Synthetic studies on mitotic kinesin Eg5 inhibitors: Synthesis and structure-activity relationships of novel 2,4,5-substituted-1,3,4-thiadiazoline derivatives

Article abstract of DOI:10.1016/j.bmcl.2014.06.034

The 2,4,5-substituted-1,3,4-thiadiazoline derivative 1a has been identified as a new class of mitotic kinesin Eg5 inhibitor. With the aim of enhancement of the mitotic phase accumulation activity, structure optimization of side chains at the 2-, 4-, and 5

Full text of DOI:10.1016/j.bmcl.2014.06.034

Bioorganic & Medicinal Chemistry Letters 24 (2014) 3961–3963
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthetic studies on mitotic kinesin Eg5 inhibitors: Synthesis
and structure–activity relationships of novel 2,4,5-substituted-1,3,
4-thiadiazoline derivatives
⇑
Junichiro Yamamoto , Nobuyoshi Amishiro, Kazuhiko Kato, Yoshihisa Ohta, Yoji Ino, Mitsuharu Araki,
Tetsuya Tsujita, Seiho Okamoto, Takeshi Takahashi, Hideaki Kusaka, Shiro Akinaga, Yoshinori Yamashita,
Ryuichiro Nakai, Chikara Murakata
Fuji Research Park, Research Division, Kyowa Hakko Kirin Co., Ltd, 1188 Shimotogari, Nagaizumi-cho, Suntou-gun, Shizuoka 411-8731, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 24 February 2014
Revised 1 June 2014
Accepted 12 June 2014
Available online 20 June 2014
The 2,4,5-substituted-1,3,4-thiadiazoline derivative 1a has been identified as a new class of mitotic
kinesin Eg5 inhibitor. With the aim of enhancement of the mitotic phase accumulation activity, structure
optimization of side chains at the 2-, 4-, and 5-positions of the 1,3,4-thiadiazoline ring of 1a was
performed. The introduction of sulfonylamino group at the side chain at the 5-position and bulky acyl
group at the 2- and 4-position contributed to a significant increase in the mitotic phase accumulation
activity and Eg5 inhibitory activity. As a result, a series of optically active compounds exhibited an
increased antitumor activity in a human ovarian cancer xenograft mouse model that was induced by oral
administration.
Keywords:
Mitotic kinesin Eg5 inhibitor
Thiadiazoline derivatives
Ó 2014 Elsevier Ltd. All rights reserved.
Antimitotic agents such as taxanes (paclitaxel, docetaxel) and
Vinca alkaloids (vincristine, vinblastine, vinorelbine) are clinically
important chemotherapeutic drugs.1 These drugs function by bind-
ing to tubulin and blocking cell cycle progression during mitosis
via the disruption of microtubule dynamics and activation of the
spindle checkpoint, which ultimately result in cell death.2 As
microtubules are also involved in many other cellular processes,
interference with their formation or depolymerization often causes
peripheral neuropathy as an adverse event.3 Antimitotic agents
that target components of the mitotic machinery other than micro-
tubules are of great interest as new generation anticancer drugs.
The mitotic kinesin Eg5, a member of the kinesin-5 family, plays
an important role in the early stages of mitosis and is one of the
most attractive target enzymes in antimitotic drug development.4
The modulation of the Eg5 activity, via the immunodepletion of
Eg5 proteins or knockdown of Eg5 mRNA by siRNA has been shown
to cause aberrant mitotic spindle formation, cell cycle arrest during
mitosis and the inhibition of proliferation of tumor cells in cul-
ture.5 Therefore, inhibiting Eg5 may be an effective strategy for
treating human cancers.
1,3,4-thiadiazoline derivatives, including compound 1a (Fig. 1), as
a mitotic kinesin Eg5 inhibitor. Compound 1a induces mitotic
arrest and cell growth inhibition in human colorectal cancer
HCT116 cells, selectively inhibited Eg5 and has no effect on micro-
tubule polymerization.6 This compound also exhibits a modest
antitumor activity against in A2780 human ovarian cancer
xenograft model of BALB/cAJcl-nu mice. Although compound 1a
was the only derivative to display activity in our compound library,
its novel structure and chemical accessibility prompted us to
conduct a systematic medicinal chemistry approach in order to
improve the potency of the mitotic phase accumulation activity.
We herein report the synthesis and evaluation of novel 2,4,5-
substituted-1,3,4-thiadiazoline derivatives as mitotic kinesin Eg5
inhibitors. The target compounds were synthesized as shown in
Schemes 1–4.7 The synthesis of derivatives with modified side
A
high-throughput morphology-based screening of our
compound library led to the identification of a novel class of
⇑
Corresponding author. Tel.: +81 42 725 2144; fax: +81 42 726 8330.
E-mail address: junichiro.yamamoto@kyowa-kirin.co.jp (J. Yamamoto).
Figure 1. Eg5 inhibitor identified via high-throughput screening (1a).
http://dx.doi.org/10.1016/j.bmcl.2014.06.034
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

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J. Yamamoto et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3961–3963
Table 1
SAR of the 2 and 4-position of the 1,3,4-thiadiazoline ring
Scheme 1. Reagents and conditions: (a) thiosemicarbazide, cÁHCl, MeOH; (b)
[R1C(O)]2O, pyridine or R1C(O)Cl, Et3N, CH2Cl2.
Compd R1
HCT16
Eg5
IC50
b
Mitotic accumulation
Growth inhibition
a,b
b
MI20 (nmol/L)
GI50 (nmol/L)
(nmol/L)
1a
4
1b
1c
1d
1e
Me 1100
1000
n.t.c
n.t.c
550
190
n.t.c
1300
n.t.c
n.t.c
n.t.c
520
n.t.c
>10,000
1700
Et
iPr
460
iBu 170
Ph
>10,000
Scheme 2. Reagents and conditions: (a) NH2NH2ÁH2O, MeOH.
a
b
c
See Ref. 9 for the assay details.
The values are presented as the mean of at least three independent assays.
n.t., not tested.
phenyl ketone 7a–c were converted to 9a–g using the same
method as that described for the synthesis of compounds 1a–e.
To reduce lipophilicity for increasing metabolic stability, we
developed a new synthetic route as shown in Scheme 4. The selec-
tive reduction of compounds 10a–b using NaBH4 in MeOH yielded
amine 11a–b. Pivaloylation of amine 11a–b then successfully
provided the desired compounds 12a–b.
The compounds were first evaluated for their mitotic accumula-
tion activity in HCT116 cells. The MI20 value was used as the test
compound concentration required for mitotic accumulation
against 20% of the cells tested and reflected the potency of the anti-
mitotic activity. Next, the compounds exhibiting potent MI20 val-
ues were evaluated for anti-proliferative activity and IC50 value
of Eg5 ATPase activity.6 The structures and mitotic accumulation
activity of the 2 and 4-position modified derivatives of 1a are
shown in Table 1. The carbamoyl group at the 2-position was found
to be important for mitotic accumulation. Compound 4, which
changed the amino group of the 2-position of 1a resulted in the
complete loss of mitotic accumulation. The propionyl analogue
1b was less potent than 1a. However, the introduction of
iso-butyryl (1c) and pivaloyl (1d) groups to the same position
resulted in more potent analogues. These compounds also
Scheme 3. Reagents and conditions: (a) Ac2O, Et3N, CH2Cl2; (b) MsNH2, NaH, DMF;
(c) thiosemicarbazide, cÁHCl, MeOH; (d) pivaloyl chloride, Et3N, CH2Cl2.
Table 2
SAR of the 5-position of the 1,3,4-thiadiazoline ring
Scheme 4. Reagents and conditions: (a) R3C(O)Cl, Et3N, CH2Cl2; (b) NaBH4, MeOH;
(c) pivaloyl chloride, Et3N, CH2Cl2.
Compd R2
HCT16
Eg5
b
chains at the 2 and 4-positions is outlined in Scheme 1, using the
same protocol as that described previously.8 The synthesis was
started using commercially available methylphenylketone (2).
Treatment of compound 2 with thiosemicarbazide under acidic
condition yielded thiosemicarbazone 3. Subsequent cyclization
using various acyl chlorides or acid anhydrides generated 1,3,4-
thiadiazoline derivatives 1a–e. The 2-amino derivative 4 was
obtained via the reduction of compound 1a (Scheme 2).
Mitotic accumulation Growth inhibition IC50
a,b
b
MI20 (nmol/L)
GI50 (nmol/L)
(nmol/L)
1d
9a
9b
9c
9d
9e
9f
Me
CN
Ms
OMe
NHAc
NHMs
170
190
n.t.c
n.t.c
560
n.t.c
17
520
n.t.c
n.t.c
n.t.c
n.t.c
44
>10,000
>10,000
430
2200
16
CH2NHMs 71
63
120
Scheme 3 shows the introduction of various side chains at the
5-position. Compounds 7d–f was prepared from phenylketone 5
and 6. Subsequently, compounds 7d–f and commercially available
a
b
c
See Ref. 9 for the assay details.
The values are presented as the mean of at least three independent assays.
n.t., not tested.

J. Yamamoto et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3961–3963
3963
Table 3
Table 4
SAR of the 4-position of the 1,3,4-thiadiazoline derivatives and their optically active
compounds
Antitumor activities of optically active (À)-12a and (À)-12b
b
Compd
Dosea (mg/kg)
T/Cmin
Mortality
1a
(À)-12a
100
0.625
5
2.5
10
0.18
0.50
0.08
0.50
0.27
0/5
0/5
0/5
0/5
0/5
(À)-12b
a
The compounds were orally administered at b.i.d. intervals for five days.
Average of tumor volume ratio treated mice to control mice.
b
Compd
R3 Mitotic
accumulation
Eg5
IC50
Growth
MLM
Aqueous
solubility
b
inhibition CLint
a,b
b
MI20 (nmol/L) (nmol/L) GI50
(nmol/L)
(L/hr/kg) (mg/mL)
to that of the racemates and improved oral absorption (mouse F:
62%).
9f
12a
tBu 69
120
111
n.t.c
64.
110
n.t.c
48
190
1.95
0.89
n.t.c
n.t.c
0.89
n.t.c
n.t.c
2.6
Et
Et
Et
114
>10,000
55
n.t.c
n.t.c
560
n.t.c
n.t.c
63
16
Finally, the antitumor effects were evaluated following oral
administration in a sc A2780 tumor xenograft model of SCID
mice (Table 4). These optically active compounds exhibited a
dose-dependent activity and more potent activity than that of 1a
without mortality nor body weight loss. Above all, (À)-12a showed
significant antitumor activity (T/Cmin: 0.08).
(+)-12a
(À)-12a
12b
(+)-12b
(À)-12b
n.t.c
94.5
41
iPr 114
iPr >10,000
iPr 40
n.t.c
142
a
b
c
See Ref. 9 for the assay details.
In summary, we herein identified a novel series of 2,4,5-
substituted-1,3,4-thiadiazoline analogs that induce potent mitotic
accumulation as Eg5 inhibitors. The introduction of a bulky acyl
group at the 2 and 4-positions of the 2,4,5-substituted-1,3,4-thi-
adiazoline ring and reduction of lipophilicity via the incorporation
of a polar group at the 5-position were effective in enhancing the
anti-mitotic effects and improving metabolic stability. In particu-
lar, optimal (À)-12b displayed significant antitumor activity and
wide therapeutic window (MTD/ED50: 8), as well as good tolerabil-
ity, in the animal xenograft models. Further optimization of the
2- and 5-positons of 12b was also carried out and will be reported
in detail in a future paper.
The values are presented as the mean of at least three independent assays.
n.t., not tested.
exhibited a more potent Eg5 inhibitory activity5 and anti-prolifer-
ative activity5 in the HCT116 cells than 1a. On the other hand the
benzoyl analogue 1e displayed no mitotic accumulation activity.
Next, for the modification of the side chain of the 5-position, 1d
was selected as the starting point (Table 2). Because the micro-
somal stability of 1d was not poor, we planned to reduce the lipo-
philicity via the introduction of polar substituents at the side chain
of the 5-position. The introduction of cyano (9a) and methylsulfo-
nyl (9b) groups abolished the mitotic accumulation. Meanwhile,
the methoxy (9c) and acethylamino (9d) derivatives induced a
decrease in mitotic accumulation. In contrast, the introduction of
a methanesulfonylamino group in this position had a dramatic
effect on the mitotic accumulation activity, Eg5 inhibitory activity
and anti-proliferative activity in the HCT116 cells. The metha-
nesulfonylamino derivatives 9e and 9f exhibited a good inhibitory
activity; however, the mouse liver microsomal stability was
insufficient.
Therefore, our results verified that the lipophilic pivaloyl group
at the 4-position is important for improving microsomal stability
(Table 3). Compounds with propionyl (12a) and iso-butyryl (12b)
substitution at the 4-positon displayed comparable potency and
good stability. Furthermore, the optical resolution of compounds
12a and 12b was conducted. Direct separation of ( )-12 by chiral
HPLC column (CHIRALPAK ADÒ) yielded (+)-12 and (À)-12. In all
cases examined, only the (À)-enantiomers demonstrated a more
potent mitotic accumulation activity and Eg5 inhibitory activity
than the racemates. For example, the (À)-enantiomer of 12b
showed a 2-fold increase in potency, while the (+)-enantiomer of
12b displayed no mitotic accumulation. In addition, the aqueous
solubility against a bile acid solution of (À)-12b was also superior
Acknowledgments
We are grateful to Mrs. Hiromi nagai for their helpful technical
assistance.
References and notes
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7. All compounds were characterized by 1H NMR. For detailed experimental
procedures, see WO2003051854 (the language of this patent is Japanese. If
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8. Kubota, S.; Fujikane, K.; Uda, M.; Yoshioka, T. Heterocycles 1976, 4, 1909.
9. Mitotic accumulation assays. HCT116 cells were treated with the test compound.
The mitotic index was determined using a fluorescence microscopic analysis of
Hoechst 33342 stained nuclear morphology. The MI20 value was used as the test
compound concentration required for mitotic accumulation against 20% of the
cells tested and reflected in the potency of the antimitotic activity.