Discovery of a potent tubulin polymerization inhibitor: Synthesis and evaluation of water-soluble prodrugs of benzophenone analog

Article abstract of DOI:10.1016/j.bmcl.2010.05.060

Prodrugs have proven to be very useful in enhancing aqueous solubility of sparingly water-soluble drugs, thereby increasing in vivo efficacy without a need of special excipients. In vitro and in vivo evaluations of a number of amino acid prodrugs of 1, a previously identified potent tubulin polymerization inhibitor and cytotoxic against various cancer cell lines led to the discovery of 3·HCl (l-valine attached) which is highly efficacious in mouse xenografts bearing human cancer. Pharmacokinetic analysis in rats revealed that compound 1 was released immediately upon administration of 3·HCl intravenously, with rapid clearance of 3·HCl indicating the effective cleavage of prodrug. Compound 3·HCl (CKD-516) has now been progressed to phase 1 clinical trial.

Full text of DOI:10.1016/j.bmcl.2010.05.060

Bioorganic & Medicinal Chemistry Letters 20 (2010) 6327–6330
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of a potent tubulin polymerization inhibitor: Synthesis
and evaluation of water-soluble prodrugs of benzophenone analog
Jaekwang Lee a, Suyeal Bae a, Seo-hee Lee a, Hojin Choi a, Young Hoon Kim a, Soo Jin Kim a, Gyu Tae Park a,
Seung Kee Moon a, Dal-Hyun Kim a, Sungsook Lee a, Soon Kil Ahn b, Nam Song Choi a, Kyung Joo Lee a,
*
a Chong Kun Dang Research Institute, CKD Pharmaceuticals Inc., PO Box 74, Chonan, Republic of Korea
b Division of Life Science, University of Incheon, Incheon 406-772, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Prodrugs have proven to be very useful in enhancing aqueous solubility of sparingly water-soluble drugs,
thereby increasing in vivo efficacy without a need of special excipients. In vitro and in vivo evaluations of
a number of amino acid prodrugs of 1, a previously identified potent tubulin polymerization inhibitor and
Received 12 February 2010
Revised 13 May 2010
Accepted 14 May 2010
Available online 20 May 2010
cytotoxic against various cancer cell lines led to the discovery of 3ÁHCl (
L-valine attached) which is highly
efficacious in mouse xenografts bearing human cancer. Pharmacokinetic analysis in rats revealed that
compound 1 was released immediately upon administration of 3ÁHCl intravenously, with rapid clearance
of 3ÁHCl indicating the effective cleavage of prodrug. Compound 3ÁHCl (CKD-516) has now been pro-
gressed to phase 1 clinical trial.
Keywords:
Tubulin
Inhibitor
Prodrug
Ó 2010 Published by Elsevier Ltd.
Aqueous solubility
Antitumor activity
Microtubules, a major type of cytoskeletal filament in cells, are
formed from tubulin subunits, that is, - and b-tubulin. Their
suitable excipients for parenteral use, most formulation techniques
suffer from disadvantages such as chemical and physical stability
and safety issue (e.g., hypersensitivity caused by Cremophor EL
used in paclitaxel).7 Thus, prodrugs often find the high chance of
success in dealing with solubility issues.8 An approach to increase
water-solubility, especially in parenteral drug delivery, is the prep-
aration of prodrugs, in which a polar group is attached to the drug,
releasing the parent drug by cleaving off in vivo via enzymatic
reactions. The polar groups that are most frequently used are phos-
phate and amino acid. Indeed, there are numerous examples of
prodrugs in which phosphate group was attached either directly
linked to parent drug or through a linker group such as formalde-
hyde, for example, fosfluconazole,9 fosphenytoin,10 fludarabine
phosphate,11 and CA-4P (combretastatin A-4 phosphate).4 Amino
acid prodrugs are also found to be useful in enhancing water-solu-
bility of parent drugs which bear either amine or alcohol function
and some examples12 are shown in Figure 1.
We have recently found 1 and its prodrug (3ÁHCl) as potent
tubulin polymerization inhibitors.13 Compound 1 is not only highly
cytotoxic against various cancer cell lines including P-gp over-
expressing cell lines with the cellular mechanism of G2/M arrest,
but also caused a significant vascular disruption leading to central
tumor necrosis. However, its low water-solubility would hamper
further development as a parenteral drug. This was circumvented
a
importance in mitosis and cell division makes microtubules an
attractive target for anticancer drug discovery.1 A number of natu-
rally occurring compounds such as paclitaxel, epothilones, vinbla-
stin, combretastatin A-4, and colchicines exert their effect by
changing dynamics of tubulin polymerization and depolymeriza-
tion.2 In addition to their direct effect on cell division, another
mechanism of anticancer activity of tubulin polymerization inhib-
itors was revealed as VDA (vascular disrupting agent).3 By affecting
the microtubule cytoskeleton, they cause morphological and func-
tional changes in endothelia cells that lead to rapid vasculature
collapse, reduction of blood flow, and ultimately to central tumor
necrosis. Representative tubulin polymerization inhibitors such
as CA-4P4 and AVE-80625 are believed to be more efficient, less
toxic, and several of them are currently undergoing clinical trials.
AVE-8062 is the serine prodrug of AVE-8063, which was found to
have more potent cytotoxicity and antivascular activities com-
pared with CA-4P and is currently undergoing several phase 3 clin-
ical trials for the treatment of solid tumors.
One of major problem in small molecule drug discovery is its
low aqueous solubility that limits its therapeutic application due
to incomplete and variable absorption after administration.6
Although sparingly water-soluble drugs can be formulated with
by the preparation of (
excellent in vivo efficacy in various human tumor xenografts mod-
el due partly to enhanced water-solubility. In this communication,
L
)-valine prodrug (3ÁHCl) demonstrating
* Corresponding author. Tel.: +82 41 529 3320; fax: +82 41 558 3004.
E-mail addresses: kjlee@ckdpharm.com, leekj20012001@yahoo.com (K.J. Lee).
0960-894X/$ - see front matter Ó 2010 Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2010.05.060

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J. Lee et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6327–6330
coupling reaction (BOP, DIPEA) of 1 with (L)-Fmoc-Ser(OtBu)-OH
proceeded in good yield, then followed by sequential deprotection
(TFA then piperidine) gave desired compound.
The antiproliferation potencies (IC50) of parent compound and
its prodrugs against human leukemia cell line (HL60) using MTT
assay15 are shown in Table 1. The IC50 values represent the com-
pound concentrations requiring in 50% decrease in cell growth
after 3 days of incubation. As already mentioned, compound 1
demonstrated potent cytotoxicity against HL60 (IC50 = 4.8 nM) as
Table 1
IC50 values of various amino acid prodrugs of compound 1 against HL60
N
N
O
N
O
O
NH2
R
O
N
S
Figure 1. Examples of amino acid prodrugs and structure of title compounds
(1 and 3).
NH
O
a
Compd
R
IC50 (lM)
we describe the detailed amino acid prodrug study of compound 1,
that is, synthesis of various prodrugs, in vitro activity, in vivo effi-
cacy, and PK (pharmacokinetics) studies leading to the discovery of
clinical candidate (3ÁHCl, CKD-516).
1
2
3
4
5
6
7
8
H
0.0048
0.027
0.110
0.089
0.005
0.098
0.076
0.086
0.74
0.92
0.28
0.013
>2
0.081
0.012
Glycine
(L
)-Valine
)-Phenylalanine
(L
The synthesis of parent compound (1) and its amino acid pro-
drugs were prepared according to Scheme 1. 1,2,4-Triazole was re-
acted with compound 1513 to provide 16, then its C-4 bromo group
was replaced with 1-ethoxyvinyl group (17) by Stille coupling
(Pd(PPh3)2Cl2, THF, reflux). Bromination of 17 with NBS afforded
compound 18 which was condensed with thiourea (EtOH, reflux)
to give parent compound (1). Since several attempts of coupling
reactions of amino acid with compound 1 (e.g., DCC, EDC, and
HBTU) were unsuccessful, Fmoc-amino acids were first converted
to acid chloride, then reacted with 1 under basic conditions at
low temperature. Final compounds (2–13) were prepared by Fmoc
Dimethylglycine
(
(
(
(
(
(
(
(
(
L
)-Proline
)-Leucine
)-Cyclohexylalanine
)-4-Fluorophenylalanine
)-4-Chlorophenylalanine
)-Phenylglycine
)-Alanine
)-4-CF3-phenylalanine
)-Serine
L
L
9
10
11
12
13
14
AVE-8063
D
D
D
L
D
L
—
a
IC50 values are the means of at least two determinations. See the text for
detailed experimental conditions.
deprotection followed by salt formation with HCl.14 For 14 (
L-Ser),
Scheme 1. Reagents and conditions: (a) 1,2,4-triazole, K2CO3, DMF, 130 °C, 12 h; (b) tributyl (1-ethoxy-vinyl)tin, Pd(PPh3)2Cl2, THF, reflux, 6 h; (c) NBS, THF/H2O v/v 3:1, 2 h;
(d) thiourea, EtOH, reflux, 2 h; (e) DMF (cat.), SOCl2, CH2Cl2, reflux, 2 h; (f) DIPEA, pyridine, CH2Cl2, 0 °C to rt, overnight; (g) piperidine, CH3CN, rt, 12 h, then, HCl.

J. Lee et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6327–6330
6329
well as several other cancer cell lines such as MDA-MB-231
(IC50 = 9.8 nM), a P-gp overexpressing MDR (multidrug resistant)
positive cell line. The cytotoxicity of 1 were consistently better
(three to sixfold) than AVE-8063 in all cell lines including MDA-
MB-231 (IC50 = 45 nM for AVE-8063) that was highly resistant to
doxorubicin (IC50 >600 nM). It is interesting to observe that cyto-
toxicity of synthesized prodrugs were quite variable ranging from
single nanomolar to micromolar, indicating the rate of hydrolysis
of prodrugs were different from each other under assay condi-
tions.8a Compounds 2 (Gly), 5 (dimethyl-Gly), and 12 (
L-Ala) were
found to have higher cytotoxicity, implying that smaller amino
acids seemed to be hydrolyzed more rapidly relative to bulkier
analogs. The same degree of antiproliferation activity with 1 was
noted in the case of dimethylglycine (5), which gave us a high
expectation in animal efficacy study. Other prodrugs, whether
hydrophobic (3, 4, 6, 7, and 8) or hydrophilic (14) were in the very
close range of cytotoxicity (70–110 nM) and this suggested the ste-
ric factor was involved in enzymatic hydrolysis of amino acid pro-
Figure 2. Antitumor efficacy of 3ÁHCl in mouse xenografts. Nude mice bearing
human colon HCT116 were treated with vehicle control or test compound (10 mg/
kg, ip, Q4DÂ4). AVE-8062 was used a comparator (80 mg/kg, ip, Q4DÂ4). Data are
means of tumor volume (mm3) at each time point (n = 6 per group). Compounds
were administered on days 2nd, 6th, 10th and 14th.
drugs in this specific case. The low cytotoxicity of prodrugs of D-
amino acids (9, 10, 11, and 13) indicated that they are not good
substrates of hydrolytic enzymes as observed in other reports.16
Although the synthesized prodrugs showed variable cytotoxic-
ity resulting from different hydrolysis rate, it is unlikely that this
trend would translate to in vivo system because of complications
arisen from numerous factors (dissolution rate, hepatic and plasma
clearance, and hydrolysis rate, etc.). Thus, as many analogs were
subjected to in vivo efficacy studies excluding some analogs that
are difficult to synthesize in large amount (e.g., 6 and 14). Nude
mice bearing two human colon cancer cell line (CX-1 and HCT-
116) were dosed ip with vehicle or test compounds on Q4DÂ4
schedules17 (preliminary results showed that ip administration of
test compounds in every 4 days (Q4D) gave the best results
compared to other schedules such as Qd, Q2D, or 2Â/wk). AVE-
8062 was included as a comparator and the results are shown in
Table 2. AVE-8062 induced modest tumor growth inhibition
(IR = 34% at 100 mg/kg) in CX-1 which is considered somewhat
aggressive form of cancer, however significantly inhibited tumor
growth in HCT116 (IR = 60% at 80 mg/kg). Although compound 2
showed better efficacy than AVE-8062 in CX-1, overt toxicity was
observed at higher doses (two deaths at 10 mg/kg). Significant
and dose-dependent tumor growth inhibition was demonstrated
with compound 3ÁHCl (IR = 28% at 5 mg/kg and 64% at 10 mg/kg)
which is much better than AVE-8062 in CX-1. Compound 4 induced
dose-dependent tumor growth inhibition which is comparable to
AVE-8062, although the efficacy is modest. Interestingly, no signif-
icant efficacy (IR ꢀ20%) was observed with compound 5 in CX-1
despite its strong in vitro cytotoxicity (Table 1) demonstrating
the importance of suitable pharmacokinetics. For HCT116 xeno-
grafts, although AVE-8062 was shown to have significant antitu-
mor activity, compound 3 displayed impressive in vivo efficacy
(Fig. 2, IR = 88% at 10 mg/kg, p <0.001). The (L)-alanine prodrug
(12) was found to induce significant tumor growth inhibition, how-
ever, it is inferior to compound 3 considering its apparent toxicity
(one death at 10 mg/kg) at higher dose.
It was evident that starting from the same parent molecule (1),
distinct profiles were achieved by attaching different amino acids.
Based on promising antitumor efficacy, compound 3ÁHCl was se-
lected for further profiling. The apparent aqueous solubility was
substantially improved (<100
l
g/ml for 1 vs 930 mg/ml for 3ÁHCl
in deionized water) as anticipated, which is quite acceptable as
parenteral administration in human use. When incubated in liver
microsomes18 from different species, 1 and 3ÁHCl was shown to
have somewhat different stability. Thus, when 3ÁHCl was incu-
bated for 1 h in human and dog liver microsomes (at 5 lM), only
a fraction of compound was remained (15%, and 10%, respectively),
Table 2
Antitumor activities of selected compounds in BALB/c micea
Compd
CX-1
B.W. change (%)
HCT116
Doses (mg/kg)
IRb (%)
Survival number
Doses (mg/kg)
B.W. change (%)
IRb (%)
Survival number
Control
AVE-8062
2
—
100
5
10
5
10
5
10
5
10
N.D.
+24.5
+22.8
+22.2
+21.0
+25.7
+14.7
+28.9
+24.5
+24.8
+26.7
—
—
—
80
N.D.
À5.7
—
—
6/6
34
40*
44*
28
64**
21
43
23
20
6/6
6/6
4/6
6/6
6/6
6/6
6/6
6/6
6/6
0
60**
3
10
+5.1
88***
6/6
4
N.D.
N.D.
5
12
5
10
À1.7
62**
6/6
5/6
+4.7
74***
N.D.: not determined.
a
Nude mice bearing human CX-1 or HCT116 cancer were dosed ip with vehicle or tested compounds on a Q4dÂ4 schedule. All data are expressed as mean values (n = 6 per
group).
b
IR (%) = (1 À T/C) Â 100; T, tumor volume (treated); C, tumor volume (untreated). IRs’ were measured on the 4th day after last dosing.
*
p <0.05.
p <0.01.
**
***
p <0.001.

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J. Lee et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6327–6330
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by incorporating (
L
)-valine into the amino group of thiazole ring
- or -form) were prepared,
in 1. Various amino acid prodrugs (
L
D
14. Selected data for 3ÁHCl: 1H NMR (400 MHz, DMSO-d6) d 13.00 (s, 1H), 9.07 (s,
1H), 8.70 (d, J = 4.4 Hz, 3H), 8.30 (d, J = 1.5 Hz, 1H), 8.16 (dd, J = 8.0, 1.6 Hz, 1H),
8.11 (s, 1H), 7.97 (s, 1H), 7.70 (d, J = 8.1 Hz, 1H), 6.85 (s, 2H), 3.94 (m, 1H), 3.71
(s, 6H), 3.69 (s, 3H), 2.25 (m, 1H), 0.98 (d, J = 6.9 Hz, 6H).; 13C NMR (100 MHz,
DMSO-d6) d 192.9, 168.0, 157.8, 152.9, 152.7, 147.5, 144.8, 142.5, 137.6, 135.8,
132.8, 131.8, 131.2, 126.2, 121.8, 112.5, 106.9, 60.6, 57.8, 56.4, 30.4, 18.8, 18.3;
HRMS calcd for C26H28N6O5S (free base) 537.1915 [M+H]+, found 537.1915. For
12: 1H NMR (400 MHz, DMSO-d6): d 12.92 (br, 1H, NH), 9.03 (s, 1H), 8.42 (br m,
3H), 8.28 (s, 1H), 8.17 (dd, 1H, J = 8.1, 1.4), 8.09 and 7.98 (2s, 2H), 7.72 (d, 1H,
J = 8.1), 6.86 (s, 2H), 3.72 (s, 6H), 3.70 (s, 3H), 1.50 (d, 3H, J = 7.0). MS (ESI) m/z
509.0 (M++1).
15. HL60 cells were seeded into 96-well plates and the compound diluents were
added. After 72 h incubation at 37 °C in a humidified 5% CO2 atmosphere, cell
viability was determined by addition of MTT (3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide, final concentration of 0.25 mg/ml).
16. Ohsumi, K.; Hatanaka, T.; Nakagawa, R.; Fukuda, Y.; Morinaga, Y.; Suga, Y.;
Nihei, Y.; Ohishi, K.; Akiyama, Y.; Tsuji, T. Anti-Cancer Drug Des. 1999, 14, 539.
17. In brief, HCT116 and CX-1 cells were implanted sc in the flanks of nude mice.
After 20–25 days, tumors from several animals were excised. The viable
portion of the tumor was fragmented and implanted sc in the flanks of nude
mice. Therapy was started after tumor volumes reached to 100–200 mm3.
18. Coleman, M. D. Human Drug Metabolism; Wiley: Bermingham, 2005. Chapter 3.
and their in vitro antiproliferation activity against HL60 then
in vivo efficacy was evaluated. Distinct efficacy profiles were ob-
served in mouse xenografts bearing human tumor (CX-1 and
HCT-116) depending on the amino acids attached. Among them,
3ÁHCl with its aqueous solubility significantly improved showed
marked antitumor efficacy and it was evident from pharmacoki-
netic analysis that upon administration of 3ÁHCl intravenously, it
was rapidly cleared from plasma with appearance of parent com-
pound (1). Phase 1 clinical trial of compound 3ÁHCl (CKD-516) is
currently underway.
Acknowledgment
Financial support from Korean ministry of Health, Welfare &
Family Affairs (MIHWAF) to CCT (Center of Excellence in Cancer
Research, Grant Number A020599) was greatly appreciated.