Biphenyl-4-carboxylic acid [2-(1 H -indol-3-yl)-ethyl]-methylamide (CA224), a nonplanar analogue of fascaplysin, inhibits cdk4 and tubulin polymerization: Evaluation of in vitro and in vivo anticancer activity

Article abstract of DOI:10.1021/jm5014743

Biphenyl-4-carboxylic acid-[2-(1H-indol-3-yl)-ethyl]-methylamide 1 (CA224) is a nonplanar analogue of fascaplysin (2) that specifically inhibits Cdk4-cyclin D1 in vitro. Compound 1 blocks the growth of cancer cells at G₀/G₁ phase of the cell cycle. It also blocks the cell cycle at G₂/M phase, which is explained by the fact that it inhibits tubulin polymerization. Additionally, it acts as an enhancer of depolymerization for taxol-stabilized tubulin. Western blot analyses of p53-positive cancer cells treated with compound 1 indicated upregulation of p53, p21, and p27 proteins together with downregulation of cyclin B1 and Cdk1. Compound 1 selectively induces apoptosis of SV40 large T-antigen transformed cells and significantly reduces colony formation efficiency, in a dose-dependent manner, of lung cancer cells. It is efficacious at 1/10th of the MTD against human tumors derived from HCT-116 and NCI-H460 cells in SCID mouse models. The promising efficacy of compound 1 in human xenograft models as well as its excellent therapeutic window indicates its potential for clinical development.

Full text of DOI:10.1021/jm5014743

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Article
Biphenyl-4-carboxylic acid [2-(1H-indol-3-yl)-ethyl]-methylamide
(CA224), a non-planar analog of fascaplysin inhibits Cdk4 and tubulin
polymerization: Evaluation of in vitro and in vivo anticancer activity
Sachin Mahale, Sandip Bibishan Bharate, sudhakar manda, prashant joshi, Sonali
S. Bharate, Paul R Jenkins, Ram A. Vishwakarma, and Bhabatosh Chaudhuri
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm5014743 • Publication Date (Web): 04 Nov 2014
Downloaded from http://pubs.acs.org on November 8, 2014
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Page 1 of 45
Journal of Medicinal Chemistry
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Biphenyl-4-carboxylic acid [2-(1H-indol-3-yl)-ethyl]-
methylamide (CA224), a non-planar analog of
fascaplysin inhibits Cdk4 and tubulin
polymerization: Evaluation of in vitro and in vivo
anticancer activity
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‡
,
, †,ǁ
†,ǁ
†, ǁ
⊥
Sachin Mahale, Sandip B. Bharate,* Sudhakar Manda, Prashant Joshi, Sonali S. Bharate, Paul
∞
, †,ǁ
,‡
R. Jenkins, Ram A. Vishwakarma,* Bhabatosh Chaudhuri*
‡
†
School of Pharmacy, De Montfort University, Leicester, LE1 9BH, UK
Medicinal Chemistry Division, CSIRꢀIndian Institute of Integrative Medicine, Canal Road, Jammuꢀ
1
80001, India
ǁ
Academy of Scientific & Innovative Research (AcSIR), CSIRꢀIndian Institute of Integrative Medicine,
Canal Road, Jammuꢀ180001, India
⊥Preformulation Laboratory, CSIRꢀIndian Institute of Integrative Medicine, Canal Road, Jammuꢀ
180001, India
∞
Department of Chemistry, University of Leicester, Leicester, LE1 7RH, UK
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ABSTRACT
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Biphenylꢀ4ꢀcarboxylic acidꢀ[2ꢀ(1Hꢀindolꢀ3ꢀyl)ꢀethyl]ꢀmethylamide 1 (CA224) is a nonꢀplanar analog of
fascaplysin (2) that specifically inhibits Cdk4ꢀcyclin D1 inꢀvitro. Compound 1 blocks growth of cancer
cells at G /G phase of the cell cycle. It also blocks at G /M phase which is explained by the fact that it
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inhibits tubulin polymerization. Besides, it acts as an enhancer of depolymerization for taxolꢀstabilized
tubulin. Westernꢀblot analyses of p53ꢀpositive cancer cells treated with compound 1 indicated upꢀ
regulation of p53, p21 and p27 proteins together with downꢀregulation of cyclin B1 and Cdk1.
Compound 1 selectively induces apoptosis in SV40 large Tꢀantigen transformed cells and significantly
reduces colony formation efficiency, in a doseꢀdependent manner of lung cancer cells. It is efficacious
th
at 1/10 the MTD, against human tumors derived from HCTꢀ116 and NCIꢀH460 cells in SCID mice
models. The promising efficacy of compound 1 in human xenograft models with an excellent
therapeuticꢀwindow indicates its potential for clinical development.
KEYWORDS
Cdk4ꢀcyclin D1, tubulin polymerization, fascaplysin, antiꢀcancer, chemotherapeutic, cell division cycle
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INTRODUCTION
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The mammalian cell division is a highly controlled process and the loss of this control results in
development of cancer phenotype. Mammalian cells undergo various stages of the cell cycle (G , G , S,
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G , Mꢀphase), and the transition of cells from one phase to another involves various checkꢀpoint
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mechanisms including cyclinꢀdependent kinases, checkpoint kinases, and their partner cyclins.
Malfunctioning of any of these checkpoint mechanisms leads to uncontrolled cell division and
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proliferation. Cyclinꢀdependent kinase 4 and its cyclin partner D1 (checkꢀpoint mechanism) controls
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ꢀ9
transition of the cells from G1 to S phase of the cell cycle. Active Cdk4ꢀcyclin D complexes
inactivate retinoblastoma protein (pRb) by phosphorylating specifically at Ser780 and Ser795 residues,
6, 10
which allows the G /S transition of cells during the cell cycle.
Hyperphosphorylation of pRb leads to
1
loss of control over gene transcription through the E2F family of transcription factors, which ultimately
turns into uncontrolled cell division. In contrast, pRb phosphorylation by Cdk2 or Cdk3 along with their
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cyclin partners A or C is not sufficient to inactivate pRb functions.
A large number of human tumors
(around 96%) lose normal cell cycle transition checkꢀpoint mechanism due to variety of genetic and
biochemical adaptations including the hyperactivity of the Cdk4 protein, downꢀregulation of Cdk4
INK4A
positive regulator p16
and mutations in pRb. The vital role of Cdk4ꢀcyclin D1 in tumor
development has been confirmed by experiments in Cdk4 and cyclin D1 knockꢀout mice which have
been shown to be resistant to tumor development. On the other hand, antisense nucleotides based
targeting of cyclin D decreases tumor size and growth inꢀvivo. Based on these experimental
observations, Cdk4ꢀcyclin D complex protein is considered as a promising target for combating
14ꢀ16
cancer.
Inhibition of Cdk4ꢀcyclin D1 with small molecules has been an area of major interest in the field of
anticancer drug discovery since last two decades. There have been numerous scientific reports
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highlighting the role of Cdk4ꢀcyclin D1 inhibitors in cancer treatment.
Pfizer's palbociclib (6ꢀacetylꢀ
8
ꢀcyclopentylꢀ5ꢀmethylꢀ2ꢀ{[5ꢀ(1ꢀpiperazinyl)ꢀ2ꢀpyridinyl]amino}pyrido[2,3ꢀd]pyrimidinꢀ7(8H)ꢀone;
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PDꢀ0332991), a selective inhibitor of Cdk4 and Cdk6 has received FDA approval for treatment of
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patients with breast cancer.
Other Cdk4/Cdk6 dual inhibitors LY2835219 (Nꢀ[5ꢀ[(4ꢀethylꢀ1ꢀ
piperazinyl)methyl]ꢀ2ꢀpyridinyl]ꢀ5ꢀfluoroꢀ4ꢀ[4ꢀfluoroꢀ2ꢀmethylꢀ1ꢀ(1ꢀmethylethyl)ꢀ1Hꢀbenzimidazolꢀ6ꢀ
yl]ꢀ2ꢀpyrimidinamine methanesulfonate) and LEE011 (7ꢀcyclopentylꢀN,Nꢀdimethylꢀ2ꢀ((5ꢀ(piperazinꢀ1ꢀ
yl)pyridinꢀ2ꢀyl)amino)ꢀ7Hꢀpyrrolo[2,3ꢀd]pyrimidineꢀ6ꢀcarboxamide) are in phase I and III clinical
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5, 26
studies for breast cancer (Source: http://clinicaltrials.gov).
The main objective of our studies was to develop potent and specific small molecule inhibitors of
Cdk4ꢀcyclin D1 based on the structure of fascaplysin (2) which is a pentacyclic quaternary salt
2
7
originally isolated from the Fijian sponge Fascaplysinopsis Bergquist sp.
It is known to possess
It also displays potent
cytotoxicity against small cell lung cancer cells and induces cell cycle arrest in G /G at lower and Sꢀ
2
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antimicrobial, antimalarial and antiꢀacetylcholinesterase activities.
0
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phase at higher concentrations, respectively. Compound 2 also showed antiꢀtumor effects in sarcoma
3
1
mice model through apoptotic and antiꢀangiogenesis pathways. Fascaplysin is one of the specific
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, 32
inhibitor of Cdk4ꢀcyclin D1 with IC value of 410 nM.
It inhibits Cdk4ꢀcyclin D1 inꢀvitro and
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0
blocks the growth of normal and cancer cells at the G /G phase of the cell cycle, which correlates with
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the accumulation of hypoꢀphosphorylated pRb, implying there is no phosphorylation at Cdk4ꢀspecific
9
serine residues. Compound 2 has also been reported to inhibit Cdc25B with an IC of 1 µg/ml (3.26
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0
3
3, 34
µM).
An analogue of fascaplysin 1ꢀdeoxysecofascaplysin A, has been reported to inhibit cell growth
3
5
of breast cancer MCFꢀ7 and ovarian cancer OVCARꢀ3 cell lines inꢀvitro.
Nonetheless, it is unlikely that fascaplysin will ever be used therapeutically as an anticancer agent
because it is highly toxic. The potential for its planar structure to intercalate with doubleꢀstranded DNA
has been suggested as a possible explanation for its unusual biological activity and toxicity. The DNA
binding property of fascaplysin is similar to structurally related DNA intercalating agents like
3
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cryptolepine and ellipticine. We have explored the possibility of separating the DNA intercalating
ability of fascaplysin from its potent Cdk4ꢀspecific inhibitory activity. The aim of the current study was
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therefore to devise nonꢀplanar (nonꢀtoxic) Cdk4ꢀcyclin D1 inhibitors based on the structure of
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fascaplysin.
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Biphenylꢀ4ꢀcarboxylic acid [2ꢀ(1Hꢀindolꢀ3ꢀyl)ꢀethyl]ꢀmethylamide 1 (CA224) was initially
identified as a Cdk4ꢀcyclin D1 specific inhibitor lacking the ability to intercalate DNA. As expected,
compound 1 manifested its Cdk4ꢀspecific inhibitory ability by blocking cancer cells at the G /G phase
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of the cell cycle. Surprisingly however, compound 1 also inhibited the G /M phase in a Cdkꢀ
2
independent manner. Further investigations showed that the compound 1 inhibited the growth of a panel
of ten cancer cell lines (some of them potentially resistant to cancer chemotherapy) at a low micromolar
range. The validity of these results was corroborated by the observation that compound 1 has the ability
to diminish the colony formation efficiency of cancer cells. The Cdkꢀindependent G /M block was
2
found to be associated with its antiꢀtubulin activity. Compound 1 inhibited the polymerization of tubulin
inꢀvitro and also showed an enhancing effect on tubulin depolymerization in live cells. In human
xenograft models, compound 1 was found to inhibit the growth of HCTꢀ116 and Hꢀ460 tumor growth at
th
1
/10 of the MTD, 100 mg/kg. Here, we present the in vitro and inꢀvivo anticancer activity of
compound 1 in detail.
RESULTS AND DISCUSSION
Chemistry. Our efforts towards discovery of nonꢀtoxic analogs of fascaplysin led to the
identification of compound 1, a nonꢀplanar analog of fascaplysin (2). Briefly, the synthesis of compound
1
involves three steps starting from commercially available tryptamine (3). The treatment of tryptamine
(3) with ethyl chloroformate produced carbamate ester 4, which further on reduction using LAH led to
formation of Nꢀmethyl tryptamine 5. Compound 5 on reaction with 4ꢀbiphenyl carbonyl chloride (6)
produced compound 1. The detailed lead optimization and SAR have been described in previous
3
7ꢀ41
publications.
The synthetic scheme for synthesis of compound 1 is shown in Scheme 1. It was
interesting to note that the nonꢀplanar analogs with opened C and D rings of fascaplysin (2), led to loss
of DNA intercalation activity. Among C and Dꢀring opened analogs, compounds with paraꢀsubstituted
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Eꢀrings (structures shown in Table 1) maintained their ability to inhibit Cdk4 in a selective manner.
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O
N
NH2
N
H
O
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H
b
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N
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H
H
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N
H
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Scheme 1. Synthesis of biphenylꢀ4ꢀcarboxylic acid [2ꢀ(1Hꢀindolꢀ3ꢀyl)ꢀethyl]ꢀmethylamide (1):
Reagents and conditions. (a) Ethyl chloroformate, NaOH 4 N, CH Cl , 3 h, 90%; (b) LiAlH , THF, N ,
2
2
4
2
reflux, 1 h, 85%; (c) NaOH 4 N, CH Cl , 3 h, 45%.
2
2
Selective inhibition of Cdk4-cyclin D1. A series of new compounds based on the structure of
fascaplysin were identified as specific inhibitors of enzyme Cdk4ꢀcyclin D1 (Table 1). Compound 1 was
found to be the most potent inhibitor of Cdk4ꢀcyclin D1 (IC = 6 µM) and selective when the Cdk4
50
IC was compared with the IC s obtained in Cdk2ꢀcyclin A, Cdk1ꢀcyclin B1 and Cdk9ꢀcyclin T1
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assays. Compound 1 was better than its other two structural analogs Nꢀ(2ꢀ(1Hꢀindolꢀ3ꢀyl)ethyl)ꢀ4ꢀ
chloroꢀNꢀmethylbenzamide (CA223, 7) and Nꢀ(2ꢀ(1Hꢀindolꢀ3ꢀyl)ethyl)ꢀ4ꢀtertꢀbutylꢀNꢀ
methylbenzamide (CA225, 8), with >6ꢀfold better activity against Cdk4ꢀcyclin D1 (Table 1). Unlike
41
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1
fascaplysin, compound 1 does not intercalate with DNA. Compound 1 was also tested against 58
represenative kinases at Millipore Bioscience Division, UK. It was found that compound 1 was inactive
at the concentration of 10 ꢀM against all kinases including Cdk5ꢀp35, Cdk6ꢀcyclin D1, Cdk7ꢀcyclin H,
EGFR, GSK3β, MAPK1, MEK1, PDGFR, Plk3, PKA, PKCα, IGFꢀ1R etc. Results are shown in
supporting information. These results support compound 1’s selective ability to inhibit Cdk4ꢀcyclin D1
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enzyme inꢀvitro while having much reduced or no affinity for other kinases tested.
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Table 1. Activity of fascaplysin (2) and its nonꢀplanar analogs 1, 7 and 8 in inꢀvitro kinase and DNA
a
binding assays
O
O
O
Cl
D
N
N
N
C N
E
E
A
B
E
Assay
A
B
A
B
E
N
A
B
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N
H
N
N
Cl
H
H
H
O
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7
1
8
Cdk4ꢀcyclin D1
Cdk2ꢀcyclin A
Cdk2ꢀcyclin E
Cdk1ꢀcyclin B1
Cdk9ꢀcyclin T1
0.41 ± 0.04
>250
38 ± 6
6.2 ± 0.9
521 ± 11
ND
49 ± 6.5
731 ± 26
ND
658 ± 23
ND
>250
>250
>500
>500
>500
>250
>1000
>1000
>1000
EtBr
5 ± 0.4
Does not displace
Does not displace
Does not displace
displacement
a
IC values are presented in µM. All the fascaplysin analogs were dissolved in 100% DMSO and were
5
0
further diluted in the kinase assay buffer or the ethidium bromide displacement assay buffer. ND: not
determined.
In order to understand the observed selectivity towards Cdk4ꢀcyclin D1 versus Cdk2ꢀcyclin A,
molecular modeling studies were carried out. The ATP binding sites of these two Cdks are well
conserved and share 45% sequence homology with each other, however compound 1 displayed varying
degree of affinity to these Cdks. These two Cdks differ from each other, primarily by these three residue
Cdk4
Cdk2
Cdk4
sequences: 94ꢀ97(GluꢀHisꢀValꢀAsp) /81ꢀ84(GluꢀPheꢀLeuꢀHis) , 101ꢀ102(ArgꢀThr) /88ꢀ89(Lysꢀ
Cdk2
Cdk4
Cdk2
Lys)
and Glu144 /Gln131 . Compound 1 interacts flexibly with ATP binding pockets of Cdk4
and Cdk2 with 84ꢀfold selectivity towards Cdk4 due to flexible conformational movement of the
compound 1 amide bond (1 interacts with Cdk4 and Cdk2 in two different conformational states), which
allows free rotation of biphenyl ring. This subsequently leads to loss of major hydrophobic interactions
with Cdk2. Two conformations of compound 1 include (a) transꢀconformation (green colored ligand in
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Figure 1a; dihedral angle, Ψ = ꢀ151.5) which was found to interact selectively with side chains of
Arg101 residue of Cdk4 by hydrophobic πꢀcation interaction. However in Cdk2, this interaction is
missing because corresponding Lys88 residue side chain is far away from compound 1 binding cavity;
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5
6
(b) cisꢀconformation (orange colored ligand in Figure 1a; dihedral angle, Ψ = 2.6) which interacts with
Cdk2. On the other hand, the transꢀconformation of compound 1 is stabilized in Cdk4 binding site
because negativelyꢀcharged Glu144 side chain oppose biphenyl aromatic ring. In case of Cdk2, the cisꢀ
conformation is stabilized because biphenyl aromatic rings are well accommodated by the
corresponding neutral Gln131. The amino acid comparison of Cdk4 and Cdk2 binding site residues are
shown in Figure 1c.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
cis-1 – Cdk2
trans-1 – Cdk4
(a)
(b)
Protein
Cdk4
Amino acid sequence
96 97
101 102 144
Glu His Val Asp Arg Thr Glu
81 82 83 84
88 89 131
Glu Phe Leu His Lys Lys Gln
94
95
Cdk2
(c)
Figure 1. Molecular modeling studies of compound 1 with Cdk4 and Cdk2. (a) Interactions of cisꢀ and
transꢀconformations of compound 1 with Cdk2 and Cdk4, respectively (cisꢀ and transꢀ conformations of
compound 1 are shown in orange and green colors, respectively. Similarly Cdk2 and Cdk4 residue
labels are shown in orange and green colors, respectively). (b) compound 1ꢀCdk4 interaction histogram
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Journal of Medicinal Chemistry
with different binding site residues during 10 ns MD simulation of compound 1ꢀCdk4 protein complex
c) amino acid comparison between Cdk4 and Cdk2 at ATP binding site (black colored residues
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(
indicates commonly interacting residues of Cdk4/Cdk2, while red colored residues are those which
differ in interaction pattern in these two Cdks).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Cancer cell growth inhibition. All the fascaplysin analogues including the most potent compound
in the series, compound 1, were tested in a panel of ten different cancer cell lines (some of them are
known to be relatively resistant to known chemotherapeutic agents) for their ability to inhibit cancer cell
growth inꢀvitro. The inhibitory effects of compounds were quantified using the MTT assay and IC s
50
were determined (i.e. the concentration, expressed in µM, of a compound at which 50% cell growth was
inhibited). The results of these cell proliferation assays indicated that compound 1 inhibits the growth of
41
cancer cells inꢀvitro at low micromolar concentrations (Table 2). Amongst all the analogues,
compound 1 was found to be the most potent molecule even at cellular level. The inhibition of cell
growth was both p53 and pRbꢀindependent, the latter indicating that Cdk4ꢀcyclin D1 inhibition is not
the only cellular target for the mechanism of action of these molecules.
Table 2. IC concentrations expressed in ꢀM for inꢀvitro cell growth inhibition induced by exposure to
5
0
4
1
compound 1, and its analogues 7-8 for 48 h.
O
O
O
Cl
N
N
N
C N
E
E
A
B
a
D
B
E
B
A
B
E
N
A
A
Cell lines
N
H
N
N
Cl
H
H
H
O
2
7
1
8
LS174T
0.88 ± 0.04
0.92 ± 0.06
ND
42 ± 2.5
47 ± 3
3.5 ± 0.9
6.2 ± 1.1
4.0 ± 0.3
3.5 ± 0.6
18 ± 1
15 ± 1.5
PCꢀ3
MiaPaCa
A549
31± 2.2
27 ± 2.5
10.2 ± 0.9
12 ± 1.8
0.69 ± 0.03
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Caluꢀ1
1.3 ± 0.1
ND
78 ± 3.0
24 ± 1.8
21 ± 0.9
26 ± 2.0
29 ± 2.4
32 ± 1.0
11.5 ± 2.5
52 ± 3.5
9.8 ± 1.0
11.5 ± 1.6
14 ± 1.4
18 ± 2.0
22 ± 1.8
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
NCIꢀH460
NCIꢀH1299
NCIꢀH358
BNL CL2
2.0 ± 0.3
2.5 ± 0.3
2.2 ± 0.6
2.6 ± 0.9
3.8 ± 0.9
ND
ND
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
ND
BNL SV A.8
ND
a
LS174T: colorectal carcinoma (p53+, pRb+); PCꢀ3: prostate (p53 null, pRb+); MiaPaCa: pancreatic
p53His273 mut, pRb+); A549: NSCLC (p53+, pRb+); Caluꢀ1: NSCLC (p53 null, pRb+); NCIꢀH460:
(
NSCLC (p53+, pRb+); NCIꢀH1299: NSCLC (p53 null, pRb+); NCIꢀH358: NSCLC (p53 null, pRb
null); BNL CL2: mouse normal hepatic cells; BNL SV A.8: mouse hepatic; SVꢀ40 mediated
transformed cells. ND: not determined.
Compound 1 retains the G /G block in serum starved p53-null Calu-1 cells. Compound 1,
0
1
although identified as a Cdk4ꢀspecific inhibitor in the inꢀvitro enzyme assay, generally tends to block
cancer cells more profoundly at G /M than G /G phase of the cell division cycle. Moreover, compound
2
0
1
1
inhibits tubulin polymerization inꢀvitro with higher potency than it inhibits enzyme Cdk4ꢀcyclin D1.
In Caluꢀ1 cells, the mitotic spindle checkpoint is impaired implying that these cells cannot be blocked at
G /M. Hence, it was decided to treat Caluꢀ1 cells with compound 1 after release from cell
2
synchronization at G /G (cells being starved of serum using 0.1% FBS for 24 h). At IC and IC
0
1
50
70
4
0
concentrations of compound 1 the G /G block was either partially or full maintained.
Since the
0
1
maintenance of G /G block after serum starvation requires Cdk4 enzyme to be inactive, these results
0
1
indicate that compound 1 probably inhibits cellular Cdk4 at these concentrations and thereby maintains
the G /G block. In the absence of any other kinase inhibitory data, the results obtained with Caluꢀ1
0
1
cells tend to confirm the Cdk4 inhibitory potential of compound 1 in live cells.
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Journal of Medicinal Chemistry
Compound 1 at the IC concentration induces profound G /M block in asynchronous cancer
50
2
1
2
cells (p53+) A549 and (p53-null) NCI-H1299 cells. Incubation of A549 (p53+) cells with compound 1
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
at IC concentration for 24 h induces a profound block at G /M as indicated by the percentage of cells
5
0
2
at G /M. As seen in Figure 2, at the IC concentration of compound 1, 89% cells appear to be arrested
2
50
in the G /M phase (Figure 2B) and at IC concentration, 91% cells blocked at G /M (not shown). Upon
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
70
2
incubation of NCIꢀH1299 (p53ꢀnull) with IC concentration of compound 1 resulted in large number of
5
0
cells (71% cells) accumulation at G /M (Figure 2D). The results confirm that a profound G /M block
2
2
can be observed in cells (A549 and NCIꢀH1299) where the mitotic spindle checkpoint is intact.
Nocodazole and paclitaxel induced G /M block is maintained by compound 1 in NCI-H358
2
lung cancer cells. NCIꢀH358 (p53ꢀnull) cells were treated with nocodazole (1 µM, a subꢀoptimal
concentration) only for 18 h in order to induce a partial block at G /M so that treated cells are minimally
2
stressed. The blocked cells were released in fresh medium when cells readily entered the cell cycle
without any apoptosis. When blocked cells were released in the presence of compound 1 for 12 h, cells
not only maintained the G /M block but also >50% of G /G and S phase cells entered G /M (compare
2
0
1
2
Figure 2EꢀH). Although only representative histograms of nocodazole treatment are shown, similar
results were obtained when paclitaxel blocked cells were released in the presence of compound 1 for 12
h. These observations suggest that, at least, in p53ꢀnull/pRbꢀnull NCIꢀH358 cells, compound 1
maintains the proꢀmetaphase block induced by nocadozole or paclitaxel during mitosis.
Compound 1 blocks NCI-H358 cells in G /M after release from hydroxyurea-mediated G /S
2
1
cell synchronization. Hydroxyurea (250 µM, 18 h) was used to block cells at G /S (77%; Figure 2J), at
1
a stage of the cell cycle where Cdk2ꢀspecific inhibitors normally act. When released in the presence of
compound 1, cells proceed from G /S, confirming that compound 1 does not inhibit cellular Cdk2. Cells
1
ultimately accumulate at G /M (72%; Figure 2L). These results again indicate that compound 1 have an
2
inherent tendency to induce block at G /M phase of the cell cycle at least in cells where mitoic spindle
2
checkpoint is normal.
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Selective killing of SV40 large T-antigen transformed normal mouse embryonic liver cells by
compound 1. SV40 large Tꢀantigen inactivates both the tumor suppressor proteins, p53 and pRb, and
thereby transforms normal cells into tumorigenic cells. We used normal mouse embryonic hepatic
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(liver) cells BNL CL2 and its SVꢀ40 large Tꢀantigen transformed counterpart BNL SV A.8 to study the
effects of compound 1. The normal cells, upon 48 h incubation with 1, exhibited prominent G /M arrest
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
at both IC and IC concentrations with less than 5% cells detected in the subꢀG /G phase (Figure 2N)
5
0
70
0
1
and more than 50% cells appearing at G /M phase of the cell cycle. Interestingly, in the SV40 large Tꢀ
2
antigen transformed cell line, significant apoptotic cell death was observed. After 48 h treatment at the
IC concentration of compound 1, 31% cells were detected in subꢀG phase (Figure 2P) indicating
5
0
1
apoptosis. The percent apoptosis increased further from 31 to 44% at the IC concentration after 48 h
7
0
incubation (data not shown). Similar results were obtained using normal and SVꢀ40 large Tꢀantigen
transformed human lung fibroblast cells, WIꢀ38 (data not shown).
A549
NCI-H1299
Compound 1, IC50, 24 h
Control
/G = 56%
S = 21%
G /M = 18%
Compound 1, IC50, 24 h
/G = 22%
Control
G /G = 49%
G /G = 3%
G
0
1
G
G
0
2
1
A
0
1
0
1
B
C
D
S = 29%
S = 4%
S = 10%
/M = 71%
G
2
/M = 22%
G
2
/M = 89%
2
PMT4 dna Lin
PMT4 dna Lin
PMT4 dna Lin
PMT4 dna Lin
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NCI-H358
Journal of Medicinal Chemistry
Nocodazole, 18 h;
Nocodazole, 18 h;
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Nocodazole, 18 h
/G = 22%
Control
Released in medium, 12 h
Released in compound 1, IC50, 12 h
G
/G
S = 21%
G /M = 18%
= 59%
G
0
1
F
G
G /G = 40%
G /G = 7%
H
E
0
1
0
1
0
1
S = 24%
S = 10%
S = 10%
G /M = 74%
2
G
2
/M = 48%
G /M = 42%
2
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
PMT4 dna Lin
PMT4 dna Lin
PMT4 dna Lin
PMT4 dna Lin
Hydroxyurea, 250 ꢀM, 18 h
Released in fresh medium, 18 h
Hydroxyurea, 250 ꢀM, 18 h
Released in compound 1, IC50, 18 h
Control
Hydroxyurea, 250 ꢀM, 18 h
I
J
K
L
G
0
/G
S = 21%
G /M = 18%
1
= 59%
G
0
/G
S =16%
G /M = 6%
1
= 77%
G
0
/G
S = 12%
G /M = 33%
1
= 50%
G
0
/G
S = 10%
G /M = 72%
1
= 16%
2
2
2
2
PMT4 dna Lin
PMT4 dna Lin
PMT4 dna Lin
PMT4 dna Lin
BNLCL2 (mousenormal hepaticcell line)
BNLSV A. 8 (mouse SV40 transformed hepaticcell line)
Control
Compound 1, IC50, 48 h
Control
Compound 1, IC , 48 h
50
G /G = 34%
N
O
G /G = 64%
P
G /G = 31%
M
G /G = 57%
0
1
0
1
0
1
0
1
S = 9%
S = 23%
S = 21%
G /M = 14%
2
S = 15%
G /M = 23%
2
G /M = 54%
G /M = 13%
2
2
Sub G
1
= 2%
Sub G = 31%
1
Sub G = 0%
Sub G1 = 0%
1
Sub G1
Sub G
1
Sub G1
Sub G1
PMT4 dna Lin
PMT4 dna Lin
PMT4 dna Lin
PMT4 dna Lin
Figure 2. Cell cycle analysis of compound 1 treated cells. (AꢀD) Response of mitotic spindle checkpointꢀproficient human
lung cancer cell lines, A549 and NCIꢀH1299 to compound 1. Flow cytometric analysis of asynchronous cells show that
majority of cells are arrested in G /M phase of cell cycle (4n DNA content) in both the cell lines. A549 untreated (A),
2
treatment with IC50 concentration of compound 1 for 24 h (B), NCIꢀH1299 untreated or control (C) and treatment with IC50
concentration of compound 1 for 24 h (D). (EꢀL) Analysis of NCIꢀH358 cells using flow cytometer. The G /M and G /S
2
1
synchronized cells by nocodazole and hydroxyurea respectively were released either in fresh medium or in fresh medium
containing IC50 concentration of compound 1 exhibit greater tendency of compound 1 to block the cell growth at G /M. For
2
nocodazole block experiment, figure show untreated or control cells (E), treated with 1 µM nocodazole for 18 h (F), treated
with 1 µM nocodazole for 18 h and released in fresh medium (G) and treated with 1 µM nocodazole for 18 h and released in
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the presence of compound 1, IC50 (H). For hydroxyurea block experiment, figure show untreated or control cells (I), treated
with 250 µM hydroxyurea for 18 h (J), treated with 250 µM hydroxyurea for 18 h and released in fresh medium (K) and
treated with 250 µM hydroxyurea for 18 h and released in the presence of compound 1, IC50 (L). (MꢀP) Selective apoptosis
in SVꢀ40 transformed cells by compound 1, analysed by FACS. BNL CL2 (mouse embryonic normal hepatic cells) when
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
exposed to compound 1 for 48 h at IC50 concentration (N), show prominent G /M arrest. BNL SV A. 8 (mouse embryonic
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
SVꢀ40 transformed hepatic cells) underwent apoptotic cell death upon incubation with compound 1. The apoptosis was
quantitated by measuring the % cells appeared in SubG peak, 31% and 44% cells were found in SubG peak after 48 h
1
1
exposure with compound 1, IC (P) concentration. The untreated cells (O) do not show any apoptosis.
5
0
CIP1/WAF1
Effect of compound 1 on the cellular level of cyclin B1, Cdk1, p53, p21
(p21) and
KIP1
p27
(p27); analyses in p53+ cells. Westernꢀblot analyses of p53+ cells, A549 and LS174T, after
treatment with compound 1 (at the IC50 concentration) for 24 h demonstrated more than 10ꢀfold
p21CIP1/WAF1
induction of p53. Thereby, probably the global Cdk inhibitor
(p21) was also induced. The
KIP1
levels of p27
(p27) were also elevated after compound 1 treatment (Figure 3). The proteins Cdk1
and cyclin B1 were downꢀregulated in the treated cells as compared with untreated control cells.
Repression of cyclin B1 and Cdk1 and elevated levels of p21 and p27 is a possible explanation of the
G /M block seen in A549 and LS174T cells that bear functional copies of the tumor supressor protein,
2
p53 (Figure 3).
CIP1/WAF1
KIP1
The effects on the cellular level of cyclin B1, Cdk1, p53, p21
(p21) and p27
(p27);
analyses in p53ꢀnegative cells were also investigated. Westernꢀblot analyses on proteins from
MIAPaCaꢀ2 cells (carrying a p53 mutation) showed that p53, p21 and p27 levels remained unchanged
indicating that the p21 and p27 induction seen in p53ꢀpositive cells is probably p53 dependent.
Interestingly cyclin B1 and Cdk1 levels were elevated and phosphorylation of Cdk1 at the residue Tyr15
remains unaffected (data not shown) indicating that Cdk1ꢀcyclin B1 is still active in p53 mutated cells,
after compound 1 treatment.
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Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
LS174T
A549
Control
Treated
Control
Treated
p53
p21
p27
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Cyclin B1
Cdk1
Actin
Figure 3. Western-blot analysis of p53+ cells, A549 and LS174T. The treatment with compound 1, IC50 concentration for
CIP1/WAF1
24 h resulted in induction of tumor suppressor protein p53 and thereby global Cdk inhibitor p21
(p21) was also
KIP1
induced. The levels of global p27
(p27) were elevated while cyclin B1 and Cdk1 levels were down regulated.
Cell-free tubulin polymerization assays in-vitro indicate inhibition of tubulin polymerization
by compound 1. Compound 1 inhibited growth of cancer cells inꢀvitro at relatively low concentrations
than it inhibited the enzyme Cdk4ꢀcyclin D1. FACS analysis and mitotic index experiments (data not
shown) had indicated that compound 1 blocked cell growth at the proꢀmetaphase of the cell cycle. In
addition to these observations, the 2ꢀ3 fold increase of compound 1 IC in cells with impaired mitotic
5
0
spindle checkpoint suggested another cellular role for compound 1, possibly as a antimicrotubule agent.
We investigated the action of compound 1 on tubulin polymerization inꢀvitro. The results indicated
strong antitubulin activity of compound 1 (Figure 4a) while fascaplysin does not show any interaction
with tubulin (data not shown). Representative polymerization curves of compound 1, paclitaxel and
nocodazole are shown in Figure 4a. Compound 1 inhibits the polymerization of tubulin which is
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concluded from the doseꢀdependent decrease in Vmax (mOD/min) and reduction of final polymer mass
Figure 4a). When tested at four different concentrations, compound 1 decreased the Vmax from 17
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(
mOD/min to 6.2, 2.1, 1.1 and 0.4 mOD/min at 2.5, 5, 10 and 25 µM respectively. As a consequence of
decreased Vmax, upto 80% reduction in final polymer mass has been observed.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Microtubules, the key components of cytoskeleton are made up of α/βꢀtubulin heterodimers.
Microtubule assembly has been targeted using number of polymerization inhibitors and inducers, by
binding at different sites including a) colchicine binding site; at interphase of the α/β tubulin
heterodimer and b) taxol and vinblastine binding site deep inside βꢀtubulin. The mechanism of tubulin
polymerization inhibitor involves binding at the interphase of the α/β tubulin and forming complex with
tubulin like colchicine. This complex is added to the microtubule assembly, where it induces
unfavorable conformational changes in tubulin dimer (Mꢀloop) and thus further polymerization process
gets stopped. Furthermore, tubulinꢀpolymerization inhibitor complex perturbs microtubule growth by
4
2ꢀ44
sterically blocking further addition of the tubulin dimers to form microtubule assembly.
Upon
molecular docking studies performed at colchicine binding site, it was observed that compound 1 binds
at α/βꢀtubulin interphase by Hꢀbonding. Compound 1 interacts with the Thr353 residue of β tubulin by
Hꢀbonding. In addition to this, the hydrophobic biphenyl ring fits in hydrophobic core of the βꢀtubulin
formed by Leu248, Ala250, Leu252, Cys241, Leu255, Ala316, Ala317 and Ala354 (Figure 4b).
Interestingly, these interactions were missing in fascaplysin. Compound 1 binding at interphase of α/βꢀ
tubulin, is supposed to induce conformational changes in protein which further perturbs tubulin
polymerization to form microtubule assembly.
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Journal of Medicinal Chemistry
control;
paclitaxel, 10 ꢀM;
nocodazole, 10 ꢀM;
1
2
Vmax = 19 mOD/min Vmax = 61 mOD/min Vmax = 4.5 mOD/min
0.8
0.8
0.7
0.6
0.5
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
Cell free tubulin
polymerization assay
in vitro
0
0
.7
.6
3
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7
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9
1
1
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.5
.4
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.2
.1
0
1000
2000
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Time (s)
Time (s)
Time (s)
0
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Compound1, 2.5 ꢀM; Compound1, 5 ꢀM;
Vmax = 6.2 mOD/min Vmax = 2.1 mOD/min Vmax = 1.1 mOD/min Vmax = 0.4 mOD/min
Compound1, 10 ꢀM; Compound1, 25 ꢀM;
0.8
0.8
0.8
0.7
0.6
0.5
0.8
0.7
0.6
0.5
0
.7
0.7
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.6
.5
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.3
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.2
.1
0.2
0.1
0
1000
2000
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2000
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4000
0
1000
2000
3000
4000
0
1000
2000
3000
4000
Time (s)
Time (s)
Time (s)
Time (s)
A
B
Figure 4. (A) Purified tubulin polymerization assay in vitro. The ability of compound 1 to inhibit tubulin polymerization in
vitro was investigated as described in materials and methods. Paclitaxel and nocodazole were used in the assay as known
enhancer and inhibitor of tubulin polymerization. Compound 1 was tested for a range of concentrations at which it show
inhibition of in vitro cell growth. The change in Vmax value was used as an indicator of tubulin/ligand interactions. The
polymerization curves indicate 2.5 µM, 5 µM, 10 µM and 25 µM of compound 1 reduced the Vmax value from 19 mOD/min
(
control) to 6.2, 2.1, 1.1 and 0.4 mOD/min respectively. The curves shown are average of three independent experiments. (b)
Compound 1 interactions at α/βꢀtubulin interphase.
Compound 1 inhibits paclitaxel-mediated tubulin polymerization and enhances tubulin
depolymerization in live cells. The tubulin polymerization experiments performed on whole cells
(
A549, NSCLC, with intact mitotic spindle checkpoint) confirmed the observation that compound 1
inhibits polymerization of tubulin. Moreover compound 1 also enhances the depolymerization of
stabilized tubulin protein. The polymerized and depolymerized (soluble) forms of tubulin were gauged
(via Westernꢀblotting) from the accumulation/ disappearence of tubulin protein from pellet and from
supernatant fractions of cell lysates treated with IC concentration of compound 1. In the first set of
5
0
experiments where cells were treated with compound 1 in the presence of microtubule stabilizing agent
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Page 18 of 45
paclitaxel, compound 1 showed prevention of tubulin polymerization (mediated by paclitaxel) in a doseꢀ
dependent manner (Figure 5, upper two panels). In a second set of experiment where intracellular
tubulin was stabilized with paclitaxel and then subjected to compound 1 treatment, results show
enhancement of tubulin depolymerization with the increasing concentrations of compound 1 (Figure 5,
lower two panels).
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
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9
0
Supernatant
simultaneous
treatmentof
paclitaxeland
compound 1
Pellet
Supernatant
paclitaxel
treatment
followedby
compound 1
Pellet
Figure 5. Westernꢀblot analysis for effect of compound 1 on tubulin polymerization . Western blots show the response of
compound 1 to tubulin polymerization in the presence of paclitaxel and the effect of compound 1 on paclitaxel stabilized
tubulin. The assay is performed in whole cells (A549) after 30 min compound treatment at the concentrations indicated in
Figure. Supernatant and pellet represent unassembled and assembled tubulin respectively. Tubulin polymerization is
detectable by the increase of tubulin in pellet and its disappearance from supernatant. Simultaneous treatment of paclitaxel
and compound 1 show inhibition of tubulin polymerization by compound 1 in a doseꢀdependent manner and resulted in
accummulation of unassembled tubulin in supernatant. It was observed that compound 1 also act as an enhancer for tubulin
depolymerization in doseꢀdependent manner when paclitaxel stabilized tubulin was subjected to compound 1 treatment.
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Journal of Medicinal Chemistry
Compound 1 inhibits colony formation efficiency of A549 and Calu-1 cells. The ability of
1
2
compound 1 in killing cancer cells was explored in the long term survival assay which monitors cancer
cells’ colonyꢀforming capacity inꢀvitro. A549 (with normal mitotic spindle checkpoint normal, pRb+)
and Caluꢀ1 (with mitotic spindle checkpoint impaired, pRb+) cells were used in colony formation
assays. The colony formation efficiency of A549 and Caluꢀ1 cells is significantly reduced after
compound 1 treatment (Figure 6). The concentration at which 50% colony formation efficiency is
observed is comaparatively lower than the IC concentration for cell growth inhibition in MTT assay
3
4
5
6
7
8
9
1
1
1
1
1
1
1
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0
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9
0
5
0
indicating that a large number of cells loose the ability to form colonies or do not survive for a long time
after compound 1 treatment. For A549 cells, 50% colony formation efficiency was observed at an
average 1.4 µM concentration of compound 1 (cell growth inhibition IC = 3.5 µM) and for Caluꢀ1
5
0
cells 50% colony formation efficiency was observed at an average of 3 µM concentration of compound
(cell growth inhibition IC = 11.5 µM) (Table 2; Figure 6) indicating that compound 1 may be quite
1
5
0
efficacious in in vivo mouse tumor models.
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1
2
3
4
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7
8
9
1
1
1
1
1
1
1
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1
1
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6
A
A549
Calu-1
a
c
b
f
g
e
j
d
h
i
0
1
2
3
4
5
6
7
8
9
0
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9
0
B
100
A549
Calu1
8
6
4
0
0
0
20
0
1
10
100
Compound 1, µM
Figure 6. Compound 1 in colony formation assay. A549 and Caluꢀ1 cells were investigated for their long term survival
efficiency after the treatment with different concentrations of compound 1. The colony formation efficiency is expressed as
the percentage of colonies formed in the treated cultures compared with untreated cultures. (A) The representative plates
show A549 untreated (a), treated with compound 1, 3.12 µM (b), treated with 1, 6.25 µM (c), treated with compound 1, 12.5
µM (d) treated with compound 2, 0.8 µM (e); Caluꢀ1 untreated (f), treated with 1, 3.12 µM (g), treated with compound 1,
6
.25 µM (h), treated with compound 1, 12.5 µM (i) treated with compound 2, 1 µM (j). (B) The curves representing colony
formation efficiencies of A549 and Caluꢀ1 cells with increasing concentrations of compound 1. All results represent the
means and standard deviations of three independent experiments.
Compound 1 induces apoptotic cell death in cancer cells. When A549 cells were treated with
increasing concentrations of compound 1 for 24 h, a doseꢀdependent induction of fragmented nuclei,
disrupted cell membrane and apoptotic cell death was observed (Figure 7Aꢀc,d,e). The induction of
apoptosis in NCIꢀH460, NCIꢀH358 and NCIꢀH1299 cells was also measured with flow cytometry
(
Figure 7B). Doseꢀdependent and timeꢀdependent increase in apoptotic cell death was observed.
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Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
A
a
b
c
e
d
10
11
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60
B
60
NCI-H460; IC50
NCI-H460; IC70
NCI-H1299; IC50
NCI-H1299; IC70
NCI-H358; IC50
NCI-H358; IC70
50
40
30
20
10
0
24h
48h
72h
time of incubation
Figure 7. Induction of apoptotic cell death analyzed by DAPI staining and FACS. Incubation of A549 cells with
increasing concentration of compound 1 for 24 h show the dose dependent induction of fragmented nuclei, distrupted cell
membrane and apoptotic cell death (A) The fluorescence microscopic images captured at 40X magnification after staing with
DAPI. (A) A549 untreated (a) treated with compound 1, 1 µM (b), treated with compound 1, 2.5 µM (c), treated with
compound 1, 5 µM (d), and treated with compound 1, 10 µM (e). The preꢀapoptotic and apoptotic cells are indicated with
arrows. (B) The percent apoptosis induced in three cancer cell lines by the treatment with compound 1 and determined by
FACS analysis. The percent apoptosis is depicted from the percent of cells appeare in Sub G peak during cell cycle analysis.
1
The apoptosis show increase with concentration used and time of incubation with compound 1.
Aqueous solubility, cytochrome P450 liability and Caco-2 permeability of compound 1. The
solubility of compound 1 in water, phosphate buffer saline (PBS), simulated gastric fluid (SGF) and
simulated intestinal fluid (SIF) was found to be 80, 125, 125 and 5 µg/ml, respectively. In cytochrome
P450 liability studies, at 10 µM, compound 1 showed 50, 14, 51 and 19% inhibition of CYP3A4,
CYP2D6, CYP2C9 and CYPC19, respectively. In permeability experiment, efflux ratio [Papp (B–A)/Papp
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(
A–B)] was 1.1, indicating that compound 1 is not a substrate of efflux pumps.
1
2
3
4
5
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8
9
1
1
1
1
1
1
1
1
1
1
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6
In vivo experiments: Pharmacokinetics and maximum tolerated dose (MTD). In order to check
the plasma exposure of the compound in animals, the pharmacokinetics of compound 1 was studied in
BALB/c mice following a single 10 mg/kg dose administration by oral route and 1.0 mg/kg dose
administration by IV route. The time versus plasma concentration profile of compound 1 is shown in
Figure 8. Compound 1 showed good plasma exposure with Cmax of 190 and 371 ng/mL (537 and 1048
nM) by PO and IV routes, respectively. The AUC0ꢀ∞ values were 182 and 189 ngꢁh/mL (514 and 534
nM), respectively.
0
1
2
3
4
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0
Parameter
t1/2,ß (h)
IV (1 mg/kg)
0.33
Oral (10 mg/kg)
1.16
IV 1 mg/Kg
1
000
PO 10 mg/Kg
AUC0ꢀt (ngꢁh/mL)
AUC0ꢀ∞ (ngꢁh/mL)
Cmax (ng/mL)
187
189
371
172
182
190
1
00
10
1
V (L/Kg)
2.52
1.76
88.3
ꢀ
nd
nd
nd
9.6%
1 – 4 h
d
Vdss (L/Kg)
CL (mL/min/kg)
Bioavailability
Time points considered 0.5 – 2 h
0
1
2
3
4
Time (h)
for t1/2,β calculation:
B
A
Figure 8. (A). Time versus plasma concentration profile of compound 1 in BALB/c mice; (B).
Pharmacokinetic parameters determined for compound 1 after IV and oral administration.
The studies which allowed determination of MTDꢀs were performed in Swiss albino mice for two
weeks. The concentration at which compound 1 would be tested in vivo was thus ascertained. The loss
in animal body weight was considered as a measure of overꢀtoxicity for the test compound. The
concentration of the compound at which >10% weight loss was observed was determined and
designated as MTD, although usually a weight loss which is below 20% of the initial weight is harmless
4
5
and animals can recover once the treatment is stopped. The toxicity results obtained from these studies
indicated that for compound 1 the MTD in mice was ~1000 mpk (mg/kg). Based on the MTD
th
experiment and pharmacokinetics data, compound 1 was tested at 1/10 of MTD concentration i.e. 100
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Journal of Medicinal Chemistry
mg/kg in inꢀvivo xenograft studies.
1
2
3
4
5
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7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
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3
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4
4
4
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5
5
5
5
5
5
5
5
6
Effects on HCT-116 and NCI-H460 tumor growth inhibition. SCID (Severe Combined Immuno
Deficient) mouse, lacking both T and B immune cells, is an established model to study in vivo efficacy
of molecules that has potential for the treatment of human cancers. When evaluated, compound 1
th
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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0
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0
showed statistically significant (p<0.05) tumor growth inhibition (Figure 9A) at 1/10 of MTD
concentration (100 mg/kg) in the HCTꢀ116 tumor model. Mice injected with compound 1 exhibited
approximately 80% tumor growth inhibition as compared to the untreated mice injected with the vehicle
solution alone. In the second set of experiments, the antiꢀtumor potential of compound 1 was confirmed
when it was proven to be highly efficacious in NCIꢀH460 tumor models at 100 mg/kg concentration
(Figure 9CꢀD). These results indicate that compound 1 has strong antiꢀcancer properties in vivo when
administered at a concentration 10 times less than the MTD. Thus, with these studies compound 1 has
shown efficacy as a new class of anticancer compound.
During this study, the effect on animal body weight due to compound treatment was also noted. The
weight loss observed in treated animals was found to be <10% of the starting weights of the animals
(Figure 9B) in the HCTꢀ116 tumor model. This loss of weight can be considered to be statistically
insignificant indicating that compound treatment caused no major toxicity or harm to the animals. The
body weight of the animals at the beginning of treatment was measured and this was considered to be
1
00%. The percentage weight loss or gain was calculated using the initial weight as a reference. Similar
results were obtained in NCIꢀH460 tumor model (data not shown). Compound 1 shows minimal toxicity
in animal models and can be tolerated up to 1000 mg/kg concentration without any significant toxicity.
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Anti-tumour activity of compound 1 in SCID mice
using human colon cancer cell, HCT116 xenografts
Statistically insignificant (< 10%) loss in body weight
(B)
(
A)
after 9 days treatment with compound 1
1
2
3
4
5
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7
8
9
1
1
1
1
1
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6
1
40
Control
Compound 1, 100 mg/kg
Control
1
010
Compound 1, 100 mg/kg
120
100
8
10
10
6
9
0%
410
8
0
2
10
0
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60
0
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10
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20
0
2
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8
10
12
14
No. of days (9-day treatment followed by a 9-day period
of no-treatment)
No. of days (9-day treatment followed by a 5-day
period of no-treatment)
Anti-tumor activity of compound 1 in SCID mice using
Controlor untreated mice with NCI-
H460 xenografts
Controlmice with NCI-H460
xenografts treated with PEG
(C)
(D)
human lung cancer NCI-H460 xenografts
2
500
2000
1500
Control
Compound 1, 100 mg/kg
1
000
Tumors removed from controloruntreated
mice (human lung cancer,NCI-H460)
500
Mice treated with compound 1,100mg/kg
0
0
2
4
6
8
10
12
No.of days
Tumors removed frommice treated
with compound1,100 mg/kg
Figure 9. (A) Tumor growth inhibition curves for compound 1 in in vivo HCT116 xenograft model. Graphs depict tumor
growth inhibition in a group of animals treated with compound 1 at the concentration 100 mg/kg which is compared with the
untreated group of animals (shown in the graphs as the control group). Tumor sizes were recorded at 2ꢀ5 day intervals.
2
Tumor weight (in mg) was estimated according to the formula for a prolate ellipsoid: [Length (mm) x [width (mm) ] x 0.5]
assuming specific gravity to be one and π to be three. Compound 1 shows statistically significant tumor growth inhibition in
vivo. p < 0.05 (Student t test was used for multiple comparisons of different groups versus control); (B). Animal weight
profile in the HCT116 SCID mice xenograft model. The body weights of untreated and treated with compound 1 (100
mg/kg) animals were monitored by taking measurements daily during the treatment schedule. By considering the body
weight at the start of treatment as 100%, the percentage weight loss was calculated on subsequent days of treatment. (C).
Tumor growth inhibition curves for compound 1 in in vivo NCIꢀH460 tumor model. Graphs depict tumor growth inhibition
in a group of animals treated with a specific compound which is compared with the untreated group of animals (shown in the
graphs as the control group). Tumor sizes were recorded at 2ꢀ5 day intervals. Tumor weight (in mg) was estimated
2
according to the formula for a prolate ellipsoid: [Length (mm) x [width (mm) ] x 0.5] assuming specific gravity to be one and
π to be three. Compound 1 shows statistically significant tumor growth inhibition in vivo. p < 0.05 (Student t test was used
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Journal of Medicinal Chemistry
for multiple comparisons of different groups versus control). (D). Control and treatment groups of SCID mice showing NCIꢀ
H460 tumor growth inhibition followed by treatment with compound 1 as labeled in the Figure. These pictures were
obtained after treatment with compound 1 at the concentration 100 mg/kg. The treatments were continued for 9 consecutive
days intraꢀperitoneally when tumor growth had reached about 4ꢀ6 mm in diameter after about 6 days followed by the tumor
cell injection.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
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4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
CONCLUSION
In summary, we have presented data that supports the notion that compound 1 has the potential of
being a novel anticancer agent. The finding of dual inhibition of Cdk4ꢀcyclin D1 and tubulin
polymerization, may address the problems of resistance which currently limit the efficacy of valuable
antiꢀmicrotubule agents. The promising efficacy of compound 1 in xenograft models (HCT116 and
NCIꢀH460 tumor models) with an excellent therapeutic window indicates promise of this compound to
develop as anticancer agent.
EXPERIMENTAL SECTION
1
13
General. All chemicals were obtained from SigmaꢀAldrich Company and used as received. H, C
and DEPT NMR spectra were recorded on BrukerꢀAvance DPX FTꢀNMR 400 MHz instrument.
Chemical data for protons are reported in parts per million (ppm) downfield from tetramethylsilane and
are referenced to the residual proton in the NMR solvent (CDCl : 7.26 ppm). Carbon nuclear magnetic
3
13
resonance spectra ( C NMR) were recorded at 100 MHz: chemical data for carbons are reported in
parts per million (ppm, δ scale) downfield from tetramethylsilane and are referenced to the carbon
resonance of the solvent (CDCl : 77.00 ppm). ESIꢀMS and HRMS spectra were recorded on Agilent
3
1
100 LCꢀQꢀTOF and HRMSꢀ6540ꢀUHD machines. IR spectra were recorded on PerkinꢀElmer IR
spectrophotometer. HPLC analysis was done on Shimadzu HPLC system (model: LCꢀ6AD) equipped
with a PDA detector (model: SPDꢀM20A) using Inertsil C8 (3.5 ꢂ, 4.6× 250 mm) column. Mobile phase
used was ACN: water (80: 20) isocratic elution at flow rate of 1 ml/min. The purity of all compounds
25
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Page 26 of 45
was determined by HPLC method. The purity of final compound 1 was determined by HPLC and
HRMS analysis. All compounds have ≥95% purity.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Synthesis of ethyl 2-(1H-indole-3-yl)ethylcarbamate (4): To the suspension of tryptamine (3, 1.2
mmol) in CH Cl (3 ml) at 0 °C was added slowly an aqueous solution of 4 N NaOH (1.2 mmol). After
2
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5 min stirring at 0 °C, ethyl chloroformate (1.2 mmol) was added dropwise. The resulting mixture was
stirred for 5 min at 0 °C and further stirred at rt for 3 h. Completion of the reaction was monitored by
TLC. After completion of the reaction, reaction mixture was diluted with CH Cl and was washed with
2
2
water. Organic layer was separated and dried over anhydrous sodium sulfate. Solvent was evaporated
under reduced pressure and crude product was purified by silica gel (#100ꢀ200) column chromatography
using EtOAc: Hexane as mobile phase to get titled compound 4. Light pink oil; yield: 90%; HPLC: t =
R
1
3
.7 min (95% purity); H NMR (400 MHz, CDCl , ppm): δ 8.15 (brs, NH, 1H), 7.60 (d, J = 7.6 Hz, 1H),
3
7
.36 (d, J = 8 Hz, 1H), 7.25 – 7.10 (m, 2H), 7.01 (s, 1H), 4.75 (brs, NH, 1H), 4.13 – 4.08 (m, 2H), 3.52
1
3
(
d, J = 6 Hz, 2H), 2.97 (t, J = 6.8 Hz, 2H), 1.22 (t, J = 7.2 Hz, 3H); C NMR (100 MHz, CDCl , ppm):
3
δ 156.85 (CO), 136.49 (C), 127.34 (C), 122.17 (CH), 122.11 (CH), 119.40 (CH), 118.72 (CH), 112.81
(C), 111.30 (CH), 60.77 (CH ), 41.25 (CH ), 25.81 (CH ), 14.66 (CH ); IR (KBr): ν 3409, 3332,
2
2
2
3
max
3
1
057, 2980, 2932, 1917, 1695, 1620, 1524, 1479, 1456, 1380, 1338, 1258, 1230, 1171, 1141, 1093,
ꢀ1
+ 39
035, 1010 cm ; ESIꢀMS: m/z 233.10 [M+H] .
Synthesis of [2-(1H-indol-3-yl)-ethyl]-methylamine (5): To the solution of carbamate ester 4 (0.1
g, 59.4 mmol) in dry THF (5 mL) under N flux at 0 °C was added portionꢀwise LAH (178 mmol). After
2
the addition was completed, the mixture was heated under reflux for 1 h. The reaction was then cooled
to 0 °C and the excess of LAH was hydrolyzed by adding successively and very carefully water (2 mL),
15% aqueous solution of NaOH (2 ml) and water (3 ml). During these steps, it was necessary to add
THF (10 ml) to avoid the mixture becoming very thick. The suspension was filtered and the white solid,
made up of LiOH and Al(OH) , was washed with THF (10 ml). The organic layer was dried (Na SO )
3
2
4
and evaporated under reduced pressure to get the titled compound 5. Beige solid, yield: 85%; HPLC: tR
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Journal of Medicinal Chemistry
1
=
3.75 min (95% purity); H NMR (400 MHz, CDCl , ppm): δ 8.81 (brs, NH, 1H), 7.60 (s, 1H), 7.30 (s,
3
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
3
1
H), 7.17 – 7.10 (m, 2H), 6.94 (s, 1H), 2.97 – 2.91 (m, 4H), 2.41 (s, 3H); C NMR (125 MHz, DMSOꢀ
d6, ppm): δ 136.19 (C), 127.18 (C), 122.57 (CH), 120.82 (CH), 118.24 (CH), 118.12 (CH), 112.24 (C),
1
11.31 (CH), 51.94 (CH ), 35.62 (CH ), 24.80 (CH ); IR (KBr): ν
3670, 3646, 3403, 2922, 2851,
max
2
3
2
ꢀ1
+ 39
1
658, 1650, 1456, 1340, 1233, 1016 cm ; ESIꢀMS: m/z 175.0 [M+H] .
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Synthesis of biphenyl-4-carboxylic acid [2-(1H-indol-3-yl)-ethyl]-methyl amide (1): To the
suspension of [2ꢀ(1Hꢀindolꢀ3ꢀyl)ꢀethyl]ꢀmethylamine (5, 1.2 mmol) in CH Cl (3 ml) at 0 °C was added
2
2
slowly an aqueous solution of 4 N NaOH (1.2 mmol). After 5 min stirring at 0 °C, 4ꢀbiphenyl
carbonychloride (6, 1.2 mmol) was added dropwise. Mixture was stirred for 5 min at 0 °C and further
stirred at rt for 3 h. Completion of the reaction was monitored by TLC. After completion of the reaction,
reaction mixture was diluted with CH Cl and was washed with water. Organic layer was separated and
2
2
dried over anhydrous sodium sulfate. Solvent was evaporated under reduced pressure and crude product
was purified by silica gel (#100ꢀ200) column chromatography using EtOAc: Hexane as mobile phase to
1
get titled compound 1. Beige solid; yield: 45%; HPLC: t = 4.62 min (98% purity); H NMR (400 MHz,
R
CDCl , ppm): δ 8.21 (brs, NH, 1H), 7.58 – 7.09 (m, 13H), 6.88 (t, J = 6.8 Hz, 1H), 3.88 (t, J = 6.4 Hz,
3
1
7
H), 3.61 (t, J = 6.8 Hz, 1H), 3.20 (s, 3H), 2.98 (t, J = 6.8 Hz, 2H); δ (distinct peaks for minor rotamer)
1
3
.73 (d, J = 7.2 Hz, 1H), 7.01 (d, J = 7.6 Hz, 1H), 2.93 (s, 1H); C NMR (100 MHz, DMSOꢀd , ppm): δ
6
(
major rotamer) 170.58 (CO), 139.42 (C), 136.18 (C), 128.99 (CH), 127.76 (CH), 127.47 (CH), 126.91
(C), 126.69 (CH), 126.41 (CH), 120.88 (C), 120.87 (CH), 118.06 (CH), 117.82 (C), 117.81 (CH),
1
1
11.34 (CH), 51.43 (CH ), 32.33 (CH ), 23.84 (CH ). δ (distinct peaks for minor rotamer) 169.55 (CO),
2 3 2
40.55 (C), 135.72 (C), 123.06 (CH), 118.28 (CH), 110.40 (C), 47.87 (CH ), 37.44 (CH ), 22.52 (CH );
2
3
2
IR (KBr): νmax 3411, 3284, 3056, 2924, 2853, 1717, 1609, 1520, 1480, 1456, 1401, 1299, 1258, 1230,
ꢀ1
+
1
076, 1018 cm ; ESIꢀMS: m/z 355.10 [M+H] ; HRMS: m/z 355.1840 calcd for C H N O +
24 23 2
+
H (355.1810).
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Kinase profiling. Compound 1 was tested against a panel of kinases at 10 ꢀM. The inꢀvitro kinase
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
assays for testing against Cdk4ꢀcyclin D1, Cdk2ꢀcyclin A, Cdk2ꢀcyclin E, Cdk1ꢀcyclin B1 and Cdk9ꢀ
3
7ꢀ41
cyclin T1 were performed in house. The methodology and results have been reported previously.
The kinase profiling for 58 representative kinases was done commercially at Millipore Bioscience
Division, UK.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
In-vitro cell proliferation assays. All ten human cancer cell lines were maintained at 37 ºC in 5%
TM
CO in RPMIꢀ1640 medium, supplemented with 10% fetal calf serum and 100 µg/ml Normocin . The
2
ten cancer cell lines used for screening were nonꢀsmall cell lung carcinoma (NSCLC; a form of cancer
+
+
+
+
which is resistant to chemotherapy) cell lines: NCIꢀH460 (pRb , p53 ), A549 (pRb , p53 ) obtained
+
+
from the Glenfield Hospital, Leicester in June 2006; and Caluꢀ1 (pRb , p53ꢀnull), NCIꢀH1299 (pRb ,
p53ꢀnull), NCIꢀH358 (pRbꢀnull, p53ꢀnull) obtained from Mr Pat Browne (Morvus Technology) in July
+
+
+
2007. The colon cancer cell line LS174T (pRb , p53 ), and the prostate cancer cell line PC3 (pRb , p53ꢀ
null) were also gifts from Mr Pat Browne (Morvus Technology) in July 2007. The pancreatic cancer cell
+
line MiaPaca (pRb , p53ꢀmutant) was kindly provided by Prof Bill Greenhalf (University of Liverpool)
in July 2007. The genotypes within brackets indicate the status of the tumor suppressor proteins pRb
and p53. The mouse embryonic normal hepatic cell line (BNL CL2) and its SVꢀ40 large Tꢀantigen
transformed counterpart cell line (BNL SV A.8) were purchased from ATCC in January 2006. The large
T antigen inactivates the tumor suppressor proteins p53 and pRb. The obtained cell lines were tested and
authenticated via (a) short tandem repeat (STR) profiling; (b) monitoring of cell morphology; (c)
karyotyping; and (d) cytochrome C oxidase I (COI) assay. The last tests were performed by ATCC
before the cell lines were bought. The cell lines were resuscitated immediately after receipt. The
4
0
detailed procedure of cell proliferation (MTT) assay and IC determination was described previously.
5
0
Flow cytometric analysis. The untreated (control) and treated (with test compounds) cells were
harvested by trypsinization, washed once with PBS and then fixed in 70% chilled (ꢀ20 ºC) ethanol for
minimum 1 h. After the fixation step, cells were centrifuged for 5 min at 3000X g at room temperature
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Journal of Medicinal Chemistry
and then the pellet was suspended in PBS containing 50 µg/ml propidium iodide (Sigma Cat No Pꢀ
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
4
170) and 0.5 mg/ml DNaseꢀfree ribonuclease (Sigma Cat No Rꢀ5503). The cells were stained for 1 h in
TM
the dark at 4 °C. Cell cycle analysis was performed on the Beckman Coulter (Epics® Altra )
fluorescenceꢀactivated cell sorter. In order to gate all the events which represent single cells, and not
cell doublets or cell clumps, the following analyses were performed on the samples. Cytograms of
propidium iodide fluorescence peak signal versus integrated fluorescence or the linear signal were
plotted. All data points on the straight line were isolated in a single gate and the gated data further used
for plotting a histogram that represents a complete cell cycle. The total number of events was not
allowed to exceed 200 events/second. Data acquisition was stopped after a minimum of 10,000 events
had been collected.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
Western-blotting. A549, LS174T and MIAPaCa cells were seeded in 25 cm tissue culture flasks in
the complete growth medium. When the culture flasks reached 40ꢀ50% confluency, cells were treated
with 1 (IC concentration) for 24 h. Cells were harvested by trypsinization, washed in iceꢀcold PBS and
5
0
then lysed in buffer (Sigma Cat No Cꢀ2978) containing protease inhibitor cocktail (Sigma Cat No
P8340). The lysates were centrifuged at 14,000 rpm for 10 min at 4 ºC and the total protein
concentrations were estimated in the clear supernatants using the Bradford method. Equal amounts of
protein (40 µg) were loaded and electrophoresed on 10% SDSꢀpolyacrylamide gels and blotted on
Immobilon–P Transfer Membrane (Millipore Cat No IPVH20200). The blots were probed with
respective primary antibodies (at 4 ºC, overnight) at the following dilutions: cdc2 (New England
Biolabs Cat No 9110) at 1:1000 to detect Cdk1; cyclin B1 (CRꢀUK Cat No V152) at 1:1500 dilution to
detect cyclin B1; Pab 1801 (Santa Cruz Biotechnology Cat No scꢀ98) at 1: 500 dilution to detect p53; Nꢀ
2
0 (SantaCruz Biotechnology Cat No scꢀ469) at 1: 500 dilution to detect p21; Cꢀ19 (SantaCruz
Biotechnology Cat No scꢀ528) at 1:250 dilution to detect p27; ACꢀ40 (SigmaꢀAldrich Cat No. A4700)
at 1:2000 dilution to detect actin. Appropriate secondary antibodies conjugated with horseradish
peroxidase were used and the protein bands were visualised by chemiluminescence using the ECL kit
29
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