pyridin-3-ylmethyl)carbamodithioate (6m)
25

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1
White solid, m. p. 182.9-184.7℃, yield: 24%, two steps. H NMR (400 MHz,
DMSO-d6) δ 12.65 (s, 1H), 10.48 (s, 1H), 8.64 (dd, J = 14.6, 2.2 Hz, 2H), 8.60 – 8.41
(m, 3H), 7.75 (d, J = 8.5 Hz, 2H), 7.69 (d, J = 7.8 Hz, 1H), 7.55 (d, J = 8.5 Hz, 2H),
7.36 (dd, J = 7.7, 4.8 Hz, 1H), 4.86 (d, J = 5.5 Hz, 2H), 3.56 (s, 2H), 3.35 (d, J = 6.8
Hz, 2H). 13C NMR (100 MHz, DMSO-d6) δ 197.5, 193.3, 149.1, 148.6, 148.4, 143.2,
137.3, 135.5, 135.4, 133.0, 132.3, 129.5, 129.1, 128.8, 127.3, 123.5, 117.7, 114.8,
47.1, 38.3, 29.3. HRMS m/z (ESI) calcd for C23H20ClN4OS2 (M+H)+: 467.0762,
found 467.0772.
4.2.14.
3-(6-Chloro-1H-pyrrolo[2,3-b]pyridin-3-yl)-3-oxopropyl
(pyridin-3-ylmethyl)carbamodithioate (6n)
1
White solid, m. p. 180.6-182.6℃, yield: 21%, two steps. H NMR (400 MHz,
DMSO-d6) δ 12.71 (s, 1H), 10.47 (t, J = 5.3 Hz, 1H), 8.57 – 8.50 (m, 2H), 8.47 (d, J =
7.9 Hz, 2H), 7.69 (d, J = 7.8 Hz, 1H), 7.46 – 7.24 (m, 2H), 4.85 (d, J = 5.4 Hz, 2H),
13
3.54 (t, J = 6.7 Hz, 2H), 3.32 (t, J = 6.7 Hz, 2H). C NMR (100 MHz, DMSO-d6) δ
197.4, 193.2, 149.1, 148.4, 147.8, 144.4, 135.5, 134.8, 132.9, 132.6, 123.5, 118.0,
116.5, 114.8, 47.1, 38.3, 29.1. HRMS m/z (ESI) calcd for C17H16ClN4OS2 (M+H)+:
391.0449, found 391.0457.
4.2.15.
3-(6-Methyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-3-oxopropyl
(pyridin-3-ylmethyl)carbamodithioate (6o)
1
White solid, m. p. 174.4-176.2℃, yield: 22%, two steps. H NMR (400 MHz,
DMSO-d6) δ 12.31 (s, 1H), 10.46 (t, J = 5.5 Hz, 1H), 8.52 (d, J = 1.6 Hz, 1H), 8.47
(dd, J = 4.7, 1.4 Hz, 1H), 8.38 (d, J = 3.1 Hz, 1H), 8.33 (d, J = 8.0 Hz, 1H), 7.69 (d, J
= 7.8 Hz, 1H), 7.36 (dd, J = 7.7, 4.7 Hz, 1H), 7.12 (d, J = 8.0 Hz, 1H), 4.85 (d, J = 5.6
13
Hz, 2H), 3.53 (t, J = 6.8 Hz, 2H), 3.28 (t, J = 6.8 Hz, 2H), 2.53 (s, 3H). C NMR
(100 MHz, DMSO-d6) δ 197.5, 193.1, 152.8, 149.1, 148.7, 148.4, 135.5, 133.5, 133.0,
129.7, 123.5, 117.9, 115.1, 114.7, 47.1, 38.1, 29.3, 24.0. HRMS m/z (ESI) calcd for
26

ACCEPTED MANUSCRIPT
C18H19N4OS2 (M+H)+: 371.0995, found 371.1002.
4.2.16.
3-Oxo-3-(pyrazolo[1,5-a]pyridin-3-yl)propyl (pyridin-3-ylmethyl)carbamodithioate
(15a)
1
White solid, m. p. 144.0-145.3℃, yield: 52%, two steps. H NMR (400 MHz,
DMSO-d6) δ 10.48 (t, J = 5.2 Hz, 1H), 8.87 (d, J = 6.8 Hz, 1H), 8.69 (s, 1H), 8.52 (s,
1H), 8.47 (d, J = 3.9 Hz, 1H), 8.23 (d, J = 8.8 Hz, 1H), 7.79 – 7.56 (m, 2H), 7.35 (dd,
J = 7.6, 4.9 Hz, 1H), 7.19 (t, J = 6.6 Hz, 1H), 4.86 (d, J = 5.4 Hz, 2H), 3.55 (t, J = 6.7
13
Hz, 2H), 3.34 (t, J = 6.9 Hz, 2H). C NMR (100 MHz, DMSO-d6) δ 197.4, 191.6,
149.1, 148.4, 145.0, 139.3, 135.4, 132.9, 129.9, 129.4, 123.5, 118.7, 115.1, 111.6, 47.1,
39.2, 29.1. HRMS m/z (ESI) calcd for C17H17N4OS2 (M+H)+: 357.0838, found
357.0848.
4.2.17.
3-Oxo-3-(pyrazolo[1,5-b]pyridazin-3-yl)propyl (pyridin-3-ylmethyl)carbamodithioate
(15b)
1
White solid, m. p. 160.0-160.9℃, yield: 18%, two steps. H NMR (400 MHz,
DMSO-d6) δ 10.50 (d, J = 5.4 Hz, 1H), 8.81 (s, 1H), 8.68 (ddd, J = 10.6, 6.7, 1.6 Hz,
2H), 8.52 (s, 1H), 8.47 (d, J = 4.6 Hz, 1H), 7.69 (d, J = 7.8 Hz, 1H), 7.58 (dd, J = 9.0,
4.5 Hz, 1H), 7.37 (dd, J = 7.8, 4.8 Hz, 1H), 4.85 (d, J = 5.5 Hz, 2H), 3.55 (t, J = 6.7
13
Hz, 2H), 3.40 (t, J = 6.7 Hz, 2H). C NMR (100 MHz, DMSO-d6) δ 197.3, 192.4,
149.1, 148.4, 144.9, 142.4, 135.5, 133.8, 132.9, 128.3, 123.5, 121.4, 111.8, 47.1, 39.4,
28.7. HRMS m/z (ESI) calcd for C16H16N5OS2 (M+H)+: 358.0791, found 358.0801.
4.2.18.
3-Oxo-3-(2-phenylpyrazolo[1,5-b]pyridazin-3-yl)propyl
(pyridin-3-ylmethyl)carbamodithioate (15c)
1
White solid, m. p. 160.2-161.0℃, yield: 30%, two steps. H NMR (400 MHz,
DMSO-d6) δ 10.55 (s, 1H), 8.69 (dd, J = 16.0, 6.6 Hz, 2H), 8.57 – 8.30 (m, 2H), 7.79
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ACCEPTED MANUSCRIPT
– 7.56 (m, 4H), 7.56 – 7.43 (m, 3H), 7.40 – 7.27 (m, 1H), 4.79 (d, J = 5.4 Hz, 2H),
13
3.38 (t, J = 6.2 Hz, 2H), 2.94 (t, J = 6.5 Hz, 2H). C NMR (100 MHz, DMSO-d6) δ
197.0, 193.3, 153.0, 149.1, 148.4, 145.0, 135.6, 135.5, 133.0, 132.5, 129.8, 129.3,
128.6, 128.4, 123.5, 121.3, 110.1, 47.0, 41.3, 28.7. HRMS m/z (ESI) calcd for
C22H20N5OS2 (M+H)+: 434.1104, found 434.1112.
4.3. Synthesis of 7-azaindole derivatives
4.3.1. General procedure for the synthesis of compounds 8g, 8h and 8l
7-Azaindole derivatives (4.2 mmol) were added to a stirred suspension of AlCl3
(21.0 mmol, 2.80 g) in DCM (40 mL) placed at ice bath. After the mixture was stirred
at room temperature for 0.5 h, acetyl chloride (21.0 mmol, 1.49 mL) was added
dropwise and the resulting mixture was reacted for 12 h at room temperature. MeOH
(20 mL) was added cautiously to quench the reaction, the solvents were removed
under reduced vacuum. Then the residue was dissolved in 40 mL water, 1N NaOH (aq)
was added to adjust the pH up to 5, and extracted with ethyl acetate (15 mL×3). The
combined organic phase was dried over anhydride sodium sulfate and concentrated
under reduced vacuum. The residue was further purified by column chromatography
on silica gel using PE/EA as eluent to afford the corresponding acylated product.
1-(4-Chloro-1H-pyrrolo[2,3-b]pyridin-3-yl)ethenone (8g) [53]. White solid, yield:
1
50%. H NMR (400 MHz, DMSO-d6) δ 12.79 (s, 1H), 8.56 (s, 1H), 8.23 (d, J = 5.1
Hz, 1H), 7.29 (d, J = 5.0 Hz, 1H), 2.50 (s, 3H).
1-(4-Bromo-1H-pyrrolo[2,3-b]pyridin-3-yl)ethenone (8h) [53]. White solid, yield:
1
52%. H NMR (400 MHz, DMSO-d6) δ 12.77 (s, 1H), 8.56 (s, 1H), 8.13 (d, J = 5.1
Hz, 1H), 7.47 (d, J = 5.0 Hz, 1H), 2.50 (s, 3H).
1-(5-Chloro-1H-pyrrolo[2,3-b]pyridin-3-yl)ethenone (8l) [53]. White solid, yield:
88%. 1H NMR (400 MHz, CDCl3) δ 10.63 (s, 1H), 8.70 (s, 1H), 8.36 (s, 1H), 8.01 (s,
28