Design, synthesis, and biological evaluation of hydantoin bridged analogues of combretastatin A-4 as potential anticancer agents

Accepted Manuscript

Design, Synthesis, and Biological Evaluation of Hydantoin Bridged Analogues

of Combretastatin A-4 as Potential Anticancer Agents

Mao Zhang, Yu-Ru Liang, Huan Li, Ming-Ming Liu, Yang Wang

PII:

S0968-0896(17)31846-1

https://doi.org/10.1016/j.bmc.2017.10.045

BMC 14050

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Reference:

Bioorganic & Medicinal Chemistry

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30 October 2017

Please cite this article as: Zhang, M., Liang, Y-R., Li, H., Liu, M-M., Wang, Y., Design, Synthesis, and Biological

Evaluation of Hydantoin Bridged Analogues of Combretastatin A-4 as Potential Anticancer Agents, Bioorganic &

Medicinal Chemistry (2017), doi: https://doi.org/10.1016/j.bmc.2017.10.045

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Design, Synthesis, and Biological Evaluation of Hydantoin

Bridged Analogues of Combretastatin A-4 as Potential

Anticancer Agents

Mao Zhang,^†Yu-Ru Liang,^†Huan Li, Ming-Ming Liu,* and Yang Wang*

School of Pharmacy, Fudan University, Shanghai 201203, China

*Corresponding authors. Tel. & Fax: +86-21-51980115. E-mail: lmm@fudan.edu.cn (M. M. Liu),

wangyang@shmu.edu.cn (Y. Wang).

^†These authors contributed equally to this work.

ABSTRACT

A series of novel hydantoin-bridged analogues of combretastatin A-4 (CA-4)

were designed, synthesized and evaluated for antiproliferative activities in vitro and in

vivo. The most potent compound 8d, showed potent cytotoxicity against four human

cancer cell lines with IC₅₀values of 0.186-0.279 µM, and possessed the efficacy of

inhibiting tubulin polymerization, disrupting in vitro vascularization, blocking cell

cycle in G₂/M phase and inducing cell apoptosis. In the nude mice xenograft model,

8d significantly inhibited the tumor growth and showed low toxicity. Further chiral

separation proved (R)-(-)-8d to be the preferential enantiomer with IC₅₀values of

0.081-0.157 µM. These results indicated that the hydantoin derivatives merit further

investigation as potential anticancer agents that inhibit tubulin polymerization.

Keywords: hydantoin; CA-4 analogue; anticancer; tubulin polymerization

1. Introduction

Microtubules are essential components of the eukaryotic cytoskeleton, which are

polymerized by α-/β- tubulin heterodimers. The dynamic equilibrium of the

polymerization and depolymerization is the physiologic basis of the microtubule

functions and plays vital roles in mitosis, maintenance of cell morphology, division,

cellular signal transduction as well as organelle formation and transportation.^1-3Thus,

the interference of the dynamic equilibrium of the microtubule-tubulin system

becomes a valid antimitotic strategy to inhibit the rapidly proliferating tumor cells.

Colchicine (Fig. 1) is the first discovered compound which can destabilize the

tubulin, binds to the interface of α- and β-subunit, resulting in the depolymerization of

α,β-heterodimers.⁴Combretastatin A-4 (CA-4), isolated from the bark of the South

African willow tree Combretum caffrum in 1980s,^5,6disrupts tubulin polymerization

by binding to the colchicine site. It exhibits remarkable in vitro inhibitory activity

against a wide range of human cancer cell lines and vascular disrupting potency.⁷Its

water soluble prodrug, CA-4P is currently under phase III clinical trials for treatment

of various solid tumors.⁸Large amount of structure-activity relationship (SAR)

studies of CA-4 and its analogues have indicated that 3,4,5-trimethoxyphenyl (A ring)

and the cis-orientation of the olefinic bond are crucial for the anticancer activity.⁹To

avoid isomerization to trans-form resulting in the decrease of both anti-tubulin

activity and cytotoxicity, many kinds of cis-restricted CA-4 analogues have been

reported, in which the -C=C- bond was replaced by rigid bridges,¹⁰especially

heterocycles, including pyridine,¹¹pyrrole,^12,13pyrazole,^8,14,15triazoles,^15-18

tetrazole,^15,19imidazole,^14,20furan,²¹oxazole,¹⁴isoxazole,^22,23furazan,²⁴

oxadiazoles,^25,26thiazole,^15,27oxazolone,²⁸β-lactam,^29,30diazepine-2-one³¹and so on.

These researches have made considerable progress in this field.

Figure 1. Colchicine and CA-4 as representative microtubule disrupters, and the design of

hydantoin bridged CA-4 analogues.

Hydantoin, namely imidazolidine-2,4-dione, was first achieved by Baeyer in

1861.³²For a long time, the moiety has shown its pharmaceutical value as

anticonvulsants drugs.³³In recent years, large numbers of biological effects of

hydantoin have been discovered, such as antiproliferative and anti-metastatic

activity,^34-37muscle relaxant,³⁸pesticide,³⁹anti-inflammatory,⁴⁰anti-obesity,^41,42

anti-diabetes,⁴³anti-HIV⁴⁴and many others. The successful clinical applications and

the multiple biological activities enlightened us to merge this privileged scaffold into

CA-4 structure to mimic its cis-double bond. Thus, as shown in Fig. 1, a series of

hydantoin bridged CA-4 analogues were designed and synthesized, and in vitro

anti-proliferative evaluation, mechanism study as well as in vivo assays were carried

out in the present study, which would definitely enrich the scope of CA-4’s structural

modification work.

2. Results and discussion

2.1. Chemistry

As shown in Scheme 1, commercially available substituted hypnones (1a-c) and

arylamines (3a-o) were used as the starting materials. The phenylglyoxal derivatives

45

(2a-c) were obtained by oxidizing the corresponding hypnones (1a-c) by SeO₂in

72-88% yields. On the other side, reactions of arylamines (3a-m) with sodium

cyanate⁴⁶gave aryl ureas (4a-m) in the yields of 75-94%. Reactions of arylamines (3n

and 3o) with CS₂/ Et₃N, sequentially with Boc₂O afforded isothiocyanates,⁴⁷which

were then treated with NH₃·H₂O to yield aryl thioureas (4n and 4o) in 61% and 73%

respectively.

Scheme 1. Synthesis of arylglyoxals (2a-c), arylureas (4a-m) and arylthioureas (4n

and 4o). Reaction conditions: (i) SeO₂(2 eq), 1,4-dioxane, reflux, 3 h, 72-88%; (ii)

NaOCN (1.5 eq), AcOH, rt, 1 h, 75-94%; (iii) (a) CS₂(5 eq), Et₃N (2 eq), EtOH, rt, 1

h; (b) Boc₂O (1.1 eq), DMAP (0.01 eq), 0^oC to rt; (c) NH₃·H₂O (10 eq), acetone, rt, 2

h; for 3 steps, 4n: 61%, 4o: 73%; (iv) TsCl (1.2 eq), NaOH (1.5 eq), TEBA (0.1 eq),

DCM, rt, 5 h; (v) H₂, Pd/C (0.1 eq), EtOH, rt, 5 h.

With 2a-c and 4a-o in hand, hydantoin derivatives (5-7) were synthesized by

condensation reactions under the condition of AcOH / HCl,^41,42as outlined in Scheme

2. The derivatives 8a-g were prepared subsequently from corresponding candidates (6)

by alkylation, reduction or fluoro sulfonylation⁴⁸with satisfactory yields of 77-95%.

O

Ar

H

O

(i)

N

NH₂

+

NH

X

Ar'

O

N

Ar

X

Ar'

2a-c

4a-o

5-7

O

N

NH

N

Ar

5a-b

a, Ar = 4-OMe-Ph-

Ar'

O

S

7a-b

a, Ar' = 4-OMe-Ph-

b, Ar = 3-OH,4-OMe-Ph-

b, Ar' = 3-OH,4-OMe-Ph-

O

NH

N R

N

Ar'

O

6a-p

8a-g

(iv)

a, Ar' = 4-MeO-Ph-

a, Ar' = 3-OSO₂F,4-MeO-Ph-, R = H

b, Ar' = 3-OH,4-MeO-Ph-

(v)

c

b

, Ar' = 3-OH,4-MeO-Ph-, R = Me-

, Ar' = Ph-

d

, Ar' = 3-OH-Ph-

(v)

e, Ar' = 3-F,4-MeO-Ph-

f, Ar' = 3-NO₂,4-MeO-Ph-

g, Ar' = 3-NH₂,4-MeO-Ph-

c, Ar' = 3-F,4-MeO-Ph-, R = Me-

(v),(ii)

(ii)

d, Ar' = 3-NH₂,4-MeO-Ph-, R = Me-

h

, Ar' = 3-NO₂,4-Me-Ph-

i

e

, Ar' = 3-NH₂,4-Me-Ph-

, Ar' = 3-NH₂,4-MeO-Ph-, R = Et-

j, Ar' = 3,4-Me-Ph-

k, Ar' = 3,4,5-MeO-Ph-

l, Ar' = 2-MeO-pyridine-5-

f, Ar' = 3-NH₂,4-MeO-Ph-, R = HOEt-

(v),

(ii)

m

g

h

, Ar' = quinoline-3-

, Ar' = 3-NH₂,4-Me-Ph-, R = Me-

, Ar' = 3,4-Me-Ph-, R = Me-

n

o

, Ar' = quinoline-6-

(v)

, Ar' = ( -Ts-indole)-5-

N

(iii)

p, Ar' = 1 -indole-5-

H

Scheme 2. Synthesis of imidazolidine-2,4-dione and 2-thioxoimidazolidin-4-one

o

derivatives (5-8). Reaction conditions: (i) AcOH / conc. HCl (30:1), 50 C, 1 h, 72 -

89%; (ii) Pd/C (0.1 eq), H₂, EtOH, rt, 5 h; (iii) (a) KOH (2 eq), MeOH, reflux; (b)

HCl (1 M) to pH = 7; 77%; (iv) SO₂F₂(g), DCM, Et₃N (10 eq), rt, 0.5 h, 91%; (v) RX

or Me₂SO₄(1.0-1.2 eq), K₂CO₃(1.5 eq), acetone, reflux, 3 h.

2.2. The In Vitro Antiproliferative Activity of Compounds 5-8

The antiproliferative activity of the compounds 5-8 was screened against four

human cancer cell lines, HeLa, A2780, HCT-116 and MDA-MB-231 with CA-4 as

positive control. As summarized in Table 1, some of the target compounds displayed

moderate to potent inhibition on cell growth. On that basis, SAR rules can be

concluded as follows: i) The connection of A ring (3,4,5-OMe-Ph-) to the

hydantoin-5-position (6 series) is beneficial to the activity, whereas to the

hydantoin-1-position (5 series) loses potency. ii) The bioisosteric replacement of

hydantoin to the 2-thioxoimidazolidin-4-one (7 series) results in the loss of activity. iii)

The -OMe (especially) or -Me group on the 4-position of B ring is crucial for the

activity. iv) The 3,4-disubstitution on B ring is beneficial for the activity, but

substitution of -NO₂(6f and 6h) and -OSO₂F (8a) on the 3-position of B ring decrease

the potency. v) Heterocycles as B ring (6l-p) result in the loss of potency. vi) The

substitution of -Me on the 3-N of hydantoin ring (8b-d, 8g and 8h) promotes the

potency, whereas groups bulkier than -Me (8e and 8f) make a decrease. Among the

target compounds, 8d possesses the most potent antiproliferative activity with IC₅₀

value ranging from 0.186 to 0.279 µM, which attracts our interest to explore its

further pharmacological properties.

Table 1. The in vitro antiproliferative activity of compounds 5-8 against four human cancer cell

lines with CA-4 reference.

IC₅₀(µM)

Compound

HeLa

＞ 50

2.818

0.665

＞ 50

0.771

4.744

0.584

5.448

0.933

0.620

＞ 50

6.761

0.429

0.516

0.251

A2780

＞ 50

2.137

0.815

＞ 50

0.745

9.705

0.283

2.891

1.054

0.627

＞ 50

8.406

＞ 50

4.523

0.353

0.555

0.186

HCT-116

ND

MDA-MB-231

ND

5a

5b

6a

6b

6c

6d

6e

6f

ND

0.945

ND

0.768

ND

0.630

ND

0.727

ND

6g

6h

6i

0.232

ND

0.311

ND

0.988

0.475

ND

1.166

0.835

ND

6j

6k

6l

ND

6m

6n

6o

6p

7a

7b

8a

8b

8c

8d

ND

0.386

0.527

0.265

0.301

0.452

0.279

3.305

3.599

0.631

0.363

0.007

1.907

6.615

0.679

0.533

0.005

ND

8e

8f

0.681

0.307

0.006

1.186

0.493

0.002

8g

8h

CA-4

ND: not determined.

To evaluate the cytotoxic selectivity of 8d between cancer and normal cells, we

tested the cytotoxicity of 8d in a normal ovarian epithelial cell line HOSE with

paclitaxel as positive control. As shown in Fig. 2, no more than 33% of HOSE cell

growth was inhibited by 8d at both 10 and 50 µM, whereas 51% and 94% inhibition

was observed when the cells were treated by paclitaxel at the same concentrations.

This result showed the good selectivity of 8d between tumor cells and normal cells.

150

paclitaxel

8d

**

100

*

50

0

2

10

50

Concentration (µM)

Figure 2. Cell growth inhibition of 8d on HOSE.

In order to further confirm the cytotoxic effects of 8d, colony formation assay

was also conducted. As shown in Fig. 3, the colonies formed by A2780 and

MBA-MD-231 cells were reduced dose-dependently due to the exposure to 8d. At a

dose of 800 nM, 8d almost completely abolished the growth of MDA-MB-231 cells.

Figure 3. Compound 8d dose-dependently suppressed cell colony formation of A2780 and

MDA-MB-231 cells.

2.3. Inhibition of Tubulin Polymerization

To confirm whether the anti-proliferative activities were related to the

destabilization of microtubule, the representative compound 8d was evaluated for

inhibitory effects on tubulin assembly by using isolated pig brain tubulin. As shown in

Fig. 4 and Table 2, 8d suppressed in vitro tubulin polymerization in a dose-dependent

manner, with IC₅₀value of 16.2 µM.

Figure 4. Effects of 8d on tubulin polymerization in vitro.

Table 2. The IC₅₀values of 8d, CA-4 and colchicine on inhibition of tubulin polymerization in

vitro.

Compound

CA-4

6.6

Colchicine

8.6

8d

IC₅₀(µM)

16.2

To validate the biological target further, we investigated the effects of the

compound 8d on the cell cycle. Hela cells were treated with increasing concentrations

of 8d for 24 h, followed by propidium iodide staining and flow cytometry analysis. As

shown in Fig. 5A and 5B, 8d induced cell cycle arrest in G₂/M phase in a

dose-dependent manner. After the exposure to 8d at 2 µM for 12 h, 91.4% cells were

distributed in G₂/M phase, compared to 17.6% of DMSO treatment. Moreover,

immunoblotting was employed to analyze the expression of cell cycle regulators

effected by 8d. As shown in Fig. 5C, the treatment of 8d induced the expression of

cyclin B1 and the phosphorylation of histone H3, which in protein level confirmed

that 8d could induce cellular mitotic arrest.

Figure 5. (A) Cell cycle distribution of Hela cells after treatment with indicated concentrations of

8d and (B) Statistic histogram shows that 8d caused the G₂/M phase arrest and (C) Western blot

analysis of cycle-related proteins.

2.4. Inducement of Cellular Apoptosis

The Annexin V-FITC / PI staining was employed to detect the apoptosis of HeLa

cells after 8d treatment. As shown in Fig. 6A and 6B, the total proportions of

apoptotic cells increased dose-dependently, being 70.5% at 2 µM of 8d. Furthermore,

immunoblotting analysis (Fig. 6C) showed that 8d up-regulated the tumor suppressor

p53 and the apoptotic marker cleaved poly (ADP-ribose) polymerase-1 (PARP-1) in a

dose-dependent manner. These results indicated that 8d could induce cell apoptosis.

Figure 6. (A) Representative flow cytometry profiles show 8d could induce apoptosis of Hela

cells dose-dependently and (B) Statistic histogram shows that 8d induced cell apoptosis and (C)

Western blot analysis of apoptosis-related proteins.

2.5. Anti-angiogenic Effects In Vitro

Tumor vasculature plays a vital role in the growth of solid tumor, so targeting

angiogenesis has been one of the strategies for increasing the efficacy of cancer

therapy. Since many tubulin binding agents, including CA-4, have been reported to

interfere with vascular formation,⁴⁹8d was also evaluated for its effects against

angiogenesis on human umbilical vein endothelial cells (HUVECs) in vitro. As shown

in Fig. 7, compound 8d effectively altered the capillary-like structure at 1 µM after 12

h incubation, and almost completely disrupted the angiogenesis at 2 µM. By contrast,

HUVECs treated with bare DMSO produced an intact cellular capillary network

structure.

Figure 7. Compound 8d disrupted capillary-like tubule formed by HUVECs.

2.6. In Vivo Anti-tumor Efficacy

Furthermore, the nude mice xenograft tumor models were established to explore

the in vivo anti-tumor efficacy of 8d. As presented in Table 3, at a dose of 20 mg/kg,

8d suppressed the tumor growth significantly with inhibition ratio being 46.9%

compared to vehicle. Meanwhile, no observable body weight loss showed (22.35 g in

vehicle and 22.05 g in 8d treated group) at the end of the observation, which indicated

the safety of compound 8d at the therapeutic dosage. All of the above data proved that

compound 8d exhibited potent anti-tumor abilities in vivo.

Table 3. Antitumor effects of compound 8d against tumor growth on xenograft mice

Body weight (g)

Animal

Group

Tumor weight (g)

Inhibition rate

46.9%

number

Beginning

End

vehicle

8

21.74 0.63

21.62 0.93

22.35 0.74

22.05 0.71

2.62 0.28

1.23 0.19

8d

2.7. Enantiomeric Separation and Antitumor Evaluation

In order to make clear the impact of stereochemistry on the activity, 8d was

submitted to chiral separation by HPLC yielding (-)-8d (ee 96%) and (+)-8d (ee 98%).

In order to determine their absolute configurations, the electronic circular dichroism

(ECD) spectra of (-)-8d and (+)-8d were measured respectively to compare with the

calculated ECD spectrum of the (R)-configuration, which was computed by quantum

chemistry software Gaussian 09. As shown in Fig. 8, (-)-8d curve (red) better matches

the (R)-ECD (blue), whereas (+)-8d (green) poses almost the opposite trend. Hence,

the (-)-8d is predicted to be the (R)-configuration. As shown in Table 4, further

antiproliferative evaluation proved the (R)-(-)-8d to be the preferential enantiomer

(IC₅₀: 0.081-0.157 µM), which is 2-5 folds more potent than (S)-(+)-8d (IC₅₀:

0.305-0.582 µM).

Table 4. Enantiomers of 8d and their antiproliferative activities.

IC₅₀(µM)

20 a

Compd.

[α]_D

ee%

HeLa

0.081

A2780

0.097

HCT-116

MDA-MB-231

0.125

-41.8

96

0.157

(R)-(-)-8d

+41.7

98

0.399

0.251

0.305

0.186

0.355

0.265

0.582

0.279

(S)-(+)-8d

8d

^aThe specific rotation data recorded: 20 ^oC, 1.0 mg/mL in MeCN.

Figure 8. Comparison of the experimental ECD spectra of two enantiomers with the calculated

spectrum of the (R)-configuration. The (-)-8d ECD curve (red) better matches the calculated R-

curve (blue), whereas the (+)-8d curve (green) poses the opposite trend.

3. Conclusion

In the present work, 28 hydantoin bridged analogues of CA-4 were designed,

synthesized and evaluated for antiproliferative activities. SAR analysis indicated the

following factors are beneficial for the potency: the substitution of Me group on the

3-N of hydantoin ring, the linkage of A ring (3,4,5-OMe-Ph-) to the 5-position and B

ring (preferably 3,4-disubstituted and with OMe on 4-position) to the 1-position of

hydantoin. Among them, compound 8d showed the highest potency (IC₅₀:

0.186-0.279 µM against four tumor cells). Mechanism study proved that 8d possessed

the efficacy of inhibiting tubulin polymerization, disrupting tumor vascularization,

blocking cell cycle in G₂/M and inducing cell apoptosis. We also established the

xenograft mice model for in vivo antitumor assay and found that 8d possessed

significant therapeutic efficacy with low toxicity. Besides, chiral separation has

proven that the (R)-(-)-8d is the preferential enantiomer (0.081-0.157 µM), which is

2-5 folds more potent than (S)-(+)-8d. These results are definitely constructive for

further study of this type of anticancer agents.

4. Experimental

4.1. Chemistry general

All reagents were purchased from suppliers and used without further purification.

Reactions were monitored by thin layer chromatography (TLC) by using silica gel (60,

GF 254) coated glass slides. Column chromatography was performed with standard

silica gel 60 (300−400 mesh). Detections were conducted under UV (254 nm).

Melting points were measured on an SGW X-4 apparatus which was uncorrected.

Nuclear magnetic resonance spectra were recorded by a Varian 400 MHz or a Bruker

600 MHz NMR spectrometer at 303 K. LRMS and HRMS data were collected on an

Agilent 6120 quadrupole LC/MS system and an Agilent 6224 TOF LC/MS system

respectively.

4.2. General synthesis of arylglyoxal analogues (2a-c)

The procedure was performed adopting literature⁴⁵with some modifications. A

mixture of hypnone derivative (1, 30 mmol) and SeO₂(3.3 g, 30 mmol) in

1,4-dioxane (50 mL) was heated under reflux for 1 h, then another 1 eq of SeO₂(3.3 g)

was added to the solution. The solution was refluxed for 2 more hours before it was

concentrated. The residue was purified by silica gel column chromatography (Eluent:

PE-EtOAc, 1:1) to give: 3,4,5-Trimethoxyphenylglyoxal (2a), white powder, yield:

72%, ESI-MS (m/z): 225.1 (M + H⁺); 4-Methoxyphenylglyoxal (2b), pale yellow

powder,

yield:

88%,

ESI-MS

(m/z):

165.1

(M

+

H⁺);

3-Hydroxy-4-methoxyphenylglyoxal (2c), yellowish syrup, yield: 75%, ESI-MS (m/z):

181.1 (M + H⁺).

4.3. General synthesis of arylurea analogues (4a-m)

The procedure was performed adopting literature⁴⁶. To a solution of ArNH₂(3, 5

mmol, dissolved in 10 mL acetic acid), was added aquatic solution of NaOCN (7.5

mmol, 7.5 mL). The mixture was allowed to be stirred at room temperature for 1 h

and then concentrated under reduced pressure. Afterwards, saturated K₂CO₃solution

was added to adjust the pH to 7-8. A large scale of precipitate was therefore formed,

which was then filtered, washed with water for several times and then recrystallized

from MeOH - DCM.

4.3.1. 4-Methoxyphenylurea (4a)

White crystal. Yield: 84%. Mp. 163-164 °C (lit.⁴⁶: 165-166 °C). ESI-MS (m/z):

167.1 (M + H⁺).

4.3.2. 3-Hydroxy-4-methoxyphenylurea (4b)

1

White crystal. Yield: 92%. Mp. 169-171 °C. ESI-MS (m/z): 183.1 (M + H⁺). H

NMR (400 MHz, DMSO-d₆) δ 8.85 (s, 1H, -OH), 8.18 (s, 1H, -NH-), 6.97 (s, 1H,

Ph-H), 6.72 - 6.64 (m, 2H, Ph-H × 2), 5.64 (s, 2H, -NH₂), 3.66 (s, 3H, -OCH₃).

4.3.3. Phenylurea (4c)

White crystal. Yield: 86%. Mp. 145-147 °C (lit.⁴⁶: 145-147 °C). ESI-MS (m/z):

137.1 (M + H⁺). ¹H NMR (400 MHz, DMSO-d₆) δ 8.49 (s, 1H, -NH-), 7.39 (d, J = 7.4

Hz, 2H, Ph-H × 2), 7.25 – 7.17 (m, 2H, Ph-H × 2), 6.89 (d, J = 7.2 Hz, 1H, Ph-H),

5.82 (s, 2H, -NH₂). The ¹H NMR data are consistent with literature⁴⁶.

4.3.4. 3-Hydroxyphenylurea (4d)

Off-white powder. Yield: 92%. Mp. 175-177 °C. ESI-MS (m/z): 153.1 (M + H⁺).

¹H NMR (400 MHz, DMSO-d₆) δ 9.16 (s, 1H, -OH), 8.37 (s, 1H, -NH-), 6.98 (s, 1H,

Ph-H), 6.93 (t, J = 7.7 Hz, 1H, Ph-H), 6.65 (d, J = 7.5 Hz, 1H, Ph-H), 6.25 (d, J = 8.0

Hz, 1H, Ph-H), 5.74 (s, 2H, -NH₂).

4.3.5. 3-Fluoro-4-methoxyphenylurea (4e)

Off-white powder. Yield: 86%. Mp. 168-171 °C. ESI-MS (m/z): 185.1 (M + H⁺).

¹H NMR (400 MHz, DMSO-d₆) δ 8.53 (s, 1H, -NH-), 7.44 (d, J = 14.1 Hz, 1H, Ph-H),

7.08–6.89 (m, 2H, Ph-H × 2), 5.83 (s, 2H, -NH₂), 3.76 (s, 3H, -OCH₃). ¹³C NMR (150

MHz, CDCl₃) δ 155.9, 151.1 (J_C-F= 240 Hz), 141.2 (J_C-F= 11 Hz), 134.3 (J_C-F= 10

Hz), 114.3, 113.3, 106.3 (J_C-F= 23 Hz), 56.2. ESI-HRMS (m/z): calcd for

C₈H₁₀FN₂O₂(M + H⁺), 185.0721; found, 185.0720.

4.3.6. 4-Methoxy-3-nitrophenylurea (4f)

Light yellow powder. Yield: 85%. Mp. 164-167 °C. ESI-MS (m/z): 212.0 (M +

1

H⁺). H NMR (400 MHz, DMSO-d₆) δ 9.36 (s, 1H, -NH-), 8.08 (s, 1H, Ph-H), 7.50 (d,

J = 9.1 Hz, 1H, Ph-H), 7.20 (d, J = 9.0 Hz, 1H, Ph-H), 6.15 (s, 2H, -NH₂), 3.81 (s, 3H,

-OCH₃). ¹³C NMR (150 MHz, CDCl₃) δ 156.1, 146.2, 138.6, 134.1, 123.6, 114.6,

113.4, 56.6. ESI-HRMS (m/z): calcd for C₈H₁₀N₃O₄(M + H⁺), 212.0666; found,

212.0667.

4.3.7. 4-Methyl-3-nitrophenylurea (4g)

Yellow powder. Yield: 90%. Mp. 138-141 °C. ESI-MS (m/z): 196.1 (M + H⁺). ¹H

NMR (400 MHz, DMSO-d₆) δ 8.94 (s, 1H, -NH-), 8.25 (s, 1H, Ph-H), 7.49 (d, J = 7.5

Hz, 1H, Ph-H), 7.33 (d, J = 7.5 Hz, 1H, Ph-H), 6.03 (s, 2H, -NH₂), 2.42 (s, 3H,

-OCH₃). ¹³C NMR (150 MHz, CDCl₃) δ 155.6, 148.6, 139.5, 132.6, 124.4, 122.2,

112.5, 18.8. ESI-HRMS (m/z): calcd for C₈H₁₀N₃O₃(M + H⁺), 196.0717; found,

196.0718.

4.3.8. 3,4-Dimethylphenylurea (4h)

Off-white powder. Yield: 90%. Mp. 152-154 °C. ESI-MS (m/z): 165.1 (M + H⁺).

¹H NMR (400 MHz, DMSO-d₆) δ 8.30 (s, 1H, -NH-), 7.15 (s, 1H, Ph-H), 7.10 (d, J =

7.4 Hz, 1H, Ph-H), 6.95 (d, J = 7.2 Hz, 1H, Ph-H), 5.74 (s, 2H, -NH₂), 2.15 (s, 3H,

-CH₃), 2.12 (s, 3H, -CH₃).

4.3.9. 3,4,5-Trimethoxyphenylurea (4i)

Yellowish powder. Yield: 82%. Mp. 164-166 °C. ESI-MS (m/z): 227.1 (M + H⁺).

¹H NMR (400 MHz, DMSO-d₆) δ 8.45 (s, 1H, -NH-), 6.74 (s, 2H, Ph-H × 2), 5.78 (s,

2H, -NH₂), 3.71 (s, 6H, -OCH₃× 2), 3.58 (s, 3H, -OCH₃).

4.3.10. 1-(6-Methoxypyridin-3-yl)urea (4j)

Pale purple powder. Yield: 78%. Mp. 182-184 °C. ESI-MS (m/z): 168.1 (M +

1

H⁺). H NMR (400 MHz, DMSO-d₆) δ 8.44 (s, 1H, -NH-), 8.10 (s, 1H, pyridine-H),

7.78 (d, J = 8.8 Hz, 1H, pyridine-H), 6.72 (d, J = 8.7 Hz, 1H, pyridine-H), 5.86 (s, 2H,

-NH₂), 3.78 (s, 3H, -OCH₃).

4.3.11. 1-(Quinolin-3-yl)urea (4k)

Pale brown powder. Yield: 94%. Mp. 201-204 °C. ESI-MS (m/z): 188.1 (M +

1

H⁺). H NMR (400 MHz, DMSO-d₆) δ 9.03 (s, 1H, -NH-), 8.75 (d, J = 12.1 Hz, 1H,

quinoline-H), 8.47 (d, J = 12.1 Hz, 1H, quinoline-H), 7.87 (s, 2H, quinoline-H × 2),

7.54 (s, 2H, quinoline-H × 2), 6.12 (s, 2H, -NH₂).

4.3.12. 1-(Quinolin-6-yl)urea (4l)

Off-white powder. Yield: 88%. Mp. 244-246 °C. ESI-MS (m/z): 188.1 (M + H⁺).

¹H NMR (400 MHz, DMSO-d₆) δ 8.89 (s, 1H, -NH-), 8.69 (s, 1H, quinoline-H), 8.19

(d, J = 8.0 Hz, 1H, quinoline-H), 8.10 (s, 1H, quinoline-H), 7.88 (d, J = 9.0 Hz, 1H,

quinoline-H), 7.64 (d, J = 7.4 Hz, 1H, quinoline-H), 7.42 (br.s, 1H, quinoline-H), 6.01

(s, 2H, -NH₂). The ¹H NMR data are consistent with literature⁵⁰.

4.3.13. 1-(1-Tosyl-1H-indol-5-yl)urea (4m)

First of all, the Ts- protected amine was prepared by employing literature⁵¹.

Briefly, to a DCM (30 mL) solution of 5-nitro-1H-indole (1.0 g, 6.2 mmol), were

added TsCl (1.4 g, 7.4 mmol), NaOH (372 mg, 9.3 mmol) and

benzyltriethylaminium·chloride (TEBA, 141 mg, 0.6 mmol). The mixture was

allowed to be stirred at room temperature for 3 h, then portioned between water (100

mL) and DCM (50 mL × 5). The combined organic layer was dried over anhydrous

Na₂SO₄and concentrated under reduced pressure. The remaining was purified by

silica gel flash chromatography (eluent: PE-EtOAc, 8:1) to give the

5-nitro-1-tosyl-1H-indole, which was then dissolved in EtOH (50 mL). To the solution,

Pd/C (10%, 53 mg, 0.05 mmol) was added. Then the system was filled with H₂by gas

exchange and capped with a balloon filled with H₂. The mixture was stirred at room

temperature for 5 h, then filtered to give a clear solution. After evaporation under

reduced pressure, the 1-tosyl-1H-indol-5-amine (3m) was thus achieved.

Afterwards, by employing the general procedure, 3m was treated with NaOCN

(605 mg, 9.3 mmol) to give the arylurea 4m as off-white powder. Yield: 55% (3 steps).

1

Mp. 198-200 °C. ESI-MS (m/z): 330.0 (M + H⁺). H NMR (400 MHz, DMSO-d₆) δ

8.54 (s, 1H, -NH-), 7.83 - 7.75 (m, 3H, Ph-H × 2, indole-H), 7.73 (s, 1H, indole-H),

7.68 (s, 1H, indole-H), 7.36 (d, J = 7.9 Hz, 2H, Ph-H × 2), 7.21 (d, J = 9.0 Hz, 1H,

indole-H), 6.75 (s, 1H, indole-H), 5.81 (s, 2H, -NH₂), 2.31 (s, 3H, -CH₃). ¹³C NMR

(150 MHz, CDCl₃) δ 155.9, 145.1, 136.5, 134.0, 130.9, 130.0, 129.2, 127.1, 126.4,

116.2, 113.0, 109.7, 20.8. ESI-HRMS (m/z): calcd for C₁₆H₁₆N₃O₃S (M + H⁺),

330.0907; found, 330.0905.

4.4. General synthesis of Arylthiourea analogues (4n, 4o)

The isothiocyanates were prepared according to literature⁴⁷. To a stirred EtOH

(20 mL) solution of arylamine (5 mmol) were added CS₂(1.5 mL, 25 mmol) and Et₃N

(1.4 mL, 10 mmol). After the precipitation (dithiocarbamate) formed, the mixture was

stirred for another 30 min and then cooled on an ice bath. Next, Boc₂O (1.2 g, 5.5

mmol) and DMAP (6 mg, 0.05 mmol) were added to the mixture. The reaction

continued for 2 h with bubbles released and precipitation disappeared gradually. Then

it was concentrated under reduced pressure to afford crude isothiocyanate, which was

sequentially treated with NH₃·H₂O (7.7 mL, 25 % aq., 50 mmol) in acetone. After

being stirred at room temperature for approximately 1 h, massive precipitation was

formed, which was then filtered and washed by acetone for several times. The aryl

thioureas were thus achieved and used in the next step without further purification.

4-Methoxyphenylthiourea (4n), white crystal, yield 61%, ESI-MS (m/z): 183.1 (M +

H⁺); 3-Hydroxy-4-methoxyphenylthiourea (4o), white crystal, yield 73%, ESI-MS

(m/z): 227.1 (M + H⁺).

4.5. General Procedure for the Synthesis of the Diaryl Imidazolidine-2,4-dione

Analogues (5-7)

The target compounds were synthesized employing literatures^41,42. Briefly, to a

stirred acetic acid (6 mL) solution of the 2-oxo-2-arylacetaldehyde (2, 1 mmol) and

arylurea or arylthiourea (4, 1 mmol), was added conc. HCl (0.2 mL). The mixture was

o

heated at 50 C for 1 h before it was concentrated. The residue was purified by

recrystallization from MeOH/DCM or by silica gel column chromatography.

4.5.1. 5-(4-Methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)imidazolidine-2,4-dione (5a)

o

Eluent: PE-EtOAc, 1:1. Pale brown crystal. Yield: 89%. Mp: 161-163 C.

ESI-MS (m/z): 373.0 (M + H⁺), 395.0 (M + Na⁺). ¹H NMR (400 MHz, CDCl₃) δ 9.08

(s, 1H, -NH-), 7.26 (d, J = 8.1 Hz, 2H, Ph-H × 2), 6.90 (d, J = 7.9 Hz, 2H, Ph-H × 2),

6.65 (s, 2H, Ph-H × 2), 5.37 (s, 1H, -*CH-), 3.78 (s, 6H, -OCH₃× 2), 3.72 (s, 6H,

-OCH₃× 2). ¹³C NMR (150 MHz, CDCl₃) δ 170.2, 159.7, 153.8, 152.6, 134.9, 131.4,

127.6, 124.1, 114.2, 98.8, 65.4, 60.2, 55.5, 54.7. ESI-HRMS (m/z): calcd for

C₁₉H₂₁N₂O₆(M + H⁺), 373.1394; found, 373.1393.

4.5.2.

5-(3-Hydroxy-4-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)imidazolidine-2,4-dione

(5b)

o

Eluent: DCM-MeOH, 10:1. White powder. Yield: 78%. Mp: 243-246 C.

ESI-MS (m/z): 389.0 (M + H⁺), 410.9 (M + Na⁺). H NMR (400 MHz, DMSO-d₆) δ

1

11.35 (s, 1H, -NH-), 9.15 (s, 1H, -OH), 6.88 (d, J = 8.3 Hz, 1H, Ph-H), 6.82 (s, 2H,

Ph-H × 2), 6.80 (dd, J = 8.3, 2.1 Hz, 1H, Ph-H), 6.74 (d, J = 2.1 Hz, 1H, Ph-H), 5.81

(s, 1H, -*CH-), 3.72 (s, 3H, -OCH₃), 3.67 (s, 6H, -OCH₃× 2), 3.57 (s, 3H, -OCH₃).

¹³C NMR (150 MHz, DMSO-d₆) δ 171.7, 154.7, 152.5, 147.9, 146.7, 134.1, 132.5,

126.5, 118.4, 113.9, 112.2, 99.2, 64.3, 59.9, 55.8, 55.5. ESI-HRMS (m/z): calcd for

C₁₉H₂₁N₂O₇(M + H⁺), 389.1343; found, 389.1342.

4.5.3. 1-(4-Methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)imidazolidine-2,4-dione (6a)

o

Eluent: PE-EtOAc, 3:1. White powder. Yield: 79%. Mp: 164-166 C. ESI-MS

(m/z): 373.0 (M + H⁺). ¹H NMR (400 MHz, CDCl₃) δ 9.26 (s, 1H, -NH-), 7.29 (d, J =

8.6 Hz, 2H, Ph-H × 2), 6.83 (d, J = 8.6 Hz, 2H, Ph-H × 2), 6.50 (s, 2H, Ph-H × 2),

13

5.33 (s, 1H, -*CH-), 3.80 (br.s, 9H, -OCH₃× 3), 3.75 (s, 3H, -OCH₃). C NMR (150

MHz, CDCl₃) δ 170.2, 156.7, 154.2, 153.2, 138.0, 128.2, 127.4, 122.9, 113.8, 103.5,

66.1, 60.2, 55.6, 54.8. ESI-HRMS (m/z): calcd for C₁₉H₂₁N₂O₆(M + H⁺), 373.1394;

found, 373.1392.

4.5.4. 1-(3-Hydroxy-4-methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)imidazolidine-2,4-

dione (6b)

o

Eluent: DCM-MeOH, 20:1. White powder. Yield: 72%. Mp: 173-175 C.

ESI-MS (m/z): 389.0 (M + H⁺), 411.0 (M + Na⁺). ¹H NMR (400 MHz, Acetone-d₆) δ

9.97 (s, 1H, -NH-), 7.70 (s, 1H, -OH), 7.17 (d, J = 2.4 Hz, 1H, Ph-H), 6.93 (dd, J =

8.8, 2.5 Hz, 1H, Ph-H), 6.83 (d, J = 8.7 Hz, 1H, Ph-H), 6.75 (s, 2H, Ph-H × 2), 5.68 (s,

1H, -*CH-), 3.79 (s, 6H, -OCH₃× 2), 3.77 (s, 3H, -OCH₃), 3.68 (s, 3H, -OCH₃). ¹³C

NMR (150 MHz, Acetone-d₆) δ 170.0, 153.7, 153.3, 145.9, 144.1, 137.9, 130.0, 129.2,

112.3, 110.8, 109.1, 104.1, 65.2, 58.9, 55.0, 54.8. ESI-HRMS (m/z): calcd for

C₁₉H₂₁N₂O₇(M + H⁺), 389.1343; found, 389.1344.

4.5.5. 1-Phenyl-5-(3,4,5-trimethoxyphenyl)imidazolidine-2,4-dione (6c)

o

Eluent: PE-EtOAc, 3:1. White powder. Yield: 88%. Mp: 205-207 C. ESI-MS

(m/z): 343.0 (M + H⁺). ¹H NMR (400 MHz, CDCl₃) δ 9.02 (s, 1H, -NH-), 7.44 (d, J =

7.9 Hz, 2H, Ph-H × 2), 7.31 (t, J = 8.0 Hz, 2H, Ph-H × 2), 7.14 (t, J = 7.4 Hz, 1H,

Ph-H), 6.52 (s, 2H, Ph-H × 2), 5.42 (s, 1H, -*CH-), 3.81 (s, 3H, -OCH₃), 3.79 (s, 6H,

-OCH₃× 2). ¹³C NMR (150 MHz, CDCl₃) δ 170.4, 154.4, 153.9, 138.5, 136.1, 129.2,

127.8, 125.4, 120.9, 103.8, 65.9, 60.8, 56.2. ESI-HRMS (m/z): calcd for C₁₈H₁₉N₂O₅

(M + H⁺), 343.1288; found, 343.1289.

4.5.6. 1-(3-Hydroxyphenyl)-5-(3,4,5-trimethoxyphenyl)imidazolidine-2,4-dione (6d)

o

Eluent: DCM-MeOH, 10:1. White powder. Yield: 80%. Mp: 196-198 C.

ESI-MS (m/z): 359.0 (M + H⁺). ¹H NMR (400 MHz, DMSO-d₆) δ 11.32 (s, 1H, -NH-),

9.50 (s, 1H, -OH), 7.06 (t, J = 8.2 Hz, 1H, Ph-H), 7.01 (s, 1H, Ph-H), 6.86 (d, J = 7.5

Hz, 1H, Ph-H), 6.61 (s, 2H, Ph-H × 2), 6.48 (d, J = 8.0 Hz, 1H, Ph-H), 5.76 (s, 1H,

-*CH-), 3.72 (s, 6H, -OCH₃× 2), 3.62 (s, 3H, -OCH₃). ¹³C NMR (150 MHz,

DMSO-d₆) δ 171.16, 157.36, 154.54, 153.00, 137.55, 137.28, 129.51, 129.20, 111.54,

111.27, 108.39, 104.25, 64.56, 59.75, 55.80. ESI-HRMS (m/z): calcd for C₁₈H₂₂N₃O₆

+

(M + NH₄), 376.1503; found, 376.1502.

4.5.7.

1-(3-Fluoro-4-methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)imidazolidine-2,4-dione

(6e)

o

Eluent: DCM-MeOH, 30:1. White powder. Yield: 85%. Mp: 128-130 C.

ESI-MS (m/z): 391.0 (M + H⁺). H NMR (400 MHz, Acetone-d₆) δ 10.03 (s, 1H,

1

-NH-), 7.56 (dd, J = 13.5, 2.3 Hz, 1H, Ph-H), 7.20 (dt, J = 8.9, 1.2 Hz, 1H, Ph-H),

7.03 (t, J = 9.2 Hz, 1H, Ph-H), 6.78 (s, 2H, Ph-H × 2), 5.73 (s, 1H, -*CH-), 3.82 (s,

3H, -OCH₃), 3.80 (s, 6H, -OCH₃× 2), 3.69 (s, 3H, -OCH₃). ¹³C NMR (150 MHz,

Acetone-d₆) δ 169.7, 153.7, 153.3, 150.9 (J_C-F= 240 Hz), 143.9 (J_C-F= 10 Hz), 137.9,

129.7 (J_C-F= 9 Hz), 128.8, 116.4, 112.8, 109.4 (J_C-F= 23 Hz), 104.1, 64.9, 58.9, 55.0.

ESI-HRMS (m/z): calcd for C₁₉H₂₀FN₂O₆(M + H⁺), 391.1300; found, 391.1300.

4.5.8.

1-(4-Methoxy-3-nitrophenyl)-5-(3,4,5-trimethoxyphenyl)imidazolidine-2,4-

dione (6f)

o

Eluent: DCM-MeOH, 30:1. Yellowish powder. Yield: 86%. Mp: 198-201 C.

1

ESI-MS (m/z): 418.0 (M + H⁺). H NMR (400 MHz, CDCl₃) δ 9.21 (s, 1H, -NH-),

8.03 (d, J = 2.7 Hz, 1H, Ph-H), 7.55 (dd, J = 9.2, 2.6 Hz, 1H, Ph-H), 6.99 (d, J = 9.2

Hz, 1H, Ph-H), 6.51 (s, 2H, Ph-H × 2), 5.40 (s, 1H, -*CH-), 3.90 (s, 3H, -OCH₃), 3.80

(s, 3H, -OCH₃), 3.79 (s, 6H, -OCH₃× 2). ¹³C NMR (150 MHz, DMSO-d₆) δ 169.5,

153.9, 153.5, 149.7, 138.5, 138.3, 128.0, 126.4, 126.3, 118.0, 113.6, 103.6, 65.6, 60.2,

+

56.1, 55.6. ESI-HRMS (m/z): calcd for C₁₉H₂₃N₄O₈(M + NH₄), 435.1510; found,

435.1508.

4.5.9.

1-(3-Amino-4-methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)imidazolidine-2,4-dione

(6g)

6f (209 mg, 0.5 mmol), Pd/C (10%, 53 mg, 0.05 mmol) and EtOH (10 mL) were

charged into a round-bottom bottle, which was filled with H₂by gas exchange and

capped with a balloon filled with H₂. The solution was allowed to be stirred at room

temperature for 5 h. Then the mixture was filtered to give a clear solution, which was

concentrated under reduced pressure. The resulting residue was purified by silica gel

column chromatography with DCM-MeOH (10:1) as eluent to give 6g as white

o

1

powder. Yield: 85%. Mp: 208-210 C. ESI-MS (m/z): 388.0 (M + H⁺). H NMR (400

MHz, DMSO-d₆) δ 11.17 (s, 1H, -NH-), 6.86 (d, J = 2.0 Hz, 1H, Ph-H), 6.67 (d, J =

8.7 Hz, 1H, Ph-H), 6.57 (s, 2H, Ph-H × 2), 6.50 (dd, J = 8.6, 2.0 Hz, 1H, Ph-H), 5.65

(s, 1H, -*CH-), 4.79 (s, 2H, -NH₂), 3.73 (s, 6H, -OCH₃× 2), 3.68 (s, 3H, -OCH₃),

3.61 (s, 3H, -OCH₃). ¹³C NMR (150 MHz, DMSO-d₆) δ 176.3, 159.4, 157.7, 148.3,

142.3, 141.9, 134.5, 134.3, 114.8, 114.4, 113.4, 109.0, 70.0, 64.5, 60.5, 59.9.

ESI-HRMS (m/z): calcd for C₁₉H₂₂N₃O₆(M + H⁺), 388.1503; found, 388.1501.

4.5.10.1-(4-Methyl-3-nitrophenyl)-5-(3,4,5-trimethoxyphenyl)imidazolidine-2,4-dione

(6h)

o

Eluent: DCM-MeOH, 30:1. White powder. Yield: 84%. Mp: 203-205 C.

ESI-MS (m/z): 402.1 (M + H⁺). ¹H NMR (400 MHz, DMSO-d₆) δ 11.55 (s, 1H, -NH-),

8.31 (s, 1H, Ph-H), 7.63 (d, J = 8.4 Hz, 1H, Ph-H), 7.42 (d, J = 8.5 Hz, 1H, Ph-H),

6.71 (s, 2H, Ph-H × 2), 5.89 (s, 1H, -*CH-), 3.73 (s, 6H, -OCH₃× 2), 3.61 (s, 3H,

-OCH₃), 2.42 (s, 3H, -CH₃). ¹³C NMR (150 MHz, DMSO-d₆) δ 206.3, 170.9, 154.7,

153.1, 148.5, 137.5, 135.3, 132.9, 128.8, 128.0, 124.9, 116.3, 104.7, 64.4, 59.8, 55.9,

+

18.8. ESI-HRMS (m/z): calcd for C₁₉H₂₃N₄O₇(M + NH₄), 419.1561; found,

419.1559.

4.5.11.

1-(3-Amino-4-methylphenyl)-5-(3,4,5-trimethoxyphenyl)imidazolidine-2,4-dione (6i)

6h (200 mg, 0.5 mmol), Pd/C (10%, 53 mg, 0.05 mmol) and EtOH (10 mL) were

charged into a round-bottom bottle, which was filled with H₂by gas exchange and

capped with a balloon filled with H₂. The solution was allowed to be stirred at room

temperature for 5 h. Then the mixture was filtered to give a clear solution, which was

concentrated under reduced pressure. The resulting residue was purified by silica gel

column chromatography with DCM-MeOH (10:1) as eluent to give 6i as white

o

1

powder. Yield: 92%. Mp: 197-200 C. ESI-MS (m/z): 372.0 (M + H⁺). H NMR (400

MHz, DMSO-d₆) δ 11.21 (s, 1H, -NH-), 6.87 - 6.74 (m, 2H, Ph-H × 2), 6.57 (s, 2H,

Ph-H × 2), 6.50 (d, J = 7.9 Hz, 1H, Ph-H), 5.67 (s, 1H, -*CH-), 4.87 (s, 2H, -NH₂),

3.72 (s, 6H, -OCH₃× 2), 3.61 (s, 3H, -OCH₃), 1.94 (s, 3H, -CH₃). ¹³C NMR (150

MHz, DMSO-d₆) δ 71.4, 154.5, 152.9, 146.5, 137.2, 134.8, 129.7, 117.7, 109.3, 107.7,

104.3, 64.9, 59.7, 55.8, 16.7. ESI-HRMS (m/z): calcd for C₁₉H₂₂N₃O₅(M + H⁺),

372.1554; found, 372.1553.

4.5.12.

1-(3,4-Dimethylphenyl)-5-(3,4,5-trimethoxyphenyl)imidazolidine-2,4-dione

(6j)

o

Eluent: DCM-MeOH, 30:1. White powder. Yield: 85%. Mp: 206-208 C.

ESI-MS (m/z): 371.1 (M + H⁺). ¹H NMR (400 MHz, DMSO-d₆) δ 11.26 (s, 1H, -NH-),

7.34 (s, 1H, Ph-H), 7.16 (d, J = 7.8 Hz, 1H, Ph-H), 7.04 (d, J = 8.1 Hz, 1H, Ph-H),

6.63 (s, 2H, Ph-H × 2), 5.80 (s, 1H, -*CH-), 3.72 (s, 6H, -OCH₃× 2), 3.60 (s, 3H,

-OCH₃), 2.15 (s, 3H, -CH₃), 2.12 (s, 3H, -CH₃). ¹³C NMR (150 MHz, DMSO-d₆) δ

171.4, 154.6, 153.0, 137.3, 136.3, 134.0, 132.4, 129.5, 129.4, 122.9, 119.0, 104.5,

64.7, 59.7, 55.8, 19.4, 18.5. ESI-HRMS (m/z): calcd for C₂₀H₂₃N₂O₅(M + H⁺),

371.1601; found, 371.1599.

4.5.13. 1,5-Bis(3,4,5-trimethoxyphenyl)imidazolidine-2,4-dione (6k)

o

Eluent: PE-EtOAc, 1:1. White powder. Yield: 72%. Mp: 205-206 C. ESI-MS

(m/z): 433.1 (M + H⁺). ¹H NMR (400 MHz, CDCl₃) δ 9.02 (s, 1H, -NH-), 6.67 (s, 2H,

Ph-H × 2), 6.54 (s, 2H, Ph-H × 2), 5.31 (s, 1H, -*CH-), 3.82 (s, 9H, -OCH₃× 3), 3.79

(s, 3H, -OCH₃), 3.74 (s, 6H, -OCH₃× 2). ¹³C NMR (150 MHz, CDCl₃) δ 169.6, 153.8,

153.4, 152.7, 138.2, 135.2, 131.4, 127.6, 103.4, 98.9, 66.1, 60.3, 60.2, 55.7, 55.5.

ESI-HRMS (m/z): calcd for C₂₁H₂₅N₂O₈(M + H⁺), 433.1605; found, 433.1603.

4.5.14. 1-(6-Methoxypyridin-3-yl)-5-(3,4,5-trimethoxyphenyl)imidazolidine-2,4-dione

(6l)

Eluent: DCM-MeOH, 10:1. Pale purple syrup. Yield: 81%. ESI-MS (m/z): 374.0

1

(M + H⁺). H NMR (400 MHz, DMSO-d₆) δ 11.44 (s, 1H, -NH-), 8.23 (s, 1H,

pyridine-H), 7.91 (d, J = 8.9 Hz, 1H, pyridine-H), 6.82 (d, J = 8.9 Hz, 1H,

pyridine-H), 6.67 (s, 2H, Ph-H × 2), 5.82 (s, 1H, -*CH-), 3.79 (s, 3H, -OCH₃), 3.74 (s,

6H, -OCH₃× 2), 3.62 (s, 3H, -OCH₃). ¹³C NMR (150 MHz, DMSO-d₆) δ 171.4, 160.3,

154.9, 153.1, 137.4, 133.9, 129.0, 127.2, 110.1, 104.8, 104.7, 65.0, 59.7, 55.9, 53.2.

ESI-HRMS (m/z): calcd for C₁₈H₂₀N₃O₆(M + H⁺), 374.1347; found, 374.1344.

4.5.15. 1-(Quinolin-3-yl)-5-(3,4,5-trimethoxyphenyl)imidazolidine-2,4-dione (6m)

o

Eluent: DCM-MeOH, 10:1. Pale yellow powder. Yield: 89%. Mp: 233-235 C.

ESI-MS (m/z): 394.0 (M + H⁺). ¹H NMR (400 MHz, DMSO-d₆) δ 11.70 (s, 1H, -NH-),

9.29 (s, 1H, quinoline-H), 8.59 (s, 1H, quinoline-H), 8.04 (d, J = 8.5 Hz, 1H,

quinoline-H), 7.99 (d, J = 8.1 Hz, 1H, quinoline-H), 7.77 (t, J = 7.7 Hz, 1H,

quinoline-H), 7.67 (t, J = 7.4 Hz, 1H, quinoline-H), 6.79 (s, 2H, Ph-H × 2), 6.06 (s,

1H, -*CH-), 3.74 (s, 6H, -OCH₃× 2), 3.58 (s, 3H, -OCH₃). ¹³C NMR (150 MHz,

DMSO-d₆) δ 171.1, 154.9, 153.1, 143.5, 141.7, 137.4, 130.6, 129.7, 128.5, 127.9,

127.7, 127.4, 127.1, 126.4, 104.8, 64.4, 59.7, 55.9. ESI-HRMS (m/z): calcd for

C₂₁H₂₀N₃O₅(M + H⁺), 394.1397; found, 394.1396.

4.5.16. 1-(Quinolin-6-yl)-5-(3,4,5-trimethoxyphenyl)imidazolidine-2,4-dione (6n)

o

Eluent: DCM-MeOH, 10:1. Off-white powder. Yield: 75%. Mp: 329-331 C.

ESI-MS (m/z): 394.0 (M + H⁺). ¹H NMR (400 MHz, DMSO-d₆) δ 11.69 (s, 1H, -NH-),

9.09 (d, J = 4.6 Hz, 1H, quinoline-H), 8.90 (d, J = 8.3 Hz, 1H, quinoline-H), 8.37 -

8.30 (m, 2H, quinoline-H × 2), 8.25 (d, J = 9.1 Hz, 1H, quinoline-H), 7.91 (dd, J = 8.1,

5.1 Hz, 1H, quinoline-H), 6.75 (s, 2H, Ph-H × 2), 6.03 (s, 1H, -*CH-), 3.79 - 3.55 (m,

9H, -OCH₃× 3). ¹³C NMR (150 MHz, DMSO-d₆) δ 170.9, 154.8, 153.1, 145.2, 142.9,

137.4, 136.8, 136.5, 128.7, 128.5, 127.2, 123.4, 122.3, 117.5, 104.7, 64.5, 59.7, 55.9.

ESI-HRMS (m/z): calcd for C₂₁H₂₀N₃O₅(M + H⁺), 394.1397; found, 394.1395.

4.5.17. 1-(1-Tosyl-1H-indol-5-yl)-5-(3,4,5-trimethoxyphenyl)imidazolidine-2,4-dione

(6o)

Eluent: PE-EtOAc, 1:1. Yellow syrup. Yield: 82%. ESI-MS (m/z): 536.0 (M +

1

H⁺). H NMR (400 MHz, CDCl₃) δ 9.40 (s, 1H, -NH-), 7.89 (d, J = 9.0 Hz, 1H,

indole-H), 7.72 (d, J = 8.2 Hz, 2H, Ph-H × 2), 7.58 (s, 1H, indole-H), 7.54 (d, J = 3.4

Hz, 1H, indole-H), 7.31 (d, J = 9.0 Hz, 1H, indole-H), 7.20 (d, J = 8.1 Hz, 2H, Ph-H ×

2), 6.56 (d, J = 3.2 Hz, 1H, indole-H), 6.51 (s, 2H, Ph-H × 2), 5.40 (s, 1H, -*CH-),

3.79 (s, 3H, -OCH₃), 3.75 (s, 6H, -OCH₃× 2), 2.32 (s, 3H, -OCH₃). ¹³C NMR (150

MHz, CDCl₃) δ 170.0, 154.3, 153.3, 144.6, 138.0, 134.4, 131.7, 131.1, 130.6, 129.4,

127.3, 126.9, 126.2, 118.2, 114.2, 113.4, 108.3, 103.5, 66.1, 60.2, 55.5,

+

20.9.ESI-HRMS (m/z): calcd for C₂₇H₂₉N₄O₇S (M + NH₄), 553.1751; found,

553.1749.

4.5.18. 1-(1H-indol-5-yl)-5-(3,4,5-trimethoxyphenyl)imidazolidine-2,4-dione (6p)

A mixture of 6o (320 mg, 0.6 mmol) and KOH (67 mg, 1.2 mmol) in MeOH (10

o

mL) was stirred and refluxed for 1 h then cooled to 0 C. HCl (1M) was added

dropwise to adjust the pH to around 7. Then the solution was portioned between water

(100 mL) and EtOAc (30 mL × 5). The combined organic layer was dried over

anhydrous Na₂SO₄and concentrated under reduced pressure. The remaining was

purified by silica gel column chromatography with DCM-MeOH (20:1) as eluent to

give 6p as yellow syrup. Yield: 77%. ESI-MS (m/z): 382.0 (M + H⁺), 404.0 (M + Na⁺).

¹H NMR (400 MHz, CDCl₃) δ 9.56 (s, 1H, -NH-), 8.72 (s, 1H, indole-NH-), 7.52 (s,

1H, indole-H), 7.17 (d, J = 8.1 Hz, 1H, indole-H), 7.09 (s, 2H, indole-H × 2), 6.52 (s,

2H, Ph-H × 2), 6.40 (s, 1H, indole-H), 5.37 (s, 1H, -*CH-), 3.76 (s, 3H, -OCH₃), 3.71

(s, 6H, -OCH₃× 2). ¹³C NMR (150 MHz, CDCl₃) δ 170.4, 154.3, 153.8, 138.6, 134.0,

128.3, 128.1, 125.6, 117.9, 115.6, 111.6, 104.3, 103.1, 67.5, 60.8, 56.2. ESI-HRMS

(m/z): calcd for C₂₀H₂₀N₃O₅(M + H⁺), 382.1397; found, 382.1396.

4.5.19. 1-(4-Methoxyphenyl)-2-thioxo-5-(3,4,5-trimethoxyphenyl)imidazolidin-4-one

(7a)

Eluent: PE-EtOAc, 1:1. Yellow syrup. Yield: 86%. ESI-MS (m/z): 389.0 (M +

1

H⁺). H NMR (400 MHz, CDCl₃) δ 9.51 (br.s, 1H, -NH-), 7.21 (d, J = 8.9 Hz, 2H,

Ph-H × 2), 6.85 (d, J = 8.9 Hz, 2H, Ph-H × 2), 6.41 (s, 2H, Ph-H × 2), 5.35 (s, 1H,

-*CH-), 3.80 (s, 3H, -OCH₃), 3.78 (s, 6H, -OCH₃× 2), 3.76 (s, 3H, -OCH₃). ¹³C NMR

(150 MHz, CDCl₃) δ 180.2, 170.8, 158.5, 153.2, 138.3, 128.7, 127.3, 126.6, 113.8,

104.3, 70.5, 60.2, 55.6, 54.8. ESI-HRMS (m/z): calcd for C₁₉H₂₁N₂O₅S (M + H⁺),

389.1166; found, 389.1164.

4.5.20.

1-(3-Hydroxy-4-methoxyphenyl)-2-thioxo-5-(3,4,5-trimethoxyphenyl)imidazolidin-4-o

ne (7b)

Eluent: DCM-MeOH, 15:1. Yellow syrup. Yield: 76%. ESI-MS (m/z): 405.0 (M

+ H⁺), 426.9 (M + Na⁺). ¹H NMR (400 MHz, CD₃OD) δ 6.93 (br.s, 1H, Ph-H), 6.89 -

6.81 (m, 2H, Ph-H × 2), 6.53 (s, 2H, Ph-H × 2), 5.65 (s, 1H, -*CH-), 3.82 (s, 3H,

-OCH₃), 3.79 (s, 6H, -OCH₃× 2), 3.72 (s, 3H, -OCH₃). ¹³C NMR (150 MHz, CD₃OD)

δ 181.17, 172.44, 152.98, 146.56, 145.66, 137.68, 129.64, 128.19, 117.26, 113.51,

110.31, 104.29, 69.93, 59.04, 54.67, 54.44. ESI-HRMS (m/z): calcd for C₁₉H₂₁N₂O₆S

(M + H⁺), 405.1115; found, 405.1112.

4.6. Synthesis of 8(a-h)

4.6.1.

Synthesis

of

5-(2,4-Dioxo-5-(3,4,5-trimethoxyphenyl)imidazolidin-1-yl)-2-methoxyphenyl

sulfurofluoridate (8a)

The preparation was performed according to literature⁴⁸. Briefly, to a DCM (5

mL) solution of 6b (194 mg, 0.5 mmol) surrounded by SO₂F₂gas, was added Et₃N

(693 µL, 5 mmol). The mixture was stirred at room temperature for 1 h, then

concentrated under reduced pressure. The resulting residue was purified by silica gel

column chromatography with DCM / MeOH (50:1) as eluent to give 8a as yellowish

syrup. Yield: 91%. ESI-MS (m/z): 470.9 (M + H⁺).¹H NMR (400 MHz, CDCl₃) δ 8.77

(br.s, 1H, -NH-), 7.61 (d, J = 1.6 Hz, 1H, Ph-H), 7.27 (dd, J = 8.1, 1.6 Hz, 1H, Ph-H),

6.95 (d, J = 9.1 Hz, 1H, Ph-H), 6.48 (s, 2H, Ph-H × 2), 5.33 (s, 1H, -*CH-), 3.86 (s,

3H, -OCH₃), 3.81 (s, 3H, -OCH₃), 3.80 (s, 6H, -OCH₃× 2). ¹³C NMR (150 MHz,

CDCl₃) δ 169.1, 153.5, 153.4, 147.9, 138.2, 137.8, 128.6, 126.6, 121.1, 115.4, 113.0,

+

103.2, 65.6, 60.2, 55.8, 55.6. ESI-HRMS (m/z): calcd for C₁₉H₂₃FN₃O₉S (M + NH₄),

488.1134; found, 488.1132.

4.6.2.

Synthesis

of

1-(3-Hydroxy-4-methoxyphenyl)-3-methyl-5-(3,4,5-trimethoxyphenyl)imidazolidine-2,

4-dione (8b)

To an acetone (15 mL) solution of 6b (194 mg, 0.5 mmol) and K₂CO₃(103 mg,

0.75 mmol), was added Me₂SO₄(47 µL, 0.5 mmol). The mixture was refluxed for 3 h

before the addition of water (50 mL). Then the mixture was heated at 60 C for

o

another 1 h. Afterwards it was extracted with EtOAc (30 mL × 5). The combined

organic layer was dry over anhydrous Na₂SO₄and concentrated under reduced

pressure. The resulting residue was purified by silica gel column chromatography

with DCM / MeOH (40:1) as eluent to give 8b as white powder. Yield: 84%. Mp:

o

1

188-190 C. ESI-MS (m/z): 403.0 (M + H⁺), 425.0 (M + Na⁺). H NMR (400 MHz,

DMSO-d₆) δ 9.12 (s, 1H, -OH), 7.03 (s, 1H, Ph-H), 6.89 - 6.75 (m, 2H, Ph-H × 2),

6.64 (s, 2H, Ph-H × 2), 5.72 (s, 1H, -*CH-), 3.72 (s, 6H, -OCH₃× 2), 3.70 (s, 3H,

13

-OCH₃), 3.61 (s, 3H, -OCH₃), 2.97 (s, 3H, -CH₃). C NMR (150 MHz, DMSO-d₆) δ

170.2, 154.5, 152.9, 146.2, 144.9, 137.3, 129.5, 129.3, 112.7, 111.9, 110.4, 104.7, 64.0,

59.7, 55.8, 55.5, 24.8. ESI-HRMS (m/z): calcd for C₂₀H₂₃N₂O₇(M + H⁺), 403.1500;

found, 403.1497.

4.6.3.

Synthesis

of

1-(3-Fluoro-4-methoxyphenyl)-3-methyl-5-(3,4,5-trimethoxyphenyl)imidazolidine-2,4-

dione (8c)

To an acetone (15 mL) solution of 6e (195 mg, 0.5 mmol) and K₂CO₃(103 mg,

0.75 mmol), was added Me₂SO₄(56 µL, 0.6 mmol). The mixture was refluxed for 3 h

before the addition of water (50 mL). Then the mixture was heated at 60 C for

o

another 1 h. Afterwards it was extracted with EtOAc (30 mL × 5). The combined

organic layer was dry over anhydrous Na₂SO₄and concentrated under reduced

pressure. The resulting residue was purified by silica gel column chromatography

with DCM / MeOH (50:1) as eluent to give 8c as white wax. Yield: 95%. Mp: 63-65

1

^oC. ESI-MS (m/z): 405.0 (M + H⁺), 427.0 (M + Na⁺). H NMR (400 MHz, CDCl₃) δ

7.37 (dd, J = 12.8, 2.4 Hz, 1H, Ph-H), 7.02 (d, J = 7.9 Hz, 1H, Ph-H), 6.84 (t, J = 9.1

Hz, 1H, Ph-H), 6.46 (s, 2H, Ph-H × 2), 5.25 (s, 1H, -*CH-), 3.83 -3.76 (m, 12H,

13

-OCH₃× 4), 3.11 (s, 3H, -CH₃). C NMR (150 MHz, CDCl₃) δ 169.2, 154.2, 153.3,

151.4 (J_C-F= 245 Hz), 144.3 (J_C-F= 11 Hz), 138.0, 129.09 (J_C-F= 9 Hz), 127.3, 115.6

(J_C-F= 2 Hz), 113.0, 109.1 (J_C-F= 23 Hz), 103.1, 64.1, 60.1, 55.8, 55.6, 24.7.

ESI-HRMS (m/z): calcd for C₂₀H₂₂FN₂O₆(M + H⁺), 405.1456; found, 405.1456.

4.6.4.

Synthesis

of

1-(3-Amino-4-methoxyphenyl)-3-methyl-5-(3,4,5-trimethoxyphenyl)imidazolidine-2,4-

dione (8d)

To an acetone (15 mL) solution of 6f (209 mg, 0.5 mmol) and K₂CO₃(103 mg,

0.75 mmol), was added Me₂SO₄(56 µL, 0.6 mmol). The mixture was refluxed for 3 h

before the addition of water (50 mL). Then the mixture was heated at 60 C for

o

another 1 h. Afterwards it was extracted with EtOAc (30 mL × 5). The combined

organic layer was dry over anhydrous Na₂SO₄and concentrated under reduced

pressure. Then the resulting product together with Pd/C (10%, 53 mg, 0.05 mmol) and

EtOH (10 mL) was charged into a round-bottom bottle, which was filled with H₂by

gas exchange and capped with a balloon filled with H₂. The solution was allowed to

be stirred at room temperature for 5 h. Then the mixture was filtered to give a clear

solution, which was concentrated under reduced pressure. The resulting residue was

purified by silica gel column chromatography with DCM-MeOH (30:1) as eluent to

give 8d as yellowish powder. Yield: 77%. Mp: 207-209 ^oC. ESI-MS (m/z): 402.0 (M +

H⁺). ¹H NMR (400 MHz, DMSO-d₆) δ 6.84 (d, J = 2.0 Hz, 1H, Ph-H), 6.69 (d, J = 8.7

Hz, 1H, Ph-H), 6.61 (s, 2H, Ph-H × 2), 6.54 (dd, J = 8.8, 2.4 Hz, 1H, Ph-H), 5.66 (s,

1H, -*CH-), 4.80 (s, 2H, -NH₂), 3.73 (s, 6H, -OCH₃× 2), 3.69 (s, 3H, -OCH₃), 3.61 (s,

3H, -OCH₃), 2.97 (s, 3H, -CH₃). ¹³C NMR (150 MHz, DMSO-d₆) δ 170.3, 154.5,

152.9, 143.7, 137.6, 137.3, 129.4, 129.4, 110.2, 109.9, 108.7, 104.7, 64.2, 59.7, 55.8,

55.2, 24.7. ESI-HRMS (m/z): calcd for C₂₀H₂₄N₃O₆(M + H⁺), 402.1660; found,

402.1657.

4.6.5.

Synthesis

of

1-(3-Amino-4-methoxyphenyl)-3-ethyl-5-(3,4,5-trimethoxyphenyl)imidazolidine-2,4-di

one (8e)

To an acetone (15 mL) solution of 6f (209 mg, 0.5 mmol) and K₂CO₃(103 mg,

0.75 mmol), was added EtBr (45 µL, 0.6 mmol). The mixture was refluxed for 3 h