OA16752A

OA16752A - armaceutical compositions.

Info

Publication number: OA16752A
Application number: OA1201400103
Authority: OA
Inventors: Yu Chen; Lan Yang; Feiyu FENG; Qiufu GE; Dianwu Guo; Yi Chen
Original assignee: Uro-Celtique S.A.
Priority date: 2011-09-18
Filing date: 2012-09-14
Publication date: 2015-12-14

Abstract

The present invention is directed to pharmaceutical compositions comprising: (a) a cyclopolysaccharide and (b) a compound of Formula (I) or its pharmaceutical acceptable salt: wherein X1, X2, Q, Z, and m are defined herein. Also disclosed is a method for treating a neoplastic disease or an immune disease with these compositions.

Description

PHARMACEUTICAL COMPOSITIONS

RELATED APPLICATIONS

This application is an international application which daims priority to and benefit of U.S. Provisional Application No. 61/536,038, filed on September 18, 2011 and and U.S. Provisional Application No. 61/602,408, filed on February 23, 2012. The entire teachings of the above applications aie incorporated herein by reference.

BACKGROUND

Cancer is one of the most life threatening diseases in which cells in a part of the body expérience out-of-control growth. According to latest data from American Cancer Society, it is estimated to have 1.6 million new cases of cancer in USA in 2011. Cancer is the second leading cause of death in the United States (second only to heart disease) and will claim more than 570,000 lives in 2011. In fact, it is estimated that 50% of ail men and 33% of ail women living in the United States will develop some type of cancer in their lifetime. Therefore cancer constitutes a major public health burden and represents a significant cost in the United States. For décades, surgery, chemotherapy, and radiation were the established treatments for various cancers. Patients usually receive a combination of these treatments depending upon the type and extent of their disease. But the chemotherapy is most important option for cancer patient when the surgery treatment is impossible.

Bendamustîne, a well known chemotherapy first synthesized in 1963, consists of an alkylating nitrogen mustard moiety and a purine-like benzimidazol moiety with a suggested purineanalog effect (Barman Balfour JA, et al, Drugs 2001; 61: 631-640). Bendamustîne has been shown to have substantial activity against low-grade lymphomas (Herold M, et al., Blood, 1999; 94, Suppl 1: 262a), multiple myelomas (Poenisch W, et al., Blood 2000; 96, Suppl 1: 759a), and several solid tumors (Kollmannsberger C, et al., Anticancer Drugs 2000; 11: 535-539). It was also reported that bendamustîne effectively induces apoptosis in lymphonia cells (Chow KU, et al., Haematologica, 2001; 86: 485-493). On March 2008, the FDA granted approval to market bendamustîne for the treatment of chronic lymphocytic leukemia (CLL). On October 2008, the FDA granted further approval to market bendamustîne for the treatment of indolent B-cell non-Hodgkin's lymphoma (NHL) that has progressed during or within six months of treatment with rituximab or a rituximabcontaining regimen.

The clinical activity of Bendamustine as a single agent and in combination with other chemotherapeutic and immunotherapeutic drugs, coupled with its potential lack of cross-resistance with inany other chemotherapy agents, make bendamustine an attractive therapy for patients with newly diagnosed and refractory hématologie malignancies. [Leoni LM, Semin Hematol. 2011 Apr;

48 Suppl 1 :S4-11 ]. Currently Bendamustine has about 75 active clinical trials for a variety of caner indications, such as leukemia, lymphoma, small cell lung cancer, multiple myeloma, MDS, ovarian cancer, breast cancer, and brain tumor. Bendamustine, marketed by Cephalon (TREANDA™), has annual sale of $393 million in US in 2010, and an sale of more than $500 million in US in 2011. The peak sale in 2015 may reach 1 billion $. Bendamustine market exclusive right in US will expire in 2015.

Although Bendamustine has made a significant contribution to cancer treatment, the doselimiting toxicities and drug résistance remain significant hurdles in its clinical use.

In recent years, histone deacetylases (HDAC) has emerged as an important disease target for cancer treatment [Minucci, S. et al., Nat Rev Cancer 2006, 6, 38-51], The human HDAC enzymes hâve 18 isoforms grouped into Class I-IV according to their sequence homology. Class I, II and IV, commonly referred to as the classical HDACs, are comprised of 11 family members. Class III HDACs consists of 7 enzymes and they are distinct from other HDAC family members, therefore are given a unique term sirtuins. The inhibition of HDAC enzyme leads to histone acétylation which is associated with the remodelling of chromatin and plays a key rôle in lhe epigenetic régulation of gene expression. In addition, HDAC inhibitors hâve been shown to evoke the acétylation of many important non-histone proteins such as HSP90, alpha-tubulîn, Ku-70, Bcl-6, importin, cortactin, p53, STAT1, E2F1, GATA-1 and NF-kB, which can aller many important signaling networks related to cancer treatment. The underlying mechanism of action of HDAC inhibitors includes the différentiation, cell cycle arrest, inhibition of DNA repair, induction of apoptosis, upregulation of tumor suppressors, down régulation of growth factors, oxidative stress and autophagy. In the last decade, a large number of structurally diverse HDAC inhibitors hâve been identified and at least 12 HDAC inhibitors are currently in human clinical trials for cancer treatments, including short-chain fatty acid (valproic acid), hydroxamates (SAHA, LBH589, PXD101, JNJ-26481585, ITF2357, CUDC-101), cyclic tetrapeptides (FK-228), benzamide (MS30 275), and several other compounds (CHR-3996, 4SC-201, SB939). Among them, SAHA and FK228 has been approved by the US FDA for the treatment of advanced cutaneous T-cell lymphoma.

In WO/2010/085377, we reported NL-101, a first-in-class dual-functional Bendamustine dérivative which potently inhibits the HDAC pathway. The structure of parental drug Bendamustien and NL-101 is shown below:

Thc biological assay showed that NL-101 potently inhibits HDAC enzyme (HDAC1 IC50 of 9 11M). NL-101 was sent to NCI (NSC# 751447) for NCI-60 cell line panel screening. The data showed that NL-101 is about x 25-100 fold more potent than Bendamustine in the NCI-60 cell lines that are représentative of a variety of human cancer type. The sixty GI₅₀ values (one for each cell line) make up the fingerprint of the NL-101 and based on this fingerprint, the COMPARE analysis was doue by using the COMPARE algorithm on the NCI DTP website. A Pearson corrélation coefficient (PCC) of >0.8 indicates >65% agreement in the sensitivity patterns of two compounds and a high likelihood of a common mechanism of action. The COMPARE resuit showed that the fingerprint of NL-101 did not strongly correlate with any of NSC synthetic compounds (>140,000). In fact, the top match compound is epidoxoform (a doxorubicin dérivative) with a PCC of 0.676. Direct comparisons among NL-101, and the conventional nitrogen mustard (e.g. bendamustine, melphalan, and chlorambucil) showed weak corrélation coefficients (PCC < 0.483). These COMPARE resuit suggested that NL-101 is not just another conventional nitrogen mustard but possesses unique mechanistic features that differentiate it from the conventional DNA alkylating agents. In another word, NL-101 is expected to be non-cross résistant to the conventional DNA alkylating agents. Therefore NL-101 might hâve wide potential applications for cancer patients who are résistant, relapse, or refractory to conventional DNA alkylating agents such as bendamustine, melphalan, cisplatin, and temozolomide.

We hâve developed a first génération formulation of NL-101 for in vivo study, which contains 6mg/ml NL-101 in buffer system (1.5% acetic acid/0.2% NaOH) with a pH value around 4.

The animal study using the first génération formulation of NL-101 shows excellent in vivo efficacy in animal models such as imatinib-resistant Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL) model and lung cancer. Ph+ ALL is a leukemia common in adults (-35% of adult ALL) and carries a poor prognosis. Vincristine (VCR), doxorubicin (Dox), cytarabine (AraC), and cyclophosphamde (CTX) are conventional chemotherapy for Ph+ ALL treatment. Our data showed that single dose of NL-101 (60mpk) has significantiy better in vivo efficacy than the bendamustine, SAHA, VCR, Dox, AraC, and CTX (each dosued at MTD) in imatinib-resistant Ph+ ALL model. Weekly dosing of NL-lOl at 60mg/kg has similar efficacy to Sprycel, which is a FDA approved 2^nd line targeted drug for Ph+ ALL treatment. However, the first génération formulation of NL-101 has unfortunately significant disadvantages, such as low pH value, potential précipitation after injection, and sériés side effects (e.g, damaged mice tail after iv injection and sometime sudden mice death after quick iv injection due to cardiotoxicity). Therefore, there is a strong need to develop a new génération formulation of NL-101 which can overcome the shortcoming of the first génération formulation, particularly the cardiotoxicity, and can be used in future human clinical trials.

SUMMARY OF THE INVENTION

The présent invention relates to a composition comprising (a) a cyclopolysaccharide and (b) a compound of Formula (I), or a pharmaceutically acceptable sait thereof:

Formula (1) ?

X>

In Formula I, m is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16; Z is deleted, C(R_aR_b), O, S, C(O), N(R_a), SO₂, OC(O), C(O)O, OSO₂, S(O₂)O, C(O)S, SC(O), C(O)C(O), C(O)N(R_a), N(R_a)C(O), S(O₂)N(R_a), N(R_a)S(O₂), OC(O)N(R_a), N(R_a)C(O)O, N(R_a)C(O)S, or N(R_a)C(O)N(R_b), in which each of R_a and R_b, independently, is H, alkyl, alkenyl, or alkynyl; X] and X₂ independently, is halo or OSO₂R_c, in which R_c is alkyl, alkenyl, or alkynyl; and Q is cycloalkyl, heterocycloalkyl, cycloaikenyl, heterocycloalkenyl, aryl, or heteroaryl, each of which, independently, is optionally substituted with alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloaikenyl, heterocycloalkenyl, aryl, heteroaryl, halo, nitro, oxo, -C=NH, cyano, alkyl-Rj, OR_d, OC(O)R_d, OC(O)OR_(I, OC(O)SR_d, SR_d, C(O)R_d, C(O)OR_(t, C(O)SR_d, C(O)NR_cR_f, SOR<i, SO₂R_d, NRcRf, or N(R_e)C(O)Rf, in which each of R_d, R_e, and Rr, independently, is H, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, halo, cyano, amine, nitro, hydroxy, or alkoxy.

One subset of the above-described compounds includes those in which X| and X2 independently, is halo; Z is deleted, CH2, O, CO, NH, SO2, OC(O), C(O)O, C(O)S, NHC(O), C(O)NH, OC(O)NH, NHC(O)O, or NHC(O)S; m is 5, 6, 7, or 8; and Q is a 9-10 membered aryl or heteroaryl.

One preferred subset of above-described compounds represented by Formula(II)

Ri R20 ,^0H

Λ-N >NII

X ix^· _N ^m

Formula (II) , in which R] and R2 independently, is H, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, halo, -C=NH, amine, cyano, hydroxy, or alkoxy.

The most preferred compound of above-described compound is represented by Formula(III) (i.e. NL-101):

In another aspect, a preferred pharmaceutically acceptable sait is a hydrochloride sait, hydrobromide sait, methanesulfonate, toluenesulfonate, acetate, fumarate, sulfate, bisulfate, succinate, citrate, phosphate, maleate, nitrate, tartrate, benzoate, biocarbonate, carbonate, sodium hydroxide sait, calcium hydroxide sait, potassium hydroxide sait, tromethamine sait, or mixtures thereof. A more preferred pharmaceutically acceptable sait is a hydrochloride sait, methanesulfonate, toluenesulfonate, acetate, succinate, citrate, maleate, tartrate, or mixtures thereof. The most preferred pharmaceutically acceptable sait is an acetate sait.

In another aspect, a preferred cyclopolysaccharide is α-cyclodextrin or a dérivative thereof, β-cyclodextrin or dérivative thereof, and γ-cyclodextrin or a dérivative thereof. A more preferred cyclopolysaccharide is β-cyclodextrin or dérivative thereof. The most preferred cyclopolysaccharide is hydroxypropyl β-cyclodextrin, or sulfobutylether β-cyclodextrin.

As shown below in Example 6, we are surprised to found that composition comprising NL101 and hydroxypropyl β-cyclodextrin can significantly reduce the cardiotoxicity in vivo. Furthermore, as shown below in Example 10, in a NSCLC xengoraft A549 model, animais treated with a composition comprising NL-101 and hydroxypropyl β-cyclodextrin showed significantly decreased tumor size compared with animais treated with the parental drug Bendamustine and the vehicle group.

The compositions of the présent invention are useful in treating a patient having a tumor. The compounds of the invention may also useful in the prévention and treatment of an immune disease.

This invention also relates to a method of treating a neoplastic disorder (e.g., cancer, myeiodysplastic syndrome, or myeloproliferative disease) by administering to a subject in need thereof an effective amount of compositions thereof described above.

Furthermore, this invention relates to a method of treating an immune disease (e.g., rheumatoid arthritis and multiple sclerosis) by administering to a subject in need thereof an effective amount of compositions thereof described above.

The details of one or more embodiments of the invention are set forth in the description below. Other features, objects, and advantages of the invention will be apparent from the description and from the claims.

DETAILED DESCRIPTION

In a first embodiment, the invention is a composition comprising (a) cyclopolysaccharide, and (b) a compound of Formula (I) îllustrated above, or its géométrie isomers, enantiomers, diastereomers, racemates, pharmaceutically acceptable salts, prodrugs and solvatés thereof

In a preferred embodiment, the invention is a composition comprising (a) cyclopolysaccharide, and (b) a compound of Formula (II) îllustrated above, or its géométrie isomers, enantiomers, diastereomers, racemates, pharmaceutically acceptable salts, prodrugs and solvatés thereof.

In a most preferred embodiment, the invention is a composition comprising (a) cyclopolysaccharide, and (b) a compound of Formula (III) îllustrated above, or its géométrie isomers, enantiomers, diastereomers, racemates, pharmaceutically acceptable salts, prodrugs and solvatés thereof.

Exemplary compounds described herein include, but are not limited, to the following:

	0. PH	Cl J	HN-OH f—^/()
	t	r Cl	»
	Q pu Vnh	r j αλτ CI\/^_N YY-N )	HN-OH —' O
	»	£	»
	O PH Y-NU	ci	HN-OH
Cl	*	S Cl	«
	un-on		HN-OH
—^{// 1} ci^_nKAn^	/—'o	F J '
r Cl	»	^or Cl

Compounds of the invention may contain one or more asymmetric carbon atoms.

Accordingly, the compounds may exist as diastereomers, enantiomers or mixtures thereof. The synthèses of the compounds may employ racemates, diastereomers or enantiomers as starting materials or as intermediates. Diastereomeric compounds may be separated by chromatographie or 5 crystallization methods. Similarly, enantiomeric mixtures may be separated using the same techniques or others known in the art. Each of the asymmetric carbon atoms may be in the R or S configuration and both of these configurations are within the scope of the invention.

It should be recognized that the compounds of the présent invention may be présent and optionally administered in the form of salts, solvatés and prodrugs that are converted in vivo into

the compounds of the présent invention. For example, it is within the scope of the présent invention to convert the compounds of the présent invention into and use them in the form of their pharmaceutically acceptable salts derived from various organic and inorganic acids and bases in accordance with procedures well known in the art.

When the compounds of the présent invention possess a free base form, the compounds can be prepared as a pharmaceutically acceptable acid addition sait by reacting the free base form of the compound with a pharmaceutically acceptable inorganic or organic acid, e.g., hydrohalides such as hydrochloride, hydrobromide, hydroiodide; other minerai acids such as sulfate, nitrate, phosphate, etc.; and alkyl and monoarylsulfonates such as ethanesulfonate, toluenesulfonate and benzenesulfonate; and other organic acids and their corresponding salts such as acetate, tartrate, maleate, succinate, citrate, benzoate, salicylate and ascorbate. Further acid addition salts of lhe présent invention include, but are not limited to: adipate, alginate, arginate, aspartate, bisulfate, bisulfite, bromide, butyrate, camphorate, camphorsulfonate, caprylate, chloride, chlorobenzoate, cyciopentanepropionate, digluconate, dihydrogenphosphate, dinitrobenzoate, dodecylsulfate, fumarate, galacterate (from mucic acid), galacturonate, glucoheptaoate, gluconate, glutamate, glycérophosphate, hemisuccinate, hemisulfate, heptanoate, hexanoate, hippurate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, iodide, isethionate, iso-butyrate, lactate, lactobionate, malate, malonate, mandelate, metaphosphate, methanesulfonate, methylbenzoate, monohydrogenphosphate, 2-naphthalenesulfonate, nicotinate, nitrate, oxalate, oleate, pamoate, pectinate, persulfate, phenylacetate, 3-phenylpropionate, phosphate, phosphonate and phthalate. It should be recognized that the free base forms will typically differ from their respective sait forms somewhat in physical properties such as solubility in polar solvents, but otherwise the salts are équivalent to their respective free base forms for tire purposes of the présent invention.

When the compounds of the présent invention possess a free acid form, a pharmaceutically acceptable base addition sait can be prepared by reacting the free acid form of the compound with a pharmaceutically acceptable inorganic or organic base. Examples of such bases are alkali métal hydroxides including potassium, sodium and lithium hydroxides; alkaline earth métal hydroxides such as barium and calcium hydroxides; alkali métal alkoxides, e.g., potassium ethanolate and sodium propanolate; and various organic bases such as ammonium hydroxide, piperidine, diethanolamine and N-methylglutamine. Also included are the aluminum salts of the compounds of the présent invention. Further base salts of the présent invention include, but are not limited to: copper, ferrie, ferrous, lithium, magnésium, manganic, manganous, potassium, sodium and zinc

salts. Organic base salts include, but are not limited to, salts of primary, secondary and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, e.g., arginine, betaine, caffeine, chloroprocaine, choline, N,N'dibenzylethyienediamine (benzathine), dicyclohexylamine, diethanolamine, 2-diethylaminoethanol,

2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethyl morpholine, N-ethyipiperidine, glucamine, glucosamine, histidine, hydrabamine, iso-propylamine, lidocaine, lysine, meglumine, Nmethyl-D-glucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethanolamine, triethylamine, trimethylamine, tripropylamine and tris(hydroxymethyl)-methylamine (tromethamine). It should be recognized that the free acid forms will typically differ from their respective sait forms somewhat in physical properties such as solubility in polar solvents, but otherwise the salts are équivalent to their respective free acid forms for the purposes of the present invention.

Compounds of the present invention that comprise basic nitrogen-containing groups may be quatemized with such agents as (Cm) alkyl halides, e.g., methyl, ethyl, iso-propyl and tert-butyl chlorides, bromides and iodides; di-(CM) alkyl sulfates, e.g., dimethyl, diethyl and diamyl sulfates; alkyl halides, e.g., decyl, dodecyl, lauryl, myristyl and stearyl chlorides, bromides and iodides; and aryl (Cm) alkyl halides, e.g., benzyl chloride and phenethyl bromide. Such salts permit the préparation of both water-soluble and oil-soluble compounds of the present invention.

Compounds of the present invention that comprise a tertiary nitrogen atoms may be oxidized by such agents as hydrogen peroxide (H₂O₂), Caro's acid or peracids like metaChloroperoxybenzoic acid (rnCPBA) to from amine oxide. Amine oxides of anti-cancer agents hâve been developed as prodrugs and may be water-soluble.

Prodrug dérivatives of compounds according to the present invention can be prepared by modifying substituents of compounds of the present invention that are then converted in vivo to a different substituent. It is noted that in many instances, the prodrugs themselves also fall within the scope of the range of compounds according to the present invention. For example, prodrugs can be prepared by reacting a compound with a carbamylating agent (e.g., 1,1acyloxyalkylcarbonochloridate, para-nitrophenyl carbonate, or the like) or an acylating agent. Further examples of methods of making prodrugs are described in Saulnier et al. (1994), Bioorganic and Médicinal Chemistry Letters, Vol. 4, p. 1985.

Cyclopolysaccharides: the cyclopolysaccharides which may employed in the practice of this invention include cyciodcxtrins, cyclomannins, cycloaltrins, cyclofructins and the like. In general,

ΙΟ cyclopolysaccharides comprising between 6 and 8 sugar units are preferred. Among the preferred cyclopolysaccharides which may be employed are cyclodextrins.

Cyclodextrins are cyclic oligomers of dextrose with a truncated cône structure consisting of a hydrophilic exterior and a hydrophobie interior cavity. A cyclodextrin can form an inclusion complex with a guest molécule by compfexing with ail or a portion of a hydrophobie guest molécule within its cavity. The size of the cavity is determined by the number of glucopyranose units in the cyclodextrin. Alpha-(a), beta-(P), and gamma-(y) cyclodextrins are the most common cyclodextrins and possess six, seven and eight glucopyranose units, respectively. Because natural cyclodextrins have relalively low aqueous solubility and are associated with toxicity, chemically modified cyclodextrin dérivatives have been developed to overcome these limitations. Such cyclodextrin dérivatives typically possess a chemical modification at one or more of the 2, 3, or 6 position hydroxyl groups. Cyclodextrin dérivatives have, for example, been described in U. S. Pat. No. 5,134,127; 5,376,645; 5,571,534; 5,874,418; 6,046,177 and 6,133,248, the contents of which are herein incorporated by reference and made a part hereof. As used herein, the terms cyclodextrin, a-cyclodextrin, β-cyclodextrin and γ-cyclodextrin are intended to encompass unmodified cyclodextrins as well as chemically modified dérivatives thereof. The compositions of the invention comprise an inclusion complex of a cyclodextrin and a compound of Formulae (I), (II), or (III).

In yet another embodiment, the composition comprises a therapeutically effective concentration of a compound of Formulae (I), (II), or (III).

In one embodiment of lhe invention, the composition comprises a cyclodextrin selected from the group consisting of a-cyclodextrin, β-cyclodextrin and γ-cyclodextrin.

In yet another embodiment, the cyclodextrin is a β-cyclodextrin and γ-cyclodextrin.

In an additional embodiment, the cyclodextrin is a β-cyclodextrin.

In a further embodiment, the cyclodextrin is selected from the group consisting of a hydroxypropyl^-cyclodextrin (Pitha et ai, J Pharm Sci, 84 (8), 927-32 (1995)) and sulfobutyl derivatized- β-cyclodextrin (described, for example, in U.S. Pat. Nos. 5,134,127; 5,376, 645; 5,874,418; 6,046,177 and 6,133,248)..

In another embodiment, tlie cyclodextrin is a hydroxypropyl β-cyclodextrin.

In yet another embodiment of the invention, the cyclodextrin is sulfobutylether-βcyclodextrin.

Other preferred cyclopolysaccharides include, but are not limited to, β-cyclodextrin substituted with 2-hydroxy-N,N,N-trimethylpropanammonium, carboxymethylated-p-cyclodextrin, O-phosphated-P-cyclodextrin, succinyl-(2-hydroxy)propyl-betacyclodexlrin, sulfopropylated-βcyclodextrin, heptakis(6amino-6-deoxy)-P-cyclodextrin, O-sulfated-p-cyclodextrin, and 65 monodeoxy-6-mono(3-hydroxy) propylamino-P-cyclodextrin;

The cyclodextrin may be included in an amount that increases the solubility of the active compound in the composition. In one embodiment, the amount of cyclodextrin included within the composition is the minimal amount needed to solubilize the drug in the composition. In a further embodiment, the composition is a parentéral formulation and the amount of cyclodextrin included 10 within the formulation is the minimal amount of cyclodextrin needed to solubilize the drug.

In order to déterminé the minimum amount of cyclodextrin needed to solubilize a compound encompassed by Formulae I-III, a plot of the compound's solubility versus cyclodextrin concentration can be carried out. By interpolating or extrapolating from the plot, a composition can be prepared that contains the minimum amount of cyclodextrin needed to dissolve the desired 15 concentration of the active compound.

In one embodiment, the composition comprises at least 2.5% (weight/volume) of a cyclodextrin. In another embodiment, the composition comprises at least 5% of a cyclodextrin. In yet another embodiment, the composition comprises at least 10% of a cyclodextrin. In a further embodiment, the composition comprises from 2.5 to 40% of a cyclodextrin. In yet another 20 embodiment, the composition comprises from 5% to 20% of a cyclodextrin. In another embodiment, the composition comprises 7.5% to 15% of a cycloextrin. In yet another embodiment, the composition comprises about 10% of a cyclodextrin.

In one embodiment, the composition comprises at least 2.5% (weight/volume) of a βcyclodextrin. In another embodiment, the composition comprises at least 5% of a β-cyclodextrin. In 25 yet another embodiment, the composition comprises at least 10% of a β-cyclodextrin. In a further embodiment, the composition comprises from 2.5 to 40% of a β-cyclodextrin. In yet another embodiment, the composition comprises from 5% to 20% of a β-cyclodextrin. In another embodiment, the composition comprises 7.5% to 15% of a β-cycloextrîn. In yet another embodiment, the composition comprises 10% of a β-cyclodextrin.

In one embodiment, the composition comprises at least 2.5% (weight/volume) of a hydroxypropyl β-cyclodextrin or sulfobutylether β-cyclodextrin. In another embodiment, the composition comprises at least 5% of a hydroxypropyl β-cyclodextrin or sulfobutylether β-

cyclodextrin. In yet another embodiment, the composition comprises at least 10% of a hydroxypropyl β-cyclodextrin or sulfobutylether β-cyclodextrin. In a further embodiment, the composition comprises from 2.5 to 40% of a hydroxypropyl β-cyclodextrin or sulfobutylether βcyclodextrin. In yet another embodiment, the composition comprises from 5% to 20% of a hydroxypropyl β-cyclodextrin or sulfobutylether β-cyclodextrin. In another embodiment, the composition comprises 7.5% to 15% of a hydroxypropyl β-cyclodextrin or sulfobutylether βcyclodextrin. In yet another embodiment, the composition comprises 10% of a hydroxypropyl βcyclodextrin or sulfobutylether β-cyciodextrin.

In one embodiment, the composition further comprises pH adjusting agents. In a further embodiment, the pH adjusting agents are one or more acids, bases, or salts. Examples of acids that may be included in the composition include inorganic acids such as hydrochloric acid, sulfuric acid, phosphoric acid, or mixtures thereof, and organic acids such as citric acid, L(-)-malic acid and L(+)tartaric acid or mixtures thereof. Examples of bases that may be included in the composition include sodium hydroxide, potassium hydroxide. calcium hydroxide, tromethamine, or mixtures thereof. Examples of sait that may be included in the composition include sodium bicarbonate, sodium carbonate, sodium citrate, or mixtures thereof. In a further embodiment, the composition comprising one or more pH adjusting agents has a pH range of 6.0-9.0, preferably 7.0-8.0.

Another embodiment of the invention is a pharmaceutical dosage form that includes a pharmaceutical composition containing 5 to about 500 mg of compound of Formula (I-III). The more preferred formula is Formula (II), and the most preferred formula is Formula (III).

In a further embodiment, the composition comprises dextran. In yet another embodiment, the composition comprises dextran in an amount of range from about 1% to about 5% weight/volume dextran. In a further embodiment, the composition comprises from about 2 to about 4% weight/volume dextran.

Any inert excipient that is commonly used as a carrier or diluent may be used in compositions of the présent invention, such as sugars, polyalcohols, soluble polymers, salts and lipids. Sugars and polyalcohols which may be employed include, without limitation, lactose, sucrose, mannitol, and sorbitol. Illustrative of the soluble polymers which may be employed are polyoxyethylene, poloxamers, polyvinylpyrrolidone, and dextran. Useful salts include, without limitation, sodium chloride, magnésium chloride, and calcium chloride. Lipids which may be employed include, without limitation, fatty acids, glycerol fatty acid esters, glycolipids, and phospholipids.

In addition, the compositions may further comprise binders (e.g., acacia, comstarch, geiatin, carbomer, ethyl cellulose, guar gum, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, povidone), disintegrating agents (e.g., comstarch, potato starch, alginic acid, silicon dioxide, , croscarmellose sodium, crospovidone, guar gum, sodium starch glycolate, Primogel), buffers (e.g., tris-HCL, acetate, phosphate) of various pH and ionic strength, additives such as albumin or geiatin to prevent absorption to surfaces, détergents (e.g., Tween 20, Tween 80, Pluronic F68, bile acid salts), protease inhibitors, surfactants (e.g., sodium lauryl sulfate), perméation enhancers, solubilizing agents (e.g., glycerol, polyethylene glycerol, cyclodextrins), a glidant (e.g., colloïdal silicon dioxide), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite, butylated hydroxyanisole), stabilizers (e.g., hydroxypropyl cellulose, hydroxypropylmelhyl cellulose), viscosity increasing agents (e.g., carbomer, colloïdal silicon dioxide, ethyl cellulose, guar gum), sweeteners (e.g., sucrose, aspartame, citric acid), flavoring agents (e.g., peppermint, methyl salicylate, or orange flavoring), preservatives (e.g., Thimerosal, benzyl alcohol, parabens), lubricants (e.g., stearic acid, magnésium stéarate, polyethylene glycol, sodium lauryl sulfate), flow-aids (e.g., colloïdal silicon dioxide), plasticizers (e.g., diethyl phthalate, triethyl citrate), emulsifiers (e.g., carbomer, hydroxypropyl cellulose, sodium lauryl sulfate), polymer coatings (e.g., poloxamers or poloxamines), coating and film forming agents (e.g., ethyl cellulose, acrylates, polymethacrylates) and/or adjuvants.

In one embodiment, the compositions are prepared with carriers thaï will protect the compound against rapid élimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery Systems. Biodégradable, biocompatible polymers can be used, such as ethylene vinyl acetate, poly anhydride s, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for préparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.

The composition of the invention may be prepared by mixing a solution of the cyclopolysaccharide with an stock solution of a compound of Formula (I). Such resulting mixture is vigorously mixed and optionally subjected to the action of ultrasound waves to obtain a homogenous and equilibrated aqueous solution. Preferably, the final composition is filtered before

use for injection. The composition may be optionally freeze-dried to produce a solid material suitable for dissolution in injection media before its use.

DEFINITIONS :

Acyl means a carbonyl containing substituent represented by the formula -C(O)-R in which R is H, alkyl, a carbocycle, a heterocycle, carbocycle-substituted alkyl or heterocyclesubstituted alkyl wherein the alkyl, alkoxy, carbocycle and heterocycle are as defined herein. Acyl groups include alkanoyl (e.g. acetyl), aroyl (e.g. benzoyl), and heteroaroyl.

“Aliphatic” means a moiety characterized by a straight or branched chain arrangement of constituent carbon atoms and may be saturated or partially unsaturated with one or more double or triple bonds.

The term “alkyl” refers to a straight or branched hydrocarbon containing l-20 carbon atoms (e.g., C]-Cio). Examples of alkyl include, but are not limited to, methyl, methylene, ethyl, ethylene, n-propyl, i-propyl, n-butyl, i-butyl, and t-butyl. The term “alkenyl” refers to a straight or branched hydrocarbon containing 2-20 carbon atoms (e.g., Cî-Cio) and one or more double bonds. Examples of alkenyl include, but are not limited to, ethenyl, propenyl, and allyl. The term “alkynyl” refers to a straight or branched hydrocarbon containing 2-20 carbon atoms (e.g., C2-C10) and one or more triple bonds. Examples of alkynyl include, but are not limited to, ethynyl, l-propynyl, 1- and 2butynyl, and l-methyl-2-butynyl. The term “alkylamino” refers to an -N(R)-alkyl in which R can be H, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, or heteroaryl. Alkoxy means an oxygen moiety having a further alkyl substituent.

Alkoxycarbonyl means an alkoxy group attached to a carbonyl group. Oxoalkyl means an alkyl, further substituted with a carbonyl group. The carbonyl group may be an aldéhyde, ketone, ester, amide, acid or acid chloride.

The term “cycloalkyl” refers to a saturated hydrocarbon ring system having 3 to 30 carbon atoms (e.g., C3-C12). Examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. The term “cycloalkenyl” refers to a non-aromatic hydrocarbon ring system having 3 to 30 carbons (e.g., C3-C12) and one or more double bonds. Examples include cyclopentenyl, cyciohexenyl, and cycloheptenyl. The term heterocycloalkyl refers to a nonaromatic 5-8 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ring system having one or more heteroatoms (such as O, N, S, P, or Se). Examples of heterocycloalkyl groups include, but are not limited lo, piperazinyl, pyrrolidinyl,

dioxanyl, morpholinyl, and tetrahydrofuranyl. The term heterocycloalkenyl refers to a nonaromatic 5-8 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ring system having one or more heteroatoms (such as O, N, S, P, or Se) and one or more double bonds.

The term “aryl” refers to a 6-carbon monocyclic, ΙΟ-carbon bicyclic, 14-carbon tricyclic aromatic ring system. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, and anthracenyl. The term “heteroaryl” refers to an aromatic 5-8 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ring system having one or more heteroatoms (such as O, N, S, P, or Se). Examples of heteroaryl groups include pyridyl, furyl, imidazolyl, benzimidazolyl, pyrimidinyl, thienyl, quinolinyl, indolyl, and thiazolyl.

Alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, alkylamino, aryl, and heteroaryl mentioned above include both substituted and unsubstituted moieties. Possible substituents on alkylamino, cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, aryl, and heteroaryl include, but are not limited to, Ci-Cæ alkyl, C2-C|q alkenyl, C2-C10 alkynyl, C3-C20 cycloalkyl, C3-C20 cycloalkenyl, C|-C₂o heterocycloalkyl, C]-C2o heterocycloalkenyl, Ci-C;₀ alkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, amino, Ci-C₁₀alkylamino, arylamino, hydroxy, halo, oxo (O=), thioxo (S=), thio, silyl, Ci-Cjo alkylthio, arylthio, C1-C10 alkylsulfonyl, arylsulfonyl, acylamino, aminoacyl, aminothioacyl, amidino, mercapto, amido, thioureido, thiocyanato, sulfonamido, guanidine, ureido, cyano, nitro, acyl, thioacyl, acyloxy, carbamido, carbamyl, carboxyl, and carboxylic ester. On the other hand, possible substituents on alkyl, alkenyl, or alkynyl include ail of the above-recited substituents except Ci-Cæ alkyl. Cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, and heteroaryl can also be fused with each other.

Amino means a nitrogen moiety having two further substituents where each substituent has a hydrogen or carbon atom alpha bonded to the nitrogen. Unless ïndicated otherwise, the compounds of the invention containing amino moieties may include protected dérivatives thereof. Suitable protecting groups for amino moieties include acetyl, tert-butoxycarbonyl, benzyloxycarbonyl, and the like.

Aromatic means a moiety wherein the constituent atoms make up an unsaturated ring system, ail atoms in the ring system are sp2 hybridîzed and the total number of pi électrons is equal 30 to 4n+2. An aromatic ring may be such that the ring atoms are only carbon atoms or may include carbon and non-carbon atoms (see Heteroaryl).

I

Carbamoyl means the radical -OC(O)NRaRb where Ra and Rb are each independently two further substituents where a hydrogen or carbon atom is alpha to the nîtrogen, It is noted that carbamoyl moieties may include protected dérivatives thereof. Examples of suitable protécting groups for carbamoyl moieties include acetyl, tert-butoxycarbonyl, benzyloxycarbonyl, and the like. It is noted that both the unprotected and protected dérivatives fall within the scope of the invention.

Carbonyl means the radical -C(O)-. It is noted that the carbonyl radical may be further substituted with a variety of substituents to form different carbonyl groups including acids, acid halides, amides, esters, and ketones.

Carboxy means the radical -C(O)O-, It is noted that compounds of the invention containing carboxy moieties may include protected dérivatives thereof, i.e., where the oxygen is substituted with a protécting group. Suitable protécting groups for carboxy moieties include benzyl, tert-butyl, and the like.

Cyano means the radical -CN.

Halo means fluoro, chloro, bromo or iodo.

Halo-substituted alkyl, as an isolated group or part of a larger group, means alkyl substituted by one or more halo atoms, as such terms are defined in this Application. Halosubstituted alkyl includes haloalkyl, dihaloalkyl, trihaloalkyl, perhaloalkyl and the like.

Hydroxy means the radical -OH.

Imine dérivative means a dérivative comprising lhe moiety —C(NR)—, wherein R comprises a hydrogen or carbon atom alpha to the nîtrogen.

Isomers mean any compound having identical molecular formulae but differing in the nature or sequence of bonding of their atoms or in the arrangement of their atoms in space. Isomers that differ in the arrangement of their atoms in space are termed stereoisomers. Stereoisomers that are not mirror images of one another are termed diastereomers and stereoisomers that are nonsuperimposable mirror images are termed enantiomers or sometimes optical isomers. A carbon atom bonded to four nonidenlical substituents is termed a chiral center. A compound with one chiral center has two enantiomeric forms of opposite chirality. A mixture of the two enantiomeric forms is termed a racemic mixture.

Nitro means the radical -NO2.

Protected dérivatives means dérivatives of inhibitors in which a reactive site or sites are blocked with protécting groups. Protected dérivatives are useful in the préparation of inhibitors or in themselves may be active as inhibitors. A comprehensive list of suitable protécting groups can be t

I7 found in T. W. Greene, Protecting Groups in Organic Synthesis, 3rd édition, John Wiley & Sons, 1999.

Substituted or unsubstituted means that a given moiety may consist of only hydrogen substituents through available valencies (unsubstituted) or may further comprise one or more nonhydrogen substituents through available valencies (substituted) that aie not otherwise specified by the name of the given moiety.

Sulfide means -S-R wherein R is H, alkyl, carbocycle, heterocycle, carbocycloalkyl or heterocycloalkyl. Particular sulfide groups are mercapto, alkylsulfide, for example methylsulfide (S-Me); arylsulfide, for example phenylsulfide; aralkylsulfide, for example benzylsulfide.

Sulfinyl means the radical -S(O)-. It is noted that the sulfinyl radical may be further substituted with a variety of substituents to form different sulfinyl groups including sulfinic acids, sulfinamides, sulfinyl esters, and sulfoxides.

Sulfonyl means the radical -S(O)(O)-. Il is noted that the sulfonyl radical may be further substituted with a variety of substituents to form different sulfonyl groups including sulfonic acids, sulfonamides, sulfonate esters, and sulfones,

Thiocarbonyl means the radical -C(S)-. It is noted that the thiocarbonyl radical may be further substituted with a variety of substituents to form different thiocarbonyl groups including thioacids, thioamides, thioesters, and thioketones.

Animal includes humans, non-human mammals (e.g., dogs, cats, rabbits, cattie, horses, sheep, goats, swine, deer, and the like) and non-mammals (e.g., birds, and the like).

Bioavailability as used herein is the fraction or percentage of an administered dose of a drug or pharmaceutical composition that reaches the systemic circulation intact. In general, when a médication is administered intravenously, its bioavailability is 100%. However, when a médication is administered via other routes (e.g., orally), its bioavailability decreases (e.g., due to incomplète absorption and first-pass metabolism). Methods to improve the bioavailability include prodrug approach, sait synthesis, particle size réduction, complexation, change in physical form, solid dispersions, spray drying, and hot-melt extrusion.

Disease specifically includes any unhealthy condition of an animal or part thereof and includes an unhealthy condition that may be caused by, or incident to, medical or veterinary therapy applied to that animal, i.e., the side effects of such therapy.

Pharmaceutically acceptable means that which is useful in preparing a pharmaceutical composition that is generally safe, non-toxic and neither biologically nor otherwise undesirable and includes that which is acceptable for veterinary use as well as human pharmaceutical use.

Pharmaceutically acceptable salts means salts of compounds of the présent invention which are pharmaceutically acceptable, as defined above, and which possess the desired pharmacological activity. Such salts include acid addition salts formed with inorganic acids, or with organic acids. Pharmaceutically acceptable salts also include base addition salts which may be formed when acidic protons présent are capable of reacting with inorganic or organic bases.

Prodrug means a compound that is convertible in vivo metabolically into an inhibitor according to the présent invention. For example, an inhibitor comprising a hydroxyl group may be administered as an ester that is converted by hydrolysis in vivo to the hydroxyl compound.

“Pharmacophore”, as defined by The International Union of Pure and Applied Chemistry, is an ensemble of steric and eiectronic features that is necessary to ensure the optimal supramolecular interactions with a spécifie biological target and to trigger (or block) its biological response. For _texample, Camptothecin is the pharmacophore of the well known drug topotecan and irinotecan. As another example, nitrogen mustard pharmacophore has a typical formula of -NtCHîCHhXjî or its N-oxide analogues in which X is a leaving group such as halo. The anti-cancer drugs containing a nitrogen mustard pharmacophore include but not limited to Melphalan, Bendamustine, Cyclophosphamide, PX-478, TH-302, PR-104, Ifofamide, and so on.

Pharmaceutically acceptable carrier means a non-toxic solvent, dispersant, excipient, adjuvant, or other material which is mixed with the compound of the présent invention in order to permit the formation of a pharmaceutical composition, i.e, a dose form capable of administration to the patient. Examples of pharmaceutically acceptable carrier includes suitable polyethylene glycol (e.g PEG400), surfactant (e.g Cremophor), or cyclopolysaccharide (e.g hydroxypropyl-βcyclodextrin or sulfobutyl ether β-cyclodextrins), polymer, liposome, micelle, nanosphere, and so on.

“Stability” in general refers to the length of time a drug retains its properties without loss of potency. Sometimes this is referred to as shelf life. Factors affecting drug stability include, among other things, the chemical structure of the drug, impurity in the formulation, pH, moisture content, as well as environmental factors such as température, oxidization, light, and relative humidity.

Stability can be improved by providing suitable chemical and/or crystal modifications (e.g., surface modifications that can change hydration kinetics; different crystals that can hâve different

properties), excipients (e.g., anything other than the active substance in the dosage form), packaging conditions, storage conditions, etc.

“Therapeutically effective amount of a composition described herein is meant an amount of the composition which confers a therapeutic effect on the treated subject, at a reasonable benefit/risk ratio applicable to any medical treatment. The therapeutic effect may be objective (i.e„ measurable by some test or marker) or subjective (i.e., subject gives an indication of or feels an effect). An effective amount of the composition described above may range from about 0.1 mg/kg to about 500 mg/Kg, preferably from about 0.2 to about 50 mg/kg. Effective doses will also vary depending on route of administration, as well as the possibility of co-usage with other agents. It will be understood, however, that the total daily usage of lhe compositions of the présent invention will be decided by the attending physician within the scope of sound medical judgment. The spécifie therapeutically effective dose level for any particular patient will dépend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the spécifie compound employed; the spécifie composition employed; the âge, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excrétion of the spécifie compound employed; the duration of the treatment; drugs used in combination or contemporaneously with the spécifie compound employed; and like factors well known in the medical arts.

As used herein, the term “treating” refers to administering a compound to a subject that has a neoplastic or immune disorder, or has a symptom of or a prédisposition toward it, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect the disorder, the symptoms of or the prédisposition toward the disorder. The term an effective amount” refers to the amount of the active agent that is required to confer the intended therapeutic effect in the subject. Effective amounts may vary, as recognized by those skilled in the art, depending on route of administration, excipient usage, and the possibility of co-usage with other agents. A “subject” refers to a human and a non-human animal. Examples of a non-human animal include ail vertebrates, e.g., mammals, such as non-human primates (particularly higher primates), dog, rodent (e.g., mouse or rat), guinea pig, cat, and non-mammals, such as birds, amphibians, reptiles, etc. In a preferred embodiment, the subject is a human. In another embodiment, the subject is an experimental animal or animal suitable as a disease model.

GENERAL

“Combination therapy” includes the administration of the subject compositions of the présent invention in further combination with other biologically active ingrédients (such as, but not limited to, a second and different antineoplastic agent) and non-drug thérapies (such as, but not limited to, surgery or radiation treatment). For instance, the compositions of the invention can be used in combination with other pharmaceutically active compounds, or non-drug thérapies, preferably compounds that are able to enhance the effect of the compositions of the invention. The compositions of the invention can be administered simultaneously (as a single préparation or separate préparation) or sequentîally to the other thérapies. In general, a combination therapy envisions administration of two or more drugs/treatments during a single cycle or course of therapy.

In one embodiment, the compositions of the invention are administered in combination with one or more of traditional chemotherapeutic agents. The traditional chemotherapeutic agents encompass a wide range of therapeutic treatments in the field of oncology. These agents are administered at various stages of the disease for the purposes of shrinking tumors, destroying remaining cancer cells Ieft over after surgery, inducing remission, maintaining remission and/or alleviating symptoms relating to the cancer or its treatment. Examples of such agents include, but are not limited to, alkylating agents such as Nitrosureas (e.g., Carmustine, Lomustine and Streptozocin), ethylenimines (e.g., thiotepa, hexamethylmelanine), Alkylsulfonates (e.g., Busulfan), Hydrazines and Triazines (e.g., Altretamine, Procarbazine, Dacarbazine and Temozolomide), and platinum based agents (e.g., Carboplatin, Cisplatin, and Oxaliplatin); plant alkaloids such as

Podophyllotoxins (e.g., Etoposide and Tenisopide), Taxanes (e.g., Paclitaxel and Docetaxel), Vinca alkaloids (e.g., Vincristine, Vinblastine and Vinorelbine); anti-tumor antibiotics such as Chromomycins (e.g., Dactinomycin and Plicamycin), Anthracyclines (e.g., Doxorubicin, Daunorubicin, Epirubicin, Mitoxanlrone, and Idarubicin), and miscellaneous antibiotics such as Mitomycin and Bleomycin; anti-metabolites such as folie acid antagonists (e.g., Methotrexate), pyrimidine antagonists (e.g.. 5-Fluorouracil, Foxuridine, Cytarabine, Capecitabine, and Gemcitabine), purine antagonists (e.g., 6-Mercaptopurine and 6-Thioguanîne) and adenosine deaminase inhibitors (e.g., Cladribine, Fludarabine, Nelarabine and Pentostatin); topoisomerase inhibitors such as topoisomerase I inhibitors(Topotecan, Irinotecan), topoisomerase II inhibitors (e.g., Amsacrine, Etoposide, Etoposide phosphate, Teniposide), and miscellaneous anti-neoplastics such as ribonucleotide reductase inhibitors (Hydroxyurea), adrenocortical steroid inhibitor (Mitotane), anti-microtubule agents (Estramustine), and retinoids (Bexarotene, Isotretinoin, Tretînoin (ATRA).

2l

In one aspect of the invention, the compositions may be administered in combination with one or more targeted anti-cancer agents that modulate protein kinases involved in various disease states. Examples of such kinases may include, but are not limited ABLl, ABL2/ARG, ACK1, AKT1, AKT2, AKT3, ALK, ALKl/ACVRLl, ALK2/ACVR1, ALK4/ACVR1B, ALK5/TGFBR1,

ALK6/BMPR1B, AMPK(A1/Bl/Gl), AMPK(Al/Bl/G2), AMPK(Al/Bl/G3), AMPK(Al/B2/Gl), AMPK(A2/Bl/Gl), AMPK(A2/B2/Gl), AMPK(A2/B2/G2), ARAF, ARK5/NUAK1, ASK1/MAP3K5, ATM, Aurora A, Aurora B , Aurora C , AXL, BLK, BMPR2, BMX/ETK, BRAF, BRK, BRSKl, BRSK2, BTK, CAMKla , CAMKlb, CAMKld, CAMKlg , CAMKIIa, CAMKIIb , CAMKIId , CAMKIIg , CAMK4, CAMKK1, CAMKK2, CDC7-DBF4, CDKl-cyclin A, CDKl10 cyclin B, CDKl-cyclin E, CDK2-cyclin A, CDK2-cyclin Al, CDK2-cyclin E, CDK3-cyclin E, CDK4-cyclin Dl, CDK4-cyclin D3, CDK5-p25, CDK5-p35, CDK6-cyciin Dl, CDK6-cyclin D3, CDK7-cyclin H, CDK9-cyclin K, CDK9-cyclin Tl, CHK1, CHK2, CKlal , CKld , CKlepsilon , CKlgl, CKlg2, CKlg3 , CK2a , CK2a2, c-KIT, CLK1 , CLK2, CLK3, CLK4, c-MER, c-MET, COT1/MAP3K8, CSK, c-SRC, CTK/MATK, DAPK1, DAPK2, DCAMKL1, DCAMKL2, DDR1,

DDR2, DLK/MAP3K12, DMPK, DMPK2/CDC42BPG, DNA-PK, DRAK1/STK17A, DYRK1/DYRK1A, DYRK1B, DYRK2, DYRK3, DYRK4, EEF2K, EGFR, EIF2AK1, E1F2AK2, EIF2AK3, EIF2AK4/GCN2, EPHA1, EPHA2, EPHA3, EPHA4, EPHA5, EPHA6, EPHA7, EPHA8, EPHB1, EPHB2, EPHB3, EPHB4, ERBB2/HER2, ERBB4/HER4, ERK1/MAPK3, ERK2/MAPK1, ERK5/MAPK7, FAK/PTK2, FER, FES/FPS, FGFR1, FGFR2, FGFR3, FGFR4,

FGR, FLT1/VEGFR1, FLT3, FLT4/VEGFR3, FMS, FRK/PTK5, FYN, GCK/MAP4K2, GRK1, GRK2, GRK3, GRK4, GRK5, GRK6, GRK7, GSK3a, GSK3b, Haspin, HCK, HGK/MAP4K4, HIPK1, HIPK2, HIPK3, H1PK4, HPK1/MAP4K1, IGF1R, IKKa/CHUK , IKKb/IKBKB, IKKe/IKBKE, IR, IRAK1, IRAK4,1RR/INSRR, ITK, JAK1, JAK2, JAK3, JNK1 , JNK2 , JNK3, KDR/VEGFR2, KHS/MAP4K5, LATS1, LATS2, LCK, LCK2/ICK, LKB1 , LIMK1, LOK/STKIO,

LRRK2, LYN, LYNB, MAPKAPK2, MAPKAPK3, MAPKAPK5/PRAK, MARK1, MARK2/PARlBa, MARK3, MARK4, MEK1, MEK2, MEKK1, MEKK2, MEKK3, MELK, MINK/MINK1, MKK4, MKK6, MLCK/MYLK, MLCK2/MYLK2, MLK1/MAP3K9, MLK2/MAP3K10, MLK3/MAP3K11, MNK1, MNK2, MRCKa/, CDC42BPA, MRCKb/, CDC42BPB, MSK1/RPS6KA5, MSK2/RPS6KA4, MSSK1/STK23, MSTI/STK4, MST2/STK3, MST3/STK24,

MST4, mTOR/FRAPl, MUSK, MYLK3, MYO3b, NEK1, NEK2, NEK3, NEK4, NEK6, NEK7, NEK9, NEK11, NIK/MAP3K14, NLK, OSR1/OXSR1, P38a/MAPK14, P38b/MAPK11, P38d/MAPK13 , P38g/MAPK12 , P70S6K/RPS6KB1, p70S6Kb/, RPS6KB2, ΡΑΚΙ, PAK2, PAK3,

PAK4, PAK5, PAK6, PASK, PBK/TOPK, PDGFRa, PDGFRb, PDK1/PDPK1, PDKl/PDHKl, PDK2/PDHK2 , PDK3/PDHK3, PDK4/PDHK4, PHKgl , PHKg2 , PI3Ka, (pl 10a/p85a), P13Kb, (pl l0b/p85a), PI3Kd, (pl 10d/p85a), PI3Kg(pl20g), PIM1, PIM2, PIM3, PKA, PKAcb, PKAcg , PKCa , PKCb l , PKCb2 , PKCd , PKCepsilon, PKCeta, PKCg , PKCiota, PKCmu/PRKDl, PKCnu/PRKD3, PKCtheta, PKCzeta, PKD2/PRKD2, PKGla , PKGlb , PKG2/PRKG2, PKN1/PRK1, PKN2/PRK2, PKN3/PRK3, PLKl, PLK2, PLK3, PLK4/SAK, PRKX, PYK2, RAFl, RET, RIPK2, RIPK3, RIPK5, ROCKl, ROCK2, RON/MST1R, ROS/ROS1, RSK1, RSK2, RSK3, RSK4, SGKl, SGK2, SGK3/SGKL, SIKl, SIK2, SLK/STK2, SNARK/NUAK2, SRMS, SSTK/TSSK6, STK16, STK22D/TSSK1, STK25/YSK1, STK32b/YANK2, STK32c/YANK3, STK33, STK38/NDR1, STK38L/NDR2, STK39/STLK3, SRPK1, SRPK2, SYK, TAK1, TAOKl, TAOK2/TAO1, TAOK3/JIK, TBK1, TEC, TESK1, TGFBR2, TIE2/TEK, TLK1, TLK2, TNIK, TNK1, TRKA, TRKB, TRKC, TRPM7/CHAK1, TSSK2, TSSK3/STK22C, TTBK1, TTBK2, TTK, TXK, TYK1/LTK, TYK2, TYRO3/SKY, ULKl, ULK2, ULK3, VRKl, VRK2, WEEl, WNK1, WNK2, WNK3, YES/YESl, ZAK/MLTK, ZAP70, Z1PK/DAPK3, KINASE, MUTANTS, ABL1(E255K), ABLl(F3l7I), ABLl(G250E), ABLl(H396P), ABLl(M35lT), ABL1(Q252H), ABLl(T3l5I), ABLl(Y253F), ALK (Cl 156Y), ALK(Ll 196M), ALK (Fl 174L), ALK (R1275Q), BRAF(V599E), BTK(E4lK), CHK2(I157T), c-Kit(A829P), c-KIT(D816H), c-KIT(D816V), cKit(D820E), c-Kit(N822K), C-Kit (T670I), c-Kit(V559D), c-Kit(V559D/V654A), cKit(V559D/T670I), C-Kit (V560G), c-KIT(V654A), C-MET(D1228H), C-MET(D1228N), CMET(Fl200I), c-MET(Ml250T), C-MET(Yl23OA), C-MET(Yl230C), C-MET(Yl230D), CMET(Y1230H), c-Src(T34lM), EGFR(G7I9C), EGFR(G719S), EGFR(L858R), EGFR(L86lQ), EGFR(T790M), EGFR, (L858R.T790M), EGFR(d746-750fr790M), EGFR(d746-750), EGFR(d747-749/A750P), EGFR(d747-752/P753S), EGFR(d752-759), FGFR1(V561M), FGFR2(N549H), FGFR3(G697C), FGFR3(K650E), FGFR3(K650M), FGFR4(N535K), FGFR4(V550E), FGFR4(V550L), FLT3(D835Y), FLT3(ITD), JAK2 (V617F), LRRK2 (G2019S), LRRK2 (I2020T), LRRK2 (R1441C), p38a(T106M), PDGFRa(D842V), PDGFRa(T674I), PDGFRa(V56lD), RET(E762Q), RET(G69lS), RET(M918T), RET(R749T), RET(R8l3Q), RET(V804L), RET(V804M), RET(Y79lF), TIF2(R849W), TIF2(Y897S), and TIF2(Yl 108F).

In another aspect of tlie invention, the subject compositions may be administered in combination with one or more targeted anti-cancer agents that modulate non-kinase biological targets, pathway, or processes. Such targets pathways, or processes include but not limited to heat shock proteins (e.g. HSP90), poly-ADP (adenosine diphosphate)-ribose polymerase (PARP), hypoxia-inducible factors(HlF), protéasome, Wnt/Hedgehog/Notch signaling proteins, TNF-alpha, matrix metalloproteinase, famesyl transferase, apoptosis pathway (e.g Bcl-xL, Bcl-2, Bcl-w), histone deacetylases (HDAC), histone acetyltransferases (HAT), and methyltransferase (e.g histone lysine methyltransferases, histone arginine methyltransferase, DNA methyltransferase, etc).

In another aspect of the invention, the compositions of the invention are administered in combination with one or more of other anti-cancer agents that include, but are not limited to, hormonal thérapies (e.g Tamoxifen, Fuivestrant, Clomifene, Anastrozole, Exemestane, Formestane, Letrozole, etc), vascular disrupting agent, gene therapy, RNAi cancer therapy, chemoprotective agents (e.g., amfostine, mesna, and dexrazoxane), antibody conjugate(e.g brentuximab vedotin, ibritumomab tioxetan), cancer immunolherapy such as lnterleukin-2, cancer vaccines(e.g., sipuleucel-T) or monoclonal antibodies (e.g., Bevacizumab, Alemtuzumab, Rituximab, Trastuzumab, etc).

In another aspect of the invention, the subject compositions are administered in combination with radiation therapy or surgeries. Radiation is commonly delivered intemaliy (implantation of radioactive material near cancer site) or externally from a machine that employs photon (x-ray or gamma-ray) or particle radiation. Where the combination therapy further comprises radiation treatment, the radiation treatment may be conducted at any suitable time so long as a bénéficiai effect from the co-action of the combination of the therapeutic agents and radiation treatment is achieved. For example, in appropriate cases, the bénéficiai effect is still achieved when the radiation treatment is temporally removed from the administration of the therapeutic agents, perhaps by days or even weeks.

In certain preferred embodiments, the compositions of the invention are administered in combination with one or more of radiation therapy, surgery, or anti-cancer agents that include, but arc not limited to, DNA damaging agents, anti-metabolites, topoisomerase inhibitors, antimicrotubule agents, EGFR inhibitors, HER2 inhibitors, VEGFR2 inhibitors, BRAF inhibitors, BcrAbl inhibitors, PDGFR inhibitors, ALK inhibitors, PLK inhibitors, MET inhibitors, epigenetic agents, HSP90 inhibitors, PARP inhibitors, CHK inhibitors, aromatase inhibitor, estrogen receptor antagonist, and antibodies targeting VEGF, HER2, EGFR, CD50, CD20, CD30, CD33, etc.

In certain preferred embodiments, the compositions of the invention are administered in combination with one or more of abarelix, abiraterone acetate, aldesleukin, alemtuzumab, altretamine, anastrozole, asparaginase, bevacizumab, bexarotene, bicalutamide, bleomycin, bortezombi, brentuximab vedotin, busulfan, capecitabine, carboplatin, carmustine, cetuximab, chlorambucil, cisplatin, cladribine, clofarabïne, clomifene, crizotinib, cyclophosphamide, dasatinib, daunorubicin liposomal, decitabine, degarelix, denileukin diftitox, denileukin diftitox, denosumab, docetaxel, doxorubicin, doxorubicin liposomal, epirubicin, eribulin mesylate, erlotinib, estramustine, etoposide phosphate, everolimus, exemestane, fludarabine, fluorouracil, fotemustine, fulvestrant, gefitinib, gemcitabine, gemtuzumab ozogamicin, goserelin acetate, histrelin acetate, hydroxyurea, ibritumomab tiuxetan, idarubicin, ifosfamide, imatinib mesylate, interferon alfa 2a, ipilimumab, ixabepilone, lapatinib ditosylate, lenalidomide, letrozole, leucovorin, leuprolide acetate, levamisole, lomustine, mechlorethamine, melphalan, methotrexate, mitomycin C, mitoxantrone, nelarabine, nilotinib, oxaliplatin, paclitaxel, paclitaxel protein-bound particle, pamidronate, panitumumab, pegaspargase, peginterferon alfa-2b, pemetrexed disodium, pentostatin, raloxifene, rituximab, sorafenib, streptozocin, sunitinib maleate, tamoxifen, temsirolimus, teniposide, thalidomide, toremifene, tositumomab, trastuzumab, tretinoin, uramustine, vandetanib, vemurafenib, vinorelbine, zolédronate, radiation therapy, or surgery.

A wide variety of administration methods may be used in conjunction with the compositions 15 of the présent invention. Compositions of the présent invention may be administered or coadministered orally, parenterally, intraperitoneally, intravenously, intraarterially, transdermally, sublingually, intramuscularly, rectally, transbuccally, inlranasally, liposomally, via inhalation, vaginally, intraoccularly, via local delivery (for example by cathéter or stent), subcutaneously, intraadiposally, intraarticularly, or intrathecally. The compositions according to the invention may also be administered or coadministered in slow release dosage forms. Compositions may be in gaseous, liquid, semi-liquid or solid form, formulated in a manner suitable for the route of administration to be used. For oral administration, suitable solid oral formulations include tablets, capsules, pills, granules, pellets, sachets and effervescent, powders, and the like. Suitable liquid oral formulations include solutions, suspensions, dispersions, émulsions, oils and the like. For parentéral administration, reconstitution of a lyophilized powder is typically used.

The invention further provides methods for the prévention or treatment of a neoplastic disease or immune disease. In one embodiment, the invention relates to a method of treating a neoplastic disease or immune disease in a subject in need of treatment comprising administering to said subject a therapeutically effective amount of a composition of the invention. In one embodiment, the invention further provides for the use of a composition of the invention in the manufacture of a médicament for halting or decreasing a neoplastic disease or immune disease.

*

The neoplastic disease includes but not limited to lung cancer, head and neck cancer, central nervous system cancer, prostate cancer, testîcular cancer, colorectal cancer, pancreatic cancer, Iiver cancer, stomach cancer, biliary tract cancer, esophageal cancer, gastrointestinal stromal tumor, breast cancer, cervical cancer, ovarian cancer, uterine cancer, leukemia, lymphomas, multiple myeloma, melanoma, basal cell carcinoma, squamous cell carcinoma, bladder cancer, rénal cancer, sarcoma, mesothelioma, thymoma, myeiodysplastic syndrome and myeloproliferative disease.

It is well known that immunosuppression is one of major side-effect of many conventional chemotherapy. For example, at low dose, cyclophosphamide can be used to treat immune diseases such as multiple sclerosis, rheumatoid arthritis and the suppression of transplant rejections. (Emadi A, et al, Nat Rev Clin Oncol. 2009 Nov; 6( 11 ):638-47; Perini P, et al. Neurol Sci. 2008 Sep; 29 Suppl 2:S233-4.) and is also widely used in bone marrow transplantation “conditioning” and “mobilization” regimens, and for the treatment of refractory severe autoimmune conditions, such as systemic lupus erythematosus (SLE), minimal change disease, severe rheumatoid arthritis, Wegener's granulomatosis (with trade name Cytoxan), scleroderma, and multiple sclerosis (with trade name Revimmune). In addition, HDAC has recently emerging as a promising target for treating immune disease [Szyf M.Clin Rev Allergy Immunol. 2010 Aug;39(l ):62-77]. Therefore it is not difficult to imagine the compositions of présent invention could be used for treatment of an immune disease.

In a preferred embodiment, the immune disease is selected from the group consisting of the rejection of transplanted organs and tissues, a graft-versus-host disease, a non-autoimmune inflammatory disease, and an autoimmue disease, wherein said autoimmue disease is selected from the group consisting of acute disseminated encephalomyelitis, addison's disease, ankylosing spondylitis, antiphospholipid antibody syndrome, autoimmune hemolytic anémia, autoimmune hepatitis, autoimmune inner ear disease, bullous pemphigoid, coeliac disease, chagas disease, chronic obstructive pulmonary disease, churg-strauss syndrome, dermatomyositis, Crohn's disease, diabètes mellitus type l, endometriosis, goodpasture's syndrome, graves' disease, guillain-barré syndrome, hashimoto’s disease, hidradenitis suppurativa, idiopathic thrombocytopénie purpura, interstitial cystitis, lupus erythematosus, morphea, multiple sclerosis, myasthenia gravis, narcolepsy, neuromyotonia, pemphigus vulgaris, pemicîous anaemia, polymyositis, primary biliary cirrhosis, psoriasis, psoriatic arthritis, rheumatoid arthritis, schizophrenia, scleroderma, temporal arteritîs, vasculitis, vitiligo, and wegener's granulomatosis.

It should be understood that the invention is not limited to the particular embodiments shown and described herein, but that various changes and modifications may be made without departing from the spirit and scope of the invention as defined by the claims.

SYNTHETIC METHODS

The compounds of the inventions may be prepared by any process known in the field. Necessary starting materials may be obtained by standard procedures of organic chemistry. The compounds and processes of the présent invention will be better understood in connection with the following représentative synthetic schemes, which are intended as an illustration only and not limiting of the scope of the invention. Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art and such changes and modifications including, without limitation, those relating to the chemical structures, substituents, dérivatives, formulations and/or methods of the invention may be made without departing from the spirit of the invention and the scope of the appended claims.

In general, compounds of ?

X,

Formula (I) xOH can be prepared according to general Scheme 1 below. X_b X₂, Q, Z, and m in general Scheme 1 are the same as those described in the Summary section above.

Step 1 ï Q } O Step 2 ^Z' ⁰ H₂, Pd/C ^{1 0} m-l

Step 3 oxirane

Step 5 X|

NH₂OH ^X2^^N , m-l H

Step 4:

SOCI₂ θ X m-l

ΖΓη\)' m-l

Scheme 1

The starting material (1), a nitro-substituted 5-10 membered ring, can couple with an appropriate carboxylic ester to give intermediate (2), which can be subsequently reduced, for example with H₂, Pd/C, to an amino-substituted intermediate (3). The resulting intermediate (3) can react with oxirane to easily afford intermediate (4), which can be converted to intermediate (5) with high yield by reaction with a chlorinating reagent such as thionyl chloride or phosphorus pentachloride. Finally the hydroxylamination of intermediate (5) in NH₂OH can afford the target compound (6).

I

X|

Formula (II)

Compounds of ^λ i can be prepared according to general Scheme 2 below. X|, Rj, R₂, Z, and m in general Scheme 2 are the same as those described in the Summary section above.

O₂N

1. Pd/C, MeOH, H₂, rt

2. HCl (37%), reflux “

NO?

e.g dimethyl sulfate MH O acetonitrile, K^CO

nh₂oh

Scheme 2 d

*i

The starting material (1 ), a substituted 2,4-dinitroaniline can couple with an appropriate acyl chloride to give a N-acylated intermediate (2). The alkylation of N-acylated intermediate (2) with an alkylation agent such as iodomethane, methyltosylate, dimethylsulfate will lead to a dinitroaromatic intermediate (3). The réduction of intermediate (3), for example with H₂, Pd/C, followed by déhydration with acid will form benzimidazole intermediate (4). The intermediate (4) can react with oxirane to easily afford intermediate (5), which can be converted to intermediate (6) with high yield by reaction with a chlorinating reagent such as thionyl chloride or phosphorus pentachloride. Finally the hydroxylamination of intermediate (6) in NH₂0H can afford the target compound represented by formula (II).

Compound of

can be prepared according to general

Scheme 3 below.

N H, ^N°² I. Pd/C, MeOH, H₂, rt _N 0 2. HCl (37%), reflux *

NH₂OH

Scheme 3

Formula (III)

Toluene, reflux oxirane dimethyl sulfate N H q acetonitrile, K₂COj

The starting material (l), a 2,4-dinitroaniline can couple with an appropriate acyl chloride to 5 give a N-acylated intermediate (2). The alkylation of N-acylated intermediate (2) with an alkylation agent such dimethylsulfate will lead to a dinitroaromatic intermediate (3). The réduction of the dinitroaromatic intermediate (3), for example with H₂, Pd/C, followed by déhydration with acid will form benzimidazole intermediate (4). The intermediate (4) can react with oxirane to easily afford intermediate (5), which can be converted to intermediate (6) with high yield by reaction with a chlorinating reagent such as thionyl chloride. Finally the hydroxylamination of intermediate (6) in

NH₂OH can afford the target compound of formula (III).

Altematively, compound of

can be prepared according to general Scheme 4 below.

oxirane

O O i

1. Pd/C, MeOH, H₂, rt

2. HCl (37%), reflux *

NO₂

Nh₂ Toluene, reflux °^2Νγ^γ^ΝΟ2 dimcthyl sulfate q acetonitrile, K₂CO* ²

SOCI₂

S

Formula (III)

HO_v/^_n

L =

Scheme 4

The starting material (1), a 2,4-dinitroaniline can couple with an appropriate acyl chloride to give a N-acylated intermediate (2), The alkylation of N-acylated intermediate (2) with an alkylation agent such dimethylsulfate will lead to a dinitroaromatic intermediate (3). The réduction of the dinitroaromatic intermediate (3), for example with H₂, Pd/C, followed by déhydration with acid > will form benzimidazole intermediate (4). The intermediate (4) can react with oxirane to easily afford intermediate (5), which can be converted to intermediate (6) with high yield by reaction with a chlorinating reagent such as thionyl chloride. The hydrolysis of intermediate (6) in concentration

HCl will lead to the carboxylic acid intermediate (7), which can couple with O-(tetrahydro-2H10 pyran-2-yl)hydroxylamine to afford intermediate (8). Finally, the hydrolysis of intermediate (8) in acid will resuit the target compound of formula (III).

Alternatively, compound of

can be prepared according to general Scheme 5 below.

Ο₂Ν^ργΝΟ₂ CU₃NH_Î°^ÎNYy^NO^ Na₂S θ^γ^ρΝΙΙ;

' ^{Cl El0H} J H Na₂CO₃/H₂O h _CHCI₃ reflux 2h

Scheme 5

The starting material (l), l-chloro-2,4-dinitrobenzene can couple with an alkylamine to give the intermediate (2), which can be reduced to intermediate (3) with yield. The intermediate (3) can be acylated to form intermediate (4), which will undergo a déhydration reaction with acid to afford benzimidazole intermediate (5). The intermediate (5) can be subsequently reduced, for example with H₂, Pd/C, to an amino-substituted intermediate (6). The resulting intermediate (6) can react with oxirane to easily afford intermediate (7), which can be converted to intermediate (8) with high yield by reaction with a chlorinating reagent such as thionyl chloride or phosphorus pentachloride. Finally the hydroxylamination of intermediate (8) in NH₂OH can afford the target compound of Formular (III).

Altematively, compound of

can be prepared according to general Scheme 6 below.

o₂n

EtOH ch₃nhj°^2N

Formula (III)

Scheme 6 li₂SO.,

70deg/ 2h

ci l

Ii Na₂CO3 ! IhO

O 40 deg/ IMPa/ 3li

H, / Pd/C / McOH

CHCI3 reflux 2h

AcOI l, AcONa

SOCI₂, CHCI3

O-rt 2h

The starting material (1), l-chloro-2,4-dinitrobenzene can couple with an alkylamine to give the intermediate (2), which can be reduced to intermediate (3) with yield. The intermediate (3) can be acylated to form intermediate (4), which will undergo a déhydration reaction with acid to afford benzimidazole intermediate (5). The intermediate (5) can be subsequently reduced, for example with H₂, Pd/C, to an amino-substituted intermediate (6). The resulting intermediate (6) can react with oxirane to easily afford intermediate (7), which can be converted to intermediate (8) with high yield by réaction with a chlorinating reagent such as thionyl chloride or phosphorus pentachloride. The hydrolysis of intermediate (8) in concentration HCl will lead to the carboxylîc acid intermediate (9), which can couple with O-(tetrahydro-2H-pyran-2-yl)hydroxylamine to afford intermediate (10). Finally, the hydrolysis of intermediate (10) în acid will resuit the target compound of formula (III).

EXAMPLES

Example 1: Préparation of the formulation of compound of Formula (III) (also called NL-101 first génération formulation):

(1) Solution 1 : préparé 50% (v/v) acetic acid solution in DI water, store in room température;

(2) Solution 2: préparé 0.20% (w/v) NaOH solution in DI water, store in room température;

(3) Solution 3: préparé 200mg/ml of compound of Formula (III) in Solution 1 (i.e. 50% acetic acid): sonication 10-30 seconds will be very helpful to dissolve the compound;

(4) Finally, add 970uL of Solution 2 into a 30uL Solution 3, leading to a 6mg/ml solution of compound of Formula (III).

Example 2: Préparation a composition of compound of Formula (III) with hydroxypropyl βcyclodextrin (also called HPpCD-based NL-IOl formulation):

(1) Solution l : préparé 50% (v/v) acetic acid solution in DI water, store in room température;

(2) Solution 2: préparé 20% (w/v) hydroxypropyl β-cyclodextrin by adding each 80mL DI water into each 20 gram of hydroxypropyl β-cyclodextrin, vortex for 5 minutes, store in room température;

(3) Solution 3: préparé 5% (w/v) NaHCOj solution in DI water, store in room température; NaHCOs is used as a pH adjusting agent;

(4) Solution 4: préparé 200mg/ml of compound of Formula(IIl) in Solution l (i.e. 50% Acetic acid): sonication 10-30 seconds will be very helpful to dissolve the compound;

(5) Solution 5: 1:1 mix of Solution 2 and Solution 3;

(6) Add 30ul of Solution 4 into 970uL of Solution 5 and mix well, leading to a 6mg/ml solution of compound of Formula (III), with 10% hydroxypropyl β-cyclodextrin, 1.5% acetic acid, 2.5% NaHCO₃, and pH of 6-7;

(7) Filtration of the Solution: the formulations of compound of formula (III) from step (6) was filtered through a 0.2um pre-sterilized filter with >98% recovery;

(8) Préparation of a lyophilisate: the formulations from Step (7) were lyophilized to form lyophilisate as a powder. The resulted lyophilisate formulation was chemically stable at following températures, -20° C, 4° C and room température for at least 2 weeks. It can be stored at 4° C. for greater than 2 weeks without décomposition;

(9) Dilution study: the formulations from Step (7) were diluted with DI water (x 10 fold) and were chemically stable and remained in solution without précipitation (>12 hours).

Example 3: Préparation a composition of compound of Formula (III) with sulfobutylether βcyclodextrin (also called Captisol™-based NL-101 formulation):

(2) Solution 2: préparé 20% (w/v) sulfobutylether β-cyclodextrin by adding each 80mL DI water into each 20 gram of sulfobutylether β-cyclodextrin, vortex for 5 minutes, store in room température;

(3) Solution 3: préparé 5% (w/v) NaHCOi solution in DI water, store in room température; NaHCCh is used here as a pH adjusting agent;

(4) Solution 4: préparé 200mg/ml of compound of Formula(III) in Solution l (i.e. 50% Acetic acid): sonication 10-30 seconds will be very helpful to dissolve the compound;

(5) Solution 5: l : l mix of Solution 2 and Solution 3;

(6) Add 30ul of Solution 4 into 970uL of Solution 5 and mix well, leading to a 6mg/ml solution of compound of Formula (III), with 10% sulfobutylether β-cyclodextrin, 1.5% acetic acid, 2.5%NaHCO₃, andpHof6-7;

(8) Préparation of a lyophilisate: the formulations from Step (7) were lyophilized to form lyophilisate as a powder. The resulted lyophilisate formulation was chemically stable at following températures, -20° C, 4° C room température for at least 2 weeks. It can be stored at 4° C. for greater than 2 weeks without décomposition;

Example 4: Tris as an alternative pH adjusting agent:

Tris (CAS #: 77-86-1) is widely used as a component of pH buffer solution. Tris is used as excipient in some FDA approved drugs. It has a pKa of 8.30. Tris-Acetic acid buffer system has a pH range of 7-8, therefore, Tris may be idea pH adjusting agent for NL-101 formulation.

We successfully developcd a Tris containing HPBCD based NL-101 formulation with 6mg/ml NL-101, 15% HPBCD, 250mM Acetic acid, 333mM Tris, pH=7.4+/- 0.2, The formulation was prepared as following:

• Solution 1: préparé 200mg/ml NL-101 in 50% Acetic acid;

• Solution 2: préparé IM Tris, then dilute to 0.6666M: (Tris Base, F.W.121.14 g/mol).

• Solution 3: préparé 30% (w/v) HPBCD in lOOmM sodium acetate buffer (pH=5.4);

• Solution 4: 1:1 mix of Solution 2 and Solution 3.

Final solution: add 970uL of solution 4 into 30ul of solution l, mix well, leading to a 6mg/ml NL-101,15% HPBCD, 250mM Acetic acid, 333mM Tris, pH=7.4+/- 0.2.

As comparing to NaHCO3 as a pH adjusting agent, it is easier to accurately control the pH value of the Tris-containing HPBCD-based NL-101 formulation within the pH range of 7-8, since 5 the Tris-containing formulation is essential a Tris-Acetic acid buffer system with a theoretic buffer range of 7-8. The neutral pH value of Tris containing HPBCD based NL-101 formulation is a clear advantage for future clinical development.

Example 5: Single Dose IV Toxicity Study in Mice with lhe NL-101 first génération formulation:

A single dose of NL-101 l^st generaton formulation (20, 40, 60, 80 or 100 mg/kg) was slowly administered (iv, injection time>30 seconds), to mice and change in body weight was measured over 14 day to assess toxicity of the various doses of NL-101. We found that up to 60mg/kg of NL101 did not resuit in a significant change in body weight.

However, we found that this first génération formulation has many disadvantages such as low pH value, potential précipitation after i.v. injection, and sériés side effects such as damaged mice tail after iv injection. More seriously, sometimes we observed that quick iv injection (e.g injection time <5 seconds) of NL-101 may lead to mice sudden death.

Example 6: Single Dose IV Toxicity Study in Mice with the HPpCD-based NL-101 formulation:

A single dose of HPpCD-based NL-101 formulation (20, 40, 60, 80, 100, or 150 mg/kg) in

10% HPpCD was administered (iv) to mice and change in body weight was measured over 14 day to assess toxicity of the various doses of NL-101. We found that up to 60mg/kg of NL-101 did not resuit in a significant change in body weight.

We are surprised to found that the HPpCD-based NL-101 formulation can significantly reduce the cardiotoxicity in vivo. The mice even can survive under quick injection (t<5 seconds) of as high as 150mg/kg NL-101. More importantly, we didn’t observe cardiorespiratory stress in mice at therapeutically effective dose of 60mg/kg. In addition, this formulation also has many other advantages, such as neutral pH, clear and stable injection solution, no precipitate issue after iv injection, and no damaged mice tail after iv injection. Therefore, HPpCD-based NL-101 formulation will be an idéal formulation to be used in NL-101 MTD, PK, in vivo efficacy study, and IND enabling study. We are actively developing the HPpCD-based NL-101 formulation for future human clinical trial.

Example 7: Single Dose IV Toxicity Study in Mice with the Captisol™-based NL-lOl formulation:

A single dose of Captisol™-based NL-101 formulation (20,40, 60, 80,100, or 150 mg/kg) in 10% Captisol™ was administered (iv) to mice and change in body weight was measured over 14 day to assess toxicity of the various doses of NL-101. We found that up to 60mg/kg of NL-101 did not resuit in a significant change in body weight.

We are glad to found that the Captisol™-based NL-101 formulation can also significantly reduce the cardiotoxicity in vivo. The mice even can survive under quick injection (t<5 seconds) of as high as 150mg/kg NL-101. More importantly, we didn’t observe cardiorespiratory stress in mice at therapeutically effective dose of 60mg/kg. In addition, this formulation also has many other advantages, such as neutral pH, clear and stable injection solution, no precipitate issue after iv injection, and no damaged mice tail after iv injection. Therefore, Captisol™-based NL-101 formulation will be also an idéal formulation to be used in NL-101 MTD, PK, in vivo efficacy study, IND enabling study, as well as in future human clinical trial.

Example 8: Multiple Doses IV Toxicity Study in Mice with the HPpCD-based NL-101 formulation:

Multiple doses of HPpCD-based NL-101 formulation (60 mg/kg) in 10% HPpCD was administered (iv) to mice and change in body weight was measured to assess toxicity of the various doses of NL-101. We found that mice can well tolerate multiple doses of 60mg/kg of NL-101 without significant change in body weight. For example, the mice can be dosed at 60mg/kg at day 1, 4, 8, 11, 18, 25. Another feasible dosing scheme is 60mg/kg at day 1, 2, 8, 15, 22, 29.

Example 9: Multiple Doses IV Toxicity Study in Mice with the Captisol™-based NL-101 formulation:

Multiple doses of Captisol™-based NL-101 formulation (60 mg/kg) in 10% Captisol™ was administered (iv) to mice and change in body weight was measured to assess toxicity of the various doses of NL-101. We found that mice can well tolerate multiple doses of 60mg/kg of NL-101 without significant change in body weight. For example, the mice can be dosed at 60mg/kg at day 1, 4, 8, 11, 18, 25. Another feasible dosing scheme is 60mg/kg at day 1, 2, 8, 15, 22, 29.

Example 10: Efficacy of HPpCD-based NL-101 formulation on Human Non-Small Cell Lung Cancer A549 Xenograft Model

I

Animal: The balb/c mice aged 5 to 6 weeks were kept 5 per cage with an air filter cover under light (12 light/dark cycle, light on at 6H00) and température (22±l°C)-controlled environment. Ail manipulations of animais were performed under a sterilized laminar hood. The animais had ad libitum access to Purina mouse chow (Pro Lab PMH 4018, Trademark of Agway, Syracuse, N.Y.) and water. These animal studies were conducted according to the Guidelines for Care and Use of Experimental Animais.

Tumor Cell Culture: Human NSCLC A549 cells were cultured in the appropriated culture medium. The cells were harvested in their logarithmic growth phase for the préparation of tumor implantation.

Tumor Implantation: human tumor cells (2.5 to 5.0xl0⁶ cells) were implanted subcutaneously in 0.2 mL of medium containing 30% Matrigel on the two flanks of balb/c nu/nu mice through a l to 2 cm long 20-gauge needle.

Treatments: 2 to 3 weeks after tumor cell implantation, animais that developed s.c. solid tumors were selected and divided into several homogeneous groups (n=6 animais per group or dose) with respect to tumor size (100-200mm³). The animais were i.v. dosed with 60mg/kg of the following formulation at day 1,4, 8, 11, 18, 25.

1. Vehicle group: 10% HPpCD, 1.5% acetic acid. 2.5% NaHCO₃;

2. NL-101 group: 6mg/ml, 10% HPpCD, 1.5% acetic acid, 2.5% NaHCO₃;

3. Bendamustine group: 6mg/ml, 10% HPpCD, 1.5% acetic acid, 2.5% NaHCO₃;

Efficacy Evaluation: subcutaneous solid tumor measurements were performed on the day of first injection and at 4-day intervals thereafter. The two largest perpendicular diameters of each tumor were measured with calîpers and tumor sizes were estimated using the formula:

TV=LxW/2 where TV: tumor volume; L: length; W: width. The body weights of animais were also noted. The results are presented in Table below.

Group	Animal (start)	Animal (end)	Body weight (g)	Tumor weight (g)	Tumor volume (mm3)	T/C (%)
Control	10	10	28.8 ±1.7	1.94 ±0.3	2080.8 ±552.8	/
NL-101	6	6	27.6 + 2.9	0.88±0.2*	772.7 ±235.6*	37.7
Bendamustine	6	6	28.6+1.9	1.97 ±0.5	1716.5±550.6	71.9

* p<0.01 vs Control group

The above data shows that HPpCD-based NL-lOl composition has excellent in vivo efficacy in A549 xenograft model without evidence of significant general cytotoxicity and cardiotoxicity.

After extensive évaluation, the HPpCD-based NL-lOl formulation has been selected for 5 IND enabling study.

Claims

What is claimed is:

1. A pharmaceutical composition comprising (a) a cyclopolysaccharide, and (b) a compound of Formula (I)

X] Formula (I) , or a pharmaceutically acceptable sait thereof:

wherein m is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16;

Z is deleted, C(R_aR_b), O, S, C(O), N(R_a), SO₂, OC(O), C(O)O, OSO₂, S(O₂)O, C(O)S, SC(O), C(O)C(O), C(O)N(R_a), N(R_a)C(O), S(O₂)N(R_a), N(R_a)S(O₂), OC(O)N(R_a), N(R_a)C(O)O, N(R_a)C(O)S, or N(R_a)C(O)N(R_b), in which each of R_a and R_b, independently, is H, alkyl, alkenyl, or alkynyl;

X, and X2 independently, is halo or OSO₂R_C, in which R_c is alkyl, alkenyl, or alkynyl; and

Q is cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, aryl, or heteroaryl, each of which, independently, is optionally substituted with alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, halo, nitro, oxo, -C=NH, cyano, alkyl-R_d, ORj, OC(O)R_(t, OCfOJORd, OC(O)SR_d, SRu, C(O)R_d, CfOJORj, C(O)SRd, C(O)NR_cR_f, SOR_d, SO₂R<j, NR_eRf, or N(R_e)C(O)Rf, in which each of R_d, R_c, and Rf, independently, is H, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, halo, cyano, amine, nitro, hydroxy, or alkoxy.
2. The composition of claim 1, wherein X| and X2 independently, is halo; Z is deleted, CH2, O, CO, NH, SO₂, OC(O), C(O)O, C(O)S, NHC(O), C(O)NH, OC(O)NH, NHC(O)O, or NHC(O)S; m is 5, 6, 7, or 8; and Q is a 9-10 membered aryl or heteroaryl.
3. The composition of claim 2 wherein the compound is represented by Formula(II)

R, *2 _o PH , in which Rj and R₂ independently, is H, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, halo, -C=NH, amine, cyano, hydroxy, or alkoxy.
4. The composition of claim 3, wherein the compound is represented by Formula(III) HNOH /

Cl Formula (111)
5. The composition of claim 1 wherein said pharmaceutically acceptable sait is a hydrochloride sait, hydrobromide sait, methanesulfonate, toluenesulfonate, acetate, fumarate, sulfate, bisulfate, succinate, citrate, phosphate, maleate, nitrate, tartrate, benzoate, biocarbonate, carbonate, sodium hydroxide sait, calcium hydroxide sait, potassium hydroxide sait, tromethamine(Tris) sait, or mixtures thereof.
6. The composition of claim 5 wherein said pharmaceutically acceptable sait is a hydrochloride sait, methanesulfonate, toluenesulfonate, acetate, succinate, citrate, maleate, tartrate, or mixtures thereof.
7. The composition of claim 6 wherein said pharmaceutically acceptable sait is an acetate sait.
8. The composition of claim 1 wherein the cyclopolysaccharide is a cyclodextrin.
9. The composition of claim 8 wherein said cyclodextrin is selected from the group consisting of α-cyclodextrin or a dérivative thereof, β-cyclodextrin or dérivative thereof, and γcyclodextrin or a dérivative thereof.

ΙΟ. The composition of claim 9 wherein said cyclodextrin is β-cyclodextrin or a dérivative thereof.

11. The composition of claim 10 wherein said β-cyclodextrin is a hydroxypropyl βcyclodextrin, or sulfobutylether β-cyclodextrin.

12. The composition of claim 1 wherein the composition further contains a pH adjusting agent.

13. The composition of claim 12 wherein said pH adjusting agent is sodium bicarbonate, sodium carbonate, sodium hydroxide, potassium hydroxide, calcium hydroxide, tromethamine (Tris), or mixtures thereof.

14. The composition of claim 13 wherein said pH adjusting agent is a sodium bicarbonate, tromethamine (Tris), or mixtures thereof.

15. A composition comprising (a) a cyclodextrin, (b) a compound of Formula (III)

HN OH /

Cl Formula (III) , or a pharmaceutically acceptable sait thereof, and (c) a pH adjusting agent.

16. The composition of claim 15 wherein said cyclodextrin is β-cyclodextrin or a dérivative thereof.

17. The composition of claim 15 wherein said β-cyclodextrin is hydroxypropyl βcyclodextrin, or sulfobutylether β-cyclodextrin.

4l

18. The composition of claim 15 wherein said pharmaceutically acceptable sait is a hydrochloride sait, methanesulfonate, toluenesulfonate, acetate, succinate, citrate, maleate, tartrate, or mixtures thereof.

5 19. The composition of claim 15 wherein said pharmaceutically acceptable sait is an acetate sait.

20. The composition of claim 15 wherein said pH adjusting agent is bicarbonate, carbonate, sodium hydroxide, calcium hydroxide, potassium hydroxide, tromethamine, or mixtures
10 thereof.

21. The composition of claim 15 wherein said pH adjusting agent is a sodium bicarbonate.
15 22. The composition of claim 15 wherein said β-cyclodextrin is a hydroxypropyl βcyclodextrin or sulfobutylether β-cyclodextrin, said pharmaceutically acceptable sait is an acetate sait, and said pH adjusting agent is sodium bicarbonate.

23. The composition of claim 15 wherein the composition has a pH range from 6.0 to 9.0.

24. The composition of claim 15 in which the pH value of the composition is range from 7.0 to 8.0.

25. The composition of claim 15 wherein the cyclodextrin is présent at a concentration 25 from 0.5% to 40% weight/volume.

26. The composition of claim 15 wherein the cyclodextrin is présent at a concentration from 2.5% to 20% weight/volume.

30 27. A pharmaceutical dosage form comprising a pharmaceutical composition according to claim 15, wherein the pharmaceutical dosage form comprises 5 mg to 500 mg of compound represented by Formula (III).

28. A lyophilized préparation of the composition according to claim 15, wherein the préparation is packaged in a vial or other pharmaceutically acceptable container.

29, A composition comprising (a) a cyclodextrin, (b) a compound of Formula (III)

HNOH i

Cl Formula (IH) , or a pharmaceutically acceptable sait thereof, and (c) a pH adjusting agent in the manufacture of a composition for treating a neoplastic disease or an immune disease said composition being for administration either alone or in combination with other thérapies, to a subject in deed thereof.

30. A use according to claim 29, wherein said neoplastic disease is lung cancer, head and neck cancer, central nervous system cancer, prostate cancer, testicular cancer, colorectal cancer, pancreatic cancer, liver cancer, stomach cancer, biliary tract cancer, esophageal cancer, gastrointestinal stromal tumor, breast cancer, cervical cancer, ovarian cancer, uterine cancer, leukemia, lymphomas, multiple myeloma, melanoma, basal cell carcinoma, squamous cell carcinoma, bladder cancer, rénal cancer, sarcoma, mesothelioma, thymoma, myelodysplastic syndrome, or myeloproliferative disease.

31. A use according to claim 29, wherein said neoplastic disease is leukemia, lymphomas, multiple myeloma, lung cancer, breast cancer, myelodysplastic syndrome, myeloproliferative disease, pancreatic cancer, liver cancer, stomach cancer, esophageal cancer, gastrointestinal stromal tumor, cervical cancer, ovarian cancer, uterine cancer, or melanoma.

32. A compound of Formula(ll) or a pharmaceutically acceptable sait thereof:

R, ο ,ΟΗ >ΝΗ

X|^_NkA-N“^^m

Formula (II) wherein each of Ri and R₂ independently, is H, alkyl other than methyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, halo, -C=NH, amine, cyano, hydroxy, or alkoxy.