Identification of N-(5-tert-Butyl-isoxazol-3-yl)-N0-{4-[7-(2-morpholin-4-yl-ethoxy)imidazo-
[2,1-b][1,3]benzothiazol-2-yl]phenyl}urea Dihydrochloride (AC220), a Uniquely Potent, Selective, and Efficacious FMS-Like Tyrosine Kinase-3 (FLT3) Inhibitor

Qi Chao, Kelly G. Sprankle, Robert M. Grotzfeld, Andiliy G. Lai, Todd A. Carter, Anne Marie Velasco, Ruwanthi N. Gunawardane, Merryl D. Cramer, Michael F. Gardner, Joyce James, Patrick P. Zarrinkar, Hitesh K. Patel, and Shripad S. Bhagwat*
Ambit Biosciences, 4215 Sorrento Valley Boulevard, San Diego, California 92121 Received May 29, 2009
Treatment of AML patients with small molecule inhibitors of FLT3 kinase has been explored as a viable therapy. However, these agents are found to be less than optimal for the treatment of AML because of lack of sufficient potency or suboptimal oral pharmacokinetics (PK) or lack of adequate tolerability at efficacious doses. We have developed a series of extremely potent and highly selective FLT3 inhibitors with good oral PK properties. The first series of compounds represented by 1 (AB530) was found to be a potent and selective FLT3 kinase inhibitor with good PK properties. The aqueous solubility and oral PK properties at higher doses in rodents were found to be less than optimal for clinical development. A novel series of compounds were designed lacking the carboxamide group of 1 with an added water solubilizing group. Compound 7 (AC220) was identified from this series to be the most potent and selective FLT3 inhibitor with good pharmaceutical properties, excellent PK profile, and superior efficacy and tolerability in tumor xenograft models. Compound 7 has demonstrated a desirable safety and PK profile in humans and is currently in phase II clinical trials.

Introduction
Acute myeloid leukemia (AMLa) is the most common and aggressive form of acute leukemias in elderly people. AML patients treated with frontline chemotherapy of a combina- tion of an anthracycline derivative such as daunorubicin or idarubicin and a nucleoside analogue such as cytarabine (Ara-C) often respond initially but relapse or are refractory to frontline therapy and ultimately succumb to the disease. A large majority of myeloid cells in AML patients are found to express FMS-like tyrosine kinase 3 (FLT3), a trans-mem- brane tyrosine kinase that belongs to the type III split-kinase domain family of receptor tyrosine kinases (RTKs).1 Binding of FLT3 ligand to the receptor triggers receptor dimerization and autophosphorylation, leading to FLT3 kinase activation and subsequent signal transduction culminating in survival and proliferation of leukemic cells. Two types of activating mutations (internal tandem duplications (ITD) in the juxta- membrane (JM) domain and point mutations at residue D835 or deletion of I836 in the activation loop) are present in approximately one-third of all AML patients and are asso- ciated with poor response to chemotherapy, resulting in adverse clinical outcome.2
Treatment of AML patients with small molecule inhibitors
3-5
of FLT3 has been explored as a viable therapy. A number of potent small molecule FLT3 inhibitors such as lestaurtinib
(CEP-701), midostaurin(PKC-412), tandutinib(MLN-518or CT 53518), sunitinib (SU-11248), and sorafenib (BAY- 43-9006), shown in Figure 1, have been investigated as
3-6 potential therapeutic agents for the treatment of AML. However, these agents are found to be less than optimal for the treatment of AML because of lack of sufficient potency, suboptimal oral PK, or lack of adequate tolerability at efficacious doses that contribute ultimately to suboptimal inhibition of FLT3 in AML patients.7
FLT3 is expressed in dendritic cells, immature hematopoie- tic progenitors, and some mature myeloid and lymphoid cells. The role of FLT3 in bone marrow derived cells is relatively more clearly understood than in dendritic cells, the primary antigen presenting cells for T- cells. Activation of FLT3 induces proliferation and differentiation of dendritic cells, and this implies a potential role of FLT3 inhibitors in immune modulation.8,9 A potent FLT3 inhibitor, lestaurtinib, was found to protect mice from disease progression in an experi- mental autoimmune encephalomyelitis (EAE) model that mimics multiple sclerosis (MS) in humans. FLT3 inhibitors, therefore, are likely to be efficacious in animal models of other autoimmune disorders. While efficacy of lestaurtinib in psor- iasis patients is being studied (www.clinicaltrials.gov), it is not clear if any of the known FLT3 inhibitors in the clinic are being evaluated in other autoimmune disorders in humans.
We have developed a series of extremely potent and highly selective FLT3 inhibitors with good oral pharmacokinetic

*To whom correspondence should be addressed. Phone: 858-344- 4924. Fax: 858-334-2198. E-mail: [email protected].
a Abbreviations: FLT3, FMS-like tyrosine kinase 3; AML, acute myeloid leukemia; RTK, receptor tyrosine kinase; ITD, internal tandem duplications.
(PK) properties. One such compound, 1 (AB-530), shown in Figure 1, was found to regress tumors in a mouse xenograft model using MV4-11 cells.10 The aqueous solubility and oral PK properties at higher doses were found to be less than

pubs.acs.org/jmc Published on Web 09/16/2009 r 2009 American Chemical Society

Figure 1. Structure of known FLT3 inhibitors.

optimal for clinical development. We reasoned that the carboxamide group in 1 may be contributing to lower solubi- lity as well as to the absorption limited PK profile and that removal of this functionality coupled with addition of a water solubilizing group at a suitable position could address the issues in the resulting molecules. We were gratified to find the des-carboxamido-derivatives of 1 with a solubilizing group not only retained the potency and selectivity of 1 but also showed improved solubility and PK profiles. We describe herein the discovery of a novel series of FLT-3 inhibitors culminating in the identification and characterization of compound 7, N-(5-tert-butyl-isoxazol-3-yl)-N0 -{4-[7-(2-mor- pholin-4-yl-ethoxy)imidazo[2,1-b][1,3]benzothiazol-2-yl]phe- nyl}urea dihydrochloride (AC220), as a uniquely potent, selective, and efficacious compound suitable for clinical development.

Scheme 1. Synthesis of Compounds 6a, 6b, 6c, 7, 8, and 9a
Chemistry
The commercially available 2-aminobenzothiazole (2a), 2-amino-6-hydroxybenzothiazole (2b), and 2c (prepared as shown in Scheme 2) were condensed with 2-bromo-40 -nitroace- tophenone in refluxing ethanol to yield 3a, 3b, and 3c having the imidazobenzothiazoleringattacheddirectlytothe p-nitrophenyl ring (Scheme1).11 Reduction of the nitro group using iron and HCl or stannous chloride gave the corresponding amines, 4, which were coupled with 5-t-butyl-isoxazole-3-isocyanate, 5, to yield the ureas 6a, 6b, and 6c. Compound 5 was prepared from the commerciallyavailable 3-amino-5-t-butyl-isoxazolebytreat- ment with triphosgene. Mitsunobu reaction of 6b with various ω-substituted alcohols gave compounds 7-9 with different water solubilizing groups. Compound 7 could be prepared using an alternate method shown in Scheme 4 (vide infra).

a Reagents and conditions: (a) 2-bromo-40 -nitroacetophenone, EtOH, reflux; (b) Fe, iPrOH, HCl, or SnCl2 3 2H2O, EtOH; (c) toluene or CHCl3, 90-110 tiC; (d) ROH, Ph3P, diisopropyl azodicarboxylate, THF.

Compound 2c was prepared by the cyclization of 1-( p- aminophenyl)-4- methylpiperazine, 10, with ammonium thiocyanate in the presence of bromine and acetic acid (Scheme 2).12

Compounds 14 and 15 werepreparedasshownin Scheme3. The phenol 3b was alkylated using 1-bromo-3-chloropropane in DMF and potassium carbonate to give 11. Substitution of the chloro group of 11 using N-methylpiperazine or metha- nesulfonylpiperazine in the presence of tetra-n-butylammo-

Scheme 2. Synthesis of Intermediate 2ca

a Reagents and conditions: (a) NH4SCN, AcOH, Br2.

Scheme 3. Synthesis of Compounds 14 and 15a
nium iodide gave 12a and 12b, respectively, which were subjected to the sequence of reactions involving reduction of the nitro group using iron and HCl followed by coupling with 5 to yield compounds 14 and 15.
Two regioisomers of 7 were prepared starting from the corresponding hydroxy-substituted 2-aminobenzothia- zoles 16a and 16b as shown in Scheme 4. Condensation of 16a and 16b with 2-bromo-40 -nitroacetophenone as before gave 17a and 17b. Alkylation of the phenolic group with 4-(2-chloroethy)morpholine hydrochloride followed by re- duction of the nitro group with iron and HCl and coupling

a Reagents and conditions: (a) Br(CH2)3Cl, K2CO3, DMF, 80 tiC; (b) HN(CH2CH2)2NSO2Me, Bu4NI, DMF, 90 ti C; (c) Fe, 10% HCl, iPrOH, 90 tiC; (d) 5, CHCl3, reflux.

Scheme 4. Synthesis of Compounds 20a, 20b, and 7a

a Reagents and conditions: (a) 2-bromo-40 -nitroacetophenone, EtOH, reflux; (b) O(CH2CH2)2NCH2CH2Cl 3 HCl, K2CO3, DMF, 90 tiC; (c) Fe, NH4Cl, EtOH; (d) 5, toluene, 120 tiC.

Scheme 5. Synthesis of Compounds 26a, 26b, 27a, 27b, 27c, and 28a

a Reagents and conditions: (a) MeOH, H2SO4. (b) (1) 2-bromo-40 -nitroacetophenone, MeOCH2CH2OH, 40 tiC, 24 h; (2) 140 ti C, 18 h. (c) 2-bromo-40 – nitroacetophenone, EtOH, reflux. (d) SnCl2 3 2H2O, EtOH, relux. (e) 5, toluene, 110 tiC. (f ) LiOH 3 H2O, THF, water. (g) HNRR, EDC, HOBt, DMF. (h) BH3 3 Me2S, THF, 95 tiC.

Scheme 6. Synthesis of Compound 34a

a Reagents and conditions: (a) (1) 2-Bromo-40 -nitroacetophenone, MeOCH2CH2OH, 24 h, 40 tiC; (2) 140 ti C, 18 h. (b) HN(CH2CH2)2NEt, EDC, HOBt, DMF. (c) SnCl2 3 2H2O, EtOH, relux. (d) BH3 3 Me2S, THF, 95 ti C. (e) 5, toluene 110 tiC.

with 5 gave 20a and 20b. Compound 7 could also be prepared using this method as shown in Scheme 4. The yield of 7 using this reaction sequence was superior to that
from Scheme 1 and hence the Experimental Section de- scribes the synthesis of 7 using the reaction sequence in Scheme 4.

Table 1. In Vitro Kinase Binding, Cell Proliferation, and Kinase Selectivity Data

FLT3 MV4-11 selectivity scored

compd
R1, R2, R3
a,b Kd
(nM)
a,c
IC50
(nM)
S(10)

1 1.6 0.44 0.050
6a H, H, H 1.9 0.35 NDe
6b OH, H, H 2.7 0.15 ND
6c 4-methylpiperazin-1-yl, H, H 1.2 0.62 ND
7 2-morpholinoethoxy, H, H 1.6 0.56 0.079
8 2-(piperidin-1-yl)ethoxy, H, H 1.5 0.88 ND
9 3-morpholinopropoxy, H, H 0.99 0.79 ND
14 3-(4-methyl)piperazin-1-yl-propoxy, H, H 1.7 0.78 0.069
15 3-(4-(methylsulfonyl)piperazin-1-yl)propoxy, H, H 9.7 1.1 0.045
20a H, H, 2-morpholinoethoxy 5.2 9.3 0.059
20b H, 2-morpholinoethoxy, H 2.3 0.56 0.040
26a CO2H, H, H 1.6 1.0 0.064
26b CH2CH2CO2H, H, H 1.3 0.41 0.074
27a 4-ethylpiperazine-1-carbonyl, H, H 0.98 0.41 0.054
27b 3-(4-ethylpiperazin-1-yl)-3-oxopropyl, H, H 2.0 0.66 0.059
27c 3-(morpholin-4-yl)-3-oxo-propyl, H, H 1.5 0.23 0.035
28 3-morpholinopropyl, H, H 1.3 0.53 0.079
34 (4-ethylpiperazin-1-yl)methyl, H, H 1.1 0.38 0.064
sunitinib 0.47 4.3 0.41
a Each experiment was run in duplicate and the values shown are the average of the two. b Kd values are for binding to FLT3.13 c IC50 values are for inhibiting the proliferation of MV4-11 cell line. d Selectivity score or S(10), calculated as described in ref 15, is the ratio of kinase targets of the compound to the total number of kinases screened (202 distinct kinases in a panel of 227) at a 10 μM compound concentration; kinases were defined as targets if the primary screen showed >90% competition. e ND = not determined.

The compounds described above have the water solubiliz- ing group attached to a heteroatom such as O or N. The corresponding analogues with carbon atom were prepared as shown in Scheme 5. The commercially available ethyl 2-aminobenzothiazole-6-carboxylate (22a) and methyl 3-(2- aminobenzothiazol-6-yl)propionate (22b), prepared by ester- ification of the corresponding commercially available car- boxylic acid (21), were condensed with 2-bromo-40 – nitroacetophenone in 2-methoxyethanol for 22a or in ethanol for 22b to give the nitro ethyl esters 23a and 23b, respectively. Reduction of the nitro group followed by coupling with 5 gave the ureas 25a and 25b. Saponification of the esters with LiOH followed by coupling with appropriate amines using EDCI and HOBt yielded the amides 27a, 27b, and 27c. Reduction of the amide 27c using borane-dimethylsulfide in THF gave the corresponding amine 28.
Compound 34 with a one carbon tether to the ethylpiper- azine ring could not be prepared using the reaction sequence used for 28 because the borane reduction conditions led to extensive decomposition of the compound. The commercially available 2-amino-benzothiazole-6-carboxylic acid, 29, was condensed with 2-bromo-40 -nitroacetophenone in ethanol to give the nitro acid 30 (Scheme 6). Amide coupling with N-ethylpiperazine gave the amide 31. Reduction of the nitro group using stannous chloride followed by reduction of the carboxamide using borane gave the amine 33. Coupling of 33 with the isoxazole 5 gave 34 with an ethylpiperazine group as the solubilizing moiety.
In Vitro Pharmacology. The compounds described herein were tested for their binding affinity to the catalytic domain (amino acids 592-969) of FLT3 in a competition binding
assay with an ATP-competitive FLT3 inhibitor bound to a solid surface13 and were found to have high binding affinity to the kinase (Table 1). The compounds were then evaluated in a cellular assay measuring the inhibition of proliferation of the human leukemia cell line MV4-11, which is FLT3 dependent and harbors a homozygous FLT3-ITD mutation. The first compound in this series, 6a, an analogue of 1 with- out the carboxamide group, was found to be equipotent to 1 in the binding as well as cell proliferation assay (Kd of 1.9 nM for 6a vs 1.6 nM for 1; IC50 of 0.35 nM for 6a vs 0.44 nM for 1). This crucial result indicated that the differ- ence in conformation between 1 and 6a affects neither the binding of 6a to the enzyme active site nor its cell perme- ability and reinforced the rationale to optimize the series further.
The compounds reported here were almost uniformly found to be more potent in the cell proliferation assay than in the in vitro binding assay. A number of factors may contribute to this observed property of the molecules includ- ing changes in enzyme conformation between the in vitro system and cells and the difference in amount of protein present in the two assays. The cell assay was carried out in the presence of 0.5% serum proteins. The IC50 for the inhibition of proliferation decreased significantly for all compounds tested in the presence of 10% serum protein. In contrast to the series of compounds reported herein, sunitinib has ∼10-fold weaker cellular activity compared to the binding potency. The likely reasons for this behavior include ineffi- cient penetration of cell membranes and/or a preference for the enzyme conformation that is not favored in cells.14 Sorafenib, on the other hand, had a profile similar to the

series of compounds reported here with Kd = 13 nM and cell IC50 = 0.87 nM (data shown in refs 15 and 16).
Compound 6b and 6c were found to be similar in potency to 6a. Addition of polar groups either as a solubilizing group or as a handle to attach one, did not adversely affect the cell permeability. This encouraging result indicated that further optimization of the series for an optimal pharmaceutical profile may be possible with water solubilizing groups on the imidazobenzothiazole ring. A number of water solubilizing groups were attached to the hydroxyl group of 6b, a few of which are shown in Table 1. Compounds 7-9, 14, and 15 were all approximately equipotent in binding as well as cell proliferation tests, with the exception of the Kd value of 15, which was about 5-fold weaker than the others. Compound 7 had a binding constant of 1.6 nM, and it inhibited prolifera- tion of MV4-11 cells with IC50 = 0.56 nM. Replacing the morpholine ring of 7 with nitrogen containing solubilizing groups such as piperidine (8) or N-methylpiperazine (14) yielded compounds with equal potency but inferior PK properties, probably due to metabolism of the N-alkyl group (data not shown). Moving the morpholinoethoxy group of 7 to the 6-position (compound 20b) had a minimal effect on binding and cell potency, while moving that group to the 5-position (compound 20a) had a profound negative effect on cellular potency (∼17-fold less potent).
Analogues of the compounds above with a carbon atom in place of the oxygen of the aryloxyalkyl group (26a-b, 27a-c, 28, and 34) were all found to be equipotent to 7 in binding as well as cellular tests. However, these compounds had inferior PK properties (vide infra). Analogues of 7 with a nitrogen atom in place of the oxygen atom of the side chain were difficult to prepare. Compound 6c with an N-methyl- piperazine group attached directly to the 7-position of the imidazobenzothiazole ring was one of the few analogues prepared with a nitrogen atom as the handle to attach water solubilizing groups.
Evaluation of SAR of a number of compounds, some of which are included in Table 1, indicated that the morpholi- noethoxy group appended at the 7-position of the imidazo- benzothiazole ring rendered optimal overall properties to the molecule. Analogues of 7 with shorter or longer linkers to the basic nitrogen atom or with terminal alkylamino group or with the solubilizing group attached at 5- or 6-position of the imidazobenzothiazole ring were found to be inferior in PK properties.
Kinase Selectivity. The compounds shown in Table 1 were tested for their kinase selectivity in a panel of 227 kinases (202 distinct kinases excluding the mutants) using the Kino- meScan technology.13 The compounds were screened at 10 μM concentration and the kinases for which >90% competition was observed in the binding assay (measured as <10% of control experiment) were designated as “kinase targets of the compound”. A selectivity score, S(10), based on this single concentration primary screen, was calculated by dividing the “kinase targets of the compound” by the total number of kinases in the panel.15 Lower S(10) scores indicate higher selectivity and vice versa. As shown in Table 1, the selectivity score of the compounds in this series was <0.1, indicating that these are highly selective FLT3 inhibitors. Compound 7, having a selectivity score of 0.079 in a panel of 227 kinases, was also screened against a kinase panel of 402 kinases (359 distinct kinases excluding the mutants) and the S(10) score was calculated to be 0.061. This result indicates that 7 does not bind to a large number of the

additional kinases in the larger panel. All of the compounds in this series were found to be highly selective compared to sunitinib, which has a S(10) score of 0.47. These compounds are also significantly more selective than lestaurtinib and midostaurin.15,16 The kinase profile of tandutinib is similar to the series of compounds in Table 1, although the former is about 100-fold less potent than 7 in the cell proliferation assay.15,16 The selectivity profile of 7 is typical of this series of compounds. The binding affinity for every kinase in the panel was determined for 7,16 and the kinases with binding affinities within 10-fold of the Kd value for FLT3 (1.6 nM)
were the closely related RTKs, Kit, PDGFRR, PDGFRβ, RET, and CSF1R (4.8, 11, 7.7, 9.9, and 12 nM respectively). The additional kinases with Kd values within 100-fold of Kd for FLT3 were the closely related RTKs, FLT1, FLT4, DDR1, and VEGFR2 (41, 41, 81, and 87 nM, respectively).
These data suggest that 7 and analogues are among the most potent inhibitors of FLT3 in binding as well as pro- liferation assays and also among the most selective in a panel of 402 kinases. Additional details of the in vitro profile, kinase selectivity, mouse PK, and in vivo pharmacology of 7 are described in a separate paper.16
An advantage of screening compounds against a large panel of kinases is that one has the choice of guiding the lead optimization based on potency as well as a desired level of selectivity, and this helps reduce the time required for lead optimization.17,18 In fact, during the lead optimization of the current series of compounds culminating in the identification of 7 and analogues, we identified potent but less selective compounds (data not shown); however, we chose to avoid these compounds to maintain a high degree of selectivity. Our approach of extensive kinase profiling during lead optimization guided us to focus on the potent and selective compounds rather than potent but less selective compounds. It is important to note that while we chose to optimize compounds for high kinase selectivity, one can choose to dial in an appropriate level of selectivity with a desired list of kinases inhibited by the compounds.
Pharmacokinetics. A large number of compounds pre- pared during this SAR campaign had similar properties in kinase binding as well as cell proliferation tests. Therefore, we studied the pharmacokinetic profile of the more potent compounds in rats and in athymic nude mice. The PK profile in mice helped us prioritize compounds for the in vivo efficacy studies in the same species.
The compounds were dosed as a solution in 22% hydro- xypropyl β-cyclodextrin in water, except for 27a, which was dosed in 0.5% methylcellulose. The compounds differen- tiated themselves in the PK profile in mice and rats. For example, 7, at an oral dose of 10 mg/kg in mice, showed the best Cmax (3.8 μM) and AUC (35 μM 3 h) in comparison with its analogues shown in Table 2. Compound 28 was the next best compound with Cmax (2.09 μM) and AUC (21.32 μM 3 h). The morpholinoethoxy and morpholi- nopropyl side chains of 7 and 28 respectively offer the better in vitro, cellular, and PK profile than the other substituents. The PK profile of 27a and other analogues with terminal alkyl amines or N-alkyl piperazines (data not shown) were found to be inferior to 7. The poor PK profile of the carboxylic acid 26a is not surprising, and it is possible that the terminal alkylamine functionality of 15 contributes to its lower Cmax and AUC in mice than 7 (but similar to 28).

Table 2. Pharmacokinetic Profile of Compounds in Micea

compd
dose
(mg/kg)
Cmax
(μM)
Tmax
(h)
AUC(0-inf )
(μM 3 h)
oral t1/2
(h)

7 10 3.8 1.5 35.04 3.6
15 10 1.58 1.7 11.72 4.6
28 10 2.09 1.7 21.32 4.5
26a 10 0.49 0.5 0.67 6.7
27a 10 2.19 2 14.58 2.3
a Compounds were dosed orally to nude mice (n = 3, semilongitu- dinal sampling). All compounds dosed as oral gavage in hydroxypropyl β-cyclodextrin, except 27a, which was dosed in 0.5% methylcellulose.

Figure 2. Pharmacokinetic profile of 7, 15, and 28 in rats. Com- pounds were dosed orally (10 mg/kg) to Sprague-Dawley rats (n = 3); all compounds dosed as oral gavage in hydroxypropyl β-cyclodextrin.
A number of compounds were also profiled in Sprague- Dawley rats upon oral and intravenous dosing for their PK characteristics. Figure 2 shows the PK curves for com- pounds 7, 15, and 28 dosed orally at 10 mg/kg. The Cmax and AUC for 7 (2.16 μM and 20.66 μM 3 h, respectively) was clearly superior to 15 and 28. The clearance of 7 in rats was 7.11 mL/min/kg, half-life was 5.5 h, and the oral bioavail- ability was found to be 48%. The PK profile of 7 was found to be dose linear in mice and rats between 1 and 100 mg/kg doses, and its PK profile in dogs and monkeys was also acceptable (data not shown).
Efficacy in Tumor Xenograft Studies. The antitumor effi- cacy of 7 was assessed in a subcutaneous flank-tumor xenograft model in athymic nude mice using the MV4-11 cell line. Once the tumors had grown to about 200 mm3 size, compounds were dosed orally once a day for 28 days. The dosing was stopped, and the animals were observed for 32 additional days. Compound 7 was dosed at 1, 3, and 10 mg/kg (Figure 3). Sunitinib, used as a positive control, was dosed at 10 and 30 mg/kg.19 At 1 mg/kg of 7, tumor growth was completely inhibited during the dosing period, after which growth resumed. At 3 and 10 mg/kg of 7, tumors regressed almost completely and the tumor volume stayed suppressed after dosing was halted. At 3 mg/kg, tumors appeared to regrow after day 49 (21 days post last dose), while there was no sign of tumor regrowth until day 60 (32 days post last dose) in the animals treated with 10 mg/kg of 7. In contrast, the 10 mg/kg dose of sunitinib inhibited tumor growth during the first 14 days of dosing, after which they started regrowing even during the dosing period. The tumor profile of animals treated with 30 mg/kg of sunitinib was similar to the animals treated with 7 at 3 mg/kg dose in terms of regression and regrowth.
No body weight loss was observed in animals treated with 7 at all doses, indicating it is well tolerated in mice at efficacious doses. These results indicate 7 is a highly
Figure 3. Efficacy of compound 7 in a mouse tumor xenograft model. Compounds were dosed orally once a day to nude mice bearing a tumor with volume of ∼200 mm3, 10 animals per treat- ment group, drug treatment for 28 days followed by 32 days of observation, sunitinib used as a positive control, compound 7 was dosed as oral gavage in 22% hydroxypropyl β-cyclodextrin, suniti- nib was dosed in an aqueous citric acid vehicle.

efficacious and well tolerated compound in inhibiting MV4-11 tumor growth in animals.

Conclusion
Lead optimization of a potent and selective FLT3 inhibitor, 1, was carried out to improve its pharmaceutical profile. Removal of the carboxamide group in 1 and adding a water solubilizing group has resulted in a novel series of highly potent and selective compounds with a significantly improved pharma- ceutical as well as PK profile. Compound 7 was identified as one of the most potent and selective FLT3 kinase inhibitors we have characterized. The good aqueous solubility and other pharma- ceutical properties, excellent PK profile in different species, and superior efficacy and tolerability in tumor xenograft models have supported the selection of 7 as a clinical candidate.

Compound 7 has demonstrated an acceptable toxicology profile in GLP studies and was tested in AML patients in a phase I study.20 The acceptable tolerability and excellent PK profile in humans combined with signs of beneficial effect in AML patients have justified the progression of 7 into phase II clinical trials.21

Experimental Section
Chemistry: General Methods. Reactions involving air or moisture sensitive reagents were carried out under an argon atmosphere. If not otherwise specified, reactions were carried out at ambient temperature. Organic extracts were usually dried over anhydrous MgSO4. Proton NMR spectra were obtained in the deuterated solvents indicated on a Bruker Avance 300 referencing to tetramethylsilane. LC-MS analyses

were carried out on Shimadzu LC-MS 2010 EV system. Chemi- cal purity for all target compounds was determined by HPLC with a C18 column (Phenomenex Luna 5 μ C18(2) 100A, 250 mm ti 4.6 mm), detected by ELSD and MS, confirming g95% purity.
2-(4-Nitrophenyl)imidazo[2,1-b][1,3]benzothiazol-7-ol (3b): General Procedure A. A mixture of 2-amino-1,3-benzothia- zol-6-ol (20.0 g, 0.12 mol) and 2-bromo-40-nitroacetophenone (29.3 g, 0.12 mol) in 600 mL of ethanol was heated to reflux overnight. The reaction mixture was cooled to 0 ti C in an ice-water bath. Filtration of the resulting precipitate pro- vided 2-(4-nitrophenyl)imidazo[2,1-b][1,3]benzothiazol-7-ol (3b) as a yellow solid (17.0 g, 46% yield). 1H NMR (DMSO- d6) δ 10 (s, 1H), 8.9 (s, 1H), 8.3 (d, J = 8.6 Hz, 2H), 8.1 (d, J = 8.6 Hz, 2H), 7.8 (d, J = 8.7 Hz, 1H), 7.4 (s, 1H), 6.9 (d, J = 8.7 Hz, 1H). LC-MS (ESI) m/z 312 (M þ H)þ.
7-(2-Morpholin-4-yl-ethoxy)-2-(4-nitrophenyl)imidazo[2,1-b][1,3]- benzothiazole (18c): General Procedure B. To a mixture of 2-(4- nitrophenyl)imidazo[2,1-b][1,3]benzothiazol-7-ol (3b) (4.67 g, 15.0 mmol) in DMF (100 mL) was added potassium carbonate (5.52 g, 40 mmol) and 4-(2-chloroethyl)morpholine hydrochloride (3.72g,20mmol).Themixturewasheatedto60 ti Cfor6hand70 tiC for 12 h. The reaction mixture was poured into water (400 mL), filtered, and washed with water and ethyl ether to give 7-(2- morpholin-4-yl-ethoxy)-2-(4-nitrophenyl)imidazo[2,1-b][1,3]- benzothiazole (18c), which was used in the next reaction without further purification. 1H NMR (DMSO-d6) δ 9.00 (s, 1H), 8.31 (d, J=8.1 Hz, 2H), 8.09 (d, J=8.1 Hz, 2H), 7.92 (d, J=8.8 Hz, 1H), 7.72 (d, J=1.5Hz, 1H), 7.2(dd, J=8.8 and 1.5Hz, 1H), 4.17 (t, J= 5.4 Hz, 2H), 3.59 (t, J = 4.2 Hz, 4H), 3.34 (overlapping with solvent, 4H), 2.73 (t, J =5.4 Hz, 2H). LC-MS (ESI) m/z 425 (M þ H)þ.
2-(4-Aminophenyl)-7-(2-morpholin-4-yl-ethoxy)imidazo[2,1-b]- [1,3]benzothiazole (19c): General Procedure C. A mixture of 7-(2- morpholin-4-yl-ethoxy)-2-(4-nitrophenyl)imidazo[2,1-b][1,3]- benzothiazole (18c) (15.0 mmol) and ammoniumchloride(2.14 g, 40 mmol) in ethanol was heated to reflux, and then iron powder (8.378 g, 150 mmol) was added. The mixture was heated to reflux for 3 h and then immediately filtered through celite and washed with hot ethanol. The filtrate was concentrated to approximately 1/5 of the original volume, neutralized with saturated NaHCO3 solution, and extracted with dichloromethane. The combined extracts was dried over MgSO4 and concentrated to give 2-(4- aminophenyl)-7-(2-morpholin-4-yl-ethoxy)imidazo[2,1-b][1,3]- benzothiazole (19c) as an orange solid (4.067 g, 69% for two steps). 1H NMR (DMSO-d6) δ 8.40 (s, 1H), 7.86 (d, J=8.3 Hz, 1H), 7.70 (s, 1H), 7.51 (d, J=7.6 Hz, 2H), 7.22 (d, J=8.3 Hz, 1H), 6.61 (d, J=7.6 Hz, 2H), 4.34 (t, J=5.6 Hz, 2H), 3.76 (m,
4H), 2.99 (m, 6H). LC-MS (ESI) m/z 395 (M þ H)þ.
N-(5-tert-Butyl-isoxazol-3-yl)-N0-{4-[7-(2-morpholin-4-yl- ethoxy)imidazo[2,1-b][1,3]benzothiazol-2-yl]phenyl}urea Dihy- drochloride (7): General Procedure D. A suspension of 2-(4-aminophenyl)-7-(2-morpholin-4-yl-ethoxy)imidazo[2,1-b]- [1,3]benzothiazole (19c) (4.06 g, 10.3 mmol) and 5-tert-butyl- isoxazole-3-isocyanate (5) (1.994 g, 12 mmol) in toluene (60 mL) was heated at 120 tiC overnight. The reaction was quenched with a mixture of dichloromethane and water containing a little methanol, and the mixture was neutralized with saturated aqu- eous NaHCO3. The aqueous phase was extracted twice with dichloromethane, and the combined organic extracts were dried over MgSO4 and filtered. The filtrate was concentrated to a volume of about 20 mL and ethyl ether was added, resulting in the formation of a solid. The precipitate was collected by filtration, washed with ethyl ether, and dried under vacuum to give the free base of 7 (2.342 g, 41%). 1H NMR (DMSO-d6) δ 9.6 (br, 1H), 8.9 (br, 1H), 8.61 (s, 1H), 7.86 (d, J=8.9 Hz, 1H), 7.76 (d, J=8.0 Hz, 2H), 7.69 (d, J=1.3 Hz, 1H), 7.51 (d, J=8.0 Hz, 2H), 7.18 (dd, J=1.3 and 8.9 Hz, 1H), 6.52 (s, 1H), 4.16 (t, J=5.7 Hz, 2H), 3.59 (t, J=4.2 Hz, 4H), 3.36 (overlapping, 4H), 2.72 (t, J=5.7 Hz, 2H), 1.30 (s, 9H).

General Procedure E for Preparation of Hydrochloride Salt. The free base was dissolved in a mixture of dichloromethane (20 mL) and methanol (1 mL). A solution of 1.0 M HCl in ethyl ether (1.1 equiv for all compounds except 7, for which 2.5 equiv were used) was added dropwise, followed by addition of ethyl ether. The precipitate was collected by filtration to give N-(5- tert-butyl-isoxazol-3-yl)-N0 -{4-[7-(2-morpholin-4-yl-ethoxy)- imidazo[2,1-b][1,3]benzothiazol-2-yl]phenyl}urea dihydrochloride (7) (2.441 g, 98%). 1H NMR (DMSO-d6) δ 11.0 (br, 1H), 9.68 (s, 1H), 9.26 (s, 1H), 8.66 (s, 1H), 7.93 (d, J=8.9 Hz, 1H), 7.78 (m, 3H), 7.53 (d, J=8.7 Hz, 2H), 7.26 (dd, J=2.4 and 8.9 Hz, 1H), 6.53 (s, 1H), 4.50 (t, J=4.1 Hz, 2H), 3.97 (m, 2H), 3.81 (t, J=12.1 Hz, 2H), 3.6 (overlapping, 4H), 3.23 (m, 2H), 1.30 (s, 9H). LC-MS
29H32N6O4S 3 2HCl) C, H, N. C: calcd 54.97; found 54.54. H: calcd 5.22; found 5.87. N: calcd 13.26; found 13.16.
Kinase Competition Binding. KinomeScan competition bind- ing assays (www.kinomescan.com) were performed as described previously.13,15 Kinases were produced displayed on T7 phage or by expression in HEK-293 cells and tagged with DNA, binding reactions were performed at room temperature for one hour, and the fraction of kinase not bound to test compound determined by capture with an immobilized affinity ligand and quantitation by quantitative PCR.
MV4-11 Cell Proliferation. MV4-11 cells were cultured in Iscove’s media with 10% FBS and RPMI complete with 10% FBS, respectively. For proliferation assays, cells were cultured overnight in low serum media (0.5% FBS) and then seeded in a 96-well plate at 40000 cells per well. Inhibitors were added to the cells and incubated at 37 tiC for 72 h. Cell viability was measured using the Cell Titer-Blue cell viability assay from Promega (Madison, WI).
Pharmacokinetics. Precatheterized (jugular vein), male Spra- gue-Dawley rats (230-300 g; Charles River, Hollister, CA) and female athymic nude mice (20-35 g; Harlan, Livermore, CA) were acclimated at the vivarium for at least three days following delivery and prior to entering a study. Rats were fasted over- night before dosing. Compounds were administered orally (PO) by gavage at 10 mg/kg in 22% hydroxypropyl-β-cyclodextrin (HPBCD) or 0.5% methylcellulose. Blood was collected into K3EDTA tubes for plasma at 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, and 24 h postdose. Three rats were used per study, however, for blood collection in mice 150 μL retro-orbital bleeds were taken semilongitudinally using nine animals, taking 2-3 time-points per animal for a total of n = 3 plasma concentration-time curves. Plasma samples, calibration, and quality control stan- dards (25 μL) were extracted with four volumes of acetonitrile containing an internal standard and analyzed using LC-MS/MS analysis (Sciex 4000 Qtrap). Sample separation was achieved on a Zorbax SBC8 5 μm column (2.1 or 4.6 mm ti 50 mm) using a 0.8 mL/min flow rate over a 3 min gradient from 5 to 95% ACN containing 0.05% formic acid. The parent compound to frag- ment mass transitions were monitored. Pharmacokinetic para- meters were calculated from the normalized LC-MS/MS peak areas using a noncompartmental model and the linear trape- zoidal estimation method with WinNonlin (Pharsight v5.2).
Efficacy in Tumor Xenograft Studies. Xenograft studies were performed at Piedmont Research Center LLC (Morrisville, NC). MV4-11 human leukemia cells (1 ti 107) that had been cultured in Iscove’s Modified Dulbecco’s Medium supplemen- ted with 10% heat-inactivated fetal bovine serum, 100 units/mL penicillin G, 100 μg/mL streptomycin sulfate, 0.25 μg/mL amphotericin B, 2 mM glutamine, 0.075% sodium bicarbonate, and 25 μg/mL gentamicin were harvested during logarithmic
7 phase growth and resuspended at a concentration of 5 ti 10 cells/mL in 50% Matrigel matrix (BD Biosciences) and 50% PBS, and implanted subcutaneously into the right flank of female athymic nude mice (nu/nu, Harlan). Sixteen days later, mice were sorted into groups, each consisting of 10 mice with individual tumor sizes of 126-221 mm3 and group mean tumor

sizes of 160 mm3. Tumor dimensions were measured by caliper and tumor volume was calculated using the formula: tumor volume (mm3)=w2 ti l/2, where w=width and l=length in mm of an MV4-11 tumor. Each dose of drug was given in a volume of 0.2 mL per 20 g of body weight (10 mL/kg) and was adjusted for the body weight of the animal. Each animal was euthanized when its tumor reached the predetermined end point size of approximately 1000 mm3 or on the last day of the study (day 60), whichever came first.

Acknowledgment. We thank Dr. Robert C. Armstrong for his assistance in the analysis and graphical representation of the tumor xenograft data. We also thank Paul Gallant, Mazen W. Karaman, Antonio Torres, and the Ambit High Through- put Screening team for kinase profiling and Kd measurements.

Supporting Information Available: Experimental data for compounds 6a, 6b, 6c, 8, 9, 14, 15, 20a, 20b, 26a, 26b, 27a, 27b, 27c, 28, and 34. This material is available free of charge via the Internet at http://pubs.acs.org.

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