Synthesis and evaluation of novel orally active p53-MDM2 interaction inhibitors

Article abstract of DOI:10.1016/j.bmc.2013.04.056

We have discovered and reported potent p53-MDM2 interaction inhibitors possessing dihydroimidazothiazole scaffold. Our lead showed strong activity in vitro, but did not exhibit antitumor efficacy in vivo for the low metabolic stability. In order to obtain orally active compounds, we executed further optimization of our lead by the improvement of physicochemical properties. Thus we furnished optimal compounds by introducing an alkyl group onto the pyrrolidine at the C-2 substituent to prevent the metabolism; and modifying the terminal substituent of the proline motif improved solubility. These optimal compounds exhibited good PK profiles and significant antitumor efficacy with oral administration on a xenograft model using MV4-11 cells having wild type p53.

Full text of DOI:10.1016/j.bmc.2013.04.056

Accepted Manuscript
Synthesis and evaluation of novel orally active p53-MDM2 interaction inhibi‐
tors
Masaki Miyazaki, Hiroyuki Naito, Yuuichi Sugimoto, Keisuke Yoshida,
Haruko Kawato, Tooru Okayama, Hironari Shimizu, Masaya Miyazaki,
Mayumi Kitagawa, Takahiko Seki, Setsuko Fukutake, Yoshinobu Shiose,
Masashi Aonuma, Tsunehiko Soga
PII:
S0968-0896(13)00382-9
DOI:
http://dx.doi.org/10.1016/j.bmc.2013.04.056
BMC 10796
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
22 March 2013
22 April 2013
23 April 2013
Please cite this article as: Miyazaki, M., Naito, H., Sugimoto, Y., Yoshida, K., Kawato, H., Okayama, T., Shimizu,
H., Miyazaki, M., Kitagawa, M., Seki, T., Fukutake, S., Shiose, Y., Aonuma, M., Soga, T., Synthesis and evaluation
of novel orally active p53-MDM2 interaction inhibitors, Bioorganic & Medicinal Chemistry (2013), doi: http://
dx.doi.org/10.1016/j.bmc.2013.04.056
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Title:
Synthesis and evaluation of novel orally active p53-MDM2 interaction inhibitors
Masaki Miyazakia,*, Hiroyuki Naitoa, Yuuichi Sugimotoa, Keisuke Yoshidaa, Haruko Kawatoa, Tooru,
Okayamaa, Hironari Shimizua, Masaya Miyazakib, Mayumi Kitagawab, Takahiko Sekib, Setsuko
Fukutakeb, Yoshinobu Shioseb, Masashi Aonumab, Tsunehiko Sogaa
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Medicinal Chemistry Research Laboratories, R&D Division, Daiichi Sankyo Co., Ltd., 1-2-58 Hiromachi, Shinagawa-ku,
Tokyo140-8710, Japan
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Oncology Research Laboratories, R&D Division, Daiichi Sankyo Co., Ltd., 1-2-58 Hiromachi, Shinagawa-ku, Tokyo
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140-8710, Japan
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* Corresponding author. Tel.: +81-3-3492-3131; fax: +81-3-5436-8563;
E-mail address: miyazaki.masaki.p3@daiichisankyo.co.jp
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Keywords:
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MDM2
p53
Dihydroimidazothiazole
Protein-protein interaction inhibitor
Abstract
We have discovered and reported potent p53-MDM2 interaction inhibitors possessing
dihydroimidazothiazole scaffold. Our lead showed strong activity in vitro, but did not exhibit antitumor
efficacy in vivo for the low metabolic stability. In order to obtain orally active compounds, we executed
further optimization of our lead by the improvement of physicochemical properties. Thus we furnished
optimal compounds by introducing an alkyl group onto the pyrrolidine at the C-2 substituent to prevent
the metabolism; and modifying the terminal substituent of the proline motif improved solubility. These
optimal compounds exhibited good PK profiles and significant antitumor efficacy with oral
administration on a xenograft model using MV4-11 cells having wild type p53.
1. Introduction
The p53 tumor suppressor protein plays important roles as a cell cycle controller in normal cells,
regulating the transcription of genes and promoting apoptosis.1-2 In about 50% of human cancers, the

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functions of p53 are lost by its mutation or degradation. The rest of the cancers have wild type p53,
whose functions are also suppressed by an overexpression or an amplification of human murine double
minute 2 (MDM2), which is a negative feedback protein of p53.3-4 MDM2 inhibits a function of cell
cycle controller of p53 by binding to the N-terminal transcriptional activation domain. MDM2 promotes
an excretion of p53 from the nucleus to cytoplasm, and advances proteasomal degradation of p53 via
ubiquitination through its E3 ligase activity.5-7 Thus, activation of p53 functions via the inhibition of
MDM2 is regarded as an effective approach in cancer therapy. From p53-MDM2 co-crystal structure
analysis, the key moieties of the interaction between p53 and MDM2 mainly consist of three
hydrophobic residues of p53, i.e. side chains of Phe19, Trp23, and Leu26.8-10 In fact, various small
molecule inhibitors with different scaffolds,11-14 exemplified by dihydroimidazoline, spirooxyindole, and
triazolopyrimidine, mimic those residues.
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We reported recently that novel dihydroimidazothiazole derivatives showed a potent p53-MDM2
interaction inhibitory activity.15 The crystallographic analysis of compound 1/MDM2 revealed that an
additional interaction was observed by induced-fitting in addition to the three hydrophobic
interactions.16 Compound 1 showed robust p53-MDM2 inhibitory activity on HTRF (Homogeneous
Time Resolved Fluorescence)-based assay (IC50 = 8.3 nM), as a consequence of the enhanced
hydrophobic interaction via the increase of contact surface area (Figure 1). Compound 1 did not display
any therapeutic effect with oral administrations in the MV4-11 xenograft model, despite the strong
activity in vitro, presumably because of its poor metabolic stability (9 % remaining after 30 min
incubation with mouse hepatic microsome). For the discovery of orally active agents, it was necessary to
improve solubility and metabolic stability, in addition to keeping the potency in vitro.
Herein, we report the discovery and biological evaluation of orally active MDM2 inhibitors based on
dihydroimidazothiazole scaffold by improving metabolic stability and solubility with keeping strong
potency.
2. Designs and Chemistry
As we reported recently, it was confirmed that the pyrrolidine moiety of compound 1 efficiently bound
to the surface of MDM2 by an induced fitting, which provided very potent inhibitory activity as depicted
in Figure 1.16 On the other hand, we presumed that the most plausible metabolic site of compound 1
would be the pyrrolidine and the terminal dimethylamide moiety. Thus, novel pyrrolidine variants
bearing alkyl groups onto the adjacent position to the pyrrolidine amide linkage were designed to
circumvent the metabolism (Figure 2). We further explored the structure activity relationship on the

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terminal proline amide moiety, together with aiming to manipulate the physicochemical properties by
the optimization. Various piperazino- or morpholino- variants at the C-2 amide position of
(5R)-alkylpyrrolidine (23-27 in Table 1) were designed at aiming to improve the solubility and
metabolic stability. While the solubility of some compounds, i.e. 23 and 24, were improved, metabolic
stabilities were decreased compared to 21 or 22 (vide infra). We hypothesized that the plausible
metabolic site would be piperazine moiety; more specifically adjacent carbon to nitrogen of the
piperazine, which was the same in the case for the pyrrolidine. Thus, further modification, i.e.
incorporation of the methyl group onto the piperazine moiety to avoid the metabolism was executed (see
the results of 28-36 in Table 1).
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Syntheses of methyl and ethyl incorporated variants including (S)- and (R)- isomers are displayed in
Scheme 1 and 2. 5-Oxo-L-proline derivatives (2 or 11) were utilized for making both (S)- and (R)-
alkylated derivatives, and each of the products were furnished through different synthetic routes.
Syntheses of (5S)-alkylpyrrolidine derivatives are depicted in Scheme 1.17 After protection of carboxylic
acid and amide of compound 2 with the conventional manner, ring-opening reaction of 3 with
alkyllithium, ring-closure via deprotection followed by stereoselective hydrogenation provided
(5S)-alkylated derivatives (5a and 5b), regioselectively. EDC mediated amidation with methylamine,
and deprotection with HCl furnished the (5S)-alkylpyrrolidine intermediates (7a and 7b) as pure
products, which were confirmed by NMR or LC/MS. Compounds 9 and 10 were finally prepared via
condensation reaction with dihydroimidazothiazole carboxylic acid (8), reported previously.16 On the
other hand, (5R)-alkylpyrrolidine derivatives were synthesized as shown in Scheme 2. The amide of
compound 12, which was prepared via the esterification using HClO4 and t-BuOAc from 11, was
reduced with LiBHEt3 to give aminal 13. Copper-catalyzed alkylation provided (5R)-alkylated
derivatives (14a and 14b) regioselectively, which were also confirmed by NMR or LC/MS.18-21
Compounds 21-36 were prepared via two different routes by using key intermediates 14a and 14b as
shown in Scheme 2.
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3. Results and Discussions
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The result of in vitro activity (IC50s), growth inhibitions (GI50s), solubility, and metabolic stability for
the final compounds is displayed in Table 1. IC50s were measured by HTRF-based assay, and GI50s were
measured by antiproliferable cells assay using MV4-11 and DLD-1 respectively having a wild type p53
(p53wt) and a mutated p53 (p53mut). By comparing the GI50s in both cells, we checked indiscriminate
cytotoxicity not resulted from p53-MDM2 inhibitory activity. Compound’s solubility was measured for
using neutral aqueous solution (pH 6.8), and metabolic stability was measured for the remaining ratio

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(% rem) after 30 min treatment with mouse hepatic microsome in vitro. All compounds with an alkyl
moiety on the C-2 position of the pyrrolidine (9, 10, 21 and 22) showed improvement of metabolic
stability compared to 1, as expected (39-70 % rem). However, solubility of (5S)-methyl variant (9)
decreased, and cellular activity of (5S)-ethyl one (10) for MV4-11 diminished. On the other hand, both
compounds having (5R)-alkyl moieties (21 and 22) showed good metabolic stabilities (more than 50%
rem) with potent cellular activities. While compounds 23 and 25 each possessing N-methylpiperazino
substituent were twice more soluble than dimethylcarbamoyl variants (21 and 22), these resulted in
lower metabolic stability. The same tendency was also observed for compound 24 and 26 having
N-acetylpiperazino moiety. The morpholino group as in compound 27 diminished both solubility and
metabolic stability.
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For further optimization to improve the metabolic stability, we executed modification of the terminal
piperazine moiety. All compounds (28-36) bearing the methyl group onto the C-3 position of piperazine
exhibited good metabolic stability (54-84 % rem). It was suggested that metabolic oxidation was
prevented by incorporating an alkyl group adjacent to the nitrogen of the cyclic amine or amide. Both
stereochemistries of the methyl group on C-3 position were considered valuable for cellular activity on
MV4-11, solubility and metabolic stability. Compounds having (5R)-ethyl group (22, 25-27, 33-36) on
the pyrrolidine moiety tend to provide somewhat increased cellular activity against DLD-1. Among
these, GI50 of 35 (for DLD-1) was 10 M, which was weakest in the series.
We selected 28 and 35 for the PK study, which exhibited high p53-MDM2 inhibitory activity,
selectivity between MV4-11 and DLD-1, and good metabolic stabilities. Although compound 28 showed
low concentrations in plasma compared with 35 because of high penetration into tissues, both
compounds showed large AUC in plasma and good exposure in the tumor with an oral administration on
mice (Table 2). We then evaluated antitumor efficacy of these compounds against an MV4-11 xenograft
model on mice. As shown in Figure 3, compounds 28 and 35 showed a significant tumor growth
inhibition (TGI) with single administration per day (200mg/kg, po). At the end of treatments (day 31),
TGIs were 71% (28) and 76 % (35) with only slight body weight loss (<5 %). Significant toxicity was
not observed in both compounds. In comparison, Nutlin-3 (racemate)11 was also evaluated as a positive
control, and we confirmed a high antitumor effect (TGI = 87%) at MTD dosage; po, 200mg/kg/day, bid
dosing. The total amount of administration was more than twice compared to compounds 28 and 35,
delivering a significant (9 %) body weight loss. In the in vivo study, we checked the p53 induction by
using Western blot analysis (Figure 4). Both 28 and 35 increased p53 as well as MDM2 and p21 in

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protein levels compared to the control (non-treated). This data indicated that potent antitumor efficacies
of the compounds were derived from re-activation of p53 functions by robust p53-MDM2 inhibition.
Co-crystal structure analysis of compound 35 and MDM2 was performed. The structure of the complex
is displayed in Figure 5. It was observed that ethylated pyrrolidine ring also generated a unique
hydrophobic interaction by induced-fitting with MDM2, which was the same as non-alkylated
pyrrolidine variant 1 in our previous research (Figure 5A). Furthermore, the stacking model of co-crystal
structures for 1, 35 and Nutlin-2 (PDB code: 3VZV, 3W69 and 1RV1) is depicted in Figure 5B. In the
orange circled area, MDM2 loop with 35 is also distorted and changed the three-dimensional structure in
a similar way to that with 1 by the induced fitting, which was not observed at Nulin-2. This should
confirm the reason why our compounds, modified on the pyrrolidine moiety, kept strong p53-MDM2
inhibitory activity. On the other hand, no interaction of the terminal piperazine moiety with MDM2 was
observed. The moiety was found to direct outside of the protein, suggesting that it would be possible to
further improve the physicochemical property by incorporating a hydrophilic moiety to the terminal site
of the molecule.
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4. Conclusion
In conclusion, we executed lead optimization of our potent lead for the improvement of
physicochemical properties. Incorporation of an alkyl group on the pyrrolidine ring at the C-2
substituent worked to avoid the metabolism. Also, further optimization by introducing piperazine
moieties and its modification improved both solubility and metabolic stability. As a result of co-crystal
structure analysis, ethylated pyrrolidine variants kept holding the extra hydrophobic affinity for binding
with MDM2 by induced-fitting. Compounds 28 and 35 exhibited good PK profiles and robust antitumor
efficacy against the MV4-11 xenograft model with oral administrations. From these results, it was
confirmed that our dihydroimidazothiazole derivatives were potent p53-MDM2 interaction inhibitors
and novel orally active anticancer agents. Having the promising lead such as 35, further investigation to
obtain more potent compounds is under investigation.
5. Experimental Section
5.1. General methods and Materials on chemistry
All reagents and solvents were purchased from commercially available suppliers and were of reagent
grade. Flash silica gel column chromatography was performed with SHOKO Scientific Purif-α2® by
using of Purif-Pack® Si or NH2 cartridges. Thin-layer chromatography (TLC) was performed on Merck

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pre-coated TLC glass sheets with Silica Gel 60 F254, and compound visualization was detected with a
UV lamp, a solution of phosphomolybdic acid in ethanol, or Wako ninhydrin spray. Melting points were
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uncorrected. H-NMR spectra were measured on JEOL JNM-EX400 spectrometers with CDCl3 or
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DMSO-d6 as solvent, and chemical shifts are given in ppm (δ) from tetramethylsilane as an internal
standard. Mass spectra (ESI/MS) were measured on Agilent 1100 series LC/MSD mass spectrometers
with an electrospray ionization source. High resolution mass spectra were measured on JEOL
JMS-T100LP spectrometer with an electrospray ionization source (HRESI/MS) or JEOL JMS-7000
spectrometer (HREI/MS). IR spectra were measured on a JASCO FT/IR-6100 spectrometer by using the
ATR method.
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5.2. 2-Benzyl 1-tert-butyl (2S)-5-oxopyrrolidine-1,2-dicarboxylate (3)
Benzyl chloride (25.3 ml, 0.22 mol) was added to a solution of 5-oxo-L-proline (2) (25.8 g, 0.20 mol)
with triethylamine (28.0 ml, 0.20 mol) in THF (260 ml). The reaction mixture was refluxed at 70°C for 5
days. After cooling the mixture to room temperature, the solvent was removed in vacuo. The residue
was diluted with water, and extracted with chloroform. The organic layer was washed with brine, and
dried over magnesium sulfate. The mixture was filtered, and the solvent was removed in vacuo to afford
the crude product as pale brown oil. To a stirred solution of the product in dichloromethane (400 ml)
was added di-tert-butyl dicarbonate (44 g, 0.20 mol), triethylamine (28 ml, 0.2 mol) and DMAP (12.2 g ,
0.10 mol) under an ice cooling bath. The solution was warmed to room temperature and stirred for 16 h.
The reaction mixture was diluted with water, and extracted with chloroform. The organic layer was
washed with brine, and dried over magnesium sulfate. The mixture was filtered, and the solvent was
removed in vacuo to afford crude product. The residue was purified by flash silica gel chromatography
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with n-hexane/EtOAc (2:1, v/v) to give the colorless solid (51.2 g, 80% yield); H-NMR (400 MHz,
CDCl3) δ: 1.42 (9H, s), 1.98-2.05 (1H, m), 2.26-2.37 (1H, m), 2.44-2.51 (1H, m), 2.57-2.66 (1H, m),
4.64 (1H, dd, J = 9.5, 2.9 Hz), 5.19 (1H, d, J = 12.0 Hz), 5.23 (1H, d, J = 12.2 Hz), 7.34-7.37 (5H, m);
ESI/MS: m/z= 342 (M+Na).
5.3. Benzyl (2S)-2-[(tert-butoxycarbonyl)amino]-5-oxohexanoate (4a)
To a stirred solution of compound 3 (11.0 g, 0.034 mol) in dry THF (100 ml) was added methyllithium
(1.04 M in Et2O, 34.0 ml, 0.034 mol) at -78°C under a nitrogen atmosphere, and the resulting solution
was warmed to room temperature and stirred for 2 h. The reaction mixture was diluted with saturated
aqueous NH4Cl under an ice cooling bath, and extracted with EtOAc. The organic layer was washed

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with brine, and dried over magnesium sulfate. The mixture was filtered, and the solvent was removed in
vacuo to afford the crude product. The residue was purified by flash silica gel chromatography with
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n-hexane/EtOAc (3:1, v/v) to give the colorless oil (9.2 g, 91% yield); H-NMR (400 MHz, CDCl3) δ:
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m), 5.12-5.21 (3H, m), 7.34-7.39 (5H, m); ESI/MS: m/z= 358 (M+Na).
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5.4. Benzyl (2S)-2-[(tert-butoxycarbonyl)amino]-5-oxoheptanoate (4b)
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Compound 4b was prepared as a colorless oil (49% yield) from 3 according to a similar procedure for
the synthesis of 4a; 1H-NMR (400 MHz, CDCl3) δ: 1.02 (3H, t, J = 7.3 Hz), 1.43 (9H, s), 1.88-1.96 (1H,
m), 2.09-2.15 (1H, m), 2.37 (2H, q, J = 7.3 Hz), 2.38-2.52 (2H, m), 4.32 (1H, br s), 5.11 (1H, br s), 5.13
(1H, d, J = 12.2 Hz), 5.19 (1H, d, J = 12.2 Hz), 7.34-7.38 (5H, m); ESI/MS: m/z= 372 (M+Na).
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5.5. (5S)-1-(tert-Butoxycarbonyl)-5-methyl-L-proline (5a)
Trifluoroacetic acid (10 ml) was added to a solution of compound 4a (4.7 g, 0.014 mol) in
dichloromethane (30 ml). The reaction mixture was stirred for 3 h at room temperature. The solvent was
removed in vacuo, and the residue was azeotroped with toluene to afford the crude product. To a
solution of the product in methanol (50 ml) was added 10% Pd/C (500 mg, 50% wetted, type AD). The
resulting mixture was stirred under a hydrogen atmosphere (1 atm) for 16 h at room temperature. The
mixture was filtered to remove the catalyst, and the filtrate was concentrated in vacuo to afford the crude
product. To a solution of the product in acetonitrile (60 ml) with water (10 ml) was added di-tert-butyl
dicarbonate (4.58 g, 21 mmol) and 1N aqueous sodium hydroxide solution (35 ml, 35 mmol). The
resulting solution was stirred for 1 h at room temperature. After removing the solvent in vacuo, the
residue was diluted with chloroform, and extracted with water. The aqueous layer was diluted with
saturated aqueous NH4Cl, and the solvent was removed in vacuo. The residue was diluted with 10%
MeOH/CHCl3 solution, and dried over magnesium sulfate. The mixture was filtered, and the solvent was
removed in vacuo to afford the crude product. Purification by flash silica gel chromatography with
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CHCl3/MeOH (15:1, v/v) provided the title compound (2.14 g, 67% yield from 4a); H-NMR (400
MHz, CDCl3) δ: 1.25 (3H, d, J = 6.3 Hz), 1.48 (9H, s), 1.63-1.68 (1H, m), 2.00-2.09 (2H, m), 2.26-2.33
(1H, m), 3.91-3.97 (1H, m), 4.30-4.34 (1H, m); ESI/MS: m/z= 252 (M+Na).
5.6. (5S)-1-(tert-Butoxycarbonyl)-5-ethyl-L-proline (5b)

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Compound 5b was prepared (80% yield) from 4b according to a similar procedure for the synthesis of
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5a; H-NMR (400 MHz, CDCl3) δ: 0.87 (3H, t, J = 7.1 Hz), 1.39-1.45 (1H, m), 1.48 (9H, s), 1.71-1.77
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(2H, m), 1.93-2.00 (1H, m), 2.09-2.11 (1H, br m), 2.33-2.35 (1H, br m), 3.79-3.82 (1H, br m), 4.32-4.34
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(1H, br m); ESI/MS: m/z= 266 (M+Na).
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5.7. tert-Butyl (2S,5S)-2-[(dimethylamino)carbonyl]-5-methylpyrrolidine-1-carboxylate (6a)
Triethylamine (280 μl, 2.02 mmol) was added to a solution of compound 5a (232 mg, 1.01 mmol) in
dichloromethane (6 ml), followed by dimethylamine hydrochloride (124 mg, 1.52 mmol), HOBt (14 mg,
0.10 mmol) and EDC/HCl (233 mg, 1.21 mmol). The solution was stirred for 16 h at room temperature.
The solvent was removed in vacuo to afford crude product. The residue was purified by flash silica gel
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chromatography with CHCl3/MeOH (50:1, v/v) to give the colorless oil (161 mg, 62% yield); H-NMR
(400 MHz, CDCl3) δ: 1.35 (3H, d, J = 6.1 Hz), 1.40 and 1.46 (9H, each s), 1.66-1.75 (1H, m), 1.87-1.93
(1H, m), 2.01-2.12 (2H, m), 2.97 (3H, s), 3.07 and 3.11 (3H, s), 3.91-3.95 and 4.02-4.07 (1H, m),
4.53-4.58 and 4.68-4.72 (1H, m); ESI/MS: m/z= 157 (M-Boc).
5.8. (5S)-N,N,5-Trimethyl-L-prolinamide hydrochloride (7a)
4N HCl/dioxane solution (2 ml) was added to a solution of compound 6a (160 mg, 0.62 mmol) in
1,4-dioxane (4 ml). The resulting solution was warmed to 50°C and stirred for 1.5 h. After cooling to
room temperature, the solvent was removed in vacuo to give the product as a colorless solid (152 mg,
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quantitative yield); H-NMR (400 MHz, DMSO-d6) δ: 1.32 (3H, d, J = 6.6 Hz), 1.49-1.58 (1H, m),
1.81-1.90 (1H, m), 2.03-2.11 (1H, m), 2.32-2.42 (1H, m), 2.89 (3H, s), 2.98 (3H, s), 3.57-3.61 (1H, m),
4.55-4.60 (1H, m); ESI/MS: m/z= 157 (M+H).
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5.9. (5S)-5-Ethyl-N,N-dimethyl-L-prolinamide hydrochloride (7b)
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Triethylamine (265 μl, 1.9 mmol) was added to a solution of compound 5b (231 mg, 0.95 mmol) in
dichloromethane (6 ml), followed by dimethylamine hydrochloride (116 mg, 1.43 mmol), HOBt (13 mg,
0.095 mmol) and EDC/HCl (220 mg, 1.14 mmol). The solution was stirred for 16 h at room temperature.
The solvent was removed in vacuo to afford crude product. The residue was purified by flash silica gel
chromatography with CHCl3/MeOH (80:1, v/v) to give the colorless oil (6b). To a stirred solution of the
product in 1,4-dioxane (4 ml) was added 4N HCl/dioxane solution (2 ml). The resulting solution was
warmed to 50°C and stirred for 1 h. After cooling to room temperature, the solvent was removed in

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vacuo to give the product as a colorless oil (144 mg, 72% yield from 5b); H-NMR (400 MHz,
DMSO-d6) δ: 0.93 (3H, t, J = 7.4 Hz), 1.47-1.55 (1H, m), 1.60-1.68 (1H, m), 1.78-1.89 (2H, m),
2.05-2.12 (1H, m), 2.29-2.39 (1H, m), 2.89 (3H, s), 2.99 (3H, s), 3.34-3.39 (1H, m), 4.54-4.61 (1H, m);
ESI/MS: m/z= 171 (M+H).
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5.10.
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(5S)-1-{[(5R,6S)-5,6-Bis(4-chlorophenyl)-3-isopropyl-6-methyl-5,6-dihydroimidazo[2,1-b][1,3]thiaz
ol-2-yl]carbonyl}-N,N,5-trimethyl-L-prolinamide (9)
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Thionyl chloride (250 μl, 3.4 mmol) and catalytic amount of DMF were added to a suspension of
compound 816 (231 mg, 0.52 mmol) in toluene (4 ml). The resulting mixture was warmed to 70°C and
stirred for 30 min. After cooling to room temperature, the solvent was removed in vacuo to afford the
crude product. The residue was dissolved in THF (6 ml), and was dropwised to a solution of compound
7a (119 mg, 0.62 mmol) with triethylamine (181 μl, 1.3 mmol) in THF (4 ml) under an ice cooling bath.
The resulting solution was warmed to room temperature, and was stirred for 30 min. The reaction
mixture was quenched by the addition of saturated aqueous sodium bicarbonate, and extracted with
EtOAc. The organic layer was washed with brine, and dried over magnesium sulfate. The mixture was
filtered, and the solvent was removed in vacuo to afford crude product. The residue was purified by
flash silica gel column chromatography with CHCl3/MeOH (30:1, v/v) to give the colorless solid (227
mg, 75% yield), which was precipitated from Et2O/n-hexane; 1H-NMR (400 MHz, CDCl3) δ: 0.89 (3H,
d, J = 7.3 Hz), 0.95 (3H, d, J = 7.1 Hz), 1.45 (3H, d, J = 6.3 Hz), 1.80 (3H, s), 1.80-1.85 (1H, m),
1.96-2.01 (1H, m), 2.02-2.10 (1H, m), 2.12-2.18 (1H, m), 2.63-2.71 (1H, m), 2.96 (3H, s), 3.11 (3H, s),
4.22-4.28 (1H, m), 4.84-4.88 (1H, m), 4.96 (1H, s), 6.69 (2H, d, J = 8.3 Hz), 7.00-7.04 (4H, m), 7.10
(2H, d, J = 8.5 Hz); ESI/MS: m/z= 585 (M+H); HRESI/MS m/z= 585.18193 (Calcd for
C30H3535Cl2N4O2S: 585.18578); IR (ATR) cm-1: 1639, 1617, 1592, 1546, 1442, 1388, 1313, 1089, 1014.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
5.11.
(5S)-1-{[(5R,6S)-5,6-Bis(4-chlorophenyl)-3-isopropyl-6-methyl-5,6-dihydroimidazo[2,1-b][1,3]thiaz
ol-2-yl]carbonyl}-5-ethyl-N,N-dimethyl-L-prolinamide (10)
Compound 10 was prepared (82% yield) as the colorless solid from 7b according to a similar procedure
for the synthesis of 9; 1H-NMR (400 MHz, CDCl3) δ: 0.88 (3H, d, J = 7.1 Hz), 0.93 (3H, t, J = 8.2 Hz),
0.96 (3H, d, J = 7.1 Hz), 1.71-1.76 (1H, m), 1.80 (3H, s), 1.85-2.17 (5H, m), 2.65-2.71 (1H, m), 2.95
(3H, s), 3.10 (3H, s), 3.96-4.03 (1H, m), 4.82-4.88 (1H, m), 4.96 (1H, s), 6.69 (2H, d, J = 8.3 Hz), 7.01

1
2
(4H, d, J = 8.5 Hz), 7.10 (2H, d, J = 8.3 Hz); ESI/MS: m/z= 599 (M+H); HRESI/MS m/z= 599.19352
(Calcd for C31H3735Cl2N4O2S: 599.20143); IR (ATR) cm-1: 1639, 1621, 1592, 1548, 1492, 1388, 1313,
1087, 1014.
3
4
5
5.12. 1-Benzyl 2-tert-butyl (2S)-5-oxopyrrolidine-1,2-dicarboxylate (12)
6
70% aqueous perchloric acid (1.65 ml, ca. 0.065 mol) was dropwised to a suspension of compound 11
in t-BuOAc (200 ml) at room temperature, and the resulting mixture was stirred for 16 h. Saturated
aqueous sodium bicarbonate (300 ml) was added to the solution gradually, and most of the t-BuOAc in
the mixture was removed in vacuo. The residue was extracted with chloroform, and the organic layer
was washed with brine. After drying over magnesium sulfate, the mixture was filtered, and the solvent
was removed in vacuo to give the colorless oil (13.9 g, 79% yield); 1H-NMR (400 MHz, CDCl3) δ: 1.39
(9H, s), 2.01-2.08 (1H, m), 2.27-2.36 (1H, m), 2.45-2.53 (1H, m), 2.59-2.68 (1H, m), 4.55 (1H, dd, J =
9.4, 2.6 Hz), 5.25 (1H, d, J = 12.4 Hz), 5.30 (1H, d, J = 12.2 Hz), 7.31-7.41 (5H, m); ESI/MS: m/z= 342
(M+Na).
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
5.13. 1-Benzyl 2-tert-butyl (2S)-5-methoxypyrrolidine-1,2-dicarboxylate (13)
To a stirred solution of compound 12 (13.9 g, 0.044 mol) in dry THF (50 ml) was added LiBHEt3 (1.01
M in THF, 100 ml, 0.101 mol) at -78°C under a nitrogen atmosphere, and the resulting solution was
stirred for 30 min. Methanol (100 ml) was dropwised to the solution, and the solvent was removed in
vacuo. The residue was diluted with saturated aqueous sodium bicarbonate, and extracted with
chloroform. The organic layer was washed with brine, and dried over magnesium sulfate. The mixture
was filtered, and the solvent was removed in vacuo to afford the crude product. To a solution of the
product in MeOH (100 ml) was added p-toluenesulfonic acid monohydrate (837 mg, 4.4 mmol) at room
temperature, and the resulting solution was stirred for 2 days. After removing the solvent in vacuo, the
residue was diluted with saturated aqueous sodium bicarbonate, and extracted with chloroform. The
organic layer was washed with brine, and dried over magnesium sulfate. The mixture was filtered, and
the solvent was removed in vacuo. The residue was purified by flash silica gel chromatography with
n-hexane/EtOAc (4:1, v/v) to give the colorless oil (11.2 g, 76% yield); 1H-NMR (400 MHz, CDCl3) δ:
1.32 and 1.44, or 1.37 and 1.47 (9H, each s), 1.76-2.12 (3H, m), 2.28-2.37 (1H, m), 3.26 and 3.43, or
3.35 and 3.47 (3H, each s), 4.22-4.30 (1H, m), 5.10-5.38 (3H, m), 7.28-7.37 (5H, m); ESI/MS: m/z= 358
(M+Na).

1
2
5.14. 1-Benzyl 2-tert-butyl (2S,5R)-5-methylpyrrolidine-1,2-dicarboxylate (14a)
To a stirred suspension of CuBr/Me2S (11.7 g, 0.058 mol) in dry Et2O (100 ml) was added
methyllithium (0.98 M in Et2O, 59 ml, 0.058 mol) at -78°C under a nitrogen atmosphere. After stirring
for 30 min at -78°C, BF3/Et2O (7.35 ml, 0.058 mmol) was added to the resulting mixture. After stirring
for 5 min at -78°C, compound 13 (9.6 g, 0.029 mol) in Et2O (30 ml) was dropwised to the mixture. Then
the reaction was warmed to room temperature, and stirred for 16 h. The reaction mixture was filtered,
and the filtrate was quenched by the addition of 1N aqueous HCl (100 ml). After stirring for 1 h, the
solution was extracted with EtOAc. The organic layer was washed with brine, and dried over
magnesium sulfate. The mixture was filtered, and the solvent was removed in vacuo to afford crude
product. The residue was purified by flash silica gel column chromatography with n-hexane/EtOAc (4:1,
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
1
v/v) to give the colorless oil (8.68 g, 94% yield); H-NMR (400 MHz, CDCl3) δ: 1.15 and 1.23 (3H,
each d, J = each 6.3 Hz), 1.33 and 1.44 (9H, each s), 1.51-1.57 (1H, m), 1.89-1.95 (1H, m), 2.09-2.29
(2H, m), 4.13-4.28 (2H, m), 5.02-5.22 (2H, m), 7.22-7.38 (5H, m); ESI/MS: m/z= 342 (M+Na).
5.15. 1-Benzyl 2-tert-butyl (2S,5R)-5-ethylpyrrolidine-1,2-dicarboxylate (14b)
Compound 14b was prepared (78% yield) as the colorless oil from 13 according to a similar procedure
1
for the synthesis of 14a; H-NMR (400 MHz, CDCl3) δ: 0.83 and 0.89 (3H, each t, each J = 7.4 Hz),
1.33 and 1.44 (9H, each s), 1.66-1.73 (2H, m), 1.83-1.94 (2H, m), 1.99-2.07 (1H, m), 2.15-2.23 (1H, m),
3.92-3.99 (1H, m), 4.24 (1H, t, J = 7.8 Hz), 5.06-5.20 (2H, m), 7.27-7.36 (5H, m); ESI/MS: m/z= 356
(M+H).
5.16. tert-Butyl (5R)-5-methyl-L-prolinate (15a)
To a solution of compound 14a (4.0 g, 0.013 mol) in methanol (50 ml) was added 10% Pd/C (400 mg,
50% wetted, type AD). The resulting mixture was stirred under a hydrogen atmosphere (1 atm) for 16 h
at room temperature. The mixture was filtered to remove the catalyst, and the filtrate was concentrated
in vacuo to afford the crude product. The residue was purified by flash silica gel column
chromatography with CHCl3/MeOH (30:1, v/v) to give the colorless solid (2.17 g, 90% yield); 1H-NMR
(400 MHz, CDCl3) δ: 1.22 (3H, d, J = 6.1 Hz), 1.38-1.43 (1H, m), 1.47 (9H, s), 1.80-1.88 (1H, m),
1.88-1.95 (1H, m), 2.23-2.31 (1H, m), 3.38-3.46 (1H, m), 3.87 (1H, dd, J = 8.7, 6.2 Hz); ESI/MS: m/z=
186 (M+H).
5.17. tert-Butyl (5R)-5-ethyl-L-prolinate (15b)

1
2
3
4
5
6
7
8
Compound 15b was prepared (94% yield) as the colorless oil from 14b according to a similar
procedure for the synthesis of 15a; 1H-NMR (400 MHz, CDCl3) δ: 1.06 (3H, t, J = 7.4 Hz), 1.51 (9H, s),
1.81-1.95 (2H, m), 1.97-2.08 (1H, m), 2.13-2.27 (2H, m), 2.48-2.56 (1H, m), 3.58-3.66 (1H, m), 4.46
(1H, t, J = 7.9 Hz); ESI/MS: m/z= 200 (M+H).
5.18.
tert-Butyl
(5R)-1-{[(5R,6S)-5,6-Bis(4-chlorophenyl)-3-isopropyl-6-methyl-5,6-dihydroimidazo[2,1-b][1,3]thiaz
ol-2-yl]carbonyl}-5-methyl-L-prolinate (16a)
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
Thionyl chloride (500 μl, 6.9 mmol) and catalytic amount of DMF were added to a suspension of
compound 8 (300 mg, 0.67 mmol) in toluene (4 ml). The resulting mixture was warmed to 70°C and
stirred for 30 min. After cooling to room temperature, the solvent was removed in vacuo to afford the
crude product. The residue was dissolved in THF (6 ml), and was dropwised to a solution of compound
15a (137 mg, 0.74 mmol) with triethylamine (224 μl, 1.6 mmol) in THF (4 ml) under an ice cooling bath.
The resulting solution was warmed to room temperature, and was stirred for 1 h. The reaction mixture
was quenched by the addition of saturated aqueous sodium bicarbonate, and extracted with EtOAc. The
organic layer was washed with brine, and dried over magnesium sulfate. The mixture was filtered, and
the solvent was removed in vacuo to afford crude product. The residue was purified by flash silica gel
column chromatography with n-hexane/EtOAc (2:1, v/v) to give the colorless solid (340 mg, 83%
yield); 1H-NMR (400 MHz, CDCl3) δ: 0.98-1.04 (6H, m), 1.20 (3H, d, J = 6.3 Hz), 1.44-1.45 (1H, br m),
1.45 (9H, s), 1.80 (3H, s), 1.98-2.00 (1H, br m), 2.25-2.28 (2H, br m), 2.62-2.65 (1H, br m), 4.51-4.54
(2H, br m), 4.95 (1H, s), 6.69 (2H, d, J = 8.1 Hz), 7.00-7.04 (4H, m), 7.13 (2H, d, J = 8.5 Hz); ESI/MS:
m/z= 614 (M+H).
5.19.
tert-Butyl
(5R)-1-{[(5R,6S)-5,6-bis(4-chlorophenyl)-3-isopropyl-6-methyl-5,6-dihydroimidazo[2,1-b][1,3]thiaz
ol-2-yl]carbonyl}-5-ethyl-L-prolinate (16b)
27
28
29
30
31
32
Compound 16b was prepared (78% yield) as the pale yellow solid from 15b according to a similar
procedure for the synthesis of 16a; 1H-NMR (400 MHz, CDCl3) δ: 0.91 (3H, t, J = 7.4 Hz), 0.96 (3H, d,
J = 7.3 Hz), 1.04 (3H, d, J = 7.1 Hz), 1.35-1.41 (1H, m), 1.45 (9H, s), 1.75-1.80 (2H, m), 1.80 (3H, s),
1.95-2.00 (1H, m), 2.12-2.23 (2H, m), 2.70-2.77 (1H, m), 4.26-4.31 (1H, m), 4.52-4.57 (1H, m), 4.94
(1H, s), 6.67 (2H, d, J = 8.3 Hz), 7.00 (2H, d, J = 8.5 Hz), 7.02 (2H, d, J = 8.5 Hz), 7.13 (2H, d, J = 8.5
Hz); ESI/MS: m/z= 628 (M+H).

1
2
5.20.
3
(5R)-1-{[(5R,6S)-5,6-Bis(4-chlorophenyl)-3-isopropyl-6-methyl-5,6-dihydroimidazo[2,1-b][1,3]thiaz
ol-2-yl]carbonyl}-5-methyl-L-proline (17a)
4
5
Trifluoroacetic acid (40 ml) and anisole (1.63 ml, 0.015 mol) were added to a solution of compound
16a (9.0 g, 0.015 mol) in chloroform (100 ml). After stirring at room temperature for 1 h, the resulting
mixture was warmed to 40°C and stirred for 1 h. After cooling to room temperature, the solvent was
removed in vacuo, and the residue was azeotroped with toluene to afford the crude product. The residue
was purified by flash silica gel column chromatography with CHCl3/MeOH (15:1, v/v) to give the
6
7
8
9
1
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
colorless solid (5.76 g, 69% yield), which was precipitated from Et2O/n-hexane; H-NMR (400 MHz,
DMSO-d6) δ: 0.92 (3H, d, J = 7.3 Hz), 0.95 (3H, d, J = 6.8 Hz), 1.16 (3H, d, J = 6.1 Hz), 1.63-1.67 (1H,
m), 1.94 (3H, s), 1.96-2.01 (1H, m), 2.04-2.10 (1H, m), 2.34-2.40 (1H, m), 2.67-2.75 (1H, m), 4.26-4.35
(1H, m), 4.52-4.59 (1H, m), 5.89 (1H, s), 6.84-6.92 (2H, m), 7.17 (4H, d, J = 8.3 Hz), 7.23 (2H, d, J =
8.5 Hz); ESI/MS: m/z= 558 (M+H).
5.21.
(5R)-1-{[(5R,6S)-5,6-Bis(4-chlorophenyl)-3-isopropyl-6-methyl-5,6-dihydroimidazo[2,1-b][1,3]thiaz
ol-2-yl]carbonyl}-5-ethyl-L-proline (17b)
Compound 17b was prepared (quantitative yield) as the pale brown solid from 16b according to a
1
similar procedure for the synthesis of 17a; H-NMR (400 MHz, DMSO-d6) δ: 0.84 (3H, t, J = 7.3 Hz),
0.91-0.97 (6H, m), 1.33-1.42 (1H, m), 1.74-1.96 (4H, m), 2.04 (3H, s), 2.26-2.38 (1H, m), 2.70-2.77 (1H,
m), 4.04-4.09 (1H, m), 4.65-4.70 (1H, m), 6.16 (1H, s), 7.19-7.27 (8H, m), 8.30 (1H, s); ESI/MS: m/z=
572 (M+H).
5.22. (5R)-1-[(Benzyloxy)carbonyl]-5-methyl-L-proline (18a)
Trifluoroacetic acid (10ml) was added to a solution of compound 14a (4.3 g, 0.013 mol) in chloroform
(30 ml), and the resulting solution was warmed to 50°C and stirred for 16 h. After cooling to room
temperature, the solvent was removed in vacuo, and the residue was azeotroped with toluene to afford
the crude product. The residue was purified by flash silica gel column chromatography with
1
CHCl3/MeOH (10:1, v/v) to give the colorless oil (3.87 g, quantitative yield); H-NMR (400 MHz,
CDCl3) δ: 1.15-1.28 (3H, m), 1.53-1.63 (1H, m), 2.05-2.34 (3H, m), 4.12-4.25 (1H, m), 4.38-4.47 (1H,
m), 5.06-5.23 (2H, m), 7.25-7.37 (5H, m); ESI/MS: m/z= 264 (M+H).

1
2
5.23. (5R)-1-[(Benzyloxy)carbonyl]-5-ethyl-L-proline (18b)
3
Compound 18b was prepared (quantitative yield) as the pale brown solid from 14b according to a
similar procedure for the synthesis of 18a; 1H-NMR (400 MHz, CDCl3) δ: 0.85 and 0.89 (3H, each t, J =
7.6 and 7.9 Hz), 1.31-1.48 (2H, m), 1.73-1.79 and 1.82-1.89 (1H, m), 2.02-2.32 (3H, m), 3.89-3.94 and
3.96-4.02 (1H, m), 4.41 (1H, dd, J = 13.5, 8.9 Hz), 5.06-5.23 (2H, m), 7.20-7.38 (5H, m); ESI/MS: m/z=
278 (M+H).
4
5
6
7
8
9
5.24.
Benzyl
10
(2S,5R)-2-{[(3S)-3,4-dimethylpiperazin-1-yl]carbonyl}-5-methylpyrrolidine-1-carboxylate (19a)
11
12
13
14
15
16
17
18
19
20
21
22
Triethylamine (443 μl, 3.18 mmol) was added to a solution of compound 18a (334 mg, 1.27 mmol) in
dichloromethane (6 ml), followed by (2S)-1,2-dimethylpiperazine dihydrochloride (285 mg, 1.52 mmol),
HOBt (17 mg, 0.13 mmol) and EDC/HCl (292 mg, 1.52 mmol). The solution was stirred for 16 h at
room temperature. The solvent was removed in vacuo to afford crude product. The residue was purified
by flash silica gel chromatography with CHCl3/MeOH (50:1, v/v) to give the colorless oil (161 mg, 51%
yield); 1H-NMR (400 MHz, CDCl3) δ: 0.99 and 1.09 (3H, each dd, J = 13.0, 6.2 and 14.4, 6.3 Hz), 1.18
and 1.25 (3H, each d, J = 6.3 and 6.6 Hz), 1.51-1.58 (1H, m), 1.75-1.88 (1H, m), 2.04-2.31 (5H, m),
2.37-2.54 (1H, m), 2.61-2.81 (2H, m), 2.96-3.07 (1H, m), 3.17-3.32 (1H, m), 3.46-3.81 (1H, m),
4.19-4.36 (2H, m), 4.61-4.75 (1H, m), 4.95-5.26 (2H, m), 7.28-7.36 (5H, m); ESI/MS: m/z= 360 (M+H).
5.25.
Benzyl
(2S,5R)-2-{[(3R)-3,4-dimethylpiperazin-1-yl]carbonyl}-5-methylpyrrolidine-1-carboxylate (19b)
23
24
25
26
27
28
29
30
31
32
Compound 19b was prepared (70% yield) as the colorless oil from 18b according to a similar
1
procedure for the synthesis of 19a; H-NMR (400 MHz, CDCl3) δ: 0.84 and 0.90 (3H, each t, J = 7.6
and 7.4 Hz), 1.00 and 1.07 (3H, each d, J = 6.3 and 5.9 Hz), 1.35-1.42 (1H, m), 2.04-2.24 (4H, m), 2.25
and 2.29 (3H, each s), 2.64-2.90 (2H, m), 3.45-3.69 (1H, m), 3.97-4.11 (1H, m), 4.15-4.34 (1H, m),
4.59-4.74 (1H, m), 4.96-5.04 (1H, m), 5.08 and 5.22 (1H, each d, J = 12.5 and 12.2 Hz), 7.27-7.35 (5H,
m); ESI/MS: m/z= 374 (M+H).
5.26. (2S)-1,2-Dimethyl-4-[(5R)-5-methyl-L-prolyl]piperazine (20a)
To a solution of compound 19a (230 mg, 0.64 mmol) in methanol (10 ml) was added 10% Pd/C (100
mg, 50% wetted, type AD). The resulting mixture was stirred under a hydrogen atmosphere (1 atm) for

1
2
24 h at room temperature. The mixture was filtered to remove the catalyst, and the filtrate was
1
concentrated in vacuo to give the pale yellow solid (153 mg, 100% yield); H-NMR (400 MHz,
3
DMSO-d6) δ: 0.96-1.02 (3H, m), 1.17 (3H, d, J = 6.6 Hz), 1.38-1.49 (1H, m), 1.61-1.73 (1H, m),
1.89-1.99 (2H, m), 2.18 (3H, s), 2.24-2.34 (1H, m), 2.68-2.74 (1H, m), 2.80-2.88 (1H, m), 3.28-3.53 (3H,
m), 3.59-3.76 (1H, m), 3.96-4.12 (1H, m), 4.22-4.31 (1H, m); ESI/MS: m/z= 226 (M+H).
4
5
6
7
5.27. (2R)-1,2-Dimethyl-4-[(5R)-5-ethyl-L-prolyl]piperazine (20b)
8
Compound 20b was prepared (quantitative yield) as the pale yellow solid from 19b according to a
1
9
similar procedure for the synthesis of 20a; H-NMR (400 MHz, DMSO-d6) δ: 0.95 (3H, t, J = 7.4 Hz),
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
1.09-1.11 (3H, m), 1.59-1.69 (2H, m), 1.78-1.88 (2H, m), 2.08-2.14 (1H, m), 2.34 (3H, br s), 2.42-2.47
(1H, m), 2.88-3.11 (5H, m), 3.46-3.55 (1H, m), 3.74-3.82 (1H, m), 4.03-4.20 (1H, m), 4.58-4.66 (1H,
m); ESI/MS: m/z= 240 (M+H).
5.28.
(5R)-1-{[(5R,6S)-5,6-Bis(4-chlorophenyl)-3-isopropyl-6-methyl-5,6-dihydroimidazo[2,1-b][1,3]thiaz
ol-2-yl]carbonyl}-N,N,5-trimethyl-L-prolinamide (21)
Diisopropylethylamine (215 μl, 1.25 mmol) was added to a solution of compound 17a (300 mg, 0.54
mmol) in DMF (6 ml), followed by Dimethylamine dihydrochloride (61 mg, 0.75 mmol), HOBt (7 mg,
0.05 mmol) and EDC/HCl (116 mg, 0.60 mmol). The solution was stirred for 16 h at room temperature.
The reaction mixture was diluted with saturated aqueous sodium bicarbonate, and extracted with EtOAc.
The organic layer was washed with brine (3 times), and dried over magnesium sulfate. The mixture was
filtered, and the solvent was removed in vacuo to afford the crude product. The residue was purified by
flash silica gel chromatography with CHCl3/MeOH (30:1, v/v) to give the pale orange solid (116 mg,
1
40% yield), which was precipitated from Et2O/n-hexane; H-NMR (400 MHz, CDCl3) δ: 0.94 (3H, d, J
= 7.1 Hz), 1.04 (3H, d, J = 7.1 Hz), 1.23 (3H, d, J = 6.3 Hz), 1.60-1.65 (1H, m), 1.79 (3H, s), 1.82-1.88
(1H, m), 2.26-2.38 (2H, m), 2.74-2.78 (1H, m), 2.91 (3H, s), 3.11 (3H, s), 4.53-4.57 (1H, m), 4.93 (1H,
s), 5.01 (1H, d, J = 8.5 Hz), 6.68 (2H, d, J = 8.3 Hz), 7.00 (2H, d, J = 8.5 Hz), 7.01 (2H, d, J = 8.5 Hz),
7.13 (2H, d, J = 8.5 Hz); ESI/MS: m/z= 585 (M+H); HRESI/MS m/z= 585.18176 (Calcd for
C30H3535Cl2N4O2S: 585.18578); IR (ATR) cm-1: 1646, 1596, 1490, 1403, 1363, 1091, 1012.

1
2
5.29.
(5R)-1-{[(5R,6S)-5,6-Bis(4-chlorophenyl)-3-isopropyl-6-methyl-5,6-dihydroimidazo[2,1-b][1,3]thiaz
ol-2-yl]carbonyl}-5-ethyl-N,N-dimethyl-L-prolinamide (22)
3
4
Compound 22 was prepared (51% yield) as the colorless solid from 17b according to a similar
1
5
procedure for the synthesis of 21; H-NMR (400 MHz, CDCl3) δ: 0.88-0.95 (6H, m), 1.05 (3H, d, J =
6
7.1 Hz), 1.37-1.43 (1H, m), 1.75-1.80 (2H, m), 1.79 (3H, s), 1.83-1.87 (1H, m), 2.21-2.28 (2H, m),
2.74-2.85 (1H, m), 2.91 (3H, s), 3.10 (3H, s), 4.34-4.40 (1H, m), 4.93 (1H, s), 4.98-5.02 (1H, m), 6.66
(2H, d, J = 8.3 Hz), 6.99-7.02 (4H, m), 7.13 (2H, d, J = 8.5 Hz); ESI/MS: m/z= 599 (M+H); HREI/MS
m/z= 598.1927 (Calcd for C31H3635Cl2N4O2S: 598.1936); IR (ATR) cm-1: 1654, 1596, 1490, 1398, 1091,
1012.
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
5.30.
(5R,6S)-5,6-Bis(4-chlorophenyl)-2-({(2R,5S)-2-methyl-5-[(4-methylpiperazin-1-yl)carbonyl]pyrroli
din-1-yl}carbonyl)-3-isopropyl-6-methyl-5,6-dihydroimidazo[2,1-b][1,3]thiazole (23)
Compound 23 was prepared (28% yield) as the colorless solid from 17a according to a similar
procedure for the synthesis of 21; 1H-NMR (400 MHz, CDCl3) δ: 0.95 (3H, d, J = 7.1 Hz), 1.03 (3H, d,
J = 7.1 Hz), 1.23 (3H, d, J = 6.3 Hz), 1.62-1.67 (1H, m), 1.79-1.88 (1H, m), 1.79 (3H, s), 2.30 (3H, s),
2.32-2.44 (4H, m), 2.50-2.56 (1H, m), 2.71-2.77 (1H, m), 3.53-3.63 (4H, m), 4.53-4.56 (1H, m), 4.93
(1H, s), 5.00-5.03 (1H, m), 6.68 (2H, d, J = 8.3 Hz), 7.00-7.02 (4H, m), 7.12 (2H, d, J = 8.5 Hz);
ESI/MS: m/z= 640 (M+H); HRESI/MS m/z= 640.22670 (Calcd for C33H4035Cl2N5O2S: 640.22797); IR
(ATR) cm-1: 1652, 1596, 1565, 1490, 1407, 1361, 1290, 1172, 1091, 1012.
5.31.
(5R,6S)-2-({(2S,5R)-2-[(4-Acetylpiperazin-1-yl)carbonyl]-5-methylpyrrolidin-1-yl}carbonyl)-5,6-bi
s(4-chlorophenyl)-3-isopropyl-6-methyl-5,6-dihydroimidazo[2,1-b][1,3]thiazole (24)
Compound 24 was prepared (37% yield) as the colorless solid from 17a according to a similar
procedure for the synthesis of 21; 1H-NMR (400 MHz, CDCl3) δ: 0.96 (3H, d, J = 7.1 Hz), 1.02 (3H, d,
J = 7.1 Hz), 1.24 (3H, d, J = 6.3 Hz), 1.66-1.70 (1H, m), 1.79 (3H, s), 1.82-1.86 (1H, m), 2.10 (3H, s),
2.22-2.28 (1H, m), 2.36-2.43 (1H, m), 2.68-2.74 (1H, m), 3.44-3.50 (4H, m), 3.66-3.74 (4H, m),
4.53-4.58 (1H, m), 4.93 (1H, s), 4.98 (1H, dd, J = 8.4, 2.6 Hz), 6.69 (2H, d, J = 8.5 Hz), 6.99-7.03 (4H,
m), 7.12 (2H, d, J = 8.5 Hz); ESI/MS: m/z= 668 (M+H); HRESI/MS m/z= 668.21809 (Calcd for
C34H4035Cl2N5O3S: 668.22289); IR (ATR) cm-1: 1646, 1596, 1413, 1361, 1224, 1091, 1012.

1
2
5.32.
3
(5R,6S)-5,6-Bis(4-chlorophenyl)-2-({(2R,5S)-2-ethyl-5-[(4-methylpiperazin-1-yl)carbonyl]pyrrolidi
n-1-yl}carbonyl)-3-isopropyl-6-methyl-5,6-dihydroimidazo[2,1-b][1,3]thiazole (25)
Compound 25 was prepared (45% yield) as the colorless solid from 17b according to a similar
procedure for the synthesis of 21; 1H-NMR (400 MHz, CDCl3) δ: 0.91-0.96 (6H, m), 1.02-1.06 (3H, m),
1.38-1.44 (1H, m), 1.66-1.85 (2H, m), 1.79 (3H, s), 2.22-2.43 (6H, m), 2.31 (3H, s), 2.51-2.59 (1H, m),
2.70-2.77 (1H, m), 3.52-3.66 (4H, m), 4.38 (1H, t, J = 8.7 Hz), 4.94 (1H, s), 4.99-5.02 (1H, m),
6.63-6.68 (2H, m), 6.99-7.03 (4H, m), 7.12-7.15 (2H, m); ESI/MS: m/z= 654 (M+H); HRESI/MS m/z=
654.24417 (Calcd for C34H4235Cl2N5O2S: 654.24363); IR (ATR) cm-1: 1650, 1596, 1567, 1490, 1444,
1357, 1290, 1172, 1091, 1012.
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
5.33.
(5R,6S)-2-({(2S,5R)-2-[(4-Acetylpiperazin-1-yl)carbonyl]-5-ethylpyrrolidin-1-yl}carbonyl)-5,6-bis(
4-chlorophenyl)-3-isopropyl-6-methyl-5,6-dihydroimidazo[2,1-b][1,3]thiazole (26)
Compound 26 was prepared (46% yield) as the colorless solid from 17b according to a similar
1
procedure for the synthesis of 21; H-NMR (400 MHz, CDCl3) δ: 0.91-0.96 (6H, m), 1.03 (3H, d, J =
7.1 Hz), 1.36-1.46 (1H, m), 1.77-1.84 (3H, m), 1.78 (3H, s), 2.10 (3H, s), 2.20-2.31 (2H, m), 2.72-2.79
(1H, m), 3.44-3.52 (4H, m), 3.61-3.75 (4H, m), 4.35-4.39 (1H, m), 4.93 (1H, s), 4.95-4.99 (1H, m), 6.67
(2H, d, J = 8.3 Hz), 6.98-7.03 (4H, m), 7.13 (2H, d, J = 8.5 Hz); ESI/MS: m/z= 682 (M+H); HRESI/MS
m/z= 682.23549 (Calcd for C35H4235Cl2N5O3S: 682.23854); IR (ATR) cm-1: 1594, 1565, 1490, 1384,
1230, 1174, 1114, 1091, 1012.
5.34.
(5R,6S)-5,6-Bis(4-chlorophenyl)-2-{[(2R,5S)-2-ethyl-5-(morpholin-4-ylcarbonyl)pyrrolidin-1-yl]car
bonyl}-3-isopropyl-6-methyl-5,6-dihydroimidazo[2,1-b][1,3]thiazole (27)
Compound 27 was prepared (62% yield) as the colorless solid from 17b according to a similar
1
procedure for the synthesis of 21; H-NMR (400 MHz, CDCl3) δ: 0.91-0.96 (6H, m), 1.05 (3H, d, J =
7.1 Hz), 1.35-1.46 (1H, m), 1.76-1.86 (3H, m), 1.79 (3H, s), 2.19-2.30 (2H, m), 2.74-2.82 (1H, m),
3.50-3.77 (8H, m), 4.34-4.40 (1H, m), 4.94 (1H, s), 4.94-5.00 (1H, m), 6.67 (2H, d, J = 8.5 Hz), 7.01
(2H, d, J = 8.5 Hz), 7.02 (2H, d, J = 8.8 Hz), 7.13 (2H, d, J = 8.5 Hz); ESI/MS: m/z= 641 (M+H);

1
2
HRESI/MS m/z= 641.20974 (Calcd for C33H3935Cl2N4O3S: 641.21199); IR (ATR) cm-1: 1646, 1596,
1490, 1415, 1359, 1282, 1174, 1091, 1012.
3
4
5.35.
5
(5R,6S)-5,6-Bis(4-chlorophenyl)-2-[((2S,5R)-2-{[(3R)-3,4-dimethylpiperazin-1-yl]carbonyl}-5-meth
ylpyrrolidin-1-yl)carbonyl]-3-isopropyl-6-methyl-5,6-dihydroimidazo[2,1-b][1,3]thiazole (28)
Compound 28 was prepared (37% yield) as the colorless solid from 17a according to a similar
procedure for the synthesis of 21; 1H-NMR (400 MHz, CDCl3) δ: 0.94 (3H, d, J = 6.3 Hz), 1.04 (3H, d,
J = 7.1 Hz), 1.05-1.08 (3H, m), 1.23 (3H, d, J = 6.3 Hz), 1.62-1.65 (1H, m), 1.79 (3H, s), 1.83-1.86 (1H,
m), 2.20-2.31 (3H, m), 2.29 (3H, s), 2.73-2.77 (2H, m), 2.85-2.88 (1H, m), 3.40-3.45 (1H, m), 3.67-3.75
(1H, m), 4.17-4.21 (1H, m), 4.32-4.37 (1H, m), 4.51-4.57 (1H, m), 4.93 (1H, s), 5.00-5.03 (1H, m), 6.68
(2H, d, J = 8.3 Hz), 6.99-7.03 (4H, m), 7.12 (2H, d, J = 8.5 Hz); ESI/MS: m/z= 654 (M+H); HRESI/MS
m/z= 654.24561 (Calcd for C34H4235Cl2N5O2S: 654.24363); IR (ATR) cm-1: 1652, 1596, 1490, 1373,
1288, 1176, 1091, 1012.
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
5.36.
(5R,6S)-5,6-Bis(4-chlorophenyl)-2-[((2S,5R)-2-{[(3S)-3,4-dimethylpiperazin-1-yl]carbonyl}-5-meth
ylpyrrolidin-1-yl)carbonyl]-3-isopropyl-6-methyl-5,6-dihydroimidazo[2,1-b][1,3]thiazole (29)
Thionyl chloride (150 μl, 2.1 mmol) and a catalytic amount of DMF were added to a suspension of
compound 8 (300 mg, 0.67 mmol) in toluene (5 ml). The resulting mixture was warmed to 70°C and
stirred for 1 h. After cooling to room temperature, the solvent was removed in vacuo to afford the crude
product. The residue was dissolved in THF (2 ml), and was dropwised to a solution of compound 20a
(144 mg, 0.64 mmol) with triethylamine (270 μl, 1.92 mmol) in THF (6 ml) under an ice cooling bath.
The resulting solution was warmed to room temperature, and was stirred for 3 h. The reaction mixture
was quenched by the addition of saturated aqueous sodium bicarbonate, and extracted with EtOAc. The
organic layer was washed with brine, and dried over magnesium sulfate. The mixture was filtered, and
the solvent was removed in vacuo to afford crude product. The residue was purified by flash silica gel
column chromatography with CHCl3/MeOH (20:1, v/v) to give the colorless solid (204 mg, 49% yield),
1
which was precipitated from Et2O/n-hexane; H-NMR (400 MHz, CDCl3) δ: 0.95 (3H, d, J = 6.8 Hz),
1.03 (3H, d, J = 7.1 Hz), 1.09-1.11 (3H, m), 1.23 (3H, d, J = 6.3 Hz), 1.61-1.64 (1H, m), 1.79 (3H, s),
1.82-1.86 (1H, m), 2.07-2.12 (1H, m), 2.22-2.27 (1H, m), 2.29 (3H, s), 2.49-2.58 (1H, m), 2.71-2.80 (2H,
m), 2.98-3.05 (1H, m), 3.27-3.33 (1H, m), 3.59-3.64 (1H, m), 3.76-3.82 (1H, m), 4.23-4.27 (1H, m),

1
2
4.52-4.58 (1H, m), 4.93 (1H, s), 5.00-5.03 (1H, m), 6.68 (2H, d, J = 8.1 Hz), 7.01 (4H, d, J = 8.5 Hz),
7.12 (2H, d, J = 8.5 Hz); ESI/MS: m/z= 654 (M+H); HRESI/MS m/z= 654.23407 (Calcd for
C34H4235Cl2N5O2S: 654.24363); IR (ATR) cm-1: 1652, 1596, 1565, 1490, 1373, 1288, 1091, 1012.
3
4
5
5.37.
6
(5R,6S)-2-{[(2S,5R)-2-{[(3R)-4-Acetyl-3-methylpiperazin-1-yl]carbonyl}-5-methylpyrrolidin-1-yl]c
arbonyl}-5,6-bis(4-chlorophenyl)-3-isopropyl-6-methyl-5,6-dihydroimidazo[2,1-b][1,3]thiazole (30)
Compound 30 was prepared (32% yield) as the colorless solid from 17a according to a similar
procedure for the synthesis of 21; 1H-NMR (400 MHz, CDCl3) δ: 0.94 (3H, d, J = 7.1 Hz), 1.02 (3H, d,
J = 7.1 Hz), 1.19-1.21 (3H, m), 1.24 (3H, d, J= 6.3 Hz), 1.65-1.71 (1H, m), 1.81 (3H, s), 1.83-1.87 (1H,
m), 2.09 (3H, s), 2.20-2.25 (1H, m), 2.41-2.47 (1H, m), 2.69-2.76 (1H, m), 2.89-2.94 (1H, m), 3.14-3.20
(1H, m), 3.44-3.50 (1H, m), 3.69-3.75 (1H, m), 3.97-4.03 (1H, m), 4.31-4.37 (1H, m), 4.49-4.55 (1H, m),
4.84-4.88 (1H, m), 4.97 (1H, s), 5.00-5.02 (1H, m), 6.69 (2H, d, J = 8.3 Hz), 7.01 (2H, d, J = 8.5 Hz),
7.02 (2H, d, J = 8.5 Hz), 7.12 (2H, d, J = 8.5 Hz); ESI/MS: m/z= 682 (M+H); HRESI/MS m/z=
682.23688 (Calcd for C35H4235Cl2N5O3S: 682.23853); IR (ATR) cm-1: 1596, 1491, 1412, 1368, 1309,
1218, 1091, 1012.
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
5.38.
(5R,6S)-2-{[(2S,5R)-2-{[(3S)-4-Acetyl-3-methylpiperazin-1-yl]carbonyl}-5-methylpyrrolidin-1-yl]ca
rbonyl}-5,6-bis(4-chlorophenyl)-3-isopropyl-6-methyl-5,6-dihydroimidazo[2,1-b][1,3]thiazole (31)
Compound 31 was prepared (42% yield) as the colorless solid from 17a according to a similar
procedure for the synthesis of 21; 1H-NMR (400 MHz, CDCl3) δ: 0.96 (3H, d, J = 7.1 Hz), 1.02 (3H, d,
J = 7.1 Hz), 1.17-1.20 (3H, m), 1.24 (3H, d, J = 6.3 Hz), 1.67-1.71 (1H, m), 1.80 (3H, s), 1.84-1.89 (1H,
m), 2.10 (3H, s), 2.13-2.16 (1H, m), 2.28-2.32 (1H, m), 2.70-2.75 (1H, m), 2.90-2.93 (1H, m), 3.30-3.34
(1H, m), 3.65-3.69 (1H, m), 3.85-3.89 (1H, m), 4.05-4.09 (1H, m), 4.35-4.40 (1H, m), 4.53-4.57 (1H, m),
4.84-4.87 (1H, m), 4.95 (1H, s), 4.98-5.01 (1H, m), 6.69 (2H, d, J = 8.3 Hz), 7.01 (2H, d, J = 8.5 Hz),
7.02 (2H, d, J = 8.5 Hz), 7.12 (2H, d, J = 8.3 Hz); ESI/MS: m/z= 682 (M+H); HRESI/MS m/z=
682.23611 (Calcd for C35H4235Cl2N5O3S: 682.23853); IR (ATR) cm-1: 1597, 1566, 1491, 1412, 1371,
1309, 1176, 1091, 1013.
5.39.

1
2
Preparation
of
(5R,6S)-5,6-Bis(4-chlorophenyl)-2-({(2S,5R)-2-[(3,3-dimethylpiperazin-1-yl)carbonyl]-5-methylpyr
rolidin-1-yl}carbonyl)-3-isopropyl-6-methyl-5,6-dihydroimidazo[2,1-b][1,3]thiazole (32)
3
4
5
5.39.1.
6
(5R,6S)-5,6-Bis(4-chlorophenyl)-2-({(2S,5R)-2-[(3,3-dimethylpiperazin-1-yl)carbonyl]-5-methylpyr
rolidin-1-yl}carbonyl)-3-isopropyl-6-methyl-5,6-dihydroimidazo[2,1-b][1,3]thiazole
Triethylamine (624 μl, 4.48 mmol) was added to a solution of compound 17a (1.0 g, 1.79 mmol) in
DMF (20 ml), followed by 2,2-dimethylpiperazine dihydrochloride22 (402 mg, 2.15 mmol), HOBt (24
mg, 0.18 mmol) and EDC/HCl (412 mg, 2.15 mmol) under an ice cooling bath. The solution was
warmed to room temperature and stirred for 20 h. The reaction was diluted with saturated aqueous
sodium bicarbonate, and extracted with EtOAc. The organic layer was washed with brine (3 times), and
dried over magnesium sulfate. The mixture was filtered, and the solvent was removed in vacuo to afford
the crude product. The residue was purified by flash NH2 silica gel chromatography with CHCl3/MeOH
(80:1, v/v) to give the colorless solid (450 mg, 38% yield), which was precipitated from Et2O/n-hexane;
1H-NMR (400 MHz, CDCl3) δ: 0.93 (3H, d, J = 7.1 Hz), 1.03 (3H, d, J = 7.1 Hz), 1.07-1.15 (6H, m),
1.23 (3H, d, J = 6.3 Hz), 1.40-1.47 (2H, m), 1.62-1.69 (1H, m), 1.79 (3H, s), 1.83-1.88 (1H, m),
2.22-2.39 (2H, m), 2.72-2.78 (1H, m), 2.87-2.96 (1H, m), 3.20-3.27 (1H, m), 3.38-3.55 (2H, m),
4.52-4.56 (1H, m), 4.93 (1H, s), 5.01-5.04 (1H, m), 6.69 (2H, d, J = 8.5 Hz), 7.01 (5H, d, J = 8.8 Hz),
7.12 (2H, d, J = 8.5 Hz); HRESI/MS m/z= 654.24351 (Calcd for C34H4235Cl2N5O2S: 654.24363); IR
(ATR) cm-1: 1647, 1596, 1491, 1408, 1372, 1293, 1175, 1091, 1013.
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
5.39.2.
(5R,6S)-5,6-Bis(4-chlorophenyl)-2-({(2S,5R)-2-[(3,3-dimethylpiperazin-1-yl)carbonyl]-5-methylpyr
rolidin-1-yl}carbonyl)-3-isopropyl-6-methyl-5,6-dihydroimidazo[2,1-b][1,3]thiazole (32)
To
a
stirred
solution
of
(5R,6S)-5,6-Bis(4-chlorophenyl)-2-({(2S,5R)-2-[(3,3-dimethylpiperazin-1-yl)carbonyl]-5-methylpyrroli
din-1-yl}carbonyl)-3-isopropyl-6-methyl-5,6-dihydroimidazo[2,1-b][1,3]thiazole (220 mg, 0.34 mmol)
in 1,4-dioxane (6 ml) was added 37% aqueous formaldehyde solution (253 μl, 3.4 mmol). After stirring
for 15 min at room temperature, sodium triacetoxyborohydride (144 mg, 0.68 mmol) was added to the
reaction solution. The resulting mixture was stirred for 16 h at room temperature. The reaction was
diluted with saturated aqueous sodium bicarbonate, and extracted with EtOAc. The organic layer was

1
2
washed with brine, and dried over magnesium sulfate. The mixture was filtered, and the solvent was
removed in vacuo to afford the crude product. The residue was purified by flash silica gel
chromatography with CHCl3/MeOH (30:1, v/v) to give the colorless solid (150 mg, 66% yield), which
3
1
4
was precipitated from Et2O/n-hexane; H-NMR (400 MHz, CDCl3) δ: 0.92 (3H, d, J = 7.1 Hz),
5
0.95-1.02 (6H, m), 1.03 (3H, d, J = 7.1 Hz), 1.23 (3H, d, J = 6.6 Hz), 1.61-1.67 (1H, m), 1.79 (3H, s),
1.82-1.86 (1H, m), 2.24 (3H, s), 2.26-2.36 (2H, m), 2.51-2.54 (1H, m), 2.73-2.76 (2H, m), 3.18-3.34 (2H,
m), 3.47-3.65 (2H, m), 4.51-4.56 (1H, m), 4.93 (1H, s), 5.02-5.05 (1H, m), 6.68 (2H, d, J = 7.8 Hz), 7.01
(4H, d, J = 8.5 Hz), 7.12 (2H, d, J = 8.3 Hz); HRESI/MS m/z= 668.25854 (Calcd for C35H4435Cl2N5O2S:
668.25927); IR (ATR) cm-1: 1650, 1595, 1566, 1491, 1451, 1373, 1291, 1173, 1091, 1012.
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
5.40.
(5R,6S)-5,6-Bis(4-chlorophenyl)-2-[((2S,5R)-2-{[(3R)-3,4-dimethylpiperazin-1-yl]carbonyl}-5-ethyl
pyrrolidin-1-yl)carbonyl]-3-isopropyl-6-methyl-5,6-dihydroimidazo[2,1-b][1,3]thiazole (33)
Compound 33 was prepared (28% yield) as the colorless solid from 8 according to a similar procedure
1
for the synthesis of 29; H-NMR (400 MHz, CDCl3) δ: 0.88-0.95 (6H, m), 1.05 (3H, d, J = 7.1 Hz),
1.05-1.08 (3H, m), 1.37-1.44 (1H, m), 1.79 (3H, s), 1.79-1.83 (3H, m), 2.17-2.26 (3H, m), 2.29 (3H, s),
2.76-2.84 (4H, m), 3.40-3.46 (1H, m), 3.66-3.74 (1H, m), 4.17-4.21 (1H, m), 4.35-4.39 (1H, m), 4.93
(1H, s), 4.99-5.02 (1H, m), 6.66 (2H, d, J = 8.1 Hz), 7.01 (4H, d, J = 8.5 Hz), 7.12 (2H, d, J = 8.5 Hz);
ESI/MS: m/z= 668 (M+H); HRESI/MS m/z= 668.26496 (Calcd for C35H4435Cl2N5O2S: 668.25928); IR
(ATR) cm-1: 1652, 1596, 1490, 1409, 1338, 1174, 1091, 1012.
5.41.
(5R,6S)-5,6-Bis(4-chlorophenyl)-2-[((2S,5R)-2-{[(3S)-3,4-dimethylpiperazin-1-yl]carbonyl}-5-ethyl
pyrrolidin-1-yl)carbonyl]-3-isopropyl-6-methyl-5,6-dihydroimidazo[2,1-b][1,3]thiazole (34)
Compound 34 was prepared (54% yield) as the colorless solid from 17b according to a similar
1
procedure for the synthesis of 21; H-NMR (400 MHz, CDCl3) δ: 0.90-0.95 (6H, m), 1.05 (3H, d, J =
7.3 Hz), 1.07-1.13 (3H, br m), 1.38-1.44 (1H, m), 1.79 (3H, s), 1.80-1.86 (3H, m), 2.07-2.26 (3H, m),
2.29 (3H, s), 2.77 (2H, d, J = 11.5 Hz), 3.00-3.07 (1H, m), 3.27-3.34 (1H, m), 3.57-3.65 (1H, m),
3.74-3.81 (1H, m), 4.18-4.31 (1H, m), 4.32-4.39 (1H, m), 4.93 (1H, s), 5.01 (1H, d, J = 6.7 Hz), 6.66
(2H, d, J = 7.9 Hz), 7.01 (4H, d, J = 8.5 Hz), 7.12 (2H, d, J= 8.5 Hz); ESI/MS: m/z= 668 (M+H);
HRESI/MS m/z= 668.25169 (Calcd for C35H4435Cl2N5O2S: 668.25928); IR (ATR) cm-1: 1652, 1596,
1490, 1409, 1338, 1174, 1091, 1012.

1
2
5.42.
3
(5R,6S)-2-[((2S,5R)-2-{[(3R)-4-Acetyl-3-methylpiperazin-1-yl]carbonyl}-5-ethylpyrrolidin-1-yl)car
bonyl]-5,6-bis(4-chlorophenyl)-3-isopropyl-6-methyl-5,6-dihydroimidazo[2,1-b][1,3]thiazole (35)
Compound 35 was prepared (60% yield) as the colorless solid from 17b according to a similar
4
5
1
6
procedure for the synthesis of 21; H-NMR (400 MHz, CDCl3) δ: 0.90-0.96 (6H, m), 1.04 (3H, d, J =
7
7.1 Hz), 1.21 (3H, br s), 1.39-1.46 (1H, m), 1.78-1.84 (3H, m), 1.79 (3H, s), 2.09 (3H, s), 2.15-2.20 (2H,
m), 2.33-2.37 (1H, m), 2.73-2.80 (1H, m), 2.89-2.96 (1H, br m), 3.12-3.19 (1H, br m), 3.41-3.49 (1H, br
m), 3.68-3.77 (1H, br m), 3.96-4.01 (1H, br m), 4.31-4.37 (2H, m), 4.94 (1H, s), 4.95-4.99 (1H, m), 6.68
(2H, d, J = 8.3 Hz), 7.00 (2H, d, J = 8.5 Hz), 7.01 (2H, d, J = 8.8 Hz), 7.12 (2H, d, J = 8.5 Hz); ESI/MS:
m/z= 696 (M+H); HRESI/MS m/z= 696.25430 (Calcd for C36H4435Cl2N5O3S: 696.25419); IR (ATR)
cm-1: 1639, 1596, 1411, 1363, 1176, 1091, 1012.
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
5.43.
(5R,6S)-2-[((2S,5R)-2-{[(3S)-4-Acetyl-3-methylpiperazin-1-yl]carbonyl}-5-ethylpyrrolidin-1-yl)car
bonyl]-5,6-bis(4-chlorophenyl)-3-isopropyl-6-methyl-5,6-dihydroimidazo[2,1-b][1,3]thiazole (36)
Compound 36 was prepared (57% yield) as the colorless solid from 17b according to a similar
1
procedure for the synthesis of 21; H-NMR (400 MHz, CDCl3) δ: 0.90-0.96 (6H, m), 1.03 (3H, d, J =
6.8 Hz), 1.33-1.43 (4H, m), 1.76-1.84 (3H, m), 1.78 (3H, s), 2.10 (3H, s), 2.21-2.28 (2H, m), 2.73-2.79
(1H, m), 2.85-2.91 (1H, m), 3.24-3.36 (2H, m), 3.60-3.68 (1H, m), 3.82-3.88 (1H, m), 4.34-4.40 (2H, m),
4.44-4.50 (1H, m), 4.93 (1H, s), 4.96-5.01 (1H, m), 6.67 (2H, d, J = 8.3 Hz), 7.00 (2H, d, J = 8.5 Hz),
7.01 (2H, d, J = 8.5 Hz), 7.12 (2H, d, J = 8.3 Hz); ESI/MS: m/z= 696 (M+H); HRESI/MS m/z=
696.25274 (Calcd for C36H4435Cl2N5O3S: 696.25419); IR (ATR) cm-1: 1644, 1596, 1411, 1363, 1176,
1091, 1012.
5.44. p53-MDM2 HTRF assay
The assay mixture (8 μl containing 2.5 nM GST-tagged MDM2 (25-108, L33E) and 2.5 nM His-tagged
p53 (1-132) with assay buffer (20 mM HEPES pH 7.4, 150 mM NaCl, and 0.1 % BSA), 4 μl of the
tested compound in 3-fold dilutions, and 8 μl antibody mixture solution containing XL665-anti-6HIS
(Schering) in a final concentration of 1.0 μg/ml and Eu-anti-GST (Schering) in a final concentration of
0.13 μg/ml with assay buffer (20 mM HEPES pH 7.4, 150 mM NaCl, 0.1 % BSA and 0.5 M KF)) was
incubated on the 384-well plate (Corning) for 1 h at 25˚C, and was detected by time-resolved

1
2
fluorescence spectroscopy (ARVOsx, PerkinElmer Life Sciences). The IC50 values were calculated from
sigmoid-fitting curve analysis by using Prism software (GraphPad).
3
4
5.45. Antiproliferative assay in vitro
5
In vitro assay was performed against MV4-11 and DLD-1 cell lines to examine the growth inhibitory
effects of our compounds. Both cell lines were purchased from American Type Culture Collection
(ATCC). The cells were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum.
The cells were plated in 96-well micro plates on Day 0 (DLD-1) or Day 1 (MV4-11), and stock
solutions serially diluted in DMSO were added to each well on Day 1. After 3 days of culture (Day 4),
the number of viable cells was determined by the luminescent cell viability assay. The concentration of
compounds that cause 50% of growth inhibition (GI50) was calculated.
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
5.46. Antitumor activity assay
In vivo antitumor activity of compounds 28 and 35 were performed on the MV4-11 (human leukemia
cells, ATCC) xenograft model on mice. Nutlin-3 (racemate) was used as a positive control compound.
MV4-11 cells (1 x 107 cells/head) were inoculated s.c. into NOD SCID mice (Charles River
Laboratories Japan Inc.) (Day 0). Drug administration was started on Day 21 with p.o. per day (28 and
35) or twice a day (Nutlin-3) every 200mg/kg doses. Administration schedules were on Days 21-23 and
27-30 (28), or Days 21-24 and 27-30 (35 and Nutlin-3). The total dose of each of the compounds was
1400 mg/kg (28), 1600 mg/kg (35) and 3200 mg/kg (Nutlin-3). Tumor volumes and bodyweights were
measured every 2 days over the course of administration. After the mice were sacrificed on Day 31,
tumor weight was assessed and tumor growth inhibition (TGI) was calculated.
23
24
5.47. Solubility
25
26
27
28
29
30
31
The solubility was determined by HPLC analysis. 10 mM of compound solution in DMSO (50 μL) was
freeze-dried. To the residue the Japanese Pharmacopoeia Second Fluid (JP2; 250 μL, pH 6.8) was added,
and the mixture was stirred by pipette operation. The mixture was saved under shading over 12 h. After
filtration of the mixture, the resulting filtrate was diluted 20 times by adding aqueous DMSO solution
(1:1, v/v) to obtain the measurement sample solution. 5 μM of compound solution in aqueous DMSO
solution (1:1, v/v) and 100 mM of compound solution in aqueous DMSO solution (1:1, v/v) were
prepared to make a calibration curve. The measurement sample solution, 5 μM solution and 100 mM

1
2
3
4
solution were assayed using HPLC methodologies (Analytical Column: X Terra® MSC18 3.5 lm, 3.0 x
30 mm, Waters; Mobile Phase: 10 mM ammonium acetate buffer (pH 4.5)/0.05% acetic acid in
acetonitrile = 95:5 to 10:90 v/v; Wave length: PDA 220–420 nm). The solubilities were analyzed using
Millenium software (Waters).
5
6
5.48. Metabolic stability
7
8
The compounds (final 1 μM) were incubated with mouse hepatic microsome in sodium phosphate
buffer (pH 7.4) for 20 min at 37°C. The microsomal protein concentration in the assay was 0.5 mg/mL.
The reaction was started by addition of the NADPH generating system at 37°C and stopped by addition
of MeOH after 30 min. After centrifuging each solution separately at 3500 rpm for 10 min at 4°C, the
corresponding loss of the parent compound was determined by LC-MS/MS.
9
10
11
12
13
5.49. Measurement of tumor concentration
14
15
16
17
18
19
20
Synthetic compound suspended in the Japanese Pharmacopoeia First Fluid (pH 1.2) was administered
orally to tumor bearing mice (100 mg/kg). Tumor samples were collected at 1, 2, 4, 6 and 24 h after oral
dosing and homogenized using Mixer Mill MM 300. Tumor concentrations for the synthetic compound
were determined by LC/MS/MS using a Shimadzu LC-20AD system (Shimadzu) coupled to the mass
spectrometer API 4000 (SCIEX). The synthetic compound was separated on a Shim-pack XR- ODS
column (Shimadzu). Respective pharmacokinetic parameters were carried out using winnonlin software
(Pharsight Corporation).
21
22
23
References and Notes
1. Oliner, J. D.; Kinzler, K. W.; Meltzer, P. S.; George, D. L. Nature 1992, 358, 80.
24
25
26
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2. Levine, A. J. Cell 1997, 88, 323.
3. Momand, J.; Zambetti, G. P.; Olson, D. C.; George, D.; Levine, A. J. Cell. 1992, 69, 1237.
4. Eymin, B.; Gazzeri, S.; Brambilla, C.; Brambilla, E. Oncogene 2002, 21, 2750.
5. Haupt, Y.; Maya, R.; Kazaz, A.; Oren, M. Nature 1997, 387, 296.
6. Kubbutat, M. H. G.; Jones, S. N.; Vousden, K. H. Nature 1997, 387, 299.
7. Iwakuma, T.; Lozano, G. Mol. Cancer Res. 2003, 1, 993.

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8. Kussie, P. H.; Gorina, S.; Marechal, V.; Elenbaas, B.; Moreau, J.; Levine, A. J.; Pavletich, N. P.
Science 1996, 274, 948.
3
4
9. Dawson, R.; Müller, L.; Dehner, A.; Klein, C.; Kessler, H.; Buchner, J. J. Mol. Biol. 2003, 332,
1131.
5
10. Espinoza-Fonseca, L. M.; Trujillo-Ferrara, J. G. Biopolymers 2006, 83, 365.
6
7
11. Vassilev, L. T.; Vu, B. T.; Graves, B.; Carvajal, D.; Podlaski, F.; Filipovic, Z.; Kong, N.;
Kammlott, U.; Lukacs, C.; Klein, C.; Fotouhi, N.; Liu, E. A. Science 2004, 303, 844.
8
9
12. Allen, J. G.; Bourbeau, M. P.; Wohlhieter, G. E.; Bartberger, M. D.; )Michelsen, K.; Hungate, R.;
Gadwood, R. C.; Gatson, R. D.; Evans, B.; Mann, L. W.; Matison, M. E.; Schneider, S.; Huang,
X.; Yu, D.; Andrews, P. S.; Reichelt, A.; Long, A. M.; Yakowec, P.; Yang, E. Y.; Lee, T. A.;
Oliner, J. D. J. Med. Chem. 2009, 52, 7044.
10
11
12
13
13. Furet, P.; Chène, P.; Pover, A. D.; Valat, T. S.; Lisztwan, J. H.; Kallen, J.; Masuya, K. Bioorg.
Med. Chem. lett. 2012, 22, 3498.
14
15
16
17
18
14. Rew, Y.; Sun, D.; Turiso, F. G.-L. D.; Bartberger, M. D.; Beck, H. P.; Canon, J.; Chen. A.; Chow,
D.; Deignan, J.; Fox, B. M.; Gustin, D.; Huang, X.; Jiang, M.; Jiao, X.; Jin, L.; Kayser, F.;
Kopecky, D. J.; Li, Y.; Lo. M.-C.; Long, A. M.; Michelsen, K.; Oliner. J. D.; Osgood, T.; Ragains,
M.; Saiki, A. Y.; Schneider, S.; Toteva, M.; Yakowec, P.; Yan, X.; Ye, Q.; Yu, D.; Zhao, X.; Zhou,
J.; Medina, J. C.; Olson, S. H. J. Med. Chem. 2012, 55, 4936.
19
20
15. Miyazaki, M.; Kawato, H.; Naito, H.; Ikeda, M.; Miyazaki, M.; Kitagawa, M.; Seki, T.; Fukutake,
S.; Aonuma, M.; Soga, T. Bioorg. Med. Chem. Lett. 2012, 22, 6338.
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22
23
16. Miyazaki, M.; Naito, H.; Sugimoto, Y.; Kawato, H.; Okayama, T.; Shimizu, H.; Miyazaki, M.;
Kitagawa, M.; Seki, T.; Fukutake, S.; Aonuma, M.; Soga, T. Bioorg. Med. Chem. Lett. 2013, 23,
728.
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25
26
27
28
17. Pei, Z.; Li, X.; Longenecker, K.; Geldern, T. W. v.; Wiedeman, P. E.; Lubben, T. H.; Zinker, B.
A.; Stewart, K.; Ballaron, S. J.; Stashko, M. A.; Mika, A. K.; Beno, D. W. A.; Long, M.; Wells,
H.; Kempf-Grote, A. J.; Madar, D. J.; McDermott, T. S.; Bhagavatula, L.; Fickes, M. G.; Pireh, D.;
Solomon, L. R.; Lake, M. R.; Edalji, R.; Fry, E. H.; Sham, H. L.; Trevillyan, J. M. J. Med. Chem.
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29
18. Hussaini, S. R.; Moloney, M. G. Syn. Commun. 2006, 35, 1129.

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19. Moloney, M. G.; Panchal, T.; Pike, R. Org. Biomol. Chem. 2006, 4, 3894.
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3
4
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5
22. Chu, D. T. W.; Claiborne, A. K.; Clement, J. J.; Plattner, J. J. Can. J. Chem. 1992, 70, 1328.

Table 1. In vitro activity for the p53-MDM2 binding interaction and physicochemical properties of
dihydroimidazothiazoles

HTRF
IC50 (µM)
MV4-11a
DLD-1b
Solubilityc
(µg/mL)
MSd
(% rem)
cmpd
1
R
R2
GI50 (µM)
*
*
*
*
*
H
0.0083
0.014
0.039
0.014
0.018
0.020
0.11
2.4
20
>50
>50
8.5
57
<3
12
29
7
9
(S)-Me
(S)-Et
(R)-Me
(R)-Et
39
47
53
70
9
10
21
22
0.09
0.13
7.4
*
(R)-Me
(R)-Me
(R)-Et
(R)-Et
0.016
0.026
0.030
0.043
0.19
0.27
9.0
16
45
79
13
34
25
13
50
38
23
24
25
26
*
*
0.083
0.13
4.0
9.3
*
*
(R)-Et
(R)-Me
(R)-Me
0.068
0.015
0.016
0.094
0.20
0.37
4.2
6.9
6.2
9
6
27
28
29
*
*
49
47
74
63
*
(R)-Me
(R)-Me
0.030
0.037
0.083
0.40
11
72
75
55
65
30
31
*
5.2
*
(R)-Me
(R)-Et
(R)-Et
0.018
0.029
0.030
0.28
0.11
0.25
6.3
3.6
3.9
30
14
16
84
76
69
32
33
34
*
*
*
(R)-Et
(R)-Et
0.058
0.059
0.22
0.12
10
25
24
59
54
35
36
*
2.6

a cell line with p53 wild type.
b cell line with mutated p53.
c pH 6.8 phosphate buffer solution.
d in vitro metabolic stability in mouse hepatic microsome (% remaining after 30 min).