NZ620434B2 - Agonists of src homology-2 containing protein tyrosine phosphatase-1 and treatment methods using the same - Google Patents

Abstract

Disclosed are biarylethers compounds of formula I which have Src homology-2 containing protein tyrosine phosphatase-1 (SHP-1) agonist activity. Also disclosed are pharmaceutical compositions comprising compounds of formula I for treating cancer. Examples of a compound of formula I are: 1-(4-Chloro-3-(trifluoromethyl)phenyl)-3-(4-(4-cyanophenoxy)phenyl)urea 1-(4-(4-cyanophenoxy)phenyl)-3-(3,4-dimethoxybenzyl)urea 4-(3-(3-(trifluoromethyl)benzen-sulfonylamino)phenoxy)benzonitrile loro-3-(trifluoromethyl)phenyl)-3-(4-(4-cyanophenoxy)phenyl)urea 1-(4-(4-cyanophenoxy)phenyl)-3-(3,4-dimethoxybenzyl)urea 4-(3-(3-(trifluoromethyl)benzen-sulfonylamino)phenoxy)benzonitrile

Description

/049446 TITLE OF THE INVENTION AGONISTS OF SRC HOMOLOGY—Z CONTAINING PROTEIN TYROSINE PHOSPHATASE-l AND ENT METHODS USING THE SAME FIELD OF THE INVENTION The present invention relates to new compounds having Src homology-2 containing n tyrosine phosphatase-l (SHP-l) agonist ty and ent methods using the same.

BACKGROUND OF THE INVENTION

[0002] SHP-l, a protein-tyrosine phosphatase with two Src homology 2 (8H2) domains, is a regulator of various intracellular signaling molecules, such as signal transducer and activator of transcription 3 (STAT3), KIT, CD22, CD5, CD72, SHPS—l, TIMP (metalloproteinases), CDK2, p27, SRC, ZAP70, IL-lO, NF-KB, Lck, 3BP2, Lyn and cyclin D1 ..

[0003] STAT3 is a transcription factor which tes cell growth and survival by modulating the sion of target genes. It acts as an oncogene which is constitutively active in many cancers including liver, lung, head and neck, prostate, and breast as well as myeloma and leukemia. A key regulator of STAT3 activity is SHP-l. From a mechanistic perspective, SHP-l exhibits protein phosphatase activity which reduces the level of Phospho— STAT3 (P-STAT) and subsequently blocks the dimerization of 3. Therefore, expression of target genes, such as cyclin D1 and survivin transcribed by STAT3, is significantly reduced. In addition, studies of SHP-l protein and SHP—l mRNA showed that expression level of SHP-l was low in most cancer cells; and genetic increase in SHP-l in cancer cells resulted in the suppression of cell growth, suggesting that the SHP-l gene acts as a tumor suppressor. From the drug discovery point of view, development of a small molecule which can reduce P-STAT3 and increase SHP-l level is a promising direction for cancer therapy. SHP-l also play an important role in bone remodeling, a process of bone— ng osteoblasts and bone-resorbing osteoclasts. Loss function of SHP—l reuslts in osteoclast and eventually leads to osteoporosis. Therefore, enhancement of SHP—l activity might be a direction for osteoporosis t. In addition, increase of SHP-l is benefit for the macrophages of multiple sclerosis ts BRIEF SUMMARY OF THE INVENTION The present ion is based on the unexpected finding that newly designed nds act as SHP-l ts and have the ability to reduce P-STAT3, and are useful for treating certain diseases, such as cancer. Specifically, the compounds of the invention do not block activity of kinases, such as Raf-1 and VEGFRZ.

Particularly, in one aspect, the invention provides a compound of formula I wherein R1, R2, and R3 are independently hydrogen, halo, hydroxyl, optionally substituted alkoxyl, optionally substituted thioalkoxy, optionally substituted alkyl, optionally tuted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally ‘10 substituted heterocycloalkyl, optionally substituted aryl, optionally tuted aralkyl, optionally substituted aryl, optionally substituted heteroarakyl, - (C)mNHC(X)NH(C)nRa‘> '(C)pNHC(X)Rb'a' (C)qNHS(O)2Rc, ~(C)1-(X)NHRd—,0r - (ClsNH(C)tRe; wherein Ra, Rb, RC, Rd and Re are ndently hydrogen, halo, hydroxyl, optionally tuted alkoxyl, optionally substituted thioalkoxy, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substitutedcycloalkyl, optionally substituted heterocycloalkyl, ally substituted aryl, ally substituted aralkyl, optionally substituted heteroaryl, optionally substituted heteroarakyl; X=O or S; and m, n, p, q, r, s, t=O, 1, or 2.

In another aspect, the present ion provides a compound of Formula 11, including a compound of Formula II(a), a compound of Formula Il(b), or a compound of Formula 11(0), Rt”; “yfifit‘tai («R , $25.,“ ;::233?“» R” 1:“ ii: , ii: (:3 “ “‘ {:5} / “RC ”M" ixa ,i; fjmk. MAC? viii». “’1’: ’3’: ii.“ NT: fig} ii N «z N 0 x mm wherein R4, R5 and R6 are independently hydrogen, halo, hydroxyl, optionally substituted alkoxyl, optionally tuted thioalkoxy, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted aralkyl, ally substituted heteroaryl, optionally substituted heteroarakyl, ~(C)mNHC(X)NH(C)n Ra": “(C)pNHC(X)RbT (C)qNHS(O)2Rc, -(C)r(X)NHRd-,Or -(C)sNH(C)tRe; wherein Ra, Rb, RC, Rd and Re are independently hydrogen, halo, hydroxyl, optionally substituted alkoxyl, optionally tuted thioalkoxy, optionally substituted alkyl, optionally substituted l, optionally substituted alkynyl, optionally substitutedcycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted aralkyl, optionally substituted heteroaryl, ally tuted heteroarakyl; X=O or S; and m, n, p, q, r, s, t=0, 1, or 2.

In a further aspect, the invention provides a compound of Formula 111 {Dev / O\ R7 111 wherein R7 is hydrogen, halo, hydroxyl, optionally substituted alkoxyl, optionally substituted koxy, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, ally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted aralkyl, optionally substituted heteroaryl, optionally substituted heteroarakyl, -(C)mNHC(X)NH(C)n Ra~, -(C)pNHC(X)Rb—,- (C)qNHS(O)2Rc, ~(C)r(X)NHRd-,0r -(C)sNH(C)tRe; wherein Ra, Rb, RC, Rd and Re are independently hydrogen, halo, hydroxyl, optionally tuted alkoxyl, optionally substituted thioalkoxy, optionally substituted alkyl, optionally tuted alkenyl, optionally substituted alkynyl, optionally tutedcycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted aralkyl, optionally tuted heteroaryl, optionally substituted heteroarakyl; X=O or S; and m, n, p, q, r, s, t=0, 1, or 2.

The present invention also provides a pharmaceutical composition comprising one or more of the above-described compounds. The pharmaceutical composition of the invention may be used for increasing expression levels or biological activity of SHP—l in a cell, or treating a disease or condition characterized by decreased expression levels or biological ty of Src homology-2 containing protein ne phosphatase-1, which includes but is not d to cancer (e.g. hepatocellular oma, leukemia, lung cancer, breast , renal cancer, thyroid , head and neck cancer, sclerosis and osteoporosis.

Also within the scope of this invention is the use of any of the above-described compounds for increasing expression levels or ical activity of SHP-l in a cell, or treating a disease or condition characterized by decreased expression levels or biological activity of SHP—l as bed herein and for the manufacture of a medicament for treating the same.

Also provided is a method for increasing SHP-l expression levels or biological ty in a cell, comprising contacting the cell with an effective amount of a compound or a pharmaceutical composition as described herein.

Further provided is a method for ng a disease or ion characterized by decreased expression levels or biological activity of SHP-l in a subject in need thereof, comprising administering to the subject an effective amount of a nd or a pharmaceutical composition as described herein.

The various embodiments of the present invention are described in details below.

Other characteristics of the present invention will be clearly presented by the following detailed ptions and drawings about the various embodiments and claims.

[0012] It is believed that a person of ry knowledge in the art where the present invention belongs can utilize the present ion to its broadest scope based on the descriptions herein with no need of further illustration. Therefore, the following descriptions should be understood as of demonstrative purpose instead of limitative in any way to the scope of the present invention.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS For the purpose of illustrating the invention, there are shown in the drawings embodiments which are tly preferred. It should be understood, however, that the invention is not limited to the red embodiments shown.

In the drawings:

[0015] Fig. 1 shows the chemical structure of sorafenib and compound SC-l.

Fig. 2 shows the general synthetic procedure for formulae I, II and III of the invention.

Fig. 3 shows Raf-l activity in the cells treated by sorafenib and compound 1, respectively. Huh-7 cells were exposed to sorafenib or compound 1 at 10 uM for 24 hours and cell s were analyzed for raf-l activity. Columns, mean; bars, SD (n = 3). *P < 0.05.

Fig. 4 shows the results of the ELISA analysis for the inhibitory s of compounds 1-25 versus sorafenib, each at 10 MM, on the IL-6 stimulated P-STAT in PLCS cells after 24 h of treatment. Columns, mean, bars, SD (N = 3).

Fig. 5 shows the results of n blot analysis for the effect of compounds 1 and 12, each at 5 MM and 10 MM on the phosporylation of P-STAT3, STAT3, cyclin D and survivin in PLCS cells in FBS-containing medium after 24 h of treatment.

Fig. 6 shows (A) the results of ELISA analysis for cell death induced by compound 1 and 12, at 5, and 10 uM, after 24 h of treatment in PLCS cells; and (B) shows the results of flow cytometry is of cell death induced by compound 1 and 12, at 5, and uM, after 24 h of treatment in PLCS cells.

Fig. 7 shows (A) the effects of sorafenib and SC-l on phospho-VEGFR2 in HUVEC cells, wherein the cells were exposed to nib or SC-l at 10 uM for 24 h; (B) the s of sorafenib and SC-l on Raf-1 activity, wherein the cells were exposed to sorafenib or 801 at 10 uM for 24 h. Points, mean; bars, SD (n = 6).

Fig. 8 shows (A) the dose-escalation effects of sorafenib and SC—l on cell ity in four HCC cell lines, wherein cells were exposed to sorafenib or SC-l at the indicated doses for 72 h and cell Viability was assessed by MTT assay; and the dose— escalation effects of sorafenib and SC-l on apoptosis in four HCC cell lines, wherein Cells were exposed to sorafenib or 801 at the indicated doses for 24 h, and cell lysates were analyzed by ometry (B), or cell death ELISA (C). Points, mean; bars, SD (n = 6).

Fig. 9 shows (A) the effects of sorafenib or 801 on STAT3~related proteins, wherein cells were treated with sorafenib or SC-l at 10 uM for 24 h; (B) the dose-escalation effects of sorafenib or SC-l on phospho-STAT3 in PLCS cells, wherein cells were treated with drugs at the indicated concentrations for 24 h; (C) the effects of sorafenib and SC—l on STAT3 activity (left, Phospho-STAT3 ELISA; Right, luciferase reporter assay of STAT3), n cells were treated with sorafenib or SC—l at 10 uM for 24 hs and phospho—STAT3 ELISA or luciferase activity was ed; (D) the protective effects of STAT3 on apoptosis induced by sorafenib in PLCS cells, wherein cells (wild type or ectopic expression of STAT3) were d to sorafenib or SC-l at 10 uM for 24 h, and apoptotic cells were analyzed by flow cytometry. Columns, mean; bars, SD (n = 3). *P < 0.05.

Fig. 10 shows inhibition of SHP—l reverses effects of sorafenib and SC-l on o-STAT3 and sis. A, left, vanadate, a non-specific phosphatase inhibitor. Right, specific SHP-l inhibitor. Columns, mean; bars, SD (n = 3). *P < 0.05. B, left, silencing SHP- 1 by siRNA reduces effects of sorafenib or SC—l on p-STAT3 in HCC cells. PLCS cells were transfected with l siRNA or SHP-l siRNA for 24 h then treated with sorafenib or 801 for another 24 h. Middle, the ty of SHP-l in PLCS cells. Columns, mean; bars, SD (n 3). *P < 0.05. Right, effects of sorafenib or SC-l on protein interactions between SHP-l STAT3. PLCS cells were treated with sorafenib or SC-l at 10 MM for 24 hours. C, knock- down of SHP~2 does not affect the effects of sorafenib or SC—l on p-STAT3 and apoptosis. D, knock-down ofPTP-lB does not affect effects of sorafenib on p—STAT3 and apoptosis.

PLC5 cells were transfected with l siRNA or SHP-2 siRNA or PTP-lB siRNA for 24 h then d with sorafenib or SC—l at 10 uM for 24 h.

Fig. 11 shows that SC-l down-regulates p—STAT3 and induces sis in HUVEC cells. A, effects of nib or SC-l on p-STAT3 (left) and sis (right) in HUVEC cells. Cells were exposed to sorafenib or SC-l at 10 uM for 24 h. Apoptotic cells were assayed by flow cytometry (sub-G1). B, effects of SC-l on TRAIL sensitization in HCC. PLC5 cells were treated with SC—l (10 uM) and/or TRAIL (100 ng/ml) for 24 h. C, silencing Raf-l does not affect the effects of the drugs on p-STAT3. PLC5 cells were transfected with control siRNA or Raf—l siRNA for 24 h then treated with sorafenib or SC—l at 10 uM for 24 h. D, effect of sorafenib and SC-l on JAK2 activity. PLC5 cells were d to nib or SC-l at lOuM for 24 h. Points, mean; bars, SD (n = 6). E, effects of sorafenib and SC-l on SOCS—l and SOCS-3. Sk—Hepl cells were pre—treated with IL-6 for 24 h then were treated with sorafenib or SC-l at the indicated doses for another 24 h in the presence of IL—6. F, effects of STAT-C on apoptosis induced by SC-l in PLC5 cells. Cells (Wild type or ectopic sion of C) were exposed to sorafenib or 801 at 10 uM for 24 h. G, effects of sorafenib and SC-l on SHP—l. Columns, mean; bars, SD (n = 3). *P < 0.05.

[0026] Fig. 12 shows in vivo effect of sorafenib and SC—l on Huh-7 xeonograft nude mice A, sorafenib shows antitumor effect on Huh—7 tumors. Left, points, mean (n = 6); bars, SE. *, P < 0.05; **, P < 0.01. Right Upper, western blot analysis ofp-STAT3 and STAT3 in Huh—7 tumors. Right Lower, the activity of SHP—l in Huh—7 tumors. B, SC-l shows a significant antitumor effect on Huh-7 tumors. Left, points, mean (n = 6); bars, SE. Right Upper, western blot analysis of p—STAT3 and STAT3 in Huh-7 tumors. Right Lower, the activity of SHP—l in Huh—7 tumors.

Fig. 13 shows the anti—proliferation effects of SC-l and SC-43 in various cancer cell lines, including breast cancer cell lines (A) MDAMB23l, (B) MDAMB468 and (C) MCF—7, and leukemia cancer cell lines (D) HL~60, (E) KG-l, and (F) ML-l.

[0028] Fig. 14 shows that Sorafenib derivatives induce significant apoptosis in a dose— dependent manner, where (A), (B), (C), (D) and (E) refer to 8043 for PLC5, HepG2, Hep3B, HA59T and SK-Hepl cells, respectively; and (F), (G), (H), (I) and (J) refer to SC-40 for PLC5, HepG2, Hep3B, HA59T and SK-Hepl cells, respectively. Points, mean; bars, SD (n = 6).

Fig. 15 shows that that SC~43 down-regulates phospho-STAT3—related signaling pathway in HCC cells, including PLC5, HepG2, Hep3B, HA59T and SK-Hepl cells.

Fig. 16 shows that SC-40 down—regulates phospho-STAT3-related signaling pathway in HCC, cells, including PLC5, HepG2, Hep3B, HA59T and SK-Hepl cells.

Fig. 17 shows that 8043 shows better inhibition of p—STAT3-related signaling pathway than sorafenib in HCC cells, (A) PLC5 and (B) Hep 3B.

Fig. 18 shows that both SC-43 and 8040 induce strong inhibition of p—STAT3 activity (A) and (B) p-STAT3 ELISA for SC-43 and SC-40, respectively, and (C) and (D) STAT3 reporter assay for SC—43 and SC-40, respectively.

[0033] Fig. 19 shows that the SC tives increase phosphatase activity of SHP-l in vitro, (A) SC-43, (B) SC-40, and (C) SC-49.

Fig. 20 shows that the SC derivatives se phosphatase activity of SHP-l in vitro, (A) SC-43 and (B) SC~40.

Fig. 21 shows that (A) the antitumor effect of SC-40 on PLC5 tumors; (B) Western blot analysis of p—STAT3 and STAT3 in PLC5 ; (C) the body weight of the animals; and (D) the tumor weight and (E) activity of SHP-l in PLC5 tumors. Points, mean (11 = 6); bars, SE Fig. 22 shows that SC—43 exhibits antitumor effect in vitro and in vivo, (A) the cytotoxicity of SC—43 in HCC cells, (B) the antitumor effect of SC—43 in HCC-bearing mice, (C) the activity of SHP-l induced by SC—43, and (D) Western blot analysis of p-STAT3 and STAT3 in HCC cells d by SC-43 (10 uM and 20 HM).

Fig. 23 shows (A) the image obtained by the non-invasive in vivo imaging system of the treated mice, (B) shows the body weight of the mice, and (C) shows the survival curve between control and treated mice.

DETAILED DESCRIPTION OF THE INVENTION Unless defined otherwise, all cal and scientific terms used herein have the art to which this ion same meaning as commonly understood by a person skilled in the belongs. All publications mentioned herein are incorporated herein by nce to disclose and describe the methods and/or materials in connection with which the publications are cited.

As used herein, the singular forms “a”, “an”, and “the” e plural referents unless the context y dictates ise. Thus, for example, reference to “a sample” includes a plurality of such samples and equivalents thereof known to those skilled in the art. 2012/049446 nib (BAY43-9006, Nexavar) has been used clinically for renal carcinoma and hepatocellular carcinoma (HCC). It is known as a multiple kinase inhibitor that represses the activity of Raf—l and other ne kinases such as VEGFRZ, VEGFR3, Flt-3, PDGFR, and FGFR-l.

In this invention, we studied the relationship between the strucrure of sorafenib and its bioactivity and modified the structure of sorafenib. We ingly developed a number of sorafenib derivatives without the ability to block the kinase activity, and unexpectedly found that these compounds exhibit good therapeutic effects in certain diseases, such as cancer, at least comparable with that of sorafenib. Accoridng to the invention, the newly ed compounds of the invention act as SHP-l agonists and are useful for treating a disease or condition characterized by decreased expression levels or ical activity of SHP-l, such as cancer (e. g. hepatocellular carcinoma, hepatocellular carcinoma, leukemia, lung cancer, breast cancer, renal cancer, thyroid cancer, head and neck cancer, sclerosis and osteoporosis). The compounds of the invention also provide a new theraptic option for patients with the resistance to kinase inhibitors. These turmors generate kinase mutation after treatment and constitutely in the phosporylated active form, even in the present of a kinase inhibitor. Therefore, upregulation of a tumor suppressor, especially SHP-l, to repress the active on form of kinases is a promising direction for chemo-resistance patients. In other words, the compounds of the invention, acting through a new ing mechanism (kinase independent), e tive therapeutic options that may be helpful in the treatment of cacner with ance to conventional medical therapeutics.

In one aspect, the t ion provides a compound of formula I R2 1 wherein R1, R2, and R3 are ndently en, halo, hydroxyl, optionally substituted alkoxyl, optionally substituted thioalkoxy, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted aralkyl, optionally substituted heteroaryl, optionally substituted heteroarakyl, — (C)mNHC(X)NH(C)nRa—, -(C)pNHC(X)Rb—,f (C)qNHS(O)2RC, ~(C)T(X)NHRd—,or - (C)tRe; wherein R3, Rb, RC, Rd and Re are independently hydrogen, halo, hydroxyl, optionally substituted alkoxyl, optionally substituted thioalkoxy, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substitutedcycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally tuted aralkyl, optionally substituted heteroaryl, optionally tuted heteroarakyl; X=O or S; and m, n, p, q, r, s, t=0, l, or 2.

In one embodiment, the compound of formula I includes those in which R1, R2, and R3 are independently hydrogen, optionally substituted lower alkyl, — (C)mNHC(X)NH(C)n Ra-, ~(C)pNHC(X)Rb-, ~(C)qNHS(O)2Rc, or ~(C)SNH(C)tRe.

In r embodiment, the compound of formula I includes those in which Ra, with 1 to 3 Rb, RC, Rd and Re are independently phenyl or naphthyl, optionally substituted of halo, optionally substituted lower alkyl (such as groups selected from the group ting halo-substituted lower alkyl, e.g. trifluoromethyl), optionally substituted alkoxyl (e.g. such as halo-substituted lower alkoxyl, e.g. trifluoromethyl) and optionally substituted aryloxy (e.g. cyano—substituted y).

[0046] In certain examples, the compound of formula I is one of the compounds SC-l, SC-48, SC-49, SC-54, SC—SS, SC—56, SC—58, SC-43, SC-44, SC—45, SC-SO, SC—Sl, SC—52, SC-59, 8060 and SC-40 as listed in table 1.

Table l NC R1 de R1 R2 R3 5N N H H SC-48 N O\ H H H H -49 0 g H H 3%“N N H H $054 ELL/\NJLN O\ H H H H 3055 AANJKN OCF3 H H H H Jok E 5N N 8056 H H? H H 8058 N H H H H 2 <3 H PgN N H H 80-44 H fir \©/CF3 H 32 OCF3 -45 H fi/\©/ H 80-50 H 0 G H gNJkN H H 8051 H $EJK© H 80-52 2 H 35” \© H 01:3 8059 H 0 @ Me H H 2 KI N N 0 80260 H Me W0 2013/020014 PCT/U82012/049446 of Formula II, In another aspect, the present invention provides a compound of formula II(b) or a nd of including a compound of formula II(a), a compound formula II(c), R R R; a,“ (ff3 my]?, R; a i“ RE “a. xterm“. (”Rd i iiZ, [f I ,w. JRi ,, ix a x”x.p»! 0R:3% (in may R’s; C} GM (anV “~14?“ N R31 “,1 aka . ”2?, :g... Rwy”) “‘11:; “L «:3 I i“ a M 11 1 t f i N Nfifj: 1%“ ”'11:?ny “a N «a in NJ J,»- x “,2 .1 at % N N? l 1 {T3 xgfif. N02 II(a) 11(b) II(c) wherein R4 ,R5and R6 are independently hydrogen, halo, yl, optionally substituted alkyl, optionally tuted l, optionally substituted thioalkoxy, optionally substituted cycloalkyl, substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted optionally substituted heterocycloalkyl, ally substituted heteroarakyl, - aralkyl, optionally substituted heteroaryl, optionally (C)mNHC(X)NH(C)n Ra-, -(C)pNHC(X)Rb-,- S(O)2Rc, —(C)1‘(X)NHRd-,or (C)SNH(C)tRe; optionally wherein Ra, Rb, RC, Rd and Re are independently hydrogen, halo, hydroxyl, substituted alkyl, optionally substituted alkoxyl, optionally substituted thioalkoxy, optionally substitutedcycloalkyl, substituted alkenyl, ally substituted alkynyl, optionally substituted aryl, optionally substituted optionally substituted cycloalkyl, optionally tuted heteroarakyl; aralkyl, optionally substituted heteroaryl, optionally X=O or S; and m, n, p, q, r, s, t=0, l, or 2.

II includes those in which R4, R5 In one embodiment, the compound of formula and R6 are independently hydrogen, optionally substituted lower alkyl, — (C)mNHC(X)NH(C)nRa, HC(X)Rb-, -(C)qNHS(O)2Rc, or ~(C)SNH(C)tRe. a II includes thosein which Ra, In another embodiment, the compound of substituted with l to 3 Rb, Re, Rd and Re are independently phenyl or naphthyl, ally lower alkyl (such as the group consisting of halo, optionally substituted groups selected from substituted alkoxyl (e.g. such as halo—substituted lower alkyl, e.-g. trifluoromethyl), optionally substituted aryloxy (e.g. halo—substituted lower alkoxyl, e.g. trifluoromethyl) and optionally cyano—substituted phenoxy).

In certain examples, the compound of a II is one of the compounds SC—3l, SC-32, SC—33, SC-34 and SC-35, as listed in Table 2.

Table 2 I H N/ N\ de R4 R5 R6 8031 H 5NJ1 Q H N F H H 3: 00F3 SC-32 H Eli/U H SC-33 H fiN/é CF3 H 80—34 H §N H 80-35 H 3N/é\© H In a further aspect, the t invention provides a compound of Formula III R7 111 wherein R7 is hydrogen, halo, hydroxyl, ally substituted alkoxyl, optionally substituted thioalkoxy, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted lkyl, optionally substituted heterocycloalkyl, ally substituted aryl, optionally substituted aralkyl, optionally substituted heteroaryl, optionally substituted heteroarakyl, -(C)mNHC(X)NH(C)n Rag -(C)pNHC(X)Rb—,- (C)qNHS(O)2RC, -(C)r(X)NHRd-,or -(C)SNH(C)tRe; wherein Ra, R1,, R0, Rd and Re are independently hydrogen, halo, hydroxyl, optionally substituted alkoxyl, optionally substituted thioalkoxy, ally tuted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substitutedcycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted aralkyl, optionally tuted heteroaryl, optionally substituted heteroarakyl; X=O or S; and m, n, p, q, r, s, t=0, l, or 2. in which In one ment, the compound of formula 111 es those wherein R7 is independently hydrogen, optionally substituted lower alkyl, - (C)mNHC(X)NH(C)I1 Ra-, —(C)pNHC(X)Rb-, ~(C)qNHS(O)2Rc, or —(C)SNH(C)tRe. those in which Ra, In another embodiment, the compound of a III includes substituted with l to 3 Rb, RC, Rd and Re are independently phenyl or naphthyl, optionally of halo, optionally substituted lower alkyl (such as groups selected from the group consisting such as halo-substituted lower alkyl, e.g. trifluoromethyl), optionally substituted l (e.g. halo—substituted lower alkoxyl, e.g. trifluoromethyl) and optionally substituted aryloxy (e.g. cyano-substituted phenoxy).

In certain examples, the compound of formula III is one of the compounds SC—36, SC—37and SC-38, as listed in Table 3.

Table 3 N\ \0 O’M/ de R7 TAUOCF3 80—36 2 CF3 sc-37 HN/ JVPV SC—38 HN/S uv+m such as The term “halo” or “halogen” alone or in combination means all halogens, e (F), chlorine (Cl), bromine (Br) or iodine (I).

The term “hydroxyl” refers to the group —OH.

The terms “thio” and “mercapto” are used interchangeably and refer to the group- SH.” The term ” alone or in combination refers to an alkane—derived l ning, unless otherwise stated, 1-20 carbon atoms (Cl-Czo), preferably 1-15 carbon atoms (C1—C1 5), more preferably 1-10 carbon atoms (Cl-Clo). It is a straight chain alkyl, branched alkyl or lkyl, preferably, straight or branched alkyl groups containing from 1—15, more preferably 1 to 8 even more preferalbly 1-6, yet more preferably 1-4 and most preferably 1-2, carbon atoms, such as methyl, ethyl , propyl, isopropyl, butyl, t-butyl and the like. The term “lower alkyl” is used herein to describe the straight chain alkyl groups as described above. Preferably, cycloalkyl groups are monocyclic, bicyclic or tricyclic ring systems of 3-8, more preferably 3-6, ring members per ring, such as cyclipropyl, cyclopentyl, cyclohexyl, adamantly and the like. Alkyl also includes a straight chain or branched aljyl froup that contains or is interrupted by a cycloalkyl portion. The straight chain or branched alkyl group is attached at any available point to produce a stable compound. Examples of this include, but are not limited to, 4—(isopropyl)—cyclohexylene or 2— methyl- cyclopropylpentyl. A substituted With 1 to 3 groups or substituents of halo, hydroxyl, alkoxy, io, alkylsulfinyl, alkylsylfinyl, acyloxy, aryloxy, heteroaryloxy, amine optionally mono- or disubstituted with alkyl, aryl or heteroaryl groups, amidino, urea optionally substituted with alkyl, aryl, heteroaryl or heterocyclyl groups, aminosulfinyl optionally N— mono— or N,N—di-substituted With alkyl, aryl or heteroaryl groups, alkylsulfonylamino, arylsulfonylamino, arylsulfonylamino, alkylcarbonylamino, arylcarbonylamino, heteroarylcarbonylamino, or the like.

The term “alkenyl” alone or in combination means a straight, ed, or cyclic hydrocarbon containing 2-20, preferably 2-17, more ably 2—10, even more preferably 2- 8, most preferably 2-4, carbon atoms and at least one, preferaly 1-3, more preferably 1-2, most preferably one, carbon to carbon double bind. In the case of a cycloalkenyl group, conjugation of more than one carbon to carbon double bond is not such as to confer aromaticity to the ring. Carbon to carbon double bonds may be either contained Within a lkyl n, with the exception of cyclopropyl, or Within a straight chain or branched portion. Examples of alkenyl groups include ethenyl, propenyl, isopronyl, butenyl, cyclohexenyl, exenylalkyl and the like. A substituted alkenyl is the straight chain alkenyl, branched alkenyl or lkenyl groups defined previously, ndently substituted with 1 to 3 groups or substituents of halo, hydroxyl, aryloxy, alkylthio, alkylsulfinyl, alkylsulfonyl, ndently substituted with alkyl, aryl or heteroaryl groups, amidino, urea optionally substituted with alkyl, aryl, heteroaryl or heterocycloyl groups, aminosulfonyl optionally N—mono—or N, N—di—substituted with alkyl, aryl or heteroaryl aryloxycarbonyl, groups, alkylsulfonylamino, alkylcarbonylamino, arylcarbinylamino, stable heteroaryloxycarbonyl, or the like attached at any available point to produce a compound.

The term “alkynyl” alone or in combination means a straight, branched, or cyclic preferably hydrocarbon containing 2-20, preferabky 2-17, more preferably 2-10, even more 1-2, 2—8, most preferably 2-4, carbon atoms and at least one, preferaly 1-3, more ably of l groups include most preferably one, carbon to carbon triple bond. Examples is the straight ethynyl, propynyl, isopropynyl, butynyl, and the like. A tuted l tuted with l chain alkynyl, ed alkynyl groups defined previously, independently to 3 groups or substituents of halo, hydroxyl, aryloxy, alkylthio, alkylsulfinyl, alkylsulfonyl, independently substituted with alkyl, aryl or heteroaryl groups, amidino, urea optionally substituted with alkyl, aryl, heteroaryl or heterocycloyl groups, aminosulfonyl optionally mono—or N, N—di—substituted with alkyl, aryl or heteroaryl groups, alkylsulfonylamino, the like alkylcarbonylamino, arylcarbinylamino, aryloxycarbonyl, aryloxycarbonyl, or attached at any available point to produce a stable compound. where R is lower The term “alkyl alkenyl’ refers to a group—R—CR’=CR”R”’, alkyl, or substituted lower alkyl, R’, R”, R’ may independently be hydrogen, halogen, substituted hetaryl lower, alkyl, substituted lower alkyl, acyl, aryl, substituted aryl, l, or as defined below. is lower alkyl, or The term “alkyl alkynyl’ refers to a group—R—CCR’, where R lower alkyl, acyl, substituted lower alkyl, R’ is en, halogen, lower, alkyl, substituted below. aryl, substituted aryl, l, or substituted hetaryl as defined substituted

[0067] The term “alkoxy” denotes the group —OR, where R is lower alkyl, lower alkyl, acyl, aryl, substituted aryl, arakyl, substituted arakyl, heteroalkyl, substituted heteroarylalkyl, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, or cycloheteroalkyl as d. where R The term “alkylthio” or “thioalkoxy” denotes the group-SR, S(O)n=1-2— R, or substituted is lower alkyl, substituted lower alkyl, acyl, aryl, substituted aryl, arakyl, arakyl as defined herein. lower alkyl, The term ”acyl” denotes groups—C(O)R, where R is hydrogen, herein. substituted lower alkyl, aryl, tuted aryl, and the like as defined substituted aryl, The term “aryloxy’ denotes groups-OAr, where Ar is an aryl, heteroaryl, or substituted heteroaryl group as defined herein.

The term “amido” denoteds the group-C(O)NRR’, where R and R’ may independently by hydrogen, lower alkyl, substituted lower alkyl, aryl, substituted aryl, and the like as defined herein.

The term xyl” denoteds the group-C(O)OR, where R is hydrogen, lower alkyl, tuted lower alkyl, aryl, substituted aryl, and the like as defined herein.

The term “aryl” alone or in combination means phenyl or napnthyl optionally carbocyclic fused with a cycloalkyl of preferably 5-7, more ably 5-6, ring members and /or optionally substituted with l to 3 groups or substituents of halo, hydroxyl, aryloxy, alkylthio, alkylsulfinyl, alkylsulfonyl, independently substituted with alkyl, aryl or heteroaryl groups, amidino, urea optionally substituted with alkyl, aryl, heteroaryl or heterocycloyl groups, aminosulfonyl ally N—mono-or N, N—di-substituted with alkyl, aryl or heteroaryl groups, ulfonylamino, alkylcarbonylamino, arylcarbinylamino, aryloxycarbonyl, heteroaryloxycarbonyl, or the like.

The term “heterocycle” refers to a saturated, unsaturated, or ic yclic group having a single ring (e.g., morpholino, pyridyl or furyl) or multiple condensed rings (e.g., naphthpyridyl, quinoxaryl, quinolinyl, indolizinyl or benzo[b]thienyl) and having at least one hetero atom, such as N, O or S, within the ring, which can optinally be unsubstituted or substituted with, e.g., halogen, lower alkyl, lower alkoxy, alkylthioi, acetylene, amino, amido, carboxyl, hydroxyl, aryl, aryloxy, heterocycle, hetaryl, substituted l, nitro, cyano, thiol, sulfamino and the like.

The term “heteroaryl “ alone or in combinations means a monocyclic aromatic ring structure containing 5 or 6 ring atoms, or a ic aromatic group having 8 to 10 atoms, containing one or more, preferably 1-4, more ably 1-3, even more preferably 1-2, heteroatoms independently selected from the group O, S, and N, and ally tuted with l to 3 groups or substituents of halo, hydroxyl, alkoxy, alkythio, alkylsulfinyl, alkylsylfinyl, acyloxy, aryloxy, heteroaryloxy, amine optionally mono— or disubstituted with alkyl, aryl or heteroaryl groups, amidino, urea optionally substituted with alkyl, aryl, heteroaryl or heterocyclyl groups, aminosulfinyl optionally N—mono- or N,N—di—substituted with alkyl, aryl or heteroaryl , alkylsulfonylamino, arylsulfonylamino, 3O heteroarylsulfonylamino, alkylcarbonylamino, arylcarbonylamino, heteroarylcarbonylamino, or the like. Heteroaryl is also intend to include oxidized S or N, such as sulfinyl, sulfonyland N—oxide of a tertiary ring nitrogen. A carbon or nitrogen atom is the point of attachement of the heteroaryl ring ure such that a stable aromatic ring is retained. Examples of heteroaryl groups are nyl, pyridazinyl, pyrazinyl, quinazolinyl, purinyl, indonyl, quinolinyl, pyrimidinyl, pyrrolyl, oxazolyl, thiazolyl, thienyl, isoxazolyl, oxathiadiazolyl, and the like. A isothiazolyl, tetrazolyl, imidazolyl, triazinyl, l, benzofuryl, indolyl carbon or nitrogen to substituted aryl contins a substituent attached an available produce a stable compound. non-aromatic cyclialkyl The term “heterocycly ” alone or in combination means a atoms in which from 1 to 3 carbon atoms in the ring are replaced group having from 5 to fused or fused heteroaryl of 5-6 ring by heteroatoms of O, S, N, and are optionally benzo is also members and / or are optionally substituted as in the case of cycloalkyl. Heterocyclyl and e of a tertiary ring intended to include oxidized S or N, such as sulfinyl, sulfonyl atom. Example of heterocyclyl nitrogen. The point of attachement is at a carbon or nitrogen pyrrolidinyl, zinyl, group are tetrahydrofuranyl, opyridinyl, piperifinyl, contains a dihydrobenzofuryl, oindolyl, and the like. A substituted heterocyclyl stable compound. substituent nitrogen attached at an available carbon or nitrogen to produce a ” refers to The term “substituted heteroary a heterocycle ally mono or poly lower alkyl, lower alkoxy, substituted with one or more functional froups, e.g., halogen, heterocycle, substituted alkylthio acetylene, amino, amido, yl, hydroxyl, aryl, aryloxy, sulfamido and the like. heterocycle, hetaryl, substituted hetaryl, nitro, cyano, thiol, and R is The term “arakyl” refers to the group-R-Ar where Ar is an arylgroup lower alkyl or substituted lower alkyl group. Aryl groups can optionally be unsubstituted or amino, amido, substituted with, e.g., halogen, lower alkyl, lower alkoxy, alkylthio acetylene, substituted carboxyl, hydroxyl, aryl, aryloxy, heterocycle, substituted heterocycle, hetaryl, hetaryl, nitro, cyano, thiol, sulfamido and the like.

Het is a heterocycle The term “heteroalkyl” refers to the group ~R-Het Where can optionally be unsubstituted or group and R is a lower alkyl group. Heteroalkyl groups amido, substituted with halogen, lower alkyl, lower alkoxy, alkylthio acetylene, amino, hetaryl, substituted carboxyl, hydroxyl, aryl, aryloxy, heterocycle, tuted cycle, hetaryl, nitro, cyano, thiol, ido and the like. where HetAr is an The term “heteroaryalkyl” refers to the group —R—Het AR heteroaryl group and R is a lower alkyl group. Heteroarylalkyl groups can optionally unsubstituted or substituted with halogen, lower alkyl, lower alkoxy, alkylthio acetylene, tuted heterocycle, hetaryl, 3O amino, amido, carboxyl, hydroxyl, aryl, aryloxy, heterocycle, substituted hetaryl, nitro, cyano, thiol, sulfamido and the like.

The term “cycloalkyl” refers to a divalent cyclic or polycyclic alkyl group containing 3 to 15 carbon atoms. one or The term “substituted cycloalkyl” refers to a cycloalkyl group sing lower alkyl, lower alkoxy, alkylthio acetylene, amino, more substituents with, e.g., halogen, amido, carboxyl, hydroxyl, aryl, aryloxy, heterocycle, substituted heterocycle, hetaryl, substituted hetaryl, nitro, cyano, thiol, sulfarnido and the like.

The term “cycloheteroalky ” refers to a cycloalkyl group wherein one or more of the ring carbon atoms is replaced with a heteroatom (e.g, N, O, S, or P).

[0084] The term “substituted cycloheteroalkyl” refers to a cycloheteroalkyl group as herein defined which contains one or more substituents, such as n, lower alkyl, lower alkoxy, alkylthio acetylene, amino, amido, carboxyl, yl, aryl, aryloxy, heterocycle, substituted heterocycle, hetaryl, substituted hetaryl, nitro, cyano, thiol, sulfamido and the like.

The term “alkyl cycloalkyl” denoted the R’-cycloalkyl where cycloalkyl is a lkyl group and R is a lower alkyl or substituted lower alkyl. Cycloalkyl groups can optionally be unsubstituted or substituted with e.g., halogen, lower alkyl, lower alkoxy, alkylthio acetylene, amino, amido, yl, hydroxyl, aryl, aryloxy, heterocycle, substituted heterocycle, hetaryl, substituted hetaryl, nitro, cyano, thiol, sulfamido and the like.

The term “alkyl cycloheteroalkyl” denoted the group-R’-cycloheteroalkyl Where R is a lower alkyl or substituted lower alkyl. Cycloheteroalkyl groups can optionally be unsubstituted or substituted with e.g., n, lower alkyl, lower alkoxy, alkylthio acetylene, amino, amido, carboxyl, hydroxyl, aryl, aryloxy, heterocycle, substituted heterocycle, hetaryl, tuted hetaryl, nitro, cyano, thiol, sulfamido and the like.

The compounds of the invention can be prepared by conventional chemical ure such as those described in es organic chemistry written by Francis Carey and Richard Sundberg and review journal “Account of Chemical research.” Particularly, the procedure shown in the general scheme as below exemplifies synthesis of n compounds of the invention.

WO 20014 Formulal R1 R2 R2 NH2 R1 F HO R3 0 R1-Cl o Re b R3 ON ON Formula ll R6 R4 R C! m o R5 R R4 Cl O - 5 \ HO R5 \ H I ———-———--—--—-—-> b \ H l N / H a N / l O O 0 Formula lll \ 2 \ ———————-» ./ \ M 'N/ 1 2 a H2N N b / H2N N ©/U\/Br O N\ \ we. in / I, ll III: a, K2003, DMF; b, pyridine, THF; c. Et3N, dioxane General tic procedure for Formula , further purified by chromatograpgy The compounds of the invention thus synthesized can be method known in the art. or crystallization or any other proer The present invention also provides a pharmaceutical composition comprising and a pharmaceutically acceptable carrier. The or more of the above-described compounds levels or ceutical composition of the invention may be used for increasing expression biological activity of SHP-l in a cell, or treating a disease or condition characterized by decreased expression levels or biological ty of SHP-l. Also within the scope of this invention is the use of any of the above—described compounds for increasing expression levels or biological activity of SHP~1 in a cell, or ng a disease or condition characterized by decreased expression levels or biological activity of SHP-l as described herein and for the manufacture of a medicament for ng the same.

The present invention also provides a method for increasing SHP-l expression levels or biological activity in a cell, comprising contacting the cell with an effective amount of a compound or a pharmaceutical composition as described herein. Further provided is a method for treating a disease or condition characterized by decreased expression levels or biological activity of SHP-l in a subject in need f, comprising administering to the subject an effective amount of a nd or a pharmaceutical composition as described herein.

The term “treating” or “treeatmet” es laxis of the specific disorder or condition, or alleviation of the symptoms associated with a specific disorder or ion and/or preventing or eliminating said symptoms.

The compounds of the present ion can be used for the treatment of diseases or conditions characterized by decreased expression levels or ical activity of SHP-l. A compound of the invention can be administered to a human t by itself or in pharmaceutical compositions where it is mixed with suitable rs or excipients at doses to treat or rate various conditions characterized by decreased expression levels or biological activity of SHP-l. Increased or decreased expression levels or biological activity of a factor (e. g. SHP-l) can be readily detected by the gene product of the factor such as a protein or RNA, in a sample from a subject (e. g. from blood or biopsy tissue) and assaying it in vitro for RNA levels, structure and/or activity of the expressed proteins and the like, using ion methods known in the art such as enzyme-linked immunosorbent assay (ELISA), Western blotting and Northen blotting. Particular exmaples of the diseases or conditions characterized by decreased expression levels or biological activity of SHP-l according to the invention include, but are not limited to, cancer (e.g. hepatocellular carcinoma, leukemia, lung cancer, breast cancer, renal cancer, and osteoporosis.

A “subject” is particularly a mammal, such as a human, but can also be a companion animal (e. g., dogs, cats, and the like), farm animals (e. g., cows, sheep, pigs, horses, and the like) or laboratory s (e.g., rats, mice, guinea pigs, and the like) in need of the treatment as described herein.

“An effective amount” as used herein refers to the amount of an active agent combination with one or required to confer therapeutic effects on a subject, either alone or in more other active agents. Effective s vary, as recognized by those skilled in the art, active agents. depending on route of administration, excipient usage, and co—usage with other Suitable routes of administration may, for example, include oral, rectal, transmucosal, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, in a depot or intravenous, intraperitoneal, intranasal, or cular injections, and optionally sustained release formulation.

[0096] The pharmaceutical compositions of the present invention may be manufactured in a manner known in the art, e.g., by means of conventional , ving, emulsifying, for use in encapsulating, entrapping, or lyophilizing processes. Pharmaceutical compositions ance with the present invention thus may be formulated in tional manner using and/or auxiliaries that one or more physiologically able carriers comprising excipients be used tate processing of the active compounds into preparations, which can pharmaceutically. As used herein, “acceptable” means that the carrier must be compatible the active with the active ingredient of the composition (and preferably, capable of stabilizing ingredient) and not deleterious to the subject to be treated. Proper formulation is dependent chosen. upon the route of administration formulated in,

[0097] In particular, for injection, the compounds of the invention may be for example, physiologically compatible buffers, such as Hank's solution, Ringer's on, or physiological saline buffer. For oral administration, the compounds of the invention may carriers be formulated by combining the active compounds with pharmaceutically able Wheat starch, rice known in this art, such as lactose, sucrose, mannitol, sorbitol, maize starch, starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl- enable the cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP), to compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, the like. For administration by inhalation, the compounds , slurries, suspensions and from of the ion can be formulated in the form of an aerosol spray presentation pressurized packs or a nebulizer, With the use of a suitable lant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.

Without further ation, it is believed that the above ption has adequately enabled the present ion. The following specific example is, therefore, to in any ued as merely illustrative, and not limitative of the remainder of the disclosure way ever. All of the publications, including patents, cited herein are hereby incorporated by reference in their entireties.

Example 1: Chemical Synthesis 1.1 Materials Proton nuclear ic resonance (1H~NMR) spectra were recorded on Bruker DPX300 (400 MHz) instruments. Chemical shifts are reported as values (ppm) downfield from internal deuterated Chloroform of the indicated organic on. Peak multiplicities are expressed as follows: s, singlet; d, doublet; t, triplet; q, quartet; dd, doublet of doublet; ddd, doublet of t of doublets; dt, doublet of triplet; brs, broad singlet; m, let. ng constants (J values) are given in hertz (Hz). Reaction progress was determined by thin layer chromatography (TLC) analysis on silica gel 60 F254 plate (Merck). tographic purification was carried on silica gel columns 60 (0063—0200 mm or 0040—0063 mm, Merck), basic silica gel. Commercial reagents and solvents were used without additional purification. Abbreviations are used as follows: CDC13, deuterated chloroform; DMSO-dé, dimethyl sulfoxide-d6; EtOAc , ethyl acetate; DMF, N,N— dimethylformamide; MeOH, methanol; THF, tetrahydrofuran; EtOH, ethanol; DMSO, dimethyl sulfoxide; NMP, N—methylpyrrolidone. High resolution mass spectra were ed on a FINNIGAN MAT 958 mass spectrometer. 1.2 s

[00103] The structural design of the compounds of the invention is described below. First, to address the relationship between Raf kinase sion and downregulation of P-STAT3 by sorafenib, we used a chemical approach to reduce the hydrogen bonding interaction between the amide group of sorafenib with Rafby replacing amido group by a phenylcyano group (compound 1, Fig. 1). We also modified SC-l based on functional groups which contain different size, hydrogen donor, hydrogen or, hydrophobic and hydrophilic ability to te a series of compounds SC-48, SC-49, SC-54, SC—55, SC-56, SC-58, SC~ 43, SC-44, SC-45, SC—50, SC—Sl, SC—52, SC—59, SC-60 and 8040. In addition, we replaced the urea functional group in the sorafenib backbone with various amide and sulfonamide yielding compounds 2-11. Further, we replaced the pyridine ring with quinoline and used it as a platform to carry out structural modification, generating a series of compounds 12—19 and 20—25. These SC~1 derivatives were synthesized ing to a general ure described above in formula 11 Fig. 2. Morevoer, we extend the length of compound by adding one phenyl ring to explore the structure activity relationship with different functional group 36-38.

[00104] 1.2.1 Synthesis procedures for compound 1 (formula I) To a 50 mL THF solution of triphosgen (0.30 g, 1.0 mmol), 4—chloro (trifluoromethyl)aniline (0.21 g, 1.1mmol) and 2 equivalent of triethyl amine were added. mixture was heated to 50 0C for 30 min. After the temperature was back to room solution was added to the temperature, 4-(4-aminophenoxy)benzonitrile in the 10 mL THF diluted with mixture and heated to 50 0C for another 30 min. The mixture was evaporated, dried over anhydrous water and extracted with EtOAc. The extract was washed with brine, magnesium sulfate, and concentrated under reduced pressure to give 1. (0.34 g, 80%) 1.2.1.1.

I—(4—Chloro—3-(triflu0r0methyl)phenyl)~3-(4—(4-cyan0phen0xy)phenyl)urea (1) NC “TH or:3 SC—1 ] 1H NMR (400 MHZ, CDCl3): 5 9.17 (s, 1H), 8.94 (s, 1H), 8.10 (s, 1H), 7.81 (d, J: 6.8 Hz), 7.05 (d, 2H, J: 6.8), 7.63-7.59 (m, 2H), 7.54 (d, 2H, J: 7.2 Hz), 7.10 (d, 2H, 151.2, 2H, J: 7.2 Hz); 13C NMR (100 MHz, methanol—d4): 6 163.7, 163.6, 154.8, 151.4, 140.1,137.7,137.4,135.3,132.9,129.7,129.4, 128.8,128.3,125.6,125.5,1254, 124.2,122.9,122.4,122.3,122.1,120.2,119.7,118.8,118.7,118.6,118.6,106.5,106.4; HRMS calculated for C21H13C1F3N302 (M+H): 48. Found: 431.0656. 1.2.1.2.

I—(3—(4-cyan0phen0xy)phenyl)~3-(4—chlor0—3—(trifluoromethprhenyDurea (43) o mkn CF3

[00111] 1H NMR (400 MHz, DMSO): 5 9.17 (s, 1H), 9.03 (s, 1H), 8.04 (d, J: 2.4 Hz, J: 7.2 Hz, 1H), 7.83 (d, J: 8.8 Hz, 2H), 7.64— 7.55 (m, 2H), 7.41- 7.32 (m, 2H), 7.23 (d, calculated for 1H), 7.11 (d, J: 8.0 Hz, 2H), 6.75 (dd, J: 8.0 Hz, 2.4 Hz, 1H); HRMS N302F3Cl [M-H]: 70. Found: 430.0576. ' 1.2.1.3.

[00113] 4—(3-(3—(trifluoromethyl)benzen~Sulfonylamin0)phenoxy)benzonitrile (44) NC<1 £1 o WKOCFSH WO 20014 1H NMR (400 MHZ, CDC13): 5 8.00 (s, 1H), 7.96 (d, J = 8.0 Hz, 1H), 7.81 (d, J = 8.0 Hz, 1H), 7.65~ 7.54 (m, 3H), 7.26 (t, J = 8.0 Hz, 1H), 7.08 (s, 1H), 7.05— 6.97 (m, 1H), 6.94- 6.86 (m, 3H), 6.84 (t, J = 2.0 HZ, 1H), 6.81 (dd, J = 8.4 Hz, 2.0 Hz, 1H); HRMS calculated for C20H12N203F3S [M-H]': 417.0521. Found: 417.0518. ] 1.2.1.4. 4—(3—(3-(trifluoromethoxy)benzylamin0)phen0xy)benzonitrile (45) NCO £10 N/UOCFSH 1H NMR (400 MHZ, CDC13): 8 7.61 (d, J= 8.8 HZ, 2H), 7.43 (t, J: 8.0 HZ, 1H), 7.33 (d, J: 8.4 HZ, 1H), 7.29- 7.16 (m, 3H), 7.04 (d, J= 8.8 HZ, 2H), 6.55 (dd, J: 8.0 HZ, 2.4 HZ, 1H), 6.46 (dd, J: 8.0 HZ, 2.0 HZ, 1H), 6.34 (t, J: 2.4 HZ, 1H), 4.41 (s, 2H); HRMS calculated for C21H16N202F3 [M+H]+: 385.1164. Found: 385.1157. 1.2.1.5. 1—(4—(4—cyan0phen0xyjpheny0—3—(3, 4-dimethoxybenZJ/Durea (48) NCONOQHL, O

[00120] 1H NMR (400 MHZ, CDC13): 8 7.56 (d, J = 6.8 HZ, 2H), 7.34 (d, J = 8.8 HZ, 2H), 6.98- 6.94 (m, 4H), 6.88—6.75 (m, 4H), 6.56 (brs, 1H), 4.36 (s, 2H), 3.84 (s, 6H); HRMS calculated for C23H20N304 [M—H]': 402.1454. Found: 402.1462. 1.2.1.6.

I-(4—chZora—3{Influoromethybphenyb—3—(4—(4—cyan0phen0xy)benzylfitrea (49) 0 <1“ “<11ch 49 1H NMR (400 MHZ, CDC13): 8 7.76 HZ(s, 1H), 7.51— 7.39 (m, 3H), 7.29 (dd, J: 8.8 HZ, 2.4 HZ, 1H), 7.13 (d, J: 8.4 HZ, 3H), 6.83 (dd, J: 8.8 HZ, 4.8 HZ, 4H), 5.93 (t, J: 6.0 HZ, 1H), 4.24 (d, J = 6.0 HZ, 2H); HRMS calculated for C22H14N302F3Cl [M-H]: 444.0727. Found: 32. ] 1.2.1.7.

I-(3—(4-cyan0phen0xy)phenyl)—3-(3-fluor0phenyl)urea (50) “<10 0 £10 N/U\N F H H — 7.34 1H NMR (400 MHZ, MeOD): 6 7.66 (d, J: 9.2 Hz, 2H), 7.60 (s, 1H), 7.41 7.10—7.02 (m, 3H), 6.71 (m, 2H), 7.22 (q, J: 8.0 Hz, 1H), 7.18 (dd, J = 8.0 Hz, 2.0 Hz, 1H), 346.0992. Found: (dd, J = 8.8 Hz, 2.4 Hz, 2H); HRMS calculated for C20H13N302 [M-H]: 346.0999. 1.2.1.8.

N—(3—(4-cyan0phen0xy)phenyl)benzamide (51) (s, 1H),

[00129] 1H NMR (400 MHz, CDC13)I 6 8.82 (s, 1H), 7.75 (d, J: 7.6 Hz, 2H), 7.53 7.46- 7.35 (m, 4H), 7.28 (t, J: 8.0 Hz, 2H), 7.22 (t, J: 8.0 Hz, 1H), 6.90 (d, J: 8.8 Hz, 2H), 6.72 (dd, J = 8.0 Hz, 2.0 Hz, 1H); HRMS calculated for C20H13N202 [M-H]: Found: 313.0971. ] 1.2.1.9.

[00131] N-(3-(4—cyan0phen0xy)phenyl)benzenesulfonamide (52) o N/ © 1H NMR (400 MHZ, CDC13)Z 5 7.79 (d, J = 8.4 Hz, 2H), 7.53 (t, J: 4.4 Hz, 3H), 2.0 Hz, 1H), 6.86— 7.42 (t, J: 8.0 Hz, 2H), 7.20 (t, J: 8.0 Hz, 1H), 6.93 (dd, J: 8.0 Hz, calculated for C19H13N2038 [M-H]': 6.83 (m, 3H), 6.73 (dd, J: 8.0 Hz, 2.0 Hz, 1H); HRMS 349.0647. Found: 2349.0643. 1.2.1.10. 4-cyan0phen0xy)benzyD—3-(3,4-dimeth0xybenzy0urea (54) ”Q fifdwg“H H O 0/ 1H NMR (400 MHZ, DMSO): 5 7.81 (d, J: 9.2 HZ, 2H), 7.33 (d, J: 8.4 HZ, 2H), 6.76 (dd, J: 8.0 HZ, 2.0 HZ, 7.06 (dd, J: 15.6 HZ, 9.2 HZ, 4H), 6.86 (d, J: 9.2 HZ, 2H), 1H), 6.45 (t, J= 6.0 HZ, 1H), 6.38 (t, J: 6.0 Hz, 1H), 4.23 (d, J: 5.2 Hz, 2H), 4.14 (d, J: .2 Hz, 2H), 3.69 (s, 6H); HRMS calculated for C24H24N304 [M+H]+: 418.1767. Found: 418.1773.

I-(4—(4—cyan0phenoxy)benzy0—3-(3-(trifluorometlz0xy)benzyl)urea (55) NCOOQAN/{LHUOCFS 1H NMR (400 MHZ, CDC13)I 5 7.49 (d, J: 9.2 Hz, 2H), 7.17 (t, J: 8.0 Hz, 1H), 7.10 (d, J: 8.4 Hz, 2H), 7.00 — 6.92 (m, 3H), 6.88 (d, J= 9.2 Hz, 2H), 6.84 (d, J= 8.4 Hz, 2H),6.17- 6.05 (m, 2H), 4.10 (m, 4H); HRMS calculated for C23H19N303F3 : 442.1379. Found: 442.1381. 1.2.1.12.

(R)—I—(4-(4—cyanophen0xy)phenyl)—3—(1-(naphthalen—1-yl)ethyl)urea (56) NC NJLN . D ] 1H NMR (400 MHZ, MeOD): 8 8.17 (d, J: 8.0 Hz, 1H), 7.87 (d, J: 8.0 Hz, 1H), 7.78 (d, J: 8.0 Hz, 1H), 7.65 (d, J: 8.8 Hz, 2H), 7.58 (d, J: 7.2 Hz, 1H), 7.53 (d, J: 7.2 Hz, 1H), 7.50— 7.45 (m, 2H), 7.41 (d, J= 8.8 Hz, 2H), 6.99 (t, J = 9.2 Hz, 4H), 5.74 (d, J = 6.8 Hz, 1H), 1.63 (d, J: 6.8 Hz, 3H); HRMS ated for C26H20N302 [M-H]: 406.1556.

Found: 406.1563. 1.2.1.13.

[00143] 1-(4-chloro(trifluoromethyl)benzyl)(4-(4-cyanophenoxy)phenyl)urea (5 8) “merwfiCI 1H NMR (400 MHz, MeOD): 6 7.74 (brs, 1H), 7.66 (d, J: 9.2 Hz, 2H), 7.56 (d, J = 2.0 Hz, 2H), 7.44 (d, J: 9.2 Hz, 2H), 7.05- 6.98 (m, 4H), 4.43 (s, 2H); HRMS calculated for C22H14N302F3Cl [M-H]: 444.0727. Found: 444.0736.

[00145] 1.2.1.14.

J-(4—chZora-3—(trij‘luoromethprhenyD—3-(3—(4—cyan0phen0xy)~4- methylphenyDurea (59) o ”“fl\fi CF3 ] 1H NMR (400 MHZ, MeOD): 5 7.87 (d, J: 2.8 Hz, 1H), 7.60 (d, J: 9.2 Hz, 2H), J: 2.4 Hz, 1H), 7.18 (d, 7.54 (dd, J: 8.4 Hz, 2.4 Hz, 1H), 7.39 (d, J= 8.8 Hz, 1H), 7.25 (d, 9.6 Hz, 2H), 2.02 (s, 3H); J: 8.0 Hz, 1H), 7.12 (dd, J: 8.0 Hz, 2.4 Hz, 1H), 6.93 (d, J: 444.0725.

HRMS calculated for C22H14N302F3C1 [M—H]: 444.0727. Found: 1.2.1.15.

I,3-bis(3—(4—cyanophenoxy)-4—methylphenyl)urea (60) homage/C“ 1H NMR (400 MHZ, DMSO): 5 8.77 (s, 2H), 7.80 (d, J: 8.0 Hz, 4H), 7.29 (s, 2H), 7.24 (d, J 2.02 (s, 6H); HRMS = 8.0 Hz, 2H), 7.13 (d, J= 8.4 Hz, 2H), 6.98 (d, J: 8.4 Hz, 4H), calculated for C29H21N4O3 [M-H]: 14. Found: 473.1619.

[00151] 1.2.2 General procedures for nd 2-25 and catalytic In a 25 mL two-necked round flask, aniline derivatives (lmmol) at room temperature. Acyl amount of pyridine were placed in ous THF (10 mL) and stirred for 2 h at chloride or sulfonyl chloride compounds were added to the mixture under vacuum and the crude residue purified room temperature. The solvent was removed eluent (1/10 to 1/2). This by chromatography on a silica gel column using EtOAc/Hexane as 70% to 95% yield. procedure afforded the expected coupling product as a white solid from 1.2.2.1.

N—Methyl-4—(4-@henylsulfonamido)phen0xy)picolinamide (2) H 9{HQ O IN/ 1H NMR (400 MHZ, CDC13): 5 8.36 (d, 1H, J= 5.6 Hz), 8.01 (brs, 1H), 7.76 (d, 2H, J: 7.6 Hz), 7.59 (s, 1H), 7.54 (t, 1H, J: 8.0 Hz), 7.46 (t, 2H, J: 8.0 Hz), 7.12 (d, 2H, J = 8.8 Hz), 6.94 (d, 2H, J: 8.8 Hz), 6.92-6.90 (m, 1H), 3.00 (d, 3H, J= 5.2 Hz); 13(2 NMR (100 MHz, : 5166.0, 164.6, 152.1, 151.0, 149.7, 138.9, 134.2, 133.0, 129.0, 127.1, 123.9, 121.6, 114.3, 109.9, 26.1; HRMS calculated for C19H17N3O4S (M+H): 383.0940.

Found: 383.0941. 1.2.2.2.

N—MethyZ—4—(4-(4—nitr0phenylsulfonamid0)phenoxy)picolinamide (3) O IN/

[00158] 1H NMR (400 MHz, CDC13)I 5 8.39 (d, 1H, J== 5.6 Hz), 8.30 (d, 2H, J: 8.8 Hz), 8.07 (brs, 1H), 7.93 (d, 2H, J: 8.8 Hz), 7.49 (s, 1H), 7.17 (d, 2H, J: 8.8 Hz), 7.01-6.98 (m, 3H), 3.00 (d, 3H, J = 5.2 Hz); HRMS calculated for C19H16N4068 (M+H): 428.0791. Found: 428.0798. 1.2.2.3. ] 4—Fluorophenylsulfonamido)phenoxy)~N—methylpicolz’namide(4) ] 1H NMR (400 MHz, CDC13): 5 8.37 (d, 1H, J: 5.6 Hz), 8.00 (brs, 1H), 7.77—7.43 (m, 2H), 7.57 (s, 1H), 7.17—7.09 (m, 4H), 6.99-6.93 (m, 4H), 3.00 (d, 3H, J: 4.8 Hz); NMR (100 MHz, CDC13): 5166.5, 165.9, 164.6, 163.9, 152.1, 151.2, 149.7, 135.0, 134.0, 130.0, 129.9, 124.1, 121.7, 116.4, 116.2, 114.5, 109.8, 26.19; HRMS calculated for C19H16FN304S (M+H): 401.0846. Found: 401.0849. 1.2.2.4. 4-(4-(4—tert—Butylphenylsub‘onamido)phen0xy)—N—methylpicolz'namide (5) H9.N‘§<>< o N/ 1H NMR (400 MHz, CD013): 5 8.33 (d, 1H, J: 6.0 Hz), 8.21 (brs, 1H), 7.79 (brs, J: 6.8 Hz), 1H), 7.69 (d, 2H, J: 6.8 Hz), 7.62 (s, 1H), 7.44 (d, 2H, J: 6.8 Hz), 7.15 (d, 2H, 6.91 (s, 2H, J: 6.8 Hz), 6.88—6.86 (m, 1H), 2.98 (d, 3H, J: 5.2 Hz);13C NMR (100 MHz, CDC13): 5166.0, 164.6,156.8,152.2, 150.8, 1497,1361, 134.4, 127.0, 126.1, 123.6, 121.6, 439.1566. Found: 114.1, 110.1, 35.1, 30.1, 26.1; HRMS calculated for C23H25N3O4S (M+H): 439.1564. ] 1.2.2.5.

N—Methyl(4-(naphthalene—Z—sulfonamido)phenoxy)picolinamide (6) H H O O N/ /NH 1H NMR (400 MHz, CDC13): 8 8.34 (s, 1H), 8.30 (d, 1H, J = 5.2 Hz), 8.05-8.02 7.16 (d, 2H, J (m, 1H), 7.89-7.83 (m, 4H), 7.74 (dd, 1H, J: 8.4, 1.6 Hz), 7.60-7.52 (m, 3H), = 8.8 Hz), 6.88 (d, 2H, J: 8.8 Hz), 6.84—6.82 (m, 1H); 13(3 NMR (100 MHz, CD013): 165.9, 164.6, 152.1, 151.0, 149.7, 135.9, 134.9, 134.2, 132.0, 129.4, 129.3, 128.9, 128.7, 127.9, 127.5, 123.9, 122.2, 121.6, 114.2, 110.1, 26.2; HRMS ated for C23H19N3O4S (M+H): 433.1096. Found: 433.1079. 1.2.2.6. 4~(4-(2—Br0m0-4—(trifluoromethyl)phenylsulfonamido)phen0xy)-N— methylpic-olinamide (7) : O O / WO 20014 2012/049446 1H NMR (400 MHz, CDC13): 5 8.35 (d, 1H, J: 5.6 Hz), 8.15 (d, 1H, J: 8.0 Hz), 7.79 (brs, 1H), 7.96 (s, 1H), 7.67 (d, 2H, J: 8.0 Hz), 7.57 (s, 1H), 7.18 (d, 2H, J: 9.2 Hz), 6.95 (d, 2H, J: 9.2 Hz), 6.90-6.88 (m, 1H), 2.98 (d, 3H, J: 5.2 Hz);13C NMR (100 MHz, CDC13): 5165.7, 164.5, 152.2, 151.6, 149.8, 141.5, 136.1, 135.8, l35.5, 135.2, 132.7, 132.2 (In), 124.9 (111), 124.1, 123.5, 121.7, 120.8, 1204,1145, 110.0, 26.1; HRMS calculated for C20H15B1F3N304S (M+H): 528.9919. Found: 528.9917. 1.2.2.7.

N—MethyZ-4—(4—(2—nitrophenylsLtb‘onamid0)phen0xy)picolinamz'de (8) H 9WK? 0o O O /

[00172] 1H NMR (400 MHz, CDC13): 5 8.37 (d, 1H, J: 6.0 Hz), 7.98 (brs, 1H), 7.86-7.83 (m, 2H), 7.72-7.68 (m, 2H), 7.55 (s, 1H), 7.24 (d, 2H, J: 8.8 Hz), 6.98 (d, 2H, J: 8.8 Hz), 6.94-6.92 (m, 1H), 2.98 (d, 3H, J = 4.8 Hz); HRMS calculated for C19H16N4O6S (M+H): 428.0791. Found: 428.0796. ] 1.2.7.8

[00174] 4—(4—(3,5-Bz's(z‘rzfluor0methyl)benzamid0)phenoxy)—N—methylpicolinamide (9) |\ F3C (31:3 0 / 1H NMR (400 MHz, CDC13): 5 9.92 (s, 1H), 8.40 (s, 1H), 8.33 (d, 1H, J: 5.6 Hz), 8.10 (q, 1H, J: 5.2 Hz), 7.90 (s, 1H), 7.71 (d, 2H, J: 8.8 Hz), 7.40 (d, 1H, J: 2.8 Hz), 6.99-6.97 (m, 1H), 6.93 (d, 2H, J: 8.8 Hz), 2.91 (d, 3H, J: 4.8 Hz); 13’0 NMR (100 MHz, methanol—d4): 5167.8, 166.8, 165.1, 153.4, 1518,1516, 138.6, 137.6, 133.6, 133.3, 133.0, 132.6, 129.4 ((1), 126.2 (111), 126.0, 124.2, 123.2, 122.4, 115.2, 110.7, 26.4; HRMS calculated for C22H15F6N3O3 (M+H): 483.1018. Found:483.1017. ] 1.2.2.9 4-(4—(5-Fluor0—2-(trifluoromethyl)benzamid0)phenoxy)—N—methylpicolinamI'd—e (10) 1H NMR (400 MHz, CD013): 5 8.66 (d, 1H, J: 12.4 Hz), 8.31—8.26 (m, 2H), 7.93 (s, 1H), 7.70—7.65 (m, 3H), 7.56 (t, 1H, J: 2.4 Hz), .19 (m, 1H), 7.02 (d, 2H, J: 6.4 13C NMR (100 MHz, methanol-d4): 6 166.2, Hz), 6.89-6.87 (m, 1H), 2.90 (d, 3H, J: 3.2 Hz); 135.0, 130.6 (m), 129.8 (m), 128.3, 164.5, 162.9, 160.4, 160.0, 159.9, 152.2, 150.4, 149.7, 128.1, 127.7, 127.4, 124.5, 122.5, 122.4, 122.3, 121.8, 121.5, 117.2, 117.0, 114.1, 110.1, Found: 433.0152. 26.1; HRMS calculated for C21H15F4N303 (M+H): 433.1050. 1.2.2.10.

N—Methyl—4—(4—(4-(trifluoromethyl)benzamid0)phenoxy)picolinamide (11) ONHYG O O / /NH 1H NMR (400 MHz, CDC13): 5 9.45 (s, 1H), 8.31 (d, 1H, J: 5.6 Hz), 8.15 (s, 7.47 (d, 2H, J: 8.0 Hz), 7.71-7.65 (m, 3H), 7.50 (d, 1H, J: 2.4 Hz), (t, 1H, J: 1H), 8.08 166.4, 8.0 Hz), .91 (m, 3H), 2.92 (d, 3H, J: 5.2 Hz); 13c NMR (100 MHz, CDC13): 164.8, 164.7, 151.8, 1499,1498, 138.8, 135.5, 131.4, 131.1, 130.8, 130.7, 130.5, 129.1, 128.1, 125.0, 124.3 (111), 122.6, 122.3, 121.2, 114.4, 109.5, 26.1; HRMS calculated for C21H16F3N303 (M+H): 44. Found: 415.1146. 1.2.2.11. 2-Nitr0-N—(4—(quinolinyloxy)phenyl)-4—(trifluoromethyl)benzenesulfonam-ide (12) 2012/049446 H 9N-§@CF3 OO O 1H NMR (400 MHZ, CDC13): 5 8.71 (d, 1H, J: 5.2 Hz), 8.28 (d, 1H, J: 8.4 Hz), 8.12 (d, 1H, J: 8.4 Hz), 8.10 (s, 1H), 8.05 (d, 1H, J= 8.4 Hz), 7.89 (d, 1H, J: 8.4 Hz), 7.80—7.76 (m, 1H), 7.61—7.57 (m, 1H), 7.31 (d, 2H, J: 8.8 Hz), 7.13 (d, 1H, J: 8.8 Hz), 6.53 (d, 1H, J = 5.2 Hz); HRMS calculated for C22H14F3N3058 (M+H): 489.0606. Found: 4890610. 1.2.2.12. 2-Nitro-N-(4—(8—niz‘r0quinolin—4—yloxy)phenyD-4—(trz'fluoromethyl)benzenesu- ide (13) QCN—§ CF3 No2 1H NMR (400 MHZ, CDC13): 5 8.78 (d, 1H, J: 5.2 Hz), 8.51 (d, 1H, J: 8.8 Hz), 8.19 (s, 1H), 8.12-8.02 (m, 3H), 7.89 (t, 1H, J= 9.6 Hz), 7.62 (t, 1H, J= 8.4 Hz), 7.34 (d, 2H, J= 9.6 Hz), 7.15 (d, 2H, J: 9.6 Hz), 6.91 (d, 1H, J: 6.8 Hz), 6.59 (d, 1H, J: 5.2 Hz), 6.55 (d, 1H, J = 6.8 Hz); HRMS calculated for C22H13F3N4O7S (M+H): 534.0457I Found: 534.0423. 1.2.2.13 2-Br0m0-N—(4—(quino[in-4—ylquy)phenyl)(trifluoromethyDbenzenesulfon— amide (14) ON“§@CF3H 9 O 2012/049446 1H NMR (400 MHZ, CDCl3): 5 8.65 (d, 1H, J: 5.2 Hz), 8.25 (d, 1H, J: 8.4 Hz), 8.18 (d, 1H, J: 8.4 Hz), 8.07 (d, 1H, J: 8.4 Hz), 7.98 (s, 1H), 7.73 (t, 1H, J: 7.6 Hz), 7.67 (d, 1H, J: 8.4 HZ), 7.54 (t, 1H, J: 7.6 Hz), 7.24 (d, 2H, J: 8.8 Hz), 7.05 (d, 2H, J: 8.8 Hz), 6.43 (d, 1H, J: 5.2 Hz);13C NMR (100 MHZ, CDC13)Z 8 161.3, 152.6, 150.9, 149.7, 141.4, 135.9, 135.6, 135.3, 132.7, 132.4, 132.2 (m), 130.3, 129.1, 126.3, 124.9 (m), 124.4, 123.5, 122.1, 122.0, 121.9, 121.6, 121.3, 120.8, 120.4, 116.3, 104.4; HRMS calculated for C22H14BrF3N2038 (M+H): 521.9861. Found: 58. 2—Br0m0—N-(4—(8—nitr0quinolin-4—yloxy)phenyD—4-(trifluoromethyl)benzene~ sulfonamide (15) 1H NMR (400 MHz, CDC13): 8 8.76 (d, 1H, J: 5.2 Hz), 8.49 (d, 1H, J: 8.4 Hz), 7.60 8.18 (d, 1H, J: 8.4 Hz), 8.04 (d, 1H, J: 7.6 HZ), 7.99 (s, 1H), 7.68 (d, 1H, J: 8.4 Hz), (t, 1H, J: 8.4 Hz), 7.25 (d, 2H, J: 8.4 Hz), 7.07 (d, 2H, J: 8.4 Hz), 6.53 (d, 1H, J: 5.2 Hz); HRMS calculated for C22H13B1F3N305S (M+H): 566.9711. Found: 566.9706. 1.2.2.15. N-(4—(Quinolin—4—yloxy)phenyl)~3,5—bis(triflu0r0methyl)benzamide (16) ON 0 N . 1H NMR (400 MHz, CDC13): 8 10.02 (s, 1H), 8.59 (d, 1H, J: 5.6 Hz), 8.37 (s, 2H), 8.34 (d, 1H, J: 8.4 Hz), 7.99 (d, 1H, J: 8.4 Hz), 7.92 (s, 1H), 7.80 (d, 2H, J: 9.2 Hz), 7.70 (t, 1H, J= 7.6 Hz), 6.56 (t, 1H, J: 7.6 Hz), 7.14 (d, 2H, J= 9.2 Hz), 6.52 (d, 1H, J= .2 Hz) 9%: NMR (100 MHz, DMSO-d6): 5 162.6, 161.0, 151.5, 150.0, 149.2, 137.0, 136.1, 131.0, 130.7, 130.4, 130.3, 130.0, 128.8, 128.5 (111), 126.4, 125.2 (111), 124.5, 122.5, 121.7, 121.5, 121.3, 120.6, 104.3; HRMS calculated for C24H14F6N202 (M+H): 476.0959. Found: 476.0958.

[00196] 1.2.2.16 N—(4-(8—Nitroquz'nolinyloxy)phenyl)-3, 5—bz's(triflu0r0methyDbenzamide (I 7) OONO | F3C CF3 1H NMR (400 MHz, CDC13): 5 9.05 (s, 1H), 8.69 (d, 1H, J= 5.0 Hz), 8.59 (d, 1H, J: 5.0 Hz), 8.35 (s, 2H), 8.06 (d, 1H, J: 7.8 Hz), 7.97 (s, 1H), 7.80 ((1, 21-1,]: 9.0 Hz), 7.63 (t, 1H, J: 8.6 Hz), 7.16 (d, 2H, J: 9.0 Hz), 6.62 (d, 1H, J= 5.0 Hz); HRMS calculated for C24H13F6N3O4 (M+H): 521.0810. Found: 521.0814. 1.2.2.17 2-Fluor0-N-(4—(quinolin—4—yloxy)phenyl)—5~(trlj‘lztoromethybbenzamz‘de (18)

[00200] 1H NMR (400 MHz, CDC13): 8 8.83 (d, 1H, J: 12.8 Hz), 8.68 (d, 1H, J: 5.2 Hz), 8.39—8.30 (m, 2H), 8.11 (d, 1H, J: 8.4 Hz), .67 (m, 4H), 7.58 (t, 1H, J = 8.0 Hz), 7.27—7.22 (m, 1H), 7.18(d, 2H, J: 9.2 Hz), 6.56 (d, 1H, J: 5.2 Hz); 13C NMR (100 MHz, methanol—d4): 5 164.4, 164.2, 163.8, 161.7, 151.9, 151.8, 149.7, 137.5, 132.1 (m), 131.2 (m), 128.9 (In), 128.3, 128.1, 127.9, 126.5, 126.3, 123.7, 1236,1230, 122.7, 122.6, 119.1, 119.8, 118.7, 118.5, 105.2; HRMS calculated for C23H14F4N202 (M+H): 426.0991. Found: 426.0991. 1.2.2.18. 2-Fluor0-N—(4—(8-nitr0quin01in—4—yloxy)phenyl)—5—(trzfiuoromethyDbenzami—de (19) ON 0 F | F30 No2 ] 1H NMR (400 MHz, CDC13): 5 8.81 (d, 1H, J: 5.2 Hz), 8.59 (d, 1H, J: 8.8 Hz), 8.53—8.47 (m, 2H), 8.06 (d, 1H, J: 7.6 Hz), 7.83-7.77 (m, 3H), 7.64 (t, 1H, J = 7.6 Hz), .2 Hz); HRMS calculated for 7.37—7.32 (m, 1H), 7.23—7.20 (m, 2H), 6.68 (d, 1H, J = C23H13F4N304 (M+H): 471.0842. Found: 471.0850. 1.2.2.19 ethyl—4—(8-nitroquz'nolin-4—ylow)phenyD—S’,5-bis(triflu0r0methyl)ben— zamide (20) \ F3C CF3 1H NMR (400 MHz, CDCl3)Z 8 9.86 (s, 1H), 8.45 (d, 1H, J= 5.2 Hz), 8.38 (s, 2H), 7.53 8.31 (d, 1H, J: 8.4 Hz), 7.92 (s, 1H), 7.89 (d, 1H, J: 8.4 Hz), 7.69—7.63 (m, 2H), 1H, J = 7.6 Hz), 7.33 (s, 1H), 7.28 (d, 1H, J: 8.4 Hz), 6.33 (d, 1H, J = 5.2 Hz); HRMS ated for C25H15F6N304 (M+H): 535.0967 Found: 535.0956. 1.2.2.20. 8—Aminoquino[in—4—yloxy)~3-methylphenyl)~3,5-bis(trzj‘lu0romethyl)be— nzamide (21) \ F30 CF3

[00209] 1H NMR (400 MHz, CDC13)I 8 8.49 (d, 1H, J: 2.0 Hz), 8.27 (s, 1H), 8.02 (s, 1H), 7.37-7.32 (m, 2H), 6.96 (d, 1H, J= 7.94 (s, 1H), 7.65 (d, 1H, J: 8.0 Hz), 7.48-7.45 (m, 2H), 505.1225. 7.6 Hz), 4.95 (S, 2H), 2.16 (s, 3H); HRMS calculated for C25H17F6N302 (M+H): Found: 505.1216. 1.2.2.21.

[00211] N—(4-(8—Acetamidoquinolinyloxy)~3—methylphenyl)—3, 5- bis(trij‘luoromethyDbenzamide (22) 90N O l F30 CF3 HN?O 1H NMR (400 MHz, CDC13): 5 9.77 (s, 1H), 9.36 (s, 1H), 8.65 (d, 1H, J: 7.2 Hz) 8.46 (s, 2H), 8.44 (d, 1H, J: 5.2 Hz), 7.97 (s, 1H), 7.87 (d, 1H, J: 8.4 Hz), 7.72 (d, 1H, J: 8.4 Hz), 7.50 (d, 1H, J: 2.0 Hz), 7.37 (t, 1H, J: 8.0 Hz), 7.28 (d, 1H, J: 8.4 Hz), 6.41 (d, 1H, J: 5.2 Hz), 2.26 (s, 3H), 2.10 (s, 3H); 13c NMR (100 MHz, CDClg): 8 169.2, 163.0, 161.2, 152.0, 148.8, 139.5, 137.2, 136.7, 133.8, 132.6, 132.3, 132.2, 132.0, 131.6, 127.8, 127.1, 126.3, 125.2 (m), 124.2, 121.5, 120.4, 118.8, 118.3, 116.7, 115.7, 113.8, 25.0, 15.4;- HRMS ated for C27H19F6N303 (M+H): 547.1331. Found: 547.1325. 1.2.2.22.

[00214] N—(3-(8—Nz'tr0quinolz'n-4—yloxy)phenyD—3,5—bis(trif’luoromethybbenzamide (23) <1 0 CF3 O N N/ CF3 ] 1H NMR (400 MHz, CDC13): 5 8.78 (d, 1H, J: 5.6 Hz), 8.58 (d, 1H, J= 8.4 Hz), 8.52 (s, 1H), 8.31 (s, 2H), 8.22 (s, 1H), 8.08-8.04 (m, 2H), 7.71 (s, 1H), 7.64 (t, 1H, J: 8.0 Hz), 7.53-7.49 (m, 2H), 7.03 (d, 1H, J: 7.2 Hz), 6.71 (d, 1H, J: 4.8 Hz); HRMS calculated for C24H13F6N304 (M+H): 521.0810. Found: 521.0821. 1.2.2.23.

N—(3-(8-Amz'n0quz'nolz'n-4—yloxy)phenyl)—3,5-bis(trz'fluoromethybbenzamide (24) Q0 CF3 O N H ‘ N/ CF3 1H NMR (400 MHz, CDC13): 5 8.55 (d, 1H, J: 4.8 Hz), 8.29 (s, 2H), 8.05 (s, 1H), 7.90 (s, 1H), 7.60 (d, 1H, J = 8.4 Hz), 7.55 (s, 1H), 7.70—7.43 (m, 2H), 7.34 (t, 1H, J: 8.0 Hz), 7.01 (d, 1H, J: 8.0 Hz), 6.96 (d, 1H, J: 7.9 Hz), 6.63 (d, 1H, J= 4.8 Hz); 13(3 NMR (100 MHz, CDCl3)I 5163.1, 161.3, 155.3, 147.9, 143.6, 139.9, 138.8, 136.5, 132.7, 132.4, 132.1, 131.7, 130.6, 127.5 ((1), 127.0, 126.8, 125.3 (m), 124.1, 122.0, 118.7, 117.4, 117.2, Found: 113.1, 111.1, 110.0, 105.4; HRMS calculated for C24H15F6N302 (M+H): 491.1068. 491.1068.

N—(3~(8-Acetamidoquinolin—4—yloagz)phenyl)-3,5—bis(trifluor0methyl)benzam-ide (25) Q0 CF3 O N N/ CF3 \H/NH 1H NMR (400 MHz, CDC13): 8 9.77 (s, 1H), 8.74 (d, 1H, J: 7.6 Hz), 8.54 (d, 1H, J: 8.4 Hz), 7.67-7.60 (m, J = 5.2 Hz), 8.48 (s, 1H), 8.39 (s, 2H), 8.04 (s, 1H), 7.87 (d, 1H, 2H), 7.50—7.43 (m, 2H), 7.37 (t, 1H, J: 8.0 Hz), 7.00 (d, 1H, J: 8.4 Hz), 6.65 (d, 1H, J: 148.7, .2 Hz), 2.30 (s, 3H); 130 NMR (100 MHz, : 5 169.3, 163.1, 161.7, 154.6, 139.6,136.6,133.8,1327,1324,132.1,131.7,130.8,127.8(d),126.9,126.5,125.3(m), calculated for 121.5,120.8, 117.7,117.3,116.8,115.7, 113.2,104.9, 25.0; HRMS C26H17F6N303 (M+H): 533.1174. Found: 533.1167. 1.2.2.25 N—(3-(trifluoromethyl)benzene—sulfonyl)—3-(3—amino—4—nitrophenoxy)benz (SC-40) {3303 a ”What: flgfiw‘w O I? i s 9 C: if? 2:,” 4k. ., . {9% It #3., ATP], “3%.?” a.“(5;: .w «,3! .3 4 7.96 (d, J: 8.0 Hz, 1H), 1HNlVIR (400 MHZ, CDC13): 8 8.06 (d, J: 9.6 Hz, 1H), 8.00 (s, 1H), 7.81 (d, J= 8.0 Hz, 1H), 7.61 (t, J: 8.0 Hz, 1H), 7.27 (t, J: 8.0 Hz,1H),6.91— 6.80 (m, 3H), 13C NMR (100 MHz, 6.19 (dd, J: 9.6 HZ, 2.4 Hz, 1H), 6.14 (d, J: 2.4 Hz, 1H), 6.10 (brs, 2H); 129.9 (m), 128.8, CDC13):5163.1, 155.5,146.7,140.0,137.6,132.0,131.6,130.9,130.3,130.0, 128.0, 124.4, 124.3, 124.2, 124.2, 124.0, 121.6, 117.8, 117.6, 113.6, 107.6,104.3;LC—MS(ESI): 452.0528. Found: 452.0529.

M/Z 452 [M—H]"; HRMS calculated for C19H13N305F3S [M-H]: 1.2.3 compound 36-38 1.2.3.1 3-(2—phenylH—imidazo[I,2—a]pyridin— y)-N—(3- (trifluoromethwgz)benzyDbenzenemine (36) 1H NMR (400 MHz, CDC13): 8 7.96 (d, J: 8.0 Hz, 1H), 7.89 (d, J: 8.0 Hz, 2H), 7.71 (s, 1H), 7.40 (t, J: 7.2 Hz, 2H), 7.35- 7.24 (m, 3H), 7.18 (s, 1H), 7.14 (t, J: 8.0 HZ, 1H), 7.09 (d, J: 7.6 Hz, 1H), 6.98 (d, J= 2.4 Hz, 1H), 6.60 (dd, J: 7.2 Hz, 2.4 HZ, 1H), 6.46- 6.40 (m, 2H), 6.32 (t, J: 2.4 Hz, 1H), 4.31 (s, 1H), 4.20 (s, 1H); HRMS calcu1ated for C27H21N302F3 [M+H]+: 476.1586. Found: 476.1592.

[0008] 1.2.3.2 N—(3-(2-phenylimidazo[1,2-a]pyrz'din—7—yloxy)phenyl)-3— (trifluoromethyl)benzenesulfonamide (37) N’ / N,s CF3 H (j 1H NMR (400 MHZ, CDC13): 5 8.03 (d, J: 6.8 Hz, 2H), 7.92 (d, J: 8.0 Hz, 1H), 7.87 (d, J= 6.8 Hz, 2H), 7.76 (d, J: 6.0 Hz, 2H), 7.60 (t, J= 7.6 Hz, 1H), 7.39 (t, J: 8.0 Hz, 2H), 7.30 (t, J: 6.4 Hz, 1H), 7.21 (d, J: 8.8 Hz, 1H), 6.96- 6.90 (m, 2H), 6.83 (dd, J= 13.6 Hz, 2.4 Hz, 1H), 6.82 (t, J: 2.0 Hz, 2H), 6.60 (dd, J: 7.2 Hz, 2.4 Hz, 1H); HRMS calculated for C26H19N303F38 [M+H]+: 510.1099. Found: 510.1100. 1.2.3.3

[0012] N-(3—(2—phenylimidazofl,2-a]pyrz'din- 7—yloxy)phenyl)benzenesulfonamz‘de (38) cw \ <1N/ / o N’S 1H NMR (400 MHz, DMSO): 5 8.52 (d, J = 7.2 Hz, 1H), 8.33 (s, 1H), 7.93 (d, J = 7.2 Hz, 2H), 7.76- 7.70 (m, 2H), 7.66- 7.54 (m, 3H), 7.43 (t, J: 7.6 Hz, 2H), 7.33— 7.26 (m, 2H), 6.92 (d, J: 7.6 Hz, 1H), 6.84- 6.76 (m, 3H), 6.64 (dd, J= 7.6 Hz, 2.4 Hz, 1H); HRMS calculated for N303S [M+H]+: 442.1225. Found: 442.1216.

Example 2: Bioassay 2.1 Materials and methods 2.1.1. Reagents and antibodies

[0017] Sorafenib (Nexavar®) was kindly ed by Bayer Pharmaceuticals (West from Cayman Chemical Haven, CT). Sodium vanadate and SHP—l inhibitor were purchased (Ann Arbor, MI). dies for immunoblotting such as Raf—1, cylcin D1, and PARP were purchased from Santa Cruz Biotechnology (San Diego, CA). Other antibodies such as anti- and STAT3 were from pVEGFRZ (Y 1 175), VEGFRZ, survivin, phospho—STAT3 (Tyr705), Cell Signaling (Danvers, MA). 2.1.2. Cell Culture The Huh-7 HCC cell line was obtained from the Health Science ch and Hep3B ces Bank (Osaka, Japan; JCRB0403). The PLC/PRF/S (PLCS), Sk—Hep—l, The cells cell lines were obtained from American Type e Collection (Manassas, VA). 100 units/mL penicillin G, 100 were maintained in DMEM mented with 10% FBS, humidified incubator in ug/mL streptomycin e and 25 ug/mL amphotericin B in a 37°C an atmosphere of 5% C02 in air. Other cell lines, including breast cancer cells e.g.

KG—l and ML-l MDAMB231, MDAMB468, MCF-7, and leukemia cancer cells e. g. HL-60, are also provided for the assays described below..

[0020] 2.1.3. Cell death detection ELISA The effect of the compounds of the invention on cell ity was assessed by Cells were treated cell death ELISA assay (Roche Applied Science. Mannheim, Germany). collected and with a test compound at 5 and lOuM for 24 h, for example. The cells were applied to the standard protocol provided by manufacture.

[0022] 2.1.4. Apoptosis analysis Apoptotic cells were measured by flow cytometry (sub-G1). After treatment with and resuspended in various compounds, cells were trypsinized, collected by centrifugation PBS. After centrifugation, the cells were washed in PBS and resuspended in potassium iodide (PI) staining solution. ens were incubated in the dark for 30 min at 37°C and then analyzed with an EPICS Profile 11 flow ter (Coulter Corp., Hialeah, FL). All ments were performed in triplicate 2.1.5. Phospho-STAT3-level A PathScan Phospho-Stat3 (Tyr705) Sandwich ELISA Kit was used for the detection of phospho—STAT3 (Cell Signaling, Danvers, MA). Cells were pre-treated with IL— 6 1 ng/ml and then exposed with various compounds at 10 uM for 24 h. After incubation with cell lysates, both non-phospho— and phospho—Stat3 ns are captured by the coated antibody. The expression of phospho-STAT3 was measured at 450 nm absorbance. 2.1.6. Western Blot

[0027] Cells were treated with various compounds at 5 and 10 uM for 24 h. Cell lysates were analyzed by western blot. 2.1.7. Gene knockdown using siRNA Smart—pool siRNA, including l (D—001810-10), Raf-1, SHP-l, SHP-2, and PTP—lB, were all purchased from Dharrnacon Inc. (Chicago, IL). The procedure has been described previously (Chen KF et al. JBioZ Chem 2009; 284:11121-11133). 2.1.8. PLCS with ectopic expression of STAT3 STAT3 cDNA (KIAA1524) and STAT3-C were purchased from Addgene plasmid repository (http://www.addgene.org/). Briefly, following transfection, cells were incubated in the presence of G418 (0.78 . After 8 weeks of selection, surviving 2O colonies, i.e., those arising from stably transfected cells, were selected and individually amplified. 2.1.9. atase and kinase activity The RediPlate 96 EnzChek® Tyrosine Phosphatase Assay Kit (R-22067) was used for SHP-l activity assay (Molecular Probes, Carlsbad, CA). The Raf-l kinase cascade assay kit te-Millipore, Billerica, MA) was used to examine the Raf-1 kinase activity.

The JAKZ kinase activity kit was sed from on Biology Corp. (Malvern, PA). 2.1.10. STAT3 reporter assay Cells were seeded in 96-well plate and pre-treated with IL-6 at the dose 10 ng/ul for 30 min. The STAT3 reporter kit was purchased from ciences (Frederick, MD).

[0036] 2.1.11. Xenograft tumor growth Male NCr athymic nude mice (5-7 weeks of age) were obtained from the National Laboratory Animal Center i, Taiwan). All experimental procedures using these mice were performed in accordance with protocols approved by National Taiwan University.

When Huh—7 tumors reached 100—200 mm3, mice received sorafenib tosylate (10 mg/kg) p.0. (oral) once daily, or SC-l(10 mg/kg) p.o. (oral) once daily. Controls received e (Chen KF et al. Cancer Res. 2008; 68:6698—6707). 2.1.12. Statistical Analysis.

Comparisons of mean values were performed using the independent samples t test KF et al. Cancer Res in SPSS for Windows 11.5 software (SPSS, Inc, Chicago, IL) (Chen 2008; 68:6698-6707). 2.2 Results 2.2.1 Compound 1 does not affect Raf kinase activity a sorafenib derivative t providing As above described, we synthesized functional group with hydrogen donor ability by replacing the pyridine ring and amide phenyl cyanide. Then, we tested compound 1 for its ability to inhibit Raf kinase activity sorafenib was able to PLCS cells, compared with that of sorafenib. As shown in Fig. 3, inhibit 50% of the Raf—l kinase activity of the untreated cells in the PLCS cells at 5 uM; The however, compound 1 d cells showed the same Raf-l activity as vehicle control. of hydrogen g y, loss of Raf-1 inhibition can presumably be attributed to the loss and amide functional group with phenyl as a result of the replacement of the ne ring cyanide. activity relationship of replacement of urea group and 2.2.2. Structure pyridine ring in cell death of sorafenib

[0044] As above described, we replaced the urea functional group linkage These compounds were with various amide and amide, generating compounds 2-11. analyzed by MTT assay for cell growth inhibition in the PLCS cells. Table 4 shows the results.

Table 4 H l de R4 IC (MM) 0 d' leomM) p R4 in H.585 cells in PLcs cells Sorafenib 8.3 /\§’0 BF 1 7.5 d’a >40 / P\S/ 2 0” \© >40 N02 /\ 9 8 >40 0”Sb \ // 3 ”8 0 \ CF3 N02 9 >40 £990 CFs F .

\/L\© >40 /\ ”0 CF3 0 >40 0 £319 The results show that none of these derivatives within the electron donating or electron withdrawing group showed greater cell toxicity than nib and compound 1.

Next, we changed the pyridine to a quinoline ring and amide linker to generate nds 12—25. These compounds were also analyzed by MTT assay for cell growth inhibition in the PLCS cells. Table 5 shows the results.

Table 5 ~- "WO 20014 (1%l / N '0 1300”“) Opel R4 in PLCS cells 0 N02 12 d’0 >40 01:3 £370 B r 14 0”Q >40 16 \J\©/ 16.0 o F 18 \JKE: 21,1 Table6 2012/049446 IC5001M) de R4 in PLC5 cells 0 NO2 13 mi £370 Br 6’0 . CFs 17 \J\©/ >40 o F 19 V51: >40 Table 6 " ' ' H R6 We UNxR4 \ ' l / / N ‘0 50 (PM) IC 50 (uM) in PLCS cells de R4 R6 R6 in PLc5 cells de R4 CF3 CFs >40 21 , ‘, Me. 25.4 Me ,’ ll CF:3 CF3 CF3 CF3 H 24 H 23 >40 190 I ’1 I, CF3 CF3 R H Oj/NH '0 50 (NM) de R4 R6 in PLC5 cells 22 7/ Me >40 I Qua H 10.8 1’ CF3 The amide linker showed different conformation from the sulfonyl linker, sulfonyl linker compounds. For example, compound 16 exhibiting better ty than showed Showed better cell toxicity than compound 12. a Compound cytotoxicity comparable to sorafenib and 1. We ded that the urea and amide linkers exhibited the most potent cell toxicity in PLC5 cells. 2.2.3. Mechanistic validation of the mode of action of sorafenib derivatives To check the dephosphorylation of STAT3 by sorafenib derivatives, we ed P-STAT3 state in PLC5 cells exposed to lOuM of each compound for 24 h by ELISA. As showed in Fig. 4, sulfonyl linker compounds showed no appreciable change in P-STAT3; however, compound 1 and some of the amide linker compounds showed a high degree of dephosphorylation of STAT3. The decreased level of P-STAT3 induced by these derivatives was correlated with cell toxicity. In the other words, these derivatives induced cell death in part through inhibition of STAT3.

In addition, we tested the downstream signal pathway after the inhibition of P— STAT3. Expression levels of the cyclin D1 and in, downstream target genes of STAT3, were assessed using compounds 1 and 12. As shown in Fig. 5, compound 1 with STAT3 inhibitory ty, was able to reduce cyclin D1 and survivin level, but compound 12 had no effect on either n. Further, DNA fragmentation and flow cytometry analysis of PLC5 cells treated with compound 1 were ted, and the s show that cell death was attributed to the inhibition of STAT3 and further induced the apoptotic signal (Fig. 6).

Our premise that sorafenib inhibition of Raf and STAT3 could be structurally iated was borne out by compound 1, which, devoid of Raf activity, exhibited the same level of downregulation of P-STAT3 as sorafenib did. We suggest that the cyanide group of compound 1 reduces its interaction with Raf. Subsequent modifications of sorafenib by changing the linker and ne ring to amide and quinoline (compounds 1, 16, and 25, respectively) resulted in a decrease in STAT3 ~repressing potency. 2.2.4. SC-I, a sorafenib tive, lacking tory on ofRaf-1 showed similar cell death effect to sorafenib in HCC cell lines.

In this experiment, we again examined the effects of sorafenib and SC-l 0n Raf-l activity. Raf—l immunoprecipitated from PLC5 or Hep3B cell extracts was incubated with MEK recombinant protein and the phospho—MEK was status assayed in the sorafenib or SC- l-treated cells. We observed a 20-40 % reduction in Raf—l kinase ty in the presence of sorafenib; however, SC-l did not inhibit the activity of Raf—l, suggesting that SC-l is not a Raf—l inhibitor (Fig. 7A). In addition, we assayed the phosphorylation of VEGFR2, a key target of sorafenib in cancer treatment. The expression of p-VEGFR2 (Tyrl 175) was decreased in PLC5 cells treated with sorafenib whereas SC-l did not have significant effect (Fig. 7B). These data suggest that SC-l derived from nib does not affect kinase inhibition. sorafenib and SC-l. Both SC- Next, we examined the anti-proliferation effects of and sorafenib decreased the Viability of various HCC cells including PLCS, SK—Hepl, 1 Huh7, and Hep3B in a dose-dependent manner (Fig. 8A). In addition, HCC cells treated with after 24 h 801 or sorafenib showed a significant increase in sub—Gl' phase population exposure (Fig. 8B). Both drugs induced cant apoptotic cell death as detected by or sorafenib-treated HCC cells ion of DNA fragmentation in SC—l (Fig. 8C). These and as potent as sorafenib in data indicate that SC—l has a significant effect on apoptosis inhibiting HCC cell growth even though 801 does not have the ability to block kinase induces apoptosis in HCC may activity, suggesting that the mechanism by which sorafenib not be related to its kinase inhibition activity. in HCC 2.2.5. STA T3 is a vital to the sensitizing effect of sorafenib and SC—I cell lines. the kinase To verify whether down—regulation of p-STAT3 is dependent on in SC-l- inhibition of sorafenib, we further assayed the STAT3-related ing pathway and resulted treated HCC cells. Given the fact that STAT3 was egulated by sorafenib in the ion of cell death, apoptotic related molecules ing Mcl—l, cyclinDl, role in mediating survivin were examined. We found that suppression of p—STAT3 plays a SC—l-induced the or sorafenib—induced cell death. SC—l reduced sion of STAT3— related ns in HCC cells. The phosphorylation of STAT3 at tyrosine 705 is critical for residue STAT3 transactivation. SC-l as well as sorafenib down—regulated p—STAT3 at Y705 Huh7, and and suppressed Mcl-l and cyclin D1 in all tested HCC cell lines including PLCS, sorafenib and SC—l Sk—Hepl (Fig. 9A). Notably, total STAT3 protein was not affected by in a dose— and time- (Fig. 9A). Moreover, 801 and sorafenib down—regulated p—STAT3 ent manner (Fig. 9B). These data further suggest that sorafenib inhibited STAT3 by a kinase—independent mechanism.

ELISA.

We also assayed the activation status of p—STAT3 by STAT3 Twenty— four hours before exposure to sorafenib or SC-l, Sk—Hepl cells were pre—treated with treated with 801 recombinant IL-6 to mimic high sion level of STAT3 and then were the presence of IL-6. SC-l or sorafenib-treated cell or sorafenib for another 24 hours under STAT3 at Y705. The ELISA extracts were incubated with antibody t phosphorylated of p-STAT3 significantly results showed that sorafenib as well as SC—l decreased the activity (Fig. 9C, left). To te the riptional activity, STAT3-binding region was cloned decreased into Luc reporter. We found that transcription activity of STAT3 was significantly in the presence of sorafenib or SC—l (Fig. 9C, right). The firefly luciferase activity was that both sorafenib and evaluated and normalized by Renilla luciferase. These results showed SC-l potently reduced the level of phosphorylation of STAT3 through the suppression of ription. We then established STAT3-overexpressed stable clone of HCC cells to validate the effect of sorafenib in HCC. As shown in Figure 9D, both nib-induced and .

SC-l-induced apoptosis were abolished in STAT3-overexpressed HCC cells as evidenced by sub~Gl analysis, suggesting that STAT3 is a major mediator of nib- and SC-l—induced apoptosis. 2.2.6. SHP—I phosphatase plays a role in the effect of sorafenib and SC—] on phospho-STAT3 and apoptosis.

To further study how sorafenib inhibits STAT3 in HCC, we examined several protein phosphatases which may involved in regulating p—STAT3. Our s showed that sodium vanadate, a general phosphatase inhibitor, decreased apoptosis and increased 13- STAT3 (Fig. 10A, left). These data suggest that sorafenib and SC-l may affect p—STAT3 by targeting STAT3—related n phosphatases. Furthermore, we found that SHP-l phosphatase—specific inhibitor reversed sorafenib-induced cell death and inhibition of p— STAT3 (Fig. 10A, right). To further verify the role of SHP-l in SC—l and sorafenib—induced inhibition of p—STAT3, we applied siRNA specific to SHP—l to examine the influence of sorafenib and SC-l. We found that silencing of SHP-l reversed sorafenib— or SC—l-induced apoptosis and inhibition of p—STAT3 (Fig. 10B, left). In addition, both sorafenib and SC-l increased SHP—l activity up to 3-fold in comparison with l cells (P < 0.05) (Fig. 10B, middle). Sorafenib or reated PLC5 cells were immunoprecipitated by SHP—l specific ' antibody, and then SHP-l-containing x underwent fluorescence—based phospho—group assay. Notably, neither sorafenib nor SC—l affected the interaction of STAT3 and SHP-l as evidenced by SHP—l immunoprecipitation (Fig. 10B, right). These data suggest that sorafenib induced cell death through SHP-l—dependent STAT3 inactivation.

[0064] In addition to SHP-l, other phosphatases such as SHP-2 and , have been reported to te p-STAT3. As shown in Fig. lOC, the effects of sorafenib on apoptosis and p—STAT3 were not reversed by silencing SHP—Z or FTP-1B, suggesting that neither SHP- 2 nor FTP—1B played a role in mediating the effect of sorafenis or SC-l on p-STAT3. 2.2.7. SC—I down-regulatesp—STA T3 and s apoptosis in HUVEC cells.

[0066] To y the effect of nib on p-VEGFRZ, a key target of sorafenib in cancer treatment, we examined the effect of sorafenib and SC—l in HUVEC cells. As shown in Fig. 11A, left, sorafenib and SC—l both down—regulated 3 in HUVEC cells and induced significant apoptotic cell death in HCC (P < 0.05). Notably, sorafenib but not 801 down—regulated the phosphorylation of VEGFR in HUVEC cells (Fig. 7A, middle). These WO 20014 2012/049446 results indicate that neither Raf—1 nor VEGFR mediates the effect of sorafenib on apoptosis and p—STAT3.

Previous study has also suggested that Mcl-l is crucial in mediating the effect of sorafenib on TRAIL-sensitization. Interestingly, our data showed that SC~l also showed r enhancement of TRAIL-induced apoptosis in HCC by the down—regulation of p- 113). To further investigate Whether inhibition of p—STAT3 by sorafenib STAT3 (Fig.

RNA. Silencing ated with Raf-l, we knocked down Raf—l by using small interference that Raf-1 did not affect the effects of sorafenib or SC-l on 3 (Fig. 11C), indicating neither sorafenib nor Raf—1 does not mediate the effect of sorafenib on p—STAT3. Notably, mediate SC-l altered the kinase ty of JAKZ (Fig. 11D), suggesting that JAKZ does not that sorafenib and SC- effects of both compounds on p-STATB. In addition, our data showed 1 did not affect the protein levels of SOCS—l and SOCS-3 (Fig. llE). Interestingly, HCC resistant to 801 cells with constitutively active STAT-3 (STAT3—C) were not completely data suggest that (Fig. 11F). As SC-l enhanced the activity of SHP—l (Fig. 11B, middle), our the effect besides STAT-3, other SHP—l-related molecules may also play a role in mediating of SC-l. To examine whether nib or SC-l targets SHP-l directly, PLCS cells were immunoprecipitated with SHP-l antibody then incubated with sorafenib or SC-l for 6 hours. in these lysates, As shown in Fig. 11G, sorafenib and 801 increase the activity of SHP-l ting that sorafenib and SC-l s SHP-l directly.

[0002] 2.2.8. Therapeutic evaluation of effect ofSC—I and nib on Huh 7-bearing mice. to HCC To verify the therapeutic effect of SC~l, we further applied SC-l received xenograft to evaluate its significance in vivo. First, earing mice daily treatment with e or sorafenib at the dose of 10 mg/kg/day orally. Sorafenib treatment animals had a significantly inhibited Huh7 xenografi tumor growth and sorafenib-treated size of less than half that of control mice (Fig. 12A, left). There were no apparent differences in body weight or toxicity in any mice (data not shown). In addition, tumor extract from vehicle and sorafenib-treated mice were immunoblotted for p-STAT3. p- STAT3 was down-regulated in sorafenib—treated tumor (Fig. 12A, right). p-STAT3/STAT3 Huh7 tumors. Furthermore, we was observed in the homogenates of three representative examined SHP—l activity in sorafenib—treated Huh7 xenografi. Sorafenib treated tumor showed significant induction of SHP~1 activity in vivo (Fig. 12A, right). Taken together, these results confirmed that sorafenib could increase SHP-l activity to repress p-STAT3 involved in tumor inhibition in the HCC xenograft model.

In addition, treatment with SC-l had a strong inhibitory effect (P < 0.05) and tumor size in this group was only 25% that of vehicle-treated mice at the end of treatment (Fig. 12B, left). Immunoblot for p-STAT3 and SHP-l activity assay were also med on a tumor sample from SC-l-treated animals. stingly, SC-1 induced significant rising of SHP-1 activity and down regulated p-STAT3 (Fig. 12B, right). These data indicate that SC- 1, a SHP~l agonist and a STAT3 inhibitor, exhibit therapeutic effects in ting tumor growth. 2.2.9. Inhibition of cancer cell growth We also examed the effects of SC-1 and SC-43 in other cancer cell lines, including breast cancer cell lines e.g. 31, MDAMB468, MCF~7, and leukemia cancer cell lines e. g. HL-60, KG—l and ML—l. Fig. 13 shows the results. These data show that the componds of the invention are effective in inhibiting the growth of cancer cells. 2.2.10 Anti—cancer effects in HCC cells HCC cells were treated with sorafenib derivatives (SC-43 or 8040) at the indicated dose for 24h. Collected cells were fixed in 75% Ethanol and stained with 20 ug/ml Propidium Iodide (PI). Sub—G1 is was performed by tometry. Fig. 14 shows that SC-43 and SC-40, sorafenib derivatives, show significant anti—cancer effects in HCC cells, (A), (B), (C), (D) and (E) refer to SC-43 for PLCS, HepG2, Hep3B, HA59T and SK— Hepl cells, respectively; and (F), (G), (H), (I) and (J) refer to SC-40 for PLCS, HepG2, Hep3B, HA59T and SK-Hepl cells, tively. Points, mean; bars, SD (n = 6). 2.2.11 Effects of sorafenib or SC-43 on STAT3-related proteins.

HCC cells treated with SC-43 (10 uM for 24 h) were collected with RIPA lysis buffer. Antibodies for immunoblotting such as cyclin D1 was sed from Santa Cruz Biotechnology. Other antibodies such as survivin, phospho—STAT3 (Tyr705), STAT3, Mel-1, SOCSl, and SOCS3 were from Cell Signaling. Fig. 15 shows that SC-43 down-regulates phospho—STAT3-related signaling pathway in HCC. 2.2.12 Effects of sorafenib or SC-40 on STAT3—related proteins.

HCC cells treated with SC—40 (10 [AM for 24 h) were collected with RIPA lysis buffer. Antibodies for immunoblotting such as cyclin D1 were sed from Santa Cruz Biotechnology. Other antibodies such as survivin, phospho-STAT3 (Tyr705), STAT3, M01— 1, SOCSl, and SOCS3 were from Cell Signaling. Fig. 16 shows that SC—40 down-regulates phospho-STAT3~re1ated signaling pathway in HCC. 2.2.13 Effects of sorafenib or SC-43 on STAT3—related proteins HCC cells d with SC-43 (10 uM for 24 h) were collected With RIPA lysis . Antibodies for immunoblotting such as cyclin D1 were purchased from Santa Cruz Biotechnology. Other antibodies such as survivin, phospho-STAT3 (Tyr705), STAT3, and Mcl-l were from Cell Signaling. Fig. 17 shows that SC-43 shows better inhibition of p— SC~43 STAT3—related signaling pathway than sorafenib in HCC, (A) PLC5 and (B) Hep 3B. than shows significant inhibition of p—STAT3 -re1ated ns at low dose treatment sorafenib. 2.2.14 s of SC-43 and SC-40 0n STAT3 activity p-STAT3 activity: PLC5 cells treated with SC derivatives were collected follows the RIPA buffer and analyzed in p-STAT3 ELISA kit. The assay protocol cturer.

[0017] STAT3 reporter assay: PLC5 cells were seeded in a 96—well plate. with Cells were nsfected with STAT3 er construct for 24 h and treated SABiosciences. derivatives for another 24 h. The STAT3 Reporter Kit was purchased from Cells were treated with SC-43 or SC~4O at 10 uM for 24 h and phospho—STAT3 ELISA or luciferase activity was measured. Fig. 18 shows that both SC—43 and SC-4O for SC—43 and induce strong inhibition of p—STAT3 activity, (A) and (B) p—STAT3 ELISA and SC-40, SC-40, respectively, and (C) and (D) STAT3 reporter assay for SC-43 respectively. 2.2.15 Effects of 40 on phosphatase activity PLC5 protein extract was incubated with anti-SHP-l antibody in immunoprecipitation buffer overnight. Protein G Sepharose 4 Fast flow (GE Healthcare Bio- with on.

Science) was added to each sample, followed by incubation for 3 hours at 4°C This SHP-l—containing protein extract were further incubated with SC compounds (10 or ) for 30 min at 4°C. RediPlate 96 EnzChek Tyrosine Phosphatase Assay kit 67) was used for SHP—l activity assay ular Probes). Fig. 19 shows that the SC derivatives SC—49. increase phosphatase activity of SHP-l in vitro, (A) SC—43, (B) SC—40, and (C) SHP— 2.2.16 Effects of SC derivatives on atase activity in recombinant RediPlate 96 EnzChek Tyrosine Phosphatase Assay kit (R—22067) was used for SHP-l activity assay (Molecular Probes). Recombinant SHP-l protein (25 ng) was then analyzed incubated with either SC—43 or SC-40 at the indicated dose for 30 minutes and by SHP-l phosphatase activity. Fig. 20 shows that the SC derivatives increase phosphatase activity of SHP—l in vitro, (A) SC-43 and (B) SC-40. 2.2.17 In vivo effect of SC-40 on PLC5-bearing xenograft.

National Male NCr athymic nude mice (5-7 weeks of age) were obtained from the Laboratory Animal Center (Taipei, ). All experimental procedures using these mice were done in accordance with protocols approved by the utional Laboratory Animal Care and Use Committee ofNational Taiwan University. Each mouse was inoculated so. in the dorsal flank with l X 106 PLC5 cells suspended in 0.1 mL of serum-free medium containing 50% Matrigel (BD Biosciences). When tumors reached 100 to 200 mm3, mice received SC-40 tosylate (10 or 20 mg/kg) orally once daily. Tumors were measured weekly using calipers, and their volumes were calculated using the following standard formula: width>< length >< height X 0.52.

Fig. 21 shows that (A) the mor effect of SC~40 on PLC5 ; points, mean (11 = 6); bars, SE; (B) Western blot analysis of p-STAT3 and STAT3 in PLC5 tumors; (C) the body weight of the animals; and (D) tumor weight and (B) activity of SHP—l in PLC5 tumors. The results show that 8040 has significant anti—tumor effect on PLC5 tumors, but do not affect body weight of the animals. The body weight has no significant differences between control and SCtreated mice. 2.2.18 mor effect of SC-43

[0027] In this example, we show that SC-43 exhibits antitumor effect in vitro and in viva.

SC—43 shows a significant xicity in HCC cells (IC50~0.5 nM). Also, SC-43 significantly causes tumor growth inhibition in HCC-bearing mice. SHP—l/STATS-related signaling pathway acts as a vital target for the umor effect of SC—43. See Fig. 22 (A) the cytotoxicity of SC-43 in HCC cells, (B) the antitumor effect of 8043 in HCC-bearing 2O mice, (C) the activity of SHP-l induced by SC-43, and (D) Western blot analysis of p- STAT3 and STAT3 in HCC cells treated by SC—43 (10 uM and 20 uM). 2.2.19 In Vivo effect of SC—43 on PLC5/Inc orthotopic model Male NCr athymic nude mice (5—7 weeks of age) were ed from the National Laboratory Animal Center (Taipei, Taiwan). All experimental procedures using these mice were done in accordance with protocols approved by the Institutional tory Animal Care and Use Committee ofNational Taiwan University. Each mouse was inoculated within liver in the dorsal flank with l X 104 PLC5/Inc cells suspended in 0.1 mL of serum-free medium containing 50% Matrigel (BD Biosciences). When tumors formed, mice ed sorafenib or SC-43 tosylate (10 mg/kg) orally once daily. Tumor growth was monitored by non-invasive in Vivo imaging system (IVIS) image system twice weekly.

SC-40 shows a significant antitumor effect on PLC5-bearing orthotopic mice.

The tumor growth was red at the indicated time by IVIS image system. Mice were treated with either vehicle, sorafenib (10 mg/kg) or SC-43 (10 mg/kg). Fig. 23 shows (A) the images of the treated mice, (B) shows the body weight of the mice, and (C) shows the survival curve between control and d mice. The results show that SC—40 shows a cant antitumor effect on PLCS-bearing orthotopic mice. The body weight has no significant differences between control and 8043 -treated mice. 701813NZ

Claims (11)

What is claimed is:

1. A compound which is represented by a I wherein R1 and R3 are independently hydrogen, halo, hydroxyl, alkoxyl, thioalkoxy, alkyl, lower alkenyl, low alkynyl, cycloalkyl, heterocycloalkyl, aryl, aralkyl, heteroaryl, or heteroarakyl; wherein R2 is independently -(C)mNHC(X)NH(C)nRa-, -(C)pNHC(X)Rb-, - (C)qNHS(O)2Rc, -(C)r(X)NHRd-,or -(C)sNH(C)tRe; wherein Ra, Rb, Rc, Rd and Re are independently en, halo, hydroxyl, alkoxyl, thioalkoxy, alkyl,lower alkenyl, low alkynyl, cycloalkyl, cycloalkyl, aryl, l, heteroaryl, arakyl; X=O or S; and m, n, p, q, r, s, t=0, 1, or 2.

2. The compound of claim 1, wherein R1 and R3 are independently hydrogen, or lower alkyl.

3. The compound of claim 1 or 2, wherein Ra, Rb, Rc, Rd and Re are independently phenyl or naphthyl, with 1 to 3 groups selected from the group consisting of halo, lower alkyl (such as halo-substituted lower alkyl, e.g. trifluoromethyl), alkoxyl (e.g. such as halo-substituted lower alkoxyl, e.g. trifluoromethyl) and aryloxy (e.g. cyano-substituted phenoxy). 701813NZ

4. The compound of claim 1, wherein R1 and R3 are ndently hydrogen; and R2 is , , , , or ; or wherein R1 is independently hydrogen; R3 is methyl; and R2 is or

5. A compound which is represented by

6. A pharmaceutical composition comprising a compound as defined in any of claim 1 to 5 and a pharmacological acceptable carrier. 701813NZ

7. A pharmaceutical composition for increasing Src homology-2 containing protein tyrosine phosphatase-1 (SHP-1) expression in a cell, comprising a compound as defined in any of claim 1 to5 and a pharmacological acceptable carrier.

8. A ceutical composition for treating a disease or condition terized by decreased Src homology-2 containing n tyrosine phosphatase-1 (SHP-1), comprising a nd as defined in any of claim 1 to 5 and a pharmacological acceptable carrier.

9. A method for increasing Src homology-2 containing protein tyrosine phosphatase-1 (SHP-1) expression in a cell in vitro, sing contacting the cell with an effective amount of a compound as defined in any of claim 1 to 5 or a pharmaceutical composition of claim 6.

10. Use of a compound as defined in any of claim 1 to 5 for manufacture of a medicament for treating a disease or condition terized by sed Src homology-2 containing protein tyrosine phosphatase-1 (SHP-1) expression, wherein the disease or condition characterized by decreased SHP-1 expression is cancer and osteoporosis.

11. Use of claim 10, wherein the cancer is hepatocellular carcinoma (e.g. hepatocellular oma, leukemia, lung cancer, breast cancer, renal cancer, thyroid cancer colon, head and neck cancer.