Efficient synthesis of RITA and its analogues: Derivation of analogues with improved antiproliferative activity via modulation of p53/miR-34a pathway

Fig. 1 p53–MDM2 inhibitors.

Scheme 1 Retrosynthetic analysis of RITA and its analogues.

MicroRNAs (miRs) are small non-coding RNAs 18–25 and we further found a p53/miR-34a mediated antiproliferative

nucleotides in length that regulate gene expression during pathway induced by RITA and its analogues.

various crucial cell processes, such as apoptosis, diﬀeren-

1

tiation and development. Alterations in miRNA expression

1

2

have been observed in a variety of human tumors. Functional

studies indicate that miRNAs act as either tumor suppressors

or oncogenes. MiR-34a is a direct downstream eﬀector of p53

Results and discussion

A novel, eﬀective synthetic route to RITA and its analogues

and is involved in p53-induced cell senescence and apoptotic Retrosynthetic analysis indicated that a Suzuki–Miyaura reac-

1

3

pathways. The level of miR-34a is down-regulated in diverse tion could be used to synthesize RITA, and this reaction was

1

4

tumors compared with normal tissue counterparts. The res- used to prepare tricyclic α-heteroaryl thiophene analogues of

1

8

toration of miR-34a expression resulted in tumor cell apopto- RITA (Scheme 1). As shown in Scheme 2, a 5-step route was

sis, cell-cycle arrest or senescence, indicating that miR-34a acts carried out to synthesize RITA. The intermediates 6 were syn-

as an important tumor suppressor in the p53 network during thesized by Suzuki coupling of the commercially available

15

cancer development.

compounds 4 and 5 at room temperature using Pd(OAc)₂–

RITA is a small molecule with promising antiproliferative X-Phos as catalysts in CH CN–H O under argon atmosphere.

3

2

activity, but the eﬃciency of current synthetic methods is Bromination of substituted 6 with NBS at −23 °C provided

1

6

18,19

limited. Chang’s group firstly developed a synthetic method respectively 7 and 8 in good yields.

to generate RITA through a four-step reaction, including for- Suzuki reaction of 8a with commercially available 3a in the

C–C bond-forming

2

0

maldehyde protection and deprotection, Stille coupling reac- presence of Pd(PPh ) –Na CO

in dioxane–H O aﬀorded the

3

4

2

3

2

tion and sodium borohydride reduction. The overall yield of precursor 10a in 16% yield (entry 1). Homocoupling of hetero-

1

7

RITA is only about 37%. R. Danae et al. reported a synthetic aryl boronic acids (CHO-SS-CHO) occurred as the main

approach to RITA’s precursor, the dialdehyde derivative, utiliz- process, and separation of 10a from the homocoupling

ing a one pot Stille coupling (yield: 35%) and formylation product was very diﬃcult due to their very similar R values.

f

(

yield: 24%, at −78 °C) reaction. However, the expensive and Diﬀerent reaction conditions including ligands, bases, and the

toxic reagents (the toxic chlorotributyltin and expensive 2,5- source of Pd in the cross-coupling reaction were investigated,

dibromofuran), harsh reaction conditions (to produce tributyl- all of which have been successfully performed in the literature.

stannanyl-thiophene-2-carbaldehyde for the Stille reaction at As shown in Table 1, the use of Pd(PPh ) –K CO as catalyst in

3

4

2

3

−

78 °C) and the low yield of products make these methods DME (1,2-dimethoxyethane) at 80 °C under argon atmos-

1

8

unsuitable for large-scale production. Therefore, it is necessary phere only aﬀorded 10a in 12% yield (entry 2). Only trace or

to develop a more practical method to synthesize RITA.

no product was detected when the reaction was carried out

In this study, we developed a novel, eﬀective synthetic route using Pd(PPh ) Cl –K CO as catalysts in DME–CH Cl at

to RITA, which was used for deriving a series of RITA ana- 80 °C. Pd(OAc) –PPh –Na CO in DME and Pd(OAc) –

logues both for testing our method and for discovering new K CO –TBAB (tetrabutylammonium bromide) in H O under

3

2

3

2

1

22

2

3

2

3

2

3

2

3

2

antiproliferative agents, particularly those with potent activity, argon atmosphere (entries 3–5) were similarly unsuccessful.

This journal is © The Royal Society of Chemistry 2012

Org. Biomol. Chem., 2012, 10, 9734–9746 | 9735

Paper

Scheme 2 Synthesis of 1. Reagents and conditions: (a) Pd(OAc)

2

(1 mol%), K

2

CO

3

, X-Phos (2 mol%), CH

3

CN–H

2

O (1.5 : 1), rt, 8–12 h; (b) NBS, DMF, −23 °C, 4 h; (c)

NBS, BPO, toluene, −23 °C, 4 h; (d) NaBH

4

, CH

2

Cl

2

–CH

3

OH, 0 °C, 30 min; (e) conditions in Table 1; (f) Pd(OAc)

2

(1 mol%), K

2

CO

3

, X-Phos (2 mol%), CH

3

2

CN–H O

(

1.5 : 1), rt, 24 h; (g) NaBH , CH Cl –CH OH, 0 °C, 30 min.

4

2

3

Table 1 Optimization of reaction conditions for the Suzuki coupling

a

Entry

Substrates

Solvent

[Pd]/Ligand

Base

Na CO

T (°C)/t [h]

Yield (%)

1

2

3

4

5

6

7

8

9

8a + 3a

2a + 3a

Dioxane–H

DME

DME–CH

2

O

Pd(PPh

)

3 4

)

3 4

)

3 2

2

3

100/12

80/20

80/2

16

12

K

2

CO

3

2

Cl

2

Cl

2

3

Trace

0

Trace

24

2

H O

Pd(OAc)

2

3

rt/2

DME

/PPh

3

Na

2

CO

3

refl/4

refl/24

rt/24

CH

3

CN–H

3

CN–H

3

CN–H

3

CN–H

2

O

/S-Phos

/X-Phos

K CO

2

K CO

2

K CO

2

K CO

2

3

O

28

62

64

2

/X-Phos

a

Isolated yield.

The reaction yield improved slightly when Pd(OAc)

2-dicyclohexylphosphino-2′,6′-dimethoxybiphenyl) or Pd(OAc)

X-Phos (2-dicyclohexyl-phosphino-2′,4′,6′-triiso-propylbiphenyl) with 3a through Suzuki reaction to furnish 9a using Pd(OAc)₂–

were used as catalysts in refluxing CH CN–H O under argon X-Phos in refluxing CH CN–H O within 24 h under argon

2

–S-Phos the homocoupling product, reduction of 8a with NaBH

4

in

(

2

–

CH Cl –CH OH at 0 °C gave 2a, which was selected to react

2

3

2

3

2

atmosphere (entries 6–7), but the main product was still atmosphere. Surprisingly, the desired 9a was obtained in 62%

CHO-SS-CHO. In order to facilitate the separation of 10a from yield (entry 9), with no CHO-SS-CHO detected (Scheme 2). To

9

736 | Org. Biomol. Chem., 2012, 10, 9734–9746

This journal is © The Royal Society of Chemistry 2012

Organic & Biomolecular Chemistry

Scheme 3 Synthesis of RITA analogues. Reagents and conditions: (a) Oxone, TEMPO, TBAB, CH

2

Cl

2

2 2 3

–acetone, rt, 1–3 h; (b) Pd(OAc) (1 mol%), K CO , X-Phos

(2 mol%), CH

3

CN–H

2

O, rt, 24 h; (c) TIPSCl, imidazole, dry THF, rt, 24 h; (d) NH

2

OH·HCl, NaOAc, MeOH, reﬂux, 2 h; (e) NaBH

4

, CH Cl –CH OH, 0 °C, 30 min.

2

3

our delight, when this reaction was carried out at room temp- structure–activity relationships (SAR): (1) these analogues

erature, the yield increased slightly to 64%. In addition, when possess notable activity against the cancer cell lines with wild-

the amount of the substrate 2a was increased from 3.64 mmol type p53 (HCT116, A549, MCF-7, and K562), but not in

/−

(

0.94 g) to 36.4 mmol (9.4 g) and the amount of 3a and Pd HCT116/p53⁻; (2) for analogues with the same side chains,

OAc) –X-Phos were increased correspondingly, the desired compounds containing sulfur atoms at X1, X3 or at X1, X2,

2

product was isolated in 64–68% yields. Reduction of 9a with and X3 possess comparatively stronger antiproliferative activity

NaBH furnished the target molecule 1a (RITA) in 5 steps and than the other derivatives, as exhibited by 1a–1f, 9a–9g, etc.;

4

an overall yield of 53%, which is higher than that in the (3) for the same combination of X1, X2, X3, analogues with

1

6

reported literature (37% ). Compared to the reported syn- the side chains with two hydroxymethyl groups showed

thetic routes, this novel and practical approach to RITA is suit- better antiproliferative activity than those with a hydroxy-

able for large-scale production with cheap commercially methyl group and an aldehyde group, followed by two alde-

available materials, high yield and mild conditions. Under the hyde groups, as demonstrated by RITA, 9a and 10a.

optimised conditions, RITA and a series of its analogues Furthermore, we also found that if one of the side chains

9

a–9g, 1b–1f were synthesized (Scheme 2). Other RITA deriva- was protected by TIPS, as in the case of compounds 13a and

tives were synthesized as outlined in Scheme 3. Oxidation of 9 15, this resulted in no antiproliferative activity, suggesting that

with Oxone–TEMPO at room temperature in the presence of the hydroxymethyl group was essential for antiproliferative

2

4

TBAB furnished the corresponding dialdehyde 10. Suzuki activity. Hence, the hydrophilic group in the side chain may be

reaction of 2 with 11 in the presence of Pd(OAc) –X-Phos in conducive to the formation of a hydrogen bond with the

CH CN–H O at room temperature within 24 h under argon target, improving pharmacokinetic properties and antitumor

2

3

2

atmosphere aﬀorded the tricyclic heteroaryl compound 12. activity.

9

1

was protected with the triisopropylsilyl (TIPS) group to give

We further measured the cytotoxic activity of these ana-

2

5

3, which was treated with NH OH·HCl, AcONa (1.5 equiv) in logues against a normal human colon mucosal epithelial cell

2

refluxing CH OH to furnish oximes 14. Reduction of 13b with line, NCM460. As shown in Table 3, RITA exhibited weak cyto-

3

NaBH

4

aﬀorded 15.

toxicity to NCM460 cells with an inhibition rate of 24.49%,

but compound 1f had much less activity against NCM460,

with an inhibition rate of 2.62% at the concentration of

Inhibition of cell growth by new RITA analogues

Using MTT assays, we measured the antiproliferative activity of 10 μM (100 fold higher than the IC50 in colon cancer cells

RITA and its analogues in HCT116/p53wt, MCF-7, A549, K562 HCT116).

and HCT116/p53⁻cells. As shown in Table 2, some ana-

/−

The antiproliferative activity of compound 1f was

logues such as compound 1f exhibited better activity than confirmed by its capability of suppressing the clonogenic

RITA, with IC50 values of 28 nM in MCF-7 cells and 100 nM in growth of HCT116 cells. At the low concentration

HCT116 cells (Fig. 2A). The data from this series of RITA ana- of 0.0625 μM, compound 1f greatly decreased the colony

logues provided us with the following information on the formation of HCT116 cells (Fig. 2B). Taken together, these

This journal is © The Royal Society of Chemistry 2012

Org. Biomol. Chem., 2012, 10, 9734–9746 | 9737

Paper

Table 2 Antiproliferation activity of compounds against HCT116, MCF-7, A549, K562, HCT116/p53⁻^/⁻cells

a

IC50 (μM)

Compound

HCT116

MCF-7

A549

K562

HCT116/p53⁻^/⁻

1

9

1

a (RITA)

b

c

d

e

f

a

b

c

d

e

f

0.10 ± 0.02

2.10 ± 0.06

>25

6.30 ± 0.14

19.40 ± 0.17

0.10 ± 0.01

0.33 ± 0.04

5.21 ± 0.08

5.00 ± 0.11

>25

0.50 ± 0.04

5.50 ± 0.12

>25

0.054 ± 0.010

>25

3.50 ± 0.08

0.40 ± 0.09

0.028 ± 0.010

0.40 ± 0.05

6.90 ± 0.13

5.70 ± 0.06

>25

0.60 ± 0.03

>25

7.30 ± 0.02

>25

6.90 ± 0.04

11.20 ± 0.06

6.81 ± 0.07

6.90 ± 0.04

5.70 ± 0.05

5.10 ± 0.09

>25

0.50 ± 0.04

>25

0.41 ± 0.03

2.79 ± 0.04

0.26 ± 0.08

2.79 ± 0.06

3.12 ± 0.09

>25

1.46 ± 0.09

>25

2.71 ± 0.20

0.41 ± 0.07

>25

16.00 ± 0.19

>25

15.90 ± 0.12

>25

7.10 ± 0.10

11.40 ± 0.16

11.60 ± 0.08

>25

12.81 ± 0.08

>25

g

0a

0b

0c

0d

2a

2b

3a

3b

3c

3d

4a

4b

4c

5

>25

5.50 ± 0.02

3.10 ± 0.07

0.42 ± 0.02

>25

a

All values are means of three experiments.

data suggest that compound 1f is a potent anticancer Compound 1f induces G /G cell cycle arrest through

0

1

analogue of RITA.

up-regulation of miR-34a

1

3b

MiR-34a is an important target of p53.

In this study, we

further examined the eﬀect of compound 1f and RITA on

miR-34a expression in HCT116 cells using real-time reverse

transcription-PCR. As shown in Fig. 5A, the expression of

miR-34a was dramatically increased by both compound 1f and

RITA in a time-dependent manner. MiR-34a functions as a key

target for p53 in the regulation of cell cycle progression and

p53-dependent apoptosis induced by RITA and compound 1f

Compound 1f was chosen for further studies based on its

potent antiproliferative activity with the use of RITA as a

control. HCT116 cells were treated with 0.1 μM RITA or com-

pound 1f for 48 h and then stained with Hoechst 33258 for flu-

orescence microscopy observations. As shown in Fig. 3A,

characteristic morphological changes typical of apoptosis,

such as membrane blebbing, chromatin condensation, and

the formation of apoptotic bodies, were observed. An annexin-

V/PI (propidium iodide) binding assay further confirmed the

apoptosis induced by compound 1f and RITA. As shown in

Fig. 3B, the upper-right quadrant (both annexin-V and PI posi-

tive) represents the late stage apoptotic cells and the lower-

cell proliferation by down-regulating the expression of cell-

13a

cycle related proteins, such as E2F1, CDK4 and MET.

Our

data showed that in response to p53 activation, miR-34a sig-

nificantly decreased CDK4 and E2F1 protein levels (Fig. 5B),

leading to G

0 1

/G cell cycle arrest (Fig. 5C).

Molecular docking

right quadrant (annexin-V positive but PI negative) indicates It was established that RITA can bind to the HDM2-p53 trans-

the early stage apoptotic cells. In RITA-treated HCT116 cells, activation domain binding cleft by computational “blind

2

6

3

0.50% cells were in early apoptosis and 36.60% in late apop- docking” studies.

tosis, whereas compound 1f showed similar activity with between compounds and their targets, molecular docking

2.90% in early apoptosis stage and 44.15% in late apoptosis. studies on RITA and compound 1f were conducted employing

To better understand the interaction

2

These results demonstrate that compound 1f could eﬃciently the Discovery Studio 3.1/Libdock protocol (Accelrys Software

induce HCT116 cell death. To determine the p53-dependence Inc.) and the X-ray structure of HDM2 in complex with the p53

27

of the apoptosis induced by compound 1f, we examined the transactivation domain (PDB code: 1YCR). Fig. 6 shows that

p53 levels in HCT116 cells. As shown in Fig. 4, compound 1f RITA docks into the HDM2-p53 transactivation domain

indeed activated p53 in the cells, indicating that compound 1f binding cleft. In this binding model, RITA is bound to the

induces apoptosis by triggering p53.

hydrophobic HDM2-p53 transactivation domain binding cleft

9

738 | Org. Biomol. Chem., 2012, 10, 9734–9746

This journal is © The Royal Society of Chemistry 2012

Organic & Biomolecular Chemistry

Fig. 3 Apoptosis induced by RITA and compound 1f. (A) Fluorescent staining

of nuclei by Hoechst 33258: HT116 cells treated with 0.1 μM RITA or compound

1f for 48 h; (B) Flow cytometric analysis of phosphatidylserine externalization

(Annexin-V binding) and cell membrane integrity (PI staining). HCT116 cells

were treated with RITA or compound 1f at 2 μM for 36 h.

Fig. 4 Eﬀect of RITA and compound 1f on p53 expression. HCT116 cells were

treated with RITA or compound 1f for 12 h, then cell extracts were subjected to

western blot analysis using the indicated antibodies.

Fig. 2 Antiproliferative activity of RITA and compound 1f. (A) Eﬀect of RITA

−/−

and compound 1f on the viability of HCT116, HCT116/p53 , K562, MCF-7

cells. (B) Eﬀect of RITA and compound 1f on colony formation of HCT116 cells.

dynamics studies also indicated that compound 1f binds to

the target with substantially better aﬃnity compared to RITA,

with a ΔG = −26.15429 kcal mol⁻vs. −11.51159 kcal mol for

1

−1

a

Table 3 Toxicity evaluation of compounds against NCM460 cells

RITA, which explains the improved antitumor activity.

Compound Inhibition rate (μM) Compound Inhibition rate (μM)

1

a

d

e

f

24.49 ± 0.03%

15.04 ± 0.02%

25.81 ± 0.06%

2.62 ± 0.01%

9a

9g

12a

12b

85.73 ± 0.02%

36.76 ± 0.10%

3.33 ± 0.02%

42.57 ± 0.03%

Conclusions

We have developed a novel synthetic method for the prep-

aration of RITA by an eﬃcient and practical palladium-cata-

lyzed C–C bond-forming Suzuki reaction as the key reaction at

room temperature with cheap commercially available

materials, which provides a valuable and convenient tool to

a

NCM460 cells were treated by compounds at 10 μM for 72 h, and all

values are means of three experiments.

and forms three hydrogen bonds with the amino acids Gln72 derive a series of substituted tricyclic α-heteroaryl (furan/thio-

O–H⋯O:2.084 Å), Val93 (O–H⋯O:2.285 Å), His96 (O– phene) analogues of RITA under mild conditions. Some of the

(

H⋯O:1.907 Å) (Fig. 6A). Compound 1f has similar interactions newly derived RITA analogues displayed promising antiproli-

with the HDM2-p53 transactivation domain binding cleft ferative activity in p53 wild-type cells and cancer cell selectivity

(Fig. 6B), forming a hydrogen bond with Gln72 (O– compared to RITA. Compound 1f also exhibited weak cytotoxi-

H⋯O:2.024 Å) and a π–π interaction between the thiophene city to human normal NCM460 cells. The structure–activity

ring and His96. In addition, the results of the molecular relationships (SAR) of these compounds were also analyzed.

This journal is © The Royal Society of Chemistry 2012

Org. Biomol. Chem., 2012, 10, 9734–9746 | 9739

Paper

Fig. 6 Molecular docking of RITA and compound 1f with HDM2. (A) RITA with

HDM2; (B) Compound 1f with HDM2.

this research not only elucidate a novel antiproliferative mech-

anism for RITA and its analogues, but also provide important

information on the synthesis and modifications of RITA and

its substituted tricyclic α-heteroaryl (furan/thiophene)

analogues.

Experimental

General experimental conditions

Unless otherwise noted, all solvents and reagents were com-

1

mercially available and used without further purification. H

NMR spectra were recorded on a Bruker AVIII-400 spec-

trometer. Chemical shifts (in ppm) were referenced to tetra-

methylsilane (δ = 0 ppm) in CDCl , d -DMSO or CD OD as an

3

6

3

1

3

internal standard. C NMR spectra were obtained by using the

same NMR spectrometers, which were calibrated with CDCl₃,

d -DMSO or CD OD. Coupling constants (J) were in hertz (Hz),

6

3

and signals were designated as follows: s, singlet; d, doublet; t,

triplet; m, multiplet, etc. Mass spectra were recorded using a

Waters Micromass Q-TOF Premier Mass spectrometer. Thin

layer chromatography (TLC) was carried out by using plate

Fig. 5 MiR-34a mediated cell cycle arrest. (A) MiR-34 expression induced by

RITA and compound 1f in HCT116 cells as assessed by Taqman q-RT PCR assay.

HCT116 cells were treated with 0.4 μM RITA or compound 1f with DMSO and silica gel F254. Melting points were determined with a SGW

DOX (Doxorubicin hydrochloride) used as controls at the indicated time points.

X-4 digital apparatus, and are uncorrected.

The total RNA (10 ng) extracted from each of the treated cells was used for real-

time reverse transcription-PCR (RT-PCR) analysis of miR-34a and RNU66

Chemistry

expression. RNU66 transcripts were used as an internal control. The data rep-

resent the average of three independent experiments ± SD. (B) The eﬀect of

GENERAL PROCEDURE FOR PREPARATION OF COMPOUNDS 6A–6D. To a

RITA and compound 1f on CDK4 and E2F1 protein levels. HCT116 cells were

solution of heteroaryl halide 4 (1.0 equiv), boronic acid 5 (1.5

treated with 1 μM RITA and compound 1f for 12 h, 24 h and 48 h, and then cell

2 3

equiv), K CO (3.0 equiv), and 2-dicyclohexylphosphino-2′,4′,6′-

extracts were subjected to western blot analysis using the indicated antibodies;

3 2

C) Analysis of cell cycle phase distribution induced by RITA and compound 1f. triisopropylbiphenyl (X-Phos, 0.02 equiv) in CH CN–H O

(

HCT116 cells were treated by 0.25 μM RITA or compound 1f for 48 h, and then (1.5 : 1) was added Pd(OAc) (0.01 equiv). The flask and its con-

2

analyzed by ﬂow cytometry.

tents were put under reduced pressure then backfilled with

argon three times. The mixture was stirred at 25 °C for 8 to

1

2 h under argon atmosphere, then extracted with CH Cl , and

2 2

The results of molecular docking studies support the view that the combined organic layer was washed with brine and dried

the antiproliferative activity of RITA and its analogues is partly (Na SO ). The solvent was removed in vacuo to aﬀord a crude

2

4

due to their interactions with the HDM2-p53 transactivation product, which was purified by flash chromatography (eluting

domain binding cleft, providing a clue for further modifi- with n-hexane–CH Cl mixtures).

2

cations of RITA. Similarly to RITA, compound 1f markedly acti-

5-(FURAN-2-YL)THIOPHENE-2-CARBALDEHYDE (6A). The general pro-

vates p53 in HCT116 cells, inducing apoptosis. Both cedure was used with 5-bromothiophene-2-carbaldehyde (4a,

compound 1f and RITA trigger miR-34a mediated G /G cell 1.09 g, 5.71 mmol) and furan-2-ylboronic acid (5b, 0.96 g,

0

1

cycle arrest by down-regulating the expression of cell cycle- 8.57 mmol). The product was purified by column chromato-

related proteins CDK4 and E2F1. In summary, the results of graphy on silica gel (n-hexane–CH Cl , 4 : 1) to provide the title

2

9

740 | Org. Biomol. Chem., 2012, 10, 9734–9746

This journal is © The Royal Society of Chemistry 2012

Organic & Biomolecular Chemistry

compound as a light brown solid (0.92 g, 90%). H NMR (400 MHz, CDCl

1

3

): δ 9.87 (s, 1H), 7.66 (d, J = 4.0 Hz, 1H), 7.18

(

7

1

400 MHz, CDCl ): δ 9.88 (s, 1H), 7.69 (d, J = 4.0 Hz, 1H), (d, J = 3.9 Hz, 1H), 7.10 (d, J = 3.9 Hz, 1H), 7.04 (d, J = 3.9 Hz,

3

1

3

.53–7.47 (m, 1H), 7.32 (d, J = 4.0 Hz, 1H), 6.75 (d, J = 3.4 Hz, 1H); C NMR (101 MHz, CDCl ): δ 182.51, 145.82, 142.02,

1

3

H), 6.51 (dd, J = 3.4, 1.8 Hz, 1H); C NMR (101 MHz, CDCl

3

): 137.44, 137.19, 131.21, 126.21, 124.38, 114.17.

1

9

δ 182.68, 148.27, 143.57, 142.77, 141.57, 137.22, 122.96,

12.37, 108.79.

2,2′-BIFURAN]-5-CARBALDEHYDE (6B). The general procedure was of 6a (1.32 g, 7.41 mmol) and BPO (24.2 mg, 0.1 mmol) in

5-(5-BROMOFURAN-2-YL)THIOPHENE-2-CARBALDEHYDE

(8A). NBS

1

(1.32 g, 7.41 mmol) was gradually added to a stirred solution

[

used with 5-bromofuran-2-carbaldehyde (1 g, 5.71 mmol) and toluene (20 mL) over 30 min at −23 °C in air in the dark. After

furan-2-ylboronic acid (0.96 g, 8.57 mmol). The product was the mixture was stirred for 3.5 h at −23 °C, the solvent was

purified by column chromatography on silica gel (n-hexane– evaporated in vacuo. The residue was purified by column

CH Cl , 3 : 1) to provide the title compound as a brown solid chromatography on silica gel (n-hexane–CH Cl , 4 : 1) to

2

1

(

7

1

0.73 g, 78%). H NMR (400 MHz, CDCl

.55–7.51 (m, 1H), 7.30 (d, J = 3.8 Hz, 1H), 6.91 (d, J = 3.4 Hz, 94%). m.p.: 137–139 °C; H NMR (400 MHz, CDCl

H), 6.74 (d, J = 3.7 Hz, 1H), 6.54 (dd, J = 3.5, 1.8 Hz, 1H);

3

): δ 9.63 (s, 1H), provide the title compound as a light brown solid (1.79 g,

1

3

): δ 9.89 (s,

1

3

C

1H), 7.68 (d, J = 4.0 Hz, 1H), 7.32 (d, J = 4.0 Hz, 1H), 6.69 (d, J =

): δ 177.00, 151.60, 151.44, 144.95, 3.5 Hz, 1H), 6.44 (d, J = 3.5 Hz, 1H); C NMR (101 MHz,

1

3

NMR (101 MHz, CDCl

44.01, 123.42, 112.14, 109.62, 107.42.

-(THIOPHEN-2-YL)FURAN-2-CARBALDEHYDE (6C). The general pro- 123.23, 114.14, 110.90.

cedure was used with 5-bromofuran-2-carbaldehyde (1 g, 5′-BROMO-[2,2′-BIFURAN]-5-CARBALDEHYDE (8B). NBS (1.32 g,

3

1

CDCl ): δ 182.62, 150.05, 141.86, 141.29, 137.04, 123.75,

3

5

1

9

5

8

.71 mmol) and thiophen-2-ylboronic acid (1.1 mg, 7.41 mmol) was gradually added to a stirred solution of 6b

.57 mmol). The product was purified by column chromato- (1.20 g, 7.41 mmol) and BPO (24.2 mg, 0.1 mmol) in

Cl , 4 : 1) to provide the title toluene (20 mL) over 30 min at −23 °C in air in the dark. After

graphy on silica gel (n-hexane–CH

compound as a brown solid (0.86 g, 84%). H NMR (400 MHz, the mixture was stirred for 3.5 h at −23 °C, the solvent was

2

1

CDCl ): δ 9.60 (s, 1H), 7.52 (d, J = 3.5 Hz, 1H), 7.40 (d, J = evaporated in vacuo. The residue was purified by column

3

4

6

1

2 2

.9 Hz, 1H), 7.29 (d, J = 3.7 Hz, 1H), 7.10 (t, J = 4.3 Hz, 1H), chromatography on silica gel (n-hexane–CH Cl , 3 : 1) to

1

3

.67 (d, J = 3.6 Hz, 1H); C NMR (101 MHz, CDCl

3

): δ 176.87, provide the title compound as a brown solid (1.61 g, 90%).

1

54.89, 151.55, 131.69, 128.20, 127.53, 126.27, 107.53.

m.p.: 112–114 °C; H NMR (400 MHz, CDCl ): δ 9.63 (s, 1H),

3

[

2,2′-BITHIOPHENE]-5-CARBALDEHYDE (6D). The general procedure 7.30 (d, J = 3.8 Hz, 1H), 6.86 (d, J = 3.5 Hz, 1H), 6.75 (d, J = 3.8

1

3

was used with 5-bromothiophene-2-carbaldehyde (1.09 g, Hz, 1H), 6.47 (d, J = 3.5 Hz, 1H); C NMR (101 MHz, CDCl

.71 mmol) and thiophen-2-ylboronic acid (1.1 g, 8.57 mmol). δ 177.01, 151.72, 150.11, 146.74, 124.36, 123.17, 114.00,

The product was purified by column chromatography on silica 111.68, 107.83.

gel (n-hexane–CH Cl , 5 : 1) to provide the title compound as a

light brown solid (1.04 g, 94%). m.p.: 44–46 °C; H NMR

3

):

5

2

1

General procedure for preparation of compounds 2a–2d

d, J = 4.5 Hz, 2H), 7.25 (s, 1H), 7.10–7.06 (m, 1H); C NMR NaBH (0.5 equiv) was added, in portions, to 8 or 7 (1 equiv)

(

3

400 MHz, CDCl ): δ 9.86 (s, 1H), 7.67 (d, J = 3.9 Hz, 1H), 7.36

13

4

(

101 MHz, CDCl ): δ 182.48, 147.19, 141.84, 137.19, 136.11, in 10 mL of CH OH–CH Cl (1 : 1) in an ice–water bath

3

2

1

28.38, 127.09, 126.17, 124.28.

for 30 min. The solvent was evaporated, then 1 N HCl

was poured into the mixture, which was then extracted

1

8

5

-(5-BROMOTHIOPHEN-2-YL)FURAN-2-CARBALDEHYDE

(7A). To

a

stirred solution of 6c (1 g, 5.62 mmol) in DMF (10 mL) in the with CH Cl . The combined organic layers were washed

2

2 4

dark, was added NBS (1 g, 5.62 mmol) at −23 °C, and the solu- with brine, dried (Na SO ), and concentrated in vacuo to

tion was then stirred for 4 h. After this time, the reaction aﬀord the title compound as a white solid. These compounds

mixture was poured on ice and stirred to room temperature were used in the next step as soon as possible due to their

and a light yellow solid precipitated, which was isolated by fil- instability.

tration. Recrystallization from DCM–n-hexane gave the pure

compound as a light yellow solid (1.37 g, 95%). H NMR procedure was used with 8a (2 g, 7.78 mmol) and NaBH₄

(5-(5-BROMOFURAN-2-YL)THIOPHEN-2-YL)METHANOL (2A). The general

1

(400 MHz, DMSO): δ 9.57 (s, 1H), 7.65 (d, J = 3.7 Hz, 1H), 7.54 (0.15 g, 3.89 mmol). Aﬀorded the title compound as a white

(

1

d, J = 3.9 Hz, 1H), 7.37 (d, J = 4.0 Hz, 1H), 7.15 (d, J = 3.7 Hz, solid (1.98 g, 99%).

1

3

H); C NMR (101 MHz, DMSO): δ 177.46, 152.37, 151.23,

(5′-BROMO-[2,2′-BIFURAN]-5-YL)METHANOL (2B). The general pro-

1

32.42, 132.02, 127.40, 125.38, 113.94, 108.60.

4

cedure was used with 8b (2 g, 8.3 mmol) and NaBH (0.16 g,

1

8

5′-BROMO-[2,2′-BITHIOPHENE]-5-CARBALDEHYDE (7B). To a stirred 4.15 mmol). Aﬀorded the title compound as a white solid

solution of 6d (1.09 g, 5.62 mmol) in DMF (10 mL) in the dark, (1.96 g, 97%).

was added NBS (1 g, 5.62 mmol) at −23 °C, and the solution

(5-(5-BROMOTHIOPHEN-2-YL)FURAN-2-YL)METHANOL (2C). The general

was then stirred for 4 h. After this time, the reaction mixture procedure was used with 7a (2 g, 7.78 mmol) and NaBH

4

was poured on ice and stirred to room temperature and a light (0.15 g, 3.89 mmol). Aﬀorded the title compound as a white

yellow solid precipitated, which was isolated by filtration. solid (1.98 g, 98%).

Recrystallization from DCM–n-hexane gave the pure compound

as a light yellow solid (1.47 g, 96%). m.p.: 135–137 °C; H NMR procedure was used with 7b (2 g, 7.33 mmol) and NaBH₄

(5′-BROMO-[2,2′-BITHIOPHEN]-5-YL)METHANOL (2D). The general

1

This journal is © The Royal Society of Chemistry 2012

Org. Biomol. Chem., 2012, 10, 9734–9746 | 9741

Paper

(0.14 g, 3.66 mmol). Aﬀorded the title compound as a white 3b (5-formylfuran-2-yl)boronic acid (0.76 g, 5.45 mmol). The

solid (1.96 g, 97%).

product was purified by column chromatography on silica gel

n-hexane–acetone, 1 : 1) to provide the title compound as a

(

General procedure for preparation of compounds 9a–9g,

1

yellow solid (0.45 g, 48%). m.p.: 178–179 °C; H NMR

400 MHz, DMSO): δ 9.59 (s, 1H), 7.68 (d, J = 3.8 Hz, 1H), 7.20

To a solution of heteroaryl halide 2 (1.0 equiv), boronic acid 3 (d, J = 3.7 Hz, 1H), 7.08 (d, J = 3.8 Hz, 1H), 6.89 (d, J = 3.7 Hz,

1.5 equiv), K CO (3.0 equiv), 2-dicyclohexylphosphino-2′,4′,6′- 1H), 6.84 (d, J = 3.3 Hz, 1H), 6.48 (d, J = 3.3 Hz, 1H), 5.38 (t, J =

triisopropylbiphenyl (X-Phos, 0.02 equiv) in CH CN–H O 5.8 Hz, 1H), 4.47 (d, J = 5.8 Hz, 2H); C NMR (101 MHz,

1

2a–12b

(

2

3

1

3

2

(1.5 : 1) was added Pd(OAc) (0.01 equiv). The flask and its con- DMSO): δ 178.00, 156.87, 151.93, 150.09, 147.47, 144.31,

tents were put under reduced pressure then backfilled with 143.60, 125.88, 112.81, 109.77, 108.98, 108.75, 108.31, 56.08;

+

argon three times. The mixture was then stirred at room temp- HRMS (ESI): m/z calcd for C H O [M + H] : 259.0528; found:

1

4

10 5

erature for 24 h under argon atmosphere, the solvent was 259.0602.

removed in vacuo to aﬀord a crude product, which was purified

by flash chromatography (eluting with n-hexane–acetone (9E). The general procedure was used with 2c (0.94 g,

mixtures). 3.64 mmol) and 3a (5-formylthiophen-2-yl)boronic acid

-(5-(5-(HYDROXYMETHYL)THIOPHEN-2-YL)FURAN-2-YL)THIOPHENE-2- (0.85 g, 5.45 mmol). The product was purified by column

CARBALDEHYDE (9A). The general procedure was used with 2a chromatography on silica gel (n-hexane–acetone, 3 : 1) to

0.94 g, 3.64 mmol) and 3a (5-formylthiophen-2-yl)boronic provide the title compound as a brown solid (0.67 g, 63%).

5′-(5-(HYDROXYMETHYL)FURAN-2-YL)-[2,2′-BITHIOPHENE]-5-CARBALDEHYDE

5

(

1

3

acid (0.85 g, 5.45 mmol). The product was purified by column m.p.: 136–138 °C; H NMR (400 MHz, CDCl ): δ 9.86 (s, 1H),

chromatography on silica gel (n-hexane–acetone, 3 : 1) to 7.68 (d, J = 4.0 Hz, 1H), 7.29 (d, J = 4.0 Hz, 1H), 7.24 (d, J = 4.0

provide the title compound as a brown solid (0.68 g, 64%). Hz, 1H), 7.20 (d, J = 4.0 Hz, 1H), 6.53 (d, J = 3.2 Hz, 1H), 6.39

1

13

m.p.: 131–133 °C; H NMR (400 MHz, CDCl

.69 (d, J = 4.0 Hz, 1H), 7.34 (d, J = 4.0 Hz, 1H), 7.22 (d, J = 3.7 182.36, 154.14, 148.54, 146.86, 141.74, 137.28, 134.96, 134.54,

Hz, 1H), 6.97 (d, J = 3.6 Hz, 1H), 6.79 (d, J = 3.6 Hz, 1H), 6.58 126.83, 124.08, 123.64, 110.26, 107.18, 57.56; HRMS (ESI): m/z

3 3

): δ 9.87 (s, 1H), (d, J = 3.2 Hz, 1H), 4.67 (s, 2H); C NMR (101 MHz, CDCl ): δ

7

1

3

+

(

d, J = 3.6 Hz, 1H), 4.85 (s, 2H); C NMR (101 MHz, CDCl

3

): δ calcd for C14

82.50, 150.37, 147.27, 144.30, 142.30, 141.52, 137.18, 132.66,

26.12, 123.50, 122.90, 111.08, 107.73, 60.15; HRMS (ESI): m/z DEHYDE (9F). The general procedure was used with 2c (0.94 g,

10

3 2

H O S [M + H] : 291.0071; found: 291.0152.

5-(5-(5-(HYDROXYMETHYL)FURAN-2-YL)THIOPHEN-2-YL)FURAN-2-CARBAL-

1

+

calcd for C14

′-(5-(HYDROXYMETHYL)THIOPHEN-2-YL)-[2,2′-BIFURAN]-5-CARBALDEHYDE 5.45 mmol). The product was purified by column chromato-

9B). The general procedure was used with 2a (0.94 g, graphy on silica gel (n-hexane–acetone, 2 : 1) to provide the

H

10

O

3

S

2

[M + H] : 291.0071; found: 291.0153.

3.64 mmol) and 3b (5-formylfuran-2-yl)boronic acid (0.76 g,

5

(

3

5

.64 mmol) and 3b (5-formylfuran-2-yl)boronic acid (0.76 g, title compound as a yellow solid (0.56 g, 56%). m.p.:

1

.45 mmol). The product was purified by column chromato- 133–135 °C; H NMR (400 MHz, CDCl ): δ 9.56 (s, 1H),

3

graphy on silica gel (n-hexane–acetone, 2 : 1) to provide the 7.68–7.66 (m, 2H), 7.42 (d, J = 4.0 Hz, 1H), 7.15 (d, J = 3.6 Hz,

title compound as a yellow solid (0.54 g, 54%). m.p.: 1H), 6.87 (d, J = 3.2 Hz, 1H), 6.45 (d, J = 3.2 Hz, 1H), 5.34 (t, J =

1

13

1

18–120 °C; H NMR (400 MHz, CDCl ): δ 9.59 (s, 1H), 7.31 (d, 5.6 Hz, 1H), 4.44 (d, J = 5.6 Hz, 2H). 4.67 (s, 2H); C NMR

3

J = 4.0 Hz, 1H), 7.22 (d, J = 3.6 Hz, 1H), 6.96–6.94 (m, 2H), 6.77 (101 MHz, CDCl

3

): δ 177.16, 155.88, 153.37, 151.03, 146.92,

1

3

(d, J = 4.0 Hz, 1H), 6.59 (d, J = 3.6 Hz, 1H), 4.84 (s, 2H);

C

134.60, 128.68, 127.84, 125.74, 123.71, 109.60, 108.44, 107.90,

+

NMR (101 MHz, DMSO): δ 176.88, 151.69, 151.14, 150.90, 55.47; HRMS (ESI): m/z calcd for C H O S [M + H] :

1

4

10 4

+

1

2

44.55, 143.79, 132.52, 126.02, 124.27, 123.64, 111.95, 107.57, 275.0300; found: 275.0372; [M + Na] : 297.0192; found:

07.48, 60.07; HRMS (ESI): m/z calcd for C14H O S [M + H] : 297.0241, [M + K] : 313.9935; found: 313.0032.

10 4

+

75.0300; found: 275.0386.

5′′-(HYDROXYMETHYL)-[2,2′:5′,2′′-TERTHIOPHENE]-5-CARBALDEHYDE

5

-(5′-(HYDROXYMETHYL)-[2,2′-BITHIOPHEN]-5-YL)FURAN-2-CARBALDEHYDE (9G). The general procedure was used with 2d (5 g, 18.2 mmol)

(9C). The general procedure was used with 2d (1 g, 3.64 mmol) and 3a (5-formylthiophen-2-yl)boronic acid (4.25 g,

and 3b (5-formylfuran-2-yl)boronic acid (0.76 g, 5.45 mmol). 27.25 mmol). The product was purified by column chromato-

The product was purified by column chromatography on silica graphy on silica gel (n-hexane–acetone, 3 : 1) to provide the

gel (n-hexane–acetone, 3 : 1) to provide the title compound as a title compound as

a

brown solid (4.9 g, 88%). m.p.:

brown solid (0.62 g, 59%). m.p.: 126–128 °C; H NMR 166–167 °C; H NMR (400 MHz, DMSO): δ 9.89 (s, 1H), 8.00 (d,

400 MHz, DMSO): δ 9.56 (s, 1H), 7.66 (dd, J = 6.9, 3.9 Hz, 2H), J = 4.0 Hz, 1H), 7.56 (dd, J = 8.0, 3.9 Hz, 2H), 7.32 (d, J =

1

(

7

.36 (d, J = 3.9 Hz, 1H), 7.29 (d, J = 3.6 Hz, 1H), 7.15 (d, J = 3.8 3.9 Hz, 1H), 7.27 (d, J = 3.6 Hz, 1H), 6.95 (d, J = 3.6 Hz, 1H),

1

3

13

Hz, 1H), 6.96 (d, J = 3.5 Hz, 1H), 5.61 (s, 1H), 4.64 (s, 2H);

C

5.61 (t, J = 5.7 Hz, 1H), 4.64 (d, J = 5.7 Hz, 2H); C NMR

NMR (101 MHz, DMSO): δ 177.17, 153.29, 151.06, 147.28, (101 MHz, DMSO): δ 183.65, 147.23, 144.99, 141.14, 139.07,

1

2

38.91, 134.09, 128.74, 127.92, 125.67, 125.05, 124.72, 124.59, 138.51, 134.08, 133.27, 127.95, 125.08, 125.03, 124.83, 124.52,

+

08.41, 58.24; HRMS (ESI): m/z calcd for C H O S [M + H] : 58.24; HRMS (ESI): m/z calcd for C H O S [M + H] :

1

4

10

3

2

14 10 2 3

91.0071; found: 291.0156.

306.9843; found: 306.9917.

5′′-(HYDROXYMETHYL)-[2,2′:5′,2′′-TERFURAN]-5-CARBALDEHYDE (9D). The

1-(5′-(5-(HYDROXYMETHYL)FURAN-2-YL)-[2,2′-BITHIOPHEN]-5-YL)ETHA-

general procedure was used with 2b (0.88 g, 3.64 mmol) and NONE (12A). The general procedure was used with 2c (0.94 g,

9

742 | Org. Biomol. Chem., 2012, 10, 9734–9746

This journal is © The Royal Society of Chemistry 2012

Organic & Biomolecular Chemistry

3

.64 mmol) and 11 [2-(5-boronothiophen-2-yl)-2-oxoethan-1- (101 MHz, DMSO): δ 156.02, 145.29, 144.80, 109.62, 107.74,

+

ylium (0.92 g, 5.45 mmol)]. The product was purified by 107.26, 56.00; HRMS (ESI): m/z calcd for C H O [M + H] :

column chromatography on silica gel (n-hexane–acetone, 5 : 1) 261.0685; found: 261.0763; [M + Na] : 283.0577; found:

to provide the title compound as a yellow solid (0.71 g, 64%). 283.0582; [M + K] : 299.0317; found: 299.0400.

m.p.: 165–167 °C; H NMR (400 MHz, DMSO): δ 7.91 (d, J =

1

4

12 5

+

1

(5-(5′-(HYDROXYMETHYL)-[2,2′-BITHIOPHEN]-5-YL)FURAN-2-YL)METHANOL

1

4

7

3

2

1

C

.0 Hz, 1H), 7.53 (d, J = 3.9 Hz, 1H), 7.46 (d, J = 4.0 Hz, 1H), (1D). Yellow solid (0.29 g, 98%). m.p.: 135–137 °C; H NMR

.36 (d, J = 3.9 Hz, 1H), 6.83 (d, J = 3.3 Hz, 1H), 6.44 (d, J = (400 MHz, DMSO): δ 7.27 (d, J = 3.8 Hz, 1H), 7.23 (d, J = 3.8 Hz,

.3 Hz, 1H), 5.35 (t, J = 5.8 Hz, 1H), 4.44 (d, J = 5.8 Hz, 2H), 1H), 7.17 (d, J = 3.6 Hz, 1H), 6.92 (d, J = 3.6 Hz, 1H), 6.73 (d, J =

1

3

.53 (s, 3H); C NMR (101 MHz, DMSO): δ 190.83, 156.28, 3.3 Hz, 1H), 6.41 (d, J = 3.3 Hz, 1H), 5.56 (s, 1H), 5.31 (s, 1H),

1

3

47.51, 144.50, 142.54, 135.59, 134.21, 127.76, 125.46, 124.28, 4.62 (s, 2H), 4.42 (s, 2H); C NMR (101 MHz, DMSO): δ 155.17,

10.10, 108.13, 63.27, 26.78; HRMS (ESI): m/z calcd for 147.29, 146.11, 135.25, 134.74, 131.25, 124.94, 124.23, 123.47,

+

15

12 3 2

H O S [M + H] : 305.0228; found: 305.0306, [M + Na] : 123.39, 109.36, 106.50, 58.23, 55.50; HRMS (ESI): m/z calcd for

+

−

3

27.012; found: 327.0126, [M + K] : 342.9860; found: 342.9943.

14 12 3 2

C H O S [M − H] : 291.0228; found: 291.0115.

1

-(5′′-(HYDROXYMETHYL)-[2,2′:5′,2′′-TERTHIOPHEN]-5-YL)ETHANONE

(5,5′-(THIOPHENE-2,5-DIYL)BIS(FURAN-5,2-DIYL))DIMETHANOL

(1E).

12B). The general procedure was used with 2d (1 g, Yellow solid (0.27 g, 97%). m.p.: 100–101 °C; H NMR

.64 mmol) and 11 [2-(5-boronothiophen-2-yl)-2-oxoethan-1- (400 MHz, DMSO): δ 7.30 (s, 2H), 6.73 (d, J = 3.3 Hz, 2H), 6.41

1

(

3

1

3

ylium (0.92 g, 5.45 mmol)]. The product was purified by (d, J = 3.2 Hz, 2H), 5.33 (s, 2H), 4.43 (s, 4H); C NMR

column chromatography on silica gel (n-hexane–acetone, 3 : 1) (101 MHz, DMSO): δ 155.74, 147.90, 131.74, 123.86, 109.92,

+

to provide the title compound as a brown solid (0.79 g, 68%). 107.09, 56.07; HRMS (ESI): m/z calcd for C14

m.p.: 206–207 °C; H NMR (400 MHz, DMSO): δ 7.91 (d, J = 277.0456; found: 277.0532.

H

12

O

4

S [M + H] :

1

4

7

3

.0 Hz, 1H), 7.50 (d, J = 3.9 Hz, 1H), 7.45 (d, J = 4.0 Hz, 1H),

[2,2′:5′,2′′-TERTHIOPHENE]-5,5′′-DIYLDIMETHANOL (1F). Yellow solid

1

.29 (d, J = 3.9 Hz, 1H), 7.24 (d, J = 3.6 Hz, 1H), 6.95 (d, J = (0.3 g, 98%). m.p.: 196–198 °C; H NMR (400 MHz, DMSO): δ

13

.6 Hz, 1H), 5.60 (s, 1H), 4.63 (s, 2H), 2.53 (s, 3H); C NMR 7.20 (s, 2H), 7.16 (d, J = 3.6 Hz, 2H), 6.92 (d, J = 3.5 Hz, 2H),

1

3

(101 MHz, DMSO): δ 190.81, 147.57, 144.43, 142.55, 138.47, 5.56 (t, J = 5.7 Hz, 2H), 4.62 (d, J = 5.7 Hz, 4H); C NMR

1

5

3

35.56, 134.75, 134.16, 127.86, 125.58, 125.47, 125.29, 124.89, (101 MHz, DMSO): δ 146.11, 135.29, 134.70, 124.92, 124.34,

+

−

8.80, 26.78; HRMS (ESI): m/z calcd for C H O S [M + H] : 123.49, 58.23; HRMS (ESI): m/z calcd for C H O S [M − H] :

1

5

12

2

3

14 12 2 3

20.9999; found: 321.0081.

306.9999; found: 306.9923.

2

4

General procedure for preparation of compounds 1a–1f

General procedure for preparation of compounds 10a–10d

(0.5 All reactions were performed on a 1.0 mmol scale. To a solu-

NBr (0.04 equiv) in CH Cl –acetone

1 : 1, for a final concentration of 0.1 M) in an ice–water bath (2 : 1, for a final concentration of 0.1 M) was added TEMPO

for 30 min. The solvent was evaporated in vacuo, CH Cl was (0.01 equiv) and Oxone (2.2 equiv). The mixture was then

poured into the mixture and, after filtration, the filter cake was stirred for 1–3 h at room temperature and the solvent was

All reactions were performed on a 1.0 mmol scale. NaBH

4

equiv) was added, in portions, to 9 (1 equiv) in CH

3

OH–THF tion of 9 (1 equiv) and Bu

4

2

(

2

washed with CH Cl to aﬀord the title compound.

removed under reduced pressure. The product was purified by

2

5

,5′-(FURAN-2,5-DIYL)BIS(THIOPHENE-5,2-DIYL))DIMETHANOL

(RITA : column chromatography on silica gel (eluting with n-hexane–

1

A). Yellow solid (0.29 g, 98%). m.p.: 158–160 °C; H NMR acetone mixtures) to provide the title compound.

(400 MHz, DMSO): δ 7.24 (d, J = 3.6 Hz, 2H), 6.95 (d, J = 3.6 Hz,

5,5′-(FURAN-2,5-DIYL)BIS(THIOPHENE-2-CARBALDEHYDE) (10A). Stirred

2

4

1

H), 6.79 (s, 2H), 5.57 (t, J = 5.7 Hz, 2H), 4.64 (d, J = 5.7 Hz, for 2 h at room temperature, purified by silica gel column

1

3

H); C NMR (101 MHz, DMSO): δ 147.80, 145.94, 131.00, chromatography (n-hexane–acetone, 6 : 1) to give a brown solid

1

24.72, 122.48, 107.29, 58.21; HRMS (ESI): m/z calcd for (0.25 g, 86%). m.p.: 186–187 °C; H NMR (400 MHz, DMSO): δ

+

C

14

H

12

O

3

S

2

[M + H] : 293.0228; found: 293.0302.

9.93 (s, 2H), 8.06 (d, J = 3.9 Hz, 2H), 7.71 (d, J = 3.9 Hz, 2H),

1

3

(

5-(5′-(HYDROXYMETHYL)-[2,2′-BIFURAN]-5-YL)THIOPHEN-2-YL)METHANOL 7.35 (s, 2H); C NMR (101 MHz, DMSO): δ 183.90, 148.34,

1

(

1B). Yellow solid (0.26 g, 95%). m.p.: 142–144 °C; H NMR 141.86, 139.70, 138.77, 125.03, 112.33; HRMS (ESI): m/z calcd

400 MHz, CD OD): δ 7.19 (d, J = 3.6 Hz, 1H), 6.94 (d, J = 3.6 for C14H O S [M + H] : 288.9915; found: 288.9993.

3 8 3 2

+

Hz, 1H), 6. 64 (d, J = 3.6 Hz, 1H), 6.62 (d, J = 3.6 Hz, 1H), 6.58

5′-(5-FORMYLTHIOPHEN-2-YL)-[2,2′-BIFURAN]-5-CARBALDEHYDE

(10B).

(

d, J = 3.2 Hz, 1H), 6.41 (d, J = 3.2 Hz, 1H), 4.73 (s, 2H), 4.56 (s, Stirred for 3 h at room temperature, purified by silica gel

1

3

2

1

3

H); C NMR (101 MHz, CD OD): δ 155.90, 150.25, 147.27, column chromatography (n-hexane–acetone: 5 : 1) to give a

1

46.87, 145.74, 134.09, 126.78, 123.58, 110.44, 108.28, 107.71, brown solid (0.22 g, 80%). m.p.: 202–204 °C; H NMR

07.09, 60.06, 57.43; HRMS (ESI): m/z calcd for C H O S (400 MHz, DMSO): δ 9.91 (s, 1H), 9.65 (s, 1H), 7.72 (d, J =

1

4

12 4

+

[M + H] : 277.04568; found: 277.0535; [M + Na] : 299.0348; 4.0 Hz, 1H), 7.43 (d, J = 4.0 Hz, 1H), 7.33 (d, J = 3.6 Hz, 1H),

+

13

found: 299.0354; [M + K] : 315.0088; found: 315.0093.

(1C). Gray

(

7.00 (d, J = 3.6 Hz, 1H), 6.87–6.86 (m, 2H); C NMR (101 MHz,

[

2,2′:5′,2′′-TERFURAN]-5,5′′-DIYLDIMETHANOL

solid DMSO): δ 182.50, 177.00, 152.14, 150.25, 149.49, 145.48,

2

8

1

0.25 g, 95%). m.p.: 186–188 °C. (lit. 190–192 °C); H NMR 142.58, 141.20, 136.89, 124.07, 123.15, 111.94, 110.90, 108.60;

400 MHz, DMSO): δ 6.74 (s, 2H), 6.68 (d, J = 3.3 Hz, 2H), 6.41 HRMS (ESI): m/z calcd for C14H O S [M + H] : 273.0143; found:

8 4

d, J = 3.3 Hz, 2H), 5.35 (s, 2H), 4.42 (s, 4H); C NMR 273.0223.

+

1

3

Org. Biomol. Chem., 2012, 10, 9734–9746 | 9743

Paper

5

-(5′-FORMYL-[2,2′-BITHIOPHEN]-5-YL)FURAN-2-CARBALDEHYDE

(10C). 9.61 (s, 1H), 7.44 (d, J = 3.9 Hz, 1H), 7.29 (d, J = 3.8 Hz, 1H),

Stirred for 2.5 h at room temperature, purified by silica gel 7.20 (d, J = 3.9 Hz, 1H), 6.66 (d, J = 3.7 Hz, 1H), 6.54 (d, J = 3.3

column chromatography (n-hexane–acetone: 6 : 1) to give a Hz, 1H), 6.34 (d, J = 3.3 Hz, 1H), 4.77 (s, 2H), 1.25–1.07 (m,

1

13

brown solid (0.23 g, 81%). m.p.: 212–214 °C; H NMR 21H); C NMR (101 MHz, CDCl

400 MHz, DMSO): δ 9.91 (s, 1H), 9.59 (s, 1H), 8.04 (d, J = 3.9 151.63, 147.86, 135.91, 129.54, 126.94, 123.48, 123.06, 109.28,

Hz, 1H), 7.74 (d, J = 3.9 Hz, 1H), 7.73–7.63 (m, 3H), 7.25 (d, J = 107.51, 107.28, 58.61, 17.99, 12.13. HR-MS(ESI): calcd. for

3

): δ 176.72, 154.97, 154.66,

(

1

3

+

3

1

.8 Hz, 1H); C NMR (101 MHz, DMSO): δ 183.88, 177.50,

52.65, 151.37, 144.18, 141.94, 139.01, 136.71, 131.74, 128.13,

26.10, 125.47, 109.45; HRMS (ESI): m/z calcd for C14

23

30 4

C H O SSi [M + H] : 431.1634; found: 431.1719.

5′′-(((TRIISOPROPYLSILYL)OXY)METHYL)-[2,2′:5′,2′′-TERFURAN]-5-CARB-

ALDEHYDE (13D). Purification via silica gel column chromato-

graphy (n-hexane–EtOAc: 70 : 1) to give a yellow solid (0.35 g,

H

8

O

3

S

2

+

[M + H] : 288.9915; found: 288.9998.

1

[

2,2′:5′,2′′-TERTHIOPHENE]-5,5′′-DICARBALDEHYDE (10D). Stirred for 84%). m.p.: 61–63 °C; H NMR (400 MHz, CDCl ): δ 9.61 (s,

3

1

.5 h at room temperature, purified by silica gel column 1H), 7.31 (d, J = 3.8 Hz, 1H), 6.96 (d, J = 3.6 Hz, 1H), 6.77 (d, J =

chromatography (n-hexane–acetone: 7 : 1) to give a brown solid 3.8 Hz, 1H), 6.67–6.60 (m, 2H), 6.36 (d, J = 3.3 Hz, 1H), 4.78 (s,

1

13

(

0.26 g, 87%). m.p.: 209–212 °C; H NMR (400 MHz, DMSO): δ 2H), 1.09 (dd, J = 12.1, 5.4 Hz, 21H); C NMR (101 MHz,

9

3

.91 (s, 2H), 8.03 (d, J = 3.9 Hz, 2H), 7.67 (s, 2H), 7.64 (d, J = CDCl ): δ 176.75, 155.15, 151.70, 151.16, 147.99, 144.87,

1

3

.9 Hz, 2H); C NMR (101 MHz, DMSO): δ = 183.85, 144.16, 143.75, 123.39, 111.61, 108.86, 107.49, 107.44, 107.13, 58.58,

+

1

41.91, 138.98, 136.35, 128.25, 126.03; HRMS (ESI): m/z calcd 17.93, 12.09; HRMS (ESI): m/z calcd for C H O Si [M + H] :

2

3

30 5

+

for C14

H

8

O

2

S

3

[M + H] : 304.9686; found: 304.9766.

415.1863; found: 415.1939.

General procedure for preparation of compounds 13a–13d

General procedure for preparation of compounds 14a–14b

All reactions were performed on a 1.0 mmol scale. Imidazole

GENERAL PROCEDURES FOR PREPARATION OF OXIMES. All reactions

(2.5 equiv) was added to a solution of 9 (1 equiv) and triisopro- were performed on a 1.0 mmol scale. A mixture of 13 (1 equiv),

pylchlorosilane (1.1 equiv) in anhydrous THF (for a final con- NH OH·HCl (1.5 equiv), AcONa (1.5 equiv) and CH OH (for a

2

3

centration of 0.1 M). The mixture was stirred for 12 h, diluted final concentration of 0.1 M) was refluxed for 2 h and, after fil-

with 5% NaHCO , extracted with EtOAc, washed with brine, tration, the filter cake was washed with EtOAc three times,

dried (Na SO ) and concentrated in vacuo. The residue was then the solvent was removed in vacuo to aﬀord the residue,

3

2

4

purified by column chromatography on silica gel (eluting with which was purified by column chromatography on silica gel

n-hexane–EtOAc mixtures) to provide the title compound as a (eluting with n-hexane–EtOAc mixtures) to provide the title

yellow solid.

compound as a yellow solid.

5

-(5-(5-(((TRIISOPROPYLSILYL)OXY)METHYL)THIOPHEN-2-YL)FURAN-2-YL)-

(E)-5′′-(((TRIISOPROPYLSILYL)OXY)METHYL)-[2,2′:5′,2′′TERTHIOPHENE]-5-

THIOPHENE-2-CARBALDEHYDE (13A). Purification via silica gel CARBALDEHYDE OXIME (14A). Purification via silica gel column

column chromatography (n-hexane–EtOAc: 70 : 1) to give a chromatography (n-hexane–EtOAc: 30 : 1) to give a yellow solid

1

yellow solid (0.39 g, 87%). m.p.: 57–59 °C; H NMR (400 MHz, (0.22 g, 46%). m.p.: 127–131 °C; H NMR (400 MHz, CDCl

CDCl ): δ 11.86–11.81 (m, 1H), 9.87 (s, 1H), 7.68 (d, J = 4.0 Hz, 8.22 (s, 1H), 7.36 (s, 1H), 7.11 (d, J = 3.8 Hz, 1H), 7.08 (q, J =

H), 7.34 (d, J = 4.0 Hz, 1H), 7.21 (d, J = 3.6 Hz, 1H), 6.87 (d, J = 3.8 Hz, 2H), 7.05 (d, J = 3.8 Hz, 1H), 7.03 (d, J = 3.6 Hz, 1H),

3

): δ

3

1

3

.6 Hz, 1H), 6.78 (d, J = 3.6 Hz, 1H), 6.56 (d, J = 3.6 Hz, 1H), 6.82 (d, J = 3.6 Hz, 1H), 4.94 (s, 2H), 1.23–1.07 (m, 21H);

.97 (s, 2H), 1.28–1.06 (m, 22H); C NMR (101 MHz, CDCl ): δ NMR (101 MHz, CDCl ): δ = 145.69, 145.06, 139.25, 137.78,

3 3

C

1

3

4

1

82.43, 150.87, 147.03, 146.44, 142.50, 141.39, 137.15, 131.42, 135.72, 135.15, 133.95, 130.54, 125.26, 124.11, 123.88, 123.50,

1

23.81, 123.40, 122.75, 111.12, 107.31, 61.28, 18.03, 12.09; 123.36, 61.30, 18.04, 12.10; HRMS (ESI): m/z calcd for

+

HRMS (ESI): m/z calcd for C23

H

30

O

3

S

2

Si [M + H] : 447.1406;

23 2 3

C H31NO S Si [M + H] : 478.1286; found: 478.1359.

found: 447.1490.

(Z)-5′′-(((TRIISOPROPYLSILYL)OXY)METHYL)-[2,2′:5′,2′′TERTHIOPHENE]-5-

5′′-(((TRIISOPROPYLSILYL)OXY)METHYL)-[2,2′:5′,2′′-TERTHIOPHENE]-5-CAR- CARBALDEHYDE OXIME (14B). Purification via silica gel column

BALDEHYDE (13B). Purification via silica gel column chromato- chromatography (n-hexane–EtOAc: 20 : 1) to give a yellow solid

1

graphy (n-hexane–EtOAc: 80 : 1) to give a yellow solid (0.40 g, (0.23 g, 48%). m.p.: 122–124 °C; H NMR (400 MHz, CDCl ): δ

3

1

8

1

4

6

6%). m.p.: 61–63 °C; H NMR (400 MHz, CDCl

3

): δ 9.85 (s, 7.69 (s, 1H), 7.28 (d, J = 3.9 Hz, 1H), 7.16 (d, J = 3.8 Hz, 1H),

H), 7.66 (d, J = 4.0 Hz, 1H), 7.25 (d, J = 3.6 Hz, 1H), 7.21 (d, J = 7.14 (d, J = 3.9 Hz, 1H), 7.06 (d, J = 3.8 Hz, 1H), 7.03 (d, J = 3.6

.0 Hz, 1H), 7.09 (d, J = 4.0 Hz, 1H), 6.06 (d, J = 3.6 Hz, 1H), Hz, 1H), 6.82 (d, J = 3.6 Hz, 1H), 4.94 (s, 2H), 1.23–1.02 (m,

1

3

13

.83 (d, J = 4.0 Hz, 1H), 4.95 (s, 2H), 1.11–1.09 (m, 21H);

C

21H); C NMR (101 MHz, CDCl

3

): δ 145.64, 143.07, 141.31,

NMR (101 MHz, CDCl

3

): δ 182.30, 145.99, 146.41, 141.59, 137.99, 135.77, 135.14, 132.73, 129.27, 125.18, 124.18, 123.90,

1

C

39.73, 137.26, 135.25, 134.18, 126.89, 124.29, 124.03, 123.94, 123.48, 122.58, 61.30, 18.03, 12.10; HRMS (ESI): m/z calcd for

+

23.89, 61.29, 18.03, 12.09; HRMS (ESI): m/z calcd for

23

(Z)-5-(5-(5-(((TRIISOPROPYLSILYL)OXY)METHYL)THIOPHEN-2-YL)FURAN-2-

YL)THIOPHENE-2-CARBALDEHYDE OXIME (14C). Purification via silica

2 3

C H31NO S Si [M + H] : 478.1286; found: 478.1360.

+

23

H

30

O

2

S

3

Si [M + H] : 463.1177; found: 463.1268.

5

-(5-(5-(((TRIISOPROPYLSILYL)OXY)METHYL)FURAN-2-YL)THIOPHEN-2-YL)-

FURAN-2-CARBALDEHYDE (13C). Purification via silica gel column gel column chromatography (n-hexane–EtOAc: 20 : 1) to give a

1

chromatography (n-hexane–EtOAc: 70 : 1) to give a yellow solid yellow solid (0.21 g, 45%). m.p.: 125–127 °C; H NMR

1

(

0.38 g, 89%). m.p.: 81–83 °C; H NMR (400 MHz, CDCl ): δ (400 MHz, CDCl ): δ 7.69 (s, 1H), 7.31 (d, J = 3.9 Hz, 1H), 7.27

3

9

744 | Org. Biomol. Chem., 2012, 10, 9734–9746

Organic & Biomolecular Chemistry

s, 1H), 7.18 (d, J = 3.6 Hz, 1H), 6.85 (d, J = 2.8 Hz, 1H), 6.66 (d, Na

(

3

VO

4

). Liquids were kept on ice for 30 min, centrifuged

J = 3.5 Hz, 1H), 6.53 (d, J = 3.5 Hz, 1H), 4.96 (s, 2H), 1.13 (dd, (20 000 rpm, 10 min at 4 °C) and supernatants were used for

1

3

J = 20.8, 6.1 Hz, 21H); C NMR (101 MHz, CDCl

3

) δ 149.80, the assay. Equal amounts of protein extracts were fractionated

47.78, 145.55, 141.68, 138.86, 132.66, 131.97, 130.33, 123.83, by SDS-PAGE and transferred to nitrocellulose membranes.

22.73, 121.52, 108.94, 107.12, 61.28, 18.03, 12.10. HRMS After blocking in 5% non-fat dried milk at room temperature

1

+

(ESI): m/z calcd for C23

H31NO

3

S

2

Si [M + H] : 462.1515; found: for 2 h, the membrane was incubated with primary antibodies

4

62.1596.

and β-actin at 4 °C overnight. The membrane was then incu-

(

5′′-(((TRIISOPROPYLSILYL)OXY)METHYL)-[2,2′:5′,2′′-TERTHIOPHEN]-5-YL)- bated with horseradish peroxidase-conjugated goat secondary

METHANOL (15). NaBH (0.5 equiv) was added, in portions, to 9 antibody for 1.5 h at room temperature, and detected with

OH–THF (1 : 1, for a final concentration of SuperSignal West Pico Chemiluminescent Substrate (Pierce,

.1 M) in an ice–water bath for 30 min. The solvent was evapor- Thermo). The image was obtained using the Chemi Doc XRS

ated in vacuo, then 1 M HCl was poured into the mixture, which imaging system (Bio-Rad).

was then extracted with CH Cl , washed with brine, dried

Na SO ), and concentrated in vacuo to aﬀord 15 (0.454 g, 98%) Real-time reverse transcription-PCR analysis of microRNA and

4

(1 equiv) in CH

3

0

2

(

2

4

1

29

as a yellow solid: m.p.: 32–34 °C; H NMR (400 MHz, CDCl

.03 (s, 2H), 7.02–6.98 (m, 2H), 6.89 (d, J = 3.6 Hz, 1H), 6.81 (d,

J = 3.6 Hz, 1H), 4.93 (s, 2H), 4.79 (s, 2H), 1.19–1.07 (m, 21H);

3

): δ mRNA expression

7

The total RNA from HCT116 was harvested using TRIzol (Invi-

trogen) according to the manufacturer’s protocol. For detecting

microRNA expression, reverse reactions were performed using

TaqMan MicroRNA Assay kit (Assay no. 4427975; Applied Bio-

systems, USA). Five micrograms of total RNA were used for the

synthesis of first-strand cDNA using the SuperScript III First

Strand cDNA Synthesis kit (Invitrogen), following the manufac-

turer’s instructions. Real-time PCR analysis was performed

using ABI prism 7500 (Applied Biosystems, USA) with a

TaqMan probe provided by the manufacturer. The TaqMan

probes used were hsa-mir-34a (Assay no. 4427975; Applied Bio-

systems, USA) and RNU66 (Assay no. 4427975; Applied Biosys-

tems, USA). The relative amounts of the mRNA and microRNA

targeted gene were normalized by the amount of RNU66

transcript.

1

3

C NMR (101 MHz, CDCl ) δ 145.25, 143.03, 137.50, 136.81,

35.94, 135.78, 126.24, 126.19, 124.29, 123.95, 123.85, 123.21,

23.18, 61.27, 60.17, 18.01, 12.06, 12.05; HRMS (ESI): m/z calcd

1

−

for C23

H

O S

32 2 3

Si [M − H] : 463.1334; found: 463.1247.

Molecular docking

Docking studies on the representative RITA and compound 1f

were performed with the molecular modeling package Discov-

ery Studio.3.1/Libdock protocol (Accelrys Software Inc.). The

protein’s three dimensional structure from the Brookhaven

Protein Data Bank (PDB) was used as a raw model for the

docking studies on the X-ray structure of HDM2 in complex

with the p53 transactivation domain (PDB code: 1YCR). The

general procedure is as follows: (1) ligand and receptor prep-

aration, (2) protocol generation, (3) docking and scoring, (4)

Acknowledgements

analysis of the results. The Discovery Studio.3.1/Standard Financial supports from the Ministry of Science and Techno-

Dynamics Cascade protocol (Accelrys Software Inc.) was used logy of China (2012ZX09506001-010, 2012CB722605,

for molecular dynamics (MD) simulations. The general pro- 2012AA020305 and 2011DFA30620) and the Chinese National

cedure for MD is as follows: (1) preparing ligand–receptor Natural Science Foundation (21272134) are gratefully

complex; (2) performing MD simulation, (3) drawing step– acknowledged.

potential energy curve and recording the lowest energy confor-

The authors would like to thank Prof. Weige Zhang and

mation, (4) calculating binding energy with the MM/PBSA Prof. Yuzong Chen for their helpful comments on the

protocol.

manuscript.

Biological assays

Notes and references

Western blot analysis

1

(a) M. R. Junttila and G. I. Evan, Nat. Rev. Cancer, 2009, 9,

821; (b) D. Menendez, A. Inga and M. A. Resnick, Nat. Rev.

Cancer, 2009, 9, 724.

The preparation of cell extracts and Western blots were both

performed according to standard procedures. Antibodies for

immunoblotting were as follows: p53 (Cell Signaling, No.

2 (a) S. Fang, J. P. Jensen, R. L. Ludwig, K. H. Vousden and

A. M. Weissman, J. Biol. Chem., 2000, 275, 8945;

(b) R. Honda and H. Yasuda, Oncogene, 2000, 19, 1473.

3 Y. Rew, D. Sun, F. Gonzalez-Lopez De Turiso,

M. D. Bartberger, H. P. Beck, J. Canon, A. Chen, D. Chow,

J. Deignan, B. M. Fox, D. Gustin, X. Huang, M. Jiang,

X. Jiao, L. Jin, F. Kayser, D. J. Kopecky, Y. Li, M. C. Lo,

9

282), CDK4 (Santa Cruz, no. sc-260), E2F1 (Santa Cruz, no.

sc-193), β-actin (Beyotime Biotechnology, China), horseradish

peroxidase-conjugated secondary antibodies (KPL, USA).

Treated cells were washed with PBS and lysed in buﬀer (10 mM

HEPES, pH 7.9, 10 mM KCl, 1 mM EDTA, 0.1% NP-40, 1 mM

DTT, 1 mM PMSF, 1 mM Pepstatin, 1 mM leupeptin, 0.5 mM

Org. Biomol. Chem., 2012, 10, 9734–9746 | 9745

Paper