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Synthetic studies on mitotic kinesin Eg5 inhibitors: Synthesis and structure–activity relationships of novel 2,4,5-substituted-1,3,4-thiadiazoline derivatives
Bioorganic & Medicinal Chemistry Letters 24 (2014) 3961–3963
Contents lists available at ScienceDirect
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
Article history: Received 24 February 2014 Revised 1 June 2014 Accepted 12 June 2014 Available online 20 June 2014 Keywords: Mitotic kinesin Eg5 inhibitor Thiadiazoline derivatives
a b s t r a c t 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. 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 binding 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 microtubules 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 culture.5 Therefore, inhibiting Eg5 may be an effective strategy for treating human cancers. A high-throughput morphology-based screening of our compound library led to the identification of a novel class of
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 microtubule 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 2,4,5substituted-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
⇑ Corresponding author. Tel.: +81 42 725 2144; fax: +81 42 726 8330. E-mail address: [email protected] (J. Yamamoto). http://dx.doi.org/10.1016/j.bmcl.2014.06.034 0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.
Figure 1. Eg5 inhibitor identified via high-throughput screening (1a).
3962
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, cHCl, MeOH; (b) [R1C(O)]2O, pyridine or R1C(O)Cl, Et3N, CH2Cl2. Compd
1a 4 1b 1c 1d 1e
Scheme 2. Reagents and conditions: (a) NH2NH2H2O, MeOH. a b c
Scheme 3. Reagents and conditions: (a) Ac2O, Et3N, CH2Cl2; (b) MsNH2, NaH, DMF; (c) thiosemicarbazide, cHCl, MeOH; (d) pivaloyl chloride, Et3N, CH2Cl2.
R1
Me Et i Pr i Bu Ph
HCT16
Eg5
Mitotic accumulation MI20a,b (nmol/L)
Growth inhibition GI50b (nmol/L)
IC50b (nmol/L)
1100 >10,000 1700 460 170 >10,000
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
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 selective 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 accumulation 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 antimitotic activity. Next, the compounds exhibiting potent MI20 values 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
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
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 1,3,4thiadiazoline derivatives 1a–e. The 2-amino derivative 4 was obtained via the reduction of compound 1a (Scheme 2). 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
1d 9a 9b 9c 9d 9e 9f a b c
R2
Me CN Ms OMe NHAc NHMs CH2NHMs
HCT16
Eg5
Mitotic accumulation MI20a,b (nmol/L)
Growth inhibition GI50b (nmol/L)
IC50b (nmol/L)
170 >10,000 >10,000 430 2200 16 71
190 n.t.c n.t.c 560 n.t.c 17 63
520 n.t.c n.t.c n.t.c n.t.c 44 120
See Ref. 9 for the assay details. The values are presented as the mean of at least three independent assays. n.t., not tested.
3963
J. Yamamoto et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3961–3963 Table 3 SAR of the 4-position of the 1,3,4-thiadiazoline derivatives and their optically active compounds
Table 4 Antitumor activities of optically active ( )-12a and ( )-12b Compd
Dosea (mg/kg)
T/Cminb
Mortality
1a ( )-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
( )-12b a
a b c
Compd
R3
Eg5 Growth Aqueous MLM Mitotic IC50b inhibition CLint solubility accumulation MI20a,b (nmol/L) (nmol/L) GI50b (L/hr/kg) (mg/mL) (nmol/L)
9f 12a (+)-12a ( )-12a 12b (+)-12b ( )-12b
tBu Et Et Et iPr iPr iPr
69 114 >10,000 55 114 >10,000 40
120 111 n.t.c 64. 110 n.t.c 48
190 n.t.c n.t.c 560 n.t.c n.t.c 63
1.95 0.89 n.t.c n.t.c 0.89 n.t.c n.t.c
2.6 16 n.t.c 94.5 41 n.t.c 142
See Ref. 9 for the assay details. 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-proliferative 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 microsomal stability of 1d was not poor, we planned to reduce the lipophilicity via the introduction of polar substituents at the side chain of the 5-position. The introduction of cyano (9a) and methylsulfonyl (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 methanesulfonylamino 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 ( )-12. In all cases examined, only the ( )-enantiomers demonstrated a more potent mitotic accumulation activity and Eg5 inhibitory activity than the racemates. For example, the ( )-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 ( )-12b was also superior
b
The compounds were orally administered at b.i.d. intervals for five days. Average of tumor volume ratio treated mice to control mice.
to that of the racemates and improved oral absorption (mouse F: 62%). 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, ( )-12a showed significant antitumor activity (T/Cmin: 0.08). In summary, we herein identified a novel series of 2,4,5substituted-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-thiadiazoline 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 particular, optimal ( )-12b displayed significant antitumor activity and wide therapeutic window (MTD/ED50: 8), as well as good tolerability, 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. Acknowledgments We are grateful to Mrs. Hiromi nagai for their helpful technical assistance. References and notes 1. Jordan, M. A.; Wilson, L. Nat. Rev. Cancer. 2004, 4, 253. 2. Taylor, S. S.; McKeon, F. Cell 1997, 89, 727. 3. Rowinsky, E. K.; Chaudhry, D. R.; Donehower, R. C. J. Natl. Cancer Inst. 1993, 15, 107. 4. Wood, K. W.; Cornwell, W. D.; Jackson, J. R. Curr. Opin. Pharmacol. 2001, 370. 5. Weil, D.; Garcon, L.; Harper, M.; Dumenil, D.; Dautry, F., et al. Biotechniques 2002, 33, 1244. 6. Nakai, R.; Iida, S.; Takahashi, T.; Tsujita, T.; Okamoto, S.; Takada, C.; Akasaka, K.; Ichikawa, S.; Ishida, H.; Kusaka, H.; Akinaga, S.; Murakata, C.; Honda, S.; Nitta, M.; Saya, H.; Yamashita, Y. Cancer Res. 2009, 69, 3901. 7. All compounds were characterized by 1H NMR. For detailed experimental procedures, see WO2003051854 (the language of this patent is Japanese. If English version is needed, see US2007015580). 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.
Contents lists available at ScienceDirect
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
Article history: Received 24 February 2014 Revised 1 June 2014 Accepted 12 June 2014 Available online 20 June 2014 Keywords: Mitotic kinesin Eg5 inhibitor Thiadiazoline derivatives
a b s t r a c t 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. 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 binding 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 microtubules 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 culture.5 Therefore, inhibiting Eg5 may be an effective strategy for treating human cancers. A high-throughput morphology-based screening of our compound library led to the identification of a novel class of
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 microtubule 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 2,4,5substituted-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
⇑ Corresponding author. Tel.: +81 42 725 2144; fax: +81 42 726 8330. E-mail address: [email protected] (J. Yamamoto). http://dx.doi.org/10.1016/j.bmcl.2014.06.034 0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.
Figure 1. Eg5 inhibitor identified via high-throughput screening (1a).
3962
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, cHCl, MeOH; (b) [R1C(O)]2O, pyridine or R1C(O)Cl, Et3N, CH2Cl2. Compd
1a 4 1b 1c 1d 1e
Scheme 2. Reagents and conditions: (a) NH2NH2H2O, MeOH. a b c
Scheme 3. Reagents and conditions: (a) Ac2O, Et3N, CH2Cl2; (b) MsNH2, NaH, DMF; (c) thiosemicarbazide, cHCl, MeOH; (d) pivaloyl chloride, Et3N, CH2Cl2.
R1
Me Et i Pr i Bu Ph
HCT16
Eg5
Mitotic accumulation MI20a,b (nmol/L)
Growth inhibition GI50b (nmol/L)
IC50b (nmol/L)
1100 >10,000 1700 460 170 >10,000
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
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 selective 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 accumulation 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 antimitotic activity. Next, the compounds exhibiting potent MI20 values 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
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
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 1,3,4thiadiazoline derivatives 1a–e. The 2-amino derivative 4 was obtained via the reduction of compound 1a (Scheme 2). 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
1d 9a 9b 9c 9d 9e 9f a b c
R2
Me CN Ms OMe NHAc NHMs CH2NHMs
HCT16
Eg5
Mitotic accumulation MI20a,b (nmol/L)
Growth inhibition GI50b (nmol/L)
IC50b (nmol/L)
170 >10,000 >10,000 430 2200 16 71
190 n.t.c n.t.c 560 n.t.c 17 63
520 n.t.c n.t.c n.t.c n.t.c 44 120
See Ref. 9 for the assay details. The values are presented as the mean of at least three independent assays. n.t., not tested.
3963
J. Yamamoto et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3961–3963 Table 3 SAR of the 4-position of the 1,3,4-thiadiazoline derivatives and their optically active compounds
Table 4 Antitumor activities of optically active ( )-12a and ( )-12b Compd
Dosea (mg/kg)
T/Cminb
Mortality
1a ( )-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
( )-12b a
a b c
Compd
R3
Eg5 Growth Aqueous MLM Mitotic IC50b inhibition CLint solubility accumulation MI20a,b (nmol/L) (nmol/L) GI50b (L/hr/kg) (mg/mL) (nmol/L)
9f 12a (+)-12a ( )-12a 12b (+)-12b ( )-12b
tBu Et Et Et iPr iPr iPr
69 114 >10,000 55 114 >10,000 40
120 111 n.t.c 64. 110 n.t.c 48
190 n.t.c n.t.c 560 n.t.c n.t.c 63
1.95 0.89 n.t.c n.t.c 0.89 n.t.c n.t.c
2.6 16 n.t.c 94.5 41 n.t.c 142
See Ref. 9 for the assay details. 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-proliferative 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 microsomal stability of 1d was not poor, we planned to reduce the lipophilicity via the introduction of polar substituents at the side chain of the 5-position. The introduction of cyano (9a) and methylsulfonyl (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 methanesulfonylamino 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 ( )-12. In all cases examined, only the ( )-enantiomers demonstrated a more potent mitotic accumulation activity and Eg5 inhibitory activity than the racemates. For example, the ( )-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 ( )-12b was also superior
b
The compounds were orally administered at b.i.d. intervals for five days. Average of tumor volume ratio treated mice to control mice.
to that of the racemates and improved oral absorption (mouse F: 62%). 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, ( )-12a showed significant antitumor activity (T/Cmin: 0.08). In summary, we herein identified a novel series of 2,4,5substituted-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-thiadiazoline 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 particular, optimal ( )-12b displayed significant antitumor activity and wide therapeutic window (MTD/ED50: 8), as well as good tolerability, 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. Acknowledgments We are grateful to Mrs. Hiromi nagai for their helpful technical assistance. References and notes 1. Jordan, M. A.; Wilson, L. Nat. Rev. Cancer. 2004, 4, 253. 2. Taylor, S. S.; McKeon, F. Cell 1997, 89, 727. 3. Rowinsky, E. K.; Chaudhry, D. R.; Donehower, R. C. J. Natl. Cancer Inst. 1993, 15, 107. 4. Wood, K. W.; Cornwell, W. D.; Jackson, J. R. Curr. Opin. Pharmacol. 2001, 370. 5. Weil, D.; Garcon, L.; Harper, M.; Dumenil, D.; Dautry, F., et al. Biotechniques 2002, 33, 1244. 6. Nakai, R.; Iida, S.; Takahashi, T.; Tsujita, T.; Okamoto, S.; Takada, C.; Akasaka, K.; Ichikawa, S.; Ishida, H.; Kusaka, H.; Akinaga, S.; Murakata, C.; Honda, S.; Nitta, M.; Saya, H.; Yamashita, Y. Cancer Res. 2009, 69, 3901. 7. All compounds were characterized by 1H NMR. For detailed experimental procedures, see WO2003051854 (the language of this patent is Japanese. If English version is needed, see US2007015580). 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.