Synthesis of propargylamine mycophenolate analogues and their selective cytotoxic activity towards neuroblastoma SH-SY5Y cell line

Article abstract of DOI:10.1016/j.bmcl.2021.128135

Twenty six propargylamine mycophenolate analogues were designed and synthesized from mycophenolic acid 1 employing a key step A³-coupling reaction. Their cytotoxic activity was examined against six cancer cell lines. Compounds 6a, 6j, 6t, 6u, a

Full text of DOI:10.1016/j.bmcl.2021.128135

Bioorg. Med. Chem. Lett. 45 (2021) 128135
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of propargylamine mycophenolate analogues and their selective
cytotoxic activity towards neuroblastoma SH-SY5Y cell line
,
Patamawadee Silalaia, Dumnoensun Pruksakornb c, Arthit Chairoungduad, Kanoknetr Suksend,
,e,*
Rungnapha Saeenga
a Department of Chemistry and Center for Innovation in Chemistry, Faculty of Science, Burapha University, Chonburi 20131, Thailand
b Musculoskeletal Science and Translational Research Center, Department of Orthopedics, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand
c Omics Center for Health Sciences, Faculty of Medicine, Chiang Mai University, Thailand
d Department of Physiology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand
e The Research Unit in Synthetic Compounds and Synthetic Analogues from Natural Product for Drug Discovery (RSND), Burapha University, Chonburi 20131, Thailand
A R T I C L E I N F O
A B S T R A C T
Keywords:
Twenty six propargylamine mycophenolate analogues were designed and synthesized from mycophenolic acid 1
employing a key step A3-coupling reaction. Their cytotoxic activity was examined against six cancer cell lines.
Compounds 6a, 6j, 6t, 6u, and 6z exhibited selective cytotoxicity towards neuroblastoma (SH-SY5Y) cancer cells
and were less toxic to normal cells in comparison to the lead compound, MPA 1 and a standard drug, ellipticine.
Molecular docking results suggested that compound 6a is fit well in the key amino acid of three proteins (CDK9,
EGFR, and VEGFR-2) as targets in cancer therapy. The propargylamine mycophenolate scaffold might be a
valuable starting point for development of new neuroblastoma anticancer drugs.
A3-coupling reaction
Cytotoxic activity
Mycophenolic Acid
Molecular docking
Neuroblastoma SH-SY5Y cells
Propargylamine mycophenolate
Cancer is an important category of human diseases accounting for an
estimated 9.6 million deaths in 2018 (WHO).1 Synthesis of selective
anticancer agents has attracted much research.2 Recently, therapies
have focused on reducing cancer toxicity to specific molecular targets
and damage to normal cells.3 Neuroblastoma, despite some successes
remains a challenging region for treatment via surgical resection,
chemotherapy, or radiation therapy.4,5 Neuroblastoma is one of the
most common solid tumors inflicting children and responsible for
approximately 9–15% of pediatric cancer deaths6 and remains one of the
most difficult cancers to cure.7 Standard treatment for neuroblastoma is
based on a combination of drugs such as doxorubicin, vincristine,
cyclophosphamide, and cisplatin.8 Although highly beneficial chemo-
therapy lacks selectivity towards cancer cells and has severe side effects.
To this end there is an urgent need for drugs with greater efficacy and
target specificity.9
nucleotide that causes a suppression of lymphocyte activity in humans
and organ rejection in patients who have undergone trans-
plantation.12,13 MPA was approved for use against organ rejection in
USA in 1995.14 Although most research has focused on their inhibitory
immunosuppressive properties,15–17 recent rekindled interest has
focused on the synthesis of novel mycophenolate derivatives in regards
their anticancer potency.18–20 IMPDH receptors are overexpressed in
cancer cells compared to normal cells. Inhibition of IMPDH can inhibit
proliferation of and induce apoptosis in cancer cells.21 This suggests
MPA and derivatives might function as improved anticancer agents.
MPA was found as a potential antiproliferative agent, however MPA
therapy is associated with gastrointestinal (GI) adverse events.22–24 The
incidence of MPA-related GI adverse events ranges from 45 to 80% in
recipients.25,26 Therefore, we designed and synthesized of new MPA
analogues by esterification of free carboxylic group of MPA 1 to myco-
phenolate 2 (Scheme 1) and propargylation at phenolic position
(Scheme 3) and their use as a structural core for further modification to a
series of propargylamine mycophenolate analogues to reduce GI toxicity
affects.
Mycophenolic acid (MPA) is an early antibiotic drug derived from
Penicillium.10 It consists of a phthalide in which an aromatic ring is
substituted of hydroxy, methyl, methoxy, and six-carbon chain with a
double bond in the trans configuration and the free carboxylic acid.11
MPA is a highly noteworthy inhibitor of inosine monophosphate dehy-
drogenase (IMPDH), a key enzyme in the de novo synthesis of guanine
The propargylamine moiety (Fig. 1) occurs in therapeutic active
agents27,28 such as rasagiline and deprenyl that show highly selective
* Corresponding author at: Department of Chemistry and Center for Innovation in Chemistry, Faculty of Science, Burapha University, Chonburi 20131, Thailand.
E-mail address: rungnaph@buu.ac.th (R. Saeeng).
https://doi.org/10.1016/j.bmcl.2021.128135
Received 4 March 2021; Received in revised form 5 May 2021; Accepted 19 May 2021
Available online 24 May 2021
0960-894X/© 2021 Elsevier Ltd. All rights reserved.

P. Silalai et al.
Bioorganic & Medicinal Chemistry Letters 45 (2021) 128135
Scheme 1. Esterification of MPA 1.
Scheme 2. A3-coupling reaction for the synthesis of propargylamine mycophenolate.
Scheme 3. Synthesis of propargylether mycophenolate 3a. aReaction conditions: a) 1 (0.0062 mol), H2SO4 (1.0 mL), CH3OH (30.0 mL), 6 h, b) propargylbromide
(3.0 equiv), K2CO3 (3.0 equiv), DCM (30.0 mL), 0 ◦C-rt, 92%.
Fig. 1. Some of the representative propargylamine analogues as excellent candidates against different cancer types.
2

P. Silalai et al.
Table 1
Bioorganic & Medicinal Chemistry Letters 45 (2021) 128135
Optimization of the synthesis of MPA analog 6aa
Entry
Catalyst
Ligand
Solvent
Yield [%]b
1
CuI
–
Toluene
Dioxane
THF
68
92
24
20
29
52
29
70
80
70
64
61
45
52
41
31
76
NR
NR
2
CuI
–
3
CuI
–
4
CuI
–
EtOH
5
CuI
–
CH3CN
DMF
6
CuI
–
7
CuI
–
DMSO
8
CuCl
–
Dioxane
Dioxane
Dioxane
Dioxane
Dioxane
Dioxane
Dioxane
Dioxane
dioxane
dioxane
dioxane
dioxane
9
CuCl2
CuBr2
Cu(OAc)2
CuSO4
CuI
–
10
11
12
13
14
15
16
17
18
19
–
–
–
2,2′-bipy
CuI
PPh3
AgNO3
AgOAc
ZnCl2
Zn powder
NiCl.2DME
–
–
–
–
–
a
Reaction conditions: 3 (0.1344 mmol), 4 (0.6720 mmol), 5a (0.1478 mmol), catalyst (50 mol%), ligand (20 mol%), in solvent (1.0 mL) at the 120 ◦C
for 1 h.
b
Isolated yields. NR = No reaction.
and noteworthy anticancer activity against neuroblastoma cell lines
(SH-SY5Y).29,30 Two synthetic propargylamines displayed a high degree
of cytotoxic selectivity (breast and pancreatic cancer cells). The levels of
selectivity give a very interesting perspective for further development of
a new anticancer agent.31
DME was used as a catalyst (entries 18–19). The reaction with CuI in
dioxane provided optimum conditions for the A3-coupling reaction with
a product yield of 92% (Table 1, entry 2).
Then we explored the scope of reaction using various amine de-
rivatives for synthesizing a series of propargylamine MPA analogues for
study the structure activity relationship. This A3-coupling reaction
provides good functional group compatibility and high yields of
designed product and water is the only byproduct. Various substituted
piperazine were introduced to compound 3 to give the first analogues
6a-6h in good to excellent yields. The hydroxy- and cyano-phenyl
piperazine gave lower yield of 6e and 6f comparing to other pipera-
zine. Chloro-benzyl and piperonyl piperazine gave the desired coupling
product 6g and 6h in high yields. Diphenylpiperazine derivatives served
as suitable amine substrates in providing N-propargylated products 6i,
6j, and 6k in excellent yields. Acetyl, hydroxyethyl, pyrimidyl, and
carbonylbenzodioxane-piperazine gave coupling products 6l, 6m, 6n,
and 6p in up to 80% yields. When this reaction condition was applied to
the piperazine bearing pyridine group, the desired product 6o was not
produced probably due to complexation of pyridine with copper (Cu) led
to a reduction of reactivity in CuI. The scope of reaction was expanded to
piperidine derivatives with various substituents including benzyl group,
carboxylic acid, methyl ester and tetrahydroquinoline led to products
6q-6u in moderate to high yields. All products were well tolerated under
the optimized conditions. Morpholine and pyrrolidine were also
employed as substrates, affording products 6v and 6w in moderate
yields. Secondary aliphatic amines provided excellent yields of products
6x-6y. Also, we found the primary amine gave bis-mycophenolate
product 6aa in moderated yields (Scheme 4).
In this work, we designed and synthesized a series of propargylamine
mycophenolate derivatives by A3-coupling reaction.32,33 The A3-
coupling methods involves the metal-catalyzed alkyne addition to im-
ines or in situ formation of imines derived from amines and aldehydes
(Scheme 2). The present study evaluated all synthetic analogues for
cytotoxic activity against six cancer cells including neuroblastoma cell
(SH-SY5Y).
Preparation of propargylether mycophenolate 3 as the precursor for
synthesis of a series of propargylamine mycophenolate is outlined in
Scheme 3. Esterification of the MPA 1 with CH3OH in the presence of
H2SO4 afforded the methyl mycophenolate 2 followed by propargylation
reaction of 2 with propargyl bromide and K2CO3 gave compound 3 in
92% yields in two steps.
The A3-coupling reaction was investigated for synthesis a series of
propargylamine mycophenolate 6 by screening several catalysts and
solvents to find the optimal condition. Propargylether mycophenolate 3,
formaldehyde 4 and phenylpiperazine 5a were employed as the model
substrates. The reaction was carried out at 120 ◦C for 1 h and detailed
optimization conditions are presented in Table 1.
The A3-coupling reaction in the presence of catalytic amounts of CuI
(50 mol%) in toluene afforded the desired product 6a in 68% yields
(Table 1, entry 1). Interestingly, by changing the solvent to dioxane, we
were able to improve product yield significantly to 92% yields (entry 2).
Encouraged by this result, reaction was investigated with other solvents
including THF, EtOH, CH3CN, DMF and DMSO (entries 3–7), however,
yields were not improved over those with dioxane. Yields with other
copper salts, CuCl, CuCl2, CuBr2, Cu(OAc)2, and CuSO4 (entries 8–12),
were 70, 80, 70, 64, and 61%. Ligands such as 2,2′-bipy, and PPh3 did
not improve yields of 6a (entries 13–14). Ag catalysts such as AgNO3 and
AgOAc produced lower yields than CuI (entries 15–16). When ZnCl2 was
replaced, coupling product 6a was produced with 76% yield (Table 1,
entry 17). No desired product was obtained when Zn powder or NiCl2.
Cytotoxic activities of mycophenolic acid (MPA 1) and synthetic
analogues were evaluated in vitro against selected cancer cell lines
including KKU-M213 (human intrahepatic cholangiocarcinoma), FaDu
(human squamous cell carcinoma), HT-29 (human colorectal adeno-
carcinoma), MDA-MB-231 (human mammary gland/breast adenocar-
cinoma), A-549 (human lung carcinoma), SH-SY5Y (human
neuroblastoma), and MMNK-1 (Highly differentiated immortalized
human cholangiocyte cell line) in Table 2. MMNK-1 is a healthy cell line
reference. Sulforhodamine
B (SRB) assay was used to evaluate
3

P. Silalai et al.
Bioorganic & Medicinal Chemistry Letters 45 (2021) 128135
Scheme 4. Substrate scope of propargylamine mycophenolate derivativesa. aReactions performed on 0.2685 mmol scale of 3. All isolated yields were based on 3.
4

P. Silalai et al.
Bioorganic & Medicinal Chemistry Letters 45 (2021) 128135
in activity. The replacement of piperazine on propargyl-MPA scaffold
with piperidine bearing benzyl, carboxylic acid and ester 6q-6s showed
the cytotoxicity results to confirm the importance of the benzyl ring on
MPA. Benzyl substitution piperidine 6q displayed greater activity than
at other piperidine analogues 6r and 6s. Hydroquinoline-MPA 6u
showed significant activity against SH-SY5Y and A549 cell lines with
IC50 values of 6.45–7.68 and exhibited cytotoxicity against all six cancer
cell lines with IC50 value below 40.0 µM. Analog 6t also demonstrated
comparable activity against SH-SY5Y and A549 cell lines to that of 6u.
Further modification to study the SAR of propargyl-MPA series by
introducing heterocyclic and aliphatic amine led to a series of novel
propargyl-MPA bearing morpholine 6v, pyrrolidine 6w, aliphatic amine
moieties 6x-6z and bis-MPA analog 6aa. Butylaniline derivative 6z
Table 2
In vitro cytotoxicity, of MPA 1, propargylether-mycophenolate 3 and a series of
newly synthesized analogues 6a-6aa.
Comp.
IC50 (µM)a
KKU-
FaDu
HT-
29
MDA-
A-
SH-
MNN-
K1
M213
MB-231
549
SY5Y
MPA 1
2
7.16
7.45
35.73
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
25.92
>50
>50
38.18
>50
22.91
48.32
1.97
6.72
6.15
15.25
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
24.55
>50
>50
30.89
>50
21.21
46.68
1.85
6.48
23.73
28.79
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
39.54
>50
46.47
>50
>50
36.71
>50
1.95
7.83
9.62
49.23
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
28.66
>50
>50
39.78
>50
19.76
48.63
2.03
1.21
0.56
7.19
5.82
25.25
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
23.29
>50
>50
35.99
36.43
18.33
48.19
1.89
0.82
0.26
3
31.79
21.97
>50
3.07
6a
9.14
6b
6c
43.62
35.78
37.70
46.10
37.79
31.24
34.80
23.74
9.90
45.30
>50
6d
6e
>50
6f
>50
showed excellent cytotoxicity with IC50 values of 6.97 and 5.96 μM
6g
>50
against A-549 cells and the SH-SY5Y cell line and lower toxicity to
normal cells than ellipticine.
6h
6i
45.48
32.68
29.06
34.08
>50
MPA 1 and mycophenolate 2 exhibited cytotoxic activity against all
cancer cell lines especially on A-549 and SH-SY5Y, however both com-
pounds were toxic also to normal cells. Introducing propargyl group led
to compound 3 which was selectively toxic to neuroblastoma (SH-SY5Y)
cancers cells with an IC50 of 3.07 µM but non-toxic to normal cell. A3-
coupling reaction of 3 with various amines led to a series of analogues 6
in which compounds 6a, 6j, 6t, 6u, and 6z showed high cytotoxic ac-
tivity on neuroblastoma (SH-SY5Y) cancers cells but no toxicity to
normal cell line (MMNK-1; Fig. 2).
6j
6k
6l
25.83
>50
6m
6n
6p
6q
6r
>50
35.67
>50
>50
>50
>50
40.69
>50
24.50
47.22
41.16
9.57
6s
40.51
18.02
7.68
6t
6u
6v
6.45
The modification of the piperazine ring by inclusion of a phenyl
group and chloro-diphenyl at N-4 (compounds 6a and 6j) significantly
improved cytotoxicity against neuroblastoma (SH-SY5Y) cancer cell
lines and was not toxic to the normal cell line (MMNK-1) relative to MPA
1, compounds 2 and 3 (Fig. 2 and Table 2). Replacement of the piper-
azine scaffold with hydroquinoline produced analogues 6t and 6u that
exhibited comparable cytotoxicity to that of 6a and 6j on the neuro-
blastoma cancer cell line. Substituted phenyl-hydroquinoline in com-
pound 6u and butylaniline 6z significantly improved their anticancer
activity against A-549, while other derivatives reduced their anticancer
activity on this cancer cell line. Synthesized compounds 6a, 6j, 6t, 6u,
and 6z effectively kill lung and neuroblastoma cancer cells at a con-
centration that is not toxic to normal cells.
>50
>50
6w
6x
35.93
30.56
43.47
6.97
31.08
21.53
36.43
5.96
6y
6z
6aa
Ellipticine
27.52
1.16
20.56
2.13
a
IC50 values (drug concentration causing 50% growth inhibition) in μM.
cytotoxicity of synthesized compounds. Analogues were dissolved in
DMSO (<0.05%). Cytotoxic potency was expressed as the concentration
that inhibited 50% of cell viability (IC50, Table 2). Ellipticine was used as
a standard drug for comparison.
The cytotoxicity results (IC50 values) of MPA 1 and synthetic prop-
argylamine mycophenolate 6a-6aa were summarized in Table 2. MPA 1
exhibited greater cytotoxic activity than ellipticine on SH-SY5Y cancer
cell lines with IC50 0.56 µM and comparable activity on A-549 cell line.
Modification by esterification and propargylation of MPA 1 to give
propargylether mycophenolate 3 led to a reduction in toxicity to normal
cells. Compound 3 showed lower toxicity to normal cells (IC50 MNN-K1
= 25.25 µM) but good inhibition against SH-SY5Y (human neuroblas-
toma) cell line with IC50 values 3.07 µM. From this result, the intro-
duction of propargyl moiety may play an important role for selective
cytotoxic activity on neuroblastoma. Further modification of this
propargyl-MPA scaffold by introducing of various amines led to ana-
logues 6. Cytotoxic activity of some analogues 6 indicated them to be
more selective towards SH-SY5Y cell line and not toxic to normal cells.
The first modified phenyl piperazine-propargyl MPA analog 6a showed
selective cytotoxic activity on SH-SY5Y cell line with IC50 values 9.14
µM and no toxicity to the normal cells. Substituted phenyl- and benzyl-
piperazine-MPA analogues 6b-6f and 6g-6h showed lower activity on
SH-SY5Y cell than 6a. However, introducing of diphenyl piperazine on
MPA led to compounds 6i-6 k which showed activity effective than the
mono-phenyl piperazine MPA series on SH-SY5Y and A-549 cancer cells.
Chlorine analog 6j demonstrated good selective toxicity on SH-SY5Y cell
line with IC50 value of 9.90 µM. Piperazine-propargyl MPA scaffolds
containing acetyl 6l, hydroxyethyl 6m, pyrimidyl 6n, and carbon-
ylbenzodioxane 6p did not show any significant activity at concentra-
tions lower than 50 µM. These results indicated the phenyl- and benzyl-
substituent on piperazine plays an important role in improving cyto-
toxicity of MPA. Analogues without these fragment displayed a decrease
Propargylamine MPA analogues was explored for the first time on
cytotoxicity to neuroblastoma (SH-SY5Y) cancer cells and confirms the
important role played by the molecular docking method in under-
standing the interaction mode of MPA derivative 6a as the most selective
inhibitor with the three proteins CDK9, EGFR and VEGFR-2. These three
proteins have been reported as targets in cancer therapy including
neuroblastoma SH-SY5Ycells.34–36 Cyclin dependent kinases (CDKs) are
the serine/threonine kinases group37 which involved in cell cycle pro-
gression and cell proliferation.38 CDK9 are regulators for the RNA
transcription of short-lived Mcl-1 proteins in neuroblastoma SH-SY5Y-
cells.39 The inhibition of CDK9 associated with blocking RNA synthesis
can lead to inhibiting neuroblastoma cancer cell growth. Next protein,
epidermal growth factor receptor (EGFR) is shown in neuronal origin
and various tissues of epithelial and plays a significant role in initiating
the signaling that directs growth, proliferation, survival, and differen-
tiation in mammalian cells.40 The widespread expression of EGFR pro-
tein was found in neuroblastoma tissues and cell lines, which is of
interested for future treatment of neuroblastoma by targeting this pro-
tein.41,42 The protein, vascular endothelial growth factor receptors
(VEGFRs) are tyrosine kinase receptors that play an essential role in
controlling the cellular proliferation of the cancer cells including neu-
roblastoma cells.43,44 The VEGFRs family consists of three members
including VEGFR-2 which plays an important role in cancer angiogen-
esis. Blocking of EGFR and VEGFR-2 signaling pathway might regulate
the growth of neuroblastoma cancer cells which are interesting ap-
proaches for cancer therapy.45,46
The docking simulations were performed using Autodock 4.2 soft-
ware as an automated tool for predicting the binding mode by the
5

P. Silalai et al.
Bioorganic & Medicinal Chemistry Letters 45 (2021) 128135
Fig. 2. The propargylamine mycophenolate derivatives showed noteworthy and selective cytotoxicity against lung (A-549) and neuroblastoma (SH-SY5Y) can-
cers cells.
Table 3
Table 5
Molecular docking analysis of CDK9/cyclin T with propargylamine-MPA 6a. The
binding energies were evaluated using AutoDock.
Molecular docking analysis of VEGFR-2 with propargylamine-MPA 6a. The
binding energies were evaluated using AutoDock.
Compound
Binding
energy
Intermolecular
Intermolecular
hydrophobic
contacts
Compound
Binding
energy
Intermolecular hydrogen
bonding
Intermolecular
hydrogen bonding
hydrophobic contacts
(kcal/mol)
(kcal/mol)
Amino acids
interaction
Distance
(Å)
Amino acids
interaction
Distance
(Å)
6a
ꢀ 13.46
Cys106
Lys151
Asn154
2.89
3.04
2.88
Ile25, Phe105,
Ala46, Leu156
6a
ꢀ 16.09
Asp1046
Lys868
3.19
3.36
Cys919, Leu1035,
Ala866, Leu889,
Val899
Propargylamine-MPA 6a exhibited the low binding energy values of
ꢀ 13.46 kcal/mol with CDK9 and showed the formation of key hydrogen
bonds of the lactone group of MPA interacted with Cys106 residues
(Fig. 3). Moreover, the methyl ester group showed hydrogen bonding
interaction with Lys151 and Asn154. Interestingly, the phenyl and
alkyne moiety in compound 6a rests in a hydrophobic pocket formed by
Ile25, Ala46, and Leu156 as a key residue. Based on the binding inter-
action, compound 6a interact with the key amino acid residues at the
active site of CDK9.
Table 4
Molecular docking analysis of EGFR with propargylamine-MPA 6a. The binding
energies were evaluated using AutoDock.
Compound
Binding
energy
Intermolecular hydrogen
bonding
Intermolecular
hydrophobic contacts
(kcal/mol)
Amino acids
interaction
Distance
(Å)
6a
ꢀ 14.06
Met793
Lys721
3.25
3.15
Leu753, Met742,
Leu834, Leu764, and
Val702
In order to rationalize the observed SARs, analog 6a was investigated
for the binding orientation with EGRF. Compound 6a displayed binding
energy value of ꢀ 14.06 Kcal/mol with EGRF (Table 4). The oxygen atom
of propargylether and the carbonyl group of ester formed the two
hydrogen bonds Met769 and Lys721 residues respectively (Fig. 4). As
expectation, the phenyl piperazine of 6a is located in the active region
and promotes hydrophobic interactions with Leu753, Met742, Leu834
and Leu764. Additionally, the propargyl group displayed interactions
with Val702.
selective analog 6a (Table 3–5) with the X-ray crystallographic structure
of CDK9, EGFR protein kinases and VEGFR-2 receptor, obtained from
the Protein Data Bank with PDB code 3LQ5, 4HJO and 4ASD,
respectively.
6

P. Silalai et al.
Bioorganic & Medicinal Chemistry Letters 45 (2021) 128135
Fig. 3. Docking of 6a into the active site of CDK9. (a) The protein structure is shown in ribbon and propargylamine-MPA 6a is shown as stick model. (b) 2D
interaction molecular docking diagrams. Hydrogen bonds are shown as green dashed lines. (c) Protein residues are in surface representation.
7

P. Silalai et al.
Bioorganic & Medicinal Chemistry Letters 45 (2021) 128135
Fig. 4. Docking of 6a into the active site of EGFR. (a) The protein structure is shown in ribbon and propargylamine-MPA 6a is shown as stick model. (b) 2D
interaction molecular docking diagrams. Hydrogen bonds are shown as green dashed lines. (c) Protein residues are in surface representation.
8

P. Silalai et al.
Bioorganic & Medicinal Chemistry Letters 45 (2021) 128135
Fig. 5. Docking of 6a into the active site of VEGFR-2. (a) The protein structure is shown in ribbon and propargylamine-MPA 6a is shown as stick model. (b) 2D
interaction molecular docking diagrams. Hydrogen bonds are shown as green dashed lines. (c) Protein residues are in surface representation.
Similarly, propargylamine-MPA 6a was investigated for the binding
orientation into the active site of VEGFR-2 and exhibited the lowest
binding energy ꢀ 16.09 kcal/mol (Table 5). Compound 6a displayed the
formation of the two hydrogen bonds of the lactone ring with Lys868
and the propargylether with the key amino acid Asp1046 (Fig.5). Other
parts of the molecule showed hydrophobic interactions with Cys919 as a
key residue, Leu1035, Ala866, Leu889, Val899. These results indicated
that propargylamine-MPA 6a had potential to be the novel VEGFR-2
inhibitors.
demonstrated the propargyl ether and substituted phenyl group of MPA
play very significant roles in cytotoxic activity against neuroblastoma
SH-SY5Y cell lines. Propargylamine-MPA analogues could serve as a
promising candidate for further studies as an anticancer agent against
neuroblastoma.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Molecular docking simulation with all three proteins supported the
observed high activity of 6a. The propargyl ether and substituted phenyl
group of MPA analog 6a showed a strong hydrogen bond and displayed
good hydrophobic interaction while compound 6v with low cytotoxicity
showed less interaction. The calculated binding potential of 6v
comparing with 6a are represented in Table S1 and Figs. S1-S3. (SI, page
S41-S43), which led to a more clear understanding of the cytotoxicity
against neuroblastoma SH-SY5Y cell lines provided by these
interactions.
Acknowledgements
This project is funded by National Research Council of Thailand
(NRCT), The Research Unit in Synthetic Compounds and Synthetic An-
alogues from Natural Product for Drug Discovery (RSND) and the Center
of Excellence for Innovation in Chemistry (PERCH-CIC). R.S. and P.S. are
grateful for support through a PhD scholarship from Science Achieve-
ment Scholarship of Thailand. The authors would like to thank Assistant
Prof. Suchaya Pongsai, Faculty of Science, Burapha University, for a
helpful discussion on docking simulations techniques. Special thanks to
Prof. Dr Frederick W. H. Beamish, Faculty of Science, Burapha Univer-
sity, for his comments and English correction.
In summary, a simple and highly efficient method for the synthesis of
propargylamine-MPA analogues through A3-coupling reaction is re-
ported. It provides good functional group compatibility, atom economy
and highly quantitative yields of designed product without a co-catalyst
or activator. Water is the only byproduct. All propargylamine MPA an-
alogues were evaluated for their in vitro cytotoxity. Among tested
compounds, phenylpiperazine MPA 6a, chloro-diphenylpiperazine MPA
6j, hydroquinoline MPA 6t and 6u, and butylaniline MPA 6z showed
selective and promising inhibition against SH-SY5Y cancer cell lines.
They exhibited little evidence of toxicity toward human normal cells
relative to MPA 1 and the drug, ellipticine. Computational studies
revealed that 6a possesses a high affinity with CDK9 (ꢀ 13.46 kcal/mol),
EGFR (-14.06 kcal/mol), and VEGFR-2 (ꢀ 16.09 kcal/mol). Our results
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bmcl.2021.128135.
9

P. Silalai et al.
Bioorganic & Medicinal Chemistry Letters 45 (2021) 128135
patients with autoimmune disease: a Phase III, open-label, single-arm, multicenter
study. Clin Exp Gastroenterol. 2015;8: 205-213. https://doi.org/10.2147/CEG.
S81922.
References
1 WHO factsheet. https://www.who.int/news-room/fact-sheets/detail/cancer, (2018).
2 Xu S, Yao H, Pei L, et al. Design, synthesis, and biological evaluation of NAD(P)H:
Quinone oxidoreductase (NQO1)-targeted oridonin prodrugs possessing
indolequinone moiety for hypoxia-selective activation. Eur J Med Chem. 2017;132:
310–321. https://doi.org/10.1016/j.ejmech.2017.03.055.
25 Budde K, Curtis J, Knoll G, et al. Enteric-coated mycophenolate sodium can be safely
administered in maintenance renal transplant patients: results of a 1-year study. Am
J Transplant. 2004;4(2): 237-243. https://doi.org/10.1046/j.1600-
6143.2003.00321.x.
26 Meier-Kriesche HU, Davies NM, Grinyo J, et al. Mycophenolate sodium does not
reduce the incidence of GI adverse events compared with mycophenolate mofetil.
Am J Transplant. 2005;5(5): 1164; author reply 1165-1166. https://doi.org/
10.1111/j.1600-6143.2005.00778.x.
3 Samutprasert P, Chiablaem K, Teeraseranee C, et al. Epigallocatechin gallate-zinc
oxide co-crystalline nanoparticles as an anticancer drug that is non-toxic to normal
cells. RSC Adv. 2018;8(14):7369–7376. https://doi.org/10.1039/C7RA10997K.
4 Ishola TA, Chung DH. Neuroblastoma. Surg Oncol. 2007;16(3):149–156. https://doi.
org/10.1016/j.suronc.2007.09.005.
27 Bolea I, Gella A, Unzeta M. Propargylamine-derived multitarget-directed ligands:
fighting Alzheimer’s disease with monoamine oxidase inhibitors. J Neural Transm
(Vienna). 2013;120(6):893–902. https://doi.org/10.1007/s00702-012-0948-y.
28 Wünsch M, Senger J, Schultheisz P, et al. Structure-Activity Relationship of
Propargylamine-Based HDAC Inhibitors. ChemMedChem. 2017;12(24):2044–2053.
https://doi.org/10.1002/cmdc.201700550.
5 Paulino AC, Ferenci MS, Chiang K-Y, Nowlan AW, Marcus RB. Comparison of
conventional to intensity modulated radiation therapy for abdominal neuroblastoma.
Pediatr Blood Cancer. 2006;46(7):739–744. https://doi.org/10.1002/pbc.
6 Wagner LM, Danks MK. New therapeutic targets for the treatment of high-risk
neuroblastoma. J Cell Biochem. 2009;107(1):46–57. https://doi.org/10.1002/jcb.
v107:110.1002/jcb.22094.
29 Inaba-Hasegawa K, Akao Y, Maruyama W, Naoi M. Rasagiline and selegiline,
inhibitors of type B monoamine oxidase, induce type A monoamine oxidase in
human SH-SY5Y cells. J Neural Transm (Vienna). 2013;120(3):435–444. https://doi.
org/10.1007/s00702-012-0899-3.
7 Smith V, Foster J. High-Risk Neuroblastoma Treatment Review. Children (Basel).
2018;5(9):114. https://doi.org/10.3390/children5090114.
8 Tibullo D, Giallongo C, Puglisi F, et al. Effect of Lipoic Acid on the Biochemical
Mechanisms of Resistance to Bortezomib in SH-SY5Y Neuroblastoma Cells. Mol
Neurobiol. 2018;55(4):3344–3350. https://doi.org/10.1007/s12035-017-0575-6.
9 Bagnyukova TV, Serebriiskii IG, Zhou Y, Hopper-Borge EA, Golemis EA, Astsaturov I.
Chemotherapy and signaling: How can targeted therapies supercharge cytotoxic
agents. Cancer Biol Ther. 2010;10(9):839–853. https://doi.org/10.4161/
cbt.10.9.13738.
30 Wu Y, Shamoto-Nagai M, Maruyama W, Osawa T, Naoi M. Rasagiline prevents
cyclosporine A-sensitive superoxide flashes induced by PK11195, the initial signal of
mitochondrial membrane permeabilization and apoptosis. J Neural Transm (Vienna).
2016;123(5):491–494. https://doi.org/10.1007/s00702-016-1531-8.
31 Martinez-Amezaga M, Giordano RA, Prada Gori DN, et al. Synthesis of
propargylamines via the A(3) multicomponent reaction and their biological
evaluation as potential anticancer agents. Org Biomol Chem. 2020;18(13):2475–2486.
https://doi.org/10.1039/D0OB00280A.
10 Ardestani F, Fatemi S-A, Yakhchali B, Hosseyni SM, Najafpour G. Evaluation of
mycophenolic acid production by Penicillium brevicompactum MUCL 19011 in
batch and continuous submerged cultures. Biochem Eng J. 2010;50(3):99–103.
11 Regueira TB, Kildegaard KR, Hansen BG, Mortensen UH, Hertweck C, Nielsen J.
Molecular basis for mycophenolic acid biosynthesis in Penicillium brevicompactum.
Appl Environ Microbiol. 2011;77(9):3035–3043. https://doi.org/10.1128/
AEM.03015-10.
32 Choi Y-J, Jang H-Y. Copper-Catalyzed A3-Coupling: Synthesis of 3-Amino-1,4-
diynes. Eur J Org Chem. 2016;2016(18):3047–3050. https://doi.org/10.1002/
ejoc.201600343.
33 Saha TK, Das R. Progress in Synthesis of Propargylamine and Its Derivatives by
Nanoparticle Catalysis via A3 coupling: A Decade Update. ChemistrySelect. 2018;3(1):
147–169. https://doi.org/10.1002/slct.201702454.
12 Shaw LM, Figurski M, Milone MC, Trofe J, Bloom RD. Therapeutic drug monitoring
of mycophenolic acid. Clin J Am Soc Nephrol. 2007;2(5):1062–1072. https://doi.org/
10.2215/CJN.03861106.
34 Li Y, Guo Q, Zhang C, et al. Discovery of a highly potent, selective and novel CDK9
inhibitor as an anticancer drug candidate. Bioorg Med Chem Lett. 2017;27(15):
3231–3237. https://doi.org/10.1016/j.bmcl.2017.06.041.
13 T.-Y. Wu Y. Peng L.L. Pelleymounter et al. Pharmacogenetics of the mycophenolic
acid targets inosine monophosphate dehydrogenases IMPDH1 and IMPDH2: gene
sequence variation and functional genomics 161 7 2010 1584 1598 10.1111/j.1476-
5381.2010.00987.x.
35 Maiti P, Nand M, Joshi T, Ramakrishnan MA, Chandra S. Identification of luteolin -7-
glucoside and epicatechin gallate from Vernonia cinerea, as novel EGFR L858R
kinase inhibitors against lung cancer: Docking and simulation-based study. J Biomol
Struct Dyn.. 2020:1–10 https://doi.org/10.1080/07391102.2020.1784791.
36 Roaiah HM, Ghannam IAY, Ali IH, et al. Design, synthesis, and molecular docking of
novel indole scaffold-based VEGFR-2 inhibitors as targeted anticancer agents. Arch
Pharm (Weinheim). 2018;351(2):1700299. https://doi.org/10.1002/ardp.
v351.210.1002/ardp.201700299.
14 Hodge EE. The role of mycophenolate mofetil in clinical renal transplantation. World
J Urol. 1996;14:249–255.
15 El-Araby ME, Bernacki RJ, Makara GM, Pera PJ, Anderson WK. Synthesis, molecular
modeling, and evaluation of nonphenolic indole analogs of mycophenolic acid.
Bioorg Med Chem. 2004;12(11):2867–2879. https://doi.org/10.1016/j.
bmc.2004.03.048.
37 Roskoski Jr R. Cyclin-dependent protein serine/threonine kinase inhibitors as
anticancer drugs. Pharmacol Res. 2019;139:471–488. https://doi.org/10.1016/j.
phrs.2018.11.035.
16 Yang Na, Wang Q-H, Wang W-Q, et al. The design, synthesis and in vitro
immunosuppressive evaluation of novel isobenzofuran derivatives. Bioorg Med Chem
Lett. 2012;22(1):53–56. https://doi.org/10.1016/j.bmcl.2011.11.078.
17 Iwaszkiewicz-Grzes D, Cholewinski G, Kot-Wasik A, Trzonkowski P, Dzierzbicka K.
Synthesis and biological activity of mycophenolic acid-amino acid derivatives. Eur J
Med Chem. 2013;69:863–871. https://doi.org/10.1016/j.ejmech.2013.09.026.
18 Dun B, Sharma A, Teng Y, et al. Mycophenolic acid inhibits migration and invasion of
gastric cancer cells via multiple molecular pathways. PLoS ONE. 2013;8(11):e81702.
https://doi.org/10.1371/journal.pone.0081702.
38 Ding L, Cao J, Lin W, et al. The Roles of Cyclin-Dependent Kinases in Cell-Cycle
Progression and Therapeutic Strategies in Human Breast Cancer. Int J Mol Sci. 2020;
21(6):1960. https://doi.org/10.3390/ijms21061960.
39 Bettayeb K, Baunbaek D, Delehouze C, et al. CDK Inhibitors Roscovitine and CR8
Trigger Mcl-1 Down-Regulation and Apoptotic Cell Death in Neuroblastoma Cells.
Genes Cancer. 2010;1(4):369–380. https://doi.org/10.1177/1947601910369817.
40 Wee P, Wang Z. Epidermal Growth Factor Receptor Cell Proliferation Signaling
Pathways. Cancers (Basel). 2017;9(5). https://doi.org/10.3390/cancers9050052.
41 Tamura S, Hosoi H, Kuwahara Y, et al. Induction of apoptosis by an inhibitor of EGFR
in neuroblastoma cells. Biochem Biophys Res Commun. 2007;358(1):226–232. https://
doi.org/10.1016/j.bbrc.2007.04.124.
19 Felczak K, Vince R, Pankiewicz KW. NAD-based inhibitors with anticancer potential.
Bioorg Med Chem Lett. 2014;24(1):332–336. https://doi.org/10.1016/j.
bmcl.2013.11.005.
20 Koonrungsesomboon N, Ngamphaiboon N, Townamchai N, et al. Phase II, multi-
center, open-label, single-arm clinical trial evaluating the efficacy and safety of
Mycophenolate Mofetil in patients with high-grade locally advanced or metastatic
osteosarcoma (ESMMO): rationale and design of the ESMMO trial. BMC Cancer.
2020;20(1). https://doi.org/10.1186/s12885-020-06751-2.
42 Zheng C, Shen R, Li K, et al. Epidermal growth factor receptor is overexpressed in
neuroblastoma tissues and cells. Acta Biochim Biophys Sin (Shanghai). 2016;48(8):
762–767. https://doi.org/10.1093/abbs/gmw064.
43 Smith Gina A, Fearnley Gareth W, Tomlinson Darren C, Harrison Michael A,
Ponnambalam S. The cellular response to vascular endothelial growth factors
requires co-ordinated signal transduction, trafficking and proteolysis. Biosci Rep.
2015;35(5). https://doi.org/10.1042/BSR20150171.
21 Quemeneur L, Flacher M, Gerland LM, Ffrench M, Revillard JP, Bonnefoy-Berard N.
Mycophenolic acid inhibits IL-2-dependent T cell proliferation, but not IL-2-
dependent survival and sensitization to apoptosis. J Immunol. 2002;169(5):
2747–2755. http://www.jimmunol.org/content/169/5/2747.
44 Beierle EA, Dai W, Langham MR, Copeland EM, Chen MK. VEGF receptors are
differentially expressed by neuroblastoma cells in culture. J Pediatr Surg. 2003;38(3):
514–521. https://doi.org/10.1053/jpsu.2003.50091.
22 Behrend M. Adverse gastrointestinal effects of mycophenolate mofetil:aetiology,
incidence and management. Drug Saf. 2001;24(9):645–663. https://doi.org/
10.2165/00002018-200124090-00002.
45 Lindsey S, Langhans SA. Epidermal growth factor signaling in transformed cells. Int
Rev Cell Mol Biol. 2015;314:1–41. https://doi.org/10.1016/bs.ircmb.2014.10.001.
46 Shibuya M. Vascular Endothelial Growth Factor (VEGF) and Its Receptor (VEGFR)
Signaling in Angiogenesis: A Crucial Target for Anti- and Pro-Angiogenic Therapies.
Genes Cancer. 2011;2(12):1097–1105. https://doi.org/10.1177/
23 Lopez-Solis R, DeVera M, Steel J, et al. Gastrointestinal side effects in liver transplant
recipients taking enteric-coated mycophenolate sodium vs. mycophenolate mofetil.
Clin Transplant. 2014;28(7):783–788. https://doi.org/10.1111/ctr.2014.28.issue-
710.1111/ctr.12379.
1947601911423031.
24 Manger B, Hiepe F, Schneider M, et al. Impact of switching from mycophenolate
mofetil to enteric-coated mycophenolate sodium on gastrointestinal side effects in
10