Design, synthesis and antiproliferative activity evaluation of a series of pyrrolo[2,1-f][1,2,4]triazine derivatives

Article abstract of DOI:10.1016/j.bmcl.2020.127194

A series of 6-aminocarbonyl pyrrolo[2,1-f][1,2,4]triazine derivatives were designed by scaffold hopping strategy. The IC₅₀ values of compound 14a against PI3Ks were measured, showing selective activity against p110α and p110δ with IC₅₀s of 122 nM and 119 nM respectively. All the synthesized compounds were evaluated for their antiproliferative activity against human cancer cells by SRB assay. Compounds 14a, 14p and 14q exhibited potent antiproliferative activity against five types of human cancer cells and the PK property of 14q was also investigated here.

Full text of DOI:10.1016/j.bmcl.2020.127194

Journal Pre-proofs
Design, synthesis and antiproliferative activity evaluation of a series of pyrro‐
lo[2,1-f][1,2,4]triazine derivatives
Hao-Yue Xiang, Yan-Hong Chen, Yi Wang, Xi Zhang, Jian Ding, Ling-Hua
Meng, Chun-Hao Yang
PII:
S0960-894X(20)30293-6
DOI:
Reference:
https://doi.org/10.1016/j.bmcl.2020.127194
BMCL 127194
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Bioorganic & Medicinal Chemistry Letters
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17 February 2020
7 April 2020
11 April 2020
Please cite this article as: Xiang, H-Y., Chen, Y-H., Wang, Y., Zhang, X., Ding, J., Meng, L-H., Yang, C-H.,
Design, synthesis and antiproliferative activity evaluation of a series of pyrrolo[2,1-f][1,2,4]triazine derivatives,
Bioorganic & Medicinal Chemistry Letters (2020), doi: https://doi.org/10.1016/j.bmcl.2020.127194
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Design, synthesis and antiproliferative
activity evaluation of a series of pyrrolo[2,1-
f][1,2,4]triazine derivatives
Hao-Yue Xianga,b , Yan-Hong Chenb Yi Wangb,Xi Zhangb, Jian Dingb, Ling-Hua Mengb* and Chun-Hao Yangb
,
1

Bioorganic & Medicinal Chemistry Letters
Design, synthesis and antiproliferative activity evaluation of a series of
pyrrolo[2,1-f][1,2,4]triazine derivatives
Hao-Yue Xianga,b , Yan-Hong Chenb Yi Wangb, Xi Zhangb, Jian Dingb, Ling-Hua Mengb* and Chun-Hao
,
Yangb
aCollege of Chemistry and Chemical Engineering, Central South University, Changsha, Hunan, 410083, P.R. China
bState Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai, 201203, P.R.
China
Corresponding author. Tel/Fax: +86-21-50806770; e-mail: lhmeng@simm.ac.cn (L. Meng); chyang@simm.ac.cn (C. Yang).
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
A series of 6-aminocarbonyl pyrrolo[2,1-f][1,2,4]triazine derivatives were designed by scaffold
hopping strategy. The IC50 values of compound 14a against PI3Ks were measured, showing
selective activity against p110α and p110δ with IC50s of 122 nM and 119 nM respectively. All the
synthesized compounds were evaluated for their antiproliferative activity against human cancer
cells by SRB assay. Compounds 14a, 14p and 14q exhibited potent antiproliferative activity
against five types of human cancer cells and the PK property of 14q was also investigated here.
Keywords:
Antiproliferative
Cancer
2009 Elsevier Ltd. All rights reserved.
Therapy
PI3K inhibitors
Pyrrolo[1,2-f][1,2,4]triazine
Kinase
Cancer, the second common cause of death following
cardiovascular diseases, is one of the most challenging global
health problems.1-3 As a result, considerable efforts have been
devoted to develop efficient drugs for cancer therapy. For a long
time, cytotoxic agents have been used in clinic as the major
chemotherapy for most cancers, but limited by a narrow
therapeutic index, remarkable toxicities and lack of specificity.4, 5
Therefore, there is a growing imperative need for further
identification of new treatment approaches to cancer. The past
decade has witnessed tremendous advance in the molecularly
targeted therapies aiming at special targets, with the development
of new compounds that interrupt specific molecular abnormalities
driving cancer initiation and progression.6
inhibitors based on LY294002 were designed and developed, such
as PI-103,15 BKM120,16 ZSTK474,17 GDC-0941,18 ETP-46992
19(Fig. 1). All these inhibitors share the aryl-morpholine
pharmacophore, which served as a valuable chemical tool for
development of many additional PI3K inhibitors.
Phosphatidylinositol 3-kinases (PI3Ks) are lipid kinases and
subdivided into three classes (class I, II, and III) based on their
sequence, homology and structural differences.7 Among of them,
Class I PI3Ks comprising four isoforms (p110α, p110β, p110δ,
and p110γ), are extensively studied and they can catalyze
phosphorylation of the 3’-hydroxyl group of phosphatidylinositol
4,5-biphosphate (PIP2) to generate phosphatidylinositol 3,4,5-
triphosphate (PIP3), a potent secondary messenger. Abundant
evidences demonstrate that PI3K pathway is hyper-activated in
numerous cancer types.8, 9 Hence, significant efforts have been
made to develop inhibitors targeting PI3K signaling pathway for
cancer therapies.10-13 In 1994, the Lilly Research Laboratory
developed the first synthetic ATP-competitive pan-PI3K
LY294002.14 Afterwards, a considerable number of PI3K
Figure 1. The structure of some aryl-morpholine type PI3K inhibitors
The scaffold hopping is a common and useful method in
medicinal chemistry to find novel proprietary hits. Using this
strategy, our group previously designed and synthesized series of
novel PI3K inhibitors based on PI-103, an aryl-morpholine type
PI3K inhibitor. Among the PI3K inhibitors reported by our group,
4-(2-(3-
hydroxylphenyl)pyrido[2',3':4,5]pyrrolo[2,1-
f][1,2,4]triazin-4-yl)morpholine 2 was found to have the IC50
2

against PI3Kα (33.6 nM) comparable to that of PI-103 (16.8 nM)
and showed anti-proliferative activity at micromolar concentration
inseveral cancer cell lines (Fig. 2).20 However, it suffered from
unfavorable properties like rapid metabolism and poor solubility.
To circumvent these problems, we tried to replace the labile 3-
hydrophenyl segment with 2-amine-4-trifluoromethylpyrdinyl
segment from BKM120. Meanwhile, a parallel scaffold hoping
strategy was adopted to replace the tricyclic core of 2 with bicyclic
core in order to improve the solubility of these compounds.
Recently,
a straightforward methodology to 2-substituted
pyrrolo[2,1-f][1,2,4]triazin-4(3H)-ones was developed in our
lab.21 We thus envisioned applying our method to the design of
novel pyrrolo[2,1-f][1,2,4]triazine bicyclic PI3K inhibitors library
(Fig. 2). In this letter, we focused on investigating the influence of
the substituents at the pyrrole ring on the activity of the newly
designed compounds to inhibit PI3K kinase and cell proliferation.
Scheme 1. Reagents and conditions: (a) NBS, DCM, rt, 83%; (b) pivaloyl chloride,
°
°
Et3N, DCM, C ~ rt, 89%; (c) n-BuLi, DMF, THF, -78 C; (d) NH4Cl, K2CO3,
NH4OH, aliguate-336, NaClO, MTBE, 0 °C ~ rt; (e) NH3 in MeOH, sealed tube, 80
°C; (f) CuCl2·2H2O, DMSO, 100 C; (g) POCl3, DMAP, 110 C; (h) morpholine,
THF, reflux; (i) 1M KOH, EtOH, reflux; (j) H2SO4, AcOH, H2O, 100 °C; (k) amines,
EDCI, HOBT, DIPEA, DMF, rt.
°
°
Table 2. Antiproliferative activities of compounds 14 against human
rhabdomyosarcoma Rh30 cells. a
Figure 2. Optimizing strategy.
Initially, a probe compound 14a was synthesized to evaluate the
potential of this novel PI3K inhibitor scaffold. As we expected,
14a showed potent activity against p110α and p110δ with IC50s
of 122 nM and 119 nM respectively, and higher concentration
was required to inhihit p110β and p110γ by 50%. Encouragingly,
IC50
(µM)
Cpd
14a
R1
H
Amino
Cpd
14l
R1
H
Amino
IC50 (µM)
1.9
(2.4±0.5)
7.8
different
6-aminocarbonyl
pyrrolo[2,1-f][1,2,4]triazine
14b
14c
H
H
3.2
3.5
14m
14n
H
H
7.3
3.5
derivatives were synthesized and evaluated in cellular assays.
Table 1. Inhibition of the kinase activity of p110α, p110δ, p110β,
p110γ by 14a.a
14d
H
2.7
14o
H
3.6
1.3
(1.6±0.3)
14e
14f
14g
H
H
H
2.7
2.8
14p
14q
14r
Me
Me
Me
IC50 [nM]
Cpd
p110α
p110δ
p110β
p110γ
1.2
(1.7±0.5)
14a
GDC0941
122
35
119
9
1293
324
663
746
a Using PI3-Kinase (human) HTRFTM Assay kit (Cat. 33-017, Millipore)
>10
4.9
3.5
A general synthetic route to prepare the designed compounds
14 was illustrated in Scheme 1.The commercially available 3 was
converted into 6 with the conventional steps. Then cyanation of 4
generated 5 with potassium hexacyanoferrate (II) using copper
catalysis. Afterwards, the desired aldehyde 6 reacted with 9 that
prepared according to the literature21 to yield the pyrrolo[2,1-
f][1,2,4]triazin-4(3H)-ones 10. Chlorination of 10, followed by
morpholine introduction gave 12. Then deprotection and
hydrolysis of 12 in one pot provided the key intermediates 13.
Finally, the carboxylic acids 13 were treated with various amines
to give the desired analogs 14.
14h
14i
H
H
>10
1.9
14s
14t
Me
Me
>10
14j
H
H
3.0
6.2
14u
Me
3.6
0.8
14k
GDC-0941
The antiproliferative activity of the final products 14 were
evaluated against human rhabdomyosarcoma Rh30 cells by the
sulforhodamine B (SRB) assay. The results were summarized in
Table 2. Firstly, compounds 14a-o without any substituent at the
3

R1 position were synthesized and examined. Most of them
displayed antiproliferative activity against Rh30 cells at
concentrations in the micromolar range. Simple alkyl amides 14a-
d exhibited potent cellular activity, with IC50 values ranging from
1.9 to 3.5 µM. Introduction of an oxygen atom into the side chain
maintained the antiproliferative activity (compounds 14e, f).
However, Compounds 14g-h bearing a more basic group in the
amide chain almost lost their antiproliferative activity, in which
the basic side chain seems to be detrimental for cell membrane
permeability. Additionally, alkynyl, cyclopentyl and aryl
substituents were tolerable (compounds 14i-j and 14n-o).
Replacing the simple alkyl side chain of the amide segment with
six-membered heterocycles, such as morpholine, thiomorpholine
and piperazine, resulted in decreased antiproliferative activity
(compounds 14k-m).
5 (i.v.)
10 (p.o.)
1702
588
-
0.5
1.7
3.0
-
0.5
1.1
-
347
20%
Abbreviations: i.v., intravenous injection; p.o., per oral; AUC, area under
the concentration-time curve; Cmax, peak plama concentration of a drug
after administratration; MRT, mean residence time; CL, plasma clearance;
T1/2, elimination half-life; F, bioavailability.
In summary,
a series of 6-aminocarbonyl pyrrolo[2,1-
f][1,2,4]triazine derivatives were designed by scaffold hopping
strategy. The synthesized compounds were evaluated against
human rhabdomyosarcoma Rh30 cells by sulforhodamine B
(SRB) assay. Most compounds displayed potent antiproliferative
activity against Rh30 at concentrations in the micromolar range.
Selected compounds 14a, 14p and 14q showed antiproliferative
activity against other four human cancer lines. Among tested
compounds, 14q exhibited superior antiproliferative activity and
thus we investigated its PK property. Despite the PK profile of 14q
was not good enough to support itself for further development as
a drug candidate, this modification may provide a useful scaffold
for further optimization of PI3K inhibitors. Furthermore,
medicinal chemistry efforts are in progress to develop molecules
in this series for a potent PI3K inhibitors with improved PK profile
and the results will be reported in due course.
Subsequently, a methyl group was introduced at R1 position of
pyrrole ring, wishing to to increase their activity by improving the
cell membrane permeability. In line with this, all the compounds
with a methyl substituent at R1 position displayed stronger
antiproliferative activity (compounds 14p-q vs 14e-f and 14r-s vs
14g-h), especially 14p and 14q with IC50 values below 2 µM.
introducing piperidine cycle with different substituent to the amide
segment led to opposite activity (14t vs 14u). Therefore, it could
be concluded from the results in Table 2 that the introduction of a
methyl group was beneficial for increasing the antiproliferative
activity of the title compounds and the basicity of the amide chain
have significant effect on their cellular activities.
Acknowledgments
This work was supported by the National Natural Science
Foundation of China (90713034 and 81703365) ，Institutes for
Drug Discovery and Development, Chinese Academy of Sciences
（CASIMM0120185009）and the State Key Laboratory of Drug
Research Program (SIMM1705KF-15).
To further verify the antiproliferative activity of this series of
designed compounds, compounds 14a, 14p and 14q were selected
to evaluate their antiproliferative activity in other four types of
human cancer cells including human breast cancer BT-474, SK-
BR-3, T47D cells, and human ovarian carcinoma SKOV-3cells.
And these compounds showed weak antiproliferative activity
against the human hepatic cell line LO2. The results presented as
IC50 values were summarized in Table 3. All the three tested
compounds showed potent antiproliferative activity against these
four human cancer lines as well. And they were more potent
against T47D cells than other tested cancer cells with the IC50
values in the nanomolar range.
References and notes
1. A. Jemal, R. Siegel, E. Ward, T. Murray, J. Xu, M.J. Thun, CA: A Cancer J
Clin, 2007, 57, 43.
2. M. S. Kinch, Drug Discov Today, 2014, 19, 1831.
3. A. Jemal, F. Bray, M. M. Center, J. Ferlay, E. Ward, D. Forman, CA Cancer
J Clin, 2011, 61, 69.
4. X. X. Xie, H. Li, J. Wang, S. Mao, M.H. Xin, S.M. Lu, Q.B. Mei, S.Q.
Zhang, Bioorg Med Chem, 2015, 23, 6477.
5. X.M. Wang, J. Xu, M.H. Xin, S.M. Lu, S.Q. Zhang, Bioorg Med Chem Lett,
2015, 25, 1730.
Table 3. Antiproliferative activities of 14a, 14p and 14q
6. M. Huang, A. Shen, J. Ding, M. Geng, Trends Pharmacol Sci, 2014, 35, 41.
7. I. Vivanco, C.L. Sawyers, Nat Rev Cancer, 2002, 2, 489.
8. J.A. Engelman, J. Luo, L.C. Cantley, Nat Rev Genet, 2006, 7, 606.
9. P. Liu, H. Cheng, T.M. Roberts, J.J. Zhao, Nat Rev Drug Discov, 2009, 8,
627.
10. C.R. McNamara, A. Degterev, Future Med Chem, 2011, 3, 549.
11. J.E. Kurtz, I. Ray-Coquard, Anticancer Res, 2012, 32, 2463.
12. Y. Jeong, D. Kwon, S. Hong, Future Med Chem, 2014, 6, 737.
13. J. Zhu, T. Hou, X. Mao, Drug Discov Today, 2015, 20, 988.
14. C.J. Vlahos, W.F. Matter, K.Y. Hui, R.F. Brown, J Biol Chem, 1994, 269,
5241.
15. K. Kojima, M. Shimanuki, M. Shikami, I.J. Samudio, V. Ruvolo, P. Corn,
N. Hanaoka, M. Konopleva, M. Andreeff, H. Nakakuma, Leukemia, 2008,
22, 1728.
16. M.T. Burger, S. Pecchi, A. Wagman, Z.J. Ni, M. Knapp, T. Hendrickson,
G. Atallah, K. Pfister, Y. Zhang, S. Bartulis, K. Frazier, S. Ng, A. Smith,
J. Verhagen, J. Haznedar, K. Huh, E. Iwanowicz, X. Xin, D. Menezes, H.
Merritt, I. Lee, M. Wiesmann, S. Kaufman, K. Crawford, M. Chin, D.
Bussiere, K. Shoemaker, I. Zaror, S.M. Maira, C.F. Voliva, ACS Med
Chem Lett, 2011, 2, 774.
IC50 (µM)
Cpd
Rh30
BT-474
2.4±1.1
1.5±0.0
1.6±0.4
SK-BR-3
2.4±0.5
1.7±0.8
1.7±0.5
SKOV-3
2.3±0.8
2.1±0.7
2.8±1.7
T47D
LO2
14a
14p
14q
2.4±0.5
1.6±0.3
1.7±0.5
0.8±0.1 10.6±0.5
0.7±0.2
0.6±0.2
6.9±0.1
9.1±0.5
Among these compounds, 14p and 14q exhibited the most
potent antiproliferative activity against five human cancer cell
lines. However, the compound 14p was more toxic to the human
hepatic cell line LO2. As a consquence, compound 14q was
selected to further evaluate its PK properties in rats (Table 4).22
The mouse PK study revealed 14q had high clearance, low oral
exposure and short half-life. The oral availability of 14q was low
(F = 20%) and probably caused by low absorption. Comparing to
our previous reported PI3K inhibitor 1, despite the solubility and
oral availability of 14q have been improved somewhat, yet its PK
profile was not good enough to support itself for its anti-tumor
efficacy in vivo. Thus further efforts are warranted to be devoted
to improve the PK profile of this series of compounds.
17. S. Yaguchi, Y. Fukui, I. Koshimizu, H. Yoshimi, T. Matsuno, H. Gouda,
S. Hirono, K. Yamazaki, T. Yamori, J Natl Cancer Inst, 2006, 98, 545.
18. A.J. Folkes, K. Ahmadi, W.K. Alderton, S. Alix, S.J. Baker, G. Box, I.S.
Chuckowree, P.A. Clarke, P. Depledge, S.A. Eccles, L.S. Friedman, A.
Hayes, T.C. Hancox, A. Kugendradas, L. Lensun, P. Moore, A.G. Olivero,
J. Pang, S. Patel, G.H. Pergl-Wilson, F.I. Raynaud, A. Robson, N. Saghir,
L. Salphati, S. Sohal, M.H. Ultsch, M. Valenti, H.J. Wallweber, N.C. Wan,
Table 4. PK properties of 14q.
Dose
AUC0-∞
(ng·h/ml)
Cmax
(ng/ml)
MRT
(h)
CL
(l/h/kg)
T1/2
(h)
F
(mg/kg)
4

C. Wiesmann, P. Workman, A. Zhyvoloup, M.J. Zvelebil, S.J.
Shuttleworth, J Med Chem, 2008, 51, 5522.
19. S. Martinez Gonzalez, A.I. Hernandez, C. Varela, M. Lorenzo, F. Ramos-
Lima, E. Cendon, D. Cebrian, E. Aguirre, E. Gomez-Casero, M.I.
Albarran, P. Alfonso, B. Garcia-Serelde, G. Mateos, J. Oyarzabal, O.
Rabal, F. Mulero, T. Gonzalez-Granda, W. Link, J. Fominaya, M.
Barbacid, J.R. Bischoff, P. Pizcueta, C. Blanco-Aparicio, J. Pastor, Bioorg
Med Chem Lett, 2012, 22, 5208.
20. J. Wang, X. Wang, Y. Chen, S. Chen, G. Chen, L. Tong, L. Meng, Y. Xie,
J. Ding, C. Yang, Bioorg Med Chem Lett, 2012, 22, 339.
21. Y. Chen, H. Xiang, C. Tan, Y. Xie, C. Yang, Tetrahedron, 2013, 69, 2714.
22. PK studies were performed on male Sprague–Dawley rats (200–220 g) with
four animals. Compound 14q was administered via the oral route at 10
mg/kg or administered via the intravenous route at 5 mg/kg respectively.
Serial specimens (0.3 mL) were collected via the retrobulbar vein and
quantified by liquid chromatography–mass spectrometry (LC-MS). PK
parameters were calculated from the mean plasma concentration by non-
compartmental analyses. The protocol for this study was reviewed and
approved by the Institutional Animal Care and Use Committee of
Shanghai Institute of Materia Medica, Chinese Academy of Sciences.
(Shanghai, China).
5