Design and synthesis of substituted pyrido[3,2-d]-1,2,3-triazines as potential Pim-1 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2016.01.032

A novel series of substituted pyrido[3,2-d]-1,2,3-triazines were designed and synthesized as Pim-1 inhibitors through scaffold hopping. Most of the derivatives showed potent in vitro Pim-1 inhibitory activities and anti-proliferative effects toward prosta

Full text of DOI:10.1016/j.bmcl.2016.01.032

Accepted Manuscript
Design and synthesis of substituted pyrido[3,2-d]-1,2,3-triazines as potential
Pim-1 inhibitors
Yin-Bo Fan, Kun Li, Min Huang, Yu Cao, Ying Li, Shu-Yu Jin, Wen-Bing Liu,
Jia-Chen Wen, Dan Liu, Lin-Xiang Zhao
PII:
S0960-894X(16)30032-4
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.01.032
BMCL 23497
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
9 July 2015
24 December 2015
12 January 2016
Please cite this article as: Fan, Y-B., Li, K., Huang, M., Cao, Y., Li, Y., Jin, S-Y., Liu, W-B., Wen, J-C., Liu, D.,
Zhao, L-X., Design and synthesis of substituted pyrido[3,2-d]-1,2,3-triazines as potential Pim-1 inhibitors,
Bioorganic & Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.01.032
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Design and synthesis of substituted pyrido[3,2-d]-1,2,3-triazines as potential
Pim-1 inhibitors
Yin-Bo Fana, Kun Lia, Min Huanga, Yu Caoa, Ying Lib, Shu-Yu Jina, Wen-Bing Liua, Jia-Chen
Wena, Dan Liua* and Lin-Xiang Zhaoa*
aKey Laboratory of Structure-Based Drugs Design & Discovery of Ministry of Education, Shenyang Pharmaceutical
University, Shenyang 110016, China
b Department of Pharmacology, Shenyang Pharmaceutical University, Shenyang 110016, China
*Corresponding authors. Tel.: +86 024 2398 6420; Fax: +86 024 2398 6420
E-mail address: sammyld@163.com (Dan Liu);

Abstract
A novel series of substituted pyrido[3,2-d]-1,2,3-triazines were designed and
synthesized as Pim-1 inhibitors through scaffold hopping. Most of the derivatives showed
potent in vitro Pim-1 inhibitory activities and anti-proliferative effects towards prostate cancer
cells. Among them, 6b, 6h and 6m showed the best Pim-1 inhibitory activity with IC50 values
of 0.69, 0.60 and 0.80 μM, respectively. Furthermore, compound 6b, 6i, 6j and 6m showed
strong inhibitory activity to human prostate cancer LNcap and PC-3 cell lines with IC50 values
at low micromolar level. Structure-activity relationship analysis revealed that appropriate
substitutions at C-6 positions contributed to the kinase inhibition and antiproliferative effects.
Moreover, western blot assay suggested that 6j could decrease the levels of p-BAD and
p-4E-BP1 in a dose-dependent manner in PC-3 cells. Docking studies showed that 3-N of the
scaffold formed a hydrogen bond with Lys67, aromatic 4-aniline formed a key π-π stack with
Phe49. Taken together, this study might provide the first sight for developing the
pyrido[3,2-d]-1,2,3-triazine scaffold as novel Pim-1 inhibitors.
Keyword: Pim-1 kinase inhibitors; Pyrido[3,2-d]-1,2,3-triazines; Anti-proliferative activity;
Prostate cancer cells.

Cancer presents to be the most threat to human health, despite of numerous investments,
the relentless and lethal nature of cancer persists, with only limited overall improvements in
treatment outcomes. The discovery of anticancer drugs is undergoing and remains a highly
challenging endeavor.1 Dysregulation of kinase function has emerged as a robust research area
for cancer therapy, as it consists of one of the major mechanisms through which cancer cells
escape from normal constraints on growth and proliferation. About 518 kinases have been
recognized in human kinome, among which only a few have been successfully targeted for
diseases like cancer and inflammation.2
Pim-1, a member of serine/threonine kinase, has been greatly involved in complex
signaling mechanisms associated with tumorigenesis and drug resistance.3-6 High expression
level of Pim-1 has often been associated with hematological malignancies and solid cancers
like prostate cancer.7-9 Increasing expression of Pim-1 leads to promotion of proliferation and
blockade of apoptosis. Pim-1 has been reported to phosphorylate lots of substrate proteins
including BAD and 4E-BP1 to enhance cell survival and proliferation.5 In addition, it
synergistically works with c-Myc in tumorigenesis.10 Thus, Pim-1 serves as a promising
therapeutic target to solve numerous unmet medical needs in cancer therapy.
There has been persistent interest for developing Pim-1 inhibitors, and typical inhibitors
reported contain rigid fused rings as critical scaffold such as Imidazopyridazines,
triazolopyridazines, pyrazolopyrimidines and triazolopyridines (Figure 1), which were
recently utilized in the design of potent Pim-1 inhibitors.11-15 Among those inhibitors,
SGI-1776 (1) was the first generation of Pim-1 inhibitor characterized with imidazopyridazine
structure. Though exhibiting excellent potency towards Pim-1 kinase, it was terminated due to

the dose limiting cardiac toxicity in phaseⅠclinical trail.13 Efforts to improve the safety files
were undergoing, it also provoked us to discover alternative novel chemotypes to be explored
as Pim-1 inhibitors.
Figure 1. The structures of reported Pim kinase inhibitor cores including Imidazopyridazines,
Triazolopyridazines, Pyrazolopyrimidine, Triazolopyridine and the representative lead
SGI-1776.
In our efforts to discover novel Pim-1 kinase inhibitors, the five-membered imidazole,
pyrazole and triazole ring in the reported structures were replaced with the six-membered ring
of triazine to accomplish a novel scaffold and template of pyrido[3,2-d]-1,2,3-triazine under
the guidance of scaffold hopping strategy in medicinal chemistry, 3-trifluoromethoxyphenyl
was changed into corresponding aniline at suitable C-4 position (Figure 2). To illuminate the
Pim-1 kinase inhibition by this unique and proprietary scaffold in a systemic way, a series of
novel pyrido[3,2-d]-1,2,3-triazine derivatives featured with various C-6 substitutions were
described, their inhibitory activities towards Pim-1 as well as the in vitro antiproliferative
activity towards two human prostatic cancer LNcap and PC-3 cell lines were studied. Finally,
the inhibition towards the phosphorylation of BAD and 4E-BP1, two well-established Pim-1
kinase substrates, were also investigated with representative compound in PC-3 cells. The

possible binding mode in the ATP pocket of Pim-1 was illustrated to find a unique and tight
binding for these compounds.
Figure 2. Design of pyrido[3,2-d]-1,2,3-triazines as new scaffold for Pim-1 inhibitors.
The construction of pyrido[3,2-d]-1,2,3-triazine scaffold was described in our earlier
work (as shown in Scheme 1).16 The requisite intermediate 2 was synthesized in a traditional
method by treating commercial available 2,6-dichloro-3-nitropyridine with cuprous cyanide at
reflux, sodium thiosulphate was utilized as reductant converting nitro into amino group to
give 3-amino-6-chloro-2-cyanpyridine 3. The synthesis of scaffold 5 was conducted through
cyclization and Dimroth rearrangement of 4, which was prepared by diazotization of 3 and
3-trifluoromethoxyaniline. Scaffold 5 was further reacted with two fold stoichiometric
saturated N-heterocycles to afford 6a-6l, de-protection of the Boc group with chlorine hydride
in EtOAc from 6l afforded 6m. Moreover, in order to evaluate the influence on Pim-1 kinase
inhibition of C-6 position substitutions at the pyrido[3,2-d]-1,2,3-triazine scaffold, linear alkyl
substituted 6n was also prepared with 5 and monoethanolamine in neutral ethanol under
reflux, nucleophilic attack of 5 with particular sodium alkoxide in corresponding alcohol
afforded alkyl ethers 6o-6q.

Scheme 1. Synthesis of compounds 6a-6q. Reagents and conditions: a) CuCN, NMP, reflux,
31%; b) Na2S2O3, CH2Cl2, H2O, 40%; c) 3-trifluoromethoxy-aniline, NaNO2, HCl, 0℃, 50%;
d) 70% EtOH(aq.), reflux, then acetic acid, reflux, 60%; e) RH, EtOH or i-PrOH, reflux,
65-90%; f) RH, correspounding sodium alkoxide, r.t., 71-77%; g) HCl in EtOAc, CH2Cl2, r.t.,
70%.

To investigate the Pim-1 kinase inhibitory potency of all synthesized compounds 6a-6q,
the IC50 values were determined for each compound with an advanced HTRF method at
Cisbio. SGI-1776 (1) exhibited a significant inhibitory effect with IC50 value of 0.048 μM in
the tested system (the reported value is 0.007 μM), 8 and the results for all target compounds
were shown in Table 1. The scaffold 5 with chlorine substituted at C-6 position exhibited
little potency (IC50>10 μM). Generally, the substitutions with various saturated
N-heterocycles at this particular position (6a-6m) were found to be more potent than 5 and
their ethanolamine (6n) or alkyl ether counterparts (6o-6q). Morpholine (6a), piperazine (6b),
isopropylpiperazine (6c) ethylpiperazine (6g) and methylpiperazine (6h) substitutions led to
strong inhibition with IC50 values 1.45, 0.69, 1.08, 2.04 and 0.60 μM, respectively, indicating
that a heteroatom is favorable at 4’-position of the heterocycles, compared with
non-heteroatom counterparts (6d and 6e: IC50>10 μM). The large substitution of heterocycles
were tolerated to the Pim-1 kinase inhibition effects, most strikingly elucidated by
di-heterocycle substituted 6i and 6j (IC50 values were 2.47 and 1.05 μM, respectively). 6k
shares the same substitution with SGI-1776, but improved potency was not observed against
Pim-1 with IC50 of 1.87 μM. It was noteworthy that 6l, which has a bulky Boc terminal group
compared with 6k, lost potency completely. Meanwhile, 6m with 1’-NH uncovered at the
piperidine showed improved activity with IC50 value of 0.80 μM. Moreover, 6n exhibited
similar potency with 6a, which could be attributed to the existence of ethanolamine group, the
hydroxyl group might be equivalent with the 4’-heteroatom of saturated N-heterocycles, as
the ethyl linker could help to expand it into a suitable position similar with the six-membered
saturated rings. For comparison, compound 6p and 6q with linear alkyl ether attached to the

bicyclic core at C-6 was detrimental for Pim-1 inhibitory activity (IC50> 10 μM), as the
flexible alkoxyl groups lack terminal heteroatom, sharply decreasing the kinase inhibition
activity. All these data confirmed that the substitutions at C-6 position were beneficial to
pledge potent Pim-1 kinase inhibitory activities of pyrido[3,2-d]-1,2,3-triazine analogues.
Piperazine (6b), methylpiperazine (6h) and piperidin-4-ylmethanamine (6m) were the best in
the series, with IC50 values of 0.69, 0.60 and 0.80 μM, respectively.
Pim-1 is often highly expressed in prostate cancer cells, the antiproliferative activity of
the target compounds against human prostate adenocarcinoma LNcap and PC-3 cell lines was
further evaluated using MTT assay, and the results fitted well with the Pim-1 kinase inhibition
data. SGI-1776 displayed good potency with IC50 values of 6.85 and 4.86 μM towards LNcap
and PC-3 cells. The target compounds demonstrated no significant difference in inhibiting the
growth between two cell lines, regardless of dependence on androgen. Generally, the
introduction of suitable saturated N-heterocycle in C-6 position of the scaffold leading to
potent Pim-1 inhibition also contributed to improved anti-proliferative activities, while alkoxy
substituted groups sharply decreased the inhibitory activities towards Pim-1 and intact cancer
cells. Di-heterocycle derivatives 6i and 6j displayed the most potent inhibitory effects towards
both cell lines with IC50 ranging from 4.90 to 10.70 μM, similar with the reported Pim-1
inhibitor SGI-1776. Mono-heterocycle derivatives 6b, 6c, 6g, 6h and 6m proved to be less
potent antiproliferative compounds, which displayed a low micromolar activity between 5.51
and 16.89 μM against both cancer cells. compound 6b and 6m were more active than 6a,
suggesting that 4’-NH as a hydrogen bond donor in the heterocycle showed some advantage
over 4’-O both in biochemical kinase assay and in cultured cells. Once the 4’-position of the

piperazine in 6a was alkylated with methyl, ethyl or isopropyl group, its potency decreased,
as can be seen from comparing 6b with 6c, 6g and 6h, even though methylpiperazine gave the
most potent compound 6h towards Pim-1 inhibition. Substituted piperidyl derivatives 6e and
6f precipitated in the testing system, while tetrahydropyrrole substituted 6d showed no
potency
(IC50>
100
μM),
indicating
that
compounds
substituted
with
mono-heteroatom-containing heterocycle was inferior to two-heteroatom-containing ones,
which was consistent with Pim-1 kinase inhibition data. 6n exhibiting potent Pim-1 inhibitory
activity failed to display expected potency in cultured cell. In comparison, the substitution of
alkoxy group led to abrogation of activities, ethoxy substituted compound 6p had no effect
towards the tested cells, while 6o and 6q precipitated during the cell effect measurement.
Though SGI-1776 demonstrated greater potency in Pim-1 inhibition, it displayed equivalent
activity with 6i and 6j against cancer cells. In conclusion, 6b, 6i, 6j and 6m with good
inhibition of Pim-1 kinase activity showed significant activity toward the two tested cancer
cell lines, and were thus regarded as the most promising candidates as potential Pim-1
inhibitors among those target compounds.
Table 1. The inhibitory activities of compounds 5, 6a-6q on Pim-1 kinase, LNcap and PC-3
cell lines.
IC50(μM)
Compd.
Pim-1a
>10
LNcapb
8.62±0.35
30.99±6.49
8.51±0.81
10.67±0.96
>100c
PC-3b
5.99±0.16
40.97±2.43
11.50±0.24
13.82±0.33
>100
5
1.45
0.69
1.08
>10
6a
6b
6c
6d
6e
6f
d
>10
-
-
1.31
-
-

2.04
0.60
2.47
1.05
1.87
>10
14.28±0.46
20.27±0.97
4.90±1.29
10.70±1.33
17.34±0.63
>100
16.89±0.88
24.04±3.13
5.58±0.30
8.16±0.57
15.76±1.42
>100
6g
6h
6i
6j
6k
6l
0.80
2.10
2.02
>10
11.52c
8.32c
6m
42.88±3.36
-
>100
6n
-
6o
>100
>100
6p
>10
-
-
6q
0.048
6.85±0.24
4.86±0.16
SGI-1776
aAll results represent the mean of duplicate experiments. bData shown are means ± SD of three independent
experiments. cData shown are the results of single experiment. dPrecipitated in the testing system.
To gain further insights into the mechanisms of action of these compounds, compound 6j,
which possessed significant Pim-1 and cellular inhibitory effects, was selected to examine the
inhibitory effect on the phosphorylation level of BAD at Ser112 with western blot analysis in
PC-3 cells. Pim-1 catalytic BAD phosphorylation results in its anti-apoptotic activity, thus the
phosphorylation level of BAD could reflect the residual activity of Pim-1 kinase indirectly.17
PC-3 cells were treated with 10 μM SGI-1776 and 6j at different concentrations or DMSO as
negative control for 24 h. The results (as shown in Figure 3) indicated that 6j, acting exactly
like SGI-1776, could dose-dependently block the BAD phosphorylation without affecting
Pim-1 expression in PC-3 cells. Meanwhile, Pim-1 regulates protein translation through
phosphorylating 4E-BP1, which releases eIF4E, a curial component involved in
cap-dependent translation to promote survival for cancer cells.3,18 Treatment of 6j for 24 h
down-regulated 4E-BP1 phosphorylation at Thr37/46 in PC-3 cells. Both of the
dephosphorylating effects of 6j might be partially caused by Pim-1 kinase inhibition just like
SGI-1776. Additionally, the level of c-Myc associated with Pim-1 kinase was observed to

decrease upon SGI-1776 and 6j treatment, suggesting a potential inhibitory effect on myc
oncogene driven oncologic events. Taken together, our cellular assays implied that the
representative 6j blocked proliferation in intact prostate cancer cells partly through inhibiting
Pim-1 enzymatic activity.
PC-3
6j (μM)
- - 5 10 20
- 10 - - -
SGI-1776 (μM)
Pim-1
p-BAD
BAD
p-4E-BP1
4E-BP1
c-Myc
β-actin
Figure 3. 6j (0, 5, 10, 20 μM) and SGI-1776 (10 μM) decreased the expression of p-BAD,
p-4E-BP1 and c-Myc in PC-3 cells.
In order to understand the binding mode of 6j within Pim-1 kinase, the docking
experiments were conducted with Discovery studio software based on the X-ray co-crystal
structure of Pim-1 with a 1,2,3-triazolo[4,5-b]pyridine derivative (PDB 4A7C).14 Supporting
the docking results, the original ligand was first docked and fitted well with that in crystal
structure, then 6j was docked with the same parameters. The binding mode (shown in Figure

4) demonstrated that 6j inserted into and occupied the Pim-1 ATP binding cleft completely.
Differently, 6j adopted a 180 degree flip manner comparing to the inherent triazolopyridine
ligand in the pocket as shown in the overlapped version (Figure 5). Consistent with hydrogen
bonds formed with Lys67 in the ligand binding mode, a key hydrogen bonds established
between Lys67and 3-N of the triazine scaffold. Despite the loss of interaction with Asp128
due to the flip effect, a π-π stack existed between the substituted aniline of 6j and Phe49 in
return, which further stabilized the binding. The side chain at C-6 position of the scaffold
extended to the entrance of the pocket. The hydrophobic segments of 6j were buried into the
hydrophobic pocket formed by Phe49, Ala65, Val52, Ile104, Leu120 and Ile185. Similar to
the original ligand, no interactions could be observed between the molecule and the hinge
region of Pim-1 (Arg122). The docking study suggested a unique and tight binding within the
ATP-binding region, distinguished from the triazolopyridine template.
Figure 4. Proposed binding mode of 6j in the ATP binding pocket of the Pim-1 kinase
domain.

Figure 5. Overlapping spectacle of 6j (carbon atoms colored in orange) and triazolopyridine
ligand (carbon atoms colored in purple) within the ATP binding pocket of the Pim-1 kinase
domain.
We report herein the discovery and synthesis of novel pyrido[3,2-d]-1,2,3-triazines as
potential Pim-1 inhibitors. Compared to the scaffold 5 and its analogues substituted with
6-alkoxyl (6o-6q), the 6-amine series (6a-6n) were identified as more potent Pim-1 kinase
inhibitors. Among which, 6b, 6h and 6m were the most potent Pim-1 inhibitor with IC50
values of 0.69, 0.60 0.80 μM, respectively; 6i and 6j exhibited a good potency to inhibit
Pim-1, with IC50 values 2.47 and 1.05 μM. They all exhibited low micromolar
anti-proliferative activity toward prostatic cancer PC-3 and LNcap cell lines. In addition, 6j
could block the phosphorylation of BAD and 4E-BP1 in a dose-dependent manner in PC-3
cells. Molecular modeling illustrated a different but strong binding effect for 6j within Pim-1
ATP-binding site, compared to the triazolopyridine ligand. Together, these results provide

compelling evidence that the antiproliferative activity of these compounds in PC-3 cells was
mediated, at least in part, by the inhibition of Pim-1 kinase, thereby enhanceing the
proapoptotic effect of BAD. Therefore, this unique pyrido[3,2-d]-1,2,3-triazine scaffold
demonstrated great potential to be further explored as potent Pim-1 inhibitors with improved
physicochemical property and potency. Efforts are ongoing to further optimize and provide a
systematic structure-activity analysis at the proper position and the results will be reported in
due course.

Acknowledgements
Financial support from the National Major Scientific and Technological Special Project
for ‘Significant New Drugs Development’ during the Eleven Five-year Plan Period (grant no.
2009ZX09103-080) is gratefully acknowledged.

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Graphical abstract