(FGFR1) Inhibitors - American Chemical Society

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Acenaphtho[1,2-b]pyrrole-Based Selective Fibroblast Growth Factor Receptors 1 (FGFR1) Inhibitors: Design, Synthesis, and Biological Activity Zhuo Chen,†,‡ Xin Wang,† Weiping Zhu,† Xianwen Cao,† Linjiang Tong,‡ Honglin Li,† Hua Xie,*,‡ Yufang Xu,*,† Shaoying Tan,† Dong Kuang,† Jian Ding,‡ and Xuhong Qian*,† †

State Key Laboratory of Bioreactor Engineering, Shanghai Key Laboratory of Chemical Biology, School of Pharmacy, East China University of Science and Technology, Shanghai 200237, China ‡ Division of Anti-Tumor Pharmacology, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China

bS Supporting Information ABSTRACT:

A novel series of acenaphtho[1,2-b]pyrrole derivatives as potent and selective inhibitors of fibroblast growth factor receptor 1 (FGFR1) were designed and synthesized. In silico target prediction revealed that tyrosine kinases might be the potential targets of the representative compound 2, which was subsequently validated by enzyme-linked immunosorbent assay (ELISA) for its selective and active FGFR1 inhibition of various tyrosine kinases. The structure
activity relationship (SAR) analysis aided by molecular docking simulation in the ATP-binding site demonstrated that acenaphtho[1,2-b]pyrrole carboxylic acid esters (2
5) are potent inhibitors of FGFR1 with IC50 values ranging from 19 to 77 nM. Furthermore, these compounds exhibited favorable growth inhibition property against FGFR-expressing cancer cell lines with IC50 values ranging from micromolar to submicromolar. Western blotting analysis showed that compounds 2, 3, and 2b inhibited activation of FGFR1 and extracellular-signal regulated kinase 1/2 (Erk1/2).

’ INTRODUCTION The fibroblast growth factor receptors (FGFRs) are a family of four structurally related receptor tyrosine kinases, namely, FGFR1
4.1 These membrane-spanning receptors are activated in response to growth factor stimulation by dimerization and result in transphosphorylation, which is followed by recruitment and phosphorylation of downstream intracellular substrates.1,2 FGFRs are precisely regulated in normal cells but are constitutively activated in transformed cells involved in various human cancers,3 such as bladder cancer, pancreatic cancer, breast cancer, and prostate cancer.1,2,4 Aberrant activation of FGFRs has been r 2011 American Chemical Society

observed in the process of tumorgenesis and tumor development. Thus, inhibition of FGFRs represents an attractive tumor therapeutic strategy.2,5,6 Deregulated FGFRs in tumor cells could be antagonized by blocking the downstream RAF-MEK1/2Erk1/2 and PI3K-AKT pathways or by targeting the upstream driving oncoproteins, FGFRs. The latter strategy is usually more efficient, as the degree of downstream pathway activation varies among tumor cell lines.2,4 Received: December 16, 2010 Published: April 25, 2011 3732

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Table 1. Tyrosine Kinase Selectivity of Compound 2 tyrosine kinase

Figure 1. Chemical structures of FGFR inhibitors and compound 1.

Dozens of small molecular inhibitors of FGFRs have been reported, and several of them are now in clinical trials.2 For example (see Figure 1), SU5402 C1, is a potent inhibitor of VEGFR2 and FGFR,7 which is now used as a research tool for studying FGFR signaling after dropping from clinical development. SU6668 C2, an indolinone analogue of C1, has entered phase I clinical trials, which could inhibit kinase activities of VEGFR2, FGFR1, and PDGFRβ both in vitro and in vivo and also had been validated to inhibit the tumor growth in human tumor xenografts and suppress tumor angiogenesis.8,9 Another example is BIBF1120 C3, which could inhibit VEGFR, PDGFR, and FGFR at the double-digit nanomolar level and has entered phase III clinical development.1,10 Previous reports showed that most existing FGFR inhibitors also inhibit VEGFRs, PDGFRs and sometimes other tyrosine kinases, on account of structure homology of catalytic domains of various kinases.2,11,12 Multiple kinase inhibitors are believed to serve as superior anticancer agents by disrupting redundant pathways. However, the other side of the coin was the side effects associated with the non specific inhibition of kinase.2 Since few synthetic molecules have been classified as selective inhibitors of FGFRs, it is of great value to discover and develop such small molecule inhibitors for cancer therapy. With the elucidation of the three-dimensional structures of kinase domains, ATP-binding pocket has been the focus of small molecular inhibitor design.13,14 Identifying new scaffolds that could be optimized to improve their potency, selectivity, and pharmacological properties15
17 is of great importance for FGFR- or RTK-targeted drug discovery.18
20 8-Oxo-8H-acenaphtho[1,2-b]pyrrolecarbonitrile (1) was previously discovered by our group and highlighted for its convenient synthesis and easy derivation.21 To date, compound 1 and its derivatives were functionalized for ion sensing,22 biomolecule imaging,23 and tumor diagnosis.24,25 The antitumor capacity of acenaphtho[1,2-b]pyrrole derivatives was confirmed recently according to the antiproliferative assays with cancer cell lines including HeLa, MCF-7, and A549.26
28 However, other potential molecular targets of acenaphtho[1,2-b]pyrroles are not well explored. We report herein acenaphtho[1,2-b]pyrrole

selectivity of 2, IC50 (μM)

FGFR1

0.074 ( 0.050

FGFR2

>10.0

FGFR3

4.6 ( 2.5

FGFR4

>10.0

VEGFR1

3.2 ( 0.3

VEGFR2

>10.0

PDGFRR

>10.0

PDGFRβ c-kit

>10.0 >10.0

RET

>10.0

EGFR2

>10.0

c-Src

>10.0

Abl

>10.0

EphA2

>10.0

EphB2

>10.0

c-Met RON

>10.0 >10.0

IGF1R

>10.0

derivatives as a novel class of FGFR1 inhibitors with favorable structure
activity relationship (SAR). Compared to other reported FGFR1 inhibitors, the acenaphtho[1,2-b]pyrrole scaffold has been used for the first time as a tyrosine kinase inhibitor.

’ RESULTS AND DISCUSSIONS Identification of Molecular Target of Compound 2. Ethyl 8-oxo-8H-acenaphtho[1,2-b]pyrrole-9-carboxylate (2), a derivative of compound 1, was synthesized in our laboratory.29 Our previous work has verified that compound 2 had great in vitro antiproliferative effects. Despite its anticancer activity against several human cancers, the exact molecular mechanism by which compound 2 exerts its effects is not yet clearly understood. To identify potential target candidates of compound 2, in silico targets screening using PharmMapper server, a reverse pharmacophore mapping approach,30,31 was performed using an inhouse pharmacophore database (PharmTargetDB). Among the predicted target candidates (Supporting Information Table S1), there are two tyrosine kinases (LCK and Src) and three serine/ threonine kinases (MEK1, CDK2, and p38 MAPK) in the top 0.6% of prediction results, which indicate that compound 2 may bind with kinases to suppress the proliferation of the tumor cells. To validate the prediction, we narrowed our efforts to tyrosine kinases because we have built an efficient in-house tyrosine kinase related drug screening platform with enzyme-linked immunosorbent assay (ELISA) approach to determine the tyrosine kinases inhibition activity for compound 2.32,33 As shown in Table 1, compound 2 demonstrated potent inhibition on FGFR1 in vitro, with an IC50 value at around 0.074 μM. As for the other members of the FGFR family, compound 2 exhibited far less potent inhibition activities. In detail, it showed approximately 60-fold less potent inhibition on FGFR3 (IC50 = 4.6 μM) and almost no inhibition activities on FGFR2 and FGFR4 even at 10 μM. Furthermore, we detected the inhibition activity of compound 2 toward the other 14 tyrosine kinases. According to Table 1, it showed moderate inhibition on VEGFR1 with an IC50 of 3.2 μM, and no notable inhibition activity was observed 3733

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Scheme 1a

Scheme 3a

a Reagents: (a) 4.0 equiv of corresponding thiols, Et3N (1.0 equiv for compounds 1a
g), methanol, 0
5 °C, 19
49%.

a

Reagents: (a) 4.0 equiv of corresponding thiols, methanol/chloroform (4:1), room temperature or 0
5 °C (for compound 2h), 39
85%.

Scheme 2a

Figure 2. Chemical structures of some garlic constituents.

a Reagents: (a) 0.83 equiv of thiol or alcohol, 0.83 equiv of Et3N, CH2Cl2, room temperature, 89
90%; (b) for compound 3, 5.0 equiv of 3-iodoprop1-ene, 1.0 equiv of K2CO3, acetonitrile, argon, 40 °C, 43%; for compounds 4 and 5, 2.5 equiv I1 or I2, K2CO3 (0.9 equiv for compound 4, 1.0 equiv for compound 5), 1.0 equiv of KI, acetonitrile, argon, reflux, 11
13%.

on the other kinases, including VEGFR2, PDGFRR, PDGFRβ, EGFR2, c-Kit, RET, c-Src, Abl, EphA2, EphB2, c-Met, RON, and IGF1R. Hence, compound 2 exhibited remarkable selectivity toward FGFR1 compared to other tested tyrosine kinases, including other FGFRs and highly homologous enzymes PDGFRs and VEGFRs. Chemistry. To clarify preliminarily the SAR of acenaphtho[1,2-b]pyrrole derivatives as FGFR1 inhibitors, target compounds were synthesized by introduction of sulfur nucleophiles at 3-position and/or by esterification with bromide/iodide at the 9-position after hydrolysis. The precursor 1 was efficiently synthesized from commercially available acenaphthalenequinone via Knoevenagel condensation and cyclization according to our previous report.21 It was converted into 3-thiol-8-oxo-8H-acenaphtho[1,2b]pyrrole-9-carbonitriles (1a
h) via SNArH reaction with corresponding thiols in absolute methanol at 0
5 °C in (for compounds 1a
g)/without (for compound 1h) the presence of Et3N as shown in Scheme 1. 8-Oxo-8H-acenaphtho[1,2-b]pyrrole-9-carboxylic acid (D1) and compound 2 were synthesized as reported.29 As shown in Scheme 2, compound 3 was obtained by the esterification of compound D1 with allylic iodide in CH3CN in the presence of K2CO3 at 40 °C under argon. Thiol and alcohol undergo nucleophilic substitution reaction with bromoacetyl bromide to give alkyl halothioacetate (I1) or fluoride-substituted alkyl haloacetate (I2), which could subsequently react with compound D1 to produce compounds 4 and 5 in CH3CN in the presence of K2CO3 and KI at the refluxing temperature under argon.

As shown in Scheme 3, acenaphtho[1,2-b]pyrrole carboxylic acid esters (2
5) were smoothly transformed into 3-thiolacenaphtho[1,2-b]pyrrole carboxylic acid esters (2a
h, 3a,b, 4a,b, 5a,b) by thiolation with corresponding thiols in the mixture of methanol and chloroform (4:1 in volume) at room temperature or at 0
5 °C (for compound 2h). Acenaphtho[1,2-b]pyrrole Derivatives as FGFR1 Inhibitors. As demonstrated above, compound 2 exhibited potent and selective FGFR1 inhibition activity. To illuminate the SAR of acenaphtho[1,2-b]pyrrole derivatives as FGFR1 inhibitors, we prepared the 3-thiol derivatives of compound 2 and its precursor 1 and evaluated their FGFR1 inhibition activity using the ELISA method. 3-Thiol derived acenaphtho[1,2-b]pyrroles were designed based on not only sulfur-containing heterocycles derived antitumor agents reported before26,27,34
36 but also the principal sulfur-containing constituents, like DAS C4, DADS C5, DATS C6, which are responsible for tumor prevention and suppression of garlic (see Figure 2).37
41 According to Table 2, by comparison with the precursor 1, compound 2 presented much more distinctive FGFR1 inhibition activity, which is a 60-fold increase. In addition, compounds 2b and 2e exhibited equally notable FGFR1 inhibition activity (IC50 of 0.485 and 0.320 μM, respectively) of all newly synthesized 3-thiol derivatives of compounds 1 and 2. Thus, we considered the introduction of a 9-ester group to the acenaphtho[1,2-b]pyrrole scaffold to be important for FGFR1 inhibition. Besides, the small changes in 3-thiol substitutes drastically alter the FGFR1 inhibition activities, e.g., 2a vs 2b. Acenaphtho[1,2-b]pyrrole carboxylic acid esters (3
5) and their 3-thiol derivatives (3a, 3b, 4a, 4b, 5a, 5b) were then prepared and evaluated of their FGFR1 inhibition activity. First, allyl, thioester, and fluorine-substituted ester were introduced to the 9-ester group. The fluorine substituent, as a result of its special effects (e.g., improving the binding affinity to the target protein), prevails in 20
25% of drugs in the pharmaceutical pipeline.42
44 According to Table 3, compounds 3
5 exhibited similarly remarkable inhibitory effects on FGFR1 with IC50 at 3734

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Table 2. SAR of Compounds 1 and 2 and Their Corresponding 3-Thiol Derivatives (1a
h and 2a
h)

double-digit nanomolar levels just like compound 2, among which compound 3 possessed the greatest FGFR1 inhibition activity, around 3-fold more potent than compounds 4, 5, and 2. Second, allylmercaptan derivatives (3b, 4b, 5b) and their saturated analogues (3a, 4a, 5a) of compounds 3
5 were synthesized. It was reported that alkenyl derivatives such as C6 were more potent anticancer agents than alkyl analogues through microtubule disassembly.45 In accordance with the 3-thiol derivatives of compound 2, compounds 3b, 4b, and 5b showed potent inhibition against FGFR1 with IC50 at the submicromolar level while compounds 3a, 4a, and 5a did not show inhibition against FGFR1 at 10 μM. The results suggested that the allylmercaptan substituted derivatives of acenaphtho[1,2b]pyrrole carboxylic acid esters (2b, 3b, 4b, and 5b) are much more potent inhibitors of FGFR1 compared to their saturated counterparts (2a, 3a, 4a, and 5a). Binding Modes Predicted by Molecular Docking Simulation within FGFR1 Kinase Domain and Multiple Sequence Alignment of Kinase Domain for FGFR. The predicted binding mode of compound 3b which was obtained by molecular docking method was used to analyze the SAR of these acenaphtho[1,2-b]pyrrole derivatives. Several interesting features of possible

interactions can be derived from the predicted binding pose (Figure 3a): (1) Compound 3b occupied the cleft of ATP binding site, and the scaffold (i.e., the planar and hydrophobic aromatic ring fragment) stretched toward the pocket and was exposed to the solution (Figure 3b). There are extensive hydrophobic interactions and van der Waals (VDW) contacts to hydrophobic residues (Val 492, Leu 484, Ala 512, and Leu 630). (2) The allyl 9-ester side chains stretched out of the binding pocket and have hydrophobic interactions with Tyr 563 and Leu 630. (3) There is an essential hydrogen bond interaction in the hinge region of kinase domain, i.e., the carbonyl oxygen atom of the 2H-pyrrol-2-one formed a hydrogen bond with the nitrogen atom of residue Ala 564, which is like most reported FGFR1 inhibitors.7 (4) The electron-rich allylmercaptan side chain stretched toward the polar surface exposed to the solvent environment. In order to rationalize the affinity observed for our inhibitors, we also study the binding poses of compounds 2, 2b, and 2e. All these compounds have similar binding modes. Interestingly, we find that compounds with terminal unsaturated side chains are more active than compounds with saturated side chains: the IC50 of compound 2b, an allylmercaptan substitute, is 485 nM, while compound 2a has virtually no activity. This can be 3735

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Journal of Medicinal Chemistry Table 3. SAR of Acenaphtho[1,2-b]pyrrole Carboxylic Acid Esters (3
5) and Their Corresponding 3-Thiol Derivatives (3a, 3b, 4a, 4b, 5a, and 5b)

also found between compounds 3a and 3b, 4a and 4b, 5a and 5b. We supposed that the 3-position electron-rich unsaturated substitute is more favorable to bind to the polar surface when exposed to the solvent environment. This rule is supported by the phenomenon that compound 2e with an electron-rich side chain also has good activity. Besides, compounds with more hydrophobic 9-ester side chains are more active, such as compounds 3, 3b vs compounds 2, 2b. We noticed that although compounds 2b, 3b, 4b, 5b have more interactions, they are less active than compounds 2, 3, 4, 5. The solubility of compounds may contribute to this fact, and CI log S values of compouds 2b, 3b, 4b, 5b are notablely lower than those of compounds 2, 3, 4, 5 (Table 4). Multiple sequence alignment of kinase domain for FGFR1
4 is shown (Figure 3d). The unconservative regions of FGFR kinase domain mainly distribute in ATP binding sites, and the differences may cause the conformational change of the ATP binding site such as the structure of FGFR2 (Figure 3c). The binding pocket of FGFR2 is almost closed compared to FGFR1 so that our inhibitors cannot bind. Therefore, the conformational changes arising from the sequence difference of the ATP binding sites may contribute to the selectivity of FGFR1 to other types of FGFR. All these results can well illustrate not only the SAR of acenaphtho[1,2-b]pyrrole derivatives as

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FGFR1 inhibitors but the selectivity of our inhibitors against FGFR1
4. Antiproliferative Activities of Acenaphtho[1,2-b]pyrrole Carboxylic Acid Ester Derivatives against FGFR-Expressing Cancer Cell Lines. As demonstrated above, the newly synthesized compounds exhibited significant FGFR1 inhibition activity and considerable SAR at the molecular level in light of ELISA and molecular docking simulation. Acenaphtho[1,2-b]pyrrole carboxylic acid ester derivatives (2
5, 2a, 2b, 3a, 3b, 4a, 4b, 5a, 5b) and their precursor 1 were predicted by QikProp 3.1 software and XLOGP3 online service with several physicochemical properties (molecular weight, aqueous solubility, apparent Caco-2 cell permeability, and octanol/water partition coefficient). As shown in Table 4, the calculated druglikeness properties of these compounds were well within the recommended ranges, which indicated the potential for further optimizations of them as drug leads.46
48 Subsequently, the in vitro growth inhibition properties (measured as IC50) of the synthesized compounds were evaluated in human cancer cells by sulforhodamine B (SRB) assay. As deregulated FGFR kinase activity is involved in pancreatic49 and bladder cancer,4 human pancreatic carcinoma cell line BxPC350 and human bladder carcinoma cell line T24 were selected for this experiment. As shown in Table 5, the ester derivatives (2
5) of 1 exhibited quite potent antitumor activities with the IC50 at single-digit to double-digit micromolar. In particular, compounds 2
4 were 2- to 5-fold more potent than compound 1, while compound 5 was almost as toxic as 1. As for the thiol derivatives of acenaphtho[1,2-b]pyrrole carboxylic acid esters, allylmercaptan analogues (e.g., 2b and 3b, with IC50 at submicromolar to single-digit micromolar) are normally more potent than their saturated counterparts (e.g., 2a and 3a, with IC50 at single-digit micromolar). The results were in agreement with their FGFR1 inhibition activity in principle. However, the derivatives of compounds 4 and 5 (4a, 4b, 5a, 5b) were not as potent as the derivatives of compounds 2 and 3 (2a, 2b, 3a, 3b), in spite of their similar FGFR1 inhibitory activity in molecular assay. And compounds 2a and 3a, which were weak FGFR1 inhibitors according to the ELISA result, showed quite potent antiproliferative activities against BxPC3 and T24 cells. Since DNA is usually considered to be a main target for acenaphtho[1,2-b]pyrrole derivatives as reported,28 the newly synthesized compounds were also tested for their interaction with DNA by circular dichroism (CD).51 As shown in Figure S1, no distinguished change in the CD spectra of calf thymus DNA (CT DNA) was observed for most of the tested compounds, which indicated that most of the tested compounds (including compounds 2a and 3a) were weaker DNA intercalators. In addition, we also detected the antiproliferation activity of acenaphtho[1,2-b]pyrrole carboxylic acid ester derivatives on the human lung fibroblast cell line WI-38. Generally, this series of compounds inhibited the growth of cancer cell lines better than normal cells (about twice the IC50). And compound 3, which possesses the most potent FGFR1 inhibition activity of all the tested compounds, exhibited the greatest inhibitory selectivity against cancer cell lines (about 4
5 times the IC50). Overall, the cell-based antiproliferative results of most of the compounds generally agree with the FGFR1 inhibition results at the molecular level. Meanwhile, we observed that the antiproliferative activity of acenaphtho[1,2-b]pyrrole derivatives is not directly related to the FGFR1 inhibitory activity, since cell antiproliferative potency reflects the compounds’ binding 3736

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Table 4. Physicochemical Properties of Acenaphtho[1,2b]pyrrole Derivatives

Table 5. Antiproliferative Activity of Acenaphtho[1,2b]pyrrole Derivatives

compd

MWa

X log P b

CI log S c

QPPCaco2d

1

230.225

2.42


3.302

330.354

BxPC3

277.279 289.290

2.92 3.20


3.221
3.519

compd

2 3

779.122 856.710

1

25.0 ( 8.4

13.3 ( 4.6

21.7 ( 1.9

4

365.403

3.91


4.419

375.072

2

4.7 ( 2.3

2.9 ( 0.8

6.2 ( 0.4

5

439.289

4.26


6.251

334.723

2a

351.419

4.33


4.812

717.110

3 4

4.8 ( 1.3 10.1 ( 4.0

3.9 ( 1.3 10.5 ( 3.4

20.6 ( 1.7 25.2 ( 4.6

2b

349.403

4.08


4.762

703.744

5

23.3 ( 12.9

19.0 ( 7.1

48.3 ( 0.7

3a

363.430

4.61


5.110

733.559

2a

5.8 ( 2.7

1.7 ( 1.0

9.6 ( 5.6

3b

361.414

5.32


5.060

667.569

2b

2.5 ( 1.0

0.7 ( 0.3

4.6 ( 2.1

4a 4b

439.543 437.527

5.32 5.07


6.010
5.960

438.205 269.615

3a

12.9 ( 9.2

13.1 ( 7.2

33.2 ( 2.6

3b

2.5 ( 1.3

6.6 ( 4.6

19.7 ( 6.0

5a

513.435

5.76


7.842

249.604

4a

56.3 ( 10.8

79.1 ( 8.5

>100

5b

511.419

5.42


7.792

192.170

4b 5a

13.7 ( 7.3 54.1 ( 2.5

71.8 ( 20.2 >100

>100 58.8 ( 17.3

5b

38.5 ( 7.0

86.4 ( 10.9

45.6 ( 7.1

a

antiproliferative activity, IC50 (μM)

b

Molecular weight. Predicted octanol/water log P calculated by XLOGP3 online service.57 c Conformation-independent predicted aqueous solubility; S in mol/L. d Predicted Caco-2 cell permeability in nm/s.

affinities to the target protein and their ability to enter the cell, for example, the cell permeability.52 To test this hypothesis, we built simple prediction models of cell antiproliferative potencies for the three cell lines by multiple linear regression (MLR) analysis with FGFR1 inhibitory activities and cell permeabilities described by QPPCaco2 (predicted Caco-2 cell permeability in nm/s) values calculated by Qikprop. Equations 1
3 indicate the significance of each parameter. pIC50 ðT24Þ ¼
2:462 þ 0:186 pIC50 ðFGFR1Þ þ 2:340 logðQPPCaco2Þ n ¼ 13;

r 2 ¼ 0:680;

SE ¼ 0:414;

ð1Þ

F ¼ 23:009

pIC50 ðBxPC3Þ ¼
0:146 þ 0:194 pIC50 ðFGFR1Þ þ 1:456 logðQPPCaco2Þ n ¼ 13;

r 2 ¼ 0:711;

ð2Þ SE ¼ 0:265;

F ¼ 27:186

pIC50 ðWI-38Þ ¼ 0:876 þ 0:069 pIC50 ðFGFR1Þ þ 1:234 logðQPPCaco2Þ n ¼ 13;

r 2 ¼ 0:467;

ð3Þ SE ¼ 0:320;

F ¼ 9:691

n is the number of acenaphtho[1,2-b]pyrrole derivatives included in the study. r2 is the squared correlation coefficient corresponding to the fraction of observed variance accounted for by the model. SE is the standard error of estimation. F is the F-test value, a statistic for assessing the overall significance of the derived equation. The cross-correlation coefficients between pIC50(FGFR1) and log(QPPCaco2) were verified from the Pearson correlation data and found to be significantly lower (0.160). As shown in Figure 3e, the correlation coefficients r2 of T24 and BxPC3 cell lines are 0.680 and 0.711, respectively, indicating a positive synergic contribution between QPPCaco2 and FGFR1 inhibitiory potencies to the antiproliferative potencies. Although the predicted QPPCaco2 values may not be as accurate as the experimentally determined ones with artificial membrane, the relative permeability potential of each compound can be

T24

WI-38

characterized qualitatively.53 Higher QPPCaco2 values imply that the compounds are more prone to penetrate the cell membrane to exhibit more potent FGFR1 inhibitory activity, even though they are moderately potent or as potent as the other analogues in the enzyme assay (like compounds 2 vs 5 and 2b vs 5b). Moreover, our inhibitors exhibit selectivity of growth for cancer cells (T24 and BxPC3) to normal cells (WI-38), which is consistent with the lower correlation coefficient r2 in the WI-38 model. All the results are in accord with our models and confirm their reliability in predicting antiproliferative potency of the tumor cell lines for the FGFR1 inhibitors synthesized in this study. Western Blotting Analysis of Compounds 2, 3, 2a, and 2b for Intracellular FGFR1 Signaling. Activation of FGFR1 and its downstream signaling molecules, including Erk and AKT, has been verified to correlate with tumor growth, angiogenesis, and poor prognosis in a number of different cancers. The effects of compounds 2, 3, 2a, and 2b on the activation of FGFR1 and downstream signaling pathways were then determined in BxPC3 cells by Western blot analysis. The results showed that bFGFinduced phosphorylation of FGFR1 was significantly reduced in a dose-dependent manner after treatment of compounds 2, 3, and 2b for 12 h. As shown in Figure 4, the bands of phophorylated FGFR1 were obviously declined after exposure to 1.25 μM compounds 2, 3 and 2b. As a contrast, compound 2a, the saturated analogue of compound 2b, caused little change in the phosphorylation of FGFR1 even at 10 μM when compared with the control. Therefore, the FGFR1 inhibitory activities in cancer cells of these compounds were quite consistent with their molecular FGFR1 inhibitory activities obtained in the present study. We then tested the effects of these compounds on the activation of Erk1/2, an important signaling molecule downstream of FGFR1, in BxPC3 cells. As shown in Figure 4, compounds 2 and 3 caused a marked and dose-dependent decrease in FGFinduced phosphorylation of Erk1/2 in cancer cells, and the band almost disappeared when the concentration of compound 2 reached 10 μM. Compound 2b exhibited weaker activities on Erk activation than compound 2. Moreover, compound 2a had little effect on the FGF-induced activation of Erk1/2. Subsequently, the activation of AKT, another key signaling molecule downstream of FGFR1, was also detected. As expected, 3737

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Figure 3. (a) Predicted binding pose of compound 3b at the ATP-binding site of FGFR1. (b) Electrostatic potential surface of FGFR1 kinase domain. The coloring for regions is described as follows: red, negative electricity region; blue, positive electricity region; white, neutral region. (c) Overlapped poses of compounds 2, 2b, 2e, and 3b at the ATP-binding site of FGFR2. The ligands are shown in sticks, and the surface is shown in light purple. The carbon atoms for each compound are colored as follows: compound 2, cyan; compound 2b, magenta; compound 2e, orange; compound 3b, green; FGFR1 kinase domain, salmon; FGFR2 kinase domain, yellow. The coloring for oxygen atoms of each compound is red. The hydrogen bonds are denoted with blue dashed lines. All structure figures were prepared using PyMol 0.99 (http://pymol.sourceforge.net/). (d) Multiple sequence alignment of kinase domain for FGFR1
4. (e) Predicted antiproliferative activity to actual antiproliferative activity against T24, BxPC3, and WI-38 cell lines.

compound 2a exhibited little effect on the FGF-induced activation of AKT in BxPC3 cells (Figure 4). Unexpectedly, however, the phosphorylation of AKT was increased, but not decreased, in

a dose-dependent manner after exposure to 2, 3, or 2b (Figure 4). The mechanisms underlying the increase of pAKT caused by compounds 2, 3, and 2b might be the result of other potential 3738

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Figure 4. Effects of componds 2, 3, 2a, and 2b on the intracellular signaling in BxPC3 cells. bFGF-induced phosphorylation of FGFR1, Erk, and AKT was determined by immunoblotting after treatment of BxPC3 cells with increasing concentrations of compounds 2, 3, 2a, and 2b (from left to right: control, bFGF, 1.25, 2.5, 5, 10 μM). GAPDH was detected as loading control.

mechanisms or the crosstalk and feedback of the signaling pathways in human cancer cells, and they deserve further investigation in our future work.

’ CONCLUSION We have discovered a novel series of acenaphtho[1,2b]pyrrole derivatives as potent FGFR1 inhibitors. Compound 2 was predicted to be tyrosine kinases inhibitor by in silico targets screening and confirmed by ELISA to be a potent and selective FGFR1 inhibitor. Newly synthesized derivatives were produced by introduction of allyl, thioester, and fluorine-substitued ester to the 9-ester group and/or sulfur nucleophiles to the 3-position of the acenaphtho[1,2-b]pyrrole scaffold. SAR study suggested acenaphtho[1,2-b]pyrrole carboxylic acid esters (2
5) to be great FGFR1 inhibitors with IC50 at the double-digit nanomolar level. By comparison with their inactive saturated analogues (2a, 3a, 4a, 5a), the allylmercaptan substituted acenaphtho[1,2b]pyrrole carboxylic acid esters (2b, 3b, 4b, 5b) were also potent FGFR1 inhibitors with submicromolar IC50 values. Calculated physiochemical properties of the newly synthesized compounds indicated their potential as drug leads. Molecular docking simulation demonstrated that the hydrophobic aromatic ring that binds to the neutral region of ATP-binding site and the 9-substituted ester side chains that bind to the negative electricity region were essential for FGFR1 inhibition. Subsequently, the acenaphtho[1,2-b]pyrrole derivatives were validated for their effective antiproliferative activity against FGFR1-expressing cancer cell lines with micromolar to submicromolar IC50 values. Western blotting analysis results of representative compounds 2,

3, 2a, and 2b were in agreement with the ELISA results, and compounds 2, 3, and 2b could reduce the phosphorylation of FGFR1 and Erk. To the best of our knowledge, we report here for the first time acenaphtho[1,2-b]pyrrole derivatives as FGFR1 inhibitors. The easy derivation, high potency, and selectivity could facilitate further optimization of acenaphtho[1,2-b]pyrrole derivatives as FGFR1 inhibitors, and this series of chemicals provided a promising starting point for further drug optimization and development as novel anticancer agents.

’ EXPERIMENTAL SECTION All chemical reagents and solvents were purchased from commercial sources and used without further purification. Thin-layer chromatography (TLC) was performed on silica gel plates. Column chromatography was performed using silica gel (Hailang, Qingdao), 200
300 mesh. Melting points were determined using a B€uchi melting point B-540 apparatus and are uncorrected. 1H and 13C NMR spectra were recorded employing a Bruker AV-400 spectrometer with chemical shifts expressed in parts per million (in DMSO-d6 or CDCl3, Me4Si as internal standard). The mass spectra were collected at the Mass Instrumentation Facility of Analysis and Research Center of ECUST. The purities of all newly synthesized compounds were analyzed by HPLC, with the purity all being higher than 95%. Analytical HPLC was performed on a Hewlett-Packard 1100 system chromatograph equipped with photodiode array detector using a Zorbax RX-C18 5 μm, 250 mm  4.6 mm column (reverse phase) to detect the purity of the products. The mobile phase was a gradient of 90
100% methanol (solvent 1) and 10 mM NH4OAc in water (pH 6.0) (solvent 2) at a flow rate of 1.0 mL/min (0
1.0 min, 90% solvent 1; 1.0
15.0 min, 90
100% solvent 1; 15.0
20.0 min, 100
90% solvent 1). 3739

dx.doi.org/10.1021/jm200258t |J. Med. Chem. 2011, 54, 3732–3745

Journal of Medicinal Chemistry

ARTICLE

General Procedure for the Preparation of Compounds 1a
g. To a stirred solution of compound 1 (200 mg, 0.87 mmol) in

3-(1-(5-Methyl-1,3-oxathiolan-5-yl)ethylthio)-8-oxo-8Hacenaphtho[1,2-b]pyrrole-9-carbonitrile (1g). The residue

absolute methanol (20 mL) was added the corresponding thiol (2.61 mmol). The mixture was cooled to 0
5 °C. Et3N (0.088 g, 0.87 mmol) was added, and then the mixture was stirred at 0
5 °C overnight. The solvent was then removed under vacuum. The residue was further purified.

was purified by column chromatography on silica gel (CH2Cl2/ petroleum ether: 3/1) to provide 1g. Reddish brown solid (yield 40%); mp 171.6
173.3 °C. Diastereomeric ratio = 11: 9. 1H NMR (400 MHz, CDCl3): δ 8.91 (d, J = 8.4 Hz, 1H), 8.78 (d, J = 7.6 Hz, 1H), 8.21
8.18 (m, 1H), 7.94
7.89 (m, 1H), 7.83
7.78 (m, 1 H), 4.38
4.33 (m, 0.55H), 4.30
4.25 (m, 0.45H), 4.17
4.03 (m, 2H), 3.19
3.11 (m, 2H), 1.81 (d, J = 4.0 Hz, 3H), 1.66 (dd, J = 8.0, 8.0 Hz, 3H). 13C NMR (100 MHz, CDCl3): δ 177.45, 152.71, 151.95, 133.82, 133.76, 133.55, 133.49, 133.37, 133.28, 130.89, 130.71, 128.74, 128.71, 128.51, 128.43, 127.25, 127.20, 124.92, 124.46, 119.83, 119.63, 117.35, 117.07, 113.19, 113.14, 112.91, 97.80, 97.74, 71.67, 71.47, 55.16, 53.89, 34.61, 34.54, 27.88, 25.61, 19.13, 18.68. HRMS (EI) calcd for C21H16N2O2S2 [Mþ] 392.0653, found 392.0658 and 392.0669.

8-Oxo-3-(propylthio)-8H-acenaphtho[1,2-b]pyrrole-9carbonitrile (1a). The residue was purified by column chromatography on silica gel (CH2Cl2/petroleum ether: 2/1) to provide 1a. Reddish brown solid (yield 23%); mp 242.8
245.1 °C. 1H NMR (400 MHz, DMSO-d6): δ 8.82 (d, J = 8.4 Hz, 1 H), 8.68 (d, J = 7.2 Hz, 1 H), 8.20 (d, J = 8.0 Hz, 1 H), 8.04 (t, J = 7.6 Hz, 1H), 7.82 (d, J = 8.4 Hz, 1H), 1.83
1.76 (m, 2H), 1.08 (t, J = 7.2 Hz, 3 H). HRMS (EI) calcd for C18H12N2OS [Mþ] 304.0670, found 304.0670.

3-(Allylthio)-8-oxo-8H-acenaphtho[1,2-b]pyrrole-9carbonitrile (1b). The residue was purified by column chromatography on silica gel (CH2Cl2/petroleum ether: 2/1) to provide 1b. Reddish brown solid (yield 21%); mp 251.3
252.0 °C. 1H NMR (400 MHz, DMSO-d6): δ 8.83 (s, 1H), 8.68 (s, 1H), 8.20 (s, 1H), 8.05 (s,1H), 7.85 (s, 1H), 6.02
5.92 (m, 1H), 5.50 (d, J = 16.8 Hz, 1H), 5.27 (d, J = 10.0 Hz, 1H), 4.10 (d, J = 6.4 Hz, 2H). HRMS (EI) calcd for C18H10N2OS [Mþ] 302.0514, found 302.0514.

3-(Isopropylthio)-8-oxo-8H-acenaphtho[1,2-b]pyrrole-9carbonitrile (1c). The residue was purified by column chromatography on silica gel (CH2Cl2/petroleum ether: 5/2) to provide 1c. Reddish brown solid (yield 25%); mp 238.5
240.3 °C. 1H NMR (400 MHz, DMSO-d6): δ 8.80 (d, J = 8.0 Hz, 1H), 8.67 (d, J = 7.6 Hz, 1H), 8.22 (d, J = 8.0 Hz, 1H), 8.03 (t, J = 8.0 Hz, 1H), 7.89 (d, J = 8.4 Hz, 1H), 4.07
4.01 (m, 1H), 1.47 (d, J = 6.4 Hz, 6H). HRMS (EI) calcd for C18H12N2OS [Mþ] 304.0670, found 304.0672.

8-Oxo-3-(2-(pyrazin-2-yl)ethylthio)-8H-acenaphtho[1,2-b] pyrrole-9-carbonitrile (1d). The residue was purified by column chromatography on silica gel (CH2Cl2/petroleum ether: 4/1) to provide 1d. Brown solid (yield 49%); mp 212.8
213.2 °C. 1H NMR (400 MHz, DMSO-d6): δ 8.75 (d, J = 8.4 Hz, 1H), 8.68
8.66 (m, 2H), 8.61 (s, 1H), 8.53 (d, J = 2.8 Hz, 1H), 8.23 (d, J = 8.0 Hz, 1H), 8.02 (t, J = 8.0 Hz, 1H), 7.92 (d, J = 8.4 Hz, 1H), 3.80 (t, J = 7.2 Hz, 2H), 3.34 (t, J = 7.2 Hz, 2H). 13C NMR (100 MHz, DMSO-d6): δ 176.59, 153.58, 150.25, 144.12, 143.37, 142.23, 133.18, 132.27, 131.78, 130.07, 128.51, 128.30, 127.52, 125.66, 122.35, 118.35, 115.77, 113.46, 112.79, 32.00, 29.04. HRMS (EI) calcd for C21H12N4OS [Mþ] 368.0732, found 368.0732.

3-(2-Hydroxyethylthio)-8-oxo-8H-acenaphtho[1,2-b] pyrrole-9-carbonitrile (1e). The residue was purified by column chromatography on silica gel (CH2Cl2 with trace methanol) to provide 1e. Reddish brown solid (yield 27%); mp 253.4
255.4 °C. 1H NMR (400 MHz, DMSO-d6): δ 8.83 (d, J = 8.4 Hz, 1H), 8.68 (d, J = 7.6 Hz, 1H), 8.19 (d, J = 8.0 Hz, 1H), 8.04 (t, J = 8.0 Hz, 1H), 7.86 (d, J = 8.0 Hz, 1H), 5.20 (s, 1H), 3.80 (t, J = 6.0 Hz, 2H), 3.46 (t, J = 6.0 Hz, 2H). HRMS (EI) calcd for C17H10N2O2S [Mþ] 306.0463, found 306.0464.

3-(2-Methyltetrahydrofuran-3-ylthio)-8-oxo-8H-acenaphtho [1,2-b]pyrrole-9-carbonitrile (1f). The residue was purified by column chromatography on silica gel (CH2Cl2/petroleum ether: 4/1) to provide 1f. Reddish brown solid (yield 42%); mp 245.7
248.3 °C. Diastereomeric ratio = 78: 22. 1H NMR (400 MHz, CDCl 3 ): δ 8.84
8.77 (m, 2H), 8.23
8.20 (m, 1H), 7.94 (t, J = 8.0 Hz, 1H), 7.62
7.58 (m, 1H), 4.47
4.41 (m, 0.78H), 4.22
4.13 (m, 1.56H), 4.10
4.04 (m, 0.22H), 4.00
3.90 (m, 1H), 3.70
3.65 (m, 0.44H), 2.80
2.67 (m, 1H), 2.27
2.19 (m, 0.78H), 2.17
2.10 (m, 0.22H), 1.47 (d, J = 6.4 Hz, 0.66H), 1.43 (d, J = 6.4 Hz, 2.34H). 13C NMR (100 MHz, DMSO-d6): δ 177.82, 151.50, 134.36, 133.54, 133.17, 131.29, 129.81, 129.60, 128.77, 126.88, 124.41, 119.60, 117.00, 114.70, 114.03, 79.67, 76.48, 66.57, 65.77, 49.05, 47.93, 33.54, 20.19, 17.13. HRMS (EI) calcd for C20H14N2O2S [Mþ] 346.0776, found 346.0776.

8-Oxo-3-(2-(thiazolidin-3-yl)ethylthio)-8H-acenaphtho[1,2-b]pyrrole-9-carbonitrile (1h). The stirred solution of compound 1 (200 mg, 0.87 mmol) in absolute methanol (20 mL) was cooled to 0
5 °C. 2-(Thiazolidin-3-yl)ethanethiol (0.390 g, 2.61 mmol) was added, and then the mixture was stirred at 0
5 °C overnight. The solvent was then removed under vacuum. The residue was purified by column chromatography on silica gel (CH2Cl2/methanol: 50/1) to provide 1h. Reddish brown solid (yield 21%); mp 206.2
206.6 °C. 1H NMR (400 MHz, DMSO-d6): δ 8.84 (d, J = 7.6 Hz, 1H), 8.67 (d, J = 6.8 Hz, 1H), 8.20 (d, J = 7.6 Hz, 1H), 8.03 (t, J = 8.0 Hz, 1H), 7.84 (d, J = 8.0 Hz, 1H), 4.14 (s, 2H), 3.55 (t, J = 6.4 Hz, 2H), 3.11 (t, J = 6.4 Hz, 2H), 2.83 (t, J = 6.0 Hz, 2H), 2.78 (t, J = 6.8 Hz, 2H). HRMS (EI) calcd for C20H15N3OS2 [Mþ] 377.0657, found 377.0642.

Allyl 8-Oxo-8H-acenaphtho[1,2-b]pyrrole-9-carboxylate (3). To a stirred solution of compound D1 (1.000 g, 4.0 mmol) and K2CO3 (0.554 g, 4.0 mmol) in absolute acetonitrile (20 mL) was added 3-iodoprop-1-ene (3.373 g, 20.1 mmol) under argon. The mixture was heated at 40 °C for 12 h. The reaction mixture was then cooled to room temperature and concentrated under vacumm. The resulting mixture was eluted by CHCl3 on diatomite column, concentrated, and then purified by column chromatography on silica gel (CH2Cl2) to provide 3. Yellow solid (yield 43%); mp 193.0
194.6 °C. 1H NMR (400 MHz, CDCl3): δ 9.12 (d, J = 7.2 Hz, 1H), 8.75 (d, J = 7.6 Hz, 1H), 8.31 (d, J = 8.0 Hz, 1H), 8.26 (d, J = 8.4 Hz, 1H), 7.87 (t, J = 8.0 Hz, 1H), 7.77 (t, J = 8.0 Hz, 1H), 5.97
5.87 (m, 1H), 5.33 (d, J = 17.2 Hz, 1H), 5.24 (d, J = 10.4 Hz, 1H), 4.32 (d, J = 5.6 Hz, 2H). 13C NMR (100 MHz, CDCl3): δ 178.65, 168.65, 166.16, 142.88, 136.91, 136.15, 133.94, 132.53, 132.08, 131.49, 131.26, 129.10, 127.86, 127.32, 125.36, 121.31, 118.28, 40.12. HRMS (ESI) calcd for C18H12NO3 [M þ H]þ 290.0817, found 290.0816.

2-Oxo-2-(propylthio)ethyl 8-Oxo-8H-acenaphtho[1,2-b] pyrrole-9-carboxylate (4). 2-Bromoacetyl bromide (2.674 g, 13.2 mmol) was dissolved in absolute CH2Cl2 (10 mL) and cooled to 0
5 °C. To the above stirred solution was added the mixture of Et3N (1.116 g, 11.0 mmol) and propane-1-thiol (0.841 g, 11.0 mmol) in CH2Cl2 (10 mL) for 1 h. The reaction proceeded at room temperature overnight. The reaction mixture was diluted with 10% NaHCO3 solution (15 mL) and then extracted with CH2Cl2 (2  20 mL). The organic layer was dried over Na2SO4 and concentrated under vacuum. The resulting mixture was purified by column chromatography on silica gel (CH2Cl2) to provide crude S-propyl 2-bromoethanethioate (I1) as a primrose yellow liquid (yield 90%). To a stirred solution of compound D1 (1.001 g, 4.0 mmol), K2CO3 (0.495 g, 3.6 mmol), and KI (0.664 g, 4.0 mmol) in absolute acetonitrile (20 mL) was added crude S-propyl 2-bromoethanethioate (1.971 g, 10.0 mmol) under argon. The mixture was heated at the refluxing temperature for 24 h. The reaction mixture was then cooled to room temperature and concentrated under vacuum. The resulting mixture was eluted by 3740

dx.doi.org/10.1021/jm200258t |J. Med. Chem. 2011, 54, 3732–3745

Journal of Medicinal Chemistry CHCl3 on diatomite column, concentrated, and then purified by column chromatography on silica gel (CH2Cl2) to provide 4. Yellow solid (yield 11%); mp 172.7
174.3 °C. 1H NMR (400 MHz, CDCl3): δ 9.01 (d, J = 7.2 Hz, 1H), 8.67 (d, J = 7.2 Hz, 1H), 8.29 (d, J = 8.0 Hz, 1H), 8.25 (d, J = 8.0 Hz, 1H), 7.83 (t, J = 7.6 Hz, 1H), 7.73 (t, J = 8.0 Hz, 1H), 4.60 (s, 2H), 2.95 (t, J = 7.2 Hz, 2H), 1.65
1.60 (m, 2H), 0.96 (t, J = 7.6 Hz, 3H). 13C NMR (100 MHz, CDCl3): δ 193.56, 178.28, 168.16, 165.43, 142.82, 137.09, 136.48, 134.13, 132.45, 132.15, 131.31, 128.95, 127.96, 127.38, 125.40, 120.97, 46.46, 30.91, 22.69, 13.29. HRMS (ESI) calcd for C20H16NO4S [M þ H]þ 366.0800, found 366.0787.

2-Oxo-2-(2,2,3,3,3-pentafluoropropoxy)ethyl 8-Oxo-8Hacenaphtho[1,2-b]pyrrole-9-carboxylate (5). To the stirred solution of 2-bromoacetyl bromide (3.380 g, 16.7 mmol) in absolute CH2Cl2 (10 mL) was added the mixture of Et3N (1.410 g, 14.0 mmol) and 2,2,3,3,3-pentafluoropropan-1-ol (2.100 g, 14.0 mmol) in CH2Cl2 (10 mL) in 1 h. The reaction proceeded at room temperature overnight. The reaction mixture was diluted with water (20 mL) and then extracted with CH2Cl2 (2  20 mL). The organic layer was dried over Na2SO4 and concentrated under vacuum. The resulting mixture was purified by column chromatography on silica gel (CH2Cl2) to provide crude 2,2,3,3,3-pentafluoropropyl 2-bromoacetate (I2) as a primrose yellow liquid (yield 89%). To a stirred solution of compound D1 (1.003 g, 4.0 mmol), K2CO3 (0.553 g, 4.0 mmol), and KI (0.667 g, 4.0 mmol) in absolute acetonitrile (20 mL) was added crude 2,2,3,3,3-pentafluoropropyl 2-bromoacetate (2.707 g, 10.0 mmol) under argon. The mixture was heated at the refluxing temperature for 24 h. The reaction mixture was then cooled to room temperature and concentrated under vacuum. The resulting mixture was eluted by CHCl3 on diatomite column, concentrated, and then purified by column chromatography on silica gel (CH2Cl2) to provide 5. Yellow solid (yield 13%); mp 186.0
188.1 °C. 1H NMR (400 MHz, CDCl3): δ 9.05 (d, J = 7.2 Hz, 1H), 8.72 (d, J = 7.2 Hz, 1H), 8.32 (d, J = 8.0 Hz, 1H), 8.27 (d, J = 8.4 Hz, 1H), 7.87 (t, J = 7.6 Hz, 1H), 7.76 (t, J = 8.0 Hz, 1H), 4.66 (t, J = 12.4 Hz, 2H), 4.58 (s, 2H). 13C NMR (100 MHz, CDCl3): δ 178.30, 168.02, 165.91, 165.30, 142.92, 137.17, 136.61, 134.15, 132.51, 132.28, 131.36, 129.02, 128.01, 127.41, 125.45, 120.97, 60.20 (t, J = 27.9 Hz, 1C), 38.33. HRMS (ESI) calcd for C20H11NO5F5 [M þ H]þ 440.0557, found 440.0536.

General Procedure for the Preparation of Compounds 2a
g, 3a, 3b, 4a, 4b, 5a, 5b. To a stirred solution of 2
5 (0.43 mmol) in methanol (12 mL) and chloroform (3 mL) was added the corresponding thiol (1.72 mmol). The mixture was stirred at room temperature overnight. The solvent was then removed under vacuum. The residue was further purified.

Ethyl 8-Oxo-3-(propylthio)-8H-acenaphtho[1,2-b]pyrrole-9carboxylate (2a). The residue was purified by column chromatography on silica gel (CH2Cl2 with trace methanol) to provide 2a. Reddish brown solid (yield 85%); mp 255.1
256.6 °C. 1H NMR (400 MHz, CDCl3): δ 8.93 (d, J = 8.0 Hz, 1H), 8.77 (d, J = 7.2 Hz, 1H), 8.69 (d, J = 8.0 Hz, 1H), 7.83 (t, J = 7.6 Hz, 1H), 7.47 (d, J = 8.0 Hz, 1H), 3.79
3.73 (m, 2H), 3.21 (t, J = 7.2 Hz, 2H), 1.95
1.89 (m, 2H), 1.31 (t, J = 7.2 Hz, 3H), 1.19 (t, J = 7.2 Hz, 3H). 13C NMR (100 MHz, CDCl3): δ 178.09, 169.03, 166.72, 150.73, 142.93, 133.04, 132.38, 132.22, 131.81, 130.71, 129.28, 127.44, 123.05, 122.14, 117.60, 34.02, 32.94, 21.63, 13.97, 13.67. HRMS (ESI) calcd for C20H18NO3S [M þ H]þ 352.1007, found 352.0999.

Ethyl 3-(Allylthio)-8-oxo-8H-acenaphtho[1,2-b]pyrrole-9carboxylate (2b). The residue was purified by column chromatography on silica gel (CH2Cl2 with trace methanol) to provide 2b. Reddish brown solid (yield 78%); mp 198.4
201.7 °C. 1H NMR (400 MHz, CDCl3): δ 8.90 (d, J = 8.0 Hz, 1H), 8.75 (d, J = 7.2 Hz, 1H), 8.67 (d, J = 8.0 Hz, 1H), 7.82 (t, J = 8.0 Hz, 1H), 7.49 (d, J = 8.0 Hz, 1H), 6.05
5.95 (m, 1H), 5.46 (d, J = 17.2 Hz, 1H), 5.32 (d, J = 9.6 Hz, 1H), 3.89 (d, J = 6.0 Hz, 2H), 3.78
3.73 (m, 2H), 1.31 (t, J = 7.2 Hz, 3H). 13C NMR

ARTICLE

(100 MHz, CDCl3): δ 178.09, 168.96, 166.65, 149.14, 142.87, 132.90, 132.35, 132.14, 131.84, 131.09, 130.79, 129.24, 127.53, 123.33, 123.12, 120.01, 118.00, 35.18, 32.96, 13.95. HRMS (ESI) calcd for C20H15NO3NaS [M þ Na]þ 372.0670, found 372.0672.

Ethyl 3-(Isopropylthio)-8-oxo-8H-acenaphtho[1,2-b]pyrrole-9carboxylate (2c). The residue was purified by column chromatography on silica gel (CH2Cl2 with trace methanol) to provide 2c. Reddish brown solid (yield 49%); mp 266.3
268.7 °C. 1H NMR (400 MHz, CDCl3): δ 8.98 (d, J = 7.6 Hz, 1H), 8.79 (d, J = 7.2 Hz, 1H), 8.74 (d, J = 8.4 Hz, 1H), 7.84 (t, J = 7.2 Hz, 1H), 7.58 (d, J = 8.0 Hz, 1H), 3.86
3.82 (m, 1H), 3.77
3.74 (m, 2H), 1.55 (d, J = 5.6 Hz, 6H), 1.31 (t, J = 6.8 Hz, 3H). 13C NMR (100 MHz, CDCl3): δ 178.18, 169.06, 166.73, 149.75, 142.95, 133.00, 132.53, 132.47, 132.31, 131.26, 129.50, 127.49, 124.05, 123.31, 117.99, 36.96, 32.98, 22.76, 13.98. HRMS (ESI) calcd for C20H17NO3NaS [M þ Na]þ 374.0827, found 374.0822.

Ethyl 8-Oxo-3-(2-(pyrazin-2-yl)ethylthio)-8H-acenaphtho [1,2-b]pyrrole-9-carboxylate (2d). The residue was purified by column chromatography on silica gel (CH2Cl2/methanol: 50/1) to provide 2d. Reddish brown solid (yield 66%); mp 215.8
217.9 °C. 1H NMR (400 MHz, CDCl3): δ 9.00 (d, J = 8.0 Hz, 1H), 8.78 (d, J = 7.6 Hz, 1H), 8.67 (d, J = 8.4 Hz, 1H), 8.59 (s, 1H), 8.53 (s, 1H), 8.50 (s, 1H),7.84 (t, J = 8.0 Hz, 1H), 7.63 (d, J = 8.0 Hz, 1H), 3.78
3.73 (m, 2H), 3.69 (t, J = 7.2 Hz, 2H), 3.35 (t, J = 7.6 Hz, 2H), 1.30 (t, J = 7.2 Hz, 3H). HRMS (ESI) calcd for C23H17N3O3NaS [M þ Na]þ 438.0888, found 438.0905.

Ethyl 3-(2-Hydroxyethylthio)-8-oxo-8H-acenaphtho[1,2b]pyrrole-9-carboxylate (2e). The residue was purified by column chromatography on silica gel (CH2Cl2/methanol: 50/1) to provide 2e. Reddish brown solid (yield 47%); mp 238.4
240.0 °C. 1H NMR (400 MHz, CDCl3): δ 8.94 (d, J = 8.0 Hz, 1H), 8.77 (d, J = 8.0 Hz, 1H), 8.72 (d, J = 8.0 Hz, 1H), 7.85 (t, J = 8.0 Hz, 1H), 7.60 (d, J = 8.0 Hz, 1H), 4.09 (t, J = 6.0 Hz, 2H), 3.80
3.75 (m, 2H), 3.48 (t, J = 6.0 Hz, 2H), 1.33 (t, J = 7.2 Hz, 3H). HRMS (ESI) calcd for C19H15NO4NaS [M þ Na]þ 376.0619, found 376.0618.

Ethyl 3-(2-Methyltetrahydrofuran-3-ylthio)-8-oxo-8H-acenaphtho[1,2-b]pyrrole-9-carboxylate (2f). The residue was purified by column chromatography on silica gel (CH2Cl2 with trace methanol) to provide 2f. Reddish brown solid (yield 65%); mp 242.5
245.1 °C. Diastereomeric ratio = 84:16. 1H NMR (400 MHz, CDCl3): δ 8.89
8.84 (m, 1H), 8.72 (d, J = 7.2 Hz, 1H), 8.66
8.61 (m, 1H), 7.80 (t, J = 8.0 Hz, 1H), 7.51 (d, J = 8.0 Hz, 1H), 4.48
4.42 (m, 0.84H), 4.20
4.14(m, 1.68H), 4.09
4.02 (m, 0.16H), 3.98
3.90 (m, 1H), 3.78
3.73 (m, 2H), 3.68
3.63 (m, 0.32H), 2.75
2.67 (m, 1H), 2.28
2.20 (m, 0.84H), 2.17
2.10 (m, 0.16H), 1.47 (d, J = 6.0 Hz, 0.48H), 1.43 (d, J = 6.0 Hz, 2.52H), 1.32 (t, J = 7.2 Hz, 3H). 13C NMR (100 MHz, CDCl3): δ 177.97, 168.88, 166.62, 149.35, 142.66, 132.82, 132.39, 132.05, 131.85, 130.71, 129.15, 127.61, 123.23, 123.16, 117.87, 76.91, 66.09, 48.25, 33.65, 32.99, 16.98, 14.00. HRMS (ESI) calcd for C22H19NO4NaS [M þ Na]þ 416.0932, found 416.0927.

Ethyl 3-(1-(5-Methyl-1,3-oxathiolan-5-yl)ethylthio)-8-oxo8H-acenaphtho[1,2-b]pyrrole-9-carboxylate (2g). The residue was purified by column chromatography on silica gel (CH2Cl2 with trace methanol) to provide 2g. Reddish brown solid (yield 59%); mp 95.6
97.9 °C. Diastereomeric ratio = 50:50. 1H NMR (400 MHz, CDCl3): δ 8.99 (d, J = 8.0 Hz, 1H), 8.86 (d, J = 8.4 Hz, 1H), 8.79 (d, J = 7.2 Hz, 1H), 7.88
7.83 (m, 1H), 7.79
7.76 (m, 1H), 4.39
4.34 (m, 0.5H), 4.26
4.21 (m, 0.5H), 4.19
3.98 (m, 2H), 3.79
3.74 (m, 2H), 3.19
3.13 (m, 2H), 1.81 (s, 3H), 1.66
1.61 (m, 3H), 1.31 (t, J = 7.2 Hz, 3H). 13C NMR (100 MHz,CDCl3): δ 178.19, 169.03, 166.72, 166.68, 149.71, 149.06, 142.83, 142.80, 133.04, 132.87, 132.83, 132.66, 132.46, 132.42, 132.14, 131.64, 131.43, 129.43, 129.39, 127.62, 127.52, 125.87, 125.20, 123.54,123,34, 118.56, 118.30, 98.01, 97.77, 77.26, 71.66, 71.44, 55.05, 53.81, 34.62, 34.53, 32.99, 27.71, 25.63, 19.10, 18.70, 14.00. HRMS (ESI) calcd for C23H21NO4NaS2 [M þ Na]þ 462.0810, found 462.0799. 3741

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Allyl 8-Oxo-3-(propylthio)-8H-acenaphtho[1,2-b]pyrrole9-carboxylate (3a). The residue was purified by column chromatog-

2-Oxo-2-(2,2,3,3,3-pentafluoropropoxy)ethyl 3-(Allylthio)-8oxo-8H-acenaphtho[1,2-b]pyrrole-9-carboxylate (5b). The

raphy on silica gel (CH2Cl2 with trace methanol) to provide 3a. Reddish brown solid (yield 76%); mp 244.9
245.7 °C. 1H NMR (400 MHz, CDCl3): δ 8.89 (d, J = 8.0 Hz, 1H), 8.76 (d, J = 7.6 Hz, 1H), 8.67 (d, J = 8.4 Hz, 1H), 7.82 (t, J = 8.0 Hz, 1H), 7.45 (d, J = 8.4 Hz, 1H), 5.97
5.87 (m, 1H), 5.32 (d, J = 17.2 Hz, 1H), 5.25 (d, J = 10.4 Hz, 1H), 4.30 (d, J = 6.0 Hz, 2H), 3.20 (t, J = 7.2 Hz, 2H), 1.96
1.87 (m, 2H), 1.19 (t, J = 7.6 Hz, 3H). 13C NMR (100 MHz, CDCl3): δ 177.97, 168.75, 166.29, 150.95, 142.82, 133.09, 132.40, 132.21, 131.80, 131.43, 130.68, 129.25, 127.46, 122.94, 122.12, 118.11, 117.49, 40.03, 34.02, 21.61, 13.66. HRMS (ESI) calcd for C21H18NO3S [M þ H]þ 364.1007, found 364.0994.

residue was purified by column chromatography on silica gel (CH2Cl2 with trace methanol) to provide 5b. Reddish brown solid (yield 49%); mp 205.3
206.4 °C. 1H NMR (400 MHz, CDCl3): δ 8.85 (d, J = 8.0 Hz, 1H), 8.76 (d, J = 7.6 Hz, 1H), 8.69 (d, J = 8.0 Hz, 1H), 7.83 (t, J = 8.0 Hz, 1H), 7.49 (d, J = 8.0 Hz, 1H), 6.04
5.97 (m, 1H), 5.47 (d, J = 16.8 Hz, 1H), 5.34 (d, J = 10 Hz, 1H), 4.65 (t, J = 12.4 Hz, 2H), 4.56 (s, 2H), 3.90 (d, J = 6.8 Hz, 2H). 13C NMR (100 MHz, CDCl3): δ 177.70, 168.10, 165.96, 165.35, 150.25, 142.81, 133.14, 132.59, 132.11, 131.99, 130.95, 130.71, 129.18, 127.68, 123.23, 122.95, 120.13, 117.57, 60.17 (t, J = 27.9 Hz, 1C), 38.28, 35.11. HRMS (ESI) calcd for C23H15NO5F5S [M þ H]þ 512.0591, found 512.0593.

Allyl 3-(Allylthio)-8-oxo-8H-acenaphtho[1,2-b]pyrrole-9carboxylate (3b). The residue was purified by column chromatogra-

Ethyl 8-Oxo-3-(2-(thiazolidin-3-yl)ethylthio)-8H-acenaphtho [1,2-b]pyrrole-9-carboxylate (2h). The stirred solution of com-

phy on silica gel (CH2Cl2 with trace methanol) to provide 3b. Brown solid (yield 69%); mp 180.1
182.0 °C. 1H NMR (400 MHz, CDCl3): δ 8.88 (d, J = 8.0 Hz, 1H), 8.74 (d, J = 7.2 Hz, 1H), 8.66 (d, J = 8.4 Hz, 1H), 7.82 (t, J = 8.0 Hz, 1H), 7.48 (d, J = 8.0 Hz, 1H), 6.05
5.87 (m, 2H), 5.46 (d, J = 16.8 Hz, 1H), 5.34
5.30 (m, 2H), 5.24 (d, J = 10.4 Hz, 1H), 4.30 (d, J = 5.6 Hz, 2H), 3.89 (d, J = 6.4 Hz, 2H). 13C NMR (100 MHz, CDCl3): δ 177.96, 168.86, 166.20, 149.33, 142.75, 132.93, 132.35, 132.13, 131.82, 131.39, 131.06, 130.77, 129.21, 127.54, 123.24, 123.11, 120.01, 118.16, 117.91, 40.04, 35.19. HRMS (ESI) calcd for C21H16NO3S [M þ H]þ 362.0851, found 362.0839.

pound 2 (0.43 mmol) in methanol (12 mL) and chloroform (3 mL) was cooled to 0
5 °C. 2-(Thiazolidin-3-yl)ethanethiol (0.257 g, 1.72 mmol) was added, and then the mixture was stirred at 0
5 °C overnight. The solvent was then removed under vacuum. The residue was purified by column chromatography on silica gel (CH2Cl2/methanol: 100/1) to provide 2h. Reddish brown solid (yield 42%); mp 217.3-219.1 °C. 1H NMR (400 MHz, CDCl3): δ 8.83
8.77 (m, 1H), 8.68
8.56 (m, 2H), 7.78 (t, J = 8.4 Hz, 1H), 7.46 (d, J = 8.0 Hz, 1H), 4.25 (m, 2H), 3.79
3.73 (m, 2H), 3.48
3.04 (m, 8H), 1.34 (t, J = 7.2 Hz, 3H). 13C NMR (100 MHz, CDCl3): δ 177.83, 168.72, 166.67, 142.53, 132.90, 132.28, 131.92, 131.61, 130.45, 128.93, 127.54, 123.05, 122.60, 122.42, 117.69, 57.94, 51.33, 49.28, 43.40, 33.00, 29.53, 14.02. HRMS (ESI) calcd for C22H20N2O3NaS2 [M þ Na]þ 447.0813, found 447.0833. In Vitro Tyrosine Kinase Inhibition Assay32. The kinase domains of VEGFR2, c-Kit, RET, c-Src, EphA2, EphB2, c-Met, RON, and IGF1R were expressed using the Bac-to-Bac baculovirus expression system (Invitrogen, Carlsbad, CA) and purified on Ni-NTA columns (QIAGEN Inc., Valencia, CA) as previously described. FGFR1, VEGFR1, PDGFRR, PDGFRβ, EGFR2, and Abl were purchased from Upstate Biotechnology Inc. (Charlottesville, VA). Poly(Glu-Tyr)4:1, the substrate of tyrosine kinase, was from Sigma-Aldrich Corp., St. Louis, MO, U.S. Monoclonal anti-phosphotyrosine (PY) antibody PY99 was purchased from Santa Cruz Biotechnology, Inc. The tyrosine kinase activities were determined in 96-well ELISA plates (Corning, NY, U.S.) precoated with 20 μg/mL poly(Glu,Tyr)4:1. First, 80 μL of 5 μM ATP solution diluted in kinase reaction buffer (50 mM HEPES, pH 7.4, 20 mM MgCl2, 0.1 mM MnCl2, 0.2 mM Na3VO4, and 1 mM DTT) was added to each well. Various concentrations of compounds diluted in 10 μL of 1% DMSO (v/v) were then added to each reaction well, with 1% DMSO (v/v) being used as the negative control. Subsequently, the kinase reaction was initiated by the addition of tyrosine kinase proteins diluted in 10 μL of kinase reaction buffer solution. Experiments at each concentration were performed in duplicate. After incubation for 60 min at 37 °C, the plate was washed three times with phosphate buffered saline (PBS) containing 0.1% Tween 20 (T-PBS). Next, 100 μL of antiphosphotyrosine antibody (PY99, 1:1000 dilution) diluted in T-PBS containing 5 mg/mL BSA was added. After 30 min of incubation at 37 °C, the plate was washed three times as before. Horseradish peroxidase conjugated goat anti-mouse IgG (100 μL) diluted 1:2000 in T-PBS containing 5 mg/mL BSA was added. The plate was reincubated at 37 °C for 30 min and then washed with PBS. Finally, 100 μL of a solution containing 0.03% H2O2 and 2 mg/mL o-phenylenediamine in 0.1 M citrate buffer, pH 5.5, was added and samples were incubated at room temperature until color emerged. The reaction was terminated by the addition of 50 μL of 2 M H2SO4, and the plate was read using a multiwell spectrophotometer (MAX190, Molecular Devices, Sunnyvale, CA, U.S.) at 490 nm. The inhibition rate (%) was calculated using the following equation: inhibition rate = [1
(A490treated/A490control)]  100. IC50 values were determined from the

2-Oxo-2-(propylthio)ethyl 8-Oxo-3-(propylthio)-8H-acenaphtho[1,2-b]pyrrole-9-carboxylate (4a). The residue was purified by column chromatography on silica gel (CH2Cl2 with trace methanol) to provide 4a. Reddish brown solid (yield 43%); mp 235.2
236.7 °C. 1H NMR (400 MHz, CDCl3): δ 8.86 (d, J = 8.4 Hz, 1H), 8.76 (d, J = 7.2 Hz, 1H), 8.68 (d, J = 8.0 Hz, 1H), 7.82 (t, J = 8.4 Hz, 1H), 7.44 (d, J = 8.0 Hz, 1H), 4.59 (s, 2H), 3.20 (t, J = 7.2 Hz, 2H), 2.94 (t, J = 7.2 Hz, 2H), 1.94
1.89 (m, 2H), 1.66
1.60 (m, 2H), 1.19 (t, J = 7.6 Hz, 3H), 0.97 (t, J = 7.2 Hz, 3H). 13C NMR (100 MHz, CDCl3): δ 193.75, 177.77, 168.36, 165.55, 151.53, 142.84, 133.28, 132.53, 132.21, 131.90, 130.65, 129.23, 127.54, 123.04, 122.08, 117.31, 46.44, 34.00, 30.88, 22.69, 21.59, 13.65, 13.29. HRMS (ESI) calcd for C23H21NO4NaS2 [M þ Na]þ 462.0810, found 462.0794.

2-Oxo-2-(propylthio)ethyl 3-(Allylthio)-8-oxo-8H-acenaphtho [1,2-b]pyrrole-9-carboxylate (4b). The residue was purified by column chromatography on silica gel (CH2Cl2 with trace methanol) to provide 4b. Reddish brown solid (yield 39%); mp 203.9
204.4 °C. 1H NMR (400 MHz, CDCl3): δ 8.84 (d, J = 8.0 Hz, 1H), 8.74 (d, J = 7.6 Hz, 1H), 8.67 (d, J = 8.4 Hz,1H), 7.82 (t, J = 8.0 Hz, 1H), 7.47 (d, J = 8.0 Hz,1H), 6.03
5.96 (m, 1H), 5.47 (d, J = 16.8 Hz, 1H), 5.33 (d, J = 10.0 Hz, 1H), 4.59 (s, 2H), 3.89 (d, J = 6.8 Hz, 2H), 2.94 (t, J = 7.2 Hz, 2H), 1.66
1.58 (m, 2H), 0.97 (t, J = 7.6 Hz, 3H). 13C NMR (100 MHz, CDCl3): δ 193.67, 177.75, 168.27, 165.48, 149.94, 142.79, 133.14, 132.49, 132.12, 131.92, 130.99, 130.71, 129.18, 127.62, 123.32, 123.00, 120.08, 117.68, 46.44, 35.14, 30.89, 22.68, 13.27. HRMS (ESI) calcd for C23H19NO4NaS2 [M þ Na]þ 460.0653, found 460.0643.

2-Oxo-2-(2,2,3,3,3-pentafluoropropoxy)ethyl 8-Oxo-3(propylthio)-8H-acenaphtho[1,2-b]pyrrole-9-carboxylate (5a). The residue was purified by column chromatography on silica gel (CH2Cl2 with trace methanol) to provide 5a. Reddish brown solid (yield 52%); mp 256.9
258.8 °C. 1H NMR (400 MHz, CDCl3): δ 8.85 (d, J = 8.4 Hz, 1H), 8.76 (d, J = 7.6 Hz, 1H), 8.69 (d, J = 8.4 Hz, 1H), 7.82 (t, J = 8.0 Hz, 1H), 7.45 (d, J = 8.0 Hz, 1H), 4.65 (t, J = 12.4 Hz, 2H), 4.57 (s, 2H), 3.21 (t, J = 7.6 Hz, 2H), 1.95
1.90 (m, 2H), 1.19 (t, J = 7.2 Hz, 3H). 13C NMR (100 MHz, CDCl3): δ 177.68, 168.16, 165.98, 165.40, 151.82, 142.83, 133.25, 132.60, 132.18, 131.95, 130.64, 129.21, 127.58, 122.94, 122.05, 117.19, 60.16 (t, J = 27.9 Hz, 1C), 38.27, 33.99, 21.58, 13.65. HRMS (ESI) calcd for C23H17NO5F5S [M þ H] þ 514.0748, found 514.0748.

3742

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Journal of Medicinal Chemistry results of at least three independent tests and calculated from the inhibition curves. The accuracy of this in vitro screening assay was confirmed by the measured IC50 value of the positive control C1, which is similar to the reported value.

Molecular Simulation and Physiochemical Properties Calculation. The crystal structure of FGFR1 was retrieved from the Protein Data Bank (PDB entry 2FGI, chain B).54 Protein preparation, after removing all water molecules, adding hydrogen atoms, and considering the ionization, was performed using the Glide 4.0 module in Maestro, version 7.5 (Schr€odinger, Inc.).55 Considering the essential hydrogen bond interaction in the kinase hinge region, we set two hydrogen bond constraints of heteroatoms in residues Ala 564 and Glu 562 so that each compound must have one hydrogen bond interaction in the kinase hinge region at least. The grid-enclosing box was formed based on the ligand in the complex structure and constraints set in the last step. The three-dimensional (3D) structures of compounds 2, 2b, 2e, 3b were drawn using the Builder tool and generated with Ligprep module. All the compounds were docked into the ATPbinding site of FGFR1 using Glide 4.0 with standard precision (SP) approach, and a scaling factor of 1.0 was set to VDW radii of the protein atoms with the partial atomic charges less than 0.25. The best one ranked by GlideScore among the top 10 binding poses was chosen as the preliminary model for the next minimization, which was sequentially minimized with PCRG (Polak
Ribiere conjugate gradient) methods by MacroModel module in Maestro, version 7.5. The OPLS 2005 was selected as force field, and the convergence criteria were set with the maximum iteration as 500 and the gradient as 0.05 kcal/mol. Moreover, the physicochemical properties of all the compounds were also calculated with QikProp module in Maestro and XLOGP3 online service. All computations were carried out on a Linux (RedHat 3.4.6-2) cluster with 2.4 GHz Intel Xeon processors. Cell Lines. BxPC3 and WI-38 were purchased from American Type Culture Collection (ATCC, Manassas, VA, U.S.). T24 cells were from Shanghai Institute of Biochemistry and Cell Biology (Shanghai, China). They were maintained in strict accordance with the supplier’s instructions and established procedures.

In Vitro Antiproliferative Assay on Human Cancer Cell Lines. Cell proliferation was evaluated using the SRB assay as previously described.56 Briefly, cancer cells were seeded into 96-well plates and grown for 24 h. The cells were then treated with increasing concentrations of compounds and grown for a further 72 h. The medium remained unchanged until the completion of the experiment. The cells were then fixed with 10% precooled trichloroacetic acid (TCA) for 1 h at 4 °C and stained for 15 min at room temperature with 100 μL of 4 mg/mL SRB solution (Sigma) in 1% acetic acid. The SRB was then removed, and the cells were quickly rinsed five times with 1% acetic acid. After cells were air-dried, protein-bound dye was dissolved in 150 μL of 10 mM Trisbase for 5 min and measured at 515 nm using a multiwell spectrophotometer (MAX190, Molecular Devices). The inhibition rate on cell proliferation was calculated as follows: inhibition rate = (1
A515treated/ A515control)  100. The IC50 values were obtained by the Logit method and were determined from the results of at least three independent tests. DNA Intercalating Assay by CD Spectra51. CT DNA was purchased from Sigma Aldrich and used without further purification. A solution of CT DNA in 20 mM Tris-HCl buffer (pH 7.4) was stored at 4 °C and used after no more 4 days. The concentration of CT DNA was determined spectrophotometrically from the molar absorption coefficient (6600 M
1 cm
1). The stock solution was attested to be sufficiently free from protein, as it gave a UV absorbance ratio at 260 and 280 nm of more than 1.8. The CD measurements were performed on a J-815 spectrophotometer (Jasco, Japan) using 1 cm quartz cell in the range of 200
400 nm. The samples containing 100 μM CT DNA and 20 μM drugs in Tris-HCl buffer were recorded in the CD spectrum, after

ARTICLE

shaking, thermal equilibration, and subtraction of the Tris-HCl buffer. CD spectra in the range of 230
300 nm were analyzed. Western Blot Analysis. Anti-Akt, phospho-Akt, Erk1/2, and phospho-Erk1/2 (T202/Y204) antibodies were from Cell Signaling Technology (Beverly, MA). Anti-FGFR1, phosphor-FGFR1 (Tyr 653/ 654) antibodies were from Santa Cruz Biotech (Santa Cruz, CA). AntiGAPDH was from Kangchen (Shanghai, China). Goat anti-mouse lgG and Goat anti-rabbit lgG were from Calbiotech (San Diego, CA). BxPC3 cells were plated into a six-well plate (3  105 cells/well). After adherence, cells were starved and incubated in serum-free medium for 24 h and then exposed to the corresponding compounds for 12 h. For analysis of FGFR1 phosphorylation and downstream signal transduction pathways, cells were stimulated with 50 ng/mL bFGF for 10 min at 37 °C at the end of compounds treatment. Whole-cell lysates were collected and boiled for 10 min in 2 SDS sample buffer, subjected to 10% SDS
PAGE, and transferred to nitrocellulose (Amersham Life Sciences). The blot was blocked in blocking buffer (5% nonfat dry milk/ 1% Tween-20 in TBS) for 1 h at room temperature and then incubated with primary antibodies (anti-FGFR1 (1:200), anti-phospho-FGFR1 (1:200), anti-Akt (1:5000), anti-phospho-Akt (1:500), anti-Erk (1:1000), anti-phospho-Akt (1:2000), and anti-GAPDH (1:10000)) in blocking buffer for 2 h at room temperature. The bands were then visualized using horseradish peroxidase-conjugated secondary antibodies (1:2000) followed by ECL (Pierce Biotech, Rockford, IL).

’ ASSOCIATED CONTENT

bS

Supporting Information. In silico target prediction of the representative compound 2 and CD spectra of CT DNA (100 μM) incubated with target compounds. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*For H.X.: phone, 86-21-50806600 ext 2422; fax, 86-21-50806722; e-mail, [email protected]. For Y.X.: phone, 86-21-64251399; fax, 86-21-64252603; e-mail, [email protected]. For X.Q.: phone, 86-21-64253589; fax, 86-21-64252603; e-mail, [email protected].

’ ACKNOWLEDGMENT This work was financially supported by the Key New Drug Creation and Manufacturing Program (Grants 2009ZX09103001 and 2009ZX09103-102), the National High Technology Research and Development Program of China (863 Program 2011AA10A207), the China 111 Project (Grant B07023), the Shanghai Leading Academic Discipline Project (Grant B507), and the Fundamental Research Funds for the Central Universities. ’ ABBREVIATIONS USED CD, circular dichroism; CT DNA, calf thymus DNA; EGFR, epidermal growth factor receptor; ELISA, enzyme-linked immunosorbent assay; Erk1/2, extracellular-signal regulated kinase 1/2; FGFR, fibroblast growth factor receptor; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; IGF1R, insulin-like growth factor 1 receptor; PDGFR, platelet-derived growth factor receptor; RTK, receptor tyrosine kinase; SAR, structure
activity relationship; VDW, van der Waals; VEGFR, vascular endothelial growth factor receptor 3743

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