ca224, a non-planar analog of fascaplysin inhibits cdk4 and tubulin polymerization: evaluation of in...

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CA224, a non-planar analog of fascaplysin inhibits Cdk4 and

tubulin polymerization: Evaluation of in vitro and in vivo anticancer activity

Journal: Journal of Medicinal Chemistry

Manuscript ID: jm-2014-01407a

Manuscript Type: Article

Date Submitted by the Author: 13-Sep-2014

Complete List of Authors: Mahale, Sachin; De Montfort University, School of Pharmacy Manda, Sudhakar; Indian Institute of Integrative Medicine (CSIR), Medicinal Chemistry Division joshi, prashant; IIIM, Bharate, Sonali; Indian Institute of Integrative Medicine (CSIR), Preformulation Laboratory Jenkins, Paul; University of Leicester, Department of Chemistry Vishwakarma, Ram; Indian Institute of Integrative Medicine, Medicinal Chemistry Division Bharate, Sandip; Indian Institute of Integrative Medicine, Medicinal

Chemistry Chaudhuri, Bhabatosh; De Montfort University, School of Pharmacy

ACS Paragon Plus Environment

Journal of Medicinal Chemistry

1

CA224, a non-planar analog of fascaplysin inhibits

Cdk4 and tubulin polymerization: Evaluation of in

vitro and in vivo anticancer activity

Sachin Mahale, ‡, Sudhakar Manda,† Prashant Joshi,† Sonali S. Bharate,⊥ Paul R. Jenkins,∞ Ram A.

Vishwakarma,‡ Sandip B. Bharate,*, † Bhabatosh Chaudhuri*,‡

‡School of Pharmacy, De Montfort University, Leicester, LE1 9BH, UK

†Medicinal Chemistry Division, Indian Institute of Integrative Medicine (CSIR), Canal Road, Jammu-

180001, India

⊥Preformulation Laboratory, Indian Institute of Integrative Medicine (CSIR), Canal Road, Jammu-

180001, India

∞Department of Chemistry, University of Leicester, Leicester, LE1 7RH, UK

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ABSTRACT

CA224 is a non-planar analog of fascaplysin that specifically inhibits Cdk4-cyclin D1 enzyme in-vitro.

It was found that CA224 blocks growth of cancer cells at G0/G1 phase of the cell division cycle. It also

blocks at G2/M phase which is explained by the fact that CA224 inhibits tubulin polymerization which

is essential for mitotic spindle assembly and chromosomal movements. Besides, it acts as an enhancer

of depolymerization for taxol-stabilized tubulin. CA224’s ability to inhibit Cdk4-cyclin D1 and thereby

block growth at the G0/G1 phase of cell-cycle proved crucial in the Calu-1 cell line which has an

impaired mitotic spindle checkpoint. Western blot analyses of p53-positive cancer cells treated with

CA224 indicated up-regulation of the p53, p21 and p27 proteins together with down-regulation of cyclin

B1 and Cdk1. CA224 selectively induces apoptosis in SV40 large T-antigen transformed cells and

significantly reduces colony formation efficiency, in a dose-dependent manner of lung cancer cells,

A549 (p53-positive) and Calu-1 (p53-null). CA224 is efficacious at 1/10th the MTD (1000 mg/kg),

against human tumors derived from HCT-116 and NCI-H460 cells in SCID mice models. These

findings may have significant implications in designing more potent molecules that simultaneously

target Cdk4-cyclin D1 inhibition and tubulin polymerization, for identification of potential candidates

for clinical development.

KEYWORDS

Cdk4-cyclin D1, tubulin polymerization, fascaplysin, anti-cancer, chemotherapeutic, cell division cycle

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INTRODUCTION

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 (G0, G1, S,

G2, M-phase), and the transition of cells from one phase to another involves various check-point

mechanisms including cyclin-dependent kinases, checkpoint kinases, and their partner cyclins.

Malfunctioning of any of these checkpoint mechanisms leads to uncontrolled cell division and

proliferation.1-5 Cyclin-dependent kinase 4 and its cyclin partner D1 (check-point mechanism) controls

transition of the cells from G1 to S phase of the cell cycle.6-9 Active Cdk4/cyclin D complexes inactivate

retinoblastoma protein (pRb) by phosphorylating specifically at Ser780 and Ser795 residues, which

allows the G1/S transition of cells during the cell cycle.6, 10 Hyperphosphorylation of pRb leads to 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

cyclin partners A or C is not sufficient to inactivate pRb functions.11-13 A large number of human tumors

(around 96%) lose normal cell cycle transition check-point mechanism due to variety of genetic and

biochemical adaptations including the hyperactivity of the Cdk4 protein, down-regulation of Cdk4

positive regulator p16INK4A and mutations in pRb. The vital role of Cdk4-cyclin D1 in tumor

development has been confirmed by experiments in Cdk4 and cyclin D1 knock-out 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 in-vivo. Based on these experimental

observations, Cdk4/cyclin D complex protein is considered as a promising target for combating

cancer.14-16

Inhibition of Cdk4-cyclin 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

highlighting the role of Cdk4-cyclin D1 inhibitors in cancer treatment.17-21 Pfizer's palbociclib, a

selective inhibitor of Cdk4 and Cdk6 has received FDA approval for treatment of patients with breast

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cancer.22 Other Cdk4/Cdk6 dual inhibitors LY2835219 and P1446A-05 are in phase I clinical studies for

breast and advanced refractory malignancies, respectively (Source: http://clinicaltrials.gov). Most of the

research has been focused on finding ATP-competitive inhibitors of Cdk4-cyclin D1 using methods of

structure-based chemical design.23

The main objective of our studies was to develop potent and specific small molecule inhibitors of

Cdk4-cyclin D1 based on the structure of fascaplysin which is a pentacyclic quaternary salt originally

isolated from the Fijian sponge Fascaplysinopsis Bergquist sp.24 It is known to possess antimicrobial

and antimalarial activity.24, 25 It also displays potent cytotoxicity against small cell lung cancer cells and

induces cell cycle arrest in G0/G1 at lower and S-phase at higher concentrations, respectively.26

Fascaplysin also showed anti-tumor effects in sarcoma mice model through apoptotic and anti-

angiogenesis pathways.27 We have reported fascaplysin as one of the specific inhibitors of Cdk4-cyclin

D1.9, 28 It inhibits Cdk4-cyclin D1 in-vitro and blocks the growth of normal and cancer cells at the

G0/G1 phase of the cell cycle, which correlates with the accumulation of hypo-phosphorylated pRb,

implying there is no phosphorylation at Cdk4-specific serine residues.9 Fascaplysin has also been

reported to inhibit Cdc25B with an IC50 of 1 µg/ml.29, 30 An analogue of fascaplysin 1-

deoxysecofascaplysin A, has been reported to inhibit cell growth of breast cancer MCF-7 and ovarian

cancer OVCAR-3 cell lines in-vitro.31

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 double-stranded 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

cryptolepine and ellipticine.32 We have explored the possibility of separating the DNA intercalating

ability of fascaplysin from its potent Cdk4-specific inhibitory activity. The aim of the current study was

therefore to devise non-planar (non-toxic) Cdk4-cyclin D1 inhibitors based on the structure of

fascaplysin.33-36

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CA224 was initially identified as a Cdk4 specific inhibitor lacking the ability to intercalate DNA.37

As expected, CA224 manifested its Cdk4-specific inhibitory ability by blocking cancer cells at the

G0/G1 phase of the cell cycle. Surprisingly however, CA224 also inhibited the G2/M phase in a Cdk-

independent manner. Further investigations showed that the CA224 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 CA224 has the ability to

diminish the colony formation efficiency of cancer cells. The Cdk-independent G2/M block was found

to be associated with its anti-tubulin activity. CA224 inhibited the polymerization of tubulin in-vitro and

also showed an enhancing effect on tubulin depolymerization in live cells. It was found in microarray

studies that genes, related to apoptosis and stress, were up-regulated more profoundly in p53-negative

(p53-) cells than in cells that were p53-positive (p53+). The apoptotic inhibitory proteins were found to

be down-regulated in both p53+ and p53- cells. In human xenograft models CA224 was found to inhibit

the growth of HCT-116 and H-460 tumor growth at 1/10th of the MTD, 100 mg/kg. Here, we present the

biological activity including efficacy in xenograft models of CA224 in detail.

RESULTS AND DISCUSSION

Chemistry. Our efforts towards discovery of non-toxic analogs of fascaplysin led to the

identification of CA224, a non-planar analog of fascaplysin. Briefly, the synthesis of CA224 involves

three steps starting from commercially available tryptamine (1). The detailed lead optimization and SAR

have been described in previous publications.33-37 The synthetic scheme for CA224 is shown in Scheme

1. It was interesting to note that the non-planar analogs with opened C and D rings of fascaplysin, lost

its DNA intercalation activity. Among C/D-ring opened analogs, compounds with p-substituted D-rings

(e.g. CA223, CA224, CA225, structure shown in Table 1) maintained their ability to inhibit Cdk4 in a

selective manner.

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NH

NH

NH

N

O

Cl

O

NH

NH

O

O

NH

NH2

1 2 3

4

a b

c

CA224 (5)

+

Scheme 1. Reagents and conditions. (a) Ethyl chloroformate, NaOH 4 N, CH2Cl2, 3 h, 90%; (b) LiAlH4,

THF, N2, reflux, 1 h, 85%; (c) NaOH 4 N, CH2Cl2, 3 h, 45%.

Selective inhibition of Cdk4-cyclin D1. A series of new compounds based on the structure of

fascaplysin were identified as specific inhibitors of enzyme Cdk4–cyclin D1 (Table 1). CA224 was

found to be the most potent inhibitor of Cdk4-cyclin D1 (IC50 = 6 µM) and selective when the Cdk4

IC50 was compared with the IC50s obtained in Cdk2-cyclin A, Cdk1-cyclin B1 and Cdk9-cyclin T1

assays. Unlike fascaplysin, CA224 does not intercalate with DNA 37. CA224 was also tested against 58

represenative kinases at Millipore Bioscience Division, UK. It was found that CA224 was inactive at the

concentration of 10 µM against all kinases including Cdk5-p35, Cdk6-cyclin D1, Cdk7-cyclin H,

EGFR, GSK3β, MAPK1, MEK1, PDGFR, Plk3, PKA, PKCα, IGF-1R etc. These results support

CA224’s selective ability to inhibit Cdk4-cyclin D1 enzyme in-vitro while having much reduced or no

affinity for other kinases tested.

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Table 1. Activity of fascaplysin and its non-planar analogs in in-vitro kinase and DNA binding assays a

Cell lines Cl

NH

N

O

A B

CD

E

Fascaplysin

NH

N

O

Cl

CA223

NH

N

O

EA B

CA224

NH

N

O

CA225

Cdk4/cyclin

D1

0.41 ± 0.04 38 ± 6 6.2 ± 0.9 49 ± 6.5

Cdk2/cyclin A >250 731 ± 26 521 ± 11 658 ± 23

Cdk2/cyclin E >250 ND ND ND

Cdk1/cyclin

B1

>250 >500 >500 >500

Cdk9/cyclin

T1

>250 >1000 >1000 >1000

EtBr

displacement

5 ± 0.4 Does not dispalce Does not dispalce Does not dispalce

aIC50 values are presented in µM. All the fascaplyin analogs were dissolved in 100% DMSO and were further diluted in the kinase assay buffer or the ethidium bromide displacement assay buffer.

In order to understand the observed selectivity towards Cdk4-cyclin D1 versus Cdk2-cyclin A,

molecular modeling studies were carried out. The ATP binding sites of these Cdks are well conserved

and share 45% sequence homology to each other, however CA224 displayed varying degree of affinity

to these Cdks. The two Cdks differ from each other by these residue sequences: 94-97(Glu-His-Val-

Asp)Cdk4/81-84(Glu-Phe-Leu-His)Cdk2,101-102(Arg-Thr)Cdk4/88-89(Lys-Lys)Cdk2 and

Glu144Cdk4/Gln131Cdk2. CA224 interacts flexibly to ATP binding pockets of Cdk4/Cdk2 with 84-fold

selectivity towards Cdk4 due to flexible conformational movement of the CA224 amide bond (CA224

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 CA224 include (a) trans-conformation (green colored ligand in Figure 1a; dihedral

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angle is Ψ = -151.5) which was found to interact selectively with side chains of Arg101 residue of Cdk4

by hydrophobic π-cation interaction; however in Cdk2, this interaction is missing because

corresponding Lys88 residue side chain orient away from CA224 binding cavity; (b) cis-conformation

(orange colored ligand in Figure 1a; dihedral angle Ψ = 2.6) which interacts with Cdk2. On another

hand, the trans-conformation of CA224 is stabilized in Cdk4 binding site because negatively-charged

Glu144 side chain oppose biphenyl aromatic ring. In case of Cdk2, the cis-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 1b and 1c.

(a) (b)

Protein Amino acid sequence

CDK4 94 95 96 97 101 102 144

Glu His Val Asp Arg Thr Glu

CDK2 81 82 83 84 88 89 131

Glu Phe Leu His Lys Lys Gln

(c)

Figure 1. Molecular modeling studies. (a) Interactions of cis/trans-conformations of CA224 with Cdk2

and Cdk4, respectively (orange conformation is with Cdk2 and green with Cdk4). (b) CA224-Ck4

interaction histogram with different binding site residues during 10 ns MD simulation of CA224-Cdk4

protein complex (c) amino acid comparision between Cdk4 and Cdk2 at ATP binding site (black

colored residues indicates commonly interacting residues of Cdk4/Cdk2, while red colored residues are

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those which differ in interaction pattern in these two Cdks).

Cancer cell growth inhibition. All the fascaplysin analogues including the most potent compound

in the series, CA224, 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

in-vitro. The inhibitory effects of compounds were quantified using the MTT assay and IC50s 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 CA224 inhibits the growth of

cancer cells in-vitro at low micromolar concentrations (Table 2). Amongst all the analogues,37 CA224

was found to be the most potent molecule even at cellular level. The inhibition of cell growth was both

p53 and pRb-independent, the latter indicating that Cdk4-cyclin D1 inhibition is not the only cellular

target for the mechanism of action of these molecules.

Table 2. IC50 concentrations expressed in µM for in-vitro cell growth inhibition induced by exposure to

CA224, and its analogues CA225 and CA22337 for 48 h.

Cell lines Fascaplysin CA223 CA224 CA225

LS174T (colorectal carcinoma; p53+, pRb+) 0.88 ± 0.04 42 ± 2.5 3.5 ± 0.9 18 ± 1

PC-3 (prostate; p53 null, pRb+) 0.92 ± 0.06 47 ± 3 6.2 ± 1.1 15 ± 1.5

MiaPaCa (pancreatic; p53His273 mut, pRb+) ND 31± 2.2 4.0 ± 0.3 10.2 ± 0.9

A549 (NSCLC; p53+, pRb+) 0.69 ± 0.03 27 ± 2.5 3.5 ± 0.6 12 ± 1.8

Calu-1 (NSCLC; p53 null, pRb+) 1.3 ± 0.1 78 ± 3.0 11.5 ± 2.5 52 ± 3.5

NCI-H460 (NSCLC; p53+, pRb+) ND 24 ± 1.8 2.0 ± 0.3 9.8 ± 1.0

NCI-H1299 (NSCLC; p53 null, pRb+) ND 21 ± 0.9 2.5 ± 0.3 11.5 ± 1.6

NCI-H358 (NSCLC; p53 null, pRb null) ND 26 ± 2.0 2.2 ± 0.6 14 ± 1.4

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BNL CL2 (mouse normal hepatic cells) ND 29 ± 2.4 2.6 ± 0.9 18 ± 2.0

BNL SV A.8 (mouse hepatic; SV-40

mediated transformed cells)

ND 32 ± 1.0 3.8 ± 0.9 22 ± 1.8

CA224 retains the G0/G1 block in serum starved p53-null Calu-1 cells. CA224, although

identified as a Cdk4-specific inhibitor in the in-vitro enzyme assay, generally tends to block cancer cells

more profoundly at G2/M than G0/G1 phase of the cell division cycle. Moreover, CA224 inhibits tubulin

polymerization in-vitro with higher potency than it inhibits enzyme Cdk4-cyclin D1. In Calu-1 cells, the

mitotic spindle checkpoint is impaired implying that these cells cannot be blocked at G2/M. Hence, it

was decided to treat Calu-1 cells with CA224 after release from cell synchronization at G0/G1 (cells

being starved of serum using 0.1% FBS for 24 h). At IC50 and IC70 concentrations of CA224 the G0/G1

block was either partially or full maintained 36. Since the maintenance of G0/G1 block after serum

starvation requires Cdk4 enzyme to be inactive, these results indicate that CA224 probably inhibits

cellular Cdk4 at these concentrations and thereby maintains the G0/G1 block. In the absence of any

other kinase inhibitory data, the results obtained with Calu-1 cells tend to confirm the Cdk4 inhibitory

potential of CA224 in live cells.

CA224 at the IC50 concentration induces profound G2/M block in asynchronous cancer cells

(p53+) A549 and (p53-null) NCI-H1299 cells. Incubation of A549 cells with CA224 at IC50

concentration for 24 h induces a profound block at G2/M as indicated by the percentage of cells at G2/M.

As seen in Figure 2, at the IC50 concentration of CA224, 89% cells appear to be arrested in the G2/M

phase (Figure 2B) and at IC70 concentration 91% cells blocked at G2/M (not shown). Upon incubation of

NCI-H1299 (p53-null) with IC50 concentration of CA224 resulted in large number of cells (71% cells)

accumulation at G2/M (Figure 2D). The results confirm that a profound G2/M block can be observed in

cells (A549 and NCI-H1299) where the mitotic spindle checkpoint is intact.

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Nocodazole and paclitaxel induced G2/M block is maintained by CA224 in NCI-H358 lung

cancer cells. NCI-H358 (p53-null) cells were treated with nocodazole (1 µM, a sub-optimal

concentration) only for 18 h in order to induce a partial block at G2/M so that treated cells are minimally

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 CA224 for 12 h, cells not

only maintained the G2/M block but also >50% of G0/G1 and S phase cells entered G2/M (compare

Figure 2E-H). Although only representative histograms of nocodazole treatment are shown, similar

results were obtained when paclitaxel blocked cells were released in the presence of CA224 for 12 h.

These observations suggest that, at least, in p53-null/pRb-null NCI-H358 cells, CA224 maintains the

pro-metaphase block induced by nocadozole or paclitaxel during mitosis.

CA224 blocks NCI-H358 cells in G2/M after release from hydroxyurea-mediated G1/S cell

synchronization. Hydroxyurea (250 µM, 18 h) was used to block cells at G1/S (77%; Figure 2J), at a

stage of the cell cycle where Cdk2-specific inhibitors normally act. When released in the presence of

CA224, cells proceed from G1/S, confirming that CA224 does not inhibit cellular Cdk2. Cells

ultimately accumulate at G2/M (72%; Figure 2L). These results again indicate that CA224 have an

inherent tendency to induce block at G2/M phase of the cell cycle at least in cells where mitoic spindle

checkpoint is normal.

Selective killing of SV40 large T-antigen transformed normal mouse embryonic liver cells by

CA224. SV40 large T-antigen inactivates both the tumor suppressor proteins, p53 and pRb, and thereby

transforms normal cells into tumorigenic cells. We used normal mouse embryonic hepatic (liver) cells

BNL CL2 and its SV-40 large T-antigen transformed counterpart BNL SV A.8 to study the effects of

CA224. The normal cells, upon 48 h incubation with CA224, exhibited prominent G2/M arrest at both

IC50 and IC70 concentrations with less than 5% cells detected in the sub-G0/G1 phase (Figure 2N) and

more than 50% cells appearing at G2/M phase of the cell cycle. Interestingly, in the SV40 large T-

antigen transformed cell line significant apoptotic cell death was observed. After 48 h treatment at the

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IC50 concentration of CA224, 31% cells were detected in sub-G1 phase (Figure 2P) indicating apoptosis.

The percent apoptosis increased further from 31 to 44% at the IC70 concentration after 48 h incubation

(data not shown). Similar results were obtained using normal and SV-40 large T-antigen transformed

human lung fibroblast cells, WI-38 (data not shown).

BControl CA224, IC50, 24 h

G0/G1 = 49% G0/G1 = 3%

S = 29% S = 4%

G2/M = 89%G2/M = 22%

A549 NCI-H1299Control CA224, IC50, 24 hG0/G1 = 56% G0/G1 = 22%

S = 21% S = 10%

G2/M = 18% G2/M = 71%

A C D

PMT4 dna Lin PMT4 dnaLin PMT4 dnaLin PMT4 dnaLin

Control Hydroxyurea, 250 µM, 18 hHydroxyurea, 250 µM, 18 h Released in fresh medium, 18 h

Hydroxyurea, 250 µM, 18 h Released in CA224, IC50, 18 h

I J K LG0/G1=59% G0/G1=77% G0/G1=50% G0/G1=16%

S=21% S=16% S=12% S=10%

G2/M=18% G2/M=6% G2/M=33% G2/M=72%

E F G H

Control

Nocodazole, 18 h;

Released in medium, 12 hNocodazole, 18 h

Nocodazole, 18 h;

Released in CA224, IC50, 12 h

G0/G1=59% G0/G1=22% G0/G1=40% G0/G1=7%

S=21% S=24% S=10% S=10%

G2/M=18% G2/M=48% G2/M=42% G2/M=74%

NCI-H358

PMT4 dna Lin PMT4 dnaLin PMT4 dna Lin PMT4 dnaLin

PMT4 dna Lin PMT4 dnaLin PMT4 dna Lin PMT4 dnaLin

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Control ControlCA224, IC50, 48 h CA224, IC50, 48 h

G0/G1 = 57% G0/G1 = 34% G0/G1 = 64% G0/G1=31%

S = 15% S=21%S = 23%S = 9%

G2/M = 23% G2/M=14%G2/M=13%G2/M = 54%

BNL CL2 (mouse normal hepatic cell line) BNL SV A. 8 (mouse SV40 transformed hepatic cell line)

Sub G1 = 2%Sub G1 = 0% Sub G1 =31%Sub G1 =0%

Sub G1 Sub G1

Sub G1Sub G1

M N O P

PMT4 dna Lin PMT4 dnaLin PMT4 dnaLin PMT4 dnaLin

Figure 2. (A-D) Response of mitotic spindle checkpoint-proficient human lung cancer cell lines, A549

and NCI-H1299 to CA224. Flow cytometric analysis of asynchronous cells show that majority of cells

are arrested in G2/M phase of cell cycle (4n DNA content) in both the cell lines. A549 untreated (A),

treatment with IC50 concentration of CA224 for 24 h (B), NCI-H1299 untreated or control (C) and

treatment with IC50 concentration of CA224 for 24 h (D). (E-L) Analysis of NCI-H358 cells using flow

cytometer. The G2/M and G1/S synchronized cells by nocodazole and hydroxyurea respectively were

released either in fresh medium or in fresh medium containing IC50 concentration of CA224 compound

exhibit greater tendency of CA224 to block the cell growth at G2/M. For 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 the presence of CA224, 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 CA224, IC50 (L). (M-P) Selective apoptosis in SV-40 transformed cells by CA224,

analysed by FACS. BNL CL2 (mouse embryonic normal hepatic cells) when exposed to CA224 for 48

h at IC50 and IC70 concentrations, show prominent G2/M arrest. As seen in the figure untreated cells

(M), treated with IC50 concentration of CA224, 48 h (N). BNL SV A. 8 (mouse embryonic SV-40

transformed hepatic cells) underwent apoptotic cell death upon incubation with CA224. The apoptosis

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was quantitated by measuring the % cells appeared in SubG1 peak, 31% and 44% cells were found in

SubG1 peak after 48 h exposure with CA224, IC50 (P) concentration. The untreated cells (O) do not

show any apoptosis.

Effects of CA224 on the cellular level of cyclin B1, Cdk1, p53, p21CIP1/WAF1

(p21) and p27KIP1

(p27); analyses in p53+ cells. Western-blot analyses of p53+ cells, A549 and LS174T, after treatment

with CA224 (at the IC50 concentration) for 24 h demonstrated more than 10-fold induction of p53.

Thereby, probably the global Cdk inhibitorp21CIP1/WAF1 (p21) was also induced. The levels of p27KIP1

(p27) were also elevated after CA224 treatment (Figure 3). The proteins Cdk1 and cyclin B1 were

down-regulated 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 G2/M block seen in A549

and LS174T cells that bear functional copies of the tumor supressor protein, p53 (Figure 3).

The effects on the cellular level of cyclin B1, Cdk1, p53, p21CIP1/WAF1 (p21) and p27KIP1 (p27);

analyses in p53-negative cells were also investigated. Western-blot analyses on proteins from

MIAPaCa-2 cells (carrying a p53 mutation) showed that p53, p21 and p27 levels remained unchanged

indicating that the p21 and p27 induction seen in p53-positive 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 Cdk1-cyclin B1 is still active in p53 mutated cells,

after CA224 treatment.

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LS174T

Control Treated Control Treated

A549

p53

p21

p27

CyclinB1

Cdk1

Actin

Figure 3. Western-blot analysis of p53+ cells, A549 and LS174T. The treatment with CA224, IC50 concentration for 24 h

resulted in induction of tumor suppressor protein p53 and thereby global Cdk inhibitor p21CIP1/WAF1 (p21) was also induced.

The levels of global p27KIP1 (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 CA224. CA224 inhibited growth of cancer cells in-vitro at relatively low concentrations than it

inhibited the enzyme Cdk4-cyclin D1. FACS analysis and mitotic index experiments (data not shown)

had indicated that CA224 blocked cell growth at the pro-metaphase of the cell cycle. In addition to

these observations, the 2-3 fold increase of CA224 IC50 in cells with impaired mitotic spindle

checkpoint suggested another cellular role for CA224, possibly as a antimicrotubule agent. We

investigated the action of CA224 on tubulin polymerization in-vitro. The results indicated strong

antitubulin activity of CA224 (Figure 4a) while fascaplysin does not show any interaction with tubulin

(data not shown). Representative polymerization curves of CA224, paclitaxel and nocodazole are shown

in Figure 4a. CA224 inhibits the polymerization of tubulin which is concluded from the dose-dependent

decrease in Vmax (mOD/min) and reduction of final polymer mass (Figure 4a). When tested at four

different concentrations, CA224 decreased the Vmax from 17 mOD/min to 6.2, 2.1, 1.1 and 0.4

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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.

Microtubules, the key components of cytoskeleton are made up of α/β-tubulin 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 β-tubulin. 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 (M-loop) and thus further polymerization process

gets stopped. Furthermore, tubulin-polymerization inhibitor complex perturbs microtubule growth by

sterically blocking further addition of the tubulin dimers to form microtubule assembly.38-40 Upon

molecular docking studies, it was observed that CA224 binds at α/β-tubulin interphase by H-bonding.

CA224 interacts with the Thr353 residue of β tubulin by H-bonding. In addition to this, the hydrophobic

biphenyl ring fits in hydrophobic core of the β-tubulin formed by Leu248, Ala250, Leu252, Cys241,

Leu255, Ala316, Ala317 and Ala354 (Figure 4b). Interestingly, these interactions were missing in

fascaplysin. CA224 binding at interphase of α/β-tubulin, is supposed to induce conformational changes

in protein which further perturbs tubulin polymerization to form microtubule assembly.

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0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0 1000 2000 3000 4000

Time (s)

M 3

40

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0 1000 2000 3000 4000

Time (s)

M 340

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0 1000 2000 3000 4000

Time (s)

M 340

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0 1000 2000 3000 4000

Time (s)

M 3

40

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0 1000 2000 3000 4000

Time (s)

M 3

40

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0 1000 2000 3000 4000

Time (s)

M 3

40

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0 1000 2000 3000 4000

Time (s)

M 3

40

Cell free Tubulin

polymerization assay

in vitro

control; Vmax = 19 mOD/min

paclitaxel, 10 µM; Vmax = 61 mOD/min

nocodazole, 10 µM; Vmax = 4.5 mOD/min

CA224, 2.5 µM; Vmax = 6.2 mOD/min

CA224, 25 µM; Vmax = 0.4 mOD/min



(a) (b)

Figure 4. (A) Purified tubulin polymerization assay in vitro. The ability of CA224 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. CA224 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 CA224 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)

CA224 interactions at α/β-tubulin interphase.

CA224 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 CA224 inhibits

polymerization of tubulin. Moreover CA224 also enhances the depolymerization of stabilized tubulin

protein. The polymerized and depolymerized (soluble) forms of tubulin were gauged (via Western-

blotting) from the accumulation/ disappearence of tubulin protein from pellet and from supernatant

fractions of cell lysates treated with IC50 concentration of CA224. In the first set of experiments where

cells were treated with CA224 in the presence of microtubule stabilizing agent paclitaxel, CA224

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showed prevention of tubuin polymerization (mediated by paclitaxel) in a dose-dependent manner

(Figure 5, upper two panels). In a second set of experiment where intracellular tubulin was stabilised

with paclitaxel and then subjected to CA224 treatment, results show enhancement of tubulin

depolymerization with the increasing concentrations of CA224 (Figure 5, lower two panels).

simultaneous treatment of paclitaxel and CA224

paclitaxel treatment followed by CA224

supernatant

pellet

pellet

supernatant

Figure 5. Tubulin polymerization assay in vivo. Western blots show the response of CA224 to tubulin polymerization in the

presence of paclitaxel and the effect of CA224 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 CA224 show inhibition of tubulin polymerization by CA224 in a

dose dependent manner and resulted in accummulation of unassembled tubulin in supernatant. It was observed that CA224

also act as an enhancer for tubulin depolymerization in dose dependent manner when paclitaxel stabilized tubulin was

subjected CA224 treatment.

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Colony formation assay. The ability of CA224 in killing cancer cells was explored in the long term

survival assay which monitors cancer cells’ colony-forming capacity in-vitro. A549 (with normal

mitotic spindle checkpoint normal, pRb+) and Calu-1 (with mitotic spindle checkpoint impaired, pRb+)

cells were used in colony formation assays. The colony formation efficiency of A549 and Calu-1 cells is

significantly reduced after CA224 treatment (Figure 6). The concentration at which 50% colony

formation efficiency is observed is comaparatively lower than the IC50 concentration for cell growth

inhibition in MTT assay indicating that a large number of cells loose the ability to form colonies or do

not survive for a long time after CA224 treatment. For A549 cells, 50% colony formation efficiency was

observed at an average 1.4 µM concentration of CA224 (cell growth inhibition IC50 = 3.5 µM) and for

Calu-1 cells 50% colony formation efficiency was observed at an average of 3 µM concentration of

CA224 (cell growth inhibition IC50 = 11.5 µM) (Table 2; Figure 6) indicating that CA224 may be quite

efficacious in in vivo mouse tumor models.

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a

c

b

h

f

d

g

je

i

A

B

0

20

40

60

80

100

1 10 100

CA224 concentration (µM)

colo

ny f

orm

atio

n ef

fici

ency

(% u

ntre

ated

cel

ls)

A549

Calu1

A549 Calu-1

Figure 6. CA224 in colony formation assay. A549 and Calu-1 cells were investigated for their long term survival

efficiency after the treatment with different concentrations of CA224. The colony formation efficiency is expressed as the

percentage of colonies formed in the treated cultures compaired with untreated cultures. (A) The representative plates show

A549 untreated (a), treated with CA224, 3.12 µM (b), treated with CA224 6.25 µM (c), treated with CA224, 12.5 µM (d)

treated with fascaplysin 0.8 µM (e); Calu-1 untreated (f), treated with CA224, 3.12 µM (g), treated with CA224 6.25 µM (h),

treated with CA224, 12.5 µM (i) treated with fascaplysin 1 µM (j). (B) The curves representing colony formation efficiencies

of A549 and Calu-1 cells with increasing concentrations of CA224. All results represent the means and standard deviations

of three independent experiments.

CA224 induces apoptotic cell death in cancer cells. When A549 cells were treated with increasing

concentrations of CA224 for 24 h, dose-dependent induction of fragmented nuclei, disrupted cell

membrane and apoptotic cell death was observed (Figure 7A-c,d,e). The induction of apoptosis in NCI-

H460, NCI-H358 and NCI-H1299 cells was also measured with flow cytometry (Figure 7B). Dose-

dependent and time-dependent increase in apoptotic cell death was observed.

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0

10

20

30

40

50

60

24h 48h 72h

time of incubation

% a

popt

osis

NCI-H460; IC50

NCI-H460; IC70

NCI-H1299; IC50

NCI-H1299; IC70

NCI-H358; IC50

NCI-H358; IC70

A

B

a b c

d e

Figure 7. Induction of apoptotic cell death analysed by DAPI staining and FACS. Incubation of A549 cells with

increasing concentration of CA224 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 CA224, 1 µM (b), treated with CA224, 2.5 µM (c), treated with CA224, 5 µM

(d), and treated with CA224, 10 µM (e). The pre-apoptotic and apoptotic cells are indicated with arrows. (B) The percent

apoptosis induced in three cancer cell lines by the treatment with CA224 and determined by FACS analysis. The percent

apoptosis is depicted from the percent of cells appeare in Sub G1 peak during cell cycle analysis. The apoptosis show

increase with concentration used and time of incubation with CA224.

Aqueous solubility, pharmacokinetics, CYP450 liability and caco-2 permeability. The solubility

of CA224 in water, PBS, SGF and SIF was found to be 80, 125, 125 and 5 µg/ml, respectively. In order

to check the plasma exposure of the compound in animals, the pharmacokinetics of CA224 was studied

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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. CA224 showed good plasma exposure with Cmax of 190 and 371 ng/mL

(537 and 1048 nM) by PO and IV routes, respectively. The AUC0-∞ values were 182 and 189 ng·h/mL

(514 and 534 nM), respectively. In CYP-liability studies, at 10 µM, CA224 showed 50, 14, 51 and 19%

inhibition of CYP3A4, CYP2D6, CYP2C9 and CYPC19, respectively. In permeability experiment,

efflux ratio [Papp (B–A)/Papp (A–B)] was 1.1, indicating that CA224 is not a substrate of Pgp.

In vivo experiments: Maximum tolerated dose (MTD). The studies which allowed determination

of MTD-s were performed in Swiss albino mice for two weeks. The concentration at which CA224

would be tested in vivo was thus ascertained. The loss in animal body weight was considered as a

measure of over-toxicity 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 and animals can recover once the treatment is stopped.

The toxicity results obtained from these studies indicated that for CA224 the MTD in mice was ~1000

mpk (mg/kg). Based on the MTD experiment and PK data, CA224 was tested at 1/10th of MTD

concentration i.e. 100 mg/kg in in-vivo xenograft studies.

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, CA224 showed

statistically significant (p<0.05) tumor growth inhibition (Figure 8A) at 1/10th of MTD concentration

(100 mg/kg) in the HCT-116 tumor model. Mice injected with CA224 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 anti-tumor potential of CA224 was confirmed when it was proven to

be highly efficacious in NCI-H460 tumor models at 100 mg/kg concentration (Figure 8C-D). These

results indicate CA224 has strong anti-cancer properties in vivo when administered at a concentration 10

times less than the MTD. Thus, with these studies CA224 has shown efficacy as a new class of

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anticancer compound. Further chemical-biological optimization could lead to the identification of a

potential candidate for clinical development.

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 8B) in the HCT-116 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

100%. The percentage weight loss or gain was calculated using the initial weight as a reference. Similar

results were obtained in NCI-H460 tumor model (data not shown). CA224 shows minimal toxicity in

animal models and can be tolerated up to 1000 mg/kg concentration without any significant toxicity.

Anti-tumour activity of CA224 in SCID mice using

human colon cancer cell, HCT116, xenografts

10

210

410

610

810

1010

0 5 10 15 20

No. of days (9-day treatment followed by a 9-day period of no-treatment)

tumour volume, mg

ControlCA224 100mpk

(A)

90%

Statistically insignificant (< 10%) loss in body weight after 9

days treatment with CA224

60

80

100

120

140

0 2 4 6 8 10 12 14

mean body weights normalised

to 100%

of starting value

Control

CA224 100 mg/kg

No. of days (9-day treatment followed by a 5-day period of no-treatment)

(B)

0

500

1000

1500

2000

2500

0 2 4 6 8 10 12

tumor volume, mg

No. of days

Anti-tumor activity of CA224 in SCID mice using human lung cancer NCI-H-460, xenografts

Control

CA224 100 mg/kg

(C)

Control or untreated mice with NCI-H460 xenografts Mice treated with CA224 100mpk

tumors removed from control or untreated

mice (human lung cancer, NCI-H460)

tumors removedfrom mice treated

with CA224 100mpk

(D)

Figure 8. (A) Tumor growth inhibition curves for CA224 in in vivo HCT116 xenograft model. Graphs depict tumor growth

inhibition in a group of animals treated with CA224 at the concentration 100 mpk which is compared with the untreated

group of animals (shown in the graphs as the control group). Tumor sizes were recorded at 2-5 day intervals. Tumor weight

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(in mg) was estimated according to the formula for a prolate ellipsoid: [Length (mm) x [width (mm)2] x 0.5] assuming

specific gravity to be one and π to be three. CA224 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 CA224 (100 mpk) 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 CA224 in in vivo NCI-H460 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 2-5 day intervals. Tumor weight (in mg) was estimated according to the formula for a prolate

ellipsoid: [Length (mm) x [width (mm)2] x 0.5] assuming specific gravity to be one and π to be three. CA224 shows

statistically significant tumor growth inhibition in vivo. p < 0.05 (Student t test was used for multiple comparisons of

different groups versus control). (D). Control and treatment groups of SCID mice showing NCI-H460 tumor growth

inhibition followed by treatment with CA224 as labeled in the figure. These pictures were obtained after treatment with

CA224 at the concentration 100 mpk. The treatments were continued for 9 consecutive days intra-peritoneally when tumor

growth had reached about 4-6 mm in diameter after about 6 days followed by the tumor cell injection.

CONCLUSION

In summary, we have presented data that supports the notion that CA224 has the potential of being a

novel anticancer agent. The finding of dual inhibitors of Cdk4-cyclin D1 and tubulin polymerization,

may address the problems of resistance which currently limit the efficacy of valuable anti-microtubule

agents. The promising efficacy of CA224 in xenograft models (HCT-116 and NCI-H-460 tumor

models) with an excellent therapeutic window indicates promise of this compound to develop as

anticancer agent.

EXPERIMENTAL SECTION

General. All chemicals were obtained from Sigma-Aldrich Company and used as received. 1H, 13C

and DEPT NMR spectra were recorded on Bruker-Avance DPX FT-NMR 500 and 400 MHz

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instruments. 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 CDCl3. Carbon nuclear

magnetic resonance spectra (13C NMR) were recorded at 125 MHz or 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 solvents (CDCl3: 77.00 ppm). ESI-MS spectra were recorded

on Agilent 1100 LC-Q-TOF machine.

Synthesis of [2-(1H-Indol-3yl-ethyl]-carbamic acid ethyl ester (2): To the suspension of

tryptamine 1 (1.2 mmol) in CH2Cl2 (3 ml) at 0 °C was added slowly an aqueous solution of 4 N NaOH

(1.2 mmol). After 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

CH2Cl2 and was washed with 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

(#100-200) column chromatography using EtOAc: Hexane as mobile phase to get title compound 2.

Light pink oil; yield: 90%; 1H NMR (400 MHz, CDCl3, ppm): δ 8.15 (1H, brs, NH), 7.60 (1H, d, J =

8Hz), 7.36 (1H, d, J = 8 Hz), 7.25 – 7.10 (2H, m), 7.01 (1H, s), 4.75 (1H,brs, NH), 4.13 – 4.08 (2H, m),

3.52 (2H, d, J = 4 Hz), 2.97 (2H, t, J = 8 Hz), 1.27 – 1.20 (3H, m); ESI-MS: m/z 233.10 [M+1]+.

Synthesis of [2-(1H-Indol-3-yl)-ethyl]-methylamine (3): To the solution of 2 (0.1 g, 59.4 mmol) in

dry THF (5 mL) under N2 flux at 0 °C was added portionwise LAH (178 mmol). After 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 (2ml) 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)3, was washed with THF (10 ml). The organic layer was dried (Na2SO4) and

evaporated under reduced pressure to give the title compound 3; Beige solid, yield: 85%; 1H NMR (400

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MHz, CDCl3, ppm): δ 8.81 (1H,brs, NH), 7.60 (1H, s), 7.30 (1H, s), 7.17 – 7.10 (2H, m), 6.94 (1H, s),

2.97 – 2.91 (4H, m), 2.41 (3H, s); ESI-MS: m/z 175.0 [M+1]+.

Synthesis of biphenyl-4-carboxylic acid [2-(1H-indol-3-yl)-ethyl]-methyl amide (CA224, 5): To

the suspension of [2-(1H-Indol-3-yl)-ethyl]-methylamine 3 (1.2 mmol) in CH2Cl2 (3 ml) at 0 °C was

added slowly an aqueous solution of 4 N NaOH (1.2 mmol). After 5 min stirring at 0 °C, 4-biphenyl

carbonychloride (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 CH2Cl2 and was washed with 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 (#100-200) column chromatography using EtOAc: Hexane as mobile phase to

get title compound 5. Beige solid ; yield: 45%; 1H NMR (400 MHz, CDCl3, ppm): δ 8.21 (1H, brs, NH),

7.58 – 7.09 (13H, m), 6.88 (1H, t, J = 8Hz), 3.88 (1H, t, J = 8Hz), 3.61 (1H, t, J = 4Hz), 3.20 (3H, s),

2.98 (2H, t, J = 4Hz); δ (distinct peaks for minor rotamer) 7.58 (1H, d, J = 8Hz), 7.01 (1H, d, J = 8Hz),

2.93 (1H, d, J = 8Hz); 13C NMR (100 MHz, DMSO-d6, ppm): δ (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), 111.34 (CH), 51.43 (CH2), 32.33

(CH3), 23.84 (CH2). δ (distinct peaks for minor rotamer) 169.55 (CO), 140.55 (C), 135.72 (C), 123.06

(CH), 118.28 (CH), 110.40 (C), 47.87 (CH2), 37.44 (CH3), 22.52 (CH2); ESI-MS: m/z 355.10 [M+1]+.

Kinase profiling. CA224 was tested against a panel of kinases at 10 µM. The in-vitro kinase assays

for testing against Cdk4-cyclin D1, Cdk2-cyclin A, Cdk2-cyclin E, Cdk1-cyclin B1 and Cdk9-cyclin T1

were performed in house. The methodology and results have been reported previously.33-37 The kinase

profiling for 58 representative kinases was done commercially at Millipore Bioscience Division, UK.

In-vitro cell proliferation assays. All ten human cancer cell lines were maintained at 37 ºC in 5%

CO2 in RPMI-1640 medium, supplemented with 10% fetal calf serum and 100 µg/ml NormocinTM. The

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ten cancer cell lines used for screening were non-small cell lung carcinoma (NSCLC; a form of cancer

which is resistant to chemotherapy) lines: NCI-H460 (pRb+, p53+), A549 (pRb+, p53+) obtained from

the Glenfield Hospital, Leicester in June 2006; and Calu-1 (pRb+, p53-null), NCI-H1299 (pRb+, p53-

null), NCI-H358 (pRb-null, p53-null) obtained from Mr Pat Browne (Morvus Technology) in July 2007.

The colon cancer line LS174T (pRb+, p53+), and the prostate cancer line PC3 (pRb+, p53-null) were also

gifts from Mr Pat Browne (Morvus Technology) in July 2007. The pancreatic cancer line MiaPaca

(pRb+, p53-mutant) 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 SV-40 large T-antigen 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

detailed procedure of cell proliferation (MTT) assay and IC50 determination was described previously.36

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 (-20ºC) ethanol for

minimum 1 h. After the fixation step, cells were centrifuged for 5 min at 3000X g at room temperature

and then the pellet was suspended in PBS containing 50 µg/ml propidium iodide (Sigma Cat No P-

4170) and 0.5 mg/ml DNase-free ribonuclease (Sigma Cat No R-5503). The cells were stained for 1 h

in the dark at 4°C. Cell cycle analysis was performed on the Beckman Coulter (Epics® AltraTM)

fluorescence-activated 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

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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.

Western-blotting. A549, LS174T and MIAPaCa cells were seeded in 25 cm2 tissue culture flasks in

the complete growth medium. When the culture flasks reached 40-50% confluency, cells were treated

with CA224 (IC50 concentration) for 24 h. Cells were harvested by trypsinization, washed in ice-cold

PBS and then lysed in buffer (Sigma Cat No C-2978) containing protease inhibitor cocktail (Sigma Cat

No P8340). The lysates were centrifuged at 14,000 rpm for 10 min at 4ºC 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% SDS-polyacrylamide 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 (CR-UK Cat No V152) at 1:1500 dilution to

detect cyclin B1; Pab 1801 (Santa Cruz Biotechnology Cat No sc-98) at 1: 500 dilution to detect p53; N-

20 (Santa Cruz Biotechnology Cat No sc-469) at 1:500 dilution to detect p21; C-19 (SantaCruz

Biotechnology Cat No sc-528) at 1:250 dilution to detect p27; AC-40 (Sigma-Aldrich 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

(SantaCruz Biotechnology Cat No sc-2048).

Tubulin polymerization assay in-vitro. The purified tubulin was obtained commercially

(Cytoskeleton Inc. Denver USA) and the polymerization assays were carried out according to the

method previously described.41, 42 Tubulin polymerization assay is based on the adaptation of the

original method of Lee and Timasheff (1977) which demonstrated that light is scattered by microtubules

to an extent that is proportional to the concentration of the microtubule polymer. The resulting

polymerization curves is representative of three phases of microtubule polymerization, namely

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nucleation, growth and steady-state equilibrium. Paclitaxel and nocodazole were used in the assay as a

known enhancer and inhibitor of tubulin polymerization, respectively. The ability of CA224 to inhibit

tubulin polymerization in-vitro was determined according to the manufacturer’s instructions. Briefly,

tubulin protein (3 mg/ml) was polymerized in GTP buffer (80 mM PIPES pH 6.9, 2 mM MgCl2, 0.5

mM EGTA, 10.2% glycerol and 1 mM GTP) in the presence of a range of CA224 concentrations at 37

ºC in a temperature-regulated Biotech spectrophotometer. The absorbance kinetic (at 340 nm) of 61

cycles for each sample was studied and the readings were recorded at intervals of 1 min.

Western-blot analysis to test effects of CA224 on tubulin polymerization and depolymerization

of stabilized tubulin in live cells. A549 cells were plated at a concentration of 10,000 cells per well in

1 ml complete growth medium in 24 well/15 mm plates. The plates were incubated for 24 h for the

stabilization. In the first set of experiments the cells were treated for 30 min with 10 nM paclitaxel and

different concentrations of CA224 (simultaneous treatment of paclitaxel and CA224). In the second set,

in order to study the effect of CA224 on the stabilised form of tubulin, the cells were treated first with

10 nM paclitaxel for 30 min. The cell monolayer was then washed twice with sterile PBS and fresh

growth medium containing different concentrations of CA224 were added. The plates were further

incubated for 30 min, the cell monolayers were washed twice with sterile PBS at room temperature and

then 100 µl tubulin extraction buffer (1 mM MgCl2, 2 mM EGTA, 0.5% NP40 and 20 mM Tris–HCl pH

6.8) supplemented with 2 mM phenylmethylsulfonyl fluoride, and a protease inhibitor cocktail (Sigma

Cat No P8340) was added per well. After a brief and vigorous vortex, the cell lysates were incubated at

room temperature for 5 min and then centrifuged at 16,000X g for 10 min in order to separate the

soluble and polymerized tubulin fractions. Each supernatant and pellet fraction was mixed with 10X

sample buffer, heated for 7 min at 95ºC and resolved on 10% SDS-polyacrylamide gels. The resolved

proteins were then subjected to Western blotting (as described above) with a specific α-tubulin antibody

B-7 (Santa Cruz Biotechnology Cat No sc-5286).

Detection of apoptosis using DAPI staining. The apoptosis and nuclear fragmentation was

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detected using DAPI staining followed by observations under fluorescence microscope.43 A549 cells

were seeded at the concentration of 50,000 cells per well in 35 mm/6 well plates in a total of 2 ml

complete growth medium. After 24 h stabilization period, cells were further incubated in the presence of

different concentrations of CA224 for 24 h. Followed by exposure to CA224, the cells along with the

floating cells were collected by trypsinization, washed in sterile PBS and fixed in ethanol:acetic acid

(3:1) for 10 min. The cell suspension was dropped on to a glass slide in order to break open the cells

allowing them to be dried in air. The smear formed on the slide was mounted in a medium containing 1

µg/ml DAPI and covered with coverslip. The slides were observed using a fluorescence microscope

(Olympus) and a minimum of 500 nuclei were counted for each sample.

Clonogenic assay. A549 and Calu-1 cells were plated at a concentration of 500 cells per well in 35

mm/6 well plates in 2 ml complete medium. The plates were incubated for 24 h stabilization and further

incubated with a range of concentrations of fascaplysin and CA224 (0.1-10 µM) for 24 h. Plates were

then gently washed with PBS, replaced with fresh medium and incubated at 37 ºC. After 10-12 days,

cells were fixed in methanol:acetic acid (2:1) for 10 min, washed, air dried and stained with 1% crystal

violet. The colonies were evaluated by visual counts. The number of colonies in treated cultures was

expressed as a percentage of the control cultures. All results represents means and standard deviations

from at least three independent experiments.

Determination of aqueous solubility by 96-well plate-based assay. The solublity was determined as

described earlier.44 Briefly, the compound was loaded into 96-well plate in the form of methanolic

solution, followed by evaporation of solvent to get 1, 2, 4, 8, 16, 25, 40, 80, 160 and 300 µg of

compound in solid form in wells. Thereafter, 200 µl of dissolution medium was added to the wells and

plates were shaken horizontally at 300 rpm for 4 h at room temperature (25±1 °C). The plates were

covered with aluminium foil and were kept overnight at room temperature for equilibration. Later, the

plates were centrifuged at 3000 rpm for 15 min (Jouan centrifuge BR4i). Supernatant (50 µl) was

withdrawn into UV 96-well plates (Corning® 96 Well Clear Flat Bottom UV-Transparent Microplate)

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for analyses with microplate reader (Molecular Devices, USA) at corresponding λmax of the sample. The

analysis was performed in triplicate for each compound. The solubility curve of concentration (µg/mL)

vs absorbance was plotted to find out saturation point and the corresponding concentration was noted.

hrCYP P450 isoenzyme assay. hrCYP P450 isoenzyme were aliquoted as per the total

concentration required to conduct the study and stored at -70 °C until use. Total assay volume was

adjusted to 200 µl and consists of three components: cofactors, inhibitor/vehicle and enzyme-substrate

(ES) mix. The 50 µl of working cofactor stock solution was dispensed to all the specified wells in a

black coloured nunc microtiter polypropylene plate. The 50 µl of diluted working concentrations of test

compounds/ positive control /vehicle were dispensed in triplicates to the specified wells as per the plate

map design. Reaction plate with cofactor and test item was preincubated at 37±1 °C in shaking

incubator for 10 min. Simultaneously, ES mix was prepared by mixing the hrCYP P450 isoenzyme.

Remaining volume was made up with the buffer and preincubated for 10 min at 37 ± 1 °C. 100 µL of

ES mix was dispensed per well as per the plate map design and incubated at 37 ± 1 °C in shaking

incubator for predetermined time. A set of controls were incubated with hrCYP P450 isoenzymes and

substrate without test or reference item. A set of blanks were incubated with substrate and test or

reference item, in the absence of hrCYP P450 isoenzymes. Reaction was terminated by adding specific

quenching solutions (for CYP1A2, CYP2C19 and CYP3A4 – 75 µl of 100% acetonitrile; for CYP2C9 –

20 µl of 0.25 M Tris in 60% methanol; for CYP2D6 – 75 µl of 0.25 M Tris in 60% methanol). The

reaction was quenched by thoroughly mixing the final contents of the wells by repeated pippeting using

multichannel pipette. The product fluorescence per well was measured using a multimode reader at

excitation and emission wavelengths of respective hrCYP P450 isoenzyme flourogenic metabolites.

Data was analyzed using Excel spreadsheet and the % inhibition was calculated.45

Caco-2 permeability assay. Permeability study was conducted with the Caco-2 monolayer cultured

for 21days (TEER full form values >500 cm2 in each well) and by adding an appropriate volume of

buffer (HBSS buffer containing 10 mM HEPES) containing test compounds to apical chamber. Test

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sample was taken from both apical and basolateral chambers at 0 and 90 min after incubation at 37ºC

and analyzed by LC-MS/MS. Same experiment was repeated by adding an appropriate volume of

buffer (HBSS buffer containing 10 mM HEPES) containing test compound to basolateral chamber. The

AUC defined the net influx and outflow of the test compound across the Caco-2 cell monolayer.

Pharmacokinetics study. Oral and intravenous (IV) pharmacokinetic studies of CA224 were

carried out in Balb C male mice of age 4-6 weeks, by administering CA224 orally and IV formulation at

dose of 10 mg/kg for oral and 1 mg/kg for IV. Plasma samples were collected at appropriate time points

between the range of 0 hours to 24 hours and analyzed by LC-MS-MS. Mean plasma concentration

calculated and data were further analyzed for PK parameters evaluation using WinNonlin 5.3 software

package.

Maximum tolerated dose (MTD) finding studies for in vivo experiments. Swiss albino mice were

used to determine the maximum tolerated dose for the compound. CA224 was weighed and mixed with

0.5 % (w/v) carboxymethylcellulose (CMC) and triturated with Tween 20 (secundum artum) with

gradual addition of water to make up the final concentration. Care was taken not to exceed > 0.25% of

Tween 20 in the final formulation of the CA224. In this study 6 animals per group were administered

with CA224 at different doses for five days (Q1D x 5) via intraperitoneal route. Animals were

monitored for weight loss, morbidity symptoms and mortality for up to two weeks, which was the end

of treatment. Significant weight loss was considered when mean animal weight dropped by >10% and

was considered highly significant when the drop was >20%.

In vivo efficacy study in SCID mice. The in vivo efficacy was determined in two xenograft

models: HCT-116 and NCI-H460 tumor models.

HCT-116 experiments: A group of 60 Severely Combined Immune-Deficient (SCID strain-

CBySmn.CB17-Prkdcscid/J, The Jackson Laboratory, Stock # 001803) male mice weighing 18-25 g and

6-8 weeks old were used for the studies. Human colon carcinoma, HCT-116 cells (ATCC Cat No CCL-

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247) were grown in McCoy’s 5A medium supplemented with 10% FBS (Sigma-Aldrich). The cultured

cells were injected subcutaneously into dorsal side of SCID mice at the dose of 6.6 × 106 cells in 0.2 ml

of suspension. When the tumor growth reached to about 4-6 mm in diameter (about 5 days), the animals

were randomly divided into eight groups, each containing 7 mice. The treatments were continued for 9

consecutive days intraperitoneally.

NCI-H460 experiments: A group of 65 Severely Combined Immune-Deficient (SCID strain-

CBySmn.CB17-Prkdcscid/J, The Jackson Laboratory, Stock # 001803) female mice weighing 15-24 g

and 6-8 weeks old were used. Human non-small-cell lung carcinoma, NCI-H460 (ATCC Cat No HTB-

177) cells were grown in RPMI-1640 medium supplemented with 10% FBS (Sigma-Aldrich). The

cultured cells were injected subcutaneously into the dorsal side of SCID mice at a tune of 5.3 × 106 cells

in 0.2 ml of suspension. When the tumor growth reached about 4-6 mm in diameter (about 6 days), the

animals were randomly divided into eight groups, each containing 6 or 7 mice. The treatments were

continued for 9 consecutive days intraperitoneally.

Tumor weight measurements: Tumor size was recorded at 2-5 day intervals. Tumor weight (mg)

was estimated according to the formula for a prolate ellipsoid: (Length (mm) x (width (mm)2) x 0.5)

assuming specific gravity to be one and π to be 3. Tumor growth in compound treated animals was

calculated as T/C (treated/control) x 100% and growth inhibition percent (% GI) was [100-% T/C]46-48.

Body weight measurements: The body weights of animals in different treatment and control groups

were monitored by taking the measurements daily during the treatment schedule. By considering the

body weight at the start of the treatment as 100%, the percent weight loss was calculated on subsequent

days of treatments.

Statistical analysis: Data from each experiment was analysed by Microsoft Excel 2000. Statistically

significant differences were identified and analyzed using student t-test for multiple comparisons versus

control group.46-48

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Molecular docking and molecular dynamic simulation. The available crystal structures of Cdk4

are in the apo-form and have several missing residues, thus they cannot be used for molecular

modeling.49 In the present study, we have used a hybrid homology model of Cdk4 described by Shafiq

et al,50 which was developed from the Cdk4/cyclin D apo-crystal structure (PDB: 2W96) by

incorporating positions of missing gaps and activation loops from Cdk2 (PDB: 1FIN).51 This hybrid

homology model was subjected to protein preparation wizard for H-bond optimization, heterogeneous

state generation, protonation and overall minimization. Grid file of docking was constructed using XYZ

co-ordinates of the N atom of Val96 residue with a binding site of 12 Å radius grid box (X = −10.521, Y

= 208.683, Z = 107.944). For Cdk2 docking, the Cdk2 apo-protein (PDB ID: 1FIN) was subjected to

protein preparation wizard for filling missing loops and side chains (using prime), ionization, H-bond

optimization, heterogeneous state generation, protonation and overall minimization. Grid file of docking

was constructed using XYZ co-ordinates of the N atom of Leu83 residue with a binding site of 12 Å

radius grid box (X = − 10.406, Y = 209.105, Z = 107.576).

For tubulin docking, the tubulin-colchicine complex (PDB ID: 1SA0) was retrieved from the

protein data bank.52 In this complex, protein is heterodimeric in nature, consisting of two α-chains (451

residues), two β-chains (452 residues) and the Stathmin like domain (142 residues). Crystal structure

was subjected to protein preparation wizard for filling missing loops and side chains (using prime),

ionization, H-bond optimization, heterogeneous state generation, protonation and overall minimization.

All other ligands, water and ions were removed except colchicine. Grid file for docking was constructed

considering colchicine ligand as centroid of grid box of 10 Å size at interphase of α/β tubulin (C and D

chains). All ligands were sketched in Maestro, prepared using ligprep and docked by Glide molecular

docking software in XP mode.

The Cdk4-CA224 docked complex obtained from XP-docking was subjected to system builder, in

which TIP4P-Ew was used as an aqueous solvent model. The cubic box of 12 Å radius was used to

define the core and overall complex was neutralized by adding one Cl- counter ion for simulation.

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Further this complex was minimized by steepest descent method followed by the Broyden–Fletcher–

Goldfarb–Shanno algorithm with convergence threshold of 2.0 Kcal/mol and overall 1000 iterations.

MD simulations were carried out at normal temperature and pressure (300°K and 1.01325 bar,

respectively). Thermostat and barostat method opted was langevin with ensemble pathway comprising

NVT (constant number of particles, volume and temperature) and isotropic coupling method. Overall

model system was relaxed before 10 ns simulation and coulombic interactions were defined by short-

range cut-off radius of 9.0 Å and by long-range smooth particle mesh Ewald tolerance to 1e-09.

AUTHOR INFORMATION.

Corresponding Author

* Tel: 44(0)116 250 7280; Fax: +44(0) 116 257 7287; E-mail: [email protected] (B.C.)

*Tel: +91-191-2569000 (Ext. 345). Fax: +91-191-2569333. E-mail: [email protected] (S.B.B.)

*Tel: +91-191-2569111. Fax: +91-191-2569333. E-mail: [email protected] (R.A.V.).

Notes

The authors declare no competing financial interest.

Author Contributions. SM (Sachin Mahale) performed all Cdk assays and in-vitro biology

experiments; PRJ designed fascaplysin analogues for chemical syntheses; BC designed all biology

experiments both in vitro and in vivo; SM (Sudhakar Manda) synthesized CA224 for pharmacokinetic

studies; PJ carried out molecular modeling studies; PJ and SBB interpreted modeling results; SSB

determined solubility of CA224 in various biological fluids; SBB, RAV and BC contributed to

manuscript writing.

ACKNOWLEDGEMENT.

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This research work was funded by Cancer Research UK (BC and PRJ) and CSIR 12th FYP BSC-0205

project.

ABBREVIATIONS.

Cdk4, cyclin-dependent kinase 4; D/w, distilled water; min, minute; mpk, milligrams per kilogram of

body weight; MTD, maximum tolerated dose; rpm, revolutions per minute; SCID, Severe Combined

Immuno Deficient; t1/2,ß. terminal half life; AUC0-t, the area under the plasma concentration-time curve

from 0 to last measurable time point; AUC0-∞, area under the plasma concentration-time curve from time

zero to infinity; Cmax, maximum observed plasma concentration; C0, extrapolated concentration at zero

time point; CL, clearance; Vd, volume of distribution; Vdss, volume of distribution at steady state; Tlast,

time at which last concentration was found; F, oral bioavailability.

ASSOCIATED CONTENT.

Supporting information. Kinase profiling results of CA224. This material is available free of charge

via the Internet at http://www.pubs.acs.org.

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TOC Graphic

Cl

NH

N

O

NH

N

O

Fascaplysin CA224

Cdk4/D1: IC50 = 0.41 µM

Cdk2/A: IC50 = >250 µM

EtBr displacement: 5 µM

Cdk4/D1: IC50 = 6.2 µM

Cdk2/A: IC50 = 521 µM

EtBr displacement: Does not displace

NCI-H460: IC50 = 2 µM

0

500

1000

1500

2000

2500

0 2 4 6 8 10 12

tumor volume, mg

No. of days

Anti-tumor activity of CA224 in SCID mice using human lung cancer NCI-H-460, xenografts

Control

CA224 100 mg/kg

(C)

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ca224, a non-planar analog of fascaplysin inhibits cdk4 and tubulin polymerization: evaluation of in...

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